A kind of Eucommia ulmoides flavonoids liposome and its preparation method and application
By using soy phospholipids and saponin carriers, targeted treatment of porcine blue ear disease is achieved, solving the shortcomings of existing vaccines and therapeutic drugs, and improving the therapeutic effect and safety.
Patent Information
- Application Number
- CN202311172092.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-12
- Publication Date
- 2025-04-11
- Estimated Expiration
- 2043-09-12
AI Technical Summary
Blue ear disease of pigs is difficult to control, the current vaccine protection rate is low, and the incidence of vaccine poisoning and post-immunization are common, resulting in lifelong poisoning of breeding pigs and affecting offspring. The tolerability of existing therapeutic drugs such as temicocin increases after long-term use and needs further improvement.
Soybean phospholipids and saponins are used as carriers, and flavonoids of Eucommia ulmoides are used as raw materials. Eucommia ulmoides are prepared by rotary evaporation and ultrasound, and are inhaled into the lungs through a nebulizer to achieve targeted treatment.
Eucommia ulmoides flavonoid liposomes can be directed in the lungs, improve the treatment effect, replace the antibiotic temicocin, which is safe and reliable, does not increase the chance of pig miscarriage, and effectively controls the pro-phage of blue ear virus to the lungs.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of veterinary drugs, and more specifically to eucommia flavonoids liposomes and a preparation method and application thereof. Background Art
[0002] Blue ear disease is an acute and severe infectious disease caused by porcine respiratory and reproductive syndrome virus (PRRSV). Since 2006, highly pathogenic blue ear disease has broken out. The blue ear disease virus has a strong tropism for macrophages, especially alveolar macrophages, and replicates in large quantities in them, destroying macrophages and causing immunosuppression. The imbalance of the body's natural protection mechanism leads to bacterial secondary infections, such as Haemophilus parasuis, Streptococcus, Salmonella, Actinobacillus pleuropneumoniae, Pasteurella and Mycoplasma pneumoniae.
[0003] The reason why blue ear disease is difficult to control is mainly because its virus is easy to mutate, the protection rate of existing vaccines is low, the phenomenon of mixed infection is serious, the vaccine virus reversion, and the phenomenon of post-immunization disease are common, which causes the breeding pigs to carry the virus for life, greatly affecting the offspring. The blue ear disease vaccine is mainly a live attenuated vaccine and an inactivated vaccine, but both vaccines have their advantages and disadvantages. Pigs immunized with live attenuated vaccines can produce complete clinical protection against attacks by homologous strong toxins, but can only produce partial protection against heterologous strong toxins; at the same time, immunization with live attenuated vaccines cannot prevent wild virus infection. Due to the diversity of prevalent strains, the use of live attenuated vaccines to immunize pigs may result in a low cross-protection rate against variant strains of blue ear disease. Inactivated vaccines usually achieve humoral immune protection through neutralizing antibodies. The blue ear disease virus is naturally unable to induce high levels of neutralizing antibodies. Therefore, blue ear prevention and control requires targeted elimination of the blue ear virus to be completely resolved. The main drug for the treatment of blue ear disease in pigs is tilmicosin, which is a new macrolide antibiotic for animals developed by Eli Lilly in the 1980s. It has significant effects on a variety of pathogens, especially respiratory diseases of livestock and poultry. Tilmicosin is often used to reduce the activity of blue ear disease in pig farms because it can target and accumulate in alveolar macrophages, which are the main host cells of blue ear virus. However, its tolerance gradually increases with long-term use. Therefore, further improvement and development are needed. Summary of the invention
[0004] In view of the various deficiencies of the prior art and in order to solve the above problems, a eucommia flavonoid liposome and a preparation method and application thereof are proposed, and the following technical solutions are provided:
[0005] The invention discloses a method for preparing eucommia flavonoid liposomes. Soybean lecithin and saponin are used as carriers and eucommia flavonoids are used as raw materials. The carriers and the raw materials are dissolved in an organic solvent and the eucommia flavonoid liposomes are obtained by rotary evaporation and ultrasound.
[0006] Furthermore, based on the mass fraction, the dosage ratio of soybean lecithin to saponin is 30-35:15-18.5.
[0007] Furthermore, based on the mass fraction, the usage ratio of saponin and eucommia ulmoides flavonoids is 15-18.5:10.
[0008] Furthermore, after the ultrasound is finished, a lyophilization protective agent is added to perform freeze drying to obtain the eucommia flavonoids liposomes, wherein the lyophilization protective agent includes at least one of amino acids, mannitol and glucose.
[0009] Furthermore, the lyoprotectant includes amino acids, mannitol and glucose, and the mass ratio of amino acids, mannitol and glucose is 1:1:2 or 2:1:1.
[0010] Furthermore, the freeze-drying process includes: pre-freezing in a freezer at -40 to -30°C for 3-5 hours, and freeze-drying in a vacuum freeze dryer for 36-42 hours.
[0011] Furthermore, the organic solvent is dichloromethane, ethanol or ether.
[0012] In addition, the present invention also provides a eucommia flavonoids liposome, which is obtained by the preparation method of the eucommia flavonoids liposome.
[0013] The invention provides an application of eucommia flavonoids liposomes. The eucommia flavonoids liposomes are used for preparing a medicine for treating blue ear disease in pigs.
[0014] The eucommia flavonoids liposomes are used to prepare an aerosol. The pigs inhale the aerosol twice a day, and the dosage of the aerosol inhaled is 0.1-0.15 mg per kilogram of body weight.
[0015] Due to the adoption of the above technical solution, the beneficial technical effects of the present invention are:
[0016] 1. The present invention uses eucommia flavonoids as raw materials and soybean lecithin and saponin as carriers. On the one hand, soybean lecithin and saponin act together as carriers of eucommia flavonoids, so that eucommia flavonoids are directedly retained in the lungs; on the other hand, eucommia flavonoids and anemarrhena saponins produce a drug synergistic effect, thereby improving the therapeutic effect of eucommia flavonoids liposomes.
[0017] 2. The present invention creatively applies eucommia flavonoid liposomes to the preparation of drugs for treating blue ear disease in pigs. The prepared drugs for treating blue ear disease in pigs can replace tilmicosin, but are safer and more reliable than the antibiotic tilmicosin and will not increase the chance of pig abortion. DETAILED DESCRIPTION
[0018] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is clearly and completely described below in conjunction with the embodiments of the present invention. Based on the embodiments in this application, other similar embodiments obtained by ordinary technicians in this field without making any creative work should all fall within the scope of protection of this application.
[0019] A method for preparing eucommia flavonoids liposomes comprises the following steps: using soybean lecithin and saponin as carriers, eucommia flavonoids as raw materials, dissolving the carrier and the raw materials in a solvent, and obtaining eucommia flavonoids liposomes by rotary evaporation and ultrasound. The present invention uses eucommia flavonoids as raw materials, and uses soybean lecithin and saponin as carriers. On the one hand, soybean lecithin and saponin act together as carriers of eucommia flavonoids, so that eucommia flavonoids are directionally retained in the lungs; on the other hand, eucommia flavonoids and anemarrhena saponins produce drug synergistic effects, thereby improving the therapeutic effect of eucommia flavonoids liposomes.
[0020] Specifically, the saponin can be selected as Anemarrhena saponin. As a polysaccharide, Anemarrhena saponin has strong anti-inflammatory, antibacterial and antiviral effects, and can synergize with Eucommia ulmoides flavonoids.
[0021] Example 1
[0022] Take 30 mg of soybean lecithin, 16.5 mg of timosaponin and 10 mg of eucommia ulmoides flavonoids, add 50 ml of dichloromethane to mix, use a rotary evaporator to remove dichloromethane in a 35°C constant temperature water bath, and form a uniform lipid film on the wall of the flask. Add 20 ml of 0.9% mannitol aqueous solution as a hydration medium to the flask, rotate to make the lipid film on the wall of the flask fall off, use a probe ultrasonic instrument to sonicate for 3 minutes to obtain a suspension, quantitatively transfer 2 ml to a 10 ml syringe bottle, add 8 ml of an aqueous solution containing amino acids, mannitol and glucose (mass ratio of 1:1:2), shake gently to dissolve and mix evenly, the aqueous solution contains 10% by mass of amino acids and mannitol and glucose composition, pre-freeze in a -40°C ultra-low temperature freezer for 3 hours, freeze-dry in a vacuum freeze dryer, reduce pressure and vacuum for 36 hours, and immediately press the cover and seal with wax after taking out.
[0023] Example 2
[0024] Take 35 mg of soybean lecithin, 18.5 mg of timosaponin and 10 mg of eucommia flavonoids, add 50 ml of ethanol, use a rotary evaporator to remove ethanol in a constant temperature water bath at 35°C, and form a uniform lipid film on the wall of the flask. Add 20 ml of 0.9% mannitol aqueous solution as a hydration medium to the flask, rotate to make the lipid film on the wall of the flask fall off, use a probe ultrasonic instrument to sonicate for 4 minutes to obtain a suspension, quantitatively transfer 2 ml to a 10 ml syringe bottle, add 8 ml of an aqueous solution containing amino acids, mannitol and glucose (2:1:1) and shake gently to dissolve and mix evenly. The aqueous solution contains a 10% mass fraction of amino acids, mannitol and glucose composition. After pre-freezing in a -35°C ultra-low temperature freezer for 4 hours, freeze-dry in a vacuum freeze dryer, reduce pressure and vacuumize for 36 hours, and immediately press the cover and seal with wax after taking out.
[0025] Example 3
[0026] Take 33 mg of soybean lecithin, 15 mg of timosaponin and 10 mg of eucommia ulmoides flavonoids, add 50 ml of ether, use a rotary evaporator to remove the ether in a constant temperature water bath at 35°C, and form a uniform lipid film on the wall of the flask. Add 20 ml of 0.9% mannitol aqueous solution as a hydration medium to the flask, rotate to make the lipid film on the wall of the flask fall off, use a probe ultrasonic instrument to sonicate for 3 minutes to obtain a suspension, quantitatively transfer 2 ml to a 10 ml syringe bottle, add 8 ml of an aqueous solution containing amino acids, mannitol and glucose (1:1:2) and shake gently to dissolve and mix evenly. The aqueous solution contains a 10% mass fraction of amino acids, mannitol and glucose composition. After pre-freezing in a -30°C ultra-low temperature freezer for 5 hours, freeze-dry in a vacuum freeze dryer, reduce pressure and vacuumize for 42 hours, and immediately cover and seal with wax after taking out.
[0027] Comparative Example 1
[0028] The eucommia flavonoids aqueous solution is prepared by dissolving eucommia flavonoids in water, and the concentration of the eucommia flavonoids aqueous solution is 25%.
[0029] Comparative Example 2
[0030] Compared with Example 3, the mass usage ratio of soybean lecithin, anemarrhena saponin and eucommia ulmoides flavonoids is 10:10:10, and the other steps are the same as those in Example 1.
[0031] Application Example 1
[0032] Evaluation of targeting of Eucommia ulmoides flavonoids liposomes to pig lungs
[0033] 1.1 Dosage regimen
[0034] The clinical atomization method was adopted, and the dosage (in terms of Eucommia flavonoids) was: 6.636 mg per time, i.e. 0.1106 mg / kg, based on 60 kg body weight. The Eucommia flavonoids liposome freeze-dried product obtained in Example 1 was added with an appropriate amount of distilled water to reconstitute the suspension and the Eucommia flavonoids aqueous solution obtained in Comparative Example 1 (same liposome drug content, 0.45 mg / ml) were prepared into atomizers, and ultrasonic atomization inhalation was performed on healthy pigs, and the atomization inhalation time was 15 minutes.
[0035] 1.2 Sample collection
[0036] 20 weaned piglets, half male and half female, were randomly divided into 2 groups, 10 in each group. One group was subjected to aerosol inhalation of the aqueous solution of Eucommia flavonoids obtained in Comparative Example 1, and the other group was subjected to aerosol inhalation of the aerosol reconstituted with the lyophilized Eucommia flavonoids liposome obtained in Example 1. Plasma and lung tissue of each test group were taken at 0, 10, 20, 30, 45, 60, 90, 120, and 150 minutes after administration to determine the concentration of Eucommia flavonoids (n=8). The plasma collection method was to remove the eyeball to take blood, put it in a heparin anticoagulant tube, centrifuge the plasma after 30 minutes, and store it at -40°C for later use. The lung tissue of the pig was taken, weighed, and a 50% lung tissue homogenate was prepared at low temperature. The supernatant was centrifuged and stored in a -40°C refrigerator for later use.
[0037] 1.3 Sample determination
[0038] The supernatant of the spare pig plasma and spare pig lung tissue homogenate was taken to determine the concentration of Eucommia flavonoids.
[0039] 1.4 Data Analysis
[0040] Based on the measured concentration-time data of Eucommia ulmoides flavonoids, the main pharmacokinetic parameters were calculated and statistically analyzed.
[0041] 1.5 Measurement results
[0042] The drug-time curves were drawn based on the average drug concentrations of eucommia flavonoids aqueous solution and eucommia flavonoids liposome aerosol in pig plasma and lung tissue, and the relative uptake rate r was calculated. e and peak concentration ratio C e .
[0043] 1.5.1 Relative uptake rate r e
[0044] Relative uptake rate e =(AUC i ) m / (AUC i ) s
[0045] Where AUC iIt is the area under the drug-time curve of an organ or tissue calculated from the concentration-time curve. The subscripts m and s represent the liposome atomizer of Eucommia ulmoides flavonoids and the aqueous solution of Eucommia ulmoides flavonoids, respectively. e If it is greater than 1, it means that the liposome is targeted to the organ or tissue. e The larger the value, the better the targeting; a value equal to or less than 1 indicates no targeting.
[0046] Table 1 Area under the curve and relative uptake rate of Eucommia flavonoids liposome aerosol and Eucommia flavonoids aqueous solution
[0047]
[0048] As shown in Table 1 above, the r e The values were all greater than 1, indicating that the Eucommia flavonoids liposome aerosol has a certain targeting effect on lung tissue and plasma; e The value is (2.8710), indicating that liposomes made of Eucommia ulmoides flavonoids have obvious lung targeting and good targeting effect.
[0049] 1.5.2 Peak concentration ratio C e
[0050] C e =(C p ) m / (C p ) s
[0051] In the formula, C p It is the highest concentration in plasma or tissue. The subscripts m and s represent eucommia flavonoids liposomes and eucommia flavonoids aqueous solution, respectively.
[0052] C in each tissue or organ e The value indicates that the greater the effect of liposomes on changing drug distribution, the more obvious the effect of changing drug distribution.
[0053] Table 2 Peak concentration ratio of Eucommia ulmoides flavonoids liposome atomizer and Eucommia ulmoides flavonoids aqueous solution
[0054]
[0055] As can be seen from Table 2 above, the Cp value of lung tissue is the largest (2.1553), indicating that making Eucommia flavonoids into liposomes can significantly change the distribution of Eucommia flavonoids in the lungs.
[0056] Application Example 2
[0057] The eucommia flavonoids liposomes obtained in Examples 1-3 and the products obtained in Comparative Examples 1-2 were prepared into aerosols for research on the treatment of blue ear disease in gilts.
[0058] 2.1 Materials and methods
[0059] The reserve sows were selected from 140 blue ear positive pigs in a farm; Tilmicosin was purchased from a famous American brand company; the blue ear antigen test was purchased from a domestic brand biological company, and the antibody test used the porcine blue ear body test kit produced by IDEXX, an American company.
[0060] 2.2 Experimental design
[0061] 140 gilts were randomly divided into control group 1 (no medication), control group 2 (tilmicosin treatment), control group 3 (eucommia flavonoids aqueous solution obtained in comparative example 1), control group 4 (product obtained in comparative example 2), experimental group 1 (eucommia flavonoids liposomes obtained in example 1), experimental group 2 (eucommia flavonoids liposomes obtained in example 2), and experimental group 3 (eucommia flavonoids liposomes obtained in example 3), a total of 7 groups, each with 20 gilts. The control group 1 was not treated and fed a basic diet. The control group 2 was fed a basic diet with 20 gilts. The pigs were treated with 300 mg / kg tilmicosin, the control group 3 was fed with drinking water with an aqueous solution of eucommia flavonoids for treatment (1 kg / 1000 kg water), the control group 4, experimental group 1, experimental group 2, and experimental group 3 were prepared into nebulizers, and the pigs were treated with the eucommia flavonoids liposome nebulizers of comparative example 2, embodiment 1, embodiment 2, and embodiment 3 by nebulization, respectively. The dosage was 0.1106 mg per kilogram of body weight each time, twice a day, and the treatment started 15 days before delivery. The experimental period was 21 days, and blood was collected 1 day before administration and the last 1 day, and the antigen and antibody were detected by serum.
[0062] 2.3 Feeding and management
[0063] The experimental sows were fed and watered freely. All experimental sows were fed by the same breeder in the same gestation house at 8:00 a.m. and 3:00 p.m. every day, with a feed of 3.5 kg / d. The room was kept well ventilated, with a suitable temperature and good environmental hygiene.
[0064] Disinfect regularly.
[0065] 2.4 Antigen and antibody detection
[0066] The separated serum is tested according to the operating steps in the instructions of the antigen detection kit and the antibody detection kit.
[0067] 2.5 Data processing
[0068] The recorded experimental data were statistically analyzed using SPSS19.0 software. The results were expressed as mean values, and P<0.05 indicated that the data within the group were statistically significant.
[0069] 2.6 Results
[0070] Table 3 Test results of the effectiveness and safety of Eucommia flavonoids liposome aerosol in the treatment of blue ear in gilts
[0071]
[0072]
[0073] As can be seen from Table 3 above, the pre-drug antibody level, pre-drug antibody dispersion, and pre-drug antigen positivity of the control group and the experimental group are at the same level. From the comprehensive perspective of the three indicators of post-drug antibody dispersion, antigen positivity, and antibody level, the control group 2 and the three experimental groups are significantly different from the control group 1 and the control group 3 (P<0.05). The above test results show that oral treatment with Eucommia flavonoids can have a certain therapeutic effect, but compared with the tilmicosin group and the Eucommia flavonoids liposome atomizer, the effect is quite different. The Eucommia flavonoids liposome atomizer can have a good therapeutic effect on blue ear disease with tilmicosin; and from the statistical results of postpartum abortion rate, the long-term use of tilmicosin has the risk of increasing the chance of abortion, and there are no aborted pigs in all experimental groups. Therefore, compared with the tilmicosin treatment group, the Eucommia flavonoids liposome is reliable in terms of effectiveness and safety, and can be widely promoted clinically. In addition, from the comparative data of the control group 4 and the implementation groups 1-3, it can be seen that only when the liposomes prepared with soybean lecithin, anemarrhena saponin and eucommia ulmoides flavonoids within a certain mass ratio range can they achieve better clinical targeted therapeutic effects.
[0074] In summary, this application creatively applies Eucommia flavonoid liposomes to the preparation of drugs for treating blue ear disease in pigs, providing a new treatment approach for blue ear disease in pigs. Compared with the antibiotic tilmicosin, it is safer and more reliable and will not increase the chance of pig miscarriage. Experiments have proven that drugs in Eucommia flavonoid liposomes have a targeted therapeutic effect on the lungs of pigs, trapping Eucommia flavonoids in the lungs and slowly releasing them to increase the drug concentration in the target organ, thereby effectively controlling the phagocytosis of blue ear virus on the lungs, and filling the gap in the targeted technology of blue ear traditional Chinese medicine.
[0075] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.
Claims
1. A method for preparing eucommia flavonoids liposomes, characterized in that: Using soybean lecithin and anemarrhena saponin as carriers and Eucommia ulmoides flavonoids as raw materials, the carriers and raw materials were dissolved in an organic solvent and subjected to rotary evaporation and ultrasound to obtain Eucommia ulmoides flavonoids liposomes; According to the weight percentage, the dosage ratio of soybean lecithin and anemarrhena saponin is 30-35:15-18.5; According to the weight percentage, the dosage ratio of timosaponin and eucommia ulmoides flavonoids is 15-18.5:10; The organic solvent is dichloromethane, ethanol or ether.
2. The method for preparing eucommia flavonoids liposome according to claim 1, characterized in that: After the ultrasound is finished, a freeze-drying protective agent is added to perform freeze drying to obtain eucommia flavonoid liposomes, wherein the freeze-drying protective agent includes at least one of amino acids, mannitol and glucose.
3. The method for preparing eucommia flavonoids liposome according to claim 2, characterized in that: The freeze-drying protective agent includes amino acids, mannitol and glucose, and the mass ratio of amino acids, mannitol and glucose is 1:1:2 or 2:1:
1.
4. The method for preparing eucommia flavonoids liposome according to claim 2, characterized in that: The freeze-drying process includes: pre-freezing in a freezer at -40 to -30°C for 3-5 hours, and freeze-drying in a vacuum freeze dryer for 36-42 hours.
5. A liposome containing eucommia flavonoids, characterized in that: The liposome is obtained by the preparation method of the eucommia flavonoids liposome described in any one of claims 1 to 4.
6. A use of the Eucommia ulmoides flavonoids liposome according to claim 5, characterized in that: The eucommia flavonoids liposome is used for preparing medicine for treating blue ear disease in pigs.
7. The use of the Eucommia flavonoids liposome according to claim 6, characterized in that: The eucommia flavonoids liposomes are used to prepare eucommia flavonoids liposome aerosols. Pigs aerosolize and inhale the eucommia flavonoids liposome aerosols twice a day, and the inhaled dosage of the aerosols is 0.1-0.15 mg per kilogram of body weight.