In this study, Insect tea primary leaf (Malus sieboldii (Regal) Rehd.) was used as the research object to investigate the protective effect of Insect tea primary extract (ITPLE) on hydrogen peroxide (H2O2)-induced oxidative damage in human embryonic kidney 293T cells (HEK 293T cells) and the mechanism of action of the main active components. The 3-(4,5-dimethyl-2-thiazolyl)- 2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to determine the toxicity of ITPLE to HEK 293T cells in vitro as well as its protective effect against (H2O2)-induced oxidative damage in HEK 293T cells. In addition, various assay kits were used to measure oxidation-related indicators in HEK 293T cells, and quantitative polymerase chain reaction (qPCR) analysis was used to determine the mRNA expression levels of oxidation-related genes in HEK 293T cells. High performance liquid chromatography (HPLC) analysis was used to characterize active components in ITPLE. The experimental results revealed that the ITPLE had no toxic effect on cells in the range of 0–200 μg/mL, and, in this range, exhibited a concentration-dependent protective effect against H2O2-induced oxidative damage in HEK 293T cells. It was also found that the ITPLE can reduce the malondialdehyde (MDA) level and increase the levels of superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-Px), and catalase (CAT)in oxidative damage HEK 293T cells. The qPCR analysis results also showed that the ITPLE upregulated the mRNA expression levels of SOD, CAT, GSH and GSH-Px in HEK 293T cells damaged by H2O2-induced oxidative stress. The HPLC analysis identified 7 bioactive components in the ITPLE, including neochlorogenic acid, cryptochlorogenic acid, rutin, kaempferin, isochlorogenic acid B, isochlorogenic acid A and hesperidin. This study reveals that ITPLE is rich in active compounds and has good antioxidant effect in vitro, thus it has the potential to be developed into a traditional Chinese medicine and functional drinks.

中文翻译：

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昆虫茶初生叶的保护作用（三叶海棠（丽）甜茶。）上ħ提取₂ ö ₂在人胚胎肾293T细胞诱导的氧化损伤

本研究以昆虫茶原叶（Malus sieboldii（Regal）Rehd。）为研究对象，研究昆虫茶原提取物（ITPLE）对过氧化氢（H2O2）诱导的人胚肾脏氧化损伤的保护作用。 293T细胞（HEK 293T细胞）的作用机理主要是活性成分。使用3-（4,5-二甲基-2-噻唑基）-2,5-二苯基-2-H-溴化四唑（MTT）方法测定ITPLE对HEK 293T细胞的体外毒性及其保护作用对（H2O2）诱导的HEK 293T细胞的氧化损伤具有抗性。此外，各种测定试剂盒用于测量HEK 293T细胞中与氧化有关的指标，定量聚合酶链反应（qPCR）分析用于确定HEK 293T细胞中与氧化有关的基因的mRNA表达水平。高效液相色谱（HPLC）分析用于表征ITPLE中的活性成分。实验结果表明，ITPLE在0-200μg/ mL范围内对细胞无毒性作用，在此范围内，对HEK 293T细胞中H2O2诱导的氧化损伤具有浓度依赖性的保护作用。还发现ITPLE可以降低HEK 293T细胞氧化损伤中的丙二醛（MDA）水平并增加超氧化物歧化酶（SOD），谷胱甘肽（GSH），谷胱甘肽过氧化物酶（GSH-Px）和过氧化氢酶（CAT）的水平。 。qPCR分析结果还表明，ITPLE上调了H2O2诱导的氧化应激损伤的HEK 293T细胞中SOD，CAT，GSH和GSH-Px的mRNA表达水平。HPLC分析确定了ITPLE中的7种生物活性成分，包括新绿原酸，隐绿原酸，芦丁，kaempferin，异绿原酸B，异绿原酸A和橙皮苷。这项研究表明，ITPLE含有丰富的活性化合物，并且在体外具有良好的抗氧化作用，因此有可能被开发为中药和功能性饮料。