KR101007515B1 - Artemisia iwayomogi extraction fractions composition including the annphenone - Google Patents

Abstract

The present invention relates to a heat extractor extract fraction composition containing an anphenone (Annphenone).

In the present invention, the extractant composition containing the anphenone (Annphenone) extracts the heat extractor ( Artemisia iwayomogi ) with methanol, extracts the methanol extract with hexane and water, and then separates the chloroform, which is a solvent having a different polarity, from the separated water layer. Then, ethyl acetate and butanol were added thereto, followed by partition extraction to prepare a solvent fraction. Among them, an ethyl acetate fraction was fractionated by chromatography, and the fraction was purified by hexane.

According to the present invention, a heat extractor extract fraction composition containing an anphenone component having an antioxidant effect and a cytoprotective effect is provided.

Anphenone, Heat Keeper, Antioxidant, Cytoprotective, Reactive Oxygen Species, Apoptosis

Description

Heat-excited extract fraction composition containing anphenone {ARTEMISIA IWAYOMOGI EXTRACTION FRACTIONS COMPOSITION INCLUDING THE ANNPHENONE}

The present invention relates to a heat extractor extract fraction containing anphenone (Annnphenone) having an antioxidant effect.

Cells normally respond to moderate oxidative stress by regulating their antioxidant defense mechanisms.

However, imbalances between free radicals and antioxidant defenses can lead to cell damage that leads to the production of free radicals related to DNA, proteins, lipids, small molecules in ecosystems, and human diseases.

Phenolic antioxidants play an important role in the oxidation process, which is selectively oxidized by aggressive radicals.

Phenolic antioxidants can behave like reducing agents, hydrogen donors, singlet oxygen quenchers, metal ion complexing agents, inherent antioxidants, etc., due to their high reduction potential.

Therefore, efforts are being made to find phenolic antioxidants to reduce reactive oxygen species (ROS), and to reduce the degenerative diseases by reducing human health benefits, oxidative stress, and inhibition of macromolecular oxidation. Much research has been done (Silva et al., 2004; Pulido et al., 2000; Tseng et al., 1997; Andreasen et al., 2001).

Conventionally, natural antioxidants have been searched for having strong pharmaceutical properties and reducing cytotoxicity.

Korean Registered Patent Publication No. 10-0768085 "Sudberry extract having a physiological activity" discloses an antioxidant and anti-inflammatory activity composition comprising the extract of the wormwood as an active ingredient.

On the other hand, a paper has been published that separates anphenone from Artenisia annua (Phytochemistry-Oxford. Oxford: Elsevier Science Ltd. Feb 1997. v. 44 (3) p.555-557). Articles on antimalarial, antibacterial, anti-inflammatory, antihypertensive, anticancer activity against primary metabolites have been published (CURRENT 48 SCIENCE, VOL. 80, NO. 1, 10 JANUARY 2001).

However, there has been no report on the antioxidant activity of phenolic acetophenone, anphenone.

In order to solve the above problems, the present invention provides a heat extractor extract fraction containing an anphenone component that inhibits the generation of reactive oxygen species (ROS) and increases the antioxidant enzyme activity. The purpose is to provide.

In addition, it provides a heat extractor extract fraction containing an anphenone component that has a cytoprotective effect that reduces DNA damage from oxidative stress, reduces apoptosis cell content, and enhances cell viability. There is also a purpose.

The present invention relates to a heat extractor extract fraction composition containing an anphenone (Annphenone).

In the present invention, the extractant composition containing the anphenone (Annphenone) extracts the heat extractor ( Artemisia iwayomogi ) with methanol, extracts the methanol extract with hexane and water, and then separates the chloroform, which is a solvent having a different polarity, from the separated water layer. Then, ethyl acetate and butanol were added thereto, followed by partition extraction to prepare a solvent fraction. Among them, an ethyl acetate fraction was fractionated by chromatography, and the fraction was purified by hexane.

When described in more detail with respect to the present invention.

The present invention relates to a heat extractor extract fraction containing anphenone (Annnphenone) having an antioxidant effect.

Of the inventors of the present invention it was repeated a number of trial and error with no number to find the natural phenolic antioxidants, (Artemisia being heat The present invention was completed by confirming that the antioxidant activity is very large from an phenone (Annphenone) which is a phenolic acetophenone isolated from iwayomogi).

Heather iwayomogi ) is also called Buduk Worm , Aegwi Rock Mugwort, Injin Mugwort, and is a perennial tree plant belonging to the Asteraceae.

Stems and leaves have been medicinal since the Goryeo Dynasty because they were called Haninjin.

The components of this herb are esculetin (esculetin-6-methylether and esculetin-7-methylether). These components have a strong bile odor and have been used as a summary of cholelithiasis, infection and jaundice. ), Soothing effects are known.

The present invention confirmed a strong antioxidant activity in the material separated from the heat collector.

First, the present invention was extracted with methanol for 3 hours at 80 ℃ dried ground portion of the heat collector to obtain a dark brown methanol extract.

The methanol extract was partitioned and extracted with hexane and water, and then chloroform, ethyl acetate and butanol, which were solvents having different polarities, were sequentially extracted.

The obtained ethyl acetate fraction was chromatographed after silica gel column chromatography using chloroform-methanol (CHCl ₃ -MeOH) mobile phase (95: 5-25: 5) to obtain 6 fractions.

Silica gel vacuum column was eluted with chloroform-methanol (CHCl ₃ -MeOH, 12: 1-7: 1), followed by chromatography to obtain 4 fractions, which were white solids.

This material was purified with high purity hexane to give pure compound.

As a result, the compound was analyzed by EI-MS, ¹ H-NMR, ¹³ C-NMR, ¹ H- ¹ H COZY, HMQC and HMBC spectroscopy, and anphenone (annphenone; 2,4-hyroxy) as shown in FIG. -6-methoxy-acetophenone 4-O-β-D-glucopyranoside).

The present invention confirmed that the above-mentioned anphenone inhibits the generation of reactive oxygen species (ROS), and has an excellent cytoprotective effect against oxidative stress.

In the present invention, it was confirmed that the anphenone reduced the catalase and glutathione peroxidase activity.

Catalase is located in the peroxysomes and converts hydrogen peroxide to intramolecular oxygen and water (del Rio et al., 1992).

Catalase plays an important role in reducing cellular damage by protecting the cytoplasm against oxidative stress (Gaetani et al., 1996; Pietarinen et al., 1995).

In addition, glutathione peroxidase is a selenium protein (selenoprotein) that reduces hydrogen peroxide and oxidative glutathione peroxide.

The activity of glutathione peroxidase is highly dependent on GSH concentration (Prabhakar et al., 2005).

Decreased activity of glutathione peroxide may result in low GSH levels or inactivation of glutathione peroxidase.

The activity of glutathione peroxidase was significantly increased in the cells treated with anphenone, indicating that anphenone increased GSH levels or induced glutathione peroxidase activity.

In addition, it has been confirmed that anphenone reduces the damage of DNA induced by lipid membranes and hydrogen peroxide, which means that anphenone has cytoprotective properties.

V79-4 cells exposed to hydrogen peroxide show unique characteristics of apoptosis, including nuclear segments, sub G ₁ -hypodiploid cells, and mitochondrial membrane potential disruption.

However, cells pretreated with anphenone significantly reduced the content of apoptosis cells.

These results indicate that anphenone, which inhibits hydrogen peroxide, inhibits reactive oxygen species, thereby reducing apoptosis cell content.

In addition, the present invention confirmed that the anphenone has a cytoprotective effect of inhibiting apoptosis (apoptosis) caused by oxidative stress.

Anphenone inhibited apoptosis induced by hydrogen peroxide (H ₂ O ₂ ) to protect hamster lung fibroblasts (V79-4) from apoptosis, which reduced apoptotic nuclear segmentation, sub-G ₁ It was confirmed by decreasing the cell population and inhibiting the reduction of mitochondrial membrane potential (ΔΨ).

Therefore, it was found that the anphenone of the present invention is excellent in the antioxidant effect of inhibiting the production of reactive oxygen species (ROS) in the cell and increasing the activity of the antioxidant enzyme (Antioxidant enzyme).

In addition, the present invention has been found that the anphenone has an antioxidant effect because it has a cytoprotective ability to reduce DNA damage from oxidative stress, decrease the content of apoptosis cells, and enhance cell viability.

As described above, the anphenone isolated from the heat collector of the present invention has excellent antioxidant and cytoprotective effects and can be very useful for food additives, beverage compositions, functional health foods, cosmetic compositions, and the like.

It may also be used as a pharmaceutical composition, may be prepared by methods known in the pharmaceutical art, and may be mixed with itself or with a pharmaceutically acceptable carrier, excipient, diluent, or the like to form a powder, granule, tablet, capsule or It may be prepared and used in the form of an injection or the like, and they may be administered orally or parenterally.

An effective dose of anphenone according to the present invention may be appropriately selected depending on the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, the severity of the disease to be treated and the like.

The present invention provides a heat extractor extract fraction containing an anphenone component that inhibits intracellular reactive oxygen species (ROS) production and increases antioxidant enzyme activity.

In addition, it contains an anphenone as an active ingredient, which contains an anphenone component having a cytoprotective effect that reduces DNA damage from oxidative stress, reduces the content of apoptosis cells, and enhances cell viability. The heat collector extract fraction composition is provided.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples, but these do not limit the scope of the present invention.

<Example 1> Preparation of heat extractor extract fraction composition of the present invention

Heather iwayomogi ) outpost was purchased from Gyeongdong market (Seoul, Korea) and prepared.

The confirmed sample of the heat keeper has been deposited with Duksung Women's University College of Pharmacy (Seoul, Korea).

1.2 kg of the dried ground portion of the heat collector were extracted with methanol at 80 ° C. for 3 hours.

83.5 g of a dark brown methanol extract was obtained from the above.

The methanol extract was partitioned and extracted with hexane and water, and then, chloroform, ethyl acetate and butanol, which were solvents having different polarities, were partitioned and sequentially extracted, and a double ethyl acetate fraction was obtained and prepared.

Example 2 Identification of Anphenone Component from the Heat Sweeper Extract Fraction Composition of the Present Invention

7.0 g of ethyl acetate fraction in Example 1 was chromatographed after silica gel column using CHCl ₃ -MeOH mobile phase (95: 5-25: 5) to obtain 6 fractions.

Silica gel vacuum column with CHCl ₃ : MeOH (12: 1-7: 1) eluting conditions and then chromatographed gave 4 fractions, which were white solids.

This material was further purified by recrystallization from high purity hexane to give 32.6 mg of pure compound.

The compound was analyzed by EI-MS, ¹ H-NMR, ¹³ C-NMR, ¹ H- ¹ H COZY, HMQC and HMBC spectroscopy as shown in FIG. 1 by anphenone (annphenone; 2,4-hyroxy-6- methoxy-acetophenone 4-O-β-D-glucopyranoside).

Example 3 Preparation of a Food Additive Composition Containing a Extraction Extract Composition

The compound prepared by the method of Example 2 of the present invention was dried and then ground to prepare a powder.

2 wt% of the above powder was used to prepare a conventional food composition.

<Example 4> Preparation of the beverage composition containing the heat extractor extract fraction composition

The compound prepared by the method of Example 2 of the present invention was prepared.

5 wt% of the above material was used to prepare a conventional beverage composition.

<Example 5> Preparation of the cosmetic composition containing the heat extractor extract fraction composition

The compound prepared by the method of Example 2 of the present invention was prepared.

10 wt% of the above-mentioned substance was added to a conventional cosmetic composition.

Experimental Example 1 Measurement of Anphenone Intracellular Oxygen Oxygen Species (ROS) Inhibitory Activity and Antioxidant Enzyme Activity

1. Experimental method

1-1. reagent

DCF-DA (2 ', 7'-dichlorodihydrofluorescein diacetate) and Hoechst 33342 were purchased from Sigma (St. Louis, MO, USA) and thibarbituric acid (TBA) was purchased from BDH Laboratories (Poole, Dorset, UK). Ready.

Primary antiphospho histone H2A.X antibodies were purchased from Upstate Biotechnology (Lake Placid, NY, USA).

1-2. Cell culture

Lung fibroblast cells are known to be sensitive to oxidative stress responses.

Hamster lung fibroblasts (V79-4) were distributed from the American Standard Cell Line Bank to test the effect of anphenone on oxidative stress.

The cell line was maintained at 37 ° C. in an incubator while maintaining a humidified atmosphere of 5% CO ₂ , and DMEM (Dulbecco's modified Eagle's medium) containing 10% inactivated fetal bovine serum and streptomycin (100 μg / kg). mL) and incubated in penicillin (100 units / mL).

1-3. Intracellular Reactive Oxygen Species (ROS)

DCF-DA method (Rosenkranz et al., 1992) was used to determine the level of free radicals in cells.

V79-4 cells were transplanted into 96 well plates at a number of 2 × 10 ⁴ cells per well, and 6 hours after transplantation, cells prepared with 1, 10, and 50 μg / ml of anphenone were treated.

After 30 minutes the plate was further treated with 1 mM hydrogen peroxide.

The cells were further incubated at 37 ° C. for 30 minutes.

After adding 25 mM of DCF-DA solution for 10 minutes, 2 ', 7'-dichlorofluorescein phosphor was detected using a fluorescence spectrometer (Perkin Elmer LS-5B).

Intracellular reactive oxygen species (ROS) inhibitory activity (%) was calculated by the following equation.

[OD value by hydrogen peroxide treatment-OD value by anphenone and hydrogen peroxide treatment] / (OD value by hydrogen peroxide treatment) × 100

Image analysis for the generation of intracellular activated cell tumors was obtained by transplanting the cells in which 6 well plates (2 × 10 ⁵ cells per well) were dispensed onto the coverslip.

Six hours after transplantation, the cells not only increased the activity of antioxidant enzymes, but also reduced lipid peroxidation and DNA damage by reducing intracellular free radical species (ROS).

These properties have demonstrated that inhibition of thiobarbituric acid reactive substance (TBAR) formation, comet tail inhibition, and phospho-H2A.X expression are reduced.

Treatment at 10 μg / ml.

After 30 minutes 1 mM hydrogen peroxide was added to the plate.

After changing medium, 100 mM DCF-DA was treated in each well and incubated for additional 30 minutes at 37 ° C.

After washing with phosphate buffer solution, the cells were fixed on a microscope slide with mounting medium (DAKO, Carpinteria, CA, USA).

Microscopic images were collected using a confocal laser scanning microscope (5 PASCAL program; Carl Zeiss, Jena, Germany).

1-4. Catalase activity

Cells were transplanted at 1 × 10 ⁵ cells / ml, and 16 hours after transplantation, the cells were treated with 10 μg / ml of anphenone for 3 hours.

The cells obtained above were suspended in 10 mM phosphate buffer (pH 7.5) and then lysed on ice with double speed ultrasound for 15 seconds.

Then, 1% of Triton X-100 was added to the lysate further incubated on ice for 10 minutes.

The lysates were centrifuged at 5,000 × g for 30 minutes at 4 ° C. to remove debris and the protein content was measured.

To detect catalase activity, 50 μg of protein was added to 50 mM phosphate buffer (pH 7) containing 100 mM hydrogen peroxide (v / v).

The reaction mixture was incubated at 37 ° C. for 2 minutes and its absorbance was monitored at 240 nm for 5 minutes.

The change in absorbance with time was proportional to the disappearance of hydrogen peroxide.

The catalase activity is expressed in units / mg protein, where 1 unit of enzyme activity is defined as the amount of enzyme required to dissipate 1 mM hydrogen peroxide (Carrillo et al., 1991).

1-5. Glutathione peroxidase activity

50 μg of protein was added to 1 mM EDTA, NaN ₃ 1 mM, 1 mM glutathione (GSH), 0.25 unit glutathione reductase, and 25 mM phosphate buffer (pH 7.5) containing 0.1 mM NADPH were added.

After incubation at 37 ° C. for 10 minutes, hydrogen peroxide was added to the reaction mixture at a final concentration of 1 mM. Absorbance was monitored at 340 nm for 5 minutes.

The activity of glutathione peroxidase was measured according to NADPH oxidation rate by the change of absorbance at 340 nm (Paglia and Valentine, 1967; Park et al., 2006).

Glutathione peroxidase activity is expressed in units / mg protein, and 1 unit of enzyme activity is defined as the amount of enzyme required to oxidize 1 mM NADPH.

2. Experimental Results

The intracellular reactive oxygen species (ROS) inhibitory activity of anphenone was identified as 18% at 0.1 μg / ml, 29% at 1 μg / ml, 57% at 10 μg / ml, and 65% at 50 μg / ml, respectively. 2).

Intracellular reactive oxygen species inhibitory effect was not significantly different at 10 ~ 50 ㎍ / ㎖, of which 10 ㎍ / ㎖ was selected for further experiments.

As shown in FIG. 3, fluorescence intensity of DCF-DA staining was measured using a confocal microscope.

Confocal microscopy analysis showed that the anphenone reduced the red fluorescence by hydrogen peroxide treatment, thus reducing the production of reactive oxygen species.

To investigate the intracellular reactive oxygen species inhibitory activity of anphenone, it was measured by the antioxidant enzymes of catalase and glutathione peroxidase (both transforming hydrogen peroxide into oxygen and water molecules). Measured.

As a result, as shown in Figure 4, the anphenone increased the catalase activity to 25 units / mg protein at a concentration of 10 ㎍ / ㎖.

The control group was compared with the above results with 12 units / mg protein.

However, the hydrogen peroxide treated group significantly inhibited the catalase activity, and the anphenone and hydrogen peroxide treated groups recovered the catalase activity to 16 units / mg protein, compared to the control group of 6 units / mg protein in the hydrogen peroxide treated group.

In addition, glutathione peroxidase activity was decreased in the hydrogen peroxide treatment group, but glutathione peroxidase activity was restored in the anphenone and hydrogen peroxide treatment groups (FIG. 5).

Therefore, the experimental results showed that anphenone has the effect of inhibiting reactive oxygen species (ROS) and increasing antioxidant enzyme activity.

Experimental Example 2 Measurement of Anphenone Lipid Peroxidation and Inhibition of Cytoplasmic DNA Damage

1. Experimental method

1-1. Lipid Peroxidation Analysis

Lipid peroxidation is analyzed by TBA reaction (Ohkawa et al., 1979).

Cells were washed with cold PBS, rubbed and homogenized with 1.15% KCl in ice.

100 ml of cell lysate was mixed with 0.2 ml of 8.1% sodium dodecylsulfate (SDS), 1.5 ml of 20% acetic acid adjusted to pH 3.5, and 1.5 ml 0.8% TBA.

The mixture was heated at 95 ° C. for 2 hours with a final volume of 4 mL with distilled water.

After cooling to room temperature, 5 ml of a butanol and pyridine mixture (15: 1, v / v) was added to each sample and shaken.

After centrifugation at 1,000 x g for 10 minutes, the supernatant was separated and the absorbance was measured spectrophotometrically at 532 nm.

The amount of TBARS is determined using the 1,1,3,3, tetrahydroxypropane standard curve.

1-2. A comet assay

Comet mushrooms were performed to assess oxidative DNA damage (Singh, 2000; Rajagopalan et al., 2003).

Cell pellets (1.5 × 10 ⁵ cells) were mixed with 100 ml of 0.5% low melting agarose (LMA) at 39 ° C., and 200 ml of 1% normal melting agarose (NMA) on a fully coated frosted microscopic slide. Was dispensed.

After coagulation of agarose the slides were covered with another 75 ml of 0.5% LMA and then dissolved (2.5 M NaCl, 100 mM Na-EDTA, 10 mM Tris, 1% Trion X-100, and 10% DMSO, pH). 10) was immersed at 4 ℃ for 1 hour.

The slides were placed on a gel electrophoresis device containing 300 mM NaOH and 10 mM Na-EDTA (pH 13) for 30 minutes.

This releases DNA and leads to alkaline unsafe damage.

The electric field was applied at 300 mA, 25 V at 4 ° C. for 20 minutes to attract negatively charged DNA toward the cathode.

After electrophoresis, the slides were washed three times for 5 minutes at 4 ° C. with nutrient buffer (0.4 M Tris, pH 7.5), and then fixed with 75 ml of ethidium bromide (20 μg / ml).

The slides were observed using a fluorescence microscope and an image analyzer (Komet, Andor Technology, Belfast, UK).

The overall fluorescence rate at the tail and the DNA tail length of 50 cells per slide were recorded.

1-3. Western Blot

Cells were collected, washed twice with PBS and lysed on ice with 100 ml of lysis buffer [120 mM NaCl, 40 mM Tris (pH 8), 0.1% NP 40] on ice, then centrifuged at 13,000 × g for 15 minutes. It was .

Supernatants were collected from the lysate and the protein concentration was measured.

An aliquot of the lysate (protein 40 μg) was heated for 5 minutes and electrophoresed with 10% sodium dodecysulfate-polyacrylamide gel.

Blots in the gels migrated on nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), which were incubated with primary antibodies.

Protein bands were detected using an ECL western blot detector (Amersham, Little Chalfont, Buckinghamshire, UK) and then exposed on X-ray film.

1-4. Immunocytochemical test ( Immunocytochemistry)

Cells on the coverslips were fixed with 4% PFA (paraformaldehyde) for 30 minutes and infiltrated with 0.1% Triton X-100 in PBS for 2.5 minutes.

The cells were treated with blocking medium (3% bovine serum albumin in PBS) for 1 hour and incubated with diluted anti-phospho histone H2A.X antibody in blocking medium for 2 hours.

Immune Response Primary phosphohistin H2A.X antibody was detected with a 1: 500 dilution of FITC conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Glove, PA, USA) 1: 500.

After washing with PBS, the fixed cells were fixed on microscope slides in mounting medium with DAPI (Vector, Burlingame, CA, USA).

Images were collected using a confocal laser scanning microscope (5 PASCAL program; Carl Zeiss, Jena, Germany).

2. Experimental Results

The activity of anphenone to inhibit membrane lipid peroxidation and cytoplasmic DNA damage in hydrogen peroxide (H ₂ O ₂ ) treated cells was examined.

Hydrogen peroxide-induced cell membrane damage is one of the major obstacles to cell viability.

As shown in FIG. 6, V79-4 cells exposed to hydrogen peroxide were found to have increased lipid peroxidation, which was confirmed by TBARS production.

However, anphenone prevented lipid peroxidation.

Cytoplasmic DNA damage reduced by hydrogen peroxide exposure was confirmed by alkaline comet analysis and phospho histone-H2A.X expression.

Cell exposure to hydrogen peroxide increases its length and DNA content at the tail of the cell.

When the cells were exposed to hydrogen peroxide, the DNA content was increased by 46% in the tail, and decreased by 32% when the anphenone was treated (FIGS. 7, 8).

Phosphorylation of nuclear histone H2A.X, a sensitive marker that destroys DNA double helices (Rogakou et al., 1988), was increased in cells treated with hydrogen peroxide.

This was confirmed by Western blot and immunofluorescence staining results of FIGS. 9 and 10.

However, the treatment of hydrogen peroxide with anphenone decreased the expression of phospho H2A.X in the nucleus.

The results indicate that anphenone reduces DNA damage by protecting cells from hydrogen peroxide.

Experimental Example 3 Experiment for Measuring Cytoprotective Effect of Anphenone

1. Experimental method

1-1. Cell viability

The effect of anphenone (10 μg / ml) on the viability of V79-4 was analyzed by [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium] bromide (MTT) assay (Carmichael et al. , 1987).

50 mL of MTT standard solution (2 μg / mL) was added to each well of the total reaction (200 mL volume).

After incubation for 4 hours, the plate was centrifuged at 800 x g for 5 minutes and the supernatant was separated.

Insoluble crystals in each well were dissolved in 150 ml of dimethylsulfoxide and identified by scanning multi-well spectrophotometer A ₅₄₀ .

1-2. Nuclear Staining with Hoechst 33342

1.5 ml of a DNA specific fluorescent stain Hoechst 33342 (stock 10 mg / ml) was added to each well and incubated at 37 ° C. for 10 minutes.

Stained cells were observed under fluorescence microscope.

At this time, a color digital camera (CoolSNAP-Pro) was installed to check the degree of nuclear condensation.

1-3. Flow cytometry analysis

Was performed to measure the amount of; (Nicoletti et al, 1991 apoptotic sub G 1 hypo-diploid cells.) Flow cytometry analysis of apoptosis that diploid cells.

Cells were prepared and fixed in 1 ml of 70% ethanol at 4 ° C. for 30 minutes.

The cells were washed twice with PBS and then incubated for 30 minutes at 37 ° C. in a dark place of PBS containing 100 μg of propidium iodide (PI) and 100 μg of RNase A.

Flow cytometric analysis was performed and the percentage of sub G ₁ hypo-diploid cells was evaluated by generating histograms using a computer program (Cell Quest and Mod-Fit).

1-4. Mitochondrial membrane potential (ΔΨ) analysis

After preparing the cells, replacing the medium, JC-1 was added to each well and incubated for 30 minutes at 37 ° C.

After washing with PBS, fixed cells were mounted on microscope slides in mounting medium (DAKO, Carpinteria, CA, USA).

Microscopic images were collected using a laser scanning microscope 5 PASCAL program (Carl Zeiss, Jena, Germany) of a confocal microscope (Cossarizza et al., 1993).

1-5. Statistical analysis

All measurements were repeated three times and all values were expressed as mean ± standard error (SE).

The results were done by analysis of the variance (ANOVA) using the Tukey test to analyze the difference.

p <0.05 is considered significant.

2. Experimental Results

The protective effect of anphenone on cells survived in hydrogen peroxide treated cells was examined.

The cells were first treated with hydrogen peroxide and then treated with 10 μg / ml anphenone for 1 hour.

Cell viability was measured after 24 hours using MTT assay.

As shown in FIG. 11, the anphenone treated group increased cell survival by 67% compared to 55% in the hydrogen peroxide treated group.

Anphenone itself had a cell viability of 105% and showed no cytotoxicity at a concentration of 10 μg / ml.

To examine the cytoprotective effect of anphenone on apoptosis, the nuclei of V79-4 cells were stained with Hoechst 33342 and observed under a microscope.

The micrographs of FIG. 12 showed that the cells treated with hydrogen peroxide showed significant nuclear segments, whereas the control cells did not have nuclear damage.

However, cells treated with hydrogen peroxide first and then treated with anphenone for 1 hour were observed to have reduced nuclear fragments.

In addition, the protective effect of anphenone to prevent apoptosis by morphological analysis was confirmed by apoptotic sub-G ₁ DNA analysis and mitochondrial membrane potential (ΔΨ) analysis.

As shown in FIG. 13, DNA content analysis was found to increase to 49% of apoptosis sub-G ₁ DNA content in hydrogen peroxide treated cells.

Moreover, treatment with 10 μg / ml of anphenone was reduced to 25% of the apoptosis sub-G ₁ DNA content.

JC-1 has been widely used to measure mitochondrial depolarization in viable cells.

In non-apoptotic cells, JC-1 accumulated in the mitochondria stained red.

On the other hand, in apoptotic cells, JC-1 disappeared without accumulation of red fluorescent material in the mitochondria (Cossarizza et al., 1993).

As shown in Figure 14, the control cells and cells treated with only anphenone showed a red fluorescent substance in the mitochondria.

However, in the hydrogen peroxide treatment group, red fluorescent substance was found to be reduced in the mitochondria.

The 10-μg / ml concentration of the anphenone treatment group prevented the decrease of the fluorescent material in the hydrogen peroxide treatment group, which means that the anphenone inhibits the drop in the membrane potential value in the hydrogen peroxide-treated cells.

1 is a chemical structural formula of anphenone

Figure 2 is a fluorescence spectrophotometer after DCF-DA treatment showing the results of the inhibitory activity of intracellular reactive oxygen species production of an phenone

Figure 3 is a confocal micrograph of the fluorescence of DCF-DA staining showing the results of the inhibitory activity of intracellular reactive oxygen species production of an phenone

4 is a graph of the results of catalase activity of anphenone

5 is a result graph of glutathione peroxidase activity of anphenone

6 is a graph of the results of the reduction of lipid peroxidation of anphenone

7 is a representative image photograph showing the results for the reduction of DNA damage of anphenone

8 is a graph showing the results of cytoplasmic DNA damage measurement by alkaline Comet assay showing the results of the DNA damage reduction of anphenone

Figure 9 is a Western blotting result picture showing the results for the reduction of DNA damage of anphenone

10 is a confocal image showing the results of immunofluorescence staining showing the results for the reduction of DNA damage of anphenone

Figure 11 is a photograph of the MTT assay results showing the results of the cytoprotective effect of anphenone

12 is a fluorescence microscope image after Hoechst 33342 staining for apoptosis showing the results of the cytoprotective effect of an phenone

Figure 13 is a flow cytometry photograph after PI staining showing the results of the cytoprotective effect of an phenone

14 is a confocal micrograph after staining of cells with JC-1 showing the results of the cytoprotective effect of anphenone

Claims (9)

In the extract fraction composition, After extracting the heat keeper ( Artemisia iwayomogi ) with methanol, and extracting this methanol extract with hexane and water, the solvent fraction was prepared by sequentially partitioning and extracting the solvent of chloroform, ethyl acetate and butanol of different polarity into the separated water layer. Thereafter, an ethyl acetate fraction was fractionated by chromatography, and this fraction was purified by hexane to form an extractor extract composition containing an anphenone component. The anphenone component inhibits the generation of reactive oxygen species (ROS) in the cell, increases the antioxidant enzyme (Antioxidant enzyme) activity, In addition, the anphenone component is characterized by reducing DNA damage from oxidative stress, apoptosis (apoptosis) content of cells, increase cell viability, showing a cytoprotective effect, Heat extractor extract fraction composition. delete delete The method of claim 1, Characterized in being included in food additives, beverage compositions, or functional health foods, Heat extractor extract fraction composition. The method of claim 1, Characterized in being included in the cosmetic composition, Heat extractor extract fraction composition. delete delete delete delete

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