A Chinese medicine composition having the effect of improving the intestinal health of rabbits and its preparation method and application

Abstract

The present invention belongs to the field of feed additives, and specifically relates to a Chinese medicine composition having the function of improving the intestinal health of rabbits, and a preparation method and application thereof. The present invention provides a Chinese medicine formula for improving the intestinal health of rabbits, comprising 2-5 parts of astragalus; 1-3 parts of evodia rutaecarpa; 1-3 parts of dried orange peel; and 1-3 parts of liquorice. The Chinese medicine composition and feed provided by the present invention can improve the growth performance of rabbits, regulate fat metabolism and energy metabolism, improve the body's immunity, enhance the digestive capacity and antioxidant capacity of the intestines, and improve the intestinal morphology.

Description

Technical Field

[0001] The invention belongs to the field of feed additives, and particularly relates to a traditional Chinese medicine composition capable of improving the intestinal health of rabbits, and a preparation method and application thereof. Background Art

[0002] With the development of animal husbandry, the use of antibiotics has become more common, but the long-term use of antibiotics will cause a series of adverse effects. Livestock and poultry will develop drug resistance and drug resistance, which will affect the treatment of diseases faced by livestock and poultry. In addition, during the metabolism of antibiotics in livestock and poultry, the drugs will remain in livestock and poultry products, causing serious food safety problems. In addition, with animal excretion, antibiotics enter the environment and cause environmental pollution. Therefore, more and more substitutes for antibiotics are being studied and utilized. The development and utilization of Chinese herbal medicine additives as livestock and poultry feed additives is one of the current research hotspots. It has the advantages of wide sources, no pollution to the environment, and no drug resistance. Many studies have shown that Chinese herbal medicine additives have the ability to replace antibiotics, such as improving immunity, promoting digestion, and promoting growth. The intestine is the largest part of digestion and absorption of nutrients, and it is also the largest immune tissue in the body. It plays an important role in maintaining animal health and promoting animal growth. The digestive system of rabbits is very fragile and prone to diarrhea and death. More than 70% of dead rabbits have diarrhea symptoms, which brings huge economic losses to the rabbit industry. However, there is no research on the use of Chinese medicine formulas for rabbit gastrointestinal health. Summary of the invention

[0003] To solve the above problems, the present invention provides a traditional Chinese medicine composition having the function of improving the intestinal health of rabbits, which is made of the following components in parts by weight: 2-5 parts of astragalus; 1-3 parts of evodia rutaecarpa; 1-3 parts of tangerine peel; and 1-3 parts of liquorice.

[0004] Further, it is made of the following components by weight: 3 parts of Astragalus; 2.5 parts of Evodia rutaecarpa; 2.5 parts of dried tangerine peel; 1.5 parts of licorice.

[0005] The present invention also provides application of the Chinese medicine composition in preparing a medicine for improving the intestinal health of rabbits.

[0006] The present invention also provides a feed additive for improving the intestinal health of rabbits, comprising the above-mentioned Chinese medicine composition.

[0007] The present invention also provides a feed for improving the intestinal health of rabbits, comprising the above-mentioned Chinese medicine composition or the above-mentioned feed additive.

[0008] Furthermore, the content of the Chinese medicine composition in the rabbit feed is 1 / 1000 to 2 / 1000 by weight.

[0009] The present invention has the following beneficial effects:

[0010] The present invention studies the effects of compound Chinese herbal medicine as feed additive on growth performance and intestinal digestive enzymes, immune function, digestive enzyme activity, intestinal morphology and intestinal microorganisms of rabbits, and provides a reference for Chinese herbal medicine as an alternative to antibiotic additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0012] Figure 1 Effects of compound Chinese herbal medicine on immune indexes of rabbits; A. Immunoglobulin M content; B. Immunoglobulin G content; C. Secretory immunoglobulin A content of intestinal mucosa in duodenum, jejunum and ileum;

[0013] Figure 2 Effects of compound Chinese herbal medicine on digestive enzyme content in rabbit intestine; A. α-amylase content; B. Trypsin content; C. Chymotrypsin content;

[0014] Figure 3 Effects of compound Chinese herbal medicine on antioxidant indexes in rabbit intestine; A. Glutathione oxidase content; B. Superoxide dismutase activity; C. Malondialdehyde content D. Protein content;

[0015] Figure 4 Effects of compound Chinese herbal medicine on cytokines in intestinal tissues of rabbits; A. IL-4 content in intestinal tissues; B. IL-6 content in intestinal tissues; C. TNF-α content in intestinal tissues; D. INF-γ content in intestinal tissues;

[0016] Figure 5 Effects of compound Chinese herbal medicine on the villus morphology of rabbit small intestine; A. Villus length; B. Crypt depth; C. Villus / crypt ratio; D. HE staining of small intestine morphology (40×);

[0017] Figure 6 Effects of compound Chinese herbal medicine on the relative expression of tight junction protein mRNA in rabbit intestinal mucosa; A. Duodenum; B. Jejunum; C. Ileum;

[0018] Figure 7 Effects of compound Chinese herbal medicine on the diversity of rabbit cecal microorganisms; A. phylum level species stacking diagram; B. genus level species stacking diagram; C. α-diversity analysis of cecal microorganisms; D. multivariate statistical PCA analysis; E. OUTVenn diagram of different treatment groups; F. Heatmap analysis of rabbit cecal microorganisms at the genus level. DETAILED DESCRIPTION

[0019] Now, various exemplary embodiments of the present invention are described in detail. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0020] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0022] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0024] Example 1 Materials and Methods

[0025] 1.1 Test materials

[0026] The compound Chinese medicine used in this experiment is made of the following components by weight: 3 parts of Astragalus; 2.5 parts of Evodia rutaecarpa; 2.5 parts of dried orange peel; 1.5 parts of Licorice. The crushing was carried out using a pulverizer, the crushing time was 4 to 5 minutes, the pulverizer model was LG-04A, and the fineness was 30-300 mesh. The Chinese herbal medicine was crushed into a yellow-brown powder, which was mixed in proportion and added to the rabbit diet. The antibiotic was kitasamycin.

[0027] 1.2 Experimental design and diet

[0028] 96 5-week-old New Zealand white rabbits with similar weight and health were randomly selected and divided into 3 groups, with 6 replicates in each group and 6 rabbits in each replicate. The treatments of each group were blank control group (BC): basal diet, positive control group (Abs): basal diet + kitasamycin 300 mg kg -1 , Compound Chinese herbal medicine group (CM): basic diet + compound Chinese herbal medicine. The main ingredients of the basic diet are corn, alfalfa meal, bran, etc. The diets of each group were made into granules, and the Chinese herbal medicine group was first added with the prepared Chinese herbal medicine and then mixed with the ingredients of the basic diet to make granules.

[0029] 1.3 Feeding and management

[0030] Before the experiment, the rabbit cages were sterilized with high temperature, and the whole environment was sterilized again with DuPont Virbac before the rabbits were introduced. The pre-trial period was 7 days, and the formal period was 42 days. No treatment measures were taken if disease problems occurred during the feeding period. During the experiment, artificial feeding was adopted, once in the morning and once in the evening, free drinking water, and feces were cleaned once a day.

[0031] 1.4 Sample collection

[0032] On the 21st and 42nd day of the experiment, one rabbit was randomly selected from each replicate for blood sampling from the middle ear artery. The whole blood was placed at 37°C and 4°C for 2 h, respectively, and then rotated at 3500 r·min. -1 , centrifuged under the conditions of 10min and the serum was aliquoted and stored at -80℃ for later use. On the 42nd day of the experiment, three portions of the duodenum, jejunum and ileum were taken from each replicate of one rabbit. One portion was fixed with paraformaldehyde and later made into paraffin sections, and one portion was washed with pre-cooled PBS to remove residual blood. The intestinal segments were weighed according to the ratio of weight (g): volume (mL) = 1:9, and 9 times the volume of pre-cooled PBS was added. It was ground under the conditions of 4℃65Hz for 120s. The homogenate was centrifuged at 5000×g for 5min. The supernatant was aliquoted and stored at -80℃ according to the required amount. It was subsequently used for the detection of intestinal mucosal digestive enzymes, intestinal mucosal antioxidant enzymes, secretory immunoglobulin A (sIgA) and cytokines. One portion was immediately stored in liquid nitrogen for the detection of the relative expression of intestinal junction protein mRNA. The contents of the cecum were aseptically taken and quickly frozen in liquid nitrogen, and then transferred to -80℃ for the detection of intestinal microorganisms.

[0033] 1.5 Testing indicators and methods

[0034] 1.5.1 Growth performance

[0035] The fasting body weight of rabbits was measured weekly from the beginning of the formal experiment to the last day of the experiment, and the diet consumed by each replicate was recorded to calculate the average daily gain (ADG), average feed intake (ADFI) and feed-to-gain ratio (F / G) of each group.

[0036] 1.5.2 Immune indicators

[0037] The serum and tissue homogenate frozen at -80°C, as well as the test kit and the sample to be tested, were placed at room temperature for 30 min, and the levels of immunoglobulin G (IgG), immunoglobulin M (IgM) and secretory immunoglobulin A (sIgA) were determined using an enzyme-linked immunosorbent assay (ELISA) kit. When measuring IgG and IgM, the serum was first diluted 500,000 times. The test kit was purchased from Wuhan Fine Biotechnology Co., Ltd.

[0038] 1.5.3 Small Intestinal Mucosal Digestive Enzymes and Antioxidant Enzymes

[0039] The samples and test kits were placed at room temperature 30 minutes in advance, and α-amylase (α-Amylase), trypsin, chymotrypsin (Chymotrypsin) (Jiangsu Enzyme Free Industry Co., Ltd.), glutathione peroxidase (Glutathione peroxidase, GSH-Px) and malondialdehyde (Malondialdehyde, MDA) (Wuhan Elaruite Biotechnology Co., Ltd.) were detected using ELISA kits. Superoxide dismutase (SOD) was detected using the WST-8 method, and the kit was purchased from Suzhou Mengxi Biotechnology Co., Ltd. The protein concentration of the sample was detected using the BCA protein concentration determination kit, which was purchased from Beijing Solebold Technology Co., Ltd.

[0040] 1.5.4 Intestinal cytokines

[0041] The samples were placed at room temperature for 30 min together with the test kit according to the required amount before testing. The levels of interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in each intestinal segment were detected by ELISA. The test kit was purchased from Wuhan Fine Biotechnology Co., Ltd.

[0042] 1.5.5 Morphology of small intestinal villi

[0043] The duodenum, jejunum, and ileum were fixed in 4% paraformaldehyde for 24 hours, dehydrated in a series of ethanol and paraffin embedding, and then HE staining was performed. The villus length (V) and crypt depth (C) of each intestinal segment in each group were measured by optical microscopy. The Image-Pro Plus 6.0 software analysis program (Image-Pro Plus 6.0 Media Cybernetics, USA) was used. At least 5 measurement points were selected for each slice, and the average value was finally taken to calculate the V / C ratio.

[0044] 1.5.6 Relative expression of enteric junction protein mRNA

[0045] Take 5-20 mg of tissue and centrifuge at 12000 rpm for 10 min at 4 ° C to extract the supernatant for total RNA extraction. Then use Nanodrop 2000 to detect RNA concentration and purity, and dilute the RNA with excessive concentration in an appropriate proportion to make its final concentration 200 ng / μL. Then reverse transcription was performed. The pipette tip and PCR reaction tube were sterile and enzyme-free. The kit was SweScript All-in-One RT SuperMix for QPCR. The reaction system was (20 μL), 4 μL 5× SweScript All-in-One SuperMix for qPCR, 1 μL gDNA Remover, 10 μL Total RNA, add Nuclease-Free Water to make up to 20 μL, mix gently and centrifuge. The reverse transcription program was set at 25 ° C 5 min, 42 ° C 30 min, 85 ° C 5 s, and reverse transcription was completed on a common PCR instrument (Beijing Dongsheng Innovation Biotechnology Co., Ltd.). Take 0.1ml PCR reaction plate, prepare the following reaction system, 7.5μL 2×Universal Blue SYBR Green qPCR Master Mix, 1.5μL 2.5μM gene primer (upstream + downstream), 2.0μL reverse transcription product (cDNA), 4.0μL Water Nuclease-Free, complete amplification on a fluorescent quantitative PCR instrument (Bio-rad), the cycle conditions are as follows: pre-denaturation at 95℃ for 30s, denaturation at 95℃ for 15s, annealing / extension at 60℃ for 30s, 40 cycles, melting curve 65℃→95℃, collect fluorescence signal every 0.5℃ rise, all samples are repeated 3 times. The relative expression of gene mRNA level was analyzed using the 2-ΔΔCt method, and glyceraldehyde-3-6 phosphate dehydrogenase (GAPDH) was the internal reference gene. All detected genes and their primers are listed in Table 1.2 Primer sequences of tight junction protein genes by Q-PCR.

[0046] 1.5.7 High-throughput sequencing analysis of cecal microbial diversity

[0047] Microbial DNA was extracted using the HiPure Soil DNA Extraction Kit (or HiPure Stool DNA Extraction Kit) (Magen, Guangzhou, China) according to the operating instructions. Specific primers F: 5'-CCTACGGGNGGCWGCAG-3', R: 5'-GGACTACHVGGGTATCTAAT-3' were used. Amplification system: 50 μL mixture, including 10 μL 5×Q5@Reaction Buffer, 10 μL 5×Q5@High GC Enhancer, 1.5 μL 2.5 mM dNTPs, 1.5 μL upstream and downstream primers (10 μM), 0.2 μL Q5@High-Fidelity DNA Polymerase, 50 ng template DNA. PCR-related reagent manufacturer (New England Biolabs, USA). The quality of the amplified product was evaluated using 2% agarose gel, and the PCR product was purified using AMPureXP Beads (Beckman, CA, USA), and quantified using Qubit 3.0. The sequencing library was constructed using the Illumina DNA Prep Kit (Illumina, CA, USA). The library quality was tested using the ABI StepOnePlus Real-Time PCR System (Life Technologies, Foster City, USA). Qualified libraries were sequenced using the Novasek 6000 PE250 mode pooling. After the sequencing data was downloaded from the machine, we selected the DADA2 method to deduplicate, correct, reduce noise, and remove chimeras to obtain ASV sequence and abundance information. Species annotation was performed based on OTU / ASV sequences, and OTU / ASV abundance information statistics were used to obtain species abundance information at each level. According to the OTU and species abundance tables, intestinal microbial diversity was evaluated by alpha diversity (Chaol, Shannon, and Pielou-e).

[0048] 1.5.8 Data Analysis

[0049] The experimental data were statistically analyzed using SPSS27.0.1, and one-way ANOVA and LSD were used for pairwise comparison. All data are expressed as mean ± standard deviation (M ± SD). P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different. GraphPad Prism9.5 was used for drawing.

[0050] Example 2 Results and Analysis

[0051] 2.1 Growth performance

[0052] The effects of adding compound Chinese herbal medicine to feed on the growth performance of rabbits are shown in Table 1. There were no significant differences among the compound Chinese herbal medicine group, antibiotic group and blank control group (P>0.05), but the average daily weight gain and feed-to-weight ratio of the compound Chinese herbal medicine group were higher than those of the antibiotic group and blank control group.

[0053] Table 1 Effects of compound Chinese herbal medicine on growth performance of rabbits

[0054]

[0055]

[0056] 2.2 Effects of compound Chinese herbal medicine on immune indicators in rabbits

[0057] Figure 1 The effect of compound Chinese herbal medicine on IgG and IgM in rabbit serum and secretory IgA (sIgA) in intestinal mucosa. The IgG content of the mid-term CM group was significantly higher than that of the BC group and the Abs group (P < 0.05) (P < 0.01), and the IgM content of the late CM group was higher than that of the BC group and the Abs group (P < 0.01). Compared with the BC group, the sIgA content of the CM group was significantly different (P < 0.01).

[0058] 2.3 Effects of compound Chinese herbal medicine on digestive enzyme content in rabbit intestine

[0059] Effects of compound Chinese herbal medicine on digestive enzyme content in rabbit intestines Figure 2 As shown in the results, the α-amylase activity in the duodenum and jejunum was significantly increased compared with the BC group (P < 0.01) and (P < 0.05). The trypsin activity in the CM group was significantly increased compared with the BC group (P < 0.05). The chymotrypsin activity in the duodenum and jejunum of the CM group was higher than that in the Abs group and the BC group, but there was no significant difference. The chymotrypsin activity in the ileum was significantly decreased compared with the BC group (P < 0.05).

[0060] 2.4 Effects of compound Chinese herbal medicine on intestinal antioxidant indexes in rabbits

[0061] Effects of adding compound Chinese herbal medicine to diet on the activity and content of antioxidant enzymes in rabbit intestine Figure 3As shown. Compared with the BC group, the compound Chinese herbal medicine significantly increased the GSH-Px content in the jejunum and ileum (P < 0.01) and (P < 0.01), and compared with the Abs group, CM significantly increased the GSH-Px content in the ileum (P < 0.01). Compared with the BC group and the Abs group, CM increased the SOD activity in the duodenum and jejunum, but there was no significant difference (P > 0.05). Compared with the BC group, CM significantly decreased the MDA content in the duodenum (P < 0.01). Compared with the BC group and the Abs group, the protein concentration of CM in the jejunum and ileum was increased, but there was no significance (P > 0.05).

[0062] 2.5 Effects of compound Chinese herbal medicine on cytokines in rabbit intestinal tissue

[0063] Effects of compound Chinese herbal medicine in feed and blank control group on cytokines INF-γ, IL-4, IL-6, and TNF-α in duodenum, jejunum, and ileum. Figure 4 As shown in the results, compared with the BC group, the IL-4 content in the duodenum and ileum of the CM group tended to increase, but there was no significant difference (P>0.05). Compared with the BC group, there was a very significant difference in the IL-6 content in the duodenum between the BC group and the CM group and the Abs group (P<0.01), and the IL-6 content in the ileum of the CM group was significantly increased (P<0.05). Compared with the BC group, the CM group significantly reduced the TNF-α content in the jejunum (P<0.05). There was no significant difference in the INF-γ content among the three intestinal segments among the groups (P>0.05).

[0064] 2.6 Effects of compound Chinese herbal medicine on the morphology of rabbit small intestinal villi

[0065] Effects of adding compound Chinese herbal medicine and antibiotics to rabbit diet on small intestinal mucosal morphology Figure 5 As shown. Compared with the BC group, the villus length of the duodenum, jejunum and ileum in the CM group was longer, with significant differences (P < 0.05). Compared with the BC group, the depth of the villus crypts in the duodenum, jejunum and ileum in the CM group was significantly different (P < 0.05). The villus length and crypt depth (V / C) of the duodenum, ileum and jejunum in the CM group showed an increasing trend, but there was no significant difference (P > 0.05). D is a local section of the duodenum and ileum HE staining. As shown in the figure, the villus length of the BC group was shorter and sparse, with obvious breaks and damage. The villus length of the Abs group was denser than that of the BC group, but there were also some breaks. The small intestinal villi of the CM group grew well, with a large number and density, and good integrity.

[0066] 2.7 Effect of compound Chinese herbal medicine on relative expression of tight junction protein mRNA in rabbit intestinal mucosa

[0067] Effects of compound Chinese herbal medicine on the relative expression of tight junction proteins (ZO-1, Occludin, Claudin) in rabbit intestine Figure 6 The results showed that compared with the BC group and the Abs group, the relative expression of ZO-1 mRNA, Occludin mRNA, and Claudin mRNA in the jejunum and ileum of the CM group had an upward trend, and the relative expression of Claudin mRNA in the ileum was significantly different (P < 0.05).

[0068] 2.8 Effects of compound Chinese herbal medicine on the diversity of rabbit cecal microorganisms

[0069] The top ten species in the OUT cluster are presented in detail, other known species are classified as others, and unknown species are marked as unclassified, such as Figure 7 As shown in A and B, Firmicutes, Bacteroidetes, and Verrucomicrobia were the main phyla in the different treatment groups.

[0070] Rikenellaceae_RC9_gut_group, NK4A214_group, V9D2013_group, Phascolarctobacterium, Lachnospiraceae_NK4A136_group, Akkermansia, and V9D2013_group were the main genera. The Rikenellaceae_RC9_gut_group flora in the CM group was significantly higher than that in the BC and Abs groups (P < 0.05), and the compound Chinese herbal medicine upregulated the relative abundance of the NK4A214_group (P < 0.05). In order to more comprehensively evaluate the Alpha diversity of the microbial community, the Chao1 index, Simpson index, and Pielou index were used to analyze the species richness and evenness among the BC, CM, and Abs groups. Figure 7 Therefore, the Chao1 diversity index of the CM group increased significantly (P = 0.022), and the Simpson index and Pielou index of the CM group showed no increasing trend (P = 0.437) (P = 0.281), indicating that the abundance of microbial flora in the CM group was higher than that in the other two treatment groups. Figure 7 D is the PCA result diagram, which shows that the intestinal microbial composition of the CM group is different from that of the other two groups. Figure 7 There were 831 core OTUs in the three treatment groups in E, 475 OTUs unique to the CM group, 447 OTUs unique to the Abs group, and 649 OTUs in the BC group. Figure 7F shows the relative abundance distribution trend of different bacterial genera. The composition of bacterial genera at the genus level in the three treatment groups is different. This indicates that the addition of compound Chinese herbal medicine to the diet has an effect on the composition of the rabbit cecal microbial structure. The closer the color is to red, the higher the richness is, and the closer to blue, the lower the richness is.

Claims

1. A Chinese medicine composition having the effect of improving the intestinal health of rabbits, characterized in that: The invention is prepared from the following components in parts by weight: 3 parts of astragalus, 2.5 parts of evodia rutaecarpa, 2.5 parts of tangerine peel and 1.5 parts of liquorice.

2. Use of the Chinese medicine composition as claimed in claim 1 in preparing a medicament for improving the intestinal health of rabbits.

3. A feed additive for improving the intestinal health of rabbits, characterized in that: Including the Chinese medicine composition according to claim 1.

4. A feed for improving the intestinal health of rabbits, characterized in that: Including the Chinese medicine composition according to claim 1 or the feed additive according to claim 3.

5. The feed according to claim 4, characterized in that The content of the Chinese medicine composition in the rabbit feed is 1 / 1000 to 2 / 1000 by weight.

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