In vitro Protective Effect of Chemically-Characterized Flower Extracts of Clitorea ternatea L. Against Hydrogen Peroxide Induced Cytotoxicity in Human Keratinocytes (HaCaT)

Clitoria ternatea (Leguminosae), commonly known as the butterfly pea, is easily recognizable by its deep blue flowers. The leaves and roots of the plants are well research. However, very little is known about the flower parts of the plant. In Malaysia and Thailand, the flower part was used as a food colorant in the traditional dish, drink as herbal tea and consumed as vegetable salad for health benefits, particularly as defence against skin aging. This study was conducted to investigate the protective effect of C. ternatea extracts against hydrogen-peroxide induced cytotoxicity in HaCaT cells (human keratinocytes) as skin cells model. Two extracts (aqueous and 70% ethanol) were prepared to mimic the traditional methods of preparation. The antioxidant potential of the extracts was determined using diphenyl-picryl hydrazine (DPPH) and 2, 2'-azino-bis (3- ethylbenzothiazoline-6-sulphonic acid (ABTS) assays, expressed as Trolox equivalent (TE)/mg extract. The protective effect against hydrogen-peroxide induced cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) assay. The antioxidant assays showed the TE/mg extract for C. ternatea ethanol extract (CTE) were significantly higher than C. ternatea water extract (CTW) (p < 0.05). HaCaT treated with CTW extract showed to have higher percentage viability compared to untreated HaCaT after exposure to 200µM hydrogen peroxide (p < 0.05). No protective effect was observed from CTE extract. High resolution liquid chromatography-mass spectrometry (LC-MS) revealed the detection of compounds assigned as flavonol glycosides (derived from quercetin and myricetin), anthocyanins (derived from delphinidin) and coumaric acid derivatives (coumaroylsucrose and coumaroylglucose) in CTE and CTW. The higher antioxidant potential in CTE, observed by higher antioxidant potential to inhibit free radicals but antagonistically prevented the protective effect against hydrogen peroxide-induced cytotoxicity as opposed to HaCaT treated with CTW may be due to variation in the relative levels of phytochemicals in the CTE and CTW extracts.