Fractionation and characterization of polyphenols rich extract from labisia pumila leaves

Abstract

Herbal plant extracts rich in polyphenols are known to exhibit high antioxidant activity. In this study, the correlation between antioxidant and polyphenols content of L. pumila (Kacip Fatimah) leaves was investigated. The polyphenol rich fractions were prepared by ultrasonic assisted extraction (UAE) and followed by solid phase extraction (SPE). The UAE extraction was carried out by using methanol, acetonitrile, water and methanol-water mixture as the solvents. The L. pumila crude extracts were subsequently fractionated using SPE using mixtures with different methanol-water ratio. The antioxidant and total polyphenol content of the crude extracts and fractions were evaluated using spectrophotometric assays. The crude extract, extracted using 60% methanol-water mixture was found to give the highest antioxidant activity of IC50 = 59.30 ± 0.12 ppm with the content of flavonoid (285.94 µg/mg dry weight of rutin equivalent, RE) and phenolic (213.5 µg/mg dry weight of gallic acid equivalent, GAE) content. Whereas the L. pumila fraction, fractionated using 40% methanol-water was found to exhibit the highest antioxidant activity (IC50 = 1109.46 ± 4.45 ppm), phenolic (266.64 ± 0.12 dry weight of gallic acid equivalent, GAE) and flavonoid (27.66 ± 0.07 µg/mg dry weight of rutin equivalent, RE). High correlation (r = 0.60-0.96) was found between the antioxidant activity, phenolic and flavonoid content based on spectrophotometric results. Based on liquid chromatography mass spectrometry (LC-MS/MS) analysis, twenty three polyphenols constituents consisted of flavonoids and phenolic acid were identified from the fraction. Among of these constituents, epigallocatechin, myricetin and rutin were identified as the key polyphenols in the fraction. The validation of these compounds was carried out with the linearity from 0.9840 to 0.9983, limit of detection (LOD) from 0.03 µg/ml to 0.11 µg/ml, limit of quantification (LOQ) from 0.11 µg/ml to 0.35 µg/ml and recoveries from 75% to 88 %. The findings showed that epigallocatechin, myricetin and rutin can be used for the quality control of this herb.