从海藻真菌木霉TrICHOdErMA SP.PT2菌体提取物中分离到一个新倍半萜糖苷类化合物TrICHOdErMOSIdE(1)。并通过1d-,2d-nMr波谱数据鉴定了化合物的结构,并应用MTT法对化合物1的细胞毒活性进行了研究。A novel sesquiterpene glucoside,trichodermoside(1),was isolated from the mycelia extract of algicolous fungus Trichoderma sp.PT2.The structure was elucidated by spectroscopic analyses,including 1D-,2D-NMR data and mass-spectrometric analyses.Compound 1 displayed weak growth inhibition against human HeLa cells.supportedbyChinaOceanResourceR&DAssociation(GrantDYXM-115-02-2-13);theNationalHighTechnologyResearchandDevelopmentProgramofChina(863;No.2006AA09Z410

李瑶瑶

杜希萍

沈月毛

郑忠辉

鲁春华

Xiamen University Institutional Repository

天然产物研究与开发 N a tP rod R es D ev 2010, 22: 544 547文章编号: 1001 6880( 2010) 04 0544 04Received December 28, 2009; A ccep ted April 21, 2010Foundation Item: Th is work w as f inancially supported by Ch ina OceanResou rce R&D A ssociat ion ( G ran t DYXM 115 02 2 13 ) and the NationalH igh Technology Research and Developm en t Program of Ch ina( 863, No. 2006AA09Z410 ).* C orrespond ing au thor Te:l 86 532 88963253; E m ai:l ahua0966@ xm u.edu. cn分离自木霉 PT2的一个新倍半萜糖苷杜希萍1, 2,李瑶瑶1,鲁春华1*,郑忠辉1,沈月毛11厦门大学生命科学学院 细胞生物学与肿瘤细胞工程教育部重点实验室 福建省药物工程实验室, 厦门 361005;2集美大学生物工程学院,集美 361021摘 要:从海藻真菌木霉 T r ichod erm a sp. PT2菌体提取物中分离到一个新倍半萜糖苷类化合物 T r ichoderm oside( 1)。并通过 1D , 2D NMR波谱数据鉴定了化合物的结构,并应用 MTT法对化合物 1的细胞毒活性进行了研究。关键词:倍半萜糖苷 ; T r ichoderm oside;木霉; 结构解析;生物活性中图分类号: R93 文献标识码: AA Novel Sesquiterpene G lucoside from Trichoderma sp. PT2DU X i p ing1, 2, L IYao yao1, LU Chun hua1, *, ZHENG Zhong hui1, SHEN Yue mao11K ey Labora tory of theM inistry of Educa tion for Cell B io logy and Tum or Cell E ngineer ing; Fujian EngineeringLaboratory for Pharmaceu ticals, School of L ife Science, X iamen University, X iamen, 361005, Ch ina;2B ioeng ineering Co llege of J im ei University, J im ei, 361021, ChinaAbstract: A nove l sesqu iterpene g lucoside, tr ichoderm oside ( 1), w as iso lated from them ycelia ex trac t o f a lg ico lous fungus T richoderm a sp. PT2. The structure was e lucida ted by spectroscop ic ana lyses, including 1D , 2D NMR data andmass spectrom e tric ana ly ses. Com pound 1 d isp layed w eak grow th inhib ition against hum an H eLa cells.Keyw ords: sesquiterpene g lucoside; T richode rmoside; T r ichoderma sp. ; structu re determ ination; cy to tox ic ityIntroductionThe fungal genus Trichoderma is a w idespread saprophy te that occurs almost ubiquitously. Because o f theusefu l secondary metabo lites produced by th is genus,many strains have rece ived considerable attention asb iocontro l agents.[ 1, 2]. Some secondary metabolites, inc ludingN acy lated peptides, pept ibo ls[ 3, 4 ], koning in insA G[ 5 8], octaketide derived compounds, tw o buteno lide compounds[ 9, 10], and demethy lso rb icillin and oxosorb icillino l[ 11]were reported as antibiot ics w ith various act iv it ies. During the course of exam in ing fung i forb io log ica lly act ive natura l products, the strain PT2 fromB lidingia m inima K ylin, co llected at Wuyu Island,Zhangzhou of Fujian Prov ince, w as iso lated. The ethy lacetate extract o f the fermentation show ed strong ant itumor act iv it ies aga instKB andR aji cell lines[ 12]. Cyclopept ides w ere the active const ituent[ 13]. Our further research on the chem ica l components ofTrichoderma sp.PT2 y ielded one new compound. H ere the iso lation,structure eluc idation and b ioactivity assay o f this compound w as described.Results and D iscussionFermenta tion ( 10 L) w as carried out at 28 for 20 dw ithout ag itat ion. A fter filtrat ion, the myce lia w ere extracted exhaust ively w ith acetone. A cetone w as evaporated in vacuo and the crude ex tractw as part itioned betw eenme thano l and petro leum ether. The methano l solut ion w as co llected and evaporated to dryness in vacuoto afford 17 g extrac.t The ex tractw as pur ified overRP18, Sephadex LH 20, and silica gel co lumns to a fford anove l sesqu iterpene g lucoside.Trichodermoside ( 1) w as obtained as a w hite pow derw ith [ ]20D + 5. 76 ( c, 0. 26, M eOH ). The molecularformula C23H 39NO8 was determ ined acco rd ing to thequsia mo lecu lar ion peaks atm /z 480. 5 [M + N a]+and 937. 3 [ 2M + N a]+in ESIM S, and NMR data.The presence of a g lucosy l N acy l moiety w as revealed by the1H NMR signals at 4. 97 ( d, 3. 5, H1 ) , 3. 88 ( dd, 10. 2, 3. 1, H 2 ) , 3. 76 ( dd, 13. 4,4 0, H 3 ) , 3 64 ( ,t 9 4, H 4 ) , 3 45 ( ,t 8 9, H5 ) , 3 78 ( d, 5 0, H 6 ), 1 98 ( s, 3H, H 8 ), andthe13C NMR signa ls at 98 8 ( C 1 ), 54 6 ( C 2 ),72 0 ( C 3 ) , 72 8 ( C 4 ) , 71 0 ( C 5 ) , 61 7 ( C6 ) , 172 9 ( C 7 ), 22 5 ( C 8 ) [ 14] TheHMBC correlat ions o fH 1 /C 3 , H 2 /C 1 , C 3 and C 7 , H3 /C 4 and C 5 , H 6 /C 5 , andH 8 /C 7 conf irmedthis conc lusion ( Table 1)Besides the g lucosyl N acyl mo iety, the13C NMRspectra of 1 ( Table 1) d isplayed three quaternary carbons, three methy lenes ( one oxygenated ), five m ethines ( tw o o lefinic and tw o oxygenated ) , and fourmethy ls, ind icat ing the presence of a sesqu iterpenemoiety The fo llow ing HMBC corre lations w ere observed: H 13( 0 97) /C 3, C 4, C 5 and C 14, H 14( 0 70) /C 3, C 4, C 5 and C 13, H 12( 1 68) /C5, C 6, and C 1 In combinat ion w ith the1H,1H COSYcorre lations betw een H 1 /H 2 and H 2 /H 3 the fragment b w as established ( F ig 1) The fragm ent c cou ldbe also postulated from HMBC experiment The HMBCcorre lations from H 8 /C 5, C 7, C 9, C 10, and C 15Fig 1 The fragm en ts a, b, and c of compound 1,and the selected HMBC corre lation s ( HC ) and 1H, 1H COSY correlationsrevea led that fragments b and c w ere joined together.Therefore, the structure o f the sesqu iterpene mo iety w asdeterm ined. The g lycosyl was located at C 2 in v iew oftheHMBC correlationH 1 ( 4. 97) / C 2 ( 81. 1).Thus, the structure of 1was determ ined as shown in figure1. The relative configuration of 1 was determ ined byNOESY cross signals: H 3 / H 5, H 3/H3 13, H 2 /H 14.Table 1 The NMR data of com pound 1 ( CDC l3, ppm, J in Hz)No. H C HMBC1H, 1H COSY1 5. 28 ( s, 1H ) 122. 0 ( d ) C 3, C 5, C 12 H 22 3. 88 ( dd, 10. 2, 3. 1, 1H ) 81. 1 ( d) C 1 H 3, H 13 3. 34 ( d, 8. 6, 1H ) 78. 7 ( d) C 2, C 4, C 5, C 13, C 14 H 24 - 40. 0 ( s) - -5 1. 73 ( br s, 1H ) 49. 7 ( d) - H 76 - 139. 4 ( s) - -7 1. 37 ( m, 1H ) , 1. 20 ( m, 1H ) 25. 8 ( t) C 6, C 8 H 88 2. 18 ( m, 1H ) , 1. 95 ( m, 1H ) 41. 1 ( t) C 5, C 7, C 9, C 10, C 15 H 79 - 138. 9 ( s) - -10 5. 35 ( t, 6. 5, 1H ) 123. 9 ( d ) C 8, C 11, C 15 H 1111 4. 09 ( d, 6. 7, 2H ) 58. 9 ( t) C 9, C 10 H 1212 1. 68 ( s, 3H ) 21. 9 ( q) C 1, C 5, C 6 -545Vol 22 DU X i p ing, et al: A N ovel Sesqu iterpene G lu coside from Trichod erm a sp. PT213 0. 97 ( s, 3H ) 24. 4 ( q) C 3, C 4, C 5, C 14 -14 0. 70 ( s, 3H ) 14. 1 ( q) C 3, C 4, C 5, C 13 -15 1. 63 ( s, 3H ) 16. 1 ( q) C 8, C 9, C 10 -1 4. 97 ( d, 3. 5, 1H ) 98. 8 ( d) C 3 , C 1 H 2 2 3. 88 ( dd, 3. 1, 10. 2, 1H ) 54. 6 ( d) C 1 , C 3 , C 7 H 1 , H 3 3 3. 76 ( dd, 13. 4, 4. 0, 1H ) 72. 0 ( d) C 4 , C 5 H 2 4 3. 64 ( t, 9. 4, 1H ) 72. 8 ( d) C 2 , C 5 H 5 5 3. 45 ( t, 8. 9, 1H ) 71. 0 ( d) C 3 , C 6 H 4 6 3. 78 ( d, 5. 0, 2H ) 61. 7 ( t) C 5 H 5 7 - 172. 9 ( s) - -8 1. 98 ( s, 3H ) 22. 5 ( q) C 7 -BioassaysThe cy totox ic activ ity aga inst theH eLa ce ll line of compound 1 w asmeasured 72 h post treatment by theMTTmethod[ 15]. Compound 1 ( 10  g /mL) disp layed weakgrow th inhib it ion ( 12% ).Experim entalGeneralPrecoated TLC p lates w ere from Q ingdao H aiyangChem ical Factory, Q ingdao, P. R. China. For co lumnchrom atography ( CC ) , silica ge l ( 200 300, and 80100 m esh; Q ingdao ) , silica ge l 60 ( M erck ) , RP 18( M erck), and Sephadex LH 20 gel (Amersham B iosciences) w ere used. Optica l rotation w as m easured on aP erkin E lmer 341 ploarim eter w ith CHC l3 as so lven.tNMR Spectra w ere recorded on a B ruker DRX 500spectrometer ( in ppm re.l to M e4 S,i J in H z) , andM S spectra on a F inn igan LCQ A dvantage andVG Auto Spec 3000mass spectrom eters.Ferm entation of the StrainThe stra in PT2 w as ident ified as Trichoderma sp. according to its ITS sequence o f rDNA ( ITS1 5. 8SITS2) . The strain w as incubated on slope of 50% seaw ater potato dex trose agar ( PDA ) m edia in a test tubea t 28 for 7 d to affo rd seed cu ltures. Then the seedcultures w ere w ashed w ith sterile w ater, and the sporesw ere transferred to 3000 mL E rlenm eyer flasks eachcontain ing 1000 mL of 50% seaw ater potato dextrosebroth ( PDB ) for static incubat ion, room temperature,20 d.Extraction and isolationThe flask cultures w ere filtered and the mycelia w ereex tracted w ith acetone exhaustive ly. The acetone so lut ion w as co llected and evapo rated in vacuo to afford acrude extrac.t Then dissolved in methano l and part it ioned betw een petro leum ether and m ethano .l Thebrow n o il ( 17 g ) w as obtained from the m ethanolphase.The ex tract ( 17 g ) w as sub jected to MPLC ( 170 g,RP 18) eluted w ithH 2O, 30% , 50% , 70% , and 100%methano ,l respectively. Then f ive fract ions ( Fr. S1 S5)w ere obtained. Fr. S3 ( 98 mg) w as sub jected to CC( 45 g Sephadex LH 20; M eOH). A ll fractions w ere analyzed by TLC ( E toAc /M eOH 1!2) , and pooled into 3portions ( Fr. S31 S33) . Fr. S33 ( 74 mg) w as furtherseperated over CC ( silica ge,l CHC l3 / acetone 10!1, 5!1, 1!1, 1!2) to y ie ld 1 ( 5mg) .Trichodermoside [ N ( ( 2S, 3S, 4R, 5S) 2 ( ( 1S, 4S,6S ) 6 hydroxy 4 ( ( E ) 5 hydroxy 3m ethy lpent 3eny l) 3, 5, 5 trim ethy lcyclohex 2 enyloxy ) tetrahydro4, 5 d ihydroxy 6 ( hydroxymethy l) 2H pyran 3 y l) acetam ide, 1] [ ]20D + 5. 76 ( c, 0. 26, M eOH ), ESIM Sm /z 480. 5 [M + N a]+and 937. 3 [ 2M + Na]+, NMRdata, see Tab le 1.Acknow ledgements Th is w ork w as financ ia lly supported by Ch ina Ocean Resource R&D A ssoc iat ion( G rant DYXM 115 02 2 13 ) and the Nat iona l H ighTechnology R esearch andDevelopment Program o fCh ina ( 863, No. 2006AA09Z410) .R eferences1 Andrade R, Ayer WA, M ebe PP. The m etabo lites of T ri546 N a tP rod Res D ev Vo l 22choderm a longibrachia tum . Part ∀ . Iso la tion o f the m etabolites and the structure o f tr ichod im ero .l Can J Chem, 1992,70: 2526 2535.2 Andrade R, AyerWA, T rifonov LS. The m etabo lites o f T r ichoderm a long ibrach ia tum. Part II. The structures of tr ichoderm o lide and so rbiqu ino .l Can J Chem, 1996, 74: 371379.3 Rebuffat S, H lim i S, P r igen t Y, et al. Iso lation and structura le luc idation o f the 11 res idue pepta ibo l antibiotic, harzianinHK V I. J Chem Soc Perk in T rans 1, 1996, 16: 2021 2027.4 Augeven Bour I, Rebuffat S, Auv in C, et al. H arzian in HB I,an 11 residue pepta ibo l from T richod erm a harz ianum: iso lation, sequence, so lution synthesis and m embrane activ ity. JChem Soc P erkin T rans 1, 1997, 10: 1587 1594.5 Dun lop RW, S imon A, S ivasitham param K, et al. Chem ica lstud ies on the cecrop iaceae: a novel A ring seco triterpenefromM usanga cecrop io ides. JN at Prod, 1989, 52: 67 74.6 Cu tlerHG, H imm e lsbach DS, Yagen B, et al. K on ing in in B: ab io log ically ac tive congener of kon ing in in A from T richoderm a koningii. J Agric F ood Chem, 1991, 39: 977 980.7 Pa rker SR, CutlerHG, Schre iner PR. Kon ing inin C: a b io log ica lly active natura l product from T r ichoderm a koningii. B iosciB io tech B iochem, 1995, 59: 1126 1127.8 Cutle rHG, Cutler SJ, Ross SA, et al. K on ing inin G, a newm etabolite from T richoderma aureov iride. J Na t P rod, 1999,62: 137 139.9 A lm assi F, Gh isalbe rti EL, Narvey M J. N ew antib io tics fromstrains ofT r ichoderma harz ianum. J Nat Prod, 1991, 54: 396402.10 Gh isalberti EL, Row land CY. Antifunga lm etabo lites from T richoderma harzianum. J Nat P rod, 1993, 56: 1799 1804.11 Abe N, Yam am oto K, H iro ta K. Nove l funga l me tabo lites,deme thy lsorb icillin and oxo sorb ic illino ,l iso lated from T richoderma sp. USF 2690. B io sci B io tech B iochem , 2000, 64:620 622.12 Zhang J, Zheng ZH, H uang YJ, et al. The an titum or activ ity o fa lg ico lous fung .i J X iam en Univ, N at Sci. 2004, 43: 551 556.13 Zhang J. The screen ing for bioactive stra ins from a lg ico lousfung i and the prelim ina ry study on the secondarym etabolitesfrom three m arine fung .i M aster Deg ree Thesis, X iam en University, Ch ina, 2004.14 Vocad lo DJ, W ithers SG. Deta iled com parative analysis of thecata ly tic m echanism s of !N Acetyg lucosam in idases fromfam ilies 3 and 20 of g lycoside hydro lases. B iochem istry,2005, 44: 12809 12818.15 Mosm ann T. Rapid co lorim e tric assay for ce llular grow th andsurv iva:l app lication to proliferation and cy totox ity assay. JImmuno lM ethod s, 1983, 65: 55 63.547Vol 22 DU X i p ing, et al: A N ovel Sesqu iterpene G lu coside from Trichod erm a sp. PT2

A Novel Sesquiterpene Glucoside from Trichoderma sp. PT2

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A Novel Sesquiterpene Glucoside from Trichoderma sp. PT2

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