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