Study On Chemical Constituents And Pharmacological Action Of Tinpspora Sinensis(lour.)Merr.

Tinpspora sinensis(Lour.)Merr.belonging to the Tinospora Miers of Menispermaceae plants,is distributed in Guangdong,Hainan,Guangxi,Yunnan,Qinghai and other places.T.sinensis is a traditional national medicine,uesd by Tibetan,Dai,Achang,Vanguard,Jingpo,Wa,Miao,Hani,Maonan,Mulam,Yao and Zhuang.It is used for the treatment of swelling and pain,bruises sprains and rheumatism.We have made a general survey of chemical constituents of T.sinensis,and found that more than 100 kinds of known chemical constituents of T.sinensis have been found,including terpenoids,flavonoids,alkaloids,phenylpropanoids,phenolic glycosides by refering the literature.Modern pharmacological studies have confirmed that T.sinensis has the activities of anti-inflammatory analgesic,anti-leishman,anti-oxidative,anti-radiation,protecting liver,regulating immunity,preventing anemia caused by cyclophosphamide and other pharmacological effects.In this study,we focuse on the chemical constituents and pharmacological effects of the T.sinensis,in order to enrich the chemical constituents and explore their pharmacological effects,and provide a theoretical basis for the development of new drugs for rheumatoid arthritis.This thesis is divided into two parts.Part I: Study on the Chemical Constituents of the T.sinensis Silica gel column chromatography,Sephadex LH-20 column chromatography,MCI column chromatography,HPLC separation methods were used,along with the UV,MS,NMR,CD identification methods,for 95% ethanol extract of stem of T.sinensis ethyl acetate extraction.25 compounds were identified from T.sinensis,containing six flavonoids compounds,four phenylalanine compounds,three terpenoids compounds,seven amide compounds,and five aromatic compounds.Six flavonoids compounds were quercetin,rutin,isorhamnetin,Narcissoside,Nicotiflorin,Tamarixetin 3-O-rutinoside.Four phenylalanine compounds were syringaresinol-O-?-D-glucopyranoside,4,4'-dimethoxy-3'-hydroxy-7,9':7,9'-diepoxylignan-3-O-?-D-glucopyranoside,7a-[(?-D-glucopyranosyl)oxy] isolariciresinol,Tinosinen.Three terpenoids compounds were Tinosineside B,Tinocordiside,tinosinenside A.Seven amide compounds were cyclo-(Val-Tyr),cyclo-(Ile-Ala),tyrosine,5-(1-methylpropyl)-3,6-dioxo-2-piperazineacetic acid,N-acetyl-1-ethyl ester glutamic acid,cyclo-(Ala-Pro),proline.Five phenolic glycosides compounds were benzyl-O-?-D-glucopyranoside,(2-hydroxyphenyl)methyl ?-D-Glucopyranoside,Tachioside,3-ethoxy-4-methoxyphenol-?-Dglucopyranoside,Salidroside.Tinosinenside A,3-ethoxy-4-methoxyphenol-?-D-glucopyranoside were the new compounds,and Narcissoside,Nicotiflorin,Tamarixetin 3-O-rutinoside,cyclo-(Val-Tyr),cyclo-(Ile-Ala),tyrosine,5-(1-methylpropyl)-3,6-dioxo-2-piperazineacetic acid,N-acetyl-1-ethyl ester glutamic acid,cyclo-(Ala-Pro),proline,benzyl-O-?-D-glucopyranoside,(2-hydroxyphenyl)methyl ?-D-Glucopyranoside,Tachioside and Salidroside were isolated from T.sinensis for the first time.N-acetyl-1-ethyl ester glutamic acid was isolated for the first time from natural products.Part ?: the Pharmacodynamics Study of the T.sinensisA collagen-induced rat rheumatoid arthritis(CIA)model was established to study the pharmacological effects of T.sinensis on the treatment of rheumatoid arthritis.In the first place,the therapeutic effect of T.sinensis on joint inflammation was evaluated using rat arthritis index,and mechanical pain threshold.The arthritis index refers to the naked eye observation of rats after foot joint swelling,and the arthritis index score of each joint judge the rats foot swelling.The degree of foot swelling is the volume measurement of the ankle joint below the ankle joint by using the Paw Volume Meter,and the foot swelling is judged by the swelling rate of hind paw.Mechanical pain threshold refers to the use of von-Frey fiber stimulation of rat paw,the numerical paw withdrawal is the pain threshold.The results show that T.sinensis can effectively alleviate the symptoms of clinical arthritis,including the relief of joint redness,foot swelling and the pain.Secondly,the effect of T.sinensis on the inflammation of rheumatoid arthritis was observed by Micro-PET imaging.Rats were injected with a short-lived radioactive 18F-deoxyglucose(18F-FDG),and the rheumatoid arthritis treatment in rats was observed by its metabolic accumulation at the joints and by SUV values.The experimental results showed that compared with the control group,the SUV value at the rat joints of T.sinensis was significantly reduced,that is,T.sinensis had a certain therapeutic effect on the inflammation of rheumatoid arthritis.After that,HE staining was used to observe the effect of T.sinensis on histopathology in the treatment of rheumatoid arthritis.We stained the hind leg of the rat and observed whether there were synovial cell proliferation,inflammatory cell infiltration,and destruction of cartilage and bone tissue.The experimental results showed that T.sinensis relieves the proliferation and inflammatory infiltration of synovial cells in CIA rats.Finally,the molecular level of rheumatoid arthritis was studied by ELISA and Western blot.The levels of TNF-?,IL-1? and IL-17 in serum were detected by ELISA.In the literature,there are inextricable links between the three inflammatory factors and rheumatoid arthritis.Western blot was used to detect the phosphorylation levels of key proteins ERK and p38 in the MAPK signaling pathway.The results showed that the treatment of rheumatoid arthritis could be achieved by inhibiting the release of proinflammatory cytokines TNF-?,IL-1 and IL-17,and effectively inhibiting the expression of ERK and p38 phosphorylation in articular tissues.