Preparation method for improving bacterial endotoxin antagonism effect of veterinary Shuanghuanglian oral liquid

Abstract

The invention discloses a preparation method for improving the bacterial endotoxin antagonistic effect of veterinary Shuanghuanglian oral liquid, which comprises the following steps: S1, weighing honeysuckle and fructus forsythiae according to the prescription amount, crushing, extracting volatile oil for 4-6 hours by adopting a water separation distillation method, and collecting the volatile oil for later use; s2, adding 8-12 times of drinking water into the distilled honeysuckle and fructus forsythiae, heating to boil, and adding a prescription amount of scutellaria baicalensis; s3, mixing Tween-80 and polyoxyethylated castor oil according to the mass ratio of (2-4): 1 to prepare a surfactant; and S4, uniformly mixing the extracting solution with the nano-emulsion. The veterinary Shuanghuanglian oral liquid prepared by the process is rich in volatile oil and complete in effective substances, the effect of antagonizing endotoxin is improved, experiments show that the prepared Shuanghuanglian oral liquid has a better effect of reducing bacterial endotoxin in vitro, and in in-vivo experiments, chickens in a Shuanghuanglian treatment group are better in mental state and excrement forming effect, and are free of toxic and side effects. Liver bleeding is obviously reduced, and inflammatory cell infiltration is obviously reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of veterinary medicine, in particular to a preparation method for improving the effect of antagonizing bacterial endotoxin of veterinary Shuanghuanglian oral liquid. BACKGROUND

[0002] Endotoxin is a component of the cell wall of gram-negative bacteria, and its source is wide. The main sources of endotoxin are the environment of livestock and poultry breeding, vaccines and antibiotics. The pathological reaction caused by endotoxin not only harms the health of animals, but also significantly reduces the production performance, causing huge economic losses to the breeding farm. Traditional Chinese veterinary medicine has the effects of antagonizing endotoxin, anti-inflammatory, repairing damage, etc., and has a significant effect on the treatment of livestock and poultry diseases caused by endotoxin. Shuanghuanglian is prepared from honeysuckle, forsythia and scutellaria, and has the effects of clearing heat and resolving toxins.

[0003] However, honeysuckle and forsythia in the prescription of Shuanghuanglian oral liquid contain rich volatile oil, and the volatile oil has obvious pharmacodynamic effect. However, the volatile oil in the medicinal flavor is not treated in the preparation process of Shuanghuanglian oral liquid at present, resulting in loss of volatile oil, which significantly reduces the clinical use effect of Shuanghuanglian. Therefore, in view of the above problems, the present application provides a preparation method for improving the effect of antagonizing bacterial endotoxin of veterinary Shuanghuanglian oral liquid. SUMMARY

[0004] The present application aims to at least solve one of the technical problems in the prior art, and provide a preparation method for improving the effect of antagonizing bacterial endotoxin of veterinary Shuanghuanglian oral liquid. The present application collects the volatile oil of honeysuckle and forsythia by water distillation method, improves the extraction rate of volatile oil, and avoids the waste of effective substances. The prepared volatile oil nanoemulsion increases the stability of volatile oil in Shuanghuanglian oral liquid, makes it uniformly distributed, and is beneficial to the absorption of livestock and poultry body.

[0005] The veterinary Shuanghuanglian oral liquid prepared by the present process is rich in volatile oil, has effective substances, and improves the effect of antagonizing endotoxin. The experiment shows that the effect of reducing bacterial endotoxin of Shuanghuanglian oral liquid prepared by the present application is better in vitro. In the in vivo experiment, the mental state and fecal forming effect of the chicken in the treatment group of Shuanghuanglian prepared by the present process are better, the liver bleeding is significantly reduced, and the inflammatory cell infiltration is obviously reduced.

[0006] The present application also provides a preparation method for improving the effect of antagonizing bacterial endotoxin of veterinary Shuanghuanglian oral liquid, which comprises the following steps: S1, weigh the prescription amount of honeysuckle and forsythia, crush and extract the volatile oil by water distillation method, the extraction time is 4-6 hours, and the volatile oil is collected for standby; S2, to the distilled honeysuckle and forsythia, add 8-12 times of drinking water, after heating and boiling, put in the prescribed amount of Huangqin, first extract 3-4 hours, the extract is transferred to the concentrator and concentrated under reduced pressure; again, add drinking water according to the mass ratio of 1:8-10, heat and boil, then keep warm for 1-2 hours, the extract is transferred to the concentrator and concentrated under reduced pressure, combine the two concentrated liquids, stand for 12-24 hours, take the supernatant and concentrate to a relative density ≥1.05; S3, prepare the surfactant by mixing Tween-80 and polyoxyethylene castor oil according to the mass ratio of 2-4:1, prepare the oil phase by mixing anhydrous ethanol and volatile oil according to the mass ratio of 4-6:1, mix the surfactant and the oil phase according to the mass ratio of 3-6:1, then add drinking water to the mixture according to the mass ratio of 1:10-20 and stir to prepare the volatile oil nanoemulsion; S4, mix the extract and the nanoemulsion uniformly to prepare the veterinary Shuanghuanglian oral liquid.

[0007] According to the preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid provided by the application, when the volatile oil is extracted by the water-separation distillation method, the distillation temperature is controlled at 95-100℃, and the condensate water temperature is ≤25℃.

[0008] According to the preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid provided by the application, when the extract is concentrated under reduced pressure, the vacuum degree is controlled at -0.06 to -0.08 MPa, and the concentration temperature is ≤60℃.

[0009] According to the preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid provided by the application, in the preparation process of the nanoemulsion, the stirring rate is 200-400 r / min, and the time for adding drinking water is controlled at 10-20 minutes.

[0010] According to the preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid provided by the application, 0.1%-0.5% mass fraction of sodium benzoate is added to the volatile oil nanoemulsion as a preservative.

[0011] According to the preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid provided by the application, the particle size of the honeysuckle and forsythia after being crushed is 20-40 mesh.

[0012] According to the preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid provided by the application, the environment temperature during the standing for 12-24 hours is 10-25℃.

[0013] According to the preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid provided by the application, the mixing uniformity is achieved by stirring at a stirring rate of 300-500 r / min for 15-30 minutes. Advantages

[0014] Compared with the prior art, the preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid of the present application collects the volatile oil of honeysuckle and forsythia by water separation distillation, improves the extraction rate of volatile oil, and avoids the waste of effective substances.

[0015] Compared with the prior art, the preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid of the present application increases the stability of volatile oil nanoemulsion in the Shuanghuanglian oral liquid, makes it uniformly distributed, and is beneficial to the absorption of livestock and poultry bodies.

[0016] Compared with the prior art, the preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid of the present application is rich in volatile oil, the effective substances are complete, the antagonistic effect of endotoxin is improved, the experiment shows that the Shuanghuanglian oral liquid prepared by the present application has better effect on reducing bacterial endotoxin in vitro, in the in vivo experiment, the mental state of the chickens in the Shuanghuanglian treatment group is better, the feces forming effect is better, the liver bleeding is significantly reduced, and the inflammatory cell infiltration is obviously reduced. BRIEF DESCRIPTION OF DRAWINGS

[0017] The present application will be further described below in combination with the drawings and examples; Figure 1 The present application is a preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid; Figure 2 The present application is a comparison chart of the antagonistic effect of endotoxin of the Shuanghuanglian oral liquid in vivo; Figure 3 The present application is a process parameter table of Example 1 of the preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid; Figure 4 The present application is a process parameter table of Example 2 of the preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid; Figure 5 The present application is a process parameter table of Example 3 of the preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid; Figure 6 The present application is a comparison table of volatile oil stability of Test 1 of the preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid; Figure 7 The present application is a table of in vitro antagonistic effect of bacterial endotoxin of Test 2 of the preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid; Figure 8The test three endotoxin antagonism effect table of the preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid of the application. DETAILED DESCRIPTION

[0018] This part will describe the specific embodiments of the application in detail, the preferred embodiments of the application are shown in the drawings, the role of the drawings is to supplement the description of the text part of the description with figures, so that people can intuitively and visually understand each technical feature and the overall technical scheme of the application, but it cannot be understood as a limitation on the protection scope of the application.

[0019] Example 1 Reference Figure 1 , 3 The preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid of the embodiment of the application comprises: The prescribed amount of honeysuckle and forsythia are weighed, crushed, and the water distillation method is used to extract volatile oil, and the extraction time is 4 hours, and the volatile oil is collected for standby.

[0020] The prescribed amount of honeysuckle and forsythia are weighed, crushed, and the water distillation method is used to extract volatile oil, and the extraction time is 4 hours, and the volatile oil is collected for standby.

[0021] The preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid of the embodiment of the application comprises:

[0022] The preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid of the embodiment of the application comprises:

[0023] Example 2 Reference Figure 1 , 4 The preparation method for improving the antagonistic effect of bacterial endotoxin of the veterinary Shuanghuanglian oral liquid of the embodiment of the application comprises: The prescribed amount of honeysuckle and forsythia are weighed, crushed, and the water distillation method is used to extract volatile oil, and the extraction time is 4 hours, and the volatile oil is collected for standby.

[0024] The distilled honeysuckle and forsythia are added with 12 times of drinking water according to the prescription, and after being heated and boiled, the prescription amount of scutellaria is added, and the first extraction is carried out for 4 hours, and the extraction liquid is transferred into a concentrator for reduced pressure concentration; then, according to the mass ratio of 1:10, drinking water is added, heated and boiled, and then kept for 2 hours, and the extraction liquid is again transferred into a concentrator for reduced pressure concentration. The two concentrated liquids are combined, and after being kept for 24 hours, the supernatant is concentrated to a relative density of greater than or equal to 1.05 for standby.

[0025] The double-huanglian volatile oil nanoemulsion is prepared as follows: according to the mass ratio of 4:1, Tween-80 and polyoxyethylene castor oil are weighed and uniformly mixed to prepare a surfactant; according to the mass ratio of 6:1, anhydrous ethanol and double-huanglian volatile oil are weighed and uniformly mixed to prepare a double-huanglian volatile oil oil phase; according to the mass ratio of 6:1, the surfactant and the double-huanglian volatile oil oil phase are weighed and uniformly mixed; and according to the mass ratio of 1:20, the mixture of the surfactant and the double-huanglian volatile oil and drinking water are weighed, and the drinking water is gradually added to the mixture while being continuously stirred and mixed uniformly.

[0026] The double-huanglian extraction liquid and the double-huanglian volatile oil nanoemulsion are uniformly mixed to obtain the double-huanglian oral liquid.

[0027] Example Three With reference to Figure 1 , 5 , the application provides a preparation method for improving the antibacterial endotoxin effect of a double-huanglian oral liquid for veterinary use, which comprises the following steps: The prescription amount of honeysuckle and forsythia is weighed, crushed, and the volatile oil is extracted by a water distillation method, and the extraction time is 5 hours, and the volatile oil is collected for standby.

[0028] The distilled honeysuckle and forsythia are added with 10 times of drinking water according to the prescription, and after being heated and boiled, the prescription amount of scutellaria is added, and the first extraction is carried out for 3.5 hours, and the extraction liquid is transferred into a concentrator for reduced pressure concentration; then, according to the mass ratio of 1:9, drinking water is added, heated and boiled, and then kept for 1.5 hours, and the extraction liquid is again transferred into a concentrator for reduced pressure concentration. The two concentrated liquids are combined, and after being kept for 18 hours, the supernatant is concentrated to a relative density of greater than or equal to 1.05 for standby.

[0029] The double-huanglian volatile oil nanoemulsion is prepared as follows: according to the mass ratio of 3:1, Tween-80 and polyoxyethylene castor oil are weighed and uniformly mixed to prepare a surfactant; according to the mass ratio of 5:1, anhydrous ethanol and double-huanglian volatile oil are weighed and uniformly mixed to prepare a double-huanglian volatile oil oil phase; according to the mass ratio of 4:1, the surfactant and the double-huanglian volatile oil oil phase are weighed and uniformly mixed; and according to the mass ratio of 1:15, the mixture of the surfactant and the double-huanglian volatile oil and drinking water are weighed, and the drinking water is gradually added to the mixture while being continuously stirred and mixed uniformly.

[0030] The double-huanglian extraction liquid and the double-huanglian volatile oil nanoemulsion are uniformly mixed to obtain the double-huanglian oral liquid.

[0031] Test 1, comparison of volatile oil solubility and stability, refer to Figure 6 The embodiment of the present application is a preparation method for improving the effect of antagonizing bacterial endotoxin of the veterinary Shuanghuanglian oral liquid: The volatile oil-rich Shuanghuanglian oral liquid is prepared according to the conventional volatile oil adding method and the method of the present application, and is observed after standing. The results show that the volatile oil-rich Shuanghuanglian oral liquid prepared by the preparation process of the present application has uniform and stable distribution of volatile oil in the oral liquid, which is beneficial to the absorption of the livestock and poultry body.

[0032] Test 2, comparison of the effect of antagonizing bacterial endotoxin in vitro, refer to Figure 7 The embodiment of the present application is a preparation method for improving the effect of antagonizing bacterial endotoxin of the veterinary Shuanghuanglian oral liquid: The Shuanghuanglian oral liquid prepared by the present application and the Shuanghuanglian oral liquid prepared by the conventional process are diluted 100 times with endotoxin-free water respectively, and after dilution, they are mixed uniformly with 1 EU unit of endotoxin respectively, and reacted at 37℃ for 1 hour. The content of endotoxin in the solution is determined according to the endotoxin detection limulus reagent kit (test tube quantitative color developing matrix method). The results show that the Shuanghuanglian oral liquid prepared by the present application has better effect of reducing bacterial endotoxin in vitro.

[0033] Test 3, antagonizing effect on endotoxin in vivo, refer to Figure 2 , 8 The embodiment of the present application is a preparation method for improving the effect of antagonizing bacterial endotoxin of the veterinary Shuanghuanglian oral liquid: The Shuanghuanglian oral liquid prepared by the present application and the Shuanghuanglian oral liquid prepared by the conventional process are taken for the following test: 40 broiler chickens of Arbor Acres are raised in an environment of 35℃ at the age of 1-7 days, and then the temperature is reduced to 24℃ at the speed of 2℃ per week, and the temperature remains unchanged thereafter. The broiler chickens are adaptively raised for 2 weeks, and are randomly divided into 4 groups, which are blank control group, endotoxin challenge group, endotoxin challenge, the present process Shuanghuanglian treatment group and endotoxin challenge group, and conventional process Shuanghuanglian treatment group. From the age of 19 days to the age of 21 days, different treatment groups are as follows: the test groups are respectively given the present process Shuanghuanglian and the conventional process Shuanghuanglian by gavage at the dose of 0.5 mL / branch, and the blank control group and the model control group are given physiological saline by gavage. Once a day for 3 consecutive days. Two hours after the last administration, the blank control group is injected with 0.5 mL / kg of physiological saline by intravenous injection under the wing, and the other three groups are injected with 0.5 mL / kg of LPS physiological saline diluent with a concentration of 2 mg / mL by intravenous injection under the wing. The clinical symptoms of the chickens are observed 6 hours after the challenge, the serum endotoxin is determined, and the lung is taken to prepare paraffin sections to observe the pathological changes.

[0034] The results show that the endotoxin challenge group of chickens has symptoms of fluffing, bunching and fever, and the feces are thin and not shaped. The conventional process Shuanghuanglian and the process Shuanghuanglian of the present application treatment group are better than the above-mentioned symptoms of the challenge group, and the mental state and the feces forming effect of the process Shuanghuanglian of the present application treatment group are better. The liver of the endotoxin challenge group of chickens has obvious bleeding points, and the HE paraffin section shows that the challenge group has liver cell damage and inflammatory cell infiltration, and the conventional process Shuanghuanglian and the process Shuanghuanglian of the present application treatment group are better than the challenge group, especially the process Shuanghuanglian treatment group, the liver bleeding is significantly reduced, and the inflammatory cell infiltration is significantly reduced.

[0035] The embodiments of the present application are described in detail above in combination with the drawings, but the present application is not limited to the above-mentioned embodiments, and various changes can be made within the knowledge range of ordinary skilled persons in the art without departing from the purpose of the present application.

Claims

1. A preparation method for improving the antagonistic effect of Shuanghuanglian oral liquid for animals on bacterial endotoxins, characterized in that: The following steps are involved: S1. Weigh the prescribed amount of honeysuckle and forsythia, crush them, and extract the volatile oil by hydrodistillation for 4 to 6 hours. Collect the volatile oil for later use; S2. Add 8-12 times the amount of drinking water to the distilled honeysuckle and forsythia, heat to boiling, add the prescribed amount of scutellaria, extract for 3-4 hours for the first time, transfer the extract to a concentrator and concentrate under reduced pressure; then add drinking water at a mass ratio of 1:8-10, heat to boiling, and keep warm for 1-2 hours, transfer the extract to a concentrator and concentrate under reduced pressure, combine the two concentrates, let stand for 12-24 hours, and take the supernatant and concentrate to a relative density ≥1.05; S3, mixing Tween-80 and polyoxyethylene castor oil in a mass ratio of 2-4:1 to prepare a surfactant, mixing anhydrous ethanol and volatile oil in a mass ratio of 4-6:1 to prepare an oil phase, mixing the surfactant and the oil phase in a mass ratio of 3-6:1, and then adding drinking water dropwise to the mixture in a mass ratio of 1:10-20 and stirring to prepare a volatile oil nanoemulsion; S4. Evenly mix the extract and the nanoemulsion to prepare Shuanghuanglian oral liquid for animals.

2. A method for preparing Shuanghuanglian oral liquid for animals for improving its antagonistic effect on bacterial endotoxins according to claim 1, characterized in that: When extracting volatile oil by the water-removing distillation method, the distillation temperature is controlled at 95-100° C. and the condensed water temperature is ≤25° C.

3. The method for preparing Shuanghuanglian oral liquid for animals for improving its antagonistic effect on bacterial endotoxins according to claim 1, characterized in that: When the extract is concentrated under reduced pressure, the vacuum degree is controlled at -0.06 to -0.08 MPa, and the concentration temperature is ≤60°C.

4. The method for preparing Shuanghuanglian oral liquid for animals for improving its antagonistic effect on bacterial endotoxins according to claim 1, characterized in that: During the nanoemulsion preparation process, the stirring rate is 200-400 r / min, and the time for adding drinking water dropwise is controlled within 10-20 minutes.

5. The method for preparing Shuanghuanglian oral liquid for animals for improving its antagonistic effect on bacterial endotoxins according to claim 1, characterized in that: 0.1% to 0.5% by mass of sodium benzoate is added to the volatile oil nanoemulsion as a preservative.

6. A preparation method for improving the antagonistic effect of Shuanghuanglian oral liquid for animals on bacterial endotoxins according to claim 1, characterized in that: The particle size of the honeysuckle and forsythia after being crushed is 20-40 meshes.

7. The method for preparing Shuanghuanglian oral liquid for animals for improving its antagonistic effect on bacterial endotoxins according to claim 1, characterized in that: The ambient temperature during the 12 to 24 hour standing period is 10-25°C.

8. The method for preparing Shuanghuanglian oral liquid for animals for improving its antagonistic effect on bacterial endotoxins according to claim 1, characterized in that: The uniform mixing method is to stir at a stirring rate of 300-500 r / min for 15 to 30 minutes.

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