KR20240000071A - Osteoclast inhibitory composition of Aster glehni Extract and its Treatment - Google Patents

Abstract

One embodiment provides an osteoclast-inhibiting composition and a therapeutic agent thereof containing an extract of Sagebrush as an active ingredient.

Description

Osteoclast inhibitory composition of Aster glehni Extract and its Treatment {Osteoclast inhibitory composition of Aster glehni Extract and its Treatment}

The present invention relates to an osteoclast-inhibiting composition of an extract of Sagebrush and a therapeutic agent thereof.

Aster glehni F.Schmidt is a perennial herb of the Asteraceae genus.

Aster glehni F.Schmidt is rich in vitamins A and C and is known to contain protein, phosphorus, and calcium. Volatile flavor components contained in sagebrush, for example, are known to be 8.5% of 7 alcohols, 2.1% of 3 esters, 2.10% of oxides, 1.65% of acids, and 0.48% of aldehydes. . The main volatile flavor components of sagebrush are alpha-pinene, limonene, gamma-elemene, beta-pinene, cis-3-hexenol, and myrcene.

It has medicinal properties similar to that of ants, and according to Sasang medical standards, it is effective for Taeeum people who have a weak respiratory system and cough or have a lot of phlegm if they eat it regularly, but it is known that it is better for Soeum people not to eat too much of it.

Scallops are fried and eaten, added to soups, and stored as muknamul (muknamul). Like other sagebrush species, it has a slightly bitter and astringent taste, but since ancient times it has been used as a substitute for spinach because it is rich in calcium and iron. There are examples of sagebrush being used not only for food but also for medicinal purposes.

For example, Korean Patent Publication No. 2018-0020798 (title of the invention: Composition containing Asterium aster extract and vitamins for reducing blood uric acid) is a blood clot containing a mixture of Asteria serrata extract and vitamin B6 as active ingredients. Disclosed is a composition for lowering urea concentration, or a pharmaceutical/food composition for preventing, improving, and/or treating gout or hyperuricemia, and a method for preventing and/or treating gout or hyperuricemia using the same.

Korean Patent Publication No. 2015-0135037 (title of the invention: Asterium aster extract, fraction thereof, and active compound isolated therefrom effective for hyperuricemia or gout) is a Asterium aster extract, a fraction thereof, and an active compound isolated from the above fraction. The compound is disclosed to be useful as an active ingredient in medicines, health foods, etc. that require the treatment, prevention, and improvement of hyperuricemia or gout, which are diseases caused by an increase in the concentration of uric acid.

In addition to being known to have pharmacological effects on coughing, diuresis, and asthma, it is also known to be effective in preventing gout or hyperuricemia by improving the effect of lowering blood uric acid concentration as mentioned above. Gout is known to be involved in joint inflammation, and unlike other joint diseases, it is mainly involved in human metabolism rather than the immune system.

The present invention provides an osteoclast inhibitory composition that effectively inhibits osteoclast differentiation and a therapeutic agent thereof.

One embodiment provides an osteoclast-inhibiting composition and a therapeutic agent thereof containing an extract of Sagebrush as an active ingredient.

The osteoclast inhibitory composition and its therapeutic agent according to one embodiment can effectively inhibit osteoclast differentiation.

Figure 1 shows the confirmation of cell viability and NO production of Aster water extract in RAW 264.7 cells.
Figure 2 shows the confirmation of inhibition of inflammatory cytokines by Aster water extract in osteoclasts.
Figure 3 shows the confirmation of suppression of genes related to osteoclast differentiation by Aster water extract in osteoclasts.
Figure 4 shows confirmation of the inhibition of osteoclast activation-related genes in osteoclasts by the Aster water extract.

Hereinafter, some embodiments of the present invention will be described in detail through illustrative drawings. When adding reference numerals to components in each drawing, it should be noted that identical components are given the same reference numerals as much as possible even if they are shown in different drawings. Additionally, when describing embodiments of the present invention, if it is determined that a detailed description of a related known configuration or function may obscure the gist of the present invention, the detailed description will be omitted.

The terms “treatment,” “inhibition,” and “attenuation” as used in the present invention mean that the composition of the present invention is used on an individual to reduce the differentiation of specific cells, the level of a specific substance, or the progression of a specific disease or condition. It means any act of delay.

The term “individual” used in the present invention refers to all animals, including humans.

Hereinafter, an osteoclast inhibitory composition for arthritis and a therapeutic agent thereof that provide excellent therapeutic effects on arthritis when used in joints according to embodiments of the present invention will be described.

Examples: Pharmaceutical compositions and therapeutic agents thereof

One embodiment provides an osteoclast-inhibiting composition and a therapeutic agent thereof containing an extract of Asterium vulgaris as an active ingredient. As can be seen through the experimental examples described later, the Asterium aster extract may be a water extract at a concentration of 31.25 μg/ml or more and 1,000 μg/ml, but is not limited thereto.

Through the experimental example described later, it can be confirmed that the Asterium vulgare extract has an osteoclast inhibitory effect. Therefore, it can be seen that the osteoclast inhibitory extract and its therapeutic agent according to one embodiment have osteoclast inhibitory effect when they include the above-mentioned Asterium vulgaris extract as an active ingredient. Since the osteoclast inhibitory extract and its therapeutic agent according to one embodiment have an osteoclast inhibitory effect, it can be seen that they exhibit arthritis treatment efficacy.

The osteoclast inhibitory treatment may further include a pharmaceutically acceptable carrier, and may be formulated with the carrier and provided as food, medicine, feed additive, drinking water additive, etc. The term “pharmaceutically acceptable carrier” as used in the present invention refers to a carrier or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the administered compound.

In the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

The osteoclast inhibitory composition of the present specification can be applied in any formulation containing it as an active ingredient, and can be prepared as an oral or parenteral formulation. The pharmaceutical formulations herein may be administered orally, rectally, nasally, topically (including cheeks and under the tongue), subcutaneously, vaginally, or parenterally (intramuscularly, subcutaneously). and forms suitable for administration (including intravenously) or forms suitable for administration by inhalation or insufflation.

Formulations for oral administration containing the composition of the present specification as an active ingredient include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, powders or granules, emulsions, hard or soft capsules, syrups or elixirs. can do. For formulation into dosage forms such as tablets and capsules, binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, and stearic acid masene. It may contain a lubricant such as calcium, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax, and in the case of a capsule formulation, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned substances.

Formulations for parenteral administration containing the composition of the present specification as an active ingredient include injection forms such as subcutaneous injection, intravenous injection, or intramuscular injection, suppository injection, or sprays such as aerosols that allow inhalation through the respiratory tract. It can be formulated as: In order to formulate a formulation for injection, the composition of the present invention can be mixed in water with a stabilizer or buffer to prepare a solution or suspension, and the composition can be formulated for unit administration in ampoules or vials. For injection as a suppository, it can be formulated into a composition for rectal administration such as a suppository or enema containing a common suppository base such as cocoa butter or other glycerides. When formulating for spraying such as an aerosol, a propellant, etc. may be mixed with additives to disperse the water-dispersed concentrate or wet powder.

The method of administration of the osteoclast inhibitory composition and its therapeutic agent of the present invention is not particularly limited, but is administered parenterally or orally, such as intravenously, subcutaneously, intraperitoneally, inhalation, skin application, patch, or topical application, depending on the desired method. It can be administered.

The dosage range varies depending on the patient's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. Daily dosage refers to the amount of therapeutic agent of the invention sufficient to treat the disease condition alleviated by administration to an individual in need of treatment. The effective amount of a therapeutic agent will depend on the particular compound, the disease state and its severity, and the individual in need of treatment, and can be routinely determined by a person skilled in the art. As a non-limiting example, the dosage for the human body of the composition according to the present invention may vary depending on the patient's age, weight, gender, dosage form, health status, and disease severity.

It can be confirmed that the osteoclast inhibitory composition and its therapeutic agent according to the above-described examples effectively inhibit osteoclast differentiation, as confirmed through the experimental examples below.

Hereinafter, the present invention will be described in more detail by describing experimental examples. However, the following experimental example is only an example of the present invention, and the content of the present invention should not be construed as limited thereto.

In the experimental examples below, the method for producing the Asterium aster extract is illustratively described, but the embodiments of the present invention generally allow the aquila extract to be prepared through a method for producing a plant extract (change of extraction solution or extraction conditions, etc.). You can. In particular, the extract of Aster aster using water is referred to as a Aster aster water extract, but a Aster aster extract can be prepared using other common extraction solvents.

Experimental Example: Pharmaceutical Composition

1. Experimental method

1) Production of sagebrush

The sagebrush grown on Ulleungdo was obtained and used. After accurately measuring the weight of 100 g of Aster, add it with 1 L of distilled water, heat and extract it for 2 hours from the time the decoction boils, and then filter the extract using filter paper No. Filtered under reduced pressure using 4. Dried powder was obtained using a freeze dryer (FDU-1200, EYELA, Tokyo, Japan), and the yield was 32%.

2) Culture of RAW 264.7 cells and differentiation into osteoclasts

RAW 264.7 osteoclast progenitor cells were purchased from the Korean Cell Line Bank. RAW 264.7 cells were cultured in a 5% CO ₂ incubator at 37°C using Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 uint/ml penicillin-stretomycin.

To differentiate osteoclast precursor cells into osteoclasts, 50 ng/mL of Receptor activator of nuclear factor kappa-B ligand (RANKL) was added to α-Minimum Essential Media (MEM) medium containing 10% FBS and 100 uint/ml penicillin-stretomycin. ) was treated and cultured.

3) Cytotoxicity analysis

Raw 264.7 cells were distributed in a 96 _well plate at a density of ¹ Cells were treated with a ml concentration of Aster water extract. After 24 hours, 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) solution was treated, and after 4 hours, the supernatant was removed and non-aqueous formazan was removed using dimethyl sulfoxide (DMSO). The product was dissolved.

Cell viability was measured at absorbance at 540 nm using an Epoch ^® microvolume spectrophotometer system (BioTek Instruments Inc., Winooski, VT, USA).

4) Nitric oxide (NO) production amount analysis

Raw 264.7 cells were distributed at a density of 1×10 ⁵ in a 24-well plate and cultured in a 37°C, 5% CO ₂ incubator for 24 hours. After pretreatment with Aster water extract and 20 μM NIL at concentrations of 0, 15.625, 31.25, 62.5, 125, 250, 500, and 1,000 μg/ml for 1 hour, treatment with 1 μg/ml LPS for 48 hours. cultured for a while. Next, the culture supernatant was separated and transferred to a 96 well plate, treated with an equal amount of Griess reagent, reacted at room temperature, and absorbance was measured at 540 nm.

5) RNA isolation and real-time PCR analysis

RAW 264.7 cells were diluted in DMEM medium to a density of 3 x 10 ⁵ and distributed in 6-well plates and cultured in an incubator at 37°C and 5% CO ₂ for 24 hours. Then, the medium was changed to α-MEM, and the cells were pretreated with Asterium astera water extract (250, 500, and 1,000 μg/ml) 30 minutes before treatment with 50 ng/mL RANKL and cultured for 3 days. Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was obtained using extracted total RNA (2 ug), d(T)16 primer, and AMV reverse transcriptase.

The relative amounts of genes were quantified by real-time RT-PCR (Real Time PCR System7500, Applied biosystems, Foster, CA) using SYBR Primix ExTaq (Takara). Ct values of IL-1β, IL-6, RANKL, RANK, TRAF6, NFATc1, c-Fos, ATP6v0d2, Cathepsin K, MMP9, and MITF genes were converted to Ct values of β-actin gene using gene express 2.0 program (Applied biosystems). It was corrected using . Additionally, the specificity of the amplified PCR product was confirmed through melting curve analysis.

6) Statistical analysis

All values are expressed as mean ± SEM (n=3). Data were analyzed using one-way analysis of variance (ANOVA) and Dunnett’s test. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS; version 19.0).

2. Experiment results

1) Measurement of cell viability and NO production of Asterium aster water extract in RAW 264.7 cells

Since the inhibitory effect of inflammatory mediators from osteoclast precursor cells may be caused by the death of RAW 264.7 cells due to cytotoxicity, MTT assay was performed to rule out this possibility.

As a result of culturing with the addition of water extract of Asterium dulcis at concentrations of 0, 15.625, 31.25, 62.5, 125, 250, 500, and 1,000 μg/ml, the survival rate of cells did not decrease below 80% at all treatment concentrations. was observed (see Figure 1A).

Furthermore, in order to confirm whether the water extract of Sagebrush suppresses inflammatory mediators in osteoclast precursor cells, the amount of NO production was checked, and the amount of NO production was significantly suppressed starting at a concentration of 31.25 μg/ml or higher (see Figure 1B). Accordingly, we attempted to confirm the ability to inhibit osteoclast differentiation by selecting concentrations of 250, 500, and 1,000 μg/ml, which showed a significant anti-inflammatory effect without cytotoxicity.

2) Confirmation of the inflammatory cytokine inhibition effect of Aster water extract on osteoclasts

In order to determine the effect of Aster water extract on inflammatory cytokines, which are important factors in the osteoclast formation process, the mRNA expression of IL-1β and IL-6 was confirmed using qRT-PCR.

As shown in Figure 2, the mRNA expression of IL-1β and IL-6 rapidly increased in the RANKL-treated group compared to the control group, while the mRNA expression of IL-1β and IL-6 was effectively suppressed by treatment with Aster water extract. It was confirmed that it was done.

3) Confirmation of the effect of suppressing genes related to osteoclast differentiation of Sagebrush on osteoclasts

In order to determine the effect of water extract of Sagebrush on the expression of genes related to osteoclast differentiation, the mRNA expression of RANKL, RANK, and TRAF6 was confirmed using qRT-PCR.

It was confirmed that the mRNA expression of RANKL, RANK, and TRAF6 was rapidly increased by treatment with RANKL, while the mRNA expression of RANKL, RANK, and TRAF6 was effectively suppressed by treatment with the Aster water extract (see Figure 3).

4) Confirmation of the effect of suppressing genes related to osteoclast activation of Sagebrush on osteoclasts

In order to determine the effect of Asterium astera water extract on the expression of genes related to osteoclast activation, qRT-PCR was used to confirm the mRNA expression of NFATc1, c-Fos, ATP6v0d2, Cathepsin K, MMP9, and MITF.

It was confirmed that the mRNA expression of NFATc1, c-Fos, ATP6v0d2, Cathepsin K, MMP9, and MITF was rapidly increased by RANKL treatment, while their expression was effectively suppressed by treatment with Aster water extract (see Figure 4).

In summary, through the experimental examples, it was found that the extract of Asterium vulgaris has an osteoclast inhibitory effect. Through experimental examples, it can be seen that the osteoclast-inhibiting extract and its therapeutic agent according to one embodiment have an osteoclast-inhibiting effect and thus exhibit an arthritis treatment effect.

Hereinafter, examples of manufacturing therapeutic agents and health foods containing pharmaceutical compositions according to examples and experimental examples will be described.

<Preparation Example 1>: Preparation of therapeutic agent

<1-1> Production of powder

300 mg of Asterium aster extract

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled into an airtight bubble to prepare a powder.

<1-2> Preparation of tablets

50 mg of Cordycepsia extract

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are manufactured by compressing them according to a typical tablet manufacturing method.

<1-3> Manufacturing of capsules

50 mg of Cordycepsia extract

Corn starch 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 typical capsule manufacturing method.

<1-4> Preparation of injections

50 mg of Cordycepsia extract

Proper amount of sterile distilled water for injection

Appropriate amount of pH adjuster

It is prepared with the above ingredients per ampoule (2 ml) according to the usual manufacturing method for injections.

<1-5> Preparation of liquid

Sagebrush extract 1,000 mg

20 g sugar

20 g isomerized sugar

Lemon flavor appropriate amount

Purified water was added to bring the total volume to 1000 ml. The above ingredients were mixed according to a typical liquid preparation method, then filled into a brown bottle and sterilized to prepare a liquid.

<Production Example 2>: Production of health food

Sagebrush extract 1,000 mg

Vitamin mixture dosage

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 ㎍

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Mineral mixture appropriate amount

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Monobasic Potassium Phosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The composition ratio of the above vitamin and mineral mixture is a mixture of components relatively suitable for health food in a preferred embodiment, but the mixing ratio may be modified arbitrarily. The above ingredients are mixed according to a typical health food manufacturing method, and then , granules can be manufactured and used to manufacture health food compositions according to conventional methods.

<Preparation Example 3>: Preparation of health drink

1000 mg of Asterium vulgaris extract

Citric acid 1000 mg

100 g oligosaccharides

2 g plum concentrate

1 g taurine

Purified water was added to make a total of 900 ml. The above ingredients were mixed according to the usual health drink manufacturing method. After stirring and heating at 85°C for about 1 hour, the resulting solution was filtered, placed in a sterilized 2-liter container, sealed, and sterilized. It is then refrigerated and used to prepare the health drink composition of the present invention.

Since the above-mentioned therapeutic agents and health foods also contain pharmaceutical compositions, it can be confirmed that they have osteoclast inhibition effects as described above in the experimental examples.

Comparative example

Korean Patent Publication No. 2018-0020798 (Title of the invention: Composition containing Asterium extract and vitamins for reducing blood uric acid) Korean Patent Publication No. 2015-0135037 (Title of the invention: Composition containing Asterium aster extract and vitamins for reducing blood uric acid) There is a difference between the Asteria extract (effective for gout, the Asterium aster extract, its fractions, and the active compound isolated therefrom) and the pharmaceutical composition according to an embodiment of the present invention in terms of a solvent extraction method and separation of specific compounds. .

In particular, Korean Patent Publication No. 2018-0020798 (title of the invention: Composition containing Asterium aster extract and vitamins for reducing blood uric acid) extract of Asterium vulgaris extract, Korean Patent Publication No. 2015-0135037 (name of the invention: Goyo) It is only stated that the uric acid lowering function of the euphorbia extract (effective for acidosis or gout, its fractions, and active compounds isolated therefrom) can be used for the treatment and prevention of gout.

efficacy treatment disease Korean Patent Publication No. 2018-0020798 (Title of the invention: Composition containing Asterium extract and vitamins for reducing blood uric acid) Korean Patent Publication No. 2015-0135037 (Title of the invention: Composition containing Asterium aster extract and vitamins for reducing blood uric acid) Aster extract (effective for gout, Aster aster extract, fractions thereof, and active compounds isolated therefrom) Uric acid lowering function gout treatment Pharmaceutical composition according to embodiments of the present invention Osteoclast inhibition effect arthritis treatment

Since the pharmaceutical composition according to an embodiment of the present invention has an osteoclast inhibitory effect, it can inhibit inflammatory cytokines and differentiation of osteoclasts, which are important in the progression of arthritis affected by osteoclasts, as described above. Confirmed.

Example of use

The pharmaceutical composition according to the above-described embodiment of the present invention can be used not only as a treatment for arthritis but also as a health food or composition for imaging diagnosis. For example, the pharmaceutical composition according to the above-described embodiment of the present invention can be used as an osteoclast inhibitory composition mixed with ultrasonic gel used in ultrasonic imaging. Through this, an additional osteoclast inhibition effect can be achieved through the ultrasound gel used during ultrasound imaging.

Although the embodiments have been described above with reference to the drawings, the present invention is not limited thereto, and the above description is merely an illustrative explanation of the technical idea of the present invention, and those skilled in the art will understand the present invention. Various modifications and variations may be made without departing from the essential characteristics of the invention. Accordingly, the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention, but are for illustrative purposes, and the scope of the technical idea of the present invention is not limited by these embodiments. The scope of protection of the present invention should be interpreted in accordance with the claims below, and all technical ideas within the equivalent scope should be construed as being included in the scope of rights of the present invention.

Claims (4)

An osteoclast-inhibiting composition containing Asterium vulgaris extract as an active ingredient. According to paragraph 1,
The osteoclast inhibitory composition is a water extract wherein the Sagebrush extract is a water extract at a concentration of 31.25 μg/ml or more and 1,000 μg/ml. A composition containing Asteria extract as an active ingredient;
An osteoclast-inhibiting therapeutic agent comprising a pharmaceutically acceptable carrier. According to paragraph 3,
The Asterium vulgaris extract is an osteoclast inhibitory treatment agent that is a water extract at a concentration of 31.25 μg/ml or more and 1,000 μg/ml.