CN116751247A - 具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽及制备方法和应用 - Google Patents
具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽及制备方法和应用 Download PDFInfo
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Abstract
本发明一种具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽及制备方法和应用,以金枪鱼心脏动脉球为原料,通过预处理、胃蛋白酶酶解,膜超滤、色谱分离纯化得到具保肝护肝功效的抗氧化肽Tyr‑Glu‑Gly‑Gly‑Asp(YEGGD),ESI‑MS测定分子量为539.49 Da。本发明制备的寡肽Tyr‑Glu‑Gly‑Gly‑Asp(YEGGD)可显著地清除DPPH自由基、羟基自由基、ABTS自由基和超氧阴离子自由基,增加急性肝损伤小鼠的体重,降低肝损伤小鼠血清中AST和ALT活性,升高肝组织匀浆中SOD、GSH‑Px活性,显著降低MDA含量,具有显著的保肝护肝功能。
Description
技术领域
本发明涉及到一种抗氧化肽,具体指涉及一种具有保肝护肝功效的金枪鱼心脏动脉球抗氧化肽及其应用,属于水产品精深加工技术领域。
背景技术
肝脏是腹腔内最大的实质性器官,在人体代谢过程中发挥合成、分解以及转化等多个方面的作用。在体内外多种因素(如病毒、酒精、药物、自身免疫性、遗传代谢性等)的刺激下,机体代谢将会产生多种有害物质,进一步导致肝脏的急性、慢性损伤,在严重的情况下甚至会导致肝脏坏死。肝病临床上分为急性和慢性肝病,病人常表现出食欲明显减退、四肢乏力、精神萎靡、右上腹隐痛、消瘦、不明原因的发热及水肿、黄疸、腹水、皮肤瘙痒、鼻出血、皮下出血等症状,若救治不及时,会进一步发展为肝功能衰竭、肝硬化、肝癌等,已成为威胁人类健康的主要疾病之一,但是急性、慢性肝损伤的发病机制复杂,其产生机理尚未得到完全阐明。因此,肝病的防治也被全世界所关注。针对肝病治疗的药物繁多,西药有多烯磷脂酰胆碱、硫普罗宁、干扰素等,中药主要有水飞蓟素、氧化苦参碱、丹参等。上述中西医存在药效短或毒性强等缺陷。因此,寻找和开发药效持久、低毒安全的保肝护肝药物是目前研究的重要方向
金枪鱼加工过程中产生约占总重量50%~70%的下脚料,这些下脚料主要是金枪鱼鱼皮,内脏,碎肉,鱼头和鱼鳞等,造成了金枪鱼资源的浪费。因此,部分研究聚焦于综合、高效利用金枪鱼下脚料制备具有医药保健价值的功能分子。基于此,申请人经过反复研究,以金枪鱼心脏动脉球为原料,利用酶解工艺和色谱制备技术制备具有保肝护肝功效的抗氧化肽,该抗氧化肽可用于制备预防和治疗肝损伤相关的功能产品。
发明内容
本发明所要解决的技术问题是针对现有技术的现状提供一种具有保肝护肝功效的金枪鱼心脏动脉球抗氧化肽及制备方法和应用,该抗氧化肽可用于制备预防和治疗肝损伤相关的功能产品。
本发明解决上述技术问题所采用的技术方案为:
本发明第一方面提供了具有保肝护肝功效的金枪鱼心脏动脉球抗氧化肽为五肽化合物,其氨基酸序列为Tyr-Glu-Gly-Gly-Asp(YEGGD),ESI-MS测定分子量为539.49Da。
本发明第二方面提供了具有保肝护肝功效的金枪鱼心脏动脉球抗氧化肽的制备方法,包括下述步骤:
1)金枪鱼心脏动脉球预处理:将金枪鱼心脏动脉球解冻,清水洗涤,去除杂质,组织粉碎,按照料液比1g:8~12mL加入到1.0mol/L 的NaCl溶液,于35~40℃搅拌3~5h小时去除球蛋白等杂蛋白,蒸馏水洗涤3次;按照金枪鱼心脏动脉球与无水乙醇的料液比为1g:5~ 8mL进行脱脂24小时,干燥、粉碎。
2)金枪鱼心脏动脉球蛋白的酶解:将上述金枪鱼心脏动脉球粉末按照料液比1g:18~25mL加入到0.2mol/L的磷酸盐缓冲液,溶液调温至35~45℃,调节pH值至2.0,加入心脏动脉球重量2.5~3.0%胃蛋白酶,水解3~4h;然后在95℃水浴中保温15min灭酶,冷却至常温,9000rmp离心25~30min,收集上清液,即金枪鱼心脏动脉球酶解液。
3)金枪鱼心脏动脉球抗氧化肽的制备:将金枪鱼心脏动脉球酶解液经截留分子量为1kDa、3.5kDa和10kDa的超滤膜进行分级,收集分级组分,测定其对DPPH自由基的清除能力,选择对DPPH自由基清除能力最强的组分冻干,得金枪鱼心脏动脉球超滤酶解物,该超滤酶解物依次经过离子交换层析、凝胶柱层析和反相高效液相色谱 (RP-HPLC)纯化,得到具有保肝护肝功效的金枪鱼心脏动脉球抗氧化肽。
作为优选,所述的步骤1)中的金枪鱼为鲣鱼Katsuwonus pelamis。
作为优选,所述步骤3)的离子交换层析、凝胶柱层析和RP-HPLC 纯化的具体过程为:
离子交换层析:将上述金枪鱼心脏动脉球超滤酶解物溶于双蒸水配成浓度为45~55mg/mL的溶液,缓慢加入到预处理的DEAE-52纤维素阴离子交换层析柱,分别用3~5倍柱体积的双蒸水、0.05M NaCl、 0.1M NaCl、0.25M NaCl洗脱,流速2.5~3.5mL/min,收集4个洗脱组分,测定4个组分对DPPH自由基的清除能力,选择对DPPH自由基清除能力最强组分,冻干,得金枪鱼心脏动脉球离子交换层析酶解物。
凝胶柱层析:将上述金枪鱼心脏动脉球离子交换层析酶解物溶于双蒸水配成浓度为25~30mg/mL的溶液,经过葡聚糖凝胶Sephadex G-25柱层析分离,用磷酸盐缓冲液(0.2M,pH 7.0)进行洗脱,流速1.0~1.5mL/min,根据220nm下的吸光值制作凝胶层析色谱图,收集各色谱峰,测定各色谱峰组分对DPPH自由基的清除能力,选择对DPPH自由基清除能力最强的色谱峰组分,冻干,得金枪鱼心脏动脉球凝胶层析酶解物。
RP-HPLC纯化:将上述金枪鱼心脏动脉球凝胶层析酶解物用双蒸水配成45~50μg/mL的溶液,利用RP-HPLC进行纯化,根据制备抗氧化肽的对DPPH自由基的清除能力,得1个高活性寡肽 Tyr-Glu-Gly-Gly-Asp(YEGGD),ESI-MS测定分子量为539.49Da。
再优选,所述RP-HPLC条件为:进样量18~20μL;色谱柱Zorbax, SB C-18(4.6×250mm,5μm);流动相:梯度洗脱,乙腈浓度在30 min内从0匀速提高至50%;洗脱速度0.8~1.2mL/min;紫外检测波长220nm。
本发明第三方面提供了上述具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽在对DPPH自由基、羟基自由基、ABTS自由基和超氧阴离子自由基清除中的应用。
本发明第四方面提供了上述具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽在制备预防和治疗肝损伤相关功能产品中的应用。
与现有技术相比,本发明所提供的具有保肝护肝功效的金枪鱼心脏动脉球抗氧化肽Tyr-Glu-Gly-Gly-Asp(YEGGD)可有效清除DPPH 自由基、羟基自由基、ABTS自由基和超氧阴离子自由;显著增加肝损伤SPF级雌性ICR小鼠的体重,明显降低肝损伤小鼠血清中AST和ALT活性,升高肝组织匀浆中抗氧化酶(SOD、GSH-Px)活性,降低氧化产物丙二醛(MDA)含量,具有显著的保肝护肝功效。因此, Tyr-Glu-Gly-Gly-Asp(YEGGD)具有安全无毒副作用、抗氧化作用强等优点,可用于制备预防和治疗肝损伤相关的功能产品。
附图说明
图1本发明的金枪鱼心脏动脉球酶解液(TCAH)和超滤组分 (TCAH-I~TSH-IV)在2.0mg/mL浓度下对DPPH自由基的清除能力;
图2为本发明的DEAE-52纤维素阴离子交换柱层析图;
图3为DEAE-52纤维素阴离子交换柱层析酶解物组分(IEC-I~IEC-IV)在2.0mg/mL浓度下对DPPH自由基的清除能力;
图4为本发明的葡聚糖凝胶Sephadex G-25层析图;
图5为葡聚糖凝胶Sephadex G-25制备酶解物组分(GPC-I~ GPC-II)在2.0mg/mL浓度下对DPPH自由基的清除能力;
图6为葡聚糖凝胶Sephadex G-25制备酶解物(GPC-II)的 RP-HPLC分析图;
图7为Tyr-Glu-Gly-Gly-Asp(YEGGD)的质谱图;
图8为Tyr-Glu-Gly-Gly-Asp(YEGGD)的结构图。
具体实施方式
以下结合具体实施例对本发明做进一步详细描述,以便更好地理解本技术方案。
一种具有保肝护肝功效的金枪鱼心脏动脉球抗氧化肽,制备工艺流程如下:金枪鱼心脏动脉球→预处理→胃蛋白酶酶解→酶解物→超滤→离子交换层析→凝胶过滤层析→高效液相色谱制备→具有保肝护肝功效的抗氧化肽→功能评价。
具体步骤为:
1)金枪鱼心脏动脉球预处理:将金枪鱼心脏动脉球解冻,清水洗涤,去除杂质,组织粉碎,按照按照料液比1g:12mL加入到1.0 mol/L的NaCl溶液,于35℃搅拌4h去除球蛋白等杂蛋白,蒸馏水洗涤3次;按照金枪鱼心脏动脉球与无水乙醇的料液比为1g:8mL进行脱脂24小时,干燥、粉碎。
2)金枪鱼心脏动脉球蛋白的酶解:将上述金枪鱼心脏动脉球粉末按照料液比1g:20mL加入到0.2mol/L的磷酸盐缓冲液(pH 2.0),溶液调温至37℃,加入心脏动脉球重量2.6%胃蛋白酶,调节溶液pH 值至2.0,水解3.2h,然后于95℃水浴保温15min灭酶,冷却至常温,9000rmp离心30min,收集上清液,即得金枪鱼心脏动脉球酶解液(TCAH)。
3)金枪鱼心脏动脉球抗氧化肽的制备:将金枪鱼心脏动脉球酶解液(TCAH)经截留分子量为1kDa、3.5kDa和10kDa的超滤膜进行分级,收集分级组分TCAH-I(MW<1.0kDa)、TCAH-II(1.0<MW<3.5 kDa)、TCAH-III(3.5<MW<10kDa)、和TCAH-IV(MW>10kDa),测定其对DPPH自由基的清除能力(见图1),TCAH-I具有最强的DPPH自由基的清除能力,即为金枪鱼心脏动脉球超滤酶解物。TCAH-I依次经过离子交换层析、凝胶柱层析和RP-HPLC纯化,得到具有保肝护肝功效的金枪鱼心脏动脉球抗氧化肽,利用质谱测定其分子量,氨基酸序列分析仪测定其氨基酸序列,具体过程如下:
①离子交换层析:将上述金枪鱼心脏动脉球超滤酶解物(TCAH-I) 溶于双蒸水配成浓度为48mg/mL的溶液,缓慢加入到预处理的 DEAE-52纤维素阴离子交换层析柱,分别用5倍柱体积的双蒸水、0.05 M NaCl、0.1M NaCl、0.25M NaCl洗脱,流速3.0mL/min,收集4个洗脱组分(IEC-I~IEC-IV)(见图2),测定4个组分(IEC-I~IEC-IV) 对DPPH自由基的清除能力(见图3),IEC-II对DPPH自由基清除能力最强,冻干,得金枪鱼心脏动脉球离子交换层析酶解物(IEC-II)。
②凝胶柱层析:将上述金枪鱼心脏动脉球离子交换层析酶解物 (IEC-II)溶于双蒸水配成浓度为28mg/mL的溶液,经葡聚糖凝胶 Sephadex G-25柱层析分离,用磷酸盐缓冲液(0.2M,pH 7.0)进行洗脱,流速1.2mL/min,根据220nm下的吸光值制作凝胶层析色谱图(见图4),收集各色谱峰(GPC-I和GPC-II),测定各色谱峰组分对 DPPH自由基的清除能力(见图5),GPC-II对DPPH自由基的清除能力最强,冻干,得金枪鱼心脏动脉球凝胶层析酶解物(GPC-II)。
③RP-HPLC纯化:将上述金枪鱼心脏动脉球凝胶层析酶解物 (GPC-II)用双蒸水配成50μg/mL的溶液,利用反相高效液相色谱(RP-HPLC)进行纯化(进样量20μL;色谱柱Zorbax,SB C-18 (4.6×250mm,5μm);流动相:梯度洗脱,乙腈浓度在30min内从0 匀速提高至50%;洗脱速度1.2mL/min;紫外检测波长220nm),根据制备寡肽对DPPH自由基的清除能力,得1个高活性抗氧化肽(TCP6) (见图6)。
④结构检测:收集具有强DPPH自由基清除能力的色谱峰TCP6,经RP-HPLC检测达到测序要求,利用ESI-MS测定分子量为539.49Da (见图7),蛋白/多肽序列分析仪测定氨基酸序列为 Tyr-Glu-Gly-Gly-Asp(YEGGD)(见图8)。
⑤功能评价:
抗氧化能力评价:根据文献[Bin Wang,Zhong-Rui Li,Chang-Feng Chi,Qi-HongZhang,Hong-Yu Luo.Preparation and evaluation of antioxidant peptides fromethanol-soluble proteins hydrolysate of Sphyrna lewini muscle[J].Peptides,2012,36(2):240–250]测定Tyr-Glu-Gly-Gly-Asp(YEGGD)的自由基清除能力,其对DPPH自由基、羟基自由基、ABTS自由基和超氧阴离子自由基的半数清除率(EC50)分别为1.06mg/mL、1.24mg/mL、0.20mg/mL和0.93mg/mL,显示出显著的抗氧化能力。
对急性肝损伤的保护作用:参照文献[刘晨晨、赵玉勤、杨最素、丁国芳。鳕鱼皮胶原蛋白肽对小鼠急性肝损伤的保护作用研究[J]。现代食品科技,2015,31(7):18-24]建立雄性ICR小鼠(体重为19-21g)急性肝损伤模型:实验周期为7天,前7天样品组 Tyr-Glu-Gly-Gly-Asp(YEGGD)和阳性药物(联苯双酯滴丸)组按照150mg/kg BW剂量每天灌胃给药1次,对照组予以等体积的生理盐水替代;第7天末次给药后,按照10mL/kg BW的标准给将各组小鼠 (除对照组外)腹腔注射0.3%浓度的四氯化碳(CCl4)花生油溶液,禁食不禁水24h后取血、肝脏。通过测定小鼠血清中ALT和AST活性、肝组织中抗氧化酶(GSH-Px和SOD)活性及丙二醛(MDA)含量评价 Tyr-Glu-Gly-Gly-Asp(YEGGD)的保肝护肝功效。试验结果表明:与模型组小鼠比较,Tyr-Glu-Gly-Gly-Asp(YEGGD)能显著增加小鼠体重(表1),降低肝损伤小鼠血清中AST和ALT活性(表2),升高肝组织匀浆中SOD、GSH-Px活性,显著降低MDA含量(表3)。因此,本发明金枪鱼心脏动脉球抗氧化肽Tyr-Glu-Gly-Gly-Asp(YEGGD) 具有显著的保肝护肝功能。
具体试验结果数据如下列表所示:
表1抗氧化肽Tyr-Glu-Gly-Gly-Asp(YEGGD)对小鼠急性肝损伤肝重指数的影响(n=15)
表2抗氧化肽Tyr-Glu-Gly-Gly-Asp(YEGGD)对小鼠急性肝损伤血清中ALT 和AST活性的影响(n=15)
表3抗氧化肽Tyr-Glu-Gly-Gly-Asp(YEGGD)对小鼠急性肝损伤肝组织中 GSH-Px、SOD活性及MDA含量的影响(n=15)
Claims (10)
1.具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽,其特征在于该抗氧化肽的氨基酸序列为Tyr-Glu-Gly-Gly-Asp,如SEQ ID NO.1所示,ESI-MS测定分子量为539.49 Da。
2.具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽的制备方法,其特征在于包括以下步骤:
1)以脱脂金枪鱼心脏动脉球粉末作为原料,加入磷酸盐缓冲液调节pH至2.0,得混合液;
2)混合液温度升至35-45℃,加入占脱脂金枪鱼心脏动脉球质量2.5-3.0%的胃蛋白酶,酶解3-4h,得酶解产物,酶解产物灭酶处理后离心,收集上清液,即得金枪鱼心脏动脉球酶解液;
3)将金枪鱼心脏动脉球酶解液进行超滤膜进行分级,收集分级组分,选择对DPPH自由基清楚能力最强的组分冻干,得金枪鱼心脏动脉球超滤酶解物,将超滤酶解物依次经过离子交换层析、凝胶柱层析和反相高效液相色谱纯化,得具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽。
3. 如权利要求2所述的具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽的制备方法,其特征在于步骤1)中脱脂金枪鱼心脏动脉球粉末的制备过程为:将金枪鱼心脏动脉球解冻,清水洗涤,去除杂质,组织粉碎,按照料液比1g:8~12mL加入到1.0mol/L 的NaCl溶液,于35~40℃搅拌3~5h小时去除杂蛋白,蒸馏水洗涤3次;按照金枪鱼心脏动脉球与无水乙醇的料液比为1g:5~8mL进行脱脂24小时,干燥、粉碎,即得脱脂金枪鱼心脏动脉球粉末。
4. 如权利要求2所述的具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽的制备方法,其特征在于步骤1)中脱脂金枪鱼心脏动脉球粉末与磷酸盐缓冲液的料液比为1g:18~25mL,磷酸盐缓冲液的浓度为0.2 mol/L。
5.如权利要求2所述的具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽的制备方法,其特征在于步骤2)中灭酶处理具体为在95℃水浴中保温15min灭酶,冷却至常温,离心条件为:9000 rmp离心25~30min。
6. 如权利要求2所述的具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽的制备方法,其特征在于步骤3)中超滤膜分级具体为:将金枪鱼心脏动脉球酶解液经截留分子量为1 kDa、3.5kDa和10 kDa的超滤膜进行分级。
7.如权利要求2所述的具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽的制备方法,其特征在于步骤3)中离子交换层析、凝胶柱层析和反相高效液相色谱纯化的具体过程为:
离子交换层析:将金枪鱼心脏动脉球超滤酶解物溶于双蒸水配成浓度为45~55mg/mL的溶液,缓慢加入到预处理的DEAE-52纤维素阴离子交换层析柱,分别用3~5倍柱体积的双蒸水、0.05M NaCl、0.1M NaCl、0.25M NaCl 洗脱,流速2.5~3.5mL/min,收集4个洗脱组分,测定4个组分对DPPH自由基的清除能力,选择对DPPH自由基清除能力最强组分,冻干,得金枪鱼心脏动脉球离子交换层析酶解物;
凝胶柱层析:将上述金枪鱼心脏动脉球离子交换层析酶解物溶于双蒸水配成浓度为25~30mg/mL的溶液,经过葡聚糖凝胶Sephadex G-25柱层析分离,用0.2M、pH7.0的磷酸盐缓冲液进行洗脱,流速1.0~1.5mL/min,根据220nm下的吸光值制作凝胶层析色谱图,收集各色谱峰,测定各色谱峰组分对DPPH自由基的清除能力,选择对DPPH自由基清除能力最强的色谱峰组分,冻干,得金枪鱼心脏动脉球凝胶层析酶解物;
反相高效液相色谱纯化:将上述金枪鱼心脏动脉球凝胶层析酶解物用双蒸水配成45~50μg/mL的溶液,利用RP-HPLC进行纯化,根据制备抗氧化肽的对DPPH自由基的清除能力,得1个高活性寡肽Tyr-Glu-Gly-Gly-Asp(YEGGD),ESI-MS测定分子量为539.49 Da。
8. 如权利要求7所述的具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽的制备方法,其特征在于反相高效液相色谱纯化条件为:进样量18~20μL;色谱柱Zorbax, SB C-18 (4.6×250mm,5µm);流动相:梯度洗脱,乙腈浓度在30min内从0匀速提高至50%;洗脱速度0.8~1.2mL/min;紫外检测波长220nm。
9.如权利要求1所述的具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽在对DPPH自由基、羟基自由基、ABTS自由基和超氧阴离子自由基清除中的应用。
10.如权利要求1所述的具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽在制备预防和治疗肝损伤相关功能产品中的应用。
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