KR102652245B1 - Composition for Prophylaxis and Treatment of Osteoporosis Comprising Piperis Longi Fructus Extract - Google Patents

Abstract

The present invention relates to the effect of the Pilbal extract on preventing or treating osteoporosis and the effect of suppressing bone loss, bone resorption, and osteoclast differentiation. The Pilbal extract is non-cytotoxic, inhibits differentiation and bone resorption of osteoclasts, and inhibits osteoclast differentiation and osteoclast differentiation. Since it suppresses the expression of differentiation transcription factors, it can be usefully used to suppress bone loss or prevent or treat bone diseases.

Description

Composition for preventing and treating osteoporosis comprising Piperis Longi Fructus Extract {Composition for Prophylaxis and Treatment of Osteoporosis Comprising Piperis Longi Fructus Extract}

The present invention relates to the effects of Pilbal extract on preventing or treating osteoporosis and the effects of inhibiting bone loss, bone resorption, and osteoclast differentiation.

Osteoclast is the only cell that absorbs bone and is activated by various factors in the living body, and high-quality bone is maintained in balance with osteoblasts. Osteoporosis is a skeletal disease caused by various causes, which reduces bone mass and causes microstructural deformation. In addition, osteoporosis is caused by low bone density, which weakens bone strength and increases the risk of fracture even from a light impact. This type of osteoporosis occurs when the balance of bone metabolism is uncontrolled due to increased activity of osteoclasts compared to osteoblasts. Therefore, suppressing osteoclast differentiation is the most important treatment method to treat osteoporosis.

Osteoclast differentiation is regulated by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL), which binds to RANK, an osteoclast receptor, and induces the activation of c-Fos. Activated c-Fos induces the expression of Nuclear Factor Of Activated T Cells 1 (NFATc1), another transcription factor, and NFATc1 regulates osteoclast differentiation and activity such as TRAP, cathepsin K (CTK), and osteoclast-associated receptor (OSCAR). Regulates the expression of factors related to

Currently, the most commonly used treatment for osteoporosis is bisphosphonates. However, it is known that treating osteoporosis with bisphosphonate agents can cause side effects such as osteonecrosis and atypical fractures. Therefore, there is a need to develop treatments derived from natural products without side effects.

Meanwhile, Piperis Longi Fructus (hereinafter referred to as PF) is the unripe fruit of Piper longum Linne belonging to the Piperaceae family and is one of the commercially, economically and medically valuable medicinal herbs. To date, many compounds have been isolated from plants of the Pepperaceae family, and pilbal contains various components such as essential oils (camphene, piperonal, sabinene, limonene, myrcene) and fatty acids (palmitic acid). In addition, Pibal is known to be used in oriental medicine for abdominal distension, vomiting, toothache, pain relief, and diarrhea, but nothing is known about its effectiveness in treating bone metabolism.

Accordingly, the present inventors confirmed the effect of Pilbal on factors related to osteoclast differentiation and confirmed that Pilbal extract has the effect of effectively suppressing the differentiation of osteoclasts, which are known to be the main cause of postmenopausal osteoporosis. The present invention was completed by confirming that it can be used to improve various bone diseases, including osteoporosis.

Korean Patent Publication No. 10-2019-0117153

An object of the present invention is to provide a composition for inhibiting bone loss.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating bone diseases.

Another object of the present invention is to provide a food composition for preventing or improving bone disease.

Another object of the present invention is to provide a composition for inhibiting osteoclast differentiation or bone resorption.

In order to achieve the above purpose,

The present invention provides a composition for inhibiting bone loss containing Piperis Longi Fructus extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating bone diseases containing Pilbal extract as an active ingredient.

Furthermore, the present invention provides a food composition for preventing or improving bone disease containing Pilbal extract as an active ingredient.

Furthermore, the present invention provides a composition for inhibiting osteoclast differentiation or bone resorption containing a pilbal extract as an active ingredient.

It was confirmed that the Pilbal extract according to the present invention can inhibit osteoclast differentiation and bone resorption by reducing RANKL-induced TRAP activity and pit area without cytotoxicity, and it was confirmed that the osteoclast differentiation transcription factor Since it inhibits expression, it can be usefully used to suppress bone loss or prevent or treat bone diseases.

Figure 1 is a graph showing the results of confirming the cytotoxicity of Philbal ethanol extract (PF).
Figure 2 is a diagram confirming osteoclast differentiation ability by treatment with essential ethanol extract (PF).
Figure 3 is a diagram confirming the bone resorption inhibition effect by Philbal ethanol extract (PF).
Figure 4 is a diagram confirming the effect of suppressing protein expression of NFATc1 and c-Fos, which are osteoclast differentiation transcription factors, by treatment with Philbal ethanol extract (PF).

Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of techniques well known to those skilled in the art may be omitted. Additionally, when describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs.

Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

The present invention relates to a composition for inhibiting bone loss containing Piperis Longi Fructus extract as an active ingredient.

The term "extract" used in the present invention refers to a preparation obtained by squeezing a herbal medicine into an appropriate leachate and evaporating the leachate to concentrate. It is not limited thereto, but is not limited to the extract, dilution or concentrate of the extract obtained by extraction treatment, It may be a dried product obtained by drying the extract, a crude product thereof, or a purified product. The above-mentioned Argentine extract or Wangbulryuhaeng extract can be prepared using general extraction, separation and purification methods known in the art. The extraction method is not limited thereto, but preferably includes boiling water extraction, hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction.

In the present invention, the extract can be prepared by extracting with an extraction solvent or fractionating the extract by adding a fractionating solvent to the extract prepared by extraction with an extraction solvent. The extraction solvent is not limited to this, but water, an organic solvent, or a mixed solvent thereof may be used. The organic solvent may be an alcohol having 1 to 4 carbon atoms, a polar solvent such as ethyl acetate or acetone, hexane, or dichloromethane. A non-polar solvent of methane or a mixed solvent thereof can be used. In addition, water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof can be preferably used, more preferably a mixed solvent of water and ethanol can be used, and even more preferably ethanol can be used. In one embodiment of the present invention, 'pilbal ethanol extract (PF)' was prepared by extracting using 100% (v/v) ethanol as the solvent and then freeze-drying.

In one embodiment, the extract of the present invention is extracted with at least one extraction solvent selected from the group consisting of water, anhydrous or hydrous alcohol having 1 to 4 carbon atoms, ethyl acetate, acetone, glycerin, ethylene glycol, propylene glycol, and butylene glycol. It can be extracted, preferably with an extraction solvent that is a mixture of water and ethanol, and more preferably with ethanol.

In one embodiment, the composition of the present invention can inhibit osteoclast differentiation or bone resorption.

In one embodiment, the composition of the present invention may include the Wangbulryuhaeng thermal water extract at a concentration of 1 to 1000 μg/ml, preferably at a concentration of 10 to 150 μg/ml, and more preferably 12.5 μg/ml. It may be included at a concentration of from 100 μg/ml, but is not limited thereto.

Additionally, the present invention relates to a pharmaceutical composition for preventing or treating bone disease containing an extract of Piperis Longi Fructus as an active ingredient.

In one embodiment, the bone disease is selected from the group consisting of osteoporosis, osteopenia, osteolytic metastasis, senile kyphosis, and Paget disease. There may be more than one, preferably osteoporosis or osteopenia, and more preferably osteoporosis.

In one embodiment, the composition of the present invention can inhibit the RANK signaling pathway.

In one embodiment, the composition of the present invention can inhibit the expression of proteins or genes related to osteoclast differentiation. The protein or gene related to osteoclast differentiation may be any one selected from the group consisting of nuclear factor of activated T cell c1 (NFATc1), c-Fos, and tartrate-resistant acid phosphatase (TRAP).

In one embodiment, the composition of the present invention can inhibit the expression of proteins or genes related to bone resorption. The protein or gene related to bone resorption may be any one selected from the group consisting of matrix methalloproteinase 9 (MMP-9), Cathepsin K (CTK), and carbonic anhydrase II (CA2).

In one embodiment, the bone disease of the present invention may be caused by hyper-differentiation or hyper-activation of osteoclasts. The differentiation or activation of osteoclasts may be induced by RANKL.

In one embodiment, the bone disease of the present invention may exhibit increased activity or increased expression of proteins or genes related to osteoclast differentiation, and the proteins or genes related to osteoclast differentiation include TRAP, NFATc1, c-Fos, and It may be selected from the group consisting of RANK.

In one embodiment, the bone disease of the present invention may exhibit increased activity or increased expression of a protein or gene related to bone resorption, and the protein or gene related to osteoclast differentiation consists of CTK, MMP-9, and CA2. It may be selected from a group.

The pharmaceutical composition of the present invention may additionally include adjuvants. Any adjuvant known in the art may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase immunity.

As used herein, the term "treatment", unless otherwise stated, means reversing, alleviating, inhibiting the progression of, or It means prevention, and the term treatment as used herein refers to the act of treating. Accordingly, treatment or therapy for bone disease in mammals may include one or more of the following:

(1) Inhibits the growth, i.e., arrests the development, of bone diseases;

(2) prevent the spread of bone disease, i.e. prevent metastasis;

(3) alleviating bone disease;

(4) preventing recurrence of bone disease; and

(5) Alleviating symptoms of bone disease (palliating)

As used herein, the term “mammal” refers to a mammal, preferably a human, that is the subject of treatment, observation or experiment.

A composition is indicated as “pharmaceutically or physiologically acceptable” if the recipient animal can tolerate administration of the composition or if administration of the composition to the animal is suitable. If the amount administered is physiologically significant, the agent may be said to have been administered in a “therapeutically effective amount.” An agent is physiologically significant if its presence causes a detectable change in the physiology of the recipient patient.

The therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as administration method, target site, patient condition, etc. Therefore, when used in the human body, the dosage must be determined as appropriate by considering both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal testing. These considerations in determining an effective amount include, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceutically effective amount" refers to 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 determined by the patient's Factors including health status, type and severity of disease, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, drugs combined or used simultaneously, and other factors well known in the field of medicine. It can be decided depending on The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The composition of the present invention may also include carriers, diluents, excipients, or combinations of two or more commonly used in biological products. Pharmaceutically acceptable carriers are not particularly limited as long as they are suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. The compounds described in, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients can be mixed and used, and if necessary, other ingredients such as antioxidants, buffers, and bacteriostatic agents. Normal additives can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate dosage forms such as aqueous solutions, suspensions, emulsions, etc., into pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods. .

As used in the present invention, the term “pharmaceutically acceptable” means that the composition exhibits non-toxic properties to cells or humans exposed to the composition.

The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives. In this case, the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, stearic acid. Calcium, white sugar, dextrose, sorbitol, and talc may be used. The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

As used in the present invention, the term "administration" means providing a predetermined substance to a patient by any suitable method, and is administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the desired method. It can be applied as an injection formulation) or orally administered, and the dosage range varies depending on the patient's weight, age, gender, health status, diet, administration time, administration method, excretion rate, and severity of the disease.

The composition of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) or orally depending on the desired method, and the dosage is determined by the subject's age, weight, gender, physical condition, etc. is selected taking into account. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the subject, and it is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 ㎍/ml, pharmaceutical activity may not appear, and if it exceeds 5,000 ㎍/ml, it may be toxic to the human body.

The pharmaceutical composition of the present invention may be formulated in various oral or parenteral dosage forms. Dosage forms for oral administration include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, granules, etc. These dosage forms contain diluents (e.g. lactose, dextrose, water) in addition to the active ingredient. crose, mannitol, sorbitol, cellulose and/or glycine), lubricants (e.g. silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). Additionally, the tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and in some cases starch, agar, alginic acid. or disintegrants such as sodium salts thereof or effervescent mixtures and/or absorbents, colorants, flavoring and sweetening agents. The formulation can be prepared by conventional mixing, granulating or coating methods. In addition, representative formulations for parenteral administration are injectable formulations, and solvents for injectable formulations include water, Ringer's solution, isotonic saline solution, or suspension. The sterile fixed oil of the injectable preparation can be used as a solvent or suspending medium, and any non-irritating fixed oil, including mono- and di-glycerides, can be used for this purpose. Additionally, the injectable preparation may use fatty acids such as oleic acid.

In the present invention, the term “prevention” refers to all actions that inhibit or delay the occurrence, spread, and recurrence of bone disease or osteoporosis by administering the pharmaceutical composition according to the present invention.

Furthermore, the present invention relates to a food composition for preventing or improving bone disease containing Piperis Longi Fructus extract as an active ingredient.

In one embodiment, the bone disease is selected from the group consisting of osteoporosis, osteopenia, osteolytic metastasis, senile kyphosis, and Paget disease. There may be more than one, preferably osteoporosis or osteopenia, and more preferably osteoporosis.

When using the composition of the present invention as a food composition, the composition can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. In addition to the active ingredients, the composition may contain food additives acceptable to the food industry, and the mixing amount of the active ingredients can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment).

The term "food supplement" used in the present invention refers to a component that can be added as an auxiliary food additive, and can be appropriately selected and used by a person skilled in the art as it is added to manufacture each type of health functional food. Examples of food supplements include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, and protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., but the types of food supplements of the present invention are not limited to the above examples.

The food composition of the present invention may include health functional foods. The term “health functional food” used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients with functional properties useful to the human body. Here, ‘functionality’ means controlling nutrients for the structure and function of the human body or obtaining useful effects for health purposes, such as physiological effects. The health functional food of the present invention can be manufactured by methods commonly used in the field of technology, and can be manufactured by adding raw materials and components commonly added in the field of technology. Additionally, the formulation of the health functional food can also be manufactured without limitation as long as it is a formulation recognized as a health functional food. The food composition of the present invention can be manufactured in various types of formulations, and unlike general drugs, it is made from food as a raw material and has the advantage of not having side effects that may occur when taking the drug for a long period of time, and is excellent in portability, so the present invention Health functional foods can be consumed as supplements to enhance the effectiveness of arthritis or osteoporosis treatments.

Additionally, there is no limitation to the types of health foods in which the composition of the present invention can be used. In addition, a composition containing the extract of the present invention or a fraction thereof as an active ingredient can be prepared by mixing known additives with other appropriate auxiliary ingredients that may be contained in health functional foods according to the selection of a person skilled in the art. Examples of foods that can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and There are vitamin complexes, etc., and they can be manufactured by adding them to juices, teas, jellies, juices, etc. prepared using the extract according to the present invention as a main ingredient.

Furthermore, the present invention relates to the use of Piperis Longi Fructus extract as an active ingredient for inhibiting osteoclast differentiation or bone resorption.

In one embodiment, the osteoclast differentiation or bone resorption may be induced by RANKL.

Since the composition of the present invention is made from natural ingredients, it may have fewer side effects compared to common synthetic compounds even when used as a pharmaceutical composition or food composition, so it can be safely included in pharmaceutical compositions and health functional foods.

The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.

<Example 1> Preparation of Pilbal extract

Pilbal (Indonesia) was purchased through OmniHerb. 3000 ml of 100% ethanol was added to 300 g of Pilbal and subjected to immersion extraction for 2 weeks. The extract was filtered through filter paper (Whatman, No 2). Afterwards, it was concentrated using a vacuum concentrator, then freeze-dried and powdered. The finally obtained dried ethanol extract of Piperis Longi Fructus (hereinafter 'PF') was stored in the refrigerator until use.

<Example 2> Confirmation of cytotoxicity

RAW 264.7 cells were cultured in a 100° dish using DMEM medium mixed with 10% FBS and 1% P/S in an incubator with 95% humidity, a temperature of 37°C, and 5% CO ₂ environmental conditions.

The cytotoxicity of the Pilbal extract was measured by performing MTS assay in RAW 264.7 cells. First, RAW 264.7 cells were distributed in a 96-well plate and cultured for 24 hours. After stabilization, the medium was removed and new medium was added, while the PF powder prepared in Example 1 was filtered through a sterile filter (pore size 0.22 μm) and added to each culture medium at a concentration of 12.5, 25, 50, or 100 μg/ml. Then, it was cultured for 24 hours. Afterwards, the cell viability was confirmed by treating the cells with the MTS assay solution and measuring the absorbance at 405 nm. Cytotoxicity was expressed as a percentage relative to the untreated control.

As a result, Philbal ethanol extract (PF) did not show cytotoxicity at any concentration (Figure 1).

<Example 3> Confirmation of osteoclast differentiation inhibition ability

To evaluate the osteoclast differentiation inhibitory ability of the ethanol extract of Philval (PF of Example 1) according to the present invention, TRAP (tartrateresistant acid phosphatase) staining and TRAP activity measurement were performed.

In order to induce differentiation of osteoclasts, as in the above example, RAW 264.7 cells were seeded in a 96-well plate and cultured for 24 hours, then replaced with medium supplemented with RANKL (100 ng/ml). At this time, the required experimental group was added PF to the above medium at a concentration of 12.5, 25, 50, or 100 μg/ml, and cultured for 5 days, replacing the same medium every 2 days. Differentiated osteoclasts were stained using a TRAP assay kit. Afterwards, the stained cells were observed using an inverted microscope, and cells with three or more nuclei were counted as multinucleated osteoclasts. Here, cells that were not treated with RANKL were confirmed as the normal group.

Then, to measure TRAP activity, TRAP solution prepared by mixing 4.93 mg of 4-Nitrophenyl phosphate disodium salt hexahydrate (Sigma aldrich, MO, USA), 850 μL of 0.5 M acetate, and 150 μL of tartrate acid solution and the cultured cell medium ( 50μ of each culture medium was dispensed into a new 96-well plate and reacted at 37°C for 60 minutes. Afterwards, the reaction was terminated by treatment with 50 μL of 0.5M NaOH, and TRAP activity was confirmed by measuring absorbance at 405 nm using an ELISA device.

As a result, the control group (group treated only with RANKL) had more TRAP-positive cells, i.e. multinucleated osteoclasts, formed compared to the normal group (untreated group with RANKL), but in the experimental group treated with PF, osteoclast formation was reduced. This was confirmed (Figure 2A). In particular, as a result of measuring TRAP enzyme activity, the control group showed an increase in TRAP enzyme activity compared to the normal group, while the PF-treated group showed a decrease in TRAP enzyme activity compared to the control group (Figure 2B).

<Example 4> Confirmation of bone resorption inhibition effect

In order to evaluate the bone resorption inhibition effect of the ethanol extract of Pilbal (PF of Example 1) according to the present invention, pit formation inhibition ability analysis was performed.

Cultured as in the above example, but 24 hours after dispensing RAW 264.7 cells on an osteo assay surface multiple well plate, RANKL (100 ng/ml) and PF 12.5, 25, 50, or 100 μg/ml were added to the culture medium. and cultured for 5 days. It was stabilized by replacing it with the added culture medium. After differentiation was completed, cells were removed by treatment with 4% NaCIO. The plate was washed with distilled water, and the pits formed by osteoclasts were observed using an inverted microscope to analyze the absorbed pit area. Pit area was measured using Image J software Ver. It was measured using 1.46 (National Institutes of Health, Washington D.C., USA), and the pit area was expressed as a percentage of the total area.

As a result, the pit area in the control group treated only with RANKL increased compared to the normal group, but in the experimental group treated with PF, the pit area decreased in a concentration-dependent manner, confirming that PF inhibits the formation of pits that indicate bone resorption. (Figure 3).

<Example 5> Confirmation of the effect of suppressing the expression of osteoclast differentiation transcription factors

The effect of the Philbal ethanol extract (PF of Example 1) according to the present invention on the protein expression of NFATc1 and c-Fos, which are key mechanisms of osteoclast differentiation, was confirmed by the following method.

As in the above example, the same amount of RAW 264.7 cells was distributed in 60π dishes, and after 24 hours, the medium was replaced with medium containing RANKL (100 ng/ml) and PF 12.5, 25, 50, or 100 μg/ml, and incubated for 1 day. Cultured. Afterwards, the supernatant of the cultured cells was removed, washed with DPBS, and then washed with RIPA buffer (50 mM Tris.Cl, 150 mM NaCl, 1 % NP-40, 0.5% Na.deoxycholate, and 0.1% SDS) to dissolve the protein and extract the protein from the cells.

The extracted protein was quantified using the BCA protein assay, then electrophoresed on a 10% polyacrylamide/SDS gel for detection by primary and secondary antibody reactions through western blot analysis, and then transferred to a nitrocellulose transfer membrane. Afterwards, the membrane was blocked with 5% skim milk dissolved in TBST (0.05% Tween-20 in Tris-buffered saline, pH 7.4), reacted with anti-NFATc1 antibody and anti-c-Fos antibody, and then reacted with secondary antibody. I ordered it. Next, the ECL solution (Santacruz, California USA) was mixed well at a ratio of 1:1 and applied on the membrane to cause expression (color development) and development on an X-ray film to confirm the level of protein expression. The protein expression levels of NFATc1 and c-Fos were quantified using actin, a housekeeping gene.

As a result, the expression of NFATc1 and c-Fos proteins in the control group treated with RANKL increased compared to the normal group, and the expression of NFATc1 and c-Fos in the experimental group treated with PF was confirmed to be significantly decreased compared to the control group ( Figure 5A). When this was quantified using actin, the expression of NFATc1 and c-Fos in the experimental group treated with PF was found to be significantly suppressed at all concentrations (p<0.05, p<0.01) (Figure 5B).

Claims (15)

A composition for inhibiting bone loss containing Piperis Longi Fructus extract extracted with 100% ethanol as an active ingredient.
The composition inhibits the expression of NFATc1 (nuclear factor of activated T cell c1), c-Fos, and TRAP (tartrate-resistant acid phosphatase), which are proteins or genes related to osteoclast differentiation. It inhibits the expression of NFATc1 (nuclear factor of activated T cell c1), c-Fos, and TRAP (tartrate-resistant acid phosphatase).
A composition characterized in that the Pilbal extract extracted with 100% ethanol is extracted by mixing Pilbal and 100% ethanol at a ratio of 1:10 (w/v)%. delete delete delete delete According to clause 1,
A composition for inhibiting bone loss, wherein the extract extracted with 100% ethanol inhibits the RANK signaling pathway. delete delete delete delete According to clause 1,
A composition for inhibiting bone loss, wherein the bone loss is caused by hyper-differentiation or hyper-activation of osteoclasts. delete delete A food composition for inhibiting osteoclast differentiation or bone resorption containing Piperis Longi Fructus extract extracted with 100% ethanol as an active ingredient.
The composition inhibits the expression of NFATc1 (nuclear factor of activated T cell c1), c-Fos, and TRAP (tartrate-resistant acid phosphatase), which are proteins or genes related to osteoclast differentiation. It inhibits the expression of NFATc1 (nuclear factor of activated T cell c1), c-Fos, and TRAP (tartrate-resistant acid phosphatase).
A composition characterized in that the Pilbal extract extracted with 100% ethanol is extracted by mixing Pilbal and 100% ethanol in a ratio of 1:10 (w/v)%. In vivro, comprising treating an extract of Piperis Longi Fructus extracted with 100% ethanol,
It inhibits the expression of NFATc1 (nuclear factor of activated T cell c1), c-Fos, and TRAP (tartrate-resistant acid phosphatase), which are proteins or genes related to osteoclast differentiation, and NFATc1 (nuclear factor), a protein or gene related to osteoclast differentiation. As a method of suppressing the expression of factor of activated T cell c1), c-Fos, and TRAP (tartrate-resistant acid phosphatase),
The method wherein the Pilbal extract extracted with 100% ethanol is extracted by mixing Pilbal and 100% ethanol at a ratio of 1:10 (w/v)%.