KR20220086870A - A composition for preventing or treating osteoporosis comprising contains the active ingredient of water extract of Auricularia auricula - Google Patents

Abstract

The present invention relates to a composition for preventing or treating osteoporosis comprising a wood ear mushroom extract as an active ingredient. According to the present invention, the wood ear mushroom extract further inhibited osteoclast differentiation and inhibited the activity of TRAP, a molecular marker of osteoclast differentiation, but did not show cytotoxicity in macrophages, precursor cells involved in osteoclast differentiation. Moreover, wood ear mushroom water extract promoted osteoblast differentiation increased by BMP-2, did not show cytotoxicity of C2C12 cells assaying osteoblast differentiation, and significantly increased the activity of ALP, a biomarker of osteoblast differentiation. was analyzed to make Therefore, the wood ear mushroom extract according to the present invention can simultaneously regulate the differentiation of osteoclasts and osteoblasts involved in osteoporosis, and thus can be usefully used as an active ingredient for the prevention or treatment of osteoporosis.

Description

A composition for preventing or treating osteoporosis comprising a wood ear mushroom extract as an active ingredient;

The present invention relates to a composition for preventing or treating osteoporosis comprising a wood ear mushroom extract as an active ingredient.

In general, osteoporosis is a metabolic bone disease in which there is no change in the chemical composition of bone, and the bone is weakened due to a decrease in bone mass and a change in microstructure, thereby increasing the risk of fracture. Bone (bone) is formed by constantly repeating the process of bone formation, which is made by osteoblasts, and the process of bone remodeling, in which aged bones are dissolved (bone resorption) by osteoclasts.

Osteoporosis occurs when the balance between bone formation and bone resorption process is disturbed. The rate of bone resorption is either too fast or too slow, so the amount of bone resorption cannot keep up with the production, resulting in a decrease in bone mass. This can easily lead to fractures.

Osteoporosis prevention and treatment agents are classified into osteoclast activity promoting agents that promote bone formation, and osteoclast inhibitory agents inhibiting osteoclasts that promote bone resorption. In particular, existing therapeutic agents mainly inhibit bone resorption by inhibiting the latter, that is, osteoclasts. However, there is an urgent need to develop a therapeutic agent that can actively promote bone formation to substantially promote bone formation effects and minimize accompanying side effects. This is because, in patients whose bone density has already been reduced below the bone threshold, bone resorption alone cannot reduce the risk of fracture, so the bone mass must be increased above the fracture threshold.

However, treatments using hormones such as fluoride, parathyroid hormone, growth factors, and anabolic steroids, which are being developed as treatments to promote bone formation, have a risk of cancer, and functional treatments developed with non-hormonal agents, such as gastrointestinal disorders There is a problem that side effects such as such occur.

Under this background, the present inventors have studied a natural material-derived material that can be easily obtained and can treat and prevent osteoporosis by promoting bone formation without side effects to the human body, and as a result, prepare and manufacture a mushroom extract from wood ear mushroom It was confirmed that the extracted wood ear mushroom extract promotes the activity of osteoblasts and inhibits the activity of osteoclasts. In addition, the present invention was completed by confirming the toxicity to progenitor cells in the cell experiment of the wood ear mushroom extract.

Republic of Korea Patent Publication No. 10-2008-0057774 Republic of Korea Patent Publication No. 10-2019-0081053 US Patent Publication No. 2001/0195926

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the treatment or prevention of osteoporosis comprising a wood ear mushroom extract as an active ingredient for the treatment and prevention of osteoporosis that promotes bone formation without side effects to the human body.

Another object of the present invention is to provide a method for preventing or treating osteoporosis comprising administering the pharmaceutical composition to a subject.

In addition, another object of the present invention is to provide a food composition for preventing or improving osteoporosis comprising a wood ear mushroom extract.

Various problems to be solved in the present invention in the above are not limited thereto, but can be clearly understood by those of ordinary skill in the art to which the present invention belongs through the examples and claims to be described later. will be.

According to the present invention to achieve the above object, there is provided a pharmaceutical composition for preventing or treating osteoporosis comprising an extract of wood ear mushroom ( Auricularia auricula ) as an active ingredient.

Here, the wood ear mushroom extract may be extracted with a solvent selected from the group consisting of water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

In addition, the wood ear mushroom extract is characterized in that it inhibits the differentiation of osteoclasts without cytotoxicity in progenitor cells.

Here, the inhibition of the differentiation of the osteoclasts is characterized by inhibiting the activity of TRAP.

In addition, the wood ear mushroom extract is characterized in that it promotes osteoblast differentiation increased by BMP-2 without cytotoxicity in progenitor cells.

Here, the promotion of osteoblast differentiation is characterized in that it promotes the activity of ALP.

In addition, the wood ear mushroom extract may be included in the total amount of 0.1 ~ 500 ㎍ / mL of the composition.

On the other hand, in an embodiment of the present invention provides a food composition for preventing or improving osteoporosis comprising an extract of wood ear mushroom ( Auricularia auricula ) as an active ingredient.

In addition, in one embodiment of the present invention, wood ear mushroom ( Auricularia auricula ) provides a health functional food composition for preventing or improving osteoporosis comprising an extract as an active ingredient.

In addition, in an embodiment of the present invention, wood ear mushroom ( Auricularia auricula ) It provides a feed composition comprising an extract.

According to the present invention as described above, the wood ear mushroom extract further inhibited osteoclast differentiation and inhibited the activity of TRAP, a molecular marker of osteoclast differentiation, but did not show cytotoxicity in macrophages, which are progenitor cells involved in osteoclast differentiation. . Moreover, wood ear mushroom water extract promoted osteoblast differentiation increased by BMP-2, did not show cytotoxicity of C2C12 cells assaying osteoblast differentiation, and significantly increased the activity of ALP, a biomarker of osteoblast differentiation. was analyzed to make Therefore, the wood ear mushroom water extract according to the present invention can simultaneously regulate the differentiation of osteoclasts and osteoblasts involved in osteoporosis, and thus can be usefully used as an active ingredient for the prevention or treatment of osteoporosis.

The effects according to the present invention in the above are not limited to the above, and other effects of the present invention can be clearly identified by those of ordinary skill in the art through the examples and claims to be described later. can be understood

1 shows the results of comparison with an electron microscope on the effect on the differentiation of osteoclasts according to the concentration of the wood ear mushroom water extract and the wood ear mushroom alcohol extract of the present invention.
Figure 2 shows the evaluation results of the TRAP activity inhibitory efficacy of the wood ear mushroom water extract of the present invention.
3 shows the results of confirming the effect of the concentration of the shiitake mushroom water extract and the shiitake mushroom alcohol extract on the differentiation of osteoclasts by electron microscopy.
4 shows the cytotoxicity evaluation results for osteoclast progenitor cells of the wood ear mushroom water extract of the present invention.
5 shows the results of comparing the effects of the concentration of the wood ear mushroom water extract and the wood ear mushroom alcohol extract on the differentiation of osteoblasts by electron microscopy of the present invention.
6 shows the results of evaluation of the ALP activity promoting efficacy of the wood ear mushroom water extract of the present invention.
7 shows the results of confirming the effect of the concentration of the shiitake mushroom water extract and the shiitake mushroom alcohol extract on the differentiation of osteoclasts by electron microscopy.
8 shows the cytotoxicity evaluation results for osteoblast progenitor cells of the wood ear mushroom water extract of the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a composition for preventing or treating osteoporosis comprising an extract of wood ear mushroom ( Auricularia auricula ).

Specifically, the present invention is easily obtainable, and as a result of studying a natural material-derived material that can treat and prevent osteoporosis by promoting bone formation without side effects to the human body, the mushroom Auricularia auricula extract was prepared, and it was confirmed that the prepared wood ear mushroom extract promotes the activity of osteoblasts and inhibits the activity of osteoclasts. In addition, toxicity to progenitor cells was confirmed in the cell experiment of the wood ear mushroom extract.

Before describing the present invention in detail, the terms used in the present invention are terms used to properly express the preferred embodiment of the present invention, which may vary depending on the intention of the user or operator or customs in the field to which the present invention belongs. . Accordingly, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

According to an embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating osteoporosis comprising an extract of wood ear mushroom ( Auricularia auricula ) as an active ingredient.

Wood ear mushroom presented in the present invention ( Auricularia auricula ) is a mushroom of the basidiomycete family Auriculariaceae and grows on an old hardwood tree. It is mainly used for cooking, and it is used visually for its soft, chewy texture and color.

In Donguibogam, it is said that it is cold in nature and non-toxic, and it is said to improve the five intestines, overcome the poisoning in the intestines, release hot blood, stop dysentery and bleeding, restore vitality and lighten the body. In addition, as a result of the study, the anticancer effect of wood ear mushroom extract, prevention of cardiovascular disease, diet and cholesterol lowering effect are known.

The method of using the wood ear mushroom in the present invention is not particularly limited, but a powder pulverized after drying may be used, or more preferably, it may be used in the form of an extract. In the present invention, it may be included in a pharmaceutical composition or a food composition in the form of a wood ear mushroom extract.

The term "extract" used in the present invention is an extract obtained by extraction treatment of wood ear mushroom, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a prepared or purified product of the extract, or a mixture thereof, etc., It includes extracts of all formulations that can be formed using the extract itself and the extract.

In the present invention, the wood ear mushroom extract can be extracted from various organs of natural, hybrid or mutated plants of the wood ear mushroom, for example, it is possible to extract from the root, stem or leaf of the wood ear mushroom as well as the wood ear mushroom tissue culture.

The extraction method of the wood ear mushroom extract of the present invention is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method that can be used in the present invention include hot water extraction, ultrasonic extraction, filtration, reflux extraction, etc., which may be performed alone or in combination of two or more methods, preferably It can be carried out using the cold extraction method.

The type of extraction solvent used for extracting the wood ear mushroom of the present invention is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water, distilled water, ethanol, methanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, n-hexane, hydrochloric acid, acetic acid, formic acid, diethyl ether and one or more solvents selected from the group consisting of cyclohexane, and the like. When alcohol is used as the solvent, preferably C1 to C4 alcohols (methanol, ethanol, propanol, butanol) may be used. In the present invention, preferably, water, alcohol, or a mixture thereof may be used as the extraction solvent of the wood ear mushroom, and more preferably water may be used.

As used herein, the term "treatment" is an approach for obtaining beneficial or desirable clinical results, whether for the purposes of the present invention beneficial or desirable clinical results are perceivable or not, and whether partial or total. alleviation of symptoms, reduction of the severity of the disease, a stabilized (ie, not getting worse) state of the disease, delaying or slowing the progression of the disease, amelioration or temporary alleviation and amelioration of the disease state. In addition, the term "prevention" as used herein includes, but is not limited to, all acts of inhibiting or delaying a disease using the pharmaceutical composition. Accordingly, the present invention refers to both therapeutic treatment and prophylactic dimensions, and those in need of treatment include not only those already having the disease, but also those in which the disease is to be prevented. In addition, it may refer to any action to improve or benefit the symptoms of a disease by administering the pharmaceutical composition of the present invention to an individual.

As used herein, the term "apoptosis" is also referred to as apoptosis, which is a kind of programmed cell death (PCD), in which abnormal cells, damaged cells, and aged cells commit suicide by themselves by a biological program that is input into our body. It is a mechanism that allows the body to maintain overall body health by dying.

In the present invention, the composition comprising the wood ear mushroom extract may be used for the prevention or treatment of osteoporosis, and may also be used for menopausal diseases that may cause osteoporosis.

In particular, the wood ear mushroom extract of the present invention as described above has the effect of inhibiting osteoclast differentiation and promoting osteoblast differentiation, thereby having therapeutic and improving effects on osteoporosis.

In the present invention, the wood ear mushroom extract may have the effect of inhibiting the activity of TRAP in relation to the inhibition of differentiation of osteoclasts, and promoting the activity of ALP in relation to the promotion of differentiation of osteoblasts.

The pharmaceutical composition of the present invention may include the wood ear mushroom extract alone as an active ingredient, but may further include a pharmaceutically acceptable carrier, excipient, diluent, etc. depending on the formulation and purpose of use.

In addition, in the case of the pharmaceutical composition of the present invention, it may further include conventional fillers, weight agents, binders, disintegrants, surfactants, anti-aggregants, lubricants, wetting agents, fragrances, emulsifiers, etc., and can be used both parenterally and orally. can In addition, according to a conventional method, it can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions. Suitable formulations known in the art are preferably those disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA. Carriers, excipients and diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil, and the like. When formulating the composition, it is usually prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the composition, for example, starch, calcium carbonate, sucrose, lactose, It is prepared by mixing gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

As used herein, the term “administration” means providing a given composition of the present invention to a subject by any suitable method.

The pharmaceutical composition of the present invention provides an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human as thought by a researcher, veterinarian, doctor or other clinician, that is, alleviation of symptoms of a disease or disorder to be treated. It can be administered in a therapeutically effective amount, which is an amount that induces It is apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration for the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of active ingredients and other components contained in the composition, the type of formulation, and the age, weight, and general health of the patient It can be adjusted according to various factors including state, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs. For a desirable effect, the pharmaceutical composition of the present invention may be administered in an amount of 1-1,000 mg/kg/day, preferably 1-200 mg/kg/day, and may be administered once a day, several times It may be administered in divided doses.

The dosage of the composition for preventing or treating osteoporosis according to the present invention may be appropriately selected by those skilled in the art in consideration of the administration method, the age, sex and weight of the user, and the severity of the disease. For example, the composition for preventing or treating a disease of the present invention may be administered in an amount of 0.0001 mg/kg to 1000 mg/kg of wood ear mushroom extract, and 0.01 mg/kg to 100 mg/kg to be more effective. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

In addition, the wood ear mushroom extract of the present invention is preferably contained in the composition in an amount of 0.001 μg/mL or more, preferably 0.1 to 500 μg/mL, and more preferably 30 to 500 μg/mL.

The pharmaceutical composition of the present invention may be administered to an individual by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection.

In addition, the composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, or biological response modifiers for the prevention or treatment of osteoporosis or complications.

On the other hand, through an embodiment of the present invention to provide a food and health functional food composition for preventing and improving osteoporosis comprising an extract of wood ear mushroom ( Auricularia auricula ) as an active ingredient. When the wood ear mushroom extract of the present invention is used as a food additive, the wood ear mushroom extract may be added as it is, or it may be appropriately used according to a conventional method such as mixing with other foods or food ingredients.

In addition, the mixing amount of the wood ear mushroom extract, which is the active ingredient, may be suitably changed depending on the purpose of use (prevention, health or therapeutic treatment), and the wood ear mushroom extract is contained in an amount of 0.01 to 95% by weight based on the total weight of the food composition. It is preferably included, and more preferably, it is included in an amount of 1 to 80% by weight. If the content is less than 0.01% by weight, the dosing efficiency may be reduced, and if it exceeds 95% by weight, there may be difficulties in formulation.

As a specific example, when preparing food or beverage, the wood ear mushroom extract of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term ingestion for health and hygiene or health control, it may be added in an amount less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range. have.

There is no particular limitation on the type of the food. Examples of foods to which the wood ear mushroom extract of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, There are tea, drinks, alcoholic beverages, vitamin complexes, etc., and includes all health foods in the ordinary sense.

When the food composition of the present invention is prepared as a beverage, it may contain additional ingredients such as various flavoring agents or natural carbohydrates like a conventional beverage. Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin or synthetic sweeteners such as saccharin and aspartame may be used. The natural carbohydrate is contained in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight, based on the total weight of the food composition of the present invention.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , a carbonation agent used in carbonated beverages, and the like. In addition, the composition of the present invention may contain the pulp for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not particularly limited, but is generally included in the range of 0.01 to 10% by weight based on the total weight of the food composition of the present invention.

In addition, the present invention provides a feed composition comprising a wood ear mushroom extract as an active ingredient. The wood ear mushroom extract may be prepared in the same manner as in the case of the pharmaceutical composition or the food composition.

The wood ear mushroom extract of the present invention exhibits an effect of preventing or improving osteoporosis by inhibiting osteoclast differentiation and promoting osteoblast differentiation, and thus may be included in a feed composition to promote growth of animals or livestock or prevent and improve disease occurrence.

Specifically, the feed containing the wood ear mushroom extract according to the present invention as an active ingredient can be prepared in various types of feed known in the art, and preferably, a concentrated feed, roughage or special feed may be included.

In the enriched feed, seed fruits containing grains such as wheat, oats, and corn, bran containing rice bran, bran, barley bran, etc. It is a concentrated product obtained by concentrating the remaining starch residues, which are the main components of starch residues, which are by-products obtained from sesame cakes, sweet potatoes, potatoes, etc., fish meal, fish residues, and fresh liquids obtained from fish. Animal feed such as dried whey, yeast, fish soluble, meat meal, blood meal, cow meal, skim milk powder, milk from cheese, whey, which is the remainder of manufacturing casein from skim milk, yeast, Chlorella, seaweed, etc.

For roughage, raw grass feed such as wild grasses, grasses, and green cuttings, turnips for feed, beets for feed, root vegetables such as rutherbearers, a type of turnip, raw herbs, green crops, grains, etc. are placed in a silo. There are silage, which is stored feed fermented with lactic acid, wild grass, hay dried after cutting grass, straw for breeding crops, and leaves from legumes.

In special feed, mineral feed such as shell and rock salt, urea feed such as urea or its derivative diureide isobutane, and natural feed ingredients are added to supplement the ingredients that are likely to be lacking when only natural feed ingredients are mixed, or to improve the storage of feed. There are feed additives, dietary supplements, etc.

In addition, the feed composition according to the present invention may include various feed additives.

In the present invention, the term "feed additive" refers to a substance added to feed for the purpose of various effects, such as nutrient supplementation and weight loss prevention, enhancement of digestibility of fiber in feed, improvement of oil quality, prevention of reproductive disorders and improvement of fertility rate, prevention of high temperature stress in summer say The feed additive of the present invention corresponds to an auxiliary feed under the Feed Management Act, and is a mineral preparation such as sodium bicarbonate (bicarbonate), bentonite, magnesium oxide, and composite minerals, and trace minerals such as zinc, copper, cobalt, and selenium. Preparations, kerotene, vitamin E, vitamins A, D, E, nicotinic acid, vitamins such as vitamin B complex, protective amino acids such as methionine and lyic acid, protective fatty acids such as fatty acid calcium salt, probiotics (lactic acid bacteria), yeast culture Water, live bacteria such as mold fermented products, yeast agents, and the like may be further included.

The feed composition according to the present invention has an effect of preventing or improving osteoporosis by inhibiting osteoclast differentiation and promoting osteoblast differentiation, so it is not particularly limited as long as it is an individual for the purpose of promoting the growth of animals or livestock or preventing and improving disease occurrence. any is applicable The subject includes animals, such as non-primates (eg, cattle, pigs, horses, cats, dogs, rats, and mice) and primates (eg, monkeys, such as cynomolgous monkeys and mammals including chimpanzees). In another embodiment, the subject is a livestock animal (eg, horse, cow, pig, etc.) or pet animal (eg, dog or cat). Also fish, such as halibut, flounder, eel, freshwater eel, sea eel, eel.

The feed amount of the feed composition according to the present invention will depend on a number of factors such as the species, size, weight, and age of the animal. In principle, typical dosages may range from 0.001 to 1,000 mg/kg per subject/day. However, the present invention is not limited thereto.

In addition, the present invention may be provided as a cosmetic composition comprising a wood ear mushroom extract as an active ingredient.

In the present invention, the wood ear mushroom extract is derived from a natural substance and may be used alone or by mixing two or more thereof.

In the present invention, as additional auxiliary or additive substances in formulations such as creams, lotions, gels, sprays, sticks, etc., mild surfactants, oil bodies, emulsifiers, hyperfatting agents, pearl luster waxes, consistency Consistency substance, thickener, polymer, silicone compound, fat, wax, stabilizer, bioactive substance, deodorant, anti-dandruff agent, film-forming agent, swelling agent, antioxidant, inorganic coloring pigment, hydrophobicity, preservative , insect repellent, self tanning agent, solubilizer, perfume oil, colorant, and the like.

According to the present invention, the wood ear mushroom extract further inhibited osteoclast differentiation and inhibited the activity of TRAP, a molecular marker of osteoclast differentiation, but did not show cytotoxicity in macrophages, precursor cells involved in osteoclast differentiation.

Moreover, wood ear mushroom water extract promoted osteoblast differentiation increased by BMP-2, did not show cytotoxicity of C2C12 cells assaying osteoblast differentiation, and significantly increased the activity of ALP, a biomarker of osteoblast differentiation. was analyzed to make

Therefore, the wood ear mushroom water extract according to the present invention can simultaneously regulate the differentiation of osteoclasts and osteoblasts involved in osteoporosis, and thus can be usefully used as an active ingredient for the prevention or treatment of osteoporosis.

Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. However, the following examples are only intended to embody the contents of the present invention, and the present invention will not be limited thereby.

<Example 1> Preparation of wood ear mushroom extract

Commercially available dried wood ear mushrooms were purchased and used, and the dried wood ear mushrooms were extracted with water and ethanol at a concentration of 20 mg/ml. Wood ear mushrooms were used by purchasing commercially available products. Ethanol extraction was extracted using 70% alcohol at room temperature and in the dark for 24 hours, and extraction was repeated three times. The water extract was added at 200 times the weight of the wood ear mushroom and extracted for 24 hours at room temperature and in the dark, and extracted three times.

Most of the ethanol and water were removed from the extracted sample using a rotary vacuum concentrator, and only the supernatant was recovered by centrifugation at 4000 rpm for 20 minutes. The concentration of the supernatant was measured using a centrifugal concentrator.

<Example 2> Osteoclast differentiation inhibitory effect of wood ear mushroom extract

2-1 Comparison of Effects of Wood Ear Mushroom Water Extract and Wood Ear Mushroom Spirit Extract

It was confirmed whether the wood ear mushroom extract obtained in Example 1 exhibits the ability to inhibit the differentiation of osteoclasts.

Specifically, osteoclast progenitor cells were treated with the wood ear mushroom extract obtained in Example 1 at various concentrations (0.3, 1, 3, 10, 30, or 100 μg/ml), and then RANKL, an osteoclast differentiation-inducing cytokine (30 ng /ml) was added to induce differentiation, and 4 days later, Tartrate resistant acid phosphatase (TRAP), a molecular marker related to the differentiation of osteoclasts, was stained and observed with an optical microscope (FIG. 1). At this time, the activity of TRAP was performed by measuring the absorbance at 405 nm using Tartrate resistant acid phosphatase reagents from Sigma Aldrich.

Figure 1 shows the results of comparing the effects of the concentration of wood ear mushroom water extract and wood ear mushroom alcohol extract on the differentiation of osteoclasts under an electron microscope. indicates

As shown in FIG. 1 , in the case of the wood ear mushroom water extract, the inhibitory effect on osteoclast activity was confirmed when 3 μg/ml or more was treated. In particular, when treated with wood ear mushroom extract of 10 ㎍ / ㎖ or more, showed the effect of inhibiting most of the osteoclast activity. On the other hand, no significant inhibition of osteoclast activity could be confirmed in the case of the ethanol extract of wood ear mushroom.

Figure 2 shows the evaluation results of the TRAP activity inhibition efficacy of the wood ear mushroom water extract. As shown in FIG. 2 , the water extract of wood ear mushroom showed an effect of inhibiting TRAP activity in a concentration-dependent manner when treated with water extract of wood ear mushroom of 0.3 μg/ml or more, and in particular, water extract of wood ear mushroom of 30 μg/ml or more When the extract was treated, it was found to inhibit TRAP activity to a degree similar to that of the normal control group.

2-2 Comparison of Effects of Wood Ear Mushroom Extract and Other Mushroom Extracts

It was confirmed to what extent the other mushroom extracts showed the ability to inhibit the differentiation of osteoclasts compared to the wood ear mushroom extract obtained in Example 1.

First, by applying the method of Example 1 to shiitake mushrooms, water extracts from pogo mushrooms and alcohol extracts from shiitake mushrooms were obtained, respectively.

Next, the obtained shiitake mushroom water extract and shiitake mushroom alcohol extract were applied to the method of Example 2-1, and the osteoclast differentiation inhibitory activity of each of these extracts was measured.

3 shows the results of confirming the effect of the concentration of the shiitake mushroom water extract and the shiitake mushroom alcohol extract on the differentiation of osteoclasts by electron microscopy.

As shown in Figure 3, it was confirmed that the osteoclast differentiation inhibitory effect was not shown in the shiitake mushroom extract even when the concentration treatment was increased. Therefore, it was confirmed that the wood ear mushroom extract provided in the present invention has a specific osteoclast differentiation inhibitory effect.

2-3 Measurement of Cytotoxicity of Wood Ear Mushroom Extract on Osteoclast Progenitor Cells

In order to confirm the cytotoxicity of the wood ear mushroom extract to osteoclasts, after extracting primary bone marrow macrophage from the bone marrow of mice, it was mixed with M-CSF (10 ng/ml) at 1 × 10 ⁴ /well in a 96-well plate. After inoculation for one day, M-CSF (30ng/ml) and wood ear mushroom water extract were treated together and cultured for 3 days to perform cytotoxicity assay. After treating the wood ear mushroom water extract at concentrations of 1, 3, 10, 30, and 100 μg/ml, the values were obtained at absorbance at 450 nm using dojindo’s cell counting kit-8 (CCK-8) reagent, and the relative cell viability (% ) is shown.

4 shows the cytotoxicity evaluation results for osteoclast progenitor cells of wood ear mushroom water extract. As a result, as shown in FIG. 4 , it was confirmed that the water extract of wood ear mushroom hardly showed cytotoxicity to osteoclasts.

<Example 3> Osteoblast differentiation promoting effect of wood ear mushroom extract

3-1 Comparison of Effects of Wood Ear Mushroom Water Extract and Wood Ear Mushroom Spirit Extract

It was confirmed whether the wood ear mushroom extract obtained in Example 1 exhibits the ability to promote differentiation of osteoblasts.

Specifically, the wood ear mushroom extract obtained in Example 1 at various concentrations (0.3, 1, 3, 10, 30 or 100 μg/ml) in C2C12 cells, which are osteoblast progenitor cells, and BMP-2, an osteoblast differentiation-inducing cytokine (100 ng/ml) to induce differentiation into osteoblasts. In order to confirm the formation and differentiation ability of osteoblasts, ALP (Alkaline phosphatase), a molecular marker related to the differentiation of osteoblasts, was stained and observed with an optical microscope. At this time, the activity of ALP was measured by using Alkaline Phosphatase Reagents from Thermo Fisher Scientific.

Figure 5 shows the results of comparing the effects of the concentration of the wood ear mushroom water extract and the wood ear mushroom alcohol extract on the differentiation of osteoblasts under an electron microscope. indicates

As shown in FIG. 5 , the water extract of wood ear mushroom promoted the differentiation of osteoblasts according to the treatment concentration, and in particular, at 30 μg/ml or more, particularly at 100 μg/ml, the effect of promoting the differentiation of osteoblasts was the highest. appeared to be On the other hand, it was confirmed that the alcohol extract of wood ear mushroom did not promote osteoblast differentiation even when the treatment concentration was increased.

6 shows the results of evaluation of the ALP activity promoting efficacy of the wood ear mushroom water extract. As shown in FIG. 6 , the effect of promoting ALP activity in a concentration-dependent manner was shown when the wood ear mushroom water extract was treated with 0.3 μg/ml or more of wood ear mushroom water extract, and in particular, 30 μg/ml or more of wood ear mushroom water extract was shown. When the water extract was treated, it was found to have an effect of promoting higher ALP activity than the positive control group.

3-2 Comparison of Effects of Wood Ear Mushroom Extract and Other Mushroom Extracts

It was confirmed to what extent the other mushroom extracts showed the ability to promote osteoblast differentiation compared to the wood ear mushroom extract obtained in Example 1.

Specifically, the shiitake mushroom water extract and the shiitake mushroom alcohol extract obtained in Example 2 were applied to the method of Example 3-1, and the osteoblast differentiation promoting activity of each extract was measured.

7 shows the results of confirming the effect of the concentration of the shiitake mushroom water extract and the shiitake mushroom alcohol extract on the differentiation of osteoblasts with an electron microscope.

As shown in FIG. 7 , it was confirmed that the osteoblast differentiation promoting effect was observed in the shiitake mushroom extract at 100 μg/ml.

3-3 Measurement of Cytotoxicity of Osteoblastic Progenitor Cells of Wood Ear Mushroom Extract

In order to confirm the cytotoxicity of the wood ear mushroom extract to osteoblasts, the C2C12 cells, which are osteoblast progenitor cells, were treated with the wood ear mushroom water extract at concentrations of 0.3, 1, 3, 10, 30, 100 μg/ml for 3 days, and then dojinodo Cell viability was measured by using the company's cell counting kit-8 (CCK-8) reagent.

8 shows the results of evaluation of cytotoxicity against osteoblastic progenitor cells of wood ear mushroom water extract. As a result, as shown in FIG. 8, cytotoxicity of the water extract of wood ear mushroom to osteoblast progenitor cells was hardly observed, and in the case of 30 μg/ml or more, no cytotoxicity was observed.

As described above, although specific embodiments of the present invention have been described in detail, those skilled in the art who understand the spirit of the present invention may add, change, delete, etc. other components within the scope of the same spirit, and other degenerate inventions However, other embodiments included within the scope of the present invention may be easily proposed. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the following claims rather than the above detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention. should be interpreted as

Claims (13)

Wood ear mushroom ( Auricularia auricula ) A pharmaceutical composition for preventing or treating osteoporosis comprising an extract as an active ingredient.
The method of claim 1,
The wood ear mushroom extract is a pharmaceutical composition, characterized in that extracted with a solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, or a mixture thereof.
The method of claim 1,
The wood ear mushroom extract is a pharmaceutical composition, characterized in that it inhibits the differentiation of osteoclasts without cytotoxicity in progenitor cells.
4. The method of claim 3,
Inhibition of differentiation of the osteoclasts is characterized in that inhibiting the activity of TRAP, a pharmaceutical composition.
The method of claim 1,
The wood ear mushroom extract is a pharmaceutical composition, characterized in that it promotes osteoblast differentiation increased by BMP-2 without cytotoxicity in progenitor cells.
6. The method of claim 5,
The promotion of osteoblast differentiation is characterized in that it promotes the activity of ALP, a pharmaceutical composition.
The method of claim 1,
The wood ear mushroom extract is a pharmaceutical composition, characterized in that it comprises 0.1 ~ 500 ㎍ / mL in the total amount of the composition.
Wood ear mushroom ( Auricularia auricula ) A food composition for preventing or improving osteoporosis, comprising an extract as an active ingredient.
Wood ear mushroom ( Auricularia auricula ) A health functional food composition for preventing or improving osteoporosis, comprising an extract as an active ingredient.
10. The method of claim 9,
Wood ear mushroom extract inhibits osteoclast differentiation and promotes osteoblast differentiation, a health functional food composition.
Wood ear mushroom ( Auricularia auricula ) A feed composition comprising an extract.
Wood ear mushroom ( Auricularia auricula ) An external preparation for the prevention or treatment of osteoporosis, comprising an extract as an active ingredient.
Wood ear mushroom ( Auricularia auricula ) A cosmetic composition comprising an extract.

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