KR20040041475A - Green onion compositions for antioxidative function containing antioxidative active substances and preparation method thereof - Google Patents

Abstract

PURPOSE: An enzyme treated welsh onion extract composition containing a natural antioxidant active material obtained by enzymatic treatment of Welsh onion(Allium fistulosum) and then hot air drying or freeze drying thereof is provided. It can be effectively used as an antioxidant functional material having disease preventing and aging inhibiting function. CONSTITUTION: A Welsh onion extract is subjected to an enzymatic treatment with Celluclast(Novo Co.) and Viscozyme(Novo Co.) and then hot air-dried or freeze-dried. The Welsh onion extract composition contains an antioxidant material such as 4-hydroxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid, 3,4-dihydroxycinnamic acid having antioxidant activity.

Description

Green onion compositions for antioxidative function containing antioxidative active substances and preparation method

The present invention relates to a method for preparing a large juice extract having an antioxidant function by preparing a large extract juice preparation by enzymatic treatment, and identifying a natural antioxidant having antioxidant activity contained in the juice preparation.

All living things, including humans, have a fundamental self-defense mechanism against the injury of free radicals generated by the use of oxygen in the air to sustain life. However, if an abnormality is caused in a biological defense device or the production of free radicals is increased by various factors such as various physical and chemical factors, oxidative damage is caused in vivo. It is known to be involved in the phenomenon and cause a disorder of the living body directly or indirectly. Active oxygen, known as noxious oxygen, is very reactive, and the free radical reaction caused by them destroys cell components such as lipids, proteins, sugars, and DNA by non-selective and irreversible reactions.

Recent studies have shown that these free radicals are involved in various diseases such as cancer, stroke, Parkinson's disease, and cardiovascular diseases such as arteriosclerosis, digestive diseases, and skin diseases. In addition, oxides produced by lipid peroxidation by these free radicals interact with the constituents of cells in the body, resulting in biochemical changes in the cells, thereby causing aging and various functional disorders, and harmful effects such as rancidity and toxic substances in foods. It is known to do. Therefore, development of antioxidants capable of removing such active oxygen is required, and antioxidants have a function of preventing disease and aging by oxidation.

Leek is a seasoning vegetable produced all over the country. According to Dongbogam, leek is said to have pharmacological effects such as eliminating chills and fever, clearing eyes, and eliminating fraud in the liver. In order to give a phosphorus function, it is added to foods raw or vegetable nature. Previous studies on the functional substances contained in leek are described in Chung et al., Journal of the Korean Society for Nutrition and Nutrition, 22, 3, 1993, The Korean Nutrition Society, Korea, Jung Kun-yong, and the Structures of Two Diosgenin Glycosides Isolated from Leek White, 313- 316 and J Agric Food Chem. 2001 Jun; 49 (6): 3101-5. Alpha-tocopherol content in 62 edible tropical plants] has been reported to contain 74.6 mg / Kg of alpha tocopherol in the green leaves, but the 4-hydroxybenzoic acid and 4-hydroxy-3-meth as found in the present invention. It has not been reported that antioxidant substances such as oxybenzoic acid, 4-hydroxycinnamic acid, 4-hydroxy-3-methoxycinnamic acid and the like are contained in the extract preparations of the leek enzyme treatment.

Korean leek is not only used as a spice vegetable in Korean food, but also used as a medicine in oriental medicine and civilian, and it is estimated to contain functional substances in leek. However, it is used as an antioxidant functional material with antioxidant and anti-aging effects. It is necessary to identify the antioxidant substances contained in. In addition, the leek has an average solids content of about 12%, but contains a large amount of viscous material, so the general juice method has a very low juice yield.

Therefore, the technical object of the present invention is to examine the juice preparation method by the enzyme treatment to produce a high yield of leek juice, to check the antioxidant activity of the juice, the cause of the antioxidant activity in the leek juice preparation The purpose of the present invention is to provide an anti-oxidation functional material having a soybean enzyme-treated juice preparation containing natural antioxidants, which has a disease prevention effect and an anti-aging function by oxidation.

In the present invention, the juice obtained by enzymatic treatment of green onion was dried to prepare a green onion enzyme-treated juice preparation in powder form. Yield was increased when the juice preparation was enzymatically treated, and the extract fraction obtained by solvent fractionation of the juice preparation with ethyl acetate was confirmed to have antioxidant activity. The extract fraction showing antioxidant activity was purified by silica gel column chromatography, and then the contents contained by GC-MS analysis were 4-hydroxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, 4-hydroxycinnamic acid. , 4-hydroxy-3-methoxycinnamic acid was identified, the content of these substances was measured, and the antioxidant activity of these identified substances was assayed to confirm that they had antioxidant activity. Natural antioxidants, such as 4-hydroxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, 4-hydroxycinnamic acid, and 4-hydroxy-3-methoxycinnamic acid, are prepared in the leek enzyme-treated juice preparation according to the present invention. It was found that these substances have antioxidant activity and that the anti-seizure juice preparations containing these antioxidant activities can be used as an antioxidant functional material that has anti-oxidation disease and anti-aging effect. Invented.

Hereinafter, the present invention will be described in detail in the following examples. However, the present invention is not limited to these examples.

Example 1 Preparation and Yield Determination of Anti-Juman Juice Preparation by Enzyme Treatment

Allium fistulosum was harvested in December from Jisan-myeon, Jindo-gun, Jeollanam-do and used as a sample. 500 g of purified water is added to 1 kg of leek and pulverized at 1,500 rpm for 3 minutes using a wet mill. Cellulast (celluclast, Novo Co.) and biscozyme (viscozyme, Novo Co.) 0.1-0.25% range, while maintaining the 500 rpm at the reaction temperature 50 ~ 60 ℃, the enzyme treatment time is variously treated under conditions such as 10-60 minutes, and then the treatment liquid to 150 mesh network After quenching, the obtained juice solution was lyophilized to investigate the juice yield by enzyme treatment. In addition, the leek was directly pulverized by the above grinding method as a control, filtered and lyophilized to compare the juice yield. As shown in Table 1, the higher the addition amount of cellulose and biscozyme, the longer the reaction time, the higher the juice yield. In other words, the enzyme preparation was not about 10g per kilogram of leek juice, but the leek enzyme-treated juice preparation 0.25% and 1 hour each for cellulast and biscozyme were 100g or more per kilogram of leek Appeared. In addition, biskozyme showed higher yields than the enzyme treated in order to increase the yield of large sesame juice preparation.

Therefore, the yield of juice during the enzyme treatment of leek is mixed with 0.25% of cellulose and biskozyme respectively, and when the juice is stirred at 500 RPM for 60 minutes at a reaction temperature of 50-60 ° C., the most leek enzyme-treated juice preparations It can be seen that, to improve the shelf life of the juice preparation can be prepared by lyophilized drying or hot air drying powdery leek enzyme-treated juice preparation.

Example 2: Determination of Antioxidant Activity of Fractions Obtained by Solvent Fraction of Soybean Enzyme Treated Juice Preparation

After treating the leek in the optimum yield conditions of Example 1, the juice preparation obtained by the enzyme treatment was subjected to solvent fractions to measure the antioxidant activity. First, the juice preparation obtained by the enzyme treatment was extracted with excess methanol and concentrated under reduced pressure to obtain a soybean enzyme-treated methanol extract. Subsequently, the methanol extract of the prepared soybean enzyme-treated juice preparation was dissolved in distilled water, adjusted to pH 3.0 with 1 M hydrochloric acid, and then partitioned into ethyl acetate (EtOAc) to give an aqueous layer and an ethyl acetate soluble acid. Neutral fraction (EtOAc-soluble acidic-neutral fraction) was obtained. The ethyl acetate soluble acidic and neutral fractions were partitioned into buffer solution (5% NaHCO ₃ , pH 8.0) and fractionated into ethyl acetate soluble neutral fraction (EtOAc-soluble neutral fraction) and water phase fraction. In addition, 1 M hydrochloric acid was added to the obtained aqueous fraction, adjusted to pH 3.0, and then partitioned with ethyl acetate to obtain an ethyl acetate soluble acidic fraction (EtOAc-soluble acidic fraction).

Antioxidant activity of the obtained fractions was measured by DPPH (1,1-diphenyl-2-picrylhydrazyl, Sigma, USA) radical scavenging activity assay. That is, the method of the door or the like [J. Agric. Food Chem., 46, 2, 1998, USA, Moon J H et al. , Antioxidant activity of caffeic acid and dihydrocaffeic acid in lard and human low-density lipoprotein, 5062-5065]. After the reaction, absorbance was measured at 517 nm, and 50% of DPPH free radical scavenging ability (SC ₅₀ ) was obtained from the measured dose-response. Alpha tocopherol (Sigma, USA) was used as a control.

As a result of measuring the antioxidant activity of these fractions by DPPH radical scavenging activity method on the two fractions obtained by the above method (FIG. 1), 50% of DPPH free radical scavenging ability (SC ₅₀ ) was etched from the soybean enzyme-treated juice preparation. Acetate soluble acid fraction was measured at 120 μg / mL, and ethylacetate soluble neutral fraction of leek enzyme treated juice preparation was 80 μg / mL, indicating a 10% DPPH free radical scavenging ability (SC ₁₀ ). Both acidic and neutral fractions of soybean enzyme-treated juice preparations showed antioxidant activity, but ethyl acetate-soluble acidic fractions of enzyme-treated extracts showed stronger activity. Therefore, it was confirmed that the leek enzyme-treated juice preparations have antioxidant activity.

Example 3 Purification of Antioxidant Substances Contained in a Leek Enzyme Treated Juice Preparation

By the method shown in Examples 1 and 2, the ethyl acetate soluble acid fraction obtained from the soybean enzyme treatment juice preparation was purified by silica gel adsorption column chromatography, respectively. Silica gel adsorption column chromatography uses silica gel (10 g, 70-230 mesh, for column chromatography, Merck, Darmstadt, Germany) in a solvent system of hexane-ethylacetate-methanol (8: 6: 1, v / v). After filling the column with a hexane-ethylacetate-methanol solvent system, the mobile phase is 8: 6: 1, 6: 8.1, 4: 10: 1, 2: 12: 1, 0: 14: 1, 0: 0: Each fraction obtained by eluting and fractionating by stepwise method of increasing polarity stepwise to 15 (v / v, 100 mL step by step) was obtained from the method of Takao et al. Biotech. Biochem., 58, 1994, Japanese Society for Agricultural Chemistry, Japan, Takao, T. et al. A simple screening method for antioxidants and isolation of several antioxidants produced by marine bacteria from fish and shellfish, 1780-1783, spotted TLC and sprayed with an ethanol solution of DPPH radicals (200 μM) onto the TLC plate. When decolorized, it was determined to have positive antioxidant activity.

Elution and fractionation of the ethyl acetate soluble acid fraction of the leek-enzyme-treated juice preparation by silica gel adsorption column chromatography was performed to assay the antioxidant activity. As a result, hexane-ethyl acetate-methanol (8: 6: 1, v / v) In the elution fraction of, the activity appeared, to obtain a purified product of the antioxidant active substance of the ethyl acetate soluble acid fraction.

Example 4 Identification of Antioxidants in Purified Active Fraction by GC-MS Analysis

As shown in Example 3, the material contained in the active fraction obtained by silica gel adsorption column chromatography was subjected to trimethylsilyl (TMS) derivatization, and then identified by gas chromatograph-mass spectrometry (GC-MS) analysis. Tried. GC-MS analysis is an instrument with a GC (Varian STAR 3400CX, Walnut Creek, CA, USA) connected to an MS (Varian SATURN 3, Walnut Creek, CA, USA) and an Rtx-1 capillary column (0.32 mm x 30 m, Varian instruments 2700, Walnut Creek, CA, USA), and the temperature was maintained at 100 ° C. for 2 minutes, and then increased to 240 ° C. at 5 ° C. per minute and maintained at 240 ° C. for 5 minutes. In addition, MS analysis was performed under an ion source temperature of 200 ° C. and an ionized bolt of 70 eV. Trimethylsilyl derivatization of the active fractions is described by Park et al. [Biosci. Biotech. Biochem., 58, Japanese Agricultural Chemistry Society, Japan, Park, KH et al. , Brassinosteroids and monoglycerides with brassinosteroid-like activity in immature seeds of Oryza sativa and Perilla frutescena and in cultured cells of Nicotiana tabacum, a reagent 224-2243 (1994), a portion of the active fractions were (10 μg) by [Anhydrous pyridine- N , O -bis (trimethylsilyl) -acetamidetrimethylchlorochlorosilane, 10: 5: 1, v / v] was added to 20 μL, mixed, and reacted at 60 ° C. for 30 minutes, followed by GC-MS analysis. As standard reagents for GC-MS analysis, 4-hydroxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, 4-hydroxycinnamic acid, 4-hydroxy-3-methoxycinnamic acid (Sigma, MO, USA) Was used.

Some active fractions purified in Example 3 were trimethylsilyl derivatized and subjected to GC-MS analysis. As a result, about 10 peaks were shown on the gas chromatogram.

The MS spectra of these peaks were searched for a Wiley6 library. From the EI-MS spectrum of the material having a peak at the retention time ( t _R ) 11.83 minutes, m / z 282 was observed as the molecular ion, and m / z 267 (base peak), 223, 193 as the fragment ion. Observed. This spectrum was searched by Willie 6 library and found to be identical to the MS spectrum of 4-hydroxybenzoic acid (C ₁₃ H ₂₂ O ₃ Si ₂ ) in which a trimethylsilyl group was introduced to a hydroxyl group and a carbonyl group. Indicated the possibility. From the EI-MS spectrum of the material having a retention time of 14.33 minutes, m / z 312 was observed as the molecular ion and m / z 282, 267, 253, and 223 as the debris ion, which was determined by Willie 6 Library Search. Possibility of 4-hydroxy-3-methoxybenzoic acid in accordance with the MS spectrum of 4-hydroxy-3-methoxybenzoic acid (C ₁₄ H ₂₄ O ₄ Si ₂ ) in which trimethylsilyl groups are introduced with hydroxyl and carboxyl groups Indicated. From the EI-MS spectrum of the material having a retention time of 17.78 minutes, m / z 308 was observed as the molecular ion and m / z 293, 249 and 219 as the debris ion, which was searched by the Willie 6 library. The possibility of 4-hydroxycinnamic acid was indicated in accordance with the MS spectrum of 4-hydroxycinnamic acid (C ₁₅ H ₂₄ O ₃ Si ₂ ) in which trimethylsilyl groups were introduced into the actual and carboxyl groups. In addition, m / z 338 was observed as the molecular ion and base peak from the MS spectrum of the retention time 20.68 min peak, and m / z 323, 279 and 249 were observed as the fragment ion. This spectrum was searched by Willie 6 library and was found to be consistent with the MS spectrum of 4-hydroxy-3-methoxycinnamic acid (C ₁₆ H ₂₇ O ₄ Si ₂ ) having a hydroxyl group and a trimethylsilyl group introduced therein. The possibility of -hydroxy-3-methoxycinnamic acid was shown. The retention times and MS data of these materials are shown in Table 2.

As a result of GC-MS analysis of trimethylsilyl derivatized by the same method as a standard of the four substances suggested by the library search, the retention time and MS spectrum of each substance was consistent with the substance contained in the juice preparation. Therefore, the four compounds contained in the ethyl acetate soluble acid fraction of the leek-enzyme-treated juice preparations are 4-hydroxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, 4-hydroxycinnamic acid, and 4-hydroxy- It was identified as 3-methoxycinnamic.

Example 5 Analysis of the Contents of the Compounds Identified in Glycoenzyme-treated Juice Preparations by GC-MS Analysis

Silica gel adsorption column chromatography was carried out on the ethyl acetate soluble acid fraction obtained by the method of 2, 3, 4, respectively, for the preparation of the enzyme preparation treatment of leek enzyme for the analysis of the content of the substance identified by the GC-MS analysis. After taking a portion of the hexane-ethylacetate-methanol (8: 6: 1, v / v) fraction from which the active substance was eluted, trimethylsilyl derivatization was performed to analyze GC-MS. After repeated analysis three times, the area of each active substance of GC obtained was determined, and the calibration curve based on the peak area of each standard was prepared and quantified under the same conditions. In addition, each standard was corrected by obtaining a recovery rate of each standard calculated through the purification process in the same manner as the sample.

The recovery yielded by the same method was 90.3% for 4-hydroxybenzoic acid, 91.2% for 4-hydroxy-3-methoxybenzoic acid, 91.3% for 4-hydroxycinnamic acid, and 4-hydroxy-3-media. The hydroxycinnamic acid was 94.8%, indicating a relatively high recovery of 90% or more. As a result of correcting the content of the contained substance by the recovery rate, the content of 4-hydroxy-3-methoxycinnamic acid was highest in the preparation of soybean enzyme treated juice (Table 2).

Example 6: Antioxidant Activity of Substances Identified in Leek Enzyme Treated Juice Preparations

Antioxidant activity was determined by the DPPH radical scavenging activity assay shown in Example 2 for four compounds contained in the leek enzyme-treated juice preparation. The activity was also compared with that of alphatocopherol, which is well known as an antioxidant. The concentration of the compound showing 50% DPPH radical scavenging activity (SC ₅₀ ) calculated from the reaction curve (FIG. 3) was 8 μg / mL for alphatocopherol and 14 μg / mL for 4-hydroxy-3-methoxycinnamic acid. Was measured. SC ₅₀ values such as 4-hydroxybenzoic acid, 4-hydroxycinnamic acid and 4-hydroxy-3-methoxybenzoic acid were determined to be at least 500 μg / mL. 4-hydroxy-3-methoxycinnamic acid showed the strongest activity and the antioxidant activity similar to that of alpha-tocopherol.

Antioxidant activity assay by DPPH radical scavenging activity confirmed that all of these substances had antioxidant activity. Therefore, the four substances contained in the soybean enzyme-treated juice preparations differ in the magnitude of activity, but all exhibit antioxidant activity, and the antioxidant activity of the soybean enzyme-treated juice preparations includes these natural antioxidant active substances. It turned out that it is expressed by.

In the present invention, it was confirmed that the enzyme treatment increases the yield of the juice preparation in the preparation of the large juice preparation, and also the 4-hydroxybenzoic acid, 4-hydroxy-3, which is a natural antioxidant in the preparation of the juice preparation. -The discovery of the presence of methoxybenzoic acid, 4-hydroxycinnamic acid, 4-hydroxy-3-methoxycinnamic acid, and the like, and the preparation of leek enzyme-treated juice containing these natural antioxidant actives prevents disease by oxidation. The use as an antioxidant functional material which has the effect and anti-aging effect is anticipated.

Claims (2)

4-hydroxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid, 3,4-dihydroxycinnamic acid having antioxidant activity Anti-Juice Juice Formulations Having Antioxidant Function, Including Substances The method of claim 1, wherein the large extract juice preparation is prepared by lyophilizing or hot-air drying the quench enzyme-treated juice obtained by the cellulose extract and biscozyme enzyme treatment, juice