4-Hydroxyisoleucine from Fenugreek: Preparation of high pharmacological strength extract and assay method development

Abstract

Our product marketed under the trade name FenuBet® was first standardized according to its trigonelline content but latest investigations showed that 4-Hydroxyisoleucine is more implicated in the antidiabetic effect of Fenugreek seeds. In this study two main topics were covered: optimization of factors leading to the preparation of an extract with high pharmacological strength using multi-stage counter-current extraction and development and validation of a new, simple, and rapid HPTLC-Scanner densitometry method for quantitative determination of 4-Hydroxyisoleucine. Extracts with plant to solvent ratio of 1/2.26 and 4-Hydroxyisoleucine concentration up to 1.28 mg/ml were obtained. The extraction yield showed that more than 82.5% of 4-Hydroxyisoleucine was recovered. The quantification method was found to give compact spots (Rf = 0.36). The minimum detectable amount was found to be 22.5 ng/spot, whereas the limit of quantitation was found to be 160 ng/spot. The linear regression analysis data for the calibration plots showed good linear relationship with 𝑟2 = 0.998±0.001 in the concentration range 22.5-160 ng/spot. %RSD for method repeatability was under 5.5% which is satisfactory.