Application of cortex juglandis mandshuricae polysaccharide in preparation of product for relieving neurotoxicity caused by ochratoxin A

Abstract

The invention discloses an application of juglans regia polysaccharide in preparation of a product for relieving neurotoxicity caused by ochratoxin A. The monosaccharide composition of the mangnolia mandshurica polysaccharide (WGP) comprises nine monosaccharides including fucose, rhamnose, arabinose, galactose, glucose and the like, and the main chain structure is formed by alternately connecting alpha-D-GalpA and alpha-L-Rhap. The WGP can significantly improve the neural behavior, effectively regulate the expression of a TLR4 / MYD88 pathway, a Wnt signal pathway and a neurotransmitter receptor gene, especially reverse the abnormal expression of AQP1, effectively relieve nerve injury, and open up a new direction for further application of the WGP.

Description

Technical Field

[0001] The present invention relates to the fields of traditional Chinese medicine and feed additives, and particularly relates to the application of a natural polysaccharide (walnut green husk polysaccharide, WGP) extracted from walnut green husks in the preparation of a product for alleviating the neurotoxicity caused by ochratoxin A (OTA). Background Art

[0002] Ochratoxin A (OTA) is a secondary metabolite produced by Aspergillus fungi (such as Penicillium and Aspergillus ochraceus), and widely contaminates grains, feeds and foods. OTA has significant neurotoxicity, hepatorenal toxicity and growth inhibitory effects on poultry such as chicks. OTA invading the body is distributed to the liver, kidney and brain tissue through blood circulation, causing multi-organ damage. In the liver and kidney, after OTA accumulates, it induces apoptosis by inducing oxidative stress and mitochondrial dysfunction; while in the nervous system, OTA can penetrate the blood-brain barrier, activate the TLR4 / NF-κB pathway in the hippocampal region, and trigger neuroinflammation and cognitive dysfunction. In addition, OTA also inhibits the proliferation of T lymphocytes and antibody production, thereby increasing the host's susceptibility to pathogens.

[0003] Currently, the intervention methods for OTA poisoning mainly rely on adsorbents (such as montmorillonite) and chemical drugs (sodium oxide, chlorine dioxide, etc.). However, the adsorbent can only physically adsorb part of OTA, and cannot reverse the damage to the intestinal barrier and flora caused by the absorbed toxin, and long-term use will interfere with the absorption of nutrients and minerals; in addition, chemical drugs are costly, have large side effects, and have limited effects. Therefore, developing a natural alternative intervention method with high efficiency, low cost and no toxic side effects, especially plant polysaccharide substances with wide sources and biological activities, has become an important direction in the current research on the prevention and treatment of mycotoxin poisoning. Summary of the Invention

[0004] Object of the Invention: The technical problem to be solved by the present invention is to provide the application of walnut green husk polysaccharide (WGP) in the preparation of a product for alleviating ochratoxin A, aiming at the deficiencies of the existing technology.

[0005] To solve the above technical problems, the present invention discloses the following technical solutions:

[0006] In the first aspect, the present invention discloses the application of walnut green husk polysaccharide in the preparation of a product for protecting nerves.

[0007] In the second aspect, the present invention discloses a product for protecting nerves, and the product includes walnut green husk polysaccharide.

[0008] Walnut green husk is a processing waste of walnuts and is also a traditional Chinese medicine called Qinglongyi, which has the advantages of wide sources and low cost. Walnut green husk polysaccharide (WGP) is a natural polysaccharide extracted from walnut green husks and has biological activities such as anti-inflammatory, antioxidant and immunomodulatory effects.

[0009] In some embodiments, the chemical structure of the polysaccharide from Prunus davidiana Carr. peel (WGP) is as follows:

[0010] (a) Molecular weight: The weight-average molecular weight (Mw) determined by GPC-MALLS is 10.8 - 14.8 kDa, such as 11.8 - 15.8 kDa, such as 12.77 kDa, and the number-average molecular weight (Mn) is 6.3 - 10.3 kDa, such as 7.3 - 9.3 kDa, such as 8.341 kDa.

[0011] (b) Monosaccharide composition: HPLC analysis shows that WGP is composed of 9 monosaccharides: fucose (Fuc), rhamnose (Rha), arabinose, galactose, glucose, xylose, mannose, galacturonic acid (GalA), and glucuronic acid. The molar ratio of each component is 0.70 - 0.90:23.92 - 25.92:2.79 - 4.79:9.72 - 11.72:4.98 - 5.98:0.39 - 0.59:0.52 - 0.72:44.57 - 48.57:5.11 - 7.11, such as 0.80:24.92:3.79:10.72:5.98:0.49:0.62:46.57:6.11, where GalA accounts for 44.5% - 48.5%, such as 46.57%, indicating that it is an acidic heteropolysaccharide.

[0012] (c) Main chain structure: NMR analysis confirms that the main chain is composed of →4)-α-D-GalpA-(1→ (51.726%) and →2)-α-L-Rhap-(1→ (9.634%) alternatingly connected, and the end is terminated by α-D-GalpA-(1→. The sugar chain structure is as follows:

[0013] In some embodiments, the polysaccharide from Prunus davidiana Carr. peel is prepared by the water extraction and alcohol precipitation method for Prunus davidiana Carr. peel, and the specific steps include:

[0014] (1) Extract Prunus davidiana Carr. peel after removing fat-soluble impurities with water to obtain an aqueous extract; concentrate the aqueous extract to obtain a concentrated solution;

[0015] (2) Precipitate the obtained concentrated solution with alcohol and centrifuge to obtain a precipitate;

[0016] (3) Redissolve the obtained precipitate in water and remove alcohol to obtain a crude polysaccharide;

[0017] (4) Remove protein impurities from the crude polysaccharide and freeze-dry to obtain the polysaccharide from Prunus davidiana Carr. peel;

[0018] (5) Preliminary purification of the obtained polysaccharide from Prunus davidiana Carr. peel by anion exchange chromatography, and collect the eluate;

[0019] (6) Further subject the polysaccharide with the highest content to gel column chromatography to obtain highly pure Qinglongyi polysaccharide.

[0020] In step (1), the walnut green peel is defatted by ethanol reflux (heating to boiling in a water bath for 2 h) to obtain Qinglongyi with fat-soluble impurities removed.

[0021] In step (1), the Qinglongyi with fat-soluble impurities removed is subjected to first water extraction with the first water to obtain a first water extract and a first residue; the obtained first residue is added with the second water for second water extraction to obtain a second water extract; the first water extract and the second water extract are combined and concentrated to obtain a concentrated solution.

[0022] Among them, control the addition amount of the first water so that the mass-volume ratio of the Qinglongyi without removing fat-soluble impurities to the first water is 1 kg: 8 - 12 L, such as 1 kg: 10 L.

[0023] Among them, control the addition amount of the second water so that the mass-volume ratio of the Qinglongyi without removing fat-soluble impurities to the second water is 1 kg: 4 - 8 L, such as 1 kg: 6 L.

[0024] Among them, control the concentration so that the volume of the concentrated solution is 10% - 14% of the total volume of the first water and the second water.

[0025] In step (2), the alcohol precipitation is carried out with a 75 - 80% ethanol solution, and the volume ratio of the ethanol solution to the concentrated solution is 1: 3 - 5; the rotation speed of the centrifugation is 7000 - 9000 rpm, the temperature of the centrifugation is 2 - 6 °C, and the time of the centrifugation is 3 - 7 min.

[0026] In step (4), protein is removed by the Sevag method (chloroform: n-butanol = 4: 1, repeated 6 times).

[0027] In step (5), the anion exchange chromatography is DEAE Sepharose Fast Flow.

[0028] In step (5), the preliminary purification of the Qinglongyi polysaccharide by anion exchange chromatography is specifically as follows: configure the Qinglongyi polysaccharide into an aqueous solution of Qinglongyi polysaccharide with a concentration of 7 - 13 mg / mL, centrifuge, take the supernatant and pass it through an anion exchange column, the flow rate of the supernatant is 3 - 5 mL / min, and gradient elution is carried out successively with pure water, 0.08 - 0.12 M, 0.18 - 0.22 M, and 0.28 - 0.32 M NaCl solutions, collect the eluate, concentrate the eluate of the polysaccharide with the highest content, remove salt with a dialysis bag, and then carry out gel column chromatography. Among them, the cut-off amount of the dialysis bag is 2900 - 3100 Da.

[0029] In step (6), the gel column chromatography is Sephadex G-100 column chromatography.

[0030] In step (6), the gel column chromatography is specifically as follows: the polysaccharide with the highest content is configured into a polysaccharide aqueous solution of 12-18 mg / mL, centrifuged, and the supernatant is passed through a gel chromatography column at a supernatant flow rate of 0.5-1.5 mL / min, eluted with pure water, and the amount of water used is 1-2 column volumes. The eluate is collected, concentrated, and freeze-dried to obtain high-purity WGP.

[0031] In some embodiments, the method for protecting neurons is for reducing the neurotoxicity caused by ochratoxin A, such as reducing the neurotoxicity of ochratoxin A to the hippocampus.

[0032] Among them, the nerve protection includes downregulating TLR4, MYD88, P65, restoring β-catenin, and restoring neurotransmitter receptor gene expression.

[0033] The WGP alleviates hippocampal damage caused by OTA poisoning through at least one of the following mechanisms:

[0034] (a) Inhibits the gene expression of TLR4, MYD88, and P65 in the TLR4 / MYD88 pathway and restores the expression of the antioxidant stress genes NRF2 and HO-1;

[0035] (b) Restoration of gene expression of Wnt4, Wnt6, β-catenin, and GSK3α in the Wnt signaling pathway;

[0036] (c) Restoration of the expression of BDNF, IGF-1, IGF-2, and their receptors IGF-1R and IGF-2R;

[0037] (d) Alleviated OTA-induced neuroinflammation, oxidative stress, and cell apoptosis, and restored the expression of antioxidant enzyme genes CAT, SOD, and GPX-1;

[0038] (e) Regulate the expression of neurotransmitter receptor genes, such as 5-HT1A and 5-HT1B;

[0039] (f) Reduce the abnormal expression of AQP1 and AQP4 and restore the normal function of water channel proteins.

[0040] In the present invention, the product has at least one of the following effects in addition to inhibiting neurotoxicity:

[0041] Improve growth performance,

[0042] Improve abnormal serum indicators,

[0043] Relieve oxidative stress.

[0044] Among them, the improvement of growth performance includes increasing body weight, reducing feed-to-meat ratio, improving metabolic disorders, and regulating endocrine balance.

[0045] Among them, the improvement of abnormal serum indicators includes lowering AST levels, restoring AST / ALT ratios, and restoring GLOB, TP, GLU and inflammatory factor levels.

[0046] In the present invention, the WGP is administered through drinking water or feed at a dosage of 400-800 mg / kg.

[0047] This invention is the first to isolate an acidic heteropolysaccharide (WGP) with a unique structure from walnut green peel, and systematically reveals its role in effectively alleviating the neurotoxicity caused by OTA poisoning, showing a significant neuroprotective effect, filling the technical gap in the field of natural polysaccharides in alleviating OTA-induced neurotoxicity.

[0048] Beneficial effects:

[0049] The research in this paper shows that WGP can effectively restore the decline in growth performance and behavioral abnormalities in chicks caused by OTA, and can significantly alleviate the neurotoxic damage caused by OTA by regulating key mechanisms such as neuroinflammation, oxidative stress, cell apoptosis, neuro-neuroendocrine system, Wnt signaling pathway, TLR4 / MYD88 pathway and growth factors, showing significant neuroprotective effects, indicating that WGP has potential application value in alleviating OTA poisoning in chicks. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0051] Figure 1Extraction, purification, and structural characterization of Qinglongyi polysaccharide (WGP). (A) Scanning electron microscopy (SEM) images of WGP at ×500 and ×20.0K magnifications; (B) Isolation and purification of WGP by anion exchange chromatography (DEAE cellulose-52); (C) Elution profile of WGP on a Sephadex G-100 column; (D) GPC-MALLS elution profile of WGP molecular weight distribution. The red line represents the multi-angle laser light scattering (LS) signal; the intensity of the scattered light is proportional to the molecular size and molecular weight of the substance; the blue line represents the differential signal (RI); the response value depends on the refractive index change of the post-column effluent and is related to the type, concentration, and molecular weight of the substance; the black line represents the molecular weight fitted by the two signals; (E–F) Monosaccharide composition of the standard mixture and WGP. (1, Fuc, fucose; 2, Rha, rhamnose; 3, Ara, arabinose; 4, Gal, galactose; 5, Glc, glucose; 6, Xyl, xylose; 7, Man, mannose; 8, Fru, fructose; 9, Rib, ribose; 10, Gal-UA, galacturonic acid; 11, Gul-UA, guluronic acid; 12, Glc-UA, glucuronic acid; 13, Man-UA, mannuronic acid).

[0052] Figure 2 Structural characterization of WGP. (A) 1 H NMR; (B) 13 C NMR; (C) 1H-1H COSY; (D) HSQC; (E) NOESY; (F) HMBC; (G) Structure of WGP.

[0053] Figure 3 Effects of different treatments on mouse body weight, average daily food intake, and cortisol levels in the examples. (A) Weight changes in OTA-WAS mice; (B) Average daily food intake in OTA-WAS mice; (C) Serum cortisol levels in OTA-WAS mice; (D) Weight changes in OTA-WGP mice; (E) Average daily food intake in OTA-WGP mice; (F) Serum cortisol levels in OTA-WGP mice. Results are mean ± standard error, *P < 0.05, **P < 0.01, ***P < 0.001, n = 8; Identical letters indicate P > 0.05 between two groups, otherwise P < 0.05.

[0054] Figure 4 The effects of WGP on abnormal changes in growth curve, feed intake, and feed-to-meat ratio in chicks induced by OTA in the examples are shown. A. Growth curve; B. Feed intake; C. Feed-to-meat ratio; Results are mean ± SE, *P < 0.05, **P < 0.01, ***P < 0.001, n = 13.

[0055] Figure 5The results of behavioral testing of WGP on OTA-treated chicks in the examples are shown. A. Time for chicks to take their first step in the open field test; B. Number of steps in the mine field test; C. Back-rolling time. Results are mean ± standard error, *P < 0.05, **P < 0.01, ***P < 0.001, n = 15.

[0056] Figure 6 Serum biochemical analysis of WGP-treated OTA chicks in the examples. A. Aspartate aminotransferase (AST); B. Alanine aminotransferase (ALT); C. AST / ALT; D. Globulin (GLOB); E. Total protein (TP); F. Albumin (ALB); G. Triglycerides (TG); H. Lactate dehydrogenase (LDH); I. Glucose (GLU); Results are mean ± standard error, *P < 0.05, **P < 0.01, ***P < 0.001, n = 15.

[0057] Figure 7 This is a comprehensive analysis of serum oxidative stress, inflammatory factors, and neuroendocrine indicators in OTA-treated chicks treated with WGP. Malondialdehyde (MDA); B. Total antioxidant capacity (T-AOC); C. Superoxide dismutase (SOD); D. Glutathione peroxidase (GSH-Px); E. Tumor necrosis factor-α (TNF-α); F. Interleukin-6 (IL-6); G. Interleukin-1β (IL-1β); H. Diamine oxidase (DAO); I. Cortisol (CORT); J. Melatonin; K. 5-hydroxytryptamine (5-HT); L. Tryptophan (TRP); Results are mean ± SE, *P < 0.05, **P < 0.01, ***P < 0.001, n = 8.

[0058] Figure 8 The expression levels of genes related to inflammatory factors, antioxidant enzymes, apoptosis, TLR4 / Myd88 pathway, Wnt pathway, AQP proteins, neurotransmitter receptors, and cell growth factors in the hippocampus of chicks treated with WGP in the examples are shown. A. Expression levels of genes related to inflammatory factors; B. Expression levels of genes related to antioxidant enzymes; C. Expression levels of genes related to apoptosis; D. Expression levels of genes related to AQP proteins and Wnt signaling pathways; E. Expression levels of genes related to TLR4 / Myd88 signaling pathways and mitochondrial function; F. Expression levels of genes related to neurotransmitter receptors and cell growth factors; Results are mean ± standard error, *P < 0.05, **P < 0.01, ***P < 0.001, * compared with the control group; #P < 0.05, ##P < 0.01, ###P < 0.001, # compared with the OTA group; n = 7-8. DETAILED DESCRIPTION

[0059] The present invention can be better understood according to the following embodiments. However, those skilled in the art can easily understand that the content described in the embodiments is only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0060] In the following embodiments, the experimental methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0061] The extraction process of walnut shell polysaccharide (WAS) described in the following embodiments is the same as that in Example 1, except that the raw material is replaced with walnut shell.

[0062] In the following Examples 2 and 3, the WGP is the high-purity WGP prepared in Example 1 unless otherwise specified.

[0063] Example 1:

[0064] 1. Extraction and purification of polysaccharide

[0065] (1) Place 1.5 kg of Qinglongyi (purchased from Bozhou Hengyi Traditional Chinese Medicine Technology Co., Ltd.) in a round-bottom flask, add sufficient industrial alcohol until Qinglongyi is completely immersed. Then, use a condensing reflux system to heat it in a water bath until boiling and continue heating for 2 hours. This step ensures the purity of the subsequent extract by repeatedly removing fat-soluble substances. After completion, discard the alcohol and dry it in an oven again. Add the dried Qinglongyi to 15 L of deionized water, heat it to boiling, and perform the first water extraction. The extract is concentrated to 4 L, and the first water extract is collected. Then, take the residue of Qinglongyi, add 9 L of deionized water, repeat the boiling extraction, concentrate it to 3 L, and filter to obtain the second water extract. Combine the two water extracts and concentrate them to 3 L by boiling to prepare a high-concentration water extract for the subsequent alcohol precipitation step.

[0066] (2) Take the concentrated water extract and perform alcohol precipitation according to the volume ratio of the water extract to the 75% ethanol solution of 4:1 to obtain relatively pure crude polysaccharide of Qinglongyi.

[0067] (3) The crude polysaccharide obtained after alcohol precipitation is centrifuged at 8000 rpm and 4 °C for 5 minutes, the supernatant is discarded, the precipitate is redissolved in water and heated to boiling using an induction cooker to volatilize the remaining ethanol, and the concentrated crude polysaccharide is obtained.

[0068] (4) Prepare Savage reagent by mixing chloroform and n-butanol in a volume ratio of 4:1 for removing proteins from crude polysaccharides of Qinglongyi. Take 400 mL of concentrated crude polysaccharides and 100 mL of Savage reagent, place them in a 1-L conical flask, seal it, shake it on a shaker at a speed of 200 rpm for 30 minutes, let it stand for 20 minutes, and then take the supernatant. Repeat this process 6 times to remove protein impurities and obtain crude polysaccharides with proteins removed. Finally, heat the supernatant after protein removal using an induction cooker until the residual organic reagent completely evaporates to finally obtain freeze-dried WGP.

[0069] (5) The obtained WGP is preliminarily purified by anion exchange chromatography (DEAE Sepharose Fast Flow). Take an appropriate amount of crude polysaccharide sample, dissolve it in pure water to prepare a polysaccharide mother liquor with a concentration of about 10 mg / mL, centrifuge at 10000 g for 10 min, take the supernatant and pass it through an ion exchange column for purification at a flow rate of 4 mL / min; successively elute with pure water, 0.1 M, 0.2 M, and 0.3 M NaCl solutions in a gradient manner, collect one tube every 15 mL, and collect all eluates. Obtain eluates containing three polysaccharides, P1, P2, and P3, and collect 30 - 40 tubes of eluates (i.e., eluates containing P2 polysaccharide) ( Figure 1 B), determine the total sugar content of the eluates in each collection tube by the sulfuric acid-anthrone method, and draw an ion purification elution curve. Combine the eluates in each collection tube corresponding to the same elution peak, rotary evaporate and concentrate to 1 / 5 of the original volume. Dialyze through a 3000 Da dialysis bag for 48 - 72 h to remove salts. Identify the content and purity of the polysaccharides after ion purification by the sulfuric acid-anthrone method.

[0070] (6) Further purify by Sephadex G-100 gel column chromatography. Take the polysaccharide sample after ion purification, add pure water to prepare a polysaccharide mother liquor with a concentration of about 15 mg / ml. Centrifuge at 10000 g for 10 min, take the supernatant and pass it through a gel chromatography column for separation and purification at a flow rate of 1 ml / min; elute with pure water for 1.5 times the column volume, collect one tube every 12 mL, and collect all eluates. Determine the total sugar content of the eluates in each collection tube by the sulfuric acid-anthrone method, and draw a gel purification elution curve. Combine the eluates in each collection tube corresponding to the same elution peak, rotary evaporate and concentrate to 1 / 5 of the original volume. Freeze-dry, and identify the content and purity of the polysaccharides after gel purification by the sulfuric acid-anthrone method, etc., to further obtain high-purity WGP( Figure 1 C, that is, P2 polysaccharide).

[0071] 2. Structure determination of high-purity WGP

[0072] The microstructure of WGP was observed using a field emission scanning electron microscope (SEM). After the sample was coated with gold, it was detected at an acceleration voltage of 5 kV. The molecular weight analysis of WGP was carried out by gel permeation chromatography - multi - angle laser light scattering (GPC - MALLS). WGP was dissolved in 0.1 M NaNO3 solution at a concentration of 1 mg / mL, filtered and then separated, and finally the molecular weight was determined. The monosaccharide composition of WGP was analyzed by high - performance liquid chromatography (HPLC), using Dionex TM CarboPac TM PA20 (150 * 3.0 mm, 10 μm) liquid chromatography column; the injection volume was 5 μL. Mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), the flow rate was 0.5 mL / min; the column temperature was 30 °C, and the elution gradient was: 0 min, A phase / B phase / C phase (95:5:0, V / V), 26 min, A phase / B phase / C phase (85:5:10, V / V), 42 min, A phase / B phase / C phase (85:5:10, V / V), 42.1 min, A phase / B phase / C phase (60:0:40, V / V), 52 min, A phase / B phase / C phase (60:40:0, V / V), 52.1 min, A phase / B phase / C phase (95:5:0, V / V), 60 min, A phase / B phase / C phase (95:5:0, V / V). Finally, the structure of WGP was analyzed by nuclear magnetic resonance (NMR). 30 mg of WGP was dissolved in 0.5 mL of D2O, and 1H, 13C one - dimensional nuclear magnetic resonance (NMR) and 1H - 1H COSY, HSQC, HMBC, NOESY two - dimensional nuclear magnetic resonance analyses were carried out using a Bruker 600 MHz NMR instrument at 25 °C.

[0073] Scanning electron microscopy (SEM) showed that WGP presented an irregular flaky aggregated structure ( Figure 1 A, magnified at ×500 and ×20K). Gel permeation chromatography - multi - angle laser light scattering (GPC - MALLS) analysis showed that the weight - average molecular weight (Mw) of WGP was 12.77 kDa, and the number - average molecular weight (Mn) was 8.341 kDa( Figure 1D), with an elution peak time of 53.00 - 69.00 minutes, which is within the molecular weight range of bioactive polysaccharides. High-performance liquid chromatography (HPLC) monosaccharide composition analysis showed that WGP was composed of fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), galacturonic acid (GalA) and glucuronic acid (GlcA), with a molar ratio of 0.80:24.92:3.79:10.72:5.98:0.49:0.62:46.57:6.11( Figure 1 E - F), in which GalA had the highest proportion (46.57%), indicating that it was an acidic polysaccharide. Nuclear magnetic resonance (NMR) spectroscopy( 1 H, 13 C, COSY, HSQC, HMBC, NOESY) analysis showed that: the anomeric hydrogen signals (δ H 5.02, 5.00, 5.08, 4.58) and corresponding anomeric carbon signals (δ C 99.01, 103.87) of WGP were assigned to H1 / C1 of sugar residues A - H( Figure 2 A - B); the COSY spectrum resolved the H2 - H5 signals (δ H 5.01 / 3.68, 3.68 / 3.92, etc.) of residue A, such as those of residues A (δ H 3.68, 3.92, 4.34, 4.69), residues B (δ H 3.67, 3.92, 4.04, 5.09) and residues C (δ H 3.94, 3.71, 3.84, 3.72, 1.18) through continuous cross - peaks, the HSQC spectrum clarified the H / C chemical shift assignments of each residue (A, B, C) through the δH / C correspondence (such as H1 / C1 of residue A = 5.01 / 99.02 to H5 / C5 = 4.69 / 71.28), and the NOESY and HMBC cross - peaks verified the - A - A -, - A - B -, - A - C -, - B - A - linkage patterns( Figure 2 A - F). Based on the above results, WGP was identified as an acidic heteropolysaccharide with a weight - average molecular weight of 12.77 kDa, and it was speculated that its main chain was composed of →4)-α - D - GalpA linked with a small amount of →2)-α - L - Rhap, and the terminal contained α - D - GalpA( Figure 2G). The high proportion of galacturonic acid (GalA, 46.57%) in the polysaccharide structure endows it with strong acidic characteristics, and the coexistence of →4)-GalpA main chain and →2)-Rhap side chain may form a specific spatial conformation. The charge characteristics and sugar chain branching patterns of acidic polysaccharides are generally considered to be related to their potential biological activities (such as binding metal ions and regulating cell surface receptor interactions). In this study, the fine structure of WGP was systematically analyzed, providing a key structural basis for subsequent exploration of its interaction mechanism with biomolecules (such as neurotransmitters).

[0074] Example 2: Mouse animal experiment

[0075] 120 six-week-old C57BL / 6 mice were divided into six groups: control group (CON), mice were gavaged with 0.1 M sodium bicarbonate and deionized water; OTA treatment group (OTA), mice were gavaged with 2 mg / kg body weight of OTA and deionized water; low-dose WGP treatment group (OTA+L-WGP), mice were gavaged with 2 mg / kg body weight of OTA and deionized water and 200 mg / kg body weight of WGP; medium-dose WGP treatment group (OTA+M-WGP), mice were gavaged with 2 mg / kg body weight of OTA and deionized water and 400 mg / kg body weight of WGP; high-dose WGP treatment group (OTA+H-WAS), mice were gavaged with 2 mg / kg body weight of OTA and deionized water and 800 mg / kg body weight of WGP. Mice were continuously gavaged for 7 days. The experimental operation of WAS was the same as above.

[0076] Effects of different treatments on mouse body weight, average daily food intake and serum cortisol level. As Figure 3 A and Figure 3 D show that compared with the control group, the body weight of the OTA treatment group decreased day by day starting from the second day, and this abnormal situation was alleviated to varying degrees after treatment with WGP and WAS, and showed a dose-dependent manner, among which the OTA+H-WGP in the WGP treatment group had the best alleviating effect. Figure 3 B and Figure 3 E indicate that the average daily food intake of the OTA treatment group was significantly lower than that of the control group, and after treatment with WGP and WAS, there was a certain recovery effect on food intake, and the recovery effect of the WGP group was better than that of the WAS group. Figure 3 C and Figure 3 F show that the cortisol level in the OTA group increased significantly, and OTA poisoning significantly increased the cortisol level in mice, indicating that it may have caused endocrine disorders through stress response or hormone regulation mechanism. The WGP group alleviated the abnormal increase in cortisol level to varying degrees, while the WAS group had no significant change. The above results show that WGP and WAS have a certain regulatory effect on the body weight change, food intake and cortisol level caused by OTA, and the effect of WGP is better than that of WAS.

[0077] Example 3: Chicken Animal Experiment

[0078] 1. Grouping

[0079] One hundred 1-day-old male Ross 308 broilers were used. All experimental procedures followed the guidelines approved by the Animal Ethics Committee of Nanjing Agricultural University (IACUC approval number: 2016YFD0500502). They were housed in the poultry breeding room of Nanjing Agricultural University and allowed to adapt to the environment for 3 days. During the entire experimental period, they had free access to water and food. They were fed a basal diet according to their different ages (the specific formula of the known diet). At the same time, the chicks were weighed, wing-tagged, and grouped. The chicks were randomly divided into 4 groups. Seven days before the experiment, the animals in each group were separately housed in a single cage. From the 8th day, each group of animals was further divided into 3 replicate cages, with 8 - 9 chickens in each cage. The chickens in the CON group were orally administered pure water every day, the chickens in the OTA group were orally administered an OTA suspension containing 50 μg / kg (diluted with pure water after being dissolved in dimethyl sulfoxide), the OTA + WGP low-dose group was orally administered 50 μg / kg OTA and 400 mg / kg WGP, and the OTA + WGP high-dose group was orally administered 50 μg / kg OTA and 800 mg / kg WGP. Among them, OTA was administered first, and WGP was administered about 1 hour later. The feed intake was monitored and recorded at 9:00 am every day, and the weight was measured every three days. The chickens were administered the drugs for 21 days. On the 21st day, they were fed normally with free access to food. On the 23rd day, behavioral tests were conducted. Finally, they were fasted for 12 h, weighed, and dissected and sampled according to the requirements of the experimental animal ethics guidelines of Nanjing Agricultural University.

[0080] 2. Testing

[0081] (1) Analysis of chick growth curve, feed intake, and feed conversion ratio

[0082] The total weekly feed intake was statistically analyzed, and the average weekly feed intake was calculated based on the number of chickens in each cage. The feed conversion ratio was obtained by recording the weight gain on the 7th and 21st days and the total feed intake during the corresponding time period.

[0083] (2) Behavioral tests

[0084] The behavioral tests included the balance beam test, open field test, and tonic immobility test. The balance beam test used an elevated narrow balance beam (15 cm wide, 150 cm long, 22 cm high) to evaluate the fear-related behaviors of chicks, and the residence time and walking distance on the balance beam were recorded. If the chicks dared not walk, the score was 0 points and the recorded time was 120 seconds. The open field test evaluated the autonomous behavior, exploratory behavior, and tension of animals in a new environment. The test site was a 60 cm × 60 cm × 30 cm square, and behavioral indicators such as the number of times the chicks entered the central area, residence time, and movement distance were recorded. The observation duration was 10 minutes. The tonic immobility test reflected the stress response of animals. The broilers were placed on their backs in a quiet environment and their chests and heads were gently pressed until they entered a rigid state, and the duration was timed and recorded, with a maximum of 3 minutes. The longer the tonic immobility time, the higher the fear level of the broilers.

[0085] (3) Biochemical parameter analysis

[0086] Using commercial kits provided by Ningbo Meikang Biotech Co., Ltd., the contents of aspartate aminotransferase (AST), alanine aminotransferase (ALT), globulin (GLOB), total protein (TP), albumin (ALB), triglyceride (TG), lactate dehydrogenase (LDH), and glucose (GLU) in serum were determined.

[0087] (4) Oxidative stress level

[0088] Using commercial kits provided by Nanjing Jiancheng Bioengineering Institute and according to the corresponding instructions, the levels of malondialdehyde (MDA, A003-1-2), superoxide dismutase (SOD, A001-3-2), total antioxidant capacity (T-AOC, A015-2-1), and glutathione peroxidase (GSH-Px, A005-1-2) activities in serum were determined.

[0089] (5) Inflammatory factor analysis

[0090] Using an enzyme-linked immunosorbent assay (ELISA) kit (Multisciences Biotech) and according to the corresponding instructions, the levels of tumor necrosis factor α (TNF-α, EK282), interleukin-6 (IL-6, EK206), and interleukin-1β (IL-1β, EK201B) in serum were determined.

[0091] (6) Neuroendocrine index analysis

[0092] Corticosterone (CORT, E-OSEL-Ch0002), Melatonin (E-EL-M0788), 5-Hydroxytryptamine (5-HT, E-EL-0033), Tryptophan (TRP, T486389-1kit), Diamine Oxidase (DAO, BC1285)

[0093] (7) qPCR was used to measure the expression levels of related mRNAs

[0094] Hippocampal tissues from the control group, OTA-treated group, and low-dose WGP-treated group were used for the experiment. Total RNA was extracted from hippocampal tissues using TRIzol reagent (Tsingke, Beijing, China). 1 μg of RNA was taken from each sample and reverse-transcribed using a commercial reverse transcription kit (Vazyme, China). Quantitative PCR was performed on a QuantStudio 6Flex system (Applied Biosystems, USA), and the cDNA was diluted at a ratio of 1:10 for analysis. The data were analyzed using the 2 -ΔΔCT method and expressed relative to the control group. The primer sequences of the target genes are shown in Table 1

[0095] Table 1 Primer sequences for RT-PCR

[0096]

[0097]

[0098] Statistical analysis

[0099] Statistical analysis of the data was performed using GraphPad Prism 8.0 (GraphPad Software, USA) and SPSS 20.0 (IBM, Armonk, NY). The data are expressed as mean ± SEM. T-tests or two-way analyses were used for differential analysis, and differences were considered statistically significant when P < 0.05

[0100] 3. Experimental results

[0101] (1) Thirteen chickens were selected from each group to measure the growth curve, feed intake, and feed conversion ratio of chicks. As Figure 4As shown in the figure, the growth curve in Figure A shows that there were no significant differences in the body weights of chicks in each group (n = 13) at the initial stage of the experiment (the 1st week). However, starting from the 2nd week, the weight gain in the OTA group was significantly lower than that in the control group. By the 21st day, compared with the control group, the body weight in the OTA group decreased significantly (P < 0.001), and the body weights in the low-dose and high-dose WGP groups increased significantly compared with the OTA group (P < 0.001), indicating that WKP at both doses effectively reversed the abnormal decrease in the body weight of chicks caused by OTA. In Figure B, there were no significant differences in the feed intakes of each group in the first week, but starting from the second week, the feed intake in the OTA group decreased significantly (P < 0.05). The feed intake in the low-dose WGP group recovered significantly (P < 0.05), while there was no effective improvement in the high-dose group (P > 0.05). Figure C shows that there were no significant differences in the feed-to-gain ratios of each group on the 7th day, but by the 21st day, the feed-to-gain ratio in the OTA group increased significantly (P < 0.05). The low-dose WGP group effectively reduced the feed-to-gain ratio (P < 0.05), while there was no obvious change in the high-dose group, but a trend of recovering the abnormal change could also be seen (P > 0.05). In summary, OTA significantly inhibited the growth of chicks, reduced the feed intake and feed conversion rate, while the low-dose WGP could significantly reverse these abnormal changes, and the effect of the high-dose group was relatively limited.

[0102] (2) Fifteen chickens were selected from each group for behavioral tests. As Figure 5 shown, the results of the open-field experiment in Figure A show that the time for chicks in the OTA group to take the first step was significantly prolonged (P < 0.01), indicating that their motor ability was inhibited. In Figure B, the number of steps in the OTA group decreased significantly (P < 0.05), suggesting that OTA had a negative impact on the motor function of chicks. The low-dose WGP group significantly recovered in both of these two indicators (P < 0.05). Although there was a certain trend of recovery in the time for the high-dose WGP group to take the first step, it did not reach a significant difference (P > 0.05), while the number of steps recovered significantly (P < 0.05), indicating that the high-dose WGP had certain potential for improving motor function. The tonic test in Figure C shows that the time for the chicks in the OTA group to recover from dorsal turning was significantly prolonged (P < 0.01), suggesting that their motor coordination ability was impaired. The dorsal turning time in the low-dose WGP group was effectively recovered (P < 0.05), approaching the level of the control group, indicating that the low-dose WGP could effectively improve the neurological dysfunction caused by OTA. Although the high-dose WGP group showed a certain trend of recovery, it did not reach a significant difference (P > 0.05). In summary, OTA significantly affected the motor ability and neurological function of chicks, while the low-dose WGP could significantly improve these behavioral abnormalities, showing a strong neuroprotective effect.

[0103] (3) Serum biochemical indicators such as Figure 6As shown, chicks treated with OTA showed significant differences in multiple serum biochemical parameters compared to the control group. Specifically, OTA treatment significantly increased AST levels (P < 0.05) and abnormally decreased ALT levels (P < 0.01), resulting in an elevated AST / ALT ratio (P < 0.001), suggesting that OTA may have caused liver damage. Furthermore, GLOB and TP levels were significantly decreased in the OTA group (P < 0.05), indicating that OTA may affect protein synthesis or accelerate metabolism, thereby disrupting normal growth and immune function in chicks. A significant decrease in GLU levels (P < 0.05) also suggests that OTA interferes with energy metabolism in chicks. Other serum levels of ALB, TG, and LDH did not show significant differences, indicating that OTA has a minimal effect on these metabolic pathways. Abnormal serum biochemical parameters were significantly improved after treatment with low-dose WGP. Compared with the OTA group, the AST level and AST / ALT ratio in the low-dose WGP group were significantly restored (P < 0.05), indicating that WGP has a protective effect in alleviating OTA-induced liver damage. GLOB, TP, and GLU levels also significantly recovered (P < 0.05), suggesting that low-dose WGP can effectively ameliorate OTA-induced protein synthesis impairment and energy metabolism abnormalities. However, the effect of low-dose WGP on ALT, ALB, TG, and LDH levels was relatively limited, indicating that its protective effect is primarily focused on the recovery of liver damage and metabolic disorders. Serum biochemical parameters also showed significant improvement in the high-dose WGP treatment group. In particular, GLOB and TP levels were significantly restored compared to the OTA group (P < 0.01), further demonstrating the important role of WGP in restoring protein synthesis and immune function.

[0104] In summary, OTA significantly altered multiple serum biochemical parameters, particularly AST, GLOB, TP, and GLU levels, suggesting an impact of OTA on liver and metabolic function. WGP was able to effectively improve these parameters, with low-dose WGP showing significant efficacy in alleviating liver damage and metabolic disorders, while high-dose WGP played an important role in resolving metabolic disorders.

[0105] (4) Systemic oxidative stress, inflammatory factors and neuroendocrine indicators such as Figure 7 shown.

[0106] In terms of oxidative stress, OTA treatment significantly increased the level of MDA in serum (P<0.001), and significantly decreased the activities of T-AOC, SOD and GSH-Px (P<0.001), indicating that OTA significantly increased the oxidative stress level of the whole body of chicks by increasing oxidative damage products and inhibiting the activities of antioxidant enzymes. Treatment with low-dose WGP significantly reversed the abnormal level of MDA (P<0.001), and significantly increased the activities of T-AOC, SOD and GSH-Px (P<0.01), indicating that low-dose WGP can effectively alleviate the oxidative stress damage caused by OTA. The high-dose WGP group showed a certain recovery trend, especially in terms of T-AOC and SOD (P<0.05), but the overall effect was not as good as that of low-dose WGP, suggesting that the recovery effect is affected by the dose.

[0107] In terms of inflammatory response, OTA significantly increased the levels of TNF-α, IL-6 and IL-1β in serum (P<0.001). After treatment with low-dose WGP, the levels of TNF-α, IL-6 and IL-1β were effectively restored (P<0.01), showing its strong anti-inflammatory effect. High-dose WGP also showed a good anti-inflammatory effect, but its effect was weaker than that of the low-dose group, suggesting that the anti-inflammatory effect of high-dose WGP is relatively mild.

[0108] In terms of endocrine, OTA significantly increased the levels of DAO and CORT in serum (P<0.001), and at the same time significantly decreased the levels of melatonin, 5-HT and TRP (P<0.001). These changes indicate that OTA can interfere with the endocrine system and affect the physiological functions and stress responses of the body by changing the levels of key hormones and metabolites, and further affect the synthesis of neurotransmitters. After treatment with low-dose WGP, these abnormal indexes were effectively reversed (P<0.001), indicating that low-dose WGP can alleviate the negative impact of OTA on the endocrine system. High-dose WGP can also significantly reverse the abnormal changes of DAO (P<0.01), and at the same time showed a certain degree of recovery effect on the levels of melatonin, CORT, 5-HT and TRP (P<0.05), indicating that high-dose WGP also has a certain role in regulating endocrine imbalance.

[0109] In summary, OTA significantly affected the physiological functions of chicks by increasing oxidative stress, inflammatory response and endocrine disorders. Low-dose WGP can significantly restore these indexes, especially in antioxidant, anti-inflammatory and endocrine regulation, showing a significant protective effect, indicating that WGP can effectively reduce the toxic damage caused by OTA and provide significant protection for the health of chicks.

[0110] (5) As Figure 8 shown, OTA treatment significantly changed the expression of multiple key genes in the hippocampal tissue of chicks.

[0111] In Figure A, OTA significantly increased the mRNA levels of TNF-α, IL-6, and IL-1β (P<0.001), indicating that OTA exerts its neurotoxic effects by activating the inflammatory response in the central nervous system. After treatment with low-dose WGP, the mRNA levels of these inflammatory factors were significantly restored (P<0.01), indicating that WGP effectively alleviated the neurotoxicity of OTA by inhibiting the inflammatory response.

[0112] In Figure B, OTA significantly inhibited the expression of antioxidant enzyme genes CAT, SOD, and GPX-1 (P<0.05), indicating that OTA can induce oxidative stress by inhibiting the function of the antioxidant system. Treatment with low-dose WGP significantly restored these abnormalities, especially the gene expression level of SOD (P<0.001), indicating that WGP has a significant antioxidant effect.

[0113] Figure C showed that OTA significantly increased the expression of apoptosis-related genes BAX and Caspase-3 (P<0.01), suggesting that OTA causes abnormal apoptosis of nerve cells and damages the nervous system by activating the apoptotic pathway. After treatment with WGP, the expression of both BAX and Caspase-3 was significantly restored (P<0.05), further supporting the protective effect of WGP in reducing apoptosis.

[0114] In Figure D, OTA significantly increased the mRNA levels of aquaporins AQP1 and AQP4 (P<0.01), with AQP1 increasing by as much as 5-fold, suggesting that edema and abnormal cell function may be one of the key mechanisms by which OTA exacerbates nerve damage. After treatment with WGP, the abnormal expression of AQP1 and AQP4 was significantly reversed (P<0.01), restoring the high expression of AQP1 from 5-fold in the OTA group to 1.2-fold in the control group. In addition, OTA significantly decreased the mRNA levels of WNT4, WNT6, β-catenin, and GSK3a in the Wnt signaling pathway (P<0.05), indicating that the Wnt signaling pathway plays an important role in the neurotoxicity caused by OTA. The Wnt signaling pathway is crucial for the development, repair, and functional maintenance of the nervous system. Inhibition of the expression of key genes in this pathway by OTA may lead to obstruction of the nerve repair mechanism and exacerbate neurotoxicity. After treatment with WGP, the expression levels of genes related to the Wnt signaling pathway were restored to the control group level (P<0.05), indicating that WGP can promote nerve repair and alleviate nerve damage caused by OTA by regulating the Wnt signaling pathway.

[0115] Figure E shows that OTA significantly increased the expression of TLR4, MYD88, and P65 (P < 0.01) and significantly decreased the expression of antioxidant-related genes NRF2 and HO-1 (P < 0.01), indicating that OTA activates the inflammatory response through the TLR4 / MYD88 signaling pathway while inhibiting the NRF2-mediated antioxidant defense mechanism. After WGP treatment, abnormal expression of TLR4, MYD88, and P65 was restored to varying degrees, with TLR4 and P65 being the most significant (P < 0.01). Abnormal expression of NRF2 and HO-1 was also effectively reversed (P < 0.05), demonstrating the regulatory effect of WGP on the TLR4 / MYD88 pathway. Furthermore, OTA significantly decreased the mRNA levels of COX5A and COX17 (P < 0.01), genes closely associated with mitochondrial function. After treatment with WGP, the expression of these genes was significantly restored (P<0.05), indicating that WGP can reduce the damage of OTA to mitochondrial function, ensure cellular energy supply, and thus alleviate neuronal apoptosis.

[0116] In Figure F, OTA treatment significantly reduced mRNA levels of 5-HT1A and 5-HT1B (P < 0.01). These two receptors play an important role in regulating the synthesis and signal transduction of the neurotransmitter 5-HT. After WGP treatment, the expression of these genes was restored (P < 0.05), suggesting that WGP may maintain interneuronal communication by improving neurotransmitter synthesis and signal transduction. Concomitantly, OTA significantly reduced the expression of growth factors such as BDNF, IGF-1, and IGF-2, as well as their receptors IGF-1R and IGF-2R (P < 0.05). BDNF plays a key role in neural development and functional maintenance. By inhibiting BDNF expression, OTA may lead to a decrease in neuroplasticity and neuroprotection, thereby exacerbating neurotoxicity. After WGP treatment, the abnormal expression of these genes was significantly restored (P < 0.05), indicating that WGP, by restoring the expression of growth factors such as BDNF, helps improve neural function and protect the nervous system, thereby alleviating the effects of OTA on hippocampal neurotoxicity.

[0117] In summary, the experiment verified that the low-dose group (400 mg / kg) was significantly superior to the high-dose group (800 mg / kg) in comprehensive efficacy, specifically: low-dose WGP significantly improved the growth performance and behavioral performance of chicks, especially in terms of body weight, feed intake and feed conversion rate, and the recovery effect was more significant. At the same time, it showed a good therapeutic effect in alleviating the neurotoxicity caused by OTA. Specifically, it had a restorative effect on inflammatory factors, antioxidant enzymes and apoptosis genes in hippocampal tissue, and also showed good regulatory ability on the TLR4 / MYD88 pathway and Wnt signaling pathway, indicating that WGP has a multi-target protective effect on hippocampal nervous system damage caused by OTA. WGP significantly alleviates the neurotoxic damage caused by OTA by regulating key mechanisms such as neuroinflammation, oxidative stress, apoptosis, neurotransmitter synthesis, Wnt signaling pathway, TLR4 / MYD88 pathway and growth factors, showing a multi-dimensional neuroprotective effect.

[0118] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Application of polysaccharide from green walnut husk in the preparation of products for protecting nerves, characterized in that, The polysaccharide from Qinglongyi is composed of the following monosaccharides: fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid.

2. A nerve protection product, characterized in that, It includes the polysaccharide from Qinglongyi, which is composed of the following monosaccharides: fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid.

3. The application according to claim 1 or the product according to claim 2, characterized in that, The use for protecting nerves is for reducing the neurotoxicity caused by ochratoxin A, preferably for reducing the neurotoxicity of ochratoxin A to hippocampal nerves.

4. The application according to claim 1 or the product according to claim 2, characterized in that, The molar ratio of fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid is 0.70 - 0.90:23.92 - 25.92:2.79 - 4.79:9.72 - 11.72:4.98 - 5.98:0.39 - 0.59:0.52 - 0.72:44.57 - 48.57:5.11 - 7.11, preferably 0.80:24.92:3.79:10.72:5.98:0.49:0.62:46.57:6.

11.

5. The application according to claim 1 or the product according to claim 2, characterized in that, The main chain structure of the polysaccharide from Qinglongyi is alternately connected by →4)-α-D-GalpA-(1→ and →2)-α-L-Rhap-(1→, and the end is terminated by α-D-GalpA-(1→. The sugar chain structure is as follows:

6. The application according to claim 1 or the product according to claim 2, characterized in that, The weight-average molecular weight of the polysaccharide from Qinglongyi is 10.8 - 14.8 kDa, preferably 11.8 - 15.8 kDa; the number-average molecular weight of the polysaccharide from Qinglongyi is 6.3 - 10.3 kDa, preferably 7.3 - 9.3 kDa.

7. The application according to claim 1 or the product according to claim 2, characterized in that, The polysaccharide from Qinglongyi is prepared by the following method: The method includes: subjecting Qinglongyi from which fat-soluble impurities have been removed to water extraction to obtain a water extract; concentrating the water extract to obtain a concentrated solution; subjecting the obtained concentrated solution to alcohol precipitation and centrifugation to obtain a precipitate; redissolving the obtained precipitate in water and removing alcohol to obtain a crude polysaccharide; removing protein impurities from the crude polysaccharide and freeze-drying to obtain the polysaccharide from Qinglongyi.

8. The application or product according to claim 6, characterized in that, Subject Qinglongyi from which fat-soluble impurities have been removed to first water extraction with the first water to obtain a first water extract and a first residue; add the second water to the obtained first residue for second water extraction to obtain a second water extract; combine the first water extract and the second water extract and concentrate to obtain a concentrated solution; control the addition amount of the first water such that the mass-to-volume ratio of Qinglongyi without removed fat-soluble impurities to the first water is 1 kg:8 - 12 L; control the addition amount of the second water such that the mass-to-volume ratio of Qinglongyi without removed fat-soluble impurities to the second water is 1 kg:4 - 8 L; Control the concentration such that the volume of the concentrated solution is 10% - 14% of the total volume of the first water and the second water; Preferably, the alcohol precipitation is carried out with a 75 - 80% ethanol solution, and the volume ratio of the ethanol solution to the concentrated solution is 1:3 - 5; the rotation speed of the centrifugation is 7000 - 9000 rpm, the temperature of the centrifugation is 2 - 6 °C, and the time of the centrifugation is 3 - 7 min.

9. The application according to claim 1 or the product according to claim 2, characterized in that, The protection of nerves includes down-regulating TLR4, MYD88, P65, restoring β-catenin, and restoring the expression of neurotransmitter receptor genes.

10. The application according to claim 1 or the product according to claim 2, characterized in that, In addition to the effect of inhibiting neurotoxicity, the product also has at least one of the following effects: Improving growth performance, Improving abnormal serum indexes, Relieving oxidative stress; The improvement of growth performance includes increasing body weight, reducing feed-to-meat ratio, improving metabolic disorders, and regulating endocrine balance; the improvement of abnormal serum indexes includes reducing AST level, restoring AST / ALT ratio, and restoring GLOB, TP, GLU and inflammatory factor levels.

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