KR20160013413A - COMPOSITION INCLUDING Schisandra chinensis semen Extracts For Antioxidant activation And Manufacturing Method thereof - Google Patents

Abstract

The present invention relates to a composition including a Schisandra chinensis seed extract for antioxidant activation, and a method for manufacturing the same. The composition of the present invention can protect cells from oxidative DNA damage induced by H_2O_2 of a C2C12 cell by inducing the expression of Nrf2 and HO-1; can induce the protection of the C2C12 cell against H_2O_2-induced oxidative stress; and thus can be advantageously used as antioxidant food and a pharmaceutical composition. The Schisandra chinensis seed extract in the present invention is essntial oil of Schisandra chinensis seeds.

Description

TECHNICAL FIELD The present invention relates to a composition for an antioxidant activity containing an extract of Schizandra chinensis and an antioxidant activity of Schizandra chinensis,

The present invention relates to a composition for antioxidant activity containing an extract of Omija seed and a method for producing the same. More specifically, the induction of Nrf2 and HO-1 expression can protect cells from H ₂ O ₂ -induced oxidative DNA damage of C2C12 cells and effectively remove ROS resulting in H ₂ O ₂ -induced oxidative stress Which is capable of enhancing cell viability and preventing apoptosis, and a method for producing the same.

Oxidative stress, a common phenomenon in the body, occurs when the balance between the production of reactive oxygen species (ROS) and their neutralization and the protective activity of the antioxidant system, which acts to eliminate it, is broken. Under normal conditions, physiologically important intracellular ROS levels are maintained at low levels by various enzymatic systems participating in redox homeostasis. However, reductions in the ability to remove interfering and / or antioxidation in the redox state of normal cells can lead to toxicity through the production of free radicals that damage peroxide production and all cellular constituents (including proteins, lipids, and nucleic acids) . Therefore, it may cause disruption in the general mechanism associated with oxidative stress cell signaling pathways. In addition, increased production of ROS can lead to increased oxidative stress, resulting in cellular dysfunction and, consequently, pathological responses that can lead to apoptosis.

ROS formation occurs naturally, and mammalian cells develop a variety of adaptive mechanisms that can detoxify ROS or limit formation. These mechanisms use antioxidant enzymes or antioxidant compounds. Nrf2 (nuclear factor erythroid-2-related factor 2) is a basic leucine zipper-transcription factor, a profound cell sensor for oxidative stress and represents a fundamental response to change in the redox state of cells. Nrf2 binds to a cis-acting enhancer present in the promoter region of a large and distinct target gene to restore antioxidant response element (ARE) and redox homeostasis to HO-1 (heme oxygenase 1) and NQO1 (NAD (P) H quinone oxidoreductase 1). However, during pathological conditions and chronic inflammation, Nrf2 activity and expression of its cytoprotective target gene may be completely degraded and often contribute to disease proliferation. Therefore, the pharmacological activation of Nrf2 is important for the protection of cells under oxidative stress conditions.

Many traditional drugs have been considered potential pharmaceutical candidates for the management of intracellular oxidation balance due to reduced cytotoxicity and potential pharmacological properties. Among them, Schizandra chinensis Baill fruits (Schisandrae fructus) are widely used as one of the famous medicinal plants in Asia, including Korea, China, Japan and Russia. Ozia is often used to increase physical processing capacity, The main compounds implicated in essential oils extracted from Omiza are monoterpene, sesquiterpene and aromatic compounds and have been known to have various pharmacological potentials such as antioxidant, antibacterial and bactericidal effects.

Nevertheless, research on the seed part of Omija has not been conducted. In addition, there has been no report on the protective activity against oxidative stress of essential oils extracted from Omija seeds. Therefore, it is necessary to study the effect of essential oil extracted from Omija seeds.

The inventors of the present invention completed the present invention by demonstrating the protective effect of extracting essential oils from Omija seeds using the ability to protect cells from hydrogen peroxide-induced cell damage and murine skeletal muscle cell lines.

KR 10-2010-0072390 A KR 10-2006-0119081 A KR 10-0854567 B1

It is an object of the present invention to provide a composition for antioxidative activity comprising an extract of Omija seed.

Another object of the present invention is to provide a production method for producing the above-mentioned Omija seed extraction essential oil.

In order to achieve the above object, the antioxidative activity composition containing the extract of Omija seed according to an embodiment of the present invention may contain an extract of Omija seed as an active ingredient and exhibit an antioxidative activity.

The Schizandra chinensis seed extract may be an Essential oil.

The composition may contain the above-mentioned Omija seed extract essential oil at a concentration of 0.01 to less than 100 占 퐂 / ml.

The Omija seed extract is prepared by inoculating at least one microorganism selected from the group consisting of Bacillus lichenifomis, Aspergillus oryzae, and Lactobacillus plantarum in Omija One

Composition for antioxidant activity containing Omija seed extract.

The health food composition for improving antioxidant activity according to another embodiment of the present invention may include a composition for antioxidant activity including the above-mentioned Omija seed extract.

According to another embodiment of the present invention, there is provided a method for preparing an extract of Schizandra chinensis extract for improving antioxidant activity, comprising the steps of: preparing a dried Schizosaccharum by drying at 150 to 210 ° C; Crushing the dried omiza to produce an omija powder; A lyophilizing step of lyophilizing the omitha powder to prepare a lyophilized omija powder; An extracting step of extracting the lyophilized Schizandra chinensis powder with an alcohol or alcohol aqueous solution, and distilling the extract at 150 ° C to 230 ° C for 1 hour to 5 hours.

The method for producing an extract of Schizandra chinensis extract for improving antioxidant activity is characterized in that Bacillus lichenifomis, Aspergillus oryzae, and Aspergillus oryzae are added to the dried omza, And Lactobacillus plantarum, and further cultivating at least one microorganism selected from the group consisting of Lactobacillus plantarum and Lactobacillus plantarum.

Hereinafter, the present invention will be described in more detail. The following description is intended to assist in a clear understanding of the present invention only and does not limit the scope of the present invention to the following contents.

The composition for an antioxidative activity comprising an extract of Omija seed according to an embodiment of the present invention may contain an extract of Omija seed as an active ingredient and exhibit an antioxidative activity.

The antioxidant active composition may be a food composition having an antioxidative activity or a pharmaceutical composition for treating or ameliorating an antioxidative activity-related disease having an antioxidative activity.

The Schizandra chinensis has unique flavor and excellent antimicrobial activity, and has been used as a medicinal crop for a long time.

The above-mentioned omiza can be used as a dried product. When the omiza is dried, there is an advantage in commercialization. Preferably, when the omija seed is dried, the active ingredient having the effect of inhibiting the MMP activity is more activated.

The above-mentioned Omiza seed extract is an extract extracted from the seeds of Omija, and preferably can be a concentrated and distilled Omija seed extract essential oil after extraction.

The essential oil can also be referred to as concentrated oil, essential oil, and the like. When using the above-mentioned essential oil for omija, the essential oil does not affect cytotoxicity more than general omija extract (see Fig. 1 below) Has an excellent antioxidative activity as compared with a general extract or a concentrate thereof. In particular, when used as an essential oil, the expression of HO-1 is induced by an effective ingredient that is not known from the extract, and ROS is effectively eliminated in the body, thereby effectively preventing or treating the progress and invention of oxidative stress-related diseases Confirmed in the present invention.

Preferably, the seed may be a germinated seed of Omija. The germinated omija seed means that the omija seed has been sown. When the germinated seeds of germination are used, the antioxidative activity of germinated seeds may be superior to that of seed extracts or essential oils as well as common germs.

In particular, the Omija seed extract essential oil can protect cells from H ₂ O ₂ -induced oxidative DNA damage of C2C12 cells by inducing expression of Nrf2 and HO-1, effectively removing ROS, and inducing H ₂ O ₂ -induced Which can induce the protection of C2C12 cells against oxidative stress, and thus can be effectively used as antioxidant foods and pharmaceutical compositions.

The extraction solvent of the Omija seed extract may be water, an alcohol, an organic solvent, or a combination thereof. Examples of the alcohol include methanol and ethanol. Examples of the organic solvent include acetone, chloroform, methylene chloride, ether, ethyl acetate , Hexane and the like can be used. Preferably, 70% to 100% ethanol, more preferably 95 to 100% ethanol, may be used. When the ethanol is used, the antioxidant active ingredient contained in the omiza can be extracted more effectively.

The composition may contain an essential oil of Omija seed extract at a concentration of 0.01 to 100 μg / ml, preferably 0.1 to 99.99 μg / ml, and most preferably 70 μg / ml To 90 < RTI ID = 0.0 > pg / ml. ≪ / RTI > It was confirmed in the examples of the present invention that the antioxidative activity was superior to that of the case of containing the omija seed extract essential oil at 70 / / ml to 90 / / ml and less than 70 / / ml, and more than 90 ㎍ / ml , There is a problem that the cytoprotective effect deteriorates the cytoprotective effect due to the antioxidative activity. Therefore, it may be preferable to use the active ingredient in the above range.

The composition for the antioxidative activity comprising the extract of Omija seeds is characterized in that the extract of Omija seeds is composed of a group consisting of Bacillus lichenifomis, Aspergillus oryzae and Lactobacillus plantarum The microorganism may be produced by inoculating any one or more selected microorganisms.

When the microorganism is inoculated, the antioxidative activity of the microorganism is increased by the fermentation of the microorganism, and the antioxidative effect is greatly increased. Particularly, the microorganism inoculation is very effective when it is fermented by inoculating germinated omija seeds.

In case of inoculation at less than 0.01% (w / v), the fermentation process does not proceed or the fermentation period becomes too long. In case of inoculating more than 0.05% (w / v) And the antioxidant effect is rather lowered.

Preferably, the inoculation of the microorganism is carried out using Lactobacillus plantarum, which is inoculated with 0.01 to 0.03% (w / v) relative to the mass of the dried Omija seed.

When the Lactobacillus plantarum is used alone, the microorganism may contain the most active ingredient having the antioxidative activity contained in the above-mentioned Omiza extract, and the antioxidative activity may be the most excellent have.

Preferably, the fermentation is performed for 1 to 7 days. When the fermentation is performed for less than 1 day, the effect of increasing the effective ingredient showing antioxidative effect depending on the microbial activity is insignificant, and when the fermentation progresses for 7 days or more, the content of the effective substance is rather reduced.

More preferably, the dried Schizandra chinensis may be one which after drying the Schizosaccharomyces sp. When the cooked Omiza seeds are used or the cooked Omiza is fermented by using the above microorganism, the antioxidative active material contained in the Omiza extract greatly increases and the antioxidant activity can be very excellent.

The method for producing the essential oil of the present invention comprises the step of drying omija at a temperature of 150 ° C to 210 ° C to produce dried omija; Crushing the dried omiza to produce an omija powder; A lyophilizing step of lyophilizing the omitha powder to prepare a lyophilized omija powder; An extraction step of extracting the lyophilized Schizandra chinensis powder with an alcohol or alcohol aqueous solution, and distilling the extract at 150 to 230 ° C for 1 to 5 hours.

The above-mentioned omiza can be used as a dried product. When the omiza is dried, there is an advantage in commercialization. Preferably, when the omija seed is dried, the active ingredient having the effect of inhibiting the MMP activity is more activated. That is, in the case of extracting and drying the omija seeds, the omija seeds are dried and extracted even when the omija seeds are used at the same concentration, as compared with the case where the omija seeds are lyophilized immediately without drying, Which is advantageous.

Particularly, when the composition is dried at a temperature of less than 150 ° C, the above effect is hardly obtained. When the temperature exceeds 210 ° C, the active ingredient of the extract of Omija seed is destroyed and may be reduced.

More preferably, the Schizosacchis seeds may be lyophilized after drying at 170 to 190 ° C. In the case of drying the seeds of Omija in the above-mentioned temperature range, the most effective ingredient is contained, and the cells are protected from H ₂ O ₂ -induced oxidative DNA damage of C2C12 cells by inducing expression of Nrf2 and HO-1 by the active ingredient And can effectively remove ROS to increase cell viability against H ₂ O ₂ -induced oxidative stress and prevent cell death.

The above-mentioned freeze-drying method can be carried out by a method commonly used in the technical field of the present invention.

The extraction solvent of the Omija seed extract may be water, an alcohol, an organic solvent, or a combination thereof. Examples of the alcohol include methanol and ethanol. Examples of the organic solvent include acetone, chloroform, methylene chloride, ether, ethyl acetate , Hexane and the like can be used. Preferably, 70% to 100% ethanol, more preferably 95 to 100% ethanol, may be used. When the ethanol is used, an effective ingredient having an antioxidative activity contained in the omiza can be effectively extracted.

The step of distilling the extracted extract may be carried out by applying steam at a temperature of 150 ° C to 230 ° C for 1 hour to 5 hours, preferably 180 ° C to 200 ° C for 2 hours to 4 hours, followed by distillation. When the distillation in the range of the present invention essential oil Nrf2 and HO-1 through the induction of the expression of C2C12 cells H ₂ O ₂ - it is possible to protect cells against induced oxidative DNA damage, removes the ROS efficiently H ₂ O ₂ -induced oxidative stress, it is possible to effectively contain an active ingredient having an effect of preventing cell death, and enhancing cell viability.

Bacillus lichenifomis, Aspergillus oryzae, and Aspergillus oryzae are added to the dried omzae which has been subjected to the above-mentioned Omija-drying step between the above-mentioned omiza drying step and the extraction step, Lactobacillus plantarum, and Lactobacillus plantarum, and further cultivating the microorganism.

Induction of expression of Nrf2 and HO-1, including the extract of Omija seeds of the present invention, can protect cells from H ₂ O ₂ -induced oxidative DNA damage of C2C12 cells, effectively remove ROS and inhibit H ₂ O ₂ - Can increase cell viability against induced oxidative stress, prevent cell death, and can be usefully used as antioxidant foods and pharmaceutical compositions.

FIG. 1 is a graph showing the cell viability of C2C12 cells against Omija seed extract essential oil. Each figure represents the mean ± SD of three independent experimental results. Student's t- test (*, p <0.05 vs. untreated control).
FIG. 2 is a graph showing the degree of inhibition of H ₂ O ₂ -induced growth and morphological changes caused by the essential oil-extracted essential oil in C2C12 cells. The results were obtained by calculating the mean ± SD in three independent experiments, and the results were expressed relative to the untreated control (*, P <0.05 compared with the control group; #, P <0.05 H2O2-treated group compare).
FIG. 3 is a graph for confirming the protective effect of ROS on cell death and cell death by the extraction of essential oil from C2C12 cells. Each figure represents the mean ± SD of three independent experimental results (*, P <0.05 compared with control; #, P <0.05 compared with H2O2-treated group).
FIG. 4 is a graph showing the effect of H ₂ O ₂ -induced DNA damage inhibition by the extract of Omija seed in C2C12 cells. X and actin were used as an internal control, and the protein was visualized as an ECL detection system.
FIG. 5 is a graph showing the effect of the extract of Omija seed extract on migration of HO-1 expression induced by C2C12 cells.
6 is a graph showing the effect of the extract of Omija seed extract on the induction of Nrf2 expression in C2C12 cells.
FIG. 7 is a graph showing the inhibitory effect of HO-1 on SSeo-mediated reduction of Omija seed extract for inhibition of H ₂ O ₂ growth and ROS formation in C2C12 cells (*, P <0.05) and comparison; #, P <0.05 compared with the group treated with Comparative H2O2-; compared to the P <treatment and 0.05 H ₂ O ₂ SSeo- group).

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[Preparation Example and Experimental Method]

1. Preparation of Omija Seed Extract

The washed Schizandra chinensis seeds were firstly boiled at 60 ° C for 40 minutes and further boiled for 2 hours at 90 ° C or lower. The cooked omelet was hot-air dried and inoculated with Lactobacillus plantarum at 0.015% (w / v) relative to the weight of the Omiza, and fermented for 3 days while maintaining the temperature at 15 to 37 ° C. The fermented Schizosaccharomyces seeds were thoroughly dried at 180 ° C and then pulverized to 0.15 to 0.2 mm on the basis of an average particle size, and completely lyophilized with a freeze dryer (Freezone 1; Labconco Corp., MO, USA). The filtrate was concentrated on a rotary vacuum evaporator (Buchi Rotavapor R-144, Switzerland) and distilled at 190 ° C for 3 hours using a Clevenger-type apparatus hydrodistillation. Essential oil was stored in a 4 ° C refrigerator in which light was blocked and used for the experiment. The oil refinery yield was 0.66% based on the dry weight of Omija seeds.

2. Cell culture

The C2C12 myoblast cell line was purchased from the American Type Culture Collection (ATCC, Manassas, Va.) And fixed in Dulbecco's modified Eagle's medium (DMEM, Gibco-BRL, Gaithersburg, MD) with 10% heat-inactivated fetal bovine ml of streptomycin, and 0.25 μg / ml of amphotericin fungizone were added to the culture supernatant and cultured at 37 ° C and 5% CO ₂ , supplemented with serum (FBS, Gibco-BRL), 100 U / ml penicillin G, 100 μg / ml streptomycin and 0.25 μg / ml amphotericin fungizone. SSeo was used as a stock solution in dimethyl sulfoxide (DMSO, Sigma-Aldrich Chemical Co., St. Paul, Minn.) And the concentration was 100 mg / ml. The stock solution was diluted to the required concentration before use.

3. Cell viability analysis and morphological image observation

C2C12 cells were administered to a 6-well plate at a density of 1 × 10 ⁵ cells per well. After incubation for 24 hours, the cells were treated with various concentrations of Omija seed extract essential oil with or without H ₂ O ₂ and / or zinc protoporphyrin IX (ZnPP, Sigma-Aldrich) . The medium was removed and the cells were incubated with 0.5 mg / ml 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) solution for 2 hours. The supernatant was discarded and formazan blue was dissolved in DMSO. Optical density was measured at 540 nm using a microplate reader (Dynatech Laboratories, Chantilly, VA). Growth inhibition was determined as 100% viability of the vehicle-treated cells and as percent survival. Morphological changes were observed with a microphotograph obtained from an inverted phase contrast microscope (Carl Zeiss, Oberkochen, Germany).

4. Flow cytometric detection of apoptosis by flow cytometry

Cells were trypsinized and washed with phosphate buffered saline (PBS), then lysed in 100 μl of binding buffer (including annexin-V-FITC and propidium iodide (PI), BD Sciences, Heidelberg, Germany) , Room temperature, dark condition. Cells were immediately counted by flow cytometry (FACS Calibur, Becton Dickinson, San Jose, Calif.). The ratio of annexin-V ⁺ cells was expressed as mean ± SD.

5. Morphological observation of nuclear changes

The cells were washed with PBS and fixed with PBS containing 3.7% paraformaldehyde at room temperature for 10 minutes. Fixed cells were washed with PBS and stained with 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) solution for 10 minutes at room temperature. Cells were observed using a fluorescence microscope (Carl Zeiss).

6. Intracellular ROS measurement

Measurement of ROS was performed using ROS sensitive 2 ', 7', - dichlorofluorescein-diacetate (H2DCFDA, Molecular Probes, Eu gene, OR). Specifically, cells were incubated with 10 [mu] M H2DCFDA for 30 minutes at room temperature and dark conditions. In order to quantify intracellular ROS, fluorescence measurement method (spectrofluorimetry analysis) was performed using a microplate reader with excitation and emission wavelengths of 488 nm and 525 nm, respectively.

7. Gene Damage Quantitative Analysis (Comet analysis) - Single-cell electrophoresis

The cell suspension was mixed with 0.5% low melting agarose (LMA) at 37 ° C, and the mixture was precoated with 1% normal melting agarose and spread on fully frozen microscope slides. The agarose was coagulated and the slides were then covered with 0.5% LMA and lysed in a lysis solution (2.5 M NaCl, 100 mM Na-EDTA, 10 mM Tris, 1% Trion X-100, 10) at 4 ° C for 1 hour. The slides were placed in an electrophoresis apparatus (containing 300 mM NaOH and 10 mM Na-EDTA, pH 13) and DNA was loosened for 40 minutes to cause alkali-labile damage, The electric field was applied at 4 [deg.] C for 30 min (300 mA, 25 V) to draw the drawn DNA to the anode. After electrophoresis, the slides were washed three times for 5 minutes with neutralization buffer (0.4 M Tris, pH 7.5) at 4 ° C and stained with 20 μg / ml PI (Sigma-Aldrich). The slide was observed with a fluorescence microscope (Carl Zeiss).

8. Protein extraction and western blot analysis

Cells were harvested with trypsin-EDTA and resuspended in lysis buffer (20 mM sucrose, 1 mM EDTA, 20 μM Tris-1, pH 7.2, 1 mM DTT, 10 mM KCl, 1.5 mM MgCl ₂ , 5 μg / ml pepstatin A, 10 μg / ml leupeptin, and 2 μg / ml aprotinin) for 30 min at 4 ° C. The mixture was centrifuged (10,000 x g) for 10 min at 4 &lt; 0 &gt; C and the supernatant was collected as whole-cell extract. Protein content was determined using a Bio-Rad protein assay reagent (Bio-Rad, Hercules, Calif.) And the concentration of protein was measured according to the manufacturer's instructions based on bovine serum albumin. After standardization, the same amount of protein was electrophoresed on sodium dodecyl sulfate (SDS) -polyacrylamide gel, followed by nitrocellulose membrane (Schleicher & Schuell, Keene, NH) using electroblotting . The blots were blocked for 1 hour at room temperature using 5% skim milk. The blot was then incubated with primary antibodies overnight and incubated with horseradish peroxidase-conjugated donkey anti-rabbit and sheep anti-mouse immunoglobulin for 1 hour. Immunoreactive bands were identified using enhanced chemiluminescence (ECL) using a commercially available ECL kit (Amersham, Arlington Heights, IL). Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Cell Signaling Technology (Danvers, MA).

9. RNA isolation and RT-PCR

Total RNA was isolated using TRIzol reagent and 2 μg of RNA was used for cDNA synthesis using M-MLV reverse transcriptase (Promega, Madison, Wis.). RT-synthesized cDNA encoding the HO-1 gene was amplified by PCR using specific primers (forward 5'-CGA CAG CAT GTC CCA GGA TT-3 'and reverse 5'-CTG GGT TCT GCT TGT TTC GC-3'Lt; / RTI &gt; The amplified cDNA product was spread on 1% agarose gel electrophoresis and stained with ethidium bromide (EtBr, Sigma-Aldrich). In the same experiment, glyceraldehyde-3-phosphate dehydrogenase, forward 5'-CGG AGT CAA CGG ATT TGG TCG TAT-3 'and reverse 5'-AGC CTT CTC CAT GGT GAA GAC- 3 ') was used as an internal control group.

10. Statistical Analysis

Results were expressed as mean ± SD. Statistical Student's t- test. p <0.05 indicates statistical significance.

[Experimental Example: Identification of Antioxidant Activity of Schizandra chinensis extract]

One. Toxicity to C2C12 cells

The effect of concentration of SSeo on the viability of C2C12 cells was measured and the results are shown in Fig.

As shown in FIG. 1, it was confirmed that the cell culture medium treated with SSeo at a concentration of 10-80 μg / ml for 24 hours was not affected by cell viability. However, it was confirmed that the concentration of 100 μg / ml induces a significant decrease in the number of cells after 24 hours. Therefore, SSeo shows toxicity to cells at the above concentrations. Therefore, the concentration of SSeo at a concentration of less than 80 μg / ml was experimentally reported to optimize the cytoprotective effect of H ₂ O ₂ -induced cell damage.

2.H ₂ O ₂ -Induced effect on inhibition of C2C12 cell growth

The protective effect of SSeo on oxidative stress-induced cytotoxicity was confirmed.

As shown in Fig. 2, cytotoxicity; In the absence of SSeo, cell viability in H ₂ O ₂ - treated cells was significantly reduced to 62.2%. However, the viability of H ₂ O ₂ -treated cells pretreated with SSeo at 80 μg / ml was 82.2%, which was higher than that without SSeo treatment. In addition, H ₂ O ₂ stimulation causes significant morphological changes. This change can be seen to include the presence of a wide range of cytoplasmic liquid saturation and irregular cell-membrane buds, as shown in Figure 2B, which can be effectively reduced by pretreatment of SSeo (Fig. 2B).

3. C2C12 cells H ₂ O ₂ -Induced inhibition of cell death and inhibition of ROS formation

To confirm the potential effects of SSeo on H ₂ O ₂ - apoptosis, cells were stained with annexin V and PI and the percentage of apoptotic cells was determined. Flow cytometric analysis showed that the percentage of apoptotic cells was significantly increased from untreated cells - 3.1% - to 30.3% in cell controls treated with 1 mM H ₂ O ₂ ; However, as shown in FIG. 3, it was confirmed that the rate of cell death increased by pretreatment of SSeo could be remarkably alleviated. Furthermore, in cells incubated with 1 mM H ₂ O ₂ , DAPI staining shows characteristic morphological changes of chromosome condensation, formation of apoptotic bodies, and apoptosis along with nuclei. On the contrary. In the control group, a very small number of apoptotic cells were observed, and the cell lines pretreated with SSeo showed a marked decrease in apoptosis (Fig. 3B).

The elimination effect of SSeo on H ₂ O ₂ -induced ROS was confirmed using the H2DCFDA analysis, and the results are shown in FIG. 3C. Specifically ROS levels H ₂ O ₂ -, but higher than the treated cells, SSeo is H ₂ O ₂ - - in the treated cells, the level of ROS ratio shows that to reduce the ROS produced by the treated cells. As a positive control, the ROS scavenging agent 10 mM N-acetyl-L-cysteine (NAC) also significantly reduced the H ₂ O ₂ -induced ROS production, which resulted in the elimination of H ₂ O ₂ -induced ROS in SSeo .

4. In C2C12 cells, H ₂ O ₂ - Identification of the effect of mediated reduction of DNA damage

H ₂ O ₂ -mediated damage to C2C12 cell DNA was confirmed using alkaline gene damage quantitation assay and western blot analysis as oxidative stress-induced DNA damage generated lesions that caused cell-viability loss , And the results are shown in Fig. 4A.

As shown in FIG. 4A, gene damage quantitative analysis revealed that longer comet tail moments (DNA shift) were increased in H ₂ O ₂ -treated cells, whereas non-treated control cells showed only typical representative nuclei.

In addition, as shown in the Western blot results of FIG. 4B, phosphorylation of the nuclear marker histone H2A.X (Ser139) (p-gamma H2A.X) - DNA for double-strand break (DSB) Levels were increased in H ₂ O ₂ -treated cells. However, when SSeo pretreatment was performed, expression of p-γH2A.X and comet tail were significantly decreased. These results show that SSeo has a protective effect against DNA damage induced by H ₂ O ₂ treatment.

5. Effect of inducing HO-1 expression in C2C12 cells

Since HO-1 plays an important role in the protection of cells against oxidative stress, SSeo, which does not induce cytotoxicity, can induce HO-1 protein expression associated with antioxidant activity. SSeo It was confirmed that treatment with C2C12 cells induces HO-1 protein expression in a concentration- and time-dependent manner, and the results are shown in FIGS. 5A and 5B. In addition, increased HO-1 expression was found to be associated with HO-1 mRNA levels in a concentration- and time-dependent manner in SSeo treated cells, and the results are shown in Figures 5C and D. These results indicate that increased HO-1 expression at the transcription level contributes to increased HO-1 protein expression.

6. Effect on Nrf2 levels in C2C12 cells

Many studies have shown that Nrf2 is an important transcription factor for the control of phase II detoxifying enzymes containing a large number of antioxidants and HO-1. Therefore, it is necessary to confirm whether SSeo can induce Nrf2 expression in C2C12 cells And the results are shown in Fig.

As shown in Fig. 6, C2C12 cells exposed to SSeo showed a gradual increase in the level of Nrf2 strongly associated with the increase in HO-1 expression in concentration- and time-dependent conditions. However, antioxidative activities such as the levels of other Nrf2 target genes, antioxidant enzyme thioredoxin reductase-1, TrxR1, and NADPH-quinone oxidoreductase 1 (NQO-1) Enzymes were maintained unchanged in SSeo treated C2C12 cells.

7. H ₂ O ₂  Of HO-1 in C2C12 cell injury induced by treatment

To investigate whether the protective effect of SSeo on oxidative stress induction in H ₂ O ₂ -treated C2C12 cells is related to the HO-1 protein, ZnPP, a selective inhibitor of HO-1, was applied. 7.

As shown in Figure 7A, ZnPP H ₂ O ₂ - it was markedly reversed the inhibition by ROS generated from the magnetic pole SSeo C2C12 cells. Also, as shown in FIG. 7B, the protective effect of SSeo on H ₂ O ₂ -induced cytotoxicity was blocked by the added ZnPP as a result of MTT analysis. These results indicate that the cytoprotective effect of SSeo is partially mediated by HO-1 induction.

[Review]

Under normal conditions, cells are aerobic organisms with a stable level of ROS, which are balanced by biochemical constant substances. However, excessive ROS production and / or antioxidant consumption under pathological conditions causes oxidative stress with mediated damage directly or indirectly to nucleic acids, proteins, and lipids. Despite cell and tissue defense systems, including various antioxidant enzymes and antioxidants for ROS, these defense systems are overwhelmed in the face of high ROS levels. ROS-induced oxidative stress may ultimately kill cells by necrosis and / or apoptosis through overlapping signal cues and overlapping cascades. Cell death or prospective cell death is a complex step characterized by cell shrinkage, chromosome condensation, DNA fragmentation between nucleosomes and necrosis formation. In addition, the addition of intrinsic H ₂ O ₂ requires a different concentration depending on the time of the cell, but is sufficient to induce a cell death process. Inhibition of oxidative stress is an effective method of managing diseases associated with ROS-induced cell damage and apoptosis. For this reason, natural antioxidants are being sought, which have stable, low cytotoxic and effective pharmacological activity.

In the present invention, a pharmaceutical anti-oxidant was found from traditional medicinal sources, from which it was obtained an extract of Schizandra chinensis and confirmed the protective effect of the essential oil on oxidative stress-induced cytotoxicity. H ₂ O ₂ was selected as an oxidizing agent in C2C12 myoblasts because of its fast permeability and high diffusion ability of H ₂ O ₂ . Mechanisms by which H ₂ O ₂ causes changes in the cell structure and regulates the fate of cells are well known.

In the present invention, exposure of H ₂ O ₂ to C2C12 cells significantly reduces cell viability and cell death, whereas H ₂ O ₂ -induced H ₂ O ₂ -induced C2C12 cells in the case of Omija seed extract essential oil Inhibited cell death, increased viability, and decreased ROS production (FIGS. 2 and 3). In addition, H ₂ O ₂ treatment increases the expression of p-γH2A.X, increases the length of the tail in the DNA of the damaged gene, and prevents the cell death and ROS-mediated DNA damage through reduction of oxidative stress .

Among the various antioxidant enzymes, the protective function of HO-1 is attracting attention as the first of heme degradation to oxidative stress and the inducible isotype of the rate-limiting enzyme. Traditional regulation of the HO-1 gene is associated with Nrf2 transcription factors, which play an important role in cell defense. Nrf2 is normally expressed at low levels in the cytoplasm and binds to the protein Kelch-like ECH-associated protein-1 (Keap1), a special molecular sensor for immediate intracellular homeostatic changes. Nrf2 on oxidative stress is released in Keap1 as a response and carries a nuclear stress signal for the activation of a variety of stress-responsive proteins, including HO-1, as well as a group of genes encoding phase II detoxifying enzymes. Therefore, the present invention confirmed the important role of the Nrf2 / HO-1 pathway in H ₂ O ₂ -induced C2C12 cell injury and oil-mediated cytotoxicity of Schizandra chinensis extract.

In the present invention, it was confirmed that HO-1 can be induced by Omija seed extract essential oil, SSe-induced HO-1 protein expression is increased under the concentration- and time-dependent conditions, and Nrf2 expression is increased incidentally (Figure 6).

Therefore, the induction of HO-1 by the above-mentioned Omija seed extraction essential oil is useful for preventing the H ₂ O ₂ -induced oxidative damage of the C2C12 cells, and the induction of HO-1 by the ozone- It is possible to induce the protection of C2C12 cells against H ₂ O ₂ -induced oxidative stress of cells, and thus it can be usefully used as antioxidant foods and pharmaceutical compositions.

In addition, the above-mentioned Omiza seed extract may be more advantageous for protecting C2C12 cells against H ₂ O ₂ -induced oxidative stress of C2C12 cells compared to Omija seed extract without pretreatment such as fermentation.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.

Claims (7)

The present invention relates to a method for producing an omega-
Which exhibit antioxidant activity
Composition for antioxidant activity containing Omija seed extract. The method according to claim 1,
Wherein the Omija seed extract is an Essential oil of Omija seed extract. 3. The method of claim 2,
Wherein said composition comprises said Omija seed extract essential oil at a concentration of from 0.01 [mu] g / ml to less than 100 [mu] g / ml.
Composition for antioxidant activity containing Omija seed extract. The method of claim 1,
The Omija seed extract is prepared by inoculating at least one microorganism selected from the group consisting of Bacillus lichenifomis, Aspergillus oryzae, and Lactobacillus plantarum in Omija One
Composition for antioxidant activity containing Omija seed extract. A composition for antioxidative activity comprising the extract of Omija seed according to any one of claims 1 to 3
A health food composition for improving antioxidant activity. Drying omiza at 150 to 210 캜 to produce dried omija;
Crushing the dried omiza to produce an omija powder;
A lyophilizing step of lyophilizing the omitha powder to prepare a lyophilized omija powder;
An extraction step of extracting the lyophilized Schizandra chinensis powder with an alcohol or alcohol aqueous solution, and
And distilling the extract at 150 ° C to 230 ° C for 1 hour to 5 hours.
A method for preparing an essential oil for the extraction of. The method according to claim 6,
Between the sham-drying step and the extraction step
The microorganism selected from the group consisting of Bacillus lichenifomis, Aspergillus oryzae, and Lactobacillus plantarum is inoculated to the dried omija which has been subjected to the above-mentioned step of drying omiza And further comprising a step of culturing Omija
A method for preparing an essential oil for the extraction of.

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