Screening Of Antioxidant Activity Equivalent Components Of Houttuynia Cordata Thunb.

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.