摘要
目的优化大孔吸附树脂分离纯化血人参总黄酮的工艺条件,研究血人参总黄酮对酪氨酸酶的抑制作用。方法以吸附率、解吸率等为考察指标,采用静态吸附-解吸试验对6种不同型号的大孔吸附树脂进行筛选,优选最佳的树脂型号。采用单因素试验考察上样液pH、上样液浓度、上样体积、上样体积流量、洗脱溶剂、洗脱液体积流量、洗脱剂体积对血人参总黄酮纯化的影响,确定最佳纯化工艺;以L-酪氨酸、L-多巴为底物,测定血人参总黄酮对酪氨酸单酚酶和二酚酶的抑制作用,并对其抑制机理和抑制类型进行分析。结果AB-8型大孔吸附树脂对血人参总黄酮的分离纯化效果最好。最佳纯化工艺参数为:上样液pH为4、上样体积为70 mL(4.7 BV)、上样体积流量为1 mL·min^(-1)、上样液浓度为5.38 mg·mL^(-1)、洗脱溶剂为70%乙醇、洗脱剂体积流量为3 mL·min^(-1)、洗脱剂体积为120 mL(8 BV),血人参总黄酮对酪氨酸单酚酶和二酚酶均有抑制作用,其最大半数抑制浓度分别为88.78 mg·L^(-1)和36.46 mg·L^(-1),对二酚酶的抑制类型为可逆非竞争性抑制。结论AB-8型大孔吸附树脂可用于纯化血人参总黄酮,纯化后血人参总黄酮的纯度为46.01%,建立的工艺条件稳定可靠。纯化后的血人参总黄酮对酪氨酸酶有抑制作用,抑制类型为可逆非竞争性抑制。
Objective To optimize the process conditions for the purification of total flavonoids from Indigofera stachyodes Lindl.and its inhibitory effect on tyrosinase.Methods Taking the adsorption and desorption rate as investigation indexes,six kinds of macroporous adsorption resins were screened by static adsorption-elution test and the best resin was selected.Single factor testes were used to investigate the sample solution pH,the concentration of sample,upper column volume,upper column flow rate,the concentration of eluent,elution flow rate and eluent volume on the purification of total flavonoids from Indigofera stachyodes Lindl.,and determine the optimal purification process parameters.Using L-tyrosine and L-levodopa as substrates,the inhibitory effect of total flavonoids from Indigofera stachyodes Lindl.on tyrosine monophenase and diphenolase were determined,and the inhibitory mechanism and types were analyzed.Results The AB-8 macroporous adsorption resin had the best effect on the purification of total flavonoids from Indigofera stachyodes Lindl.,and the optimal purification parameters were as follows:the sample solution pH was 4,upper column volume was70 mL(4.7 BV),upper column flow rate was 1 mL·min^(-1),the concentration of sample was 5.38 mg·mL^(-1),the concentration of eluent was 70%,elution flow rate was 3 mL·min^(-1)and eluent volume was 120 mL(8 BV).The total flavonoids from Indigofera stachyodes Lindl.could inhibit the both monophenase and diphenolase activities of tyrosinase,with IC50 value were 88.78 mg·L^(-1)and 36.46μg·mL^(-1)respectively,and the type of inhibition of diphenolase was reversible noncompetitive inhibition.Conclusion AB-8 macroporous adsorption resins can be used to purify the total flavonoids of Indigofera stachyodes Lindl.The purification rate of Indigofera stachyodes Lindl was 46.01%after purification,and the established process conditions were stable and reliable.The purified total flavonoids of Indigofera stachyodes Lindl had an inhibitory effect on tyrosinase and the type was reversible non-competitive inhibition.
作者
杨姗
吴静澜
傅建
徐剑
张永萍
娄华勇
王光慧
Yang Shan;Wu Jinglan;Fu Jian;Xu Jian;Zhan Yongping;Lou Huayong;Wang Guanghui(Guizhou University of Traditional Chinese Medicine,Guiyang 550025,China;Guizhou Province Rsesarch Center for Processing and Preparation Engineering Technology of Traditional Chinese Medicine and Ethnic Medicine,Guiyang 550025,China;National Research Center of Miao Medicine Engineering Technology,Guiyang 550025,China;The key laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences,Guiyang 550014,China)
出处
《世界科学技术-中医药现代化》
CSCD
北大核心
2022年第9期3384-3393,共10页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
基金
国家自然科学基金委员会贵州喀斯特中心项目(U1812403):喀斯特地区特色民族医药若干基础问题研究,负责人:郝小江
国家自然科学基金委员会青年项目(81903517):基于结核杆菌细胞壁合成途径关键酶-UDP-半乳糖变异酶抑制剂的设计、合成及抗结核活性研究,负责人:傅建
贵州省科学技术厅一般项目(黔科合支撑[2021]一般411):差示扫描荧光法快速筛选贵州特色民族药DprE1抑制剂及抗结核活性研究与示范,负责人:傅建
关键词
血人参
总黄酮
大孔吸附树脂
酪氨酸酶
非竞争性抑制
Indigofera stachyodes Lindl.
Total flavonoids
Macroporous absorption resins
Tyrosinase
Non-competitive inhibition
作者简介
通讯作者:吴静澜,硕士,教授,主要研究方向:中药及民族药物新制剂与新剂型研究;通讯作者:傅建,博士,副教授,主要研究方向:中药及民族药物新制剂与新剂型研究。
