CN118178300A - Preparation of anti-wrinkle and moisturizing freeze-dried powder composition - Google Patents
Preparation of anti-wrinkle and moisturizing freeze-dried powder composition Download PDFInfo
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- CN118178300A CN118178300A CN202410334506.XA CN202410334506A CN118178300A CN 118178300 A CN118178300 A CN 118178300A CN 202410334506 A CN202410334506 A CN 202410334506A CN 118178300 A CN118178300 A CN 118178300A
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- freeze
- dried powder
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- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
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Abstract
Description
技术领域Technical Field
本发明涉及化妆品技术领域,具体涉及一种抗皱保湿冻干粉组合物的制备。The invention relates to the technical field of cosmetics, and in particular to the preparation of an anti-wrinkle and moisturizing freeze-dried powder composition.
背景技术Background technique
冻干粉作为化妆品领域的一颗璀璨新星,正以其独特的技术优势和显著的应用效果,展现出强劲的发展趋势。As a bright new star in the cosmetics field, freeze-dried powder is showing a strong development trend with its unique technical advantages and remarkable application effects.
随着冻干技术的不断进步和成本的降低,越来越多的化妆品企业开始采用这一技术,将高活性的护肤成分以冻干粉的形式保存,从而保证了成分的纯度和稳定性。同时,随着消费者对温和、高效护肤产品的追求,冻干粉以其温和不刺激、高显效的特性,赢得了越来越多消费者的青睐。With the continuous advancement of freeze-drying technology and the reduction of costs, more and more cosmetics companies have begun to adopt this technology to store highly active skin care ingredients in the form of freeze-dried powder, thereby ensuring the purity and stability of the ingredients. At the same time, as consumers pursue mild and effective skin care products, freeze-dried powder has won the favor of more and more consumers with its mild, non-irritating and highly effective characteristics.
在化妆品中,冻干粉被广泛应用于美白、保湿、抗衰、修护等多种功效的产品中,成为实现定制化护肤的重要载体。未来,随着市场需求的不断增长和技术创新的持续推动,冻干粉在化妆品领域的应用将更加广泛,为消费者带来更多安全、有效的护肤选择。In cosmetics, freeze-dried powder is widely used in products with various functions such as whitening, moisturizing, anti-aging, and repair, becoming an important carrier for customized skin care. In the future, with the continuous growth of market demand and the continuous promotion of technological innovation, the application of freeze-dried powder in the cosmetics field will be more extensive, bringing consumers more safe and effective skin care options.
本发明目的在于提供一种抗皱保湿冻干粉组合物以及其制备方法,以解决现有护肤品需要提供最基本的保湿性能基础上,还需赋予其抗皱性能的问题。The present invention aims to provide an anti-wrinkle and moisturizing freeze-dried powder composition and a preparation method thereof, so as to solve the problem that existing skin care products need to provide anti-wrinkle properties on top of the most basic moisturizing properties.
发明内容Summary of the invention
为了实现上述发明目的,本发明提供了一种抗皱保湿冻干粉组合物,所述组合物包含交替单胞菌发酵产物提取物、可溶性胶原蛋白、猪脐带提取物以及荇菜愈伤组织细胞提取物;除此之外,其中还包括抗氧化剂抗坏血酸。In order to achieve the above-mentioned purpose of the invention, the present invention provides an anti-wrinkle and moisturizing freeze-dried powder composition, which comprises an extract of a fermentation product of Alteromonas, soluble collagen, a porcine umbilical cord extract and a Nymphoides callus cell extract; in addition, it also comprises an antioxidant ascorbic acid.
交替单胞菌(Alteromonas macleodii)发酵产物提取物,交替单胞菌发酵产物提取物,其中所含有的抗菌肽、酶类、多糖、生物碱以及酮类酚类等化合物为皮肤提供抗皱、保湿、抑菌等多种功效。Alteromonas macleodii fermentation product extract, Alteromonas macleodii fermentation product extract, the antimicrobial peptides, enzymes, polysaccharides, alkaloids, ketones, phenols and other compounds contained in it provide anti-wrinkle, moisturizing, antibacterial and other effects to the skin.
可溶性胶原蛋白(SOLUBLE COLLAGEN),一种通过基因重组和生物工程技术所获得的胶原蛋白,其具有出色的水溶性,在护肤领域广泛应用;因其氨基酸序列与人类自身胶原蛋白高度相似,因此具有良好的组织相容性,使得其更容易被人体吸收。Soluble collagen (SOLUBLE COLLAGEN), a type of collagen obtained through genetic recombination and bioengineering technology, has excellent water solubility and is widely used in the field of skin care. Because its amino acid sequence is highly similar to human collagen, it has good tissue compatibility, making it easier to be absorbed by the human body.
脐带提取物(猪)(UMBILICAL EXTRACT),一种从动物脐带中提取出来的物质,富含多种营养成分及生长因子,具有多种生物活性,被广泛应用于美容领域,其具有滋养,增强皮肤保湿能力,减少自由基对皮肤的损害,延缓皮肤衰老等诸多优点。Umbilical cord extract (pig) is a substance extracted from the umbilical cord of animals. It is rich in various nutrients and growth factors, has multiple biological activities, and is widely used in the field of beauty. It has many advantages, such as nourishing, enhancing skin's moisturizing ability, reducing free radical damage to the skin, and delaying skin aging.
荇菜(Nymphoides peltatum)愈伤组织细胞提取物,通过对荇菜叶片进行植物组培养出的分裂期愈伤细胞进行提取冻干所得到的生物活性成分,其中富含多糖,蛋白质,类黄酮,多酚,胡萝卜素等活性物质,具有显著的抗氧化。抗菌,抗炎等生物活性。Nymphoides peltatum callus cell extract is a bioactive ingredient obtained by extracting and freeze-drying callus cells in the division stage of Nymphoides peltatum leaves. It is rich in polysaccharides, proteins, flavonoids, polyphenols, carotene and other active substances, and has significant antioxidant, antibacterial, anti-inflammatory and other biological activities.
为了实现上述目的,本发明采用以下技术方案:In order to achieve the above object, the present invention adopts the following technical solutions:
一种抗皱保湿组合物,以质量份计算包括以下组分:An anti-wrinkle and moisturizing composition, comprising the following components in parts by mass:
交替单胞菌发酵产物提取物0.1-2份;0.1-2 parts of Alteromonas fermentation product extract;
可溶性胶原蛋白0.5-4份;Soluble collagen 0.5-4 parts;
猪脐带提取物0.1-0.5份;0.1-0.5 parts of pig umbilical cord extract;
荇菜愈伤组织细胞提取物0.1-1份。0.1-1 part of Nymphoides callus cell extract.
特别地,所述荇菜愈伤组织细胞提取物的制备方法为:以其叶为外植体材料,灭菌,植入培养基;具体操作如下:In particular, the preparation method of the Nymphoides callus cell extract is: using its leaves as explant materials, sterilizing, and implanting into a culture medium; the specific operations are as follows:
选取纯度高,具有荇菜典型特征,生长健壮,无伤,无病虫荇菜植株的新鲜嫩芽叶,使用75%乙醇擦拭叶面消毒后,剪切为2-6mm见方嫩芽叶组织。Select fresh tender buds and leaves of water lily plants with high purity, typical characteristics of water lily, healthy growth, no injuries, and no diseases or insects. After wiping the leaf surface with 75% ethanol for disinfection, cut the tender bud and leaf tissue into 2-6 mm square pieces.
在消毒完成的接种室内,经无菌操作程序将嫩芽叶组织接种于培养瓶中。In the disinfected inoculation room, the young bud and leaf tissues are inoculated into culture bottles using aseptic operation procedures.
将接种完毕的培养瓶置入避光,恒温培养箱中,在愈伤组织细胞处于分裂期时,取出,超声破碎,过滤提取细胞液,冻干,得到愈伤组织细胞提取物。The inoculated culture bottle is placed in a light-proof, constant-temperature incubator. When the callus cells are in the division stage, they are taken out, ultrasonically broken, filtered to extract the cell fluid, and freeze-dried to obtain a callus cell extract.
特别地,所述培养基配置方法具体操作如下:In particular, the culture medium configuration method is specifically performed as follows:
S1.称量MS培养基、蔗糖、植物生长激素;S1. Weigh MS medium, sucrose, and plant growth hormone;
S2.常压下将水加热至沸腾后,加入S1,搅拌,调节pH至5.5-6;S2. Heat water to boiling under normal pressure, add S1, stir, and adjust the pH to 5.5-6;
S3.将S2分装,灭菌,等待接种外植体。S3. Package S2, sterilize, and wait for explant inoculation.
优选地本发明提供一种驻留类皮肤外用剂,其特征在于,其包含,组合物3%份、溶媒剂97%。Preferably, the present invention provides a leave-on skin external preparation, characterized in that it comprises 3% of the composition and 97% of the solvent.
优选地,所述溶媒剂,其特征在于包含水和透明质酸钠。Preferably, the solvent is characterized by comprising water and sodium hyaluronate.
本发明还提供一种精华液制备方法,其特征在于,所述制备方法如下:The present invention also provides a method for preparing an essence, characterized in that the preparation method is as follows:
溶媒剂中加入组合物,搅拌均匀,得到精华液;Add the composition into the solvent and stir evenly to obtain an essence;
与现有技术相比,本发明的有益效果在于;本发明开创性地使用荇菜愈伤组织细胞提取物与交替单胞菌发酵产物提取物、可溶性胶原蛋白以及猪脐带提取物混合使用,荇菜愈伤组织中所富含的胡萝卜素以及酮类及多酚可以有效地改善皮肤粗糙状态,使皮肤更加细腻光滑,其次胡萝卜素作为Va前体,可在人体内转化为Va,Va对于皮肤健康至关重要,可以有效刺激黏膜细胞持续分泌黏液,覆盖细胞表面,防止细菌侵蚀,维持皮肤黏膜完整性,且Va还可增强皮肤韧性。荇菜愈伤组织提取物与可溶性胶原蛋白和交替单胞菌发酵产物连用可以在修复皮肤粗糙状态的同时提高皮肤含水率,保湿度以及提高对皮肤中弹性蛋白酶的抑制率,使得皮肤更有活力。与此同时,脐带提取物中所蕴含的硫酸软骨素,糖原,脂质和大量的蛋白质和胶原蛋白及时滋养皮肤组织,与荇菜愈伤组织细胞提取物,可溶性胶原蛋白以及交替单胞菌发酵产物提取物协同作用,使组合物功效最大化。Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention innovatively uses water lily callus cell extracts mixed with alternatormonas fermentation product extracts, soluble collagen and porcine umbilical cord extracts, and the carotene, ketones and polyphenols rich in water lily callus can effectively improve the rough skin state and make the skin more delicate and smooth. Secondly, carotene, as a Va precursor, can be converted into Va in the human body. Va is essential for skin health and can effectively stimulate mucosal cells to continuously secrete mucus, cover the cell surface, prevent bacterial erosion, maintain the integrity of the skin mucosa, and Va can also enhance skin toughness. Water lily callus extracts combined with soluble collagen and alternatormonas fermentation products can improve the skin's moisture content, moisture retention and inhibition rate of elastase in the skin while repairing the rough skin state, making the skin more energetic. At the same time, the chondroitin sulfate, glycogen, lipids and a large amount of protein and collagen contained in the umbilical cord extract nourish the skin tissue in time, and work synergistically with the water spinach callus cell extract, soluble collagen and Alteromonas fermentation product extract to maximize the efficacy of the composition.
经实验发现,所述组合物可修复各种原因引起的皮肤损伤,具有滋养、抗皱、保湿的效果,适用于各种肤质。本发明发现,在相同使用量的前提下,本发明所限定的特定比例的荇菜愈伤组织细胞提取物与交替单胞菌发酵产物提取物、可溶性胶原蛋白以及猪脐带提取物比单独使用,或者其中任意三种及以上共同使用的效果更好,产生协同增效的效果,使其滋养、抗皱、保湿功效更好。本发明还提供了所述含有抗皱保湿组合物精华液的优选配方及制备方式。It has been found through experiments that the composition can repair skin damage caused by various reasons, has the effects of nourishing, anti-wrinkle and moisturizing, and is suitable for all skin types. The present invention has found that under the premise of the same usage amount, the specific ratio of Nymphoides callus cell extract and Alteromonas fermentation product extract, soluble collagen and porcine umbilical cord extract defined in the present invention has better effects than using them alone or any three or more of them together, producing a synergistic effect, making its nourishing, anti-wrinkle and moisturizing effects better. The present invention also provides a preferred formula and preparation method of the essence containing the anti-wrinkle and moisturizing composition.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1实施例4-6HaCaT存活率。Figure 1 Example 4-6 HaCaT survival rate.
图2实施例1-3及对比例1-5透明质酸酶抑制率。FIG. 2 shows the hyaluronidase inhibition rates of Examples 1-3 and Comparative Examples 1-5.
图3实施例1-3及对比例1-5TNF-α炎症因子抑制率。FIG3 shows the inhibition rate of TNF-α inflammatory factor in Examples 1-3 and Comparative Examples 1-5.
图4实施例1-3及对比例1-5斑马鱼尾面积缩小抑制率。FIG. 4 shows the inhibition rate of zebrafish tail area reduction in Examples 1-3 and Comparative Examples 1-5.
图5实施例1-3及对比例1-5Hacat细胞迁移率。Fig. 5 shows the migration rate of Hacat cells in Examples 1-3 and Comparative Examples 1-5.
图6实施例4-6及对比例6-7相对经皮失水率。Figure 6 Relative transepidermal water loss rates of Examples 4-6 and Comparative Examples 6-7.
具体实施方式Detailed ways
为更好地说明本发明的目的、技术方案和优点,本发明通过下列实施例进一步说明。应理解,本发明实施例仅用于说明本发明技术效果,而非用于限制本发明的保护范围,实施例中,所使用方法如无特殊说明,均为常规方法。In order to better illustrate the purpose, technical scheme and advantages of the present invention, the present invention is further described by the following examples. It should be understood that the examples of the present invention are only used to illustrate the technical effects of the present invention, but not to limit the scope of protection of the present invention. In the examples, the methods used are conventional methods unless otherwise specified.
本发明中的部分原料来源如下:Some of the raw materials in the present invention come from the following sources:
交替单胞菌发酵产物提取物:购自陕西今汉生物科技有限公司。Alteromonas fermentation product extract: purchased from Shaanxi Jinhan Biotechnology Co., Ltd.
可溶性胶原蛋白:购自湖北迈德昊化工有限公司。Soluble collagen: purchased from Hubei Maidehao Chemical Co., Ltd.
猪脐带提取物:购自陕西泽朗生物科技有限公司。Pig umbilical cord extract: purchased from Shaanxi Zelang Biotechnology Co., Ltd.
荇菜:山东嘉祥水生植物种植有限公司。Nymphoides: Shandong Jiaxiang Aquatic Plant Planting Co., Ltd.
荇菜提取物:购自陕西百川生物科技有限公司。Nymphoides extract: purchased from Shaanxi Baichuan Biotechnology Co., Ltd.
MS培养基:购自西格玛奥德里奇(上海)贸易有限公司。MS medium: purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.
所述培养基配置方法具体配置步骤如下:The specific configuration steps of the culture medium configuration method are as follows:
称量药品:Weighing medicines:
S1:MS培养基4.5g,蔗糖30g,NAA(0.3mg/L)1.5mL,6-BA(1.0mg/L)5mL;S1: MS medium 4.5 g, sucrose 30 g, NAA (0.3 mg/L) 1.5 mL, 6-BA (1.0 mg/L) 5 mL;
S2:MS培养基4.5g,蔗糖30g,NAA(0.3mg/L)2mL,6-BA(1.0mg/L)5mL;S2: MS medium 4.5 g, sucrose 30 g, NAA (0.3 mg/L) 2 mL, 6-BA (1.0 mg/L) 5 mL;
S3:MS培养基4.5g,蔗糖30g,NAA(0.3mg/L)2.5mL,6-BA(1.0mg/L)5mLS3: MS medium 4.5 g, sucrose 30 g, NAA (0.3 mg/L) 2.5 mL, 6-BA (1.0 mg/L) 5 mL
将称量好的药品加入90-100℃的1L去离子水中;搅拌均匀至透明;调节pH至5.8-6.0;分装至培养瓶中,而后灭菌;等待外植体接种。Add the weighed medicine into 1L deionized water at 90-100°C; stir evenly until transparent; adjust the pH to 5.8-6.0; dispense into culture bottles and then sterilize; wait for explant inoculation.
荇菜愈伤组织提取物的制备包括以下步骤:The preparation of Nymphoides callus extract comprises the following steps:
选取纯度高,具有荇菜典型特征,生长健壮,无伤,无病虫荇菜植株的新鲜嫩芽叶,使用75%乙醇擦拭叶面消毒后,剪切为2-6mm见方嫩芽叶组织。Select fresh tender buds and leaves of water lily plants with high purity, typical characteristics of water lily, healthy growth, no injuries, and no diseases or insects. After wiping the leaf surface with 75% ethanol for disinfection, cut the tender bud and leaf tissue into 2-6 mm square pieces.
在消毒完成的接种室内,经无菌操作程序将嫩芽叶组织分别接种于含有S1、S2、S3培养基的培养瓶中。In the disinfected inoculation room, the tender shoot and leaf tissues were inoculated into culture bottles containing S1, S2, and S3 culture media respectively through aseptic operation procedures.
将接种完毕的培养瓶置入避光,恒温培养箱中,在愈伤组织细胞处于分裂期时,取出,超声破碎,过滤提取细胞液,合并S1、S2、S3提取细胞液,冻干,得到愈伤组织细胞提取物。The inoculated culture bottle is placed in a light-proof, constant temperature incubator. When the callus cells are in the division stage, they are taken out, ultrasonically broken, filtered and the cell fluid is extracted. The cell fluids extracted from S1, S2 and S3 are combined and freeze-dried to obtain callus cell extracts.
一种溶媒剂的制备包括以下步骤:The preparation of a solvent comprises the following steps:
在水中加入透明质酸钠搅拌均匀即可得到溶媒剂。The solvent can be obtained by adding sodium hyaluronate into water and stirring evenly.
一种精华液的制备,其步骤包括:A preparation method of an essence, comprising the steps of:
溶媒剂中加入组合物,搅拌均匀,得到精华液。The composition is added into the solvent and stirred evenly to obtain the essence.
组合物质量分配比具体见表1。The specific mass distribution ratio of the composition is shown in Table 1.
精华液质量分配比具体见表2。The specific mass distribution ratio of the essence is shown in Table 2.
表1组合物质量份数配比Table 1 Composition mass ratio
表2精华液质量百分比配比Table 2 Essence mass percentage ratio
安全性测试Safety Testing
以2015年《化妆品安全技术规范》为参照标准,对实施例4-6的组合物、进行化妆品刺激性评价,测试方法为皮肤贴斑试验,对年龄16-45岁随机分布的30人进行试验。With reference to the 2015 Technical Specifications for Safety of Cosmetics, the compositions of Examples 4-6 were evaluated for cosmetic irritation. The test method was a skin patch test, and 30 randomly distributed people aged 16-45 years were tested.
测试方法:将受试物放入斑试器中,用量为0.020-0.025g,将加有受试物的斑试器用无刺激布基胶带覆于受试者背部或前臂曲侧,用手掌轻压使之均匀贴附在皮肤表面,持续24小时,去除受试斑试器后30分钟,待压痕消失后观察皮肤反应。若结果为阴性,于斑贴试验后24小时和48小时分别再次进行观察。Test method: Put the test substance into the patch tester, the dosage is 0.020-0.025g, cover the patch tester with the test substance on the back or forearm of the subject with non-irritating cloth-based tape, and gently press it with the palm to make it evenly adhere to the skin surface for 24 hours. After removing the test patch tester, observe the skin reaction after the indentation disappears 30 minutes later. If the result is negative, observe again 24 hours and 48 hours after the patch test.
评价标准:evaluation standard:
0级:阴性反应;Grade 0: negative reaction;
1级:可疑反应,仅有微弱红斑;Grade 1: Suspicious reaction, only slight erythema;
2级:弱阳性反应,红斑、浸润、水肿、可有丘疹;Grade 2: weak positive reaction, erythema, infiltration, edema, and papules may be present;
3级:强阳性反应,红斑、浸润、水肿、可有丘疹、反应可超出受试区;Grade 3: Strong positive reaction, erythema, infiltration, edema, papules, and the reaction may extend beyond the test area;
4级:极强阳性反应,明显红斑、严重浸润、水肿、融合性疱疹、反应超出受试区。Grade 4: Very strong positive reaction, obvious erythema, severe infiltration, edema, confluent herpes, and reaction beyond the test area.
测试结果:所有受试者的皮肤反应均为阴性。Test results: Skin reactions were negative in all subjects.
上述测试结果表明,本发明提供的组合物对皮肤温和无刺激,使用安全。The above test results show that the composition provided by the present invention is mild and non-irritating to the skin and is safe to use.
细胞毒性测试Cytotoxicity test
取生长状态稳定的人永生化表皮细胞系(HaCaT),调整细胞浓度至1×105/mL,以每孔100μL.接种到96孔细胞培养板中(除最外周孔及空白对照孔)。在细胞培养板的最外周孔中加入PBS200μL,以减少培养基的蒸发。置二氧化碳培养箱中,在37℃±1℃,CO2浓度5.0%±1%条件下进行培养待细胞融合达到90%以上。加入样品,样品浓度为10mg/L,设置空白对照、样品组,每孔100μL。开始加样品(样品组)、PBS(空白对照)时计时,保持每两列加样时间间隔一致,保证每个受试物对细胞的干预时间是5min,5min后,依次吸去受试物,每孔加入PBS清洗受试物,清洗2次。用真空吸液泵或排枪吸去孔中液体时,小心操作,以免吸去存活细胞而影响实验结果。在每孔中分别加入100μL完全培养基和10μL CCK-8溶液,置于培养箱中培养2h左右。在450nm波长,测定吸光度(OD)值和计算细胞活力;结果如表3所示。Take the human immortalized epidermal cell line (HaCaT) with stable growth state, adjust the cell concentration to 1×10 5 /mL, and inoculate 100μL per well into a 96-well cell culture plate (except the outermost well and blank control well). Add 200μL of PBS to the outermost well of the cell culture plate to reduce the evaporation of the culture medium. Place in a carbon dioxide incubator and culture at 37℃±1℃, CO 2 concentration 5.0%±1% until the cell fusion reaches more than 90%. Add the sample, the sample concentration is 10mg/L, set up blank control and sample group, 100μL per well. Start to add the sample (sample group) and PBS (blank control) at the beginning of the time, keep the time interval between each two columns of sample addition consistent, ensure that the intervention time of each test substance on the cell is 5min, after 5min, suck out the test substance in turn, add PBS to each well to wash the test substance, wash twice. When using a vacuum suction pump or a discharge gun to suck out the liquid in the well, be careful to avoid sucking out the surviving cells and affecting the experimental results. 100 μL of complete medium and 10 μL of CCK-8 solution were added to each well, and the cells were placed in an incubator for about 2 hours. The absorbance (OD) value was measured at a wavelength of 450 nm and the cell viability was calculated; the results are shown in Table 3.
表3细胞毒性试验Table 3 Cytotoxicity test
据表3实验结果可知,细胞存活率保持95%以上。本实验搭配组合物对HaCaT细胞活力影响较小。According to the experimental results in Table 3, the cell survival rate was maintained above 95%. The combination of the present experiment had little effect on the viability of HaCaT cells.
透明质酸酶活性抑制实验Hyaluronidase activity inhibition assay
透明质酸又名玻尿酸,是一种强效保湿剂。可以吸引并锁住水分,保持皮肤湿润度,改善干燥、粗糙和缺水的皮肤状况;且透明质酸也能够增强细胞的生物活性,对于修复,保护皮肤具有较强的增效作用。因此通过对透明质酸酶的抑制,使得皮肤中的透明质酸被分解速度降低,从而实现对皮肤的长效保水保湿及修护具有重要意义。Hyaluronic acid, also known as hyaluronic acid, is a powerful moisturizer. It can attract and lock in moisture, maintain skin moisture, and improve dry, rough and dehydrated skin conditions. Hyaluronic acid can also enhance the biological activity of cells and has a strong synergistic effect on repairing and protecting the skin. Therefore, by inhibiting hyaluronidase, the decomposition rate of hyaluronic acid in the skin is reduced, which is of great significance for achieving long-term moisture retention and repair of the skin.
(1)取四组干净的试管,分别标号为A、B、C、D,在A、B试管中各加入待测样品10mg/L50μL,在C、D试管中各加入蒸馏水50μL,在A、C试管中各加入500U/mL透明质酸酶溶液50μL,在B、D试管中各加入pH=5.6的醋酸缓冲液50μL,将试管放置于37℃保温箱保温20min。(1) Take four groups of clean test tubes, label them A, B, C, and D, add 50 μL of the sample (10 mg/L) to test tubes A and B, add 50 μL of distilled water to test tubes C and D, add 50 μL of 500 U/mL hyaluronidase solution to test tubes A and C, add 50 μL of pH 5.6 acetate buffer to test tubes B and D, and place the test tubes in a 37°C incubator for 20 min.
(2)向四组试管中分别加入2.5mol/L的CaCl2溶液10μL,然后置于37℃保温箱保温20min。(2) Add 10 μL of 2.5 mol/L CaCl 2 solution to each of the four test tubes and then place them in an incubator at 37°C for 20 min.
(3)向A、C试管中加入0.5mg/mL的透明质酸钠溶液50μL,向B、D试管中加入pH=5.6的醋酸缓冲液50μL,将试管置于37℃培养箱保温40min,然后取出室温放置10min。(3) Add 50 μL of 0.5 mg/mL sodium hyaluronate solution to test tubes A and C, and add 50 μL of pH 5.6 acetate buffer to test tubes B and D. Place the test tubes in a 37°C incubator for 40 min and then take them out and place them at room temperature for 10 min.
(4)向四组试管里分别加入蒸馏水50μL、5mol/L的NaOH溶液10μL、乙酰丙酮溶液50μL,将试管置于水浴锅中沸水浴30min,然后冰浴10min,最后在室温放置10min。(4) Add 50 μL of distilled water, 10 μL of 5 mol/L NaOH solution, and 50 μL of acetylacetone solution to each of the four test tubes. Place the test tubes in a boiling water bath for 30 min, then place them in an ice bath for 10 min, and finally place them at room temperature for 10 min.
(5)向各试管中加入P-DAB试剂100μL,然后用酶标仪在530nm处测定吸光值。(5) Add 100 μL of P-DAB reagent to each test tube, and then measure the absorbance at 530 nm using an ELISA reader.
式中:A为试样溶液的OD值;B为试样空白的OD值;C为对照样品溶液的OD值;D为对照样品空白的OD值。Wherein: A is the OD value of the sample solution; B is the OD value of the sample blank; C is the OD value of the control sample solution; D is the OD value of the control sample blank.
表4透明质酸酶活性抑制率Table 4 Hyaluronidase activity inhibition rate
从表4结果分析:从实施例1-3结果可知,本发明实施例提供的组合物具有较好的透明质酸酶抑制功效,尤其是实施例2的效果较好,From the results in Table 4, it can be seen from the results of Examples 1-3 that the compositions provided by the embodiments of the present invention have good hyaluronidase inhibition effects, especially the effect of Example 2 is better.
从实施例2及对比例1-5结果来看,表明使用本发明所限定的组分存在一定的协同增效作用,缺省、替换任意一项均不能达到本发明所达到的效果。From the results of Example 2 and Comparative Examples 1-5, it is shown that there is a certain synergistic effect when using the components defined in the present invention, and the effect achieved by the present invention cannot be achieved by omitting or replacing any one of them.
炎症因子抑制试验Inflammatory factor inhibition test
TNF-α是炎症反应中最常见的炎症因子,可诱发血管内皮细胞的表型变为炎症型,导致机体代谢和血液动力学改变,促进炎症介质生成。而炎症介质又能导致血管通透性增大,从而造成局部充血、水肿,同时可能刺激神经末梢导致皮肤瘙痒,疼痛。因此对TNF-α的抑制有助于舒缓,修护皮肤。TNF-α is the most common inflammatory factor in inflammatory reactions. It can induce the phenotype of vascular endothelial cells to change to an inflammatory type, leading to changes in the body's metabolism and hemodynamics, and promoting the production of inflammatory mediators. Inflammatory mediators can increase vascular permeability, causing local congestion and edema, and may stimulate nerve endings to cause itching and pain. Therefore, the inhibition of TNF-α helps soothe and repair the skin.
将生长至对数期的RAW264.7细胞以1.5×105个/mL的密度接种在24孔板,每孔800μL细胞悬浮液,在37℃、5%CO2培养箱培养24h,待细胞生长至将近融合时,以样品浓度10mg/L 50μL进行给药,每组设3个复孔,阳性对照为10μg/mL 50μL地塞米松,1h后用1μg/mL脂多糖(LPS)刺激。在细胞培养箱孵育24h后,将细胞培养液转入离心管,5000r/min离心10min收集细胞上清液,按小鼠TNF-α酶联免疫检测试剂盒说明书进行检测。RAW264.7 cells grown to the logarithmic phase were inoculated in a 24-well plate at a density of 1.5×10 5 /mL, with 800 μL of cell suspension per well, and cultured in a 37°C, 5% CO 2 incubator for 24 hours. When the cells grew to near confluence, 50 μL of the sample concentration of 10 mg/L was administered, with 3 replicates per group, and 50 μL of 10 μg/mL dexamethasone was used as the positive control. After 1 hour, 1 μg/mL lipopolysaccharide (LPS) was used for stimulation. After 24 hours of incubation in a cell culture incubator, the cell culture medium was transferred to a centrifuge tube, centrifuged at 5000 r/min for 10 minutes, and the cell supernatant was collected and tested according to the instructions of the mouse TNF-α enzyme-linked immunosorbent assay kit.
表5RAW分泌TNF-α结果Table 5 Results of RAW secretion of TNF-α
从表5实施例1-3结果来看,组合物具有显著的抑制TNF-α炎症因子的功效,实施例2抑制TNF-α炎症因子功效最好。From the results of Examples 1-3 in Table 5, it can be seen that the composition has a significant effect of inhibiting TNF-α inflammatory factor, and Example 2 has the best effect of inhibiting TNF-α inflammatory factor.
对比实施例2与对比例1-5,以实施例2的效果最好,说明不同组分的组合物能够对抑制TNF-α炎症因子功效造成一定程度的影响,表明组合物中存在一定的协同增效作用。任意更换或缺省本发明所限定原料或组分均无法达到本发明所提出功效。Comparing Example 2 with Comparative Examples 1-5, Example 2 has the best effect, indicating that the composition of different components can have a certain degree of influence on the efficacy of inhibiting TNF-α inflammatory factors, indicating that there is a certain synergistic effect in the composition. Any replacement or omission of the raw materials or components defined in the present invention cannot achieve the efficacy proposed by the present invention.
组合物对斑马鱼保湿的效果Effect of the composition on moisturizing of zebrafish
斑马鱼表皮具有一定的渗透压耐受范围,超过耐受范围将产生失水现象。使用高渗透压溶液处理斑马鱼会导致其尾部因失水而皱缩、变小。因此,可以建立斑马鱼补水保湿的诱导模型,进而评估保湿产品的效果。The zebrafish epidermis has a certain osmotic pressure tolerance range, and water loss will occur if the tolerance range is exceeded. Using a high osmotic pressure solution to treat zebrafish will cause its tail to shrink and shrink due to water loss. Therefore, an induced model of zebrafish hydration and moisturizing can be established to evaluate the effect of moisturizing products.
(1)配鱼繁殖:按雌雄比为2:1的比例配对斑马鱼繁殖,收集鱼卵孵育过夜(28℃培育箱)。(1) Breeding: Zebrafish were paired at a male-female ratio of 2:1, and the eggs were collected and incubated overnight (28°C incubator).
(2)实验分组:实验设置空白对照组与实验组,实验组包括模型组、阳性组和样品组,每组8尾2dpf的幼鱼。(2) Experimental groups: The experiment set up a blank control group and an experimental group. The experimental groups included a model group, a positive group, and a sample group, with 8 2 dpf fry in each group.
(3)配液:精准称量1mg样品并配置待测样品溶液,以尿素(0.05%)为阳性对照进行实验,每孔加样1mg。给药完成后将6孔板放置在28℃培育箱中避光孵育24h。(3) Solution preparation: Accurately weigh 1 mg of sample and prepare the sample solution to be tested, use urea (0.05%) as the positive control for the experiment, and add 1 mg to each well. After the administration is completed, place the 6-well plate in a 28°C incubator in the dark for 24 hours.
测试结果:如下表6所示。Test results: as shown in Table 6 below.
表6斑马鱼保湿效果测试Table 6 Zebrafish moisturizing effect test
从表6结果分析:从实施例1-3结果可知,本发明实施例提供的组合物具有较好地抑制斑马鱼尾面积缩小功效,尤其是实施例2的效果较好,From the results of Table 6, it can be seen from the results of Examples 1-3 that the composition provided by the embodiments of the present invention has a good effect of inhibiting the reduction of the tail area of zebrafish, especially Example 2 has a better effect.
从实施例2及对比例1-5结果来看,表明使用本发明所限定的组分存在一定的协同增效作用,缺省、替换任意一项均不能达到本发明所达到的效果。From the results of Example 2 and Comparative Examples 1-5, it is shown that there is a certain synergistic effect when using the components defined in the present invention, and the effect achieved by the present invention cannot be achieved by omitting or replacing any one of them.
组合物对Hacat细胞划痕迁移的影响Effects of the composition on the migration of Hacat cells through scratch wound
细胞迁移率程度对伤口愈合和皮肤修复具有重要影响。在伤口愈合过程中,细胞迁移是表皮细胞覆盖伤口的重要步骤,因此细胞迁移率越高,伤口愈合的速度越快。同时,细胞迁移也参与了皮肤损伤后的修复过程,如果细胞迁移受损,会导致皮肤修复障碍,进而引发各种皮肤疾病。The degree of cell migration has an important impact on wound healing and skin repair. In the process of wound healing, cell migration is an important step for epidermal cells to cover the wound, so the higher the cell migration rate, the faster the wound heals. At the same time, cell migration is also involved in the repair process after skin damage. If cell migration is impaired, it will lead to skin repair disorders, which in turn cause various skin diseases.
按相应的接种密度接种细胞至12孔板中,37℃,CO2培养箱孵育过夜。待12孔板中细胞铺板率达到80%左右时,对样品组和溶剂对照组进行划痕。划痕结束后,用PBS清洗细胞,以去掉死细胞,再加入新鲜的无EGF角质形成细胞培养液,按照实施例1-3、对比例1-510mg/L 50μL加样,使用倒置显微镜拍照记录。37℃,5%CO2培养箱中孵育培养24h,再次拍照记录,用ImageJ软件统计分析。Inoculate cells into 12-well plates at the corresponding inoculation density and incubate overnight at 37°C in a CO2 incubator. When the cell plating rate in the 12-well plate reaches about 80%, scratch the sample group and the solvent control group. After the scratching, wash the cells with PBS to remove dead cells, add fresh EGF-free keratinocyte culture solution, add 50μL of 10mg/L according to Example 1-3 and Comparative Example 1-5, and take photos and record using an inverted microscope. Incubate in a 37°C, 5% CO2 incubator for 24h, take photos and record again, and use ImageJ software for statistical analysis.
表7Hacat细胞迁移率Table 7 Hacat cell migration rate
从表7结果分析:从实施例1-3结果可知,本发明实施例提供的组合物具有较好地提高细胞迁移率功效,尤其是实施例2的效果较好,From the results of Table 7, it can be seen from the results of Examples 1-3 that the compositions provided by the embodiments of the present invention have a good effect of improving cell migration rate, especially Example 2 has a better effect.
从实施例2及对比例1-5结果来看,表明使用本发明所限定的组分存在一定的协同增效作用,缺省、替换任意一项均不能达到本发明所达到的效果。From the results of Example 2 and Comparative Examples 1-5, it is shown that there is a certain synergistic effect when using the components defined in the present invention, and the effect achieved by the present invention cannot be achieved by omitting or replacing any one of them.
护肤品凝胶制备与稳定性测试Preparation and stability testing of skin care gel
按照上述配方设计进行制备对实施例4-6、对比例6-7进行测试。According to the above formula design, Examples 4-6 and Comparative Examples 6-7 were prepared and tested.
1、耐热测试:恒温培养箱调节到40℃,取透明玻璃瓶中,装样品量为10ml/瓶,封口后置于恒温培养箱内,三个月后取出,恢复至室温,观察外观变化。1. Heat resistance test: Adjust the constant temperature incubator to 40℃, put the sample into a transparent glass bottle with a volume of 10ml/bottle, seal it and place it in a constant temperature incubator. Take it out after three months, return it to room temperature, and observe the changes in appearance.
2、耐寒测试:恒温培养箱调节到-10℃,取透明玻璃瓶中,装样品量为10ml/瓶,封口后置于恒温培养箱内,三个月后取出,恢复至室温,观察外观变化。2. Cold resistance test: Adjust the constant temperature incubator to -10℃, put the sample into a transparent glass bottle with a volume of 10ml/bottle, seal it and place it in a constant temperature incubator. Take it out after three months, return it to room temperature, and observe the changes in appearance.
3、常温测试:取透明玻璃瓶,装样品量为10ml/瓶,封口后常温下放置6个月后,观察外观变化。3. Room temperature test: Take a transparent glass bottle, fill it with 10 ml of sample per bottle, seal it and place it at room temperature for 6 months, then observe the changes in appearance.
4、光老化测试:取透明玻璃瓶中,装样品量为10ml/瓶,封口后白炽灯光下放置三个月后,观察外观变化。4. Light aging test: Put the sample into a transparent glass bottle with a volume of 10 ml/bottle. After sealing, place it under incandescent light for three months and observe the changes in appearance.
耐热测试、耐寒测试、常温测试、光老化测试均未观察到析出、沉淀等现象,保持原有物理特性。No precipitation or settling was observed in the heat resistance test, cold resistance test, room temperature test, and light aging test, and the original physical properties were maintained.
护肤品凝胶人体即时修护效果测试Test on the immediate repair effect of skin care gel on human body
经皮失水率(TEWL)是评估皮肤屏障功能的重要参数,它表示真皮深层的水分通过表皮的蒸发散失。经皮失水率越高,表明经皮肤散失的水分越多,皮肤的保湿能力越弱。Transepidermal water loss (TEWL) is an important parameter for evaluating skin barrier function. It indicates the loss of water from the deep dermis through evaporation of the epidermis. The higher the TEWL, the more water is lost through the skin and the weaker the skin's moisturizing ability.
经皮失水率与角质层的水合作用密切相关,是反映角质层屏障功能的常用指标。在化妆品的研制过程中,通过测试皮肤水分流失的TEWL数值,可以评价保湿化妆品的功效。此外,经皮失水率也可应用于过敏性斑贴试验、接触性皮炎、物理疗法、烧伤及新生组织的监测,及时发现皮肤的保护功能是否已被破坏。The transepidermal water loss rate is closely related to the hydration of the stratum corneum and is a commonly used indicator reflecting the barrier function of the stratum corneum. In the development of cosmetics, the efficacy of moisturizing cosmetics can be evaluated by testing the TEWL value of skin water loss. In addition, the transepidermal water loss rate can also be used in allergic patch tests, contact dermatitis, physical therapy, burns and new tissue monitoring to promptly detect whether the protective function of the skin has been damaged.
因此,经皮失水率是评估皮肤健康的重要参数,对保湿产品的研发和评价具有重要意义。Therefore, transepidermal water loss rate is an important parameter for assessing skin health and is of great significance to the research, development and evaluation of moisturizing products.
选取50名年龄18-60岁之间的健康受试者,手臂内侧无损伤者,进行分组,每组均为10人,每组受试者分别为5名男性和5名女性。设置样品组与空白组(即阴性对照组:不含测试样品),测试区域随机分布于志愿者手臂内侧1cm2区域。使用医用胶带撕脱上述受试者的前臂(两个区域)20次诱导皮肤屏障功能破坏,再加5mg/mL组胺造模10min(出现明显红斑),其中一个区域使用测试产品1g,另一个区域为空白,作为阴性对照区域,在胶带撕脱前(T0)、使用产品前(即组胺后即时)(T1)、使用产品后2小时(T2),采集前臂皮肤经皮失水率(TEWL),通过使用产品前后产品处理区域与阴性对照区域皮肤指标的变化差异,评价产品对胶带撕脱及组胺诱导皮肤刺激引起的皮肤屏障破坏的短期修护功效,取平均值,得到的结果如下表8。50 healthy subjects aged 18-60 years old with no damage on the inner side of the arm were selected and divided into groups, each group had 10 people, and each group of subjects consisted of 5 males and 5 females. A sample group and a blank group (i.e., a negative control group: excluding the test sample) were set up, and the test area was randomly distributed in the 1 cm2 area on the inner side of the volunteer's arm. The forearm (two areas) of the above-mentioned subject was torn off with medical tape 20 times to induce skin barrier function damage, and then 5 mg/mL histamine was added to model for 10 min (obvious erythema appeared), one of which used 1g of the test product, and the other area was blank as a negative control area. Before the tape was torn off (T0), before using the product (i.e., immediately after histamine) (T1), and 2 hours after using the product (T2), the transepidermal water loss rate (TEWL) of the forearm skin was collected, and the difference in skin index changes in the product treatment area and the negative control area before and after using the product was used to evaluate the short-term repair effect of the product on the skin barrier damage caused by tape tearing and histamine-induced skin irritation, and the average value was taken. The results obtained are shown in Table 8 below.
表8表皮相对失水率Table 8 Relative water loss rate of epidermis
结果分析:从实施例4-6结果来看,本发明所制备精华液具有显著的保湿、抗皱功效,实施例5即有效成分为3wt%时保湿、修护功效最好。Result analysis: From the results of Examples 4-6, the essence prepared by the present invention has significant moisturizing and anti-wrinkle effects, and Example 5, i.e., when the active ingredient is 3 wt%, has the best moisturizing and repairing effects.
对比实施例5与对比例6,以实施例5的效果较好,说明本发明所制备精华液具有较好的保湿、抗皱功效。Comparing Example 5 with Comparative Example 6, Example 5 has a better effect, which indicates that the essence prepared by the present invention has better moisturizing and anti-wrinkle effects.
对比实施例5与对比例7,失水率相似,表明过高的添加量并不能有效提高保湿修复功效,因此使用本发明所限定的各组分比例所制备的精华液能够使其功效最大化。Comparing Example 5 with Comparative Example 7, the water loss rates are similar, indicating that too high an addition amount cannot effectively improve the moisturizing and repairing effect. Therefore, the essence prepared using the proportions of the components defined in the present invention can maximize its efficacy.
以上所描述的实施例是本申请一部分实施例,而不是全部的实施例。本申请的实施例的详细描述并非旨在限制要求保护的本申请的范围,而是仅仅表示本申请的选定实施例。基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本申请保护的范围。The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.
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