Application of schizonepeta essential oil in preparation of salmonella pigeonae resisting medicine
By preparing schizonepeta essential oil emulsion, the prevention and treatment problems of pigeon salmonella disease were solved, natural and environmentally friendly antibacterial effects were achieved, and drug resistance challenges were overcome.
Patent Information
- Application Number
- CN202510710344.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-29
- Publication Date
- 2025-07-11
AI Technical Summary
The prior art is difficult to effectively prevent and control pigeon salmonella disease, and the abuse of antibiotics leads to serious drug resistance problems, which requires a natural and environmentally friendly prevention and control method.
Salmonella anti-pigeon drugs are prepared using schizonepeta essential oil, and emulsification forms are made into emulsion dosage forms to inhibit the growth of Salmonella puppeta.
Schizonepeta essential oil showed significant inhibitory effects on Salmonella pigeon, solving the problems of drug residues and drug resistance, and achieving efficient antibacterial effects.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of natural products, and particularly to the application of Nepeta cataria essential oil in the preparation of drugs against Salmonella columbae. Background Art
[0002] Salmonella columbae disease, commonly known as pigeon paratyphoid, is a bacterial disease caused by Salmonella infection. It is the most common and highest-incidence bacterial disease in pigeon farms. Once it breaks out, it is extremely difficult to purify, causing huge economic losses to the pigeon breeding industry. Sick squabs are listless, with loose feathers, loss of appetite, and white watery feces, and the mortality rate is relatively high; young pigeons often have swollen joints, lameness or stand on one leg, accompanied by diarrhea, indigestion, difficulty breathing and other symptoms, and some sick pigeons show neurological symptoms such as head and neck distortion; after breeding pigeons are infected, salpingitis often occurs, the egg production decreases, and the hatching rate is significantly reduced. The liver of dead pigeons is enlarged, the texture becomes brittle, and yellowish-white necrotic foci are scattered on the surface. In severe cases, it shows bronze discoloration; the intestinal mucosa is congested and bleeding, with yellowish-green watery contents, and fibrinous enteritis can be seen in some cases; the lungs are congested or have focal necrosis, the surface of the heart is covered with fibrinous exudate (pericarditis), and the spleen is enlarged, etc.
[0003] At present, according to the serotypes of Salmonella and the differences in its somatic antigen and flagellar antigen, more than 3,000 Salmonella have been identified. Among them, the main Salmonella infecting pigeons is Salmonella typhimurium, which has a wide range of pathogenicity, can spread horizontally and vertically, and infect humans through infected animals or animal products, seriously threatening public health safety. The O antigen of Salmonella is its somatic antigen and is a component of the outer membrane lipopolysaccharide of Salmonella. Generally, IgM antibodies are produced after the O antigen stimulates the body. The flagellar antigen of Salmonella is also called the H antigen, which is unstable under high-temperature conditions. The H antigen stimulates the host body to mainly produce IgG antibodies.
[0004] With the extensive use, and even abuse and misuse, of antibiotics and other antibacterial drugs, the problem of Salmonella drug resistance has become increasingly severe. More and more rare drug-resistant genes have been detected, seriously hindering the effective prevention and control of this disease in the pigeon breeding industry. In addition, there are certain differences in the drug resistance shown by strains of different serotypes. Research shows that whether it is pigeon farms or pigeon meat sales places, the carrying rate of Salmonella is relatively high, the drug resistance phenomenon is becoming increasingly serious, and the multi-drug resistance phenomenon is also widespread, bringing new challenges to the prevention and treatment of Salmonella disease. Since January 1, 2020, China has officially prohibited the addition of all growth-promoting drug feed additives except traditional Chinese medicines to veterinary drugs; since July 1 of the same year, feed production enterprises have been prohibited from producing commercial feeds containing growth-promoting drug feed additives (except traditional Chinese medicines). Therefore, it is imperative to adopt green and healthy prevention and treatment means. Chinese herbal medicines are derived from nature, and their toxic and side effects are much smaller than those of western medicines. At the same time, they can effectively solve the problems of drug residues and drug resistance.
[0005] Schizonepeta tenuifolia essential oil is a natural substance extracted from the medicinal plant Schizonepeta tenuifolia, and its main components include menthone, limonene, β-caryophyllene, p-cymene, isoascaridole, α-terpinene, etc. Schizonepeta tenuifolia essential oil has certain safety and various biological activities. Schizonepeta tenuifolia essential oil has significant anti-inflammatory effects and can inhibit the inflammatory response in acute and chronic inflammation models; Schizonepeta tenuifolia essential oil can promote the transdermal absorption of pesticides through the pest cuticle, thereby improving the efficacy of pesticides; in addition, Schizonepeta tenuifolia essential oil also has functions such as antioxidant, anti-tumor, and promoting wound healing, and is used for daily mosquito repellent, agricultural disease control, and medical fields. Summary of the Invention
[0006] The first technical problem to be solved by the present invention is to provide an application of Schizonepeta tenuifolia essential oil in the preparation of anti-Pigeon Salmonella drugs, so as to achieve the goal of natural environmental protection while efficiently anti-Pigeon Salmonella, and effectively solve the problems of drug residues and drug resistance.
[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:
[0008] Application of Schizonepeta tenuifolia essential oil in the preparation of anti-Pigeon Salmonella drugs.
[0009] Among them, the Schizonepeta tenuifolia essential oil is prepared by the following steps:
[0010] (1) After the whole Schizonepeta tenuifolia herb is crushed, it is soaked in water and then distilled, so that the volatile oil in Schizonepeta tenuifolia enters the condenser together with water vapor for condensation;
[0011] (2) After the condensed oil and water are separated, the oil layer is taken as Schizonepeta tenuifolia essential oil.
[0012] In step (1), for the water soaking, the amount of added water is 8 to 12 times the weight of the whole Schizonepeta tenuifolia herb, and the soaking time is 1 to 2 h.
[0013] In step (1), for the distillation, the temperature is 110 to 125 °C, the distillation time is 6 to 8 h, and the steam pressure is 0.2 MPa.
[0014] In step (2), the separation is static separation.
[0015] Among them, the dosage form of the drug is an emulsion.
[0016] Among them, the drug is prepared by the following method: the Schizonepeta tenuifolia essential oil is mixed with Tween-20 solvent, and then normal saline is added and stirred until emulsified.
[0017] Preferably, the volume ratio of the Schizonepeta tenuifolia essential oil to the Tween-20 solvent is 10:1.
[0018] Preferably, the stirring is carried out by a high-speed homogenizing emulsifier.
[0019] More preferably, the high speed is 20,000 rpm.
[0020] The temperature of the steam distillation is 110 - 125°C, the distillation time is 6 - 8 h, and the steam pressure is 0.2 MPa.
[0021] Among them, the dosage form of the drug is an emulsion.
[0022] Among them, the drug is prepared by the following method: the essential oil of Schizonepeta tenuifolia is mixed with Tween - 20, and then physiological saline is added and stirred until emulsified. Preferably, it is stirred until emulsified with a high - speed homogenizing emulsifier.
[0023] Among them, the volume ratio of the essential oil of Schizonepeta tenuifolia to the Tween - 20 solvent is 10:1.
[0024] Beneficial effects: Compared with the prior art, the essential oil of Schizonepeta tenuifolia provided by the present invention shows an obvious inhibitory effect on Salmonella gallinarum in pigeons. The essential oil of Schizonepeta tenuifolia is of natural origin and has a simple preparation method, and has important application value in the preparation of anti - Salmonella gallinarum in pigeons. Brief Description of the Drawings
[0025] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above - mentioned and / or other advantages of the present invention will become clearer.
[0026] Figure 1 It is the influence of the essential oil of Schizonepeta tenuifolia on the growth curve of Salmonella gallinarum in pigeons;
[0027] Figure 2 It is the result of the influence of the essential oil of Schizonepeta tenuifolia on the cell membrane of Salmonella gallinarum in pigeons;
[0028] Figure 3 It is the result of the influence of the essential oil of Schizonepeta tenuifolia on the cell wall of Salmonella gallinarum in pigeons. Specific Embodiments
[0029] Example 1:
[0030] The preparation steps of the essential oil of Schizonepeta tenuifolia are as follows:
[0031] (1) After the whole Schizonepeta tenuifolia herb is crushed, 10 times the amount of water is added and soaked for 2 h, then heated to boiling to generate water vapor. The distillation temperature is 121°C, the distillation time is 8 h, and the steam pressure is 0.2 MPa. The water vapor passes through the raw material layer, and the volatile oil in Schizonepeta tenuifolia is distilled out to obtain an oil - gas mixture;
[0032] (2) The above - mentioned oil - gas mixture enters the condenser and is cooled into a liquid to obtain an oil - water mixture. After standing and separating, the essential oil of Schizonepeta tenuifolia can be separated.
[0033] Example 2: Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Schizonepeta tenuifolia Essential Oil against Salmonella in Pigeons
[0034] 1. Test Strains
[0035] Salmonella in pigeons X8.6 used in this invention was preserved and provided by the Animal Biotechnology Laboratory of Jinling Institute of Technology. Through serological identification, PCR amplification and sequencing analysis, etc., the results showed that Salmonella in pigeons X8.6 was Salmonella typhimurium.
[0036] 2. Preparation of Salmonella in Pigeons Bacterial Solution
[0037] Inoculate Salmonella in pigeons X8.6 into autoclaved LB broth and culture overnight at 37°C. Adjust its OD 600nm absorbance value to 0.5 with fresh LB broth medium, and then continue to dilute it at a ratio of 1:100. The diluted bacterial solution is reserved for use.
[0038] 3. Preparation of Drug Solution
[0039] Take 10 mL of Schizonepeta tenuifolia essential oil and mix it with 1 mL of Tween-20 (v:v = 10:1), then add 10 mL of PBS and stir and emulsify it with a high-speed homogenizing emulsifier (at a rate of 20000 rpm) to prepare a Schizonepeta tenuifolia essential oil emulsion of 500 μL / mL (where the content of Tween-20 accounts for 5% v / v of the total original solution), and store it in a 4°C refrigerator for later use.
[0040] 4. Determination of Minimum Inhibitory Concentration (MIC) of Schizonepeta tenuifolia Essential Oil
[0041] It is carried out by the micro-dilution method in broth. Add 100 μL of LB broth medium to the wells in columns 2 - 12 of a 96-well cell culture plate respectively. Then add 200 μL of Schizonepeta tenuifolia essential oil emulsion (concentration of 500 μL / mL) to the wells in column 1. Use a pipette to suck 100 μL of Schizonepeta tenuifolia essential oil emulsion from the wells in column 1 and add it to the wells in column 2, and mix well by repeated pipetting; take 100 μL of the mixed solution from the wells in column 2 and add it to the wells in column 3, and mix well by repeated pipetting; sequentially dilute from the wells in column 3 to the wells in column 10; take 100 μL of the mixed solution from the wells in column 10 and add it to the wells in column 11, and mix well by repeated pipetting, then suck 100 μL of the mixed solution and discard it. Then add 100 μL of the diluted Salmonella in pigeons bacterial solution respectively, mix well, culture at 37°C for 18 - 20 h, and then add 10 μL of 2,3,5-triphenyltetrazolium chloride (TTC, purchased from Beijing Solarbio Science & Technology Co., Ltd.), mix well, and culture at 37°C for 1 h. The wells in column 12 do not contain Schizonepeta tenuifolia essential oil as a control. The determination of the minimum inhibitory concentration (MIC) of Schizonepeta tenuifolia essential oil is based on the color change of TTC. Turning red indicates the growth of bacteria, and no color change indicates no bacterial growth. The concentration of Schizonepeta tenuifolia essential oil in the wells without color change is the MIC value of Schizonepeta tenuifolia essential oil against Salmonella in pigeons X8.6.
[0042] 5. Determination of the minimum bactericidal concentration (MBC) of Schizonepeta tenuifolia essential oil
[0043] According to the determination results of MIC, using Salmonella enterica serovar Gallinarum X8.6 without Schizonepeta tenuifolia essential oil as the control, 100 μL of the liquid in the wells where TTC did not change color and the wells with the lowest concentration of Schizonepeta tenuifolia essential oil and the first two columns were taken and spread on LB agar plates, and cultured upside down at 37 °C for 24 h. The MBC value was the concentration of Schizonepeta tenuifolia essential oil corresponding to a 99% reduction in bacterial growth on the LB agar plate.
[0044] 6. Results
[0045] The MIC and MBC of Schizonepeta tenuifolia essential oil against Salmonella enterica serovar Gallinarum X8.6 are shown in Table 1. It can be seen from Table 1 that both the MIC and MBC of Salmonella enterica serovar Gallinarum X8.6 against Schizonepeta tenuifolia essential oil are 1.953 μL / mL.
[0046] Table 1 Results of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Schizonepeta tenuifolia essential oil
[0047]
[0048] Example 3: Effect of Schizonepeta tenuifolia essential oil on the growth curve of Salmonella enterica serovar Gallinarum
[0049] Take the diluted Salmonella enterica serovar Gallinarum bacterial solution prepared in Example 2 in a shaking tube, and add Schizonepeta tenuifolia essential oil to the shaking tubes respectively to make its concentrations 0, 0.488, 0.977, 1.953 μL / mL. Then culture it in a shaker at 37 °C with an oscillation speed of 150 r / min. Samples are taken at 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8 h of culture respectively, and the absorbance (OD) of the bacteria is measured at 600 nm. Using the Salmonella enterica serovar Gallinarum X8.6 bacterial solution without Schizonepeta tenuifolia essential oil as the control, the experiment is repeated three times, and the results are averaged to draw the growth curve of Salmonella enterica serovar Gallinarum X8.6.
[0050] The results are as Figure 1 shown. The OD value of the control group gradually increased from the beginning of the culture and reached 0.699 at 8 h, indicating that the bacterial solution concentration increased significantly. Observing the treatment group containing 0.488 μL / mL Schizonepeta tenuifolia essential oil, it was found that the OD value from 1 to 7 h was slightly lower than that of the control group, indicating that the treatment with low concentration of Schizonepeta tenuifolia essential oil could delay the growth of bacteria. For the treatment group containing 0.977 μL / mL Schizonepeta tenuifolia essential oil, after growing slowly for a period of time, the concentration of the bacterial solution began to increase after 5 h. However, throughout the process, the OD value of the treatment group containing 1.953 μL / mL Schizonepeta tenuifolia essential oil did not change significantly, indicating that the treatment with high concentration of Schizonepeta tenuifolia essential oil could significantly inhibit the growth and reproduction of Salmonella enterica serovar Gallinarum, making it unable to reach the normal growth peak. It can be seen that Schizonepeta tenuifolia essential oil has a good inhibitory effect on Salmonella enterica serovar Gallinarum X8.6, and the inhibitory effect increases with the increase of the concentration of Schizonepeta tenuifolia essential oil.
[0051] Example 4: Effect of Schizonepeta tenuifolia essential oil on the cell membrane of Salmonella in pigeons
[0052] The cell membrane plays a crucial role in maintaining the stability of the internal environment of bacteria. When the cell membrane is damaged by adverse external environments, not only will small-molecule substances inside the cell leak, but also biological macromolecules such as nucleic acids and proteins will leak, and these macromolecules are essential for the physiological metabolism of the bacterial cells. Nucleic acids and proteins both have characteristic peaks at 260 nm and 280 nm, and the concentration is proportional to the OD value. Measuring OD 260nm and OD 280nm is one of the indicators to evaluate the cell membrane permeability.
[0053] Take the Salmonella in pigeons bacterial solution prepared in Example 2 into shaking tubes, and add Schizonepeta tenuifolia essential oil to the shaking tubes respectively, so that the concentrations are 0, 0.977, 1.953, and 3.906 μL / mL respectively. Then incubate in a shaker at 37 °C with an oscillation speed of 150 r / min. Samples are taken at 0, 2, 4, 6, and 8 h of incubation respectively, centrifuged at 8000 rpm / min at 4 °C for 5 min, and the supernatant is taken to measure OD 260nm and OD 280nm , and the experiment is repeated three times, and the results are averaged.
[0054] The results are as Figure 2 shown. Compared with the control group, with the prolongation of the treatment time, the OD 260nm and OD 280nm values of the Schizonepeta tenuifolia essential oil treatment groups at different concentrations increased significantly (P < 0.05). Among them, at 2 h, the OD 260nm and OD 280nm of the 1.953 μL / mL Schizonepeta tenuifolia essential oil treatment group increased by 27.06% and 12.21% respectively; the OD 260nm and OD 280nm of the 3.906 μL / mL Schizonepeta tenuifolia essential oil treatment group increased by 35.34% and 13.20% respectively. At 4 h, the OD 260nm and OD 280nm of the 1.953 μL / mL Schizonepeta tenuifolia essential oil treatment group increased by 30.63% and 11.96% respectively; the OD 260nm and OD 280nm of the 3.906 μL / mL Schizonepeta tenuifolia essential oil treatment group increased by 54.51% and 22.89% respectively. At 8 h, the OD 260nm and OD 280nm of the 1.953 μL / mL Schizonepeta tenuifolia essential oil treatment group increased by 25.60% and 8.20% respectively; the OD 260nm and OD 280nmThey increased by 53.83% and 19.72% respectively. It can be seen that after the essential oil of Schizonepeta tenuifolia acts on Salmonella gallinarum, the cell membrane permeability changes, and the contents of leaked proteins and nucleic acids also increase, thus affecting the normal growth and reproduction of the bacteria.
[0055] Example 5: Effect of the essential oil of Schizonepeta tenuifolia on the cell wall of Salmonella gallinarum
[0056] Under normal circumstances, alkaline phosphatase (AKP) cannot penetrate through the cell wall and leak out of the cell, and its activity cannot be detected. Therefore, its activity can be used as a basis for reflecting the integrity of the cell wall of Salmonella gallinarum X8.6.
[0057] Take the Salmonella gallinarum bacterial liquid prepared in Example 2 into a shaking tube, and add the essential oil of Schizonepeta tenuifolia to the shaking tube respectively, so that its concentrations are 0, 0.488, 0.977, 1.953 μL / mL respectively. Then, culture it in a shaking incubator at 37°C with 150 r / min oscillation, take samples at 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8 h of culture respectively, centrifuge at 8000 rpm / min at 4°C for 5 min, take the supernatant, and measure OD 520nm , and repeat the experiment three times, and take the average value of the results.
[0058] The results show that Figure 3 it can be seen that the alkaline phosphatase activity in the bacterial liquid added with the essential oil of Schizonepeta tenuifolia is higher than that in the bacterial liquid without the essential oil of Schizonepeta tenuifolia. This indicates that the essential oil of Schizonepeta tenuifolia destroys the cell wall structure of Salmonella gallinarum X8.6, releases the alkaline phosphatase in the bacteria, and thus plays an antibacterial role. From Figure 3 it can also be seen that in the culture period of 0 - 1 h, the alkaline phosphatase activity of Salmonella gallinarum X8.6 treated with the essential oil of Schizonepeta tenuifolia at each concentration shows an upward trend; in the culture period of 1 - 5 h, when the concentration of the essential oil of Schizonepeta tenuifolia is 1.953 μL / mL, the alkaline phosphatase activity of Salmonella gallinarum X8.6 shows a downward trend, while it shows an upward trend from 5 - 8 h; in the culture period of 1 - 6 h, when the concentrations of the essential oil of Schizonepeta tenuifolia are 0.488 and 0.977 μL / mL, the alkaline phosphatase activity of Salmonella gallinarum X8.6 shows a downward trend, while it shows an upward trend from 6 - 8 h. It can be seen that the essential oil of Schizonepeta tenuifolia increases the cell wall permeability of Salmonella gallinarum X8.6, resulting in the leakage of AKP.
[0059] The present invention provides the idea and method for the application of the essential oil of Schizonepeta tenuifolia in the preparation of drugs against Salmonella gallinarum. There are many methods and ways to specifically implement this technical solution. The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and retouches can be made, and these improvements and retouches should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be realized by the existing technology.
Claims
1. Application of Schizonepeta tenuifolia essential oil in preparing drugs against pigeon Salmonella.
2. The application according to claim 1, characterized in that, The Schizonepeta tenuifolia essential oil is prepared by the following steps: (1) After the whole Schizonepeta tenuifolia herb is crushed, it is soaked in water and then distilled, so that the volatile oil in Schizonepeta tenuifolia enters the condenser together with water vapor for condensation; (2) The condensed oil and water are separated, and the oil layer is taken as the Schizonepeta tenuifolia essential oil.
3. The application according to claim 1, characterized in that, In step (1), for the water soaking, the amount of water added is 8 - 12 times the weight of the whole Schizonepeta tenuifolia herb, and the soaking time is 1 - 2 h.
4. The application according to claim 1, characterized in that, In step (1), for the distillation, the temperature is 110 - 125 °C, the distillation time is 6 - 8 h, and the steam pressure is 0.2 MPa.
5. The application according to claim 1, wherein In step (2), the separation is static separation.
6. The application according to claim 1, characterized in that, The dosage form of the drug is emulsion.
7. The application according to claim 1 or 6, characterized in that, The drug is prepared by the following method: The Schizonepeta tenuifolia essential oil is mixed with Tween - 20 solvent, and then normal saline is added and stirred until emulsification.
8. The application according to claim 7, characterized in that The volume ratio of the Schizonepeta tenuifolia essential oil to the Tween - 20 solvent is 10:
1.
9. The application according to claim 7, characterized in that, The stirring is carried out by a high - speed homogenizing emulsifier.
10. The application according to claim 9, characterized in that, The high speed has a rate of 20000 rpm.