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Journal of Tropical Oceanography >

2023 , Vol. 42 >Issue 5: 38 - 44

DOI: https://doi.org/10.11978/2022225

Marine Chemistry

Study on the chemical constituents from the marine-derived actinomycete Actinoplanes sp. M4I6

  • WANG Zipeng , 1 ,
  • LI Fenfa 2 ,
  • XIE Qingyi 2 ,
  • MA Qingyun 2 ,
  • YANG Li 2 ,
  • DAI Haofu 2 ,
  • LUO Duqiang , 1 ,
  • ZHAO Youxing , 2
Expand
  • 1. College of Life Science, Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Hebei University, Baoding 071002, China
  • 2. Haikou Key Laboratory for Research and Utilization of Tropical Natural Products, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Hainan Key Laboratory for Protection and Utilization of Tropical Bioresources, Hainan Academy of Tropical Agricultural Resource, Haikou 571101, China
LUO Duqiang. email: ;
ZHAO Youxing. email:

Received date: 2022-10-23

  Revised date: 2022-12-01

  Online published: 2022-12-07

Supported by

Natural Science Foundation of Hainan Province(821RC643)

Special Project of the Ministry of Finance and the Ministry of Agriculture and Rural Affairs on the National Modern Agricultural Industrial Technology System(CARS-21)

Special Finance Project of Ministry of Agriculture and Rural Affairs(NFZX2021)

Fold

Abstract

The secondary metabolites of marine actinomycete Actinoplanes sp. M4I6 were isolated and identified, moreover their biological activities were evaluated. The compounds were separated and purified by Silica gel, C18, Sephadex LH-20 column chromatography and high performance liquid chromatography (HPLC), and their structures were identified by NMR spectroscopic method. These compounds were evaluated for anti-Bacillus subtilis (ATCC 6633) and inhibition of acetylcholinesterase activity. Nine compounds were separated and identified as (E)-prop-1-enyl-2-hydroxypropanoate(1), cyclo-(R-Pro-R-Phe)(2), cyclo-(L-Val-L-Leu)(3), cyclo-(L-Phe-L-Val)(4), cyclo-(L-Leu-L-Phe) (5), lumichrome (6), 2-(4-hydroxyphenyl)ethylacetate(7), 4-hydroxysattabacin(8), 3-hydroxybutan-2-yl 2-phenylacetate(9), compound 1 is a new natural product. Compounds 5, 6, 8 and 9 showed certain inhibitory activities against Bacillus subtilis(ATCC 6633). Compounds 4 and 6 showed weak inhibitory activities against acetylcholinesterase.

Key words： marine-derived actinomycetes; Actinoplanes sp.; chemical composition; acetylcholinesterase inhibitory activity; Anti-Bacillus subtilis activity

Cite this article

WANG Zipeng , LI Fenfa , XIE Qingyi , MA Qingyun , YANG Li , DAI Haofu , LUO Duqiang , ZHAO Youxing . Study on the chemical constituents from the marine-derived actinomycete Actinoplanes sp. M4I6[J]. Journal of Tropical Oceanography, 2023 , 42(5) : 38 -44 . DOI: 10.11978/2022225

海洋具有高盐度、高压、低光照或无光照、异乎寻常的高温或者低温等特殊环境, 生物资源极其丰富, 多样性显著(Newman et al, 2007), 海洋微生物代谢途径独特, 可产生结构新颖的活性分子(Goodfellow et al, 2013)。海洋放线菌是海洋微生物的重要组成部分, 其次级代谢产物的结构多样, 活性独特, 是海洋微生物新颖活性天然产物的主要来源之一(李越中 等, 2000), 可为药物创制提供先导化合物(吕佩帅 等, 2021)。2020年, 从海洋来源稀有放线菌小单孢菌的发酵液中分离得到抗真菌新药Turbinmicin, 缓解了全球多重耐药病原体威胁的局面(Zhang et al, 2020)。
游动属(Actinoplanes)是稀有小单孢菌科的第二大属, 在1950年由Couch首次提出(Couch, 1950)。治疗Ⅱ型糖尿病的药物阿卡波糖, 最早就是由游动放线菌Actinoplanes sp. SE50/110的代谢产物中分离得到的寡糖改造而成(Schaffert et al, 2019)。目前从该属放线菌分离出代谢产物的类型有肽类、大环内酯、氨基糖苷类、萘醌类等, 具有抗菌、抗肿瘤、抗病毒、抗寄生虫等活性(李群 等, 1976; Chu et al, 1997; Singh et al, 2002; Zhang et al, 2009; Xiang et al, 2010; Schaffert et al, 2019; Baglioni et al, 2022)。本实验室主要从海洋微生物挖掘活性天然产物, 前期已从海洋放线菌Streptomyces sp. KFD18 (Zhou et al, 2019)和海洋真菌Penicillium sp. KFD28 (Kong et al, 2019)中发现一些具有抗肿瘤和抗糖尿病的活性分子。本研究从海洋游动属放线菌M4I6的代谢产物中分离鉴定了9个化合物, 并对其测定了抗枯草芽孢杆菌(Bacillus subtilis ATCC 6633)活性和抑制乙酰胆碱酯酶活性, 为该放线菌药用价值的挖掘提供基础。

1 试验部分

1.1 仪器与材料

Bruker AV-500型超导核磁共振波谱仪, TMS (Tetramethylsilane, 四甲基硅烷)为内标和Autospec-3000质谱仪(美国Bruke公司); SUMMIT p680A戴安半制备型高效液相色谱仪(美国DIONEX公司); 安捷伦高效液相色谱仪(美国Agilent Technologies); Waters C18半制备柱(10mm×250mm, 5μm)(日本Nacalai Tesque公司); 反相材料C-18 (FU-JI公司); Sephadex LH-20凝胶(GE公司); 薄层层析硅胶板; 200~300目柱层析硅胶(青岛海洋化工厂); 乙酰胆碱酯酶(北京Solarbio公司); 碘化硫代乙酰胆碱、氨苄、二硫代硝基苯甲酸(DTNB)和他克林(Sigma公司); ELX-800酶标仪(美国宝特公司); WRX-4熔点测定仪(上海易测仪器设备有限公司); JASCO P-1020旋光仪(日本 Jasco公司)。

1.2 方法

1.2.1 菌种来源与发酵

海洋沉积物采集自希腊锡罗斯岛, 从中分离得到放线菌菌株M4I6, 经ITS序列分析鉴定为游动属(Actinoplanes sp.)放线菌。将菌株接种于平板培养基ATCC172, 在28℃下培养10d。挑取平板上孢子适量, 接种到装有200mL液体ATCC172培养基的500mL锥形瓶中, 28℃, 180r·min-1培养7d得到种子液。配制21L改良ATCC172培养基: 可溶性淀粉20.0g, 葡萄糖10.0g, 水解干酪素5.0g, 酵母膏5.0g, 细菌学蛋白胨 5.0g, KH2PO4 0.5g, CaCO3 0.1g, 微量元素溶液[Na2B4O7·10H2O 1.0g, ZnCl2·2H2O 4.0g, FeC13·6H2O 20.0g, (NH4)6Mo7O24·4H2O 1.0g, CuCl2·2H2O 1.0g, MnC12·4H2O 1.0g, ddH2O 1L] 1mL, pH 7.5, 去离子水1L。分装于500mL三角瓶中, 每瓶200mL, 经高压灭菌锅121℃灭菌25min。将种子液接种到改良ATCC172培养基中, 每瓶接种20mL种子液, 28℃, 180r·min-1培养7d。

1.2.2 提取与分离

发酵结束后采用发酵液2倍体积的乙酸乙酯将其萃取3次后, 使用旋转蒸发仪对所得到的浸提液进行减压浓缩得到浸膏15.9g。
粗浸膏经正相硅胶柱色谱(石油醚:乙酸乙酯)以10:1 ~ 1:2 (v/v)的梯度进行洗脱, 分段收集, 并通过薄层层析和高效液相色谱仪进行检测, 将相同的部分合并, 得到5个组分(Fr.1 ~ Fr.5)。Fr.2经过反相C18柱色谱[甲醇:水, 20%~100% (v/v)]梯度洗脱后, 经过凝胶柱Sephadex LH-20 (100%甲醇)纯化得到化合物1 (1.0mg), 经过凝胶柱Sephadex LH-20 (100%甲醇), 结合半制备HPLC (High Performance Liquid Chromatography, 高效液相色谱仪)[C18半制备柱, 甲醇:水=40:60 (v/v), 流速4mL·min-1]纯化得到化合物2 (1.6mg, tR=9.8min)、3 (1.5mg, tR=15.6min)。Fr.3经过反相C18柱色谱[(甲醇:水, 20%~100% (v/v)]梯度洗脱后, 采用正相硅胶柱色谱以石油醚:乙酸乙酯(8:1 ~ 5:1)梯度洗脱, 结合半制备HPLC [C18半制备柱, 甲醇:水=40:60 (v/v), 流速4mL·min-1]纯化得到化合物4 (3.0mg, tR=19.5min)、6 (1.4mg, tR=20.8min)。采用凝胶柱Sephadex LH-20 (氯仿:甲醇=1:1), 结合半制备HPLC [C18半制备柱, 甲醇:水=30:70 (v/v), 流速4mL·min-1]纯化得到化合物5 (9.0mg, tR=13.0min)。Fr.4经过反相C18柱色谱[甲醇:水, 20%~100% (v/v)]梯度洗脱后, 采用凝胶柱Sephadex LH-20 (氯仿:甲醇=1:1), 结合半制备HPLC [C18半制备柱, 甲醇:水=40:60 (v/v), 流速4mL·min-1]纯化得到化合物7 (1.0mg, tR=16.2min)。采用正相硅胶柱色谱以石油醚:乙酸乙酯(6:1 ~ 3:1)梯度洗脱和凝胶柱Sephadex LH-20 (100%甲醇), 结合半制备HPLC [C18半制备柱, 甲醇:水=20:80 (v/v), 流速4mL·min-1]纯化得到化合物8 (1.5mg, tR=14.0min)。采用凝胶柱Sephadex LH-20 (100%甲醇), 结合半制备HPLC [C18半制备柱, 甲醇:水=30:70 (v/v), 流速4mL·min-1]纯化得到化合物9 (1.5mg, tR=14.1min)。

1.2.3 生物活性测定

1) 抑制乙酰胆碱酯酶活性
化合物均用DMSO (dimethyl sulfoxide, 二甲基亚砜)进行溶解, 制成待测样品(4mmol·L-1)。取675μL配置好的PBS (phosphate buffered saline, 磷酸盐缓冲盐)(pH=8.0)溶液于离心管中, 取待测化合物45μL的该溶液加至离心管中, 最后吸取90μL乙酰胆碱酯酶溶液(0.2U·mL-1), 摇匀, 取180μL混合均匀的待测溶液于96孔板中, 做4组平行。阳性药为他克林, 阴性与空白: 取675μL配好的PBS溶液于离心管中, 再取45μL的DMSO溶液加至离心管中, 最后吸取90μL乙酰胆碱酯酶溶液, 摇匀, 取180μL混合均匀的待测溶液于96孔板中, 做4组平行。
将96孔板于37℃放置15min后, 除空白组外各组均加入20μL底物[5mmol·L-1 AICI (Acetyl-thiocholine iodide, 硫代乙酰胆碱) 10μL＋ 1mmol·L-1 DTNB [5'-二硫代双(2-硝基苯甲酸)] 10μL]; 空白组: 加入PBS缓冲液10μL + 1mmol·L-1 DTNB溶液10μL。将96孔板于37℃放置30min, 设置酶标仪于412nm波长下测量每孔的OD值吸光度(张宇 等, 2017; 王昊 等, 2020)。
计算化合物对乙酰胆碱酯酶的抑制率采用公式(1):
$\frac{\left( \text{O}{{\text{D}}_{\text{n}}}-\text{O}{{\text{D}}_{{}}} \right)}{\left( \text{O}{{\text{D}}_{\text{n}}}-\text{O}{{\text{D}}_{\text{c}}} \right)}\times 100\text{ }\!\!%\!\!\text{ }$
式中: ODn为阴性对照平均吸光值, OD为待测样品平均吸光值, ODc为空白对照平均吸光值。
2) 抗枯草芽孢杆菌活性
化合物均用甲醇进行溶解, 制成待测样品(2.56mg·mL-1)。配制LB (Luria-Bertani)液体培养基10mL置于试管中, 灭菌后将活化好的枯草芽孢杆菌株(Bacillus subtilis ATCC6633)挑取绿豆大的单菌落于试管中, 摇床上培养12h。取200μL菌液于96孔板, 测量OD600, 若OD600大于0.1, 则用LB培养基稀释到OD600为0.1后, 再稀释100倍得到测试用菌液。
取无菌96孔板, 试验组加入10μL待测样品和190μL菌液; 阴性对照加入10μL甲醇和190μL菌液; 阳性药为氨苄; 空白对照组加入10μL甲醇和190μL培养基。操作完毕后置于培养箱中培养12h后测量OD600。采用公式(1)计算化合物对枯草芽孢杆菌的抑制率。

2. 结果与讨论

2.1 化合物结构鉴定

从该放线菌的代谢产物中共分离得到了9个化合物, 根据1H、13C-NMR和MS数据鉴定了化合物1~9的结构(图1)。
图1 化合物1~9的结构

Fig. 1 The chemical structures of compounds 1~9

化合物1: 无色油状, $\left[ \alpha \right]_{\mathrm{D}}^{20}$-10° (c 0.2, MeOD), ESI-MS给出分子量为130 (m/z: 153 [M+Na]+), 分子式C6H10O3。1H-NMR (500MHz, CD3OD) δH: 6.31 (1H, m, H-1ʹ), 5.71 (1H, m, H-2ʹ), 5.25 (1H, q, J=6.0Hz, H-2), 2.06 (3H, dd, J=7.3, 1.8Hz, H-3ʹ), 1.25 (3H, d, J=6.0Hz, H-3); 13C-NMR (125MHz, CD3OD) δC: 167.4 (C, C-1), 146.0 (CH, C-1ʹ), 121.8 (CH, C-2ʹ), 69.7 (CH, C-2), 20.3 (CH3, C-3), 15.5 (CH3, C-3ʹ)。该结构已被文献报道, 结合1H-NMR、13C-NMR、MS数据鉴定化合物为(E)-prop-1-enyl-2-hydroxypropanoate (Huse et al, 2006)。
化合物2: 无色油状, $\left[ \alpha \right]_{\mathrm{D}}^{20}$ +67° (c 0.3, MeOD), ESI-MS给出分子量为244 (m/z: 267 [M+Na]+), 分子式C14H16N2O2。1H-NMR (500MHz, CD3OD) δH: 7.38~7.22 (5H, m, H-2ʹ/3ʹ/4ʹ/5ʹ/6ʹ), 4.47 (1H, t, J=5.3Hz, H-6), 4.09 (1H, dd, J=10.9, 6.5Hz, H-9), 3.56 (1H, m, H-3a), 3.41 (1H, m, H-3b), 3.21 (2H, m, H-10), 2.11 (1H, m, H-5a), 1.82 (2H, m, H-4), 1.81 (1H, m, H-5b); 13C-NMR (125MHz, CD3OD) δC: 170.9 (C, C-7), 166.9 (C, C-1), 137.3 (C, C-1ʹ), 131.1 (CH, C-3ʹ/C-5ʹ), 129.4 (CH, C-2ʹ/C-6ʹ), 128.1 (CH, C-4ʹ), 60.1 (CH, C-6), 57.7 (CH, C-9), 46.0 (CH2, C-3), 38.2 (CH2, C-10), 29.4 (CH2, C-5), 22.7 (CH2, C-4)。以上数据与文献报道基本一致, 故鉴定化合物为cyclo-(R-Pro-R-Phe)(Adamczeski et al, 1995)。
化合物3: 白色晶体, 熔点: 180~182℃, $\left[ \alpha \right]_{\mathrm{D}}^{20}$-14° (c 0.3, MeOD), ESI-MS给出分子量为212 (m/z: 235 [M+Na]+), 分子式C11H20N2O2。1H-NMR (500MHz, CD3OD) δH: 3.90 (1H, dd, J=9.3, 4.4Hz, H-6), 3.73 (1H, d, J=4.4Hz, H-3), 2.18 (1H, m, H-11), 1.83 (1H, m, H-8), 1.71 (1H, m, H-7a), 1.57 (1H, m, H-7b), 1.01 (3H, d, J=7.0Hz, H-12), 0.94 (3H, d, J=6.9Hz, H-9), 0.93 (3H, d, J=7.0Hz, H-13), 0.92 (3H, d, J=6.9Hz, H-10); 13C-NMR (125MHz, CD3OD) δC: 171.3 (C, C-1), 169.6 (C, C-4), 61.5 (CH, C-6), 54.3 (CH, C-3), 46.0 (CH2, C-7), 33.7 (CH, C-11), 25.3 (CH, C-8), 23.7 (CH3, C-9), 21.8 (CH3, C-10), 19.3 (CH3, C-12), 17.8 (CH3, C-13)。以上数据与文献报道基本一致, 故鉴定化合物为cyclo-(L-Val-L-Leu)(Ding et al, 2013)。
化合物4: 黄色粉末, 熔点: 175~177℃, $\left[ \alpha \right]_{\mathrm{D}}^{20}$ +68° (c 0.3, MeOD), ESI-MS给出分子量为246 (m/z: 269 [M+Na]+), 分子式为C14H18N2O2。1H-NMR (500MHz, CD3OD) δH: 7.25~7.16 (5H, m, H-5/6/7/8/ 9), 4.27 (1H, dd, J=5.3, 4.5Hz, H-2), 3.60 (1H, d, J=4.5Hz, H-11), 3.20 (1H, dd, J=13.7, 5.3Hz, H-3a), 2.99 (1H, dd, J=13.7, 4.5Hz, H-3b), 1.60 (1H, dq, J=7.1, 4.5Hz, H-12), 0.75 (3H, d, J=7.1Hz, H-13), 0.37 (3H, d, J=7.1Hz, H-14); 13C-NMR (125MHz, CD3OD) δC: 169.4 (C, C-1/10), 137.0 (C, C-4), 131.5 (CH, C-6/8), 129.6 (CH, C-5/9), 128.2 (CH, C-7), 61.2 (CH, C-2), 57.2 (CH, C-11), 40.0 (CH2, C-3), 33.3 (CH, C-12), 19.1 (CH3, C-13), 17.0 (CH3, C-14)。以上数据与文献报道基本一致, 故鉴定化合物为cyclo-(L-Phe-L-Val)(刘志国 等, 2018)。
化合物5: 黄色粉末, 熔点: 177~179℃, $\left[ \alpha \right]_{\mathrm{D}}^{20}$ -33° (c 0.4, MeOD), ESI-MS给出分子量为260 (m/z: 283 [M+Na]+), 分子式为C15H20N2O2。1H-NMR (500MHz, CD3OD) δH: 7.28~7.13 (5H, m, H-5/6/7/8/9), 4.27 (1H, t, J=4.0Hz, H-2), 3.62 (1H, dd, J=13.8, 4.3Hz, H-3a), 3.24 (1H, d, J=4.0Hz, H-11), 2.90 (1H, dd, J=13.8, 5.8Hz, H-3b), 1.81 (2H, m, H-12), 1.60 (1H, m, H-13), 0.96 (3H, d, J=6.6Hz, H-14), 0.64 (3H, d, J=7.1Hz, H-15); 13C-NMR (125MHz, CD3OD) δC: 170.6 (C, C-10), 168.9 (C, C-1), 136.7 (C, C-4), 131.8 (CH, C-6/8), 129.6 (CH, C-5/9), 128.5 (CH, C-7), 57.4 (CH, C-11), 54.0 (CH, C-2), 45.2 (CH2, C-12), 40.2 (CH2, C-3), 24.6 (CH, C-13), 23.4 (CH3, C-14), 21.3 (CH3, C-15)。以上数据与文献报道基本一致, 故鉴定化合物为cyclo-(L-Leu-L-Phe)(Lopez et al, 1997)。
化合物6: 黄色油状, ESI-MS给出分子量为242 (m/z: 265 [M+Na]+), 分子式为C12H10N4O2。1H-NMR (500MHz, DMSO) δH: 7.85 (1H, s, H-6), 7.65 (1H, s, H-9), 2.43 (3H, s, H-11), 2.41 (3H, s, H-12); 13C-NMR (125MHz, DMSO) δC: 160.9 (C, C-4), 150.4 (C, C-2), 146.9 (C, C-10a), 144.7 (C, C-9a), 141.8 (C, C-5a), 138.8 (C, C-8), 138.3(C, C-7), 130.4 (C, C-4a), 128.7 (CH, C-6), 126.0 (CH, C-9), 20.3 (CH3, C-11), 19.7 (CH3, C-12)。以上数据与文献报道基本一致, 故鉴定化合物为lumichrome (陈玉英 等, 2014)。
化合物7: 黄色油状, ESI-MS给出分子量为180 (m/z: 203 [M+Na]+), 分子式为C10H12O3。1H-NMR (500MHz, CD3OD) δH: 7.00 (2H, d, J=8.5Hz, H-4ʹ/8ʹ), 6.66 (2H, d, J=8.5Hz, H-5ʹ/7ʹ), 4.15 (2H, t, J=7.1Hz, H-1ʹ), 2.77 (2H, t, J=7.1Hz, H-2ʹ), 1.96 (3H, s, H-2); 13C-NMR (125MHz, CD3OD) δC: 172.9 (C, C-1), 157.1 (C, C-6ʹ), 130.9 (CH, C-4ʹ/8ʹ), 129.9 (C, C-3ʹ), 116.2 (CH, C-5ʹ/7ʹ), 66.6 (CH2, C-1ʹ), 35.2 (CH2, C-2ʹ), 20.8 (CH3, C-2)。以上数据与文献报道基本一致, 故鉴定化合物为2-(4-hydroxyphenyl)ethylacetate
(王鸿 等, 2014)。
化合物8: 黄色油状, $\left[ \alpha \right]_{\mathrm{D}}^{20}$ +15° (c 0.3, MeOD), ESI-MS给出分子量为222 (m/z: 245 [M+Na]+), 分子式为C13H18O3。1H-NMR (500MHz, CD3OD) δH: 7.01 (2H, d, J=8.4Hz, H-2/6), 6.65 (2H, d, J=8.4Hz, H-3/5), 4.16 (1H, dd, J=8.1, 4.7Hz, H-2ʹ), 2.88 (1H, dd, J=13.9, 4.7Hz, H-1ʹ-a), 2.66 (1H, dd, J=13.9, 8.1Hz, H-1ʹ-b), 2.33 (2H, d, J=6.5Hz, H-4ʹ), 2.03 (1H, m, H-5ʹ), 0.85 (3H, d, J=2.6Hz, H-6ʹ), 0.83 (3H, d, J=2.6Hz, H-7ʹ); 13C-NMR (125MHz, CD3OD) δC: 214.4 (C, C-3ʹ), 157.1 (C, C-4), 131.5 (CH, C-2/6), 129.2 (C, C-1), 116.0 (CH, C-3/5), 79.3 (CH, C-2ʹ), 49.6 (CH2, C-4ʹ), 40.1 (CH2, C-1ʹ), 25.1 (CH, C-5ʹ), 23.0 (CH3, C-6ʹ), 22.9 (CH3, C-7ʹ)。以上数据与文献报道基本一致, 故鉴定化合物为4-hydroxysattabacin (Kim et al, 2017)。
化合物9: 黄色油状, $\left[ \alpha \right]_{\mathrm{D}}^{20}$ -10° (c 0.2, MeOD), ESI-MS给出分子量为208 (m/z: 231 [M+Na]+), 分子式为C12H16O3。1H-NMR (500MHz, CD3OD) δH: 7.22~ 7.17 (5H, m, H-1/2/3/4/5), 4.67 (1H, m, H-1ʹ), 3.62 (1H, m, H-2ʹ), 3.55 (2H, s, H-7), 1.06 (3H, d, J=6.5Hz, H-4ʹ), 1.00 (3H, d, J=6.5Hz, H-3ʹ); 13C-NMR (125MHz, CD3OD) δC: 173.2 (C, C-8), 135.8 (C, C-6), 130.4 (CH, C-1/5), 129.5 (CH, C-2/4), 128.0 (CH, C-3), 75.8 (CH, C-2ʹ), 70.0 (CH, C-1ʹ), 42.2 (CH2, C-7), 18.8 (CH3, C-4ʹ), 15.9 (CH3, C-3ʹ)。以上数据与文献报道基本一致, 故鉴定化合物为3-hydroxybutan-2-yl 2-phenylacetate (李云海 等, 2017)。

2.2 化合物生物活性评价

对分离得到的9个化合物测试了其抗枯草芽孢杆菌 (Bacillus subtilis ATCC 6633) 和乙酰胆碱酯酶抑制活性, 结果显示化合物4、6对乙酰胆碱酯酶有较弱的抑制活性, 在终浓度200μmol·L-1时抑制率为(45.1±1.6)%、(38.0±2.6)% [阳性对照组他克林在终浓度2.5μmol·L-1时抑制率为(83.7±1.2)%]。化合物5、6、8、9对枯草芽孢杆菌(Bacillus subtilis ATCC 6633)有一定的抑制活性, 在终浓度128μg·mL-1时抑制率分别为(42.7±2.5)%、(52.3±1.0)%、(52.1±3.9)%、(44.6± 2.0)% [阳性对照组氨苄在终浓度8μg·mL-1时抑制率为(92.6±2.5)%]。

3. 讨论

海洋游动属放线菌是海洋活性天然产物的重要来源之一, 对于其次级代谢产物的研究可为药物先导化合物的发现提供思路。肽类化合物是游动放线菌代谢产物的主要类型之一, 如从游动放线菌PDF-1代谢产物中分离鉴定的诺西肽(李子强 等, 2021)和济南游动放线菌中发现的创新霉素(李群 等, 1976)。肽类结构的生物活性主要有抗菌、抗肿瘤、抗病毒、抗炎和免疫抑制等活性(王优 等, 2008)。抗菌肽替考拉宁和雷莫拉宁均是从游动属放线菌代谢产物中分离得到, 并且已经应用于临床(Pallanza et al, 1984; 李丹 等, 2019)。本文从海洋放线菌Actinoplanes sp. M4I6中分离得到9个单体化合物(1~9), 代谢产物的类型主要有环二肽类以及小分子酚性成分, 发现该菌在本试验条件下主要代谢产生该属报道的肽类成分, 该放线菌是否会代谢其他类型化合物后续有待改变发酵条件进一步挖掘。根据文献报道, 化合物7对肿瘤细胞PC-3有一定的抑制活性(王鸿 等, 2014)。化合物8对黑色素的生成有显著的抑制作用(Kim et al, 2017)。本文对分离得到的9个化合物测试了其抗枯草芽孢杆菌(Bacillus subtilis ATCC 6633)和乙酰胆碱酯酶抑制活性, 测试结果显示2个化合物(4、6)对乙酰胆碱酯酶有较弱的抑制活性, 4个化合物(5、6、8、9)对枯草芽孢杆菌(Bacillus subtilis ATCC 6633)有一定的抑制活性。这些鉴定的化合物是否具有其他生物活性还需要对化合物进行富集和进一步评价。

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