Temperature-sensitive sustained-release gel for treating cow mastitis and preparation method of temperature-sensitive sustained-release gel

Abstract

The invention relates to the technical field of animal husbandry, and discloses a temperature-sensitive sustained-release gel for treating cow mastitis and a preparation method thereof.The temperature-sensitive sustained-release gel is prepared from, by mass, 15%-24% of a triblock copolymer PLGA-PEG-PLGA solution, 3-7 parts of eucommia ulmoides powder, 2-5 parts of nisin, 1-3 parts of a stabilizer, 1-3 parts of a pH regulator and the balance water. One-time perfusion can be completed under the condition of a small administration volume, a stable sustained-release drug library is formed in the local part of the mammary gland, and the drug is continuously and slowly released through the diffusion effect of the gel layer, so that a lasting inhibition effect is generated on pathogenic bacteria on the surface and in the mammary gland tissue. Due to the fact that the medicine mainly plays a role in the local part of the breast, the high local medicine concentration can be maintained in the breast tissue, diffusion of the medicine to the whole body tissue is reduced, and therefore the risk that the medicine has adverse effects on non-lesion organs or tissues is reduced.

Description

Technical Field

[0001] This invention relates to the field of animal husbandry technology, and in particular to a thermosensitive sustained-release gel for treating mastitis in dairy cows and its preparation method. Background Technology

[0002] Bovine mastitis is one of the most common infectious diseases in dairy farming worldwide, and its causative factors are complex. The main pathogens causing mastitis in dairy cows include Escherichia coli, Staphylococcus aureus, Staphylococcus, Streptococcus, and Streptococcus agalactiae. In addition, it can also be caused by other microorganisms such as yeasts, viruses, and mycoplasma, with bacterial infections being the most common. Mastitis not only affects the health of dairy cows but also directly damages mammary gland tissue, thereby affecting milk yield and milk quality.

[0003] In large-scale dairy farming, mastitis has become one of the major diseases restricting production efficiency. To control the occurrence and spread of mastitis, farms typically need to invest significant human, material, and financial resources. Currently, the main treatment for mastitis in dairy cows is antibiotics. Antibiotics have long been widely used clinically for the treatment and prevention of mastitis; however, due to long-term, high-dose, and repeated use, drug-resistant strains are constantly emerging, leading to a gradual decline in treatment effectiveness. Simultaneously, antibiotic use can cause drug residue problems, which enter the food chain through cow milk, increasing potential risks to human health. Furthermore, inadequately processed milk can become a route for the transmission of zoonotic pathogens. Therefore, relying solely on antibiotics to treat mastitis in dairy cows is no longer sufficient to meet the requirements of modern animal husbandry for safety and sustainable development.

[0004] With increasing attention being paid to food quality and safety issues, researching and developing new treatment methods that are safe, pollution-free, and residue-free to replace traditional antibiotics and chemical drugs for the prevention and treatment of mastitis in dairy cows has become an urgent technical problem to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a thermosensitive sustained-release gel for treating bovine mastitis and its preparation method.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A thermosensitive sustained-release gel for treating mastitis in dairy cows, the thermosensitive sustained-release gel comprising the following components: a 15%-24% by mass solution of triblock copolymer PLGA-PEG-PLGA, 3-7 parts of Eucommia ulmoides powder, and 2-5 parts of nisin.

[0008] Preferably, the thermosensitive sustained-release gel comprises the following components: 18% by mass of a triblock copolymer PLGA-PEG-PLGA solution, 5 parts of Eucommia ulmoides powder, and 4 parts of nisin.

[0009] The above-mentioned method for preparing a thermosensitive sustained-release gel for treating bovine mastitis includes the following steps:

[0010] The triblock copolymer PLGA-PEG-PLGA was diluted with physiological saline to prepare a copolymer solution;

[0011] A mixture was obtained by mixing physiological saline containing eucommia powder and nisin with the copolymer solution.

[0012] The mixture was placed at 4°C for 24 hours to obtain the thermosensitive sustained-release gel.

[0013] The application of the above-mentioned thermosensitive sustained-release gel in the preparation of a treatment for mastitis in dairy cows.

[0014] Preferably, the treatment for bovine mastitis is administered to the cow via intramammary infusion.

[0015] Preferably, the bovine mastitis includes mastitis caused by Escherichia coli, Staphylococcus aureus and Staphylococcus, Streptococcus and Streptococcus agalactiae.

[0016] The beneficial effects of this invention are as follows:

[0017] In this invention, a temperature-sensitive sustained-release gel system is constructed using a triblock copolymer PLGA-PEG-PLGA. This allows the gel to maintain fluidity at low temperatures and gel in situ at near-body temperature, enabling drug delivery via intramammary infusion. Compared to existing treatments that require larger doses or frequent administration to maintain efficacy, this invention allows for a single infusion with a smaller delivery volume, forming a stable sustained-release drug reservoir in the breast. The drug is released continuously and slowly through diffusion within the gel layer, resulting in a sustained inhibitory effect on pathogenic bacteria on and inside the breast tissue. Because the drug primarily acts locally in the breast, a high local drug concentration is maintained within the breast tissue, reducing drug diffusion to other tissues and thus lowering the risk of adverse effects on non-pathological organs or tissues.

[0018] Furthermore, after the thermosensitive sustained-release gel forms in breast tissue, it maintains good contact with the tissue, allowing the drug encapsulated within the gel to be slowly and persistently released in the local environment to exert its therapeutic effect. This overcomes the shortcomings of conventional drug delivery methods, such as short duration of action and easy washout by breast milk. Compared to traditional treatment methods that require multiple or large doses, this invention achieves continuous treatment with a single intramammary infusion, which helps reduce the frequency of administration and improve treatment stability.

[0019] This invention introduces Eucommia ulmoides powder and nisin into a thermosensitive sustained-release gel system. This system allows for the sustained local release of antibacterial, anti-inflammatory, and antioxidant active ingredients, enabling the drug to exert its effects in mammary tissue for an extended period. This effectively overcomes the problems of short duration of action, systemic diffusion, and the need for repeated administration associated with traditional drug delivery methods. This combination ensures therapeutic efficacy while exhibiting high safety and ease of use, making it suitable for widespread application in the prevention and treatment of mastitis in dairy cows. Experimental results show that, compared to the control group without a sustained-release system, the thermosensitive sustained-release gel of this invention can improve the cure rate of mastitis in dairy cows and maintain a low recurrence rate after treatment, thereby reducing breeding costs and demonstrating high economic benefits. Simultaneously, the milk yield of treated cows was significantly higher than that of the control group, indicating that this invention effectively controls mastitis while promoting the recovery and improvement of mammary gland function, reducing the adverse effects of the disease on production performance.

[0020] Furthermore, this invention enables effective control of the drug release cycle by adjusting the mass fraction of the triblock copolymer PLGA-PEG-PLGA. Experimental results show that different copolymer concentrations correspond to different cumulative release times, allowing the sustained-release cycle to be adjusted within the range of approximately 12 to 16 days, thereby meeting the needs of different disease stages and treatment intensities. This controllable release characteristic makes this invention suitable for both treatment scenarios requiring rapid onset of action and applications requiring extended dosing intervals and reduced reperfusion, demonstrating good flexibility and practical value. Attached Figure Description

[0021] Figure 1 The cumulative release curves of a thermosensitive sustained-release gel for treating bovine mastitis, as proposed in Examples 1-3 of this invention. Detailed Implementation

[0022] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the technical solution of this invention is described in detail below, but this should not be construed as limiting the scope of implementation of this invention. Unless otherwise specified, the methods used in this invention are conventional methods in this technical field. In this invention, materials, reagents, or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0023] In one embodiment, the present invention provides a thermosensitive sustained-release gel for treating mastitis in dairy cows, the thermosensitive sustained-release gel comprising the following components: a PLGA-PEG-PLGA solution with a mass fraction of 15%-24%, 3-7 parts of Eucommia ulmoides powder, and 2-5 parts of nisin.

[0024] As a preferred embodiment of the present invention, the thermosensitive sustained-release gel comprises the following components: a 15% by mass solution of triblock copolymer PLGA-PEG-PLGA, 5 parts of Eucommia ulmoides powder, and 4 parts of nisin.

[0025] As a preferred embodiment of the present invention, the thermosensitive sustained-release gel comprises the following components: 18% by mass of triblock copolymer PLGA-PEG-PLGA solution, 5 parts of Eucommia ulmoides powder, and 4 parts of nisin.

[0026] As a preferred embodiment of the present invention, the thermosensitive sustained-release gel comprises the following components: a 23% by mass solution of triblock copolymer PLGA-PEG-PLGA, 5 parts of Eucommia ulmoides powder, and 4 parts of nisin.

[0027] This invention introduces Eucommia ulmoides powder as one of the active ingredients in a thermosensitive sustained-release gel. Eucommia ulmoides powder is rich in iridoids and flavonoids, both of which possess antioxidant, anti-inflammatory, and antibacterial properties. Specifically, iridoids and flavonoids can scavenge H2O2 free radicals, hydroxyl radicals, and superoxide anion free radicals, and can increase the activity of superoxide dismutase (SOD) in damaged cells, thus achieving an antioxidant effect. Iridoids achieve an anti-inflammatory effect by inhibiting inflammatory pathways and reducing the level of inflammatory factors; they also have a strong inhibitory effect on the growth of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella, thus achieving an antibacterial effect. Flavonoids exert their anti-inflammatory effect through multiple mechanisms, including inhibiting inflammatory exudation, inhibiting leukocyte migration, reducing capillary permeability, inhibiting granulation tissue growth, and inhibiting the release of inflammatory mediators.

[0028] The thermosensitive sustained-release gel proposed in this invention also contains nisin as another active ingredient. Nisin can effectively inhibit the growth of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus lactis, which cause mastitis in dairy cows. The main mechanism is that after nisin adsorbs onto the cell membrane, it disrupts the integrity of the cell membrane, causing cell lysis, resulting in the leakage of large amounts of cell contents and leading to cell death. Furthermore, nisin has high activity against these pathogenic bacteria and also has a strong inhibitory effect on penicillin-resistant pathogens. In addition, nisin is a natural product, characterized by being non-toxic and pollution-free. Its degradation products can be absorbed and utilized by the body as amino acids, making it suitable for the prevention and treatment of animal diseases.

[0029] The triblock copolymer PLGA-PEG-PLGA used in this invention is synthesized using ring-opening polymerization with DL-lactide, glycolide, and polyethylene glycol as raw materials. It is a thermosensitive polymer material. Both the copolymer and its raw materials are biodegradable, and the degradation products are non-toxic and harmless to organisms. Furthermore, the catalyst used in the synthesis process, stannous isooctanoate, is an approved catalyst for human use. No organic solvents are used in the synthesis and purification processes, thus effectively ensuring the safety of the copolymer as an implantable or local drug delivery material. In addition, PLGA-PEG-PLGA is compatible with both hydrophilic and hydrophobic drugs, and the preparation process does not require additional polymerization or other covalent bond formation reactions, allowing peptide and protein drugs to be administered via injection or perfusion.

[0030] By constructing a sustained-release system using the temperature-sensitive triblock copolymer PLGA-PEG-PLGA, this invention enables the active ingredient to form a gel at near-body temperature after intramammary infusion. The drug is slowly released through diffusion within the gel layer, exerting a sustained effect on pathogenic bacteria on and inside the breast tissue. Since the drug primarily acts locally within the breast, a high local drug concentration can be maintained in the breast tissue, reducing drug diffusion to other tissues and thus lowering the risk of adverse effects on non-pathological organs or tissues. Simultaneously, the drug encapsulated in the gel maintains prolonged contact with the breast tissue, overcoming the shortcomings of traditional drug delivery methods, such as short duration of action, easy systemic diffusion, and the need for multiple administrations.

[0031] In another embodiment, the present invention provides a method for preparing a thermosensitive sustained-release gel for treating bovine mastitis, the method comprising the following steps:

[0032] The triblock copolymer PLGA-PEG-PLGA was diluted with physiological saline to prepare a copolymer solution;

[0033] A mixture was obtained by mixing physiological saline containing eucommia powder and nisin with the copolymer solution.

[0034] The mixture was placed at 4°C for 24 hours to obtain the thermosensitive sustained-release gel.

[0035] In another embodiment, the present invention also proposes the application of the above-described thermosensitive sustained-release gel in the preparation of a treatment for bovine mastitis.

[0036] In a preferred embodiment of the present invention, the preparation for treating bovine mastitis is administered to the bovine via intramammary infusion.

[0037] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0038] Example 1: The triblock copolymer PLGA-PEG-PLGA was diluted with physiological saline to prepare a 15% (w / w) triblock copolymer PLGA-PEG-PLGA solution; 3 ml of physiological saline containing 5 parts of Eucommia ulmoides powder and 4 parts of nisin was mixed with 4 ml of the triblock copolymer PLGA-PEG-PLGA solution to obtain a mixture; the mixture was placed at 4°C for 24 hours to obtain 7 ml of thermosensitive sustained-release gel.

[0039] The obtained 7 ml thermosensitive sustained-release gel was used for intramammary infusion in dairy cows: First, the affected udder area was cleaned with sterile gauze, then the teats were disinfected with 75% ethanol and iodine solution respectively. Subsequently, the teats were soaked in iodine solution for about 30 seconds, and 1-2 handfuls of milk were squeezed out. Finally, 7 ml of the obtained thermosensitive sustained-release gel was infused into the udder of the affected cow through the teats, and the udder area was massaged to ensure that the drug was evenly distributed in the mammary tissue.

[0040] Example 2: The triblock copolymer PLGA-PEG-PLGA was diluted with physiological saline to prepare a PLGA-PEG-PLGA solution with a mass fraction of 18%; 3 ml of physiological saline containing 5 parts of Eucommia ulmoides powder and 4 parts of nisin was mixed with 4 ml of the PLGA-PEG-PLGA solution to obtain a mixture; the mixture was placed at 4°C for 24 hours to obtain 7 ml of thermosensitive sustained-release gel.

[0041] The obtained 7 ml thermosensitive sustained-release gel was used for intramammary instillation in dairy cows: First, the affected udder area was cleaned with sterile gauze. Then, the teats were disinfected with 75% ethanol and iodine solution in sequence. Subsequently, the teats were soaked in iodine solution for about 30 seconds. One or two handfuls of milk were squeezed out. Finally, 7 ml of the obtained thermosensitive sustained-release gel was instilled into the udder of the affected cow through the teats, and the udder area was massaged to ensure that the drug was evenly distributed in the mammary tissue.

[0042] Example 3: The triblock copolymer PLGA-PEG-PLGA was diluted with physiological saline to prepare a 23% (w / w) triblock copolymer PLGA-PEG-PLGA solution; 3 ml of physiological saline containing 5 parts of Eucommia ulmoides powder and 4 parts of nisin was mixed with 4 ml of the triblock copolymer PLGA-PEG-PLGA solution to obtain a mixture; the mixture was placed at 4°C for 24 hours to obtain 7 ml of thermosensitive sustained-release gel.

[0043] The obtained 7 ml thermosensitive sustained-release gel was used for intramammary infusion in dairy cows: First, the affected udder area was cleaned with sterile gauze, then the teats were disinfected with 75% ethanol and iodine solution respectively. Subsequently, the teats were soaked in iodine solution for about 30 seconds, and 1-2 handfuls of milk were squeezed out. Finally, 7 ml of the obtained thermosensitive sustained-release gel was infused into the udder of the affected cow through the teats, and the udder area was massaged to ensure that the drug was evenly distributed in the mammary tissue.

[0044] The triblock copolymer PLGA-PEG-PLGA in Examples 1-3 was purchased from Sigma-Aldrich, catalog number 908843.

[0045] Experimental Example 1: 140 lactating dairy cows diagnosed with mastitis by the CMT test were used as experimental animals. These cows were randomly divided into 5 groups of 35 cows each: control group, experimental group 1, experimental group 2, experimental group 3, and experimental group 4. Specifically: control group: the same volume of physiological saline as the experimental group was infused into the udder; experimental group 1: 7 ml of physiological saline containing 5 parts Eucommia ulmoides powder and 4 parts nisin was infused into the udder; experimental group 2: 15% thermosensitive sustained-release gel prepared in Example 1 was infused into the udder; experimental group 3: 18% thermosensitive sustained-release gel prepared in Example 2 was infused into the udder; and group 4: 23% thermosensitive sustained-release gel prepared in Example 3 was infused into the udder.

[0046] Apart from the different treatment methods mentioned above, the feeding and management procedures for the affected cattle in each group were the same.

[0047] The treatment effect and milk yield of each group of cattle were observed 14 days after infusion. After the thermosensitive sustained-release gel was completely absorbed, the cattle were observed for another 7 days. The recurrence of the disease was recorded, and the daily milk yield of the cattle was also recorded. The results are shown in Table 1.

[0048] Table 1

[0049] Milk production grouping Number of treatments Number of cured cure rate Number of recurrences recurrence rate Milk production (L / average per head / day) control group 35 0 0 0 0 26.39 Experimental group 1 35 11 31.4% 4 36.4% 26.41 Experimental group 2 35 31 88.6% 1 3.2% 26.55 Experimental group 3 35 35 100% 0 0 27.03 Experimental group 4 35 33 94.3% 0 0 26.88

[0050] The experimental results show that: the cure rate of experimental group 1 was 31.4%, the recurrence rate was 36.4%, and the average milk production per head increased by 0.02 L compared with the control group; the cure rate of experimental group 2 (15% thermosensitive sustained-release gel) was 88.6%, the recurrence rate was 3.2%, and the average milk production per head increased by 0.16 L compared with the control group; the cure rate of experimental group 3 (18% thermosensitive sustained-release gel) was 100%, and no recurrence was observed after cure, and the average milk production per head increased by 0.64 L compared with the control group; the cure rate of experimental group 4 (23% thermosensitive sustained-release gel) was 94.3%, and no recurrence was observed after cure, and the average milk production per head increased by 0.49 L compared with the control group.

[0051] The above results indicate that the 18% thermosensitive sustained-release gel significantly improves the cure rate of bovine mastitis, maintains a low recurrence rate after treatment, and increases milk production. After the thermosensitive sustained-release gel was completely dissolved, milk samples were tested using both the CMT and BTB methods, and the results were negative in both cases.

[0052] And by Figure 1 As shown, the 15% sustained-release gel prepared in Example 1 reached the cumulative release endpoint in 12 days, the 18% sustained-release gel prepared in Example 2 reached the cumulative release endpoint in 14 days, and the 23% sustained-release gel prepared in Example 3 reached the cumulative release endpoint in 16 days.

[0053] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A thermosensitive sustained-release gel for treating mastitis in dairy cows, characterized in that, The thermosensitive sustained-release gel comprises the following components: 18% by mass of triblock copolymer PLGA-PEG-PLGA solution, 5 parts of Eucommia ulmoides powder, and 4 parts of nisin.

2. A method for preparing a thermosensitive sustained-release gel for treating bovine mastitis as described in claim 1, characterized in that, The method includes the following steps: The triblock copolymer PLGA-PEG-PLGA was diluted with physiological saline to prepare a copolymer solution; A mixture was obtained by mixing physiological saline containing eucommia powder and nisin with the copolymer solution. The mixture was allowed to stand to obtain the thermosensitive sustained-release gel.

3. The method for preparing the thermosensitive sustained-release gel according to claim 2, characterized in that, The mixture was placed at 4°C for 24 hours to obtain a thermosensitive sustained-release gel.

4. The use of the thermosensitive sustained-release gel as described in claim 1 in the preparation of a formulation for treating mastitis in dairy cows.

5. The application according to claim 4, characterized in that, The treatment for bovine mastitis is administered to the cows via intramammary infusion.

Images