杜希萍,李瑤瑤,魯春華＊,鄭忠輝,沈月毛

1廈門大學(xué)生命科學(xué)學(xué)院細(xì)胞生物學(xué)與腫瘤細(xì)胞工程教育部重點(diǎn)實(shí)驗(yàn)室福建省藥物工程實(shí)驗(yàn)室,廈門 361005; 2集美大學(xué)生物工程學(xué)院,集美 361021

Introduction

The fungal genusTrichoder m ais a widespread saprophyte that occurs almost ubiquitously.Because of the useful secondary metabolites produced by this genus, many strains have received considerable attention as biocontrol agents.[1,2].Some secondarymetabolites,includingN-acylated peptides,peptibols[3,4],koninginins A-G[5-8],octaketide-derived compounds, two butenolide compounds[9,10],and demethylsorbicillin and oxosorbicillinol[11]were reported as antibioticswith various activities.During the course of examining fungi for biologically active natural products,the strain PT2 from B lidingia m in im aKylin,collected at Wuyu Island, Zhangzhou of Fujian Province,was isolated.The ethyl acetate extractof the fer mentation showed strong antitumor activities against KB and Raji cell lines[12].Cyclopeptideswere the active constituent[13].Our further research on the chemical components ofTrichoder m asp. PT2 yielded one new compound.Here the isolation, structure elucidation and bioactivity assay of this compound was described.

利用以上設(shè)置的相關(guān)參數(shù)，得到的內(nèi)河水域中的流場(chǎng)分布見圖2。圖2中同時(shí)標(biāo)示漂移物體在不同時(shí)刻可能位置的區(qū)間范圍，當(dāng)矩形區(qū)域與航道邊界存在交點(diǎn)時(shí)，就存在停止繼續(xù)漂移的可能性，假設(shè)物體的可能位置在相應(yīng)時(shí)刻在矩形區(qū)域內(nèi)為均勻分布，可得到物體最終漂移位置的概率分布情況。

Results and D iscussion

Fer mentation(10 L)was carried out at 28℃for 20 d without agitation.After filtration,the mycelia were extracted exhaustively with acetone.Acetone was evaporatedin vacuoand the crude extractwaspartitioned between methanol and petroleum ether.The methanol solution was collected and evaporated to drynessin vacuo to afford 17 g extract.The extractwaspurified over RP-18,SephadexLH-20,and silica gel columns to afford a novel sesquiterpene glucoside.

Trichodermoside(1)was obtained as a white powder with[α]+5.76(c,0.26,MeOH).The molecular for mula C23H39NO8was determined according to the qusia molecular ion peaks atm/z 480.5[M +Na]+and 937.3[2M +Na]+in ESI-MS,and NMR data. The presence of aα-glucosyl N-acyl moiety was revealed by the1H NMR signals atδ4.97(d,3.5,H-1′),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,H-5′),3.78(d,5.0,H-6′),1.98(s,3H,H-8′),and the13C NMR signals atδ98.8(C-1′),54.6(C-2′), 72.0(C-3′),72.8(C-4′),71.0(C-5′),61.7(C-6′),172.9(C-7′),22.5(C-8′)[14].The HMBC correlations of H-1′/C-3′,H-2′/C-1′,C-3′and C-7′,H-3′/C-4′and C-5′,H-6′/C-5′,and H-8′/C-7′confir med this conclusion(Table 1).

Besides theα-glucosyl N-acyl moiety,the13C NMR spectra of 1(Table 1)displayed three quaternary carbons,three methylenes(one oxygenated),five methines( two olefinic and two oxygenated),and four methyls,indicating the presence of a sesquiterpene moiety.The following HMBC correlations were observed:H-13(δ0.97)/C-3,C-4,C-5 and C-14,H-14 (δ0.70)/C-3,C-4,C-5 and C-13,H-12(δ1.68)/C-5,C-6,and C-1.In combination with the1H,1H-COSY correlations between H-1/H-2 and H-2/H-3 the fragment b was established(Fig.1).The fragment c could be also postulated from HMBC experiment.The HMBC correlations from H-8/C-5,C-7,C-9,C-10,and C-15 revealed that fragments b and c were joined together. Therefore,the structure of the sesquiterpene moietywas deter mined.The glycosylwas located at C-2 in view of the HMBC correlation H-1′(δ4.97)/C-2(δ81.1). Thus,the structure of 1was deter mined as shown in figure 1.The relative configuration of 1was deter mined byNOESY cross signals:H-3/H-5,H-3/H3-13,H-2/H-14.

The strain PT2 was identified asTrichoder m asp.according to its ITS sequence of rDNA (ITS1-5.8SITS2).The strain was incubated on slope of 50%seawater potato dextrose agar(PDA)media in a test tube at 28℃for 7 d to afford seed cultures.Then the seed cultureswere washed with sterile water,and the spores were transferred to 3000 mL Erlenmeyer flasks each containing 1000 mL of 50% seawater potato dextrose broth(PDB)for static incubation,room temperature, 20 d.

Fig.1 The fragments a,b,and c of compound 1, and the selected HM BC correlations(H→C)and1H,1H-COSY correlations

Table 1 The NM R data of compound 1(CDCl3,ppm,Jin Hz)

13 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′ -

Precoated TLC plates were fromQ ingdao Haiyang Chem ical Factory,Qingdao,P.R.China.For column chromatography(CC),silica gel(200-300,and 80-100 mesh;Qingdao),silica gel 60(Merck),RP-18 (Merck),and SephadexLH-20 gel(Amersham Biosciences)were used.Optical rotation was measured on a Perkin-E lm er341 ploar imeter with CHCl3as solvent. NMR Spectra were recorded on aB ruker DRX-500 spectrometer(δin ppm rel.to Me4Si,Jin Hz),and MS spectra on a Finnigan LCQ-Advantage and VGAuto-Spec-3000 mass spectrometers.

當(dāng)需要感冒藥來緩解癥狀時(shí)，盡量只吃一種藥，針對(duì)癥狀選擇含有相應(yīng)治療成分的藥品，能選擇單一成分的盡量用單一成分藥品。多個(gè)癥狀同時(shí)出現(xiàn)時(shí)，要針對(duì)癥狀選擇含有相應(yīng)治療成分的復(fù)方感冒藥。含有同類成分的不同感冒藥不要同時(shí)服用，比如“艾暢”和“惠菲寧”，同服可導(dǎo)致偽麻黃堿過量。

Exper imental

3.2 不同品種受凍程度差異較大 不同品種，因花期、花量、抗寒性存在差異，受凍后坐果情況不同。從調(diào)查結(jié)果看，富士系、元帥系等品種，受凍減產(chǎn)幅度較大；嘎拉系、秦冠、金冠系品種，因地域差別，減產(chǎn)幅度各有不同，但幼果霜環(huán)現(xiàn)象較為普遍。近年在寶雞產(chǎn)區(qū)示范推廣的瑞陽(yáng)、瑞雪、蜜脆、粉紅女士、魔迪等新品種，受凍減產(chǎn)幅度小，隴縣盛源果品公司栽培的瑞陽(yáng)、瑞雪等品種花器凍害輕，坐果率高，產(chǎn)量、質(zhì)量在大災(zāi)之年幾乎未受影響。

Bioassays

General

Fermentation of the Stra in

The cytotoxic activity against the HeLa cell line of compound 1 wasmeasured 72 h post treatment by theMTT method[15].Compound 1(10μg/mL)displayed weak growth inhibition(12%).

疣體面積減少大于50%的小組的最終治愈率為47%；疣體面積減少小于50%的小組最終治愈率為34%，P=0.133差異無統(tǒng)計(jì)學(xué)意義。

Extraction and isolation

The flask cultures were filtered and the mycelia were extracted with acetone exhaustively.The acetone solution was collected and evaporated in vacuo to afford a crude extract.Then dissolved in methanol and partitioned between petroleum ether and methanol.The brown oil(17 g)was obtained from the methanol phase.

The extract(17 g)was subjected to MPLC(170 g, RP-18)eluted with H2O,30%,50%,70%,and 100% methanol,respectively.Then five fractions(Fr.S1-S5) were obtained.Fr.S3(98 mg)was subjected to CC (45 g SephadexLH-20;MeOH).All fractionswere analyzed by TLC(EtoAc/MeOH 1∶2),and pooled into 3 portions(Fr.S31-S33).Fr.S33(74 mg)was further seperated over CC(silica gel,CHCl3/acetone 10∶1,5∶1,1∶1,1∶2)to yield 1(5 mg).

Trichodermoside[N-((2S,3S,4R,5S)-2-((1S,4S, 6S)-6-hydroxy-4-((E)-5-hydroxy-3-methylpent-3-enyl)-3,5,5-tr imethylcyclohex-2-enyloxy)-tetrahydro-4,5-dihydroxy-6-(hydroxymethyl)-2H-pyran-3-yl)acetamide,1][α+5.76(c,0.26,MeOH),ESI-MS m/z480.5[M+Na]+and 937.3[2M+Na]+,NMR data,see Table 1.

Acknowledgements This work was financially supported by China Ocean Resource R&D Association (Grant DYXM-115-02-2-13)and the National High Technology Research and Development Program of China(863,No.2006AA09Z410).

1 Andrade R,Ayer WA,Mebe PP.The metabolites ofTri-choder ma longibrachiatum.PartⅠ.Isolation of the metabolites and the structure of trichodimerol.Can J Chem,1992, 70:2526-2535.

2 Andrade R,Ayer WA,Trifonov LS.The metabolites ofTrichoder malongibrachiatum.Part II.The structures of trichoder molide and sorbiquinol.Can J Chem,1996,74:371-379.

3 Rebuffat S,Hlimi S,Prigent Y,et al.Isolation and structural elucidation of the 11-residue peptaibol antibiotic,harzianin HKV I.J Chem Soc Perkin Trans1,1996,16:2021-2027.

4 Augeven-Bour I,Rebuffat S,Auvin C,et al.Harzianin HB I, an 11-residue peptaibol fromTrichoder m a harzianum:isolation,sequence,solution synthesis and membrane activity.J Chem Soc Perkin Trans1,1997,10:1587-1594.

5 Dunlop RW,Simon A,Sivasithamparam K,et al.Chemical studies on the cecropiaceae:a novel A-ring seco triterpene fromM usanga cecropioides.J Nat Prod,1989,52:67-74.

6 Cutler HG,Himmelsbach DS,Yagen B,et al.Koninginin B:a biologically active congener of koninginin A fromTrichoderm a koningii.J Agric Food Chem,1991,39:977-980.

7 Parker SR,CutlerHG,Schreiner PR.Koninginin C:a biologically active natural product fromTrichoder m a koningii.B iosci B iotech B iochem,1995,59:1126-1127.

8 Cutler HG,Cutler SJ,Ross SA,et al.Koninginin G,a new metabolite fromTrichoder ma aureoviride.J Nat Prod,1999, 62:137-139.

9 Almassi F,Ghisalberti EL,NarveyMJ.New antibiotics from strains ofTrichoder ma harzianum.J Nat Prod,1991,54:396-402.

10 Ghisalberti EL,Rowland CY.Antifungalmetabolites fromTrichoder ma harzianum.J Nat Prod,1993,56:1799-1804.

11 Abe N,Yamamoto K,Hirota K.Novel fungal metabolites, demethylsorbicillin and oxosorbicillinol,isolated from Trichoder masp.USF-2690.B iosci B iotech B iochem,2000,64: 620-622.

12 ZhangJ,Zheng ZH,Huang YJ,et al.The antitumor activityof algicolous fungi.J Xiam en Univ,Nat Sci.2004,43:551-556.

13 Zhang J.The screening for bioactive strains from algicolous fungi and the preliminary study on the secondarymetabolites from three marine fungi.Master'Degree Thesis,Xiamen University,China,2004.

14 Vocadlo DJ,W ithers SG.Detailed comparative analysis of the catalytic mechanisms ofβ-N-Acetyglucosaminidases from families 3 and 20 of glycoside hydrolases.B iochem istry, 2005,44:12809-12818.

15 Mos mann T.Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxity assay.J ImmunolM ethods,1983,65:55-63.