Enrichment of α-glucosidase inhibitory activity containing compounds in phaleria macrocarpa plant extract by subcritical carbon dioxide sothlet fractionation

Abstract

Phaleria macrocarpa, also known as “Mahkota Dewa,” is a common medicinal plant with enhance vitality. It is widely used as anti-diabetic remedies in Malaysia. The Subcritical Carbon dioxide Soxhlet (SubCO2), an advanced extraction technology which functions at a low temperature, was investigated for the fractionation of α-glucosidase inhibitory activity of P. macrocarpa in this study. The yields; enzyme activity and contents of potent inhibitory compounds of the extracts were examined. The yields of sonication and SubCO2 extraction method were approximately 10.2±1.0% to 25.4±0.6% and 0.9±0.1% to 2.0±1.0%, respectively. However, inhibitory activity of the extract (IC50) using SubCO2 (4.0±0.3 µg/mL) was higher than the sonication method (7.4±1.7 µg/mL). In addition, inhibitory compounds extracted using SubCO2 contained approximately 5-times more inhibitory compounds compared with sonication-extraction. Overall, SubCO2 extraction represents a promising method to produce highly potent inhibitory compounds contained extracts. In this study, inhibitory ability against ?-glucosidase of different parts of P. macrocarpa was screened. Methanol and n-hexane extracts, obtained by solvent extraction, were evaluated for invitro inhibition properties. The active compounds were identified using gas chromatography-mass spectrometry (GC-MS). The methanol extract of fruit flesh had the highest yield (25.6±0.5%), whereas n-hexane extract of stem is more effective against ?-glucosidase activity (0.8±0.1 µg/mL). The fruit flesh (1.3±0.2 ?g/mL) and leaves (1.6±0.6 ?g/mL) had also well effectively. The identified metabolites are D-fructose, squalene, ?-linolenic acid, and ?-D-glucopyranoside. In this research, a rapid and simple analytical method was developed for herbal quality control to investigate inhibitory activity of P. macrocarpa extract by multi component analyses using a Fourier transform infrared (FTIR) spectroscopy based fingerprinting. A total of thirty six extracts of different ethanol concentrations were prepared and tested on inhibitory potential, and fingerprinted by FTIR spectroscopy, coupled with chemometrics of orthogonal partial least square (OPLS) at 4000-400 cm-1 frequency region and resolution 4 cm-1. The OPLS model generated the highest regression co-efficient with R2Y=0.98, Q2Y=0.70, lowest root mean square error estimation (RMSEE) = 17.17 and root mean square error of cross validation (RMSECV) = 57.29, respectively. A five components (1+4+0) predictive model was developed to correlate FTIR spectra with activity and the responsible functional groups such as -CH, -C=O, -NH, -COOH and -OH were identified for the bioactivity. A successful multivariate model was constructed using FTIR-ATR as a simple and rapid technique to predict the inhibition activity. In this study, inhibitory potential against ?-glucosidase of various ethanolic extracts (water, 20%, 40%, 60%, 80%, and 100% ethanol) of P. macrocarpa were assessed using GC-MS and multivariate data analysis (MVDA). OPLS combined with GC-MS analysis was applied to correlate the inhibition of enzyme activity of various extracts to various compounds profiles of P. macrocarpa. The obtained score scatter plot of OPLS showed a distinct and remarkable separation of 6 different ratios of ethanolic extract into 6 clusters. GC-MS along with MVDA was used to identify the metabolites that inhibit the enzyme activity. In addition, myo-inositol, squalene, palmitic acid, and ?-D-glucopyranoside metabolites were identified and exhibit the potential inhibition activity of P. macrocarpa.