HPLC-DPPH快速发现重楼果壳总黄酮中抗氧化成分及其活性初步验证

Rapid Determination and Activity Verification of Antioxidant Constituents in Total Flavonoids from Fruit Shell of Paris Polyphylla by HPLC-DPPH

目的:以1,1-二苯基-2-三硝基苯肼(DPPH)为底物,利用HPLC快速发现重楼果壳总黄酮中的抗氧化物成分,为重楼果壳资源的利用提供研究基础。方法:对重楼果壳用70%乙醇水提取进行总黄酮提取富集后,利用DPPH与重楼果壳总黄酮进行反应,采用HPLC分析反应前后色谱峰的变化,通过制备色谱、高分辨质谱、核磁共振等鉴定潜在的抗氧化成分,并进行其抗氧化活性验证。结果:与DPPH反应后,发现一个色谱峰消失,利用制备色谱对该消失的色谱峰进行制备纯化,利用质谱和核磁共振数据,鉴定为槲皮素-3-O-龙胆二糖苷,该化合物首次从重楼属植物中分离得到,在重楼果壳总黄酮中的含量为3.28%。对其抗氧化活性进行验证,发现其对DPPH自由基半数清除率(IC50)为0.0340 mg·ml^(-1),与维生素C(IC50=0.0240 mg·ml^(-1))的抗氧化活性相当。结论:该法快速从重楼果壳中发现抗氧化活性成分槲皮素-3-O-龙胆二糖苷,为重楼果壳总黄酮的进一步开发提供研究基础。

Objective:To determine the antioxidant constituents in total flavonoids from fruit shell of Paris polyphylla(TFSP)by HPLC-DPPH to promote the use exploitation of fruit shell of P.polyphylla.Methods:Total flavonoids extracted and enriched from fruit shell of P.polyphylla was reacted with DPPH.The changes of chromatographic peaks before and after the reaction were analyzed by HPLC.The potential antioxidant component was prepared and identified by preparative chromatography/HRMS/NMR,and the antioxidant activity of the potential antioxidant component was verified.Results:After reaction with DPPH,a chromatographic peak disappeared.The disappeared peak was purified by preparative chromatography and identified as quercetin-3-O-gentiobioside by HRMS and NMR.Quercetin-3-O-gentiobioside was isolated from the genus Paris for the first time,the content of quercetin-3-O-gentiobioside in TFSP was 3.28%.The IC50 value of DPPH radical scavenging rate of this compound was 0.0340 mg·ml^(-1),which was equivalent to that of vitamin C(IC50=0.0240 mg·ml^(-1)).Conclusion:Quercetin-3-O-gentiobioside can be quickly determined from TFSP by the method mentioned above,which provides basis for the further development of P.polyphylla.