Application of pharmaceutical by-products of Isatis indigotica

Abstract

This invention discloses the application of pharmaceutical by-products from Isatis indigotica, belonging to the field of livestock breeding technology. The application of these by-products involves regulating the intestinal flora of piglets, improving liver function, or improving lipid metabolism in piglets. This invention utilizes Isatis indigotica by-products to regulate the intestinal flora, improve liver function, or improve lipid metabolism in piglets. After weaning, this effectively improves intestinal flora imbalance, liver damage, and glucose and lipid metabolism disorders caused by early weaning stress in piglets, thereby effectively reducing adverse stress responses during weaning, improving piglet health, and increasing breeding efficiency.

Description

Technical Field

[0001] This invention belongs to the field of livestock breeding technology, and in particular relates to the application of pharmaceutical by-products of Isatis indigotica. Background Technology

[0002] Piglet rearing is a crucial stage in pig production. The performance of piglets directly impacts indicators such as slaughter time, slaughter rate, and slaughter weight, thus affecting the overall efficiency of pig farming. With the continuous improvement of pig production levels, large-scale pig farms typically wean piglets at 3-4 weeks of age. However, at this stage, piglets' digestive and immune systems are not yet fully developed. Combined with factors such as separation from the mother pig, changes in diet, and environmental changes, they often experience severe stress, leading to decreased feed intake, slow growth, susceptibility to diarrhea and other diseases, and liver and kidney damage, causing significant losses to the pig farming industry.

[0003] In the early stages of pig farming, domestic and foreign farmers often added antibiotics to feed pigs to improve their health and growth rate. However, with the ban on antibiotics in feed and the deepening of related scientific research, the use of antibiotics in feed has become a thing of the past, and the role of traditional Chinese medicine in animal health care has received increasing attention: compound traditional Chinese medicines such as turmeric, coptis, and phellodendron can significantly increase the total protein content, albumin content and alanine aminotransferase activity in the serum of piglets, improve liver function, and improve weaning stress syndrome in piglets (Liu Yan. Effects of traditional Chinese medicine additives on serum biochemical indicators of piglets [J]. Special Economic Animals and Plants, 2023, 26(05):13-15.); supplementing the diet of weaned piglets with olive extract can improve the growth performance of piglets, enhance liver function, antioxidant and anti-inflammatory capabilities, regulate the abundance of intestinal flora, and have health care effects that are superior to those of adding antibiotics (Zhang Yu. Effects of olive extract on production performance, serum indicators and intestinal microbiota of weaned piglets [D]. Zhejiang University, 2022.). Isatis root, a classic traditional Chinese medicine for clearing heat and detoxifying, has long been used in animal husbandry and veterinary medicine. Its formulations or extracts mainly exert pharmacological effects such as antiviral, anti-inflammatory, antibacterial, and immune-enhancing properties. However, few studies have explored the effects of Isatis root or its byproduct residue generated during pharmaceutical manufacturing on the intestinal flora and liver function of weaned piglets, and related applications are rarely reported or practically promoted. Existing products, compared to the Isatis root pharmaceutical byproducts disclosed in this patent, are more difficult to prepare and obtain, and are more expensive, potentially increasing breeding costs and the economic burden on farmers, making them unsuitable for large-scale promotion. In contrast, the pharmaceutical industry can produce hundreds or thousands of tons of Isatis root residue monthly, which is relatively low-cost and easy to obtain.

[0004] In conclusion, severe stress during weaning in piglets commonly manifests as gut microbiota dysbiosis and impaired liver and kidney function, significantly impacting the pig farming industry. To date, there are no reports of Isatis root or its pharmaceutical byproduct residue having any effect as a feed additive in weaned piglets in regulating gut microbiota, protecting the liver, or improving glucose and lipid metabolism disorders. Pharmacological studies of Isatis root primarily focus on its antiviral, anti-inflammatory, antibacterial, and immune-enhancing properties, and have not yet demonstrated any liver-protective effects as described above. Summary of the Invention

[0005] In order to overcome the problems existing in the prior art, one of the objectives of the present invention is to provide the application of Isatis indigotica pharmaceutical by-products, which can be used to regulate the intestinal flora of piglets, improve the liver function of piglets, or improve the lipid metabolism of piglets.

[0006] The second objective of this invention is to provide a piglet feed.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides the application of a pharmaceutical byproduct of Isatis indigotica, wherein the application is at least one of the following: regulating the intestinal flora of piglets, improving the liver function of piglets, or improving the lipid metabolism of piglets.

[0009] Preferably, in the application, the pharmaceutical by-product of Isatis root is the solid residue remaining after Isatis root has been extracted by decoction once or multiple times and the extract has been filtered to separate the extract; more preferably, the application also includes the steps of drying and pulverizing the solid residue.

[0010] Preferably, in the application, the water content of the Isatis root pharmaceutical by-product is ≤12wt%; more preferably, the water content of the Isatis root pharmaceutical by-product is ≤10wt%.

[0011] Preferably, in the application, the particle size of the Isatis root pharmaceutical by-product is ≤60 mesh.

[0012] Preferably, in the application, the pharmaceutical by-product of Isatis indigotica includes the following components: L-arginine, guanine, L-phenylalanine, epigallocatechin, deoxydaunocarbazine, 3-indoleacetonitrile, indigo, and indirubin.

[0013] More preferably, in the application, the pharmaceutical by-product of Isatis indigotica includes the following components in the following amounts: L-arginine 0.8-1.6 μg / g, guanine 0.6-2 μg / g, L-phenylalanine 0.1-0.4 μg / g, bicornuate 0.3-0.6 μg / g, deoxydaunocarbazone 0.1-0.3 μg / g, 3-indoleacetonitrile 0.4-0.9 μg / g, indigo 1.5-3 μg / g, and indirubin 0.1-0.4 μg / g.

[0014] Preferably, in the application, the regulation of piglet gut microbiota includes at least one of the following features:

[0015] 1) At the phylum level, reduce the relative abundance of Actinobacteriota in the gut microbiota of piglets to below 0.003;

[0016] 2) At the phylum level, reduce the relative abundance of Actinobacteria in the piglet gut microbiota to below 0.0005;

[0017] 3) At the phylum level, reduce the relative abundance of Verrucomicrobiots in the gut microbiota of piglets to below 0.0001;

[0018] 4) At the phylum level, reduce the relative abundance of Deferribacteres in the gut microbiota of piglets to below 0.00001;

[0019] 5) At the species level, the relative abundance of Lactobacillus-reuteri in the gut microbiota of piglets was reduced to below 0.01;

[0020] 6) At the species level, reduce the relative abundance of Eubacterium-coprostanoli genes in the gut microbiota of piglets to below 0.006;

[0021] 7) At the species level, reduce the relative abundance of Actinobacillus-minor in the gut microbiota of piglets to below 0.002;

[0022] 8) At the species level, the relative abundance of Ralstonia-pickettll in the gut microbiota of piglets was reduced to below 0.001;

[0023] 9) At the species level, the relative abundance of Lactobacillus-murinus in the gut microbiota of piglets was reduced to below 0.001.

[0024] More preferably, in the application, the regulation of piglet gut microbiota includes the following nine features:

[0025] 1) At the phylum level, reduce the relative abundance of Actinobacteriota in the gut microbiota of piglets to below 0.003;

[0026] 2) At the phylum level, reduce the relative abundance of Actinobacteria in the piglet gut microbiota to below 0.0005;

[0027] 3) At the phylum level, reduce the relative abundance of Verrucomicrobiots in the gut microbiota of piglets to below 0.0001;

[0028] 4) At the phylum level, reduce the relative abundance of Deferribacteres in the gut microbiota of piglets to below 0.00001;

[0029] 5) At the species level, the relative abundance of Lactobacillus-reuteri in the gut microbiota of piglets was reduced to below 0.01;

[0030] 6) At the species level, reduce the relative abundance of Eubacterium-coprostanoli genes in the gut microbiota of piglets to below 0.006;

[0031] 7) At the species level, reduce the relative abundance of Actinobacillus-minor in the gut microbiota of piglets to below 0.002;

[0032] 8) At the species level, the relative abundance of Ralstonia-pickettll in the gut microbiota of piglets was reduced to below 0.001;

[0033] 9) At the species level, reduce the relative abundance of Lactobacillus-murinus in the gut microbiota of piglets to below 0.001. Preferably, in this application, the improvement in piglet liver function includes at least one of the following characteristics:

[0034] 1) Reduce the serum alanine aminotransferase level in piglets to below 65 U / L;

[0035] 2) Reduce the serum total bile acid content of piglets to below 55 μmol / L.

[0036] More preferably, in the application, the improvement of piglet liver function includes the following two features:

[0037] 1) Significantly reduces serum alanine aminotransferase levels in piglets, more preferably, to below 65 U / L;

[0038] 2) Significantly reduces the total bile acid content in the serum of piglets, more preferably, to below 55 μmol / L.

[0039] Preferably, in the application, the improvement of lipid metabolism in piglets includes the following features:

[0040] Reduce the LDL cholesterol level in piglets to below 1.6 mmol / L.

[0041] A second aspect of the present invention provides a piglet feed as described in the first aspect of the present invention, comprising a basal diet and a pharmaceutical by-product of Isatis indigotica.

[0042] In the piglet feed, the Isatis root pharmaceutical by-product is the Isatis root pharmaceutical by-product used in the first aspect of this invention.

[0043] Preferably, in the piglet feed, the Isatis root pharmaceutical by-product is dried to a moisture content of ≤12%wt; more preferably, the moisture content is ≤10wt%.

[0044] Preferably, the mass percentage of the Isatis root pharmaceutical by-product in the piglet feed is 0.3-4.5%; more preferably 0.5-4%; and even more preferably 0.5-1%.

[0045] Preferably, in the piglet feed, the basal diet is formulated according to NRC2012.

[0046] Preferably, the piglet feed is divided into a first dosage group, a second dosage group, a third dosage group, and a fourth dosage group; in the first dosage group feed, the mass percentage of the pharmaceutical by-product of Isatis indigotica is 0.3% ≤ < 0.8%; in the second dosage group feed, the mass percentage of the pharmaceutical by-product of Isatis indigotica is 0.8% ≤ < 1.5%; in the third dosage group feed, the mass percentage of the pharmaceutical by-product of Isatis indigotica is 1.5% ≤ < 3%; and in the fourth dosage group feed, the mass percentage of the pharmaceutical by-product of Isatis indigotica is 3% ≤ 4.5%.

[0047] Preferably, the piglet feed is used to regulate the intestinal flora of piglets, improve the liver function of piglets, or improve the lipid metabolism of piglets.

[0048] Preferably, the regulation of piglet gut microbiota includes at least one of the following features:

[0049] 1) At the phylum level, reduce the relative abundance of Actinobacteriota in the gut microbiota of piglets to below 0.003;

[0050] 2) At the phylum level, reduce the relative abundance of Actinobacteria in the piglet gut microbiota to below 0.0005;

[0051] 3) At the phylum level, reduce the relative abundance of Verrucomicrobiots in the gut microbiota of piglets to below 0.0001;

[0052] 4) At the phylum level, reduce the relative abundance of Deferribacteres in the gut microbiota of piglets to below 0.00001;

[0053] 5) At the species level, the relative abundance of Lactobacillus-reuteri in the gut microbiota of piglets was reduced to below 0.01;

[0054] 6) At the species level, reduce the relative abundance of Eubacterium-coprostanoli genes in the gut microbiota of piglets to below 0.006;

[0055] 7) At the species level, reduce the relative abundance of Actinobacillus-minor in the gut microbiota of piglets to below 0.002;

[0056] 8) At the species level, the relative abundance of Ralstonia-pickettll in the gut microbiota of piglets was reduced to below 0.001;

[0057] 9) At the species level, the relative abundance of Lactobacillus-murinus in the gut microbiota of piglets was reduced to below 0.001.

[0058] Preferably, when the piglet feed is used to reduce the relative abundance of Actinobacteriota in the piglet gut microbiota to below 0.003, the feed is selected from the first dose group feed, the second dose group feed, the third dose group feed, or the fourth dose group feed; more preferably, the fourth dose group feed.

[0059] Preferably, when the piglet feed is used to reduce the relative abundance of Actinobacteria in the piglets' gut microbiota to below 0.0005, the feed is selected from the first dose group feed, the second dose group feed, the third dose group feed, or the fourth dose group feed; more preferably, the first dose group feed, the third dose group feed, or the fourth dose group feed.

[0060] Preferably, when the piglet feed is used to reduce the relative abundance of Verrucomicrobiots in the piglet gut microbiota to below 0.0001, the feed is selected from the first dose group feed, the second dose group feed, the third dose group feed, or the fourth dose group feed; more preferably, the first dose group feed, the third dose group feed, or the fourth dose group feed.

[0061] Preferably, when the piglet feed is used to reduce the relative abundance of Deferribacteres in the piglet gut microbiota to below 0.00001, the feed is selected from the first dose group feed, the second dose group feed, the third dose group feed, or the fourth dose group feed.

[0062] Preferably, when the piglet feed is used to reduce the relative abundance of Lactobacillus-reuteri in the piglet gut microbiota to below 0.01, the feed is selected from the first dose group feed, the second dose group feed, the third dose group feed, or the fourth dose group feed; more preferably, the fourth dose group feed.

[0063] Preferably, when the piglet feed is used to reduce the relative abundance of Eubacterium-coprostanoligenes in the piglet gut microbiota to below 0.006, the feed is selected from the first dose group feed, the second dose group feed, the third dose group feed, or the fourth dose group feed; more preferably, the fourth dose group feed.

[0064] Preferably, when the piglet feed is used to reduce the relative abundance of Actinobacillus-minor in the piglet gut microbiota to below 0.002, the feed is selected from the first dose group feed, the second dose group feed, the third dose group feed, or the fourth dose group feed; more preferably, the third dose group feed or the fourth dose group feed.

[0065] Preferably, when the piglet feed is used to reduce the relative abundance of Ralstonia-pickettll in the intestinal flora of piglets to below 0.001, the feed is selected from the first dose group feed, the second dose group feed, the third dose group feed, or the fourth dose group feed; more preferably, the first dose group feed or the fourth dose group feed.

[0066] Preferably, when the piglet feed is used to reduce the relative abundance of Lactobacillus-murinus in the piglet gut microbiota to below 0.001, the feed is selected from the first dose group feed, the second dose group feed, the third dose group feed, or the fourth dose group feed.

[0067] Preferably, the improvement of piglet liver function includes at least one of the following features:

[0068] 1) Reduce the serum alanine aminotransferase level in piglets to below 65 U / L;

[0069] 2) Reduce the serum total bile acid content of piglets to below 55 μmol / L.

[0070] Preferably, when the piglet feed is used to reduce the serum alanine aminotransferase (ALT) content of piglets to below 65 U / L, the feed is selected from the first dose group feed or the fourth dose group feed; more preferably, the first dose group feed.

[0071] Preferably, when the piglet feed is used to reduce the serum total bile acid (TBA) content of piglets to below 55 μmol / L, the feed is selected from the first dose group feed, the second dose group feed, or the third dose group feed; more preferably, the third dose group feed.

[0072] Preferably, the improvement of lipid metabolism in piglets includes the following features:

[0073] Reduce the LDL cholesterol level in piglets to below 1.6 mmol / L.

[0074] Preferably, when the piglet feed is used to reduce the low-density lipoprotein cholesterol (LDL-C) content of piglets to below 1.6 mmol / L, the feed is selected from the first dose group feed, the second dose group feed, the third dose group feed, or the fourth dose group feed; more preferably, the first dose group feed.

[0075] Preferably, the piglet feed is also used to improve the physiological state of the piglets; more preferably, the improvement of the piglet's physiological state includes at least one of the following characteristics:

[0076] 1) Increase the serum total protein (TP) content of piglets to above 60g / L;

[0077] 2) Increase the serum albumin (ALB) content of piglets to above 30 g / L;

[0078] 3) Reduce the serum globulin (GLB) content in piglets to below 25 g / L;

[0079] 4) Increase the serum albumin to globulin ratio (A / G) of piglets to above 1.5.

[0080] Preferably, when the piglet feed is used to increase the serum total protein (TP) content of piglets to above 60 g / L, the feed is selected from the second dose group feed, the third dose group feed, or the fourth dose group feed; more preferably, the fourth dose group feed.

[0081] Preferably, when the piglet feed is used to increase the serum albumin (ALB) content of piglets to above 30 g / L, the feed is selected from the first dose group feed, the second dose group feed, the third dose group feed, or the fourth dose group feed; more preferably, the fourth dose group feed.

[0082] Preferably, when the piglet feed is used to reduce the serum globulin (GLB) content of piglets to below 25 g / L, the feed is selected from the first dose group feed, the second dose group feed, the third dose group feed, or the fourth dose group feed; more preferably, the fourth dose group feed.

[0083] Preferably, when the piglet feed is used to increase the serum albumin to globulin ratio (A / G) of piglets to 1.5 or higher, the feed is selected from the first dose group feed, the second dose group feed, the third dose group feed or the fourth dose group feed; more preferably, the fourth dose group feed.

[0084] The beneficial effects of this invention are:

[0085] This invention utilizes pharmaceutical byproducts of Isatis indigotica to regulate the intestinal flora of piglets, improve liver function, or improve lipid metabolism. It can effectively improve intestinal flora imbalance, liver function damage, and glucose and lipid metabolism disorders caused by early weaning stress in piglets after weaning, thereby effectively reducing adverse stress responses caused by weaning, improving the health status of piglets, and increasing breeding efficiency.

[0086] Specifically, compared with the prior art, the present invention has the following advantages:

[0087] This invention discloses a byproduct of Isatis indigotica pharmaceutical production, which can be added as a functional feed additive to the antibiotic-free basal diet of weaned piglets. Without the need for antibiotics, it can effectively regulate intestinal flora, protect the liver, and improve glucose and lipid metabolism disorders. Specifically, at the phylum level, it significantly reduces the relative abundance of Ctinobacteriota, Verrucomicrobiots, Deferribacteres, and Campulobacterota; at the species level, it significantly reduces the relative abundance of Lactobacillus-reuteri, Eubacterium-coprostanoligenes, Actinobacillus-minor, Ralstonia-pickettll, and Lactobacillus-murinus; it significantly reduces serum alanine aminotransferase (ALT) and total bile acid (TBA) levels; and it significantly reduces serum glucose (GLU) and low-density lipoprotein cholesterol (LDL-C) levels. Attached Figure Description

[0088] Figure 1 The effect of Isatis root residue on the general growth of weaned piglets (ADG: average daily weight gain, kg / d; ADFI: average daily feed intake, kg / d; F / G: feed conversion ratio; diarrhea rate, %).

[0089] Figure 2The effects of Isatis root residue on serum biochemical parameters of weaned piglets (ALT: alanine aminotransferase, U / L; AST: aspartate aminotransferase, U / L; ALP: alkaline phosphatase, U / L; LDH: lactate dehydrogenase, U / L; TBA: total bile acids, μmol / L; ALB: albumin, g / L; GLB: globulin, g / L; A / G: albumin / globulin; TP: total protein, g / L; BUN: blood urea nitrogen, mmol / L; GLU: glucose, mmol / L; TC: total cholesterol, mmol / L; TG: total triglycerides, mmol / L; HDL-C: high-density lipoprotein cholesterol, mmol / L; LDL-C: low-density lipoprotein cholesterol, mmol / L).

[0090] Figure 3 A diagram showing the top 20 bacterial communities at the phylum level in the colonic contents of weaned piglets.

[0091] Figure 4 The relative abundance of differentially expressed microbiota at the phylum level in the colonic contents of weaned piglets.

[0092] Figure 5 A diagram showing the top 20 microbial community structures at the species level in the colon of weaned piglets.

[0093] Figure 6 The relative abundance of differentially expressed microbiota at the species level in the colonic contents of weaned piglets. Detailed Implementation

[0094] The following specific embodiments further illustrate the content of the present invention in detail. It should also be understood that the following embodiments are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made by those skilled in the art based on the principles described herein are all within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make selections within a suitable range based on the description herein, and are not intended to be limited to the specific data in the examples below. Unless otherwise specified, the raw materials, reagents, or apparatus used in the following embodiments and comparative examples can be obtained from conventional commercial sources or by existing known methods.

[0095] Example 1

[0096] Preparation of Isatis root pharmaceutical by-product feed additive: Isatis root residue by-products generated during the actual large-scale industrial production of Isatis root granules, provided by Guangzhou Baiyunshan Hutchison Whampoa Chinese Medicine Co., Ltd., are dried until the moisture content is below 10%, pulverized, and passed through a 20-mesh sieve. They are then added to an antibiotic-free basal diet in the following proportions for use in the rearing of weaned piglets in the examples below. The specific preparation process includes taking Isatis root, adding water and decocting twice, the first time for 2 hours and the second time for 1 hour. The decoction is filtered and used to prepare Isatis root granules. The solid residue obtained after filtration can be used to prepare Isatis root pharmaceutical by-product feed additives.

[0097] The components of the pharmaceutical by-product of Isatis indigotica obtained in Example 1 were determined using the same method as in Example 1 of CN116593595A. The results showed that the pharmaceutical by-product of Isatis indigotica contained the following components: L-arginine 1.26 μg / g, guanine 1.84 μg / g, L-phenylalanine 0.34 μg / g, epigallocatechin 0.45 μg / g, deoxydaunocarbamate 0.23 μg / g, 3-indoleacetonitrile 0.67 μg / g, indigo 2.88 μg / g, and indirubin 0.33 μg / g.

[0098] Example 2

[0099] Experiment on feeding weaned piglets with by-products of Isatis indigotica pharmaceuticals as feed additives

[0100] 1. Experimental Design and Methods

[0101] 1.1 Animal Experiments

[0102] Fifty healthy Duroc × Landrace × Large White weaned piglets of similar body condition (purchased from Hanshou Tianxin Agricultural and Animal Husbandry Co., Ltd.) at 21 days of age were randomly divided into 5 groups (n=10). The experiment began the following day and lasted for 21 days. The control group was fed a basal diet, while the groups treated with the medicinal residue were fed a basal diet supplemented with 0.5%, 1%, 2%, and 4% by weight of the Isatis root medicinal residue prepared in Example 1 (see Table 1). The medicinal residue was pulverized and passed through a 20-mesh sieve. The basal diet was prepared according to NRC 2012, and the nutritional level met or exceeded the NRC recommendations. After the experiment, blood samples were collected from all pigs, they were slaughtered, and intestinal tissue and digesta samples were separated and collected for subsequent testing.

[0103] Table 1 Experimental Groups

[0104] Grouping Daily food Blank control group (CON) Basic Diet Isatis root residue experimental group 1 (B1) Basic diet + 0.5% Isatis root residue Isatis root residue experimental group 2 (B2) Basic diet + 1% Isatis root residue Isatis root residue experimental group 3 (B3) Basic diet + 2% Isatis root residue Isatis root residue experimental group 4 (B4) Basic diet + 4% Isatis root residue

[0105] 1.2 General Growth Conditions

[0106] At the beginning and end of the formal experiment, the fasting weight of piglets was measured. The daily feed intake and remaining amount were collected and recorded, and the average daily feed intake (kg / d), average daily weight gain (kg / d), and feed conversion ratio (average daily feed intake / average daily weight gain) were calculated. Around 3:00 PM daily, the piglets' diarrhea was observed and scored. The fecal scoring criteria were: 1: Solid, hard feces; 2: Slightly soft feces; 3: Soft, partially formed feces; 4: Semi-liquid feces; 5: Separated feces and water, watery, unformed. A score below 3 was considered diarrhea.

[0107] 1.3 Serum biochemical index detection

[0108] The levels of serum total protein (TP), albumin (ALB), total bile acids (TBA), glucose (GLU), triglycerides (TG), blood urea nitrogen (BUN), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) were detected using a fully automated biochemical analyzer and its matching reagents (Shanghai Kehua Biotechnology ZY-450).

[0109] 1.4 Analysis of gut microbiome composition

[0110] The gut microbiota composition was analyzed using 16S rDNA high-throughput sequencing technology. Anterior colon contents were sent to Beijing Novogene Technology Co., Ltd. for sequencing using the Illumina HiSeq platform. 16S iRNA technology was used to determine the diversity of the colonic microbiota. DNA was extracted from the colon contents, and its quality was evaluated by 1.0% agarose gel electrophoresis. The raw sequencing data obtained from the sequencing platform were processed and quality filtered using Timomatit v0.36 and Pear V0.9.6 software. Fiash V1.2 and Pear v0.9.6 software were used to splice the two ends of the sequences according to the overlap relationship of the FE (Features in Entries) to obtain Fasta sequences. Chimeras in Fasta sequences were removed by uchime alignment to obtain valid sequences. Sequences with a similarity greater than 97% were screened to generate Operational Taxonomic Units (OTUs), and statistical analyses were performed on OTU clustering, species classification, diversity indices, and community structure.

[0111] 1.5 Data Statistics and Analysis

[0112] The experimental data were first sorted using Excel 2019, and then one-way ANOVA was performed using the ANOVA program in SPSS 26.0 statistical software, and multiple comparisons were performed using Duncan's method. The results were expressed as "mean ± standard deviation". P<0.01 indicated extremely significant differences, P<0.05 indicated significant differences, and 0.05<P<0.10 indicated a significant trend of differences.

[0113] 2. Results and Analysis <s

[0114] 2.1 General Growth Situation <s

[0115] Growth performance is an important indicator for evaluating the production efficiency of livestock and poultry. In production, higher average daily gain and lower feed-to-meat ratio are pursued. Figure 1 To study the effect of Isatis indigotica residue on the general growth of weaned piglets, including average daily gain (ADG); average feed intake (ADFI); feed-to-meat ratio (F / G). As Figure 1 shown, compared with the control group, adding 2% and 4% of Isatis indigotica residue to the diet of weaned piglets significantly reduced the average daily gain of piglets (P<0.05). In terms of diarrhea, adding 1% and 4% of Isatis indigotica residue significantly reduced the diarrhea rate of piglets (P<0.05). In addition, there were no significant differences in average feed intake, feed-to-meat ratio, and average final weight among the groups (P>0.05). To sum up, adding a certain proportion of Isatis indigotica residue to the feed of weaned piglets will not affect the growth performance of piglets, but can improve piglet diarrhea, and the effect of 1% addition is the best. [[ID=]]

[0116] 2.2 Serum Biochemical Indexes

[0117] Figure 2 To study the effect of Isatis indigotica residue on the serum biochemical indexes of weaned piglets, including total protein (TP), albumin (ALB), total bile acid (TBA), glucose (GLU), triglyceride (TG), urea nitrogen (BUN), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH). In terms of the content of serum enzymes, as Figure 2As shown, compared with the control group, adding 0.5% Isatis root residue to the diet of weaned piglets significantly reduced ALT levels (P<0.05), 2% significantly reduced TBA levels (P<0.05), 1%, 2%, and 4% significantly increased TP levels (P<0.05), 1% and 4% significantly increased TG levels, and the addition of Isatis root residue increased ALB and A / G levels (P<0.05). The 0.5% and 1% groups significantly reduced LDL-C levels (P<0.05). In conclusion, adding a certain dose of Isatis root residue to the diet of piglets can improve liver health and lipid metabolism function, alleviate liver function decline caused by weaning stress, and demonstrate hepatoprotective effects, while also improving lipid disorders. Furthermore, the increase in TP, ALB, and A / G levels suggests that it also improves nutrient absorption and promotes muscle health, which may contribute to meat quality improvement.

[0118] 2.3 Composition of Gut Microbiota

[0119] Figure 3 The diagram shows the top 20 microbiota at the phylum level in the colon contents of weaned piglets, including the blank control group (C), the Isatis root residue test group 1 (B1), the Isatis root residue test group 2 (B2), the Isatis root residue test group 3 (B3), and the Isatis root residue test group 4 (B4). Figure 4 The relative abundance of differentially expressed microbiota at the phylum level in the colonic contents of weaned piglets. For example... Figure 3 and 4 As shown, at the phylum level, compared with the control group, all groups with added Isatis indigotica residue significantly reduced the levels of ctinobacteriota, Verrucomicrobiots, Deferribacteres, and Campulobacterota (P<0.05). Compared with the control group, the 4% group significantly increased the levels of Spirochaetota and Fusobacteriota (P<0.05) and significantly decreased the level of Cyanobacteria (P<0.05).

[0120] Figure 5 The diagram shows the top 20 microbiota structures at the colonic level in weaned piglets, including the blank control group (C), Isatis root residue test group 1 (B1), Isatis root residue test group 2 (B2), Isatis root residue test group 3 (B3), and Isatis root residue test group 4 (B4). Figure 6 The relative abundance of differentially expressed microbiota at the species level in the colonic contents of weaned piglets. For example... Figure 5 and 6As shown, compared with the control group at the species level, the 4% group significantly reduced the levels of *Lactobacillus-johnsonii*, *Lactobacillus-amylovorus*, and *Lactobacillus-salivarius* (P<0.05). Compared with the control group, all groups with added *Isatis indigotica* residue significantly reduced the levels of *Lactobacillus-reuteri*, *Eubacterium-coprostanoligenes*, *Actinobacillus-minor*, *Ralstonia-pickettll*, and *Lactobacillus-murinus* (P<0.05). Compared with the control group, except for the 1% group which showed a significant decreasing trend, all other groups significantly reduced the level of *Olsenella-sp-GAM18* (P<0.05).

[0121] Comparative Example

[0122] Fifteen healthy Duroc × Landrace × Large White weaned piglets of similar body condition, aged 21 days, were randomly divided into three groups (n=5) for a 21-day experimental period. The control group was fed a basal weaned piglet diet, while experimental groups 1 and 2 were fed a basal diet supplemented with 1% and 4% Isatis root extract, respectively. All extracts were pulverized and passed through a 20-mesh sieve. The basal diets were formulated according to NRC 2012, meeting NRC recommended nutritional levels. The health status of all pigs was observed during the experiment. After the experiment, blood samples were collected from all pigs, and they were slaughtered. Intestinal tissue and digestive samples were separated and collected for subsequent testing.

[0123] The components of the Isatis root medicinal material in the comparative example were determined using the method described in Example 1 of CN116593595A. The results showed that the Isatis root medicinal material contained the following components: L-arginine 1270.60 μg / g, guanine 230.94 μg / g, L-phenylalanine 556.89 μg / g, epigallocatechin 249.38 μg / g, deoxydaunocarbamate 24.76 μg / g, 3-indoleacetonitrile 63.31 μg / g, indigo 329.19 μg / g, and indirubin 359.23 μg / g. It is evident that the content of the main components in the Isatis root medicinal material differs significantly from that in the Isatis root residue.

[0124] 1. Detection Method

[0125] 1.1 Growth performance

[0126] The fasting weights of piglets were measured at the beginning and end of the formal experiment. The daily supply and remaining amount of the diet were collected and recorded, and the average daily feed intake (kg / d), average daily weight gain (kg / d), and feed-to-weight ratio (average daily feed intake / average daily weight gain) were calculated. The diarrhea situation of piglets was observed and scored around 15:00 every day. The fecal scoring criteria were as follows: 1: Solid and hard feces; 2: Somewhat soft feces; 3: Soft feces, partially formed; 4: Semi-liquid feces; 5: Feces and water separated, watery, unformed.

[0127] 1.2 Serum biochemical indices

[0128] The contents of total protein (TP), albumin (ALB), total bile acid (TBA), glucose (GLU), triglyceride (TG), urea nitrogen (BUN), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) in serum were detected using an automatic biochemical analyzer and its supporting reagents (ZY-450, Kehua Bio, Shanghai).

[0129] 1.3 Data statistics and analysis

[0130] The experimental data were first sorted using Excel 2019, and then one-way ANOVA was performed using the ANOVA program in SPSS 26.0 statistical software, and T-test was used for difference analysis. The results were expressed as "mean ± standard deviation". P < 0.01 indicated extremely significant differences, P < 0.05 indicated significant differences, and 0.05 < P < 0.10 indicated a significant trend.

[0131] 2. Results

[0132] 2.1 Growth performance

[0133] Growth performance is an important indicator for evaluating the production efficiency of livestock and poultry. Higher average daily weight gain and lower feed-to-meat ratio are required in production. As shown in Table 2, adding 1% and 4% of Isatis indigotica Fort. in the diet of weaned piglets significantly reduced the diarrhea score of piglets (P < 0.01), but adding a relatively large amount (4%) of Isatis indigotica Fort. in the diet of weaned piglets significantly reduced the average daily weight gain of piglets (P < 0.05). Through comprehensive analysis, it can be seen that adding Isatis indigotica Fort. powder can improve piglet diarrhea, and a certain addition amount has no obvious effect on improving growth performance, while too high addition may affect growth performance.

[0134] Table 2 Growth performance

[0135] project control group 1% Isatis root 4% Isatis root Average daily feed intake (kg / d) 0.48±0.025 0.43±0.051 0.44±0.032 Average daily weight gain (kg / d) 0.26±0.029 0.22±0.021 0.22±0.013* Meat-to-fat ratio 1.9±0.25 1.9±0.26 2.2±0.27 Diarrhea score 3.9±0.24 2.9±0.31** 3.1±0.18**

[0136] * indicates P < 0.05 compared to the control group; ** indicates P < 0.01 compared to the control group.

[0137] 2.2 Serum biochemical indicators

[0138] As shown in Table 3, adding a certain proportion of Isatis root to the diet of weaned piglets can significantly increase serum ALB, A / G, and TP levels (P<0.05), indicating that it can improve nutritional metabolism, promote muscle health, and may help improve meat quality.

[0139] Table 3 Serum Biochemical Indicators

[0140]

[0141]

[0142] * indicates P < 0.05 compared to the control group; ** indicates P < 0.01 compared to the control group.

[0143] 3. Discussion

[0144] The results of this experiment show that adding Isatis root powder to weaned piglets can reduce the severity of diarrhea and improve nutritional metabolism, but it has no significant effect on growth performance, liver function, or glucose and lipid metabolism, and excessive amounts may affect growth performance.

[0145] This invention discloses a byproduct of Isatis indigotica pharmaceutical production, which can be added as a functional feed additive to the antibiotic-free basal diet of weaned piglets. Without the need for antibiotics, it can effectively regulate intestinal flora, protect the liver, and improve glucose and lipid metabolism disorders. Specifically, at the phylum level, it significantly reduces the relative abundance of Ctinobacteriota, Verrucomicrobiots, Deferribacteres, and Campulobacterota; at the species level, it significantly reduces the relative abundance of Lactobacillus-reuteri, Eubacterium-coprostanoligenes, Actinobacillus-minor, Ralstonia-pickettll, and Lactobacillus-murinus; it significantly reduces serum alanine aminotransferase (ALT) and total bile acid (TBA) levels; and it significantly reduces serum glucose (GLU) and low-density lipoprotein cholesterol (LDL-C) levels.

[0146] This invention utilizes pharmaceutical byproducts of Isatis indigotica to regulate the intestinal flora of piglets, improve liver function, or improve lipid metabolism. It can effectively improve intestinal flora imbalance, liver function damage, and glucose and lipid metabolism disorders caused by early weaning stress in piglets after weaning, thereby effectively reducing adverse stress responses caused by weaning, improving the health status of piglets, and increasing breeding efficiency.

Claims

1. The application of pharmaceutical by-products of Isatis indigotica, characterized in that, The application is the use of Isatis indigotica pharmaceutical by-products in the preparation of products for regulating the intestinal flora of piglets, improving the liver function of piglets, or improving the lipid metabolism of piglets. The pharmaceutical by-product of Isatis indigotica contains the following components in the following amounts: L-arginine 0.8~1.6 μg / g, guanine 0.6~2 μg / g, L-phenylalanine 0.1~0.4 μg / g, epigallocatechin 0.3~0.6 μg / g, deoxydaunoside ketone 0.1~0.3 μg / g, 3 -Indoleacetonitrile 0.4~0.9μg / g, Indigo 1.5~3μg / g, Indirubin 0.1~0.4μg / g; The pharmaceutical by-product of Banlangen is the solid residue remaining after Banlangen medicinal material has been extracted by decoction once or multiple times and the extract has been separated by filtration. The regulation of piglet gut microbiota includes at least one of the following characteristics: 1) At the phylum level, reduce the gut microbiota of piglets. Actinobacteriota The relative abundance was below 0.003; 2) At the phylum level, reduce the gut microbiota of piglets. Actinobacteria The relative abundance was below 0.0005; 3) At the phylum level, reduce the gut microbiota of piglets. Verrucomicrobiots The relative abundance was below 0.0001; 4) At the phylum level, reduce the gut microbiota of piglets. Deferribacteres The relative abundance is below 0.00001; 5) At the genetic level, reduce the gut microbiota of piglets. Lactobacillus-reuteri The relative abundance was below 0.01; 6) At the seed level, reduce the gut microbiota of piglets. Eubacterium-coprostanoligenes The relative abundance was below 0.006; 7) At the seed level, reduce the gut microbiota of piglets. Actinobacillus-minor The relative abundance was below 0.002; 8) At the genetic level, reduce the gut microbiota of piglets. Ralstonia-pickettll The relative abundance was below 0.001; 9) At the seed level, reduce the gut microbiota of piglets. Lactobacillus-murinus The relative abundance was below 0.001; The improvement in piglet liver function includes at least one of the following characteristics: 1) Reduce the serum alanine aminotransferase level in piglets to below 65 U / L; 2) Reduce the serum total bile acid content of piglets to below 55 μmol / L; The improvement in piglet lipid metabolism includes the following characteristics: Reduce the LDL cholesterol level in piglets to below 1.6 mmol / L.

2. The application according to claim 1, characterized in that, The product is piglet feed; the piglet feed includes a basal diet and pharmaceutical by-products of Isatis indigotica.

3. The application according to claim 2, characterized in that, The pharmaceutical by-products of Isatis indigotica are dried to a moisture content of ≤12%wt.

4. The application according to claim 2, characterized in that, The percentage by weight of the pharmaceutical by-product of Isatis indigotica in the piglet feed is 0.3-4.5%.

5. The application according to claim 4, characterized in that, The percentage of the pharmaceutical by-product of Isatis indigotica in the piglet feed is 0.5-4% by mass.

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