CN116531306A

CN116531306A - Composition containing recombinant human fibronectin and having repairing effect, preparation method and application

Info

Publication number: CN116531306A
Application number: CN202310181015.1A
Authority: CN
Inventors: 王�琦; 常鲜耀; 王璐
Original assignee: Zhuhai Yingsheng Lianke Biotechnology Co ltd
Current assignee: Zhuhai Yingsheng Lianke Biotechnology Co ltd
Priority date: 2023-02-27
Filing date: 2023-02-27
Publication date: 2023-08-04

Abstract

The invention discloses a composition containing recombinant human fibronectin and having a repairing effect, a preparation method and application thereof. The composition comprises the following components in parts by weight: 5-20 g/100g of recombinant human fibronectin, 0.3-1.0 g/100g of hyaluronic acid mixture, 0.8-1.2 g/100g of oat glucan, 0.8-1.2 g/100g of aloe extract, 0.3-0.8 g/100g of echinacea extract, 0.8-1.2 g/100g of dendrobium stem extract, 0.3-0.8 g/100g of Shu Minjia and the balance of water; wherein the amino acid sequence of the recombinant human fibronectin is shown as SEQ ID No. 1. The composition containing recombinant human fibronectin has the repairing effect, is based on specific bioactive protein recombinant human fibronectin, strengthens the functions of effective components such as hyaluronic acid, oat glucan, aloe extract and the like through the functional domain of the recombinant human fibronectin, plays a role of multiple synergism through the mutual cooperation of various raw materials, can rapidly supplement water, resist inflammation and remove free radicals, and achieves the repairing effect after injury.

Description

Composition containing recombinant human fibronectin and having repairing effect, preparation method and application

Technical Field

The invention relates to the field of biology, in particular to a composition containing recombinant human fibronectin and having a repairing effect, a preparation method and application thereof.

Background

Skin is the largest organ of the human body and is the primary barrier against heat loss, dehydration, mechanical trauma and microbial invasion. The structures of the skin from outside to inside are epidermis layer, dermis layer and subcutaneous fat layer in sequence. The epidermis is the outermost layer of the skin, and is as thin as a sheet of paper, and consists of a large number of keratinocytes and stem cells, melanocytes, immune cells (Langerhans cells, T cells), innervates nerves and phagocytises foreign substances, and is constantly self-renewing. The dermis layer is composed of connective tissue, is rich in collagen fiber, protein fiber and fibroblast, plays a role in supporting skin structure and promotes wound repair. The subcutaneous layer is rich in adipocytes and blood vessels. Healthy skin has luster and good elasticity, and forms a stable internal environment. When the skin is subjected to external stimuli such as ionizing radiation, ultraviolet rays, burns, etc., the skin barrier is destroyed, resulting in skin problems. Ultraviolet radiation causes changes in both the morphology and appearance of the skin. The appearance is mainly characterized by Sunburn (Sunburn), the skin has acute inflammatory reactions such as erythema, congestion, edema and even ulceration, the morphology is characterized by thickening of the epidermis, massive inflammatory cell infiltration and abscess formation, and collagen synthesis is reduced. Causing photo-aging phenomena, manifested by the appearance of leather, reduced elasticity and sunburn formation.

At present, two types of cosmetics for relieving and repairing are mainly used in the market, namely natural vegetable oil and Chinese herbal medicines are used as active substances for repairing skin, and biocompatible raw materials are used as active substances for repairing skin. The natural vegetable oil and the Chinese herbal medicine have complex component substances, even some sensitization components exist, and certain potential safety hazard exists. Representative of bioactive substances are hyaluronic acid and collagen. However, the product with the effect of adding hyaluronic acid and collagen, such as collagen peptide mask liquid disclosed in Chinese patent No. CN107714631A, has the problems of poor absorption and low effective utilization rate due to the barrier function (larger protein molecular weight) of skin. In addition, most of the collagen used at present comes from animal extraction, so that not only is a certain virus hidden trouble existed, but also the hydrophilicity and biocompatibility are poor. Therefore, the skin can not be repaired in time effectively aiming at inflammatory factors and subsequent immune response in early skin injury.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide a composition containing recombinant human fibronectin and having a repairing effect, a preparation method and application.

The technical problems to be solved by the invention are realized by the following technical scheme:

in a first aspect, a composition comprising recombinant human fibronectin having repair efficacy comprises the following components in parts by weight: 5-20 g/100g of recombinant human fibronectin, 0.3-1.0 g/100g of hyaluronic acid mixture, 0.8-1.2 g/100g of oat glucan, 0.8-1.2 g/100g of aloe extract, 0.3-0.8 g/100g of echinacea extract, 0.8-1.2 g/100g of dendrobium stem extract, 0.3-0.8 g/100g of Shu Minjia and the balance of water; wherein the amino acid sequence of the recombinant human fibronectin is shown as SEQ ID No. 1.

In the invention, the composition further comprises the following components in parts by weight: 0.01 to 0.02g/100g of metal ion chelating agent, 5 to 10g/100g of polyol, 2 to 3g/100g of protein stabilizer and 0.03 to 0.05g/100g of thickener.

In a second aspect, a method for preparing a composition comprising recombinant human fibronectin with repair efficacy comprises the steps of:

step 1, mixing a hyaluronic acid mixture and water, heating to a certain temperature, and uniformly mixing to obtain a mixture 1;

step 2, mixing the recombinant human fibronectin and water, and uniformly stirring and mixing at a low speed to obtain a mixture 2;

step 3, heating the echinacea purpurea extract, the dendrobium stem extract, shu Minjia and water, and uniformly stirring to obtain a mixture 3;

step 4, mixing oat glucan, aloe extract and water, adding the mixture to a certain temperature, and uniformly mixing to obtain a mixture 4;

step 5, carrying out primary homogeneous mixing treatment on the mixtures 1 and 3; then gradually adding the mixture 4 to carry out a second homogeneous mixing treatment; and finally, adding the mixture 2 to perform a third homogeneous mixing treatment to obtain the composition with repairing effect.

In a third aspect, a recombinant human fibronectin-containing composition according to the first or second aspect is provided.

Compared with the prior art, the invention has the following beneficial effects:

the composition containing recombinant human fibronectin has the repairing effect, is based on specific bioactive protein recombinant human fibronectin, and is added with hyaluronic acid mixture, shu Minjia, aloe extract, oat glucan and other substances to achieve the effects of repairing, moisturizing, anti-inflammatory and anti-sensitization. The function of the effective components such as hyaluronic acid, oat glucan, aloe extract and the like is enhanced through the recombinant human fibronectin functional domain, and the effects of supplementing water, resisting inflammation, scavenging free radicals and repairing after injury are achieved through the mutual cooperation of various raw materials.

The repairing effect composition has the advantages that the recombinant human fibronectin has smaller molecular weight, can be quickly absorbed by skin, activates and promotes cell movement, can activate skin surface cells, promote cell adherence, stretch and proliferation, recover morphological structures of damaged cells and aged cells, strengthen cell metabolism level, promote cell growth, strengthen horny layer, promote water locking capability of skin and improve external stimulus resistance capability of horny layer.

The specific active functional domain of the recombinant human fibronectin not only strengthens the function of the hyaluronic acid binding domain, but also ensures that hyaluronic acid with different molecular sizes can act on the skin, thereby increasing the absorption and locking of the skin to moisture and showing better moisturizing effect. Can also strengthen the efficacy of oat glucan and aloe extract, and has the effects of anti-inflammatory, repairing and moisturizing rapidly. The Echinacea purpurea extract not only has the functions of antioxidant, anti-inflammatory agent and humectant, but also can effectively inhibit hyaluronidase and prolong the function of hyaluronic acid.

The invention uses active substances such as plant extracts and the like, and adopts a mode of multiple homogeneous mixing in a production scheme, so that the prepared composition is mild, safe and good in skin-friendly property; and the filtering treatment is not needed, and the production process is simple and suitable for popularization and application.

Drawings

FIG. 1 is a schematic diagram of the structure of recombinant human fibronectin of the invention;

FIG. 2 is a diagram of the structure of recombinant human fibronectin of the invention;

FIG. 3 shows the cell attachment promoting effect of recombinant human fibronectin (rhFN) of example 1 of the present invention;

FIG. 4 is a graph showing the score of skin wrinkles on the back of a damaged mouse by UV irradiation;

FIG. 5 shows the skin moisture content of mice after UV irradiation.

Detailed Description

As described in the background art, the products that act by adding hyaluronic acid and collagen in the prior art have a problem of low effective utilization rate because of poor absorption due to the barrier function (large molecular weight of protein) of the skin. In addition, most of the collagen used at present comes from animal extraction, so that not only is a certain virus hidden trouble existed, but also the hydrophilicity and biocompatibility are poor. Therefore, the skin can not be repaired in time effectively aiming at inflammatory factors and subsequent immune response in early skin injury.

In order to solve the technical problems, the invention provides a composition containing recombinant human fibronectin and having a repairing effect, a preparation method and application.

The composition containing recombinant human fibronectin with repairing effect comprises the following components in parts by weight: 5-20 g/100g of recombinant human fibronectin, 0.3-1.0 g/100g of hyaluronic acid mixture, 0.8-1.2 g/100g of oat glucan, 0.8-1.2 g/100g of aloe extract, 0.3-0.8 g/100g of echinacea extract, 0.8-1.2 g/100g of dendrobium stem extract, 0.3-0.8 g/100g of Shu Minjia and the balance of water; wherein the amino acid sequence of the recombinant human fibronectin is shown as SEQ ID No. 1.

Fibronectin (abbreviated FN) was originally discovered by Morrison in 1948. Due to its property of precipitating on cooling, it was originally called cold insoluble globulin (Cold insoluble globulin). Fibronectin, an important component commonly found in extracellular matrix, is widely present in blood, body fluids and various tissues, and is a total of 20 species in the human body. The main biological functions are the regulation factors in the cell activity process, and the important scaffold proteins which maintain and guide the tissue structure and extracellular matrix composition. In the invention, the functional domain of human plasma fibronectin (Plasma fibronectin, pFN) is recombined and modified to obtain the recombinant human fibronectin (rhFN) with special functions. In the invention, recombinant human fibronectin is taken as a main active ingredient, has the functions of activating cells, enhancing the activity of the cells, and accelerating the action of hyaluronic acid by combining functional domains with hyaluronic acid with small molecular weight, thereby promoting the proliferation and differentiation of epidermal cells and scavenging oxygen free radicals. rhFN can also be combined with macromolecular hyaluronic acid to form a protective film function for protecting damaged skin.

Recombinant human fibronectin (rhFN) with an amino acid sequence shown as SEQ ID No.1,

SEQ ID No.1：

MPGCYDNGKHYQINQQWERTYLGNALVCTCYGGSRGFNCESKPEAEETCFDKYTGNTYRVGDTYERPKDSMIWDCTCIGAGRGRISCTIANRCHEGGQSYKIGDTWRRPHETGGYMLECVCLGNGKGEWTCKPIAEKCFDHAAGTSYVVGETWEKPYQGWMMVDCTCLGEGSGRITCTSRNRCNDQDTRTSYRIGDTWSKKDNRGNLLQCICTGNGRGEWKCERHGGSGHCVTDSGVVYSVGMQWLKTQGNKQMLCTCLGNGVSCQETAVTQTYGGNSNGEPCVLPFTYNGRTFYSCTTEGRQDGHLWCSTTSNYEQDQKYSFCTDHTVLVQTRGGNSNGALCHFPFLYNNHNYTDCTSEGRRDNMKWCGTTQNYDADQKFGFCPMAAHEEICTTNEGVMYRIGDQWDKQHDMGHMMRCTCVGNGRGEWTCIAYSQLRDQCIVDDITYNVNDTFHKRHEEGHMLNCTCFGQGRGRWKCDPVDQCQDSETGTFYQIGDSWEKYVHGVRYQCYCYGRGIGEWHCQPLGGSTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGIDYDISVITLINGGESAPTTLTQQTAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSQMQVTDVQDNSISVKWLPSSSPVTGYRVTTTPKNGPGPTKTKTAGPDQTEMTIEGLQPTVEYVVSVYAQNPSGESQPLVQTAVTNIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIHELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTAIP。

the structure diagram is shown in figures 1 and 2, the recombinant and the modification are carried out on the basis of the natural human plasma fibronectin, the molecular weight of the modified protein is about 117.7KD, and compared with the molecular weight of natural FN, the molecular weight is greatly reduced (220 KD), thereby being beneficial to skin absorption and biological activity exertion; too large molecular weight, poor transdermal absorption effect, too small molecular weight, inability to include functional domains, and inadequate manifestation of some specific biological activities. The recombinant human fibronectin has the following structural functional domains:

HA-binding region: heparin and fibrin binding domain can be combined with hyaluronic acid mixture to enhance absorption of hyaluronic acid and construct a protective film, and has functions of water locking and moisture retention;

collagen-binding domain: the binding sites of the type I collagen and the type II collagen have considerable curative effect on wound healing; plays a central role in cell adhesion, stabilizing the basal layer and the stratum corneum;

edb+phsrn+rgd+eda: recognizing cell integrin sites, and further promoting cell adhesion proliferation activity; regulate cell growth and regulate skin immune function.

The hyaluronic acid mixture is formed by mixing macromolecular hyaluronic acid and micromolecular hyaluronic acid according to the weight ratio of 3-5:5-7. The hyaluronic acid mixture with a fixed proportion can have the functions of protection, prevention and repair. The macromolecular hyaluronic acid (molecular weight 1800 kDa), a straight-chain macromolecular polysaccharide consisting of disaccharide units (glucuronic acid-N-ethionine glucosamine), is a naturally occurring substance in tissues. Plays an important role of a matrix in the dermis layer of human skin, and has the functions of protection and protection. Both the maintenance of tissue structures as a whole and the transport between cells have important functions. The low molecular hyaluronic acid (molecular weight of 400-1000 kDa) is an inherent component in human body, is a glucuronic acid, can promote proliferation and differentiation of epidermal cells, remove oxygen free radicals, and can prevent and repair skin injury. In the present invention, the hyaluronic acid mixture can enhance the connection and action with cells through partial functional domains (RGD, VPP, etc.) of rhFN, enhancing the function of hyaluronic acid.

Oat glucan is a non-starch polysaccharide present in the endosperm and aleurone layer cell walls of oats. Oat glucan has excellent anti-aging effect, can smooth fine wrinkles, improve skin elasticity and improve skin texture; has a unique linear molecular structure, and endows good transdermal absorption performance; promote the synthesis of collagen by fibroblasts, promote wound healing and repair damaged skin.

Aloe extract contains anthraquinone derivative, aloe radix et rhizoma Rhei, saponin, amino acids and polysaccharide. The aloe extract promotes the degradation of fibrin to NMF, thereby increasing natural moisturizing factors, increasing water content immediately, and reducing percutaneous water loss through slight film formation. Meanwhile, the biological enzyme is contained, and the biological enzyme is slowly hydrolyzed with skin to play a role in diminishing inflammation and sterilizing, and plays a role in acting with collagen in the stratum corneum under the action of amino acid to heal wounds.

Echinacea purpurea extract (ECHINACEA PURPUREA EXTRACT), which is a plant of the order of Chrysanthemum and family Compositae, has the main effects of antioxidant, antiinflammatory agent and humectant. Has a certain inhibition effect on hyaluronidase and can enhance the effect of hyaluronic acid. Therefore, the skin care product can effectively resist aging, remove wrinkles on the skin, and also can effectively whiten and resist oxidation.

The dendrobium stem extract (Dendrobium nobile Lindl) has strong antioxidant capacity, has the strongest scavenging ability to hydroxyl free radicals, has a strong effect of inhibiting DNA damage, and is obviously stronger than VC.

Shu Minjia it is prepared from Astragalus membranaceus as main ingredient, and has effects of improving adaptability of organism, scientifically combining with "monarch, minister, assistant and guide" formula principle of Chinese medicine, and scientifically combining with radix Saposhnikoviae, rhizoma Gastrodiae, herba Sidae Rhombifoliae, etc., and repairing damaged skin.

The composition also comprises the following components in parts by weight: 0.01 to 0.02g/100g of metal ion chelating agent, 5 to 10g/100g of polyol, 2 to 5g/100g of protein stabilizer and 0.03 to 0.1g/100g of thickener.

Wherein the metal ion chelating agent is selected from at least one of EDTA-2Na, EDTA and EDTA-4 Na. The polyalcohol comprises one or more of propylene glycol, EG-1 polyglycerol, butanediol, hexanediol and pentanediol. The protein stabilizer is at least one selected from sodium dihydrogen phosphate, disodium hydrogen phosphate, arginine and trehalose. The thickener comprises one or more of carboxymethyl cellulose, hydroxyethyl cellulose and methyl cellulose.

According to the technical scheme, the composition containing the recombinant human fibronectin has the repairing effect, and based on the recombinant human fibronectin of the specific bioactive protein, substances such as hyaluronic acid mixture, shu Minjia, aloe extract, oat glucan and the like are added to achieve the effects of repairing, moisturizing, anti-inflammatory and anti-allergic. The function of the effective components such as hyaluronic acid, oat glucan, aloe extract and the like is enhanced through the recombinant human fibronectin functional domain, and the effects of supplementing water, resisting inflammation, scavenging free radicals and repairing after injury are achieved through the mutual cooperation of various raw materials.

The preparation method of the composition containing recombinant human fibronectin and having the repairing effect comprises the following steps:

step 1, mixing hyaluronic acid mixture and water, heating to a certain temperature, such as below 50 ℃, and uniformly mixing to obtain a mixture 1;

step 4, mixing oat glucan, aloe extract and water, adding the mixture to a certain temperature, for example, below 50 ℃, and uniformly mixing to obtain a mixture 4;

step 5, firstly, carrying out primary homogeneous mixing treatment on the mixture 1 and the mixture 3; then gradually adding the mixture 4 to carry out a second homogeneous mixing treatment; and finally, adding the mixture 2 to perform a third homogeneous mixing treatment to obtain the composition with repairing effect.

The invention is originally to directly and homogeneously mix under the set pH condition, and the heating and dissolving effects of some components are found to be good, and the activity of the protein is reduced after heating. Moreover, the direct mixing is very easy to generate foam, and a large amount of foam is formed by stirring protein; even, the activity of rhFN is reduced by 10-20% after multi-step mixing and homogenization. Therefore, by the above technical scheme, the invention only sets the temperature of the mixture 1 and the mixture 4 to be below 50 ℃, so that the active substance rhFN is prevented from being deactivated due to the overhigh temperature, partial protein denaturation and floccule are also avoided. And various raw materials are respectively and orderly added, stirred and mixed uniformly at various different temperatures, and then are mixed uniformly step by step, so that the various raw materials can be fully mixed and reacted, and the biological activity can be kept, thereby preparing the fine and uniform composition which is mild, safe and good in skin affinity. And the filtering treatment is not needed, and the production process is simple and suitable for popularization and application.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

The invention will be further illustrated by the following examples. The recombinant human fibronectin used in the invention is a self-made product; the other raw materials are all products of the catalogue of used cosmetics raw materials and are all commercial products; the water used is water for injection for medicine.

EXAMPLE 1 test of the cell adhesion promoting effect of recombinant human fibronectin as the main active ingredient

The Hela cells grown in the flask to 80% were digested with pancreatin, washed three times by centrifugation, and then washed withDMEM (Gibco) medium was blown, resuspended and dispersed to adjust the cell number to 10 ⁶ About/ml, and 6-well culture plates were added for culturing. The cell grouping is as follows: blank (rhFN 0. Mu.g/ml), low concentration (rhFN 20. Mu.g/ml), high concentration (rhFN 50. Mu.g/m l). DMEM and DMEM with different concentrations were added in different groups, and after culturing for 8h and 24h, cell attachment and proliferation were observed, respectively.

The results are shown in FIG. 3, in the blank, no rhFN was added, and only a small amount of cells adhered to the blank at 8h, and no proliferation-expanded cells were seen. Whereas the culture group to which rhFN was added had been attached to a large number of cells at 8 hours, and a part of the cells had proliferated. The number of adherent cells in the high-concentration group is slightly larger, and the cell proliferation condition is more obvious. After 24h, the blank group was observed to have only a portion of the cells extended to adhere, and the cell morphology was irregular. The rhFN group cells proliferate obviously, the cell morphology is typical diamond, and the adherence is firm. The high-low dose group of rhFN has no obvious difference, which shows that 20 mug/ml rhFN can produce good cell adhesion promoting and proliferation effect.

The experimental results show that the rhFN has good effects of promoting cell adhesion and proliferation, can promote cell growth and metabolism, and is beneficial to skin metabolism and repair.

Example 2

The composition containing recombinant human fibronectin and having the repairing effect comprises the following raw materials: propylene glycol, metal ion chelating agent, hyaluronic acid mixture, protein stabilizer (sodium dihydrogen phosphate, disodium hydrogen phosphate, arginine and trehalose), humectant (butanediol, polyglycerol, etc.), shu Minjia, herba Avenae Fatuae dextran, aloe extract, stem extract of herba Dendrobii, echinacea purpurea extract, etc. The amounts of the various materials are specified in Table 1 below.

step 1, mixing propylene glycol, a metal ion chelating agent, a hyaluronic acid mixture and deionized water, heating to 50 ℃, and uniformly stirring at 50r/min to obtain a mixture 1;

step 2, mixing sodium dihydrogen phosphate, disodium hydrogen phosphate, arginine, trehalose and deionized water to obtain a protein stabilizer, adding recombinant fibronectin, and stirring at a low speed (10 r/min) to obtain a mixture 2;

step 3, adding Shu Minjia, dendrobium stem extract, echinacea extract and deionized water into butanediol and polyglycerol serving as matrixes, heating, and stirring uniformly at 50r/min to obtain a mixture 3;

step 4, mixing hexanediol, oat glucan, aloe extract and deionized water, adding the mixture to 50 ℃, and uniformly mixing the mixture at 50r/min to obtain a mixture 4;

step 5, the mixture 1 and the mixture 3 were first homogenized at 3000rpm for 5min. The mixture 4 was then gradually added to the mixture for a second homogenization, at 5000rpm for 5min. In the last step, the mixture 2 was added to the above homogenized mixture for a third homogenization at 1000rpm for 5min. Discharging to obtain the mixture with repairing effect.

Table 1 weight (g) of the mixture having repairing effect on the respective raw materials

Example 3

A composition having a repairing function is different from example 2 in that the mixture of recombinant human fibronectin and hyaluronic acid as active ingredients is used in an amount of 10g of rhFN and 0.3g of hyaluronic acid mixture, as shown in Table 1.

Example 4

A composition having a repairing function is different from example 2 in that the mixture of recombinant human fibronectin and hyaluronic acid as active ingredients is used in an amount of 15g of rhFN and 0.5g of hyaluronic acid as a mixture, and the other ingredients are specifically shown in Table 1.

Example 5

A composition having a repairing function is different from example 2 in that the mixture of recombinant human fibronectin and hyaluronic acid as active ingredients is used in an amount of 20g of rhFN and 0.5g of hyaluronic acid as a mixture, and the other ingredients are specifically shown in Table 1.

Comparative example 1

This comparative example 1 differs from example 2 in that: the mixture containing no rhFN and only hyaluronic acid was 0.5g, and the other components were the same as in example 2.

Comparative example 2

This comparative example 2 differs from example 2 in that: the preparation method of the composition is different. The preparation method of comparative example 2 comprises the following steps: mixing and stirring the raw materials, regulating the pH value to 7.0, directly homogenizing and mixing, and discharging.

Test repair test of damaged mouse skin by ultraviolet irradiation

30+ -2 g KM mice (medical laboratory animal center, guangdong province), mice were acclimatized for 2 days, and were randomly grouped by body weight: (1) normal group, (2) model group (model group was subjected to ultraviolet irradiation alone, and no film-sticking treatment was given), (3) example 2 group, (4) example 3 group, (5) example 4 group, (6) example 5 group, (7) comparative example 1, and (8) comparative example 2. The above groups are all made into corresponding dressing adhesive films.

And (3) carrying out back skin dehairing treatment on all mice 24 hours before irradiation, firstly shaving longer hair on the backs of the mice by using a pet shaver, and then uniformly smearing dehairing paste to remove the remaining thin and short hair so as to form a naked area with the size of about 4cm multiplied by 3 cm. Except for the normal groups, the other groups are arranged at a position 25cm below an ultraviolet therapeutic instrument, and the ultraviolet irradiation intensity is 0.35mW/cm ² The simulated daylight was irradiated 2 times per day for 30 minutes each time. The experimental period was 14 days, and the total radiation dose was 12.6J/cm ² The method comprises the steps of carrying out a first treatment on the surface of the The feed of water was stopped during the irradiation period and the recovery was performed 0.5h after the irradiation. And (3) applying the corresponding repair membrane cloth in the groups (3) to (8) for 15 minutes from the experiment day 4. The skin wrinkles on the back of the mice are scored by a macro-scale method for 7 days and 14 daysScoring is shown in table 2 below.

TABLE 2 evaluation of skin wrinkles on the back of mice

Level of	Scoring criteria
		0	Normal skin fine texture
1	Skin is fine and scratched
		2	Small amount of shallow wrinkles appear
3	More shallow wrinkles appear
		4	Rough skin with deep wrinkles
5	Has more rough wrinkles
		6	Rough wrinkles, local skin lesions

The scored mice were cleaned with pure water by dipping cosmetic cotton, and then were subjected to intraperitoneal injection of 80mg/kg of 1% sodium pentobarbital, anesthetized, sacrificed, the central skin of the back was separated, subcutaneous tissue and adhered fat were cut off, and then soaked in 4 ℃ precooled PBS to remove blood, the surface liquid was sucked by filter paper, and the skin moisture content was measured by the current day drying method. Firstly, weighing the skin mass of the back before partial drying by an electronic balance, marking and recording, drying in a 60 ℃ oven, weighing the skin mass of the back after drying by the same method, and calculating the skin moisture percentage.

Skin moisture content (%) = (mass before drying-mass after drying)/mass before drying×100

The SPSS21.0 and GraphPad Prism software 8.1.8.1 statistical analysis software treatments were used.

Results: the back of the normal group mice has fine and smooth skin texture and is smooth and full in appearance of the back exposed skin in the last day; the skin wrinkles of the mice in the model group are gradually increased along with the increase of the accumulated irradiation time, which indicates that the wrinkles are gradually changed from less to more, from shallow to deep, and the individual mice finally appear with local injury; compared with the model group, the comparative example 1 and the comparative example 2, the skin appearance of the mice in the four groups of examples is improved to different degrees, the skin is only slightly wrinkled, the epidermis is basically not thickened, no obvious pigment is seen, the appearance is closer to that of the normal group, and the scoring result is shown in figure 4.

Exposure of the skin to uv light affects the skin barrier function and moisture in the deep dermis is lost through evaporation of the epidermis, resulting in a decrease in skin moisture content. The skin moisture content of the mice in each group is shown in fig. 5, and compared with the normal group, the skin moisture content of the mice in the model group is extremely obviously reduced, which indicates that the barrier function of the skin stratum corneum of the mice is seriously damaged after ultraviolet irradiation, so that the skin moisture on the back is greatly lost; compared with the model group, the comparative example 1 and the comparative example 2, the moisture content of the skin of the mice in the example group is obviously increased (P < 0.05), which indicates that the repair patch containing the recombinant human fibronectin can effectively inhibit the moisture loss of the skin of the back of the mice in the ultraviolet irradiation process, has the protection function on the skin barrier of the mice, and the moisture content of the skin of the mice in the example 4 group is obviously increased, which indicates that the moisture-keeping and water-supplementing effects of the high-content recombinant human fibronectin on the skin are better.

The above examples only show embodiments of the present invention, and the description thereof is more specific and detailed, but should not be construed as limiting the scope of the invention, but all technical solutions obtained by equivalent substitution or equivalent transformation shall fall within the scope of the invention.

Claims

1. The composition containing recombinant human fibronectin with the repairing effect is characterized by comprising the following components in parts by weight: 5-20 g/100g of recombinant human fibronectin, 0.3-1.0 g/100g of hyaluronic acid mixture, 0.8-1.2 g/100g of oat glucan, 0.8-1.2 g/100g of aloe extract, 0.3-0.8 g/100g of echinacea extract, 0.8-1.2 g/100g of dendrobium stem extract, 0.3-0.8 g/100g of Shu Minjia and the balance of water; wherein the amino acid sequence of the recombinant human fibronectin is shown as SEQ ID No. 1.

2. The composition of claim 1, wherein the recombinant human fibronectin has a molecular weight of 117.7KD.

3. The composition with restoration effect according to claim 1, wherein the hyaluronic acid mixture is formed by mixing large-molecular hyaluronic acid and small-molecular hyaluronic acid in a weight ratio of 3-5:5-7.

4. The composition of claim 3, wherein the macromolecular hyaluronic acid is a linear macromolecular polysaccharide consisting of disaccharide units (glucuronic acid-N-ethionine glucosamine) and having a molecular weight of 1800kDa.

5. The composition of claim 3, wherein the small molecule hyaluronic acid is glucuronic acid and has a molecular weight of 400-1000 kDa.

6. The composition with restoration efficacy containing recombinant human fibronectin according to claim 1, further comprising the following components in parts by weight: 0.01 to 0.02g/100g of metal ion chelating agent, 5 to 10g/100g of polyol, 2 to 3g/100g of protein stabilizer and 0.03 to 0.05g/100g of thickener.

7. A method of preparing a composition having a restorative effect comprising recombinant human fibronectin according to claim 1, comprising the steps of:

8. The method for producing a composition having a reparative effect, comprising recombinant human fibronectin according to claim 7, wherein the certain temperature is 50 ℃ or lower.

9. The method for preparing a composition having a restoration efficacy comprising recombinant human fibronectin according to claim 7, wherein the number of homogenizing revolutions of the first and second homogenizing mixing treatments is 2000 to 3000rpm; the number of homogenizing revolutions of the third homogenizing mixing treatment is 1000-1500 rpm.

10. Use of a composition comprising recombinant human fibronectin having a repair efficacy, characterized in that the composition is a composition comprising recombinant human fibronectin having a repair efficacy according to any one of claims 1 to 6 or a composition obtained by the preparation method according to any one of claims 7 to 9.