JP6017285B2 - Heat shock protein 70 production inducer - Google Patents

Description

The present invention relates to a heat shock protein 70 production inducer. More specifically, the present invention relates to a heat shock protein 70 production inducer of a yeast cell extract containing a zinc compound.

Heat shock protein (HSP) expression can be induced by various stresses on cells, such as bacterial infection and inflammation, ethanol, active oxygen, heavy metals, ultraviolet light, hypoxia, and when cells are exposed to high temperatures. Are known. The produced nascent protein is in an unstable state and is stabilized through a process called folding. HSP functions as a molecular chaperone that controls protein folding by binding to this nascent protein, and many of the molecular chaperones are HSP. It is known that proteins that have been denatured under high-temperature conditions or nascent proteins have problems in the folding stage, and those that cannot function are bound to HSP and processed. HSP has the function of repairing proteins with such higher-order structures and inhibiting protein denaturation, and proteins that are judged to be impossible to repair are ubiquitinated and transported to an enzyme complex called the proteasome To be decomposed.

It has been reported that HSP70 is constitutively expressed in many organs such as the digestive tract and skin, and its expression level is increased by various stresses. In recent years, it has been reported that HSP70 has an anti-cell death effect and an anti-inflammatory effect, and protects cells against various stresses such as alcohol and ultraviolet rays. For this reason, research is being conducted on application of HSP70 inducers to pharmaceuticals (such as gastric mucosa protective agents) and cosmetics.

Many therapeutic agents for gastric ulcer and gastric mucosal injury have already been known to have an HSP70-inducing effect, and geranylgeranylacetone is marketed as a pharmaceutical product under the name Selvex (Eisai Co., Ltd.) (Non-patent Document 1). It is also well known that zinc compounds induce HSP70, and polaprezinc (a new zeria drug), which is a complex compound of zinc and L-carnosine, is used as an anti-ulcer drug (Non-patent Document 2).
Furthermore, it has been reported that paeoniflorin extracted from the root of glaze induces HSP70 on Hela cells (Non-patent Document 3).
Carbenoxolone, a derivative of glycyrrhetinic acid, is also known to induce HSP70 on Hela cells (Non-patent Document 4).

Regarding the cultivation of yeast containing a zinc compound, JP-A-8-332082 (Patent Document 1) increases the zinc compound in the cells by stirring the yeast non-proliferatively in a solution containing 50 ppm or more of zinc. The method of inclusion is described. This is because when yeast is cultured in a medium containing a high amount of zinc compound, the growth of the yeast is inhibited and no longer grows. In this method, a zinc compound is taken into the cells. However, in the above method, the growth of the yeast and the incorporation of the zinc compound into the yeast are carried out in two steps, which makes the production method very complicated. Furthermore, JP-A-2-295480 (Patent Document 3) isolates yeast grown by culturing at 26-30 ° C. for 2 days in a medium containing 20-100 ppm of zinc ions in Difco's Bacto YM agar medium. To do. A method for cultivating the yeast with good growth is described by culturing the isolate in a medium supplemented with 0.2 to 10 ppm of zinc ions.

Furthermore, JP-A-9-124438 (Patent Document 2) describes a cosmetic and a food containing an aqueous solvent extract of Saccharomyces yeast cultured in a medium containing a zinc compound. As cosmetics, it has melanin production inhibitory action, melanin pigment decomposition action, photoaging prevention action (skin aging prevention action), acne growth inhibition action, moisturizing action, skin roughening action, etc. Effects such as nutritional tonic and nutritional enhancement are described. However, it is described in the yeast culture medium described in the above patent that the zinc compound is cultured in a total amount of about 10 to 1,000 ppm. However, when the zinc concentration is 10 to 1,000 ppm, the zinc concentration accumulated in the microbial cells is low, so that sufficient heat shock protein 70 production induction cannot be expected, and the effect described is simply that of the yeast extract. It's just an extension.

Biochem. Biophys. Res. Commun. 353: 399-404 2007 Otaka, M., et al Dig. Dis. Sci. 47: 2799-2804, 2002 Odashima, M., et al Cell Stress Chaperones 9: 378-389, 2004 Yan, D., et al Life.Sci. 69: 2867-2873, 2001 Nagayama, S., et al

JP-A-8-332082 JP-A-9-124438 JP-A-2-295480

As in the above technical literature, heat shock protein 70 production inducers are known to include geranylgeranylacetone, zinc compounds, complex compounds of zinc and L-carnosine, paeoniflorin, and derivatives of glycyrrhetin. It has already been used as a therapeutic drug. However, when zinc compounds are added directly to epithelial cells, cytotoxicity appears strongly, which causes a problem in ingestion. Therefore, an object of the present invention is to provide a zinc-containing yeast cell composition having a low cytotoxicity and a high effect of inducing production of heat shock protein 70 by incorporating a high concentration of the zinc compound into the yeast cell.

In addition, there are many descriptions that use yeast cells cultured by adding a zinc compound to a medium for foods and cosmetics, but no description on the induction of heat shock protein 70 has been described so far. In addition, yeast cells cultured at a zinc concentration that does not inhibit the growth of yeast have no effect of inducing heat shock protein 70 production. For this reason, it is an object of the present invention to provide the production of yeast cells containing a high concentration of zinc compound without depending on a complicated culture method.

Normally, when zinc sulfate is added to the culture medium, inhibition of yeast growth is not observed up to about 5 mM (404 ppm), but at concentrations higher than this, growth inhibition begins to occur, and growth is almost observed at 10 mM (808 ppm). It becomes impossible. However, by using a yeast that has been acclimatized by repeatedly culturing in a medium in which the concentration of the zinc compound is increased stepwise, the yeast can grow even in a medium containing a zinc compound of 10 mM or more. .

Furthermore, it was revealed that the yeast cell extract cultured in a medium containing 10 mM or more of a zinc compound has a sufficient effect for induction of heat shock protein 70 production in epithelial cells.

The yeast acclimated and cultured in a medium containing a high concentration of zinc compound according to the present invention does not inhibit growth even in a medium containing a high concentration of zinc compound, and a high concentration of zinc compound in the cell body Containing. For this reason, when the bacterial cell extract is allowed to act on epithelial cells, it is possible to induce the production of heat shock protein 70 in the epithelial cells. In addition, since it is not cytotoxic as in the case where a zinc compound is allowed to act on cells, a highly safe production inducer can be developed.
Drugs that prevent gastric ulcers and gastric mucosal injury by enabling production of heat shock protein 70 in epithelial cells by drinking yeast cells containing zinc compounds at high concentrations as they are or by drinking yeast cell extracts Can be used.

Furthermore, by using the bacterial cell extract as a cosmetic, it induces the production of heat shock protein 70 in epithelial cells, which are epithelial cells, and protects against epidermal cell damage due to stress caused by ultraviolet rays and the like, resulting in inflammation of the epidermis. It becomes possible to protect from.

Band intensity obtained by Western blotting

Hereinafter, the present invention will be described in detail.
The yeast used in the present invention is a commercially available baker's yeast, or a distributed Saccharomyces cerevisiae.
Any yeast belonging to the above can be used. In addition, since yeast can be easily separated from the natural world where saccharides are present, any yeast belonging to the genus Saccharomyces can be used as long as it is a yeast isolated from plants, soil, etc. In particular, yeast belonging to Saccharomyces cerevisiae can be used. desirable.

The zinc compound can be used as long as it is a water-soluble zinc compound, and zinc chloride, zinc nitrate, zinc sulfate, zinc citrate, zinc acetate, zinc tartrate, and zinc lactate can be used as the inorganic zinc compound. These zinc compounds are added to the yeast culture medium to culture the yeast. When the concentration of the zinc compound is about 5 mM, it is difficult to inhibit the growth of the yeast. However, when the concentration is 10 mM, the yeast hardly grows. However, by culturing for a long time, yeasts acclimatized to zinc compounds grow slightly. By repeatedly culturing the grown yeast in a zinc-added medium having the same concentration, a zinc-adapted bacterium having a high growth rate can be obtained. By repeating this operation sequentially in a medium with a high zinc concentration, acclimated bacteria exhibiting a high growth ability can be obtained even in a medium to which a high concentration of zinc compound is added. The concentration of the zinc compound added to the medium can be 10-60 mM. However, the concentration of 20-30 mM is desirable in consideration of the growth of yeast.

A medium for cultivating yeast generally contains nutrients such as a carbon source typified by glucose, a nitrogen source consisting of peptides typified by peptone, trace vitamins typified by yeast extract, and minerals. If the substance is contained, it can be cultured. Generally, a YM (Yeast-Malt) medium, a YPD (Yeast-Polypeptone-Dextrose) medium, or the like made of a natural product is used. It is also possible to use a synthetic medium not containing a natural product. The medium composition may include a minimum carbon source, nitrogen source, and phosphorus source. Examples of the carbon source include ribose, arabinose, xylose, glucose, fructose, mannose, galactose, rhamnose, etc. Disaccharides such as claus, maltose, lactose, trehalose and cellobiose, and trisaccharides such as raffinose and maltotriose can be used. One or more of these may be contained. Nitrogen sources include urea, potassium nitrate as nitrate, sodium nitrate, calcium nitrate, ammonium sulfate as ammonium sulfate, ammonium chloride, amino acid as tryptophan,
Nitrogen-containing compounds such as lysine, methionine, phenylalanine, threonine, valine, isoleucine, leucine, histidine, alanine, arginine, asparagine, serine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, proline, tyrosine, etc. can be used. As long as it contains at least one kind. As the phosphorus source, phosphate can be used, and potassium monohydrogen phosphate, potassium dihydrogen phosphate, and ammonium phosphate can be used, and one or more of these may be contained.

The basic medium of the synthetic medium is sufficient if it contains the carbon source, nitrogen source and phosphorus source, but it is possible to increase the growth of yeast by adding vitamin sources and mineral sources. It is. Examples of vitamin sources include biotin, thiamine (vitamin B1), riboflavin, pyridoxine (vitamin B6), pantothenic acid, ascorbic acid, iodic acid, cyanocobalamin, inositol, nicotinic acid, choline, carnitine, paraaminobenzoic acid, nicotinamide adenidine Nucleotides and the like can be used. Examples of mineral sources include potassium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, sodium, iodine, cobalt, and the like. Specific components that can supply these include, for example, calcium nitrate. , Potassium chloride, calcium chloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, cobalt chloride, etc. These compounds can be used. Natural ingredients such as brown sugar containing abundant minerals can also be used.

It is also possible to obtain a basic medium exhibiting higher growth by appropriately adding the above substances to Czapek medium, Burkholder medium, and YNB (Yeast Nitrogen Base) medium, which are synthetic media not containing natural components.

In extracting effective components from yeast cells cultured in a zinc compound-containing medium, the cultured cells are collected and thoroughly washed with purified water, and then the cells are removed using, for example, ultrasonic waves or a French press. Destroy and add water or ethanol, butylene glycol, propylene glycol, glycerin or other aqueous solvent alone or a mixture thereof to the wet weight of the cells about 10 to 100 times, and perform operations such as centrifugation. What is necessary is just to filter after performing. In addition, about 10 to 100 times the amount of purified water is added to the wet cell weight after washing, and after self-digestion at 45 to 55 ° C. for 24 to 72 hours, operation such as centrifugation is performed. Get Qing. Furthermore, when using an enzymatic degradation method, add purified water in an amount of about 10 to 100 times the wet cell weight after washing, add a protease, react overnight at the enzyme activity temperature, and then centrifuge. Operate to obtain the supernatant. When using the acid decomposition method, add about 5 times the amount of 1-5% hydrochloric acid to the wet cell weight after washing, and hydrolyze with stirring at 45-60 ° C for 3-7 hours. . Then, after neutralizing with an alkali, operations such as centrifugation are performed to obtain a supernatant.

The yeast cell extract prepared as described above is used as a cosmetic preparation and / or a pharmaceutical preparation. The yeast cell extract is preferably in an amount of 0.001 to 10% by mass, more preferably 0.01 to 5% by mass, and most preferably 0.1 to 5% by mass as a dry matter amount with respect to the whole preparation. use. In the case of a pharmaceutical preparation, yeast cells can be used orally as they are. Usually, it is 10-100 mass%, Preferably it is 25-70 mass%, More preferably, it is 40-60 mass%.

The dosage form for administration as a pharmaceutical preparation varies depending on the disease state, its progress, and other conditions, but the heat shock protein production inducer according to the present invention may be administered as it is, or a commonly used preparation is added. To form a solid or liquid dosage form, and oral (tablets, granules, powders, fine granules, capsules, coated tablets, pills, syrups, troches, etc.), transdermal, by known methods Absorption preparations, inhalants (aerosols, powdered inhalants, liquid inhalants, etc.), external preparations (ointments, creams, liquids, etc.), emulsions, suspensions, cataplasms, lotions, etc. . It can also be mixed with food, feed, drinking water and the like.

The preparation is performed by a known preparation method, for example, by a process such as mixing, granulating, sugar-coating, dissolution, and emulsion. As a pharmaceutical formulation for oral administration, the active ingredient may be mixed with an excipient, and may be granulated, if necessary, granulated, and after adding necessary adjuvants, tablets may be obtained.

In the case of preparations such as tablets, it can be produced using excipients commonly used in the pharmaceutical field. In the case of preparations such as injections, natural surfactants such as soybean lecithin, or polysorbates, polyoxyethine hydrogenated castor oil It can be produced by emulsification or solubilization using a synthetic surfactant or the like.

The excipient is not particularly limited. For example, starch, corn starch, dextrin, glucose, lactose, sucrose, sugar alcohol (mannitol, erythritol, xylitol, etc.), hydrogenated oil, crystalline cellulose, anhydrous silicic acid, calcium silicate, second Examples thereof include calcium hydrogen phosphate.

The heat shock protein production inducer of the present invention further comprises necessary antioxidants, binders, disintegrants, lubricants, colorants, flavoring agents, flavoring agents, solubilizers, stabilizers, emulsifiers, absorption promoters. , And may be administered as a pharmaceutical composition containing a surfactant, pH adjuster, preservative, other adjuvants, and the like.

Although it does not specifically limit as antioxidant, For example, ascorbic acid, alpha-tocopherol, ethoxyquin, dibutylhydroxytoluene, butylhydroxyanisole, etc. are mentioned.

The binder is not particularly limited, and examples thereof include polyvinyl pyrrolidone, ethyl cellulose, methyl cellulose, pregelatinized starch, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose sodium, and propylene glycol.

Although it does not specifically limit as a disintegrating agent, For example, a hydroxypropyl cellulose, carboxymethylcellulose, a calcium carbonate, carboxymethylcellulose calcium etc. are mentioned.

The lubricant is not particularly limited, and examples thereof include magnesium stearate, talc, calcium stearate, sodium stearyl fumarate, polyethylene glycol 6000, and the like.

Although it does not specifically limit as a solubilizing agent, For example, physiological saline, phosphate buffered saline, lactated Ringer's solution, polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaur Rate, macrogol, castor oil fatty acid ethyl ester and the like.

Although it does not specifically limit as a pH adjuster and a buffering agent, For example, an organic acid or an inorganic acid, its salt, sodium hydroxide etc. are mentioned.

The suspending agent is not particularly limited, and examples thereof include methyl cellulose, polysorbate 80, hydroxyethyl cellulose, carboxymethyl cellulose sodium, polyoxyethylene sorbitan monolaurate and the like.

Although it does not specifically limit as a preservative, For example, methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, etc. are mentioned.

In the case of tablets, granules, and powders, a film coating such as hydroxypropylmethylcellulose may be applied as necessary.

When producing an oral liquid, a coloring agent, a flavoring agent, an antioxidant, a solubilizing agent, a stabilizer, and the like may be added as necessary.

Examples of the dosage form for administration as a cosmetic preparation include basic cosmetics such as emulsions, creams, lotions, essences, packs, facial cleansers, makeup cosmetics such as foundations and liquid foundations, facial cleansers, body shampoos, Examples include soaps and other cosmetics for cleaning, and bath agents, but are not limited to these.

In the cosmetic preparation of the present invention, in addition to the essential components composed of the above-described zinc-rich yeast extract, blending components usually used in cosmetics such as oily components, surfactants, moisturizers, thickeners, antiseptics -A disinfectant, a powder component, an ultraviolet absorber, a pigment, a fragrance, and the like can be appropriately blended as necessary.

Here, as the oil component, for example, olive oil, jojoba oil, castor oil, soybean oil, rice oil, rice germ oil, palm oil, palm oil, cacao oil, meadow foam oil, sheer butter, tea tree oil, avocado oil, Oils derived from plants such as macadamia nut oil and plant-derived squalane; Fats derived from animals such as mink oil and turtle oil; waxes such as beeswax, carnauba wax, rice wax, lanolin; liquid paraffin, petrolatum, paraffin wax, squalane, etc. Hydrocarbons: fatty acids such as myristic acid, palmitic acid, stearic acid, oleic acid, isostearic acid, cis-11-eicosenoic acid; higher alcohols such as lauryl alcohol, cetanol, stearyl alcohol; isopropyl myristate, palmitic acid Isopropyl, me Butyl phosphate, 2-ethylhexyl glycerides, higher fatty acid octyldodecyl (octyl stearate dodecyl and the like), and the synthetic esters and synthetic triglycerides such like.

Examples of the surfactant include polyoxyethylene alkyl ether, polyoxyethylene fatty acid ester, polyoxyethylene sorbitan fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyoxyethylene hydrogenated castor oil, polyoxyethylene Nonionic surfactants such as oxyethylene sorbitol fatty acid esters; fatty acid salts, alkyl sulfates, alkylbenzene sulfonates, polyoxyethylene alkyl ether sulfates, polyoxyethylene fatty acid amine sulfates, polyoxyethylene alkyl phenyl ether sulfates, Polyoxyethylene alkyl ether phosphates, α-sulfonated fatty acid alkyl ester salts, polyoxyethylene alkyl phenyl ether phosphates, etc. ON surfactant: quaternary ammonium salt, primary to tertiary fatty acid amine salt, trialkylbenzylammonium salt, alkylpyridinium salt, 2-alkyl-1-alkyl-1-hydroxyethylimidazolinium salt, N N, N-dimethyl-N-alkyl-N-carboxymethylammoniobetaine, N, N, N-trialkyl-N-, N, N-dimethyl-N-alkyl-N-carboxymethylammoniobetaine Amphoteric surfactants such as alkylene ammoniocarboxybetaine and N-acylamidopropyl-N ′, N′-dimethyl-N′-β-hydroxypropylammoniosulfobetaine can be used.

Examples of humectants include glycols such as glycerin, propylene glycol, dipropylene glycol, 1,3-butylene glycol, and polyethylene glycol, sugars such as maltitol, sorbitol, xylitol, trehalose, glucose, sodium pyrrolidonecarboxylate, and lactic acid bacteria fermentation. Rice, mucopolysaccharide (eg, hyaluronic acid and its derivatives, chondroitin and its derivatives, heparin and its derivatives), elastin and its derivatives, collagen and its derivatives, hydrolyzed silk protein, NMF related substances, lactic acid, urea, high grade Examples include fatty acid octyldodecyl, phytosterol, soybean phospholipid, cholesteryl isostearate, seaweed extract, various amino acids, and derivatives thereof.

Examples of thickeners include, for example, brown algae such as alginic acid, agar, carrageenan, fucoidan, and fartheraran, green algae or red algae-derived components, beech extract, pectin, locust bean gum, polysaccharides such as aloe polysaccharide, xanthan gum, tragacanth gum, guar gum Synthetic polymers such as gums such as carboxymethylcellulose, hydroxyethylcellulose, etc., polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, acrylic acid / methacrylic acid copolymer; hyaluronic acid and its derivatives, polyglutamic acid and its Examples thereof include sugar compounds mainly composed of derivatives, glucosyl trehalose and hydrolyzed hydrogenated starch.

Examples of the antiseptic / bactericidal agent include paraoxybenzoates such as methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate; phenoxyethanol, dichlorophene, hexachlorophene, chlorhexidine hydrochloride, benzalkonium chloride , Salicylic acid, ethanol, undecylenic acid, phenols, jarmal (imidazodenyl urea), 1,2-pentanediol, various essential oils, bark distillate, propolis extract and the like.

Examples of the powder component include sericite, titanium oxide, talc, kaolin, bentonite, zinc oxide, magnesium carbonate, magnesium oxide, zirconium oxide, barium sulfate, silicic anhydride, mica, 6- or 12-nylon powder, polyethylene powder. , Silk powder, cellulosic powder and the like.

Examples of the ultraviolet absorber include ethyl paraaminobenzoate, ethylhexyl paradimethylaminobenzoate, amyl salicylate and derivatives thereof, 2-ethylhexyl paramethoxycinnamate, octyl cinnamate, oxybenzone, 2,4-dihydroxybenzophenone, 2-hydroxy-4 -Methoxybenzophenone-5-sulfonate, 4-tertiarybutyl-4-methoxybenzoylmethane, 2- (2-hydroxy-5-methylphenyl) benzotriazole, urocanic acid, ethyl urocanate, aloe extract, etc. .

Antioxidants include, for example, superoxide dismutase, bioactive oxygen-degrading enzymes such as catalase, vitamins such as vitamin E and vitamin D and derivatives thereof, butylhydroxyanisole, butylhydroxytoluene, propyl gallate, There are ubidecaquinone (ubiquinone), rutin, rutin glucoside, γ-oryzanol and the like.

Furthermore, if necessary, other active ingredients (whitening agent, skin aging prevention / rough skin improvement agent, anti-inflammatory agent, etc.) may be blended within the range that does not impair the effects and features of the fermented product used in the present invention. Examples of such a whitening agent include tranexamic acid and its derivatives, t-cycloamino acid derivatives, kojic acid and its derivatives, ascorbic acid and its derivatives, hydroquinone derivatives, ellagic acid and its derivatives, resorcinol derivatives, placental extraction , Cysteine, Sakuhakuhi extract, Yukinoshita extract, Hamamelis extract, Itadori extract, Licorice extract, Gentian extract, Jujube extract, Peonies extract, Sugi extract, Peach extract, Green algae, Red algae or Brown algae Seaweed extracts, linoleic acid and its derivatives or processed products (eg menthol linoleate) Ester, etc.), 2,5-dihydroxybenzoic acid derivatives, etc., are components for preventing skin aging and improving rough skin, collagen derived from animals or fish and derivatives thereof, elastin and derivatives thereof, intercellular lipids such as ceramide, placenta extraction Nicotinic acid and derivatives thereof, glycyrrhizic acid and derivatives thereof (dipotassium salt, etc.), t-cycloamino acid derivatives, vitamin A precursor, vitamin A and derivatives thereof, vitamin E and derivatives thereof, allantoin, α-hydroxy acids, diisopropyl Amine dichloroacetate, γ-amino-β-hydroxybutyric acid, coenzyme Q-10, adenosine, α-lipoic acid, picoline, carnitine and its derivatives, gentian extract, licorice extract, pearl barley extract, chamomile extract, carrot extract, aloe extract, etc. Herbal medicine extract , Rice fermented extract, green algae, red algae or brown algae seaweed extract, Sakuhakuhi extract, beech extract, beetle aloe extract, mannenrou extract, ginkgo biloba extract, horsetail extract, safflower extract, ginseng extract, If it is an anti-inflammatory component such as elderberry extract, garlic extract, lotus extract, mango extract, eggshell membrane extract protein, deoxyribonucleic acid potassium salt, purple orchid root extract, purple extract, rice extract, etc. For example, azulene derivatives such as sodium guaiazulene sulfonate and ethyl guaiazulene sulfonate, glycyrrhizic acid derivatives such as dipotassium glycyrrhizinate and stearyl glycyrrhetinate, allantoin, licorice extract, cucumber extract, peonies extract, button pie extract, forsythia extract Ryuutan extract, tow calendula extract, parsley extract, Hypericum extract, and the like.

Hereinafter, the Example of the effect test of the yeast cell extract in this invention is shown. Furthermore, although the application prescription example etc. to the composition using the yeast microbial cell extract are described, it is not limited to the Example described here. In the following, all parts are parts by mass, and all% are% by mass.

The pharmaceutical preparation of the present invention is particularly useful as a therapeutic and prophylactic agent for gastric ulcer and gastric mucosal tissue injury, comprising as active ingredients a yeast cell containing a high amount of zinc compound and an extract thereof. In addition, when used as a cosmetic preparation, epidermal cell damage due to ultraviolet rays and oxidative stress from the environment (decreased production of skin moisturizing ingredients, abnormal keratinization due to abnormal differentiation of epidermal cells, associated epidermal barrier failure, etc.) It is possible that the heat shock protein 70 production inducer can repair and prevent skin roughness due to an increase in the amount of transpiration of the skin and inflammation of the epidermal tissue.

[Experiment 1]
Saccharomyces cerevisiae Yeast Collection Method A commercially available baker's yeast (Nisshin Supercamellia Dry Yeast: Nisshin Foods Co., Ltd.) was purchased and added to an Erlenmeyer flask containing 100 ml of YM medium sterilized at 120 ° C. for 15 minutes. 1 g of the suspension was suspended, and rotational culture was performed at 28 ° C. and 150 rpm for 3 days. One platinum loop was taken from the suspension and stretched on a YM agar medium to obtain a single colony, which was used in the following experiment.
Hereinafter, “g / L” or the like in the medium composition means that each g was weighed and made 1 L with purified water.
<YM medium>
Peptone 5g / L, yeast extract 3g / L, malt extract 3g / L, glucose 10g / L
<YM agar>
Prepared by adding 20 g / L agar to YM medium and dissolving with heating.

[Experiment 2]
Yeast (Yeast Nitrogen Base) with Ammonium Sulfate synthesis medium was added 2.0% glucose to YNB (Yeast Nitrogen Sulfate) synthetic medium (hereinafter referred to as YNB-SG medium in this specification). Called).
50 ml of YNB-SG medium was added to the Erlenmeyer flask and sterilized by autoclaving at 120 ° C. for 15 minutes. After cooling this medium, an aqueous solution of 1 M zinc sulfate heptahydrate, which had been sterilized separately, was added to this medium to a concentration of 10 mM (referred to as 10 mM zinc sulfate-containing YNB-SG medium). One platinum ear of the yeast obtained in Experiment 1 was planted on this 10 mM zinc sulfate-containing YNB-SG medium and cultured at 24 ° C. and 150 rpm for 14 days. For the first 3-4 days, yeast growth is hardly observed, but growth can be gradually confirmed. 500 μl of this culture is added to 50 ml of fresh 10 mM zinc sulfate-containing YNB-SG medium, and the culture is further continued. By repeating this for about 5 passages, an acclimatized strain can be obtained. Using this acclimatized strain, 15mM zinc sulfate-containing YNB-SG medium, 20mM, 30mM, 40mM, 50mM, 60mM, and YNB-SG medium with increased zinc sulfate concentration were transferred to the culture and cultured. A zinc-resistant yeast strain acclimatized to the containing YNB-SG medium was obtained. Hereinafter, it is referred to as a 20 mM zinc-resistant yeast strain according to the amount of zinc added in the final culture state. Specifically, a strain obtained by gradually increasing the zinc concentration in the medium to be acclimatized and collecting the final culture in a medium supplemented with 60 mM zinc is referred to as a 60 mM zinc-resistant yeast strain.
In addition, in this application, although the YNB-SG culture medium was used as a basic culture medium, if a yeast can be made to contain zinc, there will exist a desired effect, Therefore Other well-known culture media may be used as a basic culture medium.
<YNB-SG medium composition>
Ammonium sulfate 5,000, potassium dihydrogen phosphate 1,000, magnesium sulfate 500, sodium chloride 100, calcium chloride 100, biotin 0.002, calcium pantothenate 0.4, folic acid 0.002, inositol 2.0, niacin 0 .4, 0.2 paraaminobenzoic acid, pyridoxine hydrochloride 0.4, riboflavin 0.2, thiamine hydrochloride 0.4, boric acid 0.5, copper sulfate 0.04, potassium iodide 0.1, ferric chloride 0.2, manganese sulfate 0.4, sodium molybdate 0.2, zinc sulfate 0.4, glucose 20,000 (unit is mg / L).

[Table 1] shows the growth of yeast when cultured in a YNB-SG medium supplemented with various concentrations of zinc sulfate.
In the initial 3 days of culture, almost no growth of yeast was observed in the YNB-SG medium supplemented with 10 mM zinc sulfate.
However, by repeating the subculture by the method described in [Experiment 2], as shown in [Table 2], high growth was also achieved in the YNB-SG medium supplemented with 10 mM zinc sulfate.

[Example 1]
Preparation of yeast cell extract 1
Prepared 800 ml of 30 mM zinc sulfate-containing YNB-SG medium in a 1 L culture bottle, added 8 ml of a 60 mM zinc-resistant yeast strain dispersion as an inoculum, and cultured at 24 ° C. with an aeration rate of 1 L / min. For 5 days. . After culturing, centrifugation was performed at 3500 rpm for 10 minutes, and 50 g of purified water was added to 10 g of the obtained yeast cells (wet weight) to disperse the cells. This suspension was allowed to stand at 50 ° C. for 24 hours and self-digested. This suspension was centrifuged at 3500 rpm for 10 minutes, and the supernatant was taken to obtain a yeast cell extract. The solid content of this extract was 2.0 w / v%.

[Example 2]
Preparation of yeast cell extract 2
Prepared 800ml of 30mM zinc sulfate-containing YNB-SG medium in a 1L culture bottle, added 8ml of 50mM zinc-resistant yeast strain dispersion as inoculum, and cultured for 5 days at 24 ° C and aeration of 1L / min. . After culturing, centrifugation is performed at 3500 rpm for 10 minutes, and 50 g of purified water is added to 10 g of the obtained yeast cells (wet weight) to disperse the cells. This suspension is adjusted to PH 2.0 with 2N-hydrochloric acid solution and allowed to stand at 60 ° C. for 24 hours for acid hydrolysis. This suspension was centrifuged at 3500 rpm for 10 minutes, and the supernatant was taken to obtain a yeast cell extract. The solid content of this extract was 2.5 w / v%.

[Example 3]
Preparation of yeast cell extract 3
Prepare 800 ml of 30 mM zinc sulfate-containing YNB-SG medium in a 1-liter culture bottle, add 8 ml of a 40 mM zinc-resistant yeast strain dispersion as the inoculum, and incubate for 5 days at 24 ° C. and aeration of 1 L / min. . After culturing, after culturing, centrifugation is performed at 3500 rpm for 10 minutes, and 50 g of purified water is added to 10 g of the obtained yeast cells (wet weight) to disperse the cells. This suspension was adjusted to pH 7.0 with a 2N sodium hydroxide solution, then 1.2% (per dry cell mass) of protease YL-NL “Amano” (Amano Enzyme Co., Ltd.) and peptidase Proteax (Amano Enzyme Co., Ltd.) 2% (per dry cell mass) is added and left at 50 ° C. for 24 hours to perform enzymatic hydrolysis. This suspension was centrifuged at 3500 rpm for 10 minutes, and the supernatant was taken to obtain a yeast cell extract. The solid content of this extract was 3.5 w / v%.

[Example 4]
Preparation of yeast cell extract 4
Prepare a 1 L culture bottle with 800 ml of 40 mM zinc sulfate-containing YNB-SG medium, add 8 ml of a 50 mM zinc-resistant yeast strain as an inoculum, and incubate for 5 days at 24 ° C. and aeration of 1 L / min. . After culturing, centrifugation is performed at 3500 rpm for 10 minutes, and 50 g of purified water is added to 10 g of the obtained yeast cells (wet weight) to disperse the cells. This suspension is adjusted to PH 2.0 with 2N-hydrochloric acid solution and allowed to stand at 60 ° C. for 24 hours for acid hydrolysis. This suspension was centrifuged at 3500 rpm for 10 minutes, and the supernatant was taken to obtain a yeast cell extract. The solid content of this extract was 2.2 w / v%.

[Example 5]
<YNB-SBS medium composition>
YNB (Yeast Nitrogen Base) with Ammonium Sulfate A medium in which brown sugar is added to a synthetic medium is referred to as a YNB-SBS medium. The composition is as follows.
Ammonium sulfate 5,000, potassium dihydrogen phosphate 1,000, magnesium sulfate 500, sodium chloride 100, calcium chloride 100, biotin 0.002, calcium pantothenate 0.4, folic acid 0.002, inositol 2.0, niacin 0 4. Paraaminobenzoic acid 0.2, pyridoxine hydrochloride 0.4, riboflavin 0.2, thiamine hydrochloride 0.4, boric acid 0.5, brown sugar 20,000 (unit is mg / L).
Preparation of yeast cell extract 4
A 60 mM zinc-resistant yeast strain was used as an inoculum and cultured in a 30 mM zinc sulfate-containing YNB-SBS medium. Prepared 800 ml of 30 mM zinc sulfate-containing YNB-SBS medium in a 1 L culture bottle, added 8 ml of a 60 mM zinc-resistant yeast strain dispersion as an inoculum, and cultured at 24 ° C. with an aeration rate of 1 L / min. For 5 days. . After culturing, centrifugation was performed at 3500 rpm for 10 minutes, and 50 g of purified water was added to 10 g of the obtained yeast cells (wet weight) to disperse the cells. This suspension was allowed to stand at 50 ° C. for 24 hours and self-digested. This suspension was centrifuged at 3500 rpm for 10 minutes, and the supernatant was taken to obtain a yeast cell extract. The solid content of this extract was 2.3 w / v%.

[Experiment 3]
Determination of zinc sulfate
To 10 ml of the yeast cell extract prepared in [Example 1], 100 ml of distilled water and 2 ml of an ammonia / ammonium chloride buffer solution of PH 10.7 are added. To this, 50 mg of Eriochrome Black T / Na chloride was added as an indicator, and titrated with 0.01 mol / L edetic acid di-Na solution.
The zinc sulfate concentration in the yeast cell extract measured by titration is shown in [Table 3].
In the yeast cell extract obtained by adding 30 mM zinc sulfate to the YNB-SG medium and cultivated, 3.5 mM zinc sulfate is added in the yeast cell extract cultivated by adding 20 mM to the YNB-SG medium. Was confirmed to contain 2.8 mM zinc sulfate.

[Example 6]
Cytotoxicity of yeast cell extracts on epithelial cells
A) Cell culture
B) Cells: NHEK-Neo-Epidermal Kera (CAMBREX)
C) Medium: EpiLife KG2 (Ca concentration: 0.06 mM)
D) Added sample: zinc sulfate heptahydrate (Wako Pure Chemical Industries), yeast cell extract epithelial cells described in [Example 1] include NHEK-Neo-Epidermal Kera (normal human epidermal cells) CAMBREX) and cultured in EpiLife KG2 (Kurabo) medium. Cells were planted in a 24-well plate to about 50% confluence. The sample was added the next day, and after 48 hours, the cells were collected and the number of cells was counted.

As shown in [Table 4], when the reagent zinc sulfate was added to the cells, the number of cells was 51% at the concentration of 200 μM, 74%, and 250 μM. However, when the yeast cell extract was added, even at a zinc sulfate concentration of 250 μM in the medium, it was 97%, and no cytotoxicity was applied. As is clear from this result, when zinc sulfate is added directly to the cells, the cytotoxicity becomes stronger, but by incorporating zinc sulfate into the yeast cells, the toxicity to epithelial cells is remarkably reduced. Became clear.

[Example 7]
Confirmation of induction of heat shock protein 70 production Cell culture Normal human epidermis cells, NHEK-Neo-Epidermal Kera (CAMBREX), Epilife KG2 (Ca concentration: 0.06 mM)
Incubate on a 50cm2 plate in medium.
2. Sample Preparation The yeast cell extract prepared in [Example 1] was added to the culture medium so as to have respective concentrations to human normal epidermal cells that had become confluent. After the addition, the mixture was incubated at 37 ° C. for 4 hours, and then the cells were collected and the cell protein was extracted with RIPA buffer.
RIPA buffer: Tris-hydrochloric acid buffer (PH 8.0) 50 mM, sodium chloride 150 mM, EDTA (PH 8.0) 1 mM, Triton X-100 1%, SDS 0.1%, sodium deoxycholate 0.1%
3. Western blotting A 12% acrylamide gel was prepared, and 30 μg of the cell protein extracted above was added to the gel Lane for electrophoresis. After transfer to PVDF membrane, primary antibody Hsp70 (Mouse monoclonal: Stress gene), secondary antibody ECL ^TM Anti-Mouse IgG, Horseradish
Western blotting was performed using Peroxidase linked whole antibody (from sheep) NA931 (Amersham Bioscience UK Limited) to confirm heat shock protein 70 with a molecular weight of 70 KDa.
4). Confirmation of heat shock protein 70 protein amount The band intensity obtained by Western blotting is shown in FIG. In cells added with the reagent zinc sulfate heptahydrate and the yeast cell extract obtained in [Example 1] obtained in Example 1, production was induced when the concentration of zinc sulfate added to the medium was 100 μM. However, when the concentration of zinc sulfate added to the medium was 200 μM, strong induction of heat shock protein 70 could be confirmed.

[Example 8]
Confirmation of UV damage inhibition effect Cell Culture Normal human fibroblasts NB1-RGB are cultured in a 24-well plate in 10% FBS-containing D-MEM medium.
2. Preparation of sample The yeast cell extract prepared in [Example 1] was added to cells that became confluent about 50% so as to have respective concentrations. After the addition, the mixture was incubated at 37 ° C. for 4 hours, and then the medium was discarded and irradiated with ultraviolet UV-B at 20 mJ / cm 2. After irradiation, serum-free D-MEM was added and cultured at 37 ° C for 24 hours. The number of cells was determined by CELL Counting Kit (Dojindo Laboratories)
Measured by the absorbance value at 415 nm.
3. UV damage control effect
As shown in [Table 5], as the zinc sulfate concentration in the yeast extract increased to 0 μM, 100 μM, 150 μM, and 200 μM, the number of cells became 0.706, 0.769, 0.775, and 0.828. It has been clarified that ultraviolet damage can be suppressed by adding a yeast extract to cells in advance before performing ultraviolet irradiation. In addition, when the number of cells with a zinc sulfate concentration of 0 μM and 200 μM was statistically processed, it was found that there was a significant difference at a risk rate of 5%.

In the above effect confirmation test, only the results using the yeast cell extract prepared in Example 1 were shown, but the same effect was obtained even when the yeast cell extract prepared in Examples 1 to 5 was used. confirmed.

Next, although the formulation example of the cosmetic formulation and / or pharmaceutical formulation which mix | blended the yeast cell culture of this invention is shown, this invention is not limited to this.

Formulation example of cosmetic preparation The formulation of the yeast cell extract shown below is the amount as the extract. The amount as evaporation residue is also shown in parentheses.
(Formulation Example 1) Cosmetic cream (mass%)
a) Beeswax 2.0
b) Stearyl alcohol ... 5.0
c) Stearic acid ... 8.0
d) Squalane ... 10.0
e) Self-emulsifying glyceryl monostearate 3.0
f) Polyoxyethylene cetyl ether (20E.O.) ... 1.0
g) Yeast cell extract prepared in [Example 1] 10.0 (0.2)
h) 1,3-butylene glycol ... 5.0
i) Potassium hydroxide ... 0.3
j) Preservative / Antioxidant: appropriate amount k) Purified water: Remaining preparation methods a) to f) are heated and dissolved and kept at 80 ° C. Heat up to h) to k), keep at 80 ° C., add to a) to f), emulsify, and cool to 40 ° C. with stirring. Then g) is added and stirred to dissolve uniformly.

(Prescription Example 2) Emulsion (mass%)
a) Beeswax 0.5
b) Petrolatum 2.0
c) Squalane ... 8.0
d) Sorbitan sesquioleate ... 0.8
e) Polyoxyethylene oleyl ether (20EO) 1.2
f) Yeast cell extract prepared in [Example 1] 0.1 (0.002)
g) 1,3-butylene glycol 7.0
h) Carboxyvinyl polymer 0.2
i) Potassium hydroxide ... 0.1
j) Purified water: balance k) antiseptic / antioxidant ... appropriate amount l) ethanol ... 7.0
The production methods a) to e) are dissolved by heating and kept at 80 ° C. G) to k) are heated and dissolved, kept at 80 ° C., added to a) to e), emulsified, and cooled to 50 ° C. with stirring. Add f), l) at 50 ° C., stir to 40 ° C. and cool.

(Formulation Example 3) Lotion (mass%)
a) Yeast cell extract prepared in [Example 2] ... 5.0 (0.125)
b) Glycerin ... 5.0
c) Polyoxyethylene sorbitan monolaurate (20EO) ... 1.0
d) Ethanol ... 6.0
e) Perfume ... appropriate amount f) antiseptic / antioxidant ... appropriate amount g) purified water ... the remaining preparation methods a) to g) are mixed and dissolved uniformly.

(Formulation example 4) Face wash (mass%)
a) Stearic acid ... 12.0
b) Myristic acid 14.0
c) Lauric acid ... 5.0
d) Jojoba oil ... 3.0
e) Glycerin 10.0
f) Sorbitol ... 15.0
g) 1,3-butylene glycol ... 10.0
h) POE (20) Glycerol monostearic acid 2.0
i) Potassium hydroxide ... 5.0
j) Water ... balance k) chelating agent ... appropriate amount l) perfume ... appropriate amount m) yeast cell extract prepared in [Example 1] ... 1.0 (0.02)
The manufacturing methods a) to h) are dissolved by heating and maintained at 70 ° C. After i) is dissolved in j), it is added to a) to h) and saponified. Then, k) and l) are added and cooled with stirring. Add m) at 50 ° C., stir to 40 ° C. and cool.

(Formulation Example 5) Lotion (mass%)
a) Yeast cell extract prepared in [Example 3] ... 5.0 (0.175)
b) Glycerin ... 5.0
c) Polyoxyethylene sorbitan monolaurate (20EO) ... 1.0
d) Ethanol ... 6.0
e) Perfume ... appropriate amount f) antiseptic / antioxidant ... appropriate amount g) purified water ... the remaining preparation methods a) to g) are mixed and dissolved uniformly.

Next, the example of a formula which freeze-dried the obtained yeast cell body and utilized as it is as a tablet is shown.
(Prescription Example 5) Tablet (mass%)
1. Dry yeast cells obtained by culturing in YNB-SG medium containing 30 mM zinc sulfate 30.0
2. Lactose ... 30.0
3. Corn starch 32.0
4). Hydroxypropyl cellulose ... 6.0
5. Magnesium stearate ... 2.0

As described above in detail, according to the present invention, it is possible to obtain a heat shock protein 70 production inducer with reduced cytotoxicity. A drug containing this production inducer is a heat shock protein production inducer with low toxicity to gastric mucosal epithelial cells and skin epithelial cells, and can be used as a composition effective for repairing and preventing gastric ulcer and gastric mucosa.
Furthermore, the cosmetic composition protects against epidermal cell damage due to stress caused by ultraviolet rays or the like, and as a result, the skin can be protected from inflammation.

Claims (4)

A heat shock protein 70 production inducer comprising a yeast cell extract cultured in a medium containing 20 mM or more of a zinc compound as an active ingredient. A UV injury inhibitor comprising as an active ingredient a yeast cell extract cultured in a medium containing 20 mM or more of a zinc compound. The heat shock protein 70 production inducer according to claim 1, wherein the yeast is a zinc-adapted bacterium previously cultured in a medium containing 10 to 60 mM of a zinc compound. The ultraviolet damage inhibitor according to claim 2, wherein the yeast is a zinc-adapted bacterium previously cultured in a medium containing 10 to 60 mM of a zinc compound.