2. 上海海洋大学 食品科学与工程国家级实验教学示范中心, 上海 201306;
3. 农业农村部水产品贮藏保鲜质量安全风险评估实验室, 上海 201306
海洋来源的真菌是活性化合物的重要来源,可用于新药的研发,目前,对海洋真菌次生代谢产物的研究与陆地来源真菌相比被探索的次数较少,但从药物发现的角度,海洋真菌次生代谢产物可能更具研究价值与开发前景。例如研究者从海洋真菌Cephalosporium acremonium次生代谢产物中得到了头孢菌素C这一临床常用的抗生素药物[1]。之后,海洋真菌代谢产物的研究经历了大约30年的发展,已经逐渐形成了系统的研究体系。自此以后,从海洋真菌中分离得到的化合物的数量急剧增加[2],且仍保持上升的趋势。近年来大量来自海洋真菌的代谢产物被报道,其结构新颖且生物活性广泛,具有抗肿瘤[3]、抗菌[4]、抗病毒[5]等生物活性。据统计,有超过1 000种海洋真菌的代谢产物被用于新药的开发[6],其中一些代谢产物具有抗癌开发的潜质[7]。值得一提的是,目前已经进入临床Ⅲ期试验的二酮派嗪类药物NPI-2358就来源于海洋真菌,其主要用于治疗转移性的晚期非小细胞肺癌[8]。
据统计,近年来有大量的新化合物分离自海洋真菌次生代谢产物中,这些真菌主要分离自红树林、海藻和海绵等,占海洋真菌次生代谢产物的56%,其中近20%来源于海绵[1]。海绵真菌来源的化合物种类有生物碱、聚酮、甾体、萜类、大环内酯类、肽类等,主要的生物学活性有细胞毒[9-10]、抗菌[11-14]、抗病毒[15]、抗疟原虫[16]和降解污染物[17]等生物功能。因此,海绵来源真菌是海洋活性天然产物的重要来源之一[18]。
笔者对分离自隋氏蒂壳海绵共附生真菌(Penicillium chrysogenum)发酵产物进行化学成分的研究,从中分离鉴定得到1个脂肪酸类化合物、1个氨基酸类化合物、3个环二肽类化合物和2个甾醇类化合物。
1 材料与方法 1.1 仪器与试剂Bruker AVANCE-600核磁共振仪(瑞士Bruker公司);Waters Q-Tof micro YA019质谱仪(美国Waters公司);Waters高效液相色谱仪(Waters 1525/1998);旋转蒸发仪(EYELA-1000型);层析硅胶(200~300目,烟台江友硅胶开发有限公司);ODS填料(日本YMC公司);色谱级甲醇、乙腈(Cinc High Purity Solvents Co. Ltd.);其他有机试剂均为分析纯(中国医药集团上海化学试剂公司);氘代试剂(Cambridge Isotope Laboratories,Inc.);显色剂为12%(体积分数)硫酸香草醛乙醇溶液。
1.2 样品来源及鉴定海绵(T.swinhoei)样品于2015年4月采自中国南海西沙群岛,种属名称由青岛海洋研究所李锦和研究员鉴定,菌株Penicillium chrysogenum(581F1)由本课题组李蕾博士从海绵T.swinhoei中分离得到,海绵及微生物标本存放于上海交通大学医学院附属仁济医院海洋药物重点实验室。
供试海绵共生菌株分离自西沙隋氏蒂壳海绵T.swinhoei。将保存4 ℃的菌株接种到平板上,置于30 ℃培养箱中培养7 d,待单菌落长出后,将单菌落挑至另一平板培养基中进行纯化。
菌株P.chrysogenum一级种子培养液的获得:将平板中培养的菌株P.chrysogenum转接至装有30 mL马铃薯葡萄糖肉汤(PDB)液体培养基的100 mL三角瓶中,置于摇床上,转速180 r/min,在28 ℃的温度下培养72 h。
二级种子培养液的获得:取一级种子培养液15 mL分别加入到装有150 mL PDB培养基的500 mL三角瓶中摇床培养。
接种:取二级种子培养液60 mL分别加入到装有600 mL真菌二号培养基[甘露醇20.0 g、味精10.0 g、麦芽糖20.0 g、CaCO3 10.0 g、酵母提取物3.0 g、葡萄糖10.0 g、玉米浆1.0 g、MgSO4 0.3 g、KH2PO4 0.5 g、人工海水(ASW)1 L]的2 000 mL三角瓶中摇床培养。
发酵条件:真菌二号培养基,180 r/min摇床培养10 d,温度28 ℃,发酵体积为30 L。
1.3 样品提取与纯化对发酵后的菌体用乙酸乙酯萃取3次,合并萃取液,减压浓缩得到总浸膏4.465 7 g,将总浸膏进行正相硅胶减压柱色谱分离,依次用不同浓度梯度的石油醚-乙酸乙酯系统进行洗脱(100: 1、50: 1、20: 1、10: 1、5: 1、2: 1、1: 1),得到A~J共10个组分。D组分(0.973 2 g)经中压ODS柱色谱(10%~100%甲醇-水梯度洗脱4 h)得到4个亚组分,记为D1~D4。D1组分(328.1 mg)经正相硅胶柱色谱(以石油醚:丙酮=8: 1为流动相)洗脱得到3个次级流分,记为D2A~D2C。D2A(18.3 mg)经半制备型HPLC[xBridge® Prep Phenyl(10 mm ×250 mm,5 μm)]纯化分离得到化合物1(90%乙腈-水,tR=46 min,2.4 mg),化合物2(95%乙腈-水,tR=50 min,1.7 mg)。G组分(188.9 mg)经正相硅胶柱色谱和半制备型HPLC[xBridge® Prep Phenyl(10 mm × 250 mm,5 μm)]纯化分离得到化合物3(65%乙腈-水,tR=40 min,1.9 mg),化合物4(95%乙腈-水,tR=48 min,1.8 mg),化合物5(83%乙腈-水,tR=50 min,3.2 mg)。H组分(81.5 mg)经正相硅胶柱色谱和半制备型HPLC[xBridge® Prep Phenyl(10 mm × 250 mm,5 μm)]纯化分离得到化合物6(95%甲醇-水,tR=47 min,1.3 mg),化合物7(95%甲醇-水,tR=50 min,3.2 mg)。
2 结果 2.1 结构鉴定化合物1:白色粉末;ESI-MS m/z 227 [M+H]+,提示相对分子质量为226,结合1H-NMR和13C-NMR分析该化合物的分子式为C12H18O4,不饱和度为4;1H-NMR (600 MHz, CDCl3)谱中显示出1个活泼的羟基质子信号δH 1.45 (1H, s, 12-OH),2个双键质子信号δH 7.44 (1H, dd, J=5.7, 1.5 Hz, H-3)和δH 6.11 (1H, dd, J=5.7, 2.0 Hz, H-2),7个亚甲基质子信号δH 2.35 (2H, td, J=7.5, 2.9, H-11), δH 1.77 (1H, s, H-5a)和δH1.63 (1H, s, H-5b), δH 1.63 (2H, s, H-10), δH 1.45 (2H, s, H-6), δH 1.37-1.32 (2H, m, H-8, 9)和δH 1.25 (2H, s, H-7)。13C-NMR (150 MHz, CDCl3)再结合DEPT谱确定有12个碳原子,其中2个羰基碳信号δC 177.6 (C-12)和δC 173.3 (C-1),2个烯烃次甲基碳信号δC 156.4 (C-3)和δC121.7 (C-2),7个亚甲基碳信号δC 33.7 (C-11), δC33.3 (C-5), δC 29.2 (C-7), δC 29.1 (C-8), δC 29.0 (C-9), δC 25 (C-6)和δC 24.7 (C-10)。最后根据化合物的核磁归属数据、分子式和不饱和度,推测化合物的结构中包含1个环状结构,1个羟基,2个酮羰基,可以推断出化合物具有环状的脂肪酸类化合物的骨架。
通过HMBC谱可以看出H-3 (δH 7.44)与C-4, C-2和C-1相关,H-2 (δH 6.11)与C-4, C-3和C-1相关,H-4 (δH 5.03)与C-6, C-5, C-2和C-3,以上可知H-4和C-1相关,再结合碳谱上δC 83.5 (C-4)的化学位移可以推断出化合物的结构中含有连氧的五元环。再通过COSY谱可以发现H-3与H-4, H-5与H-4相关,可以得出C-5与五元环相连接,又从HMBC谱中得出H-5 (δH 1.77 1.63)与C-3, C-4, C-6相关,H-6 (δH 1.45)与C-7, C-8, C-10相关,H-8 (δH 1.37-1.32)与C-10相关,H-9 (δH 1.37-1.32)与C-10相关,H-10 (δH 1.63)与C-11, C-12, C-7相关,H-11 (δH 2.35)与C-10, C-8, C-12相关,再结合DEPT谱得出C-5到C-12为脂肪酸链,因此该化合物的平面结构式得以确定。其核磁归属数据如下:1H-NMR (600MHz, CDCl3) δH:7.44 (1H, dd, J=5.7, 1.5 Hz, H-3), 6.11 (1H, dd, J=5.7, 2.0 Hz, H-2), 5.03 (1H, ddt, J=7.2, 5.3, 1.7 Hz, H-4), 2.35 (2H, td, J=7.5, 2.9, H-11), 1.77 (1H, s, H-5a), 1.63 (1H, s, H-5b), 1.63 (2H, s, H-10), 1.45 (2H, s, H-6), 1.45 (1H, S, 12-OH), 1.37-1.32 (2H, m, H-8, 9), 1.25 (2H, s, H-7); 13C-NMR (150 MHz, CDCl3)δC:177.6 (C-12), 173.3 (C-1), 156.4 (C-3), 121.7 (C-2), 83.5 (C-4), 33.7 (C-11), 33.3 (C-5), 29.2 (C-7), 29.1 (C-8), 29.0 (C-9), 25 (C-6), 24.7 (C-10)。最后经SciFinder文献检索发现,该化合物只在有机合成方面有相关报道[19],未在天然分子提取分离中有相关报道,因此化合物1是从自然界中首次分离得到,是新天然产物, 故该化合物为Traumatic acid(1)。
化合物2:无色固体;ESI-MS m/z 254 [M-H]-,提示相对分子质量为255,结合1H-NMR和13C-NMR分析该化合物的分子式为C16H17NO2,不饱和度为9。1H-NMR (600 MHz, CDCl3)显示出10个芳香质子信号δH 7.67 (2H, m, H-2, 6), δH 7.49 (1H, m, H-4), δH 7.41 (2H, m, H-3, 5), δH 7.33 (2H, m, H-13, 15), δH 7.27 (2H, m, H-12, 16)和δH 7.24 (1H, m, H-14),1个次甲基质子信号δH 4.38 (1H, m, H-8),2个亚甲基质子信号δH 3.81 (1H, dd, J=11.1, 3.6 Hz, H-10a), δH 3.76 (1H, dd, J=11.1, 5.1 Hz, H-10b), δH3.01 (2H, m, H2-9)。13C-NMR (150 MHz, CDCl3)再结合DEPT谱确定有16个碳原子,包括1个酮羰基碳信号δC168.2 (C-7),2个芳香季碳信号δC137.7 (C-11)和δC134.4 (C-1),10个芳香次甲基碳信号δC131.8 (C-4), δC129.4 (C-12, 16), δC128.9 (C-13, 15), δC128.8 (C-3, 5), δC127.1 (C-2, 6)和δC127.0 (C-14),1个次甲基碳信号δC53.8 (C-8)和2个亚甲基碳信号δC64.6 (C-9)和δC37.2 (C-10)。其核磁归属数据如下:1H-NMR (600 MHz, CDCl3) δH: 7.67 (2H, m, H-2, 6), 7.49 (1H, m, H-4), 7.41 (2H, m, H-3, 5), 7.33 (2H, m, H-13, 15), 7.27 (2H, m, H-12, 16), 7.24 (1H, m, H-14), 4.38 (1H, m, H-8), 3.81 (1H, dd, J = 11.1, 3.6 Hz, H-10a), 3.76 (1H, dd, J = 11.1, 5.1 Hz, H-10b), 3.01 (2H, m, H2-9); 13C-NMR (150 MHz, CDCl3)δC: 168.2 (C-7), 137.7 (C-11), 134.4 (C-1), 131.8 (C-4), 129.4 (C-12, 16), 128.9 (C-13, 15), 128.8 (C-3, 5), 127.1 (C-2, 6), 127.0 (C-14), 64.6 (C-9), 53.8 (C-8), 37.2 (C-10)。最后通过进一步的结构解析再结合文献[20]报道,故确定该化合物为N-benzoyl-L-phenylalaninol(2)。
化合物3:淡黄色粉末;ESI-MS m/z 274[M+H]+,提示相对分子质量为273,结合1H-NMR和13C-NMR分析该化合物的分子式为C14H15N3O3,不饱和度为9。1H-NMR (600 MHz, DMSO-d6)显示出3个氨基质子信号δH 9.26 (1H, brs, 1-NH), δH7.39 (1H, m, 12-NH)和δH6.77 (1H, brs, 15-NH), 5个烯烃质子信号δH 6.98 (1H, d, J=7.6 Hz, H-4), δH 6.81 (1H, d, J=7.8 Hz, H-7), δH 5.99 (1H, s, H-2), δH 5.97 (1H, t, J=7.2 Hz, H-6)和δH 5.25 (1H, t, J = 7.0 Hz, H-5),13C-NMR (150 MHz, DMSO-d6)再结合DEPT确定有14个碳原子,其中2个酮羰基碳信号δC166.9 (C-13)和δC164.2 (C-16),3个季碳信号δC134.9 (C-8), δC110.7(C-3)和δC108.1 (C-9),5个烯烃次甲基碳信号δC126.8 (C-2), δC123.7 (C-6), δC119.2 (C-5), δC117.6 (C-4)和δC116.9 (C-7),2个次甲基碳信号δC56.9 (C-11)和δ”C54.8(C-14),2个亚甲基碳信号δC62.4 (C-17)和δC28.9 (C-10)。其核磁归属数据如下:1H-NMR (600 MHz, CDCl3) δH: 9.26 (1H, br s, 1-NH), 7.39 (1H, m, 12-NH), 6.77 (1H, br s, 15-NH), 6.98 (1H, d, J=7.6 Hz, H-4), 6.81 (1H, d, J=7.8 Hz, H-7), 5.99 (1H, s, H-2), 5.97 (1H, t, J=7.2 Hz, H-6), 5.25 (1H, t, J=7.0 Hz, H-5), 4.01 (1H, m, H-11), 3.98 (1H, m, H-14), 3.43 (1H, m, H-17a), 2.99 (1H, m, H-17b), 2.86 (1H, m, H-10a), 2.46 (1H, m, H-10b); 13C-NMR (150 MHz, CDCl3)δC:166.9 (C-13), 164.2 (C-16), 134.9 (C-8), 126.8 (C-2), 123.7 (C-6), 119.2 (C-5), 117.6 (C-4), 116.9 (C-7), 110.7(C-3), 108.1 (C-9), 62.4 (C-17), 56.9 (C-11), 54.8(C-14), 28.9(C-10)。最后通过进一步的结构解析再结合文献[21]报道,故确定该化合物为cyclo-(Trp-Ser)(3)。
化合物4:白色粉末;ESI-MS m/z 211 [M+H]+,提示相对分子质量为210,结合1H-NMR和13C-NMR分析该化合物的分子式为C11H18N2O2,不饱和度为4。1H-NMR (600 MHz, CDCl3)谱显示出1个氨基质子信号δH 5.74 (1H, brs, 8-NH),2个氨基端残基特征质子信号δH 4.12 (1H, t, J=8.3 Hz, H-6)和δH4.02 (1H, dd, J=9.7, 3.8 Hz, H-9),2个甲基质子信号δH 1.00 (3H, d, J=6.6 Hz, H-13)和δH 0.96 (3H, d, J=6.6 Hz, H-12)。13C-NMR (150 MHz, CDCl3)再结合DEPT谱确定有11个碳原子,包括2个酮羰基碳信号δC170.2 (C-1)和δC166.3 (C-7),3个次甲基碳信号δC59.1 (C-6), δC53.5 (C-9)和δC24.9 (C-10),4个亚甲基碳信号δC45.7 (C-3), δC38.8 (C-11), δC28.3 (C-5)和δC22.8(C-4)以及2个甲基碳信号δC23.5 (C-12)和δC21.3 (C-13)。其核磁归属数据如下:1H-NMR (600 MHz, CDCl3) δH: 5.74 (1H, br s, 8-NH), 4.12 (1H, t, J=8.3 Hz, H-6), 4.02 (1H, dd, J=9.7, 3.8 Hz, H-9), 3.54 (1H, m, H-3a), 3.35 (1H, m, H-3b), 2.36 (1H, m, H-10), 2.13 (1H, m, H-5a), 2.08 (1H, m, H-5b), 2.03 (2H, m, H-4), 1.90 (1H, m, H-11a), 1.73 (1H, m, H-11b), 1.00 (3H, d, J= 6.6 Hz, H-13), 0.96 (3H, d, J=6.6 Hz, H-12); 13C-NMR (150 MHz, CDCl3)δC: 170.2 (C-1), 166.3 (C-7), 59.1 (C-6), 53.5 (C-9), 45.7 (C-3), 38.8 (C-11), 28.3 (C-5), 24.9 (C-10), 23.5 (C-12), 22.8(C-4), 21.3 (C-13)。最后通过进一步的结构解析再结合文献[22]报道,故确定该化合物为cyclo-(D-Pro-D-Leu)(4)。
化合物5:淡黄色粉末;ESI-MS m/z 311[M+H]+,提示相对分子质量为310,结合1H-NMR和13C-NMR分析该化合物的分子式为C18H18N2O3,不饱和度为11。1H-NMR (600 MHz, DMSO-d6)显示出1个羟基质子信号δH 9.97 (1H, s, 19-OH),2个活泼的氨基质子信号δH 8.01 (2H, t, J=2.7Hz, 1-NH, 4-NH),9个烯烃质子信号δH 7.67 (2H, t, J=7.1 Hz, H-17, H-19), δH 7.90 (1H, t, J=7.3 Hz, H-18), δH 7.28 (2H, d, J=7.0 Hz, H-16, H-20), δH 7.01 (2H, d, J=8.3 Hz, H-9, H-13)和δH 6.96 (2H, d, J= 8.4 Hz, H-10, H-12),两个氨基端残基特征质子信号δH 4.54 (1H, m, H-3)和δH4.38 (1H, m, H-6)。13C-NMR (150 MHz, DMSO-d6)再结合DEPT谱确定有18个碳原子,其中2个酮羰基碳信号δC167.8 (C-2)和δC167.2(C-5),3个季碳信号δC156.9 (C-11), δC137.8 (C-15)和δC127.5 (C-8),9个次甲基信号δC132.1 (C-9, C-13), δC130.7 (C-16, C-20), δC128.9 (C-17, C-19), δC127.3 (C-18)和δC115.9 (C-10, C-12),2个次甲基碳信号δC56.4 (C-3)和δC56.2 (C-6),2个亚甲基碳信号δC40.9 (C-7)和δC39.2 (C-14)。其核磁归属数据如下:1H-NMR (600 MHz, CDCl3) δH: 9.97 (1H, s, 19-OH), 8.01 (2H, t, J=2.7Hz, 1-NH, 4-NH), 7.67 (2H, t, J=7.1 Hz, H-17, H-19), 7.90 (1H, t, J=7.3 Hz, H-18), 7.28 (2H, d, J=7.0 Hz, H-16, H-20), 7.01 (2H, d, J=8.3 Hz, H-9, H-13), 6.96 (2H, d, J=8.4 Hz, H-10, H-12), 4.54 (1H, m, H-3), 4.38 (1H, m, H-6), 3.12 (1H, dd, J=13.5, 4.3, H-7a), 2.78 (1H, d, J=6.6 Hz, H-7b), 2.54 (2H, dd, J=13.7, 6.6 Hz, H-14); 13C-NMR (150 MHz, CDCl3)δC: 167.8 (C-2), 167.2 (C-5), 156.9 (C-11), 137.8 (C-15), 132.1 (C-9, C-13), 130.7 (C-16, C-20), 128.9 (C-17, C-19), 127.5 (C-8), 127.3 (C-18), 115.9 (C-10, C-12), 56.4 (C-3), 56.2 (C-6), 40.9 (C-7), 39.2 (C-14)。最后通过进一步的结构解析再结合文献[23]报道,故确定该化合物为cyclo-(Phe-Tyr)(5)。
化合物6:白色粉末;ESI-MS m/z 425 [M-H]-,确定相对分子质量为426,分析1H-NMR和13C-NMR分析该化合物的分子式为C28H42O3,不饱和度为8。1H-NMR (600 MHz, CDCl3)谱显示出5个烯烃次甲基质子信号δH 6.58 (1H, d, J=8.5 Hz, H-7), δH 6.27 (1H, d, J=8.5 Hz, H-6), δH 5.41 (1H, dd, J=6.0, 1.9 Hz, H-11), δH 5.17 (1H, dd, J=15.2, 7.5 Hz, H-23)和δH 5.14 (1H, dd, J=15.2, 7.9 Hz, H-22),6个甲基质子信号δH 1.07 (3H, s, H-19), δH 0.97 (3H, d, J= 6.6 Hz, H-21), δH 0.89 (3H, d, J=6.9 Hz, H-28), δH 0.82 (3H, d, J=6.8 Hz, H-27), δH 0.80 (3H, d, J= 6.8 Hz, H-26)和δH 0.71 (3H, s, H-18)。13C-NMR (150 MHz, CDCl3)再结合DEPT谱确定有28个碳原子,包括6个烯烃碳信号δC142.9 (C-9), δC135.3 (C-6, C-22), δC132.3 (C-23), δC130.6 (C-7)和δC119.7 (C-11),2个连氧的季碳信号δC82.9 (C-5)和δC78.7 (C-8),2个季碳信号δC45.3 (C-13)和δC37.9 (C-10),6个次甲基碳信号δC66.5 (C-3), δC55.8 (C-17), δC47.9 (C-14), δC43.0 (C-24), δC39.9 (C-20)和δC33.2 (C-25),6个亚甲基碳信号δC41.1 (C-12), δC35.8 (C-4), δC32.5 (C-1), δC30.3 (C-2), δC28.7 (C-16)和δC20.9 (C-15)以及6个甲基碳信号δC23.4 (C-19), δC20.5 (C-21), δC20.0 (C-27), δC19.5 (C-26), δC17.9 (C-28)和δC12.9 (C-18)。其核磁归属数据如下:1H-NMR (600 MHz, CDCl3) δH: 6.58 (1H, d, J=8.5 Hz, H-7), 6.27 (1H, d, J=8.5 Hz, H-6), 5.44 (1H, dd, J=15.2, 7.9 Hz, H-22), 5.41 (1H, dd, J=6.0, 1.9 Hz, H-11), 5.17 (1H, dd, J=15.2, 7.5 Hz, H-23), 5.14 (1H, dd, J=15.2, 7.9 Hz, H-22), 1.07 (3H, s, H-19), 0.97 (3H, d, J=6.6 Hz, H-21), 0.89 (3H, d, J= 6.9 Hz, H-28), 0.82 (3H, d, J=6.8 Hz, H-27), 0.80 (3H, d, J=6.8 Hz, H-26), 0.71 (3H, s, H-18); 13C-NMR (150 MHz, CDCl3)δC: 142.9 (C-9), 135.3 (C-6, C-22), 132.3 (C-23), 130.6 (C-7), 119.7 (C-11), 82.9 (C-5), 78.7 (C-8), 45.3 (C-13), 37.9 (C-10), 66.5 (C-3), 55.8 (C-17), 47.9 (C-14), 43.0 (C-24), 39.9 (C-20), 33.2 (C-25), 41.1 (C-12), 35.8 (C-4), 32.5 (C-1), 30.3 (C-2), 28.7 (C-16), 20.9 (C-15), 23.4 (C-19), 20.5 (C-21), 20.0 (C-27), 19.5 (C-26), 17.9 (C-28), 12.9 (C-18)。最后通过进一步的结构解析再结合文献[24]报道,故确定该化合物为(22E, 24S)-5α, 8α-epidioxy-24-methyl-cholesta-6, 9(11), 22-trien-3β-ol(6)。
化合物7:白色粉末;ESI-MS m/z 441 [M-H]-,确定相对分子质量为442,结合1H-NMR和13C-NMR分析该化合物的分子式为C29H46O3,不饱和度为7。1H-NMR (600 MHz, CDCl3)谱显示出3个烯烃次甲基质子信号δH 6.50 (1H, d, J=8.5 Hz, H-7), δH 6.24 (1H, d, J=8.5 Hz, H-6)和δH 5.18 (1H, m, H-22),7个甲基质子信号δH 1.50 (3H, d, J=6.6 Hz, H-29), δH 0.93 (3H, d, J=6.6 Hz, H-21), δH 0.92 (3H, d, J=6.6 Hz, H-19), δH 0.88 (3H, s, H-18),δH 0.84 (6H, t, J=6.8 Hz, H3-27, H-28)和δH 0.78 (3H, d, J= 6.8 Hz, H-26)。13C-NMR (150 MHz, CDCl3)再结合DEPT谱确定有29个碳原子,4个烯烃碳信号δC135.9 (C-23), δC135.5 (C-6), δC130.9 (C-7)和δC131.2 (C-22),2个连氧的季碳信号δC82.3 (C-5)和δC79.6 (C-8),2个季碳信号δC44.7 (C-13)和δC37.1 (C-10),7个次甲基碳信号δC66.5 (C-3), δC57.1 (C-17), δC51.8 (C-9), δC51.3 (C-14), δC50.3 (C-24), δC34.2 (C-20)和δC30.9 (C-25),7个亚甲基碳信号δC39.5 (C-12), δC37.1 (C-4), δC34.8 (C-1), δC30.3 (C-2), δC28.1 (C-16), δC23.6 (C-15)和δC20.7 (C-11)以及7个甲基碳信号δC20.8 (C-28), δC20.7 (C-27), δC20.2 (C-21), δC18.3 (C-19), δC17.1 (C-26), δC13.4 (C-18)和δC13.1 (C-29)。其核磁归属数据如下:1H-NMR (600 MHz, CDCl3) δH: 6.50 (1H, d, J=8.5 Hz, H-7), 6.24 (1H, d, J=8.5 Hz, H-6), 5.18 (1H, m, H-22), 1.50 (3H, d, J=6.6 Hz, H-29), 0.93 (3H, d, J=6.6 Hz, H-21), 0.92 (3H, d, J=6.6 Hz, H-19), 0.88 (3H, s, H-18), 0.84 (6H, t, J=6.8 Hz, H-27, H-28), 0.78 (3H, d, J=6.8 Hz, H-26); 13C-NMR (150 MHz, CDCl3)δC: 135.9 (C-23), 135.5 (C-6), 130.9 (C-7), 131.2 (C-22), 82.3 (C-5), 79.6 (C-8), 44.7 (C-13), 37.1 (C-10), 66.5 (C-3), 57.1 (C-17), 51.8 (C-9), 51.3 (C-14), 50.3 (C-24), 34.2 (C-20), 30.9 (C-25), 39.5 (C-12), 37.1 (C-4), 34.8 (C-1), 30.3 (C-2), 28.1 (C-16), 23.6 (C-15), 20.7 (C-11), 20.8 (C-28), 20.7 (C-27), 20.2 (C-21), 18.3 (C-19), 17.1 (C-26), 13.4 (C-18), 13.1 (C-29)。最后通过进一步的结构解析再结合文献[25]报道,故确定该化合物为5α, 8α-epidioxy-23-methyl-(22E, 24S)-ergosta-6, 22-dien-3β-ol(7)。
3 讨论对采自中国南海西沙群岛的隋氏蒂壳海绵共附生真菌P.chrysogenum的发酵产物进行了化学成分的研究,通过采用正相硅胶柱色谱、反相ODS柱色谱和高效液相技术从中分离纯化了7个单体化合物,再采用现代核磁波谱技术鉴定它们的结构,其中包括1个脂肪酸类化合物(1)、1个氨基酸类化合物(2)、3个环二肽类化合物(3、4、5)和2个甾醇类化合物(6、7),其中化合物1是从自然界中首次分离得到,化合物2、3、4、5、6、7是在该真菌Penicillium chrysogenum内首次分离得到。同时,发现隋氏蒂壳海绵共附生真菌P.chrysogenum的发酵产物多为环二肽和甾醇类化合物,这为以后研究此类海绵提供了依据,也为以后海绵共附生真菌的研究提供了方向。
开发海绵共附生微生物的活性物质具有独特的优势:通过发酵工艺优化,解决开发海洋药物资源供给不足的问题和生态环境遭到破环的困扰,因为海洋药物的研究多局限于样品量的限制,那么微生物次生代谢产物能突破资源限制;也可以通过基因工程手段改造增加次生代谢产物的产量和优化其结构;通过微生物生物反应器,原料可取之不尽。对海绵共附生真菌次生代谢产物的研究不仅扩大了海洋天然产物的种类,也增加了发现新颖化合物的概率。该研究也进一步表明了海绵共附生真菌次生代谢产物能为海洋药物的研发提供大量先导化合物,具有药物开发的潜力,也为今后充分利用海洋资源奠定了基础。
[1] |
RATEB M E, EBEL R. Secondary metabolites of fungi from marine habitats[J]. Natural Product Reports, 2011, 28(2): 290-344. DOI:10.1039/c0np00061b |
[2] |
丁立建.三株海绵共附生真菌的次生代谢产物及其生物活性研究[D].广州: 暨南大学, 2016. DING L J. Chemical and bioactivity studies of secondary metabolites fromthree marine sponge-associated Fungi[D]. Guangzhou: Jinan University, 2016. |
[3] |
DU L, ZHU T J, FANG Y C, et al. Aspergiolide A, a novel anthraquinone derivative with naphtho[1, 2, 3-de]chromene-2, 7-dione skeleton isolated from a marine-derived fungus Aspergillus glaucus[J]. Tetrahedron, 2007, 63(5): 1085-1088. DOI:10.1016/j.tet.2006.11.074 |
[4] |
NIU S W, LIU D, PROKSCH P, et al. New polyphenols from a deep sea Spiromastix sp. fungus, and their antibacterial activities[J]. Marine Drugs, 2015, 13(4): 2526-2540. DOI:10.3390/md13042526 |
[5] |
NIU S W, SI LL, LIU D, et al. Spiromastilactones:a new class of influenza virus inhibitors from deep-sea fungus[J]. European Journal of Medicinal Chemistry, 2016, 108: 229-244. DOI:10.1016/j.ejmech.2015.09.037 |
[6] |
GOMES N G M, LEFRANC F, KIJJOAA, et al. Can some marine-derived fungal metabolites become actual anticancer agents[J]. Marine Drugs, 2015, 13(6): 3950-3991. DOI:10.3390/md13063950 |
[7] |
BUGNI T S, IRELAND C M. Marine-derived fungi:a chemically and biologically diverse group of microorganisms[J]. ChemInform, 2004, 35(17): 143-163. |
[8] |
FU Z Y, HOU Y W, JI C P, et al. Design, synthesis and biological evaluation of anti-pancreatic cancer activity of plinabulin derivatives based on the co-crystal structure[J]. Bioorganic&Medicinal Chemistry, 2018, 26(8): 2061-2072. |
[9] |
NUMATA A, AMAGATA T, MINOURA K, et al. Gymnastatins, novel cytotoxic metabolites produced by a fungal strain from a sponge[J]. Tetrahedron Letters, 1997, 38(32): 5675-5678. DOI:10.1016/S0040-4039(97)01236-7 |
[10] |
SUN L L, SHAO C L, CHEN J F, et al. New bisabolane sesquiterpenoids from a marine-derived fungus Aspergillus sp. isolated from the sponge Xestospongia testudinaria[J]. Bioorganic&Medicinal Chemistry Letters, 2012, 22(3): 1326-1329. |
[11] |
SCOPEL M, ABRAHAM W R, HENRIQUES A T, et al. Dipeptide cis-cyclo (Leucyl-Tyrosyl) produced by sponge associated Penicillium sp. F37 inhibits biofilm formation of the pathogenic Staphylococcus epidermidis[J]. Bioorganic&Medicinal Chemistry Letters, 2013, 23(3): 624-626. |
[12] |
KONG X L, MA X H, XIE Y Y, et al. Aromatic polyketides from a sponge-derived fungus Metarhiziumanisopliae mxh-99 and their antitubercular activities[J]. Archives of Pharmacal Research, 2013, 36(6): 739-744. DOI:10.1007/s12272-013-0077-7 |
[13] |
SUBRAMANI R, KUMAR R, PRASAD P, et al. Cytotoxic and antibacterial substances against multi-drug resistant pathogens from marine sponge symbiont:citrinin, a secondary metabolite of Penicillium sp.[J]. Asian Pacific Journal of Tropical Biomedicine, 2013, 3(4): 291-296. DOI:10.1016/S2221-1691(13)60065-9 |
[14] |
MEENUPRIYA J, THANGARAJ M. Isolation and molecular characterization of bioactive secondary metabolites from Callyspongia spp. associated fungi[J]. Asian Pacific Journal of Tropical Medicine, 2010, 3(9): 738-740. DOI:10.1016/S1995-7645(10)60177-0 |
[15] |
PENG J X, JIAO J Y, LI J, et al. Pyronepolyene C-glucosides with NF-κB inhibitory and anti-influenza a viral (H1N1) activities from the sponge-associated fungus Epicoccum sp. JJY40[J]. Bioorganic&Medicinal Chemistry Letters, 2012, 22(9): 3188-3190. |
[16] |
ALMEIDA C, KEHRAUS S, PRUDÊNCIO M, et al. Marilones A-C, phthalides from the sponge-derived fungus Stachylidium sp.[J]. Beilstein Journal of Organic Chemistry, 2011, 7: 1636-1642. DOI:10.3762/bjoc.7.192 |
[17] |
KIRAN G S, HEMA T A, GANDHIMATHI R, et al. Optimization and production of a biosurfactant from the sponge-associated marine fungus Aspergillus ustus MSF3[J]. Colloids and Surfaces B-Biointerfaces, 2009, 73(2): 250-256. DOI:10.1016/j.colsurfb.2009.05.025 |
[18] |
BLUNT J W, COPP B R, MUNRO M H G, et al. Marine natural products[J]. Natural Product Reports, 2011, 28(2): 196-268. DOI:10.1039/C005001F |
[19] |
GU X D, ZHANG W J, CHOI J, et al. An 1O2 route to γ-hydroxyalkenal phospholipids by vitamin E-induced fragmentation of hydroperoxydiene-derived endoperoxides[J]. Chemical Research in Toxicology, 2011, 24(7): 1080-1093. DOI:10.1021/tx200093m |
[20] |
陈苓丽, 韩娜, 王艺纯, 等. 吉祥草化学成分的分离与鉴定[J]. 沈阳药科大学学报, 2011, 28(11): 875-878. CHEN L L, HAN N, WANG Y C, et al. The chemical constituents from whole plant of Reineckia carnea(Andr.) Kunth[J]. Journal of Shenyang Pharmaceutical University, 2011, 28(11): 875-878. |
[21] |
KHIMIUK A Y, KORENNYKH A V, VAN LANGEN L M, et al. Penicillin acylase-catalyzed peptide synthesis in aqueous medium:a chemo-enzymatic route to stereoisomerically pure diketopiperazines[J]. Tetrahedron:Asymmetry, 2003, 14(20): 3123-3128. DOI:10.1016/j.tetasy.2003.08.011 |
[22] |
FDHILAF, VÁZQUEZ V, SÁNCHEZ J L, et al. dd-diketopiperazines:antibiotics active against Vibrio anguillarum isolated from marine bacteria associated with cultures of Pecten maximus[J]. Journal of Natural Products, 2003, 66(10): 1299-1301. DOI:10.1021/np030233e |
[23] |
黄忠京, 郭志勇, 杨瑞云, 等. 南海红树林内生真菌ZZF42中生物碱类代谢产物的研究[J]. 中药材, 2007, 30(8): 939-941. HUANG Z J, GUO Z Y, YANG R Y, et al. Alkaloid metabolites of mangrove endophytic fungus ZZF42 from the South China Sea[J]. Journal of Chinese Medicinal Materials, 2007, 30(8): 939-941. DOI:10.3321/j.issn:1001-4454.2007.08.014 |
[24] |
IOANNOU E, ABDEL-RAZIK A F, ZERVOU M, et al. 5α, 8α-Epidioxysterols from the gorgonian Eunicella cavolini and the ascidian Trididemnum inarmatum:isolation and evaluation of their antiproliferative activity[J]. Steroids, 2009, 74(1): 73-80. |
[25] |
XIN Z H, LI T, ZHU T J, et al. Isocoumarin derivatives from the sea squirt-derived fungus Penicillium stoloniferum QY2-10 and the halotolerant fungus Penicillium notatum B-52[J]. Archives of Pharmacal Research, 2007, 30(7): 816-819. DOI:10.1007/BF02978830 |
2. National Experimental Teaching Demonstration Center for Food Science and Engineering, Shanghai Ocean University, Shanghai 201306, China;
3. Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation, Ministry of Agriculture and Rural Affairs, Shanghai 201306, China