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

Abstract

The present invention relates to a pharmaceutical composition for promoting collagen 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 collagen synthesis, with the purpose of effectively using waste pomegranate seeds and increasing the added value thereof. [Solution] A pharmaceutical composition for promoting collagen synthesis according to the present invention is characterized by containing a pomegranate seed extract. Furthermore, a preferred embodiment of the pharmaceutical composition for promoting collagen synthesis according to the present invention is characterized in that the pharmaceutical composition inhibits the expression of the MMP-1 gene. Furthermore, a preferred embodiment of the pharmaceutical composition for promoting collagen synthesis 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, ethyl acetate and butanol and separating the supernatant.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for promoting collagen synthesis and a method for producing the pharmaceutical composition for promoting collagen synthesis, and in particular to a pharmaceutical composition for promoting collagen synthesis containing a pomegranate seed extract and a method for producing the pharmaceutical composition for promoting collagen 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 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 an effect of promoting collagen synthesis 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, found the present invention.

[0007] That is, the pharmaceutical composition for promoting collagen synthesis of the present invention is characterized by containing a pomegranate seed extract.

[0008] In a preferred embodiment of the pharmaceutical composition for promoting collagen synthesis of the present invention, the pharmaceutical composition suppresses MMP-1 gene expression.

[0009] In addition, a preferred embodiment of the pharmaceutical composition for promoting collagen synthesis of the present invention is characterized in that the pomegranate seed extract is obtained by immersing the pulverized material obtained by pulverizing pomegranate seeds in at least one solvent selected from ethanol, methanol, water, hexane, ethyl acetate and butanol, and separating the supernatant.

[0010] In a preferred embodiment of the pharmaceutical composition for promoting collagen synthesis of the present invention, the solvent is ethanol, ethyl acetate, hexane or butanol.

[0011] In a preferred embodiment of the pharmaceutical composition for promoting collagen synthesis of the present invention, the concentration of the pomegranate seed extract is 0.1 to 100 μg / mL.

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

[0013] In addition, the method for producing a pharmaceutical composition for promoting collagen synthesis 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.

[0014] In a preferred embodiment of the method for producing the pharmaceutical composition for promoting collagen synthesis of the present invention, the solvent is at least one selected from ethanol, methanol, water, hexane, ethyl acetate and butanol. Effects of the Invention

[0015] The pharmaceutical composition for promoting collagen synthesis according to the present invention has the advantageous effect of effectively utilizing pomegranate seeds that were previously disposed of as waste. In addition, the pharmaceutical composition for promoting collagen synthesis according to the present invention has the effect of promoting collagen synthesis and has the advantageous effect of effectively maintaining skin strength. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The quantitative results of hydroxyproline are shown (n=3, mean ± standard error). Details are described below. Figure 1 The pomegranate seed extract used in the experiment was one of four fractions obtained by fractionating the pomegranate seed extract using ethyl acetate, hexane, and butanol (F1: PSE-ethyl acetate-hexane-fraction 1). Figure 2 The results of MMP-1 gene expression evaluation are shown (n=4, mean ± standard error). Figure 2 The pomegranate seed extract used in the invention uses pomegranate seed-ethanol extract (PSE). DETAILED DESCRIPTION

[0017] The pharmaceutical composition for promoting collagen synthesis of the present invention is characterized by containing pomegranate seed extract. Pomegranate seed extract is an extract 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, the target cells are not particularly limited, but the cells acting on the skin are particularly effective. Therefore, in this specification, skin cells including fibroblasts are mainly described, but the present invention is not intended to be limited thereto.

[0019] In addition, in a preferred embodiment of the pharmaceutical composition for promoting collagen synthesis of the present invention, from the viewpoint that pomegranate seed extract can inhibit the expression activity of MMP-1, and then can inhibit the decomposition of collagen, it is characterized in that the MMP-1 gene expression is inhibited. MMP (extracellular matrix metalloproteinase) is an enzyme related to the decomposition of extracellular matrix proteins, and it is found that there are more than 20 types. MMP is classified into several groups such as collagenase (collagen degrading enzyme), gelatinase (gelatin degrading enzyme) according to its properties. For example, in the embodiment described later, the MMP-1 evaluated as an example is a collagenase, which participates in the decomposition of collagen fibers, but surprisingly, it is found that in the present invention, the pomegranate seed extract helps to inhibit the decomposition of collagen fibers by inhibiting the expression of the MMP-1.

[0020] In addition, a preferred embodiment of the pharmaceutical composition for promoting collagen synthesis of the present invention is characterized in that the pulverized product obtained by pulverizing pomegranate seeds is immersed in at least one solvent selected from ethanol, methanol, water, hexane, ethyl acetate and butanol, and the supernatant is separated to obtain the 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.

[0021] In a preferred embodiment of the pharmaceutical composition for promoting collagen synthesis of the present invention, the solvent is ethanol, ethyl acetate, hexane or butanol. In a preferred embodiment, the solvent is ethanol.

[0022] In a preferred embodiment of the pharmaceutical composition for promoting collagen synthesis of the present invention, the concentration of the pomegranate seed extract is 0.1 to 100 μg / mL from the viewpoint of not affecting cell viability.

[0023] In addition, the method for producing a pharmaceutical composition for promoting collagen synthesis 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. A preferred embodiment is characterized in that the solvent is at least one selected from ethanol, methanol, water, hexane, ethyl acetate and butanol.

[0024] In addition, in a preferred embodiment 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.

[0025] In a preferred embodiment of the method for producing the pharmaceutical composition 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.

[0026] 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).

[0027] Here, first, the preparation method of the pomegranate seed extract is described. First, pomegranate seeds are prepared. The pomegranate seeds are washed and dried as needed. The drying is preferably performed sufficiently. In order to make the subsequent pulverization uniform.

[0028] 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.

[0029] 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.

[0030] 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).

[0031] 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.

[0032] 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.

[0033] 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 is no particular limitation on the quasi-drug (quasi-drug) applicable to the present invention as long as the pharmaceutical composition for promoting collagen synthesis of the present invention is used as an active ingredient.

[0034] <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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] The dosage form may contain other conventional ingredients such as a storage stabilizer, a sweetener, a colorant, a flavoring agent, and the like.

[0039] <Acute toxicity test> Regarding the ingredients contained in pomegranate seed extract, since it contains linolenic acid, it is considered to be non-toxic.

[0040] 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 Example

[0041] An embodiment of the present invention will be described below, but the present invention is not limited to the following embodiment.

[0042] Example 1 First, an example of a method for extracting and fractionating samples is described below.

[0043] <Sample extraction and fractionation method> The dried pomegranate seeds were powdered with a grinder, and 10 times the amount of 100% ethanol 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 as pomegranate seed-ethanol extract (PSE: Punica granatum Seed Ethanolextract).

[0044] Next, 2.00 g of the PSE obtained above was further distributed in ethyl acetate (100 ml×3) and Mil iQ water (ultrapure water) (100 ml) using a separatory funnel, and the fraction obtained after the solvent of the ethyl acetate layer was concentrated under reduced pressure was designated as the ethyl acetate fraction (PSE-EA), and the fraction obtained after the water layer was freeze-dried was designated as the water fraction (PSE-W). PSE-EA was further distributed in hexane (100 ml×3 times) and methanol (100 ml), and the obtained hexane layer and ethyl acetate layer were concentrated under reduced pressure using an evaporator, respectively, to prepare PSE-ethyl acetate-hexane fraction (PSE-EA-H) and PSE-ethyl acetate-methanol fraction (PSE-EA-Me). Among them, PSE-EA-H with a high yield was distributed into 4 fractions by column chromatography using hexane, ethyl acetate, and methanol (100 ml), and designated as F1, F2, F3, and F4 in order of low polarity (in the order of hexane→ethyl acetate→methanol).

[0045] Specifically, as an example of a test on the skin functionality of the pomegranate seed extract, evaluation on collagen synthesis was performed.

[0046] <Research Summary> The dermis is mainly composed of proteins collectively referred to as the extracellular matrix, which are produced by intermittent fibroblasts. In the extracellular matrix of the human dermis, collagen fibers occupy about 70%, and the strength of the skin is maintained in the form of collagen fibers with low elasticity being bundled by elastic fibers. In this example, by quantifying the modified amino acid hydroxyproline unique to collagen in the culture supernatant of KMST-6 (fibroblasts), it was studied whether pomegranate seed extract promotes collagen production by fibroblasts. Hydroxyproline accounts for about 10% of all amino acids in collagen. That is, hydroxyproline is a characteristic amino acid of collagen, so it can be expected that the increase of hydroxyproline is related to the promotion of collagen fiber synthesis, so hydroxyproline was studied below.

[0047] In addition, collagen fibers are degraded by the MMP family. In order to study the effect of inhibiting the degradation of collagen fibers, gene expression evaluation of MMP-1, which is classified as collagenase, was performed.

[0048] <Experimental methods> <Cell culture> <Pre-cultivation> HaCaT (epidermal keratinocytes) were seeded on a 12-well plate at the above final concentration, and KMST-6 were seeded on 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.

[0049] <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.

[0050] <UVA (ultraviolet A wave) irradiation test (only for gene expression evaluation)> Regarding the cells used in the UVA irradiation test, the culture medium containing 1% FBS-ITES (ITES is the abbreviation of insulin, transferrin, ethanolamine, and sodium selenite, and the concentrations of each are: final concentration of insulin: 5μg / mL, final concentration of transferrin: 20μg / mL, final concentration of ethanolamine: 20μM, final concentration of sodium selenite: 25μM) was replaced with a 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 10J / 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.

[0051] <Quantification of Hydroxyproline> KMST-6 was cultured in the presence or absence of pomegranate seed extract. An equal amount of 12M HCl was mixed with the culture supernatant after 48 hours to free hydroxyproline through a hydrolysis reaction. The liquid was centrifuged at 10,000×g for 3 minutes, and 50 μL of the supernatant was added to a 96-well plate and dried overnight at 60°C to allow free hydroxyproline to adhere to the bottom of the plate. Chloramine-T was added to it to convert it into pyrrole, and a colorimetric product was formed by a condensation reaction with 4-(dimethylamino)benzaldehyde, and the absorbance at 540 nm was measured. Based on the benchmark, the concentration of hydroxyproline in the culture supernatant was calculated from the calibration curve, and the control group cultured in the absence of pomegranate seed extract was set to 100%, and each concentration was compared.

[0052] <Gene analysis> After the culture was completed by adding pomegranate seed extract, the cells were recovered for RNA extraction, cDNA synthesis, and real-time PCR. Among the intrinsic controls, MRPL19 was selected. MRPL19 is a gene used as an endogenous 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 nothing, and serves as a benchmark.

[0053] <Experimental Results> The quantitative results of hydroxyproline in the culture supernatant are as follows Figure 1 As shown. In the presence of pomegranate seed extract, the hydroxyproline concentration in the culture supernatant of KMST-6 was higher than that in the culture supernatant in the absence of pomegranate seed extract. This suggests that pomegranate seed extract can promote the formation of collagen fibers in fibroblasts.

[0054] The results of MMP-1 gene expression evaluation were as follows Figure 2 As shown. It was observed that the activation of MMP-1 expression caused by UVA irradiation tended to be inhibited by the presence of pomegranate seed extract. The MMP-1 expression level in cells with 10 μg / mL addition, which showed the highest inhibition rate, was about 60% of that in the control group.

[0055] In summary, it is believed that pomegranate seed extract helps both promote the formation of collagen fibers and inhibit the decomposition of collagen fibers. Industrial Applicability

[0056] 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 collagen synthesis, characterized in that: Contains pomegranate seed extract.

2. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition inhibits the expression of MMP-1 gene.

3. The pharmaceutical composition according to claim 1 or 2, 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 ethanol, methanol, water, hexane, ethyl acetate or butanol, and collecting the supernatant.

4. The pharmaceutical composition according to claim 3, wherein The solvent is ethanol, ethyl acetate, hexane or butanol.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein The concentration of the pomegranate seed extract is 0.1-100 μg / mL.

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 promoting collagen synthesis 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 ethanol, methanol, water, hexane, ethyl acetate or butanol.

Images