引用本文:	吴宗泽,丁国芳,杨最素,余方苗,唐云平,贾盈露,郑媛媛,陈锐.绿侧花海葵(Anthopleura anjunae)寡肽制备的关键技术与抗前列腺癌作用研究.海洋与湖沼,2017,48(5):1114-1123.

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*绿侧花海葵(Anthopleura anjunae)寡肽制备的关键技术与抗前列腺癌作用研究*
吴宗泽^1,2, 丁国芳^1,2, 杨最素¹, 余方苗¹, 唐云平¹, 贾盈露¹, 郑媛媛¹, 陈锐¹
1.浙江海洋大学食品与医药学院浙江省海洋生物医用制品工程技术研究中心舟山 316022;2.浙江省海洋水产研究所舟山 316021

摘要:

本文探讨绿侧花海葵（Anthopleura anjunae）抗前列腺癌酶解寡肽制备与工艺优化。以绿侧花海葵肉为原料进行酶解，筛选最佳蛋白酶，通过正交实验优化酶解条件，并通过超滤、阴离子交换层析、G-25凝胶过滤和反相高效液相色谱（RP-HPLC）分离纯化，通过LC-MS和氨基酸序列测定鉴定寡肽的氨基酸序列，并以MTT法检测产物对前列腺癌细胞系DU-145增殖抑制率，以确定活性最强组分，最后通过倒置显微镜观察纯化肽的抗前列腺癌活性。结果表明，碱性蛋白酶为最佳酶种；工艺条件为：最适料液比1：5、最适pH=11、最适加酶量2000U/g、最适温度35℃、最适酶解时间6h；经高效液相纯化得到由五个氨基酸组成的绿侧花海葵抗前列腺癌寡肽，其氨基酸序列为：Tyr-Val-Pro-Gly-Pro（AAP-H）；倒置显微镜结果显示经AAP-H作用24h后的DU-145细胞具有明显的凋亡形态学特征。结论：采用碱性蛋白酶酶解并通过超滤和色谱分离技术能从绿侧花海葵肉中制备抗肿瘤活性肽；且AAP-H对DU-145细胞的增殖抑制作用呈时间与剂量依赖关系，作用后细胞出现了凋亡的形态学特征。因此，AAP-H能明显抑制前列腺癌细胞DU-145增殖，可以诱导其发生凋亡而发挥抗肿瘤作用。

关键词: 绿侧花海葵酶解抗肿瘤寡肽工艺

DOI：10.11693/hyhz20161200268

分类号:

基金项目:国家自然科学基金项目，81001393号，2015GA700044号；国家海洋重大计划项目，201586-2号；浙江省科技厅重大专项，2013C03036号；浙江省自然科学基金项目，21136001115号。

附件

ENZYMATIC PREPARATION OF OLIGOPEPTIDE FROM ANTHOPLEURA ANJUNAE AND ITS ANTI-CANCER ACTIVITY OF PROSTATE CANCER CELLS

WU Zong-Ze^1,2, DING Guo-Fang^1,2, YANG Zui-Su¹, YU Fang-Miao¹, TANG Yun-Ping¹, JIA Ying-Lu¹, ZHENG Yuan-Yuan¹, CHEN Rui¹

1.School of Food Science and Pharmacy of Zhejiang Ocean University, Zhejiang Provincial Key Engineering Technology Research Center of Biomedical Products, Zhoushan 316022, China;2.Marine Fisheries Research Institute of Zhejiang, Zhoushan 316021, China

Abstract:

The enzymatic preparation of anti-prostate cancer oligopeptide from Anthopleura anjunae and its process optimization was investigated. Taking the flesh of A. anjunae as the material, the best protease for enzymatic hydrolysis was screened and the reactions were optimized in orthogonal experiments. The active oligopeptide was purified by using ultrafiltration, anion exchange chromatography, G-25 gel chromatography and high performance liquid chromatography. All the active components were detected in methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay on prostate cancer DU-145 cells. Morphologic changes of the cells were observed in inverted microscopy. The optimum alkaline enzymatic hydrolysis conditions were determined as:temperature 35℃, pH 11, solid to liquid ratio 1:5, amount of enzyme 2000U/g, and hydrolysis time 6h. The anti-prostate cancer oligopeptide was obtained and identified as Tyr-Val-Pro-Gly-Pro (AAP-H) by N-terminal amino-acid sequencing. The results demonstrate that AAP-H suppressed the proliferation of DU-145 cells in a time-and dose-dependent manner and the apoptosis morphological features of cells occurred. The anti-prostate cancer oligopeptide was obtained from the enzymatic hydrolysates of green sea anemone using ultrafiltration and chromatographic separation techniques. Our findings suggest that AAP-H can inhibit the proliferation of prostate cancer DU-145 cells and induces apoptosis.

Key words: Anthopleura anjunae enzymatic hydrolysis anti-tumor efficacy oligopeptide technology