JP2012051837A - Glutathione production promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new glutathione production promoter having the excellent glutathione production promotion effect for a cell.SOLUTION: The glutathione production promoter contains as an active constituent a celery seed extract extracted from a preemergent celery seed using a solvent that contains a lower monohydric alcohol of carbon number 1-4 (for instance, a mixed solvent of the lower monohydric alcohol and water) as an extraction solvent.

Description

  The present invention relates to a glutathione production promoter, and more particularly to a glutathione production promoter containing a celery seed extract.

  Glutathione is a tripeptide composed of three amino acids, glutamic acid, cysteine, and glycine, and is a compound having a major cysteine residue in the cell. Further, it is an antioxidant substance present in every cell, and is known to be present at the highest concentration among the antioxidant substances present in the living body.

  Such glutathione is known to have a plurality of effects on the skin, such as not only an antioxidative effect, but also a skin immunity reduction suppressing effect and an antiallergic effect. However, it is known that the expression of glutathione decreases with aging, and this is thought to reduce the oxidative defense function in the skin and cause various skin aging phenomena.

  Therefore, if it is possible to promote the production of glutathione in cells in the living body, it is thought that the antioxidant function of the skin is strengthened, leading to acquisition of protective ability against various oxidative stresses generated in the external environment and in the body. Production promoters have been proposed. For example, the following Patent Document 1 contains an extract of a gardenia genus plant, and the following Patent Document 2 contains an extract of various natural products including butcher bloom extract. Are disclosed as glutathione production promoters. However, conventionally, it has not been known that an extract of celery seed has a glutathione production promoting action.

  By the way, it has been conventionally known that an extract of celery is blended in an external preparation for skin such as cosmetics.

  For example, Patent Document 3 below discloses an extract of celery as having active oxygen scavenging ability and antioxidant action. However, this document does not disclose the use of celery seed extract. In addition, the evaluation of the active oxygen scavenging ability and the like is to evaluate the scavenging ability (capturing ability) by causing the extract to act directly on the active oxygen species, and the glutathione production promoting action on the cells is not disclosed.

  Patent Document 4 listed below discloses a celery extract as a plant extract having an antioxidant ability, and also discloses the use of celery seeds. However, in this document as well, the evaluation of antioxidant power measures POV (peroxide value), and directly evaluates the antioxidant action, and does not disclose the glutathione production promoting action. Similarly, celery seed is described in Patent Document 5 below as an example of a herbal medicine having an antioxidant effect, but there is no data showing the antioxidant effect of celery seed itself, and a glutathione production promoting action is also disclosed. Absent.

  On the other hand, Patent Document 6 below discloses a cosmetic preparation containing an extract of a germinating plant, and celery is disclosed as an example of the germinating plant. The document also discloses the use of an extract of germinating plants to increase the intracellular GSH (glutathione) concentration. However, this document only lists a wide variety of uses as cosmetic preparations including increased GSH concentrations as the use of many germinating plants containing celery, and specific data on celery is presented. Not. In addition, this document makes it essential to use a germinated seed extract based on the fact that germinated plants contain specific active substances. What is the celery seed's own glutathione production promoting effect? There is no disclosure or suggestion. That is, since the seeds are metabolized with the germination during germination, the composition of the extract naturally differs between the germinated seeds and the ungerminated seeds themselves. Therefore, Patent Document 6 directed to germinating plants describes the present invention. It does not suggest anything.

JP 2006-347934 A JP 2009-132661 A JP-A-6-024937 JP 2001-139484 A JP 2003-095915 A JP-T-2004-532269

  An object of the present invention is to provide a novel glutathione production promoter having an excellent glutathione production promoting action on cells.

  The present inventors have intensively studied in view of the above-mentioned problems, and found that an extract obtained by extracting celery seeds with a specific solvent has a remarkable glutathione production promoting action, and completed the present invention. .

  That is, the present invention relates to a glutathione production promoter containing, as an active ingredient, a celery seed extract extracted from ungerminated celery seeds with a solvent containing a lower monohydric alcohol having 1 to 4 carbon atoms.

  ADVANTAGE OF THE INVENTION According to this invention, the glutathione production promoter which exhibits the outstanding glutathione production promotion effect | action with respect to a cell can be provided. Therefore, by blending this glutathione production promoter into various compositions, a composition excellent in glutathione production promoting action can be provided, and it is particularly suitable for blending into a skin external preparation.

  Hereinafter, matters related to the implementation of the present invention will be described in detail.

  The plant used as a raw material in the present invention is celery (scientific name: Apium graveolens var. Dulce) belonging to the family Aceraceae, and an extract of the seed is used in the present invention. As the celery seeds used for extraction, ungerminated ones are used. In sprouted seeds, even if there is an effect of promoting glutathione production, it is necessary to make the germination state uniform in order to keep the composition of the extract constant, which is complicated.

  When producing a glutathione production promoter containing celery seed extract as an active ingredient, a lower monohydric alcohol-containing solvent is used for extraction of the active ingredient. As shown in the examples described later, even with celery seed extract, water extract or butylene glycol extract does not provide glutathione production promoting action, and only by extracting with a solvent containing lower monohydric alcohol, excellent glutathione production is promoted. The effect is obtained.

  The lower monohydric alcohol-containing solvent may be a lower monohydric alcohol alone or a mixed solvent of a lower monohydric alcohol and another solvent as long as it contains a lower monohydric alcohol. As a solvent used in combination with a lower monohydric alcohol, water, a polyhydric alcohol (eg, 1,3-butylene glycol, propylene glycol, dipropylene glycol, glycerin, etc.), ethers (eg, ethyl ether, propyl ether, etc.), Esters (eg, ethyl acetate, butyl acetate, etc.), ketones (eg, acetone, ethyl methyl ketone, etc.), hydrocarbons (eg, n-hexane, benzene, etc.), halogen solvents (eg, chloroform, methylene chloride, etc.) These can be used, and these can be used alone or in combination. When the lower monohydric alcohol and these other solvents are used in combination, the content of the lower monohydric alcohol is preferably 30% by mass or more, more preferably 50 to 95% by mass.

  Examples of the lower monohydric alcohol include C1-C4 lower monohydric alcohols such as methanol, ethanol, propanol, and isopropanol, which may be used alone or in combination of two or more. Good. Among these, methanol and ethanol are preferable, and ethanol is particularly preferable.

  In particular, a preferable extraction solvent is a mixed solvent of a lower monohydric alcohol having 1 to 4 carbon atoms and water. With such a mixed solvent with water, the yield during extraction from celery seeds is high, and the production time of the extract including lyophilization can be shortened. A low level of toxicity to cells is also obtained. The ratio of the lower monohydric alcohol to water is preferably 30 to 95% by mass in terms of alcohol content, more preferably 40 to 90% by mass, still more preferably 50 to 85% by mass, and still more preferably 60%. It is -85 mass%. By setting to such a ratio, the glutathione production promoting action can be enhanced.

  No special method is required as the extraction method, and the extraction can be performed using an arbitrary apparatus at room temperature or under reflux heating according to a conventional method. Specifically, in terms of extraction efficiency, the celery seed is preferably crushed in advance by grinding or the like. The celery seeds crushed in this way are placed in a treatment tank filled with the above-mentioned alcohol-containing solvent as an extraction solvent, immersed in the alcohol-containing solvent, extracted after extraction of soluble components at room temperature or under reflux, and then filtered. An extraction liquid can be obtained by removing the extraction residue.

  The obtained extract may be used as it is, but may be used after diluting, concentrating, or drying according to a conventional method to obtain a diluted solution, a concentrated solution, or a dried product. Further, purification treatment such as decolorization, deodorization, and desalting, and fractionation treatment by column chromatography or the like may be performed within a range that does not impair the glutathione production promoting action.

  The celery seed extract thus obtained has an excellent glutathione production promoting action, and therefore can be used as an active ingredient of a glutathione production promoter. In that case, the celery seed extract can be used as it is as a glutathione production promoter as it is, but can also be provided after being formulated according to a conventional method. For formulation, in order to facilitate storage and handling, pharmaceutically acceptable carriers such as dextrin and cyclodextrin and other optional auxiliaries can be added, and powder, granule, tablet, liquid It can be formulated into any dosage form.

  Glutathione production promoter containing celery seed extract as an active ingredient is a composition having a glutathione production promoting action by being incorporated into various compositions such as cosmetics, pharmaceuticals, foods, bathing agents, etc., that is, for promoting glutathione production. A composition can be obtained. The resulting composition for promoting the production of glutathione promotes the production of glutathione on the cells, thereby enhancing the antioxidant effect of the cells and exhibiting the effect of preventing skin aging, An antiallergic action can be exhibited, and a whitening effect such as a melanin production inhibitory action by glutathione and a melanin lightening action by increasing the pheomelanin / eumelanin ratio can also be exhibited.

  The amount of glutathione production promoter containing celery seed extract as an active ingredient can be appropriately adjusted according to the type and purpose of the composition, the physiological activity of the extract, etc., but is not particularly limited. The blending amount is about 0.001 to 10% by mass in terms of solid content of the extract with respect to the total amount of the composition.

  Since the glutathione production promoter containing celery seed extract as an active ingredient is excellent in safety when applied to the skin, it is particularly suitable to be blended into various skin external preparations such as cosmetics. In the external preparation for skin, only the above-mentioned glutathione production promoter may be blended, or other effective materials may be blended in combination. Although it does not specifically limit as a kind of skin external preparation, For example, in addition to cosmetics, such as a lotion, a cosmetic liquid (essence), a milky lotion, a gel, a cream, a pack, a bath agent, a foundation, an ointment, a poultice, a plaster It may be a pharmaceutical such as an agent, or a quasi-drug.

  The skin external preparation containing the glutathione production promoter may contain various main agents and auxiliary agents usually used in the production of skin external preparations and other optional auxiliary agents, as long as it does not interfere with the glutathione production promoting action. The glutathione production promoter of the present invention is not the only main ingredient. For example, fats and oils, waxes, hydrocarbons, fatty acids, alcohols, polyhydric alcohols, esters, amines / amides / metal soaps, gums / water-soluble polymer compounds, surfactants, antioxidants, Contains various skin chemicals such as vitamins, fragrances, coloring materials, antiseptic disinfectants, amino acids, UV absorbers, sequestering agents, anti-inflammatory agents, antihistamines, crude drugs, whitening agents, moisturizers be able to.

  Hereinafter, the present invention will be described in more detail with reference to examples. The scope of the present invention is not limited to these examples.

[Example 1: Celery seed / 50% ethanol extract]
50 g of an aqueous ethanol solution (ethanol / water = 50/50 (mass ratio)) as an extraction solvent was added to 50 g of a coarsely pulverized product of ungerminated celery seeds, and extracted at around 90 ° C. for 2 hours with a reflux extractor. Filtered. The obtained extract was concentrated under reduced pressure and freeze-dried to obtain 7.7 g of the extract (solid matter).

[Example 2: Celery seed / 80% ethanol extract]
To 50 g of coarsely pulverized celery seeds ungerminated, 80% by weight ethanol aqueous solution (ethanol / water = 80/20 (mass ratio)) was added as an extraction solvent, and the mixture was extracted at around 90 ° C. for 2 hours with a reflux extractor. Filtered. The obtained extract was concentrated under reduced pressure and freeze-dried to obtain 5.6 g of the extract (solid matter).

[Example 3: Celery seed / 99% ethanol extract]
99 g ethanol aqueous solution (ethanol / water = 99/1 (mass ratio)) was added as an extraction solvent to 50 g of coarsely pulverized celery seeds that had not been germinated, and the mixture was extracted at around 90 ° C. for 2 hours with a reflux extractor. Filtered. The obtained extract was concentrated under reduced pressure and freeze-dried to obtain 2.1 g of the extract. In this case, as compared with Examples 1 and 2, freeze-drying took time, and the obtained extract was paste-like and the yield was low.

[Comparative Example 1: Celery seed / water extract]
Purified water was added as an extraction solvent to 50 g of coarsely ground celery seeds that had not germinated, and the mixture was extracted with a reflux extractor at around 90 ° C. for 2 hours and filtered. The obtained extract was concentrated under reduced pressure and lyophilized to obtain 7.3 g of the extract (solid matter).

[Comparative Example 2: Celery seed / 50% BG extract]
A 50% by weight aqueous solution of butylene glycol (1,3-butylene glycol / water = 50/50 (mass ratio)) as an extraction solvent was added to 50 g of a coarsely pulverized product of ungerminated celery seeds, and the mixture was refluxed at about 90 ° C. For 2 hours and filtered. The obtained extract was concentrated under reduced pressure and freeze-dried to obtain 1.6 g of the extract. In this case, as compared with Examples 1 and 2, freeze-drying took time and the yield was also low.

[Test Example 1: Evaluation of glutathione production promoting effect]
Using each celery seed extract of Examples 1 to 3 and Comparative Examples 1 and 2 as a sample, the glutathione production promoting action was evaluated. The evaluation method is as follows.

Human normal skin fibroblasts (NB1RGB) were pre-cultured with DMEM containing 5% fetal bovine serum (Dulbecco's modified Eagle medium “Nissui” (2)), and the medium was changed as appropriate. After the preculture, the cells were collected, seeded at 1.5 × 10 ⁵ cells per 35 mm diameter dish, and cultured at 37 ° C. and 5% CO _{2 for} 24 hours. Thereafter, a sample prepared to the target concentration was added to the cells, and the cells were cultured at 37 ° C. and 5% CO _{2 for} 20 hours, and then the number of cells was calculated by the Alamar Blue method.

  After the Alamar Blue reaction, the solution was washed with phosphate buffered saline PBS (−), and glutathione was dissolved by adding 5% trichloroacetic acid. This solution was recovered and subjected to ether extraction with 0.01N hydrochloric acid saturated diethyl ether, and then the ether was sufficiently volatilized to obtain a glutathione extract.

  The amount of glutathione was determined from a phosphate buffer containing 0.7 mM 5,5′-dithiobis (2-nitrobenzoic acid), 5.0 U / mL glutathione reductase, and 0.25 mM β-NADPH. The resulting reaction solution was mixed with the glutathione extract and the absorbance at 415 nm was measured, and the glutathione production promoting ability of each sample was evaluated based on the increase rate relative to the control.

The concentration of each sample added to the cells was set to two concentrations of 50 μg / mL and 25 μg / mL. The test at each concentration was performed at N = 3, and the average value was obtained for evaluation. Glutathione production ability was evaluated by calculating the amount of glutathione per 10 ⁴ cells and using the relative value when the control was 100. In addition, the cell survival rate is a relative value when the control is set to 100 with respect to the number of cells after completion of each test (number of cells calculated by the Alamar Blue method). The control (cont.) Used purified water as a sample, and the positive control (P.cont.) Used 3.3 μg / mL hydroquinone as a sample.

  The results are as shown in Table 1. The water extract of celery seeds according to Comparative Example 1 and the butylene glycol extract according to Comparative Example 2 did not show glutathione production promoting action, but the ethanol according to Examples It was confirmed that the extract had an excellent glutathione production promoting action equivalent to or better than hydroquinone. In particular, a high glutathione production promoting action was observed in the 80% ethanol extract shown in Example 2.

[Test Example 2: Evaluation of inhibitory action on melanin production]
Using each celery seed extract of Examples 1 to 3 and Comparative Examples 1 and 2 as samples, the melanin production inhibitory action by glutathione was evaluated. The evaluation method is as follows.

B16 melanoma cells derived from mice were seeded at 1.0 × 10 ⁵ cells per 35 mm diameter dish using DMEM containing 5% fetal bovine serum (Dulbecco's modified Eagle's medium “Nissui” (2)) at 37 ° C. The cells were cultured for 24 hours at 5% CO ₂ . Thereafter, a sample prepared to the target concentration and 0.5 mM theophylline were added to the cells, cultured for 6 days, and the number of cells was calculated by the Alamar Blue method.

After the Alamar Blue reaction, the cells were washed with phosphate buffered saline PBS (−), and the cells were dissolved in 1N-NaOH. The absorbance of the cell lysate at 405 nm was measured, and the melanin production inhibition rate was calculated by the following formula. Each sample was tested at N = 3, and the average value was obtained for evaluation.
Melanin production inhibition rate (%) = {(A−B) / A} × 100
(Where, A: absorbance of control, B: absorbance at the time of sample addition)

  The cell viability is obtained by calculating a relative value with respect to the number of cells after the completion of each test (the number of cells calculated by the Alamar Blue method) when the control is 100. The control (cont.) Used purified water as a sample, and the positive control (P.cont.) Used β-arbutin as a sample.

  A result is as showing in Table 2, and it had the outstanding melanin inhibitory effect comparable to arbutin as it was the ethanol extract of the celery seed which concerns on an Example.

  Below, the formulation example of the composition which mix | blended the glutathione production promoter which consists of a celery seed extract which concerns on this invention is shown. In the following, celery seed extract (dry solid) is an extract (dry solid) obtained in Example 1, 2 or 3.

(Formulation Example 1: Lotion)
(Compounding ingredients) (wt%)
(1) Glycerin 5.0
(2) Dipropylene glycol 4.0
(3) Xanthan gum 0.02
(4) Sorbit 1.0
(5) Dipotassium glycyrrhizinate 0.1
(6) Hamelis extract 0.2
(7) Celery seed extract (dry solid) 0.002
(8) Ethanol 10.0
(9) Purified water residue (Production method)
Add (1) to (8) to (9) and dissolve at room temperature. After solubilization, filter to make the product.

(Prescription Example 2: Essence)
(Compounding ingredients) (wt%)
(1) POE oleyl alcohol ether (60EO) 1.0
(2) Olive oil 0.2
(3) Phenoxyethanol 0.5
(4) Ethanol 7.0
(5) Sorbitol 8.0
(6) 1,3-butylene glycol 5.0
(7) Hyaluronic acid 0.1
(8) Trimethylglycine 1.0
(9) Royal Jelly Extract 1.0
(10) Yeast extract 0.1
(11) Red grape leaf extract 0.1
(12) Celery seed extract (dry solid) 0.005
(13) Purified water residue (Production method)
Add (5) to (12) to (13) and dissolve at room temperature. (1) to (3) are sequentially dissolved in (4) and then solubilized in the aqueous phase. Then, it is filtered to make a product.

(Formulation Example 3: Latex)
(Compounding ingredients) (wt%)
(1) Squalane 5.0
(2) Vaseline 2.0
(3) Beeswax 0.5
(4) Sorbitan sesquioleate ester 0.8
(5) Polyoxyethylene oleyl ether (20EO) 1.2
(6) 1,2-pentanediol 5.0
(7) Dipropylene glycol 5.0
(8) Carboxyvinyl polymer 0.2
(9) Ethanol 5.0
(10) Potassium hydroxide 0.1
(11) L-ascorbyl magnesium phosphate 1.0
(12) Rosemary extract 0.5
(13) Celery seed extract (dry solid) 0.005
(14) Residue of purified water (Production method)
(7) and (9) are added to (14) and heated and mixed, and then (8) is added to 70 ° C. Separately, (1) to (6) are mixed and dissolved by heating to 70 ° C. After adding this oil phase part to the above-mentioned water phase part and performing preliminary emulsification, (10) is added and neutralized. This is uniformly emulsified with a homomixer and then cooled with a heat exchanger. When the temperature drops to 40 ° C., (11) to (13) are added to obtain a product.

(Prescription Example 4: Emollient Gel)
(Compounding ingredients) (wt%)
(1) Dipropylene glycol 10.0
(2) Carboxyvinyl polymer 0.4
(3) Xanthan gum 0.2
(4) Sorbit 1.0
(5) Glycerin 10.0
(6) Hydrogenated soybean phospholipid 0.5
(7) Macadamian nut oil 3.0
(8) Squalane 5.0
(9) Jojoba oil 3.0
(10) Potassium hydroxide 0.1
(11) Soybean extract 0.5
(12) Celery seed extract (dry solid) 0.01
(13) Purified water residue (Production method)
Add (1) to (5) to (13) and dissolve at 70 ° C. Separately, (6) to (9) are mixed and dissolved by heating to 70 ° C. After adding this oil phase part to the above-mentioned aqueous phase part and mixing, (10) is added and neutralized. Then, it is cooled, and when the temperature falls to 40 ° C., (11) to (12) are added to obtain a product.

(Formulation Example 5: Face wash)
(Compounding ingredients) (wt%)
(1) Myristic acid 15.0
(2) Palmitic acid 8.0
(3) Stearic acid 5.0
(4) Glycerin 16.0
(5) Potassium hydroxide 5.0
(6) EO (10) Lauryl ether 5.0
(7) Cetanol 3.0
(8) Methyl parahydroxybenzoate 0.1
(9) Papain 0.1
(10) Ages extract 0.5
(11) Celery seed extract (dry solid) 0.002
(12) Purified water residue (Production method)
Add (1) to (3) to (4) and dissolve at 70 ° C. To this, (12) in which (5) is dissolved is gradually added and neutralized. Thereafter, (6) to (8) are added and cooled. When the temperature drops to 40 ° C., (9) to (11) are added to make a product.

(Formulation example 6: W / O type emollient cream)
(Compounding ingredients) (wt%)
(1) Microcrystalline wax 9.0
(2) Paraffin 2.0
(3) Beeswax 3.0
(4) Vaseline 5.0
(5) Reduced lanolin 8.0
(6) Squalane 34.0
(7) Hexadecyl adipate 10.0
(8) Ceramide 0.05
(9) Lipophilic glyceryl monooleate 3.5
(10) POE sorbitan monolaurate (20EO) 1.0
(11) Methyl parahydroxybenzoate 0.1
(12) Oat extract 0.2
(13) Chamomile extract 0.2
(14) Arnica extract 0.2
(15) Celery seed extract (dry solid) 0.002
(16) Astaxanthin 0.001
(17) Dipropylene glycol 2.0
(18) Purified water residue (Production method)
Add (17) to (18) and heat to 70 ° C. Separately, (1) to (11) are mixed and dissolved by heating to 70 ° C. The above-mentioned aqueous phase part is added to this oil phase part, and the mixture is uniformly emulsified with a homomixer and then cooled with a heat exchanger. When the temperature drops to 40 ° C., (12) to (16) are added to obtain a product.

(Prescription Example 7: Hair restorer)
(Compounding ingredients) (wt%)
(1) Hinokitiol 0.01
(2) Vitamin E 0.01
(3) Vitamin B6 0.01
(4) Dipropylene glycol 2.0
(5) Ethanol 60.0
(6) Placenta extract 0.5
(7) Seaweed extract 0.5
(8) Celery seed extract (dry solid) 0.005
(9) Purified water residue (Production method)
(1) to (5) and (6) to (9) are separately dissolved at room temperature. Thereafter, these are mixed and filtered to obtain a product.

  The glutathione production promoter of the present invention can be used by blending it with various compositions such as cosmetics, pharmaceuticals, quasi-drugs, foods, bathing agents, etc., and particularly preferably blended with a skin external preparation. is there.

Claims (2)

  A glutathione production promoter containing, as an active ingredient, a celery seed extract extracted from ungerminated celery seeds with a solvent containing a lower monohydric alcohol having 1 to 4 carbon atoms.   The glutathione production promoter according to claim 1, wherein the solvent is a mixed solvent of the lower monohydric alcohol and water.