摘要
目的:建立高效液相色谱(HPLC)-1-二苯基-2-三硝基苯肼(DPPH)在线筛选系统分析杜仲叶抗氧化活性成分;应用制备型高效液相色谱(Prep-HPLC)和高速逆流色谱仪(HSCCC)建立抗氧化成分的富集和分离方法。方法:采用大孔树脂AB-8对杜仲叶的提取物进行分离,收集水洗脱组分,采用HPLC-DPPH在线检测系统筛选抗氧化活性成分;应用Prep-HPLC对筛选目标抗氧化活性成分建立富集方法,并应用HSCCC纯化抗氧化活性成分。同时,结合分子对接和体外DPPH自由基清除率对其进行抗氧化活性评价。结果:采用HPLC-DPPH在线筛选系统及Prep-HPLC建立抗氧化活性成分的筛选和富集方法,并应用HSCCC色谱进一步分离纯化5个抗氧化活性成分,分别为儿茶素(1)、绿原酸(2)、咖啡酸(3)、山柰酚-3-O-β-D-葡萄糖苷(4)和槲皮素(5);通过分子对接和体外抗氧化活性实验进一步验证该5种化合物的抗氧化活性。分子对接显示,5种成分的结合能范围为−6.4~−4.8 kcal/mol,其中儿茶素和山柰酚-3-O-β-D-葡萄糖苷的结合能最大;体外DPPH自由基清除率显示,5种成分对DPPH自由基清除能力由大到小依次为山柰酚-3-O-β-D-葡萄糖苷、儿茶素、槲皮素、咖啡酸、绿原酸。结论:应用HPLC-DPPH在线筛选系统和Prep-HPLC能够有效筛选和富集天然产物中的抗氧化活性成分,同时应用HSCCC能够实现活性成分的进一步纯化;分离得到的抗氧化活性成分中,儿茶素和山柰酚-3-O-β-D-葡萄糖苷均表现出较强的抗氧化活性。
Objective:To screen the active components of Eucommia ulmoides leaves by establishing DPPH-HPLC online screening system,and to establish the enrichment and separation methods of antioxidant components by Prep-HPLC and HSCCC.Methods:The extracts of Eucommia ulmoides leaves were isolated with macroporous resin AB-8,and the water-eluting components were collected.The antioxidant active components were screened by HPLC-DPPH online detection system.Prep-HPLC was used to establish the enrichment method for the target antioxidant active ingredients,and HSCCC was used to purify the antioxidant active ingredients.At the same time,the antioxidant activity was evaluated by molecular docking and DPPH free radical scavenging rate in vitro.Results:The HPLC-DPPH online screening system and Prep-HPLC were used to establish a screening and enrichment method for antioxidant active ingredients,and HSCCC chromatography was used to further separate and purify 5 antioxidant active ingredients,namely catechin(1),chlorogenic acid(2),caffeic acid(3),kaempferol-3-O-β-D-glucoside(4)and quercetin(5).Molecular docking showed that the binding energy of the five components ranged from−6.4 kcal/mol to−4.8 kcal/mol,of which the binding energy of catechin and kaempferol-3-O-β-D-glucoside were highest.The maximum in vitro scavenging rate of DPPH free radicals showed that the scavenging rate of 5 components on DPPH free radicals was in the descending order of kaempferol-3-O-β-D-glucoside,catechin,quercetin,caffeic acid and chlorogenic acid.Conclusion:The application of HPLC-DPPH online screening system and Prep-HPLC can effectively screen and enrich the antioxidant active ingredients in natural products,while the application of HSCCC can achieve further purification of the active ingredients.Both catechin and kaempferol-3-O-β-D-glucoside in Eucommia ulmoides leaves show strong antioxidant activity.
作者
贾羲
朱春胜
JIA Xi;ZHU Chunsheng(The First Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,China)
出处
《中医药信息》
2022年第5期39-44,共6页
Information on Traditional Chinese Medicine
基金
河南省医学科技攻关计划联合共建项目(LHGJ20190273)。
