【作者】 潘欣； 胡发远； 王斌； 丁国芳；

【Author】 Xin Pan;Fa-yuan Hu;Bin Wang;Guo-Fang Ding;School of Food and Pharmacy,Zhejiang Ocean University;

【机构】 浙江省海洋生物医用制品工程技术研究中心浙江海洋学院食品与药学学院；

【摘要】 本文以孔鳐（Rajaporosa）软骨为实验材料,采用盐酸胍抽提、丙酮分级沉淀、蛋白酶酶解、超滤、DEAE-52阴离子交换层析、Sephadex G-15凝胶层析和反相高效液相色谱（RP-HPLC）等技术,得到孔鳐软骨多肽DCPE-A,DCPE-B和DCPE-C,经氨基酸序列分析和质谱（ESI-MS）确定其结构分别为Phe-Ile-Met-Gly-Pro-Tyr（DCPE-A）和Gly-Pro-Ala-Gly-Asp-Tyr（DCPE-B）和Ile-Val-Ala-Gly-Pro-Gin（DCPE-C）,分子量分别为726.9、578.58和583.69 Da。体外抗氧化实验结果表明:DCPE-A、DCPE-B与DCPE-C对DPPH自由基(EC₅₀为3.83、4.21和4.45 mg/1)、羟自由基（EC50为3.37、3.92和4.23 mg/mL）、ABTS自由基（ECS0为0.77、1.04和1.29 mg/mL）和超氧阴离子自由基(EC₅₀为1.61、1.66和1.82mg/mL)具有良好的清除作用,DCPE-C亦显示出了良好的脂质过氧化抑制作用。抗肿瘤活性表明:孔鳐软骨多肽DCPE-A,DCPE-B和DCPE-C的对人宫颈癌HeLa细胞的半数抑制率（IC50）分别为4.81、4.86和6.26 mg/mL,DCPE-A显示出最强的增殖抑制作用。AO/EB染色观察显示DCPE-A能引起肿瘤细胞凋亡的形态学变化;通过流式细胞术结果可以看出随DCPE-A浓度的增加,HeLa细胞的早期凋亡率从4.54%增加到19.25%;Westem blotting免疫印迹表明Bcl-2/Bax的比值急剧升高,且Caspase-3的蛋白表达水平也显著增加。研究结果表明软骨多肽DCPE-A可诱导HeLa细胞发生凋亡,为其应用于临床肿瘤治疗提供理论依据。更多还原

【Abstract】 In the experiment,anti-cancer peptides from protein hydrolysates of Raja porosa cartilage were purified,and their activities were evaluated by assays of radical scavenging and lipid peroxidation inhibition,and their antitumor activities were also measured.Using ultrafiltration and consecutive chromatographic methods,three antioxidant peptides(DCPE-A,DCPE-B and DCPE-C)were isolated from the protein hydrolysate of R.porosa cartilage,and their amino acid sequences were identified as Phe-Ile-Met-Gly-Pro-Tyr(DCPE-A),Gly-Pro-Ala-Gly-Asp-Tyr(DCPE-B) and Ile-Val-Ala-Gly-Pro-Gln(DCPE-C) with molecuLar weights of 726.9,578.58 and 583.69 Da,respectively.DCPE-A,DCPE-B and DCPE-C exhibited good scavenging activity on DPPH radical(EC503.83,4.21 and 4.45 mg/1),hydroxyl radical(EC50 3.37,3.92 and 4.23mg/mL),ABTS radical(EC50 0.77,1.04 and 1.29mg/mL),and superoxide anion radical(EC50 1.61,1.66 and 1.82 mg/mL).DCPE-A was also effectively against lipid peroxidation in a linoleic acid model system.In addition,DCPE-A displayed high anti-proliferation activity on HeLa cells by inducing apoptosis.The expression of the anti-apoptotic protein Bcl-2 was decreased and the expression of the apoptogenic protein Bax was increased when HeLa cells were treated by BCP-A for 24 h,which induced the increase of the Bax/Bcl-2expression ratio.Moreover,the expression of caspase-3 in HeLa cells treated with BCP-A was upreguLated.Therefore,DCPE-A showed significantly antioxidant activity and anticancer properties.更多还原