Bioactives in traditional Malaysian vegetables and their antioxidant activities

Abstract

The aim of the present study was to develop a specific and quantitative analytical method to determine the individual concentrations of naturally occurring phenolic acids and flavonoids and their metabolites in food; then evaluate the antioxidant capacity of the quantified phenolic acids using different assays. An LC-MS/MS method for the quantification of 9 phenolic acids and 5 commonly present flavonoid compounds in plant foods was developed. Target analytes were the benzoic acid based phenolic acids - anisic, gallic, salicylic, protocatechuic, and vanillic acid, together with the cinnamic acid based phenolic acids - caffeic, p-coumaric, ferulic and sinapinic acid. As for the flavonoids, target analytes were apigenin, luteolin, kaempferol, quercetin and myricetin. These compounds were identified by retention times and comparison of electron impact mass spectra. Deuterium-labelled ferulic acid and 13C-labeled quercetin were used as the internal standards to achieve accurate quantification. Purification was accomplished using Strata® SDBL (styrene-divinylbenzene) cartridges for solid phase extraction (Phenomenex, US). Sample extracts were subjected to acid and/or alkaline hydrolysis to release the free phenolic acids and flavonoids from the ester and glycoside forms found in plant materials. The resolution and quantitation of all compounds were excellent with linear calibration curves over the range detection limit for each compound to 750 ng/µL and correlation coefficients (R2) greater than 0.9643. The intraday assay range of relative standard deviation (RSD) was 2.6-9.0 for high concentrations and 2.3-16.7 for low concentrations of a standard mixture. For interday assay, the relative standard deviation (RSD) ranges were 10.8-20.2 for high concentrations and 12.7-19.4 for low concentrations. Percentage recovery of analytes were satisfactory (72 to 128% respectively) and within the acceptable limits of bioanalytical method validation guidelines (FDA, 2001). The developed and validated method was applied to different types of Malaysian traditional vegetables, fresh (n=15). The results revealed that flavonoids are the most abundant polyphenols in plant extracts, followed by hydroxycinnamic acids of phenolic acids, mostly in bound form as esters or glycosides. Only hydrophilic antioxidant activities of all selected samples were examined using ORAC assay. In addition the antioxidant capacity of the identified free and bound phenolic acids content was measured using different assays including ORAC, ABTS, and DPPH assay (end-point assay and kinetic assay). The results indicate that the total phenolic acids content did not correlate positively with antioxidant activity, but the total flavonoid content was positively correlated with antioxidant activity. In conclusion the analytical method presented is robust, safe, and generally applicable to the analysis of free and bound phenolic acids and flavonoids in food samples.