Research Article Evaluation of the in vivo antimalarial activity of ethanolic leaf and stembark extracts of Anthocleista djalonensis Antia S. Bassey, Jude E. Okokon1, Emmanuel I. Etim2, Francis U. Umoh3, Emmanuel Bassey1 Department of Chemistry, Faculty of Science, 1Department of Pharmacology and Toxicology, Faculty of Pharmacy, 2Departments of Pharmaceutical and Medicinal Chemistry and 3Pharmacognosy and Traditional Medicine, University of Uyo, Uyo, Nigeria Received: 01.11.2008 Revised: 23.01.2009 Accepted: 09.12.2009 DOI: 10.4103/0253-7613.59924 Correspondence to: Dr. Jude E. Okokon E-mail: judeefiom@yahoo.com ABSTRACT Objective: To evaluate the in vivo antimalarial activities of ethanolic leaf and stembark extracts of Anthocleista djalonensis used traditionally as malarial remedy in Southern Nigeria in mice infected with Plasmodium berghei berghei. Methods: The ethanolic extracts of the A. djalonensis leaf (1000 – 3000 mg/kg/day) and stembark (220 – 660 mg/kg/day) were screened for blood schizonticidal activity against chloroquine-sensitive P. berghei in mice. The schizonticidal effect during early and established infections was investigated. Results: The A. djalonensis leaf extract (1000 – 3000 mg/kg/day) exhibited a significant antiplasmodial activity both in the 4-day early infection test and in the established infection with a considerable mean survival time, which was incomparable to that of the standard drug, chloroquine (5 mg/kg/day). The stembark extract (220 – 660 mg/kg/day) also demonstrated a promising blood schizontocidal activity in early and established infections. Conclusion: These plant extracts possess considerable antiplasmodial activities, which justify their use in ethnomedicine and can be exploited in malaria therapy. KEY WORDS: Antimalarials, antiplasmodial, Anthocleista djalonensis, P. berghei berghei Introduction Malaria is still a major public health problem in Nigeria and other tropical countries where transmission of the disease is rarely controlled. Poor and inadequate drainage system has encouraged breeding of mosquitoes, causing infection to spread. Herbal preparations are patronised by most people in urban areas for the treatment of malaria infections in spite of the availability of conventional antimalarial drugs. Anthocleista djalonensis A.Chev (Loganiaceae) is a medium-sized tree of West tropical Africa, 30 – 45 feet-high, with blunt spines on the unbranched, pale grey trunk and widespreading crown.[1] The stem, rootbark and leaves of A. djalonensis are used to treat malaria, jaundice, diabetes and abscesses.[1,2] The seeds, barks and roots are used in Nigeria by the Igbos as antipyretic, laxative and remedy for various stomach disorders.[3] It is used to cure epilepsy in Ghana[2,4] and in southern Nigeria, the leaves and stembark are used traditonally as malarial remedy.[4] Burkill[5] reported that the plant is used as febrifuge, abortifacient and pain killer. Okorie[6] isolated phthalide and xanthones from A. djalonensis. Onocha et al.[7] isolated monoterpene diol, djalonenol and iridoid glucoside djalonenoside (also sweroside). They also isolated a dibenzo-alpha-pyrone-djalonensone from this plant. Some of these compounds and their semisynthetic derivatives were found to be cytotoxic against the brain tumortransformed fibroblasts.[8] Reports of antibacterial and wound 258 Indian J Pharmacol | Dec 2009 | Vol 41 | Issue 6 | 258-261 healing activity[3,9] as well as in vitro anthelmintic activity[10] have been published. We have investigated the antiplasmodial activity of the ethanolic extract of the leaves and stembark to ascertain their ethnobotanical uses. Materials and Methods Plant material The leaves and stembark of A. djalonensis (A.Chev) (Loganiaceae) were collected in August, 2007, from the Nyan forest in the Uruan area of Akwa Ibom State and authenticated by Dr. Margaret Bassey, a taxonomist in the Department of Botany, University of Uyo, Uyo, Nigeria. A voucher specimen of the plant was deposited in the Faculty of Pharmacy Herbarium, University of Uyo, Uyo. The plant materials were shade-dried and then powdered. Preparation of extracts The dried and powdered leaves and stembark of A. djalonensis (1 kg each) were separately and exhaustively macerated in 70% ethanol for 72 h. The liquid extracts obtained were concentrated in vacuo at 40°C. The corresponding yields were 3.16 and 3.25%, respectively. Phytochemical screening Phytochemical screening of the extracts was carried out employing standard procedures.[11,12] Bassey, et al.: Antimalarial activity of Anthocleista djalonensis Animals Swiss albino mice (21-32 g) of both sexes were obtained from the University of Uyo animal house, Uyo, Nigeria. The animals were housed in standard cages and acclimatized for a period of 10 days. The mice were maintained on standard pelleted diet and water ad libitum. Approval for the study was obtained from the Animal Ethics Committee, University of Uyo. Acute toxicity Acute toxicity study of the extracts was carried out to determine the LD50 of the extracts using mice by the intraperitoneal route using the method of Lorke.[13] Parasite innoculation The chloroquine-sensitive Plasmoduim berghei berghei strain was obtained from the National Institute of Medical Research (NIMR), Lagos, Nigeria, and was maintained in mice. The inoculum consisted of 5 × 107 P. berghei berghei-parasitized red blood cells per milliliters. This was done by determining both the percentage of parasitaemia and the red blood cell count of the donor mice using a hemocytometer and diluting the blood with isotonic saline in the proportion indicated by both determinations. Drug administration The drugs and the extract used in this study were orally administered with the aid of a feeding cannula. Evaluation of suppressive activity on early infection A method described by Knight and Peters[14] was used. The animals were divided into eight groups of five animals each and administered with leaf extract of 1000, 2000 and 3000 mg/kg/day and stembark extract (220, 440 and 660 mg/kg) and chloroquine (phosphate salt; Sigma, USA) (5 mg/kg) was used as positive control and the negative control group received distilled water. Each mouse was inoculated on the first day (Do), intraperitoneally, with infected blood containing 1 x 107 P. berghei berghei-parasitized red blood cells. The mice were treated daily from day 0 (immediately after infection) to day 3. On the fifth day of the test (day 4), a thin blood smear was made from the tail blood sample of each mouse and stained with Giemsa and the percentage of parasitaemia was determined by counting the number of parasitized erythrocytes out of 200 erythrocytes in random fields of the microscope. The mean chemosuppression was calculated as 100 [(A—B)/A], where A is the mean percentage of parasitaemia in the negative control group and B is the mean percentage of parasitaemia in the test group. Evaluation of curative activity on established infection (Rane test) A modified method similar to Ryley and Peters[15] was used. On the first day (day 0), standard inoculum of 1 x 107 P. berghei berghei-infected erythrocytes was injected intraperitoneally into the mice. Seventy- two hours later, the mice were divided into eight groups (A H) of five animals each. Groups A, B and C were administered, respectively, with 1000, 2000 and 3000 mg/kg of A. djalonensis leaf extract. A. djalonensis stembark extract (220, 440 and 660 mg/kg) was administered to groups D, E and F, respectively. Chloroquine (phosphate salt; Sigma) (5mg/ kg) was given to group G, which served as the positive control, while the negative control, group H, was given distilled water. The drug/extract was given once daily for 5 days. Thin smears stained with Giemsa stain were prepared from the tail blood of each mouse daily for 5 days to monitor the parasitaemia level. The mean survival time for each group was determined in each group over a period of 30 days (D0D29). Statistical analysis Data are reported as mean ± standard error of the mean (SEM) and were analyzed statistically using one-way ANOVA followed by the Tukey Kramer multiple comparison test and values of P < 0.01 were considered significant. Results Phytochemical screening The results of the phytochemical screening of the plant extracts are summarized in Table 1. Acute toxicity The mice were treated intraperitoneally with a single dose of 1, 2, 3, 4 and 5 g/kg of either A. djalonensis leaf or stembark extract after being starved for 24 h. A. djalonensis leaf extract (1–5 g/kg) produced physical signs of toxicity 1 h after administration,which include writhing, decreased motor activity, gasping and body/limb tone. All the animals treated with 5.0 g/kg of the extract survived and the LD50 was calculated to be 5.0 g/kg according to Lorke’s rule. The stembark extract of A. djalonensis (1, 2, 3, 4 and 5 g/kg) produced physical signs of toxicity 30 min to1 h after administration. The signs include writhing, gasping, palpitation, ptosis, decreased respiratory rate and death. All the mice treated with 2.5 g/kg and above doses of the extract died. The LD50 was calculated according to Lorke’s rule to be 2.23 g/kg. Suppressive test Evaluation of the suppressive activity of A. djalonensis leaf extract during early infection shows that the leaf extract produced a dose-dependent chemosuppressive effect at the various doses employed in this study (1000, 2000, 3000 mg/kg/day), with a chemosuppression of 17.05%, 26.82% and 41.46%, respectively [Table 2]. Similarly, the ethanolic stembark extract of A. djalonensis produced a dose -dependent chemosuppressive effect at the different doses employed (220, 440 and 660 mg/kg), with a chemosuppression of 22.61%, 27.39% and 33.32%, respectively. The effects of these extracts Table 1 The combined results of the phytochemical screening of the extracts Alkaloids Cardiac Glycosides De-oxy- sugars Flavonoids Phlobatanins Saponins Tannins Terpenes Anthraquinones Source of extract A. djalonensis leaf extract Stembark extract + + + + + + + - + + + + + + - Indian J Pharmacol | Dec 2009 | Vol 41 | Issue 6 | 258-261 259 Bassey, et al.: Antimalarial activity of Anthocleista djalonensis were significant (P<0.05) when compared with the control. The standard drug, chloroquine (5 mg/kg/day), caused 81.37% suppression [Table 2]. Effect of extracts on established infection Treatment of the P. berghei-infected mice with the plant extracts caused a dose-dependent daily reduction in parasitaemia in the extract-treated groups similar to that of the chloroquine-treated group, while the control group showed a daily increase in parasitaemia. In the groups treated with the leaf extract of A. djalonensis 1000, 2000 and 3000 mg/kg/day, the percentage of parasitaemia on day 7 was 14.3%, 12.0% and 10.4%, respectively, while the chloroquine-treated and control groups, respectively, had percentage parasitaemia of 5.0 and 33.0% [Figure 1]. The stem bark extract-treated groups had percentage parasitaemia of 15.3, 12.9 and 11.2%, respectively, for the 220, 440 and 660 mg/kg doses of extract-treated groups [Figure 2]. Data in Table 3 show that chloroquine (5 mg/kg/day) gave Table 2 Suppressive activities of leaf and stembark extracts of A. djalonensis during early P. berghei berghei infection in mice (4- day test) Drug/ extract Dose Average % Average % (mg/kg/ day) parasitaemia suppression 1000 22.67 ± 0.40* 17.05 2000 3000 220 440 20.00 ± 0.63* 16.00 ± 0.63* 21.67 ± 0.08* 20.32 ± 0.53* 26.82 41.46 21.61 27.39 660 18.67 ± 0.70* 33.32 5 0.2ml 4.67 ± 0.53* 28.06 + 0.07 81.37 - A.djalonensis leaf extract Stembark extract Chloroquine Distilled water (control) a mean survival time of 30.0 + 0.00 (mean + SEM) days compared to 17.67 + 1.70, 20.33 + 0.47 and 21.67 + 3.86 days, respectively, observed for the groups treated with 1000, 2000 and 3000 mg/kg of ethanolic leaf extract of A. djalonensis. Animals treated with 220, 440 and 660 mg/kg of A. djalonensis stembark extract had a mean survival time value of 16.33 + 2.08, 18.38 + 1.15 and 21.33 + 0.58 days, respectively. The mice in the control group survived for 14 days only. Discussion The results show that A. djalonensis leaf and stembark extracts have moderate to negligible toxicity, as shown in their LD50 values of 5.0 g/kg and 2.23 g/kg for the leaf and stembark extract, respectively.[16] The results also show that the plant extracts possess significant antiplasmodial activity, as evident from the chemosuppressions obtained during the 4- day early-infection test. The leaf and stembark extracts also exhibited a significant curative effect during established infection less than to the standard drug, chloroquine (5 mg/kg/ day), as demonstrated in the mean survival time of the mice in the extract- and chloroquine-treated groups. Although the antimalarial activities demonstrated by the leaf and stembark extract of A. djalonensis are low, these may have resulted from the crude nature of these extract and could be enhanced by further purification of these extracts. A. djalonensis leaf has been reported above to contain some phytochemical compounds like alkaloids, terpenes (monoterpenes) and flavonoids. Figure 1: Antiplasmodial activity of the ethanolic leaf extract of Anthocleista djalonensis during early infection Data are expressed as mean ± SEM for five animals per group.*P < 0.05 when compared with control Table 3 Mean survival time of mice receiving the various doses of ethanolic extracts of leaf and stembark extracts of A. djalonensis during an established P. berghei infection in mice Drug/extract A.djalonensis Leaf extract Stembark extract Chloroquine Distilled water (control) Dose (mg/kg/ day) Mean survival time (days) 1000 2000 3000 220 440 660 5 0.2 ml 17.67 ± 0.70* 20.33 ± 0.47* 21.67 ± 0.86* 12.33 ± 0.15* 16.33 ± 0.18* 21.33 ± 0.58* 30.0 ± 0.00* 13.67 ± 0.47 Data are expressed as mean ± SEM for five animals per group.*P<0.05 when compared to control 260 Indian J Pharmacol | Dec 2009 | Vol 41 | Issue 6 | 258-261 Figure 2: Antiplasmodial activity of the ethanolic stembark extract of Anthocleista djalonensis during early infection Bassey, et al.: Antimalarial activity of Anthocleista djalonensis Antiplasmodial screening of plants has implicated alkaloids, terpenes and flavonoids in this activity.[17,18] Sesquiterpenes and monoterpenes such as limonene have been implicated in endoperoxidation, leading to plasmodicidal activity.[19] The antiplasmodial activity observed with this plant could have resulted from its phytochemical constituents. Although the mechanism of action of these extracts has not been elucidated, some plants are known to exert antiplasmodial activity either by causing red blood cell oxidation[20] or by inhibiting protein synthesis,[21] depending on their phytochemical constituents. The extract could have exerted its action through either of the two mechanisms mentioned above or by some other unknown mechanism. These compounds may be acting singly or in synergy with one another to exert the antiplasmodial activity observed in this study. Thus, the active principle needs to be identified. The results of the present study indicate that the extracts of the leaf and stembark extracts of the plant possess antimalarial activity. This confirms their use in ethnomedicine in the treatment of malaria. Therefore, it would be interesting if the active principles are isolated and characterized. Acknowledgement The authors are grateful to Mr. Nsikan Malachy of the Department of Pharmacology and Toxicology, University of Uyo, Uyo, for his technical assistance. References 1. 2. 3. 4. 5. Dalziel JM. The useful plants of West Tropical Africa. London: Crown Agents for Overseas colonies; 1995. p. 361. Oliver-Bever B. Medicinal plants in Tropical West Africa. London: Cambridge University Press; 1986. p. 109–110. Okoli AS, Iroegbu CU. Evaluation of extracts of Anthocleista djalonensis, Nauclea latifolia and Uvaria afzali for activity against bacterial isolates from cases of nongonococcal Urethritis. J Ethnopharmacol 2004;92:135–44. Iwu MM. Handbook of African medicinal plants. London: CRC Press; 2000. p. 13. Burkill HM. The useful plants of West Tropical Africa. 2nd ed. Vol. 3. Kew (London): Royal Botanic Garden; 1985. Okorie DA. A new phthalide and xanthones from Anthocleista djalonensis and Anthocleista vogelli. Phytochemistry 1976;15:1799–800. 7. Onocha PA, Okorie DA, Connolly JD, Roycroft DS. Monterpene diol, iridoid glucoside and dibenzo-alpha-pyrone from Anthocleista djalonensis. Phytochemistry 1995;40:1183-9. 8. Onocha PA, Okorie DA, Connolly JD, Krebs HC, Meier B, Habermehl GG. Cytotoxic activity of the constituents of Anthocleista djalonensis and their derivatives. Nig J Natural Products and Medicine 2003;7:58–60. 9. Chah KF, Eze CA, Emuelosi CE, Esimone CO. Antibacterial and wound healing properties of methanolic extracts of some Nigerian Medicinal Plants. J Ethnopharmacol 2006;104:164–7. 10. Nweze NE, Ngongeh LA. In vitro Anthelmintic Activity of Anthocleista djalonensis. Nig Vet J 2007; 28:9-13. 11. Sofowora A. Medicinal plant and traditional medicine in Africa. 2nd ed. Ibadan (Nigeria): Spectrum books; 1996. 12. Trease A, Evans WC. Trease and Evans Pharmacognosy. 13th ed. London: Bailliere Tindall; 1989. 13. Lorke DA. New Approach to Practical Acute Toxicity test. Arch Toxicol 1983;54:275-86. 14. Knight DJ, Peters W. The antimalarial action of N-benzyloxydihydrotriazines 1. The action of clociguanil (BRL50216) against rodent malaria and studies on its mode of action. Ann Trop Med Parasitol 1980; 74:393–404. 15. Ryley JF, Peters W. The antimalarial activity of some quinone esters. Ann Trop Med Parasitol 1970; 84:209-22. 16. Homburger F. In vivo testing in the study of toxicity and safety evaluation. In: Marquis JK, editor. A Guide to General Toxicology. 2nd ed. New York: Karger; 1989. p. 198. 17. Christensen SB, Kharazmi A. Antimalarial natural products. Isolation, characterization and biological properties. In. Tringali C, editor. Bioactive compounds from natural sources: Isolation, characterization and biological properties. London: Taylor and Francis; 2001. p. 379–432. 18. Philipson JD, Wright C. Antiprotozoal compounds from plants sources. Planta Med 1991; 57:553-9. 19. Hatzakis E, Opesenica I, Solaja BA, Stratakis M. Synthesis of novel polar derivatives of the antimalarial endoperoxides ascaridole and dihydroascaridole. ARKIVOC 2007;(Pt.VIII):124–5. 20. Etkin NL. Antimalarial plants used by Hausa in Northern Nigeria. Trop Doct 1997; 27:12–6. 21. Kirby GC, O’Neil MJ, Philipson JD, Warhurst DC. In vitro studies on the mode of action quassionoids with activity against chloroquine–resistant Plasmodium falciparum. Biochem Pharmacol 1989;38:4367–74 6. Author Help: Online submission of the manuscripts Articles can be submitted online from http://www.journalonweb.com. For online submission, the articles should be prepared in two files (first page file and article file). Images should be submitted separately. 1) 2) 3) 4) First Page File: Prepare the title page, covering letter, acknowledgement etc. using a word processor program. All information related to your identity should be included here. Use text/rtf/doc/pdf files. Do not zip the files. 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