Tibetan medicine composition for treating calf and yak diarrhea, preparation method and application thereof

Abstract

The invention relates to the field of biotechnology, and specifically to a Tibetan medicine composition for treating calf yak diarrhea, and a preparation method and application thereof. The Tibetan medicine composition comprises the following raw materials in parts by weight: 5-10 parts of Aconite, 4-8 parts of Aconitum, and 4-8 parts of Rubia cordifolia; the pharmacological and pharmacodynamic screening of the effect of treating calf yak diarrhea is reasonable, the raw materials are easily available, the preparation process is simple, the operation is simple, and the composition is safe and effective; the Tibetan medicine composition of the invention can significantly inhibit the growth of bacteria and viruses, can significantly reduce the water content in mouse feces, and has good antidiarrheal effect, certain intestinal astringent effect, good anti-inflammatory effect and analgesic effect. The Tibetan medicine composition has been verified by clinical application experiments to have a significant effect on treating calf yak diarrhea, and has broad promotion and application value in the clinical application of plateau calf yak.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a Tibetan medicine composition for treating calf yak diarrhea, and a preparation method and application thereof. Background Art

[0002] The incidence of neonatal calf diarrhea is over 20% worldwide. The high incidence poses a great threat to the healthy breeding of calves and seriously affects the economic benefits of cattle breeding. Neonatal calf diarrhea can occur in all seasons, and the incidence varies significantly from region to region. The incidence in some areas is as high as 90% to 100%, and the mortality rate is as high as 50%. After the disease occurs, it often causes serious economic losses to farmers. Yaks mainly grow in high-altitude and cold areas with an altitude of 3000 to 5000m. The environment in the growing area is harsh, and the probability of neonatal calves is relatively high. Neonatal calf diarrhea is mainly caused by three reasons: first, insufficient nutritional supply, reduced immunity and digestive ability; second, infection with pathogenic microorganisms such as Escherichia coli and rotavirus; third, neonatal calves are susceptible to stress factors and diarrhea. Neonatal calf diarrhea generally has obvious symptoms of loose stools. Calf diarrhea caused by different causes has different defecation symptoms. Generally, indigestion diarrhea discharges white porridge-like feces without obvious fishy smell. Infectious diarrhea discharges black or yellow-white feces, and the feces have a foul odor. In addition, diarrhea in newborn calves is generally accompanied by clinical symptoms such as increased body temperature, loss of appetite, depression, decreased skin elasticity, unstable standing, and sunken eyeballs.

[0003] Tibetan medicine has high medicinal value due to its high-altitude growth advantage. Its content of biologically active ingredients is generally higher than that of similar plants in other regions. It is an important natural lead compound in the development of modern new drugs. There have been relevant studies on the treatment of calf diarrhea with Tibetan medicine. For example, the invention patent CN103751673B discloses a Tibetan medicine composition for treating yak calf diarrhea and its preparation method. The Tibetan medicine composition includes 375-420 parts of Gesneria serrata, 320-350 parts of Phellodendron chinense, 145-160 parts of pomegranate peel, and 100 parts of Kaempferia galanga. The invention patent CN112603942B discloses a Tibetan medicine compound for treating calf gastrointestinal diseases and its preparation method and application. The Tibetan medicine compound includes 1-3 parts of three needles, 1-4 parts of sea buckthorn, 5 parts of Terminalia chebula, 2 parts of Ganqinglan, and 2 parts of dwarf Corydalis. However, at present, there is no research to prove that reverse A Luo, boat-shaped Aconitum and Rubia cordifolia have the effect of treating diarrhea.

[0004] The research team to which the inventor belongs has continuously collected and sorted out Tibetan veterinary prescriptions and proven recipes, and through modern pharmacological and efficacy research, has screened out new veterinary drugs with good effects and research and development prospects. This has important research significance for the prevention and treatment of calf diarrhea and the development of Tibetan veterinary drugs. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a Tibetan medicine composition for treating diarrhea in yaks and calves.

[0006] In order to achieve the above object, the technical solution provided by the present invention is:

[0007] The primary purpose of the present invention is to provide a Tibetan medicine composition for treating diarrhea in calves and yaks. The Tibetan medicine composition comprises the following raw materials in parts by weight: 5 to 10 parts of Aconitum carmichaelii, 4 to 8 parts of Aconitum natans, and 4 to 8 parts of Rubia cordifolia.

[0008] Preferably, the Tibetan medicine composition is composed of the following raw materials in parts by weight: 5-10 parts of Aconitum carmichaelii, 4-8 parts of Aconitum natans, and 4-8 parts of Rubia cordifolia.

[0009] Preferably, the Tibetan medicine composition is composed of the following raw materials in parts by weight: 5 parts of Aconitum carmichaelii, 4 parts of Aconitum natans, and 4 parts of Rubia cordifolia.

[0010] The second object of the present invention is to provide a method for preparing the Tibetan medicine composition, which comprises the following steps: (1) accurately weighing each medicine, adding ultrapure water, and soaking; (2) boiling with water twice, filtering, collecting, and combining the extracts; (3) concentrating the extracts to obtain a concentrated extract of the Tibetan medicine composition, and sealing and storing it after cooling.

[0011] The third object of the present invention is to provide a medicine, including the Chinese medicine composition.

[0012] Preferably, the dosage form of the drug includes an oral preparation or an injection preparation.

[0013] Preferably, the oral preparation includes tablets, capsules, granules, pills, syrups, extracts, oral solutions, oral suspensions or oral emulsions.

[0014] Preferably, the injection preparation includes injection solution or powder injection.

[0015] The fourth object of the present invention is to provide the use of the Tibetan medicine composition or the medicine in preparing a medicine for treating diarrhea.

[0016] The fifth object of the present invention is to provide the use of the Tibetan medicine composition or the medicine in preparing medicine for treating yak diarrhea.

[0017] The beneficial effects of the present invention are as follows: the present invention provides a Tibetan medicine composition for treating calf yak diarrhea, which is a pharmacological and efficacy screening of the effect of treating calf yak diarrhea by combining traditional Tibetan veterinary medicine prescriptions with modern technical methods. The composition is reasonable, the raw materials are easily available, the preparation process is simple, the operation is simple, and the composition is safe and effective. The Tibetan medicine composition of the present invention can significantly inhibit the growth of bacteria and viruses, and can significantly reduce the water content in mouse feces, and has good antidiarrheal effect, certain intestinal astringent effect, good anti-inflammatory effect and analgesic effect. It has been verified by clinical application experiments that the composition has a significant effect on treating calf yak diarrhea, and has broad promotion and application value in the clinical application of plateau calf yak. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Pathological histological observation of various organs of suckling mice after treatment with Tibetan medicine combination (HE staining)

[0019] Figure 2 RT-PCR results of rotavirus

[0020] Figure 3 Effect of Tibetan medicine combination on the inhibition rate of ear swelling caused by xylene

[0021] Figure 4 Effect of Tibetan medicine combination on the latency period of writhing pain induced by glacial acetic acid

[0022] Figure 5 Effect of Tibetan medicine combination on the propulsion rate of carbon powder in the small intestine

[0023] Figure 6 Antidiarrheal effect of Tibetan medicine combination on diarrhea experimental mice DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0025] Example 1 Preparation of Tibetan medicine composition

[0026] The formula of the Tibetan medicine composition for treating diarrhea in calves and yaks is: 50g of Nialuo, 40g of Aconitum carmichaelii, and 40g of Rubia cordifolia.

[0027] The preparation method is as follows: accurately weigh 50 g of Aconitum carmichaelii, 40 g of Aconitum natans, and 40 g of Rubia cordifolia, place them in a 5L cylindrical pot, add 3L of water, and soak for 40 minutes. The cylindrical pot with the Tibetan medicine components added is placed on a heater, heated at 2000 W for 40 minutes until boiling, then the heater power is reduced to 600 W, decocted for 90 minutes, filtered with gauze to obtain the filtrate of the first decoction filtration, and stored for later use; 2L ultrapure water is added to the filter residue of the first decoction filtration, heated at 2000 W for 40 minutes until boiling, then the heater power is reduced to 600 W, decocted and boiled for 30 minutes, filtered with gauze to obtain the filtrate of the second decoction filtration, and stored for later use; the filtrate of the first decoction filtration and the filtrate of the second decoction filtration are combined, heated at 2000 W for 30 minutes until boiling, then the heater power is reduced to 1200 W, boiled for 240 minutes, and 130 g of concentrated extract of the Tibetan medicine composition are obtained, and after cooling, they are sealed and stored in a 4°C refrigerator for later use.

[0028] Example 2 Preparation of Tibetan medicine composition 2

[0029] The formula of the Tibetan medicine composition for treating diarrhea in calves and yaks is: 100g of Nialuo, 80g of Aconitum carmichaelii, and 80g of Rubia cordifolia.

[0030] The preparation method of the Tibetan medicine composition is as follows: accurately weigh 100g of Aconitum carmichaelii, 80g of Aconitum natans, and 80g of Rubia cordifolia, place them in a 10L cylindrical pot, add 5L of ultrapure water, and soak for 50 minutes. Then the cylindrical pot with the Tibetan medicine components added is placed on the heater, heated for 40 minutes at 2000W until boiling, then the heater power is reduced to 600W, decocted for 105min, filtered with gauze to obtain the filtrate of the first decoction filtration, and stored for later use; 4L ultrapure water is added to the filter residue of the first decoction filtration, heated for 40 minutes at 2200W until boiling, then the heater power is reduced to 600W, decocted and boiled for 50min, filtered with gauze to obtain the filtrate of the second decoction filtration, and stored for later use; the filtrate of the first decoction filtration and the filtrate of the second decoction filtration are combined, heated for 30 minutes at 2000W until boiling, then the heater power is reduced to 1200W, boiled for 270min, and 260g of concentrated extract of the Tibetan medicine composition are obtained, and after cooling, they are sealed and stored in a 4°C refrigerator for later use.

[0031] Example 3 Preparation of Tibetan medicine composition

[0032] The formula of the Tibetan medicine composition for treating diarrhea in calves and yaks is: 250g of Nialuo, 200g of Aconitum carmichaelii, and 200g of Rubia cordifolia.

[0033] The preparation method of the Tibetan medicine composition is as follows: accurately weigh 250g of Aconitum carmichaelii, 200g of Aconitum natans, and 200g of Rubia cordifolia, place them in a 15L cylindrical pot, add 8L of ultrapure water, and soak for 60 minutes. Then the cylindrical pot with the Tibetan medicine components added is placed on a heater, heated at 2000 W for 40 minutes until boiling, then the heater power is reduced to 600 W, decocted for 90 minutes, filtered with gauze to obtain the filtrate of the first decoction filtration, and stored for later use; 6L ultrapure water is added to the filter residue of the first decoction filtration, heated at 2000 W for 40 minutes until boiling, then the heater power is reduced to 600 W, decocted and boiled for 60 minutes, filtered with gauze to obtain the filtrate of the second decoction filtration, and stored for later use; the filtrate of the first decoction filtration and the filtrate of the second decoction filtration are combined, heated at 2000 W for 30 minutes until boiling, then the heater power is reduced to 1200 W, boiled for 300 minutes, and 650g of concentrated extract of the Tibetan medicine composition are obtained, and after cooling, they are sealed and stored in a 4°C refrigerator for later use.

[0034] Example 4 Tibetan medicine composition clinical efficacy pre-test

[0035] 1. Test materials

[0036] 1.1 Trial Drugs

[0037] A Tibetan medicine composition (1 g / ml) was prepared according to the method of Example 3.

[0038] 1.2 Experimental animals

[0039] Fifteen cases of diarrhea in yak calves were collected in Nierong County and Jiali County of Nagqu City, Tibet Autonomous Region.

[0040] 2 Test methods

[0041] 2.1 Experiment on the treatment of diarrhea in yak calves with Tibetan medicine composition

[0042] The drug was administered orally to yak calves with diarrhea. 10 mL / head was administered according to body weight, twice a day, and the yak calves were allowed to eat and drink freely. Clinical symptoms such as feces, body temperature, and mental state of the yak calves were observed daily. The treatment lasted for 5 consecutive days, and the cure rate and effectiveness rate were calculated.

[0043] 2.2 Criteria for judging the therapeutic effect of Tibetan medicine composition on diarrhea in yaks

[0044] Cure: One course of treatment is 5 days. After treatment according to the medication method, the clinical symptoms of diarrhea such as loose stools, dehydration, vomiting, etc. in the yak calves all disappeared; the mental state recovered well, and the yak calves ate and drank normally without any adverse reactions.

[0045] Effective: One course of treatment is 5 days. After treatment according to the medication method, the clinical symptoms of diarrhea such as loose stools, dehydration, and vomiting in the yaks were alleviated, their mental state gradually improved, and their eating and drinking were basically restored.

[0046] Ineffective: One course of treatment is 5 days. After treatment according to the medication method, after 5 days of medication, the clinical symptoms of diarrhea such as loose stools, dehydration, vomiting, etc. in the yak calves did not alleviate or even worsened. The yak calves became emaciated, had a low mental state, and were unwilling to eat or drink.

[0047] During the trial, the recovery of the experimental yak calves was observed daily, and the therapeutic effect of the Tibetan medicine composition on the yak calves was statistically calculated after 5 days of treatment.

[0048] 3. Test results

[0049] Table 1 The therapeutic effect of Tibetan medicine composition on diarrhea in yaks

[0050]

[0051] Table 1 shows the therapeutic effect of the Tibetan medicine composition on calf yak diarrhea. It can be seen that the Tibetan medicine composition has a cure rate of 73.33% and an effective rate of 86.67% for calf yak diarrhea, and the therapeutic effect is good. Therefore, the Tibetan medicine composition has broad application value in the clinical treatment of calf yak diarrhea.

[0052] Example 5 In vitro antibacterial activity of Tibetan medicine composition

[0053] 1. Materials

[0054] 1.1 Medicinal materials and strains

[0055] Aconitum carmichaelii, Aconitum tangutorum and Rubia cordifolia were all purchased from Tibet Yalang Tibetan Medicinal Materials Sales Co., Ltd., and a Tibetan medicine composition (1 g / ml) was prepared according to the method of Example 3.

[0056] 1.2 Strains

[0057] Escherichia coli: ATCC 25922; Escherichia coli 181 (laboratory clinical isolated strain and named, from a cattle farm in Zhangye City, Gansu Province).

[0058] 1.3 Culture medium

[0059] MH broth medium (MHB, Guangdong Huankai Microbiology Technology Co., Ltd.).

[0060] 1.4 Instruments and reagents

[0061] Biological safety cabinet (HFsafe~1500, Shanghai Lishen Scientific Instrument Co., Ltd.); high pressure sterilizer (LDZS~50KBS, Shanghai Shen'an Medical Equipment Factory); low speed centrifuge (TDZ4~WS, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); constant temperature culture oscillator (HNYC~202T, Tianjin Ounuo Instrument Co., Ltd.; electric constant temperature blast drying oven (Shanghai Hongdu Electronic Technology Co., Ltd.); Waterpool water machine (Sichuan Water Treatment Co., Ltd.); CNC ultrasonic cleaner (KQ~500DB, Kunshan Ultrasonic Instrument Co., Ltd.); CO2 incubator (TABAI ESPEC CORD, Japan).

[0062] 2. Methods

[0063] MIC was determined using the microdilution method. 100 μL of sterilized MHB broth medium was added to the 1st to 10th wells and the 12th well of a sterile 96-well culture plate, 100 μL of the extract of the Tibetan medicine composition to be tested was added to the 1st well and the 11th well, and then 100 μL was discarded; 100 μL of bacterial solution was added to the 1st to 10th wells and the 12th wells, that is, the crude drug concentrations of the 1st to 10th wells were 500 mg / mL, 250 mg / mL, 125 mg / mL, 62.50 mg / mL, 31.25 mg / mL, 15.625 mg / mL, 7.81 mg / mL, 3.90 mg / mL, 1.95 mg / mL, and 1.00 mg / mL, respectively. No bacterial solution was added to the 11th well, which was a negative control, and no drug was added to the 12th well, which was a positive control. Each test was repeated three times, and the samples were placed in a 37°C incubator for 24 hours before being taken out and the test results observed. The test results are reliable when the liquid in the positive control well is uniformly turbid and the liquid in the negative control well is clear and transparent. Observe the conditions of the other wells, and the lowest dilution at which the liquid is clear and transparent is the minimum inhibitory concentration (MIC).

[0064] 3. Conclusion

[0065] As can be seen from Table 2, the MICs of the Tibetan medicine composition against the clinical isolate of Escherichia coli and the standard strain ATCC25922 are 125 mg / ml and 62.5 mg / mL, respectively.

[0066] Table 2 Determination of the minimum inhibitory concentration of Tibetan medicine composition in vitro (n=3)

[0067]

[0068] 4. Conclusion

[0069] When the minimum inhibitory concentration of the Tibetan medicine composition for the clinical strain 181 of Escherichia coli, the main pathogenic bacteria of calf diarrhea, is 125 mg / ml, the minimum inhibitory concentration for the standard strain of Escherichia coli ATCC25922 is 62.5 mg / mL. The results show that the Tibetan medicine composition has a certain in vitro antibacterial effect on Escherichia coli, the main pathogenic bacteria of calf diarrhea.

[0070] Example 5 In vitro anti-rotavirus effect of Tibetan medicine composition

[0071] 1.1 Preparation of Tibetan medicine composition

[0072] A Tibetan medicine composition (1 g / ml) was prepared according to the method of Example 3.

[0073] 1.2 Tissue culture median infectious dose determination

[0074] Take fresh virus suspension and dilute it 10 times to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 Different dilutions were incubated at 37℃ for 1h, and then inoculated into MA104 cells grown into a monolayer in a 96-well microplate. 50μL was inoculated in each well, and 8 wells were inoculated for each dilution. The cells were adsorbed for 1.5h, washed twice with PBS, and 100μL of maintenance solution was added. The cells were cultured in a CO2 incubator for 3d. The cell lesions were observed under an inverted microscope every day. After 3d, the results were judged and the TCID 50 .

[0075] TCID 50 =6+6.71, virus titer is 10 -6.71

[0076] 1.3 Toxic effects of drugs on cells (half inhibitory concentration, IC 50 )

[0077] The cell density of the 96-well cell culture plate was adjusted to 5000 cells / well. After culturing into a single layer, the culture medium was aspirated and the Tibetan medicine composition was diluted with DMEM to 40 mg / mL, 20 mg / mL, 10 mg / mL, 5 mg / mL, 2.5 mg / mL, and 1.25 mg / mL, respectively. The Tibetan medicine composition decoction was added to each well, and 8 wells were diluted in each well for 18 hours. 20 μL of MTT solution was added to each well and culture was continued for 4 hours. The culture was terminated and the culture medium in the well was carefully aspirated. 150 μL of dimethyl sulfoxide was added to each well and the well was placed on a shaker for low speed shaking for 10 minutes to fully dissolve the crystals. The absorbance value of each well was detected by an enzyme marker, and the IC value of the Tibetan medicine composition was calculated. 5018.1.1.4 Preventive effect of drugs on rotavirus

[0078] The cell density was adjusted to 5000 cells / well in a sterile 96-well cell culture plate. After culturing into a monolayer, 10 mg / mL, 5 mg / mL, and 2.5 mg / mL of the Tibetan medicine composition decoction were added, respectively, and cultured in a CO2 incubator for 1 h. 100 TCD was added 50 50 μL of rotavirus solution was added to the culture medium and cultured for 3 days. After 3 days, the cell activity was detected by MTT method. The results showed that the Tibetan medicine composition had a preventive effect on rotavirus in vitro.

[0079] Table 3 In vitro preventive effect of Tibetan medicine composition on rotavirus

[0080] Group dose OD value High dose 10mg / mL 0.789 Medium dose 5mg / mL 0.630 Low dose 2.5mg / mL 0.415 Virus Group 0 0.212 Normal group 0 1.232

[0081] 1.5 Inhibitory effect of drugs on viral proliferation

[0082] The cell density was adjusted to 5000 cells / well in a sterile 96-well cell culture plate. After culturing into a monolayer, 50 μL of 100 TCD50 rotavirus solution was added for adsorption for 1.5 hours. The rotavirus solution was discarded and the Tibetan medicine composition was added. 10 mg / mL, 5 mg / mL, and 2.5 mg / mL of the Tibetan medicine composition were added respectively and cultured for 3 days. After 3 days, the cell activity was detected by MTT method. The Tibetan medicine composition had a significant inhibitory effect on rotavirus in vitro at a concentration of 5 mg / mL.

[0083] Table 4 In vitro inhibitory effect of Tibetan medicine composition on rotavirus

[0084] Group dose OD value High dose 10mg / mL 0.884 Medium dose 5mg / mL 0.645 Low dose 2.5mg / mL 0.448 Virus Group 0 0.233 Normal group 0 1.342

[0085] 1.6 Establishment of rotavirus infection model in mice

[0086] Separate the suckling mice from the mother mice, starve them for 1 hour under a constant temperature lamp, and then attack them with the virus: gently grasp the neck of the suckling mouse with your left hand to tilt its head back, try to keep the esophagus straight, hold a disposable 1mL syringe with a homemade gavage needle in your right hand, press the root of the tongue along the right side of the baby mouse's mouth, and gently insert it. When the needle enters the throat without obstruction, gently push the virus into the stomach, wipe the baby mouse's mouth with a sterilized cotton ball, and put it back. The injection volume of the virus solution is controlled to be 200μL. After the suckling mice are attacked with the virus, observe their physical signs and abdominal curvature, and observe for several consecutive days and record them. The abdominal curvature is mainly judged by observing the morphology of the stool-like stool discharged by the stress. The scoring criteria for diarrhea are as follows:

[0087] Table 5 Scoring criteria for diarrhea in suckling mice

[0088] Diarrhea score Diarrhea grade Stool form 3 Severe diarrhea Watery stools 2 Common diarrhea Yellow soft stool 1 No diarrhea Normal solid stool

[0089] The experimental group of 7-day-old suckling mice were gavaged with a dose of virus, and the control group was gavaged with an equal volume of PBS. After the virus attack, the suckling mice in the experimental group developed varying degrees of abdominal bulging, listlessness, and reduced activity. Some suckling mice had swollen abdomens or dry and wrinkled skin. Severe diarrhea suckling mice showed obvious signs of fecal contamination around the anus. There were no significant changes in the physical signs and activity status of the suckling mice in the control group. The suckling mouse rotavirus infection model was successfully constructed and can be used for in vivo anti-rotavirus infection tests of Tibetan veterinary medicines.

[0090] 1.7 In vivo test of Tibetan medicine composition against rotavirus infection

[0091] Based on the establishment of the rotavirus infection model, the Tibetan medicine composition was used for an in vivo anti-rotavirus infection test at a concentration of 5 mg / ml. The test results showed that the Tibetan medicine composition had a certain effect in treating rotavirus infection in vivo.

[0092] Table 6 Diarrhea scores in each group

[0093] Group Diarrhea index is 3 Diarrhea index is 2 The diarrhea index is 1 Total score Tibetan medicine composition 0 3 7 13 Virus control group 9 1 0 27 Ribavirin treatment group 0 1 9 11

[0094] Pathological histological observation (HE staining) of various organs of suckling mice after treatment with Tibetan medicine composition Figure 1 As shown, the cytoplasm of the cells in the intestinal tissue of the suckling mice treated with the Tibetan medicine composition was dense, while the cells in the diseased tissue showed vacuoles, indicating cell edema; the intestinal epithelial structure of the treatment group was clear, while the diseased tissue was accompanied by interstitial edema, inflammatory cell infiltration, increased and disordered cell density, and no significant changes in other organs.

[0095] 1.8 RT-PCR verification of the inhibitory effect of Tibetan medicine composition on rotavirus proliferation

[0096] The specific gene of rotavirus was amplified by RT-PCR. Lane 1 is the result of virus control group, and lane 2 is the result of compound treatment group. Figure 2 As shown, it can be seen that the treatment group can significantly inhibit viral proliferation.

[0097] Example 6 Tibetan medicine composition anti-inflammatory, small intestinal propulsion, analgesic, antidiarrhea test

[0098] 1. Materials

[0099] 1.1 Preparation of Tibetan medicine composition

[0100] A Tibetan medicine composition (1 g / ml) was prepared according to the method of Example 3 and stored at -20°C for later use.

[0101] 1.2 Experimental animals

[0102] A total of 210 Kunming (KM) mice (Experimental Animal Certificate No.: SCXK(Gan)2020-0002) were purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, half male and half female, with free access to food and water.

[0103] 2. Methods

[0104] 2.1 Anti-inflammatory test

[0105] Fifty KM mice were randomly divided into five groups, namely, blank control group, positive drug group, low-dose, medium-dose and high-dose Tibetan medicine combination groups, with 10 mice in each group, half male and half female. Except for the positive group, which was aspirin (0.26 g / kg), the doses of 0.3033 g / kg, 0.91 g / kg and 2.73 g / kg were administered for 3 consecutive days. 30 minutes after the last administration, 20 μL of 0.9% sodium chloride solution was applied on both sides of the right ear of the blank group mice with a pipette, and 20 μL of 100% xylene was applied to the right ears of the other groups of mice to induce inflammation, and the left ears were not treated. After 30 minutes, the mice were killed by dislocating the neck, and the ear pieces were punched in the same part of the left and right ears with a puncher with a diameter of 7 mm. The wet weight of the two ear pieces was weighed on an electronic balance, and the difference in the weight of the two ears was used as the observation index of the degree of inflammation and swelling, and the ear swelling rate and inhibition rate were calculated.

[0106] Ear swelling rate = (right ear mass - left ear mass) / left ear mass × 100%

[0107] Inhibition rate = (average swelling degree of model control group - average swelling degree of drug administration group) / average swelling degree of model control group × 100%

[0108] 2.2 Analgesia test

[0109] Fifty KM mice were randomly divided into five groups, with 10 mice in each group, half male and half female. Except for the positive group, which was treated with Changyan Ning (0.90 g / kg), the animals were grouped and dosed as above, and the drugs were administered continuously for 3 days. One day after the last administration, each group of mice was intraperitoneally injected with 1% glacial acetic acid solution 10 mL / kg. After the injection, they were immediately placed in a transparent observation box to observe and record the latency of the mice torsion and the number of torsion of the mice within 20 minutes. The number of torsion of each medication group was compared with the average number of torsion of the model group, and statistical tests were performed, and the inhibition rate of torsion reaction was calculated at the same time. Observation criteria: concavity of the abdominal cavity, extension of the hind limbs, elevation of the buttocks, etc.

[0110] Torsion inhibition rate = (average torsion times of model group - average torsion times of drug administration group) / average torsion times of model group × 100%

[0111] 2.3 Small bowel propulsion test

[0112] Fifty KM mice were randomly divided into five groups: blank group, positive group (loperamide), and Tibetan medicine composition dosage groups (0.3033 g / kg, 0.91 g / kg, 2.73 g / kg), with 10 mice in each group, half male and half female. The blank group was given deionized water by gavage, the positive group was given loperamide (0.002 g / kg) by gavage, and the Tibetan medicine composition test groups were given the corresponding concentration of the test solution by gavage. The gavage volume of each group of animals was 20 mL / kg, and the drug was administered continuously for 3 days. Fasting but not water control for 24 hours before the last administration, 30 minutes after the last administration, each group of animals was given 0.2 mL of 4% activated carbon suspension (prepared with 2% carboxymethyl cellulose solution). After 20 minutes, the animals were killed by dislocating the neck, the abdominal cavity was opened and the mesentery was separated, the intestinal tube from the pylorus to the ileocecum was cut and placed on a tray covered with 0.9% sodium chloride solution, the small intestine was gently arranged into a straight line without traction, the length from the pylorus to the ileocecum was measured as the total length of the small intestine, and then the length from the pylorus to the front edge of the charcoal was measured and recorded as the charcoal advancement distance, and the charcoal advancement rate was calculated.

[0113] Carbon powder advancement rate = Carbon powder advancement distance / small intestine total length × 100%

[0114] 2.4 Antidiarrheal test

[0115] 60 KM mice were randomly divided into 6 groups: blank group, model group, positive group (Pulsatilla oral liquid), Tibetan medicine composition dose groups (0.3033g / kg, 0.91g / kg, 2.73g / kg), 10 mice in each group, half male and half female. Senna leaves were continuously gavaged for 3 days to establish a mouse diarrhea model, and the mental state, behavioral movements and stool of the mice were observed. After the diarrhea model was successfully established, the blank group and the model group were gavaged with deionized water, the positive group was gavaged with Pulsatilla oral liquid, and the test drug groups of Aconitum decoction were gavaged with the corresponding concentration of the test drug solution. The feces of each group of mice within 5 hours were weighed from 9:00 to 14:00 every day, and weighed again after oven drying, and the feces water content of each experimental group was recorded and calculated.

[0116] 3. Research Results

[0117] 3.1 Anti-inflammatory test

[0118] From Table 7 and Figure 3 It can be seen that the medium dose group of the Tibetan medicine composition can significantly inhibit the auricle swelling rate caused by xylene (P < 0.01), and has a significant anti-inflammatory effect.

[0119] Table 7 Tibetan medicine composition inhibition rate of auricle swelling caused by xylene (n=10)

[0120] Group Mean Standard error Inhibition rate Blank control group 89.84 8.41 / Positive control group 55.94 4.3 37.73％ Low dose group 75.62 6.63 15.83％ Medium dose group 59.16 6.96 34.15％ High dose group 78.90 4.37 12.18％

[0121] 3.2 Analgesia test

[0122] From Table 8 and Figure 4 It can be seen that all dose groups of the Tibetan medicine composition can prolong the latency period of acetic acid-induced writhing pain in mice and reduce the number of pain times, but the results were not significant (P>0.05).

[0123] Table 8 Tibetan medicine composition for the incubation period and pain frequency of writhing pain caused by glacial acetic acid (n=10)

[0124]

[0125]

[0126] 3.3 Small bowel propulsion test

[0127] From Table 9 and Figure 5 It can be seen that compared with the blank group, the Tibetan medicine composition in the low, medium and high dose groups can inhibit the propagation of carbon powder in the small intestine, but not significantly (P<0.05).

[0128] Table 9 The propulsion rate of the Tibetan medicine composition on carbon powder in the small intestine (n=10)

[0129] Group Small intestinal propulsion rate Blank control group 85.18±3.39％ Positive control group 65.92±4.49％ Low dose group 80.21±1.48％ Medium dose group 73.94±2.88％ High dose group 74.41±2.19％

[0130] 3.4 Antidiarrheal test

[0131] From Table 10 and Figure 6 It can be seen that there was a significant difference in fecal water content between the blank control group and other groups 3 days after modeling (P < 0.01), indicating that the experimental model was successfully established. After 3 days of treatment, the Tibetan medicine composition could significantly reduce the water content in mouse feces (P < 0.01), indicating that the Tibetan medicine composition has a significant antidiarrheal effect.

[0132] Table 10 Effect of Tibetan medicine composition on feces water content of experimental group mice (n=10)

[0133] Group Modeling 3D feces water content (g) Fecal water content after 3 days of administration (g) Blank control group 0.5817±0.0540 0.5183±0.0861 Model Group 2.0783±0.1651 3.8667±0.4498 Positive control group 2.2167±0.2529 1.5833±0.0976 Low dose group 1.9033±0.1283 1.2017±0.0892 Medium dose group 2.0400±0.2025 1.2750±0.1866 High dose group 2.3517±0.3795 1.2767±0.1816

[0134] 4. Conclusion

[0135] By establishing a mouse experimental animal model, the antidiarrhea, small intestinal propulsion, anti-inflammatory and analgesic effects of the Tibetan medicine composition were observed. The results showed that the Tibetan medicine composition could significantly reduce the water content in the feces of mice (P < 0.01), indicating that the Tibetan medicine composition had a good antidiarrhea effect; the Tibetan medicine composition in the low, medium and high dose groups could inhibit the propulsion of carbon powder in the small intestine, but not significantly (P < 0.05), indicating that the Tibetan medicine composition had a certain astringent effect; the medium dose group could significantly inhibit the ear swelling rate caused by xylene (P < 0.01), indicating that the Tibetan medicine composition had a good anti-inflammatory effect (P < 0.01), and could prolong the latency period of acetic acid-induced writhing pain in mice and reduce the number of pains, but not significantly (P > 0.05), indicating that the Tibetan medicine composition had a certain analgesic effect.

[0136] The present invention provides a Tibetan medicine composition for treating calf yak diarrhea, which is a pharmacological and pharmacodynamic screening of the effect of treating calf yak diarrhea by combining traditional Tibetan veterinary medicine prescriptions with modern technical methods. The composition has a reasonable formula, readily available raw materials, a simple preparation process, simple operation, safety and effectiveness. The Tibetan medicine composition of the present invention can significantly inhibit the growth of bacteria and viruses, can significantly reduce the water content in mouse feces, and has good antidiarrheal effect, certain intestinal astringent effect, good anti-inflammatory effect and analgesic effect. It has been verified by clinical application experiments that the composition has a significant effect on treating calf yak diarrhea, and has broad promotion and application value in the clinical application of plateau calf yak.

Claims

1. A Tibetan medicine composition for treating diarrhea in calves and yaks, characterized in that: The Tibetan medicine composition consists of the following raw materials in parts by weight: 5-10 parts of Aconitum carmichaelii, 4-8 parts of Aconitum natans and 4-8 parts of Rubia cordifolia. The diarrhea of ​​yak calves is caused by rotavirus infection.

2. The Tibetan medicine composition according to claim 1, characterized in that: The Tibetan medicine composition is composed of the following raw materials in parts by weight: 5 parts of Aconitum carmichaelii, 4 parts of Aconitum natans and 4 parts of Rubia cordifolia.

3. The method for preparing the Tibetan medicine composition according to claim 1 or 2, characterized in that: The method comprises the following steps: (1) accurately weighing each medicine, adding ultrapure water, and soaking; (2) boiling twice with water, filtering, collecting, and combining the extracts; (3) concentrating the extracts to obtain a concentrated extract of the Tibetan medicine composition, and sealing and storing it after cooling.

4. A drug, characterized in that The invention comprises the Tibetan medicine composition according to claim 1 or 2.

5. The drug according to claim 4, characterized in that The dosage form of the drug includes oral preparations or injection preparations.

6. The drug according to claim 5, characterized in that The oral preparations include tablets, capsules, granules, pills, extracts, oral solutions, oral suspensions or oral emulsions.

7. The drug according to claim 5, characterized in that The injection preparation includes injection solution or powder injection.

8. Use of the Tibetan medicine composition according to claim 1 or 2 or the medicine according to any one of claims 4 to 7 in the preparation of a medicine for treating yak diarrhea.

Images