Compound isolated from Pseudolysimachion rotundum var. subintegrum, the composition comprising the same for treating allergic disease, inflammatory disease, asthma or chronic obstructive pulmonary disease and the use thereof

Abstract

A compound (3R,5S,5aS,6R,7S,8R,8aS)-8-chloro-8a-hydroxy-5-(2-hydroxy acetoxy) hexahydro-1H-3,6-methanocyclopenta[e][1,3]dioxepin-7-yl 3,4-dihydroxybenzoate (KS534), the isomer thereof, the pharmaceutically acceptable salt or solvates thereof, a composition comprising the same as an active ingredient for treating allergic disease, inflammatory disease, asthma or chronic obstructive pulmonary disease (COPD)

Claims

1. The compound (3R,5S,5aS,6R,7S,8R,8aS)-8-chloro-8a-hydroxy-5-(2-hydroxy acetoxy) hexahydro-1H-3,6-methanocyclopenta[e][1,3]dioxepin-7-yl3,4-dihydroxybenzoate (KS534) represented by chemical formula (1), the isomer thereof, the pharmaceutically acceptable salt or solvates thereof: ##STR00002##

2. A pharmaceutical composition comprising the compound (KS534), the isomer thereof, the pharmaceutically acceptable salt or solvates thereof as set forth in claim 1 isolated from Pseudolysimachion rotundum var subintegrum to treat a disease selected from the group consisting of allergic, inflammatory, asthma and chronic obstructive pulmonary (COPD).

3. A pharmaceutical composition comprising the compound (KS534) isolated from the extract of Pseudolysimachion rotundum var subintegrum as set forth in claim 1 and a pharmaceutically acceptable carrier or excipient, for the treatment a disease selected from the group consisting of allergic, inflammatory, asthma and chronic obstructive pulmonary (COPD).

4. A method of treating or allergic disease, inflammatory disease, asthma or chronic obstructive pulmonary disease (COPD) in a mammal, wherein the method comprises administering a therapeutically effective amount of compound (KS534), the isomer thereof, the pharmaceutically acceptable salt or solvates thereof as set forth in claim 1 into the mammal suffering from a disease selected from the group consisting of allergic, inflammatory, asthma and chronic obstructive pulmonary (COPD).

5. The method of claim 4, wherein the compound (KS534), the isomer thereof, the pharmaceutically acceptable salt or solvates thereof further comprise pharmaceutically acceptable carrier or excipient.

6. A health functional food comprising a therapeutically effective amount of the compound (KS534), the isomer thereof, the pharmaceutically acceptable salt or solvates thereof as set forth in claim 1 as an active ingredient for the alleviation of a disease selected from the group consisting of allergic, inflammatory, asthma and chronic obstructive pulmonary (COPD).

7. A health care food comprising a therapeutically effective amount of the compound (KS534), the isomer thereof, the pharmaceutically acceptable salt or solvates thereof as set forth in claim 1, together with a sitologically acceptable additive for the or alleviation of a disease selected from the group consisting of allergic, inflammatory, asthma and chronic obstructive pulmonary (COPD).

Description

DESCRIPTION OF DRAWINGS

[0070] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which.

[0071] FIG. 1 shows .sup.1H-.sup.1H COSY and HMBC correlation of KS534 compound.

[0072] FIG. 2 shows the effect of test sample on the mRNA expression of MUCSAC using by qRT-PCR. Inhibitory effect of KS-534 on mRNA expression levels of MUCSAC using qRT-PCR. Statistical analysis was performed with Student's t-test. P values of *, <0.05 were considered statistically significant.

[0073] FIG. 3 shows the inhibition effect of test sample on the activity of TNF-alpha induced NF-kappa B using by luciferase reporter assay. Suppressive effects of NF-kM activity by KS-534 using luciferase assay. Statistical analysis was performed by Student's-t-test and P value (**, <0.01) was regarded as being statistical significant.

[0074] FIG. 4 presents the inhibition effect of test sample on the expression level of MUCSAC protein using by modified-ELISA method. Inhibitory effect of KS-534 on the protein expression levels of MUCSAC using modified-ELISA. Statistical analysis was performed by Student's t-test and P value (**, <0.01 and ***, <0.001) was regarded as being statistical significant.

[0075] FIG. 5 presents the effect of the inventive compound on the number of inflammatory cells in BALF. Effects of KS-534 on inflammatory cell count in BALF.

[0076] FIG. 6 presents the effect of the inventive compound on the number of inflammatory cytokines in BALF. Effects of KS-534 on cytokines in BALF.

[0077] FIG. 7 presents the effect of the inventive compound on the number of total IgE and level of OVA-specific IgE in serum. Effects of KS-534 on total IgE and OVA-specific IgE in serum.

[0078] FIG. 8 presents the effect of the inventive compound on the inflammatory response in lung tissue cell using by the histological examination. Effects of KS-534 on inflammatory responses in lung tissue.

[0079] FIG. 9 presents the effect of the inventive compound on the mucosal hypersecretion in lung tissue cell using the histological examination. Effects of KS-534 on mucus production in lung tissue.

[0080] FIG. 10 presents the effect of the inventive compound on the number of inflammatory cells in BALF. Effects of KS-534 on inflammatory cell counts in BALF.

[0081] FIG. 11 presents the effect of the inventive compound on the reproduction of ROS in BALF. Effects of KS-534 on reactive oxygen species production.

[0082] FIG. 12 presents the effect of the inventive compound on the level of BALF contents and elastase activity in BALF. Effects of KS-534 on BALF contents and elastase activity.

[0083] FIG. 13 represents the effect of the inventive compound on the level of IL-6 and TNF-alpha in BALF. Effects of KS-534 on IL-6 and TNF-α in BALF.

[0084] FIG. 14 depicts the effect of the inventive compound on the inflammatory response in lung tissue cell using the histological examination. Effects of KS-534 on inflammatory responses in lung tissue.

BEST MODE FOR CARRYING OUT THE INVENTION

[0085] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[0086] The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

EXAMPLES

[0087] The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

Reference Example 1

Analysis Apparatus

[0088] Melting point was determined with no correction by melting point determination apparatus (Koflermicrohostage); optical rotation by Jasco P-1020 polarimeter; UV data by UV-VIS 2450 spectrometer; FT-IR spectra by Jasco FT/IR-4200; NMR spectra by Varian UNITY 400 MHz FT-NMR spectrometer using by TMS as an internal standard; HRESIMS by Waters Q-TOF Premier spectrometer; and HPLC analysis by Gilson HPLC using by UV/VIS-155 detector and pump 305 in the experiment.

Example 1

Preparation of Novel Compound (KS 534) from Pseudolysimachion Rotundum var Subintegrum

1-1. Preparation of Crude Extract

[0089] 2.0 kg of dried Pseudolysimachion rotundum var subintegrum (cultivated at 244, Soi-myeon Eumseong-gun Chungcheongbuk-do in Korea according to GAP, KRIBB 0020697, plant extract bank of KRIBB, Taejeon, KOREA) cut into small pieces and mixed with 10 L of 40% ethanol. The mixture was stirred at room temperature for 24 hours and extracted with reflux extraction at 78° C. for 12 hours to collect the filtrate, three times. The extract was filtered with filter paper to remove the debris. The collected filtrate was concentrated by rotary evaporator (EYELA, N-2100, Japan) at 55-65° C. under reduced pressure and dried with freezing dryer to obtain 198.7 g of dried crude extract

1-2. Preparation of Purified Extract

[0090] 20 g of dried crude extract was dissolved in mixture solvent (25% MeOH-water) and subjected to further purification using by preparative reverse phase chromatography (Zeoprep C18, 75 μm, 200×250 mm, Zeochem, Louisville, U.S.A.). The eluting fractions (f1-f4) were collected and concentrated under vaccuo. 5.0 g of the fraction f2 was loaded to medium pressure liquid chromatography (MPLC) using by column: RP C-18 (Zeoprep C18, 20×250 mm, 10 μm, Zeochem, Louisville, U.S.A.) and eluting solvent (MeOH-water solution=2:8, 3:7, 4:6, 10:0) to afford 5 sub-fractions, f2a, f2b, f2c, f2d and f2e.

1-3. Preparation of Novel Compound KS534

[0091] 0.8 g of sub-fraction (f2c) was subjected to semi-preparative HPLC (Synergy Polar-RP 4 μm, 21.2×250 mm, Phenomenex, Torrance, Calif., U.S.A., 22% MeCN in H.sub.2O) to obtain bright brown powdered novel iridoid compound (3R,5S,5a5,6R,7S,8R,8aS)-8-chloro-8a-hydroxy-5-(2-hydroxy acetoxy) hexahydro-1H-3,6-methanocyclopenta[e][1,3]dioxepin-7-yl 3,4-dihydroxybenzoate (KS534; C.sub.22H.sub.26ClO.sub.13) showing physic-chemical property.

[0092] [α].sup.20.sub.D-30.0° (c 0.2, MeOH).

[0093] HRESIMS (observed m/z 533.1035 [M−H].sup.−): quasimolecular 3:1 ion cluster

[0094] IR spectrum: 3412 cm.sup.−1 (hydroxyl group); 1692 cm.sup.−1. (a, f1-unsaturated ester carbonyl)

[0095] .sup.1H and .sup.13C NMR spectra: Table 1

[0096] .sup.1H-.sup.1H COSY spectrum (FIG. 1)

[0097] NOESY spectrum (FIG. 1)

TABLE-US-00001 TABLE 1 .sup.1H (400 MHz) and .sup.13C (100 MHz) NMR spectroscopic data for KS-534 in DMSO-d6 KS-534 Position δ.sub.C δ.sub.H (J in Hz) Agluc 1 90.6 5.55, d (1.6) 3 93.5 5.30, d (2.4) 4 32.9 2.38, dd (13.4, 8.2) 1.94, dd (13.4, 2.4) 5 33.0 2.26, ddd (9.6, 8.8, 2.2) 6 85.7 4.96, dd (8.8, 2.2) 7 69.2 4.54, d (8.8) 8 78.3 9 46.4 2.55, br d (10.0) 10  60.8 3.56, d (12.4) 3.86, d (12.4) 11  Glc .sup. 1′ 96.8 4.50, d (7.6) .sup. 2′ 73.0 2.91, d (8.4) .sup. 3′ 76.5 3.13, m .sup. 4′ 70.1 3.04, t (9.2) .sup. 5′ 77.1 3.13, m .sup. 6′ 60.9 3.69, d (8.8) 3.43, dd (12.0, 5.6) Aroyl  1″ 119.8  2″ 116.4 7.39, d (2.1)  3″ 145.1  4″ 150.9 6.83, d (8.0)  5″ 115.4 7.37, dd (8.0, 2.1)  6″ 122.1  7″ 165.6

Experimental Example 1

Preliminary Determination of the Cytotoxicity

[0098] In order to determine the cytotoxicity of inventive compound, following preliminary test was performed by the method disclosed in the literature (Shiyama et al., Talanta, 1997, 44, 1299-1305; Tominaga et al., Anal. Commun., 1999, 36, 47-50).

1-1. Preparation of Cell Line and Cell Culture

[0099] HT1080 (CRL-12012), H292 (CRL-1848) and murine EL4 cells (TIB-39) were purchased from company (American Type Culture Collection).

[0100] HT1080 (CRL-12012) and murine EL4 cells were cultured in DMEM medium (SB30243.01, Dulbecco's modified Eagle's medium; DMEM, HyClone) supplemented with 10% FBS (Fetal Bovine Serum; Gibco) and antibiotic (100 units/ml penicillin+100 units/ml streptomycin). H292 cell was cultured in RPMI medium (SH30027.01, RPMI 1640, Gibco) supplemented with 10% FBS and antibiotic. The cells were cultures under the condition of humidified 5% CO.sub.2 atmosphere at 37° C. PMA (P8139, Phorbol 12-myristate 13-acetate) was purchased from the company (Sigma Chemical Co., St. Louis, Mo.) and TNF-α is from the company (300-01A, PeproTech, NJ USA Inc.) to use in the experiment.

1-2. Evaluation of Cytotoxicity in H292 Cell

[0101] H292 cell line, a human hung mucosal cancer cell line, was suspended in 10% FBS-supplemented RPMI medium in a concentration of 5×10.sup.4 cells/ml, and 100 μl of the suspension was inoculated into 96 well plates to adhere cell to the plate for 12 hours. Various concentrations of test sample (KS 534 compound) were treated therewith and cultured for 24 hours. 10 μL of CCK-8 solution was mixed with 90 μL of the medium and 100 μL of solution was added to each well. After reacting from 30 mins to 4 hours, the absorbance of the solution was determined at 570 nm. The cell viability was calculated according to following Math formulae 1 based on negative control group treated with 0.2% DMSO and the result was shown in Table 2.

Cell viability (%)=OD 570 nm (test group)/OD 570 nm (negative control group)×100   Math formulae 1

[0102] At the result, as shown in Table 2, it has been confirmed that KS 534 compound did not show cytotoxicity in H292 cell at less than 20 μm.

TABLE-US-00002 TABLE 2 concentration H292 cell viability Test sample (μm) (%, mean ± variation) Negative control group 0 100.00 ± 2.50 KS-534 2.5 101.88 ± 3.71 5 102.46 ± 3.96 10  99.89 ± 4.79 20  97.87 ± 2.92

Experimental Example 2

Inhibition on the Expression of MUC5AC

[0103] In order to determine the inhibitory effect on the expression of MUC5AC induced by TNF-alpha in H292 cell line, following test was performed:

2-1. determination of the Level of mRNA Expression

[0104] The isolation of total RNA, cDNA synthesis and quantitative real-time PCR were performed by the method disclosed in the literature (Kim et al., J. Cell. Biochem. 2008, 104, 1906-1917).

[0105] In summary, total RNA was isolated using by TRIzol reagent (155596-026, Life Technologies) and the first strand cDNA was synthesized using by enzyme (Omniscript Reverse Transcriptase, 205113, Qiagen, Calif.). The quantitative real-time PCR amplification was performed using by apparatus 1 (S1000 Thermal cycler, Bio-rad, USA) and apparatus 2 (2× Greenstar master mix, Bioneer, Korea) according to the manufacture's manual.

2-2. Effect on MUCSAC Expression

[0106] In order to determine the inhibitory effect on the expression of MUCSAC induced by TNF-alpha in H292 cell line, following test was performed.

[0107] The inhibitory effect of KS534 compound on MUC5AC mRNA expression was determined to verify the treating or preventing activity of inflammatory disease such as COPD.

[0108] Specifically, the reproduced amount of TNF-alpha induced MUC5AC was determined by Real-time polymerase chain reaction (real-time PCR), quantitative real time polymerase chain reaction (qPCR) and MUC5AC immunoassay. H292 cells were inoculated into 48 well plates in a concentration of 2×10.sup.4 cells/well to adhere for 24 hours and the culture medium was replaced with new 0.1% FBS containing medium to culture for 24 hours. 10 μm KS-534 was treated with therewith for 2 hours and 20 ng/ml of TNF-alpha was treated therewith to culture for 12 hours.

[0109] In order to extract total RNA, RNA was extracted using by Trizol B (Invitrogen) and cDNA was synthesized using by Omniscript RT kit (Qiagen, GmbH, Hilden, Germany) after the quantification. The synthesized cDNA was mixed with the template, MUC5AC and GAPDH primers as shown in Table 3, respectively, and performed to PCR using by PCR mix (PCR Master Mix, Bioneer, Korea) as follows: 5 mins at 94° C., 1 cycle for pre-denaturation; 30 sec at 95° C., 45 sec at 60° C. and 45 sec at 72° C., 30 cycles for denaturation; 10 mins at 72° C., 1 cycle for final extension.

TABLE-US-00003 TABLE 3 Gene (species) primer MUC5AC sense 5′-TGA TCA TCC AGC AGC AGG (Human) GCT-3′ antisense 5′-CCG AGC TCA GAG GAC ATA TGG G-3′ GAPDH sense 5′-CGG AGT CAA CGG ATTT GGT (Human) CGT AT-3′ antisense 5′-AGC CTT CTC CAT GGT GGT  GAA GAC-3′

[0110] At the result, it has been confirmed that the amount of MUC5AC was increased in the control group treated with TNF-alpha and the amount of mRNA expression of MUC5AC in the test group treated with 10 μm was decreased by 58.47% (inhibitory ratio of KS-534 on the MUCSAC expression: 41.52%) as can be seen in Table 4 and FIG. 2.

[0111] Accordingly, it has been verified that KS-534 compound inhibit the gene expression of MUCSAC and it is useful in treating COPD.

TABLE-US-00004 TABLE 4 The summary for inhibitory activity of Muc5AC mRNA expression by KS-534 Expressed level of MUC5AC mRNA (Relative % comparing with that concentration TNF-α in TNF-alpha Inhibitory sample (μm) (20 ng/ml) treatment group) ratio (%) Negative 0 − 7.02 ± 2.82 92.82 control TNF-alpha 0 + 100.00 ± 25.35  0.00 treatment KS-534 10 + 58.47 ± 18.50 41.52

Experimental Example 3

Inhibition on the activity of NF-Kappa B

[0112] In order to determine the inhibitory effect on the activity of NF-kappa B, following test was performed:

3-1. NF-Kappa B Luciferase Reporter Assay

[0113] NF-kappa B luciferase reporter assay was performed by following procedure disclosed in the literature (Pulin Che et al., Assay Drug Dev. Technol., 2012, 10, 61-68).

[0114] In order to establish the stable cells for NF-kappa B luciferase reporter assay, NF-kappa B response element was cloned into vector (pGL4.32 vector, E849A, Promega) according to the manufacture's protocol and infected to the cell (H292 cells, CRL-1848, ATCC) using by Lipofectamine 2000 (11668-019, Invitrogen). The infected cell was selected using by hygromycin (400 μg/ml) for 2 weeks and the colonies were individually cloned and proliferated. The cell was placed on the 96-well plates in a concentration of 5×10.sup.3 cell/well for 24 hours. The test sample was pre-treated in a pre-determined concentration and 20 ng/ml of TNF-alpha was treated therewith for 12 hours. The activity of luciferase enzyme was determined by E6110 kit (OneGlo Luciferase Assay kit, Promega) and DMSO was used as a vehicle control.

3-2. Inhibitory Effect on the Activation of NF-Kappa B Transcription Factor

[0115] It has been reported that TNF-alpha activate the NF-kappa B transcription factor to induce MUC5AC expression (Busse, P. J. et al., J. Allergy Clin. Immun., 2005, 116, 1256-1263; Smirnova, M. G. et al., Cytokine. 2000, 12, 1732-1736).

[0116] Based on the inhibitory effect of KS-534 compound on MUC5AC expression, the inventors have studied to confirm whether the activity of NF-kappa B transcription factor present in MUC5AC promoter is inhibited by KS-534 compound.

[0117] The stably expressed H292 cells, a normal human bronchial epithelium cell, with luciferase reporter vector pGL4.32 (luc2P/NF-KB-RE/Hygro, Promega Company) were established prior to following experiment. The cell was suspended in 10% containing DMEM medium (Hyclone Company) in a concentration of 5×10.sup.4cells/ml and each 100 μL of cell was seeded on 96 well plates to adhere for 12 hours to the plates. The medium was replaced with new 0.1% FBS containing medium to culture 12 hours and 10 μm KS-534 was treated for 2 hours. 20 ng/ml of TNF-alpha was treated therewith to culture for 12 hours. The activation degree of luciferase was determined by One-glow Luciferase Assay system (Promega Company). After removing all the culture media in well plate, the plate was washed with PBS solution twice and 60 μl of passive lysis buffer (PLB) was added to each well to lyse the cell for 30 mins. 50 μl of lysed solution of the cell was transferred to new 96 well plates and 50 μl of LAR II reagent containing luciferase substrate of fireflies was added thereto. The luminescence was determined by Microplate Luminometer LB 96V (EG&G Berthold company) for 0.5 sec and the result was shown in Table 5 and FIG. 3.

TABLE-US-00005 TABLE 5 The summary for inhibition rate of NF-kappa B activity by KS-534 NF-kappa B activity (relative % comparing Conc. TNF-alpha with TNF-alpha Inhibitory sample (μm) (20 ng/ml) treatment group) ratio (%) Negative 0 − 34.05 ± 1.99 — control TNF-alpha 0 + 100.00 ± 6.22  0.00 treatment KS-534 10 + 63.35 ± 6.18 36.65

[0118] At the result, it has been confirmed that the test group treated with 10 μm KS-534 showed a potent inhibitory effect by 63.35% (inhibitory ratio of KS-534; 36.65%) based on NF-kappa B activity induced by TNF-alpha (100%).

[0119] Accordingly, it has been verified that KS-534 compound significantly inhibited the activity of NF-kappa B transcription factor.

Experimental Example 4

Inhibition on the Reproduction of MUC5AC Protein

[0120] In order to determine the inhibitory effect on the reproduction of MUC5AC protein, following test was performed by the method disclosed in the literature (Sikder, M A. et al., Phytother. Res., 2014, 28, 62-68)

4-1. Inhibitory Effect on Reproduction of MUCSAC Protein

[0121] In order to determine the treating activity of inflammatory disease such as COPD, the inhibitory effect on the release of MUCSAC was determined as follows:

[0122] For the immunoassay on reproduced MUCSAC, 50 μL of collected supernatant in Experimental Example 3 was distributed to 96 well plate and dried in a thermostat at 50□.

[0123] After washing with 1% BSA added PBS solution, MUCSAC antibody (ab3649, abcam Co.) was added to react together at room temperature for 1 hour and then secondary antibody was added thereto to react together for 1 hour. After re-washing, 3,3′,5,5′-tetramethylbenzidine peroxide solution (54827-17-7, Sigma-aldrich) was added thereto to react together for 20 mins. After stopping the reaction by adding sulfuric acid solution, the absorbance was determined by VERSAmax microplate reader, (SMP500-14915, Molecular Devices, USA) at 450 nm (Sikder, M. A. et al., Phytother. Res., 2014, 28, 62-68; Takeyama, K. et al. Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 3081-3086). The result was shown in Table 6 and FIG. 4.

[0124] At the result, it has been confirmed that the secreted amount of MUCSAC in the group treated with TNF-alpha was significantly increased and the test group treated with 2.5, 5 and 10 μm KS-534 showed potent inhibitory effect by 93.38, 78.27 and 64.77% (inhibitory ratio of KS-534; 6.62, 21.73 and 35.23%) on the reproduction of MUCSAC based on the secreted amount of MUCSAC in the group treated with TNF-alpha (100%).

TABLE-US-00006 TABLE 6 The summary for inhibitory activity of Muc5AC secretion by KS-534 MUC5AC secretion (relative % comparing Conc. TNF-alpha with TNF-alpha Inhibitory sample (μm) (20 ng/ml) treatment group) ratio (%) Negative control 0 − 25.98 ± 0.88 — group TNF-alpha 0 + 100.00 ± 1.22  0.00 treatment KS-534 2.5 + 93.38 ± 0.90 6.62 5 + 78.27 ± 2.16 21.73 10 + 64.77 ± 1.56 35.23

Experimental Example 5

Anti-Asthma Activity in Asthma-Induced Animal Model

[0125] In order to determine the anti-asthma activity of test sample in asthma-induced animal model, following test was performed by the method disclosed in the literature (Shin, I. S. et al., Food Chem. Toxicol., 2013, 62, 506-513)

5-1. Animal Sensitization and Airway Challenge

[0126] Specific pathogen-free female BALB/c mice (about 20 g), aged 6 weeks, which were routinely screened serologically for relevant respiratory pathogens, were purchased from Samtako Co. (Seoul, Korea) and acclimated with the experimental environment under the condition controlling the temperature of 22±2° C., and relative humidity of 55±15%, at light/dark cycle for 1 week prior to experiment.

[0127] Briefly, mice were sensitized by intraperitoneal injection of 20 μg OVA (Ovalbumin; A5503, Sigma, St. Louis, Mo.), which was emulsified in 2 mg aluminum hydroxide (A8222, Sigma-Aldrich, MO. USA) in 200 μl of PBS buffer (pH 7.4), biweekly. The mice were challenged through the airways with OVA (1% in PBS) for 30 min using an ultrasonic nebulizer (NE-U12; Omron Corp., Tokyo, Japan) from the 21.sup.st day to 23.sup.rd day after the initial sensitization. 24 hrs after the antigen treatment, the airway hyperresponsiveness was determined and the mice were sacrificed 48 hrs after the last challenge. The mice were sacrificed with an overdose of pentobarbital (50 mg/kg, Entobal®, Hanil, Korea) 48 h after the last challenge, and a tracheotomy was performed. After 1.4 ml of physiological saline solution (PBS) was instilled into the lungs, bronchoalveolar lavage fluid (BALF) was obtained by aspiration three times via tracheal cannulation.

[0128] The groups were divided into several groups, i.e., (a) normal control group(NC): the groups treated or not-treated with OVA; (b) asthma-induced group(OVA): the groups treated with OVA to induce asthma; and (c) comparative group: the groups treated with positive control group (M30, montelukast; 30 mg/kg, PO, Sigma-Aldrich Co. Ltd., SML-0101+OVA) and (d) test sample group: the groups treated with KS534 compound (30 mg/kg, PO, Sigma-Aldrich Co. Ltd., SML-0101+OVA).

[0129] The test group consists of 7 mice for each group and various concentrations of the test sample were orally administrated to the mice from 21.sup.st to 23.sup.rd day after .sup.1st OVA treatment.

[0130] All the result obtained from various experiments was recorded as mean±SD (standard deviation), and the comparison between each group was determined using by one-way ANOVA test using by SPSS 10.0 program. The statistic significance between each group was determined according to Dunnett's multiple comparison tests as a post-hoc test). The result was expressed as P values: <0.05 (*) as statistically significant.

5-2. Isolation of BALF and Determination of Immunocyte

[0131] The supernatant of bronchoalveolar lavage fluid (BALF) recovered from each mice was isolated using by centrifuge and kept in refrigerator to determine the level of inflammatory cells.

[0132] BALF was loaded onto a slide and centrifuged to fix the cells onto the slide using a Cellspin machine (Cyto12.5+clip5, Hanil Science Industrial, Korea). The cells were stained by Diff-Quick® Stain reagents (Sysmex, Cat No. 38721, Switzerland) according to the manufacturer's instructions and the number of inflammatory cells in each sample was counted by microscope (×400).

[0133] As shown in FIG. 5, the total number of eosinophil and inflammatory cells in the control group treated and inhaled with OVA as an asthma induced group (OVA) was significantly increased comparing with those in the non-treatment group with OVA as a normal control group (NC).

[0134] The total number of eosinophil and inflammatory cells in the test sample group orally administrated with various concentrations of test sample was significantly reduced.

5-3. Analysis on Level of Cytokines in BALF

[0135] In order to confirm the inhibition effect of test samples on the level of Th2 cytokines (IL-4, IL-5 and IL-13) in bronchoalveolar lavage fluid (BALF), the level of Th2 cytokines (IL-4, IL-5 and IL-13) in bronchoalveolar lavage fluid (BALF) was determined using by ELISA kit (VersaMax, Molecular Devices, USA) specifically reacting with each cytokine according to the manufacture's manual.

[0136] As shown in FIG. 6, the level of Th2 cytokines (IL-4, IL-5 and IL-13) in the control group treated with OVA as an asthma induced group (OVA) was significantly increased comparing with those in the non-treatment group with OVA as a normal control group (NC).

[0137] The increased level of Th2 cytokines (IL-4, IL-5 and IL-13) in the positive control group treated with Montelukast (MO, 30 mg/kg) as well as the test sample group orally administrated with various concentrations of test samples (KS534) were significantly reduced.

5-4. Effect on the Level of IgE and OVA-Specific IgE in Blood Serum

[0138] In order to confirm the inhibition effect of test samples prepared in Examples on the level of IgE and OVA-specific IgE in blood serum, following ELISA test was performed by the method disclosed in the literature (Kay, A. B., N. Engl. J. Med., 2001, 344, 30-37).

[0139] The blood serum isolated from caudal vena cava prepared in the above was recovered to determine the level of IgE and OVA-specific IgE in blood serum.

[0140] The blood serum was added to 96-well plates (ELISA plate, 2592, Costa, USA) and coated with 0.1M NaHCO.sub.3 buffer solution (pH 8.3) containing 20 μg/ml of OVA (Sigma, MO, USA) at 4° C. for 16 hours. After inhibiting nonspecific reaction using by PBS containing 1% bovine serum albumin (P2007, Biosesang, Korea), the serum for testing was diluted to 1:400 and reacted together for 2 hours at room temperature. After washing with PBS containing 0.05% tween 20, the serum was reacted with diluted (×300) anti-mouse IgE monoclonal antibody (MCA419, Serotec, Oxford, UK) for 2 hours and with diluted (×4000) HRP-conjugated goat anti-rat IgG polyclonal A (432402, Biolegend, USA) for 1 hours at room temperature. After washing, the solution was stained with 3,3′,5,5′-tetramethylbenzidine (432402, Biolegend Ins, USA) substrate and the reaction was stopped by 2N H.sub.2SO.sub.4 to determine the absorbance using by spectroscopy (Versamax, Molecular Devices, US) at 450 nm.

[0141] As shown in FIG. 7, the level of IgE and OVA-specific IgE in blood serum in the control group treated with OVA as an asthma induced group (OVA) was significantly increased whereas those in the positive control group treated with Montelukast (MO) as well as the test sample group orally administrated with various concentrations of test samples (KS-534) were significantly reduced.

5-4. Lung Histology

[0142] In order to confirm the anti-asthmatic effect of test samples prepared in Examples, following histopathological analysis on broncho-alveolar tissue was performed by the method disclosed in the literature (Kwak Y G. et al., J. Clin. Invest., 2003, 111, 1083-1092).

[0143] The delivered lung tissues of BALB/c mice which had not perform broncho-alveolar lavage was fixed for 24 h in 10% neutral-buffered formalin. After being embedded in epoxy, then made into 4-μm thickness sections, the tissue was stained with H&E solution (hematoxylin; Sigma MHS-16 and eosin, Sigma HT110-I-32) to observe the inflammation of lung tissue and with periodic acid Schiff (PAS, IMEB Inc., USA) to observe the mucosal secretion in lung tissue. The pathological change of lung tissue was determined by optical microscopy.

[0144] As shown in FIG. 8, many inflammatory cells including eosinophils were found in bronchiolar surroundings and hyperplasia of epithelial cells as well as hypertrophy of tracheal muscle were also found in the control group treated with OVA as an asthma induced group(OVA) whereas the invasion of the inflamed cells was significantly reduced in the positive control group treated with Montelukast (MO, 30 mg/kg) as well as the test sample group orally administrated with various concentrations of test samples (KS 534).

[0145] As shown in FIG. 9, the mucosal secretion from the goblet cell of bronchial epithelial cells in lung tissue was sharply increased in the control group treated with OVA as an asthma induced group (OVA) whereas the mucosal secretion from the goblet cell of bronchial epithelial cells in lung tissue was significantly reduced in the positive control group treated with Montelukast (MO, 30 mg/kg) as well as the test sample group orally administrated with various concentrations of test samples (KS 534).

[0146] In summary, the inventive KS534 compound could prevent or treat various diseases such as asthma caused by OVA. The inventive KS534 compound effectively inhibited the filtration of inflammatory cells including eosinophilia, the reproduction of Th2 type IL-4, 5, 13 as well as reduced the level of IgE and the reproduction of OVA-specific IgE, which has been confirmed by histopathological analysis on bronchoalveolar tissue.

[0147] In conclusion, it has been confirmed that the inventive KS534 compound effectively alleviated the main syndrome of asthma such as eosinophilia, lung inflammation, mucus hypersecretion as well as inhibited the immune response and therefore, it can be useful as a potent natural treating agent of asthma since it has similar or equivalent efficacy with conventionally available chemical drug, montelukast.

Experimental Example 6

Animal Model Test

[0148] In order to determine the anti-COPD effect of inventive compounds in COPD animal model, following test was performed by using COPD induced mice as disclosed in the literature (Bhaysar, T. et al., J. Chronic Obstr. Putin. Dis., 2008, 3, 477-481).

6-1. Experiment Animal

[0149] Specific pathogen-free male C57BL/6N mouse (about 22-24 g), aged 6 weeks, which were routinely screened serologically for relevant respiratory pathogens, were purchased from Koatech Co. (koatech.co.kr, Seoul, Korea) and bred allowing to access freely to feed (antibiotic free, Samyang Oil & Feed Corp., Korea) and water in breeding room controlling the temperature of 22±2° C., and humidity of 55±15% at the light-dark cycle for 12 hours and acclimated with the experimental environment for 1 week.

6-2. Drug and Administration

(1) Test Sample

[0150] 2 kinds of test samples, i.e., KS534 (15 and 30 mg/kg), Daxas (main ingredient: Roflumilast, 10 mg/kg) were dissolved in 0.5% sodium carboxymethyl cellulose (C9481, Sigma-Aldrich, USA) and used as test samples.

[0151] (2) Administration

[0152] KS534, and Rolflumilast (ROF 11-492, HETERO, India) were orally administrated to the mice, 1 hour before prior the intratracheal instillation (i.t.).

6-3. Preparation of COPD Mouse Model

(1) Standard Cigarette

[0153] 3R4F Kentuchy Reference Cigarettes (University of California, USA) was used as a standard cigarette for generating a cigarette smoke. The cigarette containing 9.4 mg of tar, 11 mg of TPM (total particle matter) and 12 mg of carbon monooxide per piece, was used after harmonizing with the temperature of 22±1° C. and humidity of 60±2% after opening for 48-72 hrs.

(2) Procedure

[0154] The exposure of cigarette smoke was performed by using a cigarette smoke generator (DBL Co. Ltd., dhbiolink.com, Korea). The mice were divided into five groups: normal (NC), COPD (cigarette smoke with LPS intranasal instillation), Rof (10 mg/kg of roflumilast, p.o.+cigarette smoke with LPS intranasal instillation), and KS534-15 and 30 (15 mg/kg and 30 mg/kg of KS534, p.o.+cigarette smoke with LPS instillation, respectively). Exposure of cigarette smoke (one puff/min, 35 ml puff volume over 2 s, every 60 s, 8 cigarettes per day) was conducted using a cigarette smoke generator. The mice received 1 h of cigarette smoke exposure in an exposure chamber (50 cm×30 cm×30 cm) for 3 days. Roflumilast and piscroside C were administered to mice by oral lavage 1 h before cigarette smoke exposure for 3 days. LPS was intranasally instilled (10 m dissolved in 50 μL distilled water) under anesthesia 1 h after the final exposure to cigarette smoke. After the end of experiment, the blood, BALF, and pneumonocyte of each rat were isolated and collected to test.

6-4. BALF Isolation and Determination of the Number of Immunocytes

[0155] After finishing the experiment, rats were anaesthetized with Zoleti150 (3VX9, Virbac, France, p.o) and the blood was delivered through caudal veins. In order to isolate BALF from lung, the bronchus of right lung was ligated with suture and then performed to tracheostomy. IV-use catheter (16 GA, 3S-Cath, Dukwoo, Korea) was put into the bronchus, and both of bronchus and catheter (16 GA, 3S-Cath, Dukwoo, Korea) were fixed with suture. The injector containing 1 ml of DPBS 0.7 (Dubecco's phosphate-buffered saline, Invitrogen, USA) was connected thereto and DPBS was forced to circulate three times to isolate BALF. The right lung ligated with suture was isolated, fixed with 10% neutral formalin solution, and the remaining lung tissue was reserved in refrigerator at −70° C. The isolated BALF was centrifuged for 15 mins at 1500 rpm to prepare cell pellet and the supernatant was reserved in refrigerator at −70° C. for cytokine analysis. The cell pellet was suspended in DPBS, and the cell was attached to a slide glass using by cytospin centrifuge (CS03270047, Hanil, Korea). Diff-Quik staining (ZS1003, Sysmex, Japan) was performed and the cell was observed by optical microscopy (DM1000, Leica, German) to count the number of immunocyte in each test sample.

[0156] As shown in FIG. 10, the characteristic increased level of neutrophils in BALF was observed in COPD induced group. The drug control group treated with Roflumilast showed reduced level of neutrophils by about 21.1% comparing with COPD induced group. In a while, the groups treated with 15 mg/kg and 30 mg/kg of KS534 showed remarkably reduced level of neutrophils by 26.3% and 25.7%, respectively, comparing with COPD induced group.

6-5. Effect on ROS and Neutrophils Elastase Activity in BALF

[0157] The level of ROS in BALF isolated from the mice was determined by DCF-DA (dihydrodo chloro fluoresce indiacetate, 35854, Sigma-Aldrich, USA) and the final concentration of DCF-DA was adjusted to 20 μm in 200 μL to incubate 30 mins. The Fluorescence was determined by apparatus (Flexstation3, Molecular Device, USA). The activity of neutrophils elastase activity in BALF was determined by using N-succinyl-(Ala)3-p-nitroanilide(54760, Sigma-Aldrich) as a substrate and incubated for 90 mins. The resulting absorbance was determined at 405 nm.

[0158] As shown in FIG. 11, the reproduction of ROS (Reactive oxygen species) in BALF was sharply increased in COPD induced group. However, the drug control group treated with Roflumilast showed reduced level of ROS by about 22.2% comparing with COPD induced group and the groups treated with 15 mg/kg and 30 mg/kg of KS534 showed remarkably reduced level of ROS by 19.5% and 27.5% comparing with COPD induced group.

[0159] As shown in FIG. 12, in COPD induced group, the level of proteins was sharply increased in BALF. The drug control group treated with Roflumilast showed significant reduction in the level of proteins compared with COPD induced group. The groups treated with 15 mg/kg and 30 mg/kg of KS534 showed remarkably reduced level of proteins by 20.8% and 28.5% comparing with COPD induced group

[0160] As shown in FIG. 12, in COPD induced group, the activity of neutrophils elastase enzyme was sharply increased in BALF. The drug control group treated with Roflumilast showed significant reduction in the activity of neutrophils elastase enzyme compared with COPD induced group. The groups treated with 15 mg/kg and 30 mg/kg of KS534 showed remarkably reduced activity of neutrophils elastase enzyme comparing with COPD induced group.

6-6. Cytokine Analysis in BALF

[0161] The level of IL-6 (SM6000B, R&D System, USA) and TNF-alpha (555268, BD Bioscience, USA)) in BALF isolated from the mice was determined by enzyme-linked immuno-sorbent assay (ELISA). The analysis of each cytokine was performed according to the manufacturer's manual, and the absorbance was determined at 450 nm by ELISA leader (VersaMax, Molecular Devices, USA).

[0162] As shown in FIG. 13, in COPD induced group, the level of IL-6 was sharply increased in BALF. The drug control group treated with Roflumilast showed significant reduction in the level of IL-6 compared with COPD induced group and the groups treated with 15 mg/kg and 30 mg/kg of KS534 showed reduced level of IL-6 by 35.8% and 50.6% comparing with COPD induced group. The level of TNF-alpha in BALF was sharply increased in COPD induced group. The drug control group treated with Roflumilast showed significant reduction in the level of TNF-alpha compared with COPD induced group and the groups treated with 15 mg/kg and 30 mg/kg of KS534 showed reduced level of TNF-alpha by 18.3% and 27.6% comparing with COPD induced group.

[0163] As shown in FIG. 14, the invasion of the inflamed cells was significantly increased in bronchiolar surroundings and hyperplasia of epithelial cells as well as hypertrophy of tracheal muscle were also found in COPD induced group whereas the invasion of the inflamed cells was significantly reduced in the positive control group treated with Roflumilast as well as the test sample group (30 mg/kg, KS534) orally administrated with various concentrations of test samples (KS 534).

[0164] In summary, the level of IL-6 and TNF-alpha in BALF as well as the number of inflammatory cells were remarkably increased in COPD-induced group and the reproduction of ROS and activity of elastase were sharply increased in COPD-induced group. In a while, the inventive KS534 compound effectively reduced the number of inflammatory mediator such as neutrophils and inflammatory cytokines such as IL-6 and TNF-alpha, which has been confirmed by histopathological analysis on broncho-alveolar tissue.

[0165] In conclusion, it has been confirmed that the inventive KS534 compound effectively inhibited the most of pathological syndromes of COPD comparing with the conventionally available drug (Roflumilast) used as a positive control group which did not show significant reduction on elastase activity. Accordingly, the inventive KS534 compound can be useful as a potent natural treating agent of COPD.

6-7. Statistics

[0166] All the result obtained from various experiments was determined using by one-way ANOVA test and the statistical significance between respective group was verified according to Dunnett's multiple comparison test for post hoc comparison result.

Experimental Example 7

Acute Toxicity Test of Oral Administration in Rat

[0167] The acute toxicity test was performed by administrating inventive compound to 6-weeks aged SPF Sprague-Dawley rats.

[0168] 250 mg/kg, 500 mg/kg, 1000 mg/kg, 5000 mg/kg of inventive extract and compounds was orally administrated to each group consisting of 2 rats and the symptoms of rats were observed for 14 days. After administrating the extract or compounds, all the clinical changes i.e., mortality, clinical signs, body weight changes was observed and blood test such as hematological test and hematological biochemistry test was performed. The abnormal changes of abdominal organ and thoracic organ were observed after autopsy.

[0169] There did not show any changes in mortality, clinical signs, body weight changes and gross findings in any group or either gender. Furthermore, there showed any toxicity in test group treated with 5000 mg/kg of inventive extract or compounds.

[0170] Accordingly, it has been confirmed that the inventive extract and compounds prepared in the present invention was potent and safe substance showing LD.sub.50 (more than 5000 mg/kg) in oral administration.

MODE FOR INVENTION

[0171] Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

Preparation of Injection

[0172] KS534 100 mg

[0173] Sodium metabisulfite 3.0 mg

[0174] Methyl paraben 0.8 mg

[0175] Propyl paraben 0.1 mg

[0176] Distilled water for injection optimum amount

[0177] Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2 ml ample and sterilizing by conventional injection preparation method.

Preparation of Powder

[0178] KS534 500 mg

[0179] Corn Starch 100 mg

[0180] Lactose 100 mg

[0181] Talc 10 mg

[0182] Powder preparation was prepared by mixing above components and filling sealed package.

Preparation of Tablet

[0183] KS534 200 mg

[0184] Corn Starch 100 mg

[0185] Lactose 100 mg

[0186] Magnesium stearate optimum amount

[0187] Tablet preparation was prepared by mixing above components and entabletting.

Preparation of Capsule

[0188] KS534 100 mg

[0189] Lactose 50 mg

[0190] Corn starch 50 mg

[0191] Talc 2 mg

[0192] Magnesium stearate optimum amount

[0193] Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

Preparation of Liquid

[0194] KS534 1000 mg

[0195] Sugar 20 g

[0196] Polysaccharide 20 g

[0197] Lemon flavor 20 g

[0198] Liquid preparation was prepared by dissolving active component, and then filling all the components in 1000 ml ample and sterilizing by conventional liquid preparation method.

Preparation of Health Food

[0199] KS534 1000 mg

[0200] Vitamin mixture optimum amount

[0201] Vitamin A acetate 70 g

[0202] Vitamin E 1.0 mg

[0203] Vitamin B.sub.10 13 mg

[0204] Vitamin B.sub.2 0.15 mg

[0205] Vitamin B6 0.5 mg

[0206] Vitamin B1 20.2 g

[0207] Vitamin C 10 mg

[0208] Biotin 10 g

[0209] Amide nicotinic acid 1.7 mg

[0210] Folic acid 50 g

[0211] Calcium pantothenic acid 0.5 mg

[0212] Mineral mixture optimum amount

[0213] Ferrous sulfate 1.75 mg

[0214] Zinc oxide 0.82 mg

[0215] Magnesium carbonate 25.3 mg

[0216] Monopotassium phosphate 15 mg

[0217] Dicalcium phosphate 55 mg

[0218] Potassium citrate 90 mg

[0219] Calcium carbonate 100 mg

[0220] Magnesium chloride 24.8 mg

[0221] The above mentioned vitamin and mineral mixture may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention.

Preparation of Health Beverage

[0222] KS534 1000 mg

[0223] Citric acid 1000 mg

[0224] Oligosaccharide 100 g

[0225] Apricot concentration 2 g

[0226] Taurine 1 g

[0227] Distilled water 900 ml

[0228] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85° C. for 1 hour, filtered and then filling all the components in 1000 ml ample and sterilizing by conventional health beverage preparation method.

[0229] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

INDUSTRIAL APPLICABILITY

[0230] As described in the present invention, inventive KS534 compound from the extract of Pseudolysimachion rotundum var subintegrum showed potent anti-inflammatory, anti-allergy, anti-asthma and anti-COPD activity confirmed by through various in vitro tests, for example, (1) cyto-toxicity test using by HT1080, H292 and EL4 cell line, (2) an inhibition test on the expression of MUC5AC (oligomeric mucus/gel-forming) induced by TNF-alpha, (3) NF-kappa B luciferase reporter assay, (4) inhibition test on the activity of NF-kappa B transcription factor; (5) inhibition on mRNA expression of target genes such as MMP-9, MUC5AC and IL-4 by way of inhibiting the NF-kappa B activity; (6) dose-dependent inhibiting effect on MUC5AC reproduction through MUC5AC protein reproduction assay; as well as in vivo tests, for example, (7) reducing effect on the number of inflammatory cells such as eosinophils using by OVA-sensitized/challenged mouse model, (8) inhibition test on the release of IgE, inflammatory cytokines such as IL-4, IL-5, IL-13 etc in BALF fluid and invasion of inflamed cells, as well as the suppression of airway hyperresponsiveness and golblet cell hyperplasia, (9) an inhibition test using by COPD animal model (C57B/6N mouse) on the proliferation of inflammatory cells in BALF fluid, the inhibition on the reproduction of ROS (Reactive oxygen species), and activity of neutrophil elastase, the reducing effect on the level of IL-6, TNF-alpha and the infiltrated inflammatory cells etc. Therefore, it can be used as the therapeutics or functional health food for treating and preventing allergic disease, inflammatory disease, asthma or chronic obstructive pulmonary disease (COPD).