Polypeptide kallikrein accelerant and application thereof

Abstract

The invention discloses a polypeptide kallikrein accelerant and application thereof, and belongs to the technical field of cosmetics, the polypeptide kallikrein accelerant 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, and the preservative is a mixture of glycerol, ethylhexylglycerol and 1, 2-ethylene glycol. The polypeptide kallikrein accelerant provided by the invention not only can promote skin desquamation by activating KLKs, but also can delay skin aging by inhibiting MMPs gene expression, and can be used for developing products with skin renewing and anti-aging dual effects.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and particularly relates to a polypeptide kallikrein promoter and its application. Background Art

[0002] The generation of skin aging, dullness and damage is a dynamic multi-level process. With the increase of age, the metabolism of epidermal cells in the skin gradually slows down, the accumulation of free radicals and oxidative stress intensify, leading to cell damage and accelerated aging. The changes in the metabolic process will affect functions such as cell energy production and waste clearance, and further affect the aging rate. It further leads to a decrease in the production of epidermal lipids and natural moisturizing factors, a decline in the water storage capacity of the stratum corneum, a reduction in the synthesis and loss of collagen and elastin in the dermis. Exogenous factors such as ultraviolet radiation will further cause skin aging, increase the formation of wrinkles and age spots, the skin gradually loses elasticity, and the repair ability to cope with skin damage is further reduced.

[0003] At present, in the field of cosmetic technology applications, polypeptides and their compositions have been widely used in solutions for skin care applications. For example, classic polypeptides for inhibiting expression lines include acetyl hexapeptide-8, acetyl octapeptide-3, conotoxin peptide and red scorpion peptide, etc.; signal polypeptides for promoting different collagen expressions include palmitoyl tripeptide-5, palmitoyl pentapeptide-4 and palmitoyl tripeptide-1, etc.; polypeptides for promoting elastin expression include acetyl tetrapeptide-2 and palmitoyl hexapeptide-12, etc.; signal polypeptides for promoting the formation of different proteoglycans include acetyl tetrapeptide-9, acetyl tetrapeptide-11 and hyaluronic acid polypeptide, etc.; palmitoyl dipeptide-7 and other polypeptides and their various composition forms for cosmetic applications that reduce photo-damage through the endogenous antioxidant pathway. Although the current polypeptide compositions for anti-aging on the market focus on both endogenous and exogenous anti-aging, the endogenous and exogenous targets of action are relatively single, the combined effects mostly focus on the disclosed single peptide targets, there is no clear mechanism and data support for the synergistic enhancement of the combination, and there is no innovative efficacy target reflected.

[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. At present, there are already some non-peptide products reported to have the effect of promoting a single KLK, and there are also some peptides that can inhibit the expression of KLKs, but there is no report on polypeptides that can simultaneously promote multiple key KLKs in the skin. Therefore, the development of a polypeptide kallikrein promoter and its application has important innovative significance and market potential. Summary of the Invention

[0005] The object of the present invention is 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 solution adopted by the present invention to achieve the above object is as follows: Use of a polypeptide kallikrein promoter in the preparation of a cosmetic for promoting the expression of kallikrein and / or inhibiting the expression of matrix metalloproteinase; the cosmetic has the efficacy of promoting skin desquamation and / or anti-aging; kallikrein includes at least one of kallikrein 5, kallikrein 7, and kallikrein 14; the up-regulation rate of the polypeptide kallikrein promoter on the expression of kallikrein is 15-62%.

[0007] The polypeptide kallikrein promoter provided by the present invention may promote the migration of HaCaT keratinocytes, enhance the cell migration ability while promoting the expression of kallikrein 5 (KLK5), kallikrein 7 (KLK7), and kallikrein 14 (KLK14), promote skin desquamation, and help skin keratin renewal; at the same time, down-regulate the expression of matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 3 (MMP3), and matrix metalloproteinase 9 (MMP9), reduce the loss of dermal-related extracellular matrix, enhance wound healing ability, and effectively inhibit the formation of wrinkles, so as to achieve the dual effects of promoting skin regeneration and anti-aging through internal and external coordination.

[0008] Preferably, the up-regulation rate of the polypeptide kallikrein promoter on KLK5 is 18-35%.

[0009] Preferably, the up-regulation rate of the polypeptide kallikrein promoter on KLK7 is 20-38%.

[0010] Preferably, the up-regulation rate of the polypeptide kallikrein promoter on KLK14 is 22-62%.

[0011] 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 on the expression of matrix metalloproteinase is 20-86%.

[0012] More preferably, the down-regulation rate of the polypeptide kallikrein promoter on MMP1 is 20-55%.

[0013] More preferably, the down-regulation rate of the polypeptide kallikrein promoter on MMP3 is 28-80%.

[0014] More preferably, the down-regulation rate of the polypeptide kallikrein promoter on MMP9 is 38-86%.

[0015] Preferably, the polypeptide kallikrein promoter includes a polypeptide compound, a preservative, and water.

[0016] More preferably, the dosage of the polypeptide compound is equivalent to 0.001-0.05 wt% of the total amount of the polypeptide kallikrein promoter.

[0017] 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.

[0018] Even more preferably, the dosage of red scorpion toxin is equivalent to 16-100 wt% of the total amount of the polypeptide compound.

[0019] Even more preferably, the dosage of acetyl tetrapeptide-3 is equivalent to 16-100 wt% of the total amount of the polypeptide compound.

[0020] Even more preferably, the dosage of hexapeptide-11 is equivalent to 16-100 wt% of the total amount of the polypeptide compound.

[0021] Even more preferably, the dosage of hyaluronic acid polypeptide is equivalent to 16-100 wt% of the total amount of the polypeptide compound.

[0022] Even more preferably, the dosage of palmitoyl tripeptide-5 is equivalent to 16-100 wt% of the total amount of the polypeptide compound.

[0023] Even more preferably, the dosage of acetyl octapeptide-3 is equivalent to 20-100 wt% of the total amount of the polypeptide compound.

[0024] More preferably, the dosage of the preservative is equivalent to 10-30 wt% of the total amount of the polypeptide kallikrein promoter.

[0025] More preferably, the preservative is a mixture of glycerol, ethylhexylglycerin, and 1,2-ethylene glycol.

[0026] Even more preferably, the dosage of glycerol is equivalent to 85-95 wt% of the total amount of the preservative.

[0027] Even more preferably, the dosage of ethylhexylglycerin is equivalent to 0.1-1 wt% of the total amount of the preservative.

[0028] Even more preferably, the dosage of 1,2-ethylene glycol is equivalent to 4-15 wt% of the total amount of the preservative.

[0029] Since 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 glycerin, ethylhexyl glycerin, and 1,2-ethanediol, it has the following beneficial effects: When the present invention uses red scorpion toxin, acetyl tetrapeptide-2, acetyl tetrapeptide-3, or hexapeptide-11 and the preservative and water to form a polypeptide kallikrein promoter, it can significantly promote the expression of KLKs genes, and among them, the polypeptide kallikrein promoter composed of red scorpion toxin, the preservative, and water has the most significant effect; When the present invention uses acetyl tetrapeptide-2, hyaluronic acid polypeptide, or palmitoyl tripeptide-5 and the preservative and water to form a polypeptide kallikrein promoter, it can effectively inhibit the expression of MMPs genes; The polypeptide kallikrein promoter composed of acetyl tetrapeptide-2, the preservative, and water in the present invention has a dual regulatory effect, and while promoting the expression of KLKs genes, it also has a significant effect of inhibiting the expression of MMPs genes. Therefore, the present invention provides a polypeptide kallikrein promoter with excellent effects of promoting the expression of KLKs and inhibiting the expression of MMPs, which can promote the skin desquamation process by activating the KLKs genes and delay skin aging by inhibiting the expression of MMPs genes, and its synergistic action mechanism helps to develop products with dual effects of promoting skin renewal and anti-aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the up-regulation rate of the expression of KLK5. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] The present invention will be further described in detail below in conjunction with the specific embodiments. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0032] The experimental methods in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

[0033] Example 1: A polypeptide kallikrein promoter, comprising a polypeptide compound, a preservative, and water. The dosage of the polypeptide compound is 0.05 wt% of the total amount of the polypeptide kallikrein promoter, the dosage of the preservative is 22.1 wt% of the total amount of the polypeptide kallikrein promoter, and the rest is water. The polypeptide compound is red scorpion toxin; the preservative is a mixture of glycerol, ethylhexylglycerin, and 1,2-ethanediol. The dosage of glycerol is 90.5 wt% of the total amount of the preservative, the dosage of ethylhexylglycerin is 0.5 wt% of the total amount of the preservative, and the dosage of 1,2-ethanediol is 9% of the total amount of the preservative.

[0034] Example 2: This example is different from Example 1 only in that the polypeptide compound is acetyl tetrapeptide-2, and other conditions are the same as those in Example 1.

[0035] Example 3: This example is different from Example 1 only in that the polypeptide compound is acetyl tetrapeptide-3, and other conditions are the same as those in Example 1.

[0036] Example 4: This example is different from Example 1 only in that the polypeptide compound is hexapeptide-11, and other conditions are the same as those in Example 1.

[0037] Example 5: This example is different from Example 1 only in that the polypeptide compound is hyaluronic acid polypeptide, and other conditions are the same as those in Example 1.

[0038] Example 6: This example is different from Example 1 only in that the polypeptide compound is palmitoyl tripeptide-5, and other conditions are the same as those in Example 1.

[0039] Example 7: This example is different from Example 1 only in that the polypeptide compound is acetyl octapeptide-3, and other conditions are the same as those in Example 1.

[0040] Example 8: This example is different from Example 1 only in that the polypeptide compound is a mixture of red scorpion toxin, hyaluronic acid polypeptide, and acetyl octapeptide-3. The dosage of red scorpion toxin is 33 wt% of the total amount of the polypeptide compound, the dosage of hyaluronic acid polypeptide is 33 wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is 34 wt% of the total amount of the polypeptide compound. Other conditions are the same as those in Example 1.

[0041] Example 9: This example is different from Example 1 only in that the polypeptide compound is a mixture of red scorpion toxin, palmitoyl tripeptide-5, and acetyl octapeptide-3. The dosage of red scorpion toxin is equivalent to 33 wt% of the total amount of the polypeptide compound, the dosage of palmitoyl tripeptide-5 is equivalent to 33 wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 34 wt% of the total amount of the polypeptide compound. Other conditions are the same as those in Example 1.

[0042] Example 10: This example is different from Example 1 only in that the polypeptide compound is a mixture of red scorpion toxin, hyaluronic acid polypeptide, palmitoyl tripeptide-5, and acetyl octapeptide-3. The dosage of red scorpion toxin is equivalent to 25 wt% of the total amount of the polypeptide compound, the dosage of hyaluronic acid polypeptide is equivalent to 25 wt% of the total amount of the polypeptide compound, the dosage of palmitoyl tripeptide-5 is equivalent to 25 wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 25 wt% of the total amount of the polypeptide compound. Other conditions are the same as those in Example 1.

[0043] Example 11: This example is different from Example 1 only in that the polypeptide compound is a mixture of acetyl tetrapeptide-3, hyaluronic acid polypeptide, and acetyl octapeptide-3. The dosage of acetyl tetrapeptide-3 is equivalent to 33 wt% of the total amount of the polypeptide compound, the dosage of hyaluronic acid polypeptide is equivalent to 33 wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 34 wt% of the total amount of the polypeptide compound. Other conditions are the same as those in Example 1.

[0044] Example 12: This example is different from Example 1 only in that the polypeptide compound is a mixture of acetyl tetrapeptide-3, palmitoyl tripeptide-5, and acetyl octapeptide-3. The dosage of acetyl tetrapeptide-3 is equivalent to 33 wt% of the total amount of the polypeptide compound, the dosage of palmitoyl tripeptide-5 is equivalent to 33 wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 34 wt% of the total amount of the polypeptide compound. Other conditions are the same as those in Example 1.

[0045] Example 13: This example is different from Example 1 only in that the polypeptide compound is a mixture of acetyl tetrapeptide-3, hyaluronic acid polypeptide, palmitoyl tripeptide-5, and acetyl octapeptide-3. The dosage of acetyl tetrapeptide-3 is equivalent to 25 wt% of the total amount of the polypeptide compound, the dosage of hyaluronic acid polypeptide is equivalent to 25 wt% of the total amount of the polypeptide compound, the dosage of palmitoyl tripeptide-5 is equivalent to 25 wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 25 wt% of the total amount of the polypeptide compound. Other conditions are the same as those in Example 1.

[0046] Example 14: This example is different from Example 1 only in that the polypeptide compound is a mixture of hexapeptide-11, hyaluronic acid polypeptide, and acetyl octapeptide-3. The dosage of hexapeptide-11 is equivalent to 33 wt% of the total amount of the polypeptide compound, the dosage of hyaluronic acid polypeptide is equivalent to 33 wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 34 wt% of the weight of the polypeptide compound. Other conditions are the same as in Example 1.

[0047] Example 15: This example is different from Example 1 only in that the polypeptide compound is a mixture of hexapeptide-11, palmitoyl tripeptide-5, and acetyl octapeptide-3. The dosage of hexapeptide-11 is equivalent to 33 wt% of the total amount of the polypeptide compound, the dosage of palmitoyl tripeptide-5 is equivalent to 33 wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 34 wt% of the weight of the polypeptide compound. Other conditions are the same as in Example 1.

[0048] Example 16: This example is different from Example 1 only in that the polypeptide compound is a mixture of hexapeptide-11, hyaluronic acid polypeptide, palmitoyl tripeptide-5, and acetyl octapeptide-3. The dosage of hexapeptide-11 is equivalent to 25 wt% of the total amount of the polypeptide compound, the dosage of hyaluronic acid polypeptide is equivalent to 25 wt% of the total amount of the polypeptide compound, the dosage of palmitoyl tripeptide-5 is equivalent to 25 wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 25 wt% of the weight of the polypeptide compound. Other conditions are the same as in Example 1.

[0049] Example 17: This example is different from Example 1 only in that the polypeptide compound is a mixture of red scorpion toxin, acetyl tetrapeptide-3, hyaluronic acid polypeptide, and acetyl octapeptide-3. The dosage of red scorpion toxin is equivalent to 25 wt% of the total amount of the polypeptide compound, the dosage of acetyl tetrapeptide-3 is equivalent to 25 wt% of the total amount of the polypeptide compound, the dosage of hyaluronic acid polypeptide is equivalent to 25 wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 25 wt% of the weight of the polypeptide compound. Other conditions are the same as in Example 1.

[0050] Example 18: This example is different from Example 1 only in that the polypeptide compound is a mixture of red scorpion toxin, acetyl tetrapeptide-3, palmitoyl tripeptide-5, and acetyl octapeptide-3. The dosage of red scorpion toxin is equivalent to 25 wt% of the total amount of the polypeptide compound, the dosage of acetyl tetrapeptide-3 is equivalent to 25 wt% of the total amount of the polypeptide compound, the dosage of palmitoyl tripeptide-5 is equivalent to 25 wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 25 wt% of the weight of the polypeptide compound. Other conditions are the same as in Example 1.

[0051] Example 19: Compared with Example 1, the only difference in this example 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 dosage of red scorpion toxin is equivalent to 20wt% of the total amount of the polypeptide compound, the dosage of acetyl tetrapeptide-3 is equivalent to 20wt% of the total amount of the polypeptide compound, the dosage of hyaluronic acid polypeptide is equivalent to 20wt% of the total amount of the polypeptide compound, the dosage of palmitoyl tripeptide-5 is equivalent to 20wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 20wt% of the weight of the polypeptide compound. Other conditions are the same as those in Example 1.

[0052] Example 20: Compared with Example 1, the only difference in this example is that the polypeptide compound is a mixture of red scorpion toxin, hexapeptide-11, hyaluronic acid polypeptide and acetyl octapeptide-3. The dosage of red scorpion toxin is equivalent to 25wt% of the total amount of the polypeptide compound, the dosage of hexapeptide-11 is equivalent to 25wt% of the total amount of the polypeptide compound, the dosage of hyaluronic acid polypeptide is equivalent to 25wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 25wt% of the weight of the polypeptide compound. Other conditions are the same as those in Example 1.

[0053] Example 21: Compared with Example 1, the only difference in this example is that the polypeptide compound is a mixture of red scorpion toxin, hexapeptide-11, palmitoyl tripeptide-5 and acetyl octapeptide-3. The dosage of red scorpion toxin is equivalent to 25wt% of the total amount of the polypeptide compound, the dosage of hexapeptide-11 is equivalent to 25wt% of the total amount of the polypeptide compound, the dosage of palmitoyl tripeptide-5 is equivalent to 25wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 25wt% of the weight of the polypeptide compound. Other conditions are the same as those in Example 1.

[0054] Example 22: Compared with Example 1, the only difference in this example 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 dosage of red scorpion toxin is equivalent to 20wt% of the total amount of the polypeptide compound, the dosage of hexapeptide-11 is equivalent to 20wt% of the total amount of the polypeptide compound, the dosage of hyaluronic acid polypeptide is equivalent to 20wt% of the total amount of the polypeptide compound, the dosage of palmitoyl tripeptide-5 is equivalent to 20wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 20wt% of the weight of the polypeptide compound. Other conditions are the same as those in Example 1.

[0055] Example 23: This example is different from Example 1 only in that the polypeptide compound is a mixture of red scorpion toxin, acetyl tetrapeptide-3, hexapeptide-11, hyaluronic acid polypeptide and acetyl octapeptide-3. The dosage of red scorpion toxin is equivalent to 20 wt% of the total amount of the polypeptide compound, the dosage of acetyl tetrapeptide-3 is equivalent to 20 wt% of the total amount of the polypeptide compound, the dosage of hexapeptide-11 is equivalent to 20 wt% of the total amount of the polypeptide compound, the dosage of hyaluronic acid polypeptide is equivalent to 20 wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 20 wt% of the weight of the polypeptide compound. Other conditions are the same as those in Example 1.

[0056] Example 24: This example is different from Example 1 only in that the polypeptide compound is a mixture of red scorpion toxin, acetyl tetrapeptide-3, hexapeptide-11, palmitoyl tripeptide-5 and acetyl octapeptide-3. The dosage of red scorpion toxin is equivalent to 20 wt% of the total amount of the polypeptide compound, the dosage of acetyl tetrapeptide-3 is equivalent to 20 wt% of the total amount of the polypeptide compound, the dosage of hexapeptide-11 is equivalent to 20 wt% of the total amount of the polypeptide compound, the dosage of palmitoyl tripeptide-5 is equivalent to 20 wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 20 wt% of the weight of the polypeptide compound. Other conditions are the same as those in Example 1.

[0057] Example 25: This example is different from Example 1 only in 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 dosage of red scorpion toxin is equivalent to 16 wt% of the total amount of the polypeptide compound, the dosage of acetyl tetrapeptide-3 is equivalent to 16 wt% of the total amount of the polypeptide compound, the dosage of hexapeptide-11 is equivalent to 16 wt% of the total amount of the polypeptide compound, the dosage of hyaluronic acid polypeptide is equivalent to 16 wt% of the total amount of the polypeptide compound, the dosage of palmitoyl tripeptide-5 is equivalent to 16 wt% of the total amount of the polypeptide compound, and the dosage of acetyl octapeptide-3 is equivalent to 20 wt% of the weight of the polypeptide compound. Other conditions are the same as those in Example 1.

[0058] Experimental example: 1. Biosafety cytotoxicity test The cytotoxicity test of keratinocytes was carried out using the polypeptide kallikrein promoter of the present invention. The keratinocytes were provided by Guangdong Boxi Biotechnology Co., Ltd. The specific steps are as follows: According to 1×10 4The inoculation density of cells / holes: Keratinocytes were inoculated into a 96-well plate and incubated overnight in an incubator at 37°C and 5% CO2. The experiment was set up with a zero control group, a control group, a positive control group, and 25 sample groups. In the 25 sample groups, the polypeptide compounds in the kallikrein promoters of Examples 1-25 were used for treatment in sequence. The polypeptide compounds were diluted with water to 8 mass concentration gradients, namely 100%, 50%, 20%, 10%, 5%, 1%, 0.5%, and 0.1%, and 3 replicate wells were set at each concentration gradient. Administration was carried out when the cell confluence rate in the 96-well plate reached 60%. 200 μL of DMEM cell culture medium containing 10% PBS was added to each well of the control group; 200 μL of DMEM cell culture medium containing 10% DMSO was added to each well of the positive control group; 200 μL of DMEM cell culture medium containing the corresponding concentration of the sample was added to each well of the sample group; no cells were inoculated in the zero control group, and only 200 μL of DMEM cell culture medium was added. After the administration was completed, the 96-well plate was placed in an incubator at 37°C and 5% CO2 for 24 h, then the supernatant was discarded, and DMEM medium containing 0.5 mg / mL of MTT was added, and incubated at 37°C in the dark for 4 h. After the incubation was completed, the supernatant was discarded, 150 μL of DMSO was added to each well, and the OD value was read at 490 nm. Relative cell viability (%) = (OD490nm of the sample group - OD490nm of the zero control group) / (OD490nm of the control group - OD490nm of the zero control group) × 100%.

[0059] When the mass concentration of the polypeptide compounds in the kallikrein promoters of Examples 1-25 was below 20%, the relative cell viability was 100% in all cases. In the kallikrein promoters of Examples 1-25 of the present invention, the dosage of the polypeptide compound was equivalent to 0.05 wt% of the total amount of the kallikrein promoter, and it had basically no toxicity to keratinocytes and could be used normally.

[0060] 2. Detection of KLKs gene expression After treating keratinocytes with the kallikrein promoters of Examples 1-25, the KLK5, KLK7, and KLK14 gene expression levels of keratinocytes were measured. The keratinocytes were provided by Guangdong Boxi Biotechnology Co., Ltd. The specific steps were as follows: After resuscitating the keratinocytes, when the plating rate reached 60%, the keratinocytes were seeded into 6-well plates and incubated overnight in an incubator at 37°C with a CO2 concentration of 5%. A blank control group and 25 sample groups were set up, with 3 replicates for each group. When the cell plating rate in the 6-well plates reached 62%, 2 mL of cell culture medium was added to the blank control group; 2 mL of the kallikrein promoters of Examples 1-25 were sequentially added to the 25 sample groups. After the drug administration was completed, the 6-well plates were placed in an incubator at 37°C with a CO2 concentration of 5% and cultured for 24 h.

[0061] After the incubation, each group was washed twice with 1 mL of PBS, and 1 mL of RNAiso Plus (purchased from Takara Bio Inc. (Dalian)) was added to each well. After pipetting to lyse the cells, the samples were collected. After the collection of the cell culture supernatant, each well was washed twice with 1 mL of PBS, 1 mL of RNAiso Plus was added to each well, and after pipetting to lyse the cells, the cell lysates were collected. 200 μL of chloroform was added, and the mixture was shaken vigorously 25 times. After standing at room temperature for 5 min, it was centrifuged at 12,000 rpm for 15 min at 4°C. 400 μL of the upper aqueous phase was aspirated, 400 μL of isopropanol was added, and the mixture was gently shaken 7 times. After standing at room temperature for 10 min, the supernatant was carefully discarded. 1 mL of ethanol solution with a weight concentration of 75% was added, the bottom of the tube was flicked gently to wash thoroughly and suspend the precipitate. Then it was centrifuged at 12,000 rpm for 5 min at 4°C, and the supernatant was carefully discarded. After natural drying in a fume hood for 10 min, 50 μL of DEPC water was added, and it was incubated in a water bath at 60°C for 10 min to completely dissolve it, obtaining the initial RNA solution. Using DEPC water as a blank well, 2 μL of each sample was loaded, and 2 replicates were set for each RNA sample to detect the RNA concentration.

[0062] A reaction system was composed of 1 μL of the initial RNA solution, 2 μL of gDNA Eraser Mix Ver.2, and 13 μL of nuclease-free water. The reaction was carried out at 42°C for 2 min. After the reaction ended, the RNA solution was obtained and stored at 4°C. gDNA Eraser Mix Ver.2 was purchased from Hunan Aikery Biotechnology Co., Ltd.

[0063] Take 1 μL of RNA solution, 4 μL of 5×Premix reverse transcription reagent, and 16 μL of nuclease-free water to form a reaction system. React at 37 °C for 15 min, then react at 85 °C for 5 s. After the reaction, obtain the cDNA solution and store it at 4 °C. The 5×Premix reverse transcription reagent is purchased from Takara Bio Inc. (Dalian).

[0064] Add an equal volume of sterile water to the cDNA solution, shake well to obtain a cDNA dilution. Take 2 μL of the cDNA dilution, 0.8 μL of the forward primer, 0.8 μL of the reverse primer, 10 μL of SYBR Green fluorescence quantitative premix reagent, and 6.4 μL of sterilized ultrapure water to form a reaction system. React at 95 °C for 30 s, 95 °C for 5 s, 60 °C for 30 s, 67.5 °C for 5 s, 95 °C for 5 s, and perform 40 cycles. The SYBR Green fluorescence quantitative premix reagent is purchased from Hunan Aikery Biotech Co., Ltd. Use the 2 -△△CT method to calculate the KLK5, KLK7, and KLK14 gene expression levels, and then calculate the upregulation rates of the KLK5, KLK7, and KLK14 genes. The upregulation rate (%) = (sample group - blank control group) / blank control group × 100%. The results of the KLKs gene upregulation rates are shown in Table 1.

[0065] Table 1 KLKs gene upregulation rates (%)

[0066] Figure 1 is the upregulation rate of KLK5. S1 to S8 correspond to the experimental data of Examples 1 to 8 in sequence, and S9 corresponds to the experimental data of Example 10. From Figure 1From the experimental data in Table 1, it can be seen that the polypeptide kallikrein promoters in Examples 1-4 can effectively promote the up-regulation of the expression of KLK5, KLK7, and KLK14 genes, while the polypeptide kallikrein promoters in Examples 5-7 have no regulatory effect on the KLK5, KLK7, and KLK14 genes. This indicates that the polypeptide kallikrein promoter containing red scorpion toxin, acetyl tetrapeptide-2, acetyl tetrapeptide-3, or hexapeptide-11 can effectively up-regulate the expression of KLK5, KLK7, and KLK14 genes, and red scorpion toxin, acetyl tetrapeptide-2, acetyl tetrapeptide-3, or hexapeptide-11 is the key active ingredient of the polypeptide kallikrein promoter. In promoting the up-regulation of KLK5 gene expression, the potency ranking is: red scorpion toxin > acetyl tetrapeptide-2 > hexapeptide-11 > acetyl tetrapeptide-3; in promoting the up-regulation of KLK7 gene expression, the potency ranking is: red scorpion toxin > acetyl tetrapeptide-2 > acetyl tetrapeptide-3 > hexapeptide-11; in promoting the up-regulation of KLK14 gene expression, the potency 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 preservative has the best promoting effect on KLK5, KLK7, and KLK14.

[0067] In Example 1, the dosage of red scorpion toxin is equivalent to 100 wt% of the total amount of the polypeptide compound, and its up-regulation rates of the expression of KLK5, KLK7, and KLK14 genes are 35%, 38%, and 62% respectively. From this, it can be obtained that when the dosage of red scorpion toxin is equivalent to 33 wt% of the total amount of the polypeptide compound, its theoretical up-regulation rates of the expression of KLK5, KLK7, and KLK14 genes should be 11.67%, 12.67%, and 20.67% respectively; when the dosage of red scorpion toxin is equivalent to 25 wt% of the total amount of the polypeptide compound, its theoretical up-regulation rates of the expression of KLK5, KLK7, and KLK14 genes should be 8.75%, 9.5%, and 15.5% respectively. According to the experimental results of Example 8, when the dosage of red scorpion toxin is equivalent to 33 wt% and 25 wt% of the total amount of the polypeptide compound, its actual up-regulation rates of the expression of KLK5, KLK7, and KLK14 genes are significantly higher than the theoretical values. This indicates that compared with using red scorpion toxin alone as the polypeptide compound, using red crab toxin in combination with other polypeptides to prepare the polypeptide kallikrein promoter makes its promoting effect on kallikrein expression better.

[0068] 3. MMP Gene Expression Detection After treating fibroblasts stimulated by UVA with a polypeptide kallikrein promoter, the gene expression levels of MMP1, MMP3, and MMP9 were measured. The fibroblasts were provided by Guangdong Boxi Biotechnology Co., Ltd. The specific steps were as follows: After reviving the fibroblasts, when the plating rate reached 60%, the fibroblasts were seeded into a 6-well plate and incubated overnight in an incubator at 37°C with a CO2 concentration of 5%. A blank control group, a negative control group, a positive control group, and a sample group were set up, with 3 replicates in each group. When the cell plating rate in the 6-well plate reached 40%, 2 mL of cell culture medium was added to the blank control group and the negative control group; 2 mL of cell culture medium containing 100 ng / mL TGF-β1 was added to the positive control group; 2 mL of the polypeptide kallikrein promoter of the present invention was added to the sample group in sequence. After the administration was completed, the 6-well plate was placed in an incubator at 37°C with a CO2 concentration of 5% and cultured for 24 h. Except for the blank control group, the other groups were irradiated with UVA for 1820 s, and the irradiation dose was 30 J / cm 2 , and after the irradiation, it was cultured in an incubator at 37°C with a CO2 concentration of 5% for 24 h. After the incubation was completed, the cell culture supernatant was collected, and the gene expression levels of MMP1, MMP3, and MMP9 were measured using an MMP1 ELISA kit, an MMP3 ELISA kit, and an MMP9 ELISA kit respectively. According to the detection results of the gene expression levels of MMP1, MMP3, and MMP9, the down-regulation rates of the MMP1, MMP3, and MMP9 genes were calculated. The down-regulation rate (%) = (negative control group - sample group) / negative control group × 100%. The MMP1 ELISA kit, the MMP3 ELISA kit, and the MMP9 ELISA kit were all purchased from Abcam (Shanghai) Trading Co., Ltd. The results of the MMPs gene expression down-regulation rate are shown in Table 2.

[0069] Table 2 MMPs gene expression down-regulation rate (%)

[0070] From the experimental data in Table 2, it can be seen that the polypeptide kallikrein promoters in Example 2, Example 5, and Example 6 can effectively down-regulate the gene expression of MMP1, MMP3, and MMP9, and the effects of Example 2 and Example 6 are significantly better than those of Example 5. This shows that the polypeptide kallikrein promoter containing acetyl tetrapeptide-2, hyaluronic acid polypeptide, or palmitoyl tripeptide-5 can effectively down-regulate the gene expression of MMP1, MMP3, and MMP9, and acetyl tetrapeptide-2, hyaluronic acid polypeptide, and palmitoyl tripeptide-5 are the key components for inhibiting the expression of matrix metalloproteinases. Among them, the polypeptide kallikrein promoter containing acetyl tetrapeptide-2 has the best effect on down-regulating the gene expression of MMP1 and MMP9, and the polypeptide kallikrein promoter containing palmitoyl tripeptide-5 has the best effect on down-regulating the gene expression of MMP3.

[0071] In Example 5, the dosage of hyaluronic acid polypeptide is equivalent to 100 wt% of the total amount of polypeptide compounds, and the down-regulation rates of MMP1, MMP3 and MMP9 gene expressions are 33%, 29% and 55% respectively. From this, it can be obtained that when the dosage of hyaluronic acid polypeptide is equivalent to 33 wt% of the total amount of polypeptide compounds, the theoretical down-regulation rates of MMP1, MMP3 and MMP9 gene expressions should be 11%, 9.67% and 18.33% respectively; when the dosage of hyaluronic acid polypeptide is equivalent to 25 wt% of the total amount of polypeptide compounds, the theoretical down-regulation rates of MMP1, MMP3 and MMP9 gene expressions should be 8.25%, 7.25% and 13.75% respectively. According to the experimental results of Example 8, when the dosage of hyaluronic acid polypeptide is equivalent to 33 wt% and 25 wt% of the total amount of polypeptide compounds, the actual down-regulation rates of MMP1, MMP3 and MMP9 gene expressions are significantly higher than the theoretical values. This shows that compared with using hyaluronic acid polypeptide alone as a polypeptide compound, using a polypeptide kallikrein promoter prepared by compounding hyaluronic acid polypeptide with other polypeptides makes its inhibitory effect on matrix metalloproteinase expression better.

[0072] It can be seen from the experimental data in Table 1 and Table 2 that the polypeptide kallikrein promoter containing acetyl tetrapeptide-2 can not only promote the expressions of KLK5, KLK7 and KLK14 genes, but also inhibit the expressions of MMP1, MMP3 and MMP9 genes.

[0073] The conventional operations in the operation steps of the present invention are well known to those skilled in the art and will not be elaborated here.

[0074] The above-described embodiments have detailed the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used 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 protection scope of the present invention.

Claims

1. Use of a polypeptide kallikrein promoter in the preparation of a cosmetic for promoting kallikrein expression and / or inhibiting matrix metalloproteinase expression; 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 on kallikrein expression is 15-62%.

2. The application according to claim 1, wherein 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 on matrix metalloproteinase expression is 20-86%.

3. The application according to claim 1, wherein The polypeptide kallikrein promoter includes a polypeptide compound, a preservative and water.

4. The application according to claim 3, characterized in that, The dosage of the polypeptide compound is equivalent to 0.001-0.05 wt% of the total amount of the polypeptide kallikrein promoter.

5. The application according to claim 3, wherein 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.

6. The application according to claim 3, characterized in that, The dosage of the preservative is equivalent to 10-30 wt% of the total amount of the polypeptide kallikrein promoter.

7. The application according to claim 3, characterized in that, The preservative is a mixture of glycerol, ethylhexylglycerin and 1,2-ethylene glycol.

8. The application according to claim 7, wherein The dosage of the glycerol is equivalent to 85-95 wt% of the total amount of the preservative.

9. The application according to claim 7, characterized in that, The dosage of the ethylhexylglycerin is equivalent to 0.1-1 wt% of the total amount of the preservative.

10. The application according to claim 7, wherein The dosage of the 1,2-ethylene glycol is equivalent to 4-15 wt% of the total amount of the preservative.

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