Traditional Chinese medicine and Tibetan medicine compound preparation for relieving diarrhea of weaned yak calves and preparation method and application of traditional Chinese medicine and Tibetan medicine compound preparation

Abstract

The invention discloses a traditional Chinese and Tibetan medicine compound preparation for relieving diarrhea of weaned yak calves and a preparation method and application thereof, and belongs to the technical field of traditional Chinese and Tibetan medicines. The Chinese and Tibetan medicine compound preparation is prepared from the following components in parts by weight: 12-18 parts of Chinese pulsatilla root, 5-10 parts of Tibetan coptis root, 6-12 parts of artemisia adamsii, 6-12 parts of Tibetan smoked plum, 5-10 parts of Tibet inula root, 6-15 parts of Tibetan white peony root, 3-6 parts of mountain butterflybush root and 3-10 parts of liquorice. The traditional Chinese medicine and Tibetan medicine compound preparation is applied to the yak calves in the weaning period, the inflammation level in the yak calves in the weaning period can be effectively reduced, the anti-oxidative stress capacity is improved, the diversity and structure of intestinal microbiota are improved, and therefore the effects of reducing the diarrhea rate of the yak calves in the weaning period and enhancing the physique of the yak calves in the weaning period are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese and Tibetan medicines, and particularly relates to a traditional Chinese and Tibetan medicine compound preparation for relieving diarrhea in weaned yak calves, and a preparation method and application thereof. Background Art

[0002] Yaks are mainly distributed in the high-altitude areas of the Qinghai-Tibet Plateau and its surrounding regions, such as Gansu and Sichuan provinces (the altitude is usually above 3000 meters). Yaks have become an indispensable resource for local nomadic tribes, providing basic living materials such as fur, meat, and milk for herdsmen. Due to the characteristics of the Qinghai-Tibet Plateau, including high altitude, low oxygen pressure, scarce resources, low temperature, and extreme environmental conditions, yaks adapted to this environment have evolved unique physiological characteristics, enabling them to survive under such harsh conditions and becoming an important economic source for local residents.

[0003] However, the short plant growth season in the plateau area results in low forage yield and scarce forage resources, making the contradiction between the nutritional needs of yaks and the forage supply particularly prominent. In addition, some biological characteristics of yaks themselves, such as slow growth rate, long gestation period, delayed calf weaning, and relatively high prevalence and mortality rates, pose challenges to the yak breeding industry. Especially for weaned yak calves, the weaning period is the most critical and challenging stage in their life cycle. During this period, breast milk can no longer meet the increasing nutritional and energy needs of weaned yak calves, and switching to forage and other feeds helps stimulate the development of their gastrointestinal tract and enhance immunity. However, during the process of changing food, weaned yak calves may face malnutrition due to reduced intake, and at the same time, they will also experience social and psychological stress caused by separation from their mothers. These factors will lead to changes in behavior, metabolism, physiology, and immune status, increasing the risk of diseases such as oxidative stress and diarrhea, thus seriously affecting the growth and development of weaned yak calves.

[0004] Due to their low toxicity, small side effects, and affordable prices, Chinese herbal medicines are increasingly being used to maintain health and prevent or treat diseases in humans and animals. In the livestock industry, traditional Chinese herbal medicine formulas have been proven to improve the reproductive performance and antioxidant capacity of dairy cows during the perinatal period and enhance the growth performance of weaned calves. Tibetan medicine has unique resource advantages in plateau areas. It not only grows well in alpine environments but also is rich in various active ingredients and has multiple pharmacological effects such as anti-inflammatory, antioxidant, and immunomodulatory effects. These medicinal materials can comprehensively regulate the problems faced by young yaks during the weaning period, such as malnutrition, poor gastrointestinal development, decreased immunity, and increased psychological stress. Currently, agricultural farming increasingly tends to use traditional Chinese medicine or Tibetan medicine as feed additives to promote animal health and immunity and treat livestock diseases. This method can not only reduce the use of chemical drugs and lower the residue risk but also improve production performance, enhance nutrient absorption, strengthen resistance, and reduce the prevalence rate in a natural way, opening up a new way to solve problems such as scarce forage resources and slow growth of young yaks.

[0005] Therefore, it is particularly important to develop a compound preparation of traditional Chinese and Tibetan medicines for relieving diarrhea in weaned young yaks to improve the survival rate and health level of weaned young yaks. Summary of the Invention

[0006] In view of this, the present invention provides a compound preparation of traditional Chinese and Tibetan medicines for relieving diarrhea in weaned young yaks, its preparation method, and application. The compound preparation of traditional Chinese and Tibetan medicines disclosed in the present invention can effectively reduce the inflammation level in weaned young yaks, improve the antioxidant stress capacity, and improve the diversity and structure of the intestinal microbiota when applied to weaned young yaks, thereby achieving the effects of reducing the diarrhea rate of weaned young yaks and enhancing the physique of young yaks.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention discloses a compound preparation of traditional Chinese and Tibetan medicines for relieving diarrhea in weaned young yaks, and the compound preparation of traditional Chinese and Tibetan medicines is composed of the following components in parts by weight: 12 - 18 parts of Pulsatilla chinensis, 5 - 10 parts of Picrorhiza scrophulariiflora Pennell, 6 - 12 parts of Artemisia adamsii Bess., 6 - 12 parts of Prunus mume Sieb. et Zucc. var. tibetica (Batal.) T. T. Yu et Tsai, 5 - 10 parts of Aucklandia lappa Decne., 6 - 15 parts of Paeonia veitchii Lynch, 3 - 6 parts of Anisodus tanguticus (Maxim.) Pascher, and 3 - 10 parts of Glycyrrhiza uralensis Fisch.

[0009] Preferably, the compound preparation of traditional Chinese and Tibetan medicines is composed of the following components in parts by weight: 15 parts of Pulsatilla chinensis, 10 parts of Picrorhiza scrophulariiflora Pennell, 10 parts of Artemisia adamsii Bess., 10 parts of Prunus mume Sieb. et Zucc. var. tibetica (Batal.) T. T. Yu et Tsai, 10 parts of Aucklandia lappa Decne., 10 parts of Paeonia veitchii Lynch, 5 parts of Anisodus tanguticus (Maxim.) Pascher, and 10 parts of Glycyrrhiza uralensis Fisch.

[0010] The present invention provides a preparation method of the above-mentioned compound preparation of traditional Chinese and Tibetan medicines for relieving diarrhea in weaned young yaks, including the following steps:

[0011] (1) Weigh 12 - 18 parts by weight of Pulsatilla chinensis, 5 - 10 parts of Picrorhiza scrophulariiflora Pennell, 6 - 12 parts of Artemisia adamsii Bess., 6 - 12 parts of Prunus mume Sieb. et Zucc. var. buergeriana (Miq.) Yü et Lu, 5 - 10 parts of Aucklandia lappa Decne., 6 - 15 parts of Paeonia veitchii Lynch, 3 - 6 parts of Anisodus tanguticus (Maxim.) Pascher, and 3 - 10 parts of Glycyrrhiza uralensis Fisch. respectively. Then put the weighed medicinal materials into a crusher one by one for crushing, and mix all the crushed medicinal material powders evenly.

[0012] (2) Screen the evenly mixed medicinal material powders in step (1) through a sieve. After screening, put the obtained delicate medicinal material powders into a pre - prepared sealed bag or bottle for standby, that is, obtain the traditional Chinese and Tibetan medicine compound preparation.

[0013] Preferably, in step (2), the mesh number of the sieve is 30 - 50 meshes.

[0014] The present invention also provides an application of the traditional Chinese and Tibetan medicine compound preparation in preparing a feed additive for weaned yak calves.

[0015] Preferably, when adding the traditional Chinese and Tibetan medicine compound preparation to the daily feed of weaned yak calves: the mass ratio of the traditional Chinese and Tibetan medicine compound preparation to the feed of weaned yak calves is 1:(15 - 25).

[0016] Preferably, when adding the traditional Chinese and Tibetan medicine compound preparation to the daily feed of weaned yak calves: the mass ratio of the traditional Chinese and Tibetan medicine compound preparation to the feed of weaned yak calves is 1:20.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) Through network pharmacology analysis, it is found that the traditional Chinese and Tibetan medicine compound preparation provided by the present invention interacts with a variety of immune genes. These genes are closely related to the cell growth process. The interaction between metabolism - related genes and proteins determines the main targets involved in purine metabolism, blood cell metabolism, inflammatory response and the pathways crucial for the growth, immune regulation and antioxidant function of calves. This indicates that the traditional Chinese and Tibetan medicine compound preparation exerts its effects through multiple targets and multiple pathways, and can effectively promote the healthy development of weaned yak calves.

[0019] (2) The traditional Chinese and Tibetan medicine compound preparation provided by the present invention is used for supplementary feeding of weaned yak calves by adding it to the feed. Through experiments, it is found that: the diarrhea rate of weaned yak calves fed with the traditional Chinese and Tibetan medicine compound preparation is significantly reduced, the antioxidant indexes T-AOC (total antioxidant capacity), SOD (superoxide dismutase), and GSH-px (glutathione peroxidase) in the serum increase, while MDA (malondialdehyde) is significantly reduced, and the inflammation-related factors IL-6 (interleukin-6) and IL-10 (interleukin-10) are significantly reduced. It promotes the change of the intestinal flora of weaned yak calves, especially the increase in the abundance of beneficial bacteria and short-chain fatty acid-producing bacteria, while the abundance of pathogenic bacteria and inflammation-related bacteria is significantly reduced, indicating that this compound can not only enhance the antioxidant capacity and immune function of calves, but also improve the intestinal microbial environment and reduce the occurrence of diseases.

[0020] (3) After the traditional Chinese and Tibetan medicine compound preparation provided by the present invention is applied to weaned yak calves, it is safe, harmless, green and environmentally friendly. The main medicine is sourced from Tibet and is convenient to obtain. It can reduce the diarrhea of weaned yak calves and improve the physique of yak calves, providing important technical support for the breeding of calves in plateau areas. This method based on the combination of traditional Tibetan medicine knowledge and modern science and technology not only helps to solve the practical problems faced by the animal husbandry in plateau areas, but also provides valuable reference for the animal health management in other regions. Description of the Drawings

[0021] Figure 1 It is the drug-component-target network diagram of the traditional Chinese and Tibetan medicine compound preparation provided by the present invention;

[0022] Figure 2 It is the Venn diagram of the interaction between the main component genes of the traditional Chinese and Tibetan medicine compound preparation provided by the present invention and the immune / metabolism genes;

[0023] Figure 3 It is the protein-protein interaction (PPI) network diagram of the intersection proteins of the traditional Chinese and Tibetan medicine compound preparation provided by the present invention and the immune / metabolism-related genes;

[0024] Figure 4 It is the GO enrichment analysis of the active components-immune / metabolism common targets of the traditional Chinese and Tibetan medicine compound preparation provided by the present invention;

[0025] Figure 5 It is the KEGG bubble diagram of the active components-immune / metabolism common targets of the traditional Chinese and Tibetan medicine compound preparation provided by the present invention;

[0026] Figure 6 It is the effect of the traditional Chinese and Tibetan medicine compound preparation provided by the present invention on the diarrhea and body weight of weaned yak calves;

[0027] Figure 7 It is the effect of the traditional Chinese and Tibetan medicine compound preparation provided by the present invention on the oxidative stress and inflammatory response of weaned yak calves;

[0028] Figure 8 Effect of the traditional Chinese and Tibetan medicine compound preparation provided by the present invention on the composition of the rectal contents flora of weaned calf yaks (Venn diagram);

[0029] Figure 9 Effect of the traditional Chinese and Tibetan medicine compound preparation provided by the present invention on the Alpha diversity of the rectal contents flora of weaned calf yaks;

[0030] Figure 10 Effect of the traditional Chinese and Tibetan medicine compound preparation provided by the present invention on the Beta diversity of the rectal contents flora of weaned calf yaks;

[0031] Figure 11 Effect of the traditional Chinese and Tibetan medicine compound preparation provided by the present invention on the composition of the rectal contents flora of weaned calf yaks at the phylum, genus, and species levels (species distribution histogram);

[0032] Figure 12 Effect of the traditional Chinese and Tibetan medicine compound preparation provided by the present invention on the composition of the rectal contents flora of weaned calf yaks at the genus and species levels (species distribution heat map);

[0033] Figure 13 Significance analysis of the effect of the traditional Chinese and Tibetan medicine compound preparation provided by the present invention on the composition of the rectal contents flora of weaned calf yaks;

[0034] Figure 14 Effect of the traditional Chinese and Tibetan medicine compound preparation provided by the present invention on the distribution of the functions of the rectal contents flora of weaned calf yaks;

[0035] Figure 15 Effect of the traditional Chinese and Tibetan medicine compound preparation provided by the present invention on the relative abundance of the functions of the rectal contents flora of weaned calf yaks;

[0036] Figure 16 Significance analysis of the effect of the traditional Chinese and Tibetan medicine compound preparation provided by the present invention on the functional abundance of the rectal contents flora of weaned calf yaks. Detailed implementation manners

[0037] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0038] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0039] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0040] The present invention discloses a traditional Chinese and Tibetan medicine compound preparation for alleviating diarrhea in weaned calf yaks. The traditional Chinese and Tibetan medicine compound preparation is composed of the following components in parts by weight: 12-18 parts of Pulsatilla chinensis, 5-10 parts of Picrorhiza scrophulariiflora Pennell, 6-12 parts of Artemisia adamsii Bess., 6-12 parts of Prunus mume Sieb. et Zucc. var. tibetica (Batal.) T. T. Yu et Tsai, 5-10 parts of Aucklandia lappa Decne., 6-15 parts of Paeonia lactiflora Pall. var. willmottiae (Stapf) Stern, 3-6 parts of Anisodus tanguticus (Maxim.) Pascher, and 3-10 parts of Glycyrrhiza uralensis Fisch.

[0041] Among them, the most preferred traditional Chinese and Tibetan medicine compound preparation is composed of the following components in parts by weight: 15 parts of Pulsatilla chinensis, 10 parts of Picrorhiza scrophulariiflora Pennell, 10 parts of Artemisia adamsii Bess., 10 parts of Prunus mume Sieb. et Zucc. var. tibetica (Batal.) T. T. Yu et Tsai, 10 parts of Aucklandia lappa Decne., 10 parts of Paeonia lactiflora Pall. var. willmottiae (Stapf) Stern, 5 parts of Anisodus tanguticus (Maxim.) Pascher, and 10 parts of Glycyrrhiza uralensis Fisch.

[0042] The present invention provides a preparation method of the above-mentioned traditional Chinese and Tibetan medicine compound preparation for alleviating diarrhea in weaned calf yaks, including the following steps:

[0043] (1) Weigh 12-18 parts of Pulsatilla chinensis, 5-10 parts of Picrorhiza scrophulariiflora Pennell, 6-12 parts of Artemisia adamsii Bess., 6-12 parts of Prunus mume Sieb. et Zucc. var. tibetica (Batal.) T. T. Yu et Tsai, 5-10 parts of Aucklandia lappa Decne., 6-15 parts of Paeonia lactiflora Pall. var. willmottiae (Stapf) Stern, 3-6 parts of Anisodus tanguticus (Maxim.) Pascher, and 3-10 parts of Glycyrrhiza uralensis Fisch. according to the parts by weight, and put the weighed medicinal materials into a pulverizer one by one for pulverization, and then mix all the pulverized medicinal material powders evenly;

[0044] (2) Screen the evenly mixed medicinal material powders in step (1) through a sieve with a mesh size of 30-50 meshes. After screening, put the obtained fine medicinal material powders into a pre-prepared sealed bag or bottle for standby, that is, obtain the traditional Chinese and Tibetan medicine compound preparation.

[0045] The present invention also provides an application of the above-mentioned traditional Chinese and Tibetan medicine compound preparation in the preparation of feed additives for weaned calf yaks.

[0046] Among them, when adding the traditional Chinese and Tibetan medicine compound preparation to the daily feed of weaned calf yaks: the mass ratio of the traditional Chinese and Tibetan medicine compound preparation to the feed of weaned calf yaks is 1:(15-25).

[0047] When adding the above-mentioned traditional Chinese and Tibetan medicine compound preparation to the daily feed of weaned calf yaks: the most preferred mass ratio of the traditional Chinese and Tibetan medicine compound preparation to the feed of weaned calf yaks is 1:20.

[0048] The following further illustrates the technical solution of the present invention with specific examples:

[0049] Example 1

[0050] The preparation method of the traditional Chinese medicine composition for anti-ulcerative colitis in this example is as follows:

[0051] (1) Weigh 15 parts by weight of Pulsatilla chinensis, 10 parts of Picrorhiza scrophulariiflora, 10 parts of Artemisia adamsii, 10 parts of Prunus mume var. armeniacaefolia, 10 parts of Aucklandia lappa, 10 parts of Paeonia veitchii, 5 parts of Anisodus tanguticus, and 10 parts of Glycyrrhiza uralensis respectively. Then put the weighed medicinal materials into a pulverizer one by one for pulverization, and mix all the pulverized medicinal material powders evenly.

[0052] (2) Screen the evenly mixed medicinal material powders in step (1) through a sieve with a mesh size of 30 meshes. After screening, put the obtained fine medicinal material powders into a pre-prepared sealed bag or bottle for standby, that is, obtain the traditional Chinese and Tibetan medicine compound preparation.

[0053] (3) Add the traditional Chinese and Tibetan medicine compound preparation obtained in step (2) to the daily feed of weaned yak calves according to a mass ratio of 1:20 with the weaned yak calf feed.

[0054] Example 2

[0055] This example conducts network pharmacology analysis on the traditional Chinese and Tibetan medicine compound preparation prepared in Example 1. Based on the compound traditional Chinese medicine of the present invention, it studies the effects and connections of traditional Chinese medicine components, drug targets, and diseases, and explores the action mechanisms of traditional Chinese medicine in treating diseases through multiple targets and multiple pathways. The specific steps are as follows: (i) Component screening: Using the Traditional Chinese Medicine Systems Pharmacology Analysis Platform (TCMSP) and the HERB database, search for the components of the medicinal materials by inputting specific keywords such as "Pulsatilla chinensis", "Picrorhiza scrophulariiflora Pennell", "Artemisia adamsii Bess.", "Inula racemosa Hook.f.", and "Paeonia lactiflora Pall. var. intermedia (DC.) W. T. Wang & Tsai f.", and determine the active components based on the screening conditions of bioavailability (OB >= 30%) and drug-likeness (DL >= 0.18). For the medicinal materials not included in TCMSP, use the HERB database, input the keyword "Anisodus tanguticus (Maxim.) Pascher" to search for components and obtain the Pubchem ID, search for the SMILES format of the drug components through the Pubchem ID, and use the Swiss ADME database to screen the drug components. Those that meet any 2 out of 5 of Absorption high and Drug likeness (Lipinski, Ghose, Veber, Egan, Muegge) are selected. (ii) Target gene acquisition: Import the confirmed main components into the SwissTargetPrediction database to obtain the Gene names corresponding to individual components, and select the target genes with a binding probability value greater than 0. (iii) Acquisition of immune and metabolism-related genes: Obtain the list of immune-related genes from the IMMPORT database; for metabolism-related genes, use the R language toolset to extract them from the KEGG database. (iv) Construction of the component-target network: In the Cytoscape software, construct a component-target network diagram based on the active drug components and their target information in the traditional Chinese and Tibetan medicine compound preparation. (v) Use the Bioinformatics Cloud Platform of New Life Science Co., Ltd. to upload the main component genes of the drug and the immune / metabolism-related genes to the Venn diagram, and draw the Venn diagram of the targets of the compound and the immune / metabolism-related genes. The intersection is the common target genes of the two. (vi) Construction of the PPI network: Upload the intersection genes of the above-obtained active drug targets and immune / metabolism-related targets to the STRING database, and use the Cytoscape software to construct a protein-protein interaction (PPI) network. The size and color of the nodes reflect the degree value. The larger the node and the darker the color, the greater the degree value. (vii) Functional enrichment analysis: Finally, conduct gene ontology (GO) function and signaling pathway (KEGG pathway) enrichment analysis on the intersection proteins through the DAVID database, and use the Microbial Bioinformatics platform for result visualization. The specific detection and analysis are as follows.

[0056] (1) As Figure 1 and Figure 2As shown in the figure, it can be seen from the figure that: (i) The intersection of the main component genes of the traditional Chinese and Tibetan medicine compound preparation disclosed in the present invention and the immune-related genes: Through the analysis of the online Venn diagram drawing tool, it is found that there are a total of 1,793 immune-related genes between the main component genes of the drug and the immune-related genes. After removing duplicates from the obtained drug main component target genes, there are a total of 929 drug main component target genes; By drawing the mutual relationship between the drug target and the disease target through the online Venn diagram, it is found that there are 207 intersection proteins between the traditional Chinese and Tibetan medicine compound preparation and the immune-related genes; It shows that the traditional Chinese and Tibetan medicine compound preparation of the present invention may have a potential mechanism of action in immune regulation. (ii) The intersection of the main component genes of the traditional Chinese and Tibetan medicine compound preparation of the present invention and the metabolism-related genes: For the metabolism-related genes, a total of 1,732 metabolism-related genes were obtained in the study. After removing duplicates from the obtained drug main component target genes, 929 component target genes were obtained. By drawing the mutual relationship between the drug target and the disease target through the online Venn diagram, it is found that there are 191 intersection proteins between the traditional Chinese and Tibetan medicine compound preparation and the metabolism-related genes; It shows that the traditional Chinese and Tibetan medicine compound preparation disclosed in the present invention may also have an impact on the metabolic process and has a certain regulatory effect.

[0057] In summary, these results reveal the possible multi-target and multi-pathway mechanism of action of the traditional Chinese and Tibetan medicine compound preparation of the present invention in treating diseases, as well as its potential effects in immunity and metabolism.

[0058] (2) As Figure 3 shown in the figure, it can be seen from the figure that: (i) When constructing the protein-protein interaction (PPI) network for the intersection proteins of the traditional Chinese and Tibetan medicine compound preparation disclosed in the present invention with the immune-related genes and the metabolism-related genes, the analysis results show that: For the immune-related genes, a total of a PPI network containing 189 nodes and 567 edges was obtained; According to the degree value ranking, the top 10 core targets were determined as PIK3R1, PIK3CA, PIK3CB, SRC, JAK2, PTK2, HRAS, PLCG1, MAPK3, PTPN11; These targets may play a key role in the process of the traditional Chinese and Tibetan medicine compound preparation of the present invention exerting its therapeutic effect by regulating the immune system. (ii) When constructing the protein-protein interaction network for the intersection proteins of the traditional Chinese and Tibetan medicine compound preparation with the metabolism-related genes, a PPI network containing 168 nodes and 287 edges was constructed; Arranged according to the degree value, the top 10 core targets obtained include HK1, HK2, ENPP1, CYP1A1, AKR1B1, HSD17B7, AKR1B10, GLB1, CYP1B1, PTGS1; These targets reveal that the traditional Chinese and Tibetan medicine compound preparation of the present invention may achieve its biological effects by affecting the key molecules in these metabolic pathways.

[0059] In summary, through the PPI network analysis of the intersection proteins of the traditional Chinese and Tibetan medicine compound preparation of the present invention with immune and metabolism-related genes, not only multiple potential core targets are identified, but also the multi-target and multi-pathway action mechanism of the traditional Chinese and Tibetan medicine compound preparation is further clarified, providing strong support for deeply understanding the scientific basis of traditional Chinese medicine compound preparations.

[0060] (3) As Figure 4 shown, it can be seen from the figure that: for the GO (Gene Ontology) enrichment analysis of the active ingredients of the traditional Chinese and Tibetan medicine compound preparation disclosed in the present invention and the common immune targets, the top 10 are respectively selected for visualization display in the three categories of biological process (BP), cellular component (CC), and molecular function (MF). The results show that "Protein phosphorylation", "plasma membrane", and "RNA polymerase II transcription factor activity, ligand-activated sequence-specific DNA binding" are the most significantly enriched genes in BP, CC, and MF respectively, indicating that the traditional Chinese and Tibetan medicine compound preparation has potential important effects in regulating protein phosphorylation, membrane-related structures, and the activity of specific DNA-binding transcription factors. At the same time, in the GO enrichment analysis of the active ingredients of the traditional Chinese and Tibetan medicine compound preparation and the common metabolism targets, "one-carbon metabolic process", "cytosol", and "oxidoreductase activity" are determined as the most enriched genes in BP, CC, and MF, further indicating that the traditional Chinese and Tibetan medicine compound preparation may play its metabolic regulation function by affecting the one-carbon metabolic process, cytosolic localization, and the activity of oxidoreductase.

[0061] (4) As Figure 5As shown in the KEGG bubble chart of the active ingredients of the traditional Chinese and Tibetan medicine compound preparation of the present invention and the immune common targets (left figure), it can be seen that the active ingredients of the traditional Chinese and Tibetan medicine compound preparation are significantly enriched in multiple important signaling pathways related to immunity, specifically including: the PI3K-Akt signaling pathway, which plays a key role in cell growth, proliferation and survival, regulating the cell cycle process and metabolic activities; the MAPK signaling pathway, which is involved in various cell processes such as cell differentiation, apoptosis and stress response, and has an important impact on cell proliferation and survival; the TCR signaling pathway, which is closely related to the body's immune system, regulating the activation and function of T cells, and is crucial for maintaining immune balance; the enrichment of these pathways indicates that the traditional Chinese and Tibetan medicine compound preparation may have the functions of regulating cell proliferation and immune cells. As shown in the KEGG bubble chart of the active ingredients of the traditional Chinese and Tibetan medicine compound preparation of the present invention and the metabolic common targets (right figure), it can be seen that the active ingredients of the traditional Chinese and Tibetan medicine compound preparation related to metabolism also involve multiple key metabolic pathways, specifically including: folate biosynthesis and starch and sucrose metabolism, which are closely related to the absorption of nutrients and energy metabolism, and contribute to maintaining the body's energy supply and normal physiological functions; nitrogen metabolism and linoleic acid metabolism are related to amino acid metabolism and lipid metabolism respectively, and play an important role in maintaining the body's normal metabolic balance and signal transduction; the enrichment of these metabolic pathways indicates that the traditional Chinese and Tibetan medicine compound preparation may improve the physical state of calves and promote their healthy development by intervening in the metabolic process.

[0062] In summary, through the KEGG pathway enrichment analysis of the active ingredients of the traditional Chinese and Tibetan medicine compound preparation of the present invention and the immune and metabolic common targets, it is found that the compound preparation may not only regulate cell proliferation and immune function, but also comprehensively improve the health status of calves by intervening in the metabolic process, which provides an important scientific basis for the in-depth research and development of this Tibetan medicine compound.

[0063] Example 3

[0064] This example is an experiment using the traditional Chinese and Tibetan medicine compound preparation prepared in Example 1 above to relieve the stress of calves during the weaning period, improve the physical fitness of calves during the weaning period, and prevent diarrhea in weaned calves.

[0065] This experiment was conducted at the Gesangtang Yak Breeding Base in Linzhou County, Lhasa City. The yaks at the breeding base were weaned at about six months of age, separated from the cows, and then raised in a shed to ensure free access to water and a diet consisting of concentrate feed for calves, alfalfa, and oat hay. The feeding amount was gradually increased in stages every day to meet the nutritional needs of the weaned calves. Twenty healthy six-month-old weaned calves with an average weight of about 67 kg were selected for this experiment and randomly divided into an experimental group (XB) and a control group (XD), with 10 calves in each group. A 5% traditional Chinese and Tibetan medicine compound preparation was added to the feed of the weaned calves in the experimental group (XB) (i.e., the mass ratio of the traditional Chinese and Tibetan medicine compound preparation to the feed for weaned calves was 1:20), while the control group (XD) was not given any special treatment. The entire experimental period was 30 days. During this period, the health status of the calves was observed and recorded every day; in the middle of the experiment (day 15) and at the end of the experiment (day 30), all the calves were weighed and their weight changes were recorded; in addition, rectal contents were collected using a sterile fecal collection tube, and blood samples were collected using a disposable blood collection needle and a vacuum clot activator tube to obtain serum for further analysis at the middle of the experiment (day 15) and at the end of the experiment (day 30). The specific analysis results are as follows.

[0066] (1) As Figure 6 shown, it can be seen from the figure that on day 15, the average weight of the control group (XD) was 70.25 kg and that of the experimental group (XB) was 77.60 kg; on day 30, the weight of the control group (XD) increased to 74.75 kg, while that of the experimental group (XB) reached 82.35 kg; compared with the control group (XD), the experimental group (XB) with 5% traditional Chinese and Tibetan medicine compound preparation added to the forage showed significant effects during the experiment. The diarrhea rate of its weaned calves was significantly reduced, and its weight was significantly higher than that of the weaned calves in the control group (XD).

[0067] (2) As Figure 7 shown, it can be seen from the figure that after adding 5% traditional Chinese and Tibetan medicine compound preparation to the weaned calves in the experimental group (XB), significant changes were shown in the antioxidant indexes in their serum: the levels of T-AOC, SOD, and GSH-px all increased significantly (p < 0.05), while the MDA index decreased significantly (p < 0.01). In addition, the levels of inflammatory factors IL-6, IL-1β, and TNF-α decreased significantly in the experimental group (p < 0.05), and the level of the anti-inflammatory factor IL-10 increased significantly (p < 0.01); indicating that compared with the control group (XD), the experimental group (XB) showed enhanced antioxidant capacity and a lower level of inflammatory response at the same time. This shows that the traditional Chinese and Tibetan medicine compound preparation of the present invention can effectively improve the antioxidant defense mechanism of weaned calves, reduce the inflammatory response, and thus contribute to improving the overall health status of calves.

[0068] (3) As Figure 8As shown, 16S sequencing analysis of the rectal content samples of the above-mentioned groups of weaned calves showed that in the samples (Z1) on the 15th day, there were 1,202 OTUs in total between the experimental group (XB) and the control group (XD), while the experimental group (XB) had 5,688 unique OTUs; in the samples (Z2) collected on the 30th day of the experiment, the total number of OTUs in the two groups increased to 1,395, and at the same time, the number of unique OTUs in the experimental group (XB) further increased to 6,036. This indicates that the types of intestinal microorganisms in the experimental group (XB) have increased, suggesting that the traditional Chinese and Tibetan medicine compound preparation of the present invention may promote the improvement of intestinal microbial diversity, contribute to the improvement of intestinal microbial composition, and enhance the health status of the host.

[0069] (4) As Figure 9 shown, it can be seen from the figure that: as the experiment progresses, the Alpha diversity of the experimental group (XB) (added with 5% traditional Chinese and Tibetan medicine compound preparation) shows an upward trend; specifically, in the initial stage of the experiment (the 15th day), there is no significant difference in the diversity indices such as Chao1, Shannon, and Simpson between the experimental group (XB) and the control group (XD); at the end of the experiment (the 30th day), these diversity indices of the experimental group (XB) all show a significant increase. This shows that the richness and diversity of the intestinal flora in the experimental group (XB) tend to increase over time, indicating that the traditional Chinese and Tibetan medicine compound preparation of the present invention helps to improve the intestinal microbial environment and enhance the health status of the host.

[0070] (5) As Figure 10 shown, it can be seen from the figure that: over time, the sample points of the experimental group (XB) and the control group (XD) show significant changing trends at different time points. On the 15th day when the experiment started, whether it was the experimental group (XB) or the control group (XD), the sample points showed obvious aggregation and overlap, indicating that there was no obvious difference between the two groups of samples in the initial stage of the experiment. As time passed to the 30th day, the sample points of the experimental group (XB) and the control group (XD) gradually separated, and the sample points within each group began to aggregate, forming a relatively clear difference between groups. In the NMDS graph, it can be observed that the sample points of either the experimental group (XB) or the control group (XD) have been clearly separated, showing significant differences between the two groups; at the same time, the sample points within each group are also more closely aggregated, indicating enhanced within-group consistency and more obvious between-group differences. And, from the heat map of microbial relative abundance, it can be seen that as the experiment progresses, significant differences have occurred in the microbial species and relative abundances between the experimental group (XB) and the control group (XD). The relative abundances of certain specific microorganisms increase or decrease in the experimental group (XB), while the control group (XD) remains relatively stable, which may be the main factor leading to the separation of the two groups of sample points.

[0071] In summary, by comparing the PCA and NMDS plots and the heatmap of microbial relative abundances at different time points, it can be clearly seen that the sample points of the experimental group and the control group gradually separated from the initial aggregated and overlapping state, and obvious inter-group differences and intra-group consistencies were formed at 30 days, indicating that the experimental intervention had a significant impact on the samples.

[0072] (6) As Figure 11 shown, it can be seen from the figure that Firmicutes and Bacteroidetes are the bacterial phyla with the highest abundances in both groups. On the 15th day at the start of the experiment, Firmicutes and Bacteroidetes showed relatively high abundances in both groups but without obvious differences, and the abundances of Bifidobacterium and other specific genera were relatively low and similar in both groups. As time passed to the 30th day, the abundances of Firmicutes, Bacteroidetes, and Actinobacteria in the experimental group (XB) increased significantly; at the genus level, the abundances of Bifidobacterium, Cryptobacteroides, and Faecousia increased significantly in the experimental group (XB). Figure 12 is the clustering heatmap at the genus level (left) and species level (right), where the red area indicates a relatively high abundance and the blue area indicates a relatively low abundance; in the experimental group, as time passed, the red area gradually increased, indicating that the abundances of certain bacterial genera and species increased significantly. This shows that the traditional Chinese and Tibetan medicine compound preparation disclosed in the present invention has a significant impact on the intestinal microbial community of weaned yak calves and may help improve their health status and physiological functions.

[0073] From Figure 13It can be seen that at the phylum level: in the experimental group (XB), Bacteroidetes decreased, while Firmicutes and Actinobacteria increased significantly. (i) Firmicutes and Bacteroidetes are the dominant bacteria in the intestines of ruminants and are crucial for digestion, nutrient metabolism, and intestinal metabolic homeostasis; the ratio of Firmicutes to Bacteroidetes (F / B ratio) is closely related to inflammatory markers and intestinal metabolic homeostasis; the increase in the F / B ratio in the experimental group (XB) may indicate an increase in the fat deposition rate of weaned yak calves, which helps improve production performance, such as weight gain. (ii) Actinobacteria showed a higher abundance in the experimental group (XB). Although its relative abundance in the intestinal flora is low, it plays a key role in maintaining intestinal homeostasis. Many species in the genus Actinobacteria, especially the genus Bifidobacterium, are probiotics and show beneficial effects. (iii) The content of Bifidobacterium was higher in the experimental group (XB), reflecting the anti-inflammatory and immune-promoting effects of this traditional Chinese and Tibetan medicine compound preparation. In addition, the abundance of harmful bacteria such as Prevotella in the experimental group (XB) decreased, further supporting the positive impact of this traditional Chinese and Tibetan medicine compound preparation on intestinal health, helping to reduce inflammatory responses and improve intestinal health status.

[0074] Based on the above analysis, in the experimental group (XB), the addition of the traditional Chinese and Tibetan medicine compound preparation of the present invention led to a decrease in Bacteroidetes and a significant increase in Firmicutes and Actinobacteria. This change in the microbial community structure not only promoted the growth and development and production performance of weaned yak calves but also enhanced their intestinal health, anti-inflammatory ability, and immune regulation function. In particular, the increase in the F / B ratio and the increase in the content of Bifidobacterium provided better nutrient absorption efficiency and a healthier intestinal environment for weaned yak calves.

[0075] (7) As Figure 14 shown, it can be seen from the figure that on the 15th day of the start of the experiment, the sample points of the experimental group (XB) and the control group (XD) showed obvious aggregation and overlap states in the PCoA plot, indicating that at this stage, the microbial community functions between the two groups were relatively similar and there was no significant separation trend. As time passed to the 30th day, the sample points of the two groups gradually changed from the initial aggregated state to separate aggregations, and there was an obvious separation trend between the groups; indicating that there were significant differences in the microbial community functions of the two groups of samples, which showed that the experimental intervention had a significant impact on the samples.

[0076] (8) The changes in relative abundances between the two groups are as Figure 15As shown in the figure, the relative abundance distribution of metabolites under different functional categories can be seen. Among them, the metabolite abundance in the "Metabolism" category is the highest, followed by "Genetic Information Processing" and "Cellular Processes". This indicates that in the two groups of samples, biological processes related to metabolism dominate.

[0077] (9) As Figure 16 shown in the figure, it can be seen that metabolic pathways such as fatty acid metabolism, glutathione metabolism, glycerolipid metabolism, glycerophospholipid metabolism, ascorbate and aldarate metabolism, and fatty acid biosynthesis are significantly enriched in the experimental group. The enhancement of fatty acid metabolism helps to improve energy utilization efficiency and promote fat accumulation, which is related to the weight gain of calves. Glutathione is an important antioxidant, and the enrichment of its metabolic pathway indicates that this traditional Chinese and Tibetan medicine compound preparation enhances the antioxidant capacity of weaned calves and helps to reduce oxidative stress. The enhancement of these metabolic pathways such as glycerolipid and glycerophospholipid metabolism may promote the construction and maintenance of cell membranes and support the growth and development of calves. Ascorbic acid (vitamin C) is another important antioxidant, and the activation of its metabolic pathway helps to further enhance the body's antioxidant defense mechanism. The enrichment of this pathway of fatty acid biosynthesis means that the fat synthesis process in weaned calves is accelerated, which is beneficial to weight gain and energy reserve.

[0078] In summary, through the above analysis, it can be known that adding the traditional Chinese and Tibetan medicine compound preparation of the present invention to the daily feed of weaned calves can not only increase the weight of weaned calves, but also enhance their antioxidant capacity and reduce the inflammation level. At the same time, the compound preparation also shows the effect of promoting immune function and enhancing the overall physique of weaned calves. Moreover, the compound preparation can also regulate the intestinal flora structure and promote the synthesis of short-chain fatty acids, which is crucial for maintaining intestinal health. In short, the traditional Chinese and Tibetan medicine compound preparation disclosed in the present invention shows significant effects in improving the growth performance, antioxidant status, inflammatory response, immune function and intestinal microbial balance of weaned calves, and can provide a new nutritional strategy for calf breeding.

[0079] The above has introduced in detail a traditional Chinese and Tibetan medicine compound preparation for relieving diarrhea in weaned calves of the present invention, its preparation method and application. Specific examples are used in this article to elaborate on the principle and implementation manner of the present invention. The description of the above examples is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and modifications can still be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A Chinese and Tibetan medicine compound preparation for alleviating diarrhea in weaned yaks, characterized in that: The Chinese-Tibetan medicine compound preparation consists of the following components in parts by weight: 12-18 parts of Pulsatilla scabra, 5-10 parts of Tibetan Coptis chinensis, 6-12 parts of Artemisia argyi, 6-12 parts of Tibetan Prunus mume, 5-10 parts of Tibetan costusroot, 6-15 parts of Tibetan white peony root, 3-6 parts of Hyoscyamus anserina, and 3-10 parts of Licorice.

2. A Chinese-Tibetan medicinal compound preparation for alleviating diarrhea in weaned yaks according to claim 1, characterized in that: The Chinese-Tibetan medicine compound preparation consists of the following components in parts by weight: 15 parts of Pulsatilla scabra, 10 parts of Tibetan Coptis chinensis, 10 parts of Artemisia selengensis, 10 parts of Tibetan Prunus mume, 10 parts of Tibetan costusroot, 10 parts of Tibetan white peony root, 5 parts of Hyoscyamus chinensis, and 10 parts of Licorice.

3. The method for preparing the Chinese-Tibetan medicinal compound preparation for alleviating diarrhea in weaned yaks according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) Weighing Pulsatilla chinensis, Coptis chinensis, Artemisia selengensis, Prunus mume, Aucklandia lappa, Paeonia lactiflora, Hyoscyamus chinensis, and Licorice root respectively by weight, and putting the weighed medicinal materials into a grinder one by one for grinding, and then mixing all the ground medicinal material powders uniformly; (2) The medicinal material powder uniformly mixed in step (1) is screened through a sieve. After the screening is completed, the fine medicinal material powder obtained is put into a pre-prepared sealed bag or bottle for standby use, that is, a Chinese Tibetan medicine compound preparation is obtained.

4. The method for preparing the Chinese-Tibetan medicinal compound preparation for alleviating diarrhea in weaned yaks according to claim 3, characterized in that: In step (2), the mesh size of the sieve is 30 to 50 meshes.

5. Use of the Chinese-Tibetan medicinal compound preparation according to any one of claims 1 to 2 in preparing a feed additive for weaned yak calves.

6. The use according to claim 5, characterized in that: When the Chinese-Tibetan medicinal compound preparation is added into the daily feed of weaned yak calves, the mass ratio of the Chinese-Tibetan medicinal compound preparation to the weaned yak calves feed is 1:(15-25).

7. The use according to claim 6, characterized in that: When the Chinese-Tibetan medicinal compound preparation is added into the daily feed of weaned yak calves, the mass ratio of the Chinese-Tibetan medicinal compound preparation to the weaned yak calves feed is 1:20.

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