KR102512517B1 - Pharmaceutical composition for use in preventing or treating osteoporosis containing extract of pigweed as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of osteoporosis containing an amaranth extract as an active ingredient. can be used In addition, since it does not show cytotoxicity, it has the advantage of having few side effects even when taken for a long time.

Description

Pharmaceutical composition for use in preventing or treating osteoporosis containing extract of pigweed as an active ingredient}

The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis containing an amaranth extract as an active ingredient.

Osteoporosis is a disease caused by an imbalance between osteoblasts and osteoclasts, resulting in increased bone resorption rather than bone formation. At the onset of osteoporosis, calcium in the bone tissue is reduced, and the density of the bone is thinned, thereby widening the medullary cavity. Aging, lack of exercise, low weight, smoking, low calcium diet, menopause, ovarian ablation, etc. are known as causes of osteoporosis.

Osteoclasts are large multinucleated cells that destroy or absorb unnecessary bone tissue in the process of bone growth of vertebrates. If their activity is too active, bone density is low, causing diseases such as osteoporosis. In order to differentiate into these osteoclasts, RANKL (Receptor activator of nuclear factor kappa-B ligand) is required, and macrophage growth stimulating factor (M-CSF) is required for the proliferation of osteoclasts.

Differentiation of osteoclasts begins when RANKL binds to its receptor RANK. When RANKL binds to RANK, adapter molecules such as tumor necrosis factor receptor-associated factor 6 (TRAF6) are activated. TRAF2, TRAF5, and TRAF6, known as members of the tumor necrosis factor receptor-associated factor family, activate transcription factors such as NF-kB and activating protein-1 (AP-1) necessary for the differentiation of osteoclasts. When TRAF6 is activated by RANKL signal for osteoclast differentiation, phosphatitylinositol kinase 3-kinase (PI3K), transforming growth factor beta-activated kinase (TAK1), protein kinase B (Akt)/PKB, and Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), mitogen-activated protein kinases (MAPKs) including p38, NF-kB, c-Fos, Fra-1, cyclic AMP-induced gene (CREB) , NFATc1s are activated. This induction occurs by the c-Fos pathway via TRAF6-NF-kB and RANKL-RANK signals. And by them, osteoclast-specific genes such as cathepsin K, TRAP (tartrate-resistant acid phospatase), calcitonin receptor and osteoclast-associated receptor (OSCAR) are expressed. The process of attaching osteoclasts to bone and degrading bone leads to cytoskeleton reoragnization, including the formation of a sealing zone and filamentous actin ring, which are formed by attaching to the bone surface around the erosive space. induce The RANKL/RANK/TRAF6 axis and NFATc1 are essential for differentiation into mature osteoclasts, and these signaling pathways are initiated by RANKL, and molecules related to them can be targets of drugs for bone diseases.

Most osteoporosis drugs currently on the market are estrogen-based substances, and these have problems in that side effects such as cancer, gallstones, and thrombosis appear when administered for a long time. Since osteoporosis cannot be treated with only short-term drug administration and long-term drug administration is essential, it is necessary to develop a new substance that does not have the above side effects even when the drug is administered for a long time and has excellent efficacy enough to replace estrogen.

Accordingly, research and development on osteoporosis treatment using various natural substances such as chinquapin extract, sandalwood extract, galaxian root extract, and sorghum ginseng extract are continuously being conducted. For example, a natural substance, Pear extract, has been studied and developed as a drug for preventing or treating osteoporosis by significantly reducing the expression of osteoclast differentiation-related genes and the expression of transcription factors that regulate their expression (Patent Document 1).

On the other hand, amaranth ( Amaranthus mangostanus L. ) is an annual plant of the Amaranthaceae native to India. The leaves are lozenge-shaped, egg-shaped, and have long petioles. From summer to autumn, white-green small flowers form spike inflorescences, blooming at the ends of branches or at the axils of leaves, and several of them are grouped together again. The fruit is canine and has an oval shape. Since ancient times, it has been used as a medicine because it is effective for hemostasis and colic, and is also called 'longevity herb' because it contains various vitamins.

In addition, it is known that amaranth has antipyretic, detoxifying, and antipyretic effects, so it can be applied to diseases such as cold, eye disease, hemorrhoids, snakebite, and boils. However, there has been no specific study on the preventive or therapeutic effect of osteoporosis by the osteoclast inhibitory activity of the amaranth extract.

Republic of Korea Patent Registration No. 10-1242635

The inventors of the present invention confirmed that the amaranth extract inhibits the production of osteoclasts without showing cytotoxicity while trying to develop a new pharmaceutical composition that has excellent efficacy in preventing and treating osteoporosis and has few side effects even when taken for a long period of time. has been completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition and health functional food having excellent efficacy in preventing and treating osteoporosis and having few side effects even when taken for a long period of time.

To achieve the above purpose,

The present invention provides a pharmaceutical composition for preventing or treating osteoporosis containing an alcohol extract of amaranth as an active ingredient.

The composition may be characterized in that it inhibits osteoclast differentiation.

The alcohol may be characterized in that it is a C ₁ ~ C ₄ alcohol.

The composition may be formulated in the form of a powder, granule, tablet, hard capsule, soft capsule or injection.

In addition, the present invention provides a health functional food for preventing or improving osteoporosis containing an alcohol extract of amaranth as an active ingredient.

The health functional food may be characterized by inhibiting osteoclast differentiation.

The alcohol may be a C ₁ ~ C ₄ alcohol.

The pharmaceutical composition for preventing or treating osteoporosis containing the alcohol extract of amaranth according to the present invention as an active ingredient exhibits an effect of significantly inhibiting the production of osteoclasts, and thus can be widely used as a medicine or health food.

In addition, since it does not show cytotoxicity, it has the advantage of having few side effects even when taken for a long time.

1 is a result according to (1) of Experimental Example 1 confirming the cytotoxicity of the amaranth extract.
Figure 2 is a result according to (2) of Experimental Example 1 confirming the differentiation inhibitory effect of the amaranth extract into osteoclasts using the TRAP staining method.
3 is a result according to (3) of Experimental Example 1 confirming the osteoclast differentiation inhibitory mechanism of the amaranth extract.

Hereinafter, the present invention will be described in detail.

The terms or words used in this specification and claims should not be construed as being limited to ordinary or dictionary meanings, and the inventors may appropriately define the concept of terms in order to explain their invention in the best way. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

Pharmaceutical composition for preventing or treating osteoporosis

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating osteoporosis containing an alcohol extract of amaranth as an active ingredient.

The alcohol extract of the amaranth inhibits osteoclast differentiation and can be usefully used as a pharmaceutical composition for preventing or treating osteoporosis.

The alcohol may be characterized in that it is a C ₁ ~ C ₄ alcohol.

More specifically, the alcohol may be a C ₁ ~ C ₄ alcohol or a mixture thereof, preferably ethanol.

When ethanol is used as an extraction solvent, active ingredients can be more effectively extracted and the complexity of extracted compounds can be reduced, and there are advantages of being cheap, less toxic, and easy to remove.

Extraction of the alcohol extract of the amaranth may be obtained by mixing 0.1 to 20 g of amaranth powder with respect to 100 ml of alcohol, and extracting at a temperature of 20 to 60 ° C. for 12 to 36 hours. A preferred method for preparing the alcohol extract of amaranth used in the present invention will be described in more detail below.

As used herein, the term osteoporosis refers to a state in which bone strength is weakened due to a decrease in the amount and qualitative change of bones, and prevention, improvement, and treatment of osteoporosis include various diseases caused by a decrease in bone density and bone loss. interpreted as doing

The pharmaceutical composition according to the present invention can be administered orally or parenterally during clinical administration, and can be used in the form of a general pharmaceutical preparation.

The composition may be formulated in the form of a powder, granule, tablet, hard capsule, soft capsule or injection.

More specifically, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, sterile injection solutions, pre-filled injection solutions, or lyophilized It can be formulated in the form, but is not limited thereto.

When formulated, it may be 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, lactose, gelatin, etc. It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid preparations for oral administration include suspensions, internal solutions, 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, suppositories, and the like.

The composition of the present invention may contain at least one active ingredient exhibiting the same or similar function in addition to the extract.

A preferred dosage of the pharmaceutical composition of the present invention may be appropriately selected depending on the patient's condition and body weight, severity of symptoms, drug type, administration route and period. For optimal efficacy, the composition of the present invention is preferably administered at 0.2 mg/kg to 200 mg/kg of the active ingredient per day. Administration may be administered once a day or divided into several times, but is not limited thereto.

Health functional food for preventing or improving osteoporosis

In another aspect, the present invention relates to a health functional food for preventing or improving osteoporosis containing an alcohol extract of amaranth as an active ingredient.

The health functional food may be characterized by inhibiting osteoclast differentiation.

The alcohol may be a C ₁ ~ C ₄ alcohol.

More specifically, the alcohol may be a C ₁ ~ C ₄ alcohol or a mixture thereof, preferably ethanol.

When ethanol is used as an extraction solvent, active ingredients can be more effectively extracted and the complexity of extracted compounds can be reduced, and there are advantages of being cheap, less toxic, and easy to remove.

Extraction of the alcohol extract of the amaranth may be obtained by mixing 0.1 to 20 g of amaranth powder with respect to 100 ml of alcohol, and extracting at a temperature of 20 to 60 ° C. for 12 to 36 hours. A preferred method for preparing the alcohol extract of amaranth used in the present invention will be described in more detail below.

The health functional food of the present invention may be prepared in the form of various beverages, chewing gum, tea, sweets, vitamin complexes, health supplements, and the like, without limitation.

In addition, the health functional food of the present invention also includes when added to health food for the purpose of improving osteoporosis, and there is no particular limitation on the type of food. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice creams, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health foods in a conventional sense.

The preferred intake amount of the health functional food of the present invention varies depending on the condition and weight of the consumer, the severity of symptoms, the type of food, and the intake period, and can be appropriately selected. The health functional food of the present invention is preferably ingested at 0.2 mg/kg to 200 mg/kg of active ingredients per day for optimum effect.

Manufacturing method of amaranth extract for preventing or treating osteoporosis

The amaranth extract according to the present invention,

(a) obtaining powder by freeze-drying amaranth, then mixing 0.1 to 20 g of amaranth powder per 100 ml of alcohol; (b) eluting the active ingredient while stirring the mixture at a temperature of 20 to 60° C. for 12 to 36 hours; and (c) removing solids from the extraction solution, and collecting and concentrating only the supernatant.

The manufacturing method of the amaranth extract according to the present invention will be described step by step as follows.

First, (a) after obtaining powder by freeze-drying amaranth, 0.1 to 20 g of amaranth powder is mixed per 100 ml of alcohol.

The amaranth powder is obtained by freeze-drying the amaranth, and the average particle size of the amaranth powder mixed with alcohol is preferably 0.1 to 0.5 μm, more preferably about 0.25 μm. In the case of using amaranth powder having an average particle size of 0.1 to 0.5 μm, there is an advantage in that the active ingredient can be effectively extracted even at low temperature extraction in a short time while minimizing the destruction of the active ingredient for preventing osteoporosis. On the other hand, in order to control the particle size of the amaranth powder obtained by freeze-drying, a step may be added by pulverizing the powder with a grinder or passing the amaranth powder through a sieve to select only particles of a desired size.

The alcohol used as the extraction solvent may be, but is not limited to, a lower alcohol of 1 to 4 or a polyhydric alcohol, or a mixture thereof. Preferably, the alcohol may be ethanol. When ethanol is used as an extraction solvent, active ingredients can be more effectively extracted and the complexity of extracted compounds can be reduced, and there are advantages of being cheap, less toxic, and easy to remove.

The ratio of extraction solvent and amaranth powder is preferably 0.1 to 20 g of amaranth powder per 100 ml of alcohol, and 5 to 15 g of amaranth powder per 100 ml of alcohol, more preferably around 10 g.

Next, (b) the active ingredient for osteoporosis treatment is eluted from the amaranth while stirring the mixture of the extraction solvent and the amaranth powder at a temperature of 20 to 60° C. for 12 to 36 hours. At this time, the extraction temperature is more preferably 20 to 30 ° C, and it is most preferable to extract at room temperature around 25 ° C. If the extraction temperature is excessively higher than the above range, there is a risk of destroying the active ingredient, and if the extraction temperature is low, the extraction time is long, resulting in poor economic efficiency. The extraction time may vary depending on the extraction temperature, but is most preferably around 24 hours.

When the extraction is complete, (c) the solid content is removed from the extraction solution, and only the supernatant is collected and concentrated. Separation of the solid content and the supernatant may be performed using, for example, filter paper or may be performed by centrifugation.

Additionally, a step of obtaining a powder by (d) freeze-drying the concentrate of step (c) may be further included.

Hereinafter, a pharmaceutical composition for preventing or treating osteoporosis containing the amaranth extract of the present invention as an active ingredient and its application as a health functional food will be described in more detail through the following preparation examples and preparation examples. However, the following preparation examples and preparation examples are only for embodying the contents of the present invention, and the present invention is not limited by the preparation examples and preparation examples.

< manufacturing example >

manufacturing example 1. Preparation of amaranth extract

After drying the amaranth, it was powdered using a grinder. At this time, the average particle size of the amaranth powder is 0.1 to 0.5 μm. After adding 500 ml of 70 vol% aqueous ethanol solution to 100 g of amaranth powder, extracting with stirring at 25 ° C for 24 hours, and removing solids using a centrifuge (Beckman, USA) at 8,000 ° C for 30 minutes. Only the centrifuged supernatant was collected, concentrated under reduced pressure, and lyophilized to powder.

< formulation example >

formulation example One: sanjae manufacturing

Amaranth Powder (Preparation Example 1) ----------------------------------- 300 mg

Lactose ------------------------------ 100 mg

Talc ------------------------------ 10 mg

A powder is prepared by mixing the above ingredients and filling them in an airtight bag.

formulation example 2: Preparation of tablets

Amaranth powder (Preparation Example 1) ------------------------------------ 50 mg

Cornstarch ------------------------------ 100 mg

Lactose ------------------------------ 100 mg

Magnesium stearate ------------------ 2 mg

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet manufacturing method.

formulation example 3: Preparation of capsules

Amaranth Powder (Preparation Example 1) ------------------------------------- 50 mg

Cornstarch ------------------------------ 100 mg

Lactose ------------------------------ 100 mg

Magnesium stearate ------------------ 2 mg

Capsules are prepared by mixing the above ingredients and filling them into gelatin capsules according to a conventional capsule preparation method.

formulation example 4: Preparation of injections

Amaranth powder (Preparation Example 1) ------------------------------------ 50 mg

Sterile distilled water for injection --------------------------- proper amount

pH Adjuster ----------------------------- Appropriate amount

It is prepared with the above component content per ampoule (2 ml) according to the conventional method for preparing injections.

formulation example 5: liquid medicine manufacturing

Amaranth Powder (Preparation Example 1) ----------------------------------- 100 mg

Isomerized sugar ------------------------------ 10 g

Mannitol ------------------------------ 5 g

Purified water ------------------------------appropriate amount

According to the conventional manufacturing method of liquid formulations, each component is added to purified water to dissolve, lemon flavor is added in an appropriate amount, the above components are mixed, and then purified water is added to adjust the total volume to 100 ml, and then filled in a brown bottle. Sterilize to prepare a liquid formulation.

formulation example 6: of health food manufacturing

Amaranth Powder (Preparation Example 1) ---------------------------------- 1000 mg

Vitamin Blend ------------------------------ Appropriate amount

Vitamin A Acetate ------------------------- 70 μg

Vitamin E ------------------------------ 1.0 mg

Vitamin B1 ------------------------------ 0.13 mg

Vitamin B2 ------------------------------- 0.15 mg

Vitamin B6 ------------------------------ 0.5 mg

Vitamin B12 ------------------------------ 0.2 μg

Vitamin C ------------------------------ 10 mg

Biotin ------------------------------- 10 μg

Nicotinamide ------------------------------ 1.7 mg

Folic acid ---------------------------------- 50 μg

Calcium Pantothenate ------------------------------ 0.5 mg

Mineral mixture ------------------------------- proper amount

Ferrous sulfate ------------------------------ 1.75 mg

Zinc Oxide ------------------------------ 0.82 mg

Magnesium Carbonate ------------------------------ 25.3 mg

Potassium Phosphate Monobasic -------------- 15 mg

Dibasic potassium ------------ 55 mg

Potassium Citrate -------------- 90 mg

Calcium Carbonate -------------------------------- 100 mg

Magnesium Chloride ------------------------------- 24.8 mg

Although the composition ratio of the above vitamin and mineral mixture was prepared by mixing ingredients suitable for relatively healthy food in a preferred embodiment, the mixing ratio may be arbitrarily modified. , Granules can be prepared and used in the preparation of health food compositions according to conventional methods.

formulation example 7: Manufacture of health drinks

Amaranth Powder (Preparation Example 1) ---------------------------------- 1000 mg

Citric Acid ------------------------------ 1000 mg

Oligosaccharides ---------------------------- 100 g

Plum concentrate ----------------------------- 2 g

Taurine ------------------------------ 1 g

Add purified water to make the total ------------ 900 ml

After mixing the above components according to the usual health drink manufacturing method, stirring and heating at 85 ° C. for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2L container, sealed and sterilized, and then refrigerated according to the present invention. It is used in the manufacture of health beverage compositions. Although the composition ratio is a mixture of ingredients suitable for a relatively favorite beverage in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the class of demand, the country of demand, and the purpose of use.

Hereinafter, the osteoporosis treatment and preventive activity effect of the alcohol extract of amaranth according to the present invention will be described in more detail through the following experimental examples.

< Experimental Example >

Experimental Example 1. Measurement of the effect of amaranth extract on inhibition of osteoclast formation

(1) Cytotoxicity test

In order to measure the effect of the composition according to the present invention on cell survival, 5, 10, 20, 40, 100 μg/ml of amaranth extract was added to mouse-derived RAW264.7 monocyte cells (monocyte cells prior to differentiation into osteoclasts). was added and the survival rate was measured after 48 hours.

As a result, it was confirmed that the amaranth extract according to the present invention was not involved in cell survival at all (see FIG. 1).

(2) Test to confirm the osteoclast differentiation inhibitory effect of amaranth extract

In order to confirm the inhibitory effect of the composition according to the present invention on osteoclast differentiation, osteoclast differentiation was observed using a tartate resistant acid phosphate (TRAP) staining method.

To this end, RANKL (receptor activator of nuclear factor kappa-B ligand) was added to RAW264.7 monocyte cells, and after treatment with 0, 10, 20, and 40 μg/ml of amaranth extract, respectively, TRAP staining was performed.

When RAW264.7 mononuclear cells bind to RANKL, they differentiate into multinucleated osteoclasts, and the secretion of TRAP enzyme increases during bone resorption, so the degree of osteoclast differentiation can be confirmed by measuring its activity.

As a result of the experiment, it was confirmed that the production of RANKL-induced osteoclasts in RAW264.7 monocytic cells was inhibited by the amaranth extract (see FIG. 2).

In particular, when the amaranth extract of the present invention was treated at 20 or 40 μg/ml, a statistically significant difference (p < 0.05) was shown compared to the RANKL-only group (see B in FIG. 2).

(3) Test to confirm mechanism of inhibition of osteoclast differentiation of amaranth extract

To confirm the osteoclast differentiation inhibition mechanism of amaranth extract, mitogen activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), p38, and protein, which are factors related to osteoclast differentiation and proliferation, Phosphorase B (Akt) was treated with RANKL and the amaranth extract of the present invention.

As a result of the experiment, the inhibition of differentiation from Raw264.7 cells to osteoclasts by the amaranth extract of the present invention is shown by inhibiting the signals of mitogen-activated protein kinase (MAPK) and protein kinase B (Akt), which are key signals for differentiation. I was able to confirm. In particular, it was confirmed that the amaranth extract inhibited phosphorylation of extracellular signal-regulated kinase (ERK) and p38 among mitogen-activated protein kinases (MAPK), and also inhibited phosphorylation of protein kinase B (Akt) (see Fig. 3). .

Claims (7)

A pharmaceutical composition for preventing or treating osteoporosis, containing an alcohol extract of Amaranthus mangostanus as an active ingredient.
According to claim 1,
The pharmaceutical composition, characterized in that the composition inhibits osteoclast differentiation.
According to claim 1 or 2,
The alcohol is C ₁ ~ C ₄ Pharmaceutical composition, characterized in that the alcohol.
According to claim 1 or 2,
The composition is a pharmaceutical composition, characterized in that formulated in the form of powder, granules, tablets, hard capsules, soft capsules or injections.
A health functional food for preventing or improving osteoporosis containing an alcohol extract of Amaranthus mangostanus as an active ingredient.
According to claim 5,
The health functional food is a health functional food, characterized in that inhibiting osteoclast differentiation.
According to claim 5,
The alcohol is a health functional food, characterized in that C ₁ ~ C ₄ alcohol.

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