Q-markers Of Buyang Huanwu Decoction Anti-atherosclerosis And Its Pharmacokinetic Study Of Disease

Research purpose:To explore the atherosclerosis Q-markers of Buyang Huanwu Decoction and its pharmacokinetics in normal and chronic inflammation model rats.Research methods:1 Q-marker prediction of Buyang Huanwu decoction anti-atherosclerosis1.1 research on the material basis and mechanism of anti-atherosclerotic effect of Buyang Huanwu Decoction based on network pharmacologyBased on the method of network pharmacology,the core components and targets of antiatherosclerosis in Seven herbs of Buyang Huanwu Decoction,including safflower,Ligusticum chuanxiong,peach kernel,Angelica Sinensis,Radix Paeoniae Rubra,Astragalus membranaceus and earthworm,were screened out,and the mechanism of antiatherosclerotic action of Buyang Huanwu decoction was predicted by KEGG pathway analysis.1.2 Anti-atherosclerosis Q-markers of Buyang Huanwu Decoction based on Text MiningBased on the method of literature search,the measurable components of six kinds of medicinal materials in Buyang Huanwu Decoction were retrieved from CNKI database(the compound of Buyang Huanwu decoction is composed of 7 kinds of medicinal materials,including safflower,Ligusticum chuanxiong,peach kernel,Angelica Sinensis,Radix Paeoniae Rubra,Astragalus membranaceus and earthworm.Among them,earthworm is an animal medicine,and its components are difficult to distinguish from the original substances of the body after entering the blood,so the components of earthworm are not included in the selection process of compound quality markers Consider.The research frequency of the above components was measured by Bibliometric database;the correlation between the components（bibliometric≥5）and atherosclerosis was verified in CNKI and web of science database to determine the anti-atherosclerosis Q-markers of Buyang Huanwu Decoction.2 Q-markers Quality Control of Buyang Huanwu Decoction anti-atherosclerosisThe determination was carried out on an ACQUITY UPLC BEH C₁₈（2.1 mm×50 mm,1.7?m）at 40℃.the mobile phase consisted of 0.1%formic–acetonitrile（B）acid solution（A）with gradient elution at a flow rate of 0.3 m L·min^-1.Electrospray ionization（ESI）source,positive ion mode scanning,and multiple reaction monitoring mode（MRM）detection of anti-atherosclerosis Q-markers of Buyang Huanwu Decoction（hydroxysafflor yellow A,amygdalin,albiflorin,paeoniflorin,ferulic acid,Astragaloside IV,n-butylidenephthalide,Z-ligustilide）.3 Study on pharmacodynamics of Buyang Huanwu Decoction against chronic InflammationFifty nine male SD rats were randomly divided into six groups:（1）control group（n=7）,（2）chronic inflammation model group（n=11）,（3）prednisolone acetate group（n=8）,（4）low dose Buyang Huanwu Decoction group（n=11）.（5）The middle dose group of Buyang Huanwu Decoction（n=11）;（6）the high dose group of Buyang Huanwu Decoction（n=11）;in addition to the control group,the other groups were injected with low-dose lipopolysaccharide（200?g/kg）in tail vein on the first day of each week,and the control group was injected with sterile saline of equal volume,four times in total,and the administration was started the next day after the first injection,lasting for four weeks.The anal temperature,wet weight of spleen,wet weight of thymus,white blood cell count,Gran%,Mon%,Lymph%,hs-CRP,IL-6,TNF-α,plasma MDA content and SOD activity were determined to observe the inflammatory state in SD rats.4 UPLC-MS/MS determination method of anti-atherosclerotic Q-markers of Buyang Huanwu decoction in rat plasmaThe determination was carried out on an ACQUITY UPLC BEH C₁₈（2.1 mm×50 mm,1.7?m）at 40℃.the mobile phase consisted of 0.1%formic–acetonitrile（B）acid solution（A）with gradient elution at a flow rate of 0.3 m L·min^-1,Electrospray ionization（ESI）source,electrospray ionization（ESI）positive and negative ion switching mode scanning,and multiple reaction monitoring mode（MRM）detect each effect component（hydroxysafflor yellow A,amygdalin,albiflorin,paeoniflorin,ferulic acid,Astragaloside IV,n-butylidenephthalide,Z-ligustilide）in plasma.5 Q-markers of Buyang Huanwu Decoction Anti-atherosclerosis and its pharmacokinetics in normal and chronic inflammatory rats60 male SD rats were randomly divided into 6 groups,8 rats in the low,medium and high dose control groups and 12 rats in the low,medium and high dose chronic inflammation groups.After administration by gavage,blood was taken from the venous plexus at the time points of0.083,0.25,0.5,0.75,1,1.25,1.5,2,3,4,6,12 and 24 hours respectively,and centrifuged in a high-speed freezing centrifuge immediately 10 min(3500 r?min^-1),transfer the supernatant,and freeze it in the refrigerator at-80℃.The serum concentrations of 8 Q-markers were determined in method 4.The pharmacokinetic parameters of Q-markers were analyzed by win-nonlin 6.3and SPSS statistical analysis software.Research results:1 Q-marker prediction of Buyang Huanwu Decoction anti-atherosclerosis1.1 research on the material basis and mechanism of anti-atherosclerotic effect of Buyang Huanwu Decoction based on network pharmacologyIn addition to earthworm,there are 13,10,12,32,25 and 11 anti-atherosclerotic core components in six herbs,including safflower,Ligusticum chuanxiong,peach kernel,Angelica Sinensis,Radix Paeoniae Rubra,Astragalus membranaceus,They are 103 core components in total,which are the material basis of anti-atherosclerotic effect of Buyang Huanwu decoction;The analysis results of KEGG pathway of 14 core targets screened by the network pharmacology method show that Buyang Huanwu Decoction can play an anti-inflammatory and anti-oxidation role by intervening arachidonic acid metabolism pathway,prevent endothelial damage and improve atherosclerosis.1.2 Anti-atherosclerosis Q-markers of Buyang Huanwu Decoction based on Text MiningIn this study,eight effective components were identified:hydroxysafflor yellow A,amygdalin,albiflorin,paeoniflorin,ferulic acid,astragaloside IV,n-butylidenephthalide,Z-ligustilide.There is a high degree of coincidence between the eight effective components of text mining and the antiatherosclerotic active substances of Buyang Huanwu Decoction obtained based on the network pharmacology method,while the three components of hydroxysafflor yellow A,albiflorin and amygdalin,respectively,from the three herbs of safflower,Radix Paeoniae Rubra and peach kernel,are the anti-atherosclerotic drugs of Buyang Huanwu Decoction obtained based on the network pharmacology method The effective substances are not reflected.The reason is that the above three components are excluded in the process of component acquisition because they do not meet the Lipinski rule and OB≥30%in TCMSP database.2 Q-markers Quality Control of Buyang Huanwu Decoction anti-atherosclerosisHydroxysafflor yellow A,showed a good linearity in the range of 10.00?1350.00 ng?m L^-1（r=0.9999）.amygdalin,albiflorin,paeoniflorin,showed a good linearities in the range of2.00?2700.00 ng?m L^-1（r=0.9999）.ferulic acid showed a good linearities in the range of5.00?675.00 ng?m L^-1（r=0.9998）.astragaloside IV,n-butylidenephthalide,Z-ligustilide showed a good linearities in the range of 2.00?270.00 ng?m L^-1（r=0.9999）.The average recoveries were94.61%,91.85%,100.54%,96.27%,105.22%,108.15%,104.13%and 99.79%.with RSD of8.54%,8.98%,5.17%,3.71%,6.24%,3.88%,5.00%and 7.13%.The content of 8 anti-AS Q-markers in the samples of Buyang Huanwu Decoction were all RSD<5%.3 Study on Pharmacodynamics of Buyang Huanwu Decoction against chronic inflammationCompared with the normal control group,the anal temperature（P<0.05）,wet weight of the spleen（P<0.01）,wet weight of the thymus（P<0.05）,white blood cell count（P<0.05）,Gran%（P<0.05）,Mon%（P>0.05）,Lymph%（P>0.05）,hs-CRP（P<0.05）,IL-6（P<0.05）,TNF-α（P<0.01）,MDA（P<0.01）in the chronic inflammation model group increased;meanwhile,SOD（P<0.01）and Lymph%（P<0.05）of chronic model group decreased.Compared with the chronic inflammation model group,the anal temperature of rats in each administration group had an upward trend,and the anal temperature of rats in the middle-dose group of Buyang Huanwu Decoction in the fourth week decreased significantly（P<0.05）;The spleen coefficient of each administration group had a certain downward trend,and the spleen coefficient of the prednisolone acetate group decreased significantly（P<0.01）;The thymus coefficient of the low-dose group of Buyang Huanwu Decoction decreased significantly（P<0.05）,and the thymus coefficient of the prednisolone acetate group decreased significantly（P<0.01）;The white blood cell count of Buyang Huanwu Decoction has a downward trend in the low and middle-dose groups,and the prednisolone acetate group decreases significantly（P<0.01）;Gran%and Mon%of Buyang Huanwu Decoction’s middle and high-dose group and prednisolone acetate group have a downward trend;Lymph%in Buyang Huanwu Decoction’s middle and high-dose group and prednisolone acetate group has an upward trend;The hs-CRP of each administration has a downward trend,and the low and high dose groups of Buyang Huanwu Decoction decreased significantly（P<0.05）;The IL-6 of each administration group had a downward trend,and the Buyang Huanwu Decoction low,middle-dose group and prednisolone acetate group decreased significantly（P<0.01）;The TNF-αof each administration group had a downward trend.The TNF-αof the three dose groups of Buyang Huanwu Decoction decreased significantly（P<0.01）,and the TNF-αof the prednisolone acetate group was significant.Decrease（P<0.05）;The MDA of each administration group showed a downward trend.The MDA of the low-dose group of Buyang Huanwu Decoction and the prednisolone acetate group decreased significantly（P<0.05）,and the MDA of the medium-dose group of Buyang Huanwu Decoction decreased significantly（P<0.01）;The SOD activity of each administration group has a tendency to increase,and the SOD activity of the low,medium and dose groups of Buyang Huanwu Decoction and the prednisolone acetate group increases significantly（P<0.05）.SOD activity was significantly increased（P<0.01）.4 UPLC-MS/MS determination method of anti-atherosclerotic Q-markers of Buyang Huanwu decoction in rat plasmaHydroxysafflor yellow A,amygdalin showed a good linearity in the range of 5.00?600.00ng?m L^-1（r=0.9973,0.9959）.albiflorin,paeoniflorin,showed a good linearities in the range of2.00?450.00 ng?m L^-1（r=0.9948,0.9996）.ferulic acid showed a good linearities in the range of1.00?450.00 ng?m L^-1（r=0.9958）.astragaloside IV showed a good linearities in the range of0.10?60.00 ng?m L^-1（r=0.9978）.n-butylidenephthalide showed a good linearities in the range of6.00?600.00 ng?m L^-1（r=0.9999）,Z-ligustilide showed a good linearities in the range of5.00?270.00 ng?m L^-1（r=0.9999）.The average recoveries were 94.61%,91.85%,100.54%,96.27%,105.22%,108.15%,104.13%and 99.79%.with RSD of 8.54%,8.98%,5.17%,3.71%,6.24%,3.88%,5.00%and 7.13%.RSDs of 8 active components of antiatherosclerosis in Buyang Huanwu decoction were below 5%.The recovery rate of each component was 50.84%?111.86%,and the precision RSD of Intra-/inter-day was less than or equal to 10.27%,The accuracy and stability meet the requirements of in vivo drug analysis.5 Q-markers of Buyang Huanwu Decoction anti-atherosclerosis and its pharmacokinetics in normal and chronic inflammatory ratsNo two components of n-butylidenephthalide and Z-ligustilide were detected in the plasma containing drug at various time points after the administration of low,middle and high doses of Buyang Huanwu Decoction.The pharmacokinetic parameters of the non-compartment model of the remaining 6 components were analyzed.The results showed that,compared with the normal control rats,AUC_0→t of hydroxysafflor yellow-A in the plasma of rats in the low-dose group has an increasing trend;In the middle and high-dose groups,the C_max and AUC_0→t of the 6components in the plasma of the rats all showed a downward trend,and in the high-dose group,the C_max and AUC_0→t of hydroxysafflor yellow-A,amygdalin,and paeoniflorin,ferulic acid and astragaloside IV were significantly decreased（P<0.05）;The CL of the six components in the plasma of the rats in the low,middle and high dose groups all showed an increasing trend.Conclusion:1.Based on the prediction of network pharmacology,the pharmacodynamic material basis of Buyang Huanwu Decoction for anti-atherosclerosis can provide some theoretical basis for the selection of Q-markers of Buyang Huanwu Decoction for anti-atherosclerosis.Buyang Huanwu decoction may be one of the mechanisms of its anti-atherosclerosis action.2.Eight active ingredients（hydroxysafflor yellow A,amygdalin,albiflorin,paeoniflorin,ferulic acid,astragaloside IV,n-butylidenephthalide,Z-ligustilide）mined in this paper have certain specificity,measurability and pharmacodynamic relevance,which can be used as the Q-markers of Buyang Huanwu Decoction for anti-atherosclerosis and the whole process quality of Buyang Huanwu Decoction for establishment Use of control system.3.The established method for the determination of 8 main anti-atherosclerotic Q-markers in the plasma of Buyang Huanwu Decoction and Buyang Huanwu decoction has good stability,which provides a reliable method basis for the determination of the content of Buyang Huanwu Decoction and the study of pharmacokinetics in vivo.4.In this study,a stable LPS induced chronic inflammation model was established and used to study the pharmacokinetics and pharmacodynamics of Buyang Huanwu Decoction chronic inflammation rats.Through the analysis of the main pharmacokinetic parameters,we found that chronic inflammation has a certain influence on the pharmacokinetic behavior of the main effective components of Buyang Huanwu Decoction in anti-atherosclerosis.It may be that chronic inflammation changes the regulation and function of some drug transporters and drug metabolizing enzymes in the body,causing changes in the pharmacokinetic process of the effective components.At the same time,a large number of studies have shown that atherosclerosis is a chronic inflammatory disease.This study preliminarily clarified that Buyang Huanwu Decoction can interfere with lipopolysaccharide induced chronic inflammation and has a better anti-inflammatory and anti-oxidation effect,which may be one of the mechanisms of Buyang Huanwu Decoction in anti-atherosclerosis.