海洋本草软珊瑚共附生曲霉属真菌EGF7-0-1和EGF15-0-3共培养中甾体类成分研究Ⅱ
司徒美霞(1997—), 女, 广东省江门市人, 硕士研究生, 从事天然药效物质基础研究。email: |
收稿日期: 2022-11-08
修回日期: 2022-12-06
网络出版日期: 2022-12-08
基金资助
广东省自然资源厅省级促进经济高质量发展专项资金(海洋经济发展用途)项目(GDNRC[2021]48)
广东省自然资源厅省级促进经济高质量发展专项资金(海洋经济发展用途)项目(GDNRC[2020]039)
国家自然科学基金项目(82273845)
国家自然科学基金项目(81741160)
广州市青少年科技教育项目(KP市2022027)
Research on the steroids from the coculture of soft coral-associated fungi Aspergillus sp. EGF7-0-1 and EGF15-0-3
Received date: 2022-11-08
Revised date: 2022-12-06
Online published: 2022-12-08
Supported by
Special fund of Guangdong Provincial Department of Natural Resources for Promoting High-quality Economic Development (Marine Economic Development)(GDNRC[2021]48)
Special fund of Guangdong Provincial Department of Natural Resources for Promoting High-quality Economic Development (Marine Economic Development)(GDNRC[2020]039)
National Natural Science Foundation of China(82273845)
National Natural Science Foundation of China(81741160)
Guangzhou Youth Science and Technology Education Project(KP市2022027)
文章模拟微生物自然生长环境, 对两株海洋本草软珊瑚曲霉属真菌Aspergillus sp. EGF7-0-1和Aspergillus sp. EGF15-0-3进行共培养, 以期发现新颖骨架成分。采用硅胶、反相色谱、羟丙基葡聚糖凝胶和高效液相色谱等方法对其次生代谢产物进行分离、纯化; 采用高分辨质谱、核磁共振、旋光及物理常数对照等方法对所分离得到的化合物进行结构鉴定; 采用全球天然产物分子网络(Global Natural Products Social Molecular Networking, GNPS)方法对次生代谢产物进行分析。从两株软珊瑚来源的曲霉属真菌大米共培养中分离得到10个甾体类化合物: Chaxine (1)、Herbarulide (2)、dankasterones A (3)、25-hydroxyergosta-4,6,8(14),22-tetraen3-one (4)、Ganodermasides D (5)、Ergosta-4,6,8(14),22-tetraen-3-one (6)、Ergosta-4,7,22-trien-3-one (7)、Isocyathisterol (8)、Stigmasta-4,22-dien-3-one (9)和β-sitostenone (10)。化合物1~3均为高度氧化重排的甾体类化合物。化合物4、5、7和9首次从曲霉属真菌中分离得到。GNPS分析表明与菌株EGF7-0-1和EGF15-0-3单培养相比, 两株真菌共培养能诱导出更为丰富的甾体类化合物。
司徒美霞 , 雷祖发 , 杨倩茹 , 邓胜毅 , 吴科键 , 陈鸿皓 , 韦霞 , 樊浩 , 张伟 , 张翠仙 . 海洋本草软珊瑚共附生曲霉属真菌EGF7-0-1和EGF15-0-3共培养中甾体类成分研究Ⅱ[J]. 热带海洋学报, 2023 , 42(5) : 161 -170 . DOI: 10.11978/2022240
In order to simulate the growth environment of microorganisms to change or increase the production of secondary metabolites in the presence of other microorganisms and protect the ecological environment of marine herbs, soft coral-associated two fungi Aspergillus sp. EGF7-0-1 and EGF15-0-3 were cocultured on rice medium. Firstly, GNPS (Global Natural Products Social Molecular Networking) method was used to analyze secondary metabolites. Then, the secondary metabolites were separated and purified by silica gel, ODS (Octadecylsilyl), Sephadex LH-20 and HPLC (Preparative High Performance Liquid Chromatography). Their structures were identified by HR-ESI-MS (High Resolution Electrospray Ionization Mass Spectroscopy), NMR (Nuclear Magnetic Resonance), ORD (Optical Rotatory Dispersion) and the relative physical constants were compared with the literature. Ten steroids were obtained and identified as Chaxine C (1), Herbarulide (2), dankasterones A (3), 25-hydroxyergosta-4,6,8(14),22-tetraen3-one (4), Ganodermasides D (5), Ergosta-4,6,8(14),22-tetraen-3-one (6), Ergosta-4,7,22-trien-3-one (7), Isocyathisterol (8), Stigmasta-4,22-dien-3-one (9) and β-sitostenone (10). Compounds 1~3 were steroids with oxidative rearrangement and 4, 5, 7 and 9 were firstly acquired from Aspergillus sp. compared with the single culture of EGF7-0-1 and EGF15-0-3. Based on the coculture strategy, the content types of steroids were more abundant.
Key words: soft coral; Aspergillus sp.; coculture; steroid
表1 试验所用仪器与试剂信息表Tab. 1 Instruments and reagents used in the experiment |
仪器/试剂 | 型号/规格 | 品牌 |
---|---|---|
高分辨液质联用仪 | Triple TOFTM 5600+ | 美国 AB SCIEX公司 |
核磁共振波谱仪 | 400 MHz AVANCE Ⅲ型 | 德国Bruker公司 |
高效液相色谱 | QuikSep半制备型 | 北京慧德易科技有限公司 |
自动旋光仪 | MCP 500 | 安东帕公司 |
紫外透射反射仪 | WFH-201B | 上海精科实业有限公司 |
超净工作台 | SW-CJ-1FD | 苏州净化设备厂 |
立式压力蒸汽灭菌器 | LS-100H | 江阴滨江医疗设备有限公司 |
电子天平 | PWN224ZH | 奥豪斯仪器(常州)有限制造公司 |
旋转蒸发器 | XHRE-2000A | 上海宵汉实业发展有限公司 |
低温冷却液循环泵 | XHDLSB-5/25 | 上海宵汉实业发展有限公司 |
色谱柱a: Luna 分析柱 | 4.6mm×100mm, 5μm | 美国Phenomenex公司 |
色谱柱b: YMC-Pack ODS-A半制备柱 | 10mm×250mm, 5μm | 日本YMC Co.,Ltd.公司 |
色谱柱c: H& EC18半制备柱 | 10mm×250mm, 5μm | 北京慧德易科技有限公司 |
常规柱层析硅胶 | 200~300目、300~400目 | 青岛海洋化工厂 |
Sephadex LH-20 | YILIMART 500G | 瑞典 cytiva公司 |
C-18反相色谱填料 | YMC*GEL ODS-A-HG | 日本 YMC公司 |
乙酸乙酯、石油醚、甲醇、乙腈、二氯甲烷、 三氯甲烷和正丁醇 | 分析纯 | 广东光华科技股份有限公司 |
氘代氯仿 | 25G | 美国剑桥CIT公司 |
葡萄糖 | 分析纯 | 天津致远化学试剂有限公司 |
技术琼脂粉 | 分析纯 | 广东环凯微生物科技有限公司 |
土豆、大米、海盐 | 购于市场 |
表2 化合物 1~10的 1H NMR数据(400MHz, CDCl3, δH, 多重峰mult, 偶合常数J in Hz)Tab. 2 1H NMR Data of 1~10 in CDCl3 |
No. | 1 | 2 | 3 | 4 | 5 |
---|---|---|---|---|---|
4 | 6.03, d, 10.1 | 5.74, s | 6.36, s | 5.74, s | 5.88, s |
6 | 6.04, d, 9.5 | 6.12, d, 9.6 | |||
7 | 5.64, d, 2.4 | 5.72, s | 2.65, dd, 16.9, 1.5 | 6.60, d, 9.5 | 6.49, d, 9.6 |
9 | 2.81, t, 9.1 | ||||
18 | 0.87, s | 0.63, s | 0.98, s | 0.96, s | 0.96, s |
19 | 1.47, s | 1.23, s | 1.26, s | 0.99, s | 1.11, s |
21 | 1.02, d, 6.6 | 1.02, d, 6.6 | 1.09, d, 7.1 | 1.08, d, 6.7 | 1.08, d, 6.7 |
22 | 5.14, dd, 15.3, 8.4 | 5.14, dd, 15.3, 8.4 | 5.24, dd, 15.2, 6.0 | 5.34, dd, 15.2, 7.6 | 5.20, dd, 15.3, 7.9 |
23 | 5.26, dd, 15.3, 7.7 | 5.25, dd, 15.2, 7.6 | 5.28, d, 15.2, 6.1 | 5.39, dd, 15.2, 7.8 | 5.27, dd, 15.3, 7.2 |
26 | 0.84, d, 6.8 | 0.82, d, 6.8 | 0.81, d, 6.8 | 1.14, s | 0.83, d, 6.8 |
27 | 0.82, d, 6.8 | 0.82, d, 6.8 | 0.83, d, 6.8 | 1.17, s | 0.85, d, 6.8 |
28 | 0.91, d, 6.8 | 0.91, d, 6.8 | 0.91, d, 6.8 | 1.01, d, 6.7 | 0.93, d, 6.9 |
No. | 6 | 7 | 8 | 9 | 10 |
4 | 5.73, s | 5.79, d, 2.0 | 5.72, s | 5.72, s | 5.71, s |
6 | 6.03, d, 9.5 | 6.09, d, 9.6 | |||
7 | 6.61, d, 9.5 | 6.16, d, 9.6 | |||
18 | 0.96, s | 0.60, s | 1.02, s | 0.73, s | 0.70, s |
19 | 0.99, s | 1.17, s | 1.32, s | 1.18, s | 1.17, s |
21 | 1.06, d, 6.7 | 1.03, d, 6.6 | 0.99, d, 6.6 | 1.02, d, 6.6 | .91, d, 6.5 |
22 | 5.26, dd, 15.2,7.1 | 5.16, dd, 15.2, 7.7 | 5.12, dd, 15.3, 8.2 | 5.15, dd, 15.2, 8.5 | |
23 | 5.20, dd, 15.2,7.8 | 5.23, dd, 15.2, 6.9 | 5.22, dd, 15.3, 7.6 | 5.02, dd, 15.2, 8.5 | |
26 | 0.83, d, 6.8 | 0.82, d, 6.5 | 0.82, d, 6.8 | 0.84, d, 6.3 | 0.83, d, 6.8 |
27 | 0.85, d, 6.8 | 0.84, d, 6.5 | 0.83, d, 6.8 | 0.79, d, 6.3 | 0.82, d, 6.8 |
28 | 0.93, d, 6.9 | 0.91, d, 6.5 | 0.91, d, 6.8 | ||
29 | 0.80, t, 7.3 | 0.84, t, 7.3 |
表3 化合物 1~10的 13C NMR数据(100MHz, CDCl3, δC, type)Tab. 3 13C NMR Data of 1~10 in CDCl3 |
No. | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
---|---|---|---|---|---|---|---|---|---|---|
1 | 32.1, t | 33.9, t | 39.0, t | 34.1, t | 27.6, t | 34.3, t | 33.2, t | 35.2, t | 35.7, t | 35.7, t |
2 | 24.8, t | 33.2, t | 34.5, t | 34.1, t | 34.0, t | 34.3, t | 34.3, t | 33.8, t | 34.0, t | 32.1, t |
3 | 148.8, d | 198.4, s | 199.3, s | 199.7, s | 199.5, s | 199.7, t | 199.6, s | 199.9, s | 199.7, s | 199.8, s |
4 | 128.1, d | 114.7, d | 126.7, d | 123.2, d | 126.4, d | 123.1, d | 122.8, d | 125.3, d | 123.8, d | 123.7, d |
5 | 195.8, s | 173.9, s | 156.2, s | 124.7, s | 161.4, s | 124.6, s | 169.2, s | 163.5, s | 171.7, s | 171.9, s |
6 | 164.3, d | 162.4, s | 200.2, s | 124.7, d | 124.8, d | 124.6, d | 33.2, t | 128.2, d | 33.0, t | 33.0, t |
7 | 117.5, d | 113.3, d | 41.0, t | 134.1, d | 130.9, d | 134.2, d | 115.7, d | 139.4, d | 32.0, t | 33.9, t |
8 | 156.2, s | 159.5, s | 62.3, s | 164.5, s | 126.7, s | 164.5, s | 139.7, s | 71.9, s | 35.6, d | 35.6, d |
9 | 204.6, s | 47.2, d | 49.5, d | 44.5, d | 72.6, s | 44.5, d | 45.9, d | 53.3, d | 53.8, d | 53.8, d |
10 | 81.2, s | 40.4, s | 36.1, s | 36.9, s | 42.4, s | 36.9, s | 38.2, s | 36.3, s | 38.6, s | 38.7, s |
11 | 39.0, t | 25.4, t | 25.3, t | 19.0, t | 25.5, t | 19.1, t | 22.1, t | 18.0, t | 21.0, t | 21.0, t |
12 | 38.0, t | 39.1, t | 38.5, t | 35.6, t | 32.1, t | 35.7, t | 39.1, t | 40.9, t | 39.5, t | 39.4, t |
13 | 46.5, s | 47.0, s | 54.1, s | 44.2, s | 44.6, s | 44.1, s | 43.5, s | 44.2, s | 42.3, s | 42.5, s |
14 | 57.9, d | 58.1, d | 215.0, s | 155.7, s | 158.3, s | 156.3, s | 55.1, d | 57.2, d | 56.0, d | 56.0, d |
15 | 21.9, t | 22.6, t | 38.1, t | 25.3, t | 25.7, t | 25.5, t | 23.0, t | 19.1, t | 24.3, t | 24.2, t |
16 | 29.1, t | 27.7, t | 23.3, t | 27.7, t | 27.8, t | 27.9, t | 28.2, t | 28.2, t | 28.9, t | 28.2, t |
17 | 55.3, d | 56.3, d | 49.5, d | 55.6, d | 55.7, d | 55.8, d | 55.9, d | 56.6, d | 55.9, d | 55.9, d |
18 | 12.1, q | 12.5, q | 17.2, q | 19.1, q | 17.9, q | 19.1, q | 12.3, s | 14.6, q | 12.2, q | 12.0, s |
19 | 21.9, q | 20.0, q | 24.2, q | 16.8, q | 20.9, q | 16.8, q | 21.4, q | 19.0, q | 17.4, q | 19.0, s |
20 | 40.1, d | 40.2, d | 37.4, d | 39.5, d | 39.3, d | 39.4, d | 40.6, d | 39.9, d | 40.5, d | 36.1, d |
21 | 21.0, q | 21.0, q | 23.8, q | 21.2, q | 21.3, q | 21.4, q | 21.2, q | 20.7, q | 19.0, q | 18.7, q |
22 | 134.6, d | 134.7, d | 132.5, d | 138.4, d | 134.9, d | 135.2, d | 135.6, d | 135.4, d | 138.1, d | 34.0, t |
23 | 132.9, d | 132.8, d | 135.3, d | 129.9, d | 132.9, d | 132.7, d | 132.4, d | 132.3, d | 129.5, d | 29.1, t |
24 | 42.8, d | 42.8, d | 43.4, d | 48.3, d | 43.0, d | 43.0, d | 42.9, d | 43.0, d | 51.2, d | 45.8, d |
25 | 33.0, d | 33.1, d | 33.2, d | 72.5, s | 33.2, d | 33.2, d | 33.0, d | 33.2, d | 31.9, d | 26.1, d |
26 | 19.7, q | 20.0, q | 19.8, q | 27.1, q | 19.8, q | 20.1, q | 20.1, q | 19.8, q | 21.2, q | 17.4, q |
27 | 20.0, q | 19.6, q | 20.2, q | 26.6, q | 20.1, q | 19.8, q | 19.8, q | 20.1, q | 21.1, q | 19.8, q |
28 | 17.6, q | 17.6, q | 17.1, q | 15.8, q | 17.8, q | 17.8, q | 17.7, q | 17.8, q | 25.4, t | 23.1, t |
29 | 12.3, q | 12.0, q |
图2 共培养曲霉属真菌不同时间生长图片(土黄色菌落为EGF7-0-1, 灰绿色菌落为EGF15-0-3)Fig. 2 Growth pictures of co-cultured Aspergillus sp. at different times (earthy yellow is EGF7-0-1, grayish green is EGF15-0-3) |
图3 两株真菌共培养(蓝色曲线)、EGF7-0-1单培养(粉色曲线)、EGF15-0-3单培养(红色曲线)的TIC对比图Fig. 3 TIC comparison of the cocultivation, the single culture of EGF7-0-1or EGF15-0-3 (The blue curve is the cocultivation, the pink curve is the single culture of EGF7-0-1, and the red curve is the single culture of EGF15-0-3) |
图4 两株真菌共培养、EGF15-0-3单培养、EGF7-0-1单培养的GNPS分析a. 总图; b. 甾体类成分分子笼放大图, 节点里的数字表示母离子质量 Fig. 4 GNPS analysis of the cocultivation, the single culture of EGF7-0-1 and the single culture of EGF15-0-3. (a) general view; (b) enlarged view of molecular cage of isolated components, the number in the node represents parent mass |
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