Supercritical fluid extraction and chromatographic and spectroscopic analysis of bioactive compounds from pereskia bleo

Abstract

Pereskia bleo is a cactus species of primitive origin that has green leaves unlikely with other cactus species. Leaves of P. bleo are an important ingredient for herbal products. Supercritical fluid extraction (SFE) was investigated in the present study as an alternative herbal preparation method to obtain antioxidant rich extract. Multicomponent contributing bioactivity analysis of herbal products using affordable and available instruments was also evaluated. SFE was carried out with screening and optimization of the factors affecting the yield and antioxidant activity. Initially, a 12 run plackett-burman (PB) experimental study was conducted to screen the most influential factors in SFE. A total of five factors were studied: temperature, pressure, extraction time, CO2 flow rate and modifier (%) of dry weight of sample. All factors had significant effects on yield but only pressure and modifier had significant effect on IC50 value of DPPH antioxidant assay. Consequently, three significant factors were further studied by box-behnken (BB) experimental study to achieve the optimal conditions. Temperature of 52°C, pressure of 36 MPa and 31% (v/w) ethanol were found as optimal condition for both yield and IC50. Pressure and modifier were highest affecting factors to obtain antioxidant rich extract by SFE. Addition of very small amount of ethanol (1.5% of total CO2) improved the extraction yield and antioxidant activity of the extract. GC-MS analysis has shown the higher extraction efficiency of ?-tocopherol and erythritol by ethanol modified SFE. Thin layer chromatography (TLC) and fourier transformed infrared spectroscopy (FTIR) were used for multicomponent contributing bioactivity analysis of P. bleo leaves extract. TLC was developed with optimized mobile phase with “PRISMA” optimization method. Multivariate orthogonal partial least square (OPLS) model was developed consisting wavelets converted TLC image and antioxidant activity. The OPLS model was then evaluated by different validation test. The bioactivity contributing band from TLC plate was then analyzed by GC-MS and, glycerol and amine compounds were identified for contributing activity of sample. For FTIR experiment, spectrum was collected at wavenumber range of 4000–400 cm-1 at a resolution of 2 cm-1. After three processing steps, OPLS model was developed to correlate FTIR spectrum and antioxidant activity of extracts. A three components OPLS with R2Y of 0.88 and Q2 of 0.86 was constructed, which had proved its quality by two different validation tests namely, internal cross-validation and true external test. The constructed model was described to identify the responsible peaks for the activity. The -OH, -NH and –CH peaks of FTIR spectrum mostly accounts for the antioxidant activity.