Pharmaceutical composition for promoting hyaluronic acid synthesis, and method for producing said pharmaceutical composition containing pomegranate seed extract

Abstract

The present invention relates to a pharmaceutical composition for promoting hyaluronic acid synthesis and a method for producing the pharmaceutical composition containing pomegranate seed extract. [Problem] The purpose of the present invention is to provide a composition having an effect of promoting the synthesis of hyaluronic acid, with the purpose of effectively using waste pomegranate seeds and increasing the added value thereof. [Solution] The pharmaceutical composition for promoting hyaluronic acid synthesis according to the present invention is characterized by containing pomegranate seed extract and promoting hyaluronic acid synthesis in dermis. Furthermore, a preferred embodiment of the pharmaceutical composition for promoting the synthesis of hyaluronic acid according to the present invention is characterized by promoting the synthesis of hyaluronic acid in fibroblasts of the dermis. Furthermore, a preferred embodiment of the pharmaceutical composition for promoting the synthesis of hyaluronic acid according to the present invention is a pharmaceutical composition for promoting the synthesis of hyaluronic acid by increasing the expression of a hyaluronic acid synthetase, which is HAS-1 or HAS-2.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for promoting hyaluronic acid synthesis and a method for producing the pharmaceutical composition for promoting hyaluronic acid synthesis, and particularly to a pharmaceutical composition for promoting hyaluronic acid synthesis containing a pomegranate seed extract in the dermis and a method for producing the pharmaceutical composition for promoting hyaluronic acid synthesis. Background Art

[0002] Pomegranate is a fruit in which seeds account for a considerable proportion of the entire fruit, but in principle, the seeds remaining after juicing are discarded. As a few examples of using pomegranate seeds, there is known a nutritional supplement suggestion system using pomegranate seed extract. Regarding the calculation unit of the suggested supplement, when the desired item information is beauty, there is known a nutritional supplement suggestion system that outputs at least one of the following ingredients as the suggested supplement: 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, α-carotene, β-carotene, soybean saponin, royal jelly, twintose, folic acid, rose petal extract, pomegranate seed extract, black cohosh extract, soybean isoflavones, kudzu isoflavones, squalene, brewer's yeast, fenugreek extract, calcium, magnesium, copper, iron, zinc, and manganese (Patent Document 1). Prior art literature Patent Literature

[0003] Patent Document 1: Japanese Patent Application Laid-Open No. 2011-232989 Summary of the invention Problems to be solved by the invention

[0004] However, in most cases, pomegranate seeds are generally treated as waste, and except for the above-mentioned patent document, pomegranate seeds are not effectively utilized. On the other hand, the effective utilization of waste is being re-examined.

[0005] In recent years, it has been discovered that atopic dermatitis (AD) is caused by loss-of-function mutations in the keratinization-related gene filaggrin (FLG), and the importance of skin barrier function has attracted attention. In this context, if pomegranate seeds, which are waste products, are effective against various diseases, it will be more beneficial.

[0006] Therefore, an object of the present invention is to provide a composition having an effect of promoting hyaluronic acid synthesis with the goal of effectively utilizing discarded pomegranate seeds and increasing their added value. Means of solving problems

[0007] In order to achieve the above-mentioned object, the present inventors conducted intensive research on the effective utilization of pomegranate seeds, and as a result, found the present invention.

[0008] That is, the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention is characterized by containing a pomegranate seed extract and promoting the synthesis of hyaluronic acid in the dermis.

[0009] In addition, a preferred embodiment of the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention is characterized in that the synthesis of hyaluronic acid in the fibroblasts of the dermis is promoted.

[0010] In addition, a preferred embodiment of the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention is characterized in that the pharmaceutical composition promotes hyaluronic acid synthesis by increasing the expression of hyaluronic acid synthase, and the hyaluronic acid synthase is HAS-1 or HAS-2.

[0011] In addition, a preferred embodiment of the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention is characterized in that the synthesis of hyaluronic acid in the dermis is further promoted under ultraviolet irradiation.

[0012] Furthermore, the quasi-drug of the present invention is characterized in that it contains the pharmaceutical composition of the present invention as an active ingredient.

[0013] In addition, the method for producing a pharmaceutical composition for promoting hyaluronic acid synthesis in the dermis of the present invention is characterized in that it includes the following steps: a step of immersing a crushed product obtained by crushing pomegranate seeds in a solvent and then separating the supernatant; a step of obtaining a pomegranate seed extract by separating the supernatant; and a step of adjusting the obtained pomegranate seed extract to an effective amount.

[0014] In a preferred embodiment of the method for producing a pharmaceutical composition for promoting hyaluronic acid synthesis in the dermis of the present invention, the solvent is at least one selected from ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, hexane and water.

[0015] In addition, a preferred embodiment of the method for producing a pharmaceutical composition for promoting hyaluronic acid synthesis in the dermis of the present invention is characterized in that the solvent is ethanol, the pomegranate seed-ethanol extract extracted with the ethanol is further distributed between ethyl acetate and water, and the ethyl acetate layer is concentrated under reduced pressure to obtain a pomegranate seed extract as an ethyl acetate fraction.

[0016] In a preferred embodiment of the method for producing a pharmaceutical composition for promoting hyaluronic acid synthesis in the dermis of the present invention, the ethyl acetate fraction is further partitioned between hexane and methanol to obtain a pomegranate seed extract as an ethyl acetate-hexane fraction and an ethyl acetate-methanol fraction. Effects of the Invention

[0017] The pharmaceutical composition for promoting hyaluronic acid synthesis in the dermis according to the present invention has the advantageous effect of being able to effectively utilize pomegranate seeds that were previously treated as waste. In addition, the pharmaceutical composition for promoting hyaluronic acid synthesis according to the present invention has the effect of promoting hyaluronic acid synthesis in the dermis, and has the advantageous effect of effectively improving the moisturizing function. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The HA (hyaluronic acid) content in the culture supernatant of KMST-6 is shown. (n=5, mean±standard error, ***p<0.001 vs control) Figure 2 The HA (hyaluronic acid) content in the culture supernatant of KMST-6 cells irradiated (or not irradiated) with UVA (ultraviolet A wave) is shown. (n=4, mean ± standard error) Figure 3 The results of the evaluation of HAS-2 gene expression in KMST-6 are shown. (n=3, mean±standard error) Figure 4 The flow chart and yields of fraction preparation are shown. F1 to F4 are obtained by fractionating PSE-EA-H, which has a high yield. DETAILED DESCRIPTION

[0019] The pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention is a pharmaceutical composition for promoting hyaluronic acid synthesis containing a pomegranate seed extract, characterized in that it promotes hyaluronic acid synthesis in the dermis. The pomegranate seed extract is an extract derived from pomegranate seeds. The pomegranate seed extract suitable for the present invention is a pomegranate seed extract of all kinds as long as it is derived from pomegranate seeds.

[0020] The target cells are not particularly limited, but the drug effectively acts on skin cells, especially dermal cells. Therefore, the present specification mainly describes dermal cells.

[0021] In addition, a preferred embodiment of the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention is characterized in that it promotes hyaluronic acid synthesis in the dermal fibroblasts. Specifically, the pharmaceutical composition can be a pharmaceutical composition that promotes hyaluronic acid synthesis by increasing the expression of hyaluronic acid synthase in the dermal fibroblasts.

[0022] In the present invention, the dermal fibroblasts are not particularly limited, but from the viewpoint of establishing a cell line for experiments, examples thereof include KMST-6 and NHDF (Normal Human Dermal Fibroblasts).

[0023] In a preferred embodiment, from the viewpoint of the water-retaining function of the cells improving the skin, the hyaluronic acid synthase is characterized in that what is produced by fibroblasts in the dermis is HAS-2 and HAS-1, and what is produced by keratinocytes (epidermal keratinocytes) in the epidermis is HAS-3. That is, the skin is responsible for protecting the organism from the effect of external influences and the function of preventing water loss, and it is very important to keep the normal state of the skin, as the main regulator related to the barrier function and water-retaining function of the skin, corneum (CE), hyaluronic acid (HA) can be enumerated, and it is determined that by the present invention, the synthesis of the hyaluronic acid in the dermis can be promoted. That is, it is determined that: hyaluronic acid (HA) plays the effect of keeping moisture in both the epidermis and the dermis, is synthesized by keratinocytes (such as HaCaT cells) in the epidermis, and is synthesized by fibroblasts (such as KMST-6 cells) in the dermis. Therefore, as described in the embodiment below, in the dermis, it can be evaluated by implementing quantitative evaluation to fibroblasts.

[0024] In addition, there are three kinds of isozymes of HAS-1, HAS-2 and HAS-3 in the synthetase of HA, and HAS-1 and HAS-2 act on the hyaluronic acid synthesis of dermis (the expression of HAS-1 is much less than HAS-2), and HAS-3 acts on the hyaluronic acid synthesis of epidermis. HAS-2 is the main hyaluronic acid synthase in dermis, also known as master gene (Master gene) etc. Therefore, as shown in the embodiment described later, as fibroblast, for example, using KMST-6 cell, as the evaluation of the gene level promoted by HA synthesis, it is possible to focus on HAS-2 gene and evaluate.

[0025] In addition, HAS-1, HAS-2 or HAS-3 is the abbreviation of hyaluronan synthase (HAS is the abbreviation of Hyaluronic Acid Synthase). HAS-1 gene, HAS-2 gene or HAS-3 gene refers to a gene expressing the hyaluronan synthase.

[0026] In addition, the preferred embodiment of the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention is characterized in that, further under ultraviolet irradiation, the synthesis of hyaluronic acid in the dermis is promoted. This is because the ability of known fibroblasts to produce HA will decay due to the stimulation of age or ultraviolet rays, but surprisingly, in the present invention, even under ultraviolet irradiation, the hyaluronic acid synthesis ability can be maintained. That is, although ultraviolet irradiation decays the expression of the synthase of the extracellular matrix and enhances the expression of the degrading enzyme, as demonstrated in the embodiments described later, in the present invention, by irradiation of ultraviolet A waves (UVA), the amount of synthetic HA (e.g., with reference to Figure 2 ), expression of HAS-2 (for example, see Figure 3) were decreased, and as a result, they were increased by the presence of pomegranate seed extract (PSE).

[0027] In addition, a preferred embodiment of the pharmaceutical composition for promoting hyaluronic acid synthesis in the dermis of the present invention is characterized in that the crushed material obtained by crushing pomegranate seeds is immersed in at least one solvent selected from ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, water and hexane, and the supernatant is separated to obtain the above-mentioned pomegranate seed extract. For example, vibration extraction can be performed. In the oscillation extraction, for example, it can be set on a rotator in a low-temperature chamber of about 4°C, and extraction can be performed while rotating. In addition, a preferred embodiment of the pharmaceutical composition for promoting hyaluronic acid synthesis in the dermis of the present invention is characterized in that the solvent is ethanol.

[0028] In addition, the method for producing a pharmaceutical composition for promoting hyaluronic acid synthesis in the dermis of the present invention is characterized in that it includes the following steps: a step of immersing a crushed product obtained by crushing pomegranate seeds in a solvent and then separating the supernatant; a step of obtaining a pomegranate seed extract by separating the supernatant; and a step of adjusting the obtained pomegranate seed extract to an effective amount.

[0029] In addition, in a preferred embodiment of the method for producing a pharmaceutical composition for promoting hyaluronic acid synthesis in the dermis of the present invention, from the perspective of its application and suitability for food, the ethanol extract of pomegranate seeds extracted with ethanol is further partitioned between ethyl acetate and water, and the ethyl acetate layer is concentrated under reduced pressure to obtain a pomegranate seed extract as an ethyl acetate fraction.

[0030] In addition, in a preferred embodiment of the method for producing a pharmaceutical composition for promoting hyaluronic acid synthesis in the dermis of the present invention, from the viewpoint of identifying the active ingredient, the ethyl acetate fraction is further partitioned between hexane and methanol to obtain a pomegranate seed extract as an ethyl acetate-hexane fraction and an ethyl acetate-methanol fraction.

[0031] In addition, the ethyl acetate-hexane fraction (PSE-EA-H) and the ethyl acetate-methanol fraction (PSE-EA-Me) are as follows. That is, the pomegranate seed-ethanol extract (PSE) extracted with ethanol is divided into ethyl acetate and water using a separatory funnel, and the solvent of the ethyl acetate layer is concentrated under reduced pressure by an evaporator to prepare the ethyl acetate fraction (PSE-EA), and the solvent of the water layer is freeze-dried to prepare the water fraction (PSE-W). At this time, PSE-EA is further divided into hexane and methanol, and then the solvent is concentrated under reduced pressure by an evaporator, respectively, to obtain the ethyl acetate-hexane fraction (PSE-EA-H) and the ethyl acetate-methanol fraction (PSE-EA-Me).

[0032] Here, first, an example of a method for preparing a pomegranate seed extract is described. First, pomegranate seeds are prepared. The pomegranate seeds are washed and dried as needed. Drying is preferably performed sufficiently. In order to make the subsequent pulverization uniform.

[0033] Next, the pomegranate seeds are crushed. The crushing method is not particularly limited, and a known crusher such as a ball mill, a hammer mill, a roller mill, a rod mill, a sample mill, a pounding mill, a crusher, a mortar, a mixer with a cooling device, etc. can be used. It should be noted that the crushing time can be set to several seconds and repeated more than ten times in consideration of the decomposition of the pomegranate seed composition due to heat generation during crushing.

[0034] Next, the pomegranate seeds are crushed to obtain a crushed product, and then the crushed product is immersed in various solvents. The solvent at this time is not particularly limited, and the solvent can be appropriately set according to the desired effect. In addition, a preferred embodiment of the method for producing a pomegranate seed extract of the present invention is characterized in that the solvent is at least one selected from ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, hexane and water. As the solvent, polar and non-polar solvents such as ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, water, hexane, ethyl acetate, chloroform, acetone, etc. can be mentioned. Preferably, ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, methanol, ethanol, water, etc. can be mentioned.

[0035] The impregnation can be carried out under slow stirring. The above-mentioned pulverized material is impregnated in various solvents to obtain various solutions. Various solutions can be stirred according to the state of the solution, or the solution can be left as it is according to the situation. There is no particular limitation on the stirring time, and the stirring can be continued for 10 hours to 48 hours, preferably for about 1 day (24 hours).

[0036] Then, the pomegranate seed extract can be obtained by fractionating the supernatant. If necessary, the supernatant is evaporated to dryness. The evaporation to dryness can be performed using an evaporator in 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 time.

[0037] The components contained in the pomegranate seeds are extracted (extracted) by using solvents with different polarities, and separated by shaking according to their physical properties. Therefore, the types and contents of the components of the pomegranate seed extract differ depending on the solvent used.

[0038] In addition, the quasi-drug (quasi-drug) of the present invention is characterized in that the composition of the present invention is an active ingredient. There is no particular limitation on the quasi-drug (quasi-drug) applicable to the present invention as long as the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention is an active ingredient.

[0039] <Effective amount> The composition of the present invention is prepared in the form of an effective amount of pomegranate seed extract and a suitable dosage form.

[0040] The dosage of the pomegranate seed extract in the composition of the present invention can be varied according to the condition and severity of the patient, the dosage form, the selected administration route, the number of administrations per day, and the like.

[0041] The dosage of the pomegranate seed extract in the composition of the present invention can be set at 1000 mg / kg / day when administered to mice. In humans, a lower dosage is preferably used depending on the sensitivity of the human body.

[0042] In addition, the dosage form includes oral preparations (tablets, capsules, coated tablets, granules, solutions, syrups), ointments or creams for application, etc. From the perspective of skin moisture replenishment and barrier function, the subject of administration can be female, male, adult, child, etc.

[0043] The dosage form may contain other conventional ingredients such as a storage stabilizer, a sweetener, a colorant, a flavoring agent, and the like.

[0044] <Acute toxicity test> Regarding the ingredients contained in pomegranate seed extract, since it contains linolenic acid, it is considered to be non-toxic.

[0045] 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: Hyaluronan Binding Protein HAS: Hyaluronic acid synthase PBS: Phosphate buffered saline PSE: Punica gtanatumSeed 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 light Example

[0046] An embodiment of the present invention will be described below, but the present invention is not limited to the following embodiment.

[0047] Example 1 First, the skin functionality of pomegranate extract, specifically, the synthesis of hyaluronic acid in the dermis was investigated.

[0048] <Abbreviation list> PSE: Punica granatum Seed Ethanol extract FBS: Fetal Bovine Serum

[0049] Dermis is mainly composed of proteins collectively referred to as extracellular matrix, and these components are produced by intermittent fibroblasts. In the extracellular matrix of human dermis, as a factor with water retention effect, hyaluronic acid (HA) is synthesized. Because the metabolic rate of HA is large, it is necessary to make it persist in the skin, but because the ability of fibroblasts to produce HA can be attenuated due to the increase of age or the stimulation of ultraviolet rays, it is important to improve and improve the ability of fibroblasts to produce HA. HA also plays a role in retaining moisture in the epidermis composed of keratinocytes. In the present embodiment, KMST-6 is used as the fibroblasts of dermis, and the change of HA amount in the supernatant when these cells are cultivated in the presence of PSE is quantitatively evaluated. About KMST-6, the same test when irradiating UVA and the expression evaluation of the leading gene of HA synthase, i.e. HAS-2 gene, expressed in dermis are carried out.

[0050] First, the preparation of pomegranate seed extract was attempted using ethanol as a solvent. Pomegranate seeds were crushed and extracted with 100% ethanol by shaking. The seeds were concentrated to dryness and redissolved to prepare pomegranate seed-ethanol extract for the experiment. Furthermore, the pomegranate seed-ethanol extract (hereinafter also referred to as PSE) extracted with ethanol was distributed (extracted) in ethyl acetate and water using a separatory funnel, and the solvent of the ethyl acetate layer was concentrated under reduced pressure by an evaporator to prepare an ethyl acetate fraction (PSE-EA). The water layer was freeze-dried to prepare a water fraction (PSE-W). At this time, PSE-EA was distributed in hexane and methanol, and the solvents were then concentrated under reduced pressure by evaporators respectively, and the obtained fractions became ethyl acetate-hexane fractions (hereinafter also referred to as PSE-EA-H) and ethyl acetate-methanol fractions (hereinafter also referred to as PSE-EA-Me). The details are as follows.

[0051] <Preparation method of PSE (pomegranate seed-ethanol extract)> The dried pomegranate seeds were powdered using a grinder, and 10 times the amount of 100% ethanol (manufactured by Wako Pure Chemical Industries, Ltd.) was added, followed by low-temperature shaking extraction at 4 °C for 24 hours. After 24 hours, centrifugation was performed at room temperature at 3000 rpm for 20 minutes, and the supernatant was recovered. The concentrate obtained by concentrating the supernatant under reduced pressure was used as the pomegranate seed-ethanol extract (PSE: Punicagranatum Seed Ethanol extract).

[0052] <Preparation method of ethyl acetate-hexane fraction (PSE-EA-H) and ethyl acetate-methanol fraction (PSE-EA-Me)> 2.00 g of PSE was partitioned using a separating funnel in ethyl acetate (100 ml × 3) and MilliQ water (ultrapure water) (100 ml). The solvent in the ethyl acetate layer was concentrated under reduced pressure using an evaporator to prepare the ethyl acetate fraction (PSE-EA). The aqueous layer was lyophilized to prepare the aqueous fraction (PSE-W). Further, PSE-EA was partitioned in hexane (100 ml × 3) and methanol (100 ml), and then the solvents were 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 a higher yield was passed through a column (30φ × 300 mm) in the order of hexane (100 ml), a mixture of hexane and ethyl acetate (when hexane:ethyl acetate = 9:1 (hexane 90 mL, ethyl acetate 10 mL), hexane:ethyl acetate = 8:2 (hexane 80 mL, ethyl acetate 20 mL), hexane:ethyl acetate = 6:4 (hexane 40 mL, ethyl acetate 60 mL)), ethyl acetate (100 ml), and methanol (100 ml) for partitioning. The yield of the fraction was calculated according to the following formula, Figure 4 A flowchart showing the preparation of the fraction is presented.

[0053] Yield (%) = (mass of fraction (g) / mass of PSE (g)) × 100

[0054] The prepared extract was stored frozen at -20 °C until used in the experiment.

[0055] <Experimental method> <Cell culture> The conditions for cell culture are described below. For clarification, the following KMST-6 was used as the fibroblast.

[0056] · Cells used: KMST-6 · Cell density at inoculation: 4.5×10 4cells / mL (6.0×10 4 cells / mL) · Culture medium used: MEMα (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0057] <Pre-cultivation> KMST-6 was seeded into a 96-well plate at the above final concentration and cultured in a medium containing 10% FBS for about 15 hours to allow the cells to fully adhere to the bottom surface.

[0058] <Formal training> All wells were washed once with PBS, and the culture medium was replaced with a PSE-prepared medium containing 1% FBS-ITES (control without PSE), and cultured at 37°C and 5% CO2 concentration. This culture was performed for 48 hours.

[0059] <UVA irradiation test (KMST-6)> For the cells used in the UVA irradiation test, the culture medium used was changed from the PSE preparation medium containing 1% FBS-ITES to the PSE preparation medium containing 5% FBS, and the culture medium was carried out for 24 hours. Then, the culture medium was replaced with MEMα containing 1% FBS-ITES but without phenol red, and irradiated on ice at 10 J / cm 2 UVA (about 50 minutes). During this period, considering the increase in liquid temperature caused by UVA irradiation, the non-irradiated group was placed on ice in a 32°C space. After the UVA treatment, the culture plate was directly returned to the incubator and cultured at 37°C and 5% CO2 concentration, and the culture was terminated after 24 hours.

[0060] <HA quantification (competitive ELISA)> · Make HA ELISA plate Sodium hyaluronate was prepared at a concentration of 0.5 mg / ml using 50 mM carbonate buffer, added to a 96-well plate for ELISA at 100 μL / well, and immobilized at 4°C overnight or at 37°C for 1 hour. Then, it was washed 3-4 times with 0.05% Tween 20-PBS, and 5% skim milk-PBS (hereinafter referred to as blocking solution) was added at 200 μL / well, and blocked at 4°C overnight or at 37°C for 30 minutes.

[0061] Competitive reaction The HA ELISA plate was washed 3-4 times with 0.05% Tween 20-PBS, and 25 μL of the culture supernatant or 100 μL of the standard solution after the culture was completed was added to the ELISA plate together with 500 ng / mL HABP-blocking solution at 50 μL / well. The total amount was adjusted to 200 μL with the blocking solution, and the competition reaction was carried out by shaking for 1 hour.

[0062] Secondary antibody reaction and color development reaction, absorbance measurement After washing 3-4 times with 0.05% Tween 20-PBS, add 250ng / mL HRP-labeled streptavidin-blocking solution at 100μL / well and let stand for 1 hour in a light-shielded state. Then wash in the same way and perform color reaction with TMB substrate solution. After standing at 37°C for 10-15 minutes, add 0.2M sulfuric acid solution at 100μL / well as a reaction stop solution. Measure the absorbance at the main wavelength of 450nm and the secondary wavelength of 630nm using a microplate reader.

[0063] Data processing The amount of HA in the culture supernatant was calculated from the calibration curve prepared based on the standard solution. The amount of HA in the culture supernatant in the presence of PSE was compared with that in the control group (set as 100%) and a graph was prepared.

[0064] <HAS-2 gene analysis (KMST-6)> KMST-6 was inoculated into a 6-well plate, and cultured and UVA irradiated in the same manner as above. After the culture was completed, all wells were washed with PBS, the cells were recovered, RNA was extracted, cDNA was synthesized, and real-time PCR was performed. MRPL19 was selected as an intrinsic control. MRPL19 is a gene used to correct the intrinsic control. In most gene expression evaluations, GAPDH or β-actin (β-actin) is mainly used, but due to the characteristics of cell proliferation and differentiation, they are sometimes not used as intrinsic controls, and this is also the case with fibroblasts (KMST-6 cells). For this reason, MRPL19 was selected as an intrinsic control. It should be noted that MRPL19 is a gene that is always expressed regardless of what the experimenter does or does not do, and serves as a benchmark.

[0065] <Experimental Results> The quantitative results of HA of KMST-6 are as follows Figure 1 shown. Figure 1 The figure shows the amount of HA (hyaluronic acid) in the culture supernatant of KMST-6. (n=5, mean ± standard error, ***p<0.001 vs. control). In the KMST-6 cells, it was confirmed that the amount of HA in the culture supernatant increased by adding PSE. In KMST-6, 10 μg / mL PSE produced the most HA, which was about 1.5 times.

[0066] In addition, regarding KMST-6, the quantitative results of HA in the culture supernatant when irradiated with UVA are as follows: Figure 2 shown. Figure 2The HA (hyaluronic acid) content in the culture supernatant of KMST-6 cells irradiated (or not irradiated) with UVA (ultraviolet A wave) is shown. (n=4, mean ± standard error)

[0067] The amount of HA produced in the UVA irradiation group was reduced to about 60% of that in the UVA non-irradiation group, but the reduction was reduced by culturing with the addition of PSE. In particular, 10 μg / mL PSE (10 μg / mL PSE relative to the total amount of the drug composition) with a high HA production rate improved HA production to more than 90% of the UVA non-irradiation group, which is about 1.5 times the amount of the UVA irradiation control group.

[0068] The results of the evaluation of HAS-2 expression in KMST-6 are as follows Figure 3 shown. Figure 3 The results of the evaluation of HAS-2 gene expression in KMST-6 are shown. (n=3, mean±standard error)

[0069] The decrease in HAS-2 expression due to UVA irradiation and the improvement trend produced by PSE were observed, suggesting that PSE can enhance the expression of HAS-2, which is the dominant gene of HA in the dermis.

[0070] in addition, Figure 4 The flow chart and yield of fraction preparation are shown. F1 to F4 are obtained by distributing PSE-EA-H with high yield. It is explained that further fractionation of PSE-EA-H to obtain F1 to F4 means that the fractions obtained after passing PSE-EA-H through the chromatographic column according to F1 being a mixture of hexane and ethyl acetate in a ratio of 8:2, F2 being a mixture of hexane and ethyl acetate in a ratio of 6:4, F3 being 100% ethyl acetate, and F4 being 100% methanol. The same effect was obtained with these pomegranate seed extracts. Industrial Applicability

[0071] According to the present invention, it is not enough to mention the contribution to the medical industry field, the social contribution such as the effective utilization of waste is high, and the utilization value is high in a wide range.

Claims

1. A pharmaceutical composition for promoting hyaluronic acid synthesis, characterized in that: Contains pomegranate seed extract to promote hyaluronic acid synthesis in the dermis.

2. The pharmaceutical composition for promoting hyaluronic acid synthesis according to claim 1, characterized in that Stimulates hyaluronic acid synthesis in fibroblasts of the dermis.

3. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition is a pharmaceutical composition that promotes hyaluronic acid synthesis by increasing the expression of hyaluronic acid synthase, and the hyaluronic acid synthase is HAS-1 or HAS-2.

4. The pharmaceutical composition for promoting hyaluronic acid synthesis according to any one of claims 1 to 3, characterized in that Further, UV irradiation promotes hyaluronic acid synthesis in the dermis.

5. A quasi-drug, characterized in that The pharmaceutical composition according to any one of claims 1 to 4 is used as an active ingredient.

6. A method for preparing a pharmaceutical composition for promoting hyaluronic acid synthesis in dermis, characterized in that: Including the following processes: The process of immersing the crushed product obtained by crushing pomegranate seeds in a solvent and then collecting the supernatant. a step of obtaining a pomegranate seed extract by fractionating the supernatant, and A step of adjusting the obtained pomegranate seed extract to an effective amount.

7. The method according to claim 6, characterized in that 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.

8. 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 between ethyl acetate and water. The ethyl acetate layer is concentrated under reduced pressure to obtain a pomegranate seed extract as an ethyl acetate fraction.

9. The method of claim 8, wherein: The ethyl acetate fraction was further partitioned between 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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