火木层孔菌液体培养物的化学成分研究

目的:研究针层孔属火木层孔菌Phellinus igniarius液体培养物的化学成分并对其在多种体外药理模型上进行随机活性筛选.方法:分别对发酵液及菌丝体的化学成分进行研究,采用硅胶柱色谱、Sephadex LH-20柱色谱以及反相HPLC柱色谱等分离方法进行分离纯化;运用光谱数据解析鉴定化合物结构;在细胞水平模型上,筛选化合物在肿瘤细胞毒、神经保护、肝保护、抗炎和抗HIV等方面的活性.结果:从发酵液和菌丝体2部分共分离鉴定了29个化合物,分别为3个倍半萜:3S,9R,10S-3-羟基-11,12-O-异丙基血苋烷烯(1),3S,9R,10S-3,11,12-三羟基血苋烷烯(2)和3S,4S,9R,10-11,12,14-三羟基血苋烷烯(3);3个甾体:24R-麦角甾-4,6,8(14),22-四烯-3-酮(4),豆甾-7,22-二烯-3β,5α,6α-三醇(5)和麦角甾5α,8α-过氧-6,22-二烯-3β-醇(6);14个环二肽:环(L-脯氨酸-L-缬氨酸)(7),环(L-亮氨酸-D-脯氨酸)(8),环(L-亮氨酸-L-脯氨酸)(9),环(异亮氨酸-脯氨酸)(10),环(甘氨酸-亮氨酸)(11),环(苯丙氨酸-丝氨酸)(12),环(丙氨酸-脯氨酸)(13),环(丙氨酸-苯丙氨酸)(14),环(4-羟基-脯氨酸-苯丙氨酸)(15),环(L-苯丙氨酸-D-脯氨酸)(16),环(D-苯丙氨酸-D-脯氨酸)(17),环(6-羟基-脯氨酸-苯丙氨酸)(18),环(谷氨酸氨-脯氨酸)(19)和环(天冬氨酸-亮氨酸)(20);9个其他类化合物:乙酰氨基苯丙氨酸(21)、腺苷(22)、苯乙二醇(23)、邻-羟基苯乙醇(24)、苯甲酸(25)、对-甲氧基苯甲酸(26)、间-甲氧基苯甲酸(27)、十六烷酸(28)和3-吡啶羧酸(29).化合物5和8在1×10-5mol·L-1时能够有效地抑制MPP+诱导的PC12-syn细胞损伤,相对保护率分别为90.3%和87.5%,与模型组比较有显著性差异(P<0.05).在1×10-5mol·L-1浓度下,化合物12和18可较好地保护DL-半乳糖胺诱导的WB-F344细胞损伤,细胞成活率分别为25%和24%(双环醇作为对照,细胞成活率为24%).结论:化合物1-29均为首次从针层孔属火木层孔菌液体培养物中分离鉴定;化合物5和S有神经细胞保护活性,化合物12和18具有肝细胞损伤保护活性,其他化合物在测试浓度下在以上筛选模型中未显示明确活性.

Studies on constituents of cultures of fungus Phellinus igniarius

Objective : To investigate the chemical constituents of the culture of Phellinus igniarius and their phamacological activities. Method : The constituents were isolated by using a combination of various chromatographic techniques including column chromatography over silica gel, Sephadex LH-20, and reversed-phase HPLC. Structures of the isolates were identified by spectroscopic data analysis. Cytotoxic, neuroprotective, hepatoprotective, anti-inflammatory, and anti-HIV activities were screened by using cell-based models. Result : Twenty-nine constituents were isolated. Their structures were identified as three sesquiterpenes: 3S,9R,10S-3-hydroxy-11, 12-O-isopropyldrimene ( 1 ), 3S, 9R, 10S-3, 11, 12-trihydroxydrimene ( 2 ), and 3S, 4S, 9R, 10S-11, 12, 14-trihydroxydrimene ( 3 ); three steriods: 24R-ergosta-4, 6, 8(14), 22-tetraen-3-one ( 4 ), stigmasta-7, 22-diene-3b, 5a, 6a-triol ( 5 ), and 5a, 8a-epi dioxyergosta-6, 22-diene-3b-ol ( 6 ); fourteen cyclo-dipeptide: cyclo (L-Pro-L-Val) ( 7 ), cyclo (L-Leu-D-Pro) ( 8 ), cyclo (L-Leu-L-Pro) ( 9 ), cyclo (ILe-Pro) ( 10 ), cyclo (Gly-Leu) ( 11 ), cyclo (Phe-Ser) ( 12 ), cyclo (Ala-Pro) ( 13 ), cyclo (Ala-Phe) ( 14 ), cyclo (4-HyP-Phe) ( 15 ), cyclo (L-Phe-D-Pro) ( 16 ), cyclo (D-Phe-D-Pro) ( 17 ), cyclo (6-HyP-Phe) ( 18 ), cycle (Gln-Pro) (19), and cyclo (Asn-Leu) ( 20 ); and nine other compounds: N-acetyl-phenylalanine ( 21 ), adenosine ( 22 ), phenyldiethanol ( 23 ), o-hydroxy-phenylethanol ( 24 ), benzoic acid ( 25 ), p-methoxybenzoic acid ( 26 ), m-methoxybenzoic acid ( 27 ), hexadecanoic acid ( 28 ), and 3-pyridinecarboxylic acid ( 29 ). In the in vitro assays, at a concentration of 1×10^-5 mol·L^-1, compounds 5 and 8 showed neuroprotective activity against MPP⁺ induced PC12-syn cell damage, with a relative cell proliferation rate of 90.3% and 87.5% (P<0.05). At 1×10^-5 mol·L^-1, compounds 12 and 18 showed hepatoprotective activities against DL-galactosamine-induced toxicity examined in WB-F344 cell, with cell survival rates of 25% and 24%, respectivily. Conclusion : Compounds 1~29 were obtained from P. igniarius for the first time. Compounds 5 and 8 showed potent PC12-syn protective activities, while 12 and 18 showed hepato cytes (WB-F344 cells) protective activities.