Original Article Cellular antioxidant, cytotoxic and antileishmanial activities of Hippocratea africana Jude E. Okokon, Ashana Dar1, Iqbal M. Choudhary1 ABSTRACT Introduction: Antioxidants of natural origin are of great health benefits in the prevention and treatment of various diseases associated with reactive oxygen species. This investigation was aimed to evaluate the cellular antioxidant, cytotoxic and antileishmanial activities of Hippocratea africana (Willd.) Loes. ex Engl. (family-Celastraceae) syn. Loeseneriella africana (Willd.) N.Hallé. Materials and Methods: The ethanol root extract and fractions of Hippocratea africana were investigated for cytotoxicity activity against HeLa cells using sulforhodamine-B (SRB) method. Antioxidative burst activity of the extract in whole blood, neutrophils and macrophages was also investigated using luminol/ lucigenin-based chemiluminescence assay. The ethanol extract and fractions were similarly screened for antileishmanial activity against promastigotes of Leishmania major in vitro. The GC-MS analysis of the most active fraction against HeLa cells was carried out. Results: The ethanol root extract was found to exert strong cytotoxic activity with the hexane fraction exhibiting the most pronounced effect. The ethanol crude extract significantly inhibited oxidative burst activity in whole blood (P < 0.05-0.001), isolated polymorphonuclear cells (PMNs) (P < 0.01-0.001) and mononuclear cells (MNCs) (P < 0.05-0.001) when two different phagocytosis activators (serum opsonizing zymosan-A and PMA) were used. The ethanol crude extract also exhibited moderate antileishmanial activity against promastigotes of Leishmania major in vitro. GCMS analysis of active fraction revealed pharmacologically active compounds such as sabinene, α – Thujene, retinoic acid methyl ester, phenol,3,4,5-trimethoxy and others. Conclusion: These results suggest that the root extract/ fractions of Hippocratea africana possess cytotoxic, antioxidative burst and antileishmanial activities. Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria, 1 HEJ, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan Key words: Antileishmanial, antioxidant, celastraceae, cytotoxicity, Hippocratea africana INTRODUCTION Access this article online Website: www.jnatpharm.org DOI: *** Quick Response Code: Address for Correspondence: Dr. Jude E Okokon, Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria. E-mail: judeefiom@yahoo.com Members of Celastraceae family are used in folk medicine in different regions of the world to treat various diseases. However, their medicinal potentials have been ascribed to the presence of some secondary metabolites of medicinal importance such as alkaloids, lupanes, oleanes, alkaloids, flavonoids, sesquiterpenes and triterpenes, which have been implicated in the various bioactivities of members of this family like anticancer and antimicrobial.[1] The genus Hippocratea with about 115 species is distributed mainly in tropical areas of America, Africa and Asia and they are greatly used in the various areas for medicinal values. Hippocratea africana (Willd.) Loes. ex Engl. (family-Celastraceae) syn. Loeseneriella africana (Willd.) N.Hallé is Journal of Natural Pharmaceuticals, Volume 4, Issue 2, July-December, 2013 a green forest perennial climber without hairs (glabrous) and reproducing from seeds.[2] It is commonly known as African paddle-pod. The Ibibio tribe of Nigeria calls it ‘Eba enang enang’. The plant is widely distributed in tropical Africa. The root of the plant is used traditionally by the Ibibios of the Niger Delta region of Nigeria in the treatment of various ailments such as fever, malaria, body pains, diabetes and diarrhea.[3] The plant (root) has been reported to possess in vivo antiplasmodial,[3] antiinflammatory and analgesic,[4] antidiabetic and hypolipidemic[5] and antidiarrhoeal and antiulcer activities,[6] and hepatoprotective potentials.[7] Reports of scientific studies on Hippocratea africana are few so we investigated the antioxidative burst, cytotoxic and antileishmanial activities of the root extract of Hippocratea africana. 81 Okokon, et al.: Biological activities of Hippocratea africana MATERIALS AND METHODS Plants collection The plant material Hippocratea africana (roots) were collected in compounds in Uruan area, Akwa Ibom State, Nigeria in April, 2011. The plant was identified and authenticated by Dr. Margaret Bassey of Department of Botany and Ecological Studies, University of Uyo, Nigeria. Herbarium specimen was deposited at Faculty of Pharmacy Hebarium (FPUU 233), University of Uyo. Extraction The roots were washed and shade-dried for two weeks. The dried plants’ materials were further chopped into small pieces and reduced to powder. The powdered material was macerated in 70% ethanol. The liquid filtrates were concentrated and evaporated to dryness in vacuo 40°C using rotary evaporator. The crude ethanol extract (100 gm) was further partitioned successively into 1 L each of n-hexane, dichloromethane, ethyl acetate and butanol to give the corresponding fractions of these solvents. Cellular antioxidant (immunomodulatory) activity The ethanol crude extract was screened for cellular antioxidant activities in whole blood, neutrophils and macrophages using chemiluminescence assay. Briefly, luminol or lucigenin-enhanced chemiluminescence assay were performed as described earlier,[8,9] Briefly, 25 μL diluted whole blood (1:50 dilution in sterile HBSS++) or 25 μL of PMNCs (1 × 106) or MNCs (5 × 106) cells were incubated with 25 μL of serially diluted plant extract with concentration ranges between 6.25 and 100 μg/mL. Control wells received HBSS++ and cells without extract. Tests were performed in white 96 wells plates, which were incubated at 37°C for 30 min in the thermostated chamber of the luminometer. Opsonized zymosan-A or PMA 25 μL, followed by 25 μL luminol (7 × 105M) or lucigenin (0.5 mM) along with HBSS++ was added to each well to obtain a 200 μL volume/well. The luminometer results were monitored as chemiluminescence (Relative Luminescence Units, (RLU)) with peak and total integral values set with repeated scans at 30s intervals and 1s points measuring time. Cytotoxic activity The growth inhibitory and cytotoxic activities of the ethanol crude extract and fractions were evaluated against HeLa cells (Cervix cancer cell) by using the sulforhodamine-B assay.[10] The cells (10000 cells/100 μL) in 96-well plate were incubated for 24 h at 37°C in a humidified 5% CO2 incubator. The stock solutions of ethanol extract and fractions were prepared in dimethyl sulfoxide (DMSO). Various dilutions of the ethanol crude extract and fractions (0.1, 1, 10, 100 and 250 μg/mL), were added (100 μL) in each well. After 48 h of incubation, 50 μL of cold trichloroacetic acid (TCA) (50%) was added gently and left for 30 min 82 at room temperature, followed by washing with distilled water and drying overnight. To each well, 100 μL of SRB solution (0.4% wt/vol in 1% acetic acid) was added and after 10 min, the unbound stain was removed by washing with acetic acid (1%) and air-dried at room temperature. The protein bound stain was solubilized with tris base (pH 10.2), and was shaken for 5 min. Absorbance was measured at 515 nm using a microplate reader. The absorbance of the appropriate blanks, including test substance blank, and control (without drug), was used to calculate the growth inhibition, and cytotoxicity of the test compounds, and represented as GI50, TGI and LC50 (μg/mL) values. Antileishmanial activity The antileishmanial activity of the ethanol crude extract and fractions were evaluated against promastigotes of Leishmania major (Defence Science and Technology Organization, DESTO) in culture using microplates. Leishmania major promastigotes were grown in bulk, early in modified Novy-MacNeal-Nicolle (NNN) biphasic medium, using normal physiological saline. Then the promastigotes were cultured with RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum (FBS). The parasites (Leishmania major) were harvested at log phase and centrifuged at 3000 rpm for 10 min. They were washed three times with saline at same speed and time. Finally, the parasites were counted with the help of Neubauer chamber under the microscope and diluted with fresh culture medium to give a final density of 106 cells/mL. In a 96-well micro titer plate, 180 mL of the culture medium was added in different wells. The ethanol crude extract and fractions were dissolved in PBS (Phospate buffered saline, pH 7.4 containing 0.5% MeOH, 0.5% DMSO) to make a stock concentration of 1000 mg/mL. 20 μl of each extract/fraction concentration was added to the wells and serially diluted to get working concentrations ranging between 1.0 μg/mL to 100 μg/mL. 100 mL of parasite culture (final density of 106 cells/mL) was added in all wells. Two rows were left, one for negative and other for positive control. Negative controls received the medium while the positive controls received Pentamidine and amphotericin B as standard antileishmanial compounds. The plate was incubated between 21-22°C for 72 h. The culture was examined microscopically for cell viability by counting the number of motile cells on an improved neubauer counting chamber and IC50 values of compounds possessing antileishmanial activity were calculated.[11] GC-MS analysis of fractions Quantitative and qualitative data were determined by GC and GC-MS, respectively. The hexane fraction was injected onto a Shimadzu GC-17A system, equipped with an AOC-20i autosampler and a split/ splitless injector. The column used was an DB-5 (Optima-5), 30 m, 0.25 mm i.d., 0.25 μm df, coated with 5% diphenyl-95% polydimethylsiloxane, operated with the following oven Journal of Natural Pharmaceuticals, Volume 4, Issue 2, July-December, 2013 Okokon, et al.: Biological activities of Hippocratea africana temperature programme: 50°C, held for 1 min, rising at 3°C/min to 250°C, held for 5 min, rising at 2°C/min to 280°C, held for 3 min; injection temperature and volume, 250°C and 1.0 μl, respectively; injection mode, split; split ratio, 30:1; carrier gas, nitrogen at 30 cm/s linear velocity and inlet pressure 99.8 KPa; detector temperature, 280°C; hydrogen, flow rate, 50 mL/min; air flow rate, 400 mL/min; make-up (H2/air), flow rate, 50 mL/min; sampling rate, 40 ms. Data were acquired by means of GC solution software (Shimadzu). Gas chromatography-mass spectrometry analysis Agilent 6890N GC was interfaced with a VG Analytical 70-250s double-focusing mass spectrometer. Helium was used as the carrier gas. The MS operating conditions were: ionization voltage 70 eV, ion source 250°C. The GC was fitted with a 30 m × 0.32 mm fused capillary silica column coated with DB-5. The GC operating parameters were identical with those of GC analysis described above. Identification of the compounds The identification of components present in the active fraction of the plants’ extracts was based on direct comparison of the retention times and mass spectral data with those for standard compounds, and by computer matching with the Wiley and Nist Libraries attached to the machine, as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literatures.[12,13] Table 1: Anicancer acivity of crude extract and fracions of root of Hippocratea africana against HeLa cells Extract/Frac on GI50 (μg/mL) Crude extract Hexane frac on DCM frac on Ethyl acetate frac on Butanol Aqueous frac on Doxorubucin 160.0±5.00* 14.3±0.32* 37.3±2.40* 52.6±1.45* 64.3±1.20* 64.6±1.20* 0.61±0.03 μM LC50 (μg/mL) TGI (μg/mL) 241.0±2.90* 213.0±3.21* 73.3±0.87* 40.5±2.90* 84.0±1.15* 64.0±1.52* — 76.0±1.15* — 82.0±0.57* — 85.6±0.32* 7.80±0.80 μM 3.60±0.30 μM Data are represented as mean±SEM of three independent experiments. *p<0.001 rela ve to the standard drug, Values in the table are concentra ons of extract/ frac on expressed as μg/ml, GI50: Concentra on of the drug causing 50% growth inhibi on of the cells, TGI: Concentra on of the drug causing total growth inhibi on of the cells, LC50: Lethal concentra on of the drug that killed 50% of the cells Table 2: Cellular anioxidant acivity of ethanolic root extract of Hippocratea africana Cell type Concentra on (μg/mL) % Inhibi on (RLU) Whole blood Neutrophils (intracellular) Neutrophils (extracellular) Macrophages 1 10 100 0.5 5 50 0.5 5 50 0.5 5 50 IC50 (μg/ml) 6.70±0.75* 82.70±6.00*** 99.30±0.63*** 24.50±1.24** 65.00±2.83*** 99.30±0.17*** 36.30±7.57** 76.40±1.27*** 96.20±0.11*** 8.70±3.06* 74.40±3.69*** 99.70±0.00*** Table 3: Anileishmanial acivity of Hippocratea africana (EC50) Data obtained from this work were analyzed statistically using Students’ t-test and differences between means were considered significant at 1% and 5% level of significance i.e p ≤ 0.01and 0.05. Extract/Frac on EC50 (μg/ml) Crude ethanol extract Hexane frac on DCM frac on Ethyl acetate frac on Butanol frac on Aqueous frac on Pentamidine Amphotericin B 42.96±0.43* >100 >100 40.32±0.54* >100 >100 5.09±0.04 0.29±0.05 Cytotoxic activity against HeLa cells The results of cytotoxic activity of crude ethanol extract and fractions of Hippocratea africana shows prominent activity with the hexane fraction exerting highest activity than other fractions and crude ethanol extract [Table 1]. The potency order was hexane> dichloromethane> ethylacetate> butanol> aqueous> crude ethanol extract. 2.13 1.09 2.12 Data are represented as mean±SEM of 3 independent experiments.*p<0.05, **p<0.01, ***p<0.001 rela ve to control Statistical analysis and data evaluation RESULTS 3.71 Data are represented as mean±SEM of three independent experiments, *p<0.001 rela ve to the standard drugs in neutrophils when activated with PMA and 8.70 - 99.70% in macrophages [Table 2]. Antileishmanial activity Cellular antioxidant activity Ethanol crude root extract of Hippocratea africana was observed to produce significant inhibitory effect on the oxidative burst activities of the whole blood (p < 0.05-0.001) neutrophils (p < 0.01-0.001) and macrophages (p < 0.05-0.001) in a dose-dependent manner. The ethanol extract produced 6.70-99.30% inhibition in whole blood, 24.50-99.30% in neutrophils when activated with zymosan-A, 24.50-96.20% Ethanol crude extract and fractions of root extract of Hippocratea africana exerted prominent antileishmanial activity when tested against promastigotes of Leishmania major. Ethyl acetate fraction exerted a higher activity than other fractions with EC50 value of 40.32 ± 0.54 μg/ml and ethanol crude extract though incomparable to the standard drugs, pentamidine and amphotericin B [Table 3]. Journal of Natural Pharmaceuticals, Volume 4, Issue 2, July-December, 2013 83 Okokon, et al.: Biological activities of Hippocratea africana Table 4: GC – ms analysis of hexane fracion of Hippocratea africana Name of compound Benzoic acid, 4-hydroxy-3-methoxy-,ethyl ester Phenol,3,4,5-trimethoxy 4a(2H),phenanthrene carboxaldehyde 1,3,4,9,10,10a-hexahydro-6-methoxy-1, 1-dimethoxy-1,1-dimethy-7-(1-methyl ethyl),4aR-trans α – Thujene Sabinene Aspidofrac nine-3-methanol,17-methoxy-(2a,3a,5a) Re noic acid methyl ester 4a(2H)-Phenanthrenemethanol,1,3,4,9,10,10a-hexahydro-6-methoxy-1, 1-dimethyl-7-(1-methylethyl)-,(4Ar-trans)- GC-MS analysis of hexane fraction Phytochemical analysis of hexane fraction by GC-MS revealed the presence of some pharmacologically active compounds [Table 4]. DISCUSSION Hippocratea africana is use traditionally in the treatment of various ailments and diseases including malaria, diabetes, ulcer and inflammatory diseases among others. The root which has been reported to possess some pharmacological properties have been found in this study to exert pronounced cytotoxic activity against HeLa cells with the hexane fraction having the highest activity. The cytotoxic mechanism of action was found to be unrelated to DNA interaction and is likely to involve interference with cell division processes. However, the GCMS analysis revealed the presence of some pharmacologically active compounds such as retinoic acid and phenanthrene derivatives which have been implicated in the cytotoxic activity of plants.[14-16] These compounds and others are likely to be involved in the cytotoxic activity of this extract. The root extract was also observed to exhibit strong antioxidant activity in whole blood, neutrophils (extracellular and intracellular) and macrophages. This activity may have resulted from the presence of phenolic compounds such as 3, 4, 5 trimethoxy phenol and other monoterpenes (thujene and sabinene) and sesquiterpenes as revealed by GCMS analysis. These compounds have been reported to possess antioxidant activity.[17-20] Although no compound was isolated in this study, these compounds present in the root extract may have contributed to the significant antioxidant activity observed. The significant antioxidant activity of this extract explains the strong cytotoxic activity of the root extract. Generation of reactive oxygen species has been implicated in the pathogenesis of cancer and other diseases.[21] The activities of antioxidant counteract the redox state precipitated intracellularly and hence ensure cytotoxicity. This could possibly be one of the mechanisms of cytotoxic activity of this extract. 84 Concentra on (%) 4.92 7.72 3.07 Mol. Wt 196 184 314 Chemical formula C10H12O4 C9H12O4 C21H30O2 RI 437 466 874 4.84 4.16 3.51 3.20 6.30 136 136 340 314 316 C10H16 C10H16 C20H26N2O C21H30O2 C21H32O2 925 964 993 1011 1022 The root extract also demonstrated antileishmanial activity. Compounds such as terpenes (mono and sesquiterpenes) which have been implicated in antiparasitic activities of plants[22] have been found to be present in this extract. These compounds may have been responsible for the antileishmanial activity observed in this study. CONCLUSION From the results of these studies, it can be concluded that the root extract of Hippocratea africana has cytotoxic activity against HeLa cells, immunomodulatory and antileishmanial activities which are due to the phytochemical constituents of the extract and fractions. ACKNOWLEDGMENT Dr. Jude Okokon is grateful to TWAS for financial support for postdoctoral fellowship and ICCBS for providing research facilities. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. Silva S, Silva G, Barbosa L, Duarte L, Filho SA. Lupane pentacyclic triterpenes isolated from stems and branches of Maytenus imbricata (Celastraceae). Helvet Chim Acta 2005;88:1102-9. Dalziel JM. Useful Plants of West Tropical Africa. London: Crown Agents for Overseas Government; 1956. Okokon JE, Ita BN, Udokpoh AE. The in vivo antimalarial activities of Uvariae chamae and Hippocratea africana. Ann Trop Med Parasitol 2006;100:585-90. Okokon JE, Antia BS, Umoh EE. Analgesic and antiinflammatory activities of Hippocratea africana. Int J Pharmacol 2008;4:51-5. Okokon JE, Antia BS, Umoh EE, Etim EI. Antidiabetic and hypolipidaemic activities of Hippocratea africana. Int J Drug Dev Res 2010;2:501-6. Okokon JE, Akpan HD, Ekaidem I, Umoh EE. Antiulcer and antidiarrhoeal activity of Hippocratea africana. Pak J Pharm Sci 2011;24:201-5. Okokon JE, Nwafor PA, Charles U, Dar A, Choudhary MI. Antioxidative burst and hepato protective effects of ethanol root extract of Hippocratea africana against paracetamol-induced liver injury. Pharm Biol 2013;27. Helfand S, Werkmeister J, Roder J. Chemiluminescence response of human natural killer cells. I. The relationship between target cell binding, chemiluminescence, and cytolysis. J Exper Med 1982;156:492-505. Journal of Natural Pharmaceuticals, Volume 4, Issue 2, July-December, 2013 Okokon, et al.: Biological activities of Hippocratea africana 9. 10. 11. 12. 13. 14. 15. Haklar G, Ozveri ES, Yuksel M, Aktan A, Yalcin AS. Different kinds of reactive oxygen and nitrogen species were detected in colon and breast tumors. Cancer Lett 2001;165:219-24. Houghton P, Fang R, Techatanawat I, Steventon G, Hylands PJ, Lee CC. The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity. Methods 2007;42:377-87. Atta-ur-Rahman, Choudhary MI, William JT. Bioassay techniques for drug development. U.S.: Harward Academic Publisher; 1997. p. 67-8. Adams RP. Identification of essential oils by gas chromatography quadrupole mass spectrometry. Carol Stream, USA: Allured Publishing Corporation; 2001. Setzer WN, Stokes SL, Penton AF, Takaku S, Haber WA, Hansell E, et al. Cruzain inhibitory activity of leaf essential oils of neotropical lauraceae and essential oil components. Nat Prod Comm 2007;2:1203-10. Sporn MB, Roberts AB. Biological methods for analysis and assay of retinoids. In: M. B. Sporn, editor. The Retinoids, Vol. 1. New York: Academic Press; 1984. p. 235. Martirosyan AR, Rahim-Bata R, Freeman AB, Clarke CD, Howard RL, Strobl JS. Differentiation – inducing quinolones as experimental breast cancer agents in the MCF-7 human breast cancer cell model. Biochem Pharmacol 2004;68:1729-38. 16. Wang YY, Taniguchi T, Baba T, Li Y, Ishibashi H, Mukaida N. Identification of phenenthrene derivatives as a potent anticancer drug with potent Pim kinase inhibitory activity. Cancer Sci 2012;103:107-15. 17. Aderogba MA, McGaw LJ, Bezabih M, Abegaz BM. Isolation and characterisation of novel antioxidant constituents of Croton zambesicus leaf extract. Nat Prod Res 2011;25:1224-33. 18. Falah S, Katayama T, Suzuki T. Chemical constituents from Gmelina arborea bark and their antioxidant activity. J Wood Sci 2008;54:483-9. 19. Kumar PP, Kumaravel S, Lalitha C. Screening of antioxidant activity, total phenolics and GC-MS study of Vitex negundo. Afr J Biochem Res 2010;4:191-5. 20. Okokon JE, Dar A, Choudhary MI. Immunostimulatory, anticancer and antileishmanial activities of Mammea africana from Nigeria. J Nat Pharm 2012;3:105-9. 21. Halliwell B, Gutteridge JM. Free radicals in biology and medicine. 3rd ed. Oxford: Oxford University Press; 1999. 22. Uma B, Parvathavarthini R. Antibacterial effect of hexane extract of sea urchin, Temnopleurus alexandri. Inter J Pharm Tech Res 2010;2:1677-80. Cite this article as: We will update details while making issue online***. Source of Support: Nil. Conflict of Interest: None declared. Journal of Natural Pharmaceuticals, Volume 4, Issue 2, July-December, 2013 85