CN113599391A

CN113599391A - Application of carboxymethyl chitosan in promoting skin wound healing and repairing skin barrier

Info

Publication number: CN113599391A
Application number: CN202110975976.0A
Authority: CN
Inventors: 方庆秋; 徐宇帆; 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: 2021-11-05

Abstract

Use of carboxymethyl chitosan for promoting the healing of skin wounds, repairing skin barriers. Experiments show that the carboxymethyl chitosan is an effective component for promoting skin wound healing and repairing skin barriers, can achieve a remarkable skin repairing effect, particularly has good applicability to repairing of skins after medical arts, has good antibacterial and repairing effects after being applied to skin surfaces as shown by cell experiments and human body experiments, and has good skin-friendly and biocompatible effects and practical application values.

Description

Application of carboxymethyl chitosan in promoting skin wound healing and repairing skin barrier

Technical Field

The invention relates to a new application of carboxymethyl chitosan, in particular to an application of carboxymethyl chitosan in preparation of medicines for promoting skin wound healing and repairing skin barriers.

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.

The skin barrier is the largest tissue of the human body and is also the interface between the external environment and the internal environment, and is used for keeping the internal environment of the human body stable, preventing harmful substances or unwanted substances from invading the skin, and preventing various nutrient substances, water and electrolytes in the body from losing; the broad skin barrier includes microbial barrier, chemical barrier, physical barrier, and immunological barrier. Skin barrier structural and functional abnormalities can lead to the development of skin inflammation and skin disease.

The photoelectric treatment in medical and beauty comprises an ultrasonic knife, photodynamic, radio frequency, laser, visible light, intense pulsed light and the like, and can be used for treating diseases or beautifying. It can be used for repairing and reconstructing skin injury through photothermal action, photomechanical action or photomodulation.

Different targets of photoelectric action have different damage structures to skin barriers and different damage degrees. Photo-electric treatment can lead to a breakdown of the skin barrier function and may cause local adverse reactions such as inflammation, infection, scarring or pigmentation. Clinicians must properly face skin barrier damage from different photovoltaic treatments, be familiar with various post-photovoltaic treatment adverse effects, and provide optimal, effective, and timely post-treatment care that will greatly reduce the risks associated with treatment modalities.

However, at present, there is no unified standard or care product for the postoperative care of medical and aesthetic photoelectric treatment, and there is no product for the wound care and repair after photoelectric treatment in the prior art.

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 the healing of skin wounds in a non-therapeutic destination.

The invention also discloses application of the carboxymethyl chitosan serving as an effective component for promoting skin wound healing in preparation of external products.

The invention also discloses the use of carboxymethyl chitosan for repairing skin barriers in non-therapeutic destinations.

The invention also discloses application of the carboxymethyl chitosan serving as an effective component for repairing the skin barrier in preparing an external product.

The invention also discloses an external product of carboxymethyl chitosan for preparing skin problems caused by medical and aesthetic photoelectric treatment.

Preferably, in the present application, the external product refers to a product for external application to the skin surface.

Preferably, the carboxymethyl chitosan is O-carboxymethyl chitosan.

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

Preferably, 0.01 wt% to 0.2 wt% carboxymethyl chitosan is used when used in a non-therapeutic destination to promote healing of skin wounds.

Preferably, 0.01% to 0.2% by weight of carboxymethyl chitosan is used when used in the repair of skin barriers at non-therapeutic destinations.

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, 0.05 to 1 wt% of carboxymethyl chitosan is used in the external product for skin problems caused by medical and aesthetic photoelectric treatment.

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. More preferably, 0.05 wt% to 1 wt%, more preferably 0.05 wt% to 0.5 wt%.

Preferably, the carboxymethyl chitosan is O-carboxymethyl chitosan.

Preferably, 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.

Preferably, the external product is a spray, a serum cream, a lotion, a mask or a medical moist dressing.

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.

The applicant has unexpectedly found that the carboxymethyl chitosan with good skin-friendly property and biocompatibility can be used as an effective ingredient for preparing products for repairing skin, and related test results show that the carboxymethyl chitosan has remarkable skin repairing efficacy, particularly can obviously repair damaged skin barriers and promote skin wound healing, and products for repairing skin, such as products for skin surface care, such as cosmetics, formed by the carboxymethyl chitosan as an effective ingredient are very suitable for repairing skin problems caused by medical photoelectric treatment.

Drawings

FIG. 1 is a graph showing the trend of cell viability changes in the results of cytotoxicity assays using MHA-20.

FIG. 2 shows the results of morphological examination of the results of cytotoxicity assays using MHA-20.

FIG. 3 is a graph showing the trend of cell viability in the cytotoxicity assay using MHA-30.

FIG. 4 shows the results of morphological examination among the results of cytotoxicity assays using MHA-30.

FIG. 5 is a graph showing the trend of cell viability changes in the results of the cytotoxicity assay of MHA-45.

FIG. 6 shows the results of morphological examination of the results of cytotoxicity assays using MHA-45.

FIG. 7 is a line graph showing the results of cell proliferation assay.

Fig. 8 shows a photograph of normal skin tissue morphology.

Fig. 9 shows a photograph of the morphology of skin tissue in a blank wound control group.

FIG. 10 shows photographs of the morphology of skin tissue after application of MHA-20 in the present application.

FIG. 11 is a photograph showing the morphology of the stratum corneum of normal skin epidermis.

FIG. 12 is a photograph showing the morphology of stratum corneum of skin epidermis of hollow-1 in the blank control group.

FIG. 13 is a photograph showing the morphology of the stratum corneum of the skin epidermis in the sample group 1 in the samples 1-1.

FIG. 14 is a photograph showing the stratum corneum morphology of the skin corresponding to the 2-1 samples in sample group 2.

FIG. 15 is a photograph showing the morphology of the stratum corneum of the epidermis of the skin of 3-1 in comparative group 3.

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 carboxymethyl chitosan has a remarkable skin repairing effect when being externally applied to the surface of skin, and the carboxymethyl chitosan can be used as a unique effective component to prepare a product for repairing skin.

Based on human 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 human fibroblast proliferation.

The developed animal experiments and effects prove that the carboxymethyl chitosan can promote the formation of granulation tissues of wounds, activate epithelial tissues, relieve the proliferation of fibroblasts and scars, reduce or avoid scars, restore skin lesions to be flat, accelerate the healing of the wounds and promote the healing of skin wounds. Therefore, the carboxymethyl chitosan in the application can be clearly demonstrated to be used as a cosmetic raw material for skin care, can play a good role in promoting the healing of skin wounds, and especially has a good treatment effect on skin problems caused by medical and aesthetic photoelectric treatment.

The developed animal experiments and effects prove that the carboxymethyl chitosan can promote the recovery of the horny layer of the epidermis of the skin, so that the carboxymethyl chitosan can be used for repairing the barrier of the damaged skin. Therefore, the carboxymethyl chitosan in the application can be used as a cosmetic raw material for skin care, has a good effect of repairing a damaged skin barrier, and also has a good treatment effect on skin problems caused by medical and aesthetic photoelectric treatment.

The carboxymethyl chitosan in the application has the antibacterial and anti-inflammatory effects, and the skin-friendly property and biocompatibility of the carboxymethyl chitosan are good, so that other skin safety problems caused in the process of using the carboxymethyl chitosan as a repair effect component can be avoided, and therefore, the carboxymethyl chitosan in the application is very suitable for being used as a raw material of skin repair cosmetics.

Thus, applicants first provide a use of carboxymethyl chitosan for promoting healing of skin wounds in a non-therapeutic destination.

In a specific embodiment, the application of carboxymethyl chitosan as an effective ingredient for promoting the healing of skin wounds to prepare an external product is also provided. Carboxymethyl chitosan is used as a raw material ingredient in products for repairing skin, and in this case, carboxymethyl chitosan is the only active ingredient or one of the active ingredients for repairing skin.

In a particular embodiment, applicants disclose the use of carboxymethyl chitosan for repairing skin barriers in non-therapeutic destinations.

In a specific embodiment, the use of carboxymethyl chitosan as an effective ingredient for repairing skin barrier for the preparation of products for external use is also provided.

In a specific embodiment, the application of the carboxymethyl chitosan in preparing an external product suitable for skin problems caused by medical and aesthetic photoelectric treatment 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 repairing the skin can be spray, essence lotion, essence cream, facial mask, medical wet auxiliary materials and the like.

In a preferred embodiment, 0.01 wt% to 0.2 wt% carboxymethyl chitosan is used when used in a non-therapeutic destination to promote healing of skin wounds.

In a preferred embodiment, 0.01% to 0.2% by weight of carboxymethyl chitosan is used when used in the non-therapeutic destination for repairing the skin barrier.

In a preferred embodiment, 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 external product, when the external product is prepared as an effective ingredient.

In a more preferred embodiment, the carboxymethyl chitosan is added in an amount of 0.05 to 1 wt%, in a more preferred embodiment, 0.05 to 0.5 wt%, in a more preferred embodiment, 0.1 to 0.5 wt%, as in a more specific embodiment, 0.1, 0.2, 0.3, 0.4 or 0.5 wt%, based on the total mass of the external product, when the external product is prepared.

In a specific embodiment, 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 more 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 specific embodiment, the product containing carboxymethyl chitosan provided in the present application not only has the new effects of significantly promoting human fibroblast proliferation, promoting formation of wound granulation tissue, and promoting recovery of skin epidermal stratum corneum, but also combines the antibacterial and anti-inflammatory effects of the product and the characteristics of being easily dissolved in water to form a wet adjuvant, so that the product is very suitable for being applied to the repair of skin after medical and artistic activities, and not only can help to repair skin barrier and promote wound healing, but also avoids inflammation and infection in a wet environment or in a repair process.

In order to further illustrate that carboxymethyl chitosan can be used as the only effective component in the external product, the application also provides a specific external product, and the product for repairing skin at least comprises carboxymethyl chitosan and water.

In a preferred embodiment, the topical product further comprises 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, creams or medical moist dressings.

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), 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.

The following are specific experimental and performance 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.

First, fibroblast-based proliferation promotion assay.

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 assay were human fibroblasts, and cell batch number 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 BioTekEpoch, the micro-oscillator is the Linebel MM-1 thereof, and the balance is Sidolisi BSA 124S.

1.2 test methods

1.2.1 cytotoxicity detection and detection results

1.2.1.1 MTT assay

Cell inoculation: by 1 × 10⁴The human fibroblasts were seeded at a 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: waiting 24-hole plateWhen the cell plating rate reaches 40-60%, the drug is administered, the sample group is added with the test substances with different concentrations, the solvent control group is added with the culture solution, and the culture solution is incubated 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 human 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, it is believed that the sample MHA-20 was based on human fibroblasts and showed no significant cytotoxicity in the concentration range of 2 mg/mL.

TABLE 2 MHA-20MTT assay results

8 dosing concentrations of the sample MHA-30 were set, cytotoxicity detection experiments were performed on human 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, it is believed that the sample MHA-30, which is based on human fibroblasts, does not exhibit significant cytotoxicity in the concentration range of 2 mg/mL.

TABLE 3 MHA-30MTT assay results

8 dosing concentrations of the sample MHA-45 were set, cytotoxicity detection experiments were performed on human fibroblasts, the MTT detection results are shown in Table 4, and the trend of cell viability 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, it is believed that the sample MHA-45 was based on human fibroblasts and showed no significant cytotoxicity in the concentration range of 2 mg/mL.

TABLE 4 MHA-20MTT assay results

1.2.2 cell proliferation assay and assay results.

1.2.2.1 cell proliferation assay

Cell inoculation: by 4X 10³The human fibroblasts were seeded at a 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 LDMSO 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 MHA-20 sample shows remarkable effect of promoting human fibroblast proliferation at the concentration of 0.01%, 0.025% and 0.05% and the MHA-30 and MHA-45 sample at the concentration of 0.025% for 24h and 48 h.

And secondly, detecting the promotion effect experiment and effect of the carboxymethyl chitosan on wound healing.

Based on mouse experiments, the effect of carboxymethyl chitosan in the present application on the promotion of wound healing was evaluated.

2.1 testing materials

2.1.1 test animals

Guinea pigs (male/female).

2.1.2 test conditions

The laboratory temperature is 20-25 ℃, and the relative humidity is 60-70%.

2.2, test methods and results.

Selecting 5 healthy white guinea pigs, removing hair in the range of 5cm × 5cm on both sides of spinal column of each animal, performing aseptic operation with a scalpel, making a straight incision on skin on both sides, wherein the incision is measured by blood seepage, and smearing 0.025 wt% MHA-20 water solution. Animals were sacrificed 1 time a day after 5 consecutive days, dissected, and the skin incision test area was sectioned for pathology and histology.

The test results are shown in fig. 8, 9 and 10, in which fig. 8 is a photograph of a normal skin tissue morphology, fig. 9 is a photograph of a skin tissue morphology of a blank wound control group, and fig. 10 is a photograph of a skin tissue morphology after being smeared with MHA-20 of the present application.

As is obvious from the tissue form picture on the picture, the carboxymethyl chitosan MHA-20 has the functions of promoting the formation of granulation tissue of the wound, activating epithelial tissue, proliferating fibroblasts and scars in the link, reducing or avoiding scars, recovering skin lesions to be smooth and accelerating the healing of the wound.

And thirdly, performing an experiment based on the emulsion containing the carboxymethyl chitosan as one of the raw material components to evaluate the repair of the skin barrier damage by the carboxymethyl chitosan.

3.1 testing materials

In this embodiment, an emulsion is formed based on carboxymethyl chitosan, and includes the following raw materials by weight: 0.05 wt% EDTA-2Na, 0.2 wt% TR-2, 3 wt% 1, 3-butanediol, 5 wt% glycerol, 0.1 wt% xanthan gum DSM, 0.1 wt% gamma-PGA, 0.1 wt% sodium hyaluronate, 0.25 wt% MHA-20, 3 wt% MONTANOV L, 0.5 wt% TEGOCARE 165, 1 wt% cetostearyl alcohol, 5 wt% caprylic triglyceride, 5 wt% shea butter, 0.5 wt% bisabolol, 5 wt% squalane, 0.2 wt% arginine, 3 wt% SIMULGEL NS, a suitable amount of preservative (5 wt% pentanediol), and the balance water.

When in preparation, the MHA-20 can be dissolved in the pentanediol water solution and then is uniformly mixed with other raw material components.

And 3.2, repairing test and effect data.

Selecting 12 male nude mice, randomly dividing the nude mice into 3 sample groups and a blank control group in 8-10 weeks, firstly, continuously sticking and quickly tearing off the test parts of 3cm multiplied by 3cm on the skins of the left side and the right side of the nude mice by using an adhesive tape for a plurality of times until the skins have small blood spots, stopping sticking and tearing, respectively smearing each sample group, smearing once every day, and after continuously smearing for 5 days, killing the animals, dissecting the skin tissues of the test parts, and taking a photo by taking a pathological section through microscopic examination.

During the test, the laboratory temperature is 20-24 ℃, and the relative humidity is 60-70%.

The test results are shown in table 7, fig. 11, fig. 12, fig. 13, fig. 14, and fig. 15.

TABLE 7

In Table 7, sample set 1 was smeared with an emulsion containing MHA-20 as described above. Sample set 2 was smeared with an emulsion of RGF 3000U/g. Whereas comparative group 3 was applied with an emulsion not containing MHA-20, but 0.25 wt% MHA-20 was replaced with 0.25 wt% water. The blank control was not smeared with any substance. Wherein RGF is epidermal growth factor.

FIG. 11 is a photograph of the tissue morphology of the stratum corneum of normal skin epidermis; FIG. 12 is a photograph showing the appearance of horny layer of epidermis in hollow-1 of blank control group, which shows that the horny layer of epidermis is not restored; FIG. 13 is a photograph showing the morphology of the horny layer of the epidermis in the sample group 1 of samples 1-1, and it can be seen that the horny layer of the epidermis has been restored. FIG. 14 is a photograph showing the morphology of the stratum corneum of the skin corresponding to the 2-1 samples in the sample group 2, and the stratum corneum recovery of the skin can be seen from FIG. 14. FIG. 15 is a photograph showing the morphology of the horny layer of the epidermis of the skin of 3-1 in the comparative group 3, and it can be seen that the horny layer of the epidermis of the skin is not restored.

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 skin wound healing in a non-therapeutic destination;

used as effective component for promoting skin wound healing for preparing topical product;

for repairing skin barriers at non-therapeutic destinations;

the active ingredient for repairing skin barrier is used for preparing external products;

can be used for preparing topical products for treating skin problems caused by photoelectric therapy.

2. The use according to claim 1, wherein 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 0.01 to 0.2 wt.% of carboxymethyl chitosan is used; and/or, when used in non-therapeutic repair of skin barriers, 0.01 wt% to 0.2 wt% carboxymethyl chitosan is employed.

4. Use according to claim 1 or 2, characterized in that, 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.

5. Use according to claim 1 or 2, characterized in that 0.05 to 1 wt% of carboxymethyl chitosan is used in the external product for skin problems caused by medical and aesthetic photoelectric treatments.

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

7. The external product according to claim 6, wherein the carboxymethyl chitosan is contained in an amount of 0.05 to 1 wt%, preferably 0.05 to 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. The product for external use according to claim 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 product for external use according to claim 6, characterized in that the product for external use is a spray, a serum cream, a lotion, a mask or a medical moist dressing.