JP2018087177A - Composition that inhibits antibody production, and method for producing punica granatum seed extract - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition having antiallergic action to effectively utilize Punica Granatum seeds, which have been discarded, and add value to them.SOLUTION: A composition that inhibits antibody production, contains Punica Granatum seed extract. In a preferred embodiment of the composition, the Punica Granatum seed extract contains punicic acid or ellagic acid. In a preferred embodiment of the composition, the Punica Granatum seed extract is obtained by grinding Punica Granatum seeds to obtain a ground product, immersing it in at least one solvent selected from the group consisting of ethanol, methanol, water, and hexane, and fractionating a supernatant of it.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a composition for suppressing antibody production and a method for producing a pomegranate seed extract, and more particularly to a composition for inhibiting antibody production containing a pomegranate seed extract and a method for producing a pomegranate seed extract.

  In recent years, the number of allergic patients has increased rapidly, and the number of type I allergic patients, particularly bronchial asthma and allergic rhinitis, has increased remarkably. Pomegranate is a fruit in which seeds occupy a significant proportion of the whole fruit, but in principle, the seeds remaining after squeezing are discarded. As a few examples using pomegranate seeds, there is a supplement suggestion system using pomegranate seed extract, where the proposed supplement calculation means is multivitamin, multimineral, vitamin A , Vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin E, vitamin P, niacin, biotin, collagen, elastin, polyphenol, pantothenic acid, hyaluronic acid, acetylglucosamine, inositol, coenzyme Q10, alpha carotene, beta Carotene, soy saponin, royal jelly, twin-tose, folic acid, rose petal extract, pomegranate seed extract, black cohosh extract, soy isoflavone, kuzu isoflavone squalene, brewer's yeast, fenugreek extract, cal A supplement proposal system that outputs at least one of the group consisting of calcium, magnesium, copper, iron, zinc, and manganese is known (Patent Document 1).

JP2011-232989

  However, at present, in many cases, pomegranate seeds are in principle treated as waste, and other than the above-mentioned patent documents, they are not used effectively. On the other hand, the effective use of waste has been reviewed, and it is desired to search for foods having functional ingredients that are effective in preventing and improving allergic diseases.

  Accordingly, an object of the present invention is to provide a composition having an antiallergic action for the purpose of effective utilization and added value of discarded pomegranate seeds.

  In order to achieve the above object, the present inventors have intensively studied on effective utilization of pomegranate seeds, and as a result, have found the present invention.

  That is, the composition for suppressing antibody production of the present invention contains a pomegranate seed extract.

  In a preferred embodiment of the composition for suppressing antibody production of the present invention, the pomegranate seed extract contains punicic acid or ellagic acid.

  Further, in a preferred embodiment of the composition for suppressing antibody production of the present invention, the pulverized product obtained by pulverizing pomegranate seeds is used in at least one solvent selected from the group consisting of ethanol, methanol, water, and hexane. The pomegranate seed extract was obtained by dipping and separating the supernatant.

  In a preferred embodiment of the composition for suppressing antibody production of the present invention, the antibody is an IgE antibody.

  In addition, the cosmetic of the present invention is characterized in that the composition for suppressing antibody production of the present invention is an active ingredient.

  In addition, the quasi-drug of the present invention is characterized in that the composition for suppressing antibody production of the present invention is an active ingredient.

  In addition, the method for producing a pomegranate seed extract of the present invention is characterized in that a pulverized product obtained by pulverizing pomegranate seeds is immersed in a solvent, and then a supernatant is collected.

  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 ethanol, methanol, hexane, and water.

  According to the composition for suppressing antibody production of the present invention, there is an advantageous effect that it is possible to effectively use pomegranate seeds that have been treated as waste. In addition, the composition for suppressing antibody production of the present invention has an advantageous effect that it has an effective antiallergic action and is effective for various allergic diseases.

FIG. 1 is a graph showing cytotoxicity against U266 when the composition according to one embodiment of the present invention is used. The left side of the figure shows 100% ethanol extracted, and the right side shows NaPB extracted. The vertical axis shows cell viability and the horizontal axis shows sample concentration. FIG. 2 is a diagram showing evaluation of IgE antibody production inhibition when the composition according to one embodiment of the present invention is used. The left side of the figure shows 100% ethanol extracted. The vertical axis represents human IgE (relative value), and the horizontal axis represents the sample concentration. The right side of the figure shows the NaPB extracted. The vertical axis represents human IgE (relative value), and the horizontal axis represents the sample concentration. FIG. 3 is a diagram showing evaluation of suppression of IgE antibody production when the culture time in the embodiment of the present invention is 1 day, 2 days, and 3 days. The vertical axis shows the amount of human IgE production, and the horizontal axis shows the culture time.

  The composition for suppressing antibody production of the present invention comprises a pomegranate seed extract. The pomegranate seed extract is an extract derived from pomegranate seeds. As long as the pomegranate seed extract applied to the present invention is derived from pomegranate seeds, all pomegranate seed extracts are targeted.

  In addition, the target cells are not particularly limited, but examples thereof include cells such as the immune system and reproductive system, and general cells such as skin. Particularly effective are the cells of the skin. Accordingly, although skin cells are mainly described herein, the present invention is not intended to be limited thereto.

  In a preferred embodiment of the composition for suppressing antibody production of the present invention, the pomegranate seed extract contains punicic acid or ellagic acid.

  Further, in a preferred embodiment of the composition for suppressing antibody production of the present invention, the pulverized product obtained by pulverizing pomegranate seeds is used in at least one solvent selected from the group consisting of ethanol, methanol, water, and hexane. The pomegranate seed extract was obtained by dipping and separating the supernatant. For example, it can be extracted by shaking. In the shaking extraction, for example, the extraction can be performed while being set and rotated on a rotator in a low temperature chamber of about 4 ° C or the like.

  Further, in a preferred embodiment of the composition for suppressing antibody production of the present invention, the IgE antibody is an IgE antibody or IgG1 antibody from the viewpoint that the IgE antibody is deeply involved in the development of allergic diseases. .

  In addition, the method for producing a pomegranate seed extract of the present invention is characterized in that a pulverized product obtained by pulverizing pomegranate seeds is immersed in a solvent, and then a supernatant is collected.

  Here, first, a method for preparing the pomegranate seed extract will be described. First, pomegranate seeds are prepared. Pomegranate seeds are washed and dried as necessary. Drying is preferably performed sufficiently. This is because the subsequent pulverization is performed uniformly.

  Next, the pomegranate seeds are pulverized. The pulverization method is not particularly limited, and a known pulverizer such as a ball mill, a hammer mill, a roller mill, a rod mill, a sample mill, a stamp mill, a disintegrator, a mortar, and a blender with a cooling device can be used. In addition, since it may be considered that the pomegranate seed composition is decomposed due to heat generation during pulverization, the pulverization time is set to several seconds and can be repeated ten times or more.

  Next, the pomegranate seeds are pulverized to obtain a pulverized product, and then the pulverized product is immersed in various solvents. The solvent in this case is not particularly limited, and the solvent can be appropriately set 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 ethanol, methanol, hexane, and water. Examples of the solvent include polar and nonpolar solvents such as ethanol, methanol, water, hexane, ethyl acetate, chloroform, and acetone. Preferably, methanol, ethanol, water, etc. can be mentioned.

  Immersion can be performed under gentle stirring. The pulverized product is immersed in various solvents to obtain various solutions. Various solutions may be stirred according to the state of the solution, and in some cases, the solution may be left as it is. In the case of stirring, although not particularly limited, stirring can be continued for 10 hours to 48 hours, preferably about 1 day (24 hours).

  Then, a pomegranate seed extract can be obtained by fractionating the supernatant. If necessary, evaporate the supernatant to dryness. Evaporation to dryness can be carried out using an evaporator on a warm bath of 20 ° C to 60 ° C, preferably 37 ° C to 40 ° C. Pomegranate seed extract can be stored for a long time by evaporating to dryness.

  The components contained in pomegranate seeds are sorted according to their physical properties by extracting the pomegranate seeds with solvents having different polarities. Therefore, the types and contents of the pomegranate seed extract components vary depending on the solvent used.

  In addition, the cosmetic of the present invention is characterized in that the composition for suppressing antibody production of the present invention is an active ingredient. The cosmetics applicable to the present invention are not particularly limited as long as the composition for suppressing antibody production of the present invention is used as an active ingredient.

  In addition, the quasi-drug of the present invention is characterized in that the composition for suppressing antibody production of the present invention is an active ingredient. The quasi-drug applicable to the present invention is not particularly limited as long as the composition that suppresses antibody production of the present invention is an active ingredient.

<Effective amount>
The composition according to the invention is prepared in the form of an effective amount of pomegranate seed extract and a suitable dosage form.

  The dosage of the pomegranate seed extract in the composition of the present invention can be changed according to the pathology and severity of the patient to be administered, the dosage form, the selected administration route, the number of administrations per day, and the like.

  The dose of pomegranate seed extract in the composition of the present invention can be set to a dose of 1000 mg / kg / day when carried out in mice, and in humans, the dose should be lower due to differences in sensitivity, etc. Is preferred.

  Examples of dosage forms include oral preparations (tablets, capsules, coated tablets, granules, solutions, syrups), suppositories for rectal administration, injections, and the like. The administration target patient is often a chronic disease, and is preferably an oral agent from the viewpoint of the necessity of long-term administration and ease of continuous use.

  Dosage forms can include other conventional ingredients such as stabilizers, sweeteners, colorants, fragrances and the like.

<Acute toxicity test>
About the component of the pomegranate seed extract, linolenic acid is also included, so it is considered that no toxicity is observed.

  Hereinafter, one embodiment of the present invention will be described, but the present invention is not construed as being limited to the following embodiment.

Example 1
First, preparation of pomegranate seed extract was attempted using ethanol as a solvent. Pomegranate seeds were crushed and extracted with 100% ethanol by shaking. These were concentrated to dryness and redissolved, and subjected to experiments as pomegranate seed ethanol extract. Specifically, it is as follows.

<Sample preparation>
30 mL of 100% ethanol was added to 3 g of pomegranate seeds (pulverized with a mixer), and subjected to rotary extraction at 4 ° C. for 24 hours. Thereafter, centrifugation was performed at 3000 rpm for 20 minutes, and the supernatant was collected. The supernatant was concentrated to dryness with an evaporator, weighed and redissolved in 100% ethanol to a concentration of 25 mg / mL. Further, 30 mL of 10 mM NaPB (sodium phosphate buffer) was added to 3 g of pomegranate seeds (pulverized with a mixer), and subjected to rotary extraction at 4 ° C. for 24 hours. Thereafter, centrifugation was performed at 3000 rpm for 20 minutes, and the supernatant was collected. The stock solution was used in the following experiment, and the ratio of the total amount of the culture system to the sample was expressed in%.

Example 2
Next, using human multiple myeloma U266 cells that constantly produce IgE antibodies, pomegranate seed ethanol extract was added at various concentrations and cultured under serum-free conditions for 24 hours. The antiallergic action of the composition was examined. Further, the culture supernatant was collected, the amount of secreted human IgE antibody was measured by ELISA, and cytotoxicity was also confirmed by using WST-8 method. Furthermore, the expression of IgE gene in U266 cells cultured with pomegranate seed ethanol extract was examined. Specifically, it is as follows.

<Culture of U266 cells>
In a 96-well plate, U266 cells suspended in RPMI1640 medium supplemented with ITES (insulin, transferrin, ethanolamine, selenite) were seeded at a cell density of 1 × 10 5 cells / mL. U266 cells are human myeloma cells that constantly produce human IgE. Pomegranate seed ethanol extract and pomegranate seed NaPB extract were added thereto at various concentrations and cultured for 24 hours.

<Toxicity to U266 cells>
After the above cell culture, cytotoxicity and proliferation were measured using WST-8 kit (Kishida Chemical). FIG. 1 shows cytotoxicity against U266. The left side of the figure shows 100% ethanol extracted, and the right side shows NaPB extracted. The vertical axis shows cell viability and the horizontal axis shows sample concentration. In general, cytotoxicity was observed at high concentrations, and pomegranate seed ethanol extract tended to be cytotoxic at 125 μg / mL or higher. Similarly, the NaPB extract showed toxicity at 0.063% or more. That is, it can be seen from the results in FIG. 1 that the amount of IgE antibody does not decrease due to the decrease in cells.

<IgE antibody production inhibition evaluation (Dose-response)>
Human IgE antibody was quantified by ELISA. Human IgE ELISA Quantitation Set (Bethyl) was used. FIG. 2 is a diagram showing an evaluation of suppression of IgE antibody production. The left side of the figure shows 100% ethanol extracted. The vertical axis represents human IgE (relative value), and the horizontal axis represents the sample concentration. The right side of the figure shows the NaPB extracted. The vertical axis represents human IgE (relative value), and the horizontal axis represents the sample concentration.

  As a result, in the 100% ethanol extract, IgE production was suppressed in a concentration-dependent manner. In the NaPB extract, IgE production suppression with concentration dependency was observed, although slightly.

<IgE antibody production inhibition evaluation (Time-course)>
The pomegranate seed ethanol extract showed an IgE production inhibitory effect 24 hours after addition, but in order to investigate the persistence of the inhibitory effect, the culture time was 24 hours, 48 hours, and 72 hours. The amount of secreted IgE was quantified by ELISA.

  FIG. 3 is a diagram showing evaluation of IgE antibody production inhibition when the culture time is 24 hours, 48 hours, and 72 hours for pomegranate seed ethanol extracts at various concentrations (100 μg / mL, 50 μg / mL). . The vertical axis shows the amount of human IgE production, and the horizontal axis shows the culture time. As a result, it was revealed that the pomegranate seed ethanol extract maintained the suppression effect on IgE production not only for 24 hours but also for 48 hours and 72 hours.

  As a result, it was revealed that pomegranate seed ethanol extracts at various concentrations were added to U266 cells and cultured, and IgE antibody production decreased in a concentration-dependent manner. At 125 μg / ml, the most effective, IgE production was suppressed by about 70% compared to control (sterile water).

  Pomegranate seed ethanol extract was found to suppress the production of IgE antibody, which is deeply involved in the development of type I allergy, in a concentration-dependent manner. From the results of this Example, it was suggested that some components contained in pomegranate seeds, possibly components such as punicic acid or ellagic acid, exhibited IgE production inhibitory activity and also exhibited an antiallergic effect in vivo.

  According to the present invention, the contribution to the fields of the medical and beauty industries is high, and the social contribution such as the effective use of waste is high, and the utility value is high in a wide range.

Claims (8)

  A composition for suppressing antibody production, comprising a pomegranate seed extract.   The composition according to claim 1, wherein the pomegranate seed extract contains punicic acid or ellagic acid. The pulverized product obtained by pulverizing pomegranate seeds was immersed in at least one solvent selected from the group consisting of ethanol, methanol, water, and hexane, and the supernatant was separated to obtain the pomegranate seed extract. The composition according to claim 1 or 2, wherein   The composition according to any one of claims 1 to 3, wherein the antibody is an IgE antibody.   Cosmetics comprising the composition according to any one of claims 1 to 4 as an active ingredient.   A quasi-drug containing the composition according to any one of claims 1 to 4 as an active ingredient.   A method for producing a pomegranate seed extract, comprising immersing a pulverized product obtained by pulverizing pomegranate seeds in a solvent, and then collecting a supernatant.   The method according to claim 6, wherein the solvent is at least one selected from the group consisting of ethanol, methanol, hexane, and water.