An investigation of the emmunomodulatory properties of Sutherlandia Frutescens and Hypoxis Hermerocallidea

Abstract

Human immunodeficiency virus (HIV) is currently the most significant infectious pathogen and the causative agent of acquired immune deficiency syndrome (AIDS). Unfortunately, due to lack of resources, delivery of antiretroviral therapy (ART) to countries where they are most needed, such as South Africa, Botswana, Lesotho, Malawi and Swaziland, is limited and inefficient. Moreover, the short supply and high cost of antiretroviral drugs have caused researchers to turn to plants as prospective therapies in the search for alternative anti-HIV or immunomodulatory compounds. In an African context, traditional medicines are of great importance, not so much as an alternative to treatment, but in many cases as the only source of treatment. There are various South African plants used medicinally which possess phytochemical constituents that target certain mediators of inflammation and the immune system. In African regions where patients do not have access or financial capability to obtain conventional antiretroviral treatment, traditional herbal medicines are used as primary treatment of HIV/AIDS, regardless of the fact that the safety, toxicity and efficacy of these products are not yet fully understood and that a risk for adverse effects exists. Hypoxis hemerocallidea Fisch&C. A. Mey. (Hypoxidaceae) as well as Sutherlandia frutescens L. R. Br. (Leguminosae) have various effects on the immune system and due to claims about their immune boosting properties, they are two of the most common African herbal compounds being used for HIV management in South Africa. In this study, the immune modulating properties of H. hemerocallidea and S. frutescens were investigated in order to determine whether anectodal claims made about these plants could be supported. Differentiated THP-1 and U937 macrophages were treated with aqueous extracts of H. hemerocallidea and S. frutescens as well as with solutions of compound standards reputedly isolated from these plants such as beta-sitosterol, found in H. hemerocallidea, canavanine, pinitol and gammaaminobutyric acid (GABA) which are present in S. frutescens Cytotoxicity of the test compounds was determined using the 3-(4,5-dimethylthiazol- 2-yl)-2,5-dephenyl tetrazolium bromide (MTT) assay. Antioxidant capacity was assessed using the Trolox equivalence antioxidant capacity (TEAC) and Oxygen radical antioxidant capacity (ORAC) assays. Determination of prostaglandin E2 (PGE2) concentration in treated THP-1 and U937 cell culture supernatants was performed by ELISA. Concentrations of cytokines (IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, TNF-á and IFN-ϒ) in treated THP-1 and U937 cell culture supernatants were determined by flow cytometry. Curcumin, a well-known immunomodulatory compound, was used as a positive control. Results of cytotoxicity assessments showed that H. hemerocallidea (0.1 – 1.9 mg/ml), S. frutescens (0.1 – 1.6 mg/ml), beta-sitosterol (0.2 – 25 ìM), canavanine, pinitol and GABA (1.5 – 200 ìM) were not cytotoxic to THP-1 and U937 macrophages and had cytotoxicity profiles comparable to that of the positive control, curcumin (0.8 - 25 ìM). The TEAC and ORAC assays showed different results in the antioxidant capacities of the test compounds. The purported antioxidant activity of H. hemerocallidea was confirmed by the TEAC assay with antioxidant effects equivalent to 0.2 mg/ml Trolox. Canavanine showed antioxidant activity equivalent to approximately 0.17 mg/ml Trolox and comparable to that of curcumin in the ORAC assay, suggesting its involvement in the inhibition of peroxyl radical-induced oxidation. Flow cytometry results showed that curcumin (20 ìg/ml and 10 ìg/ml) and beta-sitosterol (25 ìg/ml and 12.5 ìg/ml) reduced IL-1â and IL-8 production and significantly (p<0.05) decreased the production of TNF-á. This suggests that betasitosterol could indeed possess anti-inflammatory properties, with effects comparable to the known anti-inflammatory effect of curcumin in terms of cytokine profiles. Beta-sitosterol (25 ìg/ml) and pinitol (50 ìg/ml) significantly (p<0.001) decreased extracellular PGE2 levels in U937 macrophages by 233.4 pg/ml and 281.7 pg/ml, respectively and were the only two compounds showing greater reductions in PGE2 than curcumin. In conclusion, results of this study do not provide enough evidence to support all anecdotal claims about the ‘immune boosting’ properties of S. frutescens and H. hemerocallidea, but the compounds canavanine, beta-sitosterol and pinitol were found to have modulatory effects on certain aspects of the immune system.