CN108113916B - 一种天然修复肽面膜液及其制备方法 - Google Patents
一种天然修复肽面膜液及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种天然修复肽面膜液及其制备方法,该天然修复肽面膜液的制备方法包括以下步骤:先制备浓缩多肽液,再制备银耳提取物,再制备黄芩、马齿苋和甘草混合水提液,最后将他们混合均匀,得到天然修复肽面膜液。本发明的面膜液中含有丰富的小分子活性多肽,分子量小,纯度高,还添加有黄芩、马齿苋和甘草混合水提液,修复功能强大,且有深层滋养保湿作用,对痤疮后色素沉着、新鲜瘢痕、紫外线照射等引起的皮肤损伤,修复效果好,并有紧肤、防皱、保湿、滋养和延缓衰老的美容功效,对皮肤无任何刺激,使用舒适,其制备工艺流程简单、经济、实用,生产过程无化学有机溶剂的污染,反应条件温和,可大规模工业化生产。
Description
技术领域
本发明涉及一种天然修复肽面膜液及其制备方法,属于胶原蛋白深加工技术领域。
背景技术
皮肤老化可分为自然老化和光老化。光老化是指皮肤长期受到日光照射所引起的损害,由于人的面部皮肤长期暴露在阳光中,过了30岁以后,便开始慢慢衰老,主要表现为色素异常(如老年斑、日光性黑子等)、皮肤松弛、细小皱纹、面部红血丝、毛孔扩大、皮肤颜色晦暗等,严重影响美观。目前,改善皮肤光老化的方法有很多,包括药物治疗、化妆品护理、外科治疗、注射性治疗、剥脱性治疗、非剥脱性嫩肤治疗等。其中,化妆品护理是应用最广泛的方法。然而,化妆品属于精细化工产品,其原料大多为人工合成或改性的化工产品,其抗原性和对人体合适的剂量不好掌控,部分人在使用之后,皮肤会出现瘙痒、红肿、干燥等皮肤炎症。
通过生物酶定向剪切技术从原生态深海鱼皮中提取的小分子活性肽具有低分子量和高生物活性的特点,其肽链长度约为十几个纳米,可快速透过皮肤屏障,进入细胞内,发挥多种生物学活性:1)营养性:该小分子多肽可以供给皮肤细胞所必需的养分,使皮肤中的角质形成细胞和成纤维细胞活性增强,保持角质层水分以及纤维结构的完整性,改善皮肤细胞生存环境和促进皮肤组织的新陈代谢,增加肌肤循环,有滋润皮肤延缓老化、美白、祛皱、嫩肤的作用;2)修复性:该小分子多肽具有较强的修复功能,与周围组织的亲合性好,组织修复作用强,对于疤痕、皮肤粗糙有独特的修复效果,可显著增加皮肤的平滑性和弹性;3)保湿性:该小分子多肽中含有大量的亲水基,具有良好的保湿功效,能够达到保持肌肤润泽度的目的,它可以深入皮肤深层,达到深层保湿、长效保湿效果;4)吸湿性:该小分子多肽具有强的吸湿性能,干燥的粉末暴露在空气中,会迅速吸收空气中的水分,强于透明质酸,具有补水效果;5)易吸收性:皮肤对该小分子多肽有很好的吸收作用,实验表明,该小分子多肽的皮肤吸收率几乎达到92%;6)良好的溶解性:该小分子多肽含有大量的亲水基团(-COOH、-NH2、-OH),与水分子之间的相互作用强,水溶性好;7)低免疫原性、无毒副作用:该小分子多肽来源于天然的深海鱼类,通过定向剪切酶技术得到,在制备过程中无有害物质的加入,故无毒副作用,可以长期敷用。
黄芩中含黄芩苷、汉黄芩素、黄芩新素等,其水提物有较广的抗菌谱,对多种皮肤致病性细菌、真菌,均有抑制作用,而且不产生抗药性,且具有抗氧化、抗炎、抗变态反应、抗衰老作用;马齿苋含有丰富的二羟乙胺、苹果酸、葡萄糖、钙、磷、铁以及维生素E、胡萝卜素、维生素B和维生素C等营养物质,特别富含维生素A,能促进上皮细胞的生理功能趋于正常,并能促进溃疡的愈合,且其抑菌作用强,对常见致病性皮肤细菌和真菌有明显的抑制作用;甘草含有甘草酸、甘草苷等多种成分,临床上有显著的抗过敏和抗炎、抗氧化和抗衰老作用。
发明内容
本发明的目的在于提供一种天然修复肽面膜液及其制备方法。
本发明所采取的技术方案是:
一种天然修复肽面膜液的制备方法,包括以下步骤:
1)加热变性:将海参斑鱼皮洗净,切条,加水煮沸,沸腾10~30min,过滤出鱼皮,得到浸出液;
2)鱼皮破碎:在步骤1)过滤出的鱼皮中加水,先用组织破碎机进行破碎,再用胶体磨进行破碎,得到鱼皮匀浆;
3)生物酶定向剪切酶解:将步骤1)的浸出液和步骤2)的鱼皮匀浆合并,先加入胰蛋白酶进行酶解,再加入木瓜蛋白酶进行酶解;
4)灭活离心:将步骤3)的酶解产物于95~100℃灭活30~40min,离心除去固体残渣;
5)去腥脱色:在步骤4)的离心产物中加入活性炭,去腥脱色,再进行超滤膜过滤,得到透过液;
6)浓缩:对步骤5)的透过液进行浓缩,得到浓缩多肽液;
7)银耳提取物的制备:将干银耳粉碎,加水浸胀,再90~100℃超声浸提2~4h,过滤,对滤液进行浓缩,得到银耳提取物;
8)黄芩、马齿苋和甘草混合水提液的制备:将黄芩、马齿苋和甘草混合物加水浸胀,再90~100℃超声浸提1~2h,过滤,在滤液中加入活性炭,脱色,过滤,对滤液进行浓缩,得到黄芩、马齿苋和甘草混合水提液;
9)产物的制备:将步骤7)的银耳提取物和步骤8)的混合水提液加入到步骤6)的浓缩多肽液中,混合均匀,得到天然修复肽面膜液。
所述的海参斑鱼皮、银耳、黄芩、马齿苋、甘草的质量比为1000:(80~120):(40~60):(40~60):(40~60)。
步骤1)中鱼皮和水的质量比为1:(2~10)。
步骤2)中鱼皮和水的质量比为1:(2~10)。
步骤3)中胰蛋白酶的添加量为20000~50000U/g底物蛋白,酶解温度为55~65℃,pH=7.0~7.5,酶解的时间为4~8h。
步骤3)中木瓜蛋白酶的添加量为4000~8000U/g底物蛋白,酶解温度为60~70℃,pH=7.0~7.5,酶解的时间为2~3h。
步骤5)所述的去腥脱色在55~60℃,pH=6~7的条件下进行,去腥脱色的时间为30~60min。
步骤7)中银耳和水的质量比为1:(40~60)。
步骤8)中黄芩、马齿苋和甘草混合物与水的质量比为1:(10~30)。
本发明的有益效果是:
1)本发明的天然修复肽面膜液中含有丰富的小分子活性多肽,该小分子多肽来源于天然的深海鱼类,通过定向剪切酶技术得到,分子量在100~3000Da之间,平均分子量约为800Da,其中分子量为500~1000Da的小分子多肽占约62%,多肽纯度高,且在制备过程中无酸碱、有机溶剂等物质的加入,因此,面膜液中没有有害杂质残留,绿色、安全,无毒副作用,可以长期敷用;
2)本发明的天然修复肽面膜液中添加有黄芩、马齿苋和甘草混合水提液,修复功能强大,且有深层滋养保湿作用,对痤疮后色素沉积、新鲜瘢痕、紫外线照射引起的皮肤损伤等的修复效果好,并有紧肤、防皱、保湿、滋养和延缓衰老的美容功效,且对皮肤无任何刺激,使用舒适;
3)本发明的天然修复肽面膜液的生产原料来源天然,未添加人工香料、色素、防腐剂等成份,生产工艺流程简单、经济、实用,生产过程无化学有机溶剂的污染,反应条件温和,可大规模工业化生产。
具体实施方式
一种天然修复肽面膜液的制备方法,包括以下步骤:
1)加热变性:将海参斑鱼皮洗净,切条,加水煮沸,沸腾10~30min,过滤出鱼皮,得到浸出液;
2)鱼皮破碎:在步骤1)过滤出的鱼皮中加水,先用组织破碎机进行破碎,再用胶体磨进行破碎,得到鱼皮匀浆;
3)生物酶定向剪切酶解:将步骤1)的浸出液和步骤2)的鱼皮匀浆合并,先加入胰蛋白酶进行酶解,再加入木瓜蛋白酶进行酶解;
4)灭活离心:将步骤3)的酶解产物于95~100℃灭活30~40min,离心除去固体残渣;
5)去腥脱色:在步骤4)的离心产物中加入活性炭,去腥脱色,再进行超滤膜过滤,得到透过液;
6)浓缩:对步骤5)的透过液进行浓缩,得到浓缩多肽液;
7)银耳提取物的制备:将干银耳粉碎,加水浸胀,再90~100℃超声浸提2~4h,过滤,对滤液进行浓缩,得到银耳提取物;
8)黄芩、马齿苋和甘草混合水提液的制备:将黄芩、马齿苋和甘草混合物加水浸胀,再90~100℃超声浸提1~2h,过滤,在滤液中加入活性炭,脱色,过滤,对滤液进行浓缩,得到黄芩、马齿苋和甘草混合水提液;
9)产物的制备:将步骤7)的银耳提取物和步骤8)的混合水提液加入到步骤6)的浓缩多肽液中,混合均匀,得到天然修复肽面膜液。
优选的,所述的海参斑鱼皮、银耳、黄芩、马齿苋、甘草的质量比为1000:(80~120):(40~60):(40~60):(40~60)。
进一步优选的,所述的海参斑鱼皮、银耳、黄芩、马齿苋、甘草的质量比为1000:100:50:50:50。
优选的,步骤1)中鱼皮和水的质量比为1:(2~10)。
优选的,步骤2)中鱼皮和水的质量比为1:(2~10)。
优选的,步骤3)中胰蛋白酶的添加量为20000~50000U/g底物蛋白,酶解温度为55~65℃,pH=7.0~7.5,酶解的时间为4~8h。
优选的,步骤3)中木瓜蛋白酶的添加量为4000~8000U/g底物蛋白,酶解温度为60~70℃,pH=7.0~7.5,酶解的时间为2~3h。
优选的,步骤4)中进行离心时离心机的转速为4300~4500rpm,离心时间为13~15min。
优选的,步骤5)中活性炭的添加量为0.5~0.7g每100mL离心产物。
优选的,步骤5)所述的去腥脱色在55~60℃,pH=6~7的条件下进行,去腥脱色的时间为30~60min。
优选的,步骤5)中所述的超滤膜可以截留分子量3000Da以上的物质。
优选的,步骤7)中银耳和水的质量比为1∶(40~60)。
优选的,步骤8)中黄芩、马齿苋和甘草混合物与水的质量比为1∶(10~30)。
优选的,步骤8)中活性炭的添加量为0.5~0.7g每100mL滤液。
下面结合具体实施例对本发明作进一步的解释和说明。
实施例1:
1)加热变性:将新鲜冰冻的海参斑鱼用微波炉解冻,除掉鱼身上的黑色表皮,小心取下鱼皮,得到1kg鱼皮,将鱼皮洗净,切成1cm宽的条形,加入3kg纯净水,煮沸,沸腾20min,过滤出鱼皮,得到浸出液;
2)鱼皮破碎:在步骤1)过滤出的鱼皮中加入6kg纯净水,先用组织破碎机进行破碎,再用胶体磨进行破碎,得到鱼皮匀浆;
3)生物酶定向剪切酶解:将步骤1)的浸出液和步骤2)的鱼皮匀浆合并,先加入胰蛋白酶进行酶解,胰蛋白酶的添加量为30000U/g底物蛋白,酶解温度为60℃,pH=7.0,酶解的时间为6h,再加入木瓜蛋白酶,木瓜蛋白酶的添加量为4000U/g底物蛋白,酶解温度为65℃,pH=7.0,酶解的时间为2.5小时;
4)灭活离心:将步骤3)的酶解产物于100℃灭活30min,离心除去固体残渣;
5)去腥脱色:在步骤4)的离心产物中加入活性炭,55℃去腥脱色50min,再进行超滤膜过滤,截留分子量大于3000Da的物质,得到透过液;
6)浓缩:对步骤5)的透过液进行浓缩,得到8kg浓缩多肽液;
7)银耳提取物的制备:将0.1kg干银耳粉碎成片状(约0.5cm×0.5cm),加入4kg纯净水浸泡4小时,再100℃超声浸提2小时,过滤,对滤液进行浓缩,得到1kg银耳提取物;
8)黄芩、马齿苋和甘草混合水提液的制备:将0.05kg黄芩、0.05kg马齿苋和0.05kg甘草混合物加1.5kg纯净水浸泡3小时,再100℃超声浸提1小时,过滤,在滤液中加入活性炭脱色,过滤,对滤液进行浓缩,得到1kg黄芩、马齿苋和甘草混合水提液;
9)天然修复肽面膜液制备:将步骤7)的银耳提取物和步骤8)的混合水提液加入到步骤6)的浓缩多肽液中,混合均匀,得到10kg天然修复肽面膜液,分装进25mL的耐高温的玻璃瓶,用医用高温蒸汽灭菌锅高压灭菌。
经测试(采用双缩脲法进行测试),本实施例制备的天然修复肽面膜液中分子量在100~3000Da之间的多肽的质量分数为7.7%。
临床测试表明:面膜液中小分子活性肽的含量在5%~10%之间,效果最佳。
实施例2:
1)加热变性:将新鲜冰冻的海参斑鱼用微波炉解冻,除掉鱼身上的黑色表皮,小心取下鱼皮,得到1kg鱼皮,洗净,切成1cm宽的条形,加入6kg的纯净水,煮沸,沸腾30min,过滤出鱼皮,得到浸出液;
2)鱼皮破碎:在步骤1)过滤出的鱼皮中加入9kg的纯净水,先用组织破碎机进行破碎,再用胶体磨进行破碎,得到鱼皮匀浆;
3)生物酶定向剪切酶解:将步骤1)的浸出液和步骤2)的鱼皮匀浆合并,先加入胰蛋白酶进行酶解,胰蛋白酶的添加量为45000U/g底物蛋白,酶解温度为65℃,pH=7.2,酶解的时间为4h,再加入木瓜蛋白酶,木瓜蛋白酶的添加量为6000U/g底物蛋白,酶解温度为60℃,pH=7.2,酶解的时间为3小时;
4)灭活离心:将步骤3)的酶解产物于95℃灭活30min,离心除去固体残渣;
5)去腥脱色:在步骤4)的离心产物中加入活性炭,60℃去腥脱色30min,再进行超滤膜过滤,截留分子量大于3000Da的物质,得到透过液;
6)浓缩:对步骤5)的透过液进行浓缩,得到8kg浓缩多肽液;
7)银耳提取物的制备:将0.1kg干银耳粉碎成片状(约0.5cm×0.5cm),加入5kg纯净水浸泡6小时,再95℃超声浸提3小时,过滤,在滤液中加入活性炭脱色,过滤,对滤液进行浓缩,得到1kg银耳提取物;
8)黄芩、马齿苋和甘草混合水提液的制备:将0.05kg黄芩、0.05kg马齿苋和0.05kg甘草混合物加1.5kg水浸泡5小时,再95℃超声浸提1.5小时,过滤,加入活性炭脱色,对滤液进行浓缩,得到1kg黄芩、马齿苋和甘草混合水提液;
9)天然修复肽面膜液制备:将步骤7)的银耳提取物和步骤8)的混合水提液加入到步骤6)的浓缩多肽液中,混合均匀,得到10kg天然修复肽面膜液,分装进25mL的耐高温的玻璃瓶,用医用高温蒸汽灭菌锅高压灭菌。
经测试(采用双缩脲法进行测试),本实施例制备的天然修复肽面膜液中分子量在100~3000Da之间的多肽的质量分数为8.4%。
实施例3:
1)加热变性:将新鲜冰冻的海参斑鱼用微波炉解冻,除掉鱼身上的黑色表皮,小心取下鱼皮,得到1kg鱼皮,洗净,切成1cm宽的条形,加入5kg纯净水,煮沸,沸腾25min,过滤出鱼皮,得到浸出液;
2)鱼皮破碎:在步骤1)过滤出的鱼皮中加入10kg纯净水,先用组织破碎机进行破碎,再用胶体磨进行破碎,得到鱼皮匀浆;
3)生物酶定向剪切酶解:将步骤1)的浸出液和步骤2)的鱼皮匀浆合并,先加入胰蛋白酶进行酶解,胰蛋白酶的添加量为20000U/g底物蛋白,酶解温度为55℃,pH=7.5,酶解的时间为8h,再加入木瓜蛋白酶,木瓜蛋白酶的添加量为8000U/g底物蛋白,酶解温度为70℃,pH=7.5,酶解的时间为2小时;
4)灭活离心:将步骤3)的酶解产物于98℃灭活35min,离心除去固体残渣;
5)去腥脱色:在步骤4)的离心产物中加入活性炭,58℃去腥脱色40min,再进行超滤膜过滤,截留分子量大于3000Da的物质,得到透过液;
6)浓缩:对步骤5)的透过液进行浓缩,得到8kg浓缩多肽液;
7)银耳提取物的制备:将0.1kg干银耳粉碎成片状(约0.5cm×0.5cm),加入6kg纯净水浸泡5小时,再95℃超声浸提4小时,过滤,在滤液中加入活性炭脱色,过滤,对滤液进行浓缩,得到1kg银耳提取物;
8)黄芩、马齿苋和甘草混合水提液的制备:将0.05kg黄芩、0.05kg马齿苋和0.05kg甘草混合物加2kg水浸泡4小时,再90℃超声浸提2小时,过滤,加入活性炭脱色,对滤液进行浓缩,得到1kg黄芩、马齿苋和甘草混合水提液;
9)天然修复肽面膜液制备:将步骤7)的银耳提取物和步骤8)的混合水提液加入到步骤6)的浓缩多肽液中,混合均匀,得到10kg天然修复肽面膜液,分装进25mL的耐高温的玻璃瓶,用医用高温蒸汽灭菌锅高压灭菌。
经测试(采用双缩脲法进行测试),本实施例制备的天然修复肽面膜液中分子量在100~3000Da之间的多肽的质量分数为8.8%。
测试例:临床应用测试
1)入选和排除标准
入选标准:①临床上明确诊断为面部皮炎和皮肤敏感患者,主观症状有皮肤灼热、瘙痒、疼痛、干皱感、紧胀感;客观症状有皮肤干燥、脱屑、红斑或潮红、丘疹、丘疱疹等;②年龄为18~55岁,男女不限;③愿意接受生物活性肽面膜的治疗且能遵守治疗方案;④口头知情同意。
排除标准:对多肽、银耳、黄芩、马齿苋、甘草过敏者;患有脂溢性皮炎、痤疮等影响疗效观察者;病情严重,治疗中需要接受面部湿敷或外用激素制剂治疗者;已系统使用过雷公藤制剂、激素或四环素类等药物,抗生素停药不足1个月;外用过激素、非甾体类抗炎药物,停药不足1周;有严重的心、肝、肾疾病。
2)病例
入选符合临床诊断标准的患者88例;其中男22例,女66例,平均年龄(32.88±6.62)岁(20~49岁);平均病程(20.00±7.79)个月(1~48个月)。
3)方法
治疗方法:每晚在清洁面部后外用生物活性肽面膜(由实施例1的天然修复肽面膜液制备得到)1贴,连用4周后停用。分别于应用面膜后1周(±2d)、2周(±2d)和4周(±2d)以及停药后2周(±2d)各随访1次,记录症状评分,计算疗效指数,测试结果如表1所示。
4)疗效评价:按照以下标准进行症状评分,自觉症状:灼热、瘙痒、疼痛、干皱感、紧胀感,分别按无、轻、中、重计为0~3分;客观症状中干燥、脱屑、红斑或潮红、丘疹、丘疱疹分别按无、轻、中、重计为0~3分。疗效指数=(治疗前总积分-治疗后总积分)/治疗前总积分×100%。基愈:疗效指数≥90%;显效:疗效指数为60%~89%;进步:疗效指数为20%~59%;无效:疗效指数<20%。
5)统计学方法
采用SPSS13.0软件对试验数据进行统计学分析。治疗前、后总积分比较采用t检验,P<0.05认为差异有统计学意义。
表1生物活性肽面膜治疗88例面部皮炎患者的疗效测试结果[例(%)]
时间 | 显效 | 进步 | 无效 |
治疗1周 | 22(25.0%) | 64(72.7%) | 2(2.2%) |
治疗2周 | 44(50.0%) | 44(50.0%) | 0(0) |
治疗4周 | 53(81.0%) | 34(38.6%) | 1(1.1%) |
停药后2周 | 41(46.6%) | 43(49.9%) | 4(4.5%) |
注:未见不良反应的发生。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (9)
1.一种天然修复肽面膜液的制备方法,其特征在于:包括以下步骤:
1)加热变性:将海参斑鱼皮洗净,切条,加水煮沸,沸腾10~30min,过滤出鱼皮,得到浸出液;
2)鱼皮破碎:在步骤1)过滤出的鱼皮中加水,先用组织破碎机进行破碎,再用胶体磨进行破碎,得到鱼皮匀浆;
3)生物酶定向剪切酶解:将步骤1)的浸出液和步骤2)的鱼皮匀浆合并,先加入胰蛋白酶进行酶解,再加入木瓜蛋白酶进行酶解;
4)灭活离心:将步骤3)的酶解产物于95~100℃灭活30~40min,离心除去固体残渣;
5)去腥脱色:在步骤4)的离心产物中加入活性炭,去腥脱色,再进行超滤膜过滤,得到透过液;
6)浓缩:对步骤5)的透过液进行浓缩,得到浓缩多肽液;
7)银耳提取物的制备:将干银耳粉碎,加水浸胀,再90~100℃超声浸提2~4h,过滤,对滤液进行浓缩,得到银耳提取物;
8)黄芩、马齿苋和甘草混合水提液的制备:将黄芩、马齿苋和甘草混合物加水浸胀,再90~100℃超声浸提1~2h,过滤,在滤液中加入活性炭,脱色,过滤,对滤液进行浓缩,得到黄芩、马齿苋和甘草混合水提液;
9)产物的制备:将步骤7)的银耳提取物和步骤8)的混合水提液加入到步骤6)的浓缩多肽液中,混合均匀,得到天然修复肽面膜液;
所述的海参斑鱼皮、银耳、黄芩、马齿苋、甘草的质量比为1000:(80~120):(40~60):(40~60):(40~60)。
2.根据权利要求1所述的制备方法,其特征在于:步骤1)中鱼皮和水的质量比为1:(2~10)。
3.根据权利要求1所述的制备方法,其特征在于:步骤2)中鱼皮和水的质量比为1:(2~10)。
4.根据权利要求1所述的制备方法,其特征在于:步骤3)中胰蛋白酶的添加量为20000~50000U/g底物蛋白,酶解温度为55~65℃,pH=7.0~7.5,酶解的时间为4~8h。
5.根据权利要求1所述的制备方法,其特征在于:步骤3)中木瓜蛋白酶的添加量为4000~8000U/g底物蛋白,酶解温度为60~70℃,pH=7.0~7.5,酶解的时间为2~3h。
6.根据权利要求1所述的制备方法,其特征在于:步骤5)所述的去腥脱色在55~60℃,pH=6~7的条件下进行,去腥脱色的时间为30~60min。
7.根据权利要求1所述的制备方法,其特征在于:步骤7)中银耳和水的质量比为1:(40~60)。
8.根据权利要求1所述的制备方法,其特征在于:步骤8)中黄芩、马齿苋和甘草混合物与水的质量比为1:(10~30)。
9.权利要求1~8中任意一项所述的方法制备的天然修复肽面膜液。
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