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

Background: 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), r 2 -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 X 2 -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.

Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

(Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)