阅读说明：本技术 促进皮肤创伤愈合的医药组成物及其用途 (Pharmaceutical composition for promoting healing of skin wound and application thereof ) 是由 王泰来 吴智谋 于 2019-03-06 设计创作，主要内容包括：本发明公开一种促进皮肤创伤愈合的医药组成物及其用途。提取鱼类受精卵中复合性成分,由其所具有消除自由基的抗氧化力、抑制细胞酪氨酸酶活性及促进皮肤纤维母细胞分泌大量胶原蛋白的功能促进皮肤美白、抗老化及伤口修复愈合,以达成皮肤全方位修护,作为化妆品原料及伤口敷料的用途。(The invention discloses a medical composition for promoting skin wound healing and application thereof. The compound components in fish fertilized eggs are extracted, and the functions of eliminating the antioxidant capacity of free radicals, inhibiting the activity of cell tyrosinase and promoting the secretion of a large amount of collagen by skin fibroblasts promote skin whitening, aging resistance and wound repair and healing so as to achieve the comprehensive repair of the skin and serve as cosmetic raw materials and wound dressings.)

1. A pharmaceutical composition for promoting skin wound healing, which comprises a fish fertilized egg extract obtained by low-temperature water extraction of fish fertilized eggs.

2. The pharmaceutical composition for promoting skin wound healing according to claim 1, wherein the fish is fish of the order Perciformes.

3. The pharmaceutical composition for promoting skin wound healing as recited in claim 1, wherein the fish species comprises fish species of the genus of the subfamily bass family.

4. The pharmaceutical composition for promoting skin wound healing according to claim 1, wherein the fish is selected from the group consisting of: one or a combination of the group consisting of Epinephelus lanceolatus, Epinephelus fusca, Epinephelus lanceolatus, Epinephelus malabaricus, Lateolabrax japonicus, Leopard , Epinephelus lanceolatus, Epinephelus fusca, and Epinephelus septemfasciatus.

5. The pharmaceutical composition for promoting the healing of skin wounds according to claim 1, wherein the skin wounds promoted by the pharmaceutical composition are selected from one of the group consisting of cuts, abrasions, burns, sunburn and combinations thereof.

6. The pharmaceutical composition for promoting healing of skin wound according to claim 1, wherein the concentration of the extract of fish fertilized eggs is 0.01 to 1 wt%.

7. The pharmaceutical composition for promoting healing of skin wounds of claim 1, further comprising one or more pharmaceutically acceptable carriers.

8. The pharmaceutical composition for promoting healing of skin wound according to claim 1, wherein the fish fertilized egg extract is in the form of an aqueous liquid, a powder, a granule, a gel or a cream.

9. The pharmaceutical composition for promoting healing of skin wounds according to claim 1, wherein the pharmaceutical composition comprises an oral or topical dosage form.

10. Application of fish fertilized egg extract in preparing medicine for promoting skin wound healing is provided.

11. A skin collagen proliferation promoter, which comprises using the extract of fish fertilized egg according to claim 1.

12. A cosmeceutical characterized by comprising using the fish fertilized egg extract according to claim 1.

13. Use of fish fertilized egg extract in preparing cosmetic raw material for inhibiting tyrosinase activity and promoting skin collagen hyperplasia is provided.

14. A wound dressing for promoting healing of a skin wound, comprising applying the fish zygote extract according to claim 1 to a wound.

15. Application of fish fertilized egg extract in preparing wound dressing for promoting skin wound healing is provided.

16. An antioxidant comprising the use of the fish zygote extract according to claim 1, which has a concentration of 0.21wt% and has an ability to scavenge 50% of free radicals.

Technical Field

The invention relates to a composition for healing skin wounds, in particular to a medical composition for promoting wound healing and reducing wound infection by using a fish fertilized egg extract and application thereof.

Background

The tissue wound healing (wound healing) process can be divided into three phases, physiologically defined: inflammation, proliferation and maturation, in which longer inflammation increases the chance of infection and delays wound healing. Furthermore, the recovery of wounds is divided into the hyperplasia repair of epidermis and dermis, and the outer epidermis usually heals faster, while the inner dermis takes longer time to complete the tissue remodeling and recover to the original shape.

The Asia breeding fishery is quite advanced, and the technology of breeding and domesticating wild fish species as parent fishes for breeding is unique in the whole world. The fertilized eggs produced in the breeding period of the breeding fish are hatched and released according to the market breeding demand, so that the phenomenon of excess of the fertilized eggs of the breed can be caused when the wind direction of the breed is changed in a slack season or released, and the breed can only be abandoned but is pity. Since the fertilized egg has the ability to develop into a complete individual, it also represents that the fertilized egg is rich in all the various factors that may be closely related to cell proliferation and differentiation, such as growth factors, cytokines, and the like. If the fertilized eggs can be further extracted and utilized, the fertilized eggs can be expected to be applied to wound nursing and maintenance, so that the fertilized eggs are fully used, and the added value of the aquaculture fishery can be increased.

Disclosure of Invention

The invention provides a pharmaceutical composition for promoting skin wound healing, which can promote skin wound healing and can improve the added value of aquaculture fishery.

In order to achieve the purpose, the invention adopts the technical scheme that:

a pharmaceutical composition for promoting skin wound healing comprises fish fertilized egg extract obtained by low-temperature water extraction of fish fertilized eggs.

Further, the fish is fish belonging to the order Perciformes.

Further, the fish species include those of the genera of the subfamily of bass.

Further, the fish is selected from: one or a combination of the group consisting of Epinephelus lanceolatus, Epinephelus fusca, Epinephelus lanceolatus, Epinephelus malabaricus, Lateolabrax japonicus, Leopard , Epinephelus lanceolatus, Epinephelus fusca, and Epinephelus septemfasciatus.

Further, the pharmaceutical composition promotes skin wounds selected from the group consisting of cuts, abrasions, burns, sunburn and combinations thereof.

Further, the concentration of the fish fertilized egg extract is 0.01-1 wt%.

Further, it comprises one or more pharmaceutically acceptable carriers.

Further, the extract of fish fertilized eggs is in the form of water liquid, powder, granule, gel or cream.

Further, the pharmaceutical composition includes oral or external dosage forms.

The second purpose of the invention is to provide the application of the fish fertilized egg extract in preparing the medicine for promoting the healing of skin wounds.

The third object of the present invention is to provide a skin collagen proliferation promoter comprising the extract of fish fertilized eggs.

The fourth object of the present invention is to provide a cosmeceutical comprising the use of the fish fertilized egg extract.

The fifth purpose of the invention is to provide the application of the fish fertilized egg extract in preparing cosmetic raw materials for inhibiting the activity of tyrosinase and promoting the skin collagen hyperplasia.

It is a sixth object of the present invention to provide a wound dressing for promoting the healing of skin wounds, which comprises applying the fish zygote extract to wounds.

The seventh purpose of the invention is to provide the application of the fish fertilized egg extract in preparing the wound dressing for promoting the healing of skin wound.

An eighth object of the present invention is to provide an antioxidant comprising the use of the fish fertilized egg extract having a concentration of 0.21wt% and an ability to scavenge 50% of free radicals.

The invention has the beneficial effects that:

1. the fish fertilized egg extract extracted by the invention is used on the affected part of skin wound, can promote wound healing and greatly shorten the inflammatory period; if the wound is damaged to the dermis layer, the papilla structure of the dermis layer can be remodeled, and the epidermis layer and the dermis layer of the wound are better.

2. Experiments prove that the fish fertilized egg extract has the functions of eliminating the antioxidant capacity of free radicals, inhibiting the activity of cell tyrosinase and promoting skin fibroblasts to secrete a large amount of collagen, so that the fish fertilized egg extract can be further applied to the fields of cosmetics, care products, wound dressings of medical devices and the like, not only achieves the effects of whitening and anti-aging, but also can accelerate the healing of skin wounds (such as medical and beauty minimally invasive operations, burns, scalds, cuts, abrasions or sunburns), and has a wide application range.

Drawings

FIG. 1 is a graph showing the results of the viability of a fish zygote extract in the cytotoxicity test according to the example of the present invention.

FIG. 2 is a graph showing the effect of treating human fibroblasts with a fish fertilized egg extract to promote collagen proliferation in accordance with an embodiment of the present invention.

FIG. 3 is an image of the healing of a wound of rat skin after burn and scald treatment with a fish fertilized egg extract according to an embodiment of the present invention.

FIG. 4 is a graph showing the effect of promoting wound healing after the treatment of the burn and scald wound on the rat skin with the extract of fish fertilized egg according to the embodiment of the present invention.

FIG. 5 is a section and a staining chart of skin tissue after a burn wound of rat skin is treated with a fish fertilized egg extract according to an embodiment of the present invention.

Detailed Description

To further understand the technical solution of the present invention, the following preferred embodiments are specifically illustrated in the following drawings, and the following detailed description is made in order to achieve the above objects and effects:

the present invention provides a pharmaceutical composition for promoting skin wound healing, wherein the pharmaceutical composition comprises fish fertilized egg extract. The extract of fish fertilized egg is obtained by conventional low-temperature water extraction method, which comprises homogenizing fertilized egg at low temperature (10-18 deg.C) with ultrasonic wave or high-speed homogenizer to release protein or peptide molecules in fertilized egg into water solution, filtering with multi-stage impurities (1 mm-0.1 um pore size filter/membrane) and deodorizing (1g active carbon/1L extractive solution).

It is understood by those skilled in the art that the extract of fertilized fish egg is a kind of protein summation concept, i.e. all the possible proteins and peptides related to cell proliferation and differentiation are retained in the fertilized fish egg, because many unknown Growth factors (Growth factors) or Cytokines (Cytokines) may be contained therein. However, before the sequencing of the sequence of the gene of the strain of the bass subfamily of the bass family has not been completed in the current global gene bank, the unknown growth factors or cytokines cannot be isolated and defined (no standard products are available), so the extract of the fertilized fish egg can be defined only by the concept of protein summation, and the most beneficial proteins and peptide molecules for cell proliferation and differentiation are retained as far as possible, which is expected to be applied to the fields of anti-aging cosmetic products and medical products.

Preferably, the fish is fish of the order Perciformes. Preferably, the fish species include those of the genera of the subfamily of the family Sermonidae. Preferably, the fish is selected from: one or a combination of the group consisting of Epinephelus lanceolatus, Epinephelus fusca, Epinephelus lanceolatus, Epinephelus malabaricus, Lateolabrax japonicus, Leopard , Epinephelus lanceolatus, Epinephelus fusca, and Epinephelus septemfasciatus. The fish fertilized egg may be a fertilized egg produced by crossing any two of the above-mentioned varieties, and may have excellent inheritance in both parents. The fish is captured and domesticated, and then a large number of fish seeds can be bred artificially, and the number of obtained fertilized eggs can be quantized to produce extract for market demand because of the large breeding, so that the fish has high industrial utilization value.

In the present embodiment, the extracts obtained by mixing fertilized eggs of five kinds of fishes such as epinephelus lanceolatus, epinephelus palmaris, epinephelus carinatus, and malabara malabaricus in equal proportions and extracting the mixed fertilized eggs are exemplified, but the present invention is not limited to the case where fertilized eggs of fishes are obtained from a mixture of multiple species, and fertilized eggs of a single species may be used for extraction. Those skilled in the art can understand that the extract of fertilized eggs of the same fish species has the same or similar efficacy within the range that can be presumed.

In the embodiment of the present invention, the concentration of the extract of fish fertilized eggs is substantially 0.01 to 1 wt%. The concentration range is defined such that theoretically, the higher the concentration, the better the effect should be exhibited, but the safety of skin application should be considered, so that the upper limit is 1wt% after safety evaluation of skin patch. Preferably, the extract of fish fertilized egg is in the form of aqueous liquid or gel; or freeze drying to obtain powder or granule; or emulsified into cream to meet the requirements of different dosage forms. Of course, the pharmaceutical composition for promoting the healing of skin wounds of the present invention may further comprise one or more pharmaceutically acceptable carriers, so that the dosage form is more diversified. Preferably, the pharmaceutical composition comprises an oral or external preparation, and since fertilized eggs of fish which are conventionally edible are also identified as edible materials, it can be used in oral preparations, such as oral endoconch. In the present embodiment, the external preparation in an aqueous liquid form (conventional essence preparation form) is used as an example for performing various tests.

The invention also provides application of the fish fertilized egg extract in preparing a medicine for promoting skin wound healing.

The invention further provides a medicinal cosmetic, which uses the medicinal composition for promoting skin wound healing and has the effects of accelerating skin injury repair, inhibiting tyrosinase activity and promoting skin collagen proliferation.

The invention also provides application of the fish fertilized egg extract in preparing cosmetic raw materials for inhibiting the activity of tyrosinase and promoting skin collagen proliferation.

The invention also provides a wound dressing for promoting the healing of skin wounds, which comprises the medicinal composition for promoting the healing of skin wounds and is applied to the wounds.

The invention further provides application of the fish fertilized egg extract in preparing a wound dressing for promoting skin wound healing.

The invention further provides an antioxidant, which comprises the fish fertilized egg extract, and the fish fertilized egg extract has the capacity of clearing 50% of free radicals when the concentration is 0.21 wt%.

The embodiments of the invention are illustrated as follows:

first, cytotoxicity test

Referring to fig. 1, a graph of survival rate results of a fish zygote extract on cytotoxicity test is shown in the example of the present invention. The effect of fish zygote extract on the growth of human skin fibroblasts (Hs68) was analyzed by cell viability assay (MTT assay) as understood by one of ordinary skill in the art, and observed after 24 hours of culture with different concentrations of the aforementioned fish zygote extract (0.01 wt%, 0.05wt%, 0.1 wt%, and 0.5 wt%). Wherein the total protein concentration in terms of absorbance at 280nm measured at 0.01 wt%, 0.05wt%, 0.1 wt% and 0.5 wt% is about 0.03 mg/ml, 0.15 mg/ml, 0.3 mg/ml and 1.5 mg/ml, respectively. Microscopic photography results of cell types of each group show that when the cell types reach high concentration of 0.5%, the cell appearance has no obvious change and no obvious damage, no adverse effect is caused on the growth of the cell, and the calculated survival rate of the Hs68 cell of each group is more than 90%, which shows that the fish fertilized egg extract has no toxic effect on Hs68 cells.

Second, radical scavenging ability test

In the embodiment of the invention, DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine; 1, 1-diphenyl-2-piperidinylhydrazyl) reagent which is commonly used for detecting antioxidant capacity is used for detection, and the result of triple-test shows that the free radical inhibition rate (IC) of the fish fertilized egg extract is 50% concentration (IC)₅₀) 2.11 plus or minus 0.31 mg/ml, and the conversion weight percentage concentration is about 0.21 percent, namely the capacity of eliminating 50 percent of free radicals. Therefore, the fish fertilized egg extract has the antioxidant capacity of eliminating free radicals, and can further assist the skin to resist the invasion of environmental free radicals and delay the skin aging.

Tyrosinase activity inhibition assay

In the embodiment of the invention, a test is carried out by using a mouse melanoma cell line (B16-F10; BCRC 60031) and divided into a control group (untreated) and an experimental group, wherein 0.0125wt% of the fish fertilized egg extract and cells are treated and cultured for 72 hours in the experimental group, and the cells are collected. Cells were lysed in 20 mM Tris-0.1% Triton X-100 and centrifuged, and a portion of the supernatant was first subjected to quantitative protein analysis. Adding the supernatant into L-DOPA (L-3, 4-dihydroxyphenylnine) solution, reacting at 37 deg.C for 10 min, and measuring the absorbance at 475 nm. The results obtained by conversion were as follows: the tyrosinase activity of the control group was regarded as 100%, and the tyrosinase activity in the experimental group was only 76.93%, in other words, 0.0125wt% of the extract of the aforementioned fish fertilized egg had a tyrosinase activity inhibition rate of 23.07%. The above tests prove that the fish fertilized egg extract has the effect of inhibiting tyrosinase activity, further inhibiting skin melanin generation and achieving the effect of whitening skin.

Fourth, collagen proliferation promoting test

Referring to fig. 2, the effect of promoting collagen proliferation after treating human fibroblast (Hs68) with fish fertilized egg extract according to the present invention is shown. The detection and analysis of the present invention were performed by using a conventional total collagen Assay kit (TotalCollagen Assay kit, BioVision). In the test, human fibroblasts are taken as a subject to be added with the fish fertilized egg extract for culture, and the concentration points are respectively as follows: controls (0%), 0.01%, 0.05%, 0.1% and 0.5%, each run was performed in triplicate. After 24 hours of culture, the cells were harvested, lysed with 20 mM Tris-0.1% Triton X-100, centrifuged, and the protein solution collected for assay. The absorbance at 560nm was measured individually, and it was found that 0.01%, 0.05%, 0.1%, 0.5% of the fish fertilized egg extract had collagen proliferation rates of about 24%, 48.2%, 88.2% and 126.4%, respectively, and P <0.05 (see fig. 2) in this order, respectively, compared to the control group, and it was found that there was a significant difference from the control group. Although the present embodiment is tested at four concentration points of 0.01%, 0.05%, 0.1%, 0.5%, etc., and it is reasonably speculated according to the linear rule of the data result that a better collagen proliferation rate can be expected as the concentration increases to above 0.5%, so one of ordinary skill would understand that the concentration range is not the limitation of the present invention to achieve the effect of promoting collagen proliferation. The conventional background knowledge recognizes that collagen, which is a main component of the skin extracellular matrix, maintains the elasticity of the skin, and the loss and aging of collagen lose the elasticity of the skin to cause wrinkles. Therefore, the test results show that the collagen hyperplasia in the skin is promoted, the effects of skin compaction, elasticity recovery and fine line smoothening can be further achieved, and the anti-aging effect of the skin is realized.

Fifth, burn and scald wound healing promotion test

The experiment was divided into three groups: in the control group (treated with physiological saline for injection), the test group a (0.05 wt% of the fish fertilized egg extract) and the test group B (0.05 wt% of the fish fertilized egg extract), 6 rats were each anesthetized and then contacted with the skin for 12 seconds on the back with a 92 ℃ copper bar to create a circular deep second-degree burn and scald wound with a diameter of about 2 centimeters, and a burn and scald pattern was established. Debridement, administration and dressing of the wound were resumed on days 0, 1, 2, 3, 5, 7, 9, 12, 15, 18, 21, and 24 after the wound was opened. The wounds were anesthetized with Isoflurane for 0, 1, 2, 3, 5, 7, 9, 12, 15, 18, 21, 24, 28 days, archived by camera (sticker for correction was attached near the wound), wound was drawn with a slide and scanned into a figure file, and the percentage of wound healing was calculated by analysis with Image J software. Percent wound healing% = [ (wound area on day 0-wound area on day N)/wound area on day 0 ] × 100%.

As shown in FIG. 3, the healing condition of rat skin after burn and scald wound treatment by using fish fertilized egg extract in the embodiment of the present invention is shown in an image. It is evident from the figure that the reduced wound healing occurred on day 2 after the initiation of the wound in test group a and test group B, and indeed the wound healing repair mechanism was initiated 2 to 3 days earlier than the reduced wound healing occurred on day 5 in the control group. Thus, it was confirmed that the aforementioned fish fertilized egg extract has the effects of promoting healing and advancing the burn and scald wounds.

Referring to fig. 4, the effect of promoting wound healing after the treatment of the burn and scald wound of the rat skin with the fish fertilized egg extract in the embodiment of the present invention is shown. It is evident from the figure that the aforementioned test group a and test group B do accelerate the healing of the wound earlier and faster than the control group. Of note, test a group was statistically significantly different (P <0.05) from the control group at days 3, 5, 9, 12, 15, 19 and 21 (indicated as a in the figure). From this result, we speculate that the extract obtained by mixing the fertilized eggs of the fish from multiple strains may have better efficacy than the extract obtained from a single strain.

Referring to FIG. 5, in the embodiment of the present invention, the skin tissue section and staining pattern of the rat skin after the burn wound treatment with the fish zygote extract are shown. In this test, on day 14 after the wound was opened (wound hyperplasia period), the skin of the scalded back part of each group of randomly sacrificed rats was removed, and on day 28 (wound maturation period), the skin of the scalded back part of each group of sacrificed rats was also removed, and directly immersed in 10% (v/v) formalin solution for at least 24 hours, followed by tissue sectioning and staining (H & E stain). The figures show that there was no significant difference in the appearance of the wound including scab formation (see marked places in the figure), neoepithelium (see marked places in the figure), multinucleated leukocyte infiltration, fibroblast infiltration, new blood vessels and collagen at day 14 for each group of wounds, observed under photomicrographs (40x, 200 x). On day 28, fibroblast proliferation was more pronounced in groups a and B (p <0.05), compared to the control group, indicating that a large amount of collagen was secreted, promoting neogenesis repair of epidermal and dermal skin (see points & points in the figure). It should be noted that, compared with the other two groups, the group A in the above test can see that the new epidermal tissue extends downwards to the dermis layer, and the original dermal papilla (dermal papilla) structure is reconstructed (see the arrow in the figure), so that the connection surface between the epidermis and the dermis is enlarged, which is beneficial for the firm connection between the epidermis and the dermis, and the epidermis is convenient to obtain nutrition from the blood vessels of the dermis, so that the new epidermal tissue structure is more completely recovered.

The results of the animal burn and scald healing test show that the deep second degree burn and scald, the injured epidermis and the dermis are quite serious skin wounds, and the invention proves that the invention has obvious effect on promoting the healing and the repair of the serious skin wounds. Therefore, the inventor believes that the fish zygote extract has the same effect of promoting wound healing and repair in light skin wounds (such as surgery, laser minimally invasive surgery, light burns, cuts, abrasions or sunburn) which generally only damage the epidermis layer, as would be understood by a person of ordinary skill in the art to which the invention pertains.

The combination of the above test results proves that the fish fertilized egg extract has the obvious effects of oxidation resistance, melanin generation inhibition, skin collagen hyperplasia promotion, skin wound healing promotion and the like, so that the fish fertilized egg extract has the potential of being applied to the dressing development of medical products, cosmeceuticals and medical devices, and creates a new win-win situation for fishery and consumers.

While the operation, use and efficacy of the present invention will be fully understood from the foregoing description of the preferred embodiments, it is to be understood that the present invention is not limited to the disclosed embodiments, but is intended to cover various modifications, equivalents and alternatives falling within the spirit and scope of the invention as defined by the appended claims.