Tibetan medicine composition for treating calf diarrhea as well as preparation method and application of Tibetan medicine composition

Abstract

The invention provides a Tibetan medicine composition for treating calf diarrhea. The Tibetan medicine composition is prepared from the following components in parts by weight: 10 to 30 parts of fructus chebulae, 10 to 25 parts of fructus cumin, 15 to 35 parts of pericarpium granati and 20 to 40 parts of radix puerariae. The invention also provides a preparation method of the traditional Chinese medicine composition and application of the traditional Chinese medicine composition in preparation of medicines for treating calf diarrhea. The Tibetan medicine compound disclosed by the invention not only has the effects of easing pain, resisting inflammation and inhibiting intestinal peristalsis, but also can reduce the number of intestinal inflammatory cells, protect the intestinal morphology, regulate intestinal flora, repair intestinal mucosal barriers, treat diarrhea and reduce the problems of drug residues, bacterial drug resistance and the like when antibiotics and chemical synthetic drugs are used. In addition, the compound is a pure natural medicine, bacterial drug resistance is not prone to being generated, and animal production performance is not affected.

Description

Technical Field

[0001] The invention belongs to the technical field of Tibetan medicines, and particularly relates to a Tibetan medicine composition for treating calf diarrhea, a preparation method and an application thereof. Background Art

[0002] Calf diarrhea is a common digestive tract disease in newborn calves about 10 days after birth. This disease can occur all year round. The main clinical manifestations include indigestion, diarrhea and dehydration. In severe cases, it may even lead to the death of the calf. Calf diarrhea is one of the main diseases that cause growth retardation, developmental stunting and death in newborn calves in my country. The incidence rate accounts for about 55% of calf diseases and the mortality rate is about 15%. This disease has gradually become one of the main causes of calf death, which not only seriously affects the production efficiency of the livestock industry, but also causes serious economic losses. It is called the killer of newborn calves.

[0003] The causes of calf diarrhea are mainly divided into two factors: infectious and non-infectious. Infectious causes are mainly caused by various microorganisms such as viruses, bacteria and parasites. Non-infectious causes are mainly related to nutritional imbalance and improper feeding management. At present, antibiotics are mainly used to treat this disease at home and abroad, but due to the diversity of pathogens of calf diarrhea and lack of specificity, the efficacy is limited, and it is easy to lead to the continuous enhancement of bacterial resistance and drug residue problems, which poses serious public health and safety problems. Therefore, it is very important to develop non-antibiotic treatments for calf diarrhea.

[0004] Tibetan medicine mostly grows in high-altitude areas, has a long growth cycle, and generally has high bioactive ingredients. In addition, Tibetan medicine has a complete theoretical system. The products produced under the guidance of its theory are green and safe, with multiple targets, no residue, and low side effects. They have good therapeutic effects on digestive system diseases, such as Jiebai Pills. In recent years, technicians in this field have also carried out a lot of research on calf diarrhea, using compound forms to eliminate pathogens through antibacterial and antiviral methods, as well as anti-inflammatory, analgesic, and antioxidant methods to achieve the purpose of treating both the symptoms and the root causes. However, the state of the intestine determines the health of the digestive tract and is the key to curing diarrhea. The intestinal mucosal barrier formed by intestinal epithelial cells, lower connective tissue, and a variety of symbiotic flora can prevent harmful substances such as pathogenic microorganisms and endotoxins from passing through the intestinal mucosa, while selectively absorbing nutrients in the intestine to maintain the relative stability of the body's internal environment and maintain the body's normal life activities. It plays an important role in immune defense and maintaining the integrity of intestinal function. However, there is no relevant research at present. 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 calf diarrhea and its preparation method and application. The Tibetan medicine compound of the present invention can not only repair the intestinal mucosal barrier, but also relieve pain, resist inflammation, and inhibit intestinal peristalsis, and has a good therapeutic effect on calf diarrhea.

[0006] The invention provides a Tibetan medicine composition for treating calf diarrhea. The Tibetan medicine composition consists of the following components in parts by weight: 10-30 parts of terminalia chebula, 10-25 parts of niangzezhe, 15-35 parts of pomegranate peels and 20-40 parts of kudzu root.

[0007] Preferably, the Tibetan medicine composition is composed of the following components in parts by weight: 10-25 parts of Terminalia chebula, 17-22 parts of Niangzizhe, 20-35 parts of Punica granatum peel, and 25-40 parts of Pueraria root.

[0008] Preferably, the Tibetan medicine composition is composed of the following components in parts by weight: 20 parts of Terminalia chebula, 18 parts of Niangzizhe, 24 parts of Punica granatum peel, and 30 parts of Pueraria root; or

[0009] The Tibetan medicine composition is composed of the following components in parts by weight: 25 parts of Terminalia chebula, 22 parts of Niangzizhe, 20 parts of Punica granatum peel and 25 parts of Pueraria root.

[0010] The invention also provides a preparation method of the Tibetan medicine composition, comprising the following steps: crushing a formula amount of Terminalia chebula into coarse powder, mixing the powder with a formula amount of Nianzizhe, pomegranate peel and Pueraria root to obtain a mixture; decocting the obtained mixture with water, filtering and collecting the filtrate.

[0011] Preferably, the ratio of the mixture to water is 1 g: 6-10 ml; the decoction is performed 2-3 times, each time for 1-2 hours.

[0012] Preferably, the decoctions from each batch are combined and concentrated to contain 0.8 to 1.2 g of the crude drug per ml.

[0013] The invention also provides application of the Tibetan medicine composition in preparing medicine for treating calf diarrhea.

[0014] Preferably, the dosage of the Tibetan medicine composition is: each calf is gavaged with 100-120 mL of the Tibetan medicine composition each time, twice a day, for 3-5 consecutive days.

[0015] The invention also provides a medicine, the effective components of which include the Tibetan medicine composition.

[0016] Preferably, the dosage form of the drug includes an oral solution.

[0017] Compared with the prior art, the Tibetan medicine composition of the present invention has the following advantages and significant progress:

[0018] (1) Tibetan medicines are mostly grown in high altitude areas, have long growth cycles, and generally have high bioactive components. The Tibetan medicine composition of the present invention is extracted and made into an oral liquid, which is administered orally, has a fast onset of action, good efficacy, no toxic side effects, and no residue, and can significantly reduce the use of antibiotics in calf breeding.

[0019] (2) The Tibetan medicine composition of the present invention has been strictly screened and studied for its medicinal properties. The compound has the functions of purging fire and detoxifying, drying dampness and thickening intestines, and is an important medicine for treating dysentery; Pueraria root relieves muscles and reduces fever, promotes the production of body fluids and quenches thirst, and promotes yang and stops diarrhea; Pomegranate peel has the functions of astringing intestines and stopping diarrhea, stopping bleeding, and expelling parasites; Terminalia chebula has the functions of astringing intestines and stopping diarrhea, and is commonly used for chronic diarrhea, blood in the stool and prolapse of the anus; the prescription is formulated according to the Tibetan medicine theory of clearing away heat and drying dampness, relieving exterior pathogens and clearing the interior, and comprehensively preventing and treating diarrhea pathogenic bacteria, viruses and parasites, and combined with the functions of Pueraria root in promoting the production of body fluids and quenching thirst and protecting the intestines, it takes both internal and external factors into consideration to achieve the effect of preventing and treating diarrhea in an all-round way. It has been clinically verified to have a good effect in treating calf diarrhea.

[0020] (3) The Tibetan medicine compound of the present invention not only has the effects of analgesia, anti-inflammatory and inhibiting intestinal peristalsis, but also can reduce the number of intestinal inflammatory cells, protect intestinal morphology, regulate intestinal flora, repair intestinal mucosal barrier, treat diarrhea, and reduce problems such as drug residues and bacterial resistance when using antibiotics and chemical synthetic drugs. In addition, the compound is a pure natural medicine, is not easy to produce bacterial resistance, and does not affect animal production performance.

[0021] (4) The present invention uses only four Tibetan medicinal ingredients, namely, Terminalia chebula, Niangzizhe, Punica granatum and Pueraria root, to formulate the prescription. Compared with the prior art, the raw materials are easily available, the preparation process is simple, the pharmacological effects are clear, and it is convenient for industrial production, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0023] Figure 1 To investigate the effect of Tibetan medicine compound on xylene-induced ear swelling in mice.

[0024] Figure 2 To investigate the effect of Tibetan medicine compound on carrageenan-induced paw swelling in rats.

[0025] Figure 3 This study aims to investigate the effects of a Tibetan medicine compound on serum NO, TNF-α, IL-1β, and IL-6 in rats with paw swelling.

[0026] Figure 4 To investigate the effect of Tibetan medicine compound on hot plate-induced pain in mice.

[0027] Figure 5This study is about the effect of Tibetan medicine compound on acetic acid-induced writhing reaction in mice.

[0028] Figure 6 This is the effect of Tibetan medicine compound on carbon powder advancement rate in mice.

[0029] Figure 7 The body weight changes of rats in each group.

[0030] Figure 8 The changes of loose stool rate and diarrhea index in each group of rats.

[0031] Fig. 9 Comparison of blood routine indexes of rats in each group.

[0032] Fig.10 The changes of biochemical indexes in each group of rats.

[0033] Fig.11 HE staining of small intestine and colon of rats in each group.

[0034] Fig.12 Comparison of small intestine morphology in rats of each group.

[0035] Fig.13 This study investigates the effects of Tibetan medicine compound on the mRNA expression levels of tight junction proteins Claudin-1, ZO-1, Occludin, and MUC-2 in the intestine of rats.

[0036] Fig.14 This is the immunohistochemistry of positive expression of Claudin-1, Zo-1, Occludin and Muc-2 in rat jejunum tissue.

[0037] Fig.15 This study is to investigate the effect of Tibetan medicine compound on the expression of Claudin-1, Zo-1, Occludin and Muc-2 proteins in rat jejunal tissue.

[0038] Fig.16 PAS staining of the jejunum of rats in each group.

[0039] Fig.17 The changes in the number of goblet cells in each group.

[0040] Fig.18 Analyze the petal plots for OTU species.

[0041] Fig.19 This is the result of α analysis.

[0042] Fig. 20 This is the principal coordinate analysis of the intestinal flora of rats in each group.

[0043] Fig.21 The classification composition of the intestinal microbiota of rats in each group. DETAILED DESCRIPTION

[0044] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0045] The Tibetan medicine composition for treating calf diarrhea of ​​the present invention is composed of the following components in parts by weight:

[0046] 10-30 parts of Terminalia chebula, 10-25 parts of Niangzi zhe, 15-35 parts of pomegranate peel, and 20-40 parts of Pueraria root.

[0047] The optimal formula is: 10-25 parts of Terminalia chebula, 17-22 parts of Niangzizhe, 20-35 parts of pomegranate peel, and 25-40 parts of Pueraria root.

[0048] The optimal formula is: 20 parts of Terminalia chebula, 18 parts of Niangzi, 24 parts of pomegranate peel, and 30 parts of Pueraria root.

[0049] The preparation method of a Tibetan medicine composition for treating calf diarrhea of ​​the present invention is as follows:

[0050] The formula amount of Terminalia chebula is crushed into coarse powder, and mixed with the formula amount of Nianzizhe, pomegranate peel and Pueraria root to obtain a mixture; and the obtained mixture is decocted with water.

[0051] Terminalia chebula is the dried mature fruit of the Combretaceae plant Terminalia chebula. It is large in size and hard in texture. Crushing and decocting it is beneficial to the release of its active ingredients.

[0052] In some embodiments, the mixture is soaked for 1-2 hours before the first decoction.

[0053] In some embodiments, the ratio of the mixture to water is 1 g: 6-10 ml.

[0054] In some embodiments, the mixture is decocted 2-3 times, each time for 1-2 hours.

[0055] In some embodiments, each decoction is combined and concentrated to contain 0.8-1.2 grams of crude drug per milliliter of oral solution.

[0056] In some embodiments, after concentration, high temperature sterilization and filling steps are also included.

[0057] When treating calf diarrhea, each calf is gavaged with 100-120 mL of the Tibetan medicine composition of the present invention twice a day for 3-5 consecutive days.

[0058] Example 1

[0059] A Tibetan medicine composition for treating calf diarrhea in this embodiment is composed of the following components in parts by weight:

[0060] 20 parts of Terminalia chebula, 18 parts of Niangzizhe, 24 parts of Punica granatum, and 30 parts of Pueraria root.

[0061] The preparation method of a Tibetan medicine composition for treating calf diarrhea in this embodiment is as follows:

[0062] The formula amount of Terminalia chebula is crushed into coarse powder, mixed with the formula amount of Nianzizhe, pomegranate peel and Pueraria root to obtain a mixture; water is added in a ratio of 1g:8ml of the mixture to water, decocted 3 times, each time for 1.5h, soaked for 2h before the first decoction, the three decoctions are combined, concentrated to 1g of crude drug per ml of oral liquid, filtered, sterilized at high temperature, and filled. Named Tibetan medicine compound No. 1.

[0063] Example 2

[0064] A Tibetan medicine composition for treating calf diarrhea in this embodiment is composed of the following components in parts by weight:

[0065] 25 parts of Terminalia chebula, 22 parts of Niangzizhe, 20 parts of Punica granatum, and 25 parts of Pueraria root.

[0066] The preparation method of a Tibetan medicine composition for treating calf diarrhea in this embodiment is as follows:

[0067] The formula amount of Terminalia chebula is crushed into coarse powder, mixed with the formula amount of Nianzizhe, pomegranate peel and Pueraria root to obtain a mixture; water is added in a ratio of 1g:9ml of the mixture to water, and decocted for 3 times, each time for 1.5h, soaked for 2h before the first decoction, the three decoctions are combined, concentrated to 1g of crude drug per ml of oral liquid, filtered, sterilized at high temperature, and filled. Named Tibetan medicine compound No. 2.

[0068] Example 3

[0069] A Tibetan medicine composition for treating calf diarrhea in this embodiment is composed of the following components in parts by weight:

[0070] 10 parts of Terminalia chebula, 17 parts of Niangzizhe, 35 parts of Punica granatum, and 40 parts of Pueraria root.

[0071] The preparation method of a Tibetan medicine composition for treating calf diarrhea in this embodiment is as follows:

[0072] The formula amount of Terminalia chebula is crushed into coarse powder, mixed with the formula amount of Nianzizhe, pomegranate peel and Pueraria root to obtain a mixture; water is added in a ratio of 1g:10ml of the mixture to water, and decocted for 3 times, each time for 1.5 hours, soaked for 2 hours before the first decoction, the three decoctions are combined, concentrated to 1g of crude drug per milliliter of oral liquid, filtered, sterilized at high temperature, and filled. It is named Tibetan medicine compound No. 3.

[0073] Example 4

[0074] A Tibetan medicine composition for treating calf diarrhea in this embodiment is composed of the following components in parts by weight:

[0075] 25 parts of Terminalia chebula, 25 parts of Nianzi zhe, and 40 parts of Pueraria root.

[0076] The preparation method of a Tibetan medicine composition for treating calf diarrhea in this embodiment is as follows:

[0077] The formula amount of Terminalia chebula is crushed into coarse powder, mixed with the formula amount of Nianzizhe, pomegranate peel and Pueraria root to obtain a mixture; water is added at a ratio of 1g:7ml of the mixture to water, and decocted for 3 times, each time for 1.5h, soaked for 2h before the first decoction, the three decoctions are combined, concentrated to 1g of crude drug per ml of oral liquid, filtered, sterilized at high temperature, and filled. Named Tibetan medicine compound No. 4.

[0078] Example 5

[0079] A Tibetan medicine composition for treating calf diarrhea in this embodiment is composed of the following components in parts by weight:

[0080] 30 parts of Terminalia chebula, 28 parts of Nianzizhe, and 34 parts of pomegranate peel.

[0081] The preparation method of a Tibetan medicine composition for treating calf diarrhea in this embodiment is as follows:

[0082] The formula amount of Terminalia chebula is crushed into coarse powder, mixed with the formula amount of Nianzizhe, pomegranate peel and Pueraria root to obtain a mixture; water is added in a ratio of 1g:9ml of the mixture to water, and decocted for 3 times, each time for 1.5h, soaked for 2h before the first decoction, the three decoctions are combined, concentrated to 1g of crude drug per ml of oral liquid, filtered, sterilized at high temperature, and filled. It is named Tibetan medicine compound No. 5.

[0083] Example 6

[0084] A Tibetan medicine composition for treating calf diarrhea in this embodiment is composed of the following components in parts by weight:

[0085] 26 parts of Terminalia chebula, 30 parts of pomegranate peels, and 36 parts of Pueraria root.

[0086] The preparation method of a Tibetan medicine composition for treating calf diarrhea in this embodiment is as follows:

[0087] The formula amount of Terminalia chebula is crushed into coarse powder, mixed with the formula amount of Nianzizhe, pomegranate peel and Pueraria root to obtain a mixture; water is added in a ratio of 1g:8ml of the mixture to water, decocted 3 times, each time for 1.5h, soaked for 2h before the first decoction, the three decoctions are combined, concentrated to 1g of crude drug per ml of oral liquid, filtered, sterilized at high temperature, and filled. It is named Tibetan medicine compound No. 6.

[0088] Test Example 1 Screening test of the Tibetan medicine compound of the present invention

[0089] Experimental study on the treatment of diarrhea in rats with Tibetan medicine compound

[0090] Main drugs and reagents: Tibetan medicine compound No. 1, Tibetan medicine compound No. 2, Tibetan medicine compound No. 3, Tibetan medicine compound No. 4, Tibetan medicine compound No. 5, Tibetan medicine compound No. 6, are prepared in turn according to Example 1, Example 2, Example 3, Example 4, Example 5, and Example 6. Pulsatilla oral solution was purchased from Beijing Shengtaier Technology Co., Ltd.

[0091] Experimental animals: 72 SD rats, weighing 200±20g; purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, with certificate number SCXK(Gan)2020-0002, and kept in the Dawashan Standard Animal House of Lanzhou Animal Husbandry and Veterinary Drug Research Institute, Chinese Academy of Agricultural Sciences, and adapted for one week before the experiment. Escherichia coli CVCC1527 was purchased from China Microbiological Culture Collection Center and cultured in a 37°C incubator for 24h with a concentration of 1×10 8 CFU / mL bacterial solution, stored at 4℃ for future use.

[0092] A total of 72 healthy SD male rats were randomly divided into blank control group, diarrhea model group, Tibetan medicine compound No. 1 group, Tibetan medicine compound No. 2 group, Tibetan medicine compound No. 3 group, Tibetan medicine compound No. 4 group, Tibetan medicine compound No. 5 group, Tibetan medicine compound No. 6 group, and Baitouweng decoction group, with 8 rats in each group. Except for the blank control group, the remaining groups were intraperitoneally injected with 0.5 mL of 1×10 8 CFU / mL of Escherichia coli, each drug group was gavaged with each drug at 1mL / 100g·BW in the afternoon of the 2nd to 6th day, and the blank control group and diarrhea model group rats were given the same volume of saline at the same time. The body weight of the rats was recorded every day during the experiment. After intraperitoneal injection of Escherichia coli on the 5th day, the loose stool rate, loose stool grade and diarrhea index of the rats were recorded and calculated. Loose stool rate = the number of loose stools discharged by each rat / the total number of stools per rat. The loose stool grade was divided into 4 levels according to the stain area of ​​the feces on the filter paper: (0) dry stool, that is, the feces are hard and there is no stain on the filter paper after picking it up; (1) mucous stool, that is, the feces are sticky with mucus; (2) clear water, that is, the feces are light yellow and have particles; (3) wet stool, that is, the feces are soft and moist, easy to separate from each other, and visually moist; (4) loose stool, that is, unformed feces.

[0093] Experimental Results

[0094] (1) Effect of Tibetan medicine compound on body weight of rats with diarrhea

[0095] The effect of Tibetan medicine compound on improving body weight of rats with diarrhea is shown in Table 1. Compared with the model group, each drug group improved the body weight of rats. Tibetan medicine compound No. 1, Tibetan medicine compound No. 2, and Tibetan medicine compound No. 3 had a more obvious effect on improving body weight, and were better than the positive control Baitouweng decoction group, indicating that Tibetan medicine compound can increase the body weight of rats with diarrhea, and Tibetan medicine compound No. 1, Tibetan medicine compound No. 2, and Tibetan medicine compound No. 3 have better effects.

[0096] Table 1 Body weight of rats in each group (g)

[0097]

[0098] (2) Effect of Tibetan medicine compound on loose stool rate and diarrhea index in rats with diarrhea

[0099] As can be seen from Table 2, compared with the model group, the loose stool rate and diarrhea index of rats in the Tibetan medicine compound No. 1, Tibetan medicine compound No. 2, Tibetan medicine compound No. 3, Tibetan medicine compound No. 4 and Baitouweng decoction groups were significantly reduced (P < 0.05); the results showed that the Tibetan medicine compound has a significant antidiarrheal effect, and the antidiarrheal effects of Tibetan medicine compound No. 1 and Tibetan medicine compound No. 2 are better than those of Baitouweng decoction.

[0100] Table 2 Changes of loose stool rate and diarrhea index of rats in each group

[0101]

[0102]

[0103] Test Example 2 Anti-inflammatory, analgesic and intestinal peristalsis inhibiting effects of the Tibetan medicine composition of the present invention

[0104] 1 Experimental drugs and experimental animals

[0105] 1.1 Main drugs and reagents: Tibetan medicine compound No. 1 was prepared according to the method of Example 1. Indomethacin (McLean Biochemical Technology Co., Ltd.), activated carbon (product of Yantai Shuangshuang Chemical Co., Ltd.), gum arabic (product of Beijing Solebaugh Technology Co., Ltd.), carrageenan (Sigma-Aldrich), senna (Dalin Chinese Medicinal Materials Purchasing and Sales Department, Yulin City, Yunnan Province), loperamide hydrochloride capsules (Xi'an Janssen Pharmaceutical Co., Ltd.). Glacial acetic acid, xylene (products of Sinopharm Chemical Reagent Co., Ltd.). Interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor (TNF-α), nitric oxide (NO) detection kit (Biyuntian Biotechnology Co., Ltd.).

[0106] 1.2 Experimental animals: Kunming mice, weight 20±2g; SD rats, weight 200±20g; purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, certificate number SCXK(Gan)2020-0002, raised in the Dawashan standard animal house of Lanzhou Institute of Animal Husbandry and Veterinary Drugs, Chinese Academy of Agricultural Sciences, and adaptively raised for one week before the experiment.

[0107] In the present invention, "HBJP" is the abbreviation of Tibetan medicine compound, "Tibetan medicine compound high-dose group" is referred to as "HBJP high-dose group", "Tibetan medicine compound medium-dose group" is referred to as "HBJP medium-dose group", and "Tibetan medicine compound low-dose group" is referred to as "HBJP low-dose group"; "model group" is also called "control group".

[0108] In each figure, the same letters indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0109] 2 Experimental process

[0110] 2.1 Effect of Tibetan medicine compound on xylene-induced ear swelling in mice

[0111] Healthy Kunming mice (20±2g) were randomly divided into 5 groups (n=10), namely, the Tibetan medicine compound high-dose group (5g / kg·BW), medium-dose group (2.5g / kg·BW), low-dose group (1.25g / kg·BW), model group (normal saline, 10mL / kg·BW), and positive drug control group (indomethacin, 3mg / kg·BW). The drug was administered by gavage once a day for 5 consecutive days. 1.0h after the last administration, 20μL of xylene was applied to the right ear of each mouse. 4h after application, the mice were killed, and symmetrical holes with a diameter of 8mm were taken from both ears and weighed. The ear swelling rate and ear swelling inhibition rate were calculated according to the following formula.

[0112] Ear swelling rate = (right ear mass - left ear mass) / left ear mass × 100%

[0113] Ear swelling inhibition rate = (ear swelling rate of model group - ear swelling rate of treatment group) / ear swelling rate of model group × 100%.

[0114] 2.2 Effect of Tibetan medicine compound on carrageenan-induced paw swelling in rats

[0115] Healthy SD rats (200±20g) were randomly divided into 5 groups (n=8), namely, Tibetan medicine compound high-dose group (3.5g / kg·BW), medium-dose group (1.75g / kg·BW), low-dose group (0.875g / kg·BW), model group (normal saline, 5mL / kg·BW), and positive drug control group (indomethacin, 2.08mg / kg·BW). The rats were given the drug by gavage once a day for 5 consecutive days. 1.0h after the last administration, 0.1mL of carrageenan solution (1%, V / V) was subcutaneously injected into the right hind foot of the rats. The volume below the right ankle joint was measured 1, 2, 3, and 4h after the injection of carrageenan, and the foot swelling rate and foot swelling inhibition rate were calculated according to the following formula.

[0116] Foot swelling rate = (foot volume after carrageenan administration - foot volume before administration) / foot volume before administration × 100%

[0117] Foot swelling inhibition rate = (foot swelling rate of model group - foot swelling rate of treatment group) / foot swelling rate of model group × 100%.

[0118] 2.3 Determination of serum NO, IL-1β, IL-6, and TNF-α levels in rats

[0119] After the last measurement under "2.2", blood was collected from the rat heart, and after blood coagulation, it was centrifuged at 3000r / min for 10min to collect serum. The levels of NO, IL-1β, IL-6 and TNF-α in serum were detected according to the instructions of the enzyme-linked immunosorbent assay (ELISA) kit.

[0120] 2.4 Effect of Tibetan medicine compound on hot plate-induced pain response in mice

[0121] Healthy Kunming mice (20±2g) were placed on a hot plate at (55±0.5)℃, and the time it took for the mice to touch the hot plate and lift their hind legs, i.e., the latency of pain response, was monitored and recorded. Mice with a latency of 5-30s were selected as qualified mice. The qualified mice were randomly divided into 5 groups (n=10), namely, a high-dose group of Tibetan medicine compound (5g / kg·BW), a medium-dose group (2.5g / kg·BW), a low-dose group (1.25g / kg·BW), a model group (normal saline, 10mL / kg·BW), and a positive drug control group (indomethacin, 3mg / kg·BW). The drugs were administered by gavage once a day for 5 consecutive days. The latency of pain response after treatment was recorded at 1.0, 1.5, 2.0, 3.0, and 4.0h after the last administration. For mice that did not respond for more than 60s, 60s was used as the latency of pain response of the test mice.

[0122] 2.5 Effect of Tibetan medicine compound on acetic acid-induced writhing reaction in mice

[0123] Healthy Kunming mice (20±2g) were randomly divided into 5 groups (n=10), namely, the Tibetan medicine compound high-dose group (5g / kg·BW), medium-dose group (2.5g / kg·BW), low-dose group (1.25g / kg·BW), model group (normal saline, 10mL / kg·BW), and positive drug control group (indomethacin, 3mg / kg·BW). The drug was administered by gavage once a day for 5 consecutive days. 1.0h after the last administration, 0.6% acetic acid (0.1mL / 10g·BW) was injected intraperitoneally, and the number of twists 11-30min after injection was observed, and the inhibition rate of twisting reaction was calculated. The criteria for twisting reaction: the abdomen contracted and sunken, the anterior abdominal wall was close to the cage bottom, the buttocks were raised, and the trunk and hind limbs were extended.

[0124] The writhing reaction inhibition rate = (average writhing times of the model group - average writhing times of the drug-treated group) / average writhing times of the model group × 100%.

[0125] 2.6 Effect of Tibetan medicine compound on small intestinal motility in mice with senna-induced diarrhea

[0126] Preparation of senna leaf water extract: After crushing the senna leaves, brew them with 7 times the amount of boiling water, and take the supernatant when using, that is, 1 mL of senna leaf water is equivalent to 0.143 g of crude drug.

[0127] Preparation method of ink: Mix 5g of gum arabic with 40mL of distilled water and boil until clear and transparent, then mix evenly with 2.50g of activated carbon powder and boil 3 times, cool, make up to 50mL with distilled water, and store in a refrigerator at 4℃ for later use.

[0128] Healthy Kunming mice (20±2g) were randomly divided into 6 groups (n=10), namely, Tibetan medicine compound high-dose group (5g / kg·BW), medium-dose group (2.5g / kg·BW), low-dose group (1.25g / kg·BW), model group, blank group (normal saline, 10mL / kg·BW), and positive drug control group (loperamide hydrochloride, 10mg / kg·BW). The drug was administered by gavage once a day for 5 consecutive days. The mice were fasted but not watered for 12.0h before the last administration. 30min after the last administration, except for the blank control group, the rest of the mice were gavaged with 1.5g / kg·BW senna leaves. 30min after gavage with senna leaves, each mouse was gavaged with 0.2mL ink (5%, W / V), and the mice were killed by cervical dislocation 30min later, and the abdominal cavity was opened to separate the small intestine. The length from the pylorus to the farthest end of the charcoal powder (A) and the length from the pylorus to the cecum (B) were measured, and the small intestinal propulsion rate was calculated according to the following formula.

[0129] Small intestinal propulsion rate = charcoal powder propulsion length (A) / length from pylorus to ileocecal end (B) × 100%.

[0130] 3 Data analysis

[0131] SPSS 26.0 software was used to perform statistical analysis on all data in this experiment. The data were tested using one-way ANOVA and compared pairwise. The analysis results were input into GraphPad Prism software for drawing. The experimental data were expressed as "mean ± standard deviation (Mean ± SD)". P < 0.05 in all results indicated significant differences.

[0132] 4 Experimental results

[0133] 4.1 Comparison of changes in ear swelling in mice

[0134] Compared with the control group, the low, medium and high dose groups of Tibetan medicine compound significantly inhibited the swelling of the mouse ear (P<0.05); compared with the low dose group, the medium dose group of Tibetan medicine compound had a stronger inhibitory effect on ear swelling (P<0.05); compared with the high dose group, there was no significant difference in the inhibition rate of ear swelling between the medium dose group of Tibetan medicine compound and the high dose group (P>0.05), but the swelling inhibition rate of the medium dose group was higher. The results showed that the Tibetan medicine compound had an anti-inflammatory effect on acute inflammation induced by xylene, and the medium dose group had the best effect. Figure 1 .

[0135] Figure 1 To investigate the effect of Tibetan medicine compound on xylene-induced ear swelling in mice.

[0136] 4.2 Comparison of changes in carrageenan-induced paw swelling in rats

[0137] Compared with the control group, the low, medium and high dose groups of Tibetan medicine compound had significant inhibitory effects on carrageenan-induced toe swelling in rats within 4 hours after carrageenan injection (P<0.05); compared with the low dose group, the medium dose group of Tibetan medicine compound had enhanced inhibitory effects on toe swelling at 1h, 3h and 4h after carrageenan injection (P<0.05); compared with the high dose group, there was no significant difference in carrageenan-induced toe swelling in the medium dose group of Tibetan medicine compound within 4h after carrageenan injection (P>0.05); 4h after inflammation, the inhibition rates of low, medium and high doses of Tibetan medicine compound on foot swelling were 24.88%, 41.46% and 33.66%, respectively. The results showed that Tibetan medicine compound had significant inhibitory effects on carrageenan-induced foot swelling inflammation in rats, and the medium dose group had the best effect. Results are shown in Figure 2 .

[0138] Figure 2 To investigate the effect of Tibetan medicine compound on carrageenan-induced paw swelling in rats.

[0139] 4.3 Determination of serum NO, IL-1β, IL-6 and TNF-α levels in rats

[0140] Compared with the control group, the serum NO level of rats in the low, medium, and high dose groups of Tibetan medicine compound and indomethacin group was significantly decreased (P<0.05); compared with the control group, the serum TNF-α, IL-1β, and IL-6 levels of rats in the low, medium, and high dose groups of Tibetan medicine compound and indomethacin group were significantly decreased (P<0.05); compared with the high and low dose groups of Tibetan medicine compound, the serum TNF-α, IL-1β, and IL-6 levels of rats in the medium dose group of Tibetan medicine compound were significantly decreased (P<0.05). The results showed that Tibetan medicine compound can reduce the serum NO level, as well as the serum TNF-α, IL-1β, and IL-6 levels, and play an anti-inflammatory role. Results are shown in Figure 3 .

[0141] Figure 3 This study aims to investigate the effects of a Tibetan medicine compound on serum NO, TNF-α, IL-1β, and IL-6 in rats with paw swelling.

[0142] 4.4 Effects on pain threshold of mice

[0143] Compared with the control group, the low, medium and high doses of Tibetan medicine compound had no significant effect on the pain threshold of mice 1 hour after administration (P>0.05); compared with the control group, the medium dose of Tibetan medicine compound significantly increased the pain threshold of mice 1.5h, 2h, 3h and 4h after administration (P<0.05); compared with the control group, the high dose of Tibetan medicine compound significantly increased the pain threshold of mice 2h and 4h after administration (P<0.05); The effect of the medium-dose group of the compound on increasing the pain threshold of mice was significantly higher than that of the low-dose group (P<0.05). The effect of the medium-dose group on increasing the pain threshold of mice was significantly higher than that of the high-dose group 2h and 3h after administration (P<0.05). Compared with the indomethacin group, the pain threshold of the medium-dose group of the Tibetan medicine compound increased significantly 2h and 3h after administration (P<0.05). At 4h after administration, the pain threshold of the mice in the medium-dose Tibetan medicine compound was greater than that in the indomethacin group, but the difference was not significant (P>0.05). The results showed that the Tibetan medicine compound can prolong the latency period of thermal stimulation response and inhibit the pain response of mice, and the medium dose has the best effect. Results are shown in Figure 4 .

[0144] Figure 4 To investigate the effect of Tibetan medicine compound on hot plate-induced pain in mice.

[0145] 4.5 Comparison of changes in the number of writhing times in mice

[0146] Within 15 minutes after modeling, the number of writhing times of mice in the low, medium and high dose groups of Tibetan medicine compound was significantly reduced compared with the control group (P<0.05); the writhing inhibition rate of the medium dose group of Tibetan medicine compound was 52.47%; the writhing inhibition rates of the high and low dose groups of Tibetan medicine compound were 47.27% and 40.61%, respectively. The results showed that Tibetan medicine compound can reduce the number of writhing times of mice and inhibit the pain response of mice, and the medium dose has the best effect. Figure 5 .

[0147] Figure 5 This study is about the effect of Tibetan medicine compound on acetic acid-induced writhing reaction in mice.

[0148] 4.6 Comparison of charcoal propulsion rate in mice

[0149] 30 minutes after modeling, the intestinal carbon propulsion rate of mice in the control group was significantly increased compared with the blank group, indicating that the model was successfully established (P<0.05); compared with the control group, the intestinal carbon propulsion rate of mice in the low, medium and high dose groups of Tibetan medicine compound was significantly reduced (P<0.05), and there was no significant difference between the low, medium and high dose groups of Tibetan medicine compound and the blank group (P>0.05); the medium dose group of Tibetan medicine compound significantly inhibited the intestinal carbon propulsion rate of mice, and the difference was significant compared with the low and high dose groups of Tibetan medicine compound (P<0.05). The results show that the Tibetan medicine compound can inhibit intestinal peristalsis and restore the small intestinal motility function to normal, and the medium dose group has the best effect. Results are shown in Figure 6 .

[0150] Figure 6 This is the effect of Tibetan medicine compound on carbon powder advancement rate in mice.

[0151] Experimental Example 3 Effect of the Tibetan medicine composition of the present invention on the intestinal mucosal barrier of rats

[0152] 1 Experimental drugs and experimental animals

[0153] Main drugs and reagents: Tibetan medicine compound No. 1 was prepared according to the method of Example 1. Pulsatilla oral liquid (hereinafter referred to as "Pulsatilla soup") was purchased from Beijing Shengtaier Technology Co., Ltd.; enrofloxacin soluble powder was purchased from Hebei New Century Pharmaceutical Co., Ltd. LB agar, LB broth (Guangdong Huankai Microbiological Technology Co., Ltd.); BCA protein quantification kit (Shanghai Biyuntian Biotechnology Co., Ltd.); TRIzol reagent (Takara); SPARKscriptⅡ, 2×SYBR Green (Shandong Sikojie Technology Co., Ltd.), total protein (TP), albumin (ALB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (TC), urea (urea) and creatinine (CREA) (Meikang Biotechnology Co., Ltd.).

[0154] Experimental animals: 56 SD rats, weighing 200±20g; purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, with certificate number SCXK(Gan)2020-0002, and kept in the Dawashan Standard Animal House of Lanzhou Animal Husbandry and Veterinary Drug Research Institute, Chinese Academy of Agricultural Sciences, and adapted for one week before the experiment. Escherichia coli CVCC1527 was purchased from China Microbiological Culture Collection Center and cultured in a 37°C incubator for 24h with a concentration of 1×10 8 CFU / mL bacterial solution, stored at 4℃ for future use.

[0155] 2 Experimental process

[0156] A total of 56 healthy SD male rats were randomly divided into a blank control group, a diarrhea model group, a low-dose Tibetan medicine compound group, a medium-dose Tibetan medicine compound group, a high-dose Tibetan medicine compound group, a Baitouweng decoction group, and an enrofloxacin group, with 8 rats in each group. Except for the blank group, all other groups were intraperitoneally injected with 0.5 mL of 1×10 8 CFU / mL of Escherichia coli were treated with different doses of drugs in the afternoon of the 2nd to 6th day: Tibetan medicine compound high-dose group (14g / kg·BW), Tibetan medicine compound medium-dose group (7g / kg·BW), Tibetan medicine compound low-dose group (3.5g / kg·BW), Baitouweng decoction group (15g / kg·BW), enrofloxacin group (0.3g / kg·BW), and blank group rats were given the same volume of normal saline at the same time. The body weight of rats was recorded every day during the experiment. After intraperitoneal injection of Escherichia coli on the 1st, 3rd, and 5th days, the loose stool rate, loose stool grade, and diarrhea index of the rats were recorded and calculated. Loose stool rate = the number of loose stools discharged by each rat / the total number of stools per rat. The loose stool level was divided into four levels according to the area of ​​fecal contamination on the filter paper: (0) dry stool, i.e., hard stool, with no stain on the filter paper after picking up; (1) mucous stool, i.e., mucus on the stool; (2) clear water, i.e., pale yellow stool with particles; (3) wet stool, i.e., soft and moist stool, easy to separate from each other, and moist visually; (4) loose stool, i.e., unformed stool. On the 7th day, all rats were euthanized. Anticoagulated blood and serum were collected for the detection of blood routine and blood biochemical indexes. The contents of the duodenum, jejunum, ileum, colon, and cecum were collected, and the duodenum, jejunum, ileum, and colon were immediately fixed in 10% formalin for histopathological examination. The contents of the cecum were placed in a cryovial, quickly placed in liquid nitrogen, and transferred to a -80°C refrigerator for storage for intestinal microbial identification. Jejunal tissue was obtained, quickly placed in liquid nitrogen, and stored in a -80°C refrigerator for detection of intestinal mucosa-related protein and gene expression.

[0157] 3 Experimental results

[0158] 3.1 Effect of Tibetan medicine compound on body weight of rats with diarrhea

[0159] from Figure 7 It can be seen that the Tibetan medicine compound medium-dose group has a comparable effect on improving rat body weight as the enrofloxacin group, the Tibetan medicine compound high-dose group has a comparable effect on improving rat body weight as the Baitouweng decoction group, and the Tibetan medicine compound medium-dose group has the best effect on improving rat body weight, but in the end it failed to catch up with the weight of rats in the normal group, indicating that the Tibetan medicine compound can increase the body weight of rats with diarrhea, and the medium-dose group has the best effect.

[0160] Figure 7 The body weight changes of rats in each group.

[0161] 3.2 Effect of Tibetan medicine compound on loose stool rate and diarrhea index in diarrhea rats

[0162] Depend on Figure 8 It can be seen that on the 3rd and 5th days, compared with the model group, the loose stool rate and diarrhea index of rats in the low-, medium- and high-dose Tibetan medicine compound groups, Baitouweng decoction group and enrofloxacin group were significantly reduced (P < 0.05). On the 3rd and 5th days of modeling, the loose stool rate and diarrhea index of rats in the low-dose Tibetan medicine compound group, high-dose Tibetan medicine compound group and Baitouweng decoction group were significantly higher than those in the medium-dose Tibetan medicine compound group and enrofloxacin group (P < 0.05); on the 3rd day of modeling, the diarrhea index in the medium-dose Tibetan medicine compound group was significantly lower than that in the enrofloxacin group (P < 0.05). The results show that the Tibetan medicine compound has a significant antidiarrheal effect, and the antidiarrheal effect of the medium dose is better than that of Baitouweng decoction, and is equivalent to the antidiarrheal effect of enrofloxacin.

[0163] Figure 8 The changes of loose stool rate and diarrhea index in each group of rats.

[0164] 3.3 Effect of Tibetan medicine compound on blood routine test in rats with diarrhea

[0165] Blood test results are shown in Fig. 9 . Compared with the model group, the numbers of monocytes, lymphocytes, leukocytes, and neutrophils in the blood of rats in each drug group were significantly reduced (P<0.05), and the numbers of red blood cells, platelets, and hemoglobin concentrations in the blood of rats were not significantly different (P>0.05). The numbers of monocytes, lymphocytes, leukocytes, and neutrophils in the medium-dose Tibetan medicine compound group were not significantly different from those in the blank group and the enrofloxacin group (P>0.05), but were significantly lower than those in the high-dose Tibetan medicine compound group and the low-dose Tibetan medicine compound group (P<0.05), while the numbers of red blood cells, platelets, and hemoglobin concentrations in the blood of rats were not significantly different among the drug groups (P>0.05). The numbers of lymphocytes and leukocytes in the medium-dose Tibetan medicine compound group were significantly lower than those in the Pulsatilla group (P<0.05). The results showed that the Tibetan medicine compound could restore abnormal blood routine indicators in rats with diarrhea, and the medium-dose group of the Tibetan medicine compound was better than the Baitouweng decoction group in restoring the number of lymphocytes and white blood cells in rats, and the effect was equivalent to that of the enrofloxacin group.

[0166] Fig. 9Comparison of blood routine indexes of rats in each group.

[0167] 3.4 Effect of Tibetan medicine compound on blood biochemistry in rats with diarrhea

[0168] Blood biochemical test results are shown in Fig.10 Compared with the model group, the values ​​of TP, TC, ALB, and TCH biochemical indices in the serum of rats in each drug-treated group were significantly increased (P<0.05), and the values ​​of ALT, AST, UREA, and CREA biochemical indices in the serum of rats were significantly decreased (P<0.05); compared with the medium-dose group of Tibetan medicine compound, the values ​​of TP, TC, ALB, and TCH biochemical indices in the serum of rats in the high-dose group of Tibetan medicine compound, the low-dose group of Tibetan medicine compound, and the Baitouweng decoction group were significantly decreased (P<0.05), and the values ​​of ALT, AST, UREA, and CREA biochemical indices in the serum of rats were significantly increased (P<0.05); the values ​​of TC, ALB, and TCH biochemical indices in the serum of rats in the enrofloxacin group were significantly decreased (P<0.05), and the values ​​of AST, UREA, and CREA biochemical indices in the serum of rats were significantly increased (P<0.05). The results showed that the Tibetan medicine compound could improve the abnormal blood biochemical indexes in rats with diarrhea, and the medium-dose group of the Tibetan medicine compound had a better effect on improving the blood biochemical indexes in rats than the Baitouweng decoction group and the enrofloxacin group.

[0169] Fig.10 The changes of biochemical indexes in each group of rats.

[0170] 3.5 Effects of Tibetan medicine compound on histopathological observation and morphological measurement changes of rat small intestine

[0171] Depend on Fig.11 -AⅠ showed that the duodenal mucosal epithelial cells and villi of the blank group rats were arranged neatly, with complete structure and clear outline of intestinal glands; Fig.11 -BⅠ showed that the duodenal microvilli of the rats in the model group were edematous, irregular in shape, and broken; after treatment with Tibetan medicine compound, Baitouweng decoction, and enrofloxacin, Fig.11 -CI shows that the duodenal microvilli of rats in the low-dose Tibetan medicine compound group were slightly edematous, slightly broken, and arranged sparsely; Fig.11 -DⅠ and 11-GⅠ showed that the duodenal microvilli of rats in the medium-dose Tibetan medicine compound group and the enrofloxacin group were regular in shape, arranged relatively closely, and their morphology basically returned to normal; Fig.11 -EⅠ and 11-FⅠ showed that the duodenal villi in the high-dose Tibetan medicine compound group and the Baitouweng decoction group were arranged more neatly, more sparsely, and some villi tips fell off. Fig.12-A, 12-D showed that compared with the blank group, the length of duodenal villi of rats in the model group was significantly reduced (P<0.05), and the villus-to-cryptotype ratio was significantly reduced (P<0.05); compared with the model group, the length of duodenal villi of rats in each drug-treated group was significantly increased (P<0.05), and the villus-to-cryptotype ratio was significantly increased (P<0.05); compared with the medium-dose group of Tibetan medicine compound, the length of duodenal villi of rats in the high-dose group of Tibetan medicine compound, the low-dose group of Tibetan medicine compound and the Baitouweng decoction group was significantly reduced (P<0.05), and the villus-to-cryptotype ratio was significantly reduced (P<0.05); the villus-to-cryptotype ratio of rats in the enrofloxacin group was significantly reduced (P<0.05). The results showed that the Tibetan medicine compound had a repairing effect on the pathological changes of duodenal tissue in rats with diarrhea, and the medium-dose group had the best effect, which was better than the Baitouweng decoction group and the enrofloxacin group.

[0172] Depend on Fig.11 -AⅡ shows that the jejunal tissue structure of the blank group rats is intact, the villi are long and neat, and the boundaries are clear; Fig.11 -BⅡ It can be seen that the jejunal structure of the rats in the model group was destroyed, the villi were destroyed and fell off, the glands were damaged, and the shape was irregular; after treatment with Tibetan medicine compound, Baitouweng decoction, and enrofloxacin, Fig.11 -CⅡ shows that the arrangement of jejunal microvilli of rats in the low-dose Tibetan medicine compound group is relatively sparse, and a small amount of intestinal villi are broken; Fig.11 -DⅡ and 11-GⅡ showed that the jejunal microvilli of rats in the medium-dose Tibetan medicine compound group and the enrofloxacin group were regular in shape, arranged relatively closely, and their morphology basically returned to normal; Fig.11 -EⅡ, 11-FⅡ show that the jejunal villi of the Tibetan medicine compound high-dose group and the Baitouweng decoction group are arranged more neatly and more sparsely. Fig.12 -B, 12-E showed that compared with the blank group, the length of jejunal villi of rats in the model group was significantly reduced (P<0.05), and the villus-to-cryptotype ratio was significantly reduced (P<0.05); compared with the model group, the length of jejunal villi of rats in each drug-treated group was significantly increased (P<0.05), and the villus-to-cryptotype ratio was significantly increased (P<0.05); compared with the medium-dose group of Tibetan medicine compound, the length of jejunal villi of rats in the high-dose group of Tibetan medicine compound, the low-dose group of Tibetan medicine compound and the Baitouweng decoction group was significantly reduced (P<0.05), and the villus-to-cryptotype ratio was significantly reduced (P<0.05). The results showed that the medium-dose group of Tibetan medicine compound had a repairing effect on the pathological changes of jejunal tissue in rats with diarrhea, and the medium-dose group had the best effect, which was better than the Baitouweng decoction group.

[0173] Depend on Fig.11 -AⅢ showed that the ileum of the blank group rats had a complete tissue structure, dense villi, regular and uniform arrangement, and clear boundaries between the submucosal layer and the muscular layer; Fig.11 -BⅢ showed that the ileum villi of the rats in the model group were broken; after treatment with Tibetan medicine compound, Baitouweng decoction and enrofloxacin, Fig.11 -CⅢ showed that the ileal villi of rats in the low-dose Tibetan medicine compound group were slightly defective and had gaps and breaks; Fig.11 -DⅢ and 11-GⅢ showed that the ileal microvilli of rats in the medium-dose Tibetan medicine compound group and the enrofloxacin group were regular in shape, arranged relatively closely, and their morphology basically returned to normal; Fig.11 -EⅢ, 11-FⅢ show that the ileal villi of the Tibetan medicine compound high-dose group and the Baitouweng decoction group are arranged more neatly and more sparsely. Fig.12 -C, 12-F showed that compared with the blank group, the length of ileal villi in the model group was significantly reduced (P<0.05), and the villus-cryptoproliferative ratio was significantly reduced (P<0.05); compared with the model group, the length of ileal villi in the rats in each drug-treated group was significantly increased (P<0.05), and the villus-cryptoproliferative ratio was significantly increased (P<0.05); compared with the medium-dose group of Tibetan medicine compound, the length of jejunal villi in the high-dose group of Tibetan medicine compound, the low-dose group of Tibetan medicine compound and the Baitouweng decoction group was significantly reduced (P<0.05), and the villus-cryptoproliferative ratio was significantly reduced (P<0.05). The results showed that the Tibetan medicine compound had a repairing effect on the pathological changes of the ileum tissue in rats with diarrhea, and the medium-dose group had the best effect, which was better than the Baitouweng decoction group.

[0174] Depend on Fig.11 -AⅣ showed that the structures of mucosal layer, submucosal layer, muscular layer and serosa layer of colon tissue in the blank group were relatively intact, and the intestinal glands in the lamina propria were arranged neatly without obvious degeneration or necrosis; Fig.11 -BⅣ showed that the colon mucosal epithelium of the rats in the model group was damaged and the intestinal gland structure was destroyed; Fig.11 The colon mucosal epithelium of rats in the 11-CⅣ, 11-DⅣ, 11-EⅣ, 11-FⅣ, and 11-GⅣ drug-treated groups showed different degrees of recovery.

[0175] Fig.11 HE staining of the small intestine and colon of rats in each group. A represents the blank group; B represents the model group; C represents the low-dose Tibetan medicine compound group; D represents the medium-dose Tibetan medicine compound group; E represents the high-dose Tibetan medicine compound group; F represents the Baitouweng decoction group; G represents the enrofloxacin group; I represents the duodenum; II represents the jejunum; III represents the ileum; and IV represents the colon.

[0176] Fig.12 Comparison of small intestine morphology in rats of each group.

[0177] 3.6 Effect of Tibetan medicine compound on the expression of Claudin-1, ZO-1, Occludin and MUC-2 mRNA in rat jejunum

[0178] Depend on Fig.13It can be seen that compared with the model group, the expression levels of Claudin-1, ZO-1, Occludin, and MUC-2 in the jejunum of rats were significantly upregulated after gavage with Tibetan medicine compound, Baitouweng decoction, and enrofloxacin (P < 0.05); the expression levels of Claudin-1, ZO-1, Occludin, and Muc-2 in the jejunum of rats in the medium-dose Tibetan medicine compound group were not significantly different from those in the enrofloxacin group (P > 0.05), and were significantly lower than those in the blank group (P < 0.05), but significantly higher than those in the low-dose Tibetan medicine compound group, the high-dose Tibetan medicine compound group, and the Baitouweng decoction group (P < 0.05). The results showed that Tibetan medicine compound can upregulate the expression of Claudin-1, ZO-1, Occludin, and MUC-2 related genes in the intestines of rats with diarrhea, and the improvement effect is better than that of the Baitouweng decoction group, and is equivalent to the repair effect of the enrofloxacin group.

[0179] Fig.13 This study investigates the effects of Tibetan medicine compound on the mRNA expression levels of tight junction proteins Claudin-1, ZO-1, Occludin, and MUC-2 in the intestine of rats.

[0180] 3.7 Effect of Tibetan medicine compound on tight junction protein expression in rat jejunum

[0181] Depend on Fig.14 and Fig.15 It can be seen that compared with the model group, the positive expression of Claudin-1, Zo-1, Occludin, and Muc-2 proteins in the jejunum of rats in each drug-treated group increased significantly (P < 0.05); the positive expression of Claudin-1, Zo-1, Occludin, and Muc-2 proteins in the jejunum of rats in the medium-dose Tibetan medicine compound group had no significant difference from that in the enrofloxacin group (P > 0.05), and was significantly lower than that in the blank group (P < 0.05), but significantly higher than that in the low-dose Tibetan medicine compound group, the high-dose Tibetan medicine compound group, and the Baitouweng decoction group (P < 0.05). The results showed that the Tibetan medicine compound could protect the intestinal mucosal barrier and relieve the diarrhea symptoms of rats by upregulating the expression of intestinal tight junction proteins.

[0182] Fig.14 This is the immunohistochemistry of positive expression of Claudin-1, Zo-1, Occludin, and Muc-2 in rat jejunum tissue. Among them, A represents the blank group; B represents the model group; C represents the low-dose group of Tibetan medicine compound; D represents the medium-dose group of Tibetan medicine compound; E represents the high-dose group of Tibetan medicine compound; F represents the Baitouweng decoction group; G represents the enrofloxacin group; I represents Claudin-1; II represents Zo-1; III represents Occludin; IV represents Muc-2.

[0183] Fig.15 This study is to investigate the effect of Tibetan medicine compound on the expression of Claudin-1, Zo-1, Occludin and Muc-2 proteins in rat jejunal tissue.

[0184] 3.8 Effects of Tibetan medicine compound on intestinal goblet cells

[0185] Depend on Fig.16 and 17 It can be seen that compared with the model group, the goblet cells in the jejunum of rats in each drug-treated group increased significantly (P < 0.05); compared with the medium-dose group of Tibetan medicine compound, the goblet cells in the jejunum of rats in the low-dose group of Tibetan medicine compound, the high-dose group of Tibetan medicine compound, the Baitouweng decoction group, and the enrofloxacin group decreased significantly (P < 0.05). The results show that the Tibetan medicine compound increases the number of goblet cells and protects the intestinal mucosa.

[0186] Fig.16 PAS staining of the jejunum of rats in each group. A represents the blank group; B represents the model group; C represents the low-dose Tibetan medicine compound group; D represents the medium-dose Tibetan medicine compound group; E represents the high-dose Tibetan medicine compound group; F represents the Baitouweng decoction group; and G represents the enrofloxacin group.

[0187] Fig.17 The changes in the number of goblet cells in each group.

[0188] 3.9 Effects of Tibetan medicine compound on intestinal flora of rats

[0189] 3.9.1 Operational Taxonomic Unit (OTU) species analysis

[0190] like Fig.18 As shown, the number of unique OTUs in the blank group was 1570, the number of unique OTUs in the model group was 955, the number of unique OTUs in the low-dose Tibetan medicine compound group was 991, the number of unique OTUs in the medium-dose Tibetan medicine compound group was 1490, the number of unique OTUs in the high-dose Tibetan medicine compound group was 1321, and the number of unique OTUs in the Baitouweng decoction group was 1494. The total number of OTUs in the six groups was 269. The same microbial species were found in the intestines of rats in each group, but there were also specific flora. The number of unique OTUs in rats in the model group was the least, and Tibetan medicine compound treatment could increase the OTU to approach the blank group.

[0191] Fig.18 Analyze the petal map for OTU species.

[0192] 3.9.2 Alpha Analysis

[0193] Depend on Fig.19 It can be seen that the species richness and community uniformity were the highest in the blank group, followed by the medium-dose group of the Tibetan medicine compound, and the lowest in the model group, but the differences between the groups were not significant (P>0.05); combined with the analysis results of the six groups of OTUs, it showed that the Tibetan medicine compound could regulate the intestinal mucosal microbial diversity of diarrhea rats.

[0194] Fig.19 The results of α analysis. Among them, (A) Chao1 index; (B) ACE index; (C) Simpson index; (D) Shannon index.

[0195] 3.9.3 β analysis

[0196] from Fig. 20 It can be seen that the samples in the model group are not clustered but scattered, indicating that the microbial population structure of the intestines of diarrhea rats is disturbed; the samples in the blank group, the low-dose Tibetan medicine compound group, the medium-dose Tibetan medicine compound group, and the high-dose Tibetan medicine compound group are relatively clustered, indicating that the sample compositions of the blank group and the Tibetan medicine compound group have certain similarities, the Baitouweng Decoction group is clustered on the other side, and the community compositions of the Baitouweng Decoction group and the Tibetan medicine compound group are different.

[0197] Fig. 20 This is the principal coordinate analysis of the intestinal flora of rats in each group.

[0198] 3.9.4 Tibetan medicine compound regulates the classification and composition of intestinal microbiota

[0199] At the phylum level, Firmicutes and Bacteroidetes are the most abundant bacterial groups in each group of intestinal microorganisms. Fig.21 -A shows that compared with the blank group, the relative abundance of Firmicutes and Actinobacteria in the intestinal flora of rats in the model group decreased, while the relative abundance of Bacteroidetes, Desulfobacterota, Proteobacteria, Deferribacterium, and Campylobacterota increased. After the other groups were given drug treatment, the intestinal flora of each drug-treated group changed. Fig.21 -A shows that compared with the model group, the relative abundance of Firmicutes and Actinobacteria in the intestinal flora of rats in each drug-treated group increased, while the relative abundance of Bacteroidetes, Desulfobacteria, Proteobacteria, Deferribacterium, and Campylobacter decreased; Fig.21 -B shows that the subordinate level analysis shows that compared with the blank group, the relative abundance of Bacteroides, Romboutsia, and Helicobacter in the intestinal flora of rats in the model group increased, and the relative abundance of Prevotellaceae (UCG-003) in the intestinal flora of rats decreased; compared with the model group, the relative abundance of Bacteroides, Romboutsia, and Helicobacter in the intestinal flora of rats in each drug-treated group decreased, and the relative abundance of Prevotellaceae in the intestinal flora of rats increased. The results show that the Tibetan medicine compound can increase the relative abundance of beneficial bacteria and adjust the flora composition of rats with diarrhea to normal.

[0200] Fig.21The classification composition of the intestinal microbiota of rats in each group. (A) The composition of the microbial community in rat feces at the phylum level, (B) The composition of the microbial community in rat feces at the genus level.

[0201] Test Example 4 Clinical Trial of Tibetan Medicine Composition of the Present Invention

[0202] Main drugs and reagents: Tibetan medicine compound No. 1 and Tibetan medicine compound No. 2 were prepared according to the methods of Example 1 and Example 2 respectively.

[0203] Clinical cases: The Holstein calves used in the experiment were all from a large-scale breeding farm in Gansu Province. All the calves included in the experiment were sick calves within 30 days of age and had not been treated with antibiotics. All calves had similar breeding environments, feeding conditions, and genetic backgrounds.

[0204] A total of 60 calves with diarrhea were included in the experiment, and the diarrhea scores of all the affected calves were greater than 5. The experiment was randomly divided into two groups: Tibetan medicine compound No. 1 group and Tibetan medicine compound No. 2 group.

[0205] The sick calves were treated with different Tibetan medicine compounds, which were administered orally twice a day, 100 mL each time, for 5 consecutive days. During the treatment, the clinical symptoms of the sick cattle, including mental state, body temperature, fecal morphology, etc., were observed and recorded every day. After the treatment, the treatment effect was evaluated according to the standards in Table 3. A score of 0 after treatment and a decrease of more than 5 points before and after treatment indicated cure; a score of less than 2 points after treatment and a decrease of more than 3 points before and after treatment indicated effectiveness; a score of more than 2 points after treatment indicated ineffectiveness, or a score of less than 3 points before and after treatment indicated ineffectiveness. All animal experimental procedures were approved by the Ethics Committee of Lanzhou Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences (SYXK[Gan]2019-0002).

[0206] Table 3 Clinical scoring table for calf diarrhea

[0207] integral Mental state Rectal temperature Stool form 0 Alert, sensitive, lively and active <39℃ Dry and formed 1 Poor spirits and reduced spontaneous activity 39℃～40℃ Shapeless, but not watery 2 Mental fatigue and slow movements >40℃ Runny, sticky and foul smelling

[0208] Cure rate = number of cured animals / number of sick animals treated × 100%

[0209] Total effective rate = (number of cured animals + number of effective animals) / number of sick animals treated × 100%.

[0210] Experimental results:

[0211] After 5 days of treatment with Tibetan medicine compound, 14 sick cows in Tibetan medicine compound No. 1 group recovered completely, and the clinical symptoms and signs of 13 sick cows were significantly improved; 10 sick cows in Tibetan medicine compound No. 2 group recovered completely, and the clinical symptoms and signs of 15 sick cows were significantly improved (Table 4). The cure rate and total effective rate of Tibetan medicine compound No. 1 were 46.67% and 90.00%, and the cure rate and total effective rate of Tibetan medicine compound No. 2 were 33.33% and 83.33%. It shows that Tibetan medicine compound No. 1 and Tibetan medicine compound No. 2 can treat calf diarrhea, and Tibetan medicine compound No. 1 has the best therapeutic effect.

[0212] Table 4 The therapeutic effect of Tibetan medicine compound on calf diarrhea

[0213] Heal (Head) Valid(header) void(header) Tibetan Medicine Compound No. 1 14 13 3 Tibetan Medicine Compound No. 2 10 15 5

[0214] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Tibetan medicine composition for treating calf diarrhea, characterized in that: The Tibetan medicine composition is composed of the following components in parts by weight: 10-30 parts of Terminalia chebula, 10-25 parts of Niangzizhe, 15-35 parts of Punica granatum peel and 20-40 parts of Pueraria root.

2. A Tibetan medicine composition for treating calf diarrhea according to claim 1, characterized in that: The Tibetan medicine composition is composed of the following components in parts by weight: 10-25 parts of Terminalia chebula, 17-22 parts of Niangzizhe, 20-35 parts of Punica granatum peel and 25-40 parts of Pueraria root.

3. A Tibetan medicine composition for treating calf diarrhea according to claim 2, characterized in that: The Tibetan medicine composition is composed of the following components in parts by weight: 20 parts of Terminalia chebula, 18 parts of Niangzizhe, 24 parts of Punica granatum peel, and 30 parts of Pueraria root; or The Tibetan medicine composition is composed of the following components in parts by weight: 25 parts of Terminalia chebula, 22 parts of Niangzizhe, 20 parts of Punica granatum peel and 25 parts of Pueraria root.

4. The method for preparing the Tibetan medicine composition according to any one of claims 1 to 3, characterized in that: The following steps are involved: Grinding a formula amount of Terminalia chebula into coarse powder, and mixing with a formula amount of Nianzizhe, pomegranate peel and Pueraria root to obtain a mixture; The resulting mixture was decocted with water and the filtrate was collected by filtration.

5. The preparation method according to claim 4, characterized in that: The ratio of the mixture to water is 1 g: 6-10 ml; the decoction times are 2-3 times, each time for 1-2 hours.

6. The preparation method according to claim 4, characterized in that: The decoctions from each batch were combined and concentrated to contain 0.8 to 1.2 g of crude drug per milliliter.

7. Use of the Tibetan medicine composition according to any one of claims 1 to 3 in the preparation of a medicine for treating calf diarrhea.

8. The use according to claim 7, characterized in that: The dosage of the Tibetan medicine composition is as follows: each calf is gavaged with 100-120 mL of the Tibetan medicine composition each time, twice a day, for 3-5 consecutive days.

9. A medicine, the active ingredient of which comprises the Tibetan medicine composition according to any one of claims 1 to 3.

10. The drug according to claim 9, characterized in that: The dosage form of the drug includes oral solution.

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