Isolation, Purification of Quercetin from in vitro Cell Suspension Culture of Caesalpinia pulcherrima and its Analysis by HPLC-DAD and NMR

Background: Caesalpinia pulcherrima, belongs to Caesapiniaceae, is a known medicinal plant widely distributed in India and is used in traditional medicine for the treatment of various ailments. Many phytochemicals are reported from the plant as potential source of crude drug. Materials and Methods: An efficient and simple reproducible protocol was developed for callus production using leaf explants of C. pulcherrima. The combination of 2, 4-D, kin and BA, was used for the callus induction. Subsequently, cell suspension culture and quercetin synthesis from in vitro callus was attempted. Role of effect of elicitors (Sucrose, ABA and salicylic acid) in cell suspension culture was carried in MS medium containing 2,4-D + BA + kinetin. Flavonoids was purified, fractionated by HPLC-DAD and NMR. Results: 2, 4-D (2.5 mg/L), BA (2.5 mg/L) + kin (1 mg/mL) was effective for maximum callus induction from leaf explants. Significant cell suspension culture was noticed with liquid MS medium containing 2,4-D (2 mg/L)+ BA (1mg/L)+ kinetin (1.5 mg/L). Sucrose, ABA and salicylic acid (SA) at different concentrations influenced cell biomass and quercetin accumulation. The addition of ABA/SA along with sucrose was found to have no remarkable effect on cell biomass and also quercetin synthesis. However, cells cultured in the medium fortified with 45 g/L sucrose without ABA/ SA showed the highest quercetin content (16.5 mg/g). Flavonoids was purified, fractionated by HPLC-DAD and NMR revealed the presence of 9 components such as quercetin, isoquercetin, quercetrin, rutin, quercetin 3-O-β-D-xyloside, quercetin 3-Oarabinopyranoside, quercetin 3-O- α-arabinopyranosyl (1→2) β-galactopyranoside, isorhamnetin 3-O-rutinoside and an unknown compound. Conclusion: C. pulcherima reveals significant synthesis of quercetin. Quercetin content recorded in cell suspension culture was significantly higher compared with in vivo plants grown in fields and the compounds were identified by NMR.