Research On Correlation Between "Effects Of Hemostasis With Epartly Preservationr" And Carbonizing Crafts Of Rehmanniae Radix Charcoal

Objective:This research focuses on the difference of"existence"of Rehmanniae Radix,and puts forward the hypothesis that the material basis of"hemostasis and preservation part properties of Rehmanniae Radix carbonized",which includes not only in vitro transformed substances but also metabolites in vivo,however,the changes in these substance bases depends on the working of the technology of making charcoal.Using metabolomics technology and thermal analysis technology to digitally characterize the technical parameters of Rehmanniae Radix?stir-frying,calcined?.The comparative analysis of the dynamic change process and metabolite changes of Rehmanniae Radix carbonized products in the body.It was made to further clarify the correlation between the process and the hemostasis and preservation part properties of Rehmanniae Radix carbonized.Medicine thermal analysis-metabolomics technology model for the research of processing technology and processing mechanism was established.Methods:1.Establish HPLC fingerprint chromatography of Rehmanniae Radix and its two carbonized products?stir-frying,calcined?.The extracts of 10 samples of Rehmanniae Radix and its two charcoal products were analyzed on a Hypersil GOLD aQ C₁₈ column?250 mm×4.6 mm,5?m?,the mobile phase was acetonitrile-0.1%phosphoric acid aqueous solution,gradient elution,volume flow rate was 1 mL·min^-1,column temperature was 30°C,and detection wavelength was 210 nm.The author performs principal component analysis,cluster analysis and similarity evaluation on the results.2.Preliminary determination of active ingredient groups in blood before and after Rehmanniae Radix carbonizing:Samples of water extracts of Rehmanniae Radix and its two charcoal products?stir-frying,calcined?were prepared,and the extracts were continuously gavage to rats for 3 days,and blood was collected to prepare rat-containing serum.HPLC fingerprint chromatography was used to detect the drug-containing serum of rats to determine the transition components in blood.Combined with the results of in vitro chemical composition study of before and after Rehmanniae Radix carbonizing,the preliminary basis of potential medicines for Rehmanniae Radix carbonizing was determined.3.Using thermal analysis technology,taking Rehmanniae Radix as a sample,under the condition of programmed temperature rise,by changing the thermal analysis atmosphere,that is,using air and nitrogen atmosphere respectively,to simulate the traditional carbon production.At the same time,under the same experimental conditions,the thermal analysis experiment was performed with the pharmacodynamic substance group of Rehmanniae Radix.In the end,the temperature corresponding to the maximum thermal weight loss rate of the medicinal substance group were used as the digital indicators of the technical parameters?firepower,heat?of the Rehmanniae Radix carbonizing.4.The blood heat and hemorrhage syndrome rat model was established by subcutaneous injection of 20%dry yeast combined with anhydrous ethanol.Based on the ultra-high performance liquid chromatography-mass spectrometry?UPLC-MS?metabolomics technology platform,through the systematic analysis of small molecule metabolites in serum,the search for differential metabolites and metabolic pathway.Results:1.A total of 15 chromatographic peaks were identified for the 10 batches of Rehmanniae Radix samples,a total of 11 chromatographic peaks were identified for the carbonized Rehmanniae Radix,and a total of 8 chromatographic peaks were identified for carbonized Rehmanniae Radix by calcining.The raw materials of Rehmanniae Radix and its two charcoal products?stir-frying,calcined?can achieve significant separation in principal component analysis and cluster analysis.The similarity evaluation revealed the differentiating effects of the origin on the samples.2.Gender has no significant effect on the medicinal ingredients that enter the blood of Rehmanniae Radix and its two charcoal products.The composition of the two kinds of charcoal?stir-frying,calcined?that enter the blood is similar,but the specific content is different,but whether it is caused by the method of making charcoal or other factors still needs further investigation.In addition,before and after Rehmanniae Radix carbonizing,research has shown that catalpol enters the blood in its original form,which is a biopharmaceutical indicator of Rehmanniae Radix.3.The optimal temperature of carbonized Rehmanniae Radix should be controlled before the pyrolysis of fructose and catalpol,the temperature at the peak of the maximum thermal weight loss rate of 5-HMF pyrolysis.Therefore,216.3?is the optimal temperature for it.The maximum thermal weight loss rate of catalpol is 274.3?,which is the upper limit of carbonized Rehmanniae Radix temperature.4.The optimal temperature of carbonized Rehmanniae Radix by calcining should be controlled before the pyrolysis of fructose and catalpol,which is the temperature at the peak of the maximum thermal weight loss rate of 5-HMF pyrolysis.Therefore,194.2?is the optimal temperature.The maximum thermal weight loss rate of catalpol is 276.8?,which is the upper limit of calcined carbon temperature.5.Based on blood heat and hemorrhage syndrome rat model and UPLC-MS metabolomics technology platform,different metabolites and metabolic pathways of different ways of Rehmanniae Radix carbonizing?stir-frying,calcined?were studied.?1?The blood heat and hemorrhage syndrome rat model was established by subcutaneous injection of 20%dry yeast combined with anhydrous ethanol.The lungs and stomach tissues of the model rats showed multiple bleeding points to the naked eye.The liver and kidney color deepened.The lung tissue pathological examination showed interstitial hemorrhage and inflammatory cell infiltration.Anal temperature increased significantly within 8h?P<0.05?.The TT,APTT and PT of the rats were significantly prolonged?P<0.05?,and the plasma FIB content was significantly increased?P<0.05?,ndicating that the blood heat and hemorrhage syndrome rat model have abnormal coagulation function and have a significant bleeding tendency.In addition,the positive drug group,the Rehmanniae Radix group,carbonized Rehmanniae Radix?traditional and new method?,and carbonized Rehmanniae Radix by calcining?traditional and new methods?can significantly reduce the relative content of different coagulation factors?P<0.05?,indicating that Rehmanniae Radix carbonizing?stir-frying,calcined?enhanced hemostatic effect,the hemostatic effect has a certain difference between the Rehmanniae Radix carbonizing?stir-frying,calcined?.The results also show that Rehmanniae Radix,carbonized Rehmanniae Radix and carbonized Rehmanniae Radix by calcining play a hemostatic role by enhancing the function of the body's endogenous coagulation system.?2?Model rats have not only abnormal blood coagulation function,but also abnormal blood rheology.The specific manifestations are the high,middle and low whole blood viscosities,plasma viscosity,both the high and low whole blood restore viscosity,erythrocyte rigidity index and erythrocyte aggregation index.All of them increased significantly?P<0.05?.According to the analysis,the carbonized Rehmanniae Radix can significantly improve the relevant hemorheological indexes?P<0.05?,indicating that the carbonized Rehmanniae Radix can retain its"cooling blood"effect.However,carbonized Rehmanniae Radix by calcining significantly reduced the both the high and low whole blood restore viscosity?P<0.05?,and had no significant effect on other hemorheological indexes.The remaining groups also improved the related indexes.?3?The effects of different duration and degree of heating preparation methods on the efficacy of their processed products have significant differences?P<0.05?.On the other hand,the charcoal products made from Rehmanniae Radix,which is not meet relevant standards,is not effective in cooling blood and stopping bleeding.It shows that duration and degree of heating in the processing of Chinese medicinal materials needs to be strictly controlled,otherwise it will affect the exertion of the medicinal effect in processed products.?4?The study of serum metabolomics based on the blood heat and hemorrhage syndrome rat model showed that different processing methods have a significant effect on the endogenous metabolic profile of processed products,as shown in the following:1)There are some differences in the processing methods of Rehmanniae Radix and its two charcoal products?stir-frying,calcined?.2)There is a difference between the new method and the traditional processing methods about Rehmanniae Radix and its two charcoal products?stir-frying,calcined?.3)Rehmanniae Radix of not meet relevant standards are different from their endogenous products.However,comprehensive analysis believes that no matter what kind of processing method,there is still a tendency to pull back to the blank group,which proves that it retains a certain therapeutic effect of Rehmanniae Radix.?5?Based on the non-targeted metabolomics platform,42 differential metabolites were screened between the Rehmanniae Radix and its two charcoal products?stir-frying,calcined?.The differences between 17 metabolic pathways and endogenous small molecules were further analyzed,and the biological mechanism of their effects was explored.Conclusion:In this study,in vitro pharmacodynamics of Rehmanniae Radix and its two charcoal products?stir-frying,calcined?were identified by fingerprint and serum drug analysis.Using metabolomics technology and thermal analysis technology to digitally characterize the technical parameters of Rehmanniae Radix?stir-frying,calcined?.At the same time,the comparative analysis of the dynamic change process and metabolite changes in the body was performed.The results indicated that carbonized Rehmanniae Radix had the hemostasis and blood-cooling effects,while carbonized Rehmanniae Radix by calcining only retains the function of hemostasis without significant blood-cooling effects.During the processing of traditional Chinese medicine,different duration and degree of heating preparation methods have a significant effect on the medicinal substances and metabolic profiles.42 differential metabolites were screened between the Rehmanniae Radix and its two charcoal products?stir-frying,calcined?.The differences between 17 metabolic pathways and endogenous small molecules were further analyzed,and the biological mechanism of their effects was explored.It was made to further clarify the correlation between the process and the hemostasis and preservation part properties of Rehmanniae Radix carbonized.