星点设计效应面法优化赶山鞭总黄酮提取工艺及提取物指纹图谱研究  

Optimization of extraction process of total flavonoids from Hypericum attenuatum by central composite design response surface method and fingerprint research

作　　者：井中旭 刘雨薇 姜海波 刘焱南[2] 秦国昭 冯宇飞[2] JING Zhong-xu;LIU Yu-wei;JIANG Hai-bo;LIU Yan-nan;QIN Guo-zhao;FENG Yu-fei(Heilongjiang Provincial Administration of Traditional Chinese Medicine,Harbin 150040,China;School of Pharmacy,Heilongjiang University of Traditional Chinese Medicine,Harbin 150040,China)

机构地区：[1]黑龙江省中医药管理局,黑龙江哈尔滨150040 [2]黑龙江中医药大学药学院,黑龙江哈尔滨150040

出　　处：《中草药》2022年第16期5010-5019,共10页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金面上项目(82174232);黑龙江省自然科学基金优秀青年项目(YQ2019H031);黑龙江中医药大学“优秀创新人才支持计划”项目(2018年);黑龙江省博士后研究人员落户黑龙江科研启动资助项目(2020年)。

摘　　要：目的优化赶山鞭总黄酮的提取工艺,建立赶山鞭总黄酮提取物的指纹图谱。方法通过单因素实验研究提取时间、提取次数、乙醇体积分数、料液比对赶山鞭中总黄酮含量及金丝桃苷的转移率的影响;以此为基础,采用Central-Composite试验设计原理进行3因素5水平试验设计和响应面分析法优化提取工艺。采用比色法,于500 nm处测定药材中以芦丁计总黄酮的含量,采用HPLC法测定总黄酮中金丝桃苷的含量,计算金丝桃苷的转移率。采用HPLC法建立赶山鞭总黄酮提取物的指纹图谱,采用《中药色谱指纹图谱相似度评价软件(2012年版)》进行相似度评价,确定共有峰,并采用SPSS 22.0软件进行聚类分析和主成分分析。结果赶山鞭总黄酮的最佳提取条件为以65%乙醇,料液比1∶10,提取120 min,提取2次。建立的赶山鞭药材总黄酮提取物的HPLC指纹图谱有15个共有指纹峰,指纹图谱的相似度分析结果显示相似度均大于0.61,表明10批样品的化学成分一致性较好,但各成分含量存在差异,对部分色谱峰进行了归属指认分别是金丝桃苷、槲皮素、芦丁、山柰酚。聚类分析可知欧氏距离为25时,10批样品可聚为2类,S1与S7为一类,其余聚为一类;欧氏距离为15时,可聚为5类,即S3、S5、S6、S8~S10聚为一类,S1、S2、S4、S7各为一类。经主成分分析,4个主成分因子的累积方差贡献率为88.659%,以S5样品的主成分因子综合得分最高。结论响应面法优化的赶山鞭总黄酮提取纯化条件简单可靠,建立的HPLC指纹图谱能够简便、高效地反映赶山鞭总黄酮提取物中大部分化学成分信息,为赶山鞭药材的质量评价奠定基础。Objective To optimize the extraction process of the total flavonoids of Ganshanbian(Hypericum attenuatum)and to establish the fingerprint profile of the total flavonoids of H.attenuatum.Methods Single-factor experiments were conducted to study the effects of solvent extraction time,extraction times,extraction concentration,and material-to-liquid ratio on the extraction rate and transfer rate of hyperoside of total flavonoids in H.attenuatum,on this basis,the central-composite design experimental design principle is used to carry out a three-factor five-level experimental design and response surface analysis to optimize the extraction process;on the basis of obtaining the best extraction method,on the basis of the best extraction method,the purification method of the total flavonoids extract of H.attenuatum was optimized by using the type of resin,the loading concentration of the extract,the loading volume of the extract,the elution concentration and the elution volume as the investigating indexes.After that,the fingerprints of the total flavonoids extract of H.attenuatum were established by HPLC,and SPSS 22.0 software was used for cluster analysis and principal component analysis.Results The best extraction conditions were 80%ethanol,1:10 ratio,2 h per extraction,extract 2 times.The HPLC fingerprints of the total flavonoids of H.attenuatum were determined to have 15 peaks common to all chromatograms,and the similarity analysis of the fingerprints showed that the similarity was greater than 0.61,the peaks were identified as auriculoside,quercetin,rutin and sannaphene,indicating that the chemical composition of the 10 batches of samples was consistent,but there were differences in the content of each component.The clustering analysis showed that when the Euclidean distance was 25,the 10 batches of samples could be clustered into 2 classes,S1 and S7 into one class,and the rest into one class;when the Euclidean distance was 5,they could be clustered into 5 classes,namely S3,S5,S6,S8—S10 into one class,and S1,S2,S4,

关 键 词：赶山鞭 总黄酮 提取 工艺优化 星点设计效应面法 指纹图谱 金丝桃苷 槲皮素 芦丁 山柰酚 

分 类 号：R283.6[医药卫生—中药学]