Quality Control Of Zhuang Medicine Bawei Longzuan Granules And Its Mechanisms In The Treatment Of Rheumatoid Arthritis

Rheumatoid arthritis（RA）is an autoimmune disease characterized by swelling,pain and stiffness in joints.Bawei Longzuan Granule（BLG）,a Zhuang medicine compound preparation developed on the basis of Longzuan Tongbi Decoction,is mainly used for the treatment of RA.At present,the chemical composition of BLG is not clear and lack of anti-RA pharmacodynamic evaluation and mechanism research.This topic uses UPLC-Q-TOF-MS technology to identify its chemical composition,HPLC-DAD technique to determine the content of main active components,and¹H-NMR metabolomics method to explore the mechanism of BLG in treatment of the RA.This provides a reference for quality control and clinical application of BLG.Purpose:1.Identifying the chemical composition and determining 4 active ingredients content in BLG to promote of its quality control.2.Evaluating the pharmacodynamic effect and mechanism of BLG in the treatment of RA rats.Methods:1.Ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry（UPLC-Q-TOF-MS）technology was used to identify the chemical constituents of BLG and high performance liquid chromatography-diode array detector（HPLC-DAD）technique was applied to determine the content of 4 active ingredients.2.The collagen-induced arthritis（CIA）model was induced by using bovine type II collagen and complete Freund’s adjuvant.The rats received oral administration of BLG（1.25,2.5 and 5 g/kg）for 30 days,and then various indicators were determined to evaluate its anti-arthritic activity,including body weight,paw swelling,arthritic score,spleen index and thymus index as well as knee joint pathology.Additionally,ELISA kits were used to measure the serum levels of tumor necrosis factor（TNF）-α,interferon（IFN）-γ,interleukin（IL）-1β,IL-6,IL-4 and IL-10 to explore the anti-inflammatory effect of BLG in CIA rats.3.The ¹H-NMR metabolomics combined with multivariate statistical analysis was used to systematically analyze the serum in normal group,CIA group and BLG high-dose group（BLG-H）to determine the potential differential metabolites closely related to RA and the regulation role of BLG.Then through the analysis of metabolic pathway network,the mechanism of BLG in treatment of the RA was revealed.Results:1.A total of 20 chemical constituents were identified from BLG,including coclaurine,magnoflorine,sinomenine,magnocurarine,tetrahydropalmatine and so on.The average content of sinomenine,magnoflorine,hesperidin and toddalolactone in BLG was 6.054,4.866,2.436 and 2.102 mg·g^-1,respectively.2.Compared with the normal group,the paw swelling increased significantly from the 10th day（P<0.05）in CIA group;the thymus index（P<0.05）and spleen index（P<0.001）increased significantly;the knee joint showed inflammatory cell infiltration and synovial hyperplasia;serum pro-inflammatory cytokines IL-1β,TNF-α,IL-6,IFN-γlevels were significantly increased（P<0.01）,while anti-inflammatory cytokines the level of IL-4 was significantly decreased（P<0.01）.Compared with the CIA group,BLG at middle and high doses（2.5,5 g/kg）significantly inhibited the paw swelling of CIA rats from the 25th day（P<0.05）and20 days（P<0.05）,respectively;middle and high dose can also significantly reduced thymus index（P<0.05）;significantly reduced inflammatory cell infiltration and synovial hyperplasia in CIA rats;and significantly reduced the content of TNF-α,IL-1βand IL-6（P<0.05）,also significantly increased the content of IL-4 and IL-10（P<0.05）in CIA rats serum.3.The metabolomics PLS-DA scores showed that the metabolic profiles in normal group,CIA group and BLG-H group were significantly different,suggesting that BLG interfered with some endogenous metabolism in CIA rats.Compared with the normal group,18 different endogenous metabolites were screened from the serum of CIA group.After the intervention of BLG at high dose（5 g/kg）,the levels of 16abnormal metabolites were significantly reversed,of which 10 metabolites were down-regulated（i.e.,LDL/VLDL,3-hydroxybutyrate,lipids,N-acetyl glycoproteins,O-acetyl glycoproteins,acetoacetate,phosphorylcholine,trimethylamine N-oxide,betaine and formate）,while 6 metabolites were up-regulated（i.e.,lactate,creatine,glucose,alanine,glutamine and lysine）.In this paper,4 potential metabolic pathways（Pathway Impact>0.1）were screened by MetaboAnalyst,namely synthesis and degradation of ketone bodies,alanine,aspartate and glutamate metabolism,glyoxylate and dicarboxylate metabolism,butanoate metabolism.Conclusion:1.BLG mainly contains alkaloids,and a few is flavonoids,lignans and other ingredient.The established HPLC-DAD method was simple,accurate and reproducible.It could be used to determine the content of 4 components in BLG and provide reference for its quality control.2.BLG at middle and high doses（2.5,5 g/kg）effectively attenuated the severity of CIA rats,and high dose had the best effect.BLG can inhibit the inflammatory responses by down-regulating pro-inflammatory（i.e.,IL-1β,TNF-α,IL-6 and IFN-γ）cytokines and up-regulating anti-inflammatory cytokines（i.e.,IL-4 and IL-10）.3.The mechanism in RA treatment with BLG may be closely associated with the improvement of lipid metabolism,glycolysis,amino acid metabolism,ketone body metabolism,inflammatory responses and intestinal flora metabolism.