A polypeptide kallikrein enhancer and uses thereof

Abstract

The application discloses a polypeptide kallikrein promoter and application thereof, and belongs to the technical field of cosmetics. The polypeptide kallikrein promoter comprises a polypeptide compound, a preservative and water. The polypeptide compound is at least one of red scorpion toxin, acetyl tetrapeptide-2, acetyl tetrapeptide-3, hexapeptide-11, hyaluronic acid polypeptide, palmitoyl tripeptide-5 and acetyl octapeptide-3. The preservative is a mixture of glycerol, ethylhexyl glycerin and 1,2-ethanediol. The polypeptide kallikrein promoter can not only promote skin desquamation by activating KLKs, but also delay skin aging by inhibiting MMPs gene expression, and can be used for developing products with skin renewal and anti-aging dual effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cosmetic technology, in particular to a polypeptide kallikrein promoter and application thereof. BACKGROUND

[0002] The generation of skin aging, dullness and damage is a dynamic and multi-layered process. With age, the metabolism of skin epidermal cells gradually slows down, and the accumulation of free radicals and oxidative stress intensifies, leading to cell damage and accelerated aging. Changes in metabolic processes affect cell energy production, waste removal and other functions, which in turn affect the rate of aging. Further, it leads to a decrease in the production of epidermal lipids and natural moisturizing factors, a decrease in the water storage capacity of the stratum corneum, and a decrease in the synthesis and loss of collagen and elastin in the dermis. Exogenous factors such as ultraviolet radiation can further contribute to skin aging, increasing the formation of wrinkles and pigmented spots, and the skin gradually loses its elasticity, further reducing its ability to repair skin damage.

[0003] Currently in the field of cosmetic technology applications, polypeptides and their compositions have been widely used in skin care application solutions. For example, classic expression line inhibiting polypeptides include acetyl hexapeptide-8, acetyl octapeptide-3, conotoxin and red scorpion peptide, etc.; signal polypeptides that promote the expression of different collagens include palmitoyl tripeptide-5, palmitoyl pentapeptide-4 and palmitoyl tripeptide-1, etc.; polypeptides that promote the expression of elastin include acetyl tetrapeptide-2 and palmitoyl hexapeptide-12, etc.; signal polypeptides that promote the formation of different proteoglycans include acetyl tetrapeptide-9, acetyl tetrapeptide-11 and hyaluronic acid polypeptide, etc.; and palmitoyl dipeptide-7, a polypeptide for cosmetic applications that reduces photodamage of the endogenous antioxidant pathway, and various types of compositions thereof. The polypeptide compositions on the market currently focus on endogenous and exogenous anti-aging, but the target points of the endogenous and exogenous effects are relatively single, the combined efficacy is focused on the single peptide target points that have been disclosed, there is no clear mechanism and data support for the synergistic effect of the combination, and there is no innovative efficacy target point.

[0004] Kallikreins (KLKs) are extracellular serine proteases secreted by granular keratinocytes, which can regulate the epidermal microenvironment and participate in skin desquamation through the degradation of the stratum corneum. Some non-peptide products have been reported to have the effect of promoting a single KLKs, and some peptides can inhibit the expression of KLKs, but polypeptides that can simultaneously promote multiple key KLKs in the skin have not been reported. Therefore, it is of great innovative significance and market potential to develop a polypeptide kallikrein promoter and its application. SUMMARY

[0005] The present application aims to provide a polypeptide kallikrein promoter that can promote the expression of KLKs and inhibit the expression of MMPs, and its application.

[0006] The technical scheme adopted by the present application to achieve the above-mentioned purpose is:

[0007] The polypeptide kallikrein promoter is used in the preparation of a cosmetic for promoting the expression of kallikrein and / or inhibiting the expression of matrix metalloproteinase; the cosmetic has the effects of promoting skin desquamation and / or anti-aging; the kallikrein includes at least one of kallikrein 5, kallikrein 7 and kallikrein 14; the up-regulation rate of the polypeptide kallikrein promoter to the expression of kallikrein is 15-62%.

[0008] The polypeptide kallikrein promoter provided by the present application can promote the expression of kallikrein 5 KLK5, kallikrein 7 KLK7 and kallikrein 14 KLK14 by activating the migration of HaCaT keratinocytes, improving the cell migration ability, promoting skin desquamation, and helping skin keratin renewal; at the same time, the expression of matrix metalloproteinase 1 MMP1, matrix metalloproteinase 3 MMP3 and matrix metalloproteinase MMP9 is down-regulated, the loss of dermal-related extracellular matrix is reduced, the wound healing ability is enhanced, and the formation of wrinkles is effectively inhibited, so as to achieve the dual effects of promoting skin regeneration and anti-aging.

[0009] Preferably, the up-regulation rate of the polypeptide kallikrein promoter to KLK5 is 18-35%.

[0010] Preferably, the up-regulation rate of the polypeptide kallikrein promoter to KLK7 is 20-38%.

[0011] Preferably, the up-regulation rate of the polypeptide kallikrein promoter to KLK14 is 22-62%.

[0012] Preferably, the matrix metalloproteinase includes at least one of matrix metalloproteinase 1, matrix metalloproteinase 3 and matrix metalloproteinase 9; the down-regulation rate of the polypeptide kallikrein promoter to the expression of matrix metalloproteinase is 20-86%.

[0013] More preferably, the down-regulation rate of the polypeptide kallikrein promoter to MMP1 is 20-55%.

[0014] More preferably, the down-regulation rate of the polypeptide kallikrein promoter to MMP3 is 28-80%.

[0015] More preferably, the down-regulation rate of the polypeptide kallikrein promoter to MMP9 is 38-86%.

[0016] Preferably, the polypeptide kallikrein promoter includes a polypeptide compound, a preservative and water.

[0017] More preferably, the amount of the polypeptide compound used is equivalent to 0.001-0.05 wt % of the total amount of the polypeptide kallikrein promoter.

[0018] More preferably, the polypeptide compound is at least one of red scorpion toxin, acetyl tetrapeptide-2, acetyl tetrapeptide-3, hexapeptide-11, hyaluronic acid polypeptide, palmitoyl tripeptide-5 and acetyl octapeptide-3.

[0019] More preferably, the amount of red scorpion toxin used is equivalent to 16-100 wt % of the total amount of the polypeptide compound.

[0020] More preferably, the amount of acetyl tetrapeptide-3 is equivalent to 16-100 wt % of the total amount of the polypeptide compound.

[0021] More preferably, the amount of hexapeptide-11 is equivalent to 16-100 wt % of the total amount of the polypeptide compound.

[0022] More preferably, the amount of hyaluronic acid polypeptide used is equivalent to 16-100 wt % of the total amount of the polypeptide compound.

[0023] More preferably, the amount of palmitoyl tripeptide-5 is equivalent to 16-100 wt % of the total amount of the polypeptide compound.

[0024] More preferably, the amount of acetyl octapeptide-3 is equivalent to 20-100 wt % of the total amount of the polypeptide compound.

[0025] More preferably, the amount of the preservative is equivalent to 10-30 wt % of the total amount of the polypeptide kallikrein promoter.

[0026] More preferably, the preservative is a mixture of glycerin, ethylhexylglycerin and 1,2-ethylene glycol.

[0027] More preferably, the amount of glycerol used is equivalent to 85-95 wt % of the total amount of the preservative.

[0028] More preferably, the amount of ethylhexylglycerin used is equivalent to 0.1-1 wt % of the total amount of the preservative.

[0029] More preferably, the amount of 1,2-ethylene glycol used is equivalent to 4-15 wt % of the total amount of the preservative.

[0030] The present invention uses a polypeptide compound, a preservative and water to form a polypeptide kallikrein promoter, the polypeptide compound is at least one of red scorpion toxin, acetyl tetrapeptide-2, acetyl tetrapeptide-3, hexapeptide-11, hyaluronic acid polypeptide, palmitoyl tripeptide-5 and acetyl octapeptide-3, and the preservative is a mixture of glycerol, ethylhexylglycerol and 1,2-ethylene glycol, so it has the following beneficial effects: when the present invention uses red scorpion toxin, acetyl tetrapeptide-2, acetyl tetrapeptide-3 or hexapeptide-11 with a preservative and water to form a polypeptide kallikrein promoter, it can significantly The present invention can effectively promote KLKs gene expression, among which the polypeptide kallikrein promoter composed of red scorpion toxin, preservatives and water has the most significant effect; when the polypeptide kallikrein promoter is composed of acetyl tetrapeptide-2, hyaluronic acid polypeptide or palmitoyl tripeptide-5, preservatives and water, it can effectively inhibit MMPs gene expression; the polypeptide kallikrein promoter composed of acetyl tetrapeptide-2, preservatives and water used in the present invention has a dual regulatory effect, promoting KLKs gene expression while also having a significant effect of inhibiting MMPs gene expression. Therefore, the present invention provides a polypeptide kallikrein promoter with excellent effects in promoting KLKs expression and inhibiting MMPs expression. It can promote skin desquamation by activating KLKs genes and delay skin aging by inhibiting MMPs gene expression. Its synergistic mechanism helps to develop products with the dual effects of promoting skin renewal and anti-aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the up-regulation rate of KLK5 expression. DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0034] Example 1:

[0035] A polypeptide kallikrein promoter comprising a polypeptide compound, a preservative, and water. The amount of the polypeptide compound is equivalent to 0.05wt% of the total amount of the polypeptide kallikrein promoter, the amount of the preservative is equivalent to 22.1wt% of the total amount of the polypeptide kallikrein promoter, and the remainder is water. The polypeptide compound is red scorpion toxin; the preservative is a mixture of glycerol, ethylhexylglycerol, and 1,2-ethylene glycol, with the amount of glycerol equivalent to 90.5wt% of the total amount of the preservative, the amount of ethylhexylglycerol equivalent to 0.5wt% of the total amount of the preservative, and the amount of 1,2-ethylene glycol equivalent to 9% of the total amount of the preservative.

[0036] Example 2:

[0037] The only difference between this example and Example 1 is that the polypeptide compound is acetyl tetrapeptide-2, and other conditions are the same as Example 1.

[0038] Example 3:

[0039] The only difference between this example and Example 1 is that the polypeptide compound is acetyl tetrapeptide-3, and other conditions are the same as Example 1.

[0040] Example 4:

[0041] The only difference between this example and Example 1 is that the polypeptide compound is hexapeptide-11, and other conditions are the same as Example 1.

[0042] Example 5:

[0043] The only difference between this embodiment and embodiment 1 is that the polypeptide compound is hyaluronic acid polypeptide, and other conditions are the same as those in embodiment 1.

[0044] Example 6:

[0045] The only difference between this example and Example 1 is that the polypeptide compound is palmitoyl tripeptide-5, and other conditions are the same as Example 1.

[0046] Example 7:

[0047] The only difference between this example and Example 1 is that the polypeptide compound is acetyl octapeptide-3, and other conditions are the same as Example 1.

[0048] Example 8:

[0049] The only difference between this example and Example 1 is that the polypeptide compound is a mixture of red scorpion toxin, hyaluronic acid polypeptide and acetyl octapeptide-3, the amount of red scorpion toxin is equivalent to 33wt% of the total amount of the polypeptide compound, the amount of hyaluronic acid polypeptide is equivalent to 33wt% of the total amount of the polypeptide compound, and the amount of acetyl octapeptide-3 is equivalent to 34wt% of the total amount of the polypeptide compound. Other conditions are the same as in Example 1.

[0050] Example 9:

[0051] The only difference between this example and Example 1 is that the polypeptide compound is a mixture of red scorpion toxin, palmitoyl tripeptide-5 and acetyl octapeptide-3, the amount of red scorpion toxin is equivalent to 33wt% of the total amount of the polypeptide compound, the amount of palmitoyl tripeptide-5 is equivalent to 33wt% of the total amount of the polypeptide compound, and the amount of acetyl octapeptide-3 is equivalent to 34wt% of the total amount of the polypeptide compound. Other conditions are the same as in Example 1.

[0052] Example 10:

[0053] The only difference between this example and Example 1 is that the polypeptide compound is a mixture of red scorpion toxin, hyaluronic acid polypeptide, palmitoyl tripeptide-5 and acetyl octapeptide-3, the amount of red scorpion toxin is equivalent to 25wt% of the total amount of the polypeptide compound, the amount of hyaluronic acid polypeptide is equivalent to 25wt% of the total amount of the polypeptide compound, the amount of palmitoyl tripeptide-5 is equivalent to 25wt% of the total amount of the polypeptide compound, and the amount of acetyl octapeptide-3 is equivalent to 25wt% of the total amount of the polypeptide compound. Other conditions are the same as in Example 1.

[0054] Example 11:

[0055] Compared with Example 1, the only difference between this example and Example 1 is that the polypeptide compound is a mixture of acetyl tetrapeptide-3, hyaluronic acid polypeptide and acetyl octapeptide-3, the amount of acetyl tetrapeptide-3 is equivalent to 33wt% of the total amount of the polypeptide compound, the amount of hyaluronic acid polypeptide is equivalent to 33wt% of the total amount of the polypeptide compound, and the amount of acetyl octapeptide-3 is equivalent to 34wt% of the total amount of the polypeptide compound. Other conditions are the same as in Example 1.

[0056] Example 12:

[0057] Compared with Example 1, the only difference between this example and Example 1 is that the polypeptide compound is a mixture of acetyl tetrapeptide-3, palmitoyl tripeptide-5 and acetyl octapeptide-3, the amount of acetyl tetrapeptide-3 is equivalent to 33wt% of the total amount of the polypeptide compound, the amount of palmitoyl tripeptide-5 is equivalent to 33wt% of the total amount of the polypeptide compound, and the amount of acetyl octapeptide-3 is equivalent to 34wt% of the total amount of the polypeptide compound. Other conditions are the same as in Example 1.

[0058] Example 13:

[0059] The difference between this example and Example 1 is that the polypeptide compound is a mixture of acetyl tetrapeptide-3, hyaluronic acid polypeptide, palmitoyl tripeptide-5 and acetyl octapeptide-3, the amount of acetyl tetrapeptide-3 is 25% by weight of the total polypeptide compound, the amount of hyaluronic acid polypeptide is 25% by weight of the total polypeptide compound, the amount of palmitoyl tripeptide-5 is 25% by weight of the total polypeptide compound, and the amount of acetyl octapeptide-3 is 25% by weight of the total polypeptide compound, and the other conditions are the same as in Example 1.

[0060] Example 14:

[0061] The difference between this example and Example 1 is that the polypeptide compound is a mixture of hexapeptide-11, hyaluronic acid polypeptide, and acetyl octapeptide-3, the amount of hexapeptide-11 is 33% by weight of the total polypeptide compound, the amount of hyaluronic acid polypeptide is 33% by weight of the total polypeptide compound, and the amount of acetyl octapeptide-3 is 34% by weight of the total polypeptide compound, and the other conditions are the same as in Example 1.

[0062] Example 15:

[0063] The difference between this example and Example 1 is that the polypeptide compound is a mixture of hexapeptide-11, palmitoyl tripeptide-5 and acetyl octapeptide-3, the amount of hexapeptide-11 is 33% by weight of the total polypeptide compound, the amount of palmitoyl tripeptide-5 is 33% by weight of the total polypeptide compound, and the amount of acetyl octapeptide-3 is 34% by weight of the total polypeptide compound, and the other conditions are the same as in Example 1.

[0064] Example 16:

[0065] The difference between this example and Example 1 is that the polypeptide compound is a mixture of hexapeptide-11, hyaluronic acid polypeptide, palmitoyl tripeptide-5 and acetyl octapeptide-3, the amount of hexapeptide-11 is 25% by weight of the total polypeptide compound, the amount of hyaluronic acid polypeptide is 25% by weight of the total polypeptide compound, the amount of palmitoyl tripeptide-5 is 25% by weight of the total polypeptide compound, and the amount of acetyl octapeptide-3 is 25% by weight of the total polypeptide compound, and the other conditions are the same as in Example 1.

[0066] Example 17:

[0067] The only difference between this example and Example 1 is that the polypeptide compound is a mixture of red scorpion toxin, acetyl tetrapeptide-3, hyaluronic acid polypeptide and acetyl octapeptide-3, the amount of red scorpion toxin is equivalent to 25wt% of the total amount of the polypeptide compound, the amount of acetyl tetrapeptide-3 is equivalent to 25wt% of the total amount of the polypeptide compound, the amount of hyaluronic acid polypeptide is equivalent to 25wt% of the total amount of the polypeptide compound, and the amount of acetyl octapeptide-3 is equivalent to 25wt% of the weight of the polypeptide compound. Other conditions are the same as in Example 1.

[0068] Example 18:

[0069] The only difference between this example and Example 1 is that the polypeptide compound is a mixture of red scorpion toxin, acetyl tetrapeptide-3, palmitoyl tripeptide-5 and acetyl octapeptide-3, the amount of red scorpion toxin is equivalent to 25wt% of the total amount of the polypeptide compound, the amount of acetyl tetrapeptide-3 is equivalent to 25wt% of the total amount of the polypeptide compound, the amount of palmitoyl tripeptide-5 is equivalent to 25wt% of the total amount of the polypeptide compound, and the amount of acetyl octapeptide-3 is equivalent to 25wt% of the weight of the polypeptide compound. Other conditions are the same as in Example 1.

[0070] Example 19:

[0071] The only difference between this example and Example 1 is that the polypeptide compound is a mixture of red scorpion toxin, acetyl tetrapeptide-3, hyaluronic acid polypeptide, palmitoyl tripeptide-5 and acetyl octapeptide-3, the amount of red scorpion toxin is equivalent to 20wt% of the total amount of the polypeptide compound, the amount of acetyl tetrapeptide-3 is equivalent to 20wt% of the total amount of the polypeptide compound, the amount of hyaluronic acid polypeptide is equivalent to 20wt% of the total amount of the polypeptide compound, the amount of palmitoyl tripeptide-5 is equivalent to 20wt% of the total amount of the polypeptide compound, and the amount of acetyl octapeptide-3 is equivalent to 20wt% of the weight of the polypeptide compound. Other conditions are the same as in Example 1.

[0072] Example 20:

[0073] The only difference between this example and Example 1 is that the polypeptide compound is a mixture of red scorpion toxin, hexapeptide-11, hyaluronic acid polypeptide and acetyl octapeptide-3, the amount of red scorpion toxin is equivalent to 25wt% of the total amount of the polypeptide compound, the amount of hexapeptide-11 is equivalent to 25wt% of the total amount of the polypeptide compound, the amount of hyaluronic acid polypeptide is equivalent to 25wt% of the total amount of the polypeptide compound, and the amount of acetyl octapeptide-3 is equivalent to 25wt% of the weight of the polypeptide compound. Other conditions are the same as in Example 1.

[0074] Example 21:

[0075] The only difference between this example and Example 1 is that the polypeptide compound is a mixture of red scorpion toxin, hexapeptide-11, palmitoyl tripeptide-5 and acetyl octapeptide-3, the amount of red scorpion toxin is equivalent to 25wt% of the total amount of the polypeptide compound, the amount of hexapeptide-11 is equivalent to 25wt% of the total amount of the polypeptide compound, the amount of palmitoyl tripeptide-5 is equivalent to 25wt% of the total amount of the polypeptide compound, and the amount of acetyl octapeptide-3 is equivalent to 25wt% of the weight of the polypeptide compound. Other conditions are the same as in Example 1.

[0076] Example 22:

[0077] The difference between this embodiment and Example 1 is that the polypeptide compound is a mixture of red scorpion toxin, hexapeptide-11, hyaluronic acid polypeptide, palmitoyl tripeptide-5 and acetyl octapeptide-3, the amount of red scorpion toxin is equivalent to 20wt% of the total amount of the polypeptide compound, the amount of hexapeptide-11 is equivalent to 20wt% of the total amount of the polypeptide compound, the amount of hyaluronic acid polypeptide is equivalent to 20wt% of the total amount of the polypeptide compound, the amount of palmitoyl tripeptide-5 is equivalent to 20wt% of the total amount of the polypeptide compound, and the amount of acetyl octapeptide-3 is equivalent to 20wt% of the weight of the polypeptide compound. Other conditions are the same as in Example 1.

[0078] Example 23:

[0079] The difference between this embodiment and Example 1 is that the polypeptide compound is a mixture of red scorpion toxin, acetyl tetrapeptide-3, hexapeptide-11, hyaluronic acid polypeptide and acetyl octapeptide-3, the amount of red scorpion toxin is equivalent to 20wt% of the total amount of the polypeptide compound, the amount of acetyl tetrapeptide-3 is equivalent to 20wt% of the total amount of the polypeptide compound, the amount of hexapeptide-11 is equivalent to 20wt% of the total amount of the polypeptide compound, the amount of hyaluronic acid polypeptide is equivalent to 20wt% of the total amount of the polypeptide compound, and the amount of acetyl octapeptide-3 is equivalent to 20wt% of the weight of the polypeptide compound. Other conditions are the same as in Example 1.

[0080] Example 24:

[0081] The only difference between this example and Example 1 is that the polypeptide compound is a mixture of red scorpion toxin, acetyl tetrapeptide-3, hexapeptide-11, palmitoyl tripeptide-5 and acetyl octapeptide-3, the amount of red scorpion toxin is equivalent to 20wt% of the total amount of the polypeptide compound, the amount of acetyl tetrapeptide-3 is equivalent to 20wt% of the total amount of the polypeptide compound, the amount of hexapeptide-11 is equivalent to 20wt% of the total amount of the polypeptide compound, the amount of palmitoyl tripeptide-5 is equivalent to 20wt% of the total amount of the polypeptide compound, and the amount of acetyl octapeptide-3 is equivalent to 20wt% of the weight of the polypeptide compound. Other conditions are the same as in Example 1.

[0082] Example 25:

[0083] The difference between this embodiment and embodiment 1 is only that the polypeptide compound is a mixture of red scorpion toxin, acetyl tetrapeptide-3, hexapeptide-11, hyaluronic acid polypeptide, palmitoyl tripeptide-5 and acetyl octapeptide-3, the amount of red scorpion toxin is 16% by weight of the total amount of polypeptide compound, the amount of acetyl tetrapeptide-3 is 16% by weight of the total amount of polypeptide compound, the amount of hexapeptide-11 is 16% by weight of the total amount of polypeptide compound, the amount of hyaluronic acid polypeptide is 16% by weight of the total amount of polypeptide compound, the amount of palmitoyl tripeptide-5 is 16% by weight of the total amount of polypeptide compound, and the amount of acetyl octapeptide-3 is 20% by weight of the total amount of polypeptide compound, and the other conditions are the same as in embodiment 1.

[0084] Experimental example:

[0085] 1. Biological safety cytotoxicity test

[0086] The polypeptide kallikrein enhancer of the application was used to perform a cytotoxicity test on keratinocytes provided by Guangdong Boxi Biological Technology Co., Ltd. The specific steps are as follows: the keratinocytes were inoculated into a 96-well plate at a seeding density of 1×10 4 cells / well, and incubated in an incubator at 37°C and 5% CO2 overnight. A zero setting group, a control group, a positive control group and 25 sample groups were set up. In the 25 sample groups, the polypeptide compounds in the polypeptide kallikrein enhancers of embodiments 1-25 were used in turn for treatment, and the polypeptide compounds were diluted with water to 8 concentration gradients, which were 100%, 50%, 20%, 10%, 5%, 1%, 0.5% and 0.1%, respectively, and 3 repeated wells were set up under each concentration gradient. When the cell plating rate in the 96-well plate reached 60%, the drug was given. The control group was added with 200 μL of DMEM cell culture solution containing 10% PBS per well; the positive control group was added with 200 μL of DMEM cell culture solution containing 10% DMSO per well; the sample group was added with 200 μL of DMEM cell culture solution containing the corresponding concentration of sample per well; the zero setting group was not inoculated with cells, and only 200 μL of DMEM cell culture solution was added. After the drug administration was completed, the 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 h, and then the supernatant was discarded, 0.5 mg / mL of MTT was added to the DMEM culture medium, and it was incubated at 37°C in the dark for 4 h. After incubation, the supernatant was discarded, 150 µL of DMSO was added to each well, and the OD value was read at 490 nm. The relative cell viability (%) = (sample group OD490nm-zero group OD490nm) / (control group OD490nm-zero group OD490nm) × 100%.

[0087] When the mass concentration of the polypeptide compound in the polypeptide kallikrein enhancer of embodiments 1-25 is less than 20%, the relative viability of the cells is 100%. The polypeptide kallikrein enhancer of embodiments 1-25 has a polypeptide compound in an amount of 0.05 wt% of the total amount of the polypeptide kallikrein enhancer, which is basically non-toxic to keratinocytes and can be used normally.

[0088] 2. Detection of KLKs gene expression

[0089] After the keratinocytes are treated with the polypeptide kallikrein enhancer of embodiments 1-25, the KLK5, KLK7 and KLK14 gene expression of the keratinocytes is detected, and the keratinocytes are provided by Guangdong Boxi Biotechnology Co., Ltd. The specific steps are as follows: after the keratinocytes are recovered, when the plating rate reaches 60%, the keratinocytes are inoculated into a 6-well plate, and incubated in an incubator at a temperature of 37°C and a CO2 concentration of 5% overnight. Set up a blank control group and 25 sample groups, each with 3 repeats. When the plating rate of the cells in the 6-well plate reaches 62%, 2 mL of cell culture solution is added to the blank control group; 2 mL of the polypeptide kallikrein enhancer of embodiments 1-25 is added to the 25 sample groups in turn, and after the administration is completed, the 6-well plate is placed in an incubator at a temperature of 37°C and a CO2 concentration of 5% for 24 h.

[0090] After incubation, each group is washed twice with 1 mL of PBS, 1 mL of RNAiso Plus is added to each well, the cell lysate is collected after the cells are lysed by blowing, and the cell culture supernatant is collected. After washing twice with 1 mL of PBS per well, 1 mL of RNAiso Plus is added to each well, the cell lysate is collected after the cells are lysed by blowing, 200 μL of chloroform is added, and the mixture is shaken vigorously for 25 times. After standing at room temperature for 5 min, centrifugation is performed at 12000 rpm for 15 min at 4°C, 400 μL of the upper aqueous phase is aspirated, 400 μL of isopropanol is added, and the mixture is shaken gently for 7 times. After standing at room temperature for 10 min, the supernatant is carefully discarded, 1 mL of 75% ethanol solution is added, the bottom of the tube is tapped, and the precipitate is suspended. After centrifugation at 12000 rpm for 5 min at 4°C, the supernatant is carefully discarded, and the sample is naturally dried in a fume hood for 10 min. 50 μL of DEPC water is added, and the mixture is incubated at 60°C for 10 min to completely dissolve the sample, and the initial RNA solution is obtained. Use DEPC water as a blank, add 2 μL of sample to each well, and set 2 replicate wells for each RNA sample to detect the RNA concentration.

[0091] The reaction system was prepared by combining 1 μL of RNA initial solution, 2 μL of gDNA Removal Mixture Ver. 2, and 13 μL of nuclease-free water. The reaction was incubated at 42°C for 2 min. After completion of the reaction, the RNA solution was obtained and stored at 4°C. gDNA Removal Mixture Ver. 2 was purchased from Hunan Aikerui Biotechnology Co., Ltd.

[0092] The reaction system was prepared by combining 1 μL of RNA solution, 4 μL of 5× Premix reverse transcription reagent, and 16 μL of nuclease-free water. The reaction was incubated at 37°C for 15 min, followed by incubation at 85°C for 5 s. After completion of the reaction, the cDNA solution was obtained and stored at 4°C. 5× Premix reverse transcription reagent was purchased from Takara Biotech (Dalian) Co., Ltd.

[0093] Add an equal volume of sterile water to the cDNA solution and shake to mix to obtain a cDNA dilution. Take 2 μL of cDNA dilution, 0.8 μL of front primer, 0.8 μL of back primer, 10 μL of SYBR Green fluorescent quantitative premix reagent and 6.4 μL of sterile ultrapure water to form a reaction system. React at 95°C for 30s, 95°C for 5s, 60°C for 30s, 67.5°C for 5s, and 95°C for 5s for 40 cycles. SYBR Green fluorescent quantitative premix reagent was purchased from Hunan Aikerui Bioengineering Co., Ltd. 2 -△△CT Methods: The expression levels of KLK5, KLK7, and KLK14 genes were calculated. The upregulation rates of KLK5, KLK7, and KLK14 were then calculated. Upregulation rate (%) = (sample group - blank control group) / blank control group × 100%. The results of the upregulation rates of KLKs gene expression are shown in Table 1.

[0094] Table 1 KLKs gene expression upregulation rate (%)

[0095]

[0096] Figure 1 is the expression up-regulation rate of KLK5, S1 to S8 correspond to the experimental data of Examples 1 to 8, and S9 corresponds to the experimental data of Example 10. Figure 1As can be seen from the experimental data in Table 1, the polypeptide kallikrein promoters of Examples 1-4 can effectively promote the upregulation of KLK5, KLK7 and KLK14 gene expression, while the polypeptide kallikrein promoters of Examples 5-7 have no regulatory effect on the KLK5, KLK7 and KLK14 genes. This indicates that polypeptide kallikrein promoters containing red scorpion toxin, acetyl tetrapeptide-2, acetyl tetrapeptide-3 or hexapeptide-11 can effectively upregulate the expression of KLK5, KLK7 and KLK14 genes, and red scorpion toxin, acetyl tetrapeptide-2, acetyl tetrapeptide-3 or hexapeptide-11 are the key active ingredients of polypeptide kallikrein promoters. In promoting the upregulation of KLK5 gene expression, the effectiveness ranking is: red scorpion toxin > acetyl tetrapeptide-2 > hexapeptide-11 > acetyl tetrapeptide-3; in promoting the upregulation of KLK7 gene expression, the effectiveness ranking is: red scorpion toxin > acetyl tetrapeptide-2 > acetyl tetrapeptide-3 > hexapeptide-11; in promoting the upregulation of KLK14 gene expression, the effectiveness ranking is: red scorpion toxin > hexapeptide-11 > acetyl tetrapeptide-3 > acetyl tetrapeptide-2. This shows that the polypeptide kallikrein promoter composed of red scorpion toxin and preservatives has the best promoting effect on KLK5, KLK7 and KLK14.

[0097] In Example 1, the amount of red scorpion toxin used was equivalent to 100wt% of the total amount of the polypeptide compound, and its upregulation rates for KLK5, KLK7, and KLK14 gene expression were 35%, 38%, and 62%, respectively. Thus, when the amount of red scorpion toxin used was equivalent to 33wt% of the total amount of the polypeptide compound, its theoretical upregulation rates for KLK5, KLK7, and KLK14 gene expression should be 11.67%, 12.67%, and 20.67%, respectively; when the amount of red scorpion toxin used was equivalent to 25wt% of the total amount of the polypeptide compound, its theoretical upregulation rates for KLK5, KLK7, and KLK14 gene expression should be 8.75%, 9.5%, and 15.5%, respectively. According to the experimental results of Example 8, when the amount of red scorpion toxin used was equivalent to 33wt% and 25wt% of the total amount of the polypeptide compound, its actual upregulation rates for KLK5, KLK7, and KLK14 gene expression were significantly higher than the theoretical values. This shows that compared with using red scorpion toxin alone as a polypeptide compound, using red crab toxin in combination with other polypeptides to prepare a polypeptide kallikrein promoter has a better promoting effect on kallikrein expression.

[0098] 3. MMP gene expression detection

[0099] After the fibroblasts stimulated by UVA were treated by using the polypeptide kallikrein promoter of the application, the expression amounts of MMP1, MMP3 and MMP9 genes were determined, and the fibroblasts were provided by Guangdong Boxi Biological Technology Co., Ltd. The specific steps were as follows: after the fibroblasts were recovered, when the plating rate reached 60%, the fibroblasts were inoculated into a 6-well plate, and then incubated in an incubator with a temperature of 37°C and a CO2 concentration of 5% overnight. A blank control group, a negative control group, a positive control group and a sample group were set, and each group was repeated for three times. When the plating rate of the cells in the 6-well plate reached 40%, 2 mL of cell culture solution was added to the blank control group and the negative control group; 2 mL of cell culture solution containing 100 ng / mL of TGF-β1 was added to the positive control group; and 2 mL of the polypeptide kallikrein promoter of the application was added to the sample group in sequence. After the administration was completed, the 6-well plate was placed in an incubator with a temperature of 37°C and a CO2 concentration of 5% for 24 h. Except for the blank control group, the rest of the groups were irradiated by UVA for 1820 s, and the irradiation dose was 30 J / cm2. 2 After the irradiation was completed, the 6-well plate was placed in an incubator with a temperature of 37°C and a CO2 concentration of 5% for 24 h. After the incubation was completed, the cell culture supernatant was collected, and the expression amounts of MMP1, MMP3 and MMP9 genes were determined by using an MMP1 ELISA kit, an MMP3 ELISA kit and an MMP9 ELISA kit, respectively. According to the detection results of the expression amounts of MMP1, MMP3 and MMP9 genes, the down-regulation rates of MMP1, MMP3 and MMP9 genes were calculated, and the down-regulation rate (%) = (negative control group - sample group) / negative control group x 100%. The MMP1 ELISA kit, the MMP3 ELISA kit and the MMP9 ELISA kit were purchased from Aibio (Shanghai) Trade Co., Ltd. The results of the down-regulation rates of the expression of MMPs genes are shown in Table 2.

[0100] Table 2 Down-regulation rates (%) of the expression of MMPs genes

[0101]

[0102] As can be seen from the experimental data in Table 2, the polypeptide kallikrein promoters of Examples 2, 5, and 6 can effectively downregulate the expression of MMP1, MMP3, and MMP9 genes, and the effects of Examples 2 and 6 are significantly better than those of Example 5. This shows that polypeptide kallikrein promoters containing acetyl tetrapeptide-2, hyaluronic acid polypeptide, or palmitoyl tripeptide-5 can effectively downregulate the expression of MMP1, MMP3, and MMP9 genes, and acetyl tetrapeptide-2, hyaluronic acid polypeptide, and palmitoyl tripeptide-5 are key components for inhibiting the expression of matrix metalloproteinases. Among them, the polypeptide kallikrein promoter containing acetyl tetrapeptide-2 has the best effect on downregulating the expression of MMP1 and MMP9 genes, and the polypeptide kallikrein promoter containing palmitoyl tripeptide-5 has the best effect on downregulating the expression of MMP3 genes.

[0103] In Example 5, the amount of hyaluronic acid polypeptide used was equivalent to 100wt% of the total amount of the polypeptide compound, and its downregulation rates for MMP1, MMP3, and MMP9 gene expression were 33%, 29%, and 55%, respectively. Therefore, when the amount of hyaluronic acid polypeptide used was equivalent to 33wt% of the total amount of the polypeptide compound, its theoretical downregulation rates for MMP1, MMP3, and MMP9 gene expression should be 11%, 9.67%, and 18.33%, respectively; when the amount of hyaluronic acid polypeptide used was equivalent to 25wt% of the total amount of the polypeptide compound, its theoretical downregulation rates for MMP1, MMP3, and MMP9 gene expression should be 8.25%, 7.25%, and 13.75%, respectively. According to the experimental results of Example 8, when the amount of hyaluronic acid polypeptide used was equivalent to 33wt% and 25wt% of the total amount of the polypeptide compound, the actual downregulation rates for MMP1, MMP3, and MMP9 gene expression were significantly higher than the theoretical values. This shows that compared with using hyaluronic acid polypeptide alone as a polypeptide compound, using hyaluronic acid polypeptide in combination with other polypeptides to prepare a polypeptide kallikrein promoter has a better inhibitory effect on the expression of matrix metalloproteinases.

[0104] From the experimental data in Tables 1 and 2, it can be seen that the polypeptide kallikrein promoter containing acetyl tetrapeptide-2 can not only promote the expression of KLK5, KLK7 and KLK14 genes, but also inhibit the expression of MMP1, MMP3 and MMP9 genes.

[0105] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0106] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the present invention. Any changes and modifications made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of a polypeptide kallikrein promoter in the preparation of a cosmetic for promoting kallikrein expression; the cosmetic has the efficacy of promoting skin desquamation and / or anti-aging; the kallikrein comprises at least one of kallikrein 5, kallikrein 7, and kallikrein 14; the polypeptide kallikrein promoter has an upregulation rate of 15-62% on kallikrein expression; The polypeptide kallikrein promoter comprises a polypeptide compound, a preservative and water, wherein the amount of the polypeptide compound is equivalent to 0.001-0.05 wt % of the total amount of the polypeptide kallikrein promoter; The polypeptide compound is at least one of red scorpion toxin, acetyl tetrapeptide-2 and acetyl tetrapeptide-3; or, the polypeptide compound is a mixture of red scorpion toxin, hyaluronic acid polypeptide and acetyl octapeptide-3; or, the polypeptide compound is a mixture of red scorpion toxin, hyaluronic acid polypeptide, palmitoyl tripeptide-5 and acetyl octapeptide-3.

2. Use of a polypeptide kallikrein promoter in the preparation of a cosmetic for inhibiting the expression of matrix metalloproteinases; the cosmetic has the efficacy of promoting skin desquamation and / or anti-aging; The matrix metalloproteinase comprises at least one of matrix metalloproteinase 1, matrix metalloproteinase 3 and matrix metalloproteinase 9; the polypeptide kallikrein promoter has a downregulation rate of 20-86% on the expression of matrix metalloproteinase; The polypeptide kallikrein promoter comprises a polypeptide compound, a preservative and water, wherein the amount of the polypeptide compound is equivalent to 0.001-0.05 wt % of the total amount of the polypeptide kallikrein promoter; The polypeptide compound is at least one of acetyl tetrapeptide-2, hyaluronic acid polypeptide, and palmitoyl tripeptide-5; or, the polypeptide compound is a mixture of red scorpion toxin, hyaluronic acid polypeptide and acetyl octapeptide-3; or, the polypeptide compound is a mixture of red scorpion toxin, hyaluronic acid polypeptide, palmitoyl tripeptide-5 and acetyl octapeptide-3.

3. Use of a polypeptide kallikrein promoter in the preparation of a cosmetic product for promoting kallikrein expression and inhibiting matrix metalloproteinase expression; the cosmetic product has the efficacy of promoting skin desquamation and / or anti-aging; the kallikrein comprises at least one of kallikrein 5, kallikrein 7, and kallikrein 14; the polypeptide kallikrein promoter has an upregulation rate of kallikrein expression of 15-62%; The matrix metalloproteinase comprises at least one of matrix metalloproteinase 1, matrix metalloproteinase 3 and matrix metalloproteinase 9; the polypeptide kallikrein promoter has a downregulation rate of 20-86% on the expression of matrix metalloproteinase; The polypeptide kallikrein promoter comprises a polypeptide compound, a preservative and water, wherein the amount of the polypeptide compound is equivalent to 0.001-0.05 wt % of the total amount of the polypeptide kallikrein promoter; The polypeptide compound is acetyl tetrapeptide-2; or, the polypeptide compound is a mixture of red scorpion toxin, hyaluronic acid polypeptide and acetyl octapeptide-3; or, the polypeptide compound is a mixture of red scorpion toxin, hyaluronic acid polypeptide, palmitoyl tripeptide-5 and acetyl octapeptide-3.

4. The use according to any one of claims 1 to 3, characterized in that The amount of the preservative is equivalent to 10-30 wt % of the total amount of the polypeptide kallikrein promoter.

5. The use according to any one of claims 1 to 3, characterized in that: The preservative is a mixture of glycerin, ethylhexylglycerin and 1,2-ethylene glycol.

6. The use according to claim 5, characterized in that The amount of glycerol used is equivalent to 85-95wt% of the total amount of preservatives.

7. The use according to claim 5, characterized in that The amount of ethylhexylglycerin used is equivalent to 0.1-1 wt % of the total amount of the preservative.

8. The use according to claim 5, characterized in that The amount of 1,2-ethylene glycol used is equivalent to 4-15 wt % of the total amount of the preservative.

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