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ANTIVIRAL AND CYTOTOXICITY ASSAY OF MEDICINAL PLANTS OF BOTSWANA AS POSSIBLE TREATMENTS FOR HIV/AIDS
Gatonye T.W.
200502848
Supervisor
M. M. Leteane
INTRODUCTION
HIV/AIDS is a global epidemic Over 15 000 new infections daily (Dixon,
2002) Botswana has one of the highest reported
HIV-prevalence rates in the world (De Korte et. al., 2004)
No cure just treatment
Response to the HIV epidemic
The introduction of ARV therapy in January 2002 (De Korte et. al., 2004)
Recommended treatment regimens of HAART in Botswana includes the three-line approach
Costs US$ 1500 per year (De Korte et. al., 2004)
Side effects and multiple resistance to drug
Any other options?• The local sangoma (traditional doctor).
• Herbal medicine- a less toxic alternative to conventional medicine
• About 20,000 plant species used for medicinal purposes are reported by WHO (Maregesi et.al., 2008 )
•Countries as far as China, Belgium and the U.S.A (Liu, et.al., 2002; Abd-Elazem, et.al., 2002; Cos, et.al., 2004) are already tapping into this potential.
Table 1: Plants & their Products used among HIV-infected individuals (Mukhtar et.al., 2008)
Common Name Scientific name Activity/ Comments
Aloe Aloe vera Acemannan, a complex sugar extracted from Aloe vera used for retroviral infection
Cat's claw (inner bark)
Uncaria tomentosa Study showed an increase in CD4+ counts.
Hyssop (leaves and flowers)
Hyssopus officinalis Inhibits the replication of HIV without any toxicities, in vitro.
Marijuana Cannabis sativa Helps prevent nausea and stimulates appetite.
PROBLEM STATEMENT Not much has been documented
about Botswana’s medicinal plants Not much proof of the claimed
medicinal uses (Maregesi et.al., 2008).
Questions arise related to safety of using traditional medicines.
Lack of qualified personnel in the field of medicinal plants.
AIMS / OBJECTIVES
Identify medicinal plants used by traditional doctors to alleviate symptoms related to HIV/AIDS.
Evaluation of plants for any antiviral activity
Evaluation of cytotoxic properties of these plants in an in vitro eukaryotic cell culture system.
Significance of the Project Collaboration between traditional
health practitioners and biomedical practitioners.
Lower current costs experienced by ARVs.
Eradicate lack of information on medicinal plants currently in use and determine how safe they are for human consumption.
METHODOLOGY
Retrieval of Plant Material & Extraction
Cytotoxicity Assay
Antiviral Assay
Retrieval of Plant materials
Plants from Maun near the Okavango Delta
Sampling strategies
- Purposive sampling
- Snowball sampling
Ethanol and water extractions
Cytotoxicity Assay Culture and maintenance of HEK 293
cells.
- Dulbecco’s Modified Eagle’s Medium
- viable cell count of 3x105 cells/ml
- incubate at 370C and 5% CO2
MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay (Abd-Elazem, et.al., 2002)
-cells seeded onto 96 well plates
-test samples at different concentrations were added to test for cytotoxicity
- 50µl of MTT is added to plates
- Absorbance measured by a microtiter reader at OD 540nm.
Plot a graph of absorbance against concentration.
Antiviral Assay
HIV 1c-strain MJ4 developed at the Harvard AIDS Institute (Ndung’u et.al.., 2000)
Cytopathogenic effect (CPE) protection assay will be performed.
50% HIV-1 tissue culture infectious dose (TCID50) will be determined.
Multiplicity of Infection (MOI) of 10-2 required for an inhibition assay.
Aug-Oct•Plant collection &
extraction.•Maintenance of
cell culture
November•Determining the TCID50 & MOI
of virus stock
Jan-Mar•Determining CPE & antiviral assay
April•Presentation of
results
Time Plan
BUDGET
ESTIMATED COST
MATERIALS P1 250.00
TRANSPORT P150.00
FOOD P50.00
COMMUNICATION P50.00
TOTAL P1 500.00
REFERENCES Abd-Elazem I.S., Chen H.S., Bates R.B., & Huang R.C.C. (2002). ‘Isolation of two
highly potent and non-toxic in hibitors of human immunodeficiency virus type 1 (HIV-1) intergrase from Salvia miltiorrhiza.’ Antiviral Research 55:91-106. Elsevier Science B.V.
Cos P., Maes L., Berghe D.V., Hermans N., Pieters L., & Vlietnck A., (2004). ‘Plant substances as Anti-HIV agents selected according to their Putative Mechanism of Action.’ Journal of Natural Products 67:284-293.
De Korte D., Mazonde P., & Darkoh E. (2004). Introducing ARV therapy in the Public Sector in Botswana. World Health Organization, Switzerland
Dixon P. (2002) AIDS and You. 3rd edition. Operation Mobilisation Books, Secunnderabad.
Liu S., Jiang S., Wu Z., Lv L., Zhang J., Zhu Z., & Wu S. (2002) ‘Identification of inhibitors of the HIV-1 gp41 six-helix bundle formation from extracts of Chinese medicinal herbs Prunella vulgaris and Rhizoma cibotte’ Life Sciences 71:1779-1791. Elsevier Science Inc
Ndung’u T., Renjifo B., Novitsky V.A., McLane M.F., Gaolekwe S., & Essex M. (2000) Molecular Cloning and Biological Characterization of Full-Length HIV-1 Subtype C from Botswana’ Virology 278: 390-399. Academic Press.
Maregesi S. M., Pieters L , Ngassapa O.D., Apers S., Vingerhoet S., Cos P. , Van Berghe D.A., and Vlietinck A. (2008). ‘Screening of some Tanzanian medicinal plants from Bunda district for antibacterial, antifungal and antiviral activities.’ Journal of Natural Products 67:284-293
Mukhtar M., Arshad M., Ahmad M., Pomerantz R.J , Wigdahl B., and Parveen Z. (2008). ‘Antiviral potentials of medicinal plants.’ http://www.sciencedirect.com/science