药学学报

Unit	Position	δ_C	δ_H	HMBC
Aba	1	170.4
	2	126.8
	3	125.6	7.33 (dd, J = 7.7, 1.6 Hz)	1, 5, 7
	4	124.3	7.13 (t, J = 7.5 Hz)	2, 6
	5	131.6	7.45 (td, J = 7.9, 1.6 Hz)	3, 7
	6	123.7	8.10 (d, J = 8.2 Hz)	2, 4, 7
	7	135.4
	NH		9.00 (s)	2, 6, 8
N-Me-Phe	8	168.5
	9	56.9	5.89 (dd, J = 11.3, 5.4 Hz)	8, 10, 11, 17, 18
	10	32.7	3.28 (dd, J = 15.0, 5.4 Hz) 3.05 (dd, J = 15.0, 11.4 Hz)	8, 9, 11, 12, 16
	11	136.6
	12/16	129.1	7.23 (d, J = 7.0 Hz)	10, 13, 14, 15
	13/15	128.6	7.26, overlapped	11, 12, 16
	14	126.8	7.19 (t, J = 7.0 Hz)
	17	30.4	2.85 (s)	9, 18
Ile	18	173.1
	19	54.4	4.44 (t, J = 10.2 Hz)	18, 20, 21, 23, 24
	20	36.4	1.81, overlapped
	21	25.0	0.51 (m)	19, 20, 22, 23
	22	11.0	0.62 (t, J = 7.2 Hz)	20, 21
	23	14.7	0.85 (d, J = 6.7 Hz)	19, 20, 21
	NH		7.24, overlapped	24
Ala	24	171.4
	25	50.0	4.50 (q, J = 6.9 Hz)	24, 26
	26	15.0	1.42 (d, J = 6.9 Hz)	24, 25
	NH		6.25 (d, J = 8.2 Hz)	1, 25, 26

Unit	Position	δ_C	δ_H	HMBC
Aba	1	170.4
	2	126.8
	3	125.6	7.33 (dd, J = 7.7, 1.6 Hz)	1, 5, 7
	4	124.3	7.13 (t, J = 7.5 Hz)	2, 6
	5	131.6	7.45 (td, J = 7.9, 1.6 Hz)	3, 7
	6	123.7	8.10 (d, J = 8.2 Hz)	2, 4, 7
	7	135.4
	NH		9.00 (s)	2, 6, 8
N-Me-Phe	8	168.5
	9	56.9	5.89 (dd, J = 11.3, 5.4 Hz)	8, 10, 11, 17, 18
	10	32.7	3.28 (dd, J = 15.0, 5.4 Hz) 3.05 (dd, J = 15.0, 11.4 Hz)	8, 9, 11, 12, 16
	11	136.6
	12/16	129.1	7.23 (d, J = 7.0 Hz)	10, 13, 14, 15
	13/15	128.6	7.26, overlapped	11, 12, 16
	14	126.8	7.19 (t, J = 7.0 Hz)
	17	30.4	2.85 (s)	9, 18
Ile	18	173.1
	19	54.4	4.44 (t, J = 10.2 Hz)	18, 20, 21, 23, 24
	20	36.4	1.81, overlapped
	21	25.0	0.51 (m)	19, 20, 22, 23
	22	11.0	0.62 (t, J = 7.2 Hz)	20, 21
	23	14.7	0.85 (d, J = 6.7 Hz)	19, 20, 21
	NH		7.24, overlapped	24
Ala	24	171.4
	25	50.0	4.50 (q, J = 6.9 Hz)	24, 26
	26	15.0	1.42 (d, J = 6.9 Hz)	24, 25
	NH		6.25 (d, J = 8.2 Hz)	1, 25, 26

Compound^a	EC₅₀/μg·mL^-1	LC₅₀/μg·mL^-1
1	9.75 ± 0.28	> 50
2	0.12 ± 0.28	> 50
3	0.19 ± 0.12	> 50
6	5.66 ± 0.07	> 50
7	1.23 ± 0.08	> 50
9	4.93 ± 0.08	> 50
11	12.52 ± 0.08	> 50
12	2.94 ± 0.07	> 50
SeaNine-211	1.94 ± 0.41	33.93 ± 2.06

Compound^a	EC₅₀/μg·mL^-1	LC₅₀/μg·mL^-1
1	9.75 ± 0.28	> 50
2	0.12 ± 0.28	> 50
3	0.19 ± 0.12	> 50
6	5.66 ± 0.07	> 50
7	1.23 ± 0.08	> 50
9	4.93 ± 0.08	> 50
11	12.52 ± 0.08	> 50
12	2.94 ± 0.07	> 50
SeaNine-211	1.94 ± 0.41	33.93 ± 2.06

Compound^a	MIC /μg·mL^-1
4	53.06	13.26	26.53	13.26
11	54.81	27.41	27.41	13.70
Chloramphenicol	12.50	6.25	12.50	12.50
Penicillin	25.00	6.25	12.50	12.50

Compound^a	MIC /μg·mL^-1
4	53.06	13.26	26.53	13.26
11	54.81	27.41	27.41	13.70
Chloramphenicol	12.50	6.25	12.50	12.50
Penicillin	25.00	6.25	12.50	12.50

海洋真菌Aspergillus terreus GXIMD00546中的一个环四肽及其抗污损活性

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于芹 ¹^,² , 戚梦凡 ² , 王玲芝 ² , 李思 ² , 刘锴 ² , 罗小卫 ² , 高程海 ² , 刘永宏 ¹^,² , 徐新亚 ²^,^*

药学学报 | 研究论文 2025,60(7): 2254-2262

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药学学报 |研究论文 2025 , 60 (7) : 2254 -2262

海洋真菌Aspergillus terreus GXIMD00546中的一个环四肽及其抗污损活性

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于芹¹^,², 戚梦凡², 王玲芝², 李思², 刘锴², 罗小卫², 高程海², 刘永宏¹^,², 徐新亚²^,^*

作者信息

¹沈阳药科大学无涯创新学院, 辽宁沈阳 110016

²广西中医药大学海洋药物研究院, 广西海洋药物重点实验室, 广西南宁 530200

通讯作者:

徐新亚，Tel: 86-771-4733826, E-mail: xuxy@gxtcmu.edu.cn

作者简介:

作者贡献:

于芹是本文第一作者, 负责化合物分离、结构鉴定和论文撰写; 戚梦凡和王玲芝参与本文的活性测定; 李思参与菌株的发酵和提取; 刘锴参与化合物结构鉴定; 罗小卫、高程海、刘永宏参与实验设计和指导; 徐新亚是本文通讯作者, 负责实验指导、稿件修改工作。

A new antifouling tetrapeptide from marine fungus Aspergillus terreus GXIMD00546

Qin YU¹^,², Meng-fan QI², Ling-zhi WANG², Si LI², Kai LIU², Xiao-wei LUO², Cheng-hai GAO², Yong-hong LIU¹^,², Xin-ya XU²^,^*

Affiliations

¹Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang 110016, China

²Guangxi Key Laboratory of Marine Drugs, Institute of Marine Drugs, Guangxi University of Chinese Medicine, Nanning 530200, China

出版时间: 2025-07-12 doi: 10.16438/j.0513-4870.2025-0051

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摘要

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为了阐明抗污损活性菌株Aspergillus terreus GXIMD00546的活性成分, 采用硅胶柱色谱、高效液相色谱(HPLC) 等色谱技术对菌株Aspergillus terreus GXIMD00546大米培养基的发酵产物进行分离纯化, 并通过核磁共振波谱、高分辨质谱和结构衍生化等方法鉴定为1个新化合物asperterrestide C (1) 和12个已知化合物territrem B (2)、methyl-3, 4, 5-trimethoxy-2-(2-(nicotinamido) benzamido) benzoate (3)、butyrolactone Ⅰ (4)、terrien (5)、(1R, 2S)-2-hydroxy-5-oxo-3-((E)-prop-1-en-1-yl) cyclopent-3-en-1-yl acetate (6)、(1S, 5R)-5-hydroxy-4-oxo-2-((E)-prop-1-en-1-yl) cyclopent-2-en-1-yl acetate (7)、4-hydroxykigelin (8)、monacolin K (9)、methyl ester of lactone ring-opened monacolin K (10)、butyrolactone Ⅶ (11)、aspernolide A (12)、epi-aszonalenin C (13)。新化合物asperterrestide C (1) 为环肽类成分, 具有少见的氨基甲基化修饰。化合物1~3、6、7、9、11、12具有明确的抗海洋污损生物藤壶附着活性, 抑制纹藤壶金星幼虫附着半数有效浓度(EC₅₀) 为0.12~12.5 μg·mL^-1, 且毒性较小。化合物1、2、4、9~12具有乙酰胆碱酯酶抑制活性, EC₅₀为17.16~55.77 μg·mL^-1。化合物4和11具有广谱抑菌活性, 对测试的人类致病菌和污损细菌均有明显抑制作用。

关键词

海洋真菌 / Aspergillus terreus / 环四肽 / 抗污损 / 酶抑制

Abstract

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In order to clarify the anti-fouling active components of the marine-derived fungal strain Aspergillus terreus GXIMD00546, its fermentation products of the rice medium were isolated by silica gel column chromatography and purified using HPLC. One new compound asperterrestide C (1) and 12 known compounds territrem B (2), methyl-3, 4, 5-trimethoxy-2-(2-(nicotinamido)benzamido)benzoate (3), butyrolactone Ⅰ (4), terrien (5), (1R, 2S)-2-hydroxy-5-oxo-3-((E)-prop-1-en-1-yl)cyclopent-3-en-1-yl acetate (6), (1S, 5R)-5-hydroxy-4-oxo-2-((E)-prop-1-en-1-yl)cyclopent-2-en-1-yl acetate (7), 4-hydroxykigelin (8), monacolin K (9), methyl ester of lactone ring-opened monacolin K (10), butyrolactone Ⅶ (11), aspernolide A (12), and epi-aszonalenin C (13) were identified by the detailed data analysis of NMR sepectra, high-resolution mass spectrometry spectra and the comparison with literatures. The new compound asperterrestide C (1) is a cyclopeptide with rare aminomethylation modifications. Compounds 1-3, 6, 7, 9, 11, 12 showed clear anti-fouling activities against the attachment of barnacle larvae in the EC₅₀ ranges of 0.12-12.5 μg·mL^-1 with low toxicity. Compounds 1, 2, 4, 9-12 also exhibited acetylcholinesterase inhibitory effects with EC₅₀ range of 17.16-55.77 μg·mL^-1. Compounds 4 and 11 have broad spectrum antibacterial activity against human pathogenic bacterium methicillin-resistant Streptococcus aureus (MRSA), and fouling bacteria Methanococcus janaschii (MJ), Vibrio pelagius (VP), and V. Rotiferianus (VR).

Key words

marine fungus / Aspergillus terreus / cyclotetrapeptide / antifouling / acetylcholinesterase inhibition

引用本文

于芹, 戚梦凡, 王玲芝, 李思, 刘锴, 罗小卫, 高程海, 刘永宏, 徐新亚. 海洋真菌Aspergillus terreus GXIMD00546中的一个环四肽及其抗污损活性. 药学学报, 2025 , 60 (7) : 2254 -2262 . DOI: 10.16438/j.0513-4870.2025-0051

Qin YU, Meng-fan QI, Ling-zhi WANG, Si LI, Kai LIU, Xiao-wei LUO, Cheng-hai GAO, Yong-hong LIU, Xin-ya XU. A new antifouling tetrapeptide from marine fungus Aspergillus terreus GXIMD00546[J]. Acta Pharmaceutica Sinica, 2025 , 60 (7) : 2254 -2262 . DOI: 10.16438/j.0513-4870.2025-0051

正文

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海洋微生物长期生存在高压、高盐、寡营养等特殊的生态环境下, 通常具有独特的代谢途径和次生代谢产物。曲霉属海洋真菌是新天然产物最重要的来源之一, 可以产生多种类型的次级代谢产物, 包括聚酮类、生物碱、丁烯内酯、多肽等, 并且通常具有较好的生物活性, 如细胞毒性、抗菌活性等^{[1, 2]}。北海竹林盐场是广西最大的天然海水结晶盐生产基地, 具有丰富的极端嗜盐微生物资源。作者前期从中分离得到16株真菌, 分属6科6属12种, 部分菌株发酵提取物具有显著的抗菌活性^[3]。从其中一株镰刀菌属真菌GXIMD00527中分离得到表伊快霉素, 能够有效抑制耐甲氧西林金黄色葡萄球菌的毒力因子和生物被膜的形成, 从而降低其致病性和耐药性^[4]。

海洋生物污损是指海洋污损生物的附着、侵袭造成的水下设施的损害, 如增加舰船航行阻力, 加大燃油消耗, 增加温室气体排放; 堵塞管道和线缆, 加速金属老化和腐蚀, 危害海上作业平台的安全; 堵塞海上渔业网具和养殖网箱, 造成养殖产量减少; 并且污损生物附着在舰船上, 还造成外来生物入侵。海洋生物污损每年造成15~30亿美元的直接经济损失^[5]。藤壶是我国最常见的污损生物之一。乙酰胆碱酯酶(acetylcholinesterase, AChE) 是生物神经传导中的一种关键性酶, 该酶能降解乙酰胆碱, 终止神经递质对突触后膜的兴奋作用, 保证神经信号在生物体内的正常传递。乙酰胆碱酯酶和藤壶附着密切相关, 是抗藤壶附着的关键靶点之一^{[6, 7]}。在海洋真菌活性次生代谢产物的研究过程中, 作者发现一株来自北海竹林盐场的土曲霉Aspergillus terreus GXIMD00546, 其发酵提取物具有明显的抗海洋生物污损活性, 能够抑制污损生物藤壶幼虫附着, 但活性成分不明确。进一步的化学成分研究从其发酵产物中分离得到1个环四肽类化合物(1) 和12个已知化合物(2~13, 图1), 部分化合物具有抗生物污损、抗乙酰胆碱酯酶和抗菌活性。本文将详细介绍了这些化合物的分离纯化、结构鉴定及其活性测定。

结果与讨论

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1　菌株的来源及鉴定

菌株分离纯化自广西北海盐田, 利用聚合酶链式反应(PCR) 扩增技术对菌株的内转录间隔区(internal transcribed spacer, ITS) 序列进行扩增测序(武汉擎科生物科技股份有限公司), 通过Blast检索分析发现其ITS序列与菌株Aspergillus terreus 1:1-546 (accession No.OW986430) 的相似度100%, 综合形态学特征将该真菌鉴定为土曲霉(GenBank Accession No. PV104274)。菌种保存于广西中医药大学海洋药物研究院海洋微生物资源库中, 保存编号为GXIMD00546。

2　化合物结构鉴定

化合物1为淡黄色粉末。根据高分辨电喷雾电离质谱(HR-ESI-MS) 显示其准分子离子峰m/z 465.250 6和487.232 7 (计算值[M+H]⁺ 465.249 6, [M+Na]⁺ 487.231 6), 确定化合物1的分子式为C₂₆H₃₂N₄O₄。根据¹H、¹³C NMR和HSQC数据(表1) 推测化合物含有4个甲基: [δ_H 2.85 (s, N-CH₃-17), δ_C 30.4 (CH₃-17)], [δ_H 0.62 (3H, t, J = 7.2 Hz, H-22), δ_C 11.0 (CH₃-22)], [δ_H 0.85 (3H, d, J = 6.7 Hz, H-23), δ_C 14.7 (CH₃-23)], [δ_H 1.42 (3H, d, J = 6.9 Hz, H-26), δ_C 15.0 (CH₃-26)]; 2个亚甲基[δ_H 3.28 (1H, dd, J = 15.0, 5.4 Hz, H-10a), 3.05 (1H, dd, J = 15.0, 11.4 Hz, H-10b), δ_C 32.7 (CH₂-10)], [δ_H 0.51 (2H, m, H-21), δ_C 25.0 (CH₂-21)]。13个次甲基包括9个芳香环次甲基信号: [δ_H 7.33 (1H, dd, J = 7.7, 1.6 Hz, H-3), δ_C 125.6 (CH-3)], [δ_H 7.13 (1H, t, J = 7.5 Hz, H-4), δ_C 124.3 (CH-4)], [δ_H 7.45 (1H, td, J = 7.9, 1.6 Hz, H-5), δ_C 131.6 (CH-5)], [δ_H 8.10 (1H, d, J = 8.2 Hz, H-6), δ_C 123.7 (CH-6)], [δ_H 7.23 (2H, d, J = 7.0 Hz, H-12/16), δ_C 129.1 (CH-12/16)], [δ_H 7.26 (2H, overlapped, H-13/15), δ_C 128.6 (CH-13/15)], [δ_H 7.19 (1H, t, J = 7.0 Hz, H-14), δ_C 126.8 (CH-14)] 和4个其他次甲基: [δ_H 5.89 (1H, dd, J = 11.3, 5.4 Hz, H-9), δ_C 56.9 (CH-9)], [δ_H 4.44 (1H, t, J = 10.2 Hz, H-19), δ_C 54.4 (CH-19)], [δ_H 1.81 (1H, overlapped, H-20), δ_C 36.4 (CH-20)], [δ_H 4.50 (1H, q, J = 6.9 Hz, H-25), δ_C 50.0 (CH-25)]。4个羰基碳信号[δ_C 170.4 (C-1), 168.5 (C-8), 173.1 (C-18), 171.4 (C-24)] 和3个芳香环季碳信号[δ_C 126.8 (C-2), 135.4 (C-7), 136.6 (C-11)]。这些数据均与环四肽化合物asperterrestide A极为相似^[8], 唯一的差异是H-10/C-10位的核磁数据从asperterrestide A [δ_H 5.15 (1H, d, J = 10.0 Hz, H-10), δ_C 71.4 (CH-10)] 在化合物1中向高场位移[δ_H 3.28 (dd, J = 15.0, 5.4 Hz, H-10a), 3.05 (dd, J = 15.0, 11.4 Hz, H-10b), δ_C 32.7 (CH-10)], 表明化合物1中C-10位的羟基可能缺失。二维核磁数据也证实了该推测: ¹H-¹H COSY相关信号H-13与H-12, 14相关, H-15与H-14, 16相关, H-9与H-10相关, HMBC谱图H-12与C-10, 11, 16相关, H-9与C-8, 10, 11相关, H-17与C-9相关, 表明存在N-甲基苯丙氨酸(N-Me-Phe) (图2)。从而确定该化合物由N-甲基苯丙氨酸(N-Me-Phe)、邻氨基苯甲酸(2-aminobenzoic acid, Aba)、异亮氨酸/别异亮氨酸(Ile/allo-Ile) 和丙氨酸(Ala) 构成。HMBC谱图邻氨基苯甲酸氨基氢[δ_H 9.00 (1H, s)] 与C-8相关, N-CH₃-17 [δ_H 2.85 (s)] 与C-18相关, 异亮氨酸/别异亮氨酸氨基氢[δ_H 7.24 (1H, overlapped)] 与C-24相关, 丙氨酸氨基氢[δ_H 6.25 (1H, s)] 与C-1相关, 以及ROESY谱图相关信号: 邻氨基苯甲酸氨基氢与H-9相关, N-甲基苯丙氨酸甲基氢与H-19相关, 表明环四肽的平面结构为环-(邻氨基苯甲酸-N-甲基苯丙氨酸-异亮氨酸/别异亮氨酸-丙氨酸)。氨基酸的构型通过比对氨基酸标准品确定: 将化合物1与L/D构型N-甲基苯丙氨酸、异亮氨酸/别异亮氨酸、丙氨酸标准品分别进行Marfey水解衍生化反应, 产物进行LC-MS分子离子峰和保留时间比对(图3), 确定为N-甲基L-苯丙氨酸、L-丙氨酸以及D-异亮氨酸/别异亮氨酸。D-异亮氨酸和D-别异亮氨酸无法通过LC-MS区分, 采用HPLC手性分析柱(Phenomenex Lux i-Amylose-1, 广州飞诺美), 正己烷/异丙醇(含0.1%甲酸) 3%~10%梯度分析, 根据保留时间差异确定为D-别异亮氨酸(图4)。从而确定化合物1为环-(邻氨基苯甲酸-N-甲基L-苯丙氨酸-D-别异亮氨酸-L-丙氨酸), 与化合物asperterrestide A和asperterrestide B^{[8, 9]}结构相似, 因此命名为asperterrestide C。

根据波谱数据结合文献分析, 已知化合物结构鉴定为territrem B^[10](2)、methyl-3, 4, 5-trimethoxy-2-(2-(nicotinamido)benzamido)benzoate^[11] (3)、butyrolactone Ⅰ^[12](4)、terrien^[13](5)、(1R, 2S)-2-hydroxy-5-oxo-3-((E)-prop-1-en-1-yl)cyclopent-3-en-1-yl acetate^{[13, 14]}(6)、(1S, 5R)-5-hydroxy-4-oxo-2-((E)-prop-1-en-1-yl)cyclopent-2-en-1-yl acetate^[14](7)、4-hydroxykigelin^[15](8)、monacolin K^[16](9)、methyl ester of lactone ring-opened monacolin K^[17](10)、butyrolactone Ⅶ^[18](11)、aspernolide A^[19](12)、epi-aszonalenin C^[20](13)。

3　生物活性测定

3.1　抗污损活性

测试了13个化合物抗海洋污损生物纹藤壶金星幼虫的附着活性, 从表2可以看出化合物1~3、6、7、9、11、12均显示活性, EC₅₀为0.12~12.5 μg·mL^-1, 均显示出良好的抗污损活性。化合物asperterrestide C (1) 显示中等抗污损活性, 同类型化合物asperterrestides A、B未检测出抗藤壶幼虫活性^[9], 可能是由于氨基酸构型不同以及C-10位羟基取代的缺失造成活性差异。化合物2、3、7的EC₅₀值分别为0.12、0.19、1.23 μg·mL^-1, 且半数致死浓度LD₅₀均大于50 μg·mL^-1, 毒性较小, 有望作为抗附着先导化合物开展进一步研究。

3.2　抑制乙酰胆碱酯酶活性

在乙酰胆碱酯酶抑制活性筛选试验中, 化合物1、2、4、9~12显示中等抑制活性, EC₅₀分别为31.24 ± 2.68、28.96 ± 1.89、17.16 ± 0.40、55.77 ± 2.95、40.59 ± 2.95、29.76 ± 1.52、33.99 ± 2.32 μg·mL^-1, 阳性对照石杉碱甲为EC₅₀为0.01 ± 0.003 μg·mL^-1。实验结果表明化合物1、2、9、11、12的抗藤壶幼虫附着的作用靶点可能是乙酰胆碱酯酶, 有待进一步验证。

3.3　抑菌活性

化合物4和11对一种人类致病菌和三种污损细菌均显示明显抑制作用, 显示出广谱抗菌活性, 最小抑菌浓度(MIC) 见表3。化合物4对耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA)、甲烷球菌(Methanococcus janaschii, MJ)、佩拉吉弧菌(Vibrio pelagius, VP) 和罗氏弧菌(V. rotiferianus, VR) 的MIC值分别是53.06、13.26、26.53、13.26 μg·mL^-1。化合物11对MRSA、M. jannaschii、V. Pelagius、V. rotiferianus的MIC分别是54.81、27.41、27.41、13.70 μg·mL^-1。

实验部分

收起

Eyela-1100V-W型旋转蒸发仪(日本东京理化株式会社); ZYJ-S型超净工作台(苏州净化设备公司); Bruker AVANCE-500 MHz超导核磁共振仪(瑞士布鲁克公司); MLS-3781L-PC型高压灭菌锅(普和希健康医疗器械上海有限公司); UPH-II-20TN型纯水仪(成都优普仪器设备有限公司); 中压制备色谱仪(北京赛普瑞思液相色谱仪); Prominnence LC-2030型高效液相色谱仪(日本岛津公司); Xevo G2 QTof型高分辨质谱仪(美国Waters公司); J-1500型圆二色谱仪(日本JASCO公司); SYNERGY H1型酶标仪(美国BioTek公司); 薄层色谱及柱色谱用硅胶(烟台江友硅胶有限公司)。

1　菌株的发酵与培养

在无菌环境下, 将真菌接种于含有海盐的PDA培养基上(马铃薯粉6 g、葡萄糖20 g、琼脂粉20 g、人工海水1 L), 在28 ℃的培养箱中培养3~4天进行活化。将活化好的真菌菌丝转移到液体PDB培养基(马铃薯粉6 g, 葡萄糖20 g, 人工海水1 L) 中, 在28 ℃和1 800 r·min^-1的摇床中培养3~4天, 获得真菌的种子液。将种子液转移到含有大米培养基的1 L三角瓶中(80 g大米、0.4 g酵母提取物、0.4 g葡萄糖、人工海水120 mL), 发酵200瓶, 静置培养30天。

2　发酵产物的提取与分离

发酵产物用乙酸乙酯萃取3次, 旋转蒸发仪减压蒸馏, 得到提取物245.2 g。提取物进行硅胶柱色谱分离, 采用二氯甲烷/甲醇体系(梯度100∶0~0∶100) 进行梯度洗脱, 经TLC检测合并之后得到17个馏分(Fr.1~Fr.17)。Fr.6 (52.7 g) 经过中压反相ODS柱分离, 水/乙腈体系(梯度95∶5~10∶90) 梯度分离得到28个亚组分(sFr.6.1~sFr.6.28)。sFr.6.6经半制备柱(45%乙腈/水) 分离纯化得到化合物13 (t_R = 26.8 min, 3.3 mg)。sFr.6.8经半制备柱(20%乙腈/水) 分离纯化得到化合物5 (t_R = 11.1 min, 8.9 mg)、化合物6 (t_R = 16.4 min, 12.2 mg)、化合物7 (t_R = 17.0 min, 19.9 mg)。sFr.6.9经半制备柱(44%乙腈/水) 纯化, 得到化合物8 (t_R = 17.9 min, 46.8 mg)。sFr.6.26 (1.4 g) 经过中压反相ODS柱分离, 采用水/乙腈体系(梯度95∶5~10∶90) 梯度分离得到13个亚组分(sFr.6.26.1~sFr.6.26.13), sFr.6.26.8经半制备柱(45%乙腈/水) 纯化, 得到化合物2 (t_R = 29.5 min, 20.1 mg)、化合物11 (t_R = 29.0 min, 9.8 mg) 和化合物12 (t_R = 29.8 min, 4.5 mg)。sFr.6.26.9经半制备柱(46%乙腈/水) 纯化得到化合物1 (t_R = 30.3 min, 10.8 mg)。sFr.6.26.11经半制备HPLC柱(65%乙腈/水) 纯化得到化合物9 (t_R = 35.6 min, 8.9 mg) 和化合物10 (t_R = 36.2 min; 22.0 mg)。Fr7 (21.6 g) 使用减压柱色谱分离, 采用石油醚/乙酸乙酯(100∶0~0∶100) 体系, 经TLC分析合并之后得到11个亚组分(sFr7.1~sFr7.11)。sFr.7.1经过中压反相ODS柱分离, 水/乙腈体系(梯度95∶5~10∶90) 梯度分离得到22个亚组分(sFr.7.1.1~sFr.7.1.22)。sFr.7.1.4经半制备柱(50%乙腈/水) 分离纯化得到化合物4 (t_R = 27.8 min, 236.7 mg)。sFr.7.1.10经半制备柱(45%乙腈/水) 分离纯化得到化合物3 (t_R = 26.1 min, 19.7 mg)。

3　化合物理化数据

化合物1 (asperterrestide C): 淡黄色固体, 易溶于甲醇; [α]

$ {}_{\mathrm{D}}^{21} $

= +14.4 (c 0.16, MeOH); CD (MeOH) λ_max (log ε) 211 (-10.5), 229 (+8.19), 2.49 (-3.20) nm; UV (MeOH) λ_max (log ε) 348 (2.11) nm; ¹H NMR (500 MHz, CDCl₃) 和¹³C NMR (125 MHz, CDCl₃) 见表1, HR-ESI-MS: m/z 465.250 6 [M+H]⁺, 487.232 7 [M+Na]⁺。

化合物2 (territrem B): 白色粉末, 易溶于甲醇; ¹H NMR (500 MHz, CDCl₃): δ_H 6.99 (2H, s, H-2′/6′), 6.39 (1H, s, H-8), 6.31 (1H, d, J = 10.2 Hz, H-3), 5.83 (1H, d, J = 10.2 Hz, H-2), 3.90 (6H, s, 3′/5′-OCH₃), 3.89 (3H, s, 4′-OCH₃), 3.39 (1H, d, J = 17.7 Hz, H_α-12), 2.85 (1H, d, J = 17.7 Hz, H_β-12), 2.46 (1H, td, J = 13.1, 4.8 Hz, H_α-6), 1.91 (2H, m, H-5), 1.81 (1H, m, H_β-6), 1.52 (3H, s, H-15), 1.46 (3H, s, H-16), 1.27 (3H, s, H-13), 1.18 (3H, s, H-14); ¹³C NMR (125 MHz, CDCl₃): δ_C 204.9 (C, C-1), 164.5 (C, C-11), 163.0 (C, C-7a), 158.7 (C, C-9), 153.6 (CH, C-3), 153.4 (C, C-3′/5′), 140.3 (C, C-4′), 126.9 (C, C-1′), 123.4 (CH, C-2), 102.8 (CH, C-2′/6′), 97.7 (CH, C-8), 97.4 (C, C-11a), 80.1 (C, C-6a), 79.2 (C, C-4a), 77.3 (C, C-12a), 61.1 (CH₃, 4′-OCH₃), 56.4 (CH₃, C-16), 56.4 (CH₃, 3′/5′-OCH₃), 42.8 (C, C-4), 28.6 (CH₂, C-6), 28.1 (CH₂, C-12), 25.9 (CH₂, C-5), 25.6 (CH₃, C-13), 24.0 (CH₃, C-14), 23.9 (CH₃, C-15), 22.0 (CH₃, C-16)。

化合物3 (methyl-3, 4, 5-trimethoxy-2-(2-(nicotinamido)benzamido)benzoate): 淡黄色粉末, 易溶于甲醇; ¹H NMR (500 MHz, CDCl₃): δ_H 12.27 (s, 1H, 2-NH), 9.27 (s, 1H, H-2′), 9.23 (d, J = 2.4 Hz, 1H, 2″-NH), 8.83 (1H, dd, J = 8.5, 1.2 Hz, H-3), 8.72 (1H, dd, J = 5.0, 1.6 Hz, H-4′), 8.26 (1H, ddd, J = 8.0, 2.4, 1.6 Hz, H-6′), 7.91 (1H, dd, J = 7.9, 1.6 Hz, H-6), 7.62 (1H, m, H-5′), 7.38 (1H, dd, J = 8.0, 4.8 Hz, H-4), 7.29 (1H, s, H-6″), 7.24 (1H, td, J = 7.6, 1.1 Hz, H-5), 3.99 (3H, s, 4″-OCH₃), 3.91 (6H, s, 3″/5″-OCH₃), 3.83 (3H, s, 7″-OCH₃); ¹³C NMR (125 MHz, CDCl₃): δ_C 168.2 (C, C-7″), 167.1 (C, C-7), 163.9 (C, C-7′), 152.5 (CH, C-4′), 151.4 (C, C-5″), 149.1 (CH, C-4″), 148.8 (CH, C-2′), 146.8 (C, C-3″), 140.3 (C, C-2), 135.2 (CH, C-6′), 133.6 (CH, C-4), 130.6 (C, C-1′), 127.9 (CH, C-6), 125.7 (C, C-5″), 123.7 (CH, C-5), 123.6 (CH, C-5′), 121.7 (CH, C-3), 120.3 (CH, C-1″), 118.9 (CH, C-1), 108.6 (CH, C-6″), 61.2 (CH₃, 3″/4″-OCH₃), 56.4 (CH₃, 5″-OCH₃), 52.6 (CH₃, 7″-OCH₃)。

化合物4 (butyrolactone Ⅰ): 无色油状液体, 易溶于甲醇; ¹H NMR (500 MHz, CDCl₃): δ_H 7.63 (2H, d, J = 8.6 Hz, H-2′/6′), 6.91 (2H, d, J = 8.6 Hz, H-3′/5′), 6.59 (1H, dd, J = 8.3, 2.1 Hz, H-12), 6.51 (1H, s, H-8), 6.52 (1H, d, J = 8.3 Hz, H-11), 5.08 (1H, t, J = 7.2 Hz, H-2″), 3.76 (3H, s, 6″-OCH₃), 3.55 (1H, d, J = 14.7 Hz, H_α-6), 3.50 (1H, d, J = 14.7 Hz, H_β-6), 3.13 (2H, d, J = 7.2 Hz, H-1″), 1.65 (3H, s, H-4″), 1.60 (3H, s, H-5″); ¹³C NMR (125 MHz, CDCl₃): δ_C 170.1 (C, C-6″), 169.9 (C, C-2), 156.9 (C, C-4′), 153.1 (C, C-10), 137.6 (C, C-3), 134.2 (C, C-3″), 131.9 (CH, C-8), 129.7 (CH, C-2′/6′), 129.2 (CH, C-12), 128.8 (C, C-4), 126.7 (C, C-9), 124.7 (C, C-7), 122.2 (C, C-1′), 121.6 (C, C-2″), 116.2 (CH, C-3′/5′), 115.2 (CH, C-11), 86.4 (C, C-5), 53.6 (CH₃, 6″-OCH₃), 38.7 (CH₂, C-6), 28.9 (CH₂, C-1″), 25.8 (CH₃, C-5″), 17.8 (CH₃, C-4″)。

化合物5 (terrien): 淡黄色粉末, 易溶于甲醇; ¹H NMR (500 MHz, CDCl₃): δ_H 6.82 (1H, dq, J = 14.0, 6.8 Hz, H-7), 6.38 (1H, d, J = 15.0 Hz, H-6), 5.99 (1H, s, H-5), 4.88 (1H, br s, H-2), 4.32 (1H, br s, H-3), 1.94 (3H, d, J = 6.6 Hz, H-8); ¹³C NMR (125 MHz, CDCl₃): δ_C 203.4 (C, C-1), 169.0 (C, C-4), 142.0 (CH, C-5), 125.2 (CH, C-6), 125.0 (CH, C-7), 81.9 (CH, C-2/3), 19.8 (CH₃, C-8)。

化合物6 ((1R, 2S)-2-hydroxy-5-oxo-3-((E)-prop-1-en-1-yl)cyclopent-3-en-1-yl acetate): 黄褐色油状液体, 易溶于甲醇; ¹H NMR (500 MHz, CDCl₃): δ_H 6.85 (1H, m, H-7), 6.42 (1H, d, J = 16.3 Hz, H-6), 6.05 (1H, s, H-5), 4.88 (1H, br s, H-2), 4.84 (1H, br s, H-3), 2.22 (3H, s, H-2′), 1.96 (3H, dd, J = 6.9, 1.2 Hz, H-8); ¹³C NMR (125 MHz, CDCl₃): δ_C 197.1 (C, C-1), 173.2 (C, C-1′), 168.5 (C, C-4), 142.5 (CH, C-5), 126.8 (CH, C-6), 125.2 (CH, C-7), 83.4 (CH, C-2), 75.9 (CH, C-3), 20.8 (CH₃, C-2′), 19.8 (CH₃, C-8)。

化合物7 ((1S, 5R)-5-hydroxy-4-oxo-2-((E)-prop-1-en-1-yl)cyclopent-2-en-1-yl acetate): 黄褐色油状液体, 易溶于甲醇; ¹H NMR (500 MHz, CDCl₃): δ_H 6.43 (1H, dq, J = 16.0, 6.7 Hz, H-7), 6.32 (1H, d, J = 16.0 Hz, H-6), 6.14 (1H, s, H-5), 5.71 (1H, s, H-3), 4.23 (1H, d, J = 2.2 Hz, H-2), 2.17 (3H, s, H-2′), 1.93 (3H, d, J = 6.7 Hz, H-8); ¹³C NMR (125 MHz, CDCl₃): δ_C 200.9 (C, C-1), 171.6 (C, C-1′), 164.9 (C, C-4), 140.3 (CH, C-5), 127.6 (CH, C-6), 124.7 (CH, C-7), 79.0 (CH, C-3), 79.0 (CH, C-2), 21.0 (CH₃, C-2′), 19.6 (CH₃, C-8)。

化合物8 (4-hydroxykigelin): 白色粉末, 易溶于甲醇; ¹H NMR (500 MHz, DMSO-d₆): δ_H 6.72 (1H, s, H-5), 4.68 (1H, dd, J = 6.5, 2.1 Hz, H-3), 4.44 (1H, d, J = 2.2 Hz, H-4), 3.89 (3H, s, 7-OCH₃), 3.71 (3H, s, 6-OCH₃), 1.38 (3H, d, J = 6.6 Hz, 3-CH₃); ¹³C NMR (500 MHz, DMSO-d₆): δ_C 169.0 (C, C-1), 158.1 (C, C-6), 154.6 (C, C-8), 138.3 (C, C-10), 135.4 (C, C-7), 103.2 (CH, C-5), 101.5 (C, C-9), 78.1 (CH, C-3), 65.26 (CH, C-4), 59.8 (CH₃, 6-OCH₃), 56.1 (CH₃, 7-OCH₃), 15.7 (CH₃, 3-CH₃)。

化合物9 (monacolin K): 无色油状, 易溶于甲醇; ¹H NMR (500 MHz, DMSO-d₆): δ_H 5.95 (1H, d, J = 9.6 Hz, H-4), 5.78 (1H, d, J = 9.6 Hz, H-3), 5.50 (1H, t, J = 3.5 Hz, H-5), 5.23 (1H, m, H-8), 4.46 (1H, ddt, J = 11.4, 7.2, 3.5 Hz, H-11), 4.09 (1H, m, H-13), 2.62 (1H, dd, J = 17.3, 4.5 Hz, H_α-14), 2.39 (1H, m, H_β-14), 2.35 (2H, m, H-2/H-6), 2.30 (2H, m, H-8a/H-2′), 1.93 (1H, ddd, J = 14.8, 8.0, 2.3 Hz, H_α-7), 1.82 (1H, dd, J = 15.0, 3.8 Hz, H-1), 1.75 (1H, dq, J = 13.5, 3.2, 2.8 Hz, H_α-12), 1.69 (1H, m, H_α-10), 1.60 (2H, m, H_β-7/H_β-12), 1.54 (1H, m, H_α-3′), 1.39 (1H, dd, J = 13.8, 7.0 Hz, H_α-9), 1.34 (1H, dd, J = 11.9, 5.1 Hz, H_β-3′), 1.29 (1H, dd, J = 11.6, 3.4 Hz, H_β-9), 1.25 (1H, dd, J = 12.5, 4.2 Hz, H_β-10), 1.02 (3H, d, J = 3.2 Hz, H-3″), 1.01 (3H, d, J = 3.8 Hz, H-2″), 0.86 (6H, m, H-1″/4′); ¹³C NMR (125 MHz, DMSO-d₆): δ_C 175.6 (C, C-1′), 170.2 (C, C-15), 133.1 (CH, C-3), 131.6 (C, C-4a), 129.2 (CH, C-5), 128.3 (CH, C-4), 75.8 (CH, C-11), 67.4 (CH, C-8), 61.3 (CH, C-13), 40.7 (CH, C-2′), 38.6 (CH₂, C-14), 36.4 (CH, C-8a), 36.0 (CH, C-1), 35.2 (CH₂, C-12), 32.4 (CH₂, C-10), 31.9 (CH₂, C-7), 30.1 (CH, C-2), 26.9 (CH, C-6), 26.4 (CH₂, C-3′), 23.7 (CH₂, C-9), 22.6 (CH₃, C-2″), 16.1 (CH₃, C-3″), 13.7 (CH₃, C-1″), 11.4 (CH₃, C-4′)。

化合物10 (methyl ester of lactone ring-opened monacolin K): 淡黄色油状, 易溶于甲醇; ¹H NMR (500 MHz, CDCl₃): δ_H 5.97 (1H, d, J = 9.6 Hz, H-4), 5.77 (1H, dd, J = 9.7, 6.1 Hz, H-3), 5.50 (1H, t, J = 3.3 Hz, H-5), 5.39 (1H, q, J = 3.2 Hz, H-8), 4.26 (1H, m, H-13), 3.78 (1H, m, H-11), 3.70 (3H, s, H-16), 2.49 (2H, d, J = 6.1 Hz, H_α-14/H_β-14), 2.44 (1H, m, H-6), 2.38 (1H, m, H-2), 2.34 (1H, d, J = 6.9 Hz, H-8a), 2.25 (1H, dd, J = 12.9, 2.7 Hz, H-2′), 1.92 (2H, m, H_α-7/H-1), 1.65 (2H, m, H_α-12/H_α-3′), 1.60 (1H, m, H_β-12), 1.56 (2H, m, H_β-7/H_α-10), 1.50 (1H, m, H_β-3′), 1.43 (1H, dt, J = 13.8, 7.0 Hz, H_α-9), 1.43 (1H, dt, J = 13.8, 7.0 Hz, H_β-3′), 1.26 (1H, m, H_β-9), 1.17 (1H, m, H_β-10), 1.11 (3H, d, J = 6.9 Hz, H-3″), 1.08 (3H, d, J = 7.4 Hz, H-2″), 0.88 (6H, dt, J = 7.5, 4.0 Hz, H-1″/4′); ¹³C NMR (125 MHz, CDCl₃): δ_C 177.0 (C, C-1′), 173.0 (C, C-15), 133.4 (CH, C-3), 131.8 (C, C-4a), 129.6 (CH, C-5), 128.3 (CH, C-4), 72.5 (CH, C-11), 69.2 (CH, C-8), 68.1 (CH, C-13), 52.0 (CH₃, C-16), 42.4 (CH₂, C-14), 41.6 (CH₂, C-12), 41.5 (CH, C-2′), 37.6 (CH, C-8a), 36.4 (CH, C-1), 34.9 (CH₂, C-10), 32.9 (CH₂, C-7), 30.7 (CH, C-2), 27.6 (CH, C-6), 26.9 (CH₂, C-3′), 24.3 (CH₂, C-9), 23.0 (CH₃, C-2″), 16.4 (CH₃, C-3″), 14.0 (CH₃, C-1″), 11.8 (CH₃, C-4′)。

化合物11 (butyrolactone Ⅶ): 淡黄色粉末, 易溶于甲醇; ¹H NMR (500 MHz, CDCl₃): δ_H 7.63 (2H, d, J = 8.6 Hz, H-2′/6′), 6.91 (2H, d, J = 8.6 Hz, H-3′/5′), 6.60 (1H, dd, J = 8.2, 2.2 Hz, H-12), 6.54 (1H, d, J = 8.2 Hz, H-11), 6.52 (1H, d, J = 2.2 Hz, H-8), 5.11 (1H, m, H-2″), 4.26 (2H, qt, J = 7.1, 3.3 Hz, H-7″), 3.53 (2H, m, H-6), 3.15 (2H, d, J = 7.2 Hz, H-1″), 1.70 (3H, d, J = 1.5 Hz, H-5″), 1.66 (3H, s, H-4″), 1.22 (3H, t, J = 7.1 Hz, H-8″); ¹³C NMR (125 MHz, CDCl₃): δ_C 169.4 (C, C-6″), 169.3 (C, C-2), 156.6 (C, C-4′), 153.4 (C, C-10), 137.3 (C, C-3), 134.7 (C, C-3″), 132.0 (CH, C-8), 129.8 (CH, C-2′/6′), 129.4 (CH, C-12), 128.1 (C, C-4), 126.7 (C, C-9), 124.9 (C, C-7), 122.5 (C, C-1′), 121.6 (CH, C-2″), 116.1 (CH, C-3′/5′), 115.3 (CH, C-11), 86.3 (C, C-5), 63.1 (CH₂, C-7″), 38.7 (CH₂, C-6), 29.4 (CH₂, C-1″), 25.9 (CH₃, C-5″), 17.9 (CH₃, C-4″), 14.1 (CH₃, C-8″)。

化合物12 (aspernolide A): 淡黄色粉末, 易溶于甲醇; ¹H NMR (500 MHz, CDCl₃): δ_H 7.61 (2H, d, J = 8.8 Hz, H-2′/6′), 6.92 (2H, d, J = 8.8 Hz, H-3′/5′), 6.56 (1H, s, H-8), 6.51 (2H, d, J = 1.3 Hz, H-11/12), 3.78 (3H, s, 6″-OCH₃), 3.49 (2H, m, H-6), 2.57 (2H, m, H-1″), 1.72 (2H, t, J = 6.8 Hz, H-2″), 1.27 (6H, s, H-4″/5″); ¹³C NMR (125 MHz, CDCl₃): δ_C 169.9 (C, C-6″), 169.1 (C, C-2), 156.5 (C, C-4′), 153.3 (C, C-10), 137.16 (C, C-3), 131.7 (CH, C-8), 129.8 (CH, C-2′/6′), 129.3 (CH, C-12), 127.7 (C, C-4), 123.8 (C, C-7), 122.6 (C, C-1′), 120.5 (C, C-9), 116.8 (C, C-11), 116.1 (CH, C-3′/5′), 86.2 (C, C-5), 74.3 (C, C-3″), 53.7 (C, 6″-OCH₃), 38.8 (CH₂, C-6), 32.8 (CH₂, C-2″), 27.0 (CH₃, C-4″/5″), 22.4 (CH₂, C-1″)。

化合物13 (epi-aszonalenin C): 白色油状液体, 易溶于甲醇; ¹H NMR (500 MHz, CDCl₃): δ_H 8.04 (1H, d, J = 7.9 Hz, H-21), 7.46 (2H, m, H-4/19), 7.40 (1H, br s, H-16), 7.25 (1H, overlapped, H-20), 7.10 (1H, t, J = 8.0 Hz, H-6), 6.79 (2H, d, J = 8.0 Hz, H-5/18), 6.12 (1H, s, H-2), 5.89 (1H, dd, J = 17.2, 10.8 Hz, H-2′), 5.16 (1H, d, J = 10.4 Hz, H_α-1′), 5.13 (1H, s, H_β-1′), 4.11 (1H, d, J = 9.4 Hz, H-11), 3.14 (1H, d, J = 13.3 Hz, H_α-10), 2.58 (1H, dd, J = 13.3, 9.6 Hz, H_β-10), 1.20 (3H, s, H-5′), 0.96 (3H, s, H-4′); ¹³C NMR (125 MHz, CDCl₃): δ_C 170.5 (C, C-17), 166.7 (C, C-13), 146.3 (C, C-15), 143.4 (CH, C-2′), 135.9 (C, C-8), 133.4 (CH, C-19), 131.6 (CH, C-21), 128.7 (CH, C-6), 128.7 (C, C-9), 126.9 (CH, C-4), 124.9 (C, C-14), 124.9 (CH, C-20), 120.7 (CH, C-18), 119.6 (CH, C-5), 114.7 (CH₂, C-1′), 81.3 (CH, C-2′), 61.4 (C, C-3), 57.2 (CH, C-11), 41.0 (C, C-3′), 29.7 (CH₂, C-10), 23.2 (CH₃, C-4′), 22.3 (CH₃, C-5′)。

4　Marfey水解衍生化反应

Marfey水解反应参考相关文献^[21], 在耐压反应瓶中加入160 μg的化合物1以及6 mol·L^-1的盐酸250 μL, 在110 ℃条件下, 电磁搅拌24 h之后在N₂环境下置换出残留的盐酸。将水解产物溶解在20 μL 1 mol·L^-1的碳酸氢钠溶液中, 调节pH为7~8, 之后加入80 μL的Marfey试剂(1-氟-2, 4-二硝基苯-5-L-亮氨酰胺, L-FDLA; 1% 丙酮溶液), 40 ℃环境下搅拌衍生化反应1 h, 反应结束后加入20 μL 1 mol·L^-1的盐酸中和, 用乙腈(400 µL) 稀释并过滤(0.22 µm PTFE), 然后进行HPLC分析。标准氨基酸L/D-Ala、N-CH₃-L/D-Phe、L/D-Ile采用同样的方法与Marfey试剂衍生化, 并在相同的色谱条件下进行HPLC和LC-MS分析。

5　抗藤壶幼虫附着活性

藤壶幼虫的养殖方式参照相关文献^[9], 在24孔板中加入995 μL海水(含有10~15个藤壶金星幼虫) 海水及5 μL DMSO溶解的样品并吹打均匀, 在室温光照条件下静置培养24 h。设置浓度为50 μg·mL^-1的样品进行活性初筛, 再根据初筛结果进行梯度实验, 得到样品抗藤壶金星幼虫附着的EC₅₀值。以DMSO为阴性对照、seaNine-211为阳性对照, 每个样品设置3个平行。

6　抗乙酰胆碱酯酶活性

参照文献^[22]的方法评价化合物的乙酰胆碱酯酶抑制活性。制备所需溶液: 0.1 mol·L^-1磷酸氢二钠-磷酸二氢钠缓冲溶液(PBS溶液, pH 8.0)、0.1 U·mL^-1乙酰胆碱酯酶溶液、6.25 mmol·L^-1 DTNB溶液和6.25 mmol·L^-1硫代乙酰胆碱溶液。采用96孔板法, 建立实验组、背景对照、阴性对照、空白对照和阳性对照。30 ℃孵育后, 加入DTNB和硫代乙酰胆碱等体积混合物。96孔板于30 ℃放置30 min, 在405 nm的波长下测量每个孔的吸光度(OD)。通过计算测定化合物对乙酰胆碱酯酶的抑制活性。

7　抗菌活性

采用96孔板法测定得到的单体化合物对致病菌耐甲氧西林金黄色葡萄球菌(methicillin-resistant S. aureus ATCC43300) 和三株海洋污损细菌詹氏甲烷球菌(M. janaschii)、佩拉吉弧菌(V. pelagius) 和罗氏弧菌(V. Rotiferianus) 的抑制作用^[23]。均由华南农业大学张晓勇博士惠赠。单体化合物用DMSO溶解初始浓度为10 mmol·L^-1的样品溶液。活化后的指示菌接入LB培养基中(海洋污损细菌接种到含有3%海盐的LB培养基中), 30 ℃、180 r·min^-1震荡培养至对数生长期, 调整菌悬液浓度为每毫升含有10⁶个菌落形成单位(colony forming units, CFU)。用移液枪吸取195 μL菌悬液, 加入96孔板, 再加入5 μL样品液, 同时设置阳性对照、阴性对照和空白对照, 青霉素和氯霉素为阳性对照。摇匀密封后, 于30 ℃培养箱中培养24 h, 使用酶标仪于600 nm条件下测定OD值。每个样品设置两个平行, 进行初步抑菌活性的筛选。样品抑菌活性结果用抑制率表示, 抑制率= (空白值- 样品值) / 空白值× 100%。对于有抑菌活性的单体化合物, 采用倍半稀释法继续测定MIC, 方法同上, 每组设置三个平行。

8　统计学分析

实验数据采用SPSS 16.0软件进行统计分析, 所有实验数据以三个独立实验的平均值±标准差(x ± s) 表示, 两组间数据的比较采用单因素方差分析(ANOVA) 和Dunnett比较检验进行差异分析, 当P < 0.05时, 差异具有统计显著性。

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2025年第60卷第7期

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doi: 10.16438/j.0513-4870.2025-0051

接收时间：2025-01-15
首发时间：2026-09-20
出版时间：2025-07-12

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收稿日期：2025-01-15
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¹沈阳药科大学无涯创新学院, 辽宁沈阳 110016

²广西中医药大学海洋药物研究院, 广西海洋药物重点实验室, 广西南宁 530200

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Unit	Position	δ_C	δ_H	HMBC
Aba	1	170.4
	2	126.8
	3	125.6	7.33 (dd, J = 7.7, 1.6 Hz)	1, 5, 7
	4	124.3	7.13 (t, J = 7.5 Hz)	2, 6
	5	131.6	7.45 (td, J = 7.9, 1.6 Hz)	3, 7
	6	123.7	8.10 (d, J = 8.2 Hz)	2, 4, 7
	7	135.4
	NH		9.00 (s)	2, 6, 8
N-Me-Phe	8	168.5
	9	56.9	5.89 (dd, J = 11.3, 5.4 Hz)	8, 10, 11, 17, 18
	10	32.7	3.28 (dd, J = 15.0, 5.4 Hz) 3.05 (dd, J = 15.0, 11.4 Hz)	8, 9, 11, 12, 16
	11	136.6
	12/16	129.1	7.23 (d, J = 7.0 Hz)	10, 13, 14, 15
	13/15	128.6	7.26, overlapped	11, 12, 16
	14	126.8	7.19 (t, J = 7.0 Hz)
	17	30.4	2.85 (s)	9, 18
Ile	18	173.1
	19	54.4	4.44 (t, J = 10.2 Hz)	18, 20, 21, 23, 24
	20	36.4	1.81, overlapped
	21	25.0	0.51 (m)	19, 20, 22, 23
	22	11.0	0.62 (t, J = 7.2 Hz)	20, 21
	23	14.7	0.85 (d, J = 6.7 Hz)	19, 20, 21
	NH		7.24, overlapped	24
Ala	24	171.4
	25	50.0	4.50 (q, J = 6.9 Hz)	24, 26
	26	15.0	1.42 (d, J = 6.9 Hz)	24, 25
	NH		6.25 (d, J = 8.2 Hz)	1, 25, 26

Unit

Position

δ_C

δ_H

HMBC

Aba

170.4

126.8

125.6

7.33 (dd, J = 7.7, 1.6 Hz)

1, 5, 7

124.3

7.13 (t, J = 7.5 Hz)

2, 6

131.6

7.45 (td, J = 7.9, 1.6 Hz)

3, 7

123.7

8.10 (d, J = 8.2 Hz)

2, 4, 7

135.4

9.00 (s)

2, 6, 8

N-Me-Phe

168.5

56.9

5.89 (dd, J = 11.3, 5.4 Hz)

8, 10, 11, 17, 18

32.7

3.28 (dd, J = 15.0, 5.4 Hz)
3.05 (dd, J = 15.0, 11.4 Hz)

8, 9, 11, 12, 16

136.6

12/16

129.1

7.23 (d, J = 7.0 Hz)

10, 13, 14, 15

13/15

128.6

7.26, overlapped

11, 12, 16

126.8

7.19 (t, J = 7.0 Hz)

30.4

2.85 (s)

9, 18

Ile

173.1

54.4

4.44 (t, J = 10.2 Hz)

18, 20, 21, 23, 24

36.4

1.81, overlapped

25.0

0.51 (m)

19, 20, 22, 23

11.0

0.62 (t, J = 7.2 Hz)

20, 21

14.7

0.85 (d, J = 6.7 Hz)

19, 20, 21

7.24, overlapped

Ala

171.4

50.0

4.50 (q, J = 6.9 Hz)

24, 26

15.0

1.42 (d, J = 6.9 Hz)

24, 25

6.25 (d, J = 8.2 Hz)

1, 25, 26

Compound^a	EC₅₀/μg·mL^-1	LC₅₀/μg·mL^-1
1	9.75 ± 0.28	> 50
2	0.12 ± 0.28	> 50
3	0.19 ± 0.12	> 50
6	5.66 ± 0.07	> 50
7	1.23 ± 0.08	> 50
9	4.93 ± 0.08	> 50
11	12.52 ± 0.08	> 50
12	2.94 ± 0.07	> 50
SeaNine-211	1.94 ± 0.41	33.93 ± 2.06

Compound^a

EC₅₀/μg·mL^-1

LC₅₀/μg·mL^-1

9.75 ± 0.28

> 50

0.12 ± 0.28

> 50

0.19 ± 0.12

> 50

5.66 ± 0.07

> 50

1.23 ± 0.08

> 50

4.93 ± 0.08

> 50

12.52 ± 0.08

> 50

2.94 ± 0.07

> 50

SeaNine-211

1.94 ± 0.41

33.93 ± 2.06

Compound^a	MIC /μg·mL^-1
4	53.06	13.26	26.53	13.26
11	54.81	27.41	27.41	13.70
Chloramphenicol	12.50	6.25	12.50	12.50
Penicillin	25.00	6.25	12.50	12.50

Compound^a

MIC /μg·mL^-1

MRSA

53.06

13.26

26.53

13.26

54.81

27.41

13.70

Chloramphenicol

12.50

6.25

12.50

Penicillin

25.00

6.25

12.50