KR100707988B1 - Complex antibiotic composition for bovine mastitis - Google Patents

Abstract

The present invention relates to a complex antimicrobial composition containing cytral and conventional antibiotics as an active ingredient, and more particularly, to the plant extract Citral, Enrofloxacin (Enrofloxacin), Oxytetracycline (Oxytetracycline) A new animal complex with antipathogenic bacteria, endotoxin neutralization, and anti-inflammatory activity by mixing a certain ratio with one or more antibiotics selected from Streptomycin, Sepharosporin and Colistin It relates to an antibiotic composition. This complex antibiotic composition neutralizes endotoxins secreted from Gram-negative pathogenic bacteria, and has an excellent effect on the treatment of Staphylococcus aureus, an antibiotic-resistant bacterium, and has excellent anti-inflammatory action, which is useful for the prevention and treatment of mastitis in cows. It is about.

Cytral, Enroproxacin, Colistin, Sepharoseporin, Combination Antibiotics, Mastitis

Description

Complex antibiotic composition for bovine mastitis

1 is an antimicrobial activity test procedure by the checkerboard method (Checkerboard method) method.

Figure 2 is a process chart of endotoxin neutralization test by the Limulus amebocyte lysate method.

The present invention relates to a complex antimicrobial composition containing cytral and conventional antibiotics as an active ingredient, and more particularly, to the plant extract Citral, Enrofloxacin (Enrofloxacin), Oxytetracycline (Oxytetracycline) A new animal complex with antipathogenic bacteria, endotoxin neutralization, and anti-inflammatory activity by mixing a certain ratio with one or more antibiotics selected from Streptomycin, Sepharosporin and Colistin It relates to an antibiotic composition. This antimicrobial composition neutralizes endotoxins secreted from Gram-negative pathogenic bacteria, and has an excellent effect on the treatment of Staphylococcus aureus, an antibiotic-resistant bacterium, and has an excellent anti-inflammatory effect and is useful for the prevention and treatment of mastitis in cows. It is about.

Mastitis in cows is a disease caused by bacteria or fungi that are widely distributed in the cow's breast and inflamed. It occurs most frequently in cow's disease. Etc. Economic loss is very large cow disease.

Mastitis can be divided into clinical mastitis, subclinical mastitis, and chronic mastitis. Clinical mastitis is a mastitis in which crude sediments are visible, that is, visible to the naked eye, and about 5.5% of cows are infected. The swelling of the breast, fever, pain and breast dysfunction, and milk change is easily distinguished by the naked eye. Clinical mastitis can be divided into three types. It is divided into acute mastitis, acute mastitis and subacute mastitis.

Subclinical mastitis is a condition that is infected with mastitis but cannot be visually identified in the eyes of the owner, and milk can be determined only by laboratory diagnostics, such as field diagnostics such as California mastitis (CMT) or milk culture. have. It is 15 to 40 times more likely than clinical mastitis, and most cases develop clinically. If left untreated, such a subclinical mastitis is likely to turn into a clinical form, and it usually causes great economic losses such as reduced oil production and lower oil quality. In order to control mastitis, such subclinical mastitis should be intensively searched, treated, and prevented, and it can bring about fundamental economic benefits such as improved oil quality and increased flow rate by eradication or reduction by intensive attention and effort.

Chronic mastitis refers to a condition that lasts for several months or until the next inorganic period. Scars develop in the breast tissue, causing changes in the shape and size of the breast and accompanied by decreased milk production. The bacterium lurks and develops according to stress and health conditions. The treatment seems to recover easily, but it is difficult to treat because of its recurring characteristics.

Lastly, it is best to prevent non-specific mastitis (sterile mastitis), which is a clinical or subclinical type of mastitis that cannot isolate pathogens. Among them, Korea is considered to have a clinical and subclinical form of 5.5% and 30.2, which is two to three times that of developed countries.

According to the causes of mastitis, the causative agents of mastitis are classified as infectious, opportunistic and environmental according to the infection pattern and the path of occurrence. The main habitat for infectious mastitis is the infected papilla, which is a bacterium that can grow and proliferate in the mammary gland and refers to bacteria that are transmitted to other individuals by indirect contact during milking. Staphylococcus aureus , Streptococcus dysgalactiae , Streptococcus agalactiae , etc. are the most representative bacteria, and they are well adapted to the mammary gland environment and mainly cause subclinical mastitis. As the term suggests, environmental mastitis is a major habitat for cows (top right, feces, soil), and enterobacteriaceae and environmental streptococci. These strains are frequently caused by an unclean environment regardless of the time of milking, between milking, during the dry season, or during cow's acetic acid. They usually show symptoms of clinical mastitis because they do not have an adaptive process to survive in the host. When a breast is infected, it proliferates immediately, induces the host's immune response, and quickly disappears. Therefore, about 70-80% of intestinal bacterial infections are clinical forms, about 50% of environmental streptococcal infections show clinical symptoms, and 60-70% of environmental pathogen infections have a short infection duration of 30 days or less. Opportunistic bacteria include coagulase negative Staphylococci (CNS) strains, except Staphylococcus aureus , which live around the breast of a cow and in the milker's hand. It refers to a bacterium that infects the breast opportunistically and causes mastitis at the time when the cow's immune status is lowered by the back.

Therefore, it can be helpful to manage mastitis by analyzing mastitis causative organisms and analyzing whether the form of mastitis in the ranch is contagious or environmental mastitis and where and why such causative organisms are caused. Therefore, in order to effectively manage mastitis, it is necessary to analyze the pattern of mastitis and then take appropriate preventive measures.

Antimicrobials currently used to treat mastitis in cows include bacteriostatic antibiotics such as sulfonamide, tetracycline, chloramphenicol, erythromycin, novobiocin, trimethoprim and bactericidal antibiotics such as penicillin, streptomycin and pramomycin. , Colistin, kanamycin, vancomycin, bacitracin, and cephalosporin. However, the emergence of resistant bacteria is difficult to treat a single agent. Therefore, a significant amount of antibiotics commercialized in Korea is composed of a combination.

The feasibility of co-administration of antibiotics can be found in three aspects. First, the antimicrobial range of a single antimicrobial agent can be broadened when two antimicrobials are used in combination. Second, the toxicity of one antimicrobial agent can be lowered when the two antimicrobials are used in combination. Third is the inhibition of the appearance of resistant strains. In other words, even if the resistance to one antimicrobial agent is obtained by another antimicrobial antimicrobial action, so the resistance is eventually obtained by the bacteria that is difficult to increase the effectiveness of the treatment.

The administration of such a combination has a significant effect in the treatment of pathogenic microorganisms, but in the case of Gram-negative bacteria, due to the many endotoxins secreted with antimicrobial action, there was a problem of side effects that threaten the survival of animals. In addition, resistant strains appeared due to long use of the combination agent is no longer effective.

Recently, in order to develop a cow mastitis treatment globally, we are promoting the development and prevention of mastitis using natural materials. Patent application and registered natural materials using natural materials in Korea include the prevention and treatment of mastitis of cows with chitosan or chitosan oligosaccharide as active ingredient (Korea Patent Publication No. 1999-44796), and cows based on grapefruit seed extract Formulation for the treatment of mastitis prophylaxis and ointment agent thereof (Korean Patent No. 195-2769), pharmaceutical composition for treating mastitis disease in livestock containing a situation mushroom extract (Korean Patent No. 2003-84223), chrysanthemum A method for preparing a mastitis treatment of pregnant women and cows using extracts from pregnant women and its application (Korea Patent Publication No. 2003-79185), a composition for reducing somatic cells in milk of cows and preventing or treating mastitis (Korea Publication No. 2003) -61102), Prophylactic Treatment of Cow Mastitis with Loquat Leaf Extract and Method thereof (Korean Patent Laid-Open Publication No. 2003-39152), comprising Buporin II Composition for the treatment of mastitis of oil (Korean Patent Publication No. 2003-16801), immunopotentiator having somatic reduction effect (Korean Patent Publication No. 2002-76434), nipple dipping solution for teat disinfection and prevention of mastitis of cows (Korea Publication Patent No. 1999-84251), Beta-carotene preparation for injection for the prevention of mastitis in cows (Korea Patent Publication No. 1998-441), Method for treating mastitis in cattle such as cattle, and ozone injection device for breasts (Korea Patent Publication No. 1997) -68997), the composition for the prevention and treatment of cow mastitis (Korean Patent No. 289006), the composition for oral administration with the treatment of mastitis in cows and the reduction of somatic cell count (Korean Patent No. 217559), for the prevention of mastitis in cows Beta carotene preparations for injection (Korean Patent No. 166295) and the like are known.

However, the effects on the prevention and treatment of cow mastitis using the natural substances presented above are limited. This is because the antibacterial action of natural substances is less effective than conventional antimicrobials. In addition, most of these natural materials are hydrophilic, and the distribution of milk is not present, so the effect is drastically reduced. Among the natural materials are citrus, which is used to treat cow mastitis, and cabarcrol, which is used to treat digestive diseases. However, in the case of the citrus, there is a disadvantage in that the antimicrobial activity is weak with a single administration and the distribution is not made in the breast. In the case of carbacrol, the antibacterial activity and the antimicrobial range are excellent, but due to the nature of essential oils, the strong odor and pathogenicity have limitations on the penetration of infected tissue.

Citral, a plant extract, has a broad antimicrobial range and excellent antimicrobial activity, and is a component of many foods everyday. This ingredient is approved as a food additive and is a product that is harmless to the human body (Kyungin KFDA No. 101). However, animal drugs using this ingredient have not been developed yet.

In the present invention, it was found that cytral has an excellent effect on antibiotic-resistant staphylococci, which is the causative agent of cow mastitis. In particular, when mixed with an appropriate ratio of the existing antimicrobial agent of the first group it was confirmed that more powerful antimicrobial activity than the conventional antimicrobial agent alone. In addition, when it is composed of one or more combinations of cytral-colistin, streptomycin, or cephalosporin, it has both endotoxin neutralizing ability and anti-inflammatory action that did not appear in existing antibiotics, and has new antibacterial effect against antibiotic resistance strains. A concept cow milk mastitis complex antimicrobial agent was developed.

Thus, the present inventors have studied in order to solve the above problems, when mixed with a certain ratio of the first group of antibiotics for the prevention and treatment of cow mastitis, while having a synergistic antibacterial action from Gram-negative bacteria The present invention has been completed by developing a complex antibiotic for the prevention and treatment of new functional cow mastitis, which neutralizes the released endotoxin and simultaneously suppresses anti-inflammatory action.

Therefore, an object of the present invention is to provide a composite antibiotic composition for animals for the prevention and treatment of cow mastitis comprising a certain ratio of the central.

The present invention is a combination antibiotic composition for animals, Enrofloxacin (Enrofloxacin), Oxytetracycline (Oxytetracycline), Streptomycin (Streptomycin), Sepharoporin (Cepharosporin) and Colistin (Colistin) any one or more selected from It is characterized by including antibiotics and central (Citral).

In addition, the composite antibiotic composition for animals of the present invention is characterized in that it is used for the prevention and treatment of cow mastitis.

Referring to the present invention in more detail as follows.

First, Citral is a natural substance extracted from grapefruit and various plants. It is a colorless or pale yellow liquid with a molecular weight of 152.24 and has a lemon-like scent that is used as a fragrance. In addition, it is a safe substance that can be used as a mouthwash, and has an excellent antimicrobial effect on gram-negative and gram-positive bacteria, and has a broad spectrum of antimicrobial activity. Although it is insoluble in water and impaired in utilization, it is soluble in alcohol and used as a mouthwash and a natural antimicrobial composition.

In the present invention, by mixing any one or more antibiotics selected from enroproxacin, oxytetracycline, streptomycin, cephalosporin and colistin is known to have excellent antimicrobial activity in the treatment of cytral and cow mastitis It provides an anti-inflammatory, endotoxin neutralization and antimicrobial activity against antibiotic resistant strains even at small concentrations with excellent synergy.

The mixing ratio of one or more antibiotics selected from cytral and enroproxacin, oxytetracycline, streptomycin, cephalosporin, and colistin is 1% by weight of thyroproxacin, oxytetracycline, 0.1 to 9 parts by weight, more preferably 0.4 to 3 parts by weight, even more preferably 0.6 to 1.8 parts by weight, most preferably 0.8 to 1 part by weight of at least one antibiotic selected from streptomycin, cephalosporin and colistin 1.4 parts by weight is to be used. If any one or more antibiotics selected from enroproxacin, oxytetracycline, streptomycin, cephalosporin and colistin exceeds the upper limit, the antimicrobial activity is lowered and side effects of the toxicity of the antimicrobial agent and the retention of the antimicrobial agent occur. And, if it is less than the lower limit, there is a problem that the antimicrobial activity is falling, the skin irritation toxicity and the production cost is increased by the central.

In addition, a combination antibiotic composition for animals that combines antibiotics and cytral with any one selected from enroproxacin, oxytetracycline, streptomycin, cephalosporin, and colistin is possible, but antimicrobial activity and endotoxin neutralizing ability are also possible. To satisfy at the same time, preferably, a mixture of colistin and cytral with at least one antibiotic selected from enroproxacin, oxytetracycline, streptomycin, and cephalosporin. The compounding ratio of the complex antibiotic composition is preferably 0.4 to 2.2 parts by weight of colistin and 1 or more antibiotics selected from enroproxacin, oxytetracycline, streptomycin and cephalosporin based on 1 part by weight of cytral. To 2.2 parts by weight, more preferably 0.4 to 1.2 parts by weight of colistin and 1 or more antibiotics selected from enroproxacin, oxytetracycline, streptomycin and cephalosporin based on 1 part by weight of cytral 0.7 to 1.2 parts by weight is to be used.

The active ingredient mixed with at least one antibiotic selected from cytral and enroproxacin, oxytetracycline, streptomycin, cephalosporin and colistin is 0.01 to 30% by weight, preferably in the total antimicrobial composition. It is preferable to prepare 0.1 to 10% by weight, more preferably 0.5 to 5% by weight.

On the other hand, sodium benzoate (Sodium benzonate) that can be added as an auxiliary ingredient to the composite antimicrobial composition for animals has an antiseptic effect and increases the antimicrobial activity of the composition as a drug used as a preservative. The preferred content of sodium benzoate is 0.01 to 1.0% by weight based on the total composition, there is a problem that the price increases if the upper limit.

In addition, allantoin (allantoin: 5-uraidhydantoin), which can be added as an auxiliary ingredient to the complex antimicrobial composition for animals, is a substance produced by the decomposition of uric acid by uricase, and rejuvenates the cells of wound tissue. Wound healing is promoted, synergistically with cytral to further promote the repair of inflammation. The preferred content of allantoin is 0.1 to 1.0% by weight based on the total composition, there is a problem that the toxicity of allantoin increases if the above upper limit.

In addition, sodium propionate, which can be added as an auxiliary ingredient to the composite antimicrobial composition for animals, acts as an antifungal substance to inhibit fungi causing mastitis. Its preferred content is 0.1 to 1.0% by weight based on the total composition, there is a problem of increasing the toxicity by propionic acid if the upper limit.

In addition, a steroidal anti-inflammatory agent having an anti-inflammatory effect can be added to the composite antimicrobial composition for animals, and preferably 0.005 to 0.5% by weight of dexamethasone phosphate can be used with respect to the whole composition. At this time, there is a problem of remaining outside the upper limit.

In addition, an appropriate amount of antihistamine may be added to the animal complex antimicrobial composition for vascular contraction, secretion inhibition and sedation of the inflammatory site. The antihistamines may be used both antihistamines, which are effective and safe for humans and animals, but may preferably include 0.01 to 0.1 wt% of triphenylamine hydrochloride based on the total composition. At this time, if the upper limit is out of use there is a problem of remaining when used in inorganic.

In addition, a variety of bases suitable for infusion administration through nipples, including oily ointment, may be used as the base for the complex antimicrobial composition for animals. As the oil-based ointment, petrolatum, vegetable oil can be used, and water-soluble base is used depending on the purpose of use. Preferably 1 to 10% by weight of brine or propylene glycol can be used based on the total composition. At this time, there is a problem that the antimicrobial activity is lowered if the upper limit.

Such composite antimicrobial composition for animals is used for the treatment of infectious diseases and disinfectants of resistant strains caused by the use of existing antibiotics and chemotherapeutic agents, and the treatment of infectious diseases is excellent in preventing or disinfecting or disinfecting enteritis, respiratory infections, and animal mastitis It is safe for animals and can be used as a natural antimicrobial agent that can be used without distinction between inorganic and dry organic compounds without requiring a separate drug holiday.

In addition, the composite antimicrobial composition for animals of the present invention is used as a prophylactic or therapeutic agent for mastitis, which is effective in acute, chronic and subclinical mastitis as well as latent mastitis.

The composite antimicrobial composition for animals of the present invention may be administered in various oral or parenteral formulations during actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used Or using excipients.

The content of the antibiotic in any one of the active ingredients of the present invention, selected from cytral and enroproxacin, oxytetracycline, streptomycin, cephalosporin, and colistin, is determined by the degree of absorption of the active ingredient in the body and inactivation. It may be appropriately selected depending on the rate of fire, the rate of excretion, the age and the condition. The dosage of the preparation of the present invention is 10-100 ml / 1, preferably 20-50 ml / 1, and intramammary injection can be performed 1-3 times a day.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example  One

Antibacterial activity test was carried out by checkerboard method (Checkerboard method) shake culture tryptic soybean broth overnight at 37 ℃, sterilized one day before transplanting the bacteria in the hardened TS agar medium and incubated in 37 ℃ incubator It was. The cultured strains were tested according to the method shown in FIG.

Staphylococcus aureus, a Gram-positive strain, has five types of Staphylococcus aureus, KCCM 40510, KCCM 11593, KCCM 41294, KCCM 40881 and KCCM 41291. Inoculated in Muller-Hinton) medium was pre-cultured at 37 ℃. The cultured strains were inoculated in Mullerhinton medium to make a test medium, and the following experiment was carried out to examine the interaction between cytral and enroproxacin, oxytetracycline, streptomycin, cephalosporin or colistin. .

Minimum inhibitory concentrations in vitro against cytral, enroproxacin, oxytetracycline, streptomycin, cephalosporin or colistin using five strains of Staphylococcus aureus resistant to the pathogenic bacteria as test strains Minimum inhibition concentration (MIC) was measured. In addition, in order to obtain the minimum inhibitory concentration when antibiotics are selected in combination with cytral and enroproxacin, oxytetracycline, streptomycin, cephalosporin and colistin, the combination ratio of cytral and each antibiotic The growth rate of the fungi was determined by varying. That is, using a 96 well plate, 50 μl was added by diluting 2 times from 5 mg / ml vertically and 50 μl by diluting twice from the MIC of antibiotics transversely. The last row of columns was 50 μl of sterile distilled water as a single antibiotic. 50 μl of 4-fold concentrated medium was added to the prepared plate, and 10,000 CFU / 50 μl of the cultured overnight was added thereto, and the growth of the bacteria was examined after overnight culture.

FIC index (Fractional inhibition concentration) was calculated by the following equation 1 from the MIC results of the above alone and co-administration.

The FIC index calculated in Equation 1 was defined as synergy when 1 or less, more than 1, additive when 2 or less and antagonism when greater than 2 [(King, TC et al, Rev Infec Rev, 147: 758- 764 (1981)] The antibiotic in Equation 1 is any one antibiotic selected from enroproxacin, oxytetracycline, streptomycin, cephalosporin and colistin.

Table 1 shows the results of experiments with antibiotic resistant Staphylococcus aureus KCCM 40510, Table 2 KCCM40881, Table 3 KCCM41294, Table 4 KCCM11593, and Table 5 KCCM41291.

Antibiotic Alone Concomitant administration FIC Index Interaction MIC-C (mg / ml) MIC-term (μg / ml) MIC-C (mg / ml) MIC-term (μg / ml) Enrofloxacin 5 64 2.5 32 1.00 Synergy Oxytetracycline 5 32 2.5 16 1.00 Synergy Streptomycin 5 32 2.5 8 0.75 Synergy Sepharophosphorin 5 128 2.5 8 0.56 Synergy Colistin 5 256 2.5 2 1.01 Additive

Antibiotic Alone Concomitant administration FIC Index Interaction MIC-C (mg / ml) MIC-term (μg / ml) MIC-C (mg / ml) MIC-term (μg / ml) Enrofloxacin 5 2 5 One 1.00 Synergy Oxytetracycline 5 4 2.5 2 1.00 Synergy Streptomycin 5 32 2.5 2 0.56 Synergy Sepharophosphorin 5 2 2.5 0.5 0.75 Synergy Colistin 5 64 2.5 32 1.00 Synergy

Antibiotic Alone Concomitant administration FIC Index Interaction MIC-C (mg / ml) MIC-term (μg / ml) MIC-C (mg / ml) MIC-term (μg / ml) Enrofloxacin 5 32 2.5 32 1.00 Synergy Oxytetracycline 5 64 2.5 16 0.75 Synergy Streptomycin 5 256 2.5 64 0.75 Synergy Sepharophosphorin 5 64 2.5 2 0.56 Synergy Colistin 5 64 2.5 16 1.25 Additive

Antibiotic Alone Concomitant administration FIC Index Interaction MIC-C (mg / ml) MIC-term (μg / ml) MIC-C (mg / ml) MIC-term (μg / ml) Enrofloxacin 5 One 2.5 0.5 1.00 Synergy Oxytetracycline 5 2 2.5 One 1.00 Synergy Streptomycin 5 32 2.5 2 0.56 Synergy Sepharophosphorin 5 One 2.5 0.25 0.75 Synergy Colistin 5 64 2.5 32 1.00 Synergy

Antibiotic Alone Concomitant administration FIC Index Interaction MIC-C (mg / ml) MIC-term (mg / ml) MIC-C (mg / ml) MIC-term (μg / ml) Enrofloxacin 5 32 5 0.05 0.50 Synergy Oxytetracycline 5 64 5 One 0.50 Additive Streptomycin 5 64 5 2 1.03 Additive Sepharophosphorin 5 16 5 0.025 0.50 Synergy Colistin 5 256 5 16 0.56 Synergy

According to Table 1 to Table 5, antibiotics co-administered with cytral are Gram-positive strains, and all five kinds of Staphylococcus aureus (C. Appeared to be. In particular, the synergistic results were shown for cytral-streptomycin and cytral-sephalosporin. Cytral, entroproxine, oxytetracycline, oxytetracycline, streptomycin, cephalosporin or colistin administered alone when combined with cytral and enroproxacin, oxytetracycline, streptomycin, cephalosporin or colistin It was found that even with a lower dose, there was an excellent antimicrobial activity.

Example  2

In order to determine the neutralizing capacity for endotoxin, an experiment was performed by using the method of FIG. 2 using endotoxin (Sigma) and Escherichia coli ( E. Coli ) k88ab. In the endotoxin neutralization test, the endotoxin content was measured using the Limulus amebocyte lysate method. In other words, endotoxin was fixed at a concentration of 5 EU / ㎖ and commercially available antimicrobials were made to a constant concentration of 2 ㎍ / ㎖. Tris-hydrochloric acid buffer solution (pH 8.0) was used as the reaction buffer, and the pH of the antimicrobial material was reacted by maintaining the pH of the solution containing the antimicrobial material at 8.0 with hydrochloric acid or sodium hydroxide without endotoxin. The color development generated by reacting the solution before and after the reaction with Rimurus amoeba lysate reagent in the same amount was measured at 404 nm, and the content of endotoxin was calculated from the absorption wavelength.

Results of laboratory reaction of endotoxins and antibiotics in order to see the direct endotoxin neutralization of any antibiotic selected from cytral and enroproxacin, oxytetracycline, streptomycin, cephalosporin and colistin Is shown in Table 6. Endotoxin was fixed at a concentration of 2 EU / ㎖ and physiological saline, antibiotics 5 ㎍ / ㎖ constant concentration was made. Cytral was measured and compared at a concentration of 10 mg / ml, ie 1 (w / v)%.

division Endotoxin after reaction (EU / mL) Saline solution 1.74 ± 0.23 Colistin 0.23 ± 0.14 Enrofloxacin 1.69 ± 0.42 Cephalosporins 1.76 ± 0.34 Ampicillin 1.86 ± 0.32 Oxytetracycline 1.67 ± 0.43 Streptomycin 1.21 ± 0.11 Central 1.23 ± 0.54

Colistin showed the greatest binding activity, and streptomycin and cytral showed endotoxin neutralization.

Table 7 summarizes the results of experiments conducted to determine whether the endotoxins released from bacteria react directly with antibiotics when bacteria react with antibiotics. Saline and antibiotics made a constant concentration of 5 mg / ㎖. Cytral was compared at a concentration of 10 mg / ml.

division Before reaction (EU / mL) After reaction (EU / mL) Secretion (EU / mL) Confirmation Physiological saline 173 ± 12 216 ± 18   36 ± 18 Colistin 161 ± 16   28 ± 0.90 -133 ± 12 Enrofloxacin 156 ± 14 306 ± 18  149 ± 13 Cephalosporins 172 ± 13 306 ± 24  128 ± 11 Ampicillin 164 ± 13 312 ± 27  148 ± 15 Oxytetracycline 168 ± 15 303 ± 32  134 ± 13 Streptomycin 172 ± 13 226 ± 16   56 ± 6 Central 167 ± 16 199 ± 13   23 ± 2

As shown in Table 7, the amount of endotoxin naturally occurring in E. coli k88ab isolated from animals was 36 EU / ml. However, when the strain was reacted with colistin, the antibiotic and endotoxin were combined with 139 EU / ml when the endotoxin was 161 EU / ml before the reaction. That is, it was found that 2.78 EU binds to 1 μg of colistin. The reason why the endotoxin neutralizing ability differs among antibiotics seems to be due to its structure.

The colistin is a peptide-type antibiotic that exhibits a cation, unlike enrofloxacin, which has a high lipid affinity, and acts on bacterial cell membranes and is difficult to penetrate into the intestine of the animal body. It is known that the characteristics of colistin have strong binding to lipopolysaccharide constituting the bacterial cell membrane and strong chemical binding with an endotoxin composed of anions. Therefore, antibiotics that exhibit fast bactericidal properties release large amounts of endotoxins as the bacteria die, and in combination with antibiotics that bind strongly to the endotoxins released, especially colistin, the pathogens caused by endotoxins can be prevented. Can be. Streptomycin inhibits the production of endotoxins by inhibiting the synthesis of bacterial proteins rather than binding them directly.

Thus, cytral, colistin, and streptomycin have been shown to inhibit endotoxin secretion and neutralization. In particular, colistin showed very strong endotoxin neutralization.

Example  3

Determination of neutralization of endotoxins released from E. coli k88ab when combined with antibiotics selected from cytral and enroproxacin, oxytetracycline, streptomycin, cephalosporin and colistin For one night, E. coli k88ab was incubated overnight, and then reacted for 10 hours after 10 or ⁹ CFU / mL and cytral and the other antibiotics alone or in combination, and the results are shown in Table 8. Complex antibiotics containing cytral in all samples consisted of 50:50 to make 1 (w / v)%, that is, 10 mg / ml solution, and each antibiotic was used in the experiment at 10 mg / ml concentration. It was.

In addition, in order to determine the effect on the inhibition of TNF-alpha secretion E. coli k88ab pre-incubated with 10 ⁹ CFU / ㎖ was inoculated into the RAW264.7 cell line, the results are shown in Table 8 Indicated.

division Endotoxin neutralization capacity (EU / mL) TNF-alpha (ng / L) Saline solution 210 512 Central 170 613 Enrofloxacin 312 800 Oxytetracycline 321 823 Streptomycin 167 324 Cephalosporins 367 495 Colistin 24 312 Central + Enrofloxacin 167 500 Cytral + Oxytetracycline 125 310 Cytral + Streptomycin 89 321 Cytral + Sepharoseporin 187 435 Citral + Colistin 12 210

As shown in Table 8, as a result of measuring the amount of endotoxin after the reaction, the physiological saline was 210 EU / mL, but the central salt was reduced to 81 EU to 170 EU / mL. Enlofloxacin, oxytetracycline, and cephalosporin increased endotoxin amounts to 149%, 153%, and 175%, but streptomycin decreased to 80%, indicating endotoxin neutralizing ability. In addition, it was found that the endotoxin secretion was decreased when the combination of the cytral and the antibiotic was administered alone. Endotoxin was increased to 149% when enlofloxacin was administered alone, but the combined antibiotic composition of cytral-enrofloxacin was reduced to 80%. In particular, in the case of cytral-streptomycin, endotoxins were reduced by 42%, and it was found that the addition of cytral to antibiotics effectively prevented the pathogenesis of Gram-negative bacteria.

In addition, it was confirmed that TNF-alpha secretion was suppressed in the case of combination administration of cytral and the antibiotic.

Therefore, when Citral is mixed with an antibiotic selected from enroproxacin, oxytetracycline, streptomycin, cephalosporin and colistin to make a complex antibiotic composition, both endotoxin neutralization ability and TNF-alpha have good effects. Showed. In particular, cytral-streptomycin and cytral-colistin showed the best endotoxin neutralization and anti-inflammatory effects.

Therefore, considering all of Examples 1 to 3, the optimal combination is a complex antimicrobial composition mixed with Cytral-Colistin- (Sephalosporin or Streptomycin), which is strong against Staphylococcus aureus, an antibiotic-resistant strain in the treatment of cow's breast infection. It was anticipated to have anti-inflammatory effect and prevent the treatment of mastitis of cows as a new functional antimicrobial agent that suppresses anti-inflammatory action by neutralizing endotoxins secreted from pathogenic bacteria, which are picture negative strains while exhibiting antibacterial effect.

Example  4

To find the best level of antimicrobial activity, use the following formulas: Cytral / (Sephalosporin, Streptomycin or Enrofloxacin), and Cytral / Colistin / (Sepectin, Streptomycin, Sepharosporin or Enrofloc). 1) (w / v)%, that is, 10 mg / ml solution was prepared by varying the mixing ratio (weight basis) of the reaper), and MIC was measured for Staphylococcus aureus isolated from the cow mastitis strain. Indicated.

division Minimum Growth Inhibition Concentration of Complex Antibiotic (Citral / Antibiotics) (Dilution Drainage) 100/0 80/20 70/30 50/50 40/60 30/70 0/100 CS combination antibiotic 2 32 64 1024 512 256 2 CC Complex Antibiotics 2 64 64 1024 512 32 16 CE combination antibiotic 2 32 64 1024 256 256 16 50/50/0 50/25/25 50/25/50 50/50/50 25/50/50 25/25/50 0/50/50 CCoS Antibiotics 32 128 1024 64 128 128 32 CCoC Antibiotic 64 64 2048 2048 512 128 16 CCoE Antibiotic 32 128 1024 1024 512 256 16 CCoS Antibiotics 32 64 1024 1024 256 64 16

CS complex antibiotic: cytral-streptomycin

CC Complex Antibiotics: Cytral-Sephalosporin

* CE combination antibiotic: Cytral-Enrofloxacin

* CCoS Antibiotic: Cytral-Colistin-Sfectin

CCoS Antibiotic: Cytral-Colistin-Streptomycin

* CCoC Antibiotic: Cytral-Colistin-Sephalosporin

CCoE Antibiotic: Cytral-Colistin-Enrofloxacin

The test results showed that the antimicrobial activity was significantly reduced when only the antimicrobial agent or cytral was not included. However, antibacterial agents containing cytral showed very strong antibacterial activity depending on the mixing ratio. In the above results, the compound-prescribed complexes showed the effect of increasing the antimicrobial activity of the antimicrobial agent due to the interaction with cytral even if the use of the existing antimicrobial agent is reduced. The most effective antimicrobial activity was shown by mixing cytral and antibiotics at a ratio of 50:50, or 1: 1. In addition, the combination showed the greatest antimicrobial activity when added in the same amount of colistin to supplement the endotoxin neutralizing ability.

Example  5

Cytral-Sephalosporin complex antibiotics were treated as follows to observe the timely bactericidal effect against antibiotic resistant strains. In other words, the number of cultured strains was measured, and 1 ml was inoculated into 8 ml Mullertonton liquid medium prepared in advance, and 1 ml of the combined antimicrobial agent was added at the same time, 30, 60, 120, and 24 hours before inoculation. The killing rate of was measured. As a control group, antimicrobial agents ampicillin and nofloxacin, which are currently used as mastitis agents, were compared. The number of bacteria was measured according to the National Committee for Clinical Laboratory Standards (NCCLS) method, and the results are shown in Table 10 and Table 11. Table 10 shows the killing rate of Staphylococcus KCCM 40510 and Table 11 shows the killing rate of Staphylococcus KCCM 11539, each of which is in CFU / mL. Complex antibiotic 1 is a weight ratio of cytral: sephalosporin 50:50, complex antibiotic 2 is a weight ratio of cytral: sephalosporin 5: 5.

division 0 min 30 minutes 60 minutes 120 minutes 24 hours Tween 20 1.0 × 10 ⁹ 2.05 × 10 ⁹ 3.0 × 10 ⁹ 4.5 × 10 ⁹ 3.0 × 10 ¹⁰ Central (1 mg / ml) 1.0 × 10 ⁹ 5.0 × 10 ⁶ 5.0 × 10 ⁶ 0 0 Cephalosporin (1 mg / ml) 1.0 × 10 ⁹ 1.5 × 10 ⁸ 2.5 × 10 ⁸ 2.7 × 10 ⁸ 2.7 × 10 ⁸ Colistin (1 mg / ml) 1.0 × 10 ⁹ 9.5 × 10 ⁸ 9.5 × 10 ⁸ 9.5 × 10 ⁸ 9.5 × 10 ⁸ Combination Antibiotics 1 (1 mg / ml) 1.0 × 10 ⁹ 0 0 0 0 Combination Antibiotic 2 (1 mg / mL) 1.0 × 10 ⁹ 1.5 × 10 ⁸ 1.5 × 10 ⁸ 2.5 × 10 ⁷ 2.5 × 10 ⁷ Nofloxacin (10 μg / ml) 1.0 × 10 ⁹ 9.5 × 10 ⁸ 5.4 × 10 ⁸ 3.0 × 10 ⁹ 6.5 × 10 ³ Ampicillin (100 μg / ml) 1.0 × 10 ⁹ 1.1 × 10 ⁹ 2.2 × 10 ⁸ 2.4 × 10 ⁸ 3.3 × 10 ⁹

division 0 min 30 minutes 60 minutes 120 minutes 24 hours Vegetable oil 1.0 × 10 ⁹ 3.7 × 10 ⁹ 1.4 × 10 ⁹ 2.5 × 10 ⁹ 2.1 × 10 ⁹ Central (1 mg / ml) 1.0 × 10 ⁹ 2.0 × 10 ⁷ 2.0 × 10 ³ 0 0 Cephalosporin (1 mg / ml) 1.0 × 10 ⁹ 2.5 × 10 ⁴ 1.7 × 10 ² 0 0 Colistin (1 mg / ml) 1.0 × 10 ⁹ 9.5 × 10 ⁸ 9.5 × 10 ⁸ 9.5 × 10 ⁸ 9.5 × 10 ⁸ Combination Antibiotics 1 (1 mg / ml) 1.0 × 10 ⁹ 2.2 × 10 ² 0 0 0 Combination Antibiotic 2 (1 mg / mL) 1.0 × 10 ⁹ 2.5 × 10 ² 1.3 × 10 ³ 1.7 × 10 ³ 0 Nofloxacin (10 μg / ml) 1.0 × 10 ⁹ 3.5 × 10 ⁸ 5.4 × 10 ³ 0 0 Ampicillin (100 μg / ml) 1.0 × 10 ⁹ 1.5 × 10 ⁴ 5.4 × 10 ² 0 0

As shown in Table 10 and Table 11, the most popular antibacterial agents nofloxacin and ampicillin showed no effect in treating staphylococcus aureus of the resistant strain KCCM 40510. In particular, it was confirmed that the antimicrobial activity of the complex antibiotic 1 is much better than the complex antibiotic 2. Therefore, it can be seen that there is a difference in the antimicrobial activity according to the proper mixing ratio for these components. In addition, the results showed that the use of antibiotics could be reduced, as well as the use of cytral.

Example  6

In order to compare the efficacy of the combined antimicrobial agents for the treatment of cow mastitis, the strains of cows with cow mastitis were compared. Pathogens were measured for Staphylococcus, Streptococcus, Mycoplasma and Escherichia coli. Antimicrobial susceptibility test medium was inoculated in Muller-Hinton medium and pre-cultured at 37 ℃. The incubated strain was inoculated in Müllertonton medium to make a test medium and compare the antimicrobial activity of the samples used in Table 11 of Example 5. 0.5 ml (1.0 × 109) of the pre-cultured strain solution was added to 4 ml of Mulhinton medium, and 0.5 ml of each of the samples used in Table 11 of Example 5 was added, and then the number of strains was confirmed after overnight incubation. However, Mycoplasma high opneumoniae was measured by the liquid culture method using the fris medium due to the difficulty and slow growth of the culture of the bacteria after 14 days was measured by the change of medium color.

division Staphylococcus spp . (CFU / ml) M. hyopneumoniae (CCU / ml) Streptococcus spp (CFU / ml) E. coli (CFU / ml) Central (0.2 mg / ml) 2.5 × 10 ² 2.5 × 10 ² 1.7 × 10 ² NG Cephalosporin (0.2 mg / ml) 1.5 × 10 ⁵ 1.7 × 10 ¹⁰ 2.3 × 10 ⁷ 2.3 × 10 ² Streptomycin (0.2 mg / ml) 6.5 × 10 ⁶ 2.7 × 10 ⁶ 6.5 × 10 ⁶ NG Combination Antibiotics 1 (0.2 mg / mL) NG NG NG NG Combination Antibiotic 2 (0.2 mg / mL) 1.5 × 10 ⁴ 3.7 × 10 ³ 1.5 × 10 ² NG Control 1.5 × 10 ⁹ 1.6 × 10 ⁸ 2.7 × 10 ⁸ 3.2 × 10 ⁸

NG: No growth; CFU: Colony forming unit; CCU: Color changing Unit

As shown in Table 12, the complex antibiotic showed an excellent antimicrobial effect against mastitis causative bacteria. In particular, antimicrobial compound 1 was more effective than staphylococcus aureus, a causative agent of mastitis, than single antibiotics.

Example  7: Somatic cell reduction test

Massaging was performed after injecting 10 mg of the combined antibiotic 1, the combined antibiotic 2, the cytral, or the nofloxacin of Example 6 into the breast three times at 24 intervals to 8 cows with mastitis. At this time, the syringe used was a nipple insertion syringe. 50 ml of milk was used as a control before medicinal administration and tested for 1 week after drug administration. The change in somatic cell count in milk of the milk obtained from this was measured using Fossomatic 300. Specifically, the method was performed in accordance with the method of coating the cells in the milk with a fluorescent substance Ethydium bromide and then applying a certain amount to the disk to measure the somatic cells passing through the instant with a halogen lamp, the results Somatic cell count (Cells / ㎖) was measured and measured for the reduction of somatic cells over time is shown in Table 13. Measurement of somatic cells was carried out with a somatic instrument of Fosmatic-90 (FOSSMATIC-90, Denmark).

Time (week) Antimicrobial Complex 1 (10 mg) Antimicrobial Agents 2 (10 mg) Norfloxacin 10 (10 mg) Central (10 mg) 0 100% 100% 100% 100% One 45% 56% 80% 50% 2 37% 46% 70% 60% 3 32% 53% 70% 53% 4 26% 54% 73% 55% 5 32% 51% 72% 52% 7 23% 47% 73% 46%

As shown in Table 13, the complex antimicrobial agent 1 showed a decrease in somatic cell count of about 30% in cows with mastitis. Cytral administered alone showed an excellent reduction of 50%, but antimicrobial activity was increased when antimicrobial agents were added. However, even when added in a small amount showed increased antibacterial activity than norfloxacin alone. As a result, the administration of complex antimicrobial agent 1 (50:50) showed an excellent somatic cell reduction rate than the administration of cytral or antimicrobial alone.

Example  8: Toxicity test

Sprague Dwaley (SD) males were 4 weeks of age, 40 animals, and weighed 121.0 ± 4.8 g. At the start of administration, the age was 5 weeks, the number of animals was 40, and the weight was 156.4 ± 6.9 g. SD rat females were 4 weeks old, 40 animals and weighed 109.0 ± 4.9 g. The age of initiation was 5 weeks, the number of animals was 40, and the weight was 135.2 ± 6.7 g. Purification and quarantine were purified in the animal room under test for about 1 week after animal acquisition, and only healthy animals were provided for the test by observing the general condition during the period of purification.

Environmental conditions are pharmacological toxicology, set at temperature 23 ± 3 ℃, relative humidity 50 ± 10%, ventilation frequency 13 ~ 18 times / hr, lighting time 12 hours (6 am~6pm), illuminance 150 ~ 300 Lux It was carried out in an experimental animal room. All the test workers received work training before entering the animal room and wore work clothes, hoods, masks, and gloves. During the breeding period of the test period, the temperature and humidity of the animal room were measured every hour by the automatic temperature and humidity meter. As a result of the environmental measurement, there was no change that would affect the reliability of the test. Breeding boxes, breeding densities, and identification of breeding boxes were reared at 5 / cage in polycarbonate wire mesh boxes (220W × 410L × 200H mm) during the acclimation and testing periods. The cage was affixed with an identification card containing the test number, animal number and dose. Feed and water feeding method was freely ingested by irradiation sterilization of the standard animal feed (purina Korea, # 5057), and water was freely ingested by filtration purified water.

Dose setting was set to 10 ml, the maximum single oral dose per 100 g, considering the dose per rat, and 20 ml / kg was the lowest dose group, and 40 and 80 ml / kg dose groups were set at azeotropic 2.0. In addition, a control group (complex antibiotic-free group) was placed. Complex antibiotic 1 of Example 6 was used as a sample of the toxicity test.

Group separation of animals was performed as follows. First, the weight of all the animals determined to be healthy during the acclimation period was measured and divided into 5 g body weight intervals. 40 males and females were selected for individuals close to the average body weight. Each 40 males and females selected at 5 g intervals were distributed by random method so that 5 males were evenly included in each group. Individual identification of the animals was carried out by the perforation method and the identification card identification method.

The administration route and administration method were fasted overnight before administration of the animals, and then forced oral administration was performed once in accordance with the dose set in each group using an injection tube attached with an oral needle.

In acute oral dose toxicity studies using SD rats for combination antibiotics, no dead animals were observed in all males and females during the entire test period. As a general symptom, activity loss was observed in some animals, but after 2 ~ 3 days, it was returned to normal, and there were no differences in weight and other health indices compared to the control group. Autopsy findings showed that all test animals survived the entire test period and no autopsy differences or abnormalities were observed due to low and high doses.

As described above, acute oral toxicity test of rats of the combination did not show any abnormalities in general symptoms, body weight and autopsy findings, and could not identify specific target organs. In addition, the LD ₅₀ value could not be calculated because no animals died during the entire test period, and the oral dose that could cause death is estimated to be at least 10 ml / kg. In general, dead animals are not observed even at a dose of 10 times and 100 times the manufacturer's recommended standard, so oral application of the combination is considered to be very safe. And it was confirmed that the action, such as unexpected toxicity, hardly occur in clinical applications.

Production Example  1: Preparation of mastitis injection

Injectables containing a combination antimicrobial composition for the prophylaxis and treatment of mastitis in cows are infused once in a breast or lactation into one breast with 7.5 g in one syringe as prescribed below.

The composition of the injection for the treatment of mastitis of 100 g is as follows.

(Citral: colistin or streptomycin = 50: 50) 5 g

0.05 g sodium benzoate

0.5 g of allantoin

0.2 g sodium propionate

0.01 g of triprenamine hydrochloride

3 g of salt

Water-soluble or vegetable oil

Distilled water balance

Production Example  2: Cow mastitis  Preparation of propellants for prevention and treatment

A propellant containing a complex antimicrobial composition for the prevention and treatment of cow mastitis is equipped with an automatic control device to easily disinfect every corner of the teat and the teat through the micro-injection of the drug without touching the hand. to be.

The composition ratio of the 0.5% propellant (500 ml) is as follows.

2.5 g (Citral: Sepharosporin = 50: 50)

20 g of polysorbates

Ethyl Alcohol

Distilled water balance

Production Example  3: Central  Containing or containing antimicrobial agents Cow mastitis  Antibacterial paper towel for prevention and treatment

Paper towels for the prevention and treatment of cow mastitis are papers which absorb the complex antimicrobial composition and are used to massage the breast of cows or to wash hands.

The paper towel is as follows. Area of 30 cm x 30 cm paper towel with the following propellant: It contains 1 to 10% by weight.

The composition ratio of the 1% propellant (500 ml) is as follows.

(Citral: streptomycin = 50: 50) 5 g

20 g of polysorbates

Beta glucan 1 g

Ethyl Alcohol

Distilled water balance

As described above, the antimicrobial composition of the present invention has an antimicrobial action that acts directly on and kills bacteria that primarily cause cow mastitis, and secondly, neutralizes endotoxins against Gram-negative bacteria. Anti-inflammatory action is given to significantly reduce the number of somatic cells. In particular, the inclusion of cytral shows an excellent effect on staphylococcus aureus resistant to the antimicrobial agent, thereby expanding the antimicrobial range of the existing antimicrobial agent, and can improve the mastitis healing and prevention effect of the cow as a secondary selective antimicrobial agent.

Claims (9)

At least one antibiotic selected from Enrofloxacin, Oxytetracycline, Streptomycin, Sepharosporin, and Colistin, and Ciral Animal antibiotic composition, characterized in that. The method according to claim 1, One or more antibiotics selected from Enrofloxacin, Oxytetracycline, Streptomycin, Sepharosporin, and Colistin based on 1 part by weight of cytral Combination antibiotic composition for animals comprising 0.1 to 9 parts by weight. One or more antibiotics selected from Enrofloxacin, Oxytetracycline, Streptomycin, and Sepharosporin, and Colistin and Ciral Animal antibiotic composition, characterized in that. The method according to claim 3, 0.4 to 2.2 parts by weight of the colistin and Enrofloxacin, Oxytetracycline, Streptomycin, and Sepharosporin (Cepharosporin) Animal antibiotic composition, characterized in that it comprises 0.5 to 2.2 parts by weight of the above antibiotic. The method according to any one of claims 1 to 4, Sodium benzonate 0.01 to 1% by weight, allantoin 0.1 to 1.0% by weight, sodium propionate 0.1 to 1% by weight, dexamethasone sodium phosphate 0.005 to 0.5% by weight, tripelenamine hydrochloride 0.01 to An animal composite antibiotic composition further comprising any one or more components selected from 0.1 wt%, brine 1-10 wt% and propylene glycol 1-10 wt%. The method according to any one of claims 1 to 4, Animal antibiotic antibiotic composition, characterized in that for preventing or treating mastitis of cows. Injection comprising the animal antibiotic composition of claim 6. A propellant comprising the animal complex antibiotic composition of claim 6. An antibacterial paper towel comprising the animal antibiotic composition of claim 6.

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