金樱根提取物不同极性部位的体外抗氧化活性考察及其化学成分分析

目的:考察金樱根75%乙醇提取物及其不同极性萃取部位的抗氧化活性和化学组成。方法:采用有机溶剂萃取法将金樱根75%乙醇提取物分为二氯甲烷、乙酸乙酯、正丁醇和水部位,利用1,1-二苯基-2-三硝基苯肼(DPPH)和2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)自由基清除试验、铁离子还原能力(FRAP)试验考察金樱根乙醇提取物及其不同极性萃取部位的体外抗氧化活性。采用紫外分光光度法测定各部位中总三萜、总酚、总鞣质和缩合鞣质的含量。运用SPSS24.0对各部位抗氧化活性与其化学成分含量进行Pearson相关分析,筛选活性部位及主要的活性成分;采用超高效液相色谱-四极杆飞行时间质谱法(UPLC-Q-TOF-MS/MS)对该活性部位化学成分进行分析,推测主要色谱峰的结构。结果:金樱根乙醇提取物及其不同极性萃取部位均有一定的抗氧化活性,且在一定浓度范围内呈显著剂量-效应关系,但不同部位的抗氧化活性有差异;在DPPH和ABTS自由基清除试验中,阳性药维生素C(VC)和各部位抗氧化能力排序为乙酸乙酯部位>VC>正丁醇部位>乙醇提取物>水部位>二氯甲烷部位;FRAP试验中乙酸乙酯部位的活性弱于VC,其他次序不变。含量测定结果显示,乙酸乙酯部位中总三萜、总酚、总鞣质和缩合鞣质的质量分数分别为3.81%,50.33%,3.32%,39.79%,正丁醇部位中依次为0.88%,41.42%,2.25%,23.55%,乙醇提取物中依次为2.90%,41.95%,3.43%,20.14%,水部位中依次为0,26.80%,16.90%,7.57%,二氯甲烷部位中依次为21.23%,12.90%,1.59%,6.17%。相关性分析结果显示金樱根中总酚和缩合鞣质含量与抗氧化活性呈正相关,总三萜含量与抗氧化活性呈负相关,总鞣质含量与抗氧化活性相关性不明显。经液质联用分析,从乙酸乙酯部位中共鉴定出26个化合物,包括11个缩合鞣质,4个可水解鞣质,6个三萜类,3个黄酮类,1个苯甲酸衍生物和1个绿原酸类化合物。结论:乙酸乙酯部位是金樱根发挥抗氧化作用的主要活性部位,而以缩合鞣质为主的酚类物质是其主要活性成分,可为天然抗氧化剂的开发提供实验依据。

Investigation on in Vitro Antioxidant Activity and Chemical Composition of Different Polar Parts of Extract of Rosa cymosa Roots

Objective: To investigate the antioxidant activity and chemical composition of 75%ethanol extract of Rosa cymosa roots and its different polar parts. Method: The 75% ethanol extract of R.cymosa roots was divided into dichloromethane,ethyl acetate,n-butanol and water parts by organic solvent extraction. In vitro antioxidant activity of each fraction was determined by 1,1-diphenyl-2-picrylhydrazyl(DPPH)and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)free radical scavenging assays,as well as ferric reducing antioxidant power(FRAP)test. The contents of total triterpenes,total phenols,total tannins and condensed tannins in each fraction were determined by spectrophotometry. SPSS 24.0 software was used to conduct Pearson correlation analysis between the antioxidant activity of each fraction and the content of the main components,and then the main active fraction and the main active components were determined. The chemical constituents of the active fraction was analyzed by ultra performance liquid chromatographyquadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS), and the structures of the main chromatographic peaks were predicted. Result: Each fraction of R. cymosa roots had certain antioxidant activity, and there was a significant dose-effect relationship within a certain concentration range, but the antioxidant activity of different polar parts was different. In DPPH and ABTS free radical scavenging tests,the antioxidant activity of each fraction and vitamin C(VC,the positive drug)was ranked as ethyl acetate fraction>VC>n-butanol fraction>ethanol extract>water fraction>dichloromethane fraction. In FRAP test,the activity of ethyl acetate fraction was weaker than that of VC,and the other order was unchanged. The contents of total triterpenes,total phenols,total tannins and condensed tannins in ethyl acetate fraction were 3.81%,50.33%,3.32%,and 39.79%,in n-butanol fraction were 0.88%,41.42%,2.25% and 23.55%,in ethanol extract were 2.90%, 41.95%, 3.43% and 20.14%, in water fraction were 0, 26.80%, 16.90% and 7.57%, and in dichloromethane fraction were 21.23%,12.90%,1.59%,and 6.17%,respectively. Correlation analysis results showed that the contents of total phenols and condensed tannins were positively correlated with the antioxidant activity, the contents of total triterpenes were negatively correlated with the antioxidant activity, and the correlation between total tannins and antioxidant activity was not obvious. A total of 26 compounds were identified from the ethyl acetate fraction by UPLC-Q-TOF-MS/MS, including 11 condensed tannins, 4 hydrolysable tannins,6 triterpenes,3 flavonoids,1 benzoic acid derivative and 1 chlorogenic acid analogue.Conclusion: Ethyl acetate fraction is the main antioxidant active site of R. cymosa roots,and phenols mainly composed of condensed tannins are the main active components. The results can provide experimental basis for the development of natural antioxidants.