CN110693814A - Veterinary tilmicosin nano-gel breast perfusion agent and preparation method thereof - Google Patents
Veterinary tilmicosin nano-gel breast perfusion agent and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of veterinary drug preparation. In particular to a veterinary tilmicosin nano-gel breast perfusion agent and a preparation method thereof. The key point of the invention is the preparation process of combining the nanometer preparation and the gel process preparation formulation and the product formula thereof. The breast perfusion agent comprises 0.5-10.0% of tilmicosin, 2.0-20.0% of oil phase, 1.0-5.0% of surfactant, 0.1-4.0% of gel material, 0.5-5.0% of cross-linking agent, 0.001-0.05% of preservative and the balance of water for preparation in terms of W/V. The invention overcomes the defect of single nano-drug front-end release, and can effectively avoid the phenomenon of tilmicosin nanoparticle burst release. The gel film also has the advantages of good adhesion of emerging gel mucous membrane, promotion of wound healing and maintenance of local internal environment, and the treatment effect of tilmicosin on staphylococcus aureus mastitis is obviously improved. The method is beneficial to reducing the using amount of tilmicosin and the culture cost.
Description
Technical Field
The invention belongs to the technical field of veterinary drug preparation, and particularly relates to a veterinary tilmicosin nano-gel breast perfusion agent and a preparation method thereof.
Background
Tilmicosin is a special antibiotic for livestock and poultry semi-synthesized by a hydrolysate of tylosin. The oral administration and the subcutaneous injection have the advantages of quick absorption, large apparent distribution volume, strong tissue penetrability and drug concentration in lung tissues which is dozens of times of that of serum. Can quickly and completely enter mammary gland from blood, has high drug concentration in milk which is 10-30 times of that in serum, has long maintenance time which can reach 1-2 days, and is beneficial to the exertion of the drug effect of the time-dependent antibiotic tilmicosin. The special pharmacokinetic characteristic is particularly suitable for treating infectious diseases such as pneumonia and mastitis of livestock, and is widely used for preventing and controlling respiratory diseases and mastitis of animals caused by actinobacillus pleurorum, pasteurella, mycoplasma, staphylococcus aureus and the like in clinic.
Staphylococcus aureus is the most common cause of clinical mastitis of dairy cows, and causes great economic loss to the dairy cow breeding industry all over the world. Because of the unique survival strategy, including facultative intracellular parasitism, biomembrane, low-metabolic-level mutant body formation, drug resistance and the like, the method causes great challenge to clinical treatment of staphylococcus aureus mastitis, and simultaneously, the mastitis infected by staphylococcus aureus is easy to relapse and difficult to radically cure due to the protection of cell membranes and the formation of intracellular infection reservoirs. Tilmicosin is easy to reach infected cells due to strong tissue cell permeability and has certain advantages in the aspect of treating staphylococcus aureus mastitis, but the drug concentration in the infected cells cannot reach the concentration level required by bacteriostasis due to weak accumulation capacity of tilmicosin in the cells, and clinical treatment failure of mastitis is often caused.
The carrier material used in the nano technology is mostly biological macromolecules (solid lipid nano) with good biocompatibility or a structure (liposome) similar to cells, so that the nano technology shows good compatibility with organisms and low toxicity. Meanwhile, the nanoparticles show stronger cell membrane permeability and intracellular accumulation capacity due to small particle size, large surface area effect and surface charge, so that the drug concentration of an intracellular infection target reaches more than bacteriostatic concentration, and the nanoparticles have great application prospect in intracellular bacterial infection. However, due to the excessive surface area effect, burst release is often exhibited, leading to a rapid increase in local drug concentration, or to unnecessary waste of the drug released at the site before reaching the target site of infection. Meanwhile, researches show that nonprofessional phagocytes (such as epithelial cells) have weak capability of phagocytosing macromolecules by themselves, so that the epithelial cells often show lower uptake capability to nanoparticles, and staphylococcus aureus can be settled in mammary gland epithelial cells of the dairy cows, thereby causing certain challenges for treating the mastitis of the staphylococcus aureus by using nano-drugs.
The gel technology which is started in recent years is widely used in the field of pharmaceutical preparations due to the advantages of good biocompatibility and mucous membrane adhesion of gel materials, capability of promoting wound healing, capability of serving as an excellent sustained-release matrix and the likeA domain. Meanwhile, researches report that certain gel materials can improve the permeability of cells, so that a new idea is provided for overcoming the defects of single nano-drug burst release and insufficient uptake by non-professional phagocytes. The gel material has strong slow release capacity, can slow down the burst release degree of the nano-drug, and can improve the encapsulation rate of the whole system to the drug due to the packaging of the gel. In addition, the gel material is adhered to the mammary epithelial cells of the milk cow, so that the permeability of the cells can be improved, and the opportunity for the nano-drugs to enter the cells is created. At the same time, some gel materials have antibacterial activity, such as amino (NH) with positive charge2+) The chitosan of (4). The method endows the whole drug delivery system with multiple antibacterial mechanisms (antibiotics, nanoparticles and gel materials), and the bacteria need to mutate at multiple sites simultaneously when drug resistance is generated, thereby providing a new idea for relieving the development of clinical staphylococcus aureus drug resistance.
So far, reports about veterinary tilmicosin nano-gel breast perfusion agents and preparation methods thereof are not found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a veterinary tilmicosin nano-gel breast perfusion agent which has strong slow-release performance and can promote wound healing and a preparation method thereof.
The method adopts orthogonal design, screens the type, volume and concentration of the surfactant by taking the drug-loading rate of the nanoparticles as an investigation index, screens the type and dosage of the gel material by taking the slow-release performance and bacteriostatic ability of the gel material as indexes, screens the cross-linking material by the uniformity formed by the nanogels, and screens the type and dosage of the preservative according to the physicochemical property and bacteriostatic effect of the tilmicosin breast perfusion agent. The tilmicosin nano-gel breast perfusion agent for the livestock, which has the advantages of reasonable formula, simple process and safe quality, is successfully developed. The granularity and the sedimentation volume ratio of the preparation all accord with the related regulations of animal pharmacopoeia on breast perfusion agent; the tilmicosin nano-gel breast perfusion agent can be degraded by no more than 3% under the conditions of high temperature, high humidity and strong illumination, has obvious treatment effect on cow mastitis caused by staphylococcus aureus, and has the cure rate slightly higher than that of the commercial tilmicosin injection (300 mg/breast/day). The tilmicosin breast perfusion agent provided by the invention has the advantages of obvious slow release performance, good mucosa adhesiveness, small irritation, strong transmembrane and intracellular accumulation capacities and capability of promoting wound healing. The method is beneficial to reducing the clinical use amount of tilmicosin and the treatment cost of cow mastitis.
The technical scheme of the invention is as follows:
the invention takes one or the combination of castor oil, isopropyl palmitate, cottonseed oil and lanolin as an oil phase; one or the combination of 30 benzyl, 400 polyoxyethylene monolaurate and 400 polyoxyethylene monostearate is taken as a surfactant; one or other of gelatin, sodium alginate, carbomer, chitosan and bentonite is used as a gel material; with CaCO3One or the combination of EDTA-Ca, glutaraldehyde and sodium tripolyphosphate is taken as a cross-linking agent; one or the combination of sorbic acid, methyl p-hydroxybenzoate, chlorobutanol and benzyl alcohol is used as preservative. The preparation method comprises the steps of firstly preparing tilmicosin nanoparticles by a hot-melt emulsification method, then adding a gel material and a preservative into the prepared tilmicosin nanoparticles, and carrying out spontaneous grafting assembly to prepare the veterinary tilmicosin nanogel breast perfusion agent. The invention skillfully combines the advantages of the nanotechnology and the gel technology, not only obviously improves the sudden release phenomenon of the tilmicosin nanoparticles and prepares the breast perfusion agent capable of releasing the drug for a long time, but also improves the defect that the pure tilmicosin nanoparticles are not sufficiently absorbed by mammary epithelial cells by utilizing the advantages of good biocompatibility of the gel material and capability of promoting wound healing, reduces the stimulation of the drug to mammary gland and promotes the healing of infected mammary gland. The tilmicosin can be used for preventing and treating animal mastitis in clinic.
Specifically, the detailed technical scheme of the invention is as follows:
the applicant provides a veterinary tilmicosin nano-gel breast preparation which is prepared by a method combining a drug and solid lipid nano-preparation method and a hydrogel self-assembly method technology, wherein the breast perfusion preparation comprises the following components in percentage by mass/volume:
(1) 0.5-10.0% of tilmicosin;
(2) 2.0-20.0% of an oil phase;
(3) 1.0-5.0% of a surfactant;
(4) 0.1-4.0% of gel material;
(5) 0.5 to 5.0 percent of cross-linking agent;
(6) 0.001 to 0.05 percent of preservative;
(7) the rest is water for preparation;
the preparation method comprises the following steps:
1) adding tilmicosin with the formula amount into the oil phase with the formula amount, and heating and stirring to dissolve the tilmicosin into the oil phase;
2) preheating the surfactant aqueous solution with the formula amount to 100 ℃, adding the surfactant aqueous solution into the solution obtained in the step 1), and performing ultrasonic treatment for 2-8min by using a 6mm probe to prepare 200-900nm nanometer suspension;
3) adding the gel material and the preservative in the formula amount into the nano suspension obtained in the step 2), stirring for 5min, standing for 12h at normal temperature after the nano suspension is dispersed, and allowing the nano suspension to spontaneously form gel to wrap the tilmicosin nano particles obtained in the step 2), thus obtaining the 300-900nm tilmicosin nano-gel breast perfusion agent;
wherein:
the oil phase is one or the combination of castor oil, isopropyl palmitate, cottonseed oil and lanolin;
the surfactant is one or the combination of 30 benzyl, 400 polyoxyethylene monolaurate and 400 polyoxyethylene monostearate;
the gel material is one or the combination of gelatin, sodium alginate, carbomer, chitosan and bentonite;
the cross-linking agent is CaCO3One or the combination of EDTA-Ca, glutaraldehyde and sodium tripolyphosphate;
the preservative is one or the combination of sorbic acid, methyl p-hydroxybenzoate, chlorobutanol and benzyl alcohol.
As a preferred scheme, the veterinary tilmicosin nano-gel breast preparation is prepared by combining a drug and solid lipid nanocrystallization method and a hydrogel self-assembly method, and the breast perfusion preparation comprises the following components in mass/volume:
(1) 1.0-8.0% of tilmicosin;
(2) 2.0-15.0% of an oil phase;
(3) 1.0-4.0% of a surfactant;
(4) 0.2-2.0% of gel material;
(5) 1.0 to 4.0 percent of cross-linking agent;
(6) 0.005-0.05% of preservative;
(7) the rest is water for preparation;
the preparation method comprises the following steps:
1) adding tilmicosin with the formula amount into the oil phase with the formula amount, and heating and stirring to dissolve the tilmicosin into the oil phase;
2) preheating the surfactant aqueous solution with the formula amount, adding the preheated surfactant aqueous solution into the solution obtained in the step 1), and preparing 200-900nm nanometer suspension by using a 6mm probe through ultrasound;
3) adding the gel material and the preservative in the formula amount into the nano suspension obtained in the step 2), stirring for 5min, standing for 12h at normal temperature after the nano suspension is dispersed, and allowing the nano suspension to spontaneously form gel to wrap the tilmicosin nano particles obtained in the step 2), thus obtaining the 300-900nm tilmicosin nano-gel breast perfusion agent;
wherein:
the oil phase is one or the combination of castor oil, isopropyl palmitate, cottonseed oil and lanolin;
the surfactant is one or the combination of polyoxyethylene 400 monolaurate and polyoxyethylene 400 monostearate;
the gel material is one or the combination of gelatin, sodium alginate, carbomer and chitosan;
the cross-linking agent is CaCO3One or the combination of EDTA-Ca and sodium tripolyphosphate;
the preservative is one or the combination of sorbic acid, methyl p-hydroxybenzoate and benzyl alcohol.
The best embodiment of the invention is as follows: a veterinary tilmicosin nano gel breast agent is prepared by combining a drug and solid lipid nanocrystallization method and a hydrogel self-assembly method, and the breast perfusion agent comprises the following components in mass/volume:
(1) 5.0% of tilmicosin;
(2) 10.0% of oil phase;
(3) 0.75% of surfactant;
(4) 2.0% of gel material;
(5) 3% of a cross-linking agent;
(5) 0.01 percent of preservative;
(6) the rest is water for preparation;
the preparation method comprises the following steps:
1) adding tilmicosin with the formula amount into the oil phase with the formula amount, and heating and stirring to dissolve the tilmicosin into the oil phase;
2) preheating the surfactant aqueous solution with the formula amount to 100 ℃, adding the surfactant aqueous solution into the solution obtained in the step 1), and performing ultrasonic treatment for 2-8min by using a 6mm probe to prepare 200-900nm nanometer suspension;
3) adding the gel material and the preservative in the formula amount into the nano suspension obtained in the step 2), stirring for 5min, standing for 12h at normal temperature after the nano suspension is dispersed, and allowing the nano suspension to spontaneously form gel to wrap the tilmicosin nano particles obtained in the step 2), thus obtaining the 300-900nm tilmicosin nano-gel breast perfusion agent.
Wherein:
the oil phase is selected from castor oil or isopropyl palmitate;
the surfactant is selected from polyoxyethylene 400 monolaurate or polyoxyethylene 400 monostearate;
the gel material is selected from sodium alginate or chitosan;
the cross-linking agent is CaCO3Or sodium tripolyphosphate;
the preservative is sorbic acid or methyl p-hydroxybenzoate.
The breast perfusion agent has white or light yellow appearance and the particle size of 300-900 nm.
The breast perfusion agent releases less than 40% in 48h in the environment of simulated cow breast with pH being 7.
The invention has the following beneficial effects:
the tilmicosin nano-gel breast perfusion agent prepared by the invention creatively combines a nanotechnology and a gel technology, and not only has good transmembrane and intracellular accumulation capacities, but also has a remarkable slow-release capacity. Not only solves the defect that the pure nano-drug is not absorbed by mammary epithelial cells, but also makes up the defect that the front end of the pure nano-drug is released and the slow release effect is not ideal. The tilmicosin can release the medicine in the infected mammary gland for a long time, the using times of the tilmicosin can be effectively reduced, and the exertion of the maximum drug effect of the time-dependent antibiotic tilmicosin is undoubtedly facilitated, and the culture cost is saved. Meanwhile, the gel material has good biocompatibility and mucosa adhesiveness, which is beneficial to improving the permeability of mammary epithelial cells, and is beneficial to further improving the tilmicosin nanoparticles entering the mammary epithelial cells and improving the intracellular drug concentration, thereby improving the inhibiting effect on staphylococcus aureus in the cells. And because the gel material can reduce the stimulation to the lost mucosa, absorb the tissue exudate, promote the wound healing and the like, the gel material is beneficial to improving the compliance of animals. In addition, the new preparation has multiple bacteriostasis mechanisms endowed by antibacterial drugs, nanoparticles, gel materials and the like, and is also favorable for relieving the occurrence of clinical staphylococcus aureus drug resistance.
Drawings
FIG. 1: the preparation method of the veterinary tilmicosin nanogel breast perfusion agent in the embodiments 1-5 is shown in the schematic diagram.
FIG. 2: scanning electron micrographs (x 10000) of tilmicosin nanoparticles in the veterinary tilmicosin nanogel breast perfusion agent of examples 1-5.
FIG. 3: in vitro cumulative release profiles of the veterinary tilmicosin nanogel breast infusions in examples 1-5. Description of reference numerals: in FIG. 3, TIL-SLNs represent tilmicosin nanoparticles; TIL-nanogel represents tilmicosin nanogel.
Detailed Description
Example 1 formulation of veterinary tilmicosin breast infusion 1
The formula 1 of the veterinary tilmicosin nano-gel breast perfusion agent is shown in table 1, and the uniformity of the tilmicosin nano-gel breast perfusion agent prepared by the formula 1 is shown in table 6.
TABLE 1 tilmicosin nanogel Breast perfusion formulation 1
The preparation method comprises the following steps:
1) adding 5g of tilmicosin into 5mL of castor oil and 5mL of isopropyl palmitate, and heating and stirring to dissolve the tilmicosin into the castor oil and the isopropyl palmitate;
2) adding 1g of sodium alginate into 90mL of 3.3% aqueous solution of benzyl 30, dissolving, preheating to 100 ℃, adding into the solution obtained in the step 1), and preparing 200-600nm nano suspension by using a 6mm probe ultrasonic 6min emulsification method;
3) 1g of CaCO3And adding 0.05g of sorbic acid into the nano suspension obtained in the step 2), stirring for 5min to disperse, standing for 12h, and automatically forming gel to wrap the tilmicosin nano-particles to obtain the 300-inch and 700-inch tilmicosin nano-gel breast perfusion agent.
Example 2 formulation of veterinary tilmicosin breast infusion 2
The formula 2 of the veterinary tilmicosin nano-gel breast perfusion agent is shown in table 2, and the uniformity of the tilmicosin nano-gel breast perfusion agent prepared by the formula 2 is shown in table 6.
TABLE 2 tilmicosin nanogel breast infusion formulation 2
The preparation method comprises the following steps:
1) adding 2.05g of tilmicosin into 10g of cottonseed oil, and heating and stirring to dissolve the tilmicosin in the cottonseed oil;
2) adding 10g of gelatin into 73mL of aqueous solution of 2.5 percent benzyl 30 and 1.25 percent polyoxyethylene 400 monolaurate, dissolving, preheating to 100 ℃, adding the solution obtained in the step 1), and preparing 200-600nm nano suspension by using a 6mm probe ultrasonic 6min emulsification method;
3) adding 1mL of glutaraldehyde and 0.05g of methylparaben into the nano suspension obtained in the step 2), stirring for 5min to disperse, standing for 12h, and automatically forming gel to wrap the tilmicosin nano-particles to obtain the 300-700nm tilmicosin nano-gel breast perfusion agent.
Example 3 formulation of veterinary tilmicosin breast infusion 3
The formula 3 of the veterinary tilmicosin nano-gel breast perfusion agent is shown in table 3, and the uniformity of the tilmicosin nano-gel breast perfusion agent prepared by the formula 3 is shown in table 6.
TABLE 3 tilmicosin nanogel Breast perfusion agent formulation 3
The preparation method comprises the following steps:
1) adding 5g of tilmicosin into 8g of isopropyl palmitate and 2g of lanolin, and heating and stirring to dissolve the tilmicosin in the isopropyl palmitate box lanolin;
2) adding 1g of carbomer and 1g of chitosan into 78mL of 3.75% polyoxyethylene 400 monostearate and 1.25% aqueous solution of beneze 30 for dissolving, preheating to 100 ℃, adding into the solution obtained in the step 1), and preparing 200-600nm nano suspension by using a 6mm probe for ultrasonic treatment for 6 min;
3) adding 1g of sodium tripolyphosphate and 0.05g of sorbic acid into the nano suspension obtained in the step 2), stirring for 5min to disperse, standing for 12h, and automatically forming gel to wrap the tilmicosin nano-particles to obtain the 300-700nm tilmicosin nano-gel breast perfusion agent.
Example 4 formulation of veterinary tilmicosin breast infusion 4
The formula 4 of the veterinary tilmicosin nano-gel breast perfusion agent is shown in table 4, and the uniformity of the tilmicosin nano-gel breast perfusion agent prepared by the formula 4 is shown in table 6.
TABLE 4 tilmicosin nanogel breast perfusate formulation 4
The preparation method comprises the following steps:
1) adding 5g of tilmicosin into 5g of castor oil and 5g of cottonseed oil, and heating and stirring to dissolve the tilmicosin in the castor oil and the cottonseed oil;
2) dissolving 1g of sodium alginate in 80mL of 3.75% polyoxyethylene 400 monostearate aqueous solution, preheating to 100 ℃, adding the solution obtained in the step 1), and preparing 200-600nm nano suspension by using a 6mm probe and an ultrasonic 6min emulsification method;
3) adding 1g of EDTA-Ca and 1g of benzyl alcohol into the nano suspension obtained in the step 2), stirring for 5min to disperse, standing for 12h, and automatically forming gel to wrap the tilmicosin nano-particles to obtain the 300-700nm tilmicosin nano-gel breast perfusion agent.
Example 5 formulation of veterinary tilmicosin breast infusion 5
The formula 5 of the veterinary tilmicosin nano-gel breast perfusion agent is shown in table 5, and the uniformity of the tilmicosin nano-gel breast perfusion agent prepared by the formula 5 is shown in table 6.
TABLE 5 tilmicosin nanogel breast perfusate formulation 5
The preparation method comprises the following steps:
1) adding 5g of tilmicosin into 10g of castor oil, and heating and stirring to dissolve the tilmicosin in the castor oil;
2) dissolving 1g of sodium alginate in 79mL of 3.75% polyoxyethylene 400 monostearate aqueous solution, preheating to 100 ℃, adding the solution obtained in the step 1), and preparing 200-600nm nano suspension by using a 6mm probe and an ultrasonic 6min emulsification method;
3) adding 1g of EDTA-Ca and 0.05g of methyl p-hydroxybenzoate into the nano suspension obtained in the step 2), stirring for 5min to disperse, standing for 12h, and automatically forming gel to wrap tilmicosin nano-particles to obtain the 300-700nm tilmicosin nano-gel breast perfusion agent.
EXAMPLE 6 homogeneity examination of tilmicosin nanogel Breast perfusion Agents in examples 1-5
And (3) taking a proper amount of the tilmicosin nano-gel breast perfusion agent in the examples 1 to 5, and observing whether the gel material and the cross-linking agent form gel with the generation of particles or bubbles with uneven sizes or not by eye observation. The homogeneity of the tilmicosin nanogel breast perfusate in the examples 1 to 5 is shown in table 6, and it can be seen that the tilmicosin nanogel breast perfusate prepared by the formula of the example 5 has good homogeneity and can be used for subsequent tests.
Table 6 homogeneity of tilmicosin nanogel breast infusions in examples 1-5
| Sample (I) | Uniformity of |
| Example 1 | Non-uniform and varying sized particles |
| Example 2 | Non-uniform and varying sized particles |
| Example 3 | Non-uniformity, large bubble generation |
| Example 4 | Non-uniform, with particles and bubbles of varying sizes |
| Example 5 | Uniform, no particles and bubbles of different sizes |
Example 7 treatment trials for bovine mastitis Using tilmicosin nanogel Breast perfusion formulation prepared in example 5
1. Materials and methods
1.1 medicine tilmicosin injection (10%), tilmicosin nano-gel breast perfusion agent (5%)
1.2 test animals of 48 Holstein cows, 400-
1.3 somatic cell count analyzer, super clean bench, pH meter.
1.4 test methods
1.4.1 dosing regimens
48 cows with a Somatic Cell Count (SCC) of more than 50 ten thousand/mL were randomly divided into 4 groups of a blank group, a commercially available tilmicosin injection group, a low-dose tilmicosin nanogel breast perfusate group, and a tilmicosin nanogel breast perfusate group, each group having 12 cows. The dosing schedule for each group is shown in table 7.
Table 7 dosing regimen for each group
1.4.2 test animals
The experiment is carried out on the premise of strictly complying with the animal welfare principle, and the conditions of proper temperature, sufficient feed and free drinking water are ensured in the period. Holstein cows with the weight of 48 heads of 400-kg and the somatic cell count of more than 50 ten thousand/mL are randomly divided into a blank group, a commercially available tilmicosin injection group, a low-dose nano-gel group 1 and a normal-dose nano-gel group, wherein each group has 12 heads. The cattle are respectively housed in cattle pens which can freely feed and drink water, and treatment tests are carried out for 13 days. Each group was perfused with 10mL of breast perfused with tilmicosin formulations containing different doses, and the blank group was perfused with 10mL of saline. Continuously administering for 5 days, fully sterilizing breast before administration, hot compressing with hot towel, and gently massaging breast after administration to make medicine distribution uniform. Milk samples are collected respectively on the day before administration, the day after administration and the day 7 after administration, and the number of staphylococcus aureus, the somatic cell count and the pH value of the milk samples in the milk samples are detected, so that the cure rate and the effective rate of each group are calculated.
1.5 test results
The somatic cell count, bacterial count and milk-like pH values of each group are shown in tables 8-10, respectively, and the cure rate and effective rate are shown in table 11.
Table 8 change in somatic cell count for each group after administration (Mean ± SD, n ═ 12)
TABLE 9 Change in Staphylococcus aureus counts after dosing (Mean + -SD, n-12)
Table 10 change in milk pH of each group after administration (Mean ± SD, n ═ 12)
Table 11 cure rate and effective rate for each group (n ═ 12)
From tables 8-10, it can be seen that the body cell count, staphylococcus aureus count, and milk-like pH of the cows decreased rapidly and gradually toward normal levels after treatment with tilmicosin compared to the blank group. The 7 th day after treatment in Table 9, Staphylococcus aureus was still detected, which may be related to some of the Staphylococcus aureus resistance or the formation of mutant bodies. It can be seen from table 11 that tilmicosin has an ideal therapeutic effect on mastitis with staphylococcus aureus compared to the blank group. It is worth noting that the cure rate of the low-dose nano-gel group is slightly higher than that of the tilmicosin injection group sold in the market, and the tilmicosin nano-gel breast perfusion agent prepared by the invention can obviously improve the treatment effect of tilmicosin on cow mastitis. This will undoubtedly help to reduce the clinical tilmicosin usage and the cost of farming.
Claims (6)
1. The veterinary tilmicosin nano gel breast agent is characterized in that the breast perfusion agent is prepared by the following steps, and the preparation comprises the following components in percentage by mass/volume:
(1) 0.5-10.0% of tilmicosin;
(2) 2.0-20.0% of an oil phase;
(3) 1.0-5.0% of a surfactant;
(4) 1.0-10.0% of gel material;
(5) 0.5 to 5.0 percent of cross-linking agent
(6) 0.001 to 0.05 percent of preservative;
(7) the rest is water for preparation;
the preparation method comprises the following steps:
1) adding tilmicosin with the formula amount into the oil phase with the formula amount, and heating and stirring to dissolve the tilmicosin into the oil phase;
2) preheating the surfactant aqueous solution with the formula amount to 100 ℃, adding the surfactant aqueous solution into the solution obtained in the step 1), and performing ultrasonic treatment for 2-8min by using a 6mm probe to prepare 200-900nm nanometer suspension;
3) adding the gel material and the preservative in the formula amount into the nano-suspension obtained in the step 2), stirring for 5min, standing for 12h at normal temperature after the nano-suspension is dispersed, and allowing the nano-suspension to spontaneously form gel so as to wrap the tilmicosin nano-particles in the gel, thus obtaining the 300-nm and 900-nm tilmicosin nano-gel breast perfusion agent.
Wherein:
the oil phase is one or the combination of castor oil, isopropyl palmitate, cottonseed oil and lanolin;
the surfactant is one or the combination of 30 benzyl, 400 polyoxyethylene monolaurate and 400 polyoxyethylene monostearate;
the gel material is one or the combination of gelatin, sodium alginate, carbomer, chitosan and bentonite;
the cross-linking agent is CaCO3One or the combination of EDTA-Ca, glutaraldehyde and sodium tripolyphosphate;
the preservative is one or the combination of sorbic acid, methyl p-hydroxybenzoate, chlorobutanol and benzyl alcohol.
2. The veterinary tilmicosin nano gel breast agent is characterized in that the breast perfusion agent comprises the following components in parts by mass/volume:
(1) 1.0-8.0% of tilmicosin;
(2) 2.0-15.0% of an oil phase;
(3) 1.0-4.0% of a surfactant;
(4) 0.2-2.0% of gel material;
(5) 1.0 to 4.0 percent of cross-linking agent
(6) 0.005-0.05% of preservative;
(6) the rest is water for preparation.
The preparation method comprises the following steps:
1) adding tilmicosin with the formula amount into the oil phase with the formula amount, and heating and stirring to dissolve the tilmicosin into the oil phase;
2) preheating the surfactant aqueous solution with the formula amount to 100 ℃, adding the surfactant aqueous solution into the solution obtained in the step 1), and performing ultrasonic treatment for 2-8min by using a 6mm probe to prepare 200-900nm nanometer suspension;
3) adding the gel material and the preservative in the formula amount into the nano suspension obtained in the step 2), stirring for 5min, standing for 12h at normal temperature after the nano suspension is dispersed, allowing the nano suspension to spontaneously form gel, and wrapping the tilmicosin nano particles obtained in the step 2) to obtain the 300-900nm tilmicosin nano-gel breast perfusion agent.
Wherein:
the oil phase is one or the combination of castor oil, isopropyl palmitate, cottonseed oil and lanolin;
the surfactant is one or the combination of polyoxyethylene 400 monolaurate and polyoxyethylene 400 monostearate;
the gel material is one or the combination of gelatin, alginic acid, carbomer and chitosan;
the cross-linking agent is CaCO3One or the combination of EDTA-Ca and sodium tripolyphosphate;
the preservative is one or the combination of sorbic acid, methyl p-hydroxybenzoate and benzyl alcohol.
3. A veterinary tilmicosin nano gel breast agent and a preparation method thereof are characterized in that the breast perfusion agent comprises the following components by mass/volume:
(1) 5.0% of tilmicosin;
(2) 10.0% of oil phase;
(3) 0.75% of surfactant;
(4) 2.0% of gel material;
(5) 3% of a cross-linking agent;
(6) 0.01 percent of preservative;
(7) the rest is water for preparation.
The preparation method comprises the following steps:
1) adding tilmicosin with the formula amount into the oil phase with the formula amount, and heating and stirring to dissolve the tilmicosin into the oil phase;
2) preheating the surfactant aqueous solution with the formula amount to 100 ℃, adding the surfactant aqueous solution into the solution obtained in the step 1), and performing ultrasonic treatment for 2-8min by using a 6mm probe to prepare 200-900nm nanometer suspension;
3) adding the gel material and the preservative in the formula amount into the nano suspension obtained in the step 2), stirring for 5min, standing for 12h at normal temperature after the nano suspension is dispersed, and allowing the nano suspension to spontaneously form gel to wrap the tilmicosin nano particles obtained in the step 2), thus obtaining the 300-900nm tilmicosin nano-gel breast perfusion agent.
Wherein:
the oil phase is selected from one of castor oil or isopropyl palmitate;
the surfactant is selected from polyoxyethylene 400 monolaurate or polyoxyethylene 400 monostearate;
the gel material is selected from sodium alginate or chitosan;
the cross-linking agent is CaCO3Or sodium tripolyphosphate;
the preservative is selected from sorbic acid or methyl p-hydroxybenzoate.
4. The tilmicosin nanogel breast injectant for veterinary use as claimed in claim 1, wherein the breast injectant is white or light yellow in appearance, and the particle size is 300-900 nm.
5. The veterinary tilmicosin nanogel breast perfusate according to claim 1, wherein the breast perfusate releases less than 40% in 48h in the environment of a simulated cow breast with pH of 7.
6. The method for preparing the tilmicosin nanogel breast perfusion agent for veterinary use as claimed in claim 1, 2, 3 or 4.
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| CN113171337A (en) * | 2021-04-27 | 2021-07-27 | 塔里木大学 | Veterinary cefquinome nanogel and preparation method thereof |
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