Application of dogwood extract in preparation of compound feed for relieving diarrhea and enteritis

Abstract

The invention belongs to the technical field of livestock and poultry feed additives, and particularly relates to application of a dogwood extract in preparation of a compound feed for relieving diarrhea and enteritis, and the dogwood extract is an aqueous extract of a dogwood medicinal material. 500mg-2000mg of the dogwood extract is added into each kilogram of the basal feed, so that the compound feed is obtained. The compound feed can relieve diarrhea and intestinal inflammation of weaned piglets, especially at the 21-35-day-old stage, the diarrhea occurrence rate of the weaned piglets can be remarkably reduced by 15% or above, meanwhile, the average daily gain is increased, the feed-gain ratio is reduced, the comprehensive growth performance is better than that of a control group, the level of pro-inflammatory cytokines in IPEC-J2 cells or intestinal mucosa tissues is remarkably reduced, and the weaned piglets are prevented from suffering from diarrhea and intestinal inflammation. Meanwhile, the level of anti-inflammatory cytokines is up-regulated, and excessive activation of an NF-kB key inflammatory signal channel can be inhibited.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of livestock feed additives, and particularly relates to application of an extract of dogwood in preparation of compound feed for relieving diarrhea and enteritis. BACKGROUND

[0002] The weaning stage of piglets is a key window period of pig breeding. Piglets are extremely vulnerable to invasion of external bacteria due to unstable intestinal flora, immature intestinal mucosal barrier and weaning stress response, resulting in diarrhea and intestinal inflammation, and further seriously affecting the economic benefits of the pig industry.

[0003] In the prior art, although the addition of antibiotic additives in feed can inhibit the reproduction of pathogenic bacteria in a short term, long-term use will lead to excessive drug residues in livestock products, threatening the health of consumers.

[0004] Further, the substitutes for antibiotics mainly include antibacterial peptides, microecological preparations and Chinese herbal medicine plant extracts. Among them, Chinese herbal medicine has the unique advantages of large planting amount, antibacterial, anti-inflammatory, no pollution, no public harm and no residue, and is an ideal candidate type of additive for ensuring the safety of feed and animal food and human health.

[0005] The prior art uses a composition containing dogwood as an additive to improve the survival rate and promote growth, ensuring the healthy and rapid growth of piglets and good economic benefits.

[0006] However, the composition containing dogwood also contains other traditional Chinese medicine additives, resulting in complex additive components and inability to relieve diarrhea and enteritis. SUMMARY

[0007] The application aims to provide application of an extract of dogwood in preparation of compound feed for relieving diarrhea and enteritis, so as to solve the problem that the homoeopathic plant of traditional Chinese medicine in the prior art cannot relieve diarrhea and enteritis of piglets when piglets are fed with the homoeopathic plant as a feed additive.

[0008] To achieve the above-mentioned purpose, the application adopts the following technical solutions. Application of an extract of dogwood in preparation of compound feed for relieving diarrhea and intestinal inflammation of weaned piglets, wherein the extract of dogwood is a water extract of dogwood medicinal materials.

[0009] Further, 500 mg to 2000 mg of the extract of dogwood is added to each kilogram of the compound feed.

[0010] Further, each gram of the extract of dogwood is prepared from 100 g of dogwood medicinal materials by water extraction.

[0011] Further, the base feed is prepared from the raw materials according to the following mass percentage: 27.37%~46.65% corn, 15.00%~33.00% puffed corn, 12.80%~20.30% soybean meal, 2.00%~10.00% fermented soybean meal, 1.50%~2.00% soybean oil, 1.00%~8.00% bran, 0.50%~3.50% fish meal, 2.00%~6.00% whey powder, 0.55%~0.80% stone powder, 1.15%~1.60% calcium hydrogen phosphate, 0.30% salt, 0.45%~0.50% L-lysine hydrochloride, 0.10% DL-methionine, 0.20% L-threonine, 0.05%~0.08% L-tryptophan, and 1% premix, totaling 100%.

[0012] Further, the formula of the premix added per kilogram of feed is: 10000 IU vitamin A, 2750 IU vitamin D3, 80 IU vitamin E, 2 mg vitamin K3, 0.3 mg vitamin B12, 12 mg riboflavin, 2.25 mg vitamin B6, 40 mg niacin, 25 mg d-pantothenic acid, 0.25 mg biotin, 1.6 mg folic acid, 3.0 mg thiamine, 150 mg iron element, 105 mg zinc element, 30 mg manganese element, 25 mg copper element, 0.5 mg iodine element, and 0.3 mg selenium element.

[0013] Further, the iron element is from ferrous sulfate monohydrate, the zinc element is from zinc sulfate monohydrate, the manganese element is from manganese sulfate monohydrate, the copper element is from copper sulfate pentahydrate, the iodine element is from calcium iodate, and the selenium element is from sodium selenite.

[0014] Further, the preparation method of the compound feed comprises the following steps: mixing the cornus officinalis extract and the base feed to obtain a compound feed for relieving diarrhea and enteritis of weaned piglets.

[0015] Further, the weight of the piglets fed with the compound feed is 5.11kg~5.89kg, and each piglet consumes 317.53g~373.65g of feed per day on average.

[0016] The principle of the present application is that: As a traditional medicine and food homologous plant, the active ingredients of the extract of cornus officinalis contain iridoid glycosides, polysaccharides and triterpenic acid, the present application specifically applies the cornus officinalis extract to weaned piglets, systematically evaluates the prevention and control effect of the cornus officinalis extract on weaning stress diarrhea and intestinal inflammation, and develops the cornus officinalis extract into a standardized additive product suitable for the feed industry.

[0017] Compared with the prior art, the present application has the following beneficial effects: (1) The application of Cornus officinalis extract in the preparation of compound feed to relieve diarrhea and enteritis. The compound feed containing Cornus officinalis extract can relieve diarrhea and intestinal inflammation in weaned piglets, especially in the 21-35 day age stage. It can significantly reduce the incidence of diarrhea in weaned piglets by more than 15%. At the same time, it can increase the average daily weight gain, reduce the feed conversion ratio, and the overall growth performance is better than the control group. It can significantly downregulate the level of pro-inflammatory cytokines in IPEC-J2 cells or intestinal mucosal tissue, while upregulating the level of anti-inflammatory cytokines, and can inhibit the excessive activation of the NF-κB key inflammatory signaling pathway.

[0018] (2) This invention provides a natural, safe, and residue-free antibiotic-free feed additive: overcoming the problems of drug resistance and drug residues caused by antibiotic additives, utilizing the natural anti-inflammatory and immunomodulatory activities of Cornus officinalis extract to prepare a feed additive free of antibiotics and hormones, ensuring the safety and compliance of piglet breeding. Cornus officinalis extract is of pure plant origin, is completely metabolized in animals, and poses no risk of harmful residues. Its use can completely avoid the problems of drug resistance and drug residues caused by antibiotics, conforming to the development trend of green and ecological farming.

[0019] (3) This invention opens up a new application field for Cornus officinalis extract: explore the application value of Cornus officinalis extract in the field of healthy livestock and poultry breeding, develop it into a feed additive to alleviate diarrhea and intestinal inflammation in weaned piglets, enrich the categories of antibiotic-free feed additives, and provide a new, efficient and green prevention and control solution for the breeding industry.

[0020] (4) This invention clarifies the mechanism of action of Cornus officinalis extract in the complex intestinal environment of weaned piglets, as well as the appropriate form and dosage of its addition. Attached Figure Description

[0021] Figure 1 The effects of Fce and LPS on the viability of IPEC-J2 cells.

[0022] Figure 2 This demonstrates the protective effect of Fce against LPS-damaged IPEC-J2 cells.

[0023] Figure 3 The effect of Fce on the content of inflammation-related cytokines in the supernatant of LPS-damaged IPEC-J2 cell culture medium.

[0024] Figure 4 The effect of Fce on the expression of inflammation-related genes in an LPS-induced IPEC-J2 cell injury model.

[0025] Figure 5 To investigate the effects of Fce on genes related to intestinal mucosal inflammation in weaned piglets.

[0026] Figure 6The effect of Fce on the expression of inflammation-related proteins in an LPS-induced IPEC-J2 cell injury model was investigated. In the figure, A represents Western blot analysis, B represents the quantitative results of IL-1β, C represents the quantitative results of TNF-α, and D represents the quantitative results of NF-κB p65. Significant differences are indicated between the different letters. P< 0.05 indicates that there is no significant difference between letters with the same meaning. P> 0.05.

[0027] Figure 7 In the table, A represents the quantitative results of Fce on P-NF-kB P65 in the LPS-induced IPEC-J2 cell damage model, and B represents the quantitative results of Fce on P-P65 / P65 in the LPS-induced IPEC-J2 cell damage model.

[0028] Figure 8 The effect of Fce on inflammation-related proteins in the jejunal mucosa of weaned piglets; where A represents Western blot analysis, B represents the quantitative results of IL-1β, C represents the quantitative results of TNF-α, and D represents the quantitative results of NF-κB P65. Significant differences are indicated between different letters. P< 0.05 indicates that there is no significant difference between letters with the same meaning. P> 0.05.

[0029] Figure 9 In the table, A represents the quantitative results of Fce on P-NF-kB P65 in the mucosal tissue of weaned jejunum, and B represents the quantitative results of Fce on P-P65 / P65 in the intestinal mucosa of weaned piglets.

[0030] Figure 10 The effect of Fce on inflammation-related proteins in the ileal mucosa of weaned piglets; where A represents Western blot analysis, B represents the quantitative results of IL-1β, C represents the quantitative results of TNF-α, and D represents the quantitative results of NF-κB P65. Significant differences are indicated between different letters. P< 0.05 indicates that there is no significant difference between letters with the same meaning. P> 0.05.

[0031] Figure 11 In the table, A represents the quantitative results of Fce on P-NF-kB P65 in the ileal mucosa of weaned piglets, and B represents the quantitative results of Fce on P-P65 / P65 in the intestinal mucosa of weaned piglets. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, instruments, etc. used are all commercially available. The following contents need to be specifically explained:

[0033] Cornus officinalis extract was purchased from Xi'an Aotai Biotechnology Co., Ltd., product number: AT-22082301; Cornus officinalis extract is abbreviated as Fce; lipopolysaccharide is abbreviated as LPS.

[0034] The formula for the premixed feed added per kilogram of feed is: 10000 IU vitamin A, 2750 IU vitamin D3, 80 IU vitamin E, 2 mg vitamin K3, 0.3 mg vitamin B12, 12 mg riboflavin, 2.25 mg vitamin B6, 40 mg niacin, 25 mg d-pantothenic acid, 0.25 mg biotin, 1.6 mg folic acid, 3.0 mg thiamine, 460 mg ferrous sulfate monohydrate, 290 mg zinc sulfate, 95 mg manganese sulfate monohydrate, 100 mg anhydrous copper sulfate, 0.8 mg calcium iodate, and 0.7 mg sodium selenite.

[0035] The weaned Xiangcheng piglets came from Xinhong Livestock Breeding Professional Cooperative in Shenqiu County, Zhoukou City, Henan Province, and the Xiangcheng Pig Breeding Center.

[0036] Example 1: A feed to alleviate diarrhea and enteritis in weaned piglets, the specific preparation method is as follows: The basic feed is prepared by mixing 27.37% corn, 33.00% extruded corn, 12.80% soybean meal, 10.00% fermented soybean meal, 2.00% soybean oil, 1.00% wheat bran, 3.50% fishmeal, 6.00% whey powder, 0.55% limestone powder, 1.6% dicalcium phosphate, 0.30% salt, 0.5% L-lysine hydrochloride, 0.10% DL-methionine, 0.20% L-threonine, 0.08% L-tryptophan, and 1% premix.

[0037] Add 500mg of Cornus officinalis extract to each kilogram of basic feed and mix to obtain compound feed 1 that relieves diarrhea and enteritis in weaned piglets.

[0038] Example 2: A feed to alleviate diarrhea and enteritis in weaned piglets, the specific preparation method is as follows: The basic feed is prepared by mixing 46.65% corn, 15.00% extruded corn, 20.30% soybean meal, 2.00% fermented soybean meal, 1.50% soybean oil, 8.00% wheat bran, 0.50% fishmeal, 2.00% whey powder, 0.80% limestone powder, 1.15% dicalcium phosphate, 0.30% salt, 0.45% L-lysine hydrochloride, 0.10% DL-methionine, 0.20% L-threonine, 0.05% L-tryptophan, and 1% premix.

[0039] Add 500mg of Cornus officinalis extract to each kilogram of basic feed and mix to obtain compound feed 2 that relieves diarrhea and enteritis in weaned piglets.

[0040] Example 3: A feed to alleviate diarrhea and enteritis in weaned piglets, the specific preparation method of which is as follows: Based on Example 1, 1000 mg of Cornus officinalis extract was added to each kilogram of basic feed and mixed to obtain compound feed 3 for relieving diarrhea and enteritis in weaned piglets.

[0041] Example 4: A feed to alleviate diarrhea and enteritis in weaned piglets, the specific preparation method of which is as follows: Based on Example 2, 1000 mg of Cornus officinalis extract was added to each kilogram of basic feed and mixed to obtain compound feed 4 for relieving diarrhea and enteritis in weaned piglets.

[0042] Example 5: A feed to alleviate diarrhea and enteritis in weaned piglets, the specific preparation method of which is as follows: Based on Example 1, 1500 mg of Cornus officinalis extract was added to each kilogram of basic feed and mixed to obtain compound feed 5 that relieves diarrhea and enteritis in weaned piglets.

[0043] Example 6: A feed to alleviate diarrhea and enteritis in weaned piglets, the specific preparation method of which is as follows: Based on Example 2, 1500 mg of Cornus officinalis extract was added to each kilogram of basic feed and mixed to obtain compound feed 6 that relieves diarrhea and enteritis in weaned piglets.

[0044] Example 7 A feed to alleviate diarrhea and enteritis in weaned piglets, the specific preparation method is as follows: Based on Example 1, 2000 mg of Cornus officinalis extract was added to each kilogram of basic feed and mixed to obtain compound feed 7 that relieves diarrhea and enteritis in weaned piglets.

[0045] Example 8: A feed to alleviate diarrhea and enteritis in weaned piglets, the specific preparation method of which is as follows: Based on Example 2, 2000 mg of Cornus officinalis extract was added to each kilogram of basic feed and mixed to obtain compound feed 8 that relieves diarrhea and enteritis in weaned piglets.

[0046] Comparative Example 1: A piglet feed, the specific preparation method of which is as follows: Comparative feed 1 was prepared by mixing 27.37% corn, 33.00% extruded corn, 12.80% soybean meal, 10.00% fermented soybean meal, 2.00% soybean oil, 1.00% wheat bran, 3.50% fishmeal, 6.00% whey powder, 0.55% limestone powder, 1.6% dicalcium phosphate, 0.30% salt, 0.5% L-lysine hydrochloride, 0.10% DL-methionine, 0.20% L-threonine, 0.08% L-tryptophan, and 1% premix.

[0047] Comparative Example 2: A piglet feed, the specific preparation method of which is as follows: Comparative feed 2 was prepared by mixing 46.65% corn, 15.00% extruded corn, 20.30% soybean meal, 2.00% fermented soybean meal, 1.50% soybean oil, 8.00% wheat bran, 0.50% fishmeal, 2.00% whey powder, 0.80% limestone powder, 1.15% dicalcium phosphate, 0.30% salt, 0.45% L-lysine hydrochloride, 0.10% DL-methionine, 0.20% L-threonine, 0.05% L-tryptophan, and 1% premix. The nutritional levels of the basal feed and the control feed are shown in Table 1.

[0048] Table 1 Nutritional levels of feed To verify the feed performance of the examples, the following experiments were conducted: SPSS 26.0 software was used to analyze the experimental data of each group. One-way ANOVA was used; Duncan's multiple comparison test was selected to analyze the significance of differences between groups. Results are expressed as mean ± standard error, Mean ± SEM. P< 0.05 indicates a significant difference. Data visualization analysis was performed using GraphPad Prism 9.5.0.

[0049] 1. Animal experiment design: This experiment employed a single-factor experimental design, selecting 120 weaned Xiangcheng piglets at 28 days of age with a weight of 5.50 kg ± 0.39 kg. These piglets were randomly divided into 5 groups: one control group (Con) and four experimental groups (Fce1–Fce4), as shown in Table 2. Each group had 3 replicates, with 8 piglets per replicate (half male and half female). The control group was fed the control group's feed, while the experimental groups were fed the compound feed described in the example. The experiment lasted 28 days. Throughout the experiment, all pigs had free access to food and water, and daily management, disinfection, vaccination, and deworming procedures were performed according to the pig farm's standard production procedures.

[0050] Table 2 Grouping of Piglets for Feeding After a 12-hour fast, piglets were weighed on an empty stomach in the morning of day 1 and day 29 of the experiment. The initial and final weights of piglets in each group were accurately recorded to calculate the average daily gain (ADG). Feed consumption was recorded daily throughout the experiment to calculate the average daily feed intake (ADFI) and the feed conversion ratio (F / G). All piglets were observed daily at 8:00 AM to assess diarrhea incidence based on fecal characteristics, and the diarrhea rate for each group was statistically recorded.

[0051] The results are shown in Table 3. There was no significant difference in the initial weight of weaned piglets among the groups. P> 0.05). Compared with the Con group, the average feed intake and average daily weight gain of the Fce2 and Fce3 groups were significantly increased ( P< 0.05). The F / G ratio in the Fce3 and Fce4 groups, and the DR in the Fce2, Fce3, and Fce4 groups were all significantly lower than those in the control group ( P< 0.05). Among them, Fce3 group achieved the highest FBW, ADFI, and ADG ( P< 0.05), while F / G and DR reached their lowest values ​​( P< (0.05). This indicates that adding 1500 mg / kg Cornus officinalis extract to the diet of weaned piglets significantly increased the final weight, average daily feed intake, and average daily weight gain of weaned piglets, and significantly reduced the feed conversion ratio and diarrhea rate.

[0052] Table 3 Effects of Fce on growth performance of weaned piglets Note: IBW: Initial body weight; FBW: Final body weight; ADG: Average daily weight gain; ADFI: Average daily food intake; F / G: Food intake to weight gain ratio; DR: Diarrhea rate; Different letters indicate significant differences. P< 0.05 indicates that there is no significant difference between letters with the same meaning. P> 0.05.

[0053] After the experiment, based on the principle of similar average weight, two piglets were selected from each replicate of each group, for a total of 30 piglets, half male and half female, for slaughter. The duodenum, jejunum, and ileum of the experimental pigs were dissected and separated. After rinsing with physiological saline, the intestinal mucosa was scraped off using a microscope slide and placed in a 1.5 mL sterile enzyme-free EP tube. After being rapidly frozen in liquid nitrogen, it was stored in a -80°C freezer to obtain the piglet intestinal mucosa.

[0054] 2. Cell experiment design: The porcine intestinal epithelial cell line used in this experiment, IPEC-J2, was preserved in the laboratory of the Animal Nutrition and Meat Quality Regulation Innovation Team at Henan University of Science and Technology. IPEC-J2 cells were continuously passaged in culture dishes, cultured in DMEM medium supplemented with 10% FBS and 1% antibiotics, and incubated statically at 37°C in a 5% CO2 incubator. When the cells reached 80% confluence, they were digested with trypsin and seeded into six-well cell culture plates, approximately 3 × 10⁶ cells per well. 5 Cells. After incubation overnight, the cells were used for subsequent treatments, with each treatment repeated three times.

[0055] Experiment 1 Cell viability assay The effects of different concentrations of Cornus officinalis extract and LPS on the viability of IPEC-J2 cells were investigated using the CCK8 assay. 100 μL of each extract and LPS were added to each cell. 4 Cell suspensions of 100 cells / mL were seeded into 96-well plates and cultured overnight. Cells were then treated with different concentrations of Cornus officinalis extract (0 μg / mL, 300 μg / mL, 600 μg / mL, 900 μg / mL, 1200 μg / mL, 1500 μg / mL, 1800 μg / mL, 2100 μg / mL) and LPS (0 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL) for 24 h. The medium was then replaced with standard DMEM + 10% FBS + 1% antibiotics. Afterward, 10 μL of CCK8 solution was added to each well and the cells were incubated at 37°C for 2 h. The OD value of each well was measured at 450 nm using a microplate reader to calculate cell viability and determine the optimal concentrations of Cornus officinalis extract and LPS for their effects on cell viability.

[0056] Cell viability (%) = [(OD treatment group - OD blank group) / (OD control group - OD blank group)] × 100.

[0057] The results are as follows Figure 1 As shown in Figure A, after treating IPEC-J2 cells with different concentrations of Cornus officinalis extract for 24 hours, the cell viability of IPEC-J2 cells showed a trend of first increasing and then decreasing. Among them, the cell viability of the 900 μg / mL treatment group was significantly higher than that of the control group. P< 0.05), while the cell viability of the 1800 μg / mL and 2100 μg / mL treatment groups was significantly lower than that of the control group ( P< (0.05). Therefore, this experiment selected Cornus officinalis extract ranging from 0 μg / mL to 1500 μg / mL for subsequent tests.

[0058] like Figure 1 As shown in Figure B, after 24 hours of treatment with different concentrations of LPS, the cell viability of IPEC-J2 cells showed a decreasing trend. Compared with the control group, the cell viability of the 60 μg / mL and 80 μg / mL LPS treatment groups was significantly decreased. P< (0.05). Therefore, 60 μg / mL LPS was selected for subsequent experiments in this study.

[0059] The CCK8 assay described above was used to evaluate the protective effect of Cornus officinalis extract against LPS-induced damage to IPEC-J2 cells. After overnight culture in 96-well plates, cells were treated with different concentrations (0 μg / mL, 300 μg / mL, 600 μg / mL, 900 μg / mL, 1200 μg / mL, 1500 μg / mL, and 1800 μg / mL) of Cornus officinalis extract and 60 μg / mL LPS for 24 h. The medium was then replaced with normal culture medium. Subsequently, 10 μL of CCK8 solution was added to each well, and absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated to determine the optimal concentration of Cornus officinalis extract for protecting against LPS-induced cell damage.

[0060] The results are as follows Figure 2 As shown, when IPEC-J2 cells were treated with 60 μg / mL LPS alone, the cell viability decreased significantly. P< 0.05), while when cells were treated with 300 μg / mL to 1500 μg / mL of Cornus officinalis extract and LPS, the viability of IPEC-J2 cells showed an increasing trend, and the cell viability increased significantly when treated with 600 μg / mL to 1500 μg / mL of Cornus officinalis extract. P< (0.05). Therefore, in this experiment, extracts of Cornus officinalis at concentrations of 600 μg / mL to 1500 μg / mL were selected for subsequent tests.

[0061] IPEC-J2 cells were divided into a control group (Con), a model group (Mode), and four experimental groups (Fce1-Fce4). The control group was treated with ordinary culture medium for 24 h. The model group and the four experimental groups were treated with different concentrations (0 μg / mL, 600 μg / mL, 900 μg / mL, 1200 μg / mL, and 1500 μg / mL) of Cornus officinalis extract and 60 μg / mL LPS for 24 h. The supernatant of the culture medium was collected for the determination of inflammatory cytokines. After washing with PBS, the cells were collected for subsequent qPCR and Western blot analysis.

[0062] Experiment 2: Determination of Cytokines and Immunoglobulins ELISA kits, purchased from Jiangsu Enzyme Immunoassay Co., Ltd., Jiangsu, China, were used to determine the concentrations of TNF-α, IL-1β, IL-6, and IL-10 in the supernatant of IPEC-J2 cell culture medium in each group, according to their instructions. The concentrations of IgA, IgG, IgM, TNF-α, IL-1β, IL-6, and IL-10 in the intestinal mucosa of piglets were also measured.

[0063] The results are as follows Figure 3 As shown, compared with the Con group, the levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 in the supernatant of IPEC-J2 cell culture medium in the Mode group were significantly increased.P< 0.05), while the content of the anti-inflammatory factor IL-10 decreased significantly ( P< 0.05). Compared with the Mode group, the levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 in the supernatant of IPEC-J2 cell culture medium in the Fce2, Fce3, and Fce4 groups were significantly decreased. P< 0.05), while the content of the anti-inflammatory factor IL-10 increased significantly ( P< (0.05). This indicates that 900 μg / mL to 1500 μg / mL of Fce can reverse the changes in inflammatory factors in the supernatant of IPEC-J2 cell culture medium induced by LPS, thereby alleviating the inflammatory damage to IPEC-J2 cells caused by LPS.

[0064] As shown in Table 4, the TNF-α content in the duodenal mucosa of weaned piglets was significantly lower in each experimental group than in the control group. P< 0.05). Compared with the control group, the IgA content in the Fce1-3 group was significantly increased ( P< 0.05), the IgM content in the Fce2-4 group was significantly increased ( P< 0.05), while the IL-6 content in the Fce2 group was significantly reduced ( P< 0.05).

[0065] In the jejunal mucosa tissue of weaned piglets, the levels of IL-1β and IL-6 in each experimental group were significantly lower than those in the control group. P< The IL-10 content was significantly higher than that of the control group (0.05). P< 0.05). The levels of IgG, IgM, and TNF-α in each experimental group were not significantly different from those in the control group. P> 0.05).

[0066] In the ileal mucosa tissue of weaned piglets, the levels of TNF-α and IL-6 in each experimental group were significantly lower than those in the control group. P< The levels of IgG and IgM were significantly higher than those in the control group (0.05). P< 0.05). Compared with the control group, the IL-1β level in the Fce1-3 group was significantly reduced ( P< 0.05). This indicates that Fce can, to some extent, increase the content of intestinal mucosal immunoglobulins and the level of anti-inflammatory factors, and reduce the level of pro-inflammatory factors, thereby alleviating intestinal inflammatory response and reducing weaning stress in piglets.

[0067] Table 4. Effects of Fce on intestinal mucosal immunoglobulins and inflammation-related cytokines in weaned piglets. Note: " / " indicates that the item is not present, and different letters indicate significant differences between groups.

[0068] Experiment 3 Real-time quantitative PCR Total RNA was extracted from IPEC-J2 cells and piglet intestinal mucosal tissue using TRIzol reagent, and the RNA concentration and purity were detected using a spectrophotometer. RNA was reverse transcribed into cDNA using the Prime Script RT kit. qPCR analysis was performed using SYBR Green qPCR premix and a real-time quantitative PCR system. -∆∆Ct The data were analyzed using a method that standardized the data using β-actin as an internal reference gene. The sequences of the primers used are shown in Table 5.

[0069] Table 5. qPCR primer sequence list The results are as follows Figure 4 As shown, compared with the Con group, the relative expression levels of MYD88, NF-κB, IL-1β, and TNF-α mRNA in IPEC-J2 cells of the Mode group were significantly increased. P< 0.05). Compared with the Mode group, the relative expression levels of TLR4, MYD88, NF-κB, and IL-1β mRNA in IPEC-J2 cells of the Fce1-4 group were significantly reduced. P< 0.05), the relative expression level of TNF-α mRNA in IPEC-J2 cells of the Fce4 group was significantly reduced ( P< 0.05). Among them, the relative expression levels of TLR4 and MYD88 mRNA in IPEC-J2 cells of group Fce2 were the lowest compared with those of NF-κB and IL-1β in group Fce3. P< 0.05). This indicates that Fce can reduce the expression of inflammation-related genes in IPEC-J2 cells induced by LPS, thereby alleviating the inflammatory damage to IPEC-J2 cells caused by LPS.

[0070] Depend on Figure 5 As shown in A, compared with the control group, the relative expression levels of IL-2 and IL-10 mRNA in the duodenal mucosa of weaned piglets in each experimental group were significantly increased. P< 0.05), while the relative expression level of TNF-α mRNA was significantly reduced ( P< 0.05). Furthermore, the relative expression levels of NF-κB and IL-6 mRNA in the Fce2, Fce3, and Fce4 groups were significantly lower than those in the control group ( P< 0.05). Compared with the control group, the relative expression levels of TLR4 mRNA in the Fce2 and Fce3 groups, MYD88 mRNA in the Fce2 group, and IL-1β mRNA in the Fce3 group were significantly decreased. P< 0.05).

[0071] Depend on Figure 5 As shown in B, compared with the control group, the relative expression levels of MYD88, NF-κB, IL-1β, TNF-α, and IL-6 mRNA in the jejunal mucosa of weaned piglets in each experimental group were significantly reduced. P< 0.05), while the relative expression levels of IL-2 and IL-10 mRNA were significantly increased ( P< 0.05). Meanwhile, the relative expression levels of TLR4 mRNA in the Fce2 and Fce3 groups were significantly lower than those in the control group ( P< 0.05).

[0072] Depend on Figure 5 As shown in C, the relative expression levels of IL-1β and TNF-α mRNA in the ileal mucosa of weaned piglets in each experimental group were significantly lower than those in the control group. P< 0.05), the relative expression level of IL-2 mRNA was significantly higher than that of the control group ( P< 0.05). Furthermore, the relative expression levels of NF-κB mRNA in the Fce2, Fce3, and Fce4 groups were significantly lower than those in the control group ( P< 0.05). Compared with the control group, the relative expression levels of TLR4 mRNA in the Fce1 and Fce2 groups and MYD88 mRNA in the Fce2 and Fce3 groups were significantly reduced ( P< 0.05). This study shows that adding Fce to the diet can significantly regulate the expression of genes related to the NF-κB signaling pathway in the intestinal mucosa of animals. By inhibiting the abnormal activation of this pathway, it reduces the gene expression of downstream pro-inflammatory factors, thereby effectively alleviating the inflammatory response of the intestinal mucosa.

[0073] Experiment 4 Western blot analysis 50 mg of piglet intestinal mucosal tissue was placed in a grinding tube, and RIPA lysis buffer containing 1% protease and 1% phosphatase inhibitor was added. The tissue was then homogenized for 180 s using a tissue homogenizer, followed by centrifugation at 12000 rpm / min for 10 min at 4 °C. The supernatant was collected. The total protein concentration was determined using the BCA protein assay. 400 μg of protein from the supernatant was transferred to a new centrifuge tube, followed by 25 μL of 4× protein loading buffer. RIPA lysis buffer was then added to a final volume of 100 μL, and the mixture was denatured at 99 °C for 10 min. The extracted proteins were separated on a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PDVF) membrane. The membrane was then blocked with 5% skim milk powder for 3 hours, followed by washing with PBST 5 times (6 min each time). Primary antibodies were then added: β-Actin and IL-1β from Beyotime (China); p-NF-κB p65, TNF-α, and NF-κB p65 from Abmart (Shanghai, China). The membrane was incubated overnight at 4°C. After washing with PBST 5 times (6 min each time), secondary antibodies from Beyotime (China) were added: goat anti-mouse IgG (H+L) and goat anti-rabbit IgG (H+L). The membrane was incubated for 1 hour. After washing with PBST 5 times (6 min each time), chemiluminescence signals were acquired using ECL ultrasensitive chemiluminescence buffer and an Amersham Imager 600 imaging system. ImageJ software was used to analyze the grayscale values ​​of the protein bands, and the relative expression level of the target protein was expressed as the ratio of the grayscale value of the target protein band to that of the internal control protein band.

[0074] like Figure 6 and Figure 7 As shown, compared with the Con group, the relative protein expression levels of IL-1β, TNF-α, NF-κB P65, P-NF-κB P65, and P-P65 / P65 in IPEC-J2 cells of the Mode group were significantly increased. P< 0.05). Compared with the Mode group, the relative expression levels of IL-1β, TNF-α, NF-κB P65, P-NF-κB P65, and P-P65 / P65 proteins in IPEC-J2 cells of the Fce2-4 group were significantly reduced. P< 0.05). Among them, the relative expression levels of IL-1β, NF-κB P65, P-NF-κB P65, and P-P65 / P65 were lowest in the Fce3 group and in the Fce4 group. P< 0.05). The results of this study confirm that Fce can significantly reduce the expression of inflammation-related proteins in IPEC-J2 cells stimulated by lipopolysaccharide, thereby exerting a significant protective effect against LPS-induced cellular inflammatory damage.

[0075] Depend onFigure 8~Figure 9 It can be seen that the relative expression levels of IL-1β, TNF-α, P-NF-κB P65, and P-P65 / P65 proteins in the jejunal mucosa of weaned piglets in each experimental group were significantly lower than those in the control group. P< 0.05), while the relative expression level of NF-κB P65 protein was not significantly different from that of the control group ( P> 0.05). Among them, the relative expression levels of TNF-α, P-NF-κB P65, and P-P65 / P65 in the jejunal mucosa of weaned piglets in group Fce3 were the lowest, while the relative expression levels of IL-1β protein in group Fce4 were the lowest. P< 0.05).

[0076] Depend on Figure 10 and Figure 11 It can be seen that the relative expression levels of IL-1β and NF-κB P65 proteins in the ileal mucosa of weaned piglets in each experimental group were significantly lower than those in the control group. P< 0.05). Compared with the control group, the relative expression levels of TNF-α and P-NF-κBP65 in the Fce2-4 group and P-P65 / P65 in the Fce2 and Fce3 groups were significantly reduced. P< 0.05). Among them, the relative expression levels of IL-1β, NF-κB P65, P-NF-κB P65, and P-P65 / P65 in the jejunal mucosa tissue of weaned piglets in group Fce2 were the lowest, while the relative expression levels of TNF-α protein in group Fce3 were the lowest. P< 0.05)

[0077] In summary, Cornus officinalis extract at concentrations of 900 μg / mL–1200 μg / mL reduced the levels of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 in the supernatant of LPS-induced IPEC-J2 cell culture medium, while increasing the level of the anti-inflammatory cytokine IL-10. It also reduced the relative expression levels of TLR4, MyD88, NF-κB, and IL-1β mRNA in IPEC-J2 cells, and decreased the relative expression levels of IL-1β and TNF-α proteins and the phosphorylation level of NF-κB. Cornus officinalis extract increased the content of immunoglobulin IgM in the duodenum and ileum of weaned piglets. Simultaneously, it significantly reduced the levels of pro-inflammatory cytokines IL-1β and IL-6, the relative expression levels of NF-κB, IL-1β, and TNF-α mRNA, the relative expression levels of IL-1β and TNF-α proteins, and the phosphorylation level of NF-κB in the jejunal and ileal mucosa (P<0.05). Therefore, Cornus officinalis extract can effectively alleviate the inflammatory response in the intestines of weaned piglets by inhibiting the NF-κB signaling pathway.

[0078] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. The application of Cornus officinalis extract in the preparation of compound feed to alleviate diarrhea and intestinal inflammation in weaned piglets, wherein the Cornus officinalis extract is an aqueous extract of Cornus officinalis.

2. The application according to claim 1, characterized in that, The compound feed contains 500mg~2000mg of Cornus officinalis extract per kilogram.

3. The application according to claim 1, characterized in that, Each gram of Cornus officinalis extract is prepared by water extraction from 100g of Cornus officinalis medicinal material.

4. The application according to claim 1, characterized in that, The preparation method of compound feed includes the following steps: mixing Cornus officinalis extract with basic feed to obtain a compound feed that relieves diarrhea and enteritis in weaned piglets.

5. The application according to claim 4, characterized in that, The basic feed is prepared from the following raw materials in the following mass percentages: 27.37%~46.65% corn, 15.00%~33.00% extruded corn, 12.80%~20.30% soybean meal, 2.00%~10.00% fermented soybean meal, 1.50%~2.00% soybean oil, 1.00%~8.00% wheat bran, 0.50%~3.50% fishmeal, 2.00%~6.00% whey powder, 0.55%~0.80% limestone, 1.15%~1.60% dicalcium phosphate, 0.30% salt, 0.45%~0.50% L-lysine hydrochloride, 0.10% DL-methionine, 0.20% L-threonine, 0.05%~0.08% L-tryptophan, and 1% premix, totaling 100%.

6. The application according to claim 5, characterized in that, The formula for the premixed feed added per kilogram of feed is: 10000 IU vitamin A, 2750 IU vitamin D3, 80 IU vitamin E, 2 mg vitamin K3, 0.3 mg vitamin B12, 12 mg riboflavin, 2.25 mg vitamin B6, 40 mg niacin, 25 mg d-pantothenic acid, 0.25 mg biotin, 1.6 mg folic acid, 3.0 mg thiamine, 150 mg iron, 105 mg zinc, 30 mg manganese, 25 mg copper, 0.5 mg iodine, and 0.3 mg selenium.

7. The application according to claim 6, characterized in that, The iron element comes from ferrous sulfate monohydrate, the zinc element comes from zinc sulfate monohydrate, the manganese element comes from manganese sulfate monohydrate, the copper element comes from copper sulfate pentahydrate, the iodine element comes from calcium iodate, and the selenium element comes from sodium selenite.

8. The application according to claim 1, characterized in that, The weight of piglets fed with compound feed is 5.11kg~5.89kg, and each piglet consumes an average of 317.53g~373.65g of feed per day.

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