Mechanistic and synergistic actions of the in vitro antimycobacterial and immunomodulatory properties of Knowltonia vesicatoria (L.f.) Sims

Abstract

Three South-African plant extracts, Knowltonia vesicatoria (L.f.) Sims (aerial, ethanol), Pelargonium sidoides DC. (root, ethanol) and Euclea natalensis A.DC. (root, chloroform) were subjected to in vitro antimycobacterial, cytotoxicity, intracellular antimycobacterial and immune modulating assays to investigate the possibility of plant therapies that can serve as a treatment for the depleting immune system and the virulence of M. tb in TB/HIV-coinfected sufferers. A Glutathione reductase enzyme (Gtr) assay was employed as a surrogate for the M. tb enzyme (Mycothione reductase) to screen samples for selective enzyme inhibition as the possible mechanism of action. The same set of assays was undertaken for synergistic amalgamations of the three extracts in combination with the first line drug, isoniazid (INH). Extracellular antimycobacterial tests included the radiometric BACTEC method and a colorimetric microplate dilution assay. Sample cytotoxicity on human macrophages (differentiated U937 cells) was determined using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2-Htetrazolium hydroxide (XTT) proliferation kit. Intracellular activity was established by enumeratring the colony forming units (CFU) via the colony counting system ColorQCount. To determine the immunological effects of samples on the macrophages, cytokine profiles were analyzed using cytometric bead array technology. To eliminate the possibility of tannins as the antimycobacterial actives in the K. vesicatoria extract, the extract was subjected to a solvent partitioning method to remove the polyphenols. It was confirmed that the tannin-free fraction had poor antimycobacterial activity when tested against M. smegmatis exhibiting a MIC of 2500 μg/mL and was not active against M. tb at the highest concentration tested. The activity of the whole Knowltonia extract remained superior to the fractions obtained by solvent partitioning, with a MIC of 625.0 μg/mL against M. smegmatis and a MIC of 50.00 μg/mL against M. tb. It was found that the synergistic combination of K. vesicatoria with INH exhibited the best fractional inhibitory concentration (FIC) of 0.25 and a fifty percent inhibitory concentration (IC50) of 121.0 ìg/mL on the macrophages, indicating a therapeutic index (TI) of 19.00. Two compounds, a sterol (stigmasta-5,23-dien-3-ol) and a lactone (5-(hydroxymethyl)furan-2(5H)-one) were successfully isolated from K. vesicatoria, as the first report of isolation for both compounds from this plant. The antimycobacterial properties of these compounds have not been reported earlier. The sterol was the most active sample against a drug-sensitive strain of M. tb with a minimum inhibitory concentration (MIC) of 50.00 μg/mL. The extract of K. vesicatoria was tested against two drug resistant strains of M. tb (resistant to INH, ethambutol (EMB), streptomycin (STR) and rifampicin; RIF) which resulted in a MIC of 50.00 μg/mL on both strains, indicating a mechanism of action different to INH, EMB, STR and RIF. The isolated lactone revealed 70% inhibition of Gtr. The only samples that exhibited any significant intracellular antimycobacterial activity were the crude extracts of K. vesicatoria and E. natalensis with 80 and 60% M. tb growth inhibition at concentrations of 50.00 and 8.00 μg/mL, respectively. Immunological activity of selected test samples was determined by measuring cytokine levels (IL-2, IL-4, IL-5, IL-10, IFN-ϒ and TNF-α) of TB-infected and uninfected macrophages. A slight Th1-type response was seen for infected cells treated with 7-methyljuglone and P. sidoides at their IC50 values of 18.00 μg/mL and 43.00 μg/mL, respectively. Further studies are required to confirm the immunological activities by evaluating various other cytokines such as IL-12.