Research On The Quality Evaluation Of Panax Notoginseng With Different Commercial Specifications Based On Hemostatic Efficacy

BackgroundThe traditional Chinese medicine Notoginseng Radix et Rhizoma is made of dried roots and rhizomes of Panax notoginseng(Burk.)F.H.Chen.According to the commercial specifications,Panax notoginseng is divided into 20,30,40,60,80,120,200 Tou,countless Tou,rhizome,rootlet,thick root,hair root.Ancient and modern documents record that hemostasis is one of the main effects of Panax notoginseng."New Compilation of Materia Medica"[1]records:"Panax notoginseng is a magical medicine for hemostasis."The 2020 edition of the Chinese Pharmacopoeia[2]records that "Panax notoginseng can dissipate blood stasis,stop bleeding,reduce swelling and relieve pain.Panax notoginseng can be used for hemoptysis,hematemesis,epistaxis,homatochezia,uterine bleeding,traumatic bleeding,etc."Modern clinical and pharmacological studies have also shown that Panax notoginseng can treat various bleeding diseases.Panax notoginseng powder[3,4]and dencichine[5]have good effect on gastric bleeding,and can significantly shorten the prothrombin time,thrombin time and clotting time.Modern chemical composition research shows that the hemostatic components of Panax notoginseng are dencichine,notoginsenoside Fti and total saponins.In addition,network pharmacology studies have shown that flavonoids also have hemostatic effect.So far,the hemostatic active ingredients group of Panax notoginseng is not clear,which brings difficulties to the quality evaluation of Panax notoginseng.Therefore,it is urgent to adopt a more scientific and reasonable method to screen the hemostasis effective ingredients group of Panax notoginseng,and then evaluate the quality of Panax notoginseng.At present,researches on the quality evaluation of Panax notoginseng mostly base on the chemical components or efficacy indicators.It is difficult to truly reflect the quality of Panax notoginseng.In the early study of the research group,the spectrum-effect correlation method was used to screen the active ingredients group of Panax notoginseng to promote blood circulation based on the effect of promoting blood circulation,which laid the methodological foundation for this research.The spectrum-effect correlation method is a method that combines chemical components and efficacy indicators to screen the effective ingredients of medicinal materials,which makes up for the one-sided problem of evaluating the quality of Panax notoginseng from the chemical ingredients or efficacy indicators alone.In vitro hemostasis models,plasma recalcification time is mostly used as the evaluation index.Because this model has the characteristics such as simple operation,high efficiency and economy.It is suitable for the preliminary evaluation of the hemostatic efficacy of Panax notoginseng.In vivo hemostasis model is based on the whole animal,which can reflect the medicinal effect the drug acts on the human body.Panax notoginseng enters the stomach meridian,so the rat gastric bleeding experiment was used to evaluate the hemostatic efficacy of Panax notoginseng with different commercial specifications.Combining the two sides can evaluate the hemostatic effect of Panax notoginseng more comprehensively.The biological effect evaluation method can integrate in vivo and in vitro efficacy indicators,and reflect the efficacy of medicinal materials truly and directly.TOPSIS can comprehensively evaluate the quality of medicinal materials based on the spectrum-effect correlation coefficient as the weight and the content of the effective ingredients group.ObjectiveThis study intends to use in vitro and in vivo pharmacodynamic experiments to evaluate the hemostatic biological effects of different commercial specifications of Panax notoginseng.HPLC,UPLC-Q-Exactive Orbitrap MS and Ultraviolet Spectrophotometry are used to determine the contents of saponins,dencichine and total flavonoids in different commercial specifications of Panax notoginseng.Using the spectrum-effect correlation method obtain the hemostatic effective ingredients group of Panax notoginseng.Using the TOPSIS sort the hemostatic effective ingredients group of different commercial specifications of Panax notoginseng,comprehensively evaluate the quality of different commercial specifications of Panax notoginseng,and provide a scientific basis for the formulation of the quality standard for Panax notoginseng.Methods1 Research on the hemostatic effect of different commercial specifications of Panax notoginseng.In vitro plasma calcium replication time assay was used to observe the effect of different commercial specifications of Panax notoginseng from Wenshan on plasma calcium replication time of rats,and to preliminarily evaluate the strength of hemostatic effects of different commercial specifications of Panax notoginseng from Wenshan.That rat gastric hemorrhage model was established,and the effects of different commercial specifications of Panax notoginseng on plasma prothrombin time,prothrombin time and activated partial thromboplastin time were observed to further evaluate the strength of hemostatic effect of different commercial specifications of Panax notoginseng form Wenshan.2 Research on the hemostatic bio-efficacy of Panax notoginseng with different commercial specifications.The plasma recalcification time index and the gastric bleeding index(thrombin time,prothrombin time,activated partial thrombin time)were weighted.Through the comprehensive coagulation value calculation method,the comprehensive coagulation value of Pan ax notoginseng with different commercial specifications of Wenshan were calculated.Then,the biological effect evaluation method was used to obtain the hemostatic biological valence of Panax notoginseng in different product specifications of Wenshan.3 Research on chemical ingredients of Panax notoginseng with different commercial specifications.On the basis of the previous study,the HPLC fingerprint profiles of Panax notoginseng with different commercial specifications were established and their shared peaks were analyzed quantitatively/semi-quantitatively to determine the content of saponins.UPLC-Q-Exactive Orbitrap MS was used to determine the content of dencichine in Panax notoginseng with different commercial specifications from Wenshan.UV spectrophotometric method was used to determine the total flavonoid content of Panax notoginseng.4 Screening of Panax notoginseng hemostatic effective ingredients group by spectrumeffect correlation method.Using partial least squares method,the common components of Panax notoginseng with different commodity specifications were correlated with plasma recalcification time,gastric hemorrhage comprehensive coagulation value,and comprehensive coagulation value.The hemostatic effective ingredients group based on plasma recalcification time,the hemostatic effective ingredients group based on gastric bleeding effect and the hemostatic effective ingredient group were screened.5 Using Technique for Order Preference by Similarity to Ideal Solution to evaluate the quality of Panax notoginseng comprehensively.HPLC,UPLC-Q-Exactive Orbitrap MS and Ultraviolet Spectrophotometry were used to detect the content of the hemostatic effective ingredients group of 36 batches of Wenshan different commercial specifications of Panax notoginseng,including saponins,dencichine and flavonoids.By TOPSIS combining the efficacy coefficients of each index of the hemostatic effective ingredients group,a quality evaluation model of Wenshan Panax notoginseng for different commercial specifications was constructed to comprehensively evaluate the quality of Wenshan Panax notoginseng with different commercial specifications.Results1 Research on the hemostatic effect of different commercial specifications of Panax notoginseng.The results showed that the effect of Panax notoginseng on plasma recalcification time was prior in Wenshan 30-Tou,40-Tou and 120-Tou Panax notoginseng,and not so good in Wenshan 20-Tou Panax notoginseng,and the difference between the 12 groups of Panax notoginseng and the model group was statistically significant.The effects of Panax notoginseng on gastric hemorrhage in rats were compared among different commercial specifications,and it was found that all 12 groups could protect against gastric hemorrhage to a certain extent and relief the hemorrhage symptoms,which also provided data support for the subsequent study of the spectrum-effect correlation.It also showed that the reason for the different hemostatic effects of Panax notoginseng with different commercial specifications might be related to the contents of chemical components.2 Research on the hemostatic bio-efficacy of Panax notoginseng with different commercial specifications.The evaluation results of the biological effects of 12 different commercial specifications of Panax notoginseng showed that the hemostatic biological potency range of 12 different commercial specifications of Panax notoginseng was 69.28～237.35 IU·g-1.Wenshan 80 Tou Panax notoginseng had the lowest hemostatic biological potency,and Wenshan countless Tou Panax notoginseng had the highest hemostatic biological potency.3 Research on chemical ingredients of Panax notoginseng with different commercial specifications.Eleven kinds of saponins were identified by HPLC technique:Notoginsenoside Ft1,Notoginsenoside Ri,Ginsenoside F1,Ginsenoside Rb1,Ginsenoside Rc,Ginsenoside Rd,Ginsenoside Re,Ginsenoside Rg1,Ginsenoside Rg2,Ginsenoside Rh1,Ginsenoside Rki.The contents of dencichine with 12 different commercial specifications of Panax notoginseng ranged from 0.001807%to 0.03070%by UPLC-Q-Exactive Orbitrap MS.The total flavonoid contents of Panax notoginseng in 12 different commercial specifications were obtained by UV spectrophotometric technique in the range of 0.2293%一 0.6141%.The above results indicated that there were large differences in the contents of the above three chemical components among the 12 different commercial specifications of Panax notoginseng,which can be used as data support for the subsequent spectrum-effect correlation screening.4 Screening of Panax notoginseng’s hemostatic effective ingredients group by spectrumeffect correlation method.The effective ingredients group of Panax notoginseng plasma recalcification time experiment was screened based on the spectrum-effect correlation analysis:Notoginsenoside Fti,Notoginsenoside R1,Ginsenoside Re,Ginsenoside Rg1,Ginsenoside Rg2,Ginsenoside Rh1,Ginsenoside Rk1,total flavonoids,dencichine,Unconfirmed 4,Unconfirmed 6,Unconfirmed 8,Unconfirmed 9,Unconfirmed 13,Unconfirmed 14,Unconfirmed 15.Among the designated components,dencichine,Notoginsenoside Ft1,Ginsenoside Rg2 contributed more to the reduction of plasma recalcification time.On the contrary,Ginsenoside F1,Ginsenoside Rb1,Ginsenoside Rc,Ginsenoside Rd had a negative effect on reducing plasma recalcification time.According to the spectrum-effect correlation analysis,the group of hemostatic effective ingredients that affect the gastric bleeding of Panax notoginseng was selected as:Notoginsenoside Ft1,Notoginsenoside R1,Ginsenoside F1,Ginsenoside Rb1,Ginsenoside Re,Ginsenoside Rg1,Ginsenoside Rg2,Ginsenoside Rk1,total flavonoids,dencichine,Unconfirmed 1,Unconfirmed 4,Unconfirmed 6,Unconfirmed 7,Unconfirmed 8,Unconfirmed 9,Unconfirmed 11,Unconfirmed 12,Unconfirmed 13,Unconfirmed 15.Among the designated components,dencichine,Notoginsenoside R1,Ginsenoside Rg1 had a higher contribution to reducing the comprehensive coagulation value of gastric bleeding.Ginsenoside Rc,Ginsenoside Rd,Ginsenoside Rh1 had a negative effect on reducing the comprehensive coagulation value of gastric bleeding.According to the spectrum-effect correlation analysis,the hemostatic effective ingredients group of Panax notoginseng on the comprehensive coagulation value was screened:Notoginsenoside Ft1,Notoginsenoside R1,Ginsenoside Fi,Ginsenoside Re,Ginsenoside Rg1,Ginsenoside Rg2,Ginsenoside Rh1,Ginsenoside Rki,total flavonoids,dencichine,Unconfirmed 4,Unconfirmed 6,Unconfirmed 8,Unconfirmed 9,Unconfirmed 13,Unconfirmed 14,Unconfirmed 15.Among the designated components,dencichine,Notoginsenoside Ft1,Ginsenoside Rg2 contributed more to the postive effect on hemostasis.Ginsenoside Rb1,Ginsenoside Rc,Ginsenoside Rd contributed more to the negative effect on hemostasis.5 Comprehensive evaluation of Panax notoginseng quality by Technique for Order Preference by Similarity to Ideal Solution.From the evaluation results,the relative proximity of 36 batches of Panax notoginseng with different commercial specifications ranged from 0.2085 to 0.5670,and the 36 batches of Panax notoginseng with different commercial specifications were divided into four grades(Grade I:relative proximity≥0.5000,excellent quality.Grade II:0.4200>relative proximity≥0.3800,good quality.Grade Ⅲ:0.3300>relative proximity≥0.2800,medium quality.Grade IV:0.2800>relative proximity,poor quality).The 36 batches of Panax notoginseng samples with different commercial specifications were initially graded into 4 grades.Grade I were Wenshan 120-Tou and Wenshan 80-Tou Panax notoginseng samples.Grade Ⅱ were Wenshan hair root,Wenshan countless Tou,Wenshan rootlet,Wenshan 20-Tou and Wenshan thick root Panax notoginseng samples.Grade Ⅲ were Wenshan rhizome,Wenshan 200-Tou,Wenshan 60Tou and Wenshan 40-Tou Panax notoginseng samples.Grade Ⅳ was Wenshan 30-Tou Panax notoginseng samples.Conclusions1.In this paper,we initially established a screening method for the group of hemostatic effective ingredients in Panax notoginseng with different commercial specifications based on spectrumeffect correlation,which provided a new idea to reveal the group of active ingredients of hemostatic efficacy of Panax notoginseng.2.In this study,the bio-efficacy assay was used to evaluate the hemostatic effect of Panax notoginseng with different commercial specifications by integrating the in vitro and in vivo model hemostatic pharmacodynamic indexes,which filled the research gap in the part of bioactivity evaluation of Panax notoginseng with different commercial specifications.3.In this study,based on the weight of the drug efficacy indexes and the chemical components,a quality evaluation method that can reflect the hemostatic activity of Panax notoginseng was established according to the TOPSIS.Provided reference for the quality evaluation and clinical rational use of Panax notoginseng.