CN113769066A - 一种促进创面愈合的组合物及其制备方法与用途 - Google Patents
一种促进创面愈合的组合物及其制备方法与用途 Download PDFInfo
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Abstract
本发明提供了一种促进创面愈合的组合物及其制备方法与用途,属于制剂领域。该组合物包括类贻贝粘蛋白和多肽或其复合物,所述多肽或其复合物为GHK、GHK‑Cu、GQPR中的一种或多种。该组合物中的类贻贝粘蛋白和GHK‑Cu通过协同增效的作用,使组合物具有良好的抗炎功效和促进胶原蛋白生成的作用,能够有效地促进创面的愈合,明显缩短创面愈合的时间;该组合物同时还兼具免疫原性低、吸收性好、稳定性强、安全无毒等优点,在生物医药领域具有良好的应用前景。
Description
技术领域
本发明属于制剂领域,具体涉及一种促进创面愈合的组合物及其制备方法与用途。
背景技术
皮肤创面愈合是一个复杂的生物学过程,涉及多种细胞类型、细胞外基质成分及细胞因子等共同的参与和协调。创面愈合大致分为3个阶段,主要是凝血期、炎症期和修复期。不同的细胞、细胞因子和炎症递质参与了创面愈合的各个过程。凝血期主要由血小板和纤维蛋白参与反应,炎症期主要由中性粒细胞、巨噬细胞和淋巴细胞参与反应,而修复期主要由成纤维细胞、内皮细胞、上皮细胞和胶原完成。
随着对创面愈合研究的不断深入,市面上的创面愈合材料越来越多样化。目前,市面上主打创面愈合功能的医用材料大多以壳聚糖、海藻酸盐和透明质酸等为原料,均具有一定的促进伤口愈合的功能。专利CN107496972B公开了一种促烧伤创面愈合的防粘连湿性敷料及其制备方法,这种湿性敷料包括聚丙烯无纺布和黏合在聚丙烯无纺布上的复合水凝胶,复合水凝胶的主要成分是羧甲基纤维素钠、海藻酸钠、壳聚糖和丙二醇。专利CN106492260B公开了一种海藻酸盐基水凝胶敷料及其制备方法,制得的水凝胶敷料具有良好的保湿性能、韧性和粘性,主要适用于皮肤浅表损伤。专利CN106421881B公开了一种壳聚糖止血愈创材料及其制备方法,制得的材料同时具有快速止血和促进创面愈合的功能。专利CN110038156B公开了一种止血促创面愈合的乳液及其制备方法,制得的乳液可稳固的黏附在创伤部位,能有效地止血和促进伤口愈合。但是,上述以壳聚糖、海藻酸盐和透明质酸为功能成分的创面愈合材料均存在无抗炎功能、容易引起免疫反应、吸收性不好等缺点,无法满足当前市场的需求。
贻贝粘蛋白(mussel adhesive protein,MAP)是贻贝足部分泌腺分泌出的一种蛋白复合物,近年来因其具有高强度、高韧性、防水性和可降解性等优点受到了广泛的关注,在生物医药方面得到了大力的推广和应用。贻贝粘蛋白与高等植物细胞壁中的伸展蛋白相似,是一种属于糖蛋白类型的黏液蛋白,具有特殊的组成结构以及超强的粘性和伸展性。另一方面,贻贝粘蛋白还具有生物相容性好和可生物降解的优点,伸展性强,粘性大,安全无毒,是一种具有极大优势和潜力的生物粘合剂,在人造骨骼、牙科缝合、外科手术等方面得到了广泛的应用。专利申请CN111973551A公开了一种贻贝粘蛋白抑菌凝胶及其制备方法,由以下质量百分数的原料组成:贻贝粘蛋白1%、硼酸0.2%、30万分子量的透明质酸钠0.1%、壳聚糖0.3-1.0%、薄荷脑0.1-0.5%、醋酸氯已定0.2-0.5%、卡波姆940 0.8-1.5%、乙醇0.5-1.0%、尼泊金乙酯0.01-0.05%、三乙醇胺0.05-0.1%、纯化水90-98%,以上各组份的质量百分数总和为100%。该贻贝粘蛋白抑菌凝胶是贻贝粘蛋白、透明质酸钠、壳聚糖等复方配伍制成的抑菌凝胶,具有快速修复保湿私密黏膜、抗氧化、抑菌止痒,促进伤口愈合、平衡菌群、辅助治疗***、***及各种炎症的功效。但是,该贻贝粘蛋白抑菌凝胶的活性成分包括贻贝粘蛋白、透明质酸钠和壳聚糖,原料组成复杂,制备成本较高。
为了克服目前市面上诸多创面愈合材料的缺点,亟需开发出一种组成更简单、促进创面愈合效果更好的修复材料。
发明内容
本发明的一个目的在于提供一种促进创面愈合的联合用药物;本发明的另一个目的在于提供一种促进创面愈合的组合物及其制备方法与用途。
本发明提供了类贻贝粘蛋白和多肽或其复合物在制备具有抗氧化和/或抗炎作用的联合用制剂中的用途,所述多肽或其复合物为GHK、GHK-Cu、GQPR中的一种或多种。
进一步地,所述多肽或其复合物为GHK-Cu;所述类贻贝粘蛋白和GHK-Cu的重量比为(0.5~5.0):1,优选为(1.0~2.5):1,更优选为1.25:1;
和/或,所述类贻贝粘蛋白选自mlfp-1、mlfp-2、mlfp-3、mlfp-4、mlfp-5、mlfp-6、mlfp-151、mlfp-131、mlfp-153、mlfp-351中一种或多种;mlfp-1的氨基酸序列如SEQ IDNO.1所示,mlfp-2的氨基酸序列如SEQ ID NO.2所示,mlfp-3的氨基酸序列如SEQ ID NO.3所示,mlfp-4的氨基酸序列如SEQ ID NO.4所示,mlfp-5的氨基酸序列如SEQ ID NO.5所示,mlfp-6的氨基酸序列如SEQ ID NO.6所示,mlfp-151的氨基酸序列如SEQ ID NO.7所示,mlfp-131的氨基酸序列如SEQ ID NO.8所示,mlfp-153的氨基酸序列如SEQ ID NO.9所示,mlfp-351的氨基酸序列如SEQ ID NO.10所示。
其中,mlfp-1的氨基酸序列如下:MEGIKLNLCLLCIFTFDVLGFSNGNIYNAHVSSYAGASAGAYKKLPNAYPYGTKPEPVYKPVKTSYSAPYKPPTYQQLKKKVDYRPTKSYPPTYGSKTNYLPLAKKLSSYKPIKTTYNAKTNYPPVYKPKMTYPPTYKPKPSYPPTYKSKPTYKPKITCPPTYKAKPSYPPTYKPKKTYPPTYKPKVTYPPTYKPKPSYPPIYKSKPTYKPKITYPPTYKAKPSYPPTYKAKPSYPPTYKAKPTYKAKPTYPSTYKAKPTYPPTYKAKPSYPPTYKAKPSYPPTYKAKPTYIAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKSSYPPTYKAKPTYKAKPTYPSTYKAKPSYPPTYKAKPTYKAKPTYPSTYKAKPTYPSTYKAKPSYPPTYKPKISYPPTYKAKPSYPSTYKAKSSYPPTYKAKPSYPPTYKAKPTYKAKPTYPSTYKAKPTYKAKPTYPPTYKAKPSYPPTYKPKPSYPPTYKSKSSYPSSYKPKKTYPPTYKPKLTYPPTYKPKPSYPPSYKPKITYPSTYKLKPSYPPTYKSKTSYPPTYNKKISYPSQY(SEQ IDNO.1);
mlfp-2的氨基酸序列如下:MLFSFFLLLTCTQLCLGTSPRQYDDDEDDYSPPVYKPSPSKYRPVNPCLKKPCKYNGVCKPNGGSYKCTCKGGYYGYNCNLKNACKPNQCKNKSRCIPVGKTYKCVCRNGNYGRLCEKNVCSPNPCKNKGKCAPWGKTGYKCRCSGGYTGPRCEVHACKPNPCKNNGSCKPDGKTGYKCTCVGGYSGPTCQENACKPNPCSNGGKCSADKFGDYSCECQKRYYGPECEKYVCAPNPCKNGGKCSSDGSGGYKCQCTGGYSGLTCNVNVCKPNPCKNSGRCVNKGSSYKCICKGGYSGPTCGENVCKPNPCQNKGRCYPDQSDDGFKCKCLGGYTGPTCEDKPNPCNTKPCKNGGKCSYNGKTYTCKCAYGYRGRHCTAKAYNPCASRPCKNRGKCTVKGTKYVCTCAKGYSGRYCALKSPPSYNDDDEY(SEQ ID NO.2);
mlfp-3的氨基酸序列如下:MNKFSVAVLLVLVLIGLFAVQSXAGYGYDLGYNAPWPYNNGYYGYNGYNGYHGRYGWNKGWNSGPWGGSYYGNKGYLY(SEQ ID NO.3);
mlfp-4的氨基酸序列如下:MKRGLFTILLITAVLVVVAESYGRRYGEPSGYANIGHRRYYERAISFHRHSHVHGHHLLHRHVHRHSVLHGHVHMHRVSHRIMHRHRVLHGHVHRHRVLHNHVHRHSVLHGHVHRHRVLHRHVHRHNVLHGHVHRHRVLHKHVHNHRVLHKHLHKHQVLHGHVHRHQVLHKHVHNHRVLHKHLHKHQVLHGHVHTHRVLHKHVHKHRVLHKHLHKHQVLHGHIHTHRVLHKHLHKHQVLHGHVHTHRVLHKHVHKHRVLHKHLHKHQVLHGHVHMHRVLHKHVHKHRVLHKHVHKHHVVHKHVHSHRVLHKHVHKHRVEHQHVHKHHVLHRHVHSHHVVHSHVHKHRVVHSHVHKHNVVHSHVHRHQILHRHVHRHQVVHRHVHRHLIAHRHIHSHQAAVHRHVHTHVFEGNFNDDGTDVNLRIRHGIIYFGGNTYRLSGGRRRFMTLWQECLESYGDSDECFVQLGNQHLFTVVQGHHSTSFRSDLSNDLHPDNNIEQIANDHVNDIAQSTDGDINDFADTHYNDVAPIADVHVDNIAQTADNHVKNIAQTAHHHVNDVAQIADDHVNDIGQTAYDHVNNIGQTADDHVNDIAQTADDHVNAIAQTADDHVNAIAQTADDHVNDIGDTANSHIVRVQGVAKNHLYGINKAIGKHIQHLKDVSNRHIEKLNNHATKNLLQSALQHKQQTIEREIQHKRHLSEKEDINLQHENAMKSKVSYDGPVFNEKVSVVSNQGSYNEKVPVLSNGGGYNGKVSALSDQGSYNEGYAY(SEQ ID NO.4);
mlfp-5的氨基酸序列如下:SSEEYKGGYYPGNTYHYHSGGSYHGSGYHGGYKGKYYGKAKKYYYKYKNSGKYKYLKKARKYHRKGYKKYYGGGSS(SEQ ID NO.5);
mlfp-6的氨基酸序列如下:MKSIQMILAVFVITALCGIVESGGGNYRGYCSNKGCRSGYIFYDNRGFCKYGSSSYKYDCGNYAGCCLPRNPYGRVKYYCTKKYSCPDDFYYYNNKGYYYYNDKDYFNCGSYNGCCLRSGY(SEQ ID NO.6);
mlfp-151的氨基酸序列如下:MAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKSSEEYKGGYYPGNTYHYHSGGSYHGSGYHGGYKGKYYGKAKKYYYKYKNSGKYKYLKKARKYHRKGYKKYYGGGSSAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYK(SEQ ID NO.7);
mlfp-131的氨基酸序列如下:MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPSSRSAAGTMAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKPWADYYGPKYGPPRRYGGGNYNRYGRRYGGYKGWNNGWKRGRWGRKYYGSAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKLGG(SEQ ID NO.8);
mlfp-153的氨基酸序列如下:MAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKEFSSEEYKGGYYPGNSNHYHSGGSYHGSGYHGGYKGKYYGKAKKYYYKYKNSGKYKYLKKARKYHRKGYKKYYGGGSSKLADYYGPKYGPPRRYGGGNYNRYGRRYGGYKGWNNGWKRGRWGRKYYLE(SEQ IDNO.9);
mlfp-351的氨基酸序列如下:MADYYGPKYGPPRRYGGGNYNRYGRRYGGYKGWNNGWKRGRWGRKYYLEFSSEEYKGGYYPGNSNHYHSGGSYHGSGYHGGYKGKYYGKAKKYYYKYKNSGKYKYLKKARKYHRKGYKKYYGGGSSKLAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKLE(SEQID NO.10)。
本发明还提供了一种促进创面愈合的组合物,它包括类贻贝粘蛋白和多肽或其复合物,所述多肽或其复合物为GHK、GHK-Cu、GQPR中的一种或多种,所述类贻贝粘蛋白和多肽或其复合物的重量比为(0.1~10):1。
进一步地,所述多肽或其复合物为GHK-Cu;所述类贻贝粘蛋白和GHK-Cu的重量比为(0.5~5.0):1,优选为(1.0~2.5):1,更优选为1.25:1;
和/或,所述类贻贝粘蛋白选自mlfp-1、mlfp-2、mlfp-3、mlfp-4、mlfp-5、mlfp-6、mlfp-151、mlfp-131、mlfp-153、mlfp-351中一种或多种;mlfp-1的氨基酸序列如SEQ IDNO.1所示,mlfp-2的氨基酸序列如SEQ ID NO.2所示,mlfp-3的氨基酸序列如SEQ ID NO.3所示,mlfp-4的氨基酸序列如SEQ ID NO.4所示,mlfp-5的氨基酸序列如SEQ ID NO.5所示,mlfp-6的氨基酸序列如SEQ ID NO.6所示,mlfp-151的氨基酸序列如SEQ ID NO.7所示,mlfp-131的氨基酸序列如SEQ ID NO.8所示,mlfp-153的氨基酸序列如SEQ ID NO.9所示,mlfp-351的氨基酸序列如SEQ ID NO.10所示。
进一步地,它是以类贻贝粘蛋白和多肽或其复合物为活性成分,加上药学领域或日化用品领域常用的辅料制得的制剂。
进一步地,所述药学领域或日化用品领域常用的辅料包括保湿剂、增稠剂、有机酸、溶剂中的一种或多种;
优选地,所述保湿剂为甘油、丙二醇中的一种或两种,所述增稠剂为甲基纤维素,所述有机酸为柠檬酸、乙酸、草酸、碳酸、磷酸中的一种或多种,所述溶剂为水。
进一步地,所述制剂为外用制剂,优选为凝胶剂。
进一步地,它由如下重量百分比的组分组成:类贻贝粘蛋白0.03%-0.15%,GHK-Cu 0.01%-0.1%,甘油2.5%-10%,甲基纤维素0.1%-2%,丙二醇0.5%-5%,有机酸0.1%-2%,山梨酸钾0.01%-0.5%,其余为水;
优选地,它由如下重量百分比的组分组成:类贻贝粘蛋白0.05%-0.1%,GHK-Cu0.02%-0.08%,甘油3%-5%,甲基纤维素0.5%-1.5%,丙二醇2%-4%,有机酸0.2%-0.6%,山梨酸钾0.1%-0.4%,其余为水;
更优选地,它由如下重量百分比的组分组成:类贻贝粘蛋白0.1%,GHK-Cu0.08%,甘油5%,甲基纤维素1.5%,丙二醇4%,有机酸0.6%,山梨酸钾0.4%,其余为水。
进一步地,所述组合物的制备方法包括以下步骤:
(1)将类贻贝粘蛋白溶解在有机酸中,得到A相;
(2)将GHK-Cu和水混合,得到B相;
(3)将丙二醇和水混合,得到C相;
(4)将山梨酸钾和水混合,得到D相;
(5)将甘油和水混合,充分搅拌均匀后加热至80-90℃,再加入甲基纤维素,室温条件下搅拌至溶液呈白色粘稠状,得到E相;
(6)混合A、B、C、D相,搅拌均匀,再加入E相,室温条件下搅拌30-60min,加入氢氧化钠调pH至6.0,即得。
本发明还提供了一种促进创面愈合的药物或日化用品,它是以上述组合物为原料制得的。
本发明还提供了上述组合物在制备促进创面愈合的药物或日化用品中的用途;
优选地,所述促进创面愈合的药物或日化用品能够抗炎,能够促进胶原蛋白生成。
本发明中,日化用品包括洗发水、沐浴露、护肤品、化妆品等。
GHK是一种含有三个氨基酸的活性多肽,其氨基酸序列是Gly-His-Lys。
GHK-Cu(Copper peptide,CAS号:89030-95-5)是GHK和铜离子形成的复合物,其中GHK:Cu=1:1(摩尔比)。
GQPR是一种由四个氨基酸组成的活性多肽,其氨基酸序列为Gly-Gln-Pro-Arg。它是免疫球蛋白IgG中的活性片段,能显著降低细胞中炎症因子IL-6的水平。有研究表明,GQPR可以延缓和抑制过量白介素的生成,从而抑制一些不必要的炎症反应和糖基化损伤。在照射紫外线后受损的细胞中,这种抑制白介素产生的效应更加明显,得益于其显著的抑制炎症因子的作用,GQPR能改善淋巴循环、促进胶原蛋白生成,阻止弹力蛋白流失,还能提高皮肤的紧致和弹力,减轻皱纹的加深,使皮肤恢复活力状态。
类贻贝粘蛋白是指人工制备的且具有贻贝粘蛋白氨基酸序列同源性的蛋白质。
本发明提供的类贻贝粘蛋白复合凝胶具有良好的抗炎功效和促进胶原蛋白生成的作用,能够有效地促进创面的愈合,明显缩短创面愈合的时间。
细胞划痕实验结果表明,本发明同时含GHK-Cu和类贻贝粘蛋白的复合凝胶的促细胞迁移率高于相同剂量下只含GHK-Cu的凝胶与只含类贻贝粘蛋白的凝胶的促细胞迁移率之和。说明本发明的类贻贝粘蛋白复合凝胶中,类贻贝粘蛋白和GHK-Cu发挥了协同增效的作用,共同促进创面愈合,显著缩短创面愈合时间。
促胶原生成实验结果表明,本发明的类贻贝粘蛋白复合凝胶中,类贻贝粘蛋白和GHK-Cu联合使用对促进细胞分泌胶原蛋白发挥了协同增效的作用,说明本发明提供的类贻贝粘蛋白复合凝胶具有明显提高的促进胶原蛋白生成作用,能有效地促进创面的愈合。
检测细胞炎症因子含量的实验结果表明,本发明的类贻贝粘蛋白复合凝胶中类贻贝粘蛋白和GHK-Cu联合使用对抑制细胞的炎症反应发挥了协同增效的作用,细胞炎症因子IL-1、IL-6和TNF-α含量显著降低,说明本发明提供的类贻贝粘蛋白复合凝胶具有优良的抗炎功能,有利于创面的愈合。
本发明提供的类贻贝粘蛋白复合凝胶安全、无刺激性,适合用来制备促进创面愈合的医疗用品。
与现有技术相比,本发明的有益效果为:
1.本发明制备得到的类贻贝粘蛋白复合凝胶中,类贻贝粘蛋白和GHK-Cu两者协同作用,促进创面愈合效果优于相同剂量的类贻贝粘蛋白和GHK-Cu单独使用下的凝胶;本发明制备得到的类贻贝粘蛋白复合凝胶在较低的使用浓度下就可以达到优异的促进创面愈合效果;
2.本发明将类贻贝粘蛋白、GHK-Cu和多种添加剂混合在一起制备而成的凝胶具有促进胶原蛋白生成、促进细胞增殖和抗炎等多重功效,可以加速创面的愈合,同时还兼具免疫原性低、吸收性好、安全无毒等优点;
3.本发明提供的类贻贝粘蛋白复合凝胶的制备方法简单便捷,可操作性强,有利于工业化生产。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为实施例1类贻贝粘蛋白复合凝胶的细胞划痕实验结果图。
图2为实施例2类贻贝粘蛋白复合凝胶的细胞划痕实验结果图。
图3为实施例3类贻贝粘蛋白复合凝胶的细胞划痕实验结果图。
图4为实施例4类贻贝粘蛋白复合凝胶的细胞划痕实验结果图。
图5为对比例1类贻贝粘蛋白凝胶的细胞划痕实验结果图。
图6为对比例2三肽GHK-Cu凝胶的细胞划痕实验结果图。
图7为对比例3水凝胶的细胞划痕实验结果图。
图8为MTT毒性实验结果。
图9为IL-β、IL-6和TNF-α含量测试结果。
具体实施方式
本发明所用原料与设备均为已知产品,通过购买市售产品所得。
其中,实施例1~实施例5采用的类贻贝粘蛋白为mlfp-151。GHK-Cu(Copperpeptide,CAS号:89030-95-5)是GHK和铜离子形成的复合物,其中GHK:Cu=1:1(摩尔比),为市售产品。
实施例1类贻贝粘蛋白复合凝胶的配方及制备方法
类贻贝粘蛋白复合凝胶的原料重量百分比为:类贻贝粘蛋白0.1%,GHK-Cu0.08%,甘油5%,甲基纤维素1.5%,丙二醇4%,柠檬酸0.6%,山梨酸钾0.4%,其余为去离子水。
制备方法:
(1)将类贻贝粘蛋白溶解在3%的柠檬酸溶液中,得到类贻贝粘蛋白浓度为0.5%的溶液,记为A相;
(2)将三肽GHK-Cu加入到去离子水中,得到GHK-Cu浓度为0.4%的溶液,记为B相;
(3)将丙二醇和去离子水混合,搅拌均匀,得到丙二醇浓度为20%的溶液,记为C相;
(4)将山梨酸钾溶解于去离子水中,得到山梨酸钾浓度为2%的溶液,记为D相;
(5)将甘油和去离子水混合,充分搅拌均匀后加热至80-90℃,再缓慢加入甲基纤维素,室温条件下缓慢搅拌至溶液呈白色粘稠状,得到甘油浓度25%,甲基纤维素7.5%的溶液,记为E相;
(6)按照1:1:1:1的重量比预先混合A、B、C、D相,搅拌均匀,再按4:1的重量比加入冷却至室温的E相,室温条件下缓慢搅拌30-60min,加入氢氧化钠调pH至6.0,得到类贻贝粘蛋白复合凝胶。
实施例2类贻贝粘蛋白复合凝胶的配方及制备方法
类贻贝粘蛋白复合凝胶的原料重量百分比为:类贻贝粘蛋白0.08%,GHK-Cu0.05%,甘油4%,甲基纤维素1%,丙二醇3%,柠檬酸0.4%,山梨酸钾0.2%,其余为去离子水。
制备方法:
(1)将类贻贝粘蛋白溶解在2%的柠檬酸溶液中,得到类贻贝粘蛋白浓度为0.4%的溶液,记为A相;
(2)将三肽GHK-Cu加入到去离子水中,得到GHK-Cu浓度为0.25%的溶液,记为B相;
(3)将丙二醇和去离子水混合,搅拌均匀,得到丙二醇浓度为15%的溶液,记为C相;
(4)将山梨酸钾溶解于去离子水水中,得到山梨酸钾浓度为1%的溶液,记为D相;
(5)将甘油和去离子水混合,充分搅拌均匀后加热至80-90℃,再缓慢加入甲基纤维素,室温条件下缓慢搅拌至溶液呈白色粘稠状,得到甘油浓度20%,甲基纤维素5%的溶液,记为E相;
(6)按照1:1:1:1的重量比预先混合A、B、C、D相,搅拌均匀,再按4:1的重量比加入冷却至室温的E相,室温条件下缓慢搅拌30-60min,加入氢氧化钠调pH至6.0,得到类贻贝粘蛋白复合凝胶。
实施例3类贻贝粘蛋白复合凝胶的配方及制备方法
类贻贝粘蛋白复合凝胶的原料重量百分比为:类贻贝粘蛋白0.05%,GHK-Cu0.02%,甘油3%,甲基纤维素0.5%,丙二醇2%,柠檬酸0.2%,山梨酸钾0.1%,其余为去离子水。
制备方法:
(1)将类贻贝粘蛋白溶解在1%的柠檬酸溶液中,得到类贻贝粘蛋白浓度为0.25%的溶液,记为A相;
(2)将GHK-Cu加入到去离子水中,得到GHK-Cu浓度为0.1%的溶液,记为B相;
(3)将丙二醇和去离子水混合,搅拌均匀,得到丙二醇浓度为10%的溶液,记为C相;
(4)将山梨酸钾溶解于去离子水中,得到山梨酸钾浓度为0.5%的溶液,记为D相;
(5)将甘油和去离子水混合,充分搅拌均匀后加热至80-90℃,再缓慢加入甲基纤维素,室温条件下缓慢搅拌至溶液呈白色粘稠状,得到甘油浓度15%,甲基纤维素2.5%的溶液,记为E相;
(6)按照1:1:1:1的重量比预先混合A、B、C、D相,搅拌均匀,再按4:1的重量比加入冷却至室温的E相,室温条件下缓慢搅拌30-60min,加入氢氧化钠调pH至6.0,得到类贻贝粘蛋白复合凝胶。
实施例4类贻贝粘蛋白复合凝胶的配方及制备方法
与实施例1相比,本实施例的区别仅在于:用乙酸代替柠檬酸。参照实施例1的制备方法,制得本实施例的类贻贝粘蛋白复合凝胶。
实施例5促进创面愈合的组合物
配方(重量份数):10份类贻贝粘蛋白,8份GHK-Cu。
制备方法:将配方中的类贻贝粘蛋白和GHK-Cu混合均匀,即得促进创面愈合的组合物。
以下为对照样品的制备。
对比例1类贻贝粘蛋白凝胶的制备方法
与实施例1相比,本对比例的区别仅在于:B相不加入GHK-Cu,用等量的去离子水代替。参照实施例1的制备方法,制得本对比例的类贻贝粘蛋白凝胶。
对比例2三肽GHK-Cu凝胶的制备方法
与实施例1相比,本对比例的区别仅在于:A相不加入类贻贝粘蛋白,用等量的去离子水代替。参照实施例1的制备方法,制得本对比例的三肽GHK-Cu凝胶。
对比例3水凝胶的制备方法
与实施例1相比,本对比例的区别仅在于:A相不加入类贻贝粘蛋白,用等量的去离子水代替;B相不加入GHK-Cu,用等量的去离子水代替。参照实施例1的制备方法,制得本对比例的水凝胶。
以下通过实验例证明本发明的有益效果。
实验例1细胞划痕实验
1.实验方法
(1)第一天,加入胰蛋白酶,消化预培养的小鼠成纤维细胞NIH/3T3,细胞悬液在1000rpm离心3min。用DMEM培养基重悬细胞,调整密度为1×105个细胞/ml,用移液器在6孔板的每个孔中加入1ml密度为1×105个细胞/ml的细胞悬液。
(2)细胞孵育24h(5%CO2,37℃)以形成半汇合单层,在此孵育期间确保细胞恢复、贴附至指数生长,划痕前在6孔板底部用黑色记号笔画平行线;
(3)第二天,用1ml枪头垂直于预先画好的平行线划线(2-3道划痕),孵育24h后吸出培养基,更换1ml无血清DMEM培养基;
(4)每孔分别加入1ml含实施例1-4和对比例1-3的凝胶样品或空白对照的处理培养基(凝胶样品:DMEM培养基=1:19),采用无血清培养基作为空白对照;
(5)将6孔板放置培养箱中,5%CO2,37℃条件下继续培养24h,记录不同样品处理后的细胞增殖情况,测量划痕间距。细胞迁移速率=(初始划痕长度-t小时后划痕长度)/t,t为样品处理后的细胞培养时间。
(6)根据细胞迁移速率,利用以下公式计算各凝胶样品的促细胞迁移率:
促细胞迁移率=(待测样品组细胞迁移速率-对比例3样品组细胞迁移速率)/对比例3样品组细胞迁移速率。
2.实验结果
结果如表1和图1~图7所示,与不含类贻贝粘蛋白和GHK-Cu的对比例3水凝胶相比,实施例1-4的类贻贝粘蛋白复合凝胶、对比例1的类贻贝粘蛋白凝胶和对比例2的GHK-Cu凝胶均对细胞增殖和迁移表现出一定的促进作用。具体表现为:使用实施例1的类贻贝粘蛋白复合凝胶的细胞迁移速率为40.48μm/h,明显快于对比例1的类贻贝粘蛋白凝胶的22.85μm/h和对比例2的GHK-Cu凝胶的23.61μm/h;同时,实施例2-4的类贻贝粘蛋白复合凝胶的细胞迁移速率分别为24.21μm/h、16.33μm/h和27.24μm/h,也明显快于对比例3的水凝胶的9.58μm/h。表明类贻贝粘蛋白和GHK-Cu协同使用对细胞增殖和迁移的效果显著优于类贻贝粘蛋白和GHK-Cu单组分使用。
另外,实施例1的类贻贝粘蛋白复合凝胶的促细胞迁移率为322.55%,对比例1的类贻贝粘蛋白凝胶的促细胞迁移率为138.52%,对比例2的GHK-Cu凝胶的促细胞迁移率为146.45%。也就是说,本发明同时含GHK-Cu和类贻贝粘蛋白的复合凝剂的促细胞迁移率高于只含相同剂量GHK-Cu的凝胶的促细胞迁移率与只含相同剂量类贻贝粘蛋白的凝胶的促细胞迁移率之和,本发明实施例1的类贻贝粘蛋白复合凝胶对促进细胞迁移发挥了协同增效的作用,能够显著提高促进伤口愈合的效果。
表1各样品类贻贝粘蛋白、GHK-Cu工作浓度、细胞迁移速率及促细胞迁移率
实验例2 MTT细胞毒性测定
1.实验方法
(1)第一天,加入胰蛋白酶,消化预培养的小鼠成纤维细胞NIH/3T3,细胞悬液在1000rpm离心3min。用DMEM培养基重悬细胞,调整密度为1×105个细胞/ml,用移液器将100μl培养基加到96孔板的***孔中,在其余孔中加入100μl密度为1×105个细胞/ml的细胞悬液;
(2)细胞孵育24h(5%CO2,37℃)以形成半汇合单层,在此孵育期间确保细胞恢复、贴附至指数生长;
(3)第二天,孵育24h后吸出培养基,每孔分别加入100μl含实施例1-4和对比例1-3的凝胶样品或空白对照的处理培养基(凝胶样品:DMEM培养基=1:19),采用无血清培养基作为空白对照;
(4)细胞孵育24h(5%CO2,37℃);
(5)孵育结束后,小心吸取培养基弃掉,每孔加MTT(1mg/mL)50μl,继续孵育2h;
(6)然后弃去MTT溶液,每孔中加入异丙醇100μl;
(7)培养板置于摇床上摇动,直至紫色晶体完全溶解,约30min;
(8)将板放入BIOTEK全波长酶标仪,设置检测波长570nm,参比波长650nm,进行吸光值测定并根据细胞存活率(%)=检测孔OD570*100%/空白对照孔OD570,计算细胞存活率。
2.实验结果
结果如图8所示,与不含类贻贝粘蛋白和GHK-Cu的对比例3水凝胶相比,实施例1-4的类贻贝粘蛋白复合凝胶、对比例1的类贻贝粘蛋白凝胶和对比例2的GHK-Cu凝胶均对细胞无毒性。具体表现为:实施例1的类贻贝粘蛋白复合凝胶的细胞存活率高达131.26%,明显高于对比例1的类贻贝粘蛋白凝胶的115.12%和对比例2的GHK-Cu凝胶的112.02%;同时,实施例2-4的类贻贝粘蛋白复合凝胶的细胞存活率分别为120.12%、110.84%和120.65%,明显高于对比例3的水凝胶。
实验例3促胶原功能测定
羟脯氨酸是亚氨基酸之一,是胶原组织的主要成分之一,并且是胶原中特有的氨基酸,约占胶原氨基酸总量的13%。利用羟脯氨酸在胶原蛋白中含量最高这一特点,通过血液、尿液和组织的羟脯氨酸含量测定成为衡量机体胶原组织代谢的重要指标。
1.实验方法
(1)第一天,采用酶促消化(胰蛋白酶/EDTA)将NIH/3T3细胞从培养瓶中移除,细胞悬液在1000rpm离心3min。用DMEM培养基重悬细胞,调整密度为1×105个细胞/ml,用移液器在24孔板每个孔中加入500μl密度为1×105个细胞/ml的细胞悬液(约5×104个细胞/孔);
(2)细胞孵育24h(5%CO2,37℃)以形成半汇合单层,在此孵育期间确保细胞恢复、贴附至指数生长;
(3)第二天,每孔分别加入500μl含实施例1-4和对比例1-3的凝胶样品或空白对照的处理培养基(凝胶样品:DMEM培养基=1:19),采用无血清培养基作为空白对照;
(4)加步骤(3)制得的混合物继续孵育48h后,吸取培养基上清,用于测定羟脯氨酸含量;
(5)使用南京建成生物工程研究所生产的羟脯氨酸测定试剂盒,按照说明书的步骤,进行羟脯氨酸含量测定。
其中,标准品浓度为5μg/mL。
(6)根据羟脯氨酸含量,利用以下公式计算各凝胶样品对胶原蛋白的促分泌作用:
对胶原蛋白的促分泌作用=(待测样品组羟脯氨酸含量-对比例3样品组羟脯氨酸含量)/对比例3样品组羟脯氨酸含量。
2.实验结果
结果如表2所示,与不含类贻贝粘蛋白和GHK-Cu的对比例3水凝胶相比,实施例1-4的类贻贝粘蛋白复合凝胶和对比例2的GHK-Cu凝胶均能明显刺激细胞分泌胶原蛋白。具体表现为:实施例2-4的类贻贝粘蛋白复合凝胶的促分泌作用分别为139.51%、92.93%和141.50%。更重要的是,实施例1的类贻贝粘蛋白复合凝胶对胶原蛋白的促分泌作用高达173.95%,明显高于对比例1的类贻贝粘蛋白凝胶的32.89%和对比例2的GHK-Cu凝胶的104.19%之和,说明本发明实施例1的类贻贝粘蛋白复合凝胶中,类贻贝粘蛋白和GHK-Cu联合使用对促进细胞分泌胶原蛋白发挥了协同增效的作用。
表2各样品促胶原功能测试结果
实验例4细胞炎症因子IL-1β、IL-6和TNF-α含量测定
IL-1是由巨噬细胞所分泌的多肽类细胞因子,与细胞表面的受体结合后,能趋化角质细胞、中性粒细胞和淋巴细胞,并能刺激成纤维细胞合成胶原。IL-6可以促进中性粒细胞分化、活化,促进角质细胞、肾小球细胞生长。TNF对人体皮肤成纤维细胞的增殖有明显的促进作用,并促进Ⅰ、Ⅲ型胶原和聚胺多糖的生成。
1.实验方法
(1)分Raw 264.7细胞至96孔细胞板,2×104cells/well。使用含10%FBS(胎牛血清),100U/ml青霉素,100μg/ml链霉素的DMEM培养基;
(2)将实施例1-4和对比例1-3中得到的凝胶加入到培养基(含10%FBS,100U/ml青霉素,100μg/ml链霉素的DMEM)中,混匀后加至各细胞孔中,每孔1mL,3个复孔;
(3)加步骤(2)制得的混合物2小时后,加入LPS(脂多糖,1μg/ml)继续孵育48h,吸取培养基上清,用于测定细胞因子IL-1β、IL-6和TNF-α含量;
(4)使用欣博盛生物科技有限公司生产的小鼠IL-1β、IL-6和TNF-αELISA试剂盒,按照说明书的步骤,进行IL-1β、IL-6和TNF-α含量测定。
(5)利用以下公式计算各样品对IL-1β、IL-6和TNF-α的抑制率:
对IL-1β的抑制率=(对比例3样品组IL-1β含量-待测样品组IL-1β含量)/对比例3样品组IL-1β含量;
对IL-6的抑制率=(对比例3样品组IL-6含量-待测样品组IL-6含量)/对比例3样品组IL-6含量;
对TNF-α的抑制率=(对比例3样品组TNF-α含量-待测样品组TNF-α含量)/对比例3样品组TNF-α含量。
2.实验结果
结果如图9和表3所示,与不含类贻贝粘蛋白和GHK-Cu的对比例3水凝胶相比,实施例1-4的类贻贝粘蛋白复合凝胶和对比例2的GHK-Cu凝胶均能有效抑制炎症反应。具体表现为:实施例2-4的炎症因子IL-1β、IL-6和TNF-α的含量明显低于对比例3的水凝胶。同时,使用实施例1的类贻贝粘蛋白复合凝胶的细胞中炎症因子IL-1β、IL-6和TNF-α的含量分别为421pg/ml、4712pg/ml和893pg/ml,明显低于对比例1的类贻贝粘蛋白凝胶和对比例2的GHK-Cu凝胶。
实施例1的类贻贝粘蛋白复合凝胶对IL-1β、IL-6和TNF-α的抑制率分别为43.94%、36.84%和36.21%,对比例1的类贻贝粘蛋白凝胶对IL-1β、IL-6和TNF-α的抑制率分别为7.19%、4.4%和7.29%,对比例2的GHK-Cu凝胶对IL-1β、IL-6和TNF-α的抑制率分别为29.03%、26.72%和26.07%。可以看出,本发明实施例1的类贻贝粘蛋白复合凝胶对IL-1β、IL-6、TNF-α的抑制率均明显高于对比例1和对比例2对IL-1β、IL-6、TNF-α的抑制率之和,表明本发明实施例1的类贻贝粘蛋白复合凝胶中类贻贝粘蛋白和GHK-Cu联合使用对抑制细胞的炎症反应发挥了协同增效的作用。
表3各样品IL-β、IL-6和TNF-α含量测试结果
实验例5大鼠创面愈合实验
1.实验方法
(1)试验前,将大鼠背部脊柱两侧的毛剪掉,面积约5cm×5cm,选择背部脊柱两侧各旁开1cm,距肩胛骨以下2cm处制造创面;
(2)在创面部位用8mm环钻制造伤口,然后用手术剪在注明部位将皮肤全层剪除,制成圆形创面;
(3)将上述创伤大鼠随机分成7组,每组5只,分别将实施例1-4和对比例1-3的凝胶敷在创面上。每天观察每组大鼠的创面愈合效果,计算创面愈合率和记录愈合时间。创面愈合率=(初始创面面积-未愈合创面面积)/初始创面面积×100%。
2.实验结果
结果如表4所示,与不含类贻贝粘蛋白和GHK-Cu的对比例3水凝胶相比,实施例1-4的类贻贝粘蛋白复合凝胶、对比例1的类贻贝粘蛋白凝胶和对比例2的GHK-Cu凝胶均对创面愈合有一定的促进作用。具体表现为:实施例1、4的愈合时间为5天,实施例2和3的愈合时间分别为6天和8天,均明显低于对比例3的愈合时间。而且,实施例1-4组均未出现瘙痒、粘连等现象,创面愈合情况良好且无疤痕残留。对比例1组出现了轻微的炎症反应,创面有些微红肿的现象,完全愈合的时间为7天。对比例2组未出现炎症反应,创面也无红肿粘连的现象,完全愈合时间为6天。对比例3组出现了明显的炎症反应,创面也有红肿瘙痒的现象,直至10天创面才完全愈合。
表4大鼠创面愈合情况
综上,本发明提供了一种促进创面愈合的组合物及其制备方法与用途。该组合物中的类贻贝粘蛋白和GHK-Cu通过协同增效的作用,使组合物具有良好的抗炎功效和促进胶原蛋白生成的作用,能够有效地促进创面的愈合,明显缩短创面愈合的时间;该组合物同时还兼具免疫原性低、吸收性好、稳定性强、安全无毒等优点,在生物医药领域具有良好的应用前景。
SEQUENCE LISTING
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Thr Ser Phe Arg Ser Asp Leu Ser Asn Asp Leu His Pro Asp Asn Asn
485 490 495
Ile Glu Gln Ile Ala Asn Asp His Val Asn Asp Ile Ala Gln Ser Thr
500 505 510
Asp Gly Asp Ile Asn Asp Phe Ala Asp Thr His Tyr Asn Asp Val Ala
515 520 525
Pro Ile Ala Asp Val His Val Asp Asn Ile Ala Gln Thr Ala Asp Asn
530 535 540
His Val Lys Asn Ile Ala Gln Thr Ala His His His Val Asn Asp Val
545 550 555 560
Ala Gln Ile Ala Asp Asp His Val Asn Asp Ile Gly Gln Thr Ala Tyr
565 570 575
Asp His Val Asn Asn Ile Gly Gln Thr Ala Asp Asp His Val Asn Asp
580 585 590
Ile Ala Gln Thr Ala Asp Asp His Val Asn Ala Ile Ala Gln Thr Ala
595 600 605
Asp Asp His Val Asn Ala Ile Ala Gln Thr Ala Asp Asp His Val Asn
610 615 620
Asp Ile Gly Asp Thr Ala Asn Ser His Ile Val Arg Val Gln Gly Val
625 630 635 640
Ala Lys Asn His Leu Tyr Gly Ile Asn Lys Ala Ile Gly Lys His Ile
645 650 655
Gln His Leu Lys Asp Val Ser Asn Arg His Ile Glu Lys Leu Asn Asn
660 665 670
His Ala Thr Lys Asn Leu Leu Gln Ser Ala Leu Gln His Lys Gln Gln
675 680 685
Thr Ile Glu Arg Glu Ile Gln His Lys Arg His Leu Ser Glu Lys Glu
690 695 700
Asp Ile Asn Leu Gln His Glu Asn Ala Met Lys Ser Lys Val Ser Tyr
705 710 715 720
Asp Gly Pro Val Phe Asn Glu Lys Val Ser Val Val Ser Asn Gln Gly
725 730 735
Ser Tyr Asn Glu Lys Val Pro Val Leu Ser Asn Gly Gly Gly Tyr Asn
740 745 750
Gly Lys Val Ser Ala Leu Ser Asp Gln Gly Ser Tyr Asn Glu Gly Tyr
755 760 765
Ala Tyr
770
<210> 5
<211> 76
<212> PRT
<213> 人工序列
<400> 5
Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His
1 5 10 15
Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr
20 25 30
Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys
35 40 45
Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg
50 55 60
Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser
65 70 75
<210> 6
<211> 121
<212> PRT
<213> 人工序列
<400> 6
Met Lys Ser Ile Gln Met Ile Leu Ala Val Phe Val Ile Thr Ala Leu
1 5 10 15
Cys Gly Ile Val Glu Ser Gly Gly Gly Asn Tyr Arg Gly Tyr Cys Ser
20 25 30
Asn Lys Gly Cys Arg Ser Gly Tyr Ile Phe Tyr Asp Asn Arg Gly Phe
35 40 45
Cys Lys Tyr Gly Ser Ser Ser Tyr Lys Tyr Asp Cys Gly Asn Tyr Ala
50 55 60
Gly Cys Cys Leu Pro Arg Asn Pro Tyr Gly Arg Val Lys Tyr Tyr Cys
65 70 75 80
Thr Lys Lys Tyr Ser Cys Pro Asp Asp Phe Tyr Tyr Tyr Asn Asn Lys
85 90 95
Gly Tyr Tyr Tyr Tyr Asn Asp Lys Asp Tyr Phe Asn Cys Gly Ser Tyr
100 105 110
Asn Gly Cys Cys Leu Arg Ser Gly Tyr
115 120
<210> 7
<211> 197
<212> PRT
<213> 人工序列
<400> 7
Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr
1 5 10 15
Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala
20 25 30
Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro
35 40 45
Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu
50 55 60
Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser
65 70 75 80
Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys
85 90 95
Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly
100 105 110
Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr
115 120 125
Lys Lys Tyr Tyr Gly Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro
130 135 140
Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro
145 150 155 160
Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr
165 170 175
Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr
180 185 190
Pro Pro Thr Tyr Lys
195
<210> 8
<211> 215
<212> PRT
<213> 人工序列
<400> 8
Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr
1 5 10 15
Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp
20 25 30
Pro Ser Ser Arg Ser Ala Ala Gly Thr Met Ala Lys Pro Ser Tyr Pro
35 40 45
Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys
50 55 60
Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr
65 70 75 80
Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser
85 90 95
Tyr Pro Pro Thr Tyr Lys Pro Trp Ala Asp Tyr Tyr Gly Pro Lys Tyr
100 105 110
Gly Pro Pro Arg Arg Tyr Gly Gly Gly Asn Tyr Asn Arg Tyr Gly Arg
115 120 125
Arg Tyr Gly Gly Tyr Lys Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg
130 135 140
Trp Gly Arg Lys Tyr Tyr Gly Ser Ala Lys Pro Ser Tyr Pro Pro Thr
145 150 155 160
Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser
165 170 175
Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys
180 185 190
Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro
195 200 205
Pro Thr Tyr Lys Leu Gly Gly
210 215
<210> 9
<211> 189
<212> PRT
<213> 人工序列
<400> 9
Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr
1 5 10 15
Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala
20 25 30
Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro
35 40 45
Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Glu Phe Ser
50 55 60
Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ser Asn His Tyr
65 70 75 80
His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys
85 90 95
Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn
100 105 110
Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys
115 120 125
Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser Lys Leu Ala Asp Tyr
130 135 140
Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly Asn Tyr
145 150 155 160
Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn Asn Gly
165 170 175
Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Leu Glu
180 185
<210> 10
<211> 190
<212> PRT
<213> 人工序列
<400> 10
Met Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly
1 5 10 15
Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly
20 25 30
Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Leu
35 40 45
Glu Phe Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ser
50 55 60
Asn His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly
65 70 75 80
Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys
85 90 95
Tyr Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr
100 105 110
His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser Lys Leu
115 120 125
Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro
130 135 140
Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys
145 150 155 160
Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr
165 170 175
Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Leu Glu
180 185 190
Claims (10)
1.类贻贝粘蛋白和多肽或其复合物在制备具有抗氧化和/或抗炎作用的联合用制剂中的用途,所述多肽或其复合物为GHK、GHK-Cu、GQPR中的一种或多种。
2.根据权利要求1所述的用途,其特征在于:所述多肽或其复合物为GHK-Cu;所述类贻贝粘蛋白和GHK-Cu的重量比为(0.5~5.0):1,优选为(1.0~2.5):1,更优选为1.25:1;
和/或,所述类贻贝粘蛋白选自mlfp-1、mlfp-2、mlfp-3、mlfp-4、mlfp-5、mlfp-6、mlfp-151、mlfp-131、mlfp-153、mlfp-351中一种或多种;mlfp-1的氨基酸序列如SEQ ID NO.1所示,mlfp-2的氨基酸序列如SEQ ID NO.2所示,mlfp-3的氨基酸序列如SEQ ID NO.3所示,mlfp-4的氨基酸序列如SEQ ID NO.4所示,mlfp-5的氨基酸序列如SEQ ID NO.5所示,mlfp-6的氨基酸序列如SEQ ID NO.6所示,mlfp-151的氨基酸序列如SEQ ID NO.7所示,mlfp-131的氨基酸序列如SEQ ID NO.8所示,mlfp-153的氨基酸序列如SEQ ID NO.9所示,mlfp-351的氨基酸序列如SEQ ID NO.10所示。
3.一种促进创面愈合的组合物,其特征在于:它包括类贻贝粘蛋白和多肽或其复合物,所述多肽或其复合物为GHK、GHK-Cu、GQPR中的一种或多种,所述类贻贝粘蛋白和多肽或其复合物的重量比为(0.1~10):1。
4.根据权利要求3所述的组合物,其特征在于:所述多肽或其复合物为GHK-Cu;所述类贻贝粘蛋白和GHK-Cu的重量比为(0.5~5.0):1,优选为(1.0~2.5):1,更优选为1.25:1;
和/或,所述类贻贝粘蛋白选自mlfp-1、mlfp-2、mlfp-3、mlfp-4、mlfp-5、mlfp-6、mlfp-151、mlfp-131、mlfp-153、mlfp-351中一种或多种;mlfp-1的氨基酸序列如SEQ ID NO.1所示,mlfp-2的氨基酸序列如SEQ ID NO.2所示,mlfp-3的氨基酸序列如SEQ ID NO.3所示,mlfp-4的氨基酸序列如SEQ ID NO.4所示,mlfp-5的氨基酸序列如SEQ ID NO.5所示,mlfp-6的氨基酸序列如SEQ ID NO.6所示,mlfp-151的氨基酸序列如SEQ ID NO.7所示,mlfp-131的氨基酸序列如SEQ ID NO.8所示,mlfp-153的氨基酸序列如SEQ ID NO.9所示,mlfp-351的氨基酸序列如SEQ ID NO.10所示。
5.根据权利要求3所述的组合物,其特征在于:它是以类贻贝粘蛋白和多肽或其复合物为活性成分,加上药学领域或日化用品领域常用的辅料制得的制剂。
6.根据权利要求5所述的组合物,其特征在于:所述药学领域或日化用品领域常用的辅料包括保湿剂、增稠剂、有机酸、溶剂中的一种或多种;
优选地,所述保湿剂为甘油、丙二醇中的一种或两种,所述增稠剂为甲基纤维素,所述有机酸为柠檬酸、乙酸、草酸、碳酸、磷酸中的一种或多种,所述溶剂为水。
7.根据权利要求6所述的组合物,其特征在于:所述制剂为外用制剂,优选为凝胶剂。
8.根据权利要求7所述的组合物,其特征在于:它由如下重量百分比的组分组成:类贻贝粘蛋白0.03%-0.15%,GHK-Cu 0.01%-0.1%,甘油2.5%-10%,甲基纤维素0.1%-2%,丙二醇0.5%-5%,有机酸0.1%-2%,山梨酸钾0.01%-0.5%,其余为水;
优选地,它由如下重量百分比的组分组成:类贻贝粘蛋白0.05%-0.1%,GHK-Cu0.02%-0.08%,甘油3%-5%,甲基纤维素0.5%-1.5%,丙二醇2%-4%,有机酸0.2%-0.6%,山梨酸钾0.1%-0.4%,其余为水;
更优选地,它由如下重量百分比的组分组成:类贻贝粘蛋白0.1%,GHK-Cu 0.08%,甘油5%,甲基纤维素1.5%,丙二醇4%,有机酸0.6%,山梨酸钾0.4%,其余为水。
9.一种促进创面愈合的药物或日化用品,其特征在于:它是以权利要求3~8任一项所述组合物为原料制得的。
10.权利要求3~8任一项所述组合物在制备促进创面愈合的药物或日化用品中的用途;
优选地,所述促进创面愈合的药物或日化用品能够抗炎,能够促进胶原蛋白生成。
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CN114853863B (zh) * | 2022-04-28 | 2023-11-21 | 西安德诺海思医疗科技有限公司 | 一种重组类贻贝粘蛋白纯化方法 |
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