KR20220168782A - A composition for prevention and treatment of osteoporosis comprising extract from Finger1ho - Google Patents

Abstract

The present invention relates to a food composition for preventing or alleviating osteoporosis, containing finger millet no. 1 extract; a pharmaceutical composition for preventing or treating osteoporosis, containing finger millet no. 1 extract; a method for preventing or treating osteoporosis, comprising a step of administering the pharmaceutical composition; and a feed composition for alleviating osteoporosis, containing the extract. The finger millet no. 1 extract of the present invention not only inhibits the differentiation of osteoclasts, which represent bone homeostasis disorders, but also promotes the differentiation of osteoblasts. Thus, the present invention can be used for food and medicine for the prevention, alleviation, or treatment of osteoporosis.

Description

A composition for prevention or treatment of osteoporosis comprising a Finger No. 1 extract {A composition for prevention and treatment of osteoporosis extract comprising from Finger1ho}

The present invention relates to a composition for preventing or treating osteoporosis containing an extract of Finger No. 1, and specifically, a food composition for preventing or improving osteoporosis containing an extract of Finger No. 1; A pharmaceutical composition for preventing or treating osteoporosis comprising an extract of Finger No. 1; It relates to a method for preventing or treating osteoporosis comprising administering the pharmaceutical composition, and a feed composition for improving osteoporosis comprising the extract.

Osteoporosis is one of the skeletal diseases in which the amount of bone decreases and the strength of the bone weakens, leading to easy fracture. It becomes a problem by restricting activities, and it is known to account for about 15% of deaths in the elderly.

Causes of osteoporosis include age, differences in basic bone mass, early menopause or low concentrations of estrogen, smoking, and drugs such as steroids. Bone loss progresses rapidly due to changes. That is, when reaching menopause, the estrogen concentration rapidly decreases, and accordingly, the activity of osteoclasts increases, reducing the amount of calcium contained in bone and weakening the structure maintaining bone shape.

To treat osteoporosis, hormones, alendronate, calcitonin, raloxifene, Na-F, calcitriol, or bisphosphonate preparations are administered. However, since the treatment of osteoporosis is a disease requiring a long treatment period, long-term use of conventional drugs increases the possibility of side effects such as urolithiasis, endometrial cancer, and breast cancer.

Calcium supplements used as a first-generation osteoporosis treatment have been reported to have little effect, and it has been reported that hormone therapy using estrogen or calcitonin is not safe in terms of safety (Jang JS. Osteoporotic fracture-Medical treatment. Journal of the Korean Fracture Society. 2010;23(3):326-340). In particular, although hormone therapy increases bone density, side effects such as breast cancer, myocardial infarction, and venous thrombosis have been reported (Rossouw JE et al. Postmenopausal hormone therapy and risk of cardiovascular disease by age and years since menopause. JAMA. 2007;297:1465-1477). Bisphosphonate-based bone resorption inhibitors, which are currently widely used, sometimes cause lesions in the upper respiratory tract when administered incorrectly (Jang JS. Osteoporotic fracture-Medical treatment. Journal of the Korean Fracture Society. 2010;23(3):326-340). Since osteoporosis is a disease in which long-term administration of drugs is essential, the development of new substances with safety and efficacy is urgently required.

Accordingly, there is an urgent need to develop a therapeutic agent with less toxicity and side effects, and recently, research on alternative therapies using active ingredients derived from natural products such as herbal medicines and foods that can promote bone formation while minimizing bone loss without other side effects and various extracts Manufacturing methods are being developed. However, the development of a natural pharmaceutical composition or a raw material thereof, which has an excellent effect on preventing and improving osteoporosis and has fewer side effects than existing synthetic pharmaceutical compositions, is still incomplete.

Under this background, the present inventors have made diligent research efforts to develop a therapeutic agent for osteoporosis using components derived from natural products. It was confirmed that it can be used as, and the present invention was completed.

One object of the present invention is to provide a food composition for the prevention or improvement of osteoporosis comprising the finger claw finger No. 1 extract as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of osteoporosis, containing the extract of Finger No. 1 as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating osteoporosis comprising administering the pharmaceutical composition to a non-human subject.

Another object of the present invention is to provide a feed composition for the prevention or improvement of osteoporosis comprising the finger claw finger No. 1 extract as an active ingredient.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.

As one aspect of the present invention, in order to achieve the above object, the present invention provides a food composition for the prevention or improvement of osteoporosis containing the finger claw finger No. 1 extract.

The term of the present invention, "Eleusine coracana (L.) Gaertn" is one of the flowers and small grains belonging to the millet class, and its origin is in the East African region. Major cultivation areas are mainly cultivated in semi-arid eastern and central Africa and southern India, and India has a cultivation area of 1.19 million hectares, accounting for 85% of the world's finger algae production (Sakamma et al. 2018). It represents the world's 4th largest production of millet, and the quantity varies greatly depending on the region and time. It is 0.3~1.6 t/ha in Africa, 1 t/ha in the dry season, and 2 t/ha in the rainy season in India (Dida et al. , 2008). Finger millet has excellent environmental adaptability and is adaptable to a wide range of annual average rainfall of 11~27℃, annual precipitation of 290~4290 mm, and soil pH of 5.0~8.2 (Duke, 1979). As a 'marginal crop' that endures well in harsh environments, it can be grown even with 30% rainfall of rice.

The term of the present invention, "Finger1ho" is a new lineage of finger claw, which was difficult to cultivate in Korea due to the long number of days, and is a variety that has been improved to suit the cultivation environment in Korea. And, it can be used for raw food, processing, etc. by providing one with increased calcium content. Finger No. 1 was published on May 15, 2019 as variety application publication number 'Name 2019-652', and registered as variety name with registration number 01-0039-1 on June 19, 2019 It is a variety, and it was applied on April 25, 2019 with the variety protection application number 'Application 2019-229' and was published on June 15, 2019 under the variety protection application publication number '2019-304', and on June 2, 2021 It is a cultivar that has been designated for cultivar protection. The finger number 1 may be used interchangeably with "finger finger number 1" herein, and in the present specification, all of the above-described terms may be used.

Specifically, the finger No. 1 grows in a semi-erect type, the number of days of growth is 99 days, the leaf color is green, the height is short, the stem is branched, the number of ears is 8.7 / week, the ear shape is dense, The color is dark purple, and the grain color is reddish brown.

In the present invention, finger claw finger No. 1 may be purchased and used commercially, or may be used collected or cultivated in nature, but is not limited thereto.

In the present invention, the finger claw finger No. 1 extract can be used to prevent, treat or improve osteoporosis.

As used herein, the term "extract" refers to a liquid component obtained by immersing a desired substance in various solvents and then extracting the substance at room temperature or at elevated temperature for a certain period of time, and a solid component obtained by removing the solvent from the liquid component. means In addition, in addition to the above results, it can be comprehensively interpreted as including all dilutions of the results, concentrates thereof, adjusted products, and purified products thereof. Accordingly, the finger nail finger No. 1 extract provided in the present invention is an extract obtained by extracting the finger finger finger No. 1, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, and a crude product or tablet of the extract It can be interpreted as including the extract of all formulations that can be formed using the extract itself and the extract, such as water or a mixture thereof.

For the finger claw finger No. 1 extract of the present invention, the extraction method thereof is not particularly limited, and can be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include a hot water extraction method, an ultrasonic extraction method, a filtration method, a reflux extraction method, and the like, which may be performed alone or in combination of two or more methods.

The calcium content in the finger claw finger No. 1 extract provided in the present invention may be 0.001 mg to 0.2 mg, specifically, 0.005 to 0.15 or 0.01 to 0.12, relative to 100 g of the extract, but is not limited thereto.

In one embodiment of the present invention, it was confirmed that the extract of finger claw finger No. 1 contained almost no calcium (Table 1).

Therefore, it was confirmed that the effect of preventing or treating osteoporosis of the finger claw finger No. 1 extract of the present invention was not due to calcium.

The type of solvent used for the extraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water, alcohol, or a mixed solvent thereof, and these may be used alone or in combination of one or more, and specifically, a mixed solvent of water and ethanol may be used. When alcohol is used as a solvent, alcohol having 1 to 4 carbon atoms may be specifically used. In the case of using the mixed solvent of water and ethanol, the mixing ratio of water and ethanol is specifically 100:0 to 0:100, more specifically 90:10 to 10:90, 80:20 to 20:80, 70: It may be 30 to 30:70, 60:40 to 40:60 or 50:50, but is not limited thereto.

In addition, the extract may be prepared and used in a dry powder form after extraction, but is not limited thereto.

As used herein, the term "osteoporosis" refers to a condition in which the amount of bone is reduced and the strength of bone is weakened due to qualitative changes, resulting in a high possibility of fracture.

The fingernail finger No. 1 extract provided by the present invention can be used for preventing or treating osteoporosis by inhibiting the activity of osteoclasts.

In addition, the fingernail finger No. 1 extract provided by the present invention can be used for the prevention or treatment of osteoporosis by promoting the differentiation of osteoblasts.

As used herein, the term "prevention" refers to any action that suppresses or delays the onset of osteoporosis by administering a pharmaceutical composition containing the fingernail finger No. 1 extract of the present invention.

As used herein, the term "improvement" refers to any activity that at least reduces a parameter related to a condition to be treated, for example, the severity of a symptom, by administration of a composition containing the fingernail finger No. 1 extract.

As used herein, the term "food" refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, and alcoholic beverages. , vitamin complexes, health functional foods and health foods, etc., and include all foods in the conventional sense.

Since the food composition of the present invention is derived from fingernails that can be consumed daily, a high osteoporosis improvement effect can be expected, so it can be used very usefully for health promotion purposes.

The health functional food (functional food) is the same term as food for special health use (FoSHU), and is a medicine processed to efficiently display bioregulatory functions in addition to nutritional supply, and has high medical effect. means food. Here, 'function (sex)' means obtaining useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions. The food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and ingredients commonly added in the art during the preparation. In addition, the formulation of the food may be prepared without limitation as long as the formulation is recognized as food.

The food composition of the present invention can be prepared in various types of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using natural products as raw materials, and has excellent portability, so the present invention Of the foods can be consumed as a supplement to enhance the improvement effect of osteoporosis.

The health food (health food) means a food having an active health maintenance or promotion effect compared to general food, health supplement food (health supplement food) means a food for the purpose of health supplement. In some cases, the terms health functional food, health food, and health supplement food are used interchangeably.

Specifically, the health functional food is a food prepared by adding the composition of the present invention to food materials such as beverages, teas, spices, gum, confectionery, etc., or encapsulated, powdered, or suspended, and when ingested, certain health It means to bring about an effect, but unlike general drugs, it has the advantage of not having side effects that can occur when taking drugs for a long time by using food as a raw material.

The food composition may further include a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier commonly used in the art may be used.

In addition, the food composition may include additional ingredients that are commonly used in food compositions and can improve smell, taste, and vision. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chrome (Cr); and amino acids such as lysine, tryptophan, cysteine, and valine.

In addition, the food composition may include preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder, high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butyl hydroxy Loxytoluene (BHT), etc.), coloring agents (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite, etc.), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclemate, saccharin) , sodium, etc.), flavoring (vanillin, lactones, etc.), expanding agent (alum, D-potassium hydrogen stannate, etc.), strengthening agent, emulsifier, thickener (thickener), coating agent, gum base agent, foam inhibitor, solvent, improver, etc. food May contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.

An example of the food composition of the present invention may be used as a health beverage composition, and in this case, it may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The aforementioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrins and cyclodextrins; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame may be used. The ratio of the natural carbohydrates may be generally about 0.01 to 0.04 g, specifically, about 0.02 to 0.03 g per 100 ml of the health drink composition of the present invention.

In addition to the above, the health beverage composition includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol or a carbonating agent, and the like. In addition, it may contain fruit flesh for the manufacture of natural fruit juice, fruit juice beverages, or vegetable beverages. These components may be used independently or in combination.

The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

Another aspect of the present invention for achieving the above object provides a pharmaceutical composition for the prevention or treatment of osteoporosis comprising the finger claw finger No. 1 extract as an active ingredient.

At this time, the definitions of the term, "finger nail", "finger No. 1", "extract" and "prevention" are as described above.

As used herein, the term "treatment" refers to all activities in which symptoms of osteoporosis suspected or affected individuals are improved or beneficially changed by administration of the pharmaceutical composition.

The pharmaceutical composition of the present invention may include 0.001 to 80, specifically 0.001 to 70, and more specifically 0.001 to 60% by weight of the fingernail finger No. 1 extract based on the total weight of the composition, but is not limited thereto.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent commonly used in the preparation of pharmaceutical compositions, and the carrier may include a non-naturally occurring carrier. can

The term of the present invention, "pharmaceutically acceptable" means exhibiting characteristics that are not toxic to cells or humans exposed to the pharmaceutical composition.

Specifically, the pharmaceutical composition is formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. can In the present invention, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, cal sodium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose or lactose. It is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions for internal use, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be included in addition to water and liquid paraffin, which are commonly used simple diluents. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

Prevention or treatment of osteoporosis in the present invention may be that the extract inhibits the activity of osteoclasts and induces differentiation into osteoblasts.

In one embodiment of the present invention, it was confirmed that the finger claw finger No. 1 extract has excellent osteoclast activity inhibitory ability (FIGS. 2 and 3).

In addition, in one embodiment of the present invention, it was confirmed that the finger claw finger No. 1 extract has the ability to form osteoblasts (Figs. 4 to 6).

Therefore, the fingernail finger No. 1 extract provided by the present invention can be effectively used for preventing or treating osteoporosis.

Another aspect of the present invention for achieving the above object provides a method for preventing or treating osteoporosis comprising the step of administering the pharmaceutical composition to a subject other than a human having or likely to have osteoporosis. .

At this time, the definitions of the term, "pharmaceutical composition", "prevention" and "treatment" are as described above.

As used herein, the term "administration" refers to the act of introducing a composition containing the finger claw finger No. 1 extract to a subject by an appropriate method.

As used herein, the term "subject" refers to all animals, such as rats, mice, livestock, and the like, including humans who have or may develop osteoporosis. As a specific example, it may be mammals including humans.

Specifically, the method for preventing or treating osteoporosis of the present invention may include the step of administering a pharmaceutical composition for preventing or treating osteoporosis containing a fingernail finger No. 1 extract in a pharmaceutically effective amount to a non-human subject. .

As used herein, the term "pharmaceutically effective amount" means an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is dependent on the gender of the patient. , age, weight, health condition, type of disease, severity, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other medical factors. It can be readily determined by a person skilled in the art according to factors well known in the art. Specifically, the composition of the present invention can be administered at a dose of 0.0001 to 100 mg/kg per day, more specifically, 0.001 to 100 mg/kg based on the solid content, and the administration is administered once or several times a day. You may.

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

In addition, the pharmaceutical composition may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically applied) according to the desired method, and the dosage is the patient's condition and weight, the degree of disease , Depending on the drug form, administration route and time, it can be appropriately selected by those skilled in the art.

Another aspect of the present invention for achieving the above object provides a feed composition for the prevention or improvement of osteoporosis comprising the finger claw finger No. 1 extract.

At this time, the definitions of the terms, "finger nail", "finger No. 1", "extract", "prevention" and "improvement" are as described above.

Since the fingernail finger No. 1 extract according to the present invention exhibits excellent osteoclast differentiation inhibition and osteoblast formation, it can be included in a feed composition for the purpose of preventing or improving osteoporosis, and the feed composition is not to be consumed by animals on a daily basis. Since it is possible, a high effect can be expected with respect to the prevention or improvement of osteoporosis.

As used herein, the term "feed" refers to any natural or artificial diet, meal, etc., or component of said meal, intended for or suitable for consumption by animals.

The type of feed is not particularly limited, and feeds commonly used in the art may be used. Non-limiting examples of the feed include vegetable feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal or grain by-products; Animal feeds such as proteins, inorganic materials, oils, mineral oils, oils, single-celled proteins, zooplankton, or food may be mentioned. These may be used alone or in combination of two or more.

Since the composition for preventing or treating osteoporosis containing the fingernail finger No. 1 extract of the present invention exhibits excellent osteoclast differentiation inhibition and osteoblast formation, it can be provided as a composition for preventing, improving, or treating osteoporosis.

1 is a graph showing the effect of finger claw finger No. 1 extract on cytotoxicity to bone marrow macrophages.
Figure 2 is a graph showing the ability to inhibit osteoclast differentiation of finger claw finger No. 1 extract.
Figure 3 is a table showing the inhibition rate of differentiation of osteoclasts of finger claw finger No. 1 extract.
Figure 4 is a graph showing the effect of finger claw finger No. 1 extract on cytotoxicity to human bone marrow stem cells.
Figure 5 is a photograph showing the effect of finger claw finger No. 1 extract on ALP activity, which is an early marker for osteoblast differentiation.
Figure 6 is a photograph and graph showing the effect of finger claw finger No. 1 extract on calcium deposition.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited by these examples.

Example 1. Preparation of finger claw finger No. 1 extract and soybean extract

Example 1-1. Confirmation of extraction yield and calcium content according to the solvent of finger clam finger No. 1 extract

In order to confirm the extraction yield and calcium content according to the solvent of the Finger No. 1 ethanol extract, 10 g of the powder obtained by pulverizing the Finger No. 1 seed was mixed with water, 50% ethanol, 80% ethanol, and 100% water corresponding to 10 times the solvent. 100 ml of % ethanol was added and extracted at room temperature under shaking conditions for 16 hours, and the extract was concentrated using a vacuum concentrator.

The extraction yield according to each extraction solvent is shown in Table 1 below.

extraction solvent 100% water 50% ethanol 80% ethanol 100% ethanol crude extract
transference number(%) 5.61 6.52 6.50 5.41

As a result, as shown in Table 1, it was confirmed that extraction yields were the highest at 6.52% and 6.50% when extracted using 50% ethanol and 80% ethanol, and when extracted with 100% water, the yield was 5.61% and 100 It was confirmed that extraction with % ethanol showed the lowest extraction yield of 5.41%.

In addition, the inorganic component analysis was performed by the dry method according to the AOAC method. Specifically, the inorganic component (calcium) content of the extract was analyzed using ICP (Thermo Jarrell Ash, Franklin, MA, USA).

Calcium content according to each extraction solvent is shown in Table 2 below.

Item strain Calcium content of crude extract according to extraction solvent 100% water 50% ethanol 80% ethanol 100% ethanol calcium content
(mg/100g) 322 0.12 0.03 0.01 0.02

As a result, as shown in Table 2, it was confirmed that the extract extracted using a solvent hardly contained 322 mg of calcium per 100 g of the seed. Comparing the content, when extracted with 100% water from 100 g of seeds, the extract contained 0.12 mg of calcium, when extracted with 50% ethanol, 0.03 mg, when extracted with 80% ethanol, 0.01 mg, and when extracted with 100% ethanol, 0.02 mg. It was confirmed that the crude extract contained extremely low calcium content. In particular, it was confirmed that the 80% ethanol solvent had the lowest calcium extraction yield and contained 0.01 mg (9.8 ug). Therefore, the experiment was conducted while almost completely excluding the osteoporosis preventive effect of calcium contained in the extract.

Example 1-2. Preparation of finger claw finger No. 1 extract and soybean extract

Extraction and freeze-drying of Finger Joe Finger No. 1, general soybean Seonyu No. 2, and black soybean No. 4 Cheongja were carried out by the Southern Crop Division of the National Institute of Crop Science. 19 g of each sample was extracted for 16 hours at room temperature using 100 ml of 80% ethanol. Soybean and finger algae extracts were dissolved in dimethyl sulfoxide (DMSO) at 100 mg/ml, aliquoted and stored at -80°C, and melted at room temperature each time they were used.

Example 2. Confirmation of osteoclast differentiation inhibitory effect of finger claw finger No. 1 extract

The ability of the finger claw finger No. 1 extract obtained in Example 1 and other soybean extracts to inhibit osteoclast differentiation was confirmed using bone marrow macrophages.

Specifically, the iliac bone marrow of mice aged 6 weeks or older was layered using histopaque (Sigma, St. Louis, MO) centrifugation to recover bone marrow mononuclear cells, and then 10% bovine serum and macrophage colony-stimulating factor (M- Bone marrow-derived macrophages obtained by culturing for 3 days in a medium containing CSF) were used.

Example 2-1. Confirmation of cytotoxicity of finger algae finger No. 1 extract on bone marrow macrophages

First, the cytotoxicity of finger claw finger No. 1 extract and other soybean extracts in bone marrow-derived macrophages was confirmed through MTT (Sigma-Aldrich) analysis.

Specifically, MTT analysis was performed after 3 days by treating each of the extracts at 1x10 ⁴ cell/well at concentrations of 1, 10, and 50 μg/ml. Cells were then incubated with 5 μg/ml of MTT for 2 hours and insoluble formazan was lysed with 100 μl of DMSO. Absorbance at 570 nm was analyzed with a microplate reader (Epoch, BioTek instrument, VT, USA).

As a result, as shown in FIG. 1, it was confirmed that no cytotoxicity was observed even at the highest concentration of 50 μg/ml for both finger claw finger extract No. 1 and other soybean extracts (Seonyu No. 2 extract and Celadon No. 4 extract). Therefore, the treatment concentration of the extract for analyzing the effect of regulating the differentiation of osteoclasts was determined to be 50ug/ml.

Example 2-2. Confirmation of osteoclast differentiation inhibitory activity of finger claw finger No. 1 extract

Bone marrow macrophages were treated with finger algae and soybean extracts, and TRAP staining and multinucleated cell counting were performed to evaluate the effect of inhibiting osteoclast differentiation by M-CSF and RANKL.

Specifically, bone marrow-derived macrophages were treated with the finger claw finger No. 1 extract, Seonyu No. 2 extract, and Celadon No. 4 extract obtained in Example 1 at a concentration of 50 μg/ml, and then M-CSF (10 ng/ml) and RANKL (20 ng/ml)-containing differentiation medium for 3 days to induce differentiation into multinucleated mature osteoclasts. Subsequently, the differentiation of osteoclasts was confirmed by staining the cells with a tartrate resistant acid phosphatase (TRAP) solution, which is an osteoclast-specific marker, and cells having three or more nuclei of multinucleated osteoclasts were counted as differentiated osteoclasts. .

As a result, as shown in FIGS. 2 and 3, compared to the 48.2% inhibition rate of Seonyu No. 2, which is a common bean, and the 46.8% inhibition rate of Celadon No. 4, which is a black soybean, the finger claw Finger No. 1 extract has an inhibition rate of 95.2%, which is more than twice as high as osteoclast cell It was confirmed that there is an excellent efficacy in inhibiting differentiation.

Example 3. Confirmation of osteoblast differentiation promoting effect of finger claw finger No. 1 extract

The ability of the finger claw finger No. 1 extract obtained in Example 1 and other soybean extracts to promote differentiation of osteoblasts was confirmed using human bone marrow stem cells.

Specifically, human bone marrow stem cells were used for culture and experiments using minimum essential medium Eagle-alpha modification (α-MEM) + 10% fetal bovine serum (FBS) + 1% penicillin/streptomycin, and this composition was used as control media. called Osteogenesis inducing medium (OIM) was used to induce osteoblast differentiation of human bone marrow stem cells, and the composition thereof contained 10 mM β-glycerophosphate, 50 μg/ml ascorbic acid, and 10 nM dexamethasone in control media. Human bone marrow stem cells were used in all experiments at passage 6.

Example 3-1. Confirmation of cytotoxicity of finger claw finger No. 1 extract on human bone marrow stem cells

First, the cytotoxicity of finger claw finger No. 1 extract and other soybean extracts in human bone marrow stem cells was confirmed through MTT (Sigma-Aldrich) analysis.

Specifically, MTT analysis was performed after 3 days by treating each extract at a concentration of 10, 50, 100, and 200 μg/ml in 7x10 ² cell/well. Cells were then incubated with 5 μg/ml of MTT for 2 hours and insoluble formazan was lysed with 100 μl of DMSO. Absorbance at 570 nm was analyzed with a microplate reader (Epoch, BioTek instrument, VT, USA).

As a result, as shown in Figure 4, the cell viability of the 200 μg / ml treated group was reduced by about 24.6% when compared to the control group. When compared with the control group, Celadon No. 4 extract did not noticeably differ in cytotoxicity up to a concentration of 200 μg/ml. When compared to the control group, the finger claw finger extract No. 1 was reduced by 19.9% and 22.6%, respectively, in the groups treated with 100 and 200 μg / ml, respectively, confirming that it was at an effective concentration that did not exhibit cytotoxicity.

Example 3-2. Confirmation of the effect of finger claw finger No. 1 extract on the ALP activity of human bone marrow stem cells

In order to examine the effect of finger millet and soybean extracts on the osteoblast differentiation potential of human bone marrow stem cells, each extract was treated during the induction of osteoblast differentiation of human bone marrow stem cells, and ALP activity, an early osteoblast differentiation marker, was analyzed by ALP staining. measured through

Specifically, human bone marrow stem cells were seeded in a 24-well plate at 2x10 ⁴ cells/well per well, and then stabilized in a CO ₂ incubator for 1 day. Subsequently, human bone marrow stem cells were treated with 100 μg/ml of osteoblast differentiation inducer (OIM) and respective extracts (Finger Finger No. 1 extract, Seonyu No. 2 extract, and Celadon No. 4 extract) for 7 days. After 8 days, a photograph of the cells reacted with the ALP staining solution was obtained using a BX41 microscope (Olympus, Tokyo, Japan).

As a result, as shown in Figure 5, when compared to the OIM + vehicle group, the ALP activity staining degree of the Seminyu No. 2 extract was slightly increased. Celadon No. 4 extract slightly increased the degree of ALP activity staining compared to the OIM + vehicle group. Finger No. 1 extract was confirmed to have increased the degree of ALP activity staining compared to the OIM + vehicle group.

Accordingly, it was confirmed that the finger claw finger No. 1 extract promotes the activity of ALP, an early osteoblast differentiation marker.

Example 3-3. Confirmation of the effect of finger claw finger No. 1 extract on calcium deposition in human bone marrow stem cells

In order to examine the effect of finger millet and soybean extracts on the osteoblast differentiation potential of human bone marrow stem cells, each extract was treated during the induction of osteoblast differentiation of human bone marrow stem cells, and the calcium deposition (mineral deposition) effect was analyzed by Alizarin Red S staining. It was measured through

Specifically, human bone marrow stem cells were seeded in a 24-well plate at 2x10 ⁴ cells/well per well, and then stabilized in a CO ₂ incubator for 1 day. Subsequently, human bone marrow stem cells were treated with 100 μg/ml of osteoblast differentiation inducer (OIM) and respective extracts (Finger Finger No. 1 extract, Seonyu No. 2 extract, and Celadon No. 4 extract). After 14-18 days, pictures of minerals stained with Alizarin Red S staining solution were obtained using a BX41 microscope. To quantify the degree of staining, the absorbance of the CPC solution was measured at 570 nm using a microplate reader (Epoch).

As a result, as shown in FIG. 6, it was confirmed that calcium deposition increased at 100 μg/ml when compared to the OIM + vehicle group in the general soybean extract No. 2.

When compared with the OIM + vehicle group, it was confirmed that calcium deposition increased at 100 μg/ml in the black soybean celadon No. 4 extract.

When compared to the OIM + vehicle group, it was confirmed that the calcium deposition increased at 100 μg / ml in the extract of Finger No. 1. Accordingly, it was confirmed that the finger clam Finger No. 1 extract exhibited a calcium deposition increasing effect similar to that of soybeans known to have an existing calcium deposition increasing activity.

In summary, it was confirmed that the finger claw Finger No. 1 extract promotes the activity of ALP, an early osteoblast differentiation marker, and exhibits an effect similar to that of the soybean extract in calcium deposition, which represents the final stage of osteoblast differentiation.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

Claims (7)

A food composition for preventing or improving osteoporosis, comprising an extract of Finger No. 1.
According to claim 1,
The finger claw Finger No. 1 extract is obtained by extracting Finger No. 1 with one or more solvents selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.
According to claim 1,
The finger claw finger No. 1 extract is to inhibit the differentiation of osteoclasts, food composition.
According to claim 1,
The fingernail finger No. 1 extract is to promote the differentiation of osteoblasts, food composition.
A pharmaceutical composition for the prevention or treatment of osteoporosis, comprising an extract of Finger No. 1.
A method for preventing or treating osteoporosis, comprising administering the pharmaceutical composition of claim 5 to a non-human subject.
A feed composition for preventing or improving osteoporosis, comprising an extract of Finger No. 1.

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