KR20180099543A - An antimicrobial composition comprising sialyllactose as an active ingredient - Google Patents

Abstract

The present invention relates to an antimicrobial composition against Klebsiella pneumoniae comprising sialyllactose as an active ingredient. The present invention relates to the antimicrobial composition comprising the sialyllactose as the active ingredient, wherein the antimicrobial composition has the antimicrobial activity, and promotes the phagocytosis of macrophages.

Description

An antimicrobial composition comprising cialyllactose as an active ingredient (sialyllactose as an active ingredient as an antimicrobial composition)

The present invention relates to a composition for antibacterial activity against Klebsiella pneumoniae comprising cialyllactose as an active ingredient, and more particularly to a composition for antimicrobial treatment against Klebsiella pneumoniae comprising α2,3-cialyl lactose or α2,6-cialyl lactose An antimicrobial composition for Klebsiella pneumoniae as an active ingredient, a composition for promoting the phagocyte action of macrophages against Klebsiella pneumoniae, and the like.

Direct or indirect damages caused by pathogenic microorganisms are causing economic, environmental and medical problems, and as interest in hygiene has increased recently, interest in antibacterial materials in foods, medical devices, medicines and fibers has been increasing . However, as the use of chemically synthesized antibiotics becomes more frequent, the incidence of resistance increases. Therefore, research on new antibiotics derived from natural materials is becoming an important issue.

For example, vancomycin-resistant S. aureus (VRSA), which is highly resistant to vancomycin, which is the last treatment for Staphylococcus aureus , the most common cause of human infection, (Centers for Disease Control), the so-called super-bacteria has become very popular. Since methicillin-resistant S. aureus (MRSA), which is only treated by vancomycin, has been in question since the 1970s, the vancomycin resistant Enterococcus (VRE) resistant to vancomycin in 1988 was first discovered in Europe And vancomycin intermediate-resistant S. aureus (VISA), a vancomycin resistant strain reported in Japan, the United States, France, and Korea in the late 1990s, caused a very serious problem of antimicrobial resistance And accordingly, development of antibiotics useful as natural products derived therefrom is urgently required.

Under these circumstances, the present inventors have made intensive efforts to find a new antibiotic. As a result, it has been found that a composition containing? 2,3-cialyl lactose or? 2,6-cialyl lactose is highly effective against Klebsiella pneumoniae It has been confirmed that a composition comprising the above-mentioned? 2,3-cialyl lactose or? 2,6-cialyl lactose can be effectively used as an antimicrobial composition, thus completing the present invention.

Korean Patent Registration No. 10-1725175 Korean Patent Registration No. 10-1773066

It is an object of the present invention to provide a composition for antibacterial activity against Klebsiella pneumoniae , which comprises cialyllactose as an active ingredient.

It is another object of the present invention to provide a composition for promoting phagocytosis of macrophages against Klebsiella pneumoniae comprising cialyllactose as an active ingredient.

This will be described in detail as follows. On the other hand, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. Further, the scope of the present invention is not limited by the detailed description described below.

One aspect of the present invention provides an antimicrobial composition for Klebsiella pneumoniae comprising cialyllactose as an active ingredient.

The term "cialyl lactose " in the present invention is known as a substance having the formula C ₂₃ H ₃₉ NO _19, molecular weight 633.5511 g / mol in the form of an oligosaccharide of sialic acid.

The cialyl lactose specifically includes 2,3-cialyl lactose, 3-cialyl lactose, 2,6-cialyl lactose, 6-cialyl lactose,? 2,3-cialyl lactose or? May be used in combination with lactose.

The 2,3-cialyl lactose or 2,6-cialyl lactose is a compound consisting of the following formula 1 or 2, respectively.

The present invention is not particularly limited to the method for obtaining cialyllactose, and it may be chemically synthesized by a method known in the art, or a commercially available substance may be used.

The cialyllactose of the present invention may exist in solvated as well as unsolvated forms. In addition, the cialyllactose of the present invention may exist in crystalline or amorphous form, and all such physical forms are included within the scope of the present invention.

The term "antibacterial" in the present invention means an activity of preventing or treating the infection of a bacterium by inhibiting the growth or proliferation of the bacterium containing the microorganism.

The cialyl lactose, or a composition containing the cialyl lactose, may have antibacterial activity against Klebsiella pneumoniae .

In the present invention, Klebsiella pneumoniae is a gram-negative staphylococcus, and corresponds to pathogens in the human intestinal tract and oral cavity. It is known that cephalosporin antibiotics are effective for the treatment of diseases caused by Klebsiella pneumoniae infection, but the antimicrobial activity of 2,3-cialyllactose or 2,6-cialyllactose against Klebsiella pneumoniae Is not known and was first identified by the present inventors.

In a specific example of the present invention, the antibacterial activity of cialyllactose against Klebsiella pneumoniae was examined. As a result, it was confirmed that cialyllactose promotes the phagocytosis of Klebsiella pneumoniae (FIG. 4 and FIG. 5), while it was confirmed that it had no antibacterial activity against other bacteria. Therefore, it was confirmed that cialyl lactose had a specific antimicrobial activity against Klebsiella pneumoniae strain.

In the present invention, the cialyllactose may increase the expression level of macrokinetic CXCL8 in macrophages.

The term " chemokine "as used herein refers to a basic heparin-binding low-molecular protein having a leukocyte lyophilising action and an activating action, and includes CXC (CXCL), CC (CCL), CX ₃ C (CX ₃ CL) XCL), and are now identified as more than 40 species. "Chymocaine CXCL8", also called interleukin 8 (IL8), is a chimokine produced by macrophages or epithelial cells and classified as CXC.

In one embodiment of the present invention, the treatment with cialyllactose in the macrophage group treated with LPS showed that the expression level of CXCL8 was increased about three times as compared with the group not treated with cialyllactose (see Fig. 2 ).

In the present invention, the cialyl lactose may increase the active oxygen content or lysosomal activity in macrophages.

In one specific embodiment of the present invention, in the macrophage group treated with LPS together with 2,3-cialyllactose and 2,6-cialyllactose, Rac protein producing reactive oxygen (ROS) (Fig. 8). It was confirmed that the activity of lysosomes in macrophages was increased (Fig. 8).

In the present invention, the cialyllactose may be one which promotes phagocytosis of macrophages.

Another aspect of the present invention provides a composition for promoting phagocytosis of macrophages against Klebsiella pneumoniae , which comprises sialyllactose as an active ingredient.

In the present invention, phagocytosis refers to a phenomenon in which macrophages receive and treat pathogens or foreign substances invading from the outside.

In one specific embodiment of the present invention, caffeicellum pneumoniae treated macrophage groups were treated with cialyllactose, indicating that cialyllactose activates the phagocytosis of macrophages against Klebsiella pneumoniae (Fig. 5).

In order to accomplish the above object, another aspect of the present invention provides a pharmaceutical composition for antibacterial use comprising cialyl lactose as an active ingredient. The antimicrobial pharmaceutical composition may have an antibacterial activity against Klebsiella pneumoniae .

The above-mentioned cialyl lactose, Klebsiella mennonii, and the antibacterial agent are as described above.

The composition comprising the cialyllactose of the present invention as an active ingredient can be usefully used as an antimicrobial pharmaceutical composition for inhibiting proliferation of Klebsiella species.

The pharmaceutical composition of the present invention may be used as a single agent, and may be prepared by using a pharmaceutical composition further comprising a pharmaceutical composition known to have an authorized antimicrobial effect.

The pharmaceutical composition of the present invention may be formulated into a pharmaceutical unit dosage form by adding a pharmaceutically acceptable carrier, excipient, or diluent.

The compositions of the present invention comprising a pharmaceutically acceptable carrier may be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition may be selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, ≪ / RTI >

In addition, the cialyllactose in the composition of the present invention may be contained in a pharmaceutically effective amount. The term "pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will vary depending on the species and severity, age, sex, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, As shown in FIG. Preferably, the cialyllactose is contained in the pharmaceutical composition in an amount of 0.001 to 200 μg / ml, more preferably 0.001 to 100 μg / ml.

In order to achieve the above object, another aspect of the present invention provides a quasi-drug composition for antimicrobial use comprising cialyl lactose as an active ingredient.

The above-mentioned cialyl lactose, Klebsiella mennonii, and the antibacterial agent are as described above.

The quasi-drug composition of the present invention may further contain a pharmaceutically acceptable carrier, excipient or diluent as necessary in addition to the above components. The pharmaceutically acceptable carrier, excipient or diluent is not limited as long as the effect of the present invention is not impaired, and examples thereof include fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, .

When the composition containing the cialyllactose of the present invention as an active ingredient is used as a quasi-drug, it may further contain one or more active ingredients exhibiting the same or similar functions. For example, a known antimicrobial component. The addition of an additional antimicrobial component may further increase the antimicrobial effect of the composition of the present invention. When the above components are added, the safety of compound use, easiness of formulation, and stability of effective ingredients can be considered. The quasi-drug composition may further comprise an antibacterial component known in the art.

The quasi-drug composition of the present invention can be exemplified by a disinfectant cleaner, a shower foam, a softener solution, a wet tissue, a coating agent, and the like. The formulation method, dosage, usage method, And the like.

In order to achieve the above object, another aspect of the present invention provides an antimicrobial food composition comprising cialyl lactose as an active ingredient.

The above-mentioned cialyl lactose, Klebsiella mennonii, and the antibacterial agent are as described above.

Since the food composition of the present invention contains a substance derived from a natural substance and can be taken on a daily basis, a high antimicrobial effect against Klebsiella pneumoniae can be expected, which is very useful.

The antimicrobial food composition of the present invention includes forms such as pills, powders, granules, infusions, tablets, capsules or liquid preparations. Foods to which the composition of the present invention can be added include, for example, various foods, For example, drinks, gum, tea, vitamin complex, and health supplement foods.

There are no particular restrictions on other components other than those containing the cialyl lactose or a physiologically acceptable salt thereof as an essential ingredient that can be contained in the antimicrobial food composition containing the cialyl lactose of the present invention as an active ingredient, Various herbal medicine extracts, food-aid additives, natural carbohydrates and the like as an additional ingredient.

The term " physiologically acceptable salt " of the present invention means a compound which is physiologically acceptable and which, when administered to an organism, does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, Quot; and " the "

Further, as mentioned above, the food-aid additive may be further added, and the food-aid additive includes food-aid additives customary in the art, for example, flavorings, flavors, colorants, fillers, stabilizers and the like .

Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. In addition to the above, natural flavoring agents (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used as flavoring agents.

In addition to the above, the antimicrobial food composition containing the cialyllactose of the present invention as an active ingredient may be used in various flavoring agents such as various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, Etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. In addition, it may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination.

In the present invention, the health supplement food includes health functional food, health food and the like. The term "functional food" as used herein is the same term as "food for special health use" (FoSHU). In addition to nutritional supplementation, functional foods are processed so as to efficiently show the biological control function, It means food. Here, the term "function (surname)" means that the structure and function of the human body have a beneficial effect for health use such as controlling nutrients or physiological action. The food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients which are conventionally added in the art. In addition, the formulations of the food can also be produced without restrictions as long as they are formulations recognized as food.

According to another aspect of the present invention for achieving the above object, there is provided an antibacterial method comprising treating a subject with a composition comprising cialyllactose as an active ingredient.

The above-mentioned cialyl lactose, Klebsiella mennonii, and the antibacterial agent are as described above.

The term "individual" as used herein may refer to any animal, including humans, that has been infected or susceptible to infection by Klebsiella pneumoniae. The animal may be, but is not limited to, a mammal such as a cow, a horse, a sheep, a pig, a goat, a camel, a nutrient, a dog, a cat,

The antimicrobial method of the present invention may specifically include administering or applying the composition in a pharmaceutically effective amount to a subject at risk of being infected or infected by Klebsiella spp.

The present invention includes administering a pharmaceutical composition comprising a cialyllactose in a pharmaceutically effective amount. It will be apparent to those skilled in the art that the appropriate total daily dose may be determined by the practitioner within the scope of sound medical judgment. In addition, it can be administered once or several times in divided doses. For purposes of the present invention, however, the specific therapeutically effective amount for a particular patient will depend upon the nature and extent of the reaction to be achieved, the particular composition, including whether or not other agents are used, the age, weight, Sex and diet of the patient, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used or concurrently used with the specific composition, and similar factors well known in the medical arts.

The present invention provides an antimicrobial composition comprising cialyllactose as an active ingredient, an antimicrobial composition having an excellent antimicrobial activity against Klebsiella pneumoniae strain, and a composition for promoting phagocyte action of macrophages.

1 is a graph showing the cytotoxicity of 2,3-cialyl lactose and 2,6-cialyllactose in THP-1 cells and Raw 264.7 cells.
FIG. 2 is a graph showing the expression levels of CXCL8 and TNF-? When THP-1 cells were treated with 2,3-cialyllactose and LPS alone or in combination. FIG.
FIG. 3 is a graph showing a comparison between the CXCL8 reporter vector (A) prepared using the CXCL8 promoter region containing the binding sequence of STAT, AP-1, and NF-κB and the luciferase expression gene (A), 2,3- And the degree of luciferase activity by LPS alone or in combination.
FIG. 4 shows a process for confirming the ability of 3-cialyllactose and 2,6-cialyllactose to promote the phagocytosis of Klebsiella pneumoniae .
FIG. 5 is a graph showing the ability of 2,3-cialyl lactose and 2,6-cialyllactose to promote phagocytosis of Klebsiella pneumoniae . (Unit of vertical axis: CFU; unit of horizontal axis: hour)
Fig. 6 shows the results of confirming the stimulation of phagocytosis of 2,3-cialyl lactose, 2,6-cialyl lactose, Staphylococcus aureus, and Streptococcus pseudopneumonia.
7 shows the phagocytotic activity of 2,3-cialyl lactose and 2,6-cialyl lactose through endocytosis verification of TLR4 receptor.
FIG. 8 is a diagram The effect of 2,3-cialyl lactose, 2,6-cialyl lactose on the phenomenon of Rac protein aggregation on the surface of macrophages and lysosomal activity in macrophages.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Materials and Experimental Methods

1-1. Cell culture (animal cell culture)

THP-1 (human mononuclear cells), HL-60 (neutrophil cell line) and A549 (human lung epithelial cells) were cultured in RPMI-1640 medium containing 10% FBS inactivated by heat and antibiotics and β - mercaptoethanol , Raw 264.7 mouse macrophages were cultured in heat-inactivated DMEM medium containing 10% FBS and antibiotics, and the cells were cultured in an incubator supplied with 5% CO ₂ at 37 ° C.

1-2. Test substances (reagents)

2,3-cialyl lactose and 2,6-cialyl lactose were obtained from Jin-Chem Co., Ltd., dissolved in distilled water and added to the cell culture medium.

1-3. WST assay (WST assay)

As a WST analysis, THP-1 cells and Raw 264.7 cells were seeded on a 96-well plate at a density of 5 x 10 < ³ > and cultured for a day. Then 2,3-cialyl lactose and 2,6- Cialyl lactose was treated at 0.1 uM, 1 uM, 10 uM, 100 uM, and 300 uM each for 24 hours. Cell viability was determined by measuring the OD value using a reader after placing the Ez-Cytox reagent in each well.

1-4. An enzyme-linked immunosorbent assay (ELISA)

THP-1 cells were seeded in 24-well plates at a density of 2 × 10 ⁵ cells per day, 2,3-cialyl lactose was pretreated for 3 hours, and LPS was treated for 24 hours. Cell culture supernatants were collected and placed in a 96-well plate coated with capture antibody of CXCL8 and reacted at room temperature for 2 hours. After addition of CXCL8 detection antibody and HRP for 1 hour at room temperature, TMB solution and Stop solution were added and fixed, and the degree of luminescence was measured using a reader.

1-5. Plasmid vector production

The CXCL8 promoter region containing the STAT, AP-1 and NF-κB binding sequences was amplified from the genomic DNA of the cells by PCR. The resulting PCR product was cloned into a TA vector containing the same restriction enzyme site as the pGL4-Lucifearase vector. The insert obtained from the TA vector was ligated to the Kpn I-Xho I restriction site of the pGL4 vector. The plasmid vector thus constructed was finally confirmed by sequencing.

1-6. Transfection and Luciferase Reporter Assay.

A549 cells were seeded in a 24-well plate for 8 × 10 ⁴ cells per well and plasmid expression vectors were transfected into cell lines using Attractene reagent according to the manufacturer's protocol. After that, 2,3-cialyl lactose was pretreated for 3 hours and LPS was treated for 24 hours. Lysis buffer was used to lyse the cells, luciferase reagent was added, and luciferase activity was measured using a luminometer.

1-7. The cialyl lactose, Klebsiella pneumoniae ( Klebsiella pneumoniae)  Confirming the ability to promote phagocytosis of bacteria

First, THP-1 cells were subcultured and 2 × 10 ⁶ / ml of water was dispensed into 12 well culture plates at 1 ml / well to prepare macrophage cells.

to the next, K. pneumoniae , LB broth was inoculated one day before infection, and then cultured in a shaking incubator at 37 ° C for 16 to 20 hours. The cultured K. pneumoniae was diluted to 2 x 10 ⁵ CFU in 20 μl of PBS and prepared for infection. The prepared bacteria were mixed with culture medium to adjust the number of bacteria to be 10, 50 MOI (multiplicity of infection) in the cultured THP-1 cells, and the number of internalization cells was confirmed by macrophages.

The concentration of 2,3-cialyl lactose and 2,6-cialyl lactose was adjusted to 100 nM to the cultured THP-1 cells to analyze the phagocytosis activity of the cells.

In addition, CFU (colony forming unit) was measured And to confirm the ability of the macrophage to grow. The cultured macrophage cell line, THP-1 cells were infected with K. pneumoniae , and after 0.5, 1, 2 and 3 hours, K. pneumoniae, which was not predated by macrophages, was removed by administration of the antibiotic gentamicin. At this time, the efficacy of K. pneumoniae was tested in the group administered with cialyllactose simultaneously. In order to confirm the number of microorganisms present in the cells by phagocytosis, macrophages were recovered and necrosis was performed, and colonies formed through CFU using agar plates were measured.

1-8. Identification of ability to promote phagocytosis of Staphylococcus aureus, Streptococcus pseudopneumonia

Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus aureus.

Specifically, Staphylococcus aureus and Streptococcus pseudomunoa were infected to THP-1 cells, which were cultured in the same manner as in Example 1-7, to give THP-1: bacteria = 1:50 (MOI 50) , And 3 hours later, gentamicin, an antibiotic, was administered to remove bacteria that were not predated by macrophages. At this time, the effect of the cialyl lactose on the bacterial ability of the bacteria in the group administered simultaneously was examined. In order to confirm the number of bacteria present in the cells by phagocytosis, macrophages were collected and necrosis was performed, and the number of bacteria in the cells was determined by colony generated through CFU using agar plates. And confirmed the number of bacteria that had arisen.

1-9. K. pneumoniae Endocytosis of Toll-like receptor 4, which is a receptor of Toll-like receptor 4,

The THP-1 cells treated with 2,3-cialyl lactose, 2,6-cialyl lactose and K. pneumoniae were recovered by time (0.5, 1, 2, 4 h) and washed with PBS. Paraformaldehyde ) On a slide. TLR4 antibody (sc-13593, Santa Cruz Biotechnology, Dallas, TX, USA) was reacted with a FITC-conjugated secondary antibody to stain TLR4 present on the cell surface, microscope, Carl Zeiss, Jena, Germany).

1-10. Identification of assembly phenomena and lysosomal activity on the cell surface of RAC-producing enzyme RAC

THP-1 cells were seeded on a 24-well plate at a density of 2 × 10, cultured for one day, 2,3-cialyl lactose and 2,6-cialyl lactose were each pretreated for 3 hours, Min, 120 min, 240 min. After washing the cells with PBS, the cells were fixed with paraformaldehyde without permeabilization separately to confirm only the Rac1 protein present on the cell surface. After blocking with 1% BSA, Rac1 antibody was added and reacted at room temperature for 1 hour. The secondary antibody conjugated with Alexa fluor 594 and DAPI were added and further reacted, and the stained cells were observed under an optical microscope (Rac1 localization).

In addition, the lysosomal activity measurement was performed using the LYSO-ID Red Detection Kit. Specifically, THP-1 cells were seeded on a 24-well plate at a density of 2 × 10, cultured for one day, 2,3-cialyl lactose and 2,6-cialyl lactose were each pretreated for 3 hours, Min, 60 min, 120 min. Cells were washed with assay buffer and Lyso-tracker dye was added and incubated at 37 ° C for 30 min. Cells were washed once more with assay buffer, and stained cells were observed under an optical microscope

Example 2: Confirmation of intracellular safety of cialyllactose

In order to confirm cytotoxicity of 2,3-cialyl lactose and 2,6-cialyl lactose, THP-1 cells and Raw 264.7 cells were seeded to a number of 5 × 10 ³ on a 96-well plate, Reilactose and 2,6-cialyl lactose were treated at 0.1 uM, 1 uM, 10 uM, 100 uM, and 300 uM for 24 hours, respectively, and WST assays were performed as in Example 1-3.

As a result, 2,3-cialyl lactose and 2,6-cialyl lactose showed no cytotoxicity at various concentrations (FIG. 1).

Example 3 Confirmation of Increase of Cytosine CXCL8 by Cialyl Lactose (Protein Analysis)

Expression of thymocyte in THP-1 cells was confirmed by enzyme immunoassay (ELISA) as in Example 1-4. THP-1 cells were pretreated with 2,3-cialyllactose for 3 hours, treated with LPS for 24 hours, and the expression level of CXCL8 and TNF-α was confirmed.

As a result, the expression of TNF-α was not significantly different from that of the LPS-only group, whereas the expression of CXCL8 was increased 3-fold in the 2,3-cialyllactose treatment group compared to the group treated with LPS alone synergistic effects were found (Fig. 2).

Therefore, it was confirmed that the cialyl lactose further increased the expression amount of the chemokine CXCL8 in the presence of LPS compared to the case of no LPS.

Example 4 Confirmation of Increase of Cytosine CXCL8 by Cialyllactose (Promoter Analysis)

Unlike Example 3, in which the expression of CXCL8 was confirmed by ELISA, the promoter activity was used to determine whether 2,3-cialyllactose increased the amount of CXCL8.

As in Example 1-5, the CXCL8 reporter vector was constructed using a gene recombination technique with the CXCL8 promoter region containing the binding sequence of STAT, AP-1, and NF-κB and the luciferase expression gene (FIG. 3A) . The prepared expression vector was injected into A549 cells, 2,3-cialyl lactose was pretreated for 3 hours, and LPS was treated for 24 hours to induce expression. Then, the activity of luciferase was measured and compared with that of CXCL8 The activation of the promoter was measured.

As a result of the experiment, it was confirmed that luciferase activity upon cialyllactose treatment was confirmed (FIG. 3B), and the effect of CXCL8 activity was higher in the group treated with cialyllactose than the group treated with LPS alone .

Example 5: Preparation of cialyllactose, Klebsiella pneumoniae ( Klebsiella pneumoniae)  Confirming the ability to promote phagocytosis of bacteria

The ability of the cialyllactose to promote the phagocytosis of Klebsiella pneumoniae was confirmed by the method described in Example 1-7 above.

As a result, when the number of K. pneumoniae bacteria was 1:10 (MOI 10) relative to macrophages, the phagocytic ability of macrophages was remarkably increased by addition of 2,3-cialyl lactose and 2,6-cialyl lactose for 1 hour , And it was confirmed that the predation ability of the cells was increased 4 to 5 times in the early stage (within 1 hour) through the colony formation unit (CFU) (FIG. 5).

In other words, it can be seen that the number of microorganisms existing in the cell through the activity of macrophages after 4 hours has been remarkably decreased in the predated microorganism after the microorganism existing in the cell has been removed over time.

From the above results, it can be seen that cialyl lactose has excellent antibacterial activity against Klebsiella pneumoniae strain.

Example 6 Identification of Stimulatory Action Promoting Ability for Staphylococcus aureus, Streptococcus pseudopneumonia

It was confirmed by the method described in the above Example 1-8 that the bacteria other than Klebsiella pneumoniae had the same antimicrobial activity.

Thus, the ability of cialyl lactose to stimulate phagocytosis of Staphylococcus aureus and Streptococcus pseudomunoa was confirmed. When the number of Staphylococcus pseudomunoa versus macrophages is 1:50 (MOI 50) relative to macrophages, the predatory ability of macrophages is higher than that of 2,3-cialyl lactose, 2,6-cialyl lactose As a result, it was confirmed that Staphylococcus aureus pneumoniae and Streptococcus pseudo-pneumoniae did not affect the phagocyte action (FIG. 6).

That is, the antibacterial activity of cialyl lactose varies depending on the kind of bacteria.

Example 7: K. pneumoniae Endocytosis of Toll-like receptor 4, which is a receptor of Toll-like receptor 4,

LPS-treated THP-1 cells recognize LPS through the TLR4 receptor and endocytosis occurs within the cell. One hour after the LPS treatment, most of the receptor TLR4 enters the cell and returns to the cell surface after 4 hours (endocytosis-exocytosis cycle).

Based on this, the phytopathogenic activity of Toll like receptor 4 was confirmed by endocytosis verification in Examples 1-9.

As a result, endocytosis of the TLR4 receptor was promoted in the cells to which cialyl lactose had been added, as compared to the THP-1 cell line treated with LPS. In the group treated with cialyllactose, endocytosis of the TLR4 receptor occurred within 30 minutes after the LPS treatment, and the time to return to the surface was also accelerated (Fig. 7).

From these results, it can be seen that the phagocytotic action of the macrophages on the Klebsiella spp. Strain is activated by cialyllactose.

Example 8: Confirmation of the assembly phenomenon and the lysosomal activity phenomenon on the cell surface of RAC which is an active oxygen (ROS) generating enzyme

The antimicrobial activity of cialyllactose against Klebsiella pneumoniae strain was confirmed by the method of Example 1-10.

As a result, according to FIG. 8, in the THP-1 treated with LPS, which is a surface factor of K. pneumoniae, the assembly of Rac protein to the cell surface was clearly confirmed after 60 minutes. On the other hand, in the THP-1 cell group treated with LPS together with 2,3-cialyl lactose and 2,6-cialyllactose, the phenomenon of aggregation of Rac protein on the cell surface appeared from 30 minutes. The Rac protein on the cell surface is a component of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase, which is an enzyme necessary for the production of ROS, and has an activity to rapidly remove ROS on the cell surface and to remove foreign bacteria and toxins. Bacteria and toxins invading from the outside migrate to endosomes in the cell due to phagocytosis, sterilized and removed by the ROS produced therein, and degraded by various enzymes in the lysosome.

The lysosomal activity of LPS-treated THP-1 was found to be prominent after 120 minutes, whereas LPS was treated with 2,3-cialyl lactose and 2,6-cialyl lactose , The lysosomal activity was evident at a time point of 60 minutes after a shorter time (FIG. 8).

From the above results, it was confirmed that cialyl lactose promotes the phagocytosis of macrophages against Klebsiella pneumoniae strain, rapidly increases the active oxygen content in macrophages, and lysosomal activity and thus exhibits excellent antimicrobial activity And

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (6)

A composition for antibacterial activity against Klebsiella pneumoniae , which contains sialyllactose as an active ingredient.
The composition according to claim 1, wherein the cialyl lactose is? 2,3-cialyl lactose or? 2,6-cialyl lactose.
2. The composition of claim 1, wherein the cialyl lactose increases the expression level of macrocytic CXCL8 in macrophages.
2. The composition of claim 1, wherein the cialyl lactose increases active oxygen content or lysosomal activity in macrophages.
The composition of claim 1, wherein the cialyl lactose promotes phagocytosis of macrophages.
A composition for promoting phagocytosis of macrophages against Klebsiella pneumoniae , which comprises sialyllactose as an active ingredient.

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