VALIDATION OF HEPATOPROTECTIVE AND ANTIOXIDANT ACTIVITY OF FOLK LORE KNOWLEDGE OF PURIFIED ANTHOCYANIN FROM CELL SUSPENSION CULTURE OF CLERODENDRON INFORTUNATUM L.: A SEARCH
Keywords:Clerodendron, HPLC, MS medium, hormones, anthocyanin, column chromatography, hepatoprotective, antioxidant, scavening activity, oxidative stress and CCl4
Objective: To validate the hepatoprotective and antioxidant activity of purified anthocyanin extracted from the cell suspension culture of Clerodendron infortunatum Linn.
Methods: A protocol has been developed for the induction of callus proliferation from leaf and nodal explants of C. infortunatum. The explants were inoculated on MS medium supplemented with diverse combinations of 2,4-D and BAP for triggering callus formation. Subsequently, the green compact callus has been sub-cultured in the medium fortified with 2,4-D and Kinetin for anthocyanin synthesis. Cell suspension culture was also established and the elicitor, salicylic acid was used for triggering anthocyanin synthesis. Three different chromatographic columns (solid phase extraction by Sepharose C18 column, Oasis-MCX and Amberlite XAD 7 + Sephadex LH 120 sorbents) were employed to purify the in vitro synthesized anthocyanin from cell suspension cultures. For purity evaluation HPLC and molar absorptivity assay were used. Further, hepatoprotective and antioxidant activity was evaluated comparing with silymarine, as standard in rats. In vitro antioxidant scavenging activity was analysed by DPPH, FRAP and ORAC assay.
Results: After 1 month, the leaf explants yielded remarkable green compact callus on MS medium containing 2.0 mg/l BAP and 0.5 mg/l 2,4-D. Salicylic acid enhanced anthocyanin synthesis. The mean purity values obtained by HPLC were 90.9% Â± 1.9 and 80.60% Â±2.3 for Oasis MCX, Amberlite XAD-7 + Sephadex LH-20 column respectively. However, the purity calculated by molar absorptivity was found to be less. The highest purity achieved using molar absorptivity analysis was with MCX cartidges i.e., 85.9 Â± 3.8%. HPLC yielded 12 anthocyanin fractions. Remarkable antioxidant scavenging activity was noticed as revealed by DPPH, FRAP and ORAC assay. The hepatoprotection activity (25, 50, 100 mg/100g b.w) was compared with silymarine (25 mg/kg b.w) against CCl4 induced toxicity.Â Anthocyanin extract improved the AST, ALT and recovered the activity of kidney function by decreasing the urea and creatinine content. In addition, the administration of anthocyanin significantly inhibited the oxidative stress via its scavenging of the reactive oxygen species formed by CCl4 stress. Further, a decrease in the MDA, H2O2, NO accumulation and increase of GSH content were noticed. Similarly, Â improved lipid profiles, LDL and HDL levels were also observed suggesting that anthocyanin significantly suppress the toxicity via its activation of antioxidant enzymes (GST, CAT and SOD).
Conclusion: The overall results showed that the purified anthocyanin of C. infortunatum function as an antioxidant andÂ there by hepatoprotective protection against CCl4 inducedtoxicity in animal models.Â
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