KR102088476B1 - A preparation method for extract of mulberry fruit having increased contents of resveratrol - Google Patents

Abstract

The present invention relates to a method for preparing an Audi extract having an increased content of resveratrol, and specifically, comprising adding a mixed solvent containing ethanol, HCl, and water to Audi to obtain a mixture, wherein the mixed solvent comprises ethanol and A method for preparing an Audi extract having an increased content of resveratrol, which includes HCl in a volume ratio of 6 to 8: 0.1 to 2, pharmaceutical compositions for preventing or treating bone diseases, including fractions of the extract, and bone diseases It relates to a method of prevention or treatment.
The method of extracting Audi according to the present invention can be obtained to the maximum resveratrol contained in Audi, and thus can be utilized in the production of Audi extracts with increased antioxidant activity. In addition, the resveratrol contained in the Audi extract of the present invention exhibits an activity of promoting osteoblast differentiation and inhibiting osteoclast differentiation, and thus may be useful for the prevention and treatment of bone diseases.

Description

A preparation method for extract of mulberry fruit having increased contents of resveratrol}

The present invention relates to a method for producing an Audi extract having an increased content of resveratrol, and specifically, comprising adding a mixed solvent containing ethanol, HCl, and water to Audi to obtain a mixture, wherein the mixed solvent comprises ethanol and A method for preparing an Audi extract having an increased content of resveratrol, which contains HCl in a volume ratio of 6 to 8: 0.1 to 2, a pharmaceutical composition for preventing or treating bone diseases including a fraction of the extract, and a bone disease. It relates to a method of prevention or treatment.

Resveratrol is a substance belonging to the polyphenol family, an antioxidant found in plants, and is contained in grapes, audi, and peanuts, and is especially contained in red wine. It is also known to act as an anti-cancer and powerful antioxidant and lower serum cholesterol. In addition, it is said to have antiviral, neuroprotective, anti-inflammatory, anti-aging and prolonging effects.

On the other hand, Audi is a fruit of a mulberry tree or a mountain mulberry tree, characterized by a very soft and thin rind, which is green at first and ripens to a blackish purple. When cooked, the juice becomes richer, and the taste is sweet, sour and sweet. Ingredients include glucose, fructose, citric acid, malic acid, tannin, and pectin, as well as minerals such as vitamins (A, B1, B2, D), calcium, phosphorus, and iron, as well as antioxidants such as anthocyanins and polyphenols. Audi is known as a tonic, relieves thirst, softens joints, breaks down alcohol, and relaxes the mind, which is also effective for insomnia and forgetfulness. In addition, it has hematopoietic action and is used to treat rheumatism, and has blood sugar and cholesterol lowering effects.

As described above, Audi contains various nutrients, so it may be eaten raw or soaked in juice or juice. In addition, there are various uses of Audi, such as preparing an extract of Audi, mixing it with flour dough to make a cookie, or drying it at a low temperature to eat it.

Conventional Audi extraction method is mainly based on extraction with alcohol, and the method of increasing the temperature and the method of ultrasonic crushing are carried out in parallel.However, in this method, saccharification derivatives, which are functional substances such as resveratrol and anthocyanin, are removed in the purification process, and the purification efficiency is improved. Had this falling issue. Accordingly, a method for extracting oxyresveratrol with high purity from Audi (Chinese Patent Publication No. 104803830), and an extraction method for obtaining the maximum content of nutrients and functional compounds from Audi (Korea Patent Publication No. 10-2014- 0039444) has been developed, but these methods have been made through complicated steps, or because they use enzymes or microorganisms, the extraction process is difficult.

Under these backgrounds, the present inventors have verified the optimal extraction conditions to obtain the maximum resveratrol content in Audi as a result of earnest effort research to easily produce an Audi extract with increased functional ingredient content. The obtained Audi extract or resveratrol obtained by purifying the same was confirmed to show the activity of promoting osteoblast differentiation and inhibiting osteoclast differentiation, thereby completing the present invention.

One object of the present invention includes the step of obtaining a mixture by adding a mixed solvent containing ethanol, HCl and water to Audi, the mixed solvent comprising ethanol and HCl in a volume ratio of 6 to 8: 0.1 to 2. That is, to provide a method for producing an Audi extract having an increased content of resveratrol.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bone disease, which comprises the ethyl acetate fraction of udi acid ethanol extract.

Another object of the present invention is to provide a method for preventing or treating bone disease, comprising administering the pharmaceutical composition to an individual who has or may have bone disease other than humans.

To achieve the above object, one aspect of the present invention comprises adding a mixed solvent comprising ethanol, HCl and water to Audi to obtain a mixture, the mixed solvent comprising ethanol and HCl 6 to 8: It provides a method for producing an Audi extract having an increased content of resveratrol, which is included in a volume ratio of 0.1 to 2.

The term "Audi" in the present invention is a mulberry ( Morus alba ) or a mulberry tree ( Morus bombycis ), and the fruit may include seeds. In the present invention, it was confirmed the optimal extraction conditions to obtain the maximum resveratrol content in Audi.

The term "extract" of the present invention refers to a liquid component obtained by immersing a desired substance in various solvents, and then extracting it for a period of time at a normal temperature or a warm state, and a solid component obtained by removing a solvent from the liquid component. it means. In addition, in addition to the above results, it can be comprehensively interpreted to include all of the dilutions of the above results, their concentrates, their modulators, and tablets.

The Audi extract of the present invention can be obtained by extracting the Audi raw fruit, its dried products, processed products or the like alone or in combination with water or various organic solvents. As a specific example of the extraction solvent, water, a polar solvent or a non-polar solvent may be used, more specifically water, a lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol or butanol, etc.), a mixed solvent thereof, etc. , Most specifically, a mixed solvent containing ethanol and water can be used.

For the purposes of the present invention, the extraction solvent may be a mixed solvent comprising ethanol, HCl and water. Specifically, it may include ethanol and HCl in a volume ratio of 6 to 8: 0.1 to 2, and more specifically, it may include ethanol, HCl and water in a volume ratio of 6 to 8: 0.5 to 2: 1, Most specifically, it may include ethanol, HCl, and water in a volume ratio of 7: 1: 1, but is not limited thereto. When using the above mixed solvent, it is possible to obtain the maximum content of resveratrol in Audi.

In addition, the method of the present invention, a mixture of immersed in a solvent, a dried product thereof, a processed product, etc. in the solvent, 60 to 80 ℃, more specifically at 65 to 75 ℃ for 1 to 4 hours, more specifically for 2 to 3 hours A second step of reacting may be further included, but is not limited thereto. When using the above reaction conditions, it is possible to obtain the maximum content of resveratrol in Audi.

The method for producing an audi extract of the present invention may further include a method for preparing a fraction of an audi extract by further comprising a third step of fractionating the extract with hexane and / or ethyl acetate.

The term "fraction" of the present invention means a result obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various components. In the present invention, the fraction may be interpreted as a fraction obtained by applying the Audi extract of the present invention to various fractionation methods.

The fraction of the Audi extract of the present invention can be obtained by applying the extract to various fractionation methods. As a specific example, a solvent fractionation method performed by treating various solvents, an ultrafiltration fractionation method performed by passing through an ultrafiltration membrane having a constant molecular weight cut-off value, and various chromatographies (for separation according to size, charge, hydrophobicity or affinity) It may be a chromatographic fractionation method, etc. performed), but is not limited thereto.

At this time, as the solvent used in the solvent fractionation method, a polar solvent or a non-polar solvent may be used, and specifically, a non-polar solvent may be used. In addition, the solvent fractionation method may be performed by sequentially fractionating the extract using a low solvent from a solvent having a high non-polarity level. Specifically, a method of sequentially fractionating the extract using hexane or ethyl acetate Can be used, but is not limited thereto.

In addition, the Audi extract according to the present invention may be one that does not contain oxyresveratrol.

The term "oxyresveratrol" (oxyresveratrol) of the present invention is a type of stilbenoid, and refers to aglycone of mulberroside A, a compound found in mulberry trees. Since the oxyresveratrol exhibits tyrosinase inhibitory activity, it has been reported that it can be used as a skin whitening agent (Kim, JK. Et el., Journal of industrial microbiology & biotechnology., 2010, 37 (6): 631 -637.).

In a specific embodiment of the present invention, it was confirmed that the content of resveratrol is maximized in the Audi extract using a mixed solvent containing ethanol: acid: water in a volume ratio of 7: 1: 1 (FIG. 1), When used for 2 to 3 hours at 70 ° C, it was confirmed that the content of resveratrol was maximized in the Audi extract (FIG. 2), and it was confirmed that resveratrol was included in the hexane or ethyl acetate fraction of the extract (FIG. 5). In particular, since the ethyl acetate fraction has low toxicity to cells, it was confirmed that it can exhibit a prophylactic or therapeutic effect of bone disease without side effects. On the other hand, it was confirmed that the extract does not contain oxyresveratrol.

This suggests that the method for producing Audi extract of the present invention is an optimal extraction method capable of maximally obtaining resveratrol contained in Audi.

Another aspect provides a pharmaceutical composition for the prevention or treatment of bone disease comprising an ethyl acetate fraction of audi acid ethanol extract.

At this time, the definitions of 'Audi', 'Extract' and 'Fraction' are as described above.

The term of the present invention, "acidic ethanol" means acidic ethanol, and means an extraction solvent used in the preparation of the extract of the present invention.

Specifically, the acidic ethanol may include ethanol and HCl in a volume ratio of 6 to 8: 0.1 to 2, and more specifically, ethanol, HCl and water in a volume ratio of 6 to 8: 0.5 to 2: 1 It may be, and most specifically, may include ethanol, HCl and water in a volume ratio of 7: 1: 1, but is not limited thereto.

The term "bone disease" of the present invention refers to a disease that is caused by an imbalance of osteoblasts and osteoclasts and causes a disorder of bone tissue or destroys bone tissue.

As specific examples, anorexia, fractures, rickets, osteomalacia, scoliosis, pelvic deformity, osteoporosis, rheumatoid arthritis, periodontal disease ), Hyperparathyroidism, Paget disease or metastatic bone cancers, but is not limited thereto.

The term "prevention or treatment of bone disease" refers to all actions to prevent the development of the bone-related disease or to improve or beneficially improve the symptoms caused by the development of the disease, prevention and reduction of bone disease symptoms. , Improvement, alleviation of pain in the symptoms, reduction of the incidence of bone disease, or any other change in the patient to increase treatment outcome.

In a specific embodiment of the present invention, a fraction of hexane or ethyl acetate was obtained from the Audi extract having an increased content of resveratrol prepared according to the present invention (FIGS. 3 to 5), and the resveratrol purified from the fraction is differentiation of osteoblasts It was confirmed that it exhibits an promoting effect and an inhibitory effect of osteoclasts.

This suggests that resveratrol present in the extracts and fractions of the present invention can be usefully used for the prevention and treatment of bone diseases, and the method of producing Audi extract of the present invention can obtain the maximum resveratrol contained in Audi. Therefore, it is suggested that it can be usefully used for the prevention and treatment of bone diseases.

Meanwhile, the pharmaceutical composition for preventing or treating bone disease of the present invention may further include a suitable carrier, excipient, or diluent commonly used in the manufacture of pharmaceutical compositions, and the carrier is a non-naturally occurring carrier ). In addition, the form of the carrier may include various irregular carriers, microspheres, nanofibers, and the like. As a specific example, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone (PCL), polylactic acid (PLA; Poly Lactic Acid), poly-L-lactic acid (PLLA) and mineral oil.

In addition, the pharmaceutical composition of the present invention is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. You can. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient, for example, starch, calcium carbonate in the Audi extract of the present invention and its fractions. ), Sucrose (lactose), gelatin, etc. are prepared by mixing. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solvents, emulsions, and syrups. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The content of the Audi extract or fractions thereof contained in the pharmaceutical composition of the present invention may be included in an amount of 0.0001 to 50% by weight, specifically 0.01 to 20% by weight, based on the total weight of the final composition, but is not limited thereto.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, the term, 'a pharmaceutically effective amount' is an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level refers to the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration, and the rate of excretion. It may be determined according to factors including a drug used in combination with or used with the composition of the present invention used 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, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And single or multiple administrations. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects.

The dosage of the pharmaceutical composition may be, for example, 1 to 20 mg / kg, specifically 1 to 10 mg / kg of the pharmaceutical composition of the present invention for 1 day to a mammal including a human, and the present invention The frequency of administration of the composition is not particularly limited, but may be administered once a day or divided into doses and administered several times.

Another aspect provides a method of preventing or treating bone disease, comprising administering the pharmaceutical composition to an individual who is or may have a bone disease other than humans.

As described above, since the Audi extract or a fraction thereof having an increased content of resveratrol provided in the present invention can be used as an active ingredient in a pharmaceutical composition for preventing or treating bone disease, the composition can be used to prevent or treat bone disease. have.

The term "individual" of the present invention includes, without limitation, mammals, including mice, livestock, humans, and the like, who are at or at risk of developing bone disease.

In the method for treating bone disease of the present invention, the route of administration of the pharmaceutical composition can be administered through any general route as long as it can reach the target tissue. As a specific example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, intrapulmonary administration, rectal administration, but is not limited thereto. In addition, the composition may be administered by any device capable of transporting the active substance to target cells.

The method of extracting Audi according to the present invention can be obtained to the maximum resveratrol contained in Audi, and thus can be utilized in the production of Audi extract having increased antioxidant activity.

In addition, resveratrol contained in the Audi extract of the present invention exhibits an activity of promoting osteoblast differentiation and inhibiting osteoclast differentiation, and thus may be useful for the prevention and treatment of bone diseases.

1 is a result of analyzing the resveratrol content of the Audi extract according to the solvent. Lane 1 represents 1 mM resveratrol, Lane 2 represents 1 mM oxyresveratrol, and Lanes 3 to 6 each contain ethanol: acid (HCl): water 7: 4: 1 (lane 3; Experimental Example 1); 7: 2: 1 (lane 4; Experimental Example 2); 7: 1: 1 (lane 5; Experimental Example 3); And 7: 0.5: 1 (lane 6; Experimental Example 4) extracted with a mixed solvent comprising a volume ratio of resveratrol contained in the Audi extract.
2 is a result of analyzing the resveratrol content of Audi extract according to the extraction time. Lane 1 represents 1 mM resveratrol, Lane 2 represents 1 mM oxyresveratrol, and lanes 3 to 6 are 0.5 hours each (lane 3; Experimental Example 5); 1 hour (lane 4; Experimental Example 6); 2 hours (lane 5; Experimental Example 8); And resveratrol contained in the Audi extract, extracted for 3 hours (lane 6; Experimental Example 8).
Figure 3 is a schematic diagram showing the strategy of obtaining fractions of Audi extract using hexane and ethyl acetate.
Figure 4 is an image showing the hexane or ethyl acetate fraction layer of the Audi extract.
Figure 5 is a result of confirming the resveratrol contained in the hexane fraction layer (9 to 12) of the Audi extract.
Figure 6 shows the effect of promoting the differentiation of osteoblasts (D1 ORL UVA) of the ethyl acetate fraction fractionated from the Audi extract according to an embodiment of the present invention. a is an Alizarin Red s staining image showing the degree of osteoblast differentiation according to the treatment concentration of resveratrol, and b and c are graphs and tables showing the staining degree of a. At this time, CTL or ODM refers to osteoblasts not treated with resveratrol.
7 is an image showing the inhibitory effect of osteoclast (RAW 264.7) differentiation of the ethyl acetate fraction fractionated from the Audi extract of the present invention according to one embodiment. CTL refers to osteoclasts not treated with resveratrol, RANKL refers to osteoclasts treated with RNAKL (positive control), and RES refers to osteoclasts treated with resveratrol.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Example  1. Audi extract according to the solvent Resveratrol  Content analysis

In order to prepare an Audi extract having the maximum content of resveratrol, the resveratrol content of the Audi extract according to the solvent was analyzed.

First, the conditions of the solvent as set forth in Table 1 below were set. The volume ratio of ethanol and water was fixed to 7 and 1, respectively, and the ratio of acid (HCl) was changed.

ethanol mountain water Experimental Example 1 7 4 One Experimental Example 2 7 2 One Experimental Example 3 7 One One Experimental Example 4 7 0.5 One

Thereafter, 2 g of Audi was treated with the mixed extraction solvent according to Experimental Examples 1 to 4, and reacted at a temperature of 70 ° C. for about 2 hours. After the reaction was completed, the precipitate was discarded using a centrifuge to obtain only the supernatant, and the content of resveratrol contained in the supernatant was checked using thin-layer chromatography (TLC). Specifically, after sprinkling methanol on a TLC plate and reacting in an oven at 120 ° C. for 5 minutes, resveratrol to be used as a standard material and the Audi extract sample were loaded.

As a result, as shown in FIG. 1, it was confirmed that the content of resveratrol was maximized in the Audi extract using a mixed solvent (Experimental Example 3) containing ethanol: acid: water in a volume ratio of 7: 1: 1. On the other hand, it was confirmed that the extract does not contain oxyresveratrol.

Example  2. Extraction of Audi extract according to extraction time Resveratrol  Content analysis

By checking the mixing ratio of the solvent to maximize the content of resveratrol in the Audi extract through Example 1, the resveratrol content of the Audi extract according to the extraction time was analyzed.

First, the conditions of time as set forth in Table 2 below were set. At this time, an extraction solvent containing ethanol: acid: water in a volume ratio of 7: 1: 1 was used.

division Extraction time (h) Experimental Example 5 0.5 Experimental Example 6 One Experimental Example 7 2 Experimental Example 8 3

Thereafter, 2 g of Audi was treated with an extraction solvent containing ethanol: acid: water in a volume ratio of 7: 1: 1, and reacted at a temperature of 70 ° C. for 0.5 to 3 hours according to Experimental Examples 5 to 8. After the reaction was completed, the precipitate was discarded using a centrifuge to obtain only the supernatant, and the content of resveratrol contained in the supernatant was confirmed by the method according to Example 1.

As a result, as shown in FIG. 2, it was confirmed that the content of resveratrol was maximized in the Audi extract having extraction times of 2 to 3 hours (Experimental Examples 7 and 8). On the other hand, it was confirmed that the extract does not contain oxyresveratrol.

Through this, the optimum extraction condition to obtain the maximum resveratrol content in Audi is to react for 2 to 3 hours at 70 ° C with a mixed solvent containing ethanol: acid: water in a volume ratio of 7: 1: 1. Could know.

Example  3. Included in Audi extract Resveratrol  refine

Example  3-1. Of Audi extract Fraction  Produce

In order to purify resveratrol from the Audi extract having an increased content of resveratrol prepared through Examples 1 and 2, first, a fraction of the Audi extract was obtained.

As shown in FIG. 3, for 50 g of audi, an audi extract was obtained by the extraction method identified in Examples 1 and 2 above, and hexane and ethyl acetate were used as a fractional solvent. Specifically, after dissolving 100 g of silica gel in hexane or ethyl acetate, the column was charged. After 30 minutes, sea sand was placed on the silica gel containing the solvent, and a fraction of the extract was obtained by passing the Audi extract through the column.

As a result, as can be seen in Figure 4, a fraction of hexane or ethyl acetate could be obtained.

Example  3-2. Using TLC Hexane In fractions  included Resveratrol  checking way

To confirm that resveratrol was included in the fraction of Example 3-1, TLC was first performed on the hexane fraction by the method according to Example 1.

As a result, as shown in FIG. 5, it was confirmed that all of the four hexane fractions obtained exhibited a band at the same position as resveratrol, and as a result of measuring the absorbance, it was confirmed that a compound having the same absorbance was included. .

Through this, it was found that the hexane fraction contained resveratrol.

Example  3-3. HPLC  Used Hexane In fractions  included Resveratrol  checking way

Through Example 3-2, it was confirmed that resveratrol was included in the hexane fraction, and HPLC was performed on the hexane fraction of Example 3-1 to more accurately perform this.

Specifically, conditions were set as described in Table 3 below, the separation time was 55 minutes in total, the injection volume was 10 μl, and the flow rate was 0.8 ml / min.

Mobile phase time 0.05% H3PO4 in H2O: acetonitrile = 90:10 5 minutes 0.05% H3PO4 in H2O: acetonitrile = 20:80 45 minutes 0.05% H3PO4 in H2O: acetonitrile = 20:80 5 minutes

As a result, it was confirmed that a compound having a peak at the same position as resveratrol was included in all four hexane fractions obtained.

Through this, it was found that the hexane fraction contained resveratrol.

Example  3-4. Ethyl acetate Fraction  Concentrate production

The ethyl acetate fraction obtained through Example 3-1 was concentrated using a vacuum concentrator, dissolved in ethanol, and then filtered to obtain a concentrate of the fraction containing resveratrol. It was adjusted to the corresponding concentration and used in Examples 4-1 and 4-2 below.

Example  4. Included in Audi extract Resveratrol  Confirmation of in vitro activity

Example  4-1. Resveratrol  Osteoblast differentiation promoting effect

In order to confirm the effect of promoting the osteoblast differentiation of the ethyl acetate fraction concentrate prepared through Example 3-4, the differentiation efficiency of osteoblasts according to their concentration was analyzed.

Specifically, the mouse cell line D1 ORL UVA was dispensed at a density of 3x10 cells / well in a 96-well plate, and cultured for 24 hours in an incubator at 37 ° C and 5% CO ₂ . Then, the osteoblast differentiation induction medium (ODM; Osteoblast differentiation media) containing resveratrol (0.1 nM to 1 μM), glycerophosphate, ascorbic acid, and dexamethasone of the present invention was used as the cell. To. Cells were cultured for 21 days, and medium was changed every 3 days with ODM containing resveratrol.

After the culture was completed, Alizarin Red S staining was performed. Cells cultured for 21 days were removed with a medium, washed with DPBS, and fixed for 20 minutes using 3.7% formalin. Fixed cells were washed 3 times with DPBS and stained with 2% Alizarin Red S stain for 15 minutes. The stained cells were washed again with DPBS and dried, and the degree of staining of the cells was checked using an optical microscope.

As a result, as shown in a to c of Figure 6, when resveratrol was included, it was confirmed that the differentiation of osteoblasts was promoted, and it was confirmed that it exhibits an effective differentiation promoting effect even at a low concentration of 0.1 nM.

Example  4-2. Resveratrol  Inhibition effect of osteoclast differentiation

In order to confirm the effect of inhibiting osteoclast differentiation of the ethyl acetate fraction concentrate prepared through Example 3-4, the efficiency of suppressing the differentiation of osteoclasts according to its concentration was analyzed.

Specifically, RAW 264.7 cells were dispensed in a 96-well plate at a density of 3x10 cells / well, and cultured for 24 hours in an incubator at 37 ° C and 5% CO ₂ . Thereafter, resveratrol (0.1 μM to 50 μM) of the present invention or RANKL (100 ng), known as a cytokine that promotes osteoclast differentiation, was treated, and the cells were cultured for an additional 3 days.

After the culture was completed, TRAP staining was performed. Cells cultured for 3 days were removed from the medium, washed with DPBS, and fixed for 20 minutes using 3.7% formalin. The fixed cells were washed 3 times with DPBS and stained for 30-40 minutes using a TRAP staining kit (Acid Phophate kit no.387, Sigma).

As a result, as shown in FIG. 7, when resveratrol was included, it was confirmed that the differentiation of osteoclasts was suppressed by confirming that the number of round osteoclasts was significantly reduced, and resveratrol RANKL, a known osteoclast differentiation inhibitor It was confirmed that it exhibits a better effect of suppressing differentiation.

Through this, it was found that resveratrol present in the Audi extract and fractions according to the present invention shows an effect of promoting differentiation of osteoblasts and inhibiting differentiation of osteoclasts, and thus can be usefully used for prevention and treatment of bone diseases.

From the above description, those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the following claims rather than the above detailed description and equivalent concepts thereof.

Claims (11)

And adding a mixed solvent containing ethanol, HCl, and water to Audi to obtain a mixture, wherein the mixed solvent comprises ethanol, HCl, and water in a volume ratio of 6 to 8: 0.5 to 2: 1. , Resveratrol method of producing an Audi extract with an increased content.
The method of claim 1, further comprising a second step of reacting the mixture at 60 to 80 ° C. for 1 to 4 hours.
delete The method according to claim 1, wherein the mixed solvent comprises ethanol, HCl and water in a volume ratio of 7: 1: 1.
delete The method of claim 2, further comprising a third step of fractionating the mixture with ethyl acetate.
The method of claim 1, wherein the Audi extract does not contain oxyresveratrol.
As a pharmaceutical composition for the prevention or treatment of bone disease comprising an ethyl acetate fraction of an acidic ethanol extract,
The bone diseases include growth insufficiency, fracture, rickets, osteomalacia, scoliosis, pelvic deformity, osteoporosis, rheumatoid arthritis, periodontal disease (periodontal) disease), hyperparathyroidism, paget disease and metastatic bone cancers, or one or more selected from the group consisting of metastatic bone cancers.
delete delete A method of preventing or treating bone disease, comprising the step of administering the pharmaceutical composition of claim 8 to a subject with or unlikely to develop bone disease other than humans.

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