JP2025002393A - Elastase inhibitor, anti-wrinkle agent, and agent for preventing or improving loss of skin firmness and/or elasticity - Google Patents

Abstract

To provide a novel elastase inhibitor and also provide an anti-wrinkle agent and a preventive or improving agent for the reduction of skin firmness and/or resilience.SOLUTION: The present invention provides an elastase inhibitor containing Brazilian propolis, and an elastase inhibitor containing propolis derived from Eucalyptus plants and/or propolis derived from Baccharis plants. The present invention also provides an anti-wrinkle agent and a preventive or improving agent for the reduction of skin firmness and/or resilience, each containing the elastase inhibitor.SELECTED DRAWING: None

Description

The present invention relates to an elastase inhibitor, an anti-wrinkle agent, and an agent for preventing or improving a decrease in skin firmness and/or elasticity, and more specifically, to an elastase inhibitor, an anti-wrinkle agent, and an agent for preventing or improving a decrease in skin firmness and/or elasticity, which contains propolis.

Skin aging is thought to occur due to the denaturation of collagen and elastin in the dermis, and collagenase and elastase are involved in this denaturation. Elastin in particular contributes to tissue elasticity by forming cross-links with other proteins, but it is known to denature and degrade due to exposure to ultraviolet light and aging, as a result of overexpression of elastase, an elastin-degrading enzyme. Therefore, inhibiting elastase activity would be effective in preventing skin aging, and in order to prevent wrinkles and loss of skin firmness due to aging, it is important to inhibit elastase activity and prevent the breakdown of elastin.

Propolis is a glue-like substance made by mixing tree sap, plant buds and exudates collected by honeybees with beeswax and other substances, and is known to have antibacterial, antioxidant, anti-inflammatory, antitumor and anti-allergic effects.

The components of propolis are influenced by the type and composition of the original plant from which it is made, so it is known that the types and amounts of active ingredients contained in propolis, as well as their physiological activity, can vary greatly depending on the country or region where it is harvested.

In recent years, research has been conducted into the components of propolis from different regions. For example, the main components of Brazilian propolis, which is derived from Baccharis doracunclifolia, are p-coumaric acid, artepillin C, drupanin, baccharin, and other cinnamic acid derivatives. On the other hand, the main components of propolis from China, Europe, and Australia, which is derived from poplar, are flavonoids such as chrysin and galangin.

Non-patent literature 1 reports that ethanol extracts and ethyl acetate extracts of propolis clearly inhibited human neutrophil elastase activity. The propolis used here was collected in the northeast of Brandenburg, Germany.

Neutrophil elastase is known to destroy structures in the dermis such as collagen fibers, elastin, and basement membrane, impairing skin elasticity and promoting the formation of wrinkles.

Biol. Pharm. Bull. 28(7) 1183-1186(2005)

The present invention aims to provide a novel elastase inhibitor. It also aims to provide an anti-wrinkle agent and an agent for preventing or improving a decrease in skin firmness and/or elasticity.

Because propolis is made from plants harvested by bees, the ingredients vary depending on the place of origin, and it is expected that the effects will vary depending on the place of origin. Therefore, the inventors conducted extensive research to achieve the above-mentioned object, and discovered that Brazilian propolis, which is produced in a different place of origin, as well as propolis derived from plants of the genus Eucalyptus and propolis derived from plants of the genus Baccharis, have superior elastase inhibitory effects compared to European propolis, which is the same as German propolis, and thus completed the present invention. However, activity could not be confirmed with propolis produced in other places of origin.

That is, the present invention relates to the following [1] to [7].
[1] Elastase inhibitor containing Brazilian propolis.
[2] An elastase inhibitor comprising propolis derived from a plant of the genus Eucalyptus and/or propolis derived from a plant of the genus Baccharis.
[3] An anti-wrinkle agent comprising the elastase inhibitor described in [1].
[4] An anti-wrinkle agent comprising the elastase inhibitor described in [2].
[5] An agent for preventing or improving loss of skin firmness and/or elasticity, comprising the elastase inhibitor described in [1].
[6] An agent for preventing or improving loss of skin firmness and/or elasticity, comprising the elastase inhibitor described in [2].
[7] The agent according to any one of [1] to [6], which is a food or drink, a cosmetic, a medicine, or a quasi-drug.

Brazilian propolis, as well as propolis derived from plants of the genus Eucalyptus and propolis derived from plants of the genus Baccharis, have excellent elastase inhibitory activity and are therefore useful as active ingredients in elastase inhibitors, anti-wrinkle agents, and agents for preventing or improving the loss of skin firmness and/or elasticity.

In addition, propolis has traditionally been used as a food ingredient, so it is highly safe.

The following describes in detail the mode for carrying out the present invention, but the present invention is not limited to the following embodiment.

In this specification, the word "comprise" includes the meanings "essentially consisting of" and "consisting only of."

Elastase is a proteolytic enzyme that hydrolyzes elastin, and there is no limit to the type of elastin that can be targeted as long as it inhibits the hydrolysis. Elastase inhibition means both reducing and eliminating the activity of elastase. The elastase inhibitory effect can be evaluated according to known methods, and can be confirmed, for example, by measuring the amount of degradation products after acting on a substrate. If the amount of degradation products is reduced, it can be confirmed that activity has been inhibited.

The elastase inhibitor of the present invention contains Brazilian propolis as an active ingredient.

In another embodiment, the elastase inhibitor of the present invention contains propolis derived from a plant of the genus Eucalyptus and/or propolis derived from a plant of the genus Baccharis as an active ingredient.

Propolis is a resinous or waxy substance that constitutes the walls of beehives, and is made by mixing plant shoots and resins collected by bees with the saliva of bees. Propolis can be obtained, for example, as a beekeeping product in the usual manner. In one embodiment of the present invention, propolis from Brazil is used. In addition to Brazil, propolis from other producing regions such as China, European countries, Russia, Oceania, and America may also be used in combination. Propolis can be used alone or in combination from two or more producing regions. Propolis may be of any rank, such as super green or ultra green.

The propolis used in one embodiment of the present invention is not particularly limited as long as it is collected in Brazil. In particular, propolis from Brazil that originates from plants of the genus Eucalyptus and Baccharis is preferred.

In another embodiment of the present invention, the propolis is propolis derived from a plant of the genus Eucalyptus and propolis derived from a plant of the genus Baccharis.

The propolis used in another embodiment of the present invention is not particularly limited as long as it is derived from the above-mentioned plants, and may be derived from any place of origin, such as Brazil, China, Taiwan, Russia, Oceania, America, etc., and among these, Brazilian propolis is preferred. Examples of propolis derived from Eucalyptus plants and Baccharis plants include Brazilian propolis.

The propolis in the present invention may be, for example, a propolis lump, or a processed propolis product obtained by subjecting a propolis lump to some kind of processing. The processed propolis may be, for example, a propolis lump that has been subjected to processing such as pulverization, extraction, concentrating or powdering the extract, or granulating the powder, or may be an extraction residue remaining after extraction. That is, the processed propolis may be, for example, a pulverized propolis, an extract, a concentrated extract, an extract powder, an extract granule, an extraction residue, or the like. The extraction may be, for example, water extraction, hydrophilic organic solvent extraction, supercritical extraction, or the like. Examples of hydrophilic organic solvents include ethanol, glycerin, and 1,3-butylene glycol. The propolis extract may be obtained by extraction from a propolis lump, or may be obtained by further extraction from the residue after extraction. In addition, the extraction solvent may be used alone, or two or more types may be combined in any combination to be used as a mixed solvent. The processing method may be one, or two or more may be combined. As the processed propolis product, a propolis hydrophilic organic solvent extract is preferred because it is an extract that efficiently extracts the active ingredients of propolis in a short time and in a well-balanced manner. The processed propolis product is preferably a propolis ethanol extract.

The propolis extract includes the recovered extract (including further purified extract if necessary), a concentrate obtained by concentrating the extract, and a solid obtained by removing the solvent from the extract by freeze-drying, spray-drying, or the like. Here, the concentration, freeze-drying, and spray-drying of the extract can be carried out according to conventional methods. The propolis extract of the present invention also includes a powder obtained by powdering such an extract, and a granular form obtained by granulating the powder.

Propolis or its extract may be commercially available. Specific examples of commercially available propolis include Propolis 300 (manufactured by Yamada Bee Farm Co., Ltd.) and Propolis Liquid 30 (manufactured by Yamada Bee Farm Co., Ltd.).

The content of propolis in the elastase inhibitor 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., and can be, 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 or more based on the total solid content in the elastase inhibitor. Above, 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 The following may be 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.

The elastase inhibitors of the present invention may also be administered to humans, for example, for use in inhibiting elastase in the body. The elastase inhibitors of the present invention may be administered orally or parenterally. Oral administration includes enteral administration, and parenteral administration includes topical administration, particularly transdermal administration.

The dosage of the elastase inhibitor of the present invention when administered orally or parenterally may vary depending on the form of the active ingredient and composition, and the application method and amount. For example, for an adult weighing 60 kg, the dosage may be 1 μg to 1000 mg, preferably 5 μg to 500 mg, more preferably 8 μg to 100 mg, and even more preferably 10 μg to 1 mg, of the active ingredient, propolis, per day, calculated as dry solids. The dosage may be increased or decreased as appropriate depending on factors such as the health condition of the person taking it, the administration method, the type of active ingredient, and the combination with other agents.

The elastase inhibitor of the present invention may be administered once a day, or in multiple doses, such as twice or three times a day, so long as the effective daily dose is within the above-mentioned range. The elastase inhibitor of the present invention is effective immediately after administration, but continuous administration for 1 to 4 weeks, or for one month or more, six months or more, or one year or more is preferable because the effect can be sustained longer.

The elastase inhibitor of the present invention can be used as cosmetics, food and drink (particularly food and drink intended for health, maintenance or promotion of health, etc. (e.g., health foods, functional foods, nutritional compositions, dietary supplements, supplements, health foods, foods for specified health uses, foods with nutrient functions, or foods with functional claims)), quasi-drugs, medicines, etc. The elastase inhibitor of the present invention also includes the meaning of an additive that imparts elastase inhibitory activity.

Cosmetics, foods and beverages, quasi-drugs, or pharmaceuticals that contain the elastase inhibitor of the present invention as an ingredient can be produced, for example, by adding the elastase inhibitor of the present invention to intermediate products in the manufacturing process of these products.

The elastase inhibitor of the present invention can be suitably used as an active ingredient in topical skin preparations and cosmetics that have the effect of inhibiting elastase.

In addition to the propolis, the above-mentioned cosmetics can contain ingredients that are normally used in cosmetics, such as disinfectants, preservatives, surfactants, alcohols, aqueous components, water, colorants, pH adjusters, solubilizers, abrasives, foaming agents, enzymes, flavorings, chelating agents, excipients, thickeners, bases, emulsifiers, solvents, stabilizers, oils, cleaning agents (lactic acid bacteria), whitening agents, moisturizers, antioxidants, UV absorbers, powdered components, coloring materials, various skin nutrients, etc., as needed.

The cosmetic raw material may be in any form, such as a solid, liquid, or paste. The cosmetic may be a medicated cosmetic (i.e., a quasi-drug). Cosmetics include any cosmetic that can be applied to the skin, mucous membranes, body hair, head hair, scalp, nails, teeth, facial skin, lips, and other parts of animals (including humans).

Cosmetics can take a wide range of formulations, including aqueous solutions, solubilized solutions, emulsions, powders, oils, gels, ointments, aerosols, two-layer water-oil systems, and three-layer water-oil-powder systems.

The use of the cosmetics is also arbitrary. For example, basic cosmetics include face wash, lotion, milky lotion, cream, gel, essence, beauty serum, pack, mask, mist, UV protection cosmetics, etc.; makeup cosmetics include foundation, lipstick, blusher, eye shadow, eyeliner, mascara, etc.; nail cosmetics include nail polish, base coat, top coat, nail polish remover, etc.; and other cosmetics include face wash, (paste or liquid) dentifrice, mouth rinse, oral cosmetics, massage agents, cleansing agents, aftershave lotion, preshave lotion, shaving cream, body soap, soap, shampoo, rinse, hair treatment, hair styling agent, hair tonic, hair mist, hair foam, hair liquid, hair gel, hair spray, hand cream, hand soap, hair growth agent, antiperspirant, bath agent, etc.

The above-mentioned propolis can be used as is in the above-mentioned foods and beverages, or, if necessary, can be mixed 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, solubilizers, wetting agents, etc.

Food and drink includes any food and drink that can be consumed by animals (including humans). The types of food and drink are not particularly limited, and examples include dairy products; fermented foods (yogurt, etc.); beverages (soft drinks such as coffee, juice, and tea drinks, dairy drinks, lactic acid bacteria drinks, drinks containing lactic acid bacteria, yogurt drinks, carbonated drinks, sake, Western liquor, and alcohol such as fruit wine); spreads (custard cream, etc.); pastes (fruit paste, etc.); Western confectionery (chocolate, donuts, pies, cream puffs, gum, gummy candies, jellies, candies, cookies, cakes, puddings, etc.); Japanese confectionery (daifuku, mochi, manju, castella, anmitsu, yokan, etc.); frozen desserts (ice cream, popsicles, sorbets, etc.); foods (curry, beef bowl, porridge, miso soup, soup, meat sauce, pasta, pickles, jam, etc.); and seasonings (dressings, furikake, umami seasonings, soup bases, etc.).

The method for producing the food or beverage is not particularly limited, and any known method can be used as appropriate.

The dosage unit form when used as a supplement is not particularly limited and can be selected appropriately, and examples include chewable tablets, troches, capsules, granules, liquids, powders, syrups, pastes, drinks, gummies, etc.

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

When the elastase inhibitor of the present invention is prepared as a pharmaceutical product, the propolis can be prepared together with pharmaceutical acceptable ingredients into the form of tablets (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), capsules, pills, powders (dispersed medicines), fine granules, granules, liquids, suspensions, emulsions, syrups, pastes, ointments, external liquids, sprays, creams, gels, injections (including the case where it is mixed with distilled water or infusions such as amino acid infusions or electrolyte infusions at the time of use to prepare it as a liquid), etc., to form a pharmaceutical preparation.

Medicinal products may be administered locally or systemically. There are no particular limitations on the method of administration, and they may be administered orally or parenterally. Parenteral administration routes include subcutaneous, intradermal, intramuscular, intraperitoneal, intravenous or intraarterial administration, and transdermal administration.

In addition to propolis, the pharmaceutical product of the present invention may contain, as necessary, pharma- ceutically acceptable ingredients such as excipients, binders, disintegrants, lubricants, colorants, suspending agents, thickeners, antioxidants, absorption promoters, pH adjusters, preservatives, stabilizers, surfactants, sweeteners, flavorings, and fragrances.

The cosmetics and pharmaceuticals of the present invention also include quasi-drugs.

The elastase inhibitor of the present invention described above is applicable to mammals, including humans (preferably humans).

The Brazilian propolis, or the propolis derived from Eucalyptus plants and the propolis derived from Baccharis plants, which are contained as an active ingredient in the elastase inhibitor of the present invention, have an esterase inhibitory effect as shown in the examples described below, and are therefore useful as an active ingredient of the elastase inhibitor. The Brazilian propolis, or the propolis derived from Eucalyptus plants and the propolis derived from Baccharis plants have the effect of suppressing the decrease in wrinkles, firmness, and elasticity of the skin by inhibiting the activity of esterase, and therefore can also be used as an active ingredient of an anti-wrinkle agent and an agent for preventing or improving the decrease in firmness and/or elasticity of the skin. The anti-wrinkle agent of the present invention refers to an action of improving or preventing wrinkles. The above description regarding the elastase inhibitor can be applied to the anti-wrinkle agent and the agent for preventing or improving the decrease in firmness and/or elasticity of the skin. In other words, the content and dosage of propolis in the anti-wrinkle agent and the agent for preventing or improving the decrease in firmness and/or elasticity of the skin of the present invention are the same as those described above.

The anti-wrinkle agent and agent for preventing or improving the decline in skin firmness and/or elasticity of the present invention can be used as cosmetics, food and drink (particularly food and drink intended for health maintenance, health promotion, etc. (e.g., health foods, functional foods, nutritional compositions, nutritional supplements, supplements, health foods, foods for specified health uses, nutritional functional foods, or foods with functional claims)), quasi-drugs, medicines, etc. The anti-wrinkle agent and agent for preventing or improving the decline in skin firmness and/or elasticity of the present invention also includes the meaning of additives that impart anti-wrinkle action and action of preventing or improving the decline in skin firmness and/or elasticity. The cosmetics, food and drink, quasi-drugs, and medicines are the same as those described above.

The anti-wrinkle agent and agent for preventing or improving the loss of skin firmness and/or elasticity of the present invention can be suitably used as an active ingredient in topical skin preparations and cosmetics that have the effect of preventing or improving the loss of skin firmness and/or elasticity as well as anti-wrinkle.

In addition, the elastase inhibitor, anti-wrinkle agent, and agent for preventing or improving the loss of skin firmness and/or elasticity of the present invention use propolis, which has traditionally been used as a food ingredient, and are therefore highly safe.

The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples.

Example 1 (Preparation of propolis extract)
The propolis raw mass was pulverized, and 80% by volume ethanol was added and stirred at room temperature for 24 hours to extract the propolis. The extract was filtered through a filter paper (No. 2, manufactured by Advantec Toyo Co., Ltd.) and then concentrated under reduced pressure using an evaporator. The concentrated liquid was freeze-dried to obtain a propolis extract.

Test Example 1 (Method for measuring elastase inhibitory activity)
The effect of the test products on elastase activity using N-Succinyl-Ala-Ala-Ala-p-Nitroanilide as a substrate was evaluated.

1) 50 μL of the test product or control, 50 μL of 1.25 μg/mL elastase enzyme (CAS No. 39445-21-1, Sigma-Aldrich, USA) solution, and 100 μL of N-Succinyl-Ala-Ala-Ala-p-Nitroanilide (CAS No. 52299-14-6, Sigma-Aldrich, USA) solution were added to a 96-well plate, shaken at 270 rpm for 30 seconds, and then incubated at 37°C for 15 minutes.

As a blank, 0.05M Tris-HCl buffer was used instead of elastase enzyme. Three wells were used for each treatment group.

2) The 96-well plate was shaken at 270 rpm for 10 seconds to uniformly disperse the dye in the wells, and the absorbance at 415 nm (OD415) was then measured using a microplate reader.

3) The elastase activity inhibition rate of the test product was calculated from the OD415 of the test product and the control using the following formula.
Elastase activity inhibition rate (%) = {(C-CB) - (S-SB)} / (C-CB) x 100
C: Control OD415 CB: Control blank OD415
S: OD415 of the test product SB: Blank OD415 of the test product

The results in Table 1 show that Brazilian propolis, derived from plants of the Baccharis and Eucalyptus genera, has a superior elastase inhibitory effect to Romanian and Hungarian propolis, which are produced in Europe, just like the German propolis disclosed in Non-Patent Document 1.

Claims (7)

Elastase inhibitors containing Brazilian propolis. An elastase inhibitor containing propolis derived from plants of the genus Eucalyptus and/or propolis derived from plants of the genus Baccharis. An anti-wrinkle agent comprising the elastase inhibitor according to claim 1. An anti-wrinkle agent comprising the elastase inhibitor according to claim 2. An agent for preventing or improving loss of skin firmness and/or elasticity, comprising the elastase inhibitor according to claim 1. An agent for preventing or improving loss of skin firmness and/or elasticity, comprising the elastase inhibitor described in claim 2. The agent according to any one of claims 1 to 6, which is a food or drink, a cosmetic, a medicine, or a quasi-drug.