KR100686251B1 - DISJUNCT MATTER ISOLATED THE TRIPHLORETHOL-A OF gamma;-RAY RADIOPROTECTIVE AND THE PREPARATION METHOD OF THEREOF - Google Patents

Abstract

A fraction containing triphlorethol-A for radioprotection of a subject from gamma-ray and a preparation method thereof are provided to remove reactive oxygen species produced by irradiation of gamma-ray, protect plasma membrane lipid peroxidation and cell DNA damage induced by gamma-ray, and reduce apoptosis by gamma-ray. A composition for radioprotection of a subject from gamma-ray comprises the fraction of Ecklonia cava containing triphlorethol-A, wherein the fraction of Ecklonia cava containing triphlorethol-A is prepared by pulverizing the dried Ecklonia cava, dipping Ecklonia cava powder in 80% methanol at 20-25 deg.C for 2 days and filtering it to prepare methanol extract of Ecklonia cava, fractionating the methanol extract of Ecklonia cava with ethylacetate and water, extracting the ethylacetate fraction with diethylether to prepare the diethylether fraction, and adding chloroform and methanol into the diethylether fraction and subjecting the mixture to column chromatography.

Description

Fraction containing triglycerol-A for the protection of gamma rays and its preparation method {DISJUNCT MATTER ISOLATED THE TRIPHLORETHOL-A OF γ-RAY RADIO PROTECTIVE AND THE PREPARATION METHOD OF THEREOF}

Figure 1 is a process for producing a fraction containing trichloretol-A for gamma ray protection of the present invention.

2 is a chemical formula of the triloretol-A component of the present invention.

3 is a graph showing the radical scavenging activity of the fraction containing triloretol-A on free radicals generated by gamma-ray exposure.

Figure 4 is a graph showing the effect of the fraction containing the triloretol-A of the present invention on V79-4 cell survival.

5 is a graph showing the lipid peroxidation inhibitory activity of the fraction containing the triloretol-A of the present invention

Figure 6 is a graph showing the cytoprotective effect of the fraction containing the triloretol-A of the present invention

Figure 7 is a micrograph measuring the cytoprotective effect from apoptosis induced by gamma-ray of the fraction containing the triloretol-A of the present invention

Figure 8 is a graph showing the effect on the oxidative DNA damage of the fraction containing the triglycerol-A of the present invention

The present invention relates to a fraction containing trichloretol-A for gamma ray protection and a method for producing the same.

Ecklonia cava is a plant belonging to the species Ecklonia.

Radical scavenging activity, anti-pasmin inhibitory activity, antimutagenicity, bactericidal activity, HIV reverse transcriptase and protease inhibitory effect, tyrosinase inhibitory activity against this Ecclonia spp. Several studies, such as activity, have already been reported.

Recently, studies on biological activity using active Ecklonia cava belonging to this species has been actively conducted.

Korean Patent Publication No. 10-0363112 (a novel substance isolated from Ecklonia cava, a method for extracting and purifying it, and a use thereof as an antioxidant), comprising: extracting an organic solvent by adding an organic solvent to the crushed Ecklonia cava; Solvent fractionation of the extracted material; Through the step of purifying the solvent fraction by chromatography, it is disclosed that the antioxidant activity and thermal stability of the material extracted from Ecklonia cava are.

Korean Patent Application Laid-Open No. 2002-0025358 (Hair regrowth composition and its preparation method) includes 0.1 to 10 dry weight% of Ecklonia cava, 1 to 10 dry weight% of dripping water, 5 to 30 dry weight% of jujube, 10 to 30 dry weight of walnut , 10 to 30% by weight of black sesame seeds, dry weight of 1 to 10% by weight of the core and 5 to 25% by weight of an edible binder and a method for manufacturing the same are disclosed.

As described above, studies on the antioxidant activity of the conventional Ecklonia cava extract biological activity is actively made, but research on other biological activity effects in addition to the antioxidant activity has not been made.

In order to solve the above problems, an object of the present invention is to provide a fraction containing trichloretol-A having an excellent protective effect on gamma rays and a preparation method thereof.

The present invention relates to a fraction containing trichloretol-A for gamma ray protection and a method for producing the same.

The method for preparing a fraction containing triglycerol-A for protecting gamma rays of the present invention comprises the steps of: drying Ecklonia cava , and then grinding the Ecklonia cava into a grinder to prepare Ecklonia cava powder; Immersing the Ecklonia cava powder in 80% methanol, and then filtering the resultant filter by a vacuum filter to produce a methanol extract; Solvent fractionation of the prepared methanol extract with ethyl acetate and water; Extracting the ethyl acetate fraction in the solvent fraction with diethyl ether to obtain a diethyl ether fraction; Chloroform (CHCl ₃ ) and methanol (MeOH) were mixed with the obtained diethyl ether fraction in a weight ratio of 2: 1, and then fractionated by column chromatography to obtain a fraction containing triplolorethol-A. It consists of.

Ecklonia cava , the main ingredient used in the present invention, is a seaweed belonging to the brown seaweed seaweed seaweed seaweed family, which is a brown algae plant that grows in the deep of the lunch.

Gamma rays form reactive oxygen species and induce cell damage.

Sulphidyl (-SH) -based substances such as cystine, glutathione, and beta-mercaptoethylamine and other antioxidants can cause rats to escape the lethal toxicity of radiation, which forms reactive oxygen species and induces cell damage. It is reported to protect.

Therefore, as the interest in the interrelationship between gamma-ray exposure and oxygen species has increased, much focus has been placed on developing natural antioxidants as gamma-ray protective agents.

In other words, the inventors of the present invention researched the Ecklonia cava extract, which is known to have an excellent antioxidant effect, was found to have not only an antioxidant effect but also other biologically active effects, and in particular, the gamma-ray protective effect was excellent.

In addition, as a result of various trials and errors, it was confirmed that a solvent containing a substance exhibiting a better gamma-ray protection effect when extracted using a solvent. It could be seen whether the gamma-rays have a protective effect.

In addition, it has been found that the fraction containing the triloretol-A of the present invention activates the antioxidant system of the cells and has the effect of reducing the damage of the cells by hydrogen peroxide.

This, Triphlorethol-A is an open chain terpolymer of phloroglucinol, has a polyphenol structure, and is one of phlorotannin isolated from Ecklonia spp.

This fraction containing Triphlorethol-A has the effect of eliminating intracellular reactive oxygen species (ROS) produced by gamma irradiation, and the plasma membrane, the two targets of intracellular damage induced by gamma rays. It has been shown to simultaneously protect lipid peroxidation and cellular DNA damage.

In addition, it has been shown to reduce the formation of apoptosis cells by gamma rays.

Therefore, it was found that the fraction containing Triphlorethol-A prepared by the method of the present invention exhibited an excellent gamma ray protective effect by eliminating reactive oxygen species produced by gamma rays.

Hereinafter, a process for preparing a fraction containing trichloretol-A for gamma ray protection according to the present invention will be described in detail.

<Production Process of Fractions Containing Triple-Lortol-A for Gamma Irradiation>

1. First Process: Material Preparation

Ecklonia cava ( Ecklonia cava ) is shaded.

The dried Ecklonia cava is prepared by grinding the Ecklonia cava powder.

2. Second Process: Methanol Extract Preparation

The prepared Ecklonia cava powder is immersed in 80% methanol, and filtered through a vacuum filter to prepare a methanol extract.

At this time, it is preferable to immerse the dry Ecklonia cava in methanol at 20-25 degreeC for 2 days.

3. Third process: preparing ethyl acetate fraction

Ethyl acetate and water were added to the methanol extract prepared in the second step to fractionate the solvent to prepare each fraction.

At this time, 3 L of ethyl acetate and 3 L of water are added per 200 g of the methanol extract, and the solvent is preferably fractionated.

The water fraction is discarded and the remaining ethyl acetate fraction is prepared.

4. Fourth step: preparing diethyl ether fraction

Diethyl ether was added to the ethyl acetate fraction prepared in the third step and extracted to prepare a diethyl ether fraction.

At this time, it is preferable to add 1 L of diethyl ether per 20 g of ethyl acetate fraction to extract and prepare a diethyl ether fraction.

4. Fifth Step: Preparation of Fractions Containing Triplothol-A

To the diethyl ether fraction obtained in the fourth step, chloroform (CHCl ₃ ) and methanol (MeOH) were mixed at a weight ratio of 2: 1 to prepare a mixture.

The mixture is fractionated by column chromatography to obtain a fraction containing triplolorethol-A.

Hereinafter, the fractions and the preparation method of the triloretol-A for gamma ray protection of the present invention will be described in detail with reference to Examples and Experimental Examples.

However, these do not limit the scope of the present invention.

Example 1 Preparation of Diethyl Ether Fraction of Ecklonia cava Extract 1

In May 2005, Ecklonia cava was collected and prepared in Udo, Jeju Island.

The prepared ecstasy was shaded.

5 kg of dried Ecklonia cava was ground in a grinder to prepare Ecklonia cava powder.

4 kg of the Ecklonia cava powder was added to 20 L of 80% methanol, immersed and extracted at 25 ° C. for 2 days, and filtered through a vacuum filter to obtain 1 kg of brown methanol extract.

15 kg of ethyl acetate and 15 L of water were added to 1 kg of methanol extract, and solvent fractionation was performed.

The water fraction in the solvent fraction was discarded, and the remaining ethyl acetate fraction was prepared.

230 g of the ethyl acetate fractions were mixed with 460 g of celite.

Celite mixed with an ethyl acetate fraction was charged to the column, and 14 g of a diethyl ether fraction was obtained using 9.5 L of diethyl ether as a developing solvent.

The diethyl ether fractions were mixed with 3.2 L of chloroform (CHCl ₃ ) and 1.6 L of methanol (MeOH), followed by Sephadex LH-20 column chromatography.

After that, fractions containing triplolorethol-A were obtained from the four fractions obtained, and the data were identified by comparing the literature with NMR.

The results are shown in FIG.

It was confirmed that the purity of triplorlorethol-A in the fraction containing the triglycerol-A was 90% or more through HPLC.

In addition, it was dissolved in dimethylsulfoxide (Dimethylsulfoxide, DMSO) in order to measure the gamma-rays protective effect of triploretol-A (triphlorethol-A), the final concentration was prepared not to exceed 0.1%.

<Example 2> Preparation of the diethyl ether fraction of Ecklonia cava extract 2

In May 2005, Ecklonia cava was collected and prepared in Udo, Jeju Island.

The prepared ecstasy was shaded.

5 kg of dried Ecklonia cava was ground in a grinder to prepare Ecklonia cava powder.

4 kg of the Ecklonia cava powder was added to 20 L of 80% methanol, immersed and extracted at 25 ° C. for 2 days, and filtered through a vacuum filter to obtain 1 kg of brown methanol extract.

To 1 kg of methanol extract was added 5 L of ethyl acetate and 5 L of water to obtain a solvent fraction.

The water fraction in the solvent fraction was discarded, and the remaining ethyl acetate fraction was prepared.

230 g of the ethyl acetate fractions were mixed with 460 g of celite.

Celite mixed with an ethyl acetate fraction was charged to the column, and 14 g of a diethyl ether fraction was obtained using 9.5 L of diethyl ether as a developing solvent.

Into the diethyl ether fraction, 3.2 L of chloroform (CHCl ₃ ) and 1.6 L of methanol (MeOH) were mixed, followed by Sephadex LH-20 column chromatography, followed by fractionation with triplorlorethol-A. Got.

Experimental Example 1 Determination of the Effect of Triloretol-A Fraction of the Present Invention on Intracellular Active Oxygen Species

1. Preparation of experimental materials

Fractions containing triloretol-A prepared in Example 1 were prepared.

As a reagent, DCF-DA (propidium iodide Hoechst 33342, Sigma) was purchased and prepared, and all other reagents used analytical grade.

2. Experimental method

Irradiation was performed with gamma rays generated from a ⁶⁰ Co gamma source (MDS Nordion C-188 standard source, Cheju National University) as experimental cells.

The amount of free radicals in the cells was measured using the DCF-DA experimental method.

That is, a fraction containing 30 μM tripleloritol-A was added to V79-4 cells and exposed to gamma rays at 10 Gy intensity after one hour.

After exposure it was incubated at 37 ℃ for 24 hours.

After incubation, 25μM DCF-DA solution was added, and the fluorescence intensity of 2 ', 7'-dichlorofluorescein was measured at 485 nm and the emission wavelength was measured at 485 nm using a spectrophotometer (PerkinElmer LS-5B).

The measurement was repeated three times, and the value was expressed as an average value ± S.E.

3. Experimental Results

As a result, the fraction containing triloretol-A reduced free radical species in cells induced by radiation at concentrations of 30 μM (FIGS. 3A, 3B).

Experimental Example 2 Determination of the Effect of Triloretol-A Fraction of the Present Invention on V79-4 Cell Survival

1. Preparation of experimental materials

Fractions containing triloretol-A prepared in Example 1 were prepared.

As a reagent, DCF-DA (propidium iodide Hoechst 33342, Sigma) was purchased and prepared, and all other reagents used analytical grade.

2. Experimental method

MTT assay was used to examine the effect of fraction containing triloretol-A on V79-4 cell survival.

MTT assay is based on the principle that tetrazolium salt is reduced by mitochondrial dehydrogenase in living cells.

V79-4 cells were treated with fractions containing 30 μM triplortol-A and exposed to gamma rays of 10 Gy intensity after one hour.

After exposure it was incubated at 37 ℃ for 48 hours.

After incubation, 50 μl MTT (2 mg / ml) solution was added to each well so that the final volume was 200 μl.

After incubation for 4 hours, the supernatant was removed by centrifugation for 5 minutes at 800 x g speed.

Formazan crystals of each well were dissolved in 150 μl of DMSO and absorbance was measured using a multi-well spectrophotometer at 540 nm.

The measurement was repeated three times, and the value was expressed as an average value ± S.E.

3. Experimental Results

As a result, as shown in Figure 4, when treated with 30 μM of the fraction containing the triloretol-A to the cells exposed to radiation, the survival rate increased to 74% compared to 54% untreated group survival rate.

Experimental Example 3 Measurement of Lipid Peroxidation Inhibitory Activity of the Fractions Containing Triloretol-A of the Present Invention

1. Preparation of experimental materials

Fractions containing triloretol-A prepared in Example 1 were prepared.

As a reagent, DCF-DA (propidium iodide Hoechst 33342, Sigma) was purchased and prepared, and all other reagents used analytical grade.

2. Experimental method

Ionizing radiation causes cell membrane damage and this disorder is the most important factor in lowering survival.

Peroxidation of membrane lipids is a major damaging factor in membranes.

Lipid peroxidation was measured using thiobarbituric acid reaction.

V79-4 cells were treated with fractions containing 30 μM triplortol-A and exposed to gamma rays of 10 Gy intensity after one hour.

After exposure it was incubated at 37 ℃ for 24 hours.

The cells were washed with cold PBS and then disrupted and homogenized on cold 1.15% KCl.

 100 μl of cell lysate was mixed with 8.1% SDS, 1.5 mL 20% acetic acid (to adjust pH to 3.5).

Distilled water was added to the mixture to adjust the final volume to 4 ml and heated at 95 ℃ for 2 hours.

After cooling to room temperature, 5 ml of normal-butanol and pyridine mixture (15: 1, v / v) was added and stirred.

After centrifugation at 1000 x g for 10 minutes, the supernatant was separated and the absorbance was measured using a spectrophotometer at 532 nm.

The measurement was repeated three times, and the value was expressed as an average value ± S.E.

3. Experimental Results

As a result, as shown in FIG. 5, V79-4 cells exposed to gamma rays in vitro increased lipid peroxidation, which was measured by the generation of thiobarbituric acid reactive substance (TBARS).

However, fractions containing triloretol-A prevented the peroxidation of lipids from radiation.

Experimental Example 4 Measurement of Cytoprotective Effect of Fractions Containing Triloretol-A of the Present Invention

1. Preparation of experimental materials

Fractions containing triloretol-A prepared in Example 1 were prepared.

As a reagent, DCF-DA (propidium iodide Hoechst 33342, Sigma) was purchased and prepared, and all other reagents used analytical grade.

2. Experimental method

Sub-G ₁ generated during apoptosis using flow cytometry Hypodiploid cells were measured.

V79-4 cells were treated with 30 μM of fraction containing triloretol-A and exposed to 10 Gy gamma rays after one hour.

The exposed cells were harvested by incubating at 37 ° C. for 48 hours and fixed with 1 ml 70% ethanol at 4 ° C. for 30 minutes.

Cells were washed twice with PBS and incubated in 1 ml PBS solution containing 100 μg propidium iodide and 100 μg RNase for 30 minutes at 37 ° C. in the dark.

Flow cytometry analysis was performed using a FACS Calibue flow cytometer (Becton Dickinson, Mountain View, CA, USA).

Sub-G ₁ low diploid cell ratios were analyzed using histograms generated by the Cell quest and Mod Fit computer program.

The measurement was repeated three times, and the value was expressed as an average value ± S.E.

3. Experimental Results

As a result, as shown in Figure 6, the cells exposed to radiation increased the content of sub-G1 DNA by 35% by apoptosis and only 2% of control cells without radiation treatment showed a sub-G1 DNA content.

Sub-G1 DNA content of apoptosis (18%) was shown when treated with fractions containing 30 μM tripleloritol-A.

From these results, it can be seen that the fraction containing the triloretol-A has a cell survival protection effect by inhibiting apoptosis by radiation.

Experimental Example 5 Measurement of Cytoprotective Effect from Apoptosis Induced by Radiation of Fractions Containing Triloretol-A of the Present Invention

1. Preparation of experimental materials

Fractions containing triloretol-A prepared in Example 1 were prepared.

As a reagent, DCF-DA (propidium iodide Hoechst 33342, Sigma) was purchased and prepared, and all other reagents used analytical grade.

Cell culture medium was prepared using Chinese hamster pulmonary fibroblasts (Chinese hamster V79-4, American type culture collection) at 37 ° C. and 5% CO _{2 in} humid air, 10% heat-inactivated calf serum, streptomycin (100 μg / Ml), a culture solution containing Penicillin (penicillin, 100 units / ml) (Dulbecco's Eagle) was prepared.

2. Experimental method

For nuclear staining using Hoechst33342, V79-4 cells were treated with 30 μM of a fraction containing tripleloritol-A and irradiated with 10 Gy gamma rays after one hour.

In addition, Hoechst33342, a fluorescent dye for incubating at 37 ° C. for 48 hours and staining DNA only after 24 hours, was added 1.5 μl to each well (1.5 ml) and incubated at 37 ° C. for 10 minutes.

Stained cells were observed using a fluorescence microscope equipped with a CoolSNAP-Pro color digital camera and the degree of nuclear condensation was examined.

The measurement was repeated three times, and the value was expressed as an average value ± S.E.

3. Experimental Results

As a result, in the micrograph of FIG. 7, the cell nucleus remained intact and the nucleus was fragmented in the cells exposed to radiation, which is characteristic of apoptosis.

However, it was observed that cell nucleation fragmentation was significantly reduced when exposed to radiation for 1 hour after treatment of the cells with the fraction containing tritrolletol-A.

Experimental Example 6 Determination of the Effect on Fractional Oxidative DNA of Triloretol-A-containing Fractions of the Present Invention

1. Preparation of experimental materials

Fractions containing triloretol-A prepared in Example 1 were prepared.

As a reagent, DCF-DA (propidium iodide Hoechst 33342, Sigma) was purchased and prepared, and all other reagents used analytical grade.

2. Experimental method

Single cell gel electrophoresis (Comet assay) was performed to check for oxidative DNA damage.

The cell suspension was mixed with 75 [mu] l 0.5% low melting agarose (LMA) at 39 [deg.] C. and then coated onto a cooled microscope slide after 200 [mu] l 1% normal melting agarose (NMA) had been coated in advance.

After the agarose has solidified, the slides are covered with another 75 μl 0.5% LMA and in lysis solution (2.5 M NaCl, 100 mM Na-EDTA, 10 mM Tris, 1% Trion X-100, 10 DMSO, pH 10) for one hour. It was submerged at 4 ° C.

 The slides were placed on a gel electrophoresis apparatus containing 300 mM NaOH and 10 mM Na-EDTA (pH 13) to loosen the DNA helix for 40 minutes to reveal unstable damage to the base.

The electric field was walked at 4 ° C. for 20 minutes (300 mA, 25 V) to draw DNA from the cathode to the anode.

After electrophoresis, the slides were washed three times with neutral buffer solution (0.4 M Tris, pH 7.5) at 4 ° C. for 5 minutes.

Slides were observed under fluorescence microscopy and image analysis (Kinetic Imageing, Komet 5.5, UK).

The total fluorescence percentage of Tail and tail length of 50 cells per slide were recorded.

The measurement was repeated three times, and the value was expressed as an average value ± S.E.

3. Experimental Results

As a result, FIG. 8 shows that exposure to radiation increases comet parameters such as tail length and intracellular tail DNA percentage, indicating damage by gamma rays.

The tail length decreased when cells were treated with the fraction containing triloretol-A before exposure to radiation (FIG. 8A).

The percentage of DNA in the tail increased to 50.1 ± 3.4% when cells were exposed to 10Gy radiation (FIG. 8B).

This resulted in a reduction of 29.5 ± 2.3% as a result of treatment with the fraction containing triloretol-A, indicating protection against radiation-induced DNA damage in vitro.

According to the present invention, a fraction containing trichloretol-A having an excellent gamma-ray protective effect and a method for producing the same are provided.

Claims (4)

In the composition for gamma ray protection, First step of manufacturing Ecklonia cava in the shade, then grinding the Ecklonia cava to grind the Ecklonia cava, A second step of preparing the methanol extract by immersing the Ecklonia cava powder in 80% methanol, and then filtering the same by filtration under reduced pressure; A third step of solvent fractionation of the prepared methanol extract with ethyl acetate and water, A fourth step of extracting the ethyl acetate fraction from the solvent fraction prepared in the third step by adding 1 l of diethyl ether per 25 g of ethyl acetate fraction to obtain a diethyl ether fraction; Chloroform (CHCl ₃ ) and methanol (MeOH) were mixed at a weight ratio of 2: 1 to the diethyl ether fraction obtained in the fourth step, and then fractionated by column chromatography to contain trichlorolethol (A). A composition prepared by the fifth process of obtaining a fraction obtained, A gamma-ray protective composition comprising an Ecklonia cava fraction containing triplolorethol-A as an active ingredient. The method of claim 1, When the Ecklonia cava powder of the second step is immersed in 80% methanol, it is prepared by immersing for 2 days at 20 ~ 25 ℃, A gamma-ray protective composition comprising an Ecklonia cava fraction containing triplolorethol-A as an active ingredient. delete delete

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