KR102301000B1 - Composition comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl) acetal for Preventing and treating chronic obstructive pulmonary disease - Google Patents

Abstract

The present invention relates to a composition for the treatment, prevention or improvement of chronic obstructive pulmonary disease comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient. -Hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal inhibits apoptosis of bronchial epithelial cells induced by tobacco smoke and increases the expression of ALDH2, ALDH3A1 or WRN to protect cells Thus, it can be usefully used in the development of a therapeutic agent or an ameliorating agent for chronic obstructive pulmonary disease.

Description

Composition comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl) acetal for the prevention and treatment of chronic obstructive pulmonary disease, including 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal Preventing and treating chronic obstructive pulmonary disease}

The present invention relates to a composition for treating, preventing or improving chronic obstructive pulmonary disease, comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient.

Plum (Japanese apricot, scientific name: Prunus mume ) has long been popular as a health food among Koreans, Japanese, and Chinese. Japanese apricot extract (JAE) has several beneficial biological effects, such as reducing oxidative stress and inflammation, and enhancing innate immunity.

Concentrated plum extract may prevent cardiovascular disease as an antioxidant effect on angiotensin II-induced ROS production. In addition, the triterpenoids of plum extract have anti-tumor and anti-inflammatory effects.

Tobacco smoke is a major risk factor for chronic obstructive pulmonary disease (COPD). Tobacco smoke contains an abundance of toxic chemicals including reactive aldehydes that cause DNA damage and cell death. In humans, members of the aldehyde dehydrogenase (ALDH) superfamily of 19 NAD(P) ^{+ -dependent isoenzymes promote aldehyde oxidation.} ALDH3A1, the most strongly induced isoenzyme in the ALDH superfamily, attenuates tobacco smoke-induced DNA damage and cytotoxicity. Numerous ALDH species, including ALDH2 and ALDH3A1, promote the oxidation of aliphatic, aromatic and lipid peroxidation (LPO)-induced aldehydes, including 4-hydroxy-2-nonenal (4-HNE). ALDH3A1 also attenuates LPO-mediated growth inhibition, UV-ray-induced cytotoxicity, ROS-induced protein modification, and gene toxin-induced DNA damage and apoptosis.

Cigarette smoke activates DNA damage response (DDR) mediated by phosphoinositide 3-kinase-associated protein kinase (PIKK), including ataxia teleangiectasia mutated (ATM). Upon formation of DNA double-strand breaks (DSBs), ATM is activated by autophosphorylation at the serine 1981 residue and phosphorylates the serine 139 residue (γH2AX) of the H2AX variant at the chromatin proximal DSB site. γH2AX is required to relay subsequent DDR signaling and DNA repair.

However, the potential usefulness of plum extract in chronic obstructive pulmonary disease and skin aging caused by tobacco toxicity has not yet been evaluated.

Republic of Korea Patent No. 10-0785969

It is an object of the present invention to provide a pharmaceutical composition for treating or preventing chronic obstructive pulmonary disease, comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient.

Another object of the present invention is to provide a food composition for improving or preventing chronic obstructive pulmonary disease, comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient.

The present inventors isolated a compound having a cytoprotective effect against tobacco smoke-induced toxicity from concentrated plum extract (JAE), and as a result of structural analysis of the isolated compound, 5-hydroxymethyl-2-furaldehyde bis (5-form A novel molecule of milfurfuryl)acetal (named FA-1) was successfully identified and completed the present invention.

In one aspect for achieving the above object, the present invention provides a pharmaceutical for the treatment or prevention of chronic obstructive pulmonary disease, comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient. A composition is provided.

In the present invention, "5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal" is a compound having the structure of Formula 1 and may be isolated from plum.

In an embodiment of the present invention, polysaccharides were precipitated in the concentrate of the plum extract with ethanol to separate, the hexane fraction was separated, and the chloroform fraction fractionated with a chloroform solvent was subjected to CPC chromatography using 5-hydroxymethyl-2-fur. The aldehyde bis(5-formylfurfuryl)acetal was isolated.

Therefore, the 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal may be separated from the chloroform fraction of the plum extract, and specifically, the chloroform fraction may be separated by performing CPC chromatography. .

In the present invention, "chronic obstructive pulmonary disease" is a disease in which air circulation in the bronchial tubes becomes difficult due to chronic inflammatory changes in the airways (bronchial tubes) due to an abnormal inflammatory reaction caused by inhalation of harmful particles or gases, particularly tobacco. The most common symptoms include phlegm, shortness of breath, and phlegm. Although the symptoms are similar to asthma, unlike asthma, the decrease in lung airflow is not significantly improved by drug administration. Smoking is the most common cause of chronic obstructive pulmonary disease, and factors such as air pollution and heredity are also known to have an influence. In the present invention, the chronic obstructive pulmonary disease may be emphysema or chronic bronchitis, but is not limited thereto.

As used herein, the term "prevention" refers to any action that inhibits or delays the onset of chronic obstructive pulmonary disease by administration of the pharmaceutical composition according to the present invention, and "treatment" refers to any action by administration of the pharmaceutical composition. It refers to all actions that improve or beneficially change the symptoms of the suspected or onset chronic obstructive pulmonary disease.

In one embodiment of the present invention, 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal isolated from plum extract (JAE) is immortalized in human bronchial epithelial cells (HBEC2) and normal human epidermal keratin It has been shown to protect against tobacco smoke extract (CSE)-induced cytotoxicity and DNA damage accompanied by increased levels of aldehyde dehydrogenase (ALDH)2, aldehyde dehydrogenase (ALDH)3A1 and Werner syndrome protein (WRN) expression in cells (NHEK). confirmed that.

That is, it was confirmed that 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal inhibits or protects bronchial cell damage caused by tobacco, the 5-hydroxymethyl-2-furaldehyde Bis(5-formylfurfuryl)acetal can be used as a composition for treating or preventing chronic obstructive pulmonary disease. Specifically, the chronic obstructive pulmonary disease may be caused by tobacco.

In the present invention, the treatment and prevention of chronic obstructive pulmonary disease may be to delay or inhibit the symptoms or invention of chronic obstructive pulmonary disease by specifically inhibiting tobacco-induced cell death and DNA damage.

In addition, it was confirmed that 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal increased the expression of ALDH2, AlDH3A1 and WRN in human bronchial epithelial cells in a concentration-dependent manner in the examples of the present invention, It was confirmed that the expression of WRN decreased by tobacco smoke extract was increased.

Therefore, 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal of the present invention may increase the expression of ALDH2, AlDH3A1 and WRN to inhibit or prevent cell death and damage caused by tobacco. have.

The pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease of the present invention may further include an appropriate carrier, excipient or diluent commonly used in the manufacture of the pharmaceutical composition. Specifically, the pharmaceutical composition is each 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 to be used. can In the present invention, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate , calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one of 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal. The above excipients, for example, are prepared by mixing starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are simple diluents commonly used for suspensions, solutions, emulsions, syrups, etc. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents 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, glycerogelatin, and the like can be used.

The content of the 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal included in the pharmaceutical composition of the present invention is not particularly limited thereto, but 0.0001 to 50 weights based on the total weight of the final composition %, more preferably 0.01 to 20% by weight.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" of the present invention means to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention. means an amount sufficient to The duration of treatment may be determined according to factors including drugs used in combination with or concurrently with the composition of the present invention and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects.

The dosage of the pharmaceutical composition of the present invention may be, for example, 0.1 to 2,000 mg/kg body weight of the pharmaceutical composition of the present invention to mammals including humans per day. The frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or administered several times in divided doses. The above dosage does not limit the scope of the present invention in any way.

As another embodiment for achieving the above object, the present invention is chronic obstructive pulmonary disease comprising administering the pharmaceutical composition to an individual likely to develop or develop chronic obstructive pulmonary disease in a pharmaceutically effective amount A method for preventing or treating a disease is provided.

The 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal is the same as described above.

As described above, the 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal provided in the present invention can be used as an active ingredient in a pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease. , The composition may be used to prevent or treat chronic obstructive pulmonary disease.

As used herein, the term "individual" includes, without limitation, mammals, including rats, livestock, and humans, that are likely to or have developed chronic obstructive pulmonary disease.

In the method of treating chronic obstructive pulmonary disease of the present invention, the administration route of the pharmaceutical composition may be administered through any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, intrapulmonary administration, rectal administration, etc. can be administered through In addition, the composition may be administered by any device capable of transporting the active agent to a target cell.

In another aspect, the present invention provides a food composition for preventing or improving chronic obstructive pulmonary disease comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient.

The 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal and chronic obstructive pulmonary disease are the same as described above.

When the composition of the present invention is used as a food composition, the 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal may be added as it is, or it may be used together with other foods or food ingredients. It can be used appropriately depending on the method. In addition to the active ingredient, the composition may include a food additive that is pharmaceutically acceptable, and the mixing amount of the active ingredient may be suitably determined according to the purpose of use (prevention, health or therapeutic treatment).

The term "food supplement additive" used in the present invention refers to a component that can be added to food as an auxiliary, and is added to the manufacture of health functional food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverages, etc., but the above examples are not limited to the type of food supplement additive of the present invention.

The food composition of the present invention may include a health functional food. The term "health functional food" as used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful in the human body. Here, the term 'functionality' refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and during the manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, if the formulation of the health functional food is also recognized as a health functional food, it can be prepared without limitation. The composition for food of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur during long-term administration of the drug using food as a raw material, and has excellent portability, and the present invention health functional food can be taken as a supplement to enhance the treatment effect of chronic obstructive pulmonary disease.

In addition, there is no particular limitation on the type of health functional food in which the composition of the present invention can be used. In addition, the composition comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal of the present invention as an active ingredient is known with other suitable auxiliary ingredients that may be included in health functional foods according to the selection of those skilled in the art. It can be prepared by mixing the additives of Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes and the like, and may include all health functional foods in a conventional sense, and may include foods used as feed for animals.

The present invention relates to a composition for the treatment, prevention or improvement of chronic obstructive pulmonary disease comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient. -Hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal inhibits apoptosis of bronchial epithelial cells induced by tobacco smoke and increases the expression of ALDH2, ALDH3A1 or WRN to protect cells Thus, it can be usefully used in the development of a therapeutic agent or an ameliorating agent for chronic obstructive pulmonary disease.

1 is a diagram showing that plum extract (JAE) increases WRN and ALDH proteins in immortalized human bronchial epithelial cells. (A) HBEC2 cells were cultured in the absence or presence of 0, 2, 4 and 6% tobacco smoke extract (CSE) for 24 h after treatment with JAE (0, 1, 2 and 4 mg/mL) for 24 h. . Cell viability was analyzed by MTT analysis. (B) Immortalized HBEC2 cells were treated with JAE (0, 1, 2 and 4 mg/mL) for 24 h and then cultured in the presence or presence of 1.5% CSE for 24 h. Immunoblot analysis was analyzed for WRN, ALDH2 and ALDH3A1 proteins. Data for two independent experiments using three triplicate samples are expressed as mean ± SEM.
2 is a view showing the purification process of FA-1 [5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal] from plum extract (JAE).
3 is a diagram showing the structure determination of FA-1 [5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl) acetal]. (A) ESI-TOFMS data of FA-1, (B) 1H NMR data (chemical shift, ppm; JH,H values, Hz), (C) 13C NMR data (chemical shift, ppm) and (D) HMBC correlation is a relationship
4 is a diagram showing that FA-1 increases ALDH and WRN proteins in a dose dependent manner and protects against tobacco smoke-induced cytotoxicity (MTT and flow cytometry) in immortalized human bronchial epithelial cells. (A) Expression analysis of ALDH2, ALDH3A1, and WRN as a result of treatment with FA-1 [5, 25, 50, 75 and 150 nM in DMSO (dimethylsulfoxide)] or vehicle (corresponding to 1.0% volume of DMSO) Then, HBEC2 cells were cultured. (B) MTT assay showed that HBEC2 cells were incubated with NAC (2 mM), FA-1 (150 nM) and vehicle in the presence or absence of 0 and 6% CSE for 24 h and assayed for viability. Data are expressed as mean ± SEM (**p<0.01) cytotoxicity in cultured immortalized human bronchial epithelial cells. (C) As a result of flow cytometry, annexin V and propidium iodide (PI) staining were performed and apoptosis was analyzed.
5 is a diagram showing that FA-1 attenuates tobacco smoke-induced oxidative damage and DNA damage in HBEC2 cells. HBEC2 cells were incubated with FA-1 (150 nM) in the presence or absence of 2% CSE for 24 h, and immunoblot analysis was performed for phosphorylation of 4-HNE and ATM.
6 is a diagram showing that FA-1 increases ALDH2 and WRN proteins in a dose-dependent manner and attenuates smoke-induced down-regulation of WRN proteins in normal human epidermal keratinocytes.
(A) ALDH2 and WRN immunoblot results. NHEK cells were cultured after treatment with various concentrations of FA-1 [5, 25, 50, 75 and 150 nM in DMSO (dimethylsulfoxide)) or vehicle (corresponding to 1.0% volume of DMSO).
(B) The results of immunoblot analysis of attenuation of tobacco smoke-induced downregulation. NHEK cells were incubated with FA-1 (150 nM) and vehicle in the presence or absence of 2% CSE for 24 h.
7 is a diagram showing that FA-1 prevents tobacco smoke-induced cytotoxicity and DNA damage in normal human epidermal keratinocytes. (A) NHEK MTT (3-(4,5-dimethylthia) after treatment with FA-1 [150 nM in DMSO (dimethylsulfoxide)) for 24 h in the presence or absence of 0, 2, 4 and 6% CSE Zol-2-yl)-2,5-diphenyl tetrazolium bromide)-based cell viability. Data are expressed as mean ± SEM (**p<0.01) cytotoxicity in cultured NHEK. (B) NHEK cells were treated with 150 nM of FA-1 for 24 h in the presence or absence of 2% CSE. Immunoblot analysis was performed for phosphorylation of ATM and H2AX.
8 is a cell viability analysis result for tobacco smoke extract-induced cytotoxicity of fractions F1 to F4 obtained by refractionation of the chloroform fraction.
9 to 14 are structural analysis results of the isolated active compound FA-1. Specifically, 9 is a 1H NMR spectrum, FIG. 10 is a 13C NMR spectrum, FIG. 11 is a COSY spectrum, FIG. 12 is a TOCSY spectrum, FIG. 13 is an HMBX spectrum, and FIG. 14 is a HSQC spectrum result.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

<Materials and Methods>

1. Preparation of reagents and antibodies

Chemicals were purchased from Sigma Aldrich, and proteinase and phosphatase inhibitors were purchased from Thermo Scientific. Phosphorylated ATM (serine 1981), ALDH2, and ALDH3A1 antibodies were purchased from Abcam, phosphorylated H2AX antibody from Cell signaling, 4-HNE antibody from R&D Systems, and β-actin from Sigma-Aldrich.

2. Isolation and Purification of FA-1

The concentrated plum ( Prunus mume ) extract was purchased from Fysee Inc. in Korea and stored at -20°C or lower.

FA-1 was isolated as shown in FIG. 2 . 250 g of the concentrated plum extract was dissolved in 1 L of distilled water, and 2 L of ethanol was added to precipitate and remove polysaccharide. Then, n-hexane was added to separate the hexane fraction, and then chloroform was added to separate the chloroform fraction to obtain a chloroform fraction (9.26 g). Next, four subfractions (F1, F2, F3 and F4) were prepared from the chloroform fraction using an ODS column. The cytoprotective effects of these four subfractions (F1 to F4) on tobacco smoke extract-induced cytotoxicity were analyzed. As a result of the analysis, it was confirmed that the F2 fraction had the highest protective effect against tobacco smoke extract (CSE)-induced cytotoxicity (FIG. 8).

To separate the active compound from the chloroform fraction, centrifugation chromatography was performed using two phases consisting of n-hexane : ethyl acetate (EtOAc) : methanol (MeOH) : water = 3: 7: 5: 6 (v/v). Graphography (CPC) was performed. To purify the active compound, the CPC eluent (F2, 815.25 mg) was subjected to preparative ODS thin layer chromatography (TLC) using 50% and 60% methanol (MeOH) solvents, monitored under UV (280 nm), FA-1 (18.53 mg) was successively isolated and purified from the F2 fraction.

3. Structural Analysis of FA-1

Since FA-1 provides effective protection against tobacco smoke extract (CSE) induced cytotoxicity, the structure of the FA-1 compound was analyzed. NMR spectra were recorded on an Agilent 600 MHz NMR spectrometer (Agilent Technologies, Santa Clara, USA) in 0.4 ml of CD _{3 OD at 20°C.} Spectra were referenced to the residual solvent signal with resonance at δH/C = 3.30/49.8 ppm (CDOD). Signals were assigned based on analysis of COSY, HSQC and HMBC spectra. MS analysis was recorded on ESI-TOFMS (micrOTOF-Q II, Bruker, Billerica, MA, USA).

4. Manufacture of tobacco smoke extract

Tobacco smoke extract (CSE) solution was prepared by purchasing a research cigarette (3R4F) from the University of Kentucky. The CSE solution was prepared by modifying the Blue and Janoff method. After putting 10 ml of sterile DMEM medium into a 50 ml plastic syringe, 40 ml of cigarette smoke was drawn into the syringe and mixed with the medium by vigorous shaking. Thereafter, it was filtered through a 0.22 μm filter to remove large particles to prepare a tobacco smoke extract. The tobacco smoke extract was designated as 100% concentration, and the tobacco smoke extract was used immediately after preparation.

5. Cell Culture and Cell Viability Analysis

Immortalized human bronchial epithelial cells (HBEC2) were provided by Dr. Shay and Minna of Southwestern Medical Center, Dallas, Texas.

Normal human epidermal keratinocytes (NHEK) were supplemented with 5 μg/L human recombinant epidermal growth factor and 50 mg/L bovine pituitary extract (Invitrogen) in plates coated with FNC coating mix (Athena ES). It was cultured in a medium free of keratinocyte serum.

All cells were grown under standard incubator conditions of _{5% CO 2} at 37°C. Experiments were performed in 12-well and 6-well tissue culture plates at a starting cell density of 10 x 10 ³ /cm ² .

Cell viability assay is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT) was measured and analyzed. HBEC2 and NHEK cells were cultured in 12-well plates for 24 hours and treated with 150 nM FA-1 dissolved in DMSO for 24 hours. After treatment for 24 hours, cells were exposed to Cigarette smoke extract (CSE) at a concentration of 2%, 4% or 6% for 24 hours. Absorbance was measured at 540 nm, and the relative cell viability of the tobacco smoke extract-treated cells compared to the vehicle control cells not treated with the tobacco smoke extract was measured.

6. Flow Cytometric analysis

Apoptosis was analyzed using flow cytometry. After treatment with FA-1 (150 nM) and exposure to tobacco smoke extract (CSE), cells were harvested by trypsinization. 10 ⁵ cells using 10 µL of propidium iodide (PI) and 5 µL of Annexin V-FITC in 1x binding buffer (0.01 M HEPES, pH 7.4; 0.14 M NaCl; 0.25 mM CaCl _{2 )} was dyed.

Cells were incubated at room temperature for 15 min in the dark and the percentage of FITC- and PI-positive cells was quantified using a FACS Canto-II flow cytometer and analyzed using Flowjo software (version 7.6.3).

7. Immunoblot Analysis

After treatment with FA-1 (150 nM) and exposure to tobacco smoke extract, cells were lysed using 1X RIPA buffer (Sigma-Aldrich) containing proteinase and phosphatase inhibitor. seafood was obtained. The protein concentration of the cell lysate was measured with a BCA protein assay reagent. Protein samples were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% nonfat dry milk (TBS solvent containing 0.5% Tween-20), and incubated with primary and secondary antibodies. Visualized with enhanced chemiluminescence reagent (Advansta). The blot was stripped and reprobed with an antibody against β-actin to confirm equal loading of each protein.

Relative quantification of signal intensity was determined using Image Lab software (Bio-Rad, Hercules, CA, USA) and expressed as relative concentrations.

8. Statistical Analysis

For all comparisons involving multiple treatment groups, treatment effects were confirmed using one-way analysis of variance (ANOVA). The effect of individual treatments was evaluated using the p-value based on the calculated contrast. The results were graphically expressed as mean ± SEM, and p<0.05 was considered statistically significant.

<Result>

1. Cytoprotective effect of plum concentrate against tobacco smoke extract-induced cytotoxicity

To confirm the cytotoxic protective effect of plum extract against tobacco smoke extract, cell viability and changes in WRN, ALDH2, and ADLH3A1 protein expression were analyzed.

1 is a diagram showing the increase in WRN, ALDH2, and ADLH3A1 proteins in human bronchial epithelial cells of Japanese apricot extract (JAE).

HBEC2 cells were treated with a plum extract at concentrations of 0, 1, 2 and 4 mg/mL for 24 hours, and then treated with 0, 2, 4 and 6% concentrations of tobacco smoke extract (CSE) for 24 hours and cultured. The cells were analyzed for cell viability by MTT analysis. As a result of the analysis, it was confirmed that the plum extract protected the cells from cytotoxicity caused by the tobacco smoke extract at all concentrations (FIG. 1A).

In a previous study, the present inventors reported that cigarette smoke induces cell aging and cell migration disorders through downregulation of Werner's syndrome protein (WRN). Therefore, preservation of Werner's syndrome protein (WRN) may have therapeutic effects in smoking-related diseases.

HBEC2 cells were incubated with JAE (1,2 and 4 mg/mL) or vehicle (corresponding to 1.0% volume of sterile H ₂ O), and also the effect of JAE on ALDH2, ALDH3A1 and WRN proteins was measured in vitro. did. Plum extract increased the expression of ALDH2, ADLH3A1 and WRN proteins in a dose-dependent manner ( FIG. 1B ).

That is, it was confirmed that plum extract (JAE) protects HBEC2 cells from tobacco smoke extract-induced cytotoxicity and increases ALDH2, ALDH3A1 and WRN proteins in HBEC2 cells.

2. Determination of the structure of FA-1.

Since FA-1 provides effective protection against tobacco smoke extract (CSE) induced cytotoxicity, the structure of the FA-1 compound was analyzed. 3 is an NMR analysis result of FA-1. ¹ H NMR, ¹³ C NMR spectra and the main HMBC correlation of FA-1 are shown in FIG. 3 . FA-1 has the molecular formula C ₁₈ H ₁₆ O ₈ (C ₁₈ H ₂₀ NO ₈ + 378.1183 [M + NH4] ⁺ m/z 378.1188 calculated for ESI-TOFMS) ( FIG. 3A ).

The structure of this compound was revealed as a trimer of 5-hydroxymethyl-2-furaldehyde based on their NMR spectra (1 H NMR, 13 C NMR, COSY, HSQC and HMBC) (Supplementary Figures S2-9). In the 1H NMR spectrum, two sets of similar signals with an integral ratio of almost 2:1 were displayed. Two sets of these signals are conjugated aldehyde signals (δC 178.1, δH 9.54, s), J values of 3.2 and 3.5 Hz, two sets of olefinic methine protons binding to each other, and two pairs of olefinic quaternary carbons (δC 151.8/157.4 and 155.1). /160.2), non-equivalent (δH 4.67, 4.69) and equivalent (δH 4.47) hydroxylmethyl proton signals were analyzed as 5-hydroxy-2-furaldehyde backbones. Also, one acetal signal (δC 98.1, δH 5.81) was HMBC correlated with both furan C2 and non-identical hydroxymethyl signals. The structure of this active compound, along with other major HMBC interactions shown in Figure 3d, was identified as 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal and designated FA-1. The assignment of 1H and 13C NMR signals is shown in FIGS. 3B and C, and the NMR spectra are shown in FIGS. 8 and 9 .

3. The bronchial cytoprotective effect of 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal (FA-1) and the increase in ALDH and WRN proteins

Next, in order to analyze the cytoprotective effect of 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal (FA-1) isolated from plum extract on the tobacco smoke extract-induced toxicity, human bronchial epithelium Cell viability of cells (HBEC2) and changes in ALDH and WRN protein expression were analyzed by immunoblot.

5-Hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal [5, 25, 50, 75 and 150 nM in DMSO (dimethylsulfoxide)] or vehicle (corresponding to 1.0% volume of DMSO) After treatment with various concentrations of HBEC2 cells were cultured. FA-1 increased ALDH2, ADLH3A1 and WRN protein expression in a dose-dependent manner ( FIG. 4A ).

That is, the 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal enhances ALDH2, ALDH3A1 and WRN proteins in HBEC2 cells, thereby enhancing 5-hydroxymethyl-2-furaldehyde bis(5- It was confirmed that the effect of formylfurfuryl)acetal was the same as that of the plum extract (JAE).

Therefore, in subsequent experiments, FA-1 at a concentration of 150 nM was determined and used. In addition, to determine the effect of FA-1 on tobacco smoke extract (CSE) induced cytotoxicity, HBEC2 cells were treated with 2 mM N-acetyl-L-cysteine (NAC) in vehicle in the presence or absence of 6% CSE for 24 h. ) or after incubation with 150 nM of FA-1, cell viability was analyzed. As a result of the analysis, it was confirmed that 2 mM NAC had no protective effect against 6% tobacco smoke extract (CSE)-induced cytotoxicity in HBEC2. On the other hand, FA-1 showed a cytoprotective effect against 6% tobacco smoke extract (CSE)-induced cytotoxicity (FIG. 4B).

Next, propidium iodide (PI) and Annexin V staining and flow cytometry were performed to protect the cytoprotective effect of FA-1 on tobacco smoke extract (CSE)-induced apoptosis. was analyzed. As a result of the analysis, it was confirmed that 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal reduced tobacco smoke extract (CSE)-induced apoptosis ( FIG. 4C ).

That is, it was found that 150 nM of 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal increased ALDH and WRN proteins and protected against tobacco smoke extract (CSE)-induced apoptosis in HBEC2 cells. can

4. Weakening effect on oxidative damage and DNA damage of FA-1 in human bronchial epithelial cells

To determine the effect of FA-1 on inhibiting tobacco smoke extract-induced oxidative damage and DNA damage in human bronchial epithelial cells, we analyzed changes in 4-HNE and phosphorylated ATM expression.

Cigarette smoke (CS) exposure is known to increase the protein of 4-HNE, a marker of oxidative stress and DNA damage, as evidenced by increased phosphorylation of ATM. To identify FA-1 against cigarette smoke (CS)-induced oxidative stress and DNA damage, HBEC2 cells were treated with 150 nM FA-1 in the presence or absence of 2% CSE, incubated for 24 hours, and then immunoblotted. was performed to analyze the expression of 4-HNE and phosphorylated ATM.

As a result of the analysis, it was found that FA-1 attenuated the accumulation of 4-HNE protein and the phosphorylation of ATM in CSE-exposed HBEC2 cells ( FIG. 5 ). That is, it can be seen that FA-1 protects against tobacco smoke extract (CSE)-induced oxidative stress and DNA damage in cultured HBEC2 cells.

5. Regulation of ALDH2 and WRN protein expression of FA-1 in human epidermal keratinocytes

To determine the effect of FA-1 on ALDH2 and WRN proteins, various concentrations of FA-1 [5, 25, 50, 75 and 150 nM in DMSO (dimethylsulfoxide)] or vehicle (in 1.0 vol% DMSO) NHEK cells were cultured after treatment with the corresponding). FA-1 enhanced ALDH2 and WRN protein expression in NHEK cells in a dose-dependent manner ( FIG. 6A ). Due to the low production of ALDH3A1 protein in NHEK cells, ALDH3A1 was not detected. That is, it was confirmed that FA-1 protects against tobacco smoke extract (CSE)-induced cytotoxicity, improves ALDH2 and WRN proteins in HBEC2 cells, and the effect of FA-1 is the same as that of plum extract (JAE).

To determine the effect of FA-1 on tobacco smoke extract (CSE)-induced down-regulation of WRN protein, HBEC2 cells were cultured with FA-1 (150 nM) or vehicle in the presence or absence of 1.5% CSE. FA-1 attenuated CS-induced down-regulation of WRN protein in cultured HBEC2 cells ( FIG. 6B ). That is, it was confirmed that FA-1 preserves WRN protein expression and provides resistance to CS-induced downregulation.

6. Tobacco smoke extract (CSE)-induced cytotoxicity and DNA damage protective effect of FA-1 in human epidermal keratinocytes.

To determine the effect of FA-1 on tobacco smoke extract (CSE)-induced cytotoxicity, NHEK cells were treated with FA-1 [DMSO (dimethylsulfoxide) After treatment with 150 nM), cell viability was analyzed by MTT analysis. As a result of the analysis, it was confirmed that FA-1 had a protective effect by increasing the cell viability in a concentration-dependent manner against tobacco smoke extract (CSE)-induced cytotoxicity (FIG. 7A).

Smoking activates DNA damage responses (DDRs) mediated by phosphoinositide 3-kinase-associated protein kinase (PIKK), including ataxia teleangiectasia mutated (ATM). Upon formation of DNA double-strand breaks (DSBs), ATM is activated by autophosphorylation at the serine 1981 residue and phosphorylates the serine 139 residue (γH2AX) of the H2AX variant at the chromatin proximal DSB site.

Therefore, in order to confirm the effect of inhibiting DNA damage induced by tobacco smoke extract (CSE), changes in the phosphorylation levels of ATM and H2AX related to DNA damage were analyzed. NHEK cells were treated with 150 nM of FA-1 for 24 h in the presence or absence of 2% CSE. Immunoblot analysis was performed for phosphorylation of ATM and H2AX. FA-1 reduced tobacco smoke extract (CSE)-induced phosphorylation of ATM and H2AX ( FIG. 7B ). That is, it was confirmed that FA-1 protects against tobacco smoke extract (CSE)-induced cytotoxicity and DNA damage in cultured NHEK cells.

Claims (5)

A pharmaceutical composition for treating chronic obstructive pulmonary disease, comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal. The pharmaceutical composition according to claim 1, wherein the 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal is isolated from plum. The pharmaceutical composition according to claim 1, wherein the 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal increases the expression of ALDH2, ALDH3A1 or WRN. A food composition for preventing or improving chronic obstructive pulmonary disease, comprising 5-hydroxymethyl-2-furaldehyde bis(5-formylfurfuryl)acetal as an active ingredient. The food composition for preventing or improving chronic obstructive pulmonary disease according to claim 4, wherein the food composition is a health functional food.

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