KR20190002092A - Antiobesitic composition comprising extract of Rhododendron mucronulatum - Google Patents

Abstract

The present invention relates to an antiobesity composition comprising an extract of Rhododendron mucronulatum. A pharmaceutical composition or a health functional food containing the extract of Rhododendron mucronulatum can be usefully used for a composition for the treatment of metabolic diseases such as obesity, or a health functional food for prevention and alleviation of the diseases.

Description

An antiobesity composition comprising extracts of Rhododendron mucronulatum

The present invention relates to an anti-obesity composition containing an azalea extract. More specifically, the present invention relates to an anti-obesity composition containing an alcohol extract of leaves and branches of azalea which has an inhibitory effect on adipocyte differentiation inhibition and fat accumulation inhibition and an oxidation reaction depending on adipocyte differentiation process.

In recent years, the population of obesity has been rapidly increasing due to improvements in living standards due to economic development, lack of exercise due to busy living environment, and excessive intake of nutrition. Obesity is a condition in which energy is excessively accumulated in the body due to unbalanced energy intake and consumption, resulting in an abnormally increased adipose tissue. The World Health Organization (WHO) considers Oriental BMI (body mass index) 23-25 as overweight, 25-30 obese, and over 30 as high obesity. The causes of obesity include environmental factors such as high fat, high calorie diet, lack of exercise due to busy social environment, endocrine abnormality, and genetic factors. Of these, about 50 to 70% of obesity is due to environmental factors And the rest is known to be caused by genetic factors. According to the statistics of the World Health Organization (WHO), the incidence of obesity is reported to increase year by year, especially in the OECD countries, the adult obesity population is increasing year by year. In Korea, the obesity population is increasing rapidly due to changes in the living environment, and the Korean adult obesity population with a body mass index (BMI) of 25 or more is increased by 40 million every year. As of 2005, 3 out of 10 were obese. According to data from the CDC, the obesity population in 1995 was 26.3% of the total population, but in 2001 it increased to 30.6% of the total population and to 31.5% of the total population in 2005, and is still showing a steady upward trend.

Rhododendron ( mucronulatum Turcz.) Is also called a spice or a dog. It grows in the mountains of 50 ~ 2,000m high across the country. Its height is 2 ~ 3m and many branches are split at the upper part of the stem. The small branches are light brown and scaly pieces. Leaves are alternate long, elliptical, or inverted, with a length of 4 ~ 7cm, both ends are narrow, and the edges are flat. There are scaly pieces on the surface of the leaves, dense scales on the back, no hairs, and petiole length of 6 ~ 10mm. The flowers bloom earlier than the leaves in April, and they come out one by one at the end of the branches, but two to five are also running together. The corolla is a funnel-shaped, 4-5cm in diameter, purple or light red with reddish hairs, hairs on the surface, and 5 ends. There are 10 stamens, white hairs on the underside of the operating table, 1 pistil and much longer than surgery. The fruit is capsule, 2cm in length, cylindrical shape, and the style remains at the end. The flowers are planted for ornamental purposes, and the flowers make blooming flowers in the early spring or dip azalea liquor. In one room, the flower is used as a medicinal substance called Youngsanhong, which is effective for sea water, bronchitis and headache caused by cold, and has diuretic effect. It is distributed in Korea, Japan and China. The white flowers are called white rhododendrons (for. Albiflorum), and the hairy branches of small branches and leaves are called fir azaleas (var. Ciliatum). They are often grown on the beach and high mountains. It is called "forbidden azalea" (white blossom), and it is sometimes seen near the coast. It is called a wild azalea (var. Latifolium) whose leaves are round or wide elliptical. Among the things that grow near the beach, there are shiny leaves on the leaves, and warts like warts on the both sides are called "veritable azaleas" (var. Maritimum). It is called "R.saisiuense" and it grows near the top of Mt. Halla.

The present inventors have studied various pharmacological activities of Azalea, and found that Azalea extract has an anti-obesity effect, thus completing the present invention.

Korean Patent No. 10-1367512, entitled "Natural Preservative Composition Containing Rhododendron Fermented Extract and Method for Producing the Same", Applicant: Intaglio Co., Ltd., Registered Date: Feb. 19, 2014) Korean Patent No. 10-0829722, entitled "Cosmetic Composition for Preventing Aging of Skin Containing Azalea Root Extract as Active Ingredient, Applicant: Koreana Cosmetics Co., Ltd., registered on May 08, 2008) Korean Patent Laid-Open No. 10-2011-0117876 (entitled "Antioxidative, Cosmetic Composition Containing Fermented Extract of Carriers Having Anti-inflammatory Effect", Applicant: Woongjin Coway Co., Ltd. Disclosure Date: October 28, 2011) Korean Patent Laid-Open No. 10-2016-0098567 (entitled Anti-inflammatory Antioxidative Composition Containing Tail Rhododendron Extract, Applicant: Jae Hae Jung, Publication Date: Aug. 19, 2016)

It is an object of the present invention to provide an anti-obesity composition containing an azalea extract. More specifically, it is an object of the present invention to provide an anti-obesity composition containing an alcohol extract of leaves and branches of azalea, which has an inhibitory effect on adipocyte differentiation inhibition and fat accumulation inhibition and an oxidation reaction according to an adipocyte differentiation process There is.

The present invention relates to an anti-obesity composition containing Rhododendron mucronulatum Turcz. Extract.

The azalea extract can be extracted with water, C1-C4 alcohol or a mixed solution thereof as a solvent.

The azalea extract may be one obtained by extracting leaves and twigs.

The present invention also provides an anti-obesity health functional food containing azalea extract.

Hereinafter, the present invention will be described in detail.

The present invention relates to an anti-obesity composition containing an azalea extract. When producing an azalea extract according to the present invention, it is possible to use any part such as a leaf, a branch, a twig, a stem, a bark, a woody part, a root, a fruit, etc. Preferably, The leaves and twigs extracts of Azalea have the most anti-obesity effect than the extracts of other parts. In this case, the leaves and twigs of the azalea were extracted separately, and the extracts obtained by mixing the leaves and twigs showed better efficacy. It is most preferable that the leaves and the twigs are mixed at a weight ratio of 1: 0.5 to 2.

The azalea extract can be extracted with water, a C1-C4 alcohol or a mixed solution thereof as a solvent. The C1-C4 alcohol is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol and isobutanol . The solvent may preferably be a 50 to 90% (v / v) aqueous solution of a C1 to C4 alcohol, more preferably a 50 to 90% (v / v) aqueous solution of ethanol.

The water, the C1-C4 alcohol or the mixed solution thereof used in the production of the Azalea extract may be 1 to 40 times as much as the azalea used weight (1 to 40 liters per kg), preferably 5 to 40 times the volume Can be used. The extraction conditions of the azalea extract may be 1 to 48 hours at 20 to 100 ° C. The above process can be repeated 1 to 4 times. However, each process is not limited thereto. As the extraction apparatus, a conventional extraction apparatus, an ultrasonic pulverization extractor, or a fractionator may be used. The azalea extract thus prepared can be removed by hot air drying, vacuum drying or freeze drying. In addition, the azalea extract can be purified by using column chromatography.

The azalea extract can be isolated or extracted according to a conventional method, for example, by a known method used for separation and extraction of plant components such as extraction with an organic solvent (alcohol, ether, acetone, etc.), distribution of hexane and water, And then fractionated or purified by using a combination method.

The chromatography can be carried out using silica gel column chromatography, LH-20 column chromatography, ion exchange resin chromatography, medium pressure liquid chromatography chromatography, thin layer chromatography (TLC), silica gel vacuum liquid chromatography, and high performance liquid chromatography.

The present invention also provides a pharmaceutical composition for anti-obesity containing an azalea extract. The azalea extract may be added in an amount of 0.001 to 100% by weight to the pharmaceutical composition of the present invention.

The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to conventional methods. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition 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 and 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. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

The dosage of the pharmaceutical composition of the present invention will depend on the age, sex, body weight of the subject to be treated, the particular disease or condition to be treated, the severity of the disease or condition, the route of administration and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, 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 dural or intracerebral injection. Since the compound of the present invention has little toxicity and side effects, it can be safely used even for long-term administration for preventive purposes.

The present invention also provides an anti-obesity health functional food comprising an azalea extract and a pharmaceutically acceptable food-aid additive. The azalea extract may be added in an amount of 0.001 to 100% by weight to the health functional food of the present invention. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the extract of the present invention can be added include various kinds of drinks, meat, sausage, bread, Snacks, noodles, ice cream, dairy products, soups, ionic drinks, beverages, alcoholic beverages, gums, tea and vitamin complexes.

The present invention relates to an anti-obesity composition containing an azalea extract, wherein the pharmaceutical composition or the health functional food containing the azalea extract is a composition for treating metabolic diseases such as obesity, or a health functional food As shown in Fig.

Prior art utilizing rhododendrons is disclosed in Korean Patent No. 10-1367512, in which a preservative composition technology fermented with azaleas and a skin containing a root extract of azalea such as disclosed in Korean Patent No. 10-0829722 as an active ingredient Antioxidant such as anti-aging cosmetic technology, Korean Patent Laid-Open No. 10-2011-0117876, cosmetic composition containing fermentation extract of vasectomy having anti-inflammatory effect, and the like are only confirmed. Therefore, the present invention provides azalea extract having the best bio-reactivity by preparing leaf and twig extracts of azalea, confirming the concentration of the extract without cytotoxicity, and showing the content of the extract having anti-obesity effect at this concentration .

FIG. 1A shows photographs of leaves and twigs of azaleas used in the preparation of extracts, and FIG. 1B shows results of cytotoxicity of azalea extracts.
FIG. 2 shows the result of confirming the inhibition of adipocyte differentiation by Azalea extract.
FIG. 3 shows the result of measuring the ROS scavenging activity of the azalea extract in the adipocyte differentiation process.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

≪ Example 1: Preparation of azalea extract >

The Azalea extract was prepared by immersing 100 g of a sample (blend of leaves and twigs at a weight ratio of 1: 1, refer to FIG. 1A) in 1000 ml of 70% (v / v) ethanol aqueous solution and extracting at room temperature (25 ° C) for 24 hours Respectively. The extraction procedure was repeated three times, and the obtained extracts were all combined. The extract was filtered with a filter paper (Whatman, No.3, Maidstone, Kent, UK), concentrated under reduced pressure using a rotary vacuum concentrator (EYELA, N-3000, Tokyo, Japan) and then lyophilized (Ilshinbiobase Co., Ltd.). , Ltd, Yangju, Korea).

<Example 2> Identification of anti-obesity activity of azalea extract>

Example 2-1. Preparation of sample

The azalea extract obtained in Example 1 used in the experiment was diluted to a final concentration of 25 μg / ml, 50 μg / ml and 100 μg / ml using 100% DMSO (dimethyl sulfoxide) Respectively.

Example 2-2. 3T3-L1 cell culture

3T3-L1 cells were purchased from ATCC and cultured in high-glucose DMEM (Dulbecco's modified Eagle's medium) in the presence of 10% BS (bovine serum) and 1% penicillin / streptomycin (P / S) at 37 ° C in a 5% CO ₂ incubator . When the 3T3-L1 cells in the preadipocyte state are filled to 100% in the cell culture dish, a high concentration of 10% FBS (fetal bovine serum) and 1% P / S are added for differentiation into adipocytes Glucose DMEM was used and 0.5 mM Isobutylmethylxanthine (IBMX), 1 μM Dexamethasone (DEX) and 1 μg / ㎖ insulin were added for 48 hours. Cells were cultured and tested in a 96-well plate and a 24-well plate by dividing cells according to the purpose of the experiment.

Examples 2-3. Evaluation of cytotoxicity using XTT assays

The cytotoxicity of 3T3-L1 cells was assessed using an XTT assay kit (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carboxanilide innersalt assay kit Respectively.

XTT and PMS were added to the cells to increase the bioreduction by adding PMS (phenazine methosulfate), which serves as an Election coupling agent to XTT, and then the Tetrazolium ring of XTT by mitochondrial dehydrogenase of living cells was decomposed to form Formazan crystal Formazan crystals are dissolved in aqueous solution and become yellow. This yellow was measured by ELISA (VersaMax ELISA Microplate Reader, Molecular Devices, Sunnyvale, USA) and used for cytotoxicity evaluation.

For this purpose, 3T3-L1 preadipocyte cells were plated in 96-well plates at a concentration of 1 × 10 ^{6 on the} day before the experiment and cultured for 24 hours. The concentrations of Azalea extract were 25 ㎍ / ㎖, 50 ㎍ / ㎖ and 100 ㎍ / ㎖, respectively, and cultured in a cell incubator for 24 hours. Then, 1 ㎖ of XTT and 20 ㎕ of PMS reagent were mixed, and 20% of the media volume was added. After reacting for 4 hours in a cell incubator, absorbance at 450 nm and 690 nm was measured using a spectrophotometer And the result is shown in FIG. 1B.

1B, the effect of Azalea extract on the survival rate of 3T3-L1 cells was measured by XTT assay at a concentration of 25 μg / ml, 50 μg / ml and 100 μg / ml, respectively. As a result, Lt; RTI ID = 0.0 &gt; pg / ml. &Lt; / RTI &gt;

Examples 2-4. Inhibition of adipocyte differentiation and evaluation of fat accumulation inhibition by oil red O staining

In order to measure the amount of fat accumulation in 3T3-L1 cells according to the differentiation process using oil red O staining, a sample of Azalea extract was treated to remove the culture of 3T3-L1 cells differentiated for 8 days in a 24-well plate Then, 0.5 ml of a 10% formalin solution was added, and the mixture was allowed to stand at room temperature for 5 minutes. 3T3-L1 cells differentiated with the same amount of formalin solution were left at room temperature for more than 1 hour, formalin was removed, and cells were washed with 0.5 ml of 60% isopropyl alcohol solution to completely dry the cells. The fully dried cells were stained with a pre-prepared working solution (Oil Red O: DDW = 6: 4), and the cells were washed 4 times with distilled water It was completely dried. The oily red O bound to the lipid components accumulated in the cells was eluted with a 100% isopropyl alcohol solution and absorbance was measured at 490 nm using a spectrophotometer. At this time, the degree of differentiation of 3T3-L1 cells was compared and analyzed using 5 mM NAC (N-acetyl-L-cysteine) as a positive control. The results are shown in FIG.

2, the fat accumulation was significantly decreased in a concentration dependent manner on the Azalea extract. The Azalea extract was found to be effective in inhibiting differentiation of fat cells and inhibiting fat accumulation (see con - Without extracts).

Examples 2-5. Evaluation of ROS abatement efficacy using NBT assay

To determine the amount of reactive oxygen species (ROS) produced by differentiation of adipocytes using Nitroblue tetrazolium assay, azalea extracts were added to 24-well plates for 8 days (25, 50, 100 μg / mL) to induce differentiation. By using this after 3T3-L1 After removing the culture medium of the cells with sterile PBS (phosphate-buffered saline, pH 7.4) 2 times wash, 0.2% (w / v) NBT was added to 0.2 ㎖ (nitroblue tetrazolium) solution, CO ₂ And incubated for 90 minutes in the incubator. After the staining, the plate was rinsed twice with distilled water and dried. DMSO (the ROS accumulated in the cells reacted with the NBT solution to produce dark blue formazan, which was eluted from the cells to measure the absorbance) and 1 N potassium hydroxide : 3 (v: v). The same amount of distilled water was added thereto, and the absorbance at 570 nm was measured using a spectrophotometer. The results are shown in FIG.

Referring to FIG. 3, it was shown that ROS content was significantly decreased in adipocytes treated with Azalea extract (concentration not treated with con-extract of FIG. 3) in a concentration-dependent manner.

&Lt; Formulation Example 1 >

200 g of the Azalea extract of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

<Formulation Example 2: Food Preparation>

Formulation Example 2-1. Manufacture of cooking seasonings

The azalea extract of the present invention was added to the cooking seasoning at 1 wt% to prepare a cooking sauce for health promotion.

Formulation Example 2-2. Manufacture of flour food products

The azalea extract of the present invention was added to wheat flour in an amount of 0.1 wt%, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare a food for health promotion.

Preparation Example 2-3. Manufacture of soups and gravies

The azalea extract of the present invention was added to soup and juice at 0.1 wt% to prepare health promotion soup and juice.

Formulation Example 2-4. Manufacture of dairy products

The azalea extract of the present invention was added to milk in an amount of 0.1 wt%, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 2-5. Vegetable juice manufacturing

0.5g of the azalea extract of the present invention was added to 1,000ml of tomato juice or carrot juice to prepare health promotion vegetable juice.

Formulation Example 2-6. Manufacture of fruit juice

0.1 g of the azalea extract of the present invention was added to 1,000 ml of apple juice or grape juice to prepare fruit juice for health promotion.

Claims (6)

A composition for anti-obesity characterized by containing Rhododendron mucronulatum Turcz. Extract. The method according to claim 1,
Wherein the azalea extract is extracted with water, C1-C4 alcohol or a mixed solution thereof as a solvent. The method according to claim 1,
Wherein the azalea extract is obtained by extracting leaves and twigs of azalea. An anti-obesity health functional food characterized by containing Rhododendron mucronulatum Turcz. Extract. 5. The method of claim 4,
Wherein the azalea extract is extracted with water, C1-C4 alcohol or a mixed solution thereof as a solvent. 5. The method of claim 4,
The azalea extract is characterized by extracting leaves and twigs of azalea.

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