JP2023005922A - Sirtuin activator - Google Patents

Abstract

To provide an excellent sirtuin activator.SOLUTION: An improved sirtuin activator can be provided by combining Kaempferia parviflora, a Kaempferia parviflora extract, or a polyalkoxyflavonoid compound with nicotinamide mononucleotide (NMN).SELECTED DRAWING: Figure 1

Description

The present invention relates to sirtuin activators.

Due to recent developments in medical technology, the average life expectancy of people has increased, and we have entered an age called the "era of 100-year lifespans." On the other hand, Japan is facing a declining birthrate and aging population, and it is said that by 2060, the proportion of elderly people aged 65 and over will reach approximately 40% of the Japanese population. As a result, the number of workers in the country is expected to decrease, and there is concern that this will adversely affect the development of society (see Non-Patent Documents 1 to 3). In other words, in order to develop society in the future, it is important not only to realize a longevity society, but also to extend healthy life expectancy.

Average life expectancy refers to how long a person can live from birth, and healthy life expectancy refers to the length of time a person can live without being restricted by health problems. In other words, the difference between the average life expectancy and the healthy life expectancy means the period during which daily life is limited, that is, the period during which one is not healthy. Currently, the gap between average life expectancy and healthy life expectancy is 9.13 years for men and 12.68 years for women.

Prevention of age-related diseases is considered important as one means of extending healthy life expectancy (Non-Patent Document 2). Activation of enzymes called sirtuins (hereinafter referred to as "sirtuin enzymes") is an important means for preventing age-related diseases. This sirtuin enzyme is a NAD ⁺ -dependent deacetylase, and has been reported to be involved in the suppression of neurodegeneration, improvement of glucose and lipid metabolism, and anti-obesity through various transcription factors. It is an important enzyme for suppressing aging and regulating metabolism. Therefore, it is desired to provide an anti-aging material having an excellent sirtuin activating action (Non-Patent Document 4).

By the way, black turmeric (Kaempferia parviflora) is a plant belonging to the genus Zingiberaceae of the family Zingiberaceae, and is also called black ginger, Thai ginseng, or Krachaidum. Black turmeric originates from Thailand and Laos in Southeast Asia, where it has been consumed on a daily basis for a long time, and is used for nutritional tonic, energy enhancement, lowering blood sugar levels, recovery of physical strength, and improvement of the circulatory and digestive systems. So far, it has been described in Non-Patent Document 5, Patent Documents 1 and 2 below that black turmeric extract has a function of activating sirtuins.

Black turmeric contains five characteristic polymethoxyflavonoid compounds (apigenin-5,7,4'-trimethylether), quercetin-3,5,7,3',4 '-pentamethyl ether (quercetin-3,5,7,3',4'-pentamethylether), chrysin dimethyl ether (chrysin dimethyl ether), 3,5,7,4'-tetramethoxyflavone (3,5,7,4 '-tetramethoxyflavone), 3,5,7-trimethoxyflavone (3,5,7-trimethoxyflavone)). Moreover, it has been reported that all of these five types of polymethoxyflavonoid compounds have sirtuin activating action (see, for example, Non-Patent Documents 5 and 6 below).

NMN is a precursor of nicotinamide adenine nucleotide (hereinafter referred to as "NAD+") and is also used to generate NAD+. It has been reported that NAD+ is an important electron carrier present in all species and involved in sirtuin enzymatic reactions. In addition, the following non-patent document 7 states that it is believed that the ability to generate NMN decreases with aging, and that NAD+ is thought to decrease with age.

Japanese Patent No. 6417630 U.S. Pat. No. 10,889,557

Health and Productivity Management Research Unit, Policy Alternatives Research Center, The University of Tokyo. Ministry of Health, Labor and Welfare, 2015 National Health and Nutrition Survey Report, Part 3 Lifestyle Survey Results Materials from the 11th Health Japan 21 (Second) Promotion Committee Rahman, S.; , & Islam, R. (2011). "Mammalian Sirt 1: insights on its biological functions." Cell Communication and Signaling, 9, 11. Nakata, A.; , Koike, Y.; , Matsui, H.; , Shimada, T.; , Aburada, M.; , & Yang,J. (2014). "Potent SIRT1 enzyme-stimulating and anti-glycosylation activities of polymethoxyflavonoids from Kaempferia parviflora." Natural product communications, 9(9), 14291 Zhang, M.; , Lu, P.; , Terada, T.; , Sui, M.; , Furuta, H.; , Iida, K.; , Katayama, Y.; , Lu, Y.; , Okamoto, K.; , Suzuki, M.; , Asakura, T.; , Shimizu, K.; , Hakuno, F.; , Takahashi, S.; , Shimada, N.; , Yang, J.; , Ishikawa, T.; , Tatsuzaki, J.; , Nagata, K.; (2021). "Quercetin 3,5,7,3',4',-pentamethyl ether from Kaempferia parviflora directly and effectively activates human SIRT1." Communication Biology, 4, 209. Bonkowski, M.; , Sinclair, D.; (2017). "Slowing aging by design: The rise of NAD+ and sirtuin-activating compounds." Nature Reviews Molecular Cell Biology, 17(11), 679-690.

As described above, as the aging society becomes more serious, it is desired to provide a composition having a more excellent sirtuin activating action.

However, the techniques described in Patent Documents 1 and 2, Non-Patent Documents 5, 6 and 7 are only reports using a single plant extract or plant-derived component, and there are no reports of combining these. Absent.

Therefore, in view of the above problems, an object of the present invention is to provide a composition having a superior sirtuin activation action by combining materials.

The present inventors have made intensive studies on the above problems, and found that combining black turmeric, black turmeric extract, or polyalkoxyflavonoid compounds with NMN, specifically, by simultaneously containing NMN, The inventors have discovered that an excellent sirtuin-activating action was obtained, and have completed the present invention.

That is, the sirtuin activator according to one aspect of the present invention is characterized by containing at least one of black turmeric and black turmeric (Kaempferia parviflora) extract, and nicotinamide mononucleotide (NMN). It is something to do.

（式中のＲ１～Ｒ７は、各々独立に、水素原子、水酸基、または炭素数１～６の低級アルコキシ基を表す）。 In addition, a sirtuin activator according to another aspect of the present invention contains a polyalkoxyflavonoid compound represented by the following general formula (I) and nicotinamide mononucleotide (NMN). It is characterized by

(R1 to R7 in the formula each independently represent a hydrogen atom, a hydroxyl group, or a lower alkoxy group having 1 to 6 carbon atoms).

（式中のＲ１１、Ｒ１２、Ｒ１４、Ｒ１５およびＲ１６は、各々独立に、水素原子、水酸基、またはメトキシを表し、Ｒ１３は水素原子またはメチル基を表す）。 Moreover, from this point of view, although not limited, the polyalkoxyflavonoid compound is preferably a polymethoxyflavonoid compound represented by the following general formula (II).

(R11, R12, R14, R15 and R16 in the formula each independently represent a hydrogen atom, a hydroxyl group or a methoxy group, and R13 represents a hydrogen atom or a methyl group).

In addition, from this point of view, it is preferable that at least one of black turmeric (Kaempferia parviflora) and black turmeric extract is included.

In addition, in this aspect, the compound represented by the general formula (I) or (II) includes quercetin-3,5,7,3′,4′-pentamethyl ether, although not limited thereto. It is preferable that the

A food or drink product, a health food product, a cosmetic product, or a pharmaceutical composition according to another aspect of the present invention contains any of the sirtuin activators described above.

As described above, the present invention can provide a composition having a superior sirtuin activating action.

FIG. 4 is a diagram showing the sirtuin-activating action when NMN and black turmeric extract or each alone is added in Examples. FIG. 3 is a diagram showing the sirtuin-activating action when NMN and quercetin-3,5,7,3′,4′-pentamethyl ether, or each alone is added in Examples.

Embodiments of the present invention will be described below. However, the present invention can be embodied in many different forms, and is not limited only to the specific exemplifications in the embodiments and examples given below.

One embodiment of the present invention is a composition with superior sirtuin activation. That is, it is characterized by containing black turmeric extract and NMN, or containing quercetin-3,5,7,3′,4′-pentamethyl ether and NMN. is expected to have an excellent sirtuin activation effect.

The black turmeric used in this embodiment can be the rhizome of Kaempferia parviflora, which is native to Southeast Asia such as Thailand and Laos, or cultivated in Okinawa. Black turmeric can be used raw, dried or processed from the rhizome, and the underground part of black turmeric including the rhizome may be used as it is.

In addition, the extraction method of the black ginger extract is not particularly limited, and can be carried out according to methods well known to those skilled in the art. Water or hot water, alcoholic solvents, and other organic solvents such as acetone can be used as extraction solvents. Examples of alcohol solvents include methanol, ethanol, propanol, isopropanol, butanol, isobutanol and the like. Examples of organic solvents other than acetone include esters such as ethyl acetate; polyhydric alcohols such as ethylene glycol, propylene glycol and 1,3-butylene glycol; ethers such as diethyl ether. These solvents may be used alone or in combination. The extraction solvent may be used in the form of a water-containing hydrophilic solvent by combining water and a hydrophilic organic solvent. When using a combination of solvents, the mixing ratio of the solvents can be set arbitrarily. Among these, ethanol is preferably used, and hydrous ethanol having an ethanol content of 30% to 95% (v/v) is more preferably used.

The amount of the extraction solvent is preferably 2 to 100 parts by weight based on the dry weight of the rhizome of black turmeric. The extraction temperature is preferably 4 to 90°C. The extraction time is preferably 30 minutes to 1 week. The extraction method can be any method such as stirring extraction, immersion extraction, countercurrent extraction, ultrasonic extraction, supercritical extraction, and the like.

The black ginger extract includes a filtrate obtained by filtering the obtained extract, a concentrate obtained by concentrating the filtrate, a dried product obtained by drying the concentrate, or a crude or purified product thereof. is also included. Concentration can be performed by any method such as evaporative concentration and membrane concentration. Any drying method such as vacuum drying, freeze-drying, and spray-drying can be used. An excipient such as dextrin may be included if desired. When purification is carried out, it can be carried out according to means known to those skilled in the art. For example, synthetic adsorption resins, activated carbon, ion exchange resins, Sephadex, gel filtration media such as biogel, column chromatography, recrystallization, etc. may be used alone or in combination.

R ₁ to R ₇ in the above formula each independently represent a lower alkoxy group having 1 to 6 carbon atoms, a hydrogen atom or a hydroxyl group.

A lower alkoxy group having 1 to 6 carbon atoms for R ₁ to R ₇ in general formula (I) means an alkyl group having 1 to 6 carbon atoms ether-bonded to an oxygen atom. The lower alkoxy group having 1 to 6 carbon atoms includes methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, or structural isomers thereof, preferably methoxy.
A preferred polyalkoxyflavonoid compound is a polymethoxyflavonoid compound, and a more preferred polymethoxyflavonoid compound is a compound represented by the following general formula (II).

R ₁₁ to R ₁₆ in the above formula each independently represent a hydrogen atom, hydroxyl group or methoxy, and R13 represents a hydrogen atom or a methyl group.

Further, specific examples of the compounds represented by the general formula (II) are shown in Table 1 below.

The polyalkoxyflavonoid compound represented by the above general formula (I) or (II) can be obtained by chemical synthesis or by extraction and purification from black turmeric or citrus. The synthesis method is not particularly limited, and conventionally known methods can be used. Moreover, the polyalkoxyflavonoid compound represented by the general formula (I) or (II) can be combined with at least one of black ginger and black ginger extract.

The citrus family includes Citrus depressa, C. tachibana, C. leiocarpa, C. tardiva, C. succosa, Shikaikan, C. kinokuni, and Kobeni mandarin. (C. erythrosa), Sunki (C. sunki), Chinese mandarin (C. deliciosa), King (C. nobilis), Ponkan (C. retuculata), Dancy tangerine (C. tangerina), Hanayu belonging to Yuzu Ward (C. hanayu), C. nippokoreana.

In addition, methods for purifying polyalkoxyflavonoid compounds include, for example, synthetic adsorption resins, activated carbon, ion exchange resins, Sephadex, gel filtration media such as biogel, column chromatography, recrystallization, etc., used alone or in combination. may

The structure of the polyalkoxyflavonoid compound obtained by purification can be identified according to means known to those skilled in the art. For example, proton nuclear magnetic resonance spectroscopy ( ¹ H-NMR), carbon nuclear magnetic resonance spectroscopy ( ¹³ C-NMR), mass spectrometry (MS), elemental analysis, infrared spectroscopy (IR), ultraviolet spectroscopy (UV), melting point determination, etc. may be used alone or in combination.

This black turmeric extract contains five types of polymethoxyflavonoid compounds (apigenin-5,7,4'-trimethyl ether, quercetin-3,5,7,3',4'-pentamethyl ether, chrysin dimethyl ether, 3, 5,7,4′-tetramethoxyflavone, 3,5,7-trimethoxyflavone) is preferably contained in an amount of 4% by weight or more, more preferably 10% by weight or more and 50% by weight or less. Range. By containing in this range, the effects of the present composition are preferably exhibited.

The black turmeric extract and NMN contained in the present composition are not limited, but when the weight of NMN is 1, the amount of black turmeric extract is in the range of 0.01 or more and 20 or less. It is preferably 0.05 or more and 10 or less, and still more preferably 0.1 or more and 5 or less.

In addition, the polyalkoxyflavonoid and NMN are not limited, but when the weight of NMN is 1, the amount of polyalkoxyflavonoid is preferably in the range of 0.0003 or more and 0.6 or less, and more preferably. is 0.0015 or more and 0.3 or less, more preferably 0.003 or more and 0.15 or less.

In addition, the present composition can take the form of pharmaceuticals and foods, but each extract is used for food alone, and it is expected that it will not cause great harm to the user. Confirmed. Therefore, if it is a food, it can be obtained easily without relying on a doctor's prescription, and it can take the form of a food with function claims and a health food.

Furthermore, the form of the present composition is not limited to solids, and can be in the form of beverages. Examples of the form of the composition when it is a food include the form of desserts such as drinks, candies, jellies, and gummies. , for example, tablets, hard capsules, soft capsules, granules, drinks and the like.

As described above, an excellent sirtuin activator can be provided by combining at least one of black turmeric (Kaempferia parviflora), black turmeric extract and polyalkoxyflavonoid compound with NMN.

Here, the composition according to the above embodiment was actually produced, and its effect was confirmed. The present invention will be specifically described below, but the present invention is not limited to the following examples.

(black turmeric extract)
Dried chips of rhizomes of black turmeric (Kaempferia parviflora) were pulverized with a mixer, 80% ethanol was added to the pulverized product, and after heating under reflux, the mixture was filtered to obtain an extract. The extraction residue was again heated under reflux with 80% ethanol and then filtered to obtain an extract. The first extract and the second extract were combined, concentrated under reduced pressure, added with excipients, and dried under reduced pressure to obtain a black turmeric extract (80% ethanol extract).

(Quercetin-3,5,7,3′,4′-pentamethyl ether)
The 80% ethanol extract of black turmeric obtained above was partitioned with water and ethyl acetate, and after concentration and drying, 4.58 g of an ethyl acetate phase was obtained. After the obtained ethyl acetate phase was adsorbed on silica gel, it was eluted with a methanol-chloroform mixed solvent, and the resulting fraction was further isolated and purified by reversed-phase high-performance liquid chromatography (ODS) to give Compound 1. (784 mg) was obtained.

Then, a nuclear magnetic resonance spectrum of Compound 1 obtained above was obtained. Table 2 below shows the results of proton nuclear magnetic resonance spectroscopy ( ¹ H-NMR) and carbon nuclear magnetic resonance spectroscopy ( ¹³ C-NMR). In the table, the "measured value" indicates the result measured by the present inventors, and the "literature value" refers to "Simultaneous identification and quantification of 11 flavonoid constituents in Kaempferia parviflora by gas chromatographic grapha- pharaphraphmal. , 1143:227-233, 2007.”.

Table 3 below shows physical property data of compound 1 measured by melting point measurement, infrared spectroscopy (IR), ultraviolet spectroscopy (UV) and mass spectroscopy (MS).

Chemical shift values, spectral shapes, coupling constants, coupling states, and comparisons with literature values in the nuclear magnetic resonance spectra ( ¹ H-NMR and ¹³ C-NMR) of compound 1, and physical property data such as the melting point of the compound 1 was identified as quercetin-3,5,7,3′,4′-pentamethylether.

(Evaluation test of sirtuin activation effect)
2.5×10 ⁵ cells/mL (10% fetal bovine serum (FBS)-containing Dulbecco's MEM) monocyte-macrophage cells (RAW264.7) derived from mouse ascites were seeded in a cell culture dish and incubated overnight at 37°C. Cultured in a 5% CO ₂ incubator. After the culture was completed, the medium was replaced with a new culture medium, and the culture was further continued for 1 hour. After that, 100 μL of 25, 50 or 100 μg/mL NMN was added, and the mixture was allowed to stand in a 37° C., 5% CO ₂ incubator for 6 hours. Thereafter, the NMN-containing culture medium was removed, 200 μL of cell lysis buffer (50 mM HEPES buffer pH 8, 50 mM NaCl, 1% Triton X-100) was added, and the cells were collected with a cell scraper. The recovered cells were disrupted with a homogenizer, the disrupted cell solution was centrifuged at 12,000 rpm, and three types of supernatants were recovered.

Black turmeric extract or quercetin-3,5,7,3′,4′-pentamethyl ether was added to each of the three cell supernatants obtained above, and FLUOR DE LYS (registered trademark) SIRT Using an activity assay kit, sirtuin activation was evaluated. In addition, the final concentration of black turmeric extract was adjusted to 50 µg/mL, and the final concentration of quercetin-3,5,7,3',4'-pentamethyl ether was adjusted to 2 µg/mL.

(Test result 1)
As shown in FIG. 1, when the black ginger extract and NMN were added simultaneously, a stronger sirtuin-activating action than the black ginger extract or NMN alone was exhibited. Moreover, a similar sirtuin-activating action was confirmed at any ratio of black turmeric extract to NMN of 1:2, 1:1, and 2:1. That is, according to the present results, it was confirmed that a superior synergistic sirtuin activating action can be obtained when the black turmeric extract and NMN are simultaneously added, as compared to the case of the black turmeric extract alone and the case of NMN alone. .

(Test result 2)
As shown in FIG. 2, when quercetin-3,5,7,3′,4′-pentamethyl ether and NMN were added simultaneously, a stronger sirtuin-activating action was exhibited than each alone. Thus, according to the present results, quercetin-3,5,7,3′,4′-pentamethyl ether alone and NMN alone are more effective than quercetin-3,5,7,3′,4′-pentamethyl ether. It was confirmed that an excellent synergistic sirtuin activation effect can be obtained when ether and NMN are added simultaneously.

As described above, it was confirmed that a combination of black turmeric extract or quercetin-3,5,7,3',4'-pentamethyl ether and NMN has a superior sirtuin activation effect.

A better sirtuin activator can be provided by combining black turmeric (Kaempferia parviflora) or a black turmeric extract or a polyalkoxyflavonoid compound with nicotinamide mononucleotide (NMN).

Claims (6)

A sirtuin activator characterized by containing at least one of black turmeric (Kaempferia parviflora) and black turmeric extract, and nicotinamide mononucleotide (NMN). A sirtuin activator comprising a polyalkoxyflavonoid compound represented by the following general formula (I) and nicotinamide mononucleotide (NMN).

(R1 to R7 in the formula each independently represent a hydrogen atom, a hydroxyl group, or a lower alkoxy group having 1 to 6 carbon atoms). The sirtuin activator according to claim 2, wherein the polyalkoxyflavonoid compound is a polymethoxyflavonoid compound represented by the following general formula (II):

(R11, R12, R14, R15 and R16 in the formula each independently represent a hydrogen atom, a hydroxyl group or a methoxy group, and R13 represents a hydrogen atom or a methyl group). 3. The sirtuin activator according to claim 2, comprising at least one of black turmeric (Kaempferia parviflora) and black turmeric extract. The sirtuin activator according to claim 2 or 3, wherein the compound represented by general formula (I) or (II) comprises quercetin-3,5,7,3',4'-pentamethyl ether. A food, drink, health food, cosmetic, or pharmaceutical composition comprising the sirtuin activator according to any one of claims 1 to 5.

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