JP2019094357A - Clock gene expression promoting composition - Google Patents

Abstract

To provide compositions capable of adjusting a biological clock system, the composition not having safety problems as compared with prior arts and being capable of manufacturing and utilizing in technical scale.SOLUTION: The above object of the invention can be solved by a composition containing Kaempferia parviflora and having the expression promoting effect of clock genes such as Per2, Cry1, Bmal1, and Clock, a circadian rhythm adjustment effect, and/or a sleep improving effect.SELECTED DRAWING: Figure 1

Description

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

The circadian rhythm is a rhythm with an approximately 24-hour cycle that an organism has, and is controlled by the circadian clock that the organism itself has. The biological clock causes biological diurnal fluctuation, and controls various biological functions such as eating behavior, body temperature, hormone secretion, blood pressure, metabolism, mind-body activity, as well as sleep and awakening. In addition, disorder of the biological clock due to irregular life, sleep disorders; skin diseases; psychoneurotic diseases such as depression; lifestyle-related diseases such as obesity, diabetes, metabolic syndrome; cancer; various physical and mental such as aging Abnormal risk has been shown to increase.

The biological clock is controlled by a circadian clock system including a gene group called a clock gene. As clock genes, Per, Cry, Bmal, Clock, Dec, Rev-Erb, ROR, DBP, E4BP4 and the like are known. In mammals, among these, proteins encoded by Per, Cry, Bmal and Clock play an important role in the biological clock system, and the feedback loop formed by the promotion and repression of transcription of these genes is a molecular of the biological clock. At the core of the organization.

That is, the positive transcription factors Clock protein and Bmal protein form a complex, which binds to E-Box sequences upstream of Per and Cry to promote Per and Cry transcription. On the other hand, Per protein and Cry protein are negative transcription factors, and their formation into a complex and transfer to the nucleus suppresses the formation of Clock / Bmal complex and reduces Per and Cry transcription. .

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

The biological clock includes a central clock located in the hypothalamic suprachiasmatic nucleus of the brain and peripheral clocks present in various tissues such as the liver, lungs, kidneys, heart, and muscles, and the central clock is adjusted by light On the other hand, peripheral clocks are known to be mainly adjusted by diet. Both of these show the expression of the internal clock system, ie, the clock gene. For example, the clock gene is expressed in the skin, and as with other organs, homeostasis is maintained by the rhythm of the clock gene being in synchronization with the central clock in the brain.

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

Special re-publication 2013-46272 gazette JP, 2013-181005, A

The clock gene expression promoter described in Patent Document 1 contains a synthetic compound and can not be taken on a daily basis from the viewpoint of safety. In addition, the circadian rhythm improving agent described in Patent Document 2 uses a treated product of a specific lactic acid bacteria as an active ingredient, but the means for obtaining such lactic acid bacteria is limited, and a large amount of lactic acid bacteria is cultured. And, since the culture has to be processed, it is difficult to manufacture and utilize on an industrial scale.

Therefore, the present invention is to provide a composition capable of controlling 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 an issue to be solved.

The present inventors have intensively studied to solve the above-mentioned problems, and surprisingly, the expression of the persimmon gene, Per2, Cry1, Bmal1 and Clock, which are clock genes that make up the feedback loop in the circadian clock system, is enhanced. I found that I could do it. Then, as a composition for promoting clock gene expression, a composition for adjusting circadian rhythm, and a composition for improving sleep, a composition containing black ginger has succeeded. 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, which comprises black ginger ( Kaempferia parviflora ).

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

According to another aspect of the present invention, there is provided a composition for improving sleep comprising 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, said black ginger extract is 3,5,7,3 ', 4'-pentamethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7-dimethoxyflavone, It is 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.

The composition of the present invention has a clock gene expression promoting action, a circadian rhythm regulating action and / or a sleep improving action by containing black ginger, and further, in relation to these actions, a skin disease improving action, It can be expected to exhibit a mental disease ameliorating action, a lifestyle-related disease ameliorating action, an anticancer action, an anti-aging action and the like.

Since the black ginger used in the composition of the present invention has a history of use in cosmetics and the like, the composition of the present invention is highly safe. In addition, 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 parenteral or oral form in expectation of the above-mentioned action.

FIG. 1 shows the results of expressing clock gene expression as a relative amount to a control when test sample group 1 was added, as described in the examples. FIG. 2 is a view showing the results of measurement of relative mRNA amount over time when black-baked salmon was used as a test sample, as described in the examples. FIG. 3 shows the results of examining the expression of the clock gene as a relative amount to the control when adding various concentrations of black ginger as described in the examples. FIG. 4 is a diagram showing the results of performing fractionation under HPLC condition 1 on a 95% ethanol extract of Japanese black persimmon, as described in the examples. FIG. 5: is the figure which showed the result of having investigated expression of the clock gene at the time of adding Fr-1-Fr-5 as a relative amount with respect to control as it describes in the Example. FIG. 6 shows the results of performing fractionation under HPLC condition 2 for Fr-2 and examining the expression of the clock gene of the resulting fraction as a relative amount to the control, as described in the examples. FIG. FIG. 7 shows the results of carrying out fractionation under HPLC condition 3 for Fr-4 and examining the expression of the clock gene of the resulting fraction as a relative amount to the control, as described in the Examples. FIG. FIG. 8 shows the results of FT-MS and ¹ identification of the components contained in fractions Fr-2- (1) to (6) and Fr-4- (1) and (2) as described in the examples. It is the 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 the examples. It is the figure which showed the result of having implemented the real-time reporter gene assay about Per2 gene.

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

The composition of the present invention can exhibit a circadian rhythm regulating action and a sleep improving action in addition to a clock gene expression promoting action. In addition, in relation to these effects, 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 anti-aging action and the like.

The composition of the present invention can have at least the aspect of a clock gene expression promoting composition, a circadian rhythm adjusting composition and a sleep improving composition by having the above-described action.

The black ginger ( Kaempferia parviflora ) is a plant of the ginger family Banturus spp. Which is known to grow naturally in Southeast Asia and the like. It is known that black ginger has effects such as enhancement of energy, nourishment, blood sugar reduction, strength recovery, improvement of digestive system, improvement of vaginal bandage, hemorrhoids, gonorrhea, gonorrhea, stomatitis, arthralgia and stomach pain etc. It is done. Since black ginger is a proven natural plant that has been ingested by humans for a long time and is highly safe, the composition of the present invention is highly practical and can be used parenterally as it is or by processing. It is applicable to various uses in the form of oral or oral.

The site of use of the black ginger is not particularly limited as long as it is a site containing a component contributing to the desired pharmacological action, and examples thereof include roots, stems, leaves, flowers, branches and the like, preferably 3, 5, 7 , 3 ', 4'-pentamethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7-dimethoxyflavone, 3,5,7-trimethoxyflavone, 3,5,7,4'-tetramethoxy It is a rhizome which is rich in polymethoxyflavonoids (PMFs) such as flavone.

The black ginger is, in addition to the raw ones in the collected state, for example, the processing of black ginger such as treated (dried, cut, etc.) or powder of black ginger or treated black ginger, squeezed extract, etc. Include things. Here, the extract is not particularly limited as long as it is a substance from which a component in black ginger is extracted, but, for example, an extract obtained by extracting black ginger or a treated product thereof with a solvent, a dilution liquid or a concentrate thereof Or their dried products and their powders. The black ginger used in the present invention is an extract of black ginger or an extract such as squeezed juice, considering the ease of application to a composition for parenteral use such as a composition for external use on the skin or a composition for oral use etc. It is preferred that the extract is a dried product.

The black ginger powder is obtained by, for example, drying the sliced black ginger after washing with the sun or a dryer and then cutting it as it is or by cutting it into an appropriate shape or size using a grinder. It can be obtained by crushing. As the grinding apparatus, those generally used can be widely used. For example, a grinding machine composed of a raw material hopper, a grinding machine, a classifier, a product holder and the like can be used.

The black ginger extract is obtained by extracting black ginger and its processed product with a solvent. Examples of the solvent used for the extraction include lower alcohols such as ethanol, methanol, isopropanol and butanol; lower esters such as ethyl acetate and methyl acetate; acetone; a mixed solvent of these with water and the like. Water is also mentioned as an extraction solvent, and may be hot water, warm water or the like. Since the composition of the present invention has the possibility of being orally ingested by humans, the extract of black ginger as a raw material is water alone, ethanol alone or a mixed solvent of water and ethanol It is preferable that it is extracted by ethanol). For example, it is more preferable to use water-containing ethanol at 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, etc. may be used. it can. In the case of ethanol / water, for example, 1 to 40 times, preferably 2 to 20 times the weight of the solvent is added to the dried rhizome of black ginger, continuously or discontinuously at room temperature or under heating It is preferable to extract for several minutes to several days, preferably for about 5 minutes to 10 days, by such stirring.

Although the extraction method is not particularly limited, for example, it is preferable to perform the extraction operation under as mild conditions as possible, from the viewpoint of safety, convenience and industrialization. For example, a portion of black ginger or its dried substance is crushed, crushed, shredded etc., 2 to 20 times the mass of solvent is added to this, and 5 minutes to 10 days in the range of 0 ° C. to the reflux temperature of the solvent Extraction is performed under any conditions such as standing, shaking, stirring, reflux and the like. After the extraction operation, solid-liquid separation operation such as filtration and centrifugation is performed to remove insoluble solids. An extract is obtained by performing operations such as dilution and concentration as necessary. Furthermore, the same procedure may be repeated to extract insolubles, and the extract may be used in combination with the previous extract. These extracts may be further purified and used according to the purification method commonly used by those skilled in the art.

The obtained extract can be used as it is or, for example, in the form of a liquid, a concentrate, or a dried product obtained by drying these, by concentration or the like. The drying method is not particularly limited, and may be carried out by methods commonly used by those skilled in the art, such as spray drying, lyophilization, reduced pressure drying, fluid drying and the like. Furthermore, it is possible to use the dried product obtained by the above method as a powder using methods known to those skilled in the art. In addition, as described in Examples described later, after obtaining an extract and a dried product thereof, fractionation processing may be performed 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, for example, 3,5,7,3 ', 4'-pentamethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7,5 -Black flea extract containing PMF 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 It is a black ginger extract containing one or more kinds, more preferably a black ginger extract containing two or more of them, still more preferably a black ginger extract containing all of these That.

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

Although the content of 3,5,7,3 ′, 4′-pentamethoxyflavone in the black ginger extract is not particularly limited, for example, 3,5,7,3 ′, 4′-pentamethoxyflavone relative to the total amount of extract The proportion of the amount (3,5,7,3 ′, 4′-pentamethoxyflavone content) is, for example, 0.01 wt% or more, preferably 0.1 wt% or more, more preferably 1 wt% or more. More preferably 10 to 70 wt%.

Although the content of 5,7,4'-trimethoxyflavone in the black ginger extract is not particularly limited, for example, the ratio of the amount of 5,7,4'-trimethoxyflavone to the total amount of extract (5,7,4 ' The 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%.

Although the content of 5,7-dimethoxyflavone in the black ginger extract is not particularly limited, for example, the ratio of 5,7-dimethoxyflavone amount to the total amount of 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%.

Although the content of 3,5,7-trimethoxyflavone in the black ginger extract is not particularly limited, for example, the ratio of the amount of 3,5,7-trimethoxyflavone to the total amount of 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%.

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

There are no particular limitations on the method of producing the processed and processed black ginger containing the black ginger extract. The processed and processed products of black ginger may be manufactured, for example, by referring to the above description and the description of the examples described later, or may be manufactured by methods commonly known by those skilled in the art. And may be distributed in the market.

As described in Examples described later, black ginger has a stronger clock gene expression promoting action as compared with other natural products such as black beans, mushrooms, shirogwai, broccoli, sweet potato, burdock and tomato.

Where the clock gene is known as Per, Cry, Bmal, Clock, Dec, Rev-Erb, ROR, DBP, E4BP4 etc., the clock gene whose expression is promoted by the composition of the present invention is not particularly limited. As described in the examples below, the composition of the present invention can exhibit an expression promoting action on Per2, Cry1, Bmal1 or Clock. For example, the composition of the present invention may be 3,5,7,3 ', 4'-pentamethoxyflavone, 5,7,4'-trimethoxyflavone, 5,7-dimethoxyflavone, 3,5,7-trif When it is a composition containing a black ginger, a black ginger treated product or a black ginger extract containing at least one of methoxyflavone and 3,5,7,4′-tetramethoxyflavone, the Per2 expression promoting action is Bmal 1 when it is a composition containing black ginger, a black ginger treated product or a black ginger extract containing at least one of 5,7,4′-trimethoxyflavone and 5,7-dimethoxyflavone It can exhibit an expression promoting effect.

The term "clock gene expression promoting action" as used herein refers to, 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. At least one action of In addition, the circadian rhythm adjusting action is at least one of the recovery from the abnormal rhythm to the normal rhythm and the maintenance of the normal rhythm of the circadian rhythm of about 24 hours which the organism originally has. Say one action. The sleep improving action means suppressing or delaying becoming a current or future sleep abnormality or a state considered to be sleep abnormality in a subject, or improving the state thereof. The composition of the present invention can take the form of a clock gene expression promoter, a circadian rhythm regulator and a sleep improving agent as having the above-mentioned action.

The composition of the present invention may further contain additives used in ordinary processing such as excipients, fillers, binders, thickeners, emulsifiers, colorants, flavors, perfume oils and the like. The amount of the additive used is not particularly limited as long as it does not prevent 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 a form usually used, and for example, liquid, lotion, mousse, gel, emulsion, suspension, cream, ointment, sheet, aerosol It can take various forms such as spray, stick, powder, granular, granular, tablet, rod, 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 composition of the present invention can be processed into various forms such as lotions, emulsions, gels, creams, ointments and the like, as it is or mixed with additives commonly used in cosmetic processing. . Specifically, it can be used as a lotion, cosmetic cream, milk, cream, pack, hair tonic, hair cream, shampoo, hair rinse, treatment, face wash, foundation, hair restorer, aqueous ointment, spray and the like.

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

The usage and dose of the daily usage amount, single use amount, usage period, usage interval and the like of the composition of the present invention are not particularly limited, and may be appropriately selected depending on the usage mode and the condition of the circadian clock system of the user. It can be set. However, regarding the use period and use interval, as described in the examples described later, after taking the composition of the present invention, the clock genes Per 2 and Cry 1 are relatively peaked at 4 to 12 hours after intake The composition of the present invention is, for example, any of morning, day or night, since expression is promoted early and expression is promoted relatively slowly with Bmal1 and Clock peaking at 16 to 24 hours after ingestion. It is preferable to use it for one or more times for 2 days or more.

The method for producing the composition of the present invention is not particularly limited, and a solid composition can be obtained, for example, by mixing black sugar and additives at room temperature or under heating. Alternatively, this solid composition can be dissolved in a solvent such as water to make a liquid composition. Furthermore, it can be made a liquid composition by adding and mixing the other solid-like component containing an additive to the liquid containing black ginger. The obtained liquid composition may be subjected to drying treatment and then powdered. As a method of the drying process in this case, the method used when drying said black ginger can be utilized suitably, and it does not specifically limit.

The composition of the present invention can be used as it is or mixed with other components, compositions or products, depending on the application. Thus, the composition of the present invention may be mixed or used in combination with various ones as long as the object of the present invention can be achieved, as long as the composition contains black thorn.

As a specific embodiment of the composition of the present invention, for example, 0.1 to 50 wt% 3,5,7,3 ′, 4′-pentamethoxyflavone, 0.1 to 50 wt% 5,7,4 ′ Trimethoxyflavone, 0.1 to 50 wt% of 5,7-dimethoxyflavone, 0.1 to 50 wt% of 3,5,7-trimethoxyflavone and 0.1 to 50 wt% of 3,5,7,4 A composition containing '-tetramethoxy-flavone-containing black ginger extract is 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′-tributyl Containing methoxyflavone, 1 to 25 wt% of 5,7-dimethoxyflavone, 1 to 25 wt% of 3,5,7-trimethoxyflavone and 1 to 25 wt% of 3,5,7,4'-tetramethoxyflavone A composition containing black ginger extract is mentioned.

The amount of each PMF in the specific embodiment of the composition of the present invention described above is a 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 the additive.

Hereinafter, although the example of a combination concerning the concrete mode of the composition of the present invention is shown, the present invention is not limited to these example of a combination, and the present invention takes various aspects as long as the subject of the present invention can be solved. It can be taken.

(Composition example 1: Lotion)
The whole is 100 parts by mass, 0.01 part by mass of black ginger extract, 0.01 part by mass of ginger extract 0.01 part by mass of Ketchmeishi, 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, methyl paraben 0.15 parts by mass 0.2 parts by weight of carbomer, 0.03 parts by weight of sodium hydroxide and the balance of water were mixed to prepare a composition of the present invention in the form of a lotion.

(Composition example 2: Shampoo)
The total amount is 100 parts by mass, 0.01 part by mass of black ginger extract, 0.02 parts by mass of hop extract, 0.01 parts by mass of caffeine, 7.5 parts by mass of sodium laureth sulfate, cocamidopropyl betaine 4.2 Parts by weight, 3 parts by weight of cocamide DEA, 0.1 parts by weight of 1,3-butylene glycol, 0.225 parts by weight of polyquaternium-10, 0.15 parts by weight of citric acid, 0.05 parts by weight of sodium citrate, phenoxyethanol 0. The composition of the present invention was prepared in the form of a shampoo by mixing 9 parts by weight and the balance of water.

(Composition example 3: Soap)
The whole is 100 parts by mass, 0.5 parts by mass of ground product of black ginger, 0.2 parts by mass of Meriloto extract, 0.1 parts by mass of elastin, 2 parts by mass of glycerin, 1 part by mass of olive oil, 0.1 sodium EDTA-4 The composition of the invention was prepared in the form of a soap by mixing and solidifying parts by weight, 0.2 parts by weight of tetrasodium etidronate and the balance of the soap base.

(Composition example 4: Emulsion)
The whole is 100 parts by mass, 0.1 part by mass of black pepper extract, 0.1 parts by mass of alanine, 0.1 parts 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 parts by mass of sodium hydroxide and the balance of purified water, The composition of the invention was prepared in the form of an emulsion.

(Composition example 5: cosmetic cream)
The whole is 100 parts by mass, 0.1 part by mass of black pepper extract, 0.1 part by mass of isomaltooligosaccharide, 0.1 part by mass of fructooligosaccharides, 15.0 parts by mass of squalane, 4.0 parts 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, lipophilic lipophilic 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 white beeswax, 0.1 parts by mass of d-δ-tocopherol, 0.3 parts by mass of methyl paraben Parts, C10 to C30 alkyl-modified carboxyvinyl polymer 0.2 parts by mass, carboxyvinyl polymer 0.1 parts by mass, 1, - 18.0 parts by weight butanediol, 0.1 parts by mass of sodium hydroxide and a mixture of purified water balance, the composition of the present invention was prepared in the manner of a cosmetic cream.

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

(Composition example 7: Tablet)
100 parts by weight of the whole, 10 parts by weight of black ginger powder, 8 parts by weight of ginger extract, 5 parts by weight of caffeine, 5 parts by weight of melilote, 20 parts by weight of crystalline cellulose, 50 parts by weight of lactose, 4 parts by weight of magnesium stearate The composition of the present invention was prepared in the form of a tablet by mixing and compressing the remaining portion of corn starch and corn starch.

(Composition Example 8: Tablet)
100 parts by weight of the whole, 10 parts by weight of a black-green potato powder, 8 parts by weight of ketsumeishi powder, 5 parts by weight of meriloto, 0.5 parts by weight of isomaltooligosaccharide, 0.5 parts by weight of fructooligosaccharide, 20 parts by weight of crystalline cellulose, lactose The composition of the present invention was prepared in the form of a tablet by mixing and compressing 50 parts by weight, 4 parts by weight of magnesium stearate and the balance of corn starch.

(Composition example 9: Granules)
By mixing and granulating the whole into 100 parts by mass, 10 parts by mass of black sweet potato powder, 15 parts by mass of hop powder, 5 parts by mass of Ketchmeishi extract, 10 parts by mass of lactose, 1 part by mass of calcium stearate and crystalline cellulose The composition of the present invention was prepared in the form of granules.

(Composition Example 10: Capsule)
The whole is 100 parts by mass, 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 olive oil, and the mixture is used as a content liquid as a capsule shell. The composition of the present invention was prepared in the form of a capsule by encapsulating in

(Formulation example 11: Liquid agent)
The whole is 100 parts by mass, 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 glucose 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 perfume preparation, 0.05 parts by mass of sucralose, 0.03 parts by mass of acesulfame potassium, and the balance of purified water.

Hereinafter, the present invention will be described in more detail by way of 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. Method (1) Preparation of Test Sample Group 1 A mixture of 0.5 g of dried rhizome of black ginger added to 10 mL of 50% ethanol is subjected to 30 seconds of vortex treatment and 10 seconds of sonication, and then shaken for 10 minutes. It was subjected to treatment and then to centrifugation. The supernatant obtained by centrifugation was used as a 50% ethanol extract of black potato. Similarly, 50% ethanol extract was prepared using black beans, mushrooms, shrews, broccoli, sweet potato, burdock or tomatoes instead of black ginger. These 50% ethanol extracts were used as test sample group 1 and were used for gene expression analysis described later.

(2) Preparation of Test Sample Group 2 A mixture of 100 g of dried rhizomes of black ginger with 1,000 mL of 95% ethanol was subjected to discontinuous stirring for 6 days, and then subjected to filtration. The obtained filtrate was evaporated to dryness to obtain a blackened rice bran 95% ethanol extract (6.8 g). The black ginger 95% ethanol extract was used for the fraction mentioned later.

(3) Gene expression analysis After culturing NIH-3T3 cells which are mouse fibroblasts for 3 days, they are synchronized for 2 hours with 100 nM dexamethasone, and then the test sample group 1 becomes 0.5 mg / mL. As added to NIH-3T3 cells. After addition, total RNA was extracted from NIH-3T3 cells at predetermined times. The expression of clock genes (Per2, Cry1, Bmal1 and Clock) was examined by quantitative RT-PCR. In addition, what added ethanol so that it might become 0.5% instead of test sample group 1 was set as control.

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

[HPLC condition 1]
Column: YMC-DispoPack AT ODS-25
Eluent: ACN / H ₂ O (55/45)
Flow Rate: 15mL / min
[HPLC condition 2]
Column: Wakosil-II 5 C 18 RS-Prep, 5 μm, 20 × 250 mm
Eluent: ACN / MeOH / H ₂ O (20/30/35)
Flow Rate: 13 mL / min
[HPLC condition 3]
Column: Wakosil-II 5C18 RS-Prep, 5 μm, 20 × 250 mm
Eluent: ACN / MeOH / H ₂ O (20/30/30)
Flow Rate: 13 mL / min

2. Results (1) Gene Expression Analysis Six hours after addition of the test sample group 1, the results of examining clock gene expression as a relative amount to the control are shown in FIG. The result of having compared relative mRNA quantity sequentially over time every 4 hours at the time of using a black ginger as a test sample is shown in FIG. The final concentrations of black-baked salmon are changed to 0.005, 0.01, 0.03 and 0.05 mg / mL, and 6 hours (Per2 and Cry1) or 20 hours (Bmal1) after the black-green glaze is added, The result of examining the expression of the clock gene as a relative amount to the control is shown in FIG.

As FIG. 1 shows, the test sample which used black ginger made the expression level of Per2 and Bmal1 promoted compared with the test sample which used the other natural-material raw material. As shown in FIG. 2, rhythms peaking at 4 to 6 hours after the addition of black potato were confirmed for the expression of Per2 and Cry1. 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 are promoted depending on the concentration of black ginger.

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

FIG. 5 shows the expression of the clock gene after 6 hours (Per 2 and Cry 1) or 20 hours (Bmal 1) of Fr-1 to Fr-5 added to 25 μg / mL as a relative amount with respect to the control. Shown in. As shown in FIG. 5, the expression levels of Per2, Cry1 and Bmal1 were promoted in Fr-2, and the expression levels of Per2 and Cry1 were promoted in Fr-4.

Fractionation was carried out under HPLC condition 2 for Fr-2. Also, the expression of the clock gene after 6 hours (Per 2) or 20 hours (Bmal 1) after adding the resulting 6 fractions to dryness to 25 μg / mL was examined as a relative amount to the control The These results are shown in FIG. As shown in FIG. 6, the expression levels of Per2 and Bmal1 are promoted in Fr-2- (3) and (4), and the expression level of Per2 is promoted in Fr-2- (2), (5) and (6) It was done.

Fractionation was carried out under HPLC condition 3 for Fr-4. In addition, the expression of the clock gene after 6 hours (Per 2) of adding the dried two fractions to 12.5 μg / mL 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).

Identification of components contained in Fr-2- (1) to (6) and Fr-4- (1) and (2) among fractionated fractions using FT-MS and ¹ H-NMR went. The measurement results of the content of the identified compound and the extract of black ginger are shown in FIG. From the above results, among the components of black ginger, the component involved in the regulation of expression of Per2 gene is 3,5,7,3 ′, 4′-pentamethoxyflavone, 5,7,4′-trimethoxyflavone, It was confirmed to be 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. From these facts, it has been suggested that the black ginger polymethoxyflavone has the potential to adjust the circadian clock and improve the disturbed circadian rhythm.

In addition, pSLG (PEST) -Per2 (-376 / + 48) was introduced into NIH-3T3 cells as a reporter plasmid, and the results of real-time reporter gene assay performed using Kronos AB-2500 (ATTO) as a luminometer are shown in FIG. 9 shows. As a result, expression of Per2 gene peaked 12 hours after administration by 3,5,7,3 ', 4'-pentamethoxyflavone, 5,7,4'-trimethoxyflavone and 5,7-dimethoxyflavone Was confirmed to be promoted.

The composition of the present invention contains black ginger which is applicable in any of parenteral and oral aspects, and a subject who suffers from symptoms associated with abnormalities in the internal clock system such as sleep abnormality and jet lag. And contribute to the health and well-being of such subjects.

Claims (2)

A composition for adjusting a circadian rhythm, comprising black ginger. A composition for improving sleep, comprising black ginger.