分离自木霉 PT2的一个新倍半萜糖苷

杜希萍,李瑶瑶,鲁春华＊,郑忠辉,沈月毛

1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

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.

利用以上设置的相关参数，得到的内河水域中的流场分布见图2。图2中同时标示漂移物体在不同时刻可能位置的区间范围，当矩形区域与航道边界存在交点时，就存在停止继续漂移的可能性，假设物体的可能位置在相应时刻在矩形区域内为均匀分布，可得到物体最终漂移位置的概率分布情况。

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.

当需要感冒药来缓解症状时，尽量只吃一种药，针对症状选择含有相应治疗成分的药品，能选择单一成分的尽量用单一成分药品。多个症状同时出现时，要针对症状选择含有相应治疗成分的复方感冒药。含有同类成分的不同感冒药不要同时服用，比如“艾畅”和“惠菲宁”，同服可导致伪麻黄碱过量。

Exper imental

3.2 不同品种受冻程度差异较大 不同品种，因花期、花量、抗寒性存在差异，受冻后坐果情况不同。从调查结果看，富士系、元帅系等品种，受冻减产幅度较大；嘎拉系、秦冠、金冠系品种，因地域差别，减产幅度各有不同，但幼果霜环现象较为普遍。近年在宝鸡产区示范推广的瑞阳、瑞雪、蜜脆、粉红女士、魔迪等新品种，受冻减产幅度小，陇县盛源果品公司栽培的瑞阳、瑞雪等品种花器冻害轻，坐果率高，产量、质量在大灾之年几乎未受影响。

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%).

疣体面积减少大于50%的小组的最终治愈率为47%；疣体面积减少小于50%的小组最终治愈率为34%，P=0.133差异无统计学意义。

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.