【作者】 谭涛；

【导师】 赵希娟；

【作者基本信息】 西南大学 ， 果树学， 2024， 硕士

【摘要】 佛手（Citrus medica L.var.sarcodactylis Swingle）是芸香科柑橘属植物,兼具药食两用与观赏于一身。本文测定了金华佛手和万州佛手3个不同生长期类黄酮和香豆素的含量,通过3种化学法结合RAW264.7细胞抗氧化评价方法检测了3个不同生长期佛手提取物的体外抗氧化能力,并分析了香豆素和类黄酮含量与其抗氧化能力之间的相关性。通过上述试验,得出如下结论:（1）基于超高效液相色谱-三重四极杆串联质谱（Ultra-high-performance liquid chromatography with triple quadrupole mass spectrometry,UPLC-Qq Q-MS/MS）平台建立了48种功能成分的定量方法,可在15 min内对佛手中22种香豆素和26种类黄酮进行筛查和定量分析,这是目前对佛手生物活性物质靶向筛查和定量分析报道中较全面的研究,所有样品中共检出45种物质,槲皮素、根皮素、补骨脂素无检出,对其中44种物质进行了准确定量,4’,5,6,7-四甲氧基黄酮有检出但无法定量,该方法线性关系良好,相关系数均大于0.99,灵敏度和准确度高,能很好地区分同分异构体。39种物质的检出限不高于1.00μg/L,32种物质的定量限不高于1.00μg/L,回收率在76.23～125.91%之间。不同产地或者生长期的佛手类黄酮和香豆素种类上基本一致,橙皮苷、地奥司明、芦丁、新橙皮苷、水仙苷是佛手果实中重要的类黄酮,含量最高的地奥司明在638.95～989.36μg/g之间,橙皮苷含量在627.71～974.29μg/g之间,柠檬油素、氧化前胡素、白当归脑、佛手苷内酯、滨蒿内酯、东莨菪亭是佛手果实中重要的香豆素,含量最高的柠檬油素在852.89～2078.92μg/g之间,占总香豆素含量的66.34%～84.00%,不同生长期的类黄酮和香豆素含量差异很大,在11月时含有更高的类黄酮,在万州佛手和金华佛手总类黄酮含量分别高达1873.88±45.11μg/g和2360.89±123.753μg/g。（2）采用DPPH、ABTS和FRAP三种化学方法,对不同生长期佛手提取物体外化学抗氧化活性进行评价,不同生长期的佛手提取物都具有较好的体外抗氧化活性,并呈剂量依赖性,使用APC综合排名确定各生长期的抗氧化能力强弱顺序,不同生长期化学抗氧化活性的关系为11月>7月>9月。（3）建立H₂O₂诱导RAW264.7细胞氧化应激模型,用0.004%H₂O₂处理细胞时,RAW264.7细胞的存活率达到49.93%。研究不同生长期佛手提取物低、中、高剂量组（400 mg/L,600 mg/L,1000 mg/L）对损伤细胞的保护作用,结果表明不同生长期佛手提取物均可显著抑制H₂O₂对RAW264.7细胞活性的影响（P<0.05）,显著降低细胞及细胞培养液中NO、ROS、MDA含量的同时提高SOD的活性（P<0.05）,不同生长期细胞抗氧化活性的关系为11月>7月>9月。（4）采用皮尔逊相关系数对佛手主要活性物质与其抗氧化活性进行相关性分析,分析结果显示总类黄酮含量与佛手抗氧化能力极显著正相关（P<0.01）,地奥司明、橙皮苷、水仙苷、新橙皮苷和柠檬油素是反映佛手抗氧化活性的重要特征物质。更多还原

【Abstract】 Fingered citron（Citrus medica L.var.sarcodactylis Swingle）belongs to Rutaceae family.In addition to its long medicinal history,it also has edible value and ornamental value.In this paper,the content of flavonoids and coumarins in three different growth periods of Jinhua and Wanzhou fingered citron was determined.The antioxidant ability of three different growth periods of fingered citron extracts was detected by three chemical methods combined with RAW264.7 cells.The correlation between coumarins and flavonoids and their antioxidant capacity was also analyzed.Through the above test,the following conclusions are drawn:（1）A quantitative method of 48 functional components was established based on the ultra-high-performance liquid chromatography with triple quadrupole mass spectrometry（UPLC-Qq Q-MS/MS）,which could screen and quantitatively analyze 22coumarins and 26 flavonoids in fingered citron within 15 min.This is a comprehensive study on the quantitative analysis of bioactive compounds contained in fingered citron.A total of 45 compounds were detected in all samples,quercetin,phloretin and psoralen were not detected,44 of which were accurately quantified,and 4’,5,6,7-tetramethoxyflavone were detected but could not be quantified.This method has a good linear relationship with correlation coefficients all greater than 0.99,high sensitivity and accuracy,and can distinguish isomers well.The LOD of 39 compounds was not higher than 1.00μg/L,and the LOQ of 32 compounds was was not higher than 1.00μg/L.The recoveries ranged from 76.23%to 125.91%.The types of flavonoids and coumarins in different regions or growth periods were basically the same.Hesperidin,diosmin,rutin,neohesperidin and narcissoside were important flavonoids in the fingered citron.Diosmin showed the highest content of 638.95～989.36μg/g,and the content of hesperidin was627.71～974.29μg/g.Limettin,oxypeucedanin,byakangelicol,bergapten,scoparone and scopoletin were the important coumarins in fingered citron,and limettin had the highest content of 852.89～2078.92μg/g,accounting for 66.34%～84.00%of the total coumarin content.The flavonoid and coumarin contents varied greatly in different growth periods,and the flavonoid contents in Wanzhou fingered citron and Jinhua fingered citron were higher in November.The total contents of flavonoid in Wanzhou and Jinhua fingered citron were as high as 1873.88±45.11μg/g and 2360.89±123.753μg/g,respectively.（2）DPPH,ABTS and FRAP methods were used to evaluate the antioxidant activity of fingered citron extracts at different growth periods.The extracts of fingered citron at different growth periods showed good antioxidant activity in vitro and were dose-dependent.APC ranking was used to determine the order of antioxidant capacity in each growth periods.The order of chemical antioxidant activity at different growth periods was November>July>September.（3）the oxidative stress model of RAW264.7 cells induced by H₂O₂ was established.The survival rate of RAW264.7 cells reached 49.93%when cells were treated with 0.004%H₂O₂.The protective effects of fingered citron extracts in low,medium and high dose groups（400 mg/L,600 mg/L,1000mg/L）at different growth periods were further studied.The results showed that at different growth periods,the extract could significantly inhibit the effects of H₂O₂ on the activity of RAW264.7 cells（P<0.05）,significantly reduce the contents of NO,ROS and MDA in cells and cell culture medium,and increase the activity of SOD（P<0.05）.The relationship of antioxidant activity of cells at different growth periods was November>July>September.（4）Pearson correlation coefficient was used to analyze the correlation between the main active compounds of fingered citron and their antioxidant activities.The results showed that the content of total flavonoids was significantly positively correlated with the antioxidant capacity of fingered citron（P<0.01）.Diosmin,hesperidin,narcissoside,neohesperidin and limettin were important characteristic compounds contributing to the antioxidant activity of fingered citron.更多还原