KR102427677B1 - Manufacturing method of ginsenoside Rg2, Rg4, Rg6 and Rh1 mixture - Google Patents

Abstract

The present invention relates to a method for preparing a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 enriched with ginsenoside Rg2 from ginsenoside Re, and to a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 prepared therefrom. The preparation method of the present invention can easily obtain a large amount of a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 from ginsenoside Re, and the ginsenoside mixture is a single ginsenoside Rg4, Rg6 and Rh1, ginseng extract or Compared to red ginseng extract, it is particularly selective for the effect of treating respiratory diseases caused by fine dust (decreased permeability, reduced reactive oxygen species (ROS) production, reduced white blood cell count, and reduced cytokine production in models induced by fine dust). Therefore, it is a useful manufacturing method when used as a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust or as a health functional food for preventing or improving respiratory diseases caused by fine dust.

Description

Method for preparing ginsenoside Rg2, Rg4, Rg6 and Rh1 mixture {Manufacturing method of ginsenoside Rg2, Rg4, Rg6 and Rh1 mixture}

The present invention relates to a method for preparing a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 enriched with ginsenoside Rg2 from ginsenoside Re, and to a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 prepared therefrom.

Recently, as the concentration of fine dust continues to increase, social interest in fine dust and its effects on the human body is increasing. In fine dust, various components such as carbon components (soot, organic carbon), ionic components (chlorine, nitric acid, ammonium, sodium, calcium, etc.), metal components (arsenic, lead, mercury, etc.), polycyclic aromatic hydrocarbons (benzopyrene, etc.) This is included. When fine dust increases, visibility is blurred and discomfort is felt, and serious diseases such as conjunctivitis, sinusitis, otitis media, bronchitis, asthma and chronic obstructive pulmonary disease exacerbation, pneumonia, angina, myocardial infarction, lung cancer, etc. was reported to be associated with an increase in mortality as well as the incidence of respiratory and cardiovascular diseases (Jang, A. S., J Korean Med Assoc., 57(9), 763-768, 2014).

Particulate matter smaller than 10㎛ is called fine dust, and particulate matter smaller than _10㎛ is called PM10. Among PM10, relatively large particles between 2.5㎛ and 10㎛ are mainly dust from roads or soil, and particles smaller than _2.5㎛ (referred to as ultrafine dust or PM _2.5 ) are mainly automobile exhaust gas or factory chimneys. It is dust generated in the process of burning fossil fuels such as smoke or secondary generated through chemical reaction of gaseous air pollutants in the air (Kwon, Hojang, 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 granulocytes and lymphocytes in the alveolar lavage 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, and mast cells increase the size of mucous cells, It induces an inflammatory response by increasing the amount of mucus. Among the granulocytes known to be involved in inflammation, eosinophils help the production of T cells from Th2 cells when the airway responds to antigens, and the Th2 cytokines IL-5 and eotaxin secreted from mast cells stimulate the production of eosinophils and eosinophils. Helps activate

Inhalation of fine dust causes inflammation of the bronchial tubes, and when such inflammation continues, chronic bronchitis, asthma, allergic alveolitis, etc. Chronic obstructive pulmonary disease (COPD), a representative disease caused by fine dust, is a disease in which the function of the lung tissue deteriorates due to an abnormal inflammatory response and breathing difficulties occur. In particular, among the fine dust components, diesel exhaust particles (DEPs) increase the secretion of IL-17A and exacerbate bronchial asthma. It is highly likely to cause or exacerbate various diseases such as disability. In addition, fine dust may cause respiratory tract infections by interfering with the inactivation or removal of bacteria in lung tissue.

Recently, our atmospheric environment is exposed to air pollutants including yellow dust, PM ₁₀ , PM _2.5 and ultra-fine dust, and industrial nanoparticles, whose application range is rapidly spreading to the human body. It is known that fine dust and harmful chemicals contained in fine dust cause harmful effects on the human body through bronchial inflammatory mechanisms such as oxidative stress and inflammatory response. 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 (Hyeongwoo 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 effect unlike saponins found in other plants. Ginsenoside is a neutral glycoside in which glucose, arabinose, xylose, rhamnose, etc. are bonded to the triterpenoid-type dammarane skeleton. , at present, more than 30 types of chemical structures have been identified, and depending on the characteristics of the chemical structures, protopanaxadiol (PPD)-based (19 types), protopanaxatriol (PPD)-based (10 types) and oleic acid It is classified as oleanane (type 1).

The main ginsenosides present in fresh ginseng or white ginseng are Rg1, Re, Rf, Rh1, Rb1, Rb2, Rc, Rd, etc., accounting for more than 80-90% of the total ginsenosides. In the case of rare ginsenosides, some of the sugars are separated from the ginsenosides present in a large amount, or a dehydration reaction occurs.

The pharmacological activity of ginsenosides is shown by the rare ginsenosides resulting from the ginsenosides present in large amounts, and it enhances immunity, anti-inflammatory, anti-allergy, anti-cancer, blood pressure lowering, anti-cholesterol, anti-thrombosis, anti-aging, Antioxidant, brain activity promotion, and skin beautifying effect are known as major effects (Kim, Y. S. 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, S. C. et al., Chin J Physiol., 46(1) ), 1-7, 2003).

On the other hand, as a prior document related to a method for preparing a mixture containing ginsenosides Rg2, Rg4, Rg6 and Rh1, Korean Patent No. 10-1897811 discloses that distilled water is added to ginsenoside Re and steamed to obtain from ginsenoside Re. A method for mass production of a mixture of ginsenosides Rh1, Rg4 and Rg6 has been disclosed, and Korean Patent No. 10-1625474 discloses a method for mass production of ginsenoside Rh4 by adding an organic acid to Re and steaming the ginsenoside Re.

However, the present invention is a mixture of ginsenoside Rg2, Rg4, Rg6 and Rh1 containing 30% by weight or more of ginsenoside Rg2 among ginsenoside components by steaming by adding 130 to 160 parts by weight of distilled water to 100 parts by weight of ginsenoside Re. While it relates to a manufacturing method of It is an invention related to a method for mass production of Rg4 and Rg6, and Korean Patent No. 10-1625474 is an invention related to a method for mass production of ginsenoside Rh4 by adding organic acid to ginsenoside Re and steaming. There is no mention at all about the ginsenoside Rg2 component included in a large amount as in the present invention.

Therefore, the present inventors found that the ginsenoside Rg2, Rg4, Rg6 and Rh1 mixture prepared by the method of the present invention in the process of researching ginsenoside contains a large amount of ginsenoside Rg2, which is particularly effective for treating respiratory diseases caused by fine dust. The present invention could be completed by confirming that the single ginsenosides Rg4, Rg6 and Rh1, ginseng extract or red ginseng extract were particularly selective for the effect of treating respiratory diseases caused by fine dust.

Korean Patent No. 10-1897811, mass production method of ginsenosides Rh1-S, Rh1-R, Rg4 and Rg6, registered on September 05, 2018. Korean Patent No. 10-1625474, mass production method of ginsenoside RH 4, registered on May 24, 2016.

Hojang Kwon, Fine Dust and Health, Korean Social Trends 2014, 281-287, 2014. Hyung-Woo Song et al., Respiratory protective effect of baeam tea extract against 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, Y. S. et al., Differential expression of protein kinase C subtypes during ginsenoside Rh2-induced 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. 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 method for preparing a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 enriched with ginsenoside Rg2 from ginsenoside Re and a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 prepared therefrom is doing

The present invention relates to a method for preparing a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1,

(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 at a high temperature and pressure for 4 to 6 hours at a temperature range of 110 to 140° C. and a pressure of 0.11 to 0.16 MPa; and

(Step 3) column separation of the reactants in Step 2 to obtain a mixture containing ginsenosides Rg2, Rg4, Rg6 and Rh1; includes

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

[Scheme 1]

The 1st and 2nd steps are steps of reacting at high temperature and high pressure by mixing distilled water with ginsenoside Re, more preferably mixing 140-150 parts by weight of distilled water with ginsenoside Re, and then 110-140 for 4-6 hours A mixture of ginsenoside Rg2, Rg4, Rg6 and Rh1 containing 35 wt% or more of ginsenoside Rg2 is prepared by reacting at a high temperature and high pressure in a temperature range of ℃ and a pressure condition of 0.11 to 0.16 MPa and column separation.

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

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

The ginsenoside Re is preferably prepared separately from the ginseng extract, and the ginseng extract can be prepared using everything derived from ginseng, such as roots, fruits, leaves, and stems of ginseng, but preferably a large amount from the leaves of ginseng. of ginsenoside Re can be obtained. 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 ), ginseng ( Panax notoginseng ), bamboo japonicus ( Panax japonicus ), Himalayan ginseng ( Panaxa pseudoginseng ). One 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 using ginseng water, C1 to C4 alcohol or a mixture thereof as a solvent, and the C1 to C4 alcohol is methanol, ethanol, propanol, isopropanol, from the group consisting of butanol and isobutanol. can be selected. Water, C1 to C4 alcohol or a mixture thereof used in the preparation of the ginseng extract may be used in an amount of 1 to 40 times the weight of ginseng used (1 to 40 liters based on 1 kg of ginseng), preferably 5 to 40 times can be used, and the above process can be repeated 1 to 4 times.

The synthetic adsorbent used in the column separation is 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, SP205-207, HP1MG, HP2MG, etc. may be used, and preferably GSH-20, HP-20, SP825 may be used.

In another aspect, the present invention relates to a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 prepared through steps 1 to 3, wherein the mixture is excellent in treating respiratory diseases caused by fine dust, so fine dust It can be used as a pharmaceutical composition for preventing or treating respiratory diseases or as a health functional food for preventing or improving respiratory diseases caused by fine dust.

The respiratory disease is a disease that causes chronic inflammation in the airways, bronchial tubes, and lungs accompanied by coughing, sputum, dyspnea and fever. Preferably, the respiratory disease is respiratory inflammatory lung disease, asthma, chronic obstructive disease, etc. 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 commonly used pharmaceutically acceptable carrier. “Pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans. In addition, the composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively.

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, but is not limited thereto. In the case of formulation, it is prepared using diluents or excipients such as fillers, stabilizers, binders, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the ginsenoside mixture of the present invention, for example, starch, microcrystalline cellulose, sucrose. 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 formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, etc. may be used. In order 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 wetting agent or an emulsifying agent, a salt for regulating the osmotic pressure and/or a buffer; and other therapeutically useful substances to prepare a solution or suspension by mixing in water, which may be prepared in ampoules or vial 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 envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection. The dosage may vary depending on the age, sex, weight, specific disease or pathology to be treated, the severity of the disease or pathology, administration time, administration route, absorption, distribution and excretion rate of the drug, the type of other drugs used and the prescriber's It will depend on judgment, etc. Dosage determination based on these factors is within the level of one of ordinary 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 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

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

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

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

The present invention relates to a method for preparing a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 enriched with ginsenoside Rg2 from ginsenoside Re, and to a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 prepared therefrom. The preparation method of the present invention can easily obtain a large amount of a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 from ginsenoside Re, and the ginsenoside mixture is a single ginsenoside Rg4, Rg6 and Rh1, ginseng extract or Compared to red ginseng extract, it is particularly selective for the effect of treating respiratory diseases caused by fine dust (decreased permeability, reduced reactive oxygen species (ROS) production, reduced white blood cell count, and reduced cytokine production in models induced by fine dust). Therefore, it is a useful manufacturing method when used as a pharmaceutical composition for preventing or treating respiratory diseases caused by fine dust or as a health functional food for preventing or improving respiratory diseases caused by fine dust.

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

Hereinafter, preferred embodiments 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, it is provided so that this disclosure will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

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

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

300 g of a synthetic adsorbent at least 10 times the ratio of the raw material was added to the column and thoroughly washed with an elution solvent. After pouring 5 liters of distilled water, 5 liters of fermented alcohol was flowed. At this time, be careful not to mix air bubbles in the column, and 5 L of water was again flowed to prepare the column.

The reactant treated with the ginsenoside Re for 6 hours was dissolved using fermentation alcohol (50 mL) and distilled water (300 mL), and a water fraction was prepared and poured into the column. After pouring the water fraction, about 5 liters of distilled water was flowed, and then, 20% fermented alcohol, 25% fermented alcohol, and 30% fermented alcohol solvent were sequentially flowed to remove foreign substances. Rgx 365 was obtained as a fraction in 35-70% fermentation alcohol solvent, and this was concentrated under reduced pressure to obtain 18 g of Example 1 (Rgx 365).

In addition, prepared as in Example 1, but with reference to Table 1 below, 48 ml or 40 ml of distilled water was added to 30.4 g of ginsenoside Re, and then treated at 121° C. and 0.13 MPa for 6 hours, followed by column separation. Examples 2 and 3 were obtained by 18 g each.

Among Examples 1 to 3 obtained from the above process, ginsenoside Rg2, Rg4, Rg6 and Rh1 contained in Example 1 were subjected to HPLC under the conditions of Table 2 below to confirm the content of each component, and it is shown in FIGS. 1 and 3 indicated.

mixing conditions Ginsenoside Re(g) Distilled water (ml) Example 1 30.4 45 Example 2 30.4 48 Example 3 30.4 40

HPLC performance conditions column ACE 5-C ₁₈ (250×4.6㎜) flux 1.0ml/min sample injection volume 10 μl 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 No. 10-1897811.

First, 25 μl of distilled water was mixed with 10 mg of ginsenoside Re, and then placed in a sterilizer and treated at 120° C., under a pressure of 0.13 to 0.15 Mpa for 8 hours. Next, after concentration 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.

<Comparative Example 2. Preparation of a mixture containing comparative ginsenosides Rg2, Rg4, Rg6 and Rh1 ①>

It was prepared as in Example 1, but 60 ml of distilled water added to 30.4 g of ginsenoside Re was added instead of 45 ml, and the resultant was treated at 121° C. and 0.13 MPa for 6 hours, followed by column separation.

<Comparative Example 3. Preparation of a mixture containing comparative ginsenosides Rg2, Rg4, Rg6 and Rh1②>

It was prepared as in Example 1, but 53 ml of distilled water added to 30.4 g of ginsenoside Re 30.4 g was added, and the resultant was treated at 121° C. and 0.13 MPa for 6 hours, followed by column separation.

<Comparative Example 4. Preparation of a mixture containing comparative ginsenosides Rg2, Rg4, Rg6 and Rh1 ③>

It was prepared as in Example 1, but instead of 45 ml of distilled water added to 30.4 g of ginsenoside Re, 35 ml was added, and the resultant was treated at 121° C. and 0.13 MPa for 6 hours, followed by column separation.

<Comparative Example 5. Preparation of a mixture containing comparative ginsenosides Rg2, Rg4, Rg6 and Rh1 ④>

Prepared as in Example 1, but added 30 ml of distilled water added to 30.4 g of ginsenoside Re 30.4 g, and treated at 121° C. and 0.13 MPa for 6 hours, followed by column separation.

<Experimental Example 1. Comparison of ginsenoside Rg2 content>

The content of ginsenoside Rg2 among the ginsenoside components included in the ginsenoside mixtures of Examples 1 to 3 and Comparative Examples 1 to 5 was shown in Table 4 below by HPLC under the conditions of Table 2.

Condition Ginsenoside Rg2
Content (wt%) Example 1 100 parts by weight of ginsenoside Re,
148 parts by weight of distilled water 41.1 Example 2 100 parts by weight of ginsenoside Re,
158 parts by weight of distilled water 42.5
Example 3 Ginsenoside Re 100 parts by weight,
132 parts by weight of distilled water 37.5 Comparative Example 1 Ginsenoside Re 100 parts by weight,
250 parts by weight of distilled water <10 Comparative Example 2 100 parts by weight of ginsenoside Re,
197 parts by weight of distilled water 11.5 Comparative Example 3 100 parts by weight of ginsenoside Re,
174 parts by weight of distilled water <25 Comparative Example 4 Ginsenoside Re 100 parts by weight,
115 parts by weight of distilled water 19.8 Comparative Example 5 100 parts by weight of ginsenoside Re,
100 parts by weight of distilled water <10

Looking at Table 4, when 130 to 160 parts by weight of distilled water are mixed with 100 parts by weight of ginsenoside Re as in the present invention and then steamed, ginsenoside Rg2 is added to the final prepared ginsenoside Rg2, Rg4, Rg6 and Rh1 mixture It was confirmed that the content was 35% by weight or more.

However, unlike the present invention, when the amount of distilled water added to 100 parts by weight of ginsenoside Re is less than 130 parts by weight or exceeds 160 parts by weight, ginsenoside Rg2 contained in the finally prepared ginsenoside mixture is 25 parts by weight. % or less. That is, when distilled water added to 100 parts by weight of ginsenoside Re is less than 130 parts by weight, it is confirmed that the amount of ginsenoside Re is small because the amount of remaining ginsenoside Re does not sufficiently react and the amount of ginsenoside Rg2 is small, and exceeds 160 parts by weight of distilled water. In this case, it was confirmed that the ginsenoside Rg2 did not remain in the final mixture and was converted to the ginsenoside Rg4 and Rg6, resulting in increased production of the ginsenoside Rg4 and Rg6.

Therefore, as in the present invention, mixing 130 to 160 parts by weight of distilled water with ginsenoside Re and then steaming is particularly effective for treating respiratory diseases caused by fine dust. It was confirmed that it is a useful manufacturing method when used as a pharmaceutical composition for disease prevention or treatment.

<Experimental Example 2. 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, 7-week-old male Balb/c mice (weight 27g, Orient Bio Co., Sungnam, Republic of Korea) were treated at a temperature of 20-25°C, humidity of 40-45%, and day/night intervals of 12 hours. It was allowed to acclimatize for 12 days. Mice used in the experiment were handled in accordance with the Guidelines for the Care and Use of Laboratory Animals of Kyungpook National University (IRB No. KNU 2017-101).

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

For monolayer culture of cells, endothelial cell basal 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 conditions.

<Experimental Example 3. Confirmation of cytotoxicity>

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

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

Referring to FIG. 2 , it was confirmed that Example 1 (Rgx 365) of the present invention exhibited cell viability equivalent to that of the untreated group regardless of the treatment concentration, thereby showing no cytotoxicity.

<Experimental Example 4. 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, and secretion of cytokines and chemokines, increase in the number of white blood cells, generation of reactive oxygen species in the lungs, and cell and tissue reactions by endotoxin occur. This action causes or worsens asthma, chronic bronchitis, and airway obstruction. Therefore, the anti-inflammatory effect was confirmed by measuring the permeability assay and reactive oxygen production change in mouse lung microvascular endothelial cells according to the treatment of Example 1 of the present invention.

Experimental Example 4-1. Permeability assay

For quantification of endothelial cell permeability according to the concentration of Example 1 of the present invention [Bae, J. S. et al., Blood, 118(14), 3952-3959, 2011; Jung, B. et al., BMB Rep., 49(4), 214-219, 2016], a two-chamber model (2) for the flux of Evans blue-bound albumin across functional cell monolayers. -compartment chamber model).

For the permeability assay in mouse lung microvascular endothelial cells, the cells of Experimental Example 2 were seeded into transwells (pore size, 3㎛; diameter, 12mm) 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, and then fine dust (PM _2.5 , 1 mg/ml) was treated for 6 hours. Thereafter, the permeability was measured using an ELISA plate reader, and the measurement results are shown in FIGS. 3A and 3B.

Referring to FIGS. 3A and 3B, when the present invention Example 1 (Rgx 365) was treated with microvascular endothelial cells in the mouse lung induced by fine dust inflammation, the single ginsenoside ginsenoside Re, Rg4, Rg6, Compared with the treatment of Rh1 and Comparative Example 1 mixture, 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. was found to be

Experimental Example 4-2. Check active oxygen (ROS)

Reactive oxygen (ROS) in mouse lung microvascular endothelial cells was quantified by fluorescence microscopy with reference to the 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 4-well glass chamber slides (> 90% confluent). After 30 minutes, the medium containing DCFDA was aspirated and the cells were washed, and the stained cells were confirmed by a fluorescence microscope and shown in FIGS. 4A and 4B.

Referring to FIGS. 4A and 4B , the amount of ROS generated by fine dust decreased according to the treatment concentration of Example 1 (Rgx 365) of the present invention. Therefore, it can be seen that Example 1 of the present invention is a composition having an excellent effect of reducing the inflammatory response increased by fine dust.

<Experimental Example 5. Confirmation of Inflammation Inhibitory Effect by Fine Dust in Animal Models>

Experimental Example 5-1. Permeability assay

First, 7-week-old male Balb/c mice (weight 27g, Orient Bio Co., Sungnam, Republic of Korea) were treated at a temperature of 20-25°C, humidity of 40-45%, and day/night intervals of 12 hours. It was allowed to acclimatize for 12 days. Mice used in the experiment were handled in accordance with the Guidelines for the Care and Use of Laboratory Animals of Kyungpook National University (IRB No. KNU 2017-101).

Example 1 solution was orally administered at a concentration of 0.05-0.6 g/kg for 10 days, and then fine dust (PM) with reference to a previous 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, prior 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) with physiological saline 1% Evans blue solution dissolved in , was intravenously injected into each mouse. Thereafter, the permeability was measured with an ELISA plate reader and shown in FIGS. 5A and 5B.

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

From this, it is found that using a mixture of ginsenosides Rg2, Rg4, Rg6 and Rh1 prepared by the present invention is the most effective in suppressing inflammation caused by fine dust rather than using single ginsenosides Re, Rg4, Rg6, and Rh1. Could know.

Experimental Example 5-2. Confirmation of white blood cell count and expression of inflammation-related factors

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

Referring to Figures 6A and 6B, when the present invention Example 1 (Rgx 365) was treated in mice induced with fine dust, leukocyte migration was inhibited and the number of leukocytes was significantly reduced, and the inflammation-related factors IL-6 and TNF-α The amount of produced was also significantly reduced, indicating that the level of inflammation increased by fine dust was restored when used for fine dust-induced respiratory diseases.

Experimental Example 5-3. H&E dyeing

In order to confirm the change of the mouse lung tissue induced by fine dust, proceed as in Experimental Example 5-1, but instead of injecting 1% Evans blue solution, fine dust was instilled into the trachea for 10 days, and then the mouse was sacrificed to the lungs. tissue was obtained. This was fixed with 4% formaldehyde solution in PBS at 4° C. for 20 hours, dehydrated with ethanol, and then embedded with paraffin. Thereafter, 4 μm sections were made, placed on slides, deparaffinized in an oven at 60° C., rehydrated and stained with hematoxylin. To remove excessive staining, slides were quickly dipped in 0.3% acid alcohol three times and counterstained with eosin. Excess staining was removed by washing with ethanol and xylene and the samples were placed under coverslips. Light microscopic analysis of lung specimens was performed with reference to a previous paper [Ozdulger, A. et al., Shock, 19(4), 366-372, 2003], and the results of changes in lung tissue are shown in FIG. 7 .

Referring to FIG. 7 , in the case of treatment with Example 1 (Rgx 365) of the present invention, leukocyte infiltration in mouse lung tissue caused 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 as a composition for preventing or treating respiratory diseases caused by fine dust found it to be useful.

<Formulation Example 1. Preparation of tablets>

20 g of Example 1 (Rgx 365) of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. After adding a 10% gelatin solution to this mixture, it was ground 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>

After mixing 100 mg of Example 1 (Rgx 365) of the present invention, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate, the above ingredients are mixed according to a conventional capsule preparation method and filled in a gelatin capsule to form a capsule. was prepared.

<Formulation Example 3. Preparation of Injection>

Invention 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 placed in a bottle and sterilized by heating at 20° C. for 30 minutes.

<Formulation Example 4. Manufacture of health functional food>

Example 1 of the present invention (Rgx 365) 20g, vitamin mixture appropriate amount, vitamin A acetate 70㎍, vitamin E 1.0mg, vitamin B1 0.13mg, vitamin B2 0.15mg, vitamin B6 0.5mg, vitamin B12 0.2㎍, vitamin C 10mg , biotin 10㎍, nicotinic acid amide 1.7mg, folic acid 50㎍, calcium pantothenate 0.5mg, mineral mixture appropriate amount, ferrous sulfate 1.75mg, zinc oxide 0.82mg, magnesium carbonate 25.3mg, potassium monophosphate 15mg, diphosphate dibasic Calcium 55 mg, potassium citrate 90 mg, calcium carbonate 100 mg, and magnesium chloride 24.8 mg were mixed to prepare granules, but it can be prepared by modifying it into various formulations depending on the use. In addition, the composition ratio of the vitamin and mineral mixture may be arbitrarily modified, and it 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>

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

Claims (1)

(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 at a high temperature and high pressure for 4 to 6 hours at a temperature range of 110 to 140° C. and a pressure of 0.11 to 0.16 MPa; and
(Step 3) column separation of the reactants in Step 2 to obtain a mixture containing ginsenosides Rg2, Rg4, Rg6 and Rh1;
From ginsenoside Re, characterized in comprising A method for preparing a mixture having an enhanced Rg2 content in %.

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