Pharmaceutical composition for enhancing hyaluronic acid synthesis and methods for producing pharmaceutical compositions containing pomegranate seed extract for enhancing hyaluronic acid synthesis

Abstract

To provide pharmaceutical compositions that enhance hyaluronic acid synthesis aiming at effective utilization of and value addition to pomegranate seeds to be disposed.SOLUTION: The invention provides a pharmaceutical composition for enhancing hyaluronic acid synthesis that comprises a pomegranate seed extract. In a preferred embodiment, the pharmaceutical composition for enhancing hyaluronic acid synthesis enhances the synthesis of hyaluronic acid in dermal fibroblasts. In a preferred embodiment, the pharmaceutical composition for enhancing hyaluronic acid synthesis increases the expression of a hyaluronic acid synthesizing enzyme, where the hyaluronic acid synthesizing enzyme is HAS-1 or HAS-2.SELECTED DRAWING: Figure 1

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 in particular to a pharmaceutical composition for promoting hyaluronic acid synthesis in the dermis, which contains pomegranate seed extract, and a method for producing the pharmaceutical composition for promoting hyaluronic acid synthesis. [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 that has the effect of promoting hyaluronic acid synthesis, 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 hyaluronic acid synthesis of the present invention is a pharmaceutical composition for promoting hyaluronic acid synthesis containing pomegranate seed extract, and is characterized by promoting the synthesis of hyaluronic acid in the dermis.

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

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

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

[0012] Furthermore, the quasi-drug of the present invention is characterized by containing the pharmaceutical composition of the present invention as an active ingredient.

[0013] In addition, the manufacturing method of the pharmaceutical composition for promoting hyaluronic acid synthesis in the dermis of the present invention is characterized by 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.

[0014] 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, 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.

[0015] 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, 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 pomegranate seed extract as an ethyl acetate fraction.

[0016] 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, the ethyl acetate fraction is further partitioned with hexane and methanol to obtain pomegranate seed extract as an ethyl acetate-hexane fraction and an ethyl acetate-methanol fraction. Effect of the Invention

[0017] According to the pharmaceutical composition for promoting hyaluronic acid synthesis in dermis of the present invention, it is possible to effectively utilize pomegranate seeds that have been treated as waste in the past.In addition, according to the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention, it has the effect of promoting hyaluronic acid synthesis in dermis, and is effective in improving moisturizing function. [Brief description of the drawings]

[0018] [Figure 1] Figure 1 shows the amount of hyaluronic acid (HA) in the culture supernatant of KMST-6 cells (n=5, mean ± standard error, ***p < 0.001 vs. Control). [Diagram 2] Figure 2 shows the amount of HA (hyaluronic acid) in the culture supernatant of KMST-6 cells irradiated (or not irradiated) with UVA (ultraviolet A rays) (n=4, mean ± standard error). [Diagram 3] FIG. 3 shows the results of evaluating HAS-2 gene expression in KMST-6 (n=3, mean ± standard error). [Figure 4] Figure 4 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

[0019] The pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention is a pharmaceutical composition for promoting hyaluronic acid synthesis that contains pomegranate seed extract, and is characterized by promoting the synthesis of hyaluronic acid in the dermis.Pomegranate seed extract is an extract derived from pomegranate seed.The pomegranate seed extract applied to the present invention covers all pomegranate seed extracts as long as they are derived from pomegranate seed.

[0020] Although the target cells are not particularly limited, the cells on which the present invention acts are particularly effective are skin cells, particularly dermal cells, and therefore, the present specification will mainly describe dermal cells.

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

[0022] In the present invention, the dermal fibroblasts are not particularly limited, but examples thereof include KMST-6, NHDF (Normal Human Dermal Fibroblasts), etc., which are established as experimental cell lines.

[0023] In a preferred embodiment, the hyaluronic acid synthase is characterized in that, from the viewpoint of improving the moisture retention function of cells such as skin, HAS-2 and HAS-1 are produced by fibroblasts in the dermis, and HAS-3 is produced by keratinocytes (epidermal keratinocytes) in the epidermis. That is, the skin plays a role in protecting the body from the outside world and preventing moisture loss, and it is very important to maintain the skin in a normal state. Major regulators involved in the barrier function and moisture retention function of the skin include cornified membrane (CE) and hyaluronic acid (HA), and this invention has revealed that it is possible to promote the synthesis of hyaluronic acid in the dermis. That is, hyaluronic acid (HA) plays a role in retaining moisture in both the epidermis and dermis, and it has been revealed that it is synthesized by keratinocytes (e.g., HaCaT cells) in the epidermis and fibroblasts (e.g., KMST-6 cells) in the dermis. Therefore, as in the examples described below, it is possible to evaluate the dermis by performing a quantitative evaluation of fibroblasts.

[0024] It has also been discovered that there are three isoenzymes of HA synthesis enzymes, HAS-1, 2, and 3, and that HAS-1 and 2 function in hyaluronic acid synthesis in the dermis (expression of HAS-1 is much lower than that of HAS-2), while HAS-3 functions in hyaluronic acid synthesis in the epidermis. HAS-2 is the main hyaluronic acid synthesis enzyme in the dermis, and is also known as the master gene. Therefore, as shown in the examples below, it is possible to use fibroblasts, such as KMST-6 cells, and focus on the HAS-2 gene to evaluate the gene level of HA synthesis promotion.

[0025] In addition, HAS-1, HAS-2, and HAS-3 refer to abbreviations for hyaluronic acid synthase (HAS is an abbreviation for Hyaluronic Acid Synthase), and the HAS-1 gene, HAS-2 gene, and HAS-3 gene refer to genes that express the hyaluronic acid synthase.

[0026] In addition, in a preferred embodiment of the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention, it is characterized by promoting the synthesis of hyaluronic acid in the dermis under ultraviolet irradiation.It is known that the HA production ability of fibroblasts is attenuated by aging or stimuli such as ultraviolet rays, but surprisingly, in the present invention, it is found that the hyaluronic acid synthesis ability can be maintained even under ultraviolet irradiation.That is, it is said that ultraviolet irradiation attenuates the expression of the synthesis enzyme of the extracellular matrix and enhances the expression of the decomposition enzyme, but as is clear from the examples described below, in the present invention, it is found that the amount of synthesized HA (e.g., see Figure 2) and the expression of HAS-2 (e.g., see Figure 3) are reduced by irradiation with ultraviolet A rays (UVA), while the presence of pomegranate seed extract (PSE) increases them.

[0027] In addition, in a preferred embodiment of the pharmaceutical composition for promoting hyaluronic acid synthesis in the dermis of the present invention, the pulverized product obtained by pulverizing pomegranate seeds 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, it can be extracted by shaking. In the shaking extraction, for example, it can be set on a rotator in a low-temperature room such as about 4°C and rotated while extracting. In addition, in a preferred embodiment of the pharmaceutical composition for promoting hyaluronic acid synthesis in the dermis of the present invention, it is characterized by being ethanol.

[0028] In addition, the manufacturing method of the pharmaceutical composition for promoting hyaluronic acid synthesis in the dermis of the present invention is characterized by 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.

[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 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 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 method is further characterized in that the ethyl acetate fraction is partitioned with hexane and methanol to obtain pomegranate seed extract as an ethyl acetate-hexane fraction and an ethyl acetate-methanol fraction.

[0031] 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).

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] In addition, the quasi-drug of the present invention is characterized by having the composition of the present invention as an active ingredient. The quasi-drug applicable to the present invention is not particularly limited as long as it has the pharmaceutical composition for promoting hyaluronic acid synthesis of the present invention as an active ingredient.

[0039] <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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] The dosage form may contain other conventional ingredients, such as preservatives, sweeteners, colorants, flavoring agents, and the like.

[0044] <Acute toxicity test> As for the ingredients contained in pomegranate seed extract, including linoleic acid, it is believed that there is no toxicity.

[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: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

[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, we investigated the skin functionality of pomegranate extract, specifically, the synthesis of hyaluronic acid in the dermis.

[0048] <List of abbreviations> · PSE: Punica granatum Seed Ethanol extract ·FBS: Fetal Bovine Serum

[0049] The dermis is mainly composed of proteins collectively called the extracellular matrix, and these components are produced by fibroblasts present in between. Among the extracellular matrix in human dermis, hyaluronic acid (HA) is synthesized as a factor that plays a role in water retention. Since HA has a high metabolic rate and needs to be continuously present in the skin, but the ability of fibroblasts to produce HA declines due to stimuli such as aging and ultraviolet rays, it is important to improve or enhance the HA-producing ability of fibroblasts. HA also plays a role in water retention in the epidermis composed of keratinocytes. In this example, KMST-6 was used as dermal fibroblasts, and a quantitative evaluation test was conducted on the change in the amount of HA in the supernatant when these were cultured in the presence of PSE. In addition, for KMST-6, a similar test was conducted when irradiated with UVA, and an expression evaluation was performed on the HAS-2 gene, which is the HA synthase master gene expressed in the dermis.

[0050] First, as a solvent, ethanol was used to attempt the preparation of pomegranate seed extract. The pomegranate seeds were ground and shaken and extracted with 100% ethanol. These were concentrated to dryness and redissolved, and used in the experiment as the pomegranate seed ethanol extract. Further, 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, the solvents were each concentrated under reduced pressure using an evaporator, and the obtained 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.

[0051] <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).

[0052] <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×300 mm) in the following order: hexane (100 ml), a mixture of hexane and ethyl acetate (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) were poured into the column and distributed. The fraction yield was calculated from the following formula, and the flow chart of fraction preparation is shown in Figure 4.

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

[0054] The prepared extracts were stored frozen at -20°C until use in experiments.

[0055] <Experimental Method> <Cell culture> The cell culture conditions are as follows. As the fibroblast, KMST-6 below was used.

[0056] · Cells used: KMST-6 · Cell density at seeding: 4.5×10 4 cells / mL (6.0×104 cells / mL for gene analysis) · Medium used: MEMα (FUJIFILM Wako Pure Chemical Corporation)

[0057] <Pre-culture> KMST-6 was seeded in a 96-well plate at the above final concentration. It was cultured in a medium containing 10% FBS for about 15 hours to allow sufficient adhesion to the bottom surface.

[0058] <Main culture> All wells were washed once with PBS, and the culture medium was replaced with a PSE-prepared medium containing 1% FBS-ITES (Control does not contain PSE), and cultured at 37°C and 5% CO2 concentration. This main culture was carried out for 48 hours.

[0059] <UVA irradiation test (KMST-6)> For the cells used in the UVA irradiation test, the main culture was carried out for 24 hours, and the medium used was changed from a PSE-prepared medium containing 1% FBS-ITES to a PSE-prepared medium containing 5% FBS. Then, the culture medium was replaced with a phenol red-free MEMα containing 1% FBS-ITES, and irradiated with 10 J / cm 2 of UVA (for about 50 minutes) on ice. During this period, the non-irradiated group was placed on ice in a 32°C space considering the increase in liquid temperature due to UVA irradiation. After the UVA treatment was completed, the plate was immediately returned to the incubator and cultured at 37°C and 5% CO2 concentration, and the culture was terminated at the time point 24 hours later.

[0060] <HA quantification (competitive ELISA)> · Preparation of HA ELISA plate Sodium hyaluronate was prepared with 50 mM carbonate buffer to a concentration of 0.5 mg / ml, and 100 μL / well was added to a 96-well plate for ELISA and immobilized overnight at 4°C or for 1 hour at 37°C. Then, it was washed 3 - 4 times with 0.05% Tween20-PBS, and 200 μL / well of 5% skim milk-PBS (hereinafter referred to as blocking solution) was added, and blocking was carried out overnight at 4°C or for 30 minutes at 37°C.

[0061] · Competitive reaction The HA ELISA plate was washed 3 - 4 times with 0.05% Tween20-PBS, and 25 μL of the culture supernatant after the end of culture or 100 μL of the standard solution was added to the ELISA plate together with 50 μL / well of 500 ng / mL HABP-blocking solution. After adjusting the total volume to 200 μL with the blocking solution, the mixture was shaken for 1 hour to carry out the competitive reaction.

[0062] · Secondary antibody reaction, color reaction, absorbance measurement After washing 3 - 4 times with 0.05% Tween 20-PBS, 100 μL / well of 250 ng / mL HRP-labeled streptavidin-blocking solution was added and left standing for 1 hour in the dark. Then, it was washed in the same manner, and a color reaction was carried out with the TMB substrate solution. After standing at 37°C for 10 - 15 minutes, 100 μL / well of 0.2 M sulfuric acid solution was added as a reaction stop solution. The absorbance at a main wavelength of 450 nm and a sub-wavelength of 630 nm was measured using a microplate reader.

[0063] · Data processing Based on the calibration curve prepared from the standard solution, the amount of HA in the culture supernatant was calculated. The amount of HA in the culture supernatant in the presence of PSE was compared with the Control set as 100%, and a graph was created.

[0064] <HAS-2 gene analysis (KMST-6)> KMST-6 were seeded in a 6-well plate, and cultured and irradiated with UVA in the same manner as above. After the culture was completed, all wells were washed with PBS to recover the cells, and RNA was extracted, cDNA was synthesized, and real-time PCR was performed. MRPL19 was selected as an endogenous control. MRPL19 is a gene used for correction as an endogenous control. In most gene expression evaluations, GAPDH and β-actin are mainly used, but there are cases where these cannot be used as endogenous controls due to the characteristics of cell proliferation and differentiation, and fibroblasts (KMST-6 cells) fall into this category. For this reason, MRPL19 was selected as an endogenous control. MRPL19 is a standard gene that is always expressed regardless of what the experimenter does or does not do.

[0065] <Experimental Results> The results of HA quantification for KMST-6 are shown in Figure 1. Figure 1 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 these KMST-6 cells, an increase in the amount of HA in the culture supernatant due to the addition of PSE was confirmed. In KMST-6, 10 μg / mL PSE produced the most HA, approximately 1.5 times higher.

[0066] The quantitative results of HA in the culture supernatant of KMST-6 when irradiated with UVA are shown in Figure 2. Figure 2 shows the amount of HA (hyaluronic acid) in the culture supernatant of KMST-6 irradiated (or not irradiated) with UVA (ultraviolet A rays) (n=4, mean ± standard error).

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

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

[0069] HAS-2 expression was decreased by UVA irradiation and tended to improve with PSE, suggesting that PSE may increase the expression of HAS-2, the master gene for HA in the dermis.

[0070] FIG. 4 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]

[0071] 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 hyaluronic acid synthesis, which contains pomegranate seed extract and is characterized by promoting the synthesis of hyaluronic acid in the dermis.

2. The pharmaceutical composition for promoting hyaluronic acid synthesis according to claim 1, characterized in that it promotes hyaluronic acid synthesis in dermal fibroblasts.

3. The pharmaceutical composition according to claim 1, characterized in that the pharmaceutical composition promotes hyaluronic acid synthesis by increasing 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, further comprising a step of promoting hyaluronic acid synthesis in the dermis under ultraviolet irradiation.

5. A quasi-drug comprising the pharmaceutical composition according to any one of claims 1 to 4 as an active ingredient.

6. A method for producing a pharmaceutical composition for promoting hyaluronic acid synthesis in the dermis, 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.

7. 7. The method according to claim 6, 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.

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 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.

9. 9. The method of claim 8, 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.

Images