Application of callicarpa nudiflora extract in weaned piglet breeding

Abstract

The invention provides application of a callicarpa nudiflora extract in weaned piglet breeding, and belongs to the technical field of livestock breeding. The callicarpa nudiflora extract is used as an antibiotic substitute for preparing the weaned piglet feed, so that the weight, the average daily gain and the average daily feed intake of weaned piglets are obviously improved, the intestinal cell bypass of the weaned piglets can be improved, the intestinal barrier of the weaned piglets can be enhanced, and meanwhile, the feed added with the callicarpa nudiflora extract has good safety. Therefore, the invention provides the application of the callicarpa nudiflora extract in preparation of the feed for breeding the weaned piglets.

Description

Technical Field

[0001] The invention belongs to the technical field of animal husbandry, and particularly relates to application of a Callicarpa nudiflora extract in weaned piglet breeding. Background Art

[0002] Callicarpa nudiflora Hook.et Arn. is a local specialty medicinal material in Hainan Province. It has the effect of stopping bleeding and dispersing blood stasis. It is the only newly added medicinal material in the 2020 edition of the Chinese Pharmacopoeia. It is used for acute infectious hepatitis, respiratory and digestive tract bleeding. Its main medicinal part is its dried leaves or young branches with leaves. It is a common medicinal material used by the Li people in Hainan. It is also distributed in Guangdong Province and Guangxi Zhuang Autonomous Region. It is mainly distributed in India, Vietnam and Malaysia abroad. In recent years, the medicinal dosage forms of Callicarpa nudiflora have gradually increased, including tablets, suppositories, ointments (Hainan Jiuzhitang Co., Ltd.), capsules (Jiangxi Xinglin Baima Pharmaceutical Co., Ltd.), mixtures (Hainan Jiuzhitang Zhongyuan Pharmaceutical Co., Ltd.), etc., which are widely used in dermatology, obstetrics and gynecology, postpartum hemorrhage and other hemorrhagic and surgical diseases. At present, there are few reports on the application of Callicarpa nudiflora extract as a new antibiotic substitute in weaned piglets. Summary of the invention

[0003] In view of this, the object of the present invention is to provide an application of a Callicarpa nudiflora extract in the preparation of feed for weaned piglet breeding. The Callicarpa nudiflora extract, as a new type of antibiotic, not only promotes the growth performance of weaned piglets and improves the intestinal health of weaned piglets, but also has good feed safety.

[0004] The invention provides an application of a Callicarpa nudiflora extract in preparing feed for weaned piglets. The Callicarpa nudiflora extract contains the following compounds: 0.7-1.0 mg / g caffeic acid, 5-11 mg / g forsythiaside B, 4-7 mg / g luteolin, 70-120 mg / g verbascoside, 40-60 mg / g isoverbascoside, 0.4-0.8 mg / g apigenin glycoside and 0.1-0.3 mg / g luteolin.

[0005] Preferably, the weaned piglet breeding includes promoting the growth of weaned piglets and / or maintaining the intestinal health of weaned piglets.

[0006] Preferably, the promoting the growth of weaned piglets comprises improving at least one of the following indicators: body weight, average daily weight gain and average daily feed intake of weaned piglets.

[0007] Preferably, maintaining the intestinal health of weaned piglets includes improving the intestinal paracellular pathway of weaned piglets and / or enhancing the intestinal barrier function of weaned piglets.

[0008] Preferably, in the weaned piglet breeding, the Callicarpa nudiflora extract is fed to the weaned piglets in the form of feed.

[0009] Preferably, the feed further comprises a basal diet;

[0010] The added mass of the Callicarpa nudiflora extract is not less than 140 mg / kg of the basic diet.

[0011] Preferably, the added mass of the Callicarpa nudiflora extract is 150-600 mg / kg of the basic diet.

[0012] Preferably, the added mass of the Callicarpa nudiflora extract is 150-300 mg / kg basal diet.

[0013] Preferably, the feeding amount of the feed is 380-400 g / day / each.

[0014] The invention provides a weaned piglet feed, comprising a Callicarpa nudiflora extract and a basic daily diet;

[0015] The mass percentage of the Callicarpa nudiflora extract in the basic diet is not less than 140 mg / kg;

[0016] The Callicarpa nudiflora extract contains the following compounds: 0.7-1.0 mg / g caffeic acid, 5-11 mg / g forsythiaside B, 4-7 mg / g luteolin, 70-120 mg / g verbascoside, 40-60 mg / g isoverbascoside, 0.4-0.8 mg / g apigenin glycoside and 0.1-0.3 mg / g luteolin.

[0017] The present invention provides an application of a Callicarpa nudiflora extract in preparing feed for weaned piglets, wherein the Callicarpa nudiflora extract contains the following compounds: 0.7-1.0 mg / g caffeic acid, 5-11 mg / g forsythiaside B, 4-7 mg / g luteolin, 70-120 mg / g verbascoside, 40-60 mg / g isoverbascoside, 0.4-0.8 mg / g apigenin glycoside and 0.1-0.3 mg / g luteolin. The present invention uses the Callicarpa nudiflora extract as an antibiotic substitute for weaned piglets, which can effectively promote the increase of the weight, average daily weight gain and average daily feed intake of weaned piglets, and can also improve the intestinal paracellular pathway of weaned piglets. At the same time, the Callicarpa nudiflora extract can effectively improve the tight junction barrier of the ileal mucosa of Danzhou chickens, enhance the intestinal barrier function, and enhance the ability of the ileal mucosa to resist bacterial and viral invasion. In addition, adding Callicarpa nudiflora extract to the diet had no effect on the development of the heart, liver, spleen, lungs and kidneys of weaned piglets, and the addition of Callicarpa nudiflora extract at different concentrations did not cause liver and kidney damage in piglets, indicating that it can be safely used in piglet feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the tight junction protein structure in the intestinal epithelial barrier;

[0019] Figure 2 The fingerprints of the dry leaves of Callicarpa nudiflora, the water-extracted extract and the chromatograms of the mixed reference substance are stacked, wherein A is the blank solvent chromatogram; B is the mixed reference substance chromatogram; C is the chromatogram of the dry leaves of Callicarpa nudiflora; and D is the chromatogram of the extract of Callicarpa nudiflora.

[0020] Figure 3 The results of colon microbial alpha diversity; A is Observed species, B is Shannon, C is Simpson, D is Chao1, E is Goods coverage, and F is Pielou;

[0021] Figure 4 The beta diversity results of colonic microorganisms; A is the result of principal coordinate analysis, and B is the result of non-metric multidimensional scaling analysis;

[0022] Figure 5 The results of colonic microbial community composition at the phylum (A) and genus (B) levels;

[0023] Figure 6 Heat map of species composition at the genus level for colonic bacteria;

[0024] Figure 7 The results of ileum microbial Alpha diversity; A is Observed species, B is Shannon, C is Simpson, D is Chao1, E is Goods coverage, and F is Pielou;

[0025] Figure 8 The beta diversity results of ileum microorganisms; A is the result of principal coordinate analysis, and B is the result of non-metric multidimensional scaling analysis;

[0026] Fig. 9 The results of ileal microbial flora composition at the phylum (A) and genus (B) levels;

[0027] Fig.10 Heat map of species composition of ileal bacteria at the genus level. DETAILED DESCRIPTION

[0028] The invention provides an application of a Callicarpa nudiflora extract in preparing feed for weaned piglets. The Callicarpa nudiflora extract contains the following compounds: 0.7-1.0 mg / g caffeic acid, 5-11 mg / g forsythiaside B, 4-7 mg / g luteolin, 70-120 mg / g verbascoside, 40-60 mg / g isoverbascoside, 0.4-0.8 mg / g apigenin glycoside and 0.1-0.3 mg / g luteolin.

[0029] In the present invention, the Callicarpa nudiflora extract preferably includes Callicarpa nudiflora extract. The Callicarpa nudiflora extract is purchased from Hainan Jiuzhitang Pharmaceutical Co., Ltd. with batch number G01210508. In an embodiment of the present invention, the Callicarpa nudiflora extract is detected by high performance liquid chromatography to contain the following compounds: The Callicarpa nudiflora extract contains the following compounds: 0.94 mg / g caffeic acid, 8.04 mg / g forsythiaside B, 0.14 mg / g luteolin, 91.91 mg / g verbascoside, 51.03 mg / g isoverbascoside, 0.6 mg / g apigenin glycoside and 5.17 mg / g luteolin.

[0030] In the present invention, in the weaned pig breeding, the Callicarpa nudiflora extract is preferably fed to weaned pigs in the form of feed. The feed preferably also includes a basal diet. The added mass of the Callicarpa nudiflora extract is preferably not higher than 350 mg / kg basal diet, more preferably 100-300 mg / kg basal diet, and further preferably 150-200 mg / kg basal diet. The feeding amount of the feed is preferably 380-400 g / day / pig.

[0031] The present invention does not impose any special restrictions on the type of the basic diet, and any basic diet known in the art can be used. In an embodiment of the present invention, the formula of the basic diet is shown in Table 1:

[0032] Table 1 Raw materials and nutritional components of the basic diet

[0033]

[0034]

[0035] Each kilogram of premix includes the following components: vitamin A 4800 IU, vitamin D 3480 IU, vitamin E 40 IU, vitamin K3 1.5 mg, thiamine 3 mg, riboflavin 8 mg, pyridoxine 3.5 mg, vitamin B12 0.04 mg, pantothenic acid 25 mg, niacin 35 mg, biotin 0.15 mg, and folic acid 1 mg.

[0036] In the present invention, the weaned piglet breeding preferably includes promoting the growth of weaned piglets and / or maintaining the intestinal health of weaned piglets. The promotion of the growth of weaned piglets preferably includes improving at least one of the following indicators: the weight, average daily weight gain and average daily feed intake of weaned piglets. The weaned piglets preferably refer to piglets aged 30 to 35 days for weaning. In one embodiment of the present invention, 150 mg / kg (T1), 300 mg / kg (T2), and 600 mg / kg (T3) of Callicarpa nudiflora extract were added to the basal diet to feed weaned piglets. The results showed that compared with the piglets in the control group (fed only with the basal diet), the piglets fed with Callicarpa nudiflora extract had significantly improved weight, average daily weight gain, average daily feed intake, etc., and the feed-to-weight ratio was significantly reduced.

[0037] In the present invention, the maintenance of intestinal health of weaned piglets preferably includes improving the paracellular pathway of the intestinal tract of weaned piglets and / or enhancing the intestinal barrier function of weaned piglets. The improvement of the paracellular pathway of the intestinal tract of weaned piglets is manifested in the maintenance of normal concentrations of biomarker content of paracellular permeability of the intestinal tract. D-lactic acid and endotoxin concentrations in serum are considered to be biomarkers for assessing paracellular permeability of the intestinal tract of weaned piglets (Celi, P., Verlhac, V., Pérez Calvo, E., Schmeisser, J. & Kluenter, A.-M. 2019. Biomarkers of gastrointestinal functionality in animal nutrition and health. Anim Feed Sci Technol, 250, 9-31). The increase of plasma endotoxin and D-lactic acid activity is always accompanied by damage to intestinal epithelial cells (Wang, J., Zeng, L., Tan, B., Li, G., Huang, B., Xiong, X., Li, F., Kong, X., Liu, G. & Yin, Y. 2016. Developmental changes in intercellular junctions and Kv channels in the intestine of piglets during the suckling and post-weaning periods. Journal of Animal Science and Biotechnology, 7, 4). The results of the embodiment show that there is no difference in the concentration of endotoxin and D-lactic acid in serum between the groups, which indicates that the addition of the extract of Callicarpa nudiflora does not cause damage to the intestinal barrier function of weaned piglets.

[0038] In the present invention, the enhancement of the intestinal barrier function of weaned piglets preferably includes increasing the expression of tight junction proteins in the ileal mucosa of piglets. The tight junction proteins in the ileal mucosa of piglets include claudin-2, ZO-1, and ZO-3. Tight junctions are located at the top of the cell side and are strip-like network structures. They are mainly composed of a variety of molecules such as occludin, claudins, junctional adhesion molecules (JAM), zonula occludens (ZO) and cytoskeletal filamentous actin (F-actin). A highly dynamic complex (see Figure 1 ). The function of tight junctions (TJ) is to connect adjacent intestinal epithelial cells and form a complete intestinal epithelial physical barrier to regulate the transport of substances through the cell paracellular pathway and maintain the polarity of the cell surface. TJ can not only close the intercellular gap and form a physical barrier to resist the invasion of foreign pathogens, but also has permeability. It regulates the entry and exit of water molecules, ions, solutes and other substances through the cell paracellular pathway, acts as a molecular "fence" to maintain the polarity of epithelial cells, and completely separates the top of the cell from the basolateral membrane to maintain the polarity of epithelial cells. Therefore, the integrity of the TJ structure has become an important indicator of intestinal health. Its abnormal expression and dysfunction are closely related to chicken intestinal diseases caused by bacteria, viruses, parasites or mycotoxins (Leng Laifu, Huang Jinxiu, Chen Zhihua, et al. Research progress on the regulation of tight junctions in the animal intestine by MAPK signaling pathway [J / OL]. China Animal Husbandry Journal, 1-13 [2024-08-08]..20231204-03). In the examples of the present invention, qPCR technology was used to detect the expression of tight junction-related proteins. The results showed that the mRNA expression of ileal mucosal tight junction proteins claudin-2, ZO-1, and ZO-3 in the T2 treatment group was increased, indicating that the addition of 300 mg / kg of Callicarpa nudiflora extract can effectively improve the tight junction barrier of the ileal mucosa of Danzhou chickens, enhance the intestinal barrier function, and enhance the ability of the ileal mucosa to resist bacterial and viral invasion.

[0039] The invention provides a weaned piglet feed, comprising a Callicarpa nudiflora extract and a basic daily diet;

[0040] The mass percentage of the Callicarpa nudiflora extract in the basic diet is not less than 140 mg / kg;

[0041] The Callicarpa nudiflora extract contains the following compounds: 0.7-1.0 mg / g caffeic acid, 5-11 mg / g forsythiaside B, 4-7 mg / g luteolin, 70-120 mg / g verbascoside, 40-60 mg / g isoverbascoside, 0.4-0.8 mg / g apigenin glycoside and 0.1-0.3 mg / g luteolin.

[0042] In the present invention, the feeding amount of the feed is preferably 380-400 g / day / pig. The feed has the characteristics of promoting rapid growth of weaned piglets, and can also maintain the intestinal health of weaned piglets by improving the intestinal cell paracellular pathway and strengthening the intestinal barrier.

[0043] The application of the Callicarpa nudiflora extract provided by the present invention in weaned pig breeding is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] A method for identifying active ingredients of Callicarpa nudiflora extract

[0046] 1.1 Instruments and Equipment

[0047] Agilent 1260 high performance liquid chromatograph (including G1311C quaternary pump and G1315D diode array detector, Agilent, USA).

[0048] 1.2 Materials and reagents

[0049] Callicarpa nudiflora extract (batch number G01210508, Hainan Jiuzhitang Pharmaceutical Co., Ltd.), reference substances verbascoside (batch number 111530-201914), luteolin (batch number 111720-201810), luteolin (batch number YO1D6S6815), caffeic acid (batch number: Y17D6C7672), forsythiaside B (batch number 111811-201603), isovalascoside (batch number HA0820KA29), and apigenin glycoside (batch number P20N6F6297) were all purchased from Shanghai Yuanye Biopharmaceutical Co., Ltd. with a purity of more than 98%; acetonitrile was chromatographically pure (Tianjin Kangkede Technology Co., Ltd.), and the other reagents were of analytical grade.

[0050] 2 Methods

[0051] 2.1 Chromatographic conditions

[0052] Agilent C18 chromatographic column (250 mm × 4.6 mm, 5 μm); column temperature 30 °C; mobile phase acetonitrile (A)-0.4% phosphoric acid (B), gradient elution 0-28 min, 7%-15% A; 28-43 min, 15%-20% A; 43-53 min, 20%-25% A; 53-63 min, 25%-34% A; 63-70 min, 34%-100% A; volume flow rate 1.0 mL min -1 ; Injection volume 10μL; Detection wavelength 330nm.

[0053] 2.2 Preparation of reference solution

[0054] Accurately weigh appropriate amounts of luteolin, caffeic acid, verbascoside, luteolin, forsythiaside B, isovalascoside, and apigenin glycoside reference substances, and add appropriate amounts of 70% chromatographic methanol to dissolve them to prepare a 0.00197 mg mL luteolin solution. -1 、Caffeic acid 0.00165mg·mL -1 , Verbascoside 2.02 mg·mL -1 , luteolin 0.060mg·mL -1 , Forsythiaside B 0.225mg·mL -1 , Isovale glycoside 0.249 mg·mL -1 and apigenin glycoside 0.157 mg·mL -1 of the reference substance stock solution.

[0055] 2.3 Preparation of test solution

[0056] Accurately weigh 0.20 g of Callicarpa oleifera extract powder, add 70% methanol to make up to 50 mL, filter after fully dissolved, and take the filtrate.

[0057] According to the fingerprint analysis results, the fingerprints of the common patterns of the test solutions of multiple batches of Callicarpa nudiflora dry leaves and extracts, the fingerprints of the mixed reference substances and the blank solvents were combined and compared ( Figure 2 The chromatographic peaks of seven components, including caffeic acid (peak 1), forsythiaside B (peak 2), luteolin (peak 3), verbascoside (peak 4), isoverbascoside (peak 5), apigenin glycoside (peak 6) and luteolin (peak 7), are common in the fingerprints of the dry leaves and extract of Callicarpa nudiflora.

[0058] Example 2

[0059] A method for preparing weaned piglet feed

[0060] Prepare the basic diet according to the formula in Table 1 above, add 150 mg / kg, 300 mg / kg, and 600 mg / kg of the Callicarpa nudiflora extract (Callica nudiflora extract) in Example 1 to the basic diet, respectively, mix well, and prepare weaned piglet feed with different additive contents.

[0061] Example 3

[0062] Experiment on feeding weaned piglets with feed containing Callicarpa nudiflora extract

[0063] Hainan black pigs were selected as the experimental materials. A total of 32 piglets weaned at 35 days of age with a body weight of approximately 3.79±0.51kg were randomly assigned to 4 groups (8 replicates in each group). The control group was supplemented with a basal diet, and the experimental groups were supplemented with 150mg / kg (T1), 300mg / kg (T2), and 600mg / kg (T3) of Callicarpa nudiflora extract in the basal diet. The two groups were fed 400g / day, and were fed an average of 3 times a day. The piglets were weighed at the beginning and end of the experiment, and the daily feed intake of each replicate was recorded. The average daily feed intake (ADFI), average daily weight gain (ADG), and the ratio of feed intake to body weight gain (F:G) were calculated. At the same time, visceral organs, blood, and tissue samples were taken from each replicate. The heart, liver, spleen, lungs, and kidneys were removed and weighed after fat removal. The organ index was calculated according to formula I.

[0064] Organ index = organ weight (g) / piglet live weight (g) × 100% Formula I.

[0065] 15 mL of venous blood was collected and centrifuged at 3000 × g for 10 min at 4 °C to prepare serum. The separated serum was frozen at -80 °C for analysis. The intestine was segmented to obtain the ileum for subsequent analysis. Serum parameters included alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, urea nitrogen, and creatinine. The blood samples were analyzed biochemically using an automatic biochemical analyzer (Chemray 240). Serum D-lactate and endotoxin were also measured using ELISA kits purchased from Shanghai Yuanju Biotechnology Center.

[0066] Total RNA was isolated from piglet ileum tissue using TRIzol (Sigma, Saint Louis, MO, USA) according to the RNA extraction method. 2% agarose gel electrophoresis was performed to assess the quality of RNA. The total RNA concentration and purity (A) were evaluated using an ultra-micro spectrophotometer (NanoPhotometer, Implen, Germany). 260 / A 280 ratio). Use Prime Script with gDNA Eraser TMTotal RNA from each ileum tissue was reverse transcribed using a RT kit (TaKaRa, Dalian, China). cDNA was amplified using a 2× RealStar Fast SYBR qPCR kit (GenStar, Beijing, China). The primer sequences are shown in Table 2, and the primer sequences for porcine genes were synthesized by Servicebio (Wuhan, China). Samples were processed using an ABI QuantStudio 6Flex thermal cycler (Applied Biosystems, Foster City, CA, USA), followed by one cycle at 50°C for 2 min, one cycle at 95°C for 10 min, one cycle at 95°C for 15 s, and one cycle at 60°C for 60 s, for a total of 40 cycles, one cycle at 95°C for 15 s, one cycle at 60°C for 60 s, and one cycle at 95°C for 15 s. The expression level of β-actin was used as the internal reference for normalization based on 2 -ΔΔCt Methods, calculation of relative gene mRNA expression,

[0067] Table 2 Primer information

[0068]

[0069]

[0070] Data statistical analysis: The data were first processed by Excel. SPSS16.0 was then used for one-way ANOVA analysis, and Duncan multiple comparisons were performed for statistical results with significant differences. When P < 0.05, it was considered that there was a significant difference between the groups, and when P > 0.05, it indicated that there was no significant difference between the groups.

[0071] The results are expressed as mean ± SD.

[0072] result

[0073] 1. The growth performance results are shown in Table 3.

[0074] Table 3 Effects of Callicarpa nudiflora extract on growth performance of weaned piglets

[0075] Indicator / Group CK T1 T2 T3 P-value Initial body weight (kg) 3.79±0.58 3.79±0.56 3.79±0.55 3.79±0.57 1.000 Ending weight (kg) 9.15±0.30 9.54±1.40 10.34±1.26 10.45±1.26 0.350 Average daily weight gain (g / d) 194.42±13.57 205.36±37.80 235.05±29.04 237.78±43.33 0.216 Average daily feed intake (g / d) 366.15±20.37 373.44±13.65 375.00±9.47 382.29±2.69 0.424 Material weight ratio 1.88±0.03 1.87±0.33 1.62±0.25 1.66±0.35 0.409

[0076] As shown in Table 3, there were no significant differences in the growth performance indicators of final body weight, average daily weight gain, average daily feed intake, and feed-to-weight ratio among the treatment groups (P>0.05). However, the final body weight, average daily weight gain, and average daily feed intake of the three treatment groups with Callicarpa nudiflora added increased compared with the control group.

[0077] 2. Results of the safety assessment of Callicarpa nudiflora extract for weaned piglets

[0078] The organ index results are shown in Table 4.

[0079] Table 4 Effects of Callicarpa nudiflora extract on organ indexes of weaned piglets

[0080] Indicator / Group CK T1 T2 T3 P-value Heart (g / kg) 4.53±0.57 4.24±0.73 4.02±0.58 5.02±2.16 0.478 Liver (g / kg) 29.37±2.61 28.46±3.05 30.89±4.65 29.67±3.59 0.657 Spleen (g / kg) 2.07±0.42 2.59±0.18 2.49±0.53 2.25±0.24 0.079 Lung (g / kg) 11.11±0.96 11.49±4.21 12.59±4.17 15.46±4.99 0.234 Kidney (g / kg) 7.16±0.80 6.31±1.06 6.49±0.79 7.25±1.05 0.209

[0081] As shown in Table 4, there were no significant differences in the organ indexes of liver, kidney and spleen among the treatment groups (P>0.05), indicating that the addition of Callicarpa nudiflora extract to the diet had no adverse effects on the heart, liver, spleen, lung and kidney of weaned piglets.

[0082] The results of serum biochemical indexes are shown in Table 5.

[0083] Table 5 Serum biochemical index results

[0084]

[0085] Alanine aminotransferase, aspartate aminotransferase, and albumin can indicate liver damage, and urea nitrogen and creatinine can indicate kidney damage. The test results in Table 5 show that these indicators have no significant differences among the groups, indicating that the addition of different concentrations of Callicarpa nudiflora extract did not cause liver and kidney damage in piglets, indicating that it can be safely used in piglet feed.

[0086] 3. Improve the results of intestinal paracellular pathways in weaned piglets

[0087] Table 6 Results of serum intestinal paracellular permeability index

[0088] Indicator / Group CK T1 T2 T3 P-value Endotoxin (ng / l) 87.69±3.88 89.93±11.75 88.17±11.17 85.10±4.83 0.901 D-Lactic acid (ng / ml) 0.56±0.19 0.64±0.17 0.54±0.10 0.61±0.03 0.741

[0089] The results in Table 6 show that there was no significant difference in the concentrations of endotoxin and D-lactic acid in serum among the groups (P>0.05), which means that the addition of Callicarpa nudiflora extract did not damage the intestinal barrier function of weaned piglets.

[0090] 4. Enhanced intestinal barrier function

[0091] The results of the effect of Callicarpa nudiflora extract on the gene expression of tight junction proteins in the ileal mucosa of piglets are shown in Table 7.

[0092] Table 7 Effect of Callicarpa nudiflora extract on tight junction protein gene expression in piglet ileum mucosa

[0093] Protein Type CK T1 T2 T3 P-value claudin-1 0.88±0.18 1.78±0.36 1.15±0.10 1.01±0.20 0.056 Claudin-2 <![CDATA[1.02±0.09 b ]]> 0.91±0.16b 0.97±0.21b 0.34±0.06a 0.007 occludin 1.30±0.35 1.47±0.29 1.08±0.18 0.86±0.21 0.451 ZO-1 <![CDATA[1.29±0.41 a ]]> 2.41±0.29b 2.72±0.35b 2.34±0.37ab 0.047 ZO-2 0.87±0.17 0.88±0.19 1.02±0.16 0.72±0.15 0.722 ZO-3 <![CDATA[0.85±0.12 ab ]]> 0.71±0.15a 1.34±0.30b 0.58±0.12a 0.050 JAMA 0.95±0.24 1.01±0.17 0.83±0.14 0.62±0.13 0.426 JAM2 0.91±0.20 1.24±0.12 1.47±0.19 0.81±0.19 0.058

[0094] The results in Table 7 showed that the mRNA expressions of ileal mucosal tight junction proteins claudin-2, ZO-1, and ZO-3 were increased in the T2 treatment group, indicating that the addition of 300 mg / kg of Callicarpa nudiflora extract can effectively improve the tight junction barrier of the ileal mucosa of piglets, enhance the intestinal barrier function, and enhance the ability of the ileal mucosa to resist bacterial and viral invasion.

[0095] Example 4

[0096] The contents of the cecum and ileum of piglets in different groups in Example 3 were collected and packed into two 5 ml sterile enzyme-free sputum cups, quickly frozen in liquid nitrogen and stored at -80 ° C, and used for 16S rDNA high-throughput sequencing. DNA extraction and quality inspection, PCR amplification, product purification, 16S rDNA sequencing and bioinformatics analysis were all entrusted to Suzhou Panomik Biomedical Technology Co., Ltd.

[0097] DNA extraction and PCR amplification: Microbial DNA was extracted from cecal fecal samples (n = 9 per group) using the OMEGA DNA Kit (M5635-02) (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer's instructions. Total DNA was eluted in 50 μL elution buffer and stored at -80°C before PCR detection. The extracted DNA was sized by 0.8% agarose gel electrophoresis and quantified by a UV spectrophotometer NanoDrop NC2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). PCR amplification used primers specific for the bacterial 16SrDNA V3-V4 region, 338F (5'-barcode + ACTCCTACGGGAGGCAGCA-3', SEQ ID NO: 17), 806R (5'-GGACTACHVGGGTWTCTAAT-3', SEQ ID NO: 18).

[0098] Quant-iT PicoGreen dsDNA Assay Kit was used to quantify PCR products on a Microplate reader (BioTek, FLx800). The library was quality checked using the Agilent HighSensitivity DNA Kit on an Agilent Bioanalyzer machine, and quantified using the Quant-iT PicoGreen dsDNA Assay Kit on a Promega QuantiFluor. Qualified libraries were sequenced on a NovaSeq machine using the NovaSeq 6000SP Reagent Kit platform. QIIME2 2019.4 was used to perform bioinformatics analysis on the microbiome, the Greengenes database was used to align ASV feature sequences, and QIIME2 software and R software were used to analyze the Alpha diversity and Beta diversity of microorganisms. Alpha diversity includes Chao1, Observed species, Goods coverage, Shannon, Simpson, and Pielou indices. Beta diversity includes principal coordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS).

[0099] result

[0100] 1. Alpha diversity of colonic microbes

[0101] Results Figure 3 There was no significant difference in the alpha diversity of colonic microorganisms in piglets among the treatment groups (P>0.05), indicating that the species richness and uniformity of samples in each treatment group were relatively consistent.

[0102] 2. Colonic microbial beta diversity

[0103] Results Figure 4 There was no significant difference in the Beta diversity of colonic microorganisms in piglets among the treatment groups (P>0.05).

[0104] 3. Colonic microbial flora composition

[0105] Results Figure 5. At the phylum level, there are 19 major enriched bacterial communities in the colon of piglets, and the top five are Firmicutes, Bacteroidota, Actinobacteriota, Tenericutes, and Proteobacteria. At the genus level, the top five are Lactobacillus, Prevotella, Ruminococcus, Parabacteroides, and Coprococcus.

[0106] 4. Results of bacterial detection with significant differences in colonic microbiota

[0107] The abundance data of the top 50 genera in terms of average abundance were used to draw a heat map. Figure 6 , where the red block represents that the abundance of the genus in this sample is higher than that in other samples, and the blue block represents that the abundance of the genus in this sample is lower than that in other samples.

[0108] Depend on Figure 6 It can be seen that the relative abundance of Butyricicoccus, Treponema, Roseburia, Paludibacter, Sporosarcina, Prevotella, Ruminococcus, Dorea, and Slackia was higher in the CK group; the relative abundance of L7A_E11, Sphaerochaeta, Parabacteroides, Lachnospira, rc4-4, Gemmiger, Ruminococcus, Acinetobacter, Turicibacter, and Blautia was higher in the T1 group; the relative abundance of Allobaculum, Phasc The relative abundances of olarctobacterium, Anaerostipes, SMB53, Streptococcus, Clostridium, Enterococcus, Bacteroides, Corynebacterium, Adlercreutzia, Oscillospira, Weissella, Collinsella, and Mycoplasma were relatively high; in the T3 group, the relative abundances of Bulleidia, [Eubacterium], CF231, Dehalobacterium, Lactobacillus, and Shuttleworthia were relatively high.

[0109] 5. Ileal Microbial Alpha Diversity

[0110] Results Figure 7 The alpha diversity of piglet ileum microorganisms was not significantly different among the treatment groups (P>0.05), indicating that the species richness and uniformity of samples were relatively consistent among the treatment groups.

[0111] 6. Ileal microbial Beta diversity

[0112] Results Figure 8 The beta diversity of ileal microorganisms in piglets was not significantly different among the treatment groups (P>0.05).

[0113] 7. Ileal microbial flora composition

[0114] Results Fig. 9 . At the phylum level, there are 20 major enriched bacterial communities in the ileum of piglets, and the top five are Firmicutes, Actinobacteriota, Proteobacteria, Bacteroidota, Actinobacteriota, and Chloroflexi. At the genus level, the top five bacterial communities in the ileum of piglets are Lactobacillus, Streptococcus, Corynebacterium, Actinobacillus, and Arthrobacter.

[0115] 8. Results of significantly different bacterial tests in the ileum

[0116] The abundance data of the top 50 genera in terms of average abundance were used to draw a heat map. Fig.10 , the red block represents that the abundance of the genus in this sample is higher than that in other samples, and the blue block represents that the abundance of the genus in this sample is lower than that in other samples.

[0117] Depend on Fig.10It can be seen that the relative abundance of Dietzia, Escherichia, Lactobacillus, Eubacterium, Collinsella, Gemmiger, Blautia, Coprococcus, and Prevotell was higher in the CK group; the relative abundance of Clostridium, Rothia, Klebsiella, and Lactococcus was higher in the T1 group; the relative abundance of Enterococcus, Piscicoccus, Corynebacterium, Actinomyces, Staphylococcus, Micrococcus, Rhodococcus, Turicibacter, Brachybacterium, Rhodobacterium, Parabacteroides, Pasteurella, aracoccus, Mycoplasma, and Sphingobacterium was higher in the T2 group; the relative abundance of Actinobacillus, SMB53, Prevotella, Bifidobacterium, AF12, Mucispirillum, Oscillospira, Arthrobacter, and Candidatus was higher in the T3 group. Koribacter, Allobaculum, and Rhodoplanes were relatively abundant.

[0118] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of a Callicarpa nudiflora extract in preparing feed for weaned piglets, the Callicarpa nudiflora extract containing the following compounds: 0.7-1.0 mg / g caffeic acid, 5-11 mg / g forsythiaside B, 4-7 mg / g luteolin, 70-120 mg / g verbascoside, 40-60 mg / g isovalascoside, 0.4-0.8 mg / g apigenin glycoside and 0.1-0.3 mg / g luteolin.

2. The use according to claim 1, characterized in that: The weaned piglet breeding includes promoting the growth of weaned piglets and / or maintaining the intestinal health of weaned piglets.

3. The application according to claim 2, characterized in that: The promoting the growth of weaned piglets comprises increasing at least one of the following: the body weight, average daily weight gain and average daily feed intake of the weaned piglets.

4. The application according to claim 2, characterized in that: The method of maintaining the intestinal health of weaned piglets comprises improving the intestinal paracellular pathway of weaned piglets and / or enhancing the intestinal barrier function of weaned piglets.

5. The use according to claim 1, characterized in that: In the weaned piglet breeding, the Callicarpa nudiflora extract is fed to the weaned piglets in the form of feed.

6. The use according to claim 1 or 5, characterized in that: The feed also includes a basal diet; The added mass of the Callicarpa nudiflora extract is not less than 140 mg / kg of the basic diet.

7. The use according to claim 6, characterized in that: The added mass of the Callicarpa nudiflora extract is 150-600 mg / kg of the basic diet.

8. The use according to claim 6, characterized in that: The added mass of the Callicarpa nudiflora extract is 150-300 mg / kg of the basic diet.

9. The use according to claim 6, characterized in that: The feeding amount of the feed is 380-400 g / day / each.

10. A weaned piglet feed, characterized in that: Includes Callicarpa nudiflora extract and basal diet; The mass percentage of the Callicarpa nudiflora extract in the basic diet is not less than 140 mg / kg; The Callicarpa nudiflora extract contains the following compounds: 0.7-1.0 mg / g caffeic acid, 5-11 mg / g forsythiaside B, 4-7 mg / g luteolin, 70-120 mg / g verbascoside, 40-60 mg / g isoverbascoside, 0.4-0.8 mg / g apigenin glycoside and 0.1-0.3 mg / g luteolin.

Images