Methods for treating inflammatory diseases

Abstract

The present invention provides a composition containing, as active ingredients, Panax ginseng, Adenophora triphylla, Wolfiporia extensa, Rehmannia glutinosa, and Mel for an antitussive, expectorant, or anti-inflammatory action, and a composition for preventing, alleviating, or treating a respiratory disease. The present invention provides a compositions having excellent effects compared with an existing antitussive agent and expectorant agent. The compositions of the present invention are a naturally derived material and has little cytotoxicity, and thus is expected to be safely used as a medicine or food composition having antitussive, expectorant, and anti-inflammatory effects.

Claims

1. A method for alleviating or treating an inflammatory disease, the method comprising administering, to a subject, a composition comprising Panax ginseng, Adenophora triphylla, Wolfiporia extensa, Rehmannia glutinosa, and mel, wherein the inflammatory disease is selected from the group consisting of dermatitis, edema, atopic disease, conjunctivitis, periodontitis, rhinitis, otitis media, laryngopharingitis, tonsillitis, stomach ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, tendovaqinitis, peritendinitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome and multiple sclerosis.

2. The method of claim 1, wherein the composition comprises 4-5 wt % of Panax ginseng, 4-5 wt % of Adenophora triphylla, 8-10 wt % of Wolfiporia extensa, 43-48 wt % of Rehmannia glutinosa, and 35-40 wt % of mel.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1a, 1 b, 1c, 1d, 1e, and 1f show ultra-performance liquid chromatography (UPLC) analysis results of Adenophora triphylla (AR), a composition obtained by mixing Panax ginseng, Wolfiporia extensa, Rehmannia glutinosa, and mel, without Adenophora triphylla (KOG), and a composition obtained by mixing Adenophora triphylla, Panax ginseng, Wolfiporia extensa, Rehmannia glutinosa, and mel (SKOG), respectively. FIGS. 1a, 1c and 1e show UPLC analysis results of standard materials. FIGS. 1b, 1d and 1f show UPLC analysis results of AR, KOR and SKOG, respectively.

(2) FIG. 2 is a graph showing the body weight change of experimental animals in the antitussive effect measurement by SKOG administration.

(3) FIG. 3 is a graph showing the coughing number change of experimental animals by SKOG administration.

(4) FIG. 4 shows the histopathological change of the organs of experimental animals by SKOG administration. Left regions indicate Hematoxylin-Eosin stain results and right regions indicate toluidine blue stain results. “LU”, “CA”, “CM”, and “EP” represent lumen, cartilage, submucosa, and epithelium, respectively. A: normal control group, B: NH.sub.4OH control group, C: TB 50 mg/kg administered group, D: AR 400 mg/kg administered group, E: KOG 400 mg/kg administered group, F: SKOG 400 mg/kg administered group, G: SKOG 200 mg/kg administered group, H: SKOG 100 mg/kg administered group. Scale bars indicate 120 μm.

(5) FIG. 5 shows the histopathological change of the lungs of experimental animals by SKOG administration. “SB”, “TA”, and “BR” represent secondary bronchus, alveolus-terminal bronchiole, and bronchus, respectively. A: normal control group, B: NH.sub.4OH control group, C: TB 50 mg/kg administered group, D: AR 400 mg/kg administered group, E: KOG 400 mg/kg administered group, F: SKOG 400 mg/kg administered group, G: SKOG 200 mg/kg administered group, H: SKOG 100 mg/kg administered group. Scale bars indicate 120 μm.

(6) FIG. 6 is a graph showing the body weight change of experimental animals in the expectorant effect measurement by SKOG administration.

(7) FIG. 7 shows the visual change of body surfaces of experimental animals by SKOG administration. A: normal control group, B: AM 250 mg/kg administered group, C: AR 400 mg/kg administered group, D: KOG 400 mg/kg administered group, E: SKOG 400 mg/kg administered group, F: SKOG 200 mg/kg administered group, G: SKOG 100 mg/kg administered group. Scale bars indicate 120 μm.

(8) FIG. 8 shows the mucus secretion change of experimental animals by SKOG administration.

(9) FIG. 9 shows the histopathological change of intrapulmonary secondary bronchus mucosa of experimental animals by SKOG administration. “LU” and “EP” represent lumen and epithelium, respectively. A: normal control group, B: AM 250 mg/kg control group, C: AR 400 mg/kg administered group, D: KOG 400 mg/kg administered group, E: SKOG 400 mg/kg administered group, F: SKOG 200 mg/kg administered group, G: SKOG 100 mg/kg administered group. Scale bars indicate 60 μm.

(10) FIG. 10 shows the thickness change of intrapulmonary secondary bronchus mucosa and the numerical increase change of PAS positive cells in experimental animals by SKOG administration.

(11) FIG. 11 shows the body weight change of experimental animals in the anti-inflammatory effect measurement by SKOG administration.

(12) FIG. 12 shows the visual change of ears of experimental animals by SKOG administration. A: normal control group, B: xylene control group, C: dexamethasone 1 mg/kg administered group, D: AR 400 mg/kg administered group, E: KOG 400 mg/kg administered group, F: SKOG 400 mg/kg administered group, G: SKOG 200 mg/kg administered group, H: SKOG 100 mg/kg administered group. Scale bars indicate 16 μm.

(13) FIG. 13 shows the ear weight change of experimental animals by SKOG administration.

(14) FIG. 14 shows the histopathological change of ear skin tissues of experimental animals by SKOG administration. “AS”, “EP”, “DE”, and “CA” represent anterior surface, epidermis, dermis, and cartilage, respectively. A: normal control group, B: xylene control group, C: dexamethasone 1 mg/kg administered group, D: AR 400 mg/kg administered group, E: KOG 400 mg/kg administered group, F: SKOG 400 mg/kg administered group, G: SKOG 200 mg/kg administered group, H: SKOG 100 mg/kg administered group. Scale bars indicate 120 μm.

DETAILED DESCRIPTION

(15) Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it would be obvious to those skilled in the art that the scope of the present invention is not limited by these examples.

(16) Throughout the present specification, the term “%” used to express the concentration of a specific material, unless otherwise particularly stated, refers to (wt/wt) % for solid/solid, (wt/vol) % for solid/liquid, and (vol/vol) % for liquid/liquid.

Example 1. Preparation of Composition for Antitussive, Expectorant, And Anti-Inflammatory Actions

(17) (1) Materials

(18) Pale yellow Adenophora triphylla Radix (AR, Andong, Gyeongsangbukdo, Korea), Panax ginseng (Geumsan, Chungcheongnamdo, Korea), Wolfiporia extensa (Anhui, Chinese), Rehmannia glutinosa (Andong and Gunwi, Gyeongsangbukdo, Korea), and mel (Okcheon, Chungcheongbukdo, Korea) were received from Okcheon Dang Pharmaceutical Co., Ltd. (Yeongcheon, Korea). Some test materials were stored in Medical Research center for Globalization of Herbal Formulation, Daegu Haany University, Gyeongsan, Korea) (Code No. Adenophora triphylla-AR2016Ku01, KOG-KOG2016Ku01, SKOG-SKOG2016Ku01). In addition, theobromine (TB, white powder), ambroxol (AM, white powder), and dexamethasone (DEXA, white granules) were purchased from Sigma-Aldrich (USA). AR and the above three kinds of control drugs were stored at 4° C. in a refrigerator until use.

(19) (2) Preparation Method for Adenophora triphylla Sample

(20) Adenophora triphylla was washed to remove impurities, such as soil, followed by complete removal of moisture, and then dried at 90-120° C. for 5-8 hours by constant hot-air drying or other drying methods, to a final moisture content of 5% or less. The dried product was prepared into a powder having a particle size of 80 mesh or more by using a pulverizer, such as a pin mill, a ball mill, a rod mill, an air mill, or a jet mill.

(21) (3) Preparation Method for KOG

(22) Wolfiporia extensa was washed to remove impurities, such as soil, followed by removal of moisture, and then the surface thereof was completely dried. Panax ginseng was washed against impurities, such as soil, to remove moisture, and then dried at 90-120° C. for 5-8 hours by hot-air drying or other drying methods, to a final moisture content of 5% or less. The dried Wolfiporia extensa and Panax ginseng were prepared into a powder having a particle size of 80 mesh or more by using a pulverizer, such as a pin mill, a ball mill, a rod mill, an air mill, or a jet mill. Rehmannia glutinosa was washed to remove impurities, such as soil, followed by removal of moisture, pulverized through a mechanical pulverizer, such as a blender, and then passed through a net or a filter with 70-80 mesh through a mechanical juicing device, such as a hydraulic presser to obtain a juice thereof. The juice needs to have a sugar content of 15-18 brix and a solid content of 13-16%, and needs to have a yield of 70% or more in the process of obtaining the juice through pulverization. Mel was processed to have a moisture content of 22-24% by heating at 80-85° C. KOG was prepared by mixing 4-5 wt % of the obtained Panax ginseng powder, 8-10 wt % of the Wolfiporia extensa powder, 4-5 wt % of the Adenophora triphylla powder, 35-40 wt % of mel, and 43-48 wt % of the Rehmannia glutinosa juice, followed by first aging at 94.5-96.5° C. for 72 hours, first cooling at 8-12° C. for 24 hours, second aging at 94.5-96.5° C. for 24 hours, and second cooling at room temperature for 72 hours.

(23) (4) Preparation Method for SKOG

(24) Wolfiporia extensa, Panax ginseng, Rehmannia glutinosa, and mel were prepared in the same manner as in the preparation method for KOG described above. Adenophora triphylla was washed against impurities, such as soil, to remove moisture, and then dried at 90-120° C. for 5-8 hours by hot-air drying or other drying methods, to a final moisture content of 5% or less. The dried product was prepared into a powder having a particle size of 80 mesh or more by using a pulverizer, such as a pin mill, a ball mill, a rod mill, an air mill, or a jet mill.

(25) SKOG was prepared by mixing 4-5 wt % of the obtained Panax ginseng powder, 8-10 wt % of the Wolfiporia extensa powder, 4-5 wt % of the Adenophora triphylla powder, 35-40 wt % of mel, and 43-48 wt % of the Rehmannia glutinosa juice, followed by first aging at 94.5-96.5° C. for 72 hours, first cooling at 8-12° C. for 24 hours, second aging at 94.5-96.5° C. for 24 hours, and second cooling at room temperature for 72 hours or more.

Example 2. Analysis of Specific Ingredients of AR, KOG and SKO

(26) (1) Instrument and Reagent

(27) Waters ACQUITY™ ultra performance liquid chromatography system (Waters Corporation, Milford, Mass., USA) equipped with Waters ACQUITY™ photodiode array detector (PDA; Waters Corporation, Milford, Mass., USA) and Waters ACQUITY™ BEH C18 column (1.7 μm, 2.1×100; Waters Corporation, Milford, Mass., USA) was used in ultra performance liquid chromatography (UPLC) analysis. In addition, Empower (Waters Corporation, Milford, Mass., USA) was used as analysis software, in the current analysis. A sample extractor was ultrasonicator model 8210R-DHT (Branson Ultrasonics, Danbury, Conn.). Reagents for this experiment were methanol (HPLC grade, Junsei Chemical Co., Ltd., Tokyo, Japan), acetonitrile (HPLC grade, BAKER, Center Valley, Pa., USA), and then water (Tertiary distilled water). The standard preparations of this experiment were from the Sigma-Aldrich (St. Louise, Mo., USA) or Extrasynthese (Genay Cedex, France).

(28) (2) Preparation of the Standard Solution

(29) The amount preparations of AR contain materials (lupeol, lobetyolin and syring aldehyde), Rehmanniae Radix Crudus contain materials (acteoside, catalposide and 5-hydroxymethyl-2-furfural (5H2F)) and Ginseng Radix Alba contain materials (ginsenoside Rg3 (Rg3)) were measured accurately and melted by DMSO (in lupeol) or methanol (in lobetyolin, syring aldehyde, acteoside, catalposide, 5H2F and Rg3) for standard stock solution as concentration level of 1 μg/ml. In the next, the right amounts of the standard undiluted solution were diluted with the methanol to be contained 1, 5, 10 ng/ml and they were a standard solution. A standard curve determination coefficient (R.sup.2) value of all standard materials was more than 0.999.

(30) (3) Preparation of the Test Liquid for Quantitative Analysis

(31) A test liquid for quantitative analysis was mixed with the sample equally and was measured 1 g precisely, and was added on the 30% methanol 10 ml, and then was extracted by microwave for 1 hour. This test liquid was filtered from the membrane filter of below 0.2 μm diameter, and was picked out as the test liquid.

(32) (4) Quantitation of the Ingredients

(33) The amounts of lupeol, lobetyolin, syring aldehyde acteoside, catalposide, 5H2F and Rg3 in AR, KOG or SKOG were quantified using UPLC equipped with PDA (photodiode array detector) and BEH (bridged ethylene hybrid) C18 column, and the Empower software. A temperature of the column was analyzed at the room temperature. In case of the PDA analysis wavelength, lupeol, acteoside, catalposide and 5H2F were analyzed in 280 nm, lobetyolin was analyzed in 310 nm, and then syring aldehyde was analyzed in 254 nm, respectively. A mobile phase was a mixed liquid of the acetonitrile and water which contain 0.1% formic acids as follows.

(34) TABLE-US-00001 TABLE 1 0.1% FA/Water 0.1% Time (min) (%) FA/Acetonitrile (%) Flow rate (ml/min) 0 98 2 0.40 1.0 98 2 0.40 2.0 90 10 0.40 4.0 70 30 0.40 7.0 50 50 0.40 9.0 30 70 0.40 10.0 10 90 0.40 12.0 0 100 0.40 14.0 98 2 0.40 16.0 98 2 0.40

(35) Rg3 was analyzed in 203 nm and the mobile phase was a mixed liquid of the acetonitrile and water as follows.

(36) TABLE-US-00002 TABLE 2 Time (min) Water (%) Acetonitrile (%) Flow rate (ml/min) 0 85 15 0.40 1.0 85 15 0.40 14.0 70 30 0.40 15.0 68 32 0.40 16.0 60 40 0.40 17.0 45 55 0.40 19.0 45 55 0.40 21.0 10 90 0.40 22.0 10 90 0.40 23.0 85 15 0.40

(37) The analysis condition was as in the following. The sample was injected with 2 μl, and a flow rate was 0.4 ml/min, and the result of analysis was observed qualitative checking by retention time, and then was quantified by peak area method (Table 3 and FIG. 1a to 1f).

(38) TABLE-US-00003 TABLE 3 Test materials Ingredient (mg/kg) AR KOG SKOG Lupeol 6.99 ± 0.24 — 224.52 ± 12.5  Lobetyolin 2029.00 ± 1.96   — — Syring 0.26 ± 0.03 — 0.14 ± 0.01 aldehyde 5H2F — 628.26 ± 13.2  559.50 ± 1.70  Acteoside — 0.33 ± 0.02 0.31 ± 0.01 Catalposide — 0.41 ± 0.03 0.33 ± 0.01 Rg3 — 7.27 ± 0.46 4.42 ± 0.02

(39) FIGS. 1b, 1d and 1d show UPLC analysis results of AR, KOG and SKOG, respectively.

(40) Lupeol, lobetyolin and syring aldehyde were detected as 6.99±0.24, 2029.00±1.96 and 0.26±0.03 mg/kg in AR, 5-hydroxymethyl-2-furfural (5H2F), acteoside, catalposide and Ginsenoside-Rg3 (Rg3) were detected as 628.26±13.2, 0.33±0.02, 0.41±0.03 and 7.27±0.46 mg/kg in KOG, and lupeol, syring aldehyde, 5H2F, acteoside, catalposide and Rg3 were detected as 224.52±12.5, 0.14±0.01, 559.50±1.70, 0.31±0.01, 0.33±0.01 and 4.42±0.02 mg/kg in SKOG, used in this study at UPLC analysis, respectively (Table 1 and FIGS. 1a to 1f).

Example 3. Antitussive Assay

(41) (1) Animals and Husbandry

(42) One-hundred thirty two 6-week male SPF/VAF CrljOri:CD1 [ICR] mice (OrientBio, Seungnam, Korea; body weight ranged in 29-32 g upon receipt) were prepared, and eight groups of 10 mice each were selected based on the body weights at 7 days after acclimatization based on the body weights (intact control: 34.30±1.74 g, ranged in 31.8-37.0 g; NH.sub.4OH treated mice: 34.17±1.32 g, ranged in 31.0-37.3 g), as follows.

(43) Animals were allocated four per polycarbonate cage in a temperature (20-25° C.) and humidity (50-55%) controlled room. Light:dark cycle was 12 hour: 12 hour, and s standard rodent chow (Cat. No. 38057; Purina feed, Seungnam, Korea) and water were supplied free to access. All laboratory animals were treated according to the national regulations of the usage and welfare of laboratory animals, and approved by the Institutional Animal Care and Use Committee in Daegu Haany University (Gyeongsan, Gyeongbuk, Korea) [DHU2016-034, Apr. 22, 2016; ANNEX III].

(44) Experimental groups (Eight groups, 10 mice in each group were finally sacrificed)

(45) 1. Intact vehicle control: Vehicle (distilled water) treated intact control mice

(46) 2. NH.sub.4OH control: Vehicle administered and NH.sub.4OH exposured control mice

(47) 3. TB: TB 50 mg/kg administered and NH.sub.4OH exposured mice

(48) 4. AR: AR 400 mg/kg administered and NH.sub.4OH exposured mice

(49) 5. KOG: KOG 400 mg/kg administered and NH.sub.4OH exposured mice

(50) 6. SKOG400: SKOG 400 mg/kg administered and NH.sub.4OH exposured mice

(51) 7. SKOG200: SKOG 200 mg/kg administered and NH.sub.4OH exposured mice

(52) 8. SKOG100: SKOG 100 mg/kg administered and NH.sub.4OH exposured mice

(53) (2) Test Substance Administration

(54) SKOG were suspended in distilled water as 40, 20 and 10 mg/ml concentration, and orally administered in a volume of 10 ml/kg (as equivalence to 400, 200 and 100 mg/kg), once a day for 11 days before NH.sub.4OH exposure. In addition, AR and KOG were also suspended in distilled water as 40 mg/ml concentration, and orally administered in a volume of 10 ml/kg (as equivalence to 400, 200 and 100 mg/kg), once a day for 11 days before NH.sub.4OH exposure. TB was also dissolved in distilled water as 5 mg/ml concentrations, and also orally administered in a volume of 10 ml/kg (as equivalence to 50 mg/kg), once a day for 11 days before NH.sub.4OH exposure. In intact vehicle and NH.sub.4OH control mice, distilled water 10 ml/kg was orally administered, instead of AR, KOG, SKOG or TB to provide same restrain stresses, in the present experiment.

(55) (3) Body Weight Measurements

(56) Changes of body weight were measured at once a day from 1 day before initial to end of last 11th oral administration of AR, KOG, SKOG or TB using an automatic laboratory animal weighing electronic balance (Precisa Instrument, Dietikon, Switzland). Animals were overnight fasted (about 18 hours, water was not restricted) before initial test substance administration and sacrifice to reduced individual differences from feeding, and also to reduce the individual body weight differences at start of experiment, the body weight gains during 11 days of oral administration of AR, KOG, SKOG or TB were calculated as follow Equation [1], in the current experiment.
Body weight gains during 11 days of oral administration of test substances[B−A]=Body weights at last administration[B]−Body weights at first administration[A]  Equation [1]

(57) The results are shown in Table 4 and FIG. 2.

(58) TABLE-US-00004 TABLE 4 Body weights (g) at test material Body weight Periods administration gains (g) Groups First [A] Last [B] [B − A] Controls Intact 29.34 ± 1.29 30.98 ± 1.39 1.64 ± 0.74 NH.sub.4OH 29.44 ± 1.65 31.11 ± 2.22 1.67 ± 0.84 Reference TB 50 mg/kg 29.23 ± 1.49 30.94 ± 1.77 1.71 ± 0.97 AR 400 mg/kg 29.41 ± 0.96 31.12 ± 1.74 1.71 ± 1.18 KOG 400 mg/kg 29.58 ± 1.24 31.10 ± 1.56 1.52 ± 0.61 SKOG 400 mg/kg 29.66 ± 1.07 31.35 ± 1.34 1.69 ± 0.70 200 mg/kg 29.27 ± 0.75 31.04 ± 0.62 1.77 ± 0.32 100 mg/kg 29.41 ± 0.86 30.92 ± 1.17 1.51 ± 0.88

(59) FIG. 2 shows the weight change of the animals of antitussive effect measurement by SKOG administration.

(60) As shown in Table 4 and FIG. 2, No significant changes on the body weights and gains during 11 days of continuous oral administration periods were detected in NH.sub.4OH control mice as compared with those of intact vehicle control mice, respectively. In addition, no significant changes on the body weights and gains were demonstrated in all AR and KOG 400 mg/kg, TB 50 mg/kg, SKOG 400, 200 and 100 mg/kg treated mice as compared with those of NH.sub.4OH control mice, and no significant changes on the body weights and gains were also demonstrated in SKOG 400, 200 and 100 mg/kg treated mice as compared to those of AR and KOG 400 mg/kg, in the current study.

(61) The body weight gains during 11 days of continuous oral administration periods in NH.sub.4OH control were changed as 1.83% as compared with intact vehicle control, and they were changed as 2.40, 2.40, −8.98, 1.20, 5.99 and −9.58% in TB 50 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of NH.sub.4OH control mice, respectively.

(62) (4) Coughing Inducement and Monitoring

(63) Coughing was induced by single inhalation of 25% NH.sub.4OH (Sigma-Aldrich, St. Louise, Mo., USA) 0.3 ml in 1,000 ml glass Erlenmeyer flask for 45 sec at 1 hour after last 11th test substance administration, individually. After NH.sub.4OH exposure, the numbers of coughing responses were measured during 6 min using video observation equipments, as described previously with some modifications. Individual intact vehicle control mouse was exposured to 0.3 ml of saline contained 1,000 ml glass Erlenmeyer flask for 45 sec, instead of NH.sub.4OH, in this experiment. The criteria to define cough in mice is that opening the mouth accompanying sound of coughing, contraction of thoracic and abdomen muscles, and a jerking of the front body.

(64) FIG. 3 is a graph showing the change in the number of coughs in an experimental animal in the measurement of the antitussive effect by administration of SKOG.

(65) Significant (p<0.01) increases of the numbers of coughing responses during 6 min after 45 sec exposure of NH.sub.4OH in NH.sub.4OH control mice as compared with intact vehicle control mice. However, significant (p<0.01) and dose-dependent decreases of coughing responses were observed in SKOG 400, 200 and 100 mg/kg as compared with those of NH.sub.4OH control mice, respectively. In addition, AR and KOG 400 mg/kg, TB 50 mg/kg treated mice also showed significant (p<0.01) decreases of the numbers of coughing responses as compared with those of NH.sub.4OH control mice, respectively. Especially, SKOG 400 and 200 mg/kg showed significantly (p<0.01) decreased coughing numbers as compared with those of AR and KOG 400 mg/kg, and SKOG 100 mg/kg showed similar favorable inhibitory effects on the NH.sub.4OH-induced coughing responses as compared with those of AR and KOG 400 mg/kg, respectively. In addition, AR and KOG 400 mg/kg, SKOG100 mg/kg showed similar or more favorable inhibitory effects on the NH4OH-induced coughing responses as compared to those of TB 50 mg/kg, in the present study (FIG. 3).

(66) Mean numbers of coughing responses during 6 min after 45 sec exposure of NH.sub.4OH in NH.sub.4OH control were changed as 2390.00% as compared with intact vehicle control, but they were changed as −59.04, −60.91, −59.71, −72.42, −67.34 and −59.57% in TB 50 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of NH.sub.4OH control mice, respectively.

(67) (5) Histopathology

(68) After video image acquirement, some parts of individual lung (left lateral lobes) and trachea (3 mm from thyroid cartilages) were sampled and fixed in 10% neutral buffered formalin (NBF), and crossly trimmed. Then embedded in paraffin, sectioned (3˜4 μm) and stained with Hematoxylin and eosin (H&E) for general histopathology or toluidine blue for mast cells, and after that the histopathological profiles of each sample were observed under light microscope (Model Eclipse 80i, Nikon, Tokyo, Japan). To more detail changes, mean diameters of trachea lumen (μm), thicknesses of trachea wall, epithelium and submucosa (μm), numbers of infiltrated inflammatory cells and mast on the trachea (cells/mm.sup.2), mean alveolar surface area (ASA; %/mm.sup.2), mean thicknesses of alveolar septum (μm), numbers of infiltrated inflammatory cells on the alveolar septum (cells/mm2) were analyzed using a computer-assisted image analysis program (iSolution FL ver 9.1, IMT i-solution Inc., Quebec, Canada), according to previously established methods, respectively. The histopathologist was blinds to group distribution when this analysis was made, and at least five repeated measurements in same histological specimens prepared were considered to calculate each mean histomorphometrical value, whenever possible, in this histopathological evaluation (FIG. 4).

(69) The results are shown in Tables 5 and 6, and FIGS. 4 and 5.

(70) TABLE-US-00005 TABLE 5 Index Diameter of lumen Thickness (μm) Cells (Numbers/mm.sup.2) Groups (μm) Total wall Epithelium Submucosa Inflammatory Mast Control Intact 1177.86 ± 13.95 161.47 ± 15.34 14.38 ± 2.91 26.60 ± 4.85  21.40 ± 12.21  1.20 ± 0.79 NH.sub.4OH  658.57 ± 106.44.sup.a 220.38 ± 13.45.sup.h 37.53 ± 9.43.sup.h 89.73 ± 10.46.sup.h 449.30 ± 102.75.sup.h 38.70 ± 11.66.sup.h Reference TB 50 mg/kg  860.11 ± 100.75.sup.ac 191.23 ± 10.24.sup.hi 24.21 ± 4.36.sup.hi 47.31 ± 11.39.sup.hi 167.40 ± 40.26.sup.hi 14.30 ± 4.60.sup.hi AR 400 mg/kg  934.14 ± 104.43.sup.dc 185.59 ± 10.94.sup.hi 23.07 ± 3.32.sup.hi 44.99 ± 6.09.sup.hi 162.70 ± 37.57.sup.hi  8.60 ± 2.27.sup.hi KOG 400 mg/kg  934.89 ± 83.13.sup.ac 186.21 ± 11.10.sup.hi 23.87 ± 2.38.sup.hi 40.31 ± 6.64.sup.hi 134.10 ± 20.32.sup.hi  8.40 ± 1.51.sup.hi SKOG 400 mg/kg 1087.76 ± 118.72.sup.bcdf 171.56 ± 7.07.sup.hikm 18.92 ± 2.14.sup.hikm 30.30 ± 3.24.sup.lkm  85.50 ± 19.82.sup.hikm  1.30 ± 1.03.sup.ikm 200 mg/kg 1036.55 ± 84.sup.aceg 175.45 ± 5.03.sup.hikm 19.74 ± 1.37.sup.hikm 32.71 ± 3.79.sup.hikm 120.70 ± 20.94.sup.hikm  3.70 ± 1.34.sup.hikm 100 mg/kg  936.74 ± 70.89.sup.ac 185.19 ± 7.57.sup.hi 23.76 ± 3.34.sup.hi 42.41 ± 8.60.sup.hi 177.60 ± 26.02.sup.hi  8.70 ± 2.16.sup.hi Values are expressed mean ± SD of 10 mice

(71) TABLE-US-00006 TABLE 6 Alveolar surface Index area Septum thickness inflammatory cells Groups (%) (μm) (numbers/mm.sup.2) Controls Intact 78.87 ± 9.31  7.32 ± 1.45  30.20 ± 19.63 NH.sub.4OH 30.94 ± 9.58.sup.a 72.41 ± 10.80.sup.g 1886.70 ± 394.17.sup.g Reference TB 50 mg/kg 51.13 ± 5.95.sup.ac 30.53 ± 10.06.sup.gh  492.00 ± 114.18.sup.gh AR 53.83 ± 7.88.sup.ac 27.22 ± 7.28.sup.gh  453.80 ± 104.35.sup.gh 400 mg/kg KOG 52.34 ± 8.24.sup.ac 27.98 ± 3.75.sup.gh  492.60 ± 118.49.sup.gh 400 mg/kg SKOG 400 mg/kg 71.32 ± 5.65.sup.bcdf 12.56 ± 2.52.sup.ghik  234.20 ± 42.55.sup.ghik 200 mg/kg 62.29 ± 6.45.sup.acef 20.26 ± 3.12.sup.ghik  347.60 ± 79.19.sup.ghik 100 mg/kg 53.36 ± 9.65.sup.ac 27.99 ± 6.85.sup.gh  481.90 ± 132.69.sup.gh Values are expressed mean ± SD of 10 mice.

(72) FIGS. 4 and 5 show the histopathological change of the organs and lung of experimental animals by SKOG administration, respectively.

(73) Significant (p<0.01) decreases of the diameters of trachea lumen, increases of trachea wall total, epithelium and submucosa thicknesses, the numbers of trachea infiltrated inflammatory and mast cells, decreases of ASA, increases of the alveolar septum thicknesses and the numbers of inflammatory cells between alveolar septum were observed in the trachea and lung of NH.sub.4OH control as classic allergic acute inflammation related histopathological findings. However, these NH.sub.4OH-induced allergic acute inflammation related histopathological findings were significantly (p<0.01) and dose-dependently inhibited by 11 days of continuous oral pretreatment of SKOG 400, 200 and 100 mg/kg as compared with those of NH.sub.4OH control mice, respectively. In addition, AR and KOG 400 mg/kg, TB 50 mg/kg also significantly (p<0.01) reduced the NH.sub.4OH-induced allergic acute inflammation related histopathological findings as compared with those of NH4OH control mice, respectively. Especially, SKOG400 and 200 mg/kg showed significantly (p<0.01 or p<0.05) decreased NH.sub.4OH-induced allergic acute inflammation related histopathological findings as compared with those of AR and KOG 400 mg/kg, and SKOG 100 mg/kg showed similar favorable inhibitory effects on the NH.sub.4OH-induced allergic acute inflammation related histopathological findings as compared with those of AR and KOG 400 mg/kg, respectively. In addition, AR and KOG 400 mg/kg, SKOG 100 mg/kg showed similar or more favorable inhibitory effects on the NH.sub.4OH-induced allergic acute inflammation related histopathological findings as compared to those of TB 50 mg/kg, in this study (Tables 5 and 6, FIGS. 4 and 5).

(74) Mean diameters of trachea lumen in NH.sub.4OH control were changed as −44.09% as compared with intact vehicle control, but they were changed as 30.60, 41.84, 41.96, 65.17, 57.39 and 42.24% in TB 50 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of NH.sub.4OH control mice, respectively.

(75) Mean thicknesses of trachea wall in NH.sub.4OH control were changed as 36.49% as compared with intact vehicle control, but they were changed as −13.23, −15.79, −15.51, −22.15, −20.39 and −15.97% in TB 50 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of NH.sub.4OH control mice, respectively.

(76) Mean thicknesses of trachea epithelium in NH4OH control were changed as 160.98% as compared with intact vehicle control, but they were changed as −35.48, −38.52, −36.41, −49.59, −47.39 and −36.70% in TB 50 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of NH.sub.4OH control mice, respectively.

(77) Mean thicknesses of trachea submucosa in NH.sub.4OH control were changed as 236.33% as compared with intact vehicle control, but they were changed as −47.27, −49.85, −55.07, −66.23, −63.54 and −52.73% in TB 50 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of NH.sub.4OH control mice, respectively.

(78) Mean numbers of infiltrated inflammatory cells on the trachea of NH4OH control were changed as 1999.53% as compared with intact vehicle control, but they were changed as −62.74, −63.79, −59.03, −80.97, −73.14 and −60.47% in TB 50 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of NH.sub.4OH control mice, respectively.

(79) Mean numbers of infiltrated mast cells on the trachea of NH.sub.4OH control were changed as 3125.00% as compared with intact vehicle control, but they were changed as −63.05, −77.78, −78.29, −95.35, −90.44 and −77.52% in TB 50 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of NH.sub.4OH control mice, respectively.

(80) Mean ASA in NH.sub.4OH control were changed as −60.77% as compared with intact vehicle control, but they were changed as 65.26, 73.98, 69.16, 130.52, 101.33 and 72.48% in TB 50 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of NH.sub.4OH control mice, respectively.

(81) Mean thicknesses of alveolar septum in NH.sub.4OH control were changed as 889.73% as compared with intact vehicle control, but they were changed as −57.84, −62.41, −61.37, −82.66, −72.02 and −61.34% in TB 50 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of NH.sub.4OH control mice, respectively.

(82) Mean numbers of infiltrated inflammatory cells on the alveolar septum of NH.sub.4OH control were changed as 3034.05% as compared with intact vehicle control, but they were changed as −73.92, −75.95, −73.89, −87.59, −81.58 and −74.46% in TB 50 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of NH.sub.4OH control mice, respectively.

Example 4. Expectorant Assay

(83) (1) Animals and Husbandry

(84) One-hundred twenty one 6-week male SPF/VAF CrljOri: CD1[ICR] mice (OrientBio, Seungnam, Korea; body weight ranged in 29-32 g upon receipt) were prepared, and seven groups of 10 mice each were selected based on the body weights at 7 days after acclimatization based on the body weights (Average:34.75±1.32 g, ranged in 31.6-37.4 g), as follows. Animals husbandries were conducted as same as antitussive assay. All laboratory animals were treated according to the national regulations of the usage and welfare of laboratory animals, and approved by the Institutional Animal Care and Use Committee in Daegu Haany University (Gyeongsan, Gyeongbuk, Korea) [DHU2016-035, Apr. 22, 2016; ANNEX IV].

(85) Experimental groups (Seven groups, 10 mice in each group were finally sacrificed)

(86) 1. Control: Vehicle (distilled water) treated intact control mice

(87) 2. AM: AM 250 mg/kg administered mice

(88) 3. AR: AR 400 mg/kg administered mice

(89) 4. KOG: KOG 400 mg/kg administered mice

(90) 5. SKOG400: SKOG 400 mg/kg administered mice

(91) 6. SKOG200: SKOG 200 mg/kg administered mice

(92) 7. SKOG100: SKOG 100 mg/kg administered mice

(93) (2) Test Substance Administration

(94) AR, KOG and SKOG were orally administered as same as antitussive assay, once a day for 11 days before phenol red treatment. In addition, AM was also dissolved in distilled water as 25 mg/ml concentrations, and orally administered in a volume of 10 ml/kg (as equivalence to 250 mg/kg), once a day for 11 days before phenol red treatment. In intact vehicle control mice, distilled water 10 ml/kg was orally administered, instead of AR, KOG, SKOG or AM to provide same restrain stresses, in the present experiment.

(95) (3) Body Weight Measurements

(96) Changes of body weights and gains were measured as same methods described in Example 3. The results are shown in Table 7 and FIG. 6.

(97) TABLE-US-00007 TABLE 7 Body weights (g) at test material Body weight Periods administration gains (g) Groups First [A] Last [B] [B − A] Control Intact 29.09 ± 1.23 30.96 ± 1.01 1.87 ± 0.60 Reference AM 250 mg/kg 29.23 ± 1.16 31.22 ± 1.65 1.99 ± 0.73 AR 400 mg/kg 29.40 ± 0.81 31.11 ± 1.25 1.71 ± 0.85 KOG 400 mg/kg 29.00 ± 1.60 30.69 ± 1.77 1.69 ± 0.90 SKOG 400 mg/kg 29.26 ± 1.12 31.01 ± 1.76 1.75 ± 1.02 200 mg/kg 29.20 ± 1.26 31.25 ± 1.83 2.05 ± 0.92 100 mg/kg 29.18 ± 1.04 31.23 ± 1.76 2.05 ± 1.03 Values are expressed mean ± SD of 10 mice.

(98) FIG. 6 shows the body weight change of experimental animals in the expectorant effect measurement by SKOG administration.

(99) No significant changes on the body weights and gains during 11 days of continuous oral administration periods were detected in AM 250 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg treated mice as compared with those of intact vehicle control mice, respectively. In addition, no significant changes on the body weights and gains were demonstrated in SKOG 400, 200 and 100 mg/kg treated mice as compared to those of AR and KOG 400 mg/kg, in our study (Table 7 and FIG. 6).

(100) The body weight gains during 11 days of continuous oral administration periods in AM 250 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice were changed as 6.42, −8.56, −9.63, −6.42, 9.63 and 9.63% as compared with intact vehicle control, respectively.

(101) (4) Body Surface Gross Findings

(102) FIG. 7 shows the visual change of body surfaces of experimental animals by SKOG administration.

(103) Noticeable and dose-dependent increases of body redness were demonstrated in SKOG 400, 200 and 100 mg/kg as compared with those of intact vehicle control mice, indicating increases of intraperitoneal injected phenol red uptake and secretion, respectively. In addition, AR and KOG 400 mg/kg, AM 250 mg/kg treated mice also showed dramatic increases of body redness at 30 min after intraperitoneal injection of phenol red solutions as compared with those of intact control mice, respectively. Especially, SKOG 400 and 200 mg/kg showed obvious increases of body surface redness gross signs as compared with those of AR and KOG 400 mg/kg, and SKOG 100 mg/kg showed similar body surface redness gross signs as compared with those of AR and KOG 400 mg/kg, respectively. In addition, AR and KOG 400 mg/kg, SKOG 100 mg/kg showed similar or more favorable increases of body surface redness gross signs as compared to those of AM 250 mg/kg, in the current experiment (FIG. 7).

(104) (5) Measurement of Mucous Secretions

(105) Mucous secretions were measured by single intraperitoneal injection of 5% phenol red (Junsei Chemical Co. Ltd., Tokyo, Japan) solution, dissolved in saline (w/v) 10 ml/kg at 30 min after last 11th test substance administration, and 30 min after phenol red solution injection, all mice were sacrificed by cervical dislocation without damaging the trachea, after gross image acquirement to observe body surface redness, individually. After dissected free from adjacent organs, the trachea was removed from the thyroid cartilage to the main stem bronchi. After ultrasonic for 15 min using ultrasonicator (Model 5210, Branson Ultrasonics, Danbury, Conn., USA), 1 ml NaHCO.sub.3 solution (5%, w/v) add to the normal saline, and optical density of these prepared trachea lavage fluid (TLF) were measured at 546 nm using a microplate reader (Model Sunrise, Tecan, Männedorf, Switzerland) as described previously with some modifications.

(106) FIG. 8 shows the mucus secretion change of experimental animals by SKOG administration.

(107) Significant (p<0.01) and dose-dependent increases of the TLF OD values were demonstrated in SKOG 400, 200 and 100 mg/kg as compared with those of intact vehicle control mice at 30 min after intraperitoneal injection of phenol red solutions, indicating increases of the trachea mucous secretion, respectively. In addition, AR and KOG 400 mg/kg, AM 250 mg/kg treated mice also showed significant (p<0.01) increases of the TLF OD values as compared with those of intact vehicle control mice, respectively. Especially, SKOG 400 and 200 mg/kg showed significantly (p<0.01) increased TLF OD values as compared with those of AR and KOG 400 mg/kg, and SKOG 100 mg/kg showed similar favorable mucous secretion increase effects as compared with those of AR and KOG 400 mg/kg, respectively. In addition, AR and KOG 400 mg/kg, SKOG 100 mg/kg showed similar or more favorable TLF OD values as compared to those of AM 250 mg/kg, in the present experiment (FIG. 8).

(108) The TLF OD values in AM 250 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice were changed as 33.95, 40.17, 39.29, 66.96, 58.78 and 46.27% as compared with intact vehicle control, respectively.

(109) (6) Histopathology

(110) Simultaneously, some parts of individual lung (left lateral lobes) were sampled at trachea excisions, and fixed in 10% NBF, and crossly trimmed. Then embedded in paraffin, sectioned (3˜4 μm) and stained with H&E for general histopathology or PAS (periodic acid schiff) for mucous producing cells, and after that the histopathological profiles of each sample were observed under light microscope. To more detail changes, mean thicknesses of secondary bronchus mucosa, numbers of PAS positive mucous producing cells on the secondary bronchus (cells/mm.sup.2) were analyzed using a computer-assisted image analysis program, according to previously established methods, respectively. The histopathologist was blinds to group distribution when this analysis was made, and at least five repeated measurements in same histological specimens prepared were considered to calculate each mean histomorphometrical value, whenever possible, in this histopathological evaluation.

(111) FIG. 9 shows the histopathological change of intrapulmonary secondary bronchus mucosa of experimental animals by SKOG administration.

(112) FIG. 10 shows the thickness change of intrapulmonary secondary bronchus mucosa and the numerical increase change of PAS positive cells in experimental animals by SKOG administration.

(113) Significant (p<0.01 or p<0.05) and dose-dependent increases of the intrapulmonary secondary bronchus mucosa thicknesses and PAS positive mucous producing cells were observed in SKOG 400, 200 and 100 mg/kg treated mice as compared to those of intact vehicle control mice, suggesting increases of mucous secretion or activity of bronchus mucosa, respectively. In addition, AR and KOG 400 mg/kg, AM 250 mg/kg also significantly (p<0.01) increased the intrapulmonary secondary bronchus mucosa thicknesses and PAS positive mucous producing cell numbers as compared with those of intact vehicle control mice, respectively. Especially, SKOG 400 and 200 mg/kg showed significantly (p<0.01) increased intrapulmonary secondary bronchus mucosa thicknesses and PAS positive mucous producing cell numbers as compared with those of AR and KOG 400 mg/kg, and SKOG100 mg/kg showed similar the intrapulmonary secondary bronchus mucosa thicknesses and PAS positive mucous producing cell numbers as compared with those of AR and KOG 400 mg/kg, respectively. In addition, AR and KOG 400 mg/kg, SKOG 100 mg/kg showed similar or more favorably increased the intrapulmonary secondary bronchus mucosa thicknesses and PAS positive mucous producing cell numbers as compared to those of AM 250 mg/kg, in this experiment (FIGS. 9 and 10).

(114) Mean thicknesses of secondary bronchus mucosa in AM 250 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice were changed as 41.69, 56.73, 51.59, 111.08, 91.95 and 54.99% as compared with intact vehicle control, respectively.

(115) Mean numbers of secondary bronchus epithelial PAS positive mucous producing cells in AM 250 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice were changed as 231.58, 364.91, 492.98, 1082.46, 761.40 and 498.25% as compared with intact vehicle control, respectively

Example 5. Anti-Inflammatory Assay

(116) (1) Animals and Husbandry

(117) One-hundred thirty two 6-week male SPF/VAF CrljOri:CD1 [ICR] mice (OrientBio, Seungnam, Korea; body weight ranged in 29-32 g upon receipt) were prepared, and eight groups of 10 mice each were selected based on the body weights at 7 days after acclimatization based on the body weights (intact control: 33.88±1.11 g, ranged in 31.8˜35.4 g; Xylene treated mice: 33.85±1.32 g, ranged in 31.1-36.6 g), as follows. Animals husbandries were conducted as same as antitussive and expectorant assays. All laboratory animals were treated according to the national regulations of the usage and welfare of laboratory animals, and approved by the Institutional Animal Care and Use Committee in Daegu Haany University (Gyeongsan, Gyeongbuk, Korea) [DHU2016-036, Apr. 22, 2016; ANNEX V].

(118) Experimental groups (Eight groups, 10 mice in each group were finally sacrificed)

(119) 1. Intact vehicle control: Vehicle (distilled water) treated intact control mice

(120) 2. Xylene control: Vehicle administered and xylene topically applied control mice

(121) 3. DEXA: DEXA 1 mg/kg administered and xylene topically applied mice

(122) 4. AR: AR 400 mg/kg administered and xylene topically applied mice

(123) 5. KOG: KOG 400 mg/kg administered and xylene topically applied mice

(124) 6. SKOG400: SKOG 400 mg/kg administered and xylene topically applied mice

(125) 7. SKOG200: SKOG 200 mg/kg administered and xylene topically applied mice

(126) 8. SKOG100: SKOG 100 mg/kg administered and xylene topically applied mice

(127) (2) Test Substance Administration

(128) AR, KOG and SKOG were orally administered as same as antitussive and expectorant assays, once a day for 11 days before xylene topical applications. In addition, DEXA-water soluble granules were dissolved indistilled water as 1.5 mg/ml concentrations (0.1 mg/ml based on DEXA itself), and also orally administered in a volume of 10 ml/kg (as equivalence to 1 mg/kg based on DEXA itself), once a day for 11 days before xylene topical applications. In intact vehicle and xylene control mice, distilled water 10 ml/kg was orally administered, instead of AR, KOG, SKOG or DEXA to provide same restrain stresses, in the present experiment.

(129) (3) Body Weight Measurements

(130) Changes of body weights and gains were measured as same methods described in Example 3 and 4.

(131) The results are shown in Table 8 and FIG. 11.

(132) TABLE-US-00008 TABLE 8 Body weights (g) at test material Body weight Periods administration gains (g) Groups First [A] Last [B] [B − A] Controls Intact 28.94 ± 1.06 31.04 ± 1.49 2.10 ± 0.90 Xylene 28.93 ± 1.01 30.97 ± 1.62 2.04 ± 0.79 Reference DEXA 1 mg/kg 28.72 ± 1.10 27.80 ± 1.66.sup.ab  −0.92 ± 0.76.sup.ab AR 400 mg/kg 28.99 ± 1.21 31.12 ± 2.51 2.13 ± 1.63 KOG 400 mg/kg 29.20 ± 1.11 31.02 ± 1.78 1.82 ± 1.01 SKOG 400 mg/kg 29.11 ± 1.27 31.29 ± 2.19 2.18 ± 1.34 200 mg/kg 28.95 ± 1.26 31.29 ± 1.66 2.34 ± 0.70 100 mg/kg 29.07 ± 0.72 31.08 ± 1.10 2.01 ± 0.86 Values are expressed mean ± SD of 10 mice.

(133) FIG. 11 shows the body weight change of experimental animals in the anti-inflammatory effect measurement by SKOG administration.

(134) No significant changes on the body weights and gains during 11 days of continuous oral administration periods were detected in xylene control mice as compared with those of intact vehicle control mice, respectively. In addition, no significant changes on the body weights and gains were demonstrated in all three different dosages of SKOG 400, 200 and 100 mg/kg, AR and KOG 400 mg/kg treated mice as compared with those of xylene control mice, and no significant changes on the body weights and gains were also demonstrated in SKOG 400, 200 and 100 mg/kg treated mice as compared to those of AR and KOG 400 mg/kg, respectively. But DEXA 1 mg/kg treated mice showed significant (p<0.01 or p<0.05) decreases of body weights from 2 days after initial administration as compared with those of intact vehicle and xylene control mice, and also significant (p<0.01) decreases in body weight gains during 11 days of continuous oral administration periods as compared with those of intact vehicle and xylene control mice, in our experiment (Table 8, FIG. 11).

(135) The body weight gains during 11 days of continuous oral administration periods in xylene control were changed as −2.86% as compared with intact vehicle control, and they were changed as −145.10, 4.41, −10.78, 6.86, 14.71 and −1.47% in DEXA 1 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of xylene control mice, respectively.

(136) (4) Acute Inflammation Inducement

(137) Acute inflammations were induced by single topical application of 0.03 ml of xylene (Duksan Pure Chemical Co. Ltd., Ansan, Korea) to the anterior surface of the right ear at 1 hour after last 11th test substance administration, as described previously with some modifications. Equal volume of saline was topically applied in intact vehicle mouse ears, instead of xylene, in our experiment.

(138) (5) Ear Weight Measurement

(139) Two hours after topical application of xylene, circular sections of induced ear were taken using a cork borer with a 7-mm diameter and weighed as absolute wet-weights, and then the relative weights (% of bodyweights) of the ears were calculated to reduce the differences from individual body weights, as follow Equation [2], in the current experiment.
Relative ear weights(% vs body weights)=(Absolute ear wet-weights/body weight at sacrifice)×100  EQUATION [2]

(140) FIG. 12 shows the visual change of ears of experimental animals by SKOG administration.

(141) Noticeable acute inflammatory response related ear redness and edema were observed in xylene control mice at 2 hours after xylene topical applications as compared with intact vehicle control mice. However, these gross xylene-induced redness and edema findings were dose-dependently and dramatically inhibited by 11 days of continuous oral pre-administration of SKOG 400, 200 and 100 mg/kg as compared with those of xylene control mice, respectively. In addition, AR and KOG 400 mg/kg, DEXA 1 mg/kg treated mice also showed obvious decreases of the ear redness and edema as compared with those of xylene control mice at gross inspections, respectively. Especially, SKOG 400 and 200 mg/kg showed clear decreases of ear redness and edema gross signs as compared with those of AR and KOG 400 mg/kg, and SKOG 100 mg/kg showed similar xylene-induced ear redness and edema gross signs as compared with those of AR and KOG 400 mg/kg, respectively. In addition, SKOG 400 mg/kg showed favorable decreases of ear redness and edema gross signs as comparable to those of DEXA 1 mg/kg, but AR and KOG 400 mg/kg, SKOG 200 and 100 mg/kg showed slighter inhibitory effects on the xylene-induced ear redness and edema as compared to those of DEXA 1 mg/kg, in the current gross observation (FIG. 12).

(142) FIG. 13 shows the ear weight change of experimental animals by SKOG administration.

(143) Significant (p<0.01) increases of the ear absolute and relative weights were demonstrated in xylene control mice as compared with intact vehicle control mice at 2 hours after xylene topical applications. However, significant (p<0.01) and dose-dependent decreases of the ear absolute and relative weights were observed in SKOG 400, 200 and 100 mg/kg as compared with those of xylene control mice, respectively. In addition, AR and KOG 400 mg/kg, DEXA 1 mg/kg treated mice also showed significant (p<0.01) decreases of the ear absolute and relative weights as compared with those of xylene control mice, respectively. Especially, SKOG400 and 200 mg/kg showed significant (p<0.01 or p<0.05) decreases of ear weights as compared with those of AR and KOG 400 mg/kg, and SKOG 100 mg/kg showed similar inhibitory activities against xylene induced ear weight increases as compared with those of AR and KOG 400 mg/kg, respectively. In addition, SKOG 400 mg/kg showed favorable inhibitory effects on the absolute and relative ear weight increased induced by topical application of xylene as comparable to those of DEXA 1 mg/kg, but AR and KOG 400 mg/kg, SKOG 200 and 100 mg/kg showed slighter inhibitory effects on the xylene-induced ear weight increases as compared to those of DEXA 1 mg/kg, in the present observation (FIG. 13).

(144) The absolute ear weights in xylene control were changed as 75.20% as compared with intact vehicle control, but they were changed as −48.52, −32.99, −33.45, −45.09, −40.87 and −34.25% in DEXA 1 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of xylene control mice, respectively.

(145) The relative ear weights in xylene control were changed as 76.00% as compared with intact vehicle control, but they were changed as −42.61, −33.18, −33.55, −45.62, −41.67 and −34.47% in DEXA 1 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of xylene control mice, respectively.

(146) (6) Histopathology

(147) After ear weight measurement, individual ear samples were fixed in 10% NBF, and crossly trimmed. Then embedded in paraffin, sectioned (3˜4 μm) and stained with Hematoxylin and eosin (H&E) for general histopathology or toluidine blue for mast cells, and after that the histopathological profiles of each sample were observed under light microscope. To more detail changes, mean total, epidermis and dermis thicknesses of the ear anterior surface, numbers of infiltrated inflammatory cells and mast cells on the dermis of ear (cells/mm.sup.2), collagen occupied region percentages on the dermis (%/mm2) were analyzed using a computer assisted image analysis program, according to previously established methods, respectively. The histopathologist was blinds to group distribution when this analysis was made, and at least five repeated measurements in same histological specimens prepared were considered to calculate each mean histomorphometrical value, whenever possible, in this histopathological evaluation.

(148) The results are shown in Table 9 and FIG. 14.

(149) TABLE-US-00009 TABLE 9 Index Thickness (μm) Cells (Numbers/mm.sup.2) Collagen fiber Groups Total Epidermis Dermis Inflammatory Mast (%/mm.sup.2 of dermis) Controls Intact 103.41 ± 11.47 8.98 ± 0.93  54.86 ± 11.97  15.20 ± 4.66 69.00 ± 15.48 78.31 ± 9.75 Xylene 264.48 ± 30.02.sup.f 9.07 ± 1.16 132.28 ± 22.16.sup.f 263.40 ± 55.50.sup.f  8.20 ± 4.16.sup.a 26.74 ± 6.58.sup.f Reference TB 50 mg/kg  99.55 ± 9.57.sup.h 8.50 ± 1.65  52.33 ± 13.72.sup.h  10.10 ± 10.54.sup.fh 61.30 ± 12.65.sup.c 77.43 ± 12.79.sup.h AR 400 mg/kg 166.51 ± 17.59.sup.fh 8.77 ± 0.71  75.41 ± 8.63.sup.fh  72.30 ± 14.17.sup.fh 45.20 ± 10.52.sup.ac 64.73 ± 10.85.sup.gh KOG 400 mg/kg 158.83 ± 13.35.sup.fh 8.76 ± 0.82  71.55 ± 5.05.sup.fh  69.00 ± 12.00.sup.fh 42.40 ± 6.72.sup.ac 67.59 ± 4.70.sup.gh SKOG 400 mg/kg 105.94 ± 13.74.sup.hij 9.15 ± 1.09  51.58 ± 6.97.sup.hij  29.20 ± 8.26.sup.fhij 61.00 ± 10.92.sup.cde 81.51 ± 7.58.sup.hij 200 mg/kg 128.25 ± 15.08.sup.fhij 8.28 ± 1.18  61.37 ± .03.sup.hij  48.40 ± 14.21.sup.fhij 58.20 ± 8.53.sup.bcde 76.84 ± 5.22.sup.hij 100 mg/kg 160.57 ± 18.68.sup.fh 8.73 ± 0.92  71.69 ± 10.31.sup.fh  77.70 ± 21.78.sup.fh 42.80 ± 10.09.sup.ac 66.43 ± 10.37.sup.gh Values are expressed mean ± SD of 10 mice

(150) FIG. 14 shows the histopathological change of ear skin tissues of experimental animals by SKOG administration.

(151) Significant (p<0.01) increases ear total and dermis thicknesses, the numbers of infiltrated inflammatory cells on the ear dermis, degranulation related decreases of mast cell numbers in the dermis, decreases of dermis collagen fiber occupied regions, without significant changes on the ear epidermis were observed in xylene control as classic contact acute inflammations—dermatitis related histopathological findings. However, these xylene-induced ear acute contact dermatitis related findings at histopathological inspections were significantly (p<0.01) and dose-dependently inhibited by 11 days of continuous oral pretreatment of SKOG 400, 200 and 100 mg/kg as compared with those of xylene control mice, respectively. In addition, AR and KOG 400 mg/kg, DEXA 1 mg/kg also significantly (p<0.01) reduced the xylene-induced ear acute contact dermatitis related histopathological findings as compared with those of xylene control mice, respectively. Especially, SKOG 400 and 200 mg/kg showed significantly (p<0.01) increased inhibitory effects on the xylene-induced ear acute contact dermatitis related histopathological findings as compared with those of AR and KOG 400 mg/kg, and SKOG 100 mg/kg showed similar inhibitory activities against xylene-induced ear acute contact dermatitis related histopathological findings as compared with those of AR and KOG 400 mg/kg, respectively. In addition, SKOG 400 mg/kg showed favorable inhibitory effects on the xylene-induced ear acute contact dermatitis related histopathological findings as comparable to those of DEXA 1 mg/kg, but AR and KOG 400 mg/kg, SKOG 200 and 100 mg/kg showed slighter inhibitory effects on the xylene-induced ear acute contact dermatitis related histopathological findings as compared to those of DEXA 1 mg/kg, in this observation (Table 9 and FIG. 14).

(152) Mean total ear thicknesses in xylene control were changed as 155.76% as compared with intact vehicle control, but they were changed as −62.39, −37.01, −39.95, −59.94, −51.51 and −39.29% in DEXA 1 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of xylene control mice, respectively.

(153) Mean ear epidermis thicknesses in xylene control were changed as 1.02% as compared with intact vehicle control, but they were changed as −6.35, −3.30, −3.45, 0.87, −8.69 and −3.80% in DEXA 1 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of xylene control mice, respectively.

(154) Mean ear dermis thicknesses in xylene control were changed as 141.11% as compared with intact vehicle control, but they were changed as −60.02, −42.99, −45.91, −61.01, −53.61 and −45.80% in DEXA 1 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of xylene control mice, respectively.

(155) Mean numbers of infiltrated inflammatory cells on the ear dermis in xylene control were changed as 1632.89% as compared with intact vehicle control, but they were changed as −93.13, −72.55, −73.80, −88.91, −81.62 and −70.50% in DEXA 1 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of xylene control mice, respectively.

(156) Mean numbers of infiltrated mast cells on the ear dermis in xylene control were changed as −88.12% as compared with intact vehicle control, but they were changed as 647.56, 451.22, 417.07, 643.90, 609.76 and 421.95% in DEXA 1 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of xylene control mice, respectively.

(157) Mean percentages of collagen occupied regions on the ear dermis in xylene control were changed as −65.86% as compared with intact vehicle control, but they were changed as 189.63, 142.11, 152.82, 204.88, 187.39 and 148.48% in DEXA 1 mg/kg, AR and KOG 400 mg/kg, SKOG 400, 200 and 100 mg/kg oral administered mice as compared with those of xylene control mice, respectively.