Research Of The Immunosuppressive Activity Alkaloids From Kopsia Officinalis

Kopsia officinalis Tsiang et P.T.Li is native to southern part of Yunnan Province,and has long been used as a folk medicine for the treatment of rheumatoid arthritis(RA),leprosy, pharyngitis and edema in Sipsongpanna. Various alkaloids have been isolated from K. officinalis, and the bygone research mainly focused on their bioactivity of anti-resistance, anti-tumor, analgesia and hepatoprotection, et al. We made an intensive study of the alkaloids and their immunosuppressive activity of the K. officinalis.ObjectiveTo investigate the alkaloid constituents from the branches and leaves of the K. officinalis, then test their immunosuppressive activity.Materials and MethodsThe crude alkaloid extract of K. officinalis was isolated and purified by repeated column chromatography(CC), thin-layer chromatography(TLC) and high performance liquid chromatography(HPLC) using various materials including positive phase silica gel, reverse phase silica gel, amino chemically bonded silica gel and Sephadex LH-20.The structures of the isolated compounds were elucidated by UV-VIS, IR, NMR and MS spectra. The immunosuppressive activity was tested against human T cell proliferation stimulated by anti-CD3/anti-CD28 monoclonal antibodies.ResultsAs a result, 30 compounds were extracted of K. officinalis, including 9 new and 21 known alkaloids. Eight alkaloids were extracted from K. officinalis for the first time.The immunosuppressive activity was tested against human T cell proliferation stimulated by anti-CD3/anti-CD28 monoclonal antibodies. Some of the compounds showed moderate immunosuppressive activity, Rhazinilam(25) significantly inhibited T cell proliferation. The proliferation rate of T cell decreased from 100% to 25.33% in the concentration of 1 μmol/L. The proliferation rate further dropped to 0.77% when the concentration was 10 μmol/L. The proliferation of T cell was significantly inhibited by25, IC50 = 0.83 ± 0.09 μmol/L(n= 3). It is worth mentioning that 25 showed no obvious cytotoxicity to resting T cell at 200 μmol/L.ConclusionThe obtained compounds include Aspidofractinine, Fruticosine, Kopsine,Aspidospermine, Pleiocarpamine and Isokopsin alkaloids. Nine new monoterpenoid indole alkaloids were isolated and identified as N(4)-methylkopsinine(14), 2-carboxyl-methylchanofruticosinate(16), 16,22-dihydroxykopsan(17), 12-methoxy-16,22-dihydr-oxykopsan(18), 11,12-methylenedioxy-16,22-dihydroxykopsan(19), 2-carboxyl-11,12-methylenedioxy-methylchanofruticosinate(20), 2-carboxyl-12-methoxymethylchano-fruticosinate(21), N(4)- methylkopsininate(24), 10-methoxy-demethoxycarbonyl-isok-opsin(29). Along with 21 known monoterpenoid indole alkaloids: methyldemethoxy-carbonylchanofruticosinate(1), kopsinic acid(2), methyl chanofruticosinate(3),(-)-11,12-methylenedioxykopsinaline(4), kopsinine(5),(-)-N-methoxycarbonyl-11,12-methylenedioxykopsinaline(6), 5-Oxokopsinic acid(7), 5,18-dioxokopsan(8),kopsinilam(9),(-)-N-methoxycarbonyl- 12-methoxykopsinaline(10), N-carbomethoxy-11-hydroxy-12-methoxykopsinaline(11), kopsinine N-oxide(12), 12-methoxykopsinali-ne(13), 11,12-methylenedioxykopsinaline N-oxide(15), 11,12-methylenedioxychano-fruticosinate(22), 12-methoxychanofruticosinate(23), rhazinilam(25), kopsinaline(26), 11,12-methylenedioxy-N-decarbomethoxychanofruticosinate(27), Demethoxycar-bonyl-isokopsin(28), pleiocarpamine methochloride(30).Some of the compounds showed moderate immunosuppressive activity, Rhazinilam(25) significantly inhibited T cell proliferation(IC50 = 0.83 ± 0.09 μmol/L, n = 3), and showed no obvious cytotoxicity. This fact revealed that rhazinilam(25) selectively inhibited T-cell proliferation, and it could be used as a lead compound to develop a new immunosuppressant.