KR100371091B1 - The composition of antibacterial complex for animal - Google Patents

Abstract

The present invention relates to a composite antibiotic composition for animals, and more particularly, by containing aminoglycoside antibiotics and chitosan oligosaccharides in a weight ratio of 1: 5 to 5: 1 by weight, thereby treating bacterial diseases of livestock and fish. The present invention relates to a combination antibiotic composition for animals that is effective in prevention and can replace expensive new antibiotics, thereby ultimately reducing production costs of livestock farms and efficiently minimizing residual residues in medicine.

Description

The composition of antibacterial complex for animal}

The present invention relates to a composite antibiotic composition for animals, and more particularly, by containing aminoglycoside antibiotics and chitosan oligosaccharides in a weight ratio of 1: 5 to 5: 1 by weight, thereby treating bacterial diseases of livestock and fish. The present invention relates to a combination antibiotic composition for animals that is effective in prevention and can replace expensive new antibiotics, thereby ultimately reducing production costs of livestock farms and efficiently minimizing residual residues in medicine.

Many antibacterial agents such as penicillin, cephalosporin, macrolide, tetracycline, aminoglycoside, chloramphenicol, polypeptides, polyenes, diterpenes, and quinolones are currently used to treat various bacterial infections in animals. It is used. The r-plasmid, known as a resistance to most synthetic antimicrobial agents, is widely distributed in Gram-negative enterobacteria such as Escherichia coli as a genetic factor of chromosomes and expresses resistance. Antimicrobial materials are developed and used, or existing antimicrobial agents are mixed and used. As a result, many problems such as excessive cost of medication, overdose of the drug, drug retention in livestock products, and the emergence of new resistant bacteria are practically occurring.

At present, the global research direction is focused on the development of non-toxic, non-toxic therapeutics using natural materials [Larry et al., 1989; Oram and Reiter, 1968; Ashton et al., 1968]. As a result, it is necessary to broaden the antimicrobial range by mixing with non-toxic non-toxic agents using natural natural substances with antimicrobial ability and to control the indiscriminate use by mixed use of drugs, while also extending the range of drug use in the direction to solve the residual problem. There is a need.

On the other hand, in case of livestock which are collectively reared, it is very difficult for the livestock to receive proper protection and management individually, even though the body itself is exposed to the external environment. Therefore, the use of natural natural substances that have antimicrobial and immunity-enhancing effects on the disease of the livestock itself can enhance the immunity against the disease, thereby enhancing the prevention effect against disease infection as well as promoting growth. You can expect Therefore, the use of natural natural substances and antimicrobial agents that have antimicrobial action and immune system strengthening effect on the domestic animal's disease at the same time can not only reduce the rapid recovery and treatment period from the disease, but also mix and Excessive use, increased administration costs due to long-term use, and drug retention problems can be solved to some extent, and can also be another way to ultimately increase the effectiveness of antimicrobial agents.

The most important thing in using the complex antibiotics as described above is the selection of natural substances having immune enhancing and antimicrobial activity, and then to determine the range of compounding ratio between the antimicrobials and natural substances. From this point of view, the chitosan oligosaccharide used in the present invention is a natural substance having an immune enhancing effect and an antibacterial activity, and the synergistic effect of the mixed use with gentamicin is very remarkable.

Chitosan-oligosaccharide is a low-molecular chitosan made by re-decomposing chitosan produced by deacetylation of chitin. These chitin and chitosan are non-toxic [Allan et al, 1979] and are naturally polymer cations that are involved in the synthesis and degradation of organisms but do not cause environmental pollution. Recently released, these materials are known to be used as potential waste resources, including environmental waste coagulants [Castellanos Perez, 1988], heavy metal adsorbents [Evans and Kent, 1962], seed coatings to increase production [Hadwiger et al, 1961], and wounds. It is widely applied in various industries such as therapeutic agent [Hadwiger et al, 1965], and also has excellent antibacterial action [長 洋 隆, 1980] and immune enhancing effect [Tanigawa et al, 1992]. Also used a lot. However, since chitosan is a polymer having a high molecular weight and is restricted in absorption and use in vivo, it is more preferable to convert chitosan to hydrolyze chitosan oligosaccharides rather than directly using them. The antimicrobial mechanism of chitosan claims that the amino group of chitosan specifically inhibits bacterial growth by binding to the cell wall of the pathogen [Uchida, 1995], and the effect of chitosan's antimicrobial activity on general bacteria on the cell surface structure [ Young et al., 1992] and the claim that it inhibits DNA formation during the metabolic process of bacteria [Stossel and Leuba, 1984], although it is not clear yet, it is known to be effective against intestinal microbial pathogens.

On the other hand, the immunological activity of chitosan was reported by Matheson (1984), et al., That D-glucosamine, the basic unit of chitosan, increases the activity of natural killer cells involved in immune defense, and Suzuki (1986) et al. It has been reported that hexasaccharide glucosamine exhibits high immune activity by reviving or increasing the immune system in which macrophages, lymphocytes and cytoplasm correlate. The first reported use of chitosan as feed for common animals was by Landes and Bough. They added 2.15% chitosan to precipitate cheese whey solids to obtain a chitosan-whey precipitate that was fed to rats and found no differences compared to lots fed casein or a simple whey solution. When rats were fed with 1.0, 2.5 or 5.0% chitosan instead of cellulose, no specific results were found.

On the other hand, the recent bacterial infectious diseases of animals have formed resistance to various drugs, so domestic livestock farms tend to indiscriminately administer antimicrobial substances, and the development and application of new antimicrobial substances in industrial animals is very difficult in Korea and abroad. It is true.

Considering such domestic and foreign situations, the composite antibiotic composition of the present invention contains chitosan oligosaccharides having non-resistant, antimicrobial and immune-boosting effects as natural substances together with aminoglycoside antibiotics, and thus can be widely applied in veterinary fields.

In addition, each of the composition drugs used in the present invention must be a substance used in a number of clinical fields, but the situation is rarely made at home and abroad in connection with the pharmacological study of their combinations.

The inventors of the present invention have been researching for many years to determine the optimal compounding ratio that shows synergistic therapeutic effect by the selection of natural substances having immune potentiation and antimicrobial activity and mixed use with other antibacterial agents. As a result, the present invention was completed by confirming that the aminoglycoside antibiotic and chitosan oligosaccharide (OCHT) were suitably blended to exhibit synergistic antimicrobial activity.

Accordingly, an object of the present invention is to provide a composite antibiotic composition for animals containing aminoglycoside antibiotics and chitosan oligosaccharides (OCHT) in a ratio of synergistic antimicrobial activity.

It is another object of the present invention to provide a use of the above-described complex antibiotic composition as an agent for treating and preventing bacterial infections in animals or an immunopotentiator.

The present invention relates to a composite antibiotic composition for animals in which aminoglycoside antibiotics and chitosan oligosaccharides are blended in a weight ratio of 1: 5 to 5: 1, and the combination antibiotic composition for use in treating and preventing bacterial infections in animals or as an immunopotentiator. Its use is characterized by that.

On the other hand, the animal in the present invention includes livestock, including chickens, pigs, cattle, horses, dogs, ducks, goats, sheep; Freshwater fish, including trout, carp and eel; And saltwater fish including flatfish, rockfish, bream and shrimp.

Such a present invention will be described in detail as follows:

The present invention relates to a novel combination antibiotic composition for animals having excellent antimicrobial activity against economic and pathogenic strains, particularly resistant E. coli, by containing an aminoglycoside antibiotic and chitosan oligosaccharide in an appropriate ratio. In particular, the use of chitosan oligosaccharide used in the present invention can not only reduce the rapid recovery and treatment period of the condensation by the antibacterial action and the immunity enhancing action during the disease, but also due to the mixing and overuse of various antibacterial agents, Increasing the cost of administration and the problem of drug retention can be solved to some extent and can ultimately be an alternative to the use of antimicrobial agents.

Next, the aminoglycoside antibiotics and chitosan oligosaccharides included in the complex antibiotic composition according to the present invention will be described.

The aminoglycoside antibiotics, which were found in 1944, based on streptomycin, are a series of antibiotics belonging to the basic oligosaccaride group consisting of amino acids and sugars, all of which inhibit bacterial protein synthesis. It exhibits bactericidal action. Such aminoglycoside antibiotics are commonly called basic water soluble antibiotics because of their high water solubility as basic substances. Aminoglycoside antibiotics show most antimicrobial effects against gram-negative pathogenic bacteria, showing a clear effect on digestive diseases caused by intestinal pathogenic microorganisms in animals, whereas gram-positive bacteria have a weak and anaerobic activity. Bacteria have no antimicrobial activity. In addition, resistance to susceptible bacteria is another weak point of aminoglycoside antibiotics, which is due to the r-plasmid of enterobacteria and the plasmid of staphylococci, and thus the aminoglyciside modifying enzyme. It is due to the action of enzyme). In addition, most of the resistance due to chromosome variation is due to the change of ribosomes. In addition, there is resistance by lowering or inhibiting the intracellular permeability of aminoglycosides. In addition, the major side effects caused by aminoglycoside antibiotics are neural disorders and eighth cranial nervous system disorders, so care should be taken to prevent the occurrence of toxicity and side effects when the dosage is excessive or used for a long time. As described above, the aminoglycoside antibiotics commonly used in the veterinary field have various disadvantages of their own, and thus, the development of new substances or supplementation with other antimicrobial substances to compensate for these defects. There is a need for the development of new combination antibiotic compositions that are expected to be synergistic. Representative examples of aminoglycoside antibiotics currently used in the veterinary field include streptomycin, kanamycin, amikacin, gentamicin, spectinomycin, and neomycin. ), And the like.

As another antibacterial substance used in the present invention, chitosan oligosaccharide has a molecular weight distribution of 200 to 150,000, preferably a distribution of 1 to 16 sugars of 45% or more, more preferably a molecular weight distribution of 200 to 99,000, and 1 to The distribution of 16 sugars is more than 55%. The chitosan oligosaccharides of the present invention described above exhibit strong antimicrobial effects [see Table 1], such as Escherichia coli, Salmonella, Staphylococcus, which are easily resistant to antimicrobial agents, and at the same time exhibit an immune enhancing effect. In relation to the immune-boosting effect, as a result of administering 1 mg of chitosan oligosaccharide used in the present invention per mouse, the gamma interferon (IFN-r) started to increase markedly after 1 hour of administration. IFN-r) increased up to about 6 times.

In general, the degree of deacetylation increases when chitosan is produced from chitin, but the molecular chain of chitin and chitosan is broken, and the molecular weight is very low, indicating a solubility limit. In particular, when chitosan is treated for a long time in a high temperature and alkaline solution, deacetylation is not only increased, but also the color of chitosan is not only changed but also the molecular weight is drastically decreased. In addition, the deacetylation degree of the polymer chitosan is very low, and a lot of waste fluids are generated, and many domestic and foreign scholars have reported the physical property control method [Alimuniar 1992; Arnold et al., 1976; Chang et al., 1979; Horowitz et al., 1957. As a method for producing chitosan oligosaccharides, chemical and enzymatic methods are largely used. The chemical method has a disadvantage in that hydrochloric acid, which is a reagent, may be left, and thus, a treatment technique may be required to completely remove it, and the enzymatic method has an economic problem of using an expensive chitosan degrading enzyme. Thus, in the present invention, in order to prevent the cleavage of the molecular chain during the production of chitin, the crab shell is finely ground to 2 mm or less, and then the temperature is adjusted to a temperature of 80 to 100 ° C. while adding nitrogen gas thereto, followed by dilution of 0.5 The pretreatment process removes primary calcium by using 0.5N HCl solution and the secondary treatment with 1 ~ 2N HCl solution using 1% NaOH solution at the temperature of 1 ~ 30 ℃ and removes residual calcium and minerals. At the same time, the decrease in viscosity is suppressed, followed by drying with natural light to obtain chitosan having excellent whiteness (molecular weight distribution: 30,012 to 350,448), followed by an enzymatic reaction using a cellulose hydrolase mixture of 90% or more and a viscosity of 2,200 cps. Hereinafter, the chitosan oligosaccharide whose molecular weight distribution is 200-150,000 and whose distribution of 1-16 sugar is 45% or more of the whole is manufactured and used. It is known that there is little toxicity in animals to chitosan oligosaccharides. When consuming 10 g of chitosan per kg body weight of the mouse, no toxicity was observed, and free chitosan was about 18 g and in the form of chitosan formate. Intake of 16 g daily did not show any particular adverse effects.

On the other hand, in the present invention, in order to investigate the interaction between the aminoglycoside antibiotics described above and chitosan oligosaccharides and to clarify the correlation between various compounding ratios, Gram-positive isolates from chickens, pigs and cattle affected by various bacterial diseases And antimicrobial effects of each combination of antimicrobial agents and chitosan oligosaccharides (OCTH) against negative bacteria were measured by the Minimum Inhibition Concentration (MIC). As a result, it was found that the compounding ratio of aminoglycoside antibiotics and chitosan oligosaccharides is preferably in the range of 1: 5 to 5: 1 by weight. When the compounding ratio is out of the above range, the synergistic effect between the two substances decreases, and the complex antibiotic It was confirmed that the antimicrobial activity of the treatment effect was reduced. Thus, the present invention can increase the antimicrobial range by producing a mixture of aminoglycoside antibiotics and chitosan oligosaccharides in the ratio as described above to suppress the emergence of strains resistant to the antimicrobial agents of the disease when the disease occurs Excellent effects can be expected for prevention and treatment.

Meanwhile, the complex antibiotic composition according to the present invention includes an aminoglycoside antibiotic and chitosan oligosaccharide mixed at an weight ratio of 1: 5 to 5: 1 as an active ingredient, and has a general knowledge in the art to which the present invention belongs. Depending on the method which can be easily carried out by itself, it may be formulated into veterinary injections, powders and granules. For example, in the case of animal injections and oral solutions, pharmaceutically acceptable carriers include distilled water, oleic acid ethyl, ethanol, propylene glycol, glycerin, and the like; Ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, tocopherol, etc .; Phenyl mercury nitrate is prepared using chimerosal, benzalkonium chloride, phenol, cresol, paraoxybenzoic acid methyl, benzyl alcohol, and the like as a preservative. Animal powders and granules are prepared by containing commonly used sugars such as vitamins, glucose, lactose, starch, powder, vermiculite or various powders and liquid enzymes in the range of usage.

In addition, the administration of the complex antibiotic according to the present invention is usually administered by the method of intramuscular injection applied to animals as an injection, the liquid is administered directly orally or mixed with negative water, and the powder and granules are mixed with negative and feed. . In particular, when administering the complex antibiotic composition according to the present invention to the animal may be toxic in the case of young dogs, careful application is required, and subcutaneous administration in the animal is thought to be desirable to avoid the other complex antibiotics safe in animals Acts as.

In addition, in the dosage of the complex antibiotic according to the present invention, in the case of injections and liquids, 0.5-50 mg (active ingredient basis) per kg body weight of the animal is administered for the purpose of preventing and treating diseases, and the powders and granules are negative. When administered as 30 to 2,000 g (active ingredient basis) per ton of negative water. In the case of mixing and feeding the feed, 100 to 2,000 g per tonne of feed (active ingredient) are administered based on the condition of the animal.

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

Examples 1-5 and Comparative Examples 1-8

In the antimicrobial susceptibility test medium, Muller Hinton agar medium (17.5% hydrolyzed casein with acid, 3% bee extract, 1.5% starch, 15% agar) in Table 2a, Table 2b and The concentrations of the mixtures of antibiotics and chitosan oligosaccharides (OCHT) in the mixing ratios described in Table 2c were adjusted by two-step dilution. The chitosan oligosaccharides (target, 'dark horse') used in this example had a deacetylation degree of 90% or more, a molecular weight distribution of 200 to 99,000, a distribution of 1 to 16 sugars of 55% or more, and a viscosity of 2,200 or less. Oligosaccharides were used. In addition, the strains used in the present examples are Gram-positive strains Streptococcus faeculis (hereinafter abbreviated as 'A strain') and Staphylococcus aureus R-209 (hereinafter called 'B strain'). Abbreviation) and Escherichia coli K88ac (hereinafter abbreviated as 'C strain'), Escherichia coli K88ab (abbreviated as 'D strain'), and Escherichia coli BE (hereinafter referred to as 'E strain') as gram-negative strains. And Salmonella typhymurium (hereinafter abbreviated as 'F strain') were inoculated into the medium. After incubation for 10 to 20 hours at 37 ℃ was determined as the minimum inhibitory concentration (MIC) by examining the concentration of the antimicrobial agent in the lowest concentration medium without the growth of bacteria. This was in accordance with the National Committee for Clinical Laboratory Standards (NCCLS) method.

As can be seen in Table 2, the MIC value of the composite antibiotic composition for animals of the present invention when administered as a complex antibiotic in the range of 1: 5 to 5: 1 weight ratio than when using the aminoglycoside antibiotic alone or chitosan oligosaccharide alone It was confirmed that there is a synergistic effect among these antimicrobial substances by lowering to 16 times. According to the above results, the compounding ratio of the aminoglycoside antibiotic and chitosan oligosaccharide was preferably in the range of 1: 5 to 5: 1, and it was confirmed that the synergistic effect between the two substances was significantly reduced when the compounding ratio was outside the above range.

Experimental Example

In order to perform an outdoor treatment test for livestock disease of the complex antibiotic composition of the above embodiment, it was tested by the following composition and method.

(1) 1/1 ml of experimental drug (hereinafter referred to as 'COG')

Gentamicin: 50 mg

-Excipient (5% chitosan oligosaccharides): the amount to make the total amount of COG 1ml (about 0.9ml)

(2) Experimental drug 2 / 1ml (hereinafter referred to as 'gentamicin')

Gentamicin: 50 mg

Poultry (家禽) T peuseu (salmonella gallinarum) experiment inoculation ^{(salmonella gallinarum 1 × 10 9 CFU} / ㎖, 1㎖ oral administration), a 20-week-old brown laying hens (産卵鷄) and drug treated groups can be 7 with the number 14, Seven controls were assigned. Then, 0.1 ml / kg body weight and 0.1 ml / kg body weight of COG were administered for 3 days to evaluate the therapeutic effect. The results of the treatment effect according to the administration of such poultry typhus are shown in Table 3 below.

As can be seen in Table 3, compared with the COG preparation according to the present invention and the gentamicin single administration, the survival rate is higher than the 29% administered alone alone, 71% of the COG mixed formulations can be seen that shows a high therapeutic effect .

In addition, after the drug treatment for the salmonella gallinarum inoculation group, the results for organ re-separation in the survival system group is shown in Table 4 below.

In the results of Table 4, salmonella gallinarum was not isolated from the cecum, egg yolk and liver in the treatment group, salmonella gallinarum was re-separated from the cecum in the non-drug control group.

Experimental Example 2

Toxicity test during intramuscular injection and oral administration of the composite antibiotic composition prepared in the above Examples was carried out as follows.

Drug 1 COG of Experimental Example 1 was used, and the experimental animals were Spraque Dawley rats without a specific pathogen, supplied from KAIST experimental animal center, and 20 females and 24 females of 5 to 6 weeks of age were purified for 3 days before administration. The animals were reared in a rat cage and fed freely with feed and drinking water.

The experimental group was divided into 0.1ml (recommended amount), 0.3ml (three times the recommended amount), and 0.5ml (five times the recommended amount) per kg body weight, and five oral administration groups were assigned according to the administration route. The intramuscular injection group selected 6 healthy individuals and assigned them. The dose was determined by measuring the weight before administration of the evaluation drug, and the weight before administration was 120-135 kg.

Each experimental group was administered orally with a syringe for intragastric administration for rats for 5 consecutive days once daily for oral administration. Intramuscular injection was given once a day for 3 days. Doses for each subject were based on body weight at each administration, and administration time was administered at 9-10 am each time, and the control group was fed only drinking water and feed.

Autopsy schedule (oral administration group: 1, 5, 10, 14 days after administration; intramuscular injection group: before injection, 1, 3, 5, 9 and 14 days), the weight of the lungs, kidneys, and spleens was measured by ether anesthesia, specific body weights, blood collections, and autopsies immediately followed by partial autopsy procedures. In addition, each organ including the brain was extracted and fixed in 10% neutral formalin solution, and then embedded in paraffin through a partial tissue treatment process. Hematoxylin & Eosin (H & E) staining was performed by cutting it into 3 ~ 4㎛. , The presence or absence of tissue lesions under an optical microscope and the results are shown in Tables 5-8.

In Tables 5 and 6, pathological lesions in other tissues were not observed except for mild hyperemia of the renal cortex.

In Table 7, only congestion within the range of physiological levels was seen, and no change was observed after injection.

In Table 8, it can be seen that no pathologically significant lesions are observed.

From the above results, it can be seen that the composite antimicrobial composition of the present invention has no toxicity and excellent stability when the reference dose is used according to the use criteria.

As described above, the complex antibiotic composition of the present invention is an aminoglycoside antibiotic and chitosan oligosaccharides prepared using natural substances are mixed with each other in an appropriate content ratio, which is useful for the treatment and prevention of bacterial diseases in animals, particularly livestock and fish. In addition, it can increase the therapeutic effect by the immune boosting effect of chitosan oligosaccharides, thereby replacing expensive new antimicrobial substances, ultimately reducing the production cost of livestock farmers and efficiently minimizing the residues of drugs in the shaft. It can be effected.

Claims (7)

An aminoglycoside antibiotic and chitosan oligosaccharide are combined at a weight ratio of 1: 5 to 5: 1 to have antimicrobial activity against Gram-positive and Gram-negative bacteria. According to claim 1, wherein the aminoglycoside antibiotic is a combination antibiotic composition for animals, characterized in that the gentamicin (Gentamicin). According to claim 1, wherein the chitosan oligosaccharide has a molecular weight distribution of 200 to 150,000, the distribution of 1 to 16 sugars complex antimicrobial composition for animals, characterized in that the more than 45%. According to claim 3, wherein the chitosan oligosaccharide has a molecular weight distribution of 200 to 99,000, 1 to 16 sugars of the composite antimicrobial composition, characterized in that the distribution of 55% or more. The combination antibiotic composition for animals according to claim 1, wherein the animal is at least one livestock selected from the group consisting of chicken, pig, cow, horse, dog, duck, goat and sheep. delete delete