西澳粘滑菇的化學成分研究

邵紅軍,房立真,劉吉開

1陜西師范大學食品工程與營養(yǎng)科學學院,西安 710062;2新鄉(xiāng)醫(yī)學院藥學院,新鄉(xiāng) 453003;3中國科學院昆明植物研究所植物化學與西部資源持續(xù)利用國家重點實驗室,昆明 650204

西澳粘滑菇的化學成分研究

邵紅軍1,3＊,房立真2,3,劉吉開3

1陜西師范大學食品工程與營養(yǎng)科學學院,西安 710062;2新鄉(xiāng)醫(yī)學院藥學院,新鄉(xiāng) 453003;3中國科學院昆明植物研究所植物化學與西部資源持續(xù)利用國家重點實驗室,昆明 650204

利用各種常規(guī)色譜分離技術從西澳粘滑菇 (Hebelom a westraliense)發(fā)酵液中分離得到 5個化合物,經波譜學分析鑒定為Volemolide(1)、過氧化麥角甾醇 (2)、對羥基苯甲酸 (3),對甲氧基苯乙酸 (4)和對羥基苯乙醇(5)。其中化合物 1是一個七降麥角甾醇類物質,系首次從絲膜菌科真菌中分離得到。

西澳粘滑菇;發(fā)酵液;Volemolide

擔子菌亞門絲膜菌科 (Cortinariaceae)滑銹傘屬(Hebelom a)包括多種有毒或非食用性種類,在北歐洲現已發(fā)現 45種[1]。目前僅有 5種該屬真菌的化學成分和活性得到研究[2-11]。我們曾對采自云南哀牢山的該屬真菌黃滑銹傘 (H.versipelle)的化學成分進行了研究,并從中發(fā)現了 1個具有中等抗腫瘤活性的新羊毛甾烷型三萜[12]。為了進一步尋找該屬真菌中的生理活性物質,本實驗對西澳粘滑菇[13](H.westraliense)發(fā)酵液的化學成分進行了研究。從中分離并鑒定了 5個化合物,分別是 Volemolide (1)、過氧化麥角甾醇 (2)、對羥基苯甲酸 (3)、甲氧基苯乙酸 (4)和對羥基苯乙醇 (5)。其中化合物 1系首次從絲膜菌科真菌中分離得到,是一個七降麥角甾醇類化合物。Kenji等[14]首次報道該化合物分離于多汁乳菇 (Lactarius volem us),并認為其是紅菇科、牛肝菌科等真菌化學分類的關鍵物質,其活性有待進一步研究。

1 儀器與材料

XRC-1型顯微熔點儀 (四川大學科學儀器廠),溫度計未校正;Bruker AM-400核磁共振儀,T MS為內標;VG Auto Spec-3000質譜儀測定;薄層層析硅膠和柱層析硅膠 (青島海洋化工廠);Sephadex LH-20(Merk)。顯色方法為 254、365 nm熒光、10%硫酸乙醇溶液和硫酸香草醛處理后加熱顯色及碘蒸氣顯色。

西澳粘滑菇(H.westraliense)菌株由昆明植物研究所紀大干研究員鑒定并分離。標本菌株存于昆明植物研究所隱花植物標本館。

2 發(fā)酵培養(yǎng)

西澳粘滑菇菌種采用平皿轉搖瓶液體培養(yǎng)方法。培養(yǎng)基:葡萄糖 40 g,去皮土豆 400 g,MgSO43.0 g,KH2PO46.0 g,VB120.0 mg,酵母膏 4 g;加水混合攪拌至用檸檬酸調 pH 6.5,溫度 25℃;轉速: 170 r/min;暗中培養(yǎng)發(fā)酵 25 d,共得發(fā)酵液約 12 L。

3 提取與分離

西澳粘滑菇發(fā)酵液 12 L,用乙酸乙酯萃取 3次(12 h/次,室溫),過濾。合并濾液并濃縮,得浸膏約1.15 g。浸膏用甲醇溶解后與 2 g RP-8硅膠拌樣,上 RP-8柱,用甲醇-水梯度洗脫。甲醇洗脫部分(0.383 g)經反復柱層析得到化合物 1(3 mg)、2(4 mg)。在甲醇-水 (8∶2)部分,經葡聚糖凝膠層析細分后用薄層制備得到化合物 4(4 mg)、5(3 mg)。在甲醇-水(4∶6)部分,經葡聚糖凝膠層析細分,得到化合物 3(26 mg)。

4 結構鑒定

Volemolide(1,2,3,4,5,10,19-heptanorergosta-9α-methoxy-7,22-dien-6,9-lactone,1) 無色油狀物;C22H36O3;EI-MSm/z(rel.int.):346[M]+(15),314(20),271(70),189(100),161(65),133 (45),83(67),69(96),55(90);1H NMR (400 MHz,CD3Cl)δH:5.75(d,J=1.7 Hz,H-7),1.77 (1H,m,H-11a),2.33(1H,ddd,J=13.3,3.5,2.2 Hz,H-11b),1.57(1H,m,H-12a),1.93(1H,m,H-12b),2.41(1H,ddd,J=9.4,6.0,1.7 Hz,H-14), 1.49(1H,m,H-15a),1.73(1H,m,H-15b),1.48 (1H,m,H-16a),1.90(1H,m,H-16b),1.50(1H, m,H-17),0.61(3H,s,H-18),2.06(1H,m,H-20), 1.03(3H,d,J=6.6 Hz,H-21),5.16(1H,dd,J= 15.3,7.3 Hz,H-22),5.26(1H,dd,J=15.3,7.3 Hz,H-23),1.87(1H,m,H-24),1.48(1H,m,H-25),0.82(3H,d,J=6.7 Hz,H-26),0.84(3H,d,J=6.7 Hz,H-27),0.92(3H,d,J=6.7 Hz,H-28), 3.12(3H,s,OMe);13C NMR(100MHz,CD3Cl)δC: 170.7(s,C-6),114.2(d,C-7),169.1(s,C-8), 107.8(s,C-9),34.5(t,C-11),35.1(t,C-12), 48.9(s,C-13),50.6(d,C-14),21.3(t,C-15), 28.9(t,C-16),55.5(d,C-17),11.9(q,C-18), 40.1(d,C-20),21.0(q,C-21),134.6(d,C-22), 132.9(d,C-23),42.8(d,C-24),33.0(d,C-25), 19.7(q,C-26),20.0(q,C-27),17.6(q,C-28), 50.1(s,OMe)。上述數據與文獻[14]報道化合物volemolide數據基本一致。

過氧化麥角甾醇 (ergosterol peroxide,2) 無色針晶;C28H44O3;mp.179～181°C;EI-MSm/z(rel.int.):428[M]+(5),410(8),396(10),251 (24),161(29),149(31),107(37),81(56),69 (100),55(73);1H NMR(400 MHz,CDCl3)δC: 6.49(1H,d,J=8.5 Hz),6.23(1H,d,J=8.5 Hz), 5.20(1H,dd,J=7.6,7.5 Hz),5.12(1H,dd,J= 7.6,7.5 Hz),3.94(1H,m),2.08～1.49(20H,m), 1.20(3H,s),0.98(3H,d,J=6.6 Hz),0.88(3H,d,J=6.8 Hz),0.86(3H,s),0.82(3H,d,J=3.6 Hz), 0.79(3H,d,J=3.4 Hz);13C NMR(100 MHz, CDCl3)δC:135.4,135.2,132.3,130.7,82.1,79.4, 66.5,56.2,51.7,51.1,44.6,42.7,39.7,39.4, 37.0,37.0,34.7,33.1,30.1,28.6,23.4,20.9, 20.6,20.0,19.6,18.1,17.5,12.9。以上波譜數據與文獻[15]一致。

對羥基苯甲酸 (4-hydroxybenzoic acid,3) 無色結晶,FeCl3反應陽性;mp.214～216℃;EI-MSm/z(rel.int.):138[M]+(84),121(100),93(30),65 (23)。與標準品點板對照一致,確定為對羥基苯甲酸。

對甲氧基苯乙酸 (4-methoxyphenylacetic acid, 4) 無色固體,mp.85℃;EI-MSm/z(rel.int.): 166[M]+(33),149(27),107(100),57(14);1H NMR(400 MHz,CD3OD)δH:3.56(2H,s,H-2), 6.76(2H,dd,J=8.5,1.5 Hz,H-2′,6′),7.13(2H, dd,J=8.5,1.5 Hz,H-3′,5′),3.70(3H,s,OMe);13C NMR(100 MHz,CD3OD)δC:172.7(s,C-1),40.2 (t,C-2),115.5(s,C-1′),130.4(d,C-2′,6′),115.1 (d,C-3′,5′),154.8(s,C-4′),52.1(q,OMe)。以上波譜數據與文獻[16]一致。

對羥基苯乙醇 (4-hydroxy-benzeneethanol,5)無色結晶,FeCl3反應陽性;mp.90～91℃;EI-MSm/z(rel.int.):138[M]+(28),107(100),77 (14);1H NMR(400MHz,CD3OD)δH:3.67(2H,t,J=7.2 Hz,H-1),2.70(2H,t,J=7.2 Hz,H-2),6.69 (2H,d,J=8.3 Hz,H-2′,6′),7.02(2H,d,J=8.3 Hz,H-3′,5′);13C NMR(100 MHz,CD3OD)δC:64.6 (t,C-1),39.4(t,C-2),115.3(s,C-1′),130.8(d, C-2′,6′),116.1(d,C-3′,5′),156.7(s,C-4′)。其核磁數據與文獻[17]一致。

1 JanV.FungiofNorthern Europe,Volume 3:the GenusHebeloma,DanishMycological Society,2005.

2 BernardiMD,Fronza G,GianottiMP,et al.Fungal metabo-lites XIII:New cytotoxic triterpene from Hebeloma species (Basidiomycetes).Tetrahedron Lett,1983,24:1635-638.

3 Fujimoto H,Takano Y,YamazakiM.Isolation,identification and pharmacological studieson three toxicmetabolites from a mushroom,Hebelom a spoliatum.Chem Phar m Bull,1992,40: 869-872.

4 Bocchi M,Garlaschelli L,Vidari G,et al.New farnesane fesquiterpenes fromHebelom a senescens.J Nat Prod,1992, 55:428-431.

5 GarlaschelliL,Vidari G,VirtuaniM,et al.The structures of new lanostane triterpenes from the fruiting bodies ofHebeloma senescens.J Nat Prod,1995,58:992-1002.

6 Fujimoto H,Suzuki K,Hag iwara H,et al.New toxic metabolites from a mushroom,Hebeloma vinosophyllum.I.structures of hebevinosides I,II,III, IV,and V.Chem Phar m Bull, 1986,34:88-99.

7 Fujimoto H,Hagiwara H,Suzuki K,et al.New toxic metabolites from a mushroom,Hebeloma vinosophyllum.II.Isolation and structures of hebevinosidesV I,V II,V III,I X,X,and XI.Chem Phar m Bull,1987,35:2254-2260.

8 Fujimoto H,Maeda K,Yamazaki M.New toxic metabolites from a mushroom,Hebelom a vinosophyllum.III.Isolation and structures of three new glycosides,hebevinosides XII,XIII, and X IV,and productivity of the hebevinosides at three growth stages of the mushroom.Chem Phar m Bull,1991,39: 1958-1961.

9 W ichlacz M,Ayer WA,Trifonov LS,et al.Cis-fused caryophyllenes from liquid cultures ofHebelom a longicaudum. Phytochem istry,1999,51:873-877.

10 W ichlaczM,AyerWA,Trifonov LS,et al.A caryophyllenerelated sesquiterpene and two 6,7-secocaryophyllenes from liquid cultures ofHebelom a longicaudum.J Nat Prod,1999, 62:484-486.

11 W ichlacz M,Ayer WA,Trifonov LS,et al.Two 6,7-secocaryophyllenes and an alloaromadendrane from liquid cultures ofHebeloma longicaudum.Phytochem istry,1999,52: 1421-1425.

12 Shao HJ,Qing C,Wang F,et al.A new cytotoxic lanostane triterpenoid from the basidiomyceteHebelom a versipelle.J Antibiot,2005,58:828-831.

13 YuanMS(袁明生),Sun PQ(孫佩瓊).Sichuan Mushroom (四川蕈菌),1stEd.Chengdu:Sichuan Science and Technology Press,1995.

14 Kenji K,TomonariW.Volemolide,a novel norsterol from the fungusLactarius volem us.B iosci,B iochem,B iotech,1994,58: 1542-1544.

15 Shao ZY(邵志宇),Guo Y W(郭躍偉),Yu JL(于嘉陵),et al.Studied on chemical constitution ofDysidea sp.from south China sea.Nat Prod Res Dev(天然產物研究與開發(fā)), 2004,16:19-22.

16 Gao JM(高錦明).Chemical constituents of five higher fungi.Kunming:Kunming Institute of Botany,Chinese Academy of Sciences(中國科學院昆明植物研究所),PhD,2001.

17 Wan YG(苑艷光),WangLQ(王錄全),Wu LJ(吳立軍),et al.Studies on the chemical constituents of the stems ofAcanthopanax senticosusHarms.J Shenyang Phar m Univ(沈陽藥科大學學報).2002,19:325-327.

Chem ical Constituents of Culture Broth ofHebelom a westraliense

SHAO Hong-jun1,3＊,FANGLi-zhen2,3,L IU Ji-kai3

1College of Food Engineering and Nutritional Science,ShaanxiNor m al University,Xi’an 710062,China;2School of Phar macy,Xinxiang M edical College,Xinxiang 453003,China;3State Key Laboratory of Phytochem istry and Plant Resources in W est China,Kunm ing Institute of Botany,Chinese Academ y of Sciences,Kunm ing 650204,China

Volemolide(1),a norsterolwith a heptanorergostane skeletonwas isolated from the culture broth ofHebelom a westraliense,togetherwith other four known compounds including ergosterolperoxide(2),4-hydroxybenzoic acid(3),4-methoxyphenylacetic acid(4),and 4-hydroxy-benzeneethanol(5).The structures of these compounds were elucidated by spectroscopic methods,and the compound 1 was firstly founded in the Cortinariaceae.

Hebelom a westraliense;culture broth;volemolide

R284.1;Q914.83

A

1001-6880(2010)05-0786-03

2010-03-05 接受日期:2010-05-20

國家自然科學基金(30470027)

＊通訊作者 Tel:86-29-85310517;E-mail:shaohj@snnu.edu.cn