KR20130070901A - Composition for treating or preventing bone disease comprising extract from rumex crispus l. as active ingredient - Google Patents

Abstract

PURPOSE: A composition for preventing and treating bone diseases is provided to reduce differentiation or activation of osteoclast, to suppress bone resorption, and to promote differentiation of osteoblast and osteogenesis. CONSTITUTION: A composition for preventing or treating bone diseases contains a Rumex crispus L. extract as an active ingredient. The extract is prepared using a solvent selected from the group consisting of water, hydrous or anhydrous C1-C4 alcohol, acetone, ethyl acetate, butyl acetate, dichloromethane(CH_2Cl_2), chloroform(CHCl_3), hexane, and 1,3-butylene glycol. Bone diseases are bone damage, osteoporosis, osteomalacia, richets, osteitis fibrosa, aplastic bone disease, or metabolic bone disease. The extract promotes differentiation of osteoblast. A pharmaceutical composition for preventing or treating bone diseases contains a pharmaceutically effective amount of the composition and a pharmaceutically acceptable carrier.

Description

Composition for Treating or Preventing Bone Disease Comprising Extract From Rumex crispus L. As Active Ingredient}

The present invention relates to a composition for the treatment, prevention or improvement of bone diseases comprising the extract of the extract.

Bones in our body are one of the most dynamic tissues that play an important role in controlling homeostasis of calcium and phosphorus in the body as well as physical support and protection of vital organs. Bones also undergo a continuous remodeling process in which bone is absorbed and removed by osteoclasts and subsequently new bone is formed by osteoblasts. The bone resorption process takes about 10 days during the bone remodeling process, after which the osteoblasts move, attach, proliferate, and differentiate to form bones for about 3 months (1). This remodeling process causes bone formation to grow rapidly during growth, resulting in a steady increase in bone mass, resulting in skeletal maturation, but as the body ages, the progression of bone absorption gradually increases. Loss, resulting in bone disease such as osteoporosis. Osteoporosis is a disease that is characterized by a decrease in bone mass, degeneration of bone tissue and abnormal microstructures, weakening bone strength and increasing the risk of fractures (2). In 2000, it increased by 25% to 1.6 million (3). After 2050, osteoporosis is expected to increase more than three times due to an increase in the elderly population. Osteoporosis is one of the major health problems at home and abroad. (4). Smoking (5), hypertension (6), and diabetes (7), which are known as risk factors for osteoporosis, are factors that increase oxidative stress. Recently, it has been found that oxidative stress and osteoporosis are closely related (8, 9). Several studies are underway. Garrett IR et al. (10) reported that free radicals influence bone absorption and osteoclast formation in vitro and in vivo. Furthermore, free radicals are known to increase osteoclast activity through the activity of NF-κB, a transcription factor of oxidative stress response, resulting in increased bone resorption (11). Reactive Oxygen Species (ROS) have also been reported to increase osteoclast activity and inhibit osteoblast metabolism (10,12). In women with osteoporosis, the concentration of antioxidants in the blood was reduced (13), and studies of high bone mineral density were reported with the intake of antioxidant vitamins (14). Taken together these reports suggest that the antioxidant content affects the efficacy of osteoporosis. Currently, calcium supplements, vitamin D metabolites and thiazide diuretic therapy, calcitonin therapy, bisphosphonates therapy, and estrogen therapy are used to treat osteoporosis. Among them, inhibition of osteoclast function and reduction of bone resorption are mainly used for the treatment of osteoporosis, and the most commonly used drug is bisphosphonate (15). Such bisphosphonate drugs are known to have a mechanical inhibitory effect on promoting apoptosis of osteoclasts and osteoclasts to perform bone resorption on the surface of bone resorption (16), and also to inhibit osteoclast differentiation. It was reported (17). However, despite the known effects on osteoporosis, these drugs are known for serious side effects such as mandibular necrosis (18), and efforts to find alternative drugs without side effects continue. In addition, there is no drug that can completely restore the reduced bone mass, so osteoporosis is more important than prevention (19).

On the other hand, osteoblasts, cells that form bones in our body, exist in the bone marrow and are mesenchymal stem cells (mesenchymal), which are precursor cells that can differentiate into adipocytes, chondrocytes, and myocytes. stem cell). Differentiation of mesenchymal stem cells into osteoblasts is initiated by BMP (bone morphogenetic protein) and is affected by the numerous bone matrix proteins that make up the bone matrix. These bone matrix proteins are specifically expressed at different stages of differentiation. do. Progenitor bone cells secrete bone-specific alkaline phosphatase (ALP) after cell proliferation stops, and the most essential bone matrix protein for type I collagen is 85-90% of bone protein. Proteoglycans (proteoglycans), osteocalcin (osteocalcin), osteopontin (osteopontin), osteonectin (osteonectin), BSP (bone sialro protein) will be synthesized. Activated osteoblasts, through the synthesis of enzymes such as ALP, carry and accumulate highly organized hydroxyapatite crystals of calcium and phosphoric acid salts on organic bone substrates to achieve bone formation. Therefore, ALP activity of osteoblasts has been used as an efficient assay for osteoblast differentiation (20). Osteoblasts are also responsible for regulating the activity of osteoclasts through the release of several cytokines (21). Osteoclasts are the major cells that break down bone during bone metabolism and are multinucleated giant cells with up to 50 nuclei. It also adheres to the surface of the bone and secretes various enzymes and acids, which play an important role in calcification and absorption of bone (22). Osteoclasts are differentiated by stimulation of cytokines secreted from osteoblasts. According to Takami et al. (23), the receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage-colony stimulation factor (M-CSF) are osteoblasts. It has been reported to induce differentiation of osteoclasts in the absence of environment. Therefore, RANKL and M-CSF are important factors in the differentiation process of osteoclasts. Recently reported studies show that RANKL is an important factor not only in osteoclast differentiation but also in function, survival, osteoclast fusion and resorption. It can be seen that (24).

Rumex crispus L. is a perennial herb that belongs to Polygonaceae and grows in all parts of Korea. It grows well in wet places such as fields, field banks and streams. It is said that the leaves of Pleurotus larvae are called yangjeyeop, and yangjeopyeop is good when it is attached to thin areas or boils. It has a sweet taste, slippery properties, and is cold and poisonous. In addition, in the private sector, young shoots are eaten with fresh juice or used for food, and are known to be effective for intestinal constipation, swelling, indigestion and intestinal bleeding. It is reported that quercus leaves contain quercitrin, a type of flavonoid, and quercitrin has effects on diuretic and capillaries, which are effective in atherosclerosis and hypertension (25). However, there have been few reports of the effects of Pleurotus eryngii on bone metabolism regulation and other physiological functions.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop substances for treating and preventing bone loss diseases from natural products that are recognized as safe for the human body. As a result, Rumex crispus L. extract promotes osteoblast differentiation, while inhibiting osteoclast differentiation and activity, thereby developing a functional food material for improving bone function as well as treating and preventing bone loss diseases. The present invention was completed by confirming that there is a possibility.

Accordingly, it is an object of the present invention to provide a composition for the prevention or treatment of bone diseases, including the extract of the extract.

In addition, another object of the present invention is to provide a food composition for improving bone diseases comprising the extract of the extract.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the invention is a Rumex Provided is a composition for preventing or treating bone diseases comprising crispus L.) extract as an active ingredient.

The active ingredient of the present invention " Rumx ( Rumex crispus L.) ”is a perennial plant belonging to Polygonaceae and grows in all parts of Korea. It grows well in humid places such as fields, field banks and streams. It is said that the larva leaves are called yangjeopyeop, and yangjeopyeop is good when it is attached to thin areas or boils. It is sweet and tastes slippery and cold and poisonous. In addition, in the private sector, young shoots are eaten with fresh juice or used for food, and are known to be effective for intestinal constipation, swelling, indigestion, and intestinal bleeding. It has been reported that quercus leaves contain a kind of flavonoids, quercitrin, and quercitrin, which is effective in diuretic and capillary strengthening, and is effective in atherosclerosis and hypertension.

As used herein, the term “lander extract” refers to an extract obtained from various organs or parts of the lander (eg, leaves, flowers, stems, etc.), preferably an extract obtained from the ground, most preferably the leaf. it means.

The composition of the present invention contains the extract of the oleander as an active ingredient. As used herein, the term 'extract' is used to refer to a tortoise, as well as the extraction result obtained by treating the extraction solvent in the tortoise, as well as the tortoises formulated (eg, powdered) to be administered to the animal itself. Also has a meaning to include.

In the case of extracting the extract from the extract used in the composition of the present invention by treating the extract with a extract, various extracting solvents may be used. Preferably, a polar solvent or a nonpolar solvent can be used. Suitable polar solvents include (i) water, (ii) alcohols (preferably methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxyethanol Or ethylene glycol), (iii) acetic acid, (iv) dimethylformamide (DMFO) and (v) dimethyl sulfoxide (DMSO). Suitable nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- But are not limited to, pentane, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2- chloropropane, toluene, 1- chloropropane, chlorobenzene, benzene, diethyl ether, diethylsulfide, Methane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF.

More preferably, the extraction solvent used in the present invention is (a) water, (b) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) the lower alcohol and water Mixed solvent with (d) acetone, (e) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3-butylene glycol, (i) hexane and (j) diethyl ether Include. Most preferably, the extract of the present invention is obtained by treating water, methanol, ethanol or a combination thereof with the ox.

As used herein, the term " extract " means that it is used in the art as a crude extract as described above, but broadly includes fractions obtained by further fractionating the extract. In other words, the extract is not only obtained by using the above-described extraction solvent, but also includes a further obtained by applying a purification process. For example, the extract may be further extracted and fractionated using the above-mentioned solvent, a fraction obtained by passing an ultrafiltration membrane having a constant molecular weight cut-off value, and prepared by various chromatography (size, charge, hydrophobicity or affinity). Fractions obtained through various purification methods additionally carried out, such as by separation).

The extract of the extract used in the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze drying or spray drying.

According to a preferred embodiment of the present invention, the extract is promoted osteoblast differentiation.

According to one specific embodiment below, the extract of the present invention is the expression of alkaline phosphatase, Runx 2 (Runt-related transcription factor 2), and type 2 collagen (type II collagen) in osteoblasts Increase the secretion of osteocalcin (OCN).

According to another preferred embodiment of the present invention, the extract is suppressed the activity or differentiation of osteoclasts.

According to one specific example below, the extract of the present invention inhibits the differentiation of osteoclasts induced by RANKL (receptor activator of NFκB ligand).

Extract of the extract of the present invention promotes the differentiation of osteoblasts, and by inhibiting the differentiation and activity of osteoclasts to promote the formation of bone, and through this action can treat or prevent various bone diseases.

According to a preferred embodiment of the present invention, examples of bone diseases that can be prevented or treated by the present invention include bone damage caused by bone metastasis of cancer cells, osteoporosis, osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease, Metabolic bone disease, osteolysis, leukopenia, malformations of bone, hypercalcemia or neurocompression syndrome.

In particular, the composition of the present invention shows an excellent effect in the prevention and treatment of osteoporosis. Osteoporosis can be divided into primary (primary) osteoporosis and primary (secondary) osteoporosis. Primary osteoporosis refers to osteoporosis, which can occur mainly in women and older men after menopause, inadequate intake of calcium and vitamin D, inadequate physical exercise, smoking, or smoking. Secondary osteoporosis means that osteoporosis is caused by certain diseases or drugs. In this case, osteoporosis can be induced in young people, and the bone strength decreases and the incidence of fracture increases in proportion to the severity of the disease or drug and the duration of exposure. Diseases include hyperthyroidism, parathyroidism, Cushing's syndrome (adrenal cortical hormone hypersecretion), early menopause, menopause caused by artificial surgery (ovarian resection), hypogonadism, chronic liver disease (cirrhosis), rheumatoid arthritis, chronic renal failure , Gastrectomy, etc., and drugs include steroids (corticosteroids), anticonvulsants (epilepsy drugs), heparin, and the like.

The composition of the present invention shows an excellent effect in the prevention and treatment of bone diseases caused by bone metastasis of cancer cells or cancer tissues. For example, the cancer cells or cancer tissue may be gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, rectal cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, cervical cancer, endometrial cancer, brain cancer, prostate cancer, bone cancer , But not limited to, skin cancer, thyroid cancer, leukemia, non-Hodgkin lymphadenopathy, parathyroid cancer, and ureter cancer cells or tissues.

According to an embodiment of the present invention, the composition of the present invention exhibits the effect of reducing the formation of osteoclasts and inhibiting bone resorption and bone destruction by osteoclasts.

Osteoclasts are cells produced from blood cells (hematopoietic stem cells), and have a function of destroying bones when calcium lacks in blood or when bone remodeling is required. In addition, the term "bone uptake" as used herein means that osteoclasts break down bone by secreting strong acid and protease into bone.

As demonstrated in the specific examples below, the extract of the present invention is an alkaline phosphatase, Runx 2 (Runt-related transcription factor 2), and type 2 collagen (type II collagen) in osteoblasts. ) And increase the secretion of osteocalcin (OCN). Alkaline phosphatase is a variety of isoenzymes (enzymes of the same function but slightly different structure), which are normally present in various parts of the body (especially bone and liver). It is present in osteoblasts, which form bones, and helps in diagnosing bone diseases and determining therapeutic effects. Especially in all cases where bone formation is high (for example, bone formation is promoted for recovery during bone fracture by fractures and tumors), osteoblast activity is increased, and alkaline phosphatase, an enzyme of osteoblasts, is increased. Activity is also increased. Osteocalcin (BGP) is a vitamin K dependent calcium binding protein with molecular weight of approximately 5,900 Da consisting of 49 residues of gamma-carboxylglutamate (Gla) and containing 2-3 residues of gamma-carboxylglutamate (Gla). 90% of the bone's organic components are collagen and the remaining 10% are non-caustic factors such as osteocalcin, osteonectin, α-HS glycoprotein, cyanoprotein, and fetuin. It is a collagen protein. Osteocalcin in bone plays an important physiological role in bone metabolism, such as regulating local coalification or controlling the movement of Ca2 + between bone and body fluids.

The composition of the present invention may be prepared as a pharmaceutical composition.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of the extract of the present invention described above; And (b) a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve the efficacy or activity of the above mentioned extract.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. It is not limited. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably administered orally.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Typical dosages of the pharmaceutical compositions of the present invention are in the range of 0.001-1000 mg / kg on an adult basis.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

The composition of the present invention can be provided as a food composition.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a food-effective amount of the extract of the present invention described above; And (b) a pharmaceutically acceptable carrier.

When the composition of the present invention is made of a food composition, as an active ingredient, as well as extracts from the extract, as well as components commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavors Include the first. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings. For example, when the food composition of the present invention is prepared as a drink, in addition to citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, tofu extract, jujube extract, licorice extract, etc. Can be.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention provides a composition for the prevention and treatment of bone diseases using extracts.

(Ii) The composition of the present invention exhibits great efficacy in reducing osteoclast (osteoclast) differentiation or activity to inhibit bone resorption, promoting osteoblast differentiation and promoting bone production.

(Iii) The present invention provides basic data as medicines and foods of the extracts of the extract of Pleurotus japonica, which have the efficacy of preventing and treating bone diseases caused by osteoporosis and bone metastasis of cancer cells.

1 is a Rumex using various solvents crispus L.) shows the process of extracting and fractionating the powder.
2 is a Rumex crispus L.) Ethanol extracts show cell viability and ALP activity in MC3T3-E1 cells. ALP activity was measured by extracellular enzymes. MC3T3-E1 cells were seeded at a density of 70-80% in 96-well plates and incubated for 1 day in α-MEM containing 10% FBS, vitamin C (100 μg / mL) and β-GP (5 mM). ALP activity was normalized to total protein. Viable cells were measured by MTT assay. MC3T3-E1 cells were plated and incubated for 48 hours in the presence or absence of different concentrations of white ethanol extract (5-20 μg / mL). Data are expressed as mean ± SD, percentage or fold over control. * p < 0.05, ** p < 0.01, *** p < 0.001.
FIG. 3 shows the effect of various fractions of the extract of Pleurotus on cell viability and ALP activity in MC3T3-E1 cells. ALP activity was measured by extracellular enzymes. MC3T3-E1 cells were seeded at a density of 70-80% in 96 well plates and incubated for 1 day in α-MEM containing 10% FBS, vitamin C (100 μg / mL) and β-GP (5 mM). ALP activity was normalized by total protein. Viable cells were measured by MTT assay. MC3T3-E1 cells were plated and incubated for 48 hours in the presence or absence of different concentrations of white ethanol extract (1-10 μg / mL). Data are expressed as mean ± SD, percentage or fold over control.
FIG. 4 shows the effect of a white ethanol extract on ALP of extracellular matrix in MC3T3-E1 cells. Cells were incubated in α-MEM containing 10% FBS, 100 μg / mL Vitamin C and 5 mM β-GP.
Figure 5 Rumex in MC3T3-E1 cells The result of staining for collagen is shown after 15 days treatment of crispus L.) extract. Cell matrix collagen was stained by Van Gieson's staining method. Data are expressed as mean ± SD, percentage relative to control. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 6 is the result of measuring the effect of the extract ethanol ethanol extract on osteocalcin secretion in MC3T3-E1 cells. Cells were treated for 20 days at various concentrations of extracts of 1, 5, 10, 20 μg / mL. Data are expressed as mean ± SD, percentage relative to control. * p < 0.05, ** p < 0.01, *** p < 0.001.
7 shows Rumex during differentiation of MC3T3-E1 osteoblasts. ALP (alkaline phosphatase), Runx-2 and type 2 collagen (type II collagen, T2C) mRNA levels were measured by treatment with crispus L. ethanol extract. GAPDH was used as an internal control. Data are expressed as mean ± SD, percentage relative to control. * p < 0.05, ** p < 0.01, *** p < 0.001.
8 shows Rumex during differentiation of MC3T3-E1 osteoblasts. Crispus L.) After treatment with ethanol extract, the expression level of BSP (bone sialo protein) protein was measured. β-actin was used as an internal control. Data are expressed as mean ± SD, multiples for control. * p < 0.05, ** p < 0.01, *** p < 0.001.
9 is a result of measuring the effect of the extract of ethanol ethanol on the proliferation and TRAP activity of mouse bone marrow-derived osteoclasts. Panel A shows the result of 48 hours of mouse bone marrow-derived osteoclasts with 10-40 μg / mL of the extract of ethanol. Panel B shows the results of incubating the mouse bone marrow-derived osteoclasts for 72 hours with an extract of ethanol, RANKL (100 ng / mL), and M-CSF (50 ng / mL). Data are expressed as mean ± SD, percentage relative to control. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 10 is the result of measuring the effect of the extract of ethanol ethanol extract on RANKL-induced osteoclast formation in mouse BMMs. Mouse BMMs were incubated with M-CSF (50 ng / mL) and RANKL (100 ng / mL) in the presence or absence of the extract. Cells were fixed and stained for TRAP.
Figure 11 is a result of measuring the effect of the extract from the extract of the mouse bone marrow-derived osteoclasts on the proliferation and TRAP activity. Panel A shows the results of incubation of mouse bone marrow-derived osteoclasts with 10-40 μg / mL of the subterranean fraction. Panel B shows the results of incubation of mouse bone marrow-derived osteoclasts with the larvae fraction, RANKL (100 ng / mL) and M-CSF (50 ng / mL) for 72 hours. Data are expressed as mean ± SD, percentage relative to control. * p < 0.05, ** p < 0.01, *** p < 0.001.
12 shows the results of measuring the effect on the formation of RANKL-induced osteoclasts in mouse BMMs of the ethyl acetate acetate fraction. Mouse BMMs were incubated with M-CSF (50 ng / mL) and RANKL (100 ng / mL), in the presence or absence of a white rat ethyl acetate fraction. Cells were fixed and stained for TRAP.
FIG. 13 shows the results of measuring the effect of fraction of ethyl acetate acetate on phosphorylation of MAPKs in RANKL signaling. Raw cells were pretreated with the ethyl acetate fraction of the shooter and stimulated with RANKL (100 ng / ml) for the designated time. Cell lysates were analyzed by western blotting with antibodies to c-fos, NFATc1, JNK, p-NF-κB, ERK and actin. Data are expressed as mean ± SD expressed in fold over the control group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

1. Reagents and Instruments

1.1. reagent

Ethanol, n-hexane, chloroform, ethyl acetate, and n-butanol, which are solvents used for sampling and fractionation, were prepared from JTBaker (Mallinckrodt Baker Inc., Philipsburg, USA). Α-MEM (alpha-minimum essential medium), antibiotics, trypsin-EDTA, and FBS (fetal bovine serum) used for cell culture were purchased from Gibco BRL (Gibco BRL, Grand Island, NY, USA). MTT [3- (4,5-di-methylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] reagent was used as Amresco (Amresco, Solon Ind. Ohio, USA). Reagents used for ALP enzyme measurement and staining were all prepared from Sigma Corporation (Sigma Chem. Co., St. Louis. Mo., USA), using reagents. The osteocalcin sandwich ELISA kit was prepared by Biomedical Technologies ( Biomedical Technologies Inc., MA, USA). Primers used in PCR experiments were obtained from Bioneer (Bioneer. Co., NY, USA), and reagents used to measure RNA content extraction were Trizol solution (Molecular Research Center, Inc. Ohio, USA), 1-bromo- 3-chloropropane was used by Sigma, RT-PCR measurement was used by RT premix (Bioneer. Co., NY, USA), DNA ladder purchased Promega (Promega Co., Madison, WI, USA) It was. Trisma base (trizma base), ethylenedinitrilotetraacetic acid disodium salt (EDTA), triton X-100, TEMED (N, N, N ', N'-tetra-methyl-ethylenediamine) used in immunoblotting analysis, Acrylamide, sodium dodecylsulfate (SDS), ammonium persulfate (APS), Tween-20, and bicinchoninic acid kit (BCA kit) are all available from Sigma (Sigma Chem. Co., St. Louis.Mo., USA). ECL detection kit was purchased from Amersham (Amersham Bioscience., Buckinghamshire, England). And immobilon-P transfer membrane was purchased from Millipore (Millipore, Billerica., Billerica Massachucetts, USA), BSP antibody was Abcam (Abcam plc 332 Cambridge Science Park., UK), anti-mouse IgG and anti-rabbit IgG Was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). RANKL and M-CSF, reagents used for osteoclast differentiation, were purchased from Peprotech (Peprotech Inc., NJ, USA).

1.2. device

The main instruments used in the experiments were rotary vacuum evaporators (Buchi, R-3000, Germany), microplate spectroscopy (Spectra max 340 pc, Molecular Device, USA), CO ₂ incubator (Model 3154, Forma Scientific, USA), PCR instruments ( Mygenie96, Bioneer, USA), mini-protein electrophoresis system (Bio-Rad Co., USA), power supply (Bio-Rad Co., USA), Michaeloplate shaker (Finemixer SH2000, FINEPCR, Korea) It was.

2. Sample Preparation

2.1. Whiter extract manufacturer

The leaves of Pleurotus used in the experiment were purchased from the dried state at the Yangnyeong market in Daegu. First, the cells were left incubated for 24 hours with 70% ethanol of 10 times (w / v) of the sample weight and extracted three times. The extract was filtered twice using filter paper (Whatman No. 3, England), and the supernatant was concentrated on a rotary vacuum evaporator (Buchi, R-3000, Germany) at 55 ° C. and then lyophilized to use as an ethanol extract.

2.2. Phylogenetic Fraction using Extract

As shown in FIG. 1, lyophilized Rumex crispus L. ethanol extract was dissolved in 20 times (w / v) of distilled water, and then n-hexane (hexane), chloroform (CHCl ₃ ), ethyl acetate (EtOAc) and butanol. (BuOH) was repeatedly extracted three times in sequence and fractionated for each solvent. The remaining aqueous layer was concentrated, concentrated under reduced pressure with a water fraction, and the remaining organic solvent was evaporated in vacuo and stored at -20 ° C for use in experiments.

3. Determination of Antioxidant Activity

3.1. Total polyphenol content measurement

The total polyphenol content was determined from the standard curve using tannnic acid using the Folin-Denis method (26). That is, 1 mg of each sample was dissolved in 1 mL of distilled water, and 2 mL of 2-fold diluted dilute folin was added to 2 mL of 10-fold dilution. After mixing well, the mixture was left for 3 minutes and then 10% Na ₂ CO ₃ 2 After adding the mL and reacting for 1 hour, the content was determined from the standard curve prepared by measuring the absorbance at 700 nm using a microplate spectrometer (Spectra max 340 pc, Molecular Device, USA).

3.2. DPPH (α-α-Diphenyl-β-picrylhydrazyl) radical scavenging activity

The free radical scavenging activity of the samples was measured by a partial modification of the Blois (27) method, and was measured by reducing the DPPH, a stable radical.The samples were dissolved in 99% methanol and diluted in concentrations of 160 μL and 0.15 mM in methanol. 40 μL of a DPPH solution was added thereto, and the mixture was left at room temperature for 30 minutes, and then absorbance was measured at 517 nm. The free radical scavenging activity of each sample extract was expressed as a percentage of the radical scavenging ability of the sample with respect to the control, using 99% methanol dissolved in the sample, and also required to reduce the absorbance of the control without adding the sample to 1/2. It is represented by the RC ₅₀ value, which is the concentration of. BHA was used for activity comparison.

3.3. ABTs radical cation decolorization  Active measurement

Antioxidant activity was measured by ABTs radical cation decolorization method (28). A mixture of 7 mM 2,2'-azino-bis- (3-ethylbenzothiazoline-6 -sulfonic acid) diammonium salt (ABTs) and 2.4 mM potassium persulphate was left in the cow for at least 12 hours to remove cyan ABTs radicals. After formation, the radical stock solution is diluted with PBS (phosphate buffer saline, pH 7.4) using an extinction coefficient such that the absorbance value is 0.07 (± 0.02) at 732 nm. After adding 0.01 mL of each sample solution prepared for each concentration to 0.9 mL of this solution and reacting at room temperature for 1 minute, the absorbance change of the reaction solution was measured at 732 nm. The free radical scavenging activity of each sample was RC ₅₀ , the concentration of the sample necessary to reduce the absorbance of the control group without the sample to 1/2. Respectively.

4. whiter extract and Fraction  goal Formability  Measure

4.1. Cell culture

Mouse calvaria osteoblastic cells, MC3T3-E1 cells, were distributed from ATCC (CRL-2593, USA) and used. 10% FBS and 1% antibiotics were added to the α- MEM medium and passaged every 2-3 days in a 37 ° C., 5% CO ₂ incubator, and 5 mM β -glycerol phosphate (Sigma, St. Louis, Mo, USA) and 100 μg / mL of vitamin C (Sigma, St. Louis, Mo, USA) were used as the differentiation induction medium, and the medium was changed every three days (29).

4.2. Cell proliferation measurement

MTT [3- (3,4-dimethyl-thiazolyl-2) -2,5-diphenyl tetrazolium bromide] analysis was performed according to the method of Green et al. (30) to measure cell proliferation and toxicity. MTT analysis is based on the principle that the yellow water-soluble substrate MTT is reduced to insoluble purple formazan by mitochondrial dehydrogenase action. The absorbance of the formed formazan is alive and metabolized. Reflect the concentration of the cell. Osteoblasts were cultured cells by measuring the cell number by 0.4% trypan blue staining method, and then divided into 200 μL in 96-well plate at a concentration of 1 × 10 ⁴ cells / well, and cultured for 24 hours. Removed. After adding 180 μL of fresh α-MEM medium, 20 μL of each concentration sample was added to each well. The sample was added to the culture so that the final concentration of the sample was 0, 5, 10, 20 μg / mL and incubated for 48 hours. After incubation, 10 μL of MTT reagent was added to each well at a concentration of 5 mg / mL and incubated for 4 hours. After the incubation, the supernatant was removed, and formazan crystals were dissolved by adding 100 μL of DMSO to each well, and the absorbance was measured at 550 nm using an ELISA reader (Spectra MAX M2, Molecular Device, USA). Cytotoxicity indicated the absorbance of the sample as a percentage of the absorbance of the control.

4.3. ALP ( Alkaline 포스화제 ) Osteoblast activity screening by activity assay

1 × 10 ⁴ MC3T3-E1 cells were cultured Cells / well were adjusted to 96-well plates, incubated for 24 hours, exchanged with differentiation-inducing media, and sampled. The sample extract was treated at each concentration, cells were cultured by day, washed with PBS, and then dissolved in 37 ° C. for 30 minutes by adding 20 μL of 0.1% Triton X-100. 5 μL of the supernatant of the lysed cells was used for protein quantification, and 20 μL of 0.1 N glycine and 10 μL of 100 mM ρ- NPP ( ρ- nitrophenylphosphate) were added to the remaining supernatant, followed by 37 ° C for 30 minutes. Reacted. After the reaction, the reaction was stopped with 200 μL of 0.1 N NaOH, and the absorbance was measured at 405 nm. ALP activity was determined by measuring ρ -NP ( ρ -nitrophenol) generated from ρ -NPP to prepare a standard graph for ρ -NP, and activity was derived through relative comparison with the control group (29).

4.4. ALP  Osteoblast activity test by staining method

To measure the degree of staining of the ALP enzyme, 24 wells were treated with 1 × 10 ⁵ cells / mL and the extract was treated with an appropriate concentration to measure enzyme activity over time. Staining was performed using an alkaline phosphatase kit (Sigma Chemical Co., USA). In other words, the medium was removed and stored at room temperature for about 30 seconds using a fixed solution (citrate-acetone-formaldehyde), washed with distilled water for 45 seconds, and then prepared diazonium solution (sodium nitrite: FRV-alakline). : Naphthol AS-BI alkaline solution = 1: 1: 1), incubated at room temperature for about 15 minutes, washed with distilled water for 2 minutes, dyed for 2 minutes with hematoxylin solution and stained with running water. The solution was washed thoroughly and observed under a microscope to confirm the staining site (31-33).

4.5. Collagen Content Search

Collagen synthesis capacity of osteoblasts was measured using a sirius red based colorimetric assay according to the method of Tullberg-Reinert et al. (34). First, the cultured osteoblasts were washed with PBS and fixed with bouin fluid for 1 hour. Thereafter, washed with PBS, dried, shaken for 1 hour with sirius red dye reagent, stained, washed with 0.01 N HCl, and dissolved by adding 200 μL of 0.1 N NaOH, followed by ELISA reader (Spectra MAX M2, Molecular Device). , USA) was used to measure the absorbance at 550 nm. At this time, the collagen content was expressed as a percentage of the control.

4.6. Osteocalcin  Secretion measurement

In order to measure the production of osteocalcin, a characteristic protein marker of osteoblasts according to the proliferation of the cells, it was tested using a sandwich ELISA assay kit. That is, MC3T3-E1 cells were dispensed into 96-well plates at 1 × 10 ⁴ cells / well and treated with differentiation induction medium and samples after 24 hours. In a well coated with osteocalcin, 25 μL of culture medium and 100 μL of osteocalcin antiserum were reacted for 24 hours, followed by washing with 300 μL / well phosphate-saline washing buffer. 100 μL of streptavidin-horseradish peroxidase reagent was added to the washed wells and reacted for 30 minutes. After washing, the mixture was mixed with TMB solution and hydrogen peroxide solution in a 1: 1 ratio, and then 100 μL was added thereto. Absorbance was measured.

4.7. RT - PCR Investigation of Bone Matrix Gene Expression Related to Osteoblast Differentiation

Total RNA was isolated using TRI reagent (Molecular Research Center, Inc.) by incubating osteoblasts at 1 X 10 ⁶ cells / mL in 100φ dish. That is, the medium was removed from the cultured cells, washed twice with 10 mL of PBS (pH 7.4), and then treated with TRI reagent. 1 mL of each 1.5 mL tube was added, 200 μL of 1-bromo-3-chloropropane was added, vortexed for 30 seconds, centrifuged (3,000 rpm, 15 minutes, 4 ° C), the supernatant was collected, and transferred to a new tube. The same amount of isopropanol was added thereto and left at -20 ° C for at least 2 hours, followed by centrifugation (13,000 rpm, 15 minutes, 4 ° C), and the supernatant was removed. 75% ethanol was added to the precipitate, followed by distilled water treated with DEPC (diethyl pyrocarbonate), followed by reaction for 10 minutes in a 65 ° C. water bath to dissolve the precipitate. The isolated RNA was quantified and confirmed using a spectrometer and 1% formaldehyde agarose gel electrophoresis. Total RNA 2μg, superscript II RT200 U, 0.5μg oligo dT, 0.5 mM dNTP was added to the reaction for 90 minutes at 42 ℃ and then the reaction was terminated for 10 minutes at 70 ℃. Total RNA was reverse transcribed and the resulting cDNA was amplified by PCR using the primers of Table 1 below. PCR reactions included 2.5 U Takara Ex. Taq polymerase, 2 mM MgCl ₂ and 0.2 mM dNTP were added to carry out the conditions for each primer.

Target gene Fragment size (bp) Oligonucleotides Runx-2 250 F: 5'-CAGGAAGACTGCAAGAAGGCTCTGG-3 '(SEQ ID NO: 1) R: 5'-ACAAGGTGTCACTGCGCTGAAGA-3 '(SEQ ID NO: 2) Type 2 collagen 172 F: 5'-CTCCGGCTCCTGCTCCTCTTA-3 '(SEQ ID NO: 3) R: 5'-GCACAGCACTCGCCCTCCC-3 '(SEQ ID NO: 4) GAPDH 514 F: 5'-TGAGAACGGGAAGCTTGTCA-3 '(SEQ ID NO: 5) R: 5'-GGAAGGCCATGCCAGTGA-3 '(SEQ ID NO: 6)

5. Measurement of Bone Absorption Capacity of Extract

5.1. Isolation of Bone Marrow Cells and Osteoclast Differentiation

Tibia from 4-5 week old ICR mice were harvested, cut at both ends and passed through α-MEM to collect bone marrow cells, and treated with 50 ng / mL macrophage-colony stimulation factor (M-CSF). Time incubation. Unattached cells were washed with α-MEM and then dispensed into 96 wells at 5 × 10 ⁴ cells / well and incubated in α-MEM medium to which 50 ng / mL of M-CSF was added for 3 days. Thereafter, 50 ng / mL of M-CSF and 100 ng / mL of RANKL were treated together, and samples were incubated for 4 days.

5.2. Osteoclast TRAP ( tartrate resistant acid 포스화제 ) activation

TRAP activity was measured according to the method of Hotokezaka et al. (35). 5X10 ⁴ to 96-well Cells were inoculated to cells / well and treated with differentiation factors and samples and incubated for 4 days. The medium was removed, washed with PBS, and then the substrate solution (1.36 mg / mL 4-nitrophenyl phosphate disodium salt, 10 mM tartrate, 50 mM citrate buffer pH 4.6) was added to the cells fixed with 10% formaldehyde. 100 μL was dispensed at 37 ^° C. for 30 minutes. After the reaction, the enzyme reaction solution was transferred to a new plate and the reaction was stopped by 0.1 N-NaOH to measure absorbance at 405 nm. TRAP activity expressed the absorbance of the sample as a percentage of the absorbance of the control.

5.3. Osteocell activity test by TRAP staining

TRAP staining was confirmed according to the method of Hotokezaka et al. (35). First, 100 ml of 1 mg / mL naphtol AS-MX phosphate and N, N-dimethyl-formamide were added to 10 mL of 50 mM sodium acetate buffer containing 50 mM tartrate acid. 45 μL of the staining solution was dispensed into the formaldehyde-fixed cells and left for 30 minutes in a thermostat.

5.4. Investigation of Protein Expression Related to Osteoclast Differentiation by Western Blot Method

Western blot was performed to investigate the effects of the ethanol extract and its fractions on the expression of RANKL-related gene regulators such as c-fos, NFATc1 and JAP, ERK, and MAP kinase and transcription factor NF-κB. RAW 264.7 cells (1 × 10 ⁶ cells / mL) were incubated for 24 hours in a 5% CO ₂ incubator using α-MEM medium in 6 well plates, followed by treatment with samples. After washing the cells with PBS 2-3 times, 1 mL of lysis buffer was added, lysed for 30 minutes-1 hour, and then centrifuged at 13,000 rpm for 10 minutes to remove cell membrane components. Protein concentration was quantified using the Bio-Rad Protein Assay Kit, which standardized BSA (bovine serum albumin). Supernatants centrifuged at 13,000 rpm for 10 minutes at 4 ° C were subjected to SDS-polyacrylamide gel electrophoresis at 125 V using a 10% running gel and a 4.5% stacking gel. Was carried out. The protein isolated by electrophoresis was transferred for 120 minutes at 350 mA using immobilon-P transfer membrane and transfer buffer (20% methanol, 25 mM Tris-HCl, 192 mM glycine). The membrane to which the protein was transferred was blocked with a 5% skim milk solution after confirming the presence or absence of transfer with a fast green solution. In order to examine the expression level of the primary antibody at 4 ° C, the solution was diluted 1: 1000 in a TST solution, reacted for 24 hours, and washed three times with TST. Subsequently, the secondary antibody was reacted for 2 hours and washed three times with TST again. After washing with distilled water and mixing the coloring reagents I and II of the ECL detection kit 1: 1 in a membrane, the mixed solution was applied, developed by exposure to an X-ray film, and the band concentration on the film was observed.

6. Statistical Processing

The experimental results were statistically calculated and the mean and standard deviation were calculated. The significance test between each group was compared with Duncan's multiple range test and Student's t- test of control and extract treatment groups at 5% level using SAS program. If after a statistical p-value less than 0.05 and the p value is less than 0.01, when the p value is less than 0.001 was determined that the statistical significance.

Experiment result

1. The voiceman ( Rumex crispus  Screening of Antioxidant Activity in L.)

1.1. Total polyphenol content measurement

Polyphenol compounds present in plants can act as natural antioxidants. Phenolic compounds are also known as secondary metabolites that are widely distributed in various plants, and are generally water-soluble and flavonoids, including simple phenols, phenolic acids, phenylpropanoids, and quinones. (36). Recently, such phenolic compounds have been reported to be effective in antioxidant (37), anticancer (38), diabetes prevention (39) and the like. The results of measuring the polyphenol content of the extract of ethanol and fractions using tannic acid as a reference are shown in Table 2 below. The ethanol extract was found to be 176.4 ± 5.8 mg / g, and the fractions were in the order of ethylacetate, butanol, hexane, chloroform and water. 703.9 ± 75.6 mg / g, 303.4 ± 28.1 mg / g, 206.8 ± 18.5 mg / g, 181.8 ± 13.8 mg / g and 33.5 ± 0.3 mg / g, respectively. Polyphenol contents of island fern leaves and water thistle leaves in Ulleungdo mountain wild vegetables were 120.7 and 130.2 mg / g, respectively (37). And it was found. In particular, it can be seen that the fraction of ethyl acetate acetate contains the most polyphenol compounds.

Rumex crispus L. Total polyphenols (mg / g) ¹⁾ EtOH extract 176.4 ± 5.8 ²⁾ Hexane fraction 206.8 ± 18.5 Chloroform fraction 181.8 ± 13.8 Ethyl acetate fraction 703.9 ± 75.6 Butanol fraction 303.4 ± 28.1 Water fraction 33.5 ± 0.3

¹⁾ Milligrams of total polyphenol content / g of plants based on tannic acid as standard.

²⁾ Each value is mean ± SD (n≥3)

1.2. DPPH  freedom Radical  Scavenging activity

DPPH is a water-soluble substance with chemically stabilized free radicals. When it meets an antioxidant-active substance, it gives off electrons and the radicals disappear and change color. It is widely used to search for antioxidants from various natural materials (40). The results of DPPH free radical scavenging activity of the extract of ethanol extract and fractions are shown in Table 3 below. In the ethanol extract, the RC ₅₀ value was 13.0 ± 1.1 μg / mL, and the ethyl acetate fraction had the RC ₅₀ value of 2.7 ± 0.2 μg / mL. Higher scavenging ability. In said to have a strong antioxidant activity known rosemary extract was a RC ₅₀ value appeared to 5.1 ± 0.1 μg / mL, curly dock and, like rosemary also ethyl acetate (ethyl acetate) antioxidant in this RC ₅₀ value of 3.2 ± 0.2 μg / mL fraction The ability was the highest. In addition, RC ₅₀ of ascorbic acid, an antioxidant Compared to the DPPH free radical scavenging activity of the larva extract and fractions shown in Table 3 (41) and the value reported at 7.47 ± 0.3 μg / mL, the larva is also thought to have a very high DPPH free radical scavenging ability, It is believed that the upper ethyl acetate fraction showed the highest DPPH free radical scavenging activity due to the high total polyphenol content and various pigments and phenols (42) contained in the ethyl acetate fraction.

¹⁾ Concentration required for 50% reduction of DPPH at 30 min after starting the reaction

²⁾ Each value is mean ± SD (n≥3)

1.3. ABTs  freedom Radical  Scavenging activity

When ABTs and potassium persulfate are left in the cows, ABTs radicals are produced. The antioxidant activity of the extracts eliminates the ABTs radicals, which decolorize the index-specific turquoise. As described above, the ABTs radical decolorization reaction shows the degree of elimination of free radicals already generated by absorbance, and thus the scavenging activity of the ABTs radicals can be measured in a short time because the ABTs radical decolorization reaction is completed in 1 minute. Applicable to all (43). The results of ABTs free radical scavenging activity of the extract of ethanol extract and fractions are shown in Table 4 below. Leechi ethanol extract showed 92% high ABTs radical scavenging activity at a concentration of 50 μg / mL, and RC ₅₀ value was 21.4 ± 1.2 μg / mL. The ethyl acetate and butanol fractions were 11.0 ± 1.7 μg / mL and 10.4 ± 0.9 μg / mL, respectively, and showed the best free radical scavenging ability of ABTs, and the hexane and chloroform fractions also showed good scavenging ability. The RC ₅₀ value of Trolox was 7.3 ± 0.8 μg / mL, indicating that ethyl acetate, butanol fraction and ethanol extract had relatively high ABTs radical scavenging activity. In addition, the results indicate that the other layers except for the water fraction have excellent antioxidant power, and it is estimated that the larvae contain a large amount of various antioxidants. Therefore, the extract of ethanol and ethyl acetate fraction are considered to have a strong antioxidant effect with high total polyphenol content, DPPH free radical and ABTs free radical scavenging ability.

¹⁾ Concentration required for 50% reduction of ABTs at 3 min after starting the reaction

²⁾ Each value is mean ± SD (n≥3)

2. Whiter Extract and Fraction  goal Formability  Measure

2.1. Osteoblast Proliferation and ALP Activity of Extract

MC3T3-E1 osteoblasts derived from the mouse skull (calvaria) have similar metabolic characteristics such as proliferation, differentiation and calcification of osteoblasts in vivo, and especially have a glycoprotein ALP in the cell membrane (4). ALP, which is abundant in bone tissue, is a marker that appears early in the differentiation of osteoblasts and begins to appear in the matrix maturation stage, and is expressed in the late stage of maturation stage. In this experiment, MC3T3-E1 osteoblasts were used to measure the proliferation and differentiation promoting effects of osteoblasts in the rat. In this example, the proliferation and cytotoxicity of osteoblasts were measured using ethanol extracts of the extract, and ALP activity, a differentiation marker enzyme, was measured. As a result of measuring the cell viability, as shown in Figure 2, even in 20 μg / mL concentration of the ethanol extract showed about 90% cell viability experiment was conducted in these ranges do not have toxicity to cells. In addition, to measure the ALP activity of the osteoblast differentiation marker enzyme, asperbic acid (ascorbic acid) and β -glycerol phosphate were diluted in each medium in each medium and mixed with MC3T3-E1 cells. After 14 days of treatment, the activity of ALP increased with the increase of treatment period. In particular, the treatment of D. extract with 2.85 ± 0.38 nmol / mg protein after 9 days treatment with 10 μg / mL concentration The highest ALP activity was observed, and on day 14, ALP activity was decreased to 2.1 ± 0.23 nmol / mg protein (see FIG. 2). The maximum increase in ALP expression at 9 days and the decrease at 14 days after treatment with the ethanol extract of D. chinensis showed the maximum expression of ALP in the late stage of the maturation stage (44). In calcification, ALP activity appears to decrease. Therefore, the extract of ethanol extract was found to have an effect of increasing osteoblast differentiation.

2.2. Osteoblast proliferation and ALP activity

Using the fractions obtained by sequential fractionation of ethanol extract excellent in osteoblast differentiation, the effect on osteoblast growth and the degree of ALP activity were confirmed, and the results are shown in FIG. 3. When osteoblasts were treated with each of n-hexane, chloroform, ethyl acetate, n-butanol and water fractions at concentrations of 1-10 μg / mL, the following experiments were carried out at these concentrations. . As a result of measuring the effect on the ALP activity of osteoblast cells by treatment of each fraction of the larvae, the activity of ALP increased in all treatment groups at 9 days compared to 4 days of treatment period. However, the fractions did not show a significant difference in ALP activity compared to the control. In other words, it was found that the fraction of Pleuritus did not promote osteoblast differentiation more than the Pleurotus ethanol extract. Therefore, it is thought that several components of the extract of the extract are more likely to promote osteoblast differentiation than the single component separated by the fraction.

2.3. Osteoblast Activity by ALP Staining

To measure the degree of staining of the ALP enzyme was measured using an alkaline phosphatate (AP) kit. At this time, Naphthol AS-BI phosphate substrate is hydrolyzed by basic phosphatase present in the tissue to release orthophosphate and naphthol as derivatives. The liberated naphthol immediately binds to the diazonium salt in the reaction solution to form an azo dye, indicating a red enzyme activity site. The red site is observed under a microscope. (23). As shown in FIG. 4, the control of the ALP enzyme incubated with ethanol extract 1-10 μg / mL incubated and then examined the staining degree of intracellular ALP enzyme did not form a lot of red enzyme. On the other hand, it was confirmed that the red spots of the enzyme were formed in the group treated with the sample by concentration, and also showed a tendency to increase the red enzyme by the concentration. Therefore, it was reconfirmed through staining that ethanol extract induces ALP enzyme activity in osteoblasts.

2.4. Collagen Content Measurement

In order to determine whether the ethanol extract of D. julibrissin increases osteoblast differentiation, the collagen synthesis ability of osteoblasts was measured using a sirius red based colorimetric assay. Collagen type 1, the most basic protein for bone matrix proteins, makes up 90% of the total bone matrix and maintains bone elasticity and flexibility, while also supporting structural support. The mineralization of bone refers to the deposition of calcium and minerals in the cell or extracellular matrix, which deposits calcium and phosphorus in the network structure of collagen fibers and mixes with water to form a hard inorganic substance called hydroxyapatite. Collagen is thus used as a major marker for bone formation (1). Therefore, the collagen content was measured by treating the osteoblastic cell line MC3T3-E1 cells with 15 liters of bone matrix formation period. As a result, it was observed that the staining degree of collagen increased significantly when the ethanol extract was treated by concentration, and the collagen content increased by 4 times at the concentration of 10 μg / mL. It was confirmed (Fig. 5). In Korea, essential oils, known to promote osteoblast differentiation, reported a 1.14-fold increase in comparison with the control at a concentration of 10 μg / mL (45) and at a concentration of 200 μg / mL of the extracts. Compared with the report that the collagen content was increased 1.25 times (4), it was found that the collagen content was very high when the ethanol extract was treated.

2.5. Osteocalcin Secretion Measurement

Osteocalcin (OCN), also known as BGP (bone gla protein), is a vitamin K-dependent α-carboxy glutamic acid protein that binds to calcium. OCN also binds to hydroyapatite, a representative mineral in bone, and is a protein specific for bone and dentin, which is produced in osteoblasts and accumulates in the extracellular matrix of bone (26-28). The result of measuring the content of OCN on in vitro by treating the extract from ethanol for 20 days is shown in FIG. 6. As a result, the content of OCN tended to increase when treated with the extract of ethanol, compared to the control without the sample. In addition, the OCN content of 1 μg / mL ethanol extract did not significantly increase compared to the control, but the 5-20 μg / mL treatment showed a significant increase in OCN content. In particular, when the sample was treated at a concentration of 20 μg / mL OCN content of about 1.6 times was confirmed. OCN is not expressed early in osteoblast differentiation and is known to increase rapidly as calcification begins (46). Therefore, it is thought that the treatment of ethanol extract with D. julibrissin for 20 days promoted calcification and increased OCN expression.

2.6. Investigation of Bone Matrix Gene Expression Related to Osteoblast Differentiation by RT-PCR Method

Runx-2 (runt-related transcription factor 2), one of the factors regulating osteoblasts, is known to play an important role in osteoblast differentiation and skeletal formation (47). Runx-2 binds to osteoblast specific element 2 (OSE 2) elements to regulate the expression of genes such as osteoblastin (OCN), osteopontin (OPN) and type II collagen, which are important osteoblast specific genes (44,48). ALP is an enzyme involved in calcium and phosphorus metabolism. It is found in high concentrations in the stromal vesicles of extracellular membranes and calcified tissues and shows optimal activity at basic pH 8-10 (49). The precise function of this enzyme in the calcification process by osteoblasts is not clear, but it has been reported to act as a trigger for calcification by hydrolyzing organic phosphates and locally increasing PO ₄ concentrations (50). In addition, Stein et al. (51) said that ALP activity should be measured to determine osteoblast differentiation as a marker of osteoblast differentiation. Torii (52) et al. Suggested that ALP osteoblasts using antisense RNA. Proved to be involved in calcification of Weiss et al. Reported bone tissue calcification failure in hypophosphatase due to a congenital deficiency of liver / bone / kidney type ALP genes. Fukayama and Tashjian (55) report that ALP is involved in calcium intake in several osteoblastic lines and that it is also involved in the regulation of parathyroid hormone signaling. In addition, ALP activity of osteoblasts was used as a differentiation index by various studies. In this study, ALP enzyme activity was previously measured as an indicator for examining osteoblastic activity and differentiation effect of ethanol extract of D. bracken. As a result of confirming the bone matrix protein obtained by PCR by electrophoresis, it was as shown in FIG. The expression level of the differentiation factor mRNA after 9 days of culture of MC3T3-E1 cells was shown. The housekeeping gene GAPDH (glyceraldehyde-3-phosphste dehydrogenase) was compared with the internal control group. As a result, GAPDH, which is a control group, was expressed at a constant level, whereas ALP expression was increased when treatment of ethanol extract by concentration, and expression of Runx-2 and type II collagen, bone matrix proteins, It was expressed higher than the control. As shown in the results of the ALP enzyme activity and collagen content shown above, it was confirmed that osteoblast differentiation was promoted by increasing the expression of ethanol extract.

2.7. Investigation of Protein Expression Related to Osteoblast Differentiation by Western Blot Method

The osteoblasts were cultured for 9 days to induce differentiation, and the changes in the protein expressed when the treatment of the rattan ethanol extract was examined by Western blot method. BSP (Bone sialo protein) is a phosphorus-binding glycoprotein that can bind to hydroxyapatite by diglutamic acid amino acid sequence and can mediate cell adhesion by Arg-Gly-Asp (RGD) amino acid sequence. The expression of is basically limited to the calcified connective tissue. The BSP gene plays an important role in the early calcification of bone, dentin and chalkiness, and the expression of the BSP gene is known to be high in the early stages of bone, tooth and chalky formation and to nucleate the formation of hydroxyapatite crystals (45). As a result of measuring the expression level of BSP protein by treating the ethanol extract 1 ~ 20 μg / mL in osteoblasts as shown in Figure 8, the concentration of BSP protein expression was confirmed to increase. Therefore, the extract of ethanol ethanol extract enhances ALP activity and induces the expression of bone matrix genes.

3. whiter extract and The fraction  Osteoclast bone On absorption capacity  Impact

3.1. Extract of osteoclasts TRAP  Effect on activity

TRAP is an enzyme that has increased secretion when osteoclasts act on bone resorption and has high activity in ATP and nitrophenyl phosphate, and it is a cytochemical marker enzyme of osteoclasts that can measure the degree of osteoclast differentiation. to be. Therefore, in order to investigate the effects of D. ethanol extract on osteoclast differentiation, macrophages obtained from bone marrow were treated with M-CSF and RANKL at appropriate concentrations to induce osteoclast differentiation, As a result of examining the osteoclast proliferation and TRAP enzyme activity by the treatment as shown in FIG. It was confirmed that there is no cytotoxicity to osteoclasts by treatment of the extract of ethanol extract. As a result of increasing the concentration, TRAP enzyme activity decreased in concentration dependently. In particular, the lowest concentration of 10 μg / mL showed very high TRAP enzyme inhibitory activity of 22.3 ± 1.9%, and at 20 and 40 μg / mL, they showed low TRAP enzyme activity of 16.8 ± 0.5% and 14.3 ± 0.3%, respectively. . This is compared with that of TRAP enzyme activity of 53.7 ± 4.6% and 15.3 ± 5.7%, respectively, at concentrations of 10 and 50 μg / mL of avocado rind extract (55). It was confirmed that the excellent material to reduce the differentiation of osteoclasts.

3.2. Influence on the Osteoclast Formation of the Root Extract of Ethanol

When the osteoclasts are differentiated into osteoclasts with activity in osteoclasts, osteoclasts have dehydrogenase activity and thus have specific activity of TRAP (55). By measuring the activity of this TRAP, the degree of differentiation of osteoclasts can be measured. Inducing the differentiation of mouse bone marrow cells into osteoclasts and the effect of ethanol extracts with excellent TRAP enzyme inhibitory activity against osteoclasts on osteoclast differentiation using TRAP staining. As a result, as shown in Fig. 10, in the control group induced differentiation into osteoclasts by treating only RANKL without treatment with the ethanol extract, the differentiation was induced to identify multinucleated osteoclasts. Induction of differentiation by treatment of ethanol extract with RANKL resulted in a decrease in the size and number of osteoclasts of multinucleated cells in a concentration-dependent manner compared to the control group treated with RANKL only, especially at 40 μg / mL. The osteoclasts could hardly be observed. Through this, it was found that the extract of ethanol inhibits the formation of mature osteoclasts.

3.3. Sounder The fraction  Osteoclast TRAP  Effect on activity

The results of examining the osteoclast viability and TRAP activity for the larvae fraction are shown in FIG. 11. As shown in Figure 11, it was confirmed that there is no cytotoxicity in the fraction once. In addition, the TRAP enzyme activity was measured by treatment of the fraction of the extract, and the water (water) fraction showed no inhibition of TRAP enzyme activity, and the other fractions showed that TRAP activity was inhibited in a concentration dependent manner. In particular, ethyl acetate fraction showed the highest inhibitory activity of 13.91 ± 3.12% at 20 μg / mL, and 44.75 ± 10.63% and 21.05 ± 7.33% of TRAP activity at concentrations of 5-10 μg / mL, respectively. Showed that the differentiation of osteoclasts was most suppressed (FIG. 11). The differentiation of osteoclasts showed 14.35 ± 7.68%, 23.76 ± 3.92%, and 20.02 ± 11.13%, respectively, when 20 μg / mL of hexane, chloroform and butanol fractions were used. Suppressed. Therefore, it was confirmed that TRAP activity was inhibited against osteoclasts in the remaining layers except the water fraction of the larvae. In particular, the ethyl acetate fraction was found to inhibit TRAP activity against osteoclasts the most. In other words, ethyl acetate fraction contains a lot of antioxidants such as pigments, polyphenols, and carotenoids (56). These components are thought to have influenced the inhibition of osteoclast activity.

3.4. Remarkable ethyl acetate Fraction  Effect on osteoclast formation

In order to differentiate the bone marrow cells collected from the tibia of the mouse into osteoclasts, RANKL (100 ng / mL) and M-CSF (50 ng / mL) were treated. Acetate fractions were treated at concentrations of 5, 10 and 20 μg / mL, which were not toxic to osteoclasts, and then confirmed their effects on osteoclast differentiation using TRAP staining. As a result of staining the osteoclasts through the TRAP staining and confirming the formation of the mature osteoclasts of the multinucleus, it was observed that the number of the osteoclasts of the multinucleus decreased in a concentration-dependent manner (FIG. 12). In particular, multinucleated mature osteoclasts could not be observed at the concentration of 10 μg / mL of ethyl acetate fraction, and it was confirmed that only the propagation bone cells were observed because differentiation was greatly inhibited at the concentration of 20 μg / mL.

3.5. Western Blotting  Investigation of osteoclast differentiation related protein expression

In order to investigate the effect of the noun ethyl acetate fraction on the protein expression of c-fos, NFATc1, which is essential for osteoclast differentiation, the ethyl acetate fraction was treated or not treated while inducing osteoclast differentiation using RANKL and M-CSF. In each case, the expression of c-fos and NFATc1 was measured. After induction of osteoclast differentiation, the protein expression of c-fos and NFATc1 was increased by western blotting when the ethyl acetate fraction was not treated. -fos, protein expression of NFATc1 was significantly reduced (FIG. 13). When the signal from RANKL is essential for osteoclast differentiation, the signal of differentiation of osteoclasts is transmitted through MAP kinases such as p38, JNK, and ERK and the transcription factor NF-κB, which is signaled by RANKL when ethyl acetate fraction is treated. Expression of NF-κB was decreased in the transporter, and the expression of JNK and ERK was also significantly decreased (FIG. 13). Through this, it was found that the inhibitory effect of osteoclast differentiation of the ethylacetate fraction of the larvae was achieved through the inhibition of the JNK and ERK pathways following the stimulation of RANKL. According to Schreck R et al. (11), free radicals have been reported to increase bone resorption through the activity of NF-κB, a transcription factor for oxidative stress reactions. In combination with the results of free radical scavenging activity measured earlier, the scavenging activity was very high in the ethyl acetate fraction of the larvae, thus inhibiting the activity of NF-κB through scavenging free radicals. It is thought to show the effect of inhibiting differentiation.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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<110> Keimyung University Industry Academic Cooperation Foundation <120> Composition for Treating or Preventing Bone Disease Comprising          Extract From Rumex crispus L. As Active Ingredient <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 1 caggaagact gcaagaaggc tctgg 25 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 2 acaaggtgtc actgcgctga aga 23 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 3 ctccggctcc tgctcctctt a 21 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 4 gcacagcact cgccctccc 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 5 tgagaacggg aagcttgtca 20 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 6 ggaaggccat gccagtga 18

Claims (9)

Rumex crispus L.) A composition for the prevention or treatment of bone diseases comprising the extract as an active ingredient.
The extract of claim 1, wherein the extract is water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, dichloromethane (CH ₂ Cl ₂ ), chloroform (CHCl ₃ ), hexane (Hexane). ) And an extract of a solvent selected from the group consisting of 1,3-butylene glycol.
The composition of claim 1, wherein the bone disease is bone damage caused by bone metastasis of cancer cells, osteoporosis, osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease or metabolic bone disease.
The composition of claim 1, wherein the extract is promoted in the differentiation of osteoblasts.
The method according to claim 4, wherein the extract of the larvae increases the expression of alkaline phosphatase, Runx 2 (Runt-related transcription factor 2), and type II collagen in osteoblasts, A composition, characterized by increasing the secretion of osteocalcin (Osteocalcin, OCN).
The composition of claim 1, wherein the extract is inhibiting the activity or differentiation of osteoclasts.
The composition of claim 6, wherein the extract is inhibited by the differentiation of osteoclasts induced by receptor activator of NFκB ligand (RANKL).
(a) a pharmaceutically effective amount of the composition of any one of claims 1 to 7; And (b) a pharmaceutical composition for preventing or treating bone diseases comprising a pharmaceutically acceptable carrier.
(a) a food-effective amount of the composition of any one of claims 1-7; And (b) food composition for improving bone disease comprising a food acceptable carrier.

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