KR20230066289A - A composition for bone health comprising citrus extract - Google Patents

Abstract

The present invention relates to a food and pharmaceutical composition for improving bone health including citrus raw materials as active ingredients. The composition for improving bone health including a citrus fruit extract as an active ingredient of the present invention includes a citrus fruit extract, fruit juice, or a pulverized fruit as an active ingredient. The composition according to one aspect of the present invention can be usefully used for treating bone diseases.

Description

Composition for improving bone health containing citrus extract as an active ingredient {A COMPOSITION FOR BONE HEALTH COMPRISING CITRUS EXTRACT}

The present invention relates to a composition for improving bone health comprising a citrus raw material as an active ingredient, and can be used for various purposes such as pharmaceuticals and foods.

Living organisms constantly need a lot of nutrients to sustain life, and these nutrients are always supplied from the outside in the form of food or some are synthesized in the body.

Food contains substances necessary for maintaining human life, and humans maintain life by ingesting food, digesting it in the digestive system, absorbing it, and supplying it to tissue cells in the body.

Therefore, good nutrition maintains a healthy body, and a healthy body guarantees a healthy mind.

Nutrition is a direct or indirect cause of disease and is involved in the prevention and treatment of diseases. The lack of nutrients caused by insufficiently balanced intake of nutrients necessary for the human body is also a problem, and excessive intake is also a serious problem. disease can be caused.

BACKGROUND ART Recently, as living standards have increased and dietary habits have 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 is gradually aging due to the extension of life expectancy, interest in health is gradually increasing.

Health promotion is to increase resistance to diseases, especially chronic degenerative diseases or infections, to continuous stress, and to increase daily activity. Sustainability and overall lifestyle management are required.

On the other hand, interest in height growth factors has recently increased due to the westernization of body shapes and the increase in average height.

If growth is defined in a broad sense, it will include not only an increase in height, but also an increase in the size and function of each organ in the body.

The increase in height is achieved through skeletal metabolism including synthesis of cartilage tissue, growth of skeletal length, 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, and is a connective tissue composed of various 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. During the growth period, the bone formation ability by osteoblasts reaches its peak, so that the amount of bone formation far exceeds the amount of bone resorption, so bone growth is achieved.

Growth hormone therapy, which was used for sluggish growth in a pathological state in the past, is being applied to growing children and adolescents with normal height as interest in height growth has increased in recent years.

However, the growth hormone formulation administration method shows excellent effects in the case of people lacking growth hormone, but in the majority of people with normal hormone secretion, various side effects such as acromegaly, growth hormone antibody positivity, systemic allergic reaction, hypothyroidism, etc. There are problems that can cause it.

In addition, problems such as excessive duration and cost of growth hormone therapy, cancer occurrence and disturbance of other growth factors have been found, and health supplements for promoting growth have not been scientifically verified in most cases.

Therefore, its efficacy in promoting growth is scientifically verified, and it is necessary to develop safe food materials.

The present inventors confirmed through research that the citrus 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, and an object of the present invention is to provide a functional food or drug derived from natural plants with excellent biosafety.

According to one aspect of the present invention, there is provided a food composition for improving bone health comprising a citrus extract, squeezed juice or pulverized product as an active ingredient.

In one embodiment, the citrus (citrus) may be at least one selected from the group consisting of green tangerines, tangerines, grapefruits, and oranges.

According to another aspect of the present invention, there is provided a food composition for promoting growth comprising a citrus extract, squeezed juice or pulverized product as an active ingredient.

According to another aspect of the present invention, a food composition for preventing or improving bone disease comprising a citrus extract, squeezed juice or pulverized product as an active ingredient is provided.

In one embodiment, the bone disease may be one or more selected from the group consisting of osteoporosis, osteogenesis imperfecta, osteomalacia, bone mass loss, fracture, bone defect, and hip joint loss.

According to another aspect of the present invention, a pharmaceutical composition for preventing or treating bone disease comprising a citrus extract, squeezed juice or pulverized product as an active ingredient is provided.

In one embodiment, the citrus (citrus) may be at least one selected from the group consisting of green tangerines, tangerines, grapefruits, and oranges.

In one embodiment, the bone disease may be one or more selected from the group consisting of osteoporosis, osteogenesis imperfecta, osteomalacia, bone mass loss, fracture, bone defect, and hip joint loss.

Since the composition according to one aspect of the present invention contains natural raw materials as an active ingredient and has excellent biosafety and excellent bone health improvement effects, it can be usefully used as a health functional food as well as for treating bone diseases.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the configuration of the invention described in the claims.

1 is a result of evaluating the cytotoxicity of a green tangerine extract and a pulverized product according to an embodiment of the present invention.
2 is a result of evaluating the osteoblast differentiation promoting activity of green tangerine extract, green tangerine pulverized product, and citrus sample according to an embodiment of the present invention.
3 and 4 are results of evaluating the ALP activity of green tangerine extract, green tangerine pulverized material, and citrus samples according to an embodiment of the present invention.

The terms used in this specification have been selected from general terms that are currently widely used as much as possible while considering the functions in the present invention, but these may vary depending on the intention of a person skilled in the art, precedent, or the emergence of new technologies.

In addition, in a specific case, there is also a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, not simply the name of the term.

Unless defined otherwise, 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 commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in this application, it should not be interpreted in an ideal or excessively formal meaning. don't

Numerical ranges are inclusive of the values defined therein. Every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written. Every numerical limitation given throughout this specification will include every better numerical range within the broader numerical range, as if the narrower numerical limitations were expressly written.

Hereinafter, embodiments of the present invention will be described in detail, but it is obvious that the present invention is not limited by the following examples.

According to one aspect of the present invention, a food composition for preventing or improving bone disease comprising a citrus extract, squeezed juice or lysate as an active ingredient is provided.

The dictionary meaning of “Citrus” is a general name for the three genera of Citrus, Fortunella, and Pontirus of the Tangerine family, and the Citrus is Narigin, Nargenin ( Naringenin), Hesperidin, Tangeretin, and Nobiletin.

Citrus includes citrus, kumquat, mandarin, tangerine, sweetie, citron, tangerine, cheonhyehyang, calamansi, pomelo, hallabong, orange, lemon, grapefruit, lime, citron, etc. It refers to, and is not particularly limited as long as it belongs to the Citrus plant.

In one embodiment, the citrus (citrus) may be at least one selected from the group consisting of green tangerines, tangerines, grapefruits, and oranges.

The “green tangerine” is an immature citrus fruit and refers to a fruit in a green state before the skin is colored.

The immature family may refer to an extremely early mandarin orange with a sugar content of less than 8 bricks or an early and common mandarin orange with a sugar content of less than 9 bricks.

The "citrus" is a general term for evergreen small trees belonging to the genus Rutaceae and Citrus, including C. unshiu, C. deliciosa, C. grandis, C. junos, Summer tangerine tree (C. natsudaidai), citron (C. medica), lime (C. aurantifolia), lemon (C. limon), fragrant tangerine (C. aurantium), rough melon (C. jambhiri), chestnut pumpkin (C. maxima), crust (C. aurantium), tangerine (C. tangerina), tangerine (C. leiocarpa), persimmon (C. pseudogalgul TANAKA), tangerine (C. tangerrina TANAKA), tangerine (C. leocarpa), red tangerine ( C. tachibana TANAKA), potato (C. benokoji TANAKA), tangerine (C. nippokoreana TANAKA), tangerine (C. erythrosa TANAKA), tiger kang (C. sulcata HORT. Ex. Tan), eggplant (C. natsudaidai HAY) ), may be selected from the group consisting of C. grandis OSBECK and C. hassaku HORT. Ex Y. Tan, but is not limited thereto.

The "grapefruit" is a fruit of a grapefruit tree belonging to the genus Citrus, which has a round shape like a tangerine, a diameter of 10 to 15 cm, a leathery outer skin, a smooth surface, and a yellow color.

The “orange (Citrus sinensis)” is a plant belonging to the genus Tangerine and contains abundantly useful components such as vitamin C, vitamins B1 and B2, flavonoids, beta-catonin, citric acid, and pectin.

Citrus is known to have various efficacies such as skin beauty, but no bone disease-related effects or mechanism of action have been known so far . .

The bone disease refers to a condition or disease in which an increase in bone mass is required or required by promoting the activity of osteoblasts or inhibiting the activity of osteoclasts, and may be, for example, metabolic bone disease or orthopedic bone disease. .

The "metabolic bone disease" refers to a condition or disease caused by excessive production or activity of osteoclasts, and may include a bone mass reduction disease. The bone mass reduction disease refers to a condition or disease in which a decrease in bone mass is 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 hypoplasia, osteomalacia, bone mass loss, fracture, bone defect, and hip joint loss, but is not limited thereto.

The "contained as an active ingredient" means to include an amount sufficient to provide the intended effect on the subject to be administered, and since the citrus extract has excellent biosafety, those skilled in the art can appropriately adjust the upper limit of the amount.

The "extract" refers to a solvent in which the active ingredient contained in the extraction material is transferred by contacting the solvent and the extraction material under specific conditions. Anything can be included regardless. For example, those obtained by extracting a component soluble in a solvent from a natural product using water or an organic solvent, and those obtained by extracting a specific component of a natural product, for example, only a specific component such as oil, may be included.

The extract can be obtained by pulverizing citrus raw materials (citrus fruits) after drying, or by various extraction methods known in the art, such as hot water extraction or ethanol extraction, and both peels and fruits can be used as extraction raw materials.

The extract may be extracted with water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane or a mixed solvent thereof, preferably with ethanol at a concentration of 30 to 70% (w/w).

Since the active ingredient included in the raw material may have a different extraction ratio depending on the polarity of the solvent, it may be different in consideration of the selectivity of the physiologically active substance according to the solvent.

The extract is obtained by washing the extraction raw material with water, then drying and pulverizing, extracting by a conventional method such as reflux circulation extraction, pressure extraction, ultrasonic extraction, etc. for about 1 to 24 hours with a solvent in a volume of 8 to 12 times the weight of the raw material. It can be prepared by filtration.

The extract may be obtained in powder form by additional processes such as distillation under reduced pressure or freeze drying.

The extract may also include an extract that has undergone a conventional purification process. For example, the extract is subjected to various additional purification methods such as separation using an ultrafiltration membrane having a certain molecular weight cut-off value and separation by various chromatography (made for separation according to size, charge, hydrophobicity or affinity). It may include fractions obtained through

The “squeezed juice” is obtained by separating juice from citrus raw materials. For example, the raw material can be put into a juicer such as a mixer or crusher to separate liquid components, and can also be obtained in powder form through filtering and freeze-drying processes. .

The “crushed material” may be processed into a powder form of small particles through physical force on citrus raw materials. The pulverized product can be obtained in an easy-to-ingest form by applying mechanical force such as compression, shear, or impact.

The food composition may be used to promote growth or improve bone health.

The “growth” may mean not only an increase in height, but also an increase in anatomical and morphological sizes and functions of each organ of the body, that is, development of functions and differentiation into organs.

The food composition includes a citrus extract to increase bone length and promote metabolism of growth plate chondrocytes, so that it can bring about excellent growth-stimulating effects without side effects, so it can be particularly useful as a food material for promoting growth in children.

The "bone health" means that normal bone decomposition and remodeling occurs, and when the balance of bone decomposition and remodeling is broken due to lack of calcium in the body or various external factors, bone-related diseases such as osteoporosis and osteoarthritis may occur. .

The food composition can effectively maintain joint and bone health by affecting the differentiation of osteoblasts and promoting bone regeneration through this.

The food composition may be used as a health functional food related to growth promotion or bone disease.

The health functional food refers to food manufactured and processed using raw materials or ingredients having functional properties useful for the human body in accordance with the Health Functional Food Act, and the functional food regulates nutrients with respect to the structure and function of the human body or physiologically It may mean ingestion for the purpose of obtaining useful effects for health purposes such as action.

The food composition may include conventional food additives, and the food additives are in accordance with the standards and standards for the item in accordance with the general rules of the Food Additive Code and general test methods approved by the Ministry of Food and Drug Safety, unless otherwise specified. suitability can be judged.

Items described in the food additives code include, for example, ketones, glycine, potassium citrate, chemical compounds such as nicotinic acid and cinnamic acid, natural additives such as persimmon pigment, licorice extract, crystalline cellulose, goyang pigment, guar gum, sodium L-glutamate preparations, and noodles. and mixed preparations such as added alkali agent, preservative preparation, and tar color preparation.

The food composition It can be used in a variety of foods and beverages for improving bone disease, for example, various foods, beverages, gum, tea, vitamin complexes, health functional supplements, and food additives.

In addition, the health functional food may be prepared and processed into any one formulation selected from the group consisting of tablets, granules, powders, capsules, liquid solutions, and pills for the purpose of improving bone diseases.

Specifically, the health functional food in the form of a tablet is obtained by granulating a mixture of citrus raw materials, excipients, binders, disintegrants, and other additives in a conventional manner, and then adding a lubricant or the like to compression molding or direct compression molding of the mixture. It can be manufactured by In addition, the health functional food in the form of a tablet may contain a corrigent, etc., if necessary, and may be coated with an appropriate coating agent, if necessary.

Among the health functional foods in the form of capsules, hard capsules can be prepared by filling ordinary hard capsules with a mixture of citrus raw materials and additives such as excipients, granular materials thereof, or coated granular materials, and soft capsules are prepared by filling citrus raw materials and excipients It can be prepared by filling a mixture with additives such as gelatin in a capsule base. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, and a preservative, if necessary.

The health functional food in the form of a ring can be prepared by shaping a mixture of citrus raw materials, excipients, binders, disintegrants, etc. in an appropriate way, and if necessary, it is coated with white sugar or other suitable coating agent, or starch, talc or other suitable material. You can also wear a suit with .

The health functional food in the form of granules can be prepared in a granular form by appropriately preparing a mixture of citrus raw materials, excipients, binders, disintegrants, etc., and may contain flavoring agents, flavoring agents, etc., if necessary.

In addition, definitions of terms for excipients, binders, disintegrants, lubricants, corrigents, flavoring agents, etc. are described in literature known in the art, and may include those having the same or similar functions.

According to another aspect of the present invention, a pharmaceutical composition for preventing or treating bone disease comprising a citrus extract, squeezed juice or pulverized product as an active ingredient is provided.

The "prevention" means any action that reduces the frequency or severity of pathological phenomena. Prevention can be complete or partial. In this case, it may mean a phenomenon in which symptoms caused by inflammation in the subject are reduced compared to the case where the composition is not used.

The "treatment" means any action that clinically intervenes to change the natural process of a target or cell to be treated, and can be performed while a clinical pathology is progressing or to prevent it. The desired therapeutic effect is to prevent the occurrence or recurrence of the disease, alleviate the symptoms, reduce any direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, or reduce the rate of disease progression. alleviating or palliating 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 pharmaceutically acceptable vehicle or carrier in suitable amounts to provide an appropriate dosage form.

The composition may further include carriers, excipients and diluents used in the preparation of pharmaceutical compositions. The carriers, excipients and diluents 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.

In addition, the composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions.

Solid preparations for oral administration may be tablets, pills, powders, granules, capsules, etc., and the solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose, It can be prepared by mixing lactose or gelatin. In addition to the above excipients, lubricants such as magnesium styrate and talc may be used.

Liquid preparations for oral administration may include suspensions, internal solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives in addition to simple diluents such as water and liquid paraffin.

Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used as the non-aqueous solvent or suspending agent. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin fat, and glycerogeratin may be used.

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 at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is based on the type and severity of the patient's disease, the activity of the drug, and the drug It may be determined according to factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical arts.

The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is preferable to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

The composition may be used for at least one material selected from the group consisting of artificial bone, artificial joint, bone fixation material, bone substitute, bone restoration material, bone filling material, and bone graft material.

The composition for promoting bone formation can be used for the prevention and treatment of bone disease or periodontal disease, and can be prepared in a form that is easily applied to artificial bone, bone fixing material, bone substitute, bone restoration material, bone filling material, or bone graft material. .

The bone graft material or bone substitute may be used for filling a space in a bone tissue in a narrow sense, and may be used for replacing part of a bone or tooth in a broad sense. Therefore, the composition for promoting bone formation can be used in the form of putty, paste, moldable strip, block, chip, etc. using methods such as compression, compression, pressure contact, packing, compression, and hardening, and using chemical additives. Thus, it may be formulated and used in the form of gel, granule, paste, tablet, pellet, etc., and may also be used in powder form.

Through the following examples, the present invention is further detailed, but it is obvious that the present invention is not limited by the following examples.

Example 1: Green tangerine sample preparation

Example 1-1. Green tangerine ethanol (30%) extract (HR1903-30E)

An extract was prepared by adding 50 times 30% (v/v) alcohol to green tangerine (Onju mandarin orange) and heating at 60° C. for 5 hours. After concentrating the filtered extract to 120 brix, it was powdered using a freeze dryer.

Example 1-2. Green tangerine ethanol (50%) extract (HR1903-50E)

An extract was prepared by adding 50 times 50% (v/v) alcohol to green tangerine (Onju mandarin orange) and heating at 60° C. for 5 hours. After concentrating the filtered extract to 120 brix, it was powdered using a freeze dryer.

Example 1-3. July Green tangerine freeze-dried powder (HR1903-7RF)

In July, raw green tangerine (Onju mandarin orange) was crushed and then freeze-dried and powdered.

Example 1-4. August Green tangerine freeze-dried powder (HR1903-8RF)

In August, raw green tangerine (Onju mandarin orange) was crushed and freeze-dried to powder.

Example 2: Mandarin, Grapefruit, Orange Sample Preparation

Example 2-1. Citrus hot water extract (HR1903-C-W)

An extract was prepared by adding 50 times purified water to a mature mandarin orange (Onju mandarin orange) and heating at 100 °C for 5 hours. After concentrating the filtered extract to 20 brix, it was powdered using a freeze dryer.

Example 2-2. Citrus juice powder (HR1903-C-PE)

Mature mandarin oranges (Onju mandarin oranges) were squeezed with a juicer, filtered, and powdered using a freeze dryer.

Example 2-3. Grapefruit hot water extract (HR1903-GF-W)

An extract was prepared by adding 50 times purified water to grapefruit and heating at 100 ° C. for 5 hours. After concentrating the filtered extract to 20 brix, it was powdered using a freeze dryer.

Example 2-4. Orange Juice Powder (HR1903-OR-PE)

Oranges were squeezed with a juicer, filtered, and powdered using a freeze dryer.

Experimental Example 1: Cell culture

A cell line, RAW264.7 macrophage cell, was purchased from American Type Culture Collection (ATCC, Manassas. USA) and used.

For cell culture, DMEM (Dulbecco's modified of Eagle's medium, Hyclone, USA) medium containing 10% Fetal Bovin Serum (FBS, Hyclone USA) and 100 units/mL penicillin/Streptoycin (Hyclone, USA) was used. C, cultured in an incubator (Thermo, Forma, Germany) in the presence of 5% CO2.

Experimental Example 2: Toxicity evaluation (MTT assay)

Using the change from Tetrazolium salt (WST-1) to formazan by mitochondrial dehydrogenase in living cells, cell viability was measured by measuring the concentration of formazan produced using a spectrophotometer.

MTT assay was performed according to the protocol using Ez-cytox (Dozen, Korea).

Raw264.7 cells were dispensed into 96 wells at 5x10 ³ cells/100 μL and then cultured for 24 hours in a 37°C, 5% CO 2 incubator.

Samples were treated for each concentration and then incubated for 24 hours. After incubation, the medium was replaced with a medium containing 10% MTT reagent, reacted at 37° C. for 1 hour, and measured with a microplate reader (Epoch, Biotek) at a wavelength of 450 nm.

Toxicity evaluation by cell viability for each sample was measured three times, and the average value and standard deviation thereof were calculated.

Referring to Figure 1, the samples of Example 1 and Example 2 did not show cytotoxicity in all concentration ranges (10, 100, 1000 μg / mL).

Experimental Example 3: Evaluation of osteoblast differentiation activity (MTT assay)

To investigate the bone health effect, osteoblasts (MC3T3-E1) were dispensed into 96 wells at 5x10 ³ cells/100 μL and stabilized for 24 hours.

Samples were treated by concentration and cultured in a CO2 incubator for 48 hours. After incubation, MTT (EZ-cytox, Daillab) solution was treated, CO 2 incubation was performed for 2 hours, and analysis was performed with a microplate reader (Epoch, Biotek) at a wavelength of 450 nm (Table 1).

division osteoblast differentiation activity
[Increase rate (%) compared to the control group, 1000 μg/mL] Example 1-1 29 Example 1-2 32 Example 1-3 37 Example 1-4 57 Example 2-1 19 Example 2-2 24 Example 2-3 37 Example 2-4 32

Referring to Figure 2, in the case of Example 1-1, the osteoblast differentiation activity was increased by about 20% compared to the control group at 100 μg / mL, and increased by about 29% compared to the control group at 1000 μg / mL.

In the case of Examples 1-2, osteoblast differentiation activity was increased by about 30% compared to the control group at 100 μg/mL, and increased by about 32% compared to the control group at 1000 μg/mL.

In the case of Examples 1-3, osteoblast differentiation activity was increased by about 29% compared to the control group at 100 μg/mL, and increased by about 37% compared to the control group at 1000 μg/mL.

In the case of Examples 1-4, osteoblast differentiation activity was increased by about 57% compared to the control group at 1000 μg/mL.

In the case of Example 2-1, osteoblast differentiation activity was increased by 19% compared to the control group at 1000 μg/mL.

In the case of Example 2-2, the osteoblast differentiation activity was increased by 18% compared to the control group at 100 μg/mL, and increased by 24% at 1000 μg/mL.

In the case of Examples 2-3, osteoblast differentiation activity was increased by 13% compared to the control group at 100 μg/mL, and increased by 37% at 1000 μg/mL.

In the case of Examples 2-4, the osteoblast differentiation activity was increased by 10% compared to the control group at 100 μg/mL, and increased by 32% at 1000 μg/mL.

The above results suggest that the samples of Examples 1 and 2 are effective in maintaining bone health by adsorbing minerals such as calcium because they promote the differentiation of osteoblasts.

Experimental Example 4: ALP activity evaluation (ALP assay)

In order to confirm cell differentiation, the activity of ALP (alkaline phosphatase) specifically expressed in osteoblasts was measured.

MC3T3-E1 (osteoblasts) was dispensed into 48 wells at 5x10 ³ cells/250μL and stabilized for 24 hours.

Samples were processed for each concentration and cultured in a CO2 incubator for 7 days. After washing with PBS, dissolving by adding Lysis buffer, and centrifuging at 12,000 rpm for 15 minutes.

The supernatant was collected by precipitating cell membrane components, etc., and the ALP activity in the supernatant was quantified using an ALP assay kit (Table 2).

division ALP activity
[Increase rate (%) compared to the control group, 1000 μg/mL] Example 1-1 35 Example 1-2 39 Example 1-3 40 Example 1-4 42 Example 2-1 30 Example 2-2 39 Example 2-3 43 Example 2-4 41

Referring to Figure 3, in the case of Example 1-1, the ALP activity was increased by 29% compared to the control group at 100 μg / mL, and increased by 35% at 1000 μg / mL.

In the case of Examples 1-2, the ALP activity was increased by 42% compared to the control group at 100 μg/mL, and increased by 39% at 1000 μg/mL.

In the case of Examples 1-3, the ALP activity increased by 40% compared to the control group at 100 μg/mL, and increased by 40% at 1000 μg/mL.

In the case of Examples 1-4, the ALP activity was increased by 30% compared to the control group at 100 μg/mL, and increased by 42% at 1000 μg/mL.

In the case of Example 2-1, the ALP activity increased by 21% compared to the control group at 100 μg/mL, and increased by 30% at 1000 μg/mL.

In the case of Example 2-2, the ALP activity was increased by 29% compared to the control group at 100 μg/mL, and increased by 39% at 1000 μg/mL.

In the case of Examples 2-3, the ALP activity was increased by 37% compared to the control group at 100 μg/mL, and increased by 43% at 1000 μg/mL.

In the case of Examples 2-4, the ALP activity was increased by 31% compared to the control group at 100 μg/mL, and increased by 41% at 1000 μg/mL.

The above results suggest that the samples of Examples 1 and 2 are effective in maintaining bone health by adsorbing minerals because they promote the differentiation of osteoblasts.

Experimental Example 5: ALP activity evaluation (Western blot)

The cultured cells were collected, washed twice with PBS, and lysed by adding lysis buffer. Cell membrane components and the like were removed by centrifugation at 12,000 rpm for 15 minutes.

Protein concentration was normalized using bovine serum albumin (BSA). 30μg of lysate was separated by 10% gel SDS-PAGE and transferred to a PVDF membrane at 120V for 90 minutes.

Blocking of the membrane 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 washed three times with TBS-T the next day.

As the secondary antibody, HRP (horse radish peroxidase) coupled anti-mouse was diluted 1:5000 and reacted at room temperature for 1 hour.

After washing three times with TBS-T and reacting with the ECL substrate for 3 minutes, it was analyzed through chemiluminescence (Table 3).

division ALP expression level
[Increase rate (%) compared to the control group, 1000 μg/mL] Example 1-1 89 Example 1-2 93 Example 1-3 103 Example 1-4 129 Example 2-1 66 Example 2-2 77 Example 2-3 114 Example 2-4 89

Referring to Figure 4, in the case of Example 1-1, the amount of ALP expression increased by about 67% compared to the control group at 100 μg / mL, and increased by about 89% compared to the control group at 1000 μg / mL.

In the case of Examples 1-2, the amount of ALP expression increased by about 108% compared to the control group at 100 μg/mL and by about 93% compared to the control group at 1000 μg/mL.

In the case of Examples 1-3, the ALP expression level increased by about 86% compared to the control group at 10 μg / mL, by about 92% compared to the control group at 100 μg / mL, and by about 103% compared to the control group at 1000 μg / mL.

In the case of Examples 1-4, the amount of ALP expression increased by about 74% compared to the control group at 100 μg/mL and by about 129% compared to the control group at 1000 μg/mL.

In the case of Example 2-1, the ALP expression level increased by about 45% compared to the control group at 100 μg/mL and by about 66% compared to the control group at 1000 μg/mL.

In the case of Example 2-2, the amount of ALP expression increased by about 57% compared to the control group at 100 μg/mL and by about 77% compared to the control group at 1000 μg/mL.

In the case of Examples 2-3, the amount of ALP expression increased by about 69% compared to the control group at 100 μg/mL and by about 114% compared to the control group at 1000 μg/mL.

In the case of Examples 2-4, the amount of ALP expression increased by about 57% compared to the control group at 100 μg/mL and by about 89% compared to the control group at 1000 μg/mL.

The above results suggest that the samples of Examples 1 and 2 are effective in maintaining bone health by affecting bone health-related action mechanisms and regulating the differentiation of osteoblasts.

Experimental Example 6: Evaluation of bone regeneration induction activity

In order to confirm the in vivo bone regeneration inducing effect of citrus raw materials, mice with skull defects were treated with citrus extract, BMP-2 or a control group, and micro CT (μCT) and histological analysis were performed.

According to micro-CT (μCT) analysis, Example 1 (1000 μg/mL), Example 2 (1000 μg/mL), and BMP-2 increased bone formation over the entire period of 6 weeks, whereas it was minimal in the control group. of bone formation was induced.

According to the histological examination, bone formation was confirmed in the defect area by the sample treatment of Examples 1 and 2, unlike the control area, which was mostly a fibrous tissue.

Experimental Example 7: Long bone growth activity evaluation

The efficacy of citrus raw materials on bone growth in rats was measured.

As a method for observing normal bone metabolism, the measurement method used a phenomenon in which a fluorescent factor is deposited in bone through a chelate bond with calcium.

When fluorescent factor is administered during the growth period when bone production is most active, the injected fluorescent factor is most deposited in the bone regeneration area at the bottom of the bone growth plate to form a line. was observed under a fluorescence microscope to measure the length of growth.

After the collected sections were placed on a slide glass and dried, the length of the long bones of the rats was measured by measuring the length between the growth plate and the line formed by the deposition of the fluorescent factor in the bone tissue using a fluorescence microscope.

As a result of the measurement, when the samples of Examples 1-1 to 1-4 were administered, the length growth compared to the control group increased by 16%, 17%, 21%, and 18%, respectively.

In addition, when the samples of Examples 2-1 to 2-4 were administered, the length growth compared to the control group increased by 12%, 10%, 11%, and 10%, respectively.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (7)

A food composition for improving bone health comprising a citrus fruit extract, fruit juice or crushed fruit as an active ingredient. According to claim 1,
The citrus (citrus) food composition at least one selected from the group consisting of green tangerines, tangerines, grapefruits, and oranges. A food composition for preventing or improving bone disease, comprising a citrus fruit extract, fruit juice or crushed fruit as an active ingredient. According to claim 3,
The bone disease is a food composition selected from one or more selected from the group consisting of osteoporosis, osteogenesis imperfecta, osteomalacia, bone mass loss, bone fracture, bone defect and hip joint loss. A pharmaceutical composition for the prevention or treatment of bone disease comprising a citrus fruit extract, fruit juice or crushed fruit as an active ingredient. According to claim 5,
The pharmaceutical composition wherein the citrus is at least one selected from the group consisting of green tangerines, tangerines, grapefruits, and oranges. According to claim 5,
The bone disease is a pharmaceutical composition selected from one or more selected from the group consisting of osteoporosis, osteogenesis imperfecta, osteomalacia, bone mass loss, fracture, bone defect and hip joint loss.

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