Optimization of the preparation process for oligomeric proanthocyanidins from grape seeds based on the quality by design concept and analysis of their in vitro antioxidant activity

Objective This study aimed to develop key technologies for grape seed oligomeric proanthocyanidins （GS-OPC） preparation to build a scientific and comprehensive quality control system， and evaluate the GS-OPC antioxidant activity， so as to provide a reference for further development and utilization of GS-OPC extracts.Method The concept of quality by design （QbD） was introduced in risk factor analysis， single-factor experiments， and experimental design to determine the operating space of the preparation process. The GS-OPC antioxidant activity was determined using the methods of 1,1-diphenyl-2-picryl hydrazyl （DPPH）， 2,2′-azino-bis（3-ethylbenzothiazoline-6-sulfonic acid） （ABTS）， FRAP （ferric ion reducing antioxidant potential） in experiments of oxidative stress damage induced by hydrogen pero-xide， erastin， and glutamate in PC12 cells.Result The GS-OPC preparation process consisted of three steps （ethanol extraction， resin enrichment， and ethyl acetate extraction）. The operating space for the ethanol-extraction process included 2 extraction times， ethanol volume fraction of 64%-81%， extraction temperature of 30-36 ℃. The resin enrichment process included a loading sample ratio of 6∶1 （mL/g）， ethanol volume fraction of 60%-80%， elution volume of 3.5-4 BV. In the targeted products of GS-OPC， the contents of proanthocyanidins achieved 109.15% and oligomeric proanthocyanidins 92.05%. Compared with grape seed extracts （GSE） and grape seed proanthocyanidin fractions （GSF）， GS-OPC exhibited a high antioxidant capacity， with a DPPH free radical scavenging ability of 404.8 mg/g， FRAP total antioxidant capacity of 543.5 mg/g， and an ABTS total antioxidant capacity of 617.6 mg/g. GS-OPC effectively protected cells from oxidative stress damage induced by H₂O₂， erastin， and Glu by regulating reactive oxygen species （ROS） and lipid hydroperoxide （LPO）； at a dose of 100 μg/mL， the cell survival rate was 89.03%， 83.01%， and 92.85%， which represented increases of 34.55%， 47.70%， and 31.88% （absolute values） compared to the model group， respectively.Conclusion Guided by the QbD concept， the development of GS-OPC is scientifically guided， yielding a final product characterized by accessible preparation， controllable quality， and excellent bioactivity.