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Journal of Tropical Oceanography >

2023 , Vol. 42 >Issue 2: 132 - 140

DOI: https://doi.org/10.11978/2022074

Exploitation of Marine Resources

Bioactive natural compounds from a whale bone-derived fungus Penicillium sp. S2014503

  • ZHANG Haibo , 1, 2, 3 ,
  • IMRAN Khan 1, 2 ,
  • KUMAR Saurav 4 ,
  • ZHANG Liping 1, 3 ,
  • FANG Zhuangjie 1, 2 ,
  • ZHANG Xinya 1, 2 ,
  • PENG Fang 5 ,
  • ZHANG Changsheng 1, 2, 3
Expand
  • 1. Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China
  • 2. University of Chinese Academy of Sciences, Beijing 100049, China
  • 3. Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 511458, China
  • 4. Laboratory of Algal Biotechnology-Centre-Algatech, Institute of Microbiology of the Czech Academy of Sciences, Trebon 37981, Czech Republic
  • 5. Wuhan University, China Center for Type Culture Collection, Wuhan 430072, China
ZHANG Haibo. email:

Copy editor: YIN Bo

Received date: 2022-04-11

  Revised date: 2022-05-10

  Online published: 2022-05-16

Supported by

Key Special Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou)(GML2019ZD0406)

National Natural Science Foundation of China(81872778)

National Natural Science Foundation of China(41911530078)

National Key R&D Program of China(2018YFC1406705)

National Key R&D Program of China(2018YFC1406704)

Fold

Abstract

Penicillium sp. S2014503 was isolated from a rotten whale bone sample. Constituent analysis and bioactivity evaluation of S2014503 were carried out to find more bioactive compounds from this fungus. Chemical structures of the purified compounds produced by S2014503 were determined by comprehensive analysis of NMR (nuclear magnetic resonance), MS (Mass spectrum) data of each compound. Stereochemistry of chiral compounds were elucidated by comparison of specific rotation value with the reported ones or determined by X-ray diffraction analysis. Ten compounds emodin (1), citreorosein (2), tetrahydro altersolanol B (3), conioxanthone A (4), chrysogine (5), pyramidamycin B (6), germicidin O (7), 2-(6-hydroxy-5,7-dimethylbenzefuranone-4-yl) acetaldehyde (8), astrophenone (9), and chenopodolans A (10) were found in the fermentation product of S2014503 which was cultured on a solid state rice medium. Bioactivities of all the isolated compounds were evaluated for the antibacterial activities and cytotoxicity against brine-shrimp larvae. Compounds 1 and 2 showed weak activity against Gram positive bacteria Micrococcus luteus. Compounds 1, 2 and 4 were significantly cytotoxic against brine-shrimp larvae (Artemia salina) after 6 hours of incubation at room temperature. Interestingly, compound 1 was a well-known laxative ingredient of Traditional Chinese Medicine Rheum officinale and Polygonam cuspidatum. This work is the first report on the emodin analogs from a whale bone derived fungus Penicillium sp.

Key words： Whale bone; Penicillium sp.; emodin

Cite this article

ZHANG Haibo , IMRAN Khan , KUMAR Saurav , ZHANG Liping , FANG Zhuangjie , ZHANG Xinya , PENG Fang , ZHANG Changsheng . Bioactive natural compounds from a whale bone-derived fungus Penicillium sp. S2014503[J]. Journal of Tropical Oceanography, 2023 , 42(2) : 132 -140 . DOI: 10.11978/2022074

为探索更多具有开发价值的生物活性分子, 海洋来源微生物正在逐渐成为研究开发的新资源(Liberra, 1995)。与海洋来源细菌和蓝细菌相比, 在2016—2020年海洋来源真菌是产出新化合物种类最多的微生物资源(Carroll et al, 2022)。青霉菌(Penicillium sp.)是一种广泛分布于世界各地的真菌, 可产生多种具有生物活性的分子, 如黄嘌呤类、生物碱类、多肽类和霉菌毒素类化合物(Pitt et al, 2000)。青霉属真菌中的代表性物种如产黄青霉(Penicillium chrysogenum)可以生产重要的抗生素药物——青霉素(Dayalan et al, 2011)。人们通常可以很容易地从食物、土壤和空气传播的样品中发现青霉属真菌, 一些青霉属真菌甚至可以在极端环境中生存(Wu et al, 2012)。令人注意的是, 近年来有一系列研究报道了海洋动物来源的青霉属真菌可以生产具有多种化学结构类型的活性天然产物分子。例如, 从南海软珊瑚来源的青霉菌中获得了多环稠合的混源萜类化合物penicimeroterpenoids A - C, 对酪氨酸磷酸化酶有很强的选择性抑制活性(Cheng et al, 2020)。从海鞘来源青霉菌P. antarcticum中分离到的两个衣康酸乙酯类化合物对人骨髓间充质干细胞分化显示了较强的抑制活性(Marchese et al, 2020)。本文报道了鲸鱼骨来源青霉菌Penicillium cavernicola发酵产物中发现的10种次生代谢产物的分离、结构鉴定和生物活性评价。上述10种次生代谢产物(图1)分别鉴定为大黄素(emodin) (1)、Ω-羟基大黄素(citreorosein) (2)、tetrahydroaltersolanol B (3)、conioxanthone A (4)、chrysogine (5)、pyramidamycin B (6)、germicidin O (7)、2-(6-hydroxy-5,7-dimethyl-benzefuranone-4-yl) acetaldehyde (8)、astrophenone (9)和chenopodolans A (10)。化合物1是中药大黄和虎杖中所含的著名泻药成分大黄素(Tang et al, 2007)。据报道, 从象鼻虫损伤的栗子上分离到的曲霉Aspergillus wentii Wehmer发酵提取物中也发现有大黄素的存在(Wells et al, 1975)。对大黄素的药理学研究揭示了该化合物具有多种疗效, 如泻下通便、抗菌、免疫抑制、血管舒张、心脏强张性、护肝和抗癌等生物活性(王淑玲 等, 2007)。同时, 化合物1和2对革兰氏阳性藤黄微球菌显示了弱的抗菌活性, 但是对卤虫(Artemia salina)具有明显的细胞毒性。
图1 化合物1 ~ 10的化学结构式

Fig. 1 Chemical structures of compounds 1

1. 试验材料与方法

1.1 仪器与试剂

仪器: CHEETAH 快速中压液相色谱系统(天津博纳艾杰尔科技有限公司); 硅胶色谱柱填料(100 ~ 200目; 300 ~ 400目, 烟台江友硅胶开发有限公司); 凝胶色谱柱填料(Sephadex LH-20, 40 ~ 70μm, Amersham Biosciences AB, 瑞典); 反相硅胶色谱柱填料(YMC*GEL ODS-A, 12nm S-50μm YMC Company Ltd., 日本); GF254薄层色谱硅胶板(涂层厚度0.1 ~ 0.2mm、0.3 ~ 0.4mm; 烟台江友硅胶开发有限公司); Agilent 1260 高效液相色谱仪(Agilent Inc., 美国); 反相半制备型高效液相色谱柱(Gemini C18 column, 250 mm×10mm, 5µm, Phenomenex Inc., 美国); 半制备型反相HPLC (ACE 5 Super C18, 250mm×10mm, 英国ACE公司)色谱柱; 制备型反相HPLC柱(XB-C18, 5μm, 250mm×21.2mm, 上海月旭科技股份有限公司); Bruker AMC-700核磁共振仪(Bruker Biosciences Corporation, 美国)。amaZon SL 低分辨离子阱质谱仪(Bruker Biosciences Corporation, 美国); MCP 500 旋光仪 (Anton Paar, 奥地利)。XtaLAB AFC12 (RINC) X-射线单晶衍射仪(Rigaku Corporation, 日本)。
试剂: 甲醇、氯仿、乙酸乙酯等为分析纯(广州化学试剂厂); 高效液相色谱乙腈为色谱纯(Sigma-Aldrich, Inc., 美国); 柱层析硅胶和GF254薄层层析硅胶板(烟台华阳新材料科技有限公司)。

1.2 菌株来源

真菌菌株CCTCC S2014503由武汉大学中国典型培养物保藏中心彭方教授鉴定并提供, 分离自一块腐烂的鲸鱼骨样品(62°12ʹS, 58°57ʹW)。将纯化后的真菌菌株置于马铃薯葡萄糖水培养基中进行振荡培养3d后, 取500μL菌体样本加入EP (Eppendorf)管中, 3000r·min-1离心8min, 弃上清。用无菌移液枪加入100μL十二烷基硫酸钠三羟甲基氨基甲烷盐酸盐乙二胺四乙酸(STEs)缓冲液[0.2mol·L-1 Tris-HCl, 0.5mol·L-1 NaCl, 0.1%十二烷基硫酸钠(SDS), 0.01mol·L-1 乙二胺四乙酸(ethylene diamine tetraacetic acid, EDTA), pH=8.0]和40μL三羟甲基氨基甲烷盐酸盐乙二胺四乙酸(TE)缓冲液(10mmol·L-1 Tris-HCl, 1mmol·L-1 EDTA, pH=8.0), 吹打混匀, 加入约80μL体积的玻璃珠, 在通风橱中再加入120μL抽提试剂酚氯仿异戊醇(PCI)(苯酚 : 氯仿 : 异戊醇 = 25 : 24 : 1)。放入高速漩涡振荡仪, 6m·s-1震荡1min。12000r·min-1离心5min后, 取110μL上层水相到新的EP离心管中, 加入110μL异丙醇(-20℃放置)和22μL 3mol·L-1醋酸钠溶液, 于冰箱-20℃静置20min。4℃ 12000r·min-1离心10min后, 除去大部分上清, 加入70%冰乙醇100μL, 沉降DNA。12000r·min-1离心1 ~ 2min后, 除去所有上清, 将EP管于37℃烘箱中充分干燥DNA后, 加入40μL TE缓冲液充分溶解DNA作为PCR (Polymerase chain reaction)模板备用。进行PCR扩增并测序获得ITS (internal transcribed spacer)序列(CCTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGAGGGCCCTCTGGGTCCAACCTCCCACCCGTGTTTATTTTACCTT
GTTGCTTCGGCGGGCCCGCCTTAACTGGCCGCCGGGGGGCTCACGCCCCCGGGCCCGCGCCCGCCGAAGACACCCTCGAA
CTCTGTCTGAAGATTGAAGTCTGAGTGAAAATATAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCG
ATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGC
CCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGCCCCGTCCTC
CGATTTCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTTGTCACCCGCTC
TGTAGGCCCGGCCGGCGCTTGCCGATCAACCCAAATTTTTATCCAGGTTGACCTCGGA), 基于上述ITS序列利用邻接法构建该菌的系统发育树(图2)(Tamura et al, 2004)。ITS序列系统发育树分析结果表明真菌菌株S2014503为青霉属真菌。
图2 基于 ITS基因序列构建的菌株CCTCC S2014503 系统发育树

图中数字表示1000 次自展支持率(单位: %), 括号内是Genbank 号

Fig. 2 Phylogenetic tree of CCTCC S2014503 based on the ITS gene sequence

1.3 菌株发酵培养

首先配制约4kg大米固体培养基。方法如下: 准备20个洗净的三角烧瓶(每瓶容量为2000mL), 向每个烧瓶中分别装入200g大米和240mL水。将大米与水混合后以透气的封口膜密封, 115℃消毒35min即得大米固体培养基。在超净工作台中将青霉菌S2014503孢子接种到每个烧瓶中, 室温(25℃)条件下静态发酵24d。

1.4 提取分离纯化

静态发酵24d后, 将青霉菌S201450的大米发酵培养产物用6L 体积分数70%丙酮水提取3次。在减压(60mbar)条件下利用旋转蒸发仪从提取液中蒸去丙酮, 获得含水的残留液体。将残留的水液用等体积的乙酸乙酯萃取。萃取液在减压(110mbar)条件下蒸去乙酸乙酯, 浓缩后得到22g棕色粗提物。采用中压液相色谱(Medium Pressure Liquid Chromatography, MPLC)系统对粗提物进行反相C-18柱(YMC*GEL ODS-A, 5μm, 310mm×45mm)分离(流速为20mL·min-1), 乙腈/水系统梯度洗脱(0 ~ 100%乙腈) 120min, 获得11个流份(F1 ~ F11)。
采用配有二极管阵列检测器(Diode Array Detector, DAD)的高效液相色谱仪对各流份中的化学成分进行分析之后, 将流份F7 (525mg)上样到Sephadex LH-20凝胶柱进行分离, 用混合溶剂体系(氯仿 : 甲醇 = l : 1, v/v)洗脱后得到12个亚流份。薄层色谱检测到亚流份F7-7 ~ F7-11中含有相同的主产物, 合并后蒸去溶剂得到23mg残留物。上述残留物经半制备型反相HPLC (ACE 5 Super C18, 250mm×10mm)色谱柱用体积分数40%乙腈-水等度洗脱(流速为2.5mL·min-1)作进一步纯化, 得到化合物7 (7.8mg, tR=10.2min)和化合物8 (5.4mg, tR=7.5min)。流份F8 (494mg)经LH-20凝胶柱分离(氯仿 : 甲醇 = l : 1, v/v)后得到16个亚流份。将亚流份F8-6 ~ F8-10合并得60mg样品, 之后用制备型反相HPLC柱(XB-C18, 5μm, 250mm×21.2mm)梯度洗脱30min (5% ~ 100%乙腈-水, 流速为10mL·min-1), 得到化合物1 (4.5mg, tR=21.5min)。将亚流份F8-11 ~ F8-13合并后蒸干得到61mg样品, 再次以LH-20凝胶柱分离得到4个组分。对上述4个组分用半制备型HPLC色谱柱(ACE 5 Super C18, 250mm×10mm, 流速为2.5mL·min-1)用体积分数35%乙腈-水等度洗脱, 获得化合物4 (2.3mg, tR=10.1min)、6 (2.9mg, tR=11.8min)和9 (4.1mg, tR=13.5min)。将亚流份F8-14 ~ F8-15合并后得到39mg样品, 上样至LH-20凝胶柱上, 用混合溶剂(氯仿 : 甲醇 = l : 1, v/v)洗脱后再以半制备型HPLC色谱柱(ACE 5 Super C18, 250mm×10mm, 流速为2.5mL·min-1)用体积分数45%乙腈-水等度洗脱, 得到化合物10 (11.2mg, tR=11.0min)。利用Sephadex LH-20凝胶柱将流份F9 (540mg)进行分离, 用甲醇洗脱后得到16个亚流份。以半制备型HPLC色谱柱(ACE 5 Super C18, 250mm×10mm, 流速为2.5mL·min-1)用体积分数5% ~ 100%乙腈-水梯度洗脱, 对合并的亚流份F9-5 ~ F9-10 (66.7mg)作进一步分离, 获得化合物5 (8.1mg)。亚流份F9-11 ~ F9-16合并后得到82mg样品, 利用半制备型HPLC色谱柱(ACE 5 Super C18, 250mm×10mm, 流速为2.5mL·min-1)用体积分数55%乙腈-水等度洗脱对其进行分离后得到化合物2 (2.7mg, tR=7.5min)和化合物3 (4.5mg, tR=8.5min)。

1.5 抗菌活性测试

选取3株革兰氏阳性菌和2株革兰氏阴性菌株作为指示菌株进行抗菌活性检测。3株革兰氏阳性菌株分别为藤黄微球菌(Micrococcus luteus SCSIO ML01)、枯草芽孢杆菌(Bacillus subtilis ATCC 1064)、沙米氏杆菌(Bacillus saiminsis)。2株革兰氏阴性菌株分别为铜绿假单胞菌(Pseudomonas aeruginosa ATCC 27853)和鲍曼不动杆菌(Acinetobacter baumannii ATCC 19606)。将以上指示菌株接种于Luria-Bertani (LB)培养基中培养12h, 然后取少量菌液(调整为相同麦氏浊度)涂布于水解酪蛋白胨肉汤(Muller Hinton)琼脂平板上。采用纸片扩散法测试所分离的化合物的抗菌活性。每个纸片上加入浓度为2.56mg·mL-1的样品5μL; 以万古霉素和甲氧苄啶为阳性对照, 二甲基亚砜(dimethysulfoxide, DMSO)为阴性对照。将覆盖好样品纸片的菌株平板置于恒温细菌培养箱, 37℃过夜培养后, 观察样品对细菌生长的抑制情况。

1.6 卤虫毒性测试

用96孔板测定了所分离的化合物对卤虫幼虫(Artemia Salina)的细胞毒性。在96孔板的每个孔内都注入200μL的人工海水, 其中含有约20只新孵化的卤虫幼虫。随后分别加入10μL含有不同样品的二甲基亚砜溶液(2.56mg·mL-1)并在室温下进行孵育; 显微镜下记录4h、6h、8h后的致死率。以川楝素和DMSO分别作为阳性和阴性对照。

2. 试验结果

2.1 化合物结构鉴定

化合物1: 红色细针晶, ESI-MS m/z: 271.5 [M+H]+。1H NMR (700MHz, DMSO-d6) δ: 12.10 (1H, s, OH-1), 12.03 (1H, s, OH-8), 7.50 (1H, d, J=1.4Hz, H-4), 7.18 (1H, d, J=1.4Hz, H-2), 7.12 (1H, d, J=2.1Hz, H-5), 6.60 (1H, d, J=2.1 Hz, H-7), 2.42 (3H, s, CH3-3); 13C NMR (175MHz, DMSO-d6) δ: 189.7 (C-9), 181.5 (C-10), 165.7 (C-6), 164.5 (C-8), 161.4 (C-1), 148.3 (C-3), 135.2 (C-10a), 132.9 (C-4a), 124.2 (C-2), 120.5 (C-4), 113.4 (C-9a), 109.0 (C-5), 108.9 (C-8a), 108.0 (C-7), 21.5 (CH3)。以上数据经比对与文献报道基本一致, 故化合物1被鉴定为大黄素(Ngan et al, 2017)。
化合物2: 红色无定型粉末, ESI-MS m/z: 287.6 [M+H]+。1H NMR (700MHz, DMSO-d6) δ: 7.65 (1H, s, H-4), 7.24 (1H, s, H-2), 7.06 (1H, d, J=2.1Hz, H-5), 6.47 (1H, d, J=2.1Hz, H-7), 4.60 (2H, brs, CH2OH), 12.2 (1H, s, OH-8 or OH-1); 13C NMR (175 DMSO-d6) δ: 188.5 (C-9), 181.9 (C-10), 164.8 (C-6), 161.4 (C-8), 161.4 (C-1), 152.9 (C-3), 135.0 (C-10a), 133.0 (C-4a), 120.8 (C-2), 116.9 (C-4), 114.4 (C-9a), 110.6 (C-8a), 110.1 (C-5), 107.9 (C-7), 62.1 (CH2OH)。以上数据经比对与文献报道基本一致, 故化合物2被鉴定为citreorosein (杨修伟 等, 1998), 该化合物又称ω-hydroxyemodin (Ngan et al, 2017)。
化合物3: 淡黄色粉末, ESI-MS m/z: 309.8 [M+H]+, $[\alpha ]_{\mathrm{D}}^{25}-24.5{}^\circ $ (甲醇, c 0.023)。1H NMR (700MHz, DMSO-d6) δ: 12.91 (1H, s, OH-8), 6.70 (1H, dd, J=2.8, 1.4Hz, H-5), 6.36 (1H, d, J=2.1Hz, H-7), 5.63 (1H, d, J=7.7Hz, OH-10), 4.45 (1H, d, J=7.0 Hz, OH-2), 4.28 (1H, dd, J=7.0, 3.5Hz, H-10), 3.81 (3H, s, OCH3), 3.27 (1H, m, H-2), 2.47 (1H, m, H-1a), 2.16 (1H, dd, J=14.0, 11.9Hz, H-4eq), 2.12 (1H, m, H-1eq), 1.95 (1H, m, H-4a), 1.46 (1H, m, H-1ax), 1.20 (3H, dd, J=14.0, 11.9Hz, H-4ax), 1.16 (3H, s, CH3-3); 13C NMR (175MHz, DMSO-d6) δ: 203.3 (C-9), 165.8 (C-6), 164.4 (C-8), 151.7 (C-10a), 109.2 (C-9a), 104.0 (C-5), 99.0 (C-7), 73.5 (C-2), 70.8 (C-10), 69.5 (C-3), 55.7 (OCH3), 47.0(C-1a), 41.7 (C-4a), 41.2 (C-4), 29.3 (C-1), 27.0 (CH3-3)。以上数据经比对与文献报道基本一致, 故化合物3被鉴定为Tetrahydroaltersolanol B (Okamura et al, 1996)。在甲醇中获得了化合物3的淡黄色结晶。在XtaLAB AFC12 (RINC) X-射线单晶衍射仪(Cu Kα, λ=1.54184Å)上采集了化合物3的X-射线单晶衍射数据(CCDC-2168673)(表1)。使用Olex2 (Dolomanov et al, 2009)和SHELXT (Sheldrick, 2015a)程序求解晶体结构, 并使用最小二乘法优化的SHELXL (Sheldrick, 2015b)程序包进行精修, 最终获得了化合物3的X-射线晶体图(图3)。
表1 化合物3的X-射线晶体数据

Tab. 1 Single crystal X-ray diffraction analysis data of compound 3

Identification code Tetrahydroaltersolanol B Identification code Tetrahydroaltersolanol B
Empirical formula C16H20O6 μ/mm-1 0.971
Formula weight 308.3g·mol-1 F(000) 756.0
Temperature/K 100.00 (10) Crystal size/mm3 0.02 × 0.01 × 0.01
Crystal system monoclinic Radiation/Å Cu Kα (λ=1.54184)
Space group I2 2Θ range for data collection/(°) 4.864 to 148.83
a/Å 7.88690 (10) Index ranges -9 ≤ h ≤ 9, -7 ≤ k ≤ 7,
-45 ≤ l ≤ 45
b/Å 5.80710 (10) Reflections collected 16092
c/Å 36.3377 (4) Independent reflections 3323 [Rint=0.0283, Rsigma=0.0195]
α/(°) 90 Data/restraints/parameters 3323/1/241
β/(°) 90.0340 (10) Goodness-of-fit on F2 1.047
γ/(°) 90 Final R indexes [I≥2σ (I)] R1 = 0.0247, wR2 = 0.0640
Volume/Å3 1664.27 (4) Final R indexes [all data] R1 = 0.0254, wR2 = 0.0644
Z 4 Largest diff. peak/hole / e Å-3 0.15/-0.14
ρcalc/(g·cm-3) 1.410 Flack parameter -0.06 (5)
图3 化合物3的X-射线晶体结构

图中圆圈表示氢原子, 红色部分表示氧原子, 灰色部分表示碳原子; 虚线表示氢键, 加粗实线和单实线表示化学键

Fig. 3 X-ray crystallographic structure of compound 3

化合物4: 黄色粉末, ESI-MS m/z: 317.7 [M+H]+。1H NMR (DMSO-d6, 700MHz) δ: 12.41 (1H, s, OH-1), 6.96 (1H, s, H-4), 6.89 (1H, d, J=2.1Hz, H-5), 6.80 (1H, d, J=2.8Hz, H-7), 6.73 (1H, s, H-2), 4.58 (2H, s, H2-11), 3.87 (3H, s, CH3-13); 13C NMR (175 DMSO-d6) δ: 179.0 (C-9), 168.5 (C-12), 165.3 (C-6), 160.6 (C-1), 157.9 (C-10a), 155.4 (C-4a), 153.5 (C-3), 134.9 (C-8), 113.4 (C-7), 108.5 (C-8a), 107.6 (C-2), 106.4 (C-9a), 103.9 (C-4), 103.3 (C-5), 62.3 (C-11), 52.7 (C-13)。以上数据经比对与文献报道基本一致, 故化合物4被鉴定为Conioxanthone A (Wang et al, 2010)。
化合物5: 淡黄色粉末; ESI-MS m/z: 191.3 [M+H]+。$[\alpha ]_{\mathrm{D}}^{25}$+68.8° (甲醇, c 0.05)。1H NMR (700MHz, DMSO-d6) δ: 11.99 (1H, s, NH-7), 8.57 (1H, dd, J=8.4, 1.4Hz, H-6), 8.16 (1H, s, NH-1a), 7.74 (1H, dd, J=8.4, 1.4Hz, H-3), 7.59 (1H, s, NH-1b), 7.47 (1H, m, H-5), 7.11 (1H, dt, J=7.7, 1.4Hz, H-4), 5.98 (1H, d, J=5.6Hz, OH-9), 4.10 (1H, m, H-9), 1.29 (3H, d, J=7.0Hz, CH3-10); 13C NMR (175MHz, DMSO-d6) δ: 174.1 (C-8), 170.3 (C-1), 138.7 (C-7), 131.8 (C-5), 128.5 (C-3), 122.4 (C-4), 120.9 (C-2), 119.8 (C-6), 67.9(C-9), 20.9 (C-10)。以上数据经比对与文献报道基本一致, 故化合物5被鉴定为Chrysogine (Kettering et al, 2004)。该化合物结构中的碳氮双键会和相邻的C-9位仲醇羟基形成酮式-烯醇式互变异构(Tsantrizos et al, 1994)。
化合物6: 淡黄色粉末; ESI-MS m/z: 184.5 [M+H]+。1H NMR (700MHz, DMSO-d6) δ: 13.45 (1H, brs, OH-2), 8.29 (1H, brs, NH), 7.62 (1H, brs, NH), 7.49 (1H, d, J=8.4Hz, H-6), 6.33 (1H, d, J=8.4Hz, H-5), 3.68 (3H, s, OCH3); 13C NMR (175MHz, DMSO-d6) δ: 172.5 (C-1-CO), 156.7 (C-2), 154.9 (C-4), 135.0 (C-3), 123.5 (C-6), 107.6 (C-5), 107.0 (C-1), 59.8 (OCH3)。以上数据经比对与文献报道基本一致, 故化合物6被鉴定为Pyramidamycin B (Shaaban et al, 2012)。
化合物7: 无色油状物, ESI-MS m/z: 213.4 [M+H]+, $[\alpha ]_{\mathrm{D}}^{25}$-47.4° (甲醇, c 0.056)。1H NMR (700MHz, CD3OD) δ: 3.91 (1H, m, H-10), 2.92 (1H, m, H-9), 1.99 (3H, s, CH3-8), 1.91 (3H, s, CH3-7), 1.25 (3H, d, J=6.3Hz, CH3-11), 1.15 (3H, d, J=7.0Hz, CH3-12); 13C NMR (175MHz, CD3OD) δ: 168.8(C-2), 168.7(C-4), 161.6 (C-6), 110.4 (C-5), 98.8 (C-3), 70.6 (C-10), 43.9(C-9), 21.3 (C-11), 14.9 (C-12), 10.1 (C-8), 8.9 (C-7)。以上数据经比对与文献报道基本一致, 故化合物7被鉴定为Germicidin O (Pang et al, 2019)。
化合物8: 无色油状物, ESI-MS m/z: 221.4 [M+H]+。1H NMR (700MHz, CD3OD) δ: 3.72 (2H, s, H-3), 2.54 (2H, s, H-10), 2.10 (6H, overlapped, CH3-12 and CH3-13); 13C NMR (175MHz, CD3OD) δ: 208.3(C-11), 175.8 (C-2), 158.1 (C-8), 156.2 (C-6), 132.0 (C-9), 122.1 (C-4), 118.8 (C-5), 112.2 (C-7), 37.7 (C-3), 32.2 (C-10), 12.3 (C-12), 9.0 (C-13)。以上数据经比对与文献报道基本一致, 故化合物8被鉴定为2-(6-hydroxy-5,7-dimethylbenzefuranone-4-yl) acetaldehyde (徐燕 等, 2015)。
化合物9: 淡黄色油状物; ESI-MS m/z: 319.3 [M+H]+。1H NMR (700MHz, DMSO-d6) δ: 11.50 (s, OH-2ʹ), 9.90 (s, OH-6 or OH-6ʹ), 7.38 (1H, dd, J=7.7Hz, 1.4Hz, H-3), 7.28 (1H, t, J=7.7Hz, H-4), 7.09 (1H, dd, J=7.7Hz, 1.4Hz, H-5), 6.24 (2H, s, H-3ʹ, H-5ʹ), 4.36 (2H, s, H-7ʹ), 3.67 (3H, s, OCH3-8); 13C NMR (175MHz, DMSO-d6) δ: 200.3 (C-7), 165.8 (C-8), 161.7 (C-2ʹ, C6ʹ), 153.4 (C-6), 152.3 (C-4ʹ), 133.1 (C-1), 128.6 (C-4), 127.3 (C-2), 120.0 (C-3), 119.7 (C-5), 109.5 (C-1ʹ), 104.0 (C-3ʹ, C5ʹ), 62.4 (C-7ʹ), 51.9 (OCH3-8)。以上数据经比对与文献报道基本一致, 故化合物9被鉴定为Astrophenone (Kornsakulkarn et al, 2014)。
化合物10: 黄色油状物; ESI-MS m/z: 269.5 [M+H]+, $[\alpha ]_{\mathrm{D}}^{25}$+81.8° (甲醇, c 0.066)。1H NMR (700MHz, DMSO-d6) δ: 6.51 (1H, s, H-5), 6.47 (1H, s, H-7a), 3.94 (3H, s, OCH3-3), 3.50 (1H, q, J=6.3Hz, H-11), 2.16 (3H, s, CH3-13), 1.79 (3H, s, CH3-8), 1.27 (3H, s, CH3-9), 1.03 (3H, d, J=7.0Hz, CH3-12); 13C NMR (175MHz, DMSO-d6) δ: 166.2 (C-3), 163.6 (C-2), 159.9 (C-6), 137.0 (C-5), 127.5 (C-4), 100.0 (C-3a), 92.8 (C-7a), 75.3 (C-10), 73.5 (C-11), 56.7 (OCH3-3), 25.6 (C-9), 18.0 (C-12), 12.9 (C-13), 8.6 (C-8)。以上数据经比对与文献报道基本一致, 故化合物10被鉴定为Chenopodolans A (Cimmino et al, 2013)。

2.2 生物活性测试结果

2.2.1 抗菌活性测试结果

以万古霉素为阳性对照, 采用纸片扩散法对所分离的10个化合物(1 ~ 10)以上述3株革兰氏阳性菌和2株革兰氏阴性菌株进行了抗菌活性检测(Cockerill et al, 2010)。只发现化合物1和2对藤黄微球菌具有弱的抑制活性, 其他化合物未观察到抑菌活性。

2.2.2 卤虫毒性测试结果

以96孔板法测定了所分离的化合物对卤虫幼虫(Artemia Salina)的细胞毒性(黄筱娟 等, 2015; Rajabi et al, 2015)。加入阳性对照川楝素、阴性对照二甲基亚砜及其他待测样品溶液后共孵育6h, 大黄素(1)和Ω-羟基大黄素(2)都对卤虫幼虫表现出显著的细胞毒性(致死率为97%)。此外, 化合物Conioxanthone A (4)和Germicidin O (7)也有较强的细胞毒性(致死率为90%)。其他化合物的卤虫毒性测试结果见表2。
表2 化合物1-10卤虫致死毒性百分率测试结果

Tab. 2 Lethal toxicity assay results of compounds 1-10 against Artemia salina

样品编号 1 2 3 4 5 6 7 8 9 10 阳性对照
致死百分率/% 97 97 72 90 75 81 90 65 69 61 100

3 讨论

本文从鲸鱼骨样品来源真菌Penicillium sp. (S2014503)的大米固体发酵产物中分离鉴定了10个化合物, 首次报道了青霉菌S2014503可产生一系列大黄素类活性成分(化合物1 ~ 3); 并对以上10个化合物进行了抗菌和卤虫致死毒性等生物活性评价。发现化合物1、2、4和7对卤虫幼虫有较强的致死毒性; 化合物1和2同时对条件性致病菌藤黄微球菌显示有弱的抑制活性。值得注意的是, 青霉菌S2014503所产的大黄素(化合物1)正是中药大黄和虎杖中所含的主要活性成分。经过培养条件优化, 青霉菌S2014503有作为大黄素类化合物生产菌的潜力。本文的研究结果表明海洋动物来源的微生物可产生化学结构类型丰富的生物活性成分, 可以作为有开发潜力的微生物资源为未来的药物开发提供帮助。

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