DIFFERENTIAL EFFECT OF PURIFIED QUERCETIN AND ITS DERIVATIVES FROM IN VITRO CELL SUSPENSION CULTURES OF CAESALPINIA PULCHERRIMA SW. AGAINST SELECTED CANCER CELL LINES AND ITS MODE OF ACTION
Tribal people use the floral extract of Caesalpinia pulcherrima to cure liver, stomach and skin prone disorders in traditional Indian medicine. This study aimed to evaluate the effect of purified quercetin and its derivatives from in vitro cell suspension cultures of C. pulcherrima Sw. against SW 480, HeLa, MCF-7 and MCF 10A cell lines and its mode of action. Standard protocol was developed for callus induction using leaf explants. Cytotoxic effect was evaluated against SW 480, HeLa, MCF-7 and MCF 10A cells by MTT assay. Apoptosis was evaluated via Hoechst analysis, flow cytometry, mitochondrial membrane potential and caspase 3 and 9 expression. 2, 4-D (2.5 mg/l), BAP (2.5 mg/l) + kin (1 mg/ml) was effective for remarkable callus induction. Further, cell suspension culture was established. Effect of elicitors on cell suspension culture was also carried. Sucrose, ABA and salicylic acid (SA) at different concentrations influenced cell biomass and quercetin synthesis. Cells cultured on the medium fortified with 45 g/L sucrose without ABA/SA showed the highest quercetin content (16.5 mg/g). Quercetin was purified, fractionated by HPLC-DAD and was further analyzed by NMR revealed a major fraction of quercetin (3, 5, 7, 3’, 4’-pentahydroxyflavon). Insignificant cytotoxicity was noticed in SW 480, HeLa, MCF 10A when compared to MCF-7 cell lines exposed to different concentrations of purified quercetin for 24- 48 h. Similarly, the apoptosis by nuclei staining using Hoechst 33258 revealed a concentration dependent effect on MCF 7 cells only. This was further substantiated by caspase-9 and 3 induction and mitochondrial depolarization as revealed by flow cytometry. Overall, the results showed that quercetin and its derivatives induced effective apoptosis on MCF-7 cells. Quercetin isolated from the in vitro cell suspension culture of C. pulcherrima showed significant cytotoxicity and apoptotic activity towards MCF-7 cell lines as compared to other cell lines.
Keywords: Caesalpinia pulcherrima; quercetins; suspension culture; cytotoxicity; apoptotic.
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