JP2005336137A - Method for producing active oxygen-eliminating agent and use of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an active oxygen-eliminating agent derived from a natural product, having a high active oxygen or free radical-eliminating ability, safe for a human body and useful for various uses, and a use of the same. <P>SOLUTION: This method for producing the active oxygen-eliminating comprises (A) a process of extracting a crushed lump of propolis as a raw material with a mixed solvent containing water, an organic solvent and a surfactant to be subjected to solid-liquid separation into an extract and extracted residue, (B) a process of adding water to the extracted residue separated in the process (A), agitating them to be subjected to solid-liquid separation into the aqueous organic solvent solution and a solid material, and (C) a concentrating process of concentrating the aqueous organic solvent solution separated in the process (B) or (D) a process of drying/crush the solid material separated in the process (B). The concentrated liquid obtained in the process (C) or the powder obtained in the process (D) is provided as a product. The food and cosmetic contain the active oxygen-eliminating agent obtained by the method. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a production method and use of an active oxygen scavenger. More specifically, the present invention includes a method for producing an active oxygen scavenger useful for foods, cosmetics and the like from an extraction residue of a propolis bulk pulverized product, and an active oxygen scavenger obtained by this method. It relates to food and cosmetics.

Oxygen is an indispensable substance for the maintenance of life, such as the production of energy by metabolism for the living body, but it has a variety of reaction systems inside and outside the body, that is, activities that are highly reactive due to some immune reactions, ultraviolet rays, irradiation, etc. Oxygen species (superoxide anion: O ₂ ⁻ , hydrogen peroxide: H ₂ O ₂ , hydroxyl radical: OH ·, singlet oxygen: ¹ O _2, etc.) and free radicals are produced. While these reactive oxygen species and free radicals are useful for inactivating foreign substances such as bacteria that have invaded in certain immune reactions, they react with biological components to peroxidize lipids and denature proteins and nucleic acids. It is known to cause various diseases and promotion of aging. In particular, because the skin is the outermost layer of the body, it is easily affected by reactive oxygen species and free radicals generated by external factors such as ultraviolet rays and radiation. It is known that production of abnormal pigments of skin such as spots and freckles is enhanced.

Superoxide anion O ₂ ⁻ generated by various causes in the living body is converted to H ₂ O ₂ by the enzyme superoxide dismutase (hereinafter abbreviated as SOD), and H ₂ O ₂ acts by the enzymes catalase and glutathione peroxidase. Is decomposed into water and oxygen. In addition to these enzymes, there are antioxidants derived from foods such as vitamin C and vitamin E in the body, and these form a network that suppresses radical reactions and oxidation reactions in the body to react with active oxygen and free radicals. It is thought to protect against various obstacles. However, due to changes in the surroundings and the environment in the living body, such as infection by bacteria and viruses, changes in food intake and nutritional conditions, excessive irradiation of ultraviolet rays, various stresses received from the surroundings, aging and aging, etc. If the balance of the network is lost, the metabolic balance of active oxygen and free radicals produced in the body will be lost, resulting in an increase in the amount of lipid peroxide, cosmetic disorders such as dermatitis, spots, wrinkles, eczema and buckwheat. It is known that various symptoms appear, and diseases such as rheumatoid arthritis, arteriosclerosis, diabetes, hepatitis, nephritis and cancer are caused.

  In addition, foods and food additives ingested by humans are also oxidized and oxidized by active oxygen and free radicals generated by the action of ultraviolet rays and radiation, and normal oxidation and peroxidation and modification of lipids and other components contained in them. It is well known that these oxidative and denatured products are unfavorable for health. Furthermore, it is known that some natural fats and oils and surfactants containing unsaturated lipids that may be incorporated into cosmetics and skin external preparations are susceptible to oxidation by the action of ultraviolet rays and the like. As a result, undesired phenomena such as discoloration and off-flavoring often occur. By using such a cosmetic base that is susceptible to oxidation, the amount of lipid peroxide generated in the skin due to the above-mentioned causes increases, and it is easy to amplify the above-mentioned symptoms that cause cosmetic problems. Is to be guessed.

  Conventional antioxidants added to cosmetics, foods, food additives and feeds include synthetic antioxidants such as dibutylhydroxytoluene (BHT), butylhydroxyanisole (BHA), ethoxyquin, ascorbic acid and vitamins. There are natural antioxidants such as E. The synthetic antioxidants described above have excellent antioxidant effects, but some have problems with safety such as carcinogenicity, and some of them are limited in their use. On the other hand, although the above-mentioned naturally-occurring antioxidants are evaluated for safety, they have a drawback that their antioxidant effect is considerably inferior to that of synthetic antioxidants.

  In recent years, there has been an active search for cosmetics, food sciences and pharmaceuticals for substances or compositions having an active oxygen / free radical scavenging effect and an antioxidant effect under such circumstances. A significant number of active oxygen and free radical scavengers and antioxidants have become known. For example, an active oxygen scavenger containing baicalein, which is a flavonoid component contained in urgon (see, for example, Patent Document 1), an active oxygen scavenger / removal agent comprising clove oil or its component, dehydrodioigenol (for example, a patent) Reference 2), active oxygen scavengers containing walnut shell extract as an active ingredient (for example, see Patent Document 3) and the like have been proposed.

On the other hand, propolis has been known as a natural antibacterial substance and health promoting substance. This propolis is a dark green-brown sticky substance that bees make to preserve their nests, and it secretes itself into aggregates of gums, sap, plant pigments, perfume oil, etc. collected from trees. Made by mixing things and beeswax. For the propolis, various biological activities such as antitumor activity, antioxidant activity, anti-inflammatory activity, and antibacterial activity have been reported. Examples of the group of compounds having physiological activity include cinnamic acid derivatives, flavonoids, esters, and other phenolic compounds, and the presence of terpenes in addition to subtropical propolis has been reported.
And the propolis food composition containing the extract obtained by extracting such a propolis using the mixed solvent containing water, an alcohol-type organic solvent, and surfactant is disclosed (for example, patent documents) 4).

Japanese Patent Publication No. 4-34969 JP-A-3-227938 JP 7-69912 A Japanese Examined Patent Publication No. 4-66544

  An object of the present invention is to provide a production method and use of a natural product-derived active oxygen scavenger that has a high ability to scavenge active oxygen and free radicals, is safe to the human body, and is useful for various uses. Is.

  As a result of intensive studies to achieve the above object, the inventors of the present invention have extracted active ingredients, and the active oxygen scavenging substance still remains in the extraction residue obtained by extracting the active ingredients. Focusing on this, the present invention has been completed.

That is, the present invention
(1) (A) A step of subjecting the pulverized propolis pulverized product to extraction using a mixed solvent containing water, an organic solvent and an emulsifier, and then solid-liquid separation into an extract and an extraction residue, (B) ) After adding water to the extraction residue separated in the step and stirring, the step of solid-liquid separation into an organic solvent aqueous solution and a solid, and (C) the step of concentrating the organic solvent aqueous solution separated in the step (B) A method for producing an active oxygen scavenger (hereinafter referred to as production method I), characterized in that the concentrated liquid obtained in step (C) is used as a product.
(2) (A) A step of subjecting the pulverized propolis pulverized product to extraction using a mixed solvent containing water, an organic solvent and an emulsifier, followed by solid-liquid separation into an extract and an extraction residue, (B) ) After adding water to the extraction residue separated in step) and stirring, the step of solid-liquid separation into an organic solvent aqueous solution and solid matter, and (D) drying and grinding treatment of the solid matter separated in step (B) A method for producing an active oxygen scavenger (hereinafter referred to as production method II), characterized in that the powder obtained in step (D) is used as a product.
(3) The active oxygen scavenger according to (1) or (2) above, wherein the organic solvent in the mixed solvent used in step (A) is a polyhydric alcohol compound having two or more hydroxyl groups in the molecule. Production method,
(4) The emulsifier in the mixed solvent used in the step (A) is at least selected from glycerin fatty acid ester, polyglycerin fatty acid ester, propylene glycol fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, lecithins and saponins. A method for producing an active oxygen scavenger according to item (1), (2) or (3), which is one type,
(5) A food comprising the active oxygen scavenger obtained by the method according to any one of (1) to (4) above, and (6) the above (1) to (4) A cosmetic comprising an active oxygen scavenger obtained by the method according to any one of items
Is to provide.

  According to the present invention, a method for producing an active oxygen scavenger, which has high active oxygen and free radical scavenging ability and is safe for the human body and is useful for various applications, from propolis bulk extraction residue, and this method Foods and cosmetics containing the active oxygen scavenger obtained in (1) can be provided.

In the method for producing an active oxygen scavenger according to the present invention, there are two embodiments: production method I and production method II.
The production method I includes (A) a step of subjecting the pulverized propolis mass to extraction using a mixed solvent containing water, an organic solvent, and an emulsifier, and then solid-liquid separation into an extract and an extraction residue, (B) (A) After adding water to the extraction residue separated in the step and stirring, the step of solid-liquid separation into an organic solvent aqueous solution and a solid, and (C) the organic solvent aqueous solution separated in the step (B) is concentrated. A process for producing an active oxygen scavenger using the concentrated liquid obtained in step (C) as a product.
On the other hand, the production method II includes a step of drying and pulverizing the solid separated in the step (A), (B), and (D) step (B), and in the step (D) This is a method for producing an active oxygen scavenger using the obtained powder as a product.

  In the production method I and production method II of the present invention, the pulverized propolis ingot used in the step (A) can be obtained by pulverizing the raw propolis ingot with a pulverizer. It is desirable to use the raw material propolis ingot, which is stored at a temperature of usually 15 ° C. or lower, preferably about 0 to 10 ° C., in order to efficiently perform pulverization and extraction treatment. The hole diameter of the screen attached to the pulverizer is usually 1 to 5 mm, preferably 2 to 3 mm.

  The organic solvent in the mixed solvent used for the extraction treatment is preferably a polyhydric alcohol compound having two or more hydroxyl groups in the molecule, specifically, glycerin, ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, Examples include glycerin alkylene oxide adducts, ethylene glycol alkylene oxide adducts, propylene glycol alkylene oxide adducts, glucono delta lactones, and sugar alcohols such as xylitol, hexitol, mannitol, and sorbitol. These may be used singly or in combination of two or more, but glycerin and various sugar alcohols are particularly suitable when the obtained active oxygen scavenger is used for food applications.

  On the other hand, examples of the emulsifier include glycerin fatty acid ester, polyglycerin fatty acid ester, propylene glycol fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, lecithins and saponins.

  Examples of the glycerin fatty acid ester include glycerin = monolaurate, glycerin = monopalmitate, glycerin = monostearate, glycerin = monooleate, glycerin = monolinoleate, and glycerin = monoricinoleate.

  Examples of polyglycerin fatty acid esters include diglycerin = monolaurate, diglycerin = monopalmitate, diglycerin = monostearate, diglycerin = monooleate, diglycerin = monolinoleate, diglycerin = monoricinoleate, tetraglycerin = mono. Laurate, tetraglycerin = dilaurate, tetraglycerin = monopalmitate, tetraglycerin = dipalmitate, tetraglycerin = monostearate, tetraglycerin = distearate, tetraglycerin = monooleate, tetraglycerin = dioleate, tetraglycerin = monolinoleate, tetraglycerin = Dilinoleate, tetraglycerin = monoricinoleate, tetraglycerin = diricinoleate, tetraglycerin = monobehenene , Tetraglycerin = dibehenate, pentaglycerin = monolaurate, pentaglycerin = dilaurate, pentaglycerin = monopalmitate, pentaglycerin = dipalmitate, pentaglycerin = monostearate, pentaglycerin = distearate, pentaglycerin = monooleate, pentaglycerin = Dioleate, pentaglycerin = monolinoleate, pentaglycerin = dilinoleate, pentaglycerin = monoricinoleate, pentaglycerin = diricinoleate, pentaglycerin = monobehenate, pentaglycerin = dibehenate, decaglycerin = monolaurate, decaglycerin = dilaurate, decaglycerin = Trilaurate, decaglycerin monopalmitate, decaglycerin dipalmite , Decaglycerin = tripalmitate, decaglycerin = monostearate, decaglycerin = distearate, decaglycerin = tristearate, decaglycerin = monooleate, decaglycerin = dioleate, decaglycerin = trioleate, decaglycerin = monolinoleate, decaglycerin = Dilinoleate, decaglycerin = trilinoleate, decaglycerin = monoricinoleate, decaglycerin = diricinoleate, decaglycerin = triricinoleate, decaglycerin = monobehenate, decaglycerin = dibehenate, decaglycerin = tribehenate, decaglycerin = monoisostearate, Decaglycerin = sesquiisostearate, decaglycerin = diisostearate, decaglycerin = triisostearate And decaglycerin = mono (12-hydroxy) stearate, decaglycerin = di (12-hydroxy) stearate, decaglycerin = tri (12-hydroxy) stearate, and the like.

  Examples of the propylene glycol fatty acid ester include propylene glycol = monolaurate, propylene glycol = monopalmitate, propylene glycol = monostearate, propylene glycol = monooleate, propylene glycol = monolinoleate, propylene glycol = monoricinolate, propylene glycol = mono Examples thereof include isostearate and propylene glycol = mono (12-hydroxy) stearate.

  Examples of sorbitan fatty acid esters include sorbitan monolaurate, sorbitan dilaurate, sorbitan monopalmitate, sorbitan dipalmitate, sorbitan monostearate, sorbitan distearate, sorbitan monooleate, sorbitan dioleate, sorbitan monolinoleate, sorbitan = Dilinoleate, sorbitan = monoricinoleate, sorbitan = diricinolate, sorbitan = monobehenate, sorbitan = dibehenate, sorbitan = monoisostearate, sorbitan = diisostearate, sorbitan = mono (12-hydroxy) stearate, sorbitan = di ( 12-hydroxy) stearate and the like.

  Examples of sucrose fatty acid esters include sucrose = monolaurate, sucrose = dilaurate, sucrose = monopalmitate, sucrose = dipalmitate, sucrose = monostearate, sucrose = distearate, sucrose = monooleate, sucrose Sugar = dioleate, sucrose = monolinoleate, sucrose = dilinoleate, sucrose = monoricinoleate, sucrose = diricinolate, sucrose = monoisostearate, sucrose = diisostearate, sucrose = mono (12-hydroxy ) Stearate, sucrose = di (12-hydroxy) stearate, and the like.

  Examples of lecithins include soy lecithin and other plant lecithins, and examples of saponins include quilla extract and soy saponin.

  These emulsifiers may be used alone or in combination of two or more. In addition, since the said emulsifier can be used for foodstuffs, when using the obtained active oxygen scavenger for foodstuffs, it may be contained.

  The content ratio of water, the organic solvent and the emulsifier in the mixed solvent is usually 5 to 50% by mass of water, 45 to 94.9% by mass of the organic solvent, and 0 to 0% of the emulsifier when the total amount thereof is 100% by mass. 0.1 to 10% by mass, preferably water: 5 to 30% by mass, organic solvent: 65 to 94.5% by mass, and emulsifier: 0.1 to 5% by mass.

The extraction process of the pulverized propolis ingot in the step (A) can be performed as follows.
About 3 to 7 parts by mass of an extraction solvent composed of a mixed solvent containing water, the organic solvent, and the emulsifier in the above proportions is added to 1 part by mass of the pulverized propolis mass. If the amount of the extraction solvent used is less than 3 parts by mass, the viscosity of the propolis bulk pulverized product and the extraction solvent will cause the extraction work efficiency to decrease, and if it exceeds 7 parts by mass, the content of the active ingredient in the extract will decrease. There is a risk.

  Next, extraction is performed for about 30 to 120 minutes by heating to a temperature of about 50 to 90 ° C., preferably 65 to 80 ° C. After the extraction treatment, solid-liquid separation is performed into an extract and an extraction residue by means such as filtration, centrifugation, and pressing.

  According to the study by the present inventors (Japanese Patent Application No. 2004-008862), the extract obtained in this way was found to be p-coumaric acid, 3,4-di-O—. Caffeoylquinic acid (3,4-di-O-caffeoylquinic acid), 3,5-di-O-caffeoylquinic acid, 4-hydroxy-3-prenylcinnax Acid (4-hydroxy-3-prenylcinnamic acid) and 4'-methoxy-3,5,7-trihydroxyflavanol (4'-methoxy-3,5,7-trihydroxyflavanol) are included as main active ingredients, In addition, 3,5-diprenyl-4-hydroxycinnamic acid (Artepi C) [3,5-diprenyl-4-hydroxycinnamic acid (artepillinC)], kaemferide, bethetol, chlorogenic acid, ermanin (ermanin), cricin (caffeine) acid), kaempherol and vanillin are included. Caemferide is 4'-methoxy-3,5,7-trihydroxyflavone, bethletol is 4 ', 6-dimethoxy-3,5,7-trihydroxyflavone, and ermanine is 3,4'-dimethoxy-. 5,7-dihydroxyflavone and chrysin are 5,7-dihydroxyflavone, and caemferol is 3,4 ', 5,7-tetrahydroxyflavone.

  On the other hand, since the extraction liquid contains about 40 to 60% by mass of the extraction liquid, it naturally includes each of the active ingredients present in the extraction liquid. Ingredients and other active ingredients appear to remain.

  In order to use the active ingredient contained in the extraction residue, the extraction residue is dried by a method such as hot air drying, spray drying, freeze drying, etc. Most of the moisture is removed and it is difficult to powder the residue.

  Therefore, in the production methods I and II of the present invention, first, as the step (B), water is added to the extraction residue separated in the step (A) and stirred, followed by solid-liquid separation into an organic solvent aqueous solution and a solid matter. I do. At this time, the extraction residue is pulverized by a pulverizer as needed in order to efficiently perform the organic solvent removal operation from the extraction residue. In order to efficiently perform the pulverization treatment, it is desirable to use a residue stored at a temperature of preferably 15 ° C. or lower, more preferably about 0 to 10 ° C. At this time, the diameter of the screen hole attached to the pulverizer is usually 2 to 20 mm, preferably 5 to 10 mm.

  About 3 to 7 parts by mass of water is added to 1 part by mass of the propolis bulk extraction residue and stirred. As the water, it is desirable to use ion-exchanged water, distilled water, or the like, and the water temperature is preferably room temperature or lower, more preferably 15 ° C. or lower for the purpose of suppressing the outflow of active ingredients in the residue. If the amount of water used is less than 3 parts by mass, a sufficient deorganic solvent is not performed, and if it exceeds 7 parts by mass, the active ingredient in the residue may flow out excessively. Next, solid-liquid separation is performed into an organic solvent aqueous solution and a solid by a known means.

In the production method I, in the step (C), the organic solvent aqueous solution separated in the step (B) is moderately concentrated to obtain a liquid product active oxygen scavenger.
On the other hand, in the production method II, in the step (D), the solid matter separated in the step (B) is subjected to a known method, for example, a drying treatment such as hot air drying, spray drying and freeze drying, and pulverization treatment Thus, an active oxygen scavenger for the powder product is obtained. At this time, the concentration can be adjusted by adding sugar or the like to the solid.

  The liquid or powdered active oxygen scavenger thus obtained contains a wide range of components from hydrophilic to hydrophobic, such as flavonoids, hydroxycinnamic acid derivatives, and other phenolic compounds. It has free radical scavenging ability and antioxidant ability, and can be used for, for example, food use, cosmetic use, and feed use.

  The active oxygen scavenger is directly blended into a composition such as food or cosmetics as a liquid product, or is blended into a composition such as food or feed as a powder, or the active ingredient is formulated in advance. It is possible to increase the commercial value of the composition by adding it to the composition and imparting an active oxygen scavenging effect and an antioxidant effect.

  Examples of formulation include tablets, capsules, powders, or granules. The active ingredients are starches, excipients such as lactose and mannitol, binders such as carboxymethylcellulose and hydroxypropylcellulose, crystalline cellulose and It can be formulated by formulating a disintegrating agent such as carboxymethylcellulose calcium, a lubricant such as talc or magnesium stearate, and other appropriate combinations of a wetting agent, a colorant, a fragrance and the like. The solution may be an aqueous or oily emulsion or syrup, such as simple syrup, sorbitol, suspending agents such as methylcellulose or sodium carboxymethylcellulose, egg yolk lecithin, sorbitan monofatty acid ester, lauromacrogol, An emulsifier such as castor oil, and other preservatives, preservatives, stabilizers, and the like as necessary can be formulated as appropriate. For ointments, petrolatum, paraffin, silicone, plastibase, hydrophobic bases such as vegetable oils and waxes, purified lanolin, carboxyvinyl polymer, hydrophilic bases such as propylene glycol and 1,3-butanediol, polyethanolamine Etc. can be formulated by appropriately blending an emulsifier and the like.

The present invention also provides foods and cosmetics containing the active oxygen scavenger.
As the form of the food, the active oxygen scavenger is used as it is, or added to and mixed with seasoning such as miso, soy sauce and mayonnaise, and cooking oil such as salad oil, various cooked foods; desserts, ice confectionery , Sweets such as candy, chewing gum and fruit juice, and the like. In these foods, arbitrary components can be used depending on the purpose of use. For example, in the case of ice confectionery and beverages, fruit juice, sweetener, acidulant, colorant, flavor, and the like can be appropriately blended. What is necessary is just to change suitably the addition amount to the foodstuff of the active ingredient of this invention by the form.

  On the other hand, various types of cosmetics can be mentioned, such as lotions, milky lotions, creams, facial cleansers, packs, makeup cosmetics, hair cosmetics, lip cosmetics, nail cosmetics, bath preparations. And antiperspirants. In these cosmetics, any component can be used depending on the purpose of use. For example, in the case of cream, hydrophobic bases such as petrolatum, paraffin, squalane, lanolin, propylene glycol, 1,3-butadiol, etc. Emulsifiers such as hydrophilic bases, fatty acid monoglycerides, sorbitan fatty acid esters, preservatives, pigments, fragrances, and other nutrients, moisturizers, whitening agents, UV absorbers, etc., if necessary Can do. Similarly, for other products, necessary components according to the type can be appropriately blended. What is necessary is just to change suitably the addition amount to the cosmetics of the active ingredient of this invention by the form.

  Moreover, the active oxygen scavenger of this invention can also be mix | blended with feed. As the form of the feed, various types can be mentioned, for example, a mixed feed in the form of a powder, a paste product or a pellet for livestock, poultry and fish. In these feeds, arbitrary components can be used according to the purpose of use. For example, in the case of a kneaded product-like feed, coloring agents, flavors and the like can be appropriately blended. What is necessary is just to change suitably the addition amount to the feed of the active ingredient of this invention by the form.

EXAMPLES Next, although an Example demonstrates this invention further in detail, this invention is not limited at all by these examples.
Example 1
(1) Extraction of pulverized propolis mass: 25 mass parts of pulverized propolis mass (Baccharis dracunculifolia base type from Brazil), 10 mass% of water, 89 mass% of glycerin and 1 mass% of emulsifier (glycerin = monooleate) 200 parts by mass of the mixed solution was added, extracted at 70 ° C. for 1 hour with stirring, and then filtered to obtain an extract and a propolis bulk extraction residue.
(2) Concentration of the separated liquid 4 parts by mass of ion-exchanged water is added to 1 part by mass of the propolis bulk extraction residue obtained in (1) above, and the mixture is stirred for 30 minutes and then centrifuged to separate the organic matter from the solids. A solvent aqueous solution was obtained. Next, this aqueous solution was concentrated to obtain four types of concentrated liquid samples (a), (b), (c), and (d) having different concentrations. Moreover, the extract obtained in the above (1) was prepared as a control (e).
(3) Content of propolis component Regarding the four concentrated liquid samples (a), (b), (c), (d) and the control (e) obtained in (2) above, the Japan Health and Nutrition Food Foundation By applying the Association Propolis Food Standards (Revised 1995), the total flavonoid amount (Quercetin equivalent) was measured using an absorptiometry (λ = 415 nm). The results are shown in Table 1.
Moreover, the total amount of phenolic acid (p-coumaric acid equivalent) was measured in accordance with Japan Health and Nutrition Food Association Propolis Food Standard (revised in 2001). The results are shown in Table 1.
(4) DPPH radical scavenging activity To evaluate the antioxidant activity of the four concentrated liquid samples (a), (b), (c), (d) and the control (e) obtained in (2) above. These ethanol solutions having various concentrations were prepared and mixed with the same amount of 60 μmol / L DPPH (1,1-diphenyl-2-picrylhydrazyl) (Wako Pure Chemical Industries) ethanol solution, and 30 The absorbance (λ = 520 nm) after a minute was measured, and the IC ₅₀ value of DPPH radical elimination for each sample was determined. The results are shown in Table 1.

表１から４種の試料（ａ）、（ｂ）、（ｃ）、（ｄ）は総フラボノイド量および総フェノール酸量に依存してＤＰＰＨラジカル消去活性を示すことがわかる。
また、試料（ｄ）においては対照（ｅ）に相当する測定値が得られることがわかる。

It can be seen from Table 1 that the four samples (a), (b), (c), and (d) show DPPH radical scavenging activity depending on the total flavonoid amount and the total phenolic acid amount.
It can also be seen that the sample (d) has a measured value corresponding to the control (e).

Example 2
(1) Deorganic solvent from propolis bulk extraction residue Add 5 parts by weight of ion-exchanged water to 1 part by weight of propolis bulk extraction residue obtained in Example 1 (1), stir for 30 minutes, and then perform centrifugation. The solid portion was separated and then freeze-dried to obtain a solid content.
(2) Content of propolis component About the freeze-dried product (f) obtained in (1) above and the three propolis ingots (g), (h), (i), Japan Health and Nutrition Food Association Propolis Food A standard standard (revised in 1995) was applied, and the total flavonoid amount (Quercetin equivalent) was measured using an absorptiometry (λ = 415 nm). The results are shown in Table 2.
Moreover, the total amount of phenolic acid (p-coumaric acid equivalent) was measured in accordance with Japan Health and Nutrition Food Association Propolis Food Standard (revised in 2001). The results are shown in Table 2.
(3) DPPH radical scavenging activity For the freeze-dried product (f) obtained in (1) above and the propolis bulk material (g), (h), (i) An ethanol solution with a suitable concentration was prepared and mixed with the same amount of DPPH (1,1-diphenyl-2-picrylhydrazyl) (Wako Pure Chemical Industries, Ltd.) ethanol solution with a concentration of 60 micromol / L. Absorbance (λ = 520 nm) was measured, and the IC ₅₀ value of DPPH radical elimination for each sample was determined. The results are shown in Table 2.

表２から、凍結乾燥品（ｆ）とプロポリス原塊３種（ｇ）、（ｈ）、（ｉ）を比較すると、総フラボノイド量（Ｑｕｅｒｃｅｔｉｎ当量）においてはプロポリス原塊の測定値に対し、６８〜６８６％の値を示した。
総フェノール酸量（ｐ−クマル酸当量）においてはプロポリス原塊の測定値に対し、７９〜２１１％の値を示した。

From Table 2, when comparing the freeze-dried product (f) and the propolis bulk 4 types (g), (h), (i), the total amount of flavonoids (Quercetin equivalent) is 68 A value of ˜686% was indicated.
The total amount of phenolic acid (p-coumaric acid equivalent) showed a value of 79 to 211% with respect to the measured value of the propolis bulk.

<Sample analysis>
The concentrated liquid sample (d) obtained in Example 1 (2), the lyophilized product (f) obtained in Example 2 (1), and the propolis bulk (i) were analyzed by high performance liquid chromatography (HPLC). went.
The measurement conditions of HPLC are as follows.
Column CLC-ODS (150 × 6.0 mm, id), column temperature 40 ° C., mobile phase starts at linear gradient condition 0.5% acetic acid (Wako Pure Chemical Industries) -methanol (Wako Pure Chemical Industries) After 30 minutes from (70:30 v / v), 0.5% acetic acid-methanol (20:80 v / v) is retained after 30 minutes, analysis time is 50 minutes, flow rate is 1.0 mL / min, and measurement wavelength is λ = 275 nm. Samples (d: sample amount 0.6 g) (f: 0.1 g) (i: 0.1 g) were prepared according to the Japan Health and Nutrition Food Association Propolis Food Standards (revised in 2001), and Sartorius Mini After filtration through Zalt (0.45 μm, Goettingen, Germany), 10 μL of the filtrate was used for HPLC analysis.
The chromatograms of the concentrated sample (d), the lyophilized product (f) and the propolis bulk (g) are shown in FIGS. 1, 2 and 3, respectively, and the retention time of the chromatogram and the corresponding compound names are shown in Table 3. Show.

化合物名は、ａ：クロロゲン酸（ｃｈｌｏｒｏｇｅｎｉｃ ａｃｉｄ）、ｃ：カフェー酸（ｃａｆｆｅｉｃ ａｃｉｄ）、ｄ：バニリン（ｖａｎｉｌｌｉｎ）、ｅ：ｐ−クマリン酸（ｐ−ｃｏｕｍａｒｉｃ ａｃｉｄ）、ｆ：３，５−ジ−Ｏ−カフェオイルキナ酸（３，５−ｄｉ−Ｏ−ｃａｆｆｅｏｙｌｑｕｉｎｉｃ ａｃｉｄ）、ｈ：３，４−ジ−Ｏ−カフェオイルキナ酸（３，４−ｄｉ−Ｏ−ｃａｆｆｅｏｙｌｑｕｉｎｉｃ ａｃｉｄ）、ｏ：３，５，７−トリヒドロキシ−４’−メトキシフラボノール（３，５，７−ｔｒｉｈｙｄｒｏｘｙ−４’−ｍｅｔｈｏｘｙｆｌａｖｏｎｏｌ）、ｐ：カエムフェロール（ｋａｅｍｐｈｅｒｏｌ）、ｑ：４−ヒドロキシ−３−プレニルケイ皮酸（４−ｈｙｄｒｏｘｙ−３−ｐｒｅｎｙｌｃｉｎｎａｍｉｃ ａｃｉｄ）、ｓ：クリシン（ｃｈｒｙｓｉｎ）、ｕ：カエムフェライド（ｋａｅｍｐｈｅｒｉｄｅ）、ｚ：３，５−ジプレニル−４−ヒドロキシケイ皮酸（アーテピリンＣ）［３，５−ｄｉｐｒｅｎｙｌ−４−ｈｙｄｒｏｘｙｃｉｎｎａｍｉｃ ａｃｉｄ（ａｒｔｅｐｉｌｌｉｎＣ）］である。
図１〜３のクロマトグラムより検出されるピークの保持時間は一致し、これらに対応するフラボノイド類やヒドロキシケイ皮酸誘導体類により抗酸化活性が発現するものと考えられる。
有機溶剤水溶液の濃縮液は、多種の抗酸化化合物を含むことが確認され、適当な濃度に調整することにより、例えば、食品用、化粧料用、飼料用などとして好適に用いることができる。
さらに、抽出残渣凍結乾燥粉末は、プロポリス原塊に匹敵する活性酸素消去能を有し、食品用、化粧料用、飼料用などとして好適に用いることができる。

The compound names are a: chlorogenic acid, c: caffeic acid, d: vanillin, e: p-coumaric acid, f: 3,5-di- O-caffeoylquinic acid (3,5-di-O-caffeoylquinic acid), h: 3,4-di-O-caffeoylquinic acid (o: 3,4-di-O-caffeoylquinic acid), o: 3 5,7-trihydroxy-4′-methoxyflavonol (3,5,7-trihydroxy-4′-methoxyflavonol), p: kaempherol, q: 4-hydroxy-3-prenylcinnamic acid (4- hydroxy-3-prenylcinnamic a id), s: chrysin, u: kaempheride, z: 3,5-diprenyl-4-hydroxycinnamic acid (Artepillin C) [3,5-diprenyl-4-hydroxycinnamic acid (artepillin C)] It is.
The retention times of the peaks detected from the chromatograms of FIGS. 1 to 3 are the same, and it is considered that the antioxidant activity is expressed by flavonoids and hydroxycinnamic acid derivatives corresponding to these.
The concentrated solution of the organic solvent aqueous solution is confirmed to contain various antioxidant compounds, and can be suitably used, for example, for foods, cosmetics, feeds, etc. by adjusting to an appropriate concentration.
Furthermore, the extraction residue freeze-dried powder has an active oxygen scavenging ability comparable to the propolis bulk, and can be suitably used for foods, cosmetics, feeds, and the like.

  The method of the present invention effectively uses the residue obtained by extracting the active ingredient from the propolis bulk, and the active oxygen scavenger obtained by the method of the present invention has high safety for human body and high active oxygen. -It has free radical scavenging ability and antioxidant ability, and is suitably used for foods, cosmetics, feeds, etc.

It is a chromatogram of one example of the concentrated liquid sample of the organic-solvent aqueous solution obtained by the hydrolysis process of the propolis original block extraction residue. It is a chromatogram of one example of the freeze-dried product of the solid substance obtained by the hydrolysis process of the propolis bulk lump extraction residue. It is a chromatogram of one example of a propolis original block.

Claims (6)

  (A) The process of extracting the pulverized propolis mass using a mixed solvent containing water, an organic solvent, and an emulsifier, and then separating it into an extract and an extraction residue, (B) in the step (A) Adding water to the separated extraction residue and stirring, followed by solid-liquid separation into an organic solvent aqueous solution and a solid, and (C) a step of concentrating the organic solvent aqueous solution separated in the step (B), A method for producing an active oxygen scavenger, wherein the concentrate obtained in the step (C) is used as a product.   (A) The process of extracting the pulverized propolis mass using a mixed solvent containing water, an organic solvent, and an emulsifier, and then separating it into an extract and an extraction residue, (B) in the step (A) Steps of adding water to the separated extraction residue and stirring, followed by solid-liquid separation into an organic solvent aqueous solution and a solid, and (D) a step of drying and pulverizing the solid separated in the step (B) A method for producing an active oxygen scavenger, comprising using the powder obtained in step (D) as a product.   The method for producing an active oxygen scavenger according to claim 1 or 2, wherein the organic solvent in the mixed solvent used in the step (A) is a polyhydric alcohol compound having two or more hydroxyl groups in the molecule.   The emulsifier in the mixed solvent used in the step (A) is at least one selected from glycerin fatty acid ester, polyglycerin fatty acid ester, propylene glycol fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, lecithins and saponins. A method for producing an active oxygen scavenger according to claim 1, 2 or 3.   A food comprising the active oxygen scavenger obtained by the method according to any one of claims 1 to 4. A cosmetic comprising the active oxygen scavenger obtained by the method according to any one of claims 1 to 4.

Images