Pharmaceutical composition for cell proliferation activity, and method for producing the pharmaceutical composition for cell proliferation activity

Abstract

To provide a composition that exhibits an effect in proliferating cells, targeting effectively utilizing discarded pomegranate seeds and increasing added value thereof.SOLUTION: A pharmaceutical composition for cell proliferation activity contains a pomegranate seed extract. In a preferable embodiment of the pharmaceutical composition for cell proliferation activity, a pulverized material prepared by pulverizing pomegranate seeds is immersed in at least one solvent selected from a group consisting of ethanol, methanol, water, and hexane, and a supernatant is separated to obtain the pomegranate seed extract.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for cell proliferation activity and a method for producing said pharmaceutical composition for cell proliferation activity, in particular to a pharmaceutical composition for cell proliferation activity containing pomegranate seed extract and a method for producing said pharmaceutical composition for cell proliferation activity. [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 the above-mentioned patent documents. On the other hand, effective utilization of waste materials is being reconsidered. Under such circumstances, it would be more beneficial if waste materials such as pomegranate seeds could be effective against various diseases.

[0005] Therefore, an object of the present invention is to provide a composition having a cell proliferation effect, aiming at effective utilization and added value of discarded pomegranate seeds. [Means for solving the problem]

[0006] 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.

[0007] That is, the pharmaceutical composition for cell proliferation activation of the present invention is characterized by containing pomegranate seed extract.

[0008] In addition, in a preferred embodiment of the pharmaceutical composition for cell proliferation activity of the present invention, the pomegranate seed extract is obtained by crushing pomegranate seeds, immersing the crushed product 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 separating the supernatant.

[0009] In a preferred embodiment of the pharmaceutical composition for cell proliferation activity of the present invention, the solvent is ethanol.

[0010] In a preferred embodiment of the pharmaceutical composition for cell proliferation activation of the present invention, the content of the pomegranate seed extract is 1 to 100 μg / mL relative to the total amount of the composition.

[0011] In a preferred embodiment of the pharmaceutical composition for cell proliferation activity of the present invention, the cells to be the target of the proliferation activity are epidermal and / or dermal cells.

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

[0013] In addition, the manufacturing method of the pharmaceutical composition for cell proliferation activity of the present invention is characterized by comprising the steps of crushing pomegranate seeds, immersing the crushed product 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] Furthermore, in a preferred embodiment of the method for producing a pharmaceutical composition for cell proliferation activity 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, water, and hexane. Effect of the Invention

[0015] The pharmaceutical composition for cell proliferation activation of the present invention has an advantageous effect of enabling effective use of pomegranate seeds, which have been conventionally disposed of as waste, and also has an advantageous effect of improving proliferation and migration of cells constituting the skin. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 is a graph showing the time course of changes in cell viability in HaCaT cells treated with pomegranate seed extract in a composition according to one embodiment of the present invention (n=4, mean ± standard error, *p < 0.05, **p < 0.01 vs. Control). [Diagram 2] 2 is a graph showing the change in cell viability in KMST-6 cells when a composition according to one embodiment of the present invention is used and pomegranate seed extract is administered (n=4, mean ± standard error, *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control). [Diagram 3]Figure 3 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

[0017] The pharmaceutical composition for cell proliferation activation of the present invention is characterized by containing 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.

[0018] In a preferred embodiment of the pharmaceutical composition for cell proliferation activity of the present invention, the target cells for proliferation activity are epidermal and / or dermal cells. That is, the target cells are not particularly limited, but the cells that act particularly effectively are skin cells, particularly epidermal or dermal cells. Therefore, the present specification will mainly describe skin cells.

[0019] In a preferred embodiment of the present invention, the present invention is characterized by promoting cell proliferation activity in keratinocytes constituting the epidermis or fibroblasts in the dermis.

[0020] In the present invention, the epidermal keratinocytes are not particularly limited, but examples thereof include HaCaT, which is an established cell line.

[0021] 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.

[0022] In a preferred embodiment of the pharmaceutical composition for cell proliferation activity 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 cell proliferation activity of the present invention, the solvent is ethanol.

[0023] In addition, in a preferred embodiment of the pharmaceutical composition for cell proliferation activity of the present invention, the content of the pomegranate seed extract is 1 to 100 μg / mL, more preferably 1 to 50 μg / mL, relative to the total amount of the composition, from the viewpoint of not causing a decrease in cell viability.

[0024] In addition, the manufacturing method of the pharmaceutical composition for cell proliferation activity 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.

[0025] In addition, in a preferred embodiment of the method for producing a pharmaceutical composition for cell proliferation activity 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.

[0026] In addition, in a preferred embodiment of the method for producing a pharmaceutical composition for cell proliferation activity of the present invention, from the viewpoint of identifying the active ingredient, the ethyl acetate fraction is further partitioned with hexane and methanol to obtain a pomegranate seed extract as an ethyl acetate-hexane fraction and an ethyl acetate-methanol fraction.

[0027] 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).

[0028] 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.

[0029] 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.

[0030] Next, the pomegranate seeds are pulverized to obtain a pulverized product, and the pulverized product is immersed in various solvents. The solvent in this case is not particularly limited, and can be appropriately selected according to the desired effect. In a preferred embodiment of the method for producing a pharmaceutical composition for cell proliferation activity 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] The quasi-drug of the present invention is characterized by containing 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 contains the pharmaceutical composition for cell proliferation activation of the present invention as an active ingredient.

[0035] <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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] The dosage form may contain other conventional ingredients, such as preservatives, sweeteners, colorants, flavoring agents, and the like.

[0040] <Acute toxicity test> As for the ingredients contained in pomegranate seed extract, including linoleic acid, it is believed that there is no toxicity.

[0041] 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

[0042] An embodiment of the present invention will be described below, but the present invention is not limited to the following embodiment.

[0043] Example 1

[0044] <Preparation of pomegranate seed extract> <Sample> Pomegranate seeds: Obtained from Sunny Place Co., Ltd. <Equipment> Blender: Manufactured by Iwatani Corporation Rotary evaporator: EYELA manufactured by Tokyo Rikakikai Co., Ltd. Freeze dryer FDU-1200: EYELA manufactured by Tokyo Rikakikai Co., Ltd. <Reagents> 100% Ethanol: 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 (registered trademark) 60, 64 - 210 μm): manufactured by Wako Pure Chemical Industries, Ltd. MilliQ water <Equipment> Chromatography column with filter (30φ × 300 mm): manufactured by Cosmos Bio Co., Ltd.

[0045] First, as a solvent, ethanol was used to attempt the preparation of pomegranate seed extract. Pomegranate seeds were ground and shaken and extracted with 100% ethanol. These were concentrated to dryness and redissolved, and used in the experiment as 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 of the ethyl acetate layer was concentrated under reduced pressure using an evaporator, and when the aqueous layer was freeze-dried with the solvent to obtain a water fraction (PSE-W), after partitioning PSE-EA with hexane and methanol, the solvents were each concentrated under reduced pressure using an evaporator, and the obtained fractions were used as an ethyl acetate - hexane fraction (hereinafter also referred to as PSE-EA-H) and an ethyl acetate - methanol fraction (hereinafter also referred to as PSE-EA-Me). Specifically, it is as follows.

[0046] <Preparation method of PSE (Punica granatum Seed Ethanol extract)> Dried pomegranate seeds were powdered with a miller, 10 times the amount of 100% ethanol (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and low-temperature shaking extraction was performed at 4°C for 24 hours. After 24 hours, centrifugation was performed at room temperature at 3,000 rpm for 20 minutes, and the supernatant was collected. The concentrate obtained by concentrating the supernatant under reduced pressure was used as pomegranate seed ethanol extract (PSE: Punica granatum Seed Ethanol extract).

[0047] <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.

[0048] Yield (%) = (mass of fraction (g) / mass of PSE (g)) x 100

[0049] The prepared extracts were stored frozen at -20°C until use in experiments.

[0050] The pomegranate seed extract thus prepared was used to examine its cell proliferation activity.

[0051] <List of abbreviations> · PSE: Punica granatum Seed Ethanol extract ·FBS:Fetal Bovine Serum(Sigma-Aldrich) ·ITES: Insulin (Sigma-Aldrich), Transferrin (Sigma-Aldrich), Ethanol amine (Wako Pure Chemical Industries, Ltd.), and Sodium Selenite (Wako Pure Chemical Industries, Ltd.) were mixed to give final concentrations in the culture medium of 5 μg / mL, 20 μg / mL, 20 μM, and 25 nM, respectively.

[0052] <Research overview> The proliferation and migration of cells that compose the skin are important for wound healing, improving skin troubles, and promoting turnover in the epidermis. In this experiment, we evaluated the cell proliferation activity that pomegranate seed ethanol extract can have on two types of cells: keratinocytes that compose the epidermis and fibroblasts in the dermis.

[0053] <Experimental Method> -conditions- <Keratinocytes> ·Cells used: HaCaT Cell density at seeding: 5.0×10 4 cells / mL Culture medium used: D-MEM (FUJIFILM Wako Pure Chemical Industries, Ltd.)

[0054] <Fibroblast cells> ·Cells used: KMST-6 Cell density at seeding: 3.0×10 4 cells / mL Culture medium used: MEMα (FUJIFILM Wako Pure Chemical Industries, Ltd.)

[0055] <Preculture> Each type of cell was seeded at the above final concentration in separate 96-well plates (200 μL per well). The cells were cultured in a 10% FBS-containing medium for about 15 hours to allow sufficient adhesion to the bottom surface.

[0056] <Main culture> All wells were washed once with PBS, and the culture medium was replaced with a medium containing 1% FBS-ITES, to which PSE was added.

[0057] <Measurement of cell viability> The start of the main culture was defined as 0 h, and cell viability was measured using the WST-8 method at 0, 8, 16, and 24 h. 10 μL of WST-8 kit solution diluted 2-fold with PBS was added to all wells, and formazan dye was generated by reaction with coenzymes involved in cell metabolism. After 3 hours, the absorbance at 450 nm was measured and the amount of dye was quantified. The ratio of the 0-h control to the 0-h control was calculated for all elapsed times of 0, 8, 16, and 24 h, and the cell proliferation activity of PSE was evaluated.

[0058] <Experimental Results> Cell proliferation by addition of PSE was confirmed in both HaCaT cells, which were used as human keratinocytes that make up the epidermis, and KMST-6 cells, which were used as fibroblasts that produce components of the extracellular matrix that make up the dermis.

[0059] The time course of cell viability in HaCaT cells is shown in Figure 1. Figure 1 shows the time course of cell viability in HaCaT cells (n=4, mean ± standard error, *p < 0.05, **p < 0.01 vs. Control).

[0060] The proliferation activity of cells in the presence of PSE was improved compared to the control. For cells treated with a low concentration of 1 μg / mL PSE, the proliferation rate was about 1.7 times higher than the control at 8 hours, which was particularly high in the short term, while for cells treated with a high concentration of 50 μg / mL PSE, the proliferation rate was about 1.3 times higher than the control at 24 hours, which was higher over a long period of time compared to the low concentration.

[0061] The time course of cell viability in KMST-6 is shown in Figure 2. (n=4, mean ± standard error, *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control)

[0062] As with HaCaT, proliferation activity was increased in cells treated with PSE. A concentration-dependent increase was observed at 8 and 16 h, but at 24 h, cells treated with 10 μg / mL PSE showed the highest cell proliferation.

[0063] FIG. 3 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]

[0064] 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 activating cell proliferation, comprising pomegranate seed extract.

2. The composition according to claim 1, characterized in that the pomegranate seed extract is obtained by crushing pomegranate seeds, immersing the crushed product 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 separating the supernatant.

3. 3. The composition of claim 2, wherein the solvent is ethanol.

4. The composition according to any one of claims 1 to 3, wherein the content of the pomegranate seed extract is 1 to 100 µg / mL based on the total amount of the composition.

5. The composition according to any one of claims 1 to 4, wherein the cells to be targeted for proliferation activity are epidermal and / or dermal cells.

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

7. A method for producing a pharmaceutical composition for cell proliferation activity containing pomegranate seed extract, comprising the steps of: immersing the resulting pulverized product 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.

8. 8. The method according to claim 7, wherein the solvent is at least one selected from the group consisting of ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, water, and hexane.

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