A rifaximin nanoemulsion, a preparation method and application thereof

Abstract

This invention discloses a rifaximin nanoemulsion, its preparation method, and its application, belonging to the field of veterinary drug technology. The technical solution includes a rifaximin nanoemulsion comprising rifaximin, a compound essential oil, a surfactant, a co-surfactant, and water; the compound essential oil includes oregano oil, cinnamon oil, and tea tree oil. This invention prepares a rifaximin nanoemulsion using a compound of oregano, cinnamon, and tea tree oils as the oil phase, exhibiting both high antibacterial efficiency and high safety. The three essential oils have a synergistic antibacterial effect, complementing rifaximin through multi-target action, broadening the antibacterial spectrum, and showing significant inhibitory effects against various bacteria and fungi. It can enhance uterine smooth muscle contraction function by stimulating the synthesis and secretion of prostaglandins and oxytocin, providing potential support for uterine involution and endometrial repair. After administration, it can effectively reduce the number of inflammatory cells such as leukocytes and neutrophils in experimental dairy cows, significantly reduce purulent vaginal discharge, and restore the softness and elasticity of the uterus upon rectal examination.

Description

Technical Field

[0001] This invention belongs to the field of veterinary drug technology, and in particular relates to a rifaximin nanoemulsion, its preparation method and application. Background Technology

[0002] Endometritis in dairy cows is a common reproductive system disease after calving and a major cause of infertility, resulting in significant losses for the dairy farming industry. Bacterial infection is the primary cause of endometritis in dairy cows, with Escherichia coli and Staphylococcus aureus being the main pathogens. Rifaximin is a broad-spectrum rifamycin antibiotic with strong bactericidal activity, exhibiting potent activity against Gram-positive bacteria. Clinically, it is mainly used to treat uterine inflammation. Due to its unique physicochemical properties, rifaximin is almost not absorbed by the mucous membranes, and therefore does not leave drug residues in milk after clinical use.

[0003] Rifaximin has extremely poor water solubility and low dissolution efficiency. Conventional formulations can only prepare it as a suspension. After injection into the uterus, the drug particles settle rapidly, failing to distribute evenly on the infected mucosa and maintain an effective therapeutic concentration. This results in extremely low local bioavailability and unstable efficacy. Furthermore, due to the large particle size of the suspension, drug particles inevitably cause damage to the local uterine mucosa after uterine instillation, forming scars and affecting embryo implantation after mating.

[0004] To overcome the aforementioned drawbacks of suspensions in uterine drug delivery, nanoemulsions have emerged as a potential alternative dosage form due to their excellent dispersibility and lack of damage to the uterine mucosa. However, their preparation requires precise matching of components such as the oil phase, aqueous phase, and surfactants. The preparation of stable drug-loaded emulsions typically necessitates high-energy-consuming equipment and complex processes, including high-pressure homogenization and ultrasound. Furthermore, they are unfavorable to heat-sensitive essential oil components, resulting in significant challenges in process reproducibility and large-scale production. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the technical problem to be solved by the present invention is that rifaximin is poorly soluble in water, and the suspension will inevitably cause damage to the uterine mucosa. The present invention proposes a rifaximin nanoemulsion with high solubility, high bioavailability and bactericidal efficiency, as well as its preparation method and application.

[0006] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows: This invention provides a rifaximin nanoemulsion, comprising rifaximin, a complex essential oil, a surfactant, a co-surfactant, and water; The compound essential oils include oregano oil, cinnamon oil, and tea tree oil.

[0007] Preferably, the compound essential oil comprises 2-3 parts oregano essential oil, 1-2 parts cinnamon essential oil, and 1 part tea tree essential oil.

[0008] Preferably, the surfactant is polyoxyethylene hydrogenated castor oil, and the co-surfactant is polyethylene glycol-200.

[0009] Preferably, the rifaximin nanoemulsion comprises, by mass percentage, 0.2%-0.5% rifaximin, 2%-3% compound essential oil, 19-20% surfactant, 4-5% co-surfactant, and the balance being water.

[0010] In a second aspect, the present invention provides a method for preparing the above-mentioned rifaximin nanoemulsion, comprising the following steps: A compound essential oil is obtained by mixing oregano essential oil, cinnamon essential oil and tea tree essential oil; a surfactant and co-surfactant are mixed to obtain a surfactant mixture. Rifaximin was dissolved in a compound essential oil in a water bath at 80℃-85℃ to obtain an oil phase. The obtained oil phase was then thoroughly mixed with a surfactant mixture. Water was slowly added dropwise under a high-speed shear press while continuously stirring to prepare a clear and transparent rifaximin nanoemulsion.

[0011] A third aspect of the present invention provides the use of the above-described rifaximin nanoemulsion in the preparation of products for increasing the content of prostate hormones and / or oxytocin in dairy cows.

[0012] In a fourth aspect, the present invention provides the application of the above-mentioned rifaximin nanoemulsion in the preparation of antibacterial agents.

[0013] Preferably, the antibacterial agent is capable of inhibiting Escherichia coli and / or Staphylococcus aureus.

[0014] In a fifth aspect, the present invention provides the use of the above-described rifaximin nanoemulsion in the preparation of a medicament for the prevention and treatment of bovine endometritis.

[0015] Preferably, the drug prevents and treats bovine endometritis through at least one of the following pathways: 1) Stimulates the secretion of prostaglandins in dairy cows; 2) Stimulates the secretion of oxytocin in dairy cows; 3) Reduces the number of inflammatory cells in dairy cows with endometritis; 4) Reduce purulent vaginal discharge in dairy cows with endometritis.

[0016] In a sixth aspect, the present invention provides the use of the above-described rifaximin nanoemulsion in the preparation of products for postpartum uterine repair in dairy cows.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides a rifaximin nanoemulsion, prepared using a blend of oregano, cinnamon, and tea tree oils as the oil phase. It combines highly effective antibacterial properties with high safety. Its core advantages and key technical points are as follows: First, the oil phase ratio: adding tea tree oil reduces the irritation of oregano and cinnamon oils while retaining their warming effect, increasing blood flow and promoting uterine repair. The three essential oils have a synergistic antibacterial effect, complementing rifaximin through multi-target action, broadening the antibacterial spectrum, and showing significant inhibitory effects against various bacteria and fungi. Second, the nanoemulsion process: selecting the optimal formulation ratio and essential oil dosage reduces mucosal irritation. Choosing suitable surfactants and co-surfactants makes the nanoemulsion more stable. The uniform dispersion of the nanoemulsion improves biocompatibility and suitability for mucosal drug delivery. The preparation process is simple, without complex operations, reducing production costs. Third, it offers high delivery efficiency and good stability. The nanoemulsion improves the water solubility of rifaximin and essential oils, reduces component degradation, prolongs in vivo retention time, and enhances bioavailability.

[0018] 2. The rifaximin nanoemulsion provided by this invention can enhance uterine smooth muscle contraction function by stimulating the synthesis and secretion of prostaglandins and oxytocin, thus providing potential support for uterine involution. Both prostaglandins and oxytocin play key roles in the endometrial repair process. Prostaglandins can induce rhythmic contractions of uterine smooth muscle, dilate the cervix, promote the expulsion of residual tissue from the uterine cavity, and simultaneously contribute to uterine volume reduction and endometrial wound repair. Oxytocin can significantly enhance uterine contractility and contraction frequency, accelerate the uterine involution process, and provide a good foundation for endometrial regeneration and structural reconstruction. The two work synergistically to promote the uterus's return to its normal physiological state after delivery, enhance uterine smooth muscle contraction function, accelerate uterine involution and the expulsion of residual tissue from the uterine cavity, and provide positive support for uterine involution and endometrial repair.

[0019] 3. This invention successfully prepared rifaximin nanoemulsion, which is an oil-in-water emulsion that can be infinitely diluted and has good stability and dispersibility. After administration, it can effectively reduce the number of inflammatory cells such as white blood cells and neutrophils in experimental dairy cows, significantly reduce purulent vaginal discharge, and restore the softness and elasticity of the uterus in experimental dairy cows upon rectal examination. This provides a practical theoretical basis and technical solution for the development of long-acting and stable veterinary uterine instillation antibacterial nano-preparations. Attached Figure Description

[0020] Figure 1 This is a schematic diagram showing the area of ​​the emulsion region formed by different surfactants according to an embodiment of the present invention; Figure 2 This is a schematic diagram showing the maximum absorbance of the three co-surfactants provided in the embodiments of the present invention; Figure 3Standard curves for anhydrous ethanol (left), PEG-200 (middle), and PEG-400 (right) provided in embodiments of the present invention; Figure 4 This is a schematic diagram showing the maximum concentrations of the three co-surfactants provided in the embodiments of the present invention; Figure 5 This is a schematic diagram showing the emulsion area formed by the surfactant and co-surfactant at different Km values ​​according to embodiments of the present invention. Figure 6 The following are schematic diagrams of the state of nanoemulsions after centrifugation and after centrifugation and storage for 14 days, provided in the embodiments of the present invention; wherein Figure (a) is a schematic diagram of the state of nanoemulsions after centrifugation (the left side is after centrifugation at 3000 rpm, and the right side is after centrifugation at 5000 rpm), and Figure (b) is a schematic diagram of the state of nanoemulsions after centrifugation (the left side is after centrifugation at 3000 rpm, and the right side is after centrifugation at 5000 rpm). Figure 7 This is a schematic diagram of the state of nanoemulsion after high-speed shearing, provided in an embodiment of the present invention. Figure 8 This is a schematic diagram of the state of nanoemulsions after being stored at different temperatures for 14 days, provided in an embodiment of the present invention. Figure 9 A schematic diagram showing the dispersion state of rifaximin oregano essential oil nanoemulsion in standard hard water over time, as provided in an embodiment of the present invention. Figure 10 A schematic diagram illustrating the staining state of Sudan Red (left) and Methylene Blue (right) in rifaximin oregano oil nanoemulsion provided in an embodiment of the present invention; Figure 11 This is a secondary mass spectrum of rifaximin in cow milk after drug administration, provided in an embodiment of the present invention; wherein, A: before drug administration; B: after drug administration; Figure 12 This is a secondary mass spectrometry image of rifaximin in the blood of dairy cows after drug administration, provided in an embodiment of the present invention; wherein, A: before drug administration; B: after drug administration; Figure 13 This is a high-performance liquid chromatogram of cow's milk provided in an embodiment of the present invention; wherein, A: before drug administration; B: after drug administration; Figure 14 The image shows a high-performance liquid chromatogram of bovine blood provided in an embodiment of the present invention; where A: before medication; B: after medication.

[0021] Figure 15 The graph showing the changes in prostaglandin and oxytocin levels in the blood of dairy cows before and after medication, provided in an embodiment of the present invention, shows that: A: prostaglandin content; B: oxytocin content. Detailed Implementation

[0022] The technical solutions in specific embodiments of the present invention will now be described in detail and completely with reference to the accompanying drawings. Obviously, the described embodiments are merely some specific implementations of the overall technical solution of the present invention, and not all implementations. Based on the overall concept of the present invention, all other embodiments obtained by those skilled in the art fall within the protection scope of the present invention.

[0023] The present invention provides a rifaximin nanoemulsion, comprising rifaximin, a complex essential oil, a surfactant, a co-surfactant, and water; The compound essential oils include oregano oil, cinnamon oil, and tea tree oil.

[0024] It should be noted that the existing technology for rifaximin has the disadvantages of extremely poor water solubility and low dissolution efficiency, and conventional formulations can only prepare it as a suspension. After injection into the uterus, the drug particles settle rapidly, failing to distribute evenly on the infected mucosa surface and maintain an effective therapeutic concentration, resulting in extremely low local bioavailability and unstable efficacy. Furthermore, because the suspension molecules are relatively large, they inevitably cause damage to the uterine mucosa, affecting embryo implantation after mating.

[0025] To address the problem of rifaximin's poor water solubility and its unavoidable damage to the uterine mucosa, this invention develops a rifaximin nanoemulsion. Using a compound plant essential oil as the oil phase to dissolve rifaximin effectively solves this solubility issue. The nanoemulsion exhibits good dispersibility and will not damage the uterine mucosa. The compound rifaximin nanoemulsion is not absorbed by the mucosa and does not enter the emulsion. Rifaximin can dissolve in the compound essential oils. This results in better bactericidal effects and reduced damage to the endometrium. Cinnamon and oregano essential oils have a warming effect that can stimulate increased blood flow to the endometrium, enhancing endometrial repair and renewal, shortening treatment time, and allowing for insemination after treatment.

[0026] The aforementioned technical solution further specifies that the compound essential oils include oregano, cinnamon, and tea tree oil. This is because cinnamon and oregano oils have been proven to have strong bactericidal effects against various pathogens such as Escherichia coli and Staphylococcus aureus, with their main active ingredients being cinnamaldehyde and carvacrol, respectively. While cinnamon and oregano oils have strong bactericidal properties and can act as oil-phase carriers for rifaximin to produce synergistic antibacterial effects, their main components are highly irritating to mucous membranes, and direct compounding and perfusion can easily cause mucosal damage. Tea tree oil, whose main component is terpinene-4-ol, has mild antibacterial and anti-inflammatory properties. Compounding it into the oil phase can enhance the antibacterial effect and reduce the irritation of cinnamon and oregano oils to mucous membranes. This invention explores the oil phase ratio and, while retaining the potent bactericidal activity of cinnamon and oregano essential oils, employs a dual strategy of compounding tea tree essential oil and nanoemulsion encapsulation to physically shield irritating components from direct contact with mucous membranes, thereby reducing the local irritation of the formulation to a safe and acceptable range.

[0027] In a preferred embodiment, the compound essential oil comprises 2-3 parts oregano essential oil, 1-2 parts cinnamon essential oil, and 1 part tea tree essential oil.

[0028] The above technical solution limits the blending ratio of tea tree oil, oregano oil, and cinnamon oil in the compound essential oil. Under the above ratio, the FICI index of the three can be controlled between 0.25 and 0.375, which is a strong synergistic effect.

[0029] In a preferred embodiment, the surfactant is polyoxyethylene hydrogenated castor oil, and the co-surfactant is polyethylene glycol-200.

[0030] In a preferred embodiment, the rifaximin nanoemulsion comprises, by mass percentage, 0.2%-0.5% rifaximin, 2%-3% compound essential oil, 19%-20% surfactant, 4%-5% co-surfactant, and the balance being water.

[0031] In a second aspect, the present invention provides a method for preparing the above-mentioned rifaximin nanoemulsion, comprising the following steps: A compound essential oil is obtained by mixing oregano essential oil, cinnamon essential oil and tea tree essential oil; a surfactant and co-surfactant are mixed to obtain a surfactant mixture. Rifaximin was dissolved in a compound essential oil in a water bath at 80℃-85℃ to obtain an oil phase. The obtained oil phase was then thoroughly mixed with a surfactant mixture at room temperature. Water was added drop by drop at an average rate of one second using a pipette while continuously stirring to prepare a clear and transparent rifaximin nanoemulsion.

[0032] It should be noted that the preparation of nanoemulsions requires precise matching of components such as oil phase, aqueous phase, and surfactant. The preparation of stable drug-loaded emulsions usually requires high-energy-consuming equipment and complex processes such as high-pressure homogenization and ultrasound. Furthermore, it is not conducive to heat-sensitive essential oil components, and the process reproducibility and large-scale production are difficult.

[0033] This invention provides a mild and low-energy preparation method that enables nanoemulsions to spontaneously form under simple stirring. The process is simple, the conditions are mild, the energy consumption is low, and it is easy to achieve stable and controllable industrial production.

[0034] A third aspect of the present invention provides the use of the above-described rifaximin nanoemulsion in the preparation of products for increasing the content of prostate hormones and / or oxytocin in dairy cows.

[0035] The rifaximin nanoemulsion provided by this invention can enhance uterine smooth muscle contraction by stimulating the synthesis and secretion of prostaglandins and oxytocin, thus providing potential support for uterine involution. Both prostaglandins and oxytocin play key roles in endometrial repair. Prostaglandins can induce rhythmic contractions of uterine smooth muscle, dilate the cervix, promote the expulsion of residual tissue from the uterine cavity, and simultaneously contribute to uterine volume reduction and endometrial wound repair. Oxytocin can significantly enhance uterine contractility and frequency, accelerate the uterine involution process, and provide a good foundation for endometrial regeneration and structural reconstruction. The two work synergistically to promote the uterus's return to its normal physiological state after delivery, enhance uterine smooth muscle contraction, accelerate uterine involution and the expulsion of residual tissue from the uterine cavity, and provide positive support for uterine involution and endometrial repair.

[0036] In a fourth aspect, the present invention provides the application of the above-mentioned rifaximin nanoemulsion in the preparation of antibacterial agents.

[0037] In a preferred embodiment, the antibacterial agent is capable of inhibiting Escherichia coli and / or Staphylococcus aureus.

[0038] In a fifth aspect, the present invention provides the use of the above-described rifaximin nanoemulsion in the preparation of a medicament for the prevention and treatment of bovine endometritis.

[0039] In a preferred embodiment, the drug prevents and treats bovine endometritis through at least one of the following pathways: 1) Stimulates the secretion of prostaglandins in dairy cows; 2) Stimulates the secretion of oxytocin in dairy cows; 3) Reduces the number of inflammatory cells in dairy cows with endometritis; 4) Reduce purulent vaginal discharge in dairy cows with endometritis.

[0040] In a sixth aspect, the present invention provides the use of the above-described rifaximin nanoemulsion in the preparation of products for postpartum uterine repair in dairy cows.

[0041] To provide a clearer and more detailed description of the rifaximin nanoemulsion, its preparation method, and its application provided by the embodiments of the present invention, the following description will be based on specific embodiments.

[0042] In the following embodiments and comparative examples of the present invention, the oregano essential oil was purchased from Ji'an Zhongxiang Natural Plant Co., Ltd., CAS No. 8007-11-2; Cinnamon essential oil was purchased from Ji'an Zhongxiang Natural Plant Co., Ltd., CAS No. 8007-80-5; The tea tree oil was purchased from Shanghai Yuanye Biotechnology Co., Ltd., CAS number 68647-73-4.

[0043] Example 1: Screening of Rifaximin Nanoemulsion Formulation 1. Formula screening methods 1.1 Screening of the oil phase Prepare MH broth medium: Weigh 2.1 g of MH broth medium into an Erlenmeyer flask, add 100 mL of distilled water, seal the flask with gauze and filter paper, and autoclave. While still hot, dispense the MH medium into shaker tubes. Inoculate 5 μL of each of the collected Escherichia coli and Staphylococcus aureus into the prepared MH broth medium and incubate in a shaker for 18–24 h. Prepare LB solid medium: Add 12.5 g of LB broth powder and 7.5 g of agarose to 500 mL of distilled water, seal the flask with gauze and filter paper, and autoclave. While still hot, pour the LB medium into petri dishes. After cooling and solidifying, add 100 μL of Escherichia coli and Staphylococcus aureus bacterial suspensions, spread evenly with a glass rod, and take care to sterilize to prevent cross-contamination. After spreading the solution evenly, use small tweezers to place a piece of drug sensitivity paper in the center of the petri dish. Vertically add 5 μL of either a single plant essential oil or a compound essential oil to the paper. Incubate at 37℃ for 18–24 h. Measure the diameter of the inhibition zone for both the single plant essential oil and the compound essential oil using the cross-hatching method. Repeat the experiment three times for each strain and take the average value. Calculate the FICI value using the following formula: FICI= MIC blend / MIC oregano + MIC blend / MIC cinnamon + MIC blend / MIC tea tree.

[0044] 1.2 Screening of Surfactants At room temperature, three commonly used surfactants (EL-40, RH-40, and Tween-80) with a hydrophilic-lipophilic balance (HLB) value between 8 and 16, selected from the pre-screened mixed oil phase ratio, were mixed in conical flasks at mass ratios of 9:1 to 1:9. The conical flasks were placed on a magnetic stirrer, and distilled water was added dropwise while stirring. The formation of a clear, transparent liquid was observed, and the phase transition point (the change from clear to turbid) was recorded. The mass of distilled water added at this point was also recorded. Distilled water was continued to be added dropwise until the liquid in the conical flask was clear, transparent, and did not separate into layers. A pseudo-ternary phase diagram was drawn using Origin 2021 software, with the oil phase, water phase, and surfactant as the three vertices. The surfactant with the largest emulsion area was selected.

[0045] 1.3 Screening of co-surfactants Co-surfactants are mainly medium-chain and short-chain, and should be alcohols with low or no toxicity. Therefore, anhydrous ethanol, PEG-200, and PEG-400 were selected as three co-surfactants for screening. At room temperature, excess rifaximin was dissolved in anhydrous ethanol, PEG-200, and PEG-400 respectively to prepare corresponding saturated solutions. The absorbance of the three co-emulsifiers in the range of 390 nm to 550 nm was scanned using a microplate reader, and the maximum absorbance of each was recorded. Figure 2 ).

[0046] At room temperature, 1 mg rifaximin was dissolved in 10 mL of anhydrous ethanol, 10 mL of polyethylene glycol-200, and 10 mL of polyethylene glycol-400 to prepare stock solutions of 0.1 mg / mL. The three stock solutions were then serially diluted from 0.01 to 0.1 mg / mL. The absorbance of the diluted stock solutions was scanned using a microplate reader, and the absorbance in the range of 390 nm to 550 nm was recorded. Standard curves were plotted with the concentration of each stock solution as the X-axis and the corresponding absorbance as the Y-axis. Figure 3 By substituting the maximum absorbance values ​​of the three co-emulsifiers into their respective standard curves, the maximum concentrations of the three co-emulsifiers can be obtained, thus allowing the selection of the optimal co-emulsifier.

[0047] 1.4 Determination of the surfactant / co-surfactant mass ratio (Km value) At room temperature, the screened surfactants and co-surfactants were mixed in beakers at mass ratios of 2:1, 3:1, and 4:1, respectively. These were then mixed with the screened oil phase at mass ratios of 1:9 to 9:1. The mixture was placed on a magnetic stirrer and distilled water was added dropwise under high-speed shearing while stirring. The phase transition point, i.e., the state where the system changed from clear to turbid, was recorded, along with the mass of distilled water added at this point. A pseudo-ternary phase diagram was drawn using Origin 2021 software. The Km value was determined by selecting the ratio of surfactant to co-surfactant with the largest emulsion area.

[0048] 2. Test Results: 2.1 Oil phase screening results Table 1. Average antibacterial diameter (mm) of the three essential oils

[0049] As shown in Table 1, oregano essential oil, cinnamon essential oil, and tea tree essential oil all have antibacterial effects against Escherichia coli and Staphylococcus aureus.

[0050] Table 2. Average antibacterial diameter (mm) of the three essential oils working synergistically.

[0051] Table 3. Synergistic FICI effects of three essential oils

[0052] As shown in Table 3, the three essential oils exhibit synergistic effects at ratios of 2:1:1, 2:2:1, and 3:2:1, with the strongest synergistic effect observed at a ratio of 3:2:1. Therefore, this ratio was selected as the oil phase for the preparation of rifaximin nanoemulsion in Example 2.

[0053] 2.2 Screening Results of Surfactants Depend on Figure 1 It can be seen that the emulsion area is the largest when RH-40 is used as the surfactant. Therefore, RH-40 is selected as the surfactant for the nanoemulsion.

[0054] 2.3 Screening results of co-surfactants like Figure 4 As shown, it is evident that rifaximin has a higher solubility in PEG-200 than in PEG-400 and anhydrous ethanol. Therefore, PEG-200 was chosen as the co-surfactant for the nanoemulsion.

[0055] 2.4 Mass ratio of surfactant to co-surfactant (Km value) Depend on Figure 5 It can be seen that the area of ​​the breast tissue formed is the largest when the Km ratio is 4:1. Therefore, the Km ratio of 4:1 is chosen.

[0056] The optimal formulation of compound rifaximin nanoemulsion was determined based on the drug loading, stability, and emulsion area of ​​the pseudo-ternary phase diagram.

[0057] Example 2: Preparation of rifaximin nanoemulsion 1. Reaction raw materials (mass percentage): rifaximin 0.2%, compound essential oil 2.44%, surfactant 19.51%, co-surfactant 4.88%, and the balance water; The compound essential oil consists of 3 parts oregano essential oil, 2 parts cinnamon essential oil and 1 part tea tree essential oil; 2. Preparation method: At room temperature, RH-40 and PEG-200 were weighed into a beaker at a Km ratio of 4:1 and stirred on a magnetic stirrer until the liquid in the beaker became clear and transparent, thus obtaining a mixture of RH-40 and PEG-200. Rifaximin and mixed essential oils were weighed, and rifaximin was dissolved in the essential oils in a water bath at 80°C. At room temperature, the resulting oil phase was thoroughly mixed with the RH-40 and PEG-200 mixture. Water was added drop by drop at an average rate of one second using a pipette while stirring continuously to prepare a clear and transparent rifaximin nanoemulsion.

[0058] Comparative Example 1: Preparation of Rifaximin Nanoemulsion 1. Reaction raw materials (mass percentage): rifaximin 0.2%, compound essential oil 2.44%, surfactant 19.51%, co-surfactant 4.88%, and the balance water; The blended essential oil consists of 3 parts oregano essential oil and 2 parts cinnamon essential oil; 2. Preparation method: At room temperature, RH-40 and PEG-200 were weighed into a beaker at a Km ratio of 4:1 and stirred on a magnetic stirrer until the liquid in the beaker became clear and transparent, thus obtaining a mixture of RH-40 and PEG-200. Rifaximin and mixed essential oils were weighed, and rifaximin was dissolved in the essential oils in a water bath at 80°C. At room temperature, the resulting oil phase was thoroughly mixed with the RH-40 and PEG-200 mixture. Water was added drop by drop at an average rate of one second using a pipette while stirring continuously to prepare a clear and transparent rifaximin nanoemulsion.

[0059] Comparative Example 2: Preparation of Rifaximin Nanoemulsion 1. Reaction raw materials (mass percentage): rifaximin 0.2%, mixed essential oils 2.44%, surfactant 19.51%, co-surfactant 4.88%, and the balance water; The blended essential oil consists of 3 parts oregano oil and 1 part tea tree oil; 2. Preparation method: At room temperature, RH-40 and PEG-200 were weighed into a beaker at a Km ratio of 4:1 and stirred on a magnetic stirrer until the liquid in the beaker became clear and transparent, thus obtaining a mixture of RH-40 and PEG-200. Rifaximin and mixed essential oils were weighed, and rifaximin was dissolved in the essential oils in a water bath at 80°C. At room temperature, the resulting oil phase was thoroughly mixed with the RH-40 and PEG-200 mixture. Water was added drop by drop at an average rate of one second using a pipette while stirring continuously to prepare a clear and transparent rifaximin nanoemulsion.

[0060] Comparative Example 3: Preparation of Rifaximin Nanoemulsion 1. Reaction raw materials (mass percentage): rifaximin 0.2%, mixed essential oils 2.44%, surfactant 19.51%, co-surfactant 4.88%, and the balance water; The blended essential oil consists of 2 parts cinnamon oil and 1 part tea tree oil; 2. Preparation method: At room temperature, RH-40 and PEG-200 were weighed into a beaker at a Km ratio of 4:1 and stirred on a magnetic stirrer until the liquid in the beaker became clear and transparent, thus obtaining a mixture of RH-40 and PEG-200. Rifaximin and mixed essential oils were weighed, and rifaximin was dissolved in the essential oils in a water bath at 80°C. At room temperature, the resulting oil phase was thoroughly mixed with the RH-40 and PEG-200 mixture. Water was added drop by drop at an average rate of one second using a pipette while stirring continuously to prepare a clear and transparent rifaximin nanoemulsion.

[0061] Comparative Example 4: Rifaximin Suspension The rifaximin suspension was purchased from Hubei Huisheng Biotechnology Co., Ltd., and the mass fraction of rifaximin in the rifaximin suspension was 0.2%.

[0062] Example 3: Quality evaluation of rifaximin nanoemulsion: 1. Evaluation Methods 1.1 Centrifugal stability test Centrifugal stability testing was used to examine the formulation's resistance to aggregation under mechanical stress. 10 mL of the rifaximin nanoemulsion sample prepared in Example 2 was placed in a 15 mL centrifuge tube and centrifuged at 3000 rpm and 5000 rpm (simulating centrifugal force generated by transport vibration or storage shaking) at a constant temperature of 25°C for 15 min. After centrifugation, the emulsion in the tube was immediately observed visually for stratification, turbidity, flocculent precipitation, or oil-water separation. The centrifuged sample was then observed for its appearance, paying particular attention to any visible droplet aggregation or phase separation tendency.

[0063] 1.2 High-speed shear resistance test In the quality evaluation of rifaximin nanoemulsions, the high-speed shear tolerance test was used to examine the formulation's adaptability to mechanical stress during industrial production. The specific procedure was as follows: 20 mL of the rifaximin nanoemulsion sample prepared in Example 2 was placed in a high-speed shear apparatus and continuously sheared at 12,000 rpm for 10 min at a constant temperature of 25°C. Every 2 min, the appearance of the emulsion was observed visually, and the presence of turbidity, stratification, flocculent precipitation, or phase separation was recorded. After the shearing treatment, the appearance of the sample was observed, paying particular attention to any visible droplet aggregation, oil-water separation, or abnormal viscosity changes.

[0064] 1.3 Temperature tolerance test Temperature tolerance tests are used to examine the physical stability of the formulation under different ambient temperatures. 10 mL of the rifaximin nanoemulsion sample prepared in Example 2 was placed into transparent vials, sealed, and placed in a -20°C freezer, a 4°C freezer, and a 40°C incubator for 14 days. After being allowed to recover at room temperature for 30 minutes, the appearance was observed visually, with particular attention to whether turbidity, layering, flocculent precipitation, oil droplet formation, or abnormal viscosity (such as thickening or thinning) occurred.

[0065] 1.4 Dispersion Experiment The dispersibility test is used to examine the dispersibility and uniformity of a formulation in a specific medium. Weigh 0.304 g of anhydrous calcium chloride and 0.139 g of magnesium chloride hexahydrate, dissolve them separately in a small amount of distilled water, pour both solutions into 1000 mL volumetric flasks, add distilled water to the calibration mark, and shake well to prepare standard hard water. Take 100 mL of standard hard water in a graduated cylinder, and use a micropipette to add 500 μL of rifaximin oregano essential oil nanoemulsion to the water from a distance of 5 cm from the water surface in the graduated cylinder, observing its dispersion state.

[0066] 1.5 nanometer emulsion type identification test The type of nanoemulsion was identified using Sudan III (an oil-soluble dye) and methylene blue (a water-soluble dye) staining methods. The determination was based on the dye's solubility characteristics in different emulsion phases: uniform dispersion of oil-soluble dyes indicated an oil phase (W / O type), while uniform dispersion of water-soluble dyes indicated an aqueous phase (O / W type). Two transparent glass bottles were used, and appropriate amounts of rifaximin oregano oil nanoemulsion were added to each. A suitable amount of 0.1% Sudan III anhydrous ethanol solution was added to one bottle, and a suitable amount of 0.1% methylene blue aqueous solution was added to the other. The dispersion of the dyes in the two groups of nanoemulsions was observed.

[0067] 2. Test Results 2.1 Centrifugation test like Figure 6 The results showed that after centrifugation at 3000 rpm (left) and 5000 rpm (right) for 5 min, the nanoemulsions did not show any visible layering, precipitation or turbidity, and remained a clear and transparent homogeneous system. After storage for 14 days, the appearance of each group of samples was still consistent with that before centrifugation, with no layering or abnormal viscosity changes.

[0068] 2.2 High-speed shear resistance test like Figure 7 The results showed that after the high-speed shearing process, the appearance of the rifaximin oregano essential oil nanoemulsion remained in its initial state, without any layering, turbidity or precipitation, indicating that the colloidal structure of the emulsion remained stable under high-intensity mechanical stress and long-term storage.

[0069] 2.3 Temperature tolerance test Figure 8 The results showed that the nanoemulsion remained clear and transparent at -20℃, with no ice crystal precipitation or droplet aggregation, and its appearance was consistent with the initial state after returning to room temperature. At 4℃, the sample did not show any stratification or turbidity for 14 days, and the pale orange color of the nanoemulsion was uniform and stable. Even at 40℃, a slight increase in viscosity was observed only on the 14th day (with no visible phase separation), but the viscosity reversibility decreased after returning to room temperature, and there was no significant change in appearance.

[0070] 2.4 Dispersion Test like Figure 9 The results showed that the rifaximin nanoemulsion automatically dispersed in standard hard water in a cloud-like manner, with no visible particles settling. This indicates that the rifaximin oregano oil nanoemulsion has good dispersibility and uniform dispersion.

[0071] 2.5 Nanoemulsion Type Identification Test according to Figure 10 The results showed that the system with added methylene blue rapidly and uniformly turned blue, while the Sudan Red solution only dispersed locally and did not color the entire system. Analysis indicated that methylene blue, as a water-soluble dye, dissolved uniformly in the external phase, confirming that the external phase was aqueous; Sudan Red, as an oil-soluble dye, did not disperse uniformly, indicating that the oil phase was internal. Based on these findings, the nanoemulsion was determined to be an oil-in-water (O / W) type, consistent with the formulation design expectations.

[0072] Experimental Example 1: Uterine Mucosal Stimulation Test in Mice Mice were randomly divided into 3 groups and fasted for 6 hours before the experiment. Each group contained 5 adult female mice. Groups 1 and 2 were the experimental groups, and group 3 was the control group. Treatment group 1 was instilled with rifaximin nanoemulsion prepared in Comparative Example 1, treatment group 2 was instilled with rifaximin nanoemulsion prepared in Example 2, and the control group was instilled with an equal volume of physiological saline.

[0073] The uterus was instilled with 20 μL of the drug continuously for one week. Twenty-four hours after the last administration, the uterus was euthanized by cervical dislocation to observe mucosal irritation. Scoring was performed according to the scoring method in Table 4. The average score for each group was then calculated, and the results were evaluated according to the uterine mucosal irritation intensity evaluation criteria.

[0074] Table 4. Scoring criteria for uterine mucosal irritation response

[0075] Table 5 Evaluation Criteria for Uterine Mucosal Irritation Intensity

[0076] 2. Test Results Table 6 Irritation scores of mouse uterine mucosa

[0077] As shown in Table 6, the irritation score of nanoemulsion with added tea tree oil was lower than that without added tea tree oil. Therefore, it can be concluded that adding tea tree oil nanoemulsion reduces the irritation of nanoemulsion on the uterine mucosa of mice.

[0078] Experiment Example 2: Clinical Efficacy Trial of Drug Use in Dairy Cows on a Cattle Farm 1. Test method: 1.1 Animal grouping: Cows suffering from postpartum endometritis were selected as experimental cows, with 7 cows selected in each group. The experiment was divided into five groups, as follows: Nanoemulsion group: The experimental dairy cows were treated with the drug once each at 30 days, 40 days and 50 days postpartum, and each time 100 mL of rifaximin nanoemulsion prepared in Example 2 was instilled into the uterus; Control group: The experimental dairy cows were treated with the drug once each at 30 days, 40 days and 50 days postpartum, with 100 mL of physiological saline instilled through the uterus each time; Suspension group: The experimental dairy cows were treated with the drug once each at 30 days, 40 days and 50 days postpartum, and each time 100 mL of rifaximin suspension of Comparative Example 4 was instilled through the uterus; Tea tree oregano nanoemulsion group: The experimental dairy cows were treated with the drug once each at 30 days, 40 days and 50 days postpartum, and each time 100 mL of rifaximin nanoemulsion prepared in Comparative Example 2 was instilled into the uterus. Tea tree cinnamon nanoemulsion group: The experimental dairy cows were treated with the drug once each at 30 days, 40 days and 50 days postpartum, and each time 100 mL of rifaximin nanoemulsion prepared in Comparative Example 3 was instilled into the uterus. 1.2 Test Procedure: 1.2.1 Blood routine tests, blood biochemical indicators, drug levels, and cinnamaldehyde residues in the experimental dairy cows. Before administration of the nanoemulsion drug 30 days postpartum, 8 mL of blood and 40 mL of milk were collected from each cow in the nanoemulsion group for pre-administration drug residue testing, routine pre-administration biochemical tests, and prostaglandin and oxytocin levels. One hour after administration, blood was collected to detect changes in hormone levels, and serum was separated by centrifugation. Prostaglandin and oxytocin levels were detected using enzyme-linked immunosorbent assay (ELISA). On the second day after the first administration at 30 days postpartum, 8 mL of blood and 4 mL of milk were collected from each cow for post-administration drug residue testing. On 60 days postpartum, 5 mL of blood was collected from each cow for routine post-administration biochemical tests.

[0079] The collected anticoagulated whole blood from dairy cows was analyzed using a fully automated blood analyzer. Blood samples from each group without anticoagulants were centrifuged at 5000 r / min for 10 min, and the supernatant was collected. The blood biochemical parameters of the dairy cow serum were then detected using a fully automated biochemical analyzer.

[0080] Rifaxine nanoemulsion and cinnamon drug residues in serum and milk were detected by liquid chromatography-mass spectrometry.

[0081] 1.2.2 Treatment Effects in Experimental Dairy Cows Ten days after the last administration, clinical observations were conducted on the cows' mental state, vaginal discharge, and uterine condition. Pregnancy status was also observed after the last administration.

[0082] 2. Test Results: 2.1 Effects of compound rifaximab nanoemulsion on routine blood parameters of experimental dairy cows Table 7 Effects of rifaximin nanoemulsion on routine blood parameters of experimental dairy cows

[0083] As shown in Table 7, the medication can reduce the number of inflammatory cells such as white blood cells and neutrophils.

[0084] Table 8 Effects of rifaximin nanoemulsion on blood biochemical parameters of experimental dairy cows

[0085] As shown in Table 8, rifaximin nanoemulsion did not adversely affect the functions of important organs such as liver detoxification and metabolism, kidney excretion and filtration, or the homeostasis of glucose and lipid metabolism in dairy cows, demonstrating good clinical safety and biocompatibility.

[0086] 2.2 Rifaximin residues in milk and blood after medication administration Table 9. Rifaximin Residues in Dairy Cow Milk

[0087] Table 10. Rifaximin Residues in Dairy Cow Blood

[0088] To determine whether rifaximin is absorbed by the mucous membrane, drug residues in the blood and milk of dairy cows were tested after administration. See Tables 9 and 10 for details. Figure 11 , 12 The conclusion can be drawn that rifaximin is not absorbed by the mucous membrane after uterine administration to dairy cows, and no drug residues were detected in milk or blood.

[0089] 2.3 Cinnamaldehyde drug residues in milk and blood after medication administration Table 11 Cinnamaldehyde Residues in Dairy Cow Milk

[0090] Table 12 Cinnamaldehyde drug residues in dairy cow blood

[0091] Cinnamaldehyde is volatile, has a strong odor, and affects the flavor of dairy products. Tests were conducted to detect cinnamaldehyde content in milk and blood, as shown in Tables 11 and 12. Figure 13 , 14 The conclusion can be drawn that no cinnamaldehyde drug residues were detected in either milk or blood.

[0092] 2.4 Therapeutic effects on experimental dairy cows 2.4.1 Effects of treatment on changes in stimulation in experimental dairy cows like Figure 15 As shown, after drug administration, the levels of prostaglandins and oxytocin in dairy cows were significantly increased (P<0.01), suggesting that the tested drug can stimulate the secretion of prostaglandins and oxytocin, enhance the contractile function of uterine smooth muscle, and provide potential support for uterine involution and endometrial repair.

[0093] 2.4.2 Clinical treatment effects in dairy cows Table 13 Clinical treatment effects in dairy cows

[0094] It can be seen that after the experimental dairy cows were given the medication, the purulent vaginal discharge was significantly reduced, and rectal examination showed that the uterus of the dairy cows regained its softness and elasticity.

[0095] 2.4.3 Cow conception rate Table 14 Conception rate of dairy cows under different treatments

[0096] All seven cows treated with the mixed essential oil nanoemulsion became pregnant, achieving a 100% pregnancy rate. The pregnancy rate with the suspension was 71.43%. The mixed essential oil rifaximin nanoemulsion showed significant effects on postpartum uterine repair and inflammation control in dairy cows, with a higher efficacy than the suspension group.

Claims

1. A rifaximin nanoemulsion, characterized in that, Including rifaximin, compound essential oils, surfactants, co-surfactants and water; The compound essential oils include oregano oil, cinnamon oil, and tea tree oil.

2. The rifaximin nanoemulsion according to claim 1, characterized in that, The compound essential oil comprises 2-3 parts oregano essential oil, 1-2 parts cinnamon essential oil, and 1 part tea tree essential oil.

3. The rifaximin nanoemulsion according to claim 1, characterized in that, The surfactant is polyoxyethylene hydrogenated castor oil, and the co-surfactant is polyethylene glycol-200.

4. The rifaximin nanoemulsion according to claim 1, characterized in that, The rifaximin nanoemulsion comprises, by mass percentage, 0.2%-0.5% rifaximin, 2%-3% compound essential oil, 19%-20% surfactant, 4%-5% co-surfactant, and the balance being water.

5. The method for preparing rifaximin nanoemulsion according to any one of claims 1-4, characterized in that, Includes the following steps: A compound essential oil is obtained by mixing oregano essential oil, cinnamon essential oil and tea tree essential oil; a surfactant and co-surfactant are mixed to obtain a surfactant mixture. Rifaximin was dissolved in a compound essential oil in a water bath at 80℃-85℃ to obtain an oil phase. The obtained oil phase was then thoroughly mixed with a surfactant mixture. Water was slowly added dropwise under a high-speed shear press while continuously stirring to prepare a clear and transparent rifaximin nanoemulsion.

6. The use of the rifaximin nanoemulsion according to any one of claims 1-4 in the preparation of products for increasing the content of prostate hormones and / or oxytocin in dairy cows.

7. The use of the rifaximin nanoemulsion according to any one of claims 1-4 in the preparation of antibacterial agents, characterized in that, The antibacterial agent can inhibit Escherichia coli and / or Staphylococcus aureus.

8. The use of the rifaximin nanoemulsion according to any one of claims 1-4 in the preparation of a medicament for the prevention and treatment of bovine endometritis.

9. The application according to claim 8, characterized in that, The drug prevents and treats bovine endometritis through at least one of the following pathways: 1) Stimulates the secretion of prostaglandins in dairy cows; 2) Stimulates the secretion of oxytocin in dairy cows; 3) Reduces the number of inflammatory cells in dairy cows with endometritis; 4) Reduce purulent vaginal discharge in dairy cows with endometritis.

10. The use of the rifaximin nanoemulsion according to any one of claims 1-4 in the preparation of products for postpartum uterine repair in dairy cows.

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