山楂绿原酸提取工艺优化及其对血管内皮细胞氧化损伤的保护作用

牟双; 黄亦琳; 郭杨; 毛芯雨; 朱文赫

doi:10.13386/j.issn1002-0306.2025080328

山楂绿原酸提取工艺优化及其对血管内皮细胞氧化损伤的保护作用

Optimization of Extraction Process of Chlorogenic Acid from Hawthorn and Its Protective Effect Against Oxidative Damage of Vascular Endothelial Cells

摘要

摘要: 为优化山楂绿原酸的提取工艺，探讨其抗氧化作用及其对血管内皮细胞氧化损伤的保护作用，本研究采用微波辅助提取法，通过单因素实验和正交试验考察乙醇体积分数、料液比、提取时间和微波功率4个因素对山楂绿原酸得率的影响，并建立过氧化氢（Hydrogen peroxide，H₂O₂）诱导的人脐静脉内皮细胞（Human umbilical vein endothelial cells，HUVEC）氧化损伤模型，考察山楂绿原酸（Chlorogenic acid，CGA）在保护血管内皮细胞氧化损伤中的作用。结果表明，山楂绿原酸的最佳提取工艺条件为：乙醇体积分数50%、料液比1:20 g/mL、提取时间10 min、微波功率320 W。在此条件下，绿原酸得率可达3.98%，且同一因素水平下得率波动较小，表明工艺稳定性良好。氧化损伤保护实验结果显示，与对照组相比，H₂O₂损伤组能够明显降低细胞存活率（P<0.01），细胞内活性氧（Reactive oxygen species，ROS）含量上升，细胞内超氧化物歧化酶（Superoxide dismutase，SOD）活性和谷胱甘肽过氧化物酶（Glutathione peroxidase，GSH-Px）活性下降，丙二醛（Malondialdehyde，MDA）含量上升，Cleaved caspase-3表达水平升高，Bax/Bcl-2蛋白表达比值升高（P<0.05）。与H₂O₂损伤组相比，不同CGA处理组细胞存活率显著提升（P<0.05），细胞内MDA含量下降，SOD活和GSH-Px活性升高，细胞内ROS表达下降，细胞内激活型Caspase-3表达下降，Bax/Bcl-2比值下降（P<0.05）。因此，研究表明山楂CGA对H₂O₂所致人脐静脉内皮细胞氧化损伤具有保护作用，本研究为绿原酸在功能性食品研发及心血管相关疾病辅助干预的候选成分筛选中提供了细胞水平的理论参考。

Abstract: To optimize the extraction process of chlorogenic acid from hawthorn and investigate its antioxidant effects and protective role against oxidative damage in vascular endothelial cells, this study employed microwave-assisted extraction. The effects of four factors—ethanol concentration, solid-to-liquid ratio, extraction time, and microwave power—on the yield of chlorogenic acid from hawthorn were examined through single-factor and orthogonal experiments. Additionally, an oxidative damage model of human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H₂O₂) was established to evaluate the protective effect of hawthorn chlorogenic acid (CGA) against oxidative damage in vascular endothelial cells. The results showed that the optimal extraction conditions for chlorogenic acid from hawthorn were as follows: Ethanol concentration of 50%, solid-to-liquid ratio of 1:20 (g/mL), extraction time of 10 min, and microwave power of 320 W. Under these conditions, the yield of chlorogenic acid could reach 3.98%, and the yield fluctuation was small at the same factor level, indicating good process stability. The results of the oxidative damage protection experiment showed that compared with the control group, the H₂O₂-induced damage group significantly reduced cell viability (P<0.01), increased the intracellular content of reactive oxygen species (ROS), decreased the activities of intracellular superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), increased the content of malondialdehyde (MDA), upregulated the expression level of Cleaved caspase-3, and elevated the Bax/Bcl-2 protein expression ratio (P<0.05). Compared with the H₂O₂-induced damage group, the different CGA treatment groups significantly increased cell viability (P<0.05), reduced intracellular MDA content, elevated the activities of SOD and GSH-Px, decreased intracellular ROS levels, downregulated the expression of Cleaved caspase-3, and lowered the Bax/Bcl-2 ratio (P<0.05). Therefore, this study demonstrates that CGA from hawthorn exerts a protective effect against H₂O₂-induced oxidative damage in human umbilical vein endothelial cells (HUVECs). It provides a cellular-level theoretical reference for the application of chlorogenic acid in the development of functional foods and the screening of candidate components for auxiliary intervention in cardiovascular-related diseases.