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Journal of Tropical Oceanography >

2024 , Vol. 43 >Issue 4: 189 - 197

DOI: https://doi.org/10.11978/2023132

Exploitation of Marine Resources

Chemical composition and antibacterial activities of the soft coral Lobophytum sp. from the South China Sea

  • SUN Manman , 1, 2, 3 ,
  • ZENG Yanbo , 1, 3 ,
  • XU Han 2, 4 ,
  • YAO Ligong 2 ,
  • GUO Yuewei 2 ,
  • SU Mingzhi , 2
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  • 1. Ocean College of Hebei Agricultural University, Qinhuangdao 066000, China
  • 2. Shandong Laboratory of Yantai Drug Discovery, Bohai Rim Advanced Research Institute for Drug Discovery, Yantai 264117, China
  • 3. Hainan Provincial Key Laboratory for Functional Components Research and Utilization of Marine Bio-resources, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
  • 4. School of Pharmcy, Yantai University, Yantai 264000, China
SU Mingzhi, email: ;
ZENG Yanbo, email:

Copy editor: SUN Cuici

Received date: 2023-09-04

  Revised date: 2023-10-05

  Online published: 2023-11-05

Supported by

National Natural Science Foundation of China(81991521)

National Key Research and Development Program of China(2022YFC2804100)

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Abstract

To investigate the chemical composition and biological activity of the soft coral Lobophytum sp. collected from South China Sea. A series of methods such as silica gel column chromatography, thin-layer chromatography, Sephadex LH-20 column chromatography, and semi-preparative high performance liquid chromatography were used to separate and purify. The structure of the compounds was characterized by analysis of NMR data and comparison with the data reported in literature. Twelve compounds were identified from the soft coral Lobophytum sp., including nine diterpenoids: (+)-(7R, 8R)-epoxy-7, 8-dihydrocembrene C(1), (1R, 2E, 4R, 7E, 11E)-cembra-2, 7, 11- triene-4-ol(2), (-)-(2R, 7R, 8R)-sarcophytoxide(3), entsarcophine(4), sarcotenusene C(5), (7S, 8S)-epoxy-1, 3, 11-cembratrien-16-oic methyl ester(6), sarcophytonin B(7), sarcoconvolutum D(8), laevigatol A(9); three steroids: campesterol(10), (24S)-24-methylcholesterol(11), gorgonol(12). Compound 8 was isolated from the soft coral Lobophytum sp. for the first time. The bioassay results revealed that compounds 3, 4, 5, 7 and 10 show significant antibacterial activity against fish pathogens Photobacterium damselae FP2244, oxytetracycline-resistant Streptococcus parauberis SPOF3K, and human pathogen vancomycin-resistant Enterococcus faecium.

Key words： soft coral; Lobophytum sp.; diterpenoid; steroid; antibacterial activity; drug-resistance bacteria

Cite this article

SUN Manman , ZENG Yanbo , XU Han , YAO Ligong , GUO Yuewei , SU Mingzhi . Chemical composition and antibacterial activities of the soft coral Lobophytum sp. from the South China Sea[J]. Journal of Tropical Oceanography, 2024 , 43(4) : 189 -197 . DOI: 10.11978/2023132

海洋是古老而又神秘的栖息地, 占据地球表面积的71%, 拥有多样的海洋生态系统。这种高盐、高压、缺氧和低温、低光照的极端环境赋予了数百万的海洋无脊椎动物特殊的代谢系统(肖细姬 等, 2019; 王子鹏 等, 2023), 使其能够产生独特的次级代谢产物用来抵御捕食者的猎杀和微生物的侵蚀。软珊瑚为腔肠动物门(Coelenterata)珊瑚虫纲(Anthozoa)八放珊瑚亚纲(Octocorallia)软珊瑚目(Alcyonacea)的海洋无脊椎动物(柴兴云 等, 2011), 主要栖息于热带和亚热带浅海区(Benayahu et al, 2004; 张兴旺 等, 2016), 如印度—太平洋海域, 我国南海海域也有广泛分布。软珊瑚作为固着底栖的海洋无脊椎动物, 它们缺乏坚硬的物理外壳的保护, 却可以通过独特的化学防御机制使其在危机四伏的海洋环境中生存。因此, 对其次级代谢产物的研究一直是海洋天然产物化学家们的研究热点。据文献报道, 软珊瑚的次级代谢产物种类丰富, 富含结构新颖和生物活性多样的萜类、甾体和生物碱类化合物, 并大多具有抗炎、抗菌、抗增殖、拒食等生物活性(牛燕 等, 2009; 郎俊慧 等, 2015; Peng et al, 2018; Vanisree et al, 2000)。因此, 对软珊瑚中的生物活性物质进行研究, 有利于“蓝色药库”的构建和为我国海洋新药挖掘提供新源泉(付秀梅 等, 2019)。
随着抗生素长期广泛超量使用, 使得部分细菌产生变异成为耐药菌株, 如临床上耐甲氧西林金黄色葡萄球菌、耐万古霉素葡萄球菌、耐万古霉素肠球菌等超级细菌的出现, 不仅严重危害人类的健康, 这种情况继续恶化下去很可能使人类面临感染时无药可用的境地。此外, 由于渔业养殖中也普遍存在滥用抗生素的情况, 从而也诱导衍生出了多种耐药的鱼类致病菌, 这也将对渔业经济以及人类健康造成严重威胁。因此, 开发新型抑制耐药细菌的抗生素已成为研究热点。本课题对采自中国南海的豆荚软珊瑚Lobophytum sp.中分离鉴定了12个化合物(化合物1—12)(图1), 分别为(+)-(7S, 8S)-epoxy-7, 8-dihydrocembrene C(1)、(1R, 2E, 4R, 7E, 11E)-cembra-2, 7, 11-triene-4-ol(2)、(-)-(2R, 7R, 8R)-sarcophytoxide(3)、entsarcophine(4)、sarcotenusene C(5)、(7S, 8S)-epoxy-1, 3, 11-cembratrien-16-oic methyl ester(6)、sarcophytonin B(7)、sarcoconvolutum D(8)、laevigatol A(9)、菜油甾醇(10)、(24S)-24-甲基胆甾醇(11) 和柳珊瑚甾醇(12)。并对分离的化合物进行了抗菌活性检测, 包括6种鱼类致病菌: 美人鱼发光杆菌Photobacterium damselae FP2244、耐土霉素的副乳房链球菌oxytetracycline-resistant Streptococcus parauberis SPOF3K、溶藻弧菌 Vibrio algaelyticus RZ53、杀鱼爱德华氏菌Edwardsiella piscicida TH1、杀鲑气单胞菌Aeromonas salraonicida AS42和无乳链球菌Streptococcus agalactiae WR10; 以及4株不同来源的人类致病菌: 耐万古霉素屎肠球菌vancomycin-resistant Enterococcus faecalis G1、G4、G7、G8的抗菌活性的检测。同时, 也对分离的部分化合物进行了抗细胞增殖活性检测。
图1 化合物1—12的化学结构

Fig. 1 Chemical structures of compounds 1—12

1 材料与方法

1.1 实验材料

实验样品于2021年11月采自中国南海广东省徐闻县海域, 经海南大学李秀保教授鉴定为豆荚软珊瑚Lobophytum sp.。标本(编号: S-21-XW-26)保存于烟台新药创制山东省实验室/中科环渤海(烟台)药物高等研究院。
鱼类致病菌: 美人鱼发光杆菌Photobacterium damselae FP2244、耐土霉素的副乳房链球菌oxytetracycline-resistant S. parauberis SPOF3K、杀鲑气单胞菌A. salraonicida AS42、溶藻弧菌V. algaelyticus RZ53、杀鱼爱德华氏菌E. piscicida TH1和无乳链球菌S. agalactiae WR10获赠于韩国国家渔业研究与发展研究所。
四株人类致病菌耐万古霉素屎肠球菌vancomycin-resistant E. faecalis G1、G4、G7、G8由上海交通大学医学院附属瑞金医院提供。

1.2 主要仪器与试剂

仪器: Agilent 1260高效液相色谱仪(high performance liquid chromatography, HPLC, 美国Agilent公司)、Agilent Eclipse XDB-C18半制备色谱柱(美国Agilent公司)、Bruker DRX-600型核磁共振仪(德国Bruker公司)、Sephadex LH-20凝胶(美国Pharmacia公司)、液相质谱仪(liquid chromatograph mass spectrometer, LC-MS, 美国Agilent公司)。
试剂: 薄层层析硅胶板(烟台江友硅胶开发有限公司)、柱色谱硅胶(60~80、200~300、300~400目, 青岛海湾精细化工有限公司)、分析纯试剂(国药集团化学试剂有限公司)、欧普森色谱纯试剂(烟台辉森特新材料科技有限公司)。

1.3 实验方法

1.3.1 提取与分离

样品处理: 将冷冻的豆荚软珊瑚Lobophytum sp.(干重295.2g)切碎, 放于5L的三角瓶中, 丙酮超声提取5次, 将提取液过滤, 减压浓缩, 得到丙酮提取物(30.5g)。加300mL纯水, 将丙酮提取物充分溶解, 再加入等体积的乙酸乙酯进行萃取, 萃取过程进行5次, 将萃取液减压浓缩, 得到乙酸乙酯相浸膏7.5g, 再用正丁醇进行萃取, 方法同上, 得到正丁醇萃取相浸膏1.7g。
乙酸乙酯相浸膏(7.5g)经硅胶柱色谱分离, 用石油醚-乙酸乙酯(100%~0%, V/V)和二氯甲烷-甲醇(100%~0%, V/V)两个不同的溶剂体系进行梯度洗脱共得到36个组分, 极性由低到高分别为Fr.1(0.0241g)、Fr.2 (0.0891g)、Fr.3 (0.0134g)、Fr.4 (0.48g)、Fr.5 (0.18g)、Fr.6 (0.18g)、Fr.7 (0.37g)、Fr.8 (0.20g)、Fr.9 (0.75g)、Fr.10 (0.30g)、Fr.11 (0.18g)、Fr.12 (0.54g)、Fr.13 (0.15g)、Fr.14 (0.07g)、Fr.15 (0.08g)、Fr.16 (0.08g)、Fr.17 (0.17g)、Fr.18 (0.06g)、Fr.19 (0.04g)、Fr.20 (0.1g)、Fr.21 (0.1g)、Fr.22 (0.06g)、Fr.23 0.37g)、Fr.24 (0.16g)、Fr.25 (0.12g)、Fr.26 (0.26g)、Fr.27 (0.2g)、Fr.28 (0.2g)、Fr.29 (0.1g)、Fr.30 (0.17g)、Fr.31 (0.15g)、Fr.32 (0.13g)、Fr.33 (0.12g)、Fr.34 (0.12g)、Fr.35 (0.12g)、Fr.36 (1.28g)。
组分Fr.4(0.48g)先后经Sephadex LH-20柱色谱(石油醚-二氯甲烷-甲醇, 2: 1: 1, V/V/V)、硅胶柱色谱(石油醚-乙酸乙酯, 100: 1~0: 100, V/V)和半制备HPLC[甲醇(MeOH)-H2O, 85: 15, V/V, 流速: 3mL·min-1]得到化合物1(1.6mg, tR=34.2min)和化合物11(1.3mg, tR=50min)。
组分Fr.5(0.18g)依次经硅胶柱色谱(石油醚-乙酸乙酯, 150: 1~0: 100, V/V)和半制备HPLC(MeOH-H2O, 80: 20, V/V, 流速: 3mL·min-1)得到化合物7(1.9mg, tR=7.5min)。
组分Fr.9(0.75g)依次经Sephadex LH-20柱色谱(二氯甲烷-甲醇, 1: 1, V/V)、硅胶柱色谱(石油醚-乙酸乙酯, 20: 1, V/V)和半制备HPLC(MeOH-H2O, 65: 35, V/V, 流速: 3mL·min-1)洗脱, 得到化合物3(0.7mg, tR=53.5min), 化合物4(4.2mg, tR=16.8min)和化合物9(1.1mg, tR=32.5min)。
组分Fr.18(0.06g)先经Sephadex LH-20柱色谱(石油醚-二氯甲烷-甲醇, 2: 1: 1, V/V/V)、硅胶柱色谱(二氯甲烷-甲醇, 150: 1, V/V)洗脱, 后经半制备HPLC(MeOH-H2O, 50: 50, 流速: 3mL·min-1)得到化合物8(1.1mg, tR=34.2min)。
组分Fr.20(0.1g)先后经硅胶柱色谱(石油醚-乙酸乙酯, 20: 1, V/V)和半制备HPLC(MeOH-H2O, 80: 20, V/V, 流速: 3mL·min-1)得到化合物6(1.8mg, tR= 34.2min)和化合物2(1.0mg, tR=80.0min)。
组分Fr.10(0.30g)经硅胶柱色谱(石油醚-乙酸乙酯, 40: 1~0: 40, V/V)梯度洗脱, 得到化合物10(20.1mg)。
组分Fr.13(0.15g)、Fr.14(0.07g)的主要成分相同, 将两者合到一起经Sephadex LH-20柱色谱(石油醚-二氯甲烷-甲醇, 2: 1: 1, V/V/V)洗脱除去色素, 又经半制备HPLC(MeOH-H2O, 90: 10, V/V, 流速: 3mL·min-1)分离得到化合物12(0.6mg, tR=48min)。
组分Fr.26(0.26g)先经Sephadex LH-20柱色谱(石油醚-二氯甲烷-甲醇, 2: 1: 1, V/V/V)等度洗脱, 硅胶柱色谱(石油醚-乙酸乙酯, 100: 0~1: 1, V/V)梯度洗脱, 最后通过半制备HPLC(MeOH-H2O, 68: 37, V/V, 流速: 3mL·min-1)得到化合物5(2.5mg, tR=13.5min)。

1.3.2 抗菌活性测试

采用改进的0.5 Mcfarland标准法测定化合物的最小抑菌浓度(minimal inhibit concentration, MIC)。将待测化合物用二甲基亚砜(dimethyl sulfoxide, DMSO)等比系列稀释成10个浓度, 将菌液用LB培养基调节106 cell·mL-1的菌密度, 在96孔板中加入5μL待测化合物, 再每孔接种95μL细菌培养液, 37℃孵育12h测定MIC值。为了确保溶剂载体DMSO对细菌生长没有显著影响, 每种细菌都在含有LB培养基和5%载体溶剂中进行培养, 结果显示5%DMSO对细菌生长没有影响。

1.3.3 抗增殖活性测试

将100μL对数生长期的A549肺癌细胞按照每孔3000个细胞的密度接种于96孔板中, 37℃培养12h后加入100μL相应浓度的待测化合物后继续培养72h, 然后每孔加入噻唑蓝溶液20μL, 37℃培养4h, 弃上清, 每孔加入DMSO 150μL溶解紫色结晶, 于570nm波长下测光密度值(optical density, OD)。抑制率%=1− (ODsample/ODcontrol)×100%。

2 实验结果

2.1 结构鉴定

化合物1: 无色油状; 分子式为C20H32O; [α]25D+18.3(c=0.1mg·mL−1, CH2Cl2); EI-MS m/z 288 [M]+; 1H NMR (600 MHz, CDCl3) δ 6.06(1H, d, J=10.9Hz, H-2), 6.00(1H, d, J=10.9Hz, H-3), 2.24(2H, m, H-5), 1.97~1.99(1H, m, H-6a), 2.17~2.19(1H, m, H-6b), 2.88(1H, t, J=5.5Hz, H-7), 1.67~1.68(1H, m, H-9a), 1.79~1.80(1H, m, H-9b), 1.99~2.01(2H, m, H-10), 5.10(1H, t, J=6.3Hz, H-11), 2.18~2.19(1H, m, H-13a), 2.01~2.02(1H, m, H-13b), 2.24~2.25(2H, m, H-14), 2.31~2.32(1H, m, H-15), 1.08(3H, d, J=6.9Hz, H-16), 1.09(3H, d, J=6.9Hz, H-17), 1.78(3H, s, H-18), 1.29(3H, s, H-19), 1.62(3H, s, H-20); 13C NMR(150 MHz, CDCl3) δ 148.32(C-1), 118.57(C-2), 121.57(C-3), 134.37(C-4), 35.92(C-5), 37.65(C-6), 61.78(C-7), 60.26(C-8), 25.89(C-9), 22.66(C-10), 125.70(C-11), 135.79(C-12), 39.60(C-13), 28.39(C-14), 35.06(C-15), 22.31(C-16), 22.48(C-17), 17.26(C-18), 18.00(C-19), 17.11(C-20)。以上数据与文献(Shaker et al, 2010)报道一致, 故鉴定化合物1为(+)-(7R, 8R)-epoxy-7, 8-dihydrocembrene C。
化合物2: 无色油状; 分子式为C20H34O; [ α ] D 25 -163.1(c=0.1mg·mL−1, CH2Cl2); EI-MS m/z 290 [M]+; 1H NMR (600 MHz, CDCl3) δ 5.31~5.21(1H, m, H-2), 5.72(1H, d, J=15.6Hz, H-3), 5.19(1H, t, J=7.5Hz, H-7), 5.00(1H, s, H-11), 2.01~2.19(2H, m, H-13), 2.22~2.25(2H, m, H-14), 2.29~2.31(1H, m, H-15), 0.85(3H, d, J=6.7Hz, H-16), 0.82(3H, d, J=6.8Hz, H-17), 1.34(3H, s, H-18), ) 1.61(3H, s, H-19), 1.25(3H, s, H-20); 13C NMR (150 MHz, CDCl3) δ 45.96(C-1), 128.51(C-2), 138.04(C-3), 72.67(C-4), 42.97(C-5), 23.70(C-6), 129.20(C-7), 125.21(C-8), 39.16(C-9), 22.55(C-10), 125.15(C-11), 132.40(C-12), 36.79(C-13), 27.58(C-14), 32.92(C-15), 20.48(C-16), 19.46(C-17), 14.71(C-18), 15.09(C-19), 28.07(C-20)。以上数据与文献(Carmely et al, 1981)报道一致, 故鉴定化合物2为(1R, 2E, 4R, 7E, 11E)-cembra-2, 7, 11-triene-4-ol。
化合物3: 无色油状; 分子式为C20H30O2; [ α ] D 25-102.5(c=0.2mg·mL−1, CH2Cl2); EI-MS m/z 302 [M]+; 1H NMR (600 MHz, CDCl3) δ 5.59~5.53(1H, m, H-2), 5.25(1H, d, J=9.8Hz, H-3), 2.33(2H, m, H-5), 1.9(1H, m, H-6a), 1.3(1H, m, H-6b), 2.75(1H, t, J=4.2Hz, H-7), 0.99(1H, dt, J=13.0, 4.9Hz, H-9a), 2.00(1H, m, H-9b), 1.85(1H, m, H-10a), 2.11(1H, m, H-10b), 5.14~5.08(1H, m, H-11), 1.91(2H, m, H-13), 1.65(1H, m, H-14a), 2.55(1H, m, H-14b), 4.52(2H, dp, J=4.5, 1.5Hz, H-16), 1.65(3H, s, H-17), 1.82(3H, s, H-18), 1.27(3H, s, H-19), 1.60(3H, s, H-20); 13C NMR (150 MHz, CDCl3) δ 133.37(C-1), 83.81 (C-2), 126.44(C-3), 139.47(C-4), 37.81(C-5), 25.45(C-6), 62.09(C-7), 60.03(C-8), 39.99(C-9), 23.69(C-10), 123.76(C-11), 137.01(C-12), 36.83(C-13), 26.28(C-14), 128.02(C-15), 78.56(C-16), 10.35(C-17), 15.75(C-18), 17.07(C-19), 15.23(C-20)。以上数据与文献(Bowden et al, 2004)报道一致, 故鉴定化合物3为(-)-(2R, 7R, 8R)-sarcophytoxide。
化合物4: 无色油状; 分子式为C20H28O3; [ α ] D 25-103.1(c=0.2mg·mL−1, CH2Cl2); EI-MS m/z 316 [M]+; 1H NMR (600 MHz, CDCl3) δ 5.59(1H, dd, J=9.9, 1.9Hz, H-2), 5.08~5.05(1H, m, H-3), 2.15(2H, m, H-5), 1.71(2H, m, H-6), 2.70(1H, t, J=4.4Hz, H-7), 2.47(1H, m, H-9a), 1.78(1H, m, H-9b), 1.93(1H, m, H-10a), 1.87(1H, m, H-10b), 5.16(1H, dd, J=10.1, 5.5Hz, H-11), 2.21(1H, m, H-13a), 2.09(1H, m, H-13b), 1.51(1H, m, H-14a), 2.53(1H, m, H-14b), 1.88(3H, s, H-17), 1.92(3H, s, H-18), 1.30(3H, s, H-19), 1.64(3H, s, H-20); 13C NMR (150 MHz, CDCl3) δ 162.36(C-1), 78.92(C-2), 120.78(C-3), 144.17(C-4), 36.52(C-5), 25.36(C-6), 61.58(C-7), 60.07(C-8), 39.18(C-9), 23.47(C-10), 125.06(C-11), 135.68(C-12), 37.53(C-13), 27.70(C-14), 123.09(C-15), 174.85(C-16), 9.15(C-17), 17.29(C-18), 16.27(C-19), 15.57(C-20)。以上数据与文献(Zhao et al, 2016)报道一致, 故鉴定化合物4为entsarcophine。
化合物5: 无色油状; 分子式为C20H28O4; [ α ] D 25-5.3(c=0.03mg·mL−1, CH2Cl2); EI-MS m/z 332 [M]+; 1H NMR (600 MHz, CDCl3) δ 5.40(1H, s, H-3), 1.96(1H, m, H-5a), 1.51(1H, m, H-5b), 2.09(1H, m, H-6a), 1.64(1H, m, H-6b), 2.61(2H, m, H-7), 1.82(1H, m, H-9a), 2.58(1H, m, H-9b), 2.07(1H, m, H-10a), 2.00(1H, m, H-10b), 5.01(1H, t, J=7.4Hz, H-11), 2.37(1H, m, H-13a), 2.25(1H, m, H-13b), 1.46(1H, m, H-14a), 2.55(1H, m, H-14b), 1.95(3H, s, H-17), 1.50(3H, s, H-18), 1.22(3H, s, H-19), 1.57(3H, s, H-20); 13C NMR (150 MHz, CDCl3) δ 151.88(C-1), 148.37(C-2), 116.29(C-3), 73.59(C-4), 39.74(C-5), 22.87(C-6), 60.98(C-7), 61.57(C-8), 35.86(C-9), 23.59(C-10), 128.59(C-11), 131.45(C-12), 37.89(C-13), 23.06(C-14), 124.14(C-15), 169.94(C-16), 9.14(C-17), 30.63(C-18), 18.46(C-19), 15.96(C-20)。以上数据与文献(El Sayed et al, 1998)报道一致, 故鉴定化合物5为sarcotenusene C。
化合物6: 无色油状; 分子式为C21H32O3; [α]25D+50(c=0.004mg·mL−1, CH2Cl2); EI-MS m/z 332 [M]+; 1H NMR (600 MHz, CDCl3) δ 6.21(1H, d, J=11.0Hz, H-2), 5.98(1H, dd, J=11.1, 2.0, 1.8Hz, H-3), 2.27~2.20(2H, m, H-5), 1.74(2H, m, H-6), 2.85(1H, t, J=5.6Hz, H-7), 1.92(1H, m, H-9a), 1.58(1H, m, H-9b), 2.02(2H, m, H-10), 5.06(1H, t, J=6.9Hz, H-11), 2.31(1H, m, H-13a), 2.01(1H, m, H-13b), 2.37(2H, m, H-14), 3.20(1H, q, J=7.2Hz, H-15), 1.32(3H, d, J=7.1Hz, H-17), 1.74(3H, s, H-18), 1.29(3H, s, H-19), 1.60(3H, s, H-20), 3.69(3H, s, H-21); 13C NMR (150 MHz, CDCl3) δ 139.76(C-1), 122.96(C-2), 121.03(C-3), 136.72(C-4), 35.95(C-5), 25.73(C-6), 61.35(C-7), 60.16(C-8), 37.33(C-9), 22.42(C-10), 126.21(C-11), 135.39(C-12), 38.61(C-13), 29.08(C-14), 46.38(C-15), 175.42(C-16), 16.88(C-17), 17.38(C-18), 18.19(C-19), 17.17(C-20), 51.95(C-21)。以上数据与文献(Matthée et al, 1998)报道一致, 故鉴定化合物6为(7S, 8S)-epoxy-1, 3, 11-cembratrien-16-oic methyl ester。
化合物7: 无色油状; 分子式为C20H28O2; [ α ] D 25+158(c=1.00mg·mL−1, CH2Cl2); EI-MS m/z 300 [M]+; 1H NMR (600 MHz, CDCl3) δ 5.52(1H, d, J=10.0Hz, H-2), 2.13(1H, m, H-5a), 1.71(1H, m, H-5b), 2.13(1H, m, H-6a), 1.73(1H, m, H-6b), 5.01(1H, t, J=6.9Hz, H-7), 0.98(1H, m, H-9a), 2.07(1H, m, H-9b), 2.01(1H, m, H-10a), 2.39(1H, m, H-10b), 4.86(2H, d, J=9.1Hz, H-3和H-11), 2.04(1H, m, H-13a), 2.17(1H, m, H-13b), 2.10(1H, m, H-14a), 2.96(1H, m, H-14b), 1.77(3H, s, H-17), 1.83(3H, s, H-18), 1.59(3H, s, H-19), 1.64(3H, s, H-20); 13C NMR (150 MHz, CDCl3) δ 163.13(C-1), 79.33(C-2), 119.87(C-3), 144.90(C-4), 38.94(C-5), 23.67(C-6), 124.70(C-7), 134.00(C-8), 39.99(C-9), 24.66(C-10), 125.24(C-11), 133.96(C-12), 36.54(C-13), 27.18(C-14), 122.52(C-15), 175.24(C-16), 9.05(C-17), 15.42(C-18), 16.06(C-19), 16.03(C-20)。以上数据与文献(Kobayashi et al, 1990)报道一致, 故鉴定化合物7为sarcophytonin B。
化合物8: 无色油状; 分子式为C20H28O5; [ α ] D 25+37.2(c=0.1mg·mL−1, CH2Cl2); EI-MS m/z 348 [M]+; 1H NMR (600 MHz, CDCl3) δ 5.47(1H, s, H-2), 5.01(1H, d, J=10.0Hz, H-3), 2.26(1H, m, H-5a), 2.34(1H, m, H-5b), 1.60(1H, m, H-6a), 1.82(1H, m, H-6b), 2.67~2.62(2H, m, H-7), 2.41(1H, m, H-9a), 2.46(1H, m, H-9b), 5.42(1H, m, H-10), 5.45(1H, m, H-11), 1.58(1H, m, H-13a), 1.89(1H, m, H-13b), 2.07(1H, m, H-14a), 2.27(1H, m, H-14b), 1.86(3H, s, H-17), 1.87(3H, s, H-18), 1.31(3H, s, H-19), 1.42(3H, s, H-20); 13C NMR (150 MHz, CDCl3) δ 161.72(C-1), 78.91(C-2), 121.26(C-3), 144.54(C-4), 36.76 (C-5), 24.20(C-6), 57.69(C-7), 60.15(C-8), 39.10 (C-9), 125.99(C-10), 135.62(C-11), 84.78(C-12), 36.12(C-13), 22.14(C-14), 123.74(C-15), 174.91(C-16), 9.21(C-17), 15.93(C-18), 22.35(C-19), 18.96(C-20)。以上数据与文献(Mohamed et al, 2021)报道一致, 故鉴定化合物8为sarcoconvolutum D。
化合物9: 无色油状; 分子式为C20H30O4; [ α ] D 25-23.1(c=1.00mg·mL−1, CH2Cl2)EI-MS m/z 334 [M]+; 1H NMR (600 MHz, CDCl3) δ 4.92(1H, d, J=11.0Hz, H-2), 5.41(1H, d, J=11.0Hz, H-3), 2.31(1H, m, H-5a), 2.35(1H, m, H-5b), 1.62(1H, m, H-6a), 1.91(1H, m, H-6b), 2.70~2.63(1H, m, H-7), 0.91(1H, m, H-9a), 2.12(1H, m, H-9b), 1.88(1H, m, H-10a), 2.26(1H, m, H-10b), 5.18(1H, d, J=10.4Hz, H-11), 1.93(1H, m, H-13a), 2.20(1H, m, H-13b), 1.69(1H, m, H-14a), 1.76(1H, m, H-14b), 5.30(1H, d, J=1.8Hz, H-16), 1.46(3H, s, H-17), 1.82(3H, s, H-18), 1.28(3H, s, H-19), 1.57(3H, s, H-20); 13C NMR (150 MHz, CDCl3) δ 70.75(C-1), 77.60(C-2), 124.67(C-3), 140.24(C-4), 38.02(C-5), 25.52(C-6), 62.8(C-7), 59.87(C-8), 40.28 (C-9), 23.83(C-10), 124.10(C-11), 136.17(C-12), 35.45 (C-13), 27.46(C-14), 68.47(C-15), 98.64(C-16), 11.23 (C-17), 15.65(C-18), 16.86(C-19), 15.00(C-20)。以上数据与文献(Quang et al, 2011)报道一致, 故鉴定化合物9为laevigatol A。
化合物10: 无色针状晶体; 分子式为C28H48O; EI-MS m/z 400 [M]+; 1H NMR (600 MHz, CDCl3) δ 1.87~1.77(4H, m, H-1a, H-2a, H-15a和H-16a), 1.21~1.03(7H, m, H-1b, H-12a, H-14, H-15b, H-22b, H-23a和H-24), 1.26(1H, m, H-16b), 1.61~1.32(9H, H-2b, H-7a, H-11, H-15a, H-17, H-22a, H-23b和H-25), 3.52(1H, tt, J=11.1, 4.6Hz, H-3), 1.98(1H, dq, J=10.2, 5.1, 2.3Hz, H-4a), 2.26(1H, m, H-4b), 5.35(1H, dt, J=4.9, 2.2Hz, H-6), 1.00(3H, s, H-18), 0.67(3H, s, H-19), 0.92(3H, d, J=6.6Hz, H-26), 0.77(6H, dd, J=4.2Hz, H-21和H-28), 0.85(3H, d, J=6.9Hz, H-27); 13C NMR (150 MHz, CDCl3) δ 37.40(C-1), 31.80(C-2), 71.95(C-3), 42.44(C-4), 140.91(C-5), 121.88(C-6), 30.72(C-7), 31.60 (C-8), 50.28(C-9), 36.65(C-10), 21.23(C-11), 39.91(C-12), 42.46(C-13), 56.90(C-14), 24.44(C-15), 28.34(C-16), 56.13(C-17), 12.00(C-18), 19.54(C-19), 36.33(C-20), 19.04(C-21), 33.86(C-22), 32.05(C-23), 39.21(C-24), 32.05(C-25), 17.73(C-26), 20.67(C-27), 15.59(C-28)。以上数据与文献(Koizumi et al, 1979)报道一致, 故鉴定化合物10为菜油甾醇。
化合物11: 白色固体; 分子式为C28H48O; EI-MS m/z 400 [M]+; 1H NMR (600 MHz, CDCl3) δ 1.79~ 1.87(3H, m, H-1a, H-2a和H-16a), 2.00(1H, m, H-4a), 2.29(1H, ddd, J=13.0, 5.2, 2.2Hz, H-4b), 1.17~1.01(5H, m, H-1b, H-14, H-22b, H-23a和H-24), 5.35(1H, dt, J=4.8, 2.1Hz, H-6), 1.33~1.54(9H, m, H-2b, H-7a, H-11, H-15a, H-17, H-22a, H-23b和H-25), 1.59(1H, m, H-8), 1.96(1H, q, J=2.5Hz, H-7b), 1.20(1H, ddd, J=17.7, 8.8, 4.8Hz, H-12a), 2.23(1H, m, H-12b), 1.15(1H, d, J=4.7Hz, H-15b), 1.27(1H, m, H-16b), 1.01(3H, s, H-18), 0.68(3H, s, H-19), 0.78(6H, dd, J=6.8, 4.3Hz, H-21和H-28), 0.92(3H, d, J=6.5Hz, H-26), 0.85(3H, d, J=6.8Hz, H-27); 13C NMR (150 MHz, CDCl3) δ 37.40(C-1), 31.82(C-2), 71.97(C-3), 42.46(C-4), 140.91 (C-5), 121.88(C-6), 30.72(C-7), 31.61(C-8), 50.28(C-9), 36.66(C-10), 21.23(C-11), 39.92(C-12), 56.90(C-14), 24.45(C-15), 28.34(C-16), 56.14(C-17), 12.01(C-18), 19.55(C-19), 36.33(C-20), 19.04(C-21), 33.87(C-22), 32.06(C-23), 39.22(C-24), 32.05(C-25), 17.74(C-26), 20.67(C-27), 15.59(C-28)。以上数据与文献(Deveci et al, 2019)报道一致, 故鉴定化合物11为(24S)-24-甲基胆甾醇。
化合物12: 白色固体C30H50O; EI-MS m/z 426 [M]+; 1H NMR (600 MHz, CDCl3) δ 1.82(1H, m, H-1a), 1.06(1H, m, H-1b), 1.85(1H, m, H-2a), 1.50(1H, m, H-2b), 3.51(1H, m, H-3), 2.23(1H, m, H-4a), 2.30(1H, m, H-4b), 5.35(1H, dd, J=5.1, 2.3Hz, H-6), 1.50(1H, m, H-7a), 2.02(1H, m, H-7b), 1.44(1H, m, H-11a), 1.51(1H, m, H-11b), 1.22(1H, m, H-12a), 2.20(1H, m, H-12b), 1.02(1H, m, H-14), 1.08(1H, d, J=4.8Hz, H-15a), 1.61(1H, m, H-15b), 1.32(1H, m, H-17), 0.66(3H, s, H-18), 1.01(6H, s, H-19和H-21), 0.17(1H, dq, J=8.9, 6.9Hz, H-22), 0.45(1H, m, H-24), 0.86(3H, d, J=6.6Hz, H-26), 0.94(6H, t, J=7.2Hz, H-28和H-27), 0.90(3H, s, H-29), 0.25(1H, d, J=8.9Hz, H-30a), -0.13(1H, m, H-30b); 13C NMR (150 MHz, CDCl3) δ 37.41(C-1), 32.19 (C-2), 71.98(C-3), 42.46(C-4), 140.92(C-5), 121.89(C-6), 32.05(C-7), 31.82(C-8), 50.31(C-9), 36.66(C-10), 21.29(C-11), 40.01(C-12), 42.93(C-13), 56.78(C-14), 24.70(C-15), 28.39(C-16), 58.08(C-17), 12.05(C-18), 19.56(C-19), 35.44(C-20), 22.34(C 21), 32.29(C-22), 25.95(C-23), 50.96(C-24), 32.12(C-25), 21.68(C-26), 21.32(C-27), 14.43(C-28), 15.60(C-29), 21.45(C-30)。以上数据与文献(蓝文健 等, 2003)报道一致, 故鉴定化合物12为柳珊瑚甾醇。

2.2 生物活性测试结果

2.2.1 抗菌活性测试

为探索分离得到的二萜和甾体类化合物的生物活性, 本实验开展了其对美人鱼发光杆菌P. damselae FP2244、耐土霉素的副乳房链球菌oxytetracycline-resistant S. parauberis SPOF3K、杀鲑气单胞菌A. salraonicida AS42、溶藻弧菌V. algaelyticus RZ53、杀鱼爱德华氏菌E. piscicida TH1和无乳链球菌S. agalactiae WR10的抗菌活性试验; 以及其对人类病原菌耐万古霉素屎肠球菌vancomycin-resistant E. faecalis G1、G4、G7、G8进行了抗菌活性检测, 实验结果如下表1所示。
表1 化合物的抗菌活性结果(MIC, 单位: μg·mL-1)

Tab. 1 Results of antimicrobial activity of compounds (MIC, unit: μg·mL-1)

菌种 化合物MIC
3 4 5 7 10 Tetr Oxy Lev Amp Van
vancomycin-resistant E. faecalis G1 - - - - 38.7 0.09 0.19 >39.78 >37.14 >297
vancomycin-resistant E. faecalis G4 - - - - 38.7 0.19 0.19 >39.78 >37.14 >297
vancomycin-resistant E. faecalis G7 30.2 31.6 33.2 1.5 0.48 0.09 0.09 >39.78 >37.14 18.56
vancomycin-resistant E. faecalis G8 - - - - 38.7 0.09 0.05 39.78 >37.14 74.25
A. salraonicida AS42 - - - - - 6.11 0.39 0.31 >18.57 NT
E. piscicida TH1 - - - - - 1.5 0.78 0.62 9.28 >148.5
P. damselae FP2244 30.2 31.6 33.2 30 38.7 0.02 0.02 0.02 0.02 NT
oxytetracycline-resistant S. parauberis SPOF3K 30.2 - - 30 38.7 >24.05 12.42 1.24 0.58 NT
S. agalactiae WR10 - - - - - 0.75 0.78 2.49 1.16 <0.2
V. algaelyticus RZ53 - - - - - 1.5 1.55 0.31 >37.14 18.56

注: - 表明50μg·mL-1无显著抑菌活性。NT: 未作抗菌试验。Tetr: 盐酸四环素; Oxy: 盐酸土霉素: Lev: 盐酸左氧氟沙星; Amp: 氨苄青霉素钠; Van: 盐酸万古霉素。

2.2.2 抗增殖活性测试

本研究对化合物3、4、5、7和10也进行了肿瘤细胞毒活性测试。运用MTT[5-di- phenytetrazoliumromide]法, 测试以上5种化合物对人非小细胞肺癌细胞(A549)的体外细胞毒活性, 结果表明化合物5对A549细胞具有细胞毒活性, IC50为37.89µmol·L-1。

3 讨论

豆荚软珊瑚作为软珊瑚中重要成员之一, 对其次级代谢产物进行化学和生物活性研究具有重大科学意义。本实验从豆荚软珊瑚Lobophytum sp.中分离到9个二萜类和3个甾体类化合物, 其中化合物8为首次从豆荚软珊瑚Lobophytum sp.分离得到。
本研究对分离的化合物进行了抗菌活性测试。结果表明, 化合物10对4株不同来源的耐万古霉素屎肠球菌具有较好的抑菌作用, 化合物3、4、5、7和10对耐万古霉素屎肠球菌G7, 以及对鱼类致病菌美人鱼发光杆菌FP2244和耐土霉素的副乳房链球菌SPOF3K具有显著的抑制作用。此外, 化合物5对A549细胞表现出抗细胞增殖活性, 半抑制浓度(half maximal inhibitory concentration, IC50)为37.89µmol·L-1。据文献报道, 化合物1对人脐静脉内皮细胞HUVEC和人慢性髓系白血病细胞K-562具有抗增殖活性, IC50分别为(48.4±1.2)和(53.8± 3.9)µmol·L-1, 对宫颈癌细胞系HeLa具有细胞毒活性, IC50为(51.9±0.7)µmol·L-1(Shaker et al, 2010)。化合物4对脂多糖诱导的小鼠腹腔巨噬细胞中NO的产生具有抑制作用, IC50为(24±2)µmol·L-1 (Zhao et al, 2016)。化合物6对黑穗病菌的生长具有抑制作用, 抑菌浓度为50µg·mm-1(Matthée et al, 1998)。化合物8对人非小细胞肺癌细胞A549和人口腔鳞状肿瘤细胞HSC-2具有细胞毒活性, IC50分别为49.70µmol·L-1和53.17µmol·L-1(Mohamed et al, 2021)。化合物9对TNF-α诱导的HepG2细胞中NF-κB的转录活性具有剂量依赖性的抑制作用, IC50为9.4µmol·L-1(Quang et al, 2011)。对比化合物4、5、7和8的结构及其抗肿瘤活性, 推测在C4位和C12位上的羟基取代可能增强了西松烷二萜对A549肿瘤细胞的抗增殖活性; 对比化合物3、4的结构, 它们C1、C2位构成的α, β-不饱和-γ-内酯环氧化程度是不同的, 推测C16位的羰基取代可能对西松烷二萜的抗肿瘤活性具有提升作用; 对比化合物3、4、5、7的结构及抗菌活性结果, 推测在C1、C2位形成的α, β-不饱和-γ-内酯环可能增强了西松烷二萜的抗菌活性, 与活性最好的化合物7相比, 化合物3、4、5在C7和C8位形成的环氧结构对西松烷二萜的抗菌活性有降低作用。本研究在一定程度上丰富了豆荚软珊瑚Lobophytum sp.的化学多样性, 阐明了豆荚软珊瑚用于抵御细菌的化学防御物质基础, 为海洋来源的新型抗生素的挖掘提供了依据, 同时也对西松烷二萜类化合物与其抗肿瘤活性以及抗菌活性之间构效关系的研究提供了一定的参考价值。

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