KR20200064776A - Composiotion for prevention, improvement or treatment of fracture including prunus davidiana franchet - Google Patents

Abstract

The present invention provides a composition for preventing, alleviating, and treating a fracture containing a Prunus persica seed extract. According to an aspect of the present invention, by using the Prunus persica seed extract as a component of a pharmaceutical composition or a food composition, it is possible to improve an effect of alleviating fracture diseases while minimizing side effects.

Description

A composition for preventing, improving or treating fractures containing Doin extract {COMPOSIOTION FOR PREVENTION, IMPROVEMENT OR TREATMENT OF FRACTURE INCLUDING PRUNUS DAVIDIANA FRANCHET}

The present invention relates to a pharmaceutical or food composition comprising an extract of Prunus davidiana Franchet.

The bone (bone) supports the human body's soft tissue and weight, and surrounds the internal organs to protect the internal organs from external shocks.

Bone is also an important part of the human body that not only structurally supports muscles or organs, but also stores substances such as calcium and other essential minerals in the body, such as phosphorus and magnesium.

Therefore, the bones of grown adults maintain balance by dynamically and continually regenerating the process of creation and absorption that removes old bones and replaces them with new ones. This is called bone remodeling.

Bone turnover is essential to repair and maintain the function of bone damage caused by growth and stress. In adults, about 10-30% of the skeleton is remodeled annually through remodeling of bone resorption-bone formation.

Two types of cells are involved in bone remodeling, osteoblasts that produce bones, and osteoclasts that destroy bones, and bone homeostasis is maintained through close association.

For example, osteoblasts are bones in the body by regulating the differentiation of osteoclasts responsible for bone resorption through the secretion of substances such as receptor activator of nuclear factor-κB ligand (RANKL), and its bait receptor, osteotrophegerin (OPG). Maintain homeostasis.

That is, when RANKL binds to RANK, the osteoclast progenitor cell surface receptor, osteoclast progenitor cells mature into osteoclasts, resulting in bone resorption, and when OPG binds to RANKL, the binding between RANKL and RANK is blocked, thereby inhibiting the formation of osteoclasts.

The osteoclasts formed from progenitor cells absorb or destroy old bones and release a small amount of calcium accumulated in the bones into the bloodstream to be used to maintain body function.

The metabolism is regulated by physical effects, hormonal system, and local factors, and when various factors cause bone absorption to exceed bone formation and the bone mass decreases below the limit, various bone diseases with damage to bone tissue occur.

In the past, with regard to the treatment of diseases associated with bone tissue damage, studies have mainly been conducted mainly on bone minerals, that is, metabolic abnormalities of calcium and phosphorus, and have not progressed in the investigation of the mechanism. Therefore, many studies have been conducted to develop osteoclast differentiation inhibitors.

On the other hand, the herbal medicines mainly used in Oriental medicine have emerged as a potential as a more fundamental treatment by the multi-target mechanism in the prevention and treatment of chronic and intractable diseases in which the causes and symptoms of the disease are complex.

In addition, in the case of therapeutic agents using such herbal ingredients as raw materials, it is possible to minimize various side effects that may occur due to the use of chemical agents, and thus, it is attracting attention as an alternative material for existing pharmaceutical ingredients.

William J. et al., NATURE., 423, pp. 337342, 2033

An object of the present invention is to provide a pharmaceutical composition for preventing or treating fractures comprising the extract of Prunus davidiana Franchet.

Another object of the present invention is to provide a food composition for preventing or improving fractures comprising a phosphorus extract.

One aspect of the present invention for achieving the above object is to provide a pharmaceutical composition for the prevention or treatment of fractures comprising the extract ( Prunus davidiana Franchet).

The "degree (Prunus davidiana Franchet)" in the present invention as a dry mature seed of Rosaceae copy leaves microglial mokin belonging to (Rosaceae) tree Prunus persica (L.) Batsch., And acid copies P. davidiana (Carr.) Franch. (HERBOLOGY EDITORIAL COMMITTEE OF KOREAN MEDICINE SCHOOLS. Boncho-hak [Herbology], Seoul: Younglimsa, 2004.)誌 2006; 21(2):87-93.), Atherosclerosis (尹仁漢. 미치는仁's effect on the 粥狀动脈硬化 of the family due to high Cholesterol eating. Kyungsan University Graduate School 1996.) have. In particular, it has traditionally been used in clinical practice to improve blood circulation and remove blood.

However, the improvement effect or related action mechanism or efficacy of the fracture disease of Doin has not been known to date, and the present inventors tried to investigate the effect of promoting the osteoblast differentiation of the Doin extract.

In the present invention, the "extract" is to separate the active ingredient contained in the extraction material by contacting the solvent and the extraction material under a specific condition, the extract may include the active ingredient in the raw material by an extraction process for the phosphorus.

The phosphorus extract may be extracted according to a conventional method known in the art for extracting an extract from a natural product, that is, using a conventional solvent under conditions of conventional temperature and pressure. For example, the phosphorus extract may be extracted using water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane or a mixed solvent thereof, specifically ethanol, and the concentration of the ethanol is 70 to 90% ( w/w), but is not limited thereto.

In addition, the extraction method may use a variety of methods such as hot water extraction, cold needle extraction, reflux extraction, ultrasonic extraction, but is not limited thereto.

The “fracture” refers to a state in which continuity of bone or cartilage is broken due to excessive impact or force. Treatment of fractures goes through the inflammatory phase, restoration phase, remodeling phase, and healing phases. Health functional foods and herbal medicines that promote bone union can be selected effectively according to the healing phase of the fracture.

In most bone metabolic diseases (osteoporosis, arthritis, osteonecrosis, osteopetrosis, etc.), osteoclasts are the main cause of the disease. Referring to a previously published paper (Boyce BF.The osteoclast, bone remodelling and treatment of metabolic bone diseases, 2012, Eur J Clin Invest.), osteoclast activity through various causes induces osteoporosis, arthritis and osteonecrosis. , It can be seen that osteoclast degeneration causes osteopetrosis.

However, in contrast, the treatment of fractures is very important in osteoblast activity, unlike other metabolic bone diseases. Referring to a previously published paper (Frederic Shapiro, Bone development and its relation to fracture repair.The role of mesenchymal osteoblasts and surface osteoblasts, 2008, European Cells and Materials), treatment of fracture 1) differentiates between mesenchymal stem cells. Bone regeneration through differentiation into osteoblasts and 2) the activity of osteoblasts activated on the bone surface plays the most important role in the treatment of fractures.

For this reason, it is possible to determine that the extract of Indo, which induces osteoblast activity, has the potential to specifically treat fractures, unlike conventional bone metabolism therapeutics.

In an embodiment of the present invention, it was confirmed that the differentiation of osteoblasts was promoted in a concentration-dependent manner when an extract extracted from MC3T3-E1 cells induced osteoblast differentiation was treated (FIG. 2).

In addition, it was confirmed that the expression of mitogen-activated protein kinase (MAPK), the mechanism of osteoblast differentiation, was enhanced when the MC3T3-E1 cells were treated with an extract of phosphorus (FIG. 3).

In the analysis of a fractured animal model, which is an embodiment of the present invention, it was confirmed that the recovery of the fracture site was improved by the treatment of the extract of Doin (FIG. 4).

The above results suggest that the extract of the present invention has a high osteoblast differentiation promoting activity, and thus can be usefully used as a composition for preventing, improving, and treating fractures having excellent osteosynthesis or bone regeneration promoting activity.

In the present invention, the term "prevention" refers to the reduction of the degree of pathogenesis or damage to cells or loss of cells in an animal. Prevention can be complete or partial. In this case, the occurrence of pathological cells in the individual or osteoclast proliferation may mean a phenomenon that is reduced compared to the case where the composition for preventing and treating the fracture disease is not used.

In addition, the term "treatment" means improving one or more symptoms of a fracture disease or deterring the progression of symptoms, and may include the meaning of commonly used treatment.

The “pharmaceutical composition” of the present invention may further include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" in the present invention means that it exhibits non-toxic properties to cells or humans exposed to the composition.

The composition comprising a pharmaceutically acceptable carrier may be various oral or parenteral formulations. In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are usually used.

The carrier, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, It may be one or more selected from the group consisting of polyvinyl pyrrolidone, physiological saline, methylhydroxybenzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol and liquid paraffin, It is not limited to this, and any conventional carrier, excipient, or diluent may be used. The ingredients may be added independently or in combination to the active ingredient Doin extract.

In addition, the composition may be used in the form of an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, external preparation, suppository, and sterile injectable solution.

The solid preparation for oral administration includes tablets, pills, powders, granules, capsules, and the like, and the solid preparation includes at least one excipient in the extract of the phosphorus and its fractions, such as starch, calcium carbonate, sucrose, and lactose. Or, it can be prepared by mixing gelatin or the like. In addition to the excipients, lubricants such as magnesium stearate and talc may be used.

As used herein, the term “fraction” is an operation of separating each component or a specific group from various components or mixtures, that is, each component or group separated using chromatography, electrophoresis, centrifugation, and the like. Means

As a liquid preparation for oral administration, a suspension, an intravenous solution, an emulsion, or a syrup may be used. In addition to simple diluents such as water and liquid paraffin, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. have.

As a preparation for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories may be used. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The pharmaceutical composition comprising the doin extract may be administered to a subject in a pharmaceutically effective amount.

The “pharmaceutical effective amount” means an amount sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, drug activity, and drug Sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-drugs, and other factors well known in the medical arts.

The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is preferable to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The term “improvement” means any action that at least reduces the severity of the parameters associated with the condition being treated, for example symptoms.

The “food composition” of the present invention may exemplify meat, snacks, dairy products, beverages, and the like, but is not limited thereto, and may be understood as a concept including all of the normal health functional foods.

The health functional food refers to food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Act on Health Functional Food, and the functionality controls nutrients or physiologically regulates the structure and function of the human body. It may mean taking it for the purpose of obtaining a useful effect for health purposes such as action.

The dietary supplement may include ordinary food additives, and the food additives are in accordance with the standards and standards for the item in accordance with the General Regulations and General Test Methods of Food Additives approved by the Ministry of Food and Drug Safety unless otherwise specified. It is possible to judge suitability.

Items described in the food additives revolution include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid, natural additives such as chromic pigment, licorice extract, crystalline cellulose, high color pigments, guar gum, L-sodium glutamate formulation, and noodles And mixed preparations such as added alkalis, preservatives, and tar colorants.

The health functional food may be variously used in foods and beverages for improving fractures, and may be used, for example, in various foods, beverages, gums, teas, vitamin complexes, health functional supplements, and food additives.

The health functional food may include the composition of 1.0 to 10.0% by weight based on the total weight for the purpose of improving fracture diseases. If the composition is less than 1.0% by weight, a fracture disease improvement effect may not be sufficiently implemented, and if it is more than 10.0% by weight, the product's natural quality may not be realized or cost efficiency may be reduced.

In addition, the health functional food may be manufactured and processed into any one formulation selected from the group consisting of tablets, granules, powders, capsules, liquid solutions and pills for the purpose of improving fracture diseases.

Specifically, the health functional food in the form of tablets is granulated with a mixture of Doin extract or its fractions, excipients, binders, disintegrants and other additives in a conventional manner, and then compression molded with a lubricant or the like. It can be produced by direct compression molding. In addition, the health functional food in the form of tablets may contain a mating agent or the like as necessary, and may be coated with a suitable peeling agent if necessary.

Among the health functional foods in the form of capsules, the hard capsules may be prepared by filling a conventional hard capsule with a mixture of additives such as eggplant extracts and excipients or granular or peeled granules thereof, and the soft capsule is an extract of Doin or its A mixture with an additive such as a fraction and an excipient can be prepared by filling a capsule base such as gelatin.

The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, and a preservative, if necessary.

The health functional food in the form of a ring may be prepared by molding a mixture of a phosphorus extract or a fraction thereof, an excipient, a binder, a disintegrant, etc. in an appropriate manner, and, if necessary, peeling with white sugar or other suitable peeling agents, or starch or talc. Alternatively, it may be reconciled with a suitable material.

The granular form of the health functional food may be prepared in the form of a mixture of an extract of Doin or its fractions, excipients, binders, disintegrants, etc. in a suitable manner, and may contain flavoring agents, mating agents, etc. as necessary.

In addition, the term definitions for the excipients, binders, disintegrants, lubricants, mating agents, flavoring agents, etc. are described in the literature known in the art and may include those having the same or similar functions.

According to one aspect of the present invention, by using a natural herbal extract Doin extract as a component of the pharmaceutical composition or food composition, it is possible to improve the effect of improving fracture disease while minimizing side effects.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the invention.

1 is a result of measuring the cell viability according to the phosphorus extract (PF) treatment.
2 is a result of evaluating osteoblast differentiation ability according to the concentration of the extract treatment using Alizarin S staining method.
3 is a result of measuring the effect on the expression of mitogen-activated protein kinase (MAPK) according to the treatment time of the extract of Doin using BCA analysis.
Figure 4 is a result of evaluating the degree of recovery of the fracture site according to the treatment of the extract in the fracture animal model micro-CT, (A) is a cross-sectional view of the fracture site, (B) is a front view of the fracture site .

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, the present invention is not limited by the following examples.

Preparation Example 1: Method of extracting ethanol

Doin (Heukkuk, Hebei) 500 g of Doin was added to 3000 mL of 80% ethanol and immersed for 1 week. The extract was filtered with filter paper (Whatman, No 2), and the filtrate was concentrated under reduced pressure and lyophilized to powder. The dried ethanol extract (Ethanol extract of Prunus davidiana Franchet, PF) was 13.95 g, and the yield was 2.79%. The extract of Doin was used for refrigeration, and the Doin used for cell culture was filtered through a 0.22 μm sterile filter.

Preparation Example 2: Cell culture

Dispensed MC3T3-E1 cells in α-MEM (Minimum Essential Medium Eagle-alpha modification) medium containing 10% FBS (Fetal Bovine Serum) and 1% P/S (penicillin and streptomycin) to a humidity of 95%, Cultured at 37°C and 5% CO ₂ .

Experimental Example 1: Cytotoxicity Analysis

In order to check the cytotoxicity of the extract of the phosphorus, the cell extract of the preparation example 1 was treated with the cells of the preparation example 2, and then cell proliferation was measured using an MTT assay.

Specifically, after the cells were stabilized for 24 hours, 10, 20, and 40 μg/mL of the extract extracted from the culture medium were treated, respectively, and then cultured for 24 hours in an incubator under the same conditions.

The cultured cells were measured for absorbance at 562 nm after treatment with MTT assay solution.

The cytotoxicity measurement results indicated the absorbance of the sample as a percentage of the absorbance of the control (doin extract 0 μg/mL).

As a result of the measurement, cytotoxicity was not observed when treated with 10, 20, and 40 μg/mL of the extract of Doin (FIG. 1 ).

Experimental Example 2: Osteoblast differentiation evaluation

In order to investigate the effect of the phosphorus extract on osteoblast differentiation, the cells of Preparation Example 2 were treated with ascorbic acid (25 ug/ml) and β-glycerophosphate (10 mM), which are osteoblast differentiation inducers, to induce osteoblast differentiation. , Alizarin S staining was used to analyze osteoblast differentiation potential.

When the differentiation ability of osteoblasts is increased by the inducing agent, intracellular calcium deposition is further increased, and the degree of calcium deposition can be confirmed through Alizarin S staining.

Specifically, MC3T3-E1 cells of Preparation Example 2 were treated with 25 μg/mL ascorbic acid and 10 mM β-glycerophosphate, and treated with extracts 0, 10, 20, and 40 μg/mL, respectively, for 21 days. Cultured. The differentiated cells were fixed with 80% Et-OH for 10 minutes. Then, it was stained with Alizarin red S solution for 10 minutes, and washed with DPBS. The degree of calcium deposition was compared by taking a picture of the plate directly (Fig. 2).

The group not treated with anything was set as the normal group.

2, in the normal group, there was no staining with Alizarin S, and in the control group treated only with the inducer, differentiated osteoblasts were stained with red.

In the experimental group treated with the inducer and the extract of phosphorus, the degree of calcium deposition increased in a concentration-dependent manner than when the extract was not treated with the extract.

Experimental Example 3: Analysis of MAPK expression in osteoblasts

The effect of Doin extract on the expression of mitogen-activated protein kinase (MAPK), an osteoblast differentiation mechanism, was analyzed using a BCA assay.

Specifically, after treatment with MC3T3-E1 cells of Preparation Example 2 at 40 μg/mL for 5, 15, and 30 minutes, the cells were dispensed with RIPA buffer, scraped with scraper, and reacted on ice for 30 minutes. .

Then, centrifugation was performed at 13200 rpm for 20 minutes to extract the entire protein. The extracted protein was quantified through a BCA assay, and each sample was unified to 30 μg, electrophoresed at 10% polyacrylamide/SDS gel at 100 V, and transferred to a nitrocellulose transfer membrane.

Membrane was placed in TBST with 5% skim milk and 0.5% Tween-20 (sigma aldrich, MO, USA) and reacted for 1 hour to prevent non-specific protein from sticking.

Thereafter, p-ERK, t-ERK, p-JNK, t-JNK, p-p38 and t-p38 were reacted, respectively. After that, the secondary antibody was reacted according to the origin of the primary antibody. Thereafter, the membrane was developed using an ECL solution (Santacruz, California USA) on a membrane in a dark room through a substrate reaction, and developed on an X-ray film (FIG. 3).

Referring to FIG. 3, the expression of P-ERK, P-JNK and P-p38 increased at all times of treatment with the Doin extract. Particularly, when the extract of Doin was treated for 5 and 15 minutes, the expression of P-ERK, P-JNK and P-p38 was significantly increased compared to the normal group.

Experimental Example 4: Fracture animal model analysis

To investigate the effect of the extract of phosphorus on the fracture recovery, the progress of the fracture treatment was analyzed by micro-CT of the right femur after treating the extract with SD-rat (distributed by Coretech) that caused the fracture.

Specifically, male 8-week-old SD-rats had an adjustment period of 1 week. Then, after inhalation anesthesia with isoflurane, the central portion of the lower right femur was cut using a chainsaw, and the cut femur was fixed by inserting a wire toward the knee.

Subsequently, after randomly dividing the group, antibiotics were administered for 3 days, and the wound gave a recovery period for 1 week. The experiment was conducted by classifying the following groups. The experimental animals were divided into 4 groups of 8 animals each.

1) Non-femoral ablation control group (Normal),

2) Femoral resection induced group (control),

3) Doin dose group (PF-L) at a concentration of 50 mg/kg after femur resection,

4) Doin dose group (PF-H) at a concentration of 100 mg/kg after femur resection

Each group was orally administered with 1 ml of the sample daily and kept for 4 weeks. During the experiment, dietary intake and body weight were measured at regular times each week. The thigh bone extracted after sacrifice was photographed on the fracture site and the front of the femur using a micro-CT (Skyscan) device (FIG. 4).

Referring to FIG. 4, the density and size of callus of SD-rat to which the extract was administered was increased (A).

In addition, when confirming the frontal image of the femur (B), the recovery of the fracture site was markedly improved by treatment with the Toin extract.

The above description of the present invention is for illustration only, and a person having ordinary knowledge in the technical field to which the present invention pertains can understand that it can be easily modified to other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts should be interpreted to be included in the scope of the present invention.

Claims (8)

A pharmaceutical composition for the prevention or treatment of fractures, comprising the extract of Prunus davidiana Franchet. According to claim 1,
The extract is water, ethanol, ethyl acetate, acetone, nucleic acid, dichloromethane or a pharmaceutical composition for preventing or treating fractures extracted with a mixed solvent thereof. According to claim 2,
The extract is a pharmaceutical composition for preventing or treating fractures extracted using ethanol as an extraction solvent. According to claim 3,
The extract is a pharmaceutical composition for preventing or treating fractures extracted by ethanol at a concentration of 70 to 90% (w/w). According to claim 1,
A pharmaceutical composition for preventing or treating fractures that promote osteoblast differentiation. The method of claim 5,
A pharmaceutical composition for preventing or treating fractures that promote bone union or bone regeneration. Food composition for preventing or improving fractures, including the extract of Doin. The method of claim 7,
A food composition for preventing or improving fractures showing the effect of promoting bone union or bone regeneration due to promoting osteoblast differentiation.

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