Ultrasonic-assisted extraction of fenugreek flavonoids and its geographical-based comparative evaluation using green UHPLC-DAD analysis

Abstract

This study, for the first time, reports a simultaneous determination of flavonoids; rutin (RT), quercetin (QT), luteolin (LT), and kaempferol (KF) in different origins of fenugreek seeds samples (N = 45) using a green UHPLC-DAD analysis METHODOLOGY: Ultrasound-assisted extraction (UAE) was employed to extract fenugreek flavonoids using different polarity solvents of n-hexane (n-hex), dichloromethane (DCM), and methanol (MeOH) RESULTS: The extract yield on an individual basis was observed in the range of 1.03-17.29 mg, with the highest yield (mg/sample) for the Egyptian sample (17.29 mg). The highest total extract yield (mg/origin) was observed for the Iranian sample (82.28 ± 5.38). The solvent with the highest extract yield (mg) was n-hex 169.35 ± 13.47, followed by MeOH 114.39 ± 12.27. The validated green UHPLC-DAD method resulted in a short runtime (9 min) with an accuracy of 97.86(±12.32)-101.37(±5.91), r²-values = 0.993-0.999, LOD = 2.09-4.48 ppm, and LOQ = 6.33-13.57 ppm for flavonoids analysis within the linearity range of 1-500 ppm. The general yield for flavonoids exhibited a descending order (ppm): RT (2924.55 ± 143.84) > QT (457.05 ± 34.07) > LT (82.37 ± 3.27) > KF (4.54 ± 0.00). The yield (ppm) for the flavonoids was more in MeOH solvent (3424.81 ± 235.44) constructing a descending order of MeOH > n-hex > DCM. For an individual flavonoid yield; MeOH was seen with an order of RT > QT > LT, n-hex (LT > QT), and DCM (RT > LT > QT). The statistical analysis of PCA (principle component analysis) revealed a widespread distribution of flavonoids in fenugreek seeds with a variance of 35.93% (PC1). Moreover, flavonoids extraction was prone to the nature and specificity of the solvent used (PC2: 33.34%) rather than the amount of the extract yield (P = 0.00). The K-mean cluster analysis showed the origins with higher flavonoids yield in appropriate solvent as I3M (Indian accession # 3 MeOH extract) with more QT amount, IR2M (Iranian accession # 2 MeOH extract) with more LT amount along with I2M (Indian accession # 2 MeOH extract) and Q2M (Qassim Saudi Arabia accession # 2 MeOH extract) containing high amount of RT. The outcomes are supported by KMO (Kaiser-Meyer-Olkin) and Bartlett's test value of 0.56 with X²-value of 191.87 (P = 0.00) CONCLUSION: The samples were effectively evaluated and standardized in terms of flavonoid amount suggesting a significant variation in fenugreek quality.

Keywords: Fenugreek; Flavonoids; Geographical origins; UAE; UHPLC.