CN113577112B - A kind of pharmaceutical composition for preventing and treating poultry salmonellosis and preparation method - Google Patents
A kind of pharmaceutical composition for preventing and treating poultry salmonellosis and preparation method Download PDFInfo
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- CN113577112B CN113577112B CN202111071631.9A CN202111071631A CN113577112B CN 113577112 B CN113577112 B CN 113577112B CN 202111071631 A CN202111071631 A CN 202111071631A CN 113577112 B CN113577112 B CN 113577112B
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Classifications
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/425—Thiazoles
- A61K31/429—Thiazoles condensed with heterocyclic ring systems
- A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
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- A—HUMAN NECESSITIES
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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Abstract
本发明涉及一种预防和治疗禽沙门氏菌病的药物组合物及其制备方法，该药物组合物包括以下组分：青蒿酸、氨苄西林钠、环丙沙星、多糖+益生菌微囊。本发明的药物组合物选取单体中药有效成分与抗生素联合使用，可在提高抗菌抑杀作用的前提下，减少以往中药和抗生素的使用量，提高药物生物利用度和效能。同时以免疫增强调节为辅助治疗手段，选择多糖与益生菌对患病雏鸡进行肠道菌群调节及重新构建良好的微生物菌系，从而加强雏鸡免疫功能，提高生产性能。本发明的药物组合物可通过混饲给药，适于临床应用且疗效确切，适于集约化规模养殖场应用。The invention relates to a pharmaceutical composition for preventing and treating poultry salmonellosis and a preparation method thereof. The pharmaceutical composition comprises the following components: artemisinic acid, ampicillin sodium, ciprofloxacin, polysaccharide+probiotic microcapsules. The pharmaceutical composition of the present invention selects the active ingredients of a single traditional Chinese medicine and is used in combination with an antibiotic, which can reduce the use amount of traditional traditional Chinese medicine and antibiotics in the past, and improve the bioavailability and efficacy of the medicine on the premise of improving the antibacterial and killing effect. At the same time, using immune enhancement and regulation as an adjuvant treatment method, polysaccharides and probiotics were selected to regulate the intestinal flora of sick chicks and rebuild good microbial flora, thereby strengthening the immune function of chicks and improving production performance. The pharmaceutical composition of the present invention can be administered by mixed feeding, is suitable for clinical application, has exact curative effect, and is suitable for intensive scale farm application.
Description
Technical Field
The invention belongs to the field of veterinary drugs, and particularly relates to a pharmaceutical composition for preventing and treating avian salmonellosis and a preparation method thereof.
Background
The avian salmonellosis is a bacterial disease seriously threatening the intestinal health of poultry caused by one or more salmonella in salmonella of enterobacteriaceae, and is divided into three types, namely pullorum disease, avian typhoid disease and avian paratyphoid disease according to different antigen structures of pathogens. The disease incidence and mortality of pullorum disease are the highest and epidemic, chicks within 2-3 weeks are most susceptible, and the chicks are infected to show diarrhea and discharge white and pasty thin feces, fluff around the anus is often polluted by the feces, and the feces are dried to form lime-like hard blocks; adult chickens are infected with the egg to present chronic or recessive passage, and although the infection has no clinical symptoms, the infection affects the egg laying amount and the fertilization rate, and can also be transmitted to offspring chickens through the egg. The disease rate of chicks is 20-40% in old epidemic stations, but the disease rate is obviously increased when newly introduced into the sick chicken stations, and the disease death rate is higher than that in old epidemic stations. Salmonella inducing avian paratyphi is of great importance in public health, since it can infect a wide variety of animals and humans, for example, humans most commonly in the gastroenteritis type (food poisoning) after infection. The pathogens of the disease have a plurality of serotypes, the disease is difficult to prevent by using vaccines, antibiotics such as fluoroquinolones and ampicillin can control the disease but cannot be completely eliminated, and the problems of long-term abuse of the antibiotics, easy generation of drug resistance of bacteria and increasingly prominent drug residue of livestock and poultry products are caused. Therefore, according to the sensitivity of bacteria to drugs, the antibacterial drugs are required to be used correctly in clinic, and meanwhile, a novel high-efficiency low-toxicity residue-free pharmaceutical preparation is sought for controlling the salmonellosis of the fowl.
The Chinese herbal medicine has nonspecific antibacterial effect, low residual toxicity, and no drug resistance. However, the traditional Chinese medicine preparation clinically applied to the treatment of pullorum disease at present is mainly powder, has poor palatability and low bioavailability, and cannot be applied to the development and use of modern intensive large-scale breeding industry. Earlier researches show that artemisinin synthetic precursor-artemisinic acid can cooperate with different quinolones or beta-lactam antibiotics to generate better antibacterial effects on diarrhea-causing and drug-resistant salmonella, the effect is obviously superior to that of independently using artemisinic acid monomers or independently using antibiotics, the antibacterial sensitization effect of the combined use of the artemisinin synthetic precursor and the antibiotics is synergistic, and the drug sensitivity of drug-resistant bacteria on the antibiotics and the artemisinic acid can be enhanced. Compared with the Minimum Inhibitory Concentration (MIC) value of 1 when the single medicine is used alone, the minimum concentration of the single medicine is only 1/4MIC, 1/8MIC and 1/16MIC when the single medicine is used in combination, and the antibacterial effect is doubled, so that the use amount of clinical antibiotics can be greatly reduced. In conclusion, the effective components of the monomeric traditional Chinese medicine are selected for the salmonellosis fowl to be combined with the antibiotics, so that the use amount of the traditional Chinese medicine and the antibiotics can be reduced on the premise of improving the drug effect, the bioavailability and the efficacy of the medicine are improved, and the method is an ideal research and development new way. Meanwhile, the immune enhancement adjustment is taken as an auxiliary treatment means, and polysaccharide and probiotics are selected to adjust intestinal flora of the sick chicks, so that the recovery is accelerated, a good microbial strain is reconstructed, the immune function of the chicks is comprehensively enhanced, and the production performance is improved.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the pharmaceutical composition for treating the salmonellosis of the poultry, which addresses both the symptoms and root causes and fundamentally solves the intestinal health problem of the poultry.
In order to achieve the purpose, the invention adopts a pharmaceutical composition for preventing and treating avian salmonellosis, which is characterized in that: the pharmaceutical composition comprises the following components in parts by weight: 10-30 parts of arteannuic acid; 0.001-0.002 parts of ampicillin sodium; 0.00005-0.00015 part of ciprofloxacin; 270 portions of microcapsule prepared from tremella polysaccharide, bacillus subtilis and candida utilis and 290 portions of microcapsule.
Further, the pharmaceutical composition comprises the following components in parts by weight: 20 parts of arteannuic acid; 0.00125 part of ampicillin sodium; 0.0001 part of ciprofloxacin; 280 parts of tremella polysaccharide, bacillus subtilis and candida utilis microcapsules.
Further, when the microcapsule is prepared, the microcapsule comprises the following components in parts by weight: 70 parts of tremella polysaccharide, 70 parts of bacillus subtilis and 140 parts of candida utilis.
The invention also provides a preparation method of the pharmaceutical composition for treating salmonellosis avium, which comprises the following steps:
step 1, preparing tremella polysaccharide, bacillus subtilis and candida utilis microcapsules respectively;
step 1.1, inoculating a fresh bacillus subtilis culture to a trypticase soytone liquid medium, and carrying out constant-temperature shaking culture for about 24 hours under the conditions of constant-temperature shaking: the temperature is 30-35 ℃, the rotating speed is 150r/min, and the content of the viable bacteria in the culture medium is enlarged and cultured to 9.0 multiplied by 108CFU/mL is preferred; and (3) carrying out low-temperature centrifugation on the obtained bacterial liquid, wherein the low-temperature centrifugation conditions are as follows: centrifuging for 5-6 min at 4 ℃ and 3000rpm, discarding the supernatant and collecting bottom bacterial sludge; adding PBS buffer solution with pH of 7.4, blowing and beating the mixed bacterial sludge by a liquid transfer gun, and centrifuging at low temperature to obtain clean wet bacteria; weighing 70mg of bacteriaAdjusting the cell concentration to 1.2 × 10 with physiological saline9CFU/mL, and storing at 4 ℃ for later use;
step 1.2, inoculating the candida utilis fresh rejuvenation culture to a basic culture medium, and carrying out constant-temperature oscillation culture for 24 hours under the conditions of constant-temperature oscillation: the temperature is 28 ℃, the rotating speed is 180r/min, and the content of the viable bacteria in the culture medium is expanded and cultured to 9.0 multiplied by 108CFU/mL; carrying out low-temperature centrifugation under the following conditions: centrifuging at 4 ℃ and 3000rpm for 5-6 min, and removing supernatant to collect bottom bacterial sludge; adding PBS buffer solution with pH of 7.4, blowing and beating the mixed bacterial sludge by a liquid transfer gun, and centrifuging at low temperature to obtain clean wet bacteria; weighing 140mg of bacterial sludge, and adjusting the bacterial concentration to 1.2X 10 by using normal saline9CFU/mL, and storing at 4 ℃ for later use;
step 1.3. preparation of microencapsulated required reagent solution: preparing sodium alginate water solution with mass concentration of 2.0%, and preparing CaCl with mass concentration of 2.0%2The solution is used as a first capsule forming solution, a chitosan solution with the mass concentration of 2.0 percent is used as a second capsule forming solution, and CaCl with the mass concentration of 1.0 percent is prepared2The solution is used as a washing solution; 20mL of soybean salad oil is taken as an emulsifier, and span 80 with the volume ratio of 0.1-0.4 percent is contained.
Step 1.4, taking tremella polysaccharide as an auxiliary core material and taking sodium alginate-chitosan microcapsules as a carrier to prepare microecological preparation microcapsules;
slowly adding the bacillus subtilis suspension, the candida utilis suspension and 70mg of tremella polysaccharide which are prepared in the step 1.1 and the step 1.2 into 100mL of 2.0% sodium alginate aqueous solution; adding 2.0mL of soybean salad oil as emulsifier, and homogenizing and emulsifying for 30min under the magnetic stirring effect of 400rpm at 37 deg.C to form uniform turbid emulsion;
step 1.5, the homogeneous emulsion prepared in the step 1.4 is extruded and dropped into 2.0 percent of CaCl through an injector2Coating in the solution, wherein the coating stirring speed is 100-150 rpm; after the reaction is finished, standing and curing for about 40 min; removing supernatant, and washing the microspheres with the 1.0% CaCl2 solution; reacting with 2.0% chitosan solution, mixing, and further curing for 30 min; the microspheres are deionizedWashing with water, filtering, and naturally drying or freeze drying to obtain microcapsule product coated with Tremella polysaccharide, Bacillus subtilis and Candida utilis;
step 2, preparing the pharmaceutical composition: according to the parts by weight, 20 parts of arteannuic acid; 0.00125 part of ampicillin sodium; 0.0001 part of ciprofloxacin; 280 parts of tremella polysaccharide, bacillus subtilis and candida utilis microcapsules, wherein 70 parts of tremella polysaccharide, 70 parts of bacillus subtilis and 140 parts of candida utilis; weighing in sequence, and mixing; weighing the medicinal mixture according to the dosage to be filled, subpackaging, packaging with small heat-sealed aluminum foil bags, labeling, and storing at 4 deg.C.
Furthermore, the invention also provides application of the pharmaceutical composition in preparing a medicament for preventing and treating salmonellosis fowl.
Furthermore, the invention also provides application of the pharmaceutical composition, which is used for treating diseases caused by pullorum disease, fowl typhoid and fowl paratyphoid salmonella infection.
Further, the pharmaceutical composition is oral powder, is taken by mixing with feed, and is added into the feed in a treatment dosage of 3.0g/10kg of chicken feed, and 1 treatment course comprises 3-5 days; the feed is added in a preventive amount of 1.5g/10kg, and 3-5 days are 1 course of treatment
Compared with the prior art, the pharmaceutical composition can effectively prevent and treat salmonellosis of poultry, achieves the effect of treating both principal and secondary aspects of disease, and fundamentally solves the intestinal health problem of poultry. The invention has the following beneficial effects:
(1) the pharmaceutical composition prepared by the invention can be used for treating acute diarrhea caused by salmonella of poultry, such as pullorum disease, fowl typhoid and fowl paratyphoid. When the artemisinic acid contained in the compound is combined with beta-lactam antibiotics ampicillin sodium and quinolone antibiotics ciprofloxacin, the combined antibacterial sensitization effect can be generated. The combined sensitization composition can synergistically reverse drug resistance of salmonella causing diarrhea, improve drug sensitivity of drug-resistant salmonella, and remarkably enhance the bacteriostatic action of artemisinic acid or antibiotics, and the diarrhea treatment effect is obviously superior to that of independent use of artemisinic acid or independent use of antibiotics, and the synergistic bactericidal action can be enhanced in multiples. Compared with the Minimum Inhibitory Concentration (MIC) value of 1, the minimum concentration of the single drug is only 1/4MIC, 1/8MIC and 1/16MIC when the single drug is used in combination. Therefore, the selection of the effective components of the monomer traditional Chinese medicine and the antibiotics are combined for use, so that the use amount of the traditional Chinese medicine and the antibiotics can be reduced on the premise of improving the antibacterial and killing effects, the bioavailability and the efficacy of the medicine are improved, and the method is an ideal research and development new way.
(2) The invention provides a pharmaceutical composition for treating salmonellosis of poultry, which contains tremella polysaccharide, probiotics bacillus subtilis and candida utilis, adopts an enteric microcapsule formulation, and is particularly beneficial to being taken by chicks. The microcapsule has good tightness, can avoid the inhibition and killing effect of antibiotics in the medicine composition on probiotics, can better improve the stability of the medicine, and is convenient to transport, store and use. Secondly, the medicine in the microcapsule can be dispersed, dissolved and absorbed at fixed points in the gastrointestinal tract of the poultry, is particularly beneficial to protecting the biological activity of the contained probiotics, can effectively ensure the probiotics to play a role in the intestinal tract of the sick livestock, and reduces the number of pathogenic salmonella in the intestinal tract through competitive exclusion, thereby improving the bioavailability of the medicine.
(3) According to the pharmaceutical composition provided by the invention, the tremella polysaccharide and the probiotics are combined together, intestinal flora is coordinated by the tremella polysaccharide, and the tremella polysaccharide can stimulate immune response of animal organisms and reduce stress reaction of animals by virtue of rich nutritional ingredients such as protein, vitamins and amino acids in the tremella polysaccharide and non-specific immunogenicity of the probiotics; the tremella polysaccharide can also be used as an adjuvant for certain exogenous antigens such as toxins, viruses and pathogenic bacteria, so that the titer of the antigens is increased, and the cellular and humoral immune response of an animal body is enhanced. In addition, the disease resistance of poultry can be enhanced, stress reaction caused by factors such as drastic change of temperature and invasion of microorganisms can be relieved, and the disease resistance of animal organisms can be improved.
(4) The probiotic bacillus subtilis and candida utilis contained in the microcapsule can regulate and control the micro-ecological environment of the gastrointestinal tract of animals, can selectively promote the growth and the reproduction of beneficial bacteria in the intestinal tract of the animals, form micro-ecological competitive advantages in the gastrointestinal tract, and inhibit the adhesion and the colonization of harmful exogenous bacteria and intrinsic putrefying bacteria in the intestine in the intestinal tract, thereby exerting the normal functions of normal intestinal flora in barrier, nutrition and immunity and maintaining the normal digestive tract environment. Secondly, the contained candida utilis contains rich protein, the taste of the product can be improved, the recovery can be accelerated and a good intestinal microbial strain can be established by stimulating the defense capacity of the immune system of the sick poultry, the generation of toxic substances in the intestinal tract can be reduced, the integrity of the intestinal mucosa can be protected, and the candida utilis has good promotion effect on the immune function and the production performance of the poultry organism.
The pharmaceutical composition provided by the invention is prepared by mixing artemisinic acid, antibiotics, tremella polysaccharide, bacillus subtilis and candida utilis microcapsules in certain parts by weight according to the pathogenesis inducement and clinical characteristics of the salmonellosis avium and the treatment principles of anti-inflammation and bacteriostasis, bowel clearing and diarrhea checking and immune adjustment. Firstly, the synergistic antibacterial sensitization effect between the drug monomer and the antibiotic can multiply enhance the antibacterial effect, the antidiarrheal effect is ideal, the treatment time is short, and the use amount of the antibiotic can be reduced, so that the threat of antibiotic residue on the public health of pasture grasslands is eliminated, and the social requirement of providing safe and pollution-free animal-derived food at present is met. Secondly, the tremella polysaccharide, the bacillus subtilis and the candida utilis microcapsules are enteric microcapsules, are particularly suitable for being taken by chicks (mixed with feed), and meet the convenience requirement of clinical medication; after the medicinal components in the microcapsule are dissolved and released in the intestinal tract, the propagation of pathogenic salmonella can be inhibited, the proliferation of probiotics is promoted, and the intestinal mucosa of the chick is quickly repaired. The preparation method of the pharmaceutical composition is simple, safe and reliable, convenient in material selection, low in cost and easy to form large-scale production.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below. It should be understood, however, that the description herein of specific embodiments is only intended to illustrate the invention and not to limit the scope of the invention. The test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1MIC values for artemisinic acid, ampicillin sodium and ciprofloxacin, and Combined antimicrobial FICI index determination
(1) MIC value determination of effective component and antibiotic of monomer traditional Chinese medicine
The MIC values of artemisinic acid monomer were determined using a 96-well plate broth dilution method according to the procedures recommended by the American clinical laboratory standards Committee (NCCLS). MIC values of antibiotics ampicillin sodium and ciprofloxacin for test bacteria were determined by an E-test using drug sensitive strips (Liofilchem. r.l, italy) containing the drug, reading the scale of the reading at the transverse intersection of the zone of inhibition and the strip. Antibiotic ciprofloxacin (Lot # C10532503 ≧ 98%) available from Shanghai Michelin Biotechnology ltd; ampicillin sodium (Lot.No.826H031, ≧ 85%) was purchased from Beijing Soilebao Tech Co., Ltd. Salmonella pullorum (Salmonella pullorum, BNCC124693, available from Beijing Beinana institute of Biotechnology); salmonella paratyphi B (Salmonella paratyphi B, cmcc (B)50094, available from china general microbiological culture collection center); the rest 2 salmonella strains are clinical isolates, and are preserved in the laboratory and used for experiments.
(2) FICI index determination of combination drug by checkerboard dilution
The combined scheme comprises artemisinic acid + beta-lactam antibiotics ampicillin sodium, artemisinic acid + quinolone antibiotics ciprofloxacin and artemisinic acid + ampicillin sodium + ciprofloxacin (3 drug combinations), FICI values of 4 salmonella strains (12 bacteria combined anti-sensitizers) are respectively measured, 5 dilutions of each combined anti-sensitizers (except drug controls and dilution controls) are carried out, and finally 300 combined sensitizer combinations with different dilutions are obtained. The concentration of the antibacterial drugs (monomer artemisinic acid and antibiotics) is 2 XMIC to 1/16 XMIC (6 dilutions including drug control and dilution control). And calculating the partial antibacterial concentration index (FICI) of each row according to the minimum drug concentration of the bacterial growth pores which are not found in each row after combination.
FICI interpretation criteria: FICI ≦ 0.5, synergistic; 0.5< FICI <1, partially synergistic; FICI ═ 1, additive effect; 1< FICI <4, no correlation; FICI ≧ 4, antagonistic action.
(3) Test results (Table 1)
The 2 clinical isolates of salmonella selected in the test have moderate drug resistance to ampicillin sodium and moderate sensitivity to ciprofloxacin, and the artemisinic acid and the 2 antibiotics have poor antibacterial effect when being applied independently. Through combined application of artemisinic acid and antibiotics, a good antibacterial sensitization effect can be generated on 4 strains of salmonella, the synergistic effect is obvious, the drug sensitivity of drug-resistant bacteria to the antibiotics or the artemisinic acid can be enhanced, the effect is obviously superior to that of singly using artemisinin or the artemisinic acid or singly using the antibiotics, and the generated combined antibacterial sensitization effect is the most obvious in combination of the artemisinic acid, ampicillin sodium and ciprofloxacin. The antibacterial effect is increased in times when the medicines are combined, and compared with the Minimum Inhibitory Concentration (MIC) value of 1 when the medicines are used alone, the minimum concentrations of the single medicines are only 1/4MIC, 1/8MIC and 1/16MIC when the medicines are combined.
TABLE 1 minimum FICI index results for artemisinic acid in combination with 2 antibiotics on 4 Salmonella strains
Example 2 preparation of Tremella polysaccharide + Bacillus subtilis + Candida utilis microcapsules
The experiment uses Bacillus subtilis (purchased from Beijing Naucai Chuanglian Biotechnology research institute, as feed yeast, with the strain number BNCC223758) and Candida utilis (purchased from China Industrial microbial cultures preservation management center, as feed yeast, with the strain number CICC1769) as model strains, tremella polysaccharide (food grade, purity 30%, product batch number 20200312, Saanen-Na bioengineering Co., Ltd.) as auxiliary core materials, and sodium alginate-chitosan microcapsule as a carrier to prepare the microecological preparation microcapsule.
(1) Culturing Bacillus subtilisInoculating the extract into trypticase soy peptone liquid culture medium, performing constant temperature shaking culture for about 24h (30-35 deg.C, 150r/min), and performing amplification culture to live bacteria content of 1.2 × 109CFU/mL is preferred (4.0 McLeod tube concentration). And centrifuging the obtained bacterial liquid at a low temperature (4 ℃, 3000rpm, 5-6 min), and discarding the supernatant to collect bottom bacterial sludge. Adding PBS buffer solution with pH of 7.4, blowing and beating the bacterial sludge uniformly by a liquid transfer gun, and centrifuging at low temperature under the same condition to obtain clean wet bacteria. Weighing 70mg of bacterial sludge, and adjusting the bacterial concentration to 1.2 multiplied by 10 by using normal saline9CFU/mL (4.0 McLeod tube concentration), stored at 4 ℃ for use.
(2) Inoculating the fresh rejuvenation culture of Candida utilis to a basic culture medium (including glucose 30g/L, sucrose 20g/L, peptone 30g/L, yeast extract 10g/L, and malt extract 100mL/L, KH2PO4 2.0g/L、NH4SO4g/L、MgSO41.0 g/L. Adjusting pH to 5.5, sterilizing at 115 deg.C for 20 min). Shaking and culturing at constant temperature for about 24h (28 deg.C, 180r/min), and enlarging and culturing viable bacteria content in the culture medium to 1.2 × 109CFU/mL is preferred (4.0 McLeod tube concentration). Centrifuging at 4 ℃ and 3000rpm for 5-6 min, and removing the supernatant to collect bottom bacterial sludge. Adding PBS buffer solution with pH of 7.4, blowing and beating the bacterial sludge uniformly by a liquid transfer gun, and centrifuging at low temperature under the same condition to obtain clean wet bacteria. Weighing 140mg of bacterial sludge, and adjusting the bacterial concentration to 1.2X 10 by using normal saline9CFU/mL (4.0 McLeod tube concentration), stored at 4 ℃ for use.
(3) Preparation of microencapsulated solutions of the reagents required: comprises preparing 2.0% sodium alginate aqueous solution (w/v) and 2.0% CaCl2Solution (w/v, i.e. encapsulation solution 1), 2.0% chitosan solution (w/v, encapsulation solution 2), 1.0% CaCl2Solution (w/v, wash solution); 20mL of soybean salad oil (emulsifier, containing span-80 with the volume ratio of 0.1-0.4%).
(4) Slowly adding the bacillus subtilis suspension, the candida utilis suspension and 70mg of tremella polysaccharide which are prepared in the steps (1) and (2) into 100mL of 2.0% sodium alginate aqueous solution by taking two feeding probiotics as model strains, taking tremella polysaccharide as an auxiliary core material and taking sodium alginate-chitosan microcapsule as a carrier; then 2.0mL of emulsifier soybean salad oil is added, and homogeneous emulsification is carried out for 30min under the magnetic stirring effect of 400rpm at 37 ℃ to form uniform turbid emulsion.
(5) Extruding and dripping the homogenized emulsion prepared in the step (4) into 2.0% CaCl by using an injector2Coating in the solution, wherein the coating stirring speed is 100-150 rpm; after the reaction is finished, standing and curing for about 40 min; removing supernatant, and adding 1.0% CaCl into the microsphere2Washing the solution; reacting with 2.0% chitosan solution, mixing, and further curing for 30 min; and (3) cleaning and filtering the microspheres by using deionized water, and naturally drying to obtain a microcapsule product which has an average particle size of 1.0-1.5 mm and is coated with tremella polysaccharide, bacillus subtilis and candida utilis.
Example 3 in vitro release test of polysaccharide + probiotic microcapsules
(1) Measurement of swelling degree of microcapsule
The microcapsule is spherical, and the diameter of the particle is 1.0-1.5 mm after the microcapsule is washed and dried by normal saline. After the microcapsule is soaked in 0.9% normal saline for 3h, the grain size basically has no obvious change; in the artificial simulated gastric juice, the particle size is not obviously changed after being soaked for 1h, the microcapsule begins to expand after 2h, the average diameter of the microcapsule is 1.8mm after being soaked for 3h, and the expansion rate is 120 percent; and after the artificial intestinal juice is soaked for 10min, the microcapsules begin to expand, after the artificial intestinal juice is soaked for 1h, the average diameter of the microcapsules can reach 1.8mm, and the expansion rate of the microcapsules reaches 200 percent.
The result proves that the microcapsule prepared by taking chitosan-sodium alginate as the coating material has better resistance to pepsin, and can effectively resist the action of simulated artificial gastric acid without disintegration.
(2) Calculation of embedding rate and embedding yield and research of stability test
2.1 determination of viable count on the surface of the microcapsule: directly dissolving the surface thalli of the microcapsule with PBS (buffer solution), and counting viable bacteria after dilution by multiple times.
2.2 determination of viable count in product: and (3) putting the microcapsule product into the microcapsule-dissolving liquid, stirring for 30min, shaking and uniformly mixing to completely dissolve the microcapsule, and counting after dilution by multiple times. The results are as follows:
embedding rate is 1- (micro)The number of living bacteria on the surface of the capsule/the number of living bacteria in the product) x 100 percent is 1- (3 multiplied by 10)8CFU/6.6×109CFU)×100％＝95.45％
The embedding yield (number of living bacteria in the product)/(number of added living bacteria) × 100% ═ 6.6 × 109CFU/7.2×109CFU×100％＝91.67％
2.3 the microcapsule-embedded viable bacteria preparation is stored for 30 days at room temperature, the survival rate of viable bacteria is 98.8 percent, the survival rate of viable bacteria is 90 days, the survival rate of viable bacteria is 50.6 percent, the survival rate of viable bacteria is 180 days, and the survival rate of viable bacteria is 15.7 percent; correspondingly, when the control sample which is not microencapsulated is stored at room temperature for 30 days, the survival rate of the viable bacteria is about 54.1%, and after the control sample is stored for 90 days, the survival rate of the viable bacteria is only 1.41%. The two samples are stored for 90 days under the same condition (refrigerated storage at 4 ℃), and the survival rate of viable bacteria of the samples without microencapsulation is only 66.2% of that of the samples with microencapsulation preparation.
The result shows that the sodium alginate-chitosan is used for embedding the bacillus subtilis, the candida utilis and the tremella polysaccharide to prepare the microcapsule, so that the survival rate of the bacteria is improved to a certain extent, the normal-temperature storage life of the viable bacteria is prolonged, and the immune efficacy of the probiotics and the tremella polysaccharide can be better improved.
Example 4 simulated gastric fluid, simulated intestinal fluid tolerance test of polysaccharide + probiotic microcapsules
3.1 determination of gastric juice tolerance simulated microcapsules (Table 2)
And (3) putting 0.1g of microcapsules into simulated gastric juice, shaking and uniformly mixing, incubating in a water bath at 37 ℃, taking out every 0.5h, and counting viable bacteria. Plate counting was used, 3 replicates at a time.
Table 2 results of the survival rate of the cells in simulated gastric fluid (x 10)9CFU)
After the uncoated probiotics are digested in simulated gastric juice with the pH of 2.5 for 2 hours and 3 hours, the number of the live bacteria is respectively reduced by 46.94 percent and 56.81 percent. After the microencapsulation treatment, the number of viable bacteria decreased by 9.55% and 11.36% respectively at the same time point. The result shows that the microcapsule preparation can resist gastric acid with the pH value of 2.5, the microcapsule preparation coated by the probiotics and the tremella polysaccharide by the sodium alginate-chitosan can well resist the acid environment, the damage of acid gastric juice to the probiotics is reduced, and the utilization efficiency of the bacillus subtilis can be improved after the microcapsule preparation is microencapsulated.
3.2 microcapsule simulated intestinal fluid tolerance assay (Table 3)
Washing the microcapsules treated by different simulated gastric juice with PBS for 2 times, placing the microcapsules into simulated intestinal juice, shaking and uniformly mixing, incubating in water bath at 37 ℃, sampling every 0.5h, and counting viable bacteria. The counting method and data processing are the same as above.
The result shows that after the microcapsule is continuously digested in simulated intestinal fluid with the pH of 6.8 for 1.0h, 2.0h and 3.0h, the number of viable bacteria is respectively reduced by 44.79%, 57.26% and 61.54%; while the control group had 46.94%, 53.05% and 58.52% decrease in viable cell counts at the same time points, respectively. After digestion in simulated intestinal juice for 1.0h, the reduction frequency of the total number of viable bacteria between two groups is similar, which shows that the expansion release rate of the medicine reaches the highest level after the microcapsule is soaked in the intestinal juice for 1.0h, and then the release amount gradually tends to be stable and can play a role of slow release; secondly, the microcapsule is very sensitive to the artificial intestinal juice and has enteric solubility, and after 3.0h of digestion, the reduction frequency of the total number of the live bacteria between two groups is similar without obvious difference.
Table 3 results of cell survival rate in simulated intestinal juice (× 10)9CFU)
EXAMPLE 5 preparation of pharmaceutical composition
Materials: artemisinic acid (Lot # C15H2202, ≧ 98%) available from Guangdong Wenjiang chemical reagents, Inc.; antibiotic ciprofloxacin (Lot # C10532503 ≧ 98%) available from Shanghai Michelin Biotechnology ltd; antibiotic ampicillin sodium (Lot. No.826H031, ≧ 85%) was purchased from Beijing Soilebao Tech Co., Ltd.
Formula 1:
10 parts of arteannuic acid, 0.002 part of ampicillin sodium, 0.00015 part of ciprofloxacin, 290 parts of tremella polysaccharide, bacillus subtilis and candida utilis micro-capsules
And (2) formula:
10 parts of arteannuic acid, 0.001 part of ampicillin sodium, 0.00005 part of ciprofloxacin, 290 parts of tremella polysaccharide, bacillus subtilis and candida utilis micro-capsules
And (3) formula:
artemisinic acid 20 parts, ampicillin sodium 0.002 part, ciprofloxacin 0.00015 part, tremella polysaccharide + bacillus subtilis + candida utilis microzyme microcapsule 280 parts
And (4) formula:
artemisinic acid 20 parts, ampicillin sodium 0.001 part, ciprofloxacin 0.00005 part, tremella polysaccharide + bacillus subtilis + candida utilis microzyme microcapsule 280 parts
And (5) formula:
30 parts of arteannuic acid, 0.002 part of ampicillin sodium, 0.00015 part of ciprofloxacin, 270 parts of tremella polysaccharide, bacillus subtilis and candida utilis microcapsules
And (6) formula:
30 parts of arteannuic acid, 0.001 part of ampicillin sodium, 0.00005 part of ciprofloxacin, 270 parts of tremella polysaccharide, bacillus subtilis and candida utilis micro-capsules
And (3) formula 7:
artemisinic acid 20 parts, ampicillin sodium 0.00125 parts, ciprofloxacin 0.0001 parts, tremella polysaccharide + bacillus subtilis + candida utilis microzyme microcapsule 280 parts
The preparation method comprises the following steps:
(1) the tremella polysaccharide, bacillus subtilis and candida utilis microcapsule is a microcapsule preparation prepared according to the preparation method and steps of the example 2.
(2) Preparation of pharmaceutical compositions
Weighing the raw materials of the components according to the formula proportion, sequentially weighing 100 parts by weight of the artemisinic acid, 100 parts by weight of the ampicillin sodium, 100 parts by weight of the ciprofloxacin, 100 parts by weight of the tremella polysaccharide, the bacillus subtilis and the candida utilis microcapsules, and uniformly mixing; weighing the medicine mixture according to the required dosage (10% of the total weight of each medicine mixture), subpackaging 10 parts (about 3.0 g/part), packaging in a small heat-sealed aluminum foil bag, labeling, and storing at 4 ℃ for later use.
EXAMPLE 6 efficacy of the pharmaceutical composition and test of efficacy against Experimental Chicken Salmonella
(1) The pharmaceutical composition is a preparation prepared according to the method of example 5, and specifically comprises: the pharmaceutical composition of formulation 7.
(2) Test animals and clinical drug groups
2.1 drug treatment groups and dosages
High dose group (including prevention group, treatment group): 2 parts of the pharmaceutical composition per 10kg of chicken feed (w/w), 3-5 days for 1 course of treatment.
Medium dose group (including prevention group, treatment group): 1 part of the pharmaceutical composition per 10kg of chicken feed (w/w), 3-5 days for 1 course of treatment.
Low dose group (including prevention group, treatment group): 0.5 part of the pharmaceutical composition per 10kg of chicken feed (w/w), 3-5 days for 1 treatment course.
Control drug group (treatment only group): 5% ciprofloxacin hydrochloride soluble powder (product batch: 20191226, manufactured by Luoyang Hongmu Biotech Co., Ltd.; 100 g/bag, 1.0g/1.5kg feed (w/w), 3-5 d for 1 treatment course)
2.2 strains for challenge: the strain is Salmonella pullorum (Salmonella pullorum, BNCC124693, available from Beijing Beinana union Biotechnology research institute). Inoculating a fresh culture of salmonella to a trypticase soy peptone liquid medium, shaking at a constant temperature of 30-35 ℃ for 24h, and adjusting the bacteria concentration to 1.0 multiplied by 108CFU/mL, stored at 37 ℃ for use.
2.3 test animals: the method comprises the following steps of (1) breeding a 1-day-old Liangfenghua broiler chicken purchased from Yongdeng division of white-silver Dojingzhen poultry farming Limited company in a pollution-free cage with the room temperature of 20-24 ℃ and the relative humidity of 50 +/-10%; the feed is a complete feed (non-drug compound feed) for the broiler chickens, and is purchased from Lanzhou dynamic feed Co. Feed 3 times daily and drink water freely.
2.4 test methods
When the chicks are raised to 14 days old, healthy chickens with good mental status are taken, weighed one by one and randomly divided into 9 groups, the weight difference of each chicken in each group is not more than 10g, the total weight difference of each group is not more than 200g, and each group comprises 20 chickens. After grouping, except for the healthy control group, the other groups of chickens were injected with 0.6mL of Salmonella suspension in the abdominal cavity. Each trial medication treatment group was treated with medication after clinical symptoms had occurred. The healthy control group and the infected control group were not dosed with any drug. The preventive high, medium and low dose groups were administered 3 days in advance, the same amount of bacteria were attacked at 14 days of age, and administration was continued for 5 days. The healthy control group was fed with normal drinking water and kept separately. The incidence and recovery of the chickens in each group were closely followed during the treatment period. The morbidity, mortality and cure rate of each group were observed and recorded. And (4) performing a cesarean examination on the dead chicken, aseptically collecting the liver or lung of the chicken, and performing bacteria separation and identification.
2.5 therapeutic Effect determination and Observation index
Death: the typical symptoms of pullorum disease appeared and died during the test, the autopsy had typical pathological features, and salmonella pullorum disease was isolated and cultured from liver, spleen and heart and judged to be infected with the bacterium and died. Mortality was calculated for each group based on the number of dead chickens.
And (3) curing: after administration, the chicken can recover normal spirit state and appetite, no craw symptom appears, no paste feces is discharged, and typical symptoms such as villus pollution around anus and inflammation around anus disappear; and (4) carrying out identification culture on the suspected bacterial colonies obtained by the isolation culture of the excrement sample by xylose lysine deoxycholate to obtain no salmonella typical bacterial colony which is judged to be cured.
The method has the following advantages: after administration, the above clinical symptoms of the chicken are obviously improved, diarrhea symptoms are relieved, and mental state, appetite, respiration and the like are gradually recovered to be normal, so that the chicken is judged to be effective.
Relative rate of weight gain: and calculating the average weight gain according to the weight of each chicken at the beginning and the end of the test, and then calculating the average weight gain of each group of chickens, wherein the weight gain of the blank control group is 100%, and the weight gain of each group is compared with the weight gain of each group to obtain the relative weight gain rate of each group.
2.6 test results (Table 4)
Except for the healthy control group, each group of challenge chickens all showed the typical clinical symptoms of pullorum disease. After administration, compared with an infection control group, each drug treatment group can rapidly relieve diarrhea clinical symptoms, has quick response and no abnormal reaction or toxic and side effects, and has no dead chicken in high and medium dose groups and the morbidity and mortality rate obviously lower than those of the infection control group. After the administration for 5 days, the weight increasing effect of the chicken is obvious, which shows that the pharmaceutical composition provided by the invention has good treatment effect on the salmonellosis fowl, has small toxic and side effects, and simultaneously has the function of improving the relative weight increasing rate and the production performance. After 3 days of administration, the mental state and appetite of the dead chicks in each drug treatment group are basically recovered to normal; after 5 days of administration, paste-like gray thin feces are not discharged, and the feces sample is separated and cultured by the identification culture medium, and no salmonella typical colony is detected, and the disease can be judged to be cured.
The results show that: the cure rate of the high and medium dose prevention group and the treatment group of the pharmaceutical composition is obviously higher than that of a control drug ciprofloxacin hydrochloride group with 5 percent; the cure rate of the low-dose group of the pharmaceutical composition is slightly lower than that of the control drug group. The infected control group chicken only grows slowly, and the weight gain effect and the appetite recovery condition of the high, medium and low prevention groups are obviously better than those of the high, medium and low treatment groups and the control drug group, and the material weight ratio results are basically similar. In consideration of clinical medication cost, a medium dose group (1 part of the pharmaceutical composition/10 kg of chicken feed for 3-5 days for 1 treatment course) is recommended as an optimal therapeutic dose, and a low dose group (0.5 part of the pharmaceutical composition/10 kg of chicken feed for 3-5 days for 1 treatment course) is recommended as a prophylactic dose for chicks.
TABLE 4 therapeutic effect and Productivity results of each drug treatment group on Salmonella gallinarum (n ═ 20)
The pharmaceutical composition has good effect of treating salmonellosis of poultry, takes effect quickly, has low cost, is safe and practical, can reduce the use of antibiotics, can improve the resistance of animal organisms, and is suitable for popularization and use in poultry breeding.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A pharmaceutical composition for preventing and treating avian salmonellosis is characterized in that: the pharmaceutical composition comprises the following components in parts by weight: 10-30 parts of arteannuic acid; 0.001-0.002 parts of ampicillin sodium; 0.00005-0.00015 part of ciprofloxacin; 270 portions of microcapsule prepared from tremella polysaccharide, bacillus subtilis and candida utilis and 290 portions of microcapsule.
2. The pharmaceutical composition of claim 1, wherein: the pharmaceutical composition comprises the following components in parts by weight: 20 parts of arteannuic acid; 0.00125 part of ampicillin sodium; 0.0001 part of ciprofloxacin; 280 parts of tremella polysaccharide, bacillus subtilis and candida utilis microcapsules.
3. The pharmaceutical composition according to claim 1 or 2, characterized in that: when the microcapsule is prepared, the microcapsule comprises the following components in parts by weight: 70 parts of tremella polysaccharide, 70 parts of bacillus subtilis and 140 parts of candida utilis.
4. A process for the preparation of a pharmaceutical composition according to any one of claims 1 and 2, comprising the steps of:
step 1, preparing tremella polysaccharide, bacillus subtilis and candida utilis micro-capsules;
step 1.1, inoculating a fresh bacillus subtilis culture to a trypticase soytone liquid medium, and carrying out constant-temperature shaking culture for about 24 hours under the conditions of constant-temperature shaking: the temperature is 30-35 ℃, the rotating speed is 150r/min, and the content of the viable bacteria in the culture medium is enlarged and cultured to 9.0 multiplied by 108CFU/mL is preferred; and (3) carrying out low-temperature centrifugation on the obtained bacterial liquid, wherein the low-temperature centrifugation conditions are as follows: centrifuging for 5-6 min at 4 ℃ and 3000rpm, discarding the supernatant and collecting bottom bacterial sludge; further adding PBS buffer of pH 7.4Blowing and beating the mixed bacterial sludge by a liquid transfer gun, and centrifuging at low temperature to obtain clean wet bacteria; weighing 70mg of bacterial sludge, and adjusting the bacterial concentration to 1.2 multiplied by 10 by using normal saline9CFU/mL, and storing at 4 ℃ for later use;
step 1.2, inoculating the candida utilis fresh rejuvenation culture to a basic culture medium, and carrying out constant-temperature oscillation culture for 24 hours under the conditions of constant-temperature oscillation: the temperature is 28 ℃, the rotating speed is 180r/min, and the content of the viable bacteria in the culture medium is expanded and cultured to 9.0 multiplied by 108CFU/mL; carrying out low-temperature centrifugation under the following conditions: centrifuging at 4 ℃ and 3000rpm for 5-6 min, and removing supernatant to collect bottom bacterial sludge; adding PBS buffer solution with pH of 7.4, blowing and beating the mixed bacterial sludge by a liquid transfer gun, and centrifuging at low temperature to obtain clean wet bacteria; weighing 140mg of bacterial sludge, and adjusting the bacterial concentration to 1.2X 10 by using normal saline9CFU/mL, and storing at 4 ℃ for later use;
step 1.3. preparation of microencapsulated required reagent solution: preparing sodium alginate water solution with mass concentration of 2.0%, and preparing CaCl with mass concentration of 2.0%2The solution is used as a first capsule forming solution, a chitosan solution with the mass concentration of 2.0 percent is used as a second capsule forming solution, and CaCl with the mass concentration of 1.0 percent is prepared2The solution is used as a washing solution; 20mL of soybean salad oil is taken as an emulsifier, and span 80 with the volume ratio of 0.1-0.4 percent is contained;
step 1.4, taking tremella polysaccharide as an auxiliary core material and taking sodium alginate-chitosan microcapsules as a carrier to prepare microecological preparation microcapsules;
slowly adding the bacillus subtilis suspension, the candida utilis suspension and 70mg of tremella polysaccharide which are prepared in the step 1.1 and the step 1.2 into 100mL of 2.0% sodium alginate aqueous solution; adding 2.0mL of soybean salad oil as emulsifier, and homogenizing and emulsifying for 30min under the magnetic stirring effect of 400rpm at 37 deg.C to form uniform turbid emulsion;
step 1.5, the homogeneous emulsion prepared in the step 1.4 is extruded and dropped into 2.0 percent of CaCl through an injector2Coating in the solution, wherein the coating stirring speed is 100-150 rpm; after the reaction is finished, standing and curing for about 40 min; removing supernatant, and adding 1.0% CaCl into the microsphere2Washing the solution; then withReacting 2.0% chitosan solution, mixing, and further curing for 30 min; washing the microspheres with deionized water, filtering, and naturally drying or freeze drying to obtain microcapsule product coated with Tremella polysaccharide, Bacillus subtilis, and Candida utilis;
step 2, preparing the pharmaceutical composition: according to the parts by weight, 20 parts of arteannuic acid; 0.00125 part of ampicillin sodium; 0.0001 part of ciprofloxacin; 280 parts of tremella polysaccharide, bacillus subtilis and candida utilis microcapsules, wherein 70 parts of tremella polysaccharide, 70 parts of bacillus subtilis and 140 parts of candida utilis; weighing in sequence, and mixing; weighing the medicinal mixture according to the dosage to be filled, subpackaging, packaging with small heat-sealed aluminum foil bags, labeling, and storing at 4 deg.C.
5. Use of the pharmaceutical composition according to any one of claims 1 to 3 for the preparation of a medicament for the prevention and treatment of salmonellosis fowl.
6. The use of the pharmaceutical composition according to claim 5, for the treatment of diseases caused by pullorum disease, fowl typhoid, and fowl paratyphoid salmonella infection.
7. The pharmaceutical composition is an oral powder, and is administered by mixing with feed, the dosage of the pharmaceutical composition added for treatment is 3.0g of the pharmaceutical composition per 10kg of chicken feed, and 1 treatment course lasts for 3-5 days; the prevention addition amount is 1.5g of the pharmaceutical composition per 10kg of chicken feed, and 3-5 days are 1 course of treatment.
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