KR20150105561A - Pharmaceutical composition for preventing or treating bone disease comprising Hovenia dulcis Thunb extract - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and functional health food composition containing Hovenia dulcis Thunb extracts for preventing or treating bone disease. According to the present invention, the Hovenia dulcis Thunb extracts are derived from natural ingredients, thereby having less toxicity. Also, the Hovenia dulcis Thunb extracts impede an excessive bone resorption by effectively inhibiting an osteoclast differentiation, thereby being able to be used as a novel treating and preventing material for bone diseases induced by an osteoporosis as well as a bone loss. In addition, the present invention shows dramatic inhibitory effects on the osteoclast differentiation compared to an existing restorative herb medicine by adding certain amounts of the present invention in a restorative herb medicine. Therefore, it is possible to provide a product effective on human body, portable, and easy to take, and also possible to help promote health and replenish nutrition with ease for anyone.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis involving Hovenia dulcis Thunb extract,

The present invention relates to a pharmaceutical composition and a health functional food composition for preventing or treating bone diseases containing Hovenia dulcis extract.

Bone formation in the human body is a dynamic organ that is constantly repeated throughout its lifetime, such as bone remodeling, such as bone formation and bone resorption by osteoblasts. Bone tissue constitutes the cartilage and skeletal system. It plays a role of support and muscle attachment by mechanical function, protects organism and bone marrow, and preserve calcium and phosphorus to maintain homeostasis. Bone tissue that functions like this is composed of many kinds of cells such as collagen, glycoprotein, and osteoblast, osteoclast, and osteocyte. Among them, osteoclasts are cells derived from hematopoietic stem cells, which are responsible for the absorption of aged bone. Osteoblasts are derived from bone marrow stromal cells and play a major role in osteogenesis.

The osteoclasts are differentiated into multinucleated osteoclasts by RAW264.7 mononuclear cells of the mouse by RANKL (receptor activator of nuclear factor κB (RANK) ligand). This differentiation process promotes the activation of mitogen-activated protein kinase (MAPK) by RANKL binding to RANK in the extracellular region, and this is because the transcription factor NF-κB enters into the nucleus and binds to osteoclast differentiation-related TRAP (MMP-9) and tyrosine kinase (tyrosine kinase). These multinuclear osteoclasts formed a mineralized bone, It plays a role of absorption. In addition, when RANKL binds to RANK, the activity of TRAF6 (tumor necrosis factor receptor-associated factor 6) is promoted to promote the activation of transcription factors such as MAPK or NF-κB, AP-1 and NFATc1 HH. Signal transduction by receptor activator of nuclear factor kappa B in osteoclasts. Biochem Biophys Res Commun 2003 May 30, 305, 211-4). Therefore, blocking of the signaling pathway activated by RANKL is recognized as one of the therapeutic approaches for the treatment of osteoporosis and other bone diseases.

Osteoporosis, which is a typical example of bone disease, is a disease rapidly caused by fracture due to bone loss which is caused by loss of bone calcium due to breakage of balance between reabsorption and reshaping of bone and speed of bone resorption. Therefore, in order to develop a therapeutic agent for osteoporosis, a conventional method of inhibiting osteoclast activity or selecting a substance that increases the activity of osteoblast is firstly applied to an animal having symptoms similar to osteoporosis in humans, The degree of recovery is evaluated to the extent of recovery and how much bone strength has been restored.

Osteoporosis can be thought of as a variety of causes, but the most common treatment is estrogen, vitamin D, and calcitonin, since the majority are older (aging) or hormone imbalance after menopause in women. However, administration of hormones is not a safe treatment because of the risk of inducing cancer (especially breast cancer, uterine cancer, etc.). In addition, although bisphosphonate is administered as a second-generation therapeutic agent, there is a problem that rebound occurs when administration is discontinued. Either of these methods may delay the progression of osteoporosis, but it also has the disadvantage that it is difficult to regenerate the reduced bone.

On the other hand, in order to treat bone diseases, it is necessary to control the balance between osteoclasts and osteoblasts, and bone remodeling agents and bone formation stimulators are widely used as therapeutic agents. Although bone resorption stimulants have been actively studied, bone mineral density enhancement due to osteogenic stimuli does not necessarily mean reduction of fracture. Therefore, more research is needed to be widely used for clinical use. In addition, as an agent for stimulating osteogenesis, an accelerator for activating osteoblast and an inhibitor for inhibiting bone resorption of osteoclast generally need to be administered to a patient for a long period of time. The research on this subject is urgently needed.

Accordingly, the present inventors have made intensive efforts to find a compound that is less toxic and can inhibit osteoclast differentiation, which is derived from a natural product. As a result, Hovenia dulcis extract significantly inhibited the differentiation of osteoclasts and could be used as a preventive and therapeutic agent for osteoporosis And completed the invention.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating osteoporosis comprising an extract of Hovenia dulcis as an active ingredient.

It is another object of the present invention to provide a health functional food composition for preventing or ameliorating osteopathy which contains Hovenia dulcis extract as an active ingredient.

In one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating bone diseases, comprising an extract of Hovenia dulcis as an active ingredient.

Specifically, the pharmaceutical composition for preventing or treating bone diseases of the present invention may include a hinoki extract.

In the present invention, " Hovenia dulcis ) "is a deciduous broad-leaved tree species distributed in Korea, Japan, China, etc. Recently, research on the pharmacological effect of Hovenia dulcis has been actively carried out, and it is good for liver, large intestine, stomach and kidney, , And "Party Honshu" have a new record in detoxification after drinking. In addition, Hovenia japonica is effective in relieving liver disease and thirst such as alcoholic hepatitis, fatty liver, liver cirrhosis, etc., It has been known that it is effective for eliminating bad breath and constipation, but its effect on prevention or treatment of bone disease has not been disclosed to date and it is the first time that the present inventors have clarified.

In the present invention, the term " hinoki extract "refers to a preparation which is obtained by squeezing a hinoki tree with a suitable leaching solution and evaporating the leachate. The hinoki extract is prepared by dissolving the hinoki wood pulp with water, an organic solvent, Extraction process, and may include an extract, a diluent or concentrate thereof, or a dry powder of the extract or all the forms formulated with the extract. The Hovenia dulcis extract may be extracted from various organs of natural, hybrid, and variant plants and extracted from plant tissue culture as well as root, top, stem, branch, and leaf, Can be extracted.

Further, in the extraction method, a method such as hot water extraction, hot water extraction, cold extraction, warm-up extraction, pressure extraction, reflux cooling extraction, or ultrasonic extraction may be used.

In obtaining the Hovenia dulcis extract, it may preferably be extracted using water, an organic solvent or a mixed solvent thereof. In the case of extraction using an organic solvent, an organic solvent such as methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF) (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof. The active ingredient of the herbal medicine may be extracted at room temperature or warmed under the condition that the active ingredient is not destroyed or minimized. But it is not limited thereto. The extraction efficiency and the degree of loss of the active ingredient of the medicament may differ depending on the organic solvent to be extracted. Therefore, An appropriate organic solvent can be selected and used.

The solvent extract may further comprise a step of filtering the extract to remove suspended solid particles. Nylon, etc., or ultrafiltration, freezing filtration, centrifugation, etc. may be used.

Concentration of the extract can be carried out by reduced-pressure concentration, reverse osmosis concentration, or the like.

The concentration and drying step includes freeze drying, vacuum drying, hot air drying, spray drying, vacuum drying, foam drying, high frequency drying, or infrared drying. In some cases, it may further comprise a step of pulverizing the finally dried extract.

In addition, the extract may be subjected to an additional fractionation process. For example, the extract is suspended in distilled water to obtain a nonpolar solvent soluble layer by extraction with a nonpolar organic solvent, for example, hexane, ether, dichloromethane, chloroform, ethyl acetate or a mixed solvent thereof, Concentrated and / or dried.

In the present invention, it was confirmed that Hovenia dulcis extract effectively inhibited the expression of TRAP, which is known as an osteoclast differentiation marker, and thus it can be effectively used for prevention or treatment of bone diseases.

In the present invention, "prevention" means any action that inhibits or delays the onset of bone disease by administration of the composition of the present invention, and "treatment" means that the composition of the present invention alleviates or alleviates It means all acts.

The term "bone disease" in the present invention includes all diseases caused by a decrease in bone density, and may include osteoporosis, osteogenesis, osteopenia, osteoarthritis, osteoarthritis and the like, preferably osteoporosis. The decrease in bone mineral density, a symptom of osteoporosis, is a result of imbalance of osteoclasts and osteoblasts in the bone. Excessive osteoclastogenesis induces diseases such as osteoporosis which reduce bone density.

The osteoporosis is, for example, primary osteoporosis such as osteoporosis associated with menopause in women, senile osteoporosis and osteoporosis due to oophorectomy; Secondary osteoporosis, such as glutamic acid-induced osteoporosis, thyroid glandular osteoporosis, osteoporosis induced by Cushing's syndrome and rheumatoid osteoporosis; osteoporosis, osteoporosis, heparin-induced osteoporosis, immunosuppressive osteoporosis, osteoporosis due to renal failure, inflammatory osteoporosis; And the like, but is not limited thereto.

The composition of the present invention is characterized by inhibiting osteoclast differentiation or bone resorption. Can effectively be used as a preventive or therapeutic agent for bone diseases by effectively inhibiting the expression of c-Fos, NFATc1, DC-STAMP, c-src, cathepsin K, oscar or TRAP known as differentiation markers of osteoclast.

In the present invention, "osteoclast" is a cell derived from macrophage precursor. Osteoclast precursor cells are macrophage colony stimulating factor (M-CSF), receptor of NF-κB (RANKL) or the like to form osteoclasts, which form multinucleated osteoclasts through fusion. The osteoclasts bind to the bone through αvβ3 integrin and the like to form an acidic environment while releasing various collagenase and protease to cause bone resorption. The osteoclasts do not proliferate to completely differentiated cells, and when they reach the shelf life of about 2 weeks, they cause apoptosis.

In the examples of the present invention, the composition of the present invention was administered to osteoclast-differentiated mouse mononuclear cells, and then the osteoclast differentiation marker TRAP (tarrate-resistant acid phosphate) staining showed that TRAP activity was inhibited in a concentration- (Fig. 2). As described above, the composition of the present invention effectively inhibits osteoclast differentiation through inhibition of TRAP activity, so that it is possible to prevent or treat osteopathy such as osteoporosis by increasing osteoclast activity.

The pharmaceutical compositions comprising the Hovenia dulcis extract of the present invention may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions. At this time, the content of Hovenia dulcis Thunb. Extract contained in the composition is not particularly limited, but it may be 0.01 to 100% by weight, preferably 1 to 50% by weight based on the total weight of the composition.

The pharmaceutical composition may be any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, nonaqueous solvents, suspensions, emulsions, And may be oral or parenteral formulations of various forms. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The present invention can provide a method for preventing or treating bone diseases, comprising the step of administering the pharmaceutical composition to a suspected subject of bone disease.

In the present invention, the suspected individual of the bone disease refers to all animals including a human who has developed or may develop osteopathy. By administering the pharmaceutical composition containing the Hovenia dulcis extract of the present invention to a suspected osteoporosis individual, Can be efficiently treated. The bone disease is as described above.

In the present invention, "administration" means introducing the pharmaceutical composition of the present invention into a subject suspected to have a bone disorder by any appropriate method, and the administration route can be administered through various routes of oral or parenteral administration .

The method of treatment of the present invention may comprise administering a pharmaceutically effective amount of a pharmaceutical composition comprising a Hodgkin's extract.

The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will vary depending on the species and severity, age, sex, The type of drug, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art. The preferred dosage of the composition of the present invention will depend on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, and the appropriate total daily dose may be determined by treatment, Generally, an amount of 0.001 to 1000 mg / kg, preferably 0.05 to 200 mg / kg, more preferably 0.1 to 100 mg / kg, can be administered in a single dose, divided into several times a day.

The composition of the present invention may further comprise jellyfish.

In the present invention, " kyokokogo " may include ginseng, gyeongryeong, raw persimmon, honey, and the like, which are the traditional health promotion related materials recorded in Donguibogam. Gyeongokgyo is a kind of waxy medicine that helps blood circulation and prolongs life. It is said that it is effective to maintain and revitalize the function of all the organs in the human body according to the Dongwoo Bokcheon. In addition, Gyeongokgyo is not only a medicinal efficacy as a medicinal product and energetic agent, but also more specifically, patients suffering from weakness of the stomach and frequent fatigue, patients suffering from gastrointestinal illness due to weak digestion, gastrointestinal stomach cramps, frequent abdominal pain, It is said that it is effective for a patient who suffers from a cold and suffers from breathing by bronchial asthma and has a severe coughing of the seawater. In particular, it is said that when the elderly weak in mind and body are buried, the baldness becomes blackened, , The specific and scientific basis for clinical trials has yet to be presented.

The composition of the present invention may contain hinoki extract and mushroom at a ratio of 1:50 to 50: 1, preferably 1:20 to 1: 2. In contrast, when the ratio is greater than 50: 1, the overall characteristics including the property, taste, and the like are completely different, and the effect of preventing or treating osteoporosis is different from that of the osteoporosis. Can be expressed.

Also, the mixture of hinoki extract and jelly bean ginseng comprises 5 to 10% by weight of ginseng, 10 to 20% by weight of gypsum, 0.01 to 30% by weight of zucchini, 45 to 55% by weight of honey, But is not limited to, from 0.001 to 30% by weight of Hovenia dulcis extract. Preferably from 6.5 to 8.5% by weight of ginseng, from 14.5 to 16.5% by weight of ginger, from 50 to 52% by weight of honey, from 0.1 to 26% by weight of zucchini and from 0.1 to 26% by weight of hinoki extract.

In a specific example of the present invention, Kyoikoku was added so that the content of hornbug extract (HD) was 10% by weight and 25% by weight in the mixture of Hovenia dulcis Thunb extract and Kyoikoko, and the mice (KOG-HD) As a result of biochemical analysis, histological analysis, and morphological analysis of the tibia and femur of the model, osteoclast formation inhibitory activity was significantly increased as compared with KOG, which does not contain Hovenia dulcis extracts. It was confirmed that osteoclast formation inhibitory activity was significantly increased depending on the concentration of Hovenia dulcis extract.

In another aspect, the present invention provides a health functional food composition for preventing or ameliorating bone diseases, which contains Hovenia dulcis extract as an active ingredient.

Specifically, the Hovenia dulcis extract of the present invention may be incorporated into a health functional food composition for the purpose of preventing or improving bone diseases.

The hinoki extracts and bone diseases are as described above.

In the present invention, "improvement" refers to all the actions of using the composition to alleviate the suspicion of bone disease and symptoms of an onset individual.

When the composition of the present invention is incorporated into a health functional food, the composition may be added as it is or may be used together with other health functional foods or health functional food ingredients and suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use. Generally, the composition of the present invention may be added in an amount of preferably not more than 15 parts by weight, more preferably not more than 10 parts by weight, based on the raw material, in the production of food or beverage. However, in the case of long-term intake for the purpose of health control and hygiene, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of health functional food that can contain the composition of the present invention, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gum, Dairy products, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include foodstuffs used as food for animals, which may include all health functional foods in the conventional sense.

The health functional food composition of the present invention can be used as a health functional food composition containing various nutrients, vitamins, electrolytes, flavorings, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , A carbonating agent used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

The extract of Hovenia dulcis according to the present invention is not only toxic due to its natural origin but also effectively inhibits the differentiation of osteoclasts, thereby suppressing excessive bone resorption and ultimately preventing and treating osteoporosis due to bone loss including osteoporosis Lt; / RTI >

In addition, it has a remarkable inhibitory effect on osteoclast differentiation compared to existing kyangokgol by being contained in a certain amount in Kyeongokgok, and it is a product that is excellent in the effect on the human body, is easy to carry and take, and is helpful for easy supplementation and health promotion Can be provided.

FIG. 1 is a graph showing the oocyte cell formation inhibitory effect of Hovenia dulcis Thunb extract (HD). (A) and (B) show the decrease of osteoclast according to the concentration of Hovenia dulcis extract, and (C) the cytotoxicity according to the concentration of Hovenia dulcis extract.
Fig. 2 is a graph showing the oocyte cell formation inhibitory effect of a mixture of Kyooko Kogyo (KOG) and Kyoeokogo-Hovenoko extract (KOG-HD). (A) and (B) show the decrease of osteoclast according to the concentration of KOG and KOG-HD, and (C) the cytotoxicity according to the concentration of KOG and KOG-HD.
FIG. 3 is a graph showing (A) body weight, (B) thymus weight, (C) spleen weight and (D) uterine weight of a mouse administered with a mixture of Kyoelysophygea and Hovenia dulcis extracts.
FIG. 4 is a graph showing the degree of marker expression of a mixture of Kyoikoko and Hovenia dulcis extracts as a result of measuring biochemical markers from the serum of collected blood. (A) is calcium, (B) is inorganic phosphoric acid, (C) is ALP (Alkaline phosphatase), (D) is TCHO (total cholesterol), E is estradiol and F is TRAP.
FIG. 5 is a graph showing the expression levels of CD4, CD8 and CD11c in the spleen of mice administered with a mixture of Kyoikoko and Hovenia dulcifera and CD4 & 8 in the thymus.
FIG. 6 shows morphological characteristics of the tibia and femur of a mouse administered with a mixture of Kyoelysophyceae and Hovenia dulcis extracts. (A) Bone volume / tissue volume (BV / TV) BS / TV), (C) bone surface / bone volume (BS / BV), (D) SMI, (E) trabecular bone pattern factor (Tb.Pf), (F) trabecular thickness (Tb.Th) ) trabecular number (Tb.N) and (H) trabecular separation (Tb.Sp).
Fig. 7 is a photograph of a tibia and a femur of a mouse administered with a mixture of Kiyokoko and Hinoki extracts, and CT images of the tibia and femur converted into 3D photographs.
FIG. 8A is a graph showing the results of H & E staining, TRAP staining, and Masson's trichrome staining of tibia portions of a mouse administered with a mixture of Kyoelysophyceae and Hovenia dulcis extracts.
FIG. 8B is a diagram showing the results of H & E staining, TRAP staining, and Masson's trichrome staining on the distal femur of a mouse administered with a mixture of Kyoikoko and Hovenuo extracts.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  One: Hinoki  Extracts and Kyunghee University  Preparation of composition

Example  1-1: Hinoki  Preparation of extract

Weighing 1,500 kg of the twigs with an electronic balance, 8.1 L of purified water was added and the water was boiled to 100 ° C, and the extract was extracted for 4 hours. After the extraction was completed, the filtrate was filtered through a filter, and the filtrate was transferred to a storage tank. The temperature of the storage tank was raised to 50 ° C, the solid was pressure-adjusted to be 40 Brix or more, and concentrated in vacuo at 0.08 to 0.09 MPa . After concentrating in vacuo, the properties and flavor were checked and it was confirmed that it was concentrated without problems. It was confirmed that the concentrated product had a fragrance inherent to the horseshoe branch and that the property was a dark brown liquid fluid.

Example  1-2: Hinoki  The extract and Kyunghee University  Preparation of mixtures

KOG (Kyeongok High), KOGHD 10% (Kyeongok High + 10% Hovenia dulcis THUNB) and KOGHD 25% (Kyohei + 25% Hovenia dulcis THUNB) Samples were received from JBT Company.

The raw persimmon was washed well in water and dried. Ginseng and Yulbok (gyeongryeong) were blended in proportion and dried very slowly while baijiwang was dried. It was dried with yellow rhubarb and squeezed juice, put it in ginseng and gyeongryeong powder, rubbed it again, and rinsed it with the native honey which was collected from shiryeon. It was soaked in water for 72 hours, then immersed in running water for 24 hours to allow the fire to cool, which was then added to the bath for 24 hours. After the mixture was cooled sufficiently, the mixed solution was kneaded in the form of Kiyokoko kneading. The mixture was rubbed once again and aged for about 6 months in a jar for aging. The ratios of the medicaments contained in the sample are shown in Table 1 below.

division Control
( KOG ) Experimental Section  One
( KOGHD10 %) Experimental Section  2
( KOGHD25 %) Ginseng 7.8% 7.8% 7.8% Boryeong 15.6% 15.6% 15.6% Jujube juice (12Brix) 25.6% 15.6% 0.6% honey 51.0% 51.0% 51.0% Houttuyniae extract concentrate (12Brix) - 10.0% 25.0%

The juice concentrate was the same as the juice concentrate and Brix.

Example  2: Manufacture of ovariectomized bone disease animal model

Experimental animals were fed a specific pathogen free C3H / HeN 8 week old mouse at Orient Bio (Korea, Jeollabuk-do, Iksan) and received 4 densities per polycarbonate breeding box (18 × 20 cm) And maintained. These mice were fed with sufficient water and feed for 1 to 2 weeks at a temperature of 21 ± 2 ° C and a humidity of 55 ± 10%. Day and night cycles were adjusted for 12 hours to avoid possible stress, ~ 9 weeks of mice were used in the experiment.

As an anesthetic agent, zolethyl (Rocun) and Rumpun (Bayer Korea Co., Ltd.) were mixed at a certain ratio, and the anesthetic agent was mixed at a certain ratio. After general anesthesia with intraperitoneal injection, the back central portion of the mice was incised about 1 cm. The ovaries were checked along the uterus with care to avoid damage to other organs, and the ovaries were ligated into the suture chamber, and both ovaries were excised. After ovariectomy, each organ was repositioned into the peritoneal cavity and closed with a suture. The success of ovariectomy can be confirmed by the development of blood vessels around the uterus and the measurement of the size and weight of the uterus after the sample has been administered for a fixed period of time.

OVX group in which ovariectomized Sham group and ovariectomized mouse are administered water, and administration group (KOG, KOGHD 10%, KOGHD 25%) in the concentration-dependent group, and the number of individuals per experimental group is statistically significant Were set as 5 individuals. Since the treatment period for bone disease is characterized by long-term use, water and sample (185 mg / kg) are administered once a day by oral administration .

Experimental Example  One: TRAP ( tartarate resistance acid 포스화제 ) Dyeing and activity analysis

Bone marrow cells were obtained by separating the femur and tibial bones of five - week - old mice and washing the subcortical space with a 1 cc syringe. Separated bone marrow cells were cultured in α-minimal essential medium (α-MEM) medium containing 10% FBS, antibiotics and M-CSF (30 ng / ml) for 3 days. Three days later, adherent cells were used as bone marrow macrophages (BMM). The progenitor cells were cultured by adding M-CSF (30 ng / ml) and RANKL (10 ng / ml) and cultured in the same manner as in Example 1-1 except that the hornwood extract (HD) A mixture of wood extract and mushroom was treated. After 4 days, the cultured cells were stained with TRAP (Sigma Aldrich, USA) and the red stained cells were regarded as osteoclasts.

Multinuclear osteoclasts are fixed with 3.7% formalin and reddish by treatment with TRAP solution when permeability of the membrane increases with 0.1% Triton X-100 for 10 minutes. The red-stained osteoclasts were recognized as osteoclasts only with three or more nuclei. TRAP activity was determined by treating TRAP-stained osteoclasts with TRAP buffer (100 mM sodium citrate pH 5.0, 50 mM sodium tartrate) containing 3 mM p-nitrophenyl phosphate (Sigma-Aldrich) for 5 min at 37 ° C, After transferring the solution to a new plate, the same amount of 0.1 N NaOH was added to confirm the absorbance at 405 nm.

As a result, as shown in FIGS. 1A and 1B, it was observed that the positive control group (PC) induced by RANKL formed a significantly larger number of reddish osteoclasts than the negative control group (NC). Also, it was confirmed that the treatment of Hodgkin's Extract (HD) of Example 1-1 at this time significantly reduced the number of red-stained osteoclasts while suppressing TRAP activity in a concentration-dependent manner.

In addition, as shown in Figs. 2A and 2B, the number of osteoclasts was significantly decreased by decreasing the TRAP activity in a concentration-dependent manner of the Hovenia dulcis extract containing a mixture of Hovenuo extract and Kyoikoku.

As described above, it has been found that the mixture of Hovenia dulcis extract, Hovenia dulcis Thunb extract and Kyoikoko can effectively be used for bone diseases such as osteoporosis because it effectively inhibits osteoclast differentiation.

Experimental Example  2: Cytotoxicity analysis

The progeny cells were cultured in 96-well plates at a density of 1 x 10 ⁴ cells / well in the same manner as in Example 1-1 except that the hornwood extract (HD) prepared in Example 1-1 or the Hovenia monkey extract (KOGHD) and M-CSF (30 ng / ml) were added and cultured for 3 days. After 3 days, 50 μl of CCK solution was added to each well. After incubation for 4 hours, the absorbance was confirmed at 450 nm using an ELISA reader (Molecular Devices, CA, USA).

As a result, as shown in FIG. 1C and FIG. 2C, Hovenia dulcis extract (HD) or Hovenia dulcis Thunb extract (KOGHD) containing Krypton core component was treated by concentration for 100% of the survival rate of the control group Cell viability was not significantly decreased, indicating that there was little cytotoxicity.

Experimental Example  3: Measure the length of body weight, organ weight (spleen, thymus, uterus) and bones (tibia, femur)

After 6 weeks of administration of the Hovenia dulcis extract (HD) or Hovenia dulcis extract (KOGHD) prepared in Example 1 or the mixture of Hovenuo extract and Koryo gum (KOGHD), the weight was measured using an electronic scales , Followed by cervical dislocation and weight of organs after sacrifice. The tibia and femur were extracted from both the left and the right. The lengths were measured using vernier calipers and the weights were measured using a scale.

As a result, it was found that (1) the body weight was increased in the Ovx group as compared with the Sham group, and that the increase in body weight was not suppressed in the three groups treated with the sample (FIG. (2) the weight of the thymus was not statistically significant in all groups (FIG. 3B). (3) In the case of spleen, the weight of OVX group was increased compared to that of Sham group, and the increase of spleen weight was inhibited in 25% of KOGHD group (FIG. 3C). (4) The uterine weight was significantly decreased in the OVX group compared to the Sham group, and the uterine weight was increased in the 25% KOGHD group (FIG. 3 D). It has been reported in many papers that the weight of the uterus decreases when the ovary is removed, which is an important factor in determining whether the osteoporosis model has been successfully induced. (5) The weight and length of the tibia and femur were not significantly different between the Sham group and the OVX group, but the weight was increased in the KOGHD 10% and KOGHD 25% groups (Table 2).

Experimental Example  4: Biochemical analysis

For the biochemical analysis, the mouse was anesthetized with ether, and then the orbital blood was collected by using a microheamatocrit capillary tube, and the serum was separated by centrifugation. Alkaline phosphatase (ALP), calcium (Ca), and ionogranic phosphorus ) And total cholesterol (TCHO) were measured with an automatic analyzer (Fuji Dri-Chem, Fuji, Japan) using a measurement slide (Fujifilm, Japan).

As a result, (1) calcium showed an increase pattern in the OVX group compared to the Sham group, and it was confirmed that it was decreased in the KOGHD 10% and KOGHD 25% treatment groups (FIG. 4A). (2) Inorganic phosphoric acid was increased in the 25% KOGHD group compared to the OVX group (FIG. 4B). (3) ALP was significantly inhibited by 10% KOGHD compared with OVX group (FIG. 4C), and (4) TCHO was not significantly different among the groups. Therefore, this suggests that Hovenia dulcis extract has activity of inhibiting the release of calcium or ALP into serum.

Experimental Example  5: Estradiol ( E2 )and tartrate - resistant acid 포스화제 ( TRAP ) Measure

Estradiol (E2) and tartrate-resistant acid phosphatase (TRAP) were measured by using an Estradiol ELISA kit (Calbiotech, San Diego, CA, USA) and a TRAP ELISA kit (Uscn Life Science, Wuhan, PR China) The experiment was carried out in accordance with the measuring method.

As a result, it was confirmed that estradiol, which is known as a therapeutic agent for osteoporosis, was dose-dependently increased in all the groups to which the KOGHD sample was administered (Fig. 4E). On the other hand, TRAP was increased in the OVX group, but it was inhibited in the 25% KOGHD group.

Experimental Example  6: Immunocyte population  Measure

In order to test whether osteoporosis-induced osteoporosis induction model can inhibit bone loss through regulation of lymphocytes, spleen cells and thymus were extracted and single cells were collected, blocking at 4 ℃ for 30 minutes -CD16 / CD32 (FcγⅢ / ⅱReceptor) mAb and washed with washing solution (1% FBS, 0.1% NaN 2 / PBS). The cells were stained with FITC Rat Anti-Mouse CD4, PE Rat Anti-Mouse CD8a, PE Hamster Anti mouse CD11C and FITC Anti-Mouse CD19 (BD Pharmigen), and then stained with a FACS cantoⅡ flow cytometer (BD Biosciences) Respectively.

As a result, it was confirmed that CD4, CD8 and CD11c in the spleen and CD4 and CD8 cells in the thymus were increased in the OVX group and inhibited when the three samples (KOG, KOGHD10%, and KOGHD25%) were administered ). Considering that T cells such as CD4, CD8 and CD11c secrete cytokines that promote osteoclast differentiation, a mixture of Hovenia dulcis extract and kyphokhi (KOGHD) inhibits the osteoclast action by inhibiting the expression of T cells And thus prevent progression to osteoporosis.

Experimental Example  7: Tibia and femur Shochu  Morphological analysis Micro CT  shooting)

For micro CT scans, the tibia and femur were stored in 70% EtOH. Tb, Tb, Tb, Nb, Tb, Tb, Tb, Tb, Tb, and Tb were measured using the Skyscan1172 analyzer. .Pf) and structure model index (SMI) were measured. The fifth slice of 150 slices was selected from the disappearance of the growth plate of proximal tibia and distal femur.

(1) Bone volume / tissue volume (BV / TV) was measured by bone volume density and decreased in OVX group compared to Sham group. However, all three samples (KOG, KOGHD, KOGHD 25% (Fig. 6A). (B), (3) trabecular number (Tb.N) and trabecular thickness (Tb.Th) were similar to those of BV / TV The number and thickness of shochu bone were measured, respectively. It was also decreased in the OVX group compared to the Sham group, and increased in the group to which the three samples were administered (F and G in Fig. 6).

(4) The bone surface / bone volume (BS / BV) was increased in the OVX group than in the Sham group and decreased in the KOGHD 10% group (FIG. 6C). (6) The trabecular bone pattern factor (Tb.Pf) was used to measure the degree of concavity and convexity of the bones. (Fig. 6E), which was significantly lower in the KOG and KOGHD 10% treated groups than in the OVX group, which was increased compared to the Sham group. (7) Trabecular separation (Tb.Sp) was a factor to measure the interval of the soju bone. It decreased in 10% of KOGHD in the tibia, 10% in KOGHD and 25% in KOGHD in the femur compared with the OVX group which was increased compared to the Sham group (H in Fig. 6).

A micro CT scan of the tibia and femur was used to convert the slice into a 3D image. The results are shown in photographs from the disappearance of the proximal tibia and distal femoral growth plate. There is almost no change in the sebaceous bone of the tibia and femur, and it can be confirmed that there is a change in the case of soju bone. In the Ovx group, it was found that the shoal bone disappeared much more than the Sham group, and when the KOG, KOGHD, and KOGHD were treated with 25% of the tibia and femur, the soju bone was increased (FIG. 7).

Experimental Example  8: Histological analysis of tibia and femur

The cells were fixed with 4% paraformaldehyde and then demineralized with 10% EDTA, dried, embedded in paraffin, cut into 5 ㎛ thick, and stained with hematoxylin and eosin (H & E). TRAP staining was performed with 225 μM Naphthol AS (Sigma-Aldrich), 50 mM sodium acetate, and 1% methyl green were used as the buffer solution.

Histologic analysis showed that the number, spacing, thickness, and osteoclast expression of bone destruction in the bone of the femur and tibia were observed. As a result, In the OVX group, the number and thickness were decreased, and the interval between the shoal bone and the sham group was increased. However, it was confirmed that the number of shochu grains was increased when three samples were treated (Figs. 8A and 8B).

TRAP staining was performed to identify activated osteoclasts (bone destruction cells). TRAP was more expressed in the OVX group. And decreased in the 25% KOGHD-treated group (Figs. 8A and 8B).

According to Masson's trichrome staining, the nucleus of the cell is black, the muscle tissue is red, and the collagen is blue. In bone tissue, collagen accounts for more than 90%, and bone tissue is stained blue . The KOGHD-treated group had more blue part than the OVX group, suggesting an increase in bone tissue (FIGS. 8A and 8B).

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (14)

A pharmaceutical composition for the prevention or treatment of bone diseases comprising an extract of Hovenia dulcis as an active ingredient.
The composition of claim 1, wherein the hinoki is a hinoki branch.
The composition according to claim 1, wherein the hinoki extract is extracted with water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
2. The composition of claim 1, further comprising a mushroom.
5. The composition of claim 4, wherein the gypsum ginseng comprises ginseng, bamboo, rhizome and honey.
5. The composition of claim 4, comprising 6.5 to 8.5 wt% ginseng, 14.5 to 16.5 wt% gypsum, and 50 to 52 wt% of honey based on the total weight of the mixture of jellyfish and hornwood extract.
6. The composition of claim 4, wherein the composition comprises Hovenia dulcis extract and mushroom at a ratio of 1:20 to 1: 2.
The composition according to claim 1, wherein the prevention or treatment of bone disease is by inhibiting osteoclast differentiation.
A health functional food composition for preventing or ameliorating osteoporosis comprising an extract of Hovenia dulcis as an active ingredient.
10. The composition according to claim 9, wherein the hinoki is a hinoki branch, and the hinoki extract is extracted with water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
10. The composition of claim 9, further comprising a pyrophyllite.
12. The composition of claim 11, wherein the hinoki extract and mushroom are present in a ratio of 1:20 to 1: 2.
10. The composition of claim 1 or 9, wherein the bone disease is selected from the group consisting of osteoporosis, osteomalacia, osteopenia, bone atrophy and osteoarthritis.
10. The composition of claim 1 or 9, wherein the bone disease is osteoporosis.

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