Application of sophocarpidine and composition thereof in preparation of product for improving health of mammals

Abstract

The invention discloses application of sophocarpidine in preparation of a product for improving the survival rate of mammalian cubs. The mammalian is a gestational animal infected by gram-positive bacteria or at the risk of gram-positive bacteria infection. The product is matrine; the matrine and the composition thereof can effectively improve the survival rate of mammalian cubs in the gestation period.

Description

Technical Field

[0001] The present invention belongs to the field of animal functional feed additives, and particularly relates to the application of matrine and its composition in the preparation of products for improving the health of mammals. Background Art

[0002] For a long time, antibiotics have played a crucial role in the feed industry and animal husbandry. However, the harms of extensive and long-term use of antibiotics have become increasingly apparent. It may trigger "bacterial drug resistance", accelerate the formation of "super bacteria", cause drug residues in animal products, and endanger human health. Therefore, the state has introduced relevant policies for regulation. Clostridium perfringens is a Gram-positive anaerobic bacterium widely distributed in natural environments, the intestines of animals and humans, and foods such as raw meat, dehydrated soup, raw vegetables, and spices. In the fields of livestock farming, etc., it is also an important pathogen of various diseases in mammals such as pigs, cows, and sheep, and poultry, often causing diseases such as lamb dysentery, sheep enterotoxemia, gangrenous dermatitis, enterotoxemia, and necrotic enteritis. Currently, with the rapid increase in the multiple drug resistance of Clostridium perfringens to antibiotics, the therapeutic effects of traditional antibacterial drugs such as antibiotics continue to decline, and the dosage increases year by year, seriously affecting the development of the feed industry and animal husbandry, and at the same time endangering human health. Therefore, finding a potential green, safe, environmentally friendly, and highly efficient antibiotic substitute is crucial for feed production and the livestock and poultry breeding industry.

[0003] Although some methods or components for the prevention and treatment of Clostridium perfringens have been proposed in the prior art, the related technologies have defects such as scarce raw material sources, difficulty in obtaining, and high production costs. Matrine (MT) is a natural quinolizidine alkaloid. As a herbal medicine, it has the advantages of clear chemical structure, low toxicity and side effects, and high safety. It can be extracted from the plant Sophora alopecuroides. The plant Sophora alopecuroides is cold and drought tolerant, easy to plant and manage, and has strong adaptability. The fresh grass yield per mu is about 700 - 900 kg, and the seed yield per mu is 100 - 150 kg. The natural plant resources of Sophora alopecuroides are rich, green, safe, and residue-free, and it is one of the ideal products for replacing antibiotics.

[0004] On the other hand, due to the physical differences between different species and between adult animals and young animals, some methods or components for the prevention and treatment of Clostridium perfringens proposed in the prior art only show the treatment of some related diseases. At the same time, the unclear dosage may also make it difficult for the related methods or components to achieve the treatment or prevention effect, and even cause serious liver and kidney toxicity, unable to achieve an effective antibiotic replacement effect, resulting in huge losses.

[0005] Therefore, obtaining an ideal alternative to antibiotics with clear composition, low toxicity and side effects, high safety, easy availability, and obvious dosage and therapeutic or preventive effects is still an urgent problem to be solved for the prevention and treatment of Clostridium perfringens at present. Summary of the Invention

[0006] The object of the present invention is to provide the use of matrine and its composition in the preparation of products for improving the health of mammals, aiming to solve the problems pointed out in the background technology that the dosage of existing alternative antibiotic products is not clear, it is difficult to achieve therapeutic or preventive effects, and even serious liver and kidney toxicity may be produced.

[0007] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0008] The use of matrine in the preparation of products for improving the survival rate of mammalian cubs, wherein the mammals are pregnant animals infected with Gram-positive bacteria or at risk of Gram-positive bacteria infection;

[0009] The product is matrine.

[0010] The use of matrine in the preparation of products for improving the intestinal health of mammalian cubs, wherein the mammalian cubs are cubs infected with Gram-positive bacteria or in the state of Gram-positive bacteria infection;

[0011] The product includes matrine.

[0012] Further, the Gram-positive bacteria are Clostridium perfringens.

[0013] Further, the product further includes at least one of dandelion, other edible excipients, and solvents; the edible excipients are animal feeds or pharmaceutical excipients; the solvent is CAMHB culture solution.

[0014] Further, the daily oral dose of matrine in the product is below 120 mg / kg.

[0015] Further, for every 1 mg of dandelion in the product, the corresponding matrine is 1.4 - 1.6 mg.

[0016] Further, the preparation method of the matrine:

[0017] Dry and crush the Sophora alopecuroides seeds, extract by hot reflux, and concentrate to obtain a pure extract; dissolve the pure extract with an acid solution, filter by suction to remove the residue, and obtain an acid aqueous solution; extract according to the volume ratio of dichloromethane: acid aqueous solution = 1:3 until the dichloromethane layer is clear and transparent, then recover the acidic dichloromethane part under reduced pressure; subsequently, adjust the pH value of the acidic dichloromethane part to 9 - 11 with a solid basic regulator; extract again with dichloromethane and n-butanol, and recover the solvent under reduced pressure to obtain an extract of the basic dichloromethane and n-butanol parts, and the obtained extract of the basic dichloromethane and n-butanol parts is the crude extract of Sophora alopecuroides alkaloids.

[0018] Furthermore, the mammals include farmable livestock and laboratory animals.

[0019] Furthermore, the livestock are porcine animals; the laboratory animals are rodents, non-human primates, canines, rabbits, and porcine animals.

[0020] Compared with the prior art, the present invention using the above composition has at least one of the following beneficial effects:

[0021] (1) Under the oral dosage of the present invention, the survival rate of the offspring of pregnant mammals can be effectively increased, and the number of stillborn or / and weak offspring of pregnant animals can be reduced;

[0022] (2) Under the oral dosage of the present invention, mammals infected with Clostridium perfringens, especially pregnant mammals and weaned offspring, can be effectively prevented or treated, and there is no any toxic effect, it is safe and reliable, and there is no antibiotic residue;

[0023] (3) Under the oral dosage of the present invention, it has enhanced immune performance at the same time, has the function of repairing intestinal tissue damage, reduces the abundance of Clostridium perfringens in the intestine and the content of α-toxin, has the effect of preventing diarrhea caused by Clostridium perfringens in mammals and reduces the incidence of diarrhea in offspring;

[0024] (4) The product under the oral dosage of the present invention has no toxic effect on the routine blood indexes, liver function, kidney function indexes of mammals and the morphology of mammalian visceral tissues. Therefore, matrine and the composition are safe and have no side effects on mammals. At the same time, it is found that matrine and the composition can restore the number of lymphocytes and monocytes in the blood, and no pathological clinical symptoms such as diarrhea occur when matrine and the composition act on mammals together with Clostridium perfringens;

[0025] (5) The raw materials of the matrine and composition preparation of the present invention are widely distributed, have high yields, are easy to prepare, and have low costs. They can be made into feed additives and applied to large-scale farms to scale down the incidence of diarrhea caused by Clostridium perfringens in farms and increase the economic income of farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention, and do not constitute a limitation to the present invention.

[0027] In the drawings:

[0028] Figure 1 are the HE and PAS staining results of the intestinal tissues of mice after being fed with matrine for 30 days;

[0029] Figure 2 Effect of matrine on IgG levels in pregnant and lactating female rats ( Figure 2 A) and their offspring ( Figure 2 B);

[0030] Figure 3 shows the effect of matrine on the ileal tissue morphology of pregnant and lactating female rats and their offspring. A, Effect of matrine on the ileal tissue morphology of pregnant and lactating female rats and their offspring (H&E×200); B, Effect of matrine on the mucus layer and goblet cells in the ileal tissue of pregnant and lactating female rats and their offspring (PAS×200);

[0031] Figure 4 Effect of matrine on blood liver and kidney function indexes in piglets with diarrhea caused by Clostridium perfringens;

[0032] Figure 5 Effect of matrine on inflammatory factor indexes in intestinal tissues (A) and blood (B) of piglets with diarrhea caused by Clostridium perfringens;

[0033] Figure 6 Effect of matrine on the content of α-toxin in intestinal tissues, feces, and muscles of piglets with diarrhea caused by Clostridium perfringens;

[0034] Figure 7A Analysis of the differences in the composition of intestinal flora at the phylum level in piglets with diarrhea caused by Clostridium perfringens treated with matrine;

[0035] Figure 7B Abundance of Clostridium perfringens in the intestines of piglets with diarrhea caused by Clostridium perfringens treated with matrine;

[0036] Figure 7C Differences in α-diversity among groups of intestinal flora in piglets with diarrhea caused by Clostridium perfringens treated with matrine;

[0037] Figure 7D Comparison of the LDA scores of the abundances of Bacilli, Bacteroidota, Clostridium, Lactobacillus, Muribaculaceae, and Oscillospirales in the intestinal flora of piglets;

[0038] Figure 8 Effect of matrine on the intestinal mucosal barrier in piglets with diarrhea caused by Clostridium perfringens. A, Effect of matrine on the expression levels of sIgA and MUC2 in the intestines of piglets with diarrhea caused by Clostridium perfringens; B, Effect of matrine on the morphological structure of intestinal tissues in piglets with diarrhea caused by Clostridium perfringens; C, Effect of matrine on the mucus layer structure and the number of goblet cells in the intestines of piglets with diarrhea caused by Clostridium perfringens;

[0039] Figure 9 Effect of matrine on the morphological structure of visceral tissues in piglets with diarrhea caused by Clostridium perfringens. Detailed implementation manners

[0040] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only for illustrating and explaining the present invention, and are not used to limit the present invention.

[0041] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods.

[0042] Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels.

[0043] Definitions Unless otherwise stated, the terms used herein have the following definitions.

[0044] As used herein, the term "matrine" (MT) refers to a unique class of quinolizidine alkaloids widely present in the roots of plants such as Sophora flavescens, Sophora alopecuroides, and Sophora subprostrata. Pure matrine is a white powder, which can be directly purchased through commercially available products, or can be prepared by extracting the dried roots, plants, and fruits of leguminous plants with organic solvents such as ethanol. Any extraction method that can effectively extract matrine in the prior art can be used to obtain matrine in the present invention. For example, the extraction steps of matrine disclosed in CN1119347C, CN1161121C, and CN104230932B or their combinations can be used.

[0045] Matrine can be detected or purified by one or more combinations of chemical analysis methods for matrine detection or purification disclosed in known prior arts such as chromatography (such as gas chromatography, liquid chromatography), mass spectrometry, surface imprinting materials, etc. including but not limited to, for example, the methods described in CN103245754B and CN101775152B can be used for the detection or purification of matrine in the present invention.

[0046] After the pure extracts in the matrine preparation steps are combined, they can be dissolved with hydrochloric acid or dilute sulfuric acid; the pH value can be adjusted with one or more of solid bases such as sodium hydroxide, potassium hydroxide, anhydrous sodium carbonate, sodium bicarbonate, and calcium hydroxide.

[0047] As used herein, the terms "Clostridium perfringens" and "Clostridium welchii" can be used interchangeably, and Clostridium welchii is selected from one or more of Clostridium perfringens type A, B, C, D, E, or F.

[0048] The terms "comprising", "including", or "containing" should be understood to include the specified components, but not exclude any other components.

[0049] When referring to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids with "administering" and "treating", it means contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject being treated, cell, tissue, organ, or biological fluid. "Administering" and "treating" can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and test methods. Treating cells includes contacting a reagent with the cells and contacting a reagent with a flowing fluid that contacts the cells.

[0050] As used herein, "preventing" or "treating" includes delaying the development of symptoms associated with a disease and / or reducing the severity of these symptoms that the disease will or is expected to develop. The terms also include alleviating existing symptoms, preventing additional symptoms, and slowing or preventing the underlying causes of these symptoms. Thus, the terms indicate that a beneficial result has been conferred on a mammalian subject suffering from a disease, such as a porcine animal.

[0051] Immediately, matrine of the present disclosure can also be provided in other effective forms such as salts, including salts in anionic form and salts in cationic form. Some examples of salts in anionic form include hydrochloride, citrate, chloride salt, and acetate. Preferably, the salt is acetate. Some examples of salts in cationic form include salts in which the cation is selected from the following: alkali metals (such as sodium and potassium), alkaline earth metals (such as calcium), etc.

[0052] Median lethal dose

[0053] Generally, the median lethal dose (LD50) is used as a safety index for drugs / foods, indicating the minimum number of bacteria or amount of toxin required to cause half of a certain animal of a certain body weight or age to die within a specified time through a specified infection route. LD50 reflects the magnitude of the acute toxicity of a drug and is an important manifestation of the therapeutic index (TI). Any method known in the art for determining the median lethal dose (LD50) can be used for the determination of LD50 in the present invention, including but not limited to the Bliss method, the Karber method, the modified Karber method, etc.

[0054] Premises for the application of the modified Karber method: ① The number of animals in each group is the same; ② The doses form a geometric progression; ③ The reaction conditions generally conform to a normal distribution; ④ The experimental examples include or are close to reaction rates of 0% and 100%.

[0055] The calculation formula is: LD50 = log-1[Xm - I(Σp - 0.5)]

[0056] Where: Xm = logarithm of the maximum dose; p = animal mortality rate; Σp = sum of the mortality rates of each group; I = logarithm of the ratio of two adjacent doses, with the high dose as the numerator.

[0057] In some embodiments, in any application of matrine or matrine co-formulation, the daily oral dose of matrine is about 1 mg / kg body weight, about 2 mg / kg body weight, about 3 mg / kg body weight, about 4 mg / kg body weight, about 5 mg / kg body weight, about 6 mg / kg body weight, about 7 mg / kg body weight, about 8 mg / kg body weight, about 9 mg / kg body weight, about 10 mg / kg body weight, about 20 mg / kg body weight, about 30 mg / kg body weight, about 40 mg / kg body weight, about 50 mg / kg body weight, about 60 mg / kg body weight, about 70 mg / kg body weight, about 80 mg / kg body weight, about 90 mg / kg body weight, about 100 mg / kg body weight, about 110 mg / kg body weight, about 120 mg / kg body weight, or any range and value between these values.

[0058] In some embodiments, in any application of matrine or matrine compound preparation, the daily oral dose of matrine is 12 mg / kg to 120 mg / kg.

[0059] In some embodiments, in any application of matrine or matrine compound preparation, the daily oral dose of matrine is 12 mg / kg to 30 mg / kg.

[0060] In some embodiments, in any application of the matrine compound preparation, the daily oral dose of the matrine compound preparation contains 1.4-1.6 mg of matrine per 1 mg of dandelion.

[0061] Mammals include domesticated livestock and experimental animals. Livestock include swine, cattle, horses, donkeys, and sheep; experimental animals include rodents, non-human primates, dogs, rabbits, and pigs.

[0062] In the following examples: the matrine+Clostridium perfringens group is the prevention group.

[0063] Take the experimental steps of measuring the median lethal dose (LD50) of oral drugs in mice using the modified Koch method as an example:

[0064] Experimental preparation: Select healthy mice of similar weight, generally 18-22 grams, half male and half female, and randomly divide them into groups. Prepare the oral drug to be tested into solutions of different concentrations. The solvent should be non-toxic to mice and does not affect the properties of the drug.

[0065] Preliminary experiment: Determine the dosage range: First, select 3-5 dosage groups with a larger dosage interval, such as 10-fold increments, with 3-5 mice in each group, administer the drug to the mice by gavage, and observe for 48-72 hours to find the dosage range that causes all mice to die or survive.

[0066] Formal experiment: Group the experimental mice. According to the results of the preliminary experiment, within the range of the all-dead and all-alive doses, set 5 - 7 dose groups according to the geometric progression. The number of mice in each group is generally 10. Use a gavage needle to accurately gavage the drug solution of different concentrations / doses to the mice at a volume of 0.1 - 0.2 ml / 10 g body weight. The dosing time for each dose group should be as the same as possible. Continuously observe and record the experimental results, and record the number of dead mice and the time of death in each group, etc. Calculate the mortality rate of the mice in each dose group, expressed in %, and calculate LD 50 。

[0067] During the experiment, it is necessary to strictly abide by the ethical norms of animal experiments. At the same time, the accuracy and consistency of the operation should be ensured to ensure the reliability of the experimental results.

[0068] Growth performance indicators

[0069] In the present invention, the growth performance indicators of mammals are the average daily gain (ADG), average daily feed intake (ADFI), feed conversion ratio (F / G), etc. obtained by collecting and calculating data such as the feed intake, body weight, fecal conditions, and tail conditions of piglets of mammals on the day before and the day after the application method of matrine and / or pathogenic bacteria described in the present invention. The calculation methods are as follows:

[0070] ① Average daily gain ADG (kg / d) = Total weight gain AD / Number of experimental days;

[0071] ② Total weight gain AD: Individually weigh the animals at 8 am on the morning after fasting for 12 hours on the day before the start of the experiment and on the 14th day. Subtract the initial weight from the final weight, which is the weight gain of the animal;

[0072] ③ Average daily feed intake ADFI (kg / d): Record the feed intake of each individual in each group every day. After the experiment ends, accumulate and count the weight of the feed eaten daily minus the remaining feed, and then divide by the number of animals in each pen to obtain;

[0073] ④ Feed conversion ratio F / G = ADFI / AD;

[0074] ⑤ Diarrhea rate: Number of diarrhea animals / Total number of animals × 100%;

[0075] ⑥ Diarrhea index = Sum of diarrhea scores of all animals / (Number of experimental days × Number of experimental pigs).

[0076]

[0077]

[0078] Detection of hematological indicators

[0079] 1. Sample collection and processing

[0080] (1) Blood sample collection: Collect 5 ml of anterior vena cava blood from weaned piglets in each group on the 0th, 7th, and 14th days of the experiment using the anterior vena cava blood collection method. Collect whole blood using a sterile EDTA anticoagulant blood collection tube, gently invert and mix 10 times to avoid hemolysis.

[0081] (2) Sample preservation and transportation: Immediately place the sample in an ice box at 4°C after blood collection and send it to the Animal Hospital of Northwest A&F University in Xi'an for testing within 2 hours. Avoid violent shaking during transportation to ensure the integrity of the sample.

[0082] 2. Detection items and methods

[0083] (1) Detection instrument: Use a fully automatic hematology analyzer (model: Sysmex XN-1000) for routine blood tests.

[0084] (2) Detection items: Red blood cell-related indicators, white blood cell-related indicators, and platelet-related indicators.

[0085] (3) Detection method: Combine impedance method and flow cytometry to ensure the accuracy and repeatability of the detection results. Each sample is detected 3 times, and the average value is taken as the final result.

[0086] Serum biochemical index detection

[0087] 1. Sample processing and detection indicators

[0088] Collect 2 ml of anterior vena cava blood samples from weaned piglets in each group on the 0th, 7th, and 14th days, anticoagulate with sodium heparin, and centrifuge at 4°C, 3000×g for 10 min to separate the serum. Use commercial kits from Nanjing Jiancheng Bioengineering Institute to detect the following indicators:

[0089] (1) Liver function indicators: Alanine aminotransferase (ALT, product number: C009-2-1), Aspartate aminotransferase (AST, product number: C010-1-1), Alkaline phosphatase (ALP, product number: A059-1-1)

[0090] (2) Renal function indicators: Creatinine (Cr, product number: C011-2-1), Blood urea nitrogen (BUN, product number: C013-1-1)

[0091] Operation steps

[0092] (1) Reagent preparation: Prepare standard products, buffers, and reaction substrates according to the instructions.

[0093] (2) Sample addition and reaction: Add serum samples, standard products, and blank controls to a 96-well plate respectively, and then add substrates in sequence to initiate the enzymatic reaction.

[0094] (3) Determination: Use an enzyme-linked immunosorbent assay (ELISA) reader to read the absorbance values at the specified wavelengths (ALT / AST: 505 nm; ALP: 405 nm; Cr / BUN: 546 nm).

[0095] (4) Calculation: Calculate the concentration of each index according to the standard curve, and the results are expressed in U / L (ALT / AST / ALP) or μmol / L (Cr / BUN).

[0096] 2. Detection of the expression levels of inflammatory factors in blood

[0097] ELISA was used to detect the expression levels of the inflammatory factors procalcitonin (PCT), C-reactive protein (CRP), serum amyloid A (SAA) and the expression level of α-toxin in serum. The specific operation steps are as follows:

[0098] (1) Restore to room temperature: Before the detection, the kit needs to be equilibrated at room temperature for 30 min.

[0099] (2) Prepare the washing solution: Dilute the concentrated washing solution with distilled water according to the dilution ratio and set aside for later use.

[0100] (3) Add samples: Dilute the standard products into different concentrations according to the instructions. Add 50 μL of the diluted standard products to each well, and add 50 μL of the diluent to the zero well; After diluting the samples 5-fold, add them to the wells (first add 40 μL of the diluent to each well, and then add 10 μL of the sample to be tested); After all the samples are added, add 50 μL of the biotinylated antigen working solution to each well.

[0101] (4) Incubation: Stick on the sealing film and incubate the detection plate in an incubator at 37 °C for 30 min.

[0102] (5) Washing: Tear off the sealing film, discard the liquid and shake dry. Add 250 μL of the washing solution to each well with a multi-channel pipette, let it stand for 30 s and then discard. Repeat 5 times, and pat dry on the filter paper each time.

[0103] (6) Add 50 μL of avidin-HRP to each well, stick on the sealing film, gently shake well and then incubate in an incubator at 37 °C for 30 min; Wash 5 times, same as step 5.

[0104] (7) Color development: Add 50 μL of chromogenic reagent A and 50 μL of chromogenic reagent B to each well, gently shake and mix well, and develop color in the dark in an incubator at 37 °C for 10 min.

[0105] (8) Termination: Add 50 μL of the termination solution to each well to terminate the reaction (at this time, the blue color immediately turns yellow).

[0106] (9) Detection: Zero with the blank well, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm.

[0107] (10) Data analysis: Make a standard curve based on the OD values and concentrations of each standard product, and convert the sample concentration.

[0108] 3. Histopathological observation

[0109] Place the heart, liver, lung, kidney, jejunum, ileum, and colon of piglets in each experimental group in 10% neutral formalin for fixation for more than 3 days, and prepare HE-stained tissue sections; prepare AB-stained tissue sections after fixing the jejunum, ileum, and colon. The specific steps are as follows:

[0110] 3.1 HE staining

[0111] (1) Deparaffinization to hydration: Xylene I (5 min) → Xylene II (5 min) → Absolute ethanol (5 min) → Gradient ethanol (95% → 80% → 70%, each 2 min) → Distilled water wash (2 min).

[0112] (2) Staining and differentiation: Hematoxylin (7 min) → Running water rinse → 1% hydrochloric acid ethanol differentiation (30 s) → Running water blueing (15 min) → Eosin (15 - 30 s) → Running water rinse (5 min).

[0113] Dehydration and mounting: Dehydration with gradient ethanol (95% → absolute) → Xylene transparency → Mounting with neutral gum.

[0114] 3.2 AB staining

[0115] (1) Oxidation and staining: After deparaffinization and hydration, 1% periodic acid oxidation (10 min) → Running water rinse for 5 min → Incubation with Schiff stain solution in the dark (10 min) → Running water rinse for 5 min.

[0116] (2) Counterstaining and mounting: Counterstaining with hematoxylin (1 min) → Differentiation and blueing → Gradient dehydration → Xylene transparency → Mounting with neutral gum.

[0117] 4 Detection of Clostridium perfringens load in feces

[0118] 4.1 DNA extraction

[0119] (1) Sample pretreatment: Collect about 200 mg of rectal contents from female mice and weaned piglets, immediately transfer them to a 2 ml sterile cryotube, freeze them in liquid nitrogen for 5 min, and then store them in a -80°C ultra-low temperature freezer.

[0120] (2) Lysis and purification: After thawing, weigh 50 mg of fecal sample, add 1 ml of pre-cooled sterile PBS buffer (pH 7.4), vortex vigorously for 2 min, and let stand for 5 min to precipitate large particles. Centrifuge at 4 °C (3000×g, 5 min), take the supernatant and transfer it to a new centrifuge tube. Add 200 μL of buffer GA (TIANamp Stool DNA Extraction Kit) and 20 μL of proteinase K (20 mg / ml) to the supernatant, vortex and mix well, then incubate in a water bath at 70 °C for 10 min, shaking once every 2 min during this period. After adding 200 μL of absolute ethanol and mixing well, transfer the mixture to adsorption column CR2, centrifuge at 4 °C (12000×g, 1 min), and discard the filtrate.

[0121] (3) Washing and elution: Add 500 μL of buffer GD and 600 μL of washing buffer PW to the adsorption column in sequence, centrifuge at 12000×g for 1 min each time to discard the waste liquid. Centrifuge at 12000×g for 2 min to remove residual ethanol, add 50 μL of preheated (65 °C) elution buffer TB dropwise to the center of the adsorption column, let stand for 5 min, and then centrifuge (12000×g, 2 min) to collect the DNA. Use NanoDrop 2000 to detect the DNA purity (OD260 / OD280 1.8 - 2.0), aliquot and store at -80 °C for later use.

[0122] 4.2 Real-time fluorescence quantitative PCR (qPCR)

[0123] (1) Primer design and verification: Design specific primers for the 16S rRNA gene of Clostridium perfringens (forward: 5’-CGTAGGCGGATGATTAAGT-3’; reverse: 5’-CCTCAGCGTCAGTTACAG-3’). Conventional PCR amplification conditions: pre-denaturation at 94 °C for 2 min; 35 cycles (94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s); final extension at 72 °C for 2 min. Verify a single target band of 182 bp by 1.5% agarose gel electrophoresis.

[0124] (2) Standard curve preparation: Use the genomic DNA of Clostridium perfringens standard strain (ATCC 13124) as a template, and perform serial dilution (8.7×10 10 ~8.7×10 2 copies / μL) as the standard.

[0125] (3) qPCR reaction system and procedure: Reaction system (25 μL): 12.5 μL of 2×UltraSYBR Mixture (ComWin Biotech), 1 μL each of forward and reverse primers (10 μM), 2 μL of DNA template, and 8.5 μL of ddH2O. Amplification procedure: Pre-denaturation at 95°C for 10 min; 40 cycles (95°C for 15 s, 60°C for 1 min); melting curve analysis (from 60°C to 95°C, increasing by 0.5°C each time and staying for 5 s).

[0126] (4) Data analysis: Calculate the Clostridium perfringens load (copy number / g) in fecal samples according to the standard curve (R 2 > 0.99). There is no amplification in the negative control, and the amplification efficiency of the positive control is 90% - 110%. The difference in Ct values of replicate wells < 0.5 is considered valid.

[0127] 5 Metagenomic sequencing and data analysis

[0128] To comprehensively analyze the effects of matrine intervention on the intestinal flora structure and function of weaned piglets, this study used metagenomic sequencing technology to deeply analyze the fecal microbial community. The specific process is as follows:

[0129] 5.1 Sample pretreatment and DNA extraction

[0130] (1) Sample collection: Fresh fecal samples (1.0 g per head) of piglets in each group were collected on the 0th, 7th, and 14th days of the experiment, immediately placed in sterile cryotubes, frozen in liquid nitrogen, and then transferred to -80°C for storage.

[0131] (2) DNA extraction: Use the TIANamp Stool DNA Kit (TIANGEN Biotech, product number: DP328) from Tiangen Biochemical Technology Co., Ltd. to extract total microbial DNA, and operate according to the instructions: Add 200 mg of fecal sample to 1.5 ml of lysis buffer (containing proteinase K), vortex for 5 min until fully mixed; Incubate at 65°C in a water bath for 30 min, vortexing once every 10 min during this period; Add 200 μL of buffer GB, vortex and incubate at 70°C for 10 min, centrifuge (12000×g, 4°C, 5 min) to take the supernatant; Purify DNA through the adsorption column in turn, and finally elute with 50 μL of elution buffer (Buffer TE) and store at -20°C for later use.

[0132] (3) DNA quality detection: Detect the concentration and purity.

[0133] 5.2 Library construction and sequencing

[0134] (1) DNA fragmentation and library preparation: The DNA was randomly fragmented into 350 bp fragments using a Covaris M220 sonicator (Covaris, USA). After end repair, A-tailing, and ligation of Illumina sequencing adapters, AMPure XP magnetic beads (Beckman Coulter, USA) were used to screen for target fragments (300 - 400 bp). PCR amplification (cycle number: 8 - 10 cycles) was performed using KAPA HiFi HotStart ReadyMix (Roche, Switzerland) to construct a paired-end (PE) sequencing library.

[0135] (2) Library quality control: The fragment size of the library was detected, and then the concentration was quantified.

[0136] (3) High-throughput sequencing: After the library was mixed and denatured, paired-end sequencing with PE150 was performed on the Illumina NovaSeq 6000 platform (Illumina, USA), with a target data volume of ≥10 Gb / sample (completed by Shanghai Majorbio Bio-pharm Technology Co., Ltd.).

[0137] Gel electrophoresis and Western blot

[0138] 1. Extraction of total tissue protein

[0139] The tissue sample was taken out from the -80 °C refrigerator and thawed on ice. Approximately 100 mg of tissue was weighed, rinsed 3 times with pre-cooled PBS to remove surface impurities and blood. The tissue was minced into pieces of 1 - 2 mm 3 in size and placed in a 2 ml centrifuge tube. Pre-cooled powerful tissue protein lysate (containing 1 mM PMSF) was added at a ratio of 1:10 (w / v). Using a tissue homogenizer (operated on ice), homogenize at 10000 rpm for 30 s, with a 30 s interval, and repeat 3 - 5 times until the tissue was completely broken. Transfer the homogenate to a 1.5 ml centrifuge tube and let it stand on ice for 30 min, vortexing and mixing every 10 min. Further break the cells using an ultrasonic cell disruptor to release the total protein. Centrifuge the lysate at 4 °C and 12000 × g for 15 min, and transfer the supernatant (containing total protein) to a new centrifuge tube, discarding the precipitate (cell debris and unlysed tissue).

[0140] 2. BCA assay for protein concentration and protein denaturation

[0141] (1) Dilution of standards: Dilute the standards according to the instructions;

[0142] (2) Preparation of BCA working solution: Prepare the working solution by mixing reagent A and reagent B (50:1) and mix well;

[0143] (3) Sample addition and incubation: Take 25 μL of the standard product and the sample respectively and add them into the 96-well plate. Add 200 μL of the working solution to each well, and incubate in the 37 °C incubator in the dark for 30 min;

[0144] (4) Detection: Use a multi-functional microplate reader to detect the absorbance at 562 nm;

[0145] (5) Standard curve making and sample concentration calculation: Make a standard curve based on the OD value and calculate the sample concentration;

[0146] (6) Add 5 μL of protein loading buffer according to the volume of each sample. Seal the sample centrifuge tube with a sealing film for protection. Boil it on an induction cooker for 10 - 15 min to denature it. After cooling, aliquot and store it in a -20 °C refrigerator for later use.

[0147] 3. Western-blot detection of the expression of related proteins

[0148] (1) Gel preparation: Wash the glass plates and fix them in the gel preparation clamp. Fill it with ultrapure water for leak detection for 10 - 20 min; Select a 10% separating gel according to the protein molecular weight (the molecular weights of sIgA, MUC2, and β-actin proteins are 50, 110, and 45 kDa respectively). After injecting the separating gel, add 1 mL of isopropanol on top to remove air bubbles; Let it stand for about 30 min until the separating gel solidifies. Discard the isopropanol and add 5% stacking gel on top. Insert the comb and let it stand for 30 min until it solidifies;

[0149] (2) Sample loading and electrophoresis: Add electrophoresis buffer to the electrophoresis tank. Remove the comb. Add protein samples according to the protein loading amount of 20 μg / well. Add 2 μL and 1 μL of protein Marker on both sides of the sample wells respectively; First, run the gel at 80 V for 30 - 40 min to develop the marker, and then select 120 V voltage for 60 - 70 min for gel running.

[0150] (3) Membrane transfer: Pre-cool the pre-prepared membrane transfer solution in a 4 °C refrigerator. Prepare the "sandwich" clip for membrane transfer; After electrophoresis, cut the target protein and the internal reference protein from the gel according to the band size shown by the Marker and place them on the black side of the clip. Cut the membrane according to the size of the gel, activate it in methanol for 1 min after marking, neatly cover it on the gel, use a scraper to drive away the air bubbles on it, carefully clamp the clip firmly, and place it in the membrane transfer tank, paying attention to the direction of the clip; Place the membrane transfer tank in a foam box, add ice water around it for cooling, pour in the membrane transfer solution, select a voltage of 100 V, and select the membrane transfer time according to the protein molecular weight. Transfer for as many minutes as the protein is in kDa (for example, the molecular weight of the internal reference protein β-actin is 45 kDa, so the set time length for membrane transfer is 45 min).

[0151] (4) Blocking: After the membrane transfer is completed, take the membrane out of the clip and place it in a 5% non-fat milk blocking solution, and incubate it at room temperature on a shaker for 2 h;

[0152] (5) Primary antibody incubation: Put the blocked membrane into the TBST washing solution and wash it on a shaker for 1 min to remove the excess blocking solution; put the membrane into an incubation box containing the primary antibody and incubate it overnight at 4 °C; after incubation, wash the membrane 3 times with TBST, 10 min each time;

[0153] (6) Secondary antibody incubation: Put the membrane into the diluted secondary antibody incubation box and incubate it on a shaker at room temperature for 2 h; after incubation, wash the membrane 3 times with TBST, 10 min each time;

[0154] (7) Chemiluminescent imaging: Prepare and mix the luminescent solution A and B in a volume ratio of 1:1, evenly apply 50 - 80 μL of the luminescent solution on the membrane, and perform automatic exposure for color development.

[0155] Example

[0156] Example 1 Preparation of Matrine

[0157] Preparation of crude matrine extract: Weigh Sophora alopecuroides seeds, dry and crush them, then use 95% ethanol solution for heat reflux extraction. The reflux temperature is 72 °C and the reflux time is 4 h. Recover the extraction solution and concentrate it to obtain an ethanol extract. Repeat the extraction operation 5 - 6 times; after combining the ethanol extracts, dissolve them with hydrochloric acid, filter to remove the residue, and obtain an acid aqueous solution; extract with dichloromethane:acid aqueous solution = 1:3 by volume until the dichloromethane layer is clear and transparent, recover the solvent under reduced pressure to obtain a partially acidic dichloromethane; then adjust the pH value of the acidic dichloromethane part to 9 - 11 with solid sodium hydroxide, and extract with dichloromethane and n-butanol in sequence. Recover the solvent under reduced pressure to obtain an extract of the basic dichloromethane and n-butanol parts. The obtained extract of the basic dichloromethane and n-butanol parts is the crude extraction of Sophora alopecuroides alkaloids. After detection, the content of matrine in the prepared crude extract of Sophora alopecuroides alkaloids is 3.5%.

[0158] Mix the raw material matrine with dandelion and make a matrine compound preparation in an appropriate amount of CAMHB medium. Among them, the final concentrations of matrine and dandelion in the compound preparation are 30 mg / mL and 20 mg / mL respectively.

[0159] Example 2 Acute Toxicology Study of Matrine

[0160] Select 60 8-week-old C57BL / 6 mice, randomly divide them into six groups (n = 10), and orally administer the matrine preparation to the mice in each group according to the doses in Table 1. After administration, observe the status of the mice and record the death situation of the mice. Use the improved Karber method to calculate the median lethal dose of matrine. The results are shown in Table 1.

[0161] Table 1 Calculation method of LD50 of matrine by oral administration in mice

[0162]

[0163]

[0164] The results of acute toxicology study showed that the LD50 of matrine was 600 mg / kg.

[0165] Example 3 Chronic toxicology study of matrine

[0166] C57BL / 6 mice at 8 weeks old were fed with matrine compound preparations at four doses of 1 / 5 LD50, 1 / 20 LD50, 1 / 50 LD50 and 0 for 30 d. We detected the liver function, kidney function and intestinal pathological histology of the four groups of mice respectively.

[0167] Liver function detection:

[0168] Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Serum alkaline phosphatase (ALP)

[0169] Kidney function detection: Blood urea nitrogen (BUN), Serum creatinine (SCr)

[0170] Intestinal function detection.

[0171] 3.1 Changes in liver function indexes after feeding matrine for 30 d

[0172] Table 2 Liver function indexes of four groups of mice after feeding matrine for 30 d

[0173] Dose ALT (U / L) AST (U / L) ALP (U / L) 1 / 5 LD50 23.58±3.25* 34.88±3.01* 135.99±9.55* 1 / 20 LD50 12.34±2.31 17.82±2.15 91.25±7.02 1 / 50 LD50 13.69±2.03 19.56±3.25 95.86±6.35 0 LD50 10.98±1.20 16.77±1.19 89.66±6.89

[0174] Note: *P<0.05, the comparison between the experimental group and the control group was significantly different.

[0175] As shown in Table 2, it was found that there was no significant difference in the contents of ALT, AST and ALP in the livers of mice in the 1 / 20 LD50 and 1 / 50 LD50 groups compared with the control group (P>0.05); when the dose reached 1 / 5 LD50, the contents of ALT, AST and ALP in the livers of mice began to increase.

[0176] Therefore, feeding matrine at doses below 1 / 5 LD50, especially at doses of 1 / 20 LD50 and 1 / 50 LD50, showed good safety for the livers of mammals.

[0177] 3.2 Changes in kidney function indexes after feeding matrine for 30 d

[0178] Table 3 Kidney function indexes of four groups of mice after feeding matrine for 30 d

[0179] Dose BUN (mg / L) Ser (umol / L) 1 / 5 LD50 299.87±26.98* 77.85±12.35* 1 / 20 LD50 246.41±22.14 68.21±11.25 1 / 50 LD50 235.89±24.18 63.54±10.25 0 LD50 200.01±25.69 59.68±10.36

[0180] Note: *P < 0.05 indicates a significant difference compared with the control group.

[0181] As shown in the results of Table 3, there was no significant difference in the contents of BUN and Ser in the kidneys of mice in the 1 / 20LD50 and 1 / 50LD50 groups compared with the control group (p > 0.05). Only starting from the 1 / 5LD50 group, the contents of BUN and Ser in the kidneys of mice were significantly higher than those in the control group (p < 0.05).

[0182] 3.3 Intestinal tissue morphological structure after feeding matrine for 30 days

[0183] Figure 1 It was shown that after feeding matrine at 1 / 20LD50 and 1 / 50LD50 for 30 days, there was no significant difference in the intestinal villus length, crypt depth, number of goblet cells, and thickness of the mucus layer barrier of mice compared with the control group. When the matrine feeding reached 1 / 5LD50 and the feeding time reached 30 days, slight damage occurred in the ileum segment of mice, such as shortening of the small intestinal villus length, deepening of the small intestinal crypt depth, and significant reduction in the number of goblet cells, and the intestinal mucus layer barrier disappeared.

[0184] In summary, through the above research on the liver, kidney, and intestinal toxicity tests, the results showed that when the daily application dose was below 120 mg / kg, the matrine of the present invention did not have obvious chronic toxicity to animals. Especially when applied at the dose of 1 / 20LD50 (i.e., 30 mg / kg) to 1 / 50LD50 (i.e., 12 mg / kg), the matrine of the present invention was safe and had no toxic side effects on the liver, kidney, and intestine.

[0185] Example 4 Research on the improvement of the survival rate of mammalian cubs by matrine

[0186] In order to study the effect of matrine on improving the survival rate of mammalian cubs during pregnancy infected with Clostridium perfringens, 3-month-old sexually mature healthy female mice were randomly divided into four groups (Control group, CPA group, MT group, CPA + MT group). After successful mating with healthy male mice, the Control group was given 0.2 mL of normal saline; the Clostridium perfringens (CPA) group was given 0.2 mL of Clostridium perfringens bacteria solution at 8.7×10 7 CFU / mL; the matrine (MT) group was given 0.2 mL of the matrine compound preparation; the Clostridium perfringens + matrine group (CPA + MT) was given 8.7×10 70.2 mL of Clostridium perfringens bacterial solution at CFU / mL and 0.2 mL of matrine compound preparation were used to intragastrically administer four groups of experimental female rats. A batch of female rats was sacrificed during the delivery period (marked as female rats during the delivery period); the other female rats were continuously fed according to the previous experimental treatment procedure until weaning, and a batch of female rats and pups were processed (marked as female rats and pups during the weaning period); the remaining pups were fed according to the previous experimental treatment procedure until 15 days after weaning, and then the pups were sacrificed (marked as weaned pups). On the day of the pups' birth in the control group, the birth litter weight, total litter size, number of live pups, number of stillbirths, number of weak pups, etc. of the pups were recorded; the weights of the female rats and the pups 15 days after birth and the daily diarrhea number of the pups were recorded.

[0187] The pups were weaned 21 days after birth. Blood samples of the female rats and pups were collected, and ELISA technology was used to detect the contents of immunoglobulin IgG, IgM, and IgA in the serum, and the contents of non-specific immune indexes malondialdehyde (MDA), lysozyme (LZM), catalase (CAT), and superoxide dismutase (SOD) in the blood of the pups were detected; the intestinal tissues of the pups were collected, and ELISA technology was used to detect the contents of intestinal sIgA and MUC2; HE staining was used to observe the changes in intestinal villi and tissue structure of the four groups of pups; PAS staining and immunohistochemistry technology were used to analyze the expression of MUC2 in the intestinal mucus layer.

[0188] The results are shown in Table 4 and Table 5.

[0189] Table 4 Effects of matrine compound preparation on the reproductive performance of pups

[0190] Reproductive index Control MT MT + CPA CPA P Value Litter weight of newborn pups (average) / g 23.4 22.6 22.3 22.6 0.6334 Total number of pups born 122 128 109 117 0.6748 Number of live pups born 122 128 109 112 0.6669 Number of stillborn pups 0 0 0 5 0.0001 Number of weak pups 0 1 1 13 0.0004

[0191] It can be obtained from Table 4 that after the female rats were infected with Clostridium perfringens, the effects on the birth litter weight, total number of pups born, and number of live pups were relatively small, but the number of stillbirths and weak pups after birth was significantly higher than that of the control group (P<0.05). After prevention with matrine, there was no significant difference in the number of stillbirths and weak pups between the pups and the control group (P>0.05).

[0192] Table 5 Effects of matrine compound preparation on α-toxin in the feces of diarrhea mice caused by Clostridium perfringens

[0193] Reproductive index Control MT MT + CPA CPA Positive Female rats during parturition 0.10 0.12 0.08 1.73 >0.24 Female rats during weaning 0.07 0.08 0.10 1.76 >0.24 Pups during weaning 0.08 0.08 0.08 1.10 >0.24 Pups 15 days after weaning 0.08 0.08 0.09 1.10 >0.24

[0194] Table 6 Effects of matrine compound preparation on Clostridium perfringens CT in the feces of diarrhea mice caused by Clostridium perfringens

[0195] Reproductive index Control MT MT + CPA CPA P Value Female rats during parturition 26.88 25.13 24.77 19.49 0.0334 Female rats during weaning 25.08 24.37 23.266 19.316 0.0248 Pups during weaning 28.19 22.7 22.62 20.67 0.0469 Pups 15 days after weaning 28.345 25.8 22.573 16.69 0.00321

[0196] As shown in Table 5 and Table 6, only giving the matrine compound preparation did not change the abundance of Clostridium perfringens and the content of α-toxin in the feces of female mice during parturition and weaning, and in the feces of neonatal mice and neonatal mice 15 days after weaning. There was no significant difference compared with the control group (P>0.05). However, after giving Clostridium perfringens to pregnant female mice, the abundance of Clostridium perfringens and the content of α-toxin in the feces of female mice during parturition and weaning, and in the feces of neonatal mice and neonatal mice 15 days after weaning were all significantly higher than those in the control group (P<0.05). After intragastric administration of the matrine compound preparation, the abundance of Clostridium perfringens and the content of α-toxin in the feces of female mice during parturition and weaning, and in the feces of neonatal mice and neonatal mice 15 days after weaning decreased significantly, and there was no significant difference compared with the control group (P>0.05). It can be seen that the matrine compound preparation can effectively prevent diarrhea caused by Clostridium perfringens infection in female and neonatal mice.

[0197] Meanwhile, Figure 2 as shown, there was no significant difference in the content of IgG in the bodies of female and neonatal mice when only fed with the matrine compound preparation; however, after Clostridium perfringens infection, the matrine compound preparation can significantly increase the content of IgG in neonatal mice at weaning and 15 days after weaning, and enhance the non-specific immune function of neonatal mice.

[0198] Figure 3( Figure 3A and Figure 3B ) shows that during parturition, weaning and 15 days after weaning, there were no significant pathological changes in the ileum tissues of female and neonatal mice in the matrine compound preparation group compared with the control group. At the same time, it was found that the matrine compound preparation can increase the number of goblet cells and the thickness of the intestinal mucus layer. In the Clostridium perfringens group, the intestinal villi of female and neonatal mice shrank and fell off, the intestinal crypts became shallow, the distance between intestinal villi increased, the number of goblet cells decreased, the intestinal mucus layer disappeared, and even the serosa layer of the small intestine was damaged. In the matrine compound preparation + Clostridium perfringens group, the damaged parts of the ileum tissues of female and neonatal mice caused by Clostridium perfringens were repaired, and there was no significant difference compared with the control group.

[0199] Example 5 Effect of Matrine on Preventing Clostridium perfringens Infection in Piglets

[0200] Twelve 7kg healthy piglets were purchased and randomly divided into four groups (3 piglets in each group, single pen feeding). The Clostridium perfringens (CPA) group was gavaged with 0.5mL sterile drinking water in the morning and 0.5mL 8.7×108CFU / mL CPA bacterial suspension in the afternoon; the matrine + Clostridium perfringens (MT+CPA) group was gavaged with 0.5mL matrine (30mg / kg) in the morning and 0.5mL 8.7×108CFU / mL CPA bacterial suspension in the afternoon; the matrine (MT) group was gavaged with 0.5mL MT in the morning and 0.5mL sterile drinking water in the afternoon; the control group was gavaged with 0.5mL sterile drinking water in the morning and afternoon, once a day, and the experimental period was 14 days. The growth performance indicators of piglets, such as average daily weight gain, daily feed intake, feed-to-meat ratio, mortality rate and stiff pig production rate, were calculated, and the diarrhea rate and diarrhea index were calculated.

[0201] Blood samples were collected from weaned piglets on the 0th, 7th, and 15th days of the experiment. The ELISA technique was used to detect the levels of immunoglobulins IgG, IgM, and IgA in the serum, the nonspecific immune indicators MDA, LZM, CAT, and SOD in the blood, and the liver and kidney function indicators were determined; the intestinal tissues of the piglets were collected, and the levels of intestinal sIgA and MUC2 were detected by ELISA and WB techniques; HE staining was used to observe the changes in the intestinal villi and tissue structure of the four groups of piglets; PAS staining and immunohistochemistry were used to analyze the expression of MUC2 and goblet cells in the intestinal mucus layer. Through the above detection methods, it is hoped that it can be determined whether the matrine compound preparation can effectively reduce the incidence of diarrhea caused by Clostridium perfringens in weaned piglets.

[0202] The results are as follows:

[0203] 5.1 Effect of matrine on feces of piglets with diarrhea caused by Clostridium perfringens

[0204] The results showed that the feces of piglets in the control group that only ingested antibiotics were yellow, darker in color, moderately soft and hard, and had no odor. The feces of piglets in the matrine group were similar to those in the control group. The feces of piglets in the Clostridium perfringens group were watery yellow and loose, with feces sticking to the piglets' anus, but after being fed with matrine, they were slightly softer than those in the normal group, and the feces were banana-shaped. After autopsy, there was no significant difference in the anatomical changes of the visceral and intestinal tissues of piglets in the matrine group and the Clostridium perfringens + matrine group compared with the normal group, but the Clostridium perfringens group had intestinal bloating and congestion, mesenteric hemorrhage, enlarged spleen and liver, and hemorrhage, enlargement and black mesenteric lymph nodes.

[0205] 5.2 Effects of matrine on blood routine, liver and kidney in piglets with diarrhea caused by Clostridium perfringens

[0206] It can be seen from Table 7 that the percentage of lymphocytes (Lym) in the blood of piglets in the CPA group was significantly lower than that in the Control, MT, and MT+CPA groups; the percentages of Mon, HGB, RDW-CV, and RDW-SD in the blood of piglets in the CPA group were significantly higher than those in the Control, MT, and MT+CPA groups (P<0.05). However, there was no significant difference between the MT and MT+CPA groups and the control group (P>0.05).

[0207] Variation law of routine blood index results of piglets in Table 7

[0208]

[0209]

[0210] As Figure 4 showed, the test results of liver and kidney function indexes of animals in each group showed that after 7d and 14d, the content of AST in the blood of piglets in the CPA group was extremely significantly higher than that in the Control, MT, and MT+CPA groups (P<0.01); there was no significant difference in the content of AST in the blood of piglets among the Control, MT, and MT+CPA groups (P>0.05). After 7d and 14d of the experiment, the contents of ALT, AKP, BUN, and CRE in the blood of piglets in the CPA group were significantly higher than those in the MT+CPA group, the Control group, and the MT group (P<0.05). However, the contents of ALT, AKP, BUN, and CRE in the blood of piglets in the MT+CPA group were significantly higher than those in the Control group and the MT group (P<0.05); there was no significant difference in the contents of AKP, BUN, and CRE in the blood of piglets between the Control group and the MT group (P>0.05).

[0211] From the above results, it can be seen that matrine has no toxic or harmful effects on piglets, has good safety performance, can effectively prevent and treat Clostridium perfringens infection, and can significantly restore the damage of liver and kidney functions caused by Clostridium perfringens in piglets.

[0212] 5.3 Effects of matrine on levels of blood and intestinal inflammatory factors in piglets with diarrhea caused by Clostridium perfringens

[0213] As Figure 5As shown in the figure, after 15 days of the experiment, the content of IL-4 in the jejunum of piglets in the CPA group was significantly lower than that in the Control group and the MT group (P<0.05), and there was no significant difference compared with the MT+CPA group (P>0.05); the content of IL-4 in the ileum and colon of piglets in the CPA group was significantly lower than that in the MT+CPA group, significantly lower than that in the MT group, and significantly lower than that in the Control group (P<0.05). The content of IL-10 in the jejunum, ileum and colon of piglets in the CPA group was significantly lower than that in the MT+CPA group, significantly lower than that in the MT group, and significantly lower than that in the Control group (P<0.05). The content of TGF-β in the ileum of piglets in the CPA group was significantly lower than that in the MT+CPA group, significantly lower than that in the MT group, and significantly lower than that in the Control group (P<0.05). The content of TGF-β in the jejunum and colon of piglets in the MT+CPA group was significantly lower than that in the Control group, but significantly higher than that in the CPA group (P<0.05). The content of IL-β in the jejunum of piglets in the CPA group was significantly higher than that in the MT+CPA group, significantly higher than that in the MT group, and significantly higher than that in the Control group (P<0.05). The content of IFN-γ in the ileum of CPA piglets was significantly higher than that in the MT+CPA group, significantly higher than that in the MT group, and significantly higher than that in the Control group (P<0.05). The content of TNF-α in the jejunum and ileum of piglets in the CPA group was significantly higher than that in the MT+CPA group and the MT group, and significantly higher than that in the Control group (P<0.05). There was no significant difference in the content of TNF-α in the jejunum and ileum of piglets in the MT+CPA group and the MT group (P>0.05). The content of TNF-α in the jejunum of piglets in the CPA group was significantly higher than that in the MT+CPA group, significantly higher than that in the MT group, and significantly higher than that in the Control group (P<0.05).

[0214] After 7 days and 14 days of the experiment, the contents of PCT, hs-CRP and SAA in the blood of piglets in the CPA group were significantly higher than those in the MT+CPA group, the Control group and the MT group (P<0.05); however, the contents of PCT, hs-CRP and SAA in the blood of piglets in the MT+CPA group were significantly higher than those in the Control group and the MT group (P<0.05); there was no significant difference in the contents of PCT, hs-CRP and SAA in the blood of piglets in the Control group and the MT group (P>0.05).

[0215] The above experimental results indicate that matrine can effectively reduce the inflammatory response of piglets caused by Clostridium perfringens.

[0216] 5.4 Effects of Matrine on the Expression of Clostridium perfringens and the Content of α-Toxin in the Intestinal Feces of Diarrheal Piglets Caused by Clostridium perfringens

[0217] From Table 8 and Figure 6It can be seen that the abundance of Clostridium perfringens and the content of α-toxin in the feces of piglets in the CPA group were significantly higher than those in the MT+CPA group, MT group and Control group (P<0.05). After treatment with matrine, the abundance of Clostridium perfringens and the content of α-toxin in the feces of piglets in the MT+CPA group were not significantly different from those in the MT group and Control group (P>0.05), and there was no significant difference in the abundance of Clostridium perfringens and the content of α-toxin in the feces of piglets in the MT group and Control group (P>0.05). The content of α-toxin in the jejunum, ileum, colon and muscle tissues of piglets in the CPA group was significantly higher than that in the MT+CPA group, MT group and Control group (P<0.05).

[0218] It shows that matrine can effectively inhibit the growth of Clostridium perfringens and reduce the metabolic performance of α-toxin

[0219] Table 8 Results of the expression level of Clostridium perfringens in the intestinal feces of piglets

[0220]

[0221] 5.5 Effect of matrine on the intestinal microbiological diversity of piglets with diarrhea caused by Clostridium perfringens

[0222] Combined analysis of Venn and PCoA diagrams showed that there were significant differences in the microbial communities among the four groups: control group (C), matrine group (M), Clostridium perfringens group (Cp), and Clostridium perfringens + matrine group (MCp) ( Figure 7A ,B). The relative abundance of common intestinal pathogens (mainly Firmicutes) increased in the Clostridium perfringens group and decreased after matrine prophylaxis; the relative abundance of common intestinal probiotics (mainly Bacteroidota) decreased in the Clostridium perfringens group, and the abundance of intestinal probiotics increased after matrine feeding ( Figure 7D ). Tests for differences in the α-diversity index of the intestinal flora among the four groups of piglets found significant differences in the intestinal flora among the four groups ( Figure 7C ). Matrine can increase the abundance of probiotics such as Bacteroidota (Bacteroides), Muribaculaceae, Lactobacillus (Lactobacillus), and Oscillospirales in the intestinal flora of piglets, indicating that matrine can promote the growth of intestinal probiotics. The abundance of Bacilli (Bacillus) and Clostridium in the intestines of piglets in the Clostridium perfringens group increased significantly, but after prophylaxis with matrine, the abundance of Bacilli (Bacillus) and Clostridium in the intestines of piglets decreased significantly.

[0223] It is described that matrine can improve the intestinal flora of infected animals, increase the probiotics in the intestinal flora, reduce the growth of harmful flora such as Bacillus and Clostridium, and further reduce intestinal diarrhea in piglets.

[0224] 5.6 Effects of Matrine on Intestinal Mucus Layer Barrier and Morphological Structure of Diarrheic Piglets Induced by Clostridium perfringens

[0225] The first natural immune barrier of piglet intestine is the mucus layer composed of MUC2, and its main antibody is sIgA. After extracting proteins from the intestines of four groups of piglets, we performed WB on the protein expression levels of MUC2 and sIgA.

[0226] The results showed that:

[0227] Figure 8 As shown in A, there were no significant differences in the protein expression levels of MUC2 and sIgA among the MT+CPA group, the MT group, and the Control group (P<0.05), and the protein expression levels of MUC2 and sIgA in the intestines of the three groups of piglets were significantly higher than those in the CPA group (P<0.05).

[0228] Figure 8 As shown in B, in the piglets of the Clostridium perfringens group, the intestinal villi of the jejunum, ileum, and colon tissues fell off, with a large amount of congestion, damage to the serosa layer of the small intestine, the intestinal crypts became shallower, and a large number of lymphocytes aggregated in the lamina propria of the intestinal tissue. After prevention with matrine, the damaged tissues of the jejunum, ileum, and colon ends of the piglets were restored, the number of lymphocytes in the intestinal lamina propria decreased, and the inflammatory symptoms were reduced.

[0229] Figure 8 As shown in C, in the piglets of the Clostridium perfringens group, the number of goblet cells in the jejunum, ileum, and colon decreased, and the thickness of the intestinal mucus layer decreased or disappeared. After treatment with matrine, we found that the number of goblet cells gradually increased and the thickness of the intestinal mucus layer gradually recovered.

[0230] From the above data and Figure 8 it can be seen that matrine has a good function of restoring the intestinal mucus layer defense barrier.

[0231] 5.7 Effects of Matrine on Morphological Structure of Other Organs of Piglets Infected with Clostridium perfringens

[0232] Subsequently, we further detected the heart, liver, spleen, lung, and kidney tissues of the piglets and found that there were no significant pathological changes in the heart, liver, spleen, lung, and kidney of the piglets after adding matrine to the diet ( Figure 9 ).

[0233] Compared with the group infected with Clostridium perfringens, piglets in that group showed damage to myocardial fiber tissue, necrosis of myocardial cells, and an increased gap between myocardial fibers; there was a large amount of hyperemia and congestion in the liver tissue; the area of red pulp in the spleen increased and fibrosis occurred; there was a large amount of bleeding and hyperemia in the alveoli; there was a large amount of bleeding in the kidneys and necrosis of glomeruli. In the piglets of the MT+CPA group, the damage to the heart, liver, spleen, lungs, and kidneys was repaired, similar to that of the non-infected control group, showing a trend of being in a healthy state.

[0234] It can be seen that matrine has a therapeutic and restorative effect on the damage to the heart, liver, spleen, lungs, and kidneys of piglets infected with Clostridium perfringens.

[0235] In summary, matrine improves the intestinal health of mammalian cubs in one or more of the following ways: preventing Clostridium perfringens infection or reducing the abundance of Clostridium perfringens in the digestive tract and the α-toxin to a healthy level, improving the morphological structure of the jejunum and / or ileum of weaned cubs, promoting the proliferation of goblet cells in the intestine of weaned piglets, regulating the intestinal flora of weaned piglets, preventing or treating diarrhea in piglets, improving the health of the heart, liver, kidneys, spleen, and lungs tissues of weaned cubs caused by intestinal infection, and enhancing the immunity of weaned piglets.

[0236] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Use of matrine in the preparation of a product for improving the survival rate of mammalian cubs, characterized in that: The mammalian animal is a pregnant animal infected by Gram-positive bacteria or at risk of being infected by Gram-positive bacteria; The product includes matrine.

2. Application of matrine in preparing products for improving intestinal health of mammalian cubs, characterized in that: The mammalian cub is a cub infected by Gram-positive bacteria or in the state of being infected by Gram-positive bacteria; The product includes matrine.

3. The application according to claim 1 or 2, characterized in that: The Gram-positive bacteria is Clostridium perfringens.

4. The application according to claim 1 or 2, characterized in that: The product further includes at least one of dandelion, other edible excipients, and solvents.

5. The application according to claim 4, wherein: The daily oral dose of matrine in the product is below 120 mg / kg.

6. The application according to claim 5, wherein: For every 1 mg of dandelion in the product, the corresponding amount of matrine is 1.4 - 1.6 mg.

7. The application according to claim 1 or 2, characterized in that: The preparation method of the matrine: Dry and crush the Sophora alopecuroides seeds, extract by heat reflux, and concentrate to obtain a pure extract; dissolve the pure extract with an acid solution, filter by suction to remove the residue, and obtain an acid aqueous solution; extract according to the volume ratio of dichloromethane:acid aqueous solution = 1:3 until the dichloromethane layer is clear and transparent, then recover the acidic dichloromethane part under reduced pressure; subsequently, adjust the pH value of the acidic dichloromethane part to 9 - 11 with a solid base; extract again with dichloromethane and n-butanol, and recover the solvent under reduced pressure to obtain an extract of the basic dichloromethane and n-butanol parts. The obtained extract of the basic dichloromethane and n-butanol parts is the crude extract of Sophora alopecuroides alkaloids.

8. The application according to claim 1 or 2, characterized in that: The mammalian animals include farmable livestock and laboratory animals.

9. The application according to claim 8, wherein: The livestock are porcine animals; the laboratory animals are rodents, non-human primates, canines, rabbits, and porcine animals.

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