Anti-HIV, anticancer and antioxidant activity of Fockea multiflora extracts

Dissertation (MSc (Biochemistry))--University of Pretoria, 2022.Background: Over the past four decades, the Human immunodeficiency virus (HIV) has been a great problem for public health globally. The immunosuppressive ability of HIV is associated with life-threatening opportunistic infections like the human papillomavirus (HPV) that causes cervical cancer. The development of highly active antiretroviral therapy (HAART) has transformed HIV infection into a manageable chronic disease and has improved life expectancy by providing steady viral suppression. However, HAART comes with unpleasant side effects that complicates adherence to treatment, leading to the increase in plasma viral load, the drug-resistant HIV strains development and treatment failure. To achieve the goal of eradicating HIV completely, it is imperative to develop novel antiviral agents with tolerable levels of toxicity, fewer resistance profiles and that are cost-effective. Because of the existence of plant derived natural products that have antiviral and anticancer properties, this study investigated the bioactivity of Fockea multiflora extracts with the aim of identifying anti-HIV and associated activities. The plant belongs to Apocynaceae, a family that has shown to be a vital source of beneficial bioactive compounds contained in the isolated constituents which include flavonoids, coumarins, and saponins. Methods: The chloroform, water and diethyl ether extracts of the root and leaf of F. multiflora were investigated for their cytotoxicity profile using MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] and Neutral Red Uptake (NRU) of peripheral blood mononuclear cells (PBMCs) and TZM-bl cells. This study also investigated the inhibitory effects of F. multiflora on two of the crucial enzymes of the HIV-1 life cycle; reverse transcriptase (RT) and protease (PR). The anticancer profile of F. multiflora was then monitored using MTT in a cervical cancer cell line-(HeLa) and liver cancer cell line-(HepG2). Real-time cell analysis (RTCA) was used to investigate the effects of the extracts in cancer cell proliferation in real-time. Lastly, the radical scavenging ability of F. multiflora was investigated by measuring the nitric oxide (NO) produced by HeLa and HepG2 cells. Results: Evaluating the cytotoxicity of plant extracts is important for general safety of extracts as well as for determining the treatment ranges for further investigation. Results obtained from MTT assay revealed that some F. multiflora extracts were cytotoxic towards PBMCs; extract DL (diethyl ether of leaf) gave a CC50 of 10.39 ± 0.26 µg/mL and CL (chloroform of leaf) with a CC50 of 12.14 ± 0.12 µg/mL. Natural products possess reducing agents which tend to interfere with MTT indicators and this may lead to false results unless these agents are eliminated. The NRU assay was then employed, as it involves the washing of cells which eliminates the interference. The cell viability results obtained from NRU using the TZM-bl cell line gave a dose-dependent curve for most of the extracts except for WR (water of root) and WL (water of leaf), where cells were highly viable for all concentrations with predicted CC50 values way above the tested concentrations. This confirmed the safety of the polar solvent (water) used in the extraction of these two extracts at all tested concentrations (4 – 250 µg/mL). The NRU assay revealed that none of the extracts was cytotoxic towards TZM-bl cells. The inhibition activity of Fockea multiflora extracts on HIV-1 RT resulted in a percentage inhibition of 73% at 100 µg/mL for CL, with an estimated IC50 of ˂10 µg/mL. All F. multiflora leaf extracts exhibited relatively high inhibition (>50%) and extract CR (chloroform of root) and DR (diethyl ether of root) were statistically significant (p = 0.0085 and 0.0406, respectively). A known inhibitor for HIV-1 RT, doxorubicin exhibited a percentage inhibition of 80%. The inhibitory activity of extracts on HIV-1 protease was investigated and a percentage inhibition of 70% at 100 µg/ml for CR was obtained with an estimated IC50 of 10 µg/mL (p = 0.0250). It was noted that all root extracts exhibited relatively high inhibition (>50%) and a known inhibitor for HIV-1 PR, acetyl pepstatin (AP) exhibited a percentage inhibition of 85%. The anticancer ability of F. multiflora was assessed in HeLa cells through MTT and none of the extracts exhibited inhibition activity below 50%. However, RTCA revealed lower IC50 values than those obtained in the MTT assay; extract WR for HeLa cells gave an IC50 of 35.53 µg/mL and all extract activities were found to be statistically significant (p-values ranging from 0.0001 to 0.0187). The radical scavenging ability of all F. multiflora extracts on the production of NO was observed to be higher in HepG2 cells to almost undetectable levels, with IC50 ranging from 0.001 ± 1.21 to 2.02 ± 0.09 µg/mL and all extracts were found to be statistically significant (p = 0.0001). Conclusion: This study showed that F. multiflora extracts have the potential to inhibit both HIV-1 RT and PR, with the highest percentage inhibition being close to that obtained for known inhibitors. Results also demonstrated that F. multiflora extracts are rich in antioxidants, with the greatest radical scavenging ability observed in liver cancer cell lines. These findings provide the first report on the anti-HIV-1 activity of F. multiflora extracts and supports the isolation and purification of active compounds for further studies.Faculty of Natural and Agricultural Sciences (University of Pretoria), HIV Research Group (NAS, University of Pretoria), uMlalazi Municipality (KZN)Biochemistry, Genetics and Microbiology (BGM)MSc (Biochemistry)Unrestricte