WO2021096098A1 - Composition containing winter melon extract as active ingredient for increasing bone health - Google Patents

Composition containing winter melon extract as active ingredient for increasing bone health Download PDF

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Publication number
WO2021096098A1
WO2021096098A1 PCT/KR2020/014640 KR2020014640W WO2021096098A1 WO 2021096098 A1 WO2021096098 A1 WO 2021096098A1 KR 2020014640 W KR2020014640 W KR 2020014640W WO 2021096098 A1 WO2021096098 A1 WO 2021096098A1
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Prior art keywords
bone
extract
active ingredient
copper
fruit extract
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PCT/KR2020/014640
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French (fr)
Korean (ko)
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조주현
강준철
조현덕
이다영
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주식회사 하람
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Publication of WO2021096098A1 publication Critical patent/WO2021096098A1/en

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    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105Plant extracts, their artificial duplicates or their derivatives
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20Removal of unwanted matter, e.g. deodorisation or detoxification
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/42Cucurbitaceae (Cucumber family)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00Function of food ingredients
    • A23V2200/30Foods, ingredients or supplements having a functional effect on health
    • A23V2200/306Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention

Definitions

  • the present invention relates to a composition for improving bone health comprising a copper fruit extract as an active ingredient, and can be used as a variety of uses, such as pharmaceuticals and foods.
  • Food contains substances necessary to maintain human life phenomena, and humans maintain life by ingesting food, digesting it in the digestive system, absorbing it, and supplying it to the tissue cells in the body.
  • Nutrition directly and indirectly causes diseases, and is known to be involved in the prevention and treatment of diseases.
  • the lack of nutrients caused by poorly balanced intake of nutrients necessary for the human body is a problem, and excessive intake is also a serious problem. , This can cause various diseases.
  • Health promotion is aimed at increasing resistance to diseases, especially chronic degenerative diseases or infections, continuous stress, and increasing daily activity. To this end, improving nutrition, strengthening immune function, proper exercise and rest, and mental activity. Management that affects the overall lifestyle, such as sustainability, is required.
  • growth is defined in a broad sense, it can be said that it includes not only an increase in height but also an increase in the size and function of each organ in the body.
  • the increase in kidney is achieved through skeletal metabolism, including the synthesis of cartilage tissue, skeletal length growth, and extensive proliferation of skeletal tissue by various factors such as nutrition and growth hormone.
  • Bone ( ⁇ ) tissue is a specially differentiated form of connective tissue, which is a connective tissue composed of several types of cells such as mesenchymal cells, chondrocytes, osteoblasts, osteoclasts, and bone marrow cells.
  • a balance is maintained between the amount of bone resorption by osteoclasts and the amount of bone formation by osteoblasts.
  • the bone formation ability by the osteoblasts reaches its peak and the amount of bone formation far exceeds the amount of bone resorption, so bone growth is achieved.
  • growth hormone therapy which was used for poor growth in a pathological condition in the past, has been applied to growing children and adolescents with normal height as interest in height growth has increased in recent years.
  • the administration of the growth hormone formulation has excellent effects in the case of people who are deficient in growth hormone, but in the majority of people with normal hormone secretion, various side effects such as acromegaly, positive growth hormone antibody, systemic allergic reaction, hypothyroidism, etc. There is a problem that can cause.
  • the copper fruit extract can help improve bone health by promoting the differentiation of osteoblasts involved in bone formation.
  • the present invention is to solve the problems of the prior art described above, an object of the present invention is to provide a functional food drug derived from a natural plant excellent in biosafety.
  • a food composition for improving bone health comprising a copper fruit extract as an active ingredient.
  • the extract may be extracted with water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane, or a mixed solvent thereof.
  • a food composition for promoting growth comprising a copper fruit extract as an active ingredient.
  • a food composition for preventing or improving bone disease comprising a copper fruit extract as an active ingredient.
  • the bone disease may be one or more selected from the group consisting of osteoporosis, bone insufficiency, osteomalacia, bone mass reduction, fracture, bone defect, and hip joint reduction.
  • a pharmaceutical composition for promoting growth or improving bone health comprising a copper fruit extract as an active ingredient.
  • a pharmaceutical composition for preventing or treating bone diseases comprising a copper fruit extract as an active ingredient.
  • the bone disease may be one or more selected from the group consisting of osteoporosis, bone insufficiency, osteomalacia, bone mass reduction, fracture, bone defect, and hip joint reduction.
  • composition according to an aspect of the present invention includes a natural extract as an active ingredient, so that it is not only excellent in biosafety, but also has excellent bone health improvement effects, so it can be usefully used as a health functional food as well as a bone disease treatment.
  • Figure 2 is a result of evaluating the osteoblast differentiation promoting activity of the copper fruit extract according to an embodiment of the present invention.
  • (A) is a copper and hot water extract
  • (B) is a copper and ethanol extract.
  • FIG. 3 and 4 are results of evaluating the ALP activity of the copper fruit extract according to an embodiment of the present invention.
  • (A) is a copper and hot water extract
  • (B) is a copper and ethanol extract.
  • the numerical range includes the numerical values defined in the above range. All maximum numerical limits given throughout this specification include all lower numerical limits as if the lower numerical limits were expressly written. All minimum numerical limits given throughout this specification include all higher numerical limits as if the higher numerical limits were expressly written. All numerical limits given throughout this specification will include all better numerical ranges within the wider numerical range, as if the narrower numerical limits were expressly written.
  • a food composition for preventing or improving bone disease comprising a copper fruit extract as an active ingredient.
  • the "winter melon" is an annual vine plant of the gourd family and is also called Dong-A or Fairy Tale, and its scientific name is Benincasa hispida , Benincasa cerifera , Benincasa cerifera Savi Etc.
  • the bone disease refers to a condition or disease in which an increase in bone mass is required or required through promoting the activity of osteoblasts or inhibiting the activity of osteoclasts, and may be, for example, a metabolic bone disease or an orthopedic bone disease. .
  • the “metabolic bone disease” refers to a condition or disease that appears due to excessive production or activity of osteoclasts, and may include a bone mass-lowering disease.
  • the bone mass-lowering disease refers to a condition or disease in which bone mass decreases accompanied by symptoms such as a decrease in bone density and softening of bone tissue.
  • the bone disease may be one or more selected from the group consisting of osteoporosis, bone insufficiency, osteomalacia, bone mass loss, fracture, bone defect, and hip joint loss, but is not limited thereto.
  • the "contained as an active ingredient” means containing an amount sufficient to provide a therapeutic or prophylactic effect to the individual to be administered, and the copper fruit extract is excellent in biosafety, so those skilled in the art can appropriately set the upper limit of the quantity. Can be adjusted.
  • extract refers to a solvent in which the active ingredient contained in the raw material for extraction is transferred by contacting the solvent with the raw material for extraction under specific conditions. You can include all regardless. For example, it may include extracting a component soluble in a solvent from a natural product using water or an organic solvent, and extracting a specific component of a natural product, such as extracting only a specific component such as an oil.
  • the extract can be obtained by drying and powdering the copper fruit, or by various extraction methods known in the art such as hot water extraction and ethanol extraction.
  • the extract may be extracted with water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane, or a mixed solvent thereof, and preferably may be extracted with ethanol at a concentration of 60 to 90% (w/w).
  • the extraction ratio of the active ingredient contained in the raw material may be different depending on the polarity of the solvent, it may be varied in consideration of the selectivity of the physiologically active substance according to the solvent.
  • the extract is extracted by conventional methods such as reflux extraction, pressure extraction, ultrasonic extraction, etc. for about 1 to 24 hours with a solvent having a volume of 8 to 12 times the weight of the raw material by washing the extraction raw material with water, drying and grinding, and It can be prepared by filtration.
  • the extract can be obtained in powder form by an additional process such as distillation under reduced pressure or freeze drying.
  • the extract may also include an extract that has undergone a conventional purification process.
  • the extract may be subjected to various additional purification methods, such as separation using an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatography (one prepared for separation according to size, charge, hydrophobicity, or affinity). It may contain a fraction obtained through.
  • the food composition may be used for promoting growth or improving bone health.
  • the “growth” may mean not only an increase in height, but also an increase in the anatomical and morphological size and function of each organ of the body, that is, the development of functions and differentiation into organs.
  • the food composition including copper fruit extract, can grow bone length, promote the metabolism of growth plate cartilage cells, and so forth, without side effects, and can bring about excellent growth promoting effect, so that it can be particularly useful as a food material for promoting children's growth.
  • the “bone health” refers to normal bone decomposition and remodeling, and bone-related diseases such as osteoporosis and osteoarthritis may occur if the balance of bone decomposition and remodeling is broken due to insufficient calcium in the body or due to various external factors. .
  • the food composition can effectively maintain joint and bone health by affecting the differentiation of osteoblasts and promoting bone regeneration.
  • the food composition can be used as a health functional food related to bone health improvement, growth promotion, and bone disease.
  • the health functional food refers to a food manufactured and processed using raw materials or ingredients that have useful functionality for the human body according to the Health Functional Food Act, and the functionality is to control nutrients for the structure and function of the human body or It may mean ingestion for the purpose of obtaining a useful effect for health purposes such as action.
  • the food composition may contain ordinary food additives, and the food additives are subject to the standards and standards for the relevant item according to the general rules and general test methods of food additives approved by the Ministry of Food and Drug Safety, unless otherwise specified. Whether it is appropriate or not can be determined.
  • the items described in the food additive code include, for example, ketones, glycine, potassium citrate, nicotinic acid, cinnamic acid, and other chemical compounds, dyestuffs, licorice extract, crystalline cellulose, high-volume pigments, natural additives such as guar gum, sodium L-glutamate preparations, and noodles.
  • Mixed preparations such as an added alkali agent, a preservative preparation, and a tar color preparation, are mentioned.
  • the food composition may be variously used in foods and beverages for enhancing immunity or improving bone disease, and for example, it may be used in various foods, beverages, gum, tea, vitamin complexes, health functional supplements, food additives, and the like. .
  • the health functional food may be manufactured and processed into any one formulation selected from the group consisting of tablets, granules, powders, capsules, liquid solutions, and pills for the purpose of enhancing immunity or improving bone disease.
  • a mixture of copper fruit extract, excipients, binders, disintegrants and other additives is granulated in a conventional manner, and then compression molded by putting a lubricant or the like, or directly compressing the mixture. It can be manufactured by molding.
  • the health functional food in the form of a tablet may contain a mating agent or the like, if necessary, and may be coated with a suitable coating agent if necessary.
  • hard capsules can be prepared by filling a conventional hard capsule with a mixture of additives such as copper fruit extract and excipients, granules thereof, or coated granules, and soft capsules are copper fruit extracts. And a mixture with an additive such as an excipient may be filled in a capsule base such as gelatin.
  • the soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.
  • the ring-shaped health functional food can be prepared by molding a mixture of copper fruit extract, excipient, binder, and disintegrant by an appropriate method. Substances can also be used to create a pleasing feeling.
  • the granular health functional food may be prepared in a granular form of a mixture of copper fruit extract, excipients, binders, disintegrants, and the like by a suitable method, and may contain flavoring agents, flavoring agents, and the like, if necessary.
  • a pharmaceutical composition for preventing or treating bone diseases comprising a copper fruit extract as an active ingredient.
  • prevention refers to any action that reduces the frequency or severity of pathological symptoms. Prevention can be complete or partial. In this case, it may mean a phenomenon in which symptoms caused by inflammation in the individual decrease compared to the case where the composition is not used.
  • treatment refers to any action that clinically intervenes in order to change the natural process of a target or cell to be treated, and may be performed while a clinical pathology is in progress or to prevent it.
  • the desired therapeutic effect is to prevent the occurrence or recurrence of the disease, alleviate symptoms, reduce all direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, or It may include alleviating or temporarily alleviating the condition, or improving the prognosis.
  • the composition may be administered in the form of oral delivery or parenteral delivery.
  • the composition may be administered systemically or locally, and the administration may include oral administration and parenteral administration.
  • the composition may be formulated with a suitable amount of a pharmaceutically acceptable vehicle or carrier to provide an appropriate dosage form.
  • the composition may further include a carrier, an excipient, and a diluent used in the preparation of a pharmaceutical composition.
  • the carrier, excipient and diluent 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, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil, but are not limited thereto.
  • composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile injectable solutions.
  • oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile injectable solutions.
  • Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient, such as starch, calcium carbonate, and sucrose, in the copper fruit extract and its fractions. , Lactose, or gelatin may be mixed and prepared. In addition, in addition to the excipients, lubricants such as magnesium stearate and talc may be used.
  • Liquid preparations for oral administration may include suspensions, liquid solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., may be used in addition to simple diluents such as water and liquid paraffin.
  • Formulations for parenteral administration may be sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories.
  • aqueous solutions non-aqueous solutions
  • suspensions emulsions
  • lyophilized formulations emulsions
  • suppositories emulsions
  • suppositories propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate
  • injectable ester such as ethyl oleate
  • witepsol macrogol, tween 61, cacao butter, laurin paper, and glycerogelatin may be used.
  • the pharmaceutical composition may be administered to a subject in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, drug activity, drug Sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.
  • the pharmaceutical composition may be administered as an individual therapeutic agent or administered in combination with another therapeutic agent, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is preferable to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all of the above factors, and this can be easily determined by a person skilled in the art.
  • the composition may be used for one or more materials selected from the group consisting of artificial bones, artificial joints, bone fixation materials, bone substitutes, bone repair materials, bone fillers, and bone graft materials.
  • the composition for promoting bone formation may be used for the prevention and treatment of bone disease or periodontal disease, and may be prepared in a form that is easy to be applied to artificial bone, bone fixation material, bone substitute, bone repair material, bone filler, or bone graft material. .
  • the bone graft material or bone substitute may be used to fill a space in bone tissue in a narrow way, and may be used in a broad sense to replace part of a bone or tooth. Therefore, the composition for promoting bone formation can be used in the form of putty, paste, moldable strips, blocks, chips, etc. using methods such as compression, compression, pressure contact, packing, compression, and hardening, and chemical additives are used. Thus, it may be formulated and used in the form of gels, granules, pastes, tablets, pellets, etc., and may also be used in powder form.
  • the obtained liquid extract was concentrated (20 brix) with a rotary vacuum concentrator, and then a copper fruit extract was obtained using a freeze dryer.
  • the yield of the extract obtained by powdering was 51.6% compared to the dried product.
  • the obtained liquid extract was concentrated (20 brix) with a rotary vacuum concentrator, and then a copper fruit extract was obtained using a freeze dryer.
  • the yield of the extract obtained by powdering was 53.5% compared to the dried product.
  • the cell line RAW264.7 macrophage cell was purchased and used from the American Type Culture Collection (ATCC, Manassas. USA).
  • DMEM Dulbecco's modified of Eagle's medium, Hyclone, USA
  • FBS Fetal Bovin Serum
  • penicillin/Streptoycin 100 units/mL penicillin/Streptoycin (Hyclone, USA) was used, 37 C. Incubated in an incubator (Thermo, Forma, Germany) in the presence of 5% CO 2.
  • Cell viability was measured by measuring the concentration of the produced formazan with a spectrophotometer using the change from Tetrazolium salt (WST-1) to formazan by mitochondrial dehydrogenase in living cells.
  • MTT assay was performed according to the protocol using Ez-cytox (Dozen, Korea).
  • Raw264.7 cells were dispensed into 96 wells at 5 ⁇ 10 3 cells/100 ⁇ L and incubated for 24 hours in a 37°C, 5% CO 2 incubator.
  • Toxicity evaluation based on cell viability for each sample was measured by repeating three times, and the mean value and standard deviation were calculated.
  • the copper fruit extract (Preparation Example 1), and the copper fruit ethanol extract (Preparation Example 2) showed no cytotoxicity in all concentrations (10, 100, 1000 ⁇ g/mL) range.
  • osteoblasts (MC3T3-E1) were dispensed into 96 wells at 5x10 3 cells/100 ⁇ L and stabilized for 24 hours.
  • Samples were treated by concentration and incubated in a CO 2 incubator for 48 hours. After incubation, MTT (EZ-cytox, Daillab) solution was treated and CO 2 was incubated for 2 hours, and then analyzed with a microplate reader (Epoch, Biotek) at 450 nm wavelength.
  • MTT EZ-cytox, Daillab
  • the osteoblast differentiation amount increased by 24% compared to the control at 100 ⁇ g/mL, and about 49% increased compared to the control at 1000 ⁇ g/mL.
  • the osteoblast differentiation amount increased by about 21% compared to the control at 100 ⁇ g/mL, and about 41% compared to the control at 1000 ⁇ g/mL.
  • ALP alkaline phosphatase
  • MC3T3-E1 (osteocyte) was dispensed into 48 wells at 5x10 3 cells/250 ⁇ L and stabilized for 24 hours.
  • Samples were treated by concentration and incubated in a CO 2 incubator for 7 days. After washing with PBS, lysis buffer was added to dissolve, and centrifuged at 12,000 rpm for 15 minutes.
  • the supernatant was collected by precipitating cell membrane components, and the ALP activity in the supernatant was quantified using an ALP assay kit.
  • the copper and hot water extract (Preparation Example 1) increased by 32% compared to the control at 100 ⁇ g/mL, and increased by 105% at 1000 ⁇ g/mL.
  • Protein concentration was normalized using bovine serum albumin (BSA). The lysate was separated by 30 ⁇ g 10% gel SDS-PAGE and transferred to the PVDF membrane for 120 V for 90 minutes.
  • BSA bovine serum albumin
  • Membrane blocking was performed for 1 hour in a TBS-T solution containing 5% skim milk.
  • the primary antibody (1:100 ⁇ 1000) was incubated at 4°C for one hour and then washed three times with TBS-T the next day.
  • HRP horse radish peroxidase
  • the copper and hot water extract (Preparation Example 1) increased by about 62% compared to the control at 100 ⁇ g/mL, and about 118% at 1000 ⁇ g/mL.
  • mice with a skull defect were treated with copper fruit extract, BMP-2 or a control group, and then micro-CT ( ⁇ CT) and histological analysis were performed.
  • ⁇ CT micro-CT
  • micro-CT micro-CT
  • the measurement method is a method for observing normal bone metabolism and uses a phenomenon in which a fluorescent factor is deposited on the bone through a chelate bond with calcium.
  • the administered fluorescent factor When a fluorescent factor is administered during the growth period in which bone formation is most active, the administered fluorescent factor most often deposits in the bone neoplasm under the bone growth plate to form a line. Was observed with a fluorescence microscope to measure the grown length.
  • the collected section was placed on a slide glass, dried, and the length between the growth plate and the line formed by the deposition of fluorescent factors in the bone tissue was measured using a fluorescence microscope to measure the degree of growth of the ilium of the rat.

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Abstract

The present invention relates to a food product and a pharmaceutical composition containing a winter melon extract as an active ingredient for increasing bone health.

Description

동과 추출물을 유효성분으로 함유하는 뼈 건강 개선용 조성물Composition for improving bone health containing copper fruit extract as an active ingredient
본 발명은 동과 추출물을 유효성분으로 포함하는 뼈 건강 개선용 조성물에 관한 것으로, 의약품 및 식품 등 다양한 용도로서 활용 가능하다.The present invention relates to a composition for improving bone health comprising a copper fruit extract as an active ingredient, and can be used as a variety of uses, such as pharmaceuticals and foods.
모든 생물체는 생명을 영위하기 위해 끊임없이 많은 영양소를 필요로 하며, 이러한 영양소는 식품의 형태로 항상 외부로부터 공급되거나 일부는 체내에서 합성된다.All living organisms constantly need a lot of nutrients in order to live life, and these nutrients are always supplied from the outside in the form of food or some of them are synthesized in the body.
음식물 속에는 인간의 생명현상을 유지하는데 필요한 물질들이 포함되어 있으며, 인간은 음식물을 섭취하여 소화기관에서 소화시키고 흡수하여 체내의 조직세포에 공급함으로써 생명을 유지하고 있다.Food contains substances necessary to maintain human life phenomena, and humans maintain life by ingesting food, digesting it in the digestive system, absorbing it, and supplying it to the tissue cells in the body.
따라서 좋은 영양은 건강한 신체를 유지시켜주며, 건강한 신체는 건강한 정신을 보장한다.Therefore, good nutrition maintains a healthy body, and a healthy body guarantees a healthy mind.
영양은 직접, 간접으로 질병의 원인이 되기도 하며, 질병의 예방 및 치료에도 관여하는 것으로 알려져 있는데, 인체에 필요한 영양소의 균형섭취 제대로 이루어지지 않아 생기는 영양분의 부족도 문제이고, 과잉섭취 또한 심각한 문제이며, 이로 인해 각종 질병이 유발될 수 있다.Nutrition directly and indirectly causes diseases, and is known to be involved in the prevention and treatment of diseases.The lack of nutrients caused by poorly balanced intake of nutrients necessary for the human body is a problem, and excessive intake is also a serious problem. , This can cause various diseases.
최근 생활수준이 높아지고 식생활이 서구화되면서 영양과잉, 운동부족 등으로 인한 비만, 당뇨, 고혈압 및 심장병 등과 같은 성인병 발병률이 증가하고 있다. 또한 평균수명 연장으로 인하여 인구가 점차 노령화되면서 건강에 대한 관심이 점차 높아지고 있다.Recently, as the standard of living has increased and the diet has become westernized, the incidence of adult diseases such as obesity, diabetes, high blood pressure and heart disease due to overnutrition and lack of exercise is increasing. In addition, as the population gradually ages due to the extended life expectancy, interest in health is gradually increasing.
건강증진(health promotion)은 질병, 특히 만성 퇴행성질환이나 감염증, 계속되는 스트레스에 대한 저항력의 증가, 일상의 활동력의 증대를 꾀하는 것으로 이를 위해 영양의 개선, 면역기능 강화, 적절한 운동과 휴식, 정신적 활동의 지속 등 생활양식 전반에 미치는 관리가 요구된다. Health promotion is aimed at increasing resistance to diseases, especially chronic degenerative diseases or infections, continuous stress, and increasing daily activity. To this end, improving nutrition, strengthening immune function, proper exercise and rest, and mental activity. Management that affects the overall lifestyle, such as sustainability, is required.
한편, 최근 들어 체형의 서구화 및 평균 신장의 상승으로 인하여 키 성장인자에 대한 관심이 높아지고 있다. On the other hand, in recent years, due to the westernization of body shape and an increase in average height, interest in height growth factors is increasing.
성장을 광의의 개념으로 정의하면 신장의 증가뿐 아니라 신체 각 기관의 크기와 기능의 증대를 포함하는 것이라 할 것이나, 이를 협의의 개념으로 정의하자면 신장의 증가를 의미하는 것으로 볼 수 있다.If growth is defined in a broad sense, it can be said that it includes not only an increase in height but also an increase in the size and function of each organ in the body.
신장의 증가는 영양과 성장 호르몬 등의 여러 요인에 의한 연골 조직의 합성, 골격 길이 성장 및 골격 조직의 광범위한 증식을 포함하는 골격 대사를 통하여 이루어진다.The increase in kidney is achieved through skeletal metabolism, including the synthesis of cartilage tissue, skeletal length growth, and extensive proliferation of skeletal tissue by various factors such as nutrition and growth hormone.
골(骨) 조직은 특수하게 분화된 형태의 결합 조직으로, 간엽세포, 연골세포, 조골세포, 파골세포, 골수세포 등 여러 종류의 세포로 구성된 결합조직이다.Bone (骨) tissue is a specially differentiated form of connective tissue, which is a connective tissue composed of several types of cells such as mesenchymal cells, chondrocytes, osteoblasts, osteoclasts, and bone marrow cells.
파골세포에 의한 골 흡수량과 조골세포에 의한 골 형성량 사이에는 균형이 유지되고 있는데, 성장기에는 조골세포에 의한 골 형성 능력이 최고치에 이르러 골 형성량이 골 흡수량을 훨씬 능가하게 되므로 뼈 성장이 이루어진다.A balance is maintained between the amount of bone resorption by osteoclasts and the amount of bone formation by osteoblasts. During the growth phase, the bone formation ability by the osteoblasts reaches its peak and the amount of bone formation far exceeds the amount of bone resorption, so bone growth is achieved.
특히 과거 병적인 상태의 성장부진에 쓰이던 성장호르몬 요법이 근래에 키 성장에 대한 관심이 높아져 감에 따라 정상 신장을 가지고 있는 성장기 어린이 및 청소년에게도 적용되고 있다.In particular, growth hormone therapy, which was used for poor growth in a pathological condition in the past, has been applied to growing children and adolescents with normal height as interest in height growth has increased in recent years.
그러나 상기 성장 호르몬 제제 투여법은 성장 호르몬이 부족한 사람의 경우에는 뛰어난 효과를 보이지만, 호르몬 분비가 정상적인 대다수의 사람들에게는 말단 비대증, 성장 호르몬 항체 양성, 전신 알레르기 반응, 갑상선 기능 저하증 등의 여러 가지 부작용을 야기할 수 있는 문제점이 있다. However, the administration of the growth hormone formulation has excellent effects in the case of people who are deficient in growth hormone, but in the majority of people with normal hormone secretion, various side effects such as acromegaly, positive growth hormone antibody, systemic allergic reaction, hypothyroidism, etc. There is a problem that can cause.
뿐만 아니라 성장호르몬 요법의 기간과 비용이 과도하고, 암 발생 및 다른 성장인자들의 교란을 일으키는 등의 문제점이 발견되고 있으며, 성장 촉진을 위한 건강보조식품은 과학적으로 검증되지 않은 경우가 대부분이다.In addition, problems such as excessive duration and cost of growth hormone therapy, occurrence of cancer and disturbance of other growth factors have been found, and health supplements for promoting growth are not scientifically verified.
따라서, 성장 촉진에 있어서 그 효능이 과학적으로 검증되고, 안전한 식품소재의 개발이 필요한 실정이다.Therefore, its efficacy in promoting growth has been scientifically verified, and there is a need for the development of safe food ingredients.
본 발명자들은 연구를 통해 동과 추출물이 뼈의 형성에 관여하는 조골세포의 분화를 촉진함으로써 뼈 건강 개선에도 도움을 줄 수 있음을 확인하였다.The present inventors have confirmed that through research, the copper fruit extract can help improve bone health by promoting the differentiation of osteoblasts involved in bone formation.
본 발명은 전술한 종래 기술의 문제점을 해결하기 위한 것으로, 본 발명의 목적은 생체 안전성이 우수한 천연 식물 유래의 기능성 식의약품을 제공하는 것이다.The present invention is to solve the problems of the prior art described above, an object of the present invention is to provide a functional food drug derived from a natural plant excellent in biosafety.
본 발명의 일 측면에 따르면, 동과 추출물을 유효성분으로 포함하는 뼈 건강 개선용 식품 조성물이 제공된다.According to an aspect of the present invention, there is provided a food composition for improving bone health comprising a copper fruit extract as an active ingredient.
일 실시예에 있어서, 상기 추출물은 물, 알코올, 에틸아세테이트, 아세톤, 핵산, 디클로로메탄 또는 이들의 혼합 용매로 추출될 수 있다.In one embodiment, the extract may be extracted with water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane, or a mixed solvent thereof.
본 발명의 다른 측면에 따르면, 동과 추출물을 유효성분으로 포함하는 성장 촉진용 식품 조성물이 제공된다.According to another aspect of the present invention, there is provided a food composition for promoting growth comprising a copper fruit extract as an active ingredient.
본 발명의 다른 측면에 따르면, 동과 추출물을 유효성분으로 포함하는 골 질환 예방 또는 개선용 식품 조성물이 제공된다.According to another aspect of the present invention, there is provided a food composition for preventing or improving bone disease comprising a copper fruit extract as an active ingredient.
일 실시예에 있어서, 상기 골 질환은 골다공증, 골 형성 부전증, 골연화증, 골량 감소증, 골절, 골 결손 및 고관절 감소증으로 이루어진 군에서 하나 이상 선택될 수 있다.In one embodiment, the bone disease may be one or more selected from the group consisting of osteoporosis, bone insufficiency, osteomalacia, bone mass reduction, fracture, bone defect, and hip joint reduction.
본 발명의 다른 측면에 따르면, 동과 추출물을 유효성분으로 포함하는 성장 촉진 또는 뼈 건강 개선용 약학적 조성물이 제공된다.According to another aspect of the present invention, there is provided a pharmaceutical composition for promoting growth or improving bone health comprising a copper fruit extract as an active ingredient.
본 발명의 다른 측면에 따르면, 동과 추출물을 유효성분으로 포함하는 골 질환의 예방 또는 치료용 약학적 조성물이 제공된다.According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating bone diseases comprising a copper fruit extract as an active ingredient.
일 실시예에 있어서, 상기 골 질환은 골다공증, 골 형성 부전증, 골연화증, 골량 감소증, 골절, 골 결손 및 고관절 감소증으로 이루어진 군에서 하나 이상 선택될 수 있다.In one embodiment, the bone disease may be one or more selected from the group consisting of osteoporosis, bone insufficiency, osteomalacia, bone mass reduction, fracture, bone defect, and hip joint reduction.
본 발명의 일 측면에 따른 조성물은 천연 추출물을 유효성분으로 포함하여 생체 안전성이 우수할 뿐만 아니라 및 뼈 건강 개선 효과가 우수하므로 건강기능식품뿐만 아니라 골 질환 치료 용도로서 유용하게 활용할 수 있다.The composition according to an aspect of the present invention includes a natural extract as an active ingredient, so that it is not only excellent in biosafety, but also has excellent bone health improvement effects, so it can be usefully used as a health functional food as well as a bone disease treatment.
본 발명의 효과는 상기한 효과로 한정되는 것은 아니며, 본 발명의 상세한 설명 또는 청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.
도 1은 본 발명의 일 실시예에 따른 동과 추출물의 세포 독성을 평가한 결과이다.1 is a result of evaluating the cytotoxicity of a copper fruit extract according to an embodiment of the present invention.
도 2는 본 발명의 일 실시예에 따른 동과 추출물의 조골세포 분화 촉진 활성을 평가한 결과이다. (A)는 동과 열수 추추물, (B)는 동과 에탄올 추출물이다.Figure 2 is a result of evaluating the osteoblast differentiation promoting activity of the copper fruit extract according to an embodiment of the present invention. (A) is a copper and hot water extract, and (B) is a copper and ethanol extract.
도 3 및 4는 본 발명의 일 실시예에 따른 동과 추출물의 ALP활성을 평가한 결과이다. (A)는 동과 열수 추추물, (B)는 동과 에탄올 추출물이다.3 and 4 are results of evaluating the ALP activity of the copper fruit extract according to an embodiment of the present invention. (A) is a copper and hot water extract, and (B) is a copper and ethanol extract.
본 명세서에서 사용되는 용어는 본 발명에서의 기능을 고려하면서 가능한 현재 널리 사용되는 일반적인 용어들을 선택하였으나, 이는 당 분야에 종사하는 기술자의 의도 또는 판례, 새로운 기술의 출현 등에 따라 달라질 수 있다. Terms used in the present specification have selected general terms that are currently widely used as possible while taking functions of the present invention into consideration, but this may vary according to the intention or precedent of a technician working in the field, the emergence of new technologies, and the like.
또한, 특정한 경우는 출원인이 임의로 선정한 용어도 있으며, 이 경우 해당되는 발명의 설명 부분에서 상세히 그 의미를 기재할 것이다. 따라서 본 발명에서 사용되는 용어는 단순한 용어의 명칭이 아닌, 그 용어가 가지는 의미와 본 발명의 전반에 걸친 내용을 토대로 정의되어야 한다.In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning of the terms will be described in detail in the description of the corresponding invention. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall contents of the present invention, not a simple name of the term.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다. Unless otherwise defined, all terms used herein including technical or scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in the present application. Does not.
수치 범위는 상기 범위에 정의된 수치를 포함한다. 본 명세서에 걸쳐 주어진 모든 최대의 수치 제한은 낮은 수치 제한이 명확히 쓰여져 있는 것처럼 모든 더 낮은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 최소의 수치 제한은 더 높은 수치 제한이 명확히 쓰여져 있는 것처럼 모든 더 높은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 수치 제한은 더 좁은 수치 제한이 명확히 쓰여져 있는 것처럼, 더 넓은 수치 범위 내의 더 좋은 모든 수치 범위를 포함할 것이다.The numerical range includes the numerical values defined in the above range. All maximum numerical limits given throughout this specification include all lower numerical limits as if the lower numerical limits were expressly written. All minimum numerical limits given throughout this specification include all higher numerical limits as if the higher numerical limits were expressly written. All numerical limits given throughout this specification will include all better numerical ranges within the wider numerical range, as if the narrower numerical limits were expressly written.
이하, 본 발명의 실시예를 상세히 기술하나, 하기 실시예에 의해 본 발명이 한정되지 아니함은 자명하다.Hereinafter, examples of the present invention will be described in detail, but it is obvious that the present invention is not limited by the following examples.
본 발명의 일 측면에 따르면, 동과 추출물을 유효성분으로 포함하는 골 질환 예방 또는 개선용 식품 조성물이 제공된다.According to an aspect of the present invention, there is provided a food composition for preventing or improving bone disease comprising a copper fruit extract as an active ingredient.
상기 “동과(winter melon)”는 박과의 한해살이 덩굴성 식물로 동아 또는 동화라고도 불리우며, 학명은 Benincasa hispida, Benincasa cerifera, Benincasa cerifera Savi 등이다.The "winter melon" is an annual vine plant of the gourd family and is also called Dong-A or Fairy Tale, and its scientific name is Benincasa hispida , Benincasa cerifera , Benincasa cerifera Savi Etc.
상기 동과는 열대 아시아산으로 중국을 통하여 우리나라에 전래되어 재배대기 시작한 것으로 알려져 있다. 상기 동과는 식용 또는 약용하여 왔고, 과거에는 동화선, 동화누르미, 동화적, 동화돈채 등 다양한 조리법으로 식용한 것으로 보고되고 있다. It is known that the winter fruit is produced in tropical Asia and has been introduced to Korea through China and started to grow. The copper fruit has been edible or medicated, and in the past, it has been reported to be edible in various recipes such as Donghwasun, Donghwa Nurumi, Donghwajeok, and Donghwadonchae.
생활비결서에 따르면 동과 즙액을 밤마다 얼굴에 바르고 자면 얼굴이 백옥처럼 희고 맑게 되므로 피부 미백에 탁월하다고 하였고, 검은 피부색을 바꾸는 비법이라 하였으며, 동과씨는 기미를 없애는 효능이 우수하다고 알려진다.According to the secret of living, it is said that if you apply copper fruit juice to your face every night and sleep, your face becomes white and clear like white jade, so it is excellent for skin whitening, and it is said that it is a secret method to change the dark skin color.
그러나, 상기 동과의 골 질환 관련 효과 또는 작용 기전은 현재까지 알려진 바 없으며, 본 발명자들은 상기 동과 추출물의 뼈 건강 개선 활성 및 이를 이용한 골 질환 치료 효과를 규명하고자 하였다.However, the bone disease-related effect or mechanism of action of the copper fruit has not been known until now, and the present inventors attempted to investigate the bone health improvement activity of the copper fruit extract and the effect of treating bone disease using the same.
상기 골 질환(bone disease)은 조골세포의 활성 촉진 또는 파골세포의 활성 억제를 통해 골량 증가가 필요 또는 요구되는 상태 또는 질병을 의미하는 것으로, 예컨대, 대사성 골 질환 또는 정형외과적 골 질환일 수 있다. The bone disease refers to a condition or disease in which an increase in bone mass is required or required through promoting the activity of osteoblasts or inhibiting the activity of osteoclasts, and may be, for example, a metabolic bone disease or an orthopedic bone disease. .
상기 “대사성 골 질환”은 파골세포의 과다한 생성 또는 활성으로 인해 나타나는 상태 또는 질병을 의미하며, 골량 저하 질환을 포함할 수 있다. 상기 골량 저하 질환이란 골밀도의 저하, 골조직의 연화 등의 증상을 수반하는 골량의 저하가 나타나는 상태 또는 질환을 의미한다. The “metabolic bone disease” refers to a condition or disease that appears due to excessive production or activity of osteoclasts, and may include a bone mass-lowering disease. The bone mass-lowering disease refers to a condition or disease in which bone mass decreases accompanied by symptoms such as a decrease in bone density and softening of bone tissue.
상기 골 질환은 골다공증, 골 형성 부전증, 골연화증, 골량 감소증, 골절, 골 결손 및 고관절 감소증으로 이루어진 군에서 하나 이상 선택될 수 있으나, 이에 제한되는 것은 아니다.The bone disease may be one or more selected from the group consisting of osteoporosis, bone insufficiency, osteomalacia, bone mass loss, fracture, bone defect, and hip joint loss, but is not limited thereto.
상기 “유효성분으로 포함하는”은 투여하고자 하는 개체에 대한 치료적 또는 예방적 효과를 제공하기에 충분한 양을 포함하는 것을 의미하며, 상기 동과 추출물은 생체 안전성이 우수하므로 당업자가 양적 상한을 적절히 조정할 수 있다.The "contained as an active ingredient" means containing an amount sufficient to provide a therapeutic or prophylactic effect to the individual to be administered, and the copper fruit extract is excellent in biosafety, so those skilled in the art can appropriately set the upper limit of the quantity. Can be adjusted.
상기 “추출물”은 용매와 추출 원료를 특정 조건 하에서 접촉시킴으로써 추출 원료에 함유된 유효성분이 전이된 용매를 지칭하는 것으로, 천연물로부터 원료에 함유된 성분을 분리해낸 물질이라면, 추출 방법이나 성분의 종류와 무관하게 모두 포함할 수 있다. 예컨대, 물이나 유기용매를 이용하여 천연물로부터 용매에 용해되는 성분을 추출한 것, 천연물의 특정 성분, 예컨대 오일과 같은 특정 성분만을 추출하여 얻어진 것 등을 모두 포함할 수 있다.The "extract" refers to a solvent in which the active ingredient contained in the raw material for extraction is transferred by contacting the solvent with the raw material for extraction under specific conditions. You can include all regardless. For example, it may include extracting a component soluble in a solvent from a natural product using water or an organic solvent, and extracting a specific component of a natural product, such as extracting only a specific component such as an oil.
상기 추출물은 동과를 건조 후 분말화하거나, 열수추출법, 에탄올 추출법 등 종래 알려진 다양한 추출법에 의해 수득할 수 있다.The extract can be obtained by drying and powdering the copper fruit, or by various extraction methods known in the art such as hot water extraction and ethanol extraction.
상기 추출물은 물, 알코올, 에틸아세테이트, 아세톤, 핵산, 디클로로메탄 또는 이들의 혼합 용매로 추출될 수 있고, 바람직하게는 60 내지 90%(w/w) 농도의 에탄올에 의해 추출될 수 있다.The extract may be extracted with water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane, or a mixed solvent thereof, and preferably may be extracted with ethanol at a concentration of 60 to 90% (w/w).
상기 원료에 포함된 유효성분은 용매의 극성에 따라 추출 비율이 상이해질 수 있으므로, 용매에 따른 생리 활성 물질의 선택성을 고려하여 달리할 수 있다.Since the extraction ratio of the active ingredient contained in the raw material may be different depending on the polarity of the solvent, it may be varied in consideration of the selectivity of the physiologically active substance according to the solvent.
상기 추출물은 추출 원료를 물로 수세한 후 건조 및 분쇄하여, 원료 중량의 8 내지 12배에 달하는 부피의 용매로 약 1 내지 24시간 동안 환류 순환 추출, 가압 추출, 초음파 추출 등 통상적인 방법으로 추출 및 여과하여 제조할 수 있다.The extract is extracted by conventional methods such as reflux extraction, pressure extraction, ultrasonic extraction, etc. for about 1 to 24 hours with a solvent having a volume of 8 to 12 times the weight of the raw material by washing the extraction raw material with water, drying and grinding, and It can be prepared by filtration.
상기 추출물은 감압 증류 또는 동결 건조 등과 같은 추가적인 공정에 의해 분말 상태로 수득할 수 있다.The extract can be obtained in powder form by an additional process such as distillation under reduced pressure or freeze drying.
상기 추출물은 통상적인 정제 과정을 거친 추출물도 포함할 수 있다. 예컨대, 상기 추출물은 일정한 분자량 컷-오프 값을 갖는 한외 여과막을 이용한 분리, 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리 등 추가적으로 실시된 다양한 정제 방법을 통해 얻어진 분획물을 포함할 수 있다.The extract may also include an extract that has undergone a conventional purification process. For example, the extract may be subjected to various additional purification methods, such as separation using an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatography (one prepared for separation according to size, charge, hydrophobicity, or affinity). It may contain a fraction obtained through.
상기 식품 조성물은 성장 촉진 또는 뼈 건강 개선 용도일 수 있다.The food composition may be used for promoting growth or improving bone health.
상기 “성장”이란 단순히 신장이 증가하는 것뿐 만 아니라 신체 각 기관의 해부학적, 형태학적 크기와 기능이 증가하는 것, 즉 기능의 발달 및 기관으로의 분화를 모두 의미할 수 있다.The "growth" may mean not only an increase in height, but also an increase in the anatomical and morphological size and function of each organ of the body, that is, the development of functions and differentiation into organs.
상기 식품 조성물은 동과 추출물을 포함하여 뼈 길이를 성장시키고, 성장판 연골세포의 대사를 촉진하는 등 부작용 없이 우수한 성장 촉진 효과를 가져올 수 있어 특히 어린이 성장 촉진용 식품소재로서 유용하게 사용될 수 있다.The food composition, including copper fruit extract, can grow bone length, promote the metabolism of growth plate cartilage cells, and so forth, without side effects, and can bring about excellent growth promoting effect, so that it can be particularly useful as a food material for promoting children's growth.
상기 “뼈 건강”은 정상적인 뼈의 분해와 재형성이 일어나는 것을 의미하며, 체내 칼슘이 부족하거나 여러 가지 외부 요인으로 인해 뼈의 분해와 재형성 균형이 깨지면 골다공증, 골관절염 등 골 관련 질환이 발생할 수 있다.The “bone health” refers to normal bone decomposition and remodeling, and bone-related diseases such as osteoporosis and osteoarthritis may occur if the balance of bone decomposition and remodeling is broken due to insufficient calcium in the body or due to various external factors. .
상기 식품 조성물은 조골 세포의 분화에 영향을 미치며 골 재생을 촉진함으로써 관절 및 뼈 건강을 효과적으로 유지시킬 수 있다.The food composition can effectively maintain joint and bone health by affecting the differentiation of osteoblasts and promoting bone regeneration.
상기 식품 조성물은 뼈 건강 개선, 성장 촉진, 골 질환 관련 건강기능식품으로서 사용될 수 있다.The food composition can be used as a health functional food related to bone health improvement, growth promotion, and bone disease.
상기 건강기능식품은 건강기능식품에 관한 법률에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, 상기 기능성은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미할 수 있다.The health functional food refers to a food manufactured and processed using raw materials or ingredients that have useful functionality for the human body according to the Health Functional Food Act, and the functionality is to control nutrients for the structure and function of the human body or It may mean ingestion for the purpose of obtaining a useful effect for health purposes such as action.
상기 식품 조성물은 통상의 식품 첨가물을 포함할 수 있으며, 상기 식품 첨가물은 다른 규정이 없는 한 식품의약품안전처에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 적합성 여부를 판단할 수 있다. The food composition may contain ordinary food additives, and the food additives are subject to the standards and standards for the relevant item according to the general rules and general test methods of food additives approved by the Ministry of Food and Drug Safety, unless otherwise specified. Whether it is appropriate or not can be determined.
상기 식품 첨가물 공전에 기재된 품목은 예컨대 케톤류, 글리신, 구연산칼륨, 니코틴산, 계피산 등의 화학적 합성물, 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물, L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합제제류를 들 수 있다.The items described in the food additive code include, for example, ketones, glycine, potassium citrate, nicotinic acid, cinnamic acid, and other chemical compounds, dyestuffs, licorice extract, crystalline cellulose, high-volume pigments, natural additives such as guar gum, sodium L-glutamate preparations, and noodles. Mixed preparations, such as an added alkali agent, a preservative preparation, and a tar color preparation, are mentioned.
상기 식품 조성물은 면역 증진, 또는 골 질환의 개선을 위한 식품 및 음료 등에 다양하게 이용될 수 있으며, 예컨대, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강기능성 보조 식품, 식품 첨가제 등에 사용될 수 있다.The food composition may be variously used in foods and beverages for enhancing immunity or improving bone disease, and for example, it may be used in various foods, beverages, gum, tea, vitamin complexes, health functional supplements, food additives, and the like. .
또한, 상기 건강기능식품은 면역 증진, 또는 골 질환의 개선을 목적으로 정제, 과립, 분말, 캅셀, 액상의 용액 및 환으로 이루어진 군에서 선택된 어느 하나의 제형으로 제조 및 가공될 수 있다.In addition, the health functional food may be manufactured and processed into any one formulation selected from the group consisting of tablets, granules, powders, capsules, liquid solutions, and pills for the purpose of enhancing immunity or improving bone disease.
구체적으로 상기 정제 형태의 건강기능식품은 동과 추출물, 부형제, 결합제, 붕해제 및 다른 첨가제와의 혼합물을 통상의 방법으로 과립화한 다음, 활택제 등을 넣어 압축 성형하거나, 상기 혼합물을 직접 압축 성형하여 제조할 수 있다. 또한, 상기 정제 형태의 건강기능식품은 필요에 따라 교미제 등을 함유할 수 있으며, 필요에 따라 적당한 제피제로 제피할 수도 있다.Specifically, for the health functional food in the form of tablets, a mixture of copper fruit extract, excipients, binders, disintegrants and other additives is granulated in a conventional manner, and then compression molded by putting a lubricant or the like, or directly compressing the mixture. It can be manufactured by molding. In addition, the health functional food in the form of a tablet may contain a mating agent or the like, if necessary, and may be coated with a suitable coating agent if necessary.
상기 캅셀 형태의 건강기능식품 중 경질캅셀제는 통상의 경질캅셀에 동과 추출물 및 부형제 등의 첨가제와의 혼합물 또는 그의 입상물 또는 제피한 입상물을 충진하여 제조할 수 있으며, 연질캅셀제는 동과 추출물 및 부형제 등의 첨가제와의 혼합물을 젤라틴 등 캅셀기제에 충진하여 제조할 수 있다. 상기 연질캅셀제는 필요에 따라 글리세린 또는 솔비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다.Among the capsule-type health functional foods, hard capsules can be prepared by filling a conventional hard capsule with a mixture of additives such as copper fruit extract and excipients, granules thereof, or coated granules, and soft capsules are copper fruit extracts. And a mixture with an additive such as an excipient may be filled in a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.
상기 환 형태의 건강기능식품은 동과 추출물, 부형제, 결합제, 붕해제 등의 혼합물을 적당한 방법으로 성형하여 조제할 수 있으며, 필요에 따라 백당이나 다른 적당한 제피제로 제피를, 또는 전분, 탈크 또는 적당한 물질로 환의를 입힐 수도 있다.The ring-shaped health functional food can be prepared by molding a mixture of copper fruit extract, excipient, binder, and disintegrant by an appropriate method. Substances can also be used to create a pleasing feeling.
상기 과립형태의 건강기능식품은 동과 추출물, 부형제, 결합제, 붕해제 등의 혼합물을 적당한 방법으로 입상으로 제조할 수 있으며, 필요에 따라 착향제, 교미제 등을 함유할 수 있다. The granular health functional food may be prepared in a granular form of a mixture of copper fruit extract, excipients, binders, disintegrants, and the like by a suitable method, and may contain flavoring agents, flavoring agents, and the like, if necessary.
또한, 상기 부형제, 결합제, 붕해제, 활택제, 교미제, 착향제 등에 대한 용어 정의는 당업계에 공지된 문헌에 기재된 것으로 그 기능 등이 동일 내지 유사한 것들을 포함할 수 있다.In addition, the definitions of terms for the excipients, binders, disintegrants, lubricants, flavoring agents, flavoring agents, and the like are described in documents known in the art and may include those having the same or similar functions.
본 발명의 다른 측면에 따르면, 동과 추출물을 유효성분으로 포함하는 골 질환의 예방 또는 치료용 약학적 조성물이 제공된다.According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating bone diseases comprising a copper fruit extract as an active ingredient.
상기 “예방”은 병리학적 현상의 발생 빈도 또는 정도를 감소시키는 모든 행위를 의미한다. 예방은 완전할 수 있으며 또는 부분적일 수도 있다. 이 경우에는 개체 내의 염증에 의해 발생되는 증상이 상기 조성물을 사용 하지 않은 경우와 비교하여 감소하는 현상을 의미할 수 있다.The "prevention" refers to any action that reduces the frequency or severity of pathological symptoms. Prevention can be complete or partial. In this case, it may mean a phenomenon in which symptoms caused by inflammation in the individual decrease compared to the case where the composition is not used.
상기 “치료”는 치료하고자 하는 대상 또는 세포의 천연 과정을 변경시키기 위하여 임상적으로 개입하는 모든 행위를 의미하며, 임상 병리 상태가 진행되는 동안 또는 이를 예방하기 위하여 수행할 수 있다. 목적하는 치료 효과는 질병의 발생 또는 재발을 예방하거나, 증상을 완화시키거나, 질병에 따른 모든 직접 또는 간접적인 병리학적 결과를 저하시키거나, 전이를 예방하거나, 질병 진행 속도를 감소시키거나, 질병 상태를 경감 또는 일시적 완화시키거나, 예후를 개선시키는 것을 포함할 수 있다.The "treatment" refers to any action that clinically intervenes in order to change the natural process of a target or cell to be treated, and may be performed while a clinical pathology is in progress or to prevent it. The desired therapeutic effect is to prevent the occurrence or recurrence of the disease, alleviate symptoms, reduce all direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, or It may include alleviating or temporarily alleviating the condition, or improving the prognosis.
상기 조성물은 경구적 전달, 비경구적 전달의 형태로 투여될 수 있다. 상기 조성물은 전신 또는 국소 투여될 수 있으며, 상기 투여는 경구 투여 및 비경구 투여를 포함할 수 있다. 상기 조성물은 적절한 투여 형태를 제공하도록 적합한 양의 약학적으로 허용되는 비히클 또는 담체와 함께 제형화될 수 있다.The composition may be administered in the form of oral delivery or parenteral delivery. The composition may be administered systemically or locally, and the administration may include oral administration and parenteral administration. The composition may be formulated with a suitable amount of a pharmaceutically acceptable vehicle or carrier to provide an appropriate dosage form.
상기 조성물은 약학 조성물의 제조에 사용되는 담체, 부형제 및 희석제를 더 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유를 들 수 있으나, 이에 제한되는 것은 아니다.The composition may further include a carrier, an excipient, and a diluent used in the preparation of a pharmaceutical composition. The carrier, excipient and diluent 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, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil, but are not limited thereto.
또한, 상기 조성물은 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제제화하여 사용할 수 있다.In addition, the composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile injectable solutions.
경구 투여를 위한 고형제제는 정제, 환제, 산제, 과립제, 캡슐제 등이 사용될 수 있고, 상기 고형제제는 상기 동과 추출물과 이의 분획물들에 적어도 하나 이상의 부형제, 예컨대, 전분, 칼슘카보네이트, 수크로스, 락토오스, 또는 젤라틴 등을 혼합하여 조제할 수 있다. 또한, 상기 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제가 사용될 수 있다.Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient, such as starch, calcium carbonate, and sucrose, in the copper fruit extract and its fractions. , Lactose, or gelatin may be mixed and prepared. In addition, in addition to the excipients, lubricants such as magnesium stearate and talc may be used.
경구 투여를 위한 액상 제제는 현탁제, 내용액제, 유제, 시럽제 등이 사용될 수 있고, 단순희석제인 물, 리퀴드 파라핀 외에 여러 가지 부형제, 예컨대 습윤제, 감미제, 방향제, 보존제 등이 사용될 수 있다.Liquid preparations for oral administration may include suspensions, liquid solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., may be used in addition to simple diluents such as water and liquid paraffin.
비경구 투여를 위한 제제는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 사용될 수 있다. 상기 비수성용제, 현탁제는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르가 사용될 수 있다. 상기 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴이 사용될 수 있다.Formulations for parenteral administration may be sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, and glycerogelatin may be used.
상기 약학적 조성물은 약제학적으로 유효한 양이 대상체에 투여될 수 있다. 상기 “약제학적으로 유효한 양”은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.The pharmaceutical composition may be administered to a subject in a pharmaceutically effective amount. The “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, drug activity, drug Sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.
상기 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 바람직하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition may be administered as an individual therapeutic agent or administered in combination with another therapeutic agent, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is preferable to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all of the above factors, and this can be easily determined by a person skilled in the art.
상기 조성물은 인공골, 인공관절, 골 고정재, 골 대체제, 골 수복재, 골 충진재 및 골 이식재로 이루어진 군에서 선택된 하나 이상의 물질에 사용될 수 있다. The composition may be used for one or more materials selected from the group consisting of artificial bones, artificial joints, bone fixation materials, bone substitutes, bone repair materials, bone fillers, and bone graft materials.
상기 골 형성 촉진용 조성물은 골 질환 또는 치주질환의 예방 및 치료용으로 사용될 수 있으며, 인공골, 골 고정재, 골 대체제, 골 수복재, 골 충진재 또는 골 이식재에 적용되기 용이한 형태로 제조될 수 있다.The composition for promoting bone formation may be used for the prevention and treatment of bone disease or periodontal disease, and may be prepared in a form that is easy to be applied to artificial bone, bone fixation material, bone substitute, bone repair material, bone filler, or bone graft material. .
상기 골 이식재나 골 대체체는 협의로는 골 조직 내의 공간을 충진하는 용도로 사용될 수 있고, 광의로는 뼈나 치아의 일부를 대체하는데 사용될 수 있다. 따라서, 상기 골 형성 촉진용 조성물은 압착, 압축, 가압접촉, 패킹, 압박, 굳힘 등의 방법을 사용하여 퍼티, 페이스트, 주형 가능한 스트립, 블록, 칩 등의 형태로 사용될 수 있고, 화학적 첨가물을 이용하여, 겔, 과립, 페이스트, 정제, 펠렛 등의 형태로 제형화하여 사용될 수 있으며, 분말 형태로도 사용될 수 있다.The bone graft material or bone substitute may be used to fill a space in bone tissue in a narrow way, and may be used in a broad sense to replace part of a bone or tooth. Therefore, the composition for promoting bone formation can be used in the form of putty, paste, moldable strips, blocks, chips, etc. using methods such as compression, compression, pressure contact, packing, compression, and hardening, and chemical additives are used. Thus, it may be formulated and used in the form of gels, granules, pastes, tablets, pellets, etc., and may also be used in powder form.
이하 실시예를 통해, 본 발명을 더욱 상술하나 하기 실시예에 의해 본 발명이 제한되지 아니함은 자명하다.It is apparent that the present invention is further elaborated through the following examples, but the present invention is not limited by the following examples.
제조예Manufacturing example 1 : One : 열수Hydrothermal 추출물 extract (HR1910-W) 제조(HR1910-W) manufacturing
파쇄한 동과 건조물에 100배의 정제수를 넣고 100 ℃에서 4시간 동안 가열한 후 여과하였다. 100 times of purified water was added to the crushed copper and dried product, heated at 100° C. for 4 hours, and filtered.
수득한 액상 추출물을 회전감압농축기로 농축(20 brix)한 후 동결건조기를 이용하여 동과 추출물을 수득하였다. 분말화하여 수득한 추출물의 수율은 건조물 대비 51.6%였다.The obtained liquid extract was concentrated (20 brix) with a rotary vacuum concentrator, and then a copper fruit extract was obtained using a freeze dryer. The yield of the extract obtained by powdering was 51.6% compared to the dried product.
제조예Manufacturing example 2 : 에탄올 추출물 2: ethanol extract (HR1910-70E) 제조(HR1910-70E) manufacture
파쇄한 동과 건조물에 100배의 에탄올(70% v/v)를 넣고 100 ℃에서 4시간 동안 가열한 후 여과하였다.100 times ethanol (70% v/v) was added to the crushed copper and dried product, heated at 100° C. for 4 hours, and filtered.
수득한 액상 추출물을 회전감압농축기로 농축(20 brix)한 후 동결건조기를 이용하여 동과 추출물을 수득하였다. 분말화하여 수득한 추출물의 수율은 건조물 대비 53.5%였다.The obtained liquid extract was concentrated (20 brix) with a rotary vacuum concentrator, and then a copper fruit extract was obtained using a freeze dryer. The yield of the extract obtained by powdering was 53.5% compared to the dried product.
실험예Experimental example 1 : 세포 배양 1: cell culture
세포주인 RAW264.7 macrophage cell은 American Type Culture Collection(ATCC, Manassas. USA)에서 구입하여 사용하였다.The cell line RAW264.7 macrophage cell was purchased and used from the American Type Culture Collection (ATCC, Manassas. USA).
세포의 배양을 위하여 10% Fetal Bovin Serum(FBS, Hyclone USA), 100 units/mL penicillin/Streptoycin(Hyclone, USA)이 함유된 DMEM(Dulbecco's modified of Eagle's medium, Hyclone,USA) 배지를 사용하였으며, 37℃, 5% CO 2 존재하의 incubator(Thermo, Forma, Germany)에서 배양하였다.For cell culture, DMEM (Dulbecco's modified of Eagle's medium, Hyclone, USA) medium containing 10% Fetal Bovin Serum (FBS, Hyclone USA), 100 units/mL penicillin/Streptoycin (Hyclone, USA) was used, 37 C. Incubated in an incubator (Thermo, Forma, Germany) in the presence of 5% CO 2.
실험예Experimental example 2 : 독성 평가( 2: Toxicity evaluation ( MTTMTT assay) assay)
살아있는 세포 내 미토콘드리아의 탈수소효소에 의해 Tetrazolium salt(WST-1)에서 formazan으로 변화하는 것을 이용하여, 생성된 formazan의 농도를 spectrophotometer로 측정함으로써 세포 생존률을 측정하였다.Cell viability was measured by measuring the concentration of the produced formazan with a spectrophotometer using the change from Tetrazolium salt (WST-1) to formazan by mitochondrial dehydrogenase in living cells.
Ez-cytox(Dozen, Korea)을 이용하여 프로토콜에 따라 MTT assay를 수행하였다.MTT assay was performed according to the protocol using Ez-cytox (Dozen, Korea).
Raw264.7 세포를 5x10 3 cells/100μL로 96 well에 분주한 후 37℃, 5% CO 2 배양기에서 24시간 동안 배양하였다.Raw264.7 cells were dispensed into 96 wells at 5×10 3 cells/100 μL and incubated for 24 hours in a 37°C, 5% CO 2 incubator.
농도별로 시료를 처리한 후 24시간 동안 배양하였다. 배양 후 10% MTT 시약이 포함된 배지로 교체하고 37℃에서 1시간 동안 반응시킨 후 450 nm 파장에서 microplate reader(Epoch, Biotek)로 측정하였다.After treating the samples by concentration, they were incubated for 24 hours. After incubation, it was replaced with a medium containing 10% MTT reagent, reacted at 37° C. for 1 hour, and then measured with a microplate reader (Epoch, Biotek) at 450 nm wavelength.
각 시료에 대한 세포생존율에 의한 독성 평가는 3회 반복하여 측정하였으며, 그에 대한 평균값과 표준편차를 산출하였다.Toxicity evaluation based on cell viability for each sample was measured by repeating three times, and the mean value and standard deviation were calculated.
도 1을 참조하면, 동과 열수 추출물(제조예 1), 및 동과 에탄올 추출물(제조예 2)은 모든 농도(10, 100, 1000 μg/mL) 범위에서 세포 독성이 나타나지 않았다.Referring to Figure 1, the copper fruit extract (Preparation Example 1), and the copper fruit ethanol extract (Preparation Example 2) showed no cytotoxicity in all concentrations (10, 100, 1000 μg/mL) range.
실험예Experimental example 3 : 조골세포 분화 활성 평가 3: Evaluation of osteoblast differentiation activity
뼈 건강 효능을 알아보기 위해 조골세포(MC3T3-E1)를 5x10 3 cells/100μL로 96 well에 분주하고 24시간 동안 안정화시켰다.To examine the bone health efficacy, osteoblasts (MC3T3-E1) were dispensed into 96 wells at 5x10 3 cells/100 μL and stabilized for 24 hours.
시료를 농도별로 처리하고 48시간 CO 2 배양기에 배양하였다. 배양 후 MTT(EZ-cytox, Daillab) 용액을 처리하고 2시간 동안 CO 2 배양 후 450 nm 파장에서 microplate reader(Epoch, Biotek)로 분석하였다Samples were treated by concentration and incubated in a CO 2 incubator for 48 hours. After incubation, MTT (EZ-cytox, Daillab) solution was treated and CO 2 was incubated for 2 hours, and then analyzed with a microplate reader (Epoch, Biotek) at 450 nm wavelength.
도 2를 참조하면, 동과 열수 추출물(제조예 1)의 경우 100 μg/mL에서 조골세포 분화량이 대조군 대비 24% 증가하였으며, 1000 μg/mL에서 대조군 대비 약49% 증가하였다.Referring to FIG. 2, in the case of the copper and hot water extract (Preparation Example 1), the osteoblast differentiation amount increased by 24% compared to the control at 100 μg/mL, and about 49% increased compared to the control at 1000 μg/mL.
동과 에탄올 추출물(제조예 2)의 경우 100 μg/mL에서 조골세포 분화량이 대조군 대비 약21% 증가하였으며, 1000 μg/mL에서 대조군 대비 약41% 증가하였다.In the case of the copper and ethanol extract (Preparation Example 2), the osteoblast differentiation amount increased by about 21% compared to the control at 100 μg/mL, and about 41% compared to the control at 1000 μg/mL.
상기 결과는 동과 추출물이 조골세포의 분화를 촉진하므로 칼슘과 같은 무기질을 흡착시켜 뼈 건강을 유지함에 있어서 효과적임을 시사한다.The above results suggest that copper fruit extract promotes the differentiation of osteoblasts, so it is effective in maintaining bone health by adsorbing minerals such as calcium.
실험예Experimental example 4 : ALP 활성 평가(ALP assay) 4: ALP activity evaluation (ALP assay)
세포 분화를 확인하고자 조골세포에 특이적으로 발현하는 ALP(alkaline phosphatase) 활성도를 측정하였다.To confirm cell differentiation, the activity of ALP (alkaline phosphatase) specifically expressed in osteoblasts was measured.
MC3T3-E1(조골세포)를 5x10 3 cells/250μL로 48 well에 분주하고 24시간 동안 안정화시켰다.MC3T3-E1 (osteocyte) was dispensed into 48 wells at 5x10 3 cells/250 μL and stabilized for 24 hours.
농도별로 시료를 처리하고 7일 동안 CO 2 배양기에서 배양하였다. PBS로 세척하고 Lysis buffer를 첨가하여 용해시킨 후 12,000rpm에서 15분간 원심 분리하였다. Samples were treated by concentration and incubated in a CO 2 incubator for 7 days. After washing with PBS, lysis buffer was added to dissolve, and centrifuged at 12,000 rpm for 15 minutes.
세포막 성분 등을 침전시켜 상층액을 수집하고, ALP assay kit를 이용하여 상층액 내 ALP 활성을 정량하였다.The supernatant was collected by precipitating cell membrane components, and the ALP activity in the supernatant was quantified using an ALP assay kit.
도 3을 참조하면, 동과 열수 추출물(제조예 1)의 경우 100 μg/mL에서 대조군 대비 32 % 증가하였으며, 1000 μg/mL에서 105 % 증가하였다.Referring to FIG. 3, the copper and hot water extract (Preparation Example 1) increased by 32% compared to the control at 100 μg/mL, and increased by 105% at 1000 μg/mL.
동과 에탄올 추출물(제조예 2)의 경우, 100 μg/mL에서 대조군 대비 21 % 증가하였으며, 1000 μg/mL에서 84 % 증가하였다.In the case of the copper and ethanol extract (Preparation Example 2), it was increased by 21% compared to the control at 100 μg/mL, and 84% at 1000 μg/mL.
상기 결과는 동과 추출물이 조골세포의 분화를 촉진하므로 무기질을 흡착시켜 뼈 건강을 유지함에 있어서 효과적임을 시사한다.The above results suggest that copper fruit extract promotes the differentiation of osteoblasts, so it is effective in maintaining bone health by adsorbing minerals.
실험예Experimental example 5 : 5: ALP활성ALP activity 평가(Western blot) Western blot
배양이 끝난 세포를 수집하여 PBS로 2회 세척 후 lysis buffer을 첨가하여 용해시켰다. 12,000 rpm에서 15분간 원심분리하여 세포막 성분 등을 제거하였다.After the cultured cells were collected, washed twice with PBS, lysis buffer was added and lysed. Cell membrane components were removed by centrifugation at 12,000 rpm for 15 minutes.
BSA(bovine serum albumin)를 사용하여 단백질 농도를 표준화하였다. lysate 30μg 10% gel SDS-PAGE로 분리하여 PVDF membrane에 120V, 90분 동안 transfer 하였다. Protein concentration was normalized using bovine serum albumin (BSA). The lysate was separated by 30 μg 10% gel SDS-PAGE and transferred to the PVDF membrane for 120 V for 90 minutes.
membrane의 blocking은 5% 탈지유(skim milk)가 함유된 TBS-T 용액에서 1시간 동안 실시하였다. Membrane blocking was performed for 1 hour in a TBS-T solution containing 5% skim milk.
1차 항체(1:100~1000)는 4℃에서 한 시간 동안 배양시킨 후 다음 날 TBS-T로 3회 세척하였다. The primary antibody (1:100~1000) was incubated at 4°C for one hour and then washed three times with TBS-T the next day.
2차 항체로는 HRP(horse radish peroxidase)가 결합된 항마우스(anti-mouse)를 1:5000으로 희석하여 상온에서 1시간 동안 반응시켰다.As a secondary antibody, HRP (horse radish peroxidase)-conjugated anti-mouse was diluted 1:5000 and reacted at room temperature for 1 hour.
TBS-T로 3회 세척하고 ECL 기질과 3분간 반응 후 화학 발광(chemiluminescence)을 통해 분석하였다.After washing three times with TBS-T and reacting with the ECL substrate for 3 minutes, it was analyzed by chemiluminescence.
도 4를 참조하면, 동과 열수 추출물(제조예 1)의 경우 100 μg/mL에서 대조군 대비 약 62% 증가하였으며, 1000 μg/mL에서 약 118 % 증가하였다.Referring to FIG. 4, the copper and hot water extract (Preparation Example 1) increased by about 62% compared to the control at 100 μg/mL, and about 118% at 1000 μg/mL.
동과 에탄올 추출물(제조예 2)의 경우 100 μg/mL에서 대조군 대비 약 36% 증가하였으며, 1000 μg/mL에서 약 137 % 증가하였다.In the case of the copper and ethanol extract (Preparation Example 2), it increased by about 36% compared to the control at 100 μg/mL, and about 137% at 1000 μg/mL.
상기 결과는 동과 추출물이 뼈 건강 관련 작용기전에 영향을 미치며 조골세포의 분화를 조절함으로써 뼈 건강 유지에 있어 효과적임을 시사한다.The above results suggest that copper fruit extract has an effect on bone health-related mechanisms of action and is effective in maintaining bone health by regulating the differentiation of osteoblasts.
실험예Experimental example 6 : 골 재생 유도 활성 평가 6: Evaluation of bone regeneration induction activity
in vivo에서 동과 추출물의 골 재생 유도 효과를 확인하기 위해 두개골이 결손된 생쥐에 동과 추출물, BMP-2 또는 대조군을 처리한 후, 마이크로 CT(μCT) 및 조직학 분석을 수행하였다. In order to confirm the bone regeneration-inducing effect of the copper extract in vivo , mice with a skull defect were treated with copper fruit extract, BMP-2 or a control group, and then micro-CT (μCT) and histological analysis were performed.
마이크로 CT(μCT) 분석에 따르면, 동과 추출물(1000 μg/mL) 및 BMP-2은 6주 전 구간을 걸쳐 골 형성을 증가시킨 반면, 대조군에서 극소의 골 형성이 유도되었다.According to micro-CT (μCT) analysis, copper fruit extract (1000 μg/mL) and BMP-2 increased bone formation over the entire 6-week period, whereas minimal bone formation was induced in the control group.
조직학적 시험에 따르면, 제조예 1 및 2의 동과 추출물 처리에 의해 대부분 섬유상 조직인 대조군 영역과 달리 결손 부위의 골 형성이 확인되었다. According to histological tests, bone formation at the defect site was confirmed by treatment with the copper fruit extract of Preparation Examples 1 and 2, unlike the control area, which is mostly a fibrous tissue.
실험예Experimental example 7 : 장골 길이 성장 활성 평가 7: Evaluation of iliac bone length growth activity
흰쥐의 골 성장에 대한 동과 추출물의 효능을 측정하였다.The efficacy of the extract of copper fruit on bone growth in rats was measured.
측정 방법은 정상적인 골 대사를 관찰하기 위한 방법으로 형광인자가 칼슘과 킬레이트 결합을 통해 골에 침착되는 현상을 이용한 것이다.The measurement method is a method for observing normal bone metabolism and uses a phenomenon in which a fluorescent factor is deposited on the bone through a chelate bond with calcium.
골 생성이 가장 활발한 성장기에 형광인자를 투여하면 투여된 형광인자는 골성장판 하부의 골신생부에 가장 많이 침착하여 선을 형성하므로, 시료를 1회 투여한 후 일정 기간 후에 형광선과 성장판 사이의 길이를 형광현미경으로 관찰하여 성장한 길이를 측정하였다. When a fluorescent factor is administered during the growth period in which bone formation is most active, the administered fluorescent factor most often deposits in the bone neoplasm under the bone growth plate to form a line. Was observed with a fluorescence microscope to measure the grown length.
수집한 절편을 슬라이드 글라스 위에 놓고 건조한 후 형광현미경을 이용하여 골 조직 내 형광인자의 침착으로 형성된 선과 성장판 사이의 길이를 측정함으로써 측정된 랫트의 장골 길이성장 정도를 측정하였다.The collected section was placed on a slide glass, dried, and the length between the growth plate and the line formed by the deposition of fluorescent factors in the bone tissue was measured using a fluorescence microscope to measure the degree of growth of the ilium of the rat.
측정 결과, 동과 열수 추출물(제조예 1) 및 동과 에탄올 추출물(제조예 2)을 투여한 경우 대조군 대비 길이 성장이 각각 22%, 18% 증가하였다.As a result of the measurement, when the copper and hot water extract (Preparation Example 1) and the copper and ethanol extract (Preparation Example 2) were administered, length growth was increased by 22% and 18%, respectively, compared to the control group.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 예를 들어, 단일형으로 설명되어 있는 각 구성 요소는 분산되어 실시될 수도 있으며, 마찬가지로 분산된 것으로 설명되어 있는 구성 요소들도 결합된 형태로 실시될 수 있다.The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.
본 발명의 범위는 후술하는 청구범위에 의하여 나타내어지며, 청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present invention is indicated by the claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and the concept of equivalents thereof should be construed as being included in the scope of the present invention.

Claims (8)

  1. 동과 추출물을 유효성분으로 포함하는 뼈 건강 개선용 식품 조성물. A food composition for improving bone health comprising copper fruit extract as an active ingredient.
  2. 제1항에 있어서,The method of claim 1,
    상기 추출물은 물, 알코올, 에틸아세테이트, 아세톤, 핵산, 디클로로메탄 또는 이들의 혼합 용매로 추출된 식품 조성물.The extract is a food composition extracted with water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane, or a mixed solvent thereof.
  3. 동과 추출물을 유효성분으로 포함하는 성장 촉진용 식품 조성물. A food composition for promoting growth comprising a copper fruit extract as an active ingredient.
  4. 동과 추출물을 유효성분으로 포함하는 골 질환 예방 또는 개선용 식품 조성물. A food composition for preventing or improving bone disease, comprising a copper fruit extract as an active ingredient.
  5. 제4항에 있어서,The method of claim 4,
    상기 골 질환은 골다공증, 골 형성 부전증, 골연화증, 골량 감소증, 골절, 골 결손 및 고관절 감소증으로 이루어진 군에서 하나 이상 선택된 식품 조성물.The bone disease is at least one food composition selected from the group consisting of osteoporosis, bone insufficiency, osteomalacia, bone mass reduction, fractures, bone defects, and hypochondriasis.
  6. 동과 추출물을 유효성분으로 포함하는 뼈 건강 개선 또는 성장 촉진용 약학적 조성물.A pharmaceutical composition for improving bone health or promoting growth, comprising copper fruit extract as an active ingredient.
  7. 동과 추출물을 유효성분으로 포함하는 골 질환의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for the prevention or treatment of bone diseases comprising a copper fruit extract as an active ingredient.
  8. 제7항에 있어서, The method of claim 7,
    상기 골 질환은 골다공증, 골 형성 부전증, 골연화증, 골량 감소증, 골절, 골 결손 및 고관절 감소증으로 이루어진 군에서 하나 이상 선택된 약학적 조성물.The bone disease is at least one pharmaceutical composition selected from the group consisting of osteoporosis, bone insufficiency, osteomalacia, bone mass reduction, fracture, bone defect, and hip reduction.
PCT/KR2020/014640 2019-11-15 2020-10-26 Composition containing winter melon extract as active ingredient for increasing bone health WO2021096098A1 (en)

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