KR20200089470A - Composition for promotion of skin cell growth comprising sea urchin extract - Google Patents

Abstract

The present invention relates to a composition for proliferating fibroblasts containing a sea urchin roe extract, and to a cosmetic product comprising the same and a drug. Specifically, the composition containing the sea urchin roe extract as an active ingredient of the present invention exhibits excellent fibroblast proliferation efficacy, and thus can be applied as a composition for fibroblast proliferation, a fibroblast culture method using the same, a cosmetic composition for ameliorating wrinkles and promoting skin regeneration, and a pharmaceutical composition for treating burns or wounds.

Description

Composition for promotion of skin cell growth comprising sea urchin extract

The present invention relates to a composition for skin cell proliferation comprising a sea urchin extract and a cosmetic product and a pharmaceutical product containing the same, and the composition comprising the sea urchin extract of the present invention as an active ingredient has excellent fibroblast proliferation efficacy, such as medicine and food. And cosmetic products.

The demand for functional cosmetics is increasing worldwide due to factors such as a surge in interest in beauty and an increase in life expectancy, and the cosmetics market is rapidly growing all over the world, developing into a future-oriented high-tech industry that can create high added value. have. Recently, research on cosmetic materials using natural products has been actively conducted in the cosmetics industry due to the influence of culture that favors naturalism. Currently, research on marine bio-resources as a raw material for functional cosmetics has been actively conducted mainly in EU countries and Japan. Among marine bio resources, whitening, wrinkle removal, anti-acne, anti-oxidation, and anti-cancer effects are known to exist. Therefore, it is considered that it can contribute greatly to the development of related industries, especially functional cosmetics.

Currently, cosmetics using marine natural products are at a relatively basic stage within the cosmetics industry, and commercialized products are also marine natural products with various functions as basic moisturizers, but are less useful in the functional cosmetics industry compared to terrestrial organisms. .

Meanwhile, sea urchins belong to the Phylum Echinodermata and the Class Echinoidea, and are mainly distributed from the coast to deep waters. Among the sea urchins that are distributed in the coasts of the East Sea of Korea, China, and Japan, and are used for food among sea urchins produced in the seas nearby Korea, purple sea urchin (Anthocidaris crassispina), sea urchin (Hemicentrotus pulcherrimus), and pink sea urchin (Pseudocentrotus depressus) ), and the North Sea Urchin (Strongylocentrotus internmedius). Sea urchin contains water, protein, fat, vitamin B group and vitamin C, iron, magnesium and calcium, and more protein than sea cucumber. In the case of sea urchins, the area of edible gonads (egg) is about 20%, and the remaining 80% is composed of sea urchin shells, so the average annual production of sea urchin is about 2,000 tons based on 2,500 tons, which is wasted as waste resources. Degradable shells are mostly left unattended. Furthermore, in recent years, seaweed has been depleted due to overproduction of sea urchins, and a whitening phenomenon has emerged as an environmental problem that deteriorates marine ecosystems.

Accordingly, research on cosmetic compositions using sea urchin is necessary. To date, most of the research on sea urchins is related to the composition, production, and processing of sea urchins, such as Nam (J. Korea Oil Chemists` Soc 3. 33-37 (1986)) and De la Cruz-Garcia et al. (J. Sci Food Agric 80, 1189-1192. (2000) reported the protein, amino acid and fatty acid composition of sea urchin and canned products, and Yoo et al. (Bull. Korean Fish Soc 15, 345-358 (1982)) It has been reported on the spawning and growth of. In addition, the southwestern seawater pollution test using sea urchin (Wui et al., Korean J Environ Biol 10, 92-99 (1992)) and biological evaluation of seawater (Yu and Cho, J Korean Environ Sci Soc 8, 160-164 (1999) )). In particular, the Korean inventor patent 10-1693782, which is the result of the previous study of the present inventor, revealed the antibacterial and antioxidant effects of sea urchin extract, and is not known about the fibroblast proliferation efficacy.

Fibroblasts are cells that make up an important component of animal fibrous connective tissue and are also called fibroblasts. A type of cell that synthesizes extracellular matrix and collagen, which is a stromal for animal tissues, and plays an important role in wound healing. The structure of the skin is divided into three layers: epidermis, dermis and subcutaneous tissue. Of these, the dermis is composed of extracellular matrices such as elastin, collagen and hyaluronic acid, which are important for maintaining water retention, and dermal cells, and fibroblasts synthesize extracellular matrix components in dermal cells. When the proliferation of fibroblasts in the dermis is lowered, the skin's ability to read firmness and elasticity, skin roughness, wrinkles, and the like occurs due to a decrease in the biosynthetic ability of the extracellular matrix. Therefore, by promoting the proliferation of fibroblasts, it is possible to improve the biosynthesis of collagen or hyaluronic acid to prevent wrinkle formation or skin elasticity degradation and improve skin condition. Furthermore, the effect of promoting wound healing due to trauma, burns, and surgery is expected.

The present invention provides a composition for proliferating fibroblasts comprising sea urchin extract as an active ingredient.

In addition, the present invention provides a cosmetic composition for skin regeneration or wrinkle improvement comprising the composition for proliferating fibroblasts.

In addition, the present invention provides a pharmaceutical composition for treating burns or wounds comprising the composition for proliferating fibroblasts.

The present inventors tried to develop various high-functional materials using sea urchin, and as a result, the sea urchin extract has excellent fibroblast proliferation effect, thereby promoting fibroblast proliferation, promoting skin regeneration, wrinkle improvement, as a material for the treatment of burns and wounds. By confirming that it is very useful, the present invention has been completed.

The present invention provides a composition for proliferating fibroblasts, comprising a sea urchin extract as an active ingredient.

The “sea urchin” includes purple sea urchin ( Anthocidaris crassispina ), sea urchin ( Hemicentrotus pulcherrimus ), pink sea urchin ( Pseudocentrotus depressus ) or North sea urchin ( Strongylocentrotus internmedius ), for example, purple urchin ( Anthocidaris crassispina ) or pink urchin ( Anthocidaris crassispina ) ( Pseudocentrotus depressus ).

The "sea urchin egg" is not particularly limited in the form of use, but after being separated from the sea urchin, it may be washed with water or the like as necessary, or may be a dried product appropriately, for example, a lyophilized product. Alternatively, a powder of dried sea urchin may be used.

The "fibroblasts" may be human-derived or mouse-derived cells.

The sea urchin extract of the present invention may be prepared according to a method for preparing a normal extract, and a person skilled in the art may appropriately select an extract manufacturing method in consideration of various conditions such as the state and amount of the sea urchin. .

According to an example of the present invention, the sea urchin extract may be extracted with water, a lower alcohol having 1 to 4 carbon atoms or a mixture thereof, for example, may be methanol, and 50 to 90 v/v% of It may be extracted with an aqueous solution of methanol, an aqueous solution of methanol of 60 to 80 v/v%, and an aqueous solution of methanol of 65 v/v% to 75 v/v%. When an aqueous solution of methanol exceeding 90 v/v% is used as an extraction solvent, or when an aqueous solution of methanol less than 50 v/v% is used, polyphenols, which are active ingredients of sea urchin extract, are not sufficiently extracted, and thus the concentration range is extracted. It is preferred to use a solvent.

According to one embodiment, the extraction temperature of the sea urchin extract can be carried out at 20 to 30 ℃, preferably at room temperature. The extraction time is not particularly limited, but may be 5 minutes to 2 hours, 5 minutes to 1 hour, 10 minutes to 1 hour, 10 minutes to 50 minutes, 10 minutes to 40 minutes, or 10 minutes to 30 minutes. The number of extractions can be repeated once or several times, for example, 2 to 5 times, 2 to 4 times. It can be extracted 2 to 3 times, or 3 to 4 times. According to one embodiment of the present invention, extracting was performed three times at room temperature for about 20 minutes, and extracts were prepared by mixing the extracts repeated three times.

The prepared extract can then be filtered or concentrated or dried to remove the solvent, and all filtered, concentrated and dried. Specifically, the filtration may use a filter paper (filter paper) or a filter or a vacuum filter, and the concentration may be concentrated under reduced pressure using an evaporator. The drying may be performed by, for example, a lyophilization method, but is not limited thereto.

According to an embodiment of the present invention, after adding a solvent to the sea urchin and extracting it three or more times to filter using filter paper, the solvent is removed using a reduced pressure concentrator, additional distilled water is added, and then concentrated again under reduced pressure. To prepare a concentrate. Methanol is removed by the first concentrated under reduced pressure, and methanol remaining in the extract can be completely removed by the second concentrated under reduced pressure.

The extract of the present invention is 5 to 25 parts by weight, 5 to 20 parts by weight, 5 to 15 parts by weight, 5 to 13 parts by weight, 7 to 25 parts by weight, 7 to 20 parts by weight, 7 to 20 parts by weight, based on 100 parts by weight of the solvent To 13 parts by weight, 9 to 25 parts by weight, 9 to 20 parts by weight, 9 to 13 parts by weight, or 10 to 13 parts by weight may be included and extracted.

According to one embodiment, the extract of the present invention may be prepared to contain 200 ml of solvent compared to 20 g of sea urchin eggs.

In the composition for proliferating fibroblasts according to the present invention, the content of the sea urchin extract, which is an active ingredient, can be appropriately adjusted according to the method and purpose of use of the composition for proliferation.

As an example, based on 100 parts by weight of the composition for proliferating fibroblasts of the present invention, the sea urchin extract may be included in 1 to 20 parts by weight, for example, 1 to 15 parts by weight, and 1 to 10 parts by weight.

The composition for proliferating fibroblasts of the present invention may further include a medium for culturing animal cells. A commonly used medium for animal cell culture can be used, and the type is not particularly limited. Specifically, the animal cell culture medium may be one or more selected from the group consisting of RPMI1640, DMEM, MCDB 131, MCDB 153, MDEM, IDEM, MEM, and M199, for example RPMI1640 Can be The medium may include FBS (Fetal Bovine Serum).

According to an example of the present invention, the sea urchin extract may be a dilution obtained by diluting the sea urchin extract with a dilution factor of 10 ^-1 to 10 ^-2 . Specifically, a medium is added to the prepared sea urchin extract stock solution, and diluted to 10 ^-3 to 1, 10 ^-2 to 1, 10 ^-2 to 10 ^-1 , or 10 ^-1 to 1 diluted based on the extract stock solution Can be water When the concentration of the extract is lowered to less than 10 ^-2 dilution, the proliferation effect of the sea urchin extract is insufficient, so it is preferable to use a dilution in the above range.

An example of the present invention provides a method for proliferating fibroblasts, comprising culturing fibroblasts in the composition for proliferating the fibroblasts.

The culture period may be 1 day to 10 days, 1 day to 7 days, 1 day to 5 days, for example, 2 days to 4 days, 2 days to 3 days, 3 days to 4 days, or 72 hours. Incubation temperature is 35℃ to 39℃, 36℃ to 39℃, or 36℃ to 38℃, 36℃ to 37℃, 37℃ to 38℃ Can be

According to one embodiment, the fibroblasts proliferated by the fibroblast proliferation method have an OD value measured by irradiating light (400 nm to 700 nm) in the visible region, the visible region of the fibroblasts proliferated in the culture medium for animal cells It may be 2 to 50 times, for example, 2 to 47 times, 2 to 45 times, 2.5 to 45 times as compared to the OD value measured by irradiating light. The animal cell culture medium is a medium containing no sea urchin extract and can be viewed as a control.

For example, the OD450nm value of the fibroblasts proliferated by the fibroblast proliferation method may be measured as a relative ratio of 102.5% to 145.0% compared to the fibroblasts grown in a culture medium for animal cell culture as a control.

The present invention provides a composition for promoting skin regeneration, comprising a composition for proliferating fibroblasts containing the sea urchin extract as an active ingredient.

The composition for proliferating fibroblasts of the present invention promotes the proliferation of fibroblasts, and as a result, it can exhibit not only skin regeneration, but also wrinkle improvement, skin elasticity improvement, skin aging prevention, skin moisturization improvement, blotch removal, and acne treatment have.

In addition, the present invention provides a cosmetic composition for improving wrinkles, comprising a composition for proliferating fibroblasts containing sea urchin extract as an active ingredient.

The cosmetic composition of the present invention described above can be prepared in any formulation conventionally prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactants- It may be formulated as a containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be prepared in the form of flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

The cosmetic composition may further include a blending component that may be added as an active ingredient, and such components include oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, Sterilizing agents, antioxidants, plant extracts, pH adjusting agents, alcohols, coloring agents, flavoring agents, blood circulation accelerators, cooling agents, limiting agents, purified water and the like.

In addition, the present invention can provide a pharmaceutical composition for treating burns or wounds, including the sea urchin extract as an active ingredient.

In the present invention,'wound' refers to a condition in which a living body is damaged, and tissues forming an internal or external surface of a living body, for example, skin, muscle, nerve tissue, bone, soft tissue, internal organs, or vascular tissues are divided or destroyed. It encompasses the pathological condition. Examples of wounds include, but are not limited to, non-healing traumatic wounds, tissue destruction by irradiation, abrasion, bone mass, laceration, avulsion, and penetrated wound. , Gunshot wound, cuts, burns, frostbite, bruises, skin ulcers, dry skin, keratosis, cracks, bursts, dermatitis, pain caused by dermatosis of the skin, surgical, vascular disease wounds, corneal wounds Diabetes and circulatory defects such as wounds, bedsores, spears, diabetic skin erosion, chronic ulcers, sutures after plastic surgery, spinal injury wounds, gynecological wounds, chemical wounds and acne. Damage to any part may be included.

The active ingredient of the present invention may contain 0.1-100% by weight based on the total weight of the composition, and the composition of the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. have. In addition, additives for the preparation of a solid or liquid may be used in the manufacture of a pharmaceutical composition. The additive for formulation may be either organic or inorganic.

The pharmaceutical composition for the treatment of burns or wounds of the present invention may be prepared in any formulation conventionally prepared in the art (e.g., Remington's Pharmaceutical Science, latest version; Mack Publishing Company, Easton PA), and the form of the preparation is It is not particularly limited, but may preferably be an external preparation. The external preparations of the present invention include sheet, liquid coating, spray, lotion, cream, pape, powder, penetrating pad, spray, gel, pasta, linement, ointment, aerosol, powder, suspension, transdermal A form of a conventional external preparation such as an absorbent may be included.

The pharmaceutically effective amount of the present invention may vary depending on the type of wound, the application site, the number of treatments, the treatment time, the formulation, the patient's condition, and the type of adjuvant. The amount of use is not particularly limited. The amount of the pharmaceutical composition of the present invention may include various administration routes, patient age, gender, weight, patient severity, wound type, application site, number of treatments, treatment time, formulation, patient condition, and type of adjuvant. As determined in light of the relevant factors, the effective amount should not be construed as limiting the scope of the invention in any aspect.

The sea urchin extract of the present invention has an effect of promoting the proliferation of fibroblasts, a composition for proliferating fibroblasts, a method for culturing fibroblasts using the same, a cosmetic composition for improving wrinkles and skin regeneration, a pharmaceutical composition for treating burns and wounds, etc. Can be used as

1 shows a result of measuring the cell proliferation effect according to the concentration of sea urchin extract according to Example 2 to OD450nm.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are only for illustrative purposes of the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Preparation of sea urchin extract

1-1. Preparation of extract materials, samples and devices

Raw sea urchins ( Pseudocentrotus depressus ) and Bora sea urchins ( Anthocidaris crassispina ), which are necessary for the production of sea urchin extract, were purchased from Uljin-gun, Gyeongbuk, separated from the sea urchin, washed in running water to remove salt, and then lyophilized. It was used while being powdered and stored through a grinder. Reagents such as DPPH, ABTs, and Folin reagent were purchased from Sigma (Sigma Chemical Co, USA) and the absorbometer was measured using ltiskan spectrum (Thermo, Vantaa, Finland).

1-2. Preparation of extract

A solvent extract of 70 v/v% methanol was obtained from lyophilized sea urchin eggs. After drying 20 g of sea urchin eggs with 200 ml of 70 v/v% methanol at room temperature and extracting them three times for 20 minutes each, filtering with filter paper (ADVANTEC, 2), and then removing methanol using an evaporator. Thereafter, 100 ml of tertiary distilled water was added and concentrated under reduced pressure once again to prepare a concentrate of the extract, and the concentrate was dissolved in 20 ml of distilled water.

Example 2. Confirmation of cell proliferation effect using CCK-8 kit

2-1. Mouse fibroblast culture

After the mouse fibroblast BALB/c3T3 A31 (Korea Cell Line Bank) was prepared by culturing in a 37°C, 5% CO ₂ incubator, 10% fetal bovine serum (Fetal Bovin Serum, FBS, Corning) and penicillin and streptomycin antibiotic complex (Antibiotic) -Incubated using 25T flask (SPL) in RPMI 1640 medium (Gibco) containing -Antimycotic, 100X, Gibco).

When BALB/c3T3 A31 was cultured in an area of 80 to 90% of the culture vessel, the cells were counted by washing with PBS and detaching the cells with 0.25% trypsin-EDTA (Gibco) treatment. Next, 5 x 10 ³ to 1 x 10 ⁴ cells were dispensed in 96-well plates, cultured for 24 hours after dispensing, replaced with RPMI1640 medium containing 0.4% FBS and antibiotics, and cultured for an additional 24 hours.

2-2. Confirming the effect of sea urchin extract to promote mouse fibroblast proliferation

In order to measure the cell proliferation effect of the sea urchin extract prepared in Example 1-2, Cell count kit-8 (CCK-8, DOJINDO) was used. In order to measure the degree of cell growth and proliferation, 100 μl of RPMI1640 medium containing BALB/c3T3 A31 cells and added with 0.4% FBS was used as a control, and diluted with RPMI1640 medium with 0.4% FBS added to each well. Dilutions of the sea urchin extract prepared by making the drainage into 10 ^-1 (Sample 1) and 10 ^-2 (Sample 2) of the sea urchin extract stock were dispensed by 10 μl. The remaining 90 μl was filled with RPMI1640 medium containing BALB/c3T3 A31 cells and 0.4% FBS added, and then cultured for 72 hours.

After the incubation was completed, 10 μl of the CCK-8 reagent was dispensed and reacted for 2 hours at 37° C. in a 5% CO ₂ incubator, and absorbance was measured at 450 nm using an Enzyme-Linked Immunosorbent Assay (ELISA). The average value after repeating the above experiment process three times was obtained, and the results are shown in Table 1 and FIG. 1 below.

division Concentration of extract OD450nm Standard Deviation Relative ratio (%) Control 0 1.22 0.030 100.0 Sample 1 10 ^-1 1.76 0.052 145.0 Sample 2 10 ^-2 1.25 0.033 102.5

As shown in Table 1 and Fig. 1, when a 70% methanol extract of sea urchin prepared in Example 1 was diluted 10 ^-1 times to a medium containing mouse fibroblasts, cell proliferation of mouse fibroblasts was added. It was confirmed that the improvement was 45% compared to this control group. It was confirmed that the cell proliferation of mouse fibroblasts was improved by 2.5% compared to the control group even when the extract diluted 10 ^-2 times was added. Thus, the sea urchin 70 v/v% methanol extract of the present invention promotes the proliferation of fibroblasts, and in particular, the sample 1 having the concentration of the sea urchin extract 10 ^-1 , that is, the entire composition comprising the sea urchin extract and medium 100 Compared to parts by weight, it was confirmed that the sample containing sea urchin extract in 1 part by weight has an excellent fibroblast proliferation effect.

Claims (6)

A composition for proliferating fibroblasts comprising sea urchin extract as an active ingredient. The composition of claim 1, wherein the sea urchin is purple urchin ( Anthocidaris crassispina ) or pink urchin ( Pseudocentrotus depressus ). The composition of claim 1, wherein the sea urchin extract is extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixture thereof with an extraction solvent. The composition according to claim 3, wherein the solvent is an aqueous methanol solution containing 50 to 90 v/v% methanol. A cosmetic composition for skin regeneration or wrinkle improvement, comprising the composition of any one of claims 1 to 4. A pharmaceutical composition for treating burns or wounds, comprising the composition of claim 1.

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