Bioactivity and 1H NMR-based metabolomics analysis of polyherbal formulations used traditionally for the treatment and management of sexually transmitted infections

Ethnopharmacological relevance: Sexually transmitted diseases (STDs) is a major global concern of an ever-growing population. The daily rate of infectious STDs increases by an estimate of one million infections. The development of antimicrobial resistance pathogens is a further concern. Complementary and alternative medicines (CAM) is investigated to provide aid/relief from this resistance. Medicinal plants are used traditionally as an alternative form of medicine. Aim: The aim of this study was to evaluate four medicinal plants (Dicoma anomala sbsp anomala, Elephantorrhiza elephantina, Eucalyptus cinerea and Kigelia africana) used in combination and individually for their antimicrobial activity against sexually transmitted diseases and to determine the similarities of these combinations based on their chemical constituents. Furthermore, the study determined the cytotoxicity effects and mycotoxin presence of the most active extracts. Methodology: Aqueous extracts of various different combinations and individual plant were prepared, and stored at various conditions (includes: temperatures of 4˚C, 25˚C and 37˚C for 7 and 14 days respectively). The extracts were evaluated for their antimicrobial properties against a bacterium Neisseria gonorrhoea and a fungus Candida albicans using the microtiter dilution assay. The fractional inhibitory concentration index of the combinations was also determined against both pathogens. The chemical profile of these samples was analysed using a metabolomic approach via nuclear magnetic resonance and liquid-chromatography mass spectrometry methods (1H NMR and LC-MS). Cytotoxicity studies were conducted to determine the level of toxicity of noteworthy samples against non-cancerous Vero cell lines. Furthermore, various mycotoxin levels present were determined via high-performance liquid chromatography. Results: Antimicrobial activity of polyherbal formulations containing four individual plants (Dicoma anomala sbsp anomala Elephantorrhiza elephantina, Eucalyptus cinerea and Kigelia africana) in combination, which were stored at 25˚C for 7 and 14 days respectively, exhibited noteworthy activity against N. gonorrhoea. Eight of the ninety-eight extracts displayed a minimum inhibitory concentration (MIC) value of 0.39 mg/mL. 1H NMR metabolomic analysis of all the combinations and individual plants exhibited prominent signal peaks in the aliphatic region (0.8- 3.00 ppm), carbohydrate region (3.00- 6.00 ppm) and some combinations yield signals within the aromatic region (5.00- 9.00 ppm). Thus, resulting in a similarity between phytoconstituent amongst the various different combinations irrespective of their storage conditions and antimicrobial activity. The similarity within the different types of combinations is displayed via distinct grouping on the PCA and OPLS-DA score plots. Concerning cytotoxicity studies eight polyherbal formulations were tested, cytotoxicity levels against non-cancerous Vero cell lines ranged from slightly cytotoxic to non-cytotoxic (289.12 ± 21.23 µg/mL to 866.47 ± 22.41 µg/mL). Polyherbal formulations subjected to mycotoxin analysis displayed non-detectable mycotoxin levels. Conclusion: The biological and chemical activity observed amongst the different plant combinations supports the traditional usage of polyherbal formulations in the treatment and management of sexually transmitted diseases.