CN111281922B - Compound traditional Chinese veterinary medicine oral preparation for treating diarrhea of pigs and poultry and preparation method thereof - Google Patents
Compound traditional Chinese veterinary medicine oral preparation for treating diarrhea of pigs and poultry and preparation method thereof Download PDFInfo
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- A61K36/11—Pteridophyta or Filicophyta (ferns)
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/484—Glycyrrhiza (licorice)
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- A61K36/18—Magnoliophyta (angiosperms)
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- A61K36/57—Magnoliaceae (Magnolia family)
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/63—Oleaceae (Olive family), e.g. jasmine, lilac or ash tree
- A61K36/634—Forsythia
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
- A61K36/736—Prunus, e.g. plum, cherry, peach, apricot or almond
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- A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
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- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P1/12—Antidiarrhoeals
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
Abstract
The invention discloses a compound traditional Chinese veterinary medicine oral preparation for treating porcine and avian diarrhea and a preparation method thereof, and the traditional Chinese veterinary medicine oral preparation comprises the following traditional Chinese medicinal materials in parts by weight: 5-10 parts of Chinese brake herb, 5-10 parts of vinegar schisandra chinensis, 5-10 parts of pomegranate bark, 5-10 parts of fructus forsythiae, 5-10 parts of dark plum fruit and 5-10 parts of liquorice, and the preparation method comprises the steps of crushing the Chinese medicines, mixing, adding 70% ethanol for soaking according to the material-liquid ratio of 1:20, extracting twice under reflux for 1 hour each time, combining the extracts twice, and performing rotary steaming to 1g/mL to prepare decoction or granules. The compound preparation is a pure Chinese medicinal preparation, can not generate drug resistance after long-term use, and is not easy to generate drug residue in livestock and poultry bodies. Simultaneously has the advantages of strong antibacterial activity, good antidiarrheal effect, quick response, small dosage, high effective rate and high cure rate.
Description
Technical Field
The invention relates to a compound traditional Chinese veterinary medicine preparation, relates to a traditional Chinese veterinary medicine formula for treating porcine diarrhea, and in particular relates to a compound traditional Chinese veterinary medicine oral preparation for treating porcine diarrhea and a preparation method thereof.
Background
Pathogenic Escherichia coli causes yellow and white scour and edema disease of piglets. The three diseases have high morbidity and mortality in a large-scale pig farm, and meanwhile, in recent years, the epidemic tendency, clinical symptoms and pathological changes of the three diseases are diversified, so that huge economic losses are brought to the pig raising industry. Escherichia coli has a plurality of serotypes, no ideal vaccine is used for prevention at present, effective antibacterial drugs are mostly adopted for treatment, but in the breeding link, the spreading of drug-resistant strains is caused due to the abuse of antibiotics, residues and environmental pollution are caused, and the method has more and more serious harm to the health of breeding industry and human beings. The traditional Chinese veterinary medicine can solve the problem of drug residue, partially or completely restore the sensitivity of escherichia coli to antibiotics and relieve the diffusion and transfer of drug-resistant genes. The resistance-free technology is a trend that the breeding industry and the veterinary medicine and veterinary medicine industry have to adapt to, and the traditional Chinese veterinary medicine preparation is used for replacing or partially replacing antibiotic medicines, so that the search for the traditional Chinese veterinary medicine preparation with the effects of resisting bacteria and stopping diarrhea for treating piglet diarrhea is very important.
However, most of the existing veterinary drugs and feed additives are simply processed Chinese herbal medicine mixtures, so that the traditional Chinese veterinary drugs and feed additives are large in dosage, slow in response and coarse in product, and are difficult to adapt to the needs of large-scale and intensive development of animal husbandry. Chinese veterinary drugs need to occupy a place in the fiercely competitive international market, products with independent intellectual property rights are required, the traditional process is replaced by scientific and technological means for development and application of the Chinese veterinary drugs by using modern science and technology, and the improvement of the product quality is an urgent task at present.
Disclosure of Invention
In order to solve the problems, the invention aims to provide the compound traditional Chinese veterinary medicine for treating the livestock and poultry diarrhea and the preparation method thereof, wherein the compound traditional Chinese veterinary medicine has the advantages of good effect, quick response and small dosage.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a compound traditional Chinese veterinary medicine oral preparation for treating diarrhea of pigs and poultry comprises the following traditional Chinese medicinal materials in parts by weight: 5-10 parts of Chinese brake herb, 5-10 parts of vinegar schisandra chinensis, 5-10 parts of pomegranate bark, 5-10 parts of fructus forsythiae, 5-10 parts of dark plum fruit and 5-10 parts of liquorice.
Further, the traditional Chinese veterinary medicine oral preparation comprises the following traditional Chinese medicinal materials in parts by weight: 5 parts of Chinese brake herb, 5 parts of vinegar schisandra chinensis, 5 parts of pomegranate rind, 10 parts of fructus forsythiae, 10 parts of liquorice and 5 parts of dark plum.
Further, the traditional Chinese veterinary medicine oral preparation comprises the following traditional Chinese medicinal materials in parts by weight: 6 parts of pteris multifida, 10 parts of vinegar schisandra chinensis, 6 parts of pomegranate rind, 6 parts of fructus forsythiae, 6 parts of liquorice and 6 parts of dark plum.
Further, the traditional Chinese veterinary medicine oral preparation comprises the following traditional Chinese medicinal materials in parts by weight: 9 parts of Chinese brake herb, 5 parts of vinegar schisandra chinensis, 5 parts of pomegranate rind, 5 parts of fructus forsythiae, 5 parts of liquorice and 7 parts of dark plum.
Further, the traditional Chinese veterinary medicine oral preparation comprises the following traditional Chinese medicinal materials in parts by weight: 10 parts of Chinese brake herb, 5 parts of vinegar schisandra chinensis, 10 parts of pomegranate rind, 5 parts of fructus forsythiae, 5 parts of liquorice and 10 parts of dark plum.
Preferably, the oral preparation is in the form of decoction or granules.
The preparation method of the decoction for preparing the compound traditional Chinese veterinary medicine oral preparation for treating the diarrhea of pigs and poultry specifically comprises the following steps: pulverizing the above Chinese medicinal materials, mixing, soaking in 70% ethanol at a material-to-liquid ratio of 1:20 for 0.5 hr, reflux extracting for 2 times (1 hr each time), filtering, mixing filtrates, concentrating under reduced pressure until no alcohol smell exists, concentrating to obtain medicinal liquid with concentration of 1g/mL, adding sodium benzoate, stirring and dissolving.
The preparation method of the granules for preparing the compound traditional Chinese veterinary medicine oral preparation for treating the diarrhea of pigs and poultry specifically comprises the following steps: crushing the traditional Chinese medicine raw materials, and mixing the raw materials according to a material-liquid ratio of 1: soaking 20 in 70% ethanol for 0.5 hr, reflux extracting for 2 times, each for 1 hr, filtering, mixing filtrates, concentrating under reduced pressure until no alcohol smell exists, concentrating to obtain medicinal liquid with concentration of 1g/mL, spray drying to obtain medicinal powder, and making into granule.
The use method of the compound traditional Chinese veterinary medicine oral preparation for treating the diarrhea of pigs and poultry comprises the following steps: the formula is decoction or prepared into granules, and the decoction is taken 2 times a day, and 3 days are a treatment course.
The traditional Chinese medicine components of the invention have the following medicinal properties and pharmacology:
the Pteris multifida, also known as Bidens trifoliata, is a whole plant of Pteris multifida of Pteridaceae, and has sweet, light, slightly bitter and cold properties. It enters heart, liver and large intestine meridians. Has the effects of clearing away heat, promoting diuresis, cooling blood, stopping bleeding, relieving swelling, and removing toxic substances. The product has inhibitory effect on Staphylococcus aureus, Escherichia coli, dysentery bacillus, and salmonella.
Vinegar fructus Schisandrae chinensis is sour, sweet and warm, and enters lung, heart and kidney meridians. The effects include astringency, arresting discharge, benefiting vital energy, promoting the production of body fluid, invigorating kidney and calming heart. It is mainly used for treating chronic diarrhea, chronic cough, asthma, etc.
Pomegranate rind is warm in nature, sour and astringent in taste, enters large intestine meridian, and has the effects of relieving diarrhea with astringents, stopping bleeding and expelling parasites.
Forsythia fruit, bitter in nature and slightly cold in nature, enters lung, heart and small intestine meridians. Fructus forsythiae has broad-spectrum antibacterial effect, has strong inhibitory effect on Staphylococcus aureus and Shigella dysenteriae, and also has antifungal, antiviral, antiinflammatory and antipyretic effects; can obviously antagonize inflammation, fever, swelling and pain and the like caused by endotoxin.
The liquorice has sweet and mild nature and flavor, and has the functions of invigorating the spleen and replenishing qi, clearing away heat and toxic materials, eliminating phlegm and stopping cough, relieving spasm and pain and harmonizing the medicines, and the liquorice enters heart, lung, spleen and stomach channels.
The dark plum is derived from dried nearly mature fruit of prunus mume (prunus mume) belonging to Rosaceae, and belongs to astringent drugs. Sour, astringent and neutral in nature and flavor, and entering liver, spleen, lung and large intestine meridians. The main functions are astringing lung, astringing intestine, promoting fluid production and calming ascaris.
The formula has the main effects of broad-spectrum antibiosis, diarrhea relieving with astringents, detoxification and dampness removing, and can fully play the role of each component by combining the drug effects and the proper dosage of the medicinal materials, so that the compatibility of the medicinal materials can play the drug effects to the maximum extent.
The invention has the beneficial effects that:
the compound preparation is a pure Chinese medicinal preparation, can not generate drug resistance after long-term use, and is not easy to generate drug residue in livestock and poultry bodies. The experiment shows that the traditional Chinese medicine composition can effectively inhibit the attack time of the diarrhea caused by the castor oil and reduce the diarrhea degree, has obvious treatment effect on the diarrhea caused by the escherichia coli, and has the advantages of good antibacterial effect, quick response, small dosage, high effective rate and high cure rate.
Detailed Description
The compound veterinary oral preparation of the invention is further explained by combining the test and clinical effects.
Example 1:
the compound traditional Chinese veterinary medicine oral preparation disclosed by the invention is used for the curative effect experiment of experimental diarrhea caused by castor oil:
1.1 materials and methods
1.1.1 preparation of compound traditional Chinese veterinary medicine oral preparation:
compound preparation 1 group: 9 parts of Chinese brake herb, 5 parts of vinegar schisandra chinensis, 5 parts of pomegranate rind, 5 parts of fructus forsythiae, 7 parts of dark plum fruit and 5 parts of liquorice;
compound preparation 2 groups: 5 parts of Chinese brake herb, 5 parts of vinegar schisandra chinensis, 5 parts of pomegranate rind, 10 parts of fructus forsythiae, 5 parts of dark plum fruit and 10 parts of liquorice;
compound preparation 3 groups: 10 parts of Chinese brake herb, 5 parts of vinegar schisandra chinensis, 10 parts of pomegranate rind, 5 parts of fructus forsythiae, 10 parts of dark plum fruit and 5 parts of liquorice;
accurately weighing each group of Chinese medicinal powder, packaging with filter paper into long strips, soaking in 70% ethanol solution at a ratio of 1:20 for 30min, extracting with ethanol under reflux at 50 deg.C for 2 times, each for 1 hr, mixing filtrates, and rotary evaporating at 60 deg.C to concentration of 1 g/mL.
1.1.2 preparation of single-drug extract of phoenix-tail fern:
accurately weighing 20g of herba Pteridis Multifidae powder, subpackaging into long strips with filter paper, soaking for 30min with 70% ethanol solution at a material-to-liquid ratio of 1:20, extracting with ethanol at 50 deg.C under reflux for 2 times, each for 1 hr, and mixing the filtrates. Rotary evaporation at 60 ℃ to a concentration of 1g crude drug/mL. Sucking part of the extractive solution 1g/mL, diluting with distilled water for 2 times to obtain 0.5g crude drug/mL as the medicinal liquid for intragastric administration in high dose group (the dosage of the ethanol extract of herba Pteridis Multifidae in high dose group is 5g/kg, i.e. 0.1mL/10 g), sucking part of the extractive solution 1g/mL, diluting with distilled water for 4 times to obtain 0.25g/mL as the medicinal liquid for intragastric administration in low dose group (the dosage of the ethanol extract of herba Pteridis Multifidae in low dose group is 2.5g/kg, i.e. 0.1mL/10 g).
1.1.3 grouping and administration of test animals
Kunming mice (SPF grade), 18-22g in female and male half, purchased from Experimental animals center of pharmaceutical university in Jiangxi.
Taking 70 healthy mice with the weight of 18-22g and half of each sex, randomly dividing the mice into seven groups including a normal control group, a model group, a compound 1, 2 and 3 groups and a single-drug high-dose and low-dose group of Chinese brake herb, wherein each group comprises 10 mice, 5 mice and males, feeding the mice and the males in cages, and marking (distinguishing by left upper/right upper/left lower/right lower/tail/no mark). The normal control group and the model group of mice are filled with 0.1mL/10g of gastric physiological saline, each group of compound pteris multifida is filled with 0.1mL/10g of diluted compound liquid medicine according to 5g/kg, the high-dose group and the low-dose group of ethanol extract of the pteris multifida are filled with 0.1mL/10g of diluted high-dose group and low-dose group respectively, the continuous administration is carried out for 3d, the fasting and the water prohibition are carried out for 2h before and after each administration, the last administration is carried out for 30min, and each group is filled with 0.2 mL/gastric castor oil except the normal control group which is filled with the physiological saline with the same volume.
The mouse was covered under a beaker, and a piece of filter paper was laid under the beaker, followed by observation for 6 hours, with the filter paper being changed every 1 hour. Diarrhea mice were observed for general status and behavioral changes. The mice in each group were observed for changes in mental state, behavior and stool. The data obtained in the test are processed by SPSS17.0 (the data in the first 3h of the report are taken for statistical analysis), and the test of independent samples among groups is carried out, and is expressed as x +/-s.
1.2 Observation index
Taking excrement on each piece or each pile (which means that the number of the excrement can not be separated) on the filter paper as defecation; the dried excrement is not adhered into granules; the loose stool is mostly unformed, has large water content and is internally provided with viscous liquid.
Incidence of diarrhea per hour: the ratio of the number of diarrhea per hour for each group of mice to the total number of mice in the group.
The total diarrhea rate: the ratio of the number of diarrhea in each group of mice to the total number of mice in the group over the observation time.
The rate of loose feces: the ratio of the number of loose stools to the total number of stools per mouse.
A rare excrement stage: the degree of defecation of each mouse was represented by the number of stains formed by the filter paper contaminated with the feces, which was classified into four grades: the diameter < l cm is l grade, 1.0-1.9 cm is 2 grade, 2-3 cm is 3 grade, and >3 cm is 4 grade. The diameter of the stain is measured in a regular circle; the irregular shape is measured by the longest and approximate circle diameter, and the two numbers are added together divided by 2.
Average degree of loose feces: in the statistics, the number of the loose feces in each pile of each mouse is measured one by one, then all the numbers of the loose feces of the mouse are added, and the number of the loose feces is divided by the number of the loose feces, so that the average number of the loose feces of each mouse is obtained, wherein the average number of the loose feces = the sum of the numbers of the loose feces and/or the number of the loose feces.
Sparse feces inhibition = average number of sparse feces per hour for model group-average number of sparse feces per hour for the remaining groups)/average number of sparse feces per hour for model group × 100%
1.3 test results and analysis
Except for a normal control group, after each group of the castor oil is infused with stomach for a period of time, the diarrhea mice show certain diarrhea conditions, and the diarrhea mice show mental depression, lassitude, hypodynamia, coarse hair, secretion around eyes, difficulty in opening, increased defecation frequency, yellowish brown excrement which is not fixed, increased water content of the excrement, viscous liquid in the excrement and less solid content, which shows that the model is successfully established.
The number of loose feces per hour within 0-3 h: except for the normal control group, diarrhea appeared after intragastric castor oil administration. The model group gets worse along with time within 0-3 h, the amount of excrement in the high-dose group and the low-dose group of the phoenix-tail fern is equivalent to that in the model group within 0-1 h, the amount of excrement in the high-dose group and the low-dose group of the phoenix-tail fern is more serious than that in the model group within 1-2 h, but the amount of excrement in the high-dose group and the low-dose group of the phoenix-tail fern is far lower than that in the model group within 2-3 h, which indicates that the antidiarrheal effect time of the single-drug phoenix-tail fern is 2h after the intragastric castor oil is infused; the compound 1 group has no loose feces within 0-2 h of the intragastric castor oil, and the loose feces begins to appear within 2-3 h, but the number of the loose feces is far lower than that of the model group; after the diarrhea starting time of the compound 2 group and the compound 3 group is 1 hour after the castor oil is infused, the quantity of loose stools is obviously reduced compared with that of the model group, and the results show that the compound three groups can obviously delay the time of the first diarrhea of the mice.
Incidence of diarrhea per hour: the incidence rate of diarrhea in the high-dose Chinese brake herb group within 0-3 h is lower than that in the model group, and the incidence rate of diarrhea in the three compound Chinese brake herb groups within 0-3 h is obviously lower than that in the model group (p is less than 0.05);
3h Total diarrhea Rate: the total diarrhea rate of the high-dose group, the low-dose group and the compound three groups of the phoenix-tail fern is reduced by 3h compared with that of the model group, wherein the total diarrhea rate of the model group is 90 percent (9/10), the high-dose group of the phoenix-tail fern is 40 percent (4/10), the compound 1 group and the compound 3 group are 20 percent (2/10) and the compound 2 group is 30 percent (3/10), and thus the incidence rate of diarrhea can be greatly reduced by the three compound groups.
The average stool dilution level of the high-dose group and the compound group of the Chinese brake herb is extremely remarkably reduced by 0-3 h compared with that of a model group (p is less than 0.01); the average stool dilution rate of the three compound groups is obviously different from that of the model group (p is less than 0.05); the inhibition rates of loose stool of the compound 1, 2 and 3 groups are respectively 74.36%, 56.41 and 71.79%. See table 1 for details.
TABLE 1 Effect of Compound Pteris multifida on castor oil induced diarrhea in mice (n =10, ` x. + -. s)
Note: compared to the model group, "a" indicates significant difference (p < 0.05), "a" indicates very significant difference (p < 0.01)
Castor oil is a common drug for replicating a mouse diarrhea model and is commonly used for researching the anti-diarrhea mechanism of traditional Chinese medicines. Castor oil is a stimulant laxative and is a laxative that stimulates the mucous membranes of the small intestine. Castor oil is hydrolyzed into ricinoleic acid by a lipase in the upper part of the small intestine, and intestinal smooth muscle is stimulated by the ricinoleic acid generated by hydrolysis to damage intestinal mucosal cells and cause inflammation, so that intestinal peristalsis is accelerated, absorption of water and electrolytes is inhibited, and exudative diarrhea occurs.
In conclusion, a model test of castor oil induced diarrhea in mice shows that the single-medicine pteris multifida high-dose group and the compound pteris multifida have better antidiarrheal effect, and compared with the single-medicine pteris multifida, the compound three groups have better antidiarrheal effect.
Example 2
The compound traditional Chinese veterinary medicine oral preparation has in-vitro antibacterial activity on several common pathogenic bacteria of livestock and poultry
2.1 materials
Same as 1.1.1
All the medicinal materials are crushed for later use. The medicinal materials are purchased from Yifeng big pharmacy in Nanchang city of Jiangxi province.
Escherichia coli ATCC25922 strain purchased from Guangdong collection of microorganisms.
Escherichia coli K88, Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, and animal pharmacy laboratory preservation of animal institute of agriculture university in Jiangxi.
2.2 methods
2.2.1 preparation of Pteris multifida compound
Taking the compound pteris multifida preparation extracted at 1.1.1, concentrating by a rotary evaporator, recovering ethanol, controlling the temperature at 60 ℃, concentrating into paste, and weighing. Dissolving the paste with 2mL of 20% dimethyl sulfoxide, diluting with 8 mL of sterilized double water to constant volume, filtering for sterilization, and storing in a refrigerator at 4 ℃. The concentration of each group of compound extract liquid is shown in table 2.
2.2.2 preparation of bacterial liquid
Culturing the glycerol-preserved bacteria in LB broth at 37 deg.C and 220r/min to logarithmic phase, streaking on common nutrient agar, and culturing at 37 deg.C in biochemical incubator for 18 h. Selecting 3-5 colonies with good growth and uniform size from a common agar plate by using a collunarium, inoculating the colonies in LB broth, and culturing for 12h at 37 ℃. The bacteria concentration was calculated by plate viable count method, and the bacteria concentration was adjusted to 1.5X 10 in broth just before use6CFU/mL, and refrigerating at 4 ℃ in a refrigerator for later use.
2.2.3 minimum inhibitory concentration determination of compound Pteris multifida on 5 common pathogenic bacteria
Preparing a sterile 96-well plate according to a trace two-fold dilution method recommended by CLSI (CLSI), adding 100 mu L of MH broth into each well, respectively adding 100 mu L of compound pteris multifida liquid medicine into the first well of each row, uniformly blowing and beating the mixture by a liquid transfer gun, sucking 100 mu L of the mixture from the first well to the second well, uniformly blowing and beating the mixture, repeating the above operations by the same way, sequentially diluting the mixture to the rear well in a trace two-fold manner until 100 mu L of the mixture is discarded from the twelfth well; test wells 50uL of 1.5X 10 concentration was added per well6CFU/mL of the bacterial solution so that the final concentration of bacteria per well in a 96-well plate is 5X 105CFU/mL; three replicates were done for each strain. Adding common LB broth as blank control, 5 × 105CFU/mL bacterial suspension is used as growth control group, and the drug control group is obtained by adding common LB broth into compound herba Pteridis Multifidae liquid medicine, performing static culture at 37 deg.C for 18h, and observing with naked eye.
2.3 results and analysis
The result of measuring the minimum inhibitory concentration of the compound traditional Chinese veterinary medicine oral preparation on 5 common pathogenic bacteria is shown in table 3. The experimental result shows that the three compound Chinese brake herb preparations have very good antibacterial activity on 5 common pathogens of livestock and poultry. Exhibit broad-spectrum antibacterial activity.
Example 3
Protection test of compound traditional Chinese veterinary medicine oral preparation on mouse escherichia coli infection
3.1 materials
Compound preparation 1 material: selecting 9 parts of Chinese brake herb, 5 parts of vinegar schisandra chinensis, 5 parts of pomegranate rind, 5 parts of fructus forsythiae, 7 parts of dark plum fruit and 5 parts of liquorice, and crushing for later use.
Gentamicin sulfate injection, Guangzhou Baiyunshan Tianxin pharmacy GmbH, Specification: each 4 ten thousand units/mL lot number: 20190218.
test animals: kunming mice (SPF grade), 18-22g in female and male half, purchased from Experimental animals center of pharmaceutical university in Jiangxi.
Escherichia coli K88 strain, preserved by veterinary drug research institute of Jiangxi agricultural university.
3.2 methods
3.2.1 preparation of Compound preparation 1
Accurately weighing 100g of compound 1 powder, wrapping the powder into long strips by using filter paper, soaking for 30min under the conditions of the optimal extraction process, namely ethanol concentration of 70%, liquid-material ratio of 1:20 and extraction temperature of 50 ℃, extracting twice by using an ethanol reflux extraction method under the conditions, extracting for 1h each time, combining extracting solutions twice, concentrating by using a rotary evaporator, recovering ethanol, controlling the temperature at 60 ℃, concentrating to the concentration of 1g of crude drug/mL (the administration concentration of a high-dose group of compound 1 of phoenix-tail fern), sucking out 1g/mL of liquid medicine to dilute 2 times to the concentration of 0.5g/mL (the administration concentration of a low-dose group of compound 1 of phoenix-tail fern), and freezing and storing for later use.
3.2.2 preparation of bacterial suspension of Escherichia coli K88
Inoculating glycerol-preserved Escherichia coli K88 into LB broth, culturing at 37 deg.C and 220r/min to logarithmic phase, streaking on common nutrient agar, and culturing at 37 deg.C in biochemical incubator for 18 h. Selecting 3-5 colonies with good growth and uniform size from a common agar plate by using a collunarium, inoculating the colonies in LB broth, and culturing for 12h at 37 ℃. The bacterial liquid concentration was calculated by flat viable count method, and the bacterial concentration was adjusted to 1.5X 10 with broth9CFU/mL, and refrigerating at 4 ℃ in a refrigerator for later use.
3.2.3 establishment of model for causing diarrhea in mice by Escherichia coli K88
Kunming mice were raised for 1 week and observed to be abnormal. Fasting is carried out for 2h before infection, and the other groups except the normal group are injected with 0.2mL of escherichia coli bacterial liquid in the abdominal cavity and fed normally after 2 h. The difference of the normal control group treatment is that the bacterial liquid is changed into normal saline. Animals were observed 1 time every 3h after infection and disease development was recorded. Definition of animal pathogenesis: drawing loose stool; the anus is adhered with loose stool, mucus or blood; loose hair, less movement; if the patient has little or no food intake, any one of the symptoms is the onset of the disease.
3.2.4 protective action of Fengwei compound 1 preparation on mouse experimental large intestine rod infection
Taking 50 healthy mice with half of each sex, randomly dividing the mice into a normal control group, an infected group, a drug control group and a compound high and low dose group, wherein each group comprises 10 mice, 5 mice are respectively fed with the males and the females, and marking the mice. The mice of each group are fasted and forbidden for 2 hours before infection, except for a normal control group during infection, each mouse is subjected to intraperitoneal injection of 0.2mL/10g of prepared escherichia coli liquid, after injection is finished, each group is immediately subjected to intragastric administration, the normal control group and a model group are respectively subjected to intragastric administration of 0.1mL/20g of physiological saline, a drug control group is subjected to intragastric administration of 0.1mL/20g of gentamycin injection, a compound preparation 1 with high and low dose group concentrations is subjected to intragastric administration of 0.1mL/20g of diluted compound preparation 1, administration is carried out at the same time every day, twice administration is carried out every day, 10 hours are separated, water is forbidden for 2 hours before and after each administration, continuous administration is carried out for 3 days, and the mice are killed after observation for one week. Observing the mental state of the mice, judging whether the diets are normal and the feces are normal, recording the death condition and calculating the death rate and the protection rate.
Protection rate = (number of model group deaths-number of drug group deaths)/number of model group deaths × 100%
3.3 results and analysis
After challenge, mice in an infected group are messy and dull in hair, heavy in spirit, slow in response, sleepy, refused to eat, thin, and rapid in respiration, part of the mice die due to convulsion, and escherichia coli is separated from the livers of the dead mice, so that the establishment of a model of escherichia coli diarrhea of the mice is successful. In the infection group, 8 mice died in 24h, and the specific death time is as follows: 2 death patients die after 6h and 6 death patients die after 12-24 h after the toxin is attacked. The dead mouse is dissected, the intestinal tract of the dead mouse is full of bleeding, the stomach of the dead mouse is full of gas and yellow mucus, and the liver of the dead mouse is blackened.
The mental state of mice in the gentamicin treatment group begins to be depressed and sleepy, but most of the mice begin to improve the mental state 6 hours after the administration, and most of the mice move freely and have normal appetite 2 days later; the mice in the high-dose group of the compound preparation 1 begin to have depressed mind, slow response and lethargy, but the mice begin to eat after 8 hours of administration, most mice recover from the mind and have quicker response after 2 days of administration. The compound preparation 1 reduces the death rate of the mice in a high-dose group, and the protection rate of the mice reaches 75 percent. The compound preparation 1 group of mice infected with the low-dose group of mice after toxin attack have the symptoms of depression, slow reaction and lethargy, a small amount of mice after administration have the symptoms relieved, and 6 mice die. See table 4 for details.
Example 4
The compound traditional Chinese veterinary medicine oral preparation has the clinical curative effect of treating piglet diarrhea
4.1 materials
Preparing a compound preparation of phoenix-tail fern: taking 2000 g of each of the 1.1.1 crushed and mixed compound 1, compound 2 and compound 3 powder, carrying out the same extraction method as 1.1.1, concentrating each extract filtrate by a rotary evaporator until the extract contains 1g/mL of crude drug, adding 6 g of sodium benzoate, mixing uniformly, cooling, filtering and subpackaging to obtain the compound.
Gentamicin sulfate injection, as 3.1
Test sick pig
3-35 days old piglets with diarrhea symptoms in a certain pig farm in the south and the Dingnan county of Jiangxi province are selected, the variety is a three-way hybrid white pig, and the three-way hybrid white pig is diagnosed as escherichia coli infection through clinical diagnosis, laboratory bacterial separation and biochemical identification. 4 kinds of medicines are used by sick pigs in the same colony house as much as possible by piglets.
4.2 methods
4.2.1 test grouping and treatment protocol
143 affected pigs were randomly divided into 4 groups. Each group was orally administered according to Table 5, 2 times per day for 3 consecutive days. All groups were raised under the same feeding management conditions. Adding appropriate amount of electrolytic multivitamin and oral rehydration salt to prevent organism dehydration. The mental state and diet condition of each group of pigs are observed and recorded every day, and the death condition of each group of piglets and the curative effect of each group after administration are recorded.
4.2.2 criteria
(1) And (3) curing: after the medicine is taken for 3 days, the defecation is stopped, the excrement is shaped, the ingestion, body temperature and spirit are recovered to be normal, and the disease does not relapse within one week after the medicine is stopped.
(2) The method has the following advantages: after treatment, the clinical symptoms of the affected pigs are obviously improved, the symptoms are relieved after 3 days of administration, and the appetite and the body temperature are recovered to be normal;
(3) and (4) invalidation: after treatment, the clinical symptoms of the affected pigs are not obviously changed and even die.
4.3 results and analysis
Four groups of pigs are treated by using the medicine for 3 days, the cure rate of the treatment of the compound preparation 1 group of the phoenix-tail fern is 97.4 percent, the cure rate of the treatment of the compound preparation 2 group is 93.3 percent, the cure rate of the treatment of the compound preparation 3 group is 83.3 percent, and the cure rate of the treatment of the gentamicin group is 88.9 percent. The effective rates of the compound preparation 1 and the compound preparation 2 are both 100 percent, the diarrhea symptoms and diarrhea times of the pigs after administration are reduced, and the mental state is improved. See table 5.
Test results show that the compound preparation 1, 2 and 3 groups have no significant difference (P is more than 0.05) from the gentamicin group, the Chinese brake herb compound preparation has significant treatment effect on piglet natural infection colibacillosis by being orally taken with 1 mL/kg of body weight, has no significant difference from gentamicin in effective rate and cure rate, and can be used as clinical recommended dosage. The compound preparation of the phoenix-tail fern is a pure traditional Chinese medicine preparation, can not generate drug resistance after long-term use, and is not easy to generate drug residue in livestock and poultry bodies. The experimental research has obtained obvious treatment effect, and the effect is good after the product is used in partial farms, so that the vigorous development of the product has positive promotion effect on the development of healthy breeding industry, thereby providing guarantee for the healthy and rapid development of the pig industry. By applying the traditional Chinese medicine extract, the drug resistance of the escherichia coli of the pig is necessarily reduced or delayed.
In conclusion, the compound traditional Chinese veterinary medicine oral preparation has strong in-vitro antibacterial activity and wide antibacterial spectrum on several common pathogens, has a strong antidiarrheal effect on castor oil-induced diarrhea, has a strong treatment and protection effect on escherichia coli-induced diarrhea, and can replace antibiotics to be used for preventing and treating diseases in the breeding industry under the background of 'no antibiotics' in the current breeding industry, so that the compound traditional Chinese veterinary medicine oral preparation has a wide application prospect.
Claims (8)
1. A compound traditional Chinese veterinary medicine oral preparation for treating diarrhea of pigs and poultry is characterized by comprising the following traditional Chinese medicinal materials in parts by weight: 5-10 parts of Chinese brake herb, 5-10 parts of vinegar schisandra chinensis, 5-10 parts of pomegranate bark, 5-10 parts of fructus forsythiae, 5-10 parts of dark plum fruit and 5-10 parts of liquorice.
2. The compound traditional Chinese veterinary medicine oral preparation for treating diarrhea of pigs and poultry as claimed in claim 1, is characterized by comprising the following traditional Chinese medicinal materials in parts by weight: 5 parts of Chinese brake herb, 5 parts of vinegar schisandra chinensis, 5 parts of pomegranate rind, 10 parts of fructus forsythiae, 10 parts of liquorice and 5 parts of dark plum.
3. The compound traditional Chinese veterinary medicine oral preparation for treating diarrhea of pigs and poultry as claimed in claim 1, is characterized by comprising the following traditional Chinese medicinal materials in parts by weight: 6 parts of pteris multifida, 10 parts of vinegar schisandra chinensis, 6 parts of pomegranate rind, 6 parts of fructus forsythiae, 6 parts of liquorice and 6 parts of dark plum.
4. The compound traditional Chinese veterinary medicine oral preparation for treating diarrhea of pigs and poultry as claimed in claim 1, is characterized by comprising the following traditional Chinese medicinal materials in parts by weight: 9 parts of Chinese brake herb, 5 parts of vinegar schisandra chinensis, 5 parts of pomegranate rind, 5 parts of fructus forsythiae, 5 parts of liquorice and 7 parts of dark plum.
5. The compound traditional Chinese veterinary medicine oral preparation for treating diarrhea of pigs and poultry as claimed in claim 1, is characterized by comprising the following traditional Chinese medicinal materials in parts by weight: 10 parts of Chinese brake herb, 5 parts of vinegar schisandra chinensis, 10 parts of pomegranate rind, 5 parts of fructus forsythiae, 5 parts of liquorice and 10 parts of dark plum.
6. The compound traditional Chinese veterinary medicine oral preparation for treating diarrhea of pigs and poultry as claimed in claim 1, is characterized in that the oral preparation is decoction or granules.
7. The preparation method of the decoction of the compound traditional Chinese veterinary medicine oral preparation for treating the diarrhea of pigs and birds as claimed in any one of claims 1 to 6 comprises the following steps: pulverizing the traditional Chinese medicines, mixing, and mixing according to a material-liquid ratio of 1: soaking in 70% ethanol for 0.5 hr, reflux extracting for 2 times (each for 1 hr), filtering, mixing filtrates, concentrating under reduced pressure until no alcohol smell is observed, concentrating to obtain medicinal liquid with concentration of 1g/mL, adding sodium benzoate, and stirring to dissolve.
8. The preparation method of the compound traditional Chinese veterinary medicine oral preparation granules for treating the diarrhea of pigs and poultry as claimed in any one of claims 1 to 6 comprises the following steps: crushing the traditional Chinese medicine raw materials, and mixing the raw materials according to a material-liquid ratio of 1: soaking 20 in 70% ethanol for 0.5 hr, reflux extracting for 2 times, each for 1 hr, filtering, mixing filtrates, concentrating under reduced pressure until no alcohol smell exists, concentrating to obtain medicinal liquid with concentration of 1g/mL, spray drying to obtain medicinal powder, and making into granule.
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| 中药抑菌成分及其在养猪声称中的应用前景;肖莉春等;《动物营养学报》;20121231;第24卷(第12期);第2311-2318页 * |
| 复方凤尾草胶囊治疗腹泻临床观察;胡荷斌等;《浙江中西医结合杂志》;20001231;第10卷(第11期);第668-669页 * |
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