Response Surface Methodology-Optimized Ultrasonic-Assisted Extraction Combined with Folin–Ciocalteu Assay for Total Polyphenol Determination in Grape Seeds: Development and Application

A robust Folin–Ciocalteu method, coupled with an optimized ultrasonic-assisted extraction, was established for accurate quantification of total polyphenols in high-oil grape seed matrices, where lipid interference and low extraction efficiency have been persistent challenges. Samples were first defatted with n-hexane to eliminate lipid interference. Key colorimetric parameters—Folin–Ciocalteu reagent volume, Na2CO3 concentration, reaction temperature, and time—were systematically optimized and validated for linearity, precision, and recovery. Subsequently, using defatted grape seed powder as the raw material, a four-factor, three-level Box–Behnken design combined with response surface methodology was employed to optimize the four extraction parameters: solid-to-liquid ratio, ethanol concentration, extraction temperature, and extraction time. The optimal conditions were 0.5 mL of Folin–Ciocalteu reagent, 20% Na2CO3, and reaction at 30 °C for 2.0 h, yielding a linear calibration curve (R2 = 0.9991) with satisfactory methodological validation. Optimal extraction (52% ethanol, 1:50 w/v, 68 °C, 21 min) achieved a total polyphenol content of 2.93 × 104 mg·kg−1, closely matching the predicted value (relative error = 0.34%). Analysis of seven grape seed varieties from the Hebei Province revealed significant content variation (p < 0.05), ranging from 3.24 to 7.47 × 104 mg·kg−1, with Rose grape seeds exhibiting the highest level. The developed method effectively overcame matrix interference from high oil content, offering a reliable, efficient tool for screening high-polyphenol grape seed varieties and supporting the development of value-added functional products.