KR102255000B1 - Composition for preventing, improving or treating bone disease comprising Mentha arvensis extract as effective component - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating, or treating bone diseases containing peppermint extract as an active ingredient, wherein a peppermint extract inhibits osteoclast differentiation and promotes osteoblast differentiation, increases the survival rate of osteoclast progenitors, and increases cancellous bone density and bone volume in an ovariectomized animal model. Therefore, the peppermint extract of the present invention can be usefully used as health functional food or pharmaceutical for preventing, alleviating, or treating bone diseases.

Description

Composition for preventing, improving or treating bone disease comprising Mentha arvensis extract as effective component}

The present invention relates to a composition for the prevention, improvement or treatment of bone diseases containing peppermint (Mentha arvensis) extract as an active ingredient.

The bone supports the soft tissue and weight of the human body and surrounds the internal organs to protect the internal organs from external shocks. In addition, it is one of the important parts of the human body that not only structurally supports muscles and organs, but also stores substances such as calcium or other essential minerals, such as phosphorus and magnesium, in the body.

Bone is a very complex tissue composed of cells, collagenous matrix and mineral components. They not only protect organs, but also provide basic and various functions such as providing a microenvironment necessary for hematopoiesis. In addition, it is one of the important parts of the human body that not only structurally supports muscles and organs, but also stores substances such as calcium or other essential minerals, that is, phosphorus or magnesium in the body.

The bones of adults after growth do not stop, and until the day of death, old bones are removed and replaced with new bones. The process of creation and absorption is very dynamic and constantly regenerates and maintains balance. This is called bone remodeling.

The turnover of removing old bones and replacing them with new bones is essential to repairing 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 produces bone, and the other is an osteoclast that destroys bone. Osteoblasts produce RANKL (receptor activator of nuclear factor-κB ligand) and its decoy receptor, OPG (osteoprotegerin). When RANKL binds to RANK (receptor activator of nuclear factor-κB), a receptor on the surface of osteoclast progenitor cells, osteoclast progenitor cells mature into giant multinuclear osteoclasts, resulting in bone resorption. . However, when OPG binds to RANKL, the binding between RANKL and RANK is blocked, the formation of osteoclasts is suppressed, and bone resorption more than necessary does not occur. These osteoclasts are activated to absorb or destroy old bones, which make holes in the bones and release a small amount of calcium into the bloodstream and are used to maintain body functions.

On the other hand, Mentha arvensis is a dicotyledonous plant, a perennial perennial plant in the Lamiaceae family, and has a directionality in the plant itself and is upright and branched from the top. The main ingredients of peppermint are alphapinene (+,-)), anisaldehyde, Daucosterol, Eugenol, Linalool, Menthone, (+ )-Neomenthol ((+)-Neomenthol). Mint gives off a refreshing and refreshing scent, and has sterilization and antibacterial action against microorganisms such as pathogenic fungi, E. coli, staphylococcus, etc. that cause eczema and scabies present in the skin or scalp. In addition, it has the effect of constricting the mucous membranes and blood vessels of the skin, paralyzing nerve peripherals, stopping pain, stopping itching, and so on, giving the skin a unique refreshing sensation to improve skin diseases, skin soothing and antioxidant effects. It is known to have, and menthol, which is the main component of mentha oil, is medicated as a coating agent, pain reliever, stimulant, or drying agent, and is used as a flavoring or refreshing agent for toothpaste, candy, jam, cosmetics, tobacco, etc.

As a prior art related to bone disease, a composition for preventing and treating osteoporosis containing a mixed herbal medicine extract is disclosed in Korean Patent No. 1864719, and prevention or treatment of osteoporosis containing camphen as an active ingredient in Korean Patent No. 2063962 Although a pharmaceutical composition for use is disclosed, there is no disclosed composition for the prevention, improvement or treatment of bone diseases containing the peppermint extract of the present invention as an active ingredient.

The present invention is derived from the above requirements, the present invention provides a composition for the prevention, improvement or treatment of bone diseases containing peppermint extract as an active ingredient, wherein the peppermint extract inhibits osteoclast differentiation and The present invention was completed by confirming that there is an effect of promoting differentiation, increasing the survival rate of progenitor cells of osteoclasts, and increasing the cancellous bone bone density and bone volume in an ovariectomy animal model.

In order to achieve the above object, the present invention provides a health functional food composition for the prevention or improvement of bone disease containing the extract of peppermint (Mentha arvensis) as an active ingredient.

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

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

The present invention relates to a composition for the prevention, improvement or treatment of bone diseases containing a peppermint extract as an active ingredient, and the peppermint extract has an effect of inhibiting osteoclast differentiation and promoting differentiation of osteoblasts, and has an effect on the survival rate of progenitor cells of osteoclasts It has the effect of increasing the bone density and bone volume of the cancellous bone in the animal model of ovariectomy.

1 is a result of confirming the effect of inhibiting osteoclast formation according to the treatment concentration of peppermint hot water extract (WEMA) (A) and peppermint 70% ethanol extract (EEMA) (B). ** indicates that the number of osteoclasts in the experimental group treated with the peppermint extract was statistically significantly decreased compared to the control group not treated with the peppermint extract of the present invention, p<0.01.
Figure 2 is a result of Alizarin Red S staining confirming the osteoblast differentiation promoting effect according to the treatment of peppermint hot water extract (WEMA). ** indicates that the osteoblast differentiation of the group treated with the peppermint hot water extract was statistically significantly increased compared to the control group without treatment, p<0.01.
3 is a result of confirming the cell viability of osteoclast progenitor cells according to the treatment concentration of peppermint hot water extract (WEMA) (A) and peppermint 70% ethanol extract (EEMA) (B). *, ** indicates that the cell viability of the experimental group treated with the peppermint extract was statistically significantly increased compared to the control group not treated with the peppermint extract of the present invention, * is p<0.05, ** is p<0.01.
4 is a result of confirming the change in bone density (BMD) (A) and bone volume (BV/TV) (B) of the cancellous femoral bone according to oral administration of mint hot water extract (WEMA) in an ovariectomy (OVX) mouse model. ## indicates that the bone density (BMD) and bone volume (BV/TV) of the femoral cancellous bone of the ovarian resected mouse group (OVX) were statistically significantly reduced compared to the normal mouse group (Sham) without ovarian resection. p<0.01, *, ** indicate that the cancellous bone bone density or bone volume of the group administered with 0.1 and 0.3 g/kg of peppermint hot water extract after ovariectomy was statistically significantly increased compared to the ovariectomized mouse group (OVX). , * Is p<0.05, and ** is p<0.01.

The present invention relates to a health functional food composition for the prevention or improvement of bone disease containing the peppermint (Mentha arvensis) extract as an active ingredient.

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

(1) extracting by adding an extraction solvent to mint;

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

(3) preparing an extract by concentrating the filtered extract of step (2) under reduced pressure and drying it.

In the step (1), the extraction solvent _{is preferably selected from water, a lower alcohol of C 1} to C ₄ or a mixture thereof, more preferably water or ethanol, and even more preferably water, but is not limited thereto.

In the above manufacturing 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 extracted by adding 1 to 20 times the weight of the dried mint, 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 a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably vacuum drying, vacuum drying, boiling drying, spray drying, or freeze drying, but is not limited thereto.

The mint extract is preferably extracted from mint leaves, but is not limited thereto.

The peppermint extract is characterized by inhibiting the differentiation of osteoclasts and promoting bone formation, and the bone disease can be caused by bone loss, and the bone disease is osteoporosis, osteoporotic fracture, bone loss, bone defect, nonunion Fracture, bone insufficiency, osteomalacia, osteomalacia fracture, osteogenesis disorder, neoplastic bone destruction by tumor, bone destruction by periodontal diseaes, and degenerative bone disease. It is not limiting.

The composition is preferably prepared in any one formulation selected from powders, granules, pills, tablets, capsules, candy, syrup, and beverages, but is not limited thereto.

When using the health functional food composition of the present invention as a food additive, the mint extract may be added as it is or may be used 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 use (prevention, health or therapeutic treatment). In general, when preparing 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 the purpose of health and hygiene or for the purpose of 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 above 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, gum, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and all health functional foods in the usual sense are included.

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 normal drink. The natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as texttrin 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 ratio 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 includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal heavy agents, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonic acids. It may contain a carbonation agent used in beverages and the like. In addition, the composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These ingredients may be used independently or in combination. The proportion of these additives is not very important, but 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 peppermint (Mentha arvensis) 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 diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include capsules, powders, granules, tablets, pills, and the like, and such solid preparations include at least one excipient in one or more compounds, such as starch, calcium carbonate, sucrose, or lactose ( lactose), gelatin, etc. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, emulsions, syrups, aerosols, etc., and various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., may be included in addition to water and liquid paraffin, which are commonly used simple diluents. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycero gelatin, and the like may be used. For parenteral administration, it is preferable to select an injection method for external use of the skin or intraperitoneal, rectal, intravenous, muscle, subcutaneous, intrauterine dura mater or cerebrovascular.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "a 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 the type, severity, and activity of the drug of the patient. , Sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

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

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

Mesenchymal stem cells (MSCs) in bone marrow differentiate into cartilage, bone tissue, ligaments, and bone marrow matrix, and are suitable for tissue regeneration with immunoregulatory and inhibitory functions. Mesenchymal stem cells refer to stem cells present in cartilage, bone tissue, adipose tissue, and stroma of the bone marrow differentiated from the mesoderm resulting from the division of a fertilized egg. The mesenchymal stem cells in the present invention must adhere to the bottom and grow in culture conditions, have the ability to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro, express CD73, CD90 and CD105, and c-kit , CD11b, CD19, CD14, CD34, CD45, CD14, CD79 and HLA-DR.

Promoting the differentiation of mesenchymal stem cells into osteoblasts by the composition of the present invention means that bone regeneration or bone formation can be promoted.

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

Example 1. Peppermint ( Mentha arvensis ) Extract preparation

For the hot water extract (WEMA) of peppermint, 500g of mint leaves were added to 3.5ℓ of distilled water, heated mantle for 3 hours, filtered through a qualitative filter paper (Ø185mm), and freeze-dried. In the cell experiment, peppermint hot water extract (WEMA) dissolved in distilled water was filtered through a 0.22 μl filter and used in the experiment. In animal experiments, the freeze-dried extract was dissolved in sterile distilled water and used.

Ethanol extract (EEMA) of mint leaves 500g of mint leaves in 3.5ℓ of 70% (v/v) ethanol, heating mantle for 3 hours, filtered with Qualitative Filter Paper (Ø185mm), and then rotary concentrator ( Rotary evaporator) and then freeze-dried. After dissolving with dimethyl sulfoxide, peppermint ethanol extract (EEMA) was used in the experiment.

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

It was attempted to determine whether the peppermint extract inhibits the differentiation of osteoclast progenitor cells into osteoclasts. The osteoclast progenitor cells were treated with peppermint hot water extract (WEMA) and peppermint ethanol extract (EEMA) by concentration, and the number of osteoclasts according to the treatment was analyzed.

Specifically, bone marrow cells of mice were cultured in α-MEM medium containing M-CSF (60 ng/ml) and 10% FBS for 3 days to obtain bone marrow cell-derived macrophages (BMM), which were converted into osteoclast progenitor cells. Was used. ^{To generate osteoclasts, 1×10 4} per well of the obtained BMM was put in a 96-well plate, and then cultured in a medium containing M-CSF (60 ng/ml) and RANKL (50 ng/ml) for 4 days. , Under the same culture conditions, BMM was treated with 11.1, 33.3, 100 and 200 µg/ml of peppermint hot water extract (WEMA) and 11.1, 33.3 and 100 µg/ml of ethanol extract (EEMA). After fixing the cells with 10% formalin, Tartrate-resistant acid phosphatase (TRAP) staining was performed using naphthol AS-MS phosphate and Fast red violet LB salt. Cells, osteoclast) were counted.

As a result, the effect of inhibiting osteoclast differentiation was statistically significant by treatment with 33.3, 100 and 200 µg/ml of peppermint hot water extract (WEMA), and osteoclasts were also treated with 100 µg/ml of peppermint ethanol extract (EEMA). The differentiation inhibitory effect was statistically significant (Fig. 1).

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

It was attempted to confirm whether the mint extract promotes osteoblast differentiation. The mesenchymal stem cells were treated with peppermint hot water extract (WEMA) by concentration, and the degree of differentiation into osteoblasts was analyzed by Alizarin Red S staining.

^{Specifically, 1.7×10 4} mice bone marrow-derived mesenchymal stem cells (D1 ORL UVA) per well of a 96-well plate were added and then cultured in DMEM medium containing 10% FBS for 2 days. After replacement with α-MEM medium containing 10% FBS, ascorbic acid (50 μg/ml) and β-glycerophosphate (β-glycerophosphate, 5 mM) were treated for 8 days to induce osteoblast differentiation. In culture conditions, 11.1 and 33.3 ㎍ / ㎖ of peppermint hot water extract (WEMA) was treated. After fixing the cells with 10% formalin, they were stained with Alizarin red S to check the degree of mineralization 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 the osteoblast differentiation was statistically significant by treatment with 33.3 µg/ml of peppermint hot water extract (WEMA) (Fig. 2).

Example 4. Analysis of the effect of peppermint extract on cell survival

It was attempted to confirm whether the peppermint extract inhibits the survival of the progenitor cells of osteoclasts.

Specifically, after the bone marrow cell-derived macrophages (BMM) of Example 2 ^{were added 2×10 4 per} well of a 96-well plate, peppermint hot water at various concentrations for 1 day in a medium containing M-CSF (60 ng/ml). Extract (WEMA) or peppermint ethanol extract (EEMA) sample was added and cultured. Cell viability was confirmed by measuring absorbance at 450 nm using Cell Counting Kit-8 (Dojindo Inc.).

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

In addition, the progenitor cell viability of osteoclasts was increased by treatment with 11.1, 33.3 and 100 µg/ml of peppermint ethanol extract (EEMA) (Fig. 3B).

Example 5. Analysis of the effect of peppermint hot water extract on bone loss

The bone mineral density (BMD) and bone volume (bone volume/total volume, BV/TV) of the femoral cancellous bone were analyzed by oral administration of hot water extract of peppermint to the ovarian resected mice.

Specifically, 24 6-week-old female C57BL/6J mice (Central Experimental Animal, Seoul) were acclimatized for 1 week, and then 7-week-old mice were subjected to bilateral ovarian resection (OVX, 18) and surgery (Sham, 6). I did. One week later, a low-fat feed (D12450B, Research diet) was freely ingested, and among the bilateral ovarian resection (OVX) mice, optionally, 6 mice were treated with mint hot water extract (WEMA) dissolved in sterile distilled water, 0.1 g per kg of mouse. Each was administered orally once a day for 5 weeks, and to the other 6 mice, 0.3 g of peppermint hot water extract (WEMA) dissolved in sterile distilled water was orally administered once daily for 5 weeks. Meanwhile, the hydrosurgery (Sham, 6) group and the remaining bilateral ovarian resection (OVX) control group (6) 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-CT (SkyScan 1276, Bruker) of the femur, bone mineral density (BMD) and bone volume (BV/TV) of trabecular bone were analyzed.

As a result, the bone density (BMD) and bone volume (BV/TV) of the ovariectomy (OVX) control group were statistically significantly reduced compared to the sham surgery (Sham) group without ovariectomy, 0.1 and 0.3 g compared to the ovariectomy control group. The cancellous bone bone density (BMD) and bone volume (BV/TV) of the group to which /kg of peppermint hot water extract (WEMA) was administered were statistically significantly increased (FIG. 4).

[Statistics processing]

Data obtained in Examples 2 to 4 of the present invention were expressed as mean±standard deviation, and statistical analysis obtained in Example 5 was expressed as mean±standard error. It was calculated by a one-way batch analysis of variance accompanying the Dunnett test.

Claims (11)

Peppermint ( Mentha arvensis ) health functional food composition for the prevention or improvement of any one bone disease selected from osteoporosis, bone loss, osteomalacia and osteogenesis disorders containing an extract as an active ingredient. The method of claim 1, wherein the extraction solvent of the peppermint extract is water, _{a lower alcohol of C 1} to C ₄ or a mixture thereof. Health functional food composition for prevention or improvement. According to claim 1, wherein the peppermint extract is a health functional food composition for preventing or improving any one bone disease selected from osteoporosis, bone loss, osteomalacia and bone dysplasia, characterized in that the peppermint leaf extract. According to claim 1, wherein the peppermint extract is a health functional food composition for preventing or improving any one bone disease selected from osteoporosis, bone loss, osteomalacia and bone dysplasia, characterized in that it inhibits the differentiation of osteoclasts. According to claim 1, The mint extract is a health functional food composition for the prevention or improvement of any one bone disease selected from osteoporosis, bone loss, osteomalacia and bone dysplasia, characterized in that promoting bone formation. The health functional food composition for preventing or improving any one of bone diseases according to claim 1, wherein the bone disease is caused by bone loss. delete The method of claim 1, wherein the composition is prepared in any one formulation selected from powders, granules, pills, tablets, capsules, candy, syrup, and beverages. Health functional food composition for the prevention or improvement of one bone disease. A pharmaceutical composition for the prevention or treatment of any one bone disease selected from osteoporosis, bone loss, osteomalacia, and bone dysplasia, containing a peppermint ( Mentha arvensis) extract as an active ingredient. The method of claim 9, further comprising a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient, for the prevention or treatment of any one bone disease selected from osteoporosis, bone loss, osteomalacia, and bone formation disorders. Pharmaceutical composition. A composition for differentiation of mesenchymal stem cells into osteoblasts containing an extract of Mentha arvensis as an active ingredient.

Images