Development and validation of HPTLC method for identification and quantification of sterols from leaves of Erythroxylum monogynum Roxb. and in vitro evaluation of anti-oxidant and anti-glycation activities

Abstract

The petroleum ether extract from leaves of Erythroxylum monogynum when subjected to phytochemical analysis and chromatographic purification, resulted in the isolation of two new sterols namely 4-methyl ergosta-7, 23‑dien‑3β-ol and 4-methyl ergosta-7, 24 (28)‑dien‑3β-ol. Their structure was elucidated by NMR and Mass Spectrometry. The two sterols were separated on a silica gel 60F254 HPTLC plates impregnated with AgNO3 using dichloromethane: methanol (9:1 v/v) as a mobile phase. Upon densitometric scanning at 254 nm in a reflectance mode, the two sterols separated as sharp and compact bands with Rf of 0.47±0.02 and 0.52±0.01 respectively for 4-methyl ergosta-7, 23‑dien‑3β-ol and 4-methyl ergosta-7, 24 (28)‑dien‑3β-ol. Linear regression and calibration plots constructed (n = 6) showed excellent linearity at 100–500 ng per spot with a correlation coefficient, r = 0.9979±0.0004 and 0.9974±0.0007 for 4-methyl ergosta-7,23‑dien‑3β-ol and 4-methyl ergosta-7,24 (28)‑dien‑3β-ol, respectively. The Limits of detection and quantification were found to be 21.07 ng and 57.23 ng/band for 4-methyl ergosta-7,23‑dien‑3β-ol and 25.18 ng and 63.86 ng/band for 4-methyl ergosta-7,24 (28)‑dien‑3β-ol, respectively. Both the sterols demonstrated good anti-oxidant and anti-glycation activities where, the IC50 values obtained were comparable with the positive control compounds, ascorbic acid and aminoguanidine. The 4-methyl ergosta-7,23‑dien‑3β-ol displayed 100% anti-oxidant activity at 100 µg/ml for both DPPH and H2O2 scavenging assays. In addition, both the sterols presented more than 90% inhibition of Advanced Glycation End products at 100 µg/ml concentration. The results obtained are remarkable indicating that these plant extracts could serve as a natural alternative to the conventional oral hypoglycemic molecules.