CN113621565B - Application of polypeptide in regulation and control of fibroblast in-vitro culture - Google Patents

Abstract

The invention relates to the technical field of cosmetics, in particular to application of polypeptide in regulating and controlling fibroblast in-vitro culture. The invention discovers for the first time that the polypeptide consisting of the amino acid sequence shown in the sequence SEQ ID No.1 can promote the proliferation of fibroblasts in vitro and the secretion of collagen, has obvious and stable promoting effect, has obvious effect of delaying skin aging, is hopeful to further develop a product for promoting the growth of the fibroblasts by taking the polypeptide as a main component, and has good market prospect when being used for skin care or cosmetics.

Description

Application of polypeptide in regulation and control of fibroblast in-vitro culture

Technical Field

The invention relates to the technical field of cosmetics, in particular to application of polypeptide in regulating and controlling fibroblast in-vitro culture.

Background

With the improvement of life quality, the skin aging problem is more and more emphasized. With the aging, the skin can be subjected to degenerative change, and then the functions of the skin can be gradually weakened or lost, and the functions are represented by the reduction of the proliferation capability of skin cells, the reduction of the epidermal renewal speed, the reduction of the epidermal reconstruction capability, the reduction of the dermal collagen synthesis and the like, so that the research on bioactive substances which can proliferate and repair at the cellular level and fundamentally delay the skin aging has very important significance and value for the development of the medical and cosmetic industries.

The products used for skin care or make-up are various, but due to the self-properties of chemical components in skin care products or cosmetics, there are many side effects, even damage to skin health, causing safety problems. The safe and effective skin care product or cosmetic for activating the physiological function of the skin is often expensive and cannot be popularized and applied, so that development of the safe and effective skin care or cosmetic product for activating the physiological function of the skin is urgently needed.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide an application of a polypeptide in regulating and controlling fibroblast in-vitro culture or preparing a product for regulating and controlling fibroblast growth, and provides a method for effectively promoting fibroblast proliferation and collagen secretion in vitro based on the application of the polypeptide in skin care or cosmetic products, so that the promotion effect on fibroblast proliferation and collagen secretion in skin is realized.

The invention also aims to provide a skin care product which comprises a water agent and an emulsion, takes the polypeptide as a main functional component, can activate the physiological function of skin by reasonable collocation, is safe and effective to use and is suitable for popularization.

In order to solve the technical problems and achieve the purposes, the invention provides the following technical scheme:

in a first aspect, the present invention provides an application of a polypeptide in regulating in vitro culture of fibroblasts or preparing a product for regulating in vitro culture of fibroblasts, wherein the polypeptide is composed of an amino acid sequence shown in a sequence SEQ ID No. 1;

the regulating in vitro culture of the fibroblast comprises promoting in vitro proliferation of the fibroblast and/or promoting in vitro collagen secretion of the fibroblast.

In alternative embodiments, the product comprises a cosmetic and/or skin care product.

In a second aspect, the invention provides a regulation method for in vitro culture of fibroblasts, comprising the steps of adding a polypeptide with a final concentration of 100-200 mug/mL into a fibroblast suspension;

the polypeptide is composed of an amino acid sequence shown as a sequence SEQ ID No. 1.

In a third aspect, the present invention provides a skin care product comprising a polypeptide; the polypeptide is composed of an amino acid sequence shown as a sequence SEQ ID No. 1.

In alternative embodiments, the skin care product comprises a dosage form including an aqueous solution, emulsion, cream, gel, cream, or powder.

In an alternative embodiment, the skin care product is in the form of an aqueous solution, and comprises 2% -7% of provitamin B5, 1% -2% of moisturizing factors, 25% -30% of polypeptide aqueous solution, 0.1% -0.5% of Jack horse and the balance of water according to mass percent.

In an alternative embodiment, the provitamin B5 is a 20% by mass aqueous solution of provitamin B5, the moisturizing factor comprises hyaluronic acid, collagen, amino acid or vitamin B6, and the mass percentage of polypeptide in the aqueous solution of polypeptide is 1%.

In an alternative embodiment, the skin care product is in the form of an emulsion, and comprises 9-22 parts of an oil phase, 60-70 parts of water, 30-40 parts of a polypeptide aqueous solution, 1-5 parts of an amino acid humectant, 1-5 parts of a moisturizing factor and 0.1-1 part of Jie-Ma.

In an alternative embodiment, the oil phase contains 3-7 parts of olive emulsifying wax, 3-7 parts of jojoba oil, 2-5 parts of grape seed oil and 1-3 parts of vitamin E.

In an alternative embodiment, the mass percentage of polypeptide in the aqueous polypeptide solution is 1%.

The invention discovers for the first time that the polypeptide consisting of the amino acid sequence shown in the sequence SEQ ID No.1 can promote the proliferation of fibroblasts in vitro and the secretion of collagen, has obvious and stable promoting effect, has obvious effect of delaying skin aging, is hopeful to further develop a product for promoting the growth of the fibroblasts by taking the polypeptide as a main component, and has good market prospect when being used for skin care or cosmetics.

The invention provides a regulation and control method for in vitro culture of fibroblasts, which controls the final concentration of polypeptide composed of amino acid sequences shown in sequence SEQ ID No.1 within the range of 100-200 mug/mL, and can realize proliferation promotion of the fibroblasts and promotion of collagen secretion in vitro.

The invention also provides a skin care product, which realizes the function of activating skin fibroblasts naturally by reasonably matching the polypeptide consisting of the amino acid sequence shown in the sequence SEQ ID No.1 with other functional components, is safe and effective and is suitable for popularization.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of cell proliferation assay in Experimental example 1;

FIG. 2 shows the results of the collagen secretion test in experiment 2;

FIG. 3 shows the results of the test of the effect of different anti-wrinkle components on cell proliferation in experimental example 3.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures.

In a first aspect, in a specific embodiment, the invention provides the use of a polypeptide synthesized by solid phase total synthesis technology, having an amino acid sequence of AEGADNMADMEER (as shown in SEQ ID No. 1), and a molecular weight of 1437.55Da, in modulating fibroblast in vitro culture or preparing a modulating fibroblast in vitro culture product. Proved by verification, the addition of the polypeptide can obviously promote fibroblast proliferation and collagen secretion.

Based on the first aspect, the invention provides a regulation method for in vitro culture of fibroblast, which comprises the steps of adding polypeptide with the final concentration of 100-200 mu g/mL into a fibroblast suspension;

the polypeptide is composed of an amino acid sequence shown as a sequence SEQ ID No. 1.

Based on the second aspect, the invention provides a skin care product, which contains polypeptide; the polypeptide is composed of an amino acid sequence shown as a sequence SEQ ID No. 1.

In an alternative embodiment, the polypeptide consisting of the amino acid sequence shown in the sequence SEQ ID No.1 can be obtained by a solid phase synthesis method or can be obtained by a double enzymolysis method from hirudin, and the obtained polypeptide consisting of the amino acid sequence shown in the sequence SEQ ID No.1 can be prepared into granular, powdery or water-soluble liquid and then added into conventional facial cleanser, mask and cream to prepare the whitening anti-wrinkle cosmetic.

Thus, the dosage forms of the skin care product can comprise various dosage forms such as water agent, emulsion, ointment, gel, cream or powder.

In an alternative embodiment, the skin care product is in the form of an aqueous solution, and comprises 2% -7% of provitamin B5, 1% -2% of moisturizing factors, 25% -30% of polypeptide aqueous solution, 0.1% -0.5% of Jack horse and the balance of water according to mass percent.

Wherein the provitamin B5 is a provitamin B5 aqueous solution with the mass percentage of 20%, the moisturizing factor comprises hyaluronic acid, collagen, amino acid or vitamin B6, and the mass percentage of polypeptide in the polypeptide aqueous solution is 1%.

In another alternative embodiment, the skin care product is in the form of an emulsion, and comprises 9-22 parts of an oil phase, 60-70 parts of water, 30-40 parts of a polypeptide aqueous solution, 1-5 parts of an amino acid humectant, 1-5 parts of a moisturizing factor and 0.1-1 part of jetty.

In an alternative embodiment, the oil phase contains 3-7 parts of olive emulsifying wax, 3-7 parts of jojoba oil, 2-5 parts of grape seed oil and 1-3 parts of vitamin E.

In an alternative embodiment, the mass percentage of polypeptide in the aqueous polypeptide solution is 1%.

Through researches, the inventor discovers for the first time that the polypeptide consisting of the amino acid sequence shown in the sequence SEQ ID No.1 can promote the proliferation and collagen secretion of fibroblasts in vitro, and the promotion effect is obvious and stable.

Some embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following embodiments and features of the embodiments may be combined with each other without conflict.

Example 1

The present set of examples provides polypeptides consisting of the amino acid sequence shown in SEQ ID No. 1.

Example 2

The specific compositions of the toner provided in this example are shown in Table 1.

Table 1 toner composition table in example 2

Example 3

The specific compositions of the toner provided in this example are shown in Table 2.

Table 2 toner composition table in example 3

Example 4

The specific compositions of the toner provided in this example are shown in Table 3.

TABLE 3 toner Components Table in example 4

Example 5

The specific compositions of the skin cream provided in this example are shown in Table 4.

TABLE 4 skin lotion composition table in example 5

Example 6

The specific compositions of the skin cream provided in this example are shown in Table 5.

TABLE 5 skin lotion composition table in example 6

Example 7

The specific compositions of the skin cream provided in this example are shown in Table 6.

TABLE 6 skin lotion composition table in example 7

Comparative example 1

This comparative example provides a toner which is different from example 5 in that the aqueous polypeptide solution in example 5 is replaced with 10ng/mL of basic fibroblast growth factor (bFGF).

Comparative example 2

This comparative example provides a toner which differs from example 2 in that the aqueous polypeptide solution of example 2 is replaced with a commercial oat polypeptide of equal mass percent.

Comparative example 3

The comparative example provides a toner which is different from example 2 in that the aqueous polypeptide solution in example 2 is replaced with snail stock solution of equal mass percent.

Experimental example 1 provides the effect of polypeptide on fibroblasts

(1) Cell culture: fibroblast (NIH/3T 3) density of 10 ⁵ Per mL, 100ul of cell suspension per well, 100ul of PBS per well.

(2) And (3) adding medicines: positive control basic fibroblast growth factor (bFGF) was brought to a final concentration of 10ng/mL, polypeptide was added to a final concentration of 100, 200, 400, 800 μg/mL, 5 multiplex wells were set, and a set of blank controls was set.

(3) MTT method: under light-shielding conditions, 10ul MTT per well. After four hours of incubation in the incubator, the culture broth was blotted and 100ul dmso was added to each well. OD value is measured at 570nm of the enzyme label instrument, and cell proliferation rate and survival rate are calculated.

As shown in FIG. 1, example 1 provided that NIH/3T3 cell growth was significantly promoted at a polypeptide mass concentration of 100-200. Mu.g/mL, cell proliferation rate was 26.5% + -3.68% at 200. Mu.g/mL, and NIH/3T3 cell growth was inhibited at 800. Mu.g/mL by adding 400. Mu.g/mL and 800. Mu.g/mL of the high concentration polypeptide aqueous solution.

Experimental example 2 example 1 provides the Effect of polypeptide on fibroblast collagen secretion

Hydroxyproline is 13.4% in collagen, very little in elastin, and none in other proteins. Whereas collagen is mostly distributed in skin, tendons, cartilage, blood vessels, etc., so that the amount of hydroxyproline reflects the collagen metabolism of connective tissues.

The experimental steps of the experimental example comprise:

(1) Cell culture: fibroblast (NIH/3T 3) density of 10 ⁵ Per mL, 100ul of cell suspension per well, 100ul of PBS per well.

(2) And (3) adding medicines: positive control basic fibroblast growth factor (bFGF) was brought to a final concentration of 10ng/mL, polypeptide was added to a final concentration of 50, 100, 200 μg/mL, 5 wells were set, and a set of blank controls was set. After 24h of incubation, the collagen content of each sample broth was measured using a Hydroxyproline (HYP) test box (available from Nanjing institute of biological engineering, cat# A030-1-1).

The specific method comprises the following steps: a10 mL stoppered tube was used and run in parallel as shown in Table 7. After cooling with running water, centrifuging at 3500r/min for 10min, collecting supernatant, zeroing with double distilled water at 550nm, and measuring absorbance of each tube. Calculation formula of hydroxyproline content:

TABLE 7 reagent dosage form for hydroxyproline content detection

As shown in FIG. 2, the polypeptide can promote the secretion of NIH/3T3 cell collagen, and the secretion of NIH/3T3 cell collagen gradually increases with the increase of the concentration of the polypeptide, so that the detection result is dose-dependent. When the mass concentration of the polypeptide is 200 mug/mL, the polypeptide has very obvious effect on the secretion of NIH/3T3 cell collagen, and the mass concentration of the hydroxyproline is 1.35+/-0.12 mug/mL.

Experimental example 3

Diluting oat polypeptide, snail stock solution and polypeptide with maintenance culture solution respectively, and making final concentration of the oat polypeptide, snail stock solution and polypeptide reach 50, 100 and 200 mug/ml in 96-well cell culture plate, and arranging 5 multiple wells per well. All of the above operations are performed under aseptic conditions. The other steps are operated according to the method, and the cell proliferation rate and the survival rate are calculated, and the result is shown in figure 3, wherein the snail stock solution and the polypeptide obviously promote the proliferation of NIH/3T3 cells at the concentration of 200 mug/ml, and the oat polypeptide inhibits the proliferation of NIH/3T3 cells at the high concentration. The polypeptide can obviously promote proliferation of NIH/3T3 cells at 200 mug/m, and the proliferation rate is 21.2% +/-5.3%, which indicates that the polypeptide has anti-wrinkle whitening activity.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

SEQUENCE LISTING

<110> Bohai sea aquatic products share limited university of Shandong university Weihai Industrial technology institute

Application of <120> polypeptide in regulation and control of fibroblast in-vitro culture

<130> 2021

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 13

<212> PRT

<213> hirudin

<400> 1

Ala Glu Gly Ala Asp Asn Met Ala Asp Met Glu Glu Arg

1 5 10

Claims (8)

1. Application of polypeptide in regulating and controlling fibroblast in-vitro culture, wherein the polypeptide is composed of an amino acid sequence shown as a sequence SEQ ID No. 1;

the regulation and control of the in vitro culture of the fibroblast is to promote the proliferation of the fibroblast in vitro and/or promote the collagen secretion of the fibroblast in vitro.

2. An in vitro fibroblast culture method is characterized by comprising the steps of adding polypeptide with a final concentration of 100-200 mug/mL into a fibroblast suspension;

the polypeptide is composed of an amino acid sequence shown as a sequence SEQ ID No. 1.

3. A skin care product, characterized in that the skin care product comprises a polypeptide; the polypeptide is composed of an amino acid sequence shown as a sequence SEQ ID No. 1.

4. A skin care product according to claim 3, wherein the formulation of the skin care product comprises an aqueous solution, an emulsion, a cream, a gel, a cream or a powder.

5. The skin care product according to claim 4, wherein the skin care product is in the form of an aqueous solution, and comprises 2% -7% of provitamin B5, 1% -2% of moisturizing factors, 25% -30% of polypeptide aqueous solution, 0.1% -0.5% of Jack horse and the balance of water according to mass percent;

the provitamin B5 is a provitamin B5 aqueous solution with the mass percentage of 20%;

the mass percentage of the polypeptide in the polypeptide aqueous solution is 1%.

6. The skin care product of claim 5, wherein the moisturizing factor comprises hyaluronic acid, collagen, amino acids, or vitamin B6.

7. The skin care product according to claim 4, wherein the skin care product is in the form of an emulsion, and comprises 9-22 parts of an oil phase, 60-70 parts of water, 30-40 parts of a polypeptide aqueous solution, 1-5 parts of an amino acid humectant, 1-5 parts of a moisturizing factor and 0.1-1 part of a jetty;

the mass percentage of the polypeptide in the polypeptide aqueous solution is 1%.

8. The skin care product according to claim 7, wherein the oil phase contains 3-7 parts of olive emulsifying wax, 3-7 parts of jojoba oil, 2-5 parts of grape seed oil and 1-3 parts of vitamin E.

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