Novel method for preparing antler extract

Abstract

The present invention relates to a novel method for preparing an antler extract. In addition, the present invention relates to an antler extract obtained according to the preparation method or a composition comprising the antler extract. In addition, the present invention relates to use of the antler extract or the composition comprising same for alleviating, treating, or preventing bone diseases. The antler extract obtained according to the preparation method of the present invention shows a significantly high content of indicator substances such as uracil, hypoxanthine, uridine, and inosine, and may be effectively used for alleviating, preventing, and treating bone-related diseases such as osteoporosis through promotion of bone formation and inhibition of bone resorption. Therefore, the composition of the present invention comprising the extract may be used in various fields such as foods, health functional foods, health beverages, pharmaceuticals, and quasi-drugs.

Description

Novel method for manufacturing deer antler extract

[0001] The present invention relates to a novel method for preparing deer antler extract. Furthermore, the present invention relates to a deer antler extract obtained according to the above method or a composition containing the same. Additionally, the present invention relates to the use of the deer antler extract or a composition containing the same for the improvement, treatment, or prevention of bone diseases.

[0002] Deer antlers have been primarily used in traditional Korean medicine for restoring vitality and stimulating appetite, and their efficacy is recorded in the *Donguibogam* (a classic text on Korean medicine). While deer antlers are known to possess various beneficial properties, not all of their active ingredients have been clearly identified to date. Furthermore, the types and amounts of active ingredients extracted vary depending on the processing or extraction methods, which in turn affects the degree of their efficacy.

[0003] For example, there was a problem in that the active ingredients contained in deer antlers could not be sufficiently extracted using only the traditional hot water extraction method, which involves adding water to dried deer antlers and heating them. To compensate for this, various methods have been studied to increase the efficacy of deer antler extracts or to increase the extraction yield and purity, such as extraction methods using organic solvents like hexane and chloroform (Korean Registered Patent Publication No. 10-0540941, Korean Published Patent Publication No. 10-1999-0044781), fermentation methods using microorganisms (Kim MK et al., J Korean Soc Food Sci Nutr, 2009, 38, 1237-1242), and methods for preparing hydrolyzed deer antler extracts such as treating dried deer antler powder with enzymes (Jae-Hwa Ki et al, J Korean Soc Food Sci Nutr, 2011, 40(1), 89-93). However, organic solvents such as hexane and chloroform are classified as substances of concern regarding biotoxicity, such as carcinogenicity, and environmental hazards, and are currently prohibited from use in the manufacture of pharmaceuticals. In the case of fermentation methods using microorganisms or enzymes, there were problems with complex manufacturing processes due to the difficulty in storing and managing microorganisms or enzymes, and there was a disadvantage that quality control was difficult because even a slight change in the fermentation process resulted in a completely different product.

[0004] Accordingly, there is a continuous demand for research and development on establishing efficient extraction conditions for deer antlers, standardized quality control of deer antler extracts, identification of active ingredients, and functional characteristics.

[0005] The inventors sought to establish a method for extracting deer antlers that can extract various active ingredients contained in the antlers with a high yield while using a solvent that does not exhibit toxicity to the human body. They attempted various methods and conditions, and as a result, established a method for preparing deer antler extract according to the present invention.

[0006] The method for preparing a deer antler extract according to the present invention comprises the following steps:

[0007] a) A step of extracting deer antlers with an ethanol solvent;

[0008] b-1) A step of separating the deer antler extract obtained in step a) above;

[0009] b-2) a step of hot water extraction of the deer antler extract residue obtained in step a) above; and

[0010] c) A step of mixing the product of step b-1) and the product of step b-2).

[0011] The term "velvet antler" used in this invention refers to the antlers of animals of the Cervidae family. Depending on the time of harvesting, antlers can be classified into velvet antlers and horns; velvet antlers are unossified antlers within two months of birth, while horns are fully grown antlers that have naturally fallen off after two months of birth. The velvet antlers of this invention should be interpreted to include both velvet antlers and horns. Velvet antlers can be classified sequentially from the top to the bottom into oily bone, bone powder, upper section, middle section, and lower section; however, in this invention, the entire velvet antler or any specific part may be used without limitation. Furthermore, the velvet antlers may be harvested or commercially available without limitation.

[0012] The term "extract" as used in the present invention refers to a product obtained by extracting a natural product. In the present invention, the extract is interpreted to include an extract obtained by an extraction process, a diluted or concentrated extract, a dried product obtained by drying an extract, a modified or purified product thereof, or a fraction thereof. The extract may preferably be a product obtained by extracting deer antler.

[0013] In the method for preparing a deer antler extract according to the present invention, step a) is a step of extracting deer antlers with an ethanol solvent.

[0014] In step a) above, specific parts of the deer antler, such as the oily bone, bone powder, upper, middle, or lower sections, or the entire antler, or a mixture thereof, may be used. Additionally, the deer antler may be ground or crushed before extraction with an ethanol solvent.

[0015] In step a) above, the concentration of ethanol may be 70% to 10%, for example, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%. Preferably, the concentration of ethanol is 35%, 30%, 25%, 20%, 15%, or 10%, and most preferably 20%.

[0016] In step a) above, the amount of ethanol used is 5 to 20 times the amount of raw material. Specifically, extraction can be performed using ethanol in a volume of 5 to 20 times the weight or volume of the raw deer antler as a solvent. Preferably, the amount of ethanol used is 10 to 15 times, and most preferably 15 times.

[0017] In step a) above, the extraction time may be from 1 hour to 72 hours, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, 54 hours, 60 hours, 66 hours, or 72 hours. Preferably, the extraction time is 6 hours. If there is no significant difference in the content of the indicator substance in the extracted product over time, it is preferable to extract for a shorter time, considering process costs, time, and safety such as the risk of explosion.

[0018] In step a) above, the extraction is performed at room temperature. Here, room temperature refers to the temperature of a typical indoor space, which may vary depending on the season or surrounding environment. For example, in a factory or enclosed environment where extraction is performed in large quantities, the room temperature may be higher than the outside temperature. In the present invention, the room temperature may be 20°C to 40°C.

[0019] The method for preparing a deer antler extract according to the present invention may additionally include the step of extracting the deer antler with an ethanol solvent and then filtering it in step a). The filtration may be performed 1 to 5 times and may be performed using a filter of 0.1 μm to 100 μm or a sieve of 10 to 500 mesh.

[0020] In the method for preparing a deer antler extract according to the present invention, step b-1) is a step of separating the deer antler extract obtained in step a). Step b-1) may be a step of separating the deer antler extract obtained by filtration after extracting the deer antler with an ethanol solvent in step a).

[0021] The method for preparing a deer antler extract according to the present invention may additionally include a step of concentrating the deer antler extract after separating it in step b-1). The concentration may be vacuum concentration or azeotropic concentration, but is not limited thereto. Preferably, the concentration is performed at a temperature of 55°C or lower.

[0022] In the method for preparing a deer antler extract according to the present invention, step b-2) is a step of hot water extraction of the deer antler extract residue obtained in step a). In the present invention, the deer antler extract residue is a term used to distinguish it from the deer antler extract, and refers to the solid matter remaining after extraction, which may contain some liquid.

[0023] In step b-2) above, hot water extraction is performed at 90°C to 100°C. The extraction time may be 1 hour to 5 hours. Preferably, hot water extraction is performed at 100°C for 1 hour or at 90°C for 3 hours.

[0024] In step b-2) above, the amount of water used is 5 to 20 times the amount of raw material. Specifically, hot water extraction is performed using a volume of water 5 to 20 times the weight or volume of the raw deer antler. Preferably, the amount of water used is 10 to 15 times, and most preferably 15 times. Here, it is preferable that the water be purified water.

[0025] The method for preparing a deer antler extract according to the present invention may further include a step of filtering and / or concentrating the deer antler extract residue after hot water extraction in step b-2). The filtration may be performed 1 to 5 times and may be performed using a filter of 0.1 μm to 100 μm or a sieve of 10 to 500 mesh. The concentration may be vacuum concentration or azeotropic concentration, but is not limited thereto. Preferably, the concentration is performed at a temperature of 55°C or lower.

[0026] In the method for preparing a deer antler extract according to the present invention, step c) is a step of mixing the product of step b-1) and the product of step b-2).

[0027] The method for preparing a deer antler extract according to the present invention may further include a step of concentrating the mixture obtained in step c).

[0028] The method for preparing a deer antler extract according to the present invention may further include a step of freezing or freeze-drying the mixture obtained in step c).

[0029]

[0030] Another aspect of the present invention provides a deer antler extract obtained according to the above manufacturing method.

[0031] Another aspect of the present invention provides a composition comprising the extract.

[0032] In the extracts and compositions according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0033] The extract obtained by the manufacturing method of the present invention has an increased content of active ingredients. As used herein, the term "active ingredient" refers to a component that exhibits specific activity or effect in terms of physiological, biological, pharmaceutical, cosmetic, food, quasi-pharmaceutical, etc. The term "active ingredient" may be used interchangeably with "active ingredient," "physiologically active ingredient," "useful substance," "effective substance," "active substance," and "indicator substance" in this specification.

[0034] In the present invention, the indicator substances of the deer antler extract are uracil, hypoxanthine, uridine, and inosine. These indicator substances are known to play an important role in evaluating the bone formation-promoting and bone destruction-inhibiting functions of the deer antler extract.

[0035] The above uracil is a pyrimidine base and one of the components of RNA, and promotes the differentiation and proliferation of osteoblasts. In addition, uracil is known to be a component that promotes bone matrix formation and calcium deposition by activating protein synthesis and gene expression in osteoblasts (JB Noronha-Matos et al., J Cell Physiol. 2016 Sep, 231(9):1852-61.).

[0036] The aforementioned hypoxanthine is a type of purine base that acts as a precursor to inosine and contributes to the promotion of bone formation by activating the energy metabolism of osteoblasts. In addition, hypoxanthine is also known as a component that prevents bone destruction by inhibiting the excessive activation of osteoclasts (Z Mei et al., EBioMedicine. 2020 Dec, 62:103111.; H Zhao et al., Sci Rep. 2018 Nov 7, 8(1):16456.).

[0037] The aforementioned uridine is a nucleoside in which uracil is bonded to ribose, and it promotes the differentiation and mineralization of osteoblasts. Uridine induces the activation of osteoblasts through cell signaling and increases bone density by enhancing calcium deposition in the bone matrix. In addition, uridine is known as a component that reduces bone resorption by inhibiting the formation and activation of osteoclasts (H Zhao et al., Sci Rep. 2018 Nov 7, 8(1):16456.).

[0038] The aforementioned inosine is a purine nucleoside in which hypoxanthine is bound to ribose, and it supports the function of osteoblasts through ATP synthesis and energy metabolism. In addition, inosine is known to be a component that contributes to preventing bone loss and maintaining bone density by promoting osteoblast differentiation and aiding bone tissue remodeling, while inhibiting the secretion of inflammatory mediators and thereby suppressing osteoclast activity (Z Mei et al., EBioMedicine. 2020 Dec, 62:103111.; H Zhao et al., Sci Rep. 2018 Nov 7, 8(1):16456.).

[0039] Therefore, uracil, hypoxanthin, uridine, and inosine can be used as indicator substances to evaluate whether the deer antler extract of the present invention can be effectively utilized for the prevention and treatment of bone-related diseases such as osteoporosis through the promotion of bone formation and inhibition of bone fragmentation.

[0040] The extract or composition according to the present invention has an effect of improving, preventing, or treating bone diseases. As used herein, the term "bone disease" includes all diseases induced by bone loss or a decrease in bone density resulting from a loss of balance, such as the generation and differentiation of osteoblasts and osteoclasts. Specifically, the bone disease may be one or more selected from the group consisting of osteoporosis, osteomalacia, osteopenia, bone atrophy, osteoarthritis, rheumatoid arthritis, periodontal disease, periodontitis, alveolar bone disease, osteolysis, fibrous dysplasia, and osteoogenesis imperfect, but is not limited thereto.

[0041]

[0042] Another aspect of the present invention provides a pharmaceutical composition comprising a deer antler extract obtained according to the above manufacturing method.

[0043] In the pharmaceutical composition according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0044] The pharmaceutical composition according to the present invention may treat or prevent bone disease.

[0045] As used herein, the term "treatment" refers to any act in which the symptoms of a disease are improved or completely cured by the administration or application of a composition according to the present invention. Additionally, as used herein, the term "prevention" refers to any act in which the symptoms of a disease are suppressed or delayed by the administration or application of a composition according to the present invention. Furthermore, as used herein, the term "improvement" refers to any act in which the symptoms of a disease are at least reduced or improved, or benefit, by the administration or application of a composition according to the present invention.

[0046] As used herein, the term "object" includes a human or any non-human animal, and said non-human animal may be a vertebrate, e.g., a primate, a dog, a cow, a horse, a pig, a rodent, e.g., a mouse, a rat, a guinea pig, etc. In this specification, said "object" is used interchangeably with "individual" and "patient."

[0047] A pharmaceutical composition according to the present invention contains an effective amount of an extract according to the present invention capable of exhibiting a therapeutic or preventive effect on a disease, and may be administered to a subject requiring treatment, prevention, or improvement of a disease.

[0048] The above effective amount may be a "therapeutic effective amount" or a "preventive effective amount." As used herein, the term "therapeutic effective amount" refers to any amount that, when a drug or therapeutic agent is used alone or in combination with other therapeutic agents, can result in a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or the prevention of damage or disability caused by disease suffering. As used herein, the term "preventive effective amount" refers to any amount that inhibits the occurrence or recurrence of disease in an individual. The level of the above effective amount may be determined based on factors including the severity of the subject, age, gender, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. For example, the extract of the present invention may be included in an amount of about 0.01 to 100 weight% based on the total weight of the composition.

[0049] As used herein, the term "administration" means physically introducing the pharmaceutical composition to a subject using any of the various methods and delivery systems known to a person skilled in the art. Routes of administration for the pharmaceutical composition of the present invention include, for example, oral administration routes, or intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, for example, by injection, infusion, or topical application, but are not limited thereto. The pharmaceutical composition of the present invention may be administered once or several times daily, or at intervals of several days to several months.

[0050] The pharmaceutical composition of the present invention may be prepared in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. The formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0051] The pharmaceutical composition according to the present invention can be formulated into various oral or parenteral administration forms.

[0052] Examples of the above oral formulations include tablets, pills, hard / soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, etc., and these formulations may use one or more diluents or excipients such as fillers, extenders, humectants, disintegrants, lubricants, binders, and surfactants that are commonly used in addition to the extract according to the present invention. Agar, starch, alginic acid or its sodium salt, calcium monohydrogen phosphate anhydrous, etc. may be used as disintegrants; silica, talc, stearic acid or its magnesium or calcium salt, polyethylene glycol, etc. may be used as lubricants; and magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, low-substituted hydroxypropylcellulose, etc. may be used as binders. In addition, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc. can be used as diluents, and in some cases, commonly known absorbents, coloring agents, flavoring agents, sweeteners, etc. can be used together.

[0053] In addition, the above parenteral administration formulations may be examples of injectables, creams, lotions, topical ointments, oils, moisturizers, gels, aerosols, and nasal inhalers, and these formulations may use one or more commonly used carriers, stabilizers, or preservatives in addition to the extract according to the present invention. Carriers may include water, suitable oils, saline solution, aqueous glucose, or glycol, etc. Stabilizers may include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid, etc., and preservatives may include benzalkonium chloride, methyl-paraben, propyl-paraben, or chlorobutanol, etc.

[0054] The above pharmaceutical composition may be sterilized or contain preservatives, stabilizers, hydrating agents or emulsification promoters, salts for osmotic pressure regulation, buffers, and other therapeutically useful substances, and may be formulated according to commonly used mixing, granulation, or coating methods.

[0055] The pharmaceutical composition of the present invention may be administered in combination with other therapeutic agents. In this case, the pharmaceutical composition of the present invention and the other therapeutic agents may be administered simultaneously, sequentially, or individually. The other therapeutic agents may be drugs, such as compounds or proteins, having effects of preventing, treating, and / or improving diseases, but are not limited thereto.

[0056] Furthermore, the pharmaceutical composition of the present invention may be formulated to be administered simultaneously, sequentially, or individually with other therapeutic agents. For example, the pharmaceutical composition and other therapeutic agents may be administered simultaneously as a single formulation, or simultaneously, sequentially, or individually as separate formulations. To administer simultaneously, sequentially, or individually, the pharmaceutical composition of the present invention and other therapeutic agents may be formulated separately in individual containers, or formulated together in the same container. Additionally, the pharmaceutical effective dose, administration time, administration interval, administration route, treatment duration, etc., of the pharmaceutical composition of the present invention and other therapeutic agents may be identical or different from one another.

[0057]

[0058] Another aspect of the present invention provides a food composition comprising a deer antler extract obtained according to the above manufacturing method.

[0059] In the food composition according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0060] The food composition according to the present invention may improve or prevent bone disease.

[0061] The above foods include all processed forms of natural materials, such as nutraceutical foods, medical foods, health functional foods, nutritional supplements, and food additives.

[0062] As used in this specification, the term "Nutraceutical food" refers to a food prepared from raw materials or ingredients that perform functions beneficial to a subject, and which improves or maintains the subject's health by maintaining normal functions or activating physiological functions.

[0063] As used in this specification, the term "medical food" refers to a food for special medical purposes, specifically a food in a special formulation for the specific dietary management of a subject suffering from a disease, and includes foods for special medical purposes prescribed by the Ministry of Food and Drug Safety. The medical food alleviates the subject's disease or symptoms by satisfying specific nutritional requirements that cannot be achieved by a regular diet alone, and may be consumed as part of a meal or as a meal replacement. The special formulation is a formulation that can be taken orally or via enteral nutrition, and enteral nutrition refers to supplying nutrition by inserting a nasogastric tube into the gastrointestinal tract through the nose. In this specification, the term "medical food" may be used interchangeably with the term "medical food."

[0064] As used in this specification, the term "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients that have functional properties useful to the human body. Here, "functionality" means obtaining effects useful for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0065] The types of the above foods are not specifically limited. Specific examples include yogurt, dairy products, meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., and include all foods in the conventional sense.

[0066] The food composition according to the present invention can be manufactured by methods commonly used in the art, and during such manufacturing, raw materials and ingredients commonly added in the art may be added. The added ingredients include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. The carbohydrates include all conventional sugars such as glucose, fructose, maltose, sucrose, oligosaccharides, dextrin, cyclodextrin, etc., or sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents, natural flavoring agents such as taumatin and stevia extract, or synthetic flavoring agents such as saccharin and aspartame may be used. For example, when the food composition is manufactured as a drink, in addition to the extract according to the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract, or licorice extract may be additionally included, and various natural carbohydrates, etc., may be included as additional ingredients, as in conventional beverages.

[0067] In addition, unlike general pharmaceuticals, when food is used as a raw material, there is an advantage of not having side effects that may occur during long-term use of the drug, and since it is highly portable, the food composition of the present invention can be used as an adjuvant to enhance or improve the preventive or therapeutic effects of a disease, and it is also possible to use it simultaneously or sequentially with the pharmaceutical composition, quasi-drug composition, and / or other composition or other therapy according to the present invention for the purpose of maximizing the above effects.

[0068] The content of the active ingredient included in the food composition of the present invention may be appropriately determined according to the purpose of use (prevention, improvement, or therapeutic treatment), the duration of use, the condition of the subject, etc. For example, when manufacturing food, the content of the extract according to the present invention may be included in an amount of 0.001 to 20 weight%, 0.001 to 15 weight%, or 0.001 to 10 weight% in the food composition. In the case of health drinks, it may be included in an amount of 0.01 to 2 g, specifically 0.02 to 2 g, and more specifically 0.3 to 1 g based on 100 ml. However, in the case of long-term consumption for the purpose of health and hygiene or health control, it may be used in an amount less than the above range. During the process of manufacturing the food composition of the present invention, the content of the active ingredient added to the food composition may be appropriately increased or decreased as needed.

[0069]

[0070] Another aspect of the present invention provides a quasi-drug composition comprising a deer antler extract obtained according to the above manufacturing method.

[0071] In the composition of a quasi-drug according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0072] The quasi-drug composition according to the present invention may improve, treat, or prevent bone diseases.

[0073] The quasi-drug composition according to the present invention can be manufactured in the form of general emulsion formulations and solubilized formulations, etc., using commonly known manufacturing methods. At this time, the quasi-drug composition of the present invention can be manufactured in various formulations such as patches, ointments, gels, creams, sprays, detergents, disinfectants, sanitary pads, wet wipes, masks, gauze, absorbent cotton, adhesive bandages, bandages, solid forms, liquid forms, etc., and commonly used manufacturing methods for quasi-drugs may be applied. Furthermore, the types of the above quasi-drugs are not particularly limited. Examples include toothpaste, oral spray, mouthwash, mouthwash, oral rinse, oral ointment, mouthwash, gum massage cream, cleanser, wet wipe, shower foam, soap, mask, gauze, absorbent cotton, adhesive bandage, eye mask, bandage, repellent, disinfectant, detergent, etc., and include all quasi-drugs in the conventional sense.

[0074] The quasi-drug composition according to the present invention may further include additional components in addition to the extract of the present invention to enhance efficacy. For example, there are no limitations on the additional components provided that they do not offset or reduce the efficacy of the extract according to the present invention. Optionally, carriers, excipients, diluents, etc., commonly used in the field of quasi-drugs may be further included. For example, the carrier, excipient, or diluent may be a filler, extender, binder, wetting agent, disintegrant, surfactant, lubricant, sweetener, fragrance, preservative, etc.

[0075] Optionally, ingredients typically used to add or enhance the function of the quasi-drug may also be added. For example, the quasi-drug composition may additionally include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, gelatin, glycerin, acacia gum, alginate, calcium phosphate, calcium carbonate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, propylene glycol, polyethylene glycol, vegetable oil, injectable ester, Witepsol, macrogol, Tween 61, cocoa dough, laurize, etc.

[0076]

[0077] Another aspect of the present invention provides a feed composition comprising a deer antler extract obtained according to the above manufacturing method.

[0078] In the feed composition according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0079] The feed composition according to the present invention may improve, treat, or prevent bone disease.

[0080] The above feed is a substance that supplies organic or inorganic nutrients necessary for the maintenance of life and rearing of subjects other than humans, and includes both feed and feed additives.

[0081] The types of the above feed are not particularly limited. Specific examples include grains, plant-based protein feeds, animal-based protein feeds, dairy products, nutritional supplements, digestion and absorption enhancers, growth promoters, etc., and include all feed or feed additives in the conventional sense. The above grains include, for example, ground or crushed rice, wheat, oats, barley, or corn. The above plant-based protein feed includes, for example, feeds with rapeseed (rapeseed), sunflower, or soybeans as the main components. The above animal-based protein feed includes, for example, feeds with blood meal, bone meal, meat meal, or fish meal as the main components. The above dairy products include, for example, feeds with powdered milk or whey powder as the main components.

[0082] The feed composition according to the present invention can be manufactured by a method commonly used in the art, and in addition to the active ingredient, the composition may further include a suitable carrier, preservative, stabilizer, wetting agent, emulsifier, solution accelerator, excipient, organic acid, phosphate, antioxidant, etc. commonly used in the art. Specifically, the carrier, preservative, stabilizer, wetting agent, emulsifier, solution accelerator, excipient, organic acid, phosphate, antioxidant that may be included in the feed composition may be one or more selected from, for example, corn starch, lactose, sucrose, soybean flakes, olive oil, sesame oil, peanut oil, propylene glycol, lactic acid, malic acid, citric acid, fumaric acid, adipic acid, sodium phosphate, potassium phosphate, polyphenol, vitamin C, green tea extract, licorice extract, tocopherol, tannic acid, and chitosan. The above carrier, preservative, stabilizer, wetting agent, emulsifier, solution accelerator, excipient, organic acid, phosphate, antioxidant, etc. are introduced in amounts commonly used in the industry.

[0083] The feed composition according to the present invention may be formulated using commonly known manufacturing methods. For example, it may be in the form of a powder, liquid, or granular formulation. The content of the active ingredient included in the feed composition of the present invention may be appropriately determined according to the purpose of use (prevention, improvement, or therapeutic treatment), duration of use, condition of the subject, etc. The feed composition may be used by immersion, spraying, or mixing with other feed compositions.

[0084]

[0085] Another aspect of the present invention provides a method for improving, treating, or preventing bone disease, comprising the step of administering to a subject a deer antler extract obtained according to the above manufacturing method or a composition containing the same.

[0086] In the method for improving, treating, or preventing bone disease according to the present invention, related terms are understood to have the same meaning as the terms described above, unless specifically stated otherwise.

[0087] The extract or composition according to the present invention may be administered to subjects who require improvement, treatment, or prevention of bone disease.

[0088] The extract or composition according to the present invention may be administered to a subject simultaneously, sequentially, or individually with other components or substances exhibiting effects of improving, treating, or preventing bone diseases. The other therapeutic agents may be drugs, such as compounds or proteins, having effects of preventing, treating, and / or improving bone diseases, but are not limited thereto. The term "simultaneous" administration means administering the composition of the present invention and other components as a single preparation at once, or administering the composition of the present invention and other components as separate preparations at once; in this case, the administration route of the composition of the present invention and the administration route of the other components may differ from each other. The term "sequential" administration means administering the composition of the present invention and other components relatively continuously, allowing for the minimum possible time consumed in the administration interval. The term "individual" administration means administering the composition of the present invention and other components at regular time intervals. The method of administering the composition of the present invention and other components may be appropriately selected by an expert in the art, taking into account the efficacy and side effects on the subject.

[0089]

[0090] Another aspect of the present invention provides the use of a deer antler extract obtained according to the above manufacturing method or a composition containing the same for the improvement, treatment, or prevention of bone diseases.

[0091] In the use according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0092] The deer antler extract obtained according to the manufacturing method of the present invention exhibits significantly high levels of indicator substances such as uracil, hypoxanthin, uridine, and inosine, and can be effectively utilized for the improvement, prevention, and treatment of bone-related diseases such as osteoporosis through the promotion of bone formation and inhibition of bone fragmentation. Accordingly, the composition of the present invention containing the extract can be utilized in various fields such as food, health functional food, health beverage, pharmaceuticals, and quasi-drugs.

[0093] Figures 1 to 3 are graphs showing the bone mineral density (BMD) of the whole body, tibia, and femur of a mouse, respectively.

[0094] Figures 4 to 6 are graphs showing the bone mineral content (BMC) of the whole body, tibia, and femur of a mouse, respectively.

[0095] Figures 7 to 9 are graphs showing the values ​​of TRAP 5b (tartrate-resistant acid phosphatase 5b), CTX (C-telopeptide), and NTX (N-telopeptide) in the blood of mice, respectively.

[0096] Figure 10 is a graph showing the osteocalcin levels in the blood of mice.

[0097] Figure 11 is a micrograph for the histopathological analysis of a mouse femur, and Figure 12 is a graph showing the collagen content in the femur tissue.

[0098] Figure 13 is a micrograph to confirm the change in size of fat cells within the distal femur of a mouse, and Figure 14 is a graph showing the size of the fat cells.

[0099] Figure 15 shows the mRNA expression levels of OPN (Osteopontin), BMP-2 (Bone morphogenic protein-2), and COL-II (Type II Collagen) in the mouse tibia.

[0100] Figure 16 shows a TRAP staining image of RANK-induced RAW 264.7 osteoclasts.

[0101] Figure 17 is a graph showing the Alizarin Red S staining image and the degree of calcium deposition in SaOS-2 osteoblast-like cells.

[0102] Figure 18 is a graph showing the mRNA expression levels of OPN (Osteopontin), IBSP (Integrin Binding Sialoprotein), BMP-2 (Bone Morphogenetic Protein 2), and COL1A1 (Collagen Type I Alpha 1 Chain) as osteoblast differentiation markers in SaOS-2 osteoblast-like cells.

[0103] Figure 19 is a graph showing the expression levels of ALP (Alkaline Phosphatase), OSX (Osterix), and OCN (Osteocalcin) in SaOS-2 osteoblast-like cells.

[0104] To aid in understanding the present invention, specific examples will be described below. However, the following examples are intended to explain the invention more specifically and should not be interpreted as limiting the invention, and ordinary variations are possible within the scope of the invention. Furthermore, unless otherwise specified, terms and terms described in this specification should be understood in the sense commonly used in the technical field to which the present invention belongs.

[0105]

[0106] Example 1. Preparation of deer antler extract

[0107] After crushing the whole deer antlers, they were sieved by passing them through a sieve with a mesh size of 1.4 mm; any antlers that did not pass through the sieve were re-crushed and combined. Based on the volume of the deer antler powder thus produced, circulating or stirring extraction was performed at room temperature for 6 hours using 20% ​​ethanol at a volume 15 times that of the powder. Subsequently, pressure filtration was performed using a 20 μm filter.

[0108] Accordingly, deer antler extract and deer antler extract residue were obtained.

[0109] The above deer antler extract was concentrated to 15-20 Brix by first concentrating under reduced pressure at a temperature of 55℃ or lower, and then concentrated to 45-48 Brix by adding a small amount of purified water and performing secondary azeotropic concentration (yield 18%).

[0110] The above deer antler extract residue was subjected to hot water extraction at 100°C for 1 hour using 15 times the volume of purified water based on the weight of the raw deer antler. Afterward, pressure filtration was performed using a 20 μm filter. This was added to the concentrate of the deer antler extract, and reduced pressure concentration was performed at 55°C or below. The total yield of the final product based on the raw deer antler is 55%.

[0111]

[0112] Changes in indicator substance content according to the concentration of solvent ethanol

[0113] In step a) of the above example, ethanol at a volume 15 times that of the raw material was used, and circulating or stirring extraction was performed at room temperature with varying concentrations of the solvent ethanol; the content of four indicator substances (uracil, hypoxanthin, uridine, and inosine) over time was compared. The results are shown in the table below, in the order of uracil, hypoxanthin, uridine, and inosine (unit: mg / g).

[0114] 70% Ethanol Extract 60% Ethanol Extract 35% Ethanol Extract 25% Ethanol Extract 20% Ethanol Extract 6H 0.16 0.18 0.18 0.27 0.45 12H 0.17 0.15 0.18 0.27 0.44 18H 0.18 0.21 0.18 0.28 0.45 24H 0.15 0.13 0.18 0.27 0.45 36H 0.12 0.13 0.18 0.28 0.45 48H 0.15 0.18 0.17 0.26 0.45 72H 0.15 0.13 0.18 0.27 0.46

[0115] 70% Ethanol Extract 60% Ethanol Extract 35% Ethanol Extract 25% Ethanol Extract 20% Ethanol Extract 6H 0.28 0.36 0.45 0.44 0.45 12H 0.29 0.35 0.45 0.44 0.44 18H 0.32 0.36 0.44 0.44 0.45 24H 0.31 0.35 0.45 0.45 0.46 36H 0.30 36 0.45 0.45 0.46 48H 0.29 0.40 45 0.44 0.46 72H 0.29 0.36 0.46 0.44 0.42

[0116] 70% Ethanol Extract 60% Ethanol Extract 35% Ethanol Extract 25% Ethanol Extract 20% Ethanol Extract 6H 0.07 0.09 0.17 0.22 0.3112H 0.08 0.09 0.16 0.23 0.3118H 0.07 0.09 0.16 0.23 0.3224H 0.08 0.09 0.15 0.23 0.3136H 0.08 0.09 0.12 0.23 0.3148H 0.07 0.09 0.12 0.22 0.3272H 0.08 0.09 0.14 0.22 0.34

[0117] 70% Ethanol Extract 60% Ethanol Extract 35% Ethanol Extract 25% Ethanol Extract 20% Ethanol Extract 6H 0.09 0.12 0.15 0.24 0.2512H 0.06 0.15 0.15 0.24 0.2518H 0.06 0.14 0.13 0.24 0.2524H 0.13 0.14 0.13 0.24 0.2536H 0.05 0.14 0.14 0.24 0.2548H 0.15 0.16 0.13 0.22 0.2572H 0.14 0.13 0.14 0.23 0.25

[0118] In addition, when the concentration of the solvent ethanol was used in the range of 10% to 20% in step a) of the above example, the content of four indicator substances (uracil, hypoxanthin, uridine, and inosine) was compared, and the results are shown in the table below (unit: mg / g). The content of the four indicator substances when 20% ethanol was used as a solvent in Tables 1 to 4 above and the content of the four indicator substances when 20% ethanol was used as a solvent in Table 5 below are different, because the average values ​​were different as the two experiments were conducted independently using different raw deer antler materials.

[0119] Uracil Hypoxanthine Uridine Inosine 10% Ethanol Extract 0.28 10.27 40.33 40.16 0 15% Ethanol Extract 0.28 20.28 70.36 70.17 7 20% Ethanol Extract 0.35 20.32 60.42 0 20 4

[0120]

[0121] Changes in indicator substance content according to the amount of ethanol added to the raw material

[0122] In step a) of the above example, 20% ethanol was used as the solvent to obtain the highest level of content for the four indicator substances, and the content of the four indicator substances (uracil, hypoxanthine, uridine, and inosine) was compared when the amount of ethanol added to the raw material (a multiple of the volume of the raw material) was varied. The results are shown in the table below (unit: mg / g).

[0123] UracilHypoxanthineUridineInosine 5x multiplier 0.269 0.274 0.376 0.1819x multiplier 0.281 0.286 0.386 0.18110x multiplier 0.336 0.302 0.402 0.19315x multiplier 0.352 0.326 0.420 0.20416x multiplier 0.312 0.291 0.372 0.18120x multiplier 0.323 0.311 0.398 0.192

[0124]

[0125] Changes in indicator substance content according to the type of solvent

[0126] In step a) of the above example, when 20% ethanol at 15 times the volume of the raw material was used and circulating or stirring extraction was performed at room temperature for 6 hours with different types of solvents, the content of four indicator substances (uracil, hypoxanthin, uridine, and inosine) was compared, and the results are shown in the table below (unit: mg / g).

[0127] 20% ethanol showed significantly higher values ​​for all four indicator substances compared to 20% methanol.

[0128] UracilHypoxanthineUridineInosine20% MeOH0.3070.3020.3960.19220% EtOH0.3520.3260.4200.204

[0129]

[0130] Changes in indicator substance content according to extraction temperature

[0131] When the temperature was varied during circulating or stirring extraction for 6 hours using 20% ​​ethanol at 15 times the volume of the raw material as a solvent in step a) of the above example, the content of four indicator substances (uracil, hypoxanthin, uridine, and inosine) in the product of step b-1) was compared. The results are shown in the table below (unit: mg / g).

[0132] UracilHypoxanthineUridineInosine20℃0.430.460.40.233℃0.420.460.40.240℃0.450.510.420.278℃0.460.530.420.21

[0133] In addition, the content of four indicator substances (uracil, hypoxanthin, uridine, and inosine) in the product obtained by hot water extraction of the deer antler extract residue in step b-2) was compared when the extraction temperature was varied in step a) of the above example. Here, the hot water extraction was performed at 90℃. The results are shown in the table below (unit: mg / g).

[0134] UracilHypoxanthineUridineInosine20℃ / 90℃0.040.10.040.0233℃ / 90℃0.070.120.060.0340℃ / 90℃0.060.090.01078℃ / 90℃0.060.0600

[0135]

[0136] Yield at each stage

[0137] In step a) of the above example, when circulating or stirring extraction was performed at room temperature for 6 hours using 20% ​​ethanol as a solvent at a volume 10 or 15 times that of the raw material, the yield of the deer antler extract at each step is shown in the table below.

[0138] b-1) Stage Yield b-2) Stage Yield c) Stage Yield 10x Dividend Purified Water 17.2% 32.6% 49.8% 15x Dividend Purified Water 18% 37% 55%

[0139] As examined above, it can be confirmed that when variables in the manufacturing process of deer antler extract change, the content of indicator substances necessary for the improvement, treatment, or prevention of bone diseases changes significantly. Furthermore, safety factors such as the risk of explosion due to oil vapors during the manufacturing process, as well as manufacturing costs based on the amount of ethanol and extraction time, must also be considered.

[0140]

[0141] Example 2. Confirmation of the ability of deer antler extract to improve bone diseases

[0142] Sixty 5-week-old female mice were used in this experiment after a 7-day acclimatization period. Each mouse was anesthetized, and an open laparotomy was performed to remove both ovaries. Osteoporosis was assessed 12 weeks after ovariectomy. As a result, the whole-body bone mineral density (BMD) in the normal control group (NOR group) without ovariectomy was 0.034 g / cm³. 2 It was shown to be 0.028 g / cm³ in the group in which osteoporosis was induced via ovariectomy (OVX group).2 It was found that the total bone mineral content (BMC) was 0.955 g in the normal control group (NOR group) without ovariectomy and 0.827 g in the group with osteoporosis induced by ovariectomy (OVX group), indicating a decrease of approximately 13.36% compared to the normal control group (***p<0.001). Accordingly, a mouse model in which osteoporosis is induced by ovariectomy was established.

[0143] The mice were classified into 8 groups as follows, with 10 or 20 mice assigned to each group.

[0144] - NOR group: Normal control group that underwent a sham-operation in which only a peritoneal incision was made, but the ovaries were not removed and the area was sutured.

[0145] - OVX group: Negative control group with osteoporosis induced by ovariectomy

[0146] - E2 group: Positive control group administered estradiol at 10 μg / kg to mice in the OVX group

[0147] - PKDE12 group: Experimental group administered 58 mg / kg of the deer antler extract of Example 1 to mice in the OVX group.

[0148] - PKDE24 group: Experimental group administered 117 mg / kg of the deer antler extract of Example 1 to mice in the OVX group.

[0149] - PKDE48 group: Experimental group administered 233 mg / kg of the deer antler extract of Example 1 to mice in the OVX group.

[0150] - PKDE96 group: Experimental group administered 389 mg / kg of the deer antler extract of Example 1 to mice in the OVX group.

[0151] - PKDE144 group: Experimental group in which mice in the OVX group were administered the deer antler extract of Example 1 at a dose of 700 mg / kg.

[0152] Specifically, for the PKDE12 group, 58 mg / kg of the deer antler extract finally obtained in Example 1 was diluted in distilled water and 100 μL was orally administered to mice in the OVX group at intervals of 5 times per week for a total of 20 times over 4 weeks. Similarly, for the PKDE144 group, 700 mg / kg of the deer antler extract finally obtained in Example 1 was diluted in distilled water and 100 μL was orally administered to mice in the OVX group at intervals of 5 times per week for a total of 20 times over 4 weeks.

[0153] As such, it was confirmed that no specific issues or adverse reactions were observed in mice when the deer antler extract of Example 1 was administered orally. Specifically, no abnormal values ​​were observed in any of the blood hepatotoxicity indicators ALT (Alanine Aminotransferase), AST (Aspartate Aminotransferase), and ALP (Alkaline Phosphatase), nephrotoxicity indicators blood urea nitrogen (BUN) and creatinine, and electrolyte abnormality evaluation items calcium (Ca) and phosphorus (P), and they were found to be within the normal range.

[0154] After concluding the experiment and sacrificing the mice, blood was collected via cardiac puncture and left at room temperature for about 1 hour. Subsequently, serum was separated by centrifugation at 15,000 rpm for 10 minutes, and the separated serum was used to measure the expression levels of osteoporosis-related biomarkers and analyze toxicity assessment items. In addition, the tibia and femur of the mice were separated and used to measure bone mineral density (BMD) and bone mineral content (BMC). Various experimental results are shown in Figures 1 to 19, respectively.

[0155] Figures 1 to 3 are graphs showing the bone mineral density (BMD) of the whole body, tibia, and femur of a mouse, respectively.

[0156] As can be seen in Figure 1, whole-body bone density decreased by 21.39% in the OVX group compared to the NOR group (***p<0.001), but a bone density recovery rate of about 50% was observed in all PKDE48, PKDE96, and PKDE144 groups (*p<0.05).

[0157] As can be seen in Figure 2, tibial bone density decreased by 9.21% in the OVX group compared to the NOR group (***p<0.001), but bone density recovery rates of approximately 63.48%, 76.46%, and 90.54% were observed in the PKDE48, PKDE96, and PKDE144 groups, respectively (***p<0.001). This demonstrates that the effect of the extract of the present invention is concentration-dependent.

[0158] As can be seen in Figure 3, femoral bone density decreased by 13.30% in the OVX group compared to the NOR group (***p<0.001), but the bone density recovery rates were 44.58% in the PKDE12 group, 37.92% in the PKDE24 group, 44.22% in the PKDE48 group, 45.93% in the PKDE96 group, and 43.35% in the PKDE144 group (*p<0.05, **p<0.01).

[0159] Figures 4 to 6 are graphs showing the bone mineral content (BMC) of the whole body, tibia, and femur of a mouse, respectively.

[0160] As can be seen in Figure 4, the total bone mineral content decreased by 18.15% in the OVX group compared to the NOR group (***p<0.001), but an increase in bone mineral content of approximately 52.09%, 70.07%, and 80.50% was observed in the PKDE48, PKDE96, and PKDE144 groups, respectively (*p<0.05). This demonstrates that the effect of the extract of the present invention is concentration-dependent.

[0161] As can be seen in Figure 5, the amount of tibial bone minerals decreased by 13.59% in the OVX group compared to the NOR group (***p<0.001), but an increase in bone minerals of approximately 82.14%, 111.02%, and 103.05% was observed in the PKDE48, PKDE96, and PKDE144 groups, respectively (***p<0.001).

[0162] As can be seen in Figure 6, the femoral bone mineral content decreased by 34.36% in the OVX group compared to the NOR group (***p<0.001), but an increase in bone mineral content was observed in the PKDE12, PKDE24, PKDE48, PKDE96, and PKDE144 groups to a degree similar to that of the E2 group.

[0163] Figures 7 to 9 are graphs showing the values ​​of TRAP 5b (tartrate-resistant acid phosphatase 5b), CTX (C-telopeptide), and NTX (N-telopeptide) in the blood of mice, respectively.

[0164] TRAP 5b, shown in Fig. 7, is an isoenzyme specifically secreted by osteoclasts, unlike TRAP 5a. It is an important biomarker reflecting the activation and number of osteoclasts and is known to indicate the progression of osteoporosis. Serum TRAP 5b increased more than twofold in the OVX group compared to the NOR group (***p<0.001), but decreased by approximately 30.2%, 35.6%, and 53.6% in the PKDE48, PKDE96, and PKDE144 groups, respectively (*p<0.05, ***p<0.001). This demonstrates that the extract of the present invention inhibited osteoclast activity and reduced the bone resorption process.

[0165] CTX, shown in Figure 8, is a C-terminal crosslinked telopeptide, a small fragment released into the blood when collagen is degraded during the process of bone resorption. It is known to be used to monitor the status of bone resorption as an important biomarker for evaluating bone resorption in bone metabolism. Serum CTX increased 2.11-fold in the OVX group compared to the NOR group (***p<0.001), but significant decreases of approximately 27.3% and 31.0% were observed in the PKDE96 and PKDE144 groups, respectively (*p<0.05, **p<0.01).

[0166] NTX, shown in Figure 9, is an N-terminal crosslinked telopeptide that is released into urine or blood as collagen is degraded. It is known to be an important biomarker for evaluating bone resorption in bone metabolism and is used to monitor the status of bone resorption. Serum NTX increased 1.36-fold in the OVX group compared to the NOR group (**p<0.01), but a decrease similar to that of the E2 group was observed in the PKDE12, PKDE24, PKDE48, PKDE96, and PKDE144 groups.

[0167] Figure 10 is a graph showing the osteocalcin levels in the blood of mice. Osteocalcin is a vitamin K-dependent α-carboxyglutamyl acid protein that binds to calcium in the bone structure; it is secreted by osteoblasts, and its blood concentration is known to be an indicator reflecting the degree of bone formation. Although the blood osteocalcin concentration in the OVX group decreased to less than 50% compared to the NOR group (***p<0.001), it was observed that the levels in the PKDE48, PKDE96, and PKDE144 groups increased by approximately 84.28%, 93.83%, and 77.07%, respectively, compared to the OVX group (*p<0.05, **p<0.01).

[0168] Figure 11 is a micrograph for the histopathological analysis of mouse femurs, and Figure 12 is a graph showing the collagen content in femoral tissue. The distal femur is known to be suitable for evaluating the effects of osteoporosis because it contains a relatively large amount of trabecular bone, which has a porous structure, compared to cortical bone. As a result of measuring the collagen content in bone tissue using Picrosirius Red (PSR) staining, a 60.25% decrease in collagen was observed in the OVX group compared to the NOR group (***p<0.001), while significant increases of 29.35% and 43.43% were confirmed in the PKDE96 and PKDE144 groups, respectively (**p<0.01, ***p<0.001).

[0169] Figure 13 is a micrograph confirming changes in the size of adipocytes within the distal femur of a mouse, and Figure 14 is a graph showing the size of the adipocytes. Inflammatory cytokines and adipokines secreted by adipocytes induce inhibition of osteoblast activity and promote osteoclast activity; therefore, an increase in adipocytes is known to inhibit bone formation and promote bone resorption. In the OVX group, more pores were observed within the bone compared to the NOR group, and an increase in the size of adipose tissue within the trabecular bone was observed. The adipose tissue diameter of the NOR group was 22.92 μm and the adipose tissue diameter of the OVX group was 33.30 μm, with the OVX group showing a 45.31% increase compared to the NOR group (***p<0.001). In contrast, the adipocyte sizes of the PKDE96 and PKDE144 groups decreased to 27.32 μm and 26.07 μm, respectively, compared to the OVX group (***p<0.001).

[0170] Figure 15 shows the mRNA expression levels of OPN, BMP-2, and COL-II in the mouse tibia. OPN (Osteopontin) is associated with bone formation and regeneration, BMP-2 (Bone morphogenic protein-2) is a bioactive substance for promoting bone formation that induces cartilage differentiation and bone formation, and COL-II (Type II Collagen) is one of the components of cartilage. Compared to the NOR group, OPN mRNA expression in the OVX group decreased by 64.29% (***p<0.001), BMP-2 mRNA expression decreased by 75.08% (***p<0.001), and COL-II mRNA expression decreased by 90.13% (***p<0.001). On the other hand, in the PKDE48, PKDE96, and PKDE144 groups, it was confirmed that the expression of OPN, BMP-2, and COL-II mRNA involved in osteoblasts increased as the dose increased (**p<0.01, ***p<0.001).

[0171] Figure 16 shows a TRAP staining image of RANK-induced RAW 264.7 osteoclasts. To evaluate the effect of the extract of the present invention on osteoclast differentiation, RAW 264.7 cells were simultaneously treated with the osteoclast-inducing factor RANKL (Receptor activator of nuclear factor kappa-B ligand) and the extract of the present invention to induce differentiation for 5 days, after which differentiation was induced by replacing the medium with fresh medium every 2 days. After differentiation was completed, the cells were stained using a TRAP kit and observed; as a result, cells with three or more nuclei were identified as TRAP-positive multinucleated cells. In the group treated with RANKL alone, large multinucleated osteoclasts were stained red and numerous, whereas in the group treated with the extract of the present invention (indicated as PKDE), a concentration-dependent decrease in the number of red multinucleated cells was observed.

[0172] Figure 17 shows a graph representing the degree of calcium deposition and Alizarin Red S staining in SaOS-2 osteoblast-like cells. SaOS-2 is a cell line derived from human osteosarcoma and is frequently used in bone formation and mineralization experiments due to its characteristics similar to osteoblasts. Specifically, SaOS-2 osteoblast-like cells were treated with Ascorbic Acid (AA) and β-Glycerophosphate (β-GP) to induce the bone formation process while promoting cell differentiation and mineralization. The degree of calcium deposition was determined and the ability to promote osteoblast differentiation was evaluated by staining the calcium deposited during osteoblast differentiation by Alizarin Red S. Compared to the group treated with AA and β-GP alone, 22.82% more calcium deposition was observed in the group treated with 100 μg / mL of the extract of the present invention (***p<0.001).

[0173] Figure 18 is a graph showing the mRNA expression levels of OPN (Osteopontin), IBSP (Integrin Binding Sialoprotein), BMP-2 (Bone Morphogenetic Protein 2), and COL1A1 (Collagen Type I Alpha 1 Chain) as osteoblast differentiation markers in SaOS-2 osteoblast-like cells. OPN influences bone formation by regulating the interaction between osteoblasts and osteoclasts, while IBSP is known to contribute to early mineralization, thereby assisting in the activation of osteoblasts and promoting bone formation together with OPN. In the group treated with 100 μg / mL of the extract of the present invention, OPN and IBSP mRNA expression increased 4.4-fold and 1.5-fold, respectively, compared to the group treated with AA+β-GP alone (***p<0.001). In addition, it was observed that the extract of the present invention increased COL1A1 expression by 2.6-fold in conjunction with OPN and IBSP, which are osteoblast differentiation markers (***p<0.001), demonstrating that OPN and IBSP play a role in strengthening the basic structure of bone by promoting COL1A1 expression. Furthermore, it was observed that the mRNA expression of BMP-2, which supports osteoblast differentiation and bone formation, increased by 3.53-fold in the group treated with 100 μg / mL of the extract of the present invention compared to the group treated with AA+β-GP alone (***p<0.001). This suggests the possibility that the extract of the present invention promotes osteoblast activation, contributes to the bone formation process, and acts as a key factor in regulating osteoblast differentiation and ossification.

[0174] Figure 19 is a graph showing the expression levels of ALP (Alkaline Phosphatase), OSX (Osterix), and OCN (Osteocalcin) in SaOS-2 osteoblast-like cells. The expression of ALP, a marker that can confirm activity involved in the early osteoblast process, increased 2.6-fold in the group treated with 100 μg / mL of the extract of the present invention compared to the group treated with AA+β-GP alone (***p<0.001). In addition, protein expression of OSX, which induces the expression of bone formation-related genes such as OCN and COL1A1, increased 2.8-fold at 1 μg / mL (**p<0.01), 5.5-fold at 10 μg / mL, and 7.9-fold at 100 μg / mL in a concentration-dependent manner with the extract of the present invention (***p<0.001). In addition, the protein expression level of OCN, which is a protein produced by mature osteoblasts and is considered a final marker of bone formation as it contributes to bone mineralization and bone metabolism, increased in a concentration-dependent manner with the extract of the present invention compared to the AA+β-GP single treatment group: 1.6 times at 1 μg / mL (*p<0.05), 2.1 times at 10 μg / mL, and 2.8 times at 100 μg / mL (***p<0.001). This suggests that PKDE is involved in the maturation process from the early stages of osteoblasts, as evidenced by the increase in the expression of OCN, a marker of mature osteoblasts, as well as the increase in the expression of ALP, an early factor of osteoblasts, upon treatment with the extract of the present invention.

[0175] The results confirmed through various experiments as described above demonstrate that the extract of the present invention is effective in improving bone density and bone mineral content, promotes bone formation, and is involved in the maturation process of osteoblasts from the early stages. These results prove that the extract of the present invention exhibits excellent effects in the improvement, prevention, and treatment of bone diseases by supporting bone formation through the regulation of the balance between osteoblasts and osteoclasts.

Claims

1. A method for preparing a deer antler extract comprising the following steps: a) A step of extracting deer antlers with an ethanol solvent; b-1) A step of separating the deer antler extract obtained in step a) above; b-2) a step of hot water extraction of the deer antler extract residue obtained in step a) above; and c) A step of mixing the product of step b-1) and the product of step b-2).

2. In Paragraph 1, A method for preparing a deer antler extract, characterized in that the ethanol in step a) above is 35% to 10% ethanol.

3. In Paragraph 1, A method for preparing a deer antler extract, characterized in that in step a) above, ethanol is used in a volume 5 to 20 times the weight or volume of the deer antler.

4. In Paragraph 1, A method for preparing a deer antler extract, characterized in that step a) above is performed at room temperature.

5. In Paragraph 1, A method for preparing a deer antler extract, characterized by further including the step of extracting the deer antler with an ethanol solvent and filtering it in step a) above.

6. In Paragraph 1, A method for preparing a deer antler extract, characterized in that the above step b-2) is performed at 90℃ to 100℃.

7. In Paragraph 1, A method for preparing a deer antler extract, characterized in that the water used for hot water extraction in step b-2) above is used in a volume 5 to 20 times the weight or volume of the deer antler.

8. A composition comprising a deer antler extract prepared according to the manufacturing method of claim 1, A composition containing uracil, hypoxanthin, uridine, and inosine.

9. In Paragraph 8, A composition comprising deer antler extract, characterized in that the above composition is a food composition for the improvement or prevention of bone diseases.

10. In Paragraph 8, A composition comprising a deer antler extract, characterized in that the above composition is a pharmaceutical composition for the treatment or prevention of bone diseases.

11. In Paragraph 8, A composition comprising deer antler extract, characterized in that the above composition is a quasi-drug composition for the improvement or prevention of bone diseases.

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