Studies On The Constituents Of Cynanchum Limprichtii And The Secondary Metabolites Of Pholiota Nameko

Cynanchum limprichtii Schitr is a perennial herb belong to the genus Cynanchum in the family of Asclepiadaceae,which is chiefly distributed in Sichuan Province of China.Many herbs of genus Cynanchum have been used as antitussive and expectotants in China for a long time.The chemical composition and pharmacological activity of the plant was rarely reported.As our interest in discovery of bioactive constituents from it,36 compounds were isolated from the 95%ethanol extract of C.limprichtii by means of repeated column chromatography on silica gel,Sephadex LH-20,ODS and HPLC.On the basis of physico-chemical properties and spectroscopic analysis,32 of which were identified as limproside A(L1),limproside B(L2),limproside C(L3)limproside D(L4),cynatratoside B(L5),cynatratoside A(L6),glaucogenin C 3-O-β-D-thevetopyranoside(L7),stauntoside P(L8),neocynapanoside A(L9),stauntoside T(L10),stauntoside L(L11),glaucogenin C 3-O-β-D-digitoxopyranosyl-(1→4)-β-D-3-demethyl-2-deoxythevetopyranoside(L12),glaucogenin C 3-O-α-L-cymaropyranosyl(1→4)-β-D-digitoxopyranosyl-(1→4)β-D-oleandropyranoside(L13),glaucogenin C 3-O-α-L-cymaropyranosyl-(1→4)-β-Ddigitoxopyranosyl-(1→4)-β-D-3-demethyl-2-deoxythevetopyranoside(L14),tylophosideA(L15),stauntoside M(L16),stauntoside A(L17),cynatratoside D(L18),β-daucosterol(L19),(22E,24R)-3β,5α,9α-trihydroxy-ergosta-7,22-dien-6-one(L20),7,4’-dihydroxyisoflavone(L21),4-hydroxybenzoic acid(L22),2,6-dihydroxybenzoic acid(L23),3,4-dihydroxybenzoic acid(L24),4-oxo-2-octenoic acid(L25),6-hydroxy-2,4-decadienooic acid(L26),trans-4-hydroxy-2-decenoic acid(L27),sebacic acid(L28),sucrose(L29),tianshi acid(L30),2-hydroxy-N-[1-β-D-glucopyranosyloxy-3-hydroxy-pentadec-4,8-dien-2-yl]nonadecanamide(L31),adenosine(L32).Among them,compounds L1-L4 were four new C21 steroidal glycosides.Compounds L20,L25-L27,L30-L32 were isolated from this plant for the first time.Steroidal glycosides have been proved to have antitumor activity and some of them have been previously obtained from the plant of this genus also exhibited proliferating inhibitory activity against human cancer cell line.In this paper,C21 steroidal glycosides L1-L8,L12L16 were evaluated for their in vitro cytotoxicity against three human cancer cell lines(HL60,THP-1 and Caco-2)by MTT method.Test results showed that compounds L13-L16,exhibited strong inhibitory activities against HL-60 cell line(IC50<17.9 μM);compounds L12-L16 displayed strong inhibitory activities against THP-1 cell line(with IC50 as 11.5,6.3,6.8,22.6,11.0 μM,respectively);compounds L3,L4 exhibited moderate cytotoxicity against THP-1 cell line(IC50 as 31.9,43.7 μM).Compounds L3,L4 exhibited weak inhibition against HL-60 and L13,L14 showed weak inhibition against Caco-2.Other compounds did not showed proliferating inhibitory activity against human cell line.We investigated the anti-inflammatory activity of C21 steroidal glycosides L2,L3,L7,L8,L11-L12,L14-L17 on LPS induced IEC6 activation.The NO production was measured by Griess regent.Compared with model group,pretreatment with compounds significantly suppressed the increase of NO.The results showed that LPS significantly induced NO production in IEC6 cells compared to the blank control group.The test compounds had an inhibitory effect on LPS-induced NO,but did not show significant dose dependence.Test results showed that compounds L2、L3、L7、L8、L14 exhibited good inhibitory activities at all tested concentrations,and the inhibition rates at 25 μM were 23.7%、35.33%、31.54%、25.86%and 35.75%respectively.Compound L11 showed significant inhibitory effects on NO resease at four concentrations of 100 μM,50 μM,25 μM,and 10 μM,which intibitory rates were 23.96%,32.81%,31.54%,and 21.43%,respectively.Compounds L15 and L17 showed particularly significant inhibition at a concentration of 25 μM,with inhibition rates of 37.12%and 34.17%.Pholiota nameko Ito ex lmai.,also known as pearl mushroom,phoenix mushroom,light cap parachute,belongs to Basidiomycetes fungi subclass agaricales,silk membrane bacteria family,Pholiota genus.P.nameko is a rare species native to Japan.Since it was introduced to Liaoning Province in the middle of the 1970s,it has been produced in northern Hebei,Liaoning and Heilongjiang province.Because of its delicious flavor,high nutritional value,and strong inhibitory effect on cancer cell lines,it has become a new star for medicinal and edible fungi.In order to discovery novel bioactive metabolites of it,22 compounds were isolated from the solid culture of edible mushroom Pholiota nameko Ito ex lmai.,and 20 of them were elucidated by their physico-chemical properties and spectroscopic data as follows:indolyl-3-carboxylic acid(N1),indole-3-carboxaldehyde(N2),(-)-(R)-13aαsecoantofine(N3),uridine(N4),adenosine(N5),indole-2,3-dione(N6),nicotinamide(N7),(22E,24R)-ergosta-7,22-diene-3β,5α,6β-triol(N8),6-hydroxystigmast-4-en-3-one(N9),ergosta-7,22-diene-3β;5α,6α-triol(N10),ergosta-7,22-diene-3β,5α,6β-triol,6-acetate(N11),6,9-epoxy-ergosta-7,22-dien-3β-ol(N12),(22E,24R)-3β;5α,9α-trihydroxy-ergosta-7,22-dien-6-one(N13),5,7-dimethoxy-isobenzofuran-l-one(N14),4-hydroxy-benzoic acid(N15),benzoic acid(N16),p-methoxybenzoic acid(N17),methyl-3,5-dichloro-4methoxybenzoic acid(N18),D-mannitol(N19),α-D-glucopyranose(N20).