EXTRACTION AND CHARACTERIZATION OF 4- HYDROXYISOLEUCINE FROM TRIGONELLA FOENUM GRAECUM SEEDS

The objective of the present investigation is to extract, quantify and characterize the active constituent, 4- hydroxyisoleucine in Trigonella foenum graecum seeds using HPLC and HPTLC methods. Aqueous extract of crushed seeds of Trigonella foenum graecum was prepared and subjected to HPLC and HPTLC analysis. HPLC analysis was performed using C18 reverse phase column and Fluorescence detector with an elution gradient composed of 65 mmol Sodium acetate, l.5% tetrahydrofurane (pH 5.7) and methanol. HPTLC analysis was performed using semiautomatic TLC sampler Linomat V (CAMAG) controlled by WinCATS software. A constant application rate of 80 nLs was employed with a band width of 3.0mm and distance between two bands was 6.2 mm. The plates were developed in 20 × 10 cm twin trough glass chamber containing 25mL of mobile phase as mixture of butanol: acetic acid: water (4: 1: 1, v/v/v) and detected at λ 254nm and 570 nm. HPLC analysis of Standard concentrations of 4-hydroxyisoleucine exhibited a linear plot with average retention time of 8.13 min and sample analysis revealed a retention time of 8.19 min. HPTLC analysis revealed Rf value of 0.45 for both standard and the seed extract at λ 254nm and 570 nm. Keywords: Trigonella, 4-hydroxyisoleucine, HPLC, HPTLC, Fluorescence detector, Retention time, Rf value.