Pharmaceutical compositions for enhancing collagen synthesis and methods for producing pharmaceutical compositions containing pomegranate seed extract for enhancing collagen synthesis

Abstract

To provide compositions having collagen synthesis enhancing activity to effectively use pomegranate seeds to be wasted and add values thereto.SOLUTION: The collagen synthesis enhancing pharmaceutical composition of the invention comprise a pomegranate seed extract. In a preferred embodiment of the collagen synthesis enhancing pharmaceutical compositions of the invention, the pharmaceutical composition suppresses MMP-1 gene expression. In a preferred embodiment of the collagen synthesis enhancing pharmaceutical compositions of the invention, the pomegranate seed extract is obtained as follows: pomegranate seeds are pulverized and soaked in at least one solvent selected from the group consisting of ethanol, methanol, water, hexane, ethyl acetate and butanol, and the supernatant is separated.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for promoting collagen synthesis and a method for producing a pharmaceutical composition for promoting collagen synthesis, and in particular to a pharmaceutical composition for promoting collagen synthesis containing pomegranate seed extract and a method for producing a pharmaceutical composition for promoting collagen 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 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 the effect of promoting collagen synthesis, with the aim of effectively utilizing and adding value to 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 promoting collagen synthesis of the present invention is characterized by containing pomegranate seed extract.

[0008] In a preferred embodiment of the pharmaceutical composition for promoting collagen synthesis of the present invention, the pharmaceutical composition is characterized in that it inhibits the expression of the MMP-1 gene.

[0009] In addition, in a preferred embodiment of the pharmaceutical composition for promoting collagen synthesis 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 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 according to the present invention, the concentration of the pomegranate seed extract is 0.1 to 100 μg / mL.

[0012] The quasi-drug of the present invention is characterized by containing 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 pomegranate seed extract of the present invention is characterized by comprising the steps of: crushing pomegranate seeds, immersing the resulting crushed material in a solvent, and then separating out the supernatant; obtaining pomegranate seed extract by separating out the supernatant; and adjusting the obtained pomegranate seed extract to an effective amount.

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

[0015] The medicinal composition for promoting collagen synthesis of the present invention has an advantageous effect of enabling effective use of pomegranate seeds, which have been conventionally disposed of as waste. In addition, the medicinal composition for promoting collagen synthesis of the present invention has an advantageous effect of promoting collagen synthesis and is effective in maintaining skin strength. [Brief description of the drawings]

[0016] [Figure 1] Figure 1 shows the quantitative results of hydroxyproline (n=3, mean ± standard error). Details will be described later, but the pomegranate seed extract used in Figure 1 was one of four fractions (F1: PSE-Ethyl Acetate-Hexane-Fraction 1) that were obtained by fractionating the pomegranate seed extract with ethyl acetate, hexane, and butanol. [Diagram 2] Fig. 2 shows the results of evaluating MMP-1 gene expression (n=4, mean value ± standard error). In addition, the pomegranate seed extract used in Fig. 2 was a pomegranate seed ethanol extract (PSE), which will be described in detail later. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The pharmaceutical composition for promoting collagen synthesis 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 addition, the target cells are not particularly limited, but the cells that act particularly effectively are skin cells. Therefore, although the present specification mainly describes skin cells including fibroblasts, the present invention is not intended to be limited thereto.

[0019] In a preferred embodiment of the pharmaceutical composition for promoting collagen synthesis of the present invention, the pharmaceutical composition is characterized in that pomegranate seed extract suppresses MMP-1 gene expression, from the viewpoint that it can suppress the expression activity of MMP-1 and thus suppress collagen degradation. MMP (extracellular matrix metalloprotease) is an enzyme involved in the degradation of extracellular matrix proteins, and more than 20 types have been identified. MMPs are classified into several groups, such as collagenase (collagen decomposition enzyme) and gelatinase (gelatin decomposition enzyme), depending on their properties. For example, MMP-1, which is evaluated as an example in the examples described below, is collagenase and is involved in the degradation of collagen fibers, but surprisingly, in the present invention, it has been found that pomegranate seed extract contributes to the inhibition of collagen fiber degradation by suppressing the expression of the MMP-1.

[0020] In a preferred embodiment of the pharmaceutical composition for promoting collagen synthesis of the present invention, the pomegranate seed extract is obtained by pulverizing pomegranate seeds, immersing the pulverized product in at least one solvent selected from the group consisting of ethanol, methanol, water, hexane, ethyl acetate, and butanol, and then separating the supernatant. For example, the pomegranate seed extract can be extracted by shaking. In the shaking extraction, the extract can be extracted by rotating the extract by setting it on a rotator in a low-temperature room at about 4°C.

[0021] In a preferred embodiment of the pharmaceutical composition for promoting collagen synthesis according to 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 having no effect on cell viability.

[0023] In addition, the method for producing a pharmaceutical composition for promoting collagen synthesis containing pomegranate seed extract of the present invention is characterized by comprising the steps of: immersing the pulverized product obtained by pulverizing pomegranate seeds in a solvent, separating the supernatant, obtaining a pomegranate seed extract by separating the supernatant, and adjusting the obtained pomegranate seed extract to an effective amount. In a preferred embodiment, the solvent is at least one selected from the group consisting of ethanol, methanol, water, hexane, ethyl acetate, and butanol.

[0024] In addition, in a preferred embodiment of the present invention, from the viewpoint of assuming application to food products, the pomegranate seed ethanol extract extracted with ethanol can be further partitioned with ethyl acetate and water, and the ethyl acetate layer can be concentrated under reduced pressure to obtain the pomegranate seed extract as an ethyl acetate fraction.

[0025] In addition, 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 method is further characterized in that the ethyl acetate fraction is partitioned with hexane and methanol to obtain a pomegranate seed extract as an ethyl acetate-hexane fraction and an ethyl acetate-methanol fraction.

[0026] 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).

[0027] Here, first, a method for preparing 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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 promoting collagen synthesis of the present invention as an active ingredient.

[0034] <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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] The dosage form may contain other conventional ingredients, such as preservatives, sweeteners, colorants, flavoring agents, and the like.

[0039] <Acute toxicity test> As for the ingredients contained in pomegranate seed extract, including linoleic acid, it is believed that there is no toxicity.

[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: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 EXAMPLES

[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 a sample will be described below.

[0043] <Sample extraction and fractionation method> Dried pomegranate seeds were powdered in a mill, and 10 times the amount of 100% ethanol was added, followed by low-temperature shaking extraction at 4°C for 24 hours. After 24 hours, the mixture was centrifuged at room temperature for 20 minutes at 3,000 rpm, 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).

[0044] Next, 2.00 g of the PSE obtained above was partitioned with ethyl acetate (100 ml x 3 times) and MiliQ water (100 ml) using a separatory funnel, and the solvent in the ethyl acetate layer was concentrated under reduced pressure to obtain the ethyl acetate fraction (PSE-EA), and the water layer was freeze-dried to obtain the water fraction (PSE-W). After partitioning PSE-EA with hexane (100 ml x 3 times) and methanol (100 ml), the resulting hexane layer and ethyl acetate layer were each concentrated under reduced pressure using an evaporator to obtain the PSE ethyl acetate-hexane fraction (PSE-EA-H) and PSE ethyl acetate-methanol fraction (PSE-EA-Me). Among these, the PSE-EA-H with the highest yield was partitioned into four fractions by column chromatography using hexane, ethyl acetate, and methanol (100 ml), and the fractions were named F1, F2, F3, and F4 in order of decreasing polarity (hexane → ethyl acetate → methanol).

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

[0046] <Research overview> The dermis is mainly composed of proteins collectively called extracellular matrix, and these components are produced by fibroblasts present between the dermis. Approximately 70% of the extracellular matrix in human dermis is collagen fiber, which maintains the strength of the skin by bundling it with elastic fibers (elastin fibers) as collagen fibers with low elasticity. In this example, whether pomegranate seed extract promotes collagen production by fibroblasts was examined by quantifying hydroxyproline, a modified amino acid specific to collagen, in KMST-6 (fibroblast) culture supernatant. Hydroxyproline is said to account for approximately 10% of all amino acids in collagen. In other words, since hydroxyproline is a characteristic amino acid of collagen, it can be expected that an increase in hydroxyproline will lead to the promotion of collagen fiber synthesis, so hydroxyproline was examined below.

[0047] Furthermore, collagen fibers are degraded by the MMP family. In addition to the above, in order to examine the effect related to the suppression of collagen fiber degradation, gene expression evaluation was performed on MMP-1, which is classified as collagenase.

[0048] <Experimental method> <Cell culture> <Pre-culture> HaCaT (epidermal keratinocytes) was seeded in a 12-well plate and KMST-6 was seeded in a 96-well plate at the above final concentrations. The cells were cultured in a medium containing 10% FBS for about 15 hours to allow sufficient adhesion to the bottom surface.

[0049] <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 the cells were cultured at 37 °C and 5% CO2 concentration. The main culture was carried out for 48 hours.

[0050] <UVA (ultraviolet A wave) irradiation test (only for gene expression evaluation)> 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 (ITES is the abbreviation of Insulin, Transferrin, Ethanol amine, Sodium Selenite, and the final concentrations of each are insulin - 5 μg / mL, transferrin - 20 μg / mL, ethanolamine - 20 μM, sodium selenite - 25 μM) 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 the cells were irradiated with 10 J / cm 2 of UVA on ice (for about 50 minutes). 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 when 24 hours had elapsed.

[0051] <Hydroxyproline quantification> KMST-6 was cultured in the presence or absence of pomegranate seed extract. After 48 hours, the culture supernatant was mixed with an equal amount of 12 M HCl to release hydroxyproline by hydrolysis. 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 attach the free hydroxyproline to the bottom of the plate. Chloramine-T was added to convert it to pyrrole, which was then condensed with 4-(Dimethylamino)benzaldehyde to form a colorimetric product, and the absorbance at 540 nm was measured. The hydroxyproline concentration in the culture supernatant was calculated from the standard and a calibration curve, and each concentration was compared, with the control cultured in the absence of pomegranate seed extract set at 100%.

[0052] <Genetic analysis> After the culture with pomegranate seed extract was completed, the cells were harvested, RNA was extracted, cDNA was synthesized, and real-time PCR was performed. MRPL19 was selected as the endogenous control. MRPL19 was the gene used as the 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 the endogenous control. MRPL19 is a standard gene that is always expressed regardless of what the experimenter does or does not do.

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

[0054] The results of the MMP-1 gene expression evaluation are shown in Figure 2. The activation of MMP-1 expression by UVA irradiation tended to be suppressed by the presence of pomegranate seed extract. The MMP-1 expression level in the cells treated with 10 μg / mL of pomegranate extract, which showed the greatest suppression, was about 60% of the control level.

[0055] From the above, it is believed that pomegranate seed extract contributes to both promoting the formation of collagen fibers and inhibiting their degradation. [Industrial Applicability]

[0056] 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 collagen synthesis, comprising pomegranate seed extract.

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

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

4. The composition of claim 3, wherein the solvent is ethanol, ethyl acetate, hexane, or butanol.

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

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

Images