【作者】 胡娟；

【导师】 杨占南；

【作者基本信息】 贵州师范大学 ， 分析化学， 2018， 硕士

【摘要】 中药发挥功效的有效物质辨识一直是中药现代化研究中的重点和难点,科学家们对此开展了大量的研究工作,提出了许多科学假说,并进行了实践验证。这些研究工作为传统中药发挥功效的现代科学内涵阐释、药材质量控制、新药创制等产生了显著成效。但是,随着社会经济不断发展,现有中药药效物质辨识研究现状已经远远不能满足社会发展需求。无论是药效物质辨识的理论研究,还是具体某味药材发挥功效的物质基础辨识,均需要引入新理念、新技术与新方法,开展深入研究,才有可能满足整个中药产业发展需求。因此,本论文以鱼腥草(Houttuynia cordata Thunb.)为研究对象,以“等效组分群”理论为导向,以抗氧化活性药效物质辨识为着力点,通过DPPH捕集-HPLC示踪策略,阐明鱼腥草抗氧化活性药效物质辨识基础,明确其相互作用关系,探索质量辨识模式,为其深度开发利用和质量控制提供科学依据和理论基础。主要结果如下:1.筛选鱼腥草抗氧化活性候选等效组分群。通过鱼腥草与DPPH反应后,利用HPLC示踪,分析其色谱峰的减弱或消失,辨识鱼腥草中起抗氧化活性作用的物质,结果显示绿原酸、芦丁、阿福豆苷、异槲皮苷、槲皮苷和槲皮素为鱼腥草抗氧化活性候选等效组分群。2.分析鱼腥草抗氧化活性候选等效组分群的DPPH清除能力。结果显示绿原酸、芦丁、阿福豆苷、异槲皮苷、槲皮苷和槲皮素清除DPPH的能力存在差异,DPPH先与槲皮素进行反应,且反应彻底,其次是阿福豆苷和槲皮苷,绿原酸与DPPH反应较慢,芦丁与异槲皮苷介于槲皮苷和绿原酸。当DPPH浓度较低时,各样品中DPPH能被完全消耗,说明具有相同的抗氧化效果;不同组成、含量及比例关系的候选等效组分群清除相同物质的量DPPH所消耗候选等效组分群的总消耗量存在差异,说明不同组成、含量及比例关系的候选等效组分群之间存在着某种相互作用关系。3.验证鱼腥草抗氧化活性等效组分群。通过绿原酸、芦丁、阿福豆苷、异槲皮苷、槲皮苷、槲皮素模拟鱼腥草样品,结果显示除阿福豆苷之外,绿原酸、芦丁、异槲皮苷、槲皮苷、槲皮素均与DPPH反应,且随着DPPH的浓度升高,消耗量增大。不同的模拟样品,在DPPH加入量相同的情况下,尽管各候选组分的消耗量差异显著,但各候选等效物质的总消耗量基本一致,说明它们的抗氧化效果具有等效性;通过模拟样品与实际样品的对比分析显示,在实际样品与模拟样品中槲皮素的消耗量基本一致,说明槲皮素在反应体系中,DPPH先与槲皮素反应,再与其他物质反应。绿原酸、芦丁、异槲皮苷、槲皮苷在模拟样品消耗量大于实际样品的消耗量,说明了在实际样品中还有其他组分与DPPH发生了反应。在DPPH消耗线性浓度下,每个模拟样品的总消耗量都大于实际样品,这说明实际样品中还存在其他物质具有抗氧化活性作用,且与DPPH反应的速度快。通过不同批次实际样品与模拟样品的总消耗量的比较分析显示,在每消耗50μM DPPH时,实际样品总消耗量是模拟样品总消耗量42-65%之间,说明在鱼腥草中还存在其它抗氧化组分。4.探讨基于“组分敲出”的鱼腥草抗氧化活性等效组分群中各组员的相互作用关系。结果表明在DPPH浓度为0-50μM时,绿原酸、芦丁、阿福豆苷、异槲皮苷、槲皮苷、槲皮素的消耗量与DPPH浓度成线性关系,且在DPPH加入量为50μM时,DPPH可以被消耗完全;基于每消耗50μM DPPH,所消耗抗氧化物质的量来评价抗氧化能力,如果消耗量越大说明其抗氧化活性能力越弱,反之,越强。鱼腥草的抗氧化活性等效组分的抗氧化作用强弱为:异槲皮苷>芦丁>绿原酸>槲皮苷>槲皮素>阿福豆苷;组分敲出实验显示消耗50μM DPPH时,敲出绿原酸后的剩余组分的消耗量大于未敲出组分的总消耗量,说明抗氧化能力减弱,说明绿原酸对鱼腥草抗氧化活性等效组分起到协同促进作用,反之,阿福豆苷,异槲皮苷,槲皮苷,槲皮素对鱼腥草抗氧化活性等效组分起到拮抗作用的,且拮抗作用的强弱为:阿福豆苷>槲皮素>槲皮苷>异槲皮苷>芦丁;绿原酸-阿福豆苷、芦丁-槲皮苷和异槲皮苷-槲皮苷后的剩余组分消耗量大于未敲出组分的总消耗量,说明抗氧化能力减弱,说明绿原酸-阿福豆苷、芦丁-槲皮苷和异槲皮苷-槲皮苷对鱼腥草抗氧化活性等效组分体系起到协同作用,且协同作用强弱为:芦丁-槲皮苷>绿原酸-阿福豆苷>异槲皮苷-槲皮苷,敲出其它两两组合则对体系起到拮抗作用,且拮抗作用强弱为:绿原酸-槲皮素>绿原酸-异槲皮苷>芦丁-异槲皮苷>槲皮苷-槲皮素>异槲皮苷-槲皮素>绿原酸-槲皮苷>阿福豆苷-槲皮素>阿福豆苷-异槲皮苷>绿原酸-芦丁>芦丁-槲皮素>阿福豆苷-槲皮苷>芦丁-阿福豆苷。在敲出三组分和四组分的体系中,剩余组分总消耗量都小于未敲出组分总消耗量,说明被敲出的组分都起到拮抗作用。更多还原

【Abstract】 Effective substance identification of traditional Chinese medicines has always been the focus and difficulty in modernize investigation of traditional Chinese medicine.To this end,scientists have carried out a lot of research work and put forward many scientific hypotheses and conducted practical verification,which have produced remarkable results for the modern scientific connotation and explanation of the effectiveness of traditional Chinese medicines,quality control of medicinal materials,and creation of new drugs.However,with the continuous development of the social economy,the current status of research on the identification of medicinal substances in traditional Chinese medicine is far from meeting the needs of social development.Whether it is the theoretical study of the identification of pharmacodynamic substances or the identification of the physical basis of a specific medicinal substance,new concepts,technologies and methods need to be introduced to conduct in-depth research,so that it is possible to meet the needs of the development of the entire Chinese medicine industry.Therefore,in this dissertation,H.cordata was used as the research object,with the theory of “equivalent component group” as the guide,and the identification of antioxidant active drug substance as the focal point,through the DPPH captureHPLC tracer strategy to elucidate the basis for the identification of the anti-oxidant active substances of H.cordata,to clarify its interaction relationship,to explore the mode of quality identification,and provide scientific and theoretical basis for its in-depth development and quality control.The main results are as follows:1.Screening of antioxidant activity candidate equivalent components of H.cordata.After the reaction between H.cordata and DPPH,HPLC tracer was used to analyze the attenuation or disappearance of the chromatographic peak to identify the antioxidant active substances in H.cordata.The results showed that chlorogenic acid,rutin,kaempferol-3-o-glucorhamnoside,isoquercitrin,quercitrin and quercetin were candidate antioxidant equivalent components of H.cordata.2.Analyze the DPPH scavenging capacity of the candidate equivalence components of H.cordata.The results showed differences in the scavenging DPPH capacities of chlorogenic acid,rutin,isoquercitrin,kaempferol-3-o-glucorhamnoside,quercitrin,and quercetin.DPPH reacts first with quercetin completely,followed by kaempferol-3-o-glucorhamnoside and quercitrin.The reaction of chlorogenic acid with DPPH is slow,and the reaction of rutin and isoquercitrin with DPPH are intermediate between quercitrin and chlorogenic acid.With the low concentration,DPPH was completely consumed which indicating the same antioxidant effect in each sample.The total amount of DPPH consumed by the candidate equivalent group with different compositions,contents,and ratios was different,indicating that there was some interaction between candidate equivalent groups.3.Verification of antioxidant activity candidate equivalent components of H.cordata.By simulating the H.cordata samples with the reference chlorogenic acid,rutin,kaempferol-3-o-glucorhamnoside,isoquercitrin,quercitrin,and quercetin,the results showed that chlorogenic acid,rutin,and isoquercitrin,quercetin,and quercetin reacted with DPPH in addition to kaempferol-3-o-glucorhamnoside,and the consumption increased with increasing DPPH concentration.Despite the significant difference in the consumption of the candidate components,different simulated samples,with the same amount of DPPH added,the total consumption of equivalent candidate substances are basically the same,indicating that their antioxidant effects are equivalence.The comparison between the simulated sample and the actual sample shows that the consumption of quercetin in the actual sample and the simulated sample is consistent,indicating that in the reaction system,DPPH first reacts with quercetin,and followed by other substances.The consumption of chlorogenic acid,rutin,isoquercitrin and quercitrin in simulated samples were greater than actual samples,indicating that there are other components react with DPPH in the actual sample.Under the linear concentration of DPPH consumption,the consumption of each simulated sample is larger than that of the actual sample,which indicates that other substances still have antioxidant activity in actual samples and the reaction speed with DPPH is fast.Through the comparative analysis of the total consumption of actual and simulated samples in different batches shows that the actual total sample consumption is between 42% and 65% of the total simulated sample consumption when 50 μM DPPH was consumed in each sample,indicating that there are also other antioxidant components in the H.cordata.4.To explore the interactions among team members in the antioxidant equivalent component components of H.cordata based on “component knockouts”.The results showed that the consumption of chlorogenic acid,rutin,aflavin,isoquercitrin,quercitrin,and quercetin was linearly related to the concentration of DPPH when the concentration of DPPH was 0-50 μM,and the amount of DPPH is 50 μM,the DPPH can be completely consumed.Based on the consumption of 50 μM DPPH,the amount of antioxidants consumed was used to evaluate the antioxidant capacity.The greater the consumption,the weaker its antioxidant activity,and vice versa.The antioxidant activity of the antioxidant components of H.cordata is: isoquercitrin > rutin > chlorogenic acid > quercitrin > quercetin > kaempferol-3-oglucorhamnoside.Component knock-out experiments showed that when 50 μM DPPH was consumed,the consumption of the remaining components after knocking out chlorogenic acid was greater than the total consumption of components that were not knocked out,indicating that the antioxidant capacity was reduced,further illustrating that chlorogenic acid on antioxidant activity of H.cordata equivalent components play a synergistic role.Conversely,kaempferol-3-o-glucorhamnoside,isoquercitrin,quercitrin,and quercetin could antagonize the antioxidant activity of H.cordata,and the antagonistic effect was as follows: kaempferol-3-o-glucorhamnoside > quercetin > quercitrin > isoquercitrin > rutin.The consumption of the remaining components of chlorogenic acid-kaempferol-3-o-glucorhamnoside,rutinquercitrin and isoquercitrin-quercitrin was greater than the total consumption of non-knockout components,indicating that the antioxidant capacity was reduced,further illustrating that chlorogenic acid-kaempferol-3-o-glucorhamnoside,rutin-quercitrin and isoquercitrin-quercitrin have a synergistic effect on the antioxidant component of H.cordata and the synergistic effect is: rutin-quercitrin > Chlorogenic acidkaempferol-3-o-glucorhamnoside > isoquercitrin-quercitrin.In addition,the knock-out of the other two combinations will have an antagonistic effect on the system,and the strength of the antagonism is: chlorogenic acid-quercetin > chlorogenic acid-isoquercitrin > rutin-isoquercitrin > quercitrin-quercetin > isoquercitrin-quercetin > chlorogenic acid-quercitrin > kaempferol-3-o-glucorhamnoside-quercetin > kaempferol-3-o-glucorhamnoside-isoquercitrin > Chlorogenic acid-Rutin > rutin-quercetin > kaempferol-3-o-glucorhamnoside-quercitrin > rutin-kaempferol-3-o-glucorhamnoside.In knockout threecomponent and four-component systems,the total consumption of the remaining components was less than the total consumption of the unknocked components,indicating that the knockout components all played an antagonistic role.更多还原