目的 通过响应面法优化富含车前子苷的车前草黄酮提取工艺，并探究车前草黄酮对过氧化氢（H2O2）损 伤的L02 细胞的保护作用及其作用机制。方法 通过单因素研究乙醇浓度、提取温度、液料比对车前草黄酮 得率及其中车前子苷转移率的影响，在此基础上使用Central-Composite 中心组合试验设计和响应面分析法筛选 出车前草黄酮提取得率最高，车前子苷转移率最大的提取条件。通过测定该车前草黄酮对过氧化氢诱导的人正 常肝细胞L02 存活率、细胞凋亡、线粒体膜电位的影响，及其对L02 细胞丙二醛（MDA）、谷胱甘肽（GSH）、 过氧化物歧化酶（SOD）含量，HO-1、Nrf2、SOD 蛋白表达量的影响来探究车前草黄酮对过氧化氢损伤的L02 细胞的保护作用。结果 研究结果表明，提取温度80 ℃、乙醇浓度65%、液料比为15∶1 时，提取得到的车前 草黄酮得率和车前子苷转移率均最大，分别为1.82% 和35.01%。该条件下提取得到的车前草黄酮在25 ~ 100 μg·mL-1 内可明显降低过氧化氢损伤的L02 细胞的凋亡率（P＜0.001），降低其线粒体膜电位损伤程度（P＜ 0.001），增加细胞内SOD 和GSH 的表达量（P＜0.001），降低细胞中MDA 含量（P＜0.01，P＜0.001）。此外， 该车前草黄酮可以调节过氧化氢损伤的L02 细胞中HO-1、Nrf2、SOD 的表达量，降低过氧化氢损伤肝细胞中 HO-1、Nrf2 蛋白表达水平，并增加细胞中SOD 蛋白的表达量（P＜0.05，P＜0.01）。结论 使用响应面法筛选 出的车前草黄酮提取工艺操作简便，关键有效成分车前子苷的含量丰富，可为车前草黄酮的实际生产提供参 考。该方法提取得到的车前草黄酮可降低过氧化氢损伤的肝细胞的氧化应激水平，对L02 细胞具有一定的氧 化损伤保护作用，研究结果可以为车前草黄酮作为护肝保健品的开发应用奠定基础。

Abstract：Objective To optimize the extraction process of flavonoids from Plantago asiatica L. rich in plantagoside by response surface methodology，and to explore the protective effect and mechanism of plantain flavonoids on H2O2- damaged L02 cells. Methods The effects of various parameters， including ethanol concentration， extraction temperature and the liquid-solid ratio on the yield of flavonoids from P. asiatica and the transfer rate of plantagoside were studied by single factor experiment. The central composite design （CCD） and response surface methodology （RSM） were employed on this basic to screen the optimized extraction conditions， which yielded the highest extraction rate of flavonoids from P. asiatica and the maximum transfer rate of plantagoside. The protective effect of flavonoids from P. asiatica on H2O2-damaged L02 cells was assessed through the analysis of cell survival rate， apoptosis rate，mitochondrial membrane potential，malondialdehyde （MDA） and glutathione （GSH） levels，oxide dismutase （SOD） concentration， and the expression levels of HO-1， Nrf2， and SOD proteins. Results The results showed that the highest yield of extraction rate of flavonoids from P. asiatica （1.82%） and the maximum transfer rate of plantagoside（35.01%） were achieved under the optimized extraction condition：extraction temperature of 80 ºC，ethanol concentration of 65% and liquid-solid ratio of 15∶1. Flavonoids from P. asiatica extracted under these parameters significantly reduced the apoptosis rate （P＜0.001） and the degree of mitochondrial membrane potential damage （P＜0.001）， increased the expressions of SOD and GSH （P＜0.001）， and reduced the MDA level （P＜0.01， P＜0.001） in H2O2-damaged L02 cells at concentrations ranging from 25 to 100 μg·mL-1. In addition，100 μg·mL-1 flavonoids from P. asiatica were found to regulate the expressions of HO-1，Nrf2 and SOD in H2O2-damaged L02 cells. These flavonoids decrease the expressions of HO-1 and Nrf2 proteins， while enhance the expression of SOD protein in H2O2-damaged L02 cells （P＜0.05，P＜0.01）. Conclusion The extraction process for flavonoids from P. asiatica，which was optimized using the response surface method，is operationally simple and yields a high transfer rate of the key active component，plantagoside. This approach offers valuable insights for the actual production of flavonoids from P. asiatica. Besides， flavonoids from P. asiatica could reduce oxidative stress level in H2O2-damaged liver cells，which exhibited a certain protective effect on L02 cells against oxidative damage. This study provides a foundational basis for the development and application of flavonoids from P. asiatica as liverprotective health supplements.

R285.5

国家中医药管理局2022年中医药国际合作专项（GZYIGT2022026）；国家资助博士后研究人员计划项目（GZC20230629）。