WO2006090953A1

WO2006090953A1 - Extraction method for antioxidants from pine trees using natural solvents and antioxidant extracts thereby

Info

Publication number: WO2006090953A1
Application number: PCT/KR2005/001802
Authority: WO
Inventors: Wan-Taek Han
Original assignee: Nutrapharm Co. Ltd.
Priority date: 2005-02-28
Filing date: 2005-06-14
Publication date: 2006-08-31
Also published as: KR20060095642A; KR100659945B1

Abstract

Disclosed herein is a method of extracting a natural antioxidant from the bark of pine trees using a natural solvent as an extraction medium, and an antioxidant extract prepared thereby. The method of the current invention includes extracting an antioxidant from the bark of pine trees using an alcohol solution as a natural solvent, to obtain an antioxidant extract; and separating a nonpolar material from the antioxidant extract using a natural alcohol solution and/or natural animal or vegetable oil, and is environmentally friendly and economical, thus providing a natural antioxidant having high antioxidant activity from the bark of pine trees.

Description

The antioxidant used in removing the active oxygen is largely classified into synthetic antioxidants and natural antioxidants .

The synthetic antioxidant includes, for example, BHA (butylated hydroxyanisole) , BHT (butylated hydroxytoluene) , etc. However, as these antioxidants have been proven to be toxic in recent studies, there is the need for the development of natural antioxidants having higher safety and antioxidant activity. In this regard, it is known that the bark of pine trees contains bioflavonoids as natural antioxidants able to strongly remove active oxygen, and hence, many patents related to methods of extracting an antioxidant from pine trees have been filed. In U.S. Patent No. 4,698,360, to extract and separate an antioxidant from the bark of pine trees, synthetic solvents (ethylacetate and chloroform) are repeatedly used, and inorganic salts (salt, ammonium sulfate, and sodium sulfate) are used, and thus, the preparation processes are complicated, and the extraction yield is very low (about 0.21% relative to the amount of bark extract), negating economic benefits. Further, the antioxidant extract is so unsafe that it is unsuitable for use in foods, attributable to the synthetic organic solvent remaining therein. In Korean Patent No. 10-021297'4, to extract and separate an antioxidant from the bark of pine trees, synthetic organic solvents having high volatility, such as methanol, diethylether, ethylacetate, heptane, etc., are used, decreasing the workability. Further, since the synthetic organic solvent may remain in the antioxidant extract, such an antioxidant extract is unsuitable for use in foods.

Korean Patent No. 10-0429406 discloses a method of preparing an antioxidant from pine needles, using methanol and ethylacetate as synthetic organic solvents. In addition, Korean Examined Patent Publication No. 1993-0005694 discloses a method of preparing an antioxidant from green tea leaves, comprising adding calcium carbonate to water to extract an extract, adsorbing an antioxidant using an adsorbing resin, and then separating the antioxidant using ethylacetate, methanol, acetone, etc., as synthetic organic solvents.

Further, Korean Patent No. 10-0341797 discloses a method of separating and purifying a natural antioxidant from safflower seeds, comprising separating an antioxidant using methanol, nucleic acid, ethylacetate, chloroform, etc., as synthetic organic solvents. Korean Patent No. 10-0109461 discloses a method of separating a natural antioxidant from sesame using methanol, ethylacetate, chloroform, etc., as synthetic organic solvents. In addition, Korean Patent No. 10- 0395464 discloses a method of extracting catechins from green leaves of green tea using dichloromethane, ethylacetate, acetone, etc., as synthetic organic solvents. In addition, Korean Patent No. 10-0439012 discloses a method of extracting an antioxidant from Korean raisins using methanol, ethylacetate, etc., as synthetic organic solvents.

However, the above patents are disadvantageous because a large amount of synthetic organic solvent is used to prepare. the antioxidant, and thus, safety and legal problems may arise if the extract is used as a food material. In addition, the use of the volatile organic solvent results in the requirement for more stringent working conditions. Further, a large amount of environmental pollutants may be generated, polluting air and water. In this way, various problems related to the conventional preparation processes and the quality of the antioxidant occur.

Moreover, according to conventional extraction methods, when the antioxidant is extracted and separated from the bark of pine trees, a plurality of complicated processes of extraction, concentration and separation is required, preparing the antioxidant at a very low extraction yield, decreasing the workability, and increasing production costs. In addition, since various volatile synthetic organic solvents, toxic to the human body, are used in large amounts upon the preparation of the antioxidant, work safety is decreased, and air and water pollution may be caused. Also, the synthetic organic solvent may remain in the antioxidant extract, and hence, serious problems, making it impossible to use the antioxidant extract for foods, occur.

In addition, according to the conventional methods, by using various salts, including table salt, ammonium sulfate, sodium sulfate, calcium carbonate, etc., for the extraction and separation, an additional process of completely separating the salt from the antioxidant should be further conducted, resulting in drastically decreased workability and productivity. Also, environmental problems, such as water pollution due to the use of the salt, may be generated.

Therefore, the present invention, carried out by the present inventors aiming to avoid the above problems encountered in the prior art, provides an extraction method, which is advantageous because preparation processes are simplified and the environmental treatment cost is decreased due to the generation of a minimum amount of environment pollutants, by using a fermented alcohol solution as a natural solvent having low volatility and high antioxidant extraction capability to extract antioxidants from the bark of pine trees at high efficiency, and then by using an alcohol solution and/or natural animal or vegetable oil to efficiently remove nonpolar material such as pine resin from the extract and separate and concentrate the antioxidant. According to the present invention, the antioxidant extract obtained from the bark of pine trees through extraction has high antioxidant activity and extraction yield. Thus, the extraction method of the present invention is suitable for use in highly price- competitive mass production of antioxidant from the bark of pine trees.

[Disclosure]

[Technical Problem] Accordingly, an object of the present invention is to provide a method of extracting a natural antioxidant from the bark of pine trees, comprising extracting an antioxidant from the bark using a fermented alcohol solution as a natural solvent, and then efficiently removing nonpolar material such as pine resin from the bark extract using an alcohol solution or natural animal or vegetable oil.

Another object of the present invention is to provide a method of efficiently extracting a natural antioxidant, in which conditions including the concentration of ethanol in the alcohol solution, the amount of alcohol solution used, the extraction temperature, etc., are predetermined to increase the antioxidant activity and extraction efficiency of the antioxidant extract, and also, conditions including the amount of solvent when using the alcohol solution or animal or vegetable oil, the extraction temperature, and filtration and concentration techniques are predetermined to efficiently remove the nonpolar material from the extract.

A further object of the present invention is to provide a natural antioxidant extract obtained by the above extracting method, and in particular, an extract usable as a material and an additive for food, cosmetics, medicine and feed.

According to the present invention, since only a chemically stable natural solvent is used, safety and workability are increased and environmentally friendly processes are realized. In addition, through simplified preparation processes, the extraction yield and the antioxidant activity of the antioxidant are maximized, generating economic benefits .

[Technical Solution]

The present invention pertains to a method of extracting an antioxidant from the bark of pine trees, comprising pre- treating the bark of pine trees, pulverizing the bark of pine trees, extracting an extract using an alcohol solution, concentrating or drying the extract, extracting a nonpolar material using an alcohol solution and/or animal or vegetable oil, and drying an antioxidant. In the present invention, the term 'pine tree' is defined as a plant belonging to the pine family, and includes, for example, Pinus densiflora Sieb & Zucc, Pinus thumbergii Parlatore, Pinus densiflora Sieb. & Zucc. for. erecta ϋyeki, Pinus sylvestris L., Pinus pinea Cupressus Sempervirens, Casuarina equisetifolia J.R. & G. Forst. Casuarina litorea L. Casuarinaceae or Abies pindrow.

In addition, the term 'bark of pine trees' means the external layer that may be taken from the trunk and branches but not the leaves or roots of a plant belonging to the pine family. The pre-treated bark may be pulverized to 5-100 meshes to increase solvent extraction efficiency.

In addition, the term 'alcohol solution' or 'fermented alcohol solution' used throughout the process of the present invention is defined as a fluid consisting mainly of ethanol, obtained by fermenting a material containing starch or a material containing sugar.

The alcohol solution used in the extraction of the bark of pine trees includes 1.0-95% ethanol, and preferably, 30-95% ethanol. Also, the alcohol solution used in the extraction of the nonpolar material includes 10-95% ethanol, and preferably, 40-95% ethanol. The natural animal or vegetable oil used in the extraction of the nonpolar material includes, for example, soybean oil, corn oil, canola oil, rice bran oil, sesame oil, perilla oil, safflower oil, sunflower oil, cottonseed oil, peanut oil, olive oil, palm oil, coconut oil, mixed edible oil, refined oil, shortening, red pepper seed oil, beef tallow, or lard. Below, each process of the method of the present invention is specifically described. However, these processes do not limit the technical scope of the present invention.

1. Pre-Treatment Of Bark: From the bark of pine trees, external impurities and the outermost black weathered bark portion are removed, and only a red bark portion is separated for use in the subsequent pulverizing process.

2. Pulverizing Of Bark: The separated bark is pulverized into particle or powder using a homogenizer and pulverizer, and then employed in the subsequent extraction process. Preferably, the bark is pulverized to 5-100 meshes.

3. Extraction Using Alcohol Solution: The pulverized bark powder is added with a fermented alcohol solution serving as a natural solvent, to first extract an antioxidant. As such, the alcohol solution includes 1.0-95% ethanol, and preferably, 30- 95% ethanol. The bark and the alcohol solution are mixed at a weight ratio from 1:3 to 1:50, and preferably, at a weight ratio from 1:4 to 1:20, to extract the antioxidant. The extraction process is conducted at a temperature ranging from -20 to 90^°C, and preferably, from 10 to 70°C, for a time from 5 min to 3 days, and preferably, from 30 min to 24 hr, by allowing the mixture to stand, intermittently mixing it, or continuously mixing it, to extract the antioxidant. 4. Concentration Or Drying Of Extract: After the completion of the first extraction, the extract liquid and the bark are separated using a known filtration or centrifugation technique, to remove the antioxidant extract liquid from the bark. Usable for the filtration, a filtration aid includes, for example, diatomaceous earth, china clay, bentonite, acid earth, talc, pearlite, activated carbon, etc. The alcohol solution is added to the bark filter cake in an amount 1-20 times, and preferably, 3-15 times, greater than the amount of the filter cake, so that the antioxidant remaining in the bark filter cake is further extracted once or twice. The extracts thus filtered are combined, after which ethanol and water are removed using a known vacuum concentration technique. Subsequently, the extract liquid is concentrated so that the concentrated extract liquid has 1.0-30% solid content, and preferably, 3.0-15% solid content. The concentrated extract liquid may be used unchanged, or be dried using a known drying technique, for example, freeze drying, vacuum drying, hot air drying or spray drying, and then pulverized into powder, which is then used for the subsequent separation of a nonpolar material .

5. Separation Of Nonpolar Material Using Alcohol Solution And/Or Natural Animal or Vegetable Oil: Any one of the following two processes is selectively applied to separate the nonpolar material, such as pine resin, from the concentrated extract liquid or dried extract powder, thus concentrating an antioxidant . (1) Use Of Alcohol Solution As Separation Medium: The alcohol solution for use in the separation of a nonpolar material includes 10-95% ethanol, and preferably, 40-95% ethanol. The concentrated extract liquid or extract powder and the alcohol solution are mixed at a weight ratio of 1:1-1:30, and preferably, 1:3-1:15, followed by separating the nonpolar material and concentrating the antioxidant. The extraction process is carried out at a temperature ranging from -20 to 90^°C, and preferably, from 0 to 70°C, for a time ranging from 5 min to 24 hr, and preferably from 30 min to 10 hr, by allowing the mixture to stand, intermittently mixing it, or continuously mixing it, to separate the nonpolar material. The antioxidant and the alcohol solution containing the nonpolar material may be separated using a known filtration or centrifugation technique.

(2) Use Of Natural Animal or Vegetable Oil As Separation Medium: The natural animal or vegetable oil is selected from the group consisting of soybean oil, corn oil, canola oil, rice bran oil, sesame oil, perilla oil, safflower seed oil, sunflower oil, cottonseed oil, peanut oil, olive oil, palm oil, coconut oil, mixed edible oil, refined oil, shortening, red pepper seed oil, beef tallow, lard, and mixtures thereof. The concentrated extract liquid or extract powder and the natural oil are mixed at a weight ratio of 1:0.5-1:20, and preferably, 1:1-1:10, thus separating the nonpolar material using the oil layer and concentrating the antioxidant. To separate the nonpoar material, the extraction process is carried out at a temperature ranging from 0 to 120^°C, and preferably, from 20 to

10 80^°C, for a time ranging from 30 min to 24 hr, and preferably, from 1.0 to 10 hr, by allowing the mixture to stand, intermittently mixing it, or continuously mixing it. The concentrated antioxidant extract and the natural oil containing the nonpolar material may be separated from each other using a known filtration or centrifugation technique.

Selectively, with the goal of separating the nonpolar material using the extract powder, 5.0-95% alcohol solution is added to the extract powder at a weight ratio 1.0-20 times larger than the extract powder, and the nonpolar material may be extracted using the natural oil. As such, the extraction process for the separation of the nonpolar material is carried out at a temperature ranging from -20 to 90^°C, and preferably, from 10 to 80^°C, for a time ranging from 30 min to 24 hr, and preferably, from 1.0 to 10 hr, by allowing the mixture to stand, intermittently mixing it, or continuously mixing it.

6. Drying Of Antioxidant: The concentrated antioxidant extract without the nonpolar material is prepared into concentrated liquid or solid powder using known concentration, drying and pulverizing techniques.

[Advantageous Effects]

As mentioned above, the present invention provides an environmentally friendly preparation method having high workability, which is characterized by extracting the antioxidant from the bark of pine trees using a fermented alcohol solution as a natural solvent having stable physicochemical properties, and then separating the nonpolar material, such as pine resin, from the extract using a

11 fermented alcohol solution and/or natural animal or vegetable oil, to produce an antioxidant having high antioxidant activity at a high extraction yield.

In addition, the preparation method of the present invention can enable the antioxidant to be produced at an extraction yield 2.6-21 times higher than conventional methods. Further, the antioxidant activity of the extract obtained using the present invention is higher than that of antioxidants prepared by conventional methods . This is believed to be because the conventional method includes a plurality of complicated processes, during which a large amount of antioxidant may be lost, and also, because the extraction separating process of a nonpolar material having low efficiency is applied. When the total amount of antioxidant is calculated using a total amount concept multiplying the extraction yield by the antioxidant activity of the extract, the antioxidant extract obtained by the present invention has antioxidant activity 2.6-45 times higher than that obtained by conventional methods .

[Mode for Invention] <Example 1>

1) Extraction and Filtration: From 30-year-old Pinus densiflora & Zucc. naturally grown in Samchok, Kangwon Province, Korea, the outermost layer of bark was removed. The bark thus obtained was pulverized into bark powder (30 meshes) using a homogenizer and pulverizer. 1.0 kg of the bark powder was mixed with 8.0 kg of a natural alcohol solution containing 60% ethanol, extracted in a shaken water tank at a constant 50^°C

12 for 3 hr, and then filtered. The resultant bark filter cake was added with 4.0 kg of the alcohol solution containing 60% ethanol and 40% water, extracted in a shaken water tank at a constant 50^°C for 1 hr, and then filtered. 2) Concentration and Drying: The extracted filtrates obtained in step 1) were combined, from which ethanol and water were removed using a vacuum concentrator, yielding 1,230 g of a concentrated antioxidant extract liquid, which was then spray dried, to obtain 95 g of antioxidant powder. 3) Separation of Nonpolar material and Drying of Antioxidant: 30 g of the antioxidant powder obtained in step 2) were mixed with 100 g of alcohol, after which nonpolar material was separated in a shaken water tank at a constant 30^°C for 3 hr, and then filtered. The filter cake containing the antioxidant was dried at 50^°C for 3 hr, and then pulverized, finally yielding 14.2 g of red-brownish antioxidant powder.

In the separation of nonpolar material and the drying of antioxidant of Example 1, 70 g of alcohol and 100 g of soybean oil were used instead of 100 g of alcohol, after which the nonpolar material was extracted in a shaken water tank at a constant 30°C for 5 hr to remove it. The soybean oil layer containing the nonpolar material and the alcohol layer were separated from each other using a centrifuge. The solid antioxidant without the nonpolar material was dried at 50^°C for 3 hr, and then pulverized, finally obtaining 13.2 g of red- brownish antioxidant powder.

13 <Example 3>

An antioxidant was prepared in the same manner as in

Example 1, with the exception of using alcohol containing 95% ethanol in the extraction and filtration of Example 1. Finally, 12.3 g of red-brownish antioxidant powder were obtained.

An antioxidant was prepared in the same manner as in Example 1, with the exception of using an alcohol solution containing 30% ethanol in the extraction and filtration of

Example 1. Finally, 13.5 g of red-brownish antioxidant powder were obtained.

In the separation of nonpolar material and the drying of antioxidant of Example 3, 70 g of alcohol and 100 g of soybean oil were used instead of 100 g of alcohol, after which the nonpolar material was extracted in a shaken water tank at a constant 30^°C for 5 hr to remove it. Subsequently, the soybean oil layer containing the nonpolar material and the alcohol solution layer were separated from each other using a centrifuge. The solid antioxidant without the nonpolar material was dried at 50^°C for 3 hr, and then pulverized, finally obtaining 12.1 g of red-brownish antioxidant powder.

In the separation of nonpolar material and the drying of antioxidant of Example 4, 80 g of alcohol and 120 g of soybean

14 oil were used instead of 100 g of alcohol, after which the nonpolar material was extracted in a shaken water tank at a constant 30^°C for 5 hr to remove it. Subsequently, the soybean oil layer containing the nonpolar material and the alcohol solution layer were separated using a centrifuge. The solid antioxidant without the nonpolar material was dried at 50^°C for 3 hr, and then pulverized, finally obtaining 11.3 g of red- brownish antioxidant powder.

In the separation of nonpolar material and the drying of antioxidant of Example 1, the nonpolar material was extracted in a shaken water tank at a constant 5^°C for 5 hr to remove it, and then filtered. The filter cake containing the concentrated' antioxidant was dried at 50^°C for 3 hr, and then pulverized, finally obtaining 15.7 g of red-brownish antioxidant powder.

In the separation of nonpolar material and the drying of antioxidant of Example 3, the nonpolar material was extracted in a shaken water tank at a constant 5^°C for 5 hr to remove it, and then filtered. The filter cake containing the concentrated antioxidant was dried at 50^°C for 3 hr, and then pulverized, finally obtaining 13.5 g of red-brownish antioxidant powder.

Comparative Example 1>

According to the method disclosed in U.S. Patent No. 4,698,360, the extract from the bark of pine trees was prepared as follows. As the bark of pine trees used in the extraction,

15 100 g of bark powder (30 meshes) separated from Pinus densiflora Sieb & Zucc. naturally grown in Samchok, Kangwon Province, Korea, were used. The bark powder was mixed with 500 ml of distilled water, and then heated at 100^°C for 2 hr, followed by extraction and filtration of an antioxidant. The filtrate was cooled to 20"C and saturated with a salt, and thereafter, the precipitate thus formed was filtered off. The extract filtrate was mixed with 100 ml of ethylacetate, so that the antioxidant was separated from the water layer. The separation process using ethylacetate was repeated twice, and all .ethylacetate layers were combined, added with 5.0 g of sodium sulfate, and then dehydrated. The dehydrated ethylacetate layer was concentrated in a vacuum, to remove 80% ethylacetate. The concentrated ethylacetate layer was added with 200 ml of chloroform to form a precipitate, which was then separated using a filter. The precipitate was washed with 50 ml of chloroform, and then dried at 40^°C, finally obtaining 0.21 g of antioxidant powder.

According to the method disclosed in Korean Patent No. 10- 0212974, the extract from the pine tree bark was prepared as follows. As the pine tree bark used in the extraction, 100 g of bark powder (30 meshes) separated from Pinus densiflora Sieb & Zucc. naturally grown in Samchok, Kangwon Province, Korea, were used. The bark powder was mixed with 1 L of a mixture comprising methanol and water (methanol:water = 3:1 v/v) , and extracted and then filtered at room temperature for 3 days. To increase the extraction yield, the methanol solution was

16 further added twice, and thus, the extraction and filtration were performed as mentioned above. The filtered methanol extracts were combined, and concentrated to 330 ml using a vacuum concentrator. The concentrated extract liquid was added with 330 ml of methanol, and then with 660 ml of heptane, after which the nonpolar material was removed in a mixed shaken water tank at 30^°C for 2 hr. This procedure was further performed twice using heptane, removing the nonpolar material. To further remove the nonpolar material, the methanol extract was added with 300 ml of diethylether, and the nonpolar material was removed in a mixed shaken water tank at 30^°C for 2 hr. This procedure was further performed twice using heptane, additionally removing the nonpolar material. The methanol layer without the nonpolar material was concentrated in a vacuum to remove methanol therefrom, after which the remaining water layer was dried using a spray drier, finally obtaining 2.48 g of antioxidant powder.

Experimental Example> 1) Sample Used In Measuring Antioxidant Activity: Antioxidant samples derived from the pine tree bark obtained in Examples 1 to 8 and Comparative Examples 1 and 2 were dissolved in methanol, to prepare measurement samples at various concentrations (e.g., 50, 100, 300, 500, and 700 μg/ml) . 2) Measurement Of Antioxidant Activity: The antioxidant activity of the sample was assayed by measuring DPPH (1,1- diphenyl-2-picrylhydrazine) radical scavenging activity and superoxide radical scavenging activity. As such, as standard

17 antioxidants for activity tests, BHA and Trolox as a water soluble vitamin-E analogue were used.

A) Measurement of DPPH Radical Scavenging Activity

10 μH of a sample solution for each concentration were loaded into each well of a 96-well plate, added with 90 μJl of

1.5 x 10^"4 M DPPH dissolved in ethanol, and then allowed to react for 10 min. Subsequently, absorbance was measured at 517 run using a microplate reader. The DPPH radical scavenging activity for each concentration was calculated based on the following equation. In addition, the absorbance of the sample added with ethanol instead of the DPPH solution was taken as a blank, and methanol was used instead of the sample to be a control.

DPPH Radical Scavenging Activity (%) = (Absorbance Change Of Sample Measured After 10 Min) x 100 / Absorbance Of Control

B) Measurement Of Superoxide Radical Scavenging Activity Superoxide radical scavenging activity was measured according to a riboflavin/EDTA/nitroblue tetrazolium (NBT) method. 10 fd of each sample solution and 90 pJL of a solution containing 0.03 πiM riboflavin, 1 mM EDTA, 0.6 mM methionine and 0.03 mM NBT dissolved in 50 mM potassium phosphate buffer (pH 7.8) were loaded into each well of a 96-well plate. Then, the activity of scavenging superoxide generated by a photochemical reaction was measured. To cause the superoxide-generating photochemical reaction, two fluorescent lamps provided in a light radiation box lined with aluminum foil were used, and the distance between the lamp and the reaction container was set so that the brightness was 1000 Lux. After the reaction was

18 conducted at 25^°C for 8 min, and the absorbance at 560 nm was measured using a microplate reader. The superoxide radical generated by the photochemical reaction reacted with NBT to form blue formazan, thus increasing the absorbance. When the superoxide radical was scavenged by the sample, formazan was less formed and thus the absorbance was decreased. In addition, as a control, methanol was added instead of the sample. Hence, the superoxide scavenging activity of the sample was calculated according to the following equation. Radical Scavenging Activity (%) = (1 - Absorbance Change Of Sample) x 100 / Absorbance Change Of Control

From the absorbance results of each sample obtained through the tests for the DPPH radical scavenging activity and the superoxide radical scavenging activity, the concentration of the sample required to inhibit 50% of the formation of radicals was represented by ED50 (βg/ml) . The results for antioxidant activities of samples according to the measuring methods are shown in Tables 1 and 2, below. In addition, the antioxidant activity of each sample was converted into total antioxidant activity using a total amount concept (extraction yield x relative antioxidant activity) . The results are shown in Table 3 below.

The lower the ED50 of the sample, the higher the antioxidant activity. As is apparent from Table 1, the extraction yield of the antioxidant of the present invention was 4.6-6.4%, which was 21 times higher than in U.S. Patent No. 4,698,360 (0.21%) and about 2.6 times higher than in Korean Patent No. 10-0212974 (2.5%). In addition, as is apparent from

19 Table 2, the antioxidant prepared by the present invention exhibited antioxidant activity on DPPH radicals, that was superior to materials prepared by conventional methods, and also, had antioxidant activity about 1.1-1.7 higher than Trolox. Further, although the antioxidant prepared by the present invention had superoxide antioxidant activity similar to the sample of U.S. Patent No. 4,698,360, it had activity about 1.7 times higher than the sample of Korean Patent No. 10- 0212974. In particular, the antioxidant of the present invention manifested superoxide scavenging activity about 1.7- 2.2 times higher than BHA, and about 27-35 times higher than Trolox. Thus, as is apparent from Table 3, the antioxidant prepared according to the present invention has both extraction yield and antioxidant activity much higher than those prepared using conventional methods . In the case where the activity of the antioxidant was calculated using a total amount concept multiplying the extraction yield by the antioxidant activity, total antioxidant activity thereof was found to be 3.5-45 times higher using the DPPH antioxidant activity measuring method, and was found to be 2.6-30 times higher using the superoxide antioxidant activity measuring method, compared to conventional control methods. Therefore, the antioxidant prepared according to the present invention can have both high antioxidant activity and an economical preparation process .

[Table 1] Extraction Yield of Antioxidant and DPPH Scavenging Activity

20

[Table 2] Superoxide Scavenging Antioxidant Activity of Antioxidant

[Table 3]

Antioxidant Activity of Antioxidant According To Preparation Method Using Total Amount Concept of Antioxidant Activity

21

* Total antioxidant activity = extraction yield x relative antioxidant activity (Tables 1 and 2) ; and

** The value of total antioxidant activity in Comparative Example 1 was determined to be 1.0 for the calculation of relative activity.

[industrial Applicability]

As described above, the present invention provides a method of extracting an antioxidant from the bark of pine trees using a natural solvent and an antioxidant prepared thereby. According to the present invention, a natural fermented alcohol solution and a natural animal or vegetable oil suitable for use in food may be used, thus solving various problems caused by conventionally using a variety of synthetic organic solvents, for example, danger of fire due to the use of the highly volatile solvent, worker safety, disposal cost of waste, re- treatment cost of the solvent, limited applicability of the

22 extract, containing residual organic solvents, to food, etc. Further, the preparation method of the present invention is simple and safe, and generates less environmental pollutants. In addition, the prepared antioxidant extract may be directly used in food, and a natural antioxidant having excellent activity and price competitive power and preparation processes thereof may be provided. In addition, the extract of the present invention may be used as an additive or material for cosmetics, medicine or feed. Moreover, the bark of pine trees obtainable using byproducts of wood industries and fields and mountains in Korea are utilized, to prepare a natural antioxidant having high activity, which may be then inexpensively supplied. Accordingly, the method of the present invention is advantageous because forest by-products in Korea may be recycled, and the value of pine trees and the income of forest farms in Korea may be increased. As well, various health foods, processed foods and medicines may be developed using the antioxidant of the present invention, developing food industries and improving the health of people and the quality of life. Additionally, through the export of the antioxidant of the present invention, international imports and exports may be further balanced.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing

23 from the scope and spirit of the invention as disclosed in the accompanying claims.

24

Claims

[CLAIMS] [Claim l]

A method of extracting a natural antioxidant from the bark of pine trees, comprising: extracting an antioxidant from the bark of pine trees using an alcohol solution as a natural solvent, to obtain an antioxidant extract; and separating a nonpolar material from the antioxidant extract using a natural alcohol solution and/or natural animal or vegetable oil.

[Claim 2]

The method according to claim 1, wherein the alcohol solution is a fermented alcohol solution containing 1.0-95% ethanol.

[Claim 3]

The method according to claim 1, wherein the natural animal or vegetable oil is selected from the group consisting of soybean oil, corn oil, canola oil, rice bran oil, sesame oil, perilla oil, safflower oil, sunflower oil, cottonseed oil, peanut oil, olive oil, palm oil, coconut oil, mixed edible oil, refined oil, shortening, red pepper seed oil, beef tallow, lard, and mixtures thereof.

[Claim 4]

The method according to claim 1, wherein the bark of pine trees includes a bark of a plant selected from the group

25 consisting of Pinus densiflora Sieb & Zucc, Pinus thumbergii Parlatore, Pinus densiflora Sieb. & Zucc. for. erecta Uyeki, Pinus sylvestris L., Pinus pinea Cupressus Sempervirens, Casuarina equisetifolia J. R. & G. Forst. Casuarina litorea L. Casuarinaceae, Abies pindrow, and mixtures thereof.

[Claim 5]

The method according to claim 3, wherein the separating of the nonpolar material is performed by using the natural animal or vegetable oil at a weight ratio 0.5-20 times larger than a concentrated extract liquid or dried extract.

[Claim 6]

The method according to claim 1, wherein the separating of the nonpolar material from the extract is performed by simultaneously using both the natural alcohol solution and the natural animal or vegetable oil, thus separating an antioxidant .

[Claim 7]

A natural antioxidant extract extracted from the bark of pine trees, according to the method of claim 1.

[Claim 8] An additive for food, cosmetics, medicine or feed, comprising the natural antioxidant extract extracted from the bark of pine trees of claim 7 as an effective component.

26 [Claim 9]

A material for food, cosmetics, medicine or feed, comprising the natural antioxidant extract extracted from the bark of pine trees of claim 7 as an effective component.

27