A novel compound (KS-513) isolated from Pseudolysimachion rotundum var. subintegrum, the composition comprising the same preventing or treating allergic disease, inflammatory disease, asthma or chronic obstructive pulmonary disease and the use thereof

Abstract

(1aS,1bS,2S,4S,5aR,6S,6aS)-1a-(hydroxymethyl)-4-methoxy-2-(((2S,3R,5S5,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)octahydrooxireno[2,3:4,5]cyclopenta[1,2-c]pyran-6-yl 4-hydroxybenzoate}, 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) and the use thereof.

Claims

1. (1aS,1bS,2S,4S,5aR,6S,6aS)-1a-(hydroxymethyl)-4-methoxy-2-(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)octahydrooxireno[2,3:4,5]cyclopenta[1,2-c]pyran-6-yl 4-hydroxybenzoate} (KS-513) represented by the following chemical formula (1), the isomer thereof, the pharmaceutically acceptable salt or solvates thereof: ##STR00002##

2. A pharmaceutical composition comprising the compound (KS513), the isomer thereof, the pharmaceutically acceptable salt or solvates thereof as set forth in claim 1 isolated from Pseudolysimachion rotundum var subintegrum to treat allergic disease, inflammatory disease, asthma or chronic obstructive pulmonary disease (COPD).

3. A pharmaceutical composition comprising the compound (KS513) isolated from the extract of Pseudolysimachion rotundum var subintegrum as set forth in claim 1 and the pharmaceutically acceptable carriers or excipients, for the treatment of allergic disease, inflammatory disease, asthma or chronic obstructive pulmonary disease (COPD).

4. A method of treating allergic disease, inflammatory disease, asthma or chronic obstructive pulmonary disease (COPD) in mammals, wherein the method comprises administering a therapeutically effective amount of the novel compound (KS513), the isomer thereof, the pharmaceutically acceptable salt or solvates thereof as set forth in claim 1 into the mammal suffering from allergic disease, inflammatory disease, asthma or chronic obstructive pulmonary disease (COPD).

5. (canceled)

6. A health functional food comprising a therapeutically effective amount of the compound (KS513), the isomer thereof, the pharmaceutically acceptable salt or solvates thereof as set forth in claim 1 as an active ingredients for the alleviation of allergic disease, inflammatory disease, asthma or chronic obstructive pulmonary disease (COPD).

7. A health care food comprising a therapeutically effective amount of the compound (KS513), the isomer thereof, the pharmaceutically acceptable salt or solvates thereof as set forth in claim 1, together with a sitologically acceptable additive for the alleviation of allergic disease, inflammatory disease, asthma or chronic obstructive pulmonary disease (COPD).

Description

DESCRIPTION OF DRAWINGS

[0071] The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

[0072] FIG. 1 shows the effect of test sample on the cyto-toxicity in RAW264.7 cell line;

[0073] FIG. 2 shows the effect of test sample on the mRNA expression of pro-inflammatory medicated enzyme such as iNOS or COX-2, and pro-inflammatory medicated cytokines such as IL-1beta, IL-6, TNF-alpha in RAW264.7 cell line;

[0074] FIG. 3 shows the inhibition effect of test sample on iNOS protein expression involved in NO reproduction and the level of NO reproduction using by Western blot assay and Nitric Oxide assay (Statistical analysis was performed by Student' s-t-test and P value (**, <0.05) was regarded as being statistical significant);

[0075] FIG. 4 presents the inhibition effect of test sample on the COX-2 protein expression and the reproduced level of PGE2 using by Western blot assay and enzyme immunoassay (EIA) method (Statistical analysis was performed by Student' s-t-test and P value (**, <0.01 and ***, <0.001) was regarded as being statistical significant);

[0076] FIG. 5 presents the effect of the inventive compound on the released level of IL-1beta, IL-6, TNF-alpha etc (Statistical analysis was performed by Student' s-t-test and P value (*, <0.05, (**, <0.01 and ***, <0.001) was regarded as being statistical significant)

BEST MODE FOR CARRYING OUT THE INVENTION

[0077] 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.

[0078] 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

[0079] 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

[0080] 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 513) from Pseudolysimachion rotundum var subintegrum

1-1. Preparation of Crude Extract

[0081] 4.0 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 methanol. 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, two 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 5565 C. under reduced pressure and dried with freezing dryer to obtain 397.4 g of dried crude extract

1-2. Preparation of Purified Extract

[0082] 200 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 m, 200250 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, 20250 mm, 10 m, Zeochem, Louisville, U.S.A.) and eluting solvent (MeOH-water solution=2:8, 3:7, 4:6, 10:0) to afford 5 sub-fractions, i.e., f2a, f2b, f2c, f2d and f2e.

1-3. Preparation of Novel Compound KS513

[0083] 2.3 g of sub-fraction (f2b) was subjected to semi-preparative HPLC (Synergy Polar-RP 4 m, 21.2250 mm, Phenomenex, Torrance, Calif., U.S.A., 23% MeCN in H.sub.2O) to obtain white amorphous powdered novel iridoid compound (1aS,1bS,2S,4S,5aR,6S,6aS)-1a-(hydroxymethyl)-4-methoxy-2-(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)octahydrooxireno[2,3:4,5]cyclopenta[1,2-c]pyran-6-yl4-hydroxybenzoate} (KS-513; C.sub.23H.sub.29lO.sub.13) showing following physico-chemical properties:

[0084] [].sup.20.sub.D-64.0 (c 0.2, MeOH).

[0085] HRESIMS (observed m/z 513.1609 [M-H].sup.): quasimolecular 3:1 ion cluster

[0086] IR spectrum: 3412 cm.sup.1 (hydroxyl group); 1692 cm.sup.1. (,-unsaturated ester carbonyl)

[0087] .sup.1H and .sup.13C NMR spectra: Table 1

TABLE-US-00001 TABLE 1 .sup.1H (400 MHz) and .sup.13C (100 MHz) NMR spectroscopic data for KS-513 in DMSO-d6 KS-513 Position .sub.C .sub.H (J in Hz) Agluc 1 93.2 5.10, d (7.6) 3 98.0 4.68, dd (9.2, 2.4) 4 28.2 1.72, d (13.6) 1.56, m 5 34.4 2.33, m 6 76.0 5.24, d (9.0) 7 57.9 3.68, d (9.08) 8 65.5 9 41.5 2.29, t (8.0).sup. 10 58.3 3.85, m 3.85, m 11 58.3 3.37, s.sup. Aroyl .sup.1 119.8 .sup.2 131.6 7.84, d (8.4) .sup.3 115.4 6.87, d (8.4) .sup.4 162.8 .sup.5 115.4 6.87, d (8.4) .sup.6 131.6 7.84, d (8.4) 7 165.8 Glc 1 97.4 4.59, d (8.0) 2 73.4 3.04, m 3 76.6 3.19, m 4 70.3 3.04, m 5 77.4 3.13, m 6 61.3 3.69, d (8.8) 3.43, dd (12.0, 5.6)

Experimental Example 1

Cytotoxicity Test

[0088] In order to determine the cytotoxicity of inventive compound, following cytotoxicity test using by RAW264.7 cell line, was performed by the method disclosed in the literature (Lee, S. U., et al., 2010, Anti-resorptive saurolactam exhibits in vitro anti-inflammatory activity via ERK-NK-kappa B signaling pathway, International Immunopharmacology, 10, pp 298-303).

1-1. Evaluation of Cytotoxicity in RAW264.7 Cell

[0089] RAW264.7 cell (TIB-71, ATCC), a mouse macrophage cell, was suspended in 5% FBS (Fetal Bovine Serum)-supplemented DMEM (Dulbecco's Modified Eagle Medium, Gibco) medium in a concentration of 110.sup.5 cells/ml, and 100 L of the suspension was inoculated into 96 well plates to adhere cell to the plate for 4 hours. Various concentrations of test sample (KS 513 compound) were treated therewith and cultured for 24 hours. 10 L of CCK-8 solution was mixed therewith according to the manufacture's manual kit (Dojindo Co. Ltd), reacted from 30 mins to 4 hours, and 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

[0090] At the result, as shown in FIG. 1 and Table 2, it has been confirmed that KS 513 compound showed more than 95% cell viability at less than 80 M and therefore, it did not show cytotoxicity in RAW264.7 cell.

TABLE-US-00002 TABLE 2 Summary of cell viability by KS-513 in RAW264.7 cells concentration RAW264.7 cell viability Test sample (M) (%, mean variation) Negative control group 0 100.00 4.17 KS-513 2.5 99.58 3.66 5 99.26 5.68 10 98.18 3.28 20 94.66 3.03 40 97.23 7.00 80 95.14 2.73

Experimental Example 2

Inhibition on the mRNA Expression of Pro-Inflammatory Medicated Enzyme and Pro-Inflammatory Medicated Cytokines

[0091] In order to determine the inhibitory effect on the mRNA expression of pro-inflammatory medicated enzyme such as iNOS or COX-2, and pro-inflammatory medicated cytokines such as IL-1beta, IL-6, TNF-alpha in RAW264.7 cell line induced by LPS, following test using by real-time PCR (real-time polymerase chain reaction, quantitative real time polymerase chain reaction, qPCR) was performed according to the method disclosed in the literature (Livak, K. J., Schimittgen T. D., 2001, Analysis of relative gene expression data using real-time quantitiative PCR and the 2 (-Delta Delta C(T) method, Methods 25, p 402-408):

2-1. Procedure

[0092] RAW264.7 cell was inoculated into 6-well-plates in the concentration of 110.sup.6 cell/well to incubate for 12 hours. Various concentration of KS-513 compound (2.5, 5, 10 and 20 M) was pre-treated therewith and 1 g/mL of LPS (L6529, Sigma) was added thereto to incubate for 12 hours.

[0093] To extract total RNA, RNA was extracted using by Triazol B (invitrogen) and cDNA was synthesized using by Omniscript RT kit (205113, Qiagen, Gmbh, Hilden, Germany) after the quantification. The synthesized cDNA was mixed with its template and the primers shown in Table 3 (SEQ.I.D. 1-14), performed to denaturation at 94 C. for 5 mins using by PCR mix (PCR master Mix, Bioneer, Korea), and reacted as follows: 1 cycle for pre-denaturation; at 95 C. for 30 sec, 60 C. for 45 sec, 72 C. for 45 sec, 30 cycles for denaturation; 10 mins at 72 C., 1cycle for final extension. (FIG. 2)

TABLE-US-00003 TABLE3 Primersequenceusedinthisstudy SEQ. Gene primer I.D. iNOS sense 5-CCTTGTTCAGCTACGCCTTC-3 1 antisense 5-AAGGCCAAACACAGCATACC-3 2 COX-1 sense 5-GTGGCTATTTCCTGCAGCTC-3 3 antisense 5-CAGTGCCTCAACCCCATAGT-3 4 COX-2 sense 5-AGAAGGAAATGGCTGCAGAA-3 5 antisense 5-GCTCGGCTTCCAGTATTGAG-3 6 IL-1beta sense 5-CAGGCAGGCAGTATCACTCA-3 7 antisense 5-AGGCCACAGGTATTTTGTCG-3 8 IL-6 sense 5-GTTCTCTGGGAAATCGTGGA-3 9 antisense 5-GGAAATTGGGGTAGGAAGGA-3 10 TNF- sense 5-ACGGCATGGATCTCAAAGAC-3 11 alpha antisense 5-CGGACTCCGCAAAGTCTAAG-3 12 GAPDH sense 5-AACTTTGGCATTGTGGAAGG-3 13 antisense 5-ACACATTGGGGGTAGGAACA-3 14

[0094] At the result, it has been confirmed that the expressed level of mRNA of pro-inflammatory medicated enzyme such as iNOS or COX-2, and pro-inflammatory medicated cytokines such as IL-1beta, IL-6, TNF-alpha in RAW264.7 cell line induced by LPS (1 m/m1) was increased in the control group treated with LPS and those in the test group pre-treated with various concentration of KS-513 compound was significantly decreased in a dose dependent manner comparing with those in the control group treated with LPS (100%), as can be seen in Table 4.

TABLE-US-00004 TABLE 4 The summary for inhibitory activity on mRNA expression of pro-inflammatory cytokines by KS-513 LPS Target Negative treatment KS-513 (M) gene control (1 g/ml) 2.5 5 10 20 iNOS Fold* 1.00 0.10 8.88 2.27 7.25 2.51 5.94 2.91 2.48 0.19 1.32 0.47 IR (%).sup.# 88.71 0.06 18.41 33.09 72.12 85.19 COX-2 Fold* 1.09 0.62 7.86 2.09 4.72 0.21 2.64 0.15 1.43 0.50 0.97 0.38 IR (%).sup.# 86.11 0.03 39.91 66.40 81.81 87.66 TNF- Fold* 1.00 0.08 11.21 0.48 9.60 3.33 5.34 3.27 2.72 0.99 1.49 0.77 alpha IR (%).sup.# 91.07 0.03 14.34 52.39 75.69 86.70 IL- Fold* 1.02 0.30 16.70 1.80 10.41 1.09 5.72 0.24 2.56 0.14 1.70 0.59 1beta IR (%).sup.# 93.88 0.01 37.69 65.75 84.68 89.85 IL-6 Fold* 1.15 0.81 31.15 2.30 24.73 2.26 14.24 1.44 8.57 1.23 2.35 0.81 IR (%).sup.# 96.30 0.01 20.62 54.30 72.50 92.46 *value compared with a negative group .sup.#Inhibition ratio (IR) compared with LPS treatment group

Experimental Example 3

Inhibition Effect on iNOS Protein Expression and NO Reproduction

[0095] In order to determine the inhibition effect on iNOS protein expression involved in NO reproduction and the level of NO reproduction, following test was performed:

[0096] There has been known that NO shows various biological properties such as vasodilation, blood coagulation, neurotransmission, kidney function, inflammation, anti-tumor etc by dint of NO synthetase. NO, one of active oxygen and a highly reactive biogenic substance, plays important role in causing inflammation response and is reproduced from L-arginine by NOS (Nitric oxide synthetase). In particular, iNOS (inducible NOS) is involved in inflammatory response and expressed by the stimulation of inflammatory cytokines such as TNF-alpha, LPS (lipopolysaccharide) etc (Nathan, C., 1992, Nitric oxide as a secretory product of mammalian cells, FASEB Journal: Official publication of the Federation of American Society for Experimental Biology, 6, pp. 3051-3064).

3-1. Effect on the Level of iNOS Expression

[0097] In order to determine the effect of test sample on the level of iNOS expression in RAW264.7 cell, following assay was performed by the method disclosed in the literature (Lee, S. U., et al., Anti-resorptive saurolactam exhibits in vitro anti-inflammatory activity via ERK-NF-kappabeta signaling pathway: International immunopharmacology, 10, pp 298-303).

[0098] The expressed level of iNOS in RAW264.7 cell induced by LPS (1 g/ml) was determined by using Western blot analysis. RAW264.7 cell was inoculated into 96 well plates in the concentration of 110.sup.6 cell/well to incubate for 12 hours. Various concentration of KS-513 compound (2.5, 5, 10 and 20 M) was pre-treated therewith and 1 g/mL of LPS (L6529, Sigma) was added thereto to incubate for 12 hours.

[0099] To extract protein, after washing the cell with ice-cold PBS three times, lysis buffer (50 mM Tris HCl (pH 8.0), 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS, 1 mM PMSF, and protease inhibitor cocktail tablet (Roche, Germany, 11697498001) was added to the cell, reacted at 4 C. for 30 mins and centrifuged for 10 mins at the speed of 12,000 rpm to collect the supernatant.

[0100] The equivalent amount of the protein was isolated by SDS-PAGE and the protein was transferred to nitrocellulose membrane (BR162-0145, Bio-Rad). The membrane was reacted with blocking buffer (1xPBS solution comprising 5% non-fat milk and 0.1% Tween 20, SH30258.01, Hyclone) for 1 hour to block non-specific binding of antibody. Antibody (Anti-iNOS) against the protein was added thereto for 1-2 hours, washed with TBST solution comprising 0.1%Tween 20 and reacted with the secondary antibody. The product was reacted with ECL system and the amount of protein was determined by imaging analysis apparatus (LAS-4000, Fujifilm).

[0101] The quantitative determination of protein was performed by determining the absorbance using assay kit (Bradford protein assay kit, Thermo) at 595 nm and the result was shown in FIG. 3A.

3-2. Inhibitory Effect on the NOS Reproduction

[0102] In order to determine the amount of NO reproduction in medium, following test was performed according to the method disclosed in the literature (Ding, A. H., et al., 1988, Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages, Comparison of activating cytokines and evidence for independent production, Journal of Immunology, 141, pp 2407-2412).

[0103] RAW264.7 cell was inoculated into 96 well plates in the concentration of 110.sup.5 cell/well to incubate for 18 hours. Various concentration of KS-513 compound (2.5, 5, 10 and 20 M) was pre-treated therewith and 1 g/mL of LPS (L6529, Sigma) was added thereto to incubate for 24 hours. The equivalent amount of Griess Reagent (G4410, Sigma) to the medium was added thereto and reacted at room temperature for 10 mins to determine the absorbance at 540 nm. The level of NO in the medium was determined using by standard curve expressed according to various concentration of sodium nitrite and the result was shown in Table 5 and FIG. 3.

TABLE-US-00005 TABLE 5 The summary for inhibitory activity of NO production by KS-513 NO production (relative % comparing Inhibitory Conc. LPS with LPS treatment ratio sample (M) (1 g/mL) group) (%) Negative control 0 18.66 2.01 81.44 LPS treatment 0 + 100.10 3.79 0.00 KS-513 2.5 + 91.89 4.24 8.21 5 + 83.69 3.79 16.41 10 + 80.06 2.68 20.04 20 + 72.16 3.57 27.94

[0104] At the result, it has been confirmed that the expressed level of iNOS protein in the test group treated with KS-513 was reduced in a dose dependent manner while that in the LPS treatment group was increased (FIG. 3A) and the reproduced level of NO in the test group was reduced in a dose dependent manner based on that on the LPS treatment (100%).

Experimental Example 4

Inhibition on the COX-2 Protein Expression and the Reproduced Level of PGE2

[0105] In order to determine the inhibitory effect on the COX-2 protein expression and the reproduced level of PGE2, following test was performed by the method disclosed in the literature (Chen, C, et al., 1999, Involvement of p 38 mitogen-activated protein kinase in lipopolysaccharide-induced iNOS and COX-2 expression in J774 macrophage, Immunology, 97, pp 124-129)

[0106] The expressed level of COX-2 in RAW264.7 cell induced by LPS (1 g/ml) was determined by using Western blot analysis according to the method disclosed in Experimental Example 3.

[0107] RAW264.7 cell was inoculated into 96 well plates in the concentration of 110.sup.5 cell/well to incubate for 18 hours. Various concentration of KS-513 compound (2.5, 5, 10 and 20 M) was pre-treated therewith and 1 g/mL of LPS (L6529, Sigma) was added thereto to incubate for 24 hours. The PGE2 level in the cell was determined by Enzyme immunoassay (EIA; Amersham Pharmacia) system and the result was shown in FIG. 4A, 4B and Table 6.

[0108] At the result, it has been confirmed that the expressed level of COX-2 protein in the test group treated with KS-513 was reduced in a dose dependent manner while that in the LPS treatment group was increased (FIG. 4A) and the reproduced ratio of PGE.sub.2 in the test group was reduced in a dose dependent manner based on that on the LPS treatment (100%).

TABLE-US-00006 TABLE 6 The summary for inhibitory activity of PGE.sub.2 production PGE.sub.2 production (relative % comparing Inhibitory Conc. LPS with LPS treatment ratio sample (M) (1 g/mL) group) (%) Negative control 0 14.43 0.35 85.57 LPS treatment 0 + 100.00 0.92 0.00 KS-513 2.5 + 92.68 7.29 7.31 5 + 72.87 8.78 27.13 10 + 51.65 8.44 48.35 20 + 28.23 4.72 71.77

Experimental Example 5

Inhibition on the Production of Pro-Inflammatory Cytokines

[0109] In order to determine the inhibitory effect on the production of pro-inflammatory cytokines such as IL-1beta, IL-6, TNF-alpha etc, following test was performed by the method disclosed in the literature (Lee, S. U., et al., Anti-resorptive saurolactam exhibits in vitro anti-inflammatory activity via ERK-NF-kappabeta signaling pathway: International immunopharmacology, 10, pp 298-303)

[0110] The inflammatory cytokines such as IL-1beta, IL-6, TNF-alpha etc have been closely involved in the inflammation at the various cells, for example, activated lymphocyte, macrophage etc.

[0111] RAW264.7 cell was inoculated into 96 well plates in the concentration of 110.sup.5 cell/well to incubate for 18 hours. Various concentration of KS-513 compound (2.5, 5, 10 and 20 M) was pre-treated therewith and 1 g/mL of LPS (L6529, Sigma) was added thereto to incubate for 24 hours. The level of inflammatory cytokines such as IL-1beta, IL-6, TNF-alpha etc in the cell medium was determined by ELISA kit (IL-1beta:559603, IL-6:555240, TNF-alpha:5558534, BD) and the result was shown in FIG. 5 and Table 7-9.

[0112] At the result, it has been confirmed that the level of produced pro-inflammatory cytokines such as IL-1beta, IL-6, TNF-alpha in the test group treated with KS-513 was reduced in a dose dependent manner while that in the LPS treatment group (1 g/ml) was increased (FIG. 5). The reproduced ratio of pro-inflammatory cytokines in the test group was reduced in a dose dependent manner based on that on the LPS treatment (100%).

TABLE-US-00007 TABLE 7 The summary for inhibitory activity on cytokine production of IL-6 production IL-6 production (relative % comparing Inhibitory Conc. LPS with LPS treatment ratio sample (M) (1 g/mL) group) (%) Negative control 0 0.00 0.87 100 LPS treatment 0 + 100.00 8.84 0.00 KS-513 2.5 + 75.21 2.20 24.79 5 + 71.34 5.75 28.66 10 + 51.13 2.31 48.87 20 + 39.25 2.85 60.75

TABLE-US-00008 TABLE 8 The summary for inhibitory activity on cytokine production of TNF-alpha production TNF-alpha production (relative % comparing Inhibitory Conc. LPS with LPS treatment ratio sample (M) (1 g/mL) group) (%) Negative control 0 0.00 1.08 100 LPS treatment 0 + 100.00 5.80 0.00 KS-513 2.5 + 97.38 0.58 2.62 5 + 86.12 1.14 13.88 10 + 68.03 2.51 31.97 20 + 58.44 3.50 41.56

TABLE-US-00009 TABLE 9 The summary for inhibitory activity on cytokine production of IL-1beta production IL-1beta production (relative % comparing Inhibitory Conc. LPS with LPS treatment ratio sample (M) (1 g/mL) group) (%) Negative control 0 0.00 0.49 100 LPS treatment 0 + 100.00 4.52 0.00 KS-513 2.5 + 95.17 3.68 4.83 5 + 76.69 3.40 23.31 10 + 61.56 3.18 38.44 20 + 46.10 2.51 53.9

Experimental Example 6

Acute Toxicity Test of Oral Administration in Rat

[0113] The acute toxicity test was performed by administrating inventive compound to 6-weeks aged SPF Sprague-Dawley rats.

[0114] 250 mg/kg, 500 mg/kg, 1000 mg/kg, 5000 mg/kg of inventive compound 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 haematological test and hematological biochemistry test was performed. The abnormal changes of abdominal organ and thoracic organ were observed after autopsy.

[0115] 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 compound.

[0116] Accordingly, it has been confirmed that the inventive compound 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

[0117] 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

[0118]

TABLE-US-00010 KS 513 100 mg Sodium metabisulfite 3.0 mg Methyl paraben 0.8 mg Propyl paraben 0.1 mg

Distilled Water for Injection Optimum Amount

[0119] 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

[0120]

TABLE-US-00011 KS 513 500 mg Corn Starch 100 mg Lactose 100 mg Talc 10 mg [0121] Powder preparation was prepared by mixing above components and filling sealed package.

Preparation of Tablet

[0122]

TABLE-US-00012 KS 513 200 mg Corn Starch 100 mg Lactose 100 mg Magnesium stearate optimum amount [0123] Tablet preparation was prepared by mixing above components and entabletting.

Preparation of Capsule

[0124]

TABLE-US-00013 KS 513 100 mg Lactose 50 mg Corn starch 50 mg Talc 2 mg Magnesium stearate optimum amount [0125] Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

Preparation of Liquid

[0126]

TABLE-US-00014 KS 513 1000 mg Sugar 20 g Polysaccharide 20 g Lemon flavor 20 g [0127] 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

[0128]

TABLE-US-00015 KS 513 1000 mg Vitamin mixture optimum amount Vitamin A acetate 70 g Vitamin E 1.0 mg Vitamin B.sub.10. 13 mg Vitamin B.sub.2 0.15 mg Vitamin B6 0.5 mg Vitamin B1 20.2 g Vitamin C 10 mg Biotin 10 g Amide nicotinic acid 1.7 mg Folic acid 50 g Calcium pantothenic acid 0.5 mg Mineral mixture optimum amount Ferrous sulfate 1.75 mg Zinc oxide 0.82 mg Magnesium carbonate 25.3 mg Monopotassium phosphate 15 mg Dicalcium phosphate 55 mg Potassium citrate 90 mg Calcium carbonate 100 mg Magnesium chloride 24.8 mg [0129] 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

[0130]

TABLE-US-00016 KS 513 1000 mg Citric acid 1000 mg Oligosaccharide 100 g Apricot concentration 2 g Taurine 1 g Distilled water 900 mL

[0131] 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.

[0132] 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

[0133] As described in the present invention, inventive KS513 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 tests, for example, (1) cyto-toxicity test using by RAW264.7 cell line, (2) an effect on the mRNA expression of pro-inflammatory medicated enzyme such as iNOS or COX-2, and pro-inflammatory medicated cytokines such as IL-1beta, IL-6, TNF-alpha in RAW264.7 cell line induced by LPS, (3) an inhibition effect on iNOS protein expression involved in NO reproduction and the level of NO reproduction, (4) an inhibition effect on the COX-2 protein expression and the reproduced level of PGE2, (5) an inhibition effect on the produce level of IL-1beta, IL-6, TNF-alpha 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).