JP4836923B2 - Collagen and hyaluronic acid production promoter - Google Patents

Description

The present invention relates to a production promoter for collagen and / or hyaluronic acid. Furthermore, this invention relates to the skin external preparation containing the said production promoter , and foodstuffs.

  Since various pollen such as cedar and ragweed cause hay fever, pollen is now recognized as an allergen. Moreover, since pollen stimulates the immune system, the pharmacological action of pollen on the immune system has also been studied.

  Patent Document 1 describes that a culture in which pollen is fermented with lactic acid bacteria, bifidobacteria, or yeast has a skin regeneration action, an anti-wrinkle action, and the like, and is blended in pharmaceuticals and cosmetics.

  Patent Document 2 describes that an oil body can be obtained from pollen derived from rapeseed.

  Pollen has a high nutritional value and is known to be used as a nutritional food either directly or wrapped in sugar (Non-patent Document 1).

Patent Document 3 discloses a pollen extract soft capsule made from pollen of thymosy, corn, hazel, rye, pussy willow, box willow, French peony or pine, and the capsule has an inhibitory effect on prostatitis and benign prostatic hyperplasia. It is described that it has.
JP-T-2001-504510 Japanese translation of PCT publication No. 2002-503268 Japanese Patent Laid-Open No. 9-278665 "Bees Science" 7 (1): 9-12 Honeybee Science (1986)

  An object of the present invention is to provide these new uses based on physiological activities newly found for pollen, pollen load or extracts thereof. In particular, it is an object of the present invention to provide use as a production promoter for collagen and / or hyaluronic acid, and cosmetics or foods containing the production promoter.

  As a result of examining the physiologically active action of pollen, pollen load or these extracts, the inventor of the present invention has developed pollen load extracts of cistus, tea, rapeseed, and buckwheat, particularly extracts of polar solvents such as water and alcohol. It has been found that there is a strong collagen production promoting action or hyaluronic acid production promoting action.

  The present invention has been completed based on such findings, and provides the following collagen production promoter, hyaluronic acid production promoter, skin external preparation, and food.

I. Collagen or / and hyaluronic acid production promoter (I-1) A collagen or hyaluronic acid production promoter comprising at least one selected from the group consisting of pollen, pollen cargo and extracts thereof.
(I-2) The above-mentioned pollen or pollen load is derived from a plant belonging to at least one family selected from the group consisting of Brassicaceae, Tadaceae, Camelliaaceae, and Hannitivaceae, (I-1) The production promoter described in 1.
(I-3) The production promoter according to (I-2), wherein the plant is at least one selected from the group consisting of cistus, tea, rapeseed, and buckwheat.
(I-4) The extract is an extract of pollen or pollen water, ethanol or a mixture of at least one plant selected from the group consisting of cistus, tea, rapeseed, and buckwheat (I -1) The production promoter according to any one of (I-3).
(I-5) A collagen production promoter comprising pollen or pollen load of at least one plant selected from the group consisting of cistus, tea, and rapeseed, or an extract of any of these.
(I-6) A hyaluronic acid production promoter comprising pollen or pollen of at least one plant selected from the group consisting of rapeseed and buckwheat, or an extract of any of these.

II. Skin external preparation (II-1) A skin external preparation comprising the production promoter described in any of (I-1) to (I-4) above.

In addition, the said skin external preparation can also be paraphrased as follows.
(II-a) A skin external preparation containing at least one selected from the group consisting of pollen, pollen load and extracts thereof.
(II-b) The pollen or pollen load described above is derived from a plant belonging to at least one family selected from the group consisting of Brassicaceae, Tadaceae, Camelliaaceae, and Algeriaceae, (Ia) Skin external preparation.
(II-c) The skin external preparation described in (II-b), wherein the plant is at least one selected from the group consisting of cistus, tea, rapeseed, and buckwheat.
(II-d) The extract is an extract of pollen or pollen water, ethanol or a mixture thereof of at least one plant selected from the group consisting of cistus, tea, rapeseed, and buckwheat (II -a) The external preparation for skin described in any one of (II-c).

III. Food (III-1) A food comprising the production promoter described in any of (I-1) to (I-4) above.

In addition, the said foodstuff can also be paraphrased as follows.
(III-a) A food comprising at least one selected from the group consisting of pollen, pollen load and extracts thereof.
(III-b) The above-mentioned pollen or pollen load is derived from a plant belonging to at least one family selected from the group consisting of Brassicaceae, Tadaceae, Camelliaaceae, and Alniaceae, (III-a) Foods listed in
(III-c) The food according to (III-b), wherein the plant is at least one selected from the group consisting of cistus, tea, rapeseed, and buckwheat.
(III-d) The extract is an extract of pollen or pollen water, ethanol or a mixture thereof of at least one plant selected from the group consisting of cistus, tea, rapeseed, and buckwheat (III -a) The food according to any one of (III-c).

  All of these foods can be distinguished from ordinary foods in that they are used exclusively to promote the production of at least one of collagen and hyaluronic acid. In particular, a food containing at least one selected from the group consisting of pollen, pollen load and extracts thereof of plants belonging to the Brassicaceae family such as rapeseed is used to promote the production of both collagen and hyaluronic acid It can be distinguished from ordinary food.

  Pollen, pollen load or these extracts, as shown in the test examples to be described later, in order to improve the production of collagen and hyaluronic acid, moisturize, moisturize skin, anti-aging (improve wrinkles, etc.) ), Whitening (improving dullness), and the like, and it was revealed that it is useful as a skin external preparation (especially cosmetics) and a food material. In particular, the water extract of rapeseed pollen is extremely excellent as an active ingredient of an external preparation for skin (especially cosmetics) or as a food material because it strongly promotes the production of collagen and hyaluronic acid.

(I) Collagen and / or hyaluronic acid production promoter The collagen or / and hyaluronic acid production promoter of the present invention is characterized by containing pollen, pollen cargo or an extract thereof as an active ingredient.

  Here, the pollen load is made by bees hardened with nectar, and is also called pollen dumpling. The pollen load is stored in the nest with honey. This pollen load can be easily collected.

  In the present invention, either pollen or pollen cargo may be used, but pollen cargo is preferably used because it is easily available in the above points. In addition, although pollen and pollen load can each be used independently, both can also be mixed and used.

  As a source of pollen or / and pollen load (hereinafter simply referred to as “pollen / pollen load”) of the present invention, the following table shows the family name, genus / species name, and Japanese name (name) of a suitable plant. 1 to Table 3.

  Preferable plants from which the pollen / pollen load used in the present invention are derived include Brassicaceae, Rapaceae, Camellia, and Candidae. Specifically, examples of Brassicaceae plants include araseito, rapeseed, and radish, with rapeseed being particularly preferred. An example of a plantaceae is buckwheat. As the camellia plant, sasanqua, tea, nuts, and camellia are preferably exemplified, and tea is particularly preferable. Preferred examples of the plant belonging to the family Canniaceae are cistus.

  In addition, as an active ingredient of a collagen production promoter, the cistus which is a Cannabisaceae plant, the tea which is a camellia plant, and the rapeseed which is a cruciferous plant can be mentioned suitably. Moreover, as an active ingredient of a hyaluronic acid production promoter, rapeseed, which is a cruciferous plant, and buckwheat, which is a rapeseed plant, can be preferably exemplified.

  Pollen / pollen load can be used as it is, but can also be used in the form of an extract prepared with an appropriate solvent.

  Here, the pollen / pollen load can be extracted with a solvent such as water, a water-containing solvent, and an organic solvent. Specifically, water, lower alcohol (methanol, ethanol, n-propanol, isopropanol, etc.), ketones (acetone, methyl ethyl ketone, methyl butyl ketone, etc.), esters (methyl acetate, ethyl acetate, etc.), ethers (diethyl) Ether, diisopropyl ether, etc.), aromatic hydrocarbons (toluene, xylene, etc.), halogenated hydrocarbons (methylene chloride, chloroform, etc.), glycols (ethylene glycol, propylene glycol), glycol ethers (methyl cellosolve, 1, 2 -Dimethoxyethane), glycerin, tetrahydrofuran, acetonitrile, acetic acid, propionic acid, acetamide, dimethylformamide, dimethylsulfoxide. Moreover, even if these extraction solvents are used by 1 type, 2 or more types can also be combined arbitrarily and can also be used as a liquid mixture. Preferred examples of the mixed liquid include water-containing organic solvents, particularly water-containing alcohols such as water-containing ethanol, water-containing methanol, and water-containing propanol. The extraction solvent is preferably water, ethanol, or a mixture thereof (hydrous ethanol).

  Extraction can be easily performed by adding pollen / pollen load to the solvent, immersing and extracting from 0 ° C to the boiling temperature of the solvent, usually 100 ° C or lower for 30 minutes to 24 hours, and removing insoluble matter. Can do.

  In the present invention, the pollen / pollen load extract can be used as it is as a collagen production promoter or / and hyaluronic acid production promoter, but a liquid obtained by concentrating the extract as necessary (concentration). Liquid) or a dried product such as a freeze-dried product or a spray-dried product of the extract.

  The extract thus obtained may be subjected to treatment such as decolorization, masking or deodorization as necessary in order to remove the color, taste or odor. Examples of such treatment include adsorption treatment with activated carbon and the like, purification by column chromatography, ion exchange chromatography, and the like.

  Among the pollen load extracts of various plants (Tables 1 to 3), the extract of cystus pollen load (preferably water or ethanol extract) which is a cannibalid plant, the extraction of tea pollen load of camellia plant Extract (preferably water or ethanol extract), and rapeseed pollen extract (preferably water extract), which is a cruciferous plant, has an excellent collagen production promoting action, and is effective in promoting collagen production. It can be suitably used as a component.

  Also, among the pollen load extracts of various plants (Tables 1 to 3), the extract of the rapeseed pollen load (preferably the water extract), which is a Brassicaceae plant, and the extraction of the pollen load of buckwheat, which is a radish plant The product (preferably ethanol extract) has an excellent hyaluronic acid production promoting action and can be suitably used as an active ingredient of a hyaluronic acid production promoter.

  In the collagen production promoter or / and hyaluronic acid production promoter of the present invention, the amount of pollen / pollen load or extract thereof is 0.0001 to 100% by weight, preferably 0.001 to 50% as a dry product. % By weight, more preferably 0.01 to 25% by weight.

  The collagen production promoter or / and hyaluronic acid production promoter of the present invention has a cosmetic or additive promoting effect on the production of collagen or / and hyaluronic acid, and is used for cosmetics, arthritis, gingivitis, rheumatism, etc. It can be preferably used as a medical or functional food that can reduce symptoms.

  By the way, the hyaluronic acid targeted by the present invention is an acidic mucopolysaccharide synthesized by hyaluronic acid synthase (HAS) present in the membrane of dermal fibroblasts (Y. Sugiyama et al., J. Invest. Dermatol., 110, 116 (1998)). Hyaluronic acid contributes to retention of water in the cell gap, retention of cells based on the formation of a jelly-like matrix in the tissue, and maintenance of tissue lubricity and flexibility (BIOINDUSTRY, Vol8, 346 (1991 )). Hyaluronic acid has been reported to decrease with age (K. Matsuoka et al. Aging, 1, 47 (1989)) or to denature (LAMMeyer et al. J. Invest. Dermatol., 102, 385 (1994)) Such changes contribute to skin dryness, wrinkles and sagging.

  Collagen targeted by the present invention is an important component constituting the dermal matrix. Collagen molecules are synthesized in fibroblasts and secreted extracellularly, and then regularly crosslinked to maintain the hardness of connective tissue, contributing to skin firmness and elasticity. For many years, it has been reported that collagen in the upper dermis is reduced by exposing the skin to ultraviolet rays (L.H.Kligman et al. J. Invest. Dermatol., 84, 272 (1985)). It has also been reported that collagen fibers in the dermis are denatured by UV exposure (Y. Nishimori et al. Abstract, 20th IFSCC Congress Cannus, 1, 117 (1998)).

(2) External preparation for skin, preferably cosmetics The external preparation for skin containing the collagen production promoter or / and hyaluronic acid production promoter of the present invention is the collagen production promoting action or / and hyaluronic acid production promotion of the collagen production promoter. Based on the hyaluronic acid production promoting action of the agent, it has excellent collagen production promoting action and / or hyaluronic acid production promoting action on human skin, respectively. Prevents aging, eliminates or reduces wrinkles, makes skin fresh and makes spots and dullness unnoticeable, has a beautiful skin effect, and maintains healthy skin. For this reason, the said skin external preparation is useful as cosmetics, such as whitening cosmetics, anti-aging cosmetics, beautiful skin cosmetics, or skin care cosmetics especially. Therefore, the skin external preparation as used in the present invention includes these uses as cosmetics.

  The blending amount of the pollen / pollen load or the extract thereof in the skin external preparation is 0.0005 to 20% by weight, preferably 0.001 to 10% by weight as a dry product in the total amount of the skin external preparation. If the amount is significantly less than 0.0005% by weight, the effects of the present invention tend not to be sufficiently exhibited. If the amount exceeds 20% by weight, preparation tends to be difficult. Further, it is particularly preferable that the pollen / pollen load or the extract thereof is prepared to a concentration of about 0.01 to 1% by weight and applied to the skin.

  The external preparation for skin of the present invention includes, in addition to pollen / pollen load or extracts thereof, components usually used for external preparations for skin, particularly cosmetics, such as whitening agents, moisturizers, antioxidants, oily components, ultraviolet absorption Agents, surfactants, thickeners, alcohols, powder components, color materials, aqueous components, water, various skin nutrients and the like can be appropriately blended as necessary.

  The dosage form of the external preparation for skin of the present invention is an aqueous solution system, a solubilization system, an emulsification system, a powder system, an oil liquid system, a gel system, an ointment system, an aerosol system, a water-oil two-layer system, and a water-oil-powder. A wide range of dosage forms such as a three-layer system can be adopted. For example, when the skin external preparation is a basic cosmetic, examples of the form thereof include face wash, lotion, milky lotion, cream, gel, essence, cosmetic liquid, pack, mask and the like. In addition, if the external preparation for skin is a makeup cosmetic, examples of the form include foundation, lipstick, blusher, eye shadow, eyeliner, mascara and the like. Other forms include facial cleansers, massage agents, cleansing agents, after-shave lotions, pre-shave lotions, shaving creams, body soaps, soaps, shampoos, rinses, hair treatments, hair conditioners, hair-care agents, hair restorers, antiperspirants. And hygiene products.

(3) Food The food of the present invention is a collagen production promoter or / and hyaluronic acid, which contains the pollen / pollen load or the extract thereof having the collagen production promoting action and / or hyaluronic acid production promoting action described in (1). It is characterized by blending as a production promoter. For this reason, the food of the present invention functions on the basis of these actions by the reduction of collagen / hyaluronic acid such as beauty foods and drinks for the purpose of whitening, skin beautification, moisturizing, anti-aging, etc. Food used for the purpose of alleviating the symptoms of decreasing diseases (eg arthritis, gingivitis, alveolar pyorrhea, rheumatism, etc.) is included.

  Specific examples of such foods include drinks, beverages (soft drinks, milk beverages, lactic acid bacteria beverages, yogurt beverages, carbonated beverages, alcoholic beverages, etc.), spreads, pastes, Western confectionery, Japanese confectionery, ice confectionery, retort foods, and instant foods. , Bottled / canned, jelly-like food, seasoning, bouillon, beekeeping products (honey, royal jelly, propolis, etc.), dairy products, processed fruits, processed vegetables, processed cereals, pickles, pickles, fish products, livestock products, Examples include delicacies, dried foods, side dishes, frozen foods, and fat foods. Further, the food of the present invention is a so-called health food that can be prepared by adding and blending the pollen / pollen load described in (1) or an extract thereof as a collagen production promoter or / and hyaluronic acid production promoter, Functional foods, dietary supplements, supplements, foods for specified health use, foods for the sick, combination foods for the sick (Ministry of Health, Labor and Welfare, special-purpose foods) or foods for the elderly (Ministry of Health, Labor and Welfare, special-purpose foods) In such a case, uncoated tablets, film-coated tablets, sugar-coated tablets, granules, powders, tablets, capsules (including both hard capsules and soft capsules), chewable types, syrup types, drink types, etc. You can also The preparation of a food in which the pollen / pollen load according to the present invention or an extract thereof is added and blended as a collagen production promoter or / and hyaluronic acid production promoter can be prepared by a method known per se.

  EXAMPLES Hereinafter, although an Example, a test example, and a formulation example demonstrate this invention in detail, this invention is not limited at all by these Examples.

Example 1 : Preparation of an ethanol extract of pollen cargo To each 10 g of pollen cargo (cistus, tea, rapeseed, buckwheat), add 30 ml of ethanol, knead in a mortar, transfer to another container and shake at room temperature for 16-20 hours Mix thoroughly. This was centrifuged at 1500 rpm for 5 minutes to obtain a supernatant and a precipitate. Two times the amount of ethanol was added to the resulting precipitate, kneaded again in a mortar, transferred to another container, and mixed with shaking at room temperature for 2 hours. This was centrifuged at 1500 rpm for 5 minutes to obtain a supernatant. This was combined with the previously obtained supernatant and concentrated with a rotary evaporator (manufactured by EYELA) to obtain an ethanol extract of the pollen load of each plant (cistus, tea, rapeseed, buckwheat).

Example 2: Preparation of Pollen Loaded Water Extract Three times the amount of water was added to the final precipitate obtained by the ethanol extraction of Example 1, ground and shaken at room temperature for 16-20 hours. Next, this was centrifuged at 2000 rpm for 10 minutes, and the resulting supernatant was lyophilized. This was used in the following test examples as an aqueous extract of pollen load of each plant (Cistus, tea, rapeseed, buckwheat).

Test Example Among the extracts obtained in Examples 1 and 2, a water extract and ethanol extract of cistus pollen loaded with an amount of extract sufficient for the experiment, a water extract and ethanol extract of tea pollen loaded And a rapeseed pollen water extract were subjected to a collagen production promotion test, and a rapeseed pollen water extract and a buckwheat pollen ethanol extract were subjected to a hyaluronic acid production promotion test.

Test example 1 Collagen production promotion test Normal human fibroblast cell line WI-38 cells were adjusted to 1 × 10 ⁵ cells / ml with RPMI-1640 medium containing 10% FCS (fetal calf serum) (cell suspension) 100 μl of cell suspension was added per well. The plate was then incubated overnight at 37 ° C. in 5% CO ₂ -95% air, the medium was changed with a mixture of 150 μl RPMI-1640 medium and 40 μl PBS (−), and the pollen prepared in Examples 1 and 2 Each extract of load was added to each well and incubated at 37 ° C. for 48 hours in 5% CO ₂ -95% air. The resulting culture was then centrifuged at 1500 rpm for 15 minutes to obtain a supernatant.

(1) Preparation of measurement sample:
(1-1) Pepsin treatment of collagen in culture supernatant
(i) Pepsin (manufactured by Mikuni Chemical Industry Co., Ltd.) is dissolved in a 150 mM aqueous acetic acid solution (manufactured by Kanto Chemical Co., Ltd.) to a concentration of 0.6 mg / mL.
(ii) Add 0.2 mL of the above pepsin solution to 0.2 mL of the cultured cell supernatant and stir well by inversion mixing.
(iii) Leave at 4 ° C overnight.
(iv) For neutralization, add 0.1 mL of an aqueous solution containing 200 mM Tris and 150 mM NaCl, stir well by inversion, and use this as the collagen solution for measurement.

(1-2) Pepsin treatment of collagen deposited as extracellular matrix
(i) Pepsin (manufactured by Mikuni Chemical Industry Co., Ltd.) is dissolved in a 50 mM aqueous acetic acid solution (manufactured by Kanto Chemical Co., Ltd.) to a concentration of 0.1 mg / mL.
(ii) After removing the culture solution from the petri dish, the cells are peeled off with a microcell scraper, and an equal amount of pepsin solution to the culture solution is added to the petri dish.
(iii) Shake overnight at 4 ° C.
(iv) Centrifuge at 10000 × g for 10 minutes and collect the supernatant.
(v) Add an aqueous solution containing 200 mM Tris and 150 mM NaCl in an amount one-third of the added pepsin solution and stir well by inversion to make a collagen solution for measurement.

(1-3) Preparation of measurement sample The measurement collagen solution prepared in (1-1) and (1-2) above was mixed to prepare a measurement sample.

(2) Measurement Using the measurement sample prepared by the method described above, the collagen production promotion rate was measured using a human collagen type 1 ELISA kit (manufactured by ACBio). The measuring method followed the instruction manual of the kit. The results are shown in Table 4.

  The collagen production promotion rate (increase rate) was calculated assuming that the amount of collagen when 100 μg of water was added instead of each pollen extract was 100%.

Test example 2: Hyaluronic acid production promotion test Normal human fibroblast cell line WI-38 cells were adjusted to 1 × 10 ⁵ cells / ml with 10% FCS (fetal calf serum) -containing RPMI-1640 medium (cell suspension) Solution), 100 μl of cell suspension was added per well. This plate was then incubated overnight at 37 ° C. in 5% CO ₂ -95% air, and the medium was changed with a mixture of 150 μl of RPMI-1640 medium and 40 μl of PBS (−), and obtained in Examples 1 and 2. Pollen loaded extract was added to each well and incubated at 37 ° C. for 48 hours in 5% CO ₂ -95% air. Next, the obtained culture was centrifuged at 1500 rpm for 15 minutes to obtain a supernatant. The amount of hyaluronic acid in the obtained supernatant was measured using a commercially available hyaluronic acid measurement kit (Hyaluronan Assay Kit: manufactured by Seikagaku Corporation).

  The hyaluronic acid production promotion rate (increase rate) was calculated with the amount of hyaluronic acid when 100 μl of water was added instead of each extract of pollen loaded as 100%. The results are shown in Table 5.

  From the above results, it was revealed that the pollen cargo extract targeted by the present invention has an excellent collagen production promoting effect or hyaluronic acid production promoting effect. In particular, rapeseed pollen water extract is strong in both collagen production promoting effect and hyaluronic acid production promoting effect, and is useful as a cosmetic and food material having various functions such as skin beautifying, anti-aging, whitening and moisturizing. It became clear that there was.

Formulation Example 1 : Lotion Toner lotion was produced according to a conventional method using each component shown in Table 6 below.

Formulation Example 2 : Cosmetic liquid A cosmetic liquid was produced according to a conventional method using the components shown in Table 7 below.

Formulation Example 3 : Emulsion An emulsion was prepared according to a conventional method using each component shown in Table 8 below.

Claims (5)

  A production promoter of at least one of collagen and hyaluronic acid, comprising at least one selected from the group consisting of pollen, pollen cargo and extracts thereof.   The production promoter according to claim 1, wherein the pollen or pollen load is derived from a plant belonging to at least one family selected from the group consisting of Brassicaceae, Rapaceae, Camelliaaceae, and Hannibalidae.   The production promoter according to claim 2, wherein the plant is at least one selected from the group consisting of cistus, tea, rapeseed, and buckwheat.   The production according to claim 1, wherein the extract is an extract of pollen or pollen water, ethanol or a mixture of at least one plant selected from the group consisting of cistus, tea, rapeseed, and buckwheat. Accelerator. An anti-aging skin external preparation comprising the production promoter according to any one of claims 1 to 4.