CN108813166B

CN108813166B - Use of lauroyl arginine ethyl ester derivatives as feed nutritional energy substances

Info

Publication number: CN108813166B
Application number: CN201810649022.9A
Authority: CN
Inventors: 易正芳; 邵婷; 仇文卫; 刘明耀
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2017-11-03
Filing date: 2018-06-22
Publication date: 2022-06-07
Anticipated expiration: 2038-06-22
Also published as: CN108935967A; CN108813166A

Abstract

The invention relates to a novel additive for providing animal nutrition energy, in particular to a derivative of lauroyl arginine ethyl ester ion pair compound prepared by the reaction of lauroyl arginine ethyl ester LAE and organic acid, which is used as an additive of livestock and aquatic livestock for providing nutrition energy substances, the concentration of the effective components is 0.3 to 0.9 percent of the feed of livestock and poultry or 0.01 to 1.0 percent of the feed of aquatic animals according to the total weight of the feed, which is different from the concentration of the derivative used as veterinary drug or therapeutic feed, and which compound is capable of providing a synergistic effect with respect to the original nutritional energy supplement LAE, the feed can obviously promote the growth of animals within the total weight proportion range, can effectively inhibit or kill bacteria, greatly reduces the risk of infecting the bacteria of poultry, livestock and aquatic animals, and improves the survival rate of the animals infected with pathogenic bacteria.

Description

Use of lauroyl arginine ethyl ester derivatives as feed nutritional energy substances

The invention requires Chinese patent application 201711071138.0, named as lauroyl arginine The use of ethyl esters and derivatives thereof as nutritional energy substances for feed "priority application.

Technical Field

The invention relates to an ion pair compound of lauroyl arginine ethyl ester, a preparation method and application of the ion pair compound in the aspects of antibiosis and promotion of growth of cultured animals. In particular to an ion pair compound generated by lauroyl arginine ethyl ester and organic acid and used for preparing animal nutrition energy additives, in particular to the application of the animal nutrition energy additives in the growth promoting direction of animals.

Background

Riemerella anatipestifer disease is a contact infectious disease caused by Riemerella Anatipestifer (RA), and is also called duck infectious serositis, duck septicemia, duck plague syndrome, pasteurellosis of duck disease and the like. The ducklings are mostly seen in 1-8 weeks old, and the ducklings 2-4 weeks old are most susceptible. Acute or chronic septicemia is mainly manifested by eye and nose secretion increase, asthma, cough, diarrhea, ataxia and head and neck tremor, and head and neck distortion in a few chronic cases. The pathological changes are characterized by cellulose pericarditis, perihepatitis, air sacculitis, meningitis and arthritis appearing in partial cases, and the diseases often cause a large amount of morbidity and mortality of ducklings. The morbidity of the disease can reach more than 90 percent, and the mortality is related to the age of the duck in illness, the virulence of the strain and adverse stress factors, and can reach 75 percent at most. Besides death of 1-8 weeks old ducks, salpingitis can be caused, the laying rate of adult ducks is reduced, growth of the adult ducks is retarded, and great economic loss is caused to farmers. Since the first report in 1982, the disease has become one of the most common bacterial diseases endangering the meat duck breeding industry. Under natural conditions, the disease occurs all the year round. The disease is mainly caused by contaminated feed, drinking water, dust, spray and the like entering the poultry body through respiratory tract, digestive tract or skin wound (especially webbed skin). Various kinds of ducks such as Beijing duck, cherry valley duck, Digao duck, Muscovy duck, sheldrake and the like can be infected and diseased. 48 duck farms in Jiangsu province were randomly spot checked from 7 to 8 months in 2012, wherein 45 duck farms had suspected riemerella anatipestifer disease cases, and even 12 duck farms had twice outbreaks of the disease during the spot check. In addition, if a certain duck farm suffers from the disease, the disease can be developed in the surrounding duck farms successively, so that the disease can be seen to cause serious damage to the duck farming industry.

Staphylococcus aureus is a generic term for the different diseases or types of diseases in many animals caused by Staphylococcus aureus. The pathogenic staphylococcus aureus can cause the diseases of various poultry and livestock, young livestock and poultry are most susceptible to the diseases and are mostly infected through digestive tracts, chickens can also be infected through respiratory tracts, and common symptoms comprise diarrhea, enteritis, liver necrosis and the like. Suppurative diseases are usually caused, and mastitis, arthritis, wound infection, septicemia and the like of animals are mainly caused; the other is toxic diseases, and the feed polluted by pathogenic bacteria can cause toxic enteritis of animals, toxin shock syndrome of human beings and the like. The pathogenicity of staphylococcus aureus is mainly determined by virulence pathogenic factors generated by staphylococcus aureus, and mainly comprises plasma coagulase, enterotoxin, thermostable nuclease, hemolytic toxin, leukocidin and the like.

Colibacillosis refers to the generic term for the different diseases or types of diseases in many animals caused by pathogenic E.coli. Pathogenic Escherichia coli and nonpathogenic Escherichia coli normally colonized in intestinal tracts of human and livestock are not different in morphology, staining reaction, culture characteristics, biochemical reaction and the like, but are different in antigen structure. Pathogenic Escherichia coli can cause diseases of various poultry and livestock, such as pig, cattle, sheep, horse, chicken, rabbit, etc., and young livestock and poultry are most susceptible to the diseases and are mostly infected through digestive tract, and chickens can also be infected through respiratory tract. The colibacillosis of pigs is also different in clinical diagnosis of piglets according to the growth period of piglets and the serotype of pathogenic bacteria, and can be divided into yellow dysentery type, white dysentery type and edema type. When yellow scours occur to piglets, the disease death rate is high and can reach 100% in some piglets, which is more than 90% of the normal piglets and one litter of piglets; the incidence rate of white diarrhea is 30-80%; the incidence rate of edema disease is 10-35%. Colibacillosis in chicks is commonly manifested by acute septicemia, vitelline peritonitis, ophthalmia, ballonflam head syndrome, and the like. The morbidity can reach 30-60 percent, and the fatality rate can reach 100 percent.

Aeromonas sobria is a gram-negative facultative anaerobic bacterium, is usually present in various water environments and soil environments, and is a zoonosis pathogenic bacterium. Aeromonas sobria can produce various pathogenic factors such as lysin, ectoenzyme and the like, and can cause aquatic animals to suffer from septicemia, so that the death of the aquatic animals is caused, and the economic basis of aquaculture is seriously influenced. In addition, pathogenic bacteria can infect people through aquatic products, and the patient can have symptoms such as diarrhea and the like and even develop food poisoning or septicemia.

Besides improving the feeding conditions, the application of antibiotics is a main measure for preventing and treating bacterial diseases of livestock, poultry and aquatic products. However, in recent years, common antibiotics such as aureomycin, oxytetracycline, tetracycline, chloramphenicol and the like have been widely used in livestock and poultry farming due to their disease-resistant and growth-promoting effects. According to statistics, the annual antibiotic raw material production in China is about 21 ten thousand tons, and 9.7 ten thousand tons of antibiotics are used in livestock and poultry breeding industry and account for 46.1 percent of the total production. Improper antibiotic use, no guarantee of drug quality, incomplete supervision and non-strict medication regulations lead to the aggravation of antibiotic abuse, so that a plurality of unreasonable serious problems are generated, such as the generation of bacterial drug resistance, the reduction of the immune function of animal bodies, the drug residue of meat products and the like, and the health of human beings is directly harmed. According to researches, about 75% of antibiotics cannot be absorbed and metabolized by human or animal bodies, part of the antibiotics can remain in the bodies, and 20% -50% of live chicken or frozen chicken tissues can detect the antibiotic residues; the residual antibiotics can also enter the environment along with excrement, some antibiotics can directly enter a river channel and influence the drinking water safety of downstream residents, and the residual antibiotics of oxytetracycline, tetracycline, doxycycline, amoxicillin, aureomycin and other livestock and poultry industry antibiotics are detected in tap water of many residents in rivers and cities in China. In recent years, the urine of 1000 children in Shanghai is researched to detect antibiotics, and a plurality of veterinary antibiotics (such as tylosin, chlorotetracycline, enrofloxacin and the like) which are only used in the breeding industry are detected in 58% of urine samples. More importantly, the residual antibiotics can naturally select the microorganisms in the environment or induce the genetic mutation of the microorganisms, and the bacteria with drug resistance survive and continue to breed more drug-resistant bacteria. In addition, the bacteria can also transmit own drug resistance genes to other microorganisms of different species and genera by means of conjugation, transformation, transduction, transposition and the like so as to obtain drug resistance. This makes the resistant bacteria enrich in the environment, and polluted soil and water source are easy to cause diseases and accelerate the diffusion of resistant bacteria once contacted by people or livestock. Veterinary drugs and feed additives mainly include preservatives, dust-proofing agents, antioxidants, antiprotozoal drugs, and the like, in addition to illegal addition of antibiotics or antibacterial agents. The substances can bring harm to the health of livestock and poultry and human bodies due to long-term use or improper use.

Therefore, in the cultivation of aquatic products, livestock and poultry, especially in the cultivation of poultry (such as chicken and duck), there is an urgent need for an antibacterial agent which can be rapidly sterilized, is easily degraded in vivo, has no residue, can effectively prevent the occurrence and spread of diseases in the cultivation industry, and can avoid the influence of similar antibiotic residues on human body and environment.

Lauroyl arginine Ethyl ester (LAE) is an organic matter formed by condensing fatty acid and dibasic amino acid, is a white hygroscopic solid, is stable in chemical property within the range of pH 3-7, has a melting point of 50-58 ℃, can be dispersed in 1kg of water at the temperature of 247g, has a distribution coefficient of more than 10 in water and oil, and is mainly in a water phase. Researches find that the lauroyl arginine ethyl ester LAE has the characteristics of strong antibacterial capability, low biological toxicity, good in vivo metabolism effect and high environmental compatibility. The most representative characteristic is that no residue is left in the metabolism of lauroyl arginine ethyl ester, and related researches show that the lauroyl arginine ethyl ester can be rapidly and naturally metabolized in human bodies and animal bodies to generate lauric acid and arginine which are further metabolized into ornithine, urea, carbon dioxide and water. All primary metabolites and final metabolites produced during the metabolism of lauroyl arginine ethyl ester are non-toxic and harmless, and are the same as the metabolites of food ingested daily by humans and animals in the body.

For example, Chinese patent application CN201710056593, entitled "a fruit and vegetable preservative and a preparation method and application thereof" discloses a composition taking lauroyl arginine ethyl ester hydrochloride and sodium methyl paraben as main active ingredients to be used as the fruit and vegetable preservative, which can effectively inhibit the growth of bacteria causing fruit and vegetable rot. However, the single bacteriostatic effect of the high-concentration methyl paraben sodium (2000 mug/ml) is better than that of the low-concentration LAE (1000 mug/ml) because the high-concentration methyl paraben sodium has a phenolic hydroxyl structure and the antibacterial performance is far stronger than that of benzoic acid and sorbic acid, so that on the premise of ensuring the preservative performance, the method definitely indicates that the use of the sodium methyl paraben instead of the LAE is helpful for reducing the dosage cost of the preservative.

Chinese patent application CN201510748675, entitled "method for inhibiting alcohol fermentation contaminating microorganisms by using lauroyl arginine ethyl ester" discloses a method for inhibiting alcohol fermentation contaminating microorganisms by using lauroyl arginine ethyl ester, which comprises adding LAE and salt compounds thereof into fermentation liquor of saccharomyces cerevisiae at a concentration of less than 50 μ g/ml, and can effectively inhibit the growth of lactic acid bacteria and control the growth of other contaminating microorganisms. However, this bacteriostatic slightly affects yeast growth to some extent and results in a 0.6% decrease in alcohol production.

Chinese patent application CN201610466729, entitled "a mild infant shampoo and bath bubble" discloses a mild infant shampoo and bath bubble, which is prepared by selecting disodium cocoyl glutamate, cocamidopropyl betaine, and sodium hydroxypropyl lauryl glucoside crosslinked polymer as surfactant system, selecting camellia oil, alpha-glucan oligosaccharide/inulin complex as conditioning component, and flos Chrysanthemi Indici extract and lauroyl arginine ethyl ester HCl as antiseptic system, wherein the raw materials cooperate with each other, and has good cleaning effect, mildness and no irritation.

Chinese patent application CN201280073013, entitled "synergistic antimicrobial agent", discloses that by combining an effective amount of an N- α -long chain alkanoyl dibasic amino acid alkyl ester salt with a glycerol mono fatty acid ester provides a synergistic antimicrobial composition resulting in more effective antimicrobial agents and food preservatives. Meanwhile, chinese patent application CN200810131638, entitled "microbicide composition", discloses the use of a composition of methylisothiazolinone and LAE for the preparation of antimicrobial agents and food preservatives. However, this method involves various bacteriostatic components including LAE, and the individual bacteriostatic effects of LAE have not been studied. Meanwhile, the invention only teaches the use of the composition for daily products, detergents, wound care compositions, various foods, various medical cleaning products, and the like, and does not teach how to use a single LAE component for antibacterial feeds for livestock and aquatic products.

Chinese patent application CN201280027864, entitled "cosmetic or dermatological sunscreen formulation with improved water resistance", discloses the use of LAE for the preparation of a cosmetic or dermatological sunscreen formulation comprising, in addition to a UV filter, the emulsifier polyglycerol-10 stearate.

As the closest prior art, chinese patent application CN200580051259, entitled "preservation system comprising cationic surfactant", discloses for the first time the use of LAE and its hydrochloride in preservation systems, which system comprising 0.2g/kg LAE is added in foods, cosmetics to play a role of preservation. The invention researches the antibacterial mechanism of the LAE and provides the application of the LAE in the preservative action of foods, cosmetics and the like, so that the US food safety agency approves lauroyl arginine ethyl ester for food preservatives in 2005; the european union food safety agency, australia and new zealand in 2012 also approved lauroyl arginine ethyl ester for use as a food preservative. Meanwhile, in view of the application of the invention in cosmetics for the first time, subsequent researches have found that lauroyl arginine ethyl ester can be used in products for oral care (for example, US20100330136A1, EP2361606A2, EP231603A2), such as mouthwash, toothpaste and the like, can effectively inhibit the formation of dental plaque in the oral cavity, and is compatible with other chemical components in the mouthwash and stable in chemical properties; the lauroyl arginine ethyl ester can be used in cosmetics with local therapeutic efficacy, which have the following characteristics: antibacterial effect, low toxicity, no sensitization, and no irritation to skin. Currently, researchers are developing hand lotions for cleansing and bacteriostatic agents for application to the skin surface.

Disclosure of Invention

In view of the above, the prior inventions do not teach how to use a single lauroyl arginine ethyl ester (LAE) and its derivatives as an animal nutritional energy supplement for livestock and aquatic products, nor do they disclose a suitable concentration of lauroyl arginine ethyl ester (LAE) and its derivatives as an animal nutritional energy supplement. Therefore, the invention utilizes the bacteriostatic effect of lauroyl arginine ethyl ester and derivatives thereof or hydrates thereof (preferably LAE) as preservatives to develop new LAE derivatives on the basis of the prior invention application of LAE as a feed nutritional energy substance, wherein the traditional idea for the development of the derivatives is broken through, namely the traditional idea is not limited to selecting proper forms of acid, alkali and salt/ester which are traditionally suitable for LAE or processing acid, alkali, salt or esterification groups on LEA, but an acid radical group which can enhance the bacteriostatic synergistic effect of LAE and obviously supplement the nutritional energy substance is creatively selected, and the two are combined into a new derivative, namely an ion pair compound, through strong intermolecular ionic bonds, so that the application of the LAE derivatives as the feed nutritional energy substance is obviously improved. Further, under the condition of ensuring the antibacterial effect of the LAE, the influence of the LAE serving as an energy substance in different antibacterial feeds on the nutrition growth of livestock, poultry and aquatic animals is respectively analyzed, so that the proper proportion for simultaneously realizing antibacterial and disease prevention and providing energy nutrition is determined.

The invention aims to provide the use of lauroyl arginine ethyl ester (LAE) derivatives with the structural formula shown as the following formula (I) for preparing animal nutrition energy additives:

wherein the content of the first and second substances,

x is an organic acid radical RCOO with antibacterial and energy effects^-(ii) a The RCOO^-An acid radical selected from salicylic acid, formic acid, acetic acid, diacetic acid, propionic acid, butyric acid, lactic acid, benzoic acid, sorbic acid, fumaric acid, citric acid, tartaric acid, malic acid, phosphoric acid, oxalic acid or carbonic acid;

R₁is a linear saturated fatty acid group containing 8 to 14 carbon atoms, or a linear oxoacid group containing 8 to 14 carbon atoms;

R₂is containing 1 to 18 carbon atomsOr a branched fatty acid group containing 1 to 18 carbon atoms, or an aromatic group containing 1 to 18 carbon atoms, or a straight chain alkyl group containing 1 to 4 carbon atoms;

preferably, it is a linear saturated fatty acid containing 2 carbon atoms;

R₃is one of the following structures.

n ranges from 0 to 4.

In one embodiment, the RCOO^-Acid radicals selected from salicylic acid, formic acid, acetic acid, diacetic acid, propionic acid, butyric acid, lactic acid, benzoic acid, sorbic acid, fumaric acid, citric acid, tartaric acid, malic acid, phosphoric acid, oxalic acid or carbonic acid.

In another embodiment, the organic acid radical RCOO^-Acid radical selected from nicotinic acid, tartaric acid and oxalic acid.

In one embodiment, the present invention provides lauroyl arginine ethyl ester (LAE) derivatives having the structural formula shown below in formula (III):

RCOO^-acid radicals selected from salicylic acid, formic acid, acetic acid, diacetic acid, propionic acid, butyric acid, lactic acid, benzoic acid, sorbic acid, fumaric acid, citric acid, tartaric acid, malic acid, phosphoric acid, oxalic acid or carbonic acid.

In another embodiment of the above, the organic acid radical RCOO^-Acid radical selected from nicotinic acid, tartaric acid and oxalic acid.

In any of the above embodiments, the livestock, aquatic livestock comprises: chicken, duck, goose, turkey, quail, pigeon, pig, cattle, sheep, horse, camel, cat, dog, fish, shrimp, crab.

In a preferred embodiment, the animal is a livestock animal and the effective ingredient concentration of the derivative is 0.3% to 0.9% by total weight of the feed. In a preferred embodiment, the concentration of the active ingredient is 0.6% by weight based on the total weight of the feed.

In another embodiment, the animal is an aquatic animal and the effective ingredient concentration of the derivative is 0.01% to 1% by total weight of the feed. In a preferred embodiment, the aquatic animal is a fish and the concentration of the active ingredient is 0.1% to 1% by weight based on the total weight of the feed. In a particular embodiment, the animal is selected from tilapia, the active principle of the derivative is 0.1% by weight based on the total weight of the feed, and the pathogenic microorganism is selected from pathogenic aeromonas sobria.

In any of the above embodiments, the use is the direct addition of the product to animal feed; or mixing the product with carrier to obtain premix; or mixing with other feed additive or feed raw materials to make into premix and concentrated feed for feeding animals.

The derivative can obviously promote the growth of animals in the total weight proportion range of the feed, can effectively inhibit or kill bacteria, greatly reduces the risk of infecting the bacteria of poultry, livestock and aquatic animals, and improves the survival rate of the animals infected with pathogenic bacteria.

In any of the above embodiments, the use of the derivative as an animal feed supplement for providing a nutritional energy substance refers to the use of the derivative as an animal nutritional energy supplement at a concentration which is different from the concentration at which the derivative is used as a veterinary drug or therapeutic feed and at which the derivative promotes the vegetative growth of the animal as an energy substance and prevents disease in the animal.

In any of the embodiments described above, the derivative is a LAE nicotinic acid ion pair compound.

The invention has the beneficial effects that:

the research proves that the nicotinic acid belongs to B vitamins, and the nicotinic acid is involved in lipid metabolism in vivo, an oxidation process of tissue respiration and a process of anaerobic carbohydrate decomposition. Mainly synthesized by plants and microorganisms (certain animals can realize self-synthesis through tryptophan, but the synthesis amount is far less than self-requirement), and the substances which cannot be stored in large quantities in the bodies of the animals but are indispensable for the growth of the animals. Ruminants can be supplied by microorganisms in the rumen, and poultry and swine are fed exogenously. Niacin deficiency manifests itself as follows: poultry exhibit thin feathers with loose, lusterless skin and foot dermatitis with short and thick legs and bends; livestock exhibit dermatitis, decreased appetite, and emaciation. According to the 2013 feed additive variety catalog in China, nicotinic acid belongs to a vitamin additive, can be used for various cultured animals, and is beneficial to the healthy growth of the cultured animals.

Tartaric acid is an organic acid present in many plants and also the main organic acid in wine and is commonly used as an antioxidant in food additives. Oxalic acid is also an organic acid present in many green plants, such as spinach, sweet potatoes, taro, rhubarb, etc., and cocoa beans also contain large amounts of oxalic acid. It is therefore desirable to synthesize ion pairs of LAE with these organic acids, which are non-toxic and even beneficial to animal health, to improve the disease resistance or growth promotion of LAE.

According to the invention, through experiments, organic acid which has certain bacteriostatic ability and can play a role of nutrition (such as nicotinic acid) or energy (oxalic acid) or is required by biological metabolism is selected and used as the organic acid, and a single ion pair compound is generated through condensation reaction and LAE, so that the physical and chemical properties such as monomer molecule solubility, stability and the like can be changed, respective activity can be exerted, and a synergistic effect can be generated, so that the bioavailability is improved, and the curative effect of the medicine is further improved; meanwhile, the liposome is easy to penetrate lipophilic cell membranes and improve absorption.

The present invention demonstrates that the derivatives of formula (III) also have the following advantages over LAE compounds of formula (I):

1. the derivatives represented by the formula (III) of the present invention do not inhibit the antibacterial activity of the original LAE as a single component, but rather are beneficial to the antibacterial activity. Wherein, compared with LAE, the nicotinic acid ion pair derivative has obvious bacteriostatic effect on staphylococcus aureus.

2. The derivative represented by the formula (III) does not induce a drug resistance mutation of a pathogenic microorganism when administered continuously for 30 days, and has a reduced ability to induce drug resistance of Staphylococcus aureus relative to LAE, compared to commercially available antibiotics, which induce bacterial drug resistance.

3. The derivatives of formula (III) of the present invention do not induce hemolysis of red blood cells at minimal bactericidal concentrations.

4. The derivative shown in the formula (III) can improve the survival rate of ducklings infected with riemerella anatipestifer, reduce the risk of the ducklings infected with riemerella anatipestifer, has no influence on the survival rate of healthy ducklings, and has lower dosage and better treatment effect compared with the effect of an LAE derivative

5. The effective dosage range of the derivative shown in the formula (III) as a medicament is obviously lower than the upper limit dosage range of 40mg/kg bw required by the production of LAE

6. The derivative shown in the formula (III) can restore the increase of the protein levels of inflammatory factors IL-1 beta and TNF-alpha caused by bacterial infection in animals.

7. Experiments prove that the medicine is stopped for 24 hours after the administration for 3 months, and the drug residue of heart, liver, spleen, lung, kidney, small intestine, stomach and muscle tissues of the animals in the administration group meets the requirement of European Union on the drug residue of the non-specified veterinary drugs.

8. Experiments prove that the derivative shown in the formula (III) can prevent and treat bacterial diseases of aquatic animals and can promote the vegetative growth of poultry and aquatic animals, wherein: anti-infection experimental results show that all test fishes fed with the feed not containing the derivative shown in the formula (III) die, and the survival rate of tilapia fed with the derivative shown in the formula (III) can reach 91.67 +/-2.89, which is higher than the survival rate of 88.33 +/-7.63 of LAE; for tilapia, the relative weight gain rate can be improved by 3.52%, 6.91% and 4.64% and the specific growth rate can be improved by 7.94%, 13.08% and 6.54%, the feed coefficient can be reduced by 4.76%, 5.56% and 2.38%, the relative weight gain rate, 6.23% and 3.88% and the specific growth rate are all higher than the relative weight gain rate, 12.32% and the feed coefficient can be reduced by 3.15%.

9. The lauroyl arginine ethyl ester derivative is safe to degrade in human bodies and animal bodies, has small toxic and side effects, avoids the generation of environment-resistant bacteria caused by the emission of residual antibacterial agents to the environment, achieves effective antibacterial effect and has small negative influence on the environment.

Drawings

FIG. 1: ESI mass spectrum of cation B + molecular ion peak of LAE ion pair compound;

FIG. 2: ESI mass spectrum of anion A-molecular ion peak of LAE nicotinic acid ion pair compound;

FIG. 3: peak shape and chemical shift pattern of 1H-NMR of LAE;

FIG. 4: peak shape and chemical shift pattern by 1H-NMR of nicotinic acid;

FIG. 5: peak shape and chemical shift pattern by 1H-NMR of LAE nicotinic acid ion pair;

FIG. 6: ESI mass spectrum of the anion A-molecular ion peak of the compound versus LAE tartrate.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

The first embodiment is as follows: preparation method of ion pair compound synthesized from lauroyl arginine ethyl ester hydrochloride and nicotinic acid

Dissolving 2.0g of sodium nicotinate (purchased from Taishiai (Shanghai) chemical industry development Co., Ltd.) in 50mL of water to prepare a sodium nicotinate saline solution (A); dissolving lauroyl arginine ethyl ester hydrochloride 6.8g in 40mL of water, heating to 90 ℃ until the lauroyl arginine ethyl ester hydrochloride is completely dissolved to prepare lauroyl arginine ethyl ester hydrochloride aqueous solution (B); slowly adding the sodium nicotinate aqueous solution (A) into the lauroyl arginine ethyl ester hydrochloride aqueous solution (B) at 90 ℃, continuously stirring, reacting for 2 hours, cooling to room temperature, filtering, fully washing the precipitate with purified water, and performing vacuum drying on the precipitate at 60 ℃ to obtain 7.6g of the nicotinic acid ion pair compound.

Example analysis of molecular formula and molecular weight of compound by dilauroyl arginine ethyl ester nicotinic acid ion pair

By mass spectrometry,¹H-NMR、¹³The compound obtained by C-NMR spectroscopy has the formula:

1. mass Spectrometry (ESI) analysis

Cation B⁺The molecular ion peak has m/z 385.3, see fig. 1;

mass Spectrometry detection ESI + was 124.2, see FIG. 2. The ESI-is then 122.2, i.e. anion A^-The molecular ion peak m/z is 122.2.

The theoretical calculation of the niacin ion for the cation in the compound was 507.4, and the observed value coincided with the theoretical value.

NMR analysis

Extracting lauroyl arginine ethyl ester hydrochloride (see FIG. 3), nicotinic acid¹H-NMR (see FIG. 4) and of ion-pairing compounds¹H-NMR (see FIG. 5). In the salt forming process of the LAE ion pair compound, the peak shape and chemical shift of lauroyl arginine ethyl ester in the ion pair compound are not changed greatly, but all hydrogen on nicotinic acid has shift change, and the spectral characteristics of the acid and base part are closer to the space distance compared with the original inorganic acid salt (namely LAE hydrochloride), so that the influence is generated, and the corresponding change is generated compared with the original LAE and the hydrochloride thereof, the simple superposition of the acid and base parts is not generated, for example, the solubility is changed when purified water is used for washing and precipitating, which shows that strong interaction is generated between all hydrogen nuclei of the lauroyl arginine ethyl ester and the nicotinic acid, and a stable single compound structure is formed through strong ionic bonds.

Example three: preparation method of ion pair compound synthesized by lauroyl arginine ethyl ester hydrochloride and tartaric acid

2.0g of tartaric acid (purchased from Chiese chemical industry Co., Ltd.) was dissolved in 50mL of methanol, and an equivalent amount of NaOH was added thereto, and the mixture was stirred at room temperature until a white solid was precipitated, and then the solution was filtered under suction and washed with 30mL of methanol three times to obtain a tartaric acid sodium salt. Dissolving sodium tartrate salt in 50mL of water to prepare a sodium tartrate salt aqueous solution (A); dissolving 5.6g of lauroyl arginine ethyl ester hydrochloride in 40mL of water, heating to 90 ℃ until the lauroyl arginine ethyl ester hydrochloride is completely dissolved to prepare lauroyl arginine ethyl ester hydrochloride aqueous solution (B); slowly adding the tartaric acid sodium salt aqueous solution (A) into the lauroyl arginine ethyl ester hydrochloride aqueous solution (B) at 90 ℃, continuously stirring, reacting for 2 hours, cooling to room temperature, filtering, fully washing the precipitate with purified water, and drying the precipitate in vacuum at 60 ℃ to obtain 6.3g of the tartaric acid ion pair compound.

Example analysis of molecular weight of Compounds by Artocylarginine Ethyl ester tartrate ions

Mass Spectrometry (ESI) analysis of cation B⁺Molecular ion peak m/z 385.3 (see fig. 1)

Anion A^-Molecular ion peak m/z 149.0 (see FIG. 6)

The theoretical calculation of the niacin ion for the cation in the compound was 534.3, and the observed value coincided with the theoretical value.

Example five: preparation method for synthesizing ion pair compound by using lauroyl arginine ethyl ester hydrochloride and oxalic acid

Oxalic acid (purchased from research Co., Ltd.) 1.0g was dissolved in 50mL of methanol, and an equivalent amount of NaOH was added thereto, and the mixture was stirred at room temperature until a white solid precipitated, filtered under suction and washed with 30mL of methanol three times to obtain an oxalic acid sodium salt. Dissolving sodium oxalate in 50mL of water to prepare sodium oxalate aqueous solution (A); dissolving 4.7g of lauroyl arginine ethyl ester hydrochloride in 40mL of water, heating to 90 ℃ until the lauroyl arginine ethyl ester hydrochloride is completely dissolved to prepare lauroyl arginine ethyl ester hydrochloride aqueous solution (B); slowly adding the sodium oxalate salt aqueous solution (A) into the lauroyl arginine ethyl ester hydrochloride aqueous solution (B) at 90 ℃, continuously stirring, reacting for 2 hours, cooling to room temperature, filtering, fully washing the precipitate with purified water, and drying the precipitate in vacuum at 60 ℃ to obtain 5.0g of the oxalate ion pair compound.

The results of NMR and ESI analyses performed according to the method of example two show that the ion pair compound is not a simple superposition of two acid and base portions, which are closely spaced and affect the spectral characteristics, and the spectral data of the ion pair compound is changed compared with the original LAE and its hydrochloride, for example, the solubility is changed when the precipitate is washed with purified water, which indicates that all hydrogen nuclei of lauroyl arginine ethyl ester have strong interactions with oxalic acid and form a stable single compound structure through strong ionic bonds.

Example six: preparation method of ion pair compound synthesized by lauroyl arginine ethyl ester hydrochloride and carbonic acid

1.0g of sodium carbonate (purchased from research Co., Ltd.) was dissolved in 50mL of water to prepare an aqueous sodium carbonate solution (A); dissolving 4.0g of lauroyl arginine ethyl ester hydrochloride in 40mL of water, heating to 90 ℃ until the lauroyl arginine ethyl ester hydrochloride is completely dissolved to prepare lauroyl arginine ethyl ester hydrochloride aqueous solution (B); slowly adding the sodium carbonate aqueous solution (A) into the lauroyl arginine ethyl ester hydrochloride aqueous solution (B) at 90 ℃, continuously stirring, reacting for 2 hours, cooling to room temperature, filtering, fully washing the precipitate with purified water, and drying the precipitate in vacuum at 60 ℃ to obtain 4.0g of the carbonate ion pair compound.

The results of NMR and ESI analyses performed according to the method of example two show that the ion pair compound does not have a simple superposition of two acid and base portions, the two acid and base portions are close in space distance and have an influence, and the spectral data of the ion pair compound is changed correspondingly compared with the original LAE and hydrochloride thereof, which indicates that all hydrogen nuclei of the lauroyl arginine ethyl ester have strong interaction with carbonic acid and form a stable single compound structure through strong ionic bonds.

Example seven: determination of lauroyl arginine ethyl ester ion pair compound Minimum Inhibitory Concentration (MIC) in vitro

The principle and the purpose are as follows: according to the microbubult dilution method specified by CLSI, the minimum drug concentration at which bacterial growth is inhibited after 24h of co-incubation of the drug with bacteria in a 96-well plate is the minimum inhibitory concentration of the drug.

The method comprises the following steps: diluting lauroyl arginine ethyl ester hydrochloride (LAE) and the prepared lauroyl arginine ethyl ester organic acid ion pair with Trypticase Soy Broth (TSB) to different concentrations,the drug and the bacteria are mixed and incubated in a 96-well plate, and a blank control culture medium CK1 without the bacteria, a culture medium CK2 added with LAE (1000 mu g/ml) and a normal growth control culture medium CK3 without the drug are additionally arranged. The absorbance at 625nm of each well was measured after incubating the 96-well plate in a 37 ℃ incubator for 24 hours. OD with blank control₆₂₅Wells with consistent values were considered to have no significant growth of bacteria. The lowest concentration of drug at which bacteria do not significantly grow is the minimum Inhibitory concentration mic (minimum inhibition concentration) of LAE to bacteria.

The results of comparing the antibacterial activity of various LAE derivatives (ion pair compounds) prepared with respect to the original LAE compound are shown in table 1 below.

TABLE 1 in vitro antibacterial Effect of LAE and its ion pair Compounds on three bacteria

Comparison	Escherichia coli	Staphylococcus aureus	Riemerella anatipestifer (Riemerella anatipestifer)
				LAE	16	8	16
LAE nicotinic acid ion pair	16	4	16
				LAE tartrate ion pair	16	8	16
LAE oxalate ion pair	8	8	16
				LAE carbonate ion pair	16	16	8

And (4) analyzing results:

(1) most of the ion pair compounds keep the same antibacterial activity to escherichia coli, and especially the antibacterial activity of the oxalic acid ion pair compounds is improved;

(2) most of the ion pair compounds keep the same antibacterial activity to staphylococcus aureus, the antibacterial activity of the carbonate ion pair compounds is reduced, and the antibacterial activity of the nicotinic acid ion pair compounds is obviously improved;

(3) most of the ion pair compounds keep the same antibacterial activity to the Riemerella anatipestifer, and especially the antibacterial activity of the carbonate ion pair compounds is obviously improved.

And (4) conclusion: ion pair compounds of LAE derivatives do not inhibit the antibacterial activity of the original LAE in a single component, but are beneficial to the antibacterial activity. Wherein, the nicotinic acid ion pair compound has obvious bacteriostatic effect on staphylococcus aureus.

Example eight: experiment for inducing bacterial drug resistance by lauroyl arginine ethyl ester ions to compound

The principle and the purpose are as follows: the bacteria can be induced to generate drug-resistant bacteria for the drug under the sub-bacteriostatic concentration, and the ability of the drug-induced bacteria to generate drug-resistant mutation is detected by transferring the bacteria cultured by the drug with the sub-bacteriostatic concentration into a fresh drug-containing culture medium for a long time.

The method comprises the following steps: the bacteria in log phase and the LAE organic acid ion pair solution are mixed in a glass test tube, sealed by a cotton plug and cultured for 24 hours in a constant temperature shaking table at 37 ℃ and 220 rpm. Then checking the MIC of the drug every 24h, adding the bacteria liquid with the highest drug concentration (namely the highest drug concentration lower than the MIC) capable of culturing the turbid bacteria liquid into a fresh culture medium containing different drug concentrations again according to the proportion of 1:100, culturing at 37 ℃ and 220rpm for 24h, and taking chloramphenicol as a positive control group for 30 days. The ratio of drug to bacterial MIC to initial MIC was calculated after 30 days.

TABLE 2 LAE and MIC/initial MIC ratio of its ion pair to compound after 30 days of continuous bacterial challenge

Comparison	Riemerella anatipestifer (Riemerella anatipestifer)	Escherichia coli	Staphylococcus aureus
				LAE
	1	1	2
				LAE nicotinic acid ion pair	1	1	1
LAE tartrate ion pair	1	1	1
				LAE oxalate ion pair	1	1	1
LAE carbonate ion pair	1	1	1
				Florfenicol	8	16	1
Chloromycetin	6	12	12
				Ampicillin	8	8	2

The results show that the LAE organic acid ions do not cause the bacteria to be tolerant after stimulating the bacteria for 30 days, the Riemerella anatipestifer and the Escherichia coli do not cause the bacteria to be tolerant to the LAE, and the staphylococcus aureus only generates weak tolerance resistance to a single component of the LAE. The florfenicol, chloramphenicol and ampicillin have very strong ability to induce bacterial drug resistance, and the minimum inhibitory concentration rises to 6-16 times of the original concentration in 30 days. Thus, LAE and its ion pair have low ability to induce bacterial resistance relative to commercial antibiotics, and the ability of LAE ion pair to induce s.

Example nine: effect of lauroyl arginine ethyl ester nicotinic acid ion pair compound on preventing and treating bacterial diseases of livestock

Oral treatment effect of LAE nicotinic acid ion pair on riemerella anatipestifer infected ducklings

The principle and the purpose are as follows: after ducklings are infected with bacteria, the survival condition of animals is observed by orally taking the LAE nicotinic acid ion pair, and the treatment effect of the LAE nicotinic acid ion pair on poultry suffering from bacterial diseases is studied.

The method comprises the following steps: the ducklings 50 were randomly divided into 5 groups of 10 ducks. Mixing Riemerella anatipestifer with 4 × 10⁶The amount of CFU was inoculated subcutaneously in the legs of four groups of ducklings to be an infected group, and the other group was set as a blank group without any treatment. After 12h, 4 infection groups are respectively administrated with LAE aqueous solution or blank aqueous solution control groups with different dosages in a stomach-drenching mode, the death condition is observed and recorded after 1h, 7h, 12h, 24h, 48h, 72h and 96h after the drug administration, the survival condition of 96h animals is shown in a table 3-1, and the treatment effect of LAE is shown in a table 3-2.

Table 3-1: therapeutic effect of oral LAE nicotinic acid ion on ducklings infected with listeriosis anatipestifer

Tables 3-2: therapeutic efficacy of oral LAE (see prior application 201711071138.0)

Note: the test group is animals infected with pathogenic bacteria and given LAE or nicotinic acid ion pairs thereof with different concentrations;

the blank group is animals which are not contacted with pathogenic bacteria;

the control group was animals infected with pathogenic bacteria but not administered.

The results show that:

(1) the mortality rate of the ducklings suffering from riemerella anatipestifer disease without oral administration of LAE or ion pair derivatives thereof is 100 percent;

(2) after illness, the death rate of sick ducklings infected by orally taking 4 mg per kilogram of body weight of LAE nicotinic acid ion pairs is reduced to 80 percent, and the death rate of ducklings administered with LAE is still 100 percent;

(3) the mortality rate of the ducklings affected by oral administration of 8 mg per kg of body weight of LAE nicotinic acid ion pair is only 30%, while the mortality rate of the ducklings administered with LAE is still 60%;

(4) oral administration of 16 mg/kg body weight of LAE nicotinic acid ion pair reduced the mortality to 20%, while the mortality of ducklings dosed with LAE was still 40%;

and (4) conclusion: the oral LAE nicotinic acid ion pair can treat riemerella anatipestifer disease of ducklings, and compared with the effect of an LAE compound, the oral LAE nicotinic acid ion pair has lower dosage and better treatment effect.

Control effect of LAE nicotinic acid ion on riemerella anatipestifer infection of ducklings

The principle and the purpose are as follows: before infecting bacteria, ducklings take the LAE ion pair orally, after infecting the ducklings, the LAE ion pair continues to take the oral LAE ion pair orally every day, the survival condition of animals is observed, and the effect of the LAE ion pair serving as an antibacterial agent for protecting poultry from being infected by the bacteria is researched.

The method comprises the following steps: the ducklings 50 were randomly divided into 5 groups of 10 ducks. 2 of these groups were administered with a blank aqueous solution as a gavage to give an untreated group, and 3 of these groups were administered with a LAE nicotinic acid ion pair aqueous solution as a gavage to give a test group. The Riemerella anatipestifer is administered at 4 × 10 for 8h after the first administration⁶The legs of ducklings inoculated in three experimental groups and one non-administration control group were used as infected groups by subcutaneous injection of the amount of CFU, and the other 1 non-administration group was used as a non-infected group without any treatment (i.e. without infection of bacteria and administration as blank). Blank aqueous solutions were administered in the gavage mode to the uninfected group and the infected group at 12h, 24h, and 48h after infection, respectively, and the LAE nicotinic acid ion pair aqueous solutions of different concentrations were administered to the

experiment

1, 2, and 3 groups, respectively. Death was observed and recorded at 1h, 7h, 12h, 24h, 48h, 72h, 96h post-dose, and the results for 96h survival are shown in Table 4-1. The LAE preventive effect is shown in Table 4-2.

TABLE 4-1: the oral LAE ion pair derivative has the effect of preventing and treating duck plague Lymerella anatipestifer infection.

Tables 4-2: control of Duck's resistance to Riemerella anatipestifer infection by oral LAE (see prior application 201711071138.0)

Note: the test group is animals infected with pathogenic bacteria and given ion pairs of LAE or derivatives thereof with different concentrations;

the blank group is animals which are not contacted with pathogenic bacteria and are not administrated;

And (4) analyzing results:

(1) the death rate of the ducklings infected with pathogenic bacteria and not orally taking the LAE or the ion pair derivatives thereof is 100 percent;

(2) the mortality rate of the sick ducklings orally taking 4 mg per kilogram of body weight LAE nicotinic acid ion pairs before infection is reduced to 50 percent;

(3) the mortality rate of the ducklings affected by orally taking 8 mg/kg body weight of LAE nicotinic acid ion pairs before infection is only 10 percent, while the mortality rate of the ducklings administered with LAE is still 30 percent;

(4) the mortality rate of the sick ducklings orally taking 16 mg per kilogram of body weight LAE before infection is only 20 percent, which is not as high as the prevention and treatment effect of orally taking 8 mg per kilogram of body weight LAE nicotinic acid ion pair;

(5) the mortality rate of sick ducklings orally taking 40mg per kilogram of body weight of LAE nicotinic acid ion pairs before infection is still 10 percent, while the mortality rate of the ducklings taking LAE is also 10 percent;

and (4) conclusion: oral administration of the LAE ion pair before the duckling is infected with bacteria can improve the survival rate of ducklings again compared with the administration after infection, and 8 mg of LAE compound has obtained the maximum prevention and treatment effect of the latter. Therefore, the oral dosage of LAE of 4-40mg/kg bw can effectively prevent the duckling from infecting the Riemerella anatipestifer. The continuous administration can exert the effect of preventing and treating diseases to a greater extent. All the 10 ducklings in the solvent control group die within 96h, and 9 ducklings in the administration group survive at the end of the experiment, and the survival rate reaches 90%. Considering the 4-8 day treatment cycle and the cost of LAE therapeutic solvent, the recommended dose range of LAE nicotinic acid ion pair of 10-20mg/kg bw is already the upper limit of the production requirement, significantly lower than the upper limit dose range of 40mg/kg bw required for LAE production.

Therapeutic effects of LAE ion on mice infected with gram-negative bacteria and gram-positive bacteria.

The principle and the purpose are as follows: being able to be taken orally and exert therapeutic effect is a high requirement for antibacterial agents, so we use mice as a representative of domestic animals, infect the mice with gram-negative bacteria escherichia coli, respectively, and then evaluate the therapeutic effect of the drug on animals by oral administration of LAE ion pairs.

The method comprises the following steps: 50 Balb/c mice were randomly divided into 5 groups of 10 mice each. Bacteria in log phase are treated with 10⁸The amount of CFU was injected into the abdominal cavity of 4 groups of mice, and the other group was set as an uninfected group without any treatment. After 1h, 0.5ml of physiological saline was orally administered to 1 infection group, and LAE ion pair solutions of different concentrations were orally administered to the other three infection groups. The survival of the mice after 24h was counted to obtain the results shown in tables 5-1 and 6. The therapeutic effect of LAE on gram-negative bacteria, Escherichia coli, is shown in Table 5-2.

TABLE 5-1 therapeutic Effect of oral LAE Niacin ion pair derivatives on E.coli infection in the Abdominal Cavity of mice

Tables 5-2: therapeutic Effect of oral LAE on Abdominal infection with E.coli in mice (see prior application 201711071138.0)

Note:

the test group is animals infected with pathogenic bacteria and orally taking LAE or nicotinic acid ion pairs thereof with different concentrations;

the blank group is animals which are not contacted with pathogenic bacteria;

the control group is a group infected with pathogenic bacteria but not administered.

And (4) analyzing results:

(1) after infection, LAE nicotinic acid ion pair with 0.625 mg per kilogram of body weight can remove pathogenic bacteria in mice by 25.6 percent, the effect is better than that of LAE with the same dose, and the removal rate of the LAE is only 21.9 percent;

(2) after infection, LAE nicotinic acid ion pair with 2.5 milligrams per kilogram of body weight can remove pathogenic bacteria in mice with the clearance rate of 51.2 percent, the effect is better than that of LAE with the same dosage, and the clearance rate of the LAE is only 44.21 percent;

(3) after infection, LAE nicotinic acid ion pair with 10mg per kilogram of body weight can remove pathogenic bacteria in mice by 41.6 percent, the effect is better than that of LAE with the same dose, and the removal rate of the LAE is only 33.95 percent;

and (4) conclusion: the administration of the LAE ion pair after the infection of the mice with bacteria has a better effect of removing pathogenic bacteria in vivo and a lower administration dose than the administration of the LAE compound.

The effect of LAE ion alone on the treatment of mice infected with gram-positive bacteria (Staphylococcus aureus) was tested in the same manner as described above, and the results are shown in Table 6-1. The therapeutic effect of LAE on gram-negative bacteria, Escherichia coli, is shown in Table 6-2.

TABLE 6-1 therapeutic Effect of oral LAE Niacin ion pair derivatives on mice Abdominal infection with Staphylococcus aureus

TABLE 6-2 therapeutic Effect of oral LAE on mice Abdominal infection with Staphylococcus aureus

Note:

the test group is animals infected with pathogenic bacteria and orally taking LAE nicotinic acid ion pairs with different concentrations;

the blank group is animals which are not contacted with pathogenic bacteria;

And (4) analyzing results:

(1) after infection, LAE nicotinic acid ion pair with 2.5 milligrams per kilogram of body weight can remove pathogenic bacteria in mice with 62.7 percent, the effect is better than that of LAE with the same dose, and the removal rate of the LAE is only 61.5 percent;

(2) after infection, LAE nicotinic acid ion pair with 10mg per kilogram of body weight can remove pathogenic bacteria in mice by 60.8 percent, the effect is better than that of LAE with the same dose, and the removal rate of the LAE is only 50.2 percent;

(3) after infection, LAE nicotinic acid ion with 25 mg/kg body weight is applied to ensure that the pathogenic bacteria clearance rate in mice is 67.2 percent, the effect is better than that of LAE with the same dose, and the clearance rate of the LAE is only 61 percent.

Example ten: lauroyl arginine ethyl ester ion pair compound used as feed additive for preventing diseases of mammals

Directly adding the active ingredients of the LAE nicotinic acid ion pair into the mouse feed in an amount of 0.01-1.0% by weight of the total weight of the feed; or mixing the product with carrier to obtain premix; or mixing with other feed additives or feed raw materials to make into premix and concentrated feed for feeding mice.

50 mice of 6 weeks old were selected and randomly divided into 5 treatment groups according to the principle of similar body weight. The control group is fed with corn-wheat-soybean-meal type daily ration, the test group is fed with corn-wheat-soybean-meal type daily ration, LAE ion pairs with the total weight of 0.01%, 0.1% and 1% of the total weight of the feed are added, and after the control group is fed for 7 days, the disease resistance of the mice to escherichia coli is measured. The results are shown in Table 7-1. The feeding effect of the LAE compound as a feed additive is shown in Table 7-2.

Table 7-1: disease control of mammals by adding LAE nicotinic acid ion pair derivatives into feed

Table 7-2: disease control in mammals with feed supplemented LAE (see prior application 201711071138.0)

Note:

the test group is a feed which is contacted with pathogenic bacteria and added with LAE or ion pairs thereof in different doses;

the blank group was not exposed to pathogenic bacteria;

the control group is feed without LAE or its ion pair

And (4) analyzing results:

(1) the death rate of animals infected with pathogenic bacteria without LAE or ion pair derivative feed is 100%;

(2) the animal survival rate of the feed containing 0.01 percent of the ion pair derivatives is 60 percent, which is higher than the feeding effect of LAE under the same condition by 50 percent;

(3) the animal survival rate of the feed containing 0.1 percent of the ion pair derivatives is 80 percent and is higher than the feeding effect of LAE under the same condition by 70 percent;

(4) the survival rate of animals is 90 percent and is higher than 80 percent of the feeding effect of LAE under the same condition when the feed contains 1 percent of ion pair derivatives;

and (4) conclusion: as can be seen from the above table, the survival rates of mice in all test groups are significantly increased compared with the control group, wherein the best group is the test 3 group, the survival rate is 90%, and all experimental animals in the control group, to which no LAE nicotinic acid ion pair is added, die, which indicates that LAE nicotinic acid ion pair has a prevention and treatment effect on mammalian diseases as a feed additive and has a better prevention and treatment effect compared with LAE compound as a feed additive. Considering the treatment period and the cost of the LAE nicotinic acid ion pair therapeutic agent, the effective concentration of the LAE nicotinic acid ion pair serving as a medicament or feed additive is 0.01-1%, preferably 0.1-1%, which can effectively prevent and treat the diseases and meets the production requirement.

Example eleven: LAE nicotinic acid ion pairs as nutritional energy supplements for the vegetative growth of mammals

According to the application range of the concentration of the LAE nicotinic acid ion pair determined in the fifth embodiment, on the basis of the same daily ration formula of each group, the LAE ion pair is added into the complete formula feed by 0.3%, 0.6% and 0.9% in mass ratio respectively in the groups of

experiments

1, 2 and 3, and no medicine is added into the control group. 40 Balb/c mice of 6 weeks old were randomly divided into 4 groups, and the weight of each group was weighed at day 1 at the start of the study, and the weight of the mice was weighed again after 15 days. The results are shown in Table 8-1. The results of the effect of LAE on the vegetative growth are shown in Table 8-2.

Table 8-1: effect of feed addition of LAE ion on growth of mammals

Table 8-2: effect of feed supplementation of LAE on mammalian growth (see prior application 201711071138.0)

Note:

the test group is the feed added with LAE and ion pairs thereof with different dosages;

the control group is feed without LAE or ion pair thereof

And (4) analyzing results:

(1) the average daily gain of animals fed with no LAE or ion pair derivative thereof is almost the same and is the lowest level in the same group of experiments;

(2) the daily gain weight of animals fed with 0.3%, 0.6% and 0.9% of ion pair derivative feed is slightly higher than the level of animals fed with LAE feed with the same proportion;

(3) from the point of view of the net weight gain of each individual on the 15 scales, the level of 2 groups was significantly higher than that of 1 and 3 groups in both feeding tests.

This shows that the addition of 0.6% LAE ion pair to the mouse feed significantly increased the average weight gain of the mice by 12.5% (while the level of LAE gain was only 9.64%) compared to the control group during the experiment. Therefore, the addition of the LAE ion pair to the feed can promote the growth of mammals.

Example twelve: LAE nicotinic acid ion pair as feed additive for preventing and treating diseases of ducklings

The effective component of the LAE nicotinic acid ion pair accounts for 0.01-1.0% of the total weight of the feed, and the LAE nicotinic acid ion pair is directly added into the duckling feed; or mixing the product with carrier to obtain premix; or mixing with other feed additives or feed raw materials to make into premix, concentrated feed for feeding duckling.

50 cherry ducklings of 14 days old are selected and randomly divided into 5 treatment groups according to the principle of similar weight. Each treatment was repeated 1 time (column), and each column was 10 (male and female halves). The control group is fed with corn bean pulp type daily ration, the test group is fed with corn bean pulp type daily ration, and added with LAE nicotinic acid ion pairs with the total weight of 0.01 percent, 0.1 percent and 1 percent of the total weight of the feed, and after the ducklings are fed for 7 days, the resistance of the ducklings to riemerella anatipestifer disease is measured. The results are shown in Table 9-1. The results of feeding the LAE compound as a feed additive are shown in Table 9-2.

Table 9-1: the LAE ion pair derivative added into the feed has the effects of preventing and treating diseases of ducklings:

table 9-2: disease control effect of feed additive LAE on ducklings (see prior application 201711071138.0)

Note:

the blank group was not exposed to pathogenic bacteria;

the control group is feed without LAE or ion pair thereof

And (4) analyzing results:

(2) the survival rate of animals is 70% when the feed contains 0.01% of the ion pair derivative, and the survival rate of LAE feeding under the same adding condition is 60%;

(3) the feed contains 0.1% of ion pair derivatives, the survival rate of animals is 90%, and the survival rate of LAE feeding under the same condition is 80%;

(4) the animal survival rate of the feed containing 1% of the ion pair derivatives is 90%, and the animal survival rate is the same as the animal survival rate of the feed containing LAE under the same condition;

and (4) conclusion: as can be seen from the above table, the survival rate of the ducklings in all test groups is remarkably increased compared with that of the control group, wherein the best group is the test 3 group, the survival rate is 90%, and all experimental animals in the control group without adding the LAE ion pair die, which indicates that the LAE ion pair has a prevention and treatment effect on ducklings diseases as a feed additive, and the LAE ion pair can effectively prevent and treat diseases when the effective concentration of the LAE ion pair as a medicament or a feed additive is 0.01-1% in consideration of the treatment period and the cost of the LAE ion pair as a therapeutic agent, and meets the production requirement.

EXAMPLE thirteen, LAE Niacin ion pairs as nutritional energy supplement for disease prevention and vegetative growth of ducklings

According to the application range of the concentration of the LAE nicotinic acid ion pair determined in the seventh embodiment, on the basis of the same daily ration formula of each group, the LAE ion pair is added into the complete formula feed by 0.3%, 0.6% and 0.9% in mass ratio respectively in the groups of

experiments

1, 2 and 3, and no medicine is added into the control group. Weighing the weight of each group of animals at the 1 st day when the test is started, weighing the weight again after 15 days, and measuring the disease resistance of each group of the animals to the Riemerella anatipestifer. The results are shown in Table 10-1. The feeding effect of LAE is shown in Table 10-2.

TABLE 10-1: disease prevention and treatment and vegetative growth effect of LAE nicotinic acid ion pair derivatives added into feed on ducklings

Table 10-2: the feed additive LAE has effects of preventing and treating diseases and promoting vegetative growth of ducklings (see prior application 201711071138.0)

Note:

the test groups were feeds supplemented with different amounts of LAE or its ion pair;

the control group is feed without LAE or ion pair thereof

And (4) analyzing results:

(1) the prevalence rates of animals infected with pathogenic bacteria without LAE or ion pair derivative feed are all 100%;

(2) the animal morbidity is 20 percent, and the feeding effect of the LAE is the same with that of the LAE under the same condition;

(3) the average daily gain of animals fed with no LAE or ion pair derivative thereof is almost the same and is the lowest level in the same group of experiments;

(4) the daily weight gain of animals fed with 0.3%, 0.6% and 0.9% of ion pair derivative feed is slightly higher than the level of animals fed with LAE feed with the same proportion;

(5) from the point of view of the net weight gain of each individual on a 15-balance scale, the level was significantly higher in both feeding trials in 2 groups than in 1 and 3 groups.

And (4) conclusion: therefore, compared with a control group, the average weight gain of the ducklings can be improved by adding 0.3-0.9% of LAE ion pairs into the ducklings feed in the test period, wherein the weight gain effect of 0.6% of LAE ion pairs is most obvious, and the average weight gain of the ducklings is remarkably improved by 11.5% compared with that of the control group. Therefore, the LAE ion pair can reduce the death of the duckling caused by the Riemerella anatipestifer disease, improve the growth performance of the duckling and promote the vegetative growth of the duckling.

Considering the growth cycle and the cost of the LAE ion pair feed additive, the effective concentration of the LAE ion pair as the feed additive is 0.3-0.9%, preferably 0.6-0.9%, and most preferably 0.6%, so that the disease can be effectively prevented and treated, and the production requirement is met.

Example fourteen: LAE nicotinic acid ion pair as nutritional energy additive for preventing diseases and promoting growth of aquatic animals

240 tilapia with the weight of 40.9 +/-0.18 g is selected to be divided into 4 treatment groups, LAE nicotinic acid ion pairs with different gradient levels are respectively added, each group has 3 repetitions, and 20 fishes are repeated. The effect of LAE niacin ion on the growth performance of tilapia was evaluated by 8 weeks of growth test. When the tilapia is fed with the LAE nicotinic acid ions, aeromonas sobria infection is carried out on tilapia fed with the feed for 8 weeks, and the disease resistance of the tilapia after the infection is reflected by the survival rate of the tilapia. The results are shown in Table 11-1. The effect of LAE feeding is shown in Table 11-2.

TABLE 11-1 Effect of feed addition of LAE nicotinic acid ion on the growth of Tilapia mossambica

TABLE 11-2 Effect of feed addition of LAE on the vegetative growth of Tilapia mossambica (see earlier application 201711071138.0)

Note:

the test groups are feeds added with different doses of LAE or nicotinic acid ion pairs thereof;

the control group is feed without addition of LAE or its nicotinic acid ion pair

And (4) analyzing results:

(1) the survival rate of animals infected with pathogenic bacteria without LAE or ion pair derivative feed is 0%;

(2) the feed contains 0.01 percent of ion pair derivatives, the animal survival rate is 63.66 percent, and the relative weight gain rate is 250.1 percent, which are both higher than the feeding survival rate and the relative weight gain rate of the LAE under the same condition;

(3) the feed contains 0.1% of ion pair derivatives, the animal survival rate is 80%, and the relative weight gain rate is 258.3%, which are both higher than the feeding survival rate and the relative weight gain rate of LAE under the same conditions;

(4) the survival rate of animals is 91.67%, and the relative weight gain rate is 252.8%, which are both higher than the survival rate and the relative weight gain rate of LAE fed under the same conditions;

(5) from the average individual net weight gain at 8 weeks, the level was significantly higher in both feeding trials in 2 groups than in 1 and 3 groups. And (4) conclusion: the addition of LAE ion pairs in tilapia feed can improve the relative weight gain rate by 3.52%, 6.91% and 4.64% and the specific growth rate by 7.94%, 13.08% and 6.54%, reduce the feed coefficient by 4.76%, 5.56% and 2.38%, and promote the growth of tilapia.

(6) Although the survival rate was highest in the 1% dose group of the two tests, the growth performance was not highest but in the 0.1% dose group. From the practical point of view of treatment, increasing the dosage within a certain range of the medicine can increase the curative effect, but the administration of an excessively high dosage sometimes affects the growth of the host, and the reason for this is still to be studied, and may be that the excessively high dosage has side effects on the vegetative growth.

(7) Anti-infection experimental results show that all test fishes eating the feed without the LAE ion pair die, and the survival rates of tilapia eating the feed with the LAE ion pair are 63.66%, 80% and 91.67%, respectively. Thus, the anti-infection capability of the fish can be improved by adding 0.01-1% of LAE ion pairs.

Considering the growth cycle and the cost of the LAE ion pair feed additive, the effective concentration of the LAE ion pair as the feed additive is 0.01-1%, preferably 0.1-1%, and most preferably 0.1%, so that the disease can be effectively prevented and treated, and the production requirement is met.

Example fifteen comparison of the therapeutic Effect of LAE ion pairs with antibiotics and LAE on diarrhea piglets

The applicant entrusts a large pig farm in Henan province to perform SY (LAE) and LAE ion pair experiments on 118 piglets with diarrhea in the farm.

A gentamicin administration group was also set as a parallel control.

The affected pigs were drenched 3 times a day in the morning (7:00), in the middle (12:00) and at night (18: 00).

Evaluation criteria: animals without drug administration after diarrhea treatment are counted as remaining animals, namely the test is stopped;

the animals that died inefficiently after the piglets were dosed were counted as dead animals, i.e. the test was stopped.

The results are shown in Table 12.

TABLE 12

As shown in the table above, the average cure rate for piglets treated with gentamicin was 77.5%. However, this dose has far exceeded the dose used with conventional antibiotics, suggesting that antibiotic residues in piglets will be significantly out of the norm in order to achieve this cure rate.

The cure rate of the piglet treated by the LAE is 72.2% at the minimum dose of 10mg/kg, and 78.57% at the maximum dose of 50mg/kg, which both far exceed the cure rate of the antibiotic at the same dose (the cure rate of the gentamicin at 10mg/kg is not shown), which indicates that the cure rate of the LAE at the same dose is higher than that of the gentamicin when the LAE is used for treating diarrhea of the piglet, and due to the good biological metabolism characteristic, the LAE indicates that no harmful drug residue exists in the piglet body, and provides a good direction for replacing the antibiotic for animals.

As regards the LAE ion pair, trials are ongoing in the treatment of diarrhea piglets. However, according to the test results that the LAE ion pair compound consistently showed better antibacterial property than the LAE compound in the nine to fourteen examples, the inventors could expect that the LAE ion pair compound would have better therapeutic effect on diarrhea piglets than the LAE compound, which indicates that the LAE ion pair compound can also be widely used in agricultural production instead of antibiotics.

Claims

1. Use of an ethyl Lauroyl Arginine (LAE) derivative having a structure as shown in formula (II) below for the preparation of an animal nutritional energy supplement:

wherein RCOO-is selected from nicotinic acid, and the derivative is LAE nicotinic acid ion pair compound,

when the animal is pig, cattle or sheep, the effective component concentration of the derivative is 0.6 percent based on the total weight of the feed;

when the animal is a chicken, a duck or a goose, the concentration of the effective components of the derivative is 0.6 percent based on the total weight of the feed;

when the animal is fish, shrimp or crab, the effective component concentration of the derivative is 0.1% by weight of the total weight of the feed; and (c) a second step of,

the LAE nicotinic acid ion pair compound is prepared by the following reaction:

(1) dissolving lauroyl arginine ethyl ester hydrochloride in water, heating to 90 ℃ until the lauroyl arginine ethyl ester hydrochloride is completely dissolved, and then slowly adding a sodium nicotinate solution at 90 ℃;

(2) fully stirring and uniformly mixing, and reacting under the condition of heating to 90 ℃ to obtain an LAE nicotinic acid ion pair compound;

(3) after sufficient reaction, the reaction mixture is cooled to room temperature, and is fully washed and purified by purified water and then dried in vacuum, so that the purified LAE nicotinic acid ion pair compound is prepared.

2. The use according to claim 1, wherein the animal is a pig or a duck and the concentration of the effective ingredient is 0.6% by weight based on the total weight of the feed.

3. Use according to claim 1, wherein the animal is selected from tilapia, and the effective component of the derivative is 0.1% by weight based on the total weight of the feed.

4. The use as claimed in any one of claims 1 to 3, wherein the use is the direct addition of the product to animal feed; or mixing the product with carrier to obtain premix; or mixing with other feed additive or feed raw materials to make into premix and concentrated feed for feeding animals.