Chemical Constitutes Of Erycibe Schmidtii And Their Inhibition On T Cell Proliferation And NO Releasing Of RAW 264.7 Cells

Erycibe schmidtii Craib belongs to Convolvulaeeae. It is a folk traditional Chinese medicine, possessing the efficacy of sweating, dehumidification, and alleviating tumidness and pain, etc. Clinically, it is used to treat primary glaucoma, rheumatic pain,and hemiplegia, etc. The vine part of E. schmidtii is one of the original plants of the traditional Chinese medicine “Ding Gong Teng”, which is used to treat rheumatoid arthritis. However, the biological constitutents and mechanisms of E. schmidtii to treat RA have not been clarified clearly. Up to now, only 7 compounds were isolated from E.schmidtii. Meanwhile, the anti-RA constituents were only restricted to scopoletin and scopolin, which suppress adjuvant-induced rat arthritis by inhibiting inflammation and angiogenesis. Therefore, a systematic research on the chemical constituents of of E.schmidtii has been attempted in this thesis. Since the abnormal proliferation of T cell plays a very important role in the pathogenesis of RA, accompanied by the generation of inflammation, the inhibition on T cell proliferation and NO releasing of RAW 264.7cells have been screened.ObjectiveTo investigate the chemical constitutes of E. schmidtii and their inhibition on T cell proliferation and NO releasing of RAW 264.7 cells. The vine part of E. schmidtii was extracted with 95% ethanol for 2 hours at reflux temperature to give a residue, which was suspended in H2 O, extracted with petroleum ether, Et OAc and n-Bu OH,successively. The systematic chemical constituent research on Et OAc and n-Bu OH parts has been carried out. Then, in order to explore the anti-RA constituents, Inhibiting on NO releasing of RAW 264.7 cells and T cell proliferation were tested on some of the extracts and isolated compounds.Materials and MethodsRepeated silica gel and Sephadex LH-20 column chromatography, macroporous adsorption resin and semipreparative HPLC were used to separate and purify compounds. Their structures were determined by MS, 1H-NMR, and 13C-NMR spectral methods. Relative yield of NO was used to test the inhibiting on NO releasing of RAW264.7 cells of some of the isolated compounds. The level of T cell proliferation against crude extracts and some of the pure compounds was determined by flow cytometry analysis. Human T cells were stimulated by anti-CD3/anti-CD28 monoclonal antibodies.ResultsFifteen compounds were isolated and identified as scopoletin(1), scopolin(2),chlorogenic acid(3), chlorogenic acid methyl ester(4), 4-O-caffeoylquinic acid(5),4-O-caffeoylquinic acid methyl ester(6), 4,5-di-O-caffeoylquinic acid(7),4,5-di-O-caffeoylquinic acid methyl ester(8), 3,5-di-O-caffeoylquinic acid(9),3,5-di-O-caffeoylquinic acid methyl ester(10), 3,4-di-O-caffeoylquinic acid(11), and3,4-di-O-caffeoylquinic acid methyl ester(12), caffeic acid(13),N-trans-p-hydroxyphenethyl ferolamine(14), and β-sitosterol(15). The T cell proliferation rate against Et OAc extract was 70.7% at the concentration of 20 μg/m L.The inhibition on T cell proliferation activity of compounds 1 ~ 3, 5, 6, 8, 10, 11, 13 and14 was further studied. The T cell proliferation rate of compound 14 was 77.5%(2μmol/L) and 66.8%(20 μmol/L), respectively. Other compounds did not show obvious inhibition. The anti-inflammatory experiment of compounds 1 ~ 3, 5, 6, 8, 10, 11, 13 and 14 was done by testing the NO releasing of RAW 264.7 cells. Their NO relative yield were 98.5%, 117.4%, 115.6%, 91.5%, 91.5%, 112.9%, 102.4%, 96.0%, 81.7%,and 76.1%, respectively. Compounds 13 and 14 showed some inhibitory activity of NO releasing.ConclusionThis study showed that E. schmidtii is also a rich souce of chlorogenic acid derivatives, besides scopoletin and scopolin. Compounds 5 ~ 7, 9, 11 and 12 were obtained from this genus for the first time, while compounds 4 ~ 14 were separated from this plant firstly. Et OAc extract and compound 14 exhibited certain inhibition activity on T cell proliferation. Compounds 13 and 14 showed some inhibitory activity on NO releasing of RAW 264.7 cells.