COMPOSITION FOR PREVENTING OR TREATING FINE DUST-INDUCED RESPIRATORY DISEASE COMPRISING MIXTURE (RGX-365) COMPRISING GINSENOSIDES RG2, RG4, RG6 AND RH1, AS ACTIVE INGREDIENT, AND PREPARATION METHOD THEREOF

Abstract

The present invention relates to a composition, for preventing or treating a fine dust-induced respiratory disease, comprising a mixture (Rgx 365), comprising ginsenosides Rg2, Rg4, Rg6 and Rh1, as an active ingredient, a preparation method thereof, and a functional health food, for preventing or relieving a fine dust-induced respiratory disease, comprising same. The present invention exhibits excellent effects on permeability reduction, reactive oxygen (ROS) generation reduction, leucocyte reduction and cytokine generation reduction in a model for fine dust-induced inflammatory response, and thus can be utilized for preventing and treating a fine dust-induced respiratory disease.

Claims

1. A composition comprising a mixture containing ginsenosides Rg2, Rg4, Rg6, and Rh1 as an active ingredient for preventing or treating a fine dust-induced respiratory disease.

2. The composition of claim 1, wherein the mixture contains 35-45% by weight of ginsenoside Rg2, 30-40% by weight of ginsenoside Rg4, 10-20% by weight of ginsenoside Rg6, and 1-5% by weight of ginsenoside Rh1.

3. The composition of claim 1, wherein the fine dust-induced respiratory disease is selected from the group consisting of inflammatory lung disease, asthma, chronic obstructive pulmonary disease, allergic rhinitis, cough, bronchitis, laryngopharyngitis, tympanitis, tonsillitis, sinusitis, pneumonia, pulmonary fibrosis, and laryngitis.

4. A health functional food comprising a mixture containing ginsenosides Rg2, Rg4, Rg6, and Rh1 as an active ingredient for preventing or alleviating a fine dust-induced respiratory disease.

5. The health functional food of claim 4, wherein the mixture contains 35-45 by weight of ginsenoside Rg2, 30-40% by weight of ginsenoside Rg4, 10-20% by weight of ginsenoside Rg6, and 1-5% by weight of ginsenoside Rh1.

6. The health functional food of claim 4, wherein the fine dust-induced respiratory disease is selected from the group consisting of inflammatory lung disease, asthma, chronic obstructive pulmonary disease, allergic rhinitis, cough, bronchitis, laryngopharyngitis, tympanitis, tonsillitis, sinusitis, pneumonia, pulmonary fibrosis, and laryngitis.

7. A method for preparing a mixture containing ginsenosides Rg2, Rg4, Rg6, and Rh1 from ginsenoside Re, the method comprising the steps of: (first process) mixing 100 parts by weight of ginsenoside Re with 130-160 parts by weight of distilled water; (second process) reacting the mixture of the first process for 4-6 hours at a temperature of 110-140° C. under a pressure condition of 0.11-0.16 MPa; and (third process) subjecting the reaction mixture of the second process to column separation to afford a mixture containing ginsenosides Rg2, Rg4, Rg6, and Rh1.

8. The method of claim 7, wherein the mixture contains 35-45% by weight of ginsenoside Rg2, 30-40% by weight of ginsenoside Rg4, 10-20% by weight of ginsenoside Rg6, and 1-5% by weight of ginsenoside Rh1.

9. A mixture containing ginsenosides Rg2, Rg4, Rg6, and Rh1, prepared by the method of claim 7, comprising 35-45% by weight of ginsenoside Rg2, 30-40% by weight of ginsenoside Rg4, 10-20% by weight of ginsenoside Rg6, and 1-5% by weight of ginsenoside Rh1.

10. A method for preventing or treating a fine dust-induced respiratory disease comprising administering to a subject in need thereof a therapeutically effective amount of the composition of claim 1.

11. The method of claim 10, wherein the mixture contains 35-45% by weight of ginsenoside Rg2, 30-40% by weight of ginsenoside Rg4, 10-20% by weight of ginsenoside Rg6, and 1-5% by weight of ginsenoside Rh1.

12. The method of claim 10, wherein the fine dust-induced respiratory disease is selected from the group consisting of inflammatory lung disease, asthma, chronic obstructive pulmonary disease, allergic rhinitis, cough, bronchitis, laryngopharyngitis, tympanitis, tonsillitis, sinusitis, pneumonia, pulmonary fibrosis, and laryngitis.

13. The method of claim 11, wherein the fine dust-induced respiratory disease is selected from the group consisting of inflammatory lung disease, asthma, chronic obstructive pulmonary disease, allergic rhinitis, cough, bronchitis, laryngopharyngitis, tympanitis, tonsillitis, sinusitis, pneumonia, pulmonary fibrosis, and laryngitis.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0039] FIG. 1A is a chromatogram obtained by HPLC for reference standard ginsenosides Re, Rg2, Rg4, Rg6, and Rh1, and Rh4, and FIG. 1B is an HPLC chromatogram for ginsenosides contained in the mixture (RGX-365) of Example 1.

[0040] FIG. 2 is a graph showing cell viability of mouse lung microvascular endothelial cells according to treatment concentrations of the mixture (RGX-365) of Example 1 of the present disclosure.

[0041] FIG. 3A is a graph showing permeability of fine dust-treated mouse lung microvascular endothelial cells according to treatment concentrations of the mixture (RGX-365) of Example 1 of the present disclosure and FIG. 3B is a graph showing permeability of fine dust-treated mouse lung microvascular endothelial cells according to treatment with the mixture (RGX-365) of Example 1 of the present disclosure, individual ginsenosides Re, Rg4, Rg6, Rh1, and Rg2, and the mixture of Comparative Example 1.

[0042] FIGS. 4A and 4B are fluorescent images and a graph showing changes of reactive oxygen species (ROS) production in fine dust-treated mouse lung microvascular endothelial cells with treatment concentrations of the mixture (RGX-365) of Example 1 of the present disclosure.

[0043] FIG. 5A is a graph showing dye leakage from fine dust-treated mice according to treatment concentrations of the mixture (RGX-365) of Example 1 of the present disclosure and FIG. 5B is a graph showing dye leakage from fine dust-treated mice according to treatment with the mixture (RGX-365) of Example 1 of the present disclosure, individual ginsenosides Re, Rg4, Rg6, Rh1, and Rg2, and the mixture of Comparative Example 1.

[0044] FIG. 6A is a graph showing counts of leukocytes in fine dust-treated mouse bronchoalveolar lavage fluid according to treatment concentrations of the mixture (RGX-365) of Example 1 of the present disclosure and FIG. 6B is a graph showing production changes of IL-6 and TNF-α in fine dust-treated mouse bronchoalveolar lavage fluid according to treatment concentrations of the mixture (RGX-365) of Example 1 of the present disclosure.

[0045] FIG. 7 shows leukocytic infiltration into fine dust-treated mouse lung tissues according to treatment with the mixture (RGX-365) of Example 1 of the present disclosure.

BEST MODE FOR CARRYING OUT THE INVENTION

[0046] Hereinafter, preferred embodiments of the present disclosure will be described in detail. However, the present disclosure is not limited to the embodiments described herein, but may be embodied in other forms. Rather, the embodiments is provided so that the content presented here is thorough and complete and the spirit of the present disclosure is fully understood to a person skilled in the art.

EXAMPLE 1

Preparation of Mixture (RGX-365) Containing Ginsenosides Rg2, Rg4, Rg6, and Rh1

[0047] To 30.4 g of ginsenoside Re was added 45 ml of distilled water, followed by autoclaving at 121° C. under 0.13 MPa for 6 hours and then column separation.

[0048] A synthetic adsorbent was loaded in an amount of 300 g, which was 10-fold larger than that of the material, onto the column and washed with a sufficient amount of an eluent. After 5 liters of distilled water were let to flow through a column, followed by 5 liters of fermented alcohol. Again, 5 liters of water were let to flow through the column, with a care taken to prevent incorporation of bubbles into the column.

[0049] The reaction mixture obtained by autoclaving ginsenoside Re for 6 hours was dissolved in a fermented alcohol (50 ml) and distilled water (300 ml). The water fraction was poured to the column. Then, about 5 liters of distilled water were let to flow through the column, followed by a series of 20%, 25%, and 30% fermented alcohols in that order to remove foreign matter. When 35-70% fermented alcohols were applied, an RGX 365 fraction was obtained, and concentrated in a vacuum to give 18 g of the mixture (RGX-365) of Example 1.

[0050] Mixtures of Examples 2 and 3 were each obtained in an amount of 18 g in the same manner as in Example 1, with the exception that 48 ml or 40 ml of distilled water was added to 30.4 g of ginsenoside Re, followed by autoclaving at 121° C. under 0.13 MPa for 6 hours and then column separation, as indicated in Table 1, below.

[0051] Among the mixtures of Examples 1 to 3 obtained above, the mixture of Example 1 was quantitatively analyzed for ginsenosides Rg2, Rg4, Rg6, and Rh1 by HPLC under the conditions of Table 2, below, and the contents thereof are depicted in FIG. 1 and summarized in Table 3.

TABLE-US-00001 TABLE 1 Mixing condition Ginsenoside Re (g) Distilled water (ml) Example 1 30.4 45 Example 2 30.4 48 Example 3 30.4 40

TABLE-US-00002 TABLE 2 HPLC Condition Column ACE 5-C18 (250 × 4.6 mm) Flow rate 1.0 ml/min Sample injected 10 μl Detector UV 205 nm, Agilent Technologies 1260 infinity Temp. 40° C. Solvent A; water, B; acetonitrile  0-3 min (A from 20% to 40%),  3-15 min (B from 20% to 23%), 15-20 min (B from 23% to 33%), 20-45 min (B from 33% to 40%), 45-60 min (B from 40% to 68%), 60-65 min (B from 68% to 85%), 65-70 min (B 85%), 70-73 min (B from 80% to 20%), 73-75 min (B 20%)

TABLE-US-00003 TABLE 3 Ginsenoside content g % Ginsenoside Rg2 7.4 41.1 Ginsenoside Rg4 6.4 35.4 Ginsenoside Rg6 2.6 14.2 Ginsenoside Rh1 0.8 4.4

COMPARATIVE EXAMPLE 1

Preparation of Mixture Containing Ginsenosides Rh1, Rg4, and Rg6

[0052] A mixture of Comparative Example 1 was prepared with reference to Example 1 of Korean Patent Number 10-1897811.

[0053] First, a mixture of 10 mg of ginsenoside Re and 25 μl of distilled water was treated at 120° C. under 0.13-0.15 MPa for 8 hours in an autoclave. Next, the reaction mixture was concentrated in a rotary evaporator, and the concentrated powder thus obtained was separated and purified to afford a mixture containing ginsenosides Rh1, Rg4, and Rg6 of Comparative Example 1.

COMPARATIVE EXAMPLE 2

Preparation of Comparative Mixture Containing Ginsenosides Rg2, Rg4, Rg6, and Rh1

[0054] The same procedure as in Example 1 was carried out, with the exception that 60 ml, instead of 45 ml, of distilled water was added to 30.4 g of ginsenoside Re and then autoclaved at 121° C. under 0.13 MPa for 6 hours, followed by column separation.

COMPARATIVE EXAMPLE 3

Preparation of Comparative Mixture Containing Ginsenosides Rg2, Rg4, Rg6, and Rh1

[0055] The same procedure as in Example 1 was carried out, with the exception that 53 ml, instead of 45 ml, of distilled water was added to 30.4 g of ginsenoside Re and then autoclaved at 121° C. under 0.13 MPa for 6 hours, followed by column separation.

COMPARATIVE EXAMPLE 4

Preparation of Comparative Mixture Containing Ginsenosides Rg2, Rg4, Rg6, and Rh1

[0056] The same procedure as in Example 1 was carried out, with the exception that 35 ml, instead of 45 ml, of distilled water was added to 30.4 g of ginsenoside Re and then autoclaved at 121° C. under 0.13 MPa for 6 hours, followed by column separation.

COMPARATIVE EXAMPLE 5

Preparation of Comparative Mixture Containing Ginsenosides Rg2, Rg4, Rg6, and Rh1

[0057] The same procedure as in Example 1 was carried out, with the exception that 30 ml, instead of 45 ml, of distilled water was added to 30.4 g of ginsenoside Re and then autoclaved at 121° C. under 0.13 MPa for 6 hours, followed by column separation.

EXPERIMENTAL EXAMPLE 1

Comparison of Ginsenoside Rg2 Content

[0058] Among the ginsenosides contained in the mixtures of Examples 1 to 3 and Comparative Examples 1 to 5, ginsenoside Rg2 was quantitatively analyzed by HPLC under the conditions of Table 2 and the contents thereof are summarized in Table 4, below.

TABLE-US-00004 TABLE 4 Ginsenoside Rg2 Condition Content (wt %) Ex. 1 Ginsenoside Re 100 wt. part, 41.1 Distilled water 148 wt. part Ex. 2 Ginsenoside Re 100 wt. part, 42.5 Distilled water 158 wt. part Ex. 3 Ginsenoside Re 100 wt. part, 37.5 Distilled water 132 wt. part C. Ex. 1 Ginsenoside Re 100 wt. part, <10 Distilled water 250 wt. part C. Ex. 2 Ginsenoside Re 100 wt. part, 11.5 Distilled water 197 wt. part C. Ex. 3 Ginsenoside Re 100 wt. part, <25 Distilled water 174 wt. part C. Ex. 4 Ginsenoside Re 100 wt. part, 19.8 Distilled water 115 wt. part C. Ex. 5 Ginsenoside Re 100 wt. part, <10 Distilled water 100 wt. part

[0059] As seen in Table 4, when a mixture of 100 parts by weight of ginsenoside Re and 130-160 parts by weight of distilled water was steamed according to the present disclosure, the mixture of ginsenosides Rg2, Rg4, Rg6, and Rh1 thus obtained was measured to contain ginsenoside Rg2 in an amount of 35% by weight or greater.

[0060] In contrast to the present disclosure, the content of ginsenoside Rg2 in the final ginsenoside mixture was measured to be 25% by weight or less when distilled water was added in an amount of less than 130 parts by weight or greater than 160 parts by weight to 100 parts by weight of ginsenosides Re. That is, when less than 130 parts by weight of distilled water is added to 100 parts by weight of ginsenoside Re, ginsenoside Re is not sufficiently converted, but remains significantly unreacted, with the consequent low production of ginsenoside Rg2. Given greater than 160 parts by weight of distilled water, ginsenoside Rg2 does not stay in the final mixture, but is further converted to ginsenosides Rg4 and Rg6. Thus, the final mixture increased in the content of ginsenosides Rg4 and Rg6.

[0061] Therefore, a method comprising mixing 100 parts by weight of ginsenoside Re with 130-160 parts by weight of distilled water and steaming the mixture according to the present disclosure can enrich ginsenoside Rg2, which is particularly effective for therapy of fine dust-induced respiratory diseases, and as such, is found to be a advantageous preparation method for a pharmaceutical composition for prevention or treatment of find dust-induced respiratory diseases.

EXPERIMENTAL EXAMPLE 2

Cell Culturing

[0062] Mouse lung microvascular endothelial cells (MLMVECs) were obtained as previously reported [Kovacs-Kasa, A. et al., Sci Pages Pulmonol., 1(1), 7-18, 2017].

[0063] First, 7-week-old male Balb/c mice (weighing 27 g, Orient Bio Co., Sungnam, Republic of Korea) were acclimated at a temperature of 20-25° C. and a relative humidity of 40-45% for 12 days on ah 12-hr light/12-hr dark cycle. The mice used in experiments were treated according to the guideline for the Care and use of Laboratory Animals at Kyungpook National University (IRB No. KNU 2017-101).

[0064] Lung tissues thus obtained were homogenized and then digested at 37° C. for 45-60 min with collagenase A (1 mg/ml). Epithelial cells were purified using anti-PECAM-1 monoclonal antibody magnetic beads (BD Pharmingen, San Diego, Calif.) and grown for 2 days in a growth medium.

[0065] For a monolayer culture, the cells were incubated in an endothelial cell basal medium supplemented with EGM-2 MV Bulletkit™ (Lonza, Md.) on a fibronectin-coated culture dish at 37° C. under the atmosphere of 5% CO.sub.2 and 95% air.

EXPERIMENTAL EXAMPLE 3

Cytotoxicity Assay

[0066] The mixture of Example 1 according to the present disclosure was examined for cytotoxicity in mouse lung microvascular endothelial cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay.

[0067] The mouse lung microvascular endothelial cells prepared in Experimental Example 2 were seeded at a density of 5×10.sup.3 cells/well into 96-well plates and then incubated for 24 hours with 0, 10, 25, or 50 mg/kg of the ginsenoside mixture (RGX-365) obtained in Example 1. Thereafter, the cells in each well were washed and treated for 4 hours with 100 μl of 1 mg/ml MTT solution. The formazan salt thus formed within the cells was dissolved by adding 150 μl of DMSO, followed by reading absorbance at 540 nm on a microplate reader (Tecan Austria GmbH, Austria). Cell viability was calculated from the absorbance measurements and is depicted in FIG. 2.

[0068] Referring to FIG. 2, the mixture (Rgx 365) of Example 1 according to the present disclosure allowed the cell viability equivalent to that of the non-treated group irrespective of the concentrations used, and thus was demonstrated to have no cytotoxicity.

EXPERIMENTAL EXAMPLE 4

Inhibitory Effect on Inflammation in Mouse Lung Microvascular Endothelial Cells

[0069] Exposure of the human body to fine dust causes acute inflammation, giving rise to various activities including the secretion of cytokines and chemokines, the increase of leukocytes, the generation of reactive oxygen species in the lung, the endotoxin-induced responses of cells and tissues, and so on. These activities lead to the onset or exacerbation of diseases, such as asthma, chronic bronchitis, and airway obstruction. In this context, the mixture of Example 1 according to the present disclosure was examined for inhibitory activity against inflammation through the permeability assay and the measurement of generation change of reactive oxygen species in mouse lung microvascular endothelial cells.

EXPERIMENTAL EXAMPLE 4-1

Permeability Assay

[0070] Permeability to endothelial cells of the mixture of Example 1 of the present disclosure was quantitated according to concentrations by spectrophotometric measurement of the flux of Evans blue-bound albumin across functional cell monolayers using a 2-compartment chamber model as previously described [Bae, J. S. et al., Blood, 118(14), 3952-3959, 2011; Jung, B. et al., BMB Rep., 49(4), 214-219, 2016].

[0071] For a permeability assay in the mouse lung microvascular endothelial cells, the cells of Experimental Example 2 were plated at a density of 5×10.sup.4 cells/well in a Transwell (pore size, 3 μm; diameter, 12 mm) and incubated for 3 days. When reaching confluency of monolayers in the Transwell, the cells were first treated with predetermined concentrations (1, 10, 20, 50, 100, and 200 μg/ml) of the mixture (RGX-365) of Example 1 and then with fine dust (PM.sub.2.5, 1 mg/ml) for 6 hours. For comparison, reference standard ginsenosides Re, Rg4, Rg6, Rh1, and Rg2, and the ginsenoside mixture of Comparative Example 1 were each applied at a concentration of 200 μg/ml in the same manner as described above. Subsequently, permeability was measured using an ELISA plate reader, and the measurements are depicted in FIGS. 3A and 3B.

[0072] As can be seen in FIGS. 3A and 3B, when applied to mouse lung microvascular endothelial cells having inflammation induced therein by fine dust, the mixture (Rgx 365) of Example 1 according to the present disclosure exhibited a more reductive effect on vascular permeability than the individual ginsenosides Re, Rg4, Rg6, and Rh1 and the ginsenoside mixture of Comparative Example 1.

[0073] Therefore, the mixture of ginsenosides Rg2, Rg4, Rg6, and Rh1, prepared by the method of the present disclosure, is particularly selective for the therapy of fine dust-induced respiratory diseases, so that the method is advantageous for preparing a composition for preventing or treating fine dust-induced respiratory diseases.

EXPERIMENTAL EXAMPLE 4-2

Reactive Oxygen Species (ROS)

[0074] Reactive oxygen species (ROS) in the mouse lung microvascular endothelial cells was quantitated by fluorescence microscopy as previously reported [Piao, M. J. et al., Arch Toxicol., 92(6), 2077-2091, 2018]. Primary mouse lung microvascular endothelial cells grown in 4-well glass chamber slides (>90% confluent) were treated with predetermined concentrations (0, 1, 4, 7.5, and 15 mg/kg) of the ginsenoside mixture (RGX-365) of Example 1 and then with fine dust (PM.sub.2.5, 1 mg/ml), followed by adding DCFDA (2′,7′-dichlorofluorescein diacetate, Molecular Probes, Eugene, Oreg., USA, 10 μM). After 30 min, the medium containing DCFDA was aspirated and the cells were washed. Stained cells were detected by fluorescence microscopy, and the results are given in FIGS. 4A and 4B.

[0075] As can be seen in FIGS. 4A and 4B, the ROS production increased by fine dust was reduced by the mixture (RGX 365) of Example 1 of the present disclosure in a dose-dependent manner. Thus, the data demonstrate that the mixture containing Rg2, Rg4, Rg6, and Rh1 in Example 1 of the present disclosure can be used as a composition highly inhibitory of fine dust-induced generation of reactive oxygen species (ROS).

EXPERIMENTAL EXAMPLE 5

Inhibitory Effect on Fine Dust-Induced Inflammation in Animal Model

EXPERIMENTAL EXAMPLE 5-1

Permeability Assay

[0076] First, 7-week-old male Balb/c mice (weighing 27 g, Orient Bio Co., Sungnam, Republic of Korea) were acclimated at a temperature of 20-25° C. and a relative humidity of 40-45% for 12 days on ah 12-hr light/12-hr dark cycle. The mice used in experiments were treated according to the guideline for the Care and use of Laboratory Animals at Kyungpook National University (IRB No. KNU 2017-101).

[0077] Oral administration was made of the ginsenoside mixture (RGX-365) of Example 1 at a dose of 0, 1, 2, 4, 7.5, or 15 mg/kg, or each of the reference standard ginsenosides Re, Rg4, Rg6, and Rh1 or the ginsenoside mixture of Comparative Example 1 at a dose of 15 mg/kg for 10 days to the mice to which fine dust (PM.sub.2.5, 1 mg/kg in 100 μl of saline, 10 days) was then administered intratracheally as previously reported [Wang, H. et al., Sci Rep., 7, 44256, 2017]. After 10 days of the intratracheal instillation of fine dust, the male mice anesthetized with 2% isoflurane (Forane, JW Pharmaceutical, South Korea) were each sacrificed and injected intravenously with a 1% Evans blue solution in physiological saline as previously described [Bae, J. S. et al., Blood, 118(14), 3952-3959, 2011; Jung, B. et al., BMB Rep., 49(4), 214-219, 2016]. Permeability was measured using an ELISA plate reader and the results are depicted in FIGS. 5A and 5B.

[0078] With reference to FIGS. 5A and 5B, dye leakage that was greatly induced by fine dust was reduced by treatment with the mixture (Rgx 365) of Example 1 in a dose-dependent manner. Compared with individual ginsenosides Re, Rg4, Rg6, and Rh1, the mixture of Example 1 of the present disclosure remarkably inhibited dye leakage particularly by protecting vessel walls against injury.

[0079] From the results, it is understood that the mixture containing ginsenosides Rg2, Rg4, Rg6, and Rh1 at specific contents according to the present disclosure is more effective for inhibiting fine dust-induced inflammation than individual ginsenosides Re, Rg4, Rg6, and Rh1.

EXPERIMENTAL EXAMPLE 5-2

Counts of Leukocyte and Expression of Inflammation-Related Factor

[0080] Oral administration was made of the ginsenoside mixture (RGX-365) of Example 1 at a dose of 0, 1, 2, 4, 7.5, or 15 mg/kg, or each of the reference standard ginsenosides Re, Rg4, Rg6, Rh1, and Rg2, or the ginsenoside mixture of Comparative Example 1 at a dose of 15 mg/kg for 10 days to the mice to which fine dust was intratracheally instilled for 10 days, as in Experimental Example 5-1. The mice were sacrificed before bronchoalveolar lavage fluid (BAL) was obtained therefrom. The BAL was washed with 5 ml of physiological saline and mixed with 0.38 ml of Turk's solution (0.01% crystal violet in 3% acetic acid). Leukocytes were counted under an optical microscope and are depicted in FIG. 6A. Concentrations of the inflammation-related factors IL-6 and TNF-α in the bronchoalveolar lavage fluid were measured using ELISA kits (R&D Systems, Minneapolis, MN) and are depicted in FIG. 6B.

[0081] With reference to FIGS. 6A and 6B, the ginsenoside mixture (RGX-365) of Example 1 of the present disclosure is observed to reduce the number, increased by fine dust, of leukocytes in bronchoalveolar lavage fluid in a dose-dependent manner and inhibit the fine dust-induced increase of the inflammation-related cytokines IL-6 and TNF-α in bronchoalveolar lavage fluid.

EXPERIMENTAL EXAMPLE 5-3

H&E Staining

[0082] Examination was made of histological changes in fine dust-treated mouse lung tissues and effects of the ginsenoside mixture (RGX-365) of the present disclosure thereon. In this regard, oral administration was made of the ginsenoside mixture (RGX-365) of Example 1 at a dose of 0 or 15 mg/kg for 10 days to the mice to which fine dust was intratracheally instilled for 10 days, as in Experimental Example 5-1. Then, the mice were sacrificed before lung tissues were obtained therefrom. The tissues were fixed with 4% formaldehyde solution in PBS at 4° C. for 20 hours, dehydrated with ethanol, and embedded in paraffin. Subsequently, the paraffin-embedded tissue was sectioned into 4-μm-thick slices which were mounted on slides and subjected to deparaffinization at 60° C. in an oven, rehydration, and staining with hematoxylin. The slides were quickly dipped three times in 0.3% acidic alcohol to remove an excess of the dye and then counterstained with eosin. An excess of the dye was removed by washing with ethanol and xylene and the sample were put under cover slip. Optical microscopy analysis of the lung specimen was carried out as previously reported [Ozdulger, A. et al., Shock, 19(4), 366-372, 2003], and histological changes of the lung tissue are depicted in FIG. 7.

[0083] Referring to FIG. 7, leukocytic infiltration was observed to increase in the fine dust-instilled lung tissue (PM.sub.2.5), compared to the lung tissue having no find dust instilled thereto (Control). Treatment with the mixture (RGX-365) of Example 1 according to the present disclosure remarkably reduced fine dust-induced leukocytic infiltration into mouse lung tissues.

[0084] Taken together, the data demonstrate that the ginsenoside Rg2-enriched mixture of ginsenosides Rg2, Rg4, Rg6, and Rh1, prepared in Example 1 of the present disclosure, is superb in the prevention or treatment of fine dust-induced respiratory diseases and as such, can find advantageous applications as a composition for prevention or treatment of fine dust-induced respiratory diseases.

FORMULATION EXAMPLE 1

Preparation of Tablet

[0085] With 20 g of the mixture (Rgx 365) of Example 1 according to the present disclosure were combined 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicate. The combination was added with a 10% gelatin solution, ground, and let to pass through a 14-mesh sieve. The resulting mixture was dried, and prepared, together with 160 g of potato starch, 50 g of talc, and 5 g of magnesium stearate, into a tablet.

FORMULATION EXAMPLE 2

Preparation of Capsule>

[0086] With 100 mg of the mixture (Rgx 365) of Example 1 according to the present disclosure were combined 100 mg of maize starch, 100 mg of lactose, and 2 mg of magnesium stearate. The ingredients were blended and loaded into a gelatin capsule to afford a capsule according to a typical capsule preparation method.

FORMULATION EXAMPLE 3

Preparation of Injection

[0087] Together with 0.6 g of sodium chloride and 0.1 g of ascorbic acid, 1 g of the mixture (Rgx 365) of Example 1 according to the present disclosure was dissolved in distilled water to form a final volume of 100 ml which was then charged into a bottle and heated at 20° C. for 30 min for sterilization.

FORMULATION EXAMPLE 4

Preparation of Health Functional Food

[0088] A combination of 20 g of the mixture (Rgx 365) of Example 1 according to the present disclosure, a suitable amount of vitamins including 70 μg of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 μg of vitamin B12, 10 mg of vitamin C, 10 μg of biotin, 1.7 mg of nicotinamide, 50 μg of folic acid, 0.5 mg of calcium pantothenate, and a suitable amount of minerals including 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium phosphate monobasic, 55 mg of potassium phosphate dibasic, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride were blended and prepared into granules according to uses. However, the combination may be prepared into various modified formulations. In addition, any modification may be made to the composition ratio between the vitamin and the mineral mixtures. The ingredients may be mixed and prepared into food formulations according to ordinary health food preparation method.

FORMULATION EXAMPLE 5

Preparation of Health Functional Beverage

[0089] A beverage was prepared by stirring, heating, filtering, sterilizing, and refrigerating a combination of 1 g of the mixture (Rgx 365) of Example 1 according to the present disclosure, 0.1 g of citric acid, 100 g of fructooligosaccharide, and 900 g of purified water according to an ordinary beverage preparation method.