KR20210059842A - A composition for immune enhancement comprising benicasa hispida extract - Google Patents

Abstract

The present invention relates to a food composition for immune enhancement containing a Benincasa hispida seed extract as an active component. The composition according to an aspect of the present invention contains a natural extract as an active component, thereby having excellent biosafety as well as immune enhancing activity and bone health improvement effects, so that the composition can be usefully used not only as health functional food but also as a treatment for bone diseases.

Description

A composition for enhancing immunity containing copper fruit seed extract as an active ingredient {A COMPOSITION FOR IMMUNE ENHANCEMENT COMPRISING BENICASA HISPIDA EXTRACT}

The present invention relates to a composition for enhancing immunity comprising a copper fruit seed extract as an active ingredient, and can be used as a variety of uses, such as pharmaceuticals and foods.

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.

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.

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

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.

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.

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.

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.

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.

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 (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.

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, so that the amount of bone formation far exceeds the amount of bone resorption, so bone growth occurs.

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.

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.

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.

Therefore, its efficacy in promoting growth has been scientifically verified, and there is a need for the development of safe food ingredients.

Through research, the present inventors have confirmed that the Donggwa seed extract not only enhances immune function by activating immune cells, but also helps 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.

According to an aspect of the present invention, there is provided a composition for enhancing immunity comprising a copper seed extract as an active ingredient.

In one embodiment, the extract may be extracted with water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane, or a mixed solvent thereof.

In one embodiment, the composition may further include a use for improving bone health or promoting growth.

In one embodiment, the composition may further include a use for preventing or improving bone disease.

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.

According to another aspect of the present invention, there is provided a composition for enhancing immunity comprising a roasting copper seed extract as an active ingredient.

In an embodiment, the copper seeds may be roasted at 150 to 300° C. for 5 to 50 minutes.

In one embodiment, the composition may further include a use for improving bone health or promoting growth.

The composition according to an aspect of the present invention contains a natural extract as an active ingredient, so it is not only excellent in biosafety, but also has excellent immunity enhancing activity and bone health improvement effect, so it can be usefully used as a health functional food as well as a treatment for bone diseases have.

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.

1 is a result of evaluating the cytotoxicity of the extract of Donggwa seeds according to an embodiment of the present invention. (A) is a copper fruit seed hot water extract, (B) is a copper fruit seed ethanol extract, (C) is a roasted copper fruit seed hot water extract (D) is a roasted copper fruit seed ethanol extract.
Figure 2 is a result of evaluating the immune enhancing activity (NO assay) of the Donggwa seed extract according to an embodiment of the present invention. (A) is a copper fruit seed hot water extract, (B) is a copper fruit seed ethanol extract, (C) is a roasted copper fruit seed hot water extract (D) is a roasted copper fruit seed ethanol extract.
3 and 4 are results of evaluating the immunity enhancing activity (TNF-a, IL-6 production amount) of the Dong fruit seed extract according to an embodiment of the present invention. (A) is a copper fruit seed hot water extract, (B) is a copper fruit seed ethanol extract, (C) is a roasted copper fruit seed hot water extract (D) is a roasted copper fruit seed ethanol extract.
5 is a result of evaluating the osteoblast differentiation promoting activity of the Donggwa seed extract according to an embodiment of the present invention. (A) is a copper seed ethanol extract, (B) is a roasted copper fruit seed ethanol extract.
6 and 7 are results of evaluating the ALP activity of the copper seed extract according to an embodiment of the present invention. (A) is a copper seed ethanol extract, (B) is a roasted copper fruit seed ethanol extract.

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.

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.

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.

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.

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 an aspect of the present invention, there is provided a composition for enhancing immunity comprising a copper seed extract as an active ingredient.

The “dongwa seed” refers to the mature seeds of the copper family or fairy tale as an annual vine plant of the gourd family. The winter melon is mainly distributed in the subtropical regions of Asia and the southern regions of Korea, and blooms in summer to produce long, amber-shaped oval fruits in autumn. The seeds in the fruit include saponin, linoleic acid, and oleic acid. It contains a large amount of ingredients such as (oleic acid) and unsaturated fatty acids.

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.

On the other hand, Donggwa seeds are also called Donggwaja, White Confectionery (白瓜子), and Dongguain (冬瓜仁), and they are used as medicinal materials by receiving Ryeomun seeds from late summer to early autumn and drying them in the sun.

The Donggwa seed has various effects such as expectorant, antipyretic, diuretic, anti-inflammatory, gastric ulcer prevention, histamine secretion inhibition, anti-tumor, anti-obesity, high blood pressure prevention, treatment of blood vessel damage caused by diabetes and hyperglycemia, improvement of non-alcoholic fatty liver, and skin care. Although known, the immune activity or the effect related to bone formation is unknown, and the present inventors attempted to investigate the immune enhancing activity of the Donggwa seed extract or the effect of treating bone disease using the same.

The “immunity” refers to a mechanism that protects the organism from diseases by detecting pathogens and tumor cells in an organism and neutralizing it. The “Immunostimulation” refers to various diseases such as cancer and inflammation. A set of therapeutic or prophylactic strategies to augment the body's defenses against the body may be included.

The "contained as an active ingredient" means to contain an amount sufficient to provide a therapeutic or prophylactic effect to the individual to be administered, and the copper fruit seed extract is excellent in biosafety, so that a person skilled in the art can set a quantitative upper limit. It can be adjusted appropriately.

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.

The extract may be obtained by drying and powdering the copper fruit seeds, 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).

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.

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. 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.

The composition may further include a use for preventing or improving bone diseases.

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 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 may include a copper fruit seed extract to 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 it may 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 composition can effectively maintain joint and bone health by affecting the differentiation of osteoblasts and promoting bone regeneration.

The composition may be used as a food composition, for example, as a health functional food related to immunity, growth promotion or 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 composition may contain ordinary food additives, and the food additives are appropriate in accordance with the standards and standards for the product in accordance with the general rules and general test methods of food additives approved by the Ministry of Food and Drug Safety, unless otherwise specified. You can judge whether or not.

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 composition may be variously used in foods and beverages for enhancing immunity or improving bone disease, and for example, 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 enhancing immunity or improving bone disease.

Specifically, for the health functional food in the form of a tablet, a mixture of a copper fruit seed extract, an excipient, a binder, a disintegrant, and other additives is granulated by a conventional method, and then compression molded with a lubricant or the like, or the mixture is directly It can be manufactured by compression 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.

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 seed extract and excipients, or granular or skinned granules thereof. It can be prepared by filling a mixture with additives such as seed extract and excipients 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 may be prepared by molding a mixture of copper fruit seed extract, excipient, binder, and disintegrant by an appropriate method. You can also create a ring with a suitable substance.

The granular health functional food may be prepared in a granular form of a mixture of copper fruit seed extract, excipients, binders, disintegrants, etc. in a suitable manner, and may contain flavoring agents, flavoring agents, and the like, if necessary.

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.

According to another aspect of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of bone disease comprising the extract of Donggwa seed as an active ingredient.

In addition, the composition can be usefully used in the prevention, improvement or treatment of diseases caused by immunodeficiency, immunodeficiency, or damage to the immune system by enhancing immune activity.

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.

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.

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.

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.

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 water for the copper fruit seed extract and its fractions. It can be prepared by mixing cross, lactose, or gelatin. 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. 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.

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.

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.

According to another aspect of the present invention, there is provided a composition for enhancing immunity comprising a roasting copper seed extract as an active ingredient.

The "roasting" is a method of heating without adding moisture using high dry heat of 150°C or higher. The heating device for roasting may be an oven, a gas range, a microwave oven, or the like, but is not particularly limited as long as it is a heating device capable of controlling the temperature.

Specifically, the copper seeds may be roasted at 150 to 300°C for 5 to 50 minutes, and preferably at 200 to 250°C for 10 to 15 minutes.

The roasting can increase or decrease the content of various active ingredients contained in copper seeds, and the present inventors have found that the immunity enhancing activity can be remarkably increased by the roasting process.

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.

Preparation Example 1: Preparation of hot water extract (HR1910-W)

100 times of purified water was added to the crushed dried copper fruit seeds, heated at 100° C. for 4 hours, and filtered.

After the obtained liquid extract was concentrated (20 brix) with a rotary vacuum concentrator, a copper fruit seed extract was obtained using a freeze dryer. The yield of the extract obtained by powdering was 13.3% compared to the dried product.

Preparation Example 2: Preparation of ethanol extract (HR1910-70E)

100 times ethanol (70% v/v) was added to the crushed copper seed dried product, heated at 100° C. for 4 hours, and filtered.

After the obtained liquid extract was concentrated (20 brix) with a rotary vacuum concentrator, a copper fruit seed extract was obtained using a freeze dryer. The yield of the extract obtained by powdering was 6.4% compared to the dried product.

Preparation Example 3: Preparation of roasting hot water extract (HR1910-S-W)

Heat treatment (roasting) was performed on the dried copper fruit seeds. After sufficiently preheating in advance, the roasting process was performed. The heating temperatures were 200°C, 250°C, and 300°C, and roasting was performed using a Wiswell Convection Electric Oven (CKF 4D) in the range of 10 minutes, 15 minutes, and 20 minutes at each temperature.

In each condition, the same level of activity was shown for each roasted copper fruit seed, but the activity of the copper fruit seeds roasted at 250° C. for 15 minutes was the most excellent, and thus the activity was evaluated as the sample.

100 times of purified water was added to the crushed roasted copper seed dried product, heated at 100° C. for 4 hours, and filtered.

After the obtained liquid extract was concentrated (20 brix) with a rotary vacuum concentrator, a copper fruit seed extract was obtained using a freeze dryer. The yield of the extract obtained by powdering was 16.1% compared to the dried product.

Preparation Example 4: Preparation of roasting ethanol extract (HR1910-S-70E)

Roasting was performed in the same manner as in Preparation Example 3. Activity evaluation was carried out with copper seeds roasted at 250° C. for 15 minutes.

100 times ethanol (70% v/v) was added to the crushed roasted copper seed dried product, heated at 100° C. for 4 hours, and filtered.

After the obtained liquid extract was concentrated (20 brix) with a rotary vacuum concentrator, a copper fruit seed extract was obtained using a freeze dryer. The yield of the extract obtained by powdering was 9.4% compared to the dried product.

Experimental Example 1: Cell culture

The cell line, RAW264.7 macrophage cell, was purchased and used 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), 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.

Experimental Example 2: Toxicity evaluation (MTT assay)

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 5x10 3} cells/100μL and cultured for 24 hours in a 37°C, 5% CO _{2 incubator.}

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.

Toxicity evaluation based on cell viability for each sample was measured by repeating three times, and the mean value and standard deviation were calculated.

Referring to FIG. 1, the extracts of Preparation Examples 1 to 4 did not show cytotoxicity at all concentrations (10, 100, 1000 μg/mL).

Experimental Example 3: Evaluation of immune efficacy (NO assay)

To measure the concentration of Nitric Oxide (NO), absorbance was measured at 540 nm using a grease reaction.

N-(1-naphthyl)-ethylenediamine, sulfanilamide, and NO ₂ ^- react to form azo couling, and the two ring shapes have maximum absorbance values at a wavelength of 540 nm.

Raw264.7 cells were dispensed in a 24 well plate at ^{5x10 4} cells / 250 μL, and cultured for 24 hours in _{a 37°C, 5% CO 2 incubator.}

Samples were treated by concentration and incubated in a _{CO 2 incubator for 48 hours.} Take 50 μL of the cell culture supernatant, mix 50 μL of N1 buffer, and react at room temperature for 5 to 10 minutes. After 50 μL of N2 buffer was mixed and reacted for 10 minutes, absorbance at 560 nm was measured using a microplate reader (Epoch, Biotek).

It was measured three times by the same method, and the average value and standard deviation thereof were obtained.

Referring to Figure 2, in the case of the hot water extract of copper fruit seeds (Preparation Example 1), the NO concentration was increased by about 21% compared to the control at 1000 μg/mL, and the ethanol extract of Dong fruit seeds (Preparation Example 2) was at 1000 μg/mL. It increased by about 59% compared to the control group.

In the case of the roasted copper seed hot water extract (Preparation Example 3), the NO concentration increased by about 75% at 10 μg/mL, about 355% at 100 μg/mL, and about 416% at 1000 μg/mL.

In the case of the roasted copper seed ethanol extract (Preparation Example 4), the NO concentration at 100 μg/mL increased by about 119% compared to the control group and by about 226% at 1000 μg/mL.

The above results suggest that not only the copper fruit seed extract has excellent immunity enhancing effect, but also the immunity enhancing activity may be remarkably increased by roasting.

Experimental Example 4: Evaluation of immune efficacy (evaluation of TNF-a and IL-6 production)

In order to examine the immunity increasing efficacy, Raw264.7 cells were dispensed in a 24 well plate at 5 ^{×10 4} cells/250 μL and incubated for 24 hours in _{a 37°C, 5% CO 2 incubator.}

Samples were treated by concentration _{and incubated in a CO 2} incubator for 4 hours, and then the cell suspension was centrifuged to precipitate the cells and the supernatant was collected.

The amount of TNF-a and IL-6 produced in the supernatant was quantified according to the method described in the user's manual using an ELISA kit (R&D systems Inc., Minneapolis, MN, USA).

Referring to Figure 3, in the case of the hot water extract (Preparation Example 1), the amount of TNF-a increased by about 220 pg/mL compared to the control at 100 μg/mL, and about 395 pg/mL compared to the control at 1000 μg/mL. Increased.

In the case of ethanol extract (Preparation Example 2), the amount of TNF-a was about 97 pg/mL compared to the control at 10 μg/mL, about 472 pg/mL at 100 μg/mL, and about 494 compared to the control at 1000 μg/mL. pg/mL increased.

In the case of roasted copper seed hot water extract (Preparation Example 3), the amount of TNF-a was about 296 pg/mL compared to the control at 10 μg/mL, about 429 pg/mL at 100 μg/mL, and at 1000 μg/mL compared to the control. There was an increase of about 462 pg/mL.

In the case of roasted copper seed ethanol extract (Preparation Example 4), the amount of TNF-a was about 339 pg/mL compared to the control at 10 μg/mL, about 432 pg/mL at 100 μg/mL, and at 1000 μg/mL compared to the control. There was an increase of about 473 pg/mL.

Referring to Figure 4, in the case of the hot water extract (Preparation Example 1), the amount of IL-6 increased by about 156 pg/mL compared to the control at 100 μg/mL, and about 643 pg/mL compared to the control at 1000 μg/mL. Increased.

In the case of the ethanol extract (Preparation Example 2), the amount of IL-6 was increased by about 190 pg/mL compared to the control at 100 μg/mL, and by about 709 pg/mL compared to the control at 1000 μg/mL.

In the case of the roasted copper seed hot water extract (Preparation Example 3), the amount of IL-6 increased by about 551 pg/mL compared to the control at 100 μg/mL, and by about 1378 pg/mL compared to the control at 1000 μg/mL.

In the case of the roasted copper seed ethanol extract (Preparation Example 4), the amount of IL-6 increased by about 258 pg/mL compared to the control at 100 μg/mL, and about 1143 pg/mL compared to the control at 1000 μg/mL.

When roasting the Donggwa seeds, a very high level of immunity enhancing activity was confirmed even at a particularly low concentration.

The above results suggest that not only the copper fruit seed extract has excellent immunity enhancing effect, but also the immunity enhancing activity may be remarkably increased by roasting.

Experimental Example 5: Evaluation of osteoblast differentiation activity

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.

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.

Referring to FIG. 5, in the case of the ethanol extract (Preparation Example 2), the amount of osteoblast differentiation increased by about 22% compared to the control at 100 μg/mL, and about 42% compared to the control at 1000 μg/mL.

In the case of the roasted copper seed ethanol extract (Preparation Example 4), the amount of osteoblast differentiation was increased by about 34% compared to the control at 1000 μg/mL.

In the case of the hot water extract of Donggua seeds (Preparation Example 1) and the hot water extract of roast Donggua seeds (Preparation Example 3), the osteoblast differentiation activity at the same level as the ethanol extract was confirmed.

The above results suggest that the extract of Donggwa seed promotes the differentiation of osteoblasts, so it is effective in maintaining bone health by adsorbing minerals such as calcium.

Experimental Example 6: ALP activity evaluation (ALP assay)

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

MC3T3-E1 (osteocyte) ^{was dispensed into 48 wells at 5x10 3} cells/250 μL 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.

Referring to Figure 6, in the case of the ethanol extract (Preparation Example 2) of the copper seed, ALP activity increased by 88% compared to the control at 100 μg/mL, and increased by 120% at 1000 μg/mL.

In the case of the roasted copper seed ethanol extract (Preparation Example 4), ALP activity increased by about 102% compared to the control at 1000 μg/mL.

In the case of the hot-water extract of Donggua seeds (Preparation Example 1) and the hot-water extract of roast Donggua seeds (Preparation Example 3), ALP activity at the same level as the ethanol extract was confirmed.

The above results suggest that the extract of Donggwa seed promotes the differentiation of osteoblasts, so it is effective in maintaining bone health by adsorbing minerals.

Experimental Example 7: Evaluation of ALP activity (Western blot)

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.

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.

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.

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 three times with TBS-T and reacting with the ECL substrate for 3 minutes, it was analyzed by chemiluminescence.

Referring to FIG. 7, in the case of the copper seed ethanol extract (Preparation Example 2), ALP activity increased by about 47% compared to the control at 10 μg/mL and about 136% at 100 μg/mL compared to the control.

In the case of the roasted copper seed ethanol extract (Preparation Example 4), ALP activity was increased by about 98% compared to the control at 1000 μg/mL.

In the case of the hot-water extract of Donggua seeds (Preparation Example 1) and the hot-water extract of roast Donggua seeds (Preparation Example 3), ALP activity at the same level as the ethanol extract was confirmed.

The above results suggest that the extract of Donggwa seed affects the mechanism of action related to bone health and is effective in maintaining bone health by regulating the differentiation of osteoblasts.

Experimental Example 8: Evaluation of bone regeneration induction activity

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

According to micro-CT (μCT) analysis, copper seed 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.

According to histological tests, bone formation at the defect site was confirmed by treatment of the samples of Preparation Examples 1 to 4, unlike the control area, which is mostly fibrous tissue.

Experimental Example 9: Evaluation of iliac bone length growth activity

The efficacy of the extract of Donggwa seeds on bone growth in rats was measured.

The measurement method is a method for observing normal bone metabolism and uses a phenomenon in which a fluorescent factor is deposited on bone through a chelate bond with calcium.

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. Therefore, the length between the fluorescent ray and the growth plate after a certain period of time after the sample is administered once. Was observed with a fluorescence microscope to measure the grown length.

The collected section was 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 growth of the measured iliac bone length of the rat.

As a result of the measurement, when the hot water extract of copper fruit seeds (Preparation Example 1) and ethanol extract of Dong fruit seeds (Preparation Example 2) were administered, the length growth increased by 19% and 23%, respectively, compared to the control group, and the samples of Preparation Examples 3 and 4 It was confirmed that the same level of increase in iliac length growth by

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.

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)

A composition for enhancing immunity comprising a copper fruit seed extract as an active ingredient. The method of claim 1,
The extract is a composition extracted with water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane, or a mixed solvent thereof. The method of claim 1,
A composition further comprising the use of improving bone health or promoting growth. The method of claim 1,
A composition further comprising the use of preventing or improving bone disease. The method of claim 4,
The bone disease is at least one composition selected from the group consisting of osteoporosis, bone insufficiency, osteomalacia, bone mass reduction, fracture, bone defect, and hip reduction. A composition for enhancing immunity comprising a roasting copper seed extract as an active ingredient. The method of claim 6,
The copper seeds are roasted at 150 to 300°C for 5 to 50 minutes. The method according to claim 6 or 7,
A composition further comprising the use of improving bone health or promoting growth.

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