CN112956700A - 促骨形成肽在制备促进成骨细胞增殖、分化或矿化的功能性食品、保健品或药物中的应用 - Google Patents
促骨形成肽在制备促进成骨细胞增殖、分化或矿化的功能性食品、保健品或药物中的应用 Download PDFInfo
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Abstract
本发明涉及促骨形成肽在制备促进成骨细胞增殖、分化或矿化的功能性食品、保健品或药物中的应用。该促骨形成肽的氨基酸序列为：GPAGPHGPVG。本发明提供的促骨形成肽具有较好的促进成骨细胞增殖活性，可促进成骨细胞ALP表达，有利于成骨细胞的分化和矿化，且安全无毒，在制备促进成骨细胞增殖、分化或矿化的功能性食品、保健品或药物中有较好的应用前景。
Description
技术领域
本发明属于生物材料技术领域，具体涉及促骨形成肽在制备促进成骨细胞增殖、分化或矿化的功能性食品、保健品或药物中的应用。
背景技术
我国是全球最大罗非鱼养殖加工国，2016年养殖产量达158万吨，约占同年全球总产量的13％，此中大部分罗非鱼被用于鱼片加工。据统计，罗非鱼加工过程中产生的副产物可占所用鱼体总质量的54％，然而我国目前对加工副产物合理利用率较低，不但造成了生物资源浪费，也为环境增加负担。因此，开发利用罗非鱼副产物具有丰富的原料基础和广阔的应用前景。
专利CN109265536A从罗非鱼骨架中酶解得到一种钙螯合肽，该钙螯合肽具有较好的钙螯合活性，钙螯合能力达43.018mg/g。但目前此类研究还较少。
因此，开发更多罗非鱼加工副产物的用途以对其合理利用具有重要的经济意义和应用价值。
发明内容
本发明的目的在于克服现有关于罗非鱼加工副产物的生物利用度的研究缺乏的缺陷或不足，提供促骨形成肽在制备治疗骨质疏松症药物中的应用。本发明从罗非鱼鳞中分离得到一种多肽，该多肽不仅具有较好的钙螯合能力，还具有较好的促进成骨细胞增殖活性，可促进成骨细胞ALP表达，有利于成骨细胞的分化和矿化，且安全无毒，在制备促进成骨细胞增殖、分化或矿化的功能性食品、保健品或药物中有较好的应用前景。
为实现上述发明目的，本发明采用如下技术方案：
促骨形成肽在制备促进成骨细胞增殖、分化或矿化的功能性食品、保健品或药物中的应用，所述促骨形成肽的氨基酸序列为：GPAGPHGPVG(Gly-Pro-Ala-Gly-Pro-His-Gly-Pro-Val-Gly)。
本发明在对罗非鱼鳞进行酶解时，意外发现序列为GPAGPHGPVG的多肽，其序列与专利CN109265536A中报道的钙螯合肽的序列一致。对其生物活性进行研究时，发现其不仅具有较佳的钙螯合能力，还具有较好的促进成骨细胞增殖活性，可促进成骨细胞ALP表达，有利于成骨细胞的分化和矿化，且安全无毒，在制备促进成骨细胞增殖、分化或矿化的功能性食品、保健品或药物中有较好的应用前景。
优选地，所述促骨形成肽的质荷比为414.6975。
优选地，所述促骨形成肽与EGFR的氢键作用包括ARG285、GLN8、GLY317或SER11中的一种或几种。
优选地，所述促骨形成肽与EGFR的疏水相互作用包括ASP344、GLN408、GLY39、GLY410、GLY9、HIS346、HIS409、ILE318、LEU38、LYS407、PHE380、THR10或THR406中的一种或几种。
优选地，所述促骨形成肽在制备治疗骨质疏松症药物中的应用。
优选地，所述药物包括药学上可接受的盐、载体和/或赋形剂。
优选地，所述功能性食品为奶粉或液态乳制品。
优选地，所述保健品为钙片、液态钙或口服液。
优选地，所述促骨形成肽以促骨形成肽及其衍生物或可药用盐的形式添加至功能性食品、保健品或药物中。
促骨形成肽既可按照专利CN109265536A中报道的方法分离得到，也可通过如下过程制备得到：
S1、将罗非鱼鳞用蒸馏水重复洗三次，把杂质去除；
S2、碱洗脱脂：将罗非鱼鳞放入0.1mol/L NaOH溶液(w/v1:10)搅拌24h，清洗至中性；
S3、脱钙处理：将罗非鱼鳞放入10％EDTA-2Na(pH7.2，w/v1:10)中，并置于4℃冷库中，期间进行搅拌处理5天，随后将鱼鳞清洗至中性后待用；
S4、酸处理：将脱钙后的罗非鱼鳞用质量分数4％的盐酸处理时间18h后水洗至中性，即得到罗非鱼鳞胶原蛋白；将水洗至中性的罗非鱼鳞胶原蛋白中水分滴干，随后置于55℃烘箱中烘干，取出置于密封袋中备用；
S5、酶解：选用木瓜蛋白酶进行酶解即得，料液比为8％，酶解时间为2h，酶底比为0.3％，pH为7，温度为60℃。
与现有技术相比，本发明具有如下有益效果：
本发明提供的促骨形成肽具有较好的促进成骨细胞增殖活性，可促进成骨细胞ALP表达，有利于成骨细胞的分化和矿化，且安全无毒，在制备促进成骨细胞增殖、分化或矿化的功能性食品、保健品或药物中有较好的应用前景。
附图说明
图1为EGFR几何与对接盒(PDB ID:1IVO)；
图2为GPAGPHGPVG和EGFR之间的交互作用三维图；
图3为GPAGPHGPVG和EGFR相互作用2D图；其中，绿色虚线：氢键相互作用；数：键长；红堡：疏水相互作用；
图4为不同浓度的GPAGPHGPVG的增殖效果图；
图5为GPAGPHGPVG的ALP酶活效果图；
图6为GPAGPHGPVG的矿化效果图；
图7为GPAGPHGPVG的肽谱图。
具体实施方式
下面结合实施例进一步阐述本发明。这些实施例仅用于说明本发明而不用于限制本发明的范围。下例实施例中未注明具体条件的实验方法，通常按照本领域常规条件或按照制造厂商建议的条件；所使用的原料、试剂等，如无特殊说明，均为可从常规市场等商业途径得到的原料和试剂。本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
实施例1促骨形成肽的分离制备
本实施例提供一种促骨形成肽，其氨基酸序列为：GPAGPHGPVG(Gly-Pro-Ala-Gly-Pro-His-Gly-Pro-Val-Gly)。
该促骨形成肽可从罗非鱼鳞中分离得到，具体过程如下：
(一)罗非鱼鳞的预处理
(1)将罗非鱼鳞用蒸馏水重复洗三次，把杂质去除。
(2)碱洗脱脂：将罗非鱼鳞放入0.1mol/L NaOH溶液(w/v1:10)搅拌24h，清洗至中性。
(3)脱钙处理：将罗非鱼鳞放入10％EDTA-2Na(pH7.2，w/v1:10)中，并置于4℃冷库中，期间进行搅拌处理5天，随后将鱼鳞清洗至中性后待用。
(4)酸处理：将脱钙后的罗非鱼鳞用质量分数4％的盐酸处理时间18h后水洗至中性，即得到罗非鱼鳞胶原蛋白；将水洗至中性的罗非鱼鳞胶原蛋白中水分滴干，随后置于55℃烘箱中烘干，取出置于密封袋中备用。
(二)罗非鱼鳞钙结合肽的酶解实验
在料液比，8％，酶底比0.3％，pH 7下60℃水浴酶解2h，100℃灭酶10mim后，4000r/min离心20min，取上清，冷冻干燥。
(三)罗非鱼鳞钙结合肽的富集
将冻干的罗非鱼鳞钙结合肽，加入蒸馏水形成5mg/mL体系，按肽钙比1:1加入无水氯化钙后，调节pH 8，47℃水浴下螯合42min，螯合反应结束后，加入反应体系5倍体积的无水乙醇，4000r/min离心20min，弃上清，取沉淀，冷冻干燥。
(1)质谱序列：将富集的罗非鱼鳞钙结合肽利用HPLC-MS/MS对罗非鱼鳞钙结合肽分析。
HPLC-MS/MS法处理过程及条件如下：将C18除盐柱在平衡液(50％ACN)中吹打5次，用Washing buffer(0.1％FA,2％ACN)洗柱5次，吸取样品，缓慢吹打10次，用Elutionbuffer(0.1％FA,60％ACN)洗脱样品，洗脱溶液转至新管，离心浓缩干燥，待做质谱分析。除盐后多肽样品经离心干燥后，重新溶解于Nano-LC流动相A(0.1％甲酸/水)中装瓶上样，进行在线LCMS分析。溶解后的样品以2μL的体积上样到nanoViper C18预柱上(3μm，
)，然后20ul体积冲洗脱盐。液相为Easy nLC 1200纳升液相系统(ThermoFisher,USA)，样品在预柱上脱盐保留后再经分析柱分离，分析柱规格是C18反相色谱柱(Acclaim PepMap RSLC,75μm×25cm C18-2μm
)，实验所用梯度为60min内流动相B(80％乙腈，0.1％甲酸)由5％升高至38％。质谱采用ThermoFisher Q Exactive系统(ThermoFisher,USA)结合纳升喷雾Nano Flex离子源(ThermoFisher,USA)，喷雾电压为1.9kV，离子传输管加热温度为275℃。质谱扫描方式为信息依赖的采集工作模式下(DDA，Data Dependent Analysis)，一级质谱扫描分辨率为70000，扫描范围350-2000m/z，最大注入时间100ms。每次DDA循环下最多采集20个电荷为2+到5+的二级图谱，二级质谱离子最大注入时间为50ms。碰撞室能量(高能碰撞诱导解离,HCD)设设定为28eV，适用于所有前体离子，动态排除设置为25秒。质谱采集到的原始raw图谱文件，采用PEAKS Studio 8.5(Bioinformatics Solutions Inc.Waterloo,Canada)软件进行数据加工处理和检索分析。
用HPLC-MS/MS法对罗非鱼鳞钙结合肽的氨基酸序列进行测定得到鉴定了133条肽序列，分子质量范围519-3736Da，肽段长度为5-43，其中包括序列为GPAGPHGPVG的多肽(如图7)，即为促骨形成肽。
实施例2分子对接筛选促骨形成肽
(1)分子对接
EGFR几何图形(如图1，)从RCSB蛋白数据库下载，PDD ID:1IVO。水离子和其他不相干的原子被删除，准备对接。用PyMol1.7构建肽段GPAGPHGPVG及其它肽段，然后用UFF(万向力场)分子力学力场进行能量最小化，使用Avogadro软件。与AutoDock Vina 1.2进行分子对接，对接口袋定义为一个50A*50A*50A的盒子(图1)，包括报道的所有活性位点。以上未提到的所有参数均设置为默认值。
(2)分子对接筛选单体
肽段GPAGPHGPVG与EGFR结合的能量表见表1，根据结合能可知，GPAGPHGPVG与EGFR结合非常稳定。
表1 通过HPLC-MS/MS鉴定的罗非鱼鳞钙结肽AutoDock Vina分子对接分析
(3)分子对接单体分析：
GPAGPHGPVG与EGFR的氢键作用(如图2和图3)包括ARG285、GLN8、GLY317和SER11。GPAGPHGPVG与EGFR的疏水相互作用包括ASP344、GLN408、GLY39、GLY410、GLY9、HIS346、HIS409、ILE318、LEU38、LYS407、PHE380、THR10和THR406。
(4)单体合成
从Synpeptide Co.，Ltd(南京，中国)获得纯度高于95％的合成肽GPAGPHGPVG。
实施例3促骨形成肽的活性验证
(1)成骨细胞培养：
MC3T3-E1 subclone14细胞购自中国科学院上海生命科学研究院细胞资源中心。细胞在完全培养基中培养(α-MEM中添加10％胎牛血清，1％双抗)。细胞融汇80％-90％时，用胰蛋白酶-EDTA传代。
(2)细胞增殖率：
不同浓度的GPAGPHGPVG对MC3T3-E1毒性试验。将MC3T3-E1细胞以5×103个细胞/孔的密度种植在96孔板(Costar，Corning，NY)中，并放在5％CO2培养箱中于37℃温育24小时。随后，用具有不同浓度(0、1、10、50、100、200μg/mL)的肽(GPAGPHGPVG)的100μL培养基处理细胞，并孵育24小时。孵育后，通过100μL的0.5mg/mL MTT溶液处理细胞4小时。然后，用150μL二甲基亚砜代替MTT溶液。将96孔板置于振荡器上15min。最后，使用酶标仪在570nm的波长下测量光吸收值。与对照组相比，吸光度降低10％以上的样品浓度被认为是有细胞毒性的。
图4可知，在浓度范围为1，10，50，100，200μg/mL，GPAGPHGPVG对细胞活力没有负面影响，在10μg/mL时，细胞活力达到最高值，增长率为109.88％，根据MTT试验结果，选用0，1，10，50μg/mL进行分化矿化实验。
(3)碱性磷酸酶(ALP)活性：
将MC3T3-E1细胞以1×106个细胞/孔的密度种植在6孔板(Costar，Corning，NY)中，待细胞融合后，分别更换含不同浓度(0，1，10，50μg/mL)的GPAGPHGPVG的分化完全培养液(含50μg/mL VC和10mmol/Lβ-甘油磷酸钠)培养7天。第七天后，根据ALP试剂盒(南京建成)进行ALP活性检测。
如图5，相对于空白对照，含有GPAGPHGPVG的样品组碱性磷酸酶活都显著提高，说明GPAGPHGPVG可以促进成骨细胞ALP表达，有利于成骨细胞的分化。
(4)茜素红染色：
将MC3T3-E1细胞以1×106个细胞/孔的密度种植在6孔板(Costar，Corning，NY)中，待细胞融合后，分别更换含不同浓度(0，1，10，50μg/mL)的GPAGPHGPVG的分化完全培养液(含50μg/mL VC和10mmol/Lβ-甘油磷酸钠)培养21天，前14天隔天换液，后7天每天半量换液。21天后，弃去培养基，用PBS洗细胞2次，用4％多聚甲醛固定30min，弃甲醛，蒸馏水洗2次，加入1mL茜素红染液染色5min，弃去染液，蒸馏水洗3次，PBS在37℃10min除去特异性结合，拍照。利用10％的西吡氯铵溶液，溶解钙结节，在酶标仪562nm处进行钙含量测定。
图6显示了GPAGPHGPVG对MC3T3-E1矿化有显著的影响。相对于空白组，含有GPAGPHGPVG成骨细胞矿化结节都有不同程度的增加；在钙结节定量分析中，可以直观看出，相对于空白对照，GPAGPHGPVG矿化效果好，结果说明GPAGPHGPVG可以促进成骨细胞矿化。
由上述可知，本发明提供的促骨形成肽具有较好的促进成骨细胞增殖活性，可促进成骨细胞ALP表达，有利于成骨细胞的分化和矿化，且安全无毒。
最后应当指出的是，以上实施例仅是本发明的具有代表性的例子。显然，本发明的技术方案并不限于上述实施例，还可有许多变形。本领域的普通技术人员能从本发明内容直接导出或联想到所有变形，均应认为是本发明的权利要求的保护范围。
序列表
<110> 华南农业大学
<120> 促骨形成肽在制备促进成骨细胞增殖、分化或矿化的功能性食品、保健品或药物中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 10
<212> PRT
<213> 促骨形成肽(1)
<400> 2
Gly Pro Ala Gly Pro His Gly Pro Val Gly
1 5 10
Claims (9)
1.促骨形成肽在制备促进成骨细胞增殖、分化或矿化的功能性食品、保健品或药物中的应用，其特征在于，所述罗非鱼鳞促骨形成肽的氨基酸序列为：GPAGPHGPVG。
2.根据权利要求1所述应用，其特征在于，所述促骨形成肽的质荷比为423.2168。
3.根据权利要求1所述应用，其特征在于，所述促骨形成肽与EGFR的氢键作用包括ARG285、GLN8、GLY317或SER11中的一种或几种。
4.根据权利要求1所述促骨形成肽，其特征在于，所述促骨形成肽与EGFR的疏水相互作用包括ASP344、GLN408、GLY39、GLY410、GLY9、HIS346、HIS409、ILE318、LEU38、LYS407、PHE380、THR10或THR406中的一种或几种。
5.根据权利要求1所述应用，其特征在于，所述促骨形成肽在制备治疗骨质疏松症药物中的应用。
6.根据权利要求5所述应用，其特征在于，所述药物包括药学上可接受的盐、载体和/或赋形剂。
7.根据权利要求1所述应用，其特征在于，所述功能性食品为奶粉或液态乳制品。
8.根据权利要求1所述应用，其特征在于，所述保健品为钙片及补钙制剂或口服液。
9.根据权利要求1所述应用，其特征在于，所述促骨形成肽以促骨形成肽及其衍生物或可药用盐的形式添加至功能性食品、保健品或药物中。
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