Keratinization promoting pharmaceutical composition, and method for producing keratinization promoting pharmaceutical composition containing pomegranate seed extract

Abstract

To provide a composition that exhibits an effect in promoting keratinization, targeting the efficient utilization and added value of waste pomegranate seeds.SOLUTION: A keratinization promoting pharmaceutical composition contains a pomegranate seed extract. In a preferable embodiment of the keratinization promoting pharmaceutical composition, keratinization is promoted through enhancement of an mRNA expression level in keratin protein. In another preferable embodiment of the keratinization promoting pharmaceutical composition, the keratin protein is KRT-1 or KRT-10.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for promoting keratinization and a method for producing a pharmaceutical composition for promoting keratinization, and in particular to a pharmaceutical composition for promoting keratinization in the dermis, which contains a pomegranate seed extract, and a method for producing a pharmaceutical composition for promoting keratinization. [Background technology]

[0002] Pomegranate is a fruit in which seeds account for a significant proportion of the entire fruit, but as a rule, the seeds remaining after squeezing are discarded. A few examples of using pomegranate seeds include a supplement suggestion system using pomegranate seed extract, in which a suggested supplement calculation means outputs at least one of the following as suggested supplements when the desired item information is beauty: multivitamins, multiminerals, vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin E, vitamin P, niacin, biotin, collagen, elastin, polyphenols, pantothenic acid, hyaluronic acid, acetylglucosamine, inositol, coenzyme Q10, alpha carotene, beta carotene, soybean saponin, royal jelly, twin toes, folic acid, rose petal extract, pomegranate seed extract, black cohosh extract, soybean isoflavones, kudzu isoflavone squalene, brewer's yeast, fenugreek extract, calcium, magnesium, copper, iron, zinc, and manganese (Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent Publication No. 2011-232989 DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0004] However, in most cases, pomegranate seeds are currently disposed of as waste, and are not effectively utilized except for those described in the above patent documents. On the other hand, effective utilization of waste materials is being reconsidered.

[0005] In recent years, it has been discovered that atopic dermatitis (AD) is caused by a loss-of-function mutation in the keratinization-related gene filaggrin (FLG), and the importance of skin barrier function has been drawing attention. Under these circumstances, it would be even more beneficial if pomegranate seeds, a waste product, were effective against various diseases.

[0006] Therefore, an object of the present invention is to provide a composition having the effect of promoting keratinization, with the aim of effectively utilizing and adding value to discarded pomegranate seeds. [Means for solving the problem]

[0007] In order to achieve the above object, the present inventors conducted extensive research into effective utilization of pomegranate seeds and came up with the present invention.

[0008] That is, the pharmaceutical composition for promoting keratinization of the present invention is characterized by containing pomegranate seed extract.

[0009] In a preferred embodiment of the pharmaceutical composition for promoting keratinization of the present invention, the composition is characterized in that it promotes keratinization by increasing the mRNA expression level of keratin protein.

[0010] In a preferred embodiment of the pharmaceutical composition for promoting keratinization of the present invention, the keratin protein is KRT-1 or KRT-10.

[0011] In a preferred embodiment of the pharmaceutical composition for promoting keratinization of the present invention, the composition is further characterized by promoting the formation of a cornified membrane.

[0012] In a preferred embodiment of the pharmaceutical composition for promoting keratinization of the present invention, the composition is further characterized by increasing the mRNA expression level of a gene associated with cornified membrane formation.

[0013] Furthermore, in a preferred embodiment of the pharmaceutical composition for promoting keratinization of the present invention, the cornified membrane formation-related gene is at least one selected from the group consisting of the INV gene (involucrin gene), the LOR gene (loricrin gene), and the TGM-1 gene (transglutaminase-1 gene).

[0014] Furthermore, the quasi-drug of the present invention is characterized in that it contains the pharmaceutical composition for keratinization promotion of the present invention as an active ingredient.

[0015] In addition, the manufacturing method of the pharmaceutical composition for promoting keratinization of the present invention is characterized by containing a pomegranate seed extract, which comprises the steps of crushing pomegranate seeds, immersing the crushed product in a solvent, and then separating the supernatant, obtaining a pomegranate seed extract by separating the supernatant, and adjusting the obtained pomegranate seed extract to an effective amount.

[0016] Furthermore, in a preferred embodiment of the method for producing a pharmaceutical composition for promoting keratinization of the present invention, the solvent is at least one selected from the group consisting of ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, hexane, and water.

[0017] In addition, in a preferred embodiment of the method for producing a pharmaceutical composition for promoting keratinization of the present invention, the solvent is ethanol, and the pomegranate seed ethanol extract extracted with the ethanol is further partitioned with ethyl acetate and water, and the ethyl acetate layer is concentrated under reduced pressure to obtain the pomegranate seed extract as an ethyl acetate fraction.

[0018] In addition, in a preferred embodiment of the method for producing a pharmaceutical composition for promoting keratinization of the present invention, the method is further characterized in that the ethyl acetate fraction is partitioned with hexane and methanol to obtain a pomegranate seed extract as an ethyl acetate-hexane fraction and an ethyl acetate-methanol fraction. Effect of the Invention

[0019] The pharmaceutical composition for keratinization promotion of the present invention has an advantageous effect of making effective use of pomegranate seeds, which have conventionally been disposed of as waste. In addition, the pharmaceutical composition for keratinization promotion of the present invention has an advantageous effect of having an effect of promoting keratinization, and therefore also having an effect of promoting the cornified membrane. [Brief description of the drawings]

[0020] [Figure 1] Figure 1 shows the mRNA expression level of KRT-1 in HaCaT (n=3, mean ± standard error, *p < 0.05 vs. Control). [Diagram 2] Figure 2 shows the mRNA expression level of KRT-10 in HaCaT (n=3, mean ± standard error, *p < 0.05, **p < 0.01 vs. Control). [Diagram 3] FIG. 3 shows the mRNA expression level of KRT-6 in HaCaT (n=3, mean ± standard error). [Figure 4] Figure 4 shows the evaluation of CE formation in HaCaT (n=3, mean ± standard error, *p < 0.05, **p < 0.01 vs. Control). [Diagram 5] Figure 5 shows the mRNA expression level of LOR in HaCaT (n=3, mean ± standard error, *p < 0.05 vs. Control). [Figure 6] Figure 6 shows the mRNA expression level of IVN in HaCaT (n=3, mean ± standard error, *p < 0.05 vs. Control). [Figure 7]Figure 7 shows the mRNA expression level of TGM-1 in HaCaT (n=3, mean ± standard error, *p < 0.05 vs. Control). [Figure 8] Figure 8 shows the flow chart of fraction preparation and the yields. F1 to F4 were obtained by distributing PSE-EA-H, which had a high yield. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The pharmaceutical composition for promoting keratinization of the present invention is characterized by containing a pomegranate seed extract. The pomegranate seed extract is an extract derived from pomegranate seeds. The pomegranate seed extract applied to the present invention covers all pomegranate seed extracts as long as they are derived from pomegranate seeds.

[0022] In addition, the target cells are not particularly limited, but the cells that are particularly effective are skin cells, and therefore, although the present specification will mainly describe skin cells, it is not intended to be limited thereto.

[0023] In a preferred embodiment of the pharmaceutical composition for promoting keratinization of the present invention, the keratin protein is KRT-1 or KRT-10, and the keratin protein is increased in an mRNA expression level, thereby promoting keratinization.

[0024] Keratin is produced as a cytoskeletal protein in human epithelial cell systems. At present, 21 types of keratin have been identified, and with some exceptions, it is believed that intermediate fibers are formed by forming dimers in specific pairs of relatively large molecular weight basic or type II keratins (KRT-1 to 8) and relatively small molecular weight acidic or type I keratins (KRT-9 to 21). As keratinization (differentiation) progresses in human keratinocytes, the type of keratin expressed changes, and while basal cells (undifferentiated state) are mainly composed of KRT-5 and 14, differentiated spinous cells express a pair of KRT-1 and 10 (at terminal keratinization, some of these are degraded by proteases to become fine proteins that form keratinocytes).

[0025] In the present invention, as will be apparent from the examples described below, it has surprisingly been found that the presence of pomegranate seed extract can increase the mRNA expression level of keratin proteins, such as the mRNA expression levels of KRT-1, 10, etc., and thus promote keratinization.

[0026] In a preferred embodiment of the pharmaceutical composition for promoting keratinization of the present invention, the composition is further characterized by promoting the formation of a cornified membrane, from the viewpoint of improving the barrier function of the skin.

[0027] In a preferred embodiment of the pharmaceutical composition for keratinization promotion of the present invention, the composition is further characterized in that it increases the mRNA expression level of a gene related to cornified membrane formation, from the viewpoint of improving the barrier function of the skin.

[0028] Furthermore, in a preferred embodiment of the pharmaceutical composition for promoting keratinization of the present invention, the cornified membrane formation-related gene is at least one selected from the group consisting of the INV gene (involucrin gene), the LOR gene (loricrin gene), and the TGM-1 gene (transglutaminase-1 gene).

[0029] As keratinocytes undergo cornification, the formation of the cornified envelope (CE) also progresses. Surprisingly, in the present invention, it has been found that the presence of pomegranate seed extract makes it possible to increase the mRNA expression levels of genes related to cornified envelope formation, such as the INV gene (involucrin gene), LOR gene (loricrin gene), and TGM-1 gene (transglutaminase-1 gene), thereby promoting the formation of the cornified envelope.

[0030] In a preferred embodiment of the pharmaceutical composition for keratinization promotion of the present invention, the pomegranate seeds are pulverized, the pulverized product is immersed in at least one solvent selected from the group consisting of ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, water, and hexane, and the supernatant is separated to obtain the pomegranate seed extract. For example, shaking extraction can be performed. In the shaking extraction, for example, extraction can be performed while rotating by setting on a rotator in a low-temperature room at about 4°C. In a preferred embodiment of the pharmaceutical composition for keratinization promotion in the epidermis of the present invention, the solvent is ethanol.

[0031] In addition, the manufacturing method of the pharmaceutical composition for promoting keratinization of the present invention is characterized by containing a pomegranate seed extract, which comprises the steps of crushing pomegranate seeds, immersing the crushed product in a solvent, and then separating the supernatant, obtaining a pomegranate seed extract by separating the supernatant, and adjusting the obtained pomegranate seed extract to an effective amount.

[0032] Furthermore, in a preferred embodiment of the method for producing a pharmaceutical composition for promoting keratinization of the present invention, the solvent is at least one selected from the group consisting of ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, hexane, and water.

[0033] In addition, in a preferred embodiment of the method for producing the pharmaceutical composition for promoting keratinization of the present invention, from the viewpoint of assuming application to food, the pomegranate seed ethanol extract extracted with ethanol is further partitioned with ethyl acetate and water, and the ethyl acetate layer is concentrated under reduced pressure to obtain the pomegranate seed extract as an ethyl acetate fraction.

[0034] In addition, in a preferred embodiment of the method for producing a pharmaceutical composition for promoting keratinization of the present invention, from the viewpoint of identifying the active ingredient, the method is further characterized in that the ethyl acetate fraction is partitioned with hexane and methanol to obtain a pomegranate seed extract as an ethyl acetate-hexane fraction and an ethyl acetate-methanol fraction.

[0035] The ethyl acetate-hexane fraction (PSE-EA-H) and the ethyl acetate-methanol fraction (PSE-EA-Me) are as follows: That is, when pomegranate seed ethanol extract (PSE) extracted with ethanol is partitioned between ethyl acetate and water using a separatory funnel, and the solvent of the ethyl acetate layer is concentrated under reduced pressure using an evaporator to obtain the ethyl acetate fraction (PSE-EA), and the solvent of the water layer is freeze-dried to obtain the water fraction (PSE-W), PSE-EA is partitioned between hexane and methanol, and the solvents are then each concentrated under reduced pressure using an evaporator to obtain the resulting fractions, which can be called the ethyl acetate-hexane fraction (PSE-EA-H) and the ethyl acetate-methanol fraction (PSE-EA-Me).

[0036] Here, an example of a method for preparing a pomegranate seed extract will be described. First, pomegranate seeds are prepared. The pomegranate seeds are washed and dried as necessary. It is preferable to dry the seeds thoroughly so that the subsequent grinding can be performed uniformly.

[0037] Next, the pomegranate seeds are pulverized. The pulverization method is not particularly limited, and known pulverizers such as ball mills, hammer mills, roller mills, rod mills, sample mills, stamp mills, disintegrators, mortars, and blenders with cooling devices can be used. Note that, since heat generated during pulverization may cause decomposition of the pomegranate seed composition, the pulverization time is set to several seconds, and the pulverization can be repeated a dozen times.

[0038] Next, the pomegranate seeds are crushed to obtain a crushed product, and the crushed product is immersed in various solvents. In this case, the solvent is not particularly limited, and can be appropriately selected according to the desired effect. In a preferred embodiment of the method for producing a pomegranate seed extract of the present invention, the solvent is at least one selected from the group consisting of ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, hexane, and water. Examples of the solvent include ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, water, hexane, ethyl acetate, chloroform, acetone, and other polar and non-polar solvents. Preferred examples include ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, methanol, ethanol, and water.

[0039] The immersion can be carried out under gentle stirring. The pulverized material is immersed in various solvents to obtain various solutions. The various solutions are stirred depending on the state of the solution, and in some cases the solution may be left as is. When stirring, the stirring can be continued for 10 hours to 48 hours, preferably for about one day (24 hours), although there is no particular limitation.

[0040] Thereafter, the supernatant is separated to obtain the pomegranate seed extract. If necessary, the supernatant is evaporated to dryness. The evaporation to dryness can be carried out using an evaporator on a warm bath at 20°C to 60°C, preferably 37°C to 40°C. By evaporating to dryness, the pomegranate seed extract can be stored for a long period of time.

[0041] The components contained in pomegranate seeds are extracted with solvents of different polarity and are divided according to their physical properties. Therefore, the type and content of the components in the pomegranate seed extract varies depending on the solvent used.

[0042] The quasi-drug of the present invention is characterized in that it contains the composition of the present invention as an active ingredient. There are no particular limitations on the quasi-drug applicable to the present invention, so long as it contains the pharmaceutical composition for keratinization promotion of the present invention as an active ingredient.

[0043] <Effective amount> The compositions according to the present invention are prepared by providing an effective amount of pomegranate seed extract and a suitable dosage form.

[0044] The dosage of pomegranate seed extract in the composition of the present invention can be varied depending on the pathological condition and its severity of the patient to be administered, the dosage form, the selected administration route, the number of administrations per day, and the like.

[0045] The dosage of pomegranate seed extract in the composition of the present invention can be set at 1000 mg / kg / day when tested in mice, and a lower dosage is preferable in humans due to differences in sensitivity, etc.

[0046] The dosage form may be an oral agent (tablet, capsule, coated tablet, granule, solution, syrup), or an ointment or cream for application. From the viewpoint of skin hydration and barrier function, the subject of administration may be any patient, regardless of gender, gender, adult, or child.

[0047] The dosage form may contain other conventional ingredients, such as preservatives, sweeteners, colorants, flavoring agents, and the like.

[0048] <Acute toxicity test> As for the ingredients contained in pomegranate seed extract, including linoleic acid, it is believed that there is no toxicity.

[0049] The abbreviations are as follows: ·FBS: Fetal Bovine Serum F1: PSE-Ethyl Acetate-Hexane-Fraction 1 F2: PSE-Ethyl Acetate-Hexane-Fraction 2 F3: PSE-Ethyl Acetate-Hexane-Fraction 3 F4: PSE-Ethyl Acetate-Hexane-Fraction 4 ·HA: Hyaluronic Acid ·HABP:Hyaluronic Acid Binding Protein ·HAS: Hyaluronic Acid Synthase ·PBS: Phosphate Buffered Saline · PSE: Punica granatum Seed Ethanol Extract PSE-Bu-Me: PSE-Butanol-Methanol fraction PSE-EA: PSE-Ethyl Acetate fraction PSE-EA-H: PSE-Ethyl Acetate-Hexane fraction PSE-EA-Me: PSE-Ethyl Acetate-Methanol fraction PSE-W: PSE-Water fraction UV: Ultraviolet EXAMPLES

[0050] An embodiment of the present invention will be described below, but the present invention is not limited to the following embodiment.

[0051] Example 1 <Preparation of pomegranate seed extract> <Sample> Pomegranate seeds: Obtained from Sunny Place Co., Ltd. <Machine> Millsa: Manufactured by Iwawa Industries Co., Ltd. Rotary Evaporator: Manufactured by EYELA Tokyo Rika Kikai Co., Ltd. Freeze Dryer FDU-1200: Manufactured by EYELA Tokyo Rika Kikai Co., Ltd. <Reagent> 100% Ethanol: Manufactured by Wako Pure Chemical Industries, Ltd. Ethyl Acetate: Manufactured by Wako Pure Chemical Industries, Ltd. Hexane: Manufactured by Wako Pure Chemical Industries, Ltd. Methanol: Manufactured by Wako Pure Chemical Industries, Ltd. Silica Gel (Wakasil® 60, 64 - 210μm): Manufactured by Wako Pure Chemical Industries, Ltd. MilliQ Water <Instrument> Chromatography Column with Filter (30φ × 300mm): Manufactured by Cosmos Bio Co., Ltd.

[0052] First, as a solvent, ethanol was used to attempt the preparation of pomegranate seed extract. The pomegranate seeds were crushed and extracted by shaking with 100% ethanol. These were concentrated to dryness and redissolved, and used in the experiment as pomegranate seed ethanol extract. Furthermore, the pomegranate seed ethanol extract extracted with ethanol (hereinafter also referred to as PSE) was partitioned with ethyl acetate and water using a separating funnel, the solvent in the ethyl acetate layer was concentrated under reduced pressure using an evaporator, and used as the ethyl acetate fraction (PSE-EA). The aqueous layer was freeze-dried to remove the solvent and used as the aqueous fraction (PSE-W). When PSE-EA was partitioned with hexane and methanol and then the solvents were each concentrated under reduced pressure using an evaporator, the resulting fractions were used as the ethyl acetate - hexane fraction (hereinafter also referred to as PSE-EA-H) and the ethyl acetate - methanol fraction (hereinafter also referred to as PSE-EA-Me). Specifically, it is as follows.

[0053] <Preparation Method of PSE (Punica granatum Seed Ethanol extract)> Dried pomegranate seeds were powdered in a mill, and 10 times the amount of 100% ethanol (Wako Pure Chemical Industries, Ltd.) was added and low-temperature shaking extraction was performed at 4°C for 24 hours. After 24 hours, the mixture was centrifuged at room temperature at 3,000 rpm for 20 minutes, and the supernatant was collected. The supernatant was concentrated under reduced pressure, and the resulting concentrate was named pomegranate seed ethanol extract (PSE: Punica granatum Seed Ethanol extract).

[0054] <Preparation method of ethyl acetate-hexane fraction (PSE-EA-H) and ethyl acetate-methanol fraction (PSE-EA-Me)> 2.00g of PSE was partitioned between ethyl acetate (100ml x 3) and MiliQ water (100ml) using a separatory funnel, and the solvent in the ethyl acetate layer was concentrated under reduced pressure using an evaporator to obtain the ethyl acetate fraction (PSE-EA). The solvent in the water layer was freeze-dried to obtain the water fraction (PSE-W). PSE-EA was further partitioned between hexane (100ml x 3) and methanol (100ml), and the solvents were then each concentrated under reduced pressure using an evaporator to obtain the ethyl acetate-hexane fraction (PSE-EA-H) and the ethyl acetate-methanol fraction (PSE-EA-Me). The PSE-EA-H with the highest yield was passed through a column (30φ×300mm) in the following order: hexane (100ml), a mixture of hexane and ethyl acetate (hexane:ethyl acetate=9:1 (hexane 90mL, ethyl acetate 10mL), hexane:ethyl acetate=8:2 (hexane 80mL, ethyl acetate 20mL), hexane:ethyl acetate=6:4 (hexane 40mL, ethyl acetate 60mL)), ethyl acetate (100ml), and methanol (100ml) were poured and distributed. The fraction yield was calculated from the following formula, and the flow chart of fraction preparation is shown in Figure 3.

[0055] Yield (%) = (mass of fraction (g) / mass of PSE (g)) x 100

[0056] The prepared extracts were stored frozen at -20°C until use in experiments.

[0057] Using the pomegranate seed extract thus obtained, the promotion of epidermal differentiation and CE formation were investigated as part of a study on skin functionality.

[0058] <Research overview> Activation of cell proliferation in the basal layer of the epidermis promotes keratinocyte keratinization and shortens the turnover cycle. Following proliferation of epidermal keratinocytes (HaCaT) by PSE, we evaluated the expression of genes involved in promoting keratinization of cells.

[0059] In human epithelial cell systems, keratin is produced as a cytoskeletal protein. At present, 21 types of keratin have been identified, and with some exceptions, it is believed that intermediate fibers are formed by forming dimers in specific pairs of relatively large molecular weight basic or type II keratins (KRT-1 to 8) and relatively small molecular weight acidic or type I keratins (KRT-9 to 21). As keratinization (differentiation) progresses in human keratinocytes, the type of keratin expressed changes, and while basal cells (undifferentiated state) mainly consist of KRT-5 and 14, differentiated spinous cells express a pair of KRT-1 and 10 (at the time of terminal keratinization, some of these are degraded by proteases to become fine proteins that form keratinocytes). Based on this, in this study, we evaluated the mRNA expression levels of KRT-1 and 10 as keratinization markers and examined whether their expression is enhanced by the presence of PSE. In addition, we also evaluated KRT-6, one of the three genes KRT-6, 16, and 17, which are expressed upon exposure to stimuli such as trauma.

[0060] In addition, as keratinocytes undergo keratinization, the formation of the cornified envelope (CE) also progresses. Epidermal cells eventually lose their cell membranes, cease life activities as keratinocytes, and fall off (turnover), but the barrier function of the skin is maintained by gradually forming a cornified envelope that backs the cell membrane. CE is essential to prevent the invasion of external stimuli and bacteria, but its formation ability decreases due to aging, stress, and external stimuli, so it can be said that maintaining, improving, and restoring CE formation ability is very important for keeping the skin in a healthy state. In this test, the CE formation rate in cells in the presence of PSE was measured, and the promotion activity of CE formation ability was examined. After that, the expression of the genes for loricrin and involucrin (LOR, INV), which are components of CE, and the gene for transglutaminase-1 (TGM-1), which functions to crosslink these proteins, was evaluated. These genes are also used as keratinocyte keratinization markers.

[0061] <Experimental Method> Genetic analysis: After incubation with PSE, the cells were washed with PBS and harvested, and RNA was extracted, cDNA was synthesized, and real-time PCR was performed. GAPDH was selected as an endogenous control for HaCaT, and MRPL19 was selected as an endogenous control for KMST-6.

[0062] CE formation evaluation: After the end of the PSE-added culture, the cells were harvested, 2% SDS was added, and the cell membrane was disrupted using a vortex mixer. The disrupted solution was heated at 90°C for 10 minutes, and the transmittance at 310 nm was measured. The CE formation rate was calculated by comparing with the control value.

[0063] <Experimental Results> The results of gene expression evaluation for KRT-1 are shown in Figure 1, for KRT-10 in Figure 2, and for KRT-6 in Figure 3. In cells in the presence of PSE, there was a tendency for the gene expression levels of KRT-1 and 10 to increase, and for KRT-6 to increase. The PSE concentration that caused the greatest increase in expression for both KRT-1 and 10 was 10 μg / mL, and the increase was more than two-fold in both cases.

[0064] Furthermore, the results of the evaluation of CE formation are shown in Figure 4. Promotion of CE formation was confirmed in cells cultured in the presence of 8 μg / mL or more of PSE. The results of the expression evaluation of the CE-related gene LOR are shown in Figure 5, the results for INV in Figure 6, and the results for TGM-1 in Figure 7. In the gene expression evaluation, an increase in gene expression levels was confirmed by the addition of PSE for all three types of genes: LOR, INV, and TGM-1. It is believed that CE formation is promoted by promoting the synthesis of loricrin and involucrin, which are components of CE, and by enhancing the expression of transglutaminase, which cross-links them.

[0065] It was found that PSE promotes keratinization of human keratinocytes at the genetic level, as it increased the expression of keratin markers KRT-1 and KRT-10, and CE markers LOR, INV, and TGM-1, suggesting the possibility of promoting the activation of turnover. In addition, it increased the expression of a type of keratin (KRT-6) involved in wound healing, suggesting the involvement of PSE in promoting wound healing.

[0066] FIG. 8 shows the flow chart of fraction preparation and the yield. F1 to F4 were obtained by partitioning PSE-EA-H, which had a high yield. F1 to F4 were obtained by further fractionating PSE-EA-H, meaning that F1 was obtained after running the column with a mixture of 8:2 hexane and ethyl acetate, F2 was obtained with a mixture of 6:4 hexane and ethyl acetate, F3 was obtained with 100% ethyl acetate, and F4 was obtained with 100% methanol. The same effect was obtained with these pomegranate seed extracts. [Industrial Applicability]

[0067] The present invention not only contributes to the medical industry, but also contributes greatly to society through the effective use of waste materials, and has a wide range of useful value.

Claims

1. A pharmaceutical composition for promoting keratinization, comprising pomegranate seed extract.

2. 2. The pharmaceutical composition for promoting keratinization according to claim 1, characterized in that it promotes keratinization by increasing the mRNA expression level of keratin protein.

3. 3. The pharmaceutical composition for promoting keratinization according to claim 1 or 2, characterized in that the keratin protein is KRT-1 or KRT-10.

4. The pharmaceutical composition for promoting keratinization according to any one of claims 1 to 3, further comprising a function of promoting the formation of a cornified membrane.

5. The pharmaceutical composition for promoting keratinization according to any one of claims 1 to 4, further comprising increasing the mRNA expression level of a gene related to cornified membrane formation.

6. The pharmaceutical composition for promoting keratinization according to any one of claims 1 to 5, characterized in that the cornified membrane formation-related gene is at least one selected from the group consisting of the INV gene (involucrin gene), the LOR gene (loricrin gene), and the TGM-1 gene (transglutaminase-1 gene).

7. A quasi-drug comprising the pharmaceutical composition according to any one of claims 1 to 6 as an active ingredient.

8. A method for producing a pharmaceutical composition for promoting keratinization, comprising the steps of: immersing the pulverized product obtained by pulverizing pomegranate seeds in a solvent and then separating out the supernatant; obtaining a pomegranate seed extract by separating out the supernatant; and adjusting the obtained pomegranate seed extract to an effective amount.

9. 9. The method according to claim 8, wherein the solvent is at least one selected from the group consisting of ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, hexane, and water.

10. The method according to claim 6, characterized in that the solvent is ethanol, and the pomegranate seed ethanol extract extracted with the ethanol is further partitioned with ethyl acetate and water, and the ethyl acetate layer is concentrated under reduced pressure to obtain the pomegranate seed extract as an ethyl acetate fraction.

11. 11. The method of claim 10, further comprising partitioning the ethyl acetate fraction with hexane and methanol to obtain a pomegranate seed extract as an ethyl acetate-hexane fraction and an ethyl acetate-methanol fraction.

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