Preparation method of medicine for treating bovine cholecystitis

Abstract

A method for preparing a drug for treating bovine cholecystitis belongs to the field of biological veterinary medicine. The present invention utilizes the original ecological pathogens of bovine cholecystitis, and further adds bovine blood, trace elements, and freeze-dried Escherichia coli powder, freeze-dried enterococcus powder, freeze-dried Pseudomonas aeruginosa powder, and lecithin. Weak ultrasound is used in a buffer solution to activate the activity of various pathogens, and in the intelligent cultivation process, the corresponding antimicrobial peptides and antimicrobial proteins are strongly induced in the body of the big-headed golden fly larvae. The antimicrobial peptides and antimicrobial proteins have the characteristics of high biological activity and broad spectrum of action. Clinical treatment results show that the drug prepared by the present invention has good efficacy and high efficiency. The production process of the present invention is easy to industrialize and control, the product consistency is good, energy is saved, and it is suitable for large-scale production.

Description

Technical Field

[0001] The invention belongs to the field of biological veterinary medicine preparation, and particularly relates to a method for preparing a medicine for treating bovine cholecystitis. Background Art

[0002] Cholecystitis is a disease that develops in the gallbladder of cattle. When it occurs, the mucous membranes of the eyes turn yellow and the eyelids swell. The cattle become restless and irritable, with noticeable tremors in the front shoulder blades and neck muscles. The animals also experience burning sensations in the mouth, a reddish-yellow mouth, stringy saliva, yellow discharge from the nose, yellow-red mucous membranes at the base of the tongue, dry and constipated stools, and dark yellow urine.

[0003] Niu Changkui proposed the treatment of cattle cholecystitis (bile distension) with the combination of traditional Chinese and western medicine in his paper “Treatment of cattle cholecystitis (bile distension) with the combination of traditional Chinese and western medicine” published in the 4th issue of Animal Husbandry and Veterinary Science and Technology Information in 2012, using injections of western medicines such as atropine, penicillin and analgin for treatment, while using Dahuang Chaihu Decoction composed of traditional Chinese medicines such as Bupleurum, Scutellaria, Shuanghua, Artemisia capillaris, Gardenia, Rhubarb, Corydalis yanhusuo, Curcuma aromatica, Taraxacum mongolicum, Jujube, Citrus aurantium and Gentiana scabra for treatment.

[0004] CN103055060A discloses a compound propolis composition for treating bovine cholecystitis and a preparation method thereof. The composition involved in this invention application is composed of 10 traditional Chinese medicines, including propolis, Artemisia capillaris, rhubarb, dandelion, Lysimachia scabra, Gardenia jasminoides, Alisma orientalis, Terminalia chebula, Sanguisorba officinalis, and Forsythia suspensa.

[0005] In a paper published in the Journal of Animal Science and Veterinary Medicine (January 1994), Kong Xiaoming et al., titled "Pathological Histochemistry and Ultrastructural Studies of Cholecystitis in Yellow Cattle," electron microscopic observations confirmed that when cholecystitis occurs in yellow cattle, not only does the number of glands and glandular cells increase, but the number of rough endoplasmic reticulum and secretory granules in the glandular epithelium also increases significantly. These observations suggest that chronic glandular proliferative cholecystitis in cattle bile is primarily caused by chemical pathogens, physiological factors, and other factors, with no signs of bacterial infection.

[0006] The drugs currently used to treat bovine cholecystitis are mainly chemical drugs or Chinese herbal medicines. The inflammatory reaction of bovine cholecystitis is mostly manifested as chronic glandular proliferative cholecystitis, and some are acute attacks of chronic cholecystitis. Summary of the Invention

[0007] The purpose of the present invention is to solve the problem that the existing drugs for treating bovine cholecystitis are not effective, and to provide a method for preparing the drugs for treating bovine cholecystitis.

[0008] The present invention utilizes native pathogens of bovine cholecystitis, and also adds bovine blood, trace elements, and freeze-dried Escherichia coli powder, freeze-dried Enterococcus powder, freeze-dried Pseudomonas aeruginosa powder, and lecithin. Weak ultrasound is used in a buffer solution to activate the activity of various pathogens. During the intelligent cultivation process, the corresponding antimicrobial peptides and antimicrobial proteins are strongly induced in the larvae of the large-headed golden fly. These antimicrobial peptides and antimicrobial proteins have high biological activity and a broad spectrum of action. Clinical treatment results have shown that the drug prepared by the present invention has good therapeutic efficacy and high efficiency. The production process of the present invention is easy to control industrially, the product has good consistency, saves energy, and is suitable for large-scale production.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] A method for preparing a drug for treating bovine cholecystitis, the method comprising the following specific steps:

[0011] (1) 200g-500g of cattle bile and 1g-50g of inflammatory bile were collected after slaughter, 1000g-5000g of cattle blood, 0.5g-2.0g of ZnSO4, 1.0g-2.0g of CuSO4, 5.0g-10.0g of Ca(OH)2, 0.5g-3.0g of MgSO4, 5.0g-20.0g of Escherichia coli freeze-dried powder, 2.0g-10.0g of Enterococcus freeze-dried powder, 1.0g-2.0g of Anaerobic Bacillus freeze-dried powder, 1.0g-2.0g of Pseudomonas aeruginosa freeze-dried powder and 10.0g-50.0g of lecithin were added, the pH was adjusted to 6.8-7.2 with a buffer solution, and after high-speed stirring, ultrasonic activation was performed to prepare a basic culture medium suitable for the growth of goldfly eggs and larvae;

[0012] (2) Soak 5g-15g of Chrysomyia ovata eggs in H2O2 aqueous solution at room temperature for a period of time, take them out and wash them with purified water. The sterilized Chrysomyia ovata eggs are then set aside.

[0013] (3) 3%-5% of the basic culture medium obtained in (1) is added to a culture tank, the goldfly eggs obtained in (2) are inoculated into the basic culture medium, 20-50g of protein powder is added, and the temperature is set at 20-30°C for cultivation. When the goldfly eggs metamorphose into first-instar larvae, 15%-20% of the basic culture medium obtained in (1) is added again, 3g-5g of Escherichia coli freeze-dried powder and 200-500g of protein powder are added, and the temperature is kept constant for cultivation. When the goldfly larvae grow to the second instar, all the remaining culture medium obtained in (1) is added to the culture tank, 5g-10g of Escherichia coli freeze-dried powder and 1000g-3000g of protein powder are added, the temperature is set at 20-35°C, and the culture is continued for 120h-180h.

[0014] (4) The cultured 3rd-instar chrysopidae larvae crawl out of the culture system, and the larvae collected after automatic separation are soaked in H2O2 aqueous solution for a period of time, then rinsed twice with pure water, then soaked in KMnO4 aqueous solution for a period of time, rinsed 5-10 times with pure water, and then placed in a dehydrator and dehydrated at 200r / min-750r / min for 3-5 minutes, and then filtered with a plate and frame filter at 0.2-5kg / cm 2 The mixture is squeezed under pressure, 10g-20g of solid ammonium sulfate is added as a precipitate, the precipitate is washed with 25%-40% ethanol solvent, and centrifuged to obtain a solid containing antimicrobial peptides and antimicrobial proteins. The solid is then solid-liquid separated, and the separated body fluid of the big-headed golden fly larvae is freeze-dried. The dried antimicrobial peptides and antimicrobial proteins are instantly sterilized.

[0015] The beneficial effects of the present invention compared to the prior art are:

[0016] 1. It is stimulated by mixing original ecological bovine cholecystitis bile, bovine blood and various bacteria, and has strong biological activity.

[0017] 2. The cultivation process in the cultivation system adopts intelligent control, which can better optimize various factors of the cultivation process and strongly and directionally induce the production of broad-spectrum antimicrobial peptides and antimicrobial proteins in the larvae of the big-headed golden fly. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 To develop a system diagram for intelligent online control;

[0019] Among them, 1-culture tank, 2-high-resolution probe, 3-data processing center, 4-converter, 5-basic culture medium control valve, 6-temperature controller, 7-Escherichia coli freeze-dried powder control valve, 8-protein powder control valve, 9-basic culture medium storage tank, 10-Escherichia coli freeze-dried powder storage tank, 11-protein powder storage tank. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention that does not depart from the spirit and scope of the technical solution of the present invention should be included in the scope of protection of the present invention.

[0021] The present invention collects bile from slaughtered cattle, mixes it with bile from inflammatory cattle, bovine blood, trace elements, Escherichia coli, enterococci, anaerobic bacteria, Pseudomonas aeruginosa, and lecithin, and then stirs it at high speed and activates it with weak-power ultrasonic waves to create a basic culture medium suitable for the growth of large-headed chrysophobia larvae. The large-headed chrysophobia egg culture system and culture process are intelligently controlled. Based on the growth characteristics of the large-headed chrysophobia larvae, the culture system's basic culture medium concentration, culture temperature, and the amount of pathogens and protein powder are optimized to induce the production of broad-spectrum antimicrobial peptides and proteins in the chrysophobia larvae. After being separated from the culture system, the big-headed golden fly larvae are subjected to multiple strict disinfection, cleaning, squeezing, and solid-liquid separation. The liquid is collected and added with solid ammonium sulfate to precipitate and separate the precipitate containing antimicrobial peptides and antimicrobial proteins. The precipitate is then washed with ethanol and distilled water. The purified antimicrobial peptides and antimicrobial proteins are freeze-dried, and the dried antimicrobial peptides and antimicrobial proteins are sterilized at high temperature instantaneously. The sterilized antimicrobial peptide and antimicrobial protein mixture becomes a drug raw material for treating bovine cholecystitis, and the freeze-dried powder is packaged to become a freeze-dried powder injection.

[0022] The specific culture process of the present invention is obtained in the following manner: a culture system is composed of a basic culture medium and a culture tank (1); the culture process of the big-headed golden fly larvae is operated by an intelligent online control system; 3-4 high-resolution probes (2) are respectively installed above the culture tank (1) to monitor the changes of pathogens and basic culture medium in the culture system, as well as the growth process of the big-headed golden fly eggs after they transform into larvae in real time, and the monitoring data are fed back to a data processing center (3); the data processing center processes various detection data and gives instructions to a converter (4) to optimize the concentration of the basic culture medium, regulate the temperature of the culture process, regulate the number of pathogens and regulate the amount of protein powder of the culture material, and construct an optimal process for directional and strong induction of the production of corresponding broad-spectrum antimicrobial peptides and antimicrobial proteins in the body of the big-headed golden fly larvae.

[0023] Specific embodiment 1: This embodiment describes a method for preparing a drug for treating bovine cholecystitis, and the specific steps of the method are as follows:

[0024] (1) 200g-500g of cattle bile and 1g-50g of inflammatory bile were collected after slaughter, 1000g-5000g of cattle blood, 0.5g-2.0g of ZnSO4, 1.0g-2.0g of CuSO4, 5.0g-10.0g of Ca(OH)2, 0.5g-3.0g of MgSO4, 5.0g-20.0g of Escherichia coli freeze-dried powder, 2.0g-10.0g of Enterococcus freeze-dried powder, 1.0g-2.0g of Anaerobic Bacillus freeze-dried powder, 1.0g-2.0g of Pseudomonas aeruginosa freeze-dried powder and 10.0g-50.0g of lecithin were added, the pH was adjusted to 6.8-7.2 with a buffer solution, and after high-speed stirring, ultrasonic activation was performed to prepare a basic culture medium suitable for the growth of goldfly eggs and larvae;

[0025] (2) Soak 5g-15g of Chrysomyia ovata eggs in H2O2 aqueous solution at room temperature for a period of time, take them out and wash them with purified water. The sterilized Chrysomyia ovata eggs are then set aside.

[0026] (3) 3%-5% of the basic culture medium obtained in (1) is added to a culture tank, the goldfly eggs obtained in (2) are inoculated into the basic culture medium, 20-50g of protein powder is added, and the temperature is set at 20-30°C for cultivation. When the goldfly eggs metamorphose into first-instar larvae, 15%-20% of the basic culture medium obtained in (1) is added again, 3g-5g of Escherichia coli freeze-dried powder and 200-500g of protein powder are added, and the temperature is kept constant for cultivation. When the goldfly larvae grow to the second instar, all the remaining culture medium obtained in (1) is added to the culture tank, 5g-10g of Escherichia coli freeze-dried powder and 1000g-3000g of protein powder are added, the temperature is set at 20-35°C, and the culture is continued for 120h-180h.

[0027] (4) The cultured 3rd-instar chrysopidae larvae crawl out of the culture system, and the larvae collected after automatic separation are soaked in H2O2 aqueous solution for a period of time, then rinsed twice with pure water, then soaked in KMnO4 aqueous solution for a period of time, rinsed 5-10 times with pure water, and then placed in a dehydrator and dehydrated at 200r / min-750r / min for 3-5 minutes, and then filtered with a plate and frame filter at 0.2-5kg / cm 2 The mixture is squeezed under pressure, 10g-20g of solid ammonium sulfate is added as a precipitate, the precipitate is washed with 25%-40% ethanol solvent, and centrifuged to obtain a solid containing antimicrobial peptides and antimicrobial proteins. The solid is then solid-liquid separated, and the separated body fluid of the big-headed golden fly larvae is freeze-dried. The dried antimicrobial peptides and antimicrobial proteins are instantly sterilized.

[0028] Specific embodiment 2: A method for preparing a drug for treating bovine cholecystitis as described in specific embodiment 1, (1), wherein the buffer solution is a 0.20 mol / L Na2HPO4-NaH2PO4 buffer solution; the high-speed stirring speed is 500r / min-1000r / min, and the time is 5min-10min; the power of the ultrasonic activation is 5.0W-20.0W, the frequency is 20KHz-25KHz, and the time is 1.0min-3.0min.

[0029] Specific embodiment three: Specific embodiment one is a method for preparing a drug for treating bovine cholecystitis, wherein (1) is specifically as follows: 500 g of cattle bile and 50 g of inflammatory bile are collected after slaughter, 5000 g of cattle blood, 1.0 g of ZnSO4, 2.0 g of CuSO4, 10.0 g of Ca(OH)2, 2.0 g of MgSO4, 20.0 g of Escherichia coli freeze-dried powder, 5.0 g of Enterococcus freeze-dried powder, 2.0 g of anaerobic bacillus freeze-dried powder, 2.0 g of Pseudomonas aeruginosa freeze-dried powder and 30.0 g of lecithin are added, the pH is adjusted to 7.2 with 0.20 mol / L Na2HPO4-NaH2PO4 buffer, the mixture is stirred at 1000 r / min for 10 min with a high-speed stirrer, and ultrasonically activated with a power of 20.0 W and a frequency of 20 kHz for 3 min to prepare a basic culture medium suitable for the growth of big-headed golden fly eggs and larvae.

[0030] Specific embodiment 4: In the preparation method of a drug for treating bovine cholecystitis described in specific embodiment 1, in (2), the concentration of the H2O2 aqueous solution is 0.01%-0.50%, the volume is 200mL-500mL, and the soaking time is 10min-60min; the volume of the pure water is 1000mL-5000mL.

[0031] Specific embodiment 5: The preparation method of a drug for treating bovine cholecystitis described in specific embodiment 1, wherein (2) is specifically as follows: 10g of Chrysomyia ovata eggs are soaked in 500mL of 0.20%-0.50% H2O2 aqueous solution at room temperature for 30min-50min, taken out and washed twice with 5000mL of pure water, and the sterilized Chrysomyia ovata eggs are set aside.

[0032] Specific embodiment six: a preparation method of a drug for treating bovine cholecystitis described in specific embodiment one, wherein (3) is specifically as follows: 3%-5% of the basic culture medium in the basic culture medium storage tank (9) is controlled by the basic culture medium control valve (5) to be added to the culture tank (1) to form a culture system, 10g of sterilized golden fly eggs are connected to the culture system, 20.0g-50.0g of protein powder in the protein powder storage tank (11) is controlled by the protein powder control valve (8) to be added to the culture tank (1), and the culture system is controlled by the temperature controller (6) to be maintained at 20℃-30℃ for culture; when the golden fly eggs metamorphose into first-instar worms, 15%-20% of the basic culture medium in the basic culture medium storage tank (9) is controlled by the basic culture medium control valve (5) to be added to the culture tank (1), 3.0g-5.0g of Escherichia coli freeze-dried powder in the Escherichia coli freeze-dried powder storage tank (10) is controlled by the Escherichia coli freeze-dried powder control valve (7) to be added to the culture tank (1). The freeze-dried powder is added to the culture tank (1), and the protein powder control valve (8) controls 200g-500g of protein powder in the protein powder storage tank (11) to be added to the culture tank (1), and the temperature of the culture system is controlled by the temperature controller (6) to be maintained at 20°C-30°C for cultivation; when the big-headed golden fly larvae grow to the second instar, the basic culture medium control valve (5) controls the remaining basic culture medium in the basic culture medium storage tank (9) to be added to the culture tank (1), and the Escherichia coli larvae are controlled by the basic culture medium control valve (5). The Escherichia coli freeze-dried powder control valve (7) controls the addition of 5.0 g to 10.0 g of the Escherichia coli freeze-dried powder in the Escherichia coli freeze-dried powder storage tank (10) into the culture tank (1). The protein powder control valve (8) controls the addition of 1000 g to 3000 g of protein powder in the protein powder storage tank (11) into the culture tank (1). The temperature of the culture system is controlled by the temperature controller (6) to maintain at 20° C. to 35° C. for culturing, and the culturing time is 120 h to 180 h.

[0033] Specific embodiment seven: Specific embodiment six described a method for preparing a drug for treating bovine cholecystitis, wherein (3) is specifically as follows: the basic culture medium control valve (5) controls 5% of the basic culture medium in the basic culture medium storage tank (9) to be added to the culture tank (1) to form a culture system, 10g of sterilized golden fly eggs are connected to the culture system, the protein powder control valve (8) controls 30.0g of protein powder in the protein powder storage tank (11) to be added to the culture tank (1), the temperature controller (6) controls the culture system to be maintained at 25°C-30°C for culture, and when the golden fly eggs metamorphose into first-instar worms, the basic culture medium control valve (5) controls 15% of the basic culture medium in the basic culture medium storage tank (9) to be added to the culture tank (1), the Escherichia coli freeze-dried powder control valve (7) controls 5.0g of Escherichia coli freeze-dried powder in the Escherichia coli freeze-dried powder storage tank (10) to be added to the culture tank (1). The freeze-dried powder of Escherichia coli is added to the culture tank (1), and 350g of protein powder in the protein powder storage tank (11) is added to the culture tank (1) by the protein powder control valve (8), and the temperature of the culture system is controlled by the temperature controller (6) to be maintained at 25°C-30°C for cultivation; when the big-headed golden fly larvae grow to the second instar, the remaining basic culture medium in the basic culture medium storage tank (9) is controlled by the basic culture medium control valve (5) to be added to the culture tank (1), and 10.0g of the freeze-dried powder of Escherichia coli in the freeze-dried powder storage tank (10) is controlled by the Escherichia coli freeze-dried powder control valve (7) to be added to the culture tank (1), and 3000g of protein powder in the protein powder storage tank (11) is controlled by the protein powder control valve (8) to be added to the culture tank (1), and the temperature of the culture system is controlled by the temperature controller (6) to be maintained at 28°C-35°C for cultivation, and the cultivation time is 120h.

[0034] Specific embodiment eight: In the method for preparing a drug for treating bovine cholecystitis described in specific embodiment one, in (4), the concentration of the H2O2 aqueous solution is 0.05%-0.20%, and the soaking time is 30min-60min; the concentration of the KMnO4 aqueous solution is 0.01%-0.10%, and the soaking time is 10min-30min; the centrifugal speed is 850r / min-1000r / min.

[0035] Specific embodiment nine: In the preparation method of a drug for treating bovine cholecystitis described in specific embodiment one, in (4), the specific parameters of the freeze-drying are -30℃~-50℃, vacuum degree less than 0.098mPa, and 10h-36h; the temperature of the instantaneous sterilization is 160℃-180℃, and the time is 3s-10s.

[0036] Specific embodiment ten: A method for preparing a drug for treating bovine cholecystitis according to any one of specific embodiments one to nine, the method further comprising (5): packaging the lyophilized powder into lyophilized powder injections of 50 mg / vial or 100 mg / vial.

[0037] Example 1:

[0038] 1. Take 100g-200g of gallbladder juice from an inflamed cow after slaughter, cut it open and add 1000g-5000g of cow blood; 1.0g-10.0g ZnSO4, 1.0g-5.0g CuSO4, 10.0g-50.0g Ca(OH)2, 0.5g-10.0g MgSO4; add 10.0g-20.0g of Escherichia coli freeze-dried powder, 2.0g-10.0g of Enterococcus freeze-dried powder, 1.0g-2.0g of Anaerobic Bacillus freeze-dried powder, 1.0g-2.0g of Pseudomonas aeruginosa freeze-dried powder, and 10.0g-100.0g of lecithin, adjust the pH from 6.8 to 7.2 with 0.20mol / L Na2HPO4-NaH2PO4 buffer, activate the activity of various bacteria with ultrasound at a power of 1.0W-20.0W, a frequency of 20KHz-25KHz, and a radiation time of 0.1 second-5.0 seconds, and mix thoroughly to form a basic culture medium.

[0039] 2. Soak 5g-15g of Chrysomyia ovata eggs in 200mL-500mL of 0.01%-0.50% H2O2 aqueous solution at room temperature for 10min-60min. After taking out, wash them twice with 1000mL-5000mL of pure water. The disinfected Chrysomyia ovata eggs are set aside.

[0040] The basic culture medium control valve (5) controls 3%-5% of the basic culture medium in the basic culture medium storage tank (9) to be added to the culture tank (1) to form a culture system, 10g of sterilized golden fly eggs are introduced into the culture system, 20.0g-50.0g of protein powder in the protein powder storage tank (11) is controlled by the protein powder control valve (8) to be added to the culture tank (1), and the culture system is controlled by the temperature controller (6) to be maintained at 20°C-30°C for cultivation. When the big-headed golden fly eggs metamorphose into first-instar worms, 15%-20% of the basic culture medium in the basic culture medium storage tank (9) is added to the culture tank (1) by the basic culture medium control valve (5), 3.0g-5.0g of the Escherichia coli freeze-dried powder in the Escherichia coli freeze-dried powder storage tank (10) is added to the culture tank (1) by the Escherichia coli freeze-dried powder control valve (7), and 200g-500g of the protein powder in the protein powder storage tank (11) is added to the culture tank (1) by the protein powder control valve (8). The temperature of the culture system is controlled by the temperature controller (6) to maintain at 20°C-30°C for cultivation. When the larvae grow to the second instar, the remaining basic culture medium in the basic culture medium storage tank (9) is added to the culture tank (1) under the control of the basic culture medium control valve (5), 5.0g-10.0g of Escherichia coli freeze-dried powder in the Escherichia coli freeze-dried powder storage tank (10) is added to the culture tank (1) under the control of the Escherichia coli freeze-dried powder control valve (7), 1000g-3000g of protein powder in the protein powder storage tank (11) is added to the culture tank (1) under the control of the protein powder control valve (8), and the temperature of the culture system is controlled by the temperature controller (6) to maintain at 20°C-35°C for culturing, and the culturing time is 120h-180h.

[0041] 3. The larvae of the third-instar Chrysopa spp. that have crawled out of the culture system and have been automatically separated are collected and soaked in a 0.05%-0.20% H2O2 aqueous solution for 30-60 minutes, rinsed twice with purified water, then soaked in a 0.01%-0.10% KMnO4 aqueous solution for 10-30 minutes, and rinsed 5-10 times with purified water. The Chrysopa spp. larvae are placed in a dehydrator and dehydrated at 200-750 rpm for 3-5 minutes. Then, the larvae are filtered using a plate and frame filter at 0.2-5 kg / cm 2The collected liquid is squeezed under pressure, and 10-20 g of solid ammonium sulfate is added to the collected liquid for precipitation. The precipitate is washed with 25%-40% ethanol solvent and then separated using a centrifuge at 850-1000 rpm to obtain a solid containing antimicrobial peptides and antimicrobial proteins, achieving solid-liquid separation. The obtained body fluid of the Chrysocytis megacephala larvae containing the antimicrobial peptides and antimicrobial proteins is freeze-dried at -30°C to -50°C and a vacuum degree of less than 0.098 mPa for 10-36 hours. The dried antimicrobial peptides and antimicrobial proteins are sterilized at a temperature of 160-180°C for 3-10 seconds.

[0042] 4. The sterilized lyophilized powder is divided into 50 mg / vial or 100 mg / vial lyophilized powder injections.

[0043] The lyophilized powder injection prepared in Example 1 was used in clinical trials, and the results were as follows:

[0044] 1. Diagnosis

[0045] Cholecystitis in cattle causes yellowing of the eye mucosa and swelling of the eyelids. The mouth of the affected cattle experiences burning sensation, a reddish-yellow mouth, stringy saliva, yellow-red tongue mucosa, and yellow nasal discharge. The affected cattle are restless and irritable, with noticeable tremors in the front shoulder blades and neck muscles. They also suffer from dry, constipated stools and dark yellow urine.

[0046] Routine blood examination showed significant elevation of bilirubin, serum transaminases, and alkaline phosphatase.

[0047] 2. Treatment Methods

[0048] (1) 28 cattle with clinical cholecystitis were selected;

[0049] (2) Disinfect the intended injection site with 70% medical alcohol and allow to dry;

[0050] (3) Dissolve 100 mg of the powder in 200 mL of injection-grade 0.9% NaCl saline and inject into the blood vessels near the gallbladder of cattle three times a day.

[0051] (4) The treatment cycle is 10 days.

[0052] 2. Criteria for determining efficacy

[0053] (1) Recovery: systemic symptoms disappear and return to normal;

[0054] (2) Marked effect: Systemic symptoms basically disappear, with occasional partial symptoms;

[0055] (3) Improved: The condition has improved significantly, but some symptoms still exist;

[0056] (4) Ineffective: There is no significant change in the condition, or the condition may even worsen.

[0057] 3. Clinical treatment results:

[0058]

Claims

1. A method for preparing a drug for treating bovine cholecystitis, characterized in that: The specific steps of the method are as follows: (1) Take 100g-200g of inflamed cattle gallbladder juice after slaughter, cut it open and add 1000g-5000g of cattle blood; 1.0g-10.0g ZnSO4, 1.0g-5.0g CuSO4, 10.0g-50.0g Ca(OH)2, 0.5g-10.0g MgSO4; add 10.0g-20.0g of Escherichia coli freeze-dried powder, 2.0g-10.0g of Enterococcus freeze-dried powder, 1.0g-2.0g of Anaerobic Bacillus freeze-dried powder, 1.0g-2.0g of Pseudomonas aeruginosa freeze-dried powder, and 10.0g-100.0g of lecithin, adjust the pH to 6.8-7.2 with 0.20mol / L Na2HPO4-NaH2PO4 buffer, stir at high speed, and activate the various bacterial activities with ultrasound at a power of 1.0W-20.0W, a frequency of 20kHz-25kHz, and a radiation time of 0.1second-5.0seconds, and mix thoroughly to form a basic culture medium; (2) Soak 5g-15g of Chrysomyia spp. eggs in H2O2 aqueous solution at room temperature for a period of time, take them out and wash them with purified water. The sterilized Chrysomyia spp. eggs are then set aside. (3) Take 3%-5% of the basic culture medium obtained in (1) and add it to the culture tank, inoculate the big-headed golden fly eggs in (2) into the basic culture medium, add 20-50g of protein powder, set the temperature to 20-30℃ for cultivation, when the big-headed golden fly eggs metamorphose into first-instar worms, add 15%-20% of the basic culture medium obtained in (1) again, add 3g-5g of Escherichia coli freeze-dried powder and 200-500g of protein powder, and continue to cultivate at the same temperature. When the big-headed golden fly larvae grow to the second instar, add all the remaining culture medium obtained in (1) to the culture tank, add 5g-10g of Escherichia coli freeze-dried powder and 1000g-3000g of protein powder, set the temperature to 20-35℃, and cultivate for 120h-180h; (4) The 3rd instar larvae of the cultured golden fly crawl out of the culture system, and the larvae collected after automatic separation are soaked in H2O2 aqueous solution for a period of time, then rinsed twice with pure water, and then soaked in KMnO4 aqueous solution for a period of time, rinsed 5-10 times with pure water, and then placed in a dehydrator and dehydrated at 200r / min-750r / min for 3-5 minutes, and then filtered with a plate and frame filter at 0.2-5kg / cm 2 The mixture is squeezed under pressure, 10g-20g of solid ammonium sulfate is added as a precipitate, the precipitate is washed with 25%-40% ethanol solvent, and centrifuged to obtain a solid containing antimicrobial peptides and antimicrobial proteins. The solid is then subjected to solid-liquid separation, and the separated body fluid of the big-headed golden fly larvae is freeze-dried. The dried antimicrobial peptides and antimicrobial proteins can be sterilized instantly.

2. The method for preparing a drug for treating bovine cholecystitis according to claim 1, wherein: (1), the buffer solution is 0.20 mol / L Na2HPO4-NaH2PO4 buffer solution; the high-speed stirring speed is 500 r / min-1000 r / min, and the time is 5 min-10 min; the power of the ultrasonic activation is 5.0 W-20.0 W, the frequency is 20 kHz-25 kHz, and the time is 1.0 min-3.0 min.

3. The method for preparing a drug for treating bovine cholecystitis according to claim 1, wherein: (2), the concentration of the H2O2 aqueous solution is 0.01%-0.50%, the volume is 200mL-500mL, and the soaking time is 10min-60min; the volume of the pure water is 1000mL-5000mL.

4. The method for preparing a drug for treating bovine cholecystitis according to claim 1, wherein: Said (2) is specifically as follows: 10g of Chrysomyia spp. eggs are soaked in 500mL of 0.20%-0.50% H2O2 aqueous solution at room temperature for 30min-50min, taken out and washed twice with 5000mL of pure water, and the sterilized Chrysomyia spp. eggs are set aside.

5. The method for preparing a drug for treating bovine cholecystitis according to claim 1, wherein: The (3) is specifically as follows: the basic culture medium control valve (5) controls 3%-5% of the basic culture medium in the basic culture medium storage tank (9) to be added to the culture tank (1) to form a culture system, 10g of the sterilized golden fly eggs are connected to the culture system, the protein powder control valve (8) controls 20.0g-50.0g of protein powder in the protein powder storage tank (11) to be added to the culture tank (1), and the temperature controller (6) controls the culture system to be maintained at 20℃-30℃ for culture; when the golden fly eggs metamorphose into 1st instar, the basic culture medium control valve (5) controls 15%-20% of the basic culture medium in the basic culture medium storage tank (9) to be added to the culture tank (1), the Escherichia coli freeze-dried powder control valve (7) controls 3.0g-5.0g of Escherichia coli freeze-dried powder in the Escherichia coli freeze-dried powder storage tank (10) to be added to the culture tank (1), and the protein powder control valve (8) controls the culture system to be maintained at 20℃-30℃ for culture. The powder control valve (8) controls 200 g to 500 g of protein powder in the protein powder storage tank (11) to be added to the culture tank (1), and the temperature controller (6) controls the temperature of the culture system to be maintained at 20° C. to 30° C. for cultivation; when the big-headed golden fly larvae grow to the second instar, the basic culture medium control valve (5) controls the remaining basic culture medium in the basic culture medium storage tank (9) to be added to the culture tank (1), and the Escherichia coli freeze-dried powder control valve (7) controls 5.0 g to 10.0 g of Escherichia coli freeze-dried powder in the Escherichia coli freeze-dried powder storage tank (10) to be added to the culture tank (1), and the protein powder control valve (8) controls 1000 g to 3000 g of protein powder in the protein powder storage tank (11) to be added to the culture tank (1), and the temperature controller (6) controls the temperature of the culture system to be maintained at 20° C. to 35° C. for cultivation, and the cultivation time is 120 h to 180 h.

6. The method for preparing a drug for treating bovine cholecystitis according to claim 5, wherein: The (3) is specifically as follows: the basic culture medium control valve (5) controls 5% of the basic culture medium in the basic culture medium storage tank (9) to be added to the culture tank (1) to form a culture system, 10g of sterilized golden fly eggs are connected to the culture system, the protein powder control valve (8) controls 30.0g of protein powder in the protein powder storage tank (11) to be added to the culture tank (1), the temperature controller (6) controls the culture system to be maintained at 25℃-30℃ for culture, and when the golden fly eggs metamorphose into 1st instar, the basic culture medium control valve (5) controls 15% of the basic culture medium in the basic culture medium storage tank (9) to be added to the culture tank (1), the Escherichia coli freeze-dried powder control valve (7) controls 5.0g of Escherichia coli freeze-dried powder in the Escherichia coli freeze-dried powder storage tank (10) to be added to the culture tank (1), and the temperature controller (6) controls the culture system to be maintained at 25℃-30℃ for culture. The protein powder control valve (8) controls 350 g of protein powder in the protein powder storage tank (11) to be added to the culture tank (1), and the temperature controller (6) controls the temperature of the culture system to be maintained at 25°C-30°C for cultivation; when the big-headed golden fly larvae grow to the second instar, the basic culture medium control valve (5) controls the remaining basic culture medium in the basic culture medium storage tank (9) to be added to the culture tank (1), and the Escherichia coli freeze-dried powder control valve (7) controls 10.0 g of Escherichia coli freeze-dried powder in the Escherichia coli freeze-dried powder storage tank (10) to be added to the culture tank (1), and the protein powder control valve (8) controls 3000 g of protein powder in the protein powder storage tank (11) to be added to the culture tank (1), and the temperature controller (6) controls the temperature of the culture system to be maintained at 28°C-35°C for cultivation, and the cultivation time is 120 hours.

7. The method for preparing a drug for treating bovine cholecystitis according to claim 1, wherein: (4), the concentration of the H2O2 aqueous solution is 0.05%-0.20%, and the soaking time is 30min-60min; the concentration of the KMnO4 aqueous solution is 0.01%-0.10%, and the soaking time is 10min-30min; the centrifugal speed is 850r / min-1000r / min.

8. The method for preparing a drug for treating bovine cholecystitis according to claim 1, wherein: (4), the specific parameters of the freeze-drying are -30°C to -50°C, a vacuum degree less than 0.098 mPa, and 10 hours to 36 hours; the temperature of the instantaneous sterilization is 160°C to 180°C, and the time is 3 seconds to 10 seconds.

9. A method for preparing a drug for treating bovine cholecystitis according to any one of claims 1 to 8, characterized in that: The method further comprises (5): packaging the lyophilized powder into lyophilized powder injections of 50 mg / vial or 100 mg / vial.

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