KR20000025274A - Antioxidant extracted from gulpweed and extraction thereof and refining method - Google Patents

Abstract

PURPOSE: Antioxidant extracted from gulfweed and extraction and refining thereof is provided which replaces the artificial antioxidant conventionally used by extracting and refining natural antioxidant having superior activity of anti oxidation and heat stability. CONSTITUTION: Antioxidant extracted from gulfweed and extraction and refining thereof is comprised of: extracting anti oxidation materials by using organic solvent from gulfweed; dividing active ingredients from extract by separating solvent; and refining active ingredients by chromatography.

Description

Antioxidant Extracted from Cup Leaf Hat and Its Extraction and Purification Methods

The present invention relates to an antioxidant extracted from a marine algae ( Sargassum hemiphyllum ), an algae inhabiting the waters of Korea, and more particularly, to extracting and extracting an antioxidant using an organic solvent from a mating leaf cap. The present invention relates to an antioxidant, in which an active material layer is divided through a solvent fraction, and an active fraction is purified by various chromatographic methods, and an extraction and purification method thereof.

Antioxidants are substances that can inhibit the oxidation reaction, and are used to suppress food waste and aging of human tissues. For this reason, the most widely used antioxidants are butylhydroxyanisole (BHA) or butylhydroxytoluene (BHT), which are the most widely used synthetic antioxidants in the food industry. However, such a substance is known as a carcinogen, and stability problems are emerging in advanced countries and the like.

For this reason, methods for extracting and preparing antioxidants from natural products have been developed for a long time to overcome the problems of synthetic antioxidants. In other words, β-carotene is extracted from carrots and the like. However, this method has a drawback of low commercialization due to the small amount of extraction. Recently, Shin-Ya et al. Extracted an antioxidant called naphterpin from the microbial strain Streptomyces sp. CL 190 and identified its structure, but it was not commercialized. K. Shin-Ya, S. Imai, K. Furihata, Y. Hayakawa, K. Kato, GD Vanduyne, J. Clardy and H. Seto, J. Antibiotics 43, 444 (1990)), Teshima et al. Mo and others also reported extracting antioxidants from microorganisms, but they also failed to be commercialized (Y. Teshima and K. Shin-Ya, J. Antibiotics 44, 685 (1991); CJ Mo, K. Shin-Ya). , K. Furihata, A Shimazu, Y Hayakawa and H Seto, J. Antibiotics 43, 1337 (1990)). However, researches to develop various antioxidants from agricultural products and seafood are still active.

On the other hand, efforts to extract antioxidants from physiologically active substances from seaweed began in the late 1980s, and many studies have been conducted especially in France and Japan. Takaki and Miyashita extracted natural products from twelve species of seaweeds inhabiting Japan and investigated the tocopherol content of these substances. As a result, the tocopherol present in the extracted material was found to be the main component of α-form and a small amount of β-form (K. Miyashita and T. Tagaki, Agric. Biol. Chem . 51, 315 (1987)). And Kaneiniwa et al. Also extracted antioxidants from seaweeds in the waters of Japan, which reported that 5-olefinic acid, which has antioxidant properties, was found in the lipid components of seaweeds (M. Kaneniwa). , Y. Itabashi and T Tagaki, Nippon Suisan Gakkashi 53, 861 (1987)), these compounds are not common as antioxidants. Nishibori and Nakami extracted antioxidants from hexane / ethanol mixtures of seven kinds of seaweeds, and measured their antioxidant activity. And antioxidant activity comparable to α-tocopherol (S. Nishibori and K. Namiki, 36, 17 (1985)). However, this too has not been commercialized due to the very low extraction amount.

In Korea, Park Jae-han et al. Measured the antioxidant activity of 12 kinds of seaweeds using methanol and chloroform sequentially. As a result, laver, brown seaweed, were awarded the meuljil exhibits excellent antioxidant than BHA, etc. Kelp did not become too commercialized to the problem of thermal stability (bakjaehan, ganggyuchan, baeksangbong, Lee Yoon-hyung, yigyusun, Korea Food Science and Technology 23256 (1991)).

In order to solve these problems, the present inventors conducted extensive research, and were able to extract and purify a natural antioxidant component having very high antioxidant activity and excellent thermal stability from a pair of algae, a marine algae inhabiting the waters of Korea. Completed on the basis of

Accordingly, an object of the present invention is to provide a natural antioxidant having excellent thermal stability and excellent radical scavenging activity extracted and purified from a pair of algae hatched algae in the sea near Korea.

It is another object of the present invention to provide a method for extracting and purifying a natural antioxidant having excellent radical scavenging activity and high thermal stability from a mat leaf hat.

Antioxidant of the present invention for achieving the above object is an extract obtained from a pair of hatberry.

Another antioxidant of the present invention is an organic layer fraction obtained by dispensing an extract obtained from a companion leaf cap in an organic solvent.

Another antioxidant of the present invention is a purified product obtained by purifying an organic layer fraction obtained by dispensing an extract obtained from a companion leaf cap with an organic solvent.

The method of the present invention comprises the steps of: (1) extracting the antioxidant active substance by adding an organic solvent to the crushed mat leaf hat; (2) obtaining an active ingredient from the extract obtained in step (1) using a solvent fractionation method; And (3) purifying the active ingredients obtained in step (2) by chromatography.

Hereinafter, the present invention will be described in more detail.

According to the method of the present invention, 5 kg of a pair of algae hatched algae, which are inhabited by the sea near Seongsan, Jeju Island, were collected and washed with distilled water, and then dried and crushed. Organic solvents, preferably methanol, ethanol, ethyl acetate, Obtain a reddish brown extract using acetonitrile, acetone or a water / ethanol mixture. The extract thus obtained can be used as an antioxidant.

On the other hand, the extract is selectively dissolved again in acetyl acetate and filtered to remove the remaining salt and the remaining liquid is evaporated again, concentrated. Then, it is dissolved in ethyl acetate and methanol to remove insoluble substances, and the solvent dissolved in 90% methanol and hexane is again dissolved in the substance dissolved in ethyl acetate. Through this process, the material dissolved in methanol having higher activity is fractionated using 30% methanol and isopropyl ether. In this process, the active ingredient was collected in a 30% methanol layer, and the next step was solvent fractionation using 40% methanol and chloroform. The 40% methanol fraction thus obtained can be used as an organic fraction as an antioxidant.

In the process the active ingredient is collected in the methanol layer. This is again dissolved in pure distilled water, and then passed through the membrane to divide the active ingredient, and only the highly active materials are collected and purified using chromatographic methods such as MPLC (medium pressure liquid chromatography) or HPLC (high performance liquid chromatography). The purified product thus obtained can be used as an antioxidant.

The antioxidant activity of the extracted antioxidants was determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) with free radicals attached to the molecules according to the Blois method. The radical scavenging activity of)) was measured.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to the following Examples.

Example 1

Finely pulverized 410 g of dry leaf hatcups are placed in a 1 L back bottom flask and slowly stirred with 800 ml of methanol at room temperature for 12 hours to extract antioxidants. This process is repeated several times to extract the antioxidant component with a total of 6.5 liters of methanol. Then remove the residue by centrifugation at low temperature, then remove the solvent using a rotary evaporator to obtain 64.1 g of the first extract.

Example 2

The extract extracted in Example 1 was dissolved in 4 liters of ethyl acetate, filtered to remove the residue, and then concentrated, and the insoluble residue was removed. The ethyl acetate solution thus obtained was subjected to solvent fractionation using 1 L of 90% methanol and 3.5 L of n-hexane. In this process, the active ingredient was collected in a methanol layer, and the methanol layer was subjected to solvent fractionation using 1 L of 30% methanol and 1 L of isopropyl ether again. In this step, the active ingredient is collected in a 30% methanol layer, and the 30% methanol layer is further subjected to solvent fractionation using 1 L of 40% methanol and 1 L of chloroform, followed by drying under reduced pressure of a 40% methanol solution showing antioxidant activity. Get

Example 3

The organic fraction of the 40% methanol layer obtained in the last step of Example 2 was again placed on a column filled with a 200 μm diameter ODS (octadecylsilyl) filler in a 25 × 500 mm glass tube and eluted with 30% methanol solution to obtain 850 mg of the active ingredient. This active ingredient was purified again using high performance liquid chromatography (acetonitrile: H ₂ O = 20: 80, flow rate = 2.0 ml / min, 10 × 250 mm C-18 column). .

Experimental Example 1

Antioxidant activity of the purified antioxidant substance in Example 3 was measured by radical scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) having free radicals attached to the molecule according to the Bloose method. The Blose method dissolves 20 mg of DPPH in 150 mL of ethanol to prepare a DPPH solution. To this solution, 250 μl of dimethyl sulfoxide (DMSO) is added, diluted with an appropriate amount of ethanol, shaken for 10 seconds, and the absorbance of the control at 517 nm. Set it to 0.94 to 0.97. In the same way, each sample (μg in mg) was added to 1 ml of DPPH solution with absorbance between 0.94 and 0.97, and then reacted for 10 minutes. The absorbance was measured as a DPPH radical scavenging activity. And measured. The results are shown in Table 1 below in comparison with materials used as conventional antioxidants.

Comparison of Antioxidant Activity of Extracts from Evening Leaf Caps and Other Antioxidants Antioxidant Sample BHT BHA Mat leaf hat α-tocopherol Ascorbic acid 100 µg 70.7% 100% 100% 100% 100% 20 µg 36.5% 84.6% 100% 93.9% 100% 10 µg 59.7% 100% 71.0% 100% 5 µg 100% 85.8% 2 µg 100% 67.6%

※ Compared to antioxidant activity with 10% of DPPH radical scavenging activity ΔA after 10 minutes for each concentration of young leaf cap.

Experimental Example 2

In order to investigate the thermal stability of the natural antioxidant extracted and purified from the paired maternal bed according to Example 3, the antioxidant activity was specified by time at 60 ° C. and 80 ° C., and is shown in Table 2 below. As can be seen from Table 2, the thermal stability of the antioxidant according to the present invention was found to be very excellent.

Thermal Stability Experiments of Extracts from Evening Leaf Caps Room temperature 60 ℃ 80 ℃ ΔA 15 minutes 30 minutes 60 minutes 15 minutes 30 minutes 60 minutes 0.857 0.884 0.889 0.840 0.877 0.891 0.882 stability stability stability stability stability stability

※ Sample leaf cap size: 50㎍

Table 2 shows the radical scavenging activity in the BPH DPPH assay, the higher the value, the more excellent the antioxidant, and the higher the value, the higher the thermal stability of the antioxidant component. .

As described above, the present invention extracts and purifies natural antioxidants having excellent antioxidant activity and excellent thermal stability from mating leaf caps, which are widely inhabited in Korea's waters, thereby replacing synthetic antioxidants widely used in the food industry. It can be effective. In addition, mat leaf hats are widely inhabited in the waters of Korea and are expected to greatly increase the added value of mat leaf hats, which have not had high economic value.

Claims (7)

Antioxidant from an extract obtained from companion leaf hat. Antioxidant as an organic layer fraction obtained by partitioning the extract obtained from the companion leaf cap with an organic solvent. An antioxidant as a purified product obtained by distilling an organic layer fraction obtained by distributing an extract obtained from a companion leaf cap with an organic solvent. (1) extracting an antioxidant active material by adding an organic solvent to the crushed mat leaf hat; (2) obtaining an active ingredient from the extract obtained in step (1) using a solvent fractionation method; And (3) A method for extracting and purifying antioxidants from mat leaf hats, comprising the steps of purifying the active ingredients obtained in step (2) by chromatography. The method of extracting and purifying antioxidants from a mating leaf cap according to claim 4, wherein the organic solvent is methanol, ethanol, ethyl acetate, acetonitrile, acetone or water / ethanol mixture. 5. The solvent fraction according to claim 4, wherein the solvent fraction is subjected to a first solvent fraction in 90% methanol and hexane, and then the second fraction is fractionated using 30% methanol and isopropyl ether in a substance dissolved in methanol. A method for extracting and purifying antioxidants from mat leaf hats, characterized in that a third solvent fraction is carried out using% methanol and chloroform. 5. The method of extracting and purifying antioxidants from a mating leaf cap according to claim 4, wherein the chromatographic method is MPLC (medium pressure liquid chromatography) or HPLC (high performance liquid chromatography).