KR102604848B1 - Composiotion for prevention, improvement or treatment of fracture including prunus davidiana franchet - Google Patents

Abstract

The present invention provides a composition for preventing, improving and treating fractures containing an extract of Paeonia lactiflora Pallas.

Description

Composition for preventing, improving or treating fractures comprising extracts

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

Bone supports the body's soft tissues and body weight, surrounds internal organs, and protects them from external shock.

In addition, bones are one of the important parts of the human body that not only structurally support muscles and organs, but also store substances such as calcium and other essential minerals such as phosphorus and magnesium.

Therefore, adult bones that have completed growth maintain balance by dynamically and continuously repeating the process of generation and absorption, in which old bone is removed and replaced with new bone. This is called bone remodeling.

Bone turnover is essential for recovering microscopic damage to bone caused by growth and stress and maintaining its function. In adults, approximately 10-30% of the skeleton is remodeled each year through bone resorption-osteogenesis remodeling.

Two types of cells are largely involved in bone remodeling: osteoblasts, which produce bone, and osteoclasts, which destroy bone. Bone homeostasis is maintained through close association with each other.

For example, osteoblasts regulate the differentiation of osteoclasts, which are responsible for bone resorption, through the secretion of substances such as RANKL (receptor activator of nuclear factor-κB ligand) and its decoy receptor, OPG (osteoprotegerin), thereby contributing to bone formation in the body. Maintain homeostasis.

In other words, when RANKL binds to RANK, the surface receptor of osteoclast precursor cells, the osteoclast precursor cells mature into osteoclasts and bone resorption occurs. When OPG binds to RANKL, the bond between RANKL and RANK is blocked, thereby inhibiting the formation of osteoclasts.

Osteoclasts, which are formed from progenitor cells, absorb or destroy old bone and release a small amount of calcium accumulated in the bone into the bloodstream to maintain body functions.

The metabolism is regulated by physical influences, hormonal systems, and local factors. When bone resorption exceeds bone formation due to various factors and bone mass decreases below the limit, various bone diseases accompanied by damage to bone tissue occur.

In relation to the treatment of diseases accompanied by bone tissue damage, research has been conducted in the past mainly focusing on abnormalities in the metabolism of bone minerals, that is, calcium and phosphorus, and no progress has been made in identifying the mechanisms. Therefore, much research is being conducted to develop substances that inhibit osteoclast differentiation.

Meanwhile, herbal medicines mainly used in oriental medicine have the potential to become more fundamental therapeutic agents through multi-target mechanisms in the prevention and treatment of chronic and incurable diseases with complex causes and symptoms.

In addition, therapeutic agents using these herbal medicines as raw materials are attracting attention as alternative materials to existing pharmaceutical ingredients because they can minimize various side effects that may occur due to the use of chemical agents.

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

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating fractures containing an extract of Prunus davidiana Franchet.

Another object of the present invention is to provide a food composition for preventing or improving bone fractures containing an extract of ginseng.

One aspect of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating fractures containing an extract of Prunus davidiana Franchet.

In the present invention, the " Prunus davidiana Franchet" is obtained by drying the mature seeds of Prunus persica (L.) Batsch. and P. davidiana (Carr.) Franch., which are deciduous small trees belonging to the Rosaceae family. (HERBOLOGY EDITORIAL COMMITTEE OF KOREAN MEDICINE SCHOOLS. Boncho-hak[Herbology], Seoul: Younglimsa, 2004.), Recently, anti-inflammatory effect using Chinese herbal medicine (Jo Woo-a et al. Cosmetic pharmacological activity and anti-inflammatory effect of Japanese herbal medicine bark. University of Korean Vegetable Science Association)誌 2006; 21 (2): 87-93.) there is. In particular, Taoism has traditionally been used clinically to improve blood circulation and remove stagnant blood.

However, the effect of Daoin on improving fracture disease or its related mechanism or efficacy is not known to date, and the present inventors sought to determine the effect of the Daoin extract on promoting osteoblast differentiation.

In the present invention, the “extract” is obtained by separating the active ingredients contained in the extraction material by contacting the solvent and the extraction material under specific conditions, and the extract may contain the active ingredients in the raw material through an extraction process for the extract.

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

Additionally, the extraction method may use various methods such as hot water extraction, cold extraction, reflux extraction, and ultrasonic extraction, but is not limited thereto.

The term “fracture” refers to a state in which the continuity of bone or cartilage is broken due to excessive impact or force. The treatment of fractures goes through an inflammatory phase, a restorative phase, a remodeling phase, and a healing phase. Health functional foods and herbal medicines that promote bone union can be selected effectively depending on 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 remodeling and treatment of metabolic bone diseases, 2012, Eur J Clin Invest.), osteoclast activation through various causes induces osteoporosis, arthritis, and osteonecrosis. , it can be seen that the degeneration of osteoclasts causes osteopetrosis.

However, on the contrary, unlike other metabolic bone diseases, the activity of osteoblasts is very important in the treatment of fractures. Referring to a previously published paper (Frederic Shapiro, Bone development and its relationship to fracture repair. The role of mesenchymal osteoblasts and surface osteoblasts, 2008, European Cells and Materials), treatment of fractures involves 1) differentiation from mesenchymal stem cells; Bone formation through differentiation into osteoblasts and 2) activation of osteoblasts on the bone surface play the most important role in fracture treatment.

For this reason, it can be determined that Daoin extract, which induces the activity of osteoblasts, has the potential to specifically treat fractures, unlike existing bone metabolism treatments.

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

In addition, it was confirmed that when the MC3T3-E1 cells were treated with Doin extract, the expression of MAPK (mitogen-activated protein kinase), which is a differentiation mechanism of osteoblasts, was enhanced (Figure 3).

In the analysis of the fracture animal model, which is an example of the present invention, it was confirmed that treatment with the ginseng extract improved the recovery of the fracture site (FIG. 4).

The above results suggest that the Daoyin extract of the present invention has a high differentiation-promoting activity of osteoblasts, and can be usefully used as a composition for preventing, improving, and treating fractures with excellent bone union or bone regeneration-promoting activity.

In the present invention, “prevention” means reducing the degree of occurrence of pathological cells or cell damage or loss in animals. Prevention may be complete or partial. In this case, it may mean a phenomenon in which the occurrence of pathological cells or proliferation of osteoclasts in an individual is reduced compared to the case where the composition for preventing and treating fracture diseases is not used.

In addition, the “treatment” means improving one or more symptoms of a fracture disease or preventing the progression of the symptoms, and may encompass the meaning of commonly used treatments.

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

The composition containing a pharmaceutically acceptable carrier may be in various oral or parenteral dosage forms. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, 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, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, dextrin, calcium carbonate, propylene glycol, and liquid paraffin. It is not limited to this, and all common carriers, excipients, or diluents can be used. The ingredients may be added independently or in combination with the active ingredient, Toin extract.

In addition, the composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions.

The solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are made by mixing the ginseng extract and its fractions with at least one excipient, such as starch, calcium carbonate, sucrose, and lactose. , or it can be prepared by mixing gelatin, etc. In addition to the above excipients, lubricants such as magnesium styrate and talc may also be used.

As used herein, the term "fraction" refers to the operation of separating each component or specific group from various components or mixtures, that is, each component or group separated using chromatography, electrophoresis, centrifugation, etc. means.

Liquid preparations for oral administration may include suspensions, oral solutions, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to simple diluents such as water and liquid paraffin. there is.

Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. The non-aqueous solvent or suspension may be propylene glycol, polyethylene glycol, vegetable oil such as olive oil, or injectable ester such as ethyl oleate. As a base for the suppository, witepsol, macrogol, tween 61, cacao, laurel, and glycerogenatin can be used.

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

The "pharmaceutically effective amount" means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the patient's disease, the activity of the drug, and the It can be determined based on factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

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 conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is desirable to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The term “improvement” means any action that results in at least a reduction in the severity of the parameters associated with the condition being treated, such as symptoms.

The "food composition" of the present invention may include, but is not limited to, meat, snacks, dairy products, beverages, etc., and may be understood as a concept that includes all common health functional foods.

The above health functional food refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with the Health Functional Food Act, and the functionality refers to food that regulates nutrients for the structure and function of the human body or has physiological properties. It may mean ingestion for the purpose of obtaining useful effects for health purposes, such as action.

The above-mentioned health functional food may contain common food additives, and unless otherwise specified, the above-mentioned food additives shall be subject to the specifications and standards for the relevant item in accordance with the general provisions of the Food Additives Code and General Test Methods approved by the Ministry of Food and Drug Safety. Suitability can be judged by

Items listed in the Food Additive Code include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as subchromic pigment, licorice extract, crystalline cellulose, high-lyang pigment, and guar gum, sodium L-glutamate preparations, and noodles. Examples include mixed preparations such as added alkaline agents, preservatives, and tar colorants.

The health functional food can be used in a variety of foods and beverages to improve fractures, and for example, it can be used in various foods, beverages, gum, tea, vitamin complexes, health functional supplements, food additives, etc.

The health functional food may contain 1.0 to 10.0% by weight of the composition based on the total weight for the purpose of improving fracture disease. If the composition is less than 1.0% by weight, the effect of improving fracture disease may not be sufficiently realized, and if it is more than 10.0% by weight, the original quality of the product 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 made by granulating a mixture of ginseng extract or fractions thereof, excipients, binders, disintegrants and other additives in a conventional manner, and then adding a lubricant and compression molding, or by adding the mixture. It can be manufactured by direct compression molding. In addition, the health functional food in the form of tablets may contain a flavoring agent, etc., if necessary, and may be peeled with a suitable peeling agent, if necessary.

Among the capsule-type health functional foods, hard capsules can be manufactured by filling ordinary hard capsules with a mixture of additives such as eggplant extract and excipients, or their granules or peeled granules, and soft capsules can be prepared by filling a regular hard capsule with additives such as eggplant extract and excipients, or their granules or peeled granules. It can be manufactured by filling a mixture of fractions and additives such as excipients into a capsule base such as gelatin.

The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary.

The above health functional food in the form of a pill can be prepared by molding a mixture of ginseng extract or its fractions, excipients, binders, disintegrants, etc. in an appropriate manner, and if necessary, coated with white sugar or other suitable coating agent, or added with starch or talc. Alternatively, you can dress it up with a suitable material.

The granular health functional food can be manufactured into granules from a mixture of ginseng extract or fractions thereof, excipients, binders, disintegrants, etc., by an appropriate method, and may contain flavoring agents, flavoring agents, etc. as needed.

In addition, definitions of terms such as excipients, binders, disintegrants, lubricants, corrigants, flavoring agents, etc. are described in literature known in the art and may include those with the same or similar functions.

According to one aspect of the present invention, the effect of improving bone fracture disease can be improved while minimizing side effects by using a natural herbal medicine, Pyokin extract, as a component of a pharmaceutical composition or a food composition.

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

Figure 1 shows the results of measuring the cell survival rate according to treatment with Peach extract (PF).
Figure 2 shows the results of evaluating osteoblast differentiation ability according to the concentration of Doin extract treatment using Alizarin S staining.
Figure 3 shows the results of measuring the effect on the expression of MAPK (mitogen-activated protein kinase) according to the treatment time of the ginseng extract using BCA analysis.
Figure 4 shows the results of evaluating the degree of recovery of the fracture site according to treatment with Daoin extract in a fracture animal model using micro-CT. (A) shows a cross-sectional view of the fracture site, and (B) shows a frontal view of the fracture site. .

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the present invention is not limited by the following examples.

Preparation Example 1: Doin ethanol extraction method

500 g of Doin (Bamboo soup from Hebei) was soaked and extracted by adding 3000 mL of 80% ethanol for 1 week. The extract was filtered through filter paper (Whatman, No 2), and the filtrate was concentrated under reduced pressure, freeze-dried, and powdered. The dried Ethanol extract of Prunus davidiana Franchet (PF) was 13.95 g, and the yield was 2.79%. The extract was refrigerated and used, and the extract used during cell culture was filtered through a 0.22 ㎛ sterilizing filter.

Preparation Example 2: Cell Culture

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

Experimental Example 1: Cytotoxicity analysis

In order to determine the cytotoxicity of the ginseng extract, the podin extract of Preparation Example 1 was treated with the cells of Preparation Example 2, and then cell proliferation was measured using the MTT assay.

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

The cultured cells were treated with MTT assay solution and the absorbance was measured at 562 nm.

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

As a result of the measurement, no cytotoxicity was observed when treated with 10, 20, and 40 μg/mL of Doin extract (Figure 1).

Experimental Example 2: Evaluation of osteoblast differentiation ability

In order to determine the effect of the Daoin extract on osteoblast differentiation, the cells of Preparation Example 2 were treated with osteoblast differentiation inducers ascorbic acid (25 ug/ml) and β-glycerophosphate (10 mM) to induce osteoblast differentiation. , the differentiation capacity of osteoblasts was analyzed using Alizarin S staining.

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

Specifically, the MC3T3-E1 cells of Preparation Example 2 were treated with 25 μg/mL of ascorbic acid and 10 mM β-glycerophosphate and 0, 10, 20, and 40 μg/mL of Doin extract, respectively, for 21 days. Cultured. Cells that had completed differentiation were fixed with 80% Et-OH for 10 minutes. Afterwards, it was stained with Alizarin red S solution for 10 minutes and washed with DPBS. The degree of calcium deposition was compared by directly photographing the plate (Figure 2).

The group that did not receive any treatment was set as the normal group.

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

In the experimental group treated with the inducer and the Daoin extract together, the degree of calcium deposition increased in a concentration-dependent manner compared to the case where the Daoin extract was not treated.

Experimental Example 3: Analysis of MAPK expression in osteoblasts

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

Specifically, the MC3T3-E1 cells of Preparation Example 2 were treated with 40 μg/mL of dye for 5, 15, and 30 minutes, and then RIPA buffer was dispensed into the cells, scraped off with a scraper, and reacted on ice for 30 minutes. .

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

The membrane was added to TBST dissolved in 5% skim milk and 0.5% Tween-20 (Sigma Aldrich, MO, USA) and reacted for 1 hour to prevent non-specific proteins from attaching.

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

Referring to Figure 3, the expression of P-ERK, P-JNK, and P-p38 increased at all times of treatment with the Doin extract. In particular, when treated with the Daoin extract 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

In order to determine the effect of ginseng extract on fracture recovery, SD-rats (sold by Coretech) that caused fractures were treated with ginseng extract, and the progress of fracture treatment was analyzed using micro-CT of the right femur.

Specifically, male 8-week-old SD-rats had an adaptation period of 1 week. Afterwards, after inhalation anesthesia with isoflurane, the central part of the lower right femur was cut using an electric saw, and a wire was inserted into the knee to fix the cut femur.

Afterwards, the participants were randomly divided into groups and administered antibiotics for 3 days, and the wounds were allowed to recover for 1 week. The experiment was conducted by dividing the subjects into the following groups. The experimental animals were divided into 4 groups with 8 animals each.

1) Non-femur resection control group (Normal),

2) femur resection induced group (control),

3) Doyin dose group at 50 mg/kg concentration after femoral resection (PF-L),

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

Each group was reared for 4 weeks by orally administering 1 ml of the sample every day. During the experimental period, food intake and body weight were measured at regular times every week. The femur bone removed after sacrifice was photographed using a micro-CT (Skyscan) device to image the fracture site and the front surface of the femur (Figure 4).

Referring to Figure 4, the density and size of the callus of SD-rats administered the Doin extract increased (A).

Additionally, looking at the frontal photograph of the femur (B), the recovery of the fracture site was significantly improved by treatment with Taoist extract.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as unitary may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

Claims (8)

Prunus davidiana Franchet Food composition for preventing or improving femur fractures containing 80% ethanol. According to paragraph 1,
The extract is a food composition for preventing or improving fractures that promotes differentiation of osteoblasts. According to paragraph 2,
A food composition for preventing or improving fractures, wherein the differentiation of osteoblasts promotes bone union or bone regeneration. According to paragraph 1,
The extract is a food composition for preventing or improving fractures, characterized in that it increases phosphorylation of factors selected from the group consisting of osteoblast differentiation factors ERK, JNK, and p38. A pharmaceutical composition for preventing or treating femur fractures comprising an 80% ethanol extract of Prunus davidiana Franchet. According to clause 5,
The ethanol extract is a pharmaceutical composition for preventing or treating fractures, characterized in that it promotes bone union or bone regeneration by promoting osteoblast differentiation. A method of recovering femur fractures in vitro by treating cells with 80% ethanol extract to increase phosphorylation of osteoblast differentiation factors selected from the group consisting of ERK, JNK, and p38.

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