CN106821935A - 从鲎试剂废料中提取抑菌组合物制备化妆品的方法 - Google Patents
从鲎试剂废料中提取抑菌组合物制备化妆品的方法 Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/987—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of species other than mammals or birds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/66—Enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
- A61K2800/524—Preservatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Dermatology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Zoology (AREA)
- Cosmetics (AREA)
Abstract
本发明涉及一种从鲎试剂废料中提取抑菌组合物制备化妆品的方法,包括步骤:第一步,在鲎试剂废料中提取抑菌组合物,具体包括:(1)收集鲎试剂生产工艺中丢弃的乳化物沉淀及丢弃的血浆;(2)由丢弃的乳化物沉淀制备鲎素肽粗提液;(3)由丢弃的血浆制备血蓝蛋白粗提液;(4)由丢弃的血浆制备SOD粗提液;(5)三种粗提液混合制备抑菌组合物;第二步,(1)按质量百分比抑菌组合物:30%~50%,橄榄油:2%~4%,维生素E醋酸酯:1%~2%,二乙二醇脂肪酸酯:0.5%~10.0%,赋形增稠剂:0.1%~10%,去离子水:余量配置化妆品组份,(2)按制备工艺得到化妆品成品。本发明的化妆品能有效抑制各类细菌及真菌的生长,产品安全、温和、无毒副作用,具有抗菌,消炎,润肤,保湿及防紫外线等多重功效,减缓皮肤老化。
Description
技术领域
本发明涉及化妆品技术领域,具体是一种从鲎试剂废料中提取抑菌组合物制备化妆品的方法。
背景技术
鲎,又称马蹄蟹,是一种生活在海洋中的大型底栖节肢动物,从早古生代的奥陶纪出现至今已有4亿多年的历史,是动物界具有独特进化地位的“活化石”之一。鲎是无脊椎动物血液学研究的绝好材料,也是目前鲎资源开发利用和鲎试剂研制的主要原材料,一直受到日、美等国学者的高度重视。随着对鲎血淋巴的免疫功能研究以及和其他无脊椎动物血淋巴的免疫学比较研究,尤其是从鲎血淋巴中分离出许多具有抗菌、抗病毒的活性物质。总体而言,鲎作为一种具有巨大医药开发潜力的重要资源,为世界各国高技术产业所瞩目。
目前鲎的主要用途是制备鲎试剂。鲎血细胞中的颗粒细胞中含有能与内毒素起凝集作用的酶系统。根据此原理,世界各国研究人员经不懈努力,开发出了用于药品细菌内毒素检测的鲎试剂。同时,鲎试剂还能被β-葡聚糖激活,而1-3-β-D葡聚糖是真菌细胞壁的特有成分,从而鲎试剂亦被开发应用于检测深度真菌感染。但鲎试剂的提取过程中仅利用到了鲎血细胞,剩余的其他成分并未得到有效开发利用。鲎试剂的提取过程包括:
a鲎细胞裂解液的配制:裂解液为Tris-HCl,NaCl缓冲液。其中,所述缓冲液为pH7-8,NaCl,Tris-HCl可根据实际情况调节,一般为50mmol/L NaCl,20mmol/L Tris-HCl;
b鲎细胞的裂解乳化:取全血离心后的鲎细胞,按1:5-1:8.5的比例加入鲎细胞裂解液,使用高速匀浆机匀浆5-20分钟,既得鲎细胞裂解乳化物。
c鲎细胞裂解液分离提纯:将上述乳化物低温离心25-30分钟,取上清液,分装备用,即为鲎细胞裂解液。向裂解液中以1:1-1:2的比例加入氯仿溶液,低温搅拌离心后,仔细取出上清液备用。
d制剂:取c步骤中最终上清液,各生产厂家根据自身情况,向其中添加多肽显色基质,低温搅拌分装后,真空冷冻干燥后即得鲎试剂。
另外,在鲎试剂的提取过程中作为鲎全血的另一组成部分,鲎血浆也被废弃。
化妆品在存储和使用过程中,极易受微生物的污染而导致腐败。由于微生物的作用可引起化妆品变质、腐败,在感官上其色泽和气味发生变化,从而失去商品价值。更重要的是致病微生物的污染会导致人体健康的危害。防止化妆品由于微生物污染而产生对人体皮肤的危害及产品的变质、发霉,当前最有效的办法就是添加防腐剂,防腐剂是能够抑制微生物的生长、繁殖的一类化学药剂。随着科研水平的进步,业界对防腐剂的安全性研究也越来越深入,许多传统有被使用的防腐剂,都被证实具有一定的负面作用。所以,开发安全的纯天然或是生物抗菌、防腐技术成为业界的研发方向之一。此外,在化妆品中由于其成分如主原料、防腐剂、乳化剂、香料等绝大多数都是化学合成品,使用时发生过敏反应己屡见不鲜。
发明内容
本发明的目的在于丰富目前化妆品的种类,提供一种从鲎试剂废料中提取抑菌组合物制备化妆品的方法,以实现化妆品天然绿色,且具有抗菌、防腐、滋养、修复、保湿等多重功效。
本发明实现目的的技术方案如下:
1、一种从鲎试剂废料中提取抑菌组合物制备化妆品的方法,该方法包括步骤如下:
第一步,在鲎试剂废料中提取抑菌组合物
(1)原料处理
收集鲎血细胞及血浆,作为原料备用;其中,所述鲎血细胞是鲎试剂生产工艺中丢弃的乳化物沉淀,所述血浆是鲎试剂生产过程中丢弃的血浆;
(2)鲎素肽粗提液的制备
a.酸解:将鲎细胞乳化物离心后的下层沉淀以固液比例1:6.5-8的比例加入酸液(20m mol/L),低温超声破碎,破碎条件为:100ml超声波输出功率为700W,超声波总作用时间为10min,每次超声作用时间为10S,反复超声3次,收集上清液即为细胞酸抽提液;
b.调pH去沉淀:将抽提液用NaOH调节pH至6.5,离心除沉淀,得上清液;
c.热变性处理:上清液沸水浴处理5-10min,4℃下离心弃沉淀收集上清液,上清液用0.22uM膜过滤得鲎素肽粗提液;
(3)血浆中血蓝蛋白粗提液的制备
将上述步骤(1)中的血浆经300000g超离心3.5h,即得到初步提纯的血蓝蛋白沉淀,将沉淀再溶于pH7.0,0.2mol/L磷酸缓冲液中,即为血蓝蛋白粗提液;
(4)血浆中SOD粗提液的制备
按质量份数计,取100份上述步骤(1)中的鲎血浆,加入34份0.05mol/L磷酸盐缓冲液和100份0.9%NaCl溶液,轻轻搅拌混匀,将混合液置于65℃水浴锅中,恒温20min后,急冷至10℃,离心去除沉淀,取上清液,即为SOD粗提液;
(5)粗提液的混合
将上述三种粗提液按60-80份的鲎素肽粗提液,10-20份的血蓝蛋白提取液,10-20份的SOD提取液混合,使用pH调节剂调节pH值4-5后过滤,得到最终形式的抑菌组合物。
第二步,由上述抑菌组合物制备化妆品
(1)按如下质量百分比配置化妆品包括的组份:
(2)得到化妆品成品
A.将上述化妆品组份中包括的部分油相与去离子水进行初步乳化;
b.调整上述乳化体系温度至35℃~40℃,加入橄榄油、抑菌组合物,体系经搅拌均匀降温后得成品。
而且,所述第一步的步骤(5)中的pH调节剂为柠檬酸、柠檬酸钠、乳酸、磷酸氢二钠、磷酸二氢钠、磷酸二氢钾、硼酸、硼砂中的一种或多种的组合。
而且,所述第一步的步骤(5)中抑菌组合物的鲎素肽粗提液﹕血蓝蛋白粗提液﹕SOD粗提液的最佳配比为:8﹕1﹕1或6﹕2﹕2。
而且,所述第二步的(2)步中进行初步乳化的油相组份包括维生素E醋酸酯、二乙二醇脂肪酸酯及赋形增稠剂。
本发明的优点和效果是:
1、本专利原料抑菌组合物对鲎试剂生产废料进行了充分提取。主要提取了鲎血细胞提取废料中的鲎素肽及血浆中的血蓝蛋白及超氧化物歧化酶(SOD)等成分,将其混合后用于化妆品的制备。
2、本专利方法获得的化妆品能有效的抑制各类细菌及真菌的生长。较传统化学抑菌产品而言,更为安全温和有效,无毒副作用,能避免常规抑菌产品耐药性和过敏现象的发生。
3、由于本发明的抑菌组合物应用化妆品领域,因此,鲎素肽的提取采用的是快速,便捷的低成本提取方式,提取出的鲎素肽粗提液完全能够满足日常性抗菌,消炎,防腐性用品的需要。
4、本专利方法获得的化妆品具有较强的抗紫外辐射能力。作为护肤品使用,可以抵御辐射对人体的肌肤损伤,减缓皮肤老化。
附图说明
图1是本发明化妆品在波长280-400nm范围内的紫外吸收光谱
具体实施方式
下面结合具体实施方案对本发明进一步说明,其具体实施方案应该理解为仅为举例说明,不是限定性的,不能以下述举例说明来限定本发明的保护范围。
一种从鲎试剂废料中提取抑菌组合物制备化妆品的方法,该方法包括步骤如下:
第一步,在鲎试剂废料中提取抑菌组合物
(1)原料处理
收集鲎血细胞及血浆,作为原料备用;其中,所述鲎血细胞是鲎试剂生产工艺中丢弃的乳化物沉淀,所述血浆是鲎试剂生产过程中丢弃的血浆;
(2)鲎素肽粗提液的制备
a.酸解:将鲎细胞乳化物离心后的下层沉淀以固液比例1:6.5-8的比例加入酸液(20m mol/L),低温超声破碎,破碎条件为:100ml超声波输出功率为700W,超声波总作用时间为10min,每次超声作用时间为10S,反复超声3次,收集上清液即为细胞酸抽提液;
b.调pH去沉淀:将抽提液用NaOH调节pH至6.5,离心除沉淀,得上清液;
c.热变性处理:上清液沸水浴处理5-10min,4℃下离心弃沉淀收集上清液,上清液用0.22uM膜过滤得鲎素肽粗提液;
(3)血浆中血蓝蛋白粗提液的制备
将上述步骤(1)中的血浆经300000g超离心3.5h,即得到初步提纯的血蓝蛋白沉淀,将沉淀再溶于pH7.0,0.2mol/L磷酸缓冲液中,即为血蓝蛋白粗提液;
(4)血浆中SOD粗提液的制备
按质量份数计,取100份上述步骤(1)中的鲎血浆,加入34份0.05mol/L磷酸盐缓冲液和100份0.9%NaCl溶液,轻轻搅拌混匀,将混合液置于65℃水浴锅中,恒温20min后,急冷至10℃,离心去除沉淀,取上清液,即为SOD粗提液;
(5)粗提液的混合
将上述三种粗提液按60-80份的鲎素肽粗提液,10-20份的血蓝蛋白提取液,10-20份的SOD提取液混合,使用pH调节剂调节pH值4-5后过滤,得到最终形式的抑菌组合物。
其中,所述pH调节剂为柠檬酸、柠檬酸钠、乳酸、磷酸氢二钠、磷酸二氢钠、磷酸二氢钾、硼酸、硼砂中的一种或多种的组合。
第二步,由上述抑菌组合物制备化妆品
(1)按如下质量百分比配置化妆品包括的组份:
(2)得到化妆品成品
a.上述化妆品组份中包括油相的维生素E醋酸酯、二乙二醇脂肪酸酯、赋形增稠剂及橄榄油、去离子水和本发明所述的抑菌组合物,先使化妆品原料的油相中维生素E醋酸酯、二乙二醇脂肪酸酯及赋形增稠剂和去离子水进行初步乳化;
b.调整上述乳化体系的温度,降温至35~40℃,加入橄榄油、抑菌组合物,体系经搅拌均匀,进一步降温后完成操作,然后灌装得成品。
抑菌组合物的抑菌效果实验
(1)组成
抑菌组合物由鲎素肽粗提液,血蓝蛋白提取液,SOD提取液按一定配比组合而成。
2抑菌试验
A实验材料
由鲎素肽粗提液、血蓝蛋白提取液、SOD提取液组合的抑菌组合物。
B受试菌种
细菌:金黄色葡萄球菌:(Staphylococcus aureus)ATCC 6538
大肠埃希氏菌:(Escherichia coli)ATCC 8739
铜绿假单胞菌:(Pseudomonas.aeruginosa)ATCC 9027
注:上述菌株均由中科院典型培养物保藏委员会微生物菌种库提供。
C培养基
细菌培养基:卵磷脂吐温80-营养琼脂培养基,121℃高压灭菌20min后制成斜面备用。
D菌悬液的制备
实验前将各个菌株分别接种到相应的培养基斜面,细菌(金黄色葡萄球菌,大肠埃希氏菌,铜绿假单胞菌)均于36±1℃恒温培养箱中培养36~48小时。分别挑取适量菌落于无菌生理盐水中混匀,制成一定浓度的混合菌悬液。混合细菌悬液总浓度约为1.0×108cfu/ml。置于4℃贮存备用。
E抑菌组合物抑菌液的的制备
按照正交设计法确定各提取液组合的配比,将四种提取液按表1的方案分别进行配制后,用纯净水以2:3比例进一步稀释至化妆品中添加浓度,备用。
表1复合提取液抑菌作用考察因素及水平
F提取液的抑菌试验
采用滤纸片扩散法,无菌条件下进行,将滤纸用打孔器打成直径6mm的圆形滤纸片,置于洁净干净的小烧杯,121℃干热灭菌20min后,分别放入4种不同比例提取液复配液中充分浸泡,每只试管放入8片备用。将已配制好的固体培养基融化,分别倒入培养皿中,121℃灭菌20min,待冷却凝固后,吸取0.1ml菌悬液于平板上涂布均匀。用无菌镊子分别夹取已在提取物原液中浸泡过的直径为6mm圆形滤纸片,贴在含菌平板上,每个平板内贴5片,以无菌水浸泡的滤纸片作为空白对照,将各培养皿置于37℃恒温培养箱中平板倒置培养24h,测定抑菌圈直径的大小,设3个重复,取其平均值。混合细菌悬液的抑菌圈直径见表2
表2
由抑菌圈大小可以看出,提取液配比为8:1:1或6:2:2时,抑菌作用明显。
化妆品抗紫外实验
将上述化妆品1.0g,分别置于25mL比色管中,用60%乙醇定容至刻度,用紫外可见分光光度计在波长280~400nm范围内扫描,确定其紫外吸收光谱,计算本化妆品在UVB区(280~320nm)和UVA区(320~400nm)的平均吸光度值,评价其抗紫外线辐射能力。
结果与分析
按上述方法,测得本化妆品在波长280~400nm范围内的紫外吸收光谱如图1所示。计算得该化妆品在UVB区(280~320nm)和UVA区(320~400nm)的平均吸光度值分别为0.348和0.266。可见,本化妆品在紫外线UVB区和UVA区均具有较强的抗紫外线辐射能力,即具有广谱抗紫外线能力。同时也表明,本化妆品抗UVB区紫外线的能力强于抗UVA区紫外线的能力。
Claims (4)
1.一种从鲎试剂废料中提取抑菌组合物制备化妆品的方法,其特征在于该方法包括步骤如下:
第一步,在鲎试剂废料中提取抑菌组合物
(1)原料处理
收集鲎血细胞及血浆,作为原料备用;其中,所述鲎血细胞是鲎试剂生产工艺中丢弃的乳化物沉淀,所述血浆是鲎试剂生产过程中丢弃的血浆;
(2)鲎素肽粗提液的制备
a.酸解:将鲎细胞乳化物离心后的下层沉淀以固液比例1:6.5-8的比例加入酸液(20mmol/L),低温超声破碎,破碎条件为:100ml超声波输出功率为700W,超声波总作用时间为10min,每次超声作用时间为10S,反复超声3次,收集上清液即为细胞酸抽提液;
b.调pH去沉淀:将抽提液用NaOH调节pH至6.5,离心除沉淀,得上清液;
c.热变性处理:上清液沸水浴处理5-10min,4℃下离心弃沉淀收集上清液,上清液用0.22uM膜过滤得鲎素肽粗提液;
(3)血浆中血蓝蛋白粗提液的制备
将上述步骤(1)中的血浆经300000g超离心3.5h,即得到初步提纯的血蓝蛋白沉淀,将沉淀再溶于pH7.0,0.2mol/L磷酸缓冲液中,即为血蓝蛋白粗提液;
(4)血浆中SOD粗提液的制备
按质量份数计,取100份上述步骤(1)中的鲎血浆,加入34份0.05mol/L磷酸盐缓冲液和100份0.9%NaCl溶液,轻轻搅拌混匀,将混合液置于65℃水浴锅中,恒温20min后,急冷至10℃,离心去除沉淀,取上清液,即为SOD粗提液;
(5)粗提液的混合
将上述三种粗提液按60-80份的鲎素肽粗提液,10-20份的血蓝蛋白提取液,10-20份的SOD提取液混合,使用pH调节剂调节pH值4-5后过滤,得到最终形式的抑菌组合物。
第二步,由上述抑菌组合物制备化妆品
(1)按如下质量百分比配置化妆品包括的组份:
(2)得到化妆品成品
A.将上述化妆品组份中包括的部分油相与去离子水进行初步乳化;
b.调整上述乳化体系温度至35℃~40℃,加入橄榄油、抑菌组合物,体系经搅拌均匀降温后得成品。
2.根据权利要求1所述的从鲎试剂废料中提取抑菌组合物制备化妆品的方法,其特征在于:所述第一步的步骤(5)中的pH调节剂为柠檬酸、柠檬酸钠、乳酸、磷酸氢二钠、磷酸二氢钠、磷酸二氢钾、硼酸、硼砂中的一种或多种的组合。
3.根据权利要求1所述的从鲎试剂废料中提取抑菌组合物制备化妆品的方法,其特征在于:所述第一步的步骤(5)中抑菌组合物的鲎素肽粗提液﹕血蓝蛋白粗提液﹕SOD粗提液的最佳配比为:8﹕1﹕1或6﹕2﹕2。
4.根据权利要求1所述的从鲎试剂废料中提取抑菌组合物制备化妆品的方法,其特征在于:所述第二步的(2)步中进行初步乳化的油相组份包括维生素E醋酸酯、二乙二醇脂肪酸酯及赋形增稠剂。
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CN108998427B (zh) * | 2018-07-27 | 2021-07-06 | 广东海洋大学 | 一种酚氧化酶活性蛋白的制备方法 |
CN110898080A (zh) * | 2019-11-01 | 2020-03-24 | 广西中医药大学 | 鲎血浆在促生长发育中的应用 |
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