Studies On The Constituents And Bioactivity Of Fresh Clstanche Tubulosa

Cistanche tubulosa (Schenk) Wight was reordered in the 2005 and 2010 Chinese Pharmacopoeia as an alternative of the raw materials of Herba Cistanche, and it is the major raw material of Herba Cistanche at the present time due to its relatively affluent resource. Herba Cistanch had the effect of nourishing the liver and kidney. benefiting essence-blood, relieving constipation, and so on. The people in Xiangjiang which was the main producing area of Herba Cistanch often eat fresh Herba Cistanche in order to improve the health, prolong life, and resist fatigue. In this study, by means of bioassay-guided separation, the chemical constituents and pharmacological activity of the fresh C. tubulosa was studied for the purpose of providing detailed chemical and pharmacological information for the further development of C. tubulosa and its clinical application.Using D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice, the methanol extract of the stems of C. tubulosa was found to inhibit the increase of serum aspartate aminotransaminase (sAST) and alanine aminotransaminase (sALT). Using inhibitory effects on D-GalN-induced cytotoxicity in primary cultured hepatocytes and TNF-α-induced cytotoxicity in L929 cells as a bioassay marker in vitro, the MeOH fraction eluted by Diaion HP-20 column of methanol extract from C. tubulosa was confirmed as the active site. By means of many chromatographic methods, including open column chromatography of silica gel and reversed phase ODS and pre-HPLC, fifty-three compounds were isolated from active site of C tubulosa. On the basis of physico-chemical data and spectral methods, they were identified as kankanosides H1, H2,I. J1, J2. K1, K2, L, M, N, O, P (1-12). echinacoside (13). acteoside (14). isoacteoside (15). cis-acteoside (16).2'-acetylacteoside (17), decaffeoylacteoside (18). tubuloside A (19), cistantubuloside B1, B2 (20.21), arenarioside (22), wiedemanninoside C (23). campneosideⅠ(24), campneosideⅡ(25,26). isocampneosideⅠ(27,28), salidroside (29), syringalide A 3'-α-rhamnopyranoside (30). cistantubuloside A (31). cistanoside F (32). kankanose (33,. (E)-coniferin (34), syringin (35), sinapaldehyde glucoside (36), (-)-pinoresinol O-β-D-glucopyranoside (37), eucommin A (38). isoeucommin A (39). (+)-syringaresinol O-β-D-glucopyranoside (40), bartsioside (41). gluroside (42),6-deoxycatalpol (43), kankanoside A (44). 8-epideoxyloganic acid (45), geniposidic acid (46), mussaenosidic acid (47).8-epiloganic acid (48). (2E,6E)-8-hydroxy-2,6-dimethyl-2.6-octadien-l-yl-O-β-D-glucopyranoside (49). (2E.6E)-3,7-dimethyl-8-hydroxyoctadien-l-yl-O-β-D-glucopyranoside (50). betulalbuside A (51). kankanoside E (52), (2E,6Z)-8-O-β-D-glucopyranosyloxy-2,6-dimethyl-2.6-octadienoic acid (53). Among them, compounds 1-12 were new compounds, and compounds 16,22,23,27,28. 34,36,38,39,49,50 and 51 and compound 45 were obtained for the first time from the Cistanche sp. and from the C. tubulosa. respectively.The inhibitory effects of twenty compounds isolated from C. tululosa on D-GalN-induced cytotoxicity in primary cultured hepatocytes were determined. The results indicated that seventeen phenylethanoid glycosides and one phenylacylated oligosugar had inhibitory activity. Seven phenylethanoid glycosides (echinacoside. acteoside. isoacteoside,2'-acetylacteoside. tubuloside A. tubuloside B and kankanoside G) showed strong activities. Their activities were greater than that of silybin as a positive control. The effects of echinacoside. acteoside and isoacteoside on TNF-a and NO productions from LPS-activated macrophages were determined. Three constituents showed neither TNF-a nor NO production inhibitory activities. The inhibitory effects of twenty-five compounds isolated from C. tululosa on TNF-α-induced cytotoxicity in L929 cells were examined. Nine phenylethanoid glycosides, four iridoid glycosides, one monoterpene glycoside and one lignan glycoside inhibited the decrease of the cell viability. The inhibitory activities of six phenylethanoid glycosides (echinacoside. acteoside. isoacteoside. 2'-acetylacteoside, tubuloside A and cistantubuloside B2) were stronger than that of silybin. These results suggested that the possible mechanism of hepatoprotective effect of C. tubulosa was that phenylethanoid glycosides reduced the sensitivity of hepatocytes to TNF-a induced cytotoxicity. not that the production of TNF-αwere inhibited.Finally, the effects of echinacoside, acteoside and isoacteoside. which had stronger inhibitory activities in vitro, on the D-GalN/LPS- induced liver injury in mice were examined. The results showed that three compounds significantly inhibited the increase of sAST and sALT induced by D-GalN/LPS. These results indicated that phenylethanoid glycosides were the hepatoprotective ingredients of C. tululosa.The structure-activity relationships of the inhibitory effects of phenylethanoid glycosides on cytotoxicity were elucidated as follows:(1) the aglycone part is essential for the strong activity; (2) the aglycone having the 3,4-dihydroxyl group increased the activity; (3) the 7-methoxy and 6'-O-β-D-glucopyranosyl moiety reduced the activity.A review on structural features, spectral characters and biosynthetic approach of phenyl-ethanoid glycosides in Cistanche sp. was given on the base of references and our study work.