KR20210070547A - A composition for bone health comprising narrow-leaf erecta fig extract - Google Patents

Abstract

The present invention relates to a composition for improving bone health or promoting growth containing a Ficus erecta extract as an active ingredient. According to the present invention, since a natural extract is included in the composition as an active ingredient, biostability of the composition is excellent.

Description

A composition for improving bone health containing extracts of Narrowleaf chrysanthemum as an active ingredient {A COMPOSITION FOR BONE HEALTH COMPRISING NARROW-LEAF ERECTA FIG EXTRACT}

The present invention relates to a composition for improving bone health, comprising an extract of the Narrowleaf chrysanthemum as an active ingredient, and can be used for various purposes such as medicines and food.

All living things 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 to maintain human life phenomena, 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 is known to cause diseases directly or indirectly, and is also known to be involved in the prevention and treatment of diseases. The lack of nutrients caused by inadequately balanced intake of nutrients required by the human body is also a problem, and excessive intake is also a serious problem. , which can lead to various diseases.

Recently, as the standard of living increases and the diet becomes westernized, the incidence of adult diseases such as obesity, diabetes, high blood pressure, and heart disease is increasing due to overnutrition and lack of exercise.

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, especially 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, due to the westernization of the body shape and the increase in average height in recent years, 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, but if defined in a narrow concept, it can be seen as an increase in height.

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 (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-forming ability by osteoblasts reaches the maximum, and the amount of bone formation far exceeds the amount of bone resorption, so that bone growth occurs.

In particular, growth hormone therapy, which was used for growth retardation in the past pathological condition, is being applied to children and adolescents in growth stages 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 duration 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, the efficacy of growth promotion is scientifically verified, and there is a need for the development of safe food materials.

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 product derived from a natural plant having excellent biosafety.

According to another aspect of the present invention, there is provided a food composition for improving bone health or promoting growth, comprising roasting Narrowleaf chrysanthemum extract as an active ingredient.

In one embodiment, the narrow leaf cheonseonwa may be roasted at 150 to 300 °C.

In one embodiment, the narrow leaf cheonseonwa may be roasted while controlling the temperature from 250°C to 150°C.

In one embodiment, the food composition may further include an immune enhancing use.

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

According to another aspect of the present invention, there is provided a pharmaceutical composition for improving bone health or promoting growth, comprising the roasting Narrowleaf chrysanthemum extract as an active ingredient.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating bone disease, comprising the roasting Narrowleaf chrysanthemum extract as an active ingredient.

According to another aspect of the present invention, there is provided a food composition for improving bone health and enhancing immunity, comprising roasting Narrowleaf chrysanthemum extract and hot water extract and ethanol extract as active ingredients.

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

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 the extract according to an embodiment of the present invention. (A) is Narrow-leaf C. C. and hot water extract, (B) is Narrow-leaved C. C. and ethanol extract, (C) is Narrow-leaved C. C. and hot-water extract roasted at 250°C (D) Roasted Narrow-leaved C. C. and ethanol extract (D) Roasted at 250°C and ethanol extract, (E) is an ethanol extract roasted at 200°C, (F) is an ethanol extract roasted at 200°C, (G) is a stepwise roasted extract, (H) is a stepwise roasted Narrowleaf chrysanthemum extract and ethanol extract.
2 is a result of evaluation (NO assay) of the immune enhancing activity of the extract of Narrowleaf chrysanthemum according to an embodiment of the present invention. (A) is Narrow-leaf C. C. and hot water extract, (B) is Narrow-leaved C. C. and ethanol extract, (C) is Narrow-leaved C. C. and hot-water extract roasted at 250°C (D) Roasted Narrow-leaved C. C. and ethanol extract (D) Roasted at 250°C and ethanol extract, (E) is an ethanol extract roasted at 200°C, (F) is an ethanol extract roasted at 200°C, (G) is a stepwise roasted extract, (H) is a stepwise roasted Narrowleaf chrysanthemum extract and ethanol extract.
Figure 3 is a result of evaluating the immune enhancing activity (TNF-a production amount) of the extract according to an embodiment of the present invention. (A) is Narrow-leaf C. C. and hot water extract, (B) is Narrow-leaved C. C. and ethanol extract, (C) is Narrow-leaved C. C. and hot-water extract roasted at 250°C (D) Roasted Narrow-leaved C. C. and ethanol extract (D) Roasted at 250°C and ethanol extract, (E) is an ethanol extract roasted at 200°C, (F) is an ethanol extract roasted at 200°C, (G) is a stepwise roasted extract, (H) is a stepwise roasted Narrowleaf chrysanthemum extract and ethanol extract.
4 is a result of evaluating the osteoblast differentiation promoting activity of the Narrowleaf chrysanthemum extract according to an embodiment of the present invention. (A) is Narrow-leaf T. chrysanthemum extract and hot-water extract, (B) is Narrow-leaf chrysanthemum and ethanol extract, (C) Narrow-leaf chrysanthemum roasted at 200 °C and hot water extract (D) Roasted at 200 °C and ethanol extract, (E) is a stepwise roasted extract of chrysanthemum, and hot water extract, and (F) is an ethanol extract of chrysanthemum, roasted in stages.
5 and 6 are results of evaluation of the ALP activity of the extracts of Narrowleaf chrysanthemum according to an embodiment of the present invention. (A) is Narrow-leaf T. chrysanthemum extract and hot-water extract, (B) is Narrow-leaf chrysanthemum and ethanol extract, (C) Narrow-leaf chrysanthemum roasted at 200 °C and hot water extract (D) Roasted at 200 °C and ethanol extract, (E) is a stepwise roasted extract of chrysanthemum, and hot water extract, and (F) is an ethanol extract of chrysanthemum, roasted in stages.

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, which 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.

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 improving bone health or promoting growth, comprising the extract of chrysanthemum extract as an active ingredient.

The "narrow-leaved chrysanthemum (Ficus erecta var. sieboldii )" is a plant belonging to the fig family and is also called a slender-leaf fenugreek tree, and is a tree that mainly grows on Jeju Island and the southern coast of Korea, in particular, on Jeju Island.

The narrow-leaved chrysanthemum grows at the foot of a mountain by the sea and is 2 to 4 m in height, and the bark is grayish white with dark brown bark and no hairs. Leaves are alternate phyllotaxis, narrower lanceolate, and 10-20 cm long. The tip is sharp and the edge is flat or coarsely serrated.

The side veins of the Narrow-leaved Cheonenaceae are 5 to 6 pairs, and the petiole has a length of 1 to 4 cm. The narrow-leaved cheonseonseonwae has long been known in the folklore for its effects of balancing, ripening, dryness, hwasup, strong iliac crest, small bell, hwalyeol, and detoxification. In addition, it has been used in the treatment of rheumatoid arthritis, mid-stage weakness, qi and blood loss, sadisaneon, musculoskeletal separation, bruises, transpulmonary, postpartum milk deficiency.

The above “bone health” means 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 “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, both the development of functions and differentiation into organs.

The food composition can bring excellent growth-promoting effects without side effects, such as growing the bone length and promoting the metabolism of growth plate chondrocytes, including extracts from Narrow-leaf chrysanthemum, so it can be particularly useful as a food material for promoting children's growth. .

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

The "comprising as an active ingredient" means to include an amount sufficient to provide a therapeutic or prophylactic effect to the subject to be administered, and the extract of N. chrysanthemum extract has excellent biosafety, so that those skilled in the art can limit the quantitative upper limit. can be adjusted appropriately.

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. All can be included regardless. For example, extracting a component soluble in a solvent from a natural product using water or an organic solvent, a specific component of a natural product, for example, extracting only a specific component such as oil, etc. may be included.

The extract can be obtained by a variety of conventionally known extraction methods, such as dry, powdered, or hot water extraction method, ethanol extraction method of the Narrow-leaf Cheonsonaceae.

The extract may be extracted with water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane, or a mixed solvent thereof, preferably ethanol at a concentration of 60 to 90% (w/w), more preferably 70 to 80 % concentration (w/w) of ethanol.

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 then 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 vacuum distillation 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 food composition may further include an immune enhancing activity as well as the bone health improving activity.

The “immunity” refers to a mechanism to protect a living organism from disease by detecting and neutralizing pathogens and tumor cells in an organism, and the “immunostimulation” refers to various diseases such as cancer and inflammation. It can include both therapeutic and prophylactic strategies that reinforce the body's defense mechanisms against

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

The health functional food means a food manufactured and processed using raw materials or ingredients useful for the human body according to 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 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 standards and standards for the relevant items according to the general rules and general test methods for food additives approved by the Ministry of Food and Drug Safety. can determine whether

The items described in the Food Additives Codex include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, depigmentation, licorice extract, crystalline cellulose, high pigment, natural additives such as guar gum, sodium L-glutamate preparation, noodles Mixed formulations, such as an additive alkali agent, a preservative agent, and a tar dye agent, are mentioned.

The composition may be used in various ways, such as foods and beverages for bone health and immune enhancement, 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 bone health and immunity.

Specifically, the health functional food in the form of a tablet is granulated by a conventional method by granulating a mixture of Narrowleaf chrysanthemum extract, excipients, binders, disintegrants and other additives, and then compression molding by adding a lubricant, or the mixture. It can be manufactured by direct compression molding. 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 Narrowleaf chrysanthemum and additives such as extracts and excipients, or their granules or coated granules. It can be prepared by filling a capsule base such as gelatin with a mixture of leaf chrysanthemum extract and additives such as excipients. 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 Narrowleaf chrysanthemum extract, excipients, binders, disintegrants, etc. by an appropriate method, and, if necessary, coating the skin with sucrose or other suitable skinning agent, or starch, talc Alternatively, the gown may be dressed with a suitable material.

The health functional food in the form of granules can be prepared in a granular form by a suitable method of a mixture of Narrowleaf chrysanthemum extract, excipients, binders, disintegrants, and the like, and may contain flavoring agents, flavoring agents, etc.

In addition, the term definitions 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 the extract of chrysanthemum chrysanthemum as an active ingredient. In addition, the composition can be usefully used for preventing, improving or treating diseases caused by immunodeficiency, immunosuppression, or damage to the immune system by enhancing immune activity.

The "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, metabolic bone disease or orthopedic bone disease. can

The “metabolic bone disease” refers to a condition or disease that occurs due to excessive generation or activity of osteoclasts, and may include bone loss disease. 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 "prevention" means any action that reduces the frequency or degree of occurrence of a pathological phenomenon. Prevention may be complete or partial. In this case, it may mean a phenomenon in which symptoms caused by bone disease 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, to alleviate symptoms, to reduce any direct or indirect pathological consequences of the disease, to prevent metastasis, to reduce the rate of disease progression, or to 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 can 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 may include at least one excipient, such as starch, calcium carbonate, It can be prepared by mixing sucrose, lactose, or gelatin. In addition to the 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 sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. The non-aqueous solvent and suspension 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 single or multiple. 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.

According to another aspect of the present invention, there is provided a food composition for improving bone health or promoting growth, comprising roasting Narrowleaf chrysanthemum 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 the roasting may be an oven, a gas range, a microwave oven, etc., but is not particularly limited as long as it is a heating device capable of controlling the temperature.

The narrow-leaf cheonseonwa can be roasted at 150 to 300 ℃.

Specifically, the narrow-leaf cheonseonwa may be roasted at 150 to 300° C. for 5 to 50 minutes, preferably at 200 to 250° C. for 20 to 30 minutes.

By the roasting, the content of various active ingredients contained in the Narrowleaf Cheesonaceae can be increased or decreased, and the present inventors have found that bone health and growth promoting activity can be significantly increased by the roasting process.

At this time, the narrow-leaf cheonseonwa may be roasted while changing the temperature step by step from high temperature to low temperature, for example, the narrow-leaf cheonseonwa may be roasted while controlling the temperature from 250°C to 150°C.

Specifically, in the roasting process, the temperature may be gradually lowered from 250° C. to 150° C., and roasted at 250° C., 200° C., and 150° C. for a predetermined time, respectively.

Bone health and growth promoting activity can be further improved by performing the roasting stepwise from high temperature to low temperature.

The roasting (roasting) Narrowleaf chrysanthemum extract may be extracted with ethanol.

The present inventors have confirmed that bone health improvement or growth promoting activity can be remarkably increased by extracting the roasted Narrowleaf chrysanthemum with ethanol as a solvent, and the ethanol extract is usefully used in the manufacture of foods and pharmaceuticals for bone health can be

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.

100-fold purified water was added to the crushed Narrow-leaf cheonseon and dried matter, heated at 100° C. for 4 hours, and then filtered.

The obtained liquid extract was concentrated (20 brix) with a rotary vacuum concentrator, and then an extract of Narrowleaf chrysanthemum was obtained using a freeze dryer. The yield of the extract obtained by pulverization was 36.5% compared to the dry matter.

Preparation Example 2: Preparation of ethanol extract (SW1902-70E)

100 times of ethanol (70% v/v) was added to the crushed narrow-leaf chrysanthemum and dried matter, heated at 100° C. for 4 hours, and then filtered.

The obtained liquid extract was concentrated (20 brix) with a rotary vacuum concentrator, and then an extract of Narrowleaf chrysanthemum was obtained using a freeze dryer. The yield of the extract obtained by pulverization was 27.2% compared to the dry matter.

Preparation Example 3: Preparation of roasted hot water extract (SW1902-250R-W)

Heat treatment (roasting) was performed on the narrow-leaf chrysanthemum and dried samples. After sufficiently preheating in advance, the roasting process was performed.

The heating temperature was 250° C., and roasting was performed using Biotech FEC-006 in the time range of 20 minutes, 25 minutes, and 30 minutes.

In each condition, the same level of activity was shown with each roasted Narrowleaf P., but the activity was the most excellent with Narrowleaf Prunus roasted at 250°C for 25 minutes, so the activity of the sample was evaluated.

100-fold purified water was added to the crushed roasted narrow-leaf cheonseon and dried, heated at 100° C. for 4 hours, and then filtered.

The obtained liquid extract was concentrated (20 brix) with a rotary vacuum concentrator, and then an extract of Narrowleaf chrysanthemum was obtained using a freeze dryer. The yield of the extract obtained by pulverization was 36.9% compared to the dry matter.

Preparation Example 4: Preparation of roasted ethanol extract (SW1902-250R-70E)

Roasting was performed in the same manner as in Preparation Example 3. Activity evaluation was conducted with Narrow-leaf Cheoncheongwa roasted at 250° C. for 15 minutes.

100 times of ethanol (70% v/v) was added to the crushed roasted narrow-leaf cheonseon and dried matter, heated at 100° C. for 4 hours, and then filtered.

The obtained liquid extract was concentrated (20 brix) with a rotary vacuum concentrator, and then an extract of Narrowleaf chrysanthemum was obtained using a freeze dryer. The yield of the extract obtained by pulverization was 26.2% compared to the dry matter.

Preparation Example 5: Preparation of roasted hot water extract (SW1902-200R-W)

A hot water extract was prepared in the same manner as in Preparation Example 3, but the roasting temperature was 200°C. The yield of the extract obtained by pulverization was 36.3% compared to the dry matter.

Preparation Example 6: Preparation of roasted ethanol extract (SW1902-200R-70E)

An ethanol extract was prepared in the same manner as in Preparation Example 4, but the roasting temperature was 200°C. The yield of the extract obtained by pulverization was 26.0% compared to the dry matter.

Preparation Example 7: Preparation of roasted hot water extract (SW1902-R(3STEP)-W)

A hot water extract was prepared in the same manner as in Preparation Example 3, but the roasting temperature was changed in stages.

Specifically, the roasting process was carried out in stages for 25 minutes, but at 250°C for 8.5 minutes, at 200°C for 8.5 minutes, and at 150°C for 8 minutes.

The yield of the extract obtained by pulverization was 27.8% compared to the dry matter.

Preparation Example 8: Preparation of roasted ethanol extract (SW1902-R(3STEP)-70E)

An ethanol extract was prepared in the same manner as in Preparation Example 3, but the roasting temperature was changed in stages.

Specifically, the roasting process was carried out in stages for 25 minutes, but at 250°C for 8.5 minutes, at 200°C for 8.5 minutes, and at 150°C for 8 minutes.

The yield of the extract obtained by pulverization was 23.9% compared to the dry matter.

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 Bovine Serum (FBS, Hyclone USA) and 100 units/mL penicillin/Streptomycin (Hyclone, USA) was used, 37 C, 5% CO ₂ Incubated in an incubator (Thermo, Forma, Germany) in the presence.

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 in 96 wells at 5x10 3} cells/100 μL and cultured at 37° C., 5% CO _{2 in an} incubator for 24 hours.

Samples were treated for each concentration and incubated 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.

1, the extracts of Preparation Examples 1 to 8 did not show cytotoxicity in all concentrations (10, 100, 1000 μg/mL) range.

Experimental Example 3: Immune efficacy evaluation (NO assay)

Absorbance was measured at 540 nm using a grease reaction to measure the concentration of Nitric Oxide (NO).

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

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

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

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

Referring to FIG. 2 , in the case of the Narrow-leaf chrysanthemum and hot water extract (Preparation Example 1), the NO concentration was increased by about 49% compared to the control at 1000 μg/mL, and in the case of the Narrow-leaved chrysanthemum extract (Preparation Example 2) compared to the control No significant increase was observed.

In the case of the Narrowleaf chrysanthemum and hot water extract (Preparation Example 3) roasted at 250 ° C, the NO concentration at 100 μg/mL increased by about 44% compared to the control and about 115% at 1000 μg/mL, and the ethanol extract (Preparation Example 4) In the case of , it increased by about 21% at 1000 μg/mL.

In the case of the Narrowleaf chrysanthemum roasted at 200 ° C. and the hot water extract (Preparation Example 5), the NO concentration increased by about 45% compared to the control at 1000 μg/mL, and in the case of the ethanol extract (Preparation Example 6), there was no significant increase compared to the control. .

In the case of the Narrowleaf chrysanthemum and hot water extract (Preparation Example 7) roasted at different temperatures in stages, the NO concentration at 100 µg/mL increased by about 51% compared to the control group and by about 96% at 1000 µg/mL, and the ethanol extract (Preparation Example 7) In the case of Example 8), there was no significant increase compared to the control group.

The above results suggest that not only the immunity enhancing effect of Narrowleaf chrysanthemum extract with hot water is excellent, but also that the immune enhancing activity can be remarkably increased by roasting.

Experimental Example 4: Immune efficacy evaluation (TNF-a production amount evaluation)

In order to examine the efficacy of increasing immunity, Raw264.7 cells were aliquoted at 5x10 ⁴ cells/250 μL in a 24 well plate and _{cultured at 37° C., 5% CO 2 in an} incubator for 24 hours.

The 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 collect the supernatant.

The amount of TNF-a 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 FIG. 3, in the case of the Narrowleaf chrysanthemum extract (Preparation Example 1), the amount of TNF-a increased by about 101 pg/mL compared to the control at 100 μg/mL, and at 1000 μg/mL, about 296 pg/mL compared to the control. mL increased.

On the other hand, in the case of Narrow-leaf Cheoncheon and ethanol extract (Preparation Example 2), there was no significant increase in the amount of TNF-a production compared to the control group.

The amount of TNF-a was increased by about 306 pg/mL compared to the control at 100 μg/mL, and by about 468 pg/mL compared to the control at 1000 μg/mL in the case of the Narrowleaf chrysanthemum and hot water extract (Preparation Example 3) roasted at 250°C. , In the case of the ethanol extract (Preparation Example 4), the amount of TNF-a was increased by about 68 pg/mL compared to the control at 100 μg/mL, and by about 246 pg/mL compared to the control at 1000 μg/mL.

The amount of TNF-a was increased by about 202 pg/mL compared to the control at 100 μg/mL, and by about 371 pg/mL compared to the control at 1000 μg/mL in the case of the Narrowleaf chrysanthemum and hot water extract (Preparation Example 5) roasted at 200°C. , In the case of the ethanol extract (Preparation Example 6), there was no significant increase in the amount of TNF-a production compared to the control.

In the case of the Narrowleaf chrysanthemum and hot water extract (Preparation Example 7) roasted at different temperatures in stages, the amount of TNF-a was about 307 pg/mL compared to the control at 100 μg/mL, and about 476 pg/mL compared to the control at 1000 μg/mL. mL was increased, and in the case of the ethanol extract (Preparation Example 4), the amount of TNF-a was increased by about 180 pg/mL compared to the control at 1000 μg/mL.

A high level of immune-enhancing activity was confirmed even at a particularly low concentration when the Narrowleaf Chrysanthemum was roasted.

The above results suggest that not only the immunity enhancing effect of Narrowleaf chrysanthemum extract with hot water is excellent, but also that the immune enhancing activity can be remarkably increased by roasting.

Experimental Example 5: Evaluation of osteoblast differentiation activity

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 CO ₂ incubated for 2 hours, followed by analysis with a microplate reader (Epoch, Biotek) at 450 nm wavelength.

Referring to FIG. 4 , in the case of the Narrowleaf chrysanthemum extract (Preparation Example 1) and the ethanol extract (Preparation Example 2), there was no significant increase in the amount of osteoblast differentiation compared to the control group.

In the case of the narrow-leaf cheonseon and hot water extract (Preparation Example 5) roasted at 200 ° C, there was no significant increase in the amount of osteoblast differentiation compared to the control, but in the case of the ethanol extract (Preparation Example 6), the amount of osteoblast differentiation at 100 μg/mL was the control. It increased by about 12% compared to the control, and increased by about 44% compared to the control at 1000 μg/mL.

In the case of the Narrowleaf chrysanthemum and hot water extract (Preparation Example 7) roasted at different temperatures in stages, there was no significant increase in the amount of osteoblast differentiation compared to the control group, but in the case of the ethanol extract (Preparation Example 8), osteoblasts at 100 μg/mL The amount of differentiation was increased by about 27% compared to the control group, and increased by about 66% compared to the control group at 1000 μg/mL.

The above results suggest that the extracts from chrysanthemum extract are effective in maintaining bone health by adsorbing minerals such as calcium because they promote the differentiation of osteoblasts.

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 (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 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 FIG. 5 , no significant change in ALP activity was observed in the case of the Narrowleaf chrysanthemum extract (Preparation Example 1) and the ethanol extract (Preparation Example 2) compared to the control group.

The ALP activity did not show a significant increase in the ALP activity compared to the control in the case of the Narrowleaf chrysanthemum and hot water extract (Preparation Example 5) roasted at 200 °C, but the ALP activity at 100 μg/mL in the ethanol extract (Preparation Example 6) was about 53 compared to the control. % increased, and it increased by about 81% compared to the control at 1000 μg/mL.

The ALP activity did not show a significant increase in the ALP activity compared to the control in the case of the Narrowleaf chrysanthemum and hot water extract (Preparation Example 7) roasted at different temperatures in stages, but in the case of the ethanol extract (Preparation Example 8), the ALP activity at 100 μg/mL was the control It increased by about 34% compared to the control, and increased by about 68% compared to the control at 1000 μg/mL.

The above results suggest that the extracts from chrysanthemum extracts are effective in maintaining bone health by affecting bone health-related mechanisms of action and regulating the differentiation of osteoblasts.

Experimental Example 7: ALP activity evaluation (Western blot)

After the cultured cells were collected, washed twice with PBS, and 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 bovine serum albumin (BSA). The lysate was separated by 30μg 10% gel SDS-PAGE and transferred to a 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 three times with TBS-T and reacting with ECL substrate for 3 minutes, analysis was performed by chemiluminescence.

Referring to FIG. 6 , there was no significant change in ALP activity compared to the control group in the case of the Narrowleaf chrysanthemum extract (Preparation Example 1) and the ethanol extract (Preparation Example 2).

In the case of the Narrowleaf chrysanthemum and hot water extract (Preparation Example 5) roasted at 200°C, there was no significant increase in the ALP expression level compared to the control group, but in the case of the ethanol extract (Preparation Example 6), the ALP activity at 100 μg/mL was about It increased by 65%, and it increased by about 76% compared to the control at 1000 μg/mL.

In the case of the Narrowleaf chrysanthemum and hot water extract (Preparation Example 7) roasted at different temperatures in stages, there was no significant increase in ALP activity compared to the control group, but in the case of the ethanol extract (Preparation Example 8), the ALP expression level at 100 μg/mL was the control group. It increased by about 46% compared to the control, and increased by about 96% compared to the control at 1000 μg/mL.

The above results suggest that the extracts from chrysanthemum extracts are effective in maintaining bone health by affecting bone health-related mechanisms of action and regulating the differentiation of osteoblasts.

Experimental Example 8: Evaluation of bone regeneration induction activity

To confirm the bone regeneration-inducing effect of the extract in vivo , micro-CT (μCT) and histological analysis were performed after treatment with the extract, BMP-2, or a control group in mice with a defect in the skull. .

According to micro-CT (μCT) analysis, 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 the histological test, bone formation in the defect site was confirmed, unlike the control area, which is mostly fibrous tissue, by the treatment with the extract of Narrowleaf chrysanthemum in Preparation Examples 5 to 8. In particular, according to the sample treatment of Preparation Examples 6 and 8, the formation of bone in the defect area was confirmed. Bone formation was further enhanced.

Experimental Example 9: Evaluation of long bone growth activity

Efficacy of the extracts of Narrowleaf chrysanthemum on 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 in 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, so the length between the fluorescent line and the growth plate after a certain period of time after the one time administration of the sample was observed under 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, the length growth was increased by 16% and 21%, respectively, compared to the control group, when the roasted Narrowleaf chrysanthemum and ethanol extracts (Preparation Examples 6 and 8) were administered.

The above description of the present invention 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 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)

A food composition for improving bone health or promoting growth comprising roasting Narrowleaf chrysanthemum extract as an active ingredient. According to claim 1,
A food composition roasted at 150 to 300 ℃ with the narrow leaf cheonseonwa. According to claim 1,
A food composition roasted while controlling the temperature from 250 ° C to 150 ° C. 4. The method according to any one of claims 1 to 3,
A food composition further comprising an immune enhancing use. A food composition for preventing or improving bone disease, comprising the roasting Narrowleaf chrysanthemum extract as an active ingredient. Roasting (roasting) A pharmaceutical composition for improving bone health or promoting growth comprising an extract of Narrowleaf chrysanthemum as an active ingredient. A pharmaceutical composition for the prevention or treatment of bone diseases comprising roasting Narrowleaf chrysanthemum extract as an active ingredient. A food composition for improving bone health and enhancing immunity, comprising roasting Narrowleaf chrysanthemum extract and hot water extract and ethanol extract as active ingredients.

Images