Study On The Chemical Constituents And Bioactivities Of Four Species Of Marine Invertebrates And Two Species Of Fungus Derived From Marine

Marine invertebrates have a complex metabolic system due to a long-term evolution under the special marine environment,producing unique chemical substances,which can not only be used for self-defense,but also can provide a large number of novel medicinal materials for the human diseases therapy.Since 1950 s,marine invertebrates especially sponges and corals had been a hot spot in the field of marine natural products and provided a large number of lead compounds to clinical and preclinical research.In the course of the research of marine invertebrates,more and more studies found that the real source of some secondary metabolites of invertebrates are their associated microorganisms.Marine microorganisms,which provided diverse secondary metabolites,are an important source of active substances.And the limited supply of promising compounds can be solved through fermentation.Therefore,the research of marine microorganisms,especially marine fungi,has attained rapid development in recent years.Based on the above arguments,and as a part of our group’s work,which focus on finding bioactive secondary metabolites with unique chemical structures from marine organisms,we investigated four species of marine invertebrates,namely Theonella swinhoei,Iciligorgia sp.,Lobophytum sp.,Cladiella sp.all collected from the sea area around Yongxing Island.And we also researched two marine invertebrates-associated fungi,namely,Aspergillus ochraceopetaliformis and Stemphylium lycopersici,which were selected from 15 strains by biological and chemical methods.In this investigation,105 compounds,including 31 novel compounds,were isolated by chromatography method,such as,column chromatography on silica gel,ODS and Sephadex LH-20,as well as semi-preparative HPLC.The chemical structures of these compounds were identified by spectroscopic technologies such as NMR,MS,ECD and so on,also combined with searching reported data.The chemical types of these compounds involved steroids,diterpenes,sesquiterpenes,peptides,anthraquinones,polyketides and other skeletons.These compounds were identified as: swinhoeisterol C(h1),swinhoeisterol D(h2),swinhoeisterol E(h3),swinhoeisterol F(h4),swinhoeisterol A(h5),(3S*,5S*,8S*,9S*,10S*,13R*,17R*)-17-((2R*,5R*)-5,6-dimethylheptan-2-yl)-10,13-dimethyl-4-methylenetetradecahydro-14H-8,9-epoxycyclopenta[a]phenanthrene-3,14-diol(h6),(3S*,5R*,9R*,10R*,13R*,15R*,17R*)-17-((2R*,5R*)-5,6-dimethylheptan-2-yl)-15-methoxy-10,13-dimethyl-4-methylene-2,3,4,5,6,7,9,10,11,12,13,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol(h7),15β-hydroxyconicasterol(h8),15-oxoconicasterol(h9),swinhosterol B(h10),echinohalimane A(h11),cavernosine(h12),(1S*,2S*,4R*,4a R*,5R*,6R*,9S*,12R*,12aS*)-1,9-dihydroxy-4-isopropyl-1,6-dimethyl-10-methylenetetradecahydro-5,12-epoxybenzo[10]annulene-2,6-diyl diacetate(f1),(1S*,2S*,4S*,4aR*,5R*,6R*,9S*,12R*,12aS*)-1,2,9-trihydroxy-4-(2-hydroxypropan-2-yl)-1,6-dimethyl-10-methylenetetradecahydro-5,12-epoxybenzo[10] annulen-6-yl acetate(f2),(1S*,2S*,4S*,4aR*,5R*,6R*,9S*,12R*,12aS*)-1,9-dihydroxy-4-(2-hydroxypropan-2-yl)-1,6-dimethyl-10-methylenetetradecahydro-5,12-epoxybenzo[10] annulene-2,6-diyl diacetate(f3),(1S*,4R*,5S*,5aS*,6R*,9S*,12R*,13R*,13aR*)-9-hydroxy-2,2,5,12-tetramethyl-8-methylenetetradecahydro-6,13-epoxy-1,4-methanocycl odeca[d] oxepine-5,12-diyl diacetate(f4),2-((1aR*,3S*,3aR*,4R*,5R*,8S*,11R*,11aS*,11bS*)-5-acetoxy-8-hydroxy-5,11b-dimethyl-9-methylenetetradecahydro-4,11-epoxycyclodeca[3,4] benzo[1,2-b]oxiren-3-yl)propan-2-yl acetate(f5),(6R,7R,8R,9R,12 S,14S,15 R,16R)-9,12-dihydroxy-6-isopropyl-3,9-dimethyl-13-methylene-15-oxa-tricyclo [6.6.1.0]pentadec-3-en-10-yl ester(f6),cladieunicellin B(f7),(1aR*,3R*,3aR*,4R*,5R*,8S*,9S*,11R*,11aS*,11bS*)-8,9-dihydroxy-3-isopropyl-5,9,11b-trimethyltetradecahydro-4,11-epoxycyclodeca [3,4]benzo[1,2-b]oxiren-5-yl acetate(f8),2-((1aR*,3S*,3aR*,4R*,5R*,8S*,9S*,11R*,11aS*,11bS*)-5-acet oxy-8,9-dihydroxy-5,9,11b-trimethyltetrade cahydro-4,11-epoxycyclo deca[3,4]benzo[1,2-b]oxiren-3-yl)propan-2-yl acetate(f9),(1S*,2S*,4R*,4aR*,5R*,6R*,9S*,10S*,12R*,12aS*)-1,9,10-trihydroxy-4-isopropyl-1,6,10-trimethyltetradecahydro-5,12-epoxybenzo[10]annulene-2,6-diyl diacetate(f10),(1S*,4R*,5S*,5aS*,6R*,8S*,9S*,12R*,13R*,13aR*)-8,9-dihydroxy-2,2,5,8,12-pentamethyl tetradecahydro-6,13-epoxy-1,4-methanocyclodeca[d]oxepine-5,12-diyldiacetate(f11),(1S*,2R*,4R*,4aR*,5R*,6R*,7S*,9S*,10S*,12R*,12aS*)-1,2,10-trihydroxy-4-isopropyl-9-methoxy-1,6,10-trimethyltetra decahydro-5,12-epoxybenzo[10]annulene-6,7-diyl diacetate(f12),(1S*,2R*,4R*,4aR*,5R*,6R*,9S*,10S*,12R*,12aS*)-1,2,10-trihydroxy-4-isopropyl-9-methoxy-1,6,10-trimeth yltetradecahydro-5,12-epoxybenzo [10]annulen-6-yl acetate(f13),(1S*,2R*,4S*,4aR*,5R*,6R*,9S*,10S*,12R*,12 a S*)-1,9,10-trihydroxy-4-(2-hydroxy propan-2-yl)-1,6,10-trimethyl tetradecahydro-5,12-epoxybenzo[10]annulene-2,6-diyl diacetate(f14),klysimplexin X(f15),selerophytin A(f16),selerophytin E(f17),3-acetoxycladiellin-11-ene-6,7-diol(f18),(1R*,3aR*,4R*,5R*,7aR*)-1-((2R*,5S*,E)-5,6-dimethylhept-3-en-2-yl)-4-(5-hydroxy-2-methylphenethyl)-7a-methyloctahydro-1H-indene-4,5-diol(f19),(1R*,3aR*,4R*,5R*,7aR*)-1-((2R*,5S*)-5,6-dimethylheptan-2-yl)-4-(5-hydroxy-2-methylphenethyl)-7a-methylocta hydro-1H-indene-4,5-diol(f20),Subergorgiaol C(f21),Subergorgiaol G(f22),Subergorgiaol B(f23),Subergorgiaol A(f24),Subergorgiaol D(f25),(1R*,3aR*,4R*,7aR*)-4-hydroxy-4-(5-hydroxy-2-methylphenethyl)-7a-methyl-1-((R*,E)-6-methylhept-3-en-2-yl)octahydro-5H-inden-5-one(f26),(1R*,3R*,3aS*,4R*,7aR*)-1-((2R*,5S*,E)-5,6-dimethylhept-3-en-2-yl)-3,4-dihydroxy-4-(5-hydroxy-2-methylphenethyl)-7a-methyloctahydro-5H-inden-5-one(f27),Subergorgiaol L(f28),Calicoferol F(f29),Calicoferol B(f30),Calicoferol G(f31),Astrogorgol H(f32),Calicoferol C(f33),Calicoferol E(f34),5α,6α-epoxy-(22E,24R)-ergosta-8,22-diene-3β,7β-diol(f35),meltthasterol B(f36),meltthasterol B(f37),(2S,3R)-2-((1R*,2R*,3R*)-3-((4S*,4aS*,6S*,8aS*)-4,6-dihydroxy-8a-methyl-1-oxo-1,4,4a,5,6,7,8,8a-octahydronaphth alen-2-yl)-2-(2-hydroxyethyl)-2-methylcyclopentyl)-6-methylheptan-3-yl acetate(f38),22α-acetoxy-24-methylene-3β,6α,11-trihydroxy-9,11-seco-cholest-7-en-9-one(f39),(4S,4aS,6S,8aS)-2-((1R,2R,3R)-3-((2R,5S,E)-5,6-dimethylhept-3-en-2-yl)-2-(2-hydroxy ethyl)-2-methylcyclopentyl)-4,6-dihydroxy-8a-methyl-4a,5,6,7,8,8a-hexahydronaphthalen-1(4H)-one(f40),subergorgol L(f41),9,12-seco-3β,6α,11-trihydroxy-24-methylene-5α-cholest-7-en-9-one(f42),(22E,24S)-24-Methyl-5α-cholesta-22-diene23β,5α,6β2triol(f43),5α-stigmast-22-ene-3β,5α,6β-triol(f44),Ethyl-5α-cholesta-3β,5,6β-triol(f45),(22E)-cholesta-7,22-dien-3β,5,6β-triol(f46),Pregna-1,4-diene-3,20-dione(f47),3β-hydroxy-5-en-20-one(f48),Subergorgol U(f49),(1aS*,4E,8S*,9Z*,14 a S*)-8-hydroxy-4,8,12,14atetramethyl-1a,3,6,7,8,13,14,14a-octahydrooxireno[2’,3’:5,6] cyclotetradeca[1,2-b]furan-11(2H)-one(d1),(1aS*,4E,8R*,9Z*,14aS*)-8-hydroxy-4,8,12,14a-tetramethyl-1a,3,6,7,8,13,14,14a-octahydrooxireno[2’,3’:5,6]cyclotetradeca[1,2-b]furan-11(2H)-one(d2),(6R*,10 E,14E)-7-dihydroxy-6,15a-epoxy-3,6,10,14-tetramethyl-5,6,7,8,9,12,13-octahydrocyclotetradeca[b] furan-2(4H)-one(d3),dimethyl(1Z,3E,7Z,11S*,12R*)-11,12-dihydroxy-4-isopropyl-11-methylcyclotetradeca-1,3,7-triene-1,7-dicarboxylate(d4),dimethyl(1E,3Z,7Z)-12-acetoxy-11-hydroxy-4-isopropyl-11-methylcyclotetradeca-1,3,7-triene-1,7-dicarboxylate(d5),(1aS*,4E,6S*,8E,9aR,14aS*)-6-hydroxy-4,8,12,14atetramethyl-1a,3,6,7,9a,13,14,14a-octahydrooxireno[2’,3’:5,6] cyclotetradeca[1,2-b]furan-11(2H)-one(d6),(+)-isosarcophine(d7),sarelengans B(d8),(+)-helianane(d9),(±)-(4S,8S)-foedanolide(b1),ent-4(15)-eudesmene-1?,6?-diol(b2),eudesm-11(13)-en-4b,9bdiol(b3),1?,11-dihydroxy-5-eudesmanes(b4),opposit-4(15)-ene-1β,7-diol(b5),guainanediol(b6),dihydyoxyaromadengrane-4?,10?-diol(b7),(-)-alloaromadendrane-3?,9?-diol(b8),9-chloro-8-hydroxy-8,9-deoxyasperlactone(t1),9-chloro-8-hydroxy-8,9-deoxyaspyrone(t2),6-hydroxy-5-methoxy-3-methyl-3,6-dihydro-2H-pyran-4-carboxylic acid(t3),9-chloro-8-hydroxy-8,9-deoxyasperlactone(t4),aspilactonols E/F(t5),penicillic acid(t6),3,3’-dihydroxy-5,5’-dimethyldiphenyl ether(t7),botryoisocoumarin A(t8),viomellein(t9),xanthomegnin(t10),9α,14-Dihydroxy-6β-p-nitrobenzoylcinnamolide(t11),7α,14-Dihydroxy-6β-p-nitrobenzoylconfertifolin(t12),(Z)-3-((1H-indol-3-yl)methylene)-2,3,6,7-tetrahydropyrrolo[1,2-a]pyrazine-1,4-dione(t13),cyclo[L-(4-hydroxyprolinyl)-Lleucine](t14),cyclo(L)-4-OH-Pro-(L)-Phe(t15),azonazine(t16),aspergillipeptide A(t17),macrosporin 2-O-α-D-glucopyranoside(s1),macrosporin(s2),dihydroaltersolanol B(s3),alterporriol U(s4),2-phenylethyl-α-D-glucopyranoside(s5),(S)-2-hydroxy-3-(4’-hydroxyphenyl)propanoic acid(s6),latifolicinin C(s7),methyl 4-hydroxybenzoate(s8),methyl 4-hydroxybenzoate(s9),methyl(E)-4-(5-hydroxy-3-methylpent-2-enamido)butanoate(s10).Compounds h1,h2,h3,h4,h6,h7,f1,f2,f3,f4,f5,f8,f9,f10,f11,f12,f13,f14,f19,f20,f26,f27,f38,d1,d2,d3,d4,d5,d6,s1,s10 are new compounds.Compound h11 was isolated from marine sponges for the first time,compound h12 was isolated from the genus Theonella sp.for the first time.Eunicellin type diterpenoid f15 was a new natural product.Compounds d8 and d9 were isolated from the genus Lobophytum sp.for the first time.And compounds b1,b4,b5,b6,b7 were isolated from the genus Cladiella sp.for the first time,and compound b3 was isolated from marine for the first time.As a continuing work of previous research on(h)p300 inhibitor swinhoeisterols in our group,we also evaluated new compounds h1 to h4 with the swinhoeisterols skeleton by an in vitro assay,compound h1 showed moderate activity against(h)p300,with IC50 value of 8.84 ?M.In an in vitro immune regulation activity assay,compounds f19,f20,f21,f23,f24,f25,f26,f28,f30,f31,f32,f33,f34 significantly inhibited CD4+ T cells differentiation 5 days after administration,and compounds f35,f36,f37,f43,f44,f48 inhibited CD4+ T cells differentiation 7 days after administration.While compound f38 significantly increased CD4+ T cells differentiation 7 days after treating.These interesting discovery may promote an ongoing investigation on the immunomodulatory activity of these compounds with steroid skeleton.In an in vitro assay of effects of compounds on differentiation of osteoclast,four compounds f37,f39,f47,f48 showed significant inhibition activity toward the differentiation of osteoclast,eight compounds f24,f25,f26,f40,f42,f43,f44,f45 exhibited promotion effects on the differentiation of osteoclast.These results may promote a further research of these compounds on bone metabolic diseases such as osteoporosis.In an in vitro assay of cytotoxicity towards human Huh7 stem cell-like cell using CCK-8 method,compounds f19,f20,f21,f22,f24,f25,f27,f28,f31,f33,f34,t11 exhibited different levels of growth inhibition activity,with IC50 values of 21.5 to 62.7 ?M.And the antifungal activity of 37 compunds t3~t6,t9~t14,t16,t17,f19,f22,f24~f26,f28,f32~f34,f36~f39,f43,f47,b1,b2,d7~d9 were evaluated by an in vitro antifungal activity toward Candida albicans and Candida parapsilosis.All of these compounds showed weak activity.This research expanded the amounts and chemotypes of their corresponding skeletons by providing novel compounds.And the activity screening of some obtained compounds give a guidance to the chemical modification and bioactivity study.