Pharmaceutical composition for preventing or treating respiratory disease comprising extract of <i>Justicia procumbens </i>L

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating respiratory disease, which comprises an alcohol or organic solvent extract of Justicia procumbens L. as an active ingredient, and to a pharmaceutical composition for preventing or treating respiratory disease, which comprises an alcohol or organic solvent extract of Justicia procumbens L., the extract comprising any one or more of justicidin A, justicidin B, justicidin C and phyllamyricin C, and also to a food composition for preventing or treating respiratory disease, which comprises the alcohol or organic solvent extract. The composition comprising the Justicia procumbens L. extract according to the present invention may inhibit abnormal overproliferation of splenocytes, may inhibit the secretion of allergic inflammatory cytokines, and exhibits an expectorant effect and an airway constriction inhibitory effect. Thus, it may effectively prevent, treat or improve respiratory disease.

Claims

1. A method for preventing, treating or improving a respiratory disease, comprising administering to a subject in need thereof a composition comprising an extract of Justicia procumbens L, wherein the extract is an alcohol extract or an organic solvent extract, and wherein the composition is a pharmaceutical composition or a food composition.

2. The method of claim 1, wherein the Justicia procumbens L. is one or more selected from the group consisting of a whole plant, an aerial part, a root, a leaf, a flower and a seed.

3. The method of claim 1, wherein the extract is the alcohol extract, and the alcohol is a lower alcohol having 1 to 4 carbon atoms.

4. The method of claim 1, wherein the extract is the alcohol extract, and the alcohol extract is an ethanol extract.

5. The method of claim 1, wherein the extract is the alcohol extract, and the alcohol extract is a 30% ethanol extract, a 50% ethanol extract, a 70% ethanol extract or a 100% ethanol extract.

6. The method of claim 1, wherein the extract is the organic solvent extract, and the organic solvent is one or more selected from the group consisting of hexane, ethyl acetate, dichloromethane, ether, chloroform, and acetone.

7. The method of claim 1, wherein the respiratory disease is one or more selected from the group consisting of cold, pneumonia, bronchitis, asthma, chronic obstructive pulmonary disease, and rhinitis.

8. The method of claim 7, wherein the composition further comprises a functional health food.

9. The method of claim 1, wherein the extract comprises one or more selected from the group consisting of justicidin A, justicidin B, justicidin C, and phyllamyricin C.

10. The method of claim 9, wherein the composition further comprises a functional health food.

11. The method of claim 1, wherein the extract comprises: justicidin B; and one or more selected from the group consisting of justicidin A, justicidin C and phyllamyricin C.

12. The method of claim 11, wherein the composition further comprises a functional health food.

13. The method of claim 1, wherein the extract comprises: justicidin A; and one or more selected from the group consisting of justicidin B, justicidin C and phyllamyricin C.

14. The method of claim 13, wherein the composition further comprises a functional health food.

15. The method of claim 1, wherein the extract comprises justicidin B in an amount of 1 mg/g to 200 mg/g.

16. The method of claim 15, wherein the Justicia procumbens L. is one or more selected from the group consisting of a whole plant, an aerial part, a root, a leaf, a flower and a seed.

17. The method of claim 15, wherein the extract is the alcohol extract, and the alcohol is a lower alcohol having 1 to 4 carbon atoms.

18. The method of claim 15, wherein the extract is the alcohol extract, and the alcohol extract is an ethanol extract.

19. The method of claim 15, wherein the extract is the alcohol extract, and the alcohol extract is a 30% ethanol extract, a 50% ethanol extract, a 70% ethanol extract or a 100% ethanol extract.

20. The method of claim 15, wherein the extract is the organic solvent extract, and the organic solvent is one or more selected from the group consisting of hexane, ethyl acetate, dichloromethane, ether, chloroform, and acetone.

21. The method of claim 15, wherein the respiratory disease is one or more selected from the group consisting of cold, pneumonia, bronchitis, asthma, chronic obstructive pulmonary disease, and rhinitis.

22. The method of claim 15, wherein the food composition comprises a functional health food.

23. The method of claim 1, wherein the extract comprises justicidin A in an amount of 1 mg/g to 200 mg/g.

24. The method of claim 23, wherein the composition further comprises a functional health food.

25. The method of claim 1, wherein the extract comprises justicidin B in an amount of 1 mg/g to 200 mg/g and justicidin A in an amount of 1 mg/g to 200 mg/g.

26. The method of claim 25, wherein the composition further comprises a functional health food.

27. A method for preventing, improving, or treating a respiratory disease, comprising administering to a subject in need thereof a composition comprising justicidin A, justicidin B, or a mixture thereof, wherein the composition is a pharmaceutical composition or a food composition.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the chemical structures of compounds 1 to 4 isolated from an extract of Justicia procumbens L. according to the present invention. (compound 1: justicidin B; compound 2: justicidin A; compound 3: justicidin C; and compound 4: phyllamyricin C).

(2) FIG. 2 shows the results of HPLC of a water extract and ethanol extract of Justicia procumbens L. according to the present invention.

(3) FIG. 3 shows the results of HPLC of other alcohol extracts of Justicia procumbens L. according to the present invention.

(4) FIG. 4 shows the results of airway hypersensitivity for an ethanol extract of Justicia procumbens L. according to the present invention.

(5) FIG. 5 shows the results of lung tissue staining for an ethanol extract of Justicia procumbens L. according to the present invention.

(6) FIG. 6 shows the results of lung tissue staining for a mixture of an ethanol extract of Justicia procumbens L. according to the present invention and colloidal silicon dioxide.

BEST MODE

(7) Hereinafter, preferred Preparation Examples, Examples and Formulation Examples will be described for a better understanding of the present invention. It is to be understood, however, that these Preparation Examples, Examples and Formulation Examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Preparation Example 1: Preparation of Justicia procumbens L. Extract

(8) Justicia procumbens L. used in the experiment was cultivated and harvested in Jecheon-si, Chungcheongbuk-do, South Korea, and dried and cut. The origin of the plant was identified by the National Institute of Biological Resources of the Ministry of Environment (Korea) (identification sample number: NIBRVP0000530742). Using water and organic solvents (ethanol or other alcohols) as extraction solvents, Justicia procumbens L. extracts were prepared.

Preparation Example 1-1: Preparation of Water Extract of Justicia procumbens L.

(9) The above-ground part of Justicia procumbens L. dried in the shade was cut to a size of about 1 to 2 cm, and about 10 g of the cut plant was taken exactly and extracted under reflux with 100 ml of purified water in a constant-temperature incubator (D-6, Hansol Science) at 80 C. twice (first extraction: 2 hours, and second extraction: 1 hour). The extract was naturally filtered through filter paper, and the filtrate was concentrated with a vacuum evaporator (N-1100, EYELA) at 60 C., and then dried in a vacuum oven (OV-12, JEIO Tech) at 60 C. for 12 hours, thereby obtaining 1.64 g of a water extract of Justicia procumbens L.

Preparation Example 1-2: Preparation of Ethanol Extracts and Other Alcohol Extracts of Justicia procumbens L.

(10) Ethanol extracts and other alcohol extracts of Justicia procumbens L. were prepared in the same manner as described in Preparation Example 1-1, except for the weight of herbal material (above-ground part of Justicia procumbens L.) used and extraction solvents. Specifically, ethanol extracts of Justicia procumbens L. were prepared by extracting the above-ground part of Justicia procumbens L. with varying concentrations (30%, 50%, 70%, and 100% (v/v %)) of ethanol. In addition, using isopropanol and methanol, alcohol extracts of Justicia procumbens L. were prepared.

Preparation Example 1-3: Preparation of Ethanol Extract of Each Part of Justicia procumbens L.

(11) The above-ground part of Justicia procumbens L. dried in the shade was divided into an aerial part, a stem, and a leaf and a flower, and then pulverized using a pulverizer (KSP-35, Korea Medi Co., Ltd.). About 5 g of each part of the crushed Justicia procumbens L. was taken exactly and extracted ultrasonically (UG 600, Hanil Ultrasonic) with 100 ml of ethanol, and each of the filtrates was dried in a vacuum oven (OV-12, JEIO Tech) at 60 C. for 12 hours, thereby preparing ethanol extracts.

Preparation Example 1-4: Preparation of Ethanol Extracts of Justicia procumbens L. By Various Extraction Methods

(12) The aerial part of Justicia procumbens L. dried in the shade was cut to a size of about 1 to 2 cm, and about 5 g of the cut plant was taken exactly and extracted with 100 ml of varying concentrations (30%, 50%, 70%, and 100% (v/v %)) of an ethanol solvent by ultrasonic extraction (1 hour) and dipping extraction (5 hours). Then, each of the filtrates was dried in a vacuum dryer (OV-12, JEIO Tech) at 60 C. for 12 hours, thereby preparing each extract.

Preparation Example 2: Isolation and Preparation of Active Substances

(13) 480 g of the aerial part of Justicia procumbens L., cut to a size of about 1 to 2 cm and dried in the shade, was extracted under reflux with 3 L of ethanol in a constant-temperature incubator (J-BAL, DISCO) at 80 C. twice (first extraction: 2 hours, and second extraction: 1 hour). The extract was filtered under reduced pressure at a temperature of 50 to 60 C., and then concentrated with a vacuum evaporator (N-1100, EYELA), thereby obtaining about 11.44 g (2.4% yield) of an ethanol concentrate. The ethanol concentrate was suspended in 1 L of distilled water, and then subjected to solvent fractionation three times with 1 L of n-hexane. The prepared n-hexane fraction (about 5 g) was subjected to silica gel column chromatography using a concentration gradient solvent system comprising dichloromethane and methanol as a mobile phase solvent, thereby preparing a total of 11 sub-fractions (JP-Hex-01 to 11). Among these sub-fractions, sub-fraction No. 4 (JP-Hex-04) was subjected again to silica gel column chromatography using methylene chloride and methanol as a mobile phase solvent, thereby preparing 9 sub-fractions (JP-Hex-0401 to 0409). Among them, final sub-fraction No. 4 (JP-Hex-0404) was subjected to reverse-phase preparative high-performance chromatography using 70% methanol, thereby isolating and preparing each of compound (justicidin B) with a retention time of 8.1 min, compound 2 (justicidin A) with a retention time of 10.2 min, and compound 3 (justicidin C) with a retention time of 14.3 min.

(14) In addition, sub-fraction No. 6 (JP-Hex-06) was subjected to silica gel column chromatography using n-hexane and ethyl acetate as a mobile phase solvent, thereby preparing 19 sub-fractions (JP-Hex-0601 to 0619). Among them, sub-fraction No. (JP-Hex-0606) was subjected to reverse-phase preparative high-performance chromatography using 70% methanol, thereby preparing compound 4 (phyllamyricin C) with a retention time of 14.7 min.

(15) The structural analysis of compounds 1 to 4 isolated as described above was performed, and the results were confirmed through each literature. The identified chemical structures of compounds 1 to 4 are shown in FIG. 1.

(16) Compound 1 (justicidin B): .sup.1H-NMR (CDCl.sub.3, 500 MHz) 7.70 (1H, s, H-1), 7.18 (1H, s, H-8), 7.11 (1H, s, H-5), 6.97 (1H, d, J=8.0 Hz, H-5), 6.86 (1H, d, J=1.5 Hz, H-2), 6.83 (1H, dd, J=1.5, 8.0 Hz, H-6), 6.09 (1H, d, J=22.4 Hz, OCH.sub.2O), 6.05 (1H, d, J=22.4 Hz, OCH.sub.2O), 5.38 (2H, s, H-2a), 4.05 (3H, s, 7-OCH.sub.3), 3.81 (3H, s, 6-OCH.sub.3). .sup.13C-NMR (CDCl.sub.3, 125 MHz) 170.0 (C-3a), 151.9 (C-7), 150.2 (C-6), 147.6 (C-3), 147.6 (C-4), 139.8 (C-2), 139.6 (C-4), 133.3 (C-8a), 128.9 (C-4a), 128.5 (C-1), 123.6 (C-6), 118.6 (C-3), 118.4 (C-1), 110.7 (C-2), 108.3 (C-5), 106.1 (C-8), 106.0 (C-5), 101.3 (OCH.sub.2O), 68.1 (C-2a), 56.1 (7-OCH.sub.3), 55.9 (6-OCH.sub.3). From these results, compound 1 was identified as justicidin B, and these results were consistent with the results shown in Journal of Natural Products, Vol. 58, No 2, 244-249, 1995.

(17) Compound 2 (justicidin A): .sup.1H-NMR (CDCl.sub.3, 500 MHz) 7.54 (1H, s, H-8), 7.06 (1H, s, H-5), 6.95 (1H, d, J=7.7 Hz, H-5), 6.82 (1H, d, J=1.5 Hz, H-2), 6.79 (1H, dd, J=1.5, 7.7 Hz, H-6), 6.09 (1H, d, J=22.3 Hz, OCH.sub.2O), 6.04 (1H, d, J=22.3 Hz, OCH.sub.2O), 5.54 (2H, s, H-2a), 4.13 (3H, s, 1-OCH.sub.3), 4.07 (3H, s, 7-OCH.sub.3), 3.80 (3H, s, 6-OCH.sub.3). .sup.13C-NMR (CDCl.sub.3, 125 MHz) 169.7 (C-3a), 151.7 (C-7), 150.4 (C-6), 147.9 (C-1), 147.6 (C-3), 147.5 (C-4), 134.5 (C-4), 130.7 (C-4a), 128.6 (C-1), 126.1 (C-8a), 124.6 (C-2), 123.7 (C-6), 119.4 (C-3), 110.8 (C-2), 108.3 (C-5), 106.3 (C-5), 101.3 (OCH.sub.2O), 100.7 (C-8), 66.7 (C-2a), 59.7 (1-OCH.sub.3), 56.2 (7-OCH.sub.3), 55.9 (6-OCH.sub.3). From these results, compound 2 was identified as justicidin A, and these results were consistent with the results shown in Journal of Natural Products, Vol. 62, 1056-1058, 1999.

(18) Compound 3 (justicidin C): .sup.1H-NMR (CDCl.sub.3, 500 MHz) 7.69 (1H, s, H-8), 6.98 (1H, s, H-5), 6.96 (1H, d, J=7.7 Hz, H-5), 6.81 (1H, d, J=1.7 Hz, H-2), 6.80 (1H, dd, J=1.7, 7.7 Hz, H-6), 6.09 (1H, d, J=16.4 Hz, OCH.sub.2O), 6.06 (1H, d, J=16.4 Hz, OCH.sub.2O), 5.13 (2H, s, H-3a), 4.37 (3H, s, 1-OCH.sub.3), 4.06 (3H, s, 7-OCH.sub.3), 3.83 (3H, s, 6-OCH.sub.3). .sup.13C-NMR (CDCl.sub.3, 125 MHz) 169.4 (C-2a), 155.5 (C-1), 152.5 (C-6), 149.9 (C-7), 148.4 (C-3), 147.6 (C-4), 139.1 (C-3), 133.5 (C-4a), 129.8 (C-1), 126.5 (C-4), 123.7 (C-8a), 123.0 (C-6), 109.9 (C-2), 109.5 (C-2), 109.1 (C-5), 104.2 (C-5), 102.4 (C-8), 101.5 (OCH.sub.2O), 68.9 (C-3a), 63.6 (1-OCH.sub.3), 56.2 (7-OCH.sub.3), 56.0 (6-OCH.sub.3). From these results, compound 3 was identified as justicidin C, and these results were consistent with the results shown in Tetrahedron Letters, No. 12, pp. 923-925, 1970.

(19) Compound 4 (phyllamyricin C): .sup.1H-NMR (CDCl.sub.3, 500 MHz) 7.69 (1H, s, H-5), 6.98 (1H, s, H-1), 6.96 (1H, d, J=7.8 Hz, H-5), 6.81 (1H, d, J=1.8 Hz, H-2), 6.80 (1H, dd, J=1.8, 7.8 Hz, H-6), 6.09 (1H, d, J=16.4 Hz, OCH.sub.2O), 6.06 (1H, d, J=16.4 Hz, OCH.sub.2O), 5.13 (2H, s, H-2a), 4.37 (3H, s, 6-OCH.sub.3), 4.06 (3H, s, 8-OCH.sub.3), 3.83 (3H, s, 7-OCH.sub.3). .sup.13C-NMR (CDCl.sub.3, 125 MHz) 169.3 (C-3a), 155.5 (C-6), 152.5 (C-7), 149.9 (C-8), 148.4 (C-3), 147.6 (C-4), 143.5 (C-4), 139.1 (C-2), 133.5 (C-8a), 129.8 (C-1), 126.4 (C-3), 123.7 (C-4a), 123.0 (C-6), 109.9 (C-2), 109.1 (C-5), 104.2 (C-1), 102.4 (C-5), 101.5 (OCH.sub.2O) 68.9 (C-2a), 63.6 (6-OCH.sub.3), 56.2 (8-OCH.sub.3), 56.0 (7-OCH.sub.3). From these results, compound 4 was identified as phyllamyricin C, and these results were consistent with the results shown in Journal of Natural Products, Vol. 58, No 2, 244-249, 1995.

(20) Among the isolated active ingredients, justicidin B was obtained in large amounts, and a standard (purity: 95.25%) was used after its purity was confirmed by HPLC.

Preparation Example 3: Preparation of a Mixture (1:1) of 100% Ethanol Extract of Justicia procumbens L. And Colloidal Silicon Oxide

(21) About 400 kg of Justicia procumbens L., crushed to a size of about 1 cm, was extracted by dipping in 4000 L of 100% ethanol at room temperature (25 C.) for 24 hours, and then filtered under reduced pressure. The residue was extracted again by dipping in 3200 L of 100% ethanol at room temperature (25 C.) for 24 hours, and then filtered under reduced pressure. The filtrate was concentrated under reduced pressure at 60 C., thereby obtaining about 93.85 kg (3.3% yield) of an ethanol extract of Justicia procumbens L. having a solid content of about 14%. The same weight (about 13 kg) of colloidal silicon oxide (AEROSIL 200, Evonik) as the solid weight of the ethanol extract of Justicia procumbens L. was added to the ethanol extract, stirred sufficiently, and then dried in a vacuum dryer for 72 hours. The dried mixture (1:1) of the 100% ethanol extract of Justicia procumbens L. and the colloidal silicon dioxide was powdered, thereby preparing about 26 kg of a mixture (1:1) of the 100% ethanol extract of Justicia procumbens L. and the colloidal silicon dioxide.

Example 1: High-Performance Liquid Chromatography (HPLC) Patterns and Justicidin B Contents of Justicia procumbens L. Extracts

(22) In order to confirm the active ingredients contained in the Justicia procumbens L. extracts prepared by the preparation methods of Preparation Examples 1-1 to 1-4 above, high-performance liquid chromatography (HPLC, Agilent 1260, USA) was performed under the conditions shown in Table 1 below, and the results are shown in FIGS. 2 and 3.

(23) TABLE-US-00001 TABLE 1 Detector UV absorption spectrometer Detection UV 256 nm wavelength Column Capcellpak UG C18 (4.6 250 mm, 5 m) Column 35 C. temperature <Gradient program> Time (min) % acetonitrile % water Mobile phase 0 15 85 5 15 85 40 46 54 60 55 45 70 60 40 75 40 60 76 15 85 Flow rate 0.8 mL/min Amount injected 10 l

(24) The results of HPLC analysis indicated that the active ingredients were detected in all the ethanol and other alcohol extracts of Justicia procumbens L. Specifically, as shown in FIG. 2, in the ethanol extract of Justicia procumbens L., the peaks of compounds 1 to 4, which are the active ingredients, were detected at RT 46.2 min (justicidin B), RT 49.5 min (justicidin A), and RT 53.4 min (justicidin C and phyllamyricin C C). In addition, as shown in FIG. 3, in the other alcohol extracts, the peaks of compounds 1 to 4 were detected at RT 47.6 min (justicidin B), RT 51.3 min (justicidin A), and RT 55.4 min (justicidin C and phyllamyricin C).

(25) However, justicidin B, justicidin A, justicidin C or phyllamyricin C was not detected in the water extract of Justicia procumbens L.

(26) The contents of justicidin B in the water extract and ethanol extract of Justicia procumbens L. and in the ethanol extract of each part of Justicia procumbens L. were calculated using Equation 1 below, and the results are shown in Tables 2 and 3 below.

(27) $\begin{array}{cc}\mathrm{Justicidin}B\mathrm{content}\left(\mathrm{mg}/g\right)=\frac{P_t}{P_s}\frac{C_s}{C_t}P& \left[\mathrm{Equation}1\right]\end{array}$

(28) P.sub.t and P.sub.s: peak areas of justicidin B in test sample and standard sample;

(29) C.sub.t and C.sub.s: concentrations of test sample and standard sample (test sample: 0.001 g/mL, and standard sample: 0.05 mg/mL);

(30) P: Purity (0.9525) of justicidin B.

(31) TABLE-US-00002 TABLE 2 Extraction solvent for Justicidin B content Preparation method Justicia procumbens L. (mg/g) Preparation Example 1-1 Water extract Not detected Preparation Example 1-2 30% ethanol extract 3.39 Reflux extraction 50% ethanol extract 4.76 70% ethanol extract 4.89 100% ethanol extract 49.00 Preparation Example 1-4 30% ethanol extract 1.18 Ultrasonic extraction 50% ethanol extract 4.75 70% ethanol extract 7.70 100% ethanol extract 137.83 Preparation Example 1-4 30% ethanol extract 0.95 Dipping extraction 50% ethanol extract 3.19 70% ethanol extract 6.74 100% ethanol extract 147.02

(32) TABLE-US-00003 TABLE 3 Extracted part of Justicidin B content Preparation method Justicia procumbens L. (mg/g) Preparation Example 1-3 Stem 1.95 Reflux extraction Leaf + flower 177.91 Aerial part 45.69

(33) In addition, for the 1:1 mixture of the 100% ethanol extract of Justicia procumbens L. and colloidal silicon oxide, prepared by the preparation method of Preparation Example 3 above, justicidin B was detected as described above. As a result, it was shown that the content of justicidin B was 16.87 mg/g.

Example 2: The Effects of Justicia procumbens L. Extracts on Inhibition of Spleen Cell Proliferation and Inhibition of Secretion of Th2 Inflammatory Cytokines

(34) In order to confirm the inflammation inhibitory effects of the Justicia procumbens L. extracts, splenocytes were isolated from the spleens of Balb/c mice. The isolated splenocytes were diluted in RPMI medium (containing 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 g/mL streptomycin)) at a concentration of 510.sup.6 cells/nit, and the cell dilution was dispended in a 24-well plate in an amount of 500 l/well and in a 96-well plate in an amount of 100 l/well. The plates were incubated in a 5% CO.sub.2 incubator at 37 C. for 48 hours. When the spleen cells were dispensed, the cells were treated with 5 g/mL of Concanavalin A to induce immune responses, and at the same time, the cells were treated with each of the Justicia procumbens L. extracts prepared in Preparation Examples 1-1 and 1-2, the active substances prepared in Preparation Example 2, and positive controls (dexamethasone and montelukast). The medium of the 24-well plate was used to measure the degree of secretion of Th2 inflammatory cytokines (IL4 and IL5), and the 96-well plate was used to measure the degree of cell proliferation. First, in order to measure Th2 cytokines, the medium in the 24-well plate was collected and centrifuged at 800g for 5 minutes, and the supernatant was collected. 100 l of the supernatant per well was used in IL4 or IL5 enzyme-linked immunosorbent assay (ELISA, Komabiotech Inc.) to measure the degree of secretion of these cytokines. In order to measure the degree of cell proliferation, 10 mL of CCK8 (cell counting kit 8, Donjindo) solution was added to each well of the 96-well plate, and after 4 hours, the absorbance value was measured. The degree of inhibition of spleen cell proliferation by each drug and the degree of inhibition of Th2 cytokine secretion by each drug were calculated using Equation 2 below. For drug synergistic effects, a linear equation was constructed using the concentrations of drugs administered alone as x-coordinates and using drug concentration-dependent inhibition as y-coordinates. Using this linear equation, the concentrations of drugs administered alone, which correspond to inhibition achieved when administered in combination, were calculated, and combination index (CI) was calculated using Equation 3 below. A CI value of less than 1 indicates a synergistic effect, a CI value of 1 indicates an additive effect, and a CI value of more than 1 indicates an antagonistic effect (Liang Zhao et. al., Clinical Cancer Research, 10, 7994-8004, 2004). The results of the measurement are shown in Tables 4 to 8 below.

(35) $\begin{array}{cc}\mathrm{Inhibition}\left(\%\right)=\left\{\left(\mathrm{value}\mathrm{of}\mathrm{test}\mathrm{group}-\mathrm{value}\mathrm{of}\mathrm{normal}\mathrm{group}\right)/\left(\mathrm{value}\mathrm{of}\mathrm{induced}\mathrm{group}-\mathrm{value}\mathrm{of}\mathrm{normal}\mathrm{group}\right)\right\}100& \left[\mathrm{Equation}2\right]\\ \mathrm{CI}=\frac{\mathrm{Ca}}{\mathrm{ICa}}+\frac{\mathrm{Cb}}{\mathrm{ICb}}+\mathrm{.Math.}+\frac{\mathrm{Cx}}{\mathrm{ICx}}& \left[\mathrm{Equation}3\right]\end{array}$

(36) Ca: concentration of drug a when administered in combination;

(37) Cb: concentration of drug b when administered in combination;

(38) Cx: concentration of drug x when administered in combination;

(39) ICa: concentration of drug a administered alone, which corresponds to inhibition achieved when administered in combination;

(40) ICb: concentration of drug b administered alone, which corresponds to inhibition achieved when administered in combination;

(41) ICx: concentration of drug x administered alone, which corresponds to inhibition achieved when administered in combination.

(42) TABLE-US-00004 TABLE 4 Inhibition of splenocyte proliferation (n = 3, Test group Drug concentration Mean SD) Water extract 3 g/ml 13.0 9.14 30% ethanol extract 3 g/ml 7.8 10.17 50% ethanol extract 3 g/ml 30.4 2.74 70% ethanol extract 3 g/ml 44.2 2.11 100% ethanol extract 2 g/ml 62.3 0.83 4 g/ml 99.9 1.03 100% isopropanol extract 3 g/ml 43.1 1.04 100% methanol 3 g/ml 35.8 3.06 Dexamethasone (positive 0.5 M 110.3 0.62 control) Montelukast (positive 10 M 44.1 4.34 control)

(43) TABLE-US-00005 TABLE 5 Inhibition of splenocyte proliferation (%, n = 3, Mean CI Test group Concentration (M) SD) value Justicidin A (JA) 0.5 2.7 1.84 1 9.4 4.36 2.5 56.4 18.80 5 85.0 2.43 Justicidin B (JB) 0.25 14.2 1.43 0.5 64.3 0.50 Justicidin C (JC) 0.5 3.6 4.00 5 8.4 3.20 Phyllamyricin C 0.5 0.9 2.86 (PC) 5 18.8 2.82 JB + JA 0.25 + 0.5 46.6 0.81 0.78 JB + JC 0.25 + 0.5 40.2 0.33 0.69 JB + PC 0.25 + 0.5 30.2 4.37 0.82 JB + JA + JC 0.25 + 0.5 + 0.5 39.7 6.28 0.88 JB + JA + JC + PC 0.25 + 0.5 + 0.5 + 0.5 43.9 6.69 0.88 JB + JA 0.5 + 0.5 90.1 3.93 0.49 JB + JC 0.5 + 0.5 86.2 0.65 0.42 JB + PC 0.5 + 0.5 79.6 1.80 0.45 JB + JA + JC + PC 0.5 + 0.5 + 0.5 + 0.5 88.7 4.45 0.53 Dexamethasone 0.5 118.7 0.21 (positive control) Montelukast 0.5 40.2 2.64 (positive control)

(44) TABLE-US-00006 TABLE 6 Inhibition of splenocyte Concentration proliferation (%, Test group (M) n = 3, Mean SD) CI value Justicidin A (JA) 1 43.4 1.63 5 99.0 2.35 Justicidin B (JB) 0.25 72.5 2.66 0.5 90.8 0.94 Justicidin C (JC) 1 6.7 15.08 5 31.7 12.81 phyllamyricin C 1 19.0 18.44 (PC) 5 25.0 11.37 JA + JB 1 + 0.1 94.4 0.94 0.40 1 + 0.2 94.8 2.35 0.57 JA + JC 1 + 0.1 83.5 1.90 0.26 1 + 0.2 85.9 3.19 0.26 JA + PC 1 + 0.1 84.8 2.73 0.25 1 + 0.2 86.0 2.59 0.25 JB + JC 0.2 + 0.02 84.4 1.40 0.49 0.2 + 0.04 80.5 6.38 0.56 JB + PC 0.2 + 0.02 82.5 0.84 0.52 0.2 + 0.04 82.0 4.45 0.53 100% ethanol 2 g/ml 97.0 0.91 extract of Justicia procumbens L. Dexamethasone 0.2 94.1 1.80 (positive control)

(45) As can be seen in Table 4 above, the inhibition of splenocyte proliferation almost never appeared in the case of the water extract of Justicia procumbens L., and the degree of inhibition increased as the concentration (%) of ethanol used for extraction increased. In addition, it was shown that treatment with the isopropanol extract and methanol extract of Justicia procumbens L. inhibited the proliferation of splenocytes by 43% and 36%, respectively.

(46) Meanwhile, as shown in Tables 5 and 6 above, each of justicidin A, justicidin B, justicidin C and phyllamyricin C exhibited the effect of inhibiting splenocyte proliferation, even when it was used alone. In particular, justicidin A and justicidin B showed high inhibitory activities.

(47) In addition, when treatment with justicidin A or justicidin B in combination with other three active substances exhibited higher inhibitory effects than treatment with justicidin A or justicidin B alone, and particularly, showed a CI value of less than 1, suggesting that administration of justicidin A or justicidin B in combination with other active substances shows synergistic effects.

(48) TABLE-US-00007 TABLE 7 Inhibition Inhibition of of IL5 IL4 secretion secretion Concentration (%, n = 2, Mean CI (%, n = 2, CI Test group (M) SD) value Mean SD) value Justicidin A 1 10.6 0.08 25.4 0.01 (JA) 5 54.7 0.02 56.7 0.00 Justicidin B 0.5 35.9 0.01 55.8 0.01 (JB) 1 78.2 0.01 84.4 0.00 Justicidin C 1 14.1 0.05 39.8 0.03 (JC) 5 26.1 0.04 36.4 0.02 Phyllamyricin C 1 9.8 0.05 22.9 0.01 (PC) 5 14.4 0.01 67.9 0.01 JB + JA 0.5 + 0.5 60.2 0.01 0.73 72.4 0.01 0.70 JB + JC 0.5 + 0.5 51.2 0.01 0.80 70.0 0.01 0.65 JB + PC 0.5 + 0.5 39.0 0.03 0.94 71.5 0.00 0.74 JB + JA + JC + 0.5 + 0.5 + 66.4 0.01 0.73 84.0 0.01 0.63 PC 0.5 + 0.5 100% ethanol 4 g/ml 80.7 0.01 89.2 0.00 extract of Justicia procumbens L. Dexamethasone 0.5 100.0 0.00 103.3 0.00 (positive control) Montelukast 10 42.6 0.08 70.2 0.01 (positive control)

(49) TABLE-US-00008 TABLE 8 Inhibition Inhibition of IL4 of IL5 secretion secretion Concentration (%, n = 2, (%, n = 2, Test group (mM) Mean SD) CI value Mean SD) CI value Justicidin A 1 71.6 0.02 11.7 0.27 (JA) 5 86.4 0.04 25.7 0.08 Justicidin B 0.25 81.6 0.02 41.9 0.04 (JB) 0.5 87.6 0.02 66.8 0.10 Justicidin C 1 25.3 0.15 16.9 0.06 (JC) 5 51.7 0.02 26.2 0.00 Phyllamyricin C 1 11.8 0.01 0.5 0.05 (PC) 5 51.5 0.11 31.6 0.08 JA + JB 1 + 0.1 94.4 0.00 0.27 90.1 0.01 0.18 1 + 0.2 94.8 0.00 0.39 90.5 0.00 0.31 JA + JC 1 + 0.1 91.7 0.00 0.16 86.2 0.01 0.05 1 + 0.2 91.7 0.00 0.17 87.2 0.00 0.05 JA + PC 1 + 0.1 91.0 0.00 0.17 85.8 0.02 0.05 1 + 0.2 92.0 0.00 0.18 87.4 0.03 0.06 JB + JC 0.2 + 0.02 92.2 0.00 0.29 86.0 0.01 0.29 0.2 + 0.04 92.3 0.00 0.29 82.5 0.00 0.31 JB + PC 0.2 + 0.02 92.1 0.00 0.29 85.3 0.02 0.29 0.2 + 0.04 90.5 0.00 0.33 84.3 0.00 0.30 100% ethanol 2 g/ml 95.4 0.00 91.5 0.01` extract of Justicia procumbens L. Dexamethasone 0.2 97.8 0.00 98.9 0.01 (positive control)

(50) As shown in Tables 7 and 8 above, each of justicidin A, justicidin B, justicidin C and phyllamyricin C inhibited the secretion of IL4 and IL5, even when it was used alone. In addition, when treatment with justicidin A or justicidin B in combination with other active substances exhibited higher inhibitory effects than treatment with justicidin A or justicidin B alone, and particularly, showed a CI value of less than 1, suggesting that administration of justicidin A or justicidin B in combination with other active substances shows synergistic effects. Montelukast used as a positive control did not show the effect of inhibiting IL4 secretion, and showed only the effect of inhibiting IL5 secretion.

Example 3: Test for Evaluation of Expectorant Activity

(51) In order to evaluate the expectorant activities of the water and ethanol extracts of Justicia procumbens L., prepared in Preparation Examples 1-1 to 1-2, a test for evaluation of expectorant activity was performed using male ICR mice (weighed 30 to 33 g; Orientbio). Specifically, the extracts and the comparative drug ambroxol (200 mg/kg, Boehringer Ingelheim) were administered orally to the respective groups of mice that fasted the previous day. After 30 minutes, 5% phenol red was injected intraperitoneally to the mice, and after 30 minutes, the abdominal aorta was cut to exsanguinate the animal, and the entire trachea was dissected. The isolated trachea was freeze-stored in 1 ml of physiological saline for 24 hours. Next, the trachea was sonicated, and 1N NaOH was added to the supernatant (0.1 ml of 1N NaOH per ml of the supernatant). Then, the absorbance at 546 nm was measured and the expectorant activity was measured with the concentration of phenol red. For statistical processing, calculation was performed using Equation 4 below, and the results are shown in Table 7 below.
Expectoration(%)=100{(average absorbance value of test groupaverage absorbance value of normal group)/(average absorbance value of normal group)}100[Equation 4]

(52) TABLE-US-00009 TABLE 9 Drug concentration Expectoration (%, n = 4, Test group (mg/kg) Mean SE) Normal group 0 6.79 Water extract 200 31.8 13.38 30% ethanol extract 200 27.1 23.31 50% ethanol extract 200 58.0 11.39 70% ethanol extract 200 57.3 15.26 100% ethanol extract 200 41.2 7.55 Ambroxol (positive 200 11.4 12.89 control)

(53) As can be seen in Table 9 above, the water extract of Justicia procumbens L. had no expectorant activity, whereas the 30% to 100% ethanol extracts of Justicia procumbens L. had better expectorant activities than ambroxol used as the positive control.

Example 4: Test for Evaluation of Airway Hypersensitivity

(54) In order to examine the effect of the ethanol extract of Justicia procumbens L. of Preparation Example 1-2 on the lung function, airway narrowing was induced by methacholine, and airway hypersensitivity was measured using whole-body plethysmography (DSI WBP System; DSIs Buxco Inc., USA). To this end, 6-week-old female Blab/c mice as experimental animals were purchased and randomly divided into a normal group, an induced group (negative control group) and a test group. The animals were acclimated for 1 week, and on 0 and 14 days after 1 week of acclimation, the induced group and the test group were systematically sensitized by intraperitoneally administering 0.1% ovalbumin (OVA: 1 mg/mL, Al(OH).sub.3: 20 mg/mL) in an amount of 100 l/mouse. From one week after the final systematic sensitization (21 days), 200 mg/kg of the 100% ethanol extract of Justicia procumbens L. was administered orally to each test group every day for 10 days. As a positive control, 3 mg/kg of dexamethasone was administered intraperitoneally. After one hour, 0.2% ovalbumin solution was sprayed and inhaled into the mice for 1 hour by use of a nebulizer (PARI Boy SX, Germany GmbH). After final sensitization (30 days), the test animals were stabilized in the respective chamber biases for 12 minutes, and then methacholine was inhaled for 1 minute, followed by recording for 3 minutes, thereby measuring the data of airway hypersensitivity. The concentration of methacholine increased from 0, to 12.5, 25 and 50 mg/kg, and airway hypersensitivity was evaluated in terms of PenH value. The PenH value was calculated using Equation 5 below.

(55) $\begin{array}{cc}\mathrm{PenH}=\mathrm{Pause}\frac{\mathrm{PEF}}{\mathrm{PIF}}\mathrm{Pause}=\frac{\mathrm{Te}-\mathrm{Tr}}{\mathrm{Tr}}& \left[\mathrm{Equation}5\right]\end{array}$

(56) PIF: peak inspiratory flow;

(57) PEF: peak expiratory flow;

(58) Te: expiratory time;

(59) Tr: relaxation time.

(60) As shown in FIG. 4, 100% ethanol extract of Justicia procumbens L. inhibited airway hyperresistance as much as did dexamethasone used as the positive control.

Example 5: Evaluation of Airway Constriction Inhibition by Tissue Staining of OVA-Induced Balb/c Mice

(61) 5-week-old female Balb/c mice were purchased and acclimated for 1 week. On 0 and 14 days after 1 week of acclimation, the mice were systematically sensitized by intraperitoneally administering 0.1% ovalbumin (OVA: 1 mg/mL, Al(OH).sub.3: 20 mg/mL) in an amount of 100 l/mouse. From one week after the final systematic sensitization (21 days), 200 mg/kg of the 100% ethanol extract of Justicia procumbens L. was administered orally to the mice every day for 10 days. After one hour, 0.2% ovalbumin solution was sprayed and inhaled into the mice for 1 hour by use of a nebulizer (PARI Boy SX, Germany GmbH). 5 hours after final sensitization (30 days), the mice were biopsied, and the lung tissue was taken and fixed in 10% neutral formalin. Thereafter, the lung tissue was sectioned to produce slices, and H & E staining was performed to observe the tissue. For tissue observation, the tissue was imaged with a microscope (400). The subepithelial smooth muscle thickness and epithelium thickness of the imaged tissue were measured using Image-pro Plus 6.0 program. The results are shown in Table 10 below and FIG. 5.

(62) TABLE-US-00010 TABLE 10 Justicia procumbens L. Induced group (OVA + JP) Normal group (OVA) 200 mg/kg Mean S.D. Mean S.D. Mean S.D. Subepithelial 5.62 1.15 7.35 1.10 4.77 0.36.sup.# smooth muscle thickness (m) Epithelium 15.57 1.47 36.99 6.31* 24.48 3.50*.sup.# thickness (m) *p < 0.05 vs. normal group, .sup.#p < 0.05 vs. induced group

(63) As a result, as can be seen in Table 10 above and FIG. 5, when the 100% ethanol extract of Justicia procumbens L. was administered orally, the thicknesses of subepithelial smooth muscles and epithelium, involved in airway constriction in lung tissue, decreased.

Example 6: Evaluation of Asthma Inhibitory Effect of Justicia procumbens L. Extract in Neutrophilic Asthma Balb/c Mouse Models

(64) 5-week-old female Balb/c mice were purchased and acclimated for 1 week. On 0 and 7 days after 1 week of acclimation, each of the mice was systematically sensitized by intranasally administering 75 g of ovalbumin and 10 g of lipopolysaccharide (LPS). 20, 50 or 100 mg/kg of the Justicia procumbens L. prepared in Preparation Example 3 or a positive control (10 mg/kg of dexamethasone and 1 mg/kg of montelukast) was administered orally to the mice On days 14, 15, 21, 22, 28, 29, 35, 36 and 37. One hour after administration, 50 g of ovalbumin was administered intranasally to the mice. 24 hours after final sensitization (day 37), lung lavage fluid was obtained through tracheostomy, and the lung tissue was isolated. The collected lung lavage fluid was centrifuged at 3000 rpm for minutes, and the supernatant was used to measure physiologically active substances (IL-4, IL-5, and IFN-), and the pellets were used to measure the number of inflammatory cells. The physiologically active substances (interleukin-4 (IL-4), interleukin-5 (IL-5), and interferon-gamma (IFN-)) in the isolated lung lavage fluid were measured using an enzyme-linked immunosorbent assay (ELISA, IL-4: Komabiotech #K0331144, IL-5: R&D system #M5000, IFN-: Komabiotech #K0331138) corresponding to each substance. In addition, the lung lavage fluid pellets were re-dissolved in 0.5 mL of phosphate buffered saline, and 0.1 mL of the solution was added to each well of a 96-well plate and centrifuged at 800 rpm for 5 minutes to attach the cells to the bottom (3 wells per sample). Then, each sample was stained with Diff Quik staining solution (Sysmex), and photographed with a microscope at 2-6 random sites. About 200 cells per sample were counted and the percentage (%) of inflammatory cells of each sample was calculated. The total number of inflammatory cells was measured with a microscope using a hematocytometer, and the number of eosinophils and the number of neutrophils were calculated using the percentage (%) of inflammatory cells of each sample. Staining of the lung tissue was performed in the same manner as described in Example 5 above. Statistical processing was performed using an SPSS program, and a Levene test was performed for a test for equal variance, and significance was tested by one-way analysis (ANOVA).

(65) The results are shown in Table 11 below and FIG. 6.

(66) TABLE-US-00011 TABLE 11 Lung lavage Lung Lung fluid_total lavage lavage Lung Lung Test group inflammatory fluid_eosinophil fluid_neutrophil lavage lavage (n = 5, Mean cell number number number fluid_IL-4 fluid_IL-5 S.E.) (10.sup.5) (10.sup.5) (10.sup.5) (pg/mL) (pg/mL) Normal group 0.7 0.04 0.0 0.00 0.0 0.00 36.2 4.74 15.5 9.54 Induced group 6.3 0.10** 1.3 0.21** 2.3 0.13** 256.1 95.3** 480.3 143.4** 20 mg/kg 5.1 0.30## 0.8 0.06## 1.9 0.21 83.6 1.92## 147.7 6.38## Justicia procumbens L. extract 50 mg/kg 4.0 0.13## 0.6 0.06## 1.4 0.10## 69.8 3.88## 99.0 3.45##, $$ Justicia procumbens L. extract 100 mg/kg 3.6 0.17## 0.6 0.06## 1.6 0.07## 74.0 21.77## 87.9 10.61##, $$ Justicia procumbens L. extract 10 mg/kg 4.0 0.25## 0.7 0.05## 1.5 0.07## 96.9 14.40## 194.4 23.1## montelukast (positive control) 1 mg/kg 1.3 0.07## 0.1 0.01## 0.2 0.02## 32.1 9.51## 53.0 13.59##, $$ dexamethasone (positive control) Subepithelial Lung smooth Test group lavage Epithelium muscle (n = 5, Mean fluid_IFN- thickness thickness S.E.) (pg/mL) (m) (m) Normal group 0.0 0.00 3.5 0.28 1.3 0.15 Induced group 121.3 57.57** 30.3 2.94** 11.3 0.73** 20 mg/kg 22.8 0.26##, $$ 22.3 1.23# 9.4 0.57 Justicia procumbens L. extract 50 mg/kg 11.7 1.02##, $$ 20.1 1.53##, $ 8.7 0.39#, $ Justicia procumbens L. extract 100 mg/kg 7.2 0.28##, $$ 19.7 1.51##, $ 8.4 0.40##, $ Justicia procumbens L. extract 10 mg/kg 32.2 2.13## 25.5 1.64 10.7 0.59 montelukast (positive control) 1 mg/kg 1.3 0.61##, $$ 5.0 0.38##, $$ 2.7 0.35 dexamethasone (positive control) **p < 0.01 vs. normal group, #p < 0.05 vs. induced group, ##p < 0.01 vs. induced group, $p < 0.05 vs. montelukast, $$p < 0.01 vs. montelukast.

(67) As a result, as Table 11 above and FIG. 6, in the neutrophilic asthma mouse models, the Justicia procumbens L. extract showed the effect of reducing the allergic asthma markers (IL-4, IL-5, and eosinophilic cells) in a concentration-dependent manner, and also showed the effect of reducing the non-allergic asthma markers (IFN- and neutrophilic cells). In addition, it was observed that the epithelium thickness and the subepithelial smooth muscle thickness also decreased depending on the concentration of the Justicia procumbens L. extract. Among these markers, IL-5, IFN-, the epithelium thickness and the subepithelial smooth muscle thickness more statistically significantly decreased in the group treated with the Justicia procumbens L. extract than in the group treated with the positive control montelukast.

Formulation Example 1: Preparation of Medicaments

(68) 1-1: Preparation of Powder

(69) TABLE-US-00012 Extract of a plant of the genus 100 mg Justicia or a fraction thereof Lactose 100 mg Talc 10 mg

(70) The above components are mixed with one another and filled in an airtight container, thereby preparing powder.

(71) 1-2: Preparation of Tablet

(72) TABLE-US-00013 Extract of a plant of the genus 100 mg Justicia or a fraction thereof Corn starch 100 mg Lactose 100 mg Magnesium stearate 2 mg

(73) The above components are mixed with one another, and compressed according to a conventional tablet preparation method, thereby preparing a tablet.

(74) 1-3: Preparation of Capsule

(75) TABLE-US-00014 Extract of a plant of the genus 100 mg Justicia or a fraction thereof Corn starch 100 mg Lactose 100 mg Magnesium stearate 2 mg

(76) According to a conventional capsule preparation method, the above components are mixed with one another and filled in a gelatin capsule, thereby preparing a capsule.

(77) 1-4: Preparation of Injectable Solution

(78) TABLE-US-00015 Extract of a plant of the genus 100 mg Justicia or a fraction thereof Injectable sterile distilled water q.s. pH adjusting agent q.s.

(79) According to a conventional method for preparation of an injectable solution, the above components are used per ampoule (2 ml), thereby preparing an injectable solution.

(80) 1-5: Preparation of Liquid Formulation

(81) TABLE-US-00016 Extract of a plant of the genus 100 mg Justicia or a fraction thereof Sugar 20 g Isomerized sugar 20 g Lemon fragrance q.s.

(82) Purified water is added to a total volume of 1,000 ml. According to a conventional method for preparation of a liquid formulation, the above components are mixed with one another, and then filled in a brown bottle and sterilized, thereby preparing a liquid formulation.

Formulation Example 2: Preparation of Food

(83) TABLE-US-00017 Extract of a plant of the genus 100 mg Justicia or a fraction thereof Vitamin mixture q.s. Vitamin A acetate 70 g Vitamin E 1.0 mg Vitamin B1 0.13 mg Vitamin B2 0.15 mg Vitamin B6 0.5 mg Vitamin B12 0.2 g Vitamin C 10 mg Biotin 10 g Amide nicotinate 1.7 mg Folic acid 50 g Calcium pantothenate 0.5 mg Mineral mixture q.s. Ferrous sulfate 1.75 mg Zinc oxide 0.82 mg Potassium phosphate monobasic 15 mg Potassium phosphate dibasic 55 mg Potassium citrate 90 mg Calcium carbonate 100 mg Magnesium chloride 24.8 mg

(84) Although the contents of the vitamins and the mineral mixture are preferably those suitable for health functional foods, these contents may be optionally modified. According to a conventional method for preparation of health functional food, the above components are mixed with one another, and then prepared into a health functional food (e.g., nutritional candy) according to a conventional method.

Formulation Example 3: Preparation of Beverage

(85) TABLE-US-00018 Extract of a plant of the genus 100 mg Justicia or a fraction thereof Citric acid 1000 mg Oligosaccharide 100 g Plum concentrate 2 g Taurine 1 g

(86) Purified water is added to a total volume of 900 ml. According to a conventional method for preparation of a health functional beverage, the above components are mixed with one another, and then stirred with heating at 85 C. for about 1 hour. Then, the resulting solution is filtered, and collected in a 2-liter sterilized container. Next, it is sealed, sterilized, cold-stored, and then used in the preparation of the health functional beverage composition of the present invention.

(87) Although the above composition is a preferable example of components relatively suitable for favorite beverages, the contents thereof may be optionally modified according to regional and national preferences, including consumer characteristics, consumer nations, the intended use, etc.