KR20160013410A - prevent or improvement COMPOSITION INCLUDING Schisandra chinensis semen Extracts For vascular disease And Manufacturing Method thereof - Google Patents

Abstract

The present invention relates to a composition comprising a seed extract of Schizandra chinensis for treating or alleviating vascular diseases, and to a manufacturing method thereof. The composition comprising a seed extract of Schizandra chinensis of the present invention is capable of preventing the damage in arteries from being developed as atherosclerosis by inhibiting the movement of MMP and effectively inhibiting smooth muscle cells from producing ROS substances, NO, and PEG2. In addition, the composition has antioxidant activities, is capable of inhibiting an inflammatory medium and an inflammatory reaction, and is capable of alleviating or preventing the progress of vascular diseases therethrough. Therefore, the composition can be efficiently used for treating and preventing vascular-related diseases.

Description

[0001] The present invention relates to a composition for preventing or ameliorating vascular diseases, which comprises an extract of Schizandra chinensis, and a method for preparing the same,

The present invention relates to a composition for preventing or ameliorating atherosclerosis, which comprises an extract of Omija seed, and a method for producing the same. More particularly, the present invention relates to a composition for preventing or ameliorating arteriosclerosis which is capable of inhibiting the progress of vascular disease by inhibiting MMP-9 activity, and improving, preventing or ameliorating arteriosclerosis.

Migration and proliferation of vascular smooth muscle cells (VSMC) from the vascular mesenchyme to the endothelium and subendothelial regio is a gradual pathological disorder often resulting in cardiovascular and cerebrovascular diseases and plays an important role in the progression of atherosclerosis . In the early stages of atherosclerosis or arterial wall injury, VSMCs migrate to the lining of the arterial wall and thicken the lining (Rudijanto, 2007; Hopkins, 2013). The activity of matrix metalloproteinases (MMPs) causes the development of atherosclerosis by allowing VSMC to migrate through remodeling or degradation of the extracellular matrix (ECM) surrounding the cells (Kuzuya and Iguchi, 2003, Newby, 2012; Park et al., 2013).

Among the MMPs, the gelatinase MMP-9 is not particularly good for the development of arterial atherosclerosis by controlling both migration and proliferation of VSMCs in the onset of atherosclerosis (Mason et al., 1999; Esteve et al., 2002; Sprague and Khalil, 2009). VSMC-induced cytokine tumor necrosis factor (TNF) -α induces a marked proliferation and migration of VSMCs (Doevendans and van Eys, 2002; Sprague and Khalil, 2009). Synthesis and secretion of MMP-9 in VSMC is basically low, but MMP-2 is not, but is expressed by a variety of stimuli including growth factors and cytokines such as TNF-α, transcription factor nuclear factor- (Bond et al., 2001; Knipp et al., 2004; Cheng et al., 2009; Butoi et al., 2011). NF-κB is generally present in the cytoplasm and interacts with IκB (inhibitor of NF-κB) in the inactive state. NF-κB dimers are cleaved from IκB and migrate to the nucleus by inflammatory or other stimuli that cause degradation of the IκB protein. In the nucleus, NF-kB binds to the promoter region and promotes the expression of various genes including inflammatory factors, adhesion molecules, and MMP (Palanki, 2002; Simmonds and Foxwell, 2008).

The relative contribution of reactive oxygen species (ROS) and inflammatory mediators in the vasculature remains to be seen. They integrate cell signaling associated with VSMC proliferation and migration associated with atherosclerosis. ROS quenching by antioxidant enzymes in a general pathological state is effective in maintaining recovery of antioxidant / pre-oxidative equilibrium after exposure to oxidation (Stocker and Keaney, 2004; Yung et al., 2006; Gauss et al ., 2007). However, when the ROS are overproduced, the internal antioxidant and oxidative stresses are caused by abnormal body reactions, damaging lipids, proteins and DNA.

In addition, inflammatory factors also play an important role in atherosclerosis (Sullivan et al., 2000; Touyz and Schiffrin, 2004; Aggarwal et al., 2013). Oxidative stress may also activate or increase the expression of redox-sensitive genes including pro-inflammatory factors and MMPs via the NF-κB signaling system (Siwik and Colucci, 2004; Gonzalez et al. 2011). Therefore, anti-inflammatory and oxidizing agents are believed to be able to respond to over-production of inflammatory mediators and ROS and to reduce arterial disease.

Essential oil is a hydrophobic solution that is concentrated in volatile aroma compounds and has antioxidant, antibacterial and bactericidal effects (Oussalah et al., 2004; Fratianni et al., 2010). Essential oils from various plants have beneficial effects on smooth muscle disease (Cavanagh and Wilkinson, 2002; Magalhaes et al., 2003; Coelho-de-Souza et al., 2005; Koto et al. 2007; Pinho-da-Silva et al., 2010; Karim et al., 2010; de Sousa et al., 2010; Pereira et al., 2013).

Schizandrae fructus, Schizandra chinensis (Turcz.) Baillon) is a medicinal plant and has been widely used in Korea, Japan, Taiwan, China and Russia for the treatment of various inflammatory diseases, immune diseases, cardiovascular diseases, blood sugar and hypertension Panossian and Wikman, 2008; Lu and Chen, 2009). However, the molecular scale mechanism for potential anti-atherosclerosis in seed-borne extracts is unknown.

Therefore, the present inventors have confirmed that the essential oil of Omija seed has an anti-atherosclerotic effect, thus completing the present invention.

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 pharmaceutical composition for inhibiting the progress of vascular disease by inhibiting MMP-9 activity and for improving, preventing or treating.

Another object of the present invention is to provide a method for producing the above-mentioned Omija seed extract.

In order to achieve the above object, there is provided a pharmaceutical composition for improving or treating vascular diseases, comprising the extract of Omija seed of the present invention as an active ingredient.

The vascular disease may be any one selected from the group consisting of ischemic heart disease, coronary artery disease, angina pectoris, stroke, arteriosclerosis, myocardial infarction, and combinations thereof.

The vascular disease may be arteriosclerosis.

The Omija seed extract may be an essential oil extracted from Omija seeds.

The composition may contain the above-mentioned Omija seed extract essential oil at 0.1 ng / ml to 30 ng / 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 Lt; / RTI >

The present invention also relates to a food composition for improving or preventing vascular diseases comprising an extract of Omija seed as an active ingredient.

The present invention also relates to a method for producing omija, which comprises drying omija at 150 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 an aqueous alcohol solution and a step of distilling the extract at 150 to 230 DEG C for 1 to 5 hours to prepare an essential oil extract of Schizandra chinensis for improving or preventing vascular diseases .

Bacillus lichenifomis, Aspergillus oryzae, and Lactobacillus plantarum are added to the dried omija, which has been subjected to the above-mentioned Omija-drying step, And further comprising a step of inoculating the at least one selected microorganism and culturing the microorganism.

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 pharmaceutical composition for improving or treating vascular diseases and the food composition for improving or preventing vascular diseases according to the present invention include an extract of Omija seed as an active ingredient.

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 essential oil extracted from Omiza extract, there is no influence on the cytotoxicity of common Omiza extract (see FIG. 1 below) have. In particular, it has been confirmed in the present invention that when used as an essential oil, it has remarkably excellent treatment and improvement effects on vascular diseases which could not be expected in general extracts or concentrates, by an effective ingredient not revealed in the extract.

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 activity of the active ingredient having an improving effect on the vascular diseases as well as the seed extract or the essential oil as well as the common gum can be very high.

In particular, the above-mentioned Omija seed extract essential oil has an effect of inhibiting TNF-α-induced MMP-9 activity and smooth muscle cell (HASMC) cell infiltration, effectively inhibiting the nuclear displacement of NF-κB and effectively inhibiting intracellular ROS and inflammatory mediators NO < / RTI > and PGE2. In particular, smooth muscle cell migration is an important symptom of progression of arteriosclerosis, and it can be effectively prevented, and vascular diseases, especially atherosclerosis, can be effectively prevented, prevented or treated.

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 component that inhibits vascular smooth muscle cell migration contained in the omiza can be effectively extracted.

The vascular disease includes all diseases caused by weakening and hardening of blood vessels and preferably relates to diseases caused by migration of vascular smooth muscle cells (VSMC). More preferably, it may be any one selected from the group consisting of ischemic heart disease, coronary artery disease, angina pectoris, stroke, atherosclerosis, myocardial infarction, and combinations thereof, and most preferably atherosclerosis.

In the case of the above-mentioned Omija seed extract, it is possible to prevent the development of arteriosclerosis from arteriosclerosis by effectively inhibiting the movement of MMP and effectively inhibiting smooth muscle cells from producing ROS substance, NO and PEG2, and effectively treating arteriosclerosis , ≪ / RTI >

The composition may contain the essential oil of Omija seed extract in an amount of 0.1 ng / ml to 30 ng / ml, preferably 9 ng / ml to 25 ng / ml. When the essential oil is contained in the above-mentioned concentration range, it is possible to effectively treat, ameliorate or prevent atherosclerosis without damaging the cytotoxic effect of the active ingredient.

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 Lt; / RTI >

In accordance with the microbial inoculation, the active ingredient having the effect of improving the vascular disease is increased by the fermentation of the microorganism, and the effect of improving the vascular disease is greatly enhanced. In particular, the effect of inhibiting cell infiltration in smooth muscle cells (HASMC) So that the effect of preventing or treating arteriosclerosis can be very excellent. Preferably, the microorganism inoculation is most effective in inhibiting cell infiltration in the smooth muscle cells (HASMC) when fermented by inoculation on germinated seeds of Omija.

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 inoculation exceeding 0.05% (w / v) There is a problem that the effect of improving the vascular disease is deteriorated because the activity of the active ingredient having the activity is 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 can obtain the most effective ingredient having the activity of inhibiting cell infiltration contained in the Omiza extract. When the microorganism is used in the above range, May be most effective in preventing or improving vascular diseases.

Preferably, the fermentation is performed for 1 to 7 days. When the fermentation is performed for less than 1 day, the increase effect of the active ingredient having the activity of inhibiting cell infiltration due to the microbial activity is insignificant, and when the fermentation progresses for 7 days or more, the content of the effective substance is rather decreased, There is a problem that the effect of preventing or improving the disease may be lowered.

More preferably, the dried Schizandra chinensis may be one which after drying the Schizosaccharomyces sp. When the cooked omija seeds are used or when the cooked omija is fermented by using the microorganism, the active substance inhibiting cell infiltration contained in the omija extract is greatly increased, and the prevention and improvement effect on vascular diseases can be 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. When the seeds of Omija are dried in the above-mentioned temperature range, the most effective ingredient is contained, which is effective in preventing, ameliorating or treating vascular diseases, especially atherosclerosis.

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 component that inhibits vascular smooth muscle cell migration 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. In the case of distillation in the above range, it is effective to inhibit the movement of MMP in the essential oil of the present invention and to effectively prevent the smooth muscle cells from being developed into atherosclerosis from arterial injury by effectively inhibiting production of ROS substance, NO and PEG2 The composition can be effectively contained.

The method for producing an extract of Omija seed extract is characterized in that Bacillus lichenifomis, Aspergillus oryzae and Lactobacillus spp. Are added to the dried Omija after the above- And Lactobacillus plantarum. The method of the present invention may further include a step of fermenting Omija to inoculate and cultivate at least one microorganism selected from the group consisting of Lactobacillus plantarum.

In this case, the above-mentioned Omija seed extract essential oil may contain more active ingredient having an effect for preventing or improving vascular disease, so that the prevention or improvement effect on the blood and diseases may be higher.

The composition containing the extract of Omija seed extract of the present invention inhibits the migration of MMP and effectively prevents the smooth muscle cells from producing ROS substance, NO and PEG2, thereby preventing development from arteriosclerosis to atherosclerosis, Can inhibit the mediator and inflammatory response, have an antioxidative activity, and can thereby improve or prevent the progression of the vascular disease.

FIG. 1 is a graph for confirming the cell viability of smooth muscle cells (HASMC) against essential oil extracted from Omija seeds. Each figure represents the mean ± SD of three independent experimental results.
FIG. 2 is a graph showing the degree of inhibition of TNF-α-induced MMP-9 activity by Omija seed extract essential oil in smooth muscle cells (HASMC). The results were obtained by calculating the mean ± SD in three independent experiments and the results were expressed as relative values for the untreated control (*, p <0.05 vs. untreated control; #, p <0.05 vs. TNF -α-treated HASMC).
FIG. 3 is a graph showing the inhibition of TNF-α-induced MMP-9 mRNA and protein expression by Omija seed extract essential oil in smooth muscle cells (HASMC). GAPDH and actin were used as respective internal controls for RT-PCR and Western blot analysis.
4 is a graph showing the inhibitory effect of NF-κB nuclear displacement on the TNF-α-stimulated smooth muscle cells (HASMC) by Omija seed extract essential oil
Figure 5 shows the effect of the extract of Omija seed extract on TNF-α-induced HASMC migration. (* p <0.05 vs. untreated control; # p <0.05 vs. TNF-α-treated HASMC).
FIG. 6 is a graph for confirming the effect of the extract of Omija seed extract on TNF-α-induced intracellular ROS formation in smooth muscle cells (HASMC).
7 is a graph showing the inhibitory effect of NO and PGE2 of Schizosaccharomyces essential oil extract (SSeo) in TNF-? -Treated smooth muscle cells (HASMC). * Indicates a significant difference for the TNF-α-treated group at p <0.05.
FIG. 8 shows inhibitory effects of iNOS and COX-2 on TNF-α-treated smooth muscle cells (HASMC). SSeo refers to the oil extracted from Omija seeds. GAPDH and actin were used as an internal control for RT-PCR and Western blot analysis.

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]

One. Production 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 is blocked in light 4? It was stored in the refrigerator and used for the experiment. The oil yield was 0.66% based on the dry weight of the Omija seeds.

2. Cell culture

Human aortic smooth muscle cells (HASMC) were purchased from Bio-Whittaker (Walkersville, MD, USA). The cells were cultured in SMC growth medium-2 (Gibco-BRL, Grand Island, NY, USA) at 37 ° C, 5% CO ₂ and 95% The culture medium contained 10% fetal bovine serum (FBS), 2 ng / ml human basic fibroblast growth factor, 0.5 ng / ml human epidermal growth factor, 50 μg / ml gentamicin, 50 μg / ml amphotericin- insulin.

3. Gelatin zymography

The gel permeation activity of MMP-2 and MMP-9 in the conditioned culture medium was determined by electrophoresis analysis at 4 ° C with 10% polyacrylamide gel (containing 1 mg / mL gelatin). After electrophoresis, the gel was washed for 1 hour in 2.5% Triton X-100, the active buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl 2, and 0.02% NaN ₃₎ at 37 ℃, And incubated for 24 hours. After staining with coomassie blue R-250 (10% glacial acetic acid, 30% methanol, and 1.5% Coomassie Brilliant Blue; Invitrogen Co., USA) for 2 hours, the gel was again dissolved in 10% glacial acetic acid glacial acetic acid) and 30% methanol for 1 hour. White lysis zones indicate that the gelatin is degraded, which was confirmed by staining with Coomassie brilliant blue (Lee et al., 2013).

4. RNA isolation and RT-PCR

In RT-PCR, all RNA was isolated using TRIzol reagent (Invitrogen Co.). 2 μg of RNA was used for cDNA synthesis using M-MLV reverse transcriptase (Promega, Madison, WI, USA). (RT) that encodes MMP-2, MMP-9, Tissue inhibitors of metalloproteinase (TIMP) -1, TIMP-2, inducible nitric oxide synthase (iNOS) and cyclooxygenase- -generated cDNA was amplified by PCR using specific primers. The primers were purchased from Bioneer (Seoul, Republic of Korea).

The PCR primers are as follows:

MMP-9 (5'-CGG AGC ACG GAG ACG GGT AT-3 'and 5'-TGA AGG GGA AGA CGC ACA GC-3')

MMP-2 (5'-CCC CTA TCT ACA CCT ACA CCA AGA AC-3 'and 5'-CCC CTA TCT ACA CCT ACA CCA AGA AC-3')

(5'-CTG TTG TTG CTG TGG CTG ATA-3 'and 5'-CCG TCC ACA AGC AAT GAG T-3').

TIMP-2 (5'-GTA GTG ATC AGG GCC AAA G-3 'and 5'-TTC TCT GTG ACC CAG TCC AT-3')

iNOS (5'-ATG GCT TGC CCC TGG AAG TTT CTC-3 'and 5'-CCT CTG ATG GTG CCA TCG GGC ATC TG-3')

COX-2 (5'-TTC ACC AGA CAG ATT GCT GGC-3 'and 5'-AGT CTG GAG TGG GAG GCA CTT G-3')

After amplification, the PCR reaction was electrophoresed on 1% agarose gel and visualized by staining with ethidium bromide (EtBr, Sigma-Aldrich). In the experiment, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was 5'-GAC CTG ACC TGC CGT CTA-3 'and 5'-AGG AGT GGG TGT CGC TGT-3' Were used as an internal control group.

5. Protein extraction, electrophoresis and western blot analysis

Whole-cell protein extraction from smooth muscle cells (HASMC) was performed using a cell lysis buffer (20 mM sucrose, 1 mM EDTA, 20 mM Tris-HCl, pH 7.2, 1 mM DTT, 10 mM KCl, 1.5 mM MgCl2, aprotinin) for 30 min. Protein extraction was quantitated using Bio-Rad kit (Pierce Biotechnology, Rockford, IL, USA).

For Western blot analysis, the soluble protein was digested with SDS (sodium dodecyl sulfate) - polyacrylamide gel electrophoresis and transferred to nitrocellulose transfer membranes (Schleicher & Schuell, Keene, NH, USA) Respectively. Specific proteins were detected with enhanced chemiluminescence (ECL) kits (Amersham Co., Arlington Heights, IL, USA). Cells were also washed and collected with cold phosphate-buffered saline (PBS). The cytoplasmic and nuclear proteins were then prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology). Antibodies to MMP-2, MMP-9, TIMP-1, TIMP-2, iNOS, COX-2, NF-κB p65, IκBα, nucleolin, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, Respectively. Peroxidase-labeled donkey anti-rabbit immunoglobulin and peroxidase-labeled sheep anti-mouse immunoglobulin were purchased from Amersham Co.

6. Immunofluorescence staining.

Smooth muscle cells (HASMC) were cultured on a cover glass directly on a 6-well plate for 24 hours in order to confirm the NF-κB p65 site by immunofluorescence using fluorescence microscopy. h to detect localization by immunofluorescence assay using a. After stimulation with TNF-α, the cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, and incubated for 10 min at 20 ° C. And permeated using 100% methanol. Polyclonal antibodies against anti-NF-κB p65 were incubated for 1 hour with fluorescein isothiocyanate (FITC) -conjugated donkey anti-rabbit IgG (Santa Cruz Biotechnology) for 1 hour. After washing with PBS, the nuclei were stained with 4,6-diamidino-2-phenyllindile (DAPI, Sigma-Aldrich) and fluorescence was observed using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

7. Statistical Analysis

Results were expressed as mean ± standard deviation (SD), and one - way ANOVA was used to compare various time and concentration. When statistical significance was shown by ANOVA, Duncan (multiple range test) was used to determine significant differences. p <0.05 means statistical significance.

[Experimental Example: Identification of anti-arteriosclerotic effect of Omija seed extract]

1. Cytotoxicity experiment

TNF-α and cytotoxic effects of HASMC in omija seed extract essential oil were confirmed by 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) assay. Specifically, the cells were plated at a density of 5 × 10 ³ cells / well in a 96-well culture dish and allowed to adhere for 24 hours. The cells were treated with ozone-seed extract essential oil, which had not been treated with Omija seed extract, and 100 ng / mL TNF-a, respectively. After incubation for 24 hours, MTT solution (0.5 mg / ml, Sigma-Aldrich Chemical Co., St. Louis, Mo., USA) was added to each well containing each conditioned medium, Respectively. The medium was then removed and dimethyl sulfoxide (DMSO) was added to each well. After shaking, the absorbance of soluble blue formazan was measured using a microplate reader (Dynatech MR-7000; Dynatech Laboratories, Chantilly, VA, USA) at 540 nm and the results were compared to the 100% viability of untreated cells The relative values are shown in Fig.

As shown in Fig. 1, each concentration of Omija seed extract essential oil to inhibit MMP-9 activity in smooth muscle cells (HASMC) did not cause significant changes in cell viability after treatment. These results indicate that inhibition of MMP-9 activity in TNF-α-stimulated smooth muscle cells (HASMC) is not due to the toxic activity of Omija seed extract essential oil.

2. In vitro MMP activity assay

The MMP activity in the supernatant was assayed using the MMP gelatinase activity assay kit (Chemicon International Inc., USA) according to the manufacturer's instructions. Specifically, aliquots of the culture medium were incubated with a biotin-attached gelatinase substrate, and the substrate was for dividing active MMP-2 and MMP-9 in the culture medium. The fragments were then added to a biotin-conjugated 96-well plate and incubated for 30 minutes at 37 ° C, allowing biotin-containing fragments to bind to the plate during digestion. The digested, unfrozen fragments were removed by washing, bound to the plate, and the biotin-labeled gelatinase digestion was detected by the addition of streptavidin-enzyme complex. The results were confirmed by measuring the wavelength at 540 nm in a microplate reader.

As shown in Fig. 2A, secretion of MMP-9 is increased when treated with TNF-a, even though MMP-9 has very weak activity in regulatory medium and MMP-2 has high secretion level. However, it was confirmed that there was no effect on the MMP-2 secretion level. However, Omija seed extract essential oil significantly reduces TNF-α-induced MMP-9 secretion in a dose-dependent manner. In addition, the MMP-9 substrate degradation activity assay was performed using the MMP gelatinase assay kit except for MMP-2 (FIG. 2B).

3. Confirm the effect of omija seed extract essential oil on the expression level of MMP-9

RT-PCR and Western blot analysis (Western blot analysis) were performed to confirm the effect of the extract of Omija seed extract on the expression level of MMP-9 in TNF-? -Treated smooth muscle cells (HASMC) Respectively.

As shown in Figure 3, Omija seed extraction essential oil reduces TNF- [alpha] -induced MMP-9 expression at both transcription and translation levels in a concentration-dependent manner. However, it does not affect the level of MMP-2. Next, levels of TIMP-1 and -2 were identified to confirm the effect of the extract of Omija seed extract on MMP-related endogenous inhibitors. As shown in FIG. 3, the levels of TIMP-1 and -2 mRNA and protein were not significantly changed in smooth muscle cells (HASMC) treated or not treated with TNF-α and Omija seed extract essential oils. These results indicate that inhibition of TNF-α-induced MMP-9 activity by inhibiting MMP-9 transcription levels in smooth muscle cells (HASMC) is not associated with TIMP expression.

4. Inhibition of nuclear translocation of TNF-α-induced NF-κB in smooth muscle cells (HASMC)

Nuclear translocation of NF-κB was measured in order to determine whether the inhibitory effect of Omija seed extract essential oil on TNF-α-induced MMP-9 activity was inhibited by NF-κB signaling.

Western blot analysis using cytoplasmic and nuclear fractions showed that TNF-α treatment, incidentally, enhanced the accumulation of NF-κB protein with degradation of IκB-α in the cytoplasm. However, it was confirmed that NF-κB nuclear accumulation and TNF-α-induced IκB-α degradation were significantly inhibited in smooth muscle cells (HASMC) pretreated with Omija seed extract essential oil prior to TNF-α stimulation (FIG. Immunofluorescence The nucleus accumulation of NF-κB p65 was not inducible in cells treated with Omija seed extract alone but was strongly induced after stimulation with TNF-α. The migration of NF-κB p65 to the nucleus was induced by the extraction of Omija seed extract Lt; RTI ID = 0.0 &gt; (FIG. 4B). &Lt; / RTI &gt; These results demonstrate that the inhibitory effect of Omija seed extract essential oil on the expression of TNF-α-induced MMP-9 is related to the activity of NF-κB by preventing IκB-α degradation.

5. Cell invasion assay Cell invasion assay

Matrigel infiltration assay was performed to confirm the inhibitory effect of Omija seed extract essential oil on the invasion force of smooth muscle cells (HASMC). To determine the invasion rate, smooth muscle cells (HASMC) were counted through the Matrigel and moved to the back of the Transwell membrane.

Cell migration analysis was performed using the Transwell system (Corning Costar, Cambridge, Mass., USA). Smooth muscle cells (HASMC) were resuspended in 100 μL of medium and placed on top of the Transwell plate. Cells were incubated for 8 hours and fixed with methanol. The cells were then stained with haematoxylin for 10 min and eosin Y (Sigma-Aldrich). Smooth muscle cells (HASMC) on the upper surface of the filter were removed with tissue, and the transferred cells were counted in three fields of each triple filter, the results of which are shown in FIG.

As shown in Figure 5, treatment with TNF-a significantly increased HASMC infiltration, but reduced the infiltration of cells compared with the control group in the treatment group treated with Omija seed extract essential oil. In the case of HASMC pretreated with Omija seed extract essential oil, the remarkable reduction of cellular infiltration compared to the control group indicates that MMP-9 inhibition plays a central role in the inhibition of melittin on TNF-α-induced HASMC migration .

6. Inhibition of TNF-α-induced ROS formation in Omija seed extract essential oil

The level of intracellular ROS production was measured to determine if the essential oil extract of Omija seeds could reduce the levels of TNF-α-induced oxidative stress in smooth muscle cells (HASMC).

Intracellular accumulation of ROS was confirmed using fluorescent probe 2 ', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA, Sigma-Aldrich). Smooth muscle cells (HASMC) were pretreated with 10 mM nacetylcysteine (NAC), ROS scavenger, or Omija seed extract oil for 30 min and then treated with TNF-α (100 ng / ml) for 30 min. To measure intracellular ROS, cells were incubated in PBS (20 mM H2DCFDA) at 37 ° C for 4 hours to label intracellular ROS. ROS production was measured using a flow cytometer (FACS Calibur; Becton Dickinson, San Jose, CA, USA) and Cell-Quest pro software (Jeong et al., 2012). The results are shown in FIG.

As shown in FIG. 6, TNF-.alpha. Significantly increases ROS production but significantly reduces ROS production of TNF-.alpha. -Induced cells in a concentration-dependent manner when pretreated with Omija seed extract essential oil. These results indicate that the extract of Omija seed extract can inhibit the increased level of ROS in TNF-α-treated smooth muscle cells (HASMC). For treatments treated with Omija seed extract essential oil, the ROS level can also be lowered compared to non-treatments.

7. Identification of iNOS and COX-2 expression and effects on nitrite and PEG2 production

Omija seed extract essential oil weakens TNF-α-induced inflammatory response in smooth muscle cells (HASMC), the activity of NF-κB induces the expression of pro-inflammatory mediators (Palanki, 2002; Simmonds and Foxwell, 2008) , The inhibitory effect of the extract of Omija seed extract on the TNF-α-induced inflammatory response was confirmed.

Smooth muscle cells (HASMC) pretreated or treated with Omija essential oil were stimulated with TNF-α for 24 hours and treated with smooth muscle cells (HASMC) treated with TNF-α alone to induce pro-inflammatory The median value was measured

The concentration of nitric oxide (NO) in the culture supernatant was determined by measuring the stable oxidation product of NO using Griess reagent (Sigma-Aldrich). The supernatant of the cell culture was collected, mixed with the same volume of Griess reagent, and incubated at room temperature for 10 minutes. Standard curves were prepared using NaNO ₂ and nitrite production was measured using optical density at 550 nm (Kyung et al., 2012). The results are shown in Fig. 7A).

In order to confirm the level of PGE2, a partial sample of the culture supernatant was collected and the concentration of PGE2 (pg / ml) was determined based on the concentration of the standard solution using a PGE2 enzyme-linked immunosorbent assay (ELISA) ml) were measured (Cayman Chemical Co., Ann Arbor, MI, USA). The results are shown in Fig. 7B.

The expression of iNOS and COX-2 mRNA and protein was also confirmed by RT-PCR and Western blotting. RT-PC was incubated for 1 hour with Omija seed extract essential oil at each concentration of R and incubated with TNF-α (100 ng / ml) for 6 hours. All RNAs were prepared for RT-PCR analysis of iNOS and COX-2 genes, 1% agarose gel electrophoresis, and visualized by EtBr staining. The results are shown in FIG. 8A.

Western blot was treated with TNF- [alpha] for 24 hours, cell lysates were prepared, and western blotting was performed using anti-iNOS and anti-COX-2 antibody and ECL detection system, Respectively.

As shown in Fig. 7, in the pretreatment with Omija seed extract essential oil, TNF-a function for the pro-inflammatory mediator release was lost. It was confirmed that Omija seed extract essential oil inhibited protein expression compared to TNF-α-induced iNOS and COX-2 mRNA and control (FIG. 8). These results imply that the expression of pro-inflammatory enzymes at the transcriptional level is reduced and contributes to the inhibition of the TNF-α-induced NO and PGE2 production by the omija seed extraction essential oil.

[conclusion]

The effects of TNF-α-induced MMP-9 activity and smooth muscle cells (HASMC) on cell infiltration of Omija seed extract essential oil were examined. These results demonstrate that Omija seed extract essential oil effectively inhibits the expression and secretion of MMP-9 and nuclear translocation of NF-κB in TNF-α-stimulated smooth muscle cells (HASMC). It was also confirmed that the extract of Omija seed extract can inhibit the release of TNF-α-induced intracellular ROS and the inflammatory mediators NO and PGE2.

Many studies have shown that the migration of vascular smooth muscle cells from the vascular interhemium to the subendothelial area is an important symptom in the progression of atherosclerosis following arterial injury (Rudijanto, 2007; Hopkins, 2013). Recent studies have shown that increasing the expression of MMP and degrading all types of ECM proteins in atherosclerotic lesions weakens vessel walls. These processes will also be stimulated by cytokines and inflammatory mediators, as well as cell-cell contact signals. This observation suggests that therapeutic agents targeted at inhibiting the migration of MMP-9 and VSMC would be useful in preventing atherosclerotic lesion progression. Therefore, the present invention confirmed the effect of the extract of Omija seed extract on the activity and expression of TNF-? -Induced MMP-9, and the result showed that the transcriptional activity of MMP-9 gene in smooth muscle cells (HASMC) (Figs. 1 and 3). In general, the activity of MMPs is tightly regulated by endogenous systems of transcriptional activity, complex proteolytic cascades and TIMPs. TIMPs inhibit MMPs by forming stoichiometric complexes to control matrix turnover (Lambert et al., 2004). However, the transcription and translocation of TIMP-1 and -2 were confirmed to be unchanged in the HASMC treated with Omija seed extract essential oil and TNF-α or TNF-α alone (FIG. 3). Omija seed extract essential oil does not inhibit the expression of TIMP-1 and -2 but suppresses the expression of MMP-9 by lowering the transcription of the gene.

Many studies have shown that signaling mechanisms underlying transcription factor regulation are associated with modulation of MMP-9 expression. Therefore, we define the role of NF-κB transcription factors in the down-regulated activation of MMP-9 by the extracts of Omija seed extract in TNF-α-stimulated smooth muscle cells (HASMC) The majority of intracellular NF-κB p65 is found in the cytoplasm of the nucleus and in the cytoplasm of the nucleus, . NF-κB p65 accumulation and staining in the nucleus (Fig. 4). However, the level of NF-κB p65 in the nucleus was markedly reduced by pretreatment with Omija seed extract essential oil, and the TNF-α-induced IκB-α degradation was also significantly reversed by Omija seed extract essential oil, Indicating that extractive essential oil inhibits IκB activity and inhibits NF-κB activity by inhibiting nuclear translocation of NF-κB. Furthermore, the results obtained by the Matrigel migration assay show that the essential oil extracted from Omija seed significantly inhibits the potential migration of TNF-α-induced smooth muscle cells (HASMC) (FIG. 5 ). These results demonstrate that Omija seed extract essential oil inhibits TNF-α-induced NF-κB nuclear translocation, thereby inhibiting protein expression and activity of MMP-9 and reducing HASMC migration.

Oxidative stress is a condition in which excessive ROS overwhelms the internal oxidation system. Several studies have shown that ROS is associated with the activity of NF-κB, and that inflammatory mediators are also associated with ROS products. In addition, inflammatory mediators that strongly influence the production of atherosclerotic plaques from previous results stimulate VSMC migration from the intima to the media (Stocker and Keaney, 2004; Gauss et al., 2007) . In addition, oxidative stress affects damaged ducts that progress to inflammation. In contrast, inflammation may also increase ROS in atherosclerotic lesions to control cellular responses such as VSMC proliferation and migration (Madamanchi et al., 2005; Yung et al., 2006). This vicious cycle not only causes cardiovascular disease, but also causes myocardial infarction, stroke and heart failure (Manea et al., 2012). Therefore, reducing oxidative stress and the production of inflammatory mediators is important for controlling atherosclerosis. In our experiments, TNF-α may increase intracellular ROS production in ROS smooth muscle cells (HASMC); The pretreatment of Omija seed extract essential oil can prevent the production of TNF-α-stimulated ROS, indicating that the Omija seed extract essential oil can remove the radical (FIG. 6). In addition, the Omija seed extract essential oil effectively inhibited TNF-α-induced NO and PGE2 synthesis (FIG. 7), and this inhibition is consistently associated with downregulation of iNOS and COX-2 expression (FIG. 8) .

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 (9)

Claims: 1. A pharmaceutical composition for treating or ameliorating a vascular disease, comprising an extract of Omija seed as an active ingredient. The method according to claim 1,
Wherein the vascular disease is any one selected from the group consisting of ischemic heart disease, coronary artery disease, angina pectoris, stroke, arteriosclerosis, myocardial infarction, and combinations thereof. The method according to claim 1,
Wherein said vascular disease is atherosclerosis. The method according to claim 1,
Wherein the Omija seed extract is essential oil extracted from Omija seed extract. 5. The method of claim 4,
Wherein said composition comprises said Omija seed extract essential oil at 0.1 ng / ml to 30 ng / ml.
A pharmaceutical composition for improving or treating a vascular disease. 7. The method according to any one of claims 1 to 6,
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 In fact,
A pharmaceutical composition for improving or treating a vascular disease. A food composition for improving or preventing a vascular disease comprising an extract of Omija seed as an active ingredient. Drying omiza at 150 ° C to 210 ° C to produce dried omiza;
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 ° C to 230 ° C for 1 hour to 5 hours
A method for preparing essential oil for extracting omija seeds for improving or preventing vascular diseases. 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 essential oil for extracting omija seeds for improving or preventing vascular diseases.

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