Anti-Viral Activities of Selected Kenyan Medicinal Plants against the Hepatitis - B Virus
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Introduction: Hepatitis B Virus (HBV) is a human pathogen causing serious liver disease. The virus is the major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide. It is endemic in many parts of the world especially in Asia and Africa, and an estimated 2 billion people are infected, with over 600,000 dying each year. Kenya has been considered among high endemic areas for HBV infection with upward trends of sero-prevalence among blood donors being observed. Although immunization against HBV has been widely used, current medicines for the management of HBV infection in humans are few, limited in efficacy  and relatively expensive, making them unavailable to most of the needy cases, especially those in developing countries. The resistance to these agents is also spreading fast. The search for new therapeutic agents for HBV infections is an ongoing effort and a number of researchers are now paying attention to active anti-viral compounds from natural products including plants because of their widespread use in developing countries and the large repertoire that has not been systematically investigated. The plants in this study were mainly chosen based on previous studies on their anti-viral activity. They exhibit activity against the Human Immunodeficiency Virus, Herpes Simplex Virus and Cytomegalovirus and were therefore highly postulated to have anti - HBV activity. They were also chosen based on their ethno-pharmacological use including relief of symptoms associated with viral infections.

Methods: In this study, the root barks of Carissa edulis (Forssk.) Vahl and Maytenus heterophylla (Eckl. & Zeyh.) Robson and the stem barks of Prunus africana (Hook.f.) Kalkman and Acacia mellifera (Vahl) Benth were harvested from within Kenya from among medicinal plants used widely for the management of various diseases using information obtained from literature search and their ethno - pharmacological use. Their identity was established at the School of Biological Sciences, University of Nairobi, and voucher specimens were deposited at the herbarium. These were air dried for two weeks and the water extracts of C.edulis, P.africana and M.heterophylla; methanol extracts of A.mellifera and chloroform extracts of P.africana introduced into the cell cultures of an HBV producing cell line (SNU-182) at concentrations of 1000 µg/mL, 500 µg/mL, 250 µg/mL, 125 µg/mL, 62.5 µg/mL, 31.25 µg/mL and 15.125 µg/mL. Lamivudine, Active Pharmaceutical Ingredient (API) was used as a reference drug for the investigations. Inhibition of the expression of the Hepatitis B Surface antigen (HBsAg) and HBV DNA released into the culture supernatant were used as the anti-viral indicator. A semi-quantitative Enzyme Linked Immuno Sorbent Assay (ELISA) technique was used for initial screening of the effects of the plant extracts on the expression of the Hepatitis B Surface antigen (HBsAg) while the quantity of HBV DNA was assayed by real time Polymerase Chain Reaction (PCR). The in-vitro cytotoxicity of the extracts was determined by MTT assays.

Results: Of the five plant extracts examined using the ELISA technique, three exhibited some anti - HBV activity in vitro with a CC50 of more than 100 µg/mL, suggesting the need for further investigations of their possible use in the management of HBV infections. These were the aqueous extracts from Carissa edulis (Forssk.) Vahl (Apocynaceae) and Prunus africana (Hook .f.) Kalkman (Rosaceae) and the methanol extract from Acacia mellifera (Vahl) Benth (Fabaceae). At a concentration of 200 µg/ml the extract of C.edulis exhibited the highest activity of just over 12.15 % inhibition rate relative to negative control, which was slightly below the 15 % inhibition rate of Lamivudine positive control at a concentration of 100 µg/mL. The activities of the P.africana and A.mellifera extracts of 5 % inhibition and 2.15 % inhibition respectively, relative to controls, was higher than the 2 % inhibition activity of Lamivudine positive control at a concentration of 30 µg/mL.

These results were confirmed using the quantitative real time PCR technique where the aqueous extract of C.edulis and the methanol extract of A.mellifera exhibited sustained activity over a range of plant extract concentrations above 31.25 µg/mL and 125 µg/mL. The aqueous and chloroform extracts of P.africana also exhibited activity using this technique.The evaluation of the EC50 done on the first two plant extracts exhibiting notable anti - HBV activity using this technique yielded considerably higher values than the corresponding CC50 . values. The C. edulis’ EC50 was 331.6 µg/ml while that of A.mellifera was 295.0 µg/ml. These values indicate a high level of toxicity at potent concentrations and signify the need for further investigations to identify and isolate the toxic components.

Conclusion: The results obtained in this study provide evidence of the anti-HBV activity of Carissa edulis (Forssk.) Vahl ,  Acacia mellifera (Vahl) Benth and Prunus africana (Hook .f.) Kalkman.  Further investigations are however needed to establish their possible use in the management of HBV infection and to identifty and isolate toxic components with a view to improve the bio-activity.

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