CN117379491A - Traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome and preparation method thereof - Google Patents
Traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome and preparation method thereof Download PDFInfo
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- CN117379491A CN117379491A CN202311537477.9A CN202311537477A CN117379491A CN 117379491 A CN117379491 A CN 117379491A CN 202311537477 A CN202311537477 A CN 202311537477A CN 117379491 A CN117379491 A CN 117379491A
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- traditional chinese
- chinese medicine
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- oral liquid
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Classifications
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- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
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Abstract
The invention belongs to the technical field of traditional Chinese medicine preparations, and in particular relates to a traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome and a preparation method thereof. Crushing rheum officinale, coptis chinensis, mint, radix bupleuri and schizonepeta, performing diacolation extraction on the crushed rheum officinale, coptis chinensis, mint, radix bupleuri and schizonepeta to obtain an extract A, performing water extraction on the scutellaria baicalensis, burdock, dried orange peel, fructus forsythiae, rhizoma cimicifugae, platycodon grandiflorum, liquorice, puffball, radix scrophulariae, radix isatidis, indigo naturalis, talcum powder and diacolation extraction residues, performing reflux extraction on the extract A and the extract B to obtain an extract B, performing secondary alcohol precipitation on the extract B, adding N-methylpyrrolidone, the extract A and the distillate, adding sodium benzoate and water, and adjusting the pH value. The traditional Chinese medicine oral liquid for preventing and controlling the porcine reproductive and respiratory syndrome has high content of active ingredients and high stability, and can effectively resist PRRSV virus and treat the porcine reproductive and respiratory syndrome.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine preparations, and in particular relates to a traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome and a preparation method thereof.
Background
The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection of pigs causes a high-contact infectious disease, and pigs with each day of age can be infected, and the disease incidence rate can reach 100 percent by taking the sow reproductive capacity reduction, piglet hyperpyrexia, dyspnea, acute death and the like as main characteristics. Once the pig is infected, immunosuppression can be caused to further cause other diseases, and the development and social and economic benefits of pig breeding are greatly influenced.
The pulvis is a traditional Chinese medicine powder recorded in the pharmacopoeia of the people's republic of China (the second edition of 2020), has the effects of clearing away heat and toxic materials, dispelling wind and reducing swelling, and is clinically used for preventing and treating the upward invasion of the heat toxin of livestock and poultry. In the prescription, the baikal skullcap root and the coptis root are used as monarch drugs for clearing upper-jiao heat toxin; fructus Arctii, fructus forsythiae, herba Menthae, herba Schizonepetae, bupleuri radix and cimicifugae rhizoma, which are ministerial drugs, can dispel wind and eliminate pathogenic factors; puffball, radix isatidis and indigo naturalis have the effects of detoxifying, detumescence, clearing and relieving sore throat, radix scrophulariae has the effects of nourishing yin, reducing pathogenic fire, dried orange peel has the effects of regulating qi and resolving hard mass, and talcum and rheum officinale conduct heat downwards and serve as adjuvant drugs; radix Platycodi has effects of dispersing lung qi, eliminating pathogenic factors, guiding medicine upwards, and Glycyrrhrizae radix regulating various medicines. The medicines are combined together to play the roles of clearing heat and detoxicating, dispelling wind and detumescence. The traditional Chinese medicine composition has definite curative effects on repeated fever and high fever without degeneration caused by various viruses and bacteria of livestock and poultry, and has good prevention and treatment effects on the currently popular fever type virus diseases such as swine fever, blue ear disease and the like. However, in the intensive culture mode, the traditional Chinese medicine powder has the defects of uneven material mixing, difficult quality control and the like, and cannot meet the market demand.
The disclosed preparation method of the patent CN108125912A general disinfection granule comprises the steps of extracting different active ingredients by adopting different treatment methods, dissolving the extract in ethanol, adding starch and sucrose, mixing, granulating, and precipitating impurities after dissolving in ethanol because no purification is carried out after extraction. The disclosed patent CN108079116A Puji disinfection oral liquid is prepared by preparing different extracts of coptis chinensis, radix scutellariae, dried orange peel, fructus forsythiae and the like, mixing and dissolving in purified water to prepare the oral liquid. Because coptis contains berberine, baikal skullcap root contains baicalin, and the extraction of the coptis root and the baikal skullcap root together easily causes precipitation in chemical reaction to influence the content, and simultaneously, the medicinal materials such as peppermint, schizonepeta herb and the like contain a large amount of volatile oil, and the volatile oil loss is more during the water decoction extraction, thereby influencing the efficacy of dispelling wind and reducing swelling. The disclosed patent CN106361987A is a traditional Chinese medicine composition for treating porcine reproductive and respiratory syndrome, which is prepared by adding water into papermulberry fruit, licorice root of large tree, radix scutellariae thorn and guanyin bamboo, mixing and decocting, concentrating and filtering after alcohol precipitation, and can remove most of impurities, but no preservative is added, so that the stability of the liquid medicine is easily affected. The disclosed patent CN104069478A is a traditional Chinese medicine composition for preventing and treating porcine reproductive and respiratory syndrome and a preparation method thereof, wherein the traditional Chinese medicine composition is prepared by mixing and soaking and extracting medicinal materials such as radix isatidis, folium isatidis, radix puerariae and the like by using an ethanol aqueous solution, filtering and concentrating the medicinal materials to prepare a finished product, and the extraction and preparation processes are safe, but the process is characterized in that the medicinal materials are soaked by using the ethanol aqueous solution only, the extraction rate is low, no sterilization and preservative addition are caused, and the stability of the medicinal liquid is easily influenced.
In summary, the technical problems of low purity, unstable content and quality, no volatile oil, poor efficacy and the like generally exist in the prior art.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome and a preparation method thereof. The traditional Chinese medicine oral liquid for preventing and controlling the porcine reproductive and respiratory syndrome has high content of active ingredients and high stability, and can effectively resist PRRSV virus and treat the porcine reproductive and respiratory syndrome.
In order to achieve the above object, the technical scheme of the present invention is as follows:
a preparation method of a traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome comprises the following steps:
s1, mixing rheum officinale, coptis chinensis, mint, radix bupleuri and schizonepeta, adding an ethanol aqueous solution, performing diacolation extraction, and concentrating under reduced pressure to obtain an extract A and a medicinal residue A;
s2, mixing the baical skullcap root, the great burdock achene, the tangerine peel, the weeping forsythiae capsule, the largetrifoliolious bugbane rhizome, the platycodon root, the liquoric root, the puffball, the figwort root, the Indigowoad root, the natural indigo, the talcum powder and the medicine slag A prepared in the step S1, adding water for heating reflux extraction, collecting distilled liquid, filtering the residual medicine liquid to prepare filtrate I and medicine slag B, wherein the filtrate I is extract B;
s3, carrying out alcohol precipitation treatment on the extract B prepared in the step S2 by adopting alcohol, separating, and concentrating the supernatant liquor under reduced pressure to prepare an extract C;
and S4, adding a stabilizer into the extract A prepared in the step S1, stirring uniformly, heating, then adding the extract C prepared in the step S3, stirring uniformly, cooling, adding the distillate prepared in the step S2, stirring uniformly, adding purified water and sodium benzoate, stirring for dissolution, adjusting the pH value, and filtering to obtain the product.
In the research and development process, the applicant finds that the hydrochloride of berberine in the coptis chinensis medicinal material has lower solubility in water and is easy to combine with baicalin in the baical skullcap root medicinal material to form insoluble salt, so that the content of main components and the clarity of medicinal liquid are influenced.
The medicinal materials such as peppermint, bupleurum, schizonepeta and the like contain a large amount of fat-soluble volatile oil components, wherein the volatile oil is a main substance for playing the effects of dispelling wind and reducing swelling.
Further, the preparation method of the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome comprises the steps of mixing and crushing rheum officinale, coptis chinensis, mint, radix bupleuri and schizonepeta in the step S1; the mass percentage of the ethanol water solution is 60-70%; concentrating under reduced pressure to relative density of 1.06-1.10g/ml.
Further, the preparation method of the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome comprises the following steps of: mixing radix et rhizoma Rhei, coptidis rhizoma, herba Menthae, bupleuri radix, and herba Schizonepetae, pulverizing, sieving, wetting with appropriate amount of ethanol water solution, percolating, soaking in 20 times of ethanol water solution for 3 hr, soaking for 24 hr, and concentrating under reduced pressure to obtain extract A and residue A.
Further, the preparation method of the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome further comprises the step S2 of decocting the medicine residue B, filtering, collecting filtrate II, combining filtrate I and filtrate II, and concentrating to obtain extract B.
Further, in the preparation method of the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome, in the step S2, filtering is carried out, sieving with a 200-mesh sieve, heating, refluxing and extracting for 2h, and decocting for 2h; concentrating to relative density of 1.08-1.10g/ml.
Further, in the preparation method of the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome, in the step S3, ethanol is added into a system, and the alcohol content is 75%.
Further, the alcohol precipitation treatment in the step S3 specifically includes: adding ethanol into the extract B prepared in the step S2 under stirring until the ethanol content in the system is 75%, and standing at normal temperature for 12h to obtain supernatant liquid I and precipitate I; adding 75% ethanol water solution into the precipitate I under stirring, and standing at normal temperature for 12h to obtain supernatant liquid II and precipitate II; mixing the supernatant liquid I and the supernatant liquid II, concentrating under reduced pressure to relative density of 1.06-1.10g/ml, and making into extract C. The general disinfection powder has the advantages of multiple formula components, complex ingredients and multiple impurities, and the secondary alcohol precipitation can obviously reduce the impurities in the liquid medicine and improve the content of main components.
Further, in the preparation method of the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome, the stabilizer in the step S4 is N-methylpyrrolidone. N-methyl pyrrolidone is a polar aprotic solvent, and in the formula of the invention, not only can the solubility of the main components of the traditional Chinese medicine be effectively improved, but also the stability of the traditional Chinese medicine oral liquid can be improved.
Further, the heating temperature in the step S4 is 78-82 ℃ and the cooling temperature is 37-45 ℃ in the preparation method of the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome; the pH value is regulated to 4.5-5.0.
Further, the weight dosage of each traditional Chinese medicine component in the preparation method of the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome is as follows: 1-3 parts of rheum officinale, 1-2 parts of radix scutellariae, 1-2 parts of coptis chinensis, 1 part of liquorice, 1-2 parts of puffball, 1-2 parts of mint, 1-2 parts of radix scrophulariae, 3 parts of burdock, 1-2 parts of cimicifuga foetida, 1-2 parts of radix bupleuri, 1-2 parts of platycodon grandiflorum, 1-2 parts of dried orange peel, 2 parts of fructus forsythiae, 1-2 parts of schizonepeta, 2 parts of radix isatidis, 1-2 parts of indigo naturalis, 5-6 parts of talcum powder, 0.1-0.5 part of stabilizer, 20-40 parts of purified water and 0.1-0.5 part of sodium benzoate.
The invention also provides the traditional Chinese medicine oral liquid for preventing and controlling the porcine reproductive and respiratory syndrome, which is prepared by the preparation method of the traditional Chinese medicine oral liquid for preventing and controlling the porcine reproductive and respiratory syndrome.
Compared with the prior art, the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome and the preparation method thereof have the following technical advantages:
(1) The invention is based on production practice, combines the traditional Chinese medicine theory and the modern pharmaceutical technology, develops a new product of the traditional Chinese medicine oral liquid which can be administered by drinking water, verifies the prevention and treatment effect of the product on the blue-ear disease by in-vivo and in-vitro research experiments, and provides technical support for convenient clinical medication;
(2) The invention adopts the percolation method of dynamic leaching to extract the traditional Chinese medicine raw materials, so that the solvent utilization rate is high, the leaching of the effective components is complete, and the leaching liquid can be directly collected;
(3) The traditional Chinese medicine oral liquid for preventing and controlling the porcine reproductive and respiratory syndrome provided by the invention contains the traditional Chinese medicine volatile oil, and has better curative effect on the highly pathogenic porcine reproductive and respiratory syndrome.
Drawings
FIG. 1 is a graph comparing clinical symptom scores of animals;
FIG. 2 is a graph showing comparison of weight gain of animals;
FIG. 3 shows cytotoxicity test results;
fig. 4 is an indirect immunofluorescence assay: the Chinese medicinal oral liquid has the effect of inhibiting PRRSV of different genotypes for 48 hours;
fig. 5 is an indirect immunofluorescence assay: the Chinese medicinal oral liquid has the effect of inhibiting PRRSV for 48 hours;
FIG. 6 shows the results of a virus adsorption test;
FIG. 7 is a graph showing the results of a virus penetration test;
FIG. 8 shows the results of a viral replication assay;
note that: in fig. 2, P < 0.05 indicates significant differences compared to the positive control group, and P < 0.01 indicates extremely significant differences.
Detailed Description
The present invention is further described below by way of specific embodiments, but the present invention is not limited to the following examples. Various modifications may be made by those skilled in the art in light of the basic idea of the invention, but are within the scope of the invention without departing from the basic idea of the invention.
Example 1 preparation method of Chinese medicinal oral liquid for preventing and controlling porcine reproductive and respiratory syndrome
A preparation method of a traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome comprises the following steps:
s1, mixing rheum officinale, coptis chinensis, mint, radix bupleuri and schizonepeta, crushing, sieving with a 40-mesh sieve, adding a proper amount of 65% ethanol water solution by mass percent for wetting, then filling into a percolating device, then adding 20 times of 65% ethanol water solution by mass percent for soaking for 3 hours, then soaking for 24 hours, and concentrating under reduced pressure until the relative density is 1.08g/ml to obtain an extract A and dregs A;
s2, adding 10 times of water into the baical skullcap root, the great burdock achene, the tangerine peel, the weeping forsythiae capsule, the largetrifoliolious bugbane rhizome, the platycodon root, the liquoric root, the puffball, the figwort root, the Indigowoad root, the natural indigo, the talcum powder and the dregs A prepared in the step S1, heating and reflux extracting for 2 hours, collecting distilled liquid, and sieving the residual liquid medicine with a 200-mesh sieve to prepare filtrate I and dregs B; decocting the residue B with 7 times of water for 2 hr, sieving with 200 mesh sieve, and collecting filtrate II; mixing filtrate I and filtrate II, and concentrating to relative density of 1.09g/ml to obtain extract B;
s3, adding ethanol into the extract B prepared in the step S2 under the stirring state until the alcohol content in the system is 75%, and standing for 12 hours at normal temperature to obtain a supernatant liquid I and a precipitate I; adding 75% ethanol water solution into the precipitate I under stirring, and standing at normal temperature for 12h to obtain supernatant liquid II and precipitate II; mixing the supernatant liquid I and the supernatant liquid II, concentrating under reduced pressure to relative density of 1.09g/ml to obtain extract C;
and S4, adding N-methylpyrrolidone into the extract A prepared in the step S1, uniformly stirring, heating to 80 ℃, then adding the extract C prepared in the step S3, uniformly stirring, cooling to 40 ℃, adding the distilled liquid prepared in the step S2, uniformly stirring, adding purified water and sodium benzoate, stirring for dissolving, adjusting the pH value to 4.7, and then filtering to obtain the product.
The preparation method of the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome comprises the following steps of: 2 parts of rheum officinale, 1.5 parts of scutellaria baicalensis, 1.5 parts of coptis chinensis, 1 part of liquorice, 1.5 parts of puffball, 1.5 parts of mint, 1.5 parts of radix scrophulariae, 3 parts of burdock, 1.5 parts of cimicifuga foetida, 1.5 parts of radix bupleuri, 1.5 parts of platycodon grandiflorum, 1.5 parts of dried orange peel, 2 parts of fructus forsythiae, 1.5 parts of schizonepeta, 2 parts of radix isatidis, 1.5 parts of indigo naturalis, 5.5 parts of talcum, 0.5 part of N-methylpyrrolidone, 33 parts of purified water and 0.1 part of sodium benzoate.
Example 2 preparation method of Chinese medicinal oral liquid for preventing and controlling porcine reproductive and respiratory syndrome
A preparation method of a traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome comprises the following steps:
s1, mixing rheum officinale, coptis chinensis, mint, radix bupleuri and schizonepeta, crushing, sieving with a 40-mesh sieve, adding a proper amount of 60% ethanol water solution by mass percent for wetting, then filling into a percolating device, then adding 20 times of 60% ethanol water solution by mass percent for soaking for 3 hours, then soaking for 24 hours, and concentrating under reduced pressure until the relative density is 1.06g/ml to obtain an extract A and dregs A;
s2, adding 10 times of water into the baical skullcap root, the great burdock achene, the tangerine peel, the weeping forsythiae capsule, the largetrifoliolious bugbane rhizome, the platycodon root, the liquoric root, the puffball, the figwort root, the Indigowoad root, the natural indigo, the talcum powder and the dregs A prepared in the step S1, heating and reflux extracting for 2 hours, collecting distilled liquid, and sieving the residual liquid medicine with a 200-mesh sieve to prepare filtrate I and dregs B; decocting the residue B with 7 times of water for 2 hr, sieving with 200 mesh sieve, and collecting filtrate II; mixing filtrate I and filtrate II, and concentrating to relative density of 1.08g/ml to obtain extract B;
s3, adding ethanol into the extract B prepared in the step S2 under the stirring state until the alcohol content in the system is 75%, and standing for 12 hours at normal temperature to obtain a supernatant liquid I and a precipitate I; adding 70% ethanol water solution into the precipitate I under stirring, and standing at normal temperature for 12h to obtain supernatant liquid II and precipitate II; mixing the supernatant liquid I and the supernatant liquid II, concentrating under reduced pressure to relative density of 1.06g/ml to obtain extract C;
and S4, adding N-methylpyrrolidone into the extract A prepared in the step S1, uniformly stirring, heating to 80 ℃, then adding the extract C prepared in the step S3, uniformly stirring, cooling to 40 ℃, adding the distilled liquid prepared in the step S2, uniformly stirring, adding purified water and sodium benzoate, stirring for dissolving, adjusting the pH value to 4.5, and then filtering to obtain the product.
The preparation method of the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome comprises the following steps of: 3 parts of rheum officinale, 2 parts of scutellaria baicalensis, 2 parts of coptis chinensis, 1 part of liquorice, 2 parts of puffball, 2 parts of mint, 2 parts of radix scrophulariae, 3 parts of burdock, 2 parts of cimicifugae foetidae, 2 parts of radix bupleuri, 2 parts of platycodon grandiflorum, 2 parts of dried orange peel, 2 parts of fructus forsythiae, 2 parts of schizonepeta, 2 parts of radix isatidis, 2 parts of indigo naturalis, 6 parts of talcum, 0.3 part of N-methylpyrrolidone, 40 parts of purified water and 0.4 part of sodium benzoate.
Example 3 preparation method of Chinese medicinal oral liquid for preventing and controlling porcine reproductive and respiratory syndrome
A preparation method of a traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome comprises the following steps:
s1, mixing rheum officinale, coptis chinensis, mint, radix bupleuri and schizonepeta, crushing, sieving with a 40-mesh sieve, adding a proper amount of 70% ethanol water solution by mass percent for wetting, then filling into a percolating device, then adding 20 times of 70% ethanol water solution by mass percent for soaking for 3 hours, then soaking for 24 hours, and concentrating under reduced pressure until the relative density is 1.10g/ml to obtain an extract A and dregs A;
s2, adding 10 times of water into the baical skullcap root, the great burdock achene, the tangerine peel, the weeping forsythiae capsule, the largetrifoliolious bugbane rhizome, the platycodon root, the liquoric root, the puffball, the figwort root, the Indigowoad root, the natural indigo, the talcum powder and the dregs A prepared in the step S1, heating and reflux extracting for 2 hours, collecting distilled liquid, and sieving the residual liquid medicine with a 200-mesh sieve to prepare filtrate I and dregs B; decocting the residue B with 7 times of water for 2 hr, sieving with 200 mesh sieve, and collecting filtrate II; mixing filtrate I and filtrate II, and concentrating to relative density of 1.10g/ml to obtain extract B;
s3, adding ethanol into the extract B prepared in the step S2 under the stirring state until the alcohol content in the system is 75%, and standing for 12 hours at normal temperature to obtain a supernatant liquid I and a precipitate I; adding 60% ethanol water solution into the precipitate I under stirring, and standing at normal temperature for 12h to obtain supernatant liquid II and precipitate II; mixing the supernatant liquid I and the supernatant liquid II, concentrating under reduced pressure to relative density of 1.10g/ml to obtain extract C;
and S4, adding N-methylpyrrolidone into the extract A prepared in the step S1, uniformly stirring, heating to 80 ℃, then adding the extract C prepared in the step S3, uniformly stirring, cooling to 40 ℃, adding the distilled liquid prepared in the step S2, uniformly stirring, adding purified water and sodium benzoate, stirring for dissolution, adjusting the pH value to 5.0, and then filtering to obtain the product.
The preparation method of the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome comprises the following steps of: 1 part of rheum officinale, 1 part of scutellaria baicalensis, 1 part of coptis chinensis, 1 part of liquorice, 1 part of puffball, 1 part of mint, 1 part of radix scrophulariae, 3 parts of burdock, 1 part of cimicifugae foetidae, 1 part of radix bupleuri, 1 part of platycodon grandiflorum, 1 part of dried orange peel, 2 parts of fructus forsythiae, 1 part of schizonepeta, 2 parts of radix isatidis, 1 part of indigo naturalis, 5 parts of talcum, 0.1 part of N-methylpyrrolidone, 20 parts of purified water and 0.5 part of sodium benzoate.
Comparative example 1,
This comparative example is similar to example 2, and differs from example 2 in that: in the step S1 of the comparative example, water extraction is adopted, and the specific method is as follows: mixing radix et rhizoma Rhei, coptidis rhizoma, herba Menthae, bupleuri radix, and herba Schizonepetae, pulverizing, sieving with 40 mesh sieve, adding 10 times of deionized water, decocting for 12 hr, sieving with 200 mesh sieve, concentrating the filtrate under reduced pressure to relative density of 1.06g/ml, and making into extract A and residue A.
Comparative example 2,
This comparative example is similar to example 2, and differs from example 2 in that: in the step S3 of the comparative example, one-time alcohol precipitation is adopted, and the specific method is as follows: adding ethanol into the extract B prepared in the step S2 under stirring until the ethanol content in the system is 75%, and standing at normal temperature for 12h to obtain supernatant liquid I and precipitate I; adding deionized water into the precipitate I under stirring, and standing at normal temperature for 12h to obtain supernatant liquid II and precipitate II; mixing the supernatant liquid I and the supernatant liquid II, concentrating under reduced pressure to relative density of 1.06g/ml to obtain extract C.
Comparative example 3,
This comparative example is similar to example 2, and differs from example 2 in that: in this comparative example, in step S4, an equivalent amount of sodium dodecyl sulfate was used instead of N-methylpyrrolidone.
Comparative example 4,
This comparative example is similar to example 2, and differs from example 2 in that: in the step S4 of the comparative example, the distilled liquid obtained in the step S2 was replaced with the same amount of deionized water.
Test example I, comparative study of individual extraction and mixed extraction of Coptis chinensis and Scutellaria baicalensis
The test method comprises the following steps:
chromatographic conditions: chromatographic column: agilent HC-C18 (2) (4.6 mm. Times.250 mm,5 μm); mobile phase: methanol-0.1% phosphoric acid solution (50:50), flow rate 1.0 mL.min -1 The method comprises the steps of carrying out a first treatment on the surface of the Detection wavelength: 230nm; column temperature: 30 ℃.
Preparing a reference substance solution: accurately weighing baicalin reference 2.55mg and berberine hydrochloride reference 3.09mg, respectively placing into 10ml volumetric flask, and metering with methanol to scale for use.
Sample solution preparation: pulverizing Coptidis rhizoma and Scutellariae radix into coarse powder (about 40 mesh screen), mixing Coptidis rhizoma 2g and Scutellariae radix 2g as mixed sample, extracting Coptidis rhizoma 2g as Coptidis rhizoma sample, and extracting Scutellariae radix 2g as Scutellariae radix sample. The powder is added with 10 times of 50% ethanol respectively, and extracted for 2 times each for 1 hour. Centrifuging at 4000r/min for 5min after each extraction, taking supernatant, precisely measuring 1ml to 10ml volumetric flask, metering volume to scale mark with methanol, and filtering for use. Measuring under the above chromatographic conditions, and calculating the contents of baicalin and berberine hydrochloride in the sample by external standard method
The test results are shown in Table 1.
TABLE 1 results of comparing individual and mixed extractions of coptis chinensis and scutellaria baicalensis
As can be seen from Table 1, the contents of berberine hydrochloride and baicalin are greatly reduced when the coptis chinensis and the baicalin are extracted together, which shows that the method provided by the invention can be used for extracting the coptis chinensis and the baicalin separately and effectively improving the contents of Huang Qingan and berberine hydrochloride in the traditional Chinese medicine oral liquid.
Test example II, berberine hydrochloride and baicalin content detection
Test sample: oral liquids prepared in examples 1 to 3, comparative examples 1 to 2 and comparative example 4;
the test method comprises the following steps:
chromatographic conditions: zorbax Eclipse SB C18 (250 mm. Times.4.6 mm,5 μm); mobile phase: acetonitrile (A) -0.2% phosphoric acid (B) (adding triethylamine to adjust pH value=2.5), gradient eluting (0-5 min,12% A, 5-10 min,12% -20% A, 10-20 min,20% -27% A, 20-25 min,27% -31% A, 25-30 min,31% -35% A, flow rate 1.0mL/min, detection wavelength 275nm, column temperature 25 ℃ and sample injection amount 10 μl) under the above chromatographic condition.
Preparing a reference substance: precisely weighing reference baicalin 10.21mg, dissolving in methanol, and fixing volume to 10mL; 1.21mg of berberine hydrochloride is dissolved by adding methanol, the volume is fixed to 10mL, shaking is carried out, 1mL of berberine hydrochloride is taken and then is fixed to 10mL by using methanol, and the baicalin and berberine hydrochloride concentration is 0.1021 and 0.0121mg/mL of control stock solution respectively.
Sample solution preparation: taking 1ml of three batches of samples of the Puji disinfection oral liquid, precisely weighing into a 100ml measuring flask, dissolving with a proper amount of methanol, diluting to a scale, shaking uniformly, filtering, measuring according to chromatographic conditions, and calculating the contents of baicalin and berberine hydrochloride in the sample by an external standard method.
The test results are shown in Table 2.
TABLE 2 detection results of berberine hydrochloride and baicalin
As shown in Table 2, the content of berberine hydrochloride in the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome is 1.32-1.50mg/mL, and the content of baicalin is 4.96-5.82mg/mL, wherein the content of berberine hydrochloride and baicalin in the oral liquid prepared in the embodiment 2 is the highest, and the traditional Chinese medicine oral liquid is the best embodiment of the invention.
Compared with the example 2, the water extraction method is adopted in the step S1 in the comparative example 1, but the content of berberine hydrochloride in the prepared oral liquid is greatly reduced, which shows that the extraction rate of berberine hydrochloride in coptis can be improved by adopting percolation extraction; in the comparative example 2, in the step S3, one-step alcohol precipitation is adopted, but the content of baicalin in the prepared oral liquid is greatly reduced, which proves that the step S3 adopts the secondary alcohol precipitation in the invention, so that the content of baicalin can be effectively improved; the distilled liquid prepared in the step S2 is not added in the comparative example 4, but the content of baicalin in the prepared oral liquid is greatly reduced, which shows that the extraction rate of the fat-soluble components in the medicinal materials can be obviously improved by adopting the method for extracting volatile oil through percolation extraction and then heating reflux extraction.
Test example III, oral liquid stability detection
Test sample: oral liquid prepared in examples 1-3 and comparative example 3.
The test method comprises the following steps: the test samples are sterilized and sterilized, sub-packaged in a sealed brown container, placed at 40 ℃ for 6 months, sampled at 0, 1, 2, 3, 4, 5 and 6 months respectively, and analyzed for properties, relative density, pH, microorganism limit and baicalin content.
The test results are shown in Table 3.
TABLE 3 stability test results of oral liquid
* The liquid is brown to brownish black liquid, has slightly bitter and astringent taste, has a small amount of flocculent precipitate, and is dispersed by light shaking; # is brown to brownish black liquid, has slightly bitter and astringent taste, has a large amount of sediment, and is slightly shake-free; -indicating that the microbiological detection is in compliance with the regulations; + indicates that the detection of microorganisms is out of standard.
As shown in Table 3, after the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome is placed for 6 months, the appearance shape is a tan to brownish black liquid, the taste is slightly bitter and astringent, a small amount of flocculent precipitate exists, the liquid is dispersed after being gently shaken, no obvious precipitate exists, and the relative density, the pH value and the microorganism limit are all not obviously changed and meet the requirements.
Compared with the example 2, the comparative example 3 adopts equivalent sodium dodecyl sulfate to replace N-methylpyrrolidone, but the property of the prepared oral liquid changes in the 3 rd month, the relative density is lower, the pH value change range is larger, the microorganism test exceeds the standard along with the extension of the placing time, and the baicalin content is lower, which shows that the N-methylpyrrolidone is a polar aprotic solvent, and the solubility of the main components of the traditional Chinese medicine can be effectively improved, and the stability of the traditional Chinese medicine oral liquid can be improved.
Test example four, evaluation of efficacy against porcine reproductive and respiratory syndrome
Test sample: the oral liquid prepared in example 2 (volatile oil group) and comparative example 4 (oral liquid group) and the commercial praji disinfectant powder (powder group) (the praji disinfectant powder is purchased from animal health care products factory of biological technology limited company of Guangdong Wen Shida, and the medicinal material formula can be referred to the praji disinfectant powder of the two 2020 edition of Chinese beast pharmacopoeia) are prepared by pulverizing 17 medicinal materials in the formula, sieving and uniformly mixing;
the test method comprises the following steps: the 60 pigs are purchased and randomly divided into 5 groups of 12 pigs each; at the same time as the challenge was completed, pigs of each group were fed under the same test conditions following the dosing regimen described in table 4. During the trial, clinical symptoms of each group of animals were observed and recorded in detail within 28 days after challenge and scored. Weigh and record weight gain 1 day before the start of the test and at the end of the test.
Table 4 pig administration mode
The clinical symptom scores of the animals in each group after the test are shown in figure 1, and the weight gain of the animals in each group is shown in figure 2.
As can be seen from FIG. 1, the clinical symptom scores of the pigs in the example 2 group, the comparative example 4 group and the Puji Sansan group are obviously reduced compared with those of the pigs in the positive control group after administration, wherein the clinical symptom scores of the pigs in the example 2 group are reduced more rapidly compared with those of the comparative example 4 group and the Puji Sansan group.
As can be seen from fig. 2, the weight gain of pigs in example 2, comparative example 4 and the prazix disinfectant powder group was significantly increased (P > 0.01) compared with the positive control group after the experiment was completed, wherein the weight gain of pigs in example 2 was closest to the negative control group.
The results shown in fig. 1 and fig. 2 show that the volatile oil contained in the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome has better curative effect on highly pathogenic porcine reproductive and respiratory syndrome.
Test example five, in vitro anti-PRRSV test
1. Test protocol
1.1 test materials
DMEM culture solution, fetal calf serum, PBS, CCK8 detection kit, reverse transcription kit, PCR kit, protease inhibitor, BCA protein concentration determination kit, SDS-PAGE gel configuration kit, SDS-PAGE electrophoresis buffer solution, transfer membrane solution, blocking solution, antibody dilution, PRRSV N protein monoclonal antibody, horseradish peroxidase labeled goat anti-rabbit IgG, and the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome prepared in example 2.
1.2 Test cells and viruses
African green monkey kidney Marc-145 cells, PRRSV strain: CH-1a strain, VR-2332 strain, JXA1 strain, and TF strain.
1.3 test methods
1.3.1 Resuscitation of Marc-145 cells
Taking out 1ml Marc-145 cells from liquid nitrogen tank, resuscitating in 37 deg.C water bath, placing into cleaned biosafety cabinet after dissolving, adding 6ml 10%FBS DMEM,1ml Marc-145 cells into 25T cell bottle, blowing, mixing, observing under microscope, placing into 37 deg.C, 5% CO 2 Culturing in an incubator. And (5) carrying out subculture when the saturation of the cell growth reaches 80% -90%.
1.3.2 Passage and culture of Marc-145 cells
Observing under microscope, after the cells grow to compact state, passaging Marc-145 cells, adding 1ml trypsin into 25T cell bottle to digest Marc-145 cells, washing twice, leaving 0.5ml for the third time for digestion, placing into 37 ℃ and 5% CO 2 1 mm in an incubator is digested, when the cell suspension monolayer cells are passaged, the adherent cells are visually detached into a quicksand shape, 1/3 of the cells are passaged, 3ml of DMEM (DMEM with 10% FBS) is added to terminate the digestion, and the cells are blown uniformly. Adding 17ml of DMEM containing 10% FBS into 75T cell bottle, transferring 25T 3ml into 75T cell bottle to ensure 1/3 ratio passage, observing under microscope, and placing into 37 deg.C and 5% CO 2 In an incubator.
1.3.3 Cell inoculation and culture
Taking a bottle of Marc-145 cells which grow to a compact state, regulating the concentration of the cells to 7.5x104 cells/ml by using 10% FBS culture medium, and inoculating the cells into cell culture plates of different specifications according to specific test requirements, wherein a 96-well plate is 100ul per well; a 24-well plate was used at 1ml per well; the 6-well plate was 2ml per well. For 37℃C, 5% CO 2 Culturing in an incubator for 24 hours, and performing subsequent experiments.
1.3.4 TCID50 assay for PRRSV strains
Marc-145 cells were inoculated in 96-well plates and cultured for 24h by adherenceEach well was charged with 100ul of PRRSV (CH-1 a, VR-2332, JXA1, TF) virus stock at a 10-fold dilution, for a total of 7 dilutions, 8 sub-wells. Normal cell controls were also set. At 37℃with 5% CO 2 Culturing in incubator, adding 100ul of DMEM culture solution containing 2% FBS per well, and culturing at 37deg.C and 5% CO 2 Incubate in incubator for 72h. Cytopathic effects were observed daily and TCID50 was calculated for each strain according to the Reed-Muench method.
1.3.5 Determination of Marc-145 cytotoxicity of oral liquid
Marc-145 cells were inoculated in 96-well plates, after 24h of adherent culture, 100ul of 2-fold serial dilutions of praji disinfection oral liquid diluent prepared from DMEM culture solution and 8 multiple wells were added to each well. At the same time, a cell control and a blank control were set. Placing at 37deg.C and 5% CO 2 After 48h of co-incubation in the incubator, cytotoxicity assays were performed using CCK8 kit and the maximum safe concentration of drug to cells was calculated.
1.3.6 Indirect immunofluorescence assay
Marc-145 cells were inoculated into 48-well plates, the cell concentration was adjusted to 7.5X104 cells/ml with 10% FBS medium, after 24 hours, cells were inoculated with 100 TCID50 PRRSV (CH-1 a strain, VR-2332 strain, JXA1 strain, TF strain), 250. Mu.L/well, and the cells were cultured at 37℃with 5% CO 2 After 2h incubation in incubator, the oral liquid prepared in example 2 was diluted 2-fold with DMEM medium containing 2% FBS, added to the cell plate at 1:1 equivalent volume with the virus solution, and incubated at 37deg.C with 5% CO 2 After 48h of culture in an incubator, an indirect immunofluorescence test was performed.
1.3.7 Killing effect of oral liquid on PRRSV
The maximum safe concentration of Marc-145 cells is the highest drug concentration of oral liquid, DMEM culture solution is used for serial dilution by 2 times to prepare 5 concentration Titing degrees, the two-time concentration liquid medicine diluent is mixed with 200 TCID50 PRRSV diluent in equal volume (the drug and virus concentration after mixing are halved), and the mixture is treated by 5 percent CO at 37 DEG C 2 Incubate for 2h (vortex evenly every 30 min). 500ul of each well was added to a 24-well plate of Marc-145 cells which had been subjected to adherent culture for 24 hours, and 3 wells were multiplexed. At the same time, blank control, normal cell control, ribavirin Lin Yaowu control and virus control are set. At 37℃with 5% CO 2 Incubating the incubator for 2 hours; the mixture of virus and drug in the wells was discarded, washed 2 times with PBS, and 500ul of 2% FBS medium at 37℃and 5% CO was added to each well 2 The incubator was incubated for 48 hours and observed daily for cytopathic effect (CPE). And (5) collecting samples after the completion of the detection, and performing Q-PCR and Western blot.
1.3.8 Blocking effect of oral liquid on PRRSV
The maximum safe concentration of Marc-145 cells is the highest drug concentration of the general disinfection oral liquid, the culture solution of DMEM is diluted in multiple ratio to prepare 5 concentration gradients, and 500ul of the concentration gradients are added into a 24-well plate of Marc-145 cells which are subjected to wall-mounted culture for 24 hours, and 3 multiple wells are formed. At the same time, blank control, normal cell irradiation, ribavirin drug and virus control are set. At 37℃with 5% CO 2 Incubating the incubator for 2 hours; discarding the medicinal liquid, washing with PBS for 2 times, adding 100 TCID50 PRRSV virus diluent 37 deg.C and 5% CO into each well except for fine control 2 Incubation was continued for 2h in the incubator (shaking gently every 30min to allow even distribution of virus particles). Discarding virus solution, washing with PBS for 2 times, adding 500ul of 2% FBS culture medium into each well, and adding 5% CO at 37deg.C 2 Cultures were performed in an incubator for 48 hours and observed daily for cytopathic effect (CPE). And (5) collecting samples after the completion of the detection, and performing Q-PCR and Western blot.
1.3.9 Inhibition of PRRSV by oral liquid
Taking 24h Marc-145 cell 24-well plate, adding 500ul 100 TCID50 PRRSV virus dilution per well except cell control, and culturing at 37deg.C and 5% CO 2 Incubation for 2h (every 30 min) in incubator was gently shaken to allow even distribution of virus particles. And (3) removing virus liquid, washing for 2 times by PBS, and adding 500ul of oral liquid diluent which is prepared by diluting the maximum safe concentration of the oral liquid diluent with the DMEM culture solution in a multiple ratio into each hole. Meanwhile, a blank control group, a cell control group, a ribavirin drug group and a virus control group are set. At 37℃with 5% CO 2 And (5) incubating in an incubator. Removing culture solution in the wells after 2h, washing with PBS for 2 times, adding 500ul of maintenance solution into each well, and adding 5% CO at 37deg.C 2 Incubator cultures for 24h, cytopathic Condition (CPE) was observed every day. And (5) collecting samples after the completion of the detection, and performing Q-PCR and Western blot.
2. Test results
The cytotoxicity test result is shown in figure 3, and the traditional Chinese medicine oral liquid prepared in example 2 has no toxicity to Marc-145 cells when diluted 160 times. The results of the indirect immunofluorescence test are shown in fig. 4 and 5, and the traditional Chinese medicine oral liquid reduces the virus content in PRRSV and Marc-145 cells of different gene subtypes in a dose-dependent relationship. The result of the virus adsorption test is shown in figure 6, and the traditional Chinese medicine oral liquid can inhibit the adsorption of PRRSV TF strain on Marc-145 cells. The virus penetration test result is shown in figure 7, and the traditional Chinese medicine oral liquid can inhibit penetration of PRRSV TF strain on Marc-145 cells. The result of the virus replication test is shown in figure 8, and the traditional Chinese medicine oral liquid can inhibit the replication of PRRSV TF strain on Marc-145 cells.
According to the test results, the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome provided by the invention can inhibit the proliferation of PRRSV of different gene subtypes in Marc1-145 cells in a dose-dependent manner, has no toxic effect on Marc1-145 cells when diluted 160 times, and has inhibition effects on the adsorption, penetration and replication stages of viruses.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (9)
1. The preparation method of the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome is characterized by comprising the following steps:
s1, mixing rheum officinale, coptis chinensis, mint, radix bupleuri and schizonepeta, adding an ethanol aqueous solution, performing diacolation extraction, and concentrating under reduced pressure to obtain an extract A and a medicinal residue A;
s2, mixing the baical skullcap root, the great burdock achene, the tangerine peel, the weeping forsythiae capsule, the largetrifoliolious bugbane rhizome, the platycodon root, the liquoric root, the puffball, the figwort root, the Indigowoad root, the natural indigo, the talcum powder and the medicine slag A prepared in the step S1, adding water for heating reflux extraction, collecting distilled liquid, filtering the residual medicine liquid to prepare filtrate I and medicine slag B, wherein the filtrate I is extract B;
s3, carrying out alcohol precipitation treatment on the extract B prepared in the step S2 by adopting an ethanol water solution, separating, and concentrating the supernatant liquor under reduced pressure to prepare an extract C;
and S4, adding a stabilizer into the extract A prepared in the step S1, stirring uniformly, heating, then adding the extract C prepared in the step S3, stirring uniformly, cooling, adding the distillate prepared in the step S2, stirring uniformly, adding purified water and sodium benzoate, stirring for dissolution, adjusting the pH value, and filtering to obtain the product.
2. The method for preparing the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome according to claim 1, wherein in the step S1, rheum officinale, coptis chinensis, mint, radix bupleuri and schizonepeta are mixed and crushed; the mass percentage of the ethanol water solution is 60-70%; concentrating under reduced pressure to relative density of 1.06-1.10g/ml.
3. The method for preparing the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome according to claim 1, wherein the percolation extraction in the step S1 is specifically as follows: mixing radix et rhizoma Rhei, coptidis rhizoma, herba Menthae, bupleuri radix, and herba Schizonepetae, pulverizing, sieving, wetting with appropriate amount of ethanol water solution, percolating, soaking in 20 times of ethanol water solution for 3 hr, soaking for 24 hr, and concentrating under reduced pressure to obtain extract A and residue A.
4. The method for preparing the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome according to claim 1, wherein the filtering in the step S2 is carried out by sieving with a 200-mesh sieve, and heating and reflux extracting for 2h.
5. The method for preparing the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome according to claim 1, wherein the step S2 is characterized by further comprising the steps of decocting the medicine residue B for 2 hours, filtering, collecting filtrate II, combining filtrate I and filtrate II, and concentrating to a relative density of 1.08-1.10g/ml to obtain extract B.
6. The method for preparing the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome according to claim 1, wherein the alcohol precipitation treatment in the step S3 is specifically as follows: adding ethanol into the extract B prepared in the step S2 under stirring until the ethanol content in the system is 75%, and standing at normal temperature for 12h to obtain supernatant liquid I and precipitate I; adding 75% ethanol water solution into the precipitate I under stirring, and standing at normal temperature for 12h to obtain supernatant liquid II and precipitate II; mixing the supernatant liquid I and the supernatant liquid II, concentrating under reduced pressure to relative density of 1.06-1.10g/ml, and making into extract C.
7. The method for preparing the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome according to claim 1, wherein the stabilizer in the step S4 is N-methyl pyrrolidone; the heating temperature is 78-82 ℃, and the cooling temperature is 37-45 ℃; the pH value is regulated to 4.5-5.0.
8. The method for preparing the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome, which is characterized by comprising the following traditional Chinese medicine components in parts by weight: 1-3 parts of rheum officinale, 1-2 parts of radix scutellariae, 1-2 parts of coptis chinensis, 1 part of liquorice, 1-2 parts of puffball, 1-2 parts of mint, 1-2 parts of radix scrophulariae, 3 parts of burdock, 1-2 parts of cimicifuga foetida, 1-2 parts of radix bupleuri, 1-2 parts of platycodon grandiflorum, 1-2 parts of dried orange peel, 2 parts of fructus forsythiae, 1-2 parts of schizonepeta, 2 parts of radix isatidis, 1-2 parts of indigo naturalis, 5-6 parts of talcum powder, 0.1-0.5 part of stabilizer, 20-40 parts of purified water and 0.1-0.5 part of sodium benzoate.
9. A traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome, which is prepared by the preparation method of the traditional Chinese medicine oral liquid for preventing and controlling porcine reproductive and respiratory syndrome according to any one of claims 1 to 8.
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