SIRT1 activator

Abstract

To provide a SIRT1 activator.SOLUTION: A SIRT1 activator comprises propolis produced in the Kangaroo Island, Australia.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to SIRT1 activators, and more specifically, to SIRT1 activators containing propolis.

Background Art

[0002] In previous studies on aging and lifespan control, it has been known that calorie restriction is an effective treatment for lifespan extension and anti-aging in yeast, nematodes, Drosophila, etc. And it has been clarified that Sir2 having NAD-dependent deacetylase activity is involved in the lifespan extension by those calorie restrictions (Non-Patent Document 1), and sirtuin 1 to 7 are known as mammalian homologs of Sir2. Among them, sirtuin 1 (SIRT1) which is most similar to Sir2 in structure and function has been attracting attention.

[0003] While the expression of SIRT1 decreases with aging, it is known that SIRT1 is activated by calorie restriction and also by plant polyphenols such as resveratrol which is a polyphenol in grape skins and red wine (Non-Patent Document 2). SIRT1 plays various roles in each tissue, and research on its association with aging-related diseases such as Alzheimer's dementia, osteoporosis, arteriosclerosis, sarcopenia, etc. has also been conducted. Thus, it has been clarified that SIRT1 has functions such as anti-aging effect, cognitive function improvement effect, bone formation promotion effect, neuroprotective effect, vascular endothelial protective effect, diabetes inhibitory effect, etc.

[0004] Propolis is a colloidal substance formed by mixing the sap of trees collected by bees, new shoots of plants, exudates, etc. with beeswax, etc., and is known to have antibacterial, antioxidant, anti-inflammatory, antitumor, anti-allergic effects, etc.

[0005] Because the composition of propolis is influenced by the type and proportion of the source plants from which it is made, it is known that the types, amounts, and physiological activity of active ingredients in propolis can vary considerably depending on the country or region from which it is harvested.

[0006] In recent years, research has been conducted on the components of propolis from different regions. For example, in Brazilian propolis, which uses Baccharis dracuifolia as its main source plant, cinnamic acid derivatives such as p-coumaric acid, artepillin C, dorupanin, and baccharin have been detected as the main components. On the other hand, in propolis from China, Europe, and Australia, which use poplar as their source plant, flavonoids such as chrysin and galangin have been detected as the main components.

[0007] Non-patent document 3 reports that Chinese propolis containing flavonoids (hereinafter sometimes referred to as "Chinese propolis") has the effect of increasing SIRT1 expression.

[0008] Non-patent document 4 reports that propolis from Minas Gerais state in Brazil (hereinafter sometimes referred to as "Brazilian propolis") has the effect of increasing SIRT1 expression. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] PLoS ONE 3:e1759,2008 [Non-Patent Document 2] Nature, 425:191-196 (2003) [Non-Patent Document 3] Evid Based Complement Alternat Med.2018 Jun 27;2018:4957573 [Non-Patent Document 4] BMC Complement Altern Med.2016 Aug 30;16(1):329 [Overview of the project] [Problems that the invention aims to solve]

[0010] The present invention aims to provide a SIRT1 activator. [Means for solving the problem]

[0011] It is generally known that resveratrol has a high SIRT1 activating effect. Furthermore, Non-Patent Documents 3 and 4 mentioned above have also reported that Chinese propolis and Brazilian propolis have SIRT1 activating effects. Since propolis is made from plants collected by honeybees, differences in composition are observed depending on the place of origin, and therefore it is expected that the effects will differ depending on the place of origin. Therefore, the present inventors diligently conducted research to achieve the above objective and found that propolis from Kangaroo Island, Australia (hereinafter sometimes referred to as "Kangaroo Island propolis") has a higher SIRT1 activating effect than resveratrol, Chinese propolis, and Brazilian propolis.

[0012] Based on these findings and further investigations, the present invention provides the following SIRT1 activator.

[0013] Item 1. SIRT1 activator containing propolis from Kangaroo Island, Australia. Item 2. The SIRT1 activator described in Item 1, wherein the source plant of the propolis from Kangaroo Island, Australia, is a plant of the genus Lepidosperma. Item 3. A SIRT1 activator according to item 1 or 2, used for at least one selected from the group consisting of anti-aging, cognitive function improvement, bone formation promotion, neuroprotection, vascular endothelial protection, and diabetes suppression. Item 4. A SIRT1 activator described in any of items 1 to 3, which is a food or beverage, cosmetic, pharmaceutical, or quasi-drug. [Effects of the Invention]

[0014] The propolis produced in Kangaroo Island, Australia has excellent SIRT1 activation effect, so it is useful as an active ingredient of SIRT1 activator.

[0015] In addition, since propolis has been conventionally used as a food material, it has high safety.

Mode for Carrying Out the Invention

[0016] Hereinafter, embodiments of the present invention will be described in detail.

[0017] In this specification, "comprise" includes the meaning of "essentially consist of" and the meaning of "consist of only".

[0018] The SIRT1 activator of the present invention is characterized by containing propolis produced in Kangaroo Island, Australia.

[0019] Propolis is a resinous or waxy substance that constitutes the nest wall of a honeybee hive, and is a substance made by mixing the new shoots and resins of plants collected by honeybees with the saliva of bees. In the present invention, propolis produced in Kangaroo Island, Australia is used. In addition to Kangaroo Island, Australia, propolis from other production areas such as China, Taiwan, European countries, Russia, Oceania, and the United States may be used in combination.

[0020] The propolis used in the present invention is not particularly limited as long as it is collected in Kangaroo Island, Australia. Among them, as the propolis produced in Kangaroo Island, Australia, those whose original plant is a plant of the genus Lepidosperma are preferable.

[0021] The propolis in the present invention includes, for example, propolis lumps, their pulverized products, supercritical extracts, extracts with water and / or hydrophilic organic solvents, and the like. Among them, extracts such as extracts with water and / or hydrophilic organic solvents and supercritical extracts are preferred, and extracts with water and / or hydrophilic organic solvents are more preferred. The extract with water and / or hydrophilic organic solvents is one in which the components of propolis are efficiently and balancedly extracted in a short time, and it is preferred because large-scale equipment is not required when scaling up.

[0022] Examples of the hydrophilic organic solvent include lower alcohols such as ethanol, methanol, and propanol, ketones (such as acetone, methyl ethyl ketone, and methyl butyl ketone), esters (such as methyl acetate and ethyl acetate), glycols (such as ethylene glycol and propylene glycol), glycerin, tetrahydrofuran, acetonitrile, acetic acid, propionic acid, acetamide, dimethylformamide, dimethyl sulfoxide, etc. Lower alcohols are preferred, and ethanol is particularly preferred.

[0023] These extraction solvents can be used alone, or two or more of them can be arbitrarily combined and used as a mixed solution. Preferably, it is water, a lower alcohol (preferably ethanol), or a mixed solution thereof (hydrous alcohol, preferably hydrous ethanol). When using a hydrous alcohol (preferably hydrous ethanol) as the extraction solvent, although the alcohol concentration is not limited, 50 to 100% by volume, particularly 70 to 100% by volume can be preferably cited.

[0024] For extraction, propolis lumps, propolis lumps washed with alcohol, etc. can be used, and the extraction efficiency can be improved by using propolis lumps cut or pulverized into an appropriate size. The usage volume of the extraction solvent is, for example, 1 to 20 times the mass of the propolis lumps, preferably 2 to 10 times the mass.

[0025] Extraction can be carried out by adding propolis to a solvent and setting the temperature to, for example, 0°C to the boiling point of the solvent (usually below 100°C), preferably 2 to 40°C. Extraction can be carried out at the above temperature for a predetermined time (for example, 1 hour or more) with stirring or by allowing it to stand. After extraction, the extract can be obtained by performing a process such as filtration to remove insoluble matter.

[0026] The solvent extract thus obtained can be used as is, or, if necessary, further purification can be performed to increase the content of the active ingredient by methods such as ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography (HPLC), dialysis, or a combination thereof.

[0027] Furthermore, the solvent extract can be concentrated and dried by methods such as freeze-drying or spray-drying. Here, concentration, freeze-drying, and spray-drying can be carried out according to conventional methods. The propolis extract in this invention also includes powders obtained by pulverizing such extracts, and granular forms obtained by granulating the powder.

[0028] Other known components may be appropriately incorporated into the SIRT1 activator of the present invention, as long as they do not interfere with the effects of the present invention.

[0029] The propolis content in the SIRT1 activator of the present invention is not particularly limited as long as the effects of the present invention are obtained, and can be appropriately adjusted depending on the final form, etc., relative to the total solid content of the SIRT1 activator, for example, 0.00001% by mass or more, 0.0001% by mass or more, 0.001% by mass or more, 0.01% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, 3% by mass or more, 5% by mass or more, 7% by mass or more, 10% by mass or more, 15% by mass or more, 20% by mass 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, or 90% by mass or more, 100% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass % or less, 55% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 7% by mass or less, 5% by mass or less, 3% by mass or less, 1% by mass or less, or 0.5% by mass or less.

[0030] Furthermore, propolis is preferably applied regularly to activate SIRT1, and is particularly preferably applied once a day or in several divided doses. While not particularly limited, the solid content can be used in doses of, for example, 10 μg to 1,000 mg per day for an adult weighing 60 kg, preferably 100 μg to 500 mg, and more preferably 1 mg to 300 mg. The dose can be appropriately set within the above range depending on factors such as the health condition of the person receiving the treatment, the method of administration, and combinations with other agents.

[0031] The SIRT1 activator of the present invention can be used in cosmetics, food and beverages (especially food and beverages intended for health, health maintenance, and promotion (e.g., health foods, functional foods, nutritional compositions, nutritional supplements, supplements, health foods, foods for specified health uses, nutrient function foods, or foods with functional claims)), quasi-drugs, pharmaceuticals, etc. Furthermore, the SIRT1 activator of the present invention also encompasses the meaning of an additive that imparts SIRT1 activation.

[0032] In addition to the propolis mentioned above, the above-mentioned cosmetics may contain, as needed, other ingredients commonly used in cosmetics, such as disinfectants, preservatives, surfactants, alcohols, aqueous components, water, colorants, pH adjusters, solubilizers, abrasives, foaming agents, enzymes, flavoring agents, chelating agents, excipients, thickeners, bases, emulsifiers, solvents, stabilizers, oils, cleaning agents (lactic acid bacteria), etc.

[0033] Cosmetics include all cosmetics applied to the skin, mucous membranes, body hair, head hair, scalp, nails, teeth, facial skin, lips, etc., of animals (including humans).

[0034] Cosmetic formulations can take a wide range of forms, including aqueous solutions, solubilized solutions, emulsified solutions, powders, oil-based solutions, gels, ointments, aerosols, two-layer water-oil systems, and three-layer water-oil-powder systems.

[0035] The uses of cosmetics are also arbitrary. For example, basic cosmetics include facial cleansers, toners, lotions, creams, gels, essences, serums, packs, and masks. Makeup cosmetics include foundations, lipsticks, blushes, eyeshadows, eyeliners, and mascaras. Nail cosmetics include nail polish, base coats, top coats, and nail polish removers. Other examples include facial cleansers, (paste or liquid) toothpaste, mouthwash, massage agents, cleansing agents, aftershave lotions, pre-shave lotions, shaving creams, body soaps, soaps, shampoos, conditioners, hair treatments, hair styling products, hair tonics, hair growth products, antiperspirants, and bath additives.

[0036] The above-mentioned food and beverages may use the above-mentioned propolis as is, or, as necessary, may be combined with minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, flavorings, stabilizers, gelling agents, preservatives, sustained-release regulators, surfactants, solvents, humectants, etc.

[0037] Food and beverages include all foods and beverages that can be consumed by animals (including humans). The types of food and beverages are not particularly limited and include, for example, dairy products; fermented foods (yogurt, etc.); beverages (soft drinks such as coffee, juice, and tea, milk drinks, lactic acid bacteria drinks, lactic acid bacteria drinks, yogurt drinks, carbonated drinks, sake, Western liquors, fruit wines, etc.); spreads (custard cream, etc.); pastes (fruit paste, etc.); Western sweets (chocolate, donuts, pies, cream puffs, gum, gummies, jelly, candy, cookies, cakes, puddings, etc.); Japanese sweets (daifuku, mochi, manju, castella, anmitsu, yokan, etc.); frozen desserts (ice cream, ice pops, sherbet, etc.); food products (curry, gyudon, zosui, miso soup, soup, meat sauce, pasta, pickles, jam, etc.); and seasonings (dressings, furikake, umami seasonings, soup bases, etc.).

[0038] The manufacturing method for food and beverages is not particularly limited and may be carried out according to publicly known methods as appropriate.

[0039] There are no particular restrictions on the dosage unit form when used as a supplement; it can be selected as appropriate. Examples include chewable tablets, lozenges, capsules, granules, liquids, powders, syrups, pastes, drinks, gummies, etc.

[0040] The above-mentioned pharmaceuticals may use only the propolis, or they may be used in combination with other medicinal ingredients listed in the Japanese Pharmacopoeia, such as vitamins and herbal medicines.

[0041] When preparing the SIRT1 activator of the present invention as a pharmaceutical product, the above-mentioned propolis can be prepared together with pharmaceutical-permissible components in the form of tablets (including uncoated tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, lozenges, etc.), capsules, pills, powders, granules, liquids, suspensions, emulsions, syrups, pastes, and injections (including cases where it is prepared as a liquid by mixing it with distilled water or intravenous solutions such as amino acid solutions or electrolyte solutions at the time of use) to create a pharmaceutical preparation.

[0042] The administration of pharmaceuticals may be local or systemic. There are no particular restrictions on the method of administration, and they may be administered orally or parenterally. Parenteral administration routes include subcutaneous, intradermal, intramuscular, intraperitoneal, intravenous or intra-arterial administration, and transdermal administration.

[0043] In addition to propolis, the pharmaceutical product of the present invention may contain, as needed, pharmaceutically acceptable ingredients such as excipients, binders, disintegrants, lubricants, colorants, suspending agents, thickeners, antioxidants, absorption enhancers, pH adjusters, preservatives, stabilizers, surfactants, sweeteners, flavoring agents, and fragrances.

[0044] Furthermore, the cosmetics and pharmaceuticals of this invention also include quasi-drugs.

[0045] The SIRT1 activator of the present invention described above is applicable to mammals, including humans (preferably humans).

[0046] The Australian Kangaroo Island propolis contained as an active ingredient in the SIRT1 activator of the present invention has SIRT1 activating properties, as shown in the examples described later, and is therefore useful as an active ingredient in the SIRT1 activator. In the present invention, SIRT1 activation means improving the enzyme activity of SIRT1, that is, improving the enzyme activity of SIRT1 compared to before the addition of propolis.

[0047] The SIRT1 activator of the present invention can activate SIRT1, and therefore can exert effects such as anti-aging, cognitive function improvement, bone formation promotion, neuroprotection, vascular endothelial protection, and diabetes suppression. For this reason, the SIRT1 activator of the present invention can be used for applications aimed at achieving these effects. For example, the SIRT1 activator of the present invention can be used for at least one selected from the group consisting of anti-aging, cognitive function improvement, bone formation promotion, neuroprotection, vascular endothelial protection, and diabetes suppression. Specific target diseases include, for example, Alzheimer's disease for cognitive function improvement, osteoporosis for bone formation promotion, Alzheimer's disease and Parkinson's disease for neuroprotection, arteriosclerosis for vascular endothelial protection, and type 2 diabetes for diabetes.

[0048] Furthermore, since propolis has traditionally been used as a food ingredient, it is considered highly safe. [Examples]

[0049] The present invention will be described in detail below with reference to examples. However, the present invention is not limited in any way to these examples.

[0050] Example 1 (Preparation of propolis extract) 80% ethanol (specific alcohol traceable (95% ethanol) was used) was added to the pulverized raw propolis and shaken overnight in a shaker (at room temperature). Then, it was centrifuged at 6,000 rpm and 25°C for 15 minutes, the supernatant was filtered, and concentrated under reduced pressure using an evaporator. The concentrate was freeze-dried to obtain propolis extract. The propolis extract was dissolved in DMSO to a concentration of 3 mg / ml (w / v) and filtered through 0.45 μm pore size filter paper.

[0051] Comparative Example 1 (Preparation of Resveratrol) Resveratrol (Sigma-Aldrich) was dissolved in DMSO to a concentration of 3 mg / ml and filtered through a 0.45 μm pore size filter paper.

[0052] Test Example 1 (Method for measuring SIRT1 activity) The SIRT1 Fluorometric Drug Discovery Kit (Enzo Life Sciences, Inc.: BML-AK555) was used to evaluate SIRT1 activity.

[0053] A mixture of SIRT1 0.008 U / ml, NAD 2.5 mM, and FLUOR DE LYS SIRT1,Deacetylase Substrate 0.125 mM (NAD / FLUOR DE LYS SIRT1,Deacetylase Substrate) was prepared using Assay Buffer (BML-KI286). 1xDeveloper II / 2 mM nicotinamide was prepared by mixing 760 μl Assay Buffer, 200 μl 5xDeveloper II, and 40 μl 50 mM nicotinamide in 1 ml.

[0054] A test sample was prepared by mixing 2 μl of propolis extract and resveratrol with 8 μl of ultrapure water. 5 μl of SIRT1, 2 μl of NAD / FLUOR DE LYS SIRT1, Deacetylase Substrate, 2 μl of ultrapure water, and 1 μl of the test sample (final concentration 0.06 mg / ml) were added to a microplate. The plate was sealed and left to stand at 37°C for 45 minutes. Then, 10 μl of 1xDeveloper II / 2 mM nicotinamide was added, and the mixture was left to stand at room temperature for 45 minutes. Fluorescence measurements were performed using an EnVision Multilabel Reader at an excitation wavelength of 355 nm and an fluorescence wavelength of 460 nm.

[0055] The SIRT1 activation rate was calculated using the formula: SIRT1 activation rate (%) = (AC) / (BC) × 100. A: Fluorescence measurement values ​​under conditions with the test sample added. B: Fluorescence measurement under conditions where only the enzyme and substrate reacted. C: Fluorescence measurement under conditions where 1x Developer II / 2 mM nicotinamide was added before the start of the reaction to stop the enzymatic reaction.

[0056] Resveratrol, Brazilian propolis, and Chinese propolis, all of which have been reported to have SIRT1 activity, were used as positive control components. The results are shown in Table 1.

[0057] [Table 1]

[0058] The results in Table 1 confirm that propolis from Kangaroo Island has superior SIRT1 activating activity compared to resveratrol, Brazilian propolis, and Chinese propolis.

Claims

1. A SIRT1 activator containing an 80% ethanol extract of propolis from Kangaroo Island, Australia.

2. The SIRT1 activator according to claim 1, wherein the source plant of the propolis from Kangaroo Island, Australia, is a plant of the genus Lepidosperma.

3. A SIRT1 activator according to claim 1 or 2, used for at least one selected from the group consisting of anti-aging, cognitive function improvement, bone formation promotion, neuroprotection, vascular endothelial protection, and diabetes suppression.

4. A SIRT1 activator according to claim 1 or 2, which is a food or beverage, cosmetic, pharmaceutical or quasi-drug.

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