| Liparis nervosa,a member of the Liparis genus of the Orchidaceae family,is a medicinal plant widely distributed in Jiangxi,Hunan,Fujian,Taiwan,Guangdong,Guangxi and southwest China.The plant usually grows in broad-leaved forests,particularly on moist slopes at altitudes from 850 to 1000m.L.nervosa has been used in folk medicine for a long time for the treatments of wound bleeding,detoxifying and inflammatory diseases,such as pneumonia,hemoptysis,traumatic hemorrhage,and snakebite.It has antitumor,antioxidant,antiinflammatory,antibacterial,hemostatic and antihypertensive effects according to modern pharmacological studies.The phytochemical studies on this herb have mainly led to the isolation of nervogenic acid derivatives.However,other components especially the active components with antitumor effect have not been studied in detail yet,and there is still considerable research space.Previous investigations on chemical constituents of L.nervosa in our group have reported phenanthrenes,bibenzyls and coumarins,which prompted us to undertake in-depth studies.The 95%ethanol extraction of L.nervosa was carried out in order to obtain more compounds in this dissertation,and the results can also further provide a theoretical basis for clarifying the active substances of the plant and establishing quality standards.Further chemical study on the 95%ethanol extraction of L.nervosa was performed by using advanced chromatography technology,such as silica gel column chromatography,Sephadex LH-20 column chromatography,ODS column chromatography and semi-preparative HPLC.Compounds were obtained by chromatographic isolation,and their structures were identified by morden identification technology.As a result,forty-two compounds were obtained,three(1～3)of which were novel compounds,they were named as 2,7,2’-trihydroxy-3,4,4’7’-tetramethoxy-1,1’-biphenanthrene(1),{6-O-[(4-oxo-4H-pyran-3-yloxy)-O-β-D-glucopyranosyl]}-2,2-dimethyl-8-(3-methylbut-2en-1-yl)-chromane-6-carboxylate(2)and 7-(3-methylbut-2-en-1-yl)-2,3-dihydrobenzofuran-5-carboxylic acid(3).Thirty-nine compounds were known ones,six of which were stilbenes,they were 2,5-dihydroxy-4-methoxy-9,10-dihydroxyphenanthrene(4),2,4-dihydroxy-7-methoxy-9,10-dihydroxyphenanthrene(5),7-hydroxy-2-methoxy-phenanthrene-1,4-dione(6),7-hydroxy-2-methoxy-9,10-dihy drophenanthrene-1,4-dione(7),4,4’,7,7’-tetrahydroxy-2,2’-dimethoxy-9,9’,10,10’-tetrahydro-1,1’-biphenanthrene(8)and cis-3,3’-dihydroxy-5-methoxystilbene(9).Eight nervogenic acid derivatives were elucidated as 4-hydroxy-3,5-bis(2-hydroxy-3-methyl-3-butenyl)benzoicacid(10),2,2-dimethyl-8-(3-methyl-2-butenyl)-2H-chromene-6-carboxylic acid(11),2,2-dimethyl-8-(2-isopenten-1-yl)chroman-6-carboxylic acid(12),4-hydroxy-3,5-bis(3-methyl-2-butenyl)-benzoic acid(13),4-hydroxy-3-(2-hydroxy-3-methyl-3-butenyl)-5-(3-methyl-2-butenyl)benzoic acid(14),Liparisglycoside O(15),Liparisnervoside A(16)and Liparisbenzoate C(17).Two alkaloids were Cannabisin F(18)and Thymidine(19).Twelve phenylpropanoids were identified as Auraptenol(20),Syringaresinol(21),Syringaresinol-4’-O-β-D-glucopyranoside(22),Sinapaldehyde(23),Coniferyl aldehyde(24),Syringin(25),Sinapaldehye glucoside(26),2,3-dihydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone(27),C-Veratroylglycol(28),Icariol A2(29),Erigeside 2(30)and Methylsyringin(31).Eight other compounds were Stigmasterol-3-O-β-D-glucoside(32),Daucosterol(33),(6S,7E,9R)-6,9-dihydroxymegastigma-4,7-dien-3-one-9-O-β-D-glucopyranoside(34),Butyl syringate(35),Protocatechuate(36),p-hydroxybenzaldehyde(37),Vanillic acid(38),Hexacosanoic acid(39),Shikimic acid(40),1,3,5-trimethoxybenzene(41)and Syringaldehyde(42).Compounds 5～9,11,13～14,18,20,22～29,31～32,34～36 and 38～42 were isolated from the genus Liparis for the first time,and compounds 4,15,17,21,30 and 33 were obtained from L.nervosa for the first time.The in vitro cytotoxicities of the nervogenic acid derivatives and phenanthrenes isolated were evaluated by an MTT assay.It was shown that nervogenic acid derivatives(2,3,10～17)had no inhibitory effect on A549 cell line,and all of the IC50 values were greater than 100μmol/L.However,phenanthrenes exhibited better cytotoxic activity against A549 cell line.Compounds 1,6～8 showed potent cytotoxic activity with IC50 values ranging from 10.20 to 17.56 μmol/L.Compounds 4 and 5 had moderate cytotoxic activity with IC50 values ranging from 35.73 to 42.41 μmol/L.According to the analysis of the data,phenanthraquinones(6,7)had strongly effective cytotoxic activity,which may be associated with the presence of two carbonyl groups.In addition,the results showed that biphenanthrenes exhibited stronger cytotoxicity against A549 cell than those of phenanthrenes.Taken together,the antitumor efficacy material base of L.nervosa may be phenanthrenes and their antitumor mechanisms are worth for studying in depth.Additionally,the dissertation provides theoretical foundations for further development and utilization of L.nervosa. |
