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

Abstract

The invention discloses a Tibetan veterinary medicine composition for treating yak diarrhea as well as a preparation method and application thereof. The composition comprises Tibetan coptis chinensis, berberis poiretii schneid, fructus chebulae, dracocephalum tanguticum, polygonum viviparum, malus toringoides fruits, radix rubiae, scorched hawthorn fruits, fried malt, fructus hippophae, rhizoma zingiberis, rhizoma atractylodis, wine-treated rhubarb, oxytropis verticillata, fructus quisqualis, ligularia virgaurea, rhodiola rosea and saffron crocus. The Tibetan medicine composition provided by the invention strictly follows the principle of'three-cause balance 'of Tibetan medicine, is designed aiming at the common diseases of'red Ba' heat and'bai 'cold inclusion in plateau, and has the effects of clearing heat and drying dampness, detoxifying and stopping dysentery, tonifying spleen and warming middle-jiao, and simultaneously has multiple action mechanisms of plateau stress resistance, qi and blood regulation, parasite expelling and the like. The Tibetan medicine composition is safe and free of side effects, and can inhibit intestinal inflammation reaction, protect intestinal structure, promote nutrition digestion and absorption and enhance physique of yaks.

Description

Technical Field

[0001] This invention relates to the field of Tibetan veterinary medicine technology, and in particular to a Tibetan veterinary medicine composition for treating diarrhea in yaks, its preparation method, and its application. Background Technology

[0002] The yak industry is a leading, promising, and distinctive industry in plateau regions. However, with the expansion of breeding scale and changes in farming methods, yaks are facing increasing health challenges, with diarrhea being particularly prominent. Diarrhea not only reduces yak fertility and slows growth but can also lead to secondary infections and even death, becoming a significant factor restricting the development of the yak industry.

[0003] While traditional antibiotics and chemotherapy drugs can control diseases to some extent, their long-term use leads to drug resistance and drug residue risks, which not only affect treatment effectiveness but also pose potential threats to food safety and public health.

[0004] Tibetan veterinary medicine, with its natural plant-derived characteristics and the advantages of safety and no residue, has become the preferred solution to this problem. Therefore, in view of the complex pathogenesis (chiba heat toxicity, bacon food stagnation, typhoid fever, parasitic diseases, and mixed cold and heat), and the shortcomings of existing technologies, it is an urgent technical problem for those skilled in the art to solve the problem of providing a Tibetan medicine composition that can effectively prevent and treat diseases while avoiding the many drawbacks of traditional chemical drugs, and providing a new breakthrough for achieving healthy yak breeding and livestock product safety. Summary of the Invention

[0005] In view of this, the present invention provides a Tibetan veterinary medicine composition for treating diarrhea in yaks, its preparation method and application.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] A Tibetan veterinary medicine composition for treating diarrhea in yaks, the composition comprising the following components in parts by weight: 5-15 parts of Coptis chinensis, 15-25 parts of Trichosanthes kirilowii, 20-40 g of Terminalia chebula, 10-20 parts of Cymbidium goeringii, 20-40 parts of Polygonum viviparum, 20-30 parts of Ephedra sinica, 10-25 parts of Rubia cordifolia, 30-50 parts of Crataegus pinnatifida (roasted), 10-30 parts of malt, 10-20 parts of Hippophae rhamnoides, 10-20 parts of Zingiber officinale (dried), 10-20 parts of Atractylodes lancea, 5-15 parts of Rheum palmatum (processed with wine), 20-30 parts of Oxyphyllum verticillatum, 20-30 parts of Quisqualis indica, 5-15 parts of Ligusticum ovatum, 5-15 parts of Rhodiola rosea, and 1-3 parts of Saffron.

[0008] Preferably, the composition comprises the following components in parts by weight: 10 parts Coptis chinensis, 20 parts Trifolium repens, 30 parts Terminalia chebula, 15 parts Cymbidium tangutica, 30 parts Polygonum viviparum, 30 parts Echinops latifolium, 15 parts Rubia cordifolia, 40 parts Crataegus pinnatifida, 20 parts roasted malt, 15 parts Hippophae rhamnoides, 15 parts dried ginger, 15 parts Atractylodes lancea, 10 parts rhubarb (processed with wine), 25 parts Oxyphyllum verticillatum, 25 parts Quisqualis indica, 10 parts Ligusticum ovoidense, 10 parts Rhodiola rosea, and 2 parts saffron.

[0009] Preferably, the composition comprises the following components in parts by weight: 10 g of Coptis chinensis, 10 g of Trichosanthes kirilowii, 30 g of Terminalia chebula, 15 g of Cymbidium tanguticum, 30 g of Polygonum viviparum, 30 g of Rhododendron simsii, 15 g of Rubia cordifolia, 40 g of Crataegus pinnatifida (roasted), 20 g of malt (roasted), 15 g of Hippophae rhamnoides, 15 g of Zingiber officinale (dried), 15 g of Atractylodes lancea, 10 g of Rheum palmatum (processed with wine), 25 g of Oxyphyllum verticillatum, 25 g of Quisqualis indica, 10 g of Ligusticum striatum (ovoidea), 10 g of Rhodiola rosea, and 2 g of saffron.

[0010] Preferably, the dosage form of the traditional Chinese medicine composition is an oral dosage form.

[0011] A method for preparing a Tibetan veterinary medicine composition for treating diarrhea in yaks includes the following steps:

[0012] (1) Cleaning and processing: Weigh each raw material according to the formula, clean and wash them separately, and process them according to the traditional Tibetan medicine processing method;

[0013] (2) Crushing: Crush the processed medicinal materials separately and pass them through an 80-mesh sieve to obtain coarse powder;

[0014] (3) Mixing: Place the coarse powder of each medicinal material in a mixer according to the proportion and mix at a uniform speed for 30 minutes until the color is uniform;

[0015] (4) Formulation:

[0016] Powder: The mixed powder is directly packaged to obtain the powder.

[0017] Oral liquid: Boil twice with the powder and water at a weight ratio of 1:8, 2 hours each time. Combine the filtrates, concentrate to a relative density of 1.10, add an appropriate amount of preservative, and fill and sterilize.

[0018] Granules: Mix the mixed powder with an appropriate amount of dextrin and sucrose, granulate, dry, and package.

[0019] Preferred preparations, processed according to traditional Tibetan medicine methods, include: removing the pit from Terminalia chebula; slicing dried ginger; stir-frying Atractylodes lancea with wheat bran; processing rhubarb with wine; stir-frying hawthorn and malt until charred; and washing and drying other medicinal materials such as Coptis chinensis and three needles.

[0020] Application of a Tibetan veterinary medicine composition for treating diarrhea in yaks in the prevention of diarrhea.

[0021] Application of a Tibetan veterinary medicine composition for treating diarrhea in yaks in the prevention of diarrhea in yaks.

[0022] Application of a Tibetan veterinary medicine composition for treating diarrhea in yaks.

[0023] Application of a Tibetan veterinary medicine composition for treating diarrhea in yaks.

[0024] The present invention achieves the following technical effects compared to the prior art:

[0025] (1) The formulation of this invention follows Tibetan medicine theory, with a rigorous structure of principal, assistant, adjuvant and guide herbs, taking into account multiple effects such as clearing heat, drying dampness, strengthening the spleen, expelling parasites and promoting blood circulation, treating both the symptoms and the root cause; and all ingredients are natural Tibetan medicinal materials, with no chemical additives, high safety, avoiding drug resistance and drug residues, and safe with no residues.

[0026] (2) Through in vitro experiments, this invention has confirmed that the compound has significant antioxidant and anti-inflammatory capabilities;

[0027] (3) The present invention systematically verified the overall therapeutic effect of the compound on enteritis in animal models. It has a protective effect on LPS-induced enteritis mice, can improve the overall condition of mice, reduce the disease activity index, inhibit the colonic shortening of enteritis, reduce the content of various inflammatory factors in intestinal tissue, effectively improve colonic tissue damage, and has a good anti-inflammatory effect in mice.

[0028] (4) This invention has good usability, significant curative effect, and flexible dosage form, providing a new Chinese and Tibetan medicine solution for solving the diarrhea problem of yaks and other plateau animals. Attached Figure Description

[0029] Figure 1 The antioxidant capacity of this invention (A, B, C, and D represent reducing capacity and scavenging effects on organic free radicals, hydroxyl radicals, and superoxide anions, respectively).

[0030] Figure 2 The effect of this invention on the viability of RAW264.7 cells;

[0031] Figure 3 This invention relates to the effect of LPS-induced NO release in RAW264.7 cells;

[0032] Figure 4 The images show the mouse status of each group (A, B, C, D, E, and F represent the control group, LPS model group, loperamide hydrochloride group, low-dose compound group, medium-dose compound group, and high-dose compound group, respectively).

[0033] Figure 5 This invention relates to the effect of LPS-induced enteritis on DAI in mice;

[0034] Figure 6 This invention relates to the effect of the present invention on colon length in LPS-induced enteritis mice; Figure 7 The effects of this invention on colonic tissue of LPS-induced enteritis mice (A, B, C, D, E, and F represent the effects of the control group, LPS model group, loperamide hydrochloride group, low-dose compound group, medium-dose compound group, and high-dose compound group on mouse colonic tissue, respectively).

[0035] Figure 8 The effects of this invention on the jejunal tissue of LPS-induced enteritis mice (A, B, C, D, E, and F represent the effects of the control group, LPS model group, loperamide hydrochloride group, low-dose compound group, medium-dose compound group, and high-dose compound group on the jejunal tissue of mice, respectively).

[0036] Figure 9 The present invention relates to the effects of this invention on colonic inflammatory factors in LPS-induced enteritis mice (A, B, C, D, and E represent the effects of each group on the expression levels of TNF-α, IL-1β, IL-6, IL-10, and DAO in mouse colonic tissue, respectively).

[0037] Figure 10 The effects of this invention on serum inflammatory factors in LPS-induced enteritis mice (A, B, C, D, and E represent the effects of each group on the expression levels of serum TNF-α, IL-1β, IL-6, IL-10, and DAO in mice, respectively).

[0038] Figure 11 These are photos of some yaks suffering from diarrhea. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] This invention discloses a Tibetan veterinary medicine composition for treating diarrhea in yaks. The composition comprises the following components in parts by weight: 5-15 parts of Coptis chinensis, 15-25 parts of Trichosanthes kirilowii, 20-40g of Terminalia chebula, 10-20 parts of Cymbidium goeringii, 20-40 parts of Polygonum viviparum, 20-30 parts of Ephedra sinica, 10-25 parts of Rubia cordifolia, 30-50 parts of Crataegus pinnatifida (roasted), 10-30 parts of malt, 10-20 parts of Hippophae rhamnoides, 10-20 parts of Zingiber officinale (dried), 10-20 parts of Atractylodes lancea, 5-15 parts of Rheum palmatum (processed with wine), 20-30 parts of Oxyphyllum verticillatum, 20-30 parts of Quisqualis indica, 5-15 parts of Ligusticum ovatum, 5-15 parts of Rhodiola rosea, and 1-3 parts of Saffron.

[0041] The composition consists of the following components by weight: 10 parts Coptis chinensis, 20 parts Trifolium repens, 30 parts Terminalia chebula, 15 parts Cymbidium tangutica, 30 parts Polygonum viviparum, 30 parts Echinops latifolium, 15 parts Rubia cordifolia, 40 parts Crataegus pinnatifida, 20 parts roasted malt, 15 parts Hippophae rhamnoides, 15 parts dried ginger, 15 parts Atractylodes lancea, 10 parts rhubarb (processed with wine), 25 parts Oxyphyllum verticillatum, 25 parts Quisqualis indica, 10 parts Ligusticum ovoides, 10 parts Rhodiola rosea, and 2 parts saffron.

[0042] The composition consists of the following components by weight: 10 g of Coptis chinensis, 10 g of Trichosanthes kirilowii, 30 g of Terminalia chebula, 15 g of Cymbidium goeringii, 30 g of Polygonum viviparum, 30 g of Rhododendron simsii, 15 g of Rubia cordifolia, 40 g of Crataegus pinnatifida (roasted), 20 g of malt (roasted), 15 g of Hippophae rhamnoides, 15 g of Zingiber officinale (dried), 15 g of Atractylodes lancea, 10 g of Rheum palmatum (processed with wine), 25 g of Oxyphyllum verticillatum, 25 g of Quisqualis indica, 10 g of Ligusticum striatum (ovoid), 10 g of Rhodiola rosea, and 2 g of Saffron.

[0043] The dosage form of the traditional Chinese medicine composition is an oral administration dosage form.

[0044] This invention also discloses a method for preparing a Tibetan veterinary medicine composition for treating diarrhea in yaks, comprising the following steps:

[0045] (1) Cleaning and processing: Weigh each raw material according to the formula, clean and wash them separately, and process them according to the traditional Tibetan medicine processing method;

[0046] (2) Crushing: Crush the processed medicinal materials separately and pass them through an 80-mesh sieve to obtain coarse powder;

[0047] (3) Mixing: Place the coarse powder of each medicinal material in a mixer according to the proportion and mix at a uniform speed for 30 minutes until the color is uniform;

[0048] (4) Formulation:

[0049] Powder: The mixed powder is directly packaged to obtain the powder.

[0050] Oral liquid: Boil twice with the powder and water at a weight ratio of 1:8, 2 hours each time. Combine the filtrates, concentrate to a relative density of 1.10, add an appropriate amount of preservative, and fill and sterilize.

[0051] Granules: Mix the mixed powder with an appropriate amount of dextrin and sucrose, granulate, dry, and package.

[0052] The traditional Tibetan medicine processing methods include: removing the pit from Terminalia chebula; slicing dried ginger; stir-frying Atractylodes lancea with wheat bran; processing rhubarb with wine; stir-frying hawthorn and malt until charred; and washing and drying other medicinal materials such as Coptis chinensis and three needles.

[0053] The present invention also discloses the application of a Tibetan veterinary medicine composition for treating diarrhea in yaks in the prevention of diarrhea.

[0054] The present invention also discloses the application of a Tibetan veterinary medicine composition for treating yak diarrhea in the prevention of yak diarrhea.

[0055] The present invention also discloses the application of a Tibetan veterinary medicine composition for treating diarrhea in yaks.

[0056] The present invention also discloses the application of a Tibetan veterinary medicine composition for treating yak diarrhea.

[0057] Example 1: Preparation of Tibetan medicine compound

[0058] Formula: 10 parts Coptis chinensis, 20 parts Three Needles, 30 parts Terminalia chebula, 15 parts Tangut Blue Orchid, 30 parts Polygonum viviparum, 30 parts Ossetum arvense, 15 parts Rubia cordifolia, 40 parts charred hawthorn, 20 parts roasted malt, 15 parts Hippophae rhamnoides, 15 parts dried ginger, 15 parts Atractylodes lancea, 10 parts rhubarb (processed with wine), 25 parts Oxyphyllum verticillatum, 25 parts Quisqualis indica, 10 parts Ligusticum ovoidense, 10 parts Rhodiola rosea, 2 parts saffron.

[0059] The preparation method includes the following steps:

[0060] (1) Cleaning and processing: Weigh each raw material according to the formula, clean and wash them separately, and process them according to the traditional Tibetan medicine processing method (remove the pit from Terminalia chebula; slice dried ginger; stir-fry Atractylodes lancea with wheat bran; process rhubarb with wine; stir-fry hawthorn and malt until charred; wash and dry other medicinal materials such as Coptis chinensis and three needles).

[0061] (2) Crushing: Crush the processed medicinal materials separately and pass them through an 80-120 mesh sieve to obtain coarse powder;

[0062] (3) Mixing: Place the coarse powder of each medicinal material in a mixer according to the proportion and mix at a uniform speed for 30-60 minutes until the color is uniform;

[0063] (4) Formulation:

[0064] Powder: The mixed powder is directly packaged to obtain the powder.

[0065] Oral liquid: Boil twice with the powder and water at a weight ratio of 1:8, 2 hours each time. Combine the filtrates, concentrate to a relative density of 1.10, add an appropriate amount of preservative, and fill and sterilize.

[0066] Granules: Mix the mixed powder with an appropriate amount of dextrin and sucrose, granulate, dry, and package.

[0067] Example 2: In vitro antioxidant activity study

[0068] (1) Reagents and consumables: DPPH, saffron, EDTA, potassium ferricyanide, hydrogen peroxide and trichloroacetic acid were all analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0069] (2) Main instruments: ELISA reader, UV / Vis spectrophotometer;

[0070] (3) Sample preparation: Prepare the above Tibetan medicine composition powders to concentrations of 0.0625, 0.125, 0.25, 0.5, and 1 mg / mL;

[0071] (4) Reducing power: Different concentrations of sample solution were added sequentially, followed by 1% potassium ferricyanide (1.25 ml), and kept at 50℃ for 20 min. Then, 10% trichloroacetic acid (2.5 ml) was added, and the mixture was left to stand at room temperature for 5 min. Finally, 0.1% ferric chloride (1.5 ml) was added, and the absorption wavelength was measured at 700 nm. Water was used as a control. A graph was plotted with absorbance and sample concentration as coordinates.

[0072] (5) Scavenging effect on organic free radicals (DPPH): Different concentrations of sample solution (dissolved in 50% ethanol, 3 ml) and 0.1 M DPPH ethanol solution (1 ml) were added sequentially, shaken well, and placed at room temperature for 20 min. The absorption wavelength was measured at 517 nm. The 50% ethanol solution was used as a control. A graph was plotted with scavenging rate and sample concentration as coordinates. Scavenging rate (%) = (1 - (A sample - A control) / A sample) * 100;

[0073] (6) Scavenging effect on hydroxyl radicals: Different concentrations of samples were added sequentially, 2 mM EDTA-

[0074] Iron solution (0.5 ml), 150 mM phosphate buffer (pH 7.4, 1 ml), saffron red (dissolved in phosphate buffer, 1 ml), and 3% hydrogen peroxide solution (1 ml) were incubated at 37°C for 30 min, and the absorption wavelength was measured at 520 nm. Distilled water was used instead of the sample, and phosphate buffer was used instead of hydrogen peroxide. The clearance rate was plotted against the sample concentration. Clearance rate (%) = (A sample - A blank) / (A control - A blank) * 100;

[0075] (7) Scavenging effect on superoxide anions: Different concentrations of sample solution, Tris-HCl buffer, NADH (disodium reducing coenzyme I), NBT (nitrotetrazole blue), and PMS (phenazine formic acid) were added sequentially. The mixture was incubated at room temperature for 5 min, and the absorption wavelength was measured at 560 nm. Tris-HCl buffer was used as a control. The scavenging rate and sample concentration were plotted. Scavenging rate (%) = (A sample - A control) / (A sample) * 100;

[0076] (8) Results and Analysis: such as Figure 1 As shown, the Tibetan medicine compound has significant antioxidant capacity, and its activity is clearly concentration-dependent.

[0077] When the sample concentration is in the range of 0.0625 mg / mL to 1 mg / mL, the reducing power and the scavenging ability of DPPH, hydroxyl radicals and superoxide anions are positively correlated with the concentration.

[0078] Example 3: In vitro anti-inflammatory activity study

[0079] (1) Reagents and consumables: DMEM culture medium, fetal bovine serum, penicillin-streptomycin antibiotics, phosphate buffer, dexamethasone, neutral red, NO detection kit, RAW264.7 (mouse-derived) mononuclear-macrophages;

[0080] (2) Main instruments: centrifuge; UV-Vis spectrophotometer; electric thermostatic water bath; ELISA reader; TS100 inverted microscope; carbon dioxide cell culture incubator;

[0081] (3) Culture of RAW264.7 passaged macrophages:

[0082] A. Cell resuscitation: After the cell cryopreservation solution is thawed, the cells are transferred to centrifuge tubes containing DMEM complete culture medium, centrifuged at 1000 rpm for 5 min, the upper culture medium is discarded, DMEM complete culture medium is added and gently pipetted, the cells are counted and then transferred to a new culture flask and placed in a 37℃, 5% CO2 cell culture incubator for culture.

[0083] B cell passage: After the cells have covered 80%-90% of the cell culture flask, passage them by pipetting off the adherent cells. Divide the obtained cells into two or three flasks for passage, adding 5 mL of DMEM complete medium to each flask and continuing culture;

[0084] C cell cryopreservation: When the cell density reaches 80%-90%, pipette the adherent cells and centrifuge at 1000 rpm for 5 min. After discarding the supernatant culture medium, resuspend the cells in cryopreservation solution, aliquot into cryovials, store at 4°C for 20 min, then transfer to -20°C for 40 h, and store at -80°C for later use.

[0085] (4) Determination of the maximum safe concentration of the drug: After the cells have been cultured to fill the cell flask, the adherent cells are pipetted and diluted with DMEM complete medium, and then placed into a 96-well plate at a density of 100 μL per well, with a cell density of 2 × 10⁶ cells / well. 5Each well was used for 24 h of culture. After culturing, the 96-well plate was removed, the culture medium in the wells was carefully discarded, and the plate was washed with PBS. The drug treatment groups were given 100 μL of different final concentrations of Tibetan medicine composition (4 mg / mL, 2 mg / mL, 1 mg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.625 μg / mL, diluted with DMEM complete medium), and the blank control group was given 100 μL of DMEM complete culture medium. The plates were incubated at 37°C in a 5% CO2 cell culture incubator for 36 h. Six parallel wells were set up for each concentration. After 36 h, the 96-well plate was removed, washed 2-3 times with PBS, MTT solution was added, and the plates were incubated at 37°C in a 5% CO2 cell culture incubator for 4 h. The supernatant was discarded, and DMSO (dimethyl sulfoxide) was added. The plates were shaken on a micro-oscillator for 5-10 min until all the purple crystals dissolved. The absorbance of each well was measured at 570 nm using a microplate reader. The results were recorded, and cell viability was calculated. The data were then processed and statistically analyzed. Cell viability = (OD value of drug group / OD value of blank group) × 100%.

[0086] (5) Experimental grouping and treatment: The experiment was divided into blank control group (CON), LPS modeling group (MOD), dexamethasone group (DEX), and Tibetan medicine compound (low, medium and high dose groups) (ZHLP-L, M, H), with each group repeated 5 times. The low, medium and high dose groups were determined according to the screening results of step (4). RAW264.7 was seeded into 24-well plates and cultured for 24 h. The culture medium in the wells was carefully discarded and the plates were washed with PBS. 0.5 mL of Tibetan Yellow Antidiarrheal Powder of the corresponding concentration was added to the drug group, 0.5 mL of 250 ug / mL dexamethasone was added to the dexamethasone group, and 0.5 mL of DMEM complete culture medium was added to the blank control group and LPS group. After incubation for 1 h, 0.5 mL of 1 μg / mL LPS was added to each well except for the blank group, and 0.5 mL of DMEM complete culture medium was added to the control group. After culturing for 24 h, the cell supernatant of each group was collected.

[0087] (6) Determination of NO content: After the cultured cells are plated, each group of cells is treated according to step (5). After culturing for 24 h, the supernatant is collected by centrifugation. The NO secretion of each group is determined by the Griess method according to the instructions of the detection kit.

[0088] (7) Results and Analysis: such as Figure 2 As shown, based on the results of the maximum safe concentration determination of the drug, concentrations of 1 mg / mL, 2 mg / mL, and 4 mg / mL were selected for the determination of NO content;

[0089] like Figure 3As shown, the Tibetan medicine compound has a good anti-inflammatory effect. Compared with the blank control group, the release of NO in the LPS model group was significantly increased (P < 0.001). Compared with the LPS model group, both medium and high concentrations of Tibetan medicine compound significantly inhibited the release of NO (P < 0.001).

[0090] Example 4: In vivo anti-inflammatory activity study

[0091] (1) Experimental animals: 60 half male and half female ICR mice (22-25 g), which were acclimatized for one week before the experiment. Animal feeding was strictly carried out in accordance with the requirements of experimental animal welfare ethics. Animal experiments were strictly carried out in accordance with the school's experimental animal care and use guidelines.

[0092] (2) Reagents and consumables: loperamide hydrochloride, lipopolysaccharide (LPS) solution, mouse TNF-α, IL-1β, IL-6, IL-10 and DAO ELISA kits, hematoxylin staining solution, eosin staining solution, 4% paraformaldehyde, liquid nitrogen;

[0093] (3) Main instruments: dehydrator, embedding machine, pathological slide machine, centrifuge, enzyme-linked immunosorbent assay reader, vortex analyzer, -80℃ refrigerator;

[0094] (4) Preparation of LPS suspension: Add 5 mL of sterile physiological saline to 10 mg of LPS powder, vortex for 15 min to prepare a 2 mg / mL LPS stock solution, quickly dispense, and store at -20℃. Use after incubation at 37℃ for 10 min and vortexing for 15 min.

[0095] (5) Experimental grouping and treatment: After one week of acclimatization feeding, 60 mice of equal sex were randomly divided into 6 groups of 10 each (5 females and 5 males). These groups were named blank control group, LPS modeling group, positive drug group (LOP), and different dosage groups of Tibetan medicine compound (low, medium, and high dose groups). The compound was administered according to body weight, and the dosage was converted with reference to the dosage for yak. ​​Low dose group: 0.25 g / kg, medium dose group: 0.5 g / kg, high dose group: 1 g / kg; positive drug (loperamide hydrochloride, LOP): 0.5 mg / kg, administered by gavage. The LPS modeling group and blank control group were administered the same amount of purified water by gavage, 10 mL / kg, once a day for 7 consecutive days. On the next day (D8) at 8:00 am, except for the blank group, the mice in the other groups were injected intraperitoneally with 0.3 mL of sterile LPS solution (20 mg / kg). Six hours later, the mice were weighed again and their mental state and fecal condition were observed. The mice were anesthetized with ether and the eyeballs were removed to collect peripheral blood. The mice were sacrificed and 2-3 cm of colon tissue was taken and divided into two parts. One part was fixed in 4% paraformaldehyde for HE staining and immunofluorescence, and the other part was quickly placed in liquid nitrogen and then transferred to a -80°C freezer for tissue homogenization.

[0096] (6) Disease Activity Index (DAI) assessment: The DAI score is the sum of the scores for weight loss, stool condition, and fecal occult blood. See Table 1 for detailed scoring criteria;

[0097] Table 1: DAI Scoring Criteria

[0098]

[0099] (7) Colon length measurement: Mice were euthanized by cervical dislocation, and the colon was collected to measure the distance from the anus to the ileocecal junction;

[0100] (8) Preparation of tissue homogenate: Take the frozen colon tissue from step (5) above, quickly cut off about 0.1 g and weigh it, add 500 ul 1% PMSF solution, and homogenize thoroughly with a tissue homogenizer until there is no visible precipitate. Collect the liquid, centrifuge at 3000 rpm for 10 min, collect the supernatant, quickly aliquot the supernatant into 50 ul / vial, label each vial and store at -20℃;

[0101] (9) Histopathological examination of colon tissue: After the tissue was fixed in 10% neutral formalin, it was embedded in paraffin, 5 μm thick sections were prepared, HE staining was performed, and histopathological observation and photography were carried out using a microscopic imaging system.

[0102] (10) Detection of inflammatory factors in colon tissue: The levels of TNF-α, IL-1β, IL-6, IL-10 and DAO in mouse colon tissue homogenate and serum were detected by ELISA.

[0103] (11) Results and Analysis:

[0104] like Figure 4 As shown, mice in the blank control group were active, with formed feces that were dry or moist and granular. Their anuses were clean, without redness, swelling, or debris, and the surrounding fur was clean. Mice in the LPS model group were lethargic, with watery or loose feces and frequent defecation. Their anuses were red and swollen, and the surrounding fur was contaminated with loose feces. Mice in the positive control group had significantly better mental state, activity level, and fur condition than the model group. Most of their feces were formed or soft, with occasional loose feces. The redness and swelling around their anuses had largely subsided, and the feces were relatively dry and clean, with occasional slight stains. Mice in the compound group were active, with mostly formed feces, and their anuses were dry and generally clean, without redness or swelling.

[0105] like Figure 5 As shown, compared with the blank group, the DAI index of mice in the LPS model group was significantly increased (P < 0.01). Compared with the LPS model group, the DAI index of the high-dose compound group was significantly decreased (P < 0.01), and the DAI index of the medium-dose group was significantly decreased (P < 0.05).

[0106] like Figure 6 As shown, compared with the blank group, the colon of mice in the LPS model group was significantly shortened (P < 0.001). Compared with the LPS model group, the colon length of mice in the positive drug group and the low, medium and high dose groups of the compound was significantly increased (P < 0.001).

[0107] like Figure 7 As shown, the control group had intact structures, neatly arranged crypts, numerous goblet cells, clear blood vessels, no damage to the mucosa, and normal muscle layer structure. The LPS model group showed extensive mucosal erosion (black arrows), loss of mucosal epithelial cells, complete destruction of crypt structures, and cavities in localized areas (yellow arrows). The smooth muscle layer was disordered. The positive drug group showed localized mucosal erosion (circles), cell loss, destruction of crypt structures, and scattered nuclear fragments (arrows), but other areas of the mucosa contained numerous goblet cells, and the muscle layer structure was normal. The SHLP-L group had a thicker mucosa but disordered structure (circles), with localized loss of crypt structures and cell necrosis, and occasional nuclear fragments (arrows), but numerous goblet cells, and normal deep muscle layer. The SHLP-M group showed partial mucosal erosion and (arrows) disordered structure, but overall retained a large number of intact crypts and goblet cells. The SHLP-H group showed disordered mucosal structure and loss of crypts (black arrows), but only slight ecchymosis, and normal muscle layer structure.

[0108] like Figure 8 As shown, the control group had normal structure. The intestinal villi were neatly arranged, the epithelium was intact, the crypt structure was clear, and only a few cells were necrotic. The submucosa and muscularis were normal. In the LPS model group, the intestinal villi were extensively broken and detached, a large amount of epithelium was lost, the crypt structure was destroyed, and a large number of inflammatory cells were infiltrated in the blood vessels of the mucosa (arrow). In the positive drug group, some intestinal villi were broken, local mucosal erosion and structural loss were observed, but at the same time, many nuclear mitotic figures were observed (arrow), indicating that the tissue initiated an active proliferation and repair process after injury. In the SHLP-L group, the overall structure of the intestinal villi was disordered, the crypt structure was lost, and there was inflammatory cell infiltration (arrow) and nuclear fragments. In the SHLP-M group, the intestinal villi were broken, the epithelium was lost, the blood vessels of the mucosa were congested and there were inflammatory cells infiltrated, and many cell nuclear fragments were observed locally, indicating that repair was underway. In the SHLP-H group, the intestinal villi of the mucosa were broken locally, the epithelial cells were lost, a large number of neatly arranged crypts were seen at the base, and occasional single cell necrosis was observed (arrow). No obvious pathological changes were observed in the submucosa and muscularis.

[0109] like Figure 9As shown, the Tibetan medicine compound can inhibit the release of inflammatory factors in mouse intestinal tissue and has a good in vivo anti-inflammatory effect. Compared with the blank control group, the levels of TNF-α, IL-6, IL-1β, and DAO in the intestinal tissue of mice in the LPS model group were significantly increased (P<0.001), and the level of IL-10 was significantly decreased (P<0.001). Compared with the LPS model group, the levels of TNF-α, IL-6, IL-1β, and DAO in the positive control group and the compound group were significantly decreased (P<0.001), and the level of IL-10 was significantly increased (P<0.001). Compared with the positive control group, the level of DAO in the compound group was significantly decreased (P<0.001); the levels of IL-6 in the low and medium doses of the compound group were significantly decreased (P<0.05); and the levels of IL-6 in the high dose group were significantly decreased (P<0.01) and significantly increased (P<0.05).

[0110] like Figure 10 As shown, the Tibetan medicine compound can inhibit the release of inflammatory factors in mouse serum. Compared with the blank control group, the serum levels of TNF-α, IL-6, IL-1β, and DAO in the LPS model group were significantly increased (P < 0.001), while the IL-10 level was significantly decreased (P < 0.01). Compared with the LPS model group, the levels of TNF-α, IL-6, IL-1β, and DAO in the positive control group were significantly decreased (P < 0.01 or P < 0.001), while the levels of TNF-α, IL-6, IL-1β, and DAO in the compound group were all significantly decreased (P < 0.001), and the IL-10 level in both the positive control group and the compound group was significantly increased (P < 0.05). Compared with the positive control group, the TNF-α level in the compound group was significantly decreased (P < 0.01 or P < 0.001), the levels of IL-6 at medium and high doses were significantly decreased (P < 0.001), and the level of DAO at high dose was significantly decreased (P < 0.05).

[0111] Example 5: Therapeutic effect on yak diarrhea

[0112] (1) Experimental grouping and treatment: 30 yaks with diarrhea, 10 of which were left untreated; 10 of which were given gentamicin via intramuscular injection for 3 days according to the instructions; and 10 of which were given Tibetan medicine formula, administered orally once a day at a dose of 1 g / kg of body weight for 3 to 5 consecutive days.

[0113] (2) Results and Analysis: such as Figure 11 As shown, yaks with diarrhea are lethargic, emaciated, and prefer to lie down. Their feces are unformed, often yellow or yellowish-green, white or gray, and sometimes contain blood or mucus.

[0114] The treatment outcomes for yaks are shown in Table 2. The recovery rate of the compound treatment was 80%, which was higher than that of the control group and the Western medicine group. There were no deaths.

[0115] Table 2:

[0116]

[0117] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A Tibetan veterinary medicine composition for treating diarrhea in yaks, characterized in that, The composition comprises the following components in parts by weight: 5-15 parts Coptis chinensis, 15-25 parts Three Needles, 20-40 g of Terminalia chebula, 10-20 parts of Tangut Blue Orchid, 20-40 parts of Polygonum viviparum, 20-30 parts of Ossetum arvense, 10-25 parts of Rubia cordifolia, 30-50 parts of charred Crataegus pinnatifida, 10-30 parts of roasted malt, 10-20 parts of Hippophae rhamnoides, 10-20 parts of dried ginger, 10-20 parts of Atractylodes lancea, 5-15 parts of wine-processed Rheum palmatum, 20-30 parts of Oxyphyllum verticillatum, 20-30 parts of Quisqualis indica, 5-15 parts of Ligustrum lucidum ovoidense, 5-15 parts of Rhodiola rosea, and 1-3 parts of saffron.

2. The Tibetan veterinary medicine composition for treating diarrhea in yaks according to claim 1, characterized in that, The composition comprises the following components in parts by weight: 10 parts Coptis chinensis, 20 parts Trifolium repens, 30 parts Terminalia chebula, 15 parts Cymbidium tangutica, 30 parts Polygonum viviparum, 30 parts Echinops latifolium, 15 parts Rubia cordifolia, 40 parts Crataegus pinnatifida, 20 parts roasted malt, 15 parts Hippophae rhamnoides, 15 parts dried ginger, 15 parts Atractylodes lancea, 10 parts rhubarb (processed with wine), 25 parts Oxyphyllum verticillatum, 25 parts Quisqualis indica, 10 parts Ligusticum ovoidense, 10 parts Rhodiola rosea, and 2 parts saffron.

3. The Tibetan veterinary medicine composition for treating diarrhea in yaks according to claim 1, characterized in that, The composition comprises the following components in parts by weight: 10 g of Coptis chinensis, 10 g of Trichosanthes kirilowii, 30 g of Terminalia chebula, 15 g of Cymbidium goeringii, 30 g of Polygonum viviparum, 30 g of Rhododendron simsii, 15 g of Rubia cordifolia, 40 g of Crataegus pinnatifida (roasted), 20 g of malt (roasted), 15 g of Hippophae rhamnoides, 15 g of Zingiber officinale (dried), 15 g of Atractylodes lancea, 10 g of Rheum palmatum (processed with wine), 25 g of Oxyphyllum verticillatum, 25 g of Quisqualis indica, 10 g of Ligusticum striatum (ovoid), 10 g of Rhodiola rosea, and 2 g of Saffron.

4. The Tibetan veterinary medicine composition for treating diarrhea in yaks according to claim 1, characterized in that, The dosage form of the traditional Chinese medicine composition is an oral dosage form.

5. A method for preparing a Tibetan veterinary medicine composition for treating diarrhea in yaks, characterized in that, Includes the following steps: (1) Cleaning and processing: Weigh each raw material according to the formula, clean and wash them separately, and process them according to the traditional Tibetan medicine processing method; (2) Crushing: Crush the processed medicinal materials separately and pass them through an 80-mesh sieve to obtain coarse powder; (3) Mixing: Place the coarse powder of each medicinal material in a mixer according to the proportion and mix at a uniform speed for 30 minutes until the color is uniform; (4) Formulation: Powder: The mixed powder is directly packaged to obtain the powder. Oral liquid: Boil twice with the powder and water at a weight ratio of 1:8, 2 hours each time. Combine the filtrates, concentrate to a relative density of 1.10, add an appropriate amount of preservative, and fill and sterilize. Granules: Mix the mixed powder with an appropriate amount of dextrin and sucrose, granulate, dry, and package.

6. The method for preparing a Tibetan veterinary medicine composition for treating yak diarrhea according to claim 5, characterized in that, The traditional Tibetan medicine processing method includes: removing the pit from Terminalia chebula; slicing dried ginger; stir-frying Atractylodes lancea with wheat bran; processing rhubarb with wine; stir-frying hawthorn and malt until charred; and washing and drying other medicinal materials such as Coptis chinensis and three needles.

7. The use of the Tibetan veterinary medicine composition for treating diarrhea in yaks according to any one of claims 1-4 in the prevention of diarrhea.

8. The use of the Tibetan veterinary medicine composition for treating yak diarrhea according to any one of claims 1-4 in the prevention of yak diarrhea.

9. The use of the Tibetan veterinary medicine composition for treating diarrhea in yaks according to any one of claims 1-4 in the treatment of diarrhea.

10. The use of the Tibetan veterinary medicine composition for treating yak diarrhea according to any one of claims 1-4 in the treatment of yak diarrhea.

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