柳叶腊梅叶乙醇提取物成分分析及其体外抗氧化研究

A Study on Constituents and Antioxidant Activity of the Alcohol Extract of Chimonanthus salicifolius S.Y.Hu.leaves

【目的】柳叶腊梅是我国特有品种,在民间常被畲族人民用于治疗感冒及肠胃不适,曾被誉为江西四大著名药材之一。柳叶腊梅叶中富含多种生物活性成分,其中,黄酮类化合物是柳叶腊梅叶中含量较高且具有生物活性的成分之一。通过鉴定柳叶腊梅叶乙醇提取物中的黄酮类化合物的成分组成,测定其体外抗氧化活性,确定其中主要抗氧化活性物质等,为柳叶腊梅叶的功能作用提供依据。【方法】柳叶腊梅叶经超声提取、聚酰胺树脂纯化得到柳叶腊梅叶乙醇提取物。利用LC-MS对柳叶腊梅叶乙醇提取物的成分进行分析,推测出可能的成分后,采用HPLC比对标准品,并通过加标实验确定其具体成分。体外抗氧化实验测定柳叶腊梅叶乙醇提取物清除自由基能力和还原力。利用HPLC测定柳叶腊梅叶乙醇提取物清除ABTS和DPPH自由基的主要活性物质。【结果】利用LC-MS和HPLC共定性及定量测出9种黄酮类物质,包括芦丁(2.612±0.275)μg/mg、金丝桃苷(5.308±0.093)μg/mg、异槲皮苷(13.301±0.072)μg/mg、山奈酚-3-O-芸香糖苷(1.321±0.097)μg/mg、木犀草素-5-O-葡萄糖苷(13.479±0.041)μg/mg、紫云英苷(13.959±0.025)μg/mg、阿福豆苷(9.781±0.033)μg/mg、槲皮素(45.489±1.206)μg/mg、山奈酚(51.293±0.715)μg/mg等9种黄酮单体物质。其中,山奈酚-3-O-芸香糖苷和阿福豆苷在柳叶腊梅叶总黄酮中首次发现。柳叶腊梅叶乙醇提取物具有良好的抗氧化活性,其体外清除ABTS自由基和DPPH自由基的IC50值分别为(54.588±4.117)μg/mL和(28.053±1.778)μg/mL。柳叶腊梅叶总黄酮在20μg/mL至120μg/mL的选定范围内,铁还原能力呈剂量依赖性。利用HPLC测定出柳叶腊梅叶乙醇提取物清除ABTS和DPPH自由基的主要活性物质为槲皮素和山奈酚。另外,金丝桃苷、异槲皮苷和紫云英苷3种黄酮单体物质,也具有清除ABTS自由基和DPPH自由基的作用,但活性作用明显弱于槲皮素和山奈酚。【结论】利用LC-MS和HPLC鉴定出柳叶腊梅叶乙醇提取物9种成分。柳叶腊梅叶乙醇提取物具有良好的体外抗氧化活性,并且主要活性物质为槲皮素和山奈酚。

[Objective]Chimonanthus salicifolius S.Y.Hu.is an unique variety in China.In folk,Chimonanthus salicifolius S.Y.Hu.is often used by people of She ethnic minority group to treat colds and gastrointestinal discomfort,and it was once known as one of the four famous medicinal materials in Jiangxi Province.The leaves of Chimonanthus salicifolius S.Y.Hu.are rich in a variety of bioactive ingredients and flavonoids are one of the bioactive components with a high content in the leaves of Chimonanthus salicifolius S.Y.Hu.By identifying the components of flavonoids in the ethanol extract of Chimonanthus salicifolius S.Y.Hu.leaves and determining its antioxidant activity in vitro,the main substances with antioxidant activity were identified to provide a basis for the function of Chimonanthus salicifolius S.Y.Hu.leaves.[Method]The leaves of Chimonanthus salicifolius S.Y.Hu.were extracted by ultrasonic extraction and purified by polyamide resin to obtain ethanol extract from Chimonanthus salicifolius S.Y.Hu.leaves.The components of the ethanol extract from Chimonanthus salicifolius S.Y.Hu.leaves were analyzed by high performance liquid chromatography-mass spectrometry(LC MS)to infer the possible components.The total flavonoids were compared with standards to determine the specific components through adding standards experiments by high performance liquid chromatography(HPLC).The activity of scavenging free radicals and reducing power of the total flavonoids of Chimonanthus salicifolius S.Y.Hu.leaves were evaluated in vitro and the major components of them with the activity of scavenging free radicals were determined by HPLC.[Result]Nine flavonoid monomers including rutin(2.612±0.275)μg/mg,hyperin(5.308±0.093)μg/mg,isoquercitrin(13.301±0.072)μg/mg,kaempferol-3-O-rutinoside(1.321±0.097)μg/mg,luteolin 5-O-glucoside(13.479±0.041)μg/mg,astragalin(13.959±0.025)μg/mg,afzelin(9.781±0.033)μg/mg,quercetin(45.489±1.206)μg/mg,kaempferol(51.293±0.715)μg/mg were identified and quantified by means of LC-MS and HPLC experiments.Kaempferol-3-O-rutinoside and afzelin were found for the first time in the total flavonoids of Chimonanthus salicifolius S.Y.Hu.leaves.The ethanol extract of Chimonanthus salicifolius S.Y.Hu.leaves had prominent antioxidant activity,whose IC50 values of scavenging ABTS and DPPH free radicals in vitro were(54.588±4.117)μg/mL and(28.053±1.778)μg/mL.The iron reducing capacity of the ethanol extract from Chimonanthus salicifolius S.Y.Hu.leaves in the selected range of 20μg/mg to 120μg/mg were dose-dependent.The major components of the total flavonoids of Chimonanthus salicifolius S.Y.Hu.leaves with the activity of scavenging free radicals were quercetin and kaempferol.In addition,three flavonoid monomers,such as hyperin,isoquercitrin and astragalin,also had scavenging effects on ABTS free radical and DPPH free radical,but the activity was obviously weaker than that of quercetin and kaempferol.[Conclusion]Nine components of the total flavonoids of Chimonanthus salicifolius S.Y.Hu.leaves were identified and quantified by LC MS and HPLC.The ethanol extract of Chimonanthus salicifolius S.Y.Hu.leaves had prominent antioxidant activity and the major components of the total flavonoids of Chimonanthus salicifolius S.Y.Hu.leaves with the activity of scavenging free radicals were quercetin and kaempferol.