Enrofloxacin nano-silver compound emulsion and preparation method and application thereof

Abstract

The application discloses a compound enrofloxacin-nano silver emulsion and a preparation method and application thereof, and the preparation method comprises the following steps: uniformly dispersing ganoderma extract and silver nitrate in deionized water, and reacting at constant temperature of 60-90 DEG C in dark to obtain a nano silver solution; taking enrofloxacin, lauric acid monoglyceride and oleic acid, heating and melting, and uniformly stirring to obtain an oil phase; dispersing polyglycerol-10 stearate in a polyvinyl alcohol solution, and heating and stirring to obtain an aqueous phase; under the condition of constant temperature stirring, the oil phase is transferred into the aqueous phase, and a high shear instrument is used for shearing to obtain a coarse dispersion; the coarse dispersion is treated by using a high-pressure homogenizer to obtain an enrofloxacin emulsion; the enrofloxacin emulsion is uniformly mixed with the nano silver solution, and is cooled to obtain the compound enrofloxacin-nano silver emulsion. The application can improve the solubility of enrofloxacin, has good characterization performance, the preparation cost is controllable, macro-preparation can be realized, and the compound emulsion can effectively treat animal skin infection and promote wound healing.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of pharmaceutical preparations, and particularly relates to an enrofloxacin nano-silver compound emulsion as well as a preparation method and application thereof. BACKGROUND

[0002] Skin infection seriously affects the life quality and production performance of animals. Antibiotics are still the first choice for treating skin bacterial infection, however, the increasing drug resistance problem limits the efficacy of existing antibiotic-based skin wound infection treatment drugs and preparations. In order to ensure the high efficiency of treatment, scientists continue to focus on developing alternative drugs or combining them with antibiotics. However, the preparation method of the drugs and preparations is complex, the cost is high, the encapsulation rate and drug loading capacity are low, and other problems limit their clinical application.

[0003] Nano-silver (AgNPs) generally refers to metallic silver elements with a particle size of less than 100 nm. The antibacterial properties of silver have been recognized by mankind since ancient times, and silver has strong antibacterial activity and a wide antibacterial spectrum, and can be applied to disinfectants for water, food and household appliances, and wound dressings. Nano-silver cannot easily induce bacterial drug resistance, and its anti-inflammatory, antioxidant, cell proliferation promoting and skin remodeling improving properties can improve skin wound healing.

[0004] Enrofloxacin (ENR) is a third-generation quinolone drug, which is an animal-specific antibiotic and has high antibacterial activity against gram-negative bacteria and gram-positive bacteria, and can treat deep infections and skin infections in animals, such as gastrointestinal respiratory tract infections, canine otitis externa and pyoderma. However, enrofloxacin has poor water solubility and low bioavailability, and its clinical application is limited, so it is urgent to develop a drug delivery technology that can achieve reduction and efficiency. SUMMARY

[0005] The present application aims to overcome the deficiencies in the prior art, and provides an enrofloxacin nano-silver compound emulsion with good long-term stability, which can improve the solubility of enrofloxacin and promote wound healing. Another object of the present application is to provide a preparation method of the enrofloxacin nano-silver compound emulsion, which is cost-controllable and can be prepared in large quantities. The present application further aims to provide an application of the enrofloxacin nano-silver compound emulsion in the treatment of animal skin infection.

[0006] The enrofloxacin nano-silver compound emulsion comprises the following substances in the following proportions by weight: enrofloxacin 0.1-30 parts, lauric acid monoglyceride 0.3-90 parts, oleic acid 0.7-55 parts, polyglyceryl-10 stearate 0.2-40 parts, polyvinyl alcohol 0.1-10 parts, nano-silver 100-1000 parts, and deionized water 10-1000 parts.

[0007] The preparation method of the enrofloxacin nano-silver compound emulsion comprises the following steps:

[0008] Step one, take Ganoderma extract and silver nitrate evenly dispersed in deionized water, constant temperature 60-90℃ reaction in the dark, get nanometer silver solution;

[0009] Step two, take enrofloxacin, lauric acid monoglyceride and oleic acid, heated to melt, stirring evenly to get oil phase;

[0010] Step three, take polyglycerol-10 stearate dispersed in polyvinyl alcohol solution, heated to stirring to get water phase;

[0011] Step four, under constant temperature stirring conditions, the oil phase into the water phase, with high shear instrument shear to get coarse dispersion;

[0012] Step five, with high pressure homogenizer treatment of coarse dispersion, get enrofloxacin emulsion;

[0013] Step six, mix enrofloxacin emulsion and nanometer silver solution evenly, cooling, get enrofloxacin nanometer silver compound emulsion.

[0014] NLCs are composed of solid and liquid lipids, drugs are encapsulated in the mixture of liquid and solid lipids, and stabilized by using surfactants. NLCs have high adhesion to the skin composed of lipids, can promote the formation and hydration of the membrane, while maintaining the integrity of the skin, can more effectively penetrate the skin barrier to play the drug efficacy. The application of NLCs to skin drug delivery also has many other advantages, such as protecting the drug from degradation caused by enzymes present in the skin; improve the solubility and bioavailability of the drug; prolong the drug release time, etc. In addition, lipids have emollient effect, can effectively promote skin repair and wound healing.

[0015] Further, in step one, the solution concentration of Ganoderma extract is 1g / L-4g / L, the solution concentration of silver nitrate is 1-4mM, and the constant temperature reaction in the dark is 1-4h. Preferably, the solution concentration of Ganoderma extract is 3g / L, the constant temperature reaction in the dark is 90℃, and the time is 4h.

[0016] Further, in step two, the mass ratio of lauric acid monoglyceride to oleic acid is 3 / 7-1.5, and the mass ratio of enrofloxacin to the sum of lauric acid monoglyceride and oleic acid is 1:3-10. The heating and melting temperature is 75-85℃. Preferably, the mass ratio of lauric acid monoglyceride to oleic acid is 6:4, and the mass ratio of enrofloxacin to the sum of lauric acid monoglyceride and oleic acid is 1:4, that is, the mass percentage of enrofloxacin in the total lipid (lauric acid monoglyceride and oleic acid) is 25%. The heating and melting temperature is 80℃.

[0017] Further, in step three, the heating and stirring temperature is 75-85℃, preferably 80℃.

[0018] Further, in step four, the mass ratio of the oil phase to the water phase is 1:8-10, preferably 1:10.

[0019] Further, in step five, the homogenization pressure of the high-pressure homogenizer is 400-1000 bar, and the homogenization frequency is 1-5 times.

[0020] The above enrofloxacin-nano silver compound emulsion is applied in the treatment of animal skin infection.

[0021] Preparation principle: Enrofloxacin cannot be directly dissolved in water, and the present application first dissolves it in lipids, and then disperses it in water containing emulsifiers and co-emulsifiers to prepare an oil-in-water emulsion, which improves the water solubility of enrofloxacin and improves its efficacy on skin infection pathogenic bacteria. The present application uses a high-pressure homogenization method to prepare an emulsion loaded with enrofloxacin. The lipids dissolving enrofloxacin are mixed with water containing emulsifiers and co-emulsifiers, and a high-shear machine is used to shear to form a coarse dispersion, and then a nano-particle size emulsion is formed under high pressure. After cooling at room temperature, nano-structured lipid carriers are recrystallized.

[0022] To further promote skin repair and wound healing, a nano-silver solution is introduced into the system to prepare an enrofloxacin-nano silver compound emulsion. Nano-silver can accelerate skin wound healing through anti-inflammatory effect and promote the formation of new blood vessels. VEGF and HIF-1 can promote capillary formation, in addition, Ang family proteins (Ang-I and Ang-II) have a positive effect on the protection and regeneration of wound blood vessels. Nano-silver can increase the expression levels of VEGF, HIF-1, Ang-I and Ang-II, and also increase the expression of collagen and growth factors (such as FGF and PDGF), leading to re-epithelialization, and further promoting angiogenesis and collagen deposition.

[0023] The present application uses a biosynthesis method to prepare nano-silver, i.e. using low-cost and biocompatible ganoderma extract as a reducing agent to reduce silver ion precursors to silver atoms, and then through nucleation and growth, finally growing into silver nanoparticles of a certain size. In addition, the C=O structure in ganoderma extract can non-specifically bind to silver nanoparticles, so that the ganoderma extract is coated on the surface of the silver nanoparticles to play the role of a stabilizer.

[0024] Beneficial effects: Compared with the prior art, the enrofloxacin-nano silver compound emulsion prepared by the present application has the following outstanding characteristics: the prepared enrofloxacin-nano silver compound emulsion can improve the solubility of enrofloxacin and has good performance characteristics; the preparation cost is controllable and macro-preparation can be realized; the enrofloxacin-nano silver compound emulsion can effectively treat animal skin infection and promote wound healing. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is the effect of ganoderma lucidum extract concentration on synthesis of nano-silver of the present application;

[0026] Figure 2 is the effect of reaction temperature on synthesis of nano-silver of the present application;

[0027] Figure 3 is the effect of reaction time on synthesis of nano-silver of the present application;

[0028] Figure 4 is the transmission electron microscope image of enoxacin-nano-silver compound emulsion of the present application;

[0029] Figure 5 is the hydration particle size distribution diagram of enoxacin-nano-silver compound emulsion of the present application;

[0030] Figure 6 is the zeta potential distribution diagram of enoxacin-nano-silver compound emulsion of the present application;

[0031] Figure 7 is the healing diagram of enoxacin-nano-silver compound emulsion of the present application on skin wound of rats infected with staphylococcus aureus;

[0032] Figure 8 is the effect of enoxacin-nano-silver compound emulsion of the present application on wound area of skin wound of rats;

[0033] Figure 9 is the effect of enoxacin-nano-silver compound emulsion of the present application on healing rate of skin wound of rats;

[0034] Figure 10 is the effect of enoxacin-nano-silver compound emulsion on bacterial colony count of skin wound tissue of rats;

[0035] Figure 11 is the quantitative statistical diagram of enoxacin-nano-silver compound emulsion on bacterial colony count of skin wound tissue of rats. DETAILED DESCRIPTION

[0036] In the following examples, Ganoderma lucidum extract was purchased from Shaanxi Chengqian Biological Company, containing 40wt% Ganoderma lucidum polysaccharide, silver nitrate was purchased from China National Pharmaceutical Group Corporation, enrofloxacin was purchased from Nanjing Huijie Cheng Biological Technology Co., Ltd., glyceryl monolaurate, oleic acid, polyglyceryl-10 stearate, polyvinyl alcohol were purchased from Shanghai Aladdin Biological Company. The mass percentage of polyvinyl alcohol solution was 1%. The ultraviolet-visible spectrophotometer was purchased from Japan Shimadzu Company, the model was UV-1900; the laser particle potential instrument was purchased from the United Kingdom Malvern Company, the model was Zetasizer Nano ZSE; the high shear instrument was purchased from the United States IKA Instrument Equipment Company, the model was FM-200; the high-pressure homogenizer was purchased from An Tuo Si Nanometer Technology (Suzhou) Co., Ltd., the model was AH12-300.

[0037] Examples 1-7 are the optimization of the preparation conditions of enrofloxacin-silver nanocomposite emulsion, including the concentration of Ganoderma lucidum extract, light-avoiding reaction temperature, light-avoiding reaction time, the mass ratio of glyceryl monolaurate and oleic acid, the drug-lipid ratio, the homogenization pressure and the homogenization times.

[0038] Example 1

[0039] Optimization of the concentration of Ganoderma lucidum extract:

[0040] Four 0.017g silver nitrate powders were dissolved in 50mL deionized water to prepare 2mM silver nitrate solution; 0.05g, 0.1g, 0.15g, 0.2g of Ganoderma lucidum extract powder were dissolved in 50mL deionized water to obtain 1.0, 2.0, 3.0, 4.0g / L Ganoderma lucidum extract solution. 50mL of 2mM silver nitrate solution and 50mL of 1.0, 2.0, 3.0, 4.0g / L Ganoderma lucidum extract solution were taken respectively and reacted at 80℃ in the dark for 2h, and the obtained silver nanocomposite solution was scanned at a wavelength of 300-800nm with deionized water as reference to determine the ultraviolet-visible absorption spectrum of silver nanocomposite, and the characteristic absorption peak intensity was used as the screening basis.

[0041] Since the silver ion solution and Ganoderma lucidum extract itself have no characteristic absorption peak in the ultraviolet spectrum, the ultraviolet absorption spectrum directly reflects the concentration and content of silver nanocomposite. According to the Lambert-Beer law, the characteristic absorption peak intensity of silver nanocomposite is directly proportional to the concentration of silver nanocomposite. Figure 1The results show that the intensity of the characteristic absorption peak increases with the increase of the concentration of Ganoderma extract, and the concentration of silver nanoparticles also increases with the increase of the intensity of the characteristic absorption peak. However, when the concentration of Ganoderma extract reaches 3.0 g / L, the intensity of the characteristic absorption peak decreases slightly with the further increase of the concentration of Ganoderma extract. Therefore, the concentration of Ganoderma extract solution can be selected as 3.0 g / L or 4.0 g / L. In addition, the hydrated particle size of silver nanoparticles synthesized by 3.0 g / L Ganoderma extract (78.09 ± 1.38 nm) is smaller than that of silver nanoparticles synthesized by 4.0 g / L Ganoderma extract (97.23 ± 1.33 nm). Therefore, the optimal concentration of Ganoderma extract is 3.0 g / L.

[0042] Example 2

[0043] Optimization of light-avoiding reaction temperature:

[0044] Five portions of 0.017 g silver nitrate powder were dissolved in 50 mL deionized water to prepare 2 mM silver nitrate solution. Five portions of 0.15 g Ganoderma extract powder were dissolved in 50 mL deionized water to obtain 3.0 g / L Ganoderma extract solution. 50 mL of 2 mM silver nitrate solution and 50 mL of 3.0 g / L Ganoderma extract solution were reacted at 60, 70, 80, 90, and 100 °C for 2 h in the dark, respectively. The obtained silver nanoparticle solution was scanned at a wavelength of 300-800 nm with deionized water as a reference to determine the ultraviolet-visible absorption spectrum of silver nanoparticles. The intensity of the characteristic absorption peak was used as a screening basis.

[0045] The characteristic absorption peak of the product silver nanoparticle solution was compared with the above experiment. According to the intensity of the characteristic absorption peak, the optimal reaction temperature was determined. Figure 2 The results show that the intensity of the characteristic absorption peak increases with the increase of the temperature, indicating that the concentration of silver nanoparticles increases. Since the particle size distribution of silver nanoparticles synthesized at 100 °C is not uniform and is more prone to aggregation, the optimal light-avoiding reaction temperature is 90 °C.

[0046] Example 3

[0047] Optimization of light-avoiding reaction time:

[0048] Five portions of 0.017 g silver nitrate powder were dissolved in 50 mL deionized water to prepare 2 mM silver nitrate solution. Five portions of 0.15 g Ganoderma extract powder were dissolved in 50 mL deionized water to obtain 3.0 g / L Ganoderma extract solution. 50 mL of 2 mM silver nitrate solution and 50 mL of 3.0 g / L Ganoderma extract solution were reacted at 90 °C for 1, 2, 3, 4, 5, and 6 h in the dark, respectively. The obtained silver nanoparticle solution was scanned at a wavelength of 300-800 nm with deionized water as a reference to determine the ultraviolet-visible absorption spectrum of silver nanoparticles. The intensity of the characteristic absorption peak was used as a screening basis.

[0049] The characteristic absorption peak of silver nanoparticles was compared with that of the previous experiment. According to the results, the characteristic absorption peak intensity of silver nanoparticles increased with the increase of reaction time. However, the characteristic absorption peak intensity increased slowly after the reaction proceeded for 4 h, and black elemental silver precipitated in the silver nanoparticle solution synthesized for 5 h and 6 h. Therefore, the most preferred reaction time was 4 h. Figure 3 The results showed that the characteristic absorption peak intensity increased with the increase of reaction time. However, the characteristic absorption peak intensity increased slowly after the reaction proceeded for 4 h, and black elemental silver precipitated in the silver nanoparticle solution synthesized for 5 h and 6 h. Therefore, the most preferred reaction time was 4 h.

[0050] The optimal preparation conditions of silver nanoparticle solution were as follows: concentration of Ganoderma lucidum extract: 3.0 g / L, concentration of silver nitrate: 2 mM, reaction temperature: 90°C, and reaction time: 4 h.

[0051] Example 4

[0052] Optimization of the mass ratio of monoglyceride laurate and oleic acid:

[0053] The monoglyceride laurate and oleic acid with enrofloxacin (drug-lipid ratio 20%) at a mass ratio of 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 were melted at 80°C and stirred uniformly to obtain an oil phase. The polyglyceryl-10 stearate was dispersed in a 1% polyvinyl alcohol solution and heated and stirred at 80°C to obtain an aqueous phase. At the same temperature, the oil phase was dispersed in the aqueous phase (mass ratio 1:10), and the mixed solution was sheared by a high-shear machine at 10,000 rpm for 5 min to obtain a coarse dispersion. The coarse dispersion was ultrasonically broken for 20 min at an amplitude of 300 W to form a primary emulsion. The primary emulsion was then dispersed in the pre-cooled silver nanoparticles at a volume ratio of 1:1 at 4°C, and the mixed emulsion was ice-bathed for 10 min to prepare the enrofloxacin-silver nanoparticle compound emulsion. The hydrated particle size, polydispersity coefficient, and zeta potential of the compound emulsion were determined by dynamic light scattering, and the solution state of the compound emulsion after standing for 48 h was observed. The screening was based on the above characterization parameters.

[0054] Table 1 Effect of the mass ratio of monoglyceride laurate and oleic acid on the preparation of enrofloxacin-silver nanoparticle compound emulsion

[0055]

[0056] According to the results in Table 1, with the increase of the mass ratio of monoglyceride laurate and oleic acid, the hydrated particle size and polydispersity coefficient showed an increasing trend. When the mass ratio was more than 5:5, the viscosity of the emulsion increased, and the flowability decreased after standing for 48 h. Considering the suitability of transdermal administration and obtaining smaller particle size and higher stability of the compound emulsion, the most preferred mass ratio of monoglyceride laurate and oleic acid was 6:4.

[0057] Example 5

[0058] Optimization of the mass percentage of enrofloxacin in total lipids (drug-lipid ratio):

[0059] Enrofloxacin and oleic acid (mass ratio 6:4) with enrofloxacin at a drug lipid ratio of 10%, 15%, 20%, 25% and 30% were melted at 80°C, and stirred uniformly to obtain an oil phase; polyglyceryl-10 stearate was dispersed in a 1% polyvinyl alcohol solution, and heated and stirred at 80°C to obtain an aqueous phase; at the same temperature, the oil phase was dispersed in the aqueous phase (mass ratio 1:10), and the mixed solution was sheared by a high shear machine at 10000 rpm for 5 min to obtain a coarse dispersion. The above coarse dispersion was ultrasonically broken at 300W amplitude for 20 min to form a primary emulsion, and the primary emulsion was then dispersed in an equal volume of pre-cooled nano-silver at 4°C, and the mixed emulsion was ice-bathed for 10 min to prepare an enrofloxacin-nano-silver compound emulsion. The hydrated particle size, polydispersity coefficient and zeta potential of the compound emulsion were determined by dynamic light scattering method, and the solution state of the compound emulsion after standing for 48 h was observed. The screening basis was the above characterization parameters.

[0060] Table 2 Effect of drug lipid ratio on preparation of enrofloxacin-nano-silver compound emulsion

[0061]

[0062]

[0063] According to the results in Table 2, with the increase of drug lipid ratio from 0% to 30%, the hydrated particle size and polydispersity coefficient showed a trend of first increasing and then decreasing, but the change was small. Considering the requirements of small particle size, high stability and drug content, therefore, the most preferred drug lipid ratio is 25%.

[0064] Example 6

[0065] Optimization of homogenization pressure when macro-preparing compound emulsion by high-pressure homogenizer:

[0066] Enrofloxacin and oleic acid (mass ratio 6:4) with enrofloxacin at a drug lipid ratio of 25% were melted at 80°C, and stirred uniformly to obtain an oil phase; polyglyceryl-10 stearate was dispersed in a 1% polyvinyl alcohol solution, and heated and stirred at 80°C to obtain an aqueous phase; at the same temperature, the oil phase was dispersed in the aqueous phase (mass ratio 1:10), and the mixed solution was sheared by a high shear machine at 10000 rpm for 5 min to obtain a coarse dispersion. The component system of the above coarse dispersion was expanded by 30 times in proportion, and the obtained coarse dispersion was subjected to high-pressure homogenization at a homogenization pressure of 400, 500, 600, 700, 800, 900, 1000 bar and a homogenization number of 5, and the obtained enrofloxacin emulsion was dispersed in an equal volume of pre-cooled nano-silver at 4°C to prepare an enrofloxacin-nano-silver compound emulsion. The hydrated particle size, polydispersity coefficient and zeta potential of the compound emulsion were determined by dynamic light scattering method, and the solution state of the compound emulsion after standing for 48 h was observed. The screening basis was the above characterization parameters.

[0067] According to the results of Table 3 below, the hydrated particle size of the compound emulsion decreases with the increase of the homogenization pressure, and the polydispersity coefficient also shows a decreasing trend. Especially when the homogenization pressure is increased to more than 800 bar, the hydrated particle size and the polydispersity coefficient are significantly reduced. However, considering the high energy consumption caused by excessively high homogenization pressure, the most preferred homogenization pressure is 700 bar.

[0068] Table 3 Effect of homogenization pressure on macro-preparation of enrofloxacin-nano silver compound emulsion

[0069]

[0070]

[0071] Example 7

[0072] Optimization of homogenization times in macro-preparation of compound emulsion:

[0073] The preparation process of the coarse dispersion is the same as above. The component system of the above coarse dispersion is proportionally expanded by 30 times. The obtained coarse dispersion is subjected to high pressure homogenization at a homogenization pressure of 700 bar and homogenization times of 1, 2, 3, 4 and 5, respectively. The obtained enrofloxacin emulsion is dispersed in an equal volume of nano silver pre-cooled at 4°C to prepare enrofloxacin-nano silver compound emulsion. The hydrated particle size, polydispersity coefficient and zeta potential of the compound emulsion are determined by dynamic light scattering method, and the solution state of the compound emulsion after standing for 48 h is observed. The screening basis is the above characterization parameters.

[0074] Table 4 Effect of homogenization times on macro-preparation of enrofloxacin-nano silver compound emulsion

[0075]

[0076] According to the results of Table 4, with the increase of the homogenization times, the hydrated particle size and the polydispersity coefficient of the compound emulsion show a decreasing trend. When the homogenization times is 5, the hydrated particle size and the polydispersity coefficient are the smallest, and the absolute value of the zeta potential is larger, which meets the requirements of small particle size and high stability. Therefore, the most preferred homogenization times is 5.

[0077] Example 8

[0078] This example is prepared under the optimal process parameters selected in Examples 1-7, which is the optimal example.

[0079] Materials: Ganoderma lucidum extract 0.45 g, silver nitrate 0.051 g, enrofloxacin 7.5 g, lauric acid monoglyceride 18 g, oleic acid 12 g, polyglyceryl-10 stearate 10.5 g, 1% polyvinyl alcohol solution 300 mL, nano silver 300 mL.

[0080] A preparation method of enrofloxacin-nano silver compound emulsion, comprising the following steps:

[0081] (1) 0.051 g of silver nitrate powder is dissolved in 150 mL of deionized water to prepare a 2 mM silver nitrate solution; 0.45 g of ganoderma extract powder is dissolved in 150 mL of deionized water to obtain a 3 g / L ganoderma extract solution. 150 mL of the 2 mM silver nitrate solution is reacted with 150 mL of the 3 g / L ganoderma extract solution at 90°C in the dark for 4 h to prepare a nano silver solution.

[0082] (2) 18 g of lauric acid monoglyceride, 12 g of oleic acid and 7.5 g of enrofloxacin are melted at 80°C, and stirred uniformly to obtain an oil phase.

[0083] (3) 10.5 g of polyglyceryl-10 stearate is dispersed in 300 mL of 1% polyvinyl alcohol solution, and heated and stirred at 80°C to obtain an aqueous phase;

[0084] (4) At the same temperature in step (3), the oil phase is dispersed in the aqueous phase, and the mass ratio of the oil phase to the aqueous phase is 1:10; the mixed solution is sheared by a high-shear machine at 10,000 rpm for 5 min to obtain a coarse dispersion.

[0085] (5) The coarse dispersion is homogenized by a high-pressure homogenizer at 700 bar for 5 times to obtain an enrofloxacin emulsion.

[0086] (6) The enrofloxacin emulsion is dispersed in an equal volume of nano silver pre-cooled at 4°C, and ice-bath for 10 min to obtain an enrofloxacin-nano silver compound emulsion.

[0087] Detection: The enrofloxacin-nano silver compound emulsion prepared in this example has a hydration particle size of 167.5±1.401 nm, a polydispersity coefficient of 0.140±0.013, and a zeta potential of -35.0±1.01 mV. The content of enrofloxacin in the compound emulsion is 12.40±0.68 mg / mL, and the encapsulation rate is 80.66±4.59% measured by ultraviolet spectrophotometry. The concentration of nano silver in the compound emulsion is 105.00±1.26 μg / mL measured by atomic absorption spectrometry.

[0088] Example 9

[0089] The remaining steps of this example are the same as those of example 8, and the only difference is that the homogenization pressure of the high-pressure homogenizer is replaced by 800 bar.

[0090] Detection: The enrofloxacin-nano silver compound emulsion prepared in this example has a hydration particle size of 206.8±2.570 nm, a polydispersity coefficient of 0.238±0.012, and a zeta potential of -38.0±0.141 mV.

[0091] Example 10

[0092] Materials: Ganoderma extract 0.05 g, silver nitrate 0.0085 g, enrofloxacin 0.5 g, lauric acid monoglyceride 0.3 g, oleic acid 0.7 g, polyglyceryl-10 stearate 0.2 g, 1% polyvinyl alcohol solution 10 mL, nano-silver 100 mL.

[0093] A preparation method of enrofloxacin nano-silver compound emulsion, comprising the following steps:

[0094] (1) Dissolve 0.0085 g of silver nitrate powder in 50 mL of deionized water to prepare a 1 mM silver nitrate solution; take 0.05 g of Ganoderma extract powder and dissolve it in 50 mL of deionized water to obtain a 1 g / L Ganoderma extract solution. Take 50 mL of 1 mM silver nitrate solution and 50 mL of 1 g / L Ganoderma extract solution and react at 60°C in the dark for 1 h to prepare a nano-silver solution.

[0095] (2) Take 0.3 g of lauric acid monoglyceride, 0.7 g of oleic acid, and 0.1 g of enrofloxacin and melt at 75°C, and stir uniformly to obtain an oil phase.

[0096] (3) Take 0.2 g of polyglyceryl-10 stearate and disperse it in 10 mL of 1% polyvinyl alcohol solution, and heat and stir at 75°C to obtain an aqueous phase.

[0097] (4) At the same temperature in step (3), disperse the oil phase in the aqueous phase, and the mass ratio of the oil phase to the aqueous phase is 1:8. Mix the solution and use a high shear machine to shear at 10000 rpm for 5 min to obtain a coarse dispersion.

[0098] (5) Use a high-pressure homogenizer to homogenize the coarse dispersion at 400 bar for 1 time to obtain an enrofloxacin emulsion.

[0099] (6) Disperse the enrofloxacin emulsion in an equal volume of nano-silver pre-cooled at 4°C, and ice-bath for 10 min to obtain an enrofloxacin-nano-silver compound emulsion.

[0100] Example 11

[0101] Materials: Ganoderma extract 2 g, silver nitrate 0.34 g, enrofloxacin 30 g, lauric acid monoglyceride 45 g, oleic acid 55 g, polyglyceryl-10 stearate 40 g, 1% polyvinyl alcohol solution 1000 mL, nano-silver 1000 mL.

[0102] A preparation method of enrofloxacin nano-silver compound emulsion, comprising the following steps:

[0103] (1) 0.34 g silver nitrate powder was dissolved in 500 mL of deionized water to prepare a 4 mM silver nitrate solution; 2 g of Ganoderma extract powder was dissolved in 500 mL of deionized water to obtain a 4 g / L Ganoderma extract solution. 500 mL of the 4 mM silver nitrate solution was reacted with 500 mL of the 4 g / L Ganoderma extract solution at 80°C in the dark for 2 h to prepare a nano-silver solution.

[0104] (2) 45 g of lauric acid monoglyceride, 55 g of oleic acid, and 30 g of enrofloxacin were melted at 85°C, and stirred uniformly to obtain an oil phase.

[0105] (3) 40 g of polyglyceryl-10 stearate was dispersed in 1000 mL of 1% polyvinyl alcohol solution, and heated and stirred at 85°C to obtain an aqueous phase;

[0106] (4) At the same temperature in step (3), the oil phase was dispersed in the aqueous phase, and the mass ratio of the oil phase to the aqueous phase was 1:9. The mixed solution was sheared by a high shear machine at 10000 rpm for 5 min to obtain a coarse dispersion.

[0107] (5) The coarse dispersion was homogenized by a high-pressure homogenizer at 1000 bar for 3 times to obtain an enrofloxacin emulsion;

[0108] (6) The enrofloxacin emulsion was dispersed in an equal volume of nano-silver pre-cooled at 4°C, and ice-bathed for 10 min to obtain an enrofloxacin-nano-silver compound emulsion.

[0109] Example 12

[0110] Materials: Ganoderma extract 2 g, silver nitrate 0.34 g, enrofloxacin 30 g, lauric acid monoglyceride 90 g, oleic acid 10 g, polyglyceryl-10 stearate 40 g, 1% polyvinyl alcohol solution 1000 mL, nano-silver 1000 mL.

[0111] A preparation method of an enrofloxacin-nano-silver compound emulsion, comprising the following steps:

[0112] (1) 0.34 g silver nitrate powder was dissolved in 500 mL of deionized water to prepare a 4 mM silver nitrate solution; 2 g of Ganoderma extract powder was dissolved in 500 mL of deionized water to obtain a 4 g / L Ganoderma extract solution. 500 mL of the 4 mM silver nitrate solution was reacted with 500 mL of the 4 g / L Ganoderma extract solution at 80°C in the dark for 2 h to prepare a nano-silver solution.

[0113] (2) 90 g of lauric acid monoglyceride, 10 g of oleic acid, and 30 g of enrofloxacin were melted at 85°C, and stirred uniformly to obtain an oil phase.

[0114] (3) 40 g of polyglyceryl-10 stearate was dispersed in 1000 mL of 1% polyvinyl alcohol solution, and heated and stirred at 85°C to obtain an aqueous phase;

[0115] (4) At the same temperature of step (3), the oil phase was dispersed in the water phase, the mass ratio of the oil phase to the water phase was 1:9, and the mixed solution was sheared by a high shear machine at 10000 rpm for 5 min to obtain a coarse dispersion.

[0116] (5) The coarse dispersion was homogenized by a high-pressure homogenizer at 1000 bar for 3 times to obtain an enrofloxacin emulsion;

[0117] (6) The enrofloxacin emulsion was dispersed in an equal volume of nano-silver pre-cooled at 4°C, and ice-bathed for 10 min to obtain an enrofloxacin-nano-silver compound emulsion.

[0118] Example 13

[0119] This example aims to characterize and analyze the storage stability of the enrofloxacin-nano-silver compound emulsion.

[0120] (1) Transmission electron microscopy observation of the morphology of the enrofloxacin-nano-silver compound emulsion

[0121] Take 10 μL of the enrofloxacin-nano-silver compound emulsion prepared in Example 8 and drop it on a copper mesh, and adsorb for 2 min. Use qualitative filter paper to absorb the excess sample, and air dry at room temperature for 12 h. Use transmission electron microscopy to observe the morphology and dispersion of the enrofloxacin-nano-silver compound emulsion. It can be seen from Figure 4 that the enrofloxacin-nano-silver compound emulsion is approximately spherical, with a size of about 200 nm.

[0122] (2) Measurement of the hydrated particle size, zeta potential and polydispersity coefficient of the enrofloxacin-nano-silver compound emulsion

[0123] Add 1 mL of the enrofloxacin-nano-silver compound emulsion to a sample dish, and equilibrate at 25°C for 2 min. Use a laser particle size potential instrument to measure the hydrated particle size and polydispersity coefficient, respectively. Take 0.85 mL of the enrofloxacin-nano-silver compound emulsion and place it in a capillary sample dish to measure the zeta potential. It can be seen from Figures 5-6 that the hydrated particle size of the enrofloxacin-nano-silver compound emulsion is 183.50±3.21 nm, the zeta potential is -36.00±0.20 mV, and the polydispersity coefficient is 0.181±0.025, so the enrofloxacin-nano-silver compound emulsion has good hydrated particle size and zeta potential distribution.

[0124] Comparative Example 1

[0125] The preparation method of the blank nanostructured lipid carrier is as follows:

[0126] Take 18 g of lauric acid monoglyceride and 12 g of oleic acid at 80°C, melt, stir evenly to get the oil phase; take 10.5 g of polyglyceryl-10 stearate and disperse in 300 mL of 1% polyvinyl alcohol solution, heat and stir at 80°C to get the water phase; at the same temperature, take the oil phase and disperse in the water phase (mass ratio is 1:10), mix the solution and use a high shear machine to shear at 10000 rpm for 5 min to get a coarse dispersion. Use a high-pressure homogenizer to homogenize the coarse dispersion at 700 bar for 5 times, and the obtained primary emulsion is dispersed in an equal volume of deionized water pre-cooled at 4°C to prepare a blank nanostructured lipid carrier.

[0127] Comparative Example 2

[0128] The preparation method of enrofloxacin-nanostructured lipid carrier is as follows:

[0129] Take 18 g of lauric acid monoglyceride, 12 g of oleic acid and 7.5 g of enrofloxacin at 80°C, melt, stir evenly to get the oil phase; take 10.5 g of polyglyceryl-10 stearate and disperse in 300 mL of 1% polyvinyl alcohol solution, heat and stir at 80°C to get the water phase; at the same temperature, take the oil phase and disperse in the water phase (mass ratio is 1:10), mix the solution and use a high shear machine to shear at 10000 rpm for 5 min to get a coarse dispersion. Use a high-pressure homogenizer to homogenize the coarse dispersion at 700 bar for 5 times, and the obtained primary emulsion is dispersed in an equal volume of deionized water pre-cooled at 4°C to prepare enrofloxacin nanostructured lipid carrier.

[0130] Example 14

[0131] In order to study the curative effect of enrofloxacin-nano silver compound emulsion on the skin wound infection of rats, through the model of staphylococcus aureus infection of rat skin wound, the curative effects of enrofloxacin-nano silver compound emulsion, blank nanostructured lipid carrier and enrofloxacin-nanostructured lipid carrier prepared by example 8, comparative example 1 and comparative example 2 respectively were investigated. The specific experimental steps are as follows:

[0132] (1) Test rats and feeding environment

[0133] 7-week-old male rats (specific pathogen-free level, body weight 200 g ± 20 g) were purchased from Changzhou Cavens Experimental Animal Co., Ltd. and fed in the experimental animal center of Nanjing Agricultural University. The rats were free to eat and drink water.

[0134] (2) Establishment of rat wound infection model

[0135] After rats were acclimated for 3 days in a 25±2℃ animal room, rats were anesthetized with isoflurane (respiratory anesthesia), and the dorsal skin of rats was shaved with an electric shaver to expose the epidermis. A mark was made on the dorsal spine of the rat with a marker pen, and the skin was cut to the muscle layer with a sterile forceps and ophthalmic scissors to make a circular incision with a diameter of 2 cm. 200 μL of 10 8 CFU / mL of S. aureus bacteria solution was added to the surface of the wound, and the wound was covered and fixed with a disposable indwelling membrane and simply bandaged with gauze and bandage. After the rats were anesthetized, the state of the rats was observed, and the rats were placed in a single cage for single feeding when they moved freely. After three days of continuous infection, the state of the wound of the rats was observed, and if yellow pus appeared, it was considered a successful model.

[0136] (3) Therapeutic effect of enrofloxacin-nano silver compound emulsion on skin wound infection in rats

[0137] The wound infected rats were randomly divided into 5 groups, 6 rats in each group. 40 μL of different liquid (normal saline, NLCs, AgNPs, ENR-NLCs and ENR-AgNPs) was evenly applied to the infected wound every day, and a disposable indwelling membrane was used to cover it, and a medical gauze was used to fix it. The normal saline (Control) group was used as a blank control. Dressings were changed every day, and changes in wound healing were observed. The size of the wound was measured using a 20 cm ruler, the area of the wound was calculated, and the wound healing rate was calculated by comparing the wound area A9 of each group on the 9th day with the wound area A0 on the 0th day, as follows: wound healing rate = (A0-A9) / A0 x 100%.

[0138] From Figures 7-9It was observed that on day 0 of treatment, all groups of rats showed purulent exudate and bleeding on the entire wound surface. On day 3 of treatment, the Control, NLCs, and ENR-NLCs groups still had significant bleeding and exudation on the wound surface, while the AgNPs and ENR-AgNPs groups showed scabs and fresh granulation tissue formation. On day 6 of treatment, the Control and NLCs groups still had bleeding and exudation on the wound surface, but granulation tissue formation was visible. The ENR-NLCs group showed reduced exudation, significant growth of fresh granulation tissue, and epithelial growth at the wound edge. The AgNPs and ENR-AgNPs groups showed a tendency for scab shedding, and the wound area was smaller compared to the other three groups. On day 9 of treatment, the ENR-AgNPs group wound was almost completely healed, followed by the AgNPs group. The NLCs and ENR-NLCs groups still had a small amount of scabs, but the wound area was significantly reduced. The Control group wound still had scabs and was not completely healed. The calculated wound healing rates were highest in the AgNPs group and the ENR-AgNPs group, at 94.51% and 96.49%, respectively, while the wound healing rates in the Control group, NLCs group, and ENR-NLCs group were approximately 68.36%, 80.53%, and 92.00%, respectively. In conclusion, the prepared enrofloxacin-nanosilver compound emulsion can effectively promote the healing of infected skin wounds in rats.

[0139] In addition, rats in each group were sacrificed on days 0, 3, 6, and 9 after drug administration. 50 mg of skin tissue was collected from the wound site and homogenized using a tissue homogenizer for 15 min to obtain a homogenate. The homogenate was then diluted 10 μL with sterile PBS. 3 Then, 50 μL of the diluted solution was evenly spread on tryptone soybean broth solid medium and incubated at 37°C for 22 h. The number of colonies on the plate was then counted.

[0140] Depend on Figure 10 It was observed that on day 0 of treatment, bacterial colonies grew in all groups, demonstrating the successful establishment of the rat wound infection model. On day 3 of treatment, bacterial counts decreased in all groups. The NLCs group showed no significant difference in bacterial count compared to the Control group, while the AgNPs group showed approximately a 50% reduction in bacterial count compared to the Control group. The ENR-NLCs group showed no significant difference in bacterial count compared to the ENR-AgNPs group, but the AgNPs group had slightly fewer colonies than the AgNPs group. On day 6 of treatment, the AgNPs, ENR-NLCs, and ENR-AgNPs groups had zero bacterial colonies, while the Control and NLCs groups still had relatively high colony counts, indicating that AgNPs, ENR-NLCs, and ENR-AgNPs all inhibited bacterial proliferation in skin wounds.

[0141] from Figures 7-11 It is known that enrofloxacin-nano silver compound emulsion can effectively treat skin wound infection in rats, demonstrating that it promotes skin wound healing and inhibits the proliferation of bacteria in skin wounds.

Claims

1. A method for preparing enrofloxacin nano-silver compound emulsion, characterized in that, The method comprises the following steps: Step one, uniformly disperse Ganoderma lucidum extract containing 40wt% Ganoderma lucidum polysaccharide and silver nitrate in deionized water, and react at 90℃ under constant temperature and light shielding to obtain a nano-silver solution; Step two, melt enrofloxacin, lauryl monoglyceride and oleic acid, and uniformly stir to obtain an oil phase; Step three, disperse polyglyceryl-10 stearate in polyvinyl alcohol solution, and heat and stir to obtain an aqueous phase; Step four, under constant temperature and stirring, transfer the oil phase into the aqueous phase, and shear to obtain a coarse dispersion by using a high shear instrument; Step five, treat the coarse dispersion by using a high-pressure homogenizer to obtain an enrofloxacin emulsion; Step six, uniformly mix the enrofloxacin emulsion with the nano-silver solution, cool, and obtain an enrofloxacin nano-silver compound emulsion; The enrofloxacin nano-silver compound emulsion comprises the following substances in weight percentage: enrofloxacin 7.5 parts, lauryl monoglyceride 18 parts, oleic acid 12 parts, polyglyceryl-10 stearate 10.5 parts, 1% polyvinyl alcohol 300 parts, nano-silver 300 parts, and deionized water 300 parts; In the step one, the solution concentration of the Ganoderma lucidum extract is 3 g / L, and the solution concentration of the silver nitrate is 2 mM; In the step one, the constant temperature and light shielding reaction time is 4 h; In the step two, the mass ratio of lauryl monoglyceride to oleic acid is 1.5, and the mass ratio of enrofloxacin to the sum of lauryl monoglyceride and oleic acid is 1:4; In the step five, the homogenization pressure of the high-pressure homogenizer is 700 bar, and the homogenization times is 5.

2. The preparation method of enrofloxacin nano-silver compound emulsion according to claim 1, characterized in that: In the step two, the temperature of the heating and melting is 80℃.

3. The method for preparing enrofloxacin nano-silver compound emulsion according to claim 1, characterized in that: In the step three, the temperature of the heating and stirring is 80℃.

4. The preparation method of enrofloxacin nano-silver compound emulsion according to claim 1, characterized in that: In the step four, the mass ratio of the oil phase to the aqueous phase is 1:

10.

5. The enrofloxacin nano-silver compound emulsion prepared by the preparation method of claim 1 is applied to the preparation of a medicine for treating animal skin infection.

Images