KR102144405B1 - Composition for promoting formation of new blood vessels and method for producing the same - Google Patents

Abstract

The present invention relates to a composition for promoting angiogenesis and a method for producing the same, comprising an active ingredient comprising a chicken foot-derived collagen low-molecular peptide and a yellowtail fermented extract in a weight ratio of 1:1, and comprising the composition for promoting angiogenesis The pharmaceutical composition is effective in the generation of new blood vessels and the healing of skin wounds in mammals, especially companion animals such as dogs and cats, by oral administration.

Description

Composition for promoting formation of new blood vessels and method for producing the same}

The present invention relates to a composition for promoting neovascularization and a method for producing the same, and specifically, to a composition effective for promoting the healing of skin wounds and the production of angiogenesis, and a method for producing the same.

New blood vessels play an important role in a wide range of pathophysiological processes such as wound healing, menstrual restoration, hematopoiesis, and tumor growth. Wound healing is generally used in inflammation, proliferation and It is a complex process that involves three stages of reformation. The first stage involves clotting to achieve hemostasis, replenishment of neutrophils to destroy bacteria and necrotic tissue, followed by replenishment of macrophages, and during the second stage, angiogenesis occurs and , At this time, endothelial cells move to the wound area, and at the same time, fibroblasts move to the wound area, helping to produce granulation tissue. The formation of granulation tissue allows re-epithelialization to occur. During the final stage, the level of collagen production and destruction is equalized, and the wound healed is slowly altered to achieve maximum strength.

Collagen is a fibrous structural protein of animal origin, accounting for 30% of the total protein of vertebrates and invertebrates, forming a triple helical structure, and plays a role in forming most of the skin, bones, teeth, and organic substances. Collagen fills the intercellular matrix of the dermal layer and is closely related to skin conditions such as wrinkle formation and elasticity reduction due to aging, and has been used in various fields in pharmaceuticals, cosmetics, and foods. Recently, anti-skin aging and skin elasticity improvement , And arthritis prevention.

As an example of the prior art in relation to the collagen, Patent Document 1 discloses a method for producing a fermented collagen peptide derived from fish, which is a raw material for food, cosmetic or pharmaceutical compositions. Patent Document 2 is a food composition for improving skin condition further containing at least one selected from the group consisting of elastin protein, hyaluronic acid and vitamin C in the collagen peptide, wherein the collagen peptide contains collagen tripeptide based on the total weight of the collagen peptide. To 95% by weight, and the improvement of the skin condition is obtained through at least one selected from promoting collagen synthesis in the skin, suppressing skin photoaging, increasing skin moisturization, improving the biosurvival rate of collagen peptides in the skin, and increasing cellular damage recovery. In addition, Patent Document 3 discloses a cosmetic composition comprising a natural liposome containing collagen and hyaluronic acid, which provides elasticity to the skin, improves wrinkles, and helps in moisturizing enhancement, Patent Document 4 discloses a pharmaceutical composition for preventing and treating menopausal nail disease containing rice and soybean enzyme food and collagen as an active ingredient.

The prior art is a technology that improves skin conditions such as wrinkle formation and elasticity reduction due to skin aging by using collagen or low molecular collagen as an active ingredient, and in relation to the generation of new blood vessels in the process of skin wound healing, collagen There is no suggestion for the applicability, and it has nothing to do with it.

The applicant of the present invention confirmed that the collagen low-molecular peptide and yellow pollen fermentation extract promoted the formation of new blood vessels related to wound healing of the skin, and completed the present invention.

KR 10-1254403 B KR 10-1393403 B KR 10-1842752 B KR 10-1850770 B

The object to be solved in the present invention is to provide a composition for promoting neovascularization and a method for producing the same, and in detail, for the purpose of providing a collagen-derived composition and a method for producing the same, which is effective in promoting the healing of skin wounds and neovascularization. Is to do.

More specifically, to provide a composition for promoting angiogenesis and a method for producing the same, which is effective in promoting angiogenesis, which plays an important role in the healing of skin wounds, by repeated oral administration according to the optimization of a composition containing low molecular collagen derived from chicken feet. It is for the purpose.

As a solution to the problem for achieving the object of the present invention, the composition for promoting angiogenesis is composed of an active ingredient comprising a chicken foot-derived collagen low molecular weight peptide and a yellowtail fermentation extract in a weight ratio of 1: 1.

Another aspect as a solution to the problem for achieving the object of the present invention is characterized by a composition for promoting neovascularization containing an active ingredient comprising a collagen low-molecular peptide obtained by decomposing chicken feet with enzymes and a fermentation extract obtained by fermenting yellow pollack. .

Another aspect of the present invention is a mammal comprising a composition for promoting angiogenesis comprising an active ingredient composed of a collagen low-molecular peptide derived from chicken feet and a yellow pollen fermentation extract in a weight ratio of 1: 1, especially for companion animals such as dogs and cats. It is characterized by a pharmaceutical composition for healing skin wounds.

Another aspect of the present invention is characterized by a nutrient composition comprising a composition for promoting angiogenesis, comprising an active ingredient composed of a chicken foot-derived collagen low-molecular peptide and a yellowtail fermented extract in a weight ratio of 1: 1.

And as a means of solving the problem for achieving another object of the present invention, a method of preparing a composition for promoting angiogenesis is a). Selected chicken feet are sterilized with high-pressure steam under conditions of 100 to 125°C and 1 to 2.5 atm, and then the sterilized chicken feet are decomposed with calcium hydrogen carbonate [Ca(HCO ₃ ) ₂ ], and then the fat is removed and concentrated. The first step of obtaining an acid decomposition product, b). A second step of fermenting the concentrated acid decomposition product of the first step with lactic acid bacteria to obtain a collagen low molecular weight peptide from chicken feet, c). Selected yellow pollack is sterilized with high-pressure steam under conditions of 100 to 125°C and 1 to 2.5 atm, and then the sterilized yellow pollen is fermented with lactic acid bacteria to obtain a yellow pollen fermented extract and d). It comprises a fourth step of preparing a composition for promoting neovascularization by combining the collagen low-molecular peptide obtained in the second step and the yellowtail fermentation extract obtained in the third step in a weight ratio of 1:1.

The composition for promoting angiogenesis according to the present invention is effective in promoting the generation of angiogenesis, and the composition for promoting angiogenesis is effective in healing skin wounds by repeated oral administration according to optimization.

In particular, the composition for promoting angiogenesis effective in healing skin wounds of the present invention has characteristics that can more advantageously cope with the healing of skin wounds of pets to expert groups such as pet enthusiasts and veterinarians who raise companion animals such as dogs and cats. There is this.

1 is a graph showing the amino acid composition and content by HPLC analysis of the composition for promoting angiogenesis of the present invention
2 to 6 are wound size images for the skin wound healing effect of <Test Example 1>
7 is a comparison chart of the skin wound healing effect of <Test Example 1>
8 is a blood vessel area image for the angiogenesis effect of <Test Example 2>
9 is a comparison chart of blood vessel area for the angiogenesis effect of <Test Example 2>
10 is a comparison chart of angiogenesis factor protein expression of <Test Example 2>
11 is a comparison chart of blood properties for the blood test (CBC) of <Test Example 3>

Hereinafter, the present invention will be described in more detail through specific details for the practice of the present invention, examples (production examples) and test examples, but the present invention is not limited by the following description.

When the composition for promoting angiogenesis according to the present invention is described in more detail, as an effective ingredient for promoting angiogenesis in the present invention, a collagen low molecular weight peptide derived from chicken feet and a yellow pollen fermentation extract are composed in a weight ratio of 1: 1. .

The collagen low-molecular peptide derived from chicken feet according to the present invention decomposes chicken feet with acid, specifically, calcium hydrogen carbonate [Ca(HCO ₃ ) ₂ ], separates and removes fat from the decomposition product, and ferment the concentrated decomposition product with lactic acid bacteria. It is obtained, and yellow pollen fermented extract is obtained by fermenting yellow pollack with lactic acid bacteria.

As for the yellow pollack according to the present invention, it is preferable to select commercially available yellow pollack dried pollock in a place where there is a large daily temperature difference in the winter, freezing and melting repeatedly 20 or more times in a place with a large daily temperature difference. After freezing at ∼-15℃ for 6 to 12 hours or keeping at low temperature for 6 to 12 hours, thawing while maintaining at -5 ∼ 10℃ for 3 to 6 hours, and then freezing at -20 ∼ -10℃ for 6 to 12 hours at low temperature. After holding, thaw while maintaining at -5 to 15°C for 3 to 6 hours, and thirdly, frozen at -15 to -10°C for 6 to 12 hours at low temperature, then 3 to 6 at -5 to 20°C After defrosting while maintaining time, finally, dried and aged yellow pollen prepared indoors to a moisture content of 5 to 10% may be selected, and the freezing and thawing processes, the first to third processes, are sequentially repeated twice to Can be manufactured.

In the composition for promoting angiogenesis according to the present invention, batamine C may be additionally formed in order to increase the absorption rate of the low molecular weight collagen peptide in the body in addition to the active ingredient.

The pharmaceutical composition for healing skin wounds of mammals according to the present invention includes the composition for promoting neovascularization, and promotes the creation of new blood vessels by oral administration of companion animals such as mammals, especially dogs and cats. It is effective in healing.

The pharmaceutical composition for promoting angiogenesis according to the present invention is preferably administered at a dose of 10 ml/Kg 1 to several times a day based on adult dogs.

The composition for promoting angiogenesis according to the present invention and a pharmaceutical composition comprising the same are mixed with pharmaceutically acceptable carriers, excipients, diluents, etc. to be prepared in formulations such as tablets, suspensions, granules, emulsions, capsules, syrups, etc. to be administered orally. Can be, but is not limited thereto.

In addition, the nutrient composition comprising the composition for promoting neovascularization according to the present invention can also be prepared as a health functional food by mixing a food-acceptable carrier, excipient, and diluent into tablets, suspensions, granules, emulsions, capsules, and syrups. have.

And when explaining in more detail the method for preparing the composition for promoting angiogenesis according to the present invention,

A) according to the present invention. The first step is a process of acid-decomposing selected chicken feet to obtain concentrated acid decomposition products. The selected chicken feet are sterilized with high-pressure steam under conditions of 100 to 125°C and 1 to 2.5 atm, and then calcium hydrogen carbonate [Ca( After decomposition with HCO ₃ ) ₂ ], fat is separated and removed from the decomposition product, and concentrated to obtain a concentrated acid decomposition product.

The process of obtaining the concentrated acid decomposition product is decomposed by adding calcium hydrogen carbonate [Ca(HCO ₃ ) ₂ ] to the sterilized chicken feet while bathing at 60 to 100°C with water, and fat in the process of bathing for 2 to 3 days. Is removed from time to time to obtain a concentrated acid decomposition product.

B) according to the present invention. The second step is a process of fermenting the concentrated acid decomposition product with lactic acid bacteria to obtain a collagen low molecular weight peptide, and the lactic acid bacteria selected for the fermentation are Bacillus Subtillus, Saccaromyces Cerevisiae, Two or more mixed lactic acid strains selected from Lactobacillus Acidophillus, Lactobacillus Brevis, and Lactobacillus Plantarum are selected, and at a temperature of 38 to 42°C for 2 to 3 days After fermentation, a collagen low molecular weight peptide is obtained from the fermented product.

The collagen low molecular weight peptide obtained in the second step has a molecular weight of 1,500 Da. It is the following low-molecular collagen, and its high absorption rate in the body is suitable as an active ingredient of the composition for promoting the generation of angiogenesis according to the present invention.

C) according to the present invention. The third step is the process of obtaining yellow pollack fermentation extract. Selected yellow pollack is sterilized with high-pressure steam under conditions of 100 to 125°C and 1 to 2.5 atmospheres, and then the sterilized yellow pollen is fermented with lactic acid bacteria, and then yellow pollack is fermented from the fermented product. Obtaining the extract, the lactic acid strain selected for fermentation is the same as the lactic acid bacteria fermenting the acid decomposition product in the second step, specifically Bacillus subtilus, Saccharomyces cerevisiae, Lactobacillus acidophilus, lacto Two or more mixed lactic acid strains selected from Bacillus brevis and Lactobacillus plantarum are selected and fermented for 2 to 3 days at a temperature of 38 to 42° C. to obtain a yellowtail fermented extract.

D) according to the present invention. The fourth step is a step of manufacturing a composition containing an active ingredient effective in promoting the generation of angiogenesis, and the collagen low molecular weight peptide obtained in the second step and the yellow pollen fermented extract obtained in the third step are mixed in a weight ratio of 1: 1. It is formulated to prepare a composition for promoting angiogenesis, and vitamin C, which increases absorption in the body of collagen, may be formed if necessary.

In addition, the pharmaceutical composition and nutrient composition comprising the composition for promoting angiogenesis obtained in the fourth step includes a pharmaceutical or food acceptable carrier or excipient, and can be prepared as granules, pills, capsules, etc. for oral administration. I can.

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples.

<Example>

a). 1st step process (preparation of concentrated acid decomposition product from chicken feet)

Prepare selected chicken feet by cleaning commercially purchased chicken feet, removing unnecessary parts such as claws and scales from the chicken feet, and spraying high-pressure hot water to wash them.

Put 15kg of the selected chicken feet into a prepared bowl and heat it to 120℃ and sterilize with steam for 30 minutes under 2 atm. Then, add 1.5 times the amount of drinking water to the bowl and heat it at normal pressure to boil. Add 10 g of calcium hydrogen carbonate [Ca(HCO ₃ ) ₂ ], and make a bath while heating over medium heat. During the bathing process, fat was removed from time to time, and when water was evaporated and reduced, bathing for 3 days while supplementing was performed to obtain concentrated acid decomposition products.

b). 2nd step process (Manufacture of low molecular weight collagen peptide by fermentation of lactic acid bacteria)

To 10 kg of the acid decomposition product obtained above, 20 g of a mixed strain composed of Bacillus subtilis and Saccharomyces cerevisiae was inoculated and fermented for 3 days to prepare a collagen low molecular weight peptide derived from chicken feet.

c). 3rd step process (manufacturing yellowtail fermented extract)

While washing yellow pollack, separate intestines and foreign substances, cut them into a certain size, and spray high-pressure hot water to prepare 10 kg of fresh dried yellow pollack.

The selected yellow pollack was put into a pre-prepared bath pot, heated to 120°C, sterilized with steam for 30 minutes under 2 atmospheres, cooled to room temperature, and then 20 g of a mixed strain composed of Bacillus subtilis and Saccharomyces cerevisiae was added. After inoculation and fermentation at 38° C. for 3 days, the fermented product was filtered to separate and remove foreign substances, and a yellow pollen fermented extract was obtained.

d). The fourth step process (composition manufacturing)

Combining the chicken foot-derived collagen low-molecular weight peptide obtained in the second step and the yellow pollen fermented extract obtained in the third step in a weight ratio of 1:1 to promote the generation of new blood vessels containing an effective ingredient for promoting the generation of angiogenesis A composition for use was prepared.

A graph showing the amino acid composition and content by HPLC analysis of the composition for promoting angiogenesis of the present invention prepared above is shown in [Fig. 1].

<Animal experiment>

The angiogenesis and the healing effect of skin wounds of the composition for promoting angiogenesis of the present invention prepared in the above example were tested as follows.

One). Animal and skin wound models

ICR Mouse (25 ~ 30g) males are purchased through the Hallym Laboratory Animal Research Center, and animals are supplied with feed and water freely, and temperature and light are controlled at a breeding ground (23±2℃, 12/12h light/dark cycle with lights on). at 08:00) from 3 days before the start of the experiment.

In the skin wound model, after depilating the skin on the back under anesthesia, a round wound was made on the back with a sterilized biopsy punch, a silicone ring was placed on the skin incision and fixed to the skin with surgical thread, and then Tegaderm was attached to the wound. Secondary infection was prevented.

2). Method of administering the composition for promoting angiogenesis of the present invention

The composition for promoting angiogenesis of the present invention (10 µl/g) was injected into the stomach by using a mouse zygote as an oral administration method. Administration of the composition was started 14 days before surgery, twice daily (09:00 AM, 18:00 PM), and administered until 10 days after surgery.

<Test Example 1>. Skin wound healing effect

In order to confirm the effect of healing skin wounds on the oral administration of the composition for promoting angiogenesis of the present invention, the experimental animals were treated at 0, 3, 6, 9, and 10 days after surgery with 3% N ₂ O/O ₂ gas. After respiratory anesthesia with isoflurane, the wound was photographed at the same location using a digital camera (Canon, Tokyo, Japan) and shown as [Figs. 2] to [Fig. 6], and the wound size was measured using the Image J software program. And the results are shown in [Fig. 7].

As shown in FIG. 7, the wound size on days 0 to 9 did not show any difference between the control group and the experimental group, but it was confirmed that the wound size was significantly reduced in the group to which the nutrient was administered orally on the 10th day compared to the control group.

<Test Example 2>. Angiogenesis effect

a). Blood vessel area measurement

After the mice were euthanized on the 10th day after surgery, the skin was sampled and fixed with 4% paraformaldehyde (PFA) for 2 hours at room temperature. Then, the tissue was transferred to 20% sucrose in PBS and left at room temperature for 4 hours, and then transferred to 30% sucrose in PBS and stored in refrigerator for 1 day. Freeze the skin tissue with O.C.T., cut it into 10 μm thickness using a cryostat, and place it on SuperFrost Plus slides to dry. After fixing in 4% PFA for 5 minutes, immersion in Haematoxylin and Eosin solution was sequentially dyed to raise the cover glass, and image analysis was performed with an Eclipse upright microscope (Nikon, Melville, NY, USA), and the results were [Fig. 8] and [Fig. 9] ].

b). Expression of angiogenic factor protein in the wound tissue of the skin

Dissolve skin tissue using homogenizer and Branson Sonifier and centrifuge for 40 minutes at 15,000 rpm and 4°C. Quantify using a BCA protein assay kit, and separate proteins using SDS-PAGE. Transfer the protein to the polyvinylidene difluoride membrane, block it in 5% skim milk for 40 minutes, and use the primary antibody as a rabbit anti-vascular endothelial growth factor antibody (1:1000 dilution, Santa Cruz) and rabbit anti-collagen antibody (1:2000 dilution). Thermo) overnight at 4 °C. The next day, the HRP-conjugated secondary antibodies were reacted at room temperature for 4 hours, and then image analysis was performed using the ChemiDoc™ XRS+ System (Bio-Rad, Hercules, CA, USA), and the results are shown in [Fig. 10].

As shown in [Fig. 9] and [Fig. 10], it was confirmed that one group administered orally of the composition for promoting angiogenesis according to the present invention significantly increased vascular area and angiogenesis factor compared to the control group.

<Test Example 3> Blood test (complete blood cell count; CBC)

In order to evaluate the changes in the blood of the mouse, a blood sample was extracted on the 10th day after surgery and a blood test (CBC) was performed, and the results are shown in [Fig.

As shown in Fig. 11, among the CBC items, WBC, MCV, MCH, MCHC, and PLT were not different between groups in the oral administration group of the composition for promoting angiogenesis according to the present invention compared to the control group, but RBC. HGB and HCT were significantly decreased in the oral administration group of nutrients compared to the control group.

From the results of <Test Example 1> to <Test Example 3>, it can be seen that the angiogenesis effect and the healing effect of skin wounds are improved compared to the control group by oral administration of the composition for promoting angiogenesis of the present invention. It can be predicted that the composition for promoting angiogenesis of the present invention can be provided as a pharmaceutical composition and nutritional composition that is effective for healing skin wounds, especially for pet enthusiasts and veterinarians who raise companion animals such as dogs and cats. It can be predicted that it can cope more favorably for the formation of angiogenesis and skin wound healing.

Claims (9)

delete delete delete delete delete delete a). Selected chicken feet are sterilized with high-pressure steam under conditions of 100 to 125°C and 1 to 2.5 atm, and then sterilized chicken feet are added with calcium hydrogen carbonate [Ca(HCO3)2] while bathing at 60 to 100°C with water. The first step of decomposing, removing fat by boiling water for 2 to 3 days, and concentrating to obtain a concentrated acid decomposition product,
b). A second step of fermenting the concentrated acid decomposition product of the first step with lactic acid bacteria at a temperature of 38 to 42° C. for 2 to 3 days to obtain a collagen low molecular weight peptide from chicken feet,
c). The third step of obtaining yellow pollack ferment extract by sterilizing selected yellow pollack with high-pressure steam under conditions of 100 to 125℃ and 1 to 2.5 atm, and then fermenting the sterilized yellow pollack with lactic acid bacteria at a temperature of 38 to 42℃ for 2 to 3 days. fair,
d). The fourth step of preparing a composition effective to promote neovascularization by combining the collagen low molecular weight peptide obtained in the second step and the yellowtail fermentation extract obtained in the third step in a weight ratio of 1: 1, and
e). Including a fifth step of preparing a formulation of any one of granules, pills, tablets, and capsules by blending an excipient in a composition effective to promote neovascularization obtained in the fourth step,
The lactic acid bacteria of the second and third steps are Bacillus Subtillus, Saccaromyces Cerevisiae, Lactobacillus Acidophillus, and Lactobacillus brevis. Brevis) and Lactobacillus Plantarum (Lactobacillus Plantarum) two or more mixed lactic acid strains selected from the method for producing a composition for promoting angiogenesis, characterized in that. delete A pharmaceutical composition for healing skin wounds in mammals, characterized in that it is prepared according to the manufacturing method of claim 7.

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