KR101833422B1

KR101833422B1 - Antioxidative or antiobesitic composition comprising extract of Alnus firma

Info

Publication number: KR101833422B1
Application number: KR1020150162950A
Authority: KR
Inventors: 이종석; 이사라; 손연경; 배창환; 여주홍
Original assignee: 대한민국
Priority date: 2015-11-20
Filing date: 2015-11-20
Publication date: 2018-02-28
Also published as: KR20170059101A

Abstract

The present invention relates to a composition for antioxidant or anti-obesity containing a mulberry extract, wherein the pharmaceutical composition or the health functional food containing the mulberry extract comprises a composition for treating metabolic diseases such as obesity, Can be easily used as a health functional food.

Description

An antioxidant or antiobesitic composition comprising extracts of Alnus firm,

The present invention relates to a composition for antioxidant or anti-obesity containing a mulberry extract.

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 statistics from the World Health Organization (WHO), the incidence of obesity is estimated to be around 4 billion, or about 6% of the world's population, estimated to be obesity by 2008, and by 2015 to more than 700 million . Especially in OECD countries, the adult obesity population is increasing year by year, and in the United States, one in three is obese. 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, showing a steady increase trend.

Antioxidation refers to the prevention of many oxidizing reactions in the body. When lipids or lipid proteins present as biological membranes or lipoproteins are attacked by free radicals generated in the body to form various kinds of peroxides, the structures and functions of the surrounding biomolecules are changed, It causes chronic diseases. There are many antioxidant defense mechanisms in the body that can neutralize these free radicals and protect the body. In vivo, free radicals are converted to H ₂ O ₂ by using superoxide dismutase (SOD), an antioxidant enzyme in liver and red blood cells, as a means of protecting the body from these harmful radicals. The H ₂ O ₂ produced in this case can be used to remove the antioxidant enzymes such as catalase (CAT), glutathione-peroxidase (GSH-px) and vitamin E and glutathione, (Tiidus PM et al., Sported Med., 20, pp. 12-13, 1995; Britton, C., et al., Physio. Rev., 59, pp. 527-605, 1979; Ishinaga M et al., J. Nutr. Sci. Vitaminol., 40, pp. 557-567, 1994). However, when such an antioxidant defense system declines to such an extent that free radical formation can not be controlled, excessive excess free radicals generated in the body cause an increase in lipid peroxides and protein oxides, an increase in DNA damage, (Kumar CT et al., Mol. Cell Biochem., 111, pp. 109-115, 1992). Therefore, many synthetic or natural antioxidants have been developed for the reduction of oxidative stress, but synthetic antioxidants have been reported to be vulnerable to safety (Ito N et al., J. Natl. Cancer Inst., 70, pp 343-349, 1983). In recent years, the use of carotenoids, flavonoids, and polyphenols, which are antioxidant substances such as vitamins A, C, and E, which are present in various herbal medicines and edible plant extracts, We are working hard.

The Alnus sp. Plant belongs to the Betulaceae family. There are about 30 species in the northern part of South America, and more than 17 species are known to be native to Korea such as ducks, thorns, mulberry, mulberry, have. The bark of a mulberry tree is called "red mullet" in the private and the oriental medicine. It is known that the medicinal effects known to date are effective for relieving hangover by treatment of cyanosis, strengthening action, non-hemorrhage, diarrhea, trauma hemorrhage and other folk remedies. Especially, it grows well in dry land because it grows well in the dry land. It is easy to cultivate because it grows rapidly in the dry land. It is a species that is mainly used for greening projects in Korea and the greenery project in the mountain region of Korea. .

The inventors of the present invention studied the various physiological activities of Alnus firm extract, and found that the extract of Sambang Duck had antioxidant or anti-obesity effects, thus completing the present invention.

As a prior art for the present invention, Korean Patent Laid-Open No. 10-2014-0100117, which discloses an extract of Alnus japonica and its inhibitory effect on lipid differentiation, Korean Patent No. 10-1062003, The antioxidative activity and the structural correlation activity of the compound isolated from the mulberry tree were disclosed in the thesis of Chungbuk National University, which started the antioxidant activity of the mulberry tree extract, the chemical component of the mulberry and the inhibitory activity of the adipocyte formation, Journal of Applied Physics, Vol. 34, No. 2.

Korean Patent Laid-Open Publication No. 10-2014-0100117 (composition for inhibiting lipid differentiation, comprising extract of mulberry tree or compound isolated therefrom, Elcombiaceae, 2014.08.14) Korean Patent No. 10-1062003 (Composition for prevention and treatment of diabetes mellitus comprising extract of Ascidia officinalis or a compound isolated therefrom as an active ingredient, Changwon National University Industry-University Collaboration Foundation, Aug. 29, 2011)

Dissertation of Master 's Degree in Chungbuk National University, In - Ho Choi, Antioxidant Activity of Extracts from. Ph.D Thesis, Seoul National University, Lee, Min - A, Chemical Composition and Antioxidant Activity of Oriental. Journal of Pharmacognosy, Volume 34, Issue 2 (June 2003), Antioxidant Activity and Structural Correlative Activity of Diarylheptanoid Isolated from. Britton et al., Hydroperoxide metablism in mammalian organ, Physio. Rev., 59, pp. 527-605, 1979. Kumar CT et al., Dietary supplementation of vitamin E protects heart tissue from exercise-induced oxidant stress., Mol. Cell Biochem., 111, pp. 109-115, 1992. Ishinaga M et al., Daily intake of fatty acids, sterols, and phospholipids by Japanese women and serum cholesterol., J. Nutr. Sci. Vitaminol., 40, pp. 557-567, 1994 Ito N et al., Carcinogenicity of butylated hydroxyanisole in F344 rats., J. Natl. Cancer Inst., 70, pp. 343-349, 1983. Tiidus PM et al., Vitamin E status and response to exercise training., Sported Med., 20, pp. 12-13, 1995.

It is an object of the present invention to provide a composition for antioxidant or anti-obesity containing a mulberry extract.

The present invention relates to an antioxidant or anti-obesity composition containing an Alnus firm extract.

The polyphenol content was 400 to 500 mg GAE / g, the total flavonoid content was 50 to 100 mg QE / g, the total proanthocyanidin content was 100 To 200 mg CE / g.

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

The present invention relates to a health functional food for antioxidant or anti-obesity containing an extract of Alnus firm .

Hereinafter, the present invention will be described in detail.

The present invention relates to an antioxidant or anti-obesity composition containing an Alnus firm extract. The solid content of the mulberry extract may preferably be 0.001 to 100 占 퐂 / ml, more preferably 1 to 100 占 퐂 / ml, and most preferably 25 to 100 占 퐂 / ml. The total polyphenol content of the mulberry extract is preferably 300-600 mg GAE / g, more preferably 400-500 mg GAE / g, most preferably 436.26 3.30 mg GAE / g, and the total flavonoid content preferably Preferably 10 to 200 mg QE / g, more preferably 50 to 100 mg QE / g, most preferably 73.82 ± 0.54 mg QE / g, and the total proanthocyanidin content is preferably 10 to 300 mg CE / g, more preferably 100-200 mg CE / g, most preferably 149.25 6.06 mg CE / g.

The mulberry duck extract may be extracted with water, a C1-C4 alcohol, or a mixed solution thereof as a solvent. The C1-C4 alcohol may be extracted from methanol, ethanol, propanol, isopropanol, butanol and isobutanol Can be selected. The solvent is preferably 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, most preferably a 70% v) aqueous solution.

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

The mulberry extract may be isolated or isolated by known methods used for the separation and extraction of plant components, such as extraction with an organic solvent (alcohol, ether, acetone, etc.), partition with hexane and water, And may be fractionated or purified by a suitable 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.

In the present invention, it is possible to use any part such as a leaf, a branch, a stem, a bark, a woody part, a root, a fruit and the like, preferably a leaf, Leaf extracts have the most antioxidant or anti-obesity effect than the other extracts.

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

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. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

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 a health functional food for antioxidant or anti-obesity, comprising a mulberry extract and a pharmaceutically acceptable food-aid additive. The mulberry extract may be added to the health functional food of the present invention in an amount of 0.001 to 100% by weight. 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.

As a prior art for the present invention, Korean Patent Laid-Open No. 10-2014-0100117, which discloses an extract of Alnus japonica and its inhibitory effect on lipid differentiation, Korean Patent No. 10-1062003, The antioxidative activity and the structural correlation activity of the compound isolated from the mulberry tree were disclosed in the thesis of Chungbuk National University, which started the antioxidant activity of the mulberry tree extract, the chemical component of the mulberry and the inhibitory activity of the adipocyte formation, Journal of Applied Physics, Vol. 34, No. 2. However, the extract of the present invention is more excellent in antiobesity or antioxidant activity than the extracts containing the ants of the preceding documents, including the stem, branches and roots of the ducks. In addition, the present invention extracts only the leaf of the mulberry tree separately, and confirms the concentration of the mulberry leaf extract without cytotoxicity unlike the above-mentioned prior arts. At this concentration, the total polyphenol, the total flavonoid And an appropriate content of total proanthocyanidins, thereby providing the best-bodied four-legged duck extract.

Fig. 1 shows the measurement results of the DPPH radical scavenging activity of the mulberry extract.
Fig. 2 shows the results of measurement of ABTS radical scavenging activity of the mulberry extract.
Fig. 3 shows the measurement results of reducing power of the mulberry extract.
Fig. 4 shows the results of cytotoxicity analysis of the extract of mulberry duck.
Fig. 5 shows the result of confirming the fat accumulation amount of the mulberry extract.
6 shows the result of measuring the ROS scavenging activity of adipocyte extract in adipocytes.

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.

&Lt; Example 1 > Preparation of Oriental Duck Extract >

100 g of the leaves were extracted from 1000 ml of 70% (v / v) ethanol solution and then extracted at room temperature (25 ° C) for 24 hours. 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 antioxidant activity of extracts of Oriental ducks

Example 2-1. Sample Preparation

The mulberry duck extract obtained in Example 1 was dissolved in a 94% (v / v) ethanol aqueous solution and prepared at each concentration (50, 25, 12.5, 6.25, and 3.125 占 퐂 / ml).

Ascorbic acid and BHA (2,3-butyl-4-hydroxyanisole) were also prepared for each concentration (20, 10, 5, 2.5, 1.25, 0.625 ㎍ / ㎖).

Example 2-2. Measurement of DPPH radical scavenging activity

For the measurement of DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity, 0.1 ml of sample and 0.1 ml of 0.4 mM DPPH solution were mixed and reacted in a dark place for 30 minutes. The absorbance values at 515 nm of a microplate reader (Molecular Devices, Sunnyvale, Calif., USA) were measured. The DPPH radical scavenging activity was calculated using the following equation, and the results are shown in FIG. 1 and Table 1 .

DPPH radical scavenging ability (%) = [(Ac - As) / Ac] x 100

Ac: Absorbance of blank sample, As: Absorbance of sample group

As shown in FIG. 1, the DPPH radical scavenging activity of the extract of Duck Duck was 65% at a concentration of 50 μg / ml. As shown in Table 1, the IC ₅₀ value was 28.06 ㎍ / (IC ₅₀ value of ascorbic acid used as a positive control, 7.61 / / ml, IC ₅₀ value of BHA of 14.66 / / ml).

DPPH Radical Scatters IC ₅₀ (占 퐂 / ml) Duck Duck Extract 28.06 Ascorbic acid 7.61 BHA 14.66

Examples 2-3. Measurement of ABTS radical scavenging activity

The radical scavenging activity of ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) is a measure of antioxidative activity of both fat soluble and water soluble antioxidants. The ABTS radical scavenging activity was measured by mixing 7 mM ABTS solution and 2.45 mM potassium persulfate and reacting at room temperature for 12-16 hours with light blocked to form ABTS cation And 50 μl of the sample and 150 μl of diluted ABTS solution were added and reacted at room temperature for 20 minutes, and the absorbance at 734 nm of the microplate analyzer was measured. The absorbance at 734 nm was adjusted to 0.70 ± 0.02 at 734 nm. The ABTS radical scavenging activity was calculated using the following equation and is shown in FIG. 2 and Table 2.

ABTS radical scavenging ability = [(Ac-As) / Ac] x 100

Ac: Absorbance of blank sample, As: Absorbance of sample group

As shown in FIG. 2, the ABTS radical scavenging activity of the extract of Duckweed was found to be about 90% at the concentration of 50 μg / ml. As shown in Table 2, ABTS It was confirmed that the radical scavenging activity IC ₅₀ was 25.90 μg / ml, that the ABTS radical scavenging activity IC ₅₀ of ascorbic acid as a positive control was 17.56 μg / ml, and that the ABTS radical scavenging activity IC ₅₀ of BHA was 15.53 μg / ml.

ABTS radical scavenging activity IC ₅₀ ([mu] g / ml) Duck Duck Extract 25.90 Ascorbic acid 17.56 BHA 15.53

Example 2-3. Confirm Reduction Power

In order to confirm the reducing power, the ability of Fe ₃ ⁺ (ferric-ferricyanide) to be reduced to Fe ₂ ⁺ (ferrous) is measured. 0.5 ml of sample, 0.5 ml of 0.2 M sodium phosphate buffer and 0.5 ml of 1% potassium ferricyanide were mixed and reacted at 50 ° C for 20 minutes. Then, 0.5 ml of 10% trichloroacetic acid was added thereto and centrifuged at 12,000 rpm for 10 minutes. After centrifugation, 1.0 ml of supernatant and 0.2 ml of 0.1% iron (Ⅲ) chloride were added, reacted at room temperature for 10 minutes, and absorbance was measured at 700 nm on a microplate analyzer. The results are shown in Fig. 3 The reducing power of 25 / / ml of acid was calculated as 100%).

Referring to FIG. 3, it can be seen that the reducing power of the mulberry extract is increased in a concentration-dependent manner.

Examples 2-4. Verify ORAC Acquisition

The ORAC assay is a kind of inhibition method for measuring the degree of free radical damage to fluorescent labels. To perform the ORAC assay, samples are dissolved in 75 mM phosphate buffer (pH 7.4) , 150 μl of 40 nM Fluorescein as a fluorescent substance and 25 μl of 18 mM AAPH (2,2'-azobis (2-methylpropionamidine) dihydrochloride) producing peroxyl radical were added and reacted at 37 ° C for 15 minutes. The fluorescence spectrophotometer was adjusted at 37 ° C, and then measured for 90 minutes at an excitation wavelength of 485 nm and an emission wavelength of 520 nm once every 3 minutes. The results are shown in Table 3 below. If antioxidants are present in the sample, the free radical damage is inhibited in proportion to these concentrations, thus the change (decrease) in fluorescence is suppressed. Trolox (0 ~ 10 μM) was used as the reference material and the area under the curve (AUC) of the standard reagent and the sample was measured. ORAC was expressed as μM TE / ㎎ using a regression curve between standard reagent concentration and AUC.

Sample (10 [mu] g / ml) ORAC value (占 TE / mg) Duck Duck Extract 792.01 + - 13.10

Referring to Table 3 above, it was confirmed that the extract of Orthodox ducks had a value of 792.01 ± 13.10 μM TE / mg as measured by ORAC (Oxygen radical absorbing capacity).

Examples 2-5. Measurement of NO radical scavenging ability

For the measurement of NO (nitric oxide) radical scavenging ability, 0.5 ml of 1 mM NaNO ₂ solution was added to 0.5 ml of the sample, the pH of the reaction solution was adjusted to 1.2 with 0.1 N HCl, and the final volume of the reaction solution was adjusted to 5 ml by adding distilled water. This solution was reacted at 37 ° C for 1 hour. Take 1 ml of the reaction mixture and add 5 ml of 2% acetic acid and 1 ml of Griess reagent (A: B = 1: 1, A: 1% sulfanilic acid in 30% : 1% naphthylamine in 30% acetic acid) was added and mixed well. The mixture was reacted at room temperature for 15 minutes and absorbance was measured at 520 nm. The results are shown in Table 4 below. NO radical scavenging activity was calculated using the following equation.

NO radical scavenging ability = [(Ac - As) / Ac] x 100

Ac: Absorbance of blank sample, As: Absorbance of sample group

Sample Concentration (/ / ml) NO radical scavenging ability (%) Duck Duck Extract 500 61.31 + - 0.34 200 29.24 + 1.43 100 17.20 ± 1.88 Ascorbic acid 500 64.01 + - 1.03 200 29.70 + - 6.42 100 6.72 + - 6.04

As shown in Table 4, the NO radical scavenging ability of the extracts from the mulberry ducks was NO radical scavenging ability at a concentration of 61.31% at 500 μg / ml, 29.24% at 200 μg / ml and 17.20% at 100 μg / ml And ascorbic acid, a positive control, showed 64% of NO radical scavenging activity at 500 ㎍ / ㎖, indicating that Sambang Duck Extract had similar activity to the positive control.

Examples 2-6. Total polyphenol, total flavonoid, and total proanthocyanidin content

Total phenolic contents were measured using Folin-Ciocalteu. To 500 ml of each sample, 1.25 ml of 12.5% (w / v) sodium carbonate solution and 250 μl of 1.0 M Folin-Ciocalteu's reagent were mixed and allowed to stand in a dark place for 40 minutes and the absorbance was measured at 750 nm on a microplate analyzer. Gallic acid was used as a standard and expressed as mg GAE / g sample.

The total flavonoids contents 0.5 ml of 2% (w / v) aluminum chloride was added to 0.5 ml of each sample and reacted at room temperature for 60 minutes. Absorbance was measured at 420 nm on a microplate analyzer. Quercetin was used as a standard and ㎎ QE / g respectively.

Total anthocyanin contents were measured using vanillin-hydrochloric acid (V-HCl). 0.5 mg of each sample was dissolved in 5 ml of methanol, and then 3 ml of 4% (w / v) vanillin solution was added thereto to vigorously agitate. After that, 1.5 ml of Concentrated hydrochloric acid was added to the reaction solution, reacted at room temperature for 15 minutes, and the absorbance was measured at 490 nm in a microplate analyzer. At this time, catechin was used as a reference material and expressed in ㎍ CE / g sample.

The results of each experiment are shown in Table 5 below.

Sample Total polyphenol content Total flavonoid content Total proanthocyanidin content Duck Duck Extract 436.26 + - 3.30 mg GAE / g 73.82 ± 0.54 mg QE / g 149.25 ± 6.06 mg CE / g

As shown in Table 5, total polyphenol, total flavonoid and total proanthocyanidin content of mulberry duck extract were 436.26 ± 3.30 mg GAE / g and 73.82 ± 0.54 mg QE / g, respectively, , And the total proanthocyanidin was 149.25 ± 6.06 mg CE / g, indicating that the antioxidant ability of the extract of the oriental ducks was excellent.

<Example 3> Confirmation of anti-obesity activity of extract of Oriental ducks>

Example 3-1. Preparation of sample

As the samples used in the experiment, the duckberry extract obtained in Example 1 was used at a concentration of 25 μg / ml, 50 μg / ml and 100 μg / ml using 100% DMSO (dimethyl sulfoxide).

Example 3-2. 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.

Example 3-3. Evaluation of cytotoxicity using XTT assays

The cytotoxicity of 3T3-L1 cells was assessed using the XTT assay (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 the extracts of Oriental mulberry 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.

4, the effect on the survival rate of 3T3-L1 cells was measured by the XTT assay using a concentration of 25 μg / ml, 50 μg / ml and 100 μg / ml, respectively, , And did not show cytotoxicity up to 100 μg / ml of the duck duck extract.

Example 3-4. Estimation of fat accumulation inhibitory effect by oil red O staining

To measure the amount of fat accumulation in 3T3-L1 cells according to the differentiation process using Oil red O staining, each sample 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 using 5 mM NAC (N-acetyl-L-cysteine) as a positive control, and the results are shown in FIG.

FIG. 5 shows that the fat accumulation amount was significantly decreased in the concentration-dependent manner on the mulberry extract (AF), and the mulberry dug extract was found to be effective in inhibiting fat accumulation .

Examples 3-5. Evaluation of ROS abatement efficacy using NBT assay

To measure the amount of reactive oxygen species (ROS) produced by the differentiation process using NBT assay (Nitroblue tetrazolium assay), the culture medium of 3T3-L1 cells cultured and differentiated for 8 days in 24 well plate was removed After washing twice with sterile PBS (phosphate-buffered saline, pH 7.4), 0.2 ml of 0.2% (w / v) NBT (nitroblue tetrazolium) solution was added and incubated in a CO ₂ incubator for 90 minutes. 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 was measured at 570 nm using a spectrophotometer. The results are shown in FIG.

Referring to FIG. 6, it was shown that the ROS content was significantly decreased in adipocytes treated with mulberry extract (AF) in a concentration-dependent manner (group without con-extract in FIG. 6).

&Lt; Formulation Example 1 >

Formulation Example 1-1. Manufacture of tablets

200 g of the 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 1-2. Injection preparation

1 g of the mulberry extract of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

<Formulation Example 2: Food Preparation>

Formulation Example 2-1. Manufacture of cooking seasonings

The safflower 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 bread of the present invention was added to the flour at 0.1 wt%, and bread, cake, cookies, crackers and noodles were prepared to prepare a food for health promotion.

Preparation Example 2-3. Manufacture of soups and gravies

The soup duck 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

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

Formulation Example 2-5. Vegetable juice manufacturing

The vegetable juice for health promotion was prepared by adding 0.5 g of the duckberry extract of the present invention to 1,000 ml of tomato juice or carrot juice.

Formulation Example 2-6. Manufacture of fruit juice

Healthy fruit juice was prepared by adding 0.1 g of the extract of Quercus mongolica of the present invention to 1,000 ml of apple juice or grape juice.

Claims

( Alnus firm ) leaves having a total polyphenol content of 400 to 500 mg GAE / g, a total flavonoid content of 50 to 100 mg QE / g and a total proanthocyanidin content of 100 to 200 mg CE / g A composition for an anti-obesity agent, which comprises 50 to 90% (v / v) aqueous solution of extract, which does not exhibit cytotoxicity at a solid content of 50 to 100 占 퐂 / ml and has an effect of inhibiting fat accumulation.

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( Alnus firm ) leaves having a total polyphenol content of 400 to 500 mg GAE / g, a total flavonoid content of 50 to 100 mg QE / g and a total proanthocyanidin content of 100 to 200 mg CE / g A health functional food for an anti-obesity drug characterized by containing 50 to 90% (v / v) aqueous solution extract having an anti-fat accumulation effect without exhibiting cytotoxicity at a solid content of 50 to 100 占 퐂 / ml.

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