CN113662869A

CN113662869A - Compound for promoting extracellular matrix gene expression and preparation method thereof

Info

Publication number: CN113662869A
Application number: CN202111040736.8A
Authority: CN
Inventors: 王婷; 查建生; 童莉; 刘雅芬; 郑云云
Original assignee: Spec-Chem Industry Inc
Current assignee: Spec-Chem Industry Inc
Priority date: 2021-09-06
Filing date: 2021-09-06
Publication date: 2021-11-19
Anticipated expiration: 2041-09-06
Also published as: CN113662869B

Abstract

The invention discloses a compound for promoting the gene expression of extracellular matrix and a preparation method thereof, wherein the compound comprises 1-5% of polyhydroxy acid, 1-15% of alpha hydroxy acid, 0.1-1% of carnosine, 0.01-1% of nonapeptide-1, 0.1-5% of amide, 3-8% of amino acid derivative, 10-50% of polyalcohol, 0.1-1.0% of antioxidant stabilizer and the balance of water. The compound disclosed by the invention can effectively improve the content of core components in the extracellular matrix so as to reinforce the extracellular matrix, can effectively promote the expression of collagen I, collagen VII and Laminin genes, promotes the regeneration of collagen in the extracellular matrix, further strengthens the function of the extracellular matrix, plumps and compacts the skin, and achieves the effects of smoothing static and dynamic fine lines from inside to outside and improving the skin brightness.

Description

Compound for promoting extracellular matrix gene expression and preparation method thereof

Technical Field

The invention belongs to the field of skin care products, and particularly relates to a compound for promoting extracellular matrix gene expression and a preparation method thereof.

Background

The improvement of skin condition is not determined by single factor, but is a complex result of multi-factor combined action, so that the effect of single active substance is not always as desired by the product design function because of the difference of population tolerance. Secondly, with respect to the development trend of skin care products, skin care requirements are gradually deepened from exogenous improvement to endogenous repair and nourishment. For example, whitening is not only required to be a reduction in skin color, but also to increase skin glossiness and improve a healthy skin condition, and to present skin aesthetics from inside to outside. For example, anti-aging is not only required to reduce the generation of wrinkles, but also required to inject more nourishing ingredients into the skin, so that the skin can be preferably promoted to restore and regenerate, and the skin is plump through the generation of beneficial ingredients, so that the skin can eliminate surface dynamic (light-age skin) and static (mature skin) wrinkles, and the effect of overall anti-aging is achieved.

Disclosure of Invention

The purpose of the invention is as follows: it is a first object of the present invention to provide a complex capable of effectively promoting the expression of type I collagen, type VII collagen and lamin gene in extracellular matrix (ECM); the second purpose of the invention is to provide a preparation method of the compound.

The technical scheme is as follows: the compound for promoting the gene expression of the extracellular matrix comprises the following raw materials in parts by mass: the compound comprises the following raw materials in parts by mass: 1-5% polyhydroxy acid, 1-15% alpha hydroxy acid, 0.1-1% carnosine, 0.01-1% nonapeptide-1, 0.1-5% amide, 3-8% amino acid derivative, 10-50% polyalcohol, 0.1-1.0% antioxidant stabilizer and the balance of water.

Biologically, the extracellular matrix (ECM) is a three-dimensional network of extracellular macromolecules containing collagen that provides structural and biochemical support for surrounding cells, as well as shoulder cell adhesion, cell communication, differentiation and some common functions. The collagen is used as the protein with the highest content in the extracellular matrix, and accounts for up to 90 percent. Collagen acts as a fibrin in the extracellular matrix, providing structural support for the resident cells. Based on the compound, the compound is prepared by compounding the carnosine, the nonapeptide-1, the polyhydroxy acid, the alpha hydroxy acid, the amide, the amino acid derivative, the polyalcohol and the antioxidant stabilizer, and when the compound acts on skin, the function of extracellular matrix can be strengthened by promoting the expression of related genes of the extracellular matrix and the generation of collagen, so that the skin is plump and compact, and the effects of smoothing static and dynamic fine lines from inside to outside can be achieved; in addition, the skin surface is smoother due to the increased plumpness and plumpness of the skin, and the brightness of the skin is better visually when light is emitted on the skin surface, so that the skin is bright, white and healthy. And because of the enhancement of the function of the extracellular matrix, the intercellular adhesion effect is enhanced in the skin structure similar to a brick wall-cement structure, so that the function of the epidermal barrier function is further enhanced, and the skin has more excellent self-protection function.

Further, the polyhydroxy acids employed in the complexes may include lactobionic acid, maltobionic acid, gluconolactone, gluconic acid or gluconate. Preferably, the gluconate salt may comprise zinc gluconate or copper gluconate.

Further, the alpha hydroxy acid used in the composition may include glycolic acid, lactic acid, malic acid, tartaric acid or mandelic acid.

Further, the amide employed in the complex may include ceramide or nicotinamide.

Further, the amino acid derivative used in the complex may include tranexamic acid or oxothiazolidinecarboxylic acid.

Further, the polyhydric alcohol used in the composition may include glycerin, 1, 2-propanediol, 1, 3-butanediol, 1, 2-hexanediol, or ethylhexylglycerin.

Further, the antioxidant stabilizer used in the composition may include ascorbic acid and its derivatives, tocopherol and its derivatives, polyphenol, butyl hydroxy anisole, dibutyl hydroxy toluene, tert-butyl hydroquinone or sodium metabisulfite.

The method for preparing the compound comprises the following steps: respectively adding polyalcohol, antioxidant stabilizer, polyhydroxy acid and alpha hydroxy acid into water according to mass fraction, stirring and mixing uniformly, adjusting pH to 5.5-6.5, adding carnosine, nonapeptide-1, amide and amino acid derivative, and continuously adjusting pH to 5.5-6.5 to obtain the compound.

Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: the compound can effectively improve the content of core components in the extracellular matrix so as to strengthen the extracellular matrix, and can effectively promote the expression of collagen I, collagen VII and Lamin genes so as to promote the regeneration of collagen in the extracellular matrix, namely, the compound is different from common exogenous function-improved active substances, multiple targets, multiple dimensions and channel synergistic action from the perspective of endogenous, gene expression, marker generation and accessory functions, solves the problem of multiple light aged skins, achieves the effects of resisting aging, reducing melanin generation, whitening, improving skin brightness and luster, increasing skin smoothness and relieving skin darkness; meanwhile, the raw materials of the compound are mainly natural substances, so that the compound is safe and mild to skin; in addition, the preparation method of the compound is simple and easy, and the operability is strong.

Drawings

FIG. 1 shows (fibroblast) cell viability at different concentrations tested for the complexes of the invention;

FIG. 2 is a picture of 3D human epidermal reconstructed skin model tissue treated with a negative control sample, wherein the negative control sample is DPBS buffer solution;

FIG. 3 is a picture of 3D human epidermal reconstructed skin model tissue treated with the compound of the present invention;

fig. 4 is a picture of 3D human epidermal reconstructed skin model tissue treated with a positive control sample of 5% SDS solution;

FIG. 5 is a graph of β hexosaminidase release from mast cells following the action of varying concentrations of the agents of the present invention;

FIG. 6 is a photograph showing that the complex of the present invention acts on mast cells without affecting their normal morphology; wherein: a-negative control, B-positive control, C-I different concentrations of the complex of the invention (20%, 10%, 5%, 2.5%, 1.25%, 0.625%, 0.3125%);

FIG. 7 shows the collagen content in the cell culture dish at different concentrations of the complex of the present invention;

FIG. 8 is a graph showing the inhibitory effect of the present invention on tyrosinase activity (whitening and brightening) at different concentrations;

fig. 9 is a graph of dopa-stained melanocytes (showing melanin content); wherein: a is a blank control group, B-F are the test groups of the compound of the invention at different concentrations (0.2%, 0.1%, 0.05%, 0.025% and 0.0125%);

FIG. 10 is a graph showing the effect of various concentrations of the invention on melanocyte on melanogenesis;

FIG. 11 is a graph showing the change in skin color and luminance parameter values at different times in clinical trials using the complexes of the invention.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to the following embodiments and the accompanying drawings.

Example 1

The compound raw materials added for this example are shown in table 1 below.

Table 1 feed composition of example 1

The composite of this example was prepared by the following steps: adding glycerol, sodium pyrosulfite, maltonic acid and mandelic acid into water according to mass fraction, adjusting pH to 5.5-6.5, finally adding carnosine, nonapeptide-1, nicotinamide and tranexamic acid, and adjusting to ensure that final pH is more than or equal to 5.5(5.5-6.5), thereby preparing the compound. Wherein, the pH is adjusted by using a pH adjusting agent, preferably, the pH adjusting agent can comprise sodium hydroxide, sodium citrate, triethanolamine or citric acid.

Performance detection

The compound prepared in the above example was subjected to cytotoxicity test on fibroblast, safety test on human epidermis, anti-allergy test, performance test on promoting collagen regeneration, PCR test on extracellular matrix (ECM) synthesis-related gene, tyrosinase activity inhibition, reduction of intracellular melanin production, and clinical test, respectively, and the obtained results were as follows. In the following test of the present invention, the control mentioned is a control group of non-dosed blank cells.

Performance test-1 in vitro cytotoxicity assay for fibroblasts

The complex was subjected to in vitro cytotoxicity test, and the results obtained are shown in fig. 1 below. IC of the complexes on NIH/3T3 cell line according to SN/T2328-2009 and MTT colorimetry₅₀26000ug/mL (2.6%). At the cellular level, the complex is not toxic to fibroblasts at concentrations no greater than 15600ug/mL (1.56%). If the theory is converted into the acute oral toxicity of mice, the LD50 value of the compound is 4645mg/kg (the acute oral toxicity LD50 of refined salt NaCl is about 3000mg/kg), so the compound belongs to a nontoxic substance according to the GHS classification standard and is safe to the skin.

Performance testing-2 reconstruction of human epidermal skin 3D model for testing skin mildness

The complex prepared according to the present invention was subjected to a skin mildness test, and the results obtained are shown in table 2 below and fig. 2 to 4.

TABLE 23D tissue viability results for skin models

According to the judgment standard of the in vitro skin irritation artificial skin model test, the tissue activity of the compound of the invention is 93.54 percent and more than 50 percent, so that the compound of the invention is judged to have no skin irritation. As can be seen from fig. 2 to 4, the complex does not cause adverse reactions on cells and tissues and does not affect normal morphology and tissue functions of cells on a human skin tissue model reconstructed from in vitro cells, and does not damage normal skin cells and tissues and does not cause irritation while strengthening extracellular matrix.

Performance test-3 non-allergenic (anti-allergic) test

The results obtained by subjecting the complex prepared according to the present invention to an anti-allergy test are shown in fig. 5 and 6. In FIG. 5, Negative Control, UVA irradiation and no drug administration, is Negative Control; positive Control is Compound 48/80 (polymer produced by condensation of N-methyl-p-methoxyphenethylamine and formaldehyde), and p < 0.001vs. negative Control and Positive Control, and it can be seen from the figure that the complex of the present invention does not cause increase of release of the sensitizing marker β hexosaminidase in mast cells at each concentration, and therefore does not cause sensitization reaction at an added concentration of up to 20% relative to the Positive Control. In addition, the complex of the invention has certain release inhibition effect on beta hexosaminidase which is a sensitization marker released by mast cells under the condition that the concentration is more than or equal to 1.25 percent, thereby showing certain anti-sensitization effect.

Performance test-4 detection of promotion of collagen regeneration

The results obtained by subjecting the complex prepared by the present invention to a collagen regeneration promotion assay are shown in fig. 7, in which: p < 0.001vs. control Cells (control group). The figure shows that the compound of the invention can remarkably promote the fibroblast to synthesize collagen, has a collagen synthesis promoting rate as high as 105% under the condition of extremely low addition concentration of 0.1%, has a concentration dependence effect as the addition amount is gradually increased.

Performance test-5 PCR detection of extracellular matrix (ECM) synthesis-related genes

The complex prepared by the present invention was subjected to PCR detection of ECM synthesis-associated genes, and the obtained results are shown in Table 3 and Table 4 below. By using 2^-△△CTThe results were calculated by the method, and statistical analysis was performed by the t-test method, and the amplification factor of mRNA of BC (blank non-administration) was 30344The significance of the treated group was shown by<0.01。

TABLE 3 summary of Gene testing

Wherein, P value is less than 0.01, BC is Black Control blank non-drug-given drug group.

As can be seen from Table 3, the ECM-associated Collagen I, Collagen VII genes and Laminin gene content of the complexes of the invention was significantly increased. However, the monomer active substances do not promote three ECM related genes, which indicates that the compound has a remarkable synergistic effect, and the compound has an effect of remarkably enhancing the function of the ECM based on the protein level at a test concentration of 0.625%, so that the effect of resisting wrinkles is achieved by improving the plump skin cells. As can be seen from table 4, the collagen content increased as compared to both the IOD average value and SD.

TABLE 4 summary of collagen Integrated Optical Density (IOD)/cell number

Experiment grouping	Relative IOD mean value	SD	P value
				NC control group	0.02	0.00	/
Monomer component (mandelic acid)	0.59	0.04	0.000**
				0.625% of the compound	0.8	0.09	0.000**

Wherein denotes a P value < 0.01. Control, NC is Negative Control of Negative Control, UVA irradiation and no drug.

Performance test-6 inhibition of tyrosine Activity to reduce melanogenesis

The results obtained by subjecting the complexes prepared according to the invention to a test for melanin lowering performance are shown in fig. 8, fig. 9 and fig. 10, wherein p < 0.05, p < 0.01, p < 0.001vs. As shown in FIGS. 8 to 10, the complex of the present invention has significant inhibitory effect on tyrosinase activity during melanin production, and the inhibitory rate reaches 58.5% at a concentration of 0.1%, and IC thereof is shown₅₀633 ug/mL, and the inhibitory effect was concentration-dependent. The composite of the inventionBesides the obvious inhibition effect on tyrosinase, the compound also has the obvious effect of reducing the melanin content in melanocytes, and when the addition amount of the compound is only 0.4%, the melanin generation can be reduced by 41.4%. And the melanin production-reducing action has a concentration-dependent relationship, that is, the effect is enhanced with an increase in concentration.

Performance test-7 clinical trial

The results obtained by subjecting the complexes prepared according to the invention to clinical tests are shown in figure 11, in which "L" represents the perceived brightness, "a" and "b" represent the four distinct colors of human vision: red, green, blue and yellow, ITA ° is the pigment content used to assess the overall skin color, and Δ represents the amount of change in this index. It can be seen from the figure that after 5% of the compound of the invention is used, the skin is obviously improved from 14 days to 28 days, and the compound can present the effect of white-red skin and is in a harmonious and healthy white state from inside to outside.

Example 2

The compound raw materials added for this example are shown in table 5 below.

Table 5 feed composition for example 2

Serial number	Raw materials	Content/%
			1	Lactobionic acid	5.0
2	Glycolic acid	4.0
			3	Carnosine	0.1
4	Nonapeptide-1	0.5
			5	Nicotinamide	2.0
6	Tranexamic acid	8.0
			7	1, 3-butanediol	15.0
8	Dibutylhydroxytoluene	0.2
			9	Water (W)	To 100

The composite of this example was prepared by the following steps: adding 1, 3-butanediol, dibutyl hydroxy toluene, lactobionic acid and glycolic acid into water according to mass fraction, adjusting the pH to 5.5-6.5, finally adding carnosine, nonapeptide-1, nicotinamide and tranexamic acid, and adjusting to ensure that the final pH is more than or equal to 5.5(5.5-6.5) to prepare the compound.

Example 3

The compound raw materials added for this example are shown in table 6 below.

Table 6 feed composition for example 3

Serial number	Raw materials	Content/%
			1	Gluconolactone	3.0
2	Lactic acid	8.0
			3	Carnosine	1.0
4	Nonapeptide-1	0.1
			5	Ceramide	0.2
6	Oxothiazolidinecarboxylic acids	5.0
			7	1, 2-hexanediol	15.0
8	Sodium metabisulfite	0.1
			9	Water (W)	To 100

The composite of this example was prepared by the following steps: adding 1, 2-hexanediol, sodium metabisulfite, gluconolactone and lactic acid into water according to mass fraction, adjusting pH to 5.5-6.5, finally adding carnosine, nonapeptide-1, ceramide and oxothiazolidine carboxylic acid, and adjusting to ensure that final pH is not less than 5.5(5.5-6.5), thus obtaining the compound.

Example 4

The compound materials added for this example are shown in table 7 below.

Table 7 feed composition for example 4

The composite of this example was prepared by the following steps: adding ethylhexyl glycerol, tert-butyl hydroquinone, gluconic acid and malic acid into water according to mass fraction, adjusting pH to 5.5-6.5, finally adding carnosine, nonapeptide-1, ceramide and oxothiazolidine carboxylic acid, and adjusting to ensure that final pH is more than or equal to 5.5(5.5-6.5) to obtain the compound.

Example 5

The compound materials added for this example are shown in table 8 below.

Table 8 feed composition for example 5

Serial number	Raw materials	Content/%
			1	Zinc gluconate	2.0
2	Tartaric acid	2.0
			3	Carnosine	0.5
4	Nonapeptide-1	1.0
			5	Ceramide	0.1
6	Oxothiazolidinecarboxylic acids	3.0
			7	1, 2-propanediol	10.0
8	Ascorbic acid	0.2
			9	Water (W)	To 100

The composite of this example was prepared by the following steps: adding 1, 2-propylene glycol, ascorbic acid, zinc gluconate and tartaric acid into water according to mass fraction, adjusting pH to 5.5-6.5, finally adding carnosine, nonapeptide-1, ceramide and oxothiazolidine carboxylic acid, and adjusting to ensure that final pH is more than or equal to 5.5(5.5-6.5), thus obtaining the compound.

Example 6

The compound raw materials added for this example are shown in table 9 below.

Table 9 feed composition for example 6

Serial number	Raw materials	Content/%
			1	Gluconic acid copper salt	4.0
2	Mandelic acid	1.0
			3	Carnosine	0.5
4	Nonapeptide-1	1.0
			5	Nicotinamide	5.0
6	Tranexamic acid	7.0
			7	1, 3-propanediol	30.0
8	Butylated hydroxyanisole	0.5
			9	Water (W)	To 100

The composite of this example was prepared by the following steps: adding 1, 3-propylene glycol, butyl hydroxy anisole, copper gluconate and mandelic acid into water according to mass fraction, adjusting pH to 5.5-6.5, finally adding carnosine, nonapeptide-1, nicotinamide and tranexamic acid, and adjusting to ensure that the final pH is more than or equal to 5.5(5.5-6.5) to obtain the compound.

Example 7

The compound materials added for this example are shown in table 10 below.

Table 10 feed composition for example 7

The composite of this example was prepared by the following steps: adding glycerol, tocopherol, maltobionic acid and mandelic acid into water according to mass fraction, adjusting pH to 5.5-6.5, finally adding carnosine, nonapeptide-1, nicotinamide and tranexamic acid, and adjusting to ensure that final pH is more than or equal to 5.5(5.5-6.5), thereby preparing the compound.

In addition to the above examples, the antioxidant stabilizer used in the preparation of the present invention may also be ascorbic acid derivatives, tocopherol derivatives or polyphenols. The detection effects of the embodiments 2 to 7 of the invention are the same as those of the embodiment 1, the content of core components in the extracellular matrix can be effectively increased, so that the extracellular matrix is strengthened, the expression of collagen I, collagen VII and Lamin genes can be effectively promoted, so that the regeneration of collagen in the extracellular matrix is promoted, the problems of various light aged skins are solved, and the effects of resisting aging, reducing melanin generation, whitening, improving skin brightness and luster, increasing skin smoothness and relieving skin darkness are achieved.

Claims

1. A complex for promoting the expression of extracellular matrix genes, comprising: the compound comprises the following raw materials in parts by mass: 1-5% polyhydroxy acid, 1-15% alpha hydroxy acid, 0.1-1% carnosine, 0.01-1% nonapeptide-1, 0.1-5% amide, 3-8% amino acid derivative, 10-50% polyalcohol, 0.1-1.0% antioxidant stabilizer and the balance of water.

2. The complex for promoting the expression of extracellular matrix genes according to claim 1, wherein: the polyhydroxy acids include lactobionic acid, maltobionic acid, gluconolactone, gluconic acid or gluconate.

3. The complex for promoting the expression of extracellular matrix genes according to claim 2, wherein: the gluconate comprises zinc gluconate or copper gluconate.

4. The complex for promoting the expression of extracellular matrix genes according to claim 1, wherein: the alpha hydroxy acid comprises glycolic acid, lactic acid, malic acid, tartaric acid or mandelic acid.

5. The complex for promoting the expression of extracellular matrix genes according to claim 1, wherein: the amide includes ceramide or nicotinamide.

6. The complex for promoting the expression of extracellular matrix genes according to claim 1, wherein: the amino acid derivative comprises tranexamic acid or oxothiazolidinecarboxylic acid.

7. The complex for promoting the expression of extracellular matrix genes according to claim 1, wherein: the polyhydric alcohol comprises glycerol, 1, 2-propanediol, 1, 3-butanediol, 1, 2-hexanediol, or ethylhexylglycerol.

8. The complex for promoting the expression of extracellular matrix genes according to claim 1, wherein: the antioxidant stabilizer comprises ascorbic acid and its derivatives, tocopherol and its derivatives, polyphenol, butyl hydroxy anisol, dibutyl hydroxy toluene, tert-butyl hydroquinone or sodium pyrosulfite.

9. A method for preparing a complex for promoting the expression of extracellular matrix genes according to claim 1, comprising the steps of: respectively adding polyalcohol, antioxidant stabilizer, polyhydroxy acid and alpha hydroxy acid into water according to mass fraction, stirring and mixing uniformly, adjusting pH to 5.5-6.5, adding carnosine, nonapeptide-1, amide and amino acid derivative, and continuously adjusting pH to 5.5-6.5 to obtain the compound.