西澳粘滑菇的化学成分研究

邵红军,房立真,刘吉开

1陕西师范大学食品工程与营养科学学院,西安 710062;2新乡医学院药学院,新乡 453003;3中国科学院昆明植物研究所植物化学与西部资源持续利用国家重点实验室,昆明 650204

西澳粘滑菇的化学成分研究

邵红军1,3＊,房立真2,3,刘吉开3

1陕西师范大学食品工程与营养科学学院,西安 710062;2新乡医学院药学院,新乡 453003;3中国科学院昆明植物研究所植物化学与西部资源持续利用国家重点实验室,昆明 650204

利用各种常规色谱分离技术从西澳粘滑菇 (Hebelom a westraliense)发酵液中分离得到 5个化合物,经波谱学分析鉴定为Volemolide(1)、过氧化麦角甾醇 (2)、对羟基苯甲酸 (3),对甲氧基苯乙酸 (4)和对羟基苯乙醇(5)。其中化合物 1是一个七降麦角甾醇类物质,系首次从丝膜菌科真菌中分离得到。

西澳粘滑菇;发酵液;Volemolide

担子菌亚门丝膜菌科 (Cortinariaceae)滑锈伞属(Hebelom a)包括多种有毒或非食用性种类,在北欧洲现已发现 45种[1]。目前仅有 5种该属真菌的化学成分和活性得到研究[2-11]。我们曾对采自云南哀牢山的该属真菌黄滑锈伞 (H.versipelle)的化学成分进行了研究,并从中发现了 1个具有中等抗肿瘤活性的新羊毛甾烷型三萜[12]。为了进一步寻找该属真菌中的生理活性物质,本实验对西澳粘滑菇[13](H.westraliense)发酵液的化学成分进行了研究。从中分离并鉴定了 5个化合物,分别是 Volemolide (1)、过氧化麦角甾醇 (2)、对羟基苯甲酸 (3)、甲氧基苯乙酸 (4)和对羟基苯乙醇 (5)。其中化合物 1系首次从丝膜菌科真菌中分离得到,是一个七降麦角甾醇类化合物。Kenji等[14]首次报道该化合物分离于多汁乳菇 (Lactarius volem us),并认为其是红菇科、牛肝菌科等真菌化学分类的关键物质,其活性有待进一步研究。

1 仪器与材料

XRC-1型显微熔点仪 (四川大学科学仪器厂),温度计未校正;Bruker AM-400核磁共振仪,T MS为内标;VG Auto Spec-3000质谱仪测定;薄层层析硅胶和柱层析硅胶 (青岛海洋化工厂);Sephadex LH-20(Merk)。显色方法为 254、365 nm荧光、10%硫酸乙醇溶液和硫酸香草醛处理后加热显色及碘蒸气显色。

西澳粘滑菇(H.westraliense)菌株由昆明植物研究所纪大干研究员鉴定并分离。标本菌株存于昆明植物研究所隐花植物标本馆。

2 发酵培养

西澳粘滑菇菌种采用平皿转摇瓶液体培养方法。培养基: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;加水混合搅拌至用柠檬酸调 pH 6.5,温度 25℃;转速: 170 r/min;暗中培养发酵 25 d,共得发酵液约 12 L。

3 提取与分离

西澳粘滑菇发酵液 12 L,用乙酸乙酯萃取 3次(12 h/次,室温),过滤。合并滤液并浓缩,得浸膏约1.15 g。浸膏用甲醇溶解后与 2 g RP-8硅胶拌样,上 RP-8柱,用甲醇-水梯度洗脱。甲醇洗脱部分(0.383 g)经反复柱层析得到化合物 1(3 mg)、2(4 mg)。在甲醇-水 (8∶2)部分,经葡聚糖凝胶层析细分后用薄层制备得到化合物 4(4 mg)、5(3 mg)。在甲醇-水(4∶6)部分,经葡聚糖凝胶层析细分,得到化合物 3(26 mg)。

4 结构鉴定

Volemolide(1,2,3,4,5,10,19-heptanorergosta-9α-methoxy-7,22-dien-6,9-lactone,1) 无色油状物;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)。上述数据与文献[14]报道化合物volemolide数据基本一致。

过氧化麦角甾醇 (ergosterol peroxide,2) 无色针晶;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。以上波谱数据与文献[15]一致。

对羟基苯甲酸 (4-hydroxybenzoic acid,3) 无色结晶,FeCl3反应阳性;mp.214～216℃;EI-MSm/z(rel.int.):138[M]+(84),121(100),93(30),65 (23)。与标准品点板对照一致,确定为对羟基苯甲酸。

对甲氧基苯乙酸 (4-methoxyphenylacetic acid, 4) 无色固体,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)。以上波谱数据与文献[16]一致。

对羟基苯乙醇 (4-hydroxy-benzeneethanol,5)无色结晶,FeCl3反应阳性;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′)。其核磁数据与文献[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(孙佩琼).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(郭跃伟),Yu JL(于嘉陵),et al.Studied on chemical constitution ofDysidea sp.from south China sea.Nat Prod Res Dev(天然产物研究与开发), 2004,16:19-22.

16 Gao JM(高锦明).Chemical constituents of five higher fungi.Kunming:Kunming Institute of Botany,Chinese Academy of Sciences(中国科学院昆明植物研究所),PhD,2001.

17 Wan YG(苑艳光),WangLQ(王录全),Wu LJ(吴立军),et al.Studies on the chemical constituents of the stems ofAcanthopanax senticosusHarms.J Shenyang Phar m Univ(沈阳药科大学学报).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

国家自然科学基金(30470027)

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