KR20110011846A - The method of preparing apple extract of flavonoids enhanced composition showing antioxidant, antiinflammatory, and anticancer activities - Google Patents

Abstract

PURPOSE: A producing method of an apple extract with enhanced flavonoids is provided to obtain healthy materials from apple skins using a medium pressure liquid chromatograph. CONSTITUTION: A producing method of an apple extract with enhanced flavonoids comprises the following steps: extracting materials from apple skins with water and alcohol; analyzing the resulting components with a high performance liquid chromatograph; and separating the extract with a strengthened specific component using the medium pressure liquid chromatograph.

Description

The method of preparing apple extract of flavonoids enhanced composition showing antioxidant, antiinflammatory, and anticancer activities}

The present invention relates to food additives in the health functional food industry as a manufacturing technology of apple flavonoid extracts whose antioxidant, anti-inflammatory and anticancer activity has been experimentally confirmed.

The present invention relates to substances obtained by extracting and enhancing apple flavonoids and their health functionalities.

Apples are healthy fruits, as the saying goes that eating an apple a day means you don't need to see a doctor. This is because apples contain much higher amounts of flavonoids than other fruits. Apple flavonoids are present in most apple peels. Apples contain carbohydrates next to moisture. Starch, fructose, and glucose are more ripe apples, which convert starch from inside to outside into monosaccharides. Apples are usually eaten raw for water and sugar, but the skin is usually shaved off. In addition, apple peels are often thrown away with pulp during the process of compressing apples when making commercial apple juice. The present invention attempts to create higher added value by taking health functional apple flavonoid material from apple peel which has been underestimated.

The present invention is to obtain a high value-added substance that separates the health functional substances from the apple peel, which is relatively low value of the existing, to determine the harmless and antioxidant, anti-inflammatory, anti-cancer activity to the human body and to utilize them in food additives, etc. .

In order to extract and separate health functional substances including flavonoids from apple peel, water and ethanol, which are safely used in the food industry, are sequentially extracted and analyzed by HPLC (High Performance Liquid Chromatograph High Performance Liquid Chromatograph) and analyzed by MPLC (Medium). Pressure Liquid Chromatograph) is used to separate the extract into enriched extracts. Animal testing tests the antioxidant, anti-inflammatory and anticancer activity of these extracts.

The present invention obtains an extract containing flavonoids from apple peel to obtain a flavonoid-enhanced substance by using MPLC and these substances have been proved to have antioxidant, anti-inflammatory, anti-cancer activity through animal experiments and thereby health functional food It has been shown to be useful as an additive.

The present invention relates to the identification of substances obtained by extracting and enhancing apple flavonoids and their health functionalities. Apples mainly use skins such as dark red, yellow, fuji, yoka, and reddish skins. Flavonoid fortified extracts useful in all apple species tested were obtained.

Hereinafter, preferred examples and experimental examples are presented for understanding the present invention. The following Examples and Experimental Examples are provided for easy understanding of the present invention and are not intended to limit the scope of the present invention.

Example 1: Obtaining apple flavonoid-containing extract according to the present invention

Apple peels, which contain moisture as they are originally obtained, are dissolved in distilled water, about three times the mass of the skin, to dissolve water-soluble substances such as fructose and glucose. Typically, water extraction is performed at 40 degrees twice for 4 hours per time. At this time, flavonoids such as isoquercitrin (quercetin-3-glucoside) are mostly insoluble in water and remain. After that, extract with distilled water and ethyl alcohol of Dongbu blood at 40 degrees for 4 hours. In order to analyze the extract using HPLC, the standard products such as Catechin, Caffeic acid, Quercetin-3-glucoside, Phloridzin, Quercetin, etc. were quantified to 2,000ppm, 500ppm, and 192ppm using HPLC agilent series 1200 (UV Vis DAD). The analytical conditions are 25 ^o C in a condition that flowing Eluent temperature to give a 100% initial water (0min) Conditions and concentrations so as to flow the 100% Ethyl alcohol after 60min Water and Alchol is the concentration of 0.1M acetic acid, respectively By adding it, the tailing phenomenon is reduced. Figure 1 is the ultraviolet (wavelength = 254nm) absorption line results for Fuji apple extract. The RT of 20.257 minutes is the isoquercitrin (quercetin-3-glucoside) and the peak of 21.956 corresponds to phloridzin.

Example 2: Obtaining Flavonoid Enhancing Composition Using MPLC

As a result of the HPLC experiment, it was found that a considerable amount of flavonoids such as quercetin-3-glucoside and phloridzin were present in the shell, and MPLC (Biotage Inc.) was used as a method to further strengthen the flavonoids. Isolation of flavonoid compounds from apple extracts was carried out using a 40 mm ODS column (volume = 60 ml, mass = 50 g). The operating conditions were separated under the following conditions with reference to the data obtained in the HPLC experiment. After washing with Methanol, the experiment was started after equilibration by 4 column volume under initial Eluent condition. The extract obtained in Example 1 was concentrated four times at 55 degrees Celsius, centrifuged (13500 RPM, 30 minutes, 25 degrees Celsius), and 8 mL of the clear extract obtained by removing the precipitate was loaded on the top of the column and elution under the following conditions. A 15 column volume of eluent is flowed from an aqueous 0% ethyl alcohol solution (time = 0 min) to an aqueous 100% ethyl alcohol solution. Unlike HPLC, acetic acid is not added. The apple extract loading amount is 1600 times that of 5 micro liters used for loading on HPLC. The flow rate is 35 mL / min. Therefore, the time to reach 100% ethyl alcohol condition is about 25 minutes. 2 is a chromatogram result of MPLC UV absorption lines (monitoring wavelength = 254 nm, collector wavelength = 320 nm). Where 1 fraction is the volume of 21 mL.

The red line is the wavelength of 320nm UV absorption and the blue line is the 254nm UV absorption. The results of HPLC analysis of the samples taken from the peak portion of the first monitoring wave are shown in FIG. 3, and the results of HPLC analysis of the samples taken near the second peak are shown in FIG. 4.

3 is a sample of the 12th fraction of FIG. 2 and corresponds to 12 * 21ml / 35 (ml / min) = 7 minutes of RT (residence time), and FIG. 4 is a sample of the 18th fraction of FIG. 2, 18 * 21ml / It corresponds to 35 (ml / min) = 11 components of RT, showing the Quercetin-3-glucoside and phloridzin peaks, showing a relatively enhanced composition of flavonoids.

Example 3: Antioxidant Test Using Apple Peel Extract

* Activity of Superoxid dismutase (SOD)

Superoxide dismutase (SOD) is one of the most important antioxidant enzymes known to convert superoxide anion into hydrogen peroxide and oxygen molecules. This SOD was confirmed by the method of generating nitrite. First, nitrite-generating mixture was mixed with 15mM xanthine 10mM hydroxylammonium chloride, 65mM phosphate buffer (pH 7.8), distilled water and xanthine oxidase. After mixing 100ul of the mixture solution to 100ul of the second tissue fraction, the reaction was carried out for 25 to 20 minutes. 0.5 ml of sulfanilic acid (sulfanilic acid, 3.3mg / ml) and 0.5ml alpha naphthylamine (a-naphthylamine, 1mg / ml) were added and incubated at room temperature for 20 minutes. Absorbance was measured at 530 nm. In Figure 5 was confirmed the ability to activate the reactive oxygen scavenging enzyme by measuring the concentration of Superoxide dismutase (SOD).

* Activity of Glutathione peroxidase

 In the presence of GSH, glutathione peroxidase is an enzyme that reduces hydrogen peroxide to water by converting GSH into an oxidized glutathione. 300ul of assay buffer (50mM potassium phosphate buffer pH7.0, in 0.4mM PMSF, 0.1% Triton X-100) was added to 100ul of secondary tissue fraction, and the supernatant was taken by centrifugation at 10,000rpm for 10 minutes. The reaction mixture solution was prepared by adding 1 mM EDTA, 1 mM sodium azide, 1 U / ml oxidase GSH reductase, 1 mM oxidase GSH and 0.2 mM NADPH to 50 mM potassium phosphate buffer pH7.0. Into the supernatant 100ul (microliter) was added 800ul of reaction mixture, and reacted for 20 to 5 minutes. And 100m of 2.2mM H2O2 was added and the absorbance was measured at 410nm.

-GSH quantification: The content of glutathione (GSH) was quantified using Elman's method. First, the mixture solution was mixed with 8 ml of assay buffer (100 mM potassium phosphate in 1 mM EDTA, pH 7.0), 228 microliter glutathione reductase (6 unit / ml), and 228 ul of TNB (5,5 dithiobis-2nitrobenzoic acid) for at least 3 hours. Used after incubation at room temperature. An equal amount of 5% 5-sulfosalicylic acid solution was added to 10% tissue homogenate, followed by centrifugation at 10,000 rpm for 10 minutes, and the supernatant was taken. After adding 150ul of mixture solution to 0.1ml of the supernatant and reacting at room temperature for 5 minutes, 50ul of NADPH was added and the absorbance was measured at 412nm. In Figure 6 Glutathione peroxidase activity was confirmed in Figure 7 reduced glutathione activity showed the ability to remove free radicals.

* Catalase activity

      Catalase is known to convert hydrogen peroxide produced by SOD into water that is harmless to the body. In order to measure this, 100 μl of 50 mM potassium phosphate buffer pH 7.0 containing 1 mM EDTA was added to 100 μl of the secondary tissue fraction and centrifuged at 10,000 rpm for 15 minutes to prepare a sample. After adding 30ul methanol and 100ul 100mM potassium phosphate (pH7.0) to 10ul of the prepared sample, 20ul of hydoxide peroxide (3.25mM) was added again and reacted at room temperature for 20 minutes. After adding 30ul of 10M potassium hydroxide (KOH), 30ul of purpald solution was added and shaken at room temperature for 10 minutes. After that, 10ul of potassium periodate was added and reacted at room temperature for 5 minutes, and then the absorbance was measured at 540nm to determine the amount of catalase. 8 shows the ability to remove reactive oxygen through the change in Catalase activity.

Example 4. Anti-inflammatory Activity Test Using Apple Peel Extract

p- p38 ( Inflammation-related protein activity change) Experiment: After seeding 1 × 10 ⁵ in 24well, harvest ⁵⁰⁰ µl of RIPA buffer. After Havest, let stand for 30 minutes on ice and centrifuge at 12,000 RPM for 30 minutes to separate proteins. Some of the proteins were quantified using the Bio-Rad reagent, and the other was diluted with 1x of 5x sample buffer and denaturated for 100 to 5 minutes. After 20ug loading on 12% SDS-PAGE, electrophoresis is performed at 100V. After electrophoresis is completed, transfer to PVDF membrane. After 1 hour of blocking 5% skim milk, 1st p-p38 and actin were diluted 1: 1000 and reacted for 2 hours and 30 minutes, and 2nd was reacted by diluting the anti-mouse IgG HRP at 1: 2000 for 2 hours. After washing with TBS-T (Tween 20 0.05%) three times for 15 minutes, West-zol solution was mixed 1: 1 and reacted for 1 minute and imaged with LAS-3000. As shown in Figure 9 can be confirmed the inflammatory effect.

Example 5. Anticancer Activity Test Using Apple Peel Extract

Confirmation of Cell Death in Cancer Cell Lines: After treatment with Hera ( cervical cancer) cell line, the cells were incubated for 2 days and observed for cell death by MTT assay. After treatment with the A549 ( lung cancer) cell line, the cells were cultured for 2 days, and then cell death was observed for cancer cells by MTT assay. Absorbance at 505 nm was measured using a microplate leader. It is the result of treatment with increasing concentration to 10,50,100,500ug / ml from the leftmost color column to control, bright bark, red bark, yoka bark and dark red bark extract. The bar graphs shown later use the same colored column varieties unless otherwise noted. Other experiments have shown that apple peels have health functional benefits, almost all of which have been more effective under the same conditions. 10 shows activity in Hera Cell. 11 shows the activity of removing cancer cells in A549. In Figure 12 it can be seen that there is almost no cytotoxicity in normal cells by confirming the activity in Rinm-5F.

1 shows the HPLC UV absorption line (wavelength = 254 nm) of apple (Fuji) bark extract.

Figure 2 is the result of MPLC UV absorption line (wavelength = 254nm) of apple water ethanol sequential extract

FIG. 3 shows a partial HPLC analysis (UV absorption line, wavelength = 254 nm) of the MPLC result of FIG. 2 showing flavonoid deficiency in the figure where the residence time is relatively small.

FIG. 4 shows the results of partial HPLC analysis (UV absorption line, wavelength = 254 nm) of the MPLC result of FIG. 2 showing flavonoid strengthening in the figure where the residence time is relatively large.

5 to 12 can confirm the antioxidant, anti-inflammatory and anti-cancer activity as an animal test result.

Claims (4)

Apple peel is extracted sequentially with distilled water and ethanol and concentrated, and then concentrated extract prepared from apple flavonoid fortification obtained through MPLC. Health functional food material substance with antioxidant effect as the substance of claim 1 Functional food material substance with anti-inflammatory effect as the substance of claim 1 As a substance of claim 1, a health functional food material that has no toxicity to normal cells and exhibits anticancer effect

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