| Fukeqianjin Capsule is a commonly used proprietary Chinese Gynecological medication produced by Zhuzhou Qianjin Pharmaceutical Co.,Ltd.It has the effects of clearing away heat and dehumidifying and replenishing qi and dissipating phlegm.It is widely used for the treatment of common gynecological diseases such as pelvic inflammatory disease,chronic cervicitis,and endometritis.The current quality Control in Pharmacopoeia of FKQJ is limited to the determination of andrographolide and dehydrated andrographolide,but there is no quality Control standard for other components.This quality Control model is difficult to meet the needs of modernization of traditional Chinese medicine,furthermore,it is not well correlated with clinical efficacy and safety.It is difficult to objectively,comprehensively and accurately evaluate the intrinsic quality of traditional Chinese medicine compound preparations.In order to ensure the stability of clinical efficacy,it is urgent to supplement and improve the quality Control methods associated with its clinical efficacy.At the same time,it provides ideas and reference for the research on the quality Control of other Chinese medicine preparations.The main research contents are as follows:The main chemical constituents of FKQJ Capsule were collected by using network pharmacological method,and the drug-target-disease network was constructed according to its main anti-inflammatory effect.Through the analysis of network topology parameters,64 anti-inflammatory anti-inflammatory targets of FKQJ Capsule were screened,including 37 direct acting targets and 27 indirect targets.FKQJ Capsule is mainly combined with enzymes and proteins to exert anti-inflammatory effects by activating biological processes such as transcription and inhibition of apoptosis of RNA polymerase II promoter,mainly affecting signaling pathways such as TNF,apoptosis,MAPK,and T cell receptors.The results of effector cell enrichment showed that the anti-inflammatory target of FKQJ Capsule was significantly enriched in macrophages,neutrophils and plasma cells(p<0.01).Among them,macrophages are the main anti-inflammatory effector cells,and macrophage infiltration is the main feature of chronic inflammation.Based on the above analysis,the following conclusions can be drawn: FKQJ Capsule has certain advantages in the treatment of chronic inflammation.The main anti-inflammatory effector cells are macrophages.The main targets include PGH2,NOS2 and inflammatory factors such as IL-1?,IL-6 and TNF?.The main pathways include TNF signaling pathway,NOD-like receptor signaling pathway and Toll-like receptor signaling pathway.This result has certain guiding significance for the subsequent research on the quality biological evaluation of FKQJ Capsule.The metabolic markers of FKQJ Capsule were screened by metabonomics technology.RAW264.7 cells of mouse macrophages were used as carriers to establish cell inflammation model by combined stimulation of LPS and IFN-?.The changes of metabolites of mononuclear macrophages RAW264.7 stimulated by inflammation were investigated.The results showed that the metabolic profiles of model group,Control group and model group were distinct.Obviously,Gynecological Qianjin Capsule has a great influence on the metabolic behavior of RAW264.7 cell line derived from mice.FKQJ Capsule has effects on inflammation-related pathways,amino acid metabolism pathways and energy metabolism pathways in vivo.The inflammatory metabolic pathway mainly affects the metabolism of arachidonic acid and affects the prostaglandin markers in arachidonic acid metabolism.It can be seen that its mechanism of action is related to the inhibition of COX enzyme.From the perspective of metabolomics,COX-2 can be used as an important candidate for the bio-evaluation of FKQJ Capsul,and this index is closely related to its anti-inflammatory mechanism.Therefore,from the perspective of the change of endogenous metabolites in vivo,it provides another evidence for the selection of biological evaluation indicators for the quality of Gynecological Qianjin Capsules.The anti-inflammatory biopotency method based on inhibition of COX-2 enzyme activity was optimized for the first time.The effects of the purity and dosage of the relevant reagents in the kit on the experimental results were investigated.The results showed that the background value of the reagents was significantly reduced after purification,which made the results more realistic and accurate.In order to determine the appropriate dosage,the effect of the dosage of substrate,enzyme and cofactor on the inhibition of COX-2 activity was also investigated.Through methodological investigation,it is clear that the method not only has high precision(intraday RSD=5.13%,daytime RSD=6.56%),good repeatability(RSD=7.88%),but also is convenient to e reproduce(RSD=10.15%).It is conducive to meeting the requirements of biological evaluation methods.Taking FKQJ Capsule as an exampleand RAW264.7 as a biodetector,the anti-inflammatory mechanism of Fuke Qianjin Capsule was studied by high-throughput technology of transcriptome digital gene expression profile.The differential gene expression profiles of FKQJ Capsule on LPS combined with IFN-?-induced macrophage injury in mice were compared,and the differential expression genes were screened.The signal pathway was enriched by KEGG.The microarray containing 30 anti-inflammatory index gene primers was used to verify the indicator genes of the chip and the method was applied to evaluate different herbs.In the TNF signaling pathway,FKQJ Capsule mainly plays an anti-inflammatory role by down-regulating the expression of TNF-?,IL-6,COX-2 and iNOS.This further validates the predicted results of network pharmacology and metabolomics.A method for the determination of chlorogenic acid,ferulic acid,genistein,jatrorrhizine,andrographolide,palmatine,berberine,dehydrated andrographolide and ligustilide in FKQJ Capsules by ultra-high performance liquid chromatography(UPLC)was established and the chemical fingerprints were established.The results showed that the chemical fingerprints of 20 batches of FKQJ Capsule were similar,but the contents of 9 active ingredients varied greatly.The coefficients of variation of the components between the batches were 11.34%,19.14%,20.19%,30.79%,18.19%,16.43%,36.88%,29.02%,and 16.84%,respectively.Whether better similarity of the chemical fingerprints means better consistency of biological activity will be discussed in further research.From the anti-inflammatory biopotency of COX-2 inhibition,there are significant correlations between ligustilide and palmatine,suggesting that the higher the content of ligustilide and palmatine,the higher the level of inhibition of COX-2 enzyme activity.It is suggested that ligustilide and palmatine are also effective ingredients for anti-inflammation.Therefore,the detection of ligustilide and palmatine should be considered in chemical composition accusation to ensure the consistency and stability of quality.Aiming at the deficiency of single index component in Controlling the quality of the whole preparation of FKQJ Capsule,a draft quality standard of FKQJ Capsule was preliminarily formulated based on the research of biological evaluation method based on clinical efficacy and mechanism.Compared with the existing quality standards of FKQJ Capsule,Compared with the existing quality standards of FKQJ,the similarity control index of chemical fingerprint was added,and the evaluation method of anti-inflammatory biological titer was added.The new standard has better evaluation power in evaluating the inherent quality and consistency of FKQJ.In summary,this paper takes the bio-evaluation method of FKQJ Capsule as the entry point,and constructs a quality evaluation method related to clinical efficacy and mechanism of action.This method provides a new idea and technical reference for the quality Control and evaluation of other traditional Chinese medicine compound preparations.Certainly,due to the limitation of time and energy,the evaluation method of anti-inflammatory biopotency/activity of FKQJ Capsule still has some defects,which will be further validated and optimized by the research group. |
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