| Rheumatoid arthritis(RA)belongs to the category of Bi syndrome in traditional Chinese medicine.At present,compound drugs occupy the mainstream market of RA treatment,but the safety and tolerance caused by long-term medication have become increasingly concerned issues.In contrast,traditional Chinese medicine with multi-target,multi-effect and small toxic effect has unique advantages in the treatment of RA,and is more suitable for the characteristics of RA patients who need long-term medication.Different from Western medicine,traditional Chinese medicine emphasizes dialectical legislative prescription selection and comprehensive consideration of patients’ clinical symptoms.Huangqin Qingre Chubi capsule(HQC)was made by combining a certain proportion of Radix scutellariae,Fructus gardeniae,Clematis chinensis Osbeck,Semen Coicis and Persicae semen according to the principle of the king,minister and other agents.The whole formula has the effect of clearing heat and dispelling dampness.It is often used in the treatment of RA rheumatic heat and bi syndrome.At present,studies based on HQC mainly focus on pharmacodynamic evaluation and mechanism of action of whole prescription,and lack of exploration of pharmacodynamic material basis related to clinical efficacy,and clinical efficacy is difficult to be well guaranteed.Therefore,it is urgent to establish a set of more scientific quality standards according to the characteristics of multi-component HQC,clarify the pharmacodynamic material basis of HQC to relieve RA heat syndrome,and then reveal the pharmacodynamic mechanism.Based on the principles of effectiveness,scalability,specificity and transmissibility of Quality marker(Q-Marker),this study integrated network pharmacology and pharmacokinetics to explore the dynamic quantization law and metabolic pathway of potential Q-Marker in HQC in rats with rheumatic heat bi syndrome and RA patients.Screening Q-Marker for HQC treatment of RA.1 ObjectiveIn this study,HQC was selected as the research object,and potential Q-Marker of anti-RA of HQC was screened based on network pharmacology.At the same time,the qualitative and quantitative analysis of chemical components in vitro was carried out based on potential Q-Marker.Secondly,RA patients were selected as research objects to simulate the clinical characteristics of rheumatic heat Bi syndrome,establish a rat model of rheumatic heat Bi syndrome(CIA),study the in vivo metabolic characteristics and pharmacokinetic characteristics of the active components of HQC,and initially reveal the blood-entering components in the compound.Clarify Q-Marker.2 MethodsThe relationship between HQC and RA was analyzed by network pharmacology.Through literature review and TCMSP,the chemical composition database of HQC five traditional Chinese medicine Scutellaria baicalensis,gardenia,Weilingxian,peach seed and Coix seed was established.Pharm Mpaper database predicts compound targets.Gene Cards and other databases were used to search and construct RA disease target database.The intersection targets of RA and HQC were mapped using Wayne online network and the STRING database was uploaded to analyze the interaction between proteins.The intersection targets were enriched by the GO and KEGG pathways using the Metascape platform.Finally,the active ingredient-target-pathway network was constructed by sorting and sequencing the top 20 pathways,and the targets,pathways and biological processes of the main components were predicted.To establish a quantitative analysis method for HQC multi-component Ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)spectrometry,Verify the results of HQC network pharmacological analysis.The effective components of HQC were extracted and separated by ultrasonic and Soxhlet extraction.The active components of HQC were quantified by UHPLC-MS/MS.The CIA rat model of rheumatic heat obstruction syndrome was established by injecting chicken type Ⅱ collagen emulsion into SD rats and giving them rheumatic heat environmental stimulation.The body weight,hair color and mental state of the rats were recorded.The synovial histopathology was performed to evaluate the degree of joint injury.The surface temperature of rat joints was measured by infrared thermography,and the parameters of hemorheology were measured by hemorheology.After the successful establishment of the model,the clinical equivalent dose of HQC was administered by single intragastric administration.After blood collection of fundus venous plexus and sample pretreatment,the contents of five components at different sampling time points were determined by UHPLC-MS/MS.An IDA-UHPLC-ESI-MS/MS method was established to detect the metabolites of baicalein and gardeniside in HQC in plasma and different tissues(heart,liver,spleen,lung,kidney,brain)of CIA rats with rheumatic heat bi syndrome.3 Results3.1 Pharmacodynamic substance basis and mechanism of HQC treatment for RA predicted by network pharmacologyThrough network pharmacology and text mining,a total of 95 active ingredients and 140 intersection targets were screened for each single drug by HQC.The "active ingredient-target-pathway" network predicted that HQC could achieve the therapeutic effect of RA mainly through the regulation of sphingolipid signal pathways by components such as geniposide,luteolin,amygdalin,baicalin and Coixol.3.2 Establishment of a method for in vitro analysis of HQC active ingredientsThe potential Q-Marker of HQC was quantified by UHPLC/MS/MS.Gniposide,coixol,luteolin,baicalin and amygdalin were extracted by ultrasonic method.The method of in vitro determination of HQC active ingredients conforms to the methodological requirements and reflects the testability principle of Q-Marker.3.3 Pharmacokinetics to study the dynamic changes of HQC active ingredients in vivoThe evaluation indexes of CIA rat model of rheumatic heat Bi syndrome showed that erythrocyte aggregation was enhanced,the joint surface temperature of rats gradually increased during immunization and reached a peak on day 21,and the whole blood viscosity gradually increased under the condition of low shear rate,which confirmed the successful production of CIA model of rheumatic heat Bi syndrome in line with the clinical symptoms and pharmacodynamic substance screening of RA.The pharmacokinetic experiment showed that the analysis method successfully determined the contents of five components in vivo.The blood concentration of baicalin and the area under the curve of drug duration were the largest,showing a bipeak phenomenon,reflecting the important role of its royal medicine.The blood concentration of geniposide was second only to baicalin,luteolin was absorbed rapidly and showed a bipeak phenomenon,and the content of amygdalin was low,which clarified the rationality of the compatibility of the prescriptions.These results suggest that these 5 components may be used as Q-markers for RA resistance of HQC.3.4 IDA model was used to study in vivo metabolites of HQC active componentsSeven metabolites were found in rat plasma and tissue(heart,liver,spleen,lung,kidney,brain),which were successively named as M 1,M 2,M 3,M 4,M 5,M 6,M7.The structures of the unknown metabolites were identified and speculated respectively.M 1,M 2 were detected in plasma.M 3,M 4,M 5,M 6,M 7;M1,M2,M5 and M7 were detected in the heart.M1,M4 and M5 were detected in the liver.M1,M2 and M5 were detected in the spleen.M1,M5 and M7 were detected in the lungs.M1,M2,M4,M5 and M7 were detected in the kidney.M1 and M7 were detected in the brain tissue.4 ConclusionFive active ingredients were preliminatively screened by network pharmacology as potential Q-markers for anti-Ra of HQC.Five active components in HQC were quantified by UHPLC-MS/MS.A CIA rat model stimulated by rheumatic heat environment(FSR)was established,and the dynamic quantification rules and metabolic pathways of five prototype components in rats were elucidated.It was confirmed that baicalin,luteolin,coixol,amygdalin and geniposide could be Q-markers for HQC treatment of RA from the aspects of measurability,specificity,effectiveness and transmissibility. |
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