KR102249916B1 - A pharmaceutical composition comprising ginsenoside Rg2, Rg4, Rg6 and Rh1 mixture(Rgx 365) for preventing or treating respiratory disease induced by particulate matter - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating respiratory diseases by fine dust comprising a ginsenoside Rg2, Rg4, Rg6 and Rh1 mixture (Rgx 365) enhanced with ginsenoside Rg2 from ginsenoside Re as an active ingredient will be. The ginsenoside Rg2, Rg4, Rg6 and Rh1 mixture (Rgx 365) is compared to a single ginsenoside Rg4, Rg6 and Rh1, ginseng extract or red ginseng extract, in a model in which inflammatory reaction is induced by fine dust, decreased permeability, active oxygen Since (ROS) production reduction, white blood cell count, and cytokine production reduction effect are excellent, it can be usefully used in the treatment of respiratory diseases induced by fine dust.

Description

A pharmaceutical composition comprising ginsenoside Rg2, Rg4, Rg6 and Rh1 mixture (Rgx 365) containing ginsenoside Rg2, Rg4, Rg6 and Rh1 mixture (Rgx 365) as an active ingredient. ) for preventing or treating respiratory disease induced by particulate matter}

The present invention relates to a composition for preventing or treating respiratory diseases by fine dust comprising a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 as an active ingredient.

Recently, as the concentration of fine dust continues to increase, interest in fine dust and social interest in the effect of fine dust on the human body are increasing. In fine dust, various components such as carbon components (sot, organic carbon in organisms), ionic components (chlorine, nitric acid, ammonium, sodium, calcium, etc.), metal components (arsenic, lead, mercury, etc.), polycyclic aromatic hydrocarbons (benzopyrene, etc.) This is included. Increasing fine dust not only causes blurred vision and discomfort, but also causes serious diseases such as conjunctivitis, sinusitis, otitis media, bronchitis, asthma and chronic obstructive pulmonary disease, pneumonia, angina, myocardial infarction, and lung cancer. Has been reported to be associated with increased mortality as well as incidence of respiratory and cardiovascular diseases (Jang, AS, J Korean Med Assoc., 57(9), 763-768, 2014).

Among the dust, particles with a diameter smaller than 10㎛ are called fine dust, and particulate matter smaller than 10㎛ is called _{PM 10.} Among PM ₁₀ , relatively large particles between 2.5 μm and 10 μm in size are mainly dust from roads or soil, and particles smaller than 2.5 μm ( _{referred to as ultrafine dust or PM 2.5} ) are mainly automobile exhaust gases or chimneys of factories. It is dust generated in the process of burning fossil fuels such as smoke or secondary air pollutants in the form of gases through a chemical reaction in the air (Ho-Jang Kwon, Korean Social Trends 2014, 281-287, 2014).

Fine dust particles are very small and reach the airways and alveoli and induce inflammation. Inflammation of the airways causes symptoms such as chest tightness, wheezing, and coughing. A number of respiratory diseases are associated with inflammation of the airways. In general, when inflammation occurs in the airways, the absolute total number of cells of granulocytes and lymphocytes in the alveolar lavage fluid increases and infiltrates into the airways and alveoli. In addition, inflammatory cytokines such as TNF-α, IL-4, IL-5, IL-6, IL-13, and IL-33 secreted from inflammatory cells such as eosinophils, Th2 cells, mast cells, etc. Increasing the amount of mucus induces an inflammatory response. Among granulocytes known to be involved in inflammation, eosinophils help the production of T cells in Th2 cells when the airway reacts by antigens, and IL-5 and eotaxin, Th2 cytokines secreted from mast cells, produce eosinophils and Helps revitalize.

Inhalation of fine dust causes inflammation of the bronchi, and when such inflammation persists, chronic bronchitis, asthma, allergic alveolitis, etc. are caused. Among the diseases caused by fine dust, chronic obstructive pulmonary disease (COPD) is a disease in which lung tissue function declines due to an abnormal inflammatory reaction and breathing difficulties occur. Particularly, diesel exhaust particles (DEPs) among fine dust components increase the amount of IL-17A secretion, which aggravates bronchial asthma, and diseases caused by fine dust are not only respiratory diseases, but also stroke, dementia, arteriosclerosis, high blood pressure, and blood circulation. It is likely to cause or worsen various diseases such as disability. In addition, fine dust may cause respiratory infections by interfering with the inactivation or elimination of bacteria in the lung tissue.

In recent years, our atmospheric environment is _{exposed to air pollutants including yellow dust, PM 10} , PM _2.5, and ultrafine dust, and industrial nanoparticles whose application range is rapidly spreading to the human body. It is known that harmful chemicals contained in such fine dust and fine dust cause harmful effects on the human body through inflammatory mechanisms of the bronchi such as oxidative stress and inflammatory reactions. However, since there is no clear countermeasure against the occurrence of bronchitis related to fine dust, interest in natural substances that can protect the respiratory tract when fine dust is inhaled is increasing (Song, et al., Korean J. Medicinal Crop Sci., 25(5), 269-281, 2017).

Ginsenoside is a compound word of ginseng and glycoside, and is known to have a unique chemical structure and pharmacological efficacy unlike saponins found in other plants. Ginsenoside is a neutral glycoside in which glucose, arabinose, xylose, and rhamnose are bound to the triterpenoid-based dammarane skeleton. , Currently, more than 30 types of chemical structures have been identified, and according to the characteristics of the chemical structure, protopanaxadiol (PPD) system (19 types), protopanaxatriol (PPT) system (10 types) and ole It is classified into oleanane (one type).

The main ginsenosides present in fresh or white ginseng are Rg1, Re, Rf, Rh1, Rb1, Rb2, Rc, Rd, etc., and account for more than 80-90% of the total ginsenosides. In the case of rare ginsenosides, some of the sugars are removed from the ginsenosides present in a large amount or a dehydration reaction occurs, but when ginseng is extracted with water and alcohol, it is present in trace amounts.

The pharmacological activity of ginsenosides is indicated by rare ginsenosides originating from ginsenosides present in a large amount, strengthening immunity, anti-inflammatory, anti-allergic, anti-cancer, lowering blood pressure, anti-cholesterol, anti-thrombosis, anti-aging, Antioxidation, promotion of brain activity, and skin care are known as major effects (Kim, YS et al., Arch Pharm Res., 23(5), 518-524, 2000; Shibata, S., J Korean Med Sci. , 16(suppl), S28-37, 2001; Tachikawa, E. et al., Biochem Pharmacol., 66(11), 2213-2221, 2003; Tsai, SC et al., Chin J Physiol., 46(1) ), 1-7, 2003).

Meanwhile, as a prior art document related to a composition for preventing or treating respiratory diseases caused by fine dust including a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1, prior papers [Lee, JH et al., J Ginseng Res., 42 , 476-484, 2018] discloses that ginsenoside Rg2 or ginseng extract has an inhibitory effect on pulmonary inflammation, and previous papers [Kim, JH et al., Biomedical Science Letters, 20(3), 173-179, 2014 ], it has been disclosed that red ginseng is effective in inhibiting allergic lung inflammation caused by artificial sand dust (ASD). However, there is no previous report mentioning that a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1, which is easy to mass-produce as in the present invention, has a therapeutic effect on respiratory diseases caused by fine dust.

Accordingly, in the course of researching ginsenosides, the present inventors ginsenosides Rg2, Rg4, Rg6, and Rh1 mixtures contain a large amount of ginsenosides Rg2, which are particularly effective in treating respiratory diseases caused by fine dust, so a single ginsenoside The present invention could be completed by confirming that the side Rg4, Rg6 and Rh1, ginseng extract or red ginseng extract is particularly selective for the effect of treating respiratory diseases due to fine dust compared to the extract.

Kwon Ho-jang, Fine dust and health, Korean Social Trends 2014, 281-287, 2014. Song Hyung-Woo et al. Respiratory Protection Effect of Baeam Chazgi Extract on Fine Dust-Induced Airway Inflammation, Korean J. Medicinal Crop Sci., 25(5), 269-281, 2017. Bae, J. S. et al., Activated protein C inhibits high mobility group box 1 signaling in endothelial cells, Blood, 118(14), 3952-3959, 2011. Jang, A. S., Impact of particulate matter on health, J Korean Med Assoc., 57(9), 763-768, 2014. Jung, B. et al., Anti-septic effects of dabrafenib on HMGB1-mediated inflammatory responses, BMB Rep., 49(4), 214-219, 2016. Kim, J. H. et al., Effect of Korean Red Ginseng on Artificial Sand Dust (ASD) Induced Allergic Lung Inflammation, Biomedical Science Letters, 20(3), 173-179, 2014. Kim, YS et al., Differential expression of protein kinase C subtypes during ginsenoside Rh2-lnduced apoptosis in SK-N-BE(2) and C6Bu-1 cells, Arch Pharm Res., 23(5), 518-524, 2000 . Kovacs-Kasa, A. et al., Method for the Culture of Mouse Pulmonary Microvascular Endothelial Cells, Sci Pages Pulmonol., 1(1), 7-18, 2017. Lee, J. H. et al., Anti-wrinkle Effect of Rare Ginsenosides, Produced from Ginsenoside Rd, Kor J Aesthet Cosmetol., 13(6), 909-916, 2015. Lee, J. H. et al., Ginsenosides from Korean Red Ginseng ameliorate lung inflammatory responses: inhibition of the MAPKs/NF-kB/c-Fos pathways, J Ginseng Res., 42, 476-484, 2018. Ozdulger, A. et al., The protective effect of N-acetylcysteine on apoptotic lung injury in cecal ligation and puncture-induced sepsis model, Shock, 19(4), 366-372, 2003. Piao, M. J. et al., Particulate matter 2.5 damages skin cells by inducing oxidative stress, subcellular organelle dysfunction, and apoptosis, Arch Toxicol., 92(6), 2077-2091, 2018. Shibata, S., Chemistry and cancer preventing activities of ginseng saponins and some related triterpenoid compounds, J Korean Med Sci., 16(suppl), S28-37, 2001. Tachikawa, E. et al., In vitro inhibition of adrenal catecholamine secretion by steroidal metabolites of ginseng saponins, Biochem Pharmacol., 66(11), 2213-2221, 2003. Tsai, S. C. et al., Stimulation of the secretion of luteinizing hormone by ginsenoside-Rb1 in male rats, Chin J Physiol., 46(1), 1-7, 2003. Wang, H. et al., The acute airway inflammation induced by PM2.5 exposure and the treatment of essential oils in Balb/c mice, Sci Rep., 7, 44256, 2017.

An object of the present invention is to provide a composition for preventing or treating respiratory diseases by fine dust comprising a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 as an active ingredient.

The present invention relates to a pharmaceutical composition for preventing or treating respiratory diseases by fine dust comprising a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 as an active ingredient.

The ginsenoside Rg2, Rg4, Rg6 and Rh1 mixture is included in the present invention without limitation as long as it can be prepared according to a conventional method in the art as well as a pharmaceutically acceptable salt, but preferably by the following method. Can be prepared to obtain a mixture.

(Step 1) mixing 130 to 160 parts by weight of distilled water to 100 parts by weight of ginsenoside Re;

(Step 2) reacting the mixture in Step 1 for 4 to 6 hours at a high temperature and high pressure in a temperature range of 110 to 140° C. and a pressure condition of 0.11 to 0.16 MPa; And

(Step 3) Separating the reaction product in the second step by column to obtain a mixture containing ginsenosides Rg2, Rg4, Rg6 and Rh1; Includes.

The ginsenoside Re generates ginsenosides Rg2, Rg4, Rg6 and Rh1 through the process of Reaction Scheme 1 below.

[Scheme 1]

Steps 1 and 2 are steps of mixing distilled water with ginsenoside Re to react at high temperature and high pressure, more preferably 140 to 150 parts by weight of distilled water in ginsenoside Re and then 110 to 140 for 4 to 6 hours. The reaction was carried out at high temperature and high pressure in a temperature range of °C and a pressure of 0.11 to 0.16 MPa, followed by column separation, to prepare a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 containing 35% by weight or more of ginsenoside Rg2.

Ginsenoside Rg2, Rg4, Rg6 and Rh1 mixture finally prepared from the above process is ginsenoside Rg2 35-45% by weight, ginsenoside Rg4 30-40% by weight, ginsenoside Rg6 10-20% by weight and ginseno 1-5% by weight of side Rh1 is contained.

However, if moisture is added to ginsenoside Re or reacted at high temperature and high pressure outside the above conditions, ginsenoside Re is not sufficiently reacted and the remaining amount remains large, or ginsenosides Rg2, Rg4, Rg6 and Rh1 components It does not contain all but contains only some ginsenoside components, or the content of ginsenoside Rg2 is converted to less than 35% by weight, which is not preferable.

The ginsenoside Re is preferably prepared separately from the ginseng extract, and the ginseng extract can be prepared using all of ginseng-derived ginseng, such as the root, fruit, leaf, and stem of ginseng. You can get Ginsenoside Re of. Ginseng used for the extraction of the ginsenoside Re is a perennial plant belonging to the genus Panax , Korean ginseng (Panax ginseng ), Hwagi ginseng (Panax quinquefolia ), Jeonchil ginseng (Panax notoginseng ), Jukjeolsam (Panax japonicus ), Himalayan ginseng (Panaxa pseudoginseng ). One species selected from Panax vietnamensis , Panax elegatior , Panax wangianus and Panax bipinratifidus , Panax angustifolium The above ginseng can be used, and it is most preferable to use Korean ginseng, which is known to have a high saponin content, but the type of ginseng is not particularly limited thereto.

The ginseng extract can be extracted from ginseng using water, C1 to C4 alcohol or a mixed solution thereof, and the C1 to C4 alcohol is from the group consisting of methanol, ethanol, propanol, isopropanol, butanol and isobutanol. Can be chosen. Water, C1 to C4 alcohol, or a mixed solution thereof used in the preparation of the ginseng extract may be 1 to 40 times the volume of the ginseng used (1 to 40 liters based on 1 kg of ginseng), preferably 5 to 40 times can be used, and the process can be repeated up to 1 to 4 times.

Synthetic adsorbents used in the column separation are Trilite GSH-20, GSP-07, GSP-25, GSP-50, Diaion HP-10, HP-20, HP-21, HP-30, HP-40, HP-50 , SP800, SP825, SP850, SP875, SP205 to 207, HP1MG, HP2MG, etc. may be used, preferably GSH-20, HP-20, SP825 may be used.

The respiratory disease is a disease that causes chronic inflammation in the airways, bronchi, and lungs accompanied by cough, phlegm, dyspnea and fever, and preferably, the respiratory disease is respiratory inflammatory lung disease, asthma, chronic obstructive It is selected from the group consisting of lung disease, allergic rhinitis, cough, bronchitis, sore throat, otitis media, tonsillitis, sinusitis, pneumonia, pulmonary fibrosis, and laryngitis, but is not particularly limited thereto.

The pharmaceutical composition may be formulated in a suitable form together with a generally used pharmaceutically acceptable carrier. "Pharmacologically acceptable" refers to a composition that is physiologically acceptable and, when administered to a human, does not usually cause allergic reactions or similar reactions such as gastrointestinal disorders, dizziness, and the like. In addition, the compositions may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method.

Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl paraoxybenzoate, propyl paraoxybenzoate, talc, magnesium stearate, and mineral oil may be included, but are not limited thereto. In the case of formulation, it is prepared using diluents or excipients such as generally used fillers, stabilizers, binders, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations are at least one excipient, such as starch, microcrystalline cellulose, and sucrose in the ginsenoside mixture of the present invention. Alternatively, it is prepared by mixing lactose, low-substituted hydroxypropyl cellulose, hypromellose, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used as the non-aqueous solvent and suspension. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, and the like may be used. To formulate a formulation for parenteral administration, the ginsenoside mixture or a pharmaceutically acceptable salt thereof is sterilized and/or an adjuvant such as a preservative, a stabilizer, a hydrating agent or an emulsification accelerator, a salt for controlling the osmotic pressure and/or a buffer, And other therapeutically useful substances are mixed in water to prepare a solution or suspension, which can be prepared in ampoules or vials unit dosage form.

The pharmaceutical composition may be administered to mammals such as mice, livestock, and humans by various routes. All modes of administration can be expected and may be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection. The dosage is the age, sex, weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the time of administration, the route of administration, the absorption, distribution and excretion rate of the drug, the type of other drugs used, and the prescriber's It will depend on your judgment, etc. Dosage determination based on these factors is within the level of one of skill in the art, and dosages generally range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is from 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day or may be divided several times. The above dosage does not limit the scope of the present invention in any way.

In another aspect, the present invention provides a health functional food for preventing or improving respiratory diseases by fine dust comprising a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 as an active ingredient.

The health functional food refers to a food manufactured or processed using raw materials or ingredients having useful functionalities, and includes, for example, health supplements, functional foods, nutritional supplements, supplements, and the like.

The ginsenoside mixture is preferably 0.001% by weight to 50% by weight, more preferably 0.001% by weight to 30% by weight, most preferably 0.001% by weight to 10% by weight based on the total weight of the health functional food. Can be added.

The health functional food of the present invention includes the form of tablets, capsules, pills or liquids, and foods to which the ginsenoside mixture of the present invention can be added include, for example, various foods, beverages, gum, tea, And vitamin complexes.

The present invention relates to a pharmaceutical composition for preventing or treating respiratory diseases by fine dust comprising a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 enhanced with ginsenoside Rg2 from ginsenoside Re as an active ingredient. The ginsenoside Rg2, Rg4, Rg6, and Rh1 mixture is compared with a single ginsenoside Rg4, Rg6 and Rh1, ginseng extract or red ginseng extract, in a model in which inflammatory reaction is induced by fine dust, decreases permeability, generates active oxygen (ROS) Since the effect of reducing, reducing the number of white blood cells, and reducing the amount of cytokine produced is excellent, it can be usefully used in the treatment of respiratory diseases induced by fine dust.

1A is a result of HPLC analysis using ginsenoside Re, Rg2, Rg4, Rg6, Rh1 and Rh4 standards, and FIG. 1B is a ginsenoside component contained in Example 1 (Rgx 365) analyzed by HPLC. It is the result.
2 is a result of confirming the cell viability according to the concentration treatment of Example 1 (Rgx 365) of the present invention in mouse lung microvascular endothelial cells.
Figure 3A is a result of confirming the permeability according to the concentration treatment of Example 1 (Rgx 365) of the present invention in the mouse lung microvascular endothelial cells induced fine dust, Figure 3B is the mouse lung microvascular endothelial cells induced fine dust In the present invention Example 1 (Rgx 365), ginsenosides Re, Rg4, Rg6, Rh1, Rg2, and the results of confirming the permeability according to the treatment of Comparative Example 1.
4A and 4B are graphs showing changes in the amount of active oxygen (ROS) produced according to concentration-specific treatment of the present invention Example 1 (Rgx 365) in mouse lung microvascular endothelial cells induced with fine dust with a fluorescence microscope. It is the result.
Figure 5A is the result of confirming the dye leakage according to the concentration treatment of the present invention Example 1 (Rgx 365) in the mouse in which the fine dust was induced, and Figure 5B is the present invention Example 1 (Rgx 365) in the mouse in which the fine dust was induced. ), ginsenosides Re, Rg4, Rg6, Rh1, Rg2, and the result of confirming the dye leakage according to the treatment of Comparative Example 1.
Figure 6A is a result of confirming the number of white blood cells according to the concentration treatment of Example 1 (Rgx 365) of the present invention in the mouse bronchoalveolar lavage fluid derived from fine dust, Fig. This is the result of confirming the change in the production amount of IL-6 and TNF-α according to the concentration-specific treatment of Example 1 (Rgx 365).
7 is a result of confirming leukocyte infiltration according to the treatment of Example 1 (Rgx 365) of the present invention in the mouse lung tissue in which fine dust was induced.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the content introduced herein becomes thorough and complete, and is provided to sufficiently convey the spirit of the present invention to those skilled in the art.

<Example 1. Preparation of a mixture (Rgx 365) containing ginsenosides Rg2, Rg4, Rg6 and Rh1>

Distilled water 45 ml was added to 30.4 g of ginsenoside Re, treated at 121° C. and 0.13 MPa for 6 hours, followed by column separation.

Into the column, 300 g of a synthetic adsorbent having a ratio of 10 times or more was added to the column and sufficiently washed with an elution solvent. After pouring 5ℓ of distilled water, 5ℓ of fermented alcohol was poured. At this time, taking care not to mix air bubbles in the column, 5ℓ of water was poured again to prepare the column.

The reaction product treated with the ginsenoside Re for 6 hours was dissolved with fermented alcohol (50 ml) and distilled water (300 ml), and a water fraction was prepared and poured into the column. After pouring an aliquot of water, about 5ℓ of distilled water was poured, and then the foreign matter was removed by sequentially flowing 20% fermented alcohol, 25% fermented alcohol, and 30% fermented alcohol solvent. Rgx 365 was obtained as a fraction in 35-70% fermented alcohol solvent, and this was concentrated under reduced pressure to obtain 18 g of Example 1 (Rgx 365).

In addition, ginsenosides Rg2, Rg4, Rg6 and Rh1 included in Example 1 were HPLC under the conditions of Table 1 to check the content of each component, and are shown in FIGS. 1 and 2.

HPLC performance conditions column ACE 5-C ₁₈ (250×4.6㎜) flux 1.0ml/min Sample injection volume 10µl Detector UV 205 nm, Agilent Technologies 1260 infinity Temperature 40℃ menstruum A; water, B; acetonitrile
0-3min(A from 20% to 40%),
3-15min(B from 20% to 23%),
15-20min(B from 23% to 33%),
20-45min(B from 33% to 40%),
45-60min(B from 40% to 68%),
60-65min(B from 68% to 85%),
65-70min (B 85%),
70-73min(B from 80% to 20%),
73-75min(B 20%)

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 a mixture containing ginsenosides Rh1, Rg4 and Rg6>

A mixture of Comparative Example 1 was prepared with reference to Example 1 of Korean Patent Registration No. 10-1897811.

First, 25 µl of distilled water was mixed with 10 mg of ginsenoside Re, and then put into a sterilizer and treated for 8 hours at 120°C under a pressure of 0.13 to 0.15 MPa. Next, after concentrating in a rotary concentrator, the concentrated powder was separated and purified to obtain a mixture of Comparative Example 1 containing ginsenosides Rh1, Rg4 and Rg6.

<Experimental Example 1. Cell culture>

Mouse lung microvascular endothelial cells (MLMVECs) were obtained by referring to previous papers [Kovacs-Kasa, A. et al., Sci Pages Pulmonol., 1(1), 7-18, 2017].

First, a 7-week-old male Balb/c mouse (weight 27g, Orient Bio Co., Sungnam, Republic of Korea) was treated at a temperature of 20~25℃, humidity of 40~45%, and day/night at 12 hour intervals. Acclimate for 12 days. The mice used in the experiment were handled according to the guidelines for the care and use of experimental animals of Kyungpook National University (IRB No. KNU 2017-101).

After obtaining the lung tissue in the above process, it was pulverized and digested by treatment with collagenase A (1 mg/ml) for 45 to 60 minutes at 37°C. Endothelial cells were purified using anti-PECAM-1 monoclonal antibody magnetic beads (BD Pharmingen, San Diego, CA) and grown for 2 days in growth medium.

To culture the cells in a single layer, endothelial cell basic medium containing EGM-2 MV Bulletkit™ (Lonza, MD) was placed in a fibronectin-coated culture dish and cultured at 37° C., 5% CO ₂ and 95% air.

<Experimental Example 2. Confirmation of cytotoxicity>

In mouse lung microvascular endothelial cells, cytotoxicity according to the treatment of Example 1 of the present invention was confirmed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay method.

The mouse lung microvascular endothelial cells obtained in Experimental Example 1 were aliquoted into a 96-well plate at 5×10 ³ per well and cultured for 24 hours, followed by washing the cells and adding 100 µl of 1 mg/ml MTT solution for 4 hours. Reacted. Thereafter, 150 µl of DMSO was added to dissolve the formazan salt generated in the cells, and then absorbance was measured at 540 nm with a Microplate reader (Tecan Austria GmbH, Austria). The cell viability was calculated from the absorbance measurement result and shown in FIG. 2.

Referring to FIG. 2, it was confirmed that Example 1 of the present invention (Rgx 365) exhibited a cell viability equivalent to that of the untreated group regardless of the treatment concentration, and thus did not exhibit cytotoxicity.

<Experimental Example 3. Confirmation of the effect of inhibiting inflammation in mouse lung microvascular endothelial cells>

When the human body is exposed to fine dust, it causes acute inflammation, secretion of cytokines and chemokines, an increase in the number of white blood cells, the production of active oxygen in the lungs, and the reaction of cells and tissues by endotoxins. This action causes or worsens asthma, chronic bronchitis, and airway obstruction. Accordingly, the effect of inhibiting inflammation was confirmed by measuring the permeability assay and the change in the production of reactive oxygen species in the mouse lung microvascular endothelial cells according to the treatment of Example 1 of the present invention.

Experimental Example 3-1. Permeability assay

Prior papers [Bae, J. S. et al., Blood, 118(14), 3952-3959, 2011; Jung, B. et al., BMB Rep., 49(4), 214-219, 2016], the flux of Evans blue-bound albumin across the functional cell monolayer was analyzed in a two-chamber model (2). -compartment chamber model).

For the permeability assay in mouse lung microvascular endothelial cells, the cells of Experimental Example 1 were aliquoted into a transwell (pore size, 3 μm; diameter, 12 mm) at 5×10 ⁴ cells/well and cultured for 3 days. . When the cells were filled with a monolayer in the transwell, Example 1 was first treated for 6 hours, followed by fine dust (PM _2.5 , 1 mg/ml) for 6 hours. Thereafter, the permeability was measured using an ELISA plate reader, and the measurement results are shown in FIGS. 3A and 3B.

3A and 3B, when the present invention Example 1 (Rgx 365) is treated on mouse lung microvascular endothelial cells in which inflammation is induced by fine dust, single ginsenosides ginsenosides Re, Rg4, Rg6, Compared to treatment with the mixture of Rh1 and Comparative Example 1, the effect of reducing vascular permeability was particularly superior.

Therefore, the mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 prepared by the method of the present invention is particularly selective for the effect of treating respiratory diseases caused by fine dust, so it is a useful preparation method when used as a composition for preventing or treating respiratory diseases caused by fine dust. I could see that it was.

Experimental Example 3-2. Reactive oxygen (ROS) check

Reactive oxygen (ROS) in mouse lung microvascular endothelial cells was quantified by fluorescence microscopy with reference to prior literature [Piao, M. J. et al., Arch Toxicol., 92(6), 2077-2091, 2018]. DCFDA (2′,7′-dichlorofluorescein diacetate, Molecular Probes, Eugene, OR, USA, 10 μM) was added to primary mouse lung microvascular endothelial cells grown on a 4-well glass chamber slide (> 90% confluent). After 30 minutes, the medium containing DCFDA was aspirated and the cells were washed, and the stained cells were confirmed with a fluorescence microscope and shown in FIGS. 4A and 4B.

Referring to Figures 4A and 4B, the amount of ROS generation increased by fine dust decreased according to the treatment concentration of Example 1 (Rgx 365) of the present invention. Therefore, Example 1 of the present invention was found to be a composition excellent in the effect of reducing the inflammatory response increased by fine dust.

<Experimental Example 4. Confirmation of the effect of inhibiting inflammation by fine dust in an animal model>

Experimental Example 4-1. Permeability assay

First, a 7-week-old male Balb/c mouse (weight 27g, Orient Bio Co., Sungnam, Republic of Korea) was treated at a temperature of 20~25℃, humidity of 40~45%, and day/night at 12 hour intervals. Acclimate for 12 days. The mice used in the experiment were handled according to the guidelines for the care and use of experimental animals of Kyungpook National University (IRB No. KNU 2017-101).

Example 1 After oral administration of the solution at a concentration of 0.05-0.6g/kg for 10 days, fine dust (PM) with reference to the preceding paper [Wang, H. et al., Sci Rep., 7, 44256, 2017] _2.5 , 1 mg/kg in 100 µl of saline, 10 days) was administered intratracheally. After intratracheal instillation of fine dust for 10 days, previous papers [Bae, JS et al., Blood, 118(14), 3952-3959, 2011; Jung, B. et al., BMB Rep., 49(4), 214-219, 2016] by sacrificing male mice anesthetized with 2% isoflurane (Forane, JW Pharmaceutical, South Korea) 1% Evans Blue solution dissolved in was injected intravenously to each mouse. Thereafter, the permeability was measured with an ELISA plate reader and shown in Figs.

Referring to Figures 5A and 5B, the dye leaked in a large amount by fine dust decreased according to the treatment concentration of Example 1 (Rgx 365) of the present invention. In particular, in Example 1 of the present invention, compared to treatment with single ginsenosides Re, Rg4, Rg6, Rh1 and Comparative Example 1, the effect of protecting blood vessel wall damage was excellent, and leakage of the dye was remarkably suppressed.

From this, it can be seen that rather than using a single ginsenoside Re, Rg4, Rg6, Rh1, using a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 as in the present invention is most effective in suppressing inflammation caused by fine dust. there was.

Experimental Example 4-2. Confirmation of leukocyte count and expression of inflammation-related factors

Proceed as in Experimental Example 4-1, but instead of injecting 1% Evans blue solution, fine dust was instilled in the trachea for 10 days, and then the mice were sacrificed to obtain bronchoalveolar lavage fluid (BAL). The bronchoalveolar lavage solution was washed with 5 ml of physiological saline, and 0.38 ml of Turk's solution (0.01% crystal violet in 3% acetic acid) was mixed, and then the number of white blood cells was measured with an optical microscope and shown in FIG. 6A. In addition, the concentrations of IL-6 and TNF-α among inflammation-related factors in the bronchoalveolar lavage fluid were measured with ELISA kits (R&D Systems, Minneapolis, MN) and shown in FIG. 6B.

Referring to Figures 6A and 6B, when the present invention Example 1 (Rgx 365) was treated to a mouse in which fine dust was induced, leukocyte migration was inhibited and the number of leukocytes was significantly reduced, and inflammation-related factors such as IL-6 and TNF-α It was found that the amount of produced was also remarkably decreased, thereby recovering the increased level of inflammation by fine dust in respiratory diseases induced by fine dust.

Experimental Example 4-3. H&E dyeing

To confirm the change in mouse lung tissue induced by fine dust, proceed as in Experimental Example 4-1, but instead of injecting 1% Evans blue solution, fine dust was instilled in the trachea for 10 days, and then the mouse was sacrificed to the lung. Got the tissue. This was fixed with a 4% formaldehyde solution dissolved in PBS for 20 hours at 4° C., dehydrated with ethanol, and embedded with paraffin. After that, a 4 μm section was made, placed on a slide, deparaffinized in an oven at 60° C., and then rehydrated and stained with hematoxylin. To remove excess staining, the slides were quickly dipped in 0.3% acidic alcohol 3 times and counter stained with eosin. Wash with ethanol and xylene to remove excess staining and the sample was placed under a coverslip. Optical microscopic analysis of the lung samples was performed with reference to the preceding paper [Ozdulger, A. et al., Shock, 19(4), 366-372, 2003], and the results of lung tissue changes are shown in FIG. 7.

Referring to FIG. 7, in the case of treatment of Example 1 (Rgx 365) of the present invention, leukocyte infiltration in mouse lung tissue by fine dust was significantly reduced.

From these results, the ginsenoside Rg2, Rg4, Rg6 and Rh1 mixture of the present invention with enhanced ginsenoside Rg2 is excellent in preventing or treating respiratory diseases caused by fine dust, so it is used as a composition for preventing or treating respiratory diseases due to fine dust. It was found that it could be useful.

<Formulation Example 1. Preparation of tablets>

Inventive Example 1 (Rgx 365) 20g was mixed with 175.9g lactose, 180g potato starch and 32g colloidal silicic acid. After adding a 10% gelatin solution to this mixture, it was pulverized and passed through a 14 mesh sieve. This was dried, and 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate were added thereto, and the resulting mixture was made into tablets.

<Formulation Example 2. Preparation of capsules>

Inventive Example 1 (Rgx 365) 100 mg, corn starch 100 mg, lactose 100 mg, and magnesium stearate 2 mg were mixed, and the above ingredients were mixed according to a conventional capsule preparation method, and then filled in gelatin capsules to form a capsule. Was prepared.

<Formulation Example 3. Preparation of injection>

Inventive Example 1 (Rgx 365) 1 g, sodium chloride 0.6 g, and ascorbic acid 0.1 g were dissolved in distilled water to make 100 ml. This solution was put in a bottle and sterilized by heating at 20° C. for 30 minutes.

<Formulation Example 4. Preparation of health functional food>

Invention Example 1 (Rgx 365) 20g, vitamin mixture appropriate amount, vitamin A acetate 70 ㎍, vitamin E 1.0 mg, vitamin B1 0.13 mg, vitamin B2 0.15 mg, vitamin B6 0.5 mg, vitamin B12 0.2 μg, vitamin C 10 mg , Biotin 10 ㎍, Nicotinic acid amide 1.7 mg, Folic acid 50 ㎍, Calcium pantothenate 0.5 mg, an appropriate amount of inorganic mixture, ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, potassium monophosphate 15 mg, diphosphate 55 mg of calcium, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride were mixed to form granules, but can be prepared by modifying various formulations according to the use. In addition, the composition ratio of the vitamin and mineral mixture may be arbitrarily modified, and may be prepared by mixing the above ingredients according to a conventional health functional food manufacturing method.

<Formulation Example 5. Preparation of health functional beverage>

Invention Example 1 (Rgx 365) 1g, citric acid 0.1g, fructooligosaccharide 100g, purified water 900g were mixed and stirred, heated, filtered, sterilized, and refrigerated according to a conventional beverage manufacturing method to prepare a beverage.

Claims (6)

Respiratory inflammatory lung disease, asthma, chronic obstructive pulmonary disease, allergic rhinitis, cough, bronchitis, pharyngitis, otitis media, tonsillitis, sinusitis, pneumonia, pulmonary fibrosis and A pharmaceutical composition for preventing or treating respiratory diseases by fine dust, characterized in that selected from the group consisting of laryngitis. The method of claim 1,
The mixture is fine, characterized in that it contains 35 to 45% by weight of ginsenoside Rg2, 30 to 40% by weight of ginsenoside Rg4, 10 to 20% by weight of ginsenoside Rg6 and 1 to 5% by weight of ginsenoside Rh1 A pharmaceutical composition for preventing or treating respiratory diseases caused by dust. delete Respiratory inflammatory lung disease, asthma, chronic obstructive pulmonary disease, allergic rhinitis, cough, bronchitis, pharyngitis, otitis media, tonsillitis, sinusitis, pneumonia, pulmonary fibrosis and Health functional food for preventing or improving respiratory diseases by fine dust, characterized in that selected from the group consisting of laryngitis. The method of claim 4,
The mixture is fine, characterized in that it contains 35 to 45% by weight of ginsenoside Rg2, 30 to 40% by weight of ginsenoside Rg4, 10 to 20% by weight of ginsenoside Rg6 and 1 to 5% by weight of ginsenoside Rh1 Health functional food for preventing or improving respiratory diseases caused by dust. delete

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