KR101933467B1 - Composition for improving fine dust stimulated respiratory diseases with shitake mushroom and seaweed ear complex extracts - Google Patents

Abstract

The present invention relates to a composition for alleviating respiratory diseases stimulated by fine dust, which contains a complex extract of seaweed ear and shiitake mushroom. The complex extract of sea mustard sporophyll and shiitake mushroom according to the present invention can be usefully used as a health functional food or a medicine for preventing or alleviating respiratory diseases caused by fine dust.

Description

Technical Field [0001] The present invention relates to a composition for improving fine dust-stimulating respiratory diseases, including shrimp mushrooms and seaweed ear complex extracts,

The present invention relates to a composition for improving a micro dust-stimulating respiratory disease, comprising a microsphere and a shiitake combined extract.

In the past decades, Korea has been continuously deteriorating due to rapid industrialization, industrialization and urbanization. In particular, environmental pollution in large cities is serious. Due to environmental policy enhanced regulation contaminants such as supply-up of the use of natural fuel SO _2, CO, floating dust concentration, but is gradually decreased, due to the rapid increase in the car fine dust, ozone, nitrogen oxides, hydrocarbons water pollutant But it is increasing.

In addition, China's coal dependence is more than 70%, so the smog phenomenon continues to increase, and dust, smog and high concentrations of dust from the Mongolia dry area are combined and fine dust is expected to occur and increase.

Fine particles are floating in the atmosphere of highly complex components such as sulfur oxides, nitrogen oxides, lead, ozone, and carbon monoxide, and are mostly known to be generated from automobile exhaust gas and road dust.

The size and composition of fine dust is very complicated and diverse. PM10 is called fine dust with a particle diameter (aerodynamic diameter) of 10㎛ or less. PM2.5 is called super fine dust with a particle diameter of 2.5㎛ or less. ㎛ or less, and it is known that the particle size, surface area, and chemical composition determine the health effect.

The respiratory effects of fine dusts are mainly caused by inflammation in the bronchial tubes, which causes asthma, chronic bronchitis and airway obstruction.

In addition, fine dusts may cause respiratory infections by obstructing the inactivation or elimination of bacteria in the lung tissue. In recent years, fine dusts have become important risk factors of cardiovascular diseases such as myocardial infarction, stroke, heart rate abnormality, .

It is reported that exposure of fine dust is related not only to the occurrence of respiratory and cardiovascular diseases but also to the increase in mortality rate. By age group, the influence of fine dust and yellow dust on preschool children with weak physical immune system and elderly people over 70 years .

Therefore, there is a need to prevent and improve diseases caused by such fine dusts by providing functional foods and the like using beneficial components of natural products.

Korean Patent No. 10-1466316 (November 21, 2014) Korean Patent No. 10-1516057 (April 22, 2015)

The inventors of the present invention have conducted intensive studies in order to develop useful products by searching for effective components in fine dusts using natural materials. As a result, they discovered that a combined extract of mushroom and myrtle can satisfy the above requirements. And has reached the completion of the invention.

Accordingly, an object of the present invention is to provide a composition for improving fine dust-stimulating respiratory diseases, which comprises, in one aspect, a combined extract of myrrh and a mushroom mushroom as an active ingredient.

The complex extract of Japanese mushroom and shiitake mushroom according to the present invention can be effectively used as a health functional food or medicament for preventing or improving respiratory diseases caused by fine dusts.

FIG. 1 is a graph showing the result of suppressing fine dust-induced lipid peroxidation of a mixed extract. FIG.
FIG. 2 is a graph showing the percent MDA production in the microdermic-induced lipid peroxidation test of the mixed extract. FIG.
3 is a graph showing the inhibition of fine dust-induced lipid peroxidation of the mixed extract.
FIG. 4 is a photograph and a graph showing the results of protein expression of IL-1 beta of mixed extracts in the lungs stimulated with fine dusts.
5 is a graph showing the activity of suppressing the expression of the mixed extract.
FIG. 6 is a graph showing the effect of primate extract on cell viability of a lung cancer epithelial cell line. FIG.
7 is a graph showing the effect of mushroom extract on cell viability of lung cancer epithelial cell line.
Fig. 8 is a graph showing the effect on the cell viability of the lung cancer epithelial cell line of the mixed extract of Shiso and Mushroom.
9 is a graph showing the effect of fine dust on the cell viability of a lung cancer cell line.
FIG. 10 is a graph showing the effect on the cell viability of the Raw 264.7 cell line of the nematode extract. FIG.
11 is a graph showing the effect of mushroom extract on cell viability of Raw264.7 cell line.
FIG. 12 is a graph showing the effect on the cell viability of the Raw264.7 cell line of the mixed extract of the non-wild type and the shiitake mushroom.
Figure 13 is a graph showing the effect of primate extract on the protection of PM-induced cell lethality in lung cancer epithelial cells.
14 is a graph showing the effect of mushroom extract on the protection of PM-induced cell lethality in lung cancer epithelial cells.
Fig. 15 is a graph showing the effect of the combined extract of shiso and shiitake mushroom on the protection of PM-induced cell deaths in lung cancer epithelial cells.
16 is a graph showing the cytotoxic inhibitory effect of the compound extract on lung cancer epithelial cell line.
17 is a graph showing the inhibitory effect on the PM-induced intercellular ROS of the complex extract in lung cancer cell lines.
18 is a graph showing the effect of the complex extract on cell lethal protein expression in lung cancer epithelial cell line.
19 is a graph showing the inhibitory effect of the primate extract on NO production in the PLS-stimulated lung cancer epithelial cell line.
20 is a graph showing inhibitory effect of mushroom extract on NO production in PLS-stimulated lung cancer epithelial cell line.
FIG. 21 is a graph showing inhibitory effects of the non-invertebrate shiitake mushroom extract on NO production in PLS-stimulated lung cancer epithelial cell line.
Figure 22 is a graph showing the inhibitory effect of the non-invertebrate shiitake mushroom extract on TNF-α production in the PLS-stimulated Raw264.7 cell line.
23 is a graph showing the effect of the complex extract on the expression of MMP-9 in the LPS-stimulated Raw 264.7 cell line.
24 is a graph showing the effect of the complex extract on cell penetration in the LPS-stimulated Raw264.7 cell line.

The present invention provides, in one aspect, a composition for improving fine dust-stimulating respiratory diseases, which comprises, as an active ingredient, a composite extract of mignon and shiitake mushroom.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a composition for improving respiratory diseases according to the present invention will be described in detail with reference to the accompanying drawings.

Seedlings are seaweeds belonging to the seaweeds of brown algae and contain inorganic and bioactive components such as calcium, potassium, iron, and iodine. In particular, the viscous substances and polysaccharides are effective in adsorbing and eliminating heavy metals in food contaminated with pesticides Alginic acid, an important component of brown algae cell membrane, is a copolymer of D-mannuronic acid and L-glucuronic acid. It has been reported to have cholesterol-releasing activity, suppression of Cd and Sr absorption in the body, and dressing action.

Lentinus edodes belong to the mussel, which is parasitic on the hardwood such as oak, and has a flavor component and pharmacological effect, and is widely used for edible and medicinal purposes in Korea.

Shiitake mushroom extract has been listed as a health food functional food standard in the Food and Drug Administration. However, there is no data on the respiratory protection effect on the above materials.

The shrimp and shiitake were thoroughly washed, dried, and then added with an extraction solvent 5 to 20 times the weight of the raw material. The resulting mixture was placed in a autoclave autoclave and incubated at 65 to 125 ° C for 2 to 24 hours, preferably 5 hours Extraction by heating, and filtration.

The extraction solvent may be at least one selected from water, lower alcohols having 1 to 4 carbon atoms, polyhydric alcohols, and mixtures thereof. As the lower alcohol having 1 to 4 carbon atoms, methanol, ethanol and the like can be used. As the polyhydric alcohol, butylene glycol, propylene glycol, pentylene glycol and the like can be used. Mixtures of water and lower alcohols, mixtures of water and polyhydric alcohols, mixtures of lower alcohols and polyhydric alcohols, or mixtures of water and lower alcohols and polyhydric alcohols can be used as the mixture.

Alternatively, the gingko and shiitake can be subjected to hot water extraction, cold-rolling or warm-up extraction by adding an extraction solvent. In this case, the plant material is mixed with the extraction solvent at a weight ratio of 5 to 20 times, and the mixture is extracted at 15 to 125 ° C for 2 to 6 months, and then filtered to obtain the extract.

As shown in the test examples described below, the combined extract of Shiitake mushroom and Mizuma mushroom according to the present invention inhibited lipid peroxidation reaction by micronized dust to a concentration-dependent level of 64% in MDA production inhibition test, Significantly reduced the expression of IL-1β, an inflammatory cytokine. In addition, cell viability was confirmed by MTT assay and LDH assay. As a result, the cell survival rate was increased up to 86% in the group treated with the combination of the non-wild type and shiitake mushroom extract, and the synergistic effect on the inhibitory effect on cell death was confirmed . In addition, the expression of Caspase 3 was decreased in the group treated with the mixed extract of Shigira-Shiitake mushroom, and the expression of Bcl-2 was increased as the concentration of Caspase 3 was increased, , TNF-α production and MMP-9 expression were significantly inhibited, thereby confirming the effect of the combined extract of mushroom and primate according to the present invention on fine dust-induced lung tissue.

Therefore, it can be considered that the combined extract of the microspheres and the shiitake mushroom according to the present invention can be used as an effective ingredient of medicines or health functional products for preventing or improving respiratory diseases caused by fine dusts.

In 2006, Hurst et al. Collected 90 blood samples from patients with chronic respiratory disease in general condition and patients with chronic respiratory disease when they were deteriorated by fine dust, and then plasma was separated and 36 samples including Eotaxin, IL-1, MCP and MMP9 (ROS-MAPK-NF-κB signaling pathway). In the present study, Wang J et al. J Thorac Dis. 2017, 9 (11): 4398-4412. Air pollution and cytokine responsiveness in asthmatic and non-asthmatic children. Klperper et al. Environ Res. 2015, 138: 381-390.].

In the present invention, micro dust-causing respiratory diseases include respiratory inflammatory lung disease, chronic obstructive pulmonary disease, sinusitis, allergic rhinitis, lower respiratory tract infections, chronic chronic obstructive pulmonary disease And one or more diseases selected from the group consisting of bronchitis, emphysema, pneumonia, asthma, bronchitis, bronchiectasis, sore throat, tonsillitis, and sore throat.

The medicament of the present invention can be administered by various routes. Various modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, or subcutaneous injection.

The pharmaceutical composition may be administered orally or parenterally in various clinical formulations. In the case of pharmaceutical preparation, a diluent such as a filler, a weight agent, a binder, a wetting agent, a disintegrant, a surfactant, Can be used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may be, for example, starch, calcium carbonate, sucrose or lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

The administration dose of the composition containing the complex extract of the present invention may be varied according to the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, It may be administered once to several times a day at intervals of time. For example, the daily dose may be 0.001 to 500 mg / kg, preferably 0.1 to 200 mg / kg, based on the active ingredient.

In addition, the functional food composition according to the present invention can be provided in a form including, for example, powder, granule, ring, tablet, capsule, liquid or drink form according to a conventional method in the art.

The amount of the food of the present invention is appropriately set according to the form of the preparation, the method of administration, the purpose of use, and the age, weight, and symptom of the subject to which the active ingredient is administered. Day, for example, 0.001 mg / kg to 500 mg / kg. Of course, since the dosage varies depending on various conditions, the amount may be less than the above amount, or may be more than the above range.

In addition, when the composition of the present invention is prepared from a functional food (health functional food and general food) composition, it may contain components that are commonly added to foods, and examples thereof include proteins, carbohydrates, fats, sweeteners, Wherein at least one ingredient selected from the group consisting of licorice, vitamin C, citric acid, nicotinic acid, sodium benzoate, aspartame, saccharin, pectin, maltitol, sorbitol, xylitol, guar gum, .

They are suitably used in an amount of about 0.01 to 20% by weight based on the total weight of the composition of the present invention.

For example, the beverage may contain 0.01 to 20% by weight of the complex extract, 0.01 to 2% by weight of licorice, 0.01 to 2% by weight of citric acid, 0.01 to 2% by weight of malic acid, 0.1 to 2% by weight of taurine, 0.01 to 2% by weight of vitamin B1, 0.01 to 2% by weight of biotin, 0.01 to 2% by weight of biotin, 0.01 to 20% by weight of fructose in liquid form, 0.1 to 3% by weight of polydextrose, 0.1 to 1% 0.01 to 0.5% by weight of lactose, 0.01 to 1% by weight of calcium lactate, 0.1 to 1% by weight of a flavoring agent, 0.01 to 0.05% by weight of a coloring matter, 0.01 to 0.5% by weight of sodium citrate, 0.01 to 0.5% 0.01 to 0.5% by weight, and L-menthol in an amount of 0.0001 to 0.5% by weight, alone or in combination.

<Examples>

Hereinafter, the present invention will be described more specifically by the following representative examples, but the present invention is not limited in any way by these embodiments. Each value is expressed as the mean ㅁ standard deviation of three repeated tests.

Example 1: Preparation of shiitake mushroom extract

10 Kg of shiitake mushroom was cleaned, dried, and then added with purified water of 10 times the weight of the raw material. The mixture was heated in a high-pressure sterilizing pot at 100 ° C for 5 hours, filtered and concentrated to obtain an extract. The extraction yield of shiitake mushroom was 23.8% and brix was 3.4.

Example 2: Preparation of an irregular extract

100Kg of purified water was added to 10Kg of simian, followed by extraction at 100 ° C for 5 hours, followed by filtration to obtain an irregular extract. The yield of the primate extraction was 30.5% and the brix was 4.1.

Example 3: Preparation of a mushroom spirulina extract

After washing 200 Kg of shiitake mushroom using a 1 Ton extractor, the washed shiitake mushroom was dried with hot air at a temperature of 60 ° C. Dried shiitake was added with 20 times the weight of the alcohol, and the extract was extracted at a temperature of 0.5 atm and 65 ° C for 4 hours. Subsequently, the extract was filtered with a filter paper to filter off the foreign substances, and the filtrate was concentrated at a temperature of 60 캜 under a reduced pressure of -0.08 MPA for 8 hours. The brix of solids was measured as 43. Subsequently, the concentrate was dried under vacuum at a temperature of 60 DEG C for 24 hours, pulverized into 100 mesh with a pin mill, and then subdivided into a predetermined unit.

Example 4: Preparation of a non-seaweed extract

An exudate extract was prepared according to the method of Example 3 above using 200 kg of simian. The brix of solids was measured as 43.

Example 5: Preparation of tablets

30 mg of the dried extract powder of Example 3

30 mg of the irregularly extracted powder of Example 4

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

The tablets were mixed so as to contain the above components per tablet, and then tableted according to a conventional preparation method.

Example 6: Preparation of health drinks

30 mg of the dried extract powder of Example 1

30 mg of the irregularly extracted powder of Example 2

Citric acid 20mg

Oligosaccharide 10 mg

Taurine 20mg

Purified water was added and the whole 100 ml

The beverage was prepared to contain the above content per packaging container according to the conventional health drink manufacturing method.

Test Example

In the following test examples, in vitro efficacy in respiratory tissues and inhibition of inflammatory responses of bronchial epithelial cells and macrophages were performed.

In all experiments, the combined extracts of shiitake mushroom and mugwort were evaluated for their efficacy by preparing 1: 1 ratio of mushroom hot water extract and non - hot water extract. Fine dust was added at a concentration of 50 μg / mL, which did not significantly affect cell survival. After lipid peroxidation and analysis of cytokine changes in the lung tissue after microdermal stimulation, The efficacy was evaluated.

In addition, tests for inhibition of inflammatory responses of bronchial epithelial cells and macrophages were performed by cytotoxicity test, measurement of oxidative stress markers, analysis of inhibitory effect of inflammatory factors, and MMP biomarker analysis and cell invasion assay. Each value was expressed as mean ± SD three times.

Test Example 1: Preparation of experimental animal lung tissue

Experimental animals (ICR mouse, male, 8weeks) were fasted for 24 hours before dissection and then lapped under ether anesthesia. The lung tissues were separated and stored at -70 ° C until analysis. Lung tissue was washed several times with sterile physiological saline, lysed and disrupted by adding lysis buffer, and centrifuged (10,000 x g) to prepare a homogenate of lung tissue. The protein was quantitated with BCA kit and the appropriate concentration (100 ㎍ - 1,000 ㎍) was calculated. After the sample was treated with the homogenate of lung tissue for 1 hour or the fine dust + sample, the subsequent in vitro test was performed.

Test Example 2: Lipid peroxidation analysis of a complex extract material in lung tissue

The lipid peroxidation inhibitory activity in lung tissue was evaluated in order to analyze the change of antioxidant ability of the altitude / Analysis of lipid peroxidation was done by the amount of MDA produced, and the amount of MDA produced was measured and compared in the homogenate of the sample and the control.

As a normal control, vehicle was used instead of the sample. Negative control group was used to give fine dust stimulation (50 μg / mL) to the lung tissue homogenate. As a positive control group, soluble vitamin C ascorbic acid (10 μg) (50 μg / mL), polyphenol gallic acid (10 μg) + fine dust stimulation (50 μg / mL), and the flavonoid naringin (10 μg) + fine dust stimulation (50 μg / mL). Experimental groups were treated with 5 ㎍, 10 ㎍, 20 ㎍ + fine dust stimulation (50 ㎍ / mL) as an altitude /

FIG. 1 is a graph showing the result of suppressing fine dust-induced lipid peroxidation of a mixed extract. FIG. As a result, in which MDA production of normal control group treated with buffer melted sample is 3.6257 ± 0.1088 nmol MDA / g, MDA production amount of negative control only that stimulation fine dust is 4.5623 ± 0.1369 nmol MDA / g, FeSO 4 only stimulated negative control MDA / g in the positive control group, 4.0797 ± 0.1632 nmol MDA / g in the positive control group, 4.3737 ± 0.1749 nmol MDA / g in the gallic acid + fine dust, 4.4303 in the positive control group, naringin + fine dust, and 5.2910 ± 0.1587 nmol MDA / g in the positive control ascorbic acid + ± 0.1772 nmol MDA / g in the experimental group, 4.5108 ± 0.0902 nmol MDA / g in the fine dust, 4.1884 ± 0.0838 nmol MDA / g in the fine dust, 3.9651 ± in the fine dust, 0.0793 nmol MDA / g.

FIG. 2 is a graph showing the percent MDA production in the microdermic-induced lipid peroxidation test of the mixed extract. FIG. As a result of analyzing the percentage of MDA produced in the general control group, when the MDA production amount of the normal control group treated with the buffer dissolved in the sample was 100.0%, the MDA production amount of the negative control stimulated only with fine dust was 125.83% The amount of MDA produced in the control group was 145.93%, positive control ascorbic acid + fine dust 112.52%, gallic acid + fine dust 120.63%, positive control naringin + fine dust 122.19% 124.41% in fine dust, 115.52% in 10μg + fine dust, and 109.36% in 20μg + fine dust.

3 is a graph showing the inhibition of fine dust-induced lipid peroxidation of the mixed extract. The inhibition rates of the positive control group and the experimental group were 51.53% in the positive control ascorbic acid + fine dust, 20.13% in the positive control group gallic acid + fine dust, 14.09% in the positive control group naringin + fine dust, , MDA production inhibition rate of 5 ㎍ of the mixture of the extracts of the experimental group and the microgranules + 5.50% of fine dust, 39.91% of 10 ㎍ + fine dust, and 63.76% of 20 + + fine dust.

In the lung tissue, the amount of MDA produced during the microdermost stimulation was lower than the amount of MDA produced during the FeSO ₄ stimulation. When the MDA production inhibition rate of the positive control and the experimental group was compared, Followed by ascorbic acid, medium / low density lipoprotein extract, gallic acid, polyphenol, naringin, and low / high density lipoprotein extracts. At high concentration, the extracts of highland / nematode complexes showed higher inhibition rate than MDA production inhibition rate of positive control and inhibited lipid peroxidation reaction by 5 to 64%.

Test Example 3: Cytokine analysis of complex extracts in lung tissue

IL-1 beta, an inflammatory cytokine, was used to analyze changes in cytokine expression induced by microdermal stimulation in lung tissues using a western blot test.

Western blotting analysis was performed using homogenate of lung tissue to confirm cytokine expression in lung tissues after sample treatment. Protein concentration was determined by SDS-PAGE and electrophoresis. Protein was transferred to PVDF membrane using transfer buffer respectively. After blocking with 5% non-fat skim milk solution, primary antibody and secondary antibody were attached. After the antibody reaction, the membrane was treated with ECL detection kit, exposed to X-ray film and developed. Respectively.

As a general control, the vehicle was used instead of the sample. Negative control group was used to give fine dust stimulation (50 μg / mL) to the homogenate of lung tissue, and 10 μg, 20 μg, 50 μg, 100 μg Mu] g + fine dust stimulation (50 [mu] g / mL).

FIG. 4 is a photograph and a graph showing the results of protein expression of IL-1 beta of mixed extracts in the lungs stimulated with fine dusts. As a result, the expression level of IL-1 beta expressed in the normal control group treated with the buffer dissolved in the sample was 124.47 ± 8.71% when compared with that of the control group stimulated only with fine dust at the comparison, and the experimental group, In the case of 10 μg of combined extract + fine dust, it was 133.30 ± 12.00%, that of 20 μg + fine dust was 112.77 ± 5.64%, that of 50 μg + fine dust was 88.10 ± 5.29% and that of 100 μg + fine dust was 92.03 ± 5.52%.

5 is a graph showing the activity of suppressing the expression of the mixed extract. As a result, the inhibition rate was calculated as 10 μg / 10 g of the extract of the mixture of the omega / microsphere extract +36.08% in the fine dust, 52.17% in the 20μg + fine dust, 149.04% in the 50μg + fine dust, 132.69% in the 100μg + fine dust -1 &lt; / RTI &gt;

As a result, the altitude / primate combination extract significantly reduced the expression of IL-1β, an inflammatory cytokine in lung tissue, caused by microdust stimulation at a higher concentration than at low concentrations.

Test Example 4: Measurement of cell survival rate by fine dusts of shrimp and shiitake-MTT assay

MTT assays were performed using A549 cells (Korean Cell Line Bank, 10185) and Raw264.7 cells (Korean Cell Line Bank, 40071) in order to examine the cell survival rate by shrimp and shiitake mushroom. A549 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS). PM10 (NIST, 1648a) was added when the cells were cultured at 90-100% And PM10 treatment was not performed.

Cells were seeded in 96-well plates and cultured for 12 hours. Cells were treated for 24 hours, MTT solution (final concentration: 0.5 mg / mL) After culturing, MTT was reduced and the medium was removed so that the generated formazan did not fall off the medium. 100 μl of DMSO was dispensed, and the mixture was mixed for 10 minutes, and the absorbance was measured at 540 nm.

The results are shown in Figs. FIG. 6 is a graph showing the effect of primate extract on cell survival of lung cancer epithelial cell line, FIG. 7 is a graph showing the effect of mushroom extract on cell survival of lung cancer epithelial cell line, FIG. This is a graph showing the effect of the mushroom complex extract on the cell viability of lung cancer epithelial cell line.

A549 cells were treated at 1, 5, 10, 50, 100, and 300 ㎍ / ml, and survival rates were 103, 99, 98, 99, 90 and 84% Cell viability was 100, 98, 99, 101, 89 and 81% in shiitake mushrooms. The cell survival rate was 101, 102, 100, 100, 96 and 82% when 1, 5, 10, 50, 70 and 100 ㎍ / ml were mixed with shrimp and shiitake mushrooms.

9 is a graph showing the effect of fine dust on the cell viability of a lung cancer cell line. Cell viability was determined as 81, 61, 57, 54, 51 and 44% after 10, 50, 100, 200, 300 and 500 μg / Respectively.

The cell viability was monitored after treatment with Raw264.7 at the concentrations of 1, 5, 10, 50, 100, 200, and 300 μg / ml. FIG. 10 is a graph showing the effect on the cell viability of the Raw 264.7 cell line of the primate extract, FIG. 11 is a graph showing the effect on the cell viability of the Raw 264.7 cell line of the mushroom extract, and FIG. Which is a graph showing the effect on the cell viability of the Raw264.7 cell line of the mixed extract of Mizunami and Shiitake mushroom. As a result, cell survival rate was 99, 100, 100, 101, 97, 93, 85% in the non - In shiitake mushrooms, cell viability was 99, 100, 101, 99, 100, 94 and 81%. The cell viability was 100, 101, 99, 100, 99, 90, and 74% when 1, 5, 10, 50, 100, 200 and 300μg / ml were mixed with the shrimp and shiitake mushrooms.

A549 cells were pretreated with shrimp and shiitake and MTT assay was performed to confirm the effect of suppressing the decrease of cell viability due to toxicity due to fine dust. After pretreatment for 4 hours each, the microspheres and shiitake were treated with 50 μg / ml of fine dust for 24 hours.

FIG. 13 is a graph showing the effect of primate extract on the protection of PM-induced cell death in lung cancer epithelial cells. FIG. 14 shows the effect of mushroom extract on the protection of PM-induced cell death in lung cancer epithelial cells And Fig. 15 is a graph showing the effect of the combined extract of the primate and shiitake mushroom on the protection of PM-induced cell lethality in lung cancer epithelial cells.

As a result, in the group treated with fine dust only, the number of shiitake mushrooms decreased to 58%, while in the group treated with shrimp at 5, 10, 20, 30 and 50 μg / ml, 60, 68, 73, 73 and 68% The cell viability was increased as the concentration was increased to 59, 64, 69, 70 and 74% in the groups treated with 10, 20, 30 and 50 μg / ml. The cell viability was increased to 57, 62, 70, 78, and 86% at 10, 20, 30 and 50 μg / ml, respectively.

These results showed that the synergistic effect of the combined extracts was confirmed by the high cell survival rate in the group treated with the mixed extract of the non - surrogate mushroom and shiitake mushroom, compared with the group treated with the non - shigot and the mushroom at the same concentration.

Test Example 5: Measurement of cell viability-LDH assay

Based on the results of MTT assay, the release of LDH (Lactate dehydrogenase), which is a method of measuring cytotoxic mechanism, was measured.

The cells were cultured in a CO ₂ incubator for 24 hours. After the sample was treated and cultured, 50 μl of the culture was dispensed into a new 96-well plate, and 50 μl of an LDH reagent was added to the 96-well plate After incubation at room temperature, the reaction was allowed to proceed for 20 minutes. When the reaction was completed, 100 μl of 1 N HCl as a stop solution was added to stop the reaction, and the absorbance was measured at 540 nm.

After removing the culture medium, 50 μl of a 0.5% Triton X-100 solution was added, and the cell wall was shaken at 40 rpm for 10 minutes. Then, 50 μl of the LDH reagent was added thereto in the same manner, At the end of the reaction, the reaction solution was added and the absorbance was measured at 540 nm.

The percentage of cytotoxicity by LDH was calculated as the value of LDH liberated from the culture medium for total LDH liberated from the culture medium and living cells and compared with the untreated control group.

Pretreated and shiitake were pretreated for 4 hours each and treated with 50 ㎍ / ml of fine dust for 24 hours. 16 is a graph showing the cytotoxic inhibitory effect of the compound extract on lung cancer epithelial cell line. In the group treated with 5, 10, 20, 30, and 50 μg / ml, the extracts were increased to 39, 40, 38, 32, 25 and 19% LDH release decreased.

The MTT assay and the LDH assay showed that there is a synergistic effect on the inhibitory effect on the apoptosis induced by micro dust particles.

Test Example 6: Measurement of oxidative stress-related marker

The DCF-DA method was used to determine the inhibitory effect of the combined extract on intracellular ROS production by fine dust. Based on the MTT assay results, ROS generation by micro dust was measured.

After lysing 100 μl of lysis buffer, cells were thawed and centrifuged at 12,000 rpm for 10 min at 4 ° C. Transfer only the supernatant to a clean tube and store at 70 ° C until use. Activity measurement was performed using an assay kit. The microspheres and shiitake mushrooms were pretreated for 4 hours each, and 50 μg / ml of fine dust was treated for 12 hours.

17 is a graph showing the inhibitory effect on the PM-induced intercellular ROS of the complex extract in lung cancer cell lines. In the group treated with only fine dust, it increased to about 210%. ROS production was decreased to 209, 197, 180, 168, and 149% in the group treated with 5, 10, 20, 30 and 50 μg /

Bcl-2 is known to be an antiapoptotic protein and inhibits cell death by inhibiting the migration of Bax to mitochondria. In contrast, Caspase-3 can be activated by various apoptosis stimuli, and activated Caspase-3 is cleaved , And it acts to induce apoptosis by promoting the degradation of protein PARP in cells. Western blotting of the complex extracts for the expression of Bcl-2, Caspase-3 and the like, apoptosis-related proteins, was carried out from the cells. Based on the results of MTT assay, changes in Bcl-2 and Caspase-3 expression by fine dust were measured.

18 is a graph showing the effect of the complex extract on cell lethal protein expression in lung cancer epithelial cell line. In the group treated with 5, 10, 20, 30, and 50 μg / ml of the combined extracts of the microspheres and shiitake mushrooms for 24 hours after treating microspheres with 50 μg / ml for 24 hours, Expression of Caspase 3 was found to decrease more than that of fine dust, and expression of Bcl-2 was increased as the concentration of BCl-2 was increased in the complex extract treatment group than in the fine dust treatment group. Thus, it was confirmed that the complex extract synergistically inhibited cell death by fine dust particles.

Test Example 7: Inhibitory effect on the production of inflammation-related factors

The inhibitory effect of RAW264.7 cells on NO production was measured using LPS as an inflammatory substance.

100 μl of the primary antibody of cytokine to be quantified in the precoated 96 well plate was incubated overnight at 4 ° C, washed three times for 5 minutes with 0.5% tween 20 washing solution, and then the sample to be measured and the standard solution Were added and reacted at room temperature for 2 hours. After washing as above, 100 μl of each HRP-conjugated secondary antibody was added and reacted at room temperature for 2 hours. After washing again, absorbance was measured by adding avidin / biotin to develop color. A standard curve was obtained from the absorbance of the standard and quantified from the absorbance of each sample.

19 is a graph showing the inhibitory effect of the primate extract on the NO production in the PLS-stimulated lung cancer epithelial cell line, and FIG. 20 is a graph showing the inhibitory effect of the mushroom extract on NO production in the PLS-stimulated lung cancer epithelial cell line , And FIG. 21 is a graph showing the inhibitory effect of the non-shrimp mushroom complex extract on NO production in PLS-stimulated lung cancer epithelial cell line. In the control group in which only RAW264.7 cells were cultured, NO production was increased to 180% in the LPS-treated group based on the NO concentration. In the group treated with 0, 10, 30, 50, and 70 ㎍ / ml of shrimp at concentrations of 0, 10, 20, 30, and 50 ㎍ / ml, 172, 159, 181, 172, 150, and 151%, respectively. In the group treated with 50, 10, 30, 50 and 70 ㎍ / ml, the NO production was decreased to 169, 150, 139 and 120%.

Therefore, it was confirmed that the NO production was reduced by the single extract of the mashigi or shiitake mushroom, and the NO production was significantly reduced in the group treated with the mashigi - shiitake mushroom extract, compared with the group treated with the mashigi and shiitake mushroom at the same concentration, And shiitake mushroom extracts.

In order to examine the effect of TNF-α on the inflammatory cytokine production, the inhibitory effect on production of RAW264.7 cells was measured. Figure 22 is a graph showing the inhibitory effect of the non-invertebrate shiitake mushroom extract on TNF-α production in the PLS-stimulated Raw264.7 cell line. TNF-α production was increased to 1280 pg / ml in the LPS-treated group on the basis of TNF-α production in RAW264.7 cells-only control group. In the group treated with 50, 10, 30, 50 and 70 μg / ml, the TNF-α production was decreased at 1069, 950, 739 and 520 pg / ml.

Therefore, it is possible to confirm the synergistic effect of the microsphere-shiitake complex extract on the inhibitory effect of TNF-α production by fine dusts.

Test Example 8: MMP Zymography Analysis

Cells were cultured for 24 hours and then the medium was collected and electrophoresis in sodium dodecyl sulphate (SDS) -polyacrylamide gel containing 0.25 mg / ml of gelatin. After electrophoresis, the gel was washed with washing buffer (50 mM Tris-Cl pH 7.5, 10 mM CaCl ₂ , 2.5% Triton X-100, 1.0 μM ZnCl ₂ ) , 10 mM CaCl ₂ , 0.02% NaN ₃ ) at 37 ° C for 24 hours and then stained with 0.1% Coomassie brilliant blue.

LPS-induced increase in MMP-9 expression. The complex extract was pretreated by concentration and LPS was treated for 24 hours. 23 is a graph showing the effect of the complex extract on the expression of MMP-9 in the LPS-stimulated Raw 264.7 cell line.

As a result, the expression of MMP-9 was rapidly increased by LPS treatment, and the expression of MMP-9 was decreased as the concentration of the extract increased.

Test Example 9: Cell invasion assay

Invasion assays were used to investigate the effect of complex extract on cell penetration of Raw 264.7. Invasion assays were performed using an invasion chamber coated with matrigel. After the treatment, the medium was removed and the chamber was transferred to a new 24-well plate. The cells were incubated with calcein AM for 1 hour at 37 ° C, and then stained with fluorescence microscope.

24 is a graph showing the effect of the complex extract on cell penetration in the LPS-stimulated Raw264.7 cell line. As a result, the invasion ability was increased to 240% by LPS, and decreased to 224, 193, 172 and 149% at 0, 10, 20, 30 and 50 μg / ml pretreatment. The invasion inhibitory effect of these complex extracts seems to be closely related to the inhibitory effect of MMP-9.

As can be seen from the above examples and test examples, it can be considered that the combined extract of Japanese sardine and shiitake mushroom according to the present invention can be used as a main component of health functional products for preventing or improving respiratory diseases caused by fine dusts.

It is expected that the research and development based on shrimp and shiitake mushrooms will contribute to the activation of the health food market using the agricultural products produced in Korea, and will contribute to the enhancement of the utilization and product diversification of Jangheung and Shimogye.

In addition, it is necessary to develop materials aimed at the elderly people in their 50s or older as they have entered an aging society. In the future, they will aim at functional markets such as domestic and overseas functional markets and add new raw materials processing and composition to their preferences, And shiitake mushroom materials, we can expect to improve the income of the farmers and the sales of the related companies by using the new materials.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, It will be understood that the invention may be practiced. It is therefore to be understood that one embodiment described above is illustrative in all aspects and not restrictive.

Claims (5)

And the extract of the mushroom and shiitake mushroom were thoroughly washed and dried, and then extracted with 5 to 20 times of the weight of the raw material of the plant, and the mixture was placed in a high pressure sterilizing pot. Deg.] C for 2 to 24 hours, followed by filtration and concentration. The extraction solvent is any one selected from water, lower alcohols having 1 to 4 carbon atoms, polyhydric alcohols, and mixtures thereof. Mushrooms are mixed at a weight ratio of 1: 1, and the fine dust stimulation respiratory diseases are respiratory inflammatory lung disease, chronic obstructive pulmonary disease, sinusitis, allergic rhinitis, lower respiratory tract infection, acute chronic bronchitis, emphysema, pneumonia, asthma, bronchitis, Sore throat, tonsillitis, and laryngitis. Wherein the composition is used for the preparation of a composition for improving respiratory diseases. delete delete delete delete

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