【摘要】 【目的】对大花序桉叶片黄酮类化合物进行分离鉴定和活性分析，为大花序桉叶片黄酮类化合物的开发利用提供参考依据。【方法】利用大孔树脂对大花序桉叶片黄酮类化合物进行纯化，比较8种净品级大孔树脂（X-5、ADS-17、D3520、HPD100、HPD722、HC-200、HPD400和AD201）对大花序桉叶片总黄酮的吸附和解吸特性，筛选最佳纯化树脂，并对纯化参数进行优化；应用红外光谱、超高效液相色谱-质谱联用（UHPLC-MS）技术对黄酮类化合物进行结构鉴定和定量分析，对比纯化前后黄酮类化合物的体外抗氧化活性[如1,1-二苯基-2-苦基肼自由基（DPPH）清除率、2,2-连氮基-双-3-乙基苯并噻唑啉-6-磺酸二铵盐（ABTS）清除率、总还原力]、酶抑制活性（乙酰胆碱酯酶、α-葡萄糖苷酶）和抗菌活性（大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌、欧文氏菌）。【结果】ADS-17树脂对大花序桉叶片总黄酮富集效果最佳，最优纯化工艺条件为：pH=3、洗脱剂乙醇体积分数50%、上样液质量浓度0.9 mg·mL^-1、吸附速率2.0 mL·min^-1、洗脱速率1.0 mL·min^-1、洗脱剂用量65 mL，在此条件下，总黄酮提取物纯度由20.03 mg·g^-1提高至36.31 mg·g^-1；经红外光谱扫描发现，其含有黄酮类物质特征吸收峰，进一步利用UHPLC-MS鉴定出13种黄酮类物质；与粗提物相比，纯化物黄酮类化合物的色谱峰变得更为突出，其中杨梅黄酮、杨梅苷和槲皮素纯化后的含量比纯化前分别高1.67、1.64和3.57倍；大花序桉叶片黄酮纯化物的抗氧化能力优于粗提物，其中纯化物DPPH和ABTS的半抑制质量浓度(IC₅₀)分别比粗提物降低0.77和11.31μg·mL^-1，与抗坏血酸（VC）的IC50差异不显著（P>0.05）；纯化物对乙酰胆碱酯酶和α-葡萄糖苷酶活性的抑制能力明显提高，其IC₅₀分别比粗提物降低45.26和1.60μg·mL^-1；纯化物的抑菌效果也强于粗提物，粗提物对大肠杆菌、金黄色葡萄球菌、欧文氏杆菌和枯草芽孢杆菌的最小抑菌质量浓度在12.50～25.00 mg·mL^-1范围内，而纯化物对以上菌种的最小抑菌质量浓度均小于6.25 mg·mL^-1。【结论】ADS-17大孔树脂可有效富集大花序桉叶片黄酮类化合物，纯化后的总黄酮具有良好的抗氧化、酶抑制和抑菌活性。更多还原

【Abstract】 【Objective】This study aims to provide a theoretical basis for the exploitation and utilization of Eucalyptus cloeziana leaves by isolating, and identifying the leaf flavonoids and determining their activity.【Method】In this study, the eight different macroporous resins were used to purify flavonoids from E. cloeziana leaves, and their adsorption/desorption characteristics of total flavonoids in E. cloeziana leaves were compared to select a macroporous resin suitable for purifying flavonoids from E. cloeziana leaves, and optimize the purification parameters. The structure and content of flavonoids were investigated with infrared spectroscopy and ultra-high performance liquid chromatography-mass spectrometry（UHPLC-MS）. In addition, the antimicrobial activity of flavonoids before and after purification against four bacterial strains of Staphylococcus aureus, Staphylococcus aureus,Bacillus subtilis, and Erwinia carotovora was investigated. At the same time, the in vitro antioxidant activity [such as 1,1-diphenyl-2-picrylhydrazyl（DPPH） free radical scavenging rate, 2,2-diazo-bis（3-ethyl-benzothiazole-6-sulfonic acid） diammonium（ABTS）free radical scavenging rate and total reducting capacity] and enzyme inhibitory activity（acetylcholinesterase, α-glucosidase） were also determined.【Result】The ADS-17 resin was identified as the optimum choice for its exceptional adsorption and desorption properties, and the optimal purification process conditions were as follows: pH value of 3, eluent ethanol volume fraction of 50%,concentration of flavonoids in the sample solution of 0.9 mg·mL^-1, adsorption rate of 2.0 mL·min^-1, elution rate of 1.0 mL·min^-1and eluent dosage of 65 mL. Under these conditions, the purity of the total flavonoid extract was increased from 20.03 mg·g^-1 to36.31 mg·g^-1. Through infrared spectroscopy scanning, it was shown that the extract contained characteristic absorption peaks of flavonoids. And then, a total of 13 flavonoids were identified by UHPLC-MS. Compared to the crude extract, the flavonoids peaks of the purified extract were more prominent, among which myricetin, myricitrin and quercetin were 1.67, 1.64 and 3.57 times higher than before purification, respectively. The antioxidant capacity of purified flavonoids from E. cloeziana leaves was superior to that of the crude extract. And the half inhibitory concentration(IC₅₀) values of the purified DPPH and ABTS were lower than those of the crude extract by 0.77 and 11.31 μg·mL^-1, respectively, and the difference in IC₅₀ value with VC was not significant（P>0.05）. In addition, the purified extracts showed significantly higher inhibitory ability on acetylcholinesterase and α-glucosidase activities, and their IC₅₀ values were 45.26 and 1.60 μg·mL^-1 lower than those of crude extracts, respectively. The antibacterial experiment also showed that purified extract was more effective than crude extracts, with the minimum inhibitory concentrations of the crude extracts against Escherichia coli, Staphylococcus aureus, Erwinia carotovora, and Bacillus subtilis ranging from 12.50 to25.00 mg·mL^-1, while the minimum inhibitory concentrations of the purified extracts against the above species were less than6.25 mg·mL^-1.【Conclusion】ADS-17 macroporous resin can effectively enrich the flavonoids in E. cloeziana leaves, and the purified flavonoids have good antioxidant, enzyme inhibition and bacteriostatic activities.更多还原