KR102410703B1 - Composition for preventing, improving or treating bone disease comprising Pyrrosia lingua extract as effective component - Google Patents

Abstract

The present invention relates to a composition for the prevention, improvement or treatment of bone diseases, comprising the extract of shiitake as an active ingredient. It has the effect of increasing cancellous bone density and bone volume in an ovariectomized animal model. Therefore, the stone extract of the present invention can be usefully used as a health functional food or medicine for the prevention, improvement or treatment of bone diseases.

Description

A composition for preventing, improving or treating bone disease comprising a stone extract as an active ingredient {Composition for preventing, improving or treating bone disease comprising Pyrrosia lingua extract as effective component}

The present invention relates to a composition for preventing, improving, or treating bone diseases, comprising an extract of Pyrrosia lingua as an active ingredient.

Bone (bone) supports the body's soft tissues and body weight and surrounds internal organs to protect internal organs from external impact. In addition, it is one of the important parts 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.

Bone is a very complex tissue composed of cells, collagenous matrix and mineral components. They provide basic and diverse functions, such as providing a microenvironment necessary for hematopoiesis, as well as protecting organs. In addition, it is one of the important parts 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.

Adult bones that have completed growth do not stop, and the old bone is removed and replaced with new bone until the day of death. This is called bone remodeling.

The turnover of removing old bones and replacing them with new ones is essential to restore microscopic damage to bones caused by growth and stress and to maintain proper bone function. It is known that two types of cells are largely involved in bone remodeling. One of the two cells is an osteoblast that creates bone, and the other is an osteoclast that destroys the bone. Osteoblasts produce RANKL (receptor activator of nuclear factor-κB ligand) and its inducible receptor (decoy receptor) OPG (osteoprotegerin). When RANKL binds to receptor activator of nuclear factor-κB (RANK), a receptor on the surface of osteoclast progenitor cells, the osteoclast progenitor cells mature into giant multinucleated osteoclasts (PLturation) and bone resorption occurs. . However, when OPG binds to RANKL, the binding between RANKL and RANK is blocked, inhibiting the formation of osteoclasts and preventing excessive bone resorption. The activity of these osteoclasts leads to resorption or destruction of old bone, which makes a hole in the bone and releases a small amount of calcium into the bloodstream, which is used to maintain body functions.

On the other hand, Seokwi ( Pyrrosia lingua ) is a perennial plant of the fern family, Goranaceae, and grows by attaching to tree trunks and rock surfaces. The rhizome extends laterally, is 3mm long, and is covered with red or dark brown scales. The petiole is 10-26cm in diameter, hard, grooved, and covered with hairs. The leaf body is a broad lancet-shaped or egg-shaped lancet-shaped, and both ends are narrow and thick. The front side of the leaf is dark green and hairless, but the back side has dense brown hairs and the edges are flat. The sporangial group has no membrane and is densely formed on the entire back surface. In oriental medicine, the leaves and roots are used as diuretics.

As a prior art related to bone disease, Korea Patent Publication No. 2019-0115789 discloses a drug for treating osteoporosis and a method for manufacturing the same, and Korean Patent No. 0380865 discloses an extract having an effect on preventing and treating osteoporosis. However, there has been no disclosure of a composition for preventing, improving, or treating bone diseases containing the stone extract of the present invention as an active ingredient.

The present invention has been derived from the above needs, and the present invention provides a composition for preventing, improving, or treating bone diseases, comprising a stone extract as an active ingredient, wherein the extract is used to inhibit osteoclast differentiation and reduce osteoclasts. The present invention was completed by confirming that it has an effect of promoting differentiation, increasing the survival rate of osteoclast progenitors, and increasing cancellous bone density and bone volume in an ovariectomized animal model.

In order to achieve the above object, the present invention provides a health functional food composition for preventing or improving bone disease containing an extract of Pyrrosia lingua as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of bone diseases, containing the extract of stone yam as an active ingredient.

In addition, the present invention provides a composition for differentiation of mesenchymal stem cells into osteoblasts containing the extract of shiitake as an active ingredient.

The present invention relates to a composition for the prevention, improvement or treatment of bone diseases, comprising the extract of shiitake as an active ingredient. It has the effect of increasing cancellous bone density and bone volume in an ovariectomized animal model.

1 is a result confirming the osteoclast formation inhibitory effect according to the treatment concentration of Seokwi hot water extract (WEPL) (A) and Seokwi 70% ethanol extract (EEPL) (B). ** indicates that the number of osteoclasts in the experimental group treated with the Seokwi extract of the present invention was statistically significantly decreased compared to the control group not treated with the Seokwi extract of the present invention, p<0.01.
Figure 2 is the result of Alizarin Red S staining confirming the osteoblast differentiation promoting effect according to the treatment of stone ethanol extract (EEPL). ** indicates that the osteoblast differentiation of the group treated with the Seokwi ethanol extract was statistically significantly increased compared to the control group untreated, p<0.01.
3 is a result confirming the cell viability of osteoclast progenitor cells according to the treatment concentration of Seokwi hot water extract (WEPL) (A) and Seokwi 70% ethanol extract (EEPL) (B). * and ** indicate that the cell viability of the experimental group treated with the Seokwi extract of the present invention was statistically significantly increased compared to the control group not treated with the Seokwi extract of the present invention, with * being p<0.05, and ** being p<0.01.
4 is a result confirming the changes in bone density (BMD) (A) and bone volume (BV/TV) (B) of cancellous femoral bone according to oral administration of stone ethanol extract (EEPL) in an ovariectomized (OVX) mouse model. ## indicates that the bone mineral density (BMD) and bone volume (BV/TV) of the cancellous femoral bone in the ovary resected mouse group (OVX) were statistically significantly decreased compared to the non-ovarian normal mouse group (Sham). p<0.01, * and ** indicate that the cancellous bone mineral density or bone volume in the group administered with 30 and 100 mg/kg of stone ethanol extract after ovariectomy compared with the ovariectomized mouse group (OVX) was statistically significantly increased, * is p<0.05, ** is p<0.01.

The present invention relates to a health functional food composition for preventing or improving bone disease containing an extract of Pyrrosia lingua as an active ingredient.

The stone extract may be prepared by a method comprising the following steps, but is not limited thereto:

(1) extracting by adding an extraction solvent to the stone;

(2) filtering the extract of step (1); and

(3) Concentrating the filtered extract of step (2) under reduced pressure and drying to prepare an extract.

The extraction solvent in step (1) is preferably selected from water, C ₁ to C ₄ lower alcohols or mixtures thereof, more preferably water or ethanol, and still more preferably ethanol, but is not limited thereto.

In the above preparation method, the extraction method may use all conventional methods known in the art, such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction. The extraction solvent is preferably added by adding 1 to 20 times the weight of the dried stone rock, more preferably 5 to 15 times. The extraction temperature is preferably 20 ~ 110 ℃, but is not limited thereto. In addition, the extraction time is preferably 0.5 to 10 hours, more preferably 0.5 to 5 hours, but is not limited thereto. In the above method, the vacuum concentration in step (3) is preferably, but not limited to, a vacuum vacuum concentrator or a vacuum rotary evaporator. In addition, drying under reduced pressure, vacuum drying, boiling drying, spray drying or freeze drying is preferable, but is not limited thereto.

The stone extract is preferably obtained by extracting stone leaves, but is not limited thereto.

The stone extract has the characteristics of inhibiting the differentiation of osteoclasts and promoting osteogenesis, the bone disease may be caused by bone loss, and the bone disease is osteoporosis, osteoporotic fracture, bone loss, bone defect, nonunion. It is preferable that any one selected from among fractures, osteodystrophy, osteomalacia, osteomalacia fractures, osteogenesis disorders, neoplastic bone destruction by tumors, bone destruction by periodontal diseaes and degenerative bone diseases is preferable. It is not limiting.

The composition is preferably prepared in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto.

When the health functional food composition of the present invention is used as a food additive, the stone extract may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of its use (prevention, health or therapeutic treatment). In general, in the production of food or beverage, the composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material. However, in the case of long-term intake for health and hygiene or health control, 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 greater than the above range.

There is no particular limitation on the type of the food. Examples of foods to which the extract can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health functional foods in the ordinary sense.

When the composition of the present invention is used as a health drink, it may contain various flavoring agents or natural carbohydrates as an additional component like a conventional drink. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclotenstrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as taumarin and stevia extract, synthetic sweeteners such as saccharin or aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 g of the composition of the present invention. The composition of the present invention contains various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal agents, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid It may contain a carbonation agent used in beverages, and the like. In addition, the composition of the present invention may contain the pulp for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. Although the proportion of these additives is not very important, the composition of the present invention is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.

In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of bone diseases containing the extract of Pyrrosia lingua as an active ingredient.

The composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient, and may be in various oral or parenteral formulations. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include capsules, powders, granules, tablets, pills, etc., and such solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, emulsions, syrups, aerosols, etc., and various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. may be included in addition to water and liquid paraffin, which are commonly used simple diluents. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilisates, and suppositories. As the non-aqueous solvent and the suspending solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycero gelatin, etc. may be used. For parenteral administration, it is preferable to select external or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the level of the effective amount is determined by the type, severity, and drug activity of the patient. , can be determined according to factors including sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the composition of the present invention varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease. The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

In addition, the present invention relates to a composition for differentiation of mesenchymal stem cells into osteoblasts containing the extract of shiitake as an active ingredient.

Mesenchymal stem cells (MSC) in the bone marrow differentiate into cartilage, bone tissue, ligament, and bone marrow matrix, and are stem cells suitable for tissue regeneration with immunomodulatory and inhibitory functions. Mesenchymal stem cells refer to stem cells present in cartilage, bone tissue, adipose tissue, and bone marrow stroma, which are differentiated from the live mesoderm by division of a fertilized egg. The mesenchymal stem cells in the present invention should grow attached to the bottom in culture conditions, have the ability to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro, express CD73, CD90 and CD105, and c-kit , characterized by the absence of expression of CD11b, CD19, CD14, CD34, CD45, CD14, CD79 and HLA-DR.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

Example 1. Preparation of stone extract

An extract of Pyrrosia lingua was prepared as follows.

For hot water extract (WEPL) of Seokwi, 500 g of Seokwi leaf was added to 3.5 liters of distilled water, extracted under reflux (Heating PLntle) for 3 hours, filtered through a qualitative filter paper (Qualitative Filter Paper, Ø185 mm), and freeze-dried. In the cell experiment, hot water extract (WEPL) dissolved in distilled water was filtered through a 0.22 μl filter and then used for the experiment.

For the ethanol extract (EEPL) of Seokwi, 500g of Seokwi was put in 3.5ℓ of 70% ethanol, extracted under reflux for 3 hours (Heating PLntle), filtered with Qualitative Filter Paper (Ø185mm), and then concentrated with a rotary evaporator. Then freeze-dried. After dissolving in dimethyl sulfoxide, an ethanol extract of stoneweed (EEPL) was used for the experiment. In animal experiments, the freeze-dried extract was dissolved in sterile distilled water and used.

Example 2. Analysis of the effect of Seokwi extract on osteoclast differentiation

The purpose of this study was to determine whether Seokwi extract inhibits the differentiation of osteoclast precursor cells into osteoclasts. Osteoclast progenitor cells were treated with hot water extract (WEPL) and ethanol extract (EEPL) from Seokwi by concentration, and the number of osteoclasts was analyzed accordingly.

Specifically, bone marrow cells of mice were cultured in α-MEM medium containing M-CSF (60ng/ml) and 10% FBS for 3 days to obtain bone marrow cell-derived macrophages (BMM), which were then transformed into osteoclast progenitors. was used. To generate osteoclasts, 1×10 ⁴ cells per well of a 96-well plate were put in the obtained BMM, and then cultured in a medium containing M-CSF (60 ng/ml) and RANKL (50 ng/ml) for 4 days. In the same culture conditions, 11.1, 33.3, 100 and 200 μg/ml of Seokwi hot water extract (WEPL) and 11.1, 33.3 and 100 μg/ml of ethanol extract (EEPL) were treated in BMM. After fixing the cells with 10% formalin, TRAP (tartrate-resistant acid phosphatase) was stained using naphthol AS-MS phosphate and fast red violet LB salt. cells, osteoclast) were counted.

As a result, the osteoclast differentiation inhibitory effect was statistically significant by treatment with 33.3, 100, and 200 µg/ml Seokwi hot water extract (WEPL). The differentiation inhibitory effect was found to be statistically significant.

Example 3. Analysis of the effect of Seokwi extract on osteoblast differentiation

The purpose of this study was to determine whether Seokwi extract promotes osteoblast differentiation. Mesenchymal stem cells were treated with ethanol extract (EEPL) for each concentration, and the degree of differentiation into osteoblasts was analyzed by Alizarin Red S staining.

Specifically, mouse bone marrow-derived mesenchymal stem cells (D1 ORL UVA) were placed in 1.7×10 ⁴ per well of a 96-well plate, and then cultured in DMEM medium containing 10% FBS for 2 days. After replacement with α-MEM medium containing 10% FBS, ascorbic acid (50㎍/㎖) and β-glycerophosphate (β-glycerophosphate, 5mM) were treated for 8 days to induce osteoblast differentiation, and the same In culture conditions, it was treated with a concentration of ethanol extract (EEPL) (11.1 and 33.3 ㎍ / ㎖) of Seokgwi. Cells were fixed with 10% formalin and then stained with Alizarin red S to check the degree of calcification according to osteoblast differentiation. Alizarin red S stained with 10% acetic acid was extracted and the concentration was measured.

As a result, it was confirmed that osteoblast differentiation was statistically significantly promoted by treatment with 33.3 μg/ml of stone ethanol extract (EEPL) ( FIG. 2 ).

Example 4. Analysis of Effect of Seokwi Extract on Cell Viability

The purpose of this study was to determine whether Seokwi extract inhibited the survival of osteoclast progenitor cells.

Specifically, the bone marrow cell-derived macrophages (BMM) of Example 2 were put in 2×10 ⁴ per well of a 96-well plate, and then in a medium containing M-CSF (60 ng/ml) for 1 day in various concentrations of hot water. Extracts (WEPL) or ethanol extract (EEPL) samples were added and incubated. Cell viability was confirmed by measuring absorbance at 450 nm using Cell Counting Kit-8 (Cell Counting Kit-8, Dojindo Inc.).

As a result, the progenitor cell viability of osteoclasts was increased by treatment with 11.1, 33.3, 100, and 200 μg/ml of hot water extract (WEPL) of shinoji (Fig. 3A).

In addition, the progenitor cell viability of osteoclasts was increased by treatment with 33.3 and 100 μg/ml stone ethanol extract (EEPL) ( FIG. 3B ).

Example 5. Analysis of the effect of ethanol extract of Seokwi on bone loss

Bone mineral density (BMD) and bone volume (bone volume/total volume, BV/TV) of cancellous bone of the femur were analyzed by oral administration of ethanol extract from ovariectomized mice.

Specifically, 24 6-week-old female C57BL/6J mice (Central Experimental Animal, Seoul) were acclimatized for 1 week, and bilateral ovariectomy (OVX, 18 mice) and provisional surgery (Sham, 6 mice) were performed on 7-week-old mice. did. After one week, they were allowed to freely consume a low-fat diet (D12450B, Research diet), and among the bilateral ovariectomized (OVX) mice, 30 mg/kg of ethanol extract (EEPL) dissolved in sterile distilled water was administered to 6 arbitrarily. It was orally administered once daily for 5 weeks, and to the other 6 mice, 100 mg/kg of ethanol extract (EEPL) dissolved in sterile distilled water was orally administered once daily for 5 weeks. Meanwhile, the provisional surgery (Sham, 6 animals) group and the remaining bilateral ovariectomy (OVX) control group (6 mice) were orally administered with sterile distilled water once daily for 5 weeks. Then, the femur was removed and fixed with 10% neutral formalin. After micro-computed tomography (micro-CT, SkyScan 1276, Bruker) of the femur, bone mineral density (BMD) and bone volume (BV/TV) of trabecular bone were analyzed.

As a result, as shown in Fig. 4(A), compared to the ovarian resection (OVX) control group, the ovariectomized false surgery (Sham) group and the ethanol extract (EEPL) were administered at 30 mg/kg or 100 mg/kg. There was a statistically significant increase in cancellous bone mineral density (BMD) in the group.

In addition, as shown in FIG. 4(B), compared to the ovariectomized (OVX) control group, the cancellous bone volume ( BV/TV) was significantly increased.

[Statistics processing]

The data obtained in Examples 2 to 4 of the present invention were expressed as mean ± standard deviation, and the data obtained in Example 5 were expressed as mean ± standard error. Statistical analysis was calculated by one-way ANOVA accompanied by Dunnett's test.

Claims (11)

A health functional food composition for the prevention or improvement of bone disease containing a stone leaf ( Pyrrosia lingua ) leaf extract as an active ingredient. The health functional food composition for the prevention or improvement of bone disease according to claim 1, wherein the extraction solvent of the stone leaf extract is water, a C ₁ to C ₄ lower alcohol, or a mixture thereof. delete [Claim 2] The health functional food composition for preventing or improving bone disease according to claim 1, wherein the stone leaf extract inhibits the differentiation of osteoclasts. The health functional food composition for preventing or improving bone disease according to claim 1, wherein the stone leaf extract promotes bone formation. The health functional food composition for preventing or improving bone disease according to claim 1, wherein the bone disease is caused by bone loss. The method of claim 1, wherein the bone disease is osteoporosis, osteoporotic fracture, osteoporosis, bone defect, nonunion fracture, osteogenesis imperfecta, osteomalacia, osteomalacia fracture, bone dysplasia, tumor-induced bone destruction (Neoplastic bone destruction), A health functional food composition for the prevention or improvement of bone disease, characterized in that any one selected from bone destruction and degenerative bone disease caused by periodontal disease. According to claim 1, wherein the composition is a health functional food composition for the prevention or improvement of bone disease, characterized in that it is prepared in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup and beverages. A pharmaceutical composition for the prevention or treatment of bone diseases, comprising a Pyrrosia lingua leaf extract as an active ingredient. [Claim 10] The pharmaceutical composition for preventing or treating bone disease according to claim 9, further comprising a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient. A composition for differentiation of mesenchymal stem cells into osteoblasts, comprising a stone leaf extract as an active ingredient.

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