| Prescriptions are the main form using in Traditional Chinese medicines which have been practiced clinically in China for thousands of years.It is crucial for the modernization of Chinese medicinals to elucidate the effective substance basis and mechanism of traditional Chinese medicine prescription,and to ensure its effectiveness,safety and stability.There are two prerequisites for the drug to be effective in the body:firstly,it can cross the barrier to reach the target after administration,be effectively utilized and maintained at an effective concentration;secondly,the bio-activity of the chemical form(prototypes or metabolites)of those components at the target must be related to its efficacy.Therefore,it is essential to investigate the systematic exposure of bio-active components in blood and tissues to reveal the mechanism of their efficacy.As a classical formula which consists of Astragali Radix and Angelicae Sinensis Radix at a 5:1 ratio,It has been reported that Danggui Buxue Decoction has significant effects on the protection of cardiovascular function and promotion of hematopoiesis in modern studies.However,it is not yet clear the effective substance basis of its effect,distribution and metabolism in vivo.In our group,we made some progress on the previous investigation about the potential Q-markers of Danggui Buxue Decoction by sequential metabolism.This study was conducted to reveal its effective substance basis of the distribution and transport in various target tissues from the perspective of clinical application.Research purposes:Guided by the meridian tropism and modern pharmaceutical analysis theories,Danggui Buxue Decoction was chosen as the object of this study.Based on the technology of high-resolution quadrupole/orbitrap mass spectrometer and triple quadrupole liquid tandem mass spectrometry,this study was conducted to quantify and evaluate the prescription quality,display the distribution of the prototype components and their metabolites in different tissues,elucidate the distribution kinetic differences of Danggui Buxue Decoction in different tissues and gender,and indicate the differences in blood transport and pharmacodynamics in normal and blood-deficient states.All of the above was to illuminate the distribution differences and law of the effective substance basis of Danggui Buxue Decoction in different target organs based on blood tonicity and nourishment.The study aims to provide a research strategy and data support for the quality control of the active ingredients in Danggui Buxue Decoction.Research methods and results:1.The HPLC method was established with high reliability and sensitivity to quantify 8 bio-active compounds(ferulic acid,calycosin,formononetin,calycosin-7-O-glucoside,ononin,astragaloside Ⅳ,senkyunolide I,ligustilide)in Danggui Buxue Decoction,and the sample preparation process was simple and reproducible.A qualitative analysis method for the multiple components in Danggui Buxue Decoction was established by using UPLC-Q-Exactive-MS/MS technique to achieve rapid separation and data analysis.36 chemicals in Danggui Buxue Decoction were identified and clarified into flavonoids,saponins,phthalides,organic acids,amino acids and others by metabolicpilot software,standard solution comparison,retention time,molecular weight,fragmentation peaks,relevant literature and reaxys database.The distribution of those chemicals in seven tissues was also identified by liquid-mass spectrometry,and 41 parent components or metabolites were qualitatively attributed.The transformation and metabolism were indicated to provide a reference basis for the effective substance basis of Danggui Buxue Decoction.2.Based on UPLC-TQ-MS/MS,six bio-active components were determined in seven tissues of blood-deficient rats after oral administration of the Danggui Buxue Decoction,and pharmacokinetic parameters such as Cmax AUC0～T were calculated by phoenix software.It aimed to investigate the tissue distribution tendency and kinetics of Danggui Buxue Decoction in the pathological state(blood deficiency).The comprehensive results showed that the peak time of each compound in Danggui Buxue Decoction is between 1h and 2h,and the two phthalides compounds could reach the peak faster than other compounds.The distribution of Danggui Buxue Decoction in females tended to be in the heart and uterus,which coincided with its pharmacological properties.The heart and uterus were speculated to be potential target organs for its medicinal effects.And the other tissues’ distribution level ranging from high to low was thymus>liver>lung>spleen>kidney.Based on the gender differences,the tissue distribution of Danggui Buxue Decoction in female rats and male rats was statistically analyzed by Pearson correlation analysis to investigate the tissue distribution in different gender.The results showed that gender did not affect the distribution trend in the other six tissues,but its concentration was different at individual time points.3.The rats with blood deficiency were administered high,medium and low doses of the prescription,and were observed for general signs,histomorphology and peripheral blood routine.The results showed that Danggui Buxue Decoction effectively alleviated the weight loss and tissue damage after modeling,and the values of HGB,WBC,RBC and HCT were significantly improved compared with the model group.The high-dose group had the best effect.Equilibrium dialysis was used to investigate the differences in plasma binding rate of Danggui Buxue Decoction in normal and blood-deficient rats.The plasma binding rates of Calycosin-7-O-glucoside,Senkyunolide I and Ononin were in the range of 25%～75%,which were medium to medium binding type compounds.The plasma protein binding rates of Calycosin,formononetin and Ligustilide were all higher than 75%and belonged to higher binding type compounds.The binding rates of all compounds in the model group were basically higher than those in the normal group,and the pathological microenvironment might change the blood transport of the compounds in the distribution in tissues.Conclusion:The separation and identification of complex components in Danggui Buxue Decoction were performed by high-performance liquid chromatography and ultra-performance liquid chromatography-mass spectrometry.And the qualitative and quantitative methods were successfully established for the multiple components in formulas and the distribution in different tissues in vivo.Based on the pharmacokinetics and clinical application of the formula,we determined the distribution differences between tissues and genders in blood deficiency rats.It was found that the formula distributed mostly in the heart and uterus,and the distribution trend was the same in different genders,but the concentration was different depending on the level of metabolic enzymes or the characteristics of the components in vivo.Based on the pharmacodynamic effects of Danggui Buxue Decoction,the Acetylbenzene-Cyclophosphamide complex blood deficiency model was used to detect significant improvements in peripheral blood routine,histomorphology,and general behavior in normal and blood deficiency states,at different doses of Danggui Buxue Decoction.The finding indicated that the high-dose group had the best effect.This study provides solid experimental support for the elaboration of the subsequent mechanism of Danggui Buxue Decoction acting in vivo. |
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