KR20050111667A - Soybean extract of enhancing biosynthesis of hyaluronic acid and glycosaminoglycan - Google Patents

Abstract

The present invention includes arthritis, discs comprising soy extracts for promoting the synthesis of hyaluronic acid, glycosaminoglycan, and collagen in collagen and human cells, animals, and the human body. (Intervertebral disc protrusion), skin aging and wrinkles, dry eye, wound healing, scar removal, vaginal dryness, gum disease, atopic dermatitis, dry skin prevention and treatment composition.

Description

Soybean Extract of Enhancing Biosynthesis of Hyaluronic Acid and Glycosaminoglycan (Soybean Extract of Enhancing Biosynthesis of Hyaluronic Acid and Glycosaminoglycan)

The present invention is to prevent and treat arthritis, disc (intervertebral disc prolapse), skin aging and wrinkles, dry eye, wound healing, scar removal, vaginal dryness, gum disease, atopic dermatitis and dry skin containing soy extract as an active ingredient More specifically, arthritis, discs (intervertebral discs), skin aging and soybeans containing soy extract as an active ingredient exhibiting excellent activity in promoting the biosynthesis of hyaluronic acid and glycosaminoglycans in animals and human cells, animals and the human body. Wrinkles, dry eye, wound healing, scar removal, vaginal dryness, gum disease, atopic dermatitis and dry skin preventive and therapeutic composition.

Here, glycosaminoglycan refers to polysaccharides containing amino sugars, including hyaluronic acid, chondroitin sulfate, keratan sulfate, heparin, heparan sulfate, and dermatan. Sulfuric acid (dermatan sulfate) and the like.

Hyaluronic acid and glycosaminoglycans are biopolymer polysaccharides present in all living organisms, and the human body is distributed in 56% of skin, 35% of muscle tissue, and 9% of joint synovial fluid and spinal fluid. As a substance, 1 molecule of hyaluronic acid polysaccharide combines with 218 water molecules to have both physical viscosity and elasticity, and performs various functions such as shock absorption and lubrication in muscles and joints of the skin. In addition, hyaluronic acid is an important body polysaccharide that binds to receptors of various cells such as CD44, RHAMM, and regulates life phenomena through cell differentiation, proliferation and cell signaling.

In our bodies, hyaluronic acid and glycosaminoglycans are broken down in the process of removing harmful oxygen caused by ultraviolet rays or inflammation every day, and more than 3g per day is maintained by a fast metabolism process, which is maintained again, but with age Gradually, the amount produced decreases. As the amount produced is less than the amount to be degraded, the skin loses its elasticity, wrinkles begin to form, and in the joints, shock absorption and lubrication are not performed properly, and the causes of degenerative diseases that gradually destroy cartilage and wear are continuously worsened. A vicious cycle occurs.

Until now, the development of medicines using hyaluronic acid and glycosaminoglycan has been mainly used as a surgical aid for maintaining eye shape during cataract surgery, anti-adhesion after surgery, anti-wrinkle injection, wound healing agent, sustained-release agent, and cell therapy, dry eye syndrome. As a skin moisturizer for therapeutic eye drops and tissue engineering (Scafford) and cosmetics, various products are safely used by utilizing the physicochemical properties of hyaluronic acid and glycosaminoglycan.

The present inventors have completed the present invention for the first time that soy extract has the effect of promoting hyaluronic acid and glycosaminoglycan biosynthesis in chondrocyte cells, fibroblast cells and the like.

Soybeans and soybean embryos contain functional bioactive substances, Phenolic Compounds (Isoflavone, phenolic acids), Phytic Acid, Protease Inhibitors (Pls), Lignans, Saponine, Dietary Fiber, Soy Oligosaccharide, Soy Protein, Soy Peptide, Lechitin, chiro-inositol, It is rich in vitamins and minerals.

In soybeans and soybean embryos, at least 14 phytochemicals are involved in cancer prevention, as well as anti-aging, antioxidant, osteoporosis prevention, blood pressure strengthening, antithrombosis, immune enhancement, liver function enhancement, constipation relief, anti-allergy, anti-obesity, diuretic effect, dementia prevention It is known that anti-cholesterolemia, hyperlipidemia prevention, arteriosclerosis suppression, anti-cardiovascular disease, etc. are generally effective in preventing adult diseases.

The present inventors completed the present invention by first confirming that soy extract has an effect of promoting biosynthesis of hyaluronic acid in fibroblasts and the like.

The present inventors completed the present invention by first confirming that soy extract has an effect of promoting biosynthesis of hyaluronic acid and glycosaminoglycan in fibroblasts and chondrocytes. Therefore, an object of the present invention is to arthritis, disc (intervertebral disc protrusion), skin aging and wrinkles, dry eye, scar healing, scars using the material containing the active ingredient of soy extract to induce the promotion of hyaluronic acid synthesis in animals and human cells and human body Removal, vaginal dryness, gum disease, atopic dermatitis and dry skin prophylactic and therapeutic compositions

The present invention is a soybean extract, soybean extract, soybean germ extract, soybean germ extract containing at least one compound selected from the group consisting of arthritis, disc (intervertebral disc protrusion), skin aging and wrinkles, dry eye, wound healing, scar removal, vaginal Prophylactic and therapeutic compositions for dryness, gum disease, atopic dermatitis and dry skin are provided.

In order to achieve the above object, in the present invention, in the preparation method of the extract using soybean and soybean germ, soybean and soybean germ extract of hot and cold water extract, soybean and soybean germ extract comprising a separation, precipitation, filtration process It provides a manufacturing method and soybean and soybean germ extract produced by the method.

Soybean and soybean germ extracts used in the present invention can be prepared by conventional methods.

Soybeans that can be used in the present invention include peanut, jack bean, soybean, sweet pea, lentil, lima bean, kidney bean, pea, winged bean, fava bean and their embryos and soybean meal or skim soybean which are a by-product of soybean oil production. Can be used as a raw material. These raw materials are dried, ground and powdered, and then hot water or cold soaked with about 10 times the amount of the extraction solvent, followed by centrifugation of the extracted extract solution for 1 to 10 hours to remove insoluble residues. As a coagulant, calcium sulfate, magnesium sulfate, chitosan, and glue are used. Conodelta-lactone, hydrochloric acid sulfuric acid, acetic acid, citric acid, lactic acid, malic acid, tartaric acid, phosphoric acid selected by one of the coagulation of the protein was separated and filtered and the remaining filtrate was prepared by concentrated drying.

As a result of analyzing the powder, it was composed of water-soluble protein, soy oligosaccharide (rapinose, starchiose, sugar), phytic acid, inositol, finitol, saponin, water soluble fiber, isoflavone, calcium, magnesium and the like.

The composition of the soybean and soybean germ extract, which is a hyaluronic acid and glycosaminoglycan biosynthesis accelerator, may be used as a medicine, drug additive, cosmetic or cosmetic additive, health functional food, food, food additive, beverage, or beverage additive, It can be formulated in the form of capsules, tablets, granules, powders, solutions, beverages, porridges and the like.

When the composition of the hyaluronic acid and glycosaminoglycan biosynthesis accelerator of the present invention is used as a medicine or the like, the composition may be in the form of powders, granules, tablets, capsules, pills, troches, solvents, suspensions, emulsions, and syrups. It can be used as an oral agent such as and can be used alone or in combination with each depending on the symptoms. These various preparations can be formulated using conventionally known carriers commonly used in the field of pharmaceutical preparations such as excipients, binders, preservatives, oxidative stabilizers, disintegrants, lubricants, and copulation agents, according to the purpose, in a commercial manner. have.

Usable carriers can be used without particular limitation according to the dosage form, and preferred carriers are solid carriers such as starch, lactose, mannitol, carboxymethylcellulose, corn starch, and inorganic salts, distilled water, saline solution, and aqueous glucose solution. And alcohol carriers such as ethanol, propylene glycol, and polyethylene glycol, and oily carriers such as various animal and vegetable oils, white petrolatum, paraffin and wax.

Dosage units may contain, for example, one, two, three or four times the individual dosage, or they may contain 1/2, 1/3 or 1/4 times. The individual dosage preferably contains an amount in which the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose. The effective dose of soy extract is concentration dependent but preferably 10 mg to 1 g / kg, more preferably 40 to 200 mg / kg, and may be administered 1-6 times a day.

In addition, the present invention is a health food composition for treating arthritis, disc (intervertebral disc prolapse), skin aging and wrinkles, dry eye, wound healing, scar removal, vaginal dryness, gum disease atopic dermatitis and dry skin containing soy extract as an active ingredient To provide. Health food in the present specification is a food that improves the function of the general food by adding soy extract to the general food, soy extract can be added to the general food, or can be prepared by encapsulation, powdered, suspensions and the like. When ingested, it brings a specific effect on health, and unlike the general medicine because it is made of food as a raw material has the advantage that there is no side effect that can occur when taking long-term use of the drug.

When the soybean extract of the present invention is used as a food additive, the soybean extract may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient can be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, soy extract may be added in an amount of 0.01 to 10% by weight, preferably 0.1 to 5% by weight based on the raw materials in the manufacture of food or beverage. However, in the case of prolonged ingestion for health and hygiene purposes or for health control purposes, the amount can be adjusted below the above range. In addition, when the health food of the present invention is used as the pharmaceutical composition, it is preferable to contain soy extract within the measured toxicity range.

There is no particular limitation on the kind of food. Examples of foods to which the soy extract may be added include meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, teas, drinks Alcoholic beverages and vitamin complexes. Specifically, the health food containing soy extract includes health foods and favorite products such as juice, tea, jelly, juice, etc., which are made with soy extract as a main ingredient.

Hereinafter, examples are provided to help understand the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1 Preparation of Soy Extract

After adding about 10 times the amount of purified water to raw material soybean powder and extracting it for 3 hours at 100 ° C, it was separated by passing through 100 mesh filter cloth and 5 micron filter to remove the solid residue. To allow protein to coagulate. The coagulated protein is separated using a filter cloth, and the filtrate is concentrated and dried. Drying can be used by vacuum drying, hot air drying, freeze pressure drying, spray drying.

Example 2 Recovery of Soy Water Soluble Protein

Approximately 10 times the amount of purified water compared to soybean powder, which is a raw material, was extracted for 3 hours at 100 ° C, and then separated through a 100-mesh filter cloth and a 5 micron filter to remove the solid residue, and then a calcium sulfate coagulant To allow protein to coagulate. The coagulated protein is separated using a filter cloth, microfiltration to 1um and concentrated under reduced pressure at 60 degrees. Dilute the concentrate with distilled water to 10brix, and add hydrochloric or organic acids to pH 4. Alternatively, three times the volume of ethanol in the concentrate to precipitate the protein and polymer. In order to recover the precipitated solids, the solids and the supernatant were separated by centrifugation at 3,000 rpm for 10 minutes. The recovered solid was washed 1-2 times with 95% ethanol and dried in vacuo.

Example 3 Recovery of Soy Embryonic Water Soluble Protein

About 10 times the amount of purified soybean germ powder, which is a raw material, was extracted at 100 ° C for 3 hours, and then separated through a 100 mesh filter cloth and a 5 micron filter to remove the solid residue, and then 1% by weight of calcium sulfate coagulant. To allow the protein to coagulate. The coagulated protein is separated using a filter cloth, microfiltered to 1 ~ m and concentrated under reduced pressure at 60 ° C. Dilute the concentrate with distilled water to 10brix, and add hydrochloric or organic acids to pH 4. Alternatively, ethanol is added to the concentrate at a volume of 3 times to precipitate proteins and polymers. In order to recover the precipitated solids, the solids and the supernatant were separated by centrifugation at 3,000 rpm for 10 minutes. The recovered solid was washed 1-2 times with 95% ethanol and dried in vacuo.

Example 4 Proliferation Effect of Soybean Extract in Chondrocytes

The cells were transferred to 3x10 ³ cells / well in a 96-well plate, attached to cells, replaced with 1% FBS-DMEM medium (minimizing the effects on FBS), and soybean extracts were incubated for 48 hours. Cell proliferation of soy extracts was compared with the control group using the Cell Proliferation Kit (XTT, Roche). The simple method of using KIT is to mix XTT labeling reagent and Electron coupling reagent in the same well, and then incubate for 5 hours at 5% CO ₂ , 37 ^o C and read with ELISA reader at 490nm wavelength. See FIG. 1 for the results.

Example 5 Synthesis of Hyaluronic Acid from Chondrocytes of Soybean Extracts

 1. Chondrocyte Separation

The cartilage tissue was isolated from the rabbits at 3 to 4 weeks of age and then treated with 0.2% collagenase type II at 37 ° C. for 4 hours to obtain cartilage cells by filtering with a 100? M filter. Cells were replaced every 2 days with chondrocytes (DMEM / F12, 10% FBS, Insulin, Nonessential amino acid, Ascorbic acid, penicillin, Gibco BRL products) at 37 ° C and 5% CO ₂ .

2. Make bead

Chondrocytes were mixed with 1.5% alginate solution to 2X10 ⁶ cell / ml, and then dropped into each drop of 102mM CaCl ₂ solution (pH7.4) using a syringe to make chondrocyte-bead, and finally washed with 0.15M NaCl. . Beads were changed every two days before the experiment by adding cartilage culture medium (DMEM / F12, Nonessential amino acid, Ascorbic acid, Penicillin, Gibco BRL products) and soy extract at 37 ℃, 5% CO ₂ .

3. Content of Hyaluronic Acid in Bead

3-1. Bead acquisition

Beads were cultured for 2 weeks by adding chondrocyte culture medium and soybean extract, and then washed with PBS. The beads were collected and treated in 55mM sodium citrate, 30mM EDTA, 150mM sodium chloride (pH6.8) solution at 4 ℃ for 10 ~ 15min and then Papain 20g / ml in 0.1M sodium acetate, 50mM sodium EDTA, 5mM L- Cyctein hydrochloride (pH5.53) was reacted overnight at 56-60 ° C. For enzyme inhibition, the sample obtained after heating for 10 minutes was stored at -20 ℃ until the assay.

3-2. HA assay

One day prior to assay, HA-binding protein (HABP) was coated in 96 well plates at 4 ° C overnight and blocked with 1% BSA. After binding the samples for 60 minutes at 37 ℃ washed with 0.5% PBST. After biotin-HABP was bound for 60 minutes at RT, extra-avidin alkaline phosphatase was added and then colored with p-nitrophenyl-phosphate (PNPP). The reaction is inibitioned with 3N-NaOH and the absorbance is read at 405 nm. See FIG. 2 for the results.

Example 6 GAG Synthesis in Chondrocytes from Soybean Extract

The absorbance is read at 540nm wavelength by mixing the sample titration amount with 96well and DMB (dimethylmethylene blue) dye (in formate solution). See FIG. 3 for the results.

Example 7: Wrinkle improvement effect (fibroblast proliferation assay)

Four-week-old rabbit fibroblasts (Rabbit primary fibroblasts) were incubated in 10% FBS-DMEM (Dulbecco's Modified Eagle's Medium) at 2 × 10 ³ cells / well in 96 well plates. The soy extract was replaced with 1% FBS-DMEM and soy extract was incubated at 37 ° C. for 3 days. After 3 days of culture, XTT labeling reagent {sodium 3 '-[1- (Phenylaminocarbonyl) -3,4-tetrzolium] -bis [4-methoxy-6-nitro] benzene sulfonic acid hydrate} and Electron-coupling reagent [PMS (N -methyl dibenzopyrazine methyl sulfate)] was added at 50? l / well and incubated for at least 4 hours. The XTT reagent forms a water-soluble fromazan by living cells, and the formed Formazan can be measured at 490 nm using an ELISA leader to confirm the degree of cell proliferation in a relative ratio to the control group. See FIG. 4 for the results.

Example 8 Content of Hyaluronic Acid in Fibroblasts

Rabbit fibroblasts were cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium containing 10% FBS (fetal bovine serum), and subcultured by adding 2 × 10 ⁴ cells / well to a ²⁴ well plate. The next day, soy extract was added to DMEM medium containing 5% FBS and incubated at 37 ° C. for 3 days. After 3 days of culture, the total amount of hyaluronic acid in the medium was measured by the sandwich ELIZA method of HA-binding protein (HABP) and HA-binding protein-biotin. Hyaluronic acid (SIGMA H) was used as a standard. See FIG. 5 for the results.

Example 9 Collagen Synthesis by Fibroblasts

The total collagen amount in the culture obtained by the same method as Experimental Example 8) was confirmed by the Sircol red dye bind assay method. The collagen bound with Dye was down by centrifugation, and the result was read at 540 nm by adding Alkali reagent. See FIG. 6 for the results.

By having the effect of promoting the biosynthesis of hyaluronic acid and / or glycosaminoglycans in the soybean extract according to the present invention, arthritis treatment, dry eye treatment, cosmetic aid for removing wrinkles, anti-adhesion agent after surgery, cosmetic moisturizer, artificial skin, wound It can be used for healing, scar removal, vaginal dryness and cataract surgery.

1 is a graph illustrating the proliferative effect in chondrocytes by soy extract.

2 is a graph illustrating the synthesis of hyaluronic acid in chondrocytes by soy extract.

3 is a graph illustrating GAG synthesis in chondrocytes by soy extract.

4 is a graph illustrating the anti-wrinkle effect in fibroblasts by soy extract.

5 is a graph of hyaluronic acid synthesis in fibroblasts by soy extract.

6 is a graph illustrating collagen synthesis in fibroblasts by soy extract.

Claims (16)

Hyaluronic acid, glycosaminoglycan and / or collagen biosynthesis promoting composition comprising at least one selected from the group consisting of soybean extract, soybean sprout, soybean germ extract, soybean germ pure as an active ingredient The soybean of claim 1, wherein the soybean is peanut, jack bean, soybean, sweet pea, lentil, lima bean, kidney bean. composition comprising at least one selected from the group consisting of beans, peas, winged beans, fava beans and their embryos and soybean meal or skim soybeans, which are by-products for the production of soybean oil from them. The composition according to claim 1, wherein the composition comprises arthritis, disc (intervertebral disc prolapse), dry eye, cosmetic aid for wrinkle removal, prevention of adhesion after surgery, cosmetic moisturizing, artificial skin, wound healing, scar removal, vaginal dryness A composition characterized in that it exhibits any one or more of the effects of cataract surgery, gum disease, and atopic dermatitis. According to claim 1 or 2, wherein the composition comprises a water-soluble protein, soy oligosaccharides (rapinose, starchiose, sugar), phytic acid, inositol, pinitol, saponin, water-soluble dietary fiber, isoflavones, calcium, magnesium Characteristic composition The composition of claim 1, wherein the composition comprises one or more pharmaceutically acceptable carriers or excipients. The composition according to claim 5, wherein the content of the active ingredient is administered in the range of 10 to 6,000 mg / day per 1 Kg of adult body weight. Arthritis comprising the composition of claim 5, disc (intervertebral disc prolapse), dry eye, cosmetic aid for wrinkle removal, prevention of adhesion after surgery, cosmetic moisturizing, artificial skin, wound healing, scar removal, vaginal dryness, cataract surgery aid, atopy Preparations for the Treatment of Dermatitis 8. The formulation according to claim 7, wherein the formulation comprises at least one pharmaceutically acceptable carrier or excipient. 9. The carrier according to claim 8, wherein the carrier is solid carriers such as starch, lactose, mannitol, carboxymethylcellulose, corn starch, and inorganic salts, alcohols such as distilled water, saline solution, aqueous glucose solution, ethanol, propylene glycol, and polyethylene glycol. A liquid carrier and a preparation comprising at least one carrier or excipient selected from the group consisting of various animal and vegetable oils, oily carriers such as white petrolatum, paraffin and wax. The preparation of claim 7, wherein the preparation is a powder, granule, solution, pill, troche, suspension, emulsion, syrup tablet, powder, hard or soft capsule, suspension, injectable preparation. Formulation, characterized in that the unit dosage form or several dosage forms for parenteral administration The method of claim 1, wherein the composition further comprises a pharmaceutically acceptable excipient in the form of a drug, drug additive, beverage, beverage additive or food, food additive or cosmetic, cosmetic additive. Osteoarthritis, Disc (prophylaxis), dry eye, cosmetic aid for wrinkle removal, prevention of postoperative adhesions, cosmetic moisturization, artificial skin, wound healing, scar removal, vaginal dryness, cataract surgery, atopic dermatitis improvement or treatment composition The composition of claim 11, wherein the content of the active ingredient is included in a range of 0.01 to 10% by weight. A) obtaining a crude extract by hot or cold water extraction from at least one selected from the group consisting of soybean, soybean sprout, soybean germ, soybean germ pure, b) centrifuging the crude extract to remove insoluble residue, c C) coagulating the protein by administering a coagulant, and separating and filtering the protein; and d) optionally, concentrating and drying the filtrate. The method according to claim 13, wherein the coagulant is at least one selected from calcium sulfate, magnesium sulfate, chitosan, gluconodeltalactone, hydrochloric acid, acetic acid, citric acid, lactic acid, malic acid, tartaric acid, and phosphoric acid. Arthritis, disc (vertebral disc protruding disease), dry eye, wrinkles removal molding aid, prevention of adhesion after surgery, cosmetics, characterized in that it contains soybean or soybean embryo extract prepared according to claim 13 or 14 as an active ingredient Moisturizing, artificial skin, wound healing, scar removal, vaginal dryness, cataract surgery aid, composition for the treatment of atopic dermatitis Arthritis prepared by the method according to any one of claims 13 to 14, disc (prophylaxis), dry eye, cosmetic aid for wrinkle removal, prevention of adhesion after surgery, cosmetic moisturizing, artificial skin, wound healing, scar Active fractions for removal, vaginal dryness, cataract surgery, atopic dermatitis