| Rhamnella gilgitica Mansf.Et melch.(?????????)is mainly distributed in Chayu and Mangkang of Tibet,northwest of Yunnan and southwest of Sichuan,of which the dried heartwood for medicine,calling “song sheng deng”,“sheng deng”.Jingzhu Materia Medica and Dictionary of Chinese Ethnic Medicine record that R.gilgitica.has the effect of eliminating rheumatism,drying huang shui,and is mostly used to treat RA and huang shui disease.It has been collected in Standards Issued by the Ministry.Modern research has found that R.gilgitica.has a significant therapeutic effect on RA,however,the material basis of its efficacy remains unkown,which limits the further clinical development and research of this medicine.Rheumatoid arthritis(RA)is a common systemic autoimmune disease,which seriously affects people’s health.RA is closely related to the accumulation of “huang shui” in the articular cavity of the body.Tibetan medicine regulates the balance of “three factors” by intervening “huang shui”.Therefore,with the guidance of traditional medicine theory and biological activity,this paper takes R.gilgitica.as the research object,discussing its anti-inflammatory activities,the pharmacodynamic evaluation of anti RA and the chemical constituents of its active part,and the active constituents to clarify the material basis of anti RA of R.gilgitica.,and provide basis for clinical application and development of preparations.Objectives 1.To investigate the anti-inflammatory effect of the four parts of R.gilgitica.in vitro.2.To evaluate the pharmacological effect of the ethyl acetate part of R.gilgitica.on RA model rats.3.To analyze the chemical constituents of ethyl acetate in R.gilgitica.4.To predict the active constituents of R.gilgitica.Methods 1.MTT method was used to investigate the inhibitory effects of petroleum ether,ethyl acetate,n-butanol and water parts of R.gilgitica.on the growth of RAW264.7 cells.LPS stimulated RAW264.7 cells to induce inflammation in vitro,and Griess method was used to detect NO release from different parts of R.gilgitica.2.The rat model of collagen induced arthritis(CIA)was established using bovine type II collagen and complete Freund’s adjuvant,and the extract of ethyl acetate(9.71,19.43,38.85 mg/kg)from R.gilgitica.The therapeutic effect of R.gilgitica.on CIA model rats was studied by evaluating the body weight,paw swelling,arthritis index,immune organ index,knee joint histopathology,serum cytokines expression and other indicators.3.UPLC-Q-TOF-MS technology was used to determine the chemical constituents of ethyl acetate in R.gilgitica.Data processing was carried out using unify software and other methods.Combined with the structure of literature reported,the information of mass spectrum fragments was speculated and matched to realize the rapid identification of the chemical constituents of ethyl acetate in R.gilgitica.4.The ethyl acetate extract of R.gilgitica.was separated and purified by silica gel column chromatography,Sephadex LH-20 column chromatography,RP-18 and preparative HPLC.The structure of the monomer was identified by NMR and MS.5.The network pharmacology technology was used to predict the active constituents,molecular functions,biological processes and pathways of anti-RA in R.gilgitica.,and to construct a “chemical constituents-target-pathway” network,and to analyze the potential active constituents and targets of anti-RA in R.gilgitica.6.Molecular docking technology was used to predict the interaction between 9 key active constituents and 9 key target proteins in the network,and further analysis and verification were carried out in combination with literature.Results 1.The extracts of R.gilgitica.showed anti-inflammatory activity,among which ethyl acetate and petroleum ether have the strongest anti-inflammatory activity,and the order of activity is petroleum ether ≈ ethyl acetate > n-butanol > water.2.The ethyl acetate extract of R.gilgitica.can restore the body weight of rats with CIA,reduce the arthritis index and paw swelling of rats,decrease the thymus index and spleen index of CIA rats,significantly reduce the inflammatory cell infiltration and synovial cell proliferation,inhibit the expression of IL-1 β,IL-6,IL-17,TNF-α and INF-γ cytokines in serum,and increase the expression of IL-4 and IL-10.3.By using unify software and comparing with literature,37 compounds were identified from the ethyl acetate extract of R.gilgitica.,including 26 flavonoids,6 saponins,3 phenolic acids and 2 fatty acids.4.14 compounds were isolated and identified from the ethyl acetate extract of R.gilgitica.: p-hydroxybenzoic acid(1),(-)-lyoniresinol(2),syringic acid(3),catechin(4),(-)-epicatechin(5),tristin(6),1,2-bis(4-hydroxyphenyl)ethane(7),bis(2-ethylhexyl)phthalate(8),naringenin(9),kaempferol(10),rhamnocitrin(11),apigenin(12),(+)-syringaresinol(13),episyringaresinol(14),(+)-medioresinol(15),compounds 1 ~ 8,11 ~ 15 were isolated from the plant for the first time.5.The results of the network pharmacology technology showed that 19 main constituents of R.gilgitica.may act on SRC,MTOR,IGF1 R,EGFR,MAPK8 and other key targets.262 items were enriched by GO function(P < 0.05),and 70 signal pathways were obtained by KEGG pathway enrichment and screening(P < 0.05).6.The results of molecular docking showed that the core constituents such as apigenin,rhamnocitrin,naringenin,quercetin had the strongest binding force with the target of IGF1R、AR、MAPK8.Conclusions 1.Clarified the anti-inflammatory active part and chemical constituent composition of R.gilgitica,Preliminarily clarified the material basis of anti-RA of R.gilgitica.,and provided the scientific basis for more effective evaluation of the quality and clinical rational use of the drug.2.Revealed the effect of anti-RA might be related to the down regulation of IL-1β,TNF-α,IL-6,IL-17 and up regulation of IL-4 and IL-10.3.Clarified the treatment of RA of R.gilgitica.might be related to the synergistic effect of “chemical constituents-target-pathway”,and related to naringin,quercetin,kaempferol,1,2-bis(4-hydroxyphenyl)ethane,tristin and other active constituents,and provided scientific reference for clinical research. |
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