A volatile oil emulsion of phellodendron amurense leaves, and a preparation method and application thereof
By preparing a volatile oil emulsion of wampee leaves, the problems of unpleasant odor and irritation of wampee leaf volatile oil were solved, and the stability and palatability were improved, expanding the scope of application. In particular, it has significant effects in livestock and poultry breeding and the treatment of pneumonia.
Patent Information
- Application Number
- CN202311668379.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-06
- Publication Date
- 2026-02-13
- Estimated Expiration
- 2043-12-06
AI Technical Summary
The use of volatile oil from wampee leaves is limited by their unpleasant odor and irritation, and the resource is not being used rationally, resulting in waste.
A stable emulsion of volatile oil from wampee leaves was prepared by mixing the volatile oil from wampee leaves with an emulsifier and water. The emulsion has good palatability and is suitable for veterinary drugs and pneumonia treatments.
The volatile oil emulsion of wampee leaves has reduced the irritating odor, improved stability, and expanded its application range. In particular, it can be administered through drinking water in the livestock and poultry industry to effectively inhibit Staphylococcus aureus and alleviate Staphylococcus aureus-infected pneumonia in mice, thus having an adjunctive therapeutic effect.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of agricultural and sideline products processing, and particularly relates to a Clausena lansium leaf volatile oil emulsion as well as a preparation method and application thereof. BACKGROUND
[0002] Clausena lansium (Lour.) Skeels is a plant of Rutaceae Clausena, which is a kind of medicinal and edible plant. Non-nuclear Clausena lansium is a rare fruit in Yunnan County of Yunfu City. Each part of Clausena lansium can be used as medicine, but the effects are different. Because the chemical components of different parts are different, the types and contents of volatile components are obviously different. Clausena lansium leaf is also called oil skin and oil plum, and the content of volatile components is high. However, the pruned branches and leaves of Clausena lansium after picking are mostly discarded, and there is little research on the resource utilization of Clausena lansium branches and leaves. The volatile components in Clausena lansium branches and leaves are not reasonably utilized, which causes great waste. In addition, volatile oil often has bad smell, poor palatability, strong irritation and other characteristics, which greatly limits its use. SUMMARY
[0003] The technical problem to be solved by the present application is to provide a Clausena lansium leaf volatile oil emulsion, which has good stability and good palatability.
[0004] The technical problem to be solved by the present application is to provide a Clausena lansium leaf volatile oil emulsion, which has good stability and good palatability.
[0005] The technical problem to be solved by the present application is to provide a Clausena lansium leaf volatile oil emulsion, which has good stability and good palatability.
[0006] In order to solve the above technical problems, the present application provides a Clausena lansium leaf volatile oil emulsion, which comprises the following components:
[0007] Clausena lansium leaf volatile oil 80-150 parts by weight, emulsifier 70-180 parts by volume, water 1200-2000 parts by volume;
[0008] The Clausena lansium leaf volatile oil is obtained by water vapor distillation of Clausena lansium leaf.
[0009] For example, the amount of Clausena lansium leaf volatile oil is 85 parts by weight, 90 parts by weight, 95 parts by weight, 100 parts, 105 parts by weight, 110 parts by weight, 115 parts by weight, 120 parts by weight, 125 parts by weight, 130 parts by weight, 135 parts by weight, 140 parts by weight or 145 parts by weight, but is not limited to this. Preferably, the amount is 90-120 parts by weight, and more preferably, the amount is 90-110 parts by weight.
[0010] For example, the emulsifier is used in an amount of 76 parts by volume, 82 parts by volume, 88 parts by volume, 94 parts by volume, 105 parts by volume, 114 parts by volume, 126 parts by volume, 135 parts by volume, 141 parts by volume, 153 parts by volume, 160 parts by volume or 172 parts by volume, but is not limited thereto. Preferably, the emulsifier is used in an amount of 80-120 parts by volume, and more preferably, the emulsifier is used in an amount of 90-110 parts by volume.
[0011] For example, the water is used in an amount of 1250 parts by volume, 1300 parts by volume, 1400 parts by volume, 1500 parts by volume, 1550 parts by volume, 1600 parts by volume, 1700 parts by volume, 1850 parts by volume or 1980 parts by volume, but is not limited thereto. Preferably, the water is used in an amount of 1500-2000 parts by volume, and more preferably, the water is used in an amount of 1500-1900 parts by volume.
[0012] It should be noted that the weight parts and volume parts in the present application correspond to mg, μL or both are enlarged or reduced in proportion.
[0013] The emulsifier is a non-ionic emulsifier, for example, Tween 80, Span 80 or Triton X-100, but is not limited thereto.
[0014] As an improvement of the above technical solution, the emulsifier is Tween 80 and / or Span 80.
[0015] As an improvement of the above technical solution, the ratio of the weight of the volatile oil of the leaf of Phellodendri chinensis to the volume of the emulsifier is 1 mg:(1-2) μL; for example, 1 mg:1.2 μL, 1 mg:1.4 μL, 1 mg:1.6 μL or 1 mg:1.8 μL, but is not limited thereto.
[0016] As an improvement of the above technical solution, the emulsifier is Tween 80.
[0017] The ratio of the weight of the volatile oil of the leaf of Phellodendri chinensis to the volume of the emulsifier is 1 mg:1 μL.
[0018] As an improvement of the above technical solution, the preparation method of the volatile oil of the leaf of Phellodendri chinensis is as follows:
[0019] (1) The crushed leaf of Phellodendri chinensis is soaked in water for 0.5-2 h; the ratio of the leaf of Phellodendri chinensis to water is 1 g:(10-25) mL.
[0020] (2) The intermediate product is obtained by steam distillation extraction for 1-4 h.
[0021] (3) The intermediate product is extracted with ethyl acetate for 2-3 times, and the ethyl acetate layer is combined and separated to obtain the finished product of the volatile oil of the leaf of Phellodendri chinensis.
[0022] Correspondingly, the application further discloses a preparation method of the volatile oil emulsion of the leaf of Phellodendri amurense Rupr.
[0023] The volatile oil of the leaf of Phellodendri amurense Rupr. is mixed with the emulsifier to obtain an oil phase;
[0024] The oil phase is added dropwise into water, and stirring is continuously performed until the oil phase is uniformly dispersed.
[0025] The volume ratio of the oil phase to the water is (1-3):(9-17), for example, 1:10, 1:14, 2:12, 2:16 or 3:16, but is not limited thereto.
[0026] As an improvement of the above technical solution, the volume ratio of the oil phase to the water is 1:9.
[0027] Correspondingly, the application further discloses application of the volatile oil emulsion of the leaf of Phellodendri amurense Rupr. in preparation of a veterinary drug.
[0028] As an improvement of the above technical solution, the veterinary drug is used for inhibiting Staphylococcus aureus.
[0029] Correspondingly, the application further discloses application of the volatile oil emulsion of the leaf of Phellodendri amurense Rupr. in preparation of a drug for treating pneumonia.
[0030] As an improvement of the above technical solution, the pneumonia is pneumonia caused by Staphylococcus aureus.
[0031] The drug can inhibit expression of NLRP3, ASC, IL-1β, IL-18 and caspase-1.
[0032] The application has the following beneficial effects:
[0033] The volatile oil emulsion of the leaf of Phellodendri amurense Rupr. is prepared from the volatile oil of the leaf of Phellodendri amurense Rupr., an emulsifier and water, reduces irritating odor of the volatile oil of the leaf of Phellodendri amurense Rupr., improves stability and palatability. The volatile oil emulsion of the leaf of Phellodendri amurense Rupr. can be administered through drinking water, greatly expands application in livestock and poultry breeding industry, and widens application range of the drug. The volatile oil emulsion of the leaf of Phellodendri amurense Rupr. is prepared from the leaf of Phellodendri amurense Rupr. without core, researches show that the volatile oil emulsion of the leaf of Phellodendri amurense Rupr. has obvious inhibitory effect on Staphylococcus aureus, the minimum inhibitory concentration of the volatile oil emulsion of the leaf of Phellodendri amurense Rupr. on Staphylococcus aureus is 312.50 μg / mL, can effectively inhibit formation of a biofilm of Staphylococcus aureus, cause abnormal division of bacteria, cavitation of cells and death of bacteria. In addition, the volatile oil emulsion of the leaf of Phellodendri amurense Rupr. can effectively alleviate pneumonia of a mouse infected with Staphylococcus aureus, inhibit activation of NLRP3 inflammasome and production of inflammatory factors, and therefore can have auxiliary treatment effect on bacterial pneumonia, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS
[0034] Figure 1 is the GC-MS analysis result chart of the volatile oil of the leaves of example 1;
[0035] Figure 2 is the actual photo of the change of the volatile oil emulsion of the leaves of example 2 under different conditions; in the chart, the experimental serial numbers from left to right are the test tubes 1-9 of the volatile oil emulsion of the leaves of
[0036] Figure 3 is the stability experimental result chart of the volatile oil emulsion of the leaves of example 2, in which, A is the leaves of the wampee, B is the volatile oil of the leaves of the wampee, C is the volatile oil emulsion of the leaves of the wampee, from left to right are the test tubes 1-9; D is the microscope observation result chart of the best ratio of the volatile oil emulsion of the leaves of the wampee, E is the microscope observation result chart of the best ratio of the volatile oil emulsion of the leaves of the wampee after being placed at low temperature (4℃) for 1 month, F is the microscope observation result chart of the best ratio of the volatile oil emulsion of the leaves of the wampee after being placed at high temperature (50±2℃) for 5 days, G is the microscope observation result chart of the best ratio of the volatile oil emulsion of the leaves of the wampee after the stability test according to GB / T 1603-2001;
[0037] Figure 4 is the result chart of the effect of the volatile oil emulsion of the leaves of example 2 on salmonella and staphylococcus aureus; in which, A is the time-antibacterial curve of staphylococcus aureus, B is the time-antibacterial curve of salmonella typhimurium, C is the difference chart of OD 600 of staphylococcus aureus, D is the difference chart of OD 600 of salmonella typhimurium, E is the morphology chart of salmonella typhimurium after being treated by the volatile oil emulsion of the leaves of the wampee for 24h; F is the morphology chart of staphylococcus aureus after being treated by the volatile oil emulsion of the leaves of the wampee for 24h;
[0038] Figure 5 is the result chart of the flow cytometry detection of the effect of the volatile oil emulsion of the leaves of example 2 on the death of salmonella typhimurium and staphylococcus aureus; A is the normal control group infected with staphylococcus aureus; B-D are staphylococcus aureus after being treated by 1 / 2×MIC, MIC and 4×MIC of the volatile oil emulsion of the leaves of the wampee respectively, E is the normal control group infected with salmonella typhimurium, F-H are salmonella typhimurium after being treated by 1 / 2×MIC, MIC and 4×MIC of the volatile oil emulsion of the leaves of the wampee respectively; I is the survival curve of staphylococcus aureus and salmonella typhimurium;
[0039] Figure 6 is the chart of the transmission electron microscope observation of the effect of the volatile oil emulsion of the leaves of example 2 on the microstructure of staphylococcus aureus; A is the normal control group, B is the MIC group, C is the MBC group. The red arrow indicates the bacterial cell wall blur; the blue arrow indicates the bacterial cavity; the yellow arrow indicates the inhibition of bacterial division.
[0040] Figure 7 is a graph of the influence of the leaf of phellodendri chinensis volatile oil emulsion on staphylococcus aureus biofilm formation in Example 2;
[0041] Figure 8 is a graph of the influence of the leaf of phellodendri chinensis volatile oil emulsion on the lung index and wet / dry weight ratio of staphylococcus aureus infected mice in Example 3;
[0042] Figure 9 is a graph of the influence of the leaf of phellodendri chinensis volatile oil emulsion on the bacterial load of the lung of staphylococcus aureus infected mice in Example 3;
[0043] Figure 10 is a graph of the influence of the leaf of phellodendri chinensis volatile oil emulsion on the pathological changes of the lung of staphylococcus aureus infected mice in Example 3 (HE staining 100x and 400x); wherein, A-H are blank control group, model group, Tween group, positive drug group, high-dose leaf of phellodendri chinensis volatile oil group, medium-dose leaf of phellodendri chinensis volatile oil group, low-dose leaf of phellodendri chinensis volatile oil group and leaf of phellodendri chinensis volatile oil drug control group, respectively;
[0044] Figure 11 is a graph of the influence of the leaf of phellodendri chinensis volatile oil emulsion on the activation of NLRP3 inflammasome of staphylococcus aureus infected mice in Example 3. DETAILED DESCRIPTION
[0045] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below.
[0046] Example 1 volatile oil extraction and compound detection method
[0047] Fresh leaf of phellodendri chinensis (produced in Yunfu City, Yun'an County, Guangdong Province, identified as leaf of phellodendri chinensis tree without core) is dried indoors and then ground into powder and passed through a 100 mesh sieve. 10 g of the leaf of phellodendri chinensis powder is weighed and placed in a 500 mL round-bottom flask, 200 mL of ultrapure water is added, soaked for 1 h, and then water vapor distillation is used to extract volatile oil; after 4 h of extraction, ethyl acetate is used for extraction, the upper ethyl acetate layer is taken, extraction is performed three times, anhydrous sodium sulfate is added to the extraction liquid, and after standing for 10 h, filtration is performed, ethyl acetate is evaporated in a water bath, and the volatile oil is weighed. The volatile oil sample obtained by extraction is analyzed by GC-MS. The analysis results are shown in Table 1. Figure 1
[0048] Figure 1 is the GC-MS analysis results chart of the volatile oil of the leaf of Phellodendron amurense Rupr. The relative content of each component was calculated by peak area normalization method. 37 kinds of compounds were identified, mainly including aromatic hydrocarbons, terpenoids and their oxygen derivatives, accounting for 96.49% of the total volatile oil. The main components of the volatile oil of the leaf of Phellodendron amurense Rupr. were 5,5-dimethyl-1-vinyl bicyclohexane (13.050 9%), palmitic acid (13.838 5%), β-ocimene (6.580 5%), phytol (5.772 5%), α-bisabolol (2.844 1%), caryophyllene oxide (3.077 7%), eucalyptol (1.775 1%), etc. The contents of 5,5-dimethyl-1-vinyl bicyclohexane and palmitic acid were relatively high.
[0049] Example 2 Preparation and evaluation of the volatile oil emulsion of the leaf of Phellodendron amurense Rupr.
[0050] 1) Preparation of the volatile oil emulsion of the leaf of Phellodendron amurense Rupr.
[0051] The oil phase was prepared by mixing volatile oil and emulsifier (Tween 80) in different proportions. The oil phase was added dropwise into water at 80℃, and stirred (500 r / min) with a magnetic stirrer until emulsification. (See Table 1 for test design).
[0052] Table 1 Composition design of the volatile oil emulsion of the leaf of Phellodendron amurense Rupr.
[0053]
[0054] 2) Stability test of the volatile oil emulsion of the leaf of Phellodendron amurense Rupr.
[0055] The emulsion was stored in a 4℃ refrigerator for 1 month to observe the changes in appearance under low temperature environment. The emulsion was stored in a-20℃ refrigerator for 3 days to observe whether there was precipitation and stratification. The emulsion was stored in a 50±2℃ constant temperature oven for 5 days to observe whether there was precipitation and stratification under high temperature environment. The best emulsion ratio was selected according to the above experimental results. The best ratio was selected to conduct GB / T 1603-2001 stability test (200 times dilution). Standard hard water was prepared by weighing 0.304 g of anhydrous calcium chloride and 0.139 g of magnesium chloride with crystal water into a 1000 mL volumetric flask, dissolving and diluting to the mark line with distilled water. In a 250 mL beaker, 100 mL of standard hard water was added, and an appropriate amount of emulsion sample was taken with a pipette, slowly added to the hard water under constant stirring, and made into 100 mL emulsion. After adding the emulsion, continue to stir for 30 s, immediately transfer the emulsion to a clean and dry 100 mL graduated cylinder, and place the graduated cylinder in a 30℃ constant temperature water bath, stand for 1 h, and observe the emulsion.
[0056] 3) Bacteriostatic test of the volatile oil emulsion of the leaf of Phellodendron amurense Rupr.
[0057] 3.1 Preparation of emulsion: 100 mg of volatile oil of Phellodendri chinensis leaves and 100 μL of emulsifier (Tween 80) were mixed to form an oil phase (200 μL in total). The oil phase was added dropwise into 1800 μL of water at 80°C and stirred (500 r / min) using a magnetic stirrer until emulsification, thus obtaining the volatile oil emulsion of Phellodendri chinensis leaves.
[0058] 3.2 Determination of MIC and MBC: The double dilution method was used: the drug was double diluted in LB liquid medium to different concentrations (2500.00 μg / mL, 1250.00 μg / mL, 625.00 μg / mL, 312.50 μg / mL, 156.25 μg / mL, 78.12 μg / mL), and blank control group, Tween group (2500 μL / mL, Tween 80) and positive control group were set. 10 mL of LB liquid medium was added to each test tube, followed by 0.1 mL of 10 6 CFU / mL of bacteria in the logarithmic growth phase, and the OD 600 values of the bacteria before and after growth were detected. No bacterial growth was observed by naked eye and the OD 600 values were significantly lower than those of the positive group. The minimum concentration was MIC. The test tubes with no bacterial growth were taken out, 100 μL of which was plated and cultured at 37°C for 12 h, and the minimum concentration with less than 5 colonies was MBC.
[0059] 3.3 Determination of time-bacteriostatic curve: 100 μL of 10 5 CFU / mL of Staphylococcus aureus was added to the medium containing 1 / 4 MIC, 1 / 2 MIC, MIC, 2 MIC and 4 MIC drug concentrations, respectively, and cultured at 37°C for 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h and 48 h. 100 μL of bacterial solution was taken at the above-mentioned times for dilution, and the number of bacteria was calculated.
[0060] 3.4 Scanning electron microscope observation of the morphological changes of Salmonella and Staphylococcus aureus caused by the volatile oil emulsion of Phellodendri chinensis leaves: Salmonella and Staphylococcus aureus were cultured in the medium containing MBC volatile oil emulsion of Phellodendri chinensis leaves for 24 h, fixed with glutaraldehyde fixing solution, and observed by scanning electron microscope.
[0061] 3.5 Transmission electron microscope observation of the morphological changes of Staphylococcus aureus caused by the volatile oil emulsion of Phellodendri chinensis leaves: Salmonella and Staphylococcus aureus were cultured in the medium containing MIC volatile oil emulsion of Phellodendri chinensis leaves for 6 h and 12 h, respectively, and in the medium containing MBC volatile oil emulsion of Phellodendri chinensis leaves for 6 h and 12 h, respectively. The effect of the volatile oil emulsion of Phellodendri chinensis leaves on Staphylococcus aureus was observed by transmission electron microscope.
[0062] 3.6 Effect of the volatile oil emulsion of Phellodendri Cortex on the biofilm formation of S. aureus: There were six groups in the experiment: blank control group, Tween group (emulsifier group), 1 / 8 MIC, 1 / 4 MIC, 1 / 2 MIC and MIC groups. 200 μL of LB broth containing 10 8 CFU / mL of 1 / 8 MIC, 1 / 4 MIC, 1 / 2 MIC and MIC volatile oil emulsion of Phellodendri Cortex were added to the 96-well plate, each concentration was repeated 6 times, and incubated in a constant temperature incubator at 37°C for 24 h. After 24 h, 3 times of PBS washing was used, 200 μL of methanol was added for 20 min, the methanol was removed, dried, 1% crystal violet solution was added for 20 min, 3 times of PBS washing was used after drying, 200 μL of 95% ethanol was added for 5 min, and OD 570 was measured.
[0063] 4) Experimental results
[0064] 4.1 Stability results of the volatile oil emulsion of Phellodendri Cortex
[0065] The results of the stability experiment are shown in Figure 2 and Figure 3 . As can be seen from the figure, the volatile oil of Phellodendri Cortex is golden yellow ( Figure 3 B), and has a rich aroma. The volatile oil emulsion prepared by different proportions of volatile oil, emulsifier and water was subjected to stability test, and it was found that there was no change in the appearance of the emulsion after being stored in a refrigerator at 4°C for 1 month, and there was no precipitation and stratification phenomenon after being placed in a constant temperature oven at 50°C for 5 days ( Figure 2 ). According to the results of the volatile oil stability experiment, the optimal oil phase: water phase (volume ratio) in the volatile oil emulsion of Phellodendri Cortex was determined as 1:9, and the optimal Phellodendri Cortex volatile oil (weight): emulsifier (Tween 80, volume) in the oil phase was 1:1 ( Figure 3 C). The volatile oil emulsion of Phellodendri Cortex was milky white, and the size of the emulsion droplets was below 10 μm ( Figure 3 D-G).
[0066] 4.2 Effect of the volatile oil emulsion of Phellodendri Cortex on Salmonella and S. aureus
[0067] As shown in Figure 4 , the volatile oil emulsion of Phellodendri Cortex had no obvious inhibitory effect on Salmonella ( Figure 4 B, D). The OD 600 difference of S. aureus showed that ( Figure 4 C) the emulsifier (Tween 80) in the emulsion had no effect on the growth of S. aureus, and when the drug concentration was 312.50 μg / mL, it would significantly affect the proliferation of S. aureus. Combined with the visual observation results and OD 600The difference results determined that the minimum inhibitory concentration (MIC) of the volatile oil emulsion of *Scutellaria baicalensis* leaf against *Staphylococcus aureus* was 312.50 μg / mL; according to the bacterial plating results, the MBC was 2500.00 μg / mL. The time-inhibition curve of *Scutellaria baicalensis* leaf volatile oil against *Staphylococcus aureus* was obtained from the results. Figure 4 As shown in A), the volatile oil emulsion of *Clausena lansium* leaves had a significant inhibitory effect on *Staphylococcus aureus*. Specifically, the volatile oil emulsions of *Clausena lansium* leaves at concentrations of 0.25×MIC, 0.5×MIC, and MIC showed that *Staphylococcus aureus* reached a plateau phase after 12 hours of treatment, while the inhibitory effect of the volatile oil emulsions of 2×MIC and 4×MIC gradually disappeared after 24 hours of treatment, with bacterial growth gradually reaching a plateau phase. Transmission electron microscopy results showed that, compared with the blank control group, the inhibitory effect of MBC volatile oil emulsion on *Salmonella typhimurium* was weaker after 24 hours of treatment, with fewer surface wrinkles on the bacteria, but no signs of death. Figure 4 E); however, after 24 hours, the Staphylococcus aureus cells ruptured and the bacteria died, demonstrating a significant inhibitory effect on Staphylococcus aureus. Figure 4 F).
[0068] 4.3 Flow cytometry analysis of the effect of volatile oil emulsion from *Clausena lansium* leaves on the death of *Salmonella typhimurium* and *Staphylococcus aureus*.
[0069] like Figure 5 As shown in (AD), compared with the control group, the survival rates of Staphylococcus aureus in the 1 / 2×MIC, 2×MIC, and 4×MIC groups were 84.6%, 14.5%, and 12.8%, respectively, all significantly lower than the 97.2% of the control group. Staphylococcus aureus lysed and died 24 hours after treatment with the volatile oil emulsion, hence the significantly reduced number of bacteria and stainable bacteria in the 4×MIC group. The survival rates of Salmonella typhimurium in the 1 / 2×MIC, 2×MIC, and 4×MIC groups were 96%, 95.3%, and 75.5%, respectively, similar to the control group (99.6%). Figure 5 The above results indicate that the volatile oil emulsion of *Clausena lansium* leaves has a good antibacterial effect on *Staphylococcus aureus*, and the antibacterial effect is dose-dependent, but it has no antibacterial effect on *Salmonella typhimurium*.
[0070] 4.4 Transmission electron microscopy observation of the damaging effects of volatile oil emulsion from *Clausena lansium* leaves on *Staphylococcus aureus*.
[0071] In the control group, *Staphylococcus aureus* showed a clear boundary between its cell membrane and cell wall. Some bacteria were undergoing binary fission, and the cells on either side of the septum were symmetrical. Figure 6 A). After 6 hours of treatment with MIC wampee leaf volatile oil emulsion, bacterial cavities and abnormal binary fission, including incomplete and asymmetric division, were observed; after 12 hours, the cavity size gradually increased ( Figure 6B). After treatment with MBC yellow peel leaf volatile oil emulsion for 6 hours, bacterial binary fission was significantly inhibited, no membrane structure was observed, and cavitation formation was present; after 12 hours, the distance between the bacterial cell membrane and cell wall disappeared, and the cells cavitated and died. Figure 6 C). The above results indicate that the volatile oil emulsion of wampee leaves can disrupt the cell membrane structure of Staphylococcus aureus.
[0072] 4.5 Effect of volatile oil emulsion from *Clausena lansium* leaves on biofilm formation in *Staphylococcus aureus*
[0073] like Figure 7 As shown, the concentrations of the volatile oil emulsion from the leaves of *Scutellaria baicalensis* at 2×MIC, MIC, 1 / 2MIC, 1 / 4MIC, 1 / 8MIC, 1 / 16MIC, and 1 / 32MIC significantly inhibited the formation of biofilms by *Staphylococcus aureus*.
[0074] Example 3: Effect of the volatile oil emulsion of *Clausena lansium* leaves on pneumonia in mice.
[0075] 1) Test Plan
[0076] 1.1 Preparation of the emulsion: Prepare an oil phase by mixing 100 mg of volatile oil from *Clausena lansium* leaves with 100 μL of emulsifier (Tween 80). Add the oil phase dropwise to 1800 μL of water at 80 °C and stir with a magnetic stirrer (500 r / min) until emulsification, thus obtaining the *Clausena lansium* leaf volatile oil emulsion.
[0077] 1.2 Experimental Grouping
[0078] One hundred and fifty female BALB / c mice aged 6-8 weeks were randomly divided into eight groups: blank control group (nasal PBS drops + gavage with an equal volume of PBS solution), Tween group (nasal Staphylococcus aureus drops + gavage with 5.00 mg / kg Tween 80), model group (nasal Staphylococcus aureus drops + gavage with an equal volume of PBS solution), positive control group (nasal Staphylococcus aureus drops + gavage with vancomycin 10.00 mg / kg), and high-dose group of safflower leaf volatile oil (nasal Staphylococcus aureus drops + gavage with vancomycin 10.00 mg / kg). The mice were divided into three groups: a 5.00 mg / kg emulsion of *Clausena lansium* leaf volatile oil, a medium-dose group (nasal drops of *Staphylococcus aureus* + gavage of 2.50 mg / kg emulsion of *Clausena lansium* leaf volatile oil), a low-dose group (nasal drops of *Staphylococcus aureus* + gavage of 1.25 mg / kg emulsion of *Clausena lansium* leaf volatile oil), and a control group (gavage of 2.50 mg / kg emulsion of *Clausena lansium* leaf volatile oil); each group consisted of 20 mice (10 mice in the *Clausena lansium* leaf volatile oil group). Five days after prophylactic administration, each mouse in the model group received a single intranasal drop of 30 μL (4 × 10) of the emulsion. 9 CFU / mL Staphylococcus aureus suspension was administered to mice in the non-model group via nasal drops of 30 μL PBS. The administration was continued for 2 days (total administration time 7 days) before the treatment was discontinued.
[0079] 1.3 Organ index and wet / dry ratio of mouse lungs
[0080] Carefully remove the whole lung of mice, take pictures, weigh, and calculate the lung wet / dry ratio.
[0081] Formula: Organ index = lung weight (g) / mouse weight (g)
[0082] Lung wet / dry ratio = lung weight (g) / dry lung weight (g)
[0083] 1.4 Lung pathological examination
[0084] Dissect the same part of the lung of mice in each group, fix it in 4% paraformaldehyde for 24 h, dehydrate with alcohol gradient, treat with xylene, embed in paraffin, slice, spread, and deparaffinize, then perform HE staining, alcohol gradient dehydration, and resin sealing, and finally observe the pathological features and changes of the lung tissue of mice under a microscope.
[0085] 1.5 Staphylococcus aureus load measurement
[0086] Weigh 0.05 g of lung tissue into 1 mL of pre-cooled PBS, put it into a tissue crusher, crush it, and then dilute it 10 times. Take 100 μL of each concentration of tissue homogenate to a mannitol sodium chloride agar sterile culture plate, with three repeats for each concentration. Incubate at 37°C for 24 h, record the number of colonies in the culture medium, and calculate the lung bacterial load.
[0087] 1.6 Western blot analysis of lung protein expression in mice
[0088] Mix phenylmethylsulfonyl fluoride protease inhibitor (PMSF) with cell tissue rapid lysis buffer (RIPA Lysis Buffer) at 1:100 for extracting total protein from the lung of mice. After protein electrophoresis separation, transfer the membrane to a 0.22 μm PVDF membrane at low temperature by wet method, block with 5% skimmed milk powder at room temperature for 1 h, wash with TBST for 3 times, incubate the PVDF membrane with IL-Iβ, NLRP3, IL-18, ASC, Caspasea-l, P-NF-κB p65, NF-κB p65, Claudine-3, occludin, ZO-1, and β-actin primary antibodies at room temperature for 3 h, wash with TBST for 2 times, each for 15 min, incubate with HRP-labeled goat anti-rabbit or goat anti-mouse secondary antibodies at room temperature for 1 h, wash with TBST for 2 times, each for 15 min, develop with ECL luminescent solution, and analyze the protein content by Image J software.
[0089] 2) Test results
[0090] 2.1 Volatile oil emulsion of Huangpi leaf has a protective effect on the lung of Staphylococcus aureus infected mice
[0091] Shen Figure 8 As shown, compared with the blank group, the lung indices of the mice in the model group and the Tween group were significantly increased, and the wet weight / dry weight values of the lungs in the model group and the Tween group were significantly increased (P<0.05). There was no significant difference in the lung index between the control group of Clausena lansium leaf volatile oil emulsion and the blank group. Compared with the model group, the lung indices and the ratios of wet weight / dry weight of the lungs in the positive drug group and the volatile oil emulsion group decreased (P<0.05). It shows that Clausena lansium leaf volatile oil emulsion can relieve the congestion and edema of the lungs caused by Staphylococcus aureus infection.
[0092] It can be seen from Figure 9 that, compared with the blank group, the bacterial loads in the lungs of the model group and the Tween group were significantly increased (P<0.05). Compared with the model group and the Tween group, the loads of Staphylococcus aureus in the lungs of the positive drug group and the Clausena lansium leaf volatile oil emulsion group were significantly decreased (P<0.05).
[0093] It can be seen from Figure 10 that the lungs of the mice in the blank control group were pink, with a three-dimensional and plump structure, shiny, and soft and elastic in texture; the overall staining of the mouse lungs was uniform under low magnification; under high magnification, the cell nuclei were stained blue, the cytoplasm was stained red in filaments or blocks, and the cell membranes were stained red. Compared with the blank group, the lungs of the mice in the model group and the Tween group were white, swollen in volume, dull, and harder in texture; obvious darker lesion areas were observed under low magnification, the alveolar cavities were arranged irregularly, the bronchi were filled with a large number of red blood cells and inflammatory cells, and there were also infiltrations of red blood cells and inflammatory cells in the peripheral alveolar cavities; under high magnification, the cell nuclei of the alveolar epithelial cells were enlarged, the alveolar epithelial cells proliferated, and the cell intervals were not obvious. The lungs of the mice in the positive drug group were pink, slightly larger in volume than the blank group, and the lung margins were thicker; under low magnification, some alveolar cavities were filled with red blood cells; under high magnification, a small amount of red blood cells and inflammatory cells were contained in the bronchioles. The lungs of the high-dose group of Clausena lansium leaf volatile oil were pink, slightly smaller in volume than the model group, and shiny; a small amount of alveolar hemorrhage was visible under low magnification; no obvious abnormality was seen under high magnification. The lungs of the medium-dose group of Clausena lansium leaf volatile oil were pink, slightly smaller in volume than the model group, and shiny; no obvious abnormality was seen under both low magnification and high magnification. The lungs of the low-dose group of Clausena lansium leaf volatile oil were pink, slightly smaller in volume than the model group; red blood cells were visible in some alveolar cavities under low magnification; inflammatory cells were present in the bronchioles under high magnification. The lungs of the control group of Clausena lansium leaf volatile oil were normal in color, and no obvious abnormality was seen under both low magnification and high magnification.
[0094] 2.2 Inhibition of NLRP3 inflammasome activation in the lungs of mice infected with Staphylococcus aureus by Clausena lansium leaf volatile oil emulsion and alleviation of lung inflammation
[0095] It can be seen from Figure 11It was found that the protein expression of NLRP3, ASC, IL-1β, IL-18 and caspase-1 in the model group and the Tween group were significantly increased compared with the blank group (P<0.05). Compared with the model group, the content of ASC and IL-1β in the lung of the positive drug group was significantly decreased (P<0.05); the protein expression of NLRP3, ASC, IL-1β, IL-18 and caspase-1 in the high, medium and low dose groups of the volatile oil of H. japonica was significantly decreased (P<0.05).
[0096] The above is the preferred embodiment of the application. It should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the application, and these improvements and refinements are also considered within the scope of protection of the application.
Claims
1. The application of volatile oil emulsion from *Clausena lansium* leaves in the preparation of veterinary drugs, characterized in that, The volatile oil emulsion of the yellow peel leaf comprises the following components: 80-150 parts by weight of volatile oil from the leaves of the yellow peel, 70-180 parts by volume of emulsifier, and 1200-2000 parts by volume of water; The volatile oil of the wampee leaves is the only medicinal component, which is obtained by steam distillation and ethyl acetate extraction of wampee leaves. The main components of the volatile oil of wampee leaves include: 5,5-dimethyl-1-vinylbicyclohexane, palmitic acid, β-ocimene, phytosterol, α-bisabolol, caryophyllene oxide, and eucalyptol. The emulsifier used is Tween 80; the weight ratio of the volatile oil from the yellow peel leaves to the volume ratio of the emulsifier is 1:1 to 1:
2. The volume ratio of the oil phase obtained by mixing the volatile oil of the yellow peel leaf with the emulsifier to the water is 1:8 to 1:
10. The minimum inhibitory concentration of the volatile oil emulsion of the yellow peel leaf against Staphylococcus aureus is 312.5 μg / mL; The veterinary drug is used to inhibit Staphylococcus aureus.
2. The application as described in claim 1, characterized in that, The weight ratio of the volatile oil from the wampee leaves to the volume ratio of the emulsifier is 1:
1.
3. The application as described in claim 1, characterized in that, The preparation method of the volatile oil emulsion of the wampee leaves includes: The volatile oil from the leaves of the yellow peel was mixed with an emulsifier to obtain the oil phase; The oil phase is added dropwise to water and stirred continuously until it is evenly dispersed.
4. The application as described in claim 3, characterized in that, The volume ratio of the oil phase to the water is 1:
9.
5. The application of the volatile oil emulsion of *Clausena lansium* leaves in the preparation of drugs for treating pneumonia, characterized in that... The volatile oil emulsion of the yellow peel leaf comprises the following components: 80-150 parts by weight of volatile oil from the leaves of the yellow peel, 70-180 parts by volume of emulsifier, and 1200-2000 parts by volume of water; The volatile oil of the wampee leaves is the only medicinal component, which is obtained by steam distillation and ethyl acetate extraction of wampee leaves. The main components of the volatile oil of wampee leaves include: 5,5-dimethyl-1-vinylbicyclohexane, palmitic acid, β-ocimene, phytosterol, α-bisabolol, caryophyllene oxide, and eucalyptol. The emulsifier used is Tween 80; the weight ratio of the volatile oil from the yellow peel leaves to the volume ratio of the emulsifier is 1:1 to 1:
2. The volume ratio of the oil phase obtained by mixing the volatile oil of the yellow peel leaf with the emulsifier to the water is 1:8 to 1:
10. The minimum inhibitory concentration of the volatile oil emulsion of the yellow peel leaf against Staphylococcus aureus is 312.5 μg / mL; The pneumonia mentioned is caused by Staphylococcus aureus.
6. The application as described in claim 5, characterized in that, The drug can inhibit the expression of NLRP3, ASC, IL-1β, IL-18 and caspase-1.
7. The application as described in claim 5, characterized in that, The weight ratio of the volatile oil from the wampee leaves to the volume ratio of the emulsifier is 1:
1.
8. The application as described in claim 5, characterized in that, The preparation method of the volatile oil emulsion of the wampee leaves includes: The volatile oil from the leaves of the yellow peel was mixed with an emulsifier to obtain the oil phase; The oil phase is added dropwise to water and stirred continuously until it is evenly dispersed.
9. The application as described in claim 8, characterized in that, The volume ratio of the oil phase to the water is 1:9.