KR20150056752A - Composition comprising extract of Eupatorium spp. as an effective component for prevention or treatment of metabolic bone disease - Google Patents

Abstract

The present invention relates to a Eupatorium spp. extract having anti-obesity effects as a result of decreasing adipocytes and increasing osteoblasts, as well as the effects of preventing bone disease or fractures as a result of increasing osteoblasts and of preventing osteoporosis as a result of decreasing adipocytes and increasing osteoblasts by the same proportion in mesenchymal stem cells. Extract in Eupatorium spp. may inhibit the activity of PPARγ, AP2, CD36, adiponectin C/EBPα, and LPL, which serve as significant factors for adipocyte differentiation in C3H10T1/2 cells, which are pluripotent stem cell lines, and may increase the activity of ALP, osterix, CO1I and RUNX2, which serve as significant factors for osteoblast differentiation, thereby being used as a useful material for preventing and treating osteoporosis.

Description

[0001] The present invention relates to a composition for preventing or treating bone metabolism diseases, As an effective component for prevention or treatment of metabolic bone disease,

The present invention relates to a method for the preparation of Eupatorium spp . ) Extract as an active ingredient, and the composition of the present invention can be used for the production of health functional foods or medicines for the prevention or treatment of bone metabolic diseases.

Osteoporosis refers to a condition in which fractures tend to occur as the bones become weaker as the amount and quality of the bones decrease. Osteoporosis is a particularly frequent occurrence in postmenopausal women, a disease in which the amount of bone is markedly reduced by the decreased secretion of estrogen. The reduction in bone mass is due to individual differences, or due to various other causes. However, if the amount of bone excessively decreases and falls below a predetermined value, fracture easily occurs even in a small impact. Osteoporosis can not lead to a healthy life by restricting long-term activities due to various fractures caused by weakening of the bones, especially femur fracture or vertebral fracture, rather than the symptom itself, resulting in 15% of the elderly death It is known.

Several other substances have been studied in addition to endogenous regulators for the purpose of treating or preventing osteoporosis induced by excessive osteoclast activity. Drugs currently used for this purpose exhibit a certain pharmacological action, but are known to have various side effects and difficulties in taking, and thus have a new action and drug structure, are less toxic and adverse, and are effective in preventing or treating osteoporosis Development of new materials is required. In addition, since these new substances are highly likely to be found in natural products that have not been used for a long time by folk remedies, attempts to create new drugs from natural products are being actively pursued.

So far, studies on osteoporosis have been focused on the imbalance between osteoclasts that absorb bone and osteoblasts that form bone. However, osteoporosis is involved in bone marrow-derived adipocytes as well as bone-related cells.

Most of the bone mass is produced between the ages of 12 and 18 years, with bone formation and hormones and environmental nutrition. During this period, changes in the hormone sequence or promotion of bone resorption reduce the amount of bone and increase the risk of fractures. It is important to note that childhood fractures are associated with changes in the structure of the bones due to skeletal insufficiency and changes in body composition that can lead to obesity. Bone loss due to aging has an important influence on the relationship between fat and bone. A common characteristic of aging is the inflow of bone marrow by fat. Osteoblasts and adipocytes have the same precursor and are derived from mesenchymal stem cells. In bone marrow, the number of adipocytes is reduced from mesenchymal stem cells to osteoblast differentiation, increased differentiation into adipocytes and increased with aging. Inhibition of adipocytes associated with osteoblast formation may be a goal of preventing and treating osteoporosis by inhibiting adipocyte formation or converting existing adipocytes into osteoblasts. In conclusion, the relationship between fat and bone provides a pathophysiological understanding of the loss of bone associated with aging and provides a new approach to the treatment and diagnosis of osteoporosis.

Therefore, it has been recognized that the relationship between osteoblasts and adipocytes can be used as a target for treating and preventing osteoporosis associated with aging. The present inventors have found a substance that enhances osteoblast differentiation, lowers adipocyte differentiation, And a lot of effort is being made to develop it. In particular, these substances exhibit an anti-obesity effect and are more important food / medicinal materials because they can directly affect bone diseases such as fracture for increasing osteoclast differentiation.

Meanwhile, underwater spine or distributed in the country (Eupatorium spp.) Is Eupatorium japonicum (E. japonicum), the stapes bone herb (E. lindleyanum), Bee Eupatorium japonicum (E. makinoi there is . oppisitifolium and E. rugosum are well known and are well succulent perennial plants that grow well in moist initials of the water. They are used for edible, ornamental or medicinal purposes, and are used for publication, scattering, Erythema, irritant measles, rheumatoid lumbago, and cold seawater. However, the effect on bone metabolism diseases including obesity or osteoporosis is not yet known.

It is an object of the present invention to provide a composition for the prevention or treatment of bone metabolic diseases derived from a non-toxic natural product, and to produce a health functional food and a pharmaceutical product therefrom.

The pharmaceutical composition for the prevention or treatment of bone metabolic diseases according to the present invention is characterized in that it contains Eupatorium spp . ) Extract as an active ingredient.

The health functional food for preventing or ameliorating bone metabolic diseases according to the present invention is characterized in that it contains Eupatorium spp . ) Extract as an active ingredient.

Water or spine of the present invention (Eupatorium spp.) Is Eupatorium japonicum (E. japonicum), bone Eupatorium japonicum (E. lindleyanum), bee Eupatorium japonicum (E. makinoi there is . oppisitifolium , E. rugosum and their inbred plants, preferably E. japonicum .

The extract as defined herein means extracts in water or water, including purified water, lower alcohols having 1 to 4 carbon atoms, nonpolar solvents or mixed solvents thereof, preferably methanol or ethanol, or methanol or ethanol.

Hereinafter, the present invention will be described in detail.

The spine or aqueous extract of the present invention can be prepared as follows.

The spine or water-extracted extract of the present invention may be obtained by shredding and shredding a plant, stem or flower of a plant of the spine or herbaceous plant, and then washing the plant with water at a rate of about 1 to 50 times, preferably about 10 to 40 times, 1 to 4 lower alcohols, nonpolar solvents or mixed solvents thereof at a temperature of 20 to 110 DEG C, preferably 80 to 100 DEG C for about 1 to 6 hours, preferably 2 to 4 hours, The herbal medicine extract of the present invention can be obtained by extracting, extracting, extracting with cooling water, refluxing cooling extraction, ultrasonic extraction, supercritical extraction, or the like, preferably extracting hot water, and then filtering, concentrating or drying under reduced pressure.

As the non-polar solvent, at least one of dichloromethane, chloroform, diethyl ether, ethyl acetate, hexane or supercritical fluid may be used.

When an alcohol aqueous solution is used as the mixed solvent, an alcohol aqueous solution or alcohol having a mixing ratio of water and the lower alcohol of 5 (v / v) to 100% (v / v) is used.

The composition for preventing or treating osteoporosis according to the present invention exhibits osteoblast differentiation increasing activity and adipocyte differentiation inhibiting activity, and comprises 0.1 to 50% by weight of the spinal column or aqueous extract according to the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

The composition for preventing or treating osteoporosis, including the spine or aqueous extract of the present invention, may further comprise suitable carriers, excipients and diluents conventionally used in the production of pharmaceutical compositions.

The composition containing the extract according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions, Examples of the carrier, excipient and diluent which can be contained in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at a dose of 0.01 mg / kg to 10 g / kg per day, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention provides a health functional food for osteoporosis prevention or improvement, which comprises osteoblast differentiation-enhancing activity and an adipocyte or water-based extract showing an activity of inhibiting adipocyte differentiation and a food-acceptable food supplementary additive.

The health functional food of the present invention includes various foods, gums, tea, vitamin complex, health supplement foods and the like, and can be used as powder, granule, tablet, capsule or beverage.

Since the spine or water extract of the present invention has little toxicity and side effects, it can be safely used for prolonged use even for prophylactic purposes.

When the extract of the present invention is added to food or beverage for the purpose of preventing or ameliorating osteoporosis, the amount of the extract in the food or beverage is generally from 0.01 to 15% by weight of the total food weight of the health food composition of the present invention , And the health beverage composition may be added at a ratio of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates such as ordinary beverages as an additional ingredient, as long as it contains the extract as an essential ingredient at the indicated ratio, and there is no particular limitation to the liquid ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the health functional food of the present invention may contain flavorings such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional foods of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The extracts of the spine and water of the present invention inhibit PPARγ, AP2, CD36, adiponectin C / EBPα, and LPL activities related to the differentiation of adipocytes and increase ALP, osterix and COl I genes related to osteoblast differentiation, Or osteoporosis due to loss of bones according to the present invention.

Figure 1 is a photograph of eupatorium japonicum before harvesting at Mt.
FIG. 2 is a photograph showing the effect of the extracts of each part of the spine on the C3H10T1 / 2 cell line on ALP activity after 9 days of treatment in osteoclast differentiation medium.
FIG. 3 is a graph showing relative mRNA expression levels of genes involved in the differentiation of osteoblasts after 10 days of treatment in the C3H10T1 / 2 cell line in osteoclast differentiation medium.
FIG. 4 is a graph showing relative mRNA expression levels of genes involved in the differentiation of osteoblasts after treatment with leaf extract of spikelets in C3H10T1 / 2 cell line for 9 days in osteoclast differentiation medium.
FIG. 5 is a graph showing relative mRNA expression levels of genes involved in the differentiation of osteoblasts after treatment of stem extracts of spikelets with C3H10T1 / 2 cell line for 9 days in osteoclast differentiation medium.
6 is a graph showing relative mRNA expression levels of genes involved in the differentiation of osteoblasts after 9 days of flower extracts from the C3H10T1 / 2 cell line treated with osteoblast differentiation medium.
FIG. 7 is a photograph showing inhibition of adipocyte differentiation after treatment of spinal cord herniation and extracts of each region in an adipocyte differentiation medium for 9 days in a C3H10T1 / 2 cell line.
8 is a graph showing relative mRNA expression levels of genes involved in the differentiation of adipocytes after 10 days of treatment in the adipocyte differentiation medium in the C3H10T1 / 2 cell line.
FIG. 9 is a graph showing relative mRNA expression levels of genes involved in adipocyte differentiation after 10 days treatment of leaf extract of spikelets in a C3H10T1 / 2 cell line in an adipocyte differentiation medium. FIG.
FIG. 10 is a graph showing relative mRNA expression levels of genes involved in the differentiation of adipocytes after 10 days of treatment with stem extracts of spinal herb from the C3H10T1 / 2 cell line in an adipocyte differentiation medium.
11 is a graph showing relative mRNA expression levels of genes involved in the differentiation of adipocytes after 10 days of treatment with flower extracts of spikelets in a C3H10T1 / 2 cell line in an adipocyte differentiation medium.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following examples and experimental examples are illustrative of the present invention, and the present invention is not limited thereto.

Example  One. Spine or water  Extract preparation

The spine and the water plant used in the present invention has been directly harvested from Gyeonggi Yangju Gamaksan, Eupatorium japonicum (Eupatorium japonicum ) (Fig. 1). The harvested spikelets were divided into outposts (flowers, leaves, stems), dried thoroughly for 2 days at room temperature, finely crushed to obtain spike herb powder samples, and 99.9 (v / v )% Methanol, and the mixture was extracted with shaking incubator at 120 rpm at 40 ° C for 24 hours. The supernatant was filtered with a Whatman No.1 filter paper, and the supernatant was filtered 200 ml of 99.9% methanol was added per 10 g of the sample, and the mixture was again extracted for 24 hours and filtered in the same manner as above. The filtrate was concentrated under reduced pressure at 45 ° C with a rotary vacuum evaporator to obtain a methanol extract of each part and a shrub turtle shell which had been completely blown away from the solvent.

Experimental Example 1. Measurement of synergistic effect of osteoblast differentiation in C3H10T1 / 2 cells

(One) ALP ( alkaline 포스화제 ) dyeing

The C3H10T1 / 2 cell line derived from mouse embryonic fibroblasts is a pluripotent stem cell line that can differentiate into various cell lines including osteoblasts and adipocytes. The C3H10T1 / 2 cell line was cultured in DMEM medium supplemented with 10% FBS, 1% penicillin and streptomycine at 37 ° C and 5% CO 2. Cells were cultured in a 6-well plate at a concentration of 2.5 * 10⁴ / ml with 10mM glycerophosphate and 50ug / ml ascorbic acid. The medium was replaced every 3 days with 20ug / ml and 40ug / The ALP staining was performed using 5-Bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium (BCIO / NBT) The results of ALP staining are shown in Fig. As a result, it was found that ALP activity was increased in a concentration - dependent manner in outpost, leaf, stem and flower extracts, and the leaf showed the highest ALP activity.

(2) realtime RT - PCR . osteoblast Analysis of differentiation factor expression

C3H10T1 / 2 cells were transfected every 3 days and treated with 5 ug / ml and 20 ug / ml of extracts of each region for 9 days, followed by real-time RT-PCR to determine the important factors of osteoblast formation ALP. osterix, and COl I mRNA expression was confirmed and shown in FIGS. 3 to 6.

As a result, osterix mRNA expression was higher than that of control (ctrl) at the concentration of 5 ㎍ / ml among the genes related to the osteoblast differentiation in the outpost, and compared with the control (ctrl) at the concentration of 20 ㎍ / ml mRNA expression was higher (Fig. 3). In the case of leaves, the level of ALP mRNA was significantly higher at the concentration of 5 ㎍ / ml, but not at the concentration of 20 ㎍ / ml (Fig. 4).

The expression level of ALP mRNA was higher at the 5 ㎍ / ml concentration of stem, and the expression level of ALP and COlⅠ mRNA was higher than that of the control at 20 ㎍ / ml concentration (Fig. 5) (Fig. 6).

Experimental Example  2. C3H10T1 / 2 < / RTI > adipocyte ) Measurement of inhibition of differentiation

(1) Oil Red O staining method ( Oil red O staining ) to measure adipocyte differentiation inhibition

The C3H10T1 / 2 cells were cultured for 9 days in a medium containing 1uM dexamethasone, 5 ug / ml insulin and 20 nM PPARγ and 5 ug / ml and 20 ug / ml spine herb extracts for differentiation of adipocytes at a concentration of 2.5 × 10 4 / ml Respectively. The medium was collected, fixed with 4% formaldehyde, stained with 0.5% Oil red O, and the results are shown in FIG.

As shown in Fig. 7, it was found that the outpouring, leaves, stems and flowers inhibited the differentiation of adipocytes in a concentration-dependent manner. Among them, the inhibitory effect of the flowers on the adipocytes was the highest.

(2) realtime RT - PCR (Fig. adipocyte Analysis of differentiation factor expression

A total of 10 days of C3H10T1 / 2 cells were treated with a medium containing adipocyte differentiation material and an extract of spikelets and leaves (leaf, stem, flower) for 10 days, and then real time RT-PCR was performed to determine the important factors of adipocyte formation The relative mRNA expression levels of PPARγ, AP2, CD36, adiponectin C / EBPα and LPL were confirmed and shown in FIG. 8 to FIG.

8 to 11, it was found that the outpost, leaf, stem and flower extract inhibited PPARγ, AP2, CD36, adiponectin C / EBPα and LPL activity in a concentration-dependent manner.

Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example  One. Sanje  Produce

The topical extract of Example 1        20 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

* 10 mg of the outpost extract of Example 1

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

10 mg of the outpost extract of Example 1

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

10 mg of the outpost extract of Example 1

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O 26 mg

(2 ml) per 1 ampoule according to the usual injection preparation method.

Formulation example  5. Liquid  Produce

20 mg of the outpost extract of Example 1

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

1,000 mg of the seed extract of Example 1

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

1,000 mg of the seed extract of Example 1

Citric acid 1,000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated for about 1 hour at 85 DEG C with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, ≪ / RTI >

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (7)

Eupatorium japonicum ) as an active ingredient and having an osteoblast differentiation-increasing activity and an adipocyte differentiation-inhibiting activity.
The pharmaceutical composition for preventing or treating osteoporosis according to claim 1, wherein the extract is an extract soluble in water, including purified water, a lower alcohol having 1 to 4 carbon atoms, a non-polar solvent, or a mixed solvent thereof.
The pharmaceutical composition for preventing or treating osteoporosis according to claim 2, wherein the solvent is an alcohol aqueous solution or an alcohol having a mixing ratio of the lower alcohol of 5 (v / v) to 100% (v / v).
The pharmaceutical composition according to claim 2, wherein the non-polar solvent is dichloromethane, chloroform, diethyl ether, ethyl acetate, hexane or supercritical fluid.
The method according to any one of claims 1 to 4, wherein the extract is obtained by stirring, extracting with hot water, extracting with cooling, extracting with reflux, using ultrasonic waves Extract or supercritical extraction of the compound of the present invention.
Eupatorium japonicum ) as an active ingredient and having an osteoblast differentiation increasing activity and an adipocyte differentiation inhibiting activity.
9. The health functional food for preventing or improving osteoporosis according to claim 8, wherein the health functional food is in the form of powder, granule, tablet, capsule or beverage.

Images