Abstract

SEPARATION OF POLYPHENOLS FROM HIGH YIELD POLYPHENOLIC EXTRACT (HYPE) OF PUNICA GRANATUM BY COMBINED CHROMATOGRAPHIC TECHNIQUES AND THEIR CHARACTERIZATION BY LC-MS/MS

Pooja Jaiswal*, Kajal Gupta, Tarun Kumar Dasgupta and Priscilla D. Mello

Abstract

In this study, we developed a preparative isolation process of the plant polyphenols obtained from the HYPE of Pomegranate peel (Punica granatum) which is the major part of agrowaste. Polyphenols namely catechin, myricetin and quercetin were isolated and further were characterized by using LC-MS/MS. Polyphenols were extracted from the enzyme pre-treated matrix with the help of pectinolytic and cellulolytic enzyme preparation was taken for a pre-developed gradient system. Phase- A = Acetonitrile Phase -B = Acetonitrile: water (10:90) with buffer to maintain pH = 3.25 (O-phosphoric acid). Flow rate was set at 1.0 ml/min. Preparative column with 50micron particle size reverse phase was used. The detection wave length was selected 272nm after being confirmed by a PDA detector. Under the above conditions a satisfactory separation of the targeted compounds was achieved with the standard catechin, myricetin and quercetin in crude extract. Polyphenols analysis by LC-ESIMS/ MS were carried out using an Agilent 1100 series LC and LC/MSD Trap VL mass spectrometer (Agilent Technologies, USA) equipped with electrospray ionization (ESI) interface. A good separation of polyphenols were achieved and isolated fraction of quercetin and myricetin were collected without any interference but catechin fraction further purified and analysed by MS/MS libraries. Our study demonstrates an effective isolation of polyphenols from pomegranate agro waste by gradient followed by an isocratic system. The present study may lay a foundation for large scale preparation of these plant polyphenols specially quercetin and myricetin from pomegranate waste material.

Keywords: polyphenols, quercetin, catechin, myricetin, preparative chromatography, LC-MS/MS.