Pharmaceutical composition for cell proliferative activity and method for producing same

Abstract

The present invention relates to a pharmaceutical composition for cell proliferative activity and a method for producing the same. [Problem] The purpose of the present invention is to provide a composition having a cell proliferation effect, with the purpose of effectively using waste pomegranate seeds and increasing the added value thereof. [Solution] A pharmaceutical composition for cell proliferative activity according to the present invention is characterized by containing a pomegranate seed extract. Furthermore, a preferred embodiment of the pharmaceutical composition for cell proliferative activity according to the present invention is characterized in that the pomegranate seed extract is obtained by immersing a pulverized product obtained by pulverizing pomegranate seeds in at least one solvent selected from the group consisting of ethanol, methanol, water, hexane, and the like, and separating a supernatant.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for cell proliferation activity and a method for producing the pharmaceutical composition for cell proliferation activity, and particularly to a pharmaceutical composition for cell proliferation activity containing a pomegranate seed extract and a method for producing the pharmaceutical composition for cell proliferation activity. 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

[0003] Patent Literature 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 currently treated as waste in principle, 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 reviewed. In this case, if pomegranate seeds, which are waste, are effective against various diseases, it will be more beneficial.

[0005] Therefore, an object of the present invention is to provide a composition having a cell proliferation effect with the goal of effectively utilizing discarded pomegranate seeds and increasing their added value. Means of solving problems

[0006] 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, discovered the present invention.

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

[0008] In addition, a preferred embodiment of the pharmaceutical composition for cell proliferation activity of the present invention is characterized in that it includes the following steps: immersing the crushed material obtained by crushing pomegranate seeds in at least one solvent selected from ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, water and hexane, and collecting the supernatant to obtain the above-mentioned pomegranate seed extract.

[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 activity 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 targeted for proliferation activity are epidermal and / or dermal cells.

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

[0013] In addition, the method for producing a pharmaceutical composition for cell proliferation activity of the present invention is characterized in that it includes the following steps: a step of immersing a pulverized product obtained by pulverizing 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 the pharmaceutical composition for cell proliferation activity of the present invention, the solvent is at least one selected from ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, water and hexane. Effects of the Invention

[0015] The pharmaceutical composition for cell proliferation activity of the present invention has the advantageous effect of effectively utilizing pomegranate seeds that have been disposed of as waste in the past. In addition, the pharmaceutical composition for cell proliferation activity of the present invention has the advantageous effect of enhancing the proliferation and migration of cells constituting the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure shows the time-dependent changes in cell viability in HaCaT cells caused by the pomegranate seed extract when the composition according to one embodiment of the present invention is used. (n=4, mean±standard error, *p<0.05, **p<0.01 vs control) Figure 2The figure shows the changes in cell viability in KMST-6 cells caused by the pomegranate seed extract when the composition according to one embodiment of the present invention is used. (n=4, mean±standard error, *p<0.05, **p<0.01, ***p<0.001 vs control) Figure 3 The flow chart and yield of fraction preparation are shown. F1 to F4 were obtained by partitioning PSE-EA-H, which has a high yield. DETAILED DESCRIPTION

[0017] The cell proliferation activity pharmaceutical composition of the present invention is characterized by containing a pomegranate seed extract. The pomegranate seed extract is an extract derived from pomegranate seeds. The pomegranate seed extract applicable to the present invention is all pomegranate seed extracts as long as they are derived from pomegranate seeds.

[0018] In addition, a preferred embodiment of the pharmaceutical composition for cell proliferation activity of the present invention is characterized in that the cells that become the target of proliferation activity are cells of the epidermis and / or dermis. That is, the target cells are not particularly limited, and they act particularly effectively on skin cells, especially cells of the epidermis or dermis. Therefore, in this specification, skin cells are mainly described.

[0019] In addition, a preferred embodiment of 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, epidermal keratinocytes are not particularly limited, but from the viewpoint of being established as cell lines, examples thereof include HaCaT and the like.

[0021] 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).

[0022] In addition, a preferred embodiment of the pharmaceutical composition for cell proliferation activity of the present invention is characterized in that it includes the following steps: immersing the crushed material obtained by crushing pomegranate seeds in at least one solvent selected from ethyl acetate, ethyl acetate-hexane, ethyl acetate-methanol, ethanol, methanol, water and hexane, and separating the supernatant to obtain the above-mentioned pomegranate seed extract. For example, oscillation 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 cell proliferation activity of the present invention is characterized in that the above-mentioned solvent is ethanol.

[0023] 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 method for producing a pharmaceutical composition for cell proliferation activity characterized by containing a pomegranate seed extract of the present invention is characterized in that it includes the following steps: a step of immersing a pulverized product obtained by pulverizing 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.

[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 perspective of its application and suitability for food, it is characterized in that the ethanol extract of pomegranate seeds extracted with 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.

[0026] In a preferred embodiment of the method for producing the 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 between hexane and methanol to obtain a pomegranate seed extract as an ethyl acetate-hexane fraction and an ethyl acetate-methanol fraction.

[0027] 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).

[0028] 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.

[0029] 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.

[0030] 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 pharmaceutical composition for cell proliferation activity 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.

[0031] 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).

[0032] 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.

[0033] 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.

[0034] In addition, the quasi-drug (quasi-drug) of the present invention is characterized in that the composition of the present invention is used as an active ingredient. There are no particular limitations on the quasi-drug (quasi-drug) applicable to the present invention as long as the pharmaceutical composition for cell proliferation activity of the present invention is used as an active ingredient.

[0035] <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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] The dosage form may contain other conventional ingredients such as a storage stabilizer, a sweetener, a colorant, a flavoring agent, and the like.

[0040] <Acute toxicity test> Regarding the ingredients contained in pomegranate seed extract, since it contains linolenic acid, it is considered to be non-toxic.

[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: Hyaluronan Binding Protein ●HAS: Hyaluronan 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 rays Example

[0042] The following describes an embodiment of the present invention, but the present invention is not limited to the following embodiments for interpretation.

[0043] Example 1

[0044] <Preparation of pomegranate seed extract> <Sample> Pomegranate seeds: Purchased from Sunny Place Co., Ltd. <Equipment> Grinder: Manufactured by Iwatani Corporation Rotary evaporator: Manufactured by EYELA Tokyo Rika Kikai Co., Ltd. Freeze dryer FDU-1200: Manufactured by EYELA Tokyo Rika Kikai Co., Ltd. <Reagent> 100% ethanol: Manufactured by Wako Pure Chemical Industries, Ltd. Ethyl acetate: Manufactured by Wako Pure Chemical Industries, Ltd. Hexane: Manufactured by Wako Pure Chemical Industries, Ltd. Methanol: Manufactured by Wako Pure Chemical Industries, Ltd. Silica gel (Wakasil (registered trademark) 60, 64 - 210 μm): Manufactured by Wako Pure Chemical Industries, Ltd. MilliQ water <Apparatus> Chromatographic column (with filter) (30φ × 300 mm): Manufactured by Cosmos Bio Co., Ltd.

[0045] First, using ethanol as a solvent, an attempt was made to prepare pomegranate seed extract. The pomegranate seeds were crushed and extracted by shaking with 100% ethanol. It was concentrated to dryness and redissolved to prepare a pomegranate seed-ethanol extract for experiments. Further, the pomegranate seed-ethanol extract (hereinafter also referred to as PSE) extracted with ethanol was partitioned (extracted) in ethyl acetate and water using a separating funnel, and the solvent in the ethyl acetate layer was concentrated under reduced pressure using 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 partitioned in hexane and methanol, and then the solvents were concentrated under reduced pressure using an evaporator respectively. The obtained fractions became 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 described as follows.

[0046] <Preparation method of PSE (pomegranate seed-ethanol extract)> The dried pomegranate seeds were powdered with a grinder, and 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 and 3000 rpm for 20 minutes to recover the supernatant. The supernatant was concentrated under reduced pressure to obtain a concentrate, which was used as a pomegranate seed ethanol extract (PSE: Punicagranatum Seed Ethanol Extract).

[0047] <Method for preparing ethyl acetate-hexane fraction (PSE-EA-H) and ethyl acetate-methanol fraction (PSE-EA-Me)> PSE 2.00 g was distributed in ethyl acetate (100 ml x 3) and MiliQ water (ultrapure water) (100 ml) 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 solvent of the water layer was freeze-dried to prepare a water fraction (PSE-W). PSE-EA was further distributed in hexane (100 ml x 3) and methanol (100 ml), and the solvent was then concentrated under reduced pressure by an evaporator to obtain an ethyl acetate-hexane fraction (PSE-EA-H) and an ethyl acetate-methanol fraction (PSE-EA-Me). The high-yield PSE-EA-H was distributed through a chromatographic column (30φ×300mm) in the order of 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). The fraction yield was calculated according to the following formula: Figure 3 A flow chart showing fraction preparation is shown.

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

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

[0050] The cell proliferation activity was studied using the pomegranate seed extract thus prepared.

[0051] <Abbreviation list> ●PSE: Punica granatum Seed Ethanol extract ●FBS: Fetal Bovine Serum (Sigma-Aldrich) ●ITES: Insulin (Sigma-Aldrich), transferrin (Sigma-Aldrich), ethanolamine (Wako Pure Chemical Industries, Ltd.), and sodium selenite (Wako Pure Chemical Industries, Ltd.) were mixed so that their final concentrations in the culture medium were 5 μg / mL, 20 μg / mL, 20 μM, and 25 nM, respectively.

[0052] <Research Summary> The proliferation and migration of cells constituting the skin are important in wound healing, improvement of skin problems, and promotion of epidermal renewal. In this experiment, the cell proliferation activity that can be imparted by pomegranate seed ethanol extract was evaluated on two types of cells, keratinocytes constituting the epidermis and fibroblasts of the dermis.

[0053] <Experimental methods> -condition- <Keratinocytes> ■Cells used: HaCaT ■Cell density at inoculation: 5.0×10 4 cells / mL ■ Culture medium used: D-MEM (Fujifilm Wako Pure Chemical Industries, Ltd.)

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

[0055] <Pre-cultivation> Each cell was seeded into a 96-well plate (200 μL per well) 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.

[0056] <Formal training> All wells were washed once with PBS, and the culture medium was replaced with a medium containing 1% FBS-ITES. At this time, PSE was added.

[0057] <Determination of cell viability> The start time of formal culture was set to 0h, and the cell viability was determined by the WST-8 method at 0, 8, 16, and 24h. 10 μL of WST-8 kit solution diluted 2 times with PBS was added to all wells to generate formazan pigment through the reaction with coenzymes related to cell metabolism. After 3 hours, the absorbance at 450nm was measured to quantify the pigment. For all elapsed times of 0, 8, 16, and 24h, the ratio relative to the control at 0h was calculated to evaluate the cell proliferation activity generated by PSE.

[0058] <Experimental Results> In both HaCaT cells used as human keratinocytes constituting the epidermis and KMST-6 cells used as fibroblasts producing extracellular matrix components constituting the dermis, it was confirmed that the addition of PSE resulted in cell proliferation.

[0059] The temporal changes in the viability of HaCaT cells are shown in Figure 1 . Figure 1 The time course of cell survival rate in HaCaT cells is shown. (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 increased compared to the control. For cells to which PSE was added at a low concentration of 1 μg / mL, the proliferation rate was about 1.7 times that of the control at 8 hours, and the proliferation rate was particularly high in a short period of time. For cells to which PSE was added at a high concentration of 50 μg / mL, the proliferation rate was about 1.3 times that of the control at 24 hours, and the proliferation rate was high over a long period of time compared to low concentrations.

[0061] The time-dependent changes in KMST-6 cell survival rate are shown in Figure 2 As shown. (n=4, mean±standard error, *p<0.05, **p<0.01, ***p<0.001 vs control)

[0062] As in the case of HaCaT, the proliferation activity of cells in the presence of PSE was enhanced. A concentration-dependent increase was observed at 8 and 16 hours, but after 24 hours, the cells to which 10 μg / mL of PSE was added had the highest cell proliferation.

[0063] in addition, Figure 3The flow chart and yield of fraction preparation are shown. F1 to F4 are obtained by distributing PSE-EA-H with a large 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

[0064] 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 cell proliferation activity, characterized in that Contains pomegranate seed extract.

2. The composition according to claim 1, characterized in that The pomegranate seed extract is obtained by immersing the pulverized product obtained by pulverizing pomegranate seeds 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 collecting the supernatant.

3. The composition according to claim 2, characterized in that 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 relative to the total amount of the composition.

5. The composition according to any one of claims 1 to 4, characterized in that The cells to be the subject of the proliferation activity are cells of the epidermis and / or dermis.

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

7. A method for producing a pharmaceutical composition for cell proliferation activity containing a pomegranate seed extract, 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.

8. The method according to claim 7, 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, water and hexane.

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