Unveiling the Phenolics Profile for the Fenugreek Seeds Using Green Ultrasonic Extraction with Ultra High Pressure Chromatography Analysis

This preliminary study aimed to identify and simultaneously quantify four phenolic of gallic acid (GA), scopoletin (SC), rosmarinic acid (RA), and resveratrol (RV) in the fenugreek (Trigonella foenum-graecum) seeds. Ultrasonic-assisted extraction using a dismembrator (20 kHz) was performed using the uncrushed on whole seeds with three green solvents: acetone (ACT), ethanol (EtOH), and water (H₂O). An in-house UHPLC-DAD method was developed, validated, and applied to determine phenolic profiles in 15 seed samples from Egypt, India, Qassim (Saudi Arabia), Yemen, and Iran. Method validation showed high accuracy [GA: 99.77 ± 12.33%, SC: 99.88 ± 7.94%, RA: 100.37 ± 8.02%, RV: 101.39 ± 5.39%] with r² ≥ 0.9998. Relative SD ranged from 3.04–3.90%, LOD from 10.10–13.85 ppm, and LOQ from 30.60–41.97 ppm. Extract yields (1.2–152.9 mg; mean 24.19 ± 33.12 mg, N = 45) ranked: H₂O (622.8 ± 231.4 mg) > ACT (417.8 ± 61.2 mg) > EtOH (48 ± 5.8 mg). Yemen-origin JY1 (152.9 mg), Egypt-origin JE2 (140.3 mg), and Qassim-origin RQ2 (107.3 mg) gave the highest yields. Total phenolics followed SC (2337.0 ppm) > GA (146.2 ppm) > RV (135.4 ppm) > RA (109.7 ppm). Herein, a solvent specific phenolic recovery ranked as: H₂O (2613.3 ± 502.1 ppm) > EtOH (60.99 ± 27.17 ppm) > ACT (53.6 ± 7.41 ppm), with occurrence patterns: H₂O (SC > GA > RA > RV), EtOH (RV > SC > GA), ACT (GA > SC > RV). Pearson’s correlation confirmed a positive relationship between solvent type and phenolic yield, while K-means clustering identified JQ1 (Qassim) and RE1 (Egypt) as rich in multiple phenolics. These findings offer practical insight of solvent-dependent variation in phenolic content, underlining water as the most effective green solvent for maximizing extraction yield and phenolic recovery.