WO2022060024A1 - Composition comprising citrus extract as active ingredient for improving bone health - Google Patents

Abstract

The present invention relates to a food product and a pharmaceutical composition, each comprising a citrus raw material as an active ingredient for improving bone health.

Description

Composition for improving bone health containing citrus extract as an active ingredient

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 food.

Living organisms constantly require 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 to maintain human life, and humans sustain life by ingesting food, digesting it in the digestive system, absorbing it, and supplying it to the tissue cells of the body.

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

Nutrition can directly or indirectly cause disease, and it is also involved in the prevention and treatment of disease. disease can be caused.

Recently, as living standards have increased and dietary habits have been 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 extension of life expectancy, interest in health is gradually increasing.

Health promotion aims to increase resistance to diseases, particularly chronic degenerative diseases or infectious diseases, and continuous stress, and to increase daily activity. Management that affects the overall lifestyle such as sustainability is required.

On the other hand, recently, due to the westernization of the body type and the increase in average height, interest in height growth factors is increasing.

If growth is defined as a broad concept, 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 the 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 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. During the growth phase, the ability to form bone by osteoblasts reaches the maximum, and the amount of bone formation far exceeds the amount of bone resorption, so that bone growth occurs.

Growth hormone therapy, which was used for growth retardation in the past pathological condition, 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 administration method shows excellent effects in people lacking growth hormone, but in most people with normal hormone secretion, various side effects such as acromegaly, growth hormone antibody positivity, systemic allergic reaction, hypothyroidism, etc. There are problems that may arise.

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

Therefore, there is a need to scientifically verify its efficacy in promoting growth, and to develop safe food materials.

The present inventors confirmed that the citrus extract can help improve bone health by promoting the differentiation of osteoblasts involved in bone formation through research.

The present invention is to solve the problems of the prior art, and an object of the present invention is to provide a functional food product derived from a natural plant having excellent biosafety.

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

In one embodiment, the citrus may be one or more selected from the group consisting of green tangerines, tangerines, grapefruit, and oranges.

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

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

In one embodiment, the bone disease may be one or more selected from the group consisting of osteoporosis, bone hypoplasia, osteomalacia, osteopenia, fractures, bone defects, and hip arthropathy.

According to another aspect of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of bone diseases comprising a citrus extract, a juice or a pulverized product as an active ingredient.

In one embodiment, the citrus may be one or more selected from the group consisting of green tangerines, tangerines, grapefruit, and oranges.

In one embodiment, the bone disease may be one or more selected from the group consisting of osteoporosis, bone hypoplasia, osteomalacia, osteopenia, fractures, bone defects, and hip arthropathy.

Since the composition according to one aspect of the present invention contains natural raw materials as an active ingredient, it has excellent biosafety and bone health improvement effects, so it can be usefully used not only as a health functional food but also as a treatment for bone diseases.

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

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 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 ALP activity of green tangerine extract, green tangerine pulverized product, and citrus sample according to an embodiment of the present invention.

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

In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the corresponding 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, rather than the name of a simple term.

Unless defined otherwise, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this 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 art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Numerical ranges are inclusive of the values defined in that range. Every maximum numerical limitation given throughout this specification includes all lower numerical limitations as if the lower numerical limitation were expressly written. Every minimum numerical limitation given throughout this specification includes all higher numerical limitations as if the higher numerical limitation were expressly written. Any numerical limitation given throughout this specification shall include all numerical ranges within the broader numerical range, as if the narrower numerical limitation were expressly written down.

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.

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

The dictionary meaning of the "Citrus" is a general name spanning three genera of Citrus, Fortunella, and Pontirus of the Tangerine family, and the citrus is Narginin, Nargenin ( Naringenin), hesperidin (Hesperidin), tangeretin (Tangeretin), may include various compounds such as nobiletin (Nobiletin).

The citrus is a citrus fruit, such as tangerine, kumquat, mandarin, tangerine, sweetie, citron, young tangerine, cheonhyehyang, calamansi, pomelo, hallabong, orange, lemon, grapefruit, lime, citron, and their unripe fruits. It is not particularly limited as long as it belongs to the Citrus plant.

In one embodiment, the citrus may be one or more selected from the group consisting of green tangerines, tangerines, grapefruit, and oranges.

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

The immature fruit may refer to an extremely early Onju tangerine with a sugar content of less than 8 Brix or an early and ordinary Onju tangerine with a sugar content of less than 9 Brix.

The “tangerine” is a generic term for an evergreen small tree belonging to the genus rutaceae and citrus, including C. unshiu, red tangerine (C. deliciosa), king tangerine (C. grandis), citron (C. junos), Summer tangerine (C. natsudaidai), citron (C. medica), lime (C. aurantifolia), lemon (C. limon), citrus (C. aurantium), rough melon (C. jambhiri), chestnut (C. maxima), crust (C. aurantium), tangerine (C. tangerina), tangerine (C. leiocarpa), tangerine (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 kan (C. sulcata HORT. Ex. Tan), Nasu (C. natsudaidai HAY) ), sugar yuja (C. grandis OSBECK) and palsak (C. hassaku HORT. Ex Y.Tan) may be selected from the group consisting of, but is not limited thereto.

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

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

The citrus is known to have various effects such as skin care, but the effect or mechanism of action related to bone disease is not known so far, and the present inventors The purpose of this study was to investigate the activity of improving bone health and the treatment effect of bone disease using the same.

The bone disease (bone disease) refers to a condition or disease that requires or requires an increase in bone mass through promotion of osteoblast activity or inhibition of osteoclast activity, for example, it may be a metabolic bone disease or an orthopedic bone disease. .

The “metabolic bone disease” refers to a condition or disease that occurs due to excessive generation or activity of osteoclasts, and may include a disease that decreases bone mass. The bone mass loss disease refers to a condition or disease in which a decrease in bone mass accompanied by symptoms such as a decrease in bone density and softening of bone tissue appears.

The bone disease may be one or more selected from the group consisting of osteoporosis, bone hypoplasia, osteomalacia, osteopenia, fracture, bone defect, and hip osteopenia, but is not limited thereto.

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

The “extract” refers to a solvent in which the active ingredient contained in the extraction raw material is transferred by contacting the solvent and the extraction raw material under specific conditions. They can all be included regardless. For example, extracts of components soluble in solvents from natural products using water or organic solvents, specific components of natural products, for example, products obtained by extracting only specific components such as oil may be included.

The extract can be obtained by drying and pulverizing the citrus raw material (citrus fruits), or by various conventionally known extraction methods such as hot water extraction and ethanol extraction, and both the fruit peel and the fruit can be used as the extraction raw material.

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

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

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

The extract may be obtained in a powder state 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. For example, the extract is separated using an ultrafiltration membrane having a constant molecular weight cut-off value, and various purification methods are additionally performed, such as separation by various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity). It may include a fraction obtained through

The "juice" is obtained by separating the juice from the citrus raw material, for example, by adding the raw material to a juicer such as a mixer or crusher to separate the liquid component, and can also be obtained in powder form through filtration and freeze-drying process .

The “pulverized product” may be processed in the form of small particles of powder through physical force to the citrus raw material. The pulverized product may be obtained in an easy-to-ingest form by applying a mechanical force such as compression, shearing, 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 size and function of each organ in the body, that is, development of a function and differentiation into an organ.

The food composition, including the citrus extract, can bring excellent growth promoting effects without side effects, such as growing bone length and promoting the metabolism of growth plate chondrocytes, and can be particularly useful as a food material for promoting growth in children.

The “bone health” refers to normal bone decomposition and remodeling, and if the balance of bone decomposition and remodeling is broken due to a 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 influencing 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 means a food manufactured and processed using raw materials or ingredients useful for the human body in accordance with the Health Functional Food Act, and the functionality is to control nutrients or physiologically affect the structure and function of the human body. It may refer to ingestion for the purpose of obtaining useful effects for health purposes, such as action.

The food composition may include normal food additives, and unless otherwise specified, the food additives are approved by the Ministry of Food and Drug Safety according to the general rules and general test methods of the Food Additives Code, according to the standards and standards for the item. suitability can be judged.

Items described in the Food Additives Codex include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; Mixtures, such as an added alkali agent, a preservative agent, and a tar dye agent, are mentioned.

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

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 improving bone diseases.

Specifically, the health functional food in the form of tablets is granulated with a mixture of citrus raw materials, excipients, binders, disintegrants, and other additives in a conventional manner, and then compression-molded by adding a lubricant, or the mixture is directly compression-molded. can be manufactured. In addition, the health functional food in the form of tablets may contain a mating agent, etc., if necessary, and may be coated with a suitable skinning agent if necessary.

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

The health functional food in the form of a ring can be prepared by molding a mixture of citrus raw materials, excipients, binders, disintegrants, etc. by an appropriate method, and if necessary, coating with sucrose or other suitable skinning agent, or starch, talc, or a suitable substance You can also dress up with

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

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

According to another aspect of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of bone diseases comprising a citrus extract, a juice or a pulverized product as an active ingredient.

The “prevention” refers to any action that reduces the frequency or extent of the occurrence of pathological phenomena. Prevention may be complete or partial. In this case, it may mean a phenomenon in which the symptoms caused by inflammation in the individual are reduced compared to the case where the composition is not used.

The "treatment" refers to any action that clinically intervenes to change the natural process of a subject 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 a disease, alleviate symptoms, reduce any direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, alleviating or temporarily ameliorating 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 compositions 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, excipient and diluent used in the preparation of the 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.

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

A solid preparation for oral administration may include tablets, pills, powders, granules, capsules, etc., and the solid preparation includes at least one excipient in the citrus raw material and its fractions, for example, starch, calcium carbonate, sucrose, It can be prepared by mixing lactose or gelatin. In addition to the above excipients, lubricants such as magnesium stearate and talc may be used.

Liquid formulations for oral administration may include suspensions, 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 used as sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. The non-aqueous solvent and suspension agent may be propylene glycol, polyethylene glycol, vegetable oil such as olive oil, or injectable ester such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin oil, and glycerogelatin 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 with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the patient's type, severity, drug activity, and drug Sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field.

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. Taking all of the above factors into consideration, it is preferable to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The composition may be used for one or more materials selected from the group consisting of artificial bone, artificial joint, bone fixation material, bone substitute, bone repair material, bone filler, 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 easy to apply 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 for filling a space in bone tissue in a narrow sense, and may be used to replace a part of a bone or a 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 chemical additives are used. Therefore, it can be formulated and used in the form of gels, granules, pastes, tablets, pellets, etc., and can also be used in powder form.

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

Example 1: Preparation of green tangerine samples

* 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 tangerine persimmon) and heating it at 60° C. for 5 hours. The filtered extract was concentrated to 120 brix and then 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 tangerine persimmon) and heating it at 60° C. for 5 hours. The filtered extract was concentrated to 120 brix and then powdered using a freeze dryer.

Examples 1-3. July Green Tangerine Freeze-Dried Powder (HR1903-7RF)

In July, the raw material of green mandarin orange (onju tangerine persimmon) was crushed and then freeze-dried and powdered.

Examples 1-4. August Green Tangerine Freeze-Dried Powder (HR1903-8RF)

In August, the raw material of green tangerine (onju tangerine persimmon) was crushed and freeze-dried to powder.

Example 2: Preparation of tangerine, grapefruit, and orange samples

Example 2-1. Citrus Hot Water Extract (HR1903-C-W)

An extract was prepared by adding 50-fold purified water to mature tangerine (onju citrus persimmon) and heating it at 100° C. for 5 hours. The filtered extract was concentrated to 20 brix and then powdered using a freeze dryer.

Example 2-2. Citrus Juice Powder (HR1903-C-PE)

Mature mandarin oranges (onju tangerine persimmon) were extracted 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-fold purified water to grapefruit and heating at 100° C. for 5 hours. The filtered extract was concentrated to 20 brix and then powdered using a freeze dryer.

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

After juicing the orange with a juicer, it was filtered and powdered using a freeze dryer.

Experimental Example 1: Cell Culture

The cell line, RAW264.7 macrophage cell, was purchased from the American Type Culture Collection (ATCC, Manassas. USA).

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, 37 C, and cultured in an incubator (Thermo, Forma, Germany) in the presence of 5% CO2.

Experimental Example 2: Toxicity evaluation (MTT assay)

Cell viability was measured by measuring the concentration of the generated formazan with a spectrophotometer using a 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 aliquoted into 96 wells at 5x10 ³ cells/100 μL and incubated for 24 hours at 37° C., 5% CO 2 incubator.

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

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

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

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

To examine the efficacy of bone health, 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 incubated in a CO 2 incubator for 48 hours. After incubation, MTT (EZ-cytox, Daillab) solution was treated, and after CO 2 incubation for 2 hours, 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 control group, 1000 μg/mL]
Example 1-1	29
Example 1-2	32
Examples 1-3	37
Examples 1-4	57
Example 2-1	19
Example 2-2	24
Example 2-3	37
Example 2-4	32

Referring to FIG. 2 , in Example 1-1, the osteoblast differentiation activity increased by about 20% compared to the control at 100 μg/mL, and increased by about 29% compared to the control at 1000 μg/mL. In Example 1-2 In this case, the osteoblast differentiation activity was increased by about 30% compared to the control at 100 μg/mL, and increased by about 32% compared to the control at 1000 μg/mL.

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

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

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

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

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

In the case of Example 2-4, osteoblast differentiation activity was increased by 10% compared to the control 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)

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

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

Samples were treated by concentration and cultured in a CO 2 incubator for 7 days. After washing with PBS, lysis buffer was added to dissolve, and centrifugation was performed 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 (Table 2).

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

Referring to FIG. 3 , in the case of Example 1-1, ALP activity increased by 29% compared to the control at 100 μg/mL, and increased by 35% at 1000 μg/mL. In Example 1-2, 100 μg/mL In mL, ALP activity increased by 42% compared to the control group, and increased by 39% at 1000 μg/mL.

In the case of Example 1-3, the ALP activity was increased by 40% compared to the control 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 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 at 100 μg/mL, and increased by 30% at 1000 μg/mL.

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

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

In the case of Example 2-4, ALP activity increased by 31% compared to the control 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)

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

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

The blocking of the membrane was performed for 1 hour in 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.

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

After washing 3 times with TBS-T and reacting with ECL substrate for 3 minutes, analysis was performed by chemiluminescence (Table 3).

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

4 , in the case of Example 1-1, the ALP expression level increased by about 67% compared to the control at 100 μg/mL, and by about 89% compared to the control at 1000 μg/mL. In Example 1-2, 100 At μg/mL, the ALP expression level was increased by about 108% compared to the control, and by about 93% compared to the control at 1000 μg/mL.

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

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

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

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

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

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

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

Experimental Example 6: Evaluation of bone regeneration induction activity

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

Micro CT (μCT) analysis showed that Example 1 (1000 μg/mL), Example 2 (1000 μg/mL), and BMP-2 increased bone formation over the entire 6-week interval, whereas minimal in the control group. of bone formation was induced.

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

Experimental Example 7: Evaluation of long bone growth activity

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

The measurement method was to observe normal bone metabolism, and a phenomenon in which a fluorescent factor is deposited on the bone through a chelate bond with calcium was used.

When a fluorescent factor is administered during the growth period when bone formation is the most active, the administered fluorescent factor is most deposited in the bone neoplasia under the bone growth plate to form a line. was observed with a fluorescence microscope to measure the growth length.

The collected sections were placed on a slide glass, dried, and then 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 long bone growth in rats.

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

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

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains 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, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may also 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 their equivalents should be construed as being included in the scope of the present invention.

Claims (8)

1. A food composition for improving bone health comprising a citrus extract, a juice or a pulverized product as an active ingredient.
2. The method of claim 1,

   The citrus is a food composition selected from the group consisting of green tangerines, tangerines, grapefruit, and oranges.
3. A food composition for promoting growth comprising a citrus extract, juice or pulverized product as an active ingredient.
4. A food composition for preventing or improving bone disease, comprising a citrus extract, juice or pulverized product as an active ingredient.
5. 5. The method of claim 4,

   The bone disease is one or more selected from the group consisting of osteoporosis, bone hypoplasia, osteomalacia, osteopenia, fracture, bone defect, and hip joint reduction.
6. A pharmaceutical composition for the prevention or treatment of bone diseases, comprising a citrus extract, juice or pulverized product as an active ingredient.
7. 7. The method of claim 6,

   The citrus is a pharmaceutical composition selected from the group consisting of green tangerines, tangerines, grapefruit, and oranges.
8. 7. The method of claim 6,

   The bone disease is osteoporosis, bone hypoplasia, osteomalacia, osteopenia, bone fracture, bone defect, and at least one pharmaceutical composition selected from the group consisting of hip joint atrophy.

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