CN117064787B - 一种用于受损皮肤屏障的重组ⅲ型人源化胶原蛋白贴敷料面膜及其制备方法 - Google Patents
一种用于受损皮肤屏障的重组ⅲ型人源化胶原蛋白贴敷料面膜及其制备方法 Download PDFInfo
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- CN117064787B CN117064787B CN202311119152.9A CN202311119152A CN117064787B CN 117064787 B CN117064787 B CN 117064787B CN 202311119152 A CN202311119152 A CN 202311119152A CN 117064787 B CN117064787 B CN 117064787B
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Abstract
本发明提出了一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜及其制备方法,包括如下原料:重组Ⅲ型人源化胶原蛋白0.08~0.15%、修复剂0.1~0.3%、椰子胚愈伤组织水提物0.05~0.15%、助溶剂2~4%、增稠剂0.1~0.25%和余量的水;所述修复剂为酵母β‑葡聚糖和岩藻糖基乳糖;所述椰子胚愈伤组织水提物为以椰子成熟胚为外植体,诱导培养产生不定芽后,通过水提取愈伤组织而得。本发明提供的一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜,可抑制、减少炎症反应过程中的IL‑6因子和TRPV1瘙痒调控因子的释放,起到减缓、改善皮肤干燥症的作用,有助于增强抗皮肤干燥、瘙痒的免疫力,进一步提高皮肤屏障功能。
Description
技术领域
本发明涉及护肤品技术领域,特别涉及一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜及其制备方法。
背景技术
皮肤是人体面积最大的器官,是机体的第一道屏障。皮肤由表皮、真皮和皮下组织构成,而表皮屏障则是阻止外界侵害的有力保障。当皮肤的天然屏障损伤后,导致机体的抵御能力降低,易引起炎症反应如瘙痒、红肿、刺痛等现象。皮肤干燥症(skin xerosis)是继发于其他皮肤病的一种临床常见的皮肤疾病,通常表现为皮肤表皮层的干燥,皮肤粗糙并缺少光泽,是皮肤屏障受损的最初表现。皮肤干燥症主要是因为皮肤角质层的持水能力有所下降,导致经表皮水分流失增多,不同年龄群体均可发生。持续性的皮肤干燥引起瘙痒和表皮增厚等现象,通常治疗瘙痒药物首选抗组胺药物。但由皮肤干燥症引起的瘙痒则为一种较难治疗的瘙痒症状,采用抗组胺药物治疗这种类型的瘙痒症状,疗效并不明显,恢复时间较长,增加了患者的痛苦。
发明内容
鉴于此,本发明的目的在于提出一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜及其制备方法,重组III型人源化胶原蛋白贴敷料面膜主要由重组III型人源化胶原蛋白、修复剂(酵母β-葡聚糖和岩藻糖基乳糖)、椰子胚愈伤组织水提物、羟乙基纤维素、丙二醇、己二醇、纯化水组成。本产品主要用于抑制和缓解皮炎湿疹、特应性皮炎、激素依赖性皮炎、皮肤干燥症、敏感性肌肤、痤疮,激光治疗术后等各种原因引起皮肤炎症反应,促进创面愈合与皮肤修复,缩短病程,减轻炎症后色素沉着与瘢痕形成的风险,并对上述疾病具有一定辅助治疗。
本发明的技术方案是这样实现的:
本发明的目的之一在于提供一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜,按质量百分比计,包括如下原料:重组Ⅲ型人源化胶原蛋白0.08~0.15%、修复剂0.1~0.3%、椰子胚愈伤组织水提物0.05~0.15%、助溶剂2~4%、增稠剂0.1~0.25%和余量的水;
所述修复剂为酵母β-葡聚糖和岩藻糖基乳糖组成;
所述椰子胚愈伤组织水提物为以椰子成熟胚为外植体,诱导培养产生不定芽后,通过水提取愈伤组织而得,实验表明,复配物的保湿能力好,其中,三者复配物的保湿性能更优,选用重组Ⅲ型人源化胶原蛋白、修复剂(酵母β-葡聚糖和岩藻糖基乳糖)和椰子胚愈伤组织水提物复配,具有协同效应的机制,使三者成分复配后,提高了整体复配物体系对水分子的结合力,其吸湿性能更强,在相对湿度80%的条件下,放置6h后其保湿率在40%以上。
进一步的方案是,按百分比计,包括如下原料:重组Ⅲ型人源化胶原蛋白0.08~0.1%、修复剂0.1~0.3%、椰子胚愈伤组织水提物0.07~0.1%、助溶剂2~4%、增稠剂0.1~0.25%和余量的水。
更进一步的方案是,按百分比计,包括如下原料:重组Ⅲ型人源化胶原蛋白0.1%、修复剂0.2%、椰子胚愈伤组织水提物0.09%、助溶剂2.6%、增稠剂0.15%和余量的水。
进一步的方案是,所述修复剂为质量比1~2:1~2的酵母β-葡聚糖和岩藻糖基乳糖;
所述助溶剂为己二醇和/或丙二醇;所述增稠剂为羟乙基纤维素。
进一步的方案是,所述椰子胚愈伤组织水提物的制备方法,包括:取椰子成熟胚,接种至基础培养基,振荡培养7~10d后,转接至新的基础培养基,固态培养至不定芽产生,取愈伤组织,干燥后粉碎,加入水搅拌,水浴浸提,过滤,离心,浓缩,冷冻干燥,得椰子胚愈伤组织水提物。
更进一步的方案是,所述基础培养基的配方为:在Y3培养基的基础上,添加0.4~0.6mg/LNAA、0.20~0.30mg/L 6-BA、1.0~2.0mg/L 2,4-D和55.0~65.0g/L蔗糖。
更进一步的方案是,所述振荡培养的频率为50~75r/min;所述振荡培养和固态培养的条件为:温度28~30℃,光照强度2000~3000lx,每天光照时间10~12h。
进一步的方案是,所述愈伤组织和水的料液质量体积为1g:20~30mL;所述水浴浸提的温度为70~80℃,水浴浸提的时间为2~3h。
本发明的目的之二在于提供一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜的制备方法,包括如下步骤:
S1预分散:取重组Ⅲ型人源化胶原蛋白、修复剂和椰子胚愈伤组织水提物,加入配方水总量的4~8%的水,搅拌,得溶液A;
S2溶剂:取助溶剂和增稠剂,搅拌混合,得溶剂;
S3配制:将余下配方的水边加热边加入溶剂搅拌,降温后,加入溶液A,搅拌,得溶液B,制成面膜,得目标重组Ⅲ型人源化胶原蛋白贴敷料面膜。
进一步的方案是,搅拌的速率为35~45r/min,加热的温度为75~85℃,降温至温度为55~65℃。
与现有技术相比,本发明的有益效果为:
本发明采用重组Ⅲ型人源化胶原蛋白、修复剂(酵母β-葡聚糖和岩藻糖基乳糖)和椰子胚愈伤组织水提物为有效活性成分,制备得到的一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜,可抑制、减少炎症反应过程中的IL-6因子和TRPV1瘙痒调控因子的释放,起到减缓、改善皮肤干燥症的作用,有助于增强抗皮肤干燥、瘙痒的免疫力,进一步提高皮肤屏障功能。
具体实施方式
为了更好理解本发明技术内容,下面提供具体实施例,对本发明做进一步的说明。
本发明实施例所用的实验方法如无特殊说明,均为常规方法。
本发明实施例所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
重组Ⅲ型人源化胶原蛋白购买于西安德诺海思医疗科技有限公司。
实施例1
一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜,配方如下:重组Ⅲ型人源化胶原蛋白0.1%、修复剂0.2%、椰子胚愈伤组织水提物0.09%、助溶剂2.6%、羟乙基纤维素0.15%和余量的水;
其中,修复剂为质量比1:1的酵母β-葡聚糖和岩藻糖基乳糖,助溶剂为质量比0.6:2的己二醇和丙二醇;
椰子胚愈伤组织水提物的制备方法为:取8~10个月大的红椰子种子,去壳,取出胚乳,清洗去油脂,在体积浓度75%的乙醇溶液中浸泡1min,无菌水冲洗3次,再转入质量浓度6%的次氯酸钠溶液中浸泡5min,无菌水冲洗3次,在无菌条件下取出完整的成熟胚作为外植体,接种至配方为Y3+0.5mg/L NAA+0.25mg/L 6-BA+1.5mg/L 2,4-D+60.0g/L蔗糖的培养基,以60r/min的振荡速率,28~30℃,每天光照10h,光照强度为2000~3000lx进行培养,振荡培养8d后,转接至新的配方为Y3+0.5mg/L NAA+0.25mg/L 6-BA+1.5mg/L 2,4-D+60.0g/L蔗糖+6.0g/L琼脂的培养基,以28~30℃,每天光照10h,光照强度为3000lx进行培养,得到不定芽;
取诱导不定芽的整个愈伤组织,冷冻干燥,粉碎,按料液质量体积比1g:25mL,加入水搅拌,以70~80℃水浴浸提2~3h,过滤,离心,浓缩,冷冻干燥,得椰子胚愈伤组织水提物。
上述面膜的制备方法如下:
S1预分散:取重组Ⅲ型人源化胶原蛋白、修复剂和椰子胚愈伤组织水提物,加入配制总量5%的纯化水,搅拌,使重组Ⅲ型人源化胶原蛋白、修复剂和椰子胚愈伤组织水提物溶解完全,目视为无色透明溶液,无可见异物,得溶液A;
S2溶剂:取助溶剂和羟乙基纤维素,搅拌使羟乙基纤维素分散均匀,目视为透明或半透明,无色或微黄色混悬液,无可见异物,得溶剂;
S3配制:将余下配方的纯化水,加入配液罐中,开启搅拌(40r/min±5r/min),开启加热(80℃±5℃),加入溶剂,保温搅拌20min,开启回流降温,温度降至60℃±5℃时,加入溶液A,搅拌,得无色透明溶液B,将无色透明溶液B制成面膜,得目标重组Ⅲ型人源化胶原蛋白贴敷料面膜。
对比例1
本对比例与实施例1的区别在于,修复剂替换为海藻糖,具体配方如下:重组Ⅲ型人源化胶原蛋白0.1%、海藻糖0.2%、椰子胚愈伤组织水提物0.09%、助溶剂2.6%、羟乙基纤维素0.15%和余量的水;
其中,助溶剂为质量比0.6:2的己二醇和丙二醇;
椰子胚愈伤组织水提物的制备方法和上述面膜的制备方法均同实施例1,如下:
椰子胚愈伤组织水提物的制备方法为:取8~10个月大的红椰子种子,去壳,取出胚乳,清洗去油脂,在体积浓度75%的乙醇溶液中浸泡1min,无菌水冲洗3次,再转入质量浓度6%的次氯酸钠溶液中浸泡5min,无菌水冲洗3次,在无菌条件下取出完整的成熟胚作为外植体,接种至配方为Y3+0.5mg/L NAA+0.25mg/L 6-BA+1.5mg/L 2,4-D+60.0g/L蔗糖的培养基,以60r/min的振荡速率,28~30℃,每天光照10h,光照强度为2000~3000lx进行培养,振荡培养8d后,转接至新的配方为Y3+0.5mg/L NAA+0.25mg/L 6-BA+1.5mg/L 2,4-D+60.0g/L蔗糖+6.0g/L琼脂的培养基,以28~30℃,每天光照10h,光照强度为3000lx进行培养,得到不定芽;
取诱导不定芽的整个愈伤组织,冷冻干燥,粉碎,按料液质量体积比1g:25mL,加入水搅拌,以70~80℃水浴浸提2~3h,过滤,离心,浓缩,冷冻干燥,得椰子胚愈伤组织水提物。
上述面膜的制备方法如下:
S1预分散:取重组Ⅲ型人源化胶原蛋白、海藻糖和椰子胚愈伤组织水提物,加入配制总量5%的纯化水,搅拌,使重组Ⅲ型人源化胶原蛋白、海藻糖和椰子胚愈伤组织水提物溶解完全,目视为无色透明溶液,无可见异物,得溶液A;
S2溶剂:取助溶剂和羟乙基纤维素,搅拌使羟乙基纤维素分散均匀,目视为透明或半透明,无色或微黄色混悬液,无可见异物,得溶剂;
S3配制:将余下配方的纯化水,加入配液罐中,开启搅拌(40r/min±5r/min),开启加热(80℃±5℃),加入溶剂,保温搅拌20min,开启回流降温,温度降至60℃±5℃时,加入溶液A,搅拌,得无色透明溶液B,将无色透明溶液B制成面膜,得目标重组Ⅲ型人源化胶原蛋白贴敷料面膜。
对比例2
本对比例与实施例1的区别在于,未含有椰子胚愈伤组织水提物,提高重组Ⅲ型人源化胶原蛋白的用量,具体配方如下:重组Ⅲ型人源化胶原蛋白0.2%、修复剂0.2%、助溶剂2.6%、羟乙基纤维素0.15%和余量的水;
其中,修复剂为质量比1:1的酵母β-葡聚糖和岩藻糖基乳糖,助溶剂为质量比0.6:2的己二醇和丙二醇;
上述面膜的制备方法均同实施例1,如下:
S1预分散:取重组Ⅲ型人源化胶原蛋白和修复剂,加入配制总量5%的纯化水,搅拌,使重组Ⅲ型人源化胶原蛋白和修复剂溶解完全,目视为无色透明溶液,无可见异物,得溶液A;
S2溶剂:取助溶剂和羟乙基纤维素,搅拌使羟乙基纤维素分散均匀,目视为透明或半透明,无色或微黄色混悬液,无可见异物,得溶剂;
S3配制:将余下配方的纯化水,加入配液罐中,开启搅拌(40r/min±5r/min),开启加热(80℃±5℃),加入溶剂,保温搅拌20min,开启回流降温,温度降至60℃±5℃时,加入溶液A,搅拌,得无色透明溶液B,将无色透明溶液B制成面膜,得目标重组Ⅲ型人源化胶原蛋白贴敷料面膜。
一、安全性试验
依据GB/T 16886.5-2017,评价上述面膜样品的潜在细胞毒性,将试验样品浸提液与生长旺盛的L-929细胞培养(37℃,5%CO2)24小时后,用MTT法检测结果,结果显示100%样品浸提液的存活率为80%以上,对照组结果显示本次试验结果有效。在本试验条件下,试验样品的浸提液对L-929细胞无潜在毒性影响。
依据GB/T 16886.10-2017,观察上述面膜样品潜在的皮肤刺激反应,将试验样品随机裁剪成约2.5cm×2.5cm大小,作为试验组的敷贴片,将对照样品随机裁剪成约2.5cm×2.5cm大小,用0.9%氯化钠注射液湿润,作为对照组的敷贴片,将试验组和对照组敷贴片直接贴敷在动物背部相应区域,用半封闭性绷带覆盖固定4h,去除敷贴后1h、24h、48h和72h,观察皮肤红斑和水肿等反应情况。在本次试验条件下,试验样品对兔皮肤原发性刺激指数为0,试验样品未引起皮肤刺激反应。
依据GB/T 16886.10-2017,上述面膜样品采用豚鼠封闭贴敷法致敏试验,评价其潜在的皮肤致敏反应。将试验样品随机裁剪成适宜大小,作为试验组的敷贴片;将医用纱布敷料裁剪成适宜大小,浸透阴性对照,作为对照组的敷贴片。封闭包扎固定于豚鼠左上背皮肤,试图诱发致敏。恢复期内,采用试验样品对试验组和对照组的所有动物进行激发。在本次试验条件下,试验样品未引起皮肤致敏反应,致敏阳性率为0%。
二、组分保湿性能的测定
样品A:重组Ⅲ型人源化胶原蛋白1g;
样品B:重组Ⅲ型人源化胶原蛋白1g+椰子胚愈伤组织水提物0.9g;
样品C:重组Ⅲ型人源化胶原蛋白1g+修复剂(酵母β-葡聚糖:岩藻糖基乳糖=1:1)2g+椰子胚愈伤组织水提物0.9g;
样品D:重组Ⅲ型人源化胶原蛋白1.5g+修复剂(酵母β-葡聚糖:岩藻糖基乳糖=1:1)3g+椰子胚愈伤组织水提物1.5g;
样品E:重组Ⅲ型人源化胶原蛋白0.8g+修复剂(酵母β-葡聚糖:岩藻糖基乳糖=1:1)1g+椰子胚愈伤组织水提物0.7g;
将上述样品配制成浓度为1.0g/L的水溶液,取1mL水溶液滴入玻璃片的胶带上,并将玻璃片置于有硫酸铵饱和溶液的干燥器中,在相对湿度80%的条件下放置6h,而后测定保湿率,保湿率%=(M2-M0)/(M1-M0)×100,其中,M0为玻璃片的重量,g,M1为加入样品后玻璃片的重量,g,M2为6h后玻璃片的重量,g,对照选用甘油(1.0g/L),结果如下。
表1各组样品的保湿率的结果
指标 | 样品A | 样品B | 样品C | 样品D | 样品E | 甘油 |
保湿率(%) | 30.16 | 35.42 | 42.38 | 40.81 | 41.76 | 22.86 |
由上表1的结果可知,保湿能力的大小依次为:样品C>样品E>样品D>样品B>样品A>甘油,复配物的保湿能力好,其中,三者复配物的保湿性能更优,选用重组Ⅲ型人源化胶原蛋白、修复剂(酵母β-葡聚糖和岩藻糖基乳糖)和椰子胚愈伤组织水提物复配,具有协同效应的机制,使三者成分复配后,提高了整体复配物体系对水分子的结合力,其吸湿性能更强。
三、皮肤干燥症的改善试验
选取SPF级昆明小鼠(KM),雌性,8周龄,体质量为20~24g,进行适应性喂养7周后,建立丙酮/乙醚和水诱导小鼠皮肤干燥症瘙痒的模型。小鼠皮肤干燥症瘙痒模型的建立方法为:每组10只小鼠,将小鼠颈背部位剔除面积约2×2cm区域的背毛,采用浸润丙酮/乙醚(按体积比1:1混合)溶液的棉片,均匀地涂抹裸露区域40s,而后采用浸润蒸馏水的棉片继续涂抹40s。涂抹后2h,将各组获得的溶液A,调整为质量浓度为1.0mg/mL,分别将各组溶液A涂抹于裸露区域,涂抹后6h,再次将溶液A涂抹于裸露区域,每天造模与连续涂抹6d,每天涂抹2次,最后一次涂抹6h后,将小鼠处死,剪取背部试验的皮肤组织,取0.1g皮肤组织,加入裂解液制成组织匀浆,离心,取上清液,采用ELISA试剂盒检测白细胞介素-6(Interleukin-6,IL-6)和TRPV1(transient receptor potential vanilloid 1,TRPV1)的含量,未造模的小鼠,背部涂抹蒸馏水(棉片浸润)作为空白组。
表2各组溶液A对小鼠抓挠、皮肤组织中的IL-6和TRPV1含量的结果
由上表2的结果可知,与空白组相比,模型组在建立丙酮/乙醚、水诱导小鼠皮肤干燥症瘙痒后,抓挠次数、IL-6和TRPV1含量均有所增加;实施例1的IL-6和TRPV1含量较低,表明采用重组Ⅲ型人源化胶原蛋白、修复剂(酵母β-葡聚糖和岩藻糖基乳糖)和椰子胚愈伤组织水提物为有效活性成分,制备得到的一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜,可抑制、减少炎症反应过程中的IL-6因子和TRPV1瘙痒调控因子的释放,起到减缓、改善皮肤干燥症的作用,有助于增强抗皮肤干燥、瘙痒的免疫力,进一步提高皮肤屏障功能。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜,其特征在于,按质量百分比计,包括如下原料:重组Ⅲ型人源化胶原蛋白0.08~0.15%、修复剂0.1~0.3%、椰子胚愈伤组织水提物0.05~0.15%、助溶剂2~4%、增稠剂0.1~0.25%和余量的水;
所述修复剂由酵母β-葡聚糖和岩藻糖基乳糖组成;
所述椰子胚愈伤组织水提物的制备方法,包括:取椰子成熟胚,接种至基础培养基,振荡培养7~10d后,转接至新的基础培养基,固态培养至不定芽产生,取愈伤组织,干燥后粉碎,加入水搅拌,水浴浸提,过滤,离心,浓缩,冷冻干燥,得椰子胚愈伤组织水提物;
所述基础培养基的配方为:在Y3培养基的基础上,添加0.4~0.6 mg/L NAA、0.20~0.30mg/L 6-BA、1.0~2.0 mg/L 2, 4-D和55.0~65.0 g/L蔗糖。
2.如权利要求1所述的一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜,其特征在于,按百分比计,包括如下原料:重组Ⅲ型人源化胶原蛋白0.08~0.1%、修复剂0.1~0.3%、椰子胚愈伤组织水提物0.07~0.1%、助溶剂2~4%、增稠剂0.1~0.25%和余量的水。
3.如权利要求2所述的一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜,其特征在于,按百分比计,包括如下原料:重组Ⅲ型人源化胶原蛋白0.1%、修复剂0.2%、椰子胚愈伤组织水提物0.09%、助溶剂2.6%、增稠剂0.15%和余量的水。
4.如权利要求1~3任意一项所述的一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜,其特征在于,所述修复剂为质量比1~2:1~2的酵母β-葡聚糖和岩藻糖基乳糖;
所述助溶剂为己二醇和/或丙二醇;所述增稠剂为羟乙基纤维素。
5.如权利要求1所述的一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜,其特征在于,所述振荡培养的频率为50~75r/min;所述振荡培养和固态培养的条件为:温度28~30℃,光照强度2000~3000lx,每天光照时间10~12h。
6.如权利要求1所述的一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜,其特征在于,所述愈伤组织和水的料液质量体积为1g:20~30mL;所述水浴浸提的温度为70~80℃,水浴浸提的时间为2~3h。
7.如权利要求1~6任意一项所述的一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜的制备方法,其特征在于,包括如下步骤:
S1预分散:取重组Ⅲ型人源化胶原蛋白、修复剂和椰子胚愈伤组织水提物,加入配方水总量的4~8%的水,搅拌,得溶液A;
S2溶剂:取助溶剂和增稠剂,搅拌混合,得溶剂;
S3配制:将余下配方的水边加热边加入溶剂搅拌,降温后,加入溶液A,搅拌,得溶液B,制成面膜,得目标重组Ⅲ型人源化胶原蛋白贴敷料面膜。
8.如权利要求7所述的一种用于受损皮肤屏障的重组Ⅲ型人源化胶原蛋白贴敷料面膜的制备方法,其特征在于,搅拌的速率为35~45r/min,加热的温度为75~85℃,降温至温度为55~65℃。
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