CN113559014A

CN113559014A - Application of carboxymethyl chitosan in promoting fibroblast proliferation and increasing collagen content

Info

Publication number: CN113559014A
Application number: CN202110974240.1A
Authority: CN
Inventors: 祁玲玲; 徐宇帆; 方庆秋; 何燕
Original assignee: Shanghai Taosheng Biotechnology Co ltd
Current assignee: Shanghai Taosheng Biotechnology Co ltd
Priority date: 2021-08-24
Filing date: 2021-08-24
Publication date: 2021-10-29
Anticipated expiration: 2041-08-24
Also published as: CN113559014B

Abstract

The carboxymethyl chitosan is used for promoting the proliferation of fibroblasts and increasing the content of collagen. Cell experiments and human body test results show that the carboxymethyl chitosan has the obvious effects of promoting fibroblast proliferation and improving the collagen content, can be used as an effective component for preparing external products, has good skin-friendly and biocompatibility effects, and has practical application values of improving the skin quality and resisting skin aging.

Description

Application of carboxymethyl chitosan in promoting fibroblast proliferation and increasing collagen content

Technical Field

The invention relates to a new application of carboxymethyl chitosan, in particular to an application of carboxymethyl chitosan in promoting fibroblast proliferation and improving collagen content.

Background

Carboxymethyl chitosan is a water-soluble chitosan derivative, has many characteristics, such as strong antibacterial property and fresh-keeping effect, and is an amphoteric polyelectrolyte. The chitosan derivative has various applications in the aspects of cosmetics, fresh keeping, medicines and the like, and is one of the chitosan derivatives which are researched more recently.

The carboxymethyl chitin is substituted on C6-OH of sugar residue, and a small amount of carboxymethyl is substituted on C3-OH to generate O-carboxymethyl chitin. In chitosan, the carboxymethyl group can be substituted on-OH and-NH to generate O-carboxymethyl and N-carboxymethyl chitosan, and the actual substitution conditions are as follows: C6-O-carboxymethyl, C2-N-carboxymethyl, C3-O-carboxymethyl, C6-O, C2-N-carboxymethyl and the like. Carboxymethylation at the C3 position is difficult due to steric hindrance at C3 and intramolecular hydrogen bonds between C2 and C3, so that carboxymethyl substitution on hydroxyl groups is less, C3-O carboxymethyl is less, and C6-O carboxymethyl is mainly used. For C6-OH and C2-NH, the substitution activity of carboxymethyl on hydroxyl is higher than that of amino under alkaline conditions, so that when the substitution degree is less than 1, the substitution of carboxymethyl is mainly on hydroxyl rather than on amino, and only when the substitution degree is close to 1 and higher than 1, the carboxymethyl substitution on amino can be simultaneously carried out to form O, N-carboxymethyl chitosan. The water solubility of carboxymethyl chitosan is not only soluble in water because it is a sodium carboxylate, but also because the introduction of carboxymethyl group destroys the secondary structure of chitosan molecule, greatly reducing its crystallinity, and becoming almost amorphous.

The prior art discloses the application of carboxymethyl chitosan in moisture absorption, moisture retention and bacteriostasis, but other applications are not disclosed.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a novel use of carboxymethyl chitosan.

In order to achieve the above object or other objects, the present invention is achieved by the following aspects.

The invention firstly discloses the use of carboxymethyl chitosan for promoting fibroblast proliferation in a non-therapeutic destination.

The invention also discloses application of the carboxymethyl chitosan serving as an effective component for promoting the proliferation of the fiber cells in preparing external products.

The invention also discloses the use of carboxymethyl chitosan for increasing collagen content in non-therapeutic destinations.

The invention also discloses the application of the carboxymethyl chitosan as an effective component for improving the content of the collagen in preparing external products.

The invention also discloses application of the carboxymethyl chitosan in preparing an anti-aging skin external product.

Preferably, the carboxymethyl chitosan is O-carboxymethyl chitosan.

Preferably, the number average molecular weight of the carboxymethyl chitosan is 1000 to 20000.

Preferably, in the preparation of a product for external use as an active ingredient, the carboxymethyl chitosan is added in an amount of not more than 1 wt%, preferably not more than 0.5 wt%, based on the total mass of the product for external use.

Preferably, 0.01 wt% to 0.2 wt% carboxymethyl chitosan is used when used in a non-therapeutic destination to promote fibroblast proliferation.

Preferably, 0.01 wt% to 0.2 wt% carboxymethyl chitosan is used when used to increase collagen content in non-therapeutic destinations.

Preferably, in the skin anti-aging external product, the content of the carboxymethyl chitosan is 0.05 wt% to 1 wt%.

The invention also discloses an external product at least comprising carboxymethyl chitosan and water.

Preferably, the carboxymethyl chitosan is contained in an amount of not more than 1 wt%, preferably not more than 0.5 wt%, based on the total mass of the external product.

Preferably, the carboxymethyl chitosan is O-carboxymethyl chitosan.

Preferably, the external product further comprises one or more of a thickener, a preservative, a penetrant, a humectant, an emulsifier, a skin conditioner, an antioxidant, an emollient, and a pH adjuster.

Specifically, the O-carboxymethyl chitosan is partially carboxymethylated chitosan obtained after carboxymethyl chitin is deacetylated or 6-hydroxyl hydrogen of chitosan is replaced by carboxymethyl.

More preferably, the structural formula of the O-carboxymethyl chitosan is as follows:

wherein R is₁Is CH₂COONa；R₂Is CH₂COONa or H; r₃Is CH₂COONa or H or COCH₃。

Many types of carboxymethyl chitosan exist in the prior art, but not all carboxymethyl chitosan are suitable for external application to the skin surface. Specifically, the number average molecular weight of the carboxymethyl chitosan is 1000-20000. The number average molecular weight is measured by ultraviolet-visible spectrophotometry method A according to "in pharmacopoeia of people's republic of China (four pharmacopoeias 2015) 0401₅₂₅. The principle is as follows: the acetylacetone reagent reacts with the reducing end group of standard glucosamine, carboxymethyl chitosan or hydrolysate thereof to generate chromogen; absorbance A under certain conditions₅₂₅Has a linear relationship with the molar concentration of the corresponding sugar. The multiple of the reducing end group added after hydrolysis compared with before hydrolysis is the average degree of polymerization n of the carboxymethyl chitosan, and the number average molecular mass of the saccharide can be calculated by combining the average molecular mass of the saccharide unit in the molecule.

Preferably, the external product is a spray, essence lotion, essence cream, lotion, or mask.

The applicant has unexpectedly found that the carboxymethyl chitosan with good skin-friendly property and biocompatibility can promote fibroblast proliferation and increase collagen content, and thus can be used as an effective ingredient in preparing an external product for promoting fibroblast proliferation and/or submitting collagen content, and the related test results show that the carboxymethyl chitosan has good effects of promoting fibroblast proliferation and increasing collagen content. By using the external product containing the carboxymethyl chitosan, the skin quality can be obviously improved, such as skin aging can be resisted, and particularly, the external product has obvious inhibiting and repairing effects on skin aging caused by light.

Drawings

FIG. 1 shows a trend graph of the cell viability of MHA-20 in the MMT assay.

FIG. 2 is a graph showing the results of morphological examination of MHA-20 in an MMT assay.

FIG. 3 shows a trend graph of the cell viability of MHA-30 in the MMT assay.

FIG. 4 is a graph showing the results of morphological examination of MHA-30 in an MMT assay.

FIG. 5 shows a trend graph of the cell viability of MHA-45 in the MMT assay.

FIG. 6 is a graph showing the results of morphological examination of MHA-45 in the MMT assay.

FIG. 7 is a line graph showing cell proliferation.

FIG. 8 is a photograph showing immunofluorescence detection of Collagen I in cells.

FIG. 9 is a histogram of the Collagen I integrated optical density values.

FIG. 10 shows the results of immunofluorescence assays of Collagen IV in cells.

FIG. 11 is a histogram of Collagen IV integrated optical density/cell number.

FIG. 12 is a graph showing the effect of the change in collagen fiber content in skin tissue.

Figure 13 shows a histogram of the relative area results of collagen fibrils.

FIG. 14 shows the variation of the elastic fiber content of the skin tissue.

FIG. 15 shows a bar graph of the relative area results for elastic fibers.

FIG. 16 shows the variation of the amount of Collagen IV in the skin tissue.

FIG. 17 is a bar graph showing the results of Collagen IV content.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Based on the application development and research of carboxymethyl chitosan, the applicant finds that, besides the effects of easy water solubility, skin-friendly property, antibacterial property and anti-inflammatory, carboxymethyl chitosan also has the effects of promoting fibroblast proliferation and increasing the content of collagen. Based on the two effects, when the carboxymethyl chitosan is used as an effective component in an external product, the skin quality, such as the effects of resisting aging and the like, can be greatly improved.

Based on fibroblast proliferation experiments and experimental results, the applicant finds that the carboxymethyl chitosan in the application has no cytotoxicity within a safe administration concentration, and can remarkably promote the proliferation of fibroblasts.

The developed anti-wrinkle efficacy test shows that the carboxymethyl chitosan can effectively improve the content of type I collagen and type IV collagen in fibroblasts, so that the carboxymethyl chitosan can be definitely used as a cosmetic raw material for skin care, can play a good tightening anti-wrinkle effect, and particularly has a good treatment effect on skin aging caused by light.

Thus, the applicant of the present application firstly provides the use of carboxymethyl chitosan for promoting the proliferation of fibroblasts in a non-therapeutic destination. Specifically, the carboxymethyl chitosan is used in an external product as an active ingredient of a raw material, and in this case, the carboxymethyl chitosan is the only active ingredient or one of the active ingredients for promoting fibroblast proliferation.

In a more preferred embodiment, 0.01 to 0.2 wt% carboxymethyl chitosan or a 0.01 to 0.2 wt% carboxymethyl chitosan solution is used in promoting fibroblast proliferation for non-therapeutic purposes. Such as aqueous solutions, etc.

In a more preferred embodiment, 0.01 to 0.2 wt% carboxymethyl chitosan or a carboxymethyl chitosan solution with a concentration of 0.01 to 0.2 wt% is used in increasing collagen content for non-therapeutic purposes. Such as aqueous solutions, etc.

Therefore, in the specific embodiment of the application, the application of the carboxymethyl chitosan as an effective component for promoting the proliferation of the fibroblasts to prepare the external product is also provided.

In the specific embodiment of the application, the application of the carboxymethyl chitosan as an effective component for increasing the content of the collagen in preparing the external product is also provided.

In the specific embodiment of the application, the application of the carboxymethyl chitosan in preparing the skin anti-aging external product is also provided.

In a preferred embodiment, the topical product refers to a product for external application to the skin surface. Such as cosmetics, skin care products, cosmeceuticals, and the like. Due to the good water solubility of the carboxymethyl chitosan, the product for resisting skin aging can be spray, essence lotion, essence cream, emulsion and facial mask.

In a preferred embodiment, when the carboxymethyl chitosan is used to prepare a product for external use as an effective ingredient for promoting fibroblast proliferation in a non-therapeutic destination, the carboxymethyl chitosan is added in an amount of not more than 1 wt%, such as 0.05 wt% to 1 wt%, based on the total mass of the product for external use. In a more preferred embodiment, the carboxymethyl chitosan is added in an amount of 0.05 wt% to 0.5 wt%, and in a more preferred embodiment, the carboxymethyl chitosan is added in an amount of 0.1 wt% to 0.5 wt%, such as specifically 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, or 0.5 wt%.

In a more preferred embodiment, when the active ingredient for increasing collagen content as a non-therapeutic destination is used for preparing a product for external use, the amount of carboxymethyl chitosan added is not more than 1 wt%, such as 0.05 wt% to 1 wt%, in a more preferred embodiment, 0.05 wt% to 0.5 wt%, and in a more preferred embodiment, 0.1 wt% to 0.5 wt%, such as specifically 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, or 0.5 wt%, based on the total mass of the product for external use.

In the above specific embodiments of the present application, the carboxymethyl chitosan is O-carboxymethyl chitosan. The O-carboxymethyl chitosan is partially carboxymethylated chitosan obtained after deacetylation of carboxymethyl chitin or substitution of 6-hydroxyl hydrogen of chitosan by carboxymethyl.

In a more specific embodiment, the structural formula of the O-carboxymethyl chitosan is as follows:

In a specific embodiment, the carboxymethyl chitosan has a number average molecular weight of 1000 to 20000, such as 5000, 10000, 15000 or 20000. The number average molecular weight is measured by ultraviolet-visible spectrophotometry method A according to "in pharmacopoeia of people's republic of China (four pharmacopoeias 2015) 0401₅₂₅. The principle is as follows: the acetylacetone reagent reacts with the reducing end group of standard glucosamine, carboxymethyl chitosan or hydrolysate thereof to generate chromogen; absorbance A under certain conditions₅₂₅Has a linear relationship with the molar concentration of the corresponding sugar. The multiple of the reducing end group added after hydrolysis compared with before hydrolysis is the average degree of polymerization n of the carboxymethyl chitosan, and the number average molecular mass of the saccharide can be calculated by combining the average molecular mass of the saccharide unit in the molecule. The application also specifically discloses a preparation method of the carboxymethyl chitosan, which comprises the step of degrading a carboxymethyl chitosan raw material with higher number average molecular weight by hydrogen peroxide. More preferably, the carboxymethyl chitosan material with a high number average molecular weight has a number average molecular weight of 30 to 70 ten thousand. More preferably, the carboxymethyl chitosan material with high number average molecular weight has a carboxymethyl substitution degree of 0.8-1.3. More preferably, the carboxymethyl chitosan material with higher number average molecular weight has a carboxymethyl O-position substitution degree of 0.7-1.0 and an N-position substitution degree of no more than 0.3.

The carboxymethyl chitosan raw material with higher number average molecular weight is prepared by a method disclosed by the invention patent with the domestic application number of 2013105362023, and discloses a method for preparing carboxymethyl chitosan. The method has simple process and easy control, avoids the chain breaking phenomenon caused by long-term high-temperature reaction in the deacetylation reaction of the chitosan molecular chain, obtains the carboxymethyl chitosan with high viscosity, high degree of substitution and better water solubility, and is suitable for large-scale industrial production. The pH range of the dissolved carboxymethyl chitosan with higher molecular weight is 4.5-14.0. The carboxymethyl chitosan with higher number average molecular weight can be dissolved in an acid solution with pH of 4.5-6.5. Compared with the pH range of the carboxymethyl chitosan obtained by the traditional process, which is 7.0-14.0, the carboxymethyl chitosan obtained by the method has a wider pH range of dissolution with higher number average molecular weight, and the application field of the carboxymethyl chitosan is widened.

In a preferred embodiment, the process for the degradation of hydrogen peroxide is: adding hydrogen peroxide into carboxymethyl chitosan with a high number average molecular weight under a stirring condition for degradation reaction, adding alkali in the degradation reaction process to enable the pH of the reaction system to be 8.0-8.5, and adding sodium sulfite to stop the reaction until the viscosity of the reaction system is 80-120 cp. More preferably, the reaction temperature in the degradation reaction is 25-45 ℃. And obtaining the degraded carboxymethyl chitosan through the degradation reaction.

In a more preferred embodiment, the preparation method of the carboxymethyl chitosan comprises the step of performing acid precipitation washing on the carboxymethyl chitosan after the hydrogen peroxide degradation. And the acid precipitation washing is to adjust the pH value of the degraded carboxymethyl chitosan material with hydrogen peroxide to 6-7 by using acid, add ethanol until faint yellow crystals are precipitated, repeatedly wash the mixture with ethanol, spin-dry the mixture, and dry the mixture to obtain the carboxymethyl chitosan.

In a particular embodiment, the applicant also provides the use of carboxymethyl chitosan in the preparation of a product for combating skin ageing caused by photoaging. Skin photoaging is a damage caused by long-term exposure of the skin to sunlight, and is the result of the combined action of natural aging and ultraviolet radiation. Based on experimental test results, the carboxymethyl chitosan can obviously promote the regeneration and proliferation of fibroblasts, and has obvious improvement effect on the wrinkle deepening and thickening and skin relaxation caused by photoaging.

The embodiment of the application further provides a specific external product, and the external product at least comprises carboxymethyl chitosan and water.

In a preferred embodiment, the topical product further comprises one or more of a thickener, a preservative, a humectant, an emulsifier, a skin conditioner, an antioxidant, an emollient, a chelating agent, and a pH adjuster. These ingredients are the raw materials commonly used in the field of skin cosmetics for external use to form sprays, serums, lotions or creams. In a more preferred embodiment, the thickener may be selected from one or more of transparent xanthan gum (DSM), EZ-4U, BLV, carbomer, SIMULGEL NS, cetearyl alcohol, TR-2, SIMULGEL EG, and Stabylen 30.

In a more preferred embodiment, the preservative may be selected from one or more of pentanediol, menthone, hexanediol, ethylhexyl glycerol (SC50), and caprylyl glycol.

In a more preferred embodiment, the humectant may be selected from one or more of pentanediol, 1, 3-butanediol, hexanediol, ethylhexylglycerin (SC50), caprylyl glycol, and glycerin.

In a more preferred embodiment, the emulsifier may be selected from one or more of SSE-20, alkyl glycoside emulsifiers (e.g., MONTANOV 68), alkyl glycoside emulsifiers (e.g., MONTANOV L), and self-emulsifying monoglyceride emulsifiers (e.g., TEGOCARE 165).

In a more preferred embodiment, the skin conditioning agent may be selected from one or more of carboxymethyl chitosan, arginine, poly-sodium glutamate, sodium hyaluronate, bisabolol, and tocopheryl acetate.

In a more preferred embodiment, the antioxidant may be selected from jasminodone.

In a more preferred embodiment, the emollient may be selected from one or more of squalane, shea butter, silicone oil (e.g. silsoft 034), caprylic triglyceride, and quinic triglyceride.

In a more preferred embodiment, the pH adjusting agent is selected from arginine.

In a more preferred embodiment, the chelating agent is selected from EDTA-2 Na.

The following are specific experimental and implementation effect data, and the specific schemes and the effects achieved by the schemes in the embodiments of the present application are described and supported by the following experimental and implementation effect data.

In the application, the adopted MHA-20 is carboxymethyl chitosan with the number average molecular weight of 10000, the adopted MHA-30 is carboxymethyl chitosan with the number average molecular weight of 15000, and the adopted MHA-45 is carboxymethyl chitosan with the number average molecular weight of 20000.

The test and the test effect based on the carboxymethyl chitosan for promoting the proliferation of the fibroblasts.

1.1 description of the materials and Main Equipment for testing

The carboxymethyl chitosan sample to be tested comprises: MHA-20, MHA-30 and MHA-45 are all solid powder, are white in color, have water solubility, and are stored in a dry and dark place.

Testing the model: the cells used in the test were fibroblasts, and the cell batch number is Fb 19052002.

The main reagents are as follows: PBS, MTT, DMSO, DMEM, and newborn bovine serum. Among them, specifically, PBS is supplied by Solebao, MTT and DMSO are supplied by Sigma, DMEM is supplied by Gibco, and newborn bovine serum is provided brightly in Lanzhou.

The main equipment is as follows: CO 2₂Incubator, superclean bench, inverted microscope, enzyme mark appearance, micro-oscillator, balance. Wherein, in particular, CO₂The incubator is Thermo 150I, the inverted microscope is Olympus CKX41, the microplate reader is BioTek Epoch, the micro-oscillator is its Linbel MM-1, and the balance is Sedolis BSA 124S.

1.2 test methods

1.2.1 cytotoxicity detection and detection results

1.2.1.1 MTT assay

Cell inoculation: by 1 × 10⁴Fibroblast cells were seeded at density per well into 96-well plates and incubated overnight. Incubate with incubator at 37 deg.C and 5% CO₂At the concentration.

Grouping experiments: the experiment was set up with a zero adjustment group, a solvent Control group (Control), a positive Control group (PC) and a sample group. In the sample set, 8 concentration gradients were set for each sample, and 3 replicate wells were set for each concentration gradient.

Preparing liquid: sample working fluids of different concentrations were prepared as in table 1 according to the test concentration settings.

TABLE 1 test concentration setting Table

Administration: and (3) administration is carried out when the cell plating rate in the 96-well plate reaches 40-60%. Adding 200 mu L of culture solution into each well of the solvent control group; adding 200 mu L of culture solution containing 10% DMSO into each well of the positive control group; adding 200 mu L of culture solution containing samples with corresponding concentrations into each hole of the sample group; the zero-adjusted group was seeded without cells, and only 200. mu.L of cell culture medium was added. After the administration, the 96-well plate was cultured in an incubator at 37 ℃ and 5% CO₂。

And (3) detection: after 24h of cell incubation culture, discarding the supernatant, adding 0.5mg/mL MTT working solution, incubating at 37 ℃ in a dark place for 4h, discarding the supernatant after the incubation is finished, adding 150 mu LDMSO to each well, and reading the OD value at 490 nm.

And (3) calculating the cell viability: according to the calculation of a formula,

1.2.1.2 morphological examination

Cell inoculation: a sample group and a solvent control group (SC) were set up, each group having two duplicate wells. Inoculating cells into 24-well plates at the corresponding inoculation density, and incubating overnight in an incubator at 37 deg.C with CO₂The content was 5%.

Preparing liquid: and selecting the concentration near the cell viability inflection point according to the MTT detection result, performing morphological observation, and determining the morphological observation concentration of the detection sample.

Administration: when the cell plating rate of the 24-pore plate reaches 40% -60%, the drug is administered, the samples are added with the test substances with different concentrations, the solvent contrast group is added with the culture solution, and the incubation is carried out in an incubator for 24 hours under the conditions of 37 ℃ and 5% CO₂。

And (3) cell observation: after incubation, cell morphology was observed under a microscope and photographed.

1.2.1.3 cytotoxicity assay results:

8 dosing concentrations of the sample MHA-20 were set, cytotoxicity detection experiments were performed on fibroblasts, the MTT detection results are shown in Table 2, and the trend of cell viability is shown in FIG. 1. Based on the MTT assay results, 5 concentrations were selected for morphological examination, and the morphological examination results are shown in fig. 2.

Based on MTT and morphological results, the sample MHA-20 was considered to be fibroblast-based and showed no significant cytotoxicity in the concentration range of 2 mg/mL.

TABLE 2 MHA-20 MTT assay results

8 dosing concentrations of the sample MHA-30 were set, cytotoxicity detection experiments were performed on fibroblasts, the MTT detection results are shown in Table 3, and the trend of cell viability is shown in FIG. 3. Based on the MTT assay results, 5 concentrations were selected for morphological examination, and the morphological examination results are shown in fig. 4.

Based on MTT and morphological results, the sample MHA-30 was considered to be fibroblast-based and showed no significant cytotoxicity in the concentration range of 2 mg/mL.

TABLE 3 MHA-30 MTT assay results

8 dosing concentrations of the sample MHA-45 were set, cytotoxicity detection experiments were performed on fibroblasts, the MTT detection results are shown in Table 4, and the cell viability trend is shown in FIG. 5. Based on the MTT assay results, 5 concentrations were selected for morphological examination, and the morphological examination results are shown in fig. 6.

Based on MTT and morphological results, the sample MHA-45 was considered to be fibroblast-based and showed no significant cytotoxicity in the concentration range of 2 mg/mL.

TABLE 4 MHA-20 MTT assay results

1.2.2 cell proliferation assay and assay results.

1.2.2.1 cell proliferation assay

Cell inoculation: by 4X 10³Fibroblast cells were seeded at density per well into 96-well plates and incubated overnight. Incubate with incubator at 37 deg.C and 5% CO₂At the concentration.

Preparing liquid: sample working solutions were prepared according to the experimental design as in table 5. Wherein, the concentration of the carboxymethyl chitosan in the sample concentration in the table and the following is the mass percentage concentration of the carboxymethyl chitosan aqueous solution.

TABLE 5 Experimental design

Administration: according to the experimental grouping design of table 5, when the plating rate of the cells in the 96-well plate reaches 20% -30%, the drug is administered in groups, the dose of each hole is 200 μ L, and each group is provided with 3 multiple holes. In an incubator at 37 deg.C with 5% CO₂Culturing for 24h, 48h and 72h respectively. Half-fluid change treatment is carried out every day on the groups requiring 48h and 72h of incubation culture.

And (3) detection: and after the incubation is finished, discarding the supernatant, adding 0.5mg/mL MTT working solution, incubating for 4h at 37 ℃ in a dark place, discarding the supernatant after the incubation is finished, adding 150 mu L DMSO into each hole, and reading the OD value at 490 nm. At the same time, after 48h and 72h, the MTT assay procedure described above was performed.

1.2.2.2 cell proliferation assay results.

Cell proliferation assay was performed based on the test method, and the results are summarized in table 6, and the proliferation line graph is shown in fig. 7.

Compared with the SC group, the relative cell activity of the PC group is increased, and the PC group has obvious proliferation promoting effect (p is less than 0.01), which indicates that the experimental system is effective.

Compared with SC group, MHA-20-0.01%, MHA-20-0.025%, MHA-20-0.05%, MHA-30-0.025% and MHA-45-0.025% of sample group have the function of promoting the proliferation of fibroblast in 24h and 48 h.

In the application, Graphpad Prism Program software is used for drawing, t-test statistical analysis is adopted among groups, p is less than 0.05 to indicate that the difference is obvious, and p is less than 0.01 to indicate that the difference is extremely obvious.

TABLE 6 summary of cell proliferation assay results

The cell proliferation experiments and results can show that:

compared with the SC group, the samples MHA-20 showed significant effects of promoting fibroblast proliferation at 0.01%, 0.025% and 0.05%, and MHA-30 and MHA-45 at a concentration of 0.025% for 24h and 48 h.

And secondly, detecting the protein content of Collagen I, and evaluating the in-vitro tightening and anti-wrinkle effects of the carboxymethyl chitosan.

Based on fibroblasts, detecting the content of Collagen I protein, and evaluating whether the carboxymethyl chitosan has the effects of tightening and resisting wrinkles.

2.1 testing materials

2.1.1 test System

The fibroblasts used in this test were under the batch number 19052002.

2.1.2 reagents

DMEM, fetal calf serum, PBS, 4% paraformaldehyde and Anti-Collagen I antibody. Specifically, DMEM is supplied by Gibco, fetal bovine serum is supplied by Gibco, PBS is supplied by Solebao, 4% paraformaldehyde is supplied by BioSharp, and Anti-Collagen I antibody is supplied by Abcam.

2.1.3 Main Equipment

CO₂Incubator, superclean bench, inverted microscope, fluorescence microscope. In particular, the CO₂The incubator is Thermo 150i, the super clean bench is Sujing Antai SW-CJ-1F, the inverted microscope is Olympus CKX41, and the fluorescence microscope is Leica DM 2500.

The carboxymethyl chitosan sample is MHA-20, smells solid powder, is white and has water solubility, and needs to be dried and stored away from light.

And 2.2, testing the anti-wrinkle effect.

2.2.1 test method based on anti-wrinkle efficacy of fibroblasts.

Experiment design: the experimental group specific settings are shown in table 7.

TABLE 7

Collagen I immunofluorescence assay:

cell inoculation: according to 2.5E⁴Inoculating cells to 24-well plate at a seed density per well, incubating overnight in an incubator at 37 deg.C and 5% CO₂。

Administration: according to the experimental scheme shown in Table 7, when the plating rate of the cells in the 24-well plate reaches 40% -60%, the drug is administered in groups, each well is loaded with 1mL, and each group is provided with 3 multiple wells. After the administration, the 24-well plate was placed in an incubator and incubated for 24 hours at 37 ℃ and 5% CO₂。

Collecting a sample: after the incubation culture is finished, the old solution is aspirated and discarded, 1 mL/hole PBS is washed for three times, and 1mL of 4% paraformaldehyde is added into each hole and fixed for 15min at room temperature. After fixation, the 24-well plate was stored at 4 ℃ for further use.

And (3) sealing: 200 mu L/hole of goat serum is added dropwise, and the mixture is sealed for 60min at room temperature.

And (4) incubating the primary antibody, namely discarding goat serum blocking solution, dropwise adding the diluted primary antibody (200 mu L/hole) (goat serum dilution) in a proper proportion, and incubating overnight at 4 ℃.

Incubation of secondary antibody: washing with PBS for 3 times/5 min, adding diluted fluorescent secondary antibody (200 μ L/well), and incubating at room temperature in dark for 1 h.

Nuclear dyeing: wash 3 times/5 min with PBS, add Hochest33342 (200. mu.L/well) dropwise, and incubate for 5min at room temperature.

Sealing: the slide was picked up with a needle, a drop of anti-quencher was dropped onto the slide and the slide was placed upside down on the slide.

And (4) observing and photographing: pictures were taken by fluorescence microscope within 24 h.

And (3) mapping by using Graphpad Prism Program software, wherein the difference is obvious when p is less than 0.05 and extremely obvious when p is less than 0.01 by adopting t-test statistical analysis among groups.

2.2.2 wrinkle resistance efficacy test results.

After the incubation culture, the cells were subjected to immunofluorescence assay, and the results are summarized in FIG. 8, and Table 8 is a Collagen I Integrated Optical Density (IOD)/cell number summary table. FIG. 8 is an immunofluorescent assay of Collagen I in cells. FIG. 9 is a histogram of Collagen I Integrated Optical Density (IOD) values.

TABLE 8

Sample name	Relative IOD/cell number average	SD	p value (vs BC)
				BC	1.000	0.147	/
PC	1.399	0.108	0.019
				MHA-20-0.05％	0.839	0.037	0.140
MHA-20-0.025％	0.952	0.072	0.639
				MHA-20-0.01％	1.279	0.035	0.033

From the above data it can be seen that:

compared with the BC group, the Collagen I protein content of the PC (TGF-beta 1) group is obviously increased, which indicates that the positive control detection is effective.

Compared with the BC group, the content of MHA-20-0.05 percent and MHA-20-0.025 percent Collagen I protein of the sample is not obviously increased; the content of the Collagen I protein of MHA-20-0.01 percent of the sample is obviously increased.

In conclusion, the Collagen I protein content of the MHA-20-0.01% of the sample is obviously increased, which shows that the Collagen I content of the Collagen in the fibroblast is increased in the aspect of Collagen I protein increase based on the fibroblast under the concentration of 0.01%, so that the Collagen is beneficial to improving and repairing the skin quality, and has certain effects of tightening and resisting wrinkles.

And thirdly, detecting the content of Collagen IV protein, and evaluating the in-vitro tightening and anti-wrinkle effects of the carboxymethyl chitosan.

Based on fibroblasts, detecting the content of Collagen IV protein, and evaluating whether the carboxymethyl chitosan has the effects of tightening and resisting wrinkles.

3.1 testing materials

3.1.1 test System

The fibroblasts used in this test were under batch number Fb 19052002.

3.1.2 reagents

DMEM, fetal calf serum, PBS, 4% paraformaldehyde and Anti-Collagen I antibody. Specifically, DMEM is supplied by Gibco, fetal bovine serum is supplied by Gibco, PBS is supplied by Solebone, 4% paraformaldehyde is supplied by BioSharp, and Anti-Collagen IV antibody is supplied by Abcam.

3.1.3 Main Equipment

And 3.2, testing the anti-wrinkle effect.

3.2.1 test method based on anti-wrinkle efficacy of fibroblasts.

Experiment design: the experimental grouping was specifically set up as shown in table 9.

TABLE 9

The Collagen IV immunofluorescence detection method comprises the following steps:

Administration: according to the experimental scheme shown in Table 9, when the plating rate of the cells in the 24-well plate reaches 40% -60%, the administration is carried out in groups, each well is loaded with 1mL, and each group is provided with 3 multiple wells. After the administration is finished, the 24-hole plate is placed in an incubator to be cultured for 24h and culturedThe temperature was 37 ℃ and 5% CO₂。

3.2.2 anti-wrinkle efficacy test results.

After the incubation culture, the cells were subjected to immunofluorescence assay, and the results are summarized in FIGS. 10 and 11, and Table 10 is a Collagen IV Integrated Optical Density (IOD)/cell number summary table. FIG. 10 shows the results of immunofluorescence assay of Collagen IV in cells. FIG. 11 is a Collagen IV Integrated Optical Density (IOD)/cell number histogram.

Watch 10

Sample name	Relative IOD/cell number average	SD	p value (vs BC)
				BC	1.000	0.140	/
PC	1.952	0.171	0.002
				MHA-20-0.05％	1.600	0.092	0.003
MHA-20-0.025％	1.665	0.164	0.006
				MHA-20-0.01％	1.760	0.114	0.002

From the above data it can be seen that:

compared with the BC group, the Collagen IV protein content of the PC (TGF-beta 1) group is obviously increased, which indicates that the positive control detection is effective.

Compared with the BC group, the content of the Collagen IV protein of the samples MHA-20-0.05%, MHA-20-0.025% and MHA-20-0.01% does not increase remarkably.

In conclusion, the Collagen IV protein content of the sample MHA-20-0.05%, the sample MHA-20-0.025% and the sample MHA-20-0.01% is obviously increased, which shows that the MHA-20 can be used as an effective component for increasing the Collagen content at the concentration of 0.05%, 0.025% and 0.01% based on fibroblasts, so that the skin condition can be repaired and improved, for example, a certain firming and anti-wrinkle effect can be achieved.

And fourthly, the essence containing carboxymethyl chitosan is used for in-vitro skin tissue experiments and tightening and anti-wrinkle effect evaluation.

The part takes in-vitro skin tissues cultured outside the whole body as a research object, and evaluates the effects of the essence containing carboxymethyl chitosan as the only effective anti-aging component on the aspects of tightening and anti-wrinkle through the change of the Collagen fiber content, the elastic fiber content and the Collagen IV protein content.

4.1 test materials

4.1.1 test systems: the isolated skin tissue was Ex vivo, supplied by Guangdong Boxi Biotech, Inc.

4.1.2 Main reagents: ex vivo skin tissue culture, FSK4, provided by a boxi organism, guangdong; vitamin C, supplied by Sigma; vitamin E, supplied by Sigma; 4% paraformaldehyde provided by BioSharp; xylene, provided by national medicine; the anhydrous ethanol is provided by Chinese medicines; hematoxylin is provided by Biyun Tian; Anti-Collagen IV antibody, supplied by Abcam; ABC-Peroxidase Kits, supplied by VECTASTAIN; the DAB color development kit of horseradish catalase is provided by the worker; sodium citrate is 50 x, supplied by sienna heiter organisms.

4.1.3 Main devices: CO 2₂An incubator, Thermo 150 i; the super clean bench is Suzhou Antai SW-CJ-1F; the inverted microscope was Olympus CKX 41.

4.1.4 sample information:

the information of the samples to be tested is shown in Table 11.

TABLE 11

4.2 test methods

4.2.1 test grouping

Specific dosing regimens are shown in table 12.

Table 12 test grouping settings

4.2.2 tissue treatment

Freshly obtained skin tissue was immersed in 75% alcohol, washed for 30s and then three times with sterile PBS buffer (containing double antibody); cutting skin tissue into 0.6 cm-diameter small discs with epidermis facing upward and dermis facing downward, placing into culture mold, transferring the culture mold into 6-well plate, adding 3.7mL culture solution into each well, and culturing at 37 deg.C with 5% CO₂Culturing in incubator, and changing liquid every day.

4.2.3 administration

After the isolated skin tissue was cultured for 1 day, UV irradiation and dosing were started with reference to the experimental groups and corresponding treatment conditions in table 12; the UV irradiation dose was UVA (30J/cm)²) And UVB (50 mJ/cm)²) Continuously irradiating for 4 days, replacing fresh culture solution after irradiation every day, and performing administration treatment, wherein the sample to be detected is subjected to surface administration (2 mu L). After 4 days of continuous irradiation, the three-dimensional skin tissue was cultured for another 3 days, during which no UV stimulation was performed and only the sample was administered.

4.2.4 tissue morphology detection

Fixing the skin tissue in a model by 4% paraformaldehyde after the administration, embedding the tissue, respectively carrying out Masson staining and Victoria blue staining after slicing, recovering the slicing result, taking a picture by using a microscope, and analyzing collagen fibers and elastic fibers by using Image-Pro Plus Image processing software.

4.2.5 immunohistochemical detection

1) Removing the baking sheet: and (3) placing the paraffin sections into a 70 ℃ chip baking machine, and baking the slices for 4 hours.

2) Dewaxing and hydrating: soaking the slices in xylene for 10min, replacing xylene, soaking for 10min, soaking in anhydrous ethanol for 5min, soaking in 95% ethanol for 5min, and soaking in 75% ethanol for 5 min. The cells were washed 3 times with PBS buffer for 5min each time.

3) Antigen retrieval: and (3) putting the paraffin sections into 0.01M sodium citrate antigen retrieval solution, performing high-pressure retrieval, and cooling to obtain the sections. The washing was performed 3 times for 5 min/time with PBS buffer.

4) Blocking peroxidase: adding 1 drop of 3% H into each slice₂O₂And incubated at room temperature for 30 min. The washing was performed 3 times for 5 min/time with PBS buffer.

5) Serum blocking: and (4) dropwise adding serum homologous with the secondary antibody, sealing for 60min at 37 ℃, and washing is not needed.

6) Primary antibody incubation: add the primary antibody solution dropwise and incubate overnight at 4 ℃. The washing was performed 3 times for 5 min/time with PBS buffer.

7) And (3) secondary antibody incubation: and adding a secondary antibody working solution dropwise, and incubating for 1h at room temperature. The washing was performed 3 times for 5 min/time with PBS buffer.

8) Incubation of ABC complexes: dripping ABC complex solution, and incubating at room temperature for 30 min. The washing was performed 3 times for 5 min/time with PBS buffer.

9) DAB dyeing: and adding 1 drop of DAB solution which is prepared freshly into each section, and observing for 5-30 s by using a microscope.

10) Counterdyeing: hematoxylin counterstain for 30 s.

11) And (3) dehydrating: the slices are dehydrated by gradient alcohol (75%, 95%, 100%, 100%) for 5min each, soaked in xylene for 10min, replaced with xylene and soaked for 10min again, dried, sealed with neutral resin, dried in the sun, and observed.

4.3 results of the experiment

4.3.1 collagen fiber test results

The results of collagen fiber detection of skin tissue after completion of culture are summarized in fig. 12 and 13 and table 13. Table 13 summarizes the relative areas of collagen fibers. FIG. 12 is a graph showing the effect of the change in collagen fiber content in skin tissue. Figure 13 shows a histogram of the relative area results of collagen fibrils.

TABLE 13 summary of collagen fibril relative area

When statistical analysis is performed by using the t-test method in table 13, a p value less than 0.05 indicates that the difference is significant, and a p value less than 0.01 indicates that the difference is extremely significant.

From the above data it can be seen that:

compared with the Control group, the content of the collagen fiber in the UVR group is obviously reduced, which shows that the stimulation condition of the test is effective. The collagen fiber content of skin tissue decreases after UV irradiation, which is manifested as skin relaxation and aging after illumination in real skin conditions.

Compared with a UVR group, the content of collagen fibers of the samples MHA-Repair anti-aging essence is-0.05%, the content of MHA-Repair anti-sun Repair essence is-0.1%, the content of MHA-Repair anti-aging essence is-0.2%, the content of MHA-Repair anti-aging Repair essence is-0.4%, and the area of the collagen fibers of the skin is recovered to be close to the original condition before UV irradiation of the skin by the MHA-Repair anti-aging essence-0.4%, so that the skin light aging phenomenon is effectively relieved.

4.3.2 elastic fiber test results

After the completion of the culture, the skin tissue was subjected to elastic fiber examination, and the results of the pictures are shown in FIG. 14, FIG. 15 and Table 14. FIG. 14 shows the variation of the elastic fiber content of the skin tissue. FIG. 15 shows a histogram of the relative area results of the spandex.

TABLE 14 summary of relative areas of elastic fibers

Test grouping	Average value of relative area	SD	p value
				Control	1.00	0.03	/
UVR	0.16	0.01	0.000
				MHA-Repair anti-aging essence 0.05%	1.03	0.06	0.000
MHA-Repair anti-aging essence 0.1%	1.01	0.01	0.000
				MHA-Repair anti-aging essence 0.2%	1.04	0.05	0.000
MHA-Repair anti-aging essence 0.4%	1.03	0.04	0.000

From the above data it can be seen that:

compared with the Control group, the elastic fiber content of the UVR group is obviously reduced, which indicates that the stimulation condition of the test is effective.

Compared with a UVR group, the content of collagen fibers of the samples MHA-Repair anti-aging Repair essence is obviously increased by-0.05%, the MHA-Repair anti-sun Repair essence is-0.1%, the MHA-Repair anti-aging Repair essence is-0.2%, and the MHA-Repair anti-aging Repair essence is-0.4%.

4.3.3 Collagen IV test results

After the culture is finished, the Collagen IV content of the skin tissue is detected, and the picture results are summarized as shown in figure 16, figure 17 and table 15. FIG. 16 shows the variation of the Collagen IV content in the skin tissue. FIG. 17 is a histogram of results of Collagenin IV content.

TABLE 15 Collagen IV content summary Table

From the above data it can be seen that:

compared with the Control group, the Collagen IV protein content of the UVR group is obviously reduced, which indicates that the stimulation condition of the test is effective.

Compared with a UVR group, the contents of the MHA-Repair anti-aging Repair essence, the MHA-Repair anti-solarization Repair essence, the MHA-Repair anti-aging Repair essence and the MHA-Repair anti-aging Repair essence are obviously increased by-0.05%, by-0.1%, by-0.2% and by-0.4%, of C ollagen IV protein in the samples.

Based on the experiment and the effect of the three-dimensional skin tissue, under the use amount of 0.05 wt%, 0.1 wt%, 0.2 wt% and 0.4 wt% of the four effective components, the MHA-Repair anti-aging Repair essence can effectively resist the reduction of the content of Collagen fibers, elastic fibers and Collagen IV protein caused by UVR, and shows the effects of compactness and wrinkle resistance.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Claims

1. The application of carboxymethyl chitosan is selected from any one of the following:

for promoting fibroblast proliferation in a non-therapeutic destination;

used as effective component for promoting fibroblast proliferation for preparing topical product;

for increasing collagen content in non-therapeutic destinations;

the collagen can be used as effective component for increasing collagen content for preparing topical product;

can be used for preparing skin antiaging topical product.

2. Use according to claim 1, characterized in that: the carboxymethyl chitosan is O-carboxymethyl chitosan;

and/or the number average molecular weight of the carboxymethyl chitosan is 1000-20000.

3. Use according to claim 1 or 2, characterized in that: in the case of preparing a product for external use as an active ingredient, the amount of carboxymethyl chitosan added is not more than 1 wt%, preferably not more than 0.5 wt%, based on the total mass of the product for external use.

4. Use according to claim 1 or 2, characterized in that: when the carboxymethyl chitosan is used for promoting the proliferation of fibroblasts in a non-treatment destination, 0.01-0.2 wt% of carboxymethyl chitosan is adopted; and/or, when used to increase collagen content in a non-therapeutic destination, 0.01 wt% to 0.2 wt% carboxymethyl chitosan is employed.

5. Use according to claim 1 or 2, characterized in that: in the skin anti-aging product, the content of the carboxymethyl chitosan is 0.05 wt% -1 wt%.

6. A product for external use, characterized by comprising at least carboxymethyl chitosan and water.

7. The product for external use according to claim 6, wherein: the content of the carboxymethyl chitosan is 0.05 wt% -1 wt%, preferably 0.05 wt% -0.5 wt% based on the total mass of the external product.

8. The external product according to claim 6, wherein the carboxymethyl chitosan is O-carboxymethyl chitosan; and/or the number average molecular weight of the carboxymethyl chitosan is 1000-20000.

9. A product for external use according to claim 6 or 7, wherein: the topical product further comprises one or more of a thickener, a preservative, a humectant, an emulsifier, a skin conditioner, an antioxidant, an emollient, a chelating agent, and a pH adjuster.

10. The use according to claim 1 or the external product according to any one of claims 6 to 8, wherein the external product is a spray, a serum cream, a lotion or a mask.