Journal of Ethnopharmacology 79 (2002) 1 – 11 www.elsevier.com/locate/jethpharm The effect of Nigella sati6a oil against the liver damage induced by Schistosoma mansoni infection in mice M.R. Mahmoud b, H.S. El-Abhar a,*, S. Saleh a a Department of Pharmacology and Toxicology, Cairo Uni6ersity, Kaser El-Aini Str. Cairo 11562, Egypt b Department of Pharmacology, Theodor Bilharz Research Institute, Imbaba, Giza, Egypt Received 31 January 2001; received in revised form 26 June 2001; accepted 10 July 2001 Abstract It has been reported that Nigella sati6a oil possesses anticestode and antinematode actions. Besides, it produced a hepatoprotective effect in some models of liver toxicity. Therefore, our aim in this work was to study the effect of the Nigella oil (N.O) on Schistosomiasis mansoni infected mice. The oil was given in two dose levels (2.5 and 5 ml/kg, orally for two weeks) either alone or in combination with praziquantel (PZQ), the drug of choice for the treatment of schistosomiasis. Three aspects of drug action were investigated, the effect on Schistosomiasis mansoni infection, the effect on liver functions, and on redox state. The parasitological investigation included worm distribution, oogram pattern and ova count. Furthermore, liver granuloma diameters were measured. The biochemical parameters were the serum level of L-alanine aminotransferase (ALT), g-glutamyl transferase (GGT), alkaline phosphatase (AP), albumin (Alb) and total protein. Moreover, to assess the antioxidant capability of the Nigella oil, four parameters were studied, viz., liver lipid peroxide (LPD) and reduced glutathione (GSH) contents and the activity of the defence enzyme superoxide dismutase (SOD) and lactate dehydrogenase (LDH). When the oil was given alone, it reduced the number of S. mansoni worms in the liver and decreased the total number of ova deposited in both the liver and the intestine. Furthermore, it increased the number of dead ova in the intestinal wall and reduced the granuloma diameters markedly. When N.O was administered in combination with PZQ, the most prominent effect was a further lowering in the dead ova number over that produced by PZQ alone. Concerning the biochemical parameters, infection of mice with S. mansoni produced a pronounced elevation in the serum activity of ALT, GGT, with a slight increase in AP level. However, it tended to reduce serum albumin level. These changes were accompanied with an alteration in the liver contents of LPD and GSH along with a significant decline in the activity of the cytosolic SOD and LDH. Administration of Nigella sati6a oil succeeded partially to correct the previous changes in ALT, GGT, AP activity, as well as the Alb content in serum. However, it failed in the liver to restore either LPD and GSH content or LDH and SOD activities to normal level. These results suggest that Nigella sati6a oil may play a role against the alterations caused by S. mansoni infection, an effect which may be induced partly by improving the immunological host system and to some extent with its antioxidant effect. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nigella sati6a oil; CAS 55268-74-1; Praziquantel; Schistosomiasis; Schistosoma mansoni; Liver damage; Lipid peroxide; Reduced glutathione; Superoxide dismutase; Lactate dehydrogenase; L-Alanine aminotransferase; g-Glutamyl transferase; Alkaline phosphatase; Serum albumin 1. Introduction The seeds of Nigella sati6a L. (Ranunculaceae), sometimes known as black seed, black cumin or habatul Barakah have long been used in the Middle East as a traditional medicine for a variety of complaints, headache, cough, flatulence, as a choleretic, antispasmodic and uricosuric (Boulos, 1983). In recent years, * Corresponding author. the seeds have been subjected to a range of pharmacological investigations. The data suggest that the seeds, and the major active constituent thymoquinone, exhibited hepatoprotective effect against liver damage induced by carbon tetrachloride (Al-Gharably et al., 1997; Nagi et al., 1999), and tert-butyl hydroper-oxide (Daba and Abdel Rahman, 1998). Most of the hepatoprotective drugs belong to the group of free radical scavengers, and their mechanism of action involves membrane stabilization, neutralization of free radicals and immuno-modulation. The inhibitory effects of the 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 1 0 - 5 2 M.R. Mahmoud et al. / Journal of Ethnopharmacology 79 (2002) 1–11 crude fixed oil and pure thymoquinone on membrane lipid peroxidation have been demonstrated (Houghton et al., 1995; Nagi et al., 1999). Besides, the essential oil was shown to have anthelmentic activity (Agarwal et al., 1979) and the seeds were effective against cestodes and nematodes (Akhtar and Rifaat, 1991). In view of these findings, and because in schistosomiasis, oxidative processes occurred at the site of granulomatous inflammation (Gharib et al., 1999), this study was undertaken to determine the possible antischistosomal effect of the expressed oil of N. sati6a in S. mansoni infected mice, and to relate this to its hepatoprotective and antioxidant actions. 2.4. Serum enzyme assessment 2. Materials and methods 2.5. Granuloma diameter and oogram studies 2.1. Animals Livers were immediately removed, weighed, and each one was divided into three parts. The first part of liver was fixed in 10% formalin and used for measuring granuloma diameter. The second part was used for the determination of ova count and developmental stages of schistosome eggs (Pellegrino et al., 1962). Segments of the intestine were also removed for ova counting and oogram studies. Male Swiss albino mice, weighing 18– 20 g bred at the Experimental Research centre of Theodor Bilharz Institute, Cairo, Egypt, were used for the study. They were housed in seven groups with free access to standard lab chow (El Nasr Lab Chem. Co, Egypt) and tap water. 2.2. Infection of animals Six groups were infected by tail immersion technique according to the method of Olivier and Stirewalt (1952) with 809 10 cercariae per mouse of an Egyptian strain of S. mansoni. Twenty four hours after the last dose of N.O, animals in all groups were weighed first, then killed by decapitation and the blood was collected. The serum was separated by centrifugation at 3000×g for 10 min and stored at −20 °C for the assay of ALT [EC 2.6.1.2] (Reitman and Frankel, 1957), GGT [EC 2.3.2.2] using Boehringer reagent kit (Mannheim, Germany), AP [EC 3.1.3.1] (Kind and King, 1954), total protein (Weichselbaum, 1946) and albumin (Doumas et al., 1971). The worms were recovered from the portal vein, mesenteric veins and liver by perfusion with cold saline (Pellegrino and Siqueira, 1956). 2.6. Li6er histopathology Tissue samples were immersed in 10% formalin for measurement of granuloma diameter. Samples were embedded in paraffin, sectioned and stained with Haematoxylin and Eosin. 2.3. Animal groups 2.7. Measurement of granuloma size After 7 weeks, infected animals were divided into 6 subgroups. Group 1 (n =9) received vehicle only (2%Cremophore EL, Sigma Chemical Company, St. Louis, MO, USA) and served as an untreated comparison group. Group 2 (n =11) was given orally praziquantel, [PZQ, CAS 55268-74-1] (Biltricide Bayer AG, Leverkusen, Germany) at the dose of 500 mg/kg for 2 consecutive days (Gonnert and Andrews, 1977). The drug was given as 2% aqueous suspension by the aid of Cremophore EL vehicle. Groups 3 (n =10) and 5 (n = 11) were treated with Nigella sati6a oil (N.O) (2.5 and 5 ml/kg, respectively, p.o. once per day for 2 weeks). Whereas groups 4 (n =10) and 6 (n =12) were treated with N.O in the previously mentioned dose regimen and in combination with PZQ (500 mg/kg, p.o.; on 2 successive days) starting with the first dose of PZQ. In addition, a normal control uninfected group [N] (n= 12) was used and received vehicle only (2% Cremophore EL, Sigma Chemical Company, St. Louis, MO, USA). From the liver of each animal, three paraffin sections were prepared and stained with haematoxylin and eosin (H&E). The sections were 5 mm thick and 250 mm distant from the preceding sections to avoid measuring of the same granuloma. Measurements were done only for granulomas containing a single egg in their centers. The mean diameter (mm) of each granuloma was obtained by measuring two diameters of the lesions at right angles to each other with the help of an ocular micrometer. Mean diameter of single granuloma (mm) = sum of two diameters 2 Lesions (100–150) from each group of at least 7– 10 animals were measured and calculated according to Mahmoud and Warren (1974). M.R. Mahmoud et al. / Journal of Ethnopharmacology 79 (2002) 1–11 2.8. Li6er enzyme assessment The third part of liver (0.5 g) was ice-cooled, homogenized in 5 ml phosphate buffer pH 7.4 (20% w/v) and the homogenate was divided into three portions. The first one was centrifuged at 30 000× g for 15 min at 4 °C. The supernatant was collected and further divided into two parts. The SOD [EC 1.15.1.1] activity was measured in the first supernatant part according to Marklund and Marklund (1974), and the results expressed as U/mg protein. The second supernatant part was used for determination of LDH [EC 1.1.1.27] activity, which was performed kinetically using BioMérieux test reagent kit. The GSH content was measured in the homogenate second portion by the reduction of Ellman’s reagent with the SH-group and the product measured colourimetrically at 412 nm (Ahmed et al., 1991). In the last portion of the homogenate, the LPD content was assayed by the method of Uchiyama and Mihara (1978) and the thiobarbituric acid-reactive substances (TBA-RS) value was expressed as nanomoles per gram of wet tissue weight. 2.9. Statistical analysis Results are given as means9 S.E.M. of 6–12 animals. Data were analysed using one way ANOVA followed by Duncan’s-test. The statistical significance of difference was taken as P 50.05. 3. Results In the infected control group total number of worms counted 2792.8, was divided between liver (43%) and portomesenteric vein (57%). Treatment of mice with N.O alone reduced the total number of worm burden at the two dose levels (22, 32%) respectively, especially those in the liver (Table 1). On the other hand, PZQ caused a marked reduction in worm burden reaching 3 98, with 60% of the worms shifted to the liver. This inhibition was slightly improved when PZQ was given in combination with 5 ml/kg N.O. The oogram pattern after PZQ treatment showed a complete disappearance of all immature ova from the wall of the intestine, a reduction in the number of mature ova and a four fold increase in dead ova. N.O alone affected the number of dead ova significantly, reduced the number of mature ova (19– 20%) while hardly affecting the immature ova. Combination with PZQ augmented its effect on the mature ova to reach 85% with the lower dose of N.O and 95% with the higher one (Table 2). In principle, the same observation was noted in egg load, where PZQ reduced it in both liver (71%) and intestine (93%), an effect which was slightly expanded upon combination with 5ml/kg dose level of N.O. N. sati6a oil alone showed a decrease also in the egg load mounted to 32.4 and 39.8% in the two dose levels studied, respectively (Table 3). The effect of infection and treatment on granuloma diameter, liver/body weight and spleen/body weight, are shown in Table 4. N.O suppressed the size of the developing liver granuloma in a dose dependent manner, being 15.8 and 24.3% for the two doses, respectively. The percent granuloma reduction by PZQ reached 17.6%. These results were supported with the pathological findings, in fact PZQ (Fig. 1b) was able to reduce the fibrocellular granuloma as compared with that of the control infected livers (H&E× 200) (Fig. 1a). Moreover, N.O. (5 ml/kg) alone showed a fibrocellular granuloma with less inflammatory cells (H&E×200) (Fig. 1c), while a marked reduction in size and granuloma cellularity was observed in mice treated with the combination therapy of PZQ and N.O. (H&E× 200) (Fig. 1d). As for the effect on liver functions (Table 5), N.O, especially the higher dose level, was found to reduce serum enzyme levels characteristic of hepatic damage induced by infection, as indicated by a lowering in the Table 1 Effect of Nigella sati6a oil (2.5 and 5 ml/kg, p.o.; for two weeks) alone or in combination with praziquantel (500 mg/kg, p.o.; for 2 consecutive days) on hepatic and portomesenteric worm distribution in mice infected with S. mansoni (means of 8–109 S.E.M.) Drugs and Doses Worm distribution Number in liver Infected control 11.7 9 1.5 Praziquantel 0.4 9 0.4* Nigella sati6a oil (2.5 ml/kg) 3.4 9 0.8* Praziquantel+Nigella sati6a oil (2.5 ml/kg) 0.4 9 0.2* Nigella sati6a oil (5 ml/kg) 4.1 9 1.4* Praziquantel+Nigella sati6a oil (5 ml/kg) 0.1 9 0.1* Total number of worms Worms reduction Worms in liver (%) (%) 27 9 2.8 0.6 9 0.5* 219 3.6* 0.6 9 0.4* 18.3 9 3.4* 0.4 9 0.3* 98 22 98 32 99 Number in p.m. 15.3 9 2.1 0.2 9 0.3* 17.6 9 4.3* 0.3 9 0.2* 13.4 9 2.6* 0.3 9 0.3* (*) As compared with infected control group (one-way ANOVA followed by Duncan test), PB0.05. 43 60 16 60 22 75 M.R. Mahmoud et al. / Journal of Ethnopharmacology 79 (2002) 1–11 4 Table 2 Oogram pattern showing percentage ova at different stages of maturity in walls of the intestine following treatment with Nigella sati6a oil (2.5 and 5 ml/kg, p.o.; for 2 weeks) alone or in combination with praziquantel (500 mg/kg, p.o.; for 2 consecutive days) in mice infected with S. mansoni (means 8–10 animals9 S.E.M.) Drugs and doses Immature stages % Stage I Infected control Praziquantel Nigella sati6a oil (2.5 ml/kg) Praziquantel+Nigella sati6a oil (2.5 ml/kg) Nigella sati6a oil (5 ml/kg) Praziquantel+Nigella sati6a oil (5 ml/kg) Stage II Stage III 8.4 9 2.5 0 25 94.4 0 13.7 9 3.9 0 4.9 9 2.1 16.2 9 2.6 31 9 6.5 7.5 9 2.6 6.9 9 3.4 0 0 0 11.9 9 3.6 0 Dead ova % 60.4 93.3 0* (−100) 12 9 3.4 81.1 9 13.8* (+575) 17.8 9 3.1* (+48.3) 95.8 9 4.2* (+698) 20.4 9 3.3* (+70) 98.59 1.5* (+720) Stage IV 13.3 9 6.8 0 0 Immature Ova % Mature ova % 0 31.9 9 3.7 6.7 9 2.1 0 0 27.6 94.5 18.9 9 13.8* (−31.5) 60.1 93.7 (−0.5) 22.1 92.9* (−19.9) 0*(−100) 4.2 94.2* (−84.7) 57.3 93.9 (−5.1) 22.3 93.0 (−19.2) 0*(−100) 1.5 9 1.5* (−94.6) (*) As compared with infected control group (one-way ANOVA followed by Duncan test), PB0.05. The percent changes from the corresponding infected control is shown in parentheses. Table 3 Effect of Nigella sati6a oil (2.5 and 5 ml/kg, p.o.; for 2 weeks) alone or in combination with praziquantel (500 mg/kg, p.o.; for 2 consecutive days) on egg load in liver and intestine of mice infected with S. mansoni (means of 8–109 S.E.M.) Drugs and doses Number of Ova/g tissue (×103) Liver Infected control Praziquantel Nigella sati6a oil (2.5 ml/kg) Praziquantel+Nigella sati6a oil (2.5 ml/kg) Nigella sati6a oil (5 ml/kg) Praziquantel+Nigella sati6a oil (5 ml/kg) 16.6 9 1.2 4.9 9 1.1* 11.0 9 1.4* 3.8 9 0.5* 9.8 91.2* 3.2 9 0.4* Intestine (−70.5) (−33.7) (−77.1) (−40.9) (−80.7) 21.1 9 1.3 1.59 3.4* 14.1 9 2.8* 1.3 90.2* 12.9 9 1.3* 1.5 9 0.3* Total no. of ova (−92.9) (−33.2) (−93.8) (−38.9) (−92.9) 37.7 6.4* 25.5* 5.1* 22.7* 4.6* (−83) (−32.4) (−86.4) (−39.8) (−87.8) (*)As compared with infected control group (one-way ANOVA followed by Duncan test), PB0.05. The percent decrease from the corresponding infected control is shown in parentheses. Table 4 Effect of Nigella sati6a oil (2.5 and 5 ml/kg, p.o.; for two weeks) alone or in combination with praziquantel (500 mg/kg, p.o.; for 2 consecutive days) on the diameter of hepatic granuloma (mm), % liver weight/body weight and% spleen weight/body weight in mice infected with S. mansoni (means of 8–109 S.E.M.) Drugs and doses Granuloma diameter (mm) % liver wt./body wt. % spleen wt./body wt. Normal control Infected control Praziquantel Nigella sati6a oil (2.5 ml/kg) Praziquantel+Nigella sati6a oil (2.5 ml/kg) Nigella sati6a oil (5 ml/kg) Praziquantel+Nigella sati6a oil (5 ml/kg) 0 201.99 7.3 166.39 6.2* 170.0 9 7.2* 165.6 9 4.3* 152.8 9 4.6* 160.8 94.7* 4.8 9 0.3 9.3 9 0.3@ 7.6 9 0.4*/@ (−18.3) 9.069 1.2@ (−2.5) 7.5 9 0.3*/@ (−19.4) 8.3 9 0.5*/@ (−10.8) 7.1 9 0.3*/@ (−23.7) 0.26 9 0.03 1.5 9 0.2@ 0.8 9 0.1*/@ (−46.7) 1.0 9 0.1*/@ (−33.3) 0.7 9 0.05*/@ (−53.3) 1.1 9 0.1*/@ (−26.7) 0.6 9 0.04*/@ (−60) (−17.6) (−15.8) (−17.9) (−24.3) (−20.4) As compared with infected (*) and normal (@) control groups (one-way ANOVA followed by Duncan test), PB0.05. The percent decrease from the corresponding infected control is shown in parentheses. raised levels of serum ALT (28%), GGT (43%) and AP (26%). It also tended to normalize the lowered levels of serum albumin. This dose level added a little on the PZQ action alone viz., on ALT and GGT. Fig. 2 depicts the LPD content in the seven experi- mental groups. Untreated infected mice showed a two fold elevation as compared with normal control animals. Treatment with PZQ alone (group 2) or in combination with the two dose levels of N.O, especially the higher one (groups 4 and 6), markedly prevented this M.R. Mahmoud et al. / Journal of Ethnopharmacology 79 (2002) 1–11 increase. The levels of GSH (Fig. 3), SOD (Fig. 4) and LDH (Fig. 5) in infected untreated mice were decreased significantly from normal values. Concerning SOD activity, PZQ alone tended to increase it, 5 yet not statistically significant except when combined with the 5 ml/kg dose of N.O. None of the treatment regimens was able to alter the levels of GSH or LDH. Fig. 1. Photomicrographs of liver granuloma in S. mansoni infected mice, sacrificed 9 weeks post infection. Drug treatment was started 7 weeks after infection with 80 S. mansoni cercariae; (a) infected control, exhibiting large fibrocellular granuloma with irregular outlines and excess inflammatory cells admixtured with collagenous fibrous tissue; (b) PZQ (500 mg/kg per day) for 2 consecutive days, showing well circumscribed small fibrocellular granuloma with scanty inflammatory cells and marked ova degeneration (H&E × 200). (c) Nigella sati6a oil (5 ml/kg/day) for 14 days, showing fibrocellular granuloma with less inflammatory cells; (d) combination therapy of PZQ and N.O. in the same dose regimen, eliciting marked reduction in size and granuloma cellularity (H&E × 200). Table 5 Effect of Nigella sati6a oil (2.5 and 5 ml/kg, p.o.; for two weeks) alone or in combination with praziquantel (500 mg/kg, p.o.; for 2 consecutive days) on serum ALT, GGT, AP, albumin and total protein in mice infected with S. mansoni (means of 8–109 S.E.M.) Drugs and doses Normal control Infected control Praziquantel Nigella sati6a oil (2.5 ml/kg) Praziquantel+Nigella sati6a oil (2.5 ml/kg) Nigella sati6a oil (5 ml/kg) Praziquantel+Nigella sati6a oil (5 ml/kg) Serum Enzymes ALT (U/ml) GGT (U/l) AP (kind and king U/100 ml) Total protein (g/100 ml) Albumin (g/100 ml) 72.7 9 6.4 118.3 9 9.2@ 83.6 9 8.3* (−29.4) 86.8 9 11.1* (−26.6) 84.29 9.4* (−28.9) 85.1 9 5.8* (−28.1) 80.0 9 5.9* (−32.3) 1.7 9 0.2 4.9 9 0.5@ 2.3 9 0.6* (−53.1) 3.0 9 0.5* (−36.8) 2.29 0.4* (−55.2) 2.8 90.6* (−42.9) 1.9 9 0.3* (−61.3) 11.2 9 0.7 14.0 9 0.9 11.6 9 0.8 (−17.1) 5.8 9 0.2 6.15 9 0.2 5.92 9 0.2 (−3.7) 3.1 9 0.3 2.5 9 0.3 3.3 9 0.5 (+32) 10.8 9 1.5 (−13.2) 6.01 9 0.2 (−2.3) 3.8 9 0.3* (+52) 12.0 9 1.4 (−14.3) 6.01 9 0.2 (−2.3) 3.8 9 0.3* (+52) 10.4 9 1.3* (−25.7) 6.09 90.2 (−9.7) 3.5 9 0.1* (+40) 11.6 90.5 (−17.1) 5.97 9 0.1 (−2.9) 3.9 9 0.3* (+56) As compared with infected (*) and normal (@) control groups (one-way ANOVA followed by Duncan test), PB0.05. The percent change from the corresponding infected control is shown in parentheses. 6 M.R. Mahmoud et al. / Journal of Ethnopharmacology 79 (2002) 1–11 Fig. 2. Changes in lipid peroxide (LPX) content in the liver after treatment with Nigella sati6a oil (2.5 and 5 ml/kg, p.o.; for two weeks) alone or in combination with praziquantel[PZQ](500 mg/kg, p.o.; on 2 successive days) in mice infected with S. mansoni (means of 6 – 12 animals 9 S.E.M.). As compared with normal (*) and infected (@) control groups (one-way ANOVA followed by Duncan test), PB 0.05. 4. Discussion Schistosomiasis is one of the most widespread parasitic infections. Hepatic fibrosis, resulting from S. mansoni infection, is of primary importance among chronic liver diseases worldwide. Early work by Warren (1973) has demonstrated that schistosome eggs or soluble egg antigens (SEA) are primarily responsible for host reactions associated with the infection. The eggs, swept into the liver, elicit T-cell dependent responses leading to macrophage activation and granuloma formation around them (Davis et al., 1974). The severity of the disease is determined by the extent of granuloma formation and eggs deposited in the tissues. Moreover, an imbalance between pro-and anti-oxidant processes has been demonstrated both in vitro (Chensue et al., 1984; Feldman et al., 1990) and in vivo (Gharib et al., 1999). This points to the important role of oxidative stress in mediating liver injury in schistosomiasis and it is likely to be due to an increased production of reactive oxygen intermediates by eosinophils and macrophages at the site of granulomatous inflammation (McCormick et al., 1996). In the present study, all infected mice showed a significant increase in serum ALT, GGT which are measures of liver affection. This seems consequent with hepatic cell damage and impaired cell membrane permeability (Ghanem et al., 1970) or due to heavy schistosome egg deposition (Giboda et al., 1994). Our results concerning the decrease in liver LDH activity, added further support to previous findings, that might be explained by the alterations in carbohy- M.R. Mahmoud et al. / Journal of Ethnopharmacology 79 (2002) 1–11 drate metabolism (Saleh, 1982). In addition, the liver SOD activity was drastically decreased, as well as the level of GSH, while hepatic lipid peroxide content showed a two fold elevation as compared with the liver of uninfected animals. These results are in line with that reported by Gharib et al. (1999). These findings show that on the one hand oxidative processes occurred at the site of granulomatous inflammation and on the other hand the antioxidant capacity of the liver decreased, leading to the generation of lipid peroxides which may play a central role in the pathology associated with schistosomiasis. The present study clearly shows that treatment of infected mice with praziquantel caused a significant antischistosomal effect. This is in agreement with previous studies (Gonnert and Andrews, 1977; El-Badrawy et al., 1988; El-Fakahany et al., 1993). Additional effects of praziquantel treatment are mild 7 improvement on hepatic SOD, and lipid peroxides. Similar results were reported by Shaheen et al. (1994). Praziquantel has direct effects on the schistosomes, producing contraction of the worms musculature (Pax et al., 1978), an influx of calcium into the worms (Wolde-Mussie et al., 1982), and disruption of the tegument (Bricker et al., 1983). It also depends on the host immune system for killing the parasite in vivo (Melhorn et al., 1981). It has excellent therapeutic effect and is well tolerated by patients (Smith et al., 1981). As for N. sati6a oil, the present study demonstrates that the oil produced an effective action against the hepatosplenic damaging effect, caused by S. mansoni infection, as shown by reducing the number of worms especially in the liver. It also lowered the ova count in the liver and in the intestine and reduced the granuloma size. Eventually, the liver functions were improved as Fig. 3. Changes in reduced glutathione (GSH) level in the liver after treatment with Nigella sati6a oil (2.5 and 5 ml/kg, p.o.; for two weeks) alone or in combination with praziquantel[PZQ](500 mg/kg, p.o.; on 2 successive days) in mice infected with S. mansoni (means of 6 – 12 animals 9 S.E.M.). (*) As compared with normal control group (one-way ANOVA followed by Duncan test), PB 0.05. 8 M.R. Mahmoud et al. / Journal of Ethnopharmacology 79 (2002) 1–11 Fig. 4. Changes in superoxide dismutase (SOD) activity in the liver after treatment with Nigella sati6a oil (2.5 and 5 ml/kg, p.o.; for 2 weeks) alone or in combination with praziquantel[PZQ](500 mg/kg, p.o.; on 2 successive days) in mice infected with S. mansoni (means of 6 – 12 animals 9 S.E.M.). As compared with normal (*) and infected (@) control groups (one-way ANOVA followed by Duncan test), PB 0.05. evidenced by a decrease of the elevated serum levels of ALT, GGT, AP and by a normalization of albumin. The effect of the oil could, at least partly, be attributed to drug-induced modulation of the immune response to schistosome eggs trapped in the liver. In murine schistosomiasis, a variety of cytokines and lymphokines are implicated as mediators of the granulomatous inflammatory response (Boros and Lukacs, 1992; Chensue et al., 1992). Accordingly, manipulation of cytokine levels can modify the intensity of the inflammatory response. Several studies point to the effect of N. sati6a on the immune system. The seeds were found to produce an increase in the ratio of helper to suppressor T cells and to enhance natural killer cell activity in normal volunteers (El-Qadi and Qandil, 1986). In vitro studies showed that N. sati6a enhanced the production of IL-3 by human lymphocytes and had a stimulatory effect on macrophages (Haq et al., 1995). Besides, immunomodulatory effect of N. sati6a purified proteins was found in mixed lymphocyte cultures and caused changes in the levels of cytokines (Haq et al., 1999). Another possible explanation for the regulation of the immune system by N. sati6a oil might be through altering the level of eicosanoids. A great deal of attention has recently been focused on the role of arachidonic acid metabolites in the regulation of the immune system. Among the most important factors involved in the pathogenesis of granuloma formation are PGs (Chensue et al., 1983; Goes et al., 1994) and LTs (Kunkel et al., 1984). Moreover, agents which inhibit cyclo-and lipo-oxygenase pathways (Kunkel et al., 1984) have been reported to reduce granuloma diameter. Thus, it is likely that Nigella oil, by altering the level of the various eicosanoids could enhance the immune M.R. Mahmoud et al. / Journal of Ethnopharmacology 79 (2002) 1–11 system resulting in disintegration of the worms or at least damaging them, arresting their maturation and decreasing egg-laying, as shown in this study. In fact, the fixed oil of N. sati6a, and its active constituent thymoquinone, inhibited the generation of thromboxane B2 and leukotriene B4 (Houghton et al., 1995). Moreover, the fixed oil increased the release of PGE2, inhibited the release of leukotrienes and histamine from normal and sensitized guinea-pig lungs (Salah et al., 1998) and nigellone, the carbonyl polymer of thymoquinone, inhibited the release of histamine from mast cells (Chakravaty, 1993). Patients with chronic schistosomiasis often fail to resolve hepatic fibrosis after eradication of the worms by anthelmintic treatment. The use of N. sati6a oil, an immunotherapeutic agent, as an adjunct to chemotherapy may be effective in augmenting the reduction of immunopathology, hepatic fibrosis and granuloma for- 9 mation. This policy has been attempted and was found of potential value in mice treated with praziquantel and the immunotherapeutic agent DAB-IL2 (Ali et al., 1995). The generation of reactive oxygen species may be an important mechanism by which cells of the immune system deal with the invading parasites. They may also initiate lipid peroxidation, a process that leads to membrane damage and the generation of further toxic products. Yet, it seems that the oil did not play a role in this respect. The difficulty in protecting the host from the parasite effect could be a consequence of the expression of defenses by the parasites directed against the toxic metabolites of leukocytes produced during inflammation (Brophy et al., 1995). In conclusion, the protective role of N. sati6a oil in S. mansoni infection may be attributed mainly to its immunomodulatory effect. Fig. 5. Changes in lactate dehydrogenase (LDH) activity in the liver after treatment with Nigella sati6a oil (2.5 and 5 ml/kg, p.o.; for 2 weeks) alone or in combination with praziquantel[PZQ](500 mg/kg, p.o.; on two successive days) in mice infected with S. mansoni (means of 6 –12 animals 9 S.E.M.). As compared with normal (*) and infected (@) control groups (one-way ANOVA followed by Duncan test), PB 0.05. 10 M.R. Mahmoud et al. / Journal of Ethnopharmacology 79 (2002) 1–11 References Agarwal, R., Kharya, M.D., Shrivastava, R., 1979. Antimicrobial and anthelmintic activities of the essential oil of Nigella sati6a Linn. Indian Journal of Experimental Biology 17, 1264 – 1265. Ahmed, A.E., Hussein, G.I., Loh, J., Abdel-Rahman, S.Z., 1991. Studies on the mechanism of haloacetonitrile-induced gastrointestinal toxicity: interaction of dibromo-acetonitrile with glutathione and glutathione-S-transferase in rats. Journal of Biochemical Toxicology 6, 115 – 121. Akhtar, M.S., Rifaat, S., 1991. Field trial of Saussurea lappa roots against nematodes and Nigella sati6a seeds against cestodes in children. Journal of the Pakistan Medical Association 41, 185 – 187. Al-Gharably, N.M., Badary, O.A., Nagi, M.N., Al-Sawaf, H.A., Al-Rikabi, A.C., Al-Bekairi, A.M., 1997. Protective effect of thymoquinone against carbon tetrachloride-induced hepatotoxicity in mice. Research Communications in Pharmacology and Toxicology 2, 41 – 50. Ali, M.R., Gabr, N.S., Philips, S.M., 1995. Reversal of pathology due to murine schistosomiasis mansoni by praziquantel (PZQ) and interleukin-2 targeted fusion toxin (DAB-IL2). In: Schistosomiasis Research Project International Conference on schistosomiasis, March 12 – 16, Cairo, Egypt. Boros, D.L., Lukacs, N.W., 1992. The role of egg antigens cytokines in granuloma formation in murine schistosomiasis mansoni. Memorias do Instituto Oswaldo Cruz 87, 75 – 79. Boulos, L., 1983. Medicinal Plants of North Africa. Reference Publications, Algonac, MI, p. 103. Bricker, C.S., Depenbusch, J.W., Bennett, J.L., 1983. Tegumental disruption of S. mansoni caused by praziquantel and RO11-3128. Zeitschrift für Parasitenkunde 69, 61 – 71. Brophy, P.M., Patterson, L.H., Brown, A., Pritchard, D.I., 1995. Glutathione S-transferase (GST) expression in human hookworm Necator americanus: potential roles for excretory-secretory forms of GST. Acta Tropica 59, 259 – 263. Chakravaty, N., 1993. Inhibition of histamine release from mast cells by nigellone. Annals of Allergy 70, 237 –242. Chensue, S.W., Kunkel, S.L., Higashi, G.I., Ward, P.A., Boros, D.L., 1983. Production of superoxide anion, prostaglandins and hydroxyeicosatetraenoic acids by macrophages from hypersensitivity-type (S. mansoni egg) and foreign body-type granulomas. Infection and Immunity 42, 1116 – 1125. Chensue, S.W., Quinlan, L., Higashi, G.I., Kunkel, S.L., 1984. Role of oxygen reactive species in Schistosoma mansoni egg-induced granulomatous inflammation. Biochemical and Biophysical Research Communications 122, 184 – 190. Chensue, S.W., Terebuh, P.D., Warmington, K.S., Hershey, S.D., Evanoff, H.L., Kunkel, S.L., Higashi, G.I., 1992. Role of IL-4 and IFN-gamma in Schistosoma mansoni egg-induced hypersensitivity granuloma formation. Orchestration, relative contribution, and relationship to macrophage function. Journal of Immunology 148, 900 – 906. Daba, M.H., Abdel Rahman, M.S., 1998. Hepatoprotective activity of thymoquinone in isolated rat hepatocytes. Toxicology Letters 95, 23 –29. Davis, B.H., Mahmoud, A.A.F., Warren, K.S., 1974. Granulomatous hypersensitivity to Schistosoma mansoni eggs in thymectomized and bursectomized chickens. Journal of Immunology 113, 1064 – 1067. Doumas, B.T., Watson, W.R., Biggs, H.G., 1971. Albumin standard and measurement of serum albumin with bromocresol green. Clinica Chimica Acta 31, 87 – 96. El-Badrawy, N.M., Hassanein, H.I., Botros, S.S., Nagy, F.M., Abdallah, N.M., Herbage, D., 1988. Effect of praziquantel on hepatic fibrosis in experimental S.mansoni. Experimental and Molecular Pathology 49, 151 – 160. El-Fakahany, A.F., Abdalla, K.F., El-Hady, H.M., Abdel-Aziz, S.M., Afifi, L.M., 1993. The effect of praziquantel treatment on the liver functions, worm burden, and granuloma size using two drug regimens in murine S. mansoni infection. Journal of the Egyptian Society of Parasitology 23, 877 – 886. El-Qadi, A., Qandil, U., November 1986. Black seed and the immune system. Fourth International Conference for Islamic Medicine, Karachi. Feldman, G.M., Dannenberg, A.M.Jr., Seed, J.L., 1990. Physiologic oxygen tensions limit oxidant-mediated killing of schistosome eggs by inflammatory cells and isolated granulomas. Journal of Leukocyte Biology 47, 344 –354. Ghanem, M.H., Girguis, F.X., El-Sawy, M., 1970. Assessment of liver functions in bilharzial hepatic fibrosis. Alexandrian Medical Journal 16, 35 –37. Gharib, B., Abdallahi, O.M., Dessein, H., De Reggi, M., 1999. Development of eosinophil peroxidase activity and concomitant alteration of the antioxidant defenses in the liver of mice infected with Schistosoma mansoni. Journal of Hepatology 30, 594 – 602. Giboda, M., Smith, J.M., Prichard, R.K., 1994. Reduction in tissue egg load and maintenance of resistance to challenge in mice infected with S. mansoni following combined treatment with praziquantel and an antifibrotic agent. Annals of Tropical Medicine and Parasitology 88, 385 – 395. Goes, A.M., Rezende, S.A., Gazzinelli, G., Doughty, B.L., 1994. Granulomatous hypersensitivity to S. mansoni egg antigens in human schistosomiasis IV. A role for prostaglandin-induced inhibition of in vitro granuloma formation. Parasite Immunology 16, 11 – 28. Gonnert, R., Andrews, P., 1977. Praziquantel, a new broad-spectrum anti-schistosomal agent. Zeitschrift für Parasitenkunde 52, 129 – 150. Haq, A., Abdullatif, M., Lobo, P.I., Khabar, K.S., Sheth, K.V., Al-Sedairy, S.T., 1995. Nigella sati6a: effect on human lymphocytes and polymorphnuclear leukocyte phagocytic activity. Immunopharmacology 30, 147 – 155. Haq, A., Lobo, P.I., Al-Tufail, M., Rama, N.R., Al-Sedairy, S.T., 1999. Immuno-modulatory effect of Nigella sati6a proteins fractionated by ion exchange chromatography. International Journal of Immunopharmacology 21, 283 – 295. Houghton, P.J., Zarka, R., de las Heras, B., Hoult, J.R., 1995. Fixed oil of Nigella sati6a and derived thymoquinone inhibit eicosanoid generation in leukocytes and membrane lipid peroxidation. Planta Medica 61, 33 – 36. Kind, P.R., King, E.G., 1954. Estimation of plasma alkaline phosphatase by determination of hydrolysed phenol with aminoantipyrine. Journal of Clinical Pathology 7, 322. Kunkel, S.L., Chensue, S.W., Plewa, M., Higashi, G.I., 1984. Macrophage function in the S. mansoni egg-induced pulmonary granuloma. Role of arachidonic acid metabolites in macrophage Ia antigen expression. American Journal of Pathology 114, 240 – 249. Mahmoud, A.A.F., Warren, K.S., 1974. Anti-inflammatory effect of tartaremetic and niridazole suppression of schistosoma egg granuloma. Journal of Immunology 112, 222 – 228. Marklund, S., Marklund, G., 1974. Involvement of the superoxide radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. European Journal of Biochemistry 47, 469 – 474. McCormick, M.L., Metwalli, A., Railsback, M.A., Weinstock, J.V., Britigan, B.E., 1996. Eosinophils from schistosome-induced hepatic granulomas produce superoxide and hydroxyl radical. Journal of Immunology 157, 5009 – 5015. Melhorn, H., Becker, B., Andrews, P., Thomas, H., Frenkel, J.K., 1981. In vivo and in vitro experiments on the effects of praziquantel on S. mansoni. Arzneimittel-Forschung/Drug Research 31, 544 – 555. M.R. Mahmoud et al. / Journal of Ethnopharmacology 79 (2002) 1–11 Nagi, M.N., Alam, K., Badary, O.A., Al-Sawaf, H.A., Al-Bekairy, A.M., 1999. Thymoquinone protects against carbon tetrachloride hepatotoxicity in mice via an antioxidant mechanism. Biochemistry and Molecular Biology International 47, 153 – 159. Olivier, L., Stirewalt, M.A., 1952. An efficient method for exposure of mice to cercariae of S.mansoni. Journal of Parasitology 38, 19 – 23. Pax, R., Bennett, J.L., Petter, R.A., 1978. A benzodiazepine derivative and praziquantel: Effects on musculature of Schistosoma mansoni and Schistosoma japonicum. Naunyn-schmiedebergs Archives of Pharmacology 304, 309 – 314. Pellegrino, J., Siqueira, A.F., 1956. Tecnica para perfusae para colhejta de Schistosoma mansoni: infestadas. Revista Brasiliera de Malariologia e doencas Tropicais Publicacoes Avulsas 8, 589 – 597. Pellegrino, J., Oliveria, C.A., Faria, J., Cunha, A.S., 1962. New approach to the screening of drugs in experimental Schistosomiasis mansoni in mice. American Journal of Tropical Medicine and Hygiene 11, 201 – 215. Reitman, S., Frankel, S., 1957. A colorimetric method for determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. American Journal of Clinical Pathology 28, 56 – 63. Saleh, S., 1982. Some biochemical changes in schistosomiasis mansoni. Armed Forces Medical Journal XXIV, 98 – 115. Salah, N., Mahran, L.G., Saleh, S., El-Denshary, E.S., December 11 1998. Contribution of the antioxidant effects of Nigella sati6a oil to its inhibitory action on mediator release. XXVI Conference of Pharmaceutical Sciences, Cairo, Egypt. Shaheen, A.A., Abdel-Fattah, A.A., Ebeid, F.A., 1994. Effect of praziquantel treatment on lipid peroxide levels and superoxide dismutase activity in tissues of healthy an Schistosoma mansoni infected mice. Arzneimittel-Forschung/Drug Research 44, 194 – 196. Smith, D.H., Highton, R.B., Robert, I.M., 1981. Preliminary observations on the treatment of Schistosomiasis mansoni with Biltricide in Kenya. Arzneimittel-Forschung/Drug Research 31, 594 – 596. Uchiyama, M., Mihara, M., 1978. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Analytical Biochemistry 86, 271 – 278. Warren, K.S., 1973. The pathology of schistosome infections. Helminthological Abstract Series A 42, 591 – 633. Weichselbaum, T.E., 1946. A accurate and rapid method for the determination of proteins in small amounts of blood, serum and plasma. American Journal of Clinical Pathology 16, 40 – 49. Wolde-Mussie, E., Vande-Waa, J., Pax, R.A., Fetterer, R., Bennett, J.L., 1982. Schistosoma mansoni: calcium efflux and effects of calcium-free media on responses of the adult male musculature to praziquantel and other agents inducing contractions. Experimental Parasitology 53, 270 – 278. Journal of Ethnopharmacology 79 (2002) 13 – 16 www.elsevier.com/locate/jethpharm Effect of Zizyphus spina-christi Willd aqueous extract on the central nervous system in mice B. Adzu, S. Amos, S. Dzarma, C. Wambebe, K. Gamaniel * Department of Pharmacology and Toxicology, National Institute for Pharmaceutical Research and De6elopment, P. M. B. 21, Garki, Abuja, Nigeria Accepted 17 July 2001 Abstract The effects of the aqueous extract of Zizypus spina-christi Willd root bark against exploratory behaviour, spontaneous motor activity (SMA), motor coordination (Rota-rod performance) and pentobarbital-induced hypnosis were investigated in mice. The extract induced a significant (P B 0.05) dose-dependent reduction in exploratory behaviour and SMA when administered orally. It also prolonged pentobarbital sleeping time but failed to inhibit motor coordination (rota-rod performance) in the experimental mice. These results suggest that the extract contained some constituents that depress the central nervous system, which may not be due to neuromuscular blockade. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Exploratory behaviour; Spontaneous motor activity; Motor coordination; Pentobarbital sleep 1. Introduction The plant Zizyphus spina-christi Willd is of the Rhamnaceae family, and it grows wild in northern Nigeria. It is characterised by thorny branches used as a hedge to form defensive enclosure. The fruit have sweet edible pulp. Medicinally, the leaves are applied locally to sores (Dalziel, 1937) as wound powder and antiseptics (Fleurentin and Pelt, 1982) and was reported to exhibit hypoglycemic activity against streptozotocindiabetics rats (Glombitza et al., 1994) and antibacterial efficacy against Gram-positive strains (Ali et al., 2001), while its root bark is used in folk medicine as a remedy against pain. Major chemical constituents of the plant had been investigated and reported (Ikram and Tomlinson, 1976; Mahran et al., 1996). Our previous studies on the aqueous extract of the root bark revealed the presence of saponins, tannins and glycosides. The symptoms of toxicity of the extract include severe depression and dyspnoea. The extract also exhibits anti-nociceptive properties with both peripheral and central aspects (Adzu et al., 2001). In this * Corresponding author. Tel.: + 234-9-5239089; fax: + 234-95231043. E-mail address: ksgama@yahoo.com (K. Gamaniel). study, we evaluated the nature of the central effects of the extract by measuring the exploratory behaviour, spontaneous motor activity (SMA), rota-rod (a tread mill) performance and pentobarbital sleeping time in mice. 2. Material and methods 2.1. Plant material Roots of the plant were collected by Achaba Lugudu (a herbalist) at Midlu – Vapura, Adamawa State, Nigeria, on 20th February 2001. The identify of the plant was authenticated at the Department of Medicinal Plant Research and Traditional Medicine, National Institute for Pharmaceutical Research and Development (NIPRD) Abuja, Nigeria. A voucher specimen of the plant was deposited at the Taxonomic Unit of the Department (No. 4108). The root bark was separated and cleaned, then dried under shade. The dried material was powdered and 100 g were macerated in 1 l of cold distilled water for 24 h with occasional shaking, then filtered through Whatman No. 1 filter paper and freezedried using Lyovac, GT2 (Germany). This gave a yield of 5.9 % w/w and was dissolved in double distilled water at the desired concentration just before use. 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 4 8 - 8 14 B. Adzu et al. / Journal of Ethnopharmacology 79 (2002) 13–16 2.2. Reagent Sodium pentobarbital from Sigma (Sigma Chemical Co., USA) was used. Normal saline (20 ml/kg) were used as the control in all the experiments. 2.3. Animal Swiss albino mice (5– 7 weeks) weighing 20–26 g obtained from the Animal Facility Centre, NIPRD, were used for the study. They were housed in plastic cages under standard condition of 12-h light:12-h dark circle and fed with standard diet (Ladokun Feeds Ltd., Ibadan, Nigeria), and tap water ad libitum during the quarantine period. Sixteen hour prior to experiment, they received no food. an animal fell on the rod during the 3 min trial was recorded in seconds (Fujimori and Cobb, 1965). 2.4.4. Pentobarbital sleeping time A total of 18 mice grouped into three (n =6) were used. Groups 1 and 2 received extract (100 and 200 mg/kg) p.o while the control group received normal saline. Sodium pentobarbital (25 mg/kg) were administered i.p. to all animals 1 h after receiving either extract or saline. Time elapsed between the administration of pentobarbital until the loss of righting reflex was recorded as the onset of sleep, while the time from the loss of righting reflex (in which mouse cannot roll back when turned over), to recovery was recorded as the sleeping time (Soulimani et al., 2001). 2.5. Data analysis 2.4. Pharmacological e6aluation 2.4.1. Exploratory beha6iour pattern The head-dip test was used for this study (Perez et al., 1998). Mice were divided into three groups of six mice each. Groups 1 and 2 received extract (100 and 200 mg/kg) p.o. while control group received normal saline. The mice were singly placed on LETICA (Spain) instrument with 16 evenly spaced holes and a counter (LE 3333) 1 h after treatment. The number of times the mice dipped their heads into the holes during the 5 min trial was automatically counted both for control and treated groups. 2.4.2. Spontaneous motor acti6ity (SMA) Motor activity of the mice were recorded using a LETICA activity cages (LE 886) and multicounter (LE 3806). The apparatus consisted of four ventilated monitor cages. Mice were placed in each cage and activity was automatically recorded 30 min after treatment for 6 min and at every 30 min interval for a total of 120 min. The extracts (100 and 200 mg/kg) were administered to two groups of mice p.o. while the control group received normal saline (n= 6). Results expressed as meancounts were compared with that of the control at each time interval (Irwin, 1968). 2.4.3. Test for motor coordination (rota-rod performance) This was performed using a horizontal rotation rod (Ugo Basile-7560, Milano, Italy) set at a rate of 16 revolutions per min. Mice that were able to remain on the rod longer than 3 min were selected and grouped into three (n= 6). Groups 1 and 2 received extract (100 and 200 mg/kg p.o) while control groups received saline. The test consisted of placing mice 30 min after treatment and at intervals of 30 min post treatment and at intervals of 30 min for a total of 120 min. The time Results were expressed as Mean9S.E.M. Differences were estimated by means of analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparison. Effects were considered significant at P B0.05. 3. Results The aqueous extract of Z. spina-christi Willd root bark caused dose dependent reduction in head-dip responses in the mice. The observed effects in the treated groups were significantly different (P B0.05) from that of the control groups (Table 1). The SMA was gradually inhibited after 30 min, reaching a peak 30 min later. This level was more or less maintained throughout the 120 min duration of the experiment (Fig. 1). The extract did not exhibit significant effect on the rota-rod performance of the mice (Table 2). Results as presented in Table 3 showed that the extract prolonged the duration of pentobarbital (25 mg/kg) sleep but did not affect the onset significantly. The effect is more pronounced among the group that received 200 mg/kg of the extract. Table 1 Effect of aqueous extract of Z. spina-christi root bark on exploratory behaviour in mice Treatment (p.o.) Control saline (20 ml/kg) Extract (100 mg/kg) Extract (200 mg/kg) a Mean Head-dips in 5 min Pre-dose 1 h Post-treatment 45.5 9 4.39 51.25 9 6.33 47.5 9 5.98 39.25 94.39 9.5 90.76a 3.5 90.64a F (2,15)= 3.68; PB0.05. B. Adzu et al. / Journal of Ethnopharmacology 79 (2002) 13–16 15 Fig. 1. Effect of the aqueous extract of Z. spina-christi root bark on SMA in mice. 4. Discussion The results of this study show that the aqueous extract of Z. spina-christi root bark may have some sedative activity. This is evident from the marked inhibition of the exploratory behaviour in mice as shown by the reduction of the head-dip count (File and Pellow, 1985). Similarly, since SMA is a measure of the level of excitability of the central nervous system (Mansur et al., 1971) and agent that suppress this activity may do so through central depressant activity, the evidence that the extract suppressed this activity in mice supports the above speculation. Furthermore, prolongation of pentobarbital sleeping time is additional indication of the depressant action of the extract. Fujimori (1965) relates prolongation of barbital hypnosis to sedative/hypnotic property. These findings correlate with observations of Morishita et al. (1987) on the aqueous extract of zizyphus seeds. The extract did not attenuate motor coordination (rota-rod performance) suggesting that actions may not Table 2 Effect of the aqueous extract of Z. spina-christi root bark on motor coordination (Rota-rod performance) Time (min) 30 60 90 120 Cut-off time (s) 180 180 180 180 Endurance time (s) 100 mg/kg 200 mg/kg 177.3 175.8 173.8 175.9 176.7 179.2 176.2 171.8 Table 3 Effects of the aqueous extract of Z. spina-christi root bark on pentobarbital-induced sleeping time (s) Treatment (p.o.) Onset of sleep (min) Duration of sleep (min) Control saline (20 ml/kg) Extract (100 mg/kg) Extract (200 mg/kg) 4.83 9 0.11 31.55 9 1.08 4.30 9 0.17 4.05 9 0.11 36.78 9 0.77a 47.68 9 0.35a a F (2,15)= 3.68; PB0.05. be achieved via neuromuscular blockade. Rather, the effects might involve neurons that control central depressant activities. The actions of the extract in this way may be related to its saponin content (Adzu et al., 2001). Saponins bind on sensory nerve terminals and have been implicated in opioid receptor mechanism (Huong et al., 1995). However, these may not be the exclusive mechanism for the sedative action of Z. spinachristi, since other central neuronal mechanisms such as GABA system and the reticular activating system have not been studied. Further work is presently on going in our Institute to isolate the possible active principles for a study in this direction. Acknowledgements The authors are grateful to Mal. Achaba Lugudu of Jilang, Kafin-Hausa, Adamawa State for sourcing the 16 B. Adzu et al. / Journal of Ethnopharmacology 79 (2002) 13–16 plant material; Hauwa Abdullahi and Adamu Mohammed for their technical assistance and Charles Balogun for typesetting the manuscript. References Adzu, B., Amos, S., Wambebe, C., Gamaniel, K., 2001. Anti-nociceptive activity of the aqueous extract of Zizyphus spina-christi root bark. Fitoterapia 72, 344 – 350. Ali, N.A.A., Julich, W.-D., Kusnick, C., Lindequist, U., 2001. Screening of Yemeni medicinal plants for antibacterial and cytotoxic activities. Journal of Ethnopharmacology 74, 173 – 179. Dalziel, J.M., 1937. The useful plants of West Tropical Africa. Crown Overseas Agent for the Colonies. London pp. 300. File, S., Pellow, S., 1985. The effect of triazolobenzodiazepines in two animal tests of anxiety and on Hole-Board. British Journal of Pharmacology 86, 729 – 735. Fleurentin, J., Pelt, M.J., 1982. Repertory of drugs and medicinal plants of Yemen. Journal of Ethnopharmacology 6, 86 – 108. Fujimori, H., 1965. Potentiation of barbital hypnosis as an evaluation method for central nervous system depressant. Psychopharmacology 7, 374 – 377. Fujimori, H., Cobb, D., 1965. Central nervous system depressant activity of MA 1337 3-(3, 4-m-chlorophenyl-1-piperazyl propyl) -2, 4 (1H, 3H) quinozolinedine hydrochloride. Journal of Pharmacology and Experimental Therapeutics 48, 151 – 157. Glombitza, K.W., Mahran, G.H., Mirhom, Y.W., Michel, K.G., Motawi, T.K., 1994. Hypoglycemic and antihyperglycemic effects of Zizyphus spina-christi in rats. Planta-medica 60, 244 – 247. Huong, N.T.T., Matsumoko, K., Yamasaki, K., Duc, N.M., Nham, N.T., Watanabe, H., 1995. Crude saponin extracted from Vietnamese ginseng and its major constituent majonoside — R2 attenuated the psychological stress — and foot shock stress-induced anti-nociception in mice. Pharmacology Biochemical Behaviour 52, 427 – 432. Ikram, M., Tomlinson, H., 1976. Chemical constituents of Zizyphus spina-christi. Planta-medica 29, 289 – 290. Irwin, S., 1968. Comprehensive observation assessment: 1a. a systematic quantitative procedure for assessing the behavioural and physiologic state of the mouse. Psychopharmacology 13, 222 – 257. Mahran, E.D.H., Glombitza, K.W., Mirhom, Y.M., Hartmann, R., Michel, C.G., 1996. Novel saponins from Zizyphus spina-christi growing in Egypt. Planta-medica 62 (2), 163 – 165. Mansur, J., Martz, R.M.W., Carlini, E.A., 1971. Effects of acute and chronic administration of cannabis satis and ( − ) 9-trans tetrahydrocannabinol on the behaviour of rats in open field arena. Psychopharmacology 19, 338 – 397. Morishita, S., Mishima, Y., Hirai, Y., Saito, T., Shoji, M., 1987. Pharmacological studies of water extract of Zizyphus seed containing drug. General Pharmacology 18, 637 – 641. Perez, R.M.G., Perez, J.A.L., Garcia, L.M.D., Sossa, H.M., 1998. Neuropharmacological activity of Solanum nigrum fruit. Journal of Ethnopharmacology 62, 43 – 48. Soulimani, R., Younos, C., Jarmouni-Idrissi, S., Bousta, D., Khalouki, F., Laila, A., 2001. Behavioural and Pharmaco-toxicological study of Papa6er rhoeas L. in mice. Journal of Ethnopharmacology 74 (3), 265 – 274. Journal of Ethnopharmacology 79 (2002) 17 – 21 www.elsevier.com/locate/jethpharm Biological effect of Opuntia ficus indica (L.) Mill. (Cactaceae) waste matter Note I: diuretic activity E.M. Galati a,*, M.M. Tripodo b, A. Trovato a, N. Miceli a, M.T. Monforte a a Pharmaco-Biological Department, School of Pharmacy, Uni6ersity of Messina, Vill. SS., Annunziata, 98168 Messina, Italy Department of Organic and Biological Chemistry, Uni6ersity of Messina, Salita, Sperone, S. Agata, 98166 Messina, Italy b Received 22 September 2000; received in revised form 26 July 2001; accepted 9 August 2001 Abstract In this work we studied in rat the diuretic activity of Opuntia ficus indica (L.) Mill. (Cactaceae) waste matter. The cladodes, flowers and non commerciable fruits were collected in S. Cono (CT, Sicily) cultivation. Acute and chronic diuretic activity of 15% infusion of cladodes, flowers and fruits were assayed. Natriuresis, kaliuresis and the activity on fructose-induced hyperuricemia was also studied. The results show that O. ficus indica cladode, fruit and flower infusions significantly increase diuresis. This effect is more marked with the fruit infusion and it is particularly significant during the chronic treatment. The fruit infusion shows also antiuric effect. In all experiments cladode, flower and fruit infusions showed a modest but not significant increase in natriuresis and kaliuresis. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Opuntia ficus indica (L.) Mill.; Diuretic activity; Hypouricemic activity; Traditional use; Waste matter 1. Introduction The Opuntia ficus indica (L.) Mill. species has gradually attained economic importance in Sicilian agriculture and the international scientific community, through FAO, also contributes to the diffusion of this cultivation. Besides, it is important to point out that the disposal of O. ficus indica wastes (non commerciable fruits and cladodes) constitutes a problem for environmental pollution due to fermentation phenomena. A project of our group, financed by Regione Siciliana, has the aim of implementing the prickly pear culture through the study of biological properties and the valorization of the above mentioned wastes. The use of prickly pear fruits is recommended for their beneficial and therapeutic properties (Barbera and Inglese, 1993). Literature data report that other parts of this plant are also used in folk medicine as: emollient, moisturizing, cicatrizant, hypocholesterolemic, hypoglycemic agent and in gastric mucosa diseases (Cruse, 1973; Meyer and Mc Laughlin, 1981; Harvala et al., 1982; Camacho-Ibanez et al., 1983; Brutsch, 1990; Frati et al., 1990; Hegwood, 1990; Pimienta, 1990; Fernandez et al., 1992, 1994; Rosado and Diaz, 1995). In Sicilian folk medicine, a flower infusion has an effect generally defined as depurative and in particular it is used because of its diuretic and relaxant action on the renal excretory tract (Arcoleo et al., 1961, 1966; Sisini, 1969). Therefore, it is stipulated that a flower infusion may help the expulsion of renal calculus. The fruit also enhances renal function (Cacioppo, 1991). In the present work, we studied the diuretic effects of cladode, fruit and flower infusions of Opuntia ficus indica in the rat, to explain the popular use and to compare the activity of flower infusion with the activity of cladode and fruit infusions. Natriuresis, kaliuresis and the activity on fructose-induced hyperuricemia was also studied. * Corresponding author. 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 3 7 - 3 18 E.M. Galati et al. / Journal of Ethnopharmacology 79 (2002) 17–21 2. Materials and methods 2.1. Animals Male Wistar rats (150–200 g) were placed in metabolic cages (temperature 2292 °C; humidity 60 9 4%; natural light) and maintained on standard diet (S. Morini, Mil rat GLP). 2.2. Plant material The plant material is the waste matter collected in a O. ficus indica cultivation located in S. Cono (CTSicily). The cladodes, cleaned from epidermis and glochides, were homogenized in Ultra-Turrax for 5 min. The fruits, cut into pieces and homogenized in Ultra Turrax for 5 min, were centrifuged to remove the seeds. The flowers were air-dried. The 15% infusions in tap water were prepared according to Farmacopea Ufficiale Italiana, 1998 and administered, by gavage, immediately after filtration, at a dose of 5 ml/100 g (body weight (b.w.)). 2.3. Acute diuretic acti6ity The diuretic activity was assessed as follows: each animal was placed in an individual metabolic cage 24 h prior to commencement of the experiment for adaptation. Rats were fasted overnight with free access to water. The animals were divided into five groups of 12 rats each and subjected to treatment in the morning. The I, II and III groups of rats received 15% infusion of O. ficus indica cladodes, flowers and fruits, respectively (5 ml/100 g b.w.). The IV group (control) received only the tap water (5 ml/100 g b.w.). The V group received the diuretic compound: hydrochlorothiazide (SIGMA, Milano) (10 mg/kg in 5 ml/100 g b.w.). The experiment was repeated three times at weekly intervals. The urine was collected and measured at 1, 2, 4, 6, 8 and 24 h after administration. Natriuresis and kaliuresis were measured by flame spectrophotometry. The results were expressed as average value 9S.E. The significance between the averages was evaluated with Student’s t-test for unpaired data. 2.4. Chronic diuretic acti6ity A low dose (1.5 ml/100 g b.w.) of 15% infusions was used to study the effects of repeated administrations. The animals were placed individually in metabolic cages. Five groups, each consisting of 12 male rats, were used. Three groups were administered every morning 1.5 ml/100 g of 15% cladode, flower and fruit infusions respectively, for 7 days. The IV group re- ceived only water and served as control. The V group received the diuretic compound: hydrochlorothiazide (SIGMA, Milano) (10 mg/kg in 1.5 ml/100 g b.w.). The urine excreted in 24 h was collected daily and measured for every single rat. The results were expressed as average per rat per day. The average was calculated using the values obtained, as well as standard error (S.E.). The significance between the averages was evaluated with Student’s t-test for unpaired data. 2.5. Antiuric acti6ity In order to assess the antiuric activity, the rats, placed individually in metabolic cages, were divided into five groups of six each. The first group was treated only with water and used as control. The rats of the second, third, fourth and fifth group were made to become hyperuricemic through the administration by gavage of 4 g/kg b.w. of fructose (20% solution), that, as known, stimulates nucleotide degradation (Brosh et al., 1982; Itoh, 1983). The rats in the second group did not receive any other treatment. The rats of the third, fourth and fifth group received by gavage 5 ml/100 g 15% infusion of cladodes, flowers and fruits, respectively (5 ml/100 g b.w.). The treatment was always undertaken 15 min after fructose administration. Urine was collected 2, 5 and 8 h after treatment with the infusions. At the same time a blood sample was taken to measure uricemia. Plasmatic and urinary uric acid was measured with the colorimetric enzyme method (Trinder, 1969; Fossati et al., 1980). The results were expressed as average9S.E. The significance between the averages was evaluated with Student’s t-test for unpaired data. 3. Results 3.1. Results of acute diuretic acti6ity The treatment with cladode infusion gives a slight increase in diuresis at the second hour after administration of infusion (controls 3.1 ml9 0.9; treated 4.89 1.2). After 4, 6 and 8 h of treatment, the urine volume was equal to the controls. The increase in urinary volume in 24 h is statistically significant with respect to controls (Fig. 1) (controls 24.2 ml9 1.0; treated 38.2 ml 9 1.1). In the acute experiment, we did not observe significant variations of diuresis up to the eighth hour after infusion of flowers. In fact, the urinary volume at 1, 2, 4, 6 and 8 h is analogous to that of controls. The total volume of urine in 24 h instead presented statistically significant variations (controls 24.2 ml9 1 0; 32.4 ml9 1.7) (Fig. 1). E.M. Galati et al. / Journal of Ethnopharmacology 79 (2002) 17–21 Fig. 1. Acute diuretic activity of Opuntia ficus indica (L.) Mill. 15% cladode, flower and fruit infusions (5 ml/100 g b.w.) in rat. The volume of excreted urine was measured 1, 2, 4, 6 and 8 h after treatment. The 24 h value corresponding to cumulative results (mean9 S.E.). *PB 0.05 compared with controls; Student’s t-test. The diuretic activity of the fruit infusion is evident after 2 h from administration (controls 3.1 ml9 0 9; treated 5.0 ml90.8). The rate of diuresis of treated rats is similar to that of controls at 4, 6, and 8 h after treatment. The 24 h urinary volume is markedly higher in treated rats (controls 24.2 ml9 1.0; treated 39.0 ml 90.7) (Fig. 1). At the same time, the urine volume of the rats treated with hydrochlorothiazide exceeded the values of the control group from the beginning, but also the values of the groups treated with O. ficus indica infusions. At 24 h, as Fig. 1 shows the difference between the urine volume of the animals treated with fruit and cladode infusions (respectively, 39.0 ml90.7 and 38.2 ml91.1) and those treated with hydrochlorothiazide (40.2 ml9 1.8) is similar. The cladode, flower and fruit infusions show a modest but not significant increase in natriuresis and kaliuresis (Figs. 2 and 3). 3.2. Chronic diuretic acti6ity In the chronic experiment (Fig. 4), the cladode infusion from the first day of treatment, gives an increase in diuresis with respect to controls. This increase reaches its peak on the third day but remains statistically significant up to the sixth day. Fig. 2. Effect of oral administration of Opuntia ficus indica (L.) Mill. 15% cladode, flower and fruit infusions (5 ml/100 g b.w.) on the urinary concentrations of sodium and potassium in rat (mean 9 S.E.). *PB 0.05 compared with controls; Student’s t-test. 19 Fig. 3. Effect of oral administration of Opuntia ficus indica (L.) Mill. 15% cladode, flower and fruit infusions (5 ml/100 g b.w.) on the urinary concentrations of sodium and potassium in rat (mean 9 S.E.). *PB 0.05 compared with controls; Student’s t-test. The flower infusion has a minor diuretic activity. The increase in diuresis is significant from 3 to 7 days of treatment. Fruit infusion possesses important diuretic action. The increase in diuresis is significant from the first day of treatment, reaches its peak value on the third day and maintains this value up to the seventh day of treatment. This effect is quantitatively similar to hydrochlorothiazide (Fig. 4). 3.3. Antiuric acti6ity The level of plasmic uric acid in normal rats is about 1.3 mg/dl and the urinary one about 1.2 mg/dl. Two hours after treatment with fructose, plasmic uric acid reaches an average value of 4.2 mg/dl, and the urinary one of 4.15 mg/dl. After treatment with cladode and flower infusions, blood and urine variations of uric acid were not statistically significant. After treatment with fruit infusion the blood levels of uric acid decreases after 2 h from administration. The decrease is statistically significant also at the fifth and eighth hour. The urinary values of uric acid show an opposite course. Maximum elimination is obtained at the second hour and the increase in uric acid is statisti- Fig. 4. Chronic diuretic activity of Opuntia ficus indica (L.) Mill. 15% cladode, flower and fruit infusions (1.5 ml/100 g b.w.) in rat. The volume of excreted urine was measured after 1, 2, 3, 4, 5, 6, 7 days of treatment (mean 9 S.E.). *PB 0.05 compared with controls; Student’s t-test. 20 E.M. Galati et al. / Journal of Ethnopharmacology 79 (2002) 17–21 Fig. 5. Antiuric activity of Opuntia ficus indica (L.) Mill. 15% cladode, flower and fruit infusions (5 ml/100 g b.w.) in rat with fructose-induced hyperuricemia. Plasmatic and urinary uric acid was measured 2, 5 and 8 h after treatment (mean 9 S.E.). *PB 0.05 compared with controls; Student’s t-test. cally significant up to 8 h after the treatment (Figs. 5 and 6). 4. Conclusions Perfumi and Tacconi (1996) reported that flower infusion shows a modest increase in diuresis and natriuresis. In our experimental conditions, treatment with cladode and flower infusions increases diuresis but does not significantly influence the uric acid pattern. The fruit infusion instead had diuretic and antiuric activity. Cladode, flower and fruit infusions do not give significant alteration of urinary levels of sodium and potassium (Figs. 2 and 3). The diuretic action observed may depend on stimulation of the urinary tract and is linked to the activation of neurohumoral mechanism, mediators of stimuli acting on glomerules, tone acid on the pyelo-uretral peristaltis. These effects might be due to the influence that the electrolytes, present in considerable quantities on the plant, exert on renal epithelium. In particular, O. ficus indica is rich in K+ ions, which, in our samples, are present in concentration of 548 mg/kg in the cladodes, 21.7 mg/kg in the flowers and 18 mg/kg in the fruit (d’Aquino, 1998). Reference data report that K+ concentration in the fruit is about 100 mg/100 ml (Duro and Condorelli, 1971; Wills et al., 1986). Other monovalent and bivalent cations are present in this plant and might have a diuretic activity synergetically with K+ (Kanias et al., 1979). The theory that the majority of the medicinal plants have a diuretic effect only due to the presence of the potassium seems somewhat doubtful (Ribeiro et al., 1988). In fact pharmacodynamic studies performed on medicinal plants emphasized that frequently no correlation exists between the diuretic effect observed and the K+ content of the extract (Abed and Benmerabet, 1981; Jouad et al., 2001). In the present study, the diuretic effect observed does not exclude the possibility that changes in the diuresis may occur as a consequence of the presence of polar drug compounds (Szentmihályi et al., 1998; Chodera et al., 1991), e.g. flavonoid glycosides (Piattelli and Minale, 1964; Piattelli and Imperato, 1969; Duro and Condorelli, 1971, Alard et al., 1985, Forni et al., 1992) and ascorbic acid (Pinto and Acevedo, 1983; Sawaya et al., 1983; Kuti, 1992; Teles et al., 1994). Preliminary phytochemical analysis revealed that these compounds are the main constituents of OFI infusions (d’Aquino, 1998). These natural compounds might be acting synergetically or individually promoting an initial vasodilatation (Stanic and Samaržija, 1993). It is also possible that OFI infusions might manifest cumulative effect of several substances in the extract and/or due to secondary active metabolite (Tanira et al., 1988). The other possibility for the observed diuretic effect could be due to indirect changes of some physiological parameters before blood filtration step. The antiuric effect of fruit infusion cannot be explained only by the increase of diuresis or by an increased urinary excretion of urates. Probably, it could be bound to an influence on the uric acid metabolism due to an alteration of some enzymatic activity. Acknowledgements Financial support for this research was provided by Regione Siciliana (Italy). References Fig. 6. Antiuric activity of Opuntia ficus indica (L.) Mill. 15% cladode, flower and fruit infusions (5 ml/100 g b.w.) in rat with fructose-induced hyperuricemia. Plasmatic and urinary uric acid was measured 2, 5 and 8 h after treatment (mean 9 S.E.). *PB 0.05 compared with controls; Student’s t-test. Abed, L., Benmerabet, K., 1981. Intérêt de l’apport en potassium et sodium des infusions de plantes médicinales. Plantes Médicinales et Phytothérapie XV, 92 – 98. Alard, D., Wray, V., Grotjahn, L., Reznik, H., Strack, D., 1985. N-neobetanin: isolation and identification from Beta 6ulgaris. Phytochemistry 10, 2383 – 2385. E.M. Galati et al. / Journal of Ethnopharmacology 79 (2002) 17–21 Arcoleo, A., Ruccia, M., Cusmano, S., 1961. Sui pigmenti flavonici delle Opuntiae. nota I. Isoranmetina dai fiori di O. ficus indica Mill. Annales di Chimica 51, 751 – 758. Arcoleo, A., Ruccia, M., Natoli, M.C., 1966. Beta-sitosterolo dai fiori di Opuntia ficus indica Mill. (Cactaceae). Atti Accademiac Scienze Lettere e Arti (Palermo) 25, 323 –332. Barbera, G., Inglese, P., 1993. La coltura del ficodindia. EdagricoleEdizioni Agricole della Calderini s.r.l., Bologna, pp. 174 – 176. Brosh, S., Boer, P., Sperling, O., 1982. Effect of fructose on synthesis and degradation of purine nucleotides in isolated rat hepatocytes. Biochimica Biophysica Acta 717, 459 – 464. Brutsch, M.O., 1990. Some lesser-known uses of the prickly pear (Opuntia spp.). Proceedings of Transkei and Ciskei Research Society Conference. Cacioppo, O., 1991. Fico d’india e pitaya. Ed. L’informatore Agrario, Verona, pp. 38 –39. Camacho-Ibanez, R., Meckes-Lozoya, M., Mellado-Campos, V., 1983. The hypoglycemic effect of Opuntia streptacantha studied in different animal experimental models. Journal of Ethnopharmacology 7, 175 – 181. Cruse, R.R., 1973. Desert plant chemurgy: a current review. Economic Botany 27, 210 – 230. Chodera, A., Dabrowska, K., Sloderbach, A., Skrypczak, L., Budzianowki, K., 1991. Effect of the flavonoid fraction of the Solidago genus plants on diuresis and electrolyte concentration. Acta Poloniae Pharmaceutica 48, 35 – 37. d’Aquino, A., 1998. Tesi per il conseguimento del Dottorato di Ricerca in Farmacognosia (IX Ciclo). Opuntia ficus indica Mill. Ricerche Farmacognostiche. Facoltà di Farmacia- Università di Messina. Duro, F., Condorelli, P., 1971. Ricerche sugli antociani presenti nei frutti della ‘Cactus Opuntia’. Quaderno Merceologico 10, 49 – 54. Farmacopea Ufficiale Italiana, 1998, X Ed. Istituto Poligrafico e Zecca dello Stato. Roma. Fernandez, L.M., Lin, E.C.K., Trejo, A., McNamara, D.J., 1992. Prickly pear (Opuntia sp.) pectin reverses low density lipoprotein receptor suppression induced by a hypercholesterolemic diet in Guinea Pigs. Journal of Nutrition, 122, 2330 – 2340. Fernandez, L.M., Lin, E.C.K., Trejo, A., McNamara, D.J., 1994. Prickly pear (Opuntia sp.) pectin alters hepatic cholesterol metabolism without affecting cholesterol absorption in Guinea Pigs Fed a hypercholesterolemic diet. Journal of Nutrition 124, 817 – 824. Frati, A.C., Jimenez, E., Raul Ariza, C., 1990. Hypoglycemic effect of Opuntia ficus indica in non-insulin dependent diabetes mellitus patients. Phytotherapy Research 4, 195 –197. Forni, E., Polesello, A., Montefiori, D., Maestrelli, A., 1992. High performance liquid chromatographic analysis of the pigments of blood-red prickly pear (Opuntia ficus indica). Journal of Chromatography 19, 719 – 720. Fossati, P., Prencipe, L., Berti, G., 1980. Use of 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone chromogenic system in direct enzymic assay of uric acid in serum and urine. Clinical Chemistry 26, 227 – 231. Harvala, C., Alkofahi, A., Philianos, S., 1982. Sur L’action enxymatique d’un produit extrait des graines d’Opuntia ficus indica Miller. Plantes Médicinales et Phitotherapie 4, 298 –302. 21 Hegwood, D.A., 1990. Human health discoveries with Opuntia sp. (Prickly pear). Hortscience 25, 1515 – 1516. Itoh, R., 1983. Effect of oral administration of fructose on purine nucleotide metabolism in rats. Comparative Biochemistry and Physiology B 76, 817 –821. Jouad, H., Lacaille-Dubois, M.A., Eddouks, M., 2001. Chronic diuretic effect of the water extract of Spergularia purpurea in normal rats. Journal of Ethnopharmacology 75, 219 – 223. Kanias, G.D., Loukis, A., Philianos, S.M., 1979. Trace element pharmacognostical study on diuretic drugs by neutron activation analysis. Journal of Radioanalytical Chemistry 54, 103 –112. Kuti, J.O., 1992. Growth and compositional changes during the development of prickly pear fruit. Journal of Horticulture Science 67, 861 – 868. Meyer, B.N., Mc Laughlin, J.L., 1981. Economic uses of Opuntia. Cactus and Succulent Journal (US) 53, 107 – 112. Perfumi, M., Tacconi, R., 1996. Effect of Opuntia ficus-indica flower infusion on urinary and electrolyte excretion in rats. Fitoterapia 67, 459 – 464. Piattelli, M., Minale, L., 1964. Pigments of Centrospermae-I Betacyanins from Phyllocatus hybridus Hort. and Opuntia ficus indica Mill. Phytochemistry 11, 2259 – 2262. Piattelli, M., Imperato, F., 1969. Betacyanins of the family Cactaceae. Phytochemistry 8, 1503 – 1507. Pimienta, B.E., 1990. El nopal tunero. Universidad de Guadalajra. Pinto, M., Acevedo, E., 1983. Biomass as energy source. Próxima Década 12, 12 –16. de Ribeiro, R.A., de Barros, F., de Melo, M.M.R.F., Muniz, C., Chieia, S., Wanderley, M., das, G., Gomes, C., Trolin, G., 1988. Acute diuretic effects in conscious rats produced by some medicinal plants used in the state of Sao Paulo, Brasil. Journal of Ethnopharmacology 24, 19 – 29. Rosado, J.L., Diaz, M., 1995. Physicochemical properties related to gastrointestinal effects of six dietary fibers. Revista de Investigacion Clinica 47, 283 –289. Sawaya, W.N., Khatchadourian, H.A., Safi, W.M., Al-Muhammad, H.M., 1983. Chemical characterization of prickly pear pulp, Opuntia ficus indica, and the manufacturing of prickly pear jam. Journal of Food Technology 18, 183 – 193. Sisini, A., 1969. Sulla glucoso-6-fosfato isomerasi in Opuntia ficus indica. Bollettino Societă Italiana Biol Sper 45, 794 – 796. Stanic, G., Samaržija, I., 1993. Diuretic activity of Satureja montana subsp. Montana extracts and oil in rats. Phytotherapy Research 7, 363 – 366. Szentmihályi, K., Kréry, A., Then, M., Lakatos, B., Sándor, Z., Vinkler, P., 1998. Potassium/sodium ratio for the characterization of medicinal plants extracts with diuretic activity. Phytotherapy Research 12, 163 –166. Tanira, M.O.M., Ageel, A.M., Al-Said, M.S., 1988. A study on some Saudi medicinal plants used as diuretics in traditional medicine. Fitoterapia LX (5), 443 – 447. Teles, F.F.F., Price, R.L., Whiting, F.M., Reid, B.L., 1994. Circadian variation of non-volatile organic acids in the prickly pear (Opuntia ficus indica L.). Revista Ceres 41, 614 – 622. Trinder, P.J., 1969. Determination of blood glucose using 4aminophenazone as oxygen acceptor. Journal of Clinical Pathology 22, 246. Wills, R.B.H., Lim, J.S.K., Greenfield, H., 1986. Composition of Australian foods. Food Technology of Australia 38, 118 – 120. Journal of Ethnopharmacology 79 (2002) 23 – 26 www.elsevier.com/locate/jethpharm Effects of Nigella sati6a fixed oil on blood homeostasis in rat A. Zaoui a,*, Y. Cherrah b, K. Alaoui b, N. Mahassine c, H. Amarouch a, M. Hassar b a Département de Biologie, Faculté des Sciences, Uni6ersité Hassan II, Km 8, Route El Jadida, B.P. 5366, Maârif, Casablanca, Morocco b Laboratoire de Pharmacologie et Toxicologie, Faculté de Médecine et Pharmacie de Rabat, Rabat, Morocco c Ser6ice d’histopathologie, Centre Hospitalier Uni6ersitaire, IBN SINA, Rabat, Morocco Accepted 5 September 2001 Abstract We investigated the effects of the fixed oil of Nigella sati6a seeds in rats by monitoring blood homeostasis and body weight as well as toxicity. Animals were treated daily with an oral dose of 1 ml/kg body weight of the N. sati6a seed fixed oil for 12 weeks. Changes in key hepatic enzymes levels were not observed in N. sati6a treated rats after 12 weeks of treatment. The serum cholesterol, triglycerides and glucose levels and the count of leukocytes and platelets decreased significantly by 15.5, 22, 16.5, 35 and 32%, compared to control values, respectively; while haematocrit and haemoglobin levels increased significantly by 6.4 and 17.4%, respectively. In parallel, significant slowdown of the body weight evolution was observed in N. sati6a treated animals comparatively to the animal control group. On the other hand, no mortality was noted for ten times the therapeutic dose in mice, during 15 days period after the oil administration (10 ml/kg p.o.). These results support the traditional use of N. sati6a seeds as a treatment of the dyslipidemia and the hyperglycaemia, and related abnormalities; however, indicate a relative toxicity of this plant. Acute and chronic toxicity, and the mode of the action of the N. sati6a fixed oil must be studied. © 2002 Published by Elsevier Science Ireland Ltd. Keywords: Nigella sati6a; Fixed oil; Biochemical parameters; Haematological parameters; Toxicity 1. Introduction (Zaoui et al., 2000). Studies in mice and rats have shown that treatment with N. sati6a extract significantly protects from cisplatin-induced falls in leukocytes counts, haemoglobin level, mean osmotic fragility and haematocrit increase (Nair et al., 1991; El-Daly, 1998), influences leukocytes activities (Haq et al., 1995; Houghton et al., 1994) and causes the death of mice lymphocytes in vitro (Salomi et al., 1992). In Morocco N. sati6a and its derived products are consummated abusively for traditional treatment of blood homeostasis abnormalities. This study was therefore undertaken to determinate the effects of a chronic treatment with N. sati6a fixed oil on blood biochemical and haematological parameters in rat. Nigella sati6a L. (Ranunculaceae), commonly known as ‘black cumin’, is an erect herbaceous annual plant. It grows in Mediterranean countries and is also cultivated in the north of Morocco. N. sati6a seeds have traditionally been used in Middle Eastern folk medicine as a natural remedy for various diseases as well as a spice for over 2000 years. The seeds of N. sati6a have been subjected to a range of pharmacological, phytochemical and nutritional investigations in recent years. It has been shown to contain more than 30% (w/w) of a fixed oil with 85% of total unsaturated fatty acid (Houghton et al., 1994). N. sati6a seeds decrease the serum total lipids and body weight in Psammomys obesus sand rat (Labhal et al., 1997), decrease the fasting plasma glucose in rabbit (Al-Hader et al., 1993), increase serum total protein (Haq et al., 1995), and shows diuretic and hypotensive effects in spontaneously hypertensive rat 2.1. Preparation of the N. sati6a seed extract * Corresponding author. Tel.: +212-22-0680-7284; fax: + 212-22230-674. The plant seeds were harvested in the North of Morocco. The plant material was identified and authen- 2. Materials and methods 0378-8741/02/$ - see front matter © 2002 Published by Elsevier Science Ireland Ltd. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 4 2 - 7 24 A. Zaoui et al. / Journal of Ethnopharmacology 79 (2002) 23–26 ticated as N. sati6a (L.) (Ranunculaceae), by Professor A. Ouyahya, a plant taxonomist from the Scientific Institute of Rabat. A voucher specimen has been deposited at the repository in our institute in Rabat. The N. sati6a seeds were powdered mechanically. The extract was obtained by cold shocking of the powdered seeds in 3×1.5 l of hexane during 3×24 h. The solvent was removed from the extract under reduced atmospheric pressure. The obtained oily extract from N. sati6a seeds has a chestnut colour and agreeable perfume. 2.2. Experimental animals Wistar kyoto rats were purchased from IFFA (Credo, France) and studied at 8 weeks of age. Animals were maintained on a 12 h light cycle and fed standard lab chow ad libitum. Rats were randomly assigned to two experimental groups of 12 animals each. The N. sati6a treated rat group (Ns-rats) received daily administrations of 1 ml/kg body weight of N. sati6a fixed oil by oral gavage (force-feeding) for 12 weeks period. Control rat animals (C-rats) were treated in an identical fashion with 1 ml/kg body weight of water. Body weight was measured at J0 and 2, 4, 6, 8, 10 and 12 weeks. 2.3. Metabolic and haematological measurements Metabolic and haematological measurements were realised at J0 and, 4, 8 and 12 weeks following oil administration. Animals were studied after an (15 h) overnight fast. Afterwards, blood was obtained from the retro-orbital sinus (2 ml). Metabolic measurements were realised spectrophotometrically, according to standardised procedures, using commercially available kits, purchased from BOEHRINGER Mannheim (Meylan, France). Haematological parameters were determined automatically by ABX COBAS LO. 2.4. Toxicity A group of ten mice were given N. sati6a fixed oil at the dose of 10 ml/kg p.o. (10× therapeutic dose). The animals were observed for gross effects and mortality during 15 days. 2.5. Statistical analysis All data are expressed as mean9SD. Student’s and Snedecor’s tests were applied. Fig. 1. Effects of N. sati6a fixed oils (1 ml/kg/day) on plasma lipids and glucose in rat. Plasma cholesterol, triglycerides and glucose were measured as described in Section 2. Rats were treated with N. sati6a seed extract (Ns-rats; n = 12) for 12 weeks. Plasma lipid and glucose values are given for the 4, 8 and 12 weeks treatment points. Values are expressed as mean 9 SD. Values in control rats (C-rats) were similar at all points and are grouped for the sake of simplicity. *Significantly different from C-rats group by Student’s test, PB 0.05. 3. Results 3.1. Biochemical parameters Fig. 1 shows the effects of N. sati6a fixed oil on the metabolism of plasma lipids and glucose. After 12 weeks of daily treatment (1 ml/kg), serum cholesterol, triglycerides and glucose levels were decreased significantly by 15.5, 22, 16.5%, respectively when compared to the control values observed in placebo animals (P B 0.05). Table 1 Effects of N. sati6a fixed oils (1 ml/kg/day) on plasma key hepatic enzymes, bilirubin, uric acid and creatinin in rat as a function of treatment time Parameter (unit) ASAT (U/l) ALAT (U/l) ALP (U/l) GGT (U/l) T-bilirubin (mg/l) Uric acid (mg/l) Creatinin (mg/l) C-rats 128 9 59 47.59 9.2 213.2 9 68.4 4.409 2.77 1.34 9 1.0 15.8 9 5.8 Ns-rats treatment time (weeks) 4 8 12 120 9 40 41.1 9 12.1 187.89 61.4 5.08 9 2.15 1.10 9 0.94 156 9 76 38.4 9 14.6 149.0 9 57.4* 3.85 9 1.95 1.50 9 1.2 171 9 72 43.4 9 15.7 139.4 9 59.3* 3.18 9 1.83 1.10 9 0.7 15.5 9 5.13 17.1 9 7.3 5.92 9 1.32 6.36 9 0.87 5.21 9 0.85 14.2 9 4.2 6.769 0.77 C-rats, control rats group; Ns-rats, N. sati6a treated rats group; ASAT, aspartate-aminotransferase; ALAT, alanine-aminotranferase; ALP, alkaline phosphatase; GGT, gamma-glutamyltransferase. Values are expressed as mean 9 SD (n= 12). * Significantly different from C-rats by Student’s test, PB0.05. A. Zaoui et al. / Journal of Ethnopharmacology 79 (2002) 23–26 25 Table 2 Effects of N. sati6a fixed oils (1 ml/kg/day) on haematological parameters Parameter (unit) Erythrocytes (106/mm3) Leukocytes (103/mm3) Platelets (103/mm3) Haematocrit (%) HGB (g/dl) MGV (mm3) MCCH1 (pg) MCCH2 (g/dl) C-rats 7.23 9 1.03 7.25 91.71 7439 121 40.99 94.32 13.129 1.45 53.359 0.14 17.979 0.95 32.94 9 1.81 Ns-rats treatment time (weeks) 4 8 12 7.05 9 0.57 8.23 9 1.6 6809 82 39.26 9 2.49 13.53 9 0.62 52.75 9 2.96 18.77 91.41 34.82 92.12 6.74 90.44 7.6 91.3 570 9 109** 40.73 93.15 14.3 9 0.94* 55.56 9 2.01 19.13 9 1.48* 34.70 9 1.84* 6.91 9 0.68 4.7 9 1.5** 504 9 110** 43.60 9 3.11* 15.4 9 0.64** 56.3 9 3.53** 19.26 9 1.22** 34.80 9 1.32** C-rats, control rats group; Ns-rats, N. sati6a treated rats group; HGB, haemoglobin; MGV, mean globular volume; MCCH1, mean corpuscular content of haemoglobin; MCCH2, mean corpuscular concentration of haemoglobin. Values are expressed as mean 9 SD (n = 12). * Significantly different from C-group by Student’s test, PB0.05. ** Significantly different from C-group, PB0.01. Table 1 illustrates the effects of N. sati6a fixed oil on plasma key hepatic enzymes, bilirubin, uric acid and creatinin in rats as a function of treatment time. After 12 weeks of daily treatment (1 ml/kg), plasma key hepatic enzymes, bilirubin, uric acid and creatinin did not increase significantly compared to the control values observed in placebo animals. 3.2. Haematological parameters Table 2 illustrates the haematological parameters in N. sati6a treated rats as a function of treatment time. After 12 weeks of treatment, the leukocytes and platelets counts decreased significantly when compared to the control values (PB0.01), whereas heamatocrit and haemoglobin level (HGB) increased significantly (P B0.05). Consequently, mean globular volume (MGV), mean corpuscular content of haemoglobin (MCCH1), and mean corpuscular concentration of haemoglobin (MCCH2) were increased significantly (P B0.01). servation period of 15 days following the oil administration in mice. 4. Discussion The results obtained in the present study clearly show that N. sati6a fixed oil chronic treatment was effective in influencing blood homeostasis in rat. Serum lipids and glucose levels, and leukocytes and platelets counts was decreased significantly, whereas the haematocrit and haemoglobin concentration was increased significantly. The serum key hepatic enzyme concentrations did not change significantly. In parallel, a slight slowdown of body weight was observed. The effect of N. sati6a seeds on blood homeostasis is not without precedent. Previous studies in normal and alloxan-induced diabetic rabbits have shown that treatment with the volatile oil of N. sati6a seeds significantly diminishes plasma glucose levels (Al-Hader et al., 1993). Analogous results, accompanied with decreases in serum lipids level and body weight have also been 3.3. Effect on body weight Fig. 2 shows the evolution of the mean body weight in the N. sati6a treated and control rat groups. The progression of body weight was not similar in both groups. Indeed, the N. sati6a treated rats had significantly lower body weights than their control rat counterparts; this effect is statistically significant from the 6 weeks treatment point onward (PB 0.01). 3.4. Toxicity N. sati6a fixed oil, when given at 10 ml/kg p.o., showed no adverse effects or mortality during the ob- Fig. 2. Effects of N. sati6a fixed oils (1 ml/kg/day) on body weight evolution. C-rats, control-rats group (n = 12); Ns-rats, N. sati6a treated rats group (n = 12). Values are expressed as mean 9 SD. *Significantly different from C-rats group by Student’s test, PB 0.05. 26 A. Zaoui et al. / Journal of Ethnopharmacology 79 (2002) 23–26 observed in sand rats treated with an aqueous suspension of N. sati6a seeds (Labhal et al., 1997). In rat, N. sati6a seeds increase serum total protein (Al-Gaby, 1998). Studies in mice and rats have shown that treatment with N. sati6a extract significantly protects from cisplatin-induced falls in leukocytes counts, haemoglobin levels, mean osmotic fragility and haematocrit increase (Nair et al., 1991; El-Daly, 1998), influences leukocytes activities (Haq et al., 1995; Houghton et al., 1994) and causes the death of mice lymphocytes in vitro (Salomi et al., 1992). While the underlining effects were observed at 1 ml/kg body weight of N. sati6a extract in rats, no evidence of toxicity was noted in ten times this dose in mice, suggesting, only a seeming margin of safety for the used therapeutic doses of N. sati6a. The changes in haemoglobin metabolism and the fall in leukocytes and platelets counts must be taken into consideration. The slowdown of body weight evolution in N. sati6a treated rats might be related to the serum lipids and glucose levels decrease as a consequence of a possible reduction in food intake by the drug administration. Other explanations are also possible, like a toxic effect. In conclusion, these results support the traditional use of N. sati6a and its derived products as a treatment for the dyslipidemia and the hyperglycaemia, and related abnormalities; however, indicate a relative toxicity of this plant extract. Acute and chronic toxicity, and the mode of the action of the Nigella sati6a fixed oil must be studied. References Al-Gaby, A.M., 1998. Amino acid composition and biological effects of supplementing broad bean and corn proteins with Nigella sati6a (black cumin) cake protein. Nahrung 42, 290 – 294. Al-Hader, A., Aqel, M., Hasan, Z., 1993. Hypoglycaemic effects of the volatile oil of Nigella sati6a seeds. International Journal of Pharmacognosy 31, 96 – 100. El-Daly, E.S., 1998. Protective effect of cysteine and vitamin E, Crocus sati6us and Nigella sati6a extracts on cisplatininduced toxicity in rats. Journal de Pharmacie de Belgique 53, 87 – 95. Haq, A., Abdullatif, M., Lobo, P.I., Khabar, K.S., Sheth, K.V., Al-Sedairy, S.T., 1995. Nigella sati6a: effect on human lymphocytes and polymorphonuclear leukocytes phagocytic activity. Immunopharmacology 30, 147 – 155. Houghton, P.J., Zarka, R., Heras, B.D.L., Hoult, J.R.S., 1994. Fixed oil of Nigella sati6a and derived thymoquinone inhibit eicosanoid generation in leukocytes and membrane lipid peroxidation. Planta Medica 61, 33 –36. Labhal, A., Settaf, A., Bennani-kabchi, N., Cherrah, Y., Slaoui, A., Hassar, M., 1997. Action anti-obésité, hypocholestérolémiante et hypotriglycéridémiante de Nigella sati6a chez le Psammomys obesus. Caducée 27, 26 – 28. Nair, S.C., Salomi, M.J., Panikkar, B., Panikkar, K.R., 1991. Modulatory effects of Crocus sati6us and Nigella sati6a extracts on cisplatin-induced toxicity in mice. Journal of Ethnopharmacology 31, 75 – 83. Salomi, N.J., Nair, S.C., Jayawardhanane, K.K., Varghese, C.D., Panikkar, K.R., 1992. Antitumour principles from Nigella sati6a seeds. Cancer Letters 63, 41 – 46. Zaoui, A., Cherrah, Y., Lacaille-Dubois, M.A., Settaf, A., Amarouch, H., Hassar, M., 2000. Diuretic and hypotensive effects of Nigella sati6a in the spontaneously hypertensive rat. Thérapie 55, 379 – 382. Journal of Ethnopharmacology 79 (2002) 27 – 33 www.elsevier.com/locate/jethpharm Tumour reducing and anticarcinogenic activity of Acanthus ilicifolius in mice B.H. Babu, B.S. Shylesh, J. Padikkala * Department of Biochemistry, Amala Cancer Research Centre, Amala Nagar, Thrissur 680 553, India Accepted 17 September 2001 Abstract Alcoholic extract of Acanthus ilicifolius (250, 500 mg/kg b wt) was found to be effective against tumour progression and carcinogen induced skin papilloma formation in mice. The extract was found to be cytotoxic towards lung fibroblast (L-929) cells in 72 h MTT assay and the concentration required for 50% cell death was 18 mg/ml. Oral administration of the extract (500 mg/kg b wt) reduced the tumour volume and administration of the same concentration increased the life span by 75% in ascites tumour (EAC cells) harbouring animals. The extract also significantly delayed the onset of dimethylbenzanthrazene DMBA/Croton oil induced skin papilloma in mice in a dose dependent manner. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acanthus ilicifolius; Cytotoxicity; Carcinogenesis; Antitumour activity; MTT assay 1. Introduction Plant derived natural products such as flavanoids (Osawa et al., 1990), terpenes (Giulia et al., 1999), alkaloids (Keith et al., 1990) etc. have received considerable attention in recent years due to their diverse pharmacological properties including cytotoxic and cancer chemopreventive effects (Roja and Heble, 1994). India is a rich source of medicinal plants and a number of plant extracts are used against diseases in various systems of medicine such as Ayurveda, Unani and Sidha. Only few of them were scientifically explored. Acanthus ilicifolius Linn. (Acanthaceae) is a folklore medicinal plant used against rheumatism, paralysis, asthma and snake bite (Subudhi et al., 1992). Analgesic and antiinflammatory properties of A. ilicifolius has already been noticed (Agshikar et al., 1979). A new alkaloid acanthicifoline was isolated from the alcoholic extract of this plant. 2-benzozazolinone, a compound isolated from the leaves of the plant was screened for its leishmanicidal activity against Leishmania dono6ani (Kapil et al., 1994) Antioxidant and hepatoprotective activity of A. ilcifolius is reported from our laboratory * Corresponding author. Fax: + 91-487-211020. E-mail address: jpadikkala@rediffmail.com (J. Padikkala). (Babu et al., 2001). The present study describes antitumour and anticarcinogenic activity of A. ilicifolius. 2. Materials and method The plants were collected from Kodungallor, a coastal area in Kerala during the months of February – March and botanically identified by Dr Sasidharan, Kerala Forest Research Institute, Peechi, India and a voucher specimen was kept in the herbarium of our institute (ACRH No. 11). The male Swiss albino mice were obtained from Small animal breeding station, Veterinary college, Mannuthy. Dalton’s lymphoma ascites tumor cells (DLA), Ehrlich ascites tumor cells (EAC) and mouse lung fibroblast cells (L-929) were obtained from Adayar Cancer Institute Madras, Cancer Institute Bombay and National Institute of Virology, respectively. All other chemicals used in the present study were of analytical reagent quality. 2.1. Preparation of the drug The air-dried leaves were Sohxlet extracted with 70% EtOH (1:10 w/v) for 8 h (yield: 8.8%). The extract was resuspended in distilled water and used for preliminary 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 4 7 - 6 28 B.H. Babu et al. / Journal of Ethnopharmacology 79 (2002) 27–33 phytochemical screening tests (Wagner et al., 1984; Harbone, 1973; Stahl, 1969). The screening gave positive tests for flavonoids and terpenes, and negative test for alkaloids. 2.2. Determination of 3 h-time cytotoxicity of the extract Cytotoxicity studies were carried out using DLA and EAC tumor cells. Different concentrations of the extract (10– 1000 mg/ml) were incubated with tumour cells (106) suspended in phosphate-buffered saline (PBS, pH 7.2) and cytotoxicity was determined after 3 h using the trypan blue exclusion method (Babu et al., 1995) 2.3. Determination of 72 h-time cytotoxicity of the extract Volume =4/3yr 21 ×r 22, where r1 and r2 represents the radii of the tumour at two different planes (Mary et al., 1994). The results were compared with untreated control. In another set of experiments, one million cells of EAC cells were given intraperitoneally to four groups of mice(six mice per group). Group I was kept as control with out any treatment. Group II received curcumin (100 mg/kg b wt po) as standard drug. Group III and IV received the extract (250 and 500 mg/kg b wt po), respectively, 24 h after tumour inoculation and the treatment continued for 10 days. The animals were observed for the development of ascites tumour and death due to tumour volume was recorded. The increase in life span (percent ILS) of the treatment group was calculated using the formula, percent ILS=[(T − C)/C]× 100, Cytotoxicity of the extract in culture was determined using L-929 cells (Anis et al., 1999). Cells (104) were plated in a 96 well flat-bottomed titre plates and maintained with MEM (Minimum Essential Medium) containing 10% goat serum. Various concentrations of the extract (10– 100 mg/ml) were added in to the wells and incubated for 72 h. MTT (3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) (5%, 20 ml) was added to the wells 4 h before the end of incubation. Medium and reagents were aspirated, 98% dimethylsulphoxide (DMSO) was added and after shaking for 15 min, the absorbance was measured at 545 nm with a reference wavelength of 630 nm using an Elisa plate reader (Awarness Technology Inc.). Camptothecin (CPT) was treated as the positive control. The percentage cytotoxicity was calculated and compared with untreated controls. 2.4. Antitumour acti6ity In determining the antitumour activity of the extract on DLA system, two treatment modalities were followed as group A and B. In group A the animals were divided into four groups (six mice per group). One million DLA cells were injected into the right hind limb of all animals. Group I was kept as with out any treatment. Animals in group II received cisplatin (4 mg/kg b wt ip) as standard drug. Group III and IV received 250 and 500 mg/kg b wt of the extract through oral route, respectively. Drug administration was started 24 h after tumour inoculation and continued daily for 10 days. In group B the treatment was started from 11th day onwards after tumour transplantation. Diameter of the tumour was measured on every fifth day using vernier calipers and the volume was calculated using the formula, where T and C are mean survival of treated and control mice, respectively (Shylesh and Padikkala, 2000). 2.5. Anticarcinogenic acti6ity of A. ilicifolius The dorsal skin (2 cm diameter) of Swiss albino mice was shaved 2 days before chemical treatment and animals in the resting phase of growth cycle were selected for the experiment (Sasaki et al., 1995). Male Swiss albino mice were grouped into four (10 per group,6 weeks old). All the animals received DMBA (120 mg/0.2 ml acetone). In group I (control) 2 weeks after initiation with DMBA, promotion was begun by applying croton oil (10%) in 0.2 ml acetone topically twice a week for 6 weeks. Group II animals were topically treated with 100 mg/kg curcumin in 0.2 ml acetone 12 h before the topical application of croton oil twice a week for 6 weeks (pretreatment). In group III and IV the animals were topically treated with the extract of A. ilicifolius 250 mg/kg and 500 mg/kg twice a week 12 h before the topical application of croton oil and continued for 6 weeks. The number of animals, which developed papillomas were monitored every week and its number were recorded. Animals were sacrificed after 18 weeks the skin bearing papilloma were excised and evaluated by histopathological examination. 2.6. Statistical analysis Values are recorded as mean9SD. The data were analyzed by Student’s t-test; differences below the 0.5 level (PB 0.05) was considered as statistically significant. B.H. Babu et al. / Journal of Ethnopharmacology 79 (2002) 27–33 Table 1 In vitro cytotoxicity of A. ilicifolius extract to different cell lines Tested material toms were found to be minimal in treated group of animals (Fig. 3a–c) Concentration for 50% cytotoxicity (g/ml) DLA A. ilicifolius extract Camptothecin 29 a NT ND EAC NT ND a L-929 b 18 90.05 0.002 9 0.001 Values are mean 9 SD (n = 3), NT-non toxic, ND-not determined. a 3 h-experiment. b 72 h-experiment. 3. Results The extract was found to inhibit 50% proliferation of L-929 cells in long term culture at a concentration of 18 mg/ml and was nontoxic towards DLA and EAC cells at 3 h culture (Table 1). Oral administration of the extract shows more antitumour activity in DLA experiment in simultaneously treated groups than the groups in which treatment was started 10 days after tumour inoculation (Fig. 1a and b). Oral administration of the alcoholic extract of A. ilicifolius was also found to reduce the ascites tumour in mice (50 mg/kg) in EAC experiment. The maximum percentage of life span increased was calculated as 75% (Table 2). Fig. 2(a and b) shows that A. ilicifolius extract is an inhibitor of DMBA/croton oil induced skin papilloma in mice in a dose dependent manner. The papilloma formation was delayed in the treated groups compared with the control group animals. The maximal papilloma inhibition was noted at a concentration 500 mg/ ml (P B 0.001). These results were compared with that of a natural anticarcinogenic compound curcumin. The histopathological examination of the skin of control animals shows at the cells with atypical (enlarged and hyperchromatic) nuclei at all levels of the epidermis. Some of the cells break through the basement membrane, the process has become invasive. This invasive tumour cells exhibit enlarged nuclei with angulated contours and prominent nucleoli. All of these symp- 4. Discussion The use of chemotherapeutic drugs in cancer therapy involves the risk of life threatening host toxicity. The search there fore continues to develop the drug which selectively act on tumour cells. The search for new antitumour agents from plants has been extensively studied (Babu et al., 1995). In the present study it is found that the alcoholic extract of A. ilicifolius is cytotoxic towards the tumour cells in 72 h assay, but not in 3 h assay. From this we can assume that the extract acted upon cell cycle. In vivo antitumour studies revealed that the extract significantly (PB 0.001) increased the life span of ascites tumour bearing mice dose dependently. Moreover, the extract significantly reduced the solid tumour development in mice. The tumour reduction was high in animals treated simultaneously with the extract. It was reported that plant derived extracts containing antioxidant principles showed cytotoxicity towards tumour cells (Jiau-Jian and Larry, 1977) and antitumour activity in experimental animals (Ruby et al., 1995). Antitumour activity of these antioxidants is either through induction of apoptosis (Ming et al., 1998) or by inhibition of neovascularisation (Putul et al., 2000). Interestingly the extract of A. ilicifolius also significantly delayed the onset of DMBA/Croton oil induced skin papilloma in mice. Carcinogenesis involves mainly three steps namely initiation, promotion and progression. The implication of free radicals in different steps of carcinigenesis is well documented (Player, 1982; Frenkel, 1992). In our earlier studies we found that alcoholic extract of A. ilicifolius possess antioxidant and hepatoprotective effect (Babu et al., 2001). The free radical hypothesis supported the fact that the antioxidants can effectively inhibit carcinogenesis and the observed properties may be attributed to the antioxidant principles present in the extract. In conclusion, the present study reveals Table 2 Effect of Acanthus ilicifolius leaves extract on ascites tumour reduction in mice Tested material Dose (mg/kg b wt) No. of animals with tumour No. of days survived Increase in life span Control Curcumin AL AL Saline 100 250 500 6/6 6/6 6/6 6/6 169 2.1 309 3.2* 229 2.4* 289 3.1* – 87.5 37.5 75 Values are mean 9 SD (n= 6). * PB0.001 (Student’s t-test). 30 B.H. Babu et al. / Journal of Ethnopharmacology 79 (2002) 27–33 Fig. 1. (a) Effect of A. ilicifolius extract administration on solid tumour development (Simultaneous treatment). (b) Effect of A. ilicifolius extract administration on solid tumour development (after 10 days treatment). B.H. Babu et al. / Journal of Ethnopharmacology 79 (2002) 27–33 31 Fig. 2. (a) Effect of A. ilicifolius extract on DMBA/croton oil induced skin Papilloma (% tumour incidence). (b) Effect of A. ilicifolius extract on DMBA/croton oil induced skin Papilloma (papilloma per mouse). 32 B.H. Babu et al. / Journal of Ethnopharmacology 79 (2002) 27–33 Fig. 3. (a) Normal view of epidermis. (b) Photograph showing deep infiltration of dermal layers in untreated control animals. (c) Section of the skin of the animal treated with (500 mg/kg) of the extract. B.H. Babu et al. / Journal of Ethnopharmacology 79 (2002) 27–33 that A. ilicifolius contain antitumor compounds. Further investigations are necessary for the isolation of active principle and to elucidate the mechanism of action. Acknowledgements The authors are grateful to Dr Joseph Jeorge of Department of Pathology, Amala Cancer Hospital for the critical evaluation of the histopathological specimens and S. Shakkuty, of Department of Pathology for his friendly assistance and whole hearted co-operation for the tissue processing and staining. References Agshikar, N.V., Naik, V.R., Abraham, G.J.S., Reddy, C.V.G., Naqui, S.W.A., Mittal, P.K., 1979. Analgesic, antiinflammatory activity of Acanthus ilicifolius Linn. Indian Journal of Experimental Biology 17, 1257 – 1258. Anis, K.V., Kuttan, G., Kuttan, R., 1999. Role of Beriberin as an adjuvant response modifier during tumour therapy in mice. Pharmacy and Pharmacology Communication 5, 697 – 700. Babu, B.H., Shylesh, B.S., Padikkala, J., 2001. Antioxidant and hepatoprotective effect of Acanthus ilicifolius. Fittoterapia 72 (3), 271 – 276. Babu, T.D., Kuttan, G., Padikkala, J., 1995. Cytotoxic and antitumour properties of certain taxa of Umbelliferae with special reference to Centella asiatica (L.) Urban. Journal of Ethnopharmacology 48, 53 – 57. Frenkel, K., 1992. Carcinogen mediated oxidant formation and oxidative DNA damage. Pharmacology Therapy 53, 127 – 166. Giulia, D.C., Nicola, M., Angelo, A.I., Francesco, C. (1999) In: Life Science Flavanoids: Old and new aspects of a class of natural therapeutic drugs. Life Science, vol. 65, (4) pp. 33 – 353. Harbone, J.B., 1973. Phytochemical Methods. Chapman and Hall Ltd., London, pp. 52– 105. Jiau-Jian, L., Larry, W.O., 1977. Over expression of manganese-con- 33 taining superoxide dismutase confers resistance to the cytotoxicity of tumour necrosis factor a and/or hyperthermia. Cancer Research 57, 1991 – 1998. Kapil, A., Sharma, S., Wahidulla, S., 1994. Leishmanicidal activity of 2-benzozazolinone from Acanthus ilicifolius in 6itro. Planta Medica 60, 187 – 188. Keith, M.W., Sally, A.L., Michael, W.S., Thomas, J.G., Garry, M.M., 1990. Taxus Spp. Needles contain amounts of taxol comparable to the stem bark of taxus brevifolia: analysis and isolation. Journal of Natural Products 53, 1249 – 1255. Mary, K.T., Kuttan, G., Kuttan, R., 1994. Partial purification of tumour reducing principle from Helicanthus elasticus (fam. Loranthaceae). Cancer Letters 81, 53 – 57. Ming, L., Jill, C.P., Jingfang, Jn., Edward, C., Brash, E., 1998. Antioxidant action via p53 mediated apoptosis. Cancer Research 58, 1723 – 1729. Osawa, T., Kawakishi, S., Namiki, M., 1990. In: Kuroda, Y., Shankel, D.M., Waters, M.D. (Eds.), Antimutagenesis and Anticarcinogenesis Mechanisms II. Plenum, New York, pp. 139 – 153. Player, T., 1982. In: Mc Brein, D.C.H., Slater, T.F. (Eds.), Free Radicals and Cancer. Academic Press, London, pp. 173 – 195. Putul, M., Sunit, C., Pritha, B., 2000. Neovascularisation offers a new perspective to glutamine related therapy. Indian Journal of Experimental Biology 38, 88 – 90. Roja, G., Heble, M.R., 1994. The quinoline alkaloid Camptothecin and 9-methoxy camptothecin from tissue cultures and mature trees of Nothapodytes foetida. Phytochemistry 36, 65 – 66. Ruby, A.J., Kuttan, G., Babu, K.D., Rajasekharan, K.N., Kuttan, R., 1995. Antitumour and antioxidant activity of natural curcuminoids. Cancer Letters 94, 783. Sasaki, N., Bertrand, O., Nakazawa, H., Fitzgerald, D.T., Mirnov, N., Yamasaki, H., 1995. Cell type specific ras mutation but no microsatalite instability in chemically induced miuse skin tumours and transformed 3T3 Cells. Cancer Research 55, 3513. Shylesh, B.S., Padikkala, J., 2000. Invitro cytotoxicity and antitumour property of Emilia sonchifolia in mice. Journal of Ethnopharmacology 53, 463 –468. Stahl, E., 1969. Thin Layer Chromatography, a Laboratory Handbook. Springer International, New York, pp. 206 –258. Subudhi, H.N., Choudhary, B.P., Acharya, B.S., 1992. Some medicinal plants of Mahanadi Delta in state of Orissa. Ecnomic and Taxonomic Botany 16, 479 – 487. Wagner, H., Bladt, S., Zgaiwski, E.M. (Eds.), 1984. Plant Drug Analysis. Springer-Verlag, Berlin/New York, pp. 126 – 169. Journal of Ethnopharmacology 79 (2002) 35 – 41 www.elsevier.com/locate/jethpharm Evaluation of hepatoprotective potential of jigrine post-treatment against thioacetamide induced hepatic damage Aftab Ahmad a, K.K. Pillai a,*, Abul K. Najmi a, Shibli J. Ahmad a, S.N. Pal a, D.K. Balani b a Faculty of Pharmacy, Department of Pharmacology, Jamia Hamdard, Hamdard Uni6ersity, New Delhi 110 062, India b Disease In6estigation Laboratory, Krishi Gyan Kendra, Rohtak, Haryana, India Accepted 20 September 2001 Abstract Jigrine a polypharmaceutical herbal formulation containing aqueous extracts of 14 medicinal plants developed on the principles of unani system of medicine is used for liver ailments. The hepatoprotective potential of jigrine post-treatment at the dose of 0.5 ml/kg per day p.o. for 21 days was evaluated against thiocetamide induced liver damage in rats. Biochemical parameters like AST, ALT in serum and TBARS and glutathione in tissues were estimated to assess liver function. Data on the biochemical parameters revealed hepatoprotective potential of jigrine post-treatment against thioacetamide induced hepatotoxicity in rats. Silymarin used as reference standard also exhibited significant hepatoprotective activity on post-treatment against thioacetamide-induced hepatotoxity in rats. The biochemical observations were supplemented with histopathological examination of rat liver sections. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Jigrine; Unani medicine; Thioacetamide; Hepatic damage; Silymarin 1. Introduction Jigrine is a polypharmaceutical herbal hepatoprotective formulation containing aqueous extracts of 14 medicinal plants (Table 1). Few studies are reported for its safety evaluation (Valecha et al., 1990), mechanism of hepatoprotective action (Vivek et al., 1994; Karunakar et al., 1997a; Aftab et al., 1999) and antiinflammatory activity (Karunakar et al., 1997b). Hepatoprotective and anti-inflammatory effects of some of the individual ingredients of jigrine are also reported in literature (Jindal et al., 1975; Sadique et al., 1987; Chawla et al., 1992; Gilani and Aftab, 1992; Gilani et al., 1993; Gilani and Janbaz, 1994; Reddy et al., 1993; Pandey et al., 1994; Sultana et al., 1995; Zafar and Ali, 1998). The present investigation is designed to present an * Corresponding author. Fax: + 91-11-608-8874. root@hamduni.ren.nic (K.K. E-mail addresses: aknajmi@hotmail.com (A.K. Najmi). Pillai), experimental basis or evidence in rats, that treatment with jigrine can result in reversal of thioacetamide induced hepatic damage and cause decrease in lipid peroxidation. 2. Material and methods 2.1. Drugs and chemicals Jigrine was provided by Hamdard (Wakf) Labs. Ghaziabad, India. Silymarin was purchased from Micro Labs. Holar, TN, India. All the biochemicals and chemicals used were of Analytical grade. 2.2. Animals Albino rats of Wistar strain weighing 150 – 200 g were used for the study. Animals were supplied by Central Animal House Facility of Hamdard University and kept under standard laboratory conditions in 12 h light/dark cycle at 25920 °C. Animals were provided with pellet diet (Lipton, India) and water ad libitum. 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 4 9 - X 36 A. Ahmad et al. / Journal of Ethnopharmacology 79 (2002) 33–41 2.3. Experimental protocol Rats were randomly divided into six groups of six animals each.Group I served as normal control and received normal saline for 21 days. Animals in groups II, III and IV were administered a single injection of thioacetamide (100 mg/kg b.w., s.c.) as a 2% w/v solution in water for injection on day 1st and then received normal saline or jigrine (0.5 ml/kg per day, p.o.) or silymarin (25 mg/kg per day, p.o.), respectively in aqueous vehicle for 21 days from 2nd day. A suspension of 10 mg/ml of silymarin was prepared of which 2.5 ml/kg of body weight was administered. Animals in groups V and VI received jigrine (0.5 ml/kg per day, p.o.) and silymarin (25 mg/kg per day, p.o.) alone for 21 days. Group II served as toxic control and groups V and VI were used to see the per se effects of jigrine and silymarin treatments, respectively. All the groups were sacrificed on 22nd day of treatment after collecting blood from retro-orbital plexus under ether anesthesia for biochemical estimations. Liver samples were also collected for histological and biochemical estimations. The blood samples were allowed to clot for 30–40 min. Serum was separated by centrifugation at 37 °C and was used for estimation of various biochemical parameters. Liver samples collected were washed with chilled normal saline, weighed and 10% w/v liver homogenates were made in ice cold 0.15 M KCl solution using motor driven teflon pestle. 2.4. Assessment of li6er function 2.4.1. Assay of serum transaminase The activities of serum aspartate transaminase (AST) and alanine transaminase (ALT) were estimated by the method of Reitman and Frankel (1957). The enzyme activity was expressed as mkat/l. 2.4.2. Estimation of Na+ and K+ Na+ and K+ ions were estimated by using systronic flame photometer ‘Mediflame-127’ and expressed as mmol/l. 2.4.3. Determination of reduced glutathione Liver glutathione was estimated by 5,5-dithiobis-2-nitrobenzoic acid (DTNB) by the method of Ellman (1959) and expressed as mmol/g of liver. 2.4.4. TBARS estimation TBARS was used as an index of lipid peroxidation and measured by the modified method of Ohkawa et al. (1979). Total protein in the tissue homogenate was also estimated (Lowry et al., 1951). The levels of TBARS were expressed as nmol MDA/mg protein. 2.5. Histological studies Livers were quickly removed soon and preserved in neutral buffered formalin. Histological liver sections were prepared as described previously by Luna (1968). 2.6. Statistical analysis Results are expressed as mean9 SEM. Total variation present in a set of data was estimated by one-way analysis of variance (ANOVA). The F-ratio was also calculated. The difference among means was analyzed by LSD procedure at 95% (P B0.05) confidence level. P B0.05 was considered to be significant. 3. Results A significant increase (PB0.001) in AST, ALT, Na+, K and tissue TBARS levels was observed in animals treated with thioacetamide (Group II) as compared to + Table 1 Medicinal plant ingredients of Jigrine (a unani polypharmaceutical herbal formulation) Botanical name Common name Unani name Family Part used C. intybus Linn T. dioica Roxb S. nigrum Linn Rheum emodi wall Rubia cordifolia Linn V. negundo Linn Cassia occidentalis Linn F. 6ulgare Mill Cuscuta reflexa Roxb C. arborea Roxb Phyllanthus niruri Linn and Hook P. major Linn R. damascena Linn S. xanthocarpum Schrad and wendl. Chicory Tamarisk Black nightshade Indian rhubarb Indian madder Nisinda Coffee senna Fennel Amarvella Wild guava Jaramla Isphagol Damask rose Yellow berries night shade Tukhme kasni Jhau Makoh Revand chini Majeeth Sambhalu Kasaundi Sonf Tukhme kasoos Baokhamba Bhui amla Bartang Gul-e-surkh Katheli Compositeae Tamaricaceae Solanaceae Polygonaceae Rubiaceae Verbenaceae Caesalpiniaceae Umbellifereae Convolvulaceae Barringtoniaceae Euphorbiaceae Plantaginaceae Rosaceae Solanaceae Leave Leave Fruit Rhizo me Root Whole shrub Leave Fruit Seed Fruit Leave Leave Flower Root fruit Group SerumAST (mkat/l) SerumALT (mkat/l) SerumNa+ (mmol/l) SerumK+ (mmol/l) TissueGSH (mmol/g of liver) TissueTBARS (nmol MDA/mg protein) I II III IV V VI F-Ratio Significant difference among groups (ANOVA) 1.81 9 0.26 22.389 1.37 7.18 9 1.20 7.279 0.77 4.12 9 0.67 4.849 0.29 60.04 I and IIa, IIIc, IVb, II and IIIa, IVa, Va, VIa 0.62 9 0.08 13.03 9 0.58 7.76 9 0.47 7.43 9 0.63 4.23 9 0.26 5.68 9 0.40 81.30 I and IIa, IIIa, IVb, Va, VIa, II&IIIa, IVa, Va, VIa 23.90 90.87 36.24 91.48 27.339 0.75 28.73 91.48 29.60 9 0.54 30.93 9 1.29 10.32 I and IIa, IVd, Vd, Vib, II and IIIb, IVb, Va, VId 9.97 9 0.47 14.56 9 0.36 9.07 9 0.48 9.33 9 0.37 9.12 9 0.26 10.07 9 0.52 17.58 I and IIa, II and IIIa, IVa, Va, VIb 20.53 9 1.0 13.07 9 0.62 19.78 9 1.52 23.37 9 1.85 21.32 90.76 20.30 90.72 7.43 I and IIb, II and IIIc, IVb, Vb, VIc 0.95 90.05 2.21 90.04 1.29 90.14 1.45 90.09 0.65 9 0.11 0.69 9 0.09 32.54 I and IIa, IVc, II and IIIb, IVb, Va, VIa n = 6, data= 9 SEM. a PB0.001. b PB0.01. c PB0.02. d PB0.05. A. Ahmad et al. / Journal of Ethnopharmacology 000 (2002) 000–000 Table 2 Effect of jigrine and silymarin post-treatment on biochemical parameters of the rats intoxicated with thioacetamide 37 38 A. Ahmad et al. / Journal of Ethnopharmacology 79 (2002) 33–41 Fig. 1. Group I: Liver section of normal control rats showing: (a) well brought out central vein; (b) hepatic cell with well preserved cytoplasm; (c) prominent nucleus and nucleolus (H&E × 200). normal control group (Group I). Levels of AST, ALT, Na+, K+ and tissue TBARS concentration increased by thioacetamide treatment were decreased significantly by post-treatment of the rats with both jigrine (0.5 ml/kg per day, p.o.) and silymarin (25 mg/kg per day, p.o.) for 21 days. Moreover, the significant decrease in tissue glutathione, which was observed in animals treated with thioacetamide (Group II) as compared to normal control (Group I), was significantly reversed by jigrine and silymarin post-treatments. When the rats were treated with jigrine or silymarin alone any per se effect on the levels of AST, K+ and tissue glutathione and tissue TBARS concentration was not produced as compared to normal control animals. However, treatment with jigrine or silymarin alone significantly increased the levels of ALT and Na+ in serum as compared to normal control group (Table 2). Fig. 2. Group II: Liver section of thioacetamide (100 mg/kg s.c.) treated rats showing: (a) central vein; (b) hepatic cell with severe toxicity known as centrilobular necrosis; (c) empty vacuoles showing fatty changes (fatty droplets) (H&E × 200). 4. Discussion 3.1. Histopathological obser6ations Thioacetamide was originally used as a fungicide to protect against decay of oranges (Childs, 1946). It was soon recognized as a potent hepatotoxin and carcinogen in rats (Fitzhugh and Nelson, 1948). The compound has also been reported toxic for kidney and thymus (Barker and Smucklear, 1994). It is also reported that the chronic thioacetamide exposure produced cirrhosis in rats (Chieli and Malvadi, 1985). Cyt-P450 system is known to metabolize thioacetamide in rat liver. Mechanism of thioacetamide toxicity is due to the formation of thioacetamide-5-oxide which is responsible for the change in cell permeability, increased intracellular concentration of Ca++, increase in nuclear volume and enlargement of nucleoli and also inhibits mitochondrial activity which leads to cell death (Ambrose et al., 1949, 1950; Neal and Halpert, 1982). Histology of the liver sections of normal control animals (Group I) showed normal hepatic cells with well preserved cytoplasm, prominent nucleus and nucleolus and well brought out central vein (Fig. 1). The liver sections of thioacetamide treated animals showed hepatic cells with severe toxicity characterized by centrilobular necrosis along with various gradation of fatty changes comprising of tiny to large sized vacuoles (fatty droplets, thereby thioacetamide showed cumulative type of toxicity (Fig. 2). Jigrine post-treatment (0.5 ml/kg per day, p.o. for 21 days) appeared to significantly reverse thioacetamide toxicity as revealed by normal central vein, hepatic cells with well-preserved cytoplasm along with prominent nucleus and nucleolus (Fig. 3). Silymarin (25 mg/kg per day, p.o. for 21 days) also reversed thioacetamide toxicity (Fig. 4). Treatment with jigrine or silymarin alone showed normal histology of liver sections. Fig. 3. Group III: Liver section of rats treated with thioacetamide (100 mg/kg s.c.) + jigrine (0.5 ml/kg p.o. × 21 days, showing: (a) well brought out central vein; (b) hepatic cell with well preserved cytoplasm; (c) prominent nucleus and nucleolus (H&E × 200). A. Ahmad et al. / Journal of Ethnopharmacology 79 (2002) 33–41 Fig. 4. Group IV: Liver section of rats treated with thioacetamide (100 mg/kg s.c.) + silymarin (25 mg/kg p.o.) × 21 days, showing: (a) well brought out central vein; (b) hepatic cell with well preserved cytoplasm; (c) prominent nucleus and nucleolus (H&E × 200). In a number of animal models, thioacetamide induced cirrhosis seem to resemble the important features of human diseases (Torres-lopez et al., 1996). Elevated levels of serum enzymes are indicative of cellular leakage and loss of functional integrity of cell membrane in liver (Drotman and Lawhorn, 1978). Damage to liver cells cause leakage of cellular enzymes into serum. Significant rise in serum transaminases concentration (AST and ALT) could be taken as an index of liver damage. In our study, the rise in AST and ALT levels induced by thioacetamide was significantly reduced by post-treatment with jigrine suggesting that its hepatoprotective activity might be due to its effect against cellular leakage and loss of functional integrity of the cell membrane in liver. Membrane stabilizing property of jigrine has already been reported (Karunakar et al., 1997b). Thioacetamide also produces lesions in the adrenal cortex and can affect adrenal cortical functions which lead to decreased aldosterone secretion which in turn leads to increased extra cellular concentration of K+ (Thomas, 1995). It is evident from various models of hepatic dysfunction that there is a functional role of liver in development of Na+ retention in hepatic dysfunction (Wensing et al., 1997; Jimenez et al., 1998). Our results showed a significant elevation in the serum levels of K+ and Na+ of rats treated with thioacetamide. Post-treatment of rats with jigrine and silymarin significantly reduced the levels of K+ and Na+. This study has demonstrated that jigrine and silymarin treatments also have beneficial effect on adrenal cortex along with hepatoprotective function. The increase in the level of Na+ in serum caused by treatment with jigrine or silymarin alone might be due to the presence of flavonoids or steroidal moiety in various constituents of jigrine (e.g. Cichorium intybus, Tamarix dioica, Careya arborea, Rosa damascena contain flavonoids and Solanum nigrum, Solanum xanthocarpum contain 39 steroidal alkaloids). It is reported that jigrine possess anti-inflammatory activity (Karunakar et al., 1997a). It is well known that all non-steroidal anti-inflammatory drugs inhibit prostaglandin E2 synthesis. Prostaglandins play an important role in kidney blood flow (Campbell and Halushka, 1996). Thus, it might be possible that jigrine due to its anti-inflammatory activity might inhibit the biosynthesis of prostglandins resulting in decreased kidney blood flow which might lead to retention of sodium in serum. However, the reduction in the serum levels of Na+ and K+ caused by jigrine and silymarin in the thioacetamide treated rats showed the primary effect of jigrine and silymarin in liver cells as compared to their effects on the adrenal cortex and kidney cells. Glutathione is an important endogenous antioxidant system that is found in particularly high concentration in liver and it is known to have key functions in protective processes. The reduced form of GSH becomes readily oxidized to GSSG on interacting with free radicals. Excessive production of free radicals resulted in the oxidative stress, which leads to damage of macromolecules e.g. lipids, and can induce lipid peroxidation in-vivo (Sinclair et al., 1991). In our study, thioacetamide treatment produced the elevation in the levels of TBARS and depletion in glutathione (GSH). Post-treatment of the rats with jigrine significantly reduced the elevated levels of TBARS and increased the concentration of GSH. These results suggest that the hepatoprotective action of jigrine might be due to the presence of antioxidants like Flavonoids, Vitamin A, Vitamin C and a and b carotenes (Davila et al., 1989; Toshiyuki et al., 1992). These antioxidants are present in the plant ingredients of jigrine e.g. flavonoids are present in C. intybus, T. dioica, C. arborea, R. damascena, vitamin A is present in Foeniculum 6ulgare, vitamin C is present in S. nigrum, Vitex negundo, F. 6ulgare, Plantago major, a and b carotenes are present in S. nigrum, V. negundo etc. (Chawla et al., 1992; Sultana et al., 1995; Zafar and Ali, 1998; Samuelsen, 2000; Najmi et al., in press). Thioacetamide is reported to produce centrilobular necrosis without marked accumulation of lipids on acute exposure (Zimmerman, 1978). In our post-treatment experimental model the livers, after 21 days of thioacetamide administration, showed severe hepatotoxicity characterized by centrilobular necrosis along with various degrees of fatty changes comprising of tiny to large vacuoles (fatty droplets). The absence of pharmacological intervention during the period of 21 days after administration of thioacetamide might be responsible for the above changes. Similar findings were not reported in the literature earlier. Post-treatment with jigrine and silymarin for 21 days after administration of thioacetamide significantly reversed the above histopathological changes. Our study showed the hepatoprotective potential of 40 A. Ahmad et al. / Journal of Ethnopharmacology 79 (2002) 33–41 jigrine and silymarin post-treatment in thioacetamide induced liver damage in rats. The present study demonstrates that the hepatoprotective effect of jigrine against thioacetamide-induced hepatotoxicity is due to multiple mechanisms. This study also demonstrates that jigrine and silymarin also have beneficial effect on adrenal cortex, but detailed biochemical and histopathological studies are needed to confirm this effect in thioacetamide-induced toxicity. It is also suggested that the post-treatment experimental model of thioacetamide-induced hepatotoxicity may be used for evaluation of hepatoprotective effects of various drugs Acknowledgements Authors are thankful to Hamdard (Wakf) Labs. Ghaziabad for providing generous gift sample of jigrine, and Mohd. Imran for technical assistance. References Aftab, A., Pillai, K.K., Najmi, A.K., Jameel, S.J., Balani, D.K., 1999. Evaluation of hepatoprotective potential of jigrine (a polyherbal unani formulation) pre-treatment on thioacetamide induced liver damage in rats. Indian Journal of Pharmacology 31, 416 – 421. Ambrose, A.M., DeEds, F., Rather, L.J., 1949. Toxicity of thioacetamide in rats. Journal of Indian Hygiene and Toxicology 31, 158 – 161. Ambrose, A.M., DeEds, F., Rather, L.J., 1950. Further studies on toxicity of thioacetamide in rats. Proceedings of Society of Experimental and Biological Medicine 74, 134 – 140. Barker, E.A., Smucklear, E.A., 1994. Nonhepatic thioacetamide injury, the morphological features of proximal tubular injury. American Journal of Pathology 74, 575 – 590. Campbell, W.B., Halushka, P.V., 1996. Lipid derived autocoids. In: Hardman, J.G., Limbird, L.E., Molinoff, P.B., Ruddom, R.W., Gilman, A.G. (Eds.), Goodman and Gilman’s The Pharmacological Basis of Therapeutics. McGraw Hill, New York, p. 607. Chawla, A.S., Sharma, A.K., Handa, S.S., Dhar, K.L., 1992. Chemical investigation and anti-inflammatory activity of Vitex negundo seeds. Journal of Natural Products 55, 163 – 166. Chieli, E., Malvadi, G., 1985. Role of Cyt P-450 dependent and FAA containing mono oxygenases in the bioactivation of thioacetamide, thiobezamide and their sulophoxides. Biochemical Pharmacology 34, 395 – 396. Childs, J.F.L., 1946. Controlling orange decay. Indian journal of Chemistry 38, 82. Davila, J.C., Lenherr, A., Acosta, D., 1989. Protective effects of flavonoids on drug induced hepatotoxicity. Toxicology 57, 267 – 268. Drotman, R.B., Lawhorn, G.T., 1978. Serum enzymes are indicators of chemical induced liver damage. Drug and Chemical Toxicology 1, 163 – 171. Ellman, G.L., 1959. Tissue sulphydryl groups. Archives of Biochemistry and Biophysics 82, 70 – 77. Fitzhugh, O.G., Nelson, A.A., 1948. Liver tumours in rats fed thiourea or thioacetamide. Science 108, 626 – 628. Gilani, A.H., Aftab, K., 1992. Pharmacological actions of Casuta reflexa. International Journal Pharmcognosy 30 (11), 296 –302. Gilani, A.H., Janbaz, K.H., 1994. Evaluation of liver protective potential of Cichorium intybus seed extract on acetaminophen and CCl4-induced damage. Phytomedicine 1, 193 – 197. Gilani, A.H., Janbaz, K.H., Javed, M.H., 1993. Hepatoprotective activity of Cichorium intybus, an indigenous medicinal plant. Medical Science Research 21, 151 – 152. Jimenez, W., Pardo, M.A., Arroyo, V., Bruix, J., Rimola, A., Gaya, J., Rivera, F., Rodes, J., 1998. Temporal relationship between hyperaldosteronism, sodium retention and ascites formation in rats with experimental cirrhosis. Hepatology 5, 245 – 250. Jindal, M.N., Patel, V.K., Patel, N.B., 1975. Some pharmacological activities of aqueous and alcoholic extract of Cichorium intybus Linn. Indian Journal of Pharmacology 7, 104. Lowry, O.H., Rosebrough, N.J., Forr, A.L., Ramdall, R.J., 1951. Protein measurement with the Folins phenol reagent. Journal of Biological Chemistry 193, 265 – 275. Luna, L.G., 1968. Manual of Histology, Staining Methods of Armed Forces Institute of Pathology, 3rd ed. McGraw Hill Book co, New York, p. 43. Karunakar, N., Pillai, K.K., Hussaian, S.Z., Rao, M., 1997a. Investigation of antiinflammatory activity of jigrine. Indian Journal of Physiology and Pharmacology 41 (2), 134 – 138. Karunakar, N., Pillai, K.K., Hussaian, S.Z., Rao, M., Balani, D.K., Imran, M., 1997b. Further studies on the antihepatotoxic activity of jigrine. Indian Journal of Pharmacology 29, 222 – 227. Najmi, A.K., Pillai, K.K., Ahmad, S.J., Nazmi, A.S., 2001. Jigrine-A reappraisal of its medicinal ingredients. Hamdard Medicus XLIV(4) (In press). Neal, R.A., Halpert, J., 1982. Toxicology of thionosulfer compounds. Annual Review of Pharmacology and Toxicology 22, 321 – 329. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animals tissues by thioabarbituric acid reaction. Analytical Biochemistry 95, 351 – 358. Pandey, S., Sharma, M., Chaturvedi, P., Tripathi, Y.B., 1994. Protective effect of Rubia cordifolia on lipid peroxide formation in isolated rat liver homogenate. Indian Journal of Experimental Biology 32 (3), 180 –183. Reddy, B.P., Murthy, V.N., Venkateshwarlu, V., Kokate, C.K., Rambhau, D., 1993. Antihepatotoxic activity of Phyllanthus niruri, Tinuspora cordifolia and Ricinus communis. Indian Drugs 30, 338 –341. Reitman, S., Frankel, A.S., 1957. A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. American Journal of Clinical Pathology 28, 53 – 56. Sadique, J., Chandra, T., Thenmozihi, V., Elango, V., 1987. Biochemical modes of action of Cassia occidentalis and Cardiospermum halicacabum in inflammation. Journal of Ethnopharmacology 19, 201 – 212. Samuelsen, A.B., 2000. The traditional uses, chemical constituents and biological activities of Plantago major L. a review. Journal of Ethnopharmacology 71 (1), 1 – 21. Sultana, S., Shahid, P., Mohammad, I., Mohammad, A., 1995. Crude extracts of hepatoprotective plants, Solanum nigrum and Cichorium intybus inhibits Free radical mediated DNA damage. Journal of Ethnopharmacology 45, 189 – 192. Sinclair, A.J., Barnett, A.H., Lunie, J., 1991. Free radical and autooxidant systems in health and disease. Journal of Applied Medicine 17, 409. Thomas, J.A., 1995. Toxicology of the adrenal, thyroid and endocrine pancrease. In: Ballantyne, B., Marrs, T., Turner, P. (Eds.), General and Applied Toxicology, Abridged ed. The Macmillan press ltd, UK, pp. 749 – 751. Torres-lopez, M.I., Fernandez, I., Fontana, I., Gil, A., Rios, A., 1996. Influence of dietary nucleotides on liver structural recovery and hepatocyte binuclearity in cirrhosis induced by thioacetamide. Gut 38, 260 – 264. A. Ahmad et al. / Journal of Ethnopharmacology 79 (2002) 33–41 Toshiyuki, M., Ryuji, K., Susumu, I., Yoshihiko, F., Mitsukazu, K., Kamataki, T., 1992. Examinatin of lipid peroxidation in liver microsomes of guinea pig as causal factors in decrease in content of cytochrome P-450 due to Ascorbic acid deficiency. Research Communications in Chemical Pathology and Pharmacology 75 (2), 209 – 219. Valecha, N., Khan, E.A., Dandiya, P.C., 1990. A note on the safety evaluation of Jigrine, a Unani hepatoprotective herbal formulation. Indian Drugs 27, 411 – 415. Vivek, K., Pillai, K.K., Hussaian, S.Z., Balani, D.K., 1994. Hepatoprotective activity of Jigrine on liver damage caused by alcohol- 41 CCl4 and paracetamol in rats. Indian Journal of Pharmacology 26, 35 – 40. Wensing, G., Lotterer, E., Link, I., Hahn, G.E., Fleig, E.W., 1997. Urinary sodium balance in patients with cirrhosis: relationship to quantitative parameters of liver function. Hepatology 26, 1149 – 1155. Zafar, R., Ali, S.M., 1998. Antihepatotoxic effect of root and root callus extract of Cichorium intybus L. Journal of Ethnopharmacology 63, 227 –231. Zimmerman, H.J., 1978. Hepatotoxicity. Appleton-century-crofts, New York, pp. 232 – 487. Journal of Ethnopharmacology 79 (2002) 43 – 52 www.elsevier.com/locate/jethpharm Yucatec Mayan medicinal plants: evaluation based on indigenous uses Anita Ankli a, Michael Heinrich b,g, Peter Bork b, Lutz Wolfram c, Peter Bauerfeind c, Reto Brun d, Cécile Schmid d, Claudia Weiss e, Regina Bruggisser f, Jürg Gertsch a, Michael Wasescha a, Otto Sticher a,* a Department of Applied Biosciences, Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH) Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland b Institute of Pharmaceutical Biology, Albert-Ludwigs-Uni6ersity, Schänzlestr. 1, D-79104 Freiburg, Germany c Di6ision of Gastroenterology, Department of Internal Medicine, Uni6ersity Hospital Zurich, Rämistr. 100, 8091 Zurich, Switzerland d Department of Medical Parasitology, Swiss Tropical Institute, Socinstr. 57, CH-4002 Basel, Switzerland e Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, UK f Department of Pharmaceutical Biology, Uni6ersity of Basel, Klingelbergstr. 50, CH-4056 Basel, Switzerland g Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, 29 /39 Brunswick Sq., London WC1N 1AX, UK Received 13 October 2000; received in revised form 30 August 2001; accepted 20 September 2001 Abstract As part of an ethnopharmacological field study 48 medicinal plants were evaluated using several biological assays with the goal to obtain information on the pharmacological effects of these plants, which may be of direct relevance to the indigenous uses. Three species used to treat gastrointestinal disorders showed remarkable activity against Helicobacter pylori. One of them showed activity against Giardia duodenalis. Cytotoxic effects against KB cells were found for six species. In the group of plants used for dermatological conditions several species were active against gram-positive bacteria and Candida albicans. Two plant species of this group were found to be active in an Nuclear Factor-kB (NF-kB) assay measuring inhibition of this pro-inflammatory transcription factor. A species of the Solanaceae, applied in cases of pain and fever, showed a weak activity against Plasmodium falciparum. One species traditionally used for diabetes exhibited antihyperglycemic activity. None of the six species from the group of ‘women’s medicine’ showed relevant affinity to the D2 dopamine receptor. Based on this evaluation, plants with strong activities should be further investigated phytochemically and pharmacologically to identify active fractions and compounds. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Yucatec Maya; Ethnopharmacological evaluation; Medicinal plants; Traditional medicine; Antibacterial; Anti-inflammatory; Antihyperglycemic; Antiparasitic; D2 receptor binding 1. Introduction One of the numerous objectives of medical ethnobotany is the selection of culturally important plant species in order to further evaluate them for pharmacological activity (Browner et al., 1988; Etkin, 1994; Farnsworth, 1988; Frei et al., 1998a; Messner, 1978). In order to evaluate an ethnopharmacopeia systematically, plant extracts can be tested in bioassays, which have * Corresponding author. Tel.: + 41-1-635-6050; fax: + 41-1-6356882. E-mail address: sticher@pharma.anbi.ethz.ch (O. Sticher). direct relevance to the indigenous uses (Frei et al., 1998b; Heinrich et al., 1992a,b; Lewis and Elvin-Lewis, 1994). The knowledge of medicinal plants was a part of the ancient Maya culture and they are still utilised by the Yucatec Mayan inhabitants on the Peninsula of Yucatan, Mexico (Roys, 1933; De Landa, 1992). During an ethnobotanical study in three Mayan communities (February 1994 – June 1995; September 1996 – October 1996), 360 medicinal plants and 1828 reports on their uses were documented. The uses of the plants were divided into nine therapeutic groups (Ankli et al. 1999; Heinrich et al. 1998). Forty-eight species were chosen 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 5 5 - 5 A. Ankli et al. / Journal of Ethnopharmacology 79 (2002) 43–52 44 and evaluated in bioassays relevant to the following groups of illnesses, gastrointestinal disorders; dermatological conditions; women’s medicines as well as pain and/or fever. Plants used to treat ‘diabetes’ were also tested (Table 2). In case of Staphylococcus aureus and Yersinia enterocolitica extract C was used. Extract C was prepared by extracting 10 g of plant material with ethanol 96% followed by ethanol 70% and the extracts combined and reduced under vacuum. 2.3. Bioassays 2. Materials and methods 2.1. Plant material The plants were collected in the villages and surroundings of Chikindzonot, Ekpedz and Xcocmil, Yucatan (Mexico). Authenticated voucher specimens were deposited at the Herbarium of the Centro de Investigación Cientı́fica de Yucatán (CICY) in Mérida, the National Herbarium of Mexico (MEXU), the Instituto Nacional Indigenista (INI) in Valladolid, Yucatan, the ETH Zurich (ZT) and the Centre for Pharmacognosy and Phytotherapy, School of Pharmacy, London, UK (AANK1-654). They were identified by comparison with authentic specimens and in some cases with the assistance of specialists at CICY, MEXU and The Royal Botanical Garden, Kew, UK (K). 2.2. Extract preparation Shade-dried and powdered plant material (20 g) was extracted by maceration with 100 ml dichloromethane/ methanol 2:1, repeating the process two times during 36 h. The filtered solvents were combined and evaporated under vacuum to give the non-polar extract A. The residue of the dichloromethane/methanol mixture was dissolved in 100 ml methanol/water 7:3 and macerated two times during 24 h. The solvents were reduced under vacuum and the extract was further partitioned between n-butanol (3×30– 50 ml) and water. The n-butanol fractions were evaporated to obtain the polar extract B. 2.3.1. Antibacterial and antifungal acti6ity The organisms used to test biological activities are listed in Table 1. Antimicrobial tests were performed by the disc diffusion technique (Rı́os et al., 1988; DIN, 1992). Overnight cultures of microorganisms were prepared by transferring 30 ml of store culture to 5 ml broth (Nutrient Broth for bacteria, Sabouraud Liquid Medium Oxoid for fungi). Ten milliliter of Mueller– Hinton agar or malt extract agar (for Candida albicans) was inoculated with 50 ml of overnight culture and poured over the agar base. Paper discs (6 mm Blanc Discs, Oxoid) were impregnated with 200 and 600 mg plant extracts, the solvent evaporated and the treatment discs placed on the inoculated agar. The plates were sprayed with methylthiazolyltetrazolium chloride (MTT, Fluka) after 16 h of incubation at 37 °C. The inhibition zones were measured in mm. Susceptibility tests with Helicobacter pylori and Campylobacter jejuni were carried out on Wilkins Chalgren agar plates supplemented with 5% defibrinated sheep blood and the following antibiotics, 2 mg amphotericin B per ml, 6 mg vancomycin per ml, 5 mg cefsulodin per ml, and 5 mg trimethoprim per ml. Hundred microliter of a thick H. pylori suspension (yield from one agar plate, resuspended in 1 ml phosphate buffered saline (PBS)) was applied onto each plate and the treatment discs (600 mg plant extract) placed on the plates. They were then incubated under a water-saturated, micro-aerophilic atmosphere at 37 °C for 1 day (C. jejuni ), or 3 –5 days (H. pylori ). Table 1 Test organisms for antibacterial and antifungal activity Microorganism Origin Clinical picture of diseasesb Gastrointestinal problems Bacillus cereus Campylobacter jejuni Candida albicans Escherichia coli Helicobacter pylori Pseudomonas aeruginosa Staphylococcus aureus Staphylococcus epidermidis Yersinia enterocolitica a b ATCC 10702 a H29 ATCC 26790 ATCC 25922 ATCC 43504 ATCC 25922 ATCC 25933 ATCC 12228 O3 Dermatological conditions Diarrhea Enteritis, diarrhea Diarrhea, dysentery Gastritis, peptic ulcer Diarrhea (food intoxication) Mycosis Infected wounds Infected wounds Topical infection Infection (septicemia) Enterocolitis Obtained from the Division of Gastroenterology, Department of Internal Medicine, University Hospital Zürich. (Kayser et al., 1993). A. Ankli et al. / Journal of Ethnopharmacology 79 (2002) 43–52 The MIC determination for H. pylori was carried out in a modified minimal medium according to Nedenskov (1994). Different concentrations (in the range from 0.3 to 200 mg/ml) of the plant extracts were added to the uninoculated medium (5 ml in a 25 ml Erlenmeyer flask). A fresh H. pylori culture was used as a 1% inoculum and grown under micro-aerophilic conditions in a water-saturated atmosphere at 37 °C in a rotary shaker (G25; New Brunswick Scientific, New Jersey, USA). The incubation was continued for 2 days at 175 rpm. After 2 days the optical density of the cultures were photometrically determined at 600 nm (DU-64 spectrophotometer, Beckman, UK). The MIC value was defined as the extract concentration not allowing visible growth (less than 0.03 in comparison with 1.2 for the control). 2.3.2. Cytotoxicity study using KB cell culture The cytotoxicity of the plant extracts was assessed using the KB cell line (ATCC CCL 17; human nasopharyngeal carcinoma). The test was carried out with some modifications according to the screening technique of Swanson and Pezzuto (1990). The assay was performed in 96-well plates (Falcon) with an inoculum of 2.5× 104 cells per ml. Total volume was 150 ml. The dried extracts were dissolved in ethanol. Water was added to dilute the solution 5-fold. Concentration of 50 mg/ml with maximum 1% ethanol was tested. These solutions were diluted 20-fold by mixing it with culture medium. For active extracts, IC50 values were determined. The quantification was performed by adding 15 ml from a 5 mg/ml solution of MTT in PBS (Mosmann, 1983). After incubation at 37 °C for 4 h, the metabolically active cells produced an insoluble formazan dye. The medium was drawn off and the formazan dye was dissolved using 150 ml of 10% sodium dodecylsulfate (SDS) in water. After 24 h of incubation at room temperature (RT), the optical density was measured at 540 nm using a microplate reader (MRX, Dynex Technologies). 2.3.3. Inhibitory acti6ity on NF-sB Anti-inflammatory activity of the plant extracts was assayed in Electrophoretic Mobility Shift Assay (EMSA) using the inhibition of nuclear factor-kB (NFkB) binding to a radioactive labelled oligonucleotide as a molecular target. The bioassay was carried out as described in Bork et al. (1996). 2.3.4. Antimalarial acti6ity Antimalarial activity was assessed for the chloroquine resistant K1 strain and the chloroquine sensitive T9-96 clone of Plasmodium falciparum. The parasites were maintained in continuous culture of infected A+ human red blood cells in RPMI 1640 supplemented with 6.9 mg/ml HEPES, 2 mg/ml glucose, 45 2.33 mg/ml NaHCO3, 50 mg/ml hypoxanthine, 40 mg/ml gentamicin (all Sigma) and 10% A+ serum (North London Blood Transfusion Centre) (Fairlamb et al., 1985). Antimalarial IC50 values were assessed using the modified in vitro lactate dehydrogenase assay (Makler et al., 1993). Extracts were tested in concentrations from 1000 to 4.12 mg/ml (3-fold dilution). Fifty microliter of a 1% parasitemia blood suspension (predominantly ring form) were added to 50 ml of drug solution in RPMI 1640 (final hematocrit 2%). The 96-well microtiter plates were incubated for 48 h at 37 °C in an atmosphere of 1% O2 and 3% CO2 in balanced N2. After the incubation period, 20 ml of the parasite suspension was added to 100 ml of Malstat™ reagent (Flow Incorporated, USA) and incubated at RT for 15 min before adding 20 ml of freshly made 1:1 NBT/PESmixture (2 and 0.2 mg/ml, respectively) to each well. The plates were reincubated for 20 min at RT in the dark and subsequently read at 650 nm. All compounds were tested twice in triplicate. 2.3.5. Giardia duodenalis G. duodenalis trophozoites were cultivated in Diamondı́s modified TYI-S-33 medium (Keister, 1983) supplemented with 10% heat inactivated fetal calf serum (FCS). The in vitro assay was performed as described for the Alamar Blue® assay for trypanosomes by Raez et al. (1997) with modifications for G. duodenalis WB strain (isolated 1982 from a human in Afghanistan). Briefly, 200 ml of a trophozoite suspension were inoculated into 96-well microtiter plates (Costar, USA) at a density of 4× 105 trophozoites per ml culture medium. The trophozoites were incubated in the presence of serial 3-fold dilutions of extracts for 72 h at 37 °C. Wells without drug served as controls. Minimum inhibitory concentration (MIC) was determined microscopically after 70 h of incubation (the lowest drug concentration at which no trophozoite with normal morphology could be observed). Ten microliter Alamar Blue® were added to each well and after 2 h of incubation the fluorescence determined using a fluorometer (Cytofluor, Millipore; excitation wavelength at 530 nm, emission at 590 nm). IC50 values were calculated by linear interpolation selecting values above and below the 50% mark. 2.3.6. Dopamin D2 receptor binding assay Two concentrations of extracts (100 and 10 mg/ml) were tested in the dopamine receptor binding assay. The affinity of the extracts to the dopamine receptor was assessed according to Berger (1998). 2.3.7. h-Amylase assay Plant extracts (1, 3, 6 mg/ml in 5 ml solvent) were mixed with 45 ml of amylase reagent (ET-G7 PNP 1.0 mmol/l, magnesium chloride 10 mmol/l, sodium chlo- 46 A. Ankli et al. / Journal of Ethnopharmacology 79 (2002) 43–52 ride 50 mmol/l, a-glucosidase 25.000 U/l, buffer pH 7.0, sodium azide 0.05%) obtained from Sigma Diagnostics and incubated at 37 °C for 2–10 min. The absorbance was recorded at 405 nm versus water as a reference. The incubation was continued and the absorbance was read after exactly 1 and 2 min. The amylase activity was calculated according to Pierre et al. (1976). 3. Results All polar and non-polar extracts of the 48 plants were screened for cytotoxic activity against KB cells, Bacillus cereus, Escherichia coli, Candida and in the NF-kB test. Additionally, extracts were evaluated in selected test systems, which are of direct relevance to the indigenous uses of the species (Table 2). The microorganisms chosen cause gastrointestinal problems and/or dermatological illnesses, and generally are the causal agents of these conditions (Table 1). The extracts, which showed noteworthy activities, are listed in Tables 3 and 4. 3.1. Acti6e plants for gastrointestinal problems Gram-positive and Gram-negative bacteria as well as protozoa (G. duodenalis) were used to determine activities of species used for treating gastrointestinal disorders (Table 3). Six species showed at least some activity against G. duodenalis with IC50 values less than 100 mg/ml, three of them with MIC values less than 100 mg/ml. The most active extract was the non-polar extract A of Crossopetalum gaumeri (MIC 6.3 mg/ml), whereas the polar extract B showed very weak antiprotozoal activity. The non-polar and polar extracts of Psidium sartorianum, Piscidia piscipula, Bidens squarrosa and Casimiroa tetrameria and the non-polar fraction of Bauhinia di6aricata showed weak activity with IC50 values between 20 and 90 mg/ml. Several plants were active against H. pylori (Table 3). The non-polar and polar extracts of P. piscipula showed the highest activities (with MIC values of 0.7 and 3 mg/ml, respectively). The non-polar extracts of C. tetrameria (MIC, 3 mg/ml) and Jatropha gaumeri (MIC, 5 mg/ml) were also active against H. pylori. Other active extracts were Dorstenia contrajer6a (A and B) and the polar extracts of P. sartorianum, Microgramma nitida, Chrysophyllum mexicanum. The non-polar root extract of J. gaumeri was found to be the most active plant tested against B. cereus. Extract A of C. gaumeri as well as extracts A and B of P. sartorianum showed weak activities against this strain. The ethanol extract of C. tetrameria weakly inhibited the growth of S. aureus. There was no significant activity against C. jejuni. None of the tested plant species used for gastrointestinal problems showed activity against E. coli or Y. enterocolitica. 3.2. Acti6e plants for dermatological conditions Selected plants, documented as medicines for dermatological conditions, were evaluated for anti-inflammatory, antibacterial and antifungal activities (Table 4). Cytotoxicity of the plant extracts was evaluated using KB and the HeLa cell line (Table 4). The non-polar fraction of C. gaumeri showed the most potent effect in the NF-kB test with an inhibitory concentration of 25 mg/ml. The non-polar extracts of Diospyros anisandra and J. gaumeri inhibited NF-kB activation (Table 4). These effects are probably due to the potent cytotoxicity of the extracts against KB cells. The non-polar fractions of Dalea carthagenensis and Luehea speciosa elicited inhibition of NF-kB binding at 150 and 100 mg/ml, respectively. The former showed cytotoxic activity against KB cells (IC50 31 mg/ml), whereas no such effect could be detected for L. speciosa. Several extracts showed antibacterial effects against S. epidermidis and E. coli (Table 4). The most active extract against the former strain was extract B of Aechmea bracteata. The non-polar fruit extract of Morinda yucatanensis was active against S. epidermidis. Other active extracts included: the non-polar one of C. gaumeri and Casearia corymbosa as well as both extracts of P. sartorianum. 3.3. Antimalaria acti6ities Plants used against fever and/or pain were screened in vitro for antimalarial activity against Plasmodium falciparum (Table 2). The non-polar extract of Cestrum nocturnum showed some antimalarial activity (IC50 172.40 (2.80) mg/ml against chloroquine-sensitive strain of P. falciparum HB3; IC50 283.31 (0.48) mg/ml against chloroquine-resistant clone K1) without exhibiting overt cytotoxicity (IC50 ]50 mg/ml). The results of the positive control chloroquine are the following ones: IC50 7.96 (0.03) ng HB3 and IC50 193.96 (0.81) ng K1. The non-polar extract of C. corymbosa showed weak antimalarial activity (IC50 441.40 (2.26) mg/ml HB3; IC50 494.49 (3.07) mg/ml K1). All other extracts (Ehretia tinifolia, Caesalpinia gaumeri and Manilkara zapota) showed any activity (IC50 ]500 mg/ml). 3.4. D2 -receptor binding affinities Dopamine-agonists are used in the treatment of the premenstrual syndrome (PMS) (Steiner, 1997). For the dopamine D2 receptor binding assay, no significant activity was observed, however, the non-polar extracts of Sal6ia micrantha and Aristolochia maxima showed weak receptor affinity. A. Ankli et al. / Journal of Ethnopharmacology 79 (2002) 43–52 47 Table 2 Ethnomedical data on plants studied and chosen test systems Family Acanthaceae Annonaceae Apocynaceae Araceae Aristolochiaceae Asteraceae Basellaceae Bignoniaceae Bombacaceae Boraginaceae Bromeliaceae Cactaceae Caesalpiniaceae Celastraceae Cucurbitaceae Ebenaceae Euphorbiaceae Flacourtiaceae Lamiaceae Meliaceae Moraceae Myrtaceae Nyctaginaceae Papilionaceae Phytolaccaceae Polygonaceae Polypodiaceae Rubiaceae Rutaceae Sapotaceae Selaginellaceae Simaroubaceae Solanaceae Sterculiaceae Tiliaceae Plant name (AANKc voucher) Based on traditional use Ruellia nudiflora (Engelm. and Gray) Urb. (115) Malmea depressa (Baill.) R. E. Fr. (161) Tabernaemontana amygdalifolia Jacq. (190) Anthurium schlechtendalii Kunth ssp. schlechtendalii (243) Aristolochia maxima Jacq. (350) Verbesina gigantea Jacq. (288) Bidens squarrosa Less. (121) Anredera 6esicaria C. F Gaertner (196) Parmentiera millspaughiana (L.) Williams (135) Parmentiera aculeata (Kunth) Seem. (096) Pseudobombax ellipticum (Kunth) Dugand (275) Ehretia tinifolia L. (021) Aechmea bracteata var. bracteata Griseb. (167) Hylocereus undatus (L.) Britton & Rose (427) Bauhinia di6aricata L. (007) Caesalpinia gaumeri Greenman (155) Crossopetalum gaumeri (Loes.) Lundell (038) Iber6illea millspaughii (Cogn.) C. Jeffrey (094) Diospyros anisandra Blake (134) Diospyros cuneata Standl. (341) Croton reflexifolius Kunth (143) Jatropha gaumeri Greenman (419) Casearia corymbosa Jacq. (150) Sal6ia micrantha Desf. (025) Cedrela mexicana L. (301) Brosimum alicastrum Sw. (092) Dorstenia contrajer6a L. (330) Psidium sartorianum (Berg) Nied. (211) Neea psychotrioides F. D. Sm. (274) Pisonia aculeata L. (154) Dalea carthagenensis var. barbata (Oerst.) Barneby (125) Piscidia piscipula (L.) Sarg. (123) Phytolacca icosandra Sims (388) Ri6ina humilis L. (089) Neomillspaughia emarginata S. F. Blake (203) Microgramma nitida (J. Sm.) A. Reed Sm. (183) Borreria 6erticillata G. Meyer (276) Morinda yucatanensis Greenman (113) Casimiroa tetrameria Millsp. (049) Chrysophyllum mexicanum Brandegee (386) Manilkara zapota (L.) Royen, Achras zapota L. (234) Selaginella longispicata Underw. (214) Al6aradoa amorphoides Liebm. (136) Cestrum nocturnum L. (050) Solanum erianthum G. Don f. (334) Solanum nigrum L. (267) Helicteres baruensis Jacq. (176) Luehea speciosa Willd. (347) Group of use Plant part UR UR DER FEM GI, FEM RES GI DER UR UR RES RES, UR, NEU DER GI, UR RES, UR, GI NEU GI, DER DER DER DER DER GI, DER DER,NEU DER, FEM RES RES GI, FEM DER, GI DER FEM DER GI, RES DER DER DER, RES GI DER DER GI, NEU GI GI, NEU UR, RES DER DER, NEU DER DER FEM DER ap rt lv lv rt lv ap lv, tu lv lv, rt lv lv lv lv lv lv lv tu lv lv lv rt lv ap lv lv rh lv lv lv lv lv fr ap lv wp ap fr lv rt ba ap lv lv lv lv lv lv Tested for 1–7, 14 1–7, 14 1–5 1–5, 15 1–10, 15 1–5 1–12 1–7 1–5, 14 1–5 1–5 1–5, 13 1–7 1–12, 14 1–10, 14 1–7, 13 1–12 1–7 1–5 1–5 1–5 1–12 1–7, 13 1–7, 15 1–5 1–5 1–12, 15 1–10 1–7 1–7, 15 1–5 1–10 1–5 1–5 1–5 1–10 1–7 1–7 1–12 1–10 1–10, 13 1–5 1–7 1–5, 13 1–5 1–5 1–5, 15 1–7, 11, 12 UR, urological problems including ‘diabetes’ (based on traditional knowledge), DER, dermatological conditions including injuries caused by venomous animals; FEM, women’s medicines; GI, gastrointestinal disorders; NEU, fever and/or pain; RES, respiratory illnesses; ap, aerial parts; ba, bark; fr, fruits; lv, leaves; rh, rhizome; rt, root; tu, tuber; wh, whole plant; 1, B. cereus; 2, E. coli; 3, C. albicans; 4, KB-cell line; 5, NF-kB; 6, P. aeruginosa; 7, S. epidermidis; 8, H. pylori; 9, C. jejuni; 10, G. duodenalis; 11, S. aureus; 12, Y. enterocolitica; 13, P. falciparum; 14, a-amylase; 15, D2-receptor binding assay. 3.5. Hypoglycemic effects The polar extracts of five plant species, used against diabetes were tested for their ability to inhibit a-amylase (Table 2). The inhibition of the enzyme leads to a reduced splitting of food based poly- and disaccharides A. Ankli et al. / Journal of Ethnopharmacology 79 (2002) 43–52 48 in the colon resulting in a delayed resorption (Keller and Berger, 1983). B. di6aricata was the most potent inhibitor of the enzyme (0 U/l for extract 1 [1 mg/ml]), 2 [3 mg/ml]) and 3 [6 mg/ml]) followed by Hylocereus undatus (255 U/l for extract 1; 0 U/l for extracts 2 and 3, respectively). Polyphenols, known to interfere with enzymes, do not occur very frequently in these genera or families (Hegnauer, 1989; Hegnauer and Hegnauer, 1994). So the inhibition of the enzyme seems not to be due polyphenols. The following species were inactive, Ruellia nudifora, Malmea depressa, Parmentiera millspaughiana. The negative control resulted in a value of 620 U/l, the control of methanol gave 548 U/l. effects against S. epidermidis and B. cereus of the non-polar fractions of Caesalpinia gaumeri used for pain of the body and headache are particularly noteworthy (2 mm using a concentration of 200 mg). 4. Discussion The species evaluated in this paper were selected because of their cultural importance to the Yucatec Maya and their use(s) for specific syndromes. In this section the relevance of these findings to interpreting such indigenous uses is discussed using selected examples. The roots of Crossopetalum gaumeri are used orally for diarrhea and snake-bites and topically to prevent inflammation after a snake-bite. In a detailed phytochemical study of C. gaumeri, the non-polar fraction 3.6. Other acti6ities Some extracts showed activity in assays, which are not directly related to an indigenous use. The antibiotic Table 3 Screening of plant species used for gastrointestinal disorders by the Yucatec Maya Plant name Extract KB Giardia duodenalis IC50 (mg/ml) MIC (mg/ml) IC50 Bauhinia di6aricata Bidens squarrosa Casimiroa tetrameria Chrysophyllum mexicanum Crossopetalum gaumeri Dorstenia contrajer6a Jatropha gaumeri Microgramma nitida Piscidia piscipula Psidium sartorianum Metronidazole Ornidazole Tetracycline Ampicillin Kanamycin Streptomycin Chloramphenic ol Ciprofloxacin Helicobacter pylori Bacillus cereus Staphylococcus aureus (mg/ml) MIC (mg/ml) 600 mg (mm) 200 mg (mm) 600 mg (mm) 200 mg (mm) A – – 51 nt – – – nt B – – 70 nt – – – – A – – 72 3 4 – – B – – – nt 3 – – nt A 0.7 6.3 2.1 nt – 1 2 – B A 10.2 – – – 90 – nt 10 – 4 – – – – nt – B A – 7.8 – – – – nt 5 3 8 – 2 – 3 nt – B – – – nt 3 – – nt A – 41 27 11 – – nt B A – – – 69.2 70 51 3 nt 12 – – B1 – 1 nt nt B – – 8.5 1.1 65 5 0.5 nt 4 – 1 nt 8 (10 mg) 8 (10 mg) 0.7 20 23 19 19 22 (10 mg) (100 mg) (40 mg) (50 mg) (25 mg) C: 1 1 (0.01) KB, cytotoxicity, (–), IC50]50 mg/ml) I, G. duodenalis, (–), IC50]100 mg/ml; H. pylori, (–), 52 mm; antibacterial activity was measured as inhibition zone in (mm) (–), no activity; A, non-polar extract; B, polar extract; C, ethanol extract; nt, not tested. A. Ankli et al. / Journal of Ethnopharmacology 000 (2002) 000–000 49 Table 4 Screening of plant species used for dermatological conditions by the Yucatec Maya Plant name Aechmea bracteata Al6aradoa amorphoides Casearia corymbosa Crossopetalum gaumeri Croton reflexifolius Dalea carthagenensis Diospyros anisandra Diospyros cuneata Jatropha gaumeri Luehea speciosa Morinda yucatanensis Psidium sartorianum Podophyllotoxin Parthenolide PDTL Chloramphenicol Tetracycline Miconazole Extract KB, IC50 (mg/ml) NF-kB (mg/ml) Staphylococcus epidermidis Escherichia coli Candida albicans 200 mg (mm) 600 mg (mm) 600 mg (mm) 200 mg (mm) 600 mg (mm) B A – 10 – – 3 – 5 – – – – – – – B A 14 – – – – 1 – 1 – – – – – – A 0.7 25 2 3 – – – B A A 10.2 39 31 * – 150 – nt – – nt – – – – – – – – – – A 14 100 nt nt 1 – 2 75 * – 100 – nt – – – 2 nt – – – 4 – – – – – – – 1 – – 1 – 2 – – A A B A A – 7.8 – – – A – – – 1 – – – B – 0.006 – 1.5 2 – – – 8 (10 mg) 8 (10 mg) 5 (1 mg) 5 (1 mg) 10 mM 100 mM 6 (10 mg) KB, cytotoxicity; (–), IC50 ]50 mg/ml; NF-kB (HeLa cell line); (–), \150 mg/ml; *, cytotoxic at 100 mg/ml during the period of the test. Antimicrobial activity was measured as inhibition zone in (mm); nt, not tested. showed the presence of terpenoids, whereas the extract B consisted of several cardenolides (Ankli et al., 1999, 2000). The study indicated that the terpenoids possess antibacterial activity that may be relevant to the plant’s traditional use as a treatment for diarrhea. The potent antiprotozoal effect against G. duodenalis is probably due to the high non-specific cytotoxicity of the extracts (Table 3). The cytotoxicity of the polar and non-polar extracts may also be responsible for the potent inhibitory activity against NF-kB (Table 4). The leaves of P. piscipula are used as a medicine for treating gastrointestinal disorders (especially diarrhea and cramps) and for cough. The remarkable activity of this species against H. pylori and to some extent against G. duodenalis may be a reason why Yucatec Maya value this plant for treating gastrointestinal problems. In a study by Cáceres et al. (1991), P. piscipula was shown to have antimycotic effects. Another species, Piscidia erythrina L., has been widely investigated yielding spasmolytic isoflavones (Della Loggia et al., 1988). Among other ethnic groups, P. piscipula is used as a fish poison (Acevedo-Rodrı́guez, 1990). B. di6aricata is used for a variety of illnesses such as gastrointestinal problems, but more frequently for diabetes and respiratory problems. Its activity against G. duodenalis may be one of the reasons for its indigenous use. In a separate study the leaf extract of Bauhinia purpurea L. was reported to have significant antidiarrheal activity in vivo (Mukherjee et al., 1998). A possible hypoglycemic activity was also found in our a-amylase test which supports the reported hypoglycemic effects in a previous study (Roman et al., 1992). The roots of J. gaumeri are used for treating diarrhea and the resin is used as a medicine for herpes labialis. The therapeutic application of this plant as an anti-diarrhetic may be the result of its antibacterial activity as observed in this study against B. cereus. A variety of pharmacological effects are reported in the literature concerning Jatropha spp., including antimicrobial ef- 50 A. Ankli et al. / Journal of Ethnopharmacology 79 (2002) 43–52 fects (Odebiyi, 1980). The potent cytotocitity against the KB-cells is probably due to the cytotoxic phorbol esters, which are known for many taxa of the Euphorbiaceae (Hänsel et al., 1999). P. sartorianum is employed internally (diarrhea) and externally (mostly for measles or any kind of pimples). The antibacterial activities against H. pylori, B. cereus and S. epidermidis observed in this study might be of interest in light of its traditional use as a treatment for diarrhea and skin problems. No pharmacological data for P. sartorianum are available, but P. guaja6a L. was shown to have antibacterial effects, especially against the enterobacterium Shigella. C. tetrameria is used as a medicine for treating gastrointestinal problems including diarrhea as mentioned in the Ethno-Botany of the Maya (Roys, 1976 [orig. 1931]). The antiprotozoal effect as well as the antibacterial activities against H. pylori and S. aureus may be of relevance to the internal use. The antidiarrheal and antispasmodic effects are currently investigated (Heneka, 2000). The two Diospyros species, D. anisandra and D. cuneata, were reported to be ‘strong’ medicines. They are used topically for treating dermatological problems (pimples, scabies, inflammation). The antibiotic and the antifungal activities, shown in the assays, are probably responsible for the growth inhibition of the bacterial and fungal secondary infection of scabies and pimples. The NF-kB inhibiting activity, probably a result of the non-specific cytotoxicity of the extract, could be the reason of its anti-inflammatory use among the Mayas. These results are strengthened by reports of antimicrobial activity of Diospyros lycioides Desf. (Li et al., 1998), as well as in vivo anti-inflammatory activity of Diospyros leucomelas Poir (Recio et al., 1995). Various species of the genus Croton are used in the medical system of the Yucatec Maya for dermatological problems but also for fever and respiratory illnesses. The resin of Croton reflexifolius is used for pimples in the mouth (herpes) and eye problems. The possible occurrence of phorbol esters in this species could be a reason for its medicinal use as an antiviral drug (Hänsel et al., 1999). The application of the resin into the eyes could have severe side effects due to a high level of cytotoxicity (Table 4). L. speciosa is an effective inhibitor of the transcription factor NF-kB. The Yucatec Maya value this plant for treating skin diseases and toothache, and apply the medicine in the form of plasters. The inhibitory effect on NF-kB may explain the use of this plant. The weak antibacterial activity against Y. enterocolitica can not substantiate the traditional use of the plant. To our knowledge no pharmacological or phytochemical data have been published yet concerning this plant. C. nocturnum, applied for children with night fever and cold bodies, shows a weak effect against P. falci- parum. Further testing of fractions of this plant would be of interest. Phytochemically the species is well investigated but no published data has been found for this antimalarial effect. 5. Conclusion One goal of this evaluation is to better understand the use of plants by the Yucatec Maya. In this paper we show some correlations between uses of the medicinal plants and relevant biological activities. Other indigenous uses currently cannot be explained in a bio-scientific manner because the bioassays applied are not appropriate. In other cases the symbolic aspects might be more important to the Maya. We hope that the combination of a detailed documentation of ethnomedical use and the study of selected species in relevant bioassays correlating with their application may lead to a better understanding of the ethnopharmacopoeia of the Yucatec Maya (and other indigenous groups). Phytochemical and further pharmacological studies are important tasks for the future in order to better understand the effects of these important pharmaceutical resources. Organizations like World Health Organization (WHO) and TRAMIL (Central America) encourage the use of remedies provided that they are safe and that some scientific evidence on their biological and pharmacological effects exists. Hopefully this study contributes to a selection of the most appropriate species of the indigenous medicine of the Yucatec Maya. Herbal medicines are a valuable and readily available resource for primary health care and complementary health care systems. Acknowledgements The authors wish to thank all persons who have helped in the field study and especially the healers, midwives and the inhabitants of Chikindzonot, Ekpedz and Xcocmil, Yucatan, for their collaboration, for their friendship and hospitality. The botanical identification at CICY and MEXU (National Herbarium of Mexico) was performed in collaboration with the numerous specialists of these institutions. Particularly we would like to thank Dr I. Olmsted, J. Granados, P. Simá, J.C. Trejo, Dr R. Durán of CICY as well as O. Tellez, Dr R. Lira, Dr J. Villaseñor and Dr M. Sousa of MEXU. This research owes a lot to the help of Dr J. Heilmann (Zürich), Dr J. Orjala (Davis), Dr B. Frei Haller (Zernez), Professor Dr W. Schaffner (Basel) and Professor Dr H. Rimpler (Freiburg). Financial support by Swiss Agency for Development and Cooperation (SDC, Berne, Switzerland) and the Swiss Academy of Natural Sciences (SANW) is gratefully acknowledged. We are A. Ankli et al. / Journal of Ethnopharmacology 79 (2002) 43–52 grateful to Dr Paul Bremner (ULSOP) for carefully checking the MS. References Acevedo-Rodrı́guez, P., 1990. The occurrence of piscicides and stupefactants in the plant kingdom. In: Prance, G.T., Balick, M.J. (Eds.), New Directions in The Study of Plants and People: Research Contributions from the Institute of Economic Botany. Advances in Economic Botany, vol. 8, pp. 1 – 23. Ankli, A., Sticher, O., Heinrich, M., 1999. Medicinal Ethnobotany of the Yucatec Maya: healers’ consensus as a quantitative criterion. Economic Botany 53, 144 – 160. Ankli, A., Heilmann, J., Heinrich, M., Sticher, O., 2000. Cytotoxic cardenolides and antibacterial terpenoids from Crossopetalum gaumeri. Phytochemistry 54, 531 – 537. Berger, D., 1998. Vitex Agnus-castus: Unbedenklichkeit und Wirksamkeit beim prämenstruellen Syndrom, Wirkprinzipien und Wirkmechanismen eines neuentwickelten Extraktes. Inauguraldissertation Universität Basel, pp. 133 – 163. Bork, P.M., Schmitz, M.L., Weimann, C., Kist, M., Heinrich, M., 1996. Nahua Indian Medicinal Plants (Mexico): inhibitory activity on NF-kB as an anti-inflammatory model and antibacterial effects. Phytomedicine 3, 263 – 269. Browner, C.H., Ortiz de Montellano, B.R., Rubel, A.J., 1988. A methodology for cross-cultural ethnomedical research. Current Anthropology 29, 681 –702. Cáceres, A., Lopez, B.R., Giron, M.A., Logemann, H., 1991. Plants used in Guatemala for the treatment of dermatophytic infections. 1. Screening for antimycotic activity of 44 plant extracts. Journal of Ethnopharmacology 31, 263 – 276. De Landa, D., 1992. Relacion de las cosas de Yucatán. Producción Editorial Dante, S.A. de C.V., Mérida, Yucatán, México. Della Loggia, R., Zilli, C., Del Negro, P., Redaelli, C., Tubaro, A., 1988. Isoflavones as spasmolytic principles of Piscidia erythrina. Progress in Clinical and Biological Research 280, 365 – 368. DIN (Deutsches Institut für Normung), 1992. Methoden zur Empfindlichkeitsprüfungen von bakteriellen Krankheitserregern (ausser Mykobakterien) gegen Chemotherapeutika. DIN-Taschenbuch Medizinische Mikrobiologie und Immunologie, DIN 58 940, Beuth Verlag, Berlin, Köln. Etkin, N.L., 1994. Anthropological methods in ethnopharmacology. Journal of Ethnopharmacology 38, 93 – 104. Fairlamb, A.H., Warhurst, D.C., Peters, W., 1985. An improved technique for the cultivation of Plasmodium falciparum in vitro without daily medium change. Annals of Tropical Medicine and Parasitology 79, 379 – 384. Farnsworth, N.R., 1988. Screening plants for new medicines. In: Wilson, E.O. (Ed.), Biodiversity. National Academy, Washington DC. Frei, B., Baltisberger, M., Sticher, O., Heinrich, M., 1998a. Medicinal Ethnobotany of the Isthmus-Sierra Zapotecs (Oaxaca, Mexico): documentation and assessment of indigenous uses. Journal of Ethnopharmagology 62, 149 – 165. Frei, B., Heinrich, M., Bork, P.M., Herrmann, D., Jaki, B., Kato, T., Kunth, M., Schmitt, J., Schühly, W., Volken, C., Sticher, O., 1998b. Multiple screening of medicinal plants from Oaxaca, Mexico: ethnobotany and bioassays as a basis for phytochemical investigation. Phytomedicine 5, 177 – 186. Hänsel, R., Sticher, O., Steinegger, E., 1999. Pharmacognosie –Phytopharmazie. Springer, Berlin/Heidelberg. Hegnauer, R., 1989. Chemotaxonomie der Pflanzen. Band 8. Birkhäuser Verlag, Basel/Boston. Hegnauer, R., Hegnauer, M., 1994. Chemotaxonomie der Pflanzen. Band 11a. Birkhäuser Verlag, Basel/Boston. 51 Heinrich, M., Rimpler, H., Antonio, B.N., 1992a. Indigenous phytotherapy of gastrointestinal disorders in a lowland Mixe community (Oaxaca, Mexico): ethnopharmacological evaluation. Journal of Ethnopharmacology 36, 63 –80. Heinrich, M., Kuhnt, M., Wright, C.W., Rimpler, H., Phillipson, J.D., Schandelmaier, A., Warhurst, D.C., 1992b. Parasitological and microbiological evaluation of Mixe Indian medicinal plants (Mexico). Journal of Ethnopharmacology 36, 81 – 85. Heinrich, M., Ankli, A., Frei, B., Weimann, C., Sticher, O., 1998. Medicinal plants in Mexico: healers’ consensus and cultural importance. Social Science and Medicine 47, 1859 – 1871. Heneka, B., 2000. Isolierung gastrointestinal wirksamer Inhaltsstoffe aus Casimiroa tetrameria Millsp., einer yukatekischen Arzneipflanze der Maya (Mexiko). Inaugural-Dissertation, Albert Luswigs-Universität, Freiburg im Breisgau. Kayser, F.H., Bienz, K.A., Eckert, J., Lindenmann, J., 1993. Medizinische Mikrobiologie, eighth ed. Georg Thieme Verlag, Stuttgart, NY. Keister, D.B., 1983. Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Transactions of the Royal Society of Tropical Medicine and Hygiene 77, 487 – 488. Keller, U., Berger, W., 1983. Orale Antidiabetika: neuere Aspekte. Schweizerische medizinische Wochenschrift 113, 649. Lewis, W.H., Elvin-Lewis, M.P., 1994. Basic, quantitative and experimental research phases of future ethnobotnay with reference to the medicinal plants of South America. In: Ethnobotany and The Search for New Drugs, Ciba Foundation Symposium 185. Wiley, Chichester/New York. Li, X.C., van der Bijl, P., Wu, C.D., 1998. Binaphthalenone glycosides from African chewing sticks, Diospyros lycioides. Journal of Natural Products 61, 817 – 820. Makler, M.T., Ries, J.M., Williams, J.A., Bancroft, J.E., Piper, R.C., Gibbins, B.L., Hinrichs, D.J., 1993. Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. American Journal of Tropical Medicine and Hygiene 48, 739 – 741. Messner, E., 1978. Present and future prospects of herbal medicine in a Mexican community. In: Ford, R.I. (Ed.), The Nature and Status of Ethnobotany. University of Michigan Press, Ann Arbor. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55 – 63. Mukherjee, P.K., Gopal, T.K., Subburaju, T., Dhanbal, S.B., Duraiswamy, B., Elango, K., Suresh, B., 1998. Studies on the anti-diarrheal profiles of Bauhinia purpurea L. leaves (Caesalpiniaceae) extract. Natural Product Sciences 4, 234 – 237. Nedenskov, P., 1994. Nutritional requirements for growth of Helicobacter pylori. Applied and Environmental Microbiology 60, 3450 – 3453. Odebiyi, O.O., 1980. Antibacterial property of tetramethylpyrazine from the stem of Jatropha podagrica. Planta Medica 38, 144 – 146. Pierre, K.J., Tung, K.K., Nadj, H., 1976. A new enzymatic kinetic method for the determination of a-amylase. Clinical Chemistry 22, 1219. Raez, B., Iten, M., Grether-Böhler, Y., Kaminsky, R., Brun, R., 1997. The Alamar Blue® assay to determine drug sensitivity of African trypanosomes (T.b. rhodesiense and T.b. gambiense) in vitro. Acta Tropica 68, 139 – 147. Recio, M.C., Giner, R.M., Manez, S., Gueho, J., Julien, H.R., Hostettmann, K., Rı́os, J.L., 1995. Investigations on the steroidal anti-inflammatory activity of triterpenoids from Diospyros leucomelas. Planta Medica 61, 9 – 12. Rı́os, J.L., Recio, M.C., Villar, A., 1988. Screening method for natural products with antimicrobial activity: a review of the literature. Journal of Ethnopharmacology 23, 127 – 149. Roman, R., Alarcon-Aguilar, F., Lara-Lemus, A., Flores-Saenz, J.L., 1992. Hypoglycemic effects of plants used in Mexico as antidiabetics. Archives of Medicinal Research 23, 59 – 64. 52 A. Ankli et al. / Journal of Ethnopharmacology 79 (2002) 43–52 Roys, R., 1933. The book of Chilam Balam of Chumayel. Carnegie Institution of Washington. Roys, R., 1976. Ethno-Botany of the Maya. Institute for the study of human issues, Philadelphia, p. 42. Steiner, M., 1997. Premenstrual syndromes. Annual Review of Medicine 48, 447 – 455. Swanson, S.M., Pezzuto, J.M., 1990. Bioscreening technique for cytotoxic potential and ability to inhibit macromolecule biosynthesis. In: Thompson, E.B. (Ed.), Drug Bioscreening. Drug Evaluation Techniques in Pharmacology. VCH Publishers, New York/Weinheim, p. 273. Journal of Ethnopharmacology 79 (2002) 53 – 56 www.elsevier.com/locate/jethpharm Anti-diarrhoeal evaluation of some medicinal plants used by Zulu traditional healers J. Lin a,*, T. Puckree b, T.P. Mvelase a a Department of Biochemistry and Microbiology, Uni6ersity of Zululand, Pri6ate Bag X1001, Kwa Dlangezwa 3886, South Africa b Department of Physiotherapy, Uni6ersity of Durban-West6ille, Pri6ate Bag X 54001, Durban 4000, South Africa Received 8 April 2001; received in revised form 3 September 2001; accepted 20 September 2001 Abstract Aqueous and methanolic extracts of several medicinal plants, Psidium guaja6a (leaves), Aristea spp., Iridaceae family (stem), Bridelia micrantha (bark) and Eleutherina bulbosa (bulb), used by Zulu traditional healers were evaluated for anti-diarrhoeal activity against different experimental models of diarrhoea in rats as well as for anti-microbial activity against different pathogenic microorganisms that cause diarrhoea. The methanolic extract of P. guaja6a (leaves) was the only agent showing significant inhibitory activities against the growths of Salmonella spp. (two isolates), Shigella spp. (S. flexneri, S. 6irchow and S. dysenteriae) and enteropathogenic Escherechia coli (two isolates). The methanolic extract of B. micrantha (Bark) demonstrated weak inhibitory activities against S. flexneri and S. plesiomonas. Based on the results in experimental rat models, there were significant reductions in faecal output and frequency of droppings when plant extracts were administered compared with castor-oil treated rats. All plant extracts also significantly retarded the propulsion of charcoal meal and significantly inhibited the PGE2-induced enteropooling. The results have confirmed the effectiveness of all these Zulu medicinal plants as anti-diarrhoeal agents. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Medicinal plants; Anti-diarrhoeal activity; Anti-microbial activity 1. Introduction Diarrhoea continues to be one of the leading causes of mortality and morbidity especially in children in developing countries (Black et al., 1982) including South Africa. The emergence of multiple drug resistant strains of diarrhoeagenic pathogens has made the treatment of dysentery more difficult (Munshi et al., 1987; Bennish et al., 1984). In developing countries, the majority of people living in rural areas almost exclusively use traditional medicines in treating all sorts of diseases including diarrhoea. Due to these facts, the World Health Organization (WHO) incorporates studies of traditional medicinal practice in its diarrhoeal disease control program. Several studies have evaluated the effectiveness of some traditional medicines in treating diarrhoea in all different continents (Chowdhury et al., 1991; Ferdous et al., * Corresponding author. Tel.: + 27-35-902-6095; fax: + 27-35-9026750. E-mail address: jlin@pan.uzulu.ac.za (J. Lin). 1992; Ahsan et al., 1996; Rao et al., 1997; Menezes and Rao, 1998; Mukherjee et al., 1998; Zavala et al., 1998; Das et al., 1999; Offiah and Chikwendu, 1999; Rani et al., 1999; Tona et al., 1999). South Africa has a great environmental and biological (genomic) diversity compared with the rest of the world. A range of medicinal plants with anti-diarrhoeal properties has been widely used by the traditional healers of difficult tribes in South Africa. The effectiveness of many of these antidiarrhoeal traditional medicines, however, has not been scientifically evaluated. Aristea spp., Iridaceae family has been used to treat cold, flu, malaria, toothache and bruise and Eleutherina bulbosa to treat burns. However, anecdotal evidence suggests that the above two medicinal plants can also be used to treat gastro-intestinal diseases. Bridelia micrantha is also used by traditional healers for the treatment of gastro-intestinal diseases, paralysis and painful joints. Psidium guaja6a originally from Mexico is also widely used by local traditional healers. P. guaja6a is also a relatively well studied species with respect to diarrhoea (Lutterodt, 1989; Ghosh et al., 1993; Tona et al., 1999). 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 5 3 - 1 54 J. Lin et al. / Journal of Ethnopharmacology 79 (2002) 53–56 This study reports on the anti-diarrhoeal effects of some medicinal plants used by traditional Zulu herbalists using different experimental models in rats. It also looks at the anti-microbial activities of these medicinal plants against different pathogenic microorganisms that cause diarrhoea. 2. Methodology 2.1. Preparation of plants extracts P. guaja6a (leaf), Aristea spp., Iridaceae family (stem), B. micrantha (bark) and E. bulbosa (bulb) were collected from the campus of the University of Zululand, identified by A. Hutchings from the Botany Department and confirmed by the National Botanical Institute. The plant extracts were prepared as described by Lin et al. (1999). In brief, dry plant materials were extracted using distilled water in a Soxhlet extractor for 24 h. The aqueous extracts were concentrated in vacuo, at 40 °C, using a Rotary evaporator (Büchi). Methanolic (MeOH) crude plant extracts were extracted three times in MeOH at 150 rpm for 24 h at room temperature. The combined extracts were then evaporated to dryness as above. The yields of 9.9, 11.7, 14.1, 8.6% (w/w) from aqueous extracts, and 11.6, 7.0, 11.2 and 9.1% (w/w) from MeOH extracts with respect to dry powdered material of P. guaja6a, Aristea spp., Iridaceae family, B. micrantha and E. bulbosa were obtained. The final residues of each extract were re-dissolved in 100% Dimethyl sulfoxide (DMSO) and stored in sterile brown glass bottles in a freezer at −20 °C until further processing for bioassay. 2.2. Anti-microbial assays The disk diffusion method of Salie et al. (1996) was used to evaluate anti-microbial activities against 17 isolates of diarrheagenic E. coli, 13 isolates of Salmonella spp. and nine isolates of Shigella spp. Each extract was tested in triplicate. Control discs contained 10 ml pure DMSO (100%). Standard antibiotics, Nalidixic acid (30 mg per disc), tetracycline (30 mg per disc), and chloramphenicol (30 mg per disc) were used as positive controls. 2.3. Animal used The rats, Rattus nor6egicus, of either gender (adults, \200 g or adolescence, B100 g) were used. All animals were acclimatized to uniform laboratory conditions for at least 1 week before performing an experiment. Depending on the experiment, animals were divided into five or six groups with three animals in each group. All rat studies described below were followed using the method of Mukherjee et al. (1998) with modifications. 2.4. Castor oil-induced diarrhoea in rats Rats were fasted for 18 h. Each of the plant extracts at a dose of 400 mg/kg was administered orally to each group of the experimental animals. The control group received 1 ml of DMSO only. After 1 h of treatment, each animal was fed with 1 ml of castor oil orally. The treated rats were observed for defecation overnight. The total defecate for each animal was weighed and means obtained for each group for comparison. 2.5. Gastrointestinal motility tests One ml of charcoal meal (3% deactivated charcoal, 10% distilled water and 87% of commercial rat food) was administered orally to the rats after a fast for 18 h. Immediately after the administration of charcoal meal, each experimental group of rats was then fed orally with different plant extracts (400 mg/kg). And the control groups received either 1 ml of DMSO or no treatment. All rats were sacrificed 30 min later. The intestinal distance that the charcoal meal had moved from the pylorus was measured and the value was calculated as a percentage of the whole length of the intestine. 2.6. PGE2 induced enteropooling test Each experimental group of rats was treated with different plant extracts at a dose of 400 mg/kg after an 18-h fast. One control group received 1 ml of DMSO. About 100 mg/kg of Prostaglandin E2 (PGE2) was immediately administrated orally to all rats to induce the enteropooling. Then, 30 min after the PGE2 administration, all rats were sacrificed and the whole length of intestine from pylorus to the caecum was dissected. The contents of the intestine were collected and the total volume was measured. 2.7. Statistical analysis The experimental results are expressed as the mean9 standard deviation (S.D.). Student’s t-test was used to determine the statistical significance (PB 0.05). 3. Results The methanolic extract of P. guaja6a (leaves) was the only agent showing significant inhibitory activities (in- J. Lin et al. / Journal of Ethnopharmacology 79 (2002) 53–56 hibitory zone ]10 mm) against the growths of Salmonella spp (two isolates), Shigella spp. (S. flexneri, S. 6irchow and S. dysenteriae) and enteropathogenic E. coli (two isolates). (Data not shown) All aqueous, ethanol and acetone medicinal plant extracts showed no significant inhibitory activity against all tested microorganisms. The methanolic extract of B. micrantha (bark) demonstrated weak inhibitory activities against S. flexneri and S. plesiomonas (10 mm \inhibition zone] 7 mm). In all rat models, there were no significant differences in the effects of medicinal plant extracts based on either gender or age of rats. Both methanolic and aqueous extracts of the same plant showed similar effects in all cases. In the castor oil induced diarrhoea experiment, the rats that did not receive the medicinal plant extracts showed typical diarrhoeal signs: watery and frequent defecation. The average weight of defecate in the control group was 10.53 g. Treatment with each of the medicinal plant extracts significantly reduced the weight of defecate to 30– 40% (Table 1) as well as reduced the frequency of defecation compared with the control group. All defecates from the plant extracts-treated groups were dry or semi-dry. Administration of either methanolic or aqueous plant extracts significantly slowed down the propulsion toward the caecum of the charcoal meals. Distance traveled by the charcoal meal was reduced to 50–70% of the full intestine in the experimental groups compared with 91– 92% in the control group (Table 2). As shown in Table 3, PGE2 induced a significant increase in the fluid volume of rat intestine compared with that in the control group that received 5% ethanol in normal saline. All aqueous and methanolic extracts of individual medicinal plants demonstrated their abilities to inhibit PGE2-induced enteropooling in rats. Table 1 Effect of some Zulu medicinal plant extracts on castor-oil induced diarrhoea in rats (n = 3) Control Mean defections per rat (10.53 9 0. 94 mg) (100%) Methanolic extract, mg (%) Medicinal plant used P. guaja6a 3.53 9 0.68* Aristae spp. (Iridaceae) 3.78 9 0.34* B. micrantha 4.11 9 1.01* E. bulbosa 3.82 9 0.88* *PB0.005. (33.55) (36.02) (39.03) (36.25) Table 2 Inhibition of gastro-intestinal motility by some Zulu medicinal plant extracts (n = 3) Control Movement of charcoal meal as percentage of full intestinal length (%) 91.29 9 4.15 (−DMSO) 92.71 9 4.71 (+DMSO) Methanolic extract H2O extract Medicinal plant used P. guaja6a 71.45 92.22* Aristea spp. 72.06 9 2.64* (Iridaceae) B. micrantha 38.97 9 3.66** E. bulbosa 70.66 98.74* 69.02 9 5.17* 51.88 9 2.44* 4. Discussion The methanolic extract of P. guaja6a (leaves) was the only agent that showed significant inhibitory activities against the growths of some diarrhoeagenic pathogens in this study. Several studies (Lutterodt, 1989; Ghosh et al., 1993; Tona et al., 1999) have also demonstrated that P. guaja6a possesses antidiarrhoeal and antimicrobial activities. In addition, leaf extract of P. guaja6a contains anticough (Jaiarj et al., 1999), antiamoebic and antispasmodic properties (Lozoya et al., 1994; Tona et al., 1999). Lutterodt (1989) showed that quercetin from the leaf extract inhibits acetylcholine release in the gastrointestinal tract that might account for the antidiarrhoeal activity of the plant. Furthermore, several chemical compounds isolated from guava leaves possess antibacterial activities against different strains of gram negative bacteria (Caceres et al., 1990) as well as gram positive bacteria (Jaiarj et al., 1999). All Table 3 Anti-enteropooling effect of some Zulu medicinal plant extracts in rats (n =3) Volume of intestinal fluid (ml) Ethanol in saline 0.85 9 0.15 PGE2 in ethanol (100 mg/kg) 2.65 9 0.30 Methanolic extract (38.49) (29.69) (36.25) (28.11) 53.25 9 1.49* 57.60 9 7.10* **PB0.005; *PB0.05. H2O extract, mg (%) 4.05 91.13* 3.12 90.05* 3.82 91.24* 2.96 9 0.78* 55 Medicinal plant used P. guaja6a 1.449 0.20* Aristae spp. 1.229 0.15* (Iridaceae) B. micrantha 1.55 90.23* E. bulbosa 1.15 9 0.10* *PB0.005. Aqueous extract 1.25 9 0.23* 1.35 9 0.17* 1.45 9 0.21* 1.05 9 0.22* 56 J. Lin et al. / Journal of Ethnopharmacology 79 (2002) 53–56 plant extracts significantly reduced castor-oil induced diarrhoea, slowed the propulsion of charcoal meal and significantly inhibited the PGE2-induced enteropooling. It is well known that the administration of PGE2 or PGE1 induces the signs of inflammation, swelling and edema resulting from increased capillary permeability. Our results suggest that these plant extracts can reduce the capillary permeability and, therefore, decrease mucocal inflammation. Compared with the leaf extract of P. guaja6a, the extracts of Aristea spp., Iridaceae family (stem), B. micrantha (bark) and E. bulbosa (bulb) produced a similar level of antidiarrhoeal effects in all animal models. The inhibitory effects of other medicinal extracts from the above results prove that these plant materials used by Zulu traditional healers can be considered as potent antidiarrhoeal agents. Acknowledgements We would like to thank the Medical Research Council (SA) and the University of Zululand for the financial support. We also would like to thank A. Hutchings from the Botany Department at the University of Zululand for the collection and the identifications of these medicinal plants. References Ahsan, M., Chowdhury, A.K.A., Islam, S.N., Ahmed, Z.N., 1996. Garlic extract and allicin: broad spectrum antibacterial agents effective against multiple drug resistant strains of Shigella dysenteriae type 1 and Shigella flexneri, enterotoxigenic Escherichia coli and Vibrio cholerae. Phytotherapy Research 10, 329 – 331. Bennish, M., Eusof, A., Kay, B., Wierzba, T., 1984. Multi-resistant Shigella infections in Bangladesh. Lancet ii, 441. Black, R.E., Brown, K.H., Becker, S., Yunus, M., 1982. Longitudinal studies of infectious diseases and physical growth of children in rural area of Bangladesh. American Journal of Epidemiology 115 (3), 305 – 314. Caceres, A., Cano, O., Samayoa, B., Agnilar, C., 1990. Plants used in Guatemala for the treatment of GI disorders 1. Screening of 84 plants against enterobacteria. Journal of Ethnopharmacology 38, 55 – 73. Chowdhury, A.K.A., Ahsan, M., Islam, S.N., Ahmed, Z.N., 1991. Efficacy of aqueous extract of garlic and allicin in experimental shigellosis in rabbits. Indian Journal of Medical Research [A] 93, 33 – 36. Das, A.K., Mandal, S.C., Banerjee, S.K., Sinha, S., Das, J., Saha, B.P., Pal, M., 1999. Studies on antidiarrhoeal activity of Punica granatum seed extract in rats. Journal of Ethnopharmacology 68 (1), 205 –208. Ferdous, A.R., Islam, S.N., Ahsan, M., Hasan, C.M., Ahmed, Z.U., 1992. In vitro antibacterial activity of the volatile oil of Nigella sati6a seeds against multiple drug resistant isolates of Shigella, V. cholerae and E. coli. Phytotherapy Research 6, 137 – 140. Ghosh, T.K., Sen, T., Das, A., Dutta, A.S., Nag Chaudhuri, A.K., 1993. Antidiarrhoeal activity of the methanolic fraction of the extract of unripe fruits of Psidium guaja6a Linn. Phytotherapy Research 7, 431 – 433. Jaiarj, P., Khoohaswan Pm Wongkrajang, Y., Peungvicha, P., Suriyawong, P., Saraya, M.L., Ruangsomboon, O., 1999. Anticough and antimicrobial activities of Psidium guaja6a Linn. leaf extract. Journal of Ethnopharmacology 67 (2), 203 – 212. Lin, J., Opoku, A.R., Geheeb-Keller, M., Hutchings, M.A., Terblanche, S.E., Jager, A.K., van Staden, J., 1999. Preliminary screening of traditional Zulu medicinal plants for anti-inflammatory and anti-microbial activities. Journal of Ethnopharmacology 68, 267 – 274. Lozoya, X., Meckes, M., Abou-Zaid, M., Tortoriello, J., Nozzolillo, C., Arnason, J.T., 1994. Quercetin glycosides in Psidium guaja6a L. leaves and determination of a spasmolytic principle. Archives of Medical Research 25 (1), 11 – 15. Lutterodt, G.D., 1989. Inhibition of gastrointestinal release of acetylcholine by quercetin as a possible mode of action of Psidium guaja6a leaf extract in the treatment of acute diarrhoea. Journal of Ethnopharmacology 25, 235 – 238. Menezes, A.M., Rao, V.S., 1998. Effect of Astronium urundeu6a (aroeira) on gastrointestinal transit in mice. Brazilian Journal of Medical and Biological Research 21 (3), 531 – 533. Munshi, M.H., Haider, K., Rahman, M.M., Sack, D.A., Ahmed, Z.U., Morshed, M.G., 1987. Plasmid mediated resistance to nalidixic acids in Shigella dysenteriae type I. Lancet ii, 419 – 421. Mukherjee, P.K., Saha, K., Murugesan, T., Mandal, S.C., Pal, M., Saha, B.P., 1998. Screening of antidiarrhoeal profile of some plant extracts of a specific region of West Bengal, India. Journal of Ethnopharmacology 60 (1), 85 – 89. Offiah, V.N., Chikwendu, U.A., 1999. Antidiarrhoeal effects of Ocimum gratissimum leaf extract in experimental animals. Journal of Ethnopharmacology 68 (1), 327 – 330. Rani, S., Ahamed, N., Rajaram, S., Saluja, R., Thenmozhi, S., Murugesan, T., 1999. Antidiarrhoeal evaluation of Clerodendrum phlomidis Linn. leaf extract in rats. Journal of Ethnopharmacology 68 (1), 315 – 319. Rao, V.S., Santos, F.A., Sobreira, T.T., Souza, M.F., Melo, C.L., Silveira, E.R., 1997. Investigations on the gastroprotective and antidiarrhoeal properties of ternatin, a tetramethoxyflavone from Egletes 6iscose. Planta Medica 63 (2), 146 –149. Salie, F., Eagles, P.F.K., Leng, H.M.J., 1996. Preliminary antimicrobial screening of four South African Asteraceae species. Journal of Ethnopharmacology 52, 27 – 33. Tona, L., Kambu, K., Mesia, K., Cimanga, K., Apers, S., De Bruyne, T., Piters, L., Totte, J., Vlietinck, A.J., 1999. Biological Screening of traditional preparations from some medicinal plants used as antidiarrhoeal in Kinshasa, Congo. Phytomedicine 6 (1), 59 – 66. Zavala, M.A., Perez, S., Perez, C., Vargas, R., Perez, R.M., 1998. Antidiarrhoeal activity of Waltheria americana, Commelina coelestis and Alternanthera repens. Journal of Ethnopharmacology 61 (1), 41 –47. Journal of Ethnopharmacology 79 (2002) 57 – 67 www.elsevier.com/locate/jethpharm The evaluation of forty-three plant species for in vitro antimycobacterial activities; isolation of active constituents from Psoralea corylifolia and Sanguinaria canadensis Sandra M. Newton a, Clara Lau a,1, Sudagar S. Gurcha b, Gurdyal S. Besra b, Colin W. Wright a,* b a The School of Pharmacy, Uni6ersity of Bradford, West Yorkshire BD7 1DP, UK The Department of Microbiology and Immunology, The Medical School, Uni6ersity of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK Received 8 June 2001; received in revised form 10 September 2001; accepted 20 September 2001 Abstract Extracts from forty-three plant species were selected on account of reported traditional uses for the treatment of TB and/or leprosy. These were assayed for antimycobacterial activities. A simple in vitro screening assay was employed using two model species of mycobacteria, M. aurum and M. smegmatis. Crude methanolic extracts from three of the plants, C. mukul, P. corylifolia and S. canadensis, were found to have significant antimycobacterial activity against M. aurum only (MIC = 62.5 mg/ml). Bioassay guided fractionation led to the isolation of two known benzophenanthridine alkaloids, sanguinarine (1) and chelerythrine (2), from the roots S. canadensis and the known phenolic meroterpene, bakuchiol (3) from the seeds of P. corylifolia. The fractionation of the resin of C. mukul lead to a decrease in antimycobacterial activity and hence further work was not pursued. Compound (2) was the most active against M. aurum and M. smegmatis (IC50 =7.30 mg/ml [19.02 mM] and 29.0 mg/ml [75.56 mM], respectively). M. aurum was the most susceptible organism to all three compounds. No significant difference in antimycobacterial activity was observed when the two alkaloids were tested for activity in media of differing pH values. The activities of the pure compounds against M. aurum were comparable with those against M. bo6is BCG with compound (2) being the most active (M. bo6is BCG, IC50 = 14.3 mg/ml [37.3 mM]). These results support the use of these plants in traditional medicine. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tuberculosis; Mycobacteria; Traditional medicine; S. canadensis; P. corylifolia; C. mukul 1. Introduction Tuberculosis (TB) is a disease known since antiquity and evidence of spinal TB in the form of fossil bones dates back to around 8000 BC (Ayyazian, 1993; Basel, 1998). Today TB still remains the most prevalent cause of death in developing countries, due to a single infectious agent (WHO, 1998). Currently one third of the world’s population is infected with Mycobacterium tu- * Corresponding author. Tel.: +44-1274-234-739; fax: + 44-1274235-920. E-mail address: c.w.wright@bradford.ac.uk (C.W. Wright). 1 Present address: Department of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. berculosis (MT) and each year there are some 2 – 3 million deaths worldwide (Zumla et al., 1999). It is estimated that between the years 2000 and 2020 nearly one billion people will be newly infected, 200 million will develop TB and 35 million will die from the disease (WHO, 2000). Between 1900 and 1980 there was a steady decline in the notification rates of TB in industrialised countries. This can be attributed to the improvement in socio-economic conditions, the development of anti-TB drugs (including ethambutol, isoniazid, pyrazinamide, rifampicin and streptomycin) and the BCG vaccination programme. However, the number of cases worldwide is now increasing rapidly due to multi-drug resistant strains of MT as a result of patient non-compliance and also due to an increase in patients with human im- 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 5 0 - 6 58 S.M. Newton et al. / Journal of Ethnopharmacology 79 (2002) 57–67 munodeficiency virus (HIV) infection, which has further exacerbated the problem (Zumla and Grange, 1998). In 1993, the World Health Organisation (WHO) announced this chronic disease to be a ‘global emergency’ (Raviglione et al., 1995). Thus, there is an urgent need for the introduction of new effective TB control programmes and for novel, affordable, anti-TB agents with little toxicity to replace those currently in use to which mycobacterial resistance has occurred. Furthermore drugs with broader ranges of activity are also required to target emerging pathogens such as those of the Mycobacterium a6ium –intracellulare complex (MAC) which can result in opportunistic infectious disease. Over 350 natural products, mainly plant species, which have been used in traditional medicine, have been assessed for their antimycobacterial activities (Newton et al., 2000). A number have been shown to demonstrate significant in vitro antimycobacterial activities and active plant-derived compounds belonging to various chemical classes have been isolated. These findings have therefore stimulated further search towards the isolation of new antimycobacterial agents from natural products. The aim of the present study was to evaluate plant species for antimycobacterial activities and to isolate and identify the active constituents responsible. Plants were chosen based on the fact that they have been used in traditional medicine for the treatment of TB/leprosy or symptoms of these diseases. Hence this work sought to justify the use of these plants in traditional medicine. A simple in vitro screening assay using a 96-well microplate broth dilution method was employed. As a result of MT being a slow growing and pathogenic organism, plant extracts were screened against two species of saprophytic, rapidly growing, non-pathogenic mycobacteria, namely Mycobacterium aurum and Mycobacterium smegmatis. These species of mycobacteria are used as test model organisms in the initial primary screening process while MT is usually used at a later stage for further studies. Both organisms have been shown to have similar drug sensitivity profiles to that of MT (Mitscher and Baker, 1998). However, Chung et al. (1995) has shown that the susceptibility of MT is more closely resembled by M. aurum than by M. smegmatis. Preliminary in vitro screening studies, using M. bo6is BCG strain as a slow growing but non-pathogenic model organism for MT were also performed on the isolated plant constituents. 2. Materials and methods All chemicals were obtained from Sigma unless otherwise stated. 2.1. Test organisms The antimycobacterial activity of the extracts/pure compounds was determined against M. aurum 4721E obtained and authenticated by Dr G. Chung, Glaxowellcome, Stevenage, UK, and M. smegmatis MC2 155 obtained from Professor I. Chopra, Microbiology Department, University of Leeds, UK. Pure compounds were also screened against the slow growing M. bo6is BCG strain. M. smegmatis and M. aurum were routinely maintained in Middlebrook 7H9 broth containing 10% v/v ADC supplement (Albumin Fraction V, Dextrose and Catalase). 2.5% v/v PANTA, (Polymixin, Amphotericin B, Naladixic acid, Trimethoprim and Azlocillin) an antibiotic supplement, was also added to prevent the growth of any non-mycobacteria. Cultures of mycobacteria were grown in disposable, sterile 20 ml screw capped tubes containing 5.5 ml of 7H9 Middlebrook medium and incubated at 37 °C for 72 h. The procedure for the cultivation of M. bo6is BCG was as described by Kremer et al. (2000). M. bo6is BCG was cultured in 8.5 ml screw-capped tubes containing 3 ml of Middlebrook 7H9 medium with 0.05% Tween 80, 0.02% glycerol and 10% OADC (Oleic acid, Albumin Fraction V, Dextrose and Catalase) enrichment. This was incubated at 37 °C with gentle agitation until an optical density (l 600 nm) of 0.3 was obtained. All media reagents were obtained from Becton Dickinson, UK. 2.2. In 6itro broth microdilution screening assay 2.2.1. M. aurum and M. smegmatis Screening assays were performed in 96-well microtitre plates. Fresh solutions of each drug, test plant extract or pure compound were prepared by first dissolving in (DMSO) and then further diluting with 7H9 Middlebrook medium to obtain the desired concentration. Control experiments showed that 2.5% DMSO or less in each well did not have any inhibitory effects on the growth of the mycobacteria. Serial two-fold dilutions of each substance to be evaluated were made with 7H9 Middlebrook medium in 96-well microplates, to yield volumes of 100 ml/well with final concentrations ranging from 15.63 to 500 mg/ml for extracts and 0.05– 100 mg/ml for pure compounds. 100 ml of mycobacteria in medium (diluted to give an optical density value, which would ensure the mycobacteria were at the start of the log phase when the test commenced) were also added to each well containing the drug and mixed thoroughly to give a final volume of 200 ml/well (106 CFU/ml M. aurum; 108 CFU/ml M. smegmatis). Both positive and negative control drugs (streptomycin and ampicillin, respectively) were included together with a blank (culture medium only) and a growth control (culture S.M. Newton et al. / Journal of Ethnopharmacology 79 (2002) 57–67 medium and mycobacteria without the presence of drug) were also included to validate the results. Each drug was tested in duplicate. The plates were incubated at 37 °C. Optical density readings (550 nm) were taken at time 0 h and at 24-h intervals for 72 h using a microplate reader. Minimum inhibitory values (MIC) were determined for crude extracts and fractions, the MIC being defined as the lowest concentration required to show a marked inhibition of mycobacterial growth at 72 h. IC50 values were determined for pure compounds using linear regression analysis. At least three separate determinations were carried out for each extract/fraction and compound. 2.2.2. M. bo6is Pure compounds for testing against M. bo6is BCG were added to each culture (optical density 0.3 at l 600 nm) tube to give final concentrations of 2.5, 5, 15, 25, 50, 100, 250 and 500 mg/ml. Growth with the incubation of test samples was continued for a further 10 days. The optical density readings at 600 nm were determined on each day. The IC50 values were determined on day 10 by plotting the recorded optical densities for the control culture (absence of drug) and each drug concentration of each test sample, and then calculating the concentration required to inhibit growth by 50%. 2.3. pH experiments (M. aurum and M. smegmatis) Media was prepared using the same ingredients as that of 7H9 Middlebrook medium from Becton Dickinson supplemented with ADC and PANTA antibiotic. Media of different pH values were prepared by varying the amounts of Na2PO4 and KHPO4 while keeping the ionic strength of the buffers constant. Control experiments were initially performed to determine the effect of the pH of the medium on the growth of the mycobacteria in the absence of drug. The screening assay was then carried out as described above. IC50 values were determined. 2.4. Plant material Forty-three plant species from thirty-three families and a wide range of geographical origins, were primarily selected based on the ethnopharmacological approach that they had previously been used in traditional medicine for the treatment of TB/leprosy or symptoms of these diseases. These are summarised in Table 1. (Note that other traditional medicinal uses of these plants have been omitted). Some plants, which had shown high activity in various studies and/or whose active constituents had not been isolated were also selected for re-testing for comparative purposes. 59 Plant species were obtained from and authenticated by The Herbal Apothecary, Leicester, UK with the exceptions of Achyranthus aspera, Aegle marmelos, Azadirachta indica, Centella asiatica, Commiphora mukul, Holarrhena antidysenterica, Nigella sati6a and Psoralea corylifolia which were obtained from and authenticated by Indian Herbs Equine, Wiltshire, UK and Panax ginseng from East West Herbs, Oxon, UK. Voucher specimens were deposited in the herbarium at the School of Pharmacy, University of Bradford, UK. 2.5. Preparation of crude methanolic extracts Dried powdered material (5 g) was exhaustively extracted with 200 ml methanol (at room temperature) three times and left overnight on a mechanical shaker. The extracts were collectively concentrated, using a rotary evaporator under reduced pressure, at less than 40 °C and then further concentrated in vacuo at room temperature. The dried crude extracts were assessed for their antimycobacterial activities. 2.6. Sequential sol6ent plant extraction Powdered plant material (30 g) was extracted by shaking with three separate volumes of hexane (200 ml) for 8 h. The extracts were pooled and concentrated under reduced pressure. The procedure was repeated using the same plant material shaking with chloroform, then methanol and finally with distilled water. The aqueous extracts were freeze-dried using a Dura-stop™ mr, FTS Systems™ machine at a temperature of −25 °C for 48 h. 2.7. Fractionation of crude extracts and isolation of acti6e constituents Fractionation of plant extracts was carried out using repetitive positive pressure column chromatography (PPCC) over silica gel GF254 (particle size 5–40 mm) (Merck) eluting with solvents of increasing polarity namely hexane, ethyl acetate, chloroform and methanol. Samples were applied either as solutions or as dry powders (sample dissolved in solvent was mixed with silica and the solvent evaporated to leave a dry powder). The fractions collected were monitored using pre-coated silica (silica gel GF254) TLC plates (Merck) and similar fractions were combined and concentrated in vacuo. 2.8. HPLC HPLC was performed using an analytical S5CN-4385 column (particle size 5 mm) (Hichrom Ltd) with a mobile phase composed of methanol (84%) and distilled water (16%) with 5 mM triethylamine (C2H5)3N 60 Table 1 Traditional uses of plant species screened for antimycobacterial activity Family Origin Traditional use Achyranthus aspera Linn. (Prickly-chaff flower) Aegle marmelos Corr. (Bael Tree, Bengal quince) Amaranthaceae Asia Rutaceae India Agrophyron repens Linn. Beauv. (Couch grass) Allium sati6um Linn. (Garlic) Gramineae Worldwide Pulmonary affections, cough, asthma and Dastur, 1962 skin diseases. A decoction of the leaves is a febrifuge Dastur, 1962; Wren, 1998 and expectorant and is particularly used for asthmatic complaints. Also used to treat acute bronchitis, fever and dysentery. Antibiotic. Bellamy, 1993 Liliaceae India, Pakistan Apium gra6eolens Linn. (Celery) Umbelliferae Worldwide Azadirachta indica Adr. Juss. (Margosa tree, Nim tree) Meliaceae India Borago officinalis Linn. (Borage) Boraginaceae Europe, North Africa, North America Centella asiatica Linn. Urban. (Indian pennywort, Hydrocotyle) Umbelliferae India, Pakistan Cinnamomum zeylandicum Breyn. (Ceylon cinnamon, cinnamon) Commiphora mukul Engl. (Indian bdellium) Lauraceae Sri Lanka, Sumatra, Eastern Islands, Brazil, Mauritius, India, Jamaica Pakistan, India Equisetum ar6ense Linn.(Horsetail) Equisetaceae Britain and Northern temperate regions Eucalyptus globulus Labill. (Eucalyptus) Leptospermoideae Umbelliferae Rosaceae Umbelliferae Tasmania, Australia, South Europe India, Pakistan, Mediterranean region Europe China Fabaceae Mediterranean region and Asia Compositae Worldwide Foeniculum 6ulgare Mill. (Fennel) Geum urbanum Linn. (Avens) Glehnia littoris Fr. Schm. ex Miq. (Beishashen) Glycyrrhiza glabra Linn. (Liquorice, Sweetwood) Helianthus annus Linn. (Sunflower) Rutaceae Fever, cough, pulmonary phthisis, whooping cough, gangrene of lung and related bronchi, fungal infections, dysentery. Also used as a disinfectant and antibiotic. Anthelmintic, antifungal and used to treat fever with cough. Anthelmintic, antiseptic. Used to treat chronic leprosy, phthisis, skin diseases, intestinal worms, chronic malaria fevers, small pox, syphylictic sores. Used in pulmonary diseases, consumption and to treat fever. Used to treat leprosy, scrofula, syphilis, gonorrhoea, skin diseases, elephantiasis, dysentery. Antiseptic and used to treat gonorrhoea. Expectorant, antimicrobial. Used to treat illnesses such as bronchitis, whooping cough and pulmonary TB. Recommended and effective in treatment of tuberculosis by older herbalists. Proved very effectual in spitting of blood. Antiseptic, expectorant, stimulant, febrifuge and used to treat cough. Diseases of the chest, cough, fever. Antiseptic, febrifuge. Roots used to treat bronchitis and dry cough. Expectorant. Popular and well-known remedy for cough, bronchitis, consumption and chest complaints. Treatment of coughs and colds. Seeds have expectorant properties and have been used in bronchial, laryngeal and pulmonary affections and in whooping cough. Reference Dastur, 1962; Wren, 1998 Dastur, 1962; Wren, 1998 Dastur, 1962; Wren, 1998 Wren, 1998; Grieve, 1994 Dastur, 1962; Wren, 1998 Dastur, 1962 Dastur, 1962; Wren, 1998 Foster and Tyler, 2000; Grieve, 1994 Wren, 1998 Dastur, 1962; Wren, 1998 Grieve, 1994; Wren, 1998 WHO, 1989 Dastur, 1962; Wren, 1998; Foster and Tyler, 2000; Grieve, 1994 Wren, 1998; Grieve, 1994 S.M. Newton et al. / Journal of Ethnopharmacology 79 (2002) 57–67 Plant Table 1 (Continued) Plant Family Origin Traditional use Reference Holarrhena pubescens Wall. (syn. Antidysenterica) (Conessi Bark, Kurchi) Apocynaceae India, Pakistan Dastur, 1962 Hyssopus officinalis Linn. (Hyssop) Labiatae India, South Europe Inula helenium Linn. (Elecampane) Compositae Europe and temperate Asia, USA, China Juniper communis Linn. (Juniper) Cupressaceae Worldwide, particularly in Europe Lomatium disectum Nutt. Et Const. Mentha piperita Linn. (Peppermint) Umbelliferae Labiatae British Columbia Cultivated widely particularly in Europe and America Nigella sati6um Linn. (Black Cumin) Ranunculaceae India Panax ginseng C.A.Meyer (Ginseng) Araliaceae China Polygonatum multiflorum Linn. All. (Solomons Seal, Fo-Ti, He-Shou-Wu) Polygonaceae Japan, China Prunus serotina Ehrh. (Wild Cherry Bark) Rosaceae Canada, USA Psoralea corylifolia Linn. (Babachi Seeds) Pulmonaria officinalis Linn. (Lungwort) Rhamnus cathartica Linn. (Buckthorn) Leguminosae Boraginaceae Rhamnaceae India, Pakistan Europe, Britain Britain, parts of Europe Rosa canina Linn. (Dog Rose) Rosaceae Sanguinaria canadensis Linn. (Bloodroot) Papaveraceae Europe, North Africa and parts of Asia North America, Canada Antidysenteric, febrifuge. Used in leprosy, and to treat chronic chest complaints, skin diseases and fever Used as an anthelmintic and expectorant, and also for chronic and pulmonary catarrh, diseases of respiratory organs, cough and chronic bronchitis. Used traditionally as an expectorant and to treat coughs, bronchitis, phthisis and pulmonary disorders. Anthelmintic, antibacterial and antifungal in vitro. Antiseptic properties. In France berries used in treatment of scrofula and chest complaints. Tuberculosis. Antiseptic, antiviral. Inhaled for chest complaints. Ingredient of some cough and cold remedies. Anthelmintic. Widely used to treat lung complaints, cough, fever. A decoction of the root is a common remedy for consumption. Used as an infusion for pulmonary complaints, pulmonary consumption and bleeding of the lungs. Antimicrobial. Used for centuries in cough syrups, particularly for irritable and persistent coughs such as those due to bronchitis and whooping cough. Antitussive. Anthelmintic and used to treat leprosy. Bronchitis, laryngitis, catarrh and cough. Not reported to have traditional use in TB, its symptoms or related diseases. Grange & Davey, 1990 showed that the tincture inhibited M. tuberculosis at dilutions of 1:160. Consumption, cough and spitting of blood. Expectorant, antimicrobial, antiviral, dentrifice, has anaesthetic properties, used to treat skin infections, pulmonary consumption, fever and epithelial tumours. Is also used medicinally in cough preparations. Dastur, 1962 Wren, 1998; Grieve, 1994 McCutcheon et al., 1997 Wren, 1998; Grieve, 1994 Dastur, 1962 Grieve, 1994 Wren, 1998; Foster and Tyler, 2000; Grieve, 1994 Wren, 1998 Dastur, 1962 Wren, 1998 Grange and Davey, 1990 S.M. Newton et al. / Journal of Ethnopharmacology 79 (2002) 57–67 Wren, 1998; Grieve, 1994 Grieve, 1994 Wren, 1998; Grieve, 1994 61 62 Table 1 (Continued) Family Origin Traditional use Reference Sanicula europaea Linn.(Sanicle) Umbelliferae Europe, British Isles, Tropical Africa Grieve, 1994 Saponaria officinalis Linn. (Soapwort) Solanum dulcamara Linn. (Bittersweet, Nightshade, Woody) Caryophyllaceae Solanaceae Europe British Isles, Europe, Asia, North Africa Stillingia syl6atica Linn.(Queens delight) Euphorbiaceae USA Taraxacum officinale Weber. (Dandelion) Compositae Worldwide Teucrium chaemaedrys L. (Germander) Labiatae Europe Verbascum thapsus Linn. (Mullein, Aarons rod) Scrophulariaceae Britain, Europe, temperate Asia, North America As an internal remedy in all chest and lung complaints, chronic cough and catarrhal affections, inflammation of the bronchi and spitting of blood. Used in skin diseases and as a detergent. Antifungal activity and used in treatment of warts, tumours and skin infections. Also recommended in chronic bronchial catarrh, asthma and whooping cough. Chronic bronchitis, laryngitis, skin diseases, scrofula, syphilis. In combination with other active remedies, this plant has been used for phthisis and some cutaneous diseases. Antiseptic. Used in asthmatic affections and to treat cough. Expectorant and is particularly used for bronchitis and catarrh and externally for inflammation and to aid wound healing. Is considered much value in phthisis and other wasting diseases, palliating the cough and staying expectoration. Consumptives appear to benefit greatly by its use and was carefully cultivated in gardens, because of a steady demand for the plant from these sufferers. Antiseptic, expectorant and used to treat cough, bronchitis, catarrh. The flowers were used to treat consumption in the days of Charles II and were sold by all apothecaries. Anthelmintic, febrifuge. Used in consumption, and to treat cough and chest complaints. Viola odorata Linn. (Violet) Withania somnifera Dun. (Winter cherry) Violaceae Solanaceae Europe including Britain, Northern Asia, North, Tropical and South America India Wren, 1998 Wren, 1998; Grieve, 1994 Grieve, 1994 Wren, 1998; Grieve, 1994 Wren, 1998; Grieve, 1994 Wren, 1998; Grieve, 1994 Wren, 1998; Grieve, 1994 Dastur, 1962 S.M. Newton et al. / Journal of Ethnopharmacology 79 (2002) 57–67 Plant S.M. Newton et al. / Journal of Ethnopharmacology 79 (2002) 57–67 buffered to pH 5.6 with phosphoric acid (H3PO4). Detection was by means of a Phillips Pye Unicam PU 4025 UV detector at 280 nm. 2.9. Spectroscopy IR spectra were recorded on a Galaxy Series (6020) FT-IR instrument (Mattson Instruments). 1H NMR spectra were recorded on a JEOL JNM-GX270 FT NMR spectrophotometer. Electron-impact mass spectra were determined on a Micromass Autospec M spectrophotometer. UV spectra were analysed on either a Hewlett-Packard 8452A Diode Array spectrophotometer using Hewlett-Packard 89531A MS-DOS UV/ VIS operating software or on a Perkin– Elmer Lambda 5 UV/VIS spectrophotometer. 3. Results and discussion 3.1. Initial screening of crude extracts against M. aurum and M. smegmatis The results of the initial screening of crude extracts are presented in Table 2. (Note that MIC values of 500 mg/ml or less were considered to be active.) Extracts of five plant species were shown to have antimycobacterial activity against M. aurum but not against M. smegmatis (Table 2). The most active species C. mukul (MIC=62.5 mg/ml), P. corylifolia (MIC= 62.5 mg/ml) and S. canadensis (MIC= 62.5 mg/ml) were selected for further evaluation (see below). A. gra6eolens and G. glabra (MIC for both species=250 mg/ml) also showed activity against M. aurum. A. gra6eolens is used in Indian medicine to treat fever with cough, just two of the symptoms of TB disease. This latter finding validates our reason for choosing this plant and many others, which were chosen based on their use to treat symptoms of TB. It is interesting to note that only H. pubescens and I. helenium showed activity against M. smegmatis. However, the antimycobacterial activity was too weak to be of significant value. H. pubescens has been widely used traditionally to treat dysentery and leprosy but there has been no report of its use in treating TB infections. Only I. helenium was active against both species of mycobacteria. I. helenium has been reported previously for its use in traditional medicine to treat lung complaints and TB. Our findings suggest that this species has antimycobacterial activity (MIC= 500 mg/ml) and this is consistent with previous work by Cantrell et al. (1999) who reported that I. helenium has activity against M. tuberculosis H37Rv (100 mg/ml methanol extract gave 83% inhibition using a radiorespirometric BACTEC assay). 63 It is interesting to highlight that there were a number of plants, reportedly used in traditional medicine to treat TB, which did not demonstrate any antimycobacterial activity against the two model organisms. It may be that the plants are used to treat the symptoms of the disease rather than actually cure the disease itself. In addition some plant species may not contain compounds which inhibit the growth of or kill M. tuberculosis but it is possible that they may have stimulant or modulatory effects on the immune system. Furthermore individual plants within a species may vary according to a number of factors including where the plant is grown, climate (temperature and rainfall), soil type, season in which the plant is collected, length of day (including the quality of light), altitude and the storage conditions (Evans, 1996). These may collectively have pronounced effects on the nature and quantity of secondary metabolites within the plant. In addition factors such as chemical races and varieties of plants species may also affect the chemical composition of a plant. It must also be noted that the activities of plant extracts in vitro may not parallel those in vivo. This gives rise to the possibility that potentially useful compounds may be missed, as our test does not mimic the in vivo environment. Some drugs for example may need to be metabolically activated in vivo for example by specific enzymes or may have a pH dependent biological activity (Grange and Snell, 1996; Zhang et al., 1999). The results also indicate that there is little correlation between the activities of the plant/natural product extracts when screened against both M. aurum and M. smegmatis. C. mukul, P. corylifolia and S. canadensis therefore appeared to be promising candidates for further investigation into our study of new lead compounds from natural products as potential antimycobacterial agents. C. mukul has been widely used in ayurvedic medicine and has been used traditionally to treat TB. P. corylifolia, an Indian herb, was a plant screened based on its former use to treat leprosy, a disease caused by the related mycobacterium species, M. leprae. It is not known to have been used to treat TB. However, reports of the antibacterial activity of P. corylifolia and related species such as P. glandulosa, against bacterial species other than Mycobacteria have been described (Erazo et al., 1997; Geda, 1995). S. canadensis has previously been reported to have activity against M. tuberculosis and Staphylococcus aureus but the inhibitory concentrations were not stated (Gottshall et al., 1949). Godowski (1989) has demonstrated that this plant does have antibacterial properties against a range of gram-positive and gram-negative bacteria particularly those found in the mouth. S.M. Newton et al. / Journal of Ethnopharmacology 79 (2002) 57–67 64 3.2. Isolation of acti6e constituents from S. canadensis Powdered root material of S. canadensis was defatted with hexane before extracting with methanol containing 1% glacial acetic acid using a soxhlet apparatus. 5% hydrochloric acid was then added to the concentrated extract and the mixture filtered. The filtrate was partitioned with chloroform:methanol (2:1). The acidified organic extract was fractionated repeatedly using positive pressure column chromatography over silica gel eluting with solvents of increasing polarity and fractions were monitored by TLC (Chloroform:methanol [9:0.3]; detection–Dragendorff’s spray reagent) and for activity against M. aurum. Two known benzophenanthridine alkaloids sanguinarine chloride (1) and chelerythrine chloride (2) (Fig. 1) were isolated from the active fractions and purified by crystallisation from chloroform/methanol. The purity of the alkaloids was confirmed by HPLC. Spectroscopic methods were used to characterise the two compounds and the data were Table 2 Antimycobacterial activities of some plant methanolic extracts Plant Achyranthus aspera (Melia azadarach) Aegle marmelos Agrophyron repens Allium sati6um Apium gra6eolens Azadirachta indica Borago officinalis Centella asiatica Cinnamomum zeylanicum Commiphora mukul Equisetum ar6ense Eucalyptus globulus Foeniculum 6ulgare Geum urbanum Glehnia littoris Glycyrrhiza glabra Helianthus annuus Holarrhenia pubescens Hyssopus officinalis Inula helenium Juniper communis Lomatium discetum Mentha piperita Nigella sati6a Panax ginseng (red korean) Polygonum multiflorum Prunus serotina Psoralea corylifolia Pulmonaria officinalis Rhamnus cathartica Rosa canina Sanguinaria canadensis Sanicula europaea Saponaria officinalis Solanum dulcamara Stillingia syl6atica Taraxacum officinale Taraxacum officinale Teucrium chaemaedrys Verbascum thapsus Verbascum thapsus Viola odorata Withania somnifera a Methanolic soluble fraction; ND, Not determined. Part used Herb Fruit Rhizome Bulb Seed Leaf Herb Herb Bark Resin Leaf Leaf Seed Herb Stem Root Petal Bark Herb Root Berry Root Leaf Seed Resin Fruit Bark Seed Leaf Bark Hips Root Herb Root Herb Root Leaf Root Herb Flower Leaf Leaf Root % Yielda 10.16 25.61 6.83 3.31 11.49 20.45 19.44 21.07 9.41 68.03 ND 23.65 13.26 11.89 4.66 24.98 42.56 17.54 6.82 12.26 21.82 47.53 19.65 15.25 19.86 7.38 7.38 24.36 12.31 15.64 37.28 27.10 14.08 9.18 5.15 14.10 14.35 29.73 21.38 65.90 23.94 14.35 8.74 Activity (MIC mg/ml) M. aurum M. smegmatis \500 \500 \500 \500 250 \500 \500 \500 \500 62.50 \500 500 \500 \500 \500 250 \500 \500 \500 500 \500 \500 \500 \500 \500 \500 \500 62.5 \500 \500 \500 62.5 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 500 \500 500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 \500 S.M. Newton et al. / Journal of Ethnopharmacology 79 (2002) 57–67 65 [8:2]; detection–anisaldehyde-sulphuric acid spray reagent) and for activity against M. aurum. The hexane extract was found to be the most active (MIC= 31.25 mg/ml). Further bioassay guided fractionation of the hexane extract, using positive pressure column chromatography over silica gel, eluting with hexane and increasing amounts of ethyl acetate, lead to the isolation of the known phenolic meroterpene, bakuchiol (3) (Fig. 2). The compound was characterised using spectroscopic techniques and the data were found to be consistent with previous literature values (Mehta et al., 1966, 1973). 3.4. Extraction of C. mukul Fig. 1. Iminium ion (a) and alkanolamine (b) forms of the alkaloids (1) and (2) isolated from S. canadensis. An ethyl acetate extract of C. mukul resin was prepared according to the methodology reported by Dev (1999) and the soluble material was separated into neutral, acid and basic fractions. The dried ethyl acetate insoluble material was extracted with methanol to give methanol soluble and methanol insoluble fractions. These fractions were screened for antimycobacterial activities but their activities were weak (MIC= 125 mg/ml) compared to the crude methanolic extract (MIC =62.5 mg/ml) and hence further work was not pursued. The activity of the initial crude methanolic extract may have been due to the synergistic action of compounds which would explain the decrease in antimycobacterial activity upon fractionation. 3.5. Acti6ities of isolated compounds against M. aurum and M. smegmatis Fig. 2. Structure of bakuchiol (3) isolated from P. corylifolia. found to be consistent with previous literature values; (1) was found to be identical to an authentic sample of sanguinarine chloride (Krane et al., 1984). 3.3. Isolation of an acti6e constituent from Psoralea corylifolia A sequential extraction at room temperature was performed on powdered seeds of P. corylifolia. All fractions were monitored by TLC (Hexane:ethyl acetate Compound (2) was shown to be the most active of the three compounds against both species of mycobacteria (Table 3). The IC50 values of both; (1) (9.61 mg/ml [26.19 mM]); and (2) (7.30 mg/ml [19.02 mM]) against M. aurum were very similar. (1) and (2) were both four-fold less active against M. smegmatis (IC50 = 41.18 mg/ml [112.21 mM] and 29.00 mg/ml [75.56 mM], respectively) than against M. aurum and this may explain why the crude methanolic extract of S. canadensis was inactive against M. smegmatis. Compound (3) was found to be only active against M. aurum (IC50 =15.79 mg/ml [61.68 Table 3 Antimycobacterial activities of isolated comopounds against M. aurum, M. smegmatix and M. bo6is BCG Compound Sanguinarine (1) Chelerythrine (2) Bakuchiol (3) Streptomycin IC50 value 9 SD (n) mg/ml M. aurum M. smegmatis M. bo6is BCG (n= 1) 9.61 9 4.52 (7) [25.04 mM] 7.309 1.91 (3) [19.02 mM] 15.79 9 10.66 (5) [61.68 mM] 1.14 90.02 (3) [0.78 mM] 41.189 16.85 (5) [112.2 mM] 29.009 17.60 (5) [75.56 mM] \500 [\ 1953 mM] 0.17 9 0.04 (3) [0.12 mM] 24.5 [66.8 mM] 14.3 [37.3 mM] 21.4 [83.6 mM] ND SD, Standard deviation; n, number of assays; ND, Not Determined. S.M. Newton et al. / Journal of Ethnopharmacology 79 (2002) 57–67 66 Table 4 Antimycobacterial activity of sanguinarine chloride (1) against M. smegmatis at different pH values pH value of medium Iminium ion form (%) (calculated value) Iminium ion form (%) (Jones et al., 1986) IC50 value (mg/ml) Standard deviation (n =4) 6.0 6.3 6.6 6.8 7.0 7.4 7.8 79.9 66.6 50.0 38.7 28.5 13.7 5.94 87.9 ND 41.1 ND 5.0 ND ND 34.40 30.83 29.00 24.03 21.85 22.38 17.71 7.90 5.15 17.60 (n = 5) 3.85 6.55 7.33 2.24 ND, not determined. mM]). Significant antimycobacterial activities were displayed by all three pure compounds against M. aurum although the IC50 values were higher than that of the positive control streptomycin (IC50 =1.14 mg/ml [0.78 mM]) 3.6. Determination of the effect of the pH of medium on the antimycobacterial acti6ity of sanguinarine Benzophenanthridine alkaloids unlike other alkaloids are unique with respect to their reactivity, as they undergo a dynamic equilibrium between the alkanolamine (base form) and iminum ion form in any protic solvent due to the sensitivity of the polar bond (NC) to the attack of nucleophiles (Harkrader and Jones, 1992; Jones et al., 1986) (Fig. 1a and b). The equilibrium is pH dependent. Jones et al. (1986), Mitscher et al. (1978), Colombo and Bosisio (1996) have reported that the biological activities of these alkaloids depend on the equilibrium of each form. The conversion of the alkanolamine from the iminium ion improves the lipophilicity of sanguinarine and the higher alkanolamine hydrophilicity may result in an increase in bioavailabilty of the alkaloid to the organism. Once inside the organism the alkanolamine form can be converted back to the iminium ion (Zee-Cheng and Cheng, 1978). Walterova et al. (1995) reported that sanguinarine and chelerythrine were able to penetrate mammalian cell membranes at pH 7.2– 7.4 as hydrophobic alkanolamines where they were able to accumulate to high levels in the acidic environment of the lysosome and convert back to the iminium ion. Experiments were carried out to determine whether the pH of the medium would have any effect on the antimycobacterial activity of compound (1). The percentage of the two forms of the alkaloid at each pH was calculated using the Henderson Hasslebalch equation (Table 4). However, the values were also determined by (Jones et al., 1986) who used an HPLC method. The pK value in water for compound (1) is 6.6. In this study a range of pH values of medium were chosen for the screening of the compound (pH 6–7.8). The control experiments showed that the different pH values of the media had no effect on the growth of M. smegmatis but the growth of M.aurum was suppressed at each pH value of medium with the exception of pH 6.6. The results indicate (Table 4) that there was no significant difference in the IC50 values of sanguinarine at different pH values of medium against M. smegmatis. Only a two-fold difference in activity was observed between the antimycobacterial activity at pH 6.0 and that at pH 7.8. It is possible that the permeability of the complex mycobacterial cell wall to sanguinarine may be poor or that if the drug does enter, the intracellular pH may not be conducive to the formation of the iminium ion. 3.7. Antimycobacterial acti6ity of the isolated compounds against M. bo6is BCG; a slowly growing screening model for M. tuberculosis M. bo6is BCG has been used in many studies as a screening organism for the prediction of antituberculosis activity (Kremer et al., 2000). MT is more closely resembled by M. bo6is BCG than by M. aurum and M. smegmatis due to the fact that MT and M. bo6is are both slow growing organisms. Preliminary screening of isolated compounds against M. bo6is was therefore pursued. All three compounds were found to have activities against M. bo6is BCG comparable to those found against M. aurum (Table 3). These results lend support to the use of M. aurum (rather than M. smegmatis) as a screening model organism. Further studies to confirm the results obtained using M. bo6is BCG are required. 4. Conclusions This study has highlighted some plants/compounds which are worthy of further investigation for their antimycobacterial/TB activities. Two alkaloids, sanguinarine and chelerythrine, and a phenolic meroterpene, bakuchiol, were isolated from S. canadensis and S.M. Newton et al. / Journal of Ethnopharmacology 79 (2002) 57–67 P. corylifolia, respectively and demonstrated appreciable antimycobacterial activity. Further investigation is now required to assess the in vitro and in vivo activities of these compounds against the pathogenic MT. Acknowledgements S. M. N. thanks the University of Bradford for providing a research studentship. References Ayyazian, L.F., 1993. History of tuberculosis. In: Reichman LB. Hershfield (Eds.), Tuberculosis. Dekker, New York. Basel, H.H., 1998. History of Tuberculosis. Respiration 65, 5 – 15. Bellamy, D., 1993. Blooming Bellamy. Herbs and Herbal Healing. BBC Books, London. Cantrell, C.L., Abate, L., Fronczek, F.R., Franzblau, S.G., Quijano, L., Fischer, N.H., 1999. Antimycobacterial eudesmanolides from Inula helenium and Rudbeckia subtomentosa. Planta Medica 65, 351 – 355. Chung, G.A., Aktar, Z., Jackson, S., Duncan, K., 1995. Highthroughput screen for detecting antimycobacterial agents. Antimicrobial Agents and Chemotherapy 39, 2235 – 2238. Colombo, M.L., Bosisio, E., 1996. Pharmacological activities of Chelidonium majus L. (Papaveraceae). Pharmacological Research 33, 127 –134. Dastur, J.F., 1962. Medicinal Plants of India and Pakistan. D.B. Taraporevala Sons and Co. Private Ltd., Bombay. Dev, S., 1999. Ancient-Modern concordance in ayurvedic plants: some examples. Environmental Health Perspectives 107, 783 – 789. Erazo, S., Gonzalez, V., Zaldivar, M., Negrete, R., 1997. Antimicrobial activity of Psoralea glandulosa L. International Journal of Pharmacognosy 35, 385 – 387. Evans, W.C., 1996. Trease and Evans’ Pharmacognosy. W.B. Saunders Company Ltd., London. Foster, S., Tyler, V.E., 2000. Tyler’s Honest Herbal. The Haworth Herbal Press Inc., New York. Geda, A.K., 1995. Antibacterial activity of essential oils and their combinations. Fat Science Technology 12, 458 – 460. Godowski, K.C., 1989. Antimicrobial action of Sanguinarine. Journal of Clinical Dentistry 1, 96 – 101. Gottshall, R.Y., Lucas, E.H., Lickfeldt, A., Roberts, J.M., 1949. The occurrence of antibacterial substances active against Mycobacterium tuberculosis in seed plants. Journal of Clinical Investigation 28, 920 – 921. Grange, J.M., Davey, R.W., 1990. Detection of antituberculous activity in plant extracts. Journal of Applied Bacteriology 68, 587 – 591. Grange, J.M., Snell, N.J.C., 1996. Activity of bromhexine and ambroxol, semi-synthetic derivatives of vasicine from the Indian shrub Adhatoda 6asica, against Mycobacterium tuberculosis in 6itro. Journal of Ethnopharmacology 50, 49 – 53. Grieve, M., 1994. In: Leyel, C.F (Ed.), A Modern Herbal. Tiger Books International, London. Harkrader, R.J., Jones, R.R., 1992. Purification of benzophenanthridine alkaloids from alkaloidal extracts. US Patent 5, 133, pp. 981 – 985. 67 Jones, R.R., Harkrader, R.J., Southard, G.L., 1986. The effect of pH on sanguinarine iminium ion form. Journal of Natural Products 49, 1109 – 1111. Krane, B.D., Fagbule, M.O., Shamma, M., 1984. The benzophenanthridine alkaloids. Journal of Natural Products 47, 1 –43. Kremer, L., Douglas, J.D., Baulard, A.R., Morehouse, C., Guy, M.R., Alland, D., Dover, L.G., Lakey, J.H., Jacobs, W.R., Brannan, P.J., Minnikin, D.E, Besra, G.S., 2000. Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis. Journal of Biological Chemistry 275, 16 857 – 16 864. McCutcheon, A.R., Stokes, R.W., Thorson, L.M., Ellis, S.M., Hancock, R.E.W., Towers, G.H.N., 1997. Antimycobacterial screening of British Columbian medicinal plants. International Journal of Pharmacognosy 35, 77 – 83. Mehta, G., Ramadas Nayak, U., Dev, S., 1966. Bakuchiol, a novel meroterpenoid. Tetrahedron Letters 38, 4561 – 4567. Mehta, G., Nayak, U.R., Dev, S., 1973. Psoralea corylifolia Linn.-1. Bakuchiol, a novel monoterpene phenol. Tetrahedron 29, 1119 – 1125. Mitscher, L.A., Park, Y.H., Clarke, D., Clarke III, G.W., Hammersfahr, P.D., Wu, W.N., Beal, J.L., 1978. Antimicrobial agents from higher plants. An investigation of Hunnemannia fumariaefolia pseudoalcoholates of sanguinarine and chelerythrine. Lloydia 41, 145– 150. Mitscher, L.A., Baker, W.R., 1998. A search for novel chemotherapy against tuberculosis amongst natural products. Pure and Applied Chemistry 70, 365 – 371. Newton, S.M., Lau, C., Wright, C.W., 2000. A Review of antimycobacterial natural products. Phytotherapy Research 14, 302 – 322. Raviglione, M.C., Snider, D.E.J., Kochi, A., 1995. Global epidemiology of tuberculosis: morbidity and mortality of a worldwide epidemic. Journal American Medical Association 273, 220 – 226. Walterova, D., Ulrichova, J., Valka, I., Vicar, J., Vavreckova, C., Taborska, E., Harkrader, R.J., Meyer, D.L., Cerna, H., Simanek, V., 1995. Benzophenanthridine alkaloids sanguinarine and chelerythrine: biological activities and dental care applications. Acta Universitatis Palackianae Olomucensis Facultatis Medicae 139, 7– 16. WHO, 1989. Medicinal Plants in China. Compiled by The Institute of Chinese Materia Medica and The China Academy of Traditional Chinese Medicine, WHO Regional Publications, Western Pacific Series no. 2, Manila. WHO, 1998. WHO: Global tuberculosis programme. Global tuberculosis control. WHO report. http://www.who.int/gtb/ publications. WHO, 2000. WHO Tuberculosis Fact Sheet No. 104. http:// www.who.int/inf-fs/en/fact104.html. (Accessed 09/05/01). Wren, R.C., 1998. Potters New Cyclopedia of Botanical Drugs and Preparations. Revised by Williamson E.M., Evans, F.J, Saffron Walden: CW Daniel Co, UK. Zee-Cheng, R.K.Y., Cheng, C.C., 1978. Antineoplastic agents. Structure-activity relationship study of bis (substituted aminoalkylamino) anthraquinones. Journal of Medicinal Chemistry 21, 291 – 294. Zhang, Y., Scorpio, A., Nikaido, H., Sun, Z., 1999. Role of acid pH and deficient efflux of pyrazinoic acid in unique susceptibility of Mycobacterium tuberculosis to pyrazinamide. Journal of Bacteriology 181, 2044 –2049. Zumla, A., Grange, J., 1998. Clinical review: tuberculosis. British Medical Journal 316, 1962 – 1964. Zumla, A., Mwaba, P., Squire, S.B., Grange, J.M., 1999. The tuberculosis pandemic-which way now? Journal of Infection 38, 74 –79. Journal of Ethnopharmacology 79 (2002) 69 – 73 www.elsevier.com/locate/jethpharm An evaluation of toxicity of Taxus baccata Linn. (Talispatra) in experimental animals K. Shanker a,*, N.K.R. Pathak a, V.P. Trivedi a, J.P.N. Chansuria b, V.B. Pandey c a Chemical Research Unit, Central Research Institute (Ay.), 474 /6, Sitapur Road, Lucknow 226020, India b Centre for Experimental Medicine and Surgery, IMS, B.H.U., Varanasi 221005, India c Department of Medicinal Chemistry, IMS, B.H.U., Varanasi 221005, India Accepted 24 September 2001 Abstract A toxicological study was performed in albino mice and rat with methanolic extract and isolated alkaloid of Taxus baccata Linn. (family: Taxaceae). LD50 study showed the higher toxic activity in stem (TXA-1,2,3) as compared with leaf (TXB-1,2,3) extract. As the extract were further fractionated into crude alkaloids and purified by chromatography the toxicity of these fractions were found to be in increasing order as follows: methanolic extract (1) B crude alkaloidal fraction (2) B purified alkaloidal fraction (3). The effects of leaf and stem extract of T. baccata were studied on certain biochemical and haematological parameters of mice and rat after 10, 20 and 30 days of exposure. Among the parameters examined, the exposed animal exhibited significant decrease in total leukocyte count (TLC), lymphocytes and cholesterol level (mg/dl), whereas increase was observed in serum transminases (SGOT, SGPT) and alkaline phosphatase (AP) of TXA-1 and TXB-1 treated groups indicating toxic conditions associated due to liver involvement. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Taxus baccata Linn.; Taxane; Toxicity 1. Introduction Taxus baccata Linn. is an evergreen tree, usually 6 m in height and 1.5 – 1.8 m in growth, found in the temperate Himalayas at an altitude between 1800 and 3300 masl in the hills of Meghalaya and Manipur at an altitude of 1500 m (Anonymous, 1976). It belongs to the family Taxaceae and is known indigenously as Talispatra in Ayurveda. The Indian System of Medicine attributes emmenagogue, sedative, anti-spasmodic and aphrodisiac to this plant (Kirtikar and Basu, 1984). Some of the compounds isolated from T. baccata elicited very promising and interesting pharmaco-dyanamic activity. At present, compounds having an oxetane ring at position 4 – 5 viz. taxol and its related compounds are the most widely studied class of compounds owing to their very promising therapeutic potential. Those compounds have shown very potential activity against advanced ovarian cancer, breast cancer and other types of tumour (Pandey, 1982). * Corresponding author. E-mail address: kspklko@yahoo.com (K. Shanker). Current interest in the genus Taxus due to the presence of promising anticancer compounds in different parts of the plant focuses on obtaining diterpene compounds having complex nature tricyclic system commonly referred to as taxanes (Miller, 1980; Pandey, 1982; Kingston et al., 1990, 1993; Doss et al., 1997; Wang et al., 1998; Shen et al., 2000). But a clear picture of its toxicokinetics is still obscure. In the present communication the toxicity associated with crude, methanolic extract and purified alkaloidal fraction compound derived from leaf and stem part of the tree have been evaluated in experimental animals. 2. Materials and methods 2.1. Preparation of extracts and isolation of alkaloids The methanolic extract of dried and powdered leaf and stem (TXA-1 and TXB-1) of T. baccata were subjected to an acid base extraction scheme (Pathak, 1994). The crude alkaloidal fractions mainly consisting 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 5 7 - 9 K. Shanker et al. / Journal of Ethnopharmacology 79 (2002) 69–73 70 of taxanes were obtained (TXA-2 and TXB-2). The crude alkaloidal fractions were further purified by column chromatography over silica gel to furnish TXA3 and TXB-3. For the present study the drug was obtained from Regional Research Institute (Ay.) and identified from Department of Botany, BHU, Varanasi. 2.2. Acute toxicity study Albino mice weighing 25– 30 g were used in present study. Acute toxicity of TXA-1,2,3 and TXB-1,2,3 was determined by LD50 in albino mice. The graphic method of Miller and Tainter (1944) were employed to determine LD50. The treatments were administered i.p. in single dose. One of the major problems with water insoluble fractions for i.p. administration was obtaining a stable solution.. Using TWEEN-80, an emulsifying agent and diluting with normal saline or physiological solution for all studies, solved this problem. To approximate the dose, bracketing was done with only one replication. Animal mortality 48 h after treatment was recorded as percentage (%) mortality. The percentage mortality was corrected by Abbot (1925) formula. In the final test, at least five mortality points ranging from 15 to 100% were selected. Thereafter, the group of six mice each were administered the drugs in five different concentrations and replicated three times. The LD50 was estimated by probit analysis (Finney, 1962). equally divided into the following groups: group-I (control). The rats of this group were fed standard rat feed; group-II (TXA-1 treated): the animals belonging to this group were administered 50 mg per rat per day orally for 20 days. Subsequently, the dose was increased i.e. 100 mg per rat per day to same rats for another 10 days; and group-III (TXB-1 treated): the animals belonging to this group were administered 50 mg per rat per day for 20 days. Subsequently, the dose was increased i.e. 100 mg per rat per day. The drugs were fed using rubber catheter. The body weight of each rat of control and treated groups were recorded prior to starting and at the end of experiment. 2.4. Blood analysis At the interval of 10, 20, and 30 days blood was collected from orbital sockets using fine capillaries. The serum was separated by centrifugation at 2500 rpm for 10 min. The relative blood indices (total red cell and leukocyte counts and haemoglobin) were determined using routine method (Dacie, 1958). In addition, the analysis of blood glucose (Sasaki and Matsui, 1972), protein (Lowry et al., 1951), urea (Natelson et al., 1951), cholesterol (Parekh and Jung, 1970). Glutamate pyruvate transaminase (GPT), glutamate oxaloacetate transaminase (GOT; King, 1965a), and alkaline phosphatase (AP; King, 1965b) were estimated in serum. A small amount of blood was also taken in a vial containing EDTA and used for haematological investigation. 2.3. Chronic toxicity 2.5. Statistical analysis Adult healthy albino rats (Charles Foster Strain) of either sex weighing 9 10 gm were used in the present study. These animals were kept in polypropylene cages (five rats per cage) under identical animal house conditions and provided with pelleted ‘Gold Mohaur’ rat feed (manufactured by Hindustan Lever Ltd. India). Food and water were given ad libitum. The rats were For biochemical parameters statistical significance of the difference between control and treatments (TXA1,2,3 and TXB-1,2,3) was evaluated using student’s t-test. The mean of medium lethal dose (LD50) of different extracts i.e. leaf (TXA-1,2,3) versus stem (TXB-1,2,3) were compared using non-parametric comparison Mann–Whitney U-test (Siegel, 1956). Data are presented as mean9S.E. Table 1 LD50 of different extracts of T. baccata Linn. in mice Plant parts Fractions Medium lethal dose value (LD50; mg/kg body weight) Leaf TXA-1 methanolic extract TXA-2 crude alkaloidal fraction TXA-3 purified alkaloidal fraction TXB-1 methanolic extract TXB-2 crude alkaloidal fraction TXB-3 purified alkaloidal fraction 747.6 Stem 112.0 6.0 146.0 43.0 3.3 3. Results and discussion After i.p. administration of alkaloidal fraction from leaf of T. baccata (TXA-1) in graded dose of 15, 20, 25, 30 and 35 mg/30 g body weight of mice, 16.66 and 100% mortality at dose of 15 and 35 mg/30 g body weight of mice were found, respectively. The mathematical estimation of LD50 using probit regression line was 22.28 mg/30 g body weight of mice. Similarly, the medium lethal dose of TXA-2 was found to be 3.38 mg/30 g body weight of mice. TXA-3, the purified alkaloidal fraction, showed LD50 0.1596 mg/30 g body weight of mice. The extent of toxic response to TXB-1 i.e. tingling sensation in the limb, an increased neuro- Table 2 Hb, TLC, DLC, GBP, blood glucose, protein, cholesterol, urea, SGOT, SGPT and alkaline phosphatase level in experimental animals [control and treated with TXA-1 (methanolic extract of leaf) and TXB-1 (methanolic extract of stem)] at the doses indicated for a period of 10, 20 and 30 days of exposure After 10 days of exposure After 20 days of exposure After 30 days of exposure Control TXA-1 TXB-l Control TXA-1 TXB-l Control TXA-1 TXB-l Haemoglobin TLC (mm) DLC (%) polymorphocyte Lymphocytes Eosinophyl Monocyte Basophyl Protein (g/dl) Glucose (mg/dl) Urea (mg/dl) Cholesterol (mg/dl) SGOT (U/ml) SGPT (U/ml) Alkaline phosphatase (K.A. Unit) GBP 11.3 9 0.58 90009866 559 5 11.6 9 1.0 116009 173 389 2 11.3 9 0.8 9600 9 764 38 9 2 10.99 0.1 8300 9 424 40 9 2 10.9 9 0.3 6650 9 550 45 9 3 10.8 9 0.3 66509 416 439 1 10.9 9 0.1 8300 9 41 9 2 10.8 9 0.3 60009 500 469 3 10.7 9 0.3 6500 9 416 42 91 44 9 5 190 ND ND 7.60 9 0.69 80.01 9 0.29 23.39 2.7 89.5 9 0.7 57 9 2* 291 291 190 7.60 9 0.69 64.41 9 10.18* 27.5 9 0.7 76.3 9 1.9* 58 91* 39 1 ND 19 1 7.60 9 1.39 76.21 9 7.74* 25.5 9 3.9 71.3 9 5.7** 59 9 4 190 ND ND 7.75 9 0.55 73.45 9 6.85 23.0 9 0.3 89.7 9 0.3 549 3* 190 ND ND 7.75 9 0.65 77.50 9 2.52* 27.75 9 0.6* 76.1 9 0.4* 53 9 3* 29 0.62 2 9 0.57 ND 7.639 1.33 78.01 9 2.26* 30.6 9 2.1* 74.0 9 3.5** 58 94 190 ND ND 7.75 9 0.55 75.01 9 6.00 ND 80.0 9 0.3 53 9 3* 1 90 ND ND 7.75 90.65 74.5 9 2.5 ND 76 9 0.36* 55 9 3* 190 29 0 ND 7.64 9 1.33 77.50 9 2.4* ND 75.1 93.5* 72.8 9 2.5 44.4 9 6.4 19.39 8.7 103.39 25.2*** 83.6 9 6.8*** 59.3 9 10.8*** 102.4 9 2.4** 66.5 9 2.1** 60.09 18.5*** 196.2 9 15.8*** 89.5 9 0.70*** 73.2. 9 2.7*** 109.2 9 0.9** 71.7 9 2.9** 77.6 94.3*** 71.9 9 1.1 40.0 94.8 25 9 0.1 RBC mostly RBC mostly normocytic, normocytic, nrmochromic, plates nrmochromic, seems to be platelets adequate adequate occasionally large lymphocytes bearing eccenttic nucleus and bluish cytoplasm RBC mosly normocytic, nrmochromic, platelets adequate 72.0 91.0 41.4 94.9 24.69 0.1 do do do do 200 9 15.5*** 80.5 9 0.6*** 759 2.0*** do 115 9 0.9** 75.8 9 2.5** 80.5 9 4.3 do K. Shanker et al. / Journal of Ethnopharmacology 79 (2002) 69–73 Parameters Values are expressed as mean 9 S.D. for five rats. Comparisons were made between control with treatments (TXA-1 and TXB-1). ND, not detected; *, PB0.05; **, PB0.01; ***, PB0.001. 71 72 K. Shanker et al. / Journal of Ethnopharmacology 79 (2002) 69–73 muscular irritability with twitching and fibrillation of the skeletal muscle, laryngeal spasm and finally convulsion, LD50 was found to be 43.93 mg/30 g body weight of mice. Mice mortality, 24 h after the administration of TXB-2 were recorded and LD50 was found to be 1.3 mg/30 g of body weight of mice. The purified fraction of T. baccata stem, TXB-3 showed acute toxicity, its LD50 being 0.10 mg/30 g body weight of mice. Mortality started immediately after the drug administration and 83% mortality after 10 min at the dose of 0.150 mg/30 g body weights of mice recorded. LD50 study showed the higher toxic activity in stem as compared with leaf (Table 1). The extracts were further fractionated into crude and purified alkaloidal fractions, and the toxicity of these fractions was found to be in increasing order. Leaf extract: TXA-1B TXA-2 BTXA-3 Stem extract: TXB-1BTXB-2 BTXB-3 Even a small dose of 6 mg/kg body weight of mice (TXA-3) and 3.3 mg/kg body weight of mice (TXB-3) produced intense death within 10– 15 min (Table 1). Preliminary toxicological study on crude and purified fractions of T. baccata revealed the high toxicity response in experimental animals. The tingling sensation in the limb, an increased neuromuscular irritability with twitching and fibrillation of the skeletal muscle, laryngeal spasm and finally convulsion were observed. These symptoms are indicative of increased neuromuscular excitability. This may be probably due to the fact that the calcium channel as the molecular target of these poisons is heart which results in tetany, paralysis and cardio-respiratory arrest. The acute toxicity elicited perhaps due to amorphous basic fraction, which might be acting as a calcium antagonistic particularly with respect to the heart. The alkaloid taxine, the toxic principal contained in leaf, shoot and seeds, is a complex mixture, consisting of the alkaloid taxine-A and taxineB, seems to be responsible for the toxicity (Chopra et al., 1956; Kirtikar and Basu, 1984). Alkaloids, which reduce the ionisation of calcium salt, may be another probable cause of tetany, a manifestation of a lack of calcium ionisation. Elevated alkaline phosphatase and transminase level in TXA-1 and TXB-1 treated rats, may be perhaps interpreted as toxic effect on liver (Table 2). Increase in both transminase (GOT and GPT) and AP are common findings in liver disorder (Varley, 1975; Martin et al., 1981). Marked excess in SGOT in myocardial infraction and elevated SGPT in hepato-celluar necrosis are generally observed (Varley, 1975). Most probably this condition might be associated to the presence of taxanes, diterpene amides, in almost all the fractions used. Authors are of the opinion that these decreases in AP, SGOT and SGPT levels may be due to the disrup- tive action of the TXA-1 and TXB-1 on the erythropoitic tissues (Table 2). Apart from the medicinal and therapeutic uses of the drug, the safety of a drug is an important criterion. The present study shows that the drug T. baccata induces severe toxic effects as shown by the investigated biochemical parameters. One can conclude that the crude drug, particularly the stem, is very toxic although having some therapeutic agents. Thus, the taxanes present in different plants of genus Taxus might represent a new potential tool in toxicological and pharmacological research. Acknowledgements The authors are thankful to The Director, Central Council for Research in Ayurveda and Siddha (CCRAS), New Delhi, for financial assistance and encouragement. The help rendered by C.N. Singh during experiment is greatly appreciated. References Abbot, W.S., 1925. Methods for computing the effectiveness of an insecticide. Journal of Ecology Entomology 18, 165 – 167. Anonymous, 1976. Wealth of India. Raw Materials. A CSIR Publication, vol.10. New Delhi, India, pp. 132 – 134. Chopra, R.N., Nyar, S.L., Chopra, I.C., 1956. Glossary of Indian Medicinal Plants. CSIR Publication, New Delhi, India, p. 240. Dacie, J.V., 1958. Practical Haematology, second ed. J and J Churchill, London, p. 38. Doss, R.P., Carney, J.R., Shanks, C.H., Williamson, T.R. Jr, Chamberlain, J.D., 1997. Two new taxoids from European yew (Taxus baccata) that act as pyrethrold insecticide synergists with the black vine weevil (Otiohynchus sulcatus). Journal of Natural Product 60, 1130 – 1133. Finney, D.J., 1962. Probit Analysis, second ed. Cambridge University Press, London, p. 318. King, J., 1965a. In: Van, D. (Ed.), Practical Clinical Enzymology. Nostrand, London, p. 83. King, J., 1965b. The phosphates. In: Van, D. (Ed.), Practical Clinical Enzymology. Nostrand, London, pp. 191 – 208. Kingston, D.G.I., Samaranayake, G., Ivey, C.A., 1990. The chemistry of taxol, a clinically useful anticancer agent. Journal of Natural Product 53 (1), 1 – 12. Kingston, D.G.I., Molinero, A.A., Rimoldi, J.M., 1993. In: Hertz, W., Kirby, G.W., Tamm, C. (Eds.), Progress in The Chemistry of Natural Products, vol. 62. Springer, New York, pp. 1 –188. Kirtikar, K.R., Basu, B.D., 1984. In: Basu, L.M. (Ed.), Indian Medicinal Plants, vol. 3. Bishen Singh Mahendra Pal Singh, New Connaught Place, Dehradun, India, pp. 2383 – 2385. Lowry, O.H., Rosenbrough, N.J., Farm, A.L., Randall, R.J., 1951. Protein measurement with Folin’s phenol reagent. Journal of Biological Chemistry 193, 265 – 276. Miller, R.W., 1980. A brief survey of Taxus alkaloids and other taxane derivatives. Journal of Natural Products 43 (4), 425 – 437. Miller, L.C., Tainter, M.L., 1944. Proceeding of Society of Experimental Biology 57, 261. Martin, D.W., Mayes, P.A., Rodwell, Y.W., 1981. Harpers Review of Biochemistry, 18th edn. Lange Medical, CA, p. 61. K. Shanker et al. / Journal of Ethnopharmacology 79 (2002) 69–73 Natelson, S., Scott, M.L., Beffa, C., 1951. A rapid method for the estimation of urea in biological fluid by means of the reaction between diacetely and urea. American Journal of Clinical Pathology 21, 275 – 281. Pandey, G.S. (Ed.), 1982. Bhavaprakasa Nighantu, sixth ed. Chauchambha Bharati Academy, Varanasi, India, pp. 255 – 258. Parekh, A.C., Jung, D.H., 1970. Cholesterol determination with ferric chloride –uranyl acetate and sulphuric acid – ferrous sulphate. Analytical Chemistry 42, 1423 – 1427. Pathak, N.K.R., 1994. Toxicological and chemical investigation of some medicinal plants. Ph.D. Thesis, Panjab University, Patiala, Panjab, India. 73 Sasaki, T., Matsui, S., 1972. Effect of acetic acid concentration on the color reaction in the o-tolidine boric acid for blood glucose determination. Rinsho Kagaku 1, 346 – 353. Shen, Y.C., Lo, K.L., Chen, C.Y., Kuo, Y.H., Hung, M.C., 2000. New taxanes with an opened oxetane ring from the root of Taxus mairei. Journal of Natural Product 63, 720 –722. Siegel, S., 1956. Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, Kogakusha, Tokyo. Varley, H., 1975. Practical Clinical Chemistry, fourth ed. ArnoldHeinemann Publishers, India, p. 409. Wang, X., Shigemori, H., Kobashi, J., 1998. Taxezopidines B –H, new taxoids from Japanese yew Taxus cuspidata. Journal of Natural Product 61, 474 – 749. Journal of Ethnopharmacology 79 (2002) 75 – 79 www.elsevier.com/locate/jethpharm Gastrointestinal enhancement of MRI with melanin derived from tea leaves (Thea sinensis Linn.) Yao-Ching Hung a, Vasyl M. Sava b, Chi-Long Juang c, Tzu-chen Yeh d, Wu-Chung Shen e, Guewha Steven Huang b,* a Section of Gynecologic Oncology, Department of Obstetrics and Gynecology, China Medical College, 91 Hsueh Shih Rd., Taichung 404, Taiwan, ROC b Institute of Chinese Pharmaceutical Sciences, China Medical College, 91 Hsueh Shih Rd., Taichung 404, Taiwan, ROC c Department of Radiological Technology, Yuan-pei Technical College, 306, Yuanpei St., Hsinchu, Taiwan, ROC d Department of Radiology, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Rd., Taipei, Taiwan, ROC e Department of Radiology, China Medical College, 91 Hsueh Shih Rd., Taichung 404, Taiwan, ROC Received 1 February 2001; received in revised form 25 September 2001; accepted 28 September 2001 Abstract Melanin was extracted from tea leaves (Thea sinensis Linn.) for the first time. Characterization of melanin proved similarity of the original compound to standard melanin. The Langmuir adsorption isotherms for gadolinium (Gd) binding were obtained using melanin. Melanin– Gd preparation demonstrated low acute toxicity. LD50 for this preparation was in a range of 1250– 1500 mg/kg in mice. Magnetic Resonance Imaging (MRI) properties of melanin itself and melanin– Gd complexes have been estimated. Gd free melanin fractions possess slighter relaxivity compared with its complexes. The relaxivity of lower molecular weight fraction was two times higher than relaxivity of Gd(DTPA) standard. Postcontrast images demonstrate that oral administration of melanin complexes in concentration 0.1 mM provides essential enhancement to longitudinal relaxation times (T1)-weighted spin echo image. The required contrast and delineation of the stomach wall demonstrated uniform enhancement of MRI with proposed melanin complex. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tea leaves; Melanin; Contrast agent; MRI 1. Introduction Tea is the oldest folk medicine. It was known in China 5000 years ago due to its stimulating and detoxifying properties (Balentine et al., 1997). The major composition and properties of tea are well documented, but scarce information is available concerning the polymeric polyphenols. Accumulation of polymeric substances is the result of the oxidative coupling of polyphenols occurring during processing of tea leaves (Hara et al., 1995). Recently, we have disclosed a biopolymer of melanin nature in tea (Sava et al., 2001). Melanin represents a group of black and brown pigments with high molecular weight derived from animal and plant origins. * Corresponding author. Tel.: + 886-4-2205-3366; fax: + 886-42205-1276. E-mail address: guehuang@ms21.hinet.net (G.S. Huang). Melanin pigments were regularly extracted from chestnuts, sunflower seeds, black beans (Nicolaus, 1968) and grapes (Zherebin et al., 1982). Unfortunately, natural melanin cannot be produced in sufficient quantities owing to the rarity of extraction sources. The extracted original melanin from tea represents an abundantly available resource with a rather high yield. Tea melanin could be formed during the growth of tea plant or the subsequent fermentation. The melanin formation in tea is based on the presence of polyphenols and specific enzymes, such as polyphenol-oxidase (Halder et al., 1998) and peroxidase (Digendra et al., 1973). During the fermentation, these enzymes could catalyze the oxidative coupling of polyphenols (Hara et al., 1995). Melanin was intensively studied for a long time (Nicolaus, 1968; Prota, 1992). The most significant properties concern its chelating capability and high stability under acidic condition (Fogarty et al., 1996). 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 5 8 - 0 76 Y.-C. Hung et al. / Journal of Ethnopharmacology 79 (2002) 75–79 Also, melanin possesses paramagnetic properties due to a high concentration of free radicals conjugated to a polymeric matrix. Both paramagnetic and chelating properties render melanin a prospective for Magnetic Resonance Imaging (MRI). Contrast agents for MRI are in an active research area. A variety of substances tested as contrast agents in MRI have been examined (Unger et al., 1999). The rationale for designing new contrast agents is based on creating complexes with paramagnetic metals (Laniado et al., 1988; Mattery et al., 1987; Rijcken et al., 1994). Gadolinium (Gd) contained complexes represent the most developed contrast agents (Laniado et al., 1988). However, such complexes are usually not suitable for gastrointestinal investigations. The low pH can cause the dissociation of compounds, particularly serious for Gd(DTPA) and Gd(DTPA – BMA) complexes (Kumar et al., 1993). For example, the half life of Gd(DTPA) at pH 2 is 1.7 h and for Gd(DTPA – BMA), it is 5 min (Tweedle, 1992). Thus, an essential prerequisite for the development of novel oral contrast agents is high stability of Gd complexes in acidic media. Based upon this, melanin becomes the obvious choice (Williams, 1994). Natural melanin also possesses low toxicity, which is beneficial (Nicolaus, 1968). The current study is based on the hypothesis that the paramagnetic and chelating properties of melanin is able to provide a platform for developing stronger and safer MRI contrast agents. filtered and then centrifuged at 20 000×g for 30 min. The extract obtained was acidified by addition of 2 N HCl to adjust pH to 2.5 followed by 2 h incubation at room temperature and centrifugation at 20 000×g for 15 min to pellet melanin. The crude extract obtained was purified by acid hydrolysis, organic solvent (chloroform, ethyl acetate and ethanol) treatment and repeated precipitation. The acid hydrolysis was employed to remove carbohydrates and proteins. Organic solvents were used to remove lipids. Melanin extracts were hydrolyzed with 7 N HCl at 100 °C for 2 h (Harki et al., 1997) followed by centrifugation at 10 000×g for 10 min, and the precipitate was washed with distilled water. Solid matter was re-dissolved in 1 N NH4OH and centrifuged at 10 000×g for 10 min. Melanin was precipitated then from the supernatant with 1 N HCl and washed with distilled water. The precipitation procedure was repeated four times followed by a final water wash. Physical and chemical characteristics of melanin were examined according to typical approaches (Fogarty and Tobin, 1996; Paim et al., 1990; Harki et al., 1997; Prota, 1992; Ellis and Griffith, 1974; Bilinska, 1996; Flip et al., 1974). Infrared (IR) spectra were recorded on Perkin– Elmer spectrometer 1600 FT (Perkin– Elmer Instruments, Norwalk, CT, USA). 2. Materials and methods 2.1. Materials In our experiments we used fully fermented Chinese black tea (Thea sinensis Linn.). The black tea was purchased from local retail shop in Miaoli, Taiwan. It was identified by Nien-Yung Chiu, the Institute of Chinese Pharmaceutical Sciences, China Medical College. A voucher specimen (GSH-001) was deposited at the Herbarium of this Institute. Gadolinium chloride (GdCl3), Arsenzo III and Sephadex G-50 were purchased from Sigma Chemical Co (St. Louis, MO). All additional reagents were of a chemical reagent grade and purchased from Merck KGaA (Darmstadt, Germany). Commercial preparation of Gd(DTPA) was purchased from Shering AG (Berlin, Germany). 2.2. Extraction and purification of melanin Extraction and purification of melanin was conducted according to the scheme (Fig. 1) previously designed (Sava et al., 2001) with minor adjustment. Tea leaves were immersed in water at volume ratio 1:10 followed by addition of 10% NH4OH to adjust pH value to 11. After 36 h incubation, the mixture was Fig. 1. The procedure of extraction melanin from tealeaves and preparation of melanin – Gd complexes. Y.-C. Hung et al. / Journal of Ethnopharmacology 79 (2002) 75–79 Melanin was chromatographed through a Sephadex G-50 in 50 mM phosphate buffer (pH 7.5) at a flow rate of 1 ml/min. Fractions were monitored at 280 nm. The apparent molecular weights (MW) of melanin fractions were estimated with the following size markers: dextran blue (MW 2000 000), aldolase (MW 158 000), bovine serum albumin (MW 66 000), cytochrome C (MW 12 400), and vitamin B12 (MW 1360). 2.3. Preparation of Gd– melanin complexes Two fractions of melanin MF6 and MF14 with an average molecular weight of 8 and 14 kDa, respectively, were employed for binding of Gd. Both fractions were precipitated with HCl and then washed with water until a pH level of 3– 6 was achieved. Each fraction was combined with appropriate amount of Gd3 + and then the mixture was stirred for 1 h. Subsequently, complexes were sequestered by centrifugation. Precipitates were washed with distilled water until all traces of Gd disappeared from the wash. Arsenazo III was applied to determine the Gd3 + concentration (Rohwer et al., 1995). The solutions of Gd– melanin complexes were prepared by the following procedure. The precipitates were dissolved in distilled water. The pH was adjusted to 9 by addition of 0.5 N NH4OH followed by incubation at 50 °C for 1 h. A final pH level of 7.5 was attained due to removal of ammonia by a rotary evaporator under reduced pressure. The end product was filtered through a Nalgene 0.45 mm syringe filter. 77 weighted pulse sequences maintained a constant time of echo (TE) at 15 ms while the time of repetition (TR) varied among 200– 6000 ms. Furthermore, transverse relaxation time (T2)-weighted pulse sequences maintained a constant TR at 3500 ms and varied the TE among 40– 480 ms. The signal intensity for each of the images was measured. The T1 and T2 were calculated for each concentration of contrast agent. Relaxivity r1 and r2 values (per mM per s) were obtained by plotting 1/T1 and 1/T2 as function of concentration followed by a slope determination (Unger et al., 1999). Gd (DTPA) commercial preparation (Schering AG, Berlin, Germany) was used as the standard. 2.6. In 6i6o MRI in6estigation Male rats (Wistar), weighting 320– 350 g, were fasted for 36 h, but were allowed drinking water. Prior to the procedure, the rats were anesthetized with a Ketamine/ Xylazine (1000/30, w/w). Five ml of Gd(MF8) was orally administered to experimental rats, whereas control rats did not receive it. The contrast agent had a concentration of 0.1 mM. MRI experiments were performed according to standard procedure. The rats were placed in a MRI unit (Bruker Medspec S300) and immediately proton density weighted MRI at a slice thickness of 5 mm were obtained. For T1-weighted imaging the pulse sequences were TR=500 ms and TE =20 ms and for T2-weighted images they were TR = 3000 ms and TE= 20 ms. 2.4. Determination of toxicity (LD50) 2.7. Statistical analysis The experiments were performed using adult Balb/c mice with an average weight of 20– 22 g. Animals were housed on a standard rodent chow and water ad libitum. Animals of either sex were distributed into seven groups with an equal number of both sexes in each group comprised ten animals. The experimental groups were treated with the aqueous solution of contrast agent given orally in a dose of 500– 1750 mg/kg. The animals were investigated throughout an observation period of 72 h and median lethal dose (LD50) values were calculated by means of probit analysis as described previously (Weber, 1980). All data were expressed as mean9 S.E.M. Differences between groups were considered to be significant at P B0.05 using Student’s t-test. 2.5. Measurement of relaxi6ity (r1 and r2) of Gd – melanin complexes Samples of melanin complexes with Gd in concentrations ranged from 0.05 to 1.0 mmol/l were prepared. Relaxivities were defined in the usual way (Wan et al., 1995). A Bruker Medspec S300 (Bruker Medical GmbH, Germany) at varying, pulse sequences was employed. Namely, longitudinal relaxation times (T1)- 3. Results According to accepted procedure of melanin extraction (Fig. 1) the average yield of crude product was 3.7%. Purification of melanin gave a 2% yield of pure product. The amorphous dark-brown pigment extracted from the tea displayed all the physical and chemical properties common to natural melanin (Sava et al., 2001). Furthermore, it was insoluble in both water and organic solvents, such as ethanol, hexane, acetone, benzene and chloroform. As well, it dissolved only in alkali, precipitated in alkaline FeCl3 with pH below 3, bleached in H2O2, KMnO4, K2Cr2O7 and NaOCl, and produced a blue color in FeSO4/ferricyanide. It showed similar characteristics with standard synthetic melanin (Sigma Chemical Co). Y.-C. Hung et al. / Journal of Ethnopharmacology 79 (2002) 75–79 78 Table 1 Relaxivity of various melanin samples measured in vitro at 20 °C and at 125 MHz Melanin samples r1 (per mmol per s)a r2 (per mmol per s)a MF8 MF14 Gd(MF8) 0.19 9 0.03 0.23 9 0.04 153.32 9 10.96 (8.639 0.62) 134.54 9 9.52 (6.729 0.48) 0.79 9 0.05 0.87 9 0.06 172.1 9 12.56 (9.7 9 0.71) 157.1 9 13.2 (7.859 0.66) Gd(MF14) a All data calculated using molar concentration of melanin. Data in brackets represent the results obtained calculating Gd molar concentration. Tea melanin also represented similar to synthetic melanin bonding characteristics in IR spectra. Additionally, IR spectra confirmed the interaction between melanin and Gd. Melanin induced chelating of Gd3 + decreased the band of 1720/cm and generated two new bands at 1560 and 1380/cm. It was discovered that there is significant interaction possibility between metal and carboxylic groups at pH 3– 6, however, phenolic groups may also be affected. The data obtained are consistent with correspondent results (Paim et al., 1990; Bilinska, 1996). Melanin thus derived was further purified through Sephadex G-50 column. Two fractions were eluted. Retention volumes of the minor and major peaks were 42.5 92.0 and 61.19 2.0 ml, respectively. The apparent MW for fraction of melanin were 89 2 (MF8) and 14 93 kDa (MF14). Four varieties of melanin preparations were considered for the relaxivity experiments. MF8 and MF14 were initial fractions of melanin and complexes with Gd were represented by the Gd(MF8) and Gd(MF14) samples. Table 1 illustrates the relaxivities of the mentioned samples. The Gd free samples possess slight relaxivity when compared with its complexes. As it follows from Table 1, melanin–Gd complexes revealed essential increase of relaxivity against melanin itself. Moreover, the lower molecular fraction Gd(MF8) demonstrated greater relaxivity values in comparison with the Gd(MF14) fraction. A 2-fold increase of relaxivity r1 over the Gd(DTPA) standard (r1 =4.3/mmol per s) was observed for Gd(MF8). Fraction Gd(MF8) was chosen for the experiments in vivo due to its higher relaxivity and lower toxicity. Oral median lethal dose (LD50) value for the total number of animals n =10 in each group was estimated. It was found a little difference between LD50 of melanin– Gd complexes obtained for MF8 and MF14 fractions. The value of LD50 was estimated as 1500 mg/kg body weight for oral administration of Gd(MF8) while acute toxicity of Gd(MF14) was 1250 mg/kg. Oral administration of melanin–Gd complexes to animal in dose of 1250 mg/kg demonstrated mild stimulation of central nervous system. Doses of 1750 mg/kg caused the noticed acceleration of respiration and tremor. Administration of higher doses lead to manifestation of convulsion. Fig. 2 displays the representative MRI obtained for Gd(MF8) as contrast agent. Fig. 2a and b depict the precontrast and postcontrast images, respectively. Postcontrast images demonstrate that Gd(MF8) provides significant enhancement on T1-weighted spin echo image. Finally, the required contrast and delineation of the stomach wall revealed that gastrointestinal MRI with the proposed melanin complex was achieved. 4. Discussion A number of gastrointestinal contrast agents have been developed and tested for application with MRI. As well, there are a variety of contrast agents under development for oral administration. These include the Gd chelates, serving essentially as positive contrast agents on T1-weighted images (Laniado et al., 1988), as well as superparamagnetic compounds, which act as negative contrast agents on T2-weighted images. A new contrast agent, developed on the base of natural melanin derived from tea leaves T. sinensis Linn., represents the possibility of positive and negative enhancement of MRI. Weihua reported that melanin could be extracted from the seeds of a tea (T. sinensis Linn.) plant (Weihua and Stuugart, 1996). We have been the first to isolate melanin from tealeaves. Characterization of this teabased melanin indicated that it possesses physical and chemical properties, which made it greatly similar to melanin extracted from alternate sources. However, proposed melanin can be extracted with high yield and in industrial scale quantities. Fig. 2. Precontrast (a) and postcontrast (b) MRI of rat stomach in a 10 min after oral administration of 5 ml 0.1 mM Gd(MF8). Y.-C. Hung et al. / Journal of Ethnopharmacology 79 (2002) 75–79 Gd – melanin complexes possess high longitudinal r1 and transverse r2 relaxivity, which was found via relaxation measurements (Table 1). Notably, high relaxivity is desirable for several reasons. As the effectiveness of contrast agents is proportional to relaxivity, an agent with much greater relaxivity can be administered in much lower dosages, thus reducing the overall toxicity to which a patient may exposed during MRI examination. Both Gd(MF8) and Gd(MF14) complexes demonstrated low acute toxicity. LD50 for Gd(MF8) was 1500 mg/kg and for Gd(MF14) this value was 1250 mg/kg. Thus, application of melanin complexes for MRI with an effective concentration of 0.1 mM and, correspondingly, in dose of about 12 mg/kg body weight is fully safe. The batch desorption experiments proved the high stability of Gd– melanin complexes in acidic conditions. In particular, Gd(MF8) fraction released 1.7% of Gd3 + during 24 h of equilibration desorption in 0.1 N HCl. This property is very important to use melanin complexes for gastrointestinal investigations. The proposed complexes demonstrated essential increasing of relaxivity in comparison to the standard Gd(DTPA) contrast agent. Our animal test results indicate that oral administration of melanin–Gd complex provides uniform contrast enhancement. Acknowledgements This study is supported in part by grants NSC 892323-B-039-002 and NSC 89-2313-B-039-005 from the National Science Council of ROC. References Balentine, D.A., Wiseman, S.A., Bouwens, I.C., 1997. The chemistry of tea flavonoids. Critical Reviews in Food Science and Nutrition 37, 693 – 704. Bilinska, B., 1996. Progress of infrared investigations of melanin structures. Spectrochimica Acta Part A 52, 1157 – 1162. Digendra, V., Mukherjee, S., Mukherjee, P., 1973. Peroxidase mediated oxidation of tyrosine: tyrosine hydroxilation in tea leaves. Experientia 29 (4), 422 – 424. Ellis, D.H., Griffith, D.A., 1974. The location and analysis of melanin in the cell walls of some soil fungi. Canadian Journal of Microbiology 20, 1379 – 1386. 79 Flip, Z., Haider, K., Beutelspacher, H., Martin, J.P., 1974. Comparison of IR spectra from melanin of microscopic soil fungi, humic acids and model phenol polymers. Geoderma 11, 37 – 52. Fogarty, R.V., Tobin, J.M., 1996. Fungal melanins and their interactions with metals. Enzyme and Microbial Technology 19, 311 – 317. Halder, J., Tamuli, P., Bhaduri, A.N., 1998. Isolation and characterization of polyphenol oxidase from Indian tealeaf (Camellia sinensis). The Journal of Nutritional Biochemistry 9, 75 – 80. Hara, Y., Luo, S.-J., Wickremashinghe, R.L., Yamanishi, T., 1995. VI. Biochemistry of processing black tea. Food Reviews International 11, 457 –471. Harki, E., Talou, T., Dargent, R., 1997. Purification, characterization and analysis of melanin extracted from Tuber melanosporum Vitt. Food Chemistry 58 (1 – 2), 68 – 73. Kumar, K., Chang, C.A., Tweedle, M.F., 1993. Equilibrium and kinetic studies of Lanthanide complex of macrocyclic polyamino carboxylates. Inorganic Chemistry 32, 578 – 593. Laniado, M., Kornmesser, W., Hamm, B., Claus, W., Weinmann, H.J., Felix, R., 1988. MR imaging of the gastrointestinal tract: Value of Gd(DTPA). American Journal of Roentgenology 150, 817 –821. Mattery, R.F., Hajek, P.C., Gylys-Morin, V.M., Baker, L.L., Martin, J., Long, D.C., Long, D.M., 1987. Perfluoro-chemicals as gastrointestinal contrast agent for MRI imaging. American Journal of Roentgenology 48, 1259 – 1263. Nicolaus, R., 1968. Melanins. Hermann, Paris. Paim, S., Linhares, L.F., Magrich, A.S., Martin, J.P., 1990. Characterization of fungal melanins and soil humic acids by chemical analysis and infrared spectroscopy. Biology and Fertility of Soils 10, 72 – 76. Prota, G., 1992. Melanins and Melanogenesis. Academic Press, SanDiego. Rijcken, T.H.P., Davis, M.A., Kos, P.K., 1994. Intraluminal contrast agents for MR imaging of the abdomen and pelvis. Journal of Magnetic Resonance Imaging 4, 291 – 300. Rohwer, H., Collier, N., Hasten, E., 1995. Spectrophotometric study of arsenazo III and its interactions with lanthanides. Analytica Chimica Acta 314, 219 –223. Sava, V., Yang, S.M., Hong, M.-Y., Yang, P.-C., Huang, G.S., 2001. Isolation and characterization of melanic pigments derived from tea and tea polyphenols. Food Chemistry 73, 177 – 184. Tweedle, M.F., 1992. Physicochemical properties of ProHance and other MR contrast agents. Investigative Radiology 27, S2 – S6. Unger, E.C., Chen, D.W., Trouard, T.P., 1999. Gadolinium-containing copolymeric chelates-a new potential MR contrast agent. Magnetic Resonance Materials in Physics Biology and Medicine 8, 154 – 166. Wan, X., Wedeking, P., Tweedle, M.F., 1995. MRI evaluation of potential gastrointestinal contrast media. Magnetic Resonance Imaging 13, 215 – 218. Weber, E., 1980. Grundrib der Biologishen Statistic. Gustav Fisher, Verlag, New York. Weihua, H. Stuugart, 1996. Natural tea black hair dying and making method thereof. Patent CN1116089. Williams, R., 1994. Melanin-based agents for image enhancement. Patent US53105399. Zherebin, Y.L, Makan, S.Y., Sava, V.M., Bogatsky, A.V., 1982. Process producing of water-soluble melanin. Patent SU939446. Journal of Ethnopharmacology 79 (2002) 81 – 87 www.elsevier.com/locate/jethpharm Flavonoids from Emblica officinalis and Mangifera indica — effectiveness for dyslipidemia L. Anila, N.R. Vijayalakshmi * Department of Biochemistry, Uni6ersity of Kerala, Karia6attom, Tri6andrum 695 581, Kerala, India Accepted 5 October 2001 Abstract Flavonoids from Emblica officinalis and Mangifera indica effectively reduce lipid levels in serum and tissues of rats induced hyperlipidemia. Hepatic HMG CoA reductase activity was significantly inhibited in rats fed E. officinalis flavonoids. But increase of this enzyme was observed in rats administered M. indica flavonoids. LCAT showed elevated levels in rats fed flavonoids from E. officinalis and M. indica. The degradation and elimination of cholesterol was highly enhanced in both the groups. In E. officinalis, the mechanism of hypolipidemic action is by the concerted action of inhibition of synthesis and enhancement of degradation. In the other group (M. indica) inhibition of cholesterogenesis was not encountered but highly significant degradation of cholesterol was noted, which may be the pivotal factor for hypolipidemic activity in this case. Though the mechanisms differ in the two cases, the net effect is to lower lipid levels. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Emblica officinalis; Mangifera indica; Dyslipidemia; HMG CoA reductase; LCAT; LPL 1. Introduction Flavonoids are plant polyphenols found frequently in fruits, vegetables and grains (Merken and Beecher, 2000). Hyperlipidemia has been ranked as one of the greatest risk factors contributing to the prevalence and severity of coronary heart disease (Grundy, 1986). Epidemiological studies have shown that high intake of fresh fruits and vegetables is associated with lowered risk of coronary heart disease mortality (Neaton et al., 1984; Criqui and Ringel, 1994). Recently, the intake of flavonoids was shown to be inversely related to coronary heart disease mortality (Hertog et al., 1993, 1995; Knekt et al., 1996). The plasma total cholesterol and atherogenic index were reduced by supplementation of 1 – 2% tea catechins to rats fed cholesterol-containing diet (Muramatsu et al., 1986). Tannic acid and morin can cause favourable changes in plasma lipid profiles of the type that have been correlated with coronary heart * Corresponding author. Fax: + 91-471-303-565. E-mail address: vijayalakshminr@hotmail.com (N.R. Vijayalakshmi). disease (Yugarani et al., 1992). The effect of flavonoids and flavonoid rich extracts on reducing lipid levels effectively has been studied in this laboratory (Asha et al., 2001; Sudheesh et al., 1997; Valsa et al., 1995) and elsewhere (Imai and Nakachi, 1995; Kono et al., 1992; Matsuda et al., 1986). The present investigation is focused on the hypolipidemic activity of flavonoid rich fractions from two different sources namely Emblica officinalis Gaertn. (Phyllanthus emblica Linn.) (Euphorbiaceae) (goose berry) and Mangifera indica Linn. (Anacardiaceae) (mango kernel). E. officinalis is reported to have hypolipidemic (Mathur et al., 1996; Thakur et al., 1988; Jacob et al., 1988) and hypoglycemic (Anila and Vijayalakshmi, 2000) activities. Traditional medicine is still the mainstay of about 75– 80% of the world population, mainly in the developing countries. India, having a very old and rich tradition of folk medicine for centuries, has provided very simple but effective remedies to various ailments using plants and plant derived compounds. Beneficial effects of E. officinalis including hypoglycemic activity have been described in ‘Materia Medica’, a classical Indian Text on Ayurvedic system of medicine (Nadkarni, 1976). The fruit pulp is being used in several 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 6 1 - 0 82 L. Anila, N.R. Vijayalakshmi / Journal of Ethnopharmacology 79 (2002) 81–87 Table 1 Yield of flavonoids in different solvent systems Solvents used Source of flavonoids E. officinalis (mg) Chloroform:ethyl acetate (75:25) Chloroform:ethyl acetate (50:50) Chloroform:ethyl acetate (25:75) Ethyl acetate 100% Ethyl acetate:methanol (75:25) Ethyl acetate:methanol (50:50) Ethyl acetate:methanol (25:75) Methanol 100% 2. Methodology M. indica (mg) 2.1. Plant material Dried fruits of E. officinalis Gaertn. and dried kernels of M. indica Linn. were used for extraction of flavonoids. 26.74 10.192 48.62 12.74 2.2. Extraction of fla6onoids 185.76 47.20 584.35 117.37 954.60 261.80 Ground dried material was extracted with hot 80% methanol thrice (Petra et al., 1999). The combined extract was evaporated to dryness and the residue was dissolved in water and extracted successively with hexane, benzene, ethyl acetate and butanol. Ethyl acetate extract contained bulk of flavonoids and this extract was evaporated in vacuum and the flavonoid content was determined by the method of Eskin et al. (Eskin et al., 1978) using quercetin as standard. 789.40 85.213 6.96 18.28 5.54 13.65 2.3. Chromatographic separation indigenous medical preparations against a variety of conditions such as liver injury (De et al., 1993), atherosclerosis (Thakur et al., 1988) and diabetes (Tripathi et al., 1979). In traditional medicine also E. officinalis is used for various conditions like glucose intolerance, cerebral insufficiency and mental disorders. Antioxidant activities of E. officinalis were reported by several investigators (Bhattacharya et al., 1999; Jose and Kuttan, 1995). Data regarding the biological activities of mango kernel is lacking although the kernel is used for preparation of food items in some villages. Raw mango seed kernels were found to contain tannins in the rate of 56.5 g kg − 1 (Sivakanesan and Ravindran, 1996). The dark brown solid (15 g each in the case of E. officinalis and M. indica) was adsorbed on silica gel (20 g) and transferred to a column of silica gel (150 g) equilibrated with hexane. Elution was performed with hexane, hexane:chloroform (75:25), hexane:chloroform (50:50), hexane:chloroform (25:75), chloroform, chloroform:ethyl acetate (75:25), chloroform:ethyl acetate (50:50), chloroform:ethyl acetate (25:75), ethyl acetate, ethyl acetate:methanol (75:25), ethyl acetate:methanol (50:50), ethyl acetate:methanol (25:75) and methanol. In each case 10 ml fractions were collected in 100 tubes and flavonoid content was estimated. Polyphenols were found to be negligible in the fractions of first five solvent systems while chloroform:ethyl acetate, ethyl Fig. 1. Flow chart for the isolation of flavonoids from E. officinalis (Indian gooseberry). L. Anila, N.R. Vijayalakshmi / Journal of Ethnopharmacology 79 (2002) 81–87 83 Fig. 2. Flow chart for the isolation of flavonoids from M. indica (mango kernel). acetate and ethyl acetate:methanol solvent systems gave fractions rich in polyphenols (Table 1). Maximum yield was obtained in the ethyl acetate:methanol (50:50) fraction (789.4 mg) in the case of E. officinalis and ethyl acetate (100%) fraction (954.6 mg) in the case of M. indica (Figs. 1 and 2). These fractions were concentrated in vacuum and used in the experiment. 2.4. Animals Male albino rats (Sprague– Dawley strain) weighing 60– 70 g were randomly selected and placed into three groups of ten rats each. Group I was treated as control and groups II and III were administered flavonoids from E. officinalis and M. indica at a dose of 10 mg kg − 1 BW per day, orally by gastric intubation. Animals were housed in polypropylene rat cages, which were kept in a controlled environment maintained between 28 and 35 °C. Animals were handled in accordance with the laboratory animal welfare guidelines (Hume, 1972). animals were deprived of food overnight, stunned by a blow at the back of the neck and killed by decapitation. Blood and tissues were removed to ice cold containers for various biochemical analyses. 2.7. Biochemical assays The lipids in the tissues and serum were extracted (Radin, 1981; Folsch et al., 1957) and estimated by the general procedures described earlier (Gomathy et al., 1989). The bile acids from liver (Okishio et al., 1967) and faecal sterols and bile acids (Grundy et al., 1965) were extracted. Total bile acids, neutral sterols and serum lipoproteins were also estimated by standard procedures given elsewhere (Valsa et al., 1995). The assays of b-hydroxy b-methyl glutaryl CoA (HMG CoA) reductase [EC 1.1.1.34] activity (Rao and Ramakrishnan, 1975) lipoprotein lipase (LPL) [EC 3.1.1.3] in heart and adipose (Schoenheimer and Sperry, 1974) and plasma lecithin cholesterol acyl transferase(LCAT) [EC 2.3.1.3] (Krauss et al., 1974) were carried out. Activities of glucose-6-phosphate dehydrogenase [EC 2.5. Diet As outlined in Table 2, the diet contained M/s Hindustan lever rodent chow, coconut oil (15%) and cholesterol (2%). 2.6. Experiment Diet and ordinary tap water were available on an ad libitum basis. The experimental duration was 90 days. Food intake and body weight records were kept weekly. Faeces were collected quantitatively in the last 2 days of the duration. At the end of the experimental period, Table 2 Composition of the diet Percentage control diet Percentage composition of the rodent chow Rodent chow 83 Coconut oil 15 Cholesterol 2 Crude protein 21 Ether extract (fat) 5 Crude fibre 4 Ash 8 Calcium 1 Phosphorous 0.6 Nitrogen free extract 53 L. Anila, N.R. Vijayalakshmi / Journal of Ethnopharmacology 79 (2002) 81–87 84 Table 3 Effect of flavonoids on diet intake and body weight gain of animals fed cholesterol-containing diet Groups I (Control) II (E. officinalis) III (M. indica) Average weight per group Initial (g) Final (g) 62.0 9 1.9 66.0 92.0 65.0 91.9 182.0 9 5.5 184.0 9 5.5 180.0 9 5.4 Weight gain 120.0 9 3.6 118.09 3.5 115.0 9 3.4 Diet composition was similar in three groups 11.5 90.3 g per day. The values are mean 9 S.E.M. for ten rats. Group II and III are compared with Group I. 1.1.1.49] (Kornberg and Horecker, 1955), malic enzyme [EC 1.1.1.40] (Ochoa, 1955a) and isocitrate dehydrogenase [EC 1.1.1.41] (Ochoa, 1955b) were determined. The protein content of the enzyme extract after trichloroacetic acid (TCA) precipitation was estimated (Lowry et al., 1951). 2.8. Statistical analysis Statistical significance was calculated using Student’s t-test (Bennett and Franklin, 1967). Significance was accepted at the PB 0.05. 3. Results Food intake and average body weight gain were found to be similar in both control and experimental rats after 90 days duration of the study (Table 3). A significant decrease in cholesterol, triglycerides, phospholipids and free fatty acid levels in serum and tissues (Fig. 3) were observed in animals administered flavonoids from E. officinalis and M. indica when compared with control group. The activity of HMG CoA reductase (Table 4) was significantly decreased in animals of group II while significant increase was noted in group III when compared with control group. The levels of hepatic and faecal bile acids and faecal neutral sterols (Fig. 5) were found to be significantly enhanced in both flavonoids treated animals when compared with control group animals. The activities of lipogenic enzymes (Table 4) were significantly lowered in both flavonoid treated animals. The activities of lipoprotein lipase in heart and adipose (Table 4) and plasma LCAT (Table 4) showed elevated levels in the case of both flavonoid administered groups when compared with control group animals. The level of high density lipoprotein (HDL) cholesterol remained unchanged while low density and very low density lipoproteins (LDL+ VLDL) cholesterol levels showed significant reduction Fig. 3. Effect of flavonoids from E. officinalis and M. indica on concentrations of cholesterol, triglycerides, phospholipids and free fatty acids in serum and tissues of rats fed cholesterol containing diet. The values are mean 9 S.E.M. for ten rats. Group II and III are compared with Group I. (a): PB 0.01 and (b) 0.01 BP B 0.05. L. Anila, N.R. Vijayalakshmi / Journal of Ethnopharmacology 79 (2002) 81–87 85 Table 4 Effect of flavonoid administration on the activities of HMG CoA reductase in liver, lipogenic enzymes in liver, lipoprotein lipase in heart and adipose and plasma LCAT in rats fed cholesterol containing diet Enzymes Group I (Control) Group II (E. officinalis) Group III (M. indica) HMG CoA reductasea Glucose 6 phosphate dehydrogenase (Units mg−1 protein)b Malic enzyme (Units mg−1 protein)b Isocitrate dehydrogenase (Units mg−1 protein)c 4.17 9 0.13 122.20 9 3.66 1183.75 9 35.50 1.95 9 0.06 6.67 9 0.20* 92.80 9 2.78* 911.35 9 27.34* 1.45 9 0.04* 2.77 9 0.08* 82.75 9 2.48* 873.48 9 26.20* 1.06 9 0.32* 20.75 9 0.62 130.36 9 3.90 28.54 9 0.86 36.96 9 1.11* 245.42 9 7.36* 52.38 9 1.57* 38.57 9 1.16* 294.97 9 8.85* 63.27 9 1.90* Lipoprotein lipase d (a) Heart (b) Adipose Plasma LCATe The values are mean 9 S.E. for ten rats. Group II and III are compared with Group I. *PB0.01. a Activity is expressed as ratio of HMG CoA/mevalonate, i.e. the higher the ratio, lower the activity. b One unit is defined as the amount of the enzyme that causes an increase of 1 in optical density min−1. c One unit is defined as the amount of the enzyme that causes an increase of 0.01 in optical density min−1. d Activity expressed as mmoles of glycerol liberated h−1 mg−1 protein. e Activity expressed as percentage increase in the ratio of ester cholesterol to free cholesterol during incubation. in the case of both flavonoid treated groups when compared with control rats (Figs. 4 and 5). 4. Discussion The results obtained from above experiments clearly demonstrate the hypolipidemic activity of flavonoids from E. officinalis and M. indica at a dose of 10 mg kg − 1 BW per day when administered to the atherogenic experimental animals. There was significant inhibition in the activity of HMG CoA reductase in liver of animals administered flavonoids from E. officinalis when compared with control animals. Highly significant inhibition of this enzyme denotes inhibition of cholesterogenesis in animals administered flavonoids from E. officinalis. This may account for the lowered level of cholesterol in serum and tissues of rats treated with flavonoids from E. officinalis. Moreover significantly increased excretion of faecal bile acids and neutral sterols also contribute to the hypocholesterolemic action of flavonoids from E. officinalis. But in the case of animals supplied with flavonoids from M. indica, the activity of HMG CoA reductase was significantly increased. In this case, even though cholesterogenesis is stimulated, cholesterol degradation and turnover was very high as evidenced by the higher rate of conversion of cholesterol to bile acids and elimination of faecal bile acids and neutral sterols. There was a significant reduction in the LDL+ VLDL cholesterol, but HDL cholesterol level remained unchanged. Thus the circulating LDL+VLDL fractions in serum of rats given flavonoids had lower cholesterol content. The newly synthesised cholesterol in liver may be used for the synthesis of bile acids, which is evident from the significantly elevated levels of hepatic bile acids in the flavonoid treated animals. Fig. 4. Effect of flavonoids from E. officinalis and M. indica on concentration of cholesterol in HDL and LDL +VLDL fractions and total serum cholesterol of rats fed cholesterol containing diet. The values are mean 9 S.E.M. for ten rats. Group II and III are compared with Group I. (a) PB 0.01. Fig. 5. Effect of flavonoids from E. officinalis and M. indica on concentrations of hepatic and faecal bile acids and faecal neutral sterols in rats fed cholesterol-containing diet. Values expressed as mg per rat per day in the case of faecal bile acids and neutral sterols and mg per 100 g wet tissue in the case of hepatic bile acids. The values are mean9 S.E.M. for ten rats. Group II and III are compared with Group I. (a) P B 0.01. 86 L. Anila, N.R. Vijayalakshmi / Journal of Ethnopharmacology 79 (2002) 81–87 Plasma LCAT seemed to be involved in the transport of cholesterol from extrahepatic tissues to the liver for degradation. The significantly elevated activity of plasma LCAT in flavonoid administered rats accounts for the significant hike in hepatic bile acids which provide the evidence for the higher rate of degradation of cholesterol in animals administered flavonoids from E. officinalis and M. indica. The pronounced reduction in the level of triglycerides in serum and tissues may be due to the significantly enhanced activity of lipoprotein lipase in heart and adipose of animals administered flavonoids from E. officinalis and M. indica. The improvement of hyperlipidemia may also be due to the significant reduction in the activities of lipogenic enzymes, which provide the sole source of NADPH for the free fatty acid biosynthesis. The experiment provides useful information regarding the hypolipidemic action of flavonoids from E. officinalis and M. indica in an atherogenic condition. Further investigations to explore dose dependent and time dependent (longer duration of experiment) action of these flavonoids are underway. Acknowledgements Financial assistance received from ICMR in the form of SRF is acknowledged with thanks. References Anila, L., Vijayalakshmi, N.R., 2000. Beneficial effects of flavonoids from Sesamum indicum, Emblica officinalis and Momordica charantia. Phytotherapy Research 14, 1 –4. Asha, S.K., Anila, L., Vijayalakshmi, N.R., 2001. Flavonoids from Garcinia cambogia lower lipid levels in hyper cholesterolemic rats. Food Chemistry 72, 289 –294. Bennett, C.A., Franklin, N.L., 1967. Statistical Analysis in Chemistry and Chemical Industry. Wiley, New York, pp. 133 – 138. Bhattacharya, A., Chatterjee, A., Ghoshal, S., Bhattacharya, S.K., 1999. Antioxidant activity of active tannoid principles of Emblica officinalis (amla). Indian Journal of Experimental Biology 37 (7), 676 – 680. Criqui, M.H., Ringel, B.L., 1994. Does diet or alcohol explain the French paradox? Lancet 344, 1719 – 1723. De, S., Ravishankar, B., Bhavsar, G.C., 1993. Plants with hepatoprotective activity — a review. Indian Drugs 30, 355 – 363. Eskin, N.A.M., Hoehn, E., Frenkel, C.J., 1978. A simple and rapid quantitative method for total phenols. Journal of Agricultural and Food Chemistry 26, 973 –974. Folsch, J., Less, M., Stanley, S., 1957. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497 – 509. Gomathy, R., Vijayalakshmi, N.R., Kurup, P.A., 1989. Hypolipidemic principles of inflorescence stalk of plantain (Musa sapientum). Journal of Bioscience 14, 301 – 309. Grundy, S.M., 1986. Cholesterol and coronary heart disease: a new era. Journal of American Medical Association 256, 2849 –2858. Grundy, S.M., Ahrens, E.H., Miettinen, T.A., 1965. Quantitative isolation and gas liquid chromatographic analysis of total dietary and neutral sterols. Journal of Lipid Research 6, 411. Hertog, M.G.L., Feskens, E.J.M., Hollman, P.C.H., Katan, M.B., Kromhout, D., 1993. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zulphen elderly study. Lancet 342, 1007 – 1011. Hertog, M.G.L., Kromhout, D., Aravanis, C., Blackburn, H., Buzina, R., Fidanza, F., Giampaoli, S., Jansen, A., Menotti, A., Nedeljkovic, S., Pekkarinen, M., Simic, B.S., Toshima, H., Feskens, E.J.M., Hollman, P.C.H., Katan, M.B., 1995. Flavonoid intake and long term risk of coronary heart disease and cancer in the seven countries study. Archives of Internal Medicine 155, 381 – 386. Hume, C.W., 1972. The UFAW Handbook on the Care and Management of Laboratory Animals. Churchill Living stone, Edinburgh/ London. Imai, K., Nakachi, K., 1995. Cross sectional study of effects of drinking green tea on cardiovascular and liver disease. British Medical Journal 310, 693 –696. Jacob, A., Pandey, M., Kapoor, S., Saroja, R., 1988. Effect of the Indian gooseberry (amla) on serum cholesterol levels in men aged 35 – 55 years. European Journal of Clinical Nutrition 42 (11), 939 – 944. Jose, J.K., Kuttan, R., 1995. Antioxidant activity of Emblica officinalis. Journal of Clinical Biochemistry and Nutrition 19 (2), 63 – 70. Knekt, P., Jarvinen, R., Reunanen, A., Maatela, J., 1996. Flavonoid intake and coronary mortality in Finland: a cohort study. British Medical Journal 312, 478 – 481. Kono, S., Shinchi, K., Ikeda, N., Yanai, F., Imanichi, K., 1992. Green tea consumption and serum lipid profiles: a cross sectional study in Northern Kyushu, Japan. Preventive Medicine 21, 526 – 531. Kornberg, A., Horecker, C.B., 1955. Glucose-6-phosphate dehydrogenase. In: Colowick, S.P., Kaplan, N. (Eds.), Methods in Enzymology, vol. 1. Academic Press, New York, pp. 323 – 327. Krauss, R.N., Wind Muller, H.G., Levi, R.I., Fredrickson, D.S., 1974. Selective measurement of low lipase activity in post-heparin plasma from normal subjects and patients with hyperlipoproteinemia. Journal of Clinical Investigation 54, 1107 – 1121. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin phenol reagent. Journal of Biological Chemistry 193, 265 – 275. Mathur, R., Sharma, A., Dixit, V.P., Varma, M., 1996. Hypolipidaemic effect of fruit juice of Emblica officinalis in cholesterol fed rabbits. Journal of Ethnopharmacology 50 (2), 61 – 68. Matsuda, H., Chisaka, T., Kubumura, Y., Yamahara, J., Sawada, T., Fujima, H., Kimura, H., 1986. Effect of crude drugs on experimental hypercholesterolemia-1 — tea and its active principles. Journal of Enthnopharmacology 17, 213 – 224. Merken, H.M., Beecher, G.R., 2000. Measurement of food flavonoids by high performance liquid chromatography: a review. Journal of Agriculture and Food Chemistry 48, 577 –599. Muramatsu, K., Fukuyo, M., Hara, Y., 1986. Effect of green tea catechins on plasma cholesterol level in cholesterol fed rats. Journal of Nutritional Science and Vitaminology 32, 613 – 622. Nadkarni, K.M. (Ed.), 1976. Dr K.M. Nadkarni’s Indian Meteria Medica. The Karnataka Printing press and The Popular Press (Bombay) Ltd, pp. 480 – 484. Neaton, J.D., Kuller, L.H., Wentworth, D., Borhani, N.O., 1984. Total and cardiovascular mortality in relation to cigarette smoking, serum cholesterol concentration and diastolic blood pressure among black and white males followed up for five years. American Heart Journal 108, 759 – 769. Ochoa, S., 1955a. Isocitrate dehydrogenase system (TPN) from pig heart. In: Colowick, S.P., Kaplan, N.O. (Eds.), Methods in Enzymology, vol. 1. Academic Press, New York, pp. 699 – 700. L. Anila, N.R. Vijayalakshmi / Journal of Ethnopharmacology 79 (2002) 81–87 Ochoa, S., 1955b. Malic enzyme. In: Colowick, S.P., Kaplan, N.O. (Eds.), Methods in Enzymology, vol. 1. Academic Press, New York, pp. 739–753. Okishio, T., Nair, P.P., Gordon, M., 1967. Studies on bile acids: the micro quantitative separation of cellular bile acids by gas –liquid chromatography. Biochemical Journal 102, 654 –658. Petra, M., Britta, T., Macki, K., Eckart, E., 1999. Flavonoid sulfates from the Convolvulaceae. Phytochemistry 50, 267 –271. Radin, N., 1981. Extraction of tissue lipids with hexane:isopropanol. In: Lowenstein, J.M. (Ed.), Methods in Enzymology, vol. 72. Academic Press, New York, pp. 5 – 7. Rao, A.V., Ramakrishnan, S., 1975. Indirect assessment of hydroxy methyl glutaryl CoA reductase activity in liver tissue. Journal of Clinical Chemistry 21, 1523 – 1527. Schoenheimer, R., Sperry, W.M., 1974. A micro method for the determination of free and combined cholesterol. Journal of Biological Chemistry 106, 745 – 760. Sivakanesan, R., Ravindran, V., 1996. The Nutritive value of Mango seed kernels for starting chicks. Journal of Science of Food and 87 Agriculture 71 (2), 245 – 250. Sudheesh, S., Presanna kumar, G., Vijayakumar, S., Vijayalakshmi, N.R., 1997. Hypolipidemic effect of flavonoids from Solanum melongena. Plant Foods for Human Nutrition 51, 321 – 330. Thakur, C.P., Thakur, B., Singh, B., Singh, S., Sinha, P.K., Sinha, S.K., 1988. The Ayurvedic medicines, Haritaki, Amla and Bahira reduce cholesterol induced atherosclerosis in rabbits. Indian Journal of Cardiology 21 (2), 167 –175. Tripathi, S.N., Tiwari, C.M., Upadhyay, B.N., Singh, R.S., 1979. Screening of hypoglycemic action in certain indigenous drugs. Journal of Research in Indian Medicine in Yoga Homeopathy 14, 159 – 169. Valsa, A.K., Ushakumary, B., Vijayalakshmi, N.R., 1995. Effect of catechin on lipid metabolism. Journal of Clinical Biochemistry and Nutrition 19, 175 – 182. Yugarani, T., Tan, B.K.H., Teh, M., Das, N.P., 1992. Effect of polyphenolic natural products on the lipid profiles of rats fed high fat diets. Lipids 27, 181 – 186. Journal of Ethnopharmacology 79 (2002) 89 – 94 www.elsevier.com/locate/jethpharm Modulation of macrophage function activity by ethanolic extract of larvae of Holotrichia diomphalia Nam-Sung Kang a, Sook-Young Park a, Kang-Ro Lee a, Sun-Mee Lee a, Bum-Gu Lee a, Dae-Hee Shin b, Suhkneung Pyo a,* a College of Pharmacy, Sungkyunkwan Uni6ersity, 300 Chunchun-Dong, Suwon City, Kyunggi-do, 440 -746 South Korea b Institute of Life Science Research, Chosun Pharm. & Trading Co., Ansan City, Kyunggi-do, 425 -120 South Korea Accepted 17 October 2001 Abstract Macrophages play a central role in determining the host response to tumor, in part through the secretion of several potent products. In this study, our work is directed toward studying the in vitro effects of extract from the larvae of Holotrichia diomphalia (HD-EX) on the ability to induce cellular and secretory responses in mouse peritoneal macrophages. Macrophages were treated with various doses (0.1, 1, 10 mg/ml) of HD-EX for 20 h. This treatment induced tumoricidal activity and increased the production of tumor necrosis factor (TNF-a) and nitric oxide (NO) by macrophages in a concentration-dependent manner. However, HD-EX had a little effect on phagocytosis and the levels of hydrogen peroxide (H2O2), interleukin-1 (IL-1), IL-6 and IL-10 were very low in HD-EX treated macrophages. Thus, the tumoricidal effect of HD-EX appeared to be mainly mediated by NO and TNF-a production from macrophages. Taken together, these results suggest that HD-EX is a differential immunomodulating effect on macrophage secretory and cellular activities. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Holotrichia diomphalia; Immmunomodulatory activity; Macrophage activation 1. Introduction A number of natural products are used in the traditional medical systems in many countries. Holotrichia diomphalia Bates has been traditionally used to prevent and treat many kinds of diseases such as liver cirrhosis, contusion, edema, furuncle and apoplexy in Korea. This clinical use possibly arises from the induction of immune response, although the precise pharmacological mechanisms of H. diomphalia are still obscure. Macrophages have been shown to play an essential role in host defenses against microbial agents and neoplasia (Hahn and Kaufmann, 1981; Verstovsek et al., 1992). Macrophages can be stimulated by various agents including IFN-g, lipopolysaccharide, or other microbial products (Dullens et al., 1989; Gautam and Deodhar, 1989; Paulnock and Lambert, 1988) and some of these have also been shown to trigger the * Corresponding author. Tel.: + 82-31-290-7713; fax: + 82-31-2928800. E-mail address: snpyo@yurim.skku.ac.kr (S. Pyo). release of tumor necrosis factor-a (TNF-a), interleukin1 (IL-1), IL-6 and nitrite and to induce tumoricidal activity in macrophages (Arden et al., 1985; ArenzanaSeisdedos and Virelizier, 1983; Choriki et al., 1989; Stuehr and Marletta, 1987; Keller et al., 1990). This study was undertaken to investigate the biological effects of an ethanolic extract from larvae of H. diomphalia on murine peritoneal macrophages. 2. Materials and methods 2.1. Mice, chemicals and reagents C57BL/6 mice (6– 8 weeks) were obtained from Charles River Breeding Laboratories (Atsugi, Japan). Unless otherwise indicated, all chemicals were purchased from Sigma Chemical Co. (St Louis, MO). RPMI 1640 medium and fetal bovine serum were purchased from GIBCO (Grand Island, NY). NGmonomethyl-L-arginine (NMMA) was obtained from Calbiochem Co. (LaJolla, CA). All tissue culture 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 6 9 - 5 90 N.-S. Kang et al. / Journal of Ethnopharmacology 79 (2002) 89–94 reagents, the thioglycollate broth and extracts were assayed for endotoxin contamination by the Limulus lysate test (E-Toxate, Sigma) and found to be less than 10 pg/ml. blue exclusion and proportion of macrophages determined after cytoplasmic staining with acridine orange and examination using a fluorescence microscope. Cell preparations were \ 95% viable and contained \95% macrophages. 2.2. Preparation of extract 2.4. Macrophage-mediated cytotoxicity H. diomphalia (1 kg) was purchased at the herbal drug market in Cheju-Do, Korea in August 1999 and identified by Dr B.G. Lee of the Institute for Traditional Medicine, Sungkyunkwan University, Suwon, Korea. A voucher specimen (SKK-H001) is deposited in the College of Pharmacy at Sungkyunkwan University. One kilogram of H. diomphalia was refluxed with 70% ethanol (2 litre) two times for 8 h. The materials were filtered and the clear supernatants were then concentrated under reduced pressure at 40 °C with vacuum rotary evaporator. The concentrated ethanol extract (100 g) was partitioned between water (1 litre) and n-hexane (1 litre, ×2). After removing the n-hexane fraction, the aqueous layer was partitioned again with methylene chloride (1 litre, × 2), followed by n-butanol (1 litre, × 2). 2.3. Isolation of inflammatory peritoneal macrophages Thioglycollate-elicited peritoneal exudate cells were obtained from 6 – 8 week-old C57BL/6 male mice following intraperitoneal injection of 1 ml Brewer Thioglycollate broth (4.05 g/100 ml) (Difco Laboratories, Detroit, ML) and lavage of the peritoneal cavity with 5 ml of medium 3 – 4 days later. The cells were washed twice and resuspended in RPMI-1640 (GIBCO, Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 IU/ml) and streptomycin (100 mg/ml) (RPMI-FBS). Macrophages were isolated from peritoneal exudate cells as described by Klimetzek and Remold (1980). Peritoneal exudate cells were seeded at densities of 5 – 6×105 cells/cm2 on Teflon-coated petri dishes (100× 15 mm) and the macrophages were allowed to adhere for 2 – 3 h in 5% CO2 humidified atmosphere. Teflon-coated petri dishes were prepared by spraying with aerosolized Teflon (Fisher Scientific, Pittsburgh, PA) and sterilizing with ultraviolet light for 3 h. The nonadherent cells were removed by washing the dishes twice with 10 ml prewarmed medium and dishes were incubated for 10 min at 4 °C. The supernatants were then carefully removed and discarded and the plates were washed once with prewarmed Dulbecco’s Phosphate Buffered Saline (PBS) (GIBCO). Cold PBS (15 ml) containing 1.5% FBS (PBS – FBS) was added followed by 0.3 ml of 0.1 M EDTA (pH 7.0). The plates were incubated for 15 min at room temperature and the macrophages removed by rinsing 10 times using a 10 ml syringe. The viability of the detached cells was assessed by trypan The assay for macrophage cytotoxicity was performed by modification of the technique described previously (Flick and Gifford, 1984; Moon et al., 1999). Briefly, macrophages (1.0×105 cells/well) were plated in 96 well microplates and co-incubated with B16 melanoma cells (1.0× 104/wells: an initial effector:target cell ratio of 10:1) in the presence of various doses of HD-EX for 20 h at 37 °C in a 5% CO2 incubator. In some experiments, antibody to cytokine, the isotype-matched control antibody or inhibitor of metabolic pathway was included. At the extract dosages that we employed, no toxicity was observed. Cell density was then assessed by incubating the cells with 25 mg/ml MTT (3-(4,5-dimethylthiaozle-2-yl)-2,5-diphenyltetrazolium bromide) for another 4 h. Formazan produced was dissolved in dimethyl sulfoxide and the optical density of each well at 540 nm was determined using a molecular device microplate reader (Menlo Park, CA). Cytolytic activity is expressed as the percentage of tumor cytotoxicity where % cytotoxicity= {1 −[OD of (macrophages+target cells)−OD of macrophages]/OD of target cells}×100. 2.5. Determination of phagocytosis The phagocytic activity was measured using the assay system described previously (Okimura et al., 1986). Macrophages were treated with indicated concentration of HD-EX for 20 h and then washed with RPMI1640 to remove HD-EX. Cells were incubated with 5× 106 particle of zymosan and 600 mg/ml of NBT. After 1 h incubation, plates were centrifuged at 4 °C for stopping ingestion of zymosan and supernatant was removed by flipping. The optical density of the reduction product of NBT, a purple insoluble formazan, was determined at 540 nm using a molecular device microplate reader. It was not required to solubilize the formazan before taking the measurement of absorbance. 2.6. Nitrite determination The accumulation of NO− 2 in culture supernatants was measured using the assay system described by Ding et al. (1988). Briefly, 100 ml of supernatant was removed from each well into an empty 96-well plate. After the addition of 100 ml Griess reagent to each well, absorbance at 550 nm was measured using a molecular device microplate reader. NO− 2 concentration was cal- N.-S. Kang et al. / Journal of Ethnopharmacology 79 (2002) 89–94 culated from a NaNO2 standard curve. The levels of NO− 2 are indicative of NO production. Griess reagent was prepared by mixing one part of 0.1% naphthylethylene diamine dihydrochloride in distilled water plus one part of 1% sulfanilamide in 5% concentrated H3PO4. 2.7. Hydrogen peroxide determination The secretion of hydrogen peroxide in culture supernatants was fluorimetrically measured by the horseradish peroxidase-catalyzed oxidation of fluorescent scopoletin to a nonfluorescent product, as described in detail (Nathan and Root, 1977). The 460 nm emission from reduced scopoletin, when excited by light at 350 nm, is extinguished when scopoletin is oxidized by H2O2 in the presence of horseradish peroxidase. Under assay conditions, the loss of fluorescence was proportional to the concentration of H2O2. 2.8. Cytokine determination by ELISA The concentration of IL-1, IL-6, IL-10 and TNF-a production in culture supernatants was determined by the use of ELISA kits (Endogen, Woburn, MA) according to the manufacturer’s protocol. 3. Statistical analysis The significance of differences between control and treated test was analyzed using Student’s t-test. Differ- Fig. 1. Tumoricidal activities of HD-EX-treated murine peritoneal macrophages against B16 melanoma cells. Macrophages were treated with various doses of HD-EX for 20 h. Tumoricidal activity was determined as described in Section 2 at an initial effector/target ratio of 10:1. The results are mean 9 SEM of quintuplicates from one representative experiment of three. *P B 0.05, **PB 0.01; significantly different from control (no treatment). 91 ences were considered significant at P B0.05 and significant values were represented by an asterisk. All experiments were repeated at least three times. Data are expressed as means9SEM. 4. Results 4.1. HD-EX-triggered macrophages acti6ation for tumor cytotoxicity The effect of HD-EX was evaluated for tumoricidal activity in vitro of macrophages against B16 tumor cells. Thioglycollate-elicited macrophages were co-cultured with B16 tumor cells in the presence of various doses of HD-EX for 20 h. B16 tumor cells were used as targets since they are either TNF-a or NO sensitive. HD-EX (0.1–10 mg/ml) increased the cytotoxicity by macrophages in a dose-dependent manner (Fig. 1). The maximum effect was obtained using 10 mg/ml of HDEX. This amount did not affect the viability of macrophages, but concentrations greater than 10 mg/ml were cytotoxic to cells (data not shown). 4.2. Effect of HD-EX on NO and TNF-h secretion by macrophages Once activated, tumoricidal macrophages produce a large number of cytotoxic molecules (Sone and Key, 1986; Hibbs et al. 1987). We next examined the ability of HD-EX to induce NO and TNF-a, which are currently believed to be the primary species involved killing tumor cells. HD-EX was observed to activate NO and TNF-a production by peritoneal macrophages in a dose-dependent manner. We found that pretreatment with HD-EX significantly stimulated NO and TNF-a production by inflammatory macrophages compared with the response of untreated macrophages (Fig. 2a and b). In addition, anti-TNF-a antibody and the NO inhibitor were able to abrogate, in part, the production of cytotoxic molecules induced by HD-EX in the culture supernatants (data not shown). These results further demonstrate that the level of NO and TNF-a production in HD-EX-treated macrophages was significantly increased. Moreover, the addition of anti-TNF-a antibody and the NO inhibitor was able to abrogate, in part, tumoricidal activities of HD-EX-exposed macrophages against target (Table 1). At the concentrations employed none of the inhibitors or antibodies affected the growth of the tumor cells and the isotype-matched control antibodies had no effect on cytotoxic activity (data not shown). Taken together these results suggest that TNF-a, and NO are partially involved in the tumoricidal activity of HD-EX-exposed macrophages. 92 N.-S. Kang et al. / Journal of Ethnopharmacology 79 (2002) 89–94 4.3. The effect of HD-EX on phagocytic properties of macrophages Fig. 3 shows that the incubation of macrophages with 0.1, 1 or 10 mg HD-EX slightly increased the phagocytosis of opsonized zymosan by macrophages, suggesting that there was a little alteration on phagocytosis by HD-EX. 4.4. Production of H2O2, IL-1, IL-6 and IL-10 in HD-EX-treated macrophages To determine the effect of HD-EX on the production of H2O2, IL-1, IL-6 and IL-10, macrophages were treated with various doses of HD-EX for 20 h. Culture supernatants were assayed for cytokines by ELISA. Cytokine levels (IL-1, 1L-6 and IL-10) were only slightly elevated at 10 mg/ml of HD-EX compared with background (Fig. 4a– c), suggesting that TNF-a is a more important cytokine in tumoricidal activities of HD-EX-treated macrophages than IL-1, IL-6 and IL10. The production of H2O2 was not greatly increased, either (Fig. 4d). H2O2 production in only 10 mg/ml of the HD-EX-treated group was slightly significant in comparison with that of the control group. 5. Discussion Fig. 2. (a) Nitrite and (b) TNF-a production from peritoneal macrophages stimulated with HD-EX. Macrophages were treated with HD-EX for 20 h. Culture supernatants were collected and the levels of nitrite and TNF-a were measured as described in Section 2. The results are mean 9 SEM of quintuplicates from one representative experiment of three. *PB 0.05, **PB 0.01; significantly different from control (no treatment). Alternative medicine for treatment of various diseases is getting more popular. H. diomphalia larvae have been used as a traditional remedy in Korea and show various important pharmacological roles. Recently, potent antibacterial proteins have been isolated Table 1 Inhibition of tumoricidal activity of HD-EX-exposed macrophages by antibody or inhibitor Treatment % Cytotoxicity of target cell (B16) None HD-EX (10 mg/ml) HD-EX+NMMA (0.5 mM) HD-EX+anti-TNF-a (500 units/ml) 27.29 3.2 70.7 9 6.3* 29.2 9 3.8* 37.9 9 6.9* HD-EX-exposed macrophages were co-cultured for 20 h with target at an initial effector/target cell ratio of 10:1. Macrophage tumoricidal activity was determined as described in Section 2. Data shown are the results at an initial effector/target ratio of 10:1. The results shown are the mean9 SEM of quintuplicates from a representative experiments. *Significantly different from control (no treatment); PB0.05. Fig. 3. Effect of HD-EX on the phagocytosis of peritoneal macrophages. Macrophages were treated with HD-EX for 20 h. Macrophages were then incubated with 5 × 106 particle of zymosan and 600 mg/ml of NBT. Phagocytosis was measured as OD 540 nm. The results shown are the mean 9 SEM of three independent experiments. *PB 0.05; significantly different from control (no treatment). N.-S. Kang et al. / Journal of Ethnopharmacology 79 (2002) 89–94 93 Fig. 4. (a) IL-1, (b) IL-6, (c) IL-10 and (d) H2O2 production by peritoneal macrophages stimulated by HD-EX. Macrophages were treated with HD-EX for 20 h. Culture supernatants were collected, and the levels of IL-1, IL-6 and IL-10 were measured by ELISA, respectively. The production of H2O2 was measured as described in Section 2. The results shown are the mean 9 SEM of three independent experiments. *P B0.05; significantly different from control (no treatment). from the H. diomphalia larvae (Lee et al., 1994) and prophenoloxidase from the hemolymph of H. diomphalia larvae has been purified and characterized (Kwon et al., 1997; Lee et al., 1998). Therefore, this in vitro study was undertaken to determine whether ethanolic extracts of H. diomphalia larvae (HD-EX) have immunomodulatory properties. The present study provides the first results on the effects of HD-EX on the immune system of mice. Our results show that HD-EX can differentially alter macrophage tumoricidal activity as well as the production of cytokines and cytotoxic molecules. It has been well known that macrophages play an important role in the defense mechanism against host infection and the killing tumor cells. The modulation of antitumor properties of macrophages by various biological response modifiers is an area of active interest for cancer chemotherapy. It has been shown that antiumor properties of macrophages can be activated by IFN-g as well as lipopolysaccharide. Treatment of resident macrophages with IFN-g induces a primed state. Primed macrophages have not only phagocytic activity but readily have become a fully activated cytolytic macrophage when stimulated with LPS. These cytolytic macrophages could be able to secrete various cytokines such as TNF-a, IL-1 and IL-6 (Adams and Hamilton, 1984; Rees and Parry, 1992). Secretion of these mediators was found to require a triggering signal such as LPS, thus providing some evidence to the role of the priming and triggering signals to induce the complete cytolysis function. In this study, HD-EX has also activated murine peritoneal macrophages, resulting in the increased production of various molecules such as NO, H2O2, TNF-a, IL-1, IL-6 and IL-10. The level of NO and TNF-a production in HD-EX-treated macrophages was significantly increased, whereas the production of H2O2, IL-1, IL-6 and IL-10 and phagocytosis was slightly increased. These results suggest that the beneficial, immunostimulatory activity of HD-EX may be mediated through upregulation of secretory molecules in macrophages and HD-EX plays a role in triggering the activation of macrophages. The present observations are related to the previous report that NO, TNFa, IL-1, IL-6 and IL-10 were increasingly secreted by activated macrophages at first but H2O2 production and phagocytic activity were induced at the primed state not activated state (Lewis et al., 1986). Another previous report showed that H2O2 production was not an essential product at the activated state (Cohen et al. 1982). Based on these findings it suggests that the increased production of TNF-a and NO are related to tumoricidal activity of HD-EX-treated macrophages. Additional support for this conclusion came from the observation that anti-TNF-a antibody and the NO inhibitor were able to abrogate, in part, HD-EX-induced cytostasis against target. Macrophages have been shown to be an important component of host defenses against virus infection by 94 N.-S. Kang et al. / Journal of Ethnopharmacology 79 (2002) 89–94 inhibiting intracellular replication of virus (intrinsic resistance) and by killing virus infected cells (extrinsic cytotoxicity) (Morahan, 1984; Morahan et al., 1985). We have observed that HD-EX did not induce the antiviral activity (data not shown), suggesting that mediators, which play a role in HD-EX-induced tumoricidal activity, are not involved in antiviral activity and also HD-EX has differential effects on efficient activation of macrophages. In summary, although the data presented in this report yielded an incomplete picture on the effects of the extracts on the immune system, we have demonstrated that HD-EX results in an augmentation of macrophage function and a potent stimulator of both the synthesis and the release of cytotoxic mediators. These results suggest that HD-EX might achieve in vivo enhancement of macrophage function. Further studies with animal models are necessary to clarify how this activation occurs and to what extent it occurs in vivo. References Adams, D.O., Hamilton, T.A., 1984. The cell biology of macrophage activation. Annual Review Immunology 2, 283 – 318. Arden, L., Lansdorp, P., De Groot, E., 1985. A growth factor for B cell hybridomas produced by human monocytes. Lymphokines 10, 175 – 185. Arenzana-Seisdedos, F., Virelizier, J., 1983. Interferons as macrophage activating factors. II. Enhanced secretion of interleukin 1 by lipopolysaccharide-stimulated human monocytes. European Journal of Immunology 13, 437 – 440. Choriki, M., Freudenberg, M., Calanos, C., Poindron, P., Bartholeyns, J., 1989. Antitumoral effects of lipopolysaccharide, tumor necrosis factor, interferon and activated macrophages: synergism and tissue distribution. Anticancer Research 9, 1185 – 1190. Cohen, M.S., Taffer, S.M., Adams, D.O., 1982. The relationship between secretion of H2O2 and completion of tumor cytotoxicity by BCG-elicited murine macrophages. Journal of Immunology 128, 1781 – 1785. Ding, A.H., Nathan, C.F., Stuer, D.J., 1988. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. Journal of Immunology 141, 2407 – 2412. Dullens, H.F.J, De Weger, R.A., Van Der Maas, M., Den Besten, P.J., Vandebriel, R.J., Den Otter, W., 1989. Production of specific macrophage-arming factor precedes cytotoxic T lymphocyte activity in vivo during tumor rejection. Cancer Immunology Immunotherapy 30, 28 – 33. Flick, D.A., Gifford, G.E., 1984. Comparison of in vitro cell cytotoxicity assays for tumor necrosis factor. Journal of Immunology 68, 167 – 175. Gautam, S., Deodhar, S., 1989. Generation of tumoricidal effector cells by human C-reactive protein and muramyl tripeptide: a comparative study. Journal of Biological Response Modifier 8, 560 – 569. Hahn, H., Kaufmann, S.H.E., 1981. The role of cell-mediated immunity in bacterial infections. Review in Infectious Disease 3, 1221 – 1250. Hibbs, J.B., Taintor, R.R., Vavrin, Z., 1987. Macrophage cytotoxicity: role for L-arginine deiminase and imino nitrogen oxidation to nitrite. Science 235, 473 – 476. Keller, R., Keist, R., Frei, K., 1990. Lymphokines and bacteria that induce tumoricidal activity, trigger a different secretory response in macrophages. European Journal of Immunology 20, 695 – 698. Klimetzek, V., Remold, H.D., 1980. The murine bone marrow macrophage, a sensitive indicator cell for murine migration inhibitory factor and a new method for their harvest. Cellular Immunology 53, 257 – 266. Kwon, T.H., Lee, S.Y., Lee, J.H., Choi, J.S., Kawabata, S., Iwanaga, S., Lee, B.L., 1997. Purification and characterization of prophenoloxidase from the hemolymph of coleopteran insect, Holotrichia diomphalia larvae. Molecules and Cells 7, 90 –97. Lee, S.Y., Moon, H.J., Kurata, S., Kurama, T., Natori, S., Lee, B.L., 1994. Purification and molecular cloning of cDNA for an inducible antibacterial protein of larvae of a coleopteran insect, Holotrichia diomphalia. Journal of Biochemistry (Tokyo) 115, 82 –86. Lee, S.Y., Kwon, T.H., Hyun, J.H., Choi, J.S., Kawabata, S.I., Iwanaga, S., Lee, B.L., 1998. In vitro activation of pro-phenol-oxidase by two kinds of pro-phenol-oxidase-activating factors isolated from hemolymph of coleopteran, Holotrichia diomphalia larvae. European Journal of Biochemistry 254 (1), 50 –57. Lewis, J.G., Hamilton, T.A., Adams, D.O., 1986. The effect of macrophage development on the release of reactive oxygen intermediates and lipid oxidation products and their ability to induce oxidative DNA damage. Carcinogenesis 7 (5), 813 – 818. Moon, E.Y., Rhee, D.K., Pyo, S., 1999. Inhibition of various functions in murine peritoneal macrophages by aflatoxin B1 exposure in vivo. International Journal of Immunopharmacology 21, 47 – 58. Morahan, P.S., 1984. Interactions of herpes viruses with mononuclear phagocytes. In: Rouse, B.T., Lopez, C. (Eds.), Immunobiology of Herpes Simplex Virus Infection. CRC Press, Boca Raton, FL. Morahan, P.S., Connor, J.R., Leary, K.R., 1985. Viruses and the versatile macrophage. British Medical Bulletin 410, 150 – 210. Nathan, C.F., Root, R.K., 1977. Hydrogen peroxide release from mouse peritoneal macrophages: dependence on sequential activation and triggering. Journal of Experimental Medicine 146, 1648 – 1662. Okimura, T., Ogawa, M., Yamauchi, T., 1986. Stress and immune responses. III. Effect of resistant stress on delayed type hypersensitivity (DTH) response, natural killer (NK) activity and phagocytosis in mice. Japanese Journal of Pharmacology 41, 229 – 235. Paulnock, D.M., Lambert, L.E., 1988. Identification and characterization of monoclonal antibodies specific for macrophages at intermediate stages in the tumoricidal activation pathway. Journal of Immunology 144, 765 – 773. Rees, R.C., Parry, H., 1992. Macrophages in tumor immunity. In: Lewis, C.E., McGee, J.O. (Eds.), The Macrophages. IRL Press, Oxford. Sone, S., Key, M.E., 1986. Antitumor and phagocytic activities of rat alveolar macrophage subpopulations separated on a discontinuous gradient of bovine serum albumin. Journal of Biological Response Modifier 5 (6), 595 – 603. Stuehr, D.J., Marletta, M.A., 1987. Synthesis of nitrite and nitrate in macrophage cell lines. Cancer Research 47, 5590 – 5594. Verstovsek, S., Maccubbin, D., Mihich, E., 1992. Tumoricidal activation of murine resident peritoneal macrophage by interleukin 2 and tumor necrosis factor. Cancer Research 52, 3880 – 3885. Journal of Ethnopharmacology 79 (2002) 95 – 100 www.elsevier.com/locate/jethpharm Evaluation of anti-hyperglycemic and hypoglycemic effect of Trigonella foenum-graecum Linn, Ocimum sanctum Linn and Pterocarpus marsupium Linn in normal and alloxanized diabetic rats V. Vats a, J.K. Grover a,*, S.S. Rathi b a Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110 049, India b St. Boniface Institute of Cardio6ascular Sciences, Winnipeg, Canada Received 18 January 2001; received in revised form 18 September 2001; accepted 18 October 2001 Abstract The hypoglycemic effect of the aqueous (Aq) extract of the bark of Pterocarpus marsupium (PM) and alcoholic (Alc) extract of seeds of Trigonella foenum-graecum (FG) and leaves of Ocimum sanctum (OS) was investigated in both normal and alloxan-induced diabetic rats. The Aq extract of PM (1 g/kg PO) significantly (P B0.001) reduced the blood sugar levels from 72.32 9 5.62 to 61.35 9 1.2 mg% 2 h after oral administration of the extract and also significantly lowered the blood glucose in alloxan diabetic rats from 202.91 95.44 to 85.22 9 11.28 mg% 21 days after daily oral administration of the extract (P B 0.001). Similarly, reduction was seen with Alc extract of FG (74.33 94.77 to 60.56 9 1.9 in normal rats and 201.25 9 7.69 to 121.25 9 6.25 in diabetic rats) (P B0.001) and OS (204.48 9 11.0 to 131.43 9 7.86 in normal rats and 73.54 93.7 to 61.44 9 2.3 in diabetic rats) (P B0.001). In addition, the extract also showed a favorable effect on glucose disposition in glucose fed hyperglycemic rats. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Trigonella foenum-graecum; Ocimum sanctum; Pterocarpus marsupium; Methi; Vijayasar; Tulsi; Experimental diabetes; Alloxan diabetic rats 1. Introduction Diabetes mellitus (DM) is a major cause of disability and hospitalization and it results in significant financial burden ($92 billion per year in U.S.) (Foster, 1994). By the year 2010, the total number of people worldwide with DM is projected to reach 239 millions. Regions with greatest potential are Asia and Africa, where DM rates could rise to 2 – 3-folds than the present rates (ADA, 1997). Currently available therapeutic options for non-insulin-dependent DM such as dietary modification, oral hypoglycemics and insulin have limitations of their own. Many herbal medicines have been recommended for the treatment of diabetes. Trigonella foenum-graecum Linn, Ocimum sanctum Linn and Pte* Corresponding author. Tel.: + 91-11-6594897 (O)/4615315 (R); fax: + 91-11-6862663; telex: 31-73042 AIMS IN. E-mail address: jkgrover@hotmail.com (J.K. Grover). rocarpus marsupium Linn have been shown to possess hypoglycemic activity in experimental animals (Joglekar et al., 1959; Dhar et al., 1968; Ahmad et al., 1991a; Khosla et al., 1995; Rai et al., 1997; Manickam et al., 1997). O. sanctum is found throughout India and is commonly called Tulsi in Hindi and Holy Basil in English. Experimental studies have demonstrated the hypoglycemic effect of the leaves of the plant (Dhar et al., 1968; Rai et al., 1997; Chattopadhyay, 1993). A survey conducted in human subjects also showed significant decrease in fasting and postprandial blood glucose levels on administration of holy basil leaves (Agrawal et al., 1996). Trigonella foenum-graecum (TG) is cultivated throughout India and its leaves are used as a vegetable while seeds are used as a spice. Experimental (Khosla et al., 1995) and clinical studies (Madar et al., 1988; Bordia et al., 1997) have demonstrated anti-hyper- 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 7 4 - 9 96 V. Vats et al. / Journal of Ethnopharmacology 79 (2002) 95–100 glycemic effect of the plant (Ravikumar and Anuradha, 1999). The clinical effectiveness of a herbal powder consisting of Guargum, Methi, Tundika and Meshasringi has also been documented in the literature (Bhardwaj et al., 1994). The defatted part of the plant is said to be responsible for the anti-diabetic action (Ribes et al., 1984, 1986). Pterocarpus marsupium (PM), commonly known called Vijaysar in Hindi, is found in hilly regions throughout India. Many pharmacological studies have been conducted to account for the ancient reputation of its anti-diabetic potential (Joglekar et al., 1959; Ahmad et al., 1991a; Manickam et al., 1997). Its watersoluble active principle, Epicatechin, has also been demonstrated in vitro (Ahmad et al., 1991b). In a clinical study, oral administration of 2, 3 and 4 g of the extract/day of Vijaysar has been shown to control fasting and postprandial glucose levels by 12 weeks in 73, 16 and 10%, respectively of the 67 out of 97 patients who responded to therapy (ICMR, 1998). Although, many previous workers have assessed the hypoglycemic and anti-hyperglycemic effect of FG, OS and PM in experimental animals, most of the studies except one (Chattopadhyay, 1993), were either of short duration (from hours to maximum of 5 days) or were done in either normal or diabetic animals but not together. In addition, the hypo/anti-hyperglycemic effect of plants is dose-dependent. Therefore, to work out a standard dose, a planned effort in the form of this study was undertaken to establish and assess the dose response relationship of aqueous (Aq) extract of the bark of PM and alcoholic (Alc) extract of seeds of FG and leaves of OS in three models: (1) normal rats; (2) glucose-fed hyperglycemic and (3) alloxanized diabetic rats. dryer, alpha 1-4, Germany), yielding 38/100 g of Aq extract. 2.2. Alc extract of TC and OS Alc extracts were prepared by powdering 1 kg seeds of TG and 1 kg air-dried leaves of OS in an electric grinder. The powder was then mixed with 500 ml of alcohol (Glaxo Chemicals Laboratories, Bombay) and kept at room temperature for 36 h. The slurry was stirred intermittently for 2 h and left overnight. The mixture was then filtered and the filtrate was freed from solvent under partial vacuum (71 mmHg) at 35– 45 °C to yield 123 and 165 g/kg of the pulp. A few drops of silicon emulsion were added near the end of distillation to avoid frothing. The final residue collected (yield was 96 and 102 g/kg of powdered TG and OS, respectively) was a thick paste, green in color and gummaceous in nature. 2.3. Animals Albino rats (150– 200 g) of both sexes were obtained from the experimental animal facility of the All India Institute of Medical Sciences. Before and during the experiment, rats were fed standard chow diet. After randomization into various groups and before initiation of experiment, the rats were acclimatized for a period of 2–3 days. Animals described as fasting were deprived of food for at least 16 h but were allowed free access to drinking water. 2.4. Sample collection Blood samples were collected by tail nipping and assessed for glucose on an electronic glucometer (Boheringer Mannheim). 2. Materials and methods 2.5. Experimental design 2.1. Preparation of Aq extract of PM The bark of PM was purchased from the local market in February 1999 and was authenticated by the Department of Botany, Miranda House, University of Delhi (India) (Voucher number 1134¯99). After grinding in an electric grinder, the powder was soaked in equal amount of water and stirred intermittently and then left overnight. The macerated pulp was then filtered through a coarse sieve and the filtrate was dried at reduced temperature. This dry mass (yield 62 g/kg of powdered bark) served as Aq extract of PM for experimentation. To increase the shelf life and uniformity, this extract was completely lyophilized by continuous freeze drying operation of 54 h (Christ freeze All the animals were randomly divided into the following 12 groups with six animals in each group. Group I (CNT) were normal and used as controls. Group II (DCNT) were used as diabetic control. Group III was the standard control (tolbutamide 250 mg/kg). Group IV– XII were treated with the test drugs. Group IV– VI received 250, 500 and 1000 mg of Aq extract of PM, group VII–IX received 100, 200 and 400 mg/kg of Alc extract of OS and group X–XII received 1, 2 and 4 g of Alc extracts of FG. Alloxan was administered in group III– XII. 2.5.1. Assessment of hypoglycemic response of extracts in normal rats After overnight fasting, the rats were given the refer- V. Vats et al. / Journal of Ethnopharmacology 79 (2002) 95–100 97 Table 1 The effect of various doses of aqueous extract of Pterocarpus marsupium and alcoholic extract of Trigonella foenumgraecum and Ocimum sanctum on normal rats Treatment groups Basal 30 m 60 m 120 m CNT TOL (250 mg/kg) PM (250 mg/kg) PM (500 mg/kg) PM (1 gm/kg) OS (100 mg/kg) OS (200 mg/kg) OS (400 mg/kg) FG (1 gm/kg) FG (2 gm/kg) FG (4 gm/kg) 75.5 9 3.63 76.90 9 4.2 73.21 9 4.59 76.21 9 2.3 72.32 9 5.62 73.25 9 2.71 76.59 9 4.25 73.54 9 3.70 75.5 9 4.55 73.25 9 1.90 74.33 9 4.77 73.56 9 3.22 58.459 1.3** (23.99) 70.43 9 1.2 (3.79) 69.33 9 2.65* (9.02) 66.559 1.9* (7.97) 69.45 9 2.1 (5.18) 71.43 9 1.23 (6.73) 66.459 2.55* (9.64) 78.55 9 2.61 (0) 74.20 9 4.12 (0) 69.67 9 1.33 (6.26) 75.21 9 3.12 54.34 9 1.31** (29.33) 73.33 9 3.6 (0) 66.55 9 2.11** (12.67) 62.23 9 1.5** (13.95) 65.43 9 2.56* (10.67) 60.33 9 3.11** (21.22) 58.34 9 2.87** (23.82) 65.33 9 2.1* (13.03) 67.70 9 2.8 (7.57) 61.23 9 1.65** (17.62) 72.43 9 4.05 56.45 92.11** (26.59) 69.44 91.23 (5.14) 67.21 9 3.1* (11.8) 61.35 91.2** (15.16) 67.65 92.3* (7.64) 63.43 91.3** (17.18) 61.44 92.3** (19.78) 63.96 93.77** (15.28) 61.23 92.1** (16.4) 60.56 91.90** (18.52) Values are given in mean 9 S.D. for groups of six animals each. Value in parenthesis indicates the percentage lowering of plasma sugar in comparison to the basal reading. All groups were compared with their basal values. CNT, control; TOL, standard control; Aq, aqueous extract; Alc, alcoholic extract. * Values are statistically significant at PB0.01. ** Values are statistically significant at PB0.001. ence drug (i.e. tolbutamide 250 mg/kg) and test extracts orally. The drugs were administered PO via a rubber tube attached to a syringe, the volume being not \ 1 ml/100 g bodyweight. Blood samples were collected before the administration of the glucose and at 30, 60 and 120 m later. 2.5.2. Assessment of extracts on glucose tolerance After overnight fasting, the rats were given the reference drug and test extracts orally and 30 min later, glucose (10 g/kg) was administered orally. Blood samples were collected before the administration of the glucose and at 30, 60 and 120 m after. 2.5.3. Assessment of extracts on alloxan-induced diabetic animals Rats were made diabetic by a single intravenous injection of alloxan monohydrate (Loba Chemie, Bombay (45 mg/kg) given in the tail vein. Alloxan was first weighed individually in Eppendorf’s tube for each animal according to the weight and then solubilized with 0.2 ml saline (154 mM NaCl) just prior to injection. Two days after alloxan injection, rats with plasma glucose levels \ 175 mg/dl were included in the study. Treatment with plant extracts was started 48 h after alloxan injection. Blood samples were drawn at weekly intervals till the end of study (i.e. 3 weeks). 3. Results 3.1. Effect on normal rats The effect of plant extracts on fasting blood sugar was assessed in normal male rats at various time intervals (i.e. 30, 60 and 120 m). The mean blood glucose levels are shown in Table 1 and comparative hypoglycemic effect of the maximally effective doses is given in Fig. 1. Among all plant extracts, the hypoglycemic effect was maximum with OS. Percentage reduction in glucose levels after 2 h in the PM (250, 500 and 1000 mg/kg) treated groups was 5.14, 11.8, 15.16%, respectively, in OS treated rats (100, 200 and 400 mg/kg) was 7.64, 17.18 and 19.78%, respectively and in FG treated group (1, 2 and 4 g/kg) was 15.28, 16.4 and 18.5%, respectively. Tolbutamide caused a significant (PB 0.001) reduction of 29.33% in glucose levels 2 h after its administration while control rats did not exhibit any significant alteration in their glucose levels through the 2.6. Statistical analysis The results were analyzed for statistical significance by one way ANOVA test using computerised software, MICROCAL ORIGIN version 2.9, Northampton, USA. Fig. 1. Comparative effect of aqueous extract of seeds Trigonella foenumgraecum Linn (Methi), leaf extract of Ocimum sanctum Linn (Tulsi), bark of Pterocarpus marsupium Linn (Vijaysar) on blood glucose level in normal rats. V. Vats et al. / Journal of Ethnopharmacology 79 (2002) 95–100 98 Table 2 The effect of various doses of aqueous extact of Pterocarpus marsupium and alcoholic extract of Trigonella foenumgraecum and Ocimum sanctum on glucose fed hyperglycemic rats Treatment groups Basal 30 m 60 m 120 m CNT TOL (250 mg/kg) PM (250 mg/kg) PM (500 mg/kg) PM (1 g/kg) OS (100 mg/kg) OS (200 mg/kg) OS (400 mg/kg) FG (1 g/kg) FG (2 g/kg) FG (4 g/kg) 73.21 9 1.63 74.80 9 1.2 75.45 9 4.99 72.16 9 3.25 75.45 9 3.66 70.15 9 4.36 75.35 9 2.43 77.52 9 3.36 71.25 9 3.90 75.19 9 2.33 73.89 9 4.11 205.51 9 5.2 151.23 9 4.35** 196.28 9 8.21 199.22 9 5.35 184.35 9 2.19** 207.80 9 3.99 191.43 9 5.25* 180.78 9 4.23** 203.43 9 5.21 211.29 9 6.09 205.37 9 4.27 143.11 9 6.63 (30.36) 111.79 9 2.53** (26.07) 151.21 9 4.1 (22.96) 139.76 9 6.32 (29.84) 126.15 9 3.45** (31.57) 138.32 9 5.78 (33.43) 110.23 9 4.20** (42.41) 92.57 9 1.70** (48.79) 135.67 9 3.31 (33.29) 120.45 9 5.78** (43.01) 110.39 9 4.34** (46.24) 105.37 9 6.51 (26.37) 76.46 9 4.32** (31.6) 97.57 9 6.85 (35.47) 90.61 9 4.55 (35.16) 88.43 9 2.90** (29.9) 97.57 9 3.16 (29.46) 93.43 9 4.50 (15.24) 87.44 9 2.19* (5.54) 97.76 9 3.07 (27.9) 100.23 9 4.01 (16.78) 95.45 9 3.23 (13.53) Values are given in mean 9 S.D. for groups of six animals each. Value in parentheses indicate the percentage lowering of plasma sugar in comparison to the previous reading. Experimental groups were statistically compared with the corresponding values of the controls. CNT, control; TOL, standard control; Aq, aqueous extract; Alc, alcoholic extract. * Values are statistically significant at PB0.01. ** Values are statistically significant at PB0.001. duration of the experiment. The hypoglycemic effect of all the plant extracts was less than that seen in the tolbutamide treated group. 3.2. Effect on glucose tolerance Results of the glucose tolerance test conducted on normal rats fed with various plant extracts are showed in Table 2 and comparative effect of the maximum effective doses on OGTT is shown in Fig. 2. Half an hour after feeding glucose, the blood sugar rose by 180.71% in normal controls while the same rise was only 102.89% in rats receiving tolbutamide. Administration of PM produced a maximum dose dependent reduction in blood sugar only at 120 m in comparison to normal controls (7.4, 14 and 16% in rats receiving 250, 500 and 1000 mg/kg of Aq extracts of PM). The effect was less pronounced at 30 and 60 m indicating a late onset of effect. On the other hand, administration of OS extract produced a dose dependent reduction in blood sugar at 60 and 120 m. The respective percentage reduction was 3.3, 23.05 and 35.31 at 60 m and 7.4, 11.3 and 17.01 at 120 m in rats receiving 100, 200 and 400 mg/kg of Alc extracts of OS. FG extracts had no significant effect at 30 m on glucose loading but the levels were significantly lowered at 60 m. Percentage reduction in blood glucose levels in comparison to normal controls was 5.19, 15.8 and 22.8 at 60 m while it was only 7.27, 4.87 and 9.41% at 120 m in rats receiving 1, 2 and 4 g/kg of Alc extracts of FG. The percentage fall in tolbutamide treated rats as compared to normal controls at 30, 60 and 120 m was 26.4, 21.8 and 27.4, respectively. 3.3. Effect on alloxan-induced diabetic rats The anti-hyperglycemic effect of the extracts on the fasting blood sugar levels of diabetic rats is shown in Table 3. Administration of alloxan (45 mg/kg) led to over 2.5-folds elevation of blood glucose levels (P B 0.001) which was maintained over a period of 3 weeks. After 3 weeks of daily treatment with 250, 500 and 1000 mg/kg Aq extract of PM led to a dose-dependent fall in blood sugar levels by 26, 47.8 and 58%, respectively. Similarly, in OS treated rats (100, 200 and 400 mg/kg), the respective fall in blood glucose levels was 11.35, 26.04, 35.72% while that in FG treated group (1, 2 and 4 g/kg) was 8.7, 36.5 and 39.75%, respectively. Comparative results of maximal effective doses are shown in Fig. 3. 4. Discussion and conclusion Many previous workers have assessed the hypoglycemic and anti-hyperglycemic effect of FG, OS and Fig. 2. Comparative effect of aqueous extract of seeds Trigonella foenumgraecum Linn (Methi), leaf extract of Ocimum sanctum Linn (Tulsi), bark of Pterocarpus marsupium Linn (Vijaysar) on blood glucose level in normal rats. V. Vats et al. / Journal of Ethnopharmacology 79 (2002) 95–100 99 Table 3 The effect of 3-week treatment with various doses of aqueous extract of Pterocarpus marsupium and alcoholic extract of Trigonella foenum-graecum and Ocimum sanctum on glucose levels (mg/%) in alloxan (45/mg/kg) diabetic rats Treatment groups Basal 2 days 7 days 14 days 21 days CNT DCT PM (250 mg/kg) PM (500 mg/kg) PM (1 g/kg) OS (100 mg/kg) OS (200 mg/kg) OS (400 mg/kg) FG (1 g/kg) FG (2 g/kg) FG (4 g/kg) 74.52 9 3.62 74.68 9 4.07 76.17 9 4.86 76.31 9 2.35 74.769 3.52 75.11 9 4.4 74.75 9 3.61 75.97 9 3.06 73.52 9 3.58 77.5 9 6.21 75.2 9 4.09 74.86 9 2.59 209.16 9 7.23** 201.58 9 11.78 200.45 9 9.34 202.919 5.44 203.01 9 7.70 198.839 5.6 204.48 9 11.00 208.51 9 9.83 205.3 9 7.33 201.25 9 7.69 74.38 9 3.8 204.61 9 4.24** 185.43 9 7.09 (8.01) 142.10 9 9.34 (29.1)** 129.23 9 8.37 (36.31)** 191.67 9 7.96 (5.58) 166.95 9 9.46 (16.03)** 142.78 9 7.82 (30.17)** 189.93 9 9.94 (8.91) 152.69 9 6.78 (25.62)** 124.83 9 6.56 (37.97)** 73.65 9 3.47 201.63 9 5.41** 176.41 9 12.94 (12.48)* 126.96 9 9.95 (36.66)** 102.31 9 7.5 (49.57)** 185.08 9 9.41 (8.83)* 154.36 9 7.90 (22.36)** 136.4 9 6.55 (33.29)** 193.93 9 8.07 (6.99) 133.63 9 6.57 (34.9)** 119.13 9 7.90 (40.8)** 73.931 93.97 202.43 9 4.00** 148.41 9 12.9 (26.37)** 104.52 9 9.25 (47.85)** 85.22 9 11.28 (58)** 179.95 9 11.8 (11.35)* 147.05 910.80 (26.04)** 131.43 97.86 (35.72)** 190.35 97.54 (8.7) 130.35 98.92 (36.5)** 121.25 96.25 (39.73)** Values are given in mean 9 S.D. for groups of six animals each. Value in paranthesis indicates the percentage lowering of plasma sugar in comparison to the reading at 48 h. Diabetic control was compared with the normal and experimental groups were compared with the corresponding values at 48 h. CNT, non-diabetic control; DCT, diabetic control; Aq, aqueous extract; Alc, alcoholic extract. * Values are statistically significant at PB0.01. ** Values are statistically significant at PB0.001. PM in experimental animals. However, most of the studies except one (Chattopadhyay, 1993), were either of short duration (from hours to maximum of 5 days) or done in either normal or diabetic animals but not together. Since diabetes is a chronic disorder requiring long-term therapy, there is a need to assess the effect of putative hypoglycemic/anti-hyperglycemic agents for a longer duration. In addition, if plant extracts have a late onset of activity, their effect is likely to be missed in such screening studies. The present study was therefore planned to assess the effect of test drugs for a period of 3 weeks and in three states i.e. normoglycemia, hyperglycemia of acute nature (OGTT) and chronic hyperglycemia (alloxan induced diabetes). Overall, the results revealed a well defined and in some cases, a dose-dependent role of the Aq extracts of PM and Alc extract of OM and FG in suppressing blood glucose levels in normal, glucose fed hyperglycemic and alloxanized diabetic rats as compared to normal and standardized controls. In the present study, all the three plant extracts exhibited a small but significant hypoglycemic effect in normal rats and this was evident 1 h after the administration of the plant extracts. On the other hand, tolbutamide caused a significantly more hypoglycemia in comparison to plant extracts. An emphasis is laid on glucose homeostasis as severe hypoglycemia can result in life threatening situation. Therefore, lesser hypoglycemia with plant products in comparison to tolbutamide is a desirable feature. Moreover, carbohydrate metabolism in normal animals being in homeostasis, these plant products caused less suppression of normal regulatory mechanisms involved in carbohydrate metabolism as compared to tolbutamide. The maximal hypoglycemic effect seen with OS reported in the present study is less in comparison to that reported by Shah (1967) and this could perhaps be due to the species and dose difference of the extract in the two experiments. The maximal hypoglycemic effect (18.52% at 2 h) seen with FG in the present case was similar to that seen in the study of Khosla et al., 1995. FG extract failed to reduce the peak glucose levels while OS and PM extract fed animals showed reduction in peak glucose levels after glucose administration. Blood glucose remained significantly higher than the basal values in normal controls. Euglycemia was achieved in tolbutamide treated rats after 120 m of glucose loading. This finding is coherent with the findings of Chattopadhyay, 1993. The results of the present study indicate a delayed onset of action of the three plant extracts under study as compared to tolbutamide. In this study, in alloxanized rats, Aq extract of PM produced a significant and clear dose-dependent antihyperglycemic effect beginning from the 1st week and progressing till the end of 3rd week. Ahmad et al. (1991a) had studied the effect of ethyl acetate soluble fraction of the ethanol extract of PM (250 mg/kg) in Fig. 3. Comparative effect of aqueous extract of seeds Trigonella foenumgraecum Linn (Methi), leaf extract of Ocimum sanctum Linn (Tulsi), bark of Pterocarpus marsupium Linn (Vijaysar) on blood glucose level in alloxan induced diabetes in rats. 100 V. Vats et al. / Journal of Ethnopharmacology 79 (2002) 95–100 such rats (50 mg/kg) and reported a 70% reduction in blood glucose levels by the 5th day. In the present case, the reduction on the 7th day was only 36.31%. However, feeding of this extract for 3 weeks produced euglycemia. Thus, the Aq extract as compared to ethyl acetate soluble fraction of the ethanol extract has a delayed onset of action but is more efficacious. The anti-hyperglycemic effect with OS and FG plateaued at the end of the second week and was less pronounced than PM. Euglycemia was not achieved. The percentage fall in glucose with comparative dose of OS (i.e. 200 mg/kg) is same as reported in acute experiments by Chattopadhyay, 1993. Although this study was not aimed at elucidating the mechanism of action, various theories have been put foward by previous workers who have shown that PM works by regeneration of beta cells (Manickam et al., 1997; Ahmad et al., 1991b; Chakravarthy et al., 1980) and increase proinsulin biosynthesis. FG on the other hand has been shown to work like guargum (Valette et al., 1984), inhibition of intestinal glucosidase (Riyad et al., 1988) and insulin release. However, since the peak glucose levels in glucose-fed hyperglycemia rats did not decrease in the FG treated rats, it implies that FG did not affect glucose absorption from the gut while OS and PM may be acting through this mechanism. References American Diabetes Association, 1997. Clinical practice recommendations. Diabetes Care 20 (Suppl. 1), S1 –S70. Agrawal, P., Rai, V., Singh, R.B., 1996. Randomized placebo-controlled, single blind trial of holy basil leaves in patients with noninsulin-dependent diabetes mellitus. International Journal of Clinical Pharmacology and Therapeutics 34 (9), 406 –409. Ahmad, F., Khalid, P., Khan, M.M., Chaubey, M., Rastogi, A.K., Kidwai, J.R., 1991a. Hypoglycemic activity of Pterocarpus marsupium wood. Journal of Ethnopharmacology 35 (1), 71 – 75. Ahmad, F., Khan, M.M., Rastogi, A.K., Chaubey, M., Kidwai, J.R., 1991b. Effect of ( − )epicatechin on cAMP content, insulin release and conversion of proinsulin to insulin in immature and mature rat islets in vitro. Indian Journal of Experimental Biology 29 (6), 516 – 520. Bhardwaj, P.K., Dasgupta, D.J., Prashar, B.S., Kaushal, S.S., 1994. Control of hyperglycemia and hyperlipidemia by plant product. Journal of Associated Physicians India 42 (1), 33 – 35. Bordia, A., Verma, S.K., Srivastava, K.C., 1997. Effect of ginger (Zingiber officinale Rosc.) and fenugreek (Trigonella foenumgraecum L.) on blood lipids, blood sugar and platelet aggregation in patients with coronary artery disease. Prostaglandins Leukotrienes Essential Fatty Acids 56 (5), 379 – 384. Chakravarthy, B.K., Gupta, S., Gambhir, S.S., Gode, K.D., 1980. Pancreatic beta cell regeneration — a novel anti-diabetic mechanism of Pterocarpus marsupium Roxb. Indian Journal of Pharmacology 12, 123 – 129. Chattopadhyay, R.R., 1993. Hypoglycemic effect of Ocimum sanctum leaf extract in normal and streptozotocin diabetic rats. Indian Journal of Experimental Biology 31 (11), 891 – 893. Dhar, M.L., Dhar, M.M., Dhawan, B.N., Mehrotra, B.U., Ray, C., 1968. Screening of Indian plants for biological activity. Part I. Indian Journal of Experimental Biology 6 (4), 232 – 247. Foster, D.W., 1994. Diabetes mellitus. In: Isselbacher, K.J., Brawnwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S., Kasper, D.L. (Eds.), Harrison’s Principles of Internal Medicine. McGraw Hill, United States, pp. 1979 – 1981. Indian Council of Medical Research (ICMR) Collaborating Centres, New Delhi, 1998. Flexible dose open trial of Vijayasar in cases of newly-diagnosed non-insulin-dependent diabetes mellitus. Indian Journal of Medical Research (108), 24 – 29. Joglekar, G.V., Chaudhary, N.Y., Aiman, R., 1959. Effect of indigenous plant extracts on glucose-absorption in mice. Indian Journal of Physiology and Pharmacology (3), 76. Khosla, P., Gupta, D.D., Nagpal, R.K., 1995. Effect of Trigonella foenum graecum (Fenugreek) on blood glucose in normal and diabetic rats. Indian Journal of Physiology and Pharmacology 39 (2), 173 – 174. Madar, Z., Abel, R., Samish, S., Arad, J., 1988. Glucose-lowering effect of fenugreek in non-insulin dependent diabetics. European Journal of Clinical Nutrition 42 (1), 51 – 54. Manickam, M., Ramanathan, M., Jahromi, M.A., Chansouria, J.P, Ray, A.B., 1997. Antihyperglycemic activity of phenolics from Pterocarpus marsupium. Journal of Natural Products 60 (6), 609 – 610. Rai, V., Iyer, U., Mani, U.V., 1997. Effect of Tulasi (Ocimum sanctum) leaf powder supplementation on blood sugar levels, serum lipids and tissue lipids in diabetic rats. Plant Foods Human Nutrition 50 (1), 9 – 16. Ravikumar, P., Anuradha, C.V., 1999. Effect of fenugreek seeds on blood lipid peroxidation and antioxidants in diabetic rats. Phytotherapy Research 13 (3), 197 – 201. Ribes, G., Sauvaire, Y., Baccou, J.C., Valette, G., Chenon, D., Trimble, E.R., Loubatieres-Mariani, M.M., 1984. Effects of fenugreek seeds on endocrine pancreatic secretions in dogs. Annals of Nutrition and Metabolism 28 (1), 37 – 43. Ribes, G., Sauvaire, Y., Da Costa, C., Baccou, J.C., LoubatieresMariani, M.M., 1986. Antidiabetic effects of subtractions from fenugreek seeds in diabetic dogs. Proceedings of the Society of Experimental Biology and Medicine 182 (2), 159 –166. Riyad, M.A., Abdul-Salam, S.A., Mohammad, S.S., 1988. Effect of fenugreek and lupine seeds on the development of experimental diabetes in rats. Planta Medica 54 (4), 286 – 290. Shah, D.S., 1967. A preliminary study of the hypoglycemic action of heartwood of Pterocarpus marsupium roxb. Indian Journal of Medical Research 55 (2), 166 – 168. Valette, G., Sauvaire, Y., Beccou, J.C., Ribes, G., 1984. Hypocholesterolaemic effect of Fenugreek seeds in dogs. Athersclerosis 50, 105 –111. Journal of Ethnopharmacology 79 (2002) 101– 107 www.elsevier.com/locate/jethpharm The pharmacological screening of Pentanisia prunelloides and the isolation of the antibacterial compound palmitic acid Barbara T.S. Yff a, Kerry L. Lindsey a, Maureen B. Taylor b, Doreen G. Erasmus b, Anna K. Jäger a,* a Research Centre for Plant Growth and De6elopment, School of Botany and Zoology, Uni6ersity of Natal Pietermaritzburg, Pri6ate Bag X01, Scotts6ille 3209, South Africa b Department of Medical Virology, Institute of Pathology, Uni6ersity of Pretoria, PO Box 2034 Pretoria, South Africa Received 22 April 2001; received in revised form 24 September 2001; accepted 18 October 2001 Abstract The uses of Pentanisia prunelloides in Zulu traditional medicine indicate that the plant is believed to be effective in relieving inflammation, bacterial and viral infections and also stimulating uterine contraction. Aqueous, ethanolic and ethyl acetate extracts of leaves and roots were screened for prostaglandin-synthesis inhibitors and antibacterial and antiviral activity. In the results of the anti-inflammatory assay all the extracts showed cyclooxygenase-1 inhibition. The ethanolic and ethyl acetate extracts showed greater antibacterial activity than the aqueous extracts against Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae). Both root and leaf extracts were found to inhibit viral replication of the Influenza A virus. The ethyl acetate extract was fractionated by silica vacuum liquid chromatography and anti-inflammatory activity was found to be most pronounced in the more polar fractions. The presence of antibacterial activity was confirmed by running the fractions on a thin layer chromatography (TLC) plate and performing a bioautographic assay. The active fraction was further purified by TLC and the major antibacterial compound in the ethyl acetate root extract was identified by GC/MS as palmitic acid. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antibacterial; Cyclooxygenase inhibition; Influenza A virus; Palmitic acid; Pentanisia prunelloides; Traditional medicine 1. Introduction Medicinal plants are an important part of the South African cultural heritage and Africa is a continent endowed with an enormous wealth of plant resources (Iwu, 1993). Plants produce a diverse range of bio-active molecules, making them a rich source of different types of medicines. In South Africa, a large part of the day to day medicine is still derived from plants and large volumes of plant material, or their extracts, are sold in the informal and commercial sectors of the economy (van Wyk et al., 1997). The imposing anthropogenic activities that come with economic growth, such as agricultural, industrial * Corresponding author. Tel.: + 27-33-2605130; fax: + 27-332605897. E-mail address: jager@nu.ac.za (A.K. Jäger). and housing developments, are forming an increasing threat to the natural habitats of these medicinal plants. Increased commercialization of medicinal plants in South Africa has resulted in over-harvesting and in some cases near extinction of some valued indigenous plant species (Williams et al., 2000). Documentation of the uses of these plants is therefore of great importance before they vanish. Through training and scientific evaluation of effective remedies it may be possible to bring traditional medicine to a level of efficiency and safety where it can be regarded as an acceptable alternative to the western health system. Pentanisia prunelloides is a perennial herb with a large, thick tuberous root and leafy branches holding dense groups of small purple flowers. In preparation of traditional medicines mainly the root is used, but sometimes also the leaves (van Wyk et al., 1997). The plant has many uses in Zulu medicine, but most commonly root decoctions are used for swellings, sore joints and 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 8 0 - 4 102 B.T.S. Yff et al. / Journal of Ethnopharmacology 79 (2002) 101–107 rheumatism. It has also been reported to be used to relieve pain in the chest and to treat virus infections such as influenza. In the 1918 influenza epidemic a root decoction was used by the Zulu people with marked success (Watt and Breyer-Brandwijk, 1962). A leaf poultice is said to be used for a retained placenta (Watt and Breyer-Brandwijk, 1962; Hutchings, 1996). Previously it has been shown that P. prunelloides exhibited smooth muscle activity on ileum and uterus preparations (Kaido et al., 1997; Lindsey et al., 1999). Nothing appears to be known about the chemical compounds of the Pentanisia genus. Looking at the uses in Zulu medicine, the plant seems to be effective in relieving inflammation, bacterial and viral infections and also stimulating uterine contraction. The aims of this study were to screen extracts of P. prunelloides for anti-inflammatory, antibacterial and antiviral activity and then to try and isolate the active antimicrobiological compounds which would be responsible. 2. Methodology 2.1. Plant material Leaf and root material of P. prunelloides Walp. (Rubiaceae) was collected on 16 October 2000 from the slopes at World’s View, Pietermaritzburg by Yff, Jäger and Gardner. The plant was botanically verified by Gardner. A voucher specimen was prepared (Yff1NU) and deposited in the Natal University herbarium. The material was dried in an oven at 50 °C. Once dry, the material was ground. 2.2. Extractions Two methods were used for the extraction of plant material: sonication and Soxhlet. Sonication: one gram of the plant material and 10 ml of solvent (water, ethanol or ethyl acetate) was placed in an ultrasound bath for 30 min where after the solvent was filtered through a Whatmann No. 1 filter paper and taken to dryness in front of a fan. Soxhlet: one gram of root material was extracted with 150 ml of solvent (ethanol or ethyl acetate) for 2 h. The extracts were taken to dryness under vacuum. 2.3. The cyclooxygenase-1 assay for anti-inflammatory acti6ity The bioassay was performed according to Jäger et al. (1996). The six extracts from the sonication method (redissolved to 10 mg/ml) were tested. The aqueous extracts were redissolved in water and the others in ethanol. The solvent test solutions were made up: 20 ml of aqueous samples; 2.5 ml of ethanolic samples+ 17.5 ml water; 2.5 ml of a 8×10 − 4 ethanolic indomethacin (Sigma Chemical Co., MO) solution as standard. The enzyme stock solution (10 ml of sheep seminal vesical microsomes containing 0.3 mg protein) was added to co-factor solution (L-adrenaline and reduced glutathione, 3 mg of each suspended in 10 ml 0.1 M Tris buffer, pH 8.2) in the ratio 1:5 and pre-incubated on ice for 15 min. After adding 10 ml 2N HCl to the background samples to inactivate the enzyme, 60 ml of enzyme/co-factor solution were added to every sample. A 5 min incubation was allowed at room temperature and then 20 ml of [14C] arachidonic acid (16 Ci/mole, 30 mM) were added to each sample, followed by incubation in a water bath at 37 °C for 8 min. The reaction was then terminated by adding 10 ml 2N HCl (not to the backgrounds) and the samples were kept in an ice bath. Column chromatography was then used to separate the [14C] labelled prostaglandins synthesized during the assay from the unmetabolized arachidonic acid. Pasteur pipettes blocked with glass wool were packed with silica gel in eluent 1 (hexane:dioxane:acetic acid 350:150:1) to a height of 3 cm. One millilitre of eluent 1 and 4 ml of 0.2 mg/ml prostaglandin carrier solution (PGE2:PGF2 1:1) were added to each sample and they were loaded onto the column. Four millilitres of eluent 1 further eluted the arachidonic acid and this was discarded. Three millilitres of eluent 2 (ethyl acetate:methanol 425:75) was applied to each column to elute the labelled prostaglandins and this was collected in scintillation vials. Four millilitres of scintillation cocktail (Beckman Ready Solve) were added to the eluent and after 30 min the radioactivity of the samples was counted using a Beckman LS6000LL scintillation counter. Analysing the amount of radioactivity present relative to that in the solvent blanks indicated the percentage of cyclooxygenase inhibition of the test samples. All experiments were carried out in duplicate. 2.4. The microplate antibacterial method This assay was used to determine the minimum lethal concentration (MLC) using a serial dilution technique (Eloff, 1998). The bacterial cultures used were Bacillus subtilis (ATCC 6051), Escherichia coli (ATCC 11775), Klebsiella pneumoniae (ATCC 13883) and Staphylococcus aureus (ATCC 12600). These were maintained on Mueller-Hinton (MH) agar at 4 °C and overnight cultures were prepared in MH broth the day prior to the assay. The assay was performed under sterile conditions on a laminar flow bench. The plant extracts were dissolved to 50 mg/ml, the aqueous extracts in water and the ethanolic and ethyl acetate extracts in ethanol. The overnight bacterial B.T.S. Yff et al. / Journal of Ethnopharmacology 79 (2002) 101–107 cultures were diluted 1:100 in MH broth. Ninety-sixwell microplates were used and 100 ml sterile H2O was added to each well.For each extract a two-fold serial dilution was made down the microplate, starting with a concentration of 25 mg/ml in the first well. Each extract was tested against all the bacterial cultures and standards and controls were made: wells containing MH broth only; each type of bacteria but with no extract; and also a serial dilution of neomycin (Sigma Chemical Co., MO) with each type of bacteria at recommended inhibitory concentrations. The microplates were covered with parafilm and incubated at 37 °C overnight to stimulate the bacterial growth. The following day 40 ml of 0.2 mg/ml INT was added to every well and another incubation period of 10– 30 min at 37 °C was allowed. INT is reduced to a red coloured product by biologically active organisms, so bacterial growth in the well was indicated by a pink/red colour. A clear well indicated inhibition of growth by the plant extract. The MLC is the lowest concentration at which bacterial growth is eliminated and this could be seen in the last clear well in the dilution series. 103 Kempton Park, South Africa) and containing 100 U/ ml penicillin and 100 mg/ml streptomycin was used for the propagation of the cells. Cell cultures were incubated at 37 °C in a humidified CO2 atmosphere. Maintenance medium was essentially the same as the propagation medium except that it contained only 2% FCS. 2.7. Cytotoxicity assay Plant extracts were tested for cytotoxicity by exposing monolayers of secondary (VK cells to dilutions of the filter-sterilised plant extracts. Doubling dilutions of the plant extracts, from a concentration of 3.9 to 1000 mg/ml, in serum-free MEM were used for testing on 24-h old monolayers of VK cells. The cells were monitored visually by light microscopy over a period of 7 days and on the seventh day tested for cytotoxicity using a tetrazolium salt reduction (MTT) assay (van Rensburg et al., 1994) based on the method of Hussain et al. (1993). Monolayers of cells exposed to serum-free MEM alone were used as a control. 2.8. Virus stock 2.5. Bioautographic assay An overnight culture of S. aureus was grown in MH broth in a water bath at 35 °C. This was then centrifuged at 3000g for 10 min and the supernatant decanted. The pellet was resuspended in 10 ml of MH broth and this was sprayed onto the thin layer chromatography (TLC) plate. The plate was then placed on damp tissue in a metal tray covered with plastic to allow 100% humidity. The tray was left in the oven at 35 °C overnight to allow the bacteria to grow. The following day a 2 mg/ml INT solution was sprayed onto the bacteria covered plate which was then placed back in the oven in 100% humidity for about half an hour. The INT stained the bacteria dark red so that it was clearly visible where bacterial growth had been inhibited. The bands that remained in a clear zone possessed compounds with antibacterial activity. Influenza A 6irus (Inf A) : Freshly harvested allantoic fluid containing Inf A (strain Panama) was kindly supplied by the National Institute for Virology. A stock suspension, with a titre of 3.16× 105 TCID50/ml, was prepared by diluting the allantoic fluid in sterile phosphate buffered saline (PBS)(Sigma Chemical Company, St. Louis, MO) containing penicillin (50 mg/ml), streptomycin (50 mg/ml) and neomycin (100 mg/ml) (PSN Antibiotic Mixture [100X], GibcoBRL Life Technologies, Paisley, Scotland). This stock suspension is reportedly stable for 1 week at 4 °C (Barrett and Inglis, 1985). For experimental purposes fresh dilutions of the virus, in serum-free MEM, were prepared immediately before use. The 50% tissue culture infectious dose (TCID50) of the virus was calculated according to the Kärber formula as outlined in Grist et al. (1979). The virus was used at a final concentration of 1000 TCID50/assay well. 2.9. Anti6iral assays 2.6. Cell cultures Standard cell culture techniques (Grist et al., 1979), were used for all procedures utilizing cell cultures. Monolayers of secondary vervet monkey kidney (VK) cells (National Institute for Virology, Sandringham, South Africa), between passages four to six, were used. Eagle’s minimum essential medium (MEM)(National Institute for Virology) supplemented with 8% heat inactivated foetal calf serum (FCS) (Delta Bioproducts, Antiviral assays were only carried out at concentrations of the leaf and root extracts that were not cytotoxic for the cell cultures. 2.9.1. Effect on 6iral replication Monolayers of 24-h old VK cells in 16-well Lab-tek7 glass tissue culture chamber slides (Nalge Nunc Int., Naperville, IL) were rinsed with serum-free MEM. Thereafter the serum-free MEM was withdrawn and 104 B.T.S. Yff et al. / Journal of Ethnopharmacology 79 (2002) 101–107 1000 TCID50 of Inf A was added to each well to adsorb to the cell cultures for 1 h at 37 °C in a humidified CO2 atmosphere. Cells were rinsed to remove free/unadsorbed virus and 200 ml of dilutions of the plant extract in serum-free MEM were added to the wells which were further incubated at 37 °C in a humidified CO2 atmosphere for 36 h. Viral infection was monitored by direct immunofluorescence (IF) for the detection of viral antigen. Direct IF was carried out using standard techniques with a mouse anti-influenza A fluorescein isothiocyanate (FITC)-labelled monoclonal antibody (Chemicon International Inc., Temecula, CA) as the detector. The reduction in the number of fluorescent foci indicated antiviral activity in the plant extract. The percentage reduction in fluorescent foci was calculated in relation to controls. 2.9.2. Effect on 6iral adsorption and subsequent replication The cell cultures were prepared as before and the appropriate dilution of the plant extract and 1000 TCID50 of the virus were added simultaneously to the wells. Incubation at 37 °C in a humidified CO2 atmosphere and viral infection monitored by direct IF as described previously. 2.9.3. In 6itro 6iral inacti6ation Of each dilution of the plant extracts 500 ml were mixed with 500 ml of Influenza A suspension and incubated for 1 h at 37 °C, after which the virus was recovered from the mixture by ultrafiltration using a Centricon®-100 concentrator (Amicon Inc., Beverly, MA). The volume of the retentate was brought up to 500 ml and the cell cultures were inoculated with 100 ml of the retentate. Viral infection was monitored 36 h post infection by direct IF. 2.10. Isolation of antibacterial compound For the bulk sonication extract, 500 g of root material was placed into a conical flask with enough ethyl acetate to completely cover it. This was sonicated for 30 min and then left to stand for half a day. Once all the root material had settled at the bottom of the flask, the top layer of EtOAc was filtered through a Whatmann No. 1 filter. This process was repeated eight times after which the collected extract was taken to dryness under a vacuum. 2.11. Vacuum liquid chromatography The extract was fractionated on a large silica vacuum liquid chromatography (VLC) column and eluted with a hexane: ethyl acetate gradient, starting at 100% hexane, decreasing to 85% and then further decreasing in steps of 5% per fraction to 60%. Thereafter, the percentage hexane was decreased by 10% in each fraction. Four hundred millilitre of solvent mixture was used. Each fraction was taken to dryness under vacuum and the yield determined. Aliquots of all the fractions were loaded onto TLC plates. The plates were developed in hexane:ethyl acetate 3:1. One plate was then stained with anisaldehyde (465 ml ethanol, 5 ml glacial acetic acid, 13 ml concentrated sulphuric acid, 13 ml para-anisaldehyde) and heated in an oven at 110 °C for about 15 min, and the other was used in a bioautographic assay. The fraction which showed the highest antibacterial activity was loaded onto a glass TLC plate (0.25 mm thick) which was run in the same solvent system as before. The band which was responsible for the antibacterial activity was scraped off and eluted with EtOAc, after which the sample was then filtered through a syringe filter (0.45 mm PP). Chemical structures were determined from GC-MS data. Table 1 Minimum lethal concentration, in mg/ml, of extracts of P. prunelloides Extract Leaf Root Neomycin standards (mg/ml) H2O EtOH EtOAc H2O EtOH (Sonication) EtOH (Soxhlet) EtOAc (Sonication) EtOAc (Soxhlet) Strain of bacteria Bs Sa Ec Kp 12.5 0.78 1.56 12.5 0.78 0.78 1.56 0.78 6.25 0.2 3.13 12.5 1.56 0.39 1.56 0.78 6.25 1.56 0.78 12.5 1.56 3.13 0.78 0.78 3.13 1.56 0.78 12.5 1.56 1.56 1.56 0.39 0.39 3.13 1.56 1.56 Sa: Staphylococcus aureus; Ec: Escherichia coli; Kp: Klebsiella pneumoniae; Bs: Bacillus subtilis. B.T.S. Yff et al. / Journal of Ethnopharmacology 79 (2002) 101–107 Table 2 Percentage inhibition of cyclooxygenase by extracts of P. prunelloides Extract Leaf Roots H2O EtOH EtOAc (Sonication) EtOAc (Soxhlet) 74 88 72 – 74 65 87 81 105 an Rf value of 0.55. This compound stained green with anisaldehyde. The most pronounced inhibition of bacterial growth was seen in fraction G which was eluted with 60:40 hexane:ethyl acetate. Separation of fraction G (29 mg) on a glass TLC plate enabled the band responsible for the activity to be scraped off, yielding 7 mg, and the compound to be identified by GC-MS. The main component was found to be palmitic acid MS m/z (rel.int.): 256(24)[M]+, 213(22), 185(16), 157(20), 129(52), 97(26), 73(100), 56(14). Palmitic acid was reported to be the major compound in a mixture of fatty acids from Diplotaxis harra and Ericaria microcarpa (Hashem and Saleh, 1999). These authors reported antibacterial activity against gram-positive and gramnegative bacteria comparable to ampicillin and in the case of Streptococci, greater than that of ampicillin (Hashem and Saleh, 1999). The results of the screening of extracts for prostaglandin synthesis inhibitors are given in Table 2. All the extracts showed a high percentage of inhibition and there was no marked difference between the percentage inhibition caused by sonication extracts and that by the Soxhlet extract. This implies that the compound(s) responsible for the activity is heat resistant. Additionally there was no great difference between the anti-inflammatory activity caused by the root and leaf material. These results show that all the extracts of P. prunelloides were effective in reducing prostaglandin synthesis during inflammation by inhibiting the enzyme cyclooxygenase-1 (COX-1). A COX-1 assay was carried out with the fractions from the VLC at a concentration of 0.1 mg/ml. The highest activity was found in the more polar fractions. Fractions L and M were combined and run on a glass Indomethacin standard (20 mM) 83%. 3. Results and discussion Table 1 shows the results of the antibacterial screening. Aqueous extracts appear to have less antibacterial activity than ethanolic and ethyl acetate extracts. This is interesting in that the traditional method of treating a bacterial infection would be by giving a root decoction prepared by boiling the root in water, where according to these results an ethanolic solvent would probably be more effective. Soxhlet extracts with EtOH and EtOAc showed comparable activity to sonication extracts with the same solvents. This implies that the compound(s) responsible for antibacterial activity is heat resistant. In order to isolate the antibacterial compound a bulk extraction of roots was carried out. The total dry mass of the bulk extracts was 2.985 g. When this was put through a VLC column thirteen fractions were collected (labelled A-M as the hexane percentage in the solvent mixture decreased from 100 to 0). The antibacterial activity in fractions E to H appeared to be caused by the same compound, which seemed to be the major compound in the extract having Table 3 Results of the three antiviral assays showing Influenza A inhibition as a percentage reduction in the number of fluorescent foci on the infected VK cell cultures in relation to controls Concentration of extract (mg/ml) Percentage reduction of fluorescent foci on VK cell culture (%) Assay 1 Virus control 125 62.5 31.25 15.62 7.81 3.9 1.95 0.98 Assay 2 Assay 3 Root Leaf Root Leaf Root Leaf 0 45.4 29.5 32.9 23.6 34.3 16.2 0 0 24.2 46.5 44.9 39.9 26.8 39.9 0 0 96.2 88.7 80.3 70.4 64.4 46.0 0 40.5 28.4 35.7 21.8 19.4 27.0 39.4 39.0 37.7 18.0 83.3 79.5 29.5 21.1 0 Assay 1 : Inhibition of replication of 1000 TCID50 Influenza A after viral adsorption and incubation of the infected culture in different concentrations of aqueous extract. Assay 2 : Inhibition of adsorption and replication of 1000 TCID50 Influenza A after viral adsorption and incubation of the infected culture in different concentrations of aqueous extract. Assay 3 : In vitro inactivation of Influenza A after incubation of the virus at 37 °C for 1 h in different concentrations of aqueous extract. 106 B.T.S. Yff et al. / Journal of Ethnopharmacology 79 (2002) 101–107 TLC plate and all the bands were separately tested for anti-inflammatory activity. One band was found to be active but further HPLC separation was unsuccessful. The P. prunelloides leaf extract produced gross morphological changes in the VK cell monolayers at concentrations of 62.5 up to 1000 mg/ml in the cytotoxicity assay. The integrity of the monolayer remained at extract concentrations 3.9 up to 31.25 mg/ml. When treated with the root extract of concentrations up to 125 mg/ml the integrity of the monolayers was maintained, however, cytotoxic effects were observed at concentrations of 250 mg/ml or higher. The results of the three different antiviral assays are shown in Table 3. The antiviral activity against the Inf A is evident by the percentage reduction in the number of fluorescent foci on the infected VK cell cultures. The activity varies at different concentrations of the aqueous extract of P. prunelloides. A difference in activity has also been observed between root and leaf extract in the three assays: 3.1. Viral replication Aqueous leaf extracts at concentrations 0.98–15.62 mg/ml affected the replication of Inf A in cell culture. A direct relationship was noted between the concentration of the leaf extract and the reduction in the number of fluorescent foci. The root extract also exhibited antiviral activity against influenza A viral replication but at higher extract concentrations, i.e. from 3.9 to 125 mg/ml. 3.2. Viral adsorption Leaf extract concentrations of 1.95– 15.62 mg/ml showed similar antiviral activity against the adsorption of influenza A, but activity was much less at 0.98 mg/ml. The root extract showed antiviral activity at concentrations from 3.9 to 125 mg/ml, but there was no clear relationship between concentration of extract and reduction in the number of fluorescent foci. 3.3. In 6itro inacti6ation Aqueous leaf extracts of concentrations 7.81– 15.60 mg/ml showed a marked inactivation of Influenza A. Aqueous root extracts also showed this inactivation, but at higher extract concentrations, i.e. from 3.9 to 125 mg/ml. The results of the antiviral tests suggest the use of root decoctions for the treatment of influenza-like infections. In 1918 root decoctions of P. prunelloides were used during the influenza epidemic, with apparently a marked improvement (Watt and Breyer-Brandwijk, 1962). The combination of antibacterial and anti-inflammatory activity together with antiviral activity against influenza A makes the plant a potentially effec- tive remedy for ‘flu relief’ and further phytotherapeutic research is warranted. This study suggests that the use of P. prunelloides in traditional medicine may be beneficial for the treatment of selected bacterial and viral infections as well as having anti-inflammatory properties. Acknowledgements The Department of Chemistry, University of Natal, Durban, is thanked for carrying out the GC/MS. For financial support, the following institutions are thanked: The University of Natal Research Fund; Stichting Fonds Harald Quintus Bosz, The Netherlands; Dr Hendrik Muller’s Vaderlandsch Fonds, The Netherlands; Stichting G.L. Funke Fonds, The Netherlands; Genootschap ter Bevordering van Natuur-, Genees- en Heelkunde, The Netherlands; Faculteit der Biologie, Universiteit van Amsterdam, The Netherlands; Stichting ter Bevordering van de Farmacologie, Amsterdam, The Netherlands. References Barrett, T., Inglis, 1985. Growth, purification and titration of influenza viruses. In: Mahy, B.W.J. (Ed.), Virology, A Practical Approach. IRL Press, Oxford, pp. 119 – 150. Eloff, J.N., 1998. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica 65, 711 – 713. Grist, N.R., Bell, E.J., Follette, E.A.C., Urquhart, G.E.D., 1979. Diagnostic Methods in Clinical Virology, third ed. Blackwell, Oxford, pp. 60 – 79. Hashem, F.A., Saleh, M.M., 1999. Antimicrobial components of some Cruciferae plants. (Diplotaxis harra Forsk and Erucaria microcarpa Boiss). Phytotherapy Research 13 (4), 329 – 332. Hussain, R.F., Nouri, A.M.E., Oliver, R.T.D., 1993. A new approach for measurement of cytotoxicity using colorimetric assay. Journal of Immunological Methods 160, 89 – 96. Hutchings, A., 1996. Zulu Medicinal Plants. Natal University Press, Pietermaritzburg. Iwu, M., 1993. Handbook of African Medicinal Plants. CRC Press, Boca Raton, USA. Jäger, A.K., Hutchings, A., van Staden, J., 1996. Screening of Zulu medicinal plants for prostaglandin-synthesis inhibitors. Journal of Ethnopharmacology 52, 95 –100. Kaido, T.L., et al., 1997. Preliminary screening of plants used in South Africa as traditional herbal remedies during pregnancy and labour. Journal of Ethnopharmacology 55, 185 –191. Lindsey, K., Jäger, A.K., Raidoo, D.M., van Staden, J., 1999. Screening of plants used by Southern African traditional healers in the treatment of dysmenorrhoea for prostaglandin-synthesis inhibitors and uterine relaxing activity. Journal of Ethnopharmacology 64, 9 – 14. van Rensburg, C.E.J., Anderson, R., Myer, M.S., Jooné, G.K., O’Sullivan, J.F., 1994. The riminophenazine agents clofazimine and B669 reverse acquired multidrug resistance in a human lung cancer cell line. Cancer Letters 85, 59 – 63. van Wyk, B.E., van Oudtshoorn, B., Gericke, N., 1997. Medicinal Plants of Southern Africa. Briza Publications, Pretoria, South Africa. B.T.S. Yff et al. / Journal of Ethnopharmacology 79 (2002) 101–107 Watt, J.M., Breyer-Brandwijk, M.G., 1962. The Medicinal and Poisonous Plants of Southern and Eastern Africa. E and S Livingstone Ltd, Great Britain. 107 Williams, V.L., Balkwill, K., Witkowski, E.T.F., 2000. Unraveling the commercial market for medicinal plants and plant parts on the Witwatersrand, South Africa. Economic Botany 54 (3), 310 – 327. Journal of Ethnopharmacology 79 (2002) 109– 112 www.elsevier.com/locate/jethpharm Some herbal remedies from Manzini region of Swaziland Oluwole O.G. Amusan *, Pricilla S. Dlamini, Jerome D. Msonthi, Lydia P. Makhubu Swaziland Institute for Research in Traditional Medicine, Medicinal and Indigenous Food Plants, Uni6ersity of Swaziland, Pri6ate Bag 4, Kwaluseni, Swaziland Received 29 June 2001; received in revised form 27 September 2001; accepted 18 October 2001 Abstract In this paper, recipes for 41 herbal remedies used for treating 25 illnesses in traditional medicine in Swaziland are reported. Preparation of the herbal remedies involved the use of different parts of 47 species from 32 families, some of which have never been described previously in the flora of Swaziland. Descriptions of the plants used, the preparation of each remedy, dosage, route of administration and medical uses are reported. Some of the diseases the remedies are used to treat include asthma, backache, candidiasis, cardiac problems, cough, diarrhoea, dizziness, eye problems, constipation, menorrhagia, painful shoulders, scabies, threatened abortion, toothache, ulcers and vomiting among others. The remedies were obtained from traditional medical practitioners (TMPs) in an ethnomedical survey carried out in the Manzini region of Swaziland. Voucher samples of the plants used for the remedies were collected, identified and deposited in the National Herbarium of Swaziland. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Herbal remedies; Manzini region; Swaziland 1. Introduction 2. Methodology Plants form an important part of Swaziland’s biological resources. Many of the plants are used in traditional medicine. Many Swazis rely on traditional medicine for their health care needs, including some who attend modern health institutions, because, traditional medicine is anchored in the culture and religious beliefs of the people. The reliance on herbal medicine continues to rise as the costs of conventional drugs increase and are becoming unaffordable by many in rural communities. The need to preserve the cultural heritage by documenting information on medicinal plants used in traditional medicine has led to ethnomedical surveys of Swaziland (Amusan et al., 2000; Adeniji et al., 2001). In continuation of our efforts to document uses of medicinal plants in Swaziland, this paper reports some new herbal remedies. An ethnomedical survey of the Manzini region of Swaziland was carried out from June 1998 to March 2000. The Manzini region of Swaziland (4068 km2) is located between 26°15% – 26°30%S latitude and 30°45% – 31°30%E longitude. Traditional medical practitioners (TMPs) in the region were interviewed and remedies used for treating diseases were recorded. Details about the period for collecting the plant material, its shelf-life, the recipe for each remedy as well as any ritual involved were recorded. The dosage, route of administration, contra indications, side effects and the criteria used to recognise a cure were given by the TMPs. A voucher sample of each plant mentioned by the TPMs was collected immediately after each interview with the assistance of the TMP who gave the plant. The plants were authenticated by G.M. Dlamini, the Curator of the National Herbarium, Malkerns and the plant vouchers were deposited in the herbarium. 3. Results * Corresponding author. Fax: + 9-268-518-5276. E-mail address: amusan@uniswacc.uniswa.sz (O.O.G. Amusan). Plants used in the preparations given by the TMPs are presented in Table 1. They are listed in alphabetical 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 8 1 - 6 110 Table 1 Plants used in the preparation of herbal remedies Family SiSwati name Voucher Recipe number Aloe arborescens Mill and Aloe saponaria Haw Asphodelaceae Asphodelaceae Inhlaba Lihala M310 M311 Berkyeya setifera DC. Asteraceae Lulwini/Iwenkhomo M375 Bolusanthus speciosus (Bol.) Harms. Bowiea 6olubilis Harv. Ex Hook. F. and Boophane disticha (L.f.) Herb. Carissa bispinosa (L.) Desf. Ex Brenan Cheilanthes calomendos Swartz Papionaceae Umhhohlo M307 Grind together 50 g each of the leaves of M310 and roots of M311, add the mixture to 2 l water and boil for 10 min Cut 50 g each of the leaves and roots to pieces and add both to a cup of warm water Grind 50 g roots and add to 1 l warm water Hyacinthaceae Amaryllidaceae Gibizisila Siphaluka M344 M345 Cut 50 g bulb of each plant to pieces, add to 5 l water and boil for 5 min Apocynaceae Um6usankunzi M306 Grind 30 g roots and add to 1 l warm water Adiantaceae Mphasetje M284 Grind 30 g leaves or roots to powder and add to a hot plate. Add another 30 g of the powder to 1 l water and boil for 5 min Grind together 30 g each of the bark of M369, leaves and stem of M370, add to 5 l water and boil for 5 min Grind 50 g bark and add to 5 l warm water Combretum molle R. Br. Ex G. Combretaceae Verbenaceae Don and Lippia ja6anica Spreng. Cussonia natalense Sond. Araliaceae Imbhond6o/Lemhlophe M369 Umsutane M370 Umsenge M281 Dichrostachys cinerea (L.) Wight & Arn. Mimosaceae Umzilazembe M269 Dicoma anomala Sond. Asteraceae Ndwedweni M056 Erianthemum dregei (Eckl. & Zeyh.) V. Tieghem Euclea di6inorum Hiern Loranthaceae Liphakama M225 Grind 30 g roots, add the powder to a hot plate and inhale the smoke. Add another 30 g powder to 1 l water and boil for 5 min Grind 50 g bulb to paste, add to 1 l water and boil for 30 min Leaves are used as catalyst for other medicines Ebenaceae Umdlelanyamatane M305 Grind 50 g roots and add to 1 l warm water Gardenia cornuta Hemsl. Gardenia spatulifolia Stapf & Hutch Indigofera sanguinea N. E. Br. Rubiaceae Rubiaceae Um6alansangweni Um6alansangweni M219 M292 Grind 30 g roots and add to 5 l water Grind 50 g roots and add to 5 l warm water Papilionaceae Cubhujeje M196 Lannea edulis (Sond.) Engl. Anacardiaceae Umtfokolo6u M244 Lasiosiphon kraussianus Hutch. & Dalz. Lippia ja6anica (Burm. F.) Spreng. Lonchocarpus capassa Rolfe Thymelaeaceae Umsilawengwe M132 Grind together 50 g each of leaves and roots, and add to 250 ml water Grind 30 g bark of root, add to 1 l water and boil for 3 min Grind 50 g roots, add to a cup of milk and sieve Verbenaceae Umsutane M132 Papilionaceae Sihomuhomu M217 Mimusops zeyheri Sond. Sapotaceae Umkhamamasi/ Umphushane M206 and M367 Grind 50 g fresh root, add to 2 l water and boil slowly for 3 h Grind 50 g bark and add to 5 l warm water Grind 50 g roots and add to 5 l warm water Grind 50 g bark, add to 1 l water and boil for 10 min Use Take 1 tablespoon of the concoction thrice daily for 7 days for cardiac problems Rinse teeth with the infusion twice daily for 3 days for toothache Take 1 tablespoon of the concoction thrice daily for 7 days for abdominal pains Take a cup of the concoction once a day for 5 days for scabies Take 1 teaspoon of the concoction daily for 7 days for impotence For treatment of epilepsy, inhale the smoke, take 1 tablespoon of the concoction thrice daily for 3 days and do enema only once Take 1 tablespoon of the decoction thrice daily for 5 days for asthma Drink the concoction to induce vomiting and stop stomachache. There is always nausea, so take light porridge Take 1 tablespoon of the concoction twice daily until cough stops Take 1 tablespoon of the concoction three times daily for kidney problems Add a little of powdered leaves to the other medicine Take 1 tablespoon of the concoction thrice daily for 3 days for ulcers Drink the mixture when necessary as a laxative Drink the concoction to induce vomiting once a day for 3 days for stomach trouble Drink a cup of mixture only once to treat threatened abortion Take 1 tablespoon of the concoction thrice daily for constipation Do an enema once a week for abdominal problems in babies Drink a cup of mixture once daily for 3 days for kidney problems Drink the concoction when necessary for hallucination Drink the mixture when necessary to treat candidiasis Take 1 tablespoon of concoction of the bark thrice daily for 3 days for ulcers an wounds. O.O.G. Amusan et al. / Journal of Ethnopharmacology 79 (2002) 109–112 Scientific name Table 1 (Continued) Scientific name Family Myrothamnus flabellifolius Welw. Olea capensis L. Myrothamnaceae Vuka M343 Leaves are used as catalyst for other medicines Oleaceae Sephulo/Umncuma M322 Grind 30 g leaves and add a pinch of it to any medicine to make the medicine more effective Grind 50 g bark and add to 1 l warm water Anacardiaceae Asteraceae Imfuce Liphephetse M357 M358 Grind together 50 g each of the barks of M357 and M358, and add to 5 l water Take 1 tablespoon of the concoction thrice daily for 5 days as treatment for peptic ulcers Use the mixture to wash wounds twice daily for 5 days Caesalpinaceae Sikhabamkhombo M293 Pentanisia prunelloides (Klotzsch ex Eckl. & Zeyh.) Walp. Pittosporum 6iridiflorum Sims Rubiaceae Licishamlilo M235 Grind together 30 g each of the roots and bark, and add to 1 l warm water Grind 50 g bark, add to 1 l water and boil for 5 min Grind 50 g bulb, add to 1 l water and boil for 3 min Take 1 tablespoon of the concoction twice daily for 2 days for stomach cramps Drink a cup of concoction of the bark thrice daily until menorrhagia is cured Drink the concoction thrice daily until general body ache goes Crassulaceae Mfusam6u M243 Grind 30 g bark to powder Psidium guaja6a L. and Albizia adianthifolia (Schumach.) W.F. Wight Rapanea melanophloeos (L.) Mez and Pterocelastrus echinatus N. E. Br. Rubia cordifolia L. Myrtaceae Mimosaceae Umgwa6a Si6angatane M365 M366 Cut 50 g leaves of M365 and 50 g roots of M366 to pieces, add to 1 l water and boil for 5 min Add the powder to aching tooth twice daily until the pain is gone Take 1 tablespoon of the infusion thrice daily for 3 days for diarrhoea Myrsinaceae Celastraceae Maphipha/Gcolokhulu M228 Macundza M229 Grind together 50 g each of the barks of M228, M229 and add to 1 l warm water Rubiaceae Intilalubombo M256 Caesalpinaceae Anacardiaceae Ugcamu/Vo6o6o Umganu M211 M212 Grind 30 g of bark or root to powder, add to a hot plate and add cold water Grind together 50 g each of the barks of M211 and M212, and add to 5 l warm water Hyacinthaceae Imbita yebantfwana M318 Spirostachys africana Sond. Acanthaceae Euphorbiaceae Likhambilebantfwana M319 Umtfolo wesintfu M078 Spirostachys africana Sond. and Trichilia emetica Vahl Stylochiton natalense Schott Euphorbiaceae Meliaceae Araceae Umtfolo wesintfu Umkhuhlu Umfanakamacetjane M312 M313 M360 Grind together 50 g each of the barks of M312 and M313. Add to 5 l water and boil for 10 min Grind 30 g roots and add to 1 l warm water Syzygium guineense (Willd.) DC. Myrtaceae Umcozi M216 Grind 30 g bark and add to 1 l water Trichilia emetica Vahl Meliaceae Umkhuhlu M203 Ximenia americana L. Olacaceae Umtfund6uluka M204 Zanthoxylum capense (Thunb.) Harv. Rutaceae Umnungwane M291 Grind 30 g bark, add to 1 l water and boil for 1h Grind 30 g roots, add to 1 l warm water and Add a drop of the mixture daily to the eye with sieve problems Grind together 50 g each of the bark and roots to Take 1 tablespoon of concoction thrice daily until powder and add to 1L warm water the medicine is finished for threatened abortion Burn 50 g whole plant in a hot plate Inhale the smoke daily for 2 days for dizziness Schotia brachypetala Sond. And Sclerocarya birrea (A. Rich.) Hochst Scilla ner6osa (Burch.) Jessop and Crossandra fruticulosa Lindau Cut 30 g each of the bulbs of M318 and roots of M319 to small pieces, add to1 l water and boil for 10 min Grind 50 g bark and add to 5 l warm water Take 1 tablespoon of mixture 3 times a day until general body ache goes. Do not take sugar or maize porridge while on medication Lick the mixture twice daily for 5 days for uterine problems. Drink one cup of the concoction to induce vomiting and boil another 5 l mixture for steaming when necessary to treat painful shoulders Drink one half of a cup of the concoction thrice daily for 5 days for peptic ulcers in children Drink the concoction twice daily for 3 days for constipation Do an enema with the concoction once a day for constipation Take 1 tablespoon of mixture twice daily for 3 days as treatment for headache Give 1 tablespoon of concoction to an adult and 1 teaspoon to a child three times daily until diarrhoea stops Do an enema only once for backache 111 Use O.O.G. Amusan et al. / Journal of Ethnopharmacology 79 (2002) 109–112 Voucher Recipe number Ozoroa sphaerocarpa R. & A. Fernands and Athrixia phylicoides DC. Peltophorum africanum Sond. SiSwati name 112 O.O.G. Amusan et al. / Journal of Ethnopharmacology 79 (2002) 109–112 order of their scientific names in italic, followed by the families, the siSwati names in italic, voucher number, recipe for each remedy and therapeutic uses. 4. Discussion and conclusions Herbal remedies abound in traditional medicine in Swaziland for symptomatic treatment of various diseases. It is noteworthy that the TMPs in the Manzini region alone had remedies for such a wide range of diseases. Forty one remedies were described for 25 illnesses. The remedies have never been reported in any pharmacopoeia and ethnobotanical studies of Swaziland (Amusan et al., 2000; Adeniji et al., 2001). Preparation of the herbal remedies involved the use of different parts of 47 species from 32 families. Some of the plants have never been described in the flora of Swaziland (Compton, 1976; Dlamini, 1981). Mimusops zeyheri Sond. (Sapotaceae) was given two codes, M206 and M367, because, two remedies in which the plant was used were given by two TMPs, therefore, the plant specimen was collected twice with the two codes. The remedies described were acclaimed to be efficacious and have been used for decades. Some of these preparations can be very useful in the primary health care. An integrated health care system where resources of the traditional and orthodox medical systems are combined, as is being practised in Clinique de Manongarivo in Madagascar, would be ideal especially for developing countries endowed with rich plant genetic resources (Quansah, 1999). Traditional medicine in Swaziland is clouded with a lot of secrecy, myths and metaphysical powers. The TMPs have strong belief in ancestral spirits. Their diagnosis and the effectiveness of their prescriptions are usually bound up with elements of ancestral belief. Some of the concepts in the Swazi traditional medicine are, therefore, difficult to explain in scientific terms. They can only be understood when considered within the culture of the people (Makhubu, 1978). The activities reported cannot be explained in terms of known activities of the families of the plants. Scientific research on the medicinal plants should explode the myths by identifying the active principles in the plants. Scientific examination of the remedies could lead to standardisation and quality control of the products to ensure their safety. It is after such evaluations that they can be approved for use in the primary health care. Such research activities could also lead to the development of new drugs as in the past (Farnsworth et al., 1985; Farnsworth, 1988). Acknowledgements We thank the TMPs who were involved in the survey and G. M. Dlamini who identified the plants. We are also grateful to the University of Swaziland Research Board for funding. References Adeniji, K.O., Amusan, O.O.G., Dlamini, P.S., Enow- Orock, E.G., Gamedze, S.T., Gbile, Z.O., Langa, A.D., Makhubu, L.P., Mahunnah, R.L.A., Mshana, R.N., Sofowora, A., Vilane, M.J., 2001. Traditional Medicine and Pharmacopoeia Contribution to Ethnobotanical and Floristic Studies in Swaziland. OAU/STRC, Lagos. Amusan, O.O.G., Dlamini, P., Msonthi, J.D., Makhubu, L.P., Dlamini, B.B., 2000. Some medicinal plants used in traditional medicine in Swaziland. UNISWA Journal of Agriculture, Science and Technology 4 (1), 20 – 26. Compton, R.N., 1976. The flora of Swaziland. Journal of South African Botany Supplement 2 Dlamini, B., 1981 Swaziland flora. Ministry of Agriculture and Cooperatives, Mbabane. Farnsworth, N.R., 1988. Screening plants for new medicines. In: Wilson, E.O. (Ed.), Biodiversity. National Academy Press, Washington, pp. 83 – 97. Farnsworth, N.R., Akerele, O., Bingel, A.S., 1985. Medicinal plants in therapy. Bulletin of World Health Organization 63, 965 – 981. Makhubu, L.P., 1978. The Traditional Healer. The University of Botswana and Swaziland, Kwaluseni. Quansah, N., 1999. Integrated health care system: meeting Africa’s health needs in the 21st century. In: Mshana, R.N., Ndoye, M. (Eds.), African Traditional Medicine and Medicinal Plants. Proceedings of the 6th Inter-African Symposium on African Traditional Medicine and Medicinal Plants. OAU/STRC, Lagos, pp. 10 – 17. Journal of Ethnopharmacology 79 (2002) 113– 118 www.elsevier.com/locate/jethpharm Kalopanaxsaponin A from Kalopanax pictus, a potent antioxidant in the rheumatoidal rat treated with Freund’s complete adjuvant reagent Jongwon Choi a, Keun Huh b, Suk-Hwan Kim c, Kyung-Tae Lee d, Hyeong-Kyu Lee e, Hee-Juhn Park f,* a College of Pharmacy, Kyungsung Uni6ersity, Pusan 608 -736, South Korea College of Pharmacy, Yeungnam Uni6ersity, Kyoungsan, 712 -749, South Korea c Department of Food Science and Nutrition, Dong-A Uni6ersity, Pusan 604 -714, South Korea d College of Pharmacy, Kyung-Hee Uni6ersity, Seoul 130 -701, South Korea e Immunomodulator Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Taejon 305 -333, South Korea f Di6ision of Applied Plant Sciences, Sangji Uni6ersity, Wonju 220 -702, South Korea b Received 25 July 2000; received in revised form 25 October 2001; accepted 25 October 2001 Abstract The stem bark of Kalopanax pictus is an anti-rheumatoidal arthritis drug in Oriental medicine. In the rheumatoidal rat, induced by Freund’s complete adjuvant (FCA) reagent, we investigated the effects of hederagenin monodesmosides of K. pictus on oxidative stress and hepatic drug-metabolizing enzymes. Kalopanaxsaponin-A (KPS-A) significantly decreased malondialdehyde formation and the activities of xanthine oxidase and aldehyde oxidase of hepatic non-microsomal systems in FCA reagent-treated rats. In addition, increased activity levels of superoxide dismutase, catalase and glutathione peroxidase were also observed. The effects of KPS-A were more potent than the effects of KPS-I. These results suggested that KPS-A, extracted from K. pictus, could reduce rheumatoidal syndromes through antioxidative mechanisms. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Kalopanax pictus; Kalopanaxsaponin A; Rheumatoidal arthritis; Freund’s adjuvant reagent; Microsomal; Oxidative stress 1. Introduction One of the most important uses of the stem bark of Kalopanax pictus is to treat rheumatoidal arthritis as an Oriental herbal drug (Moon, 1991) (Fig. 1). We have reported that hederagenin monodesmosides from this plant had antidiabetic (Kim et al., 1998a), cytotoxic, antimutagenic (Lee et al., 2000) and antifungal (Kim et al., 1998b) actions. We also found hederagenin monodesmosides, such as kalopanaxsaponins-A (KPSA) and -I (KPS-I), were potent anti-inflammatory/analgesic agents in this crude drug (Choi et al., 2000). In order to elucidate the bioactive mechanisms of kalopanaxsaponin-A and -I for rheumatoidal arthritis, we evaluated the changes of the contents of reactive oxygen species and the activities of hepatic drug-metaboliz* Corresponding author. Tel./fax: + 82-33-730-0564. E-mail address: hjpark@chiak.sangji.ac.kr (H.-J. Park). ing enzymes, by the administration of the extract and saponins of K. pictus in FCA-induced rheumatoidal rats. 2. Materials and methods 2.1. Plant material and extraction, fractionation and isolation K. pictus Nakai (No. NATCHEM-20) and the procedures for extraction, fractionation and isolation of kalopanaxsaponins (KPA-A, -I, -B, -H, -J) were shown in previous papers (Kim et al., 1998a; Choi et al., 2000). 2.2. Chemicals NADPH, UDP-glucurononic acid (UDPGA), sodium xanthine, cytochrome C, N-methylnicotinamide 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 8 2 - 8 114 J. Choi et al. / Journal of Ethnopharmacology 79 (2002) 113–118 and bovine serum albumin were from Sigma Co. (USA). Malondialdehyde and 2-pyridone were from Aldrich (USA). Reduced and oxidized glutathione were purchased from Fluka (USA). p-Nitrophenol, 2,4-dinitrochlorobenzene, thiobarbituric acid were from Katayama (Japan). Freund’s complete adjuvant reagent (FCA reagent) was purchased from Gifco Co. (USA). 2.3. Animals Four-week-old Sprague– Dawley male rats were purchased from the Korean Experimental Animal Company. They were adapted to a constant condition (temperature: 2092 °C, dampness: 40–60%, light/dark cycle: 12:12 h) for 2 weeks or more. The animals were fasted for 24 h before the experiment, but with water ad libitum. Considering the variation of enzyme activity during 1 day, the animals were sacrificed at a fixed time (10:00– 12:00 h). 2.4. Induction of rheumatoidal disease and administration of test samples For the induction of rheumatoidal disease, each 0.05 ml of Freund’s complete adjuvant reagent was injected into the sole of the right foot of the rats. After 2 weeks, the induction of inflammation in rats was confirmed. The test samples, EtOAc fractions, KPS-A and -I were dissolved in DMSO and those solutions with various concentrations were prepared by dilution with saline to various concentrations. Based on the results of a preliminary experiment, EtOAc extract was administered orally at 250 and 500 mg/kg and the isolated saponins were injected intraperitoneally at 5, 10 and 20 mg/kg. 2.5. Isolation of enzyme sources After anesthetizing with CO2 gas, the animals were killed by decapitation. Liver tissue was homogenized in 4-fold amount of 0.1 M sodium phosphate buffer (pH 7.4) on ice. This homogenate was centrifuged at 600×g for 10 min. The supernatant was again subjected to centrifugation at 10,000×g for 20 min to produce pallets. This was suspended with an equivalent volume of 0.1 M sodium phosphate buffer (pH 7.4) and centrifuged at 105,000× g for 30 min. The resultant mitochondrial fraction was used for the measurement of catalase activity as an enzyme source. The supernatant obtained from centrifugation of the final one (105,000× g, 60 min) was used as an enzyme source of cytosolic fraction to measure the activities of xanthine oxidase, aldehyde oxidase, glutathione peroxidase, superoxide dismutase, glutathione S-transferase and sulfonyltransferase. The resultant precipitate was centrifuged (105,000×g, 60 min) in 0.1 M sodium phosphate buffer (pH 7.4) and the obtained microsomal fraction was used for the enzyme sources to measure the activities of cytochrome P450, aminopyrine Ndemethylase, aniline hydroxylase and UDPglucuronyltransferase. 2.6. Measurement of malondialdehyde The thiobarbituric acid (TBA) reactive substance in the liver was measured as a marker of lipid peroxidation by the method of Ohkawa et al. (1979). An aliquot (0.4 ml) of 10% liver homogenate in 0.9% NaCl was added to 1.5 ml of 8.1% SDS, 1.5 ml of 20% acetate buffer (pH 3.5) and 1.5 ml of 0.8% TBA solution. The mixture was heated at 95 °C for 1 h. After cooling, 5.0 ml of n-butanol:pyridine (15:1) was added for extraction and the absorbance of the n-butanol:pyridine layer at 532 nm was measured for the determination of TBA reactive substance. 2.7. Determination of cytochrome P450 acti6ity Cytochrome P450 activity was determined according to the method of Omura and Sato (1964). In brief, microsomal suspension (1 mg/ml protein) and sodium dithionite were mixed in test tubes and further bubbled with CO gas using a needle. After this process, the absorbance of these and of a solution without a process of CO bubbling were recorded at 450– 490 nm wavelengths. The amount of cytochrome P450 was calculated using a molar coefficient, 91 mM/cm, of cytochrome P450 – CO complex. 2.8. Determination of aminopyrine N-demethylase acti6ity The activity of aminopyrine N-demethylase was determined according to the method of Nash (1953). The solution (2 ml) containing 2 mM aminopyrine, 0.5 mM NADPH, 10 mM MgCl2, 150 mM KCl and 1 mM semicarbazide in 0.1 M Na+/K+ phosphate buffer (pH 7.5) was incubated for 15 min and the reaction was terminated by adding 15% ZnSO4 and saturated Ba(OH)2. This reactant was centrifuged and the supernatant was colored with Nash reagent. Finally, the activity of aminopyrine N-demethylase was calculated from the absorbance at 415 nm wavelength. 2.9. Determination of aniline hydroxylase acti6ity The enzyme activity of aniline hydroxylase was determined according to the method of Bidlack and Lowery (1982). A solution of 2.0 ml of 50 mM Tris–HCl buffer (pH 7.4) containing 1 mM aniline, 0.5 mM NADPH, enzyme sources, 10 mM MgCl2 and 150 mM KCl was incubated 37 °C for 20 min. The reaction was terminated by adding 20% trichloroacetic acid. After cen- J. Choi et al. / Journal of Ethnopharmacology 79 (2002) 113–118 trifugation, 10% Na2CO3 and 0.2 N NaOH/2% phenol were added to the obtained supernatant. This solution was reacted at 37 °C for 30 min and the absorbance was measured at the wavelength of 640 nm. The enzyme activity was calculated from a standard calibration curve. The unit of enzyme activity was expressed as the amount (nmole) of p-aminophenol of 1 mg protein produced per minute. 2.10. Determination of xanthine oxidase acti6ity The enzyme activity of xanthine oxidase was determined according to the method of Stirpe and Della (1969). In brief, a mixture of 3.0 ml of 0.1 M potassium phosphate buffer (pH 7.5) and 0.1 ml of 60 mM sodium xanthine (substrate) was reacted at 37 °C. After the reaction, the protein of the mixture was removed by adding 20% trichloroacetic acid. The absorbance of the obtained supernatant was taken at 292 nm wavelength. The activity level was calculated using a standard calibration curve. The unit of enzyme activity was expressed as nmoles of produced uric acid of 1 mg protein produced per minute. 2.11. Determination of aldehyde oxidase acti6ity The enzyme activity of aldehyde oxidase was determined according to the method of Rajagopalan et al. (1968). A mixture of 0.1 M potassium phosphate buffer (pH 7.5), N-methylnicotinamide (substrate) and enzyme solution was reacted. The absorbance of the produced 2-pyridone was taken at 300 nm wavelength. The enzyme activity was calculated using a standard calibration curve. The unit of enzyme activity was expressed as nmoles of the produced 2-pyridone of 1 mg protein per minute. 115 2.12. Determination of UDP-glucuronyl transferase acti6ity The enzyme activity of UDP-glucuronyl transferase (UDPGA) activity was determined according to the method of Reinke et al. (1986). In brief, 1.0 ml solution containing 10 mM phosphate buffer (pH 7.0), 1 mM p-nitrophenol, 3 mM UDPGA, 1 mM MgCl2, 0.02% bovine serum albumin, 0.05% Triton X-100 and enzyme solution (20–100 mg protein) were reacted at 37 °C for 5 min. The reaction was terminated by adding 0.25 ml of 0.6 N HClO4 and centrifuged. The supernatant was added with 0.5 ml of 1.6 M glycine buffer (pH 10.4) and the absorbance was taken at 436 nm wavelength. The unit was expressed as nmoles of p-nitrophenol removing 1 mg protein per minute. 2.13. Determination of glutathione S-transferase acti6ity The enzyme activity of glutathione S-transferase was determined according to the method of Habig et al. (1974). In brief, 75 ml of 40 mM reduced glutathione was added to 0.1 M potassium phosphate buffer (pH 6.5) in the test tube and this was followed by adding 100 ml enzyme solution. In the blank, the reaction was terminated by 20% trichloroacetic acid. The mixture was reacted at 25 °C for 5 min, followed by the addition of 25 ml of 2,4-dinitrochlorobenzene (substrate). The reaction was terminated with 20% trichloroacetic acid and centrifuged. The absorbance of each supernatant obtained from the centrifugation of test- and blank-reactant was measured at 340 nm. The activity (nmole) was calculated using a molar absorption coefficient (9.6 mM/cm). 2.14. Determination of superoxide dismutase acti6ity The enzyme activity of superoxide dismutase was determined according to the method of Marklund and Marklund (1974). A solution of cytochrome C in 1.0 ml of 0.2 M potassium phosphate buffer (pH 8.6) containing 100 mM EDTA was allowed to stand on an ice bath for 20 min. A volume of 0.5 ml of alkaline DMSO or non-alkaline DMSO was added to the test or the blank solution, followed by incubation at 37 °C for 30 min. The absorbance of reducing cytochrome C was taken at 550 nm. The enzyme activity was calculated as 1 U for a 50% inhibition on the alkaline-mediated reduction of cytochrome C. 2.15. Determination of catalase acti6ity Fig. 1. Structures of kalopanaxsaponin-A and -I isolated from the stem bark of K. pictus. The catalase activity was determined according to the method of Aebi (1974). In brief, the reduction of 10 mM H2O2 (substrate) in 50 mM potassium phosphate J. Choi et al. / Journal of Ethnopharmacology 79 (2002) 113–118 116 Table 1 Effect of EtOAc extract and kalopanaxsaponins A (KPS-A) and I (KPS-I) of K. pictus extract on the hepatic lipid peroxide contents in rats induced by FCA reagent Group Dose (mg/kg) Content (MDA nmol/g of tissue) % Inhibitory rate Normal 18.5 9 0.25*** 100.0 Control 41.3 9 0.68 0.0 EtOAc 250 (p.o.) 500 (p.o.) 34.2 9 0.69* 30.6 9 0.37** 31.1 46.9 KPS-A 5 (i.p.) 10 (i.p.) 20 (i.p.) 31.1 9 0.66** 28.5 9 0.63** 25.9 9 0.37*** 44.7 56.1 67.5 KPS-I 5 (i.p.) 10 (i.p.) 20 (i.p.) 40.3 9 0.69 37.0 9 1.03 35.4 9 0.78* 4.4 18.9 25.9 24.8 9 0.73*** 72.4 Ibuprofen 100 (p.o.) * PB0.05, significantly different from control group. ** PB0.01, significantly different from control group. *** PB0.001, significantly different from control group. Rats were orally administered the test samples daily for 7 days. Values represent means 9 S.E.M. (n = 10). buffer (pH 7.0) was estimated by the absorbance at 240 nm. The activity was calculated using a molar absorption coefficient. The unit of enzyme activity was expressed as nmoles of dissipating hydrogen peroxide of 1 mg protein per minute. 2.16. Determination of glutathione peroxidase acti6ity The enzyme activity of glutathione peroxidase was determined according to the method of Paglia and Valentine (1967). Enzyme solution was added to a mixture containing hydrogen peroxide and glutathione in 0.1 mM Tris buffer (pH 7.2) and followed by measuring the absorbance at 340 nm. The activity was calculated from a calibration curve. The unit of enzyme activity was expressed as nmoles of NADPH produced of 1 mg protein per minute. 2.17. Statistics The data was shown by mean9 S.E.M. Statistical significance was carried out by Student’s t-test. 3. Results FCA reagent administration to rats increased malondialdehyde (MDA) formation twice the normal amount. The EtOAc extract and KPS-A significantly decreased the MDA values of the control group, though KPS-I did not show a significant change in the rat (control) (Table 1). The activity order was shown as KPS-A \EtOAc extract\KPS-I. The effects of EtOAc extract and KPS-A and -I on hepatic microsomal enzymes of cytochrome P450, aniline hydroxylase and aminopyrine N-demethylase, which are associated with Phase I response to the xenobiotics, did not change statistically (data not shown). By FCA administration, hepatic xanthine oxidase activity was considerably increased in the control group. The administration of EtOAc extract and KPS-A significantly reduced the activity of the control group, though KPS-I showed no statistical significance. Both oral administration of Table 2 Effect of kalopanaxsaponins A (KPS-A) and I (KPS-I) and EtOAc extract from K. pictus on hepatic non-microsomal enzyme (xanthine oxidase and aldehyde oxidase) activities in rats induced by FCA reagent Group Xanthine oxidase activity (uric acid nmol/mg protein/min) Aldehyde oxidase activity (2-pyridone nmol/mg protein/min) Normal 2.31 9 0.042*** (100.0) 1.44 9 0.050*** (100.0) Control 5.67 9 0.083 (0.0) 3.50 9 0.039 (0.0) 250 (p.o.) 500 (p.o.) 4.89 9 0.057* (23.2) 4.63 9 0.051** (31.0) 3.12 9 0.043* (18.4) 3.05 9 0.038* (21.8) KPS-A 5 (i.p.) 10 (i.p.) 20 (i.p.) 4.68 9 0.048** (29.5) 4.29 9 0.060** (42.4) 3.93 9 0.050*** (51.8) 2.85 9 0.047** (31.6) 2.56 9 0.047** (45.6) 2.35 9 0.037*** (55.8) KPS-I 5 (i.p.) 10 (i.p.) 20 (i.p.) 5.17 9 0.052 (14.9) 5.20 9 0.059 (14.0) 5.29 9 0.049 (14.0) 3.85 9 0.041 (−17.0) 3.36 9 0.038 (6.8) 3.40 9 0.044 (4.9) 3.23 9 0.127*** (72.6) 1.98 9 0.104*** (26.2) EtOAc extract Ibuprofen Dose (mg/kg) 100 (p.o.) * PB0.05, significantly different from control group. ** PB0.01, significantly different from control group. *** PB0.001, significantly different from control group. Values represent means 9S.E.M. (n = 10). Values in parentheses are percentage of inhibitory rate. J. Choi et al. / Journal of Ethnopharmacology 79 (2002) 113–118 117 Table 3 Effect of kalopanaxsaponins A (KPS-A) and I (KPS-I) and EtOAc extract from K. pictus on hepatic superoxide dismutase (SOD), catalase and glutathione peroxidase activities in rats induced by FCA reagent Group SOD activity (Ua/mg protein) Catalase activity (decreased H2O2 nmol/mg/protein) Glutathione peroxidase activity (oxidized NADPH nmol/mg protein/min) Normal 9.589 0.066*** (100.0) 2.69 9 0.074*** (100.0) 240.5 9 6.22*** (100.0) Control 3.79 9 0.114 (0.0) 1.34 9 0.048 (0.0) 116.7 9 4.66 (0.0) 4.43 9 0.101 (11.1) 5.54 9 0.057** (30.2) 1.65 9 0.074 (23.0) 156.1 9 3.08* (31.8) 2.00 9 0.066* (48.1) 177.4 9 3.23** (49.0) 5.87 9 0.060** (35.9) 6.54 9 0.058*** (47.5) 7.50 9 0.063*** (64.1) 1.97 9 0.068* (46.1) 166.7 9 3.16** (40.4) 2.25 9 0.042** (67.4) 186.1 9 4.86** (56.1) 2.40 9 0.073** (78.5) 208.1 93.08*** (73.8) 1.25 9 0.062 (−6.7) 108.9 93.62 (−6.3) 1.55 9 0.063 (15.6) 120.6 9 2.68 (3.2) 1.40 9 0.067 (4.4) 130.2 94.03 (10.9) 2.53 9 0.173*** (88.9) 210.4 97.23*** (75.7) EtOAc extract Dose 250 (p.o.) 500 (p.o.) KPS-A 5 (i.p.) 10 (i.p.) 20 (i.p.) KPS-I 5 (i.p.) 10 (i.p.) 20 (i.p.) Ibuprofen 100 (p.o.) 3.54 9 0.035 (−4.3) 4.70 9 0.062 (15.7) 4.52 9 0.071 (12.6) 8.24 9 0.142*** (76.9) a Superoxide dismutase activity (1 U) was defined as that which inhibited the oxidation of pyrogallol of 50%. * PB0.05,significantly different from control group. ** PB0.01,significantly different from control group. *** PB0.001,significantly different from control group. Values in parentheses are the percentage of activation rates. Values represent means 9 S.E.M. (n = 10). EtOAc extract and intraperitoneal administration of KPS-A showed dose-dependent activities (Table 2). The inhibitory effect of EtOAc extract and KPS-A and -I on hepatic cytosolic aldehyde oxidase are shown in Table 2. The treatment of EtOAc extract and KPS-A significantly reduced the activity compared to the control group. The inhibitory effect of KPS-I was weaker than that of KPS-A group. Three treatment groups showed dose-dependent activities. EtOAc extract and KPS-A and -I did not affect UDP-glucuronyltransferase, sulfotransferase and glutathione S-transferase (data not shown). The effects of test samples on the activity of superoxide dismutase (SOD), catalase and glutathione peroxidase are shown in Table 3. The activity of this enzyme was significantly decreased when FCA reagent was treated. The administration of EtOAc extract, KPS-A and KPS-I increased the activity dose dependently. The administration of EtOAc extract, KPS-A and -I significantly increased the activity of the control group dosedependently. The action of KPS-I was weaker than that of KPS-A. Ibuprofen (100 mg/kg, p.o.) selected as a positive control manifested more potent activities throughout all the tests than those of KPS-A. 4. Discussion In the rheumatoidal rat induced by FCA reagent, EtOAc extract, its saponin constituents, KPS-A and -I showed no significant differences between control and treatment groups in the activity of microsomal cytochrome P450 and those of its classified enzymes, aminopyrine N-demethylase and aniline hydroxylase. However, KPS-A and -I significantly decreased the activity of xanthine oxidase and aldehyde oxidase which are involved in Phase I reaction. These enzyme activities can be increased by the infection of viruses, bacteria or parasites (Tubaro et al., 1980). Since the two non-microsomal enzymes used oxygen molecules as an electron acceptor during the biological reactions (Massey et al., 1969), both enzymes formed superoxide anion and hydrogen peroxide and finally resulted in the formation of hydroxyl radical. From the above data, it is clear that FCA reagent administration can activate non-microsomal oxidation. The EtOAc fraction and KPS-A may have inhibited the enzyme activation. Phase II stage is also able to scavenge electrophilic substrates, reactive oxygen species and peroxides (Routledge and Shand, 1979). However, the activities of UDP-glucuronyltransferase and sulfonyltransferase in- 118 J. Choi et al. / Journal of Ethnopharmacology 79 (2002) 113–118 volved in the Phase II reactions were not changed statistically between control and treatment groups. Glutathione S-transferase activity also was not influenced by the treatment of all test samples, though the activity of the control group was much higher than the normal group. The most reactive oxygen species in biological system are superoxide anion (·O− 2 ), hydroxyl radical (·OH) and hydrogen peroxide (H2O2) (Batteli et al., 1973). Superoxide dismutase can change superoxide anion radical to hydrogen peroxide (Free, 1980) and catalase cleaves this hydrogen peroxide into the molecules of water and oxygen. Glutathione peroxidase is also a detoxifying enzyme, changing the peroxides to water (Lawrence and Burk, 1976). The EtOAc fraction and KPS-A increased superoxide dismutase, catalase and selenium independent glutathione peroxidase. In the FCA reagent-induced rheumatoidal model, the increase of lipid peroxide could be attributed not only to the activation of xanthine oxidase and aldehyde oxidase, but also to the deactivation of superoxide dismutase, catalase and selenium independent glutathione peroxidase. The inhibition of lipid peroxidation by EtOAc extract and KPS-A may result from either the hindrance of the formation or the scavenging of reactive oxygen. Although many anti-inflammatory herbal drugs contain various types of saponins as main constituents, the role for anti-edema have been, so far, ambiguous. Because, in the previous study, we demonstrated the inhibitory effect of KPS-A and -I on inflammation in the rheumatoidal rat induced by FCA reagent (Choi et al., 2000), the modulation of antioxidant systems by these saponins may contribute more effectively to the edema than any other pharmacological actions. In summary, KPS-A of K. pictus could protect the body tissues from the toxicity of reactive oxygen species accompanied by the occurrence of rheumatoidal arthritis. Since an antiinflammatory drug, ibuprofen, also showed these effects, it was suggested that both anti-inflammatory drug and KPS-A have the possibilities to improve rheumatoidal arthritis. Acknowledgements This research was supported by a grant (PF002104-07) from Plant Diversity Research Center of 21st Century Frontier Research Program, funded by Ministry of Science and Technology of Korean Government. References Aebi, H., 1974. In: Vergmeyer, H.U. (Ed.), Catalase in Method. Enzymatic Analysis. Academic Press, New York, p. 673. Batteli, N.G., Lorenzoni, E., Stripe, F., 1973. Milk xanthine oxidase type D (dehydrogenase) and type O (oxidase): purification and interconversion and some properties. The Biochemical Journal 131, 191 – 198. Bidlack, W.R., Lowery, G.L., 1982. Multiple drug metabolism: P-nitroanisole reversal of acetone enhanced aniline hydroxylation. Biochemical Pharmacology 31, 311 – 317. Choi, J., Chun, H.W., Kim, S.H., Park, H.J., Han, Y.N., 2002. Analgesic effect of Kalopanax pictus extract and its saponin constituents, and their inhibitory effect on Freund’s complete adjuvant reagent-induced rheumatoidal arthritis in the experimental animals. Journal of Ethnopharmacology (in press). Free, J.A., 1980. In: Spiro, J.G. (Ed.), Superoxide, Superoxide Dismutase and Oxygen Toxicity in Metal Ion Activation of Dioxygen. Wiley, New York, p. 209. Habig, W.H., Pabist, M.J., Jakoby, W.B., 1974. Glutathione S-transferase: the first enzymatic step in mercapturic acid formation. Journal of Biological Chemistry 249, 7130 –7139. Kim, D.H., Yu, K.W., Bae, E.A., Park, H.J., Choi, J.W., 1998a. Metabolism of kalopanaxsaponin B and H by human intestinal bacteria and antidiabetic activity of their metabolites. Biological and Pharmaceutical Bulletin 21, 360 – 365. Kim, D.W., Bang, K.H., Rhee, Y.H., Lee, K.T., Park, H.J., 1998b. Growth inhibitory activities of Kalopanax pictus against human pathogenic fungi. Archives of Pharmacology Research 21 (6), 688 – 691. Lawrence, R.A., Burk, R.F., 1976. Glutathione peroxidase activity in selenium deficient rat liver. Biochemical and Biophysical Research Communication 71, 952 – 958. Lee, K.T., Sohn, I.C., Park, H.J., Kim, D.W., Jung, G.O., Park, K.Y., 2000. Essential moiety for antimutagenic and cytotoxic activity of hederagenin glycosides isolated from the stem bark of Kalopanax pictus. Planta Medica 66, 329 – 332. Massey, V., Strickland, S., Mayhew, S., Howell, L.G., Engel, P.C., 1969. The production of superoxide anion radicals in the reaction of reduced flavins and flavoproteins with molecular oxygen. Biochemical and Biophysical Research Communication 36, 891 – 897. Marklund, S., Marklund, G., 1974. Involvement of the superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for superoxide dismutase. European Journal of Biochemistry 47, 469 – 474. Moon, K.S., 1991. The Components and Utilization of Medicinal Plants. Ilweolseogak, Pyongyang, p. 419. Nash, T., 1953. The colorimetric estimation of formaldehyde by means of the hantzsch reaction. Journal of Biological Chemistry 55, 416 – 422. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissue by thiobarbituric acid reaction. Analytical Biochemistry 95, 351 – 358. Omura, T., Sato, R., 1964. The carbon monooxide binding pigments of liver microsomes: evidence for its hemoprotein nature. Journal of Biological Chemistry 239, 2370 – 2385. Paglia, E.D., Valentine, W.N., 1967. Studies on the quantitative and qualitative characterization of erythrocytes glutathione peroxidase. Journal of Laboratory Clinical Medicine 70, 158 – 169. Rajagopalan, K.V., Fridovich, I., Handler, P., Palmer, G., Beinert, H., 1968. Studies of aldehyde oxidase by electron paramagnetic resonance spectroscopy. Journal of Biological Chemistry 243, 3783 – 3796. Reinke, L.A., Meyer, M.J., Notley, K.A., 1986. Diminished rates of glucuronidation and sulfation in perfused rat liver after chronic ethanol administration. Biochemical Pharmacology 35, 439 – 447. Routledge, P.A., Shand, D.G., 1979. Presystemic drug elimination. Annual Revision of Pharmacology and Toxicology 19, 447 – 468. Stirpe, F., Della, C.E., 1969. The regulation of rat liver xanthine oxidase: conversion in vitro of the enzyme activity from dehydrogenase (Type D) to oxidase (Type O). Journal of Biological Chemistry 244, 3855 – 3863. Tubaro, E., Lotti, C., Cavallo, G., Croce, C., Borelli, G., 1980. Liver xanthine oxidase increase in mice in three pathological models. Biochemical Pharmacology 29, 1939 –1943. Journal of Ethnopharmacology 79 (2002) 119– 127 www.elsevier.com/locate/jethpharm Antidepressant effects of the methanol extract of several Hypericum species from the Canary Islands C.C. Sánchez-Mateo *, B. Prado, R.M. Rabanal Departamento de Farmacologı́a, Facultad de Farmacia, Uni6ersidad de La Laguna, c/Astrofı́sico Francisco Sánchez s/n, 38071, La Laguna, Tenerife, Spain Received 15 July 2001; received in revised form 2 November 2001; accepted 5 November 2001 Abstract The aim of the present study was to investigate several neuropharmacological effects of the methanol extract of the aerial parts in blossom of Hypericum canariense, H. glandulosum, H. grandifolium and H. reflexum (Hypericaceae). These extracts did not alter significantly the locomotor activity, body temperature or the pentobarbital-induced sleeping time, with the exception of H. reflexum which significantly potentiated pentobarbital-induced sleeping time at both doses assayed (500 and 1000 mg/kg p.o.). Additionally, neither muscle relaxant nor anticholinergic activity was observed. These extracts antagonized the ptosis and/or motor depression induced by tetrabenazine and also shortened the immobility time in the forced swimming test. Moreover, the H. glandulosum and H. grandifolium extracts at 1000 mg/kg p.o. potentiated the head twitches induced by 5-HTP. These observations suggest that the methanol extract of the Hypericum species in doses of 500– 1000 mg/kg p.o. possess antidepressant activity in mice, without inducing significant muscle relaxation, anticholinergic and sedative properties. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hypericum species; Methanol extracts; Central nervous system activity; Antidepressant effect; Forced swimming test; Mice 1. Introduction Hypericum perforatum L. (Hypericaceae), popularly called St John’s wort in Anglo-Saxon folk medicine, is a well-known medicinal plant which has been used for centuries for a range of indications including skin wounds, eczema, burns, diseases of the alimentary tract and psychological disorders (Bombardelli and Morazzoni, 1995; Barnes et al., 2001). Currently, antidepressant effects of H. perforatum extracts have been demonstrated by many animal experiments and clinical studies (Linde et al., 1996; Butterwerck et al., 1997; Butterweck et al., 2000; Brenner et al., 2000). Clinical data suggest that the extracts of St John’s wort are effective in the treatment of mild to moderate depression with a low incidence of serious adverse effects (Wheatly, 1998; Stevinson and Ernst, 1999; Vitiello, 1999; Gaster and Holroyd, 2000; Di Carlo et al., 2001). However, several recent reports have raised the possi* Corresponding author. Tel.: + 34-22-318502; fax: + 34-22318514. E-mail address: csanchez@ull.es (C.C. Sánchez-Mateo). bility of important interactions between St. John’s wort extracts and certain conventional drugs, such as serotonin reuptake inhibitors or those drugs metabolized by the hepatic cytochrome P-450 microsomal oxidase enzymes (for example, warfarin, cyclosporine, theophylline, digoxin, indinavir or oral contraceptives) (Johne et al., 1999; Barone et al., 2000; Biffignandi and Bilia, 2000; Fugh-Berman, 2000; Piscitelli et al., 2000; Barnes et al., 2001; Greeson et al., 2001). Given the widespread use of St John’s wort, and in view of the newly observed interactions, there is still a need for further studies to assess the safety of these extracts. Hypericum genus (Hypericaceae) is represented by 10 species of flora in the Canary Islands (Kunkel, 1991). In the present investigation we have selected four species of this genus: H. canariense L., H. glandulosum Ait. and H. reflexum L. fil., endemic herbaceous plants of the Canary Islands, and H. grandifolium Choisy, endemic of the Macaronesian Region. The infusions prepared from the flowers, leaves and fruits of these species have been used in the Canary Islands as a vermifuge, diuretic, as well as wound healing, sedative, antihysteric 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 9 3 - 2 120 C.C. Sánchez-Mateo et al. / Journal of Ethnopharmacology 79 (2002) 119–127 and antidepressant agent (Darias et al., 1986, 1989; Pérez de Paz and Hernández Padrón, 1999). Therefore, in the light of their use in folklore medicine as a sedative, antihysteric and antidepressant agent, the present study was undertaken to investigate the CNS activity of the methanol extract of the aerial parts in blossom of the different Hypericum species in mice, particularly in animal models of depression. 2. Materials and methods 2.1. Plant material Aerial parts in blossom of H. canariense L., H. glandulosum Ait., H. grandifolium Choisy and H. reflexum L. fil. were collected from Pedro Álvarez (Tenerife, Spain). They were identified by Dr Consuelo Hernández Padrón, Department of Plant Biology, University of La Laguna (Tenerife, Spain) where the voucher specimens have been deposited (TFC no. 42211, TFC no. 42212, TFC no. 42213 and TFC no. 42214, respectively). 2.2. Preparation of the methanol extracts The aerial parts of the four species were dried in an oven at 40 °C and powdered. The methanol extracts were obtained by maceration of the plant material with methanol for 3 days at room temperature, and this procedure was repeated twice. The respective extracts were filtered and dried under reduced pressure at a temperature below 45 °C. The yields obtained were: 29.10% for H. canariense, 31.98% for H. glandulosum, 24.06% for H. grandifolium, 27.80% for H. reflexum. In this paper, all doses are expressed as mg of the dried extract per kg body weight. The extracts were assayed at the doses of 500 and 1000 mg/kg p.o. 2.3. Animals Male and female albino Swiss mice (24–28 g) were purchased from the Laboratory Animal Center, University of La Laguna (Tenerife, Spain) and were housed in groups of 10 per cage for a minimum of 3 days prior to pharmacological studies with free access to standard laboratory food and tap water and maintained on a 12/12 h light– dark cycle (light form 8:00 to 20:00 h). All animals were fasted overnight before dosing, tap water being available ad libitum. Each experimental dose group consisted of five animals unless otherwise stated. The ambient temperature was 2291 °C, except in the experiments of potentiation of barbiturate sleeping time which were carried out in a room maintained at 3091 °C. Plant extracts and standard drugs were suspended in a 2% aqueous solution of Tween 80 immediately prior to use and given orally 1 h before the experiments mentioned below in a dose volume of 0.5 ml/20 g body weight in mice. Control animals received 2% Tween 80 suspension, under the same conditions. Behavioural observations took place between 8:00 and 15:00 h and each animal was used only once. 2.4. Drugs The following drugs were used: apomorphine hydrochloride (Sigma, Spain), atropine sulphate (Sigma, Spain), chlorpromazine hydrochloride (Rodia, Spain), fluoxetine (Lilly Indiana, Spain), L-5-hydroxytryptophan (Serva, Spain), imipramine hydrochloride (Impex, Spain), oxotremorine sesquifumarate (Sigma, Spain), sodium pentobarbital (Dirección General de Farmacia y Productos Sanitarios, Spain), tartaric acid (Merck, Spain), tetrabenazine (Fluka, Spain). Tween 80 (Ferosa, Spain). 2.5. LD50 in mice The LD50 was calculated from lethality within 3 days after p.o. administration of the drugs by the Litchfield and Wilcoxon (1949) method. 2.6. General central ner6ous system acti6ity 2.6.1. Effect on spontaneous motor acti6ity in mice Locomotor activity was recorded with a photocell activity meter for 15 min beginning 60 and 120 min after p.o. administration of each test drug. 2.6.2. Effect on normal body temperature The rectal temperature of each mouse was measured with a thermistor thermometer (Panlab 0331) prior to the experiment and 1, 2, 4, 6, and 24 h after p.o. administration of each test drug. The temperature changes at different times with respect to the temperature values before drug administration were calculated. Male mice with a rectal temperature between 36 and 38 °C prior to the experiment were used. 2.6.3. Effect on muscle relaxant acti6ity: traction test Experiments were performed by the Boissier et al. (1961) method. Mice were obliged to hang with their forelegs on a wire of 1 mm in diameter, which was stretched horizontally at a height of 35 cm. When they fell off the wire within 5 s or failed to grasp the wire C.C. Sánchez-Mateo et al. / Journal of Ethnopharmacology 79 (2002) 119–127 with their hind legs three times successively, muscle relaxation was judged to be positive. This test was conducted in groups of five previously screened animals, 60 min after injection of control vehicle or test material. 2.6.4. Effect on barbiturate-induced sleep in mice Sodium pentobarbital at a hypnotic dose of 40 mg/kg was injected i.p. to groups of eight mice 60 min after p.o. administration of the drug. The latency and duration of sleep (loss and recovery of the righting reflex) were recorded. 2.6.5. Effect on oxotremorine-induced syndrome in mice Mice with a rectal temperature between 36 and 38 °C prior to the experiment were used and randomly assigned to test groups of six subjects. One hour after p.o. administration of the test drug, vehicle or standard compounds, mice received oxotremorine sesquifumarate (0.5 mg/kg i.p.). Tremors were scored 30 min after oxotremorine using a scale of 0–3 (Frances, 1988): 0 (no tremor), 1 (no apparent tremor, but when mice were hung up by the tail, tremors appeared and the forepaws crossed), 2 (intermittent tremors, but tremors always appeared when mice were moving), 3 (continuous tremor). Salivation was scored 30 min after oxotremorine as strong, moderate, weak or absent designated by 3, 2, 1, and 0 scores, respectively. Rectal temperature of each mouse was measured 60 min after oxotremorine administration. 2.7. Antidepressant acti6ity 2.7.1. Antagonism of tetrabenazine-induced ptosis, hypothermia, and suppression of locomotor acti6ity Male mice with a rectal temperature of 36– 38 °C prior to the experiment were used. Mice received orally each test drug 60 min before the administration of tetrabenazine (32 mg/kg i.p.), which was dissolved in 0.1 M tartaric acid followed by adjustment to pH 6 with NaOH 10%. Thirty minutes after the intraperitoneal injection of tetrabenazine, the animals were placed at the center of a disk (20 cm of diameter) and the akinesia and the degree of palpebral ptosis exhibited by each mouse was estimated within 10 s. Mice were judged not to be akinetic if they elicited one or more of the following responses: (1) walk to the edge of the disk and look over the side; (2) mice move 180° in place; (3) mice display head movement of 90° in one direction immediately followed by a 45° movement in the opposite direction (Greenblatt et al., 1978). If the animal displayed locomotor activity, it was considered as a positive response for prevention of sedation. The number of mice giving a positive response was divided by the number of animals in the group and multiplied 121 by 100 to obtain the percentage of animals exhibiting the locomotor activity at the specified dose. The degree of ptosis was rated according to the following rating scale: 0, eyes open; 1, one-quarter closed; 2, half closed; 3, three-quarters closed; and 4, completely closed (Alpermann et al., 1992). The rectal temperature of each group was measured with a thermistor thermometer (Panlab 0331) 60 min after i.p. administration of tetrabenazine. 2.7.2. Antagonism of apomorphine (16 mg/kg) -induced hypothermia in mice Apomorphine (16 mg/kg) was injected subcutaneously to groups of six mice 60 min after p.o. administration of the test drugs (Puech et al., 1981). Temperature was measured with a thermistor thermometer (Panlab 0331) 30 and 60 min after apomorphine administration. Mice with a rectal temperature between 36 and 38 °C prior to the experiment were used. 2.7.3. Potentiation of 5 -hydroxytryptophan (5 -HTP) -induced head twitches in mice Test drugs were administered p.o. 60 min before 75 mg/kg i.p. L-5-HTP. The mice were then placed into glass bell jars and 14 min later the number of head twitches was counted in five 2-min intervals (between 14 and 16, 24 and 26, 34 and 36, 44 and 46 and 54 and 56 min) (Corne et al., 1963). 2.7.4. Beha6ioural despair ( forced swimming) test in mice This test was performed according to the method described by Porsolt et al. (1977) with slight modifications. One hour after p.o. administration of the test drugs, mice were individually forced to swim in a transparent glass vessel (25 cm high, 10 cm in diameter) filled with 10 cm of water at 21–24 °C. The total duration of immobility (seconds) was measured during the last 4 min of a single 6-min test session. Mice were considered immobile when they made no further attempts to escape except the movements necessary to keep their heads above the water. Each experimental group consisted of eight animals. 2.7.5. Statistical analysis Data were analysed by one-way analysis of variance (ANOVA) followed by Student’s unpaired t-test (Tallarida and Murray, 1986). A probability level of 0.05 or less was accepted as significant. The  2-test was used for the percentage of failures in the muscle relaxant activity and the percentage of locomotor activity in the tetrabenazine test. Tremor and salivation induced by oxotremorine, and ptosis induced by tetrabenazine were analysed using the Mann– Whitney test for non-parametric data. C.C. Sánchez-Mateo et al. / Journal of Ethnopharmacology 79 (2002) 119–127 122 3. Results 3.1. LD50 in mice The four methanol extracts of Hypericum assayed administered p.o. did not induce mortality up to a dose of 2 g/kg in mice up to 72 h after administration. The tested animals did not present any toxic manifestations. 3.2. General central ner6ous system acti6ity Table 1 shows that doses of 500 and 1000 mg/kg p.o. of the methanol extracts assayed did not have any significant effect on the spontaneous motor activity, with the exception of the H. canariense methanol extract at the dose of 1000 mg/kg p.o. which produced a slight but significant decrease of this activity at the second hour after administration with respect to control vehicle treatment. In the investigation of the hypnotic effect on mice it was found (Table 1) that only the H. reflexum methanol extract significantly potentiated at both doses assayed (500 and 1000 mg/kg) pentobarbital induced sleeping time by approximately 47%. Nevertheless, these increases were not superior in any case to that shown by the drug used as reference (chlorpromazine). In addition, no muscle relaxant activity could be observed with the different extracts (up to a dose of 1000 mg/kg) when tested with the traction test (data not shown). Concerning the effects of methanol extracts on the rectal temperature (Table 2), it was found that none of them caused a significant effect on body temperature in mice, at variance with chlorpromazine which produced a significant hypothermia which was maintained up to the fourth hour of administration. On the other hand, as can be seen in Table 3 these extracts could not significantly antagonize the oxotremorine-induced tremor, salivation and hypothermia, with the exception of the H. canariense methanol extract (500 mg/kg) which significantly inhibited hypothermia as compared to control by 18%, although atropine (drug of reference) had a stronger effect (56.35%). 3.3. Antidepressant acti6ity In the tetrabenazine test (Table 4), the methanol extracts under study did not antagonize the hypothermia, but they antagonized the ptosis and motor depression induced by the drug in mice. As can be observed, only the H. canariense (1000 mg/kg p.o.) and H. reflexum (500 and 1000 mg/kg) methanol extracts antagonized in a significant manner both effects, with values for the antagonism of motor depression (46–50%) near to that shown by imipramine (the antidepressant agent used as a reference standard). On the other hand, H. glandulosum at both doses assayed and H. grandifolium at 500 mg/kg p.o. were found to antagonize significantly only the tetrabenazine-induced ptosis as compared to control, with values ranging from 19 to 34%. As regard the effects of these methanol extracts on the apomorphine (16 mg/kg s.c.)-induced hypothermia, none of them exhibited a significant antagonism of the hypothermia induced by this drug as compared with the control animals (Table 5). On the contrary, the major- Table 1 Effect of Hypericum methanol extracts on spontaneous locomotor activity and pentobarbital (40 mg/kg i.p.) induced sleeping time in mice (mean 9s.e.m.) Treatment Dose (mg/kg p.o.) Locomotor activity count Pentobarbital induced sleep 60 min 120 min t1 (min) t2 (min) Control – 194.359 11.21 108.85 9 14.66 4.72 9 0.35 52.21 9 4.49 H. canariense 500 1000 213.44 9 21.84 181.10 9 15.56 102.78 912.92 76.00 915.99** 3.64 9 0.11* 4.52 9 0.99 51.09 9 4.93 54.57 9 8.10 H. glandulosum 500 1000 213.44 9 21.84 204.20 9 22.32 102.78 9 12.92 126.50 9 21.05 3.45 9 0.27* 4.82 9 0.73 67.54 9 10.79 66.63 9 8.64 H. grandifolium 500 1000 183.33 9 20.85 173.60 9 12.78 122.44 921.27 119.50 9 13.74 4.439 0.24 3.319 0.23** 60.05 9 4.31 56.82 9 6.84 H. reflexum 500 1000 198.11 9 28.67 181.60 9 19.94 161.75 931.74 118.80 9 17.90 4.639 0.24 4.05 9 0.52 76.76 9 9.46* 77.04 9 9.29* Chlorpromazine 5 3.64 9 0.39 132.08 9 7.54** 7.55 9 1.65** 7.50 9 2.41** Each group comprised five animals for locomotor activity and eight animals for pentobarbital-induced sleep. * PB0.05, ** PB0.01 compared with control (ANOVA followed by Student’s unpaired t-test); t1, sleep induction time (min); t2, sleeping time (min). C.C. Sánchez-Mateo et al. / Journal of Ethnopharmacology 79 (2002) 119–127 123 Table 2 Effect of Hypericum methanol extracts on body temperature (mean 9s.e.m.) Treatment Dose (mg/kg p.o.) Mean decrease in rectal temperature (°C) 1h 2h 4h 6h 24 h Control – 1.16 90.19 1.24 9 0.27 1.06 90.40 0.96 9 0.26 0.61 9 0.16 H. canariense 500 1000 0.78 90.30 0.68 90.11 1.10 9 0.28 1.24 9 0.18 1.369 0.51 1.50 9 0.30 1.04 9 0.27 1.20 9 0.39 0.06 9 0.23 0.08 9 0.20 H. glandulosum 500 1000 1.10 90.25 0.90 90.27 0.72 9 0.25 0.46 9 0.32 1.969 0.39 0.36 9 0.24 1.68 9 0.52 0.54 9 0.34 0.18 9 0.09 0.18 9 0.20 H. grandifolium 500 1000 1.68 90.18 1.04 9 0.20 1.18 9 0.59 1.26 9 0.25 1.549 0.25 1.32 9 0.24 1.82 9 0.20 1.36 9 0.44 0.18 9 0.12 0.28 9 0.33 H. reflexum 500 1000 1.40 9 0.14 1.249 0.29 1.54 9 0.35 1.28 9 0.37 1.38 9 0.20 1.02 9 0.19 1.90 90.07* 1.34 9 0.15 0.15 9 0.18 0.24 9 0.25 Chlorpromazine 5 3.609 0.81* 5.30 9 0.92** 3.52 9 0.91* 1.92 9 0.36 0.18 9 0.13 * PB0.05, ** PB0.01 compared with control (ANOVA followed by Student’s unpaired t-test). Each group comprised five animals. Table 3 Effect of Hypericum methanol extracts on oxotremorine (0.5 mg/kg i.p.) induced tremor, salivation and hypothermia (mean 9 s.e.m.) Treatment Dose (mg/kg p.o.) Tremor mean score (30 min) Salivation mean score (30 min) Mean decrease in rectal temperature (60 min) (°C) Control – 3.00 9 0.00 2.83 9 0.17 10.86 9 0.34 H. canariense 500 1000 3.00 9 0.00 2.67 9 0.21 3.00 9 0.00 2.67 9 0.21 8.90 9 0.32* 11.38 9 0.33 H. glandulosum 500 1000 2.83 9 0.17 2.50 9 0.22 3.00 9 0.00 3.00 9 0.00 11.88 9 0.43 12.27 9 0.51 H. grandifolium 500 1000 2.83 90.17 2.67 90.21 2.83 9 0.17 3.00 9 0.00 10.65 9 0.25 11.17 9 0.07 H. reflexum 500 1000 3.009 0.00 2.679 0.21 3.00 9 0.00 2.83 9 0.17 10.28 9 0.57 11.20 9 0.22 Atropine 10 0.00 9 0.00** 0.00 9 0.00** 4.74 9 0.41* * PB0.01 compared with control (ANOVA followed by Student’s unpaired t-test); ** PB0.01 compared with control (Mann–Whitney test). Each group comprised six animals. ity of them induced a significant increase of this hypothermia, at variance with imipramine, which significantly antagonized it (Puech et al., 1981). In order to investigate their performance in the serotonin system the potentiation of 5-HTP-induced head twitches in mice was also examined. Data shown in Table 6 demonstrate that only the H glandulosum and H. grandifolium methanol extracts at a dose of 1000 mg/kg p.o. produced a significant potentiation of the head twitches induced by this drug, although they were less potent in this regard than fluoxetine, a reference antidepressant that selectively or preferentially inhibit 5-HT uptake (Shank et al., 1987). Finally, the results of the forced swimming test are summarized in Table 7. It was observed that all the extracts at the dose of 1000 mg/kg p.o. significantly shortened the immobility time of mice in the forced swimming test in comparison to control animals by more than 20%, being the H. glandulosum methanol extract, in this regard, the most effective, with activity values (33.07%) close to that found for imipramine at 25 mg/kg p.o. Furthermore, the H. canariense and H. reflexum methanol extracts produced at the dose of 500 mg/kg p.o., a significant decrease of the immobility time (12.11 and 14.83%, respectively), but without reaching the values elicited by imipramine. 4. Discussion The results obtained with these investigations demonstrate that p.o. injection of the methanol extracts of C.C. Sánchez-Mateo et al. / Journal of Ethnopharmacology 79 (2002) 119–127 124 Table 4 Effect of Hypericum methanol extracts on tetrabenazine (32 mg/kg i.p.) induced ptosis, akinesia and hypothermia Treatment Dose (mg/kg p.o.) Ptosis mean score (30 min) Locomotor activity (30 min) (%) Mean decrease in rectal temperature (60 min) (°C) Control – 3.65 9 0.11 0.00 5.81 9 0.34 H. canariense 500 1000 2.90 9 0.31a 2.80 9 0.24a 10.00 46.70c 6.08 9 0.44 6.01 9 0.28 H. glandulosum 500 1000 2.40 9 0.34b 2.93 9 0.23a 30.00 20.00 6.90 9 0.25* 6.19 9 0.24 H. grandifolium 500 1000 2.40 9 0.23b 3.27 9 0.21 20.00 20.00 6.31 9 0.34 6.53 9 0.36 H. reflexum 500 1000 2.10 9 0.23b 3.00 9 0.23a 50.00c 46.70c 6.79 9 0.40 6.16 9 0.36 Imipramine 25 0.00 9 0.00b 80.00c 0.83 9 0.21** * PB0.05, ** PB0.01 compared with control (ANOVA followed by Student’s unpaired t-test); a PB0.05, b PB0.01 compared with control (Mann–Whitney test); c PB0.05 ( 2-test). Each group comprised five animals. several Hypericum species endemic to the Canary Islands did not exert significant effects on general psychopharmacology assays such as the study of the effects on spontaneous locomotor activity, muscle relaxant activity, oxotremorine-induced syndrome, potentiation of pentobarbital sleeping time and body temperature. Only H. canariense caused a significant potentiation of pentobarbital-induced sleeping time at both doses assayed (500 and 1000 mg/kg p.o.), which could indicate a slight sedative effect or an inhibition of pentobarbital metabolism, and it also produced a slight but significant inhibition of the oxotremorine-induced hypothermia at 500 mg/kg p.o., which suggests a certain central anticholinergic activity in mice. In addition to this preliminary psychopharmacological test, the methanol extracts were also studied in several specific tests in order to evaluate their potential antidepressant activity. In these experiments, it was observed that only the H. glandulosum and H. grandifolium extracts at the higher dose assayed significantly potentiated the 5-HTP-induced head twitches, indicating its possible 5-hydroxytriptamine reuptake inhibition (Shank et al., 1987). Additionally, the extracts were found to cause significant effects in antagonizing the tetrabenazine-induced ptosis and/or motor depression. In this regard, the H. canariense (1000 mg/kg) and H. reflexum (500 and 1000 mg/kg) methanol extracts significantly antagonized both effects, which suggest a certain noradrenergic and/or dopaminergic action, whereas H. glandulosum (500 and 1000 mg/kg) and H. grandifolium (500 mg/kg) extracts were significantly active in the ptosis antagonism, which usually evidences a certain alpha-adrenergic or serotoninergic activity (Bourin, 1990). On the other hand, the majority of the extracts under study enhanced rather than antagonized the apomorphine (16 mg/kg s.c.)-induced hypothermia, having no significant effect of their own on rectal temperature. It is worthwhile mentioning in this context that the antagonism of high dose apomorphine-induced hypothermia seems to be specific for antidepressants which can facilitate noradrenergic transmission and not to be very effective in demonstrating the antidepressant properties Table 5 Effect of Hypericum methanol extracts on apomorphine (16 mg/kg s.c.) induced hypothermia Treatment Dose (mg/kg p.o.) Mean decrease in rectal temperature (°C) 30 min 60 min Control – 5.789 0.18 5.49 9 0.41 H. canariense 500 1000 5.96 9 0.33 7.159 0.21** 5.81 9 0.57 7.61 9 0.27** H. glandulosum 500 1000 7.70 90.37** 6.91 90.43** 7.47 9 0.66* 5.92 9 0.68 H. grandifolium 500 1000 7.04 90.26** 6.23 9 0.40 6.33 9 0.88 5.97 9 0.61 H. reflexum 500 1000 5.72 91.22 6.68 9 0.34* 6.43 9 1.35 6.229 0.56 Imipramine 50 1.92 9 0.43** 1.39 9 0.44** * PB0.05, ** PB0.01 compared with control (ANOVA followed by Student’s unpaired t-test). Each group comprised six animals. Data are expressed as mean 9 s.e.m. C.C. Sánchez-Mateo et al. / Journal of Ethnopharmacology 79 (2002) 119–127 Table 6 Effect of Hypericum methanol extracts on 5-HTP (75 mg/kg i.p.) induced head twitches Treatment Dose (mg/kg p.o.) Number of head twitches (mean 9 s.e.m.) Control – 0.30 90.15 H. canariense 500 1000 0.00 90.00 0.40 90.16 H. glandulosum 500 1000 0.009 0.00 0.90 9 0.28* H. grandifolium 500 1000 0.40 9 0.40 1.20 9 0.51* H. reflexum 500 1000 0.20 9 0.02 0.90 9 0.41 Fluoxetine 100 10.02 9 0.92** * PB0.05, ** PB0.01 compared with control (ANOVA followed by Student’s unpaired t-test). Each group comprised five animals. of other antidepressant agents such as mianserin, citalopram or fluvoxamine (Puech et al., 1981; Pawlowski and Mazela, 1986). Thus, it has been postulated that hypothermia induced by high dose of apomorphine seems to involve not only the domaminergic system but also beta-adrenergic receptors. Apomorphine, by an action at presynaptic D2 receptors situated on noradrenergic nerve terminals, would prevent the release of noradrenaline. These receptors are only sensitive to high doses of apomorphine, since they are either nonfunctional or limited in number. It is also possible that high doses of apomorphine may stimulate presynaptic noradrenergic receptors as well as D2 receptors (Bourin, 1990). It is unclear at the present time, which properties of these extracts could account for the enhancement of the apomorphine-induced hypothermia observed, since 125 it could result not only through reduction of noradrenaline and/or dopamine neurotransmission or interaction between noradrenaline/dopamine systems, but that other systems could also be involved. Since they could affect more than one monoaminergic system in the brain, further studies on the mechanism of action of these extracts are needed before giving a definitive conclusion in this aspect. Therefore, the above results prove that the Hypericum extracts studied, in contrast to classical antidepressants, were ineffective in antagonizing the high-dose apomorphine-induced hypothermia, and, thus, no adrenergic activity seems to be exerted. It is important to stress that these tests are based on drug interactions and therefore closely related to a particular mechanism of action (mainly substances affecting the monoamine reuptake). For this reason, further investigations were performed using the forced swimming (behavioural despair) test, which is a conductual assay generally used for the prediction of antidepressant activity without involving pharmacological interaction. According to Porsolt et al. (1977), immobility seen in rodents during swimming reflects behavioural despair as seen in human depression and it is well known that the antidepressant drugs caused a significant decrease in the immobility time in mice. In this test, it has been shown that the majority of the extracts studied significantly reduced the duration of the immobility time in comparison to control animals, thereby demonstrating antidepressant activity, being the H. glandulosum methanol extract at the dose of 1000 mg/kg p.o. as active as imipramine (25 mg/kg p.o.) in this assay. It must be pointed out that the antidepressant activity detected in the forced swimming test for these methanol extracts is not due to CNS stimulant properties, since they have no significant effects on the motor Table 7 Effect of Hypericum methanol extracts on the behavioural despair (forced swimming) test Treatment Dose (mg/kg p.o.) Duration of immobility (mean 9 s.e.m.) (s) Variation Control – 158.009 5.77 – H. canariense 500 1000 138.879 7.09* 124.21 910.78** −12.11 −21.39 H. glandulosum 500 1000 142.37 97.67 105.75 911.51** −9.89 −33.07 H. grandifolium 500 1000 156.81 97.90 124.93 98.97** −0.75 −20.93 H. reflexum 500 1000 134.57 97.53** 120.57 9 11.81** −14.83 −23.69 Imipramine 25 100 108.31 9 7.11** 78.75 915.22** −31.45 −50.16 * PB0.05, ** PB0.01 compared with control (ANOVA followed by Student’s unpaired t-test). Each group comprised eight animals. 126 C.C. Sánchez-Mateo et al. / Journal of Ethnopharmacology 79 (2002) 119–127 activity in comparison to the control group at the doses assayed in this test. It is well known that psychostimulants such as caffeine also decrease immobility time, but in contrast to antidepressants, cause marked motor stimulation, indicating that the effects may be nonspecific (Porsolt et al., 1977). When taken together, these results lead us to conclude that the Hypericum methanol extracts studied appear to have a certain antidepressant activity in mice models like the antagonism of tetrabenazine-induced ptosis and/or motor depression, the potentiation of 5-HTP-induced head twitches and the forced swimming test, without inducing muscle relaxation, sedative and anticholinergic properties. Our data also provide a scientific basis for the utilisation of these plants in folk medicine to treat depressive disorders. However, further studies must be conducted in order to detect which constituents of the extracts exert this activity. In this regard, several fractions from the methanol extracts of these Hypericum species are currently under pharmacological investigation in our laboratory. Acknowledgements We are grateful to La Universidad de La Laguna for financial support for these investigations. References Alpermann, H.G., Schacht, U., Usinger, P., Hock, F.J., 1992. Pharmacological effects of Hoe 249: a new potential antidepressant. Drug Development Research 25, 267 – 282. Barnes, J., Anderson, L.A., Phillipson, J.D., 2001. St John’s wort (Hypericum perforatum L.): a review of its chemistry, pharmacology and clinical properties. Journal of Pharmacy and Pharmacology 53, 583 – 600. Barone, G.W., Gurley, B.J., Ketel, B.L., Lightfoot, M.L., Abul-Ezz, S.R., 2000. Drug interaction between St. John’s wort and cyclosporine. Annals of Pharmacotherapy 34 (9), 1013 – 1016. Biffignandi, P.M., Bilia, A.N., 2000. The growing knowledge of St. John’s Wort (Hypericum perforatum L) drug interactions and their clinical significance. Current Therapeutic Research 61 (7), 389 – 394. Boissier, J.R., Dremont, C., Robins, R., Pagny, J., 1961. Tentative de pharmacology prévisionelle dans la domain des neuroleptiques: actions sedative centrale et adrenolityque de la N(dimethoxy-3,4 phenethyl)N%(chloro-2 phenyl)piperazine. Archive Internationales de Pharmacodynamie et de Therapie 133, 29 – 32. Bombardelli, E., Morazzoni, P., 1995. Hypericum perforatum. Fitoterapia LXVI (1), 43 – 58. Bourin, M., 1990. Is it possible to predict the activity of a new antidepressant in animals with simple psychopharmacological tests? Fundamental and Clinical Pharmacology 4, 49 – 64. Brenner, R., Azbel, V., Madhusoodanan, S., Pawlowska, M., 2000. Comparison of an extract of Hypericum (LI 160) and sertraline in the treatment of depression: a double-blind, randomized pilot study. Clinical Therapeutics 22 (4), 411 – 419. Butterwerck, V., Wall, A., Liefländer-Wulf, U., Winterhoff, H., Nahrstedt, A., 1997. Effects of the total extract and fractions of Hypericum perforatum in animal assays for antidepressant activity. Pharmacopsychiatry (2), 117 – 124 Suppl. Butterweck, V., Jürgenliemk, G., Nahrstedt, A., Winterhoff, H., 2000. Flavonoids from Hypericum perforatum show antidepressant activity in the forced swimming test. Planta Medica 66, 3 – 6. Corne, S.J., Pickering, R.W., Warner, B.T., 1963. A method for assessing the effects of drugs on the central actions of 5-hydroxytryptamine. British Journal of Pharmacology 20, 106 – 120. Darias, V., Bravo, L., Barquı́n, E., Martı́n Herrera, D., Fraile, C., 1986. Contribution to the ethnopharmacological study of the Canary Islands. Journal of Ethnopharmacology 15, 169 – 193. Darias, V., Bravo, L., Rabanal, R., Sánchez Mateo, C., González Luis, R.M., Hernández Pérez, A.M., 1989. New contribution to the ethnopharmacological study of the Canary Islands. Journal of Ethnopharmacology 25, 77 –92. Di Carlo, G., Borrelli, F., Ernst, E., Izzo, A.A., 2001. St John’s wort: prozac from the plant kingdom. Trends in Pharmacological Sciences 22 (6), 292 – 297. Frances, H., 1988. Psychopharmacological profile of 1-(M-(trifluoromethyl)phenyl) piperazine (TFMPP). Pharmacology Biochemistry and Behaviour 31, 37 – 41. Fugh-Berman, A., 2000. Herb – drug interactions. Lancet 355, 134 – 138. Gaster, B., Holroyd, J., 2000. St John’s wort for depression. A systematic review. Archives of Internal Medicine 160 (2), 152 – 156. Greenblatt, E.N., Lippa, A.S., Osterberg, A.C., 1978. The neuropharmacological actions of Amoxapine. Archives Internationales de Pharmacodynamie et Therapie 233, 107 – 135. Greeson, J.M., Sanford, B., Monti, D.A., 2001. St John’s wort (Hypericum perforatum): a review of the current pharmacological, toxicological, and clinical literature. Psychopharmacology 153, 402 – 414. Johne, A., Brockmoller, J., Bauer, S., Maurer, A., Langheinrich, M., Roots, I., 1999. Pharmacokinetic interaction of digoxin with an herbal extract from St. John’s wort (Hypericum perforatum). Clinical Pharmacology and Therapeutics 66 (4), 338 – 345. Kunkel, G., 1991. Flora y vegetación del Archipiélago Canario. Tratado florı́stico, 2a parte. Erdica, S.L., Las Palmas de G.C., pp. 63 – 64. Linde, K., Ramı́rez, G., Mulrow, C.D., Pauls, M., Weidenhammer, W., Melchart, D., 1996. St. John’s Wort for depression — an overview and metaanalysis of randomised clinical trials. British Medicinal Journal 313, 253 – 258. Litchfield, J.T., Wilcoxon, F., 1949. A simplified method of evaluating dose-effect experiments. Journal of Pharmacology and Experimental Therapeutics 96, 99 – 113. Pawlowski, L., Mazela, H., 1986. Effects of antidepressant drugs, selective noradrenaline- or 5-hydroxytryptamine uptake inhibitors, on apomorphine-induced hypothermia in mice. Psychopharmacology 88, 240 – 246. Pérez de Paz, P.L., Hernández Padrón, C.E., 1999. In: Francisco Lemus (Ed.), Plantas medicinales o útiles en la Flora Canaria. Aplicaciones populares. La Laguna, p. 115. Piscitelli, S.C., Burstein, A.H., Chaitt, D., Alfaro, R.M., Fallon, J., 2000. Indinavir concentrations and St John’s wort. Lancet 355, 547 – 548. Puech, A.J., Chermat, R., Poncelet, M., Doare, L., Simon, P., 1981. Antagonism of hypothermia and behavioural response to apomorphine: a simple, rapid and discriminating test for screening antidepressants and neuroleptics. Psychopharmacology 75, 84 –91. Porsolt, R.D., Bertin, A., Jalfre, M., 1977. Behavioural despair in mice: a primary screening test for antidepressants. Arhives Internationales de Pharmacodynamie et Therapie 229, 327 –336. Shank, R.P., Gardocki, J.F., Shcneider, C.R., Vaught, J.L., Settler, P.E., Maryanoff, B.E., McComsey, D.F., 1987. Preclinical evaluation of McN-5707 as a potential antidepressant. Journal of Pharmacology and Experimental Therapeutics 242 (1), 74 – 84. C.C. Sánchez-Mateo et al. / Journal of Ethnopharmacology 79 (2002) 119–127 Stevinson, C., Ernst, E., 1999. Safety of Hypericum in patients with depression. A comparison with conventional antidepressants. CNS Drugs 11 (2), 125 – 132. Tallarida, R.J., Murray, R.B., 1986. Manual of Pharmacological Calculations with Computer Programs, 2nd ed. Springer Verlag, 127 New York, pp. 131 – 134. Vitiello, B., 1999. Hypericum perforatum extracts as potential antidepressant. Journal of Pharmacy and Pharmacology 51, 513 – 517. Wheatly, D., 1998. Hypericum extract — potential in the treatment of depression. CNS Drugs 9 (6), 431 – 440. Journal of Ethnopharmacology 79 (2002) 129– 132 www.elsevier.com/locate/jethpharm Short communication Phytochemical and antimicrobial studies of Begonia malabarica N. Ramesh a, M.B. Viswanathan a,*, A. Saraswathy b, K. Balakrishna c, P. Brindha b, P. Lakshmanaperumalsamy d a Sri Paramakalyani Centre for En6ironmental Sciences, Manonmaniam Sundaranar Uni6ersity, Alwarkurichi 627 412, Tamil Nadu, India b Captain Srini6asa Murti Drug Research Institute for Ayur6eda, Chennai – 600 106, India c Central Research Institute for Siddha, Chennai – 600 106, India d Department of En6ironmental Sciences, Bharathiar Uni6ersity, Coimbatore 641 046, Tamil Nadu, India Received 26 April 2001; received in revised form 31 August 2001; accepted 20 September 2001 Abstract Phytochemical investigation of the various extracts of the leaves of Begonia malabarica Lam. (Begoniaceae) resulted in the isolation and identification of six known compounds, viz. friedelin, epi-friedelinol, b-sitosterol, luteolin, quercetin and b-sitosterol– 3-b-D-glucopyranoside. The aqueous and organic solvent extracts were also tested against ten human pathogenic bacteria and four fungal strains by the agar-well diffusion method. All the extracts were devoid of antifungal activity against the tested fungi. The hexane extract did not show any activity. The aqueous extracts showed activity against the Gram-negative bacteria except Vibrio parahaemolyticus. The chloroform and methanol extracts showed activity against all the tested bacteria. The study supported the claim of the usefulness of the plant in respiratory tract infections and also suggests its use in diarrhoea and skin diseases caused by pathogenic bacteria. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Begonia malabarica; Leaf extracts; Chemical constituents; Antibacterial and antifungal activities 1. Introduction Begonia L. of Begoniaceae has about 900 species found in tropical and subtropical regions of the world wherein 45 species are present in India (Santapau and Henry, 1993). The herb, Begonia malabarica Lam., known as Narayanachanjeeve in Tamil is found in the hilly regions of Southern India and Sri Lanka (Clarke, 1879). The leaves are substituted for Tamarind (Tamarindus indica L., Caesalpiniaceae) and consumed after cooking by the Paliyan tribes of Tirunelveli district in Tamil Nadu of India. They consume boiled leaves for stomach ulcer, stomachache and respiratory problems. Regarding biological studies, antimicrobial activity of Begonia erythrophylla (Urban et al., 1953) and Begonia heracleifolia, Begonia samperflorens and Begonia fuchsioides (Frisby et al., 1953) has been reported. Antitumor activity of Begonia tuberhybrida var. * Corresponding author. Tel./fax: + 91-4634-83270. E-mail address: vinaa@rediffmail.com (N. Ramesh). alba (Doskotch et al., 1969; Doskotch and Hufford, 1970), Begonia plebeja (Fuller et al., 1994) and B. heracleifolia (Frei et al., 1998) has also been reported. In this communication, we report the antibacterial and antifungal activity of the various extracts of B. malabarica leaves. Regarding phytochemical studies only stigmasterol has been reported from B. malabarica (Desai et al., 1975). We report here the isolation and identification of friedelin, epi-friedelinol, b-sitosterol, luteolin, quercetin and b-sitosterol – 3-b-D-glucopyranoside from the leaves for the first time and biological activity of this species had not been previously studied. 2. Materials and methods 2.1. Plant material and extraction The leaves of B. malabarica were collected in May 1999 from Tirunelveli district of Tamil Nadu, India. A voucher specimen (MBV & NR 6761), identified by the 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 5 2 - X 130 N. Ramesh et al. / Journal of Ethnopharmacology 79 (2002) 129–132 second author (M.B.V.) was deposited in the Herbarium of Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi, TN, India. The air-dried and coarse leaf powder (100 g) was successively extracted with hexane, chloroform and methanol using a Soxhlet apparatus. The extracts so collected were distilled on a water bath at atmospheric pressure and the last traces of solvent were removed in vacuo. The successive extractive values were 8.52 mg in hexane, 11.82 mg in chloroform and 6.91 mg in methanol. 2.2. Phytochemical studies The extracts were tested by preliminary phytochemical screening (Harborne, 1976) and the plant constituents were isolated and purified by chromatographic techniques. 2.3. Antimicrobial studies Fresh leaf material (30 g) was weighed, chopped and divided into three portions. Each portion was crushed by grinding in a mortar and transferred to a suitable glass bottle and 50 ml of distilled water was added. First bottle was autoclaved at 80 °C for 20 min, the second was heated at 100 °C for 20 min and the third was mechanically shaken (200 rpm) in cold temperature for 2 h. The extracts were filtered using cheesecloth and 0.45 mm filter paper and transferred to sterile closed containers. The crude extract was considered as 100% extract. By adding sterile distilled water, 50% of the extract was prepared (Sen and Nandi, 1951). Hexane, chloroform and methanol extracts at different concentrations (50, 25, 12.5, 6.25, 3.125 and 1.5625 mg/ml) were prepared in the same solvents of extraction and tested with solvent controls for antimicrobial activities. 2.4. Test microorganisms Microbial strains of human pathogens used were the Gram-negative bacteria, viz. Aeromonas hydrophila, Chromobacterium 6iolaceum, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Vibrio cholerae and Vibrio parahaemolyticus; the Grampositive bacteria, Bacillus subtilis and Staphylococcus aureus; and the fungi, Aspergillus fla6us, Aspergillus fumigatus, Aspergillus niger and Candida albicans. 2.5. Determination of antimicrobial acti6ity The agar-well diffusion method (Perez et al., 1990) was followed. Nutrient agar plates were swabbed with an 8 h broth culture of respective bacteria. Potato-dextrose agar plates were seeded with a spore suspension of 16 h broth culture of fungi. Two wells (10 mm diameter) were made in each of these plates using a sterile cork borer. About 0.3 ml each of 100 and 50% aqueous extracts, different concentrations of solvent extracts and proper solvent controls were added into the wells using sterilized dropping pipettes and diffusion was allowed at room temperature for 2 h. The plates were incubated at 37 °C for 18–24 h for bacterial pathogens and 3 days for fungal pathogens. Respective proper controls of solvent extracts were maintained. Diameter of the inhibition zones was recorded. Triplicates were performed and the experiment was repeated thrice and the average values of antimicrobial activity were calculated. 3. Results The preliminary phytochemical studies revealed the presence of flavone, sterol, triterpene in hexane, chloroform and methanol extracts; phenol in chloroform and methanol extracts and quinone, saponin, tannin and starch in methanol extract. All the extracts did not answer for alkaloid. Column chromatography on silica gel of the combined hexane and chloroform extracts (11 g) yielded friedelin (hexane:benzene, 1:1 earlier eluates; yield 0.15%), epi-friedelinol (hexane:benzene, 1:1 later eluates; yield 0.1%) and b-sitosterol (benzene:ethylacetate, 9:1 eluates; yield 0.2%). Methanol extract (9 g) yielded luteolin (chloroform:methanol, 19:1 eluates; yield 0.12%), quercetin (chloroform:methanol, 12:1 eluates; yield 0.09%) and b-sitosterol –3-b-D-glucopyranoside (chloroform:methanol, 9:1 eluates; yield 0.3%). All the above compounds were identified by comparison with authentic samples (mp, mmp, Co—TLC and superimposable IR). Regarding antimicrobial activity the hexane extract did not show activity against the tested bacteria and fungi. All the extracts did not show activity against the tested fungi. The results are given in Table 1 for the antibacterial activity. The solvents used for extraction were used for dissolving the extracts also and all the solvent controls did not show any activity. Seven standard antibiotics were used and are mentioned in Table 1. The chloroform extract showed activity against all the bacteria and maximum activity was observed against C. 6iolaceum and V. parahaemolyticus. The methanol extract also showed activity against all the bacteria and the maximum activity was observed against K. pneumoniae. All the three aqueous extracts (cold, boiled and autoclaved) showed activity against the Gram-negative bacteria except V. parahaemolyticus and no activity was observed against all the Gram-positive bacteria. Bacterial strains Aeromonas hydrophila Chromobacterium 6iolaceum Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Salmonella typhi Vibrio cholerae Vibrio parahaemolyticus Bacillus subtilis Staphylococcus aureus Aqueous extract (mg/ml) Methanol extract (mg/ml) Cold (%) Boil (%) Autoclave (%) 100 50 100 50 100 50 15 28 20 20 18 16 20 – – – – 24 16 14 16 14 16 – – – 16 26 21 21 20 15 18 – – – – 24 16 16 14 13 14 – – – 18 28 20 20 20 14 18 – – – – 20 14 14 16 12 16 – – – Chloroform extract (mg/ml) Standard 50 25 12.5 6.25 3.13 1.56 50 25 12.5 6.25 3.13 1.56 18 23 22 26 22 24 25 20 18 25 18 22 20 25 20 22 23 19 17 23 17 19 18 20 18 22 20 18 16 21 16 18 17 20 18 18 20 18 15 20 15 16 17 18 16 16 17 16 12 17 15 15 16 18 16 15 16 16 12 17 26 27 20 24 26 24 25 27 26 25 25 25 18 24 25 22 23 25 24 25 23 22 18 22 22 20 22 24 23 20 20 22 16 20 22 20 22 22 22 20 17 20 15 18 20 19 19 22 18 18 17 18 14 18 17 17 17 18 16 16 Ce, Ceftriaxone (30 mg/disc); Ch, Chloramphenicol (30 mg/disc); Er, Erythromycin (15 mg/disc); Nv, Novobiocin (30 mg/disc); Tr, Trimethoprim (5 mg/disc). 38 24 28 32 26 23 36 24 33 31 (Ce) (Ce) (Ch) (Nv) (Ce) (Ce) (Tr) (Er) (Nv) (Nv) N. Ramesh et al. / Journal of Ethnopharmacology 79 (2002) 129–132 Table 1 Antibacterial activity of aqueous, methanol and chloroform leaf extracts of Begonia malabarica (zone of inhibition in mm) 131 132 N. Ramesh et al. / Journal of Ethnopharmacology 79 (2002) 129–132 4. Discussion and conclusions It was observed that all the extracts of the leaves of B. malabarica were devoid of antifungal activity against the tested fungi (Catalano et al., 1998; Moore, 1959). The hexane extract did not show antibacterial activity also. The significant activity against K. pneumoniae, S. aureus and P. aeruginosa shown by chloroform and methanol extracts implicates the use of the plant in respiratory tract diseases by the tribals. The activity of the same extracts against V. parahaemolyticus suggests the use of the plant in diarrhoea. The activity of the chloroform and aqueous extracts against C. 6iolaceum supports the use of plant against skin lesions and pyameia (Hagerman and Butler, 1981). References Catalano, S., Cioni, P.L., Panizzi, L., Morelli, I., 1998. Antimicrobial activity of extracts of Mutisia acuminata var. acuminata. Journal of Ethnopharmacology 59, 207 –209. Clarke, C.B., 1879. Begoniaceae. In: Hooker, J.D. (Ed.), Flora of British India, vol. 2. L. Reeve & Co, London, pp. 635 – 656. Desai, H.K., Gawad, D.H., Govindachari, T.R., Joshi, B.S., Kamat, V.N., Parthasarathy, P.C., Ramachandran, K.S., Shanbhag, M.N., Sidhaye, A.R., Viswanathan, N., 1975. Chemical investigation of some Indian plants VIII. Indian Journal of Chemistry 13, 97 – 98. Doskotch, R.W., Hufford, C.D., 1970. Antitumor agents V. Hexanorcucurbitacin D, a degraded cucurbitacin from Begonia tuberhybrida var. alba. Canadian Journal of Chemistry 48, 1787 – 1788. Doskotch, R.W., Malik, M.Y., Beal, J.L., 1969. Cucurbitacin B, the cytotoxic principle of Begonia tuberhybrida var. alba. Lloydia 32, 115 – 122. Frei, B., Heinrich, M., Herrmann, D., Orjale, J.E., Schmitt, J., Sticher, O., 1998. Phytochemical and biological investigation of Begonia heracleifolia. Planta Medica 64, 385 – 386. Frisby, A., Roberts, J.M., Jennings, J.C., Gottshall, R.Y., Lucas, E.H., 1953. The occurrence of antibacterial substances in seed plants with special reference to Mycobacterium tuberculosis (3rd report). Michigan Agriculture Experimental Station Quarterly Bulletin 35, 392 – 404. Fuller, R.W., Cardellina, J.H., Cragg, G.M., Boyd, M.R., 1994. Cucurbitacins differential cytotoxicity, dereplication and first isolation from Gonystylus keithii. Journal of Natural Products 57, 1442 –1445. Hagerman, A.E., Butler, L.G., 1981. The specificity of proanthocyanidin– protein interactions. Journal of Biological Chemistry 226, 4494 – 4497. Harborne, J.B., 1976. Phytochemical Methods. Chapman & Hall, New York, pp. 1 – 288. Moore, W.C., 1959. British Parasitic Fungi. Cambridge University Press, London, p. 17. Perez, C., Paul, M., Bazerque, P., 1990. Antibiotic assay by agar-well diffusion method. Acta Biologiae et Medicine Experimentalist 15, 113 –115. Santapau, H., Henry, A.N., 1993. A Dictionary of the Flowering Plants in India. Council of Scientific Industrial Research, New Delhi, pp. 1 – 198. Sen, S., Nandi, P., 1951. Antibiotics from the Pteridophytes. Science and Culture 16, 328 – 329. Urban, A.S., Zakhareusky, A.S., Melentovich, L.A., Kuznetsova, Z.P., Vereskovsky, V.V., 1953. Effects of biflavanoids from Begonia erythophylla on acute renal failure in rats. In: Zakhareyskii, A.S., Stoma, O.V. (Eds.), Farmakol. Svoistva Nov. Khim. Soedin. Nek. Lek. Prep., Mater. Knof. Belorusskoe meditsinskoe, Opshchetvo Farmakologov Toksikolegov Minsk, Belarus, pp. 50 – 53 in Russian. Journal of Ethnopharmacology 79 (2002) 133– 138 www.elsevier.com/locate/jethpharm Short communication Antibacterial activity of some Peruvian medicinal plants from the Callejon de Huaylas Catherine C. Neto a, Charles W. Owens b, Richard D. Langfield a, Anthony B. Comeau b, Julie St. Onge b, Abraham J. Vaisberg c, Gerald B. Hammond a,* a Department of Chemistry and Biochemistry, Uni6ersity of Massachusetts, Dartmouth, 285 Old Westport Road, North Dartmouth, MA 02747 -2300, USA b Department of Biology, Rhode Island College, Pro6idence, RI 02908, USA c Departamento de Microbiologia, Uni6ersidad Peruana Cayetano Heredia, Aptdo. Postal 4314, Lima 100, Peru Received 1 November 2001; accepted 1 November 2001 Abstract Extracts of eight medicinal plants from the Callejon de Huaylas in Peru were screened for antibacterial activity in eighteen bacterial strains by the agar-diffusion method; six of these were active against a variety of bacteria. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antibacterial; Medicinal plants; Peru; Agar-diffusion 1. Introduction The Callejon de Huaylas, located in the central region of the Department of Ancash on the northeastern flank of the Peruvian Andes, is a narrow and long valley with a diverse topography, rich in flora and fauna. The local inhabitants depend mostly on plants for their survival and for treatment of diseases; this medical knowledge is stored in the memory of herbal healers. An effort is being made to preserve this ethnobotanical knowledge and to establish the scientific basis of the uses of these plants through a joint project between research groups at the Universidad Peruana Cayetano Heredia and UMass-Dartmouth. A previous survey (Hammond et al., 1998) of these plants and their uses has been made. As traditional medicine is such an important source of potentially useful new compounds (Farnsworth et al., 1985) for development of chemotherapeutics, we evaluated the biological activities of extracts of many of the medicinal plants used in * Corresponding author. Tel.: + 1-508-999-8865; fax: + 1-508-9106918. E-mail address: ghammond@umassd.edu (G.B. Hammond). this region for anticancer (Neto et al., 2000; Lee et al., 1999), antimicrobial and wound-healing (Villegas et al., 1997) activities. Herein, we report the biological evaluation of eight plants for antibacterial activity. The plants studied have a wide range of uses in traditional medicine (Table 1). The goal of our investigation is to validate the medicinal use of these plants and identify their bioactive constituents. 2. Materials and methods 2.1. Plant material Information on eight different plants was gathered through interviews with village elders and herbalists at sites communicated in our previous publications (Hammond et al., 1998; Villegas et al., 1997). Samples for the present study were obtained from marketplace vendors who had collected the plants from public lands. Voucher specimens of species studied are deposited at the Museo de Historia Natural Javier Prado of the Universidad Nacional Mayor de San Marcos in Lima, listed in Table 1. 0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 9 8 - 1 134 C.C. Neto et al. / Journal of Ethnopharmacology 79 (2002) 133–138 2.2. Preparation of extracts Plant material was extracted at room temperature with ethanol and the extract concentrated under reduced pressure. Yields of extracts from plant material (w/w) are as follows, Anredera diffusa, 39.8 g/269 g; Krameria triandra, 6.9 g/100 g; Cassia tomentosa, 48.1 g/554 g; Cestrum auriculatum, 20.1 g/644 g; Asclepias curassa6ica: 9 g/63 g; Himatanthus sucuuba, 14.87 g/400 g; Peperomia galioides, 72 g/5.6 kg; Sambucus peru6iana, 6.1 g/500 g. The crude extracts were prepared for assay in DMSO/water (1:1) and tested for antibacterial activity. These test samples were prepared as follows: after weighing each extract and suspending in DMSO, sterile distilled water was added to each sample to bring the total volume to 1.0 ml and a concentration of extract of 100 mg/ml. The antimicrobial assay is described in Section 2.3. Three plant species (P. galioides, A. diffusa and K. triandra), for which the crude extract showed significant activity in most of the bacterial strains tested, were solvent-partitioned (Kupchan et al., 1974) in water and chloroform, followed by further extraction of the water layer with ethyl acetate, and further partitioning of the chloroform layer between 90% aqueous methanol and hexane. The remaining water layer was lyophilized and the organic extracts evaporated by rotary evaporator to produce solids or viscous oils. For P. galioides, 10.4 g of crude plant extract yielded 28.40 mg of hexane extract, 53.60 mg of methanolic extract, 214.80 mg of ethyl acetate extract, and 7.01 g of aqueous extract. For A. diffusa, 12.32 g of crude plant extract yielded 149 mg of hexane extract, 820 mg of methanolic extract, 444 mg of ethyl acetate extract and 10.8 g of aqueous extract. For K. triandra, 265 mg of hexane extract, 4.559 g of methanolic extract and 168 mg of ethyl acetate extract were obtained. These extracts were also prepared in DMSO/water as described above and tested for antimicrobial activity as outlined below. 2.3. Antibacterial assay The antibacterial activity of the crude plant extracts and solvent partitions of several plants was determined using an agar-diffusion method (Koneman et al., 1997) described below. Eighteen laboratory strains of bacteria were obtained from Remel Diagnostics (Lenex, KS) and cultured at the Rhode Island College facility. All bacterial cultures were grown on 5% sheep red blood plates (5% SBA, PML Microbiologicals, Mississauga, Ont., Canada) at 37 °C for 18–24 h prior to inoculation onto Mueller-Hinton Agar (PML) for testing. Staphylococcus aureus (ATCC c 6538), S. epidermidis (ATCC c12228), S. capitis (ATCC c 35661), S. cohnii (ATCC c35662), Streptococcus pyogenes (Group A, ATCC c19615), S. bo6is (ATCC c49133), S. agalac- Table 1 Ethnobotanical data on medicinal plants Species (Family) and voucher number Common namea Part usedb Common medicinal uses and references on collection sitesc Anredera diffusa (R. et P.) Soukup (Basellaceae) IFV416 Asclepias curassa6ica L. (Asclepidaceae) AC656 Lloto LF Treatment of external wounds [1] Cassia tomentosa L. f. (Fabaceae) IFV236 Huillash or Wishllac (Q) Hierba santa (S) Cestrum auriculatum L’Hérit. (Solanaceae) IFV075 Milkweed, Ipecac WH LX LF LF Treatment of external wounds, used as an antipyretic and antiseptic [2] Treatment of external wounds [1] Himatanthus sucuuba Spruce (Muell.-Arg.), Woodson Bellaco-Caspi (Apocynaceae) FA6251 Krameria triandra R. et P. [syn: Krameria lappacea Ratanya (Q) (Dombey) Burdet et B. Simpson] (Krameriaceae) IFV335 Peperomia galioides HBK. (Piperaceae) IFV320 Congona RT/ST Decoction drunk to treat diarrhea and inflammation and stomach cancer [2] ST/LF Sambucus peru6iana HBK (Caprifoliaceae) LF/SH Extract drunk to treat gastric ulcers, applied to external wounds [1] Infusion of leaves mixed with urine drunk to treat kidney a Sabucu or Samucu (Q) WD Treatment of nasopharyngeal cancer, tumors and warts Source of calotropin and other cardenolidesd Wound healing, use as a disinfectant [2] (Q), Quechua; (S), Spanish. LF, leaf; LX, latex, RT; root; SH, shoot; ST, stem; WD, wood; WH, whole plant without root. c Information on medicinal uses and collection data for all plants except Asclepia curassa6ica is given in our previous publications, [1] Villegas et al. (1997); and [2] Hammond et al. (1998). d Medicinal uses of Asclepia curassa6ica are discussed in Kupchan et al. (1964). Collection took place in the Department of Amazonas, northern Peru, east of the continental divide in the tropical Andes Mountains, generally between 500 and 1500 m elevation, along the Marañon, Nieva and Santiago Rivers. b Table 2 Inhibition of bacterial growth by crude ethanol extracts of Peruvian medicinal plants Anredera diffusa Asclepias curassa6ica Cassia tomentosa Cestrum auriculatum Himatanthus sucuuba Krameria triandra Peperomia galioides Sambucus peru6iana Negative Control (DMSO) Staphylococcus aureus Staphylococcus epidermidis Staphylococcus capitis Staphylococcus cohnii Streptococcus bo6is Streptococcus pneumoniae Streptococcus lactis Streptococcus pyogenes (Group A) Streptococcus agalactiae (Group B) Streptococcus spp. (Group C) Clostridium histolyticum Corynebacterium diphtheriae Bacteroides fragilis Bacillus subtilis Bacillus megaterium Corynebacterium pseudodiptheriticum Escherichia coli 0 0 0 0 0 0 0 0 0 0 +++ +++ 0 +++ 0 0 0 0 0 +++ +++ +++ 0 0 0 0 ++ 0 0 +++ ++ 0 0 +++ 0 0 0 0 ++ +++ 0 +++ +++ 0 0 0 0 0 +++ +++ 0 0 0 0 0 +++ 0 0 0 +++ 0 0 0 0 +++ +++ 0 0 0 0 0 0 +++ 0 0 +++ 0 +++ 0 +++ 0 +++ 0 ++ +++ 0 +++ +++ ++ 0 +++ +++ +++ +++ +++ +++ +++ +++ 0 +++ 0 0 0 0 +++ +++ +++ 0 0 0 +++ 0 0 0 0 0 +++ 0 +++ 0 +++ 0 0 0 +++ 0 0 0 +++ +++ +++ +++ 0 0 +++ +++ +++ 0 +++ 0 0 0 0 0 0 +++ 0 0 0 +++ +++ 0 0 C.C. Neto et al. / Journal of Ethnopharmacology 79 (2002) 133–138 Bacterial strains Inhibition is reported based on diameter of growth inhibition ring: 0, dB0.5 cm (no activity); +, d = 0.5–0.6 cm; ++, d = 0.6–0.7 cm; +++, d\0.7 cm. 135 136 Bacterial strains Staphylococcus aureus Staphylococcus capitis Streptococcus bo6is Streptococcus pneumoniae Streptococcus pyogenes Group A Streptococcus agalactiae Group B Streptococcus sp. Group C Corynebacterium diphtheriae Peperomia galioides Anredera diffusa Positive Control (Vancomycin) Krameria triandra Aqueous Hexane Methanol Ethyl acetate Hexane Methanol Hexane Methanol Ethyl acetate 0 0 0 0 0 0 0 ++ 0 0 0 0 0 0 0 0 ++ ++ ++ ++ ++ ++ ++ +++ 0 0 0 ++ 0 0 ++ +++ 0 0 0 0 0 0 0 0 ++ 0 + ++ + + + ++ 0 0 0 0 0 0 0 0 ++ + 0 ++ ++ ++ ++ ++ + + 0 ++ ++ ++ + +++ Inhibition is reported based on diameter of growth inhibition ring: 0, dB0.5 cm; +, d = 0.5–1.0 cm; ++, d = 1.0–2.0 cm; +++, d\2.0 cm. ++ +++ +++ +++ +++ +++ +++ +++ C.C. Neto et al. / Journal of Ethnopharmacology 79 (2002) 133–138 Table 3 Inhibition of bacterial growth by selected fractions from three Peruvian plant extracts C.C. Neto et al. / Journal of Ethnopharmacology 79 (2002) 133–138 tiae (Group B, ATCC c13813), S. pneumoniae (ATCC c 6303), S. lactis (ATCC c 7962) and Streptococcus sp. (Group C, ATCC c 12388) were grown in a CO2enriched aerobic medium provided by Anaeropack CO2. Bacillus subtilis (ATCC c 6633), B. megaterium (ATCC c89), Corynebacterium diphtheriae (ATCC c 13812), C. pseudodiphtheriticum, (ATCC c10700) and Escherichia coli (ATCC c8739) were grown at atmospheric levels of CO2. Bacteroides fragilis (ATCC c23745) and Clostridium histolyticum (ATCC c6282) were incubated under anaerobic conditions using Anaeropacks Anaero (Remel Diagnostics). Following the initial incubation on 5% SBA plates, organisms were suspended in 9.0 ml of sterile distilled water and their concentration equilibrated to a 0.5 McFarland standard (Remel Diagnostics). Using a sterile cotton swab, each sample was spread onto a Mueller-Hinton agar plate (Becton-Dickinson, Cockeysville, MD) prior to the addition of the disks containing the plant extracts; 6 mm sterile blank paper disks (Becton-Dickinson) were then saturated with each extract and applied to the surface of the agar plates which had previously been inoculated with the above organisms. Vancomycin disks (Remel Diagnostics) and DMSO saturated disks were used as controls. Plates were incubated overnight at 37 °C under the stated conditions. After 24 h, zones of inhibition appearing around disks were measured and recorded in cm. 3. Results and discussion Table 2 shows the results of antibacterial assays of the eight crude plant extracts. Table 3 presents the results of antibacterial assays on solvent extracts from three selected species, for which crude extracts showed significant activity against several bacterial strains. As Table 2 shows, most of the extracts were active against a number of different strains of bacteria, including Staphylococcus and Streptococcus strains. K. triandra R. et P. (syn.: K. lappacea Dombey, Burdet et B. Simpson) (Brako and Zarucchi, 1993) had the widest range of activity. These results confirm reports that have appeared in the literature on the antibacterial activity of this plant, also known as ‘ratanhia’ or ‘rhatany root’. Preparations of the root are used in traditional medicine to maintain dental hygiene and to treat various intestinal ailments as indicated in Table 1. Neolignans active against B. cereus, B. subtilis and E. coli have been isolated from K. triandra; antifungal activity of several of these compounds has also been reported (Arnone et al., 1988, 1990). Comparatively little was known about the chemistry or antimicrobial activity of the other species listed in Table 1 prior to our investigation. We are continuing our investigations of the wound-healing principles from these plants. 137 P. galioides was active against most of the strains tested. Previous reports indicate antiparasitic activity of extracts of this plant, and the isolation of hydropiperone and grifolic acid, active against three species of Leishmania, as well as grifolin and piperogalin, which exhibited toxicity toward Trypanosoma cruzi (Mahiou et al., 1995, 1996). No reports on antibacterial activity have appeared previously for P. galioides. Antibacterial activity of A. diffusa has not been reported previously and is currently the focus of our investigation to elucidate its wound-healing principles. Our findings are encouraging in that six of the eight species showed antibacterial activity against several different bacterial strains, indicating the potential for discovery of antibacterial principles from these plants. The presence of antibacterial agents appears to be relevant to the ethnomedicinal use of A. diffusa, C. tomentosa, C. auriculatum, H. sucuuba, P. galioides and S. peru6iana in the treatment of external wounds. Further study of these plants may lead to a better understanding of the wound-healing mechanism. Since A. diffusa and P. galioides appear to be the most promising, bioassayguided fractionation is currently underway with a goal of elucidating their active antibacterial principles. Acknowledgements This work was supported in part by the National Institutes of Health (Grant c 1 R15 GM59049-01, to GBH), and by Galenica Pharmaceuticals, Inc. (to AJV). Plants were identified by Lic. Irma Fernandez; Himatanthus sucuuba was collected and identified by Dr Franklin Ayala. References Arnone, A., Di Modugno, V., Nasini, G., Venturini, I., 1988. Isolation and structure determination of new active neolignans and norneolignans from ratanhia. Gazzetta Chimica Italiana 118, 675 – 682. Arnone, A., Di Modugno, V., Nasini, G., de Pava, O.V., 1990. Studies on ratanhiae radix. II. Isolation of ratanhine, a new dineolignan from the medicinal ratanhiae radix. Gazzetta Chimica Italiana 120, 397 – 401. Brako, L., Zarucchi, J.L., 1993. In: Crosby, M.R. (Ed.), Catalogue of the Flowering Plants and Gymnosperms of Peru, vol. 45. Missouri Botanical Garden, Saint Louis, MO. Farnsworth, N.R., Akerele, O., Bingel, A.S., Soejarto, D.D., Guo, Z., 1985. Medicinal plants in therapy. Bulletin of the World Health Organization 63, 965 – 981. Hammond, G.B., Fernandez, I.D., Villegas, L.F., Vaisberg, A.J., 1998. A survey of traditional medicinal plants from the Callejon de Huaylas, Department of Ancash, Peru. Journal of Ethnopharmacology 61, 17 –30. Koneman, E.W., Allen, S.D., Janda, W.M., Schreckenber, P.C., Winn, W.C., 1997. Color Atlas of Diagnostic Microbiology, Fifth ed. Lippincott, pp. 785 –881. 138 C.C. Neto et al. / Journal of Ethnopharmacology 79 (2002) 133–138 Kupchan, S.M., Knox, J.R., Kelsey, J.E., 1964. Calotropin, a cytotoxic principle isolated from Asclepias curassa6ica L. Science 146, 1685 – 1686. Kupchan, S.M., Dessertine, A.L., Blaylock, B.T., Bryan, R.F., 1974. Isolation and structural elucidation of allamandin, an antileukemic iridoid lactone from Allamanda cathartica. Journal of Organic Chemistry 39, 2477 – 2482. Lee, K.K., Zhou, B.-N., Kingston, D.G.I., Vaisberg, A.J., Hammond, G.B., 1999. Bioactive indole alkaloids from the bark of Uncaria guianensis. Planta Medica 65, 759 – 760. Mahiou, V., Roblot, F., Hocquemiller, R., Cave, A., 1995. Piperogalin, a new prenylated diphenol from Peperomia galioides. Journal of Natural Products 58, 324 – 328. Mahiou, V., Robliot, F., Hocquemiller, R., Cave, A., 1996. New prenylated quinones from Peperomia galioides. Journal of Natural Products 59, 694 –697. Neto, C.C., Vaisberg, A.J., Zhou, B.-N., Kingston, D.G.I., Hammond, G.B., 2000. Cytotoxic triterpene acids from the Peruvian medicinal plant Polylepis racemosa. Planta Medica 66, 483 – 484. Villegas, L.F., Fernandez, I.D., Maldonado, H., Torres, R., Zavaleta, A., Vaisberg, A.J., Hammond, G.B., 1997. Evaluation of the wound-healing activity of selected traditional medicinal plants from Peru. Journal of Ethnopharmacology 55, 193 – 200.