Study On The Component Analysis,Blood-enriching Effect And Metabolic Mechanism Of Steamed Panax Notoginseng

1 OBJECTIVEPanax notoginseng is the dried root and rhizome of Panax notoginseng(Burk.)in the family Panax.Commonly used clinically have raw P.notoginseng and steamed P.notoginseng,both of which have different curative effects.Raw Panax notoginseng is mainly used to dissipate blood stasis and stop bleeding,reduce swelling and relieve pain,while steamed Panax notoginseng is good at tonifying qi and blood and tonifying body building.At present,the chemical composition and pharmacological action of raw P.notoginseng are widely studied,but lack of ripe notoginseng system comprehensive study,and mechanism of"Sheng da shu bu"of Panax notoginseng remains to be further studied.Based on the chemical composition of raw P.notoginseng and steamed P.notoginseng,the paper studied the blood tonifying effect of P.notoginseng,clarified the change rule of the substance of Panax notoginseng before and after processing,and verified the scientific nature of the theory of"shu bu".On this basis,the metabolic mechanism of the tonifying effect of steamed P.notoginseng was preliminarily discussed through metabolomics.Based on the above research results of the chemical composition change obviously before and after,and efficacy of ginsenoside Rg₃using porous material load improve its dissolution studies,laying the groundwork for new drug development.2 METHODS2.1 Ultra high performance liquid chromatography coupled with time-of-flight mass spectrometry to identify the difference chemical between raw and steamed P.notoginsengThe components of raw and steamed P.notoginseng were qualitatively analyzed by UPLC-Q-TOF-MS/MS in positive and negative ion mode.The precise mass number determination,ion data of pyrolysis fragments and literature comparison were used to identify the chemical constituents of raw and steamed P.notoginseng.Then,the chemical constituents of raw and steamed P.notoginseng were compared by using UPLC-Q-TOF-MS/MS.The SIMCA-P14.1 software,PCA,PLS-DA and OPLS-DA pattern recognition analysis method were used to discover the different components between raw and steamed P.notoginseng.2.2 Comparative study on blood deficient of raw and steamed P.notoginsengSD rats were grouped into six groups:control group,model group,compound E Jiao Jiang group,raw P.notoginseng group,steamed P.notoginseng group,ginsenoside Rg₃group.Except for the control group,the other groups were used to intervene the hemolytic blood deficiency rats induced by N-acetyl phenyl hydrazine(APH),with the rats'physical signs,weight,organ index,blood routine,biochemical indexes as the evaluation index,to investigate the blood deficient of raw and steamed P.notoginseng.2.3 Study on blood deficient mechanism of steamed P.notoginseng based on metabolomics methodSD rats were grouped into three groups:control group,model group,steamed P.notoginseng group.Metabolomic fingerprints of rat plasma samples were determined by UPLC-Q-TOF-MS/MS,establish the plasma metabolic patterns.The data sets were established and analyzed by PCA,PLS-DA,OPLS–DA,find the difference metabolites between the groups,analysis of differences between the differences in composition of metabolic pathways.The aim of the study was to investigate the possible Study on blood deficient mechanism of steamed P.notoginseng based on metabolomics method mechanisms of steamed P.notoginseng.2.4 Enhanced dissolution rate of the poorly water soluble ginsenoside Rg₃with ordered meso/macroporous silicaThe ordered meso/macorporous Si O₂was synthesized by colloidal crystal templates,and solvent natural evaporation was used to prepare Rg₃-ordered meso/macorporous silica.Rg₃-meso/macorporous Si O₂and meso/macorporous Si O₂were characterized by scanning electron microscope,transmission electron microscopy,nitrogen sorption,X-ray diffraction and fourier transform IR spectroscopy.In vitro ginsenoside Rg₃release studies from m/M-Si O₂/Rg₃powder were performed using the Turbidimetric-kinetic method of 2020"Chinese Pharmacopoeia".3 RESULTS3.1 Ultra high performance liquid chromatography coupled with time-of-flight mass spectrometry to identify the difference chemical between raw and steamed P.notoginsengThe components of raw and steamed P.notoginseng were qualitatively analyzed by UPLC-Q-TOF-MS/MS in positive and negative ion mode.The possible element composition and mass error were analyzed with the Sciex OS software,and the possible chemical formula was retrieved in the Sci Finder to screen out the possible chemical composition.According to the comparison of reference materials,accurate molecular weight and mass spectrum secondary fragmentation information,and combined with the comparison reported in the literature,a total of 47 compounds were identified from raw P.notoginseng and 53 compounds were identified from steamed P.notoginseng.The error of the precise mass obtained by the first stage mass spectrometry and the theoretical value calculated are within 5 ppm.It included proginsentriol saponins,proginsendiol saponins,other structural saponins,flavonoids,polypargynol,volatiles and other compounds.The mass spectrometry pyrolysis rules of ginsenoside Rg₃,ginsenoside Rh₁,notoginsenoside R₁,ginsenoside Rg₁and other compounds were deduced.Using SIMCA-P14.1software analysis,the principal component analysis results showed that the two groups of samples of born raw P.notoginseng and steamed P.notoginseng can be clearly distinguished,indicating that there are significant differences in the types and contents of chemical components between the two groups of samples.The orthogonal partial least-discriminant analysis of supervised analysis was used to model the distribution of raw and steamed P.notoginseng in different regions,so the saponins with great difference could be found directly and quickly.Through analysis,the difference between raw and steamed P.notoginseng products is saponins R₁,ginsenosides Rg₁,ginsenosides Rg₃,ginsenosides 20S-Rg₃,ginsenosides Rb₁,ginsenosides Re,ginsenosides Rh₁,ginsenosides 20S-Rh₁,ginsenosides Rk₃.3.2 Comparative study on blood deficient of raw and steamed P.notoginsengThe rat blood deficiency induced by N-acetyl phenyl hydrazine was selected as the animal model.We evaluated the animal model by using the rats'physical signs,weight,organ index,blood routine,biochemical indexes,to investigate the blood tonifying effect of raw and steamed P.notoginseng.Compared with the control group,the rats in the model group had shaggy hair,no luster,mental tiredness,slow action,dim eyes,pale ears and tail,and liked to huddle and curl.The body weight of rats in model group decreased significantly and the symptoms of poor diet appeared obviously.Liver index and spleen index of model group were significantly increased,while thymus index was significantly decreased.The activity of ATPase,the content of G₆PD,the content of GPX and GR in the blood of model group rats were significantly decreased.Compared with the control group,the content of methemoglobin in the blood of rats in the model group was significantly increased.After drug intervention,raw P.notoginseng,steamed P.notoginseng and ginsenoside Rg₃groups could increase the body weight and improve the organ index of model rats.The activity of ATPase,the contents of G₆PD,GPX and GR in the blood of rats were increased,and the content of methemoglobin was decreased.Among them,these results showed that the improvement effect of steamed P.notoginseng was better than that of raw P.notoginseng.3.3 Study on blood deficient mechanism of steamed P.notoginseng based on metabolomics methodPlasma metabolites in control group,model group and steamed P.notoginseng treatment groups were analyzed by UPLC-Q-TOF-MS/MS.The original plasma data detected in positive and negative ion modes were pretreated and imported into the software SIMCA-P14.1 for discriminant analysis.PCA,PLS and OPLS-DA pattern recognition analysis results illustrate that the normal group and the model group can be well separated,indicating that there are significant differences in plasma metabolites between the controland the model group.PCA analysis of control group,model group and steamed P.notoginseng group were conducted in positive and negative ion modes.The sample points of the three groups were separated to a certain extent,while the samples of the intervention group showed a trend of approaching to the control group in different degrees.These results indicated that steamed P.notoginseng had effective intervention effect on metabolism of rats with blood deficiency.In the control group,model group and steamed P.notoginseng group in rat plasma identified 11 kinds of joint endogenous metabolites,which have a significant difference between groups.The 11differences metabolites import Metabo Analyst 3.0 analysis platform,analysis done notoginseng blood deficient related article 4 metabolic pathways,respectively is:tryptophan metabolism,valine,leucine and isoleucine biosynthesis,linoleic acid metabolism,glycerolipid metabolism.3.4 Enhanced dissolution rate of the poorly water soluble ginsenoside Rg₃with ordered meso/macroporous silicaOrdered mesoporous silica porous materials were prepared by double template method using PS colloidal crystal as template and methyl orthosilicate as inorganic source and ginsenoside Rg₃-ordered macroporous-mesoporous silica carrier drug was prepared by natural evaporation method.The morphology of the two kinds of porous Si O₂were investigated by SEM and TEM.For m/M-Si O₂,the sample possesses ordered macropores with an average pore size of 270 nm.More importantly,due to the shrinkage of the PS microspheres and condensation of silica framework during the calcination process,the adjacent macropores are interconnected by open pores with diameter of 90 nm.The internal structure of the m/M-Si O₂carrier was studied by transmission electron microscopy.It was found that the macroporous silica framework of m/M-Si O₂carrier contained a large number of highly ordered mesopores.The nitrogen adsorption–desorption X-ray diffraction and Fourier infrared spectroscopy were conducted further to investigate m/M-Si O₂and m/M-Si O₂/Rg₃.The results indicate that Rg₃in the m/M-Si O₂channel existed in an amorphous state,and ginsenoside Rg₃did not react with silicon oxide,but physically adsorbed on the surface of silicon oxide.The amount of dissolved Rg₃can be calculated by the high performance liquid chromatography.For the pure Rg₃,only 21%of Rg₃was dissolved into the solution,demonstrating its intrinsically poor dissolution in water.However,the dissolution rate of Rg₃from m-Si O₂/Rg₃and m/M-Si O₂/Rg₃reached 78%and 81%,respectively within the same duration,a significant enhancement to the pure Rg₃.4 CONCLUSIONIn this study,the main different chemical components between raw and steamed P.notoginseng were clarified,and the scientific nature of"shu bu"of Panax notoginseng was verified by drug efficacy experiment in vivo.The 4kinds of amino acids and metabolic pathways related to the blood deficient of steamed P.notoginseng were determined by UPLC-MS.For the first time,ordered macroporous mesoporous silicon oxide porous material loaded with differential component ginsenoside Rg₃was used to improve its dissolution rate in vitro.This paper comprehensively elucidates the mechanism of"shu bu",and provides scientific basis for the clinical application of Panax notoginseng and its processed products.