Composition comprising <i>Sargassum horneri </i>extract for alleviating lung injury or respiratory disease

Abstract

Disclosed is a composition comprising a Sargassum horneri extract for alleviating lung injury or respiratory disease.

Claims

1. A tablet or capsule for treating lung injury in a human in need thereof consisting essentially of therapeutically effective amounts of a Sargassum horneri extract, honey and bamboo shoots.

2. The tablet or capsule of claim 1, wherein the lung injury is lung injury caused by particulate matter.

3. The tablet or capsule of claim 1, wherein the Sargassum horneri extract is an extract obtained by extraction with water, a lower alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof.

4. The tablet or capsule of claim 1, wherein the therapeutically effective amount of a Sargassum horneri extract improves lung function in the human in need thereof.

5. The tablet or capsule of claim 1, wherein the therapeutically effective amount is also for alleviating a respiratory disease in said human.

6. The tablet or capsule of claim 5, wherein the respiratory disease is respiratory disease caused by particulate matter.

7. The tablet or capsule of claim 5, wherein the respiratory disease is asthma, chronic obstructive pulmonary disease, tracheitis, bronchitis, or rhinitis.

8. The tablet or capsule of claim 5, wherein the respiratory disease is chronic obstructive pulmonary disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A and 1B show the results of evaluating the effects of a Sargassum horneri extract on cytotoxicity and cell proliferation rate in lung epithelial cells.

(2) FIGS. 2A, 2B, and 2C show the results of evaluating the effects of a Sargassum horneri extract on cytotoxicity and cell proliferation rate in lung epithelial cells damaged by particulate matter.

(3) FIG. 3 shows the results of evaluating the effect of a Sargassum horneri extract on oxidative stress in lung epithelial cells damaged by particulate matter.

(4) FIG. 4 shows the results of evaluating the effect of a Sargassum horneri extract on lipid peroxidation in lung epithelial cells damaged by particulate matter.

(5) FIGS. 5A, 5B, 5C and 5D show the results of evaluating the effect of a Sargassum horneri extract on oxidative stress-induced DNA damage in lung epithelial cells damaged by particulate matter.

(6) FIGS. 6A, 6B, 6C, 6D, 7A, 7B and 7C show the results of evaluating the effects of a Sargassum horneri extract on the expression of inflammatory cytokines and chemokines and inflammatory mediators in lung epithelial cells damaged by particulate matter.

(7) FIGS. 8A, 8B, 8C and 8D show the results of evaluating the effects of a Sargassum horneri extract on the mechanism of secretion of inflammatory cytokines in lung epithelial cells damaged by particulate matter.

(8) FIG. 9 schematically shows the construction of test groups in experimental animal models, the time of sample administration, the period of sample administration, etc.

(9) FIGS. 10A, 10B, 10C, 11A and 11B show the results of evaluating the effect of a Sargassum horneri extract on differential cell count in the blood of particulate matter-inhaled animal models.

(10) FIGS. 12A, 12B, 12C, 13A and 13B show the results of evaluating the effect of a Sargassum horneri extract on differential cell count in the bronchoalveolar lavage fluid (BALF) of particulate matter-inhaled animal models.

(11) FIGS. 14A and 14B show the results of evaluating the effect of a Sargassum horneri extract on the histopathological changes of tracheas and lungs in particulate matter-inhaled animal models. (0, normal; 1, few cells observed; 2, a ring of inflammatory cells one cell layer deep; 3, a ring of inflammatory cells 2-4 cells deep; and 4, a ring of inflammatory cells >4 cells deep).

(12) FIG. 15 shows the results of evaluating the effect of a Sargassum horneri extract on a change in the expression of 8-OHdG in the lungs of particulate matter-inhaled animal models.

(13) FIGS. 16A and 16B show the results of evaluating the effect of a Sargassum horneri extract on a change in the expression of Gr-1 in the tracheas and lungs of particulate matter-inhaled animal models.

(14) FIGS. 17A and 17B show the results of evaluating the effect of a Sargassum horneri extract on the infiltration of eosinophilic leukocytes into trachea and lung tissue in particulate matter-inhaled animal models.

(15) FIGS. 18A and 18B show the results of evaluating the effect of a Sargassum horneri extract on the infiltration of mast cells into trachea in particulate matter-inhaled animal models.

(16) FIGS. 19A, 19B, 20A and 20B show the results of evaluating the effects of a Sargassum horneri extract on mucus secretion and goblet cell proliferation in the tracheas and lungs of particulate matter-inhaled animal models.

(17) FIGS. 21A, 21B, 21C and 21D show the results of evaluating the effects of a Sargassum horneri extract on cell population changes in the lung tissue of particulate matter-inhaled animal models.

DETAILED DESCRIPTION

(18) Hereinafter, the present disclosure will be described with reference to examples and experimental examples. However, the scope of the present disclosure is not limited to these examples and experimental examples.

Example: Preparation of Sargassum horneri Extract

(19) Sargassum horneri (whole plant) produced in Korea was washed three times with purified water for 30 minutes, and then dried to a water content of 10% or less by a hot-air dryer at 50 C. for 24 hours, and ground with a pin mill to a size of 40 to 50 mesh. 10 to 15% (w/v) of the Sargassum horneri powder was added to 70% alcohol and extracted using a circulation extractor at 70 C. for 12 hours, after which it was treated with white clay for food, stirred at 60 rpm for 2 hours, and centrifuged at 12,000 rpm, and the supernatant was collected. The collected supernatant was vacuum-concentrated to a volume of using a vacuum concentrator at 60 C., and a 3-fold volume of 95% alcohol was added to the concentrate. Then, the solution was allowed to stand for 12 hours, and then the supernatant was collected by centrifugation and filtration. The collected extract was concentrated to a volume of using a vacuum concentrator, and then dried with a freeze dryer, thereby obtaining a Sargassum horneri ethanol extract powder (SHE).

EXPERIMENTAL EXAMPLES: EXPERIMENTS ON RESPIRATORY DISEASE-ALLEVIATING ACTIVITY OF SARGASSUM HORNERI EXTRACT

Experimental Example 1: Effects of Sargassum horneri Extract on Cytotoxicity and Cell Proliferation Rate in Lung Epithelial Cells

(20) (1) Lactose dehydrogenase (LDH), a cytosolic enzyme, is released extracellularly when the cell membrane is damaged. LDH catalyzes the dehydrogenation of lactic acid to produce pyruvate and NADH, and the NADH reduces tetrazolium salt (INT) to red formazan by the catalytic action of diaphorase (Part Fibre Toxicol 2017, 14:39). The activity of LDH was measured to evaluate the effect of the SHE on murine lung epithelial cell line 12 (hereinafter referred to as MLE-12). In addition, using the amount of LDH released, IC.sub.50 (at which 50% of cells lose their viability by the SHE) was calculated. In addition, the extent of incorporation of .sup.3H-thymidine was evaluated to evaluate cell proliferation rate.

(21) (2) MLE-12 Cells

(22) MLE-12 cells were added to Ham's F12 medium (supplemented with 0.05 mg/ml of insulin, 0.01 mg/ml of transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM -estradiol, 10 mM HEPES, 2 mM L-glutamine, and 2% fetal bovine serum), and dispensed in a 96-well plate at a density of 110.sup.3 cells/well. The cells were incubated in a 5% CO.sub.2 incubator at 37 C. for 12 hours, and then treated with various concentrations (0 to 500 g/ml) of the SHE, and incubated for 48 hours. Then, the cytotoxicity was analyzed using a LDH Cytotoxicity Detection Kit (Takara Bio Inc., Japan), and the IC.sub.50 (half maximal inhibitory concentration) was analyzed using the SigmaPlot V10.0 (Systat Software, Inc., Richmond, Calif., USA) program. Meanwhile, MLE-12 cells were dispensed in a 96 well plate at a density of 210.sup.4 cells/well, and incubated in a 5% CO.sub.2 incubator at 37 C. for 12 hours, after which the cells were treated with various concentrations (0 to 500 g/ml) of the SHE, and incubated in a 5% CO.sub.2 incubator at 37 C. for 54 hours. Next, each well was treated with 1 Ci of .sup.3H-thymidine (42 Ci/nmol, Amersham Life Science, Arling-ton Heights, Ill., USA), and incubated in a 5% CO.sub.2 incubator at 37 C. for 18 hours, after which the cells were captured on a glass fiber filter, and the amount of radioisotopes therein was measured using a radioactivity meter (Wallac MicroBeta TriLux, Perkin Elmer, Waltham, Mass., USA).

(23) (3) The results are shown in FIG. 1. When the MLE-12 cells were treated with various concentrations (0 to 500 g/ml) of the SHE, SHE-induced cytotoxicity did not appear at a concentration of 0 to 125 g/ml (FIG. 1A). However, treatment with a high concentration (250 or 500 g/ml) of the SHE showed cytotoxicity (FIG. 1A). In addition, treatment with 0 to 125 g/ml of the SHE had no effect on the cell proliferation rate, but treatment with 250 and 500 g/ml of the SHE reduced the cell proliferation rate by 11.0% and 21.2%, respectively (FIG. 1B).

Experimental Example 2: Effects of Sargassum horneri Extract (SHE) on Cytotoxicity and Proliferation Rate in MLE-12 Cells Damaged by Particulate Matter

(24) (1) Lactose dehydrogenase (LDH), a cytosolic enzyme, is released extracellularly when the cell membrane is damaged. LDH catalyzes the dehydrogenation of lactic acid to produce pyruvate and NADH, and the NADH reduces tetrazolium salt (INT) to red formazan by the catalytic action of diaphorase. The activity of LDH was measured to determine the degree of particulate matter-induced cell damage in MLE-12 cells and to evaluate the effect of the SHE on protection from cell damage. In addition, using the amount of LDH released, IC.sub.50 (at which 50% of cells lose their viability by particulate matter and the SHE) was calculated. In addition, the extent of incorporation of .sup.3H-thymidine was evaluated to evaluate cell proliferation rate. In addition, the extent of incorporation of .sup.3H-thymidine was evaluated to evaluate cell proliferation rate.

(25) (2) MLE-12 cells were added to Ham's F12 medium (supplemented with 0.05 mg/ml of insulin, 0.01 mg/ml of transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM -estradiol, 10 mM HEPES, 2 mM L-glutamine, and 2% fetal bovine serum), and dispensed in a 96-well plate at a density of 110.sup.3 cells/well. The cells were incubated in a 5% CO.sub.2 incubator at 37 C. for 12 hours, and then treated with various concentrations (31.3 and 125 500 g/ml) of the SHE and various concentrations (3.9 to 250 g/ml) of particulate matter, and incubated for 48 hours. Then, the cytotoxicity was analyzed using a LDH Cytotoxicity Detection Kit (Takara Bio Inc., Japan), and the IC.sub.50 was analyzed using the SigmaPlot V10.0 (Systat Software, Inc., Richmond, Calif., USA) program. Meanwhile, MLE-12 cells were dispensed in a 96 well plate at a density of 210.sup.4 cells/well, and incubated in a 5% CO.sub.2 incubator at 37 C. for 12 hours, after which the cells were treated with various concentrations (31.3 and 125 g/ml) of the SHE and various concentrations (3.9 to 250 g/ml) of particulate matter, and incubated in a 5% CO.sub.2 incubator at 37 C. for 54 hours. Next, each well was treated with 1 Ci of .sup.3H-thymidine (42 Ci/nmol, Amersham Life Science, Arling-ton Heights, Ill., USA), and incubated in a 5% CO.sub.2 incubator at 37 C. for 18 hours, after which the cells were captured on a glass fiber filter, and the amount of radioisotopes therein was measured using a radioactivity meter (Wallac MicroBeta TriLux, Perkin Elmer, Waltham, Mass., USA).

(26) (3) The results are shown in FIG. 2. When the MLE-12 cells were treated with various concentrations (0 to 250 g/ml) of particulate matter, the particulate matter showed concentration-dependent cytotoxicity from a concentration of 15.6 g/ml (FIG. 2A), and at a particulate matter treatment concentration of 44.15 g/ml, 50% of the MLE-12 cells lost their viability (IC.sub.50=44.15 g/ml) (FIG. 2B). However, when the cells were treated with particulate matter and 31.3 and 62.5 g/ml of the SHE, the high-concentration particulate matter-treated group (125 or 250 g/ml) which has shown cytotoxicity showed a concentration-dependent decrease in cytotoxicity (P; *<0.05, <0.05) (FIG. 2A). In addition, as the particulate matter treatment concentration (0 to 250 g/ml) increased, the cell proliferation rate significantly decreased (P; *<0.05, <0.05). However, when the cells were treated with the SHE and 3.9 to 250 g/ml of particulate matter, the cell proliferation rate significantly increased (P; *<0.05, **<0.005, <0.05) (FIG. 2C).

Experimental Example 3: Effect of Sargassum horneri Extract (SHE) on Oxidative Stress in MLE-12 Cells Damaged by Particulate Matter

(27) (1) Reactive oxygen species (ROS) convert non-fluorescent DCF-DA to the fluorescent substance DCF by oxidation. In order to evaluate the antioxidant effect of the Sargassum horneri extract (SHE) on MLE-12 cells damaged by particulate matter, the fluorescence intensity of DCF was measured.

(28) (2) MLE-12 cells were dispensed in a 24-well plate at a density of 1.510.sup.5 cells/well and incubated for 12 hours. Then, the cells were treated with the SHE (62.5, 125 and 250 g/ml) and particulate matter (62.5 and 125 g/ml) and incubated in a 5% CO.sub.2 incubator at 37 C. for 48 hours. Next, the cells were harvested, washed with Dulbecco's phosphate-buffered saline (DPBS), and then treated with 25 M DCF-DA and incubated in a 5% CO.sub.2 incubator at 37 C. for 30 minutes, after which the fluorescence intensity of DCF was measured using a CytoFLEX flow cytometer (Beckman coulter, Inc., Brea, Calif., USA).

(29) (3) The results are shown in FIG. 3. When the MLE-12 cells were treated with various concentrations (62.5 and 125 g/ml) of particulate matter showing cytotoxicity, the particulate matter increased ROS production in a concentration-dependent manner, and when the cells were treated with particulate matter and various concentrations (62.5, 125, and 250 g/ml) of the SHE, ROS production showed a tendency to decrease. In particular, when the cells were treated with 125 g/ml of particulate matter and 125 and 250 g/ml of the SHE, ROS production decreased by 5.3-fold and 4.0-fold, respectively (FIGS. 3A and 3B).

Experimental Example 4: Effect of Sargassum horneri Extract (SHE) on Lipid Peroxidation in MLE-12 Cells Damaged by Particulate Matter

(30) (1) The content of malondialdehyde (hereinafter referred to as MDA), a measure of lipid peroxidation, was measured to evaluate the lipid peroxidation inhibitory effect of the Sargassum horneri extract (SHE) in MLE-12 cells damaged by particulate matter.

(31) (2) MLE-12 cells were dispensed in a 10-cm dish at a density of 110.sup.6 cells, and then incubated in a 5% CO.sub.2 incubator at 37 C. for 12 hours to induce cell adhesion. Then, the cells were treated with SHE (62.5 and 125 g/ml) and particulate matter (62.5 and 125 g/ml) and incubated in in a 5% CO.sub.2 incubator at 37 C. for 6, 24 and 48 hours. Next, the cells were harvested, washed cold DPBS, and added to the NE-PER Nuclear and Cytoplasmic extraction kit (Thermo scientific, Rockford, USA), and protease inhibitors (2 mM Na.sub.3VO.sub.4, 1 mM PMSF, 10 g/ml aprotinin, and 10 g/ml leupeptin) were added to the kit to extract protein. 10% (w/v) trichloroacetic acid was added to the extracted protein and allowed to react on ice for 10 minutes, and then the supernatant was separated by centrifugation at 2000 g and 4 C. for 15 minutes. The same amount of 0.67% (w/v) thiobarbituric acid (TBA, 0.67% (w/v) solution in 50 mM TBA) was added to the supernatant and heated at 100 C. for 10 minutes, followed by centrifugation at 1000 g and 4 C. for 10 minutes. Then, the absorbance of the supernatant at 532 nm was measured using an ELISA plate reader.

(32) (3) The results are shown in FIG. 4. When the MLE-12 cells were incubated with 125 g/ml of particulate matter for 6, 24 and 48 hours, the content of MDA significantly increased. In particular, when the cells were incubated for 24 hours, the content of MDA increased by 2.5-fold compared to the control group not treated with particulate matter. In addition, when the cells were treated with particulate matter and various concentrations (62.5 and 125 g/ml) of the SHE, the content of MDA significantly decreased in a concentration-dependent manner at 6, 24 and 48 hours (FIG. 4).

Experimental Example 5: Effect of Sargassum horneri Extract (SHE) on Oxidative Stress-Induced DNA Damage in MLE-12 Cells Damaged by Particulate Matter

(33) (1) 8-Hydroxy-2-deoxyguanosine (8-OHdG), a marker of oxidative stress-induced DNA damage, is produced when the hydroxyl group at position 8 of a guanine molecule among DNA bases is oxidized (Particle and fibre toxicology, 2017, 14(38)). 8-OHdG is used as a marker for evaluating the degree of DNA damage caused by reactive oxygen species (ROS) that causes oxidative stress. Thus, MLE-12 cells were treated with particulate matter, and the degree of DNA damage caused by particulate matter and the DNA damage inhibitory effect of the Sargassum horneri extract (SHE) were evaluated by immunocytochemistry (ICC).

(34) (2) MLE-12 cells were dispensed in 12-well plates containing a coated coverslip at a density of 810.sup.4 cells per well, and then incubated in a 5% CO.sub.2 incubator at 37 C. for 12 hours to induce cell adhesion. Next, the cells were treated with the SHE (62.5 and 125 g/ml) and particulate matter (125 g/ml) and incubated in a 5% CO.sub.2 incubator at 37 C. for 24 hours. Then, the cells were washed, fixed in 4% paraformaldehyde (in PBS, pH 7.4), and then washed and subjected to immunocytochemistry (ICC). To analyze the expression of 8-OHdG in the MLE-12 cells, the cells were allowed to react with an anti-8-OHdG antibody (1:1000) at room temperature for 1 hour. After completion of the reaction, the cells were allowed to react with biotinylated anti-goat IgG at room temperature for 30 minutes and developed with 3,3-diaminobenzidine (DAB), and positive cells were counterstained with a hematoxylin solution, and then observed with an optical microscope.

(35) The results of observation with the optical microscope are shown in FIG. 5. The results of analyzing the expression of 8-OHdG indicated that the expression of 8-OHdG in the MLE-12 cells increased in the group treated with particulate matter alone (Dust only group) compared to the untreated control group (FIGS. 5A and 5B). It was observed that when the cells were treated with particulate matter and the SHE, the expression of 8-OHdG decreased in the group treated with the SHE compared to the group treated with particulate matter alone (Dust only group), and the expression of 8-OHdG effectively decreased as the concentration of the SHE increased (FIGS. 5C and 5D).

Experimental Example 6: Effect of Sargassum horneri Extract (SHE) on Expression of Cytokines and Chemokines in MLE-12 Cells Damaged by Particulate Matter

(36) (1) Using quantitative reverse transcription real-time PCR (qRT-PCR), the mRNA expression levels of inflammatory cytokines (IL-1, TNF-, IL-6, and IL-8) and chemokines (MCP1, and CCL5) were measured to evaluate the anti-inflammatory effect of the Sargassum horneri extract (SHE) on the MLE-12 cells damaged by particulate matter.

(37) (2) MLE-12 cells were dispensed in a 10-cm dish at a density of 510.sup.6 cells, and then incubated in a 5% CO.sub.2 incubator at 37 C. for 12 hours to induce cell adhesion. Next, the cells were treated with the SHE (62.5 and 125 g/ml) and particulate matter (125 g/ml) and incubated in a 5% CO.sub.2 incubator at 37 C. for 3, 24 and 48 hours. Thereafter, the cells were harvested and washed with DPBS, and RNA was extracted using Trizol reagent (Life technologies). Then, 500 l of chloroform (Sigma-Aldrich) was added to and mixed well with the RNA and centrifuged at 15,000 g for 15 minutes, and the supernatant was collected and isopropanol was added thereto, followed by centrifugation at 12,000 g and 4 C. for 10 minutes. After centrifugation, the RNA pellet was washed with 70% EtOH, dried at room temperature, and synthesized into cDNA using the Promega A3500 cDNA synthesis kit (St Louis, Cam USA). The cDNA was subjected to PCR using the StepOnePlus real-time PCR system (Applied Biosystems, Foster City, Calif.) and the Power SYBR Green PCR Master Mix (Applied Biosystems, USA). The primers used in this experiment were as follows: IL-1: 5-GCT ACC TGT GTC TTT CCC GTC G-3, 5-TTG TCG TTG CTT GGT TCT CCT TG-3; TNF-: 5-GGC AGC TTC TGT CCC TTT CAC TC-3, 5-CAC TTG GTG GTT TGC TAC GAC G-3; MCP1: 5-AACTGAAGCTCGCACTCTCG-3, 5-TCAGCACAGATCTCCTTGGC-3; CCL5: 5-GGAGTATTTCTACACCAGCAGCAAG-3, 5-GGCTAGGACTAGAGCAAGCAATGAC-3; COX2: 5-GCA AAT CCT TGC TGT TCC AAT C-3, 5-GGA GAA GGC TTC CCA GCT TTT G-3; GAPDH: 5-AAC GAC CCC TTC ATT GAC C-3, 5-TCA GAT GCC TGC TTC ACC C-3.

(38) (3) The results are shown in FIGS. 6 and 7. When the MLE-12 cells were treated with 125 g/ml of particulate matter and incubated for 3, 24 and 48 hours, the mRNA expression expressions of IL-1, TNF-, IL-6, IL-8, MCP1, CCL5 and COX-2 all increased compared to those in the group not treated with particulate matter. When the cells were treated with particulate matter and 62.5 and 125 g/ml of the SHE, the mRNA expression expressions of IL-1, TNF-, IL-6, IL-8, MCP1, CCL5 and COX-2 significantly decreased.

Experimental Example 7: Effect of Sargassum horneri Extract (SHE) on the Mechanism of Secretion of Inflammatory Cytokines in MLE-12 Cells Damaged by Particulate Matter

(39) (1) The expression levels of MAP-kinase p38, c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK) in a mitogen-activated protein kinase (MAPK) pathway, which is a cell signaling mechanism involved in various intracellular mechanisms, including cell growth, cell differentiation and cell death, were measured to evaluate the effect of the Sargassum horneri extract (SHE) on the MAPK signaling pathway in MLE-12 cells damaged by particulate matter.

(40) (2) MLE-12 cells were dispensed in a 10-cm dish at a density of 110.sup.7 cell, and then incubated in a 5% CO.sub.2 incubator at 37 C. for 12 hours to induce cell adhesion. Next, the cells were treated with SHE (62.5 and 125 g/ml) and particulate matter (125 g/ml) and incubated in a 5% CO.sub.2 incubator at 37 C. for 3, 24 and 48 hours. The cytosolic protein in the MLE-12 cells was isolated using the NE-PER Nuclear and Cytoplasmic extraction kit (Thermo scientific, Rockford, USA). 40 g of the cytosolic protein was electrophoresed on SDS-PAGE (10-12%), and the separated protein was transferred to a nitrocellulose membrane at 100V for 120 minutes. Next, to inhibit non-specific reactions, the membrane was allowed to react with 2% skim milk (Maeil, South of Korea) at room temperature for 1 hour, and allowed to react with p-38 (1:1000, Cell signaling Technology, MA, USA), phospho-p38 (1:1000, Cell signaling Technology, MA, USA), ERK (1:1000, Cell signaling Technology, MA, USA), phospho-ERK (1:1000, Cell signaling Technology, MA, USA), JNK (1:200, Santa Cruz Biotechnology, CA, USA), and p-JNK (1:200, Santa Cruz Biotechnology, CA, USA) antibodies for hour, and then allowed to react at 4 C. overnight. Next, the membrane was allowed to react with horseradish peroxidase (HRP)-conjugated anti-mouse IgG, anti-rabbit IgG (1:2000, Invitrogen, Carlsbad, Calif., USA) for 45 minutes. Color development was performed using Westzol (tNtRON Biotechnology, Sungnam, Korea), and band images were obtained using Fusion Solo (Vilber Lourmat, Eberhardzell, Germany). The band images were analyzed using the Image J software (v1.46), and intensity was calculated by comparing the expression level with -actin (1:2000, Sigma, Saint Louis, USA).

(41) (3) The results are shown in FIG. 8. When the MLE-12 cells were treated with 125 g/ml of particulate matter and incubated for 3 hours, the phosphorylation of ERK, p38 and JNK increased. When the MLE-12 cells were treated with 125 g/ml of particulate matter and incubated for 3 hours, the phosphorylation of ERK increased by 3.4-fold compared to that in the untreated group, and when the cells were treated with particulate matter and 62.5 g/ml of the SHE, the phosphorylation of ERK decreased by 2.0-fold, and when the cells were treated with particulate matter and 125 g/ml of the SHE, the phosphorylation of ERK decreased by 14.0-fold (FIGS. 8A and 8B). When the MLE-12 cells treated with 125 g/ml of particulate matter and incubated for 3 hours, the phosphorylation of p38 increased by 1.1-fold compared to that in the untreated group, and when the cells were treated with particulate matter and 62.5 g/ml of the SHE, the phosphorylation of p38 decreased by 1.3-fold, and when the cells were treated with particulate matter and 125 g/ml of the SHE, the phosphorylation of p38 decreased by 1.4-fold (FIGS. 8A and 8C). When the MLE-12 cells treated with 125 g/ml of particulate matter and incubated for 24 hours, the phosphorylation of p38 increased by 2.4-fold compared to that in the untreated group, and when the cells were treated with particulate matter and 62.5 g/ml of the SHE, the phosphorylation of p38 decreased by 2.5-fold, and when the cells were treated with particulate matter and 125 g/ml of the SHE, the phosphorylation of p38 decreased by 4.6-fold (FIGS. 8A and 8C). When the MLE-12 cells were treated with 125 g/ml of particulate matter and incubated for 3 hours, the phosphorylation of JNK increased by 3.0-fold compared to that in the untreated group, and when the cells were treated with particulate matter and 62.5 g/ml of the SHE, the phosphorylation of JNK decreased by 1.2-fold, and when the cells were treated with particulate matter and 125 g/ml of the SHE, the phosphorylation of JNK decreased by 1.1-fold (FIGS. 8A and 8D). In addition, when the MLE-12 cells were treated with 125 g/ml of particulate matter and incubated for 24 hours, the phosphorylation of JNK increased by 1.4-fold compared to that in the untreated group, and when the cells were treated with particulate matter and 62.5 g/ml of the SHE, the phosphorylation of JNK decreased by 1.3-fold, and when the cells were treated with particulate matter and 125 g/ml of the SHE, the phosphorylation of JNK decreased by 1.4-fold (FIGS. 8A and 8D).

Experimental Example 8: Experimental Animal Models

Experimental Example 8-1: Construction of Experimental Groups and Administration of Sample

(42) As experimental animals, four 7-8-week-old BALB/c mice per group were used. The experimental groups used were divided into a normal control group (hereinafter referred to as healthy control group), a group inhaled with particulate matter alone (hereinafter referred to as dust only group), an ovalbumin (hereinafter referred to as OVA)-sensitized group (hereinafter OVA only), an OVA sensitized+particulate matter-inhaled group (hereinafter referred to as OVA+dust group), an OVA sensitized+particulate matter-inhaled and sample-administered group (hereinafter referred to as OVA+dust+200 mg/kg SHE group or OVA+dust+400 mg/kg SHE group), and an OVA-sensitized+particulate matter-inhaled and allergic respiratory inflammation anti-inflammatory drug (Prednisone)-administered group (OVA+dust+5 mg/kg Prednisone group). 10 g of OVA and 2 mg of Al(OH).sub.3 were mixed well with 200 l of saline, and then intraperitoneally (i.p.) administered once to BALB/c mice. After 15 days, particulate matter was inhaled daily into the particulate matter-inhaled group at a concentration of 5 mg/m.sup.3 by nebulizer for 30 minutes during a period of 7 days. Together with inhalation of particulate matter, 200 or 400 mg/kg of the SHE was orally administered daily to the SHE-administered group, and 5 mg/kg of prednisone was orally administered daily to the allergic respiratory inflammation-treated group (FIG. 9).

Experimental Example 8-2: Effect of Sargassum horneri Extract (SHE) on Differential Cell Count in Blood of Particulate Matter-Inhaled Animal Models

(43) (1) The BALB/c mice were euthanized, and then blood was sampled by cardiac puncture using a heparin syringe, thereby obtaining mouse blood for each group. Then, the blood was plated on a slide glass to a thin thickness, and then stained with Diff Quick solution, and neutrophils, lymphocytes, monocytes, eosinophils, and basophils were counted under a microscope and compared.

(44) (2) The results of the differential cell counting are shown in FIGS. 10 and 11. The infiltration of neutrophils, eosinophils and basophils significantly increased in the OVA+dust group compared to the healthy control group (FIGS. 10A, 11A and 11B). The infiltration of eosinophils decreased by 6.6-fold in the OVA+dust+200 mg/kg SHE group administered with the SHE, and decreased by 10.0-fold in the OVA+dust+400 mg/kg SHE group (FIG. 11A). In addition, in the OVA+dust+400 mg/kg SHE group, the infiltration of neutrophils decreased by 1.5-fold (FIG. 10A), and the infiltration of basophils decreased by 3.0-fold (FIG. 11B). However, the infiltration of lymphocytes and monocytes was not changed by OVA and particulate matter (FIGS. 10B and 10C).

Experimental Example 8-3: Effect of Sargassum horneri Extract (SHE) on Differential Cell Count in Bronchoalveolar Lavage Fluid (BALF) of Particulate Matter-Inhaled Animal Models

(45) (1) Since cells contained in bronchoalveolar lavage fluid reflect the inflammatory state of lung tissue and bronchi (J Toxicol Environ Health A 2017, 80(4), 197-207), changes in differential cell counts of bronchoalveolar lavage fluid were observed to evaluate the effect of the Sargassum horneri extract (SHE) on pulmonary and bronchial inflammations caused by particulate matter inhalation.

(46) (2) The euthanized, and then bronchoalveolar lavage fluid was collected by injecting DPBS into the airway by an intra-tracheal catheter. The collected bronchoalveolar lavage fluid was centrifuged at 3000 g and 4 C. for 5 minutes, and then fixed with methanol and attached to a slide. The cell-attached slide was stained with Diff-Qick solution, and neutrophils, lymphocytes, monocytes, eosinophils and basophils were counted under a microscope and compared.

(47) (3) The results of the differential cell counting are shown in FIGS. 12 and 13. The infiltration of lymphocytes and eosinophils significantly increased in the OVA+dust group compared to the healthy control group (FIGS. 12B and 13A). In the OVA+dust+400 mg/kg SHE group administered with the SHE and the allergic respiratory inflammation anti-inflammatory drug-administered group, the infiltration of lymphocytes and eosinophils significantly decreased, and the infiltration of basophils showed a tendency to decrease without significance (FIGS. 12B, 13A and 13B). However, the infiltration of neutrophils and monocytes was not changed by OVA and particulate matter (FIGS. 12A and 12C).

Experimental Example 8-4: Effect of Sargassum horneri Extract (SHE) on Histopathological Changes of Tracheas and Lungs of Particulate Matter-Inhaled Animal Models

(48) (1) The BALB/c mice were euthanized and autopsied, and the tracheas and lungs were collected, and then paraffin blocks were prepared according to a conventional method. The paraffin blocks were sectioned to 3 m, attached to slides, deparaffinized, dehydrated, stained with Mayer's hematoxylin solution and eosin solution, dehydrated, cleared, and then mounted.

(49) (2) The results are shown in FIG. 14. The infiltration of inflammatory cells significantly increased in the tracheas of the OVA+dust group compared to the healthy control (FIG. 14A), whereas the infiltration of inflammatory cells decreased in the tracheas of the OVA+dust+200 mg/kg SHE administered with the SHE. In particular, it was confirmed that the infiltration of inflammatory cells in the OVA+dust+400 mg/kg SHE group effectively decreased to a level similar to that in the allergic respiratory inflammation anti-inflammatory drug-administered group (FIG. 14A). In the lung tissue, similarly to the trachea, the infiltration of inflammatory cells in the OVA+dust group significantly increased compared to that in the healthy control group (FIG. 14B), whereas the infiltration of inflammatory cells into lung tissue in the OVA+dust+200 mg/kg SHE and 400 mg/kg SHE groups administered with the SHE significantly decreased in a SHE concentration-dependent manner (FIG. 14B). In the graph of FIG. 14, 0, normal; 1, few cells observed; 2, a ring of inflammatory cells one cell layer deep; 3, a ring of inflammatory cells 2-4 cells deep; and 4, a ring of inflammatory cells >4 cells deep.

Experimental Example 8-5: Effect of Sargassum horneri Extract (SHE) on Change in 8-OHdG Expression in Lungs of Particulate Matter-Inhaled Animal Models

(50) (1) 8-Hydroxy-2-deoxyguanosine (8-OHdG), a marker of oxidative stress-induced DNA damage, is produced when the hydroxyl group at position 8 of a guanine molecule among DNA bases is oxidized. 8-OHdG is used as a marker for evaluating the degree of DNA damage caused by reactive oxygen species (ROS) that causes oxidative stress (Particle and fibre toxicology, 2017, 14(38)). Thus, the degree of oxidative stress caused by particulate matter inhalation and the antioxidant effect of the Sargassum horneri extract (SHE) were evaluated by immunocytochemistry (ICC).

(51) (2) The BALB/c mice were euthanized and autopsied, and the lung tissue was collected, and then prepared into a paraffin block according to a conventional method. The paraffin block was sectioned to 3 m, and the section was attached to a slide, and then subjected to immunohistochemistry (IHC). To analyze the expression of 8-OHdG, the section was allowed to react with an anti-8-OHdG antibody (1:2000) at room temperature for 1 hour. After completion of the reaction, the section was allowed to react with biotinylated anti-goat IgG at room temperature for 30 minutes and developed with 3,3-diaminobenzidine (DAB), and positive cells were counterstained with a hematoxylin solution, and then observed with an optical microscope.

(52) (3) The results of analyzing the expression of 8-OHdG are shown in FIG. 15. In neutrophils, eosinophils and epithelial cell nuclei, 8-OHdG-positive cells significantly increased in the dust only group, the OVA only group and the OVA+dust group compared to the healthy control group, but 8-OHdG-positive cells significantly decreased in the OVA+dust+200 mg/kg SHE group and the OVA+dust+400 mg/kg SHE group. In particular, 8-OHdG-positive cells in the OVA+dust+400 mg/kg SHE group significantly decreased to levels similar to those in the OVA+dust+5 mg/kg Prednisone.

Experimental Example 8-6: Effect of Sargassum horneri Extract (SHE) on Changes in Expression of Gr-1 in Tracheas and Lungs of Particulate Matter-Inhaled Animal Models

(53) (1) Gr-1, a granulocyte marker involved in the differentiation and maturation of granulocytes, is known to act on immune mechanisms by secreting cytokines when infection or the like occurs (Immunity 2012, 36(3), 451-63). Thus, the effect of the Sargassum horneri extract (SHE) on the particulate matter inhalation-induced infiltration of granulocytes into trachea and lung tissue was examined.

(54) (2) The BALB/c mice were euthanized and autopsied, and the trachea and lung tissue were collected, and then prepared into paraffin blocks according to a conventional method. The paraffin tissues were sectioned to 3 m, attached to slides, deparaffinized, dehydrated, and then immersed in 0.3% hydrogen peroxide solution in order to inhibit endogenous peroxidase in the tissue. Next, in order to nonspecific immune responses, the section was allowed to react with blocking rat serum for 30 minutes, and then allowed to react with mouse Ly-6G/Ly-6C (1:200, R&D Systems) at 4 C. overnight. After completion of the reaction, the section was allowed to react with biotinylated anti-rabbit serum at room temperature for 45 minutes, and then developed with 3,3-diaminbenzidine (DAB, Vector) and counterstained with a hematoxylin solution. Between the steps, the section was washed sufficiently with phosphate buffered saline (PBS), 0.3% PBS-triton X100, dehydrated, cleared, and then mounted.

(55) (3) The results are shown in FIG. 16. The granulocyte infiltration of trachea in the OVA+dust group significantly increased by 5.2-fold compared to that in the healthy control, whereas the infiltration of granulocytes in the OVA+dust+200 mg/kg SHE and 400 mg/kg SHE groups administered with the SHE decreased. In particular, the infiltration of granulocytes in the group administered with 400 mg/kg of the SHE significantly decreased by 2.2-fold, which was similar that in the allergic respiratory inflammation anti-inflammatory drug-administered group (FIG. 16A). In the lung tissue, similarly to the trachea, the infiltration of granulocytes into trachea in the OVA+dust group significantly increased by 5.6-fold compared to that in the healthy control group, whereas the infiltration of granulocytes in the OVA+dust+200 mg/kg SHE and 400 mg/kg SHE groups administered with the SHE significantly decreased by 1.5-fold and 1.7-fold, respectively (FIG. 16B). This decrease in the infiltration of granulocytes was similar to that in the allergic respiratory inflammation anti-inflammatory drug-administered group (2.0-fold decrease in granulocyte infiltration compared to the healthy control group), suggesting that administration of the SHE effectively inhibited the particulate matter-induced infiltration of granulocytes into trachea and lung tissue.

Example 8-7: Effect of Sargassum horneri Extract (SHE) on Infiltration of Eosinophilic Leukocytes into Trachea and Lung Tissue in Particulate Matter-Inhaled Animal Models

(56) (1) The characteristic pathologic findings of chronic allergic respiratory inflammatory disease are increased eosinophilic leukocytes, and eosinophils containing inflammatory proteins play an important role in the onset of allergic respiratory inflammation by inducing airway epithelial cell damage, increasing airway hypersensitivity, and inducing degranulation of mast cells (World Allergy Organ J 2016, 9, 7). Thus, the effect of the Sargassum horneri extract (SHE) on the particulate matter inhalation-induced infiltration of eosinophilic leukocytes into trachea and lung tissue was examined.

(57) (2) The BALB/c mice were euthanized and autopsied, and the trachea and lung tissue were collected, and then prepared into paraffin blocks according to a conventional method. The paraffin blocks were sectioned to 3 m and attached to slides, and then the tissue section was stained with 0.05% Congo red in 50% EtOH solution and mounted.

(58) (3) The results are shown in FIG. 17. The infiltration of eosinophilic leukocytes into trachea in the OVA+dust group significantly increased by 4.4-fold compared to that of the healthy control group, whereas the infiltration of eosinophilic leukocytes in the OVA+dust+200 mg/kg SHE and 400 mg/kg SHE groups administered with the SHE showed a tendency to decrease. In particular, the infiltration of eosinophilic leukocytes in the group administered with 400 mg/kg of the SHE significantly decreased by 1.9-fold (FIG. 17A). In the lung tissue, similarly to the trachea, the infiltration of eosinophilic leukocytes in the OVA+dust group significantly increased compared to that in the healthy control group, and the infiltration of eosinophilic leukocytes in the group administered with the SHE showed a tendency to decrease in a concentration-dependent manner. In particular, the infiltration of eosinophils in the OVA+dust+400 mg/kg SHE group decreased by 1.7-fold (FIG. 17B).

Experimental Example 8-8: Effect of Sargassum horneri Extract (SHE) on Infiltration of Mast Cells into Trachea in Particulate Matter-Inhaled Animal Models

(59) (1) Mast cells are mainly found in connective tissue and mucosa, and are characterized by having granules in the cytoplasm. Granulocytes contain chemical mediators, such as histamine, prostaglandin and cytokines, and are released when mast cells are activated by external stimuli, and thus these granulocytes are involved in inflammatory responses, hypersensitive responses, etc., causing bronchial constriction, increased mucus secretion, etc. (World Allergy Organ J 2016, 9, 7). Thus, the effect of the Sargassum horneri extract (SHE) on the particulate matter inhalation-induced infiltration of mast cells into trachea was examined.

(60) (2) The BALB/c mice were euthanized and autopsied, and the trachea was collected, and then prepared into a paraffin block according to a conventional method. The paraffin block was sectioned to 3 m and attached to a slide, and then the tissue section was stained with 0.05% Toluidine blue solution and mounted.

(61) (3) The results are shown in FIG. 18. The infiltration of mast cells into trachea in the OVA+dust group significantly increased compared to the healthy control group, and also significantly increased compared to that in the OVA only group and the dust only group (FIG. 18A). However, the infiltration of mast cells into trachea in the OVA+dust+200 mg/kg SHE and 400 mg/kg SHE groups administered with the SHE significantly decreased (FIG. 18A). In addition, the degranulation rate of mast cells in the OVA+dust group increased compared to that in the healthy control group (FIG. 18B), and significantly increased in the OVA only group and the dust only group (FIG. 18B). However, the degranulation rate of mast cells in the OVA+dust+200 mg/kg SHE and 400 mg/kg SHE groups administered with the SHE decreased, and particularly, the degranulation rate of mast cells in the OVA+dust+400 mg/kg SHE group effectively decreased compared to that in the OVA+dust+5 mg/kg Prednisone group (FIG. 18B).

Experimental Example 8-9: Effect of Sargassum horneri Extract (SHE) on Mucus Secretion and Goblet Cell Proliferation in Particulate Matter-Inhaled Animal Models

(62) (1) In the case of exposure to repeated airway inflammation due to foreign substances, airway remodeling occurs, and symptoms, such as increased goblet cells and mucous gland hyperplasia, appear (Chest 2018, 154(1), 169-176). Thus, the effect of the Sargassum horneri extract (SHE) on the mucus secretion and goblet cell proliferation caused by particulate matter inhalation was examined.

(63) (2) The BALB/c mice were euthanized and autopsied, and the trachea and lung tissue were collected, and then prepared into paraffin blocks according to a conventional method. The paraffin blocks were sectioned to 3 m, attached to slides, and then stained with periodic acid of Schiff (PAS). In addition, for staining with anti-mucin 5AC antibody (R&D Abcam), the paraffin blocks were sectioned to 3 m, attached to slides, and then deparaffinized, dehydrated, and immersed in 0.3% hydrogen peroxide solution in order to inhibit endogenous peroxidase in the tissue. Next, in order to inhibit nonspecific reactions, the section was allowed to react with blocking horse serum for 30 minutes, and then allowed to react with anti-mucin 5AC antibody (1:500, R&D Abcam) at 4 C. overnight. After completion of the reaction, the section was allowed to react with biotinylated anti-mouse serum at room temperature for 45 minutes, and then developed with 3, 3-diaminbenzidine (DAB, Vector) and counterstained with a hematoxylin solution. Between the steps, the section was washed sufficiently with phosphate buffered saline (PBS), 0.3% PBS-triton X100, and dehydrated, cleared, and then mounted.

(64) (3) The results are shown in FIGS. 19 and 20. In the results of trachea PAS and immunohistochemical staining for the mucin-5AC antibody, mucus secretion in the OVA+dust group increased compared to that in the healthy control, and excessive goblet cell proliferation was observed in the OVA+dust group. However, in the group administered with the SHE, mucus secretion and goblet cell proliferation decreased. In particular, mucus secretion and goblet cell proliferation in the OVA+dust+400 mg/kg SHE group decreased to levels similar to those in the OVA+dust+5 mg/kg Prednisone group (FIGS. 19A and 19B). Meanwhile, mucus secretion into trachea in the OVA+dust group significantly increased compared to those in the OVA only group and the dust only (FIG. 19B).

(65) (4) In addition, mucus secretion in the lung tissue of the OVA+dust group increased compared to that in the healthy control group, and excessive proliferation of goblet cells was observed in the OVA+dust group. However, mucus secretion and goblet cell proliferation in the OVA+dust+200 mg/kg SHE and 400 mg/kg SHE groups administered with the SHE decreased to levels similar to those in the OVA+dust+5 mg/kg Prednisone group in a concentration-dependent manner (FIGS. 20A and 20B).

Experimental Example 8-10: Effect of Sargassum horneri Extract (SHE) on Cell Population Changes in Lung Tissue of Particulate Matter-Inhaled Animal Models

(66) (1) Allergic respiratory inflammatory diseases are chronic inflammatory diseases in which the immune system is involved, and are characterized by the infiltration of T cells and eosinophils and the activation of mast cells and basophils (World Allergy Organ J 2016, 9, 7). Thus, in order to evaluate the effect of the Sargassum horneri extract (SHE) on particulate matter inhalation-induced changes in immune cell populations, analysis was performed using flow cytometry.

(67) (2) The BALB/c mice were euthanized and autopsied, and the lung was collected, sectioned finely, allowed to react with 0.4 mg/ml of collagenase at 37 C. for 30 minutes, and stirred well at 8-10-minute intervals. Next, PBS was added thereto, and the solution was stirred well at 1500 rpm for 5 minutes. Next, for leukocyte lysis, ACK solution was added, and the mixture was allowed to react at room temperature for 10 minutes, and then washed with DPBS. The pulmonary single cell suspension was treated with an Fc blocker at 4 C. for 15 minutes to reduce nonspecific reactions. Next, the cells were allowed to react with FITC- or PE-, PerCp-CyTM5.5-, BV421-, AF700-labeled CD3e (145-2C11), CD4 (H129.19), CD8a (53-6.7), CD45 (30-F11), CD11b (M1/70), and CD11c (HL3) antibodies at 4 C. for 15 minutes. Next, the cells were washed with PBS and analyzed using the CytoFLEX flow cytometer (Beckman Coulter, Inc., Kraemer Blvd, Calif., USA).

(68) (3) The results are shown in FIG. 21. The number of CD3e.sup.+CD4.sup.+ helper T cells in the OVA+dust group increased by 1.3-fold compared to that in the healthy control group, and the number of CD3e.sup.+CD4.sup.+ helper T cells in the OVA+dust+400 mg/kg SHE administered with the SHE significantly decreased by 1.2-fold (FIG. 21A). In addition, the number of CD3e.sup.+CD8.sup.+ cytotoxic T cells significantly increased in the OVA+dust group compared to the healthy control group, and the numbers of CD3e.sup.+CD8.sup.+ cytotoxic T cells in the OVA+dust+200 mg/kg SHE and 400 mg/kg SHE groups administered with the SHE significantly decreased by 1.5-fold and 2.0-fold, respectively, in a concentration-dependent manner (FIG. 21B). The number of CD45.sup.+CD11c.sup.+ dendritic cells in the OVA+dust group increased by 4.0-fold compared to that in the healthy control group, and the numbers of CD45.sup.+CD11c.sup.+ dendritic cells in the OVA+dust+200 mg/kg SHE and 400 mg/kg SHE groups administered with the SHE significantly decreased by 1.3-fold and 1.4-fold, respectively, in a concentration-dependent manner (FIG. 21C). In addition, the number of CD11.sup.c+CD11b.sup.+ macrophages in the OVA+dust group showed a tendency to increase compared to that in the healthy control group, and the number of CD11c.sup.+CD11b.sup.+ macrophages significantly decreased in the OVA+dust+200 mg/kg SHE and 400 mg/kg SHE groups administered with the SHE (FIG. 21D).

(69) Statistical Processing

(70) Each experiment was repeated 3 times or more (n=3 or more per group). The experimental results were expressed as meanstandard deviation, and significance was analyzed using Student's t-test (Microsoft Office Excel) (**; P<0.005, ***; P<0.0005, ; P<0.005, ; P<0.0005, #; P<0.05).

(71) As described above, according to the present disclosure, there may be provided a composition for alleviating lung injury caused by particulate matter or the like and a composition for alleviating respiratory disease, in which the composition contains a Sargassum horneri extract. The composition of the present disclosure may be commercialized as a functional health food or a pharmaceutical product.