Research On The Quality Evaluation Of Panax Notoginseng Based On The Effect Of Promoting Blood Circulation Spectrum

ObjectiveThe traditional Chinese medicine Notoginseng Radix et Rhizoma is made of dried roots and rhizomes of Panax notoginseng(Burk.)F.H.Chen.It can remove blood stasis,stop bleeding,reduce swelling and relieve pain.It is more and more widely used in medical care owing to its bidirectional effect of promoting blood circulation and stopping bleeding.The method of quality evaluation for traditional Chinese medicine quality evaluation is an effective way to control the quality and guarantee the curative effect of medicines.With the development of science and technology,the quality evaluation of Notoginseng Radix et Rhizoma is more and more deeply,which solves most of the problems in Notoginseng Radix et Rhizoma quality control,some difficult problems are waited to be solved.For example,the active ingredient groups of Notoginseng Radix et Rhizoma has not been completely clear,the currently established quality evaluation method based on the content of chemical ingredients cannot fully reflect the medical efficacy of Notoginseng Radix et Rhizoma.The commodity specifications classification method based on traditional characters of Notoginseng Radix et Rhizoma lacks the support of systematic modern chemistry and pharmacology.Through previous research and literature review,we learn that Panax Notoginseng Saponins(PNS)act as the active ingredients of Notoginseng Radix et Rhizoma when promoting blood circulation.However,the specific composition of total saponins of Notoginseng Radix et Rhizoma has not been fully explained,and the relationship between the content of PNS and the characteristics of Notoginseng Radix et Rhizoma has not been uniformly concluded.Therefore,there are some deficiencies in the quality control method based on the monomer content of Notoginseng Radix et Rhizoma.Dividing the commodity specification grade of Notoginseng Radix et Rhizoma according to its characteristic is lack of scientific basis.Based on the background above,this study intends to collect 50 batches of Notoginseng Radix et Rhizoma with different producing areas and different commercial specifications.Taking the blood circulation effect as an entry point,use the spectrum-effect correlation method to deeply study the composition of the active component group of Notoginseng Radix et Rhizoma when playing an important role in promoting blood circulation.Based on the content of the active component group of Notoginseng Radix et Rhizoma,combined with the spectrum-effect relationship of Notoginseng Radix et Rhizoma,using the grey correlation analysis method to established a quality evaluation method which can reflect the blood circulation efficacy of Notoginseng Radix et Rhizoma,we established a new method to classify the commodity specifications of Notoginseng Radix et Rhizoma based on the efficacy of promoting blood circulation.Methods1 Resource survey and sample preparation of Notoginseng Radix et RhizomaDetermine the scope of Notoginseng Radix et Rhizoma resource survey and experimental sample collection through literature search.Adopt the methods consist of literature survey,field survey and visit survey to understand the distribution of Notoginseng Radix et Rhizoma planting resources and market acquisition.Collect Notoginseng Radix et Rhizoma with clear producing area information.The collected samples of Notoginseng Radix et Rhizoma were extracted by 70%ethanol as solvent and by the means of heating to reflux to be ready for the follow-up research.2 Study on the drug spectrum of Notoginseng Radix et Rhizoma14 representative batches of Notoginseng Radix et Rhizoma were selected to prepare 14 batches of Notoginseng Radix et Rhizoma samples with obvious spectrum differences.Base on the previous research,the HPLC fingerprint of Notoginseng Radix et Rhizoma was established by optimizing the chromatographic conditions,improving the ability of instrumental analysis as well as detection of chemical components.And the common peaks were quantitatively or semi-quantitatively analyzed to obtain the drug spectrum of Notoginseng Radix et Rhizoma.3 Study on activating blood circulation pharmacodynamic spectrum of Notoginseng Radix et RhizomaAn in vitro capillary coagulation experiment was used to observe the effect of 14 representative batches of Notoginseng Radix et Rhizoma on capillary coagulation time in rats to preliminary evaluate the effect from different batches on promoting blood circulation.The rat model of middle cerebral artery occlusion(MCAO)ischemia-reperfusion injury was established by thread occlusion.The effects of 14 representative batches of Notoginseng Radix et Rhizoma on the percentage of cerebral infarction area,rate of brain water loss and index of brain divided by body in model rats were observed to further evaluate the effect of Notoginseng Radix et Rhizoma on promoting blood circulation in vivo.Finally,the pharmacodynamic spectrum of the time of anti-capillary coagulation and of the effect of anti-cerebral ischemia-reperfusion of Notoginseng Radix et Rhizoma were obtained.4 Screen of effective constituent groups of Notoginseng Radix et Rhizoma for promoting blood circulationThe entropy methods were used to calculate the comprehensive efficacy index of MCAO ischemia-reperfusion experiment injury in rats and the comprehensive efficacy index of Notoginseng Radix et Rhizoma activat blood circulation.By Partial Least Squares Regression analysis method,the drug spectrum were correlated with single efficacy index and comprehensive efficacy index respectively,thus the effective component groups of anti-capillary coagulation,anti-MCAO cerebral ischemia-reperfusion injury and promoting blood circulation of Notoginseng Radix et Rhizoma were screened.5 Establishment of the quality evaluation method of Notoginseng Radix et Rhizoma based on promoting blood circulation efficacyActive ingredient groups of 50 batches Notoginseng Radix et Rhizoma with different producing area information and different product specifications were detected by HPLC,and the Notoginseng Radix et Rhizoma quality evaluation model was constructed through grey correlation method combined with the pharmacodynamic coefficients of each index from blood circulation active components to comprehensively evaluate Notoginseng Radix et Rhizoma quality.According to the relative weighted degree of correlation,the product specification level threshold was set,the Notoginseng Radix et Rhizoma product specification level divided,Notoginseng Radix et Rhizoma quality evaluation and product specification level division method was established.Results1 Resource survey and sample preparation of Notoginseng Radix et RhizomaThe planting areas of Notoginseng Radix et Rhizoma are mainly distributed in Wenshan Prefecture,Honghe Prefecture and Yuxi District in Yunnan Province.Only a small number of plants are planted in Debao and Pohong in Guangxi Province.Commercially available Notoginseng Radix et Rhizoma is mainly produced in Yunnan Province then distributed in Wenshan Prefecture,Yunnan Province.A total of 50 batches of Notoginseng Radix et Rhizoma samples with different producing area information and different product specifications were collected in the experiment.The collected samples were extracted by 70%ethanol,and it was found that the collected samples has a paste extraction rate range from 24.41%to 50.31%,indicating that there is a certain difference in the content or type of the chemical composition of Notoginseng Radix et Rhizoma in each batch of the medicinal materials,which can be used as materials in subsequent experiments.2 Study on the drug spectrum of Notoginseng Radix et RhizomaThrough HPLC analysis of 14 batches of Notoginseng Radix et Rhizoma,HPLC fingerprints of Notoginseng Radix et Rhizoma were established,23 common components be seen,among which 12 common components identified.All identified components are saponins,they are N-R1,G-Rg1,G-Re,(R)N-R2,G-Rg2,G-Rh1,G-Rb1,G-Rc,G-Rb2,G-Rd,N-Ft1,20(R)G-Rh2.Quantitative and semi-quantitative analysis were performed on the designated 12 common components and 11 unspecified components,respectively,and the drugs spectrum of Notoginseng Radix et Rhizoma was obtained.The contents of 12 common saponins in 14 batches Notoginseng Radix et Rhizoma were 0.61%～1.29%,3.90%～5.90%,0.42%～1.23%,0.06%～0.11%,0.21%～0.61%,0.14%～0.27%,1.82%～3.70%,0.03%～0.07%,0.08%～0.21%,0.75%～1.30%,0.03%～0.15%,0.04%～0.15%.The results above indicate that 14 batches of Notoginseng Radix et Rhizoma have large differences in chemical composition content,and the subsequent experimental research can be continued.3 Study on activating blood circulation pharmacodynamic spectrum of Notoginseng Radix et RhizomaThe results show that there is a significant difference among the effect of promoting blood circulation in 14 batches of Notoginseng Radix et Rhizoma.In vitro capillary coagulation test show that the range of clotting time of 14 batches of Notoginseng Radix et Rhizoma was 59.17～97.08 s,which means it could significantly prolong the capillary coagulation time of rats compared with the blank group experimented rats.The results of cerebral ischemia-reperfusion experiment in rats showed that the percentage of cerebral ischemic area,the rate of brain water loss and the index of brain divided by body of 14 batches of Notoginseng Radix et Rhizoma were 5.87%～32.08%、79.76%～81.45%and 0.59%～0.66%respectively,the differences statistically significant compared with the model group.The results above provide rich pharmacodynamic information for the effective component group of Notoginseng Radix et Rhizoma in spectrum-effiect correlation studies.4 Screen of effective constituent groups of Notoginseng Radix et Rhizoma for promoting blood circulationUsing the entropy method,the comprehensive pharmacodynamic spectrums of anti-MCAO cerebral ischemia-reperfusion injury and blood circulation effect of Notoginseng Radix et Rhizoma were established respectively.The drug spectrum-anticapillary coagulation efficacy index correlation results show that out of 23 compounds in common,20 compounds play a positive effect on prolonging capillary coagulation time.Among the identified compounds,G-Rb1,G-Rb2 and G-Re have the highest contribution to prolonging capillary coagulation time,while(R)N-R2 and G-Rh1 play a negative role.The correlation between drug spectrum and effect indexes of anti-MCAO cerebral ischemia-reperfusion injury shows that out of 23 common compounds,17 components play a positive role in reducing the cerebral ischemic area of rats;1 5 components play a positive effect on reducing the rate of brain water loss in rats;9 components play a positive effect on reducing the index of brain divided by body in rats.Correlation between pharmacospectrum and anti-cerebral ischemia-reperfusion injury comprehensive drug efficacy spectrum shows that 18 out of 23 common ingredients in Notoginseng Radix et Rhizoma play a positive role in reducing cerebral ischemia-reperfusion injury.Among the identified compounds,G-Rb2,G-Rc,and G-Rd contribute the most to the resistance to cerebral ischemia-reperfusion injury,while(R)N-R2,G-Rh1,and N-Ft1 play a negative effect on cerebral ischemia-reperfusion injury effect.The correlation results of the drug spectrum and the comprehensive pharmacodynamic index of blood circulation show that 17 compounds out of 23 common components play a positive effect on the blood circulation comprehensive drug efficacy index.Among the identified compounds,G-Rb2,G-Rc,G-Rd,N-R1 and G-Rb1 contribute to the effect on the blood circulation of Notoginseng Radix et Rhizoma greatly,while G-Rh1,(R)N-R2 and N-Ft1 have a significant negative effect on the blood circulation.Regardless of in vitro or in vivo experiments,G-Rh1 and(R)N-R2 show a significant negative effect on the blood circulation index all along,and N-Ft1 also play a negative significant effect on cerebral ischemia-reperfusion injury and comprehensive drug efficacy impact.It indicates that G-Rh1,(R)N-R2 and N-Ft1 may have potential effect of coagulation promoting.5 Establishment of the quality evaluation method of Notoginseng Radix et Rhizoma based on promoting blood circulation efficacyQuantitative and semi-quantitative analysis of the active ingredients of blood circulation efficacy in 50 batches Notoginseng Radix et Rhizoma were carried out by HPLC and the gray correlation model of quality assessment was successfully established,thus establishing a new method for the classification of Notoginseng Radix et Rhizoma product specifications.The relative weighted relation grade of Notoginseng Radix et Rhizoma in 50 batches samples range from 0.4170 to 0.5210.A greater relative weighted relation grade means a better quality for Notoginseng Radix et Rhizoma test.Artificially setting the threshold value of Notoginseng Radix et Rhizoma commodity grades,and divide 50 batches of Notoginseng Radix et Rhizoma into 4 commodity grades(Grade Ⅰ:Relative weighted relation grade≥0.5000;Grade Ⅱ:0.5000>Relative weighted relation grade≥0.4600;Grade Ⅲ:0.4600>Relative weighted relation grade≥0.4200;Grade Ⅳ:0.4200>Relative weighted relation grade).Among the 50 batches of tested samples,there were 8 batches belonging to Grade Ⅰ,14 batches Grade Ⅱ,26 batches Grade Ⅲ,and 2 batch Grade Ⅳ.Conclusions1.This study established a rapid screening method based on the spectrum-effect correlation of active ingredient group of Notoginseng Radix et Rhizoma for blood circulation efficacy.It laid foundation for the full disclosure of the effective component group of Notoginseng Radix et Rhizoma,and also provided a reference for the identification of the effective component groups of other Chinese medicinal materials.2.For the first time,the entropy method was used to synthesize the in vitro and in vivo blood circulation efficacy indexes,and the spectrum-effect correlation method was used to clarify the composition of the active ingredient group of Notoginseng Radix et Rhizoma for blood circulation.It provides a reference for Notoginseng Radix et Rhizoma quality evaluation research,rational clinical use,better resource development and utilization3.Established a new method of quality evaluation and commodity specification grade classification of Notoginseng Radix et Rhizoma by taking the pharmacodynamic coefficients of each index from blood circulation active components as weight and the active component group content as a basis,as well as used the gray-correlation analysis method.It provides a reference for the research on the quality evaluation method of multi-index component Chinese medicinal materials,Notoginseng Radix et Rhizoma included.