Simultaneous Estimation of Glycosidic Isoflavones in Fermented and Unfermented Soybeans by TLC-Densitometric Method

Abstract

A simple, accurate and rapid high-performance thin-layer chromatographic (HPTLC) method for the simultaneous quantification of three glycosidic isoflavones (daidzin, genistin and glycitin) in soybean (Glycine max L.) has been established and validated. Chromatography was performed on aluminum foil-backed silica gel 60 F254 HPTLC plates and found compact spots for daidzin, genistin and glycitin (Rf value of 0.39, 0.51 and 0.32, respectively) with mobile phase toluene : ethyl acetate : formic acid : acetic acid in the ratio of 1 : 8 : 1 : 0.5, v/v/v/v. Ultraviolet detection was performed densitometrically at the maximum absorbance wavelength, 260 nm. The method was validated for precision, recovery, robustness, specificity, limit of detection (LOD) and limit of quantification (LOQ), in accordance with the ICH guidelines. The LOD (2.9, 19.3 and 3.5 µg mL(-1)), LOQ (9.03, 58.6 and 10.7 µg mL(-1)), recovery (95.9-106.66, 86.97-106.56 and 98.54-105.65%) and precision (≤2.12, ≤0.722 and ≤0.066) were satisfactory for glycosidic form of isoflavones daidzin, genistin and glycitin, respectively. Soybean variety Kh-09 bragg was found to have relatively higher amount of glycosidic isoflavones, namely daidzin, genistin and glycitin 278, 597.5 and 109.4 µg g(-1), respectively, and after fermentation the glycosidic isoflavones concentration in soybean fermented with Bacillus subtilis strain were decreased significantly after 24 h of incubation; conversely, aglycone isoflavones were increased significantly. The method for quantification of isoflavones in unfermented and fermented soybeans, with good resolution has been developed.

Comparative Study of Natural Antioxidants from Glycine max, Anethum graveolensand Pimpinella anisum Seed and Sprout Extracts Obtained by Ultrasound-Assisted Extraction

“…In the second chromatogram daidzein and genistein were separated from the extracts and could be identified in greater concentration in anise seeds, soy seeds and dill sprout extracts. Therefore, the presence of these compounds in the analyzed samples was confirmed, and was in accordance with the results from the literature [60][61][62].…”

“…Bands at 3500-3300 cm −1 , related to the asymmetric and symmetrical stretching vibrations of the hydroxyl groups, were identified, and also bands related to the vibration of the C=O groups at 1700-1600 cm −1 . The bands at 2980-3100, and also 1400 cm −1 , could be due to the stretching vibrations of the aromatic bonds υ-C=C-H [61,63]. The bands appearing in the region of 1000-1100 cm −1 appeared due to O-C vibrations bond (Table 1).…”

Analysis of Isoflavones in Foods

“…Thin layer chromatography (TLC) is used practically for preparative purposes (Zhao and others ), as is centrifugal partition chromatography (CPC) (Jeon and others ). High‐performance thin‐layer chromatography (HPTLC) has been used for quantification of the glycosidic isoflavones Di, Gi, and Gly in soybean (Puri and Panda ). Chromatographic separation was performed on aluminum foil‐backed silica gel 60 F254 HPTLC with toluene:ethyl acetate:formic acid:acetic acid (1:8:1:0.5, v/v/v/v) as mobile phase and densitometric UV detection at 260 nm.…”

A brief history and spectroscopic analysis of soy isoflavones

“…Through TLC analysis, glycitein was first discovered from soybean in 1973 (Naim et al, 1973;. Today, a highperformance TLC has been introduced, but it is less appropriate than other instrumental methods (GC and LC) when handling many samples through automated procedures (Puri and Panda, 2015).…”