KR20200139458A - Antibacterial composition comprising methyl gallate and tylosin - Google Patents

Abstract

The present invention relates to an anti-bacterial composition comprising methyl gallate which inhibits the survival of Salmonella exposed to a macrolide anti-bacterial agent. The composition of the present invention has excellent anti-bacterial activity against pathogenic strains even at low concentrations compared to single use by mixing methyl gallate and tylosin in an optimal mixing ratio, that is, 1:10-10:1 weight ratio, and it has an excellent effect of remarkably inhibiting adhesion to host cells, invasion and intracellular survival without the problem of causing antibiotic-resistant bacteria. Therefore, the composition of the present invention can be used not only in medicine, but also in various fields such as feed addition, disinfection, cleaning and preservation.

Description

Antimicrobial composition comprising methyl gallate and tylosin {Antibacterial composition comprising methyl gallate and tylosin}

The present invention relates to an antimicrobial composition for inhibiting the survival of pathogenic strains exposed to macrolide-based antimicrobial agents, and more specifically to an antimicrobial composition comprising methyl gallate and tilosin.

Salmonella (Salmonella) is a facultative anaerobic bacteria belonging to gram-negative enteric bacteria and a bacillus which does not form a spore. Salmonella is a pathogenic microorganism that infects not only humans, but also various livestock including pigs, cows, chickens, etc., and causes diseases. It infects humans, causing food poisoning, and causing various salmonellosis in animals. Salmonella enterica is known as Salmonella Typhi, which causes typhoid fever by serological classification, Salmonella Typhimurium, which is the causative agent of Zut typhus, Salmonella Enteritidis, which is an enteritis bacteria , and poultry. serum contains species such as Salmonella Galina volume (Salmonella gallinarum) the causative agents of typhoid, chubaekri causative agent of salmonellosis flow column (Salmonella Pullorum), acute septicemia and salmonella can be a causative agent of cholera hog cholera device (Salmonella choleraesuis). Salmonella, depending on the serotype, contains a common source of infection that can cause diseases not only in humans but also in livestock. Salmonella Choleraesuis ( Salmonella Choleraesuis) is a fungus known as swine cholera because it can infect pigs and cause pig cholera. It is a fungus that infects both humans and animals. This fungus causes acute sepsis caused by Salmonella along with Salmonella Typhisuis, and the acute or chronic gastrointestinal infectious disease of pigs caused by Salmonella infection is called porcine paratyphoid, and is accompanied by gastroenteritis and sepsis. It mainly occurs during the fattening period. Diseases caused by this fungus are common seasonally, mainly in summer, and acute septicemia is common in young pigs aged 2 to 4 months. The main symptoms are elevated body temperature (41~42℃), loss of energy and loss of appetite, Cyanosis occurs around the ears and in the leg area, and most of them die within 2 to 4 days of onset. Acute enteritis occurs mainly in fattening livestock. Appetite is not constant. It is accompanied by severe watery diarrhea, high fever, weakness, pneumonia, and neurological symptoms. In severe cases, skin discoloration occurs. Chronic enteritis is when livestock is severely thin due to continuous diarrhea, and gradually changes from yellowish-white or grayish-white soft stool to gelatinous mucous diarrhea, and the diarrhea contains necrotic fragments of intestinal epithelial cells and sometimes contains blood. There is also. In Korea, it has been reported that the incidence of diseases caused by the above fungi is very low, but when a disease occurs, infectiousness and damage can be greatly observed. In addition, as in most cases where Salmonella is transmitted, contaminated feedstock, water, or adult pigs carrying the bacteria without any symptoms can be an important source of infection.

Currently, antimicrobial agents that are effective against Gram-negative strains are mainly used to prevent and treat Salmonella infection, but when salmonella infects an animal, it penetrates into cells, proliferates and infects, so antibacterial agents, drugs, and other probiotics penetrate and act. Is difficult, and the problem of antibiotic resistance has occurred in many fields recently, and the cause of this has been identified as an antibiotic misuse, and antibiotics have not been sufficiently used. In particular, when macrolide-based antimicrobial agents, which are antimicrobial agents used for respiratory bacteria, are administered to animals and humans, they are exposed to misuse of macrolide-based antibacterial agents in case of mixed infection or non-expressive infection with intestinal pathogenic bacteria. Intestinal pathogenic bacteria exposed to macrolides, especially Salmonella strains, have genetic or phenotypic changes, invasion into host cells, etc., making clinical treatment difficult and developing antimicrobial resistance.

Domestic patent application number: 10-1999-0013597 Domestic patent application number: 10-2008-0027279 Domestic patent application number: 10-2013-0122095

Therefore, the present inventors continued research to develop antibiotic resistance for obtaining antibiotic resistance of Salmonella strains unintentionally exposed to macrolide antibiotics and for controlling the survival of Salmonella in host cells.As a result, a certain ratio of tilosin and methyl gallate For the first time, it was confirmed that co-administration is a new co-administration agent capable of inhibiting the spread of antimicrobial resistant bacteria. Specifically, the present inventors, when combined with tilosine and methyl gallate in a mixing ratio of 1:10-10:1, inhibits the survival and biofilm formation of Salmonella strains without causing antibiotic resistance problems, and adheres to host cells and It was first confirmed that invasion was remarkably suppressed, and the present invention was completed by confirming that it has excellent antibacterial effects against various pathogenic strains as well as Salmonella.

Accordingly, an object of the present invention is to provide an antimicrobial composition comprising methyl gallate and tylosine, and a use thereof.

In order to achieve the above object, the present invention provides an antibacterial composition comprising methyl gallate and tilosin.

In addition, the present invention provides a composition for inhibiting intestinal adhesion or invasion of pathogenic strains containing methyl gallate and tilosin.

In addition, the present invention provides a combination preparation for preventing or treating infectious diseases caused by pathogenic strains containing methyl gallate and tylosine.

In addition, the present invention provides an antibacterial feed additive composition containing methyl gallate and tilosin.

In addition, the present invention provides an antibacterial disinfectant composition comprising methyl gallate and tilosin.

In addition, the present invention provides a detergent composition for antibacterial comprising methyl gallate and tilosin.

In addition, the present invention provides an antimicrobial antiseptic composition comprising methyl gallate and tilosin.

The composition of the present invention has excellent antimicrobial activity against various pathogenic strains, including Salmonella strains, even at a lower concentration than when used alone by using methyl gallate with tylosine, and adhesion and invasion into host cells without the problem of causing antibiotic-resistant bacteria And it has an excellent effect of remarkably inhibiting the intracellular survival, the composition of the present invention can be used in a variety of fields, such as feed addition, disinfection, cleaning and preservative as well as medicine.

1 is a diagram showing the MIC concentration (A) for Salmonella typhimurium strain (ATCC14028 strain) and the cell adhesion inhibitory activity (B) to Caco-2 cells according to the mixing ratio of methyl gallate and tilosin.
Figure 2 is a diagram showing the survival curves of Salmonella typhimurium ATCC14028 strain (A) and LVPP-STI15 strain (B) (MG: methyl gallate, Ty: tylosine, MT: mixture of methyl gallate and tylosine).
3 is a result of observing the membrane integrity of Salmonella typhimurium ATCC14028 strain with a confocal microscope (MG: methyl gallate, Ty: tilosin, MT: mixture of methyl gallate and tilosin).
4 is a result of observing the membrane integrity of Salmonella typhimurium ATCC14028 strain by SEM (MG: methyl gallate, Ty: tilosin, MT: mixture of methyl gallate and tilosin).
5 is a diagram showing the effect of each test substance on the membrane potential of Salmonella typhimurium ATCC14028 strain (MG: methyl gallate, Ty: tylosine, MT: a mixture of methyl gallate and tylosine).
6A and 6B are diagrams showing the effect of each experimental substance on the biofilm formation of Salmonella typhimurium ATCC14028 strain (A) and LVPP-STI15 strain (B) as a result of crystal violet staining. 6C and D are diagrams showing the EC50 of MT in Salmonella typhimurium ATCC14028 strain (C) and LVPP-STI15 strain (D).
7 is a diagram showing the effect of methyl gallate, tylosine, or a mixture thereof on the survival of RAW 264.7 (A) and Caco-2 cells (B) (MG: methyl gallate, Ty: tylosine, MT: methyl A mixture of gallate and tylosine).
8A and B are diagrams showing the effect of each test substance on the adhesion of Salmonella typhimurium ATCC14028 strain (A) and LVPP-STI15 strain (B) to Caco-2 cells. 8C and D are diagrams showing the effect of each test substance on the invasion of Salmonella typhimurium ATCC14028 strain (C) and LVPP-STI15 strain (D) into Caco-2 cells (MG: methyl gallate, Ty: Tyrosine, MT: Mixture of methyl gallate and Tyrosine).
9 is a diagram showing the effect of each experimental substance on the survival of the Salmonella typhimurium ATCC14028 strain (A) and the LVPP-STI15 strain (B) in macrophages (MG: methyl gallate, Ty: tylosine, MT: Mixture of methyl gallate and tylosine).
Figure 10 is a comparison of the antimicrobial activity of methyl gallate (M), tylosine (T) or methyl gallate and tilosin (MT) formulated in a weight ratio of 1:2 for Salmonella typhimurium ATCC14028 strain (A) Is also. (G: no addition control, 1/2M methyl gallate 128 ug/ml: M: methyl gallate 256 ug/ml, 2M: methyl gallate 512 ug/ml 1/2T: Tyrosine 256 ug/ml, T: Tilo Sine 1024 ug/ml, T: Tyrosine 512 ug/ml, 1/2MT: a mixture of methyl gallate 128 ug/ml and Tyrosine 256 ug/ml, MT: methyl gallate 256 ug/ml and Tyrosine 512 ug /ml of a mixture, 2MT: a mixture of 512 ug/ml of methyl gallate and 1024 ug/ml of tylosine)

Hereinafter, the present invention will be described in detail.

The present invention provides an antimicrobial composition comprising methyl gallate and tilosin.

In addition, the present invention provides a composition for inhibiting intestinal adhesion or invasion of pathogenic strains containing methyl gallate and tilosin.

The composition is preferably an antimicrobial composition. The term “antimicrobial composition” refers to an agent that is provided to an individual in the form of a drug to kill bacteria, and refers to preservatives, fungicides, antibiotics and antibacterial agents.

In the present invention, the pathogenic strain refers to all strains that can invade the intestine, specifically Salmonella strain, Escherichia coli ), Staphylococcus aureus , Pasteurella multocida ( Pasteurella multocida ), Mycoplasma hyopneumoniae hyopneumoniae ) and Bordetella bronchiseptica ( Bordetella bronchiseptica ) may be one or more selected from the group consisting of, but is not limited thereto.

In the present invention, the salmonella strain is Salmonella typhimurium (Salmonella Typhimurium), Salmonella choleraesuis could device (Salmonella Choleraesuis), Salmonella Entebbe utility disk (Salmonella Enteritidis), live Monet LAL Galina volume (Salmonella Gallinarum), Salmonella tie blood (Salmonella Typhi) and Salmonella Florum ( Salmonella Pullorum) may be one or more selected from the group consisting of, preferably Salmonella Typhimurium ( Salmonella Typhimurium), but is not limited thereto.

Methyl gallate and tilosin according to the present invention are preferably contained in the composition at a low concentration that alone does not significantly affect the survival of the pathogenic strain itself. Specifically, methyl gallate and talosin may be included in a contact concentration in a weight ratio of 1:10-10:1, preferably in a contact concentration in a weight ratio of 1:4-4:1, more preferably It may be included in a contact concentration of a weight ratio of 1:2. When the mixing ratio of methyl gallate and tylosine is 1:10 or more, it is difficult to use due to the appearance of cytotoxicity and antimicrobial resistance of tylosine.If the mixing ratio of methyl gallate and tylosine is 10:1 or more, the production price of methyl gallate and It is difficult to use due to its cytotoxicity and low antimicrobial activity.

The composition of the present invention has excellent antibacterial activity, such as inhibiting the survival of pathogenic strains and inhibiting biofilm formation, even at a lower concentration than when used alone, by using methyl gallate together with tylosine, and without the problem of causing antibiotic-resistant bacteria. It has an excellent effect of remarkably inhibiting adhesion, invasion and survival in cells. Therefore, the composition of the present invention can be used not only in medicine, but also in various fields such as feed addition, disinfection, cleaning and preservation.

Thus, as an aspect, the present invention provides a combination preparation for preventing or treating infectious diseases caused by pathogenic strains containing methyl gallate and tilosin. The formulation may be a pharmaceutical formulation.

Infectious diseases caused by the pathogenic strain include Salmonella strain, Escherichia coli , Staphylococcus aureus , Pasteurella multocida , Mycoplasma hyopneumoniae , and Bordetella. Bordetella bronchiseptica ) refers to a disease caused by one or more selected from the group consisting of.

Infectious diseases caused by the Salmonella strain according to the present invention are, for example, epidemic or acute infectious diseases, including food poisoning, gastritis, enteritis, pneumonia, sepsis, pig cholera, and chubaekri, in addition to salmonellosis.

Salmonellosis is a generic term for symptoms accompanying fever, headache, diarrhea, vomiting, etc. due to Salmonella infection, and salmonellosis is broadly classified into sepsis type showing symptoms such as typhoid fever and acute gastroenteritis type showing symptoms of food poisoning, enteritis, food poisoning. , Acute bacteremia, etc.

The term “prevention” of the present invention refers to any action that suppresses or delays the onset of disease by administration of the composition.

The term "treatment" of the present invention refers to all actions in which the symptoms of the disease are improved or the disease is suppressed or alleviated and beneficially changed by the administration of the composition.

The pharmaceutical formulation of the present invention (mixed with the pharmaceutical composition) may further include a pharmaceutically acceptable carrier in addition to the active ingredient.

In the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions are sterilized and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary.

In addition, the composition of the present invention may be formulated as an injectable formulation such as an aqueous solution, suspension, emulsion, pill, capsule, granule or tablet by additionally adding a diluent, a dispersant, a surfactant, a binder and a lubricant.

Formulations for oral administration comprising the pharmaceutical composition of the present invention may be formulated as, for example, tablets, troches, lozenges, water-soluble or oily suspensions, powders or granules, emulsions, hard or soft capsules, syrups or elixirs. I can. For formulation into tablets and capsules, etc., a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, and masne stearate Lubricating oils such as calcium, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax may be included. In the case of a capsule formulation, a liquid carrier such as fatty oil may be further included in addition to the above-mentioned substances.

Formulations for parenteral administration containing the pharmaceutical composition of the present invention include injection forms such as subcutaneous injection, intravenous injection, or intramuscular injection, suppository injection, or spray, such as an aerosol that allows inhalation through the respiratory tract. It can be formulated. In order to formulate a formulation for injection, the composition of the present invention may be prepared as a solution or suspension by mixing in water together with a stabilizer or a buffer, and formulated for unit administration of ampoules or vials. When formulated for spraying such as an aerosol, a propellant or the like may be blended together with an additive so that the aqueous concentrated or wet powder is dispersed.

The composition of the present invention may further contain one or more known active ingredients having a preventive or therapeutic effect on infectious diseases.

In the present invention, the term "administering" means providing a given composition of the present invention to a subject by any suitable method.

In the method of the present invention, the route of administration of the composition may be administered through various routes, oral or parenteral, as long as it can reach the target tissue, and specifically, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, arterial It can be administered in a conventional manner through internal, transdermal, intranasal, inhalation, and the like.

Suitable dosages of the pharmaceutical composition of the present invention include formulation method, mode of administration, age, weight, sex, degree of disease symptoms, food, administration time, route of administration, excretion rate and response sensitivity of the subject animal and patient. It varies by factors, and usually an experienced physician or veterinarian can easily determine and prescribe an effective dosage for the desired treatment.

In another aspect, the present invention provides an antibacterial feed additive composition comprising methyl gallate and tilosin.

Feed-added antibiotics used in livestock and fisheries industries are used for the purpose of preventing and treating diseases, and administration of antibiotics for this purpose increases the possibility of developing resistant bacteria and is a problem because antibiotics remaining in livestock can be delivered to humans. When antibiotics are absorbed into the body through meat, they can cause antibiotic resistance, which can lead to the spread of disease. In addition, since there are many types of antibiotics mixed with feed and this has a problem that the probability of multidrug resistant bacteria increases, it is more natural-friendly, but it is a new level of antibiotic resistance suppression that solves the problems arising from the use of existing antibiotics. The composition of the present invention can be used.

In addition, the present invention can provide a feed comprising the composition for feed addition, and methyl gallate and tylosine, which are active ingredients of the present invention, are separately prepared in the form of feed additives and mixed with feed, or are directly effective when preparing feed. It can be prepared by adding ingredients. The active ingredient in the feed of the present invention may be in a liquid or dry state, preferably in a dry powder form. The drying method may be ventilated drying, natural drying, spray drying, and freeze drying, but is not limited thereto. The active ingredient of the present invention may be mixed in a powder form in an ingredient ratio of 0.05 to 10% by weight, preferably 0.1 to 2% by weight of the feed weight. In addition, the feed may further include conventional additives that can increase the preservability of the feed in addition to the active ingredient of the present invention.

Other non-pathogenic microorganisms may be additionally added to the composition for feed addition of the present invention. Microorganisms that can be added include Bacillus subtilis , which can produce proteases, lipolytic enzymes, and sugar converting enzymes, and Lactobacillus, which has physiological activity and ability to degrade organic matter under anaerobic conditions such as the stomach of cattle Strain ( Lactobacillus sp.), filamentous fungi such as Aspergillus oryzae and Saccharomyces cerevisiae showing the effect of increasing the weight of livestock, increasing milk production and increasing the digestion and absorption rate of feed. ) May be selected from the group consisting of yeast.

The feed of the present invention includes cereals, root fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, meals, grain by-products, etc. as vegetable, and protein, inorganic logistics as animal , Oils and fats, minerals, single cell proteins, zooplanktons, food leftovers, etc., but are not limited thereto.

The composition for feed addition of the present invention may contain binders, emulsifiers, preservatives, etc. added to prevent quality deterioration, and amino acids, vitamins, enzymes, probiotics, flavoring agents, non-proteinaceous agents added to the feed to increase utility Nitrogen compounds, silicates, buffers, colorants, extractants, oligosaccharides, etc. may be included, and in addition, feed mixing agents may be additionally included.

In another aspect, it provides an antibacterial food additive composition or a drinking water additive composition comprising methyl gallate and tilosin.

The food additive composition of the present invention may be added as it is to the composition containing methyl gallate and tilosine, or may be used together with other food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use. In general, methyl gallate and tilosine of the present invention are added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less based on the raw material.

The drinking water additive composition of the present invention may be used in a manner in which the composition containing methyl gallate and tylosine is separately prepared in the form of a drinking water additive and used in a manner that is mixed with drinking water or directly added when preparing drinking water. By mixing and supplying the drinking water as described above, there is an effect of continuously suppressing the survival of the strain and reducing adhesion and invasion to host cells.

In the present invention, drinking water is not particularly limited, and drinking water commonly used in the art may be used.

In another aspect, the present invention provides an antibacterial disinfectant composition comprising methyl gallate and tilosine.

The disinfectant composition according to the present invention can be usefully used as a disinfectant for hospitals and health care to prevent infection, and a disinfectant for general life, disinfectant for food and cooking places and facilities, buildings such as poultry farms, livestock houses, livestock, drinking water, litter, It can be used for disinfection of various growth products such as Nanja, transport vehicles, and tableware.

In another aspect, the present invention provides an antibacterial detergent composition comprising methyl gallate and tilosin.

Since methyl gallate and tilosin of the present invention have the effect of inhibiting the survival of pathogenic strains and inhibiting adhesion and invasion into host cells, the skin surface or parts of the body of livestock exposed to or likely to be exposed to pathogenic strains, etc. It can also be used for washing.

As another aspect, the present invention provides an antimicrobial preservative composition comprising methyl gallate and tilosin.

The preservative composition may include food preservatives, cosmetic preservatives or pharmaceutical preservatives. The food preservatives, cosmetic preservatives and pharmaceutical preservatives are additives used to prevent deterioration, spoilage, discoloration, and chemical change of pharmaceuticals. These include fungicides and antioxidants, and inhibit the growth of microorganisms such as bacteria, mold, and yeast. And functional antibiotics, such as inhibiting the growth of decaying microorganisms or sterilizing in medicines, are also included. Ideal conditions for such an antiseptic composition should be non-toxic and should be effective even in trace amounts.

In addition, the composition of the present invention can be used as an active ingredient in quasi-drugs. The quasi-drug composition of the present invention is not limited thereto, but preferably, it may be a disinfectant cleaner, a shower foam, a gagrin, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment, a patch, or a filter filler.

Terms not otherwise defined in the specification have the meanings commonly used in the art to which the present invention belongs.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Preparation example 1. Preparation of test material

Methyl gallate, tilosine, and compounds and kits used in the following experiments were purchased from Sigma (Sigma, St. Louis, MO, USA) unless otherwise specified. Stock formulations of methyl gallate and tilosine were 50%. It was prepared by diluting in ethanol, and the ratio of ethanol in the final diluted solution does not exceed 1% (v/v).

Preparation example 2. Culture of Salmonella strains and cells

Salmonella typhimurium (LVPP-STI15) isolated from pigs disclosed in the previous study and Salmonella typhimurium (ATCC 14028) purchased from ATCC were used. Salmonella strains were cultured in 37° C., LB (Luria-Bertani) medium (Difco, BD, USA).

Caco-2 cells, a human colon epithelial cell line, and RAW 264.7 cells, a mouse macrophage, were distributed from the Korea Cell Line Bank. Caco-2 cells were cultured in MEM (minimum essential medium) medium (Gibco, USA) containing 1% non-essential amino acids, 20% FBS and 1% penicillin/streptomycin (P/S). RAW 264.7 cells were cultured in RPMI 1640 medium containing 10% FBS (fetal bovine serum) and 1% P/S. Cell culture was performed at 37° C. and 5% CO ₂ conditions, and the medium was changed once every two days.

Example One. methyl Gallate And Tyrosine Selection of optimal mixing ratio

Two experiments were performed as follows in order to find the optimal mixing ratio of methyl gallate and tilosin.

First, in order to confirm the effect of Salmonella typhimurium on MIC, methyl gallate and tylosine were used as 0:0, 1:0, 1:1, 1:2, 1:4, 1:10, 1:20, The mixture was mixed at a weight ratio of 0:1, 2:1, 4:1, 10:1, and 20:1 to prepare a mixture of 1 mg/ml. For MIC measurement, MHB was used in a 96-well plate and diluted from an initial concentration of 2,560 μg/mL by a two-step dilution method. Salmonella typhimurium cultured in MHB the day before the experiment was diluted with 1,000 times MHB to each well diluted by concentration, and 100 μl of Salmonella typhimurium strain (6×10 ⁵ CFU/mL) was dispensed. After overnight cultivation, the minimum inhibitory concentration of the wells where the bacteria did not grow was confirmed. The results are shown in Fig. 1A.

As shown in Fig. 1A, when methyl gallate and tylosine are mixed in a weight ratio of 1-10:10-1, the MIC is significantly lower than that of methyl gallate or tylosine treated alone. Was confirmed. In particular, when methyl gallate and tylosine were mixed in a weight ratio of 1-4:10-1, excellent antibacterial effects were shown.

Next, an experiment was conducted to confirm the effect of Salmonella typhimurium on the degree of inhibition of invasion in host cells, which is a mechanism for survival. First, Caco-2 cells (10 ⁵ /mL) were completely grown in 24-well plates under antibiotic-free medium. Methyl gallate and tylosine were added to the Caco-2 cells 0:0, 1:0, 1:1, 1:2, 1:4, 1:10, 1:20, 0:1, 2:1, 4: Each well was treated at a weight ratio of 1, 10:1, and 20:1. At this time, all mixing ratios were at a concentration of 256 μg/mL, and were treated for 45 minutes. Salmonella typhimurium was centrifuged at 10,000 g for 15 minutes, washed twice with 1×PBS, and then suspended in an antibiotic-free medium. The Caco-2 cells were inoculated with Salmonella typhimurium strain (10 ⁹ CFU/mL), and then cultured at 37° C. for 45 minutes. This was centrifuged at 1,000 g for 2 minutes to remove the supernatant, and unattached bacteria were removed with 1×PBS. The cells were dissolved with 1% Triton x-100. The eluate was serially diluted, spread on an LB agar plate, and cultured overnight to count the number of bacteria attached to the cells. The results are shown in FIG. 1B.

As shown in B of FIG. 1, when methyl gallate and tilosin were mixed in a weight ratio of 1-10:10-1, it was confirmed that adhesion to host cells was inhibited. In particular, when methyl gallate and tylosine were mixed in a weight ratio of 1-4:4-1, excellent antibacterial effects were shown.

Through the above experimental results, it was confirmed that among various mixing ratios, the most excellent synergistic effect was exhibited when metal gallate and tylosine were mixed at a weight ratio of 1:2. When the mixing ratio of methyl gallate and tylosine is 1:10 or more, it is difficult to use due to the appearance of cytotoxicity and antimicrobial resistance of tylosine.If the mixing ratio of methyl gallate and tylosine is 10:1 or more, the production price of methyl gallate and It is difficult to use due to its cytotoxicity and low antimicrobial activity. Therefore, in the following examples, an experiment was performed using a group in which metal gallate and tilosin were mixed in a weight ratio of 1:2.

Example 2. methyl Gallate And Tyrosine Analysis of the effect of mixed treatment on the survival of Salmonella strains

In order to confirm the effect of the mixed treatment of methyl gallate and tilosin on the survival of Salmonella typhimurium, the following experiment was performed. First, in MHB Salmonella typhimurium (6×10 ⁵ CFU/mL) methyl gallate (MG = 32, 64, or 128 μg/mL), tilosin (Ty = 64, 128 or 256 μg/mL) or Mixture of methyl gallate and tylosine (¼× MT = 32 μg/mL MG and 64 μg/mL Ty; ½× MT = 64 μg/mL MG and 128 μg/mL Ty; 1× MT = 128 μg/mL MG and 256 μg/mL Ty) were each treated, and then incubated at 37 °C. After 1, 2, 4, 8, 12 and 24 hours of cultivation, 100 μL each was removed and serially diluted 10 times in agar saline. 20 μL of the dilution was spread on an LB agar plate, and then incubated at 37 °C for 24 hours, and CFU was measured. The results are shown in FIG. 2.

As shown in FIG. 2, it was confirmed that both strains of Salmonella typhimurium showed remarkably superior survival inhibitory effects in the mixed treatment group compared to the treatment with methyl gallate or tilosin alone. In particular, methyl gallate and tilosin did not show a concentration-dependent effect when treated alone, so only the highest concentration treatment group was shown in the graph, and Salmonella typhi due to synergistic effects even in the mixed treatment group mixed at a lower concentration than each treatment group alone. It can be seen that the effect of inhibiting the survival of the Murium appears.

Example 3. methyl Gallate And Tyrosine Analysis of the effect of mixing treatment on the cell membrane of Salmonella strains

In order to analyze the effect of the mixed treatment of methyl gallate and tilosin on the cell membrane of Salmonella strains, a LIVE/DEAD BacLight™ kit (Molecular Probes, Eugene, OR, USA) was used. Specifically, Salmonella typhimurium (10 ⁶ CFU / mL) methyl gallate (MG = 128 μg / mL), tylosine (Ty = 256 μg / mL) or a mixture of methyl gallate and tilosin (¼ × MT = 32 μg/mL MG and 64 μg/mL Ty; ½ × MT = 64 μg/mL MG and 128 μg/mL Ty; 1 × MT = 128 μg/mL MG and 256 μg/mL Ty), respectively, Incubated at 37° C. for 4 hours. A mixture of 3 μl of SYTO9 and PI (propidium iodide) (1:1) was added to the strain culture solution, followed by incubation in the dark for 15 minutes. The strain was visualized using a Zeiss confocal microscope (LSM700, Carl-Zeiss, Jena, Germany), and SYTO9 and PI staining were confirmed at excitation/emission wavelengths of 480/500 and 490/635 nm, respectively. The results are shown in FIG. 3

As shown in FIG. 3, when the Salmonella typhimurium strain was mixed with methyl gallate and tilosin, it was confirmed that the cell membrane of the strain was damaged (indicated by red fluorescent staining).

Additionally, the effect on the cell membrane integrity of the strain was confirmed using SEM. Specifically, Salmonella typhimurium (10 ⁷ CFU / mL) in methyl gallate (MG = 128 μg / mL), tyrosine (Ty = 256 μg / mL) or a mixture of methyl gallate and tilosin (¼ × MT = 32 μg/mL MG and 64 μg/mL Ty; ½ × MT = 64 μg/mL MG and 128 μg/mL Ty; 1 × MT = 128 μg/mL MG and 256 μg/mL Ty), respectively, Incubated at 37° C. for 6 hours. The culture solution was centrifuged at 10000g for 15 minutes, washed twice with 0.9% saline, resuspended in 2.5% glutaraldehyde, and maintained at -4°C for 12 hours. This was centrifuged again at 10000 g for 8 minutes, and then dehydrated in ethanol (30, 50, 70, 80, 90 and 100%). After drying it, it was observed with SEM (SU8200 Hitachi, Tokyo, Japan). The results are shown in FIG. 4.

As shown in FIG. 4, when methyl gallate and tylosine are mixed with Salmonella typhimurium strain, morphological modifications such as rough surface and thin bleeding occur, and significant membrane degradation due to cytoplasmic leakage compared to the single treatment group It was confirmed that appears.

Example 4. methyl Gallate And Tyrosine Analysis of the effect of mixing treatment on the membrane potential of Salmonella strains

Based on the results of Example 3, in order to analyze the effect of the mixed treatment of methyl gallate and tilosin on the membrane potential (MP) of Salmonella strains, a BacLight™ bacterial MP kit (Molecular Probes) was used. Specifically, after diluting Salmonella typhimurium (10 ⁷ CFU/mL) in PBS, methyl gallate (MG = 128 μg/mL), tilosin (Ty = 256 μg/mL) or methyl gallate and tilosin Mixture (¼× MT = 32 μg/mL MG and 64 μg/mL Ty; ½× MT = 64 μg/mL MG and 128 μg/mL Ty; 1× MT = 128 μg/mL MG and 256 μg/mL Ty) Were each treated for 2 hours. Then, 10 μL (3 mM) of DiOC (3,3-diethyloxacarbocyanine iodide) was added to 1 mL of the strain suspension, followed by mixing. 10 μL of 500 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was added to the unpolarized control, and the same volume of PVS was added to the non-treated control. After incubation for 15 minutes at room temperature, the samples were analyzed in excitation and emission filters of 488 and 515 nm, respectively, using a flow cytometer (BD Biosciences, San Jose, CA, USA). MP was determined based on the ratio of red to green fluorescence intensity, and the results are shown in FIG. 5.

As shown in FIG. 5, when the Salmonella typhimurium strain was mixed with methyl gallate and tylosine, it was confirmed that the ratio of red to green fluorescence intensity was significantly reduced compared to the single treatment group.

Example 5. methyl Gallate And Tyrosine Mixed treatment of Salmonella strains Biofilm Analysis of impact on formation

To confirm the effect of MG (2-128 μg/mL), Ty (2-256 μg/mL) and MT (1/128× MT-1/4× MT) on Salmonella typhimurium biofilm, Experiments were conducted that modified the study. Specifically, Salmonella typhimurium grown in TSB (tryptic soy broth) medium was normalized to have an OD600 of 0.8. 200 μL of a 1:100 dilution of the normalized culture solution (diluted in TSB medium) was dispensed into a 96-well plate, treated with different concentrations of MG, Ty or MT, and cultured under static conditions at 30°C. The control plate was not treated with any test material. After incubation for 24 hours, the bacterial growth of plankton was confirmed on microplates (optical density at 600 nm). The plate was washed with sterile double distilled water, fixed at 60° C. for 1 hour, and stained with 0.1% crystal violet (CV). After incubation for 15 minutes at room temperature, CV was solubilized using 30% acetic acid, and absorbance was measured at 550 nm. The concentration of 50% inhibition of maximum biofilm formation (EC ₅₀ ) was analyzed, and the effect of MT on biofilm dispersion was confirmed by further culturing for 24 hours after biofilm formation. The results are shown in FIG. 6

As shown in Figure 6, it was confirmed that the mixed treatment group (MT) of methyl gallate and tilosin reduced the biofilm formation of Salmonella typhimurium in a concentration-dependent manner. On the other hand, the group treated with methyl gallate or tylosine alone did not show a significant effect (not shown in the figure).

Further, the result of calculating the EC50 of the MT, Salmonella typhimurium _{^{(LVPP-STI15) 1/10}} × MT (a combination of 12.8 mg / mL MG and 25.6 mg / mL Ty) a half-maximal inhibition of biofilm formation of the strain The EC50 of Salmonella typhimurium (ATCC 14028) strain was ¹ / _12.5 × MT (a combination of 10.24 mg/mL MG and 20.5 mg/mL Ty). In contrast, the maximal biofilm inhibition by methyl gallate and tilosine was confirmed to be 9.0% and 5.5%, respectively. In addition, all test materials did not disperse the preformed biofilm.

Example 6. methyl Gallate And Tyrosine Cytotoxicity assay

To confirm the effect of the mixed treatment of methyl gallate and tylosine on Caco-2 and RAW 264.7 cells, MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide ) Analysis was performed. First, cells were seeded in a 96 well plate at a concentration of 10 ⁵ cells/mL, and then cultured for 24 hours. The medium was removed and fresh medium containing various concentrations of MG, Ty and MT was added, followed by incubation overnight. After 24 hours, the supernatant was removed, replaced with a medium containing 0.5 mg/mL of MTT, and cultured for 4 hours. After removing the supernatant again, DMSO (100 μL) was added, and absorbance was measured at 570 nm using VersaMax (Molecular Devices, Sunnyvale, CA, USA). Cell viability was calculated using the following calculation formula, and the results are shown in FIG. 7.

Survival rate=

As shown in FIG. 7, significant cytotoxicity was not observed for Caco-2 cells, which are human colon epithelial cell lines, and RAW 264.7 cells, which are mouse macrophages, in both the methyl gallate and tilosin alone and mixed treatment groups.

Example 7. methyl Gallate And Tyrosine Analysis of the effect of mixed treatment on cell adhesion and invasion of Salmonella strains

In order to confirm the effect of the mixed treatment of methyl gallate and tilosin on cell adhesion of Salmonella strains, the following experiment was performed. First, Caco-2 cells (10 ⁵ /mL) were completely grown in 24-well plates under antibiotic-free medium. The cells MG (8, 16 and 32 mg / mL), Ty ( 16, 32 and ^{64 mg / mL), 1/} 16 Х MT (8 mg / mL MG and ^{16 mg / mL Ty), 1} /8 Х the MT (16 mg / mL mG and 32 mg / mL Ty) and _{^{1/4 Х MT (32 mg}} / mL mG and 64 mg / mL Ty) were respectively treated for 45 min. Salmonella typhimurium was centrifuged at 10000 g for 15 minutes, washed twice with 1×PBS, and then suspended in a medium without antibiotics. The cells were inoculated with Salmonella typhimurium (10 ⁷ CFU/mL) strain, centrifuged at 1000 g for 2 minutes, and cultured again at 37° C. for 45 minutes. The supernatant was removed, and unattached bacteria were removed with 1×PBS. The cells were dissolved with 1% Triton x-100. The eluate was serially diluted, spread on an LB agar plate, and cultured overnight to count the number of bacteria attached to the cells. The results are shown in A and B of FIG. 8.

As shown in A and B of FIG. 8, it was confirmed that in both strains of Salmonella typhimurium, the mixed treatment group significantly inhibited the adhesion of the Salmonella strain to host cells compared to the treatment with methyl gallate or tylosine alone.

Next, in order to confirm the effect of the mixed treatment of methyl gallate and tilosin on the cell invasion of the Salmonella strain, the Caco-2 cells treated with the test substance were inoculated with the Salmonella typhimurium strain in a similar method to the above adhesion test. Then, it was incubated at 37°C for 60 minutes. The supernatant was removed and washed three times with 1 Х PBS to remove non-contact bacteria, and then 300 mL of gentamicin (100 μg/mL) was added. After further incubation for 60 minutes, the cells were dissolved with 1% Triton x-100. The eluate was serially diluted, spread on an LB agar plate, and cultured overnight to count the number of bacteria in the cells. The results are shown in C and D of FIG. 8.

As shown in C and D of FIG. 8, it was confirmed that in both strains of Salmonella typhimurium, the mixed treatment group significantly inhibited the invasion of the Salmonella strain into host cells compared to the treatment with methyl gallate or tilosin alone.

Example 8. methyl Gallate And Tyrosine Analysis of the effect of mixing treatment on the intracellular survival of Salmonella strains

In order to confirm the effect of the mixed treatment of methyl gallate and tilosin on the intracellular survival of Salmonella strains, experiments were performed by modifying the conventional method known by Lu et al. Specifically, RAW 264.7 cells (10 ⁵ cells/mL) were grown in a 24-well plate without antibiotics, and after treating the test material in a manner similar to the invasion test of Example 7, Salmonella typhimurium strain was inoculated. . After incubation with gentamicin (100 mg/mL) at 37° C. for 60 minutes, the cells were washed with 1×PBS, and 25 mg/mL gentamicin in MEM medium was added. Cells were thawed after 2, 4, and 8 hours of additional culture and the total number of viable bacteria was counted. The results are shown in FIG. 9.

As shown in FIG. 9, there was no significant difference between groups when cultured for 2 hours, but the survival of Salmonella strains in host cells significantly decreased by mixing treatment with methyl gallate and tylosine during culture for 4 hours and 8 hours. Confirmed that.

Example 9. methyl Gallate And Tyrosine Antibacterial effect test according to combination treatment

In order to confirm the antimicrobial effect of methyl gallate and tilosin on Salmonella typhimurium strains, methyl gallate (MG = 128, 256 or 512 μg/mL) in Salmonella typhimurium, Tilosin (Ty = 256, 512 or 1024 μg/mL) or a mixture of methyl gallate and tylosine (½MT = 128 μg/mL MG and 256 μg/mL Ty; MT = 256 μg/mL MG and 512 μg/mL Ty; 2MT = 512 μg/mL MG and 1024 μg/mL Ty) were each treated, and then the number of bacteria was confirmed over time. The results are shown in FIG. 10.

As shown in FIG. 10, it was confirmed that the combined treatment group (MT) of methyl gallate and tilosin exhibited a bactericidal effect after sterilizing for a certain time. On the other hand, methyl gallate (M) or tilosin (T) alone treatment group did not completely inhibit the survival of Salmonella typhimurium strains.

Example 10. Against pathogenic bacteria methyl Gallate And Tyrosine Antibacterial effect test according to combination treatment

In order to confirm whether the combined treatment of methyl gallate and tylosine exhibits antibacterial effects against other pathogenic bacteria other than Salmonella strains, Escherichia coli), Staphylococcus aureus (Staphylococcus aureus), La paseuturel the water Toshi (Pasteurella multocida), M. Playa jimeu Hebrews ohnyu ammonia (Mycoplasma hyopneumoniae ) and Bordetella bronchiseptica strains were tested.

Specifically, an MT combination drug in which methyl gallate and tilosin are mixed in a weight ratio of 1:2, methyl gallate (M), tilosin (T), enrofloxacin (ENRF), tetracycline (TC), streptomycin The minimum inhibitory concentration (MIC) of (SM), bacitracin (BAC), and ampicillin (AMP) was compared. MIC was performed by the broth microdilution method. Each experimental substance was diluted in a binary method so that the final concentration was 0.2-256 μg/ml. The bacterial solution was adjusted to 0.5 McFarland (10 ⁵ cfu/ml), diluted 100 times, and dispensed 50 µl per well to obtain a final 100 µl. All plates had positive control wells and negative control wells. After incubation at 37°C for 18-24 hours, the concentration of the test substance in the wells in which proliferation was completely inhibited was determined by MIC by visually and simultaneously checking with a microplate reader. The results are shown in Table 1.

As shown in Table 1, all pathogenic bacteria used in the experiment ( Escherichia coli , Staphylococcus aureus , Salmonella spp , Pasteurella multocida , Mycoplasma hyopneumoniae , Bordetella bronchiseptica ), it was confirmed to exhibit excellent antibacterial effect (1-64ug/ml).

As described above, macrolide antibiotics prescribed as targets for respiratory pathogenic bacteria are unintentionally exposed to intestinal pathogenic microorganisms, Salmorela strain, and antibiotic-resistant strains may occur. The present inventors have discovered a combination ratio of methyl gallate and tilosin that inhibits the appearance of antibiotic-resistant strains unintentionally exposed, blocks the survival mechanism of Salmorela, and enhances antibacterial activity. In particular, when methyl gallate and tilosin are treated in combination in a 1:10-10:1 weight ratio, the survival and biofilm formation of Salmonella strains are suppressed by synergistic effects between the two substances, and the host cells of the Salmonella strains without the problem of causing antibiotic-resistant bacteria. It was confirmed that adhesion to the furnace, invasion into cells, and survival in cells can be inhibited. Overall, the combination of methyl gallate and tilosin is not only a pharmaceutical composition for the prevention or treatment of infectious diseases caused by various pathogenic strains including Salmonella, but also for feed addition, drinking water, disinfection, cleaning and preservative use. It can be used in various fields such as.

Claims (11)

Antibacterial composition comprising methyl gallate and tilosin.
The composition of claim 1, wherein the methyl gallate and tilosine are mixed in a weight ratio of 1:10-10:1
The method of claim 1, wherein the composition is Salmonella strain, Escherichia coli), Staphylococcus aureus (Staphylococcus aureus), La paseuturel the water Toshi (Pasteurella multocida), M. Playa jimeu Hebrews ohnyu ammonia (Mycoplasma hyopneumoniae ) and Bordetella bronchiseptica ( Bordetella bronchiseptica ) that is for antibacterial use for at least one selected from the group consisting of.
The method of claim 3, wherein the Salmonella strain is Salmonella Typhimurium, Salmonella Choleraesuis, Salmonella Enteritidis, Salmonella Gallinarum, Salmonella Gallinarum, and Salmonella Typhimurium. ( Salmonella Typhi) and Salmonella Florum ( Salmonella Pullorum) at least one member selected from the group consisting of, a composition.
As a composition for inhibiting intestinal adhesion or invasion of pathogenic strains containing methyl gallate and tylosine,
The pathogenic strains include Salmonella strain, Escherichia coli , Staphylococcus aureus , Pasteurella multocida , Mycoplasma hyopneumoniae , and Bordetella bronchiseptica ( Bordetella bronchiseptica ) that is one or more selected from the group consisting of, the composition.
As a combination preparation for the prevention or treatment of infectious diseases caused by pathogenic strains containing methyl gallate and tylosine,
The infectious disease is Salmonella strain, Escherichia coli , Staphylococcus aureus , Pasteurella multocida , Mycoplasma hyopneumoniae ( Mycoplasma hyopneumoniae ) and Bordetella bronchiseptica ( Bordetella bronchiseptica ) that is an infectious disease caused by at least one selected from the group consisting of, a combination formulation.
The combination preparation according to claim 6, wherein the infectious disease is at least one selected from the group consisting of salmonellosis, food poisoning, gastritis, enteritis, pneumonia, sepsis, pig cholera and chubaek-ri.
Antibacterial feed additive composition containing methyl gallate and tilosin.
Antibacterial disinfectant composition comprising methyl gallate and tilosin.
Antibacterial detergent composition containing methyl gallate and tilosin.
Antimicrobial preservative composition comprising methyl gallate and tylosine.

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