KR20100023073A - A skin external composition for wound healing - Google Patents

Abstract

PURPOSE: A skin external composition containing 4-(3,4-dihydroxyphenyl)-3-buten-2-one is provided to promote cell migration and TGF-β(transforming growth factor beta) secretion. CONSTITUTION: A skin external composition for wound healing contains 0.01-10 weight% of 4-(3,4-dihydroxyphenyl)-3-buten-2-one. The 4-(3,4-dihydroxyphenyl)-3-buten-2-one is derived from Hypochoeris radicata L. or mushroom. The skin external composition is cosmetic composition or pharmaceutical composition. A method for isolating the 4-(3,4-dihydroxyphenyl)-3-buten-2-one comprises: a step of pulverizing mushroom and extracting 100% of methanol; a step of concentrating and extracting with 80% of methanol; a step of extracting with n-hexane to remove non-polar material; a step of adding 30% methanol and chloroform to fraction; a step of collecting chloroform layer and fractioning 1%, 40%, 60%, 80%, and 100% of methanol; and a step of collecting 40% of methanol fraction and performing chromatography. The composition is used in the form of liquid phase, cream phase, paste phase, or solid phase.

Description

A skin external composition for wound healing

The present invention relates to an external composition for wound healing, and relates to an external composition for skin comprising 4- (3,4-dihydroxyphenyl) -3-buten-2-one as an active ingredient.

The skin is composed of three layers such as epidermis, dermis and subcutaneous fat. The skin tissue of each layer is composed of various cells and substances surrounding the various functions of the skin. If the skin is damaged, the damaged area is invaded by bacteria and external toxins, and the skin's defense against this may cause inflammation. Therefore, for wound healing, it is important to promote the regeneration of damaged skin and to suppress inflammation caused by external factors such as bacteria.

Wound healing is a complex biological process that involves chemotaxis, differentiation and replication of cells, synthesis of lipid proteins, angiogenesis, etc., as a tissue recovery process that responds to tissues on damaged skin.

One of the representative substances that regulate the wound healing process is growth factors, which play an important role in regulating wound healing by transmitting signals for intercellular interaction such as cell growth, differentiation and metabolism throughout the wound healing process. In charge. These growth factors are polypeptides, which can be secreted by various active cells in the wound site to stimulate or inhibit cell proliferation, cell migration, or cell biosynthesis.

Representative of these are transforming growth factor beta (TGF-β) and platelet-derived growth factor (PDGF) that are produced by specific cells and affect surrounding target cells. Among them, TGF-β is a substance involved in the growth and differentiation of various cells, and it is reported to be responsible for various complex functions such as growth regulation, immune response control, and wound healing promotion. It is secreted and induces wound healing by drawing various cells related to wound healing such as macrophages and fibroblasts into the wound site. Therefore, the development of wound treatments utilizing this mechanism is being actively carried out around the world.

Collagen is a major substrate protein produced by skin fibroblasts. It is an important protein that accounts for 30% of the total weight of biological proteins. Its main functions are known as mechanical strength of skin, resistance of connective tissue and tissue binding, and support of cell adhesion. (Van der Rest et al., 1990). In addition, many recent studies have reported that if collagen metabolism is activated by promoting the synthesis of collagen in the skin, the effect of skin strengthening and wound healing can be expected. Conventionally, products that combine collagen with cosmetics have been released for the purpose of supplying collagen to skin tissues. However, due to the characteristics of cosmetics applied to the skin surface, percutaneous absorption of collagen, which is a polymer, can hardly be expected and thus cannot be said to be an essential skin function improvement. Conventional collagen synthesis promoting chemicals such as retinoic acid and chlorella extract (JP 9-40523, JP 10-36283) are used.However, retinoic acid is chemically unstable. In addition, the chlorella extract and the like are insignificant, and thus, the collagen synthesis of the skin can be substantially promoted to improve skin function. Therefore, there is an urgent need for the development of a new collagen synthesis promoter that is safer in vivo and more effective than a substance that promotes collagen synthesis.

The tissue repair process for damaged skin is a combination of cell proliferation and cell migration to repair the damaged skin. In other words, in order to repair damaged skin tissues, skin cells are actively generated and newly formed cells are moved to damaged areas to form new skin tissues (Cai AQ, Landman KA, and Hughes BD, Multi-scale modeling of a wound-healing cell migration assay, Journal of Theoretical Biology , Vol. 245: 576-594, 2006). Therefore, in order to heal wounds, it is necessary to use drugs that promote the proliferation of skin cells and regulate the movement of generated skin cells to damaged skin areas.

Conventional wound treatments in the domestic market are mainly steroid ointments or nonsteroidal anti-inflammatory ointments, but these studies have been used to suppress the inflammatory phenomena associated with wound injuries, but they slow wound healing and wound healing process. It acts to inhibit collagen synthesis from fibroblasts, and careful use is necessary. As an alternative to this, moisturizing dressing, which has been commercialized recently, has the advantage of keeping wounds moist and smoothing cell movement and adding some growth factors to heal wounds. There is inconvenience in using such as need to replace accordingly. In addition, currently used ointments widely used for fine wounds are using oils for skin regeneration and imported medicines called Centella asiatica, but TGF-β has a direct effect on wound healing. There was no product using medicinal herbs that stimulated the secretion of C and promoted collagen biosynthesis and activated cell migration to damaged sites.

Accordingly, an object of the present invention is to solve the problems of the conventional wound healing agent, and directly act on wound healing to promote the secretion of TGF-β and at the same time to promote collagen biosynthesis and to activate cell migration to the damaged site. To provide a skin external preparation for.

In addition, another object of the present invention is to provide a skin external preparation for wound healing using a natural product-derived wound healing component with fewer side effects to the human body.

The inventors of the present invention, while searching for a medicine that has the effect of promoting the secretion of TGF-β in the medicine mainly used in Oriental medicine 4- (3,4-dihydroxyphenyl) contained in mushrooms such as ants and chaga -3-buten-2-one [4- (3,4-dihydroxyphenyl) -3-buten-2-one] promotes the release of TGF-β in human fibroblasts and plays a key role in wound healing By promoting human skin cell proliferation and cell migration (cell migration) was found to be effective in wound healing and completed the skin external composition of the wound healing effect characterized by this.

The present inventors evaluated the collagen synthesis efficacy evaluation for 4- (3,4-dihydroxyphenyl) -3-buten-2-one showing excellent TGF-β production promoting effect, and as a result, the 4- (3, It was confirmed that 4-dihydroxyphenyl) -3-buten-2-one showed an excellent collagen synthesis promoting effect.

For wound healing, as described above, it is essential to promote the release of TGF-β in fibroblasts, and cell migration to repair damaged skin along with collagen synthesis (McDougall S., Dallon J., Sherratt J). ., Maini P., Fibroblast migration and collagen deposition during dermal wound healing:. mathematical modelling and clinical implications Philos Transact A Math Phys Eng Sci . 364: 1385-1405, 2006).

Accordingly, the present inventors have carried out cell migration to restore damaged skin to 4- (3,4-dihydroxyphenyl) -3-buten-2-one having excellent TGF-β production and collagen synthesis efficacy. As a result of experiments, it was found that this component has the effect of promoting cell migration as well, thus completing the external preparation for wound healing skin containing this component as the main active ingredient.

The external preparation composition for skin of the present invention exhibits excellent efficacy in wound recovery by promoting the secretion of TGF-β1, promoting collagen synthesis, and promoting cell migration to the damaged area, and thus can be used for wound healing.

The present invention is the result of searching for ingredients that promote TGF-β secretion, which plays a key role in the wound healing process, in medicines used in oriental medicine, in particular, mushrooms, such as ganoderma lucidum, chaga, etc. 4- (3,4- Dihydroxyphenyl) -3-buten-2-one has been shown to enhance the secretion of TGF-β in human fibroblasts, and it is important for promoting collagen synthesis as well as cell migration during wound healing. Has said.

First of all, the present inventors selected about 200 kinds of physiologically active medicinal herbs mainly used in oriental medicine, and selected mushroom extracts and dandelion extracts through experiments promoting the secretion of TGF-β. As a result of evaluating the efficacy of the isolated components in subsequent experiments, 4- (3,4-dihydroxyphenyl) -3-buten-2-one acts as a collagen synthesis and cell migration promoter along with the promotion of TGF-β secretion. Final confirmation.

The present invention provides a topical composition for wound healing containing 4- (3,4-dihydroxyphenyl) -3-buten-2-one.

The present invention is characterized in that the composition contains 0.01 to 10% by weight of 4- (3,4-dihydroxyphenyl) -3-buten-2-one on a dry weight basis. If less than 0.01% by weight, the effect is insignificant, if it exceeds 10% by weight is difficult to formulate and the effect of increasing the content is insignificant.

The present invention is characterized in that the composition promotes TGF-β production, collagen biosynthesis and cell migration in skin cells.

The present invention is characterized in that the topical skin composition is a cosmetic composition or a pharmaceutical composition.

In addition, the present invention is characterized in that the composition is a liquid, cream, paste or solid phase.

The composition of the present invention may be formulated by a method known in the pharmaceutical art, and may be prepared by the 4- (3,4-dihydroxyphenyl) -3-buten-2-one component itself or a pharmaceutically acceptable carrier or excipient. And the like can be formulated into conventional pharmaceutical preparations such as liquids, syrups, capsules, granules, powders, ointments, emulsions, gels, creams, and the like, which can be administered by several routes.

There is no particular limitation on the dosage of the composition of the present invention, but the preferred dosage depends on the condition and weight of the patient, the extent of the disease, the drug form, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. For the preferred effect, the extract of the present invention is usually administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration can be administered once to several times daily.

The present invention also provides a composition for external application for skin, including a cosmetic composition comprising the 4- (3,4-dihydroxyphenyl) -3-buten-2-one as an active ingredient. The coating agent which uses the composition of this invention as an active ingredient can be easily manufactured in any form according to the conventional manufacturing method. For example, in preparing a cream coating agent, a 4- (3,4-dihydroxyphenyl) -3-butene-2 of the present invention may be applied to a cream base of a general oil-in-water type (O / W) or water-in-oil type (W / O). It can contain a warmer and use fragrances, chelating agents, pigments, antioxidants, preservatives, etc. as needed, and can also use synthetic or natural materials such as proteins, minerals, vitamins, etc. for the purpose of improving the properties.

In addition, the composition of the present invention can be used as a cosmetic, pH additives, fragrances, emulsifiers, preservatives, etc. may be added as needed, and in the usual cosmetic preparation method, lotion, gel, water-soluble powder, fat-soluble powder, water-soluble liquid, cream or It may be formulated as an essence or the like.

4- (3,4-dihydroxyphenyl) -3-buten-2-one, which is the main active ingredient of the composition of the present invention, has a chemical structure as shown in FIG.

4- (3,4-dihydroxy-phenyl) -3-butene-2-one is phenylbutazone cannabinoid (phenylbutanoid) pieces of a material of a series dandelion (Hypochoeris Radicata ) (Phytochemistry, 1989) and Chaga (KP10-0609486-0000) have been isolated, but there are no studies on the biological activity of this substance.

Therefore, the present inventors have investigated the secretive effect of 4- (3,4-dihydroxyphenyl) -3-buten-2-one on the production of TGF-β and collagen synthesis. And it was evaluated whether there is an effector in the cell migration, and as a result, it was found that the effect of 4- (3,4-dihydroxyphenyl) -3-buten-2-one is excellent and completed the present invention.

Hereinafter, the configuration of the present invention through the embodiment in more detail. However, it will be apparent to those skilled in the art that the scope of the present invention is not limited only to the description of the embodiments.

Example 1 Separation and Purification of 4- (3,4-dihydroxyphenyl) -3-buten-2-one

600 g of mushrooms such as chaga were crushed with a grinder to form a powder, followed by repeated extraction three times with 100% methanol (4 L) at room temperature for 24 hours. After extraction, the filtrate was concentrated using a rotary evaporator. All 10 g of the methanol extract was taken, dissolved in 80% methanol, solvent-distributed with n-hexane to remove nonpolar material. The extra active fraction 80% methanol layer was further divided into 30% methanol and chloroform layer, where the concentrated amount of chloroform layer was about 1.9 g. 1.9 g of chloroform layer was subjected to reverse phase flash chromatography [mobile phase; 0%, 40%, 60%, 80%, 100% methanol (MeOH)] was divided into five fractions. 40% methanol fraction of this was subjected to a series of silica flash chromatography [CHCl ₃ / MeOH / H ₂ O (9: 1: 0.1)] and size exclusion chromatography (sephadexTM LH-20) [n -hexane / CH ₂ Cl ₂ ) / MeOH (4: 2: 1)] was used to obtain 10 mg of pure single component compound 4- (3,4-dihydroxyphenyl) -3-buten-2-one. Molecular weight was measured by EI-MS, and ¹ H-NMR and ¹³ C-NMR analysis were performed to obtain purified material as shown in FIG. 3, 4- (3,4-dihydroxyphenyl) -3-buten-2-one. It was confirmed that.

ESI-MS (m / z) 179 (M + H), 163, 145, 135, 117, 89, 77

¹ H-NMR (CDCl ₃ , 400 MHz) 2.33 (3H, s), 6.54 (H, d, 16.4), 6.78 (H, d, 8.4), 6.99 (H, dd, 2, 8.4), 7.07 (H , d, 2), 7.51 (H, d, 16.4),

¹³ C-NMR (CDCl ₃ , 400 MHz) 27.17, 115.37, 116.63, 123.58, 124.79, 127.81, 146.86, 146.94, 150.01, 201.59

Example 2 Evaluation of Wound Healing Effect

Experiment 1: TGF -β secretion promoting effect evaluation

Human-derived fibroblasts were dispensed into 48-well plates containing DMEM (Dulbecco's Modified Earles Medium) medium containing 10% fetal calf serum and incubated for 24 hours at 37 ° C. and 5% CO ₂ conditions. Serum was collected 48 hours after replacing with DMEM medium without serum and adding 4- (3,4-dihydroxyphenyl) -3-buten-2-one.

The amount of TGF-β contained in the cell culture was measured by absorbance at a wavelength of 450 nm using an ELISA kit. The experimental results were prepared by using a standard solution, preparing a standard curve, substituting the measured absorbance into the standard curve, and calculating the amount of TGF-β1 in the cell culture solution to which each sample was added. TGF-β1 production promoting rate was determined by substituting the following equation. Experimental results were 100% that the sample was not added, the results are summarized in Table 1.

TGF-β1 production promotion rate (%) = (A / B × 100) -100

[A, TGF-β1 Production Amount of Cell Culture Added Samples; B, TGF-β1 Production in Cell Culture without Addition of Samples]

sample TGF-β1 production amount TGF-β1 increase rate Control group (no addition) 127 ± 17 pg / ml 100% 4- (3,4-dihydroxyphenyl) -3-buten-2-one (50 ug / ml) 248 ± 38 pg / ml 195% 4- (3,4-dihydroxyphenyl) -3-buten-2-one (25 ug / ml) 227 ± 19 pg / ml 179% 4- (3,4-dihydroxyphenyl) -3-buten-2-one (10 ug / ml) 181 ± 19 pg / ml 143%

Referring to Table 1, 4- (3,4-dihydroxyphenyl) -3-buten-2-one promotes production of TGF-β1 in human-derived fibroblasts in a concentration-dependent manner, especially at 50 μg / ml. It showed an excellent effect such as 95% increase in the production of TGF-β1.

Experiment 2. Promoting Collagen Biosynthesis

Collagen biosynthesis promoting effect was tested for the effect of collagen synthesis at the cellular level by adding 4- (3,4-dihydroxyphenyl) -3-buten-2-one to the culture of human-derived fibroblasts. The measurement of biosynthesized collagen was applied to Martens' method (Martens MF, Huyben CM, Hendriks T., Collagen synthesis in fibroblasts from human colon: regulatory aspects and differences with skin fibroblasts. Gut 33: 1664-1670, 1992). That is, human-derived fibroblasts were dispensed into a 24-well plate for cell culture and cultured in a medium containing 10% fetal bovine serum for 24 hours at 37 ° C. and 5% CO ₂ , followed by PBS (phosphate buffer saline). Washed twice. The medium containing 1% fetal calf serum was treated with 0.001% of 4- (3,4-dihydroxyphenyl) -3-buten-2-one obtained in Example 1 above, and then labeled with a radioisotope. 2 µCi of one amino acid (2,3- ³ H proline) and 30 µg / ml of ascorbic acid were added. Incubation was terminated after incubation for 24 hours at 37 ° C. and 5% CO ₂ . After completion of the culture, the cells and the culture medium were divided into 1/2 each, treated with collagenase in only one fraction, and the protein was precipitated with TCA (trichloroacetic acid) to measure the amount of collagen biosynthesized from the difference in radioactivity of the two fractions. It was. Experimental results were 100% that the sample was not added, the results are summarized in Table 2.

sample Biosynthetic amount of collagen (cpm) Increase Control group (no addition) 486 ± 82 100% 4- (3,4-dihydroxyphenyl) -3-buten-2-one (50 ug / ml) 782 ± 117 161% 4- (3,4-dihydroxyphenyl) -3-buten-2-one (25 ug / ml) 630 ± 121 130% 4- (3,4-dihydroxyphenyl) -3-buten-2-one (10 ug / ml) 564 ± 93 116%

Referring to Table 2, 4- (3,4-dihydroxyphenyl) -3-buten-2-one showed an effect of promoting collagen biosynthesis in human-derived fibroblasts in a concentration-dependent manner.

Experiment 3. Cell Movement ( cell migration ) evaluation

After scratching human fibroblasts cultured in the laboratory, the 4- (3,4-dihydroxyphenyl) -3-buten-2-one of the present invention moves to fill the scratched cell site. Experiments were performed using an in vitro scratch assay (Nat. Protoc. 2007). In other words, primary fibroblasts from primary skins obtained from newborn boys were cultured with DMEM containing 10% fetal bovine serum, and when the culture density reached 80%, the medium was removed and a yellow tip under a microscope tip) to scratch. In the test group, medium containing 50 μg / ml of 4- (3,4-dihydroxyphenyl) -3-buten-2-one was added to DMEM without fetal bovine serum. After culturing for 24 hours with only excluded DMEM, cell migration in the scratched area was observed under a microscope. The result is the same as FIG.

As shown in the drawing, in case of the test cell containing 4- (3,4-dihydroxyphenyl) -3-buten-2-one, it was confirmed that the cell movement to the scratched portion was more active than the control group. It was.

[Production Examples 1 to 4 and Comparative Example 1]

An example of a prescription for external application of skin containing 4- (3,4-dihydroxyphenyl) -3-buten-2-one is as follows.

Composition Preparation Example (wt%) Comparative Example (wt%) One 2 3 4 One 4- (3,4-Dihydroxyphenyl) -3-buten-2-one -diethyl sebacate -lead -polyoxyethylene oleyl ether phosphate -sodium benzoate -petrolatum 10 8 5 6 Suitable to 100 1 8 5 6 Suitable to 100 0.1 8 5 6 Suitable to 100 0.01 8 5 6 Suitable to 100 -8 5 6 Suitable to 100

Production Example 5-8 and Comparative Example 2

Prescription examples of creams in cosmetics containing 4- (3,4-dihydroxyphenyl) -3-buten-2-one are as follows.

Composition Preparation Example (wt%) Comparative Example (wt%) 5 6 7 8 One -4- (3,4-dihydroxyphenyl) -3-buten-2-one -stearic acid -cetanol -potassium hydroxide -glycerin -propylene glycol -preservative -fragrance -purified water 10 15 1 0.7 5 3 Proper amount to 100 1 15 1 0.7 5 3 Correct quantity to 100 0.1 15 1 0.7 5 3 Correct quantity to 100 0.01 15 1 0.7 5 3 Correct quantity to 100 -15 1 0.7 5 3 Correct Volume to 100

Experiment 4. Wound healing  Effect evaluation

The ointment agents of Preparation Example 1-4 and Comparative Example 1 were compared and evaluated for wound healing effects. To compare the wound healing effect, four wounds were made with a biopsy punch on the back of 30 New Zealand white rabbits and five experimental groups were applied according to the ointment contents applied to each wound. Separately, the formulations of Preparation Example and Comparative Example were applied by 1g per day and tested for 3 weeks. Experimental results were analyzed by using an image analyzer to determine the size of the wound on the 0, 7, 14, 21 days after the start of the experiment. Wound shrinkage was obtained by substituting Equation 2 below, and the experimental results were 100% without adding a sample, and the results are summarized in Table 5.

Wound Shrinkage (%) = (A-B) / A × 100

[A, wound area at the start of the experiment; B, wound area of measurement day]

sample 7 days 14 days 21st Preparation Example 1 37 ± 8% ^* 65 ± 9% ^* 82 ± 4% ^* Preparation Example 2 32 ± 6% 61 ± 9% 79 ± 5% ^* Production Example 3 30 ± 7% 59 ± 8% 76 ± 6% ^* Preparation Example 4 27 ± 5% 56 ± 7% 75 ± 4% ^* Comparative Example 1 25 ± 5% 53 ± 6% 66 ± 4%

As a result of the experiment, the preparation example 1 containing 10% of 4- (3,4-dihydroxyphenyl) -3-buten-2-one showed a significant level of excellent wound healing effect from the 7th day of the experiment. The formulation containing 3,4-dihydroxyphenyl) -3-buten-2-one showed superior wound healing effects compared to the control.

1 shows the chemical structure of 4- (3,4-dihydroxyphenyl) -3-buten-2-one.

FIG. 2 and FIG. 3 are photographs of a microscopic observation of the results of scratch experiments on the effect of 4- (3,4-dihydroxyphenyl) -3-buten-2-one on cell migration of human fibroblasts. . Figure 2 is a control picture, Figure 3 is a test group picture.

Claims (10)

A skin external preparation composition for wound healing containing 4- (3,4-dihydroxyphenyl) -3-buten-2-one. The method of claim 1, The composition is an external composition for wound healing, characterized in that it contains 0.01 to 10% by weight of a dry powder of 4- (3,4-dihydroxyphenyl) -3-buten-2-one. The method according to claim 1 or 2, The 4- (3,4-dihydroxyphenyl) -3-buten-2-one is a wound external composition for wound healing, characterized in that it is derived from dandelion or mushrooms. The method according to claim 1 or 2, The composition is an external composition for wound healing, characterized in that to promote the production of TGF-β in skin cells. The method according to claim 1 or 2, The composition for external application for wound healing, characterized in that to promote collagen biosynthesis in skin cells. The method according to claim 1 or 2, The composition is a skin external composition for wound healing, characterized in that to promote cell migration (cell migration) in the skin cells. The method according to claim 1 or 2, The external preparation composition for wound healing external skin composition, characterized in that the cosmetic composition or pharmaceutical composition. The method of claim 3, The 4- (3,4-dihydroxyphenyl) -3-buten-2-one was crushed mushrooms, 100% methanol extraction, concentrated, 80% methanol was added to extract, n-hexane extraction After removing the non-polar material, fractions were added by adding 30% methanol and chloroform, fractionated with 0%, 40%, 60%, 80%, and 100% methanol by taking a layer of chloroform, followed by 40% methanol fraction. The external preparation composition for skin. The method of claim 7, wherein The composition for external application for wound healing, characterized in that the liquid, cream, paste or solid phase. The method of claim 7, wherein The cosmetic composition is a skin external composition for wound healing, characterized in that the lotion, gel, water-soluble liquid, cream or essence formulation.

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