Purification, Fractionation and Characterization of Anthocyanin from in vitro Culture of Bridelia retusa (L.) Spreng.

Abstract

Bridelia retusa is traditionally used as an astringent and for treating rheumatic pains. Present study revealed that 2,4-dichlorophenoxyacetic acid alone or in combination with kinetin-fortified Murashige and Skoog medium showed good response in terms of callus from leaf explants of B. retusa. Growth hormones, pH, light, and carbon source influenced secondary metabolic pathways. The highest callus induction was 98.9 % with 2.5 mg/l N6-benzyladenine+2 mg/l 2,4-dichlorophenoxyacetic acid. The optimal fresh and dry weights of the calli were 1.9±0.04 and 0.45±0.03 g, respectively. The calli incubated in light on Murashige and Skoog medium with 4 % glucose containing 2.5 mg/l benzyladenine and 2 mg/l 2,4-dichlorophenoxyacetic acid at pH 3.5 yielded 2.8 mg/g of anthocyanins. Murashige and Skoog medium with glucose was optimal compared to sucrose for anthocyanin synthesis. Addition of kinetin inhibited anthocyanin accumulation. Pigmented calli transferred to half strength Murashige and Skoog medium with NH+/NO3 (1:4 ratio), 70 g/l sucrose and supplementation with benzyladenine+2,4-dichlorophenoxyacetic acid produced remarkable biomass production with anthocyanin synthesis when compared with the initial culture conditions. Suspension cultures of Murashige and Skoog medium containing 2.5 mg/l 2,4-dichlorophenoxyacetic acid and 2 mg/l benzyladenine at pH 5.0 induced anthocyanin synthesis into the medium with pH 4.4–4.6. HCl-water and HCl-ethanol extractions for 90 min was attempted to obtain the maximum amount of anthocyanin content. Temperature factors and pH against anthocyanin extraction and stability was analyzed. Enhancement in the degradation rate constant with a corresponding decline in the t1/2 values was seen with the increasing temperature at pH 1 and 4. Fractionation of anthocyanin was carried by high performance liquid chromatography coupled mass spectrometry revealed 09 fractions comprising acylated cyanidins and two peonidins. The major compounds were cyanidin 3-p-coumaroyl and feruloyl diglucoside-5-glucosides. Thus, the calli from leaf explants could be a good source for anthocyanin synthesis.