KR20150068186A - Compositions for preventing or treating tuberculosis comprising ajoene - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating tuberculosis, which contains ajoene as an active ingredient. According to the present invention, ajoene, if applied to a cell which is infected with Mycobacterium tuberculosis, is able to induce phosphorylation of CHOP, Bip/GRP78 and eIF2α or to induce an endoplasmic reticulum stress by increasing active oxygen. The increased endoplasmic reticulum stress can induce an apoptosis of infected cells, thereby inhibiting the growth of Mycobacterium tuberculosis. Accordingly, an ajoene compound of the present invention can be used as a more effective therapeutic agent for treating tuberculosis.

Description

[0001] The present invention relates to a composition for preventing or treating tuberculosis containing azoenes,

The present invention relates to a composition for preventing or treating tuberculosis containing azoenes, and more particularly to a composition for preventing or treating tuberculosis containing azoenes which induce apoptosis of cells infected with Mycobacterium tuberculosis as an active ingredient .

Based on data from the World Health Organization in 2010, there were approximately 8.8 million patients with tuberculosis per year. In Korea, about 30% of the population is reported to be infected with tuberculosis, and the number of new patients is also increasing. Unlike the recognition that it is a disease of the backward country, Korea has the disgrace that it occupies the first place in both the mortality rate and the incidence rate among the OECD member countries (Disease Control Headquarters. In most cases, only 5-10% of infected people will develop tuberculosis. The use of attenuated Mycobacterium bovis BCG for the prevention of tuberculosis and the recent emergence of multidrug-resistant Mycobacterium tuberculosis and super Mycobacterium tuberculosis require research on the development of new anti-tuberculosis drugs and preventive vaccines. (Somoskovia A, et al., Respiratory research . 2001; 2 (3): 164-168). In addition, if the host's defense immune system fails to respond effectively to respiratory-infected tubercle bacilli, the tubercle bacilli will remain and proliferate in macrophages (Van Crevel R, et al., Clinical microbiology reviews . : 294-309).

Several natural products have been reported from the past that exhibit antifungal, antiviral, and antibacterial effects. Propolis, made by bees, is a typical example. The ethanol extracts of propolis were staphylococcus ( Staphylococcus aureus ) and the synergistic action of drug therapy in the treatment of multidrug resistant Mycobacteria ( Ipomoea) , one of morning glory species, Resin glycosides extracted from leptophylla have also been shown to have anti-TB effects (Barnes CC, et al., Journal of natural products . Nov 2003; 66 (11): 1457-1462).

Garlic has been used in many diseases in Egypt as well as its medicinal effect in Chinese medicine books 2000 years ago. Ajoene is one of the garlic extracts known as E-azoenes and Z-azo enantiomers (Kaschula CH, et al., BioFactors . Jan-Feb 2010; 36 (1): 78-85).

On the other hand, the endoplasmic reticulum (ER) is a cell organelle in which secretion or cell protein is synthesized, and functions as glycosylation, disulfide bonds, folding and assembly of proteins. The situation in which abnormal proteins accumulate in the endoplasmic reticulum due to insufficient protein folding function due to nutritional deficiency, changes in redox balance, calcium ion homeostatic degradation, post-translational modification or simple secretory protein synthesis is called " The response to this stress is called the unfolded protein response (UPR). This unfolded protein reaction inhibits protein synthesis and accumulation in the endoplasmic reticulum (Kaufman RJ. The Journal of clinical investigation . Nov 2002; 110 (10): 1389-1398). This function leads to the survival of cells by inducing the vesicle chaperone protein to prevent the accumulation of unfolded proteins and to maintain the homeostasis of the endoplasmic reticulum (Rath E, et al., European journal of nutrition . Jun 2011; 50 (4): 219-233). Molecules such as PERK (Pancreatic ER kinase), IRE1 (Inositol-requiring enzyme 1), and ATF6 (Activating transcription factor 6) are essential for the unfolded protein reaction of the endoplasmic reticulum. PERK and IRE1 are released by dimerization and are activated to activate the lower signal pathway. ATF6 is released from the endoplasmic reticulum and migrates to the Golgi body, which is activated through cleavage. Thus, the unfolded protein of the endoplasmic reticulum (Rutkowski DT, et al., PLoS biology . Nov 2006; 4 (11): e374). However, long-term sustained or excessive stress of these endoplasmic reticulum causes death through mitochondria-dependent or mitochondrial independent apoptosis pathways (Szegezdi E, et al., EMBO reports . Sep 2006; 7 (9): 880-885). Recently, it has been known that the endoplasmic reticulum stress is also related to inflammation. Inflammation is characterized by the production of large quantities of proteins such as cytokines and chemokines, and the unfolded protein response of the endoplasmic reticulum (NF-κB), JNK (c-Jun N-terminal kinases) receptors such as the production of reactive oxygen species (ROS) mediated by receptors (Deng J, et al., Molecular and cellular biology . Dec 2004; 24 (23): 10161-10168).

The inventors of the present invention confirmed that the azoene compound contained in the garlic extract can inhibit the growth of Mycobacterium tuberculosis by inducing the stress of the endoplasmic reticulum in the cells infected with the Mycobacterium tuberculosis.

Accordingly, it is an object of the present invention to provide a composition for treatment of tuberculosis containing azoin that inhibits the proliferation of Mycobacterium tuberculosis by inducing apoptosis of cells infected with Mycobacterium tuberculosis.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating tuberculosis containing an ajoene or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment of the present invention, the azoenes can be separated from garlic or can be prepared by organic synthesis.

In one embodiment of the present invention, the azoenes may inhibit the proliferation of Mycobacterium tuberculosis by inducing apoptosis of macrophages infected with Mycobacterium tuberculosis.

In one embodiment of the invention, the apoptosis can be induced through macrophage stress in macrophages.

In one embodiment of the present invention, said endoplasmic reticulum stress may be induced through phosphorylation of CHOP, Bip / GRP78 and eIF2a, and / or increase of reactive oxygen species.

In one embodiment of the present invention, the azoenes may reduce the survival rate of mycobacteria existing in the cells.

In one embodiment of the present invention, the tuberculosis is selected from the group consisting of tuberculosis, skin tuberculosis, adrenal tuberculosis, renal tuberculosis, tuberculosis tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, Tuberculosis, lymphadenopathy, pulmonary hypertension, breast tuberculosis or spinal tuberculosis.

According to the present invention, azoin can induce endoplasmic reticulum through phosphorylation of CHOP, Bip / GRP78 and eIF2a, and / or increase of reactive oxygen during treatment of cells infected with Mycobacterium tuberculosis, It is possible to induce apoptosis and inhibit proliferation of Mycobacterium tuberculosis. Therefore, the azone compound of the present invention can be used as a more effective therapeutic agent for tuberculosis.

FIG. 1 is a graph showing the inhibitory effect of azoin against intracellular mycobacterial cells on macrophage-infected M. tuberculosis.
FIG. 2 is a diagram illustrating that apoptosis is induced upon treatment with azoenes in macrophages infected with Mycobacterium tuberculosis.
FIG. 3 is a graph showing the phosphorylation of CHOP, Bip / GRP78 and eIF2? Associated with endoplasmic reticulum stress upon treatment with azoenes in macrophages.
FIG. 4 is a graph showing the production of reactive oxygen species that induces endoplasmic reticulum stress upon treatment with azoenes in macrophages.

The present invention provides a pharmaceutical composition for preventing or treating tuberculosis containing azoene as an active ingredient.

In the pharmaceutical composition of the present invention, the azoenes used as the active ingredient are represented by the following formula (1) or (2).

In the present invention, the azoene is a compound which is composed of two isomers of E-ajoene (Formula 1) and Z-ajoene (Formula 2) and can be separated from ground or crushed garlic. In the process of producing azoin from garlic, the alliinase stored in the garlic vacuole is liberated when the tissue is destroyed, converting the allyl to allylsulfenic acid and producing allysine from these two molecules. Allysine is converted to a stable compound azoenes by sulfoxide rearrangement.

The azoenes used in the present invention can be isolated from garlic by a known method or can be obtained by synthesis. For example, Hunter et al. [ Bioorg Med Chem Lett ., 2008, 18: 5277-5279], and can be produced and separated from garlic by the following method.

Specifically, for example, the following process is possible. The ground or garnished garlic or the filtrate obtained by filtration is allowed to stand at room temperature or at 37 ° C for 12 hours or longer to allow the alginin of garlic to pass through the allysine to form azoenes. Then, a polar solvent such as methanol or ethanol is added thereto, For 7 days, preferably 24 hours to 72 hours, at 10 to 100 DEG C, and the mixture is concentrated under reduced pressure. The concentrate is dispersed in water and fractionated with a solvent such as diethyl ether. From the obtained fractions, an azone-enriched fraction can be obtained through silica gel chromatography, and further chromatography can be carried out to separate pure azoenes.

The azoenes used in the present invention may also be commercially purchased and used as purely purified forms of compounds by conventional methods.

In the pharmaceutical composition of the present invention, the azoin inhibits the proliferation of Mycobacterium tuberculosis by inducing apoptosis of macrophages infected with Mycobacterium tuberculosis. In particular, apoptosis may be induced through macrophage stress in macrophages, which can be induced through the phosphorylation of CHOP, Bip / GRP78 and eIF2a, and / or the increase of reactive oxygen species.

According to the embodiment of the present invention, the effect of inhibiting proliferation of Mycobacterium tuberculosis was confirmed by inducing apoptosis through treatment with azoenes in macrophages infected with Mycobacterium tuberculosis (see Fig. 1). It has also been found that azoin reduces the survival rate of Mycobacterium tuberculosis bacteria present in cells infected with Mycobacterium tuberculosis (see Fig. 2). Therefore, it can be understood that the pharmaceutical composition containing azoenes of the present invention can be effectively applied to prevent or treat tuberculosis through reduction of the survival rate of Mycobacterium tuberculosis or death of cells infected with Mycobacterium tuberculosis.

According to another embodiment of the present invention, apoptosis of macrophages infected with M. tuberculosis due to treatment with azoenes has been shown to be induced by an increase in endoplasmic reticulum stress, and this endoplasmic reticulum stress increases the phosphorylation of CHOP, Bip / GRP78 and eIF2? (See Fig. 3). Also, it has been found that active oxygen, one of the important factors inducing ER stress, is caused by azoene treatment (see FIG. 4).

In this regard, the accumulation of unfolded proteins in the cell organellar endoplasmic reticulum due to various causes of cell stress causes the unfolded response (UPR) of the endoplasmic reticulum to fail to regulate the protein folding function. The Bip / GRP78 is a chaperone molecule present in the endoplasmic reticulum and binds to the unfolded protein to help protein folding, which increases the expression level during the initial endoplasmic reticulum stress response. The phosphorylation of eIF2a reduces the transcriptional process of protein synthesis and reduces the production of new proteins Bring to work to reduce protein accumulation overload of the endoplasmic reticulum. The present inventors have also reported that phosphorylation of eIF2? plays a very important role in the cell survival of Mycobacterium tuberculosis (Lim YJ et al., PLoS One 2011). When the UPR initiated by accumulation of the early unfolded protein is not adequately resolved, induction of excessive ER stress response induces apoptosis by the expression of CHOP, a sub-molecule of ER stress signal. By the expression of CHOP protein, (Lim YJ et al., PLoS One, 2011).

Accordingly, the pharmaceutical composition of the present invention can be used for the treatment of tuberculosis, skin tuberculosis, adrenal tuberculosis, renal tuberculosis, epididymal tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, , Pulmonary hypertension, breast tuberculosis and spinal tuberculosis.

When the azo compound, which is an active ingredient of the present invention, is manufactured from a pharmaceutical composition, it may contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier to be contained in the pharmaceutical composition of the present invention is one usually used in the present invention and may be selected from lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, no.

The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous infusion, subcutaneous injection, muscle injection, intraperitoneal injection, percutaneous administration, mucosal administration, and topical administration.

A suitable dose of the pharmaceutical composition of the present invention may be variously prescribed by factors such as the formulation method, the administration method, the age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient . Preferably, the dosage of the pharmaceutical composition of the present invention is 0.0001 to 100 mg / kg (body weight) on an adult basis.

The pharmaceutical composition of the present invention may be prepared in a unit dosage form by formulating it with a pharmaceutically acceptable carrier or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs Into a capacity container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example  1. Cell culture

The mouse-derived macrophage cell line, Raw 264.7 cells, was cultured in DMEM (Dulbecco's modified eagle's medium) containing 10% FBS, penicillin (100 IU / ml) and streptomycin (100 μg / ml). Cells (1 × 10 ⁵ cells / ml) were plated on a polypropylene tissue culture plate for attachment and cultured for 18 hours at 37 ° C in a 5% CO ₂ incubator. Bone marrow-drived macrophages (BMDM) were isolated from the femur and tibia of 6-8 week-old C57BL / 6 mice and cultured in medium containing M-CSF (macrophage colony-stimulating factor; 25 / / For 4 days.

Example  2. Azoen  compound

The azo compounds were prepared as follows. First, ground garlic (Allium sativum L., Garlic) was refluxed for 5 hours so that the active material could be eluted with 10 times the amount of alcoholic solvent, and then the alcoholic solvent extract was concentrated under reduced pressure. The concentrated fraction was suspended in 50-fold amount of distilled water and subjected to solvent fractionation three times with an equal amount of ethyl acetate as an organic solvent to obtain a crude extract containing the active substance. In order to separate the active substance of the crude extract obtained above, the RP-18 column was eluted with 500 ml of the developing solvent from 20% MeOH to 100% MeOH, and the eluate was concentrated under reduced pressure. To purify the concentrated active fraction, HPLC was carried out using 30% MeOH-40% MeOH as elution solvent on one column (Shiseido Capsule pack reverse phase C18 column; 10 x 250 mm size; particle size 5 μm) and elution rate of 1.8 ml / min to obtain 254 nm Gp-2-3 was isolated by measuring the absorbance at the wavelength of 1 GHz. To investigate the chemical structure of the separated Gp-2-3, 1 H-NMR spectra and 13 C-NMR spectra were measured and analyzed. As a result, (Ajoene). The extracted azoin was dissolved in ethanol (Merck) and DMSO (dimethyl sulfoxide, Bioniche Pharma), and each solution was prepared at a concentration of 10 mg / ml. Each extract was stimulated with Raw 264.7 cells and BMDM cells at an appropriate concentration, and DMSO and ethanol, which were used as solvents, were used as negative control, respectively.

Example  3. Samples and Drugs

Staurosporine (500 nM, 6 hr) was used as a positive control for apoptosis using tunicamycin (TM) (500 ng / ml, 6 hr) as a positive control for ER stress response. Lt; / RTI > H ₂ O ₂ (1 mM, 30 min) was used as a positive control for intracellular reactive oxygen measurement. As a negative control, DMSO used as a solvent was adjusted to the same concentration as the extract.

Example  4. Mycobacterial infections

A homogeneous suspension of Mycobacterium tuberculosis used for infection experiments was prepared by the following method. After incubation for about 3 weeks, Mycobacterium tuberculosis H37Rv (ATCC 27294) was inoculated on Ogawa medium. After incubation for 3 weeks, 10% OADC (oleic acid, albumin, dextrose, actalase), 5% glycerol, 0.05% Tween- (Middlebrook 7H9 broth), and cultured at 37 DEG C for about 7 weeks. Then, the cells were collected by centrifugation at 5000 rpm for 30 minutes. Cells were washed three times with phosphate-buffered saline (PBS) at 4 ° C. 50 ㎕ each of the collected cells was dispensed, and then stored at -70 캜.

Mycobacteria prepared by the above method were infected with macrophages for 3 hours by adjusting the number of multiplication of infection (MOI) to 1: 1 and washed twice with phosphate buffer solution. After incubating for an appropriate time in a 5% CO ₂ incubator at 37 ° C, cells or supernatants were collected and used in the experiments.

Example  5. Mycobacteria in the cell Survival  Measure

In order to confirm the viability of the Mycobacterium tuberculosis in the cells, the infected cells were cultured for the appropriate time by the above method, and then the medium was removed and washed twice with phosphate buffer solution. After 200 μl of sterile distilled water was added, the cells were allowed to react for 5 minutes to induce the release of Mycobacterium tuberculosis in the cells. After inoculation on a medium of Middlebrook 7H10 solid medium, the cells were cultured for about 14 days.

Example  6. Western Blot  analysis

The macrophages cultured in the above manner were treated according to the experimental conditions and cultured at 37 ° C in 5% CO ₂ Western blot analysis was carried out by collecting only the cells by centrifuging at 13,000 rpm for 10 seconds during the incubation in the incubator.

To the obtained cells were added 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 2 mM sodium fluoride, 2 mM EDTA, And dissolved in a RIPA buffer (radio-immunoprecipitation assay buffer) containing a protease inhibitor cocktail. After centrifugation at 13,000 × g for 10 minutes at 4 ° C., the protein in the upper layer was collected and the protein concentration was measured at 595 nm using a Bradford (250 μl) reagent. The protein amount of all samples was adjusted to 20 To 30 [mu] g.

The total volume was made equal using the sample buffer and the RIPA buffer, then heated at 95 ° C for 10 minutes and allowed to stand on ice for 15 minutes. Which was electrophoresed on a 12% or 15% SDS-PAGE gel at 80V for 30 minutes and at 120V for 1.5 hours. This SDS-PAGE gel was transferred to a PVDF membrane (polyvinyl difluoride membrane) under the condition of 90V for 70 minutes. TBS-T (tris-buffered saline tween; 20 mM tris, 137 mM sodium chloride, 0.1% tween-20, pH 8.0) for 1 hour at room temperature.

For the primary antibody reaction, each antibody was reacted at 4 ° C for 18 hours and then washed with TBS-T for 3 to 6 times for 10 minutes each. The secondary antibody reaction was performed using HRP (horseradish peroxidase) labeled anti-rabbit IgG (anti-rabbit IgG) antibody, anti-mouse IgG (anti-mouse IgG) goat IgG; Santa Cruz Biotechnology) antibody was diluted 2000-fold with TBS-T containing 5% skim milk, reacted at room temperature for 1 hour, and then washed 3 to 6 times with TBS-T for 10 minutes each. Protein expression bands were detected by exposure to X-ray film with ECL (Amercham-Pharmacia) solution.

Example  7. Measurement of active oxygen in the cell

The production level of intracellular reactive oxygen species (ROS) was measured using DHE (dihydroethidium) assay. After the antigens were treated according to the above method, macrophages were incubated for an appropriate period of time and stained with 2 μM of DHE at 37 ° C. for 30 minutes. The cells were washed three times with HBSS (Hank's balanced salt solution), fixed with 4% paraformaldehyde and analyzed for the level of active oxygen production using a FACS system (FACS Canto II system; BD Biosciences, USA).

Example  8. Cytotoxicity measurement using cell cycle analysis

Ajoene-treated macrophages were cultured at different time intervals, collected, centrifuged at 1500 rpm for 5 minutes, washed once with phosphate buffer solution, and washed with 70% ethanol for 30 minutes in a dark place on ice Respectively. After washing the cells with phosphate buffered saline twice, ethanol was removed and the cells were incubated with phosphate buffered saline containing 100 μg / ml of DNase-free RNase A (Sigma, St. Louis, Mo., USA) And then stained with PI (propidium iodide; Sigma) at a room temperature for 10 minutes. The cell cycle was analyzed using a FACS system. Each 10,000 single cells per sample was analyzed.

Statistical analysis ( StatisticalAnalyses )

All experiments were repeated 3 times independently and similar results were obtained. The results obtained from the experiments were expressed as mean and standard deviation. Statistical significance was verified using Student's t- test with the SPSS statistical program (version 11.5, Chicago, IL). p <0.05 was considered statistically significant.

Experimental Results 1. Azoen  It inhibits the proliferation of infected M. tuberculosis in macrophages.

Raw 264.7 cells and mouse BMDMs cells were prepared, and the cells were infected with Mtb H37Rv for 3 hours. Then, the cells were washed with phosphate buffer solution and incubated at various concentrations (1, 3, 5 μg / Ml) of azoin was treated and the cells were cultured for 24 hours. The cultured cells were collected, and the number of colonies of Mycobacterium tuberculosis was counted after inoculation into Middlebrook 7H10 solid medium and incubation at 37 ° C for about 3 weeks.

FIG. 1 is a graph showing the inhibitory effect of azoin against intracellular mycobacterial cells on macrophage-infected M. tuberculosis. As can be seen from FIG. 1, 64.6% at the concentration of 1 μg / ml of azoin, 51.2% at the concentration of 3 μg / ml, and 5 μg / ml at the concentration of 100% The survival rate was 41.0%. When BMDMs cells, primary cultured cells, were subjected to the same experiment, the cell survival rate was 77.7% at 1 ㎍ / ㎖, 68.4% at 3 ㎍ / ㎖ and 74.3% at 5 ㎍ / ㎖, respectively. These results indicate that the stimulation of azoenes against mouse macrophages reduces the survival of intracellular Mycobacterium tuberculosis.

These results indicate that the number of tubercle bacilli in the cells decreased significantly by treatment with azoenes. Although there is a slight difference in the reduction of BMDM and cell line Raw 264.7 cells, the number of Mycobacterium tuberculosis is significantly reduced in both cell types, .

Results 2. Azoen  Of macrophage infected with M. tuberculosis Apoptosis  .

Apoptosis plays an important role in the proliferation of intracellular infected M. tuberculosis. Therefore, in order to confirm whether treatment with azoenes induces apoptosis in mouse macrophages infected with Mycobacterium tuberculosis, the degree of apoptosis was analyzed by measuring cells corresponding to the sub-G1 phase in the cell cycle through cell cycle analysis.

FIG. 2 is a diagram illustrating that apoptosis is induced upon treatment with azoenes in macrophages infected with Mycobacterium tuberculosis. The number of cells corresponding to the sub-G1 phase increased with the concentration of azoenes, which was more noticeable in the cells treated with azoenes after infection with M. tuberculosis than with azone alone . Therefore, treatment of azoenes in mouse macrophages infected with Mycobacterium tuberculosis can be considered to be involved in the proliferation of Mycobacterium tuberculosis by inducing apoptosis.

Experimental results 3. Azoen  Effectively induces endoplasmic reticulum stress.

ER stress is closely related to apoptosis and is also known to be one of the host defense mechanisms against M. tuberculosis. Therefore, in order to confirm that azoenes induce ER stress, mouse macrophages Raw 264.7 cells and BMDMs cells were treated with 3 μg / ml of azoenes.

FIG. 3 is a graph showing the phosphorylation of CHOP, Bip / GRP78 and eIF2? Associated with endoplasmic reticulum stress upon treatment with azoenes in macrophages. In Fig. 3, phosphorylation of CHOP, Bip / GRP78 and eIF2? Also increased from 3 hours to 6 hours after stimulation with azoin. Therefore, it was confirmed that the stimulation of azoenes induced ER stress in mouse macrophage cell line Raw264.7 cell (A) and BMDM (B).

Results 4. Azoen  Thereby inducing the production of active oxygen.

The active oxygen is one of the important factors inducing the stress of the endoplasmic reticulum. The active oxygen is stimulated with azoene (3 μg / ml) and cultured for 15 minutes to 3 hours in order to confirm the generation of reactive oxygen species (ROS) After the cells were stained with DHE (dihydroethidium), the level of reactive oxygen species was measured using a flow cytometer.

FIG. 4 is a graph showing the production of reactive oxygen species that induces endoplasmic reticulum stress upon treatment with azoenes in macrophages. In Fig. 4, the production of active oxygen by azogenic stimulation increased from 15 minutes to 1 hour after treatment with azoenes, and the highest level of active oxygen was 30 minutes, which was 98.89% as compared with the negative control (50.97% To about 1.9 times. It can be seen that the azoenes increase the production of ROS.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (7)

A pharmaceutical composition for preventing or treating tuberculosis containing as an active ingredient azone or a pharmaceutically acceptable salt thereof.
2. The pharmaceutical composition according to claim 1, wherein the azone is separated from garlic.
The pharmaceutical composition according to claim 1, wherein the azoin inhibits proliferation of Mycobacterium tuberculosis by inducing apoptosis of macrophages infected with Mycobacterium tuberculosis.
The pharmaceutical composition according to claim 3, wherein the apoptosis is induced through macrophage stress in macrophages.
5. The pharmaceutical composition according to claim 4, wherein said endoplasmic reticulum stress is induced through phosphorylation of CHOP, Bip / GRP78 and eIF2a, or increase of reactive oxygen species.
2. The pharmaceutical composition according to claim 1, wherein the azoenes reduce the survival rate of mycobacteria existing in the cells.
The method of claim 1, wherein the tuberculosis is selected from the group consisting of tuberculosis, skin tuberculosis, adrenal tuberculosis, renal tuberculosis, epidemic tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, Wherein the tuberculosis is at least one selected from the group consisting of tuberculosis, lymphadenopathy, pulmonary hypertension, breast tuberculosis and spinal tuberculosis.

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