KR20020010999A - The Extracts of Rosa davurica Pall Containing High Antioxidative Activity and Preparation Process of Flavan 3-ol Derivatives Using Their Extracts - Google Patents

Abstract

PURPOSE: A process of preparing catechin as flavane-based compounds(formula) by extracting Rosa davurica PALL. is provided. The extract containing a large amount of catechin exhibits higher activity than conventional antioxidant while exhibiting DPPH free radical scavenging activity, lipoperoxidation inhibitory activity and antioxidant effect on oxidation of low density lipoprotein. CONSTITUTION: Finely cut Rosa davurica PALL. is dried in the shade, added with 20 times of alcohol such as methanol and precipitated at 20 to 80deg.C for 3 to 5 days. The precipitate is added with 20 times of methanol and then re-extracted. The above two extracts are then mixed, concentrated under reduced pressure and solvent-fractionated successively.

Description

The extract of Rosa davurica Pall Containing High Antioxidative Activity and Preparation Process of Flavan 3-ol Derivatives Using Their Extracts}

The present invention relates to a method for producing catechin, which is an extract of bionymph tree having antioxidant activity and a flavan compound. To be more specific, the extract is extracted from alcohols such as methanol from bio-wood, or from organic solvents such as ethyl acetate, and used as an antioxidant including lipid peroxidation inhibitory activity and low density lipoprotein inhibitory activity. The present invention relates to a method for isolating extracts and catechin compounds having high antioxidant activity therefrom, and may be usefully used as pharmaceuticals and quasi-drugs, cosmetic additives, food additives, beverage additives, and dietary supplements.

Rosa davurica Pall. Is a perennial plant belonging to the family Rosaceae and is a deciduous broad-leaved shrub distributed in central and northern Korea, Japan, Manchuria, and Siberia. Its height is 1 ~ 1.5m and the stem is straight, reddish brown and spiny. The leaf is divided into right upper lobes, elliptical or oblong, sharp at both ends, 1 ~ 3 cm in length, with fine teeth on the edge and occupied on the back. The flowers are rose-colored and fragrant. Fruit is spherical and ripens in yellow in September. The petals are luminescent and the ends are closed. It is used for food in the private sector, and roots and fruits are used for dry stomach and sheep's landscaping, and it has been used for the treatment of indigestion, antiexpectation, stomach pain, menstrual dysfunction [Yook Chang-soo, (1989) . In addition, the civilian has been used as a vitamin deficiency, increased resistance to various diseases, hot water medicine, and liver function protection medicine. According to the recent research results, it is reported that biodeer fruit and leaves contain a large amount of vitamin C (Shin, Kuk-Hyun et al., (1998) Korean Medicinal Crop Science Society, 6, 6-10), attracting attention as a medicinal plant resource. . However, until now, it was not known which biologically active ingredients contained these biological activities.

Recently, as theories have been recognized that aging and various adult diseases are caused by reactive oxygen species, studies are being actively conducted to develop natural antioxidants capable of regulating oxygen-derived reactive oxygen species. Substances that can control reactive oxygen species (superoxide anion, hydroxyl radical, peroxyl radical, alkoxyl radical, etc.) are called antioxidants, which include superoxide dismutase and glutathione peroxidase. Antioxidant enzymes such as glutathione peroxidase and catalase, and low-molecular nonenzymatic antioxidants such as tocopherol, glutathione, ascorbate, and flavonoid compounds Doing. Synthetic antioxidants such as BHT (bytylated hydroxytoluene) and BHA (butylated hydroxyanisole) have been developed and used in the food field. However, as mutagenicity and toxicity are pointed out, studies on the development of safer and more active natural antioxidants are actively conducted. Richard, MJ, et al ., (1992) J. Chromarography, 577, 9-18; Lissi, E., et al ., (1995) Free Rad ic. Biol. Med., 18, 153-158; Ghiselli, A., et al. , (1995) Free Radic. Biol. Med., 18, 29-36; Hsiao, G., et al ., (1996) Biochim. Biophys. Acta, 1298, 119-130; Frei, B., et al ., (1998) 85, 9748-9752. However, the antioxidants developed to date do not surpass the existing synthetic antioxidants in terms of efficacy and cost, and there is an urgent need for the development of new natural antioxidants with excellent antioxidant activity and safety.

Therefore, the inventors continued to develop new natural antioxidants from plant resources. As a result, the present inventors confirmed strong antioxidant activity from the biogas, extracted with methanol, and then suspended the extract in water, followed by hexane, chloroform, and ethyl acetate. After fractionation with an organic solvent such as butanol, the catechin compound having excellent antioxidant activity was separated from the ethyl acetate fraction and the butanol fraction, and the present invention was completed by identifying the structure.

The present invention provides a method for preparing catechin, a flavanic compound having antioxidant activity, which is extracted from bio-curved oak tree with antioxidant activity, or the extract is extracted with an organic solvent, and an antioxidant and lipid peroxidation using these extracts as an active ingredient. An object of the present invention is to provide raw materials such as food additives, pharmaceuticals and quasi-drugs, cosmetic additives, beverage additives, or functional health supplements for inhibitors or low-density lipoprotein oxidation inhibitors (arteriosclerosis inhibitors).

1 shows the ultraviolet spectrum of Formula 1 in the specification,

Figure 2 shows the hydrogen nuclear magnetic resonance spectrum of the formula (1) in the specification,

Figure 3 shows the carbon nuclear magnetic resonance spectrum of the formula (1) in the specification,

Figure 4 shows a chromatogram of high pressure liquid chromatography (HPLC) of the formula (1) in the specification,

Figure 5 shows the antioxidant effect of the barberry extract on low density lipoprotein (LDL) oxidation.

In the present invention, in the process for developing a natural antioxidant from native plant resources in Gangwon-do, the antioxidant activity was confirmed in the extract and fraction of the bark, and the antioxidant material was separated therefrom. Specifically, the present invention extracts the biogas from methanol, and the extract is suspended again in water, and then sequentially fractionated with organic solvents such as hexane, chloroform, ethyl acetate, butanol, and extracts and fractions. The extracts and fractions of the bionymph tree were used to investigate the antioxidant activity by DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging assay. At this time, among the extracts of the bark tree, the bark roots had the highest antioxidant activity. Among the fractions, the ethyl acetate and butanol fractions of the bark roots showed the most excellent antioxidant activity. In addition, the present invention is carried out by sequentially performing the adsorption chromatography, Sephadex LH-20, and high-pressure liquid chromatography (HPLC) using the ethyl acetate fraction of the root of the bark of the tree, to isolate the compound of formula 1 excellent in antioxidant activity, Purified.

(Formula 1)

In this case, it is preferable to use silica gel column chromatography, which is an adsorption chromatography, and a mixed solvent of chloroform: methanol as the developing solvent. In addition, in the case of biodeer extract, it is preferable to fractionate with an organic solvent such as ethyl acetate, and then perform high pressure liquid chromatography (HPLC) to purify the compound of Chemical Formula 1. Physical and chemical properties of Chemical Formula 1 of the present invention were examined by a spectrophotometer, carbon nuclear magnetic resonance spectrum, hydrogen nuclear magnetic resonance spectrum, and the like to confirm that the compound was catechin. The present invention is to investigate the antioxidant activity of the compound of Formula 1 and bio-extract extract, 1.1-diphenyl-2-picrylhydrayl containing free radicals (1,1-diphenyl-2-picrylhydra zyl, DPPH) to measure antioxidant activity, thiobarbituric acid reactive substances (TBARS) to measure lipid peroxide inhibitory activity, copper (lower density fat protein (low) The method to measure the antioxidant effect on the oxidation of density lipoprotein) was used. As a result, it was confirmed that the compound of the present invention and the extract of bionymph tree showed DPPH free radical scavenging activity, lipid peroxidation inhibitory activity, and strong antioxidant effect on low-density lipoprotein.

In order to use the extract of the present invention prepared by the above process as a therapeutic medicament, it can be prepared by a known method in the pharmaceutical field, itself or a pharmaceutically acceptable carrier, excipient (forming) agents), diluents and the like can be prepared and used in the form of powders, granules, tablets, capsules, or injections. In addition, they may be administered orally or parenterally. The dosage of the active ingredient according to the present invention is appropriately selected according to the absorption of the active ingredient in the body, the rate of water activation and excretion, the age, sex and condition of the patient, the severity of the disease to be treated, but in general, one day for adults About 30-300 mg is administered, but it is preferable to administer about 50-200 mg. Therefore, the unit dosage form of the present invention is prepared in an amount of 20 to 100 mg of the active substance of the present invention in consideration of the above-mentioned effective amount range. Preferably formulated to contain about 10 to 50 mg. The unit dosage form thus formulated may be administered several times at regular intervals or by using specialized dosing methods according to the judgment of the expert who monitors or observes the administration of the drug as needed and the needs of the individual.

Hereinafter, examples and experimental examples of the present invention will be described in detail. The following examples and experimental examples specifically illustrate the present invention, and the content of the present invention is not limited to the examples and experimental examples.

Example 1 Preparation of Bio-Deer Tree Extract

The present invention, in order to develop a natural antioxidant using bio-heated oak tree, harvesting the native bio-wood in the area of Jeongseon-gun, Gangwon-do, finely chopped and shaded, and then put 20g into the flask attached to the reflux cooler for each part , 20 times the sample weight of water, ethanol, methanol, and chloroform solvent were added, and the extract was warmed for 24 hours in a water bath. After filtration, the extracts obtained by darkening and concentration were subjected to preliminary experiments on the yield and antioxidant activity by DPPH free radical scavenging method, and the results are shown in Tables 1 and 2 below. Antioxidant activity of the extracts of the bark tree was investigated using free radical 1.1-diphenyl-2-picrylhydrazyl (1,1-diphenyl-2-picrylhydrazyl, DPPH) [Yoshida, T., et al ., (1989) Chem. Phar m. Bull., 37, 1919-1921. 1 ml of the DPPH (0.2 mM in methanol) solution was added to a 4 ml solution of the methanol solution diluted to an appropriate concentration (0 to 0.2 mg) in a glass test tube, and reacted at room temperature for 30 minutes, and then the absorbance was measured at 514 nm. At this time, the antioxidant activity [IC _50: ㎍ / ml] was expressed as the concentration of the compound to reduce the absorbance value of the control group without addition of 50%. As a result, the antioxidant activity [IC ₅₀ : 2.9㎍] of the methanol extract of the root of the bark tree was the highest. In addition, all extracts showed a very high antioxidant activity in the order of bio-curls, leaves, and fruits, and water extracts and ethanol extracts also exhibited high antioxidant activities except for extracts of bio-curls chloroform.

Table 1. Extraction yields of wild rosewood by solvents.

menstruum Extraction yield (%, weight / weight) Fruit leaf stem Root ethanol 3.1 15.2 3.7 7.5 Methanol 4.2 23.3 7.3 15.3 chloroform 0.9 5.5 0.8 0.4 water 8.6 26.0 8.0 13.0

Table 2. Antioxidative Effect of Bio-Deer Tree Extracts by DPPH Free Radical Scavenging Assay.

extract Antioxidant activity [IC ₅₀ : μg / ml] ^* ethanol Methanol Chlorform Ml Fruit 21.5 13.9 〉 200.0 9.6 leaf 11.5 6.4 〉 200.0 4.8 stem 5.6 3.9 21.2 5.6 Root 3.7 2.9 76.9 5.2 Antioxidant BHT 5.4 Vitamin C 1.7 Alpha-tocopherol 4.2

^* Antioxidant activity [IC _50: ㎍ / ml] was expressed as the concentration of the extract to reduce the absorbance value of the control group without the extract 50%.

<Example 2>: Solvent fractionation of fresh oak tree extract by organic solvent

In order to obtain a new substance having antioxidant activity from the extract as a bio-tree, the same procedure as in Example 1, to obtain a methanol extract of the root of the bio-tree having the most excellent antioxidant activity. In other words, finely chopped and dried the dry bark roots (1kg) by adding 20 liters of methanol (collectively) for 3 days, extracting the residue with 20 liters (L) of methanol, and then extracting all the extracts under reduced pressure to extract methanol (MeOH ext.) Was obtained. The methanol extract was suspended with water, followed by solvent fractionation in the order of hexane, chloroform, ethyl acetate and butanol to obtain each fraction, and the antioxidant activity of each fraction was investigated by DPPH free radical scavenging method described in Example 1 above. The results are shown in Table 3 below. As a result, the ethyl acetate fraction had the highest antioxidant activity [IC ₅₀ : 1.7 μg], followed by the butanol fraction [IC ₅₀ : 2.0 μg]. Ethyl acetate fractions showed similar or superior antioxidant activity to the well-known antioxidants BHT, vitamin C, or alpha-tocopherol.

Table 3. Antioxidant Effects of Fractions of Fresh Oak Trees by DPPH Free Radical Scavenging Assay.

Fraction Antioxidant activity [IC ₅₀ : μg / ml] ^* BHT 5.4 Vitamin C 1.7 Alpha-tocopherol 4.2 Methanol extract 2.9 Hexane fraction > 400.0 Chloroform fraction 8.0 Ethyl acetate fraction 1.7 Butanol fraction 2.0 Water fraction 11.3

^* Antioxidant activity [IC _50: ㎍ / ml] was expressed as the concentration of the extract to reduce the absorbance value of the control group without the extract 50%.

Example 3 Isolation of the Antioxidant Active Ingredient Catechin Compound

In order to separate the antioxidant active ingredient from the bio-wood, 10.38 g of the ethyl acetate fraction (40.52 g) obtained in Example 2 was subjected to silica gel column chromatography using a chloroform: methanol mixed solvent to obtain four fractions. In addition, 210 mg of the compound of Formula 1 was obtained by performing Sephadex LH-20 column chromatography using methanol as a solvent for one of the active fractions (0.32 g). The structure of this active material is described in Davis, AL, et al ., (1996) Magnetic Resonance in Chemistry, 34, 887-890; Kwon, Yong-Soo et al. (1997) Korean Journal of Medicinal Crop Science, 5 (4), 302-306; Yonghwa Choi, et al. (1998) Journal of Pharmacognosy, 29 (2), 79-85; Moon et al., (199 9) Korean Journal of Agricultural Chemistry, 42, 170-175], were identified as catechin, a flavanic compound, and the results of nuclear magnetic resonance spectra, physical and chemical properties of the material are as follows.

(1) Compound of Formula 1: 3 ', 4', 5,7-tetrahydroxyflavan-3-ol

(3 ', 4', 5,7-tetrahydroxyflavan-3-ol)

(2) Appearance of material: white powder

(3) Molecular weight: 290

(4) Molecular Formula: C ₁₅ H ₁₄ O ₆

(5) UV absorption spectrum: UVλmax ^MeOH nm: 212, 280

The ultraviolet absorption spectrum is as shown in FIG.

(6) Hydrogen nuclear magnetic resonance spectrum: ¹ H NMR (200 MHz, acetone- d ₆ ) δ: 6.92 (1H, J = 1 .6 Hz, H-2 '), 6.83 (1H, d, J = 8.0 Hz , H-5 '), 6.72 (1H, dd, J = 8.4 Hz, 1.9 Hz, H-6'), 6.05 (1H, d, J = 2.4 Hz, H-6), 5.90 (1H, d, J = 2.4 Hz, H-8), 4.58 (1H, d, J = 7.4 Hz, H-2), 4.02 (1H, m, H-3), 2.93 (1H, dd, J = 16.2 Hz, 5.1 Hz, H-4 _a ), 2.55 (1H, dd, J = 16.2 Hz, 8.4 Hz, H-4 _b ).

Hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectra were investigated using duacetone (acetone- d ₆ ) as a solvent and tetramethylsilane (TMS) as a standard. The result is as shown in FIG.

(7) Carbon nuclear magnetic resonance spectra: ¹³ C-NMR (acetone- d ₆ ) δ 156.08 (C-9), 155.58 (C-5), 155.28 (C-7), 144.06 (C-4 '), 143.98 (C-3 '), 130.52 (C-1'), 118.44 (C-6 '), 114.07 (C-5'), 113.59 (C-2 '), 98.98 (C-10), 94.43 (C- 6), 93.76 (C-8), 81.02 (C-2), 66.65 (C-3), 28.96 (C-4).

Carbon nuclear magnetic resonance ( ¹³ C-NMR) spectra were investigated using duacetone (acetone- d ₆ ) as a solvent and tetramethylsilane (TMS) as a standard. The result is as shown in FIG.

(8) Solubility: Soluble in methanol, acetone, etc.

(9) Rf value by thin layer chromatography (TLC): When silica gel (Kieselgel 60F ₂₅₄ , Merk) is used as the adsorbent, and the chloroform: methanol (5: 1) mixed solvent is used as a developing solvent, the Rf value is 0.31. .

(10) Chemical formula: same as electricity

Example 4 Determination of Active Ingredient Catechin

This material could be quantified from extracts of roots (roots, stems, leaves, fruits) using high pressure liquid chromatography (HPLC). Grind the hot ears with a grinder for each part, and add 20 ml of methanol to the sample, add 400 ml of methanol, extract them at room temperature for 3 days, filter, concentrate, and dry, and then extract the extract. Then, take about 10 mg of the extract, and then move the mobile phase (H ₂ O-CH ₃ CN). -TFA = 80: 20: 0.1) 10 ml of solvent was added, dissolved, and filtered to obtain a sample solution of HPLC. The column was μ Bondapak ^TM C ₁₈ (3.9 × 300mm), mobile phase was H ₂ O-CH ₃ CN-TFA = 80: 20: 0.1, the detector was UV 280 nm, the flow rate was carried out at 1.0ml / min. At this time, the standard catechin and the compound of Formula 1 showed a retention time of 4.223 minutes. The HPLC chromatogram of Chemical Formula 1 is shown in FIG. 4.

<Example 5>: Content analysis of active ingredient catechin using HPLC

Using the method specified in Example 4, the content of the active ingredient in the extracts and fractions of the bark tree (root, stem, leaf, and fruit) was measured. The results are shown in Table 4, and the methanol and ethanol extracts of the stems and roots of the hawthorn tree contained large amounts of the active ingredient catechin. Among the extracts of the stems and roots of the hawthorn tree, the active ingredient catechin contained 18.1 ± 0.3 and 25.0 ± 1.5%, respectively. In particular, it was found that the ethyl acetate acetate fraction contained very large amounts of catechin (33.4 ± 0.2%). Therefore, it is assumed that the main antioxidant active ingredient of the bio-tree is the formula (1).

Table 4. Content of Active Ingredient Catechin in Bio-Deer Tree Extract

extract % Content, mean ± standard deviation Methanol ethanol water Fruit 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 leaf 0.0 ± 0.0 1.4 ± 0.1 0.0 ± 0.0 stem 18.1 ± 0.3 8.9 ± 0.2 3.8 ± 0.3 Root 25.0 ± 1.5 13.3 ± 1.1 0.0 ± 0.0 Ethyl Acetate Fraction 33.4 ± 0.2 Butanol fraction 10.5 ± 0.1

Experimental Example 1 Antioxidant Effect of DPPH Free Radical Scavenging Activity of Chemical Formula 1

Antioxidant activity represented by the compound represented by Formula 1 of the present invention was measured by using free radicals, 1.1-diphenyl-2-picrylhydrazyl (1,1-diphenyl-2-picrylhydrazyl, DPPH). Investigate. 1 ml of the DPPH (0.2 mM in metha nol) solution was added to a 4 ml solution of methanol solution diluted to an appropriate concentration (0 to 0.1 mg) in a glass test tube, and reacted at room temperature for 30 minutes, and then absorbance was measured at 514 nm. . At this time, the antioxidant activity [IC _50: ㎍ / ml] was expressed as the concentration of the compound to reduce the absorbance value of the control group without addition of 50%. As a result, as shown in Table 5, the compound of the present invention showed higher activity than the comparative reagents BHT (butylated hydroxytoluene), alpha-tocopherol (α-tocopherol), catechin (catechin), in particular, 3.2 times than BHT It was confirmed that the antioxidant activity was 2.5 times higher than that of alpha tocopherol.

Table 5. Antioxidative Effect of Compounds of Formula 1 by DPPH Free Radical Scavenging Activity

compound Antioxidant activity [IC ₅₀ : μg / ml] ^* Compound of Formula 1 1.7 BHT 5.4 Vitamin C 1.7 Alpha-tocopherol 4.2 Catechin 2.5

^* Antioxidant activity [IC ₅₀ : μg / ml] was expressed as the concentration of the extract to reduce the absorbance value of the control group without the extract 50%.

Experimental Example 2 Lipid Peroxidation Inhibitory Effect of Root Extract and Fractions of Bio-Deer Tree

Inhibition of lipid peroxidation activity by TBA (thiobarbituric acid) was determined by the method of measurement in Enzymology, 52, 302-310. ; Ohkawa, H., et al., (1979) Anal. Biochem., 95, 351-358. Extract or fraction (0 to 300 µg) was added to the reaction system containing 1 mM Fe SO ₄ and 1 mM ascorbate, and 0.2 ml of liver equalizer (1 mg / ml) and buffer solution were added to the reaction solution to 1 ml. After reacting at 37 ° C for 1 hour, 3 ml of TCA-TBA-HCl (15% trichloroacetic acid; 0.375% thiobarbit uric acid; 0.25N HCl) solution was added to the resulting lipid peroxide, followed by reaction at 95 ° C for 15 minutes, followed by centrifugation. The supernatant was separated and the absorbance was measured at 532 nm. At this time, the lipid peroxidation inhibitory effect [IC _50: ㎍ / ml] was expressed as the concentration of the extract to reduce the value of the control group without the extract 50%. As a result, as shown in Table 6, the methanol extract and the ethyl acetate extract of the bark of Root bark showed a strong lipid peroxidation inhibitory effect. In particular, in the case of ethyl acetate fraction [IC _50: 25㎍], it was confirmed that the lipid peroxidation inhibitory effect 8.8 times higher than the well-known natural antioxidant vitamin C, 6.5 times higher than alpha-tocopherol.

Table 6. Lipid Peroxidation Inhibitory Effect of Root Extracts and Fractions of Bio-Deer Tree

Fraction Lipid Peroxidation Inhibitory Effect [IC ₅₀ : ㎍ / ml] ^* BHT 0.33 Vitamin C 221 Alpha-tocopherol 162 Methanol extract 58 Hexane fraction 129 Chloroform fraction 154 Ethyl acetate fraction 25 Butanol fraction 121 Water fraction > 300

^* Lipid peroxidation inhibitory effect [IC ₅₀ : ㎍ / ml] was expressed as the concentration of the extract to reduce the value of the control group without the extract 50%.

Example 3 Antioxidative Effect of Bio-Deer Oak Extract on Low Density Lipoprotein Oxidation

In order to investigate the inhibitory effect of arterial sclerosis of methanol extracts from the roots of the bark tree, which showed excellent antioxidant effect, the antioxidant effects of low-density lipoprotein (LDL) oxidation were measured in the bark tree extract and fractions obtained above [Yan, L.-J., et al., (1995) Biochem. Biophys. Res. Comm., 212, 360-366]. To the low-density lipoprotein (LDL, 65 µg), CuSO ₄ (20 µM), and extracts or fractions (0 to 100 µg), a total volume of 1 ml was added to the solution (phosphate buffer, pH 7.4). After reacting for 8 hours, 20 µl of EDTA (1 mM) was added to the reaction. Oxidation fat mass (the amount of malondialdehyde) in the LDL was measured by TBARS (thiobarbituric acid reactive substances) method. The results of measuring the antioxidant effect on the low-density lipoproteins of the barley oak fractions are shown in FIG. As a result, the ethyl acetate fraction and butanol fraction showed the highest antioxidant effect. At 30 μg / ml fraction, the butanol fraction was 93.2% (2.6 nmol / mg), the ethyl acetate fraction was 85.3% (5.6 nmol / mg), and the chloroform fraction was 84.0% (6.1 nmol / mg). Value). Small fraction concentrations showed strong antioxidant effects on LDL oxidation. This result shows that the extract of bionymph tree has potent DPPH free radical scavenging activity, lipid peroxidation inhibitory effect and antioxidant effect on low density lipoprotein.

The extract of the bark of the present invention contains a large amount of flavine-based catechins, and simultaneously exhibits DPPH free radical scavenging activity, lipid peroxidation inhibitory effect, and antioxidant effect (lower arteriosclerosis effect) on low density lipoprotein oxidation. Since it shows higher activity than conventional antioxidants, it can be usefully widely used as medicines and quasi-drugs, food additives, cosmetic additives, beverage additives, and functional health supplements.

Claims (7)

Finely chopped the dry oak tree, dry it, add 20 times alcohol, such as methanol, cool it, precipitate and extract it, and obtain the bio-ear extract, and add 20 times methanol to the residue, and extract the fresh heat extract. Obtaining step, the extract of bio-earwood extract or fractions, characterized in that the concentrated extract of bio-heat extract with methanol, concentrated under reduced pressure, suspended with water, and then sequentially fractionated solvent with an organic solvent. The method according to claim 1, wherein 20-fold 50-75% ethanol is added to the fresh heat wood, followed by chilling at 20-80 ° C. for 3 days to 5 days, or 20-fold ethanol is added to the residue. Raspberry extract or fraction The method according to claim 1, wherein the organic solvent is selected from hexane, chloroform, ethyl acetate or butanol, and extracting the solvent extract fractions, characterized in that the solvent is suspended by adding water to 1% with respect to the concentration of bio-extract extract extract Performing silica gel column chromatography on the above ethyl acetate fraction using a chloroform: methanol mixed solvent to obtain an active fraction, Sephadex LH-20 column chromatography, high performance liquid using methanol as a solvent in the active fraction of the former. Method for preparing a flavan compound using the antioxidant extract of bio-barberry extract, characterized in that to obtain a flavan compound of the formula (1) comprising the step of performing one or more of chromatography or gas chromatography (Formula 1) 5. The method of claim 4, wherein the mixed solvent of chloroform and methanol is mixed at 5: 1 to use a mixed solvent five times the column bed volume, and methanol is used five times the column bed volume. Method for preparing flavan compound using bio-curd extract of antioxidant bark Method of using as an active ingredient, antioxidants, lipid peroxidation inhibitors or low-density lipoprotein oxidation inhibitors as an active ingredient 7. The method according to claim 6, wherein the biodeer extract or fraction is mixed with a pharmaceutically or food acceptable carrier or excipient and used as a pharmaceutical, cosmetic, food, beverage or functional dietary supplement.