JP7849021B2 - SIRT1 activator - Google Patents

Description

This invention relates to a SIRT1 activator, and more specifically, to a SIRT1 activator containing propolis.

Previous research on aging and lifespan control has shown that calorie restriction is an effective treatment for extending life and preventing aging in yeast, nematodes, and fruit flies. Furthermore, it has been revealed that Sir2, which possesses NAD-dependent deacetylase activity, is involved in achieving life extension through calorie restriction (Non-Patent Literature 1), and sirtuins 1-7 are known as mammalian homologs of Sir2. Among these, sirtuin 1 (SIRT1), which is most similar in structure and function to Sir2, is attracting attention.

SIRT1 expression decreases with age, but is activated by calorie restriction. It is also known to be activated by plant-derived polyphenols, including resveratrol, a polyphenol found in grape skins and red wine (Non-Patent Literature 2). SIRT1 plays various roles in different tissues, and research is being conducted on its association with age-related diseases such as Alzheimer's disease, osteoporosis, arteriosclerosis, and skeletal muscular degeneration. Thus, SIRT1 has been shown to possess functions such as anti-aging effects, cognitive function improvement effects, bone formation promotion effects, neuroprotective effects, vascular endothelial protection effects, and diabetes suppression effects.

Propolis is a gelatinous substance formed by honeybees mixing tree sap, plant buds, and extracts with beeswax and other materials. It is known for its antibacterial, antioxidant, anti-inflammatory, antitumor, and anti-allergic properties.

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

In recent years, research into the components of propolis from different regions has been progressing. For example, Brazilian propolis, which uses Baccharis dracuifolia as its primary source plant, is primarily composed of cinnamic acid derivatives such as p-coumaric acid, artepillin C, dorupanin, and baccharin. On the other hand, propolis from China, Europe, and Australia, which use poplar as their source plant, is primarily composed of flavonoids such as chrysin and galangin.

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

Non-patent document 4 reports that propolis from Minas Gerais, Brazil (hereinafter sometimes referred to as "Brazilian propolis") has the effect of increasing SIRT1 expression.

PLoS ONE 3: e1759, 2008 Nature, 425:191-196 (2003) Evid Based Complement Alternat Med. 2018 Jun 27;2018:4957573 BMC Complement Alternate Med. 2016 Aug 30;16(1):329

The present invention aims to provide a SIRT1 activator.

It is generally known that resveratrol has a high SIRT1 activating effect. Furthermore, Non-Patent Documents 3 and 4 mentioned above have reported that Chinese and Brazilian propolis also have SIRT1 activating effects. Since propolis is made from plants collected by honeybees, differences in composition are observed depending on the origin, and therefore, it is expected that the effects will differ depending on the origin. Therefore, the inventors of this invention conducted extensive 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.

This invention was completed based on these findings and further research, and provides the following SIRT1 activator.

Item 1. SIRT1 activator containing propolis from Kangaroo Island, Australia.
Item 2. The SIRT1 activator according to 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.

Propolis from Kangaroo Island, Australia, possesses excellent SIRT1 activating properties, making it useful as an active ingredient in SIRT1 activators.

Furthermore, since propolis has been used as a food ingredient for a long time, it is highly safe.

The embodiments of the present invention will be described in detail below.

Furthermore, in this specification, "comprise" encompasses both the meanings of "essentially consisting of" and "consisting only of."

The SIRT1 activator of the present invention is characterized by containing propolis from Kangaroo Island, Australia.

Propolis is a resinous or waxy substance that makes up the walls of a beehive. It is a substance created by mixing plant buds and resins collected by bees with their saliva. In this invention, propolis from Kangaroo Island, Australia, is used. In addition to Kangaroo Island, propolis from other regions such as China, Taiwan, European countries, Russia, Oceania, and the United States may also be used in combination.

The propolis used in this invention is not particularly limited as long as it is collected on Kangaroo Island, Australia. Among propolis from Kangaroo Island, Australia, those whose source plant is a plant of the genus Lepidosperma are preferred.

In this invention, propolis includes, for example, raw propolis, its pulverized form, supercritical fluid extract, and extract with water and/or hydrophilic organic solvent. Among these, extracts with water and/or hydrophilic organic solvent and supercritical fluid extracts are preferred, with extracts with water and/or hydrophilic organic solvent being more preferred. Water and/or hydrophilic organic solvent extracts are preferred because they efficiently and balancedly extract the components of propolis in a short time, and because they do not require large-scale equipment for scale-up.

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

These extraction solvents can be used individually or in combination of two or more to form a mixed solution. Preferably, they are water, lower alcohols (preferably ethanol), or a mixture thereof (hydrated alcohol, preferably hydrated ethanol). When using hydrated alcohol (preferably hydrated ethanol) as the extraction solvent, the alcohol concentration is not limited, but 50 to 100% by volume, particularly 70 to 100% by volume, is preferable.

For extraction, raw propolis, alcohol-washed raw propolis, etc., can be used. Extraction efficiency can be improved by using raw propolis cut or crushed to an appropriate size. The volume of extraction solvent used is, for example, 1 to 20 times the mass of the raw propolis, preferably 2 to 10 times.

Extraction can be carried out by adding propolis to a solvent and heating it to a temperature between 0°C and the boiling point of the solvent (usually below 100°C), preferably between 2 and 40°C. Extraction can be performed at the above temperature for a predetermined time (e.g., 1 hour or more), either 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.

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 combinations thereof.

Furthermore, the solvent extract can be concentrated and dried by methods such as freeze-drying or spray-drying. 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.

The SIRT1 activator of the present invention may contain other known components as appropriate, provided that they do not interfere with the effects of the present invention.

The propolis content in the SIRT1 activator of the present invention is not particularly limited as long as the effects of the present invention can be obtained, and can be appropriately adjusted according to the final form, etc., relative to the total amount of solids in 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 mass% or less, 50 mass% or less, 40 mass% or less, 30 mass% or less, 20 mass% or less, 15 mass% or less, 10 mass% or less, 7 mass% or less, 5 mass% or less, 3 mass% or less, 1 mass% or less, or 0.5 mass% or less.

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 recipient, the method of administration, and combinations with other agents.

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 term "SIRT1 activator" of the present invention also encompasses the meaning of an additive that imparts SIRT1 activation.

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.

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

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.

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; and other products 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.

The above-mentioned food and beverages can 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.

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, dairy 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 confectionery (chocolate, donuts, pies, cream puffs, gum, gummies, jelly, candy, cookies, cakes, puddings, etc.); Japanese confectionery (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.).

The manufacturing method for food and beverages is not particularly limited and may follow any known method as appropriate.

The dosage form for supplements is not particularly limited and can be selected as appropriate. Examples include chewable tablets, lozenges, capsules, granules, liquids, powders, syrups, pastes, drinks, and gummies.

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

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 fluids such as amino acid solutions or electrolyte solutions at the time of use) to create pharmaceutical formulations.

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

In addition to propolis, the pharmaceutical product of this 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.

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

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

The Australian Kangaroo Island propolis, which is contained as the active ingredient in the SIRT1 activator of the present invention, has SIRT1 activating activity, as shown in the examples described later, and is therefore useful as the 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.

The SIRT1 activator of the present invention can activate SIRT1, thereby exhibiting effects such as anti-aging, cognitive function improvement, bone formation promotion, neuroprotection, vascular endothelial protection, and diabetes suppression. Therefore, the SIRT1 activator of the present invention can be used for applications targeting 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.

Furthermore, since propolis has traditionally been used as a food ingredient, it is highly safe.

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.

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.

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.

Test Example 1 (Method for measuring SIRT1 activity)
SIRT1 activity was evaluated using the SIRT1 Fluoromeric Drug Discovery Kit (Enzo Life Sciences, Inc.: BML-AK555).

Using Assay Buffer (BML-KI286), 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. 1x Developer II/2 mM nicotinamide was prepared by mixing 760 μl Assay Buffer, 200 μl 5x Developer II, and 40 μl 50 mM nicotinamide in 1 ml.

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 1x Developer 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.

The SIRT1 activation rate was calculated as follows: SIRT1 activation rate (%) = (A - C) / (B - C) × 100.
A: Fluorescence measurement 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 reaction started to stop the enzyme reaction.

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.

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

Claims (4)

A SIRT1 activator containing an 80% ethanol extract of propolis from Kangaroo Island, Australia. 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. 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. A SIRT1 activator according to claim 1 or 2, which is a food or beverage, cosmetic, pharmaceutical, or quasi-drug.