| Millettia Nitida Benth.var.Hirsutissima Z.Wei is a traditional Chinese medicine used in the south of China,and its medicinal part is dry rattan stem.It produced in Jiangxi,Fujian,Guangdong,Guangxi,Hunan.And has the functions of activating blood,replenishing blood and clearing collaterals,mainly treating numbness of limbs,paralysis,waist and knee pain,menoxenia and paralysis in clinical practice.The main ingredients are flavonoids,which play a biological activity role and have a variety of pharmacological effects such as anti-inflammatory and immune regulation.In the long history of drug use,although the effectiveness and reliability of Millettia Nitida Benth.var.Hirsutissima Z.Wei has been fully verified,the modern research is very scarce.In order to promote the modernization of this variety and obtain new effective anti-rheumatism drugs,it is very necessary to study the basis of anti-rheumatism active substance and its mechanism of Millettia Nitida Benth.var.Hirsutissima Z.Wei.ObjectiveThe chemical constituents of dichloromethane extract from Millettia Nitida Benth.var.Hirsutissima Z.Wei were identified by ultra-high performance liquid chromatographyquadrupole-time-of-flight mass spectrometry,and to investigate the pharmacodynamic effects of anti-rheumatoid arthritis,to elucidate the material basis of anti-rheumatoid arthritis effects and the anti-inflammatory mechanism of anti-rheumatoid arthritis.In vitro cell model was used to observe the effect of seven monomer compounds in Millettia Nitida Benth.var.Hirsutissima Z.Wei on the proliferation of human rheumatoid arthritis synovial cells.Methods1.The chemical constituents of dichloromethane extract from Millettia Nitida Benth.var.Hirsutissima Z.Wei were identified by UPLC-Q-TOF-MS-MS spectrometry,and the main chemical constituents in this part were identified.2.In vivo experiment:The CIA rat model of bovine type ? collagen induced arthritis was established,sixty SD rats were randomly divided into 6 groups.Blank group,model group,methotrexate group,Millettia Nitida Benth.var.Hirsutissima Z.Wei dichloromethane high dose group(220mg/kg),middle dose group(110mg/kg),low dose group(55mg/kg),Millettia Nitida Benth.var.Hirsutissima Z.Wei administration group was given intragastrically for 35 days.The dose of methotrexate group was 1mg/kg,which was given to the stomach once a week,but the model control group and the normal control group were not given special drug treatment.During the treatment,the activity status,AI index,weight change and toe swelling of all rats were observed and recorded weekly.After administration,blood was taken from abdominal aorta of rats in each group,serum was collected,thymus and spleen of rats were called major rats,and ankle joint and liver tissue of hind limbs of rats were fixed in 4% paraformaldehyde.The contents of IL-1? and TNF-? in serum of rats in each group were determined by ELISA,and the pathological sections of joint and liver were observed.3.In vitro experiment:through experiments in vitro isolated from Millettia Nitida Benth.var.Hirsutissima Z.Wei part of the compound of RA-FLS proliferation of synovial membrane cells,with different concentrations of seven monomer compounds,Millettia Nitida Benth.var.Hirsutissima Z.Wei dichloromethane extract and methotrexate role respectively in RA-FLS in vitro culture,The proliferation of the cells was detected by CCK-8 colorimetric assay,and the activity of anti-rheumatoid arthritis was evaluated.Result1.In this study,the UPLC-Q-TOF-MS-MS method was used.According to the precise molecular weight and molecular formula of the compound,combined with the second-order mass spectrometry data,referring to the mass spectrometry pyrolysis law of the reference substance,under the guidance of references,34 compounds were identified from dichloromethane extract Millettia Nitida Benth.var.Hirsutissima Z.Wei.Among them,twenty six flavonoids,three organic acid,one coumarin,two anthraquinone,one triterpene and one steroid.2.CIA rats with bovine type ? collagen and incomplete freund's adjuvant began to develop joint redness and swelling on the 3rd day after booster immunization.the AI index score and toe swelling were observed.there were significant differences between the blank group and the model group.It shows that the model is established successfully.3.In the pathogenesis of CIA rats,the expression of pro-inflammatory factors was significantly increased,and the expression levels of inflammatory factors IL-1? and TNF-? in serum of the model group were significantly higher than those in the blank group(P < 0.01),and the expression of pro-inflammatory factors in the model group was significantly higher than that in the blank control group(P < 0.01).Dichloromethane extract of Millettia Nitida Benth.var.Hirsutissima Z.Wei can down-regulate the secretion of IL-1? and TNF-?.The inhibitory effect of 220mg/kg on IL-1? and TNF-? was similar to that of MTX,but low dose could decrease the expression of inflammatory factors,but there was no significant difference.4.The dichloromethane extract of Millettia Nitida Benth.var.Hirsutissima Z.Wei has therapeutic effects on the condition of CIA rats,significantly inhibiting the swelling of rat toes and reducing inflammatory reactions such as synovial infiltration and fibrous hyperplasia of joints.And can restore its immunity ability to a certain extent.It is a potential anti-rheumatic effective fraction and can restore its immune ability to a certain extent.There is no obvious abnormality in liver pathology of CIA model rats and normal rats.5.The anti-rheumatoid arthritis activity of some monomer compounds isolated from Millettia Nitida Benth.var.Hirsutissima Z.Wei(formononetin,genistein,ononin,calycosin,sissotrin,daidzein,epiplupeol)and extract were tested.The results showed that the cell proliferation rate of the positive control group was lower than that of the blank group and the difference was statistically significant(P<0.01),which proved that methotrexate as a positive control drug met the requirements of this experiment.Except epiplupeol compounds,the proliferation of RA-FLS cells was inhibited to a certain extent(P< 0.05 or P<0.01).Among them,the IC50 values of dichloromethane extract,formononetin,genistein,and ononin were small,which had a significant inhibitory effect on the proliferation of synovial cells,suggesting a good anti-RA activity.However,the IC50 values of daidzein,calycosin and sissotrin were above the maximum administration concentration.Within the concentration range set in this experiment,the inhibitory effect of genistein on synovial cell proliferation increased with the increase of drug concentration in a dose-dependent manner.Among the monomer compounds,genistein has the best anti-RA effect(with higher cell proliferation inhibition effect and lower IC50 value).ConclusionDichloromethane in Millettia Nitida Benth.var.Hirsutissima Z.Wei has a good anti-rheumatoid arthritis effect in vivo.It plays an important role by inhibiting the expression of pro-inflammatory factors TNF-? and IL-1? in the serum of CIA rats.It is a potential candidate drug for anti-rheumatoid arthritis.The effective components that inhibit the proliferation of RA-FLS cells in Millettia Nitida Benth.var.Hirsutissima Z.Wei are flavonoids,The main active components are formononetin,genistein,ononin,calycosin,sissotrin,daidzein.Inhibition of synovial cell proliferation is also one of the ways to treat rheumatoid arthritis. |
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