海带中抑藻活性化合物的分离及抑藻活性分析

孙颖颖; 朱文轩; 周静; 庄丽雯; 毛奕淋; 赵秀芳; 王长海

doi:10.12284/hyxb2022042

海带中抑藻活性化合物的分离及抑藻活性分析

doi: 10.12284/hyxb2022042

孙颖颖^{1, 2, 3,},
朱文轩^{1, 4},
周静⁴,
庄丽雯¹,
毛奕淋¹,
赵秀芳¹,
王长海⁵

1.
江苏海洋大学江苏省海洋生物技术重点实验室，江苏连云港 222005
2.
江苏海洋大学江苏省海洋生物产业技术协同创新中心, 江苏连云港 222005
3.
江苏省海洋资源开发研究院，江苏连云港 222000
4.
连云港市质量技术综合检验检测中心，江苏连云港 222005
5.
南京农业大学资源与环境科学学院，江苏南京210095

基金项目: 江苏省第五期“333工程”科研资助项目（BRA2020263）；江苏省优势学科建设工程资助项目；江苏省研究生实践创新计划（SJCX20_1317）；连云港市“海燕计划”；2018年江苏高校优秀中青年教师和校长境外研修计划。

详细信息

作者简介:
孙颖颖（1978—），女，博士，教授，从事海洋生化工程研究。E-mail: syy-999@163.com

中图分类号: X55；S917.3
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- 文章访问数: 261
- HTML全文浏览量: 84
- PDF下载量: 34
- 被引次数: 0
出版历程
- 收稿日期: 2021-07-31
- 修回日期: 2021-10-28
- 网络出版日期: 2021-12-08
- 刊出日期: 2022-02-01

Isolation, purification and analysis of antialgal activities of antialgal compounds from Laminaria japonica

1.
Jiangsu Provincial Key Laboratory of Marine Biotechnology, Jiangsu Ocean University, Lianyungang 222005, China
2.
Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang 222005, China
3.
Jiangsu Institute of Marine Resources Development, Lianyungang 222005, China
4.
Lianyungang Quality and Technology Comprehensive Inspection and Testing Center, Lianyungang 222005, China
5.
College of Resources and Environment Sciences, Nanjing Agricultural University, Nanjing 210095, China

摘要

摘要: 以2 000 g海带为原料、米氏凯伦藻为测试微藻，采用甲醇浸提、液液萃取、硅胶柱层析、Sephadax LH-20凝胶柱层析和硅胶薄层层析等分离方法，纯化可能具有抑藻活性的化合物，制备到5个薄层纯样品，B₂₁（1.39 g）、B₁₃₁（0.51 g）、B₁₃₂（0.48 g）、C₂₁₁（0.016 g）和C₂₁₂（0.020 g）。经鉴定，B₂₁、B₁₃₁、B₁₃₂、C₂₁₁和C₂₁₂依次为7-羟基庚酸、十四酸、十六酸、十八酸和油酸，均是第一次从海带中分离获得。此5种脂肪酸对塔玛亚历山大藻、强壮前沟藻、赤潮异弯藻、米氏凯伦藻、球形棕囊藻和中肋骨条藻的生长表现出选择性抑制作用，其中7-羟基庚酸具有较为广泛的抑藻活性。除塔玛亚力山大藻之外，在浓度为50 μg/mL时，7-羟基庚酸对其他5种赤潮微藻生长的抑制作用与硫酸铜和重铬酸钾对它们生长的抑制作用接近，生长抑制率超过71%（第4天）；十四酸在浓度为50 μg/mL时对强壮前沟藻、米氏凯伦藻和中肋骨条藻生长也有明显的抑制作用，第4天时对它们的生长抑制率在57%以上。在此基础上，第一次确定了7-羟基庚酸、十四酸、十六酸和十八酸对强壮前沟藻、赤潮异弯藻、米氏凯伦藻、球形棕囊藻和中肋骨条藻生长的半抑制效应浓度（EC_{50~96 h}），7-羟基庚酸对塔玛亚历山大藻生长的EC_{50~96 h}也是第一次被确定。与硫酸铜和重铬酸钾对每种赤潮微藻生长的EC_{50~96 h}比较，7-羟基庚酸和十四酸在抑制强壮前沟藻上具有明显的抑藻优势。
- 抑藻活性化合物 /
- 分离 /
- 海带 /
- 纯化 /
- 赤潮微藻
Abstract: Using 2 000 g kelp as raw material and Karenia mikimotoi as the test microalgae, methanol extraction, liquid-liquid extraction, silica gel column chromatography, sephadax LH-20 gel column chromatography and silica gel thin layer chromatography are used to purify compounds that might inhibit algal activity. Five samples with thin layer purity, B₂₁ (1.39 g), B₁₃₁ (0.51 g), B₁₃₂ (0.48 g), C₂₁₁ (0.016 g) and C₂₁₂ (0.020 g), are prepared. Structural identification shows that B₂₁, B₁₃₁, B₁₃₂, C₂₁₁ and C₂₁₂ are 7-hydroxyheptanic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, and oleic acid, respectively. They are isolated from Laminaria japonica for the first time. The results show that these five compounds have selective inhibition effects on the growth of six species of red tide microalgae (Alexandrium tamarense, Amphidinium carterae, Heterosigma akashiwo, Karenia mikimotoi, Phaeocystis globsa, and Skeletonema costatum). Among them, 7-hydroxyheptanoic acid has a wide range of antialgal activities. Except for Alexandrium tamarense, at the concentration of 50 μg/mL, the growth inhibition of 7-hydroxyheptanic acid against the other five species of red tide microalgae is close to that of copper sulfate and potassium dichromate, and the growth inhibition is more than 71% (the 4th day). Tetradecanoic acid also shows obvious inhibition on the growth of Amphidinium carterae, Karenia mikimotoi, and Skeletonema costatum. On 4th day, the growth inhibition of tetradecanoic acid for the three species of red tide microalgae is more than 57%. Furthermore, EC_{50−96 h} values of 7-hydroxyheptanic acid, tetradecanoic acid, hexadecanoic acid, and octadecanoic acid for Amphidinium carterae, Heterosigma akashiwo, Karenia mikimotoi, Phaeocystis globsa, and Skeletonema costatum are obtained for the first time, respectively. EC_{50−96 h} value of 7-hydroxyheptanic acid for Alexandrium tamarense is also determined for the first time. It is found that 7-hydroxyheptanic acid and tetradecanoic acid has the advantage of inhibiting Amphidinium carterae.
- antialgal compound /
- isolation /
- Laminaria japonica /
- purification /
- red tide microalgae

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图 1 可抑制赤潮微藻的大型褐藻的目类归属分布

数据来源于1960−2019年Web of Science、Springer、Google Scholar和CNKI数据库收录的研究

Fig. 1 Distribution of brown marine macroalgae with antialgal activity against red tide microalgae according the orders

Data extracted from related studies in Web of Science, Springer, Google Scholar and CNKI between 1960 and 2019

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图 2 海带甲醇提取物对米氏凯伦藻生长的影响

Fig. 2 Effect of the methanol extracts of Laminaria japonica on the growth of Karenia mikimotoi

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图 3 液液萃取分离组分对米氏凯伦藻生长的影响

Fig. 3 Effect of several liquid-liquid extraction components from the methanol extracts on the growth of Karenia mikimotoi

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图 4 海带中抑藻活性化合物的分离纯化

Fig. 4 Isolation and purification of the antialgal compounds from Laminaria japonica

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图 5 不同化合物对6种赤潮微藻生长的抑制作用

图中数据为3个平行样的平均值

Fig. 5 Growth inhibition of the different compounds against six species of red tide microalgae

The data in the figure is the average of the three parallel samples

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图 6 7-羟基庚酸对4种赤潮微藻细胞形态的影响

a1−a4. 阴性对照组；b1−b4. 添加7-羟基庚酸实验组；光学显微镜（×400）

Fig. 6 Effect of 7-hydroxyheptanic acid on the photographs of the four species of red tide microalgae

a1−a4. Negative control group; b1−b4. groups added 7-hydroxyheptanic acid; light microscope (×400)

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表 1 组分B和组分C的分离纯化过程中抑藻活性检测

Tab. 1 Antialgal activity test of the fractions B and C in the isolation and purification process

浓度	组分B		组分C
浓度	硅胶柱层析分离（3.0 cm×40 cm）		硅胶柱层析分离（5.0 cm×80 cm）
1.0 mg/mL（Sephadax LH-20凝胶柱层析（2.0 cm²×25 cm））	组分${\rm B}_1^{++} $	组分${\rm B}_2^{++} $	组分${\rm C}_1^{-} $	组分${\rm C}_2^{++} $	组分${\rm C}_3^{-} $
0.25 mg/mL（硅胶薄层层析制备）	${\rm{B}}_{11}^- $、${\rm{B}}_{12}^- $、${\rm{B}}_{13}^{++} $	${\rm{B}}_{21}^{++} $	${\rm{C}}_{21}^{++} $、${\rm{C}}_{22}^{-} $
0.05 mg/mL	${\rm{B}}_{131}^{++} $、${\rm{B}}_{132}^{++} $		${\rm{C}}_{211}^{++} $、${\rm{C}}_{212}^{++} $
注：−表示没有抑藻活性；+表示微弱的抑藻活性，生长抑制率为20%～50%；++表示强烈的抑藻活性，生长抑制率大于50%。

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表 2 不同化合物对6种赤潮微藻生长的半抑制效应浓度（EC_{50~96 h}，单位：μg/mL）

Tab. 2 Different compounds isolated from Laminaria japonica for the six species of red tide microalgae (EC_{50−96 h}，unit：μg/mL)

藻类	硫酸铜	7-羟基庚酸	十四酸	十六酸	十八酸	油酸
塔玛亚历山大藻	6.76	7.39	10.6	36.8	115.4	−
强壮前沟藻	4.16	2.19	3.54	54.4	−	−
赤潮异弯藻	6.42	13.9	15.4	37.3	154.3	−
米氏凯伦藻	4.43	11.1	11.4	12.6	215.9	−
球形棕囊藻	11.2	18.5	20.1	95.9	361.0	−
中肋骨条藻	6.99	18.0	18.5	32.5	292.6	−
注：−表示没有计算得出。

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