基于质量源于设计理念优化葡萄籽低聚原花青素制备工艺及体外抗氧化活性分析

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

    摘要
  • 摘要: 目的 突破葡萄籽低聚原花青素（GS-OPC）制备的关键技术，建立科学、完善的质量控制体系，并评价其抗氧化活性，为葡萄籽原花青素提取物进一步开发利用提供参考依据。方法 引入质量源于设计（QbD）理念，通过风险因素分析、单因素试验及试验设计的综合运用获得制备工艺的操作空间；通过1,1-二苯基-2-三硝基苯肼（DPPH）法、铁离子还原抗氧化能力（FRAP）法、2,2′-联氮双（3-乙基苯并噻唑啉-6-磺酸）二铵盐（ABTS）法，以及过氧化氢（H2O2）、Erastin和谷氨酸（Glu）诱导的PC12细胞氧化应激损伤试验，考察GS-OPC的抗氧化活性。结果 GS-OPC的制备工艺涉及乙醇提取、树脂富集和乙酸乙酯萃取3个步骤，建立的乙醇提取工艺操作空间为提取次数2次、乙醇体积分数64%~81%、提取温度30~36 ℃；树脂富集工艺操作空间为上样量6∶1（mL/g）、乙醇体积分数60%~80%，洗脱体积3.5~4 BV；目标产品GS-OPC中原花青素含量高达109.15%，低聚原花青素含量为92.05%；相较于葡萄籽提取物（GSE）和葡萄籽原花青素富集物（GSF），GS-OPC显示出优异的抗氧化能力，其DPPH自由基清除能力、FRAP总抗氧能力和ABTS总抗氧能力分别为404.8、543.5和617.6 mg/g；GS-OPC还可通过调节活性氧（ROS）和脂质过氧化物（LPO）水平，有效保护细胞免受H2O2、Erastin和Glu引起的氧化应激损伤，在100 μg/mL剂量下，细胞存活率分别为89.03%、83.01%和92.85%，较模型组分别提高34.55%、47.70%和31.88%（绝对值）。结论 以QbD理念科学指导GS-OPC的开发，获得的终产品具有制备可及、质量可控、活性优良的特征。

     

    Abstract: 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.

     

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