Studies On Anti-tumor Constituents Of Inonotus Obliquus And Metabolites Of Its Main Components In Rats

Inonotus obliquus(Pers.:Fr.)Pil.is a medicinal white-rot fungus belonging to the family Hymenochaetaceae,widely distributed in Europe,Asia,and North America.This mushroom has been used as a folk medicine for cancer for more than four centuries in Russia and western Siberia.In our research for bioactive compounds from I.obliquus,35 compounds were isolated from its sclerotia by silica gel,ODS,Sephadex LH-20 and HPLC chromatograph.Their structures were identified by 1D and 2D NMR,ESI-MS,HR-ESI-MS,LC-MS/MS,IR and UV as ffollows:(3β,21S,24R)-21,24-cyclolanosta-8-en-3,21,25-triol(inonotusane A,1),3β,21S,24S)-21,24-cyclolanosta-8-en-3,21,25-triol(inonotusane B,2),3β-hydroxy-25,26,27-trinorlanosta-8,22E-dien-24-al(inonotusane C,3),3β-hydroxy-25,26,27-trinorlanosta-8,22E-dien-24-oic acid(4),3β-hydroxy-24,25,26,27-tetranorlanosta-8-en-22-one(inonotusane D,5),(21R,24S)-3β,12β,15α,21,25-pentahydroxy-21,24-cyclopentalanosta-7,9(11)-diene(inonotusane E,6),3β-hydroxylanosta-8,25-dien-24-one-21-oic acid(inonotusane F,7),lanosta-8-en-3β,22,24,25-tetraol-25-methyl oxide(inonotusane G,8),inonotsutriol A(9),inotodiol(10),lanosterol(11),3/3-hydroxylanosta-8,24-diene-21-al(12),trametenolic acid(13),3β-hydroxy lanosta-7,9(11),24-trien-21-oic acid(14),3β,22-dihydroxylanosta-7,9(11),24-triene(15),inonotsutriol E(16),inonotsutriol D(17),inonotsuoxide B(18),inonotsuoxide A(19),betulin(20),betulinic acid(21),oleanolic acid(22),fuscoporianol C(23),3β,22α-dihydroxylanosta-8,25-diene-24-one(24),inotolactone B(25),inotolactone A(26),ganodecochlearin A(27),saponaceoic acid I(28),inonotusol A(29),inonotusol C(30),inonotusol B(31),ergosterol peroxide(32),9,11-dehydroergosterol peroxide(33),4-(3,4-dihydroxyphenyl)-(E)-3-buten-2-one(34)and 3,4-dihydroxybenzaldehyde(35).Among them,there were 28 lanostane-type tetracyclic triterpenoids(1-19,23-31),3 pentacyclic triterpenoids(20-22),2 steroids(32-33)and 2 phenolic compounds(34-35).Compounds 1-3 and 5-8 are new compounds,while 4 is new natural product,which was mentioned one time in 1974,during the side-chain degradation of lanosterol,but without any spectroscopic data.To our knowledge,the presence of 25,26,27-trinorlanostanes(3 and 4)is unusual in the family Hymenochaetaceae.And our results could contribute to chemotaxonomic studies of the Inonotus genus.Furthermore.5 is the first 24，25,26，27-tetranorlanostane-type triterpenoid from fungus.And until now,oleanane-type triterpene(22)has been isolated from this genus for the first time.Compounds 27 and 28 are both new isolates of I.obliquus.MTT tests showed 9,16，17 and 24 exhibited strong cytotoxicity against A549 tumor cell line,with IC50 2.34,1.63,8.39 and 5.39 μM,respectively.5,28 and 32-33 exhibited strong cytotoxicity against the 4T1 tumor cell line,with IC50 9.40,7.79,9.06 and 9.31 μM.28 and 32-33 also showed strong inhibitory activities against the MCF-7 tumor cell line,with IC508.35、9.01 and 8.40 μM.While the cytotoxicity of 3,6-8,15,17-20,24,27 and 34-35 were moderate against some tested cell lines,with IC50 10-30 μM,such as 3,1,20.34 and 35 against A549,18 against HT29,3,7,17-19 and 24 against Hela,7 and 18 against L1210,6 against HepG2,7 against MCF-7,6-8,27 and 34-35 against 4T1 tumor cell line.The metabolites of inotodiol(10),trametenolic acid(13)and inonotsuoxide A(19),three main triterpenoids in Inonotus obliquus in rats were investigated by high-perf ormance liquid chromatography coupled with electrospray ionization tandem mass spect rometry(UPLC/ESI-Q-Tof-MS).Totally 9 metabolites(D1-D9),besides the parent com pound(D0),were characterized after oral administration of inotodiol.The phase I met abolites were desaturated derivatives(D5 and D9),desaturated and hydroxylated deriva tives(D1,D6 and D8).The phase Ⅱ metabolites were the glucuronides of inotodiol(D7),its desaturated derivative(D3),its hydroxylated derivative(D4),and its desaturat ed and hydroxylated derivative(D2).Totally 10 metabolites(T1-T10),besides the pare nt compound(T0),were characterized after oral administration of trametenolic acid.T he phase I metabolites were hydroxylated derivatives(T4,T5 and T10),desaturated a nd hydroxylated derivatives(T3,T6,T8 and T9)and hydroxylated and hydrated deriv atives(T1 and T2).The phase Ⅱ metabolite was the glucuronide of trametenolic acid(T7).Totally 4 metabolites(A1-A4),besides the parent compound(A0),were charact erized after oral administration of inonotsuoxide A.The phase Ⅰ metabolites were desa turated derivatives(A3 and A4),desaturated and hydroxylated derivatives(A1).The p hase Ⅱ metabolite was the glucuronide of inonotsuoxide A(A2).The biosynthetic pathways of 1-5 and other lanostane-type tetracyclic triterpenoids,and the structure-activity relationships on cytotoxicity against the tested tumor cell lines are discussed in the text.The metabolic transformation of inotodiol(10),trametenolic acid(13)and inonotsuoxide A(19)in rats included desaturation,hydroxylation and glucuronidation.