JP6999907B2 - Composition for promoting clock gene expression - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act 21st Annual Meeting of the Japanese Society for Chronobia, 2014 November 8-9, 2014 "Chronobiology" Vol. 20, No. 2

Application of Article 30, Paragraph 2 of the Patent Law 21st Annual Meeting of the Japanese Society for Chronobial Biology, pages 81 and 120 (issued on October 31, 2014)

Application of Article 30, Paragraph 2 of the Patent Act Japan Society for Bioscience and Biotechnology 2015 Annual Meeting March 28, 2015 Japan Society for Bioscience and Biotechnology 2015 Annual Meeting Abstracts (Lecture No. 3F45p04) (issued March 5, 2015)

The present invention relates to a clock gene expression promoting composition, a circadian rhythm adjusting composition, and a sleep improving composition containing a specific active ingredient.

The circadian rhythm is a rhythm with a cycle of about 24 hours possessed by the organism itself, and is controlled by the body clock (circadian lock) possessed by the organism itself. The biological clock causes biological diurnal fluctuations and controls various biological functions such as sleep and wakefulness, feeding behavior, body temperature, hormone secretion, blood pressure, metabolism, and psychosomatic activity. In addition, due to disturbance of the body clock due to irregular life, sleep abnormalities; skin diseases; neuropsychiatric disorders such as depression; lifestyle-related diseases such as obesity, diabetes and metabolic syndrome; cancer; various physical and mental disorders such as aging. It has become clear that the risk of abnormalities increases.

The biological clock is controlled by a circadian clock system containing a group of genes called clock genes. As clock genes, Per, Cry, Bmal, Block, Dec, Rev-Erb, ROR, DBP, E4BP4 and the like are known. In mammals, among these, proteins encoded by Per, Cry, Bmal and Locke play important roles in the biological clock system, and feedback loops formed by promoting and suppressing transcription of these genes are molecules of the biological clock. It forms the core of the mechanism.

That is, the positive transcription factors, the Lockk protein and the Bmal protein, form a complex, which binds to the E-Box sequence upstream of Per and Cry to promote the transcription of Per and Cry. On the other hand, Per protein and Cry protein are negative transcription factors, and when they form a complex and translocate to the nucleus, the formation of the Lock / Bmal complex is suppressed and the transcription of Per and Cry is reduced. ..

As described above, the poor expression of clock genes such as Per, Cry, Bmal, and Clock causes disturbance in the biological clock, resulting in the above-mentioned biological abnormalities.

In addition, the biological clock includes a central clock located in the suprachiasmatic nucleus of the hypothalamus of the brain and a peripheral clock present in various tissues such as the liver, lungs, kidneys, heart, and muscles, and the central clock is adjusted by light. In contrast, peripheral clocks are known to be regulated primarily by diet. Both of these show the expression of the biological clock system, the clock gene. For example, the clock gene is also expressed in the skin, and like other organs, the clock gene keeps rhythm in synchronization with the central clock in the brain, so that biological homeostasis is maintained.

In order to maintain the biological clock normally, it is effective to promote the expression of clock genes involved in the biological clock system. As such, for example, in Patent Document 1, a clock gene expression promoter containing lavender oil or eucalyptus oil and an alkylene oxide derivative is known. Further, in Patent Document 2, a circadian rhythm improving agent containing a cell-treated product of a specific lactic acid bacterium as an active ingredient is known.

Special Republication 2013-46272 Gazette Japanese Unexamined Patent Publication No. 2013-181005

The clock gene expression promoter described in Patent Document 1 contains a synthetic compound and cannot be ingested on a daily basis from the viewpoint of safety. Further, the circadian rhythm improving agent described in Patent Document 2 contains a cell-treated product of a specific lactic acid bacterium as an active ingredient, but the means for obtaining such a lactic acid bacterium is limited, and a large amount of the lactic acid bacterium is further cultured. Moreover, since the culture must be processed, it is difficult to manufacture and use it on an industrial scale.

Therefore, it is an invention of the present invention to provide a composition capable of adjusting a biological clock system, which has no problem in safety and can be manufactured and used on an industrial scale as compared with the above-mentioned prior art. Is the problem to be solved.

As a result of diligent studies to solve the above problems, the present inventors surprisingly promote the expression of Per2, Cry1, Bmal1 and Lockk, which are clock genes constituting the feedback loop in the biological clock system. I found it possible. Then, they succeeded in creating a composition containing black ginger as a composition for promoting clock gene expression, a composition for adjusting circadian rhythm, and a composition for improving sleep. The present invention is an invention completed based on such findings and successful examples.

Therefore, according to the present invention, there is provided a composition for promoting clock gene expression containing black ginger ( Kaempferia parviflora ).

According to another aspect of the present invention, there is provided a circadian rhythm adjusting composition containing black ginger.

According to another aspect of the present invention, there is provided a sleep improving composition containing black ginger.

Preferably, in the composition of the present invention, the clock gene is at least one clock gene selected from the group consisting of Per2, Cry1, Bmal1 and Clock.

Preferably, in the composition of the present invention, the black ginger is a black ginger extract.

Preferably, in the composition of the present invention, the black ginger extract is 3,5,7,3', 4'-pentamethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7-dimethoxyflavone, A black ginger extract containing at least one polymethoxyflavone selected from the group consisting of 3,5,7-trimethoxyflavone and 3,5,7,4'-tetramethoxyflavone.

By containing black ginger, the composition of the present invention has a clock gene expression promoting action, a circadian rhythm adjusting action and / or a sleep improving action, and further, in connection with these actions, a skin disease improving action, It can be expected to show an effect of improving mental illness, an effect of improving lifestyle-related diseases, an anticancer effect, an anti-aging effect, and the like.

Since the black ginger used in the composition of the present invention has been used in cosmetics and the like, the composition of the present invention is highly safe. Further, since black ginger is commercially available, the composition of the present invention can be easily manufactured and used on an industrial scale. Therefore, the composition of the present invention can be used in a parenteral or oral form in anticipation of the above-mentioned effects.

FIG. 1 is a diagram showing the results of expressing the expression of the clock gene as a relative amount to the control when the test sample group 1 is added, as described in the examples. FIG. 2 is a diagram showing the results of measuring the relative mRNA amount over time when black ginger was used as a test sample, as described in Examples. FIG. 3 is a diagram showing the results of examining the expression of the clock gene as a relative amount to the control when various concentrations of black ginger were added, as described in the examples. FIG. 4 is a diagram showing the results of fractionation of black ginger 95% ethanol extract under HPLC condition 1 as described in Examples. FIG. 5 is a diagram showing the results of examining the expression of the clock gene when Fr-1 to Fr-5 were added as a relative amount to the control, as described in the examples. FIG. 6 shows the results of fractionation of Fr-2 under HPLC condition 2 as described in Examples, and the expression of the clock gene of the obtained fraction was examined as a relative amount to the control. It is a figure shown. FIG. 7 shows the results of fractionating Fri-4 under HPLC condition 3 and examining the expression of the clock gene in the obtained fraction as a relative amount to the control, as described in Examples. It is a figure shown. FIG. 8 shows the identification of the components contained in the fractions Fr-2- (1) to (6) and Fr-4- (1) and (2) as described in Examples with FT-MS and ¹ . It is a figure which showed the result performed using 1 H-NMR. FIG. 9 shows the case where 3,5,7,3', 4'-pentamethoxyflavone, 5,7,4'-trimethoxyflavone and 5,7-dimethoxyflavone are added as described in Examples. It is a figure which showed the result of having performed the real-time reporter gene assay for Per2 gene.

Hereinafter, the present invention will be described in detail.
The composition of the present invention contains at least black ginger ( Kaempferia parviflora ) as an active ingredient that promotes clock gene expression.

The composition of the present invention may exhibit a circadian rhythm adjusting action and a sleep improving action in addition to the clock gene expression promoting action. Further, in relation to these actions, the composition of the present invention may exhibit a skin disease improving action, a psychiatric disease improving action, a lifestyle-related disease improving action, an anticancer action, an antiaging action and the like.

By having the above-mentioned actions, the composition of the present invention may at least take the form of a clock gene expression promoting composition, a circadian rhythm adjusting composition, and a sleep improving composition.

Kaempferia parviflora is a plant belonging to the genus Kaempferia of the family Zingiberaceae, which is known to grow naturally in Southeast Asia and the like. It is known that black ginger has effects such as energy enhancement, nourishing tonic, lowering blood sugar level, recovery of physical strength, improvement of digestive system, subvaginal band, hemorrhoids, hemorrhoids, nausea, stomatitis, arthralgia, and stomach pain. Has been done. Since black ginger is a natural plant that has been ingested by humans for a long period of time and is highly safe, the composition of the present invention is highly practical and is parenteral as it is or by processing. It can be applied to various uses in the form of a target or an oral form.

The site where the black ginger is used is not particularly limited as long as it contains a component that contributes to a desired pharmacological action, and examples thereof include roots, stems, leaves, flowers, and branches, but 3, 5, and 7 are preferable. , 3', 4'-pentamethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7-dimethoxyflavone, 3,5,7-trimethoxyflavone, 3,5,7,4'-tetramethoxy It is a rhizome rich in polymethoxyflavonoids (PMF) such as flavones.

Black ginger can be obtained by processing black ginger such as processed products (dried products, cut products, etc.) or powders, juices, and extracts of black ginger and processed black ginger products, in addition to the raw products collected. Including things. Here, the extract is not particularly limited as long as it is an extract of the components of black ginger, but for example, an extract obtained by extracting black ginger or a processed product thereof with a solvent, a diluted solution or a concentrated solution thereof. , Or their dried products and powders thereof. The black ginger used in the present invention is an extract of black ginger or an extract such as juice, or an extract thereof, considering the ease of application to a parenteral composition such as an external composition for skin or an oral composition. It is preferably a dried product of the extract.

For black ginger powder, for example, a processed product obtained by drying sliced black ginger after washing in the sun or using a dryer and then cutting it as it is or into an appropriate shape and size using a crusher. It can be obtained by crushing. As the crushing device, a commonly used one can be widely used, and for example, a crusher composed of a raw material hopper, a crusher, a classifier, a product holder, and the like can be used.

Black ginger extract is obtained by extracting black ginger and its processed products with a solvent. Examples of the solvent used for extraction include lower alcohols such as ethanol, methanol, isopropanol and butanol; lower esters such as ethyl acetate and methyl acetate; acetone; and a mixed solvent of these with water. Water is also mentioned as an extraction solvent, and may be hot water, hot water, or the like. Since the composition of the present invention may be orally ingested by humans, the black ginger extract as a raw material may be water alone, ethanol alone, or a mixed solvent of water and ethanol (so-called water content). It is preferably extracted with ethanol). For example, it is more preferable to use hydrous ethanol having a concentration of 20 to 95 vol% as a solvent.

When a mixed solvent is used as the solvent, for example, an acetone / water (2/8 to 8/2, volume ratio) mixture, an ethanol / water (2/8 to 8/2, volume ratio) mixture, or the like can be used. can. In the case of ethanol / water, for example, to the dried rhizome of black ginger, a solvent having a mass of 1 to 40 times, preferably 2 to 20 times the mass thereof is added, and the solvent is continuously or discontinuously added at room temperature or under heating. It is preferable to extract for several minutes to several days, preferably about 5 minutes to 10 days by gentle stirring.

The extraction method is not particularly limited, but for example, from the viewpoint of safety, convenience and industrialization, it is preferable to perform the extraction operation under as lenient conditions as possible. For example, the part of black ginger and its dried product are crushed, crushed, shredded, etc., and 2 to 20 times the mass of the solvent is added thereto, and the temperature ranges from 0 ° C. to the reflux temperature of the solvent for 5 minutes to 10 days. Extraction is performed under any conditions such as standing, shaking, stirring, and reflux. After the extraction work, solid-liquid separation operations such as filtration and centrifugation are performed to remove insoluble solids. An extract is obtained by performing operations such as dilution and concentration as necessary. Further, the insoluble matter may be extracted by repeating the same operation, and the extract may be used in combination with the previous extract. These extracts may be further purified and used by a purification method usually used by those skilled in the art.

The obtained extract can be used as it is or by concentrating it, for example, in the form of a liquid, a concentrate, or a dried product obtained by drying them. The drying method is not particularly limited, and can be performed by methods usually used by those skilled in the art, such as spray drying, freeze drying, vacuum drying, and fluid drying. Further, the dried product obtained by the above method can be powdered and used by a method known to those skilled in the art. Further, as described in Examples described later, after obtaining an extract or a dried product thereof, a fractionation treatment may be carried out in order to obtain a fraction containing a specific component at a high concentration.

The black ginger extract used in the composition of the present invention is not particularly limited, and is, for example, 3,5,7,3', 4'-pentamethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7. Examples include black ginger extract containing PMFs such as -dimethoxyflavone, 3,5,7-trimethoxyflavone and 3,5,7,4'-tetramethoxyflavone, preferably 3,5,7,3'. , 4'-Pentamethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7-dimethoxyflavone, 3,5,7-trimethoxyflavone and 3,5,7,4'-tetramethoxyflavone A black ginger extract containing one or more of these, more preferably a black ginger extract containing two or more of these, and even more preferably a black ginger extract containing all of these.

The content of PMF in the black ginger extract is not particularly limited, but for example, the ratio of the amount of PMF to the total amount of the extract (PMF content) is 0.1 wt% or more, preferably 1 to 99 wt%, and more preferably. It is 5 to 90 wt%.

The content of 3,5,7,3', 4'-pentamethoxyflavone in the black ginger extract is not particularly limited, but for example, 3,5,7,3', 4'-pentamethoxyflavone with respect to the total amount of the extract. The amount ratio (3,5,7,3', 4'-pentamethoxyflavone content) is, for example, 0.01 wt% or more, preferably 0.1 wt% or more, and more preferably 1 wt% or more. Yes, more preferably 10 to 70 wt%.

The content of 5,7,4'-trimethoxyflavone in the black ginger extract is not particularly limited, but for example, the ratio of the amount of 5,7,4'-trimethoxyflavone to the total amount of the extract (5,7,4'). -Trimethoxyflavone content) is, for example, 0.01 wt% or more, preferably 0.1 wt% or more, more preferably 1 wt% or more, and more preferably 10 to 70 wt%.

The content of 5,7-dimethoxyflavone in the black ginger extract is not particularly limited, but for example, the ratio of the amount of 5,7-dimethoxyflavone to the total amount of the extract (5,7-dimethoxyflavone content) is, for example, 0. It is 01 wt% or more, preferably 0.1 wt% or more, more preferably 1 wt% or more, and more preferably 10 to 70 wt%.

The content of 3,5,7-trimethoxyflavone in the black ginger extract is not particularly limited, but for example, the ratio of the amount of 3,5,7-trimethoxyflavone to the total amount of the extract (3,5,7-trimethoxy). The flavone content) is, for example, 0.01 wt% or more, preferably 0.1 wt% or more, more preferably 1 wt% or more, and more preferably 10 to 70 wt%.

The content of 3,5,7,4'-tetramethoxyflavone in the black ginger extract is not particularly limited, but for example, the ratio of the amount of 5,7-dimethoxyflavone to the total amount of the extract (5,7-dimethoxyflavone content). Is, for example, 0.01 wt% or more, preferably 0.1 wt% or more, more preferably 1 wt% or more, and more preferably 10 to 70 wt%.

The method for producing a processed product and a processed product of black ginger containing a black ginger extract is not particularly limited. The processed and processed products of black ginger may be produced, for example, by referring to the above description and the description of Examples described later, or may be produced by other methods usually known to those skilled in the art. However, it may be one that is on the market.

As described in the examples described below, black ginger exhibits a stronger clock gene expression promoting effect than other natural materials such as black beans, mushrooms, water chestnuts, broccoli, sweet potatoes, burdock, and tomatoes.

As clock genes, Per, Cry, Bmal, Clock, Dec, Rev-Erb, ROR, DBP, E4BP4 and the like are known, and the clock gene whose expression is promoted by the composition of the present invention is not particularly limited. As described in Examples described later, the composition of the present invention may exhibit an expression-promoting effect on Per2, Cry1, Bmal1 or Clock. For example, the composition of the present invention is 3,5,7,3', 4'-pentamethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7-dimethoxyflavone, 3,5,7-tri. When the composition contains black ginger, a treated black ginger product or a black ginger extract containing at least one of methoxyflavone and 3,5,7,4'-tetramethoxyflavone, it has a Per2 expression promoting action. Shown; Bmal1 if the composition contains black ginger, black ginger treated product or black ginger extract containing at least one of 5,7,4'-trimethoxyflavone and 5,7-dimethoxyflavone. It may show an expression promoting effect.

As used herein, the term "clock gene expression promoting action" means, for example, promoting the expression level of a clock gene in a subject, increasing the translation amount of a protein encoded by the clock gene, and activating the protein. It means the action of at least one of them. The circadian rhythm adjusting action is at least one of recovering the circadian rhythm originally possessed by the organism from an abnormal rhythm to a normal rhythm and maintaining a normal rhythm. Refers to two actions. The sleep improving action means suppressing or delaying or improving the current or future sleep abnormality or a state considered to be a sleep abnormality in the subject. The composition of the present invention may take the form of a clock gene expression promoting agent, a circadian rhythm adjusting agent, and a sleep improving agent, as having the above-mentioned actions.

The compositions of the present invention may further contain additives used in conventional processing such as excipients, bulking agents, binders, thickeners, emulsifiers, colorants, fragrances and perfume oils. The amount of the additive used is not particularly limited as long as it does not interfere with the solution of the problem of the present invention, and is appropriately adjusted.

The composition of the present invention is not particularly limited as long as it is in a commonly used form, and is, for example, liquid, lotion, mousse, gel, milky, suspension, cream, ointment, sheet, aerosol. , Spray, stick, powder, granule, granule, tablet, stick, plate, block, solid, paste, capsule, caplet and the like.

The composition of the present invention can be used as a parenteral composition, for example, in a form suitable for cosmetics. That is, one aspect of the composition of the present invention is a cosmetic composition. For example, the compositions of the invention can be processed into various forms such as lotions, emulsions, gels, creams, ointments, etc., either as is or in admixture with additives commonly used in the processing of cosmetics. .. Specifically, it can be used as a lotion, a cosmetic cream, a milky lotion, a cream, a facial mask, a hair tonic, a hair cream, a shampoo, a hair rinse, a treatment, a facial cleanser, a foundation, a hair restorer, an aqueous ointment, and a spray.

The content of black ginger contained in the composition of the present invention is not particularly limited as long as it has an effect of promoting clock gene expression, but is a dry mass, for example, a parenteral composition with respect to the entire composition. As a substance, it is 0.00001 wt% or more, preferably 0.0001 wt% or more; and as an oral composition, it is 0.0001 wt% or more, preferably 0.001 wt% or more.

The daily usage amount, one-time usage amount, usage period, usage interval, and other usage and dosage of the composition of the present invention are not particularly limited, and are appropriately determined according to the usage mode and the state of the user's body clock system. Can be set. However, regarding the period of use and the interval of use, as described in Examples described later, after ingesting the composition of the present invention, the clock genes Per2 and Cry1 are relatively peaked at 4 to 12 hours after ingestion. Since the expression is promoted at an early stage and the expression of Bmal1 and Clock is promoted relatively slowly with a peak of 16 to 24 hours after ingestion, the composition of the present invention can be used, for example, in the morning, noon or night. It is preferable to use it once or more for 2 days or more.

The method for producing the composition of the present invention is not particularly limited, and for example, a solid composition can be obtained by mixing black ginger and additives at room temperature or heating. Alternatively, this solid composition can be dissolved in a solvent such as water to obtain a liquid composition. Further, a liquid composition can be obtained by adding another solid component containing an additive to a liquid material containing black ginger and mixing the liquid material. The obtained liquid composition may be powdered after undergoing a drying treatment. As the method of the drying treatment in this case, the method used for drying the black ginger described above can be appropriately used and is not particularly limited.

The composition of the present invention can be used as it is or mixed with other components, compositions or products depending on the intended use. As described above, the composition of the present invention may be mixed or used in combination with various kinds as long as it contains black ginger, as long as the object of the present invention can be achieved.

Specific embodiments of the compositions of the present invention include, for example, 0.1-50 wt% 3,5,7,3', 4'-pentamethoxyflavone, 0.1-50 wt% 5,7,4'. -Trimethoxyflavone, 0.1-50 wt% 5,7-dimethoxyflavone, 0.1-50 wt% 3,5,7-trimethoxyflavone and 0.1-50 wt% 3,5,7,4 '-Compositions containing a black ginger extract containing a tetramethoxyflavone can be mentioned.

As another specific embodiment of the composition of the present invention, for example, 1 to 25 wt% 3,5,7,3', 4'-pentamethoxyflavone, 1 to 25 wt% 5,7,4'-tri. Contains 1-25 wt% 5,7-dimethoxyflavone, 1-25 wt% 3,5,7-trimethoxyflavone and 1-25 wt% 3,5,7,4'-tetramethoxyflavone. Examples thereof include compositions containing a black ginger extract.

The amount of each PMF in the specific embodiment of the composition of the present invention described above indicates the ratio to the amount of black ginger extract. That is, the ratio of each PMF described above indicates the ratio of each PMF to the amount of black ginger extract excluding additives.

Hereinafter, formulation examples relating to specific embodiments of the composition of the present invention will be shown, but the present invention is not limited to these formulation examples, and the present invention has various embodiments as long as the problems of the present invention can be solved. Can be taken.

(Formulation example 1: Toner)
With 100 parts by mass as a whole, 0.01 part by mass of black ginger extract, 0.01 part by mass of ginger extract, 0.01 part by mass of ketsumeishi, 10 parts by mass of glycerin, 3 parts by mass of diglycerin, 1,3-butylene glycol 12 parts by mass, pentylene glycol 3 parts by mass, sodium hyaluronate 0.1 parts by mass, citric acid 0.01 parts by mass, sodium citrate 0.02 parts by mass, xanthan gum 0.1 parts by mass, methylparaben 0.15 parts by mass , 0.2 parts by mass of carbomer, 0.03 part by mass of sodium hydroxide and the balance of water were mixed to prepare the composition of the present invention in the form of cosmetic water.

(Formulation example 2: shampoo)
With 100 parts by mass as a whole, 0.01 part by mass of black ginger extract, 0.02 part by mass of hop extract, 0.01 part by mass of caffeine, 7.5 parts by mass of sodium laureth sulfate, 4.2 by mass of cocamidopropyl betaine. By mass, 3 parts by mass of cocamide DEA, 0.1 part by mass of 1,3-butylene glycol, 0.225 parts by mass of polyquaternium-10, 0.15 parts by mass of citric acid, 0.05 part by mass of sodium citrate, phenoxyethanol 0. 9 parts by mass and the balance of water were mixed to prepare the composition of the present invention in the form of shampoo.

(Formulation example 3: Soap)
With 100 parts by mass as a whole, 0.5 parts by mass of crushed black ginger, 0.2 parts by mass of melilot extract, 0.1 parts by mass of elastin, 2 parts by mass of glycerin, 1 part by mass of olive oil, 0.1 parts of EDTA-4 sodium. The composition of the present invention was prepared in the form of soap by mixing and solidifying parts by mass, 0.2 parts by mass of tetrasodium ethidronate and a residue of soap base.

(Formulation Example 4: Emulsion)
With 100 parts by mass as a whole, 0.1 part by mass of black ginger extract, 0.1 part by mass of alanine, 0.1 part by mass of arginine, 3 parts by mass of sucrose fatty acid ester, 12 parts by mass of glycerin, 6 parts by mass of squalane, dimethyl. Mix 24 parts by mass of silicone oil, 1 part by mass of polypropylene glycol, 0.06 parts by mass of thickener, 0.2 parts by mass of phenoxyethanol, 5 parts by mass of ethanol, 0.01 part by mass of sodium hydroxide and the balance of purified water. The composition of the present invention was prepared in the form of a milky lotion.

(Combination example 5: Cosmetic cream)
With 100 parts by mass as a whole, 0.1 part by mass of black ginger extract, 0.1 part by mass of isomaltooligosaccharide, 0.1 part by mass of fructo-oligosaccharide, 15.0 part by mass of squalane, 4.0 part by mass of octyldodecyl myristate. , Hydrogenated soybean phospholipid 0.2 parts by mass, butyl alcohol 2.4 parts by mass, hardened oil 1.5 parts by mass, stearic acid 1.5 parts by mass, pro-oil type glycerin monostearate 1.5 parts by mass, mono 0.5 parts by mass of polyglyceryl stearate, 0.8 parts by mass of behenyl alcohol, 0.7 parts by mass of polyglyceryl monomyristate, 0.3 parts by mass of Sarashimitsurou, 0.1 parts by mass of d-δ-tocopherol, 0.3 parts by mass of methylparaben. 0.2 parts by mass of C10-30 alkyl modified carboxyvinyl polymer, 0.1 part by mass of carboxyvinyl polymer, 18.0 part by mass of 1,3-butanediol, 0.1 part by mass of sodium hydroxide and the balance of purified water. They were mixed to prepare the composition of the present invention in the form of a cosmetic cream.

(Formulation example 6: Packing agent)
6. With 100 parts by mass as a whole, 0.1 part by mass of black ginger extract, 0.01 part by mass of gallic acid, 20.0 parts by mass of polyvinyl alcohol, 5.0 parts by mass of glycerin, 20.0 parts by mass of ethanol, and kaolin. The composition of the present invention was prepared in the form of a packing agent by mixing 0 parts by mass, 0.2 parts by mass of the preservative, 0.1 parts by mass of the fragrance and the balance of purified water.

(Formulation Example 7: Tablet)
With 100 parts by mass as a whole, 10 parts by mass of black ginger powder, 8 parts by mass of ginger extract, 5 parts by mass of caffeine, 5 parts by mass of melilot, 20 parts by mass of crystalline cellulose, 50 parts by mass of lactose, 4 parts by mass of magnesium stearate. And the composition of the present invention was prepared in the form of a tablet by mixing and tableting the rest of the corn starch.

(Formulation Example 8: Tablet)
With 100 parts by mass as a whole, 10 parts by mass of black ginger powder, 8 parts by mass of ketsumeishi powder, 5 parts by mass of melilot, 0.5 part by mass of isomaltooligosaccharide, 0.5 part by mass of fructo-oligosaccharide, 20 parts by mass of crystalline cellulose, lactose The composition of the present invention was prepared in the form of a tablet by mixing and tableting 50 parts by mass, 4 parts by mass of magnesium stearate and the balance of corn starch.

(Formulation Example 9: Granules)
By mixing and granulating 10 parts by mass of black ginger powder, 15 parts by mass of hop powder, 5 parts by mass of lactose extract, 10 parts by mass of lactose, 1 part by mass of calcium stearate and the balance of crystalline cellulose, with the whole as 100 parts by mass. , The composition of the present invention was prepared in the form of granules.

(Formulation Example 10: Capsule)
A capsule shell prepared by mixing 10 parts by mass of black ginger extract, 20 parts by mass of ginger extract, 5 parts by mass of elastin, 8 parts by mass of lecithin and the balance of olive oil with the whole as 100 parts by mass. The composition of the present invention was prepared in the form of a capsule by encapsulating in.

(Formulation Example 11: Liquid)
With 100 parts by mass as a whole, 0.84 parts by mass of black ginger extract powder, 1 part by mass of arginine, 1 part by mass of alanine, 10 parts by mass of fructose-dextrose liquid sugar, 1 part by mass of citric acid, 0.02 parts by mass of sodium benzoate, The composition of the present invention was prepared in the form of a liquid preparation by mixing 2 parts by mass of a fragrance preparation, 0.05 parts by mass of sclarose, 0.03 parts by mass of acesulfam potassium, and the balance of purified water.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples, and the present invention can take various aspects as long as the problems of the present invention can be solved. ..

1. 1. Method (1) Preparation of test sample group 1 0.5 g of dried rhizome of black ginger added to 10 mL of 50% ethanol was subjected to vortex stirring treatment for 30 seconds and ultrasonic treatment for 10 seconds, and then shaken for 10 minutes. It was subjected to treatment and then centrifuge treatment. The supernatant obtained by centrifugation was used as a 50% ethanol extract of black ginger. Similarly, a 50% ethanol extract was prepared using black beans, mushrooms, water chestnuts, broccoli, sweet potatoes, burdock or tomatoes instead of black ginger. These 50% ethanol extracts were used as the test sample group 1 and used for the gene expression analysis described later.

(2) Preparation of Test Sample Group 2 100 g of dried rhizome of black ginger added to 1,000 mL of 95% ethanol was subjected to discontinuous stirring treatment for 6 days, and then subjected to filtration treatment. The obtained filtrate was evaporated to dryness to obtain a 95% black ginger ethanol extract (6.8 g). The black ginger 95% ethanol extract was used for the fraction described later.

(3) Gene expression analysis NIH-3T3 cells, which are mouse fibroblasts, were cultured for 3 days and synchronized with 100 nM dexamethasone for 2 hours, and then the test sample group 1 became 0.5 mg / mL. Was added to NIH-3T3 cells. After the addition, total RNA was extracted from NIH-3T3 cells at a predetermined time. The expression of clock genes (Per2, Cry1, Bmal1, and Lockk) was examined by quantitative RT-PCR. In addition, instead of the test sample group 1, ethanol was added so as to be 0.5%, which was used as a control.

(4) Fractionation and identification In order to identify the components having a clock gene regulation function in black ginger, fractionation was performed by preparative HPLC using a 95% ethanol extract of black ginger. The HPLC conditions were as follows. Further, among the fractions fractionated, the fractions containing a component having a clock gene regulation function were identified using FT-MS and ¹ H-NMR.

[HPLC Condition 1]
Volume: YMC-DispoPack AT ODS-25
Source: ACN / H ₂ O (55/45)
Flow Rate: 15mL / min
[HPLC Condition 2]
Color: Wakosil-II5C18 RS-Prep, 5 μm, 20x250 mm
Source: ACN / MeOH / H ₂ O (20/30/35)
Flow Rate: 13mL / min
[HPLC Condition 3]
Volume: Wakosil-II 5C18 RS-Prep, 5 μm, 20x250 mm
Source: ACN / MeOH / H ₂ O (20/30/30)
Flow Rate: 13mL / min

2. 2. Results (1) Gene expression analysis FIG. 1 shows the results of examining the expression of the clock gene as a relative amount to the control 6 hours after the addition of the test sample group 1. FIG. 2 shows the results of comparing the relative mRNA amounts over time every 4 hours when black ginger was used as the test sample. The final concentration of black ginger was varied to 0.005, 0.01, 0.03 and 0.05 mg / mL and 6 hours (Per2 and Cry1) or 20 hours (Bmal1) after the addition of black ginger. The results of examining the expression of the clock gene as a relative amount to the control are shown in FIG.

As shown in FIG. 1, the test sample using black ginger promoted the expression levels of Per2 and Bmal1 as compared with the test sample using other natural product materials. As shown in FIG. 2, it was confirmed that the expression of Per2 and Cry1 had a peak rhythm 4 to 6 hours after the addition of black ginger. On the other hand, it was confirmed that the expression of Bmal1 and Clock was promoted 20 to 24 hours after the addition. As shown in FIG. 3, it was confirmed that the expression levels of Per2, Cry1 and Bmal1 were promoted depending on the concentration of black ginger.

(2) Fractionation and identification The 95% ethanol extract of black ginger was fractionated under HPLC condition 1. The results are shown in FIG. As shown in FIG. 4, five fractions from Fr-1 to Fr-5 were obtained, and by drying these, 0.154 g, 0.537 g, 0.012 g, 0.041 g and 0.066 g, respectively, were obtained. Obtained a dried product. The total recovery rate of these was 81%.

The results of examining the expression of the clock gene 6 hours (Per2 and Cry1) or 20 hours (Bmal1) after adding Fr-1 to Fr-5 to 25 μg / mL as a relative amount to the control are shown in FIG. Shown in. As shown in FIG. 5, Fr-2 promoted the expression levels of Per2, Cry1 and Bmal1, and Fr-4 promoted the expression levels of Per2 and Cry1.

Fractionation was carried out for Fr-2 under HPLC condition 2. In addition, the expression of the clock gene 6 hours (Per2) or 20 hours (Bmal1) after adding the dried 6 fractions to 25 μg / mL was examined as a relative amount to the control. rice field. These results are shown in FIG. As shown in FIG. 6, the expression levels of Per2 and Bmal1 were promoted in Fr-2- (3) and (4), and the expression levels of Per2 were promoted in Fr-2- (2), (5) and (6). Was done.

Fractionation was carried out for Fr-4 under HPLC condition 3. In addition, the expression of the clock gene 6 hours after adding the dried fractions to 12.5 μg / mL (Per2) was examined as a relative amount to the control. These results are shown in FIG. As shown in FIG. 7, the expression level of Per2 was promoted in Fr-4- (1).

Of the fractions fractionated, the components contained in Fr-2- (1) to (6) and Fr-4- (1) and (2) were identified using FT-MS and ¹ H-NMR. went. The measurement results of the contents of the identified compound and black ginger extract are shown in FIG. From the above results, among the components of black ginger, the components involved in the regulation of Per2 gene expression are 3,5,7,3', 4'-pentamethoxyflavone, 5,7,4'-trimethoxyflavone, It was confirmed that they were 5,7-dimethoxyflavone, 3,5,7-trimethoxyflavone and 3,5,7,4'-tetramethoxyflavone. It was confirmed that 5,7-dimethoxyflavone and 5,7,4'-trimethoxyflavone are involved in the regulation of Bmal1 gene expression. These results suggest that black ginger polymethoxyflavone may regulate the body clock and improve the disturbed circadian rhythm.

The results of a real-time reporter gene assay in which pSLG (PEST) -Per2 (-376 / + 48) was introduced into NIH-3T3 cells as a reporter plasmid and Kronos AB-2500 (ATTO) was used as a luminometer are shown. Shown in 9. As a result, the expression of Per2 gene peaked 12 hours after administration by 3,5,7,3', 4'-pentamethoxyflavone, 5,7,4'-trimethoxyflavone and 5,7-dimethoxyflavone. It was confirmed that it was promoted to.

The composition of the present invention contains black ginger that can be applied in both parenteral and oral embodiments, and is a subject suffering from symptoms associated with abnormalities in the body clock system such as dyssomnia and jet lag. It is useful for such subjects and contributes to the health and welfare of such subjects.

Claims (1)

A composition for adjusting circadian rhythm containing black ginger (excluding a core body temperature increasing agent).

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