Abstract:
Tea and Cocoa Kombucha are fermented beverages produced by the action of a symbiotic culture of bacteria and yeasts. Their health benefits have been hailed and staunchly declared by Kombucha drinkers worldwide. However, there is minimal scientific evidence on their anti-malarial properties. This research aimed to further investigate the anti-plasmodial properties and possible mechanism of action of tea Kombucha and cocoa Kombucha by determining the inhibitory concentrations, evaluate the stage specificity, assess the inhibition of Plasmodium falciparum erythrocyte invasion and determine the antioxidant and cytotoxic activities of Kombucha extracts and fractions. Kombucha black and green tea and Kombucha cocoa were prepared, neutralized and fractionated using three different solvents (hexane, ethyl acetate and water) to obtain the respective extracts for in-vitro assays. The growth inhibition effects of the various extracts on in vitro P. falciparum were assessed using the SYBR Green I assay. Ring stage survival assays and schizont stage specific assays were evaluated after 72 hours using flow cytometry. Inhibition of P. falciparum invasion of human red blood cells (RBCs) treated with Kombucha extracts/fractions were also assessed using flow cytometry. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) method was used to assess antioxidant activity of Kombucha on human peripheral blood mononuclear cells (PBMCs). The MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) method was used to assess cytotoxicity profiles of Kombucha extracts and their fractions. The neutralized ethyl acetate fraction of cocoa Kombucha exhibited over 20 times (20x) the inhibitory activity of the corresponding unneutralized ethyl acetate fraction on Dd2 P. falciparum, and on NF54, the activity of neutral cocoa was almost 6 times (5.8x) its unneutralized counterpart. The ethyl acetate fraction of the neutralized GTK was 6x less potent than the non-neutralized ethyl acetate fractionof green tea Kombucha in inhibiting the ring stage of P. falciparum. The ethyl acetate fraction of green tea extract demonstrated schizonticidal activity at concentrations above its IC50(2mg/ml). The aqueous CTK extract displayed more potency in inhibiting invasion (46.6%) of P. falciparum Dd2 at its IC50 (280 μg/ml). The neutral cocoa Kombucha (NCTK) was 66% more effective at radical scavenging than the fermented CTK extract. The ethyl acetate NCTK fraction displayed the highest selective index (4.21). Generally, ethyl acetate selectively extracted bioactive components of Kombucha that exhibited anti-plasmodial and antioxidant activities coupled with good selectivity index compared to all other extracts and fractions. Cocoa Kombucha and its fractions exhibited the most active anti-plasmodial and stage specific activity on P. falciparum and could be explored as a food supplement for malaria prophylaxis.
