Studies On Chemical Constituents And Chemical Resistance Of H5N1

Tupistra chinensis(Liliaceae), mainly distributed in southwestern China, is a rich source of steroidal sapogenins, saponins, flavans and cardenolide. These components exhibit diverse biological activities such as anti inflammatory, immune stimulating, anti fungal and anti tumor properties. As a folk medicine, this species has usually been used for treatment of rheumatic diseases, carbuncles, ameliorate pharyngitis and snake bite. The significant bioactivities of the extract of Tupistra chinensis Baker have attracted many pharmacologists’ interests in China. They have mainly studied T. chinensis Baker, T. wattii Hook. f, T. aurantiaca wall et Baker, Tupistra yunnanensis and T. tui wang et Liang, and some compounds from the rhizomes of Tupistra chinensis Baker and their cytotoxic and antivirus activities have been reported. Based on the review on the chemical constituents and bioactivities research of Tupistra chinensis Baker, chemical constituents of T. chinensis were studied systematically and deeply, their anti H5N1 virus and antitumor activities were tested to obtain compounds with novel structures or significant bioactivities. Sixty six kinds of Chinese herb were selected for their cytotoxic activities against MDCK cell lines infected by H5N1 in vitro using the cytopathic effect(CPE) method, and 7 of them were found to possess potent cytotoxicity. They are Tupistra chinensis Baker, Myrsine africana L., Lunate Peltate Sundew Herb, Reineckia carnea(Andr.) Kunth, Hydrangea strigosa Rehd, Veronicastrum cauloptera Hance and Helicteres angustifolia L, respectively. In our screening for cytotoxic agents from these plants, the ethanol extract from the rhizomes of Tupistra chinensis Baker showed the most inhibitory effect towards H5N1 virus. Therefore, Tupistra chinensis Baker was chosen to be studied. By means of many different chromatographic methods such as AB 8 macroporous, silica gel CC, reversed phase C18, Sphadex LH 20 and preparative HPLC etc, 58 compounds have been isolated from the ethanol extract of Tupistra chinensis Baker, and 53 compounds were elucidated on the basis of chemical and spectral evidence(IR, MS, 1H NMR, 13 C NMR, 1H 1H COSY, HSQC, HMBC, NOESY), including 34 steroidal saponins, 13 flavones, 4 cardenolides, 1 androstanes and 1other kind of compound. They are as follows: tupistroside G(1), tupistroside H(2), tupistroside I(3), tupistroside J(4), tupichiside A(5), tupichigenin G(6), tupichigenin H(7), tupichigenin I(8),(25S) 1β,3β,5β triol spirost 3 O β D glucopyranoside(9), 25(27) ene 1β,3β diol spirost 3 O β D glucopyranoside(10),(24S,25S) 1β,3β diol spirost 24 O β D glucopyranoside(11),(25R) 5α spirost 2α,3β, 5α triol 3 O β D glucopyranoside(12), rhodeasapogenin 3 O β D glucopyranoside(13), isorhodeasapogenin 3 O β D glucopyranoside(14), Isoepiruscogenin 3 O β D glucopyranoside(15), epiruscogenin 3 O β D glucopyranoside(16), polyphyllin Ⅵ(17), isopolyphyllin Ⅵ(18), tupichigenin 5 O β D glucopyranoside(19),(25R) tupichigenin 5 O β D glucopyranoside(20),(25S) 26 O β D glucopyranosyl furost 1β,3β,22α,26 tetraol 3 O β D glucoside(21),(25S) furost 22 methoxy 1β,3β,26 triol 26 O β D glucopyranoside(22),(25S) 1β,3β,22α,26 tetraol furost 26 O β D glucop yranoside(23), glucodigifucoside(24), digitoxigenin 3 O β D glucopyranosyl(1 4) α L digitoxopyranosyl(25), digitoxigenin 3 O α L fucopyranose(26), 25(27) ene 1β,2β,3β,4β,5β,7α hexaol spirost 6 one(27),(25S) spirost 1β,2β,3β,4β,5β,7α hexahy droxyl 6 one(28), spirost 25(27) ene 1β,2β,3β,4β,5β,6β,7α heptol(29), wattigenin B(30), pentologenin(31), ranmogenin A(32), convallagenin B(33),(25S) 1β,3β,24β triolspirost(34), convallagenin A(35),(25S) 5(6) ene 3β,4β,7α triolspirost(36), iso epiruscogenin(37), epiruscogenin(38), digitoxigenin(39), 5 en 3β,15α dihydroxyl androst 17 one(40), 8 methylluteolin(41), orobol(42), 6 methyl 7 O methylarma dendrin(43), isoliquiritigenin(44), 4,4’ dihydroxychalcone(45), 4 en 1,3 bis(4 hy droxyphenyl) 1 one(46), tupichinol B(47),(+) catechin(48), tupichinol A(49),(2R,3R) 3,4′ dihydroxy 7 methoxyflavane(50),(2R) 7,4′ dihydroxy 8 methylflavane(51), tupichinol C(52) and 1,2 bis(4 hydroxyphenyl) cyclopentane(53). Compounds 1 8 were new compounds, and compounds 10, 12, 14, 17-20, 22-26, 34, 36, 39-46, 48, 50, 51 and 53 were isolated from Tupistra genus for the first time, in addition, compounds 13, 28, 30, 32, 33, 37 and 38 were first obtained from Tupistra chinensis Baker.In screening for anti H5N1 virus cytotoxic agents from some compounds of Tupistra chinensis Baker, compounds 40 and 5 exhibited potent anti H5N1 virus activity in MDCK cell cultures which did not show obvious cytopathic effect on cell viability, proliferation or metabolism. At the effective dose of 50.0 and 25.0 g/m L, respectively. Compound 40 was a kind of steroidal saponin, and compound 5 was a kind of flavan. The exact effect or mechanism should be further proven by hemagglutination test and in vivo studies.Thirty compounds from Tupistra chinensis Baker were tested for cytotoxicity against BGC 823 and Human Lo Vo cell lines in vitro using the MTT assay method. The cytotoxicity of the compounds against the BGC 823 and Lo Vo cell lines was compared with the respective positive control group Cisplatin, and six of them were found to possess potent cytotoxicity. The new compound 2 exhibited potent toxicity effects against Lo Vo and BGC 823 cell lines, with IC50 values of 0.267 and 0.327 M, respectively. Meanwhile, compounds 15 and 16 also showed significantly cytotoxicity against the selected cells, and the IC50 values were much lower than that of Cisplatin on BGC 823 and Lo Vo cell lines. Comparison of the cytotoxic activities and structures of these compounds suggested that the ethylenic linkage at C 5 position increased their cytotoxic activities. In addition, the flavane aglycone tupichinol A possesses more cytotoxic activity than its glucoside tupichiside A.