A soluble powder for treating glyphosate-induced liver damage in domestic animals and a method for preparing the same

Abstract

The present application belongs to pharmaceutical preparation characterized by form, and particularly relates to a soluble powder for treating glyphosate-induced liver injury of livestock and a preparation method thereof. The soluble powder comprises sucrose, a liver protection compound, and a detoxification degradation agent. The liver protection compound is obtained by modifying epigallocatechin gallate on fresh spirulina after removing the smell of the spirulina. The detoxification degradation agent is obtained by modifying laccase with rosmarinic acid, and then further encapsulating the obtained compound with chitosan and trehalose. The liver protection compound and the detoxification degradation agent can synergistically play a role. The soluble powder of the present application is suitable for being used and prepared before livestock eat forage / feed, and is added into drinking water, so that the glyphosate-induced liver injury of livestock and its degradation products can be reduced, and the function of the liver antioxidant system can be maintained as much as possible, and the oxidative stress caused by glyphosate can be effectively resisted.

Description

Technical Field

[0001] The present invention belongs to a pharmaceutical preparation characterized by form, and particularly relates to a soluble powder for treating glyphosate-induced liver damage in livestock and a preparation method thereof. Background Art

[0002] Glyphosate's chemical name is N-(phosphomethyl)glycine, with a chemical formula of C3H8NO5P and a relative molecular mass of 169.07. The pure product is a white solid with a solubility of 1.2% in water at 25°C and low solubility in organic solvents. Glyphosate is more soluble in its salt form, such as glyphosate isopropylamine, glyphosate sodium, and glyphosate potassium. As a systemic, broad-spectrum herbicide, glyphosate can kill common weeds in agriculture and animal husbandry. Glyphosate has a strong systemic property and is rapidly absorbed by plant roots, stems, and leaves after application. It migrates to and accumulates in areas of high metabolic activity, inhibiting the synthesis of 5-enolpyruvylshikimate-3-phosphate, preventing the conversion of shikimate into aromatic amino acids (such as phenylalanine, tryptophan, and tyrosine). This interferes with protein synthesis, impacts plant metabolism, and ultimately leads to plant death. Due to its good herbicidal performance, glyphosate has not yet been completely banned, but its toxicity is inevitable. Its oral LD50 in rats is 50 The oral LD500 in mice is 5000 mg / kg. 50 The LD50 in goats is 10000 mg / kg. 50 The acute skin LD50 of rabbits is 3530 mg / kg. 50 >2000mg / kg.

[0003] Existing research has focused less on the potential safety and health risks of livestock, which rely on forage as their primary source of nutrition, consuming forage treated with glyphosate. Some evidence suggests that these risks may be underestimated. Based on glyphosate residues in forage and livestock forage consumption, it is estimated that glyphosate concentrations in pig intestines are approximately 0.005-0.02 mg / mL, and in cattle intestinal contents are approximately 0.002-0.003 mg / mL. Further consideration of practical factors, such as the general unwashing of forage fed to livestock, the fluctuation of actual forage consumption with livestock growth, the livestock's health, and the availability of seasonal fresh forage, suggests that livestock may ingest higher amounts of glyphosate during their growth and be exposed to the harmful effects of excessive glyphosate. Most toxic substances in forage are metabolized by the liver, which transforms them through oxidation, reduction, and hydrolysis. This makes the liver a primary target organ for exogenous toxicants. Excessive glyphosate intake by livestock can induce liver damage. In addition, livestock feed prepared from forage will also bring the above problems. Due to the complex source of forage, improper harvesting management, and rough production process, livestock face the problem of glyphosate-induced liver damage.

[0004] Liver damage in livestock can lead to decreased immune function, metabolic dysfunction, and malnutrition, seriously impacting the livestock's life cycle and, in turn, affecting the production and economic benefits of livestock farming. Therefore, it is necessary to develop a drug for treating glyphosate-induced liver damage in livestock. Summary of the Invention

[0005] To address the above issues, the present invention provides a soluble powder for treating glyphosate-induced liver damage in livestock. The soluble powder comprises sucrose, a liver-protective complex, and a detoxifying and degrading agent. The liver-protective complex is obtained by removing the fishy smell from fresh spirulina and then modifying it with epigallocatechin gallate. The detoxifying and degrading agent is obtained by modifying laccase with rosmarinic acid, and the resulting complex is further encapsulated with chitosan and trehalose. The sucrose acts as an appetite inducer. The liver-protective complex and the detoxifying and degrading agent work synergistically. The soluble powder can be prepared immediately and added to drinking water to alleviate glyphosate-induced liver damage in livestock, maintain the function of the liver's antioxidant system to the greatest extent possible, and effectively resist the oxidative stress caused by glyphosate.

[0006] The invention provides a soluble powder for treating glyphosate-induced liver damage in livestock. The powder comprises the following raw materials in parts by weight: 0.5-1.5 parts of sucrose, 2-5 parts of a liver protection complex, and 3-5 parts of a detoxifying and degrading agent.

[0007] The preparation method of the liver protection complex is as follows:

[0008] L1. Fresh spirulina was washed and blanched in 80°C water for 1-2 minutes, followed by air drying to obtain a spirulina sample. Ginger charcoal was crushed and sieved to obtain a ginger charcoal powder with a particle size of 80-100 μm. The ginger charcoal powder was dispersed in water to obtain a ginger charcoal suspension with a concentration of 4-8% by weight. The spirulina sample was completely immersed in the ginger charcoal suspension and allowed to stand at room temperature for 2-3 hours. After completion, the sample was washed, dried completely, and ground to obtain a spirulina powder with a particle size of 10-20 μm.

[0009] L2. Weigh the spirulina powder, xanthan gum aqueous solution, and epigallocatechin gallate obtained in step L1 in a mass ratio of 8-10:10-15:1. Mix the spirulina powder and xanthan gum aqueous solution and ultrasonically disperse them until uniform. Then, add epigallocatechin gallate, adjust the pH to 7-8, and heat to 30-40°C for 4-8 hours to obtain Solution 1.

[0010] L3. The solution 1 obtained in step L2 is dialyzed at 4°C for 10-12h using a dialysis bag with a molecular weight cutoff of 9-12kDa. After the dialysis, the material in the dialysis bag is vacuum freeze-dried to obtain a liver protection complex.

[0011] Preferably, the mass percentage concentration of the xanthan gum aqueous solution in step L2 is 3-5%.

[0012] Preferably, the dialysis medium is replaced 2-3 times during dialysis in step L3, the dialysis medium is deionized water, and vacuum freeze drying is preferably performed at -60°C pre-cooling, a baffle temperature of -50°C, and a vacuum degree of 5Pa.

[0013] The preparation method of the detoxification degradation agent is as follows:

[0014] S1. Weigh rosmarinic acid and sodium hydroxide solution in a mass volume ratio of 40-50 mg: 6-9 mL, stir until uniformly dispersed, and adjust the pH to 7-7.5 to obtain solution 2;

[0015] S2. Weigh EDC, NHS, and solution 2 obtained in step S1 in a mass-volume ratio of 100-120 mg: 60-80 mg: 10-15 mL, heat to 30-40 ° C, stir until uniformly dispersed, and then stand at room temperature for 20-30 minutes to obtain solution 3;

[0016] S3. Laccase and solution 3 obtained in step S2 were weighed at a mass-to-volume ratio of 80-100 mg:10-15 mL. The mixture was heated to 32-36°C and stirred at 100-150 rpm for 18-20 hours. The mixture was then centrifuged at 5000-6000 g for 8-10 minutes. The supernatant was concentrated to 0.4-0.5 times its original volume and freeze-dried in vacuo to obtain a complex.

[0017] S4. Dissolve chitosan in acetic acid solution to obtain a chitosan solution with a mass percentage concentration of 3-5%, adjust the pH to 5-6, add the complex obtained in step S3, and add the complex in an amount of 8-10 mg / mL. Stir at a speed of 150-200 rpm at room temperature for 20-30 minutes. After the stirring, centrifuge at a centrifugal force of 8000-10000g for 10-15 minutes. The obtained precipitate is dispersed in the trehalose solution to obtain a mixture. After spray drying the mixture, a detoxification and degradation agent is obtained.

[0018] Preferably, the concentration of the sodium hydroxide solution in step S1 is 3 mol / L, and hydrochloric acid solution is preferably used to adjust the pH.

[0019] Preferably, in step S2, EDC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, CAS No. 1892-57-5, and NHS is N-hydroxysuccinimide, CAS No. 6066-82-6.

[0020] Preferably, the laccase in step S3 is derived from Coriolus versicolor, and the enzyme activity is preferably 200-300 U / g.

[0021] Preferably, the mass percentage concentration of the acetic acid solution in step S4 is preferably 2%, the mass volume concentration of the precipitate in the trehalose solution is preferably 1-3 mg / mL, the mass percentage concentration of the trehalose solution is preferably 6%, and the spray drying is preferably carried out at an air flow rate of 1.5-2 m 3 / min, air inlet temperature 70-80℃, air outlet temperature 50-60℃.

[0022] The present invention also provides a method for preparing a soluble powder for treating glyphosate-induced liver damage in livestock, the specific steps of which are as follows:

[0023] V1. Weigh sucrose, a hepatoprotective complex, and a detoxifying and degrading agent according to their respective weights, dissolve the sucrose in water to obtain a sucrose solution with a mass percentage concentration of 5-8%. Mix the sucrose solution with the detoxifying and degrading agent, spray-dry, and freeze the resulting micropowder to obtain the core contents.

[0024] V2. Dissolve the liver protection complex in water to obtain solution 4 with a mass percentage concentration of 10-15%. Add the core contents obtained in step V1 to solution 4, rapidly stir until uniformly dispersed, and vacuum freeze-dry to obtain a soluble powder for treating glyphosate-induced liver damage in livestock.

[0025] Preferably, the spray drying in step V1 is preferably carried out at an air flow rate of 2-3m 3 / min, air inlet temperature 50-60℃, air outlet temperature 30-40℃, freezing below 0℃, preferably freezing at -20℃ for 48h.

[0026] Preferably, the vacuum freeze-drying in step V2 is performed under pre-cooling at -20°C, a partition temperature of -10°C, and a vacuum degree of 8Pa.

[0027] The beneficial effects of the present invention are as follows:

[0028] The present invention prepares a soluble powder, which is preferably dissolved in water and administered to livestock before they consume forage / feed, and can effectively alleviate liver damage in livestock induced by glyphosate and its degradation products. The soluble powder of the present invention comprises sucrose, a liver protection complex, and a detoxification and degradation agent, wherein the sucrose acts as an appetite inducer. The liver protection complex is obtained by removing the fishy smell from fresh spirulina and then modifying it with epigallocatechin gallate, ultimately retaining the low-molecular-weight portion. The detoxification and degradation agent is obtained by modifying laccase with rosmarinic acid, and the obtained complex is further encapsulated with chitosan and trehalose. The soluble powder of the present invention has a shell-core structure, with the liver protection complex as the shell material and the detoxification and degradation agent as the inner core material. In actual application, the liver protection complex is first released into water, and the detoxification and degradation agent is subsequently released. The soluble powder of the present invention can be completely dissolved within 60 seconds without leaving any solid residue. Toxicological experiments have shown that it is highly safe and does not affect the normal life of mice. Livestock experiments using white-feathered chickens showed that adding the soluble powder to drinking water after immediate use can reduce glyphosate-induced liver damage, maintain the function of the liver's antioxidant system as much as possible, and effectively resist the oxidative stress caused by glyphosate. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0030] Figure 1 This is a graph showing the dissolution time results of the liver protection complex, detoxification and degradation agent, and soluble powder;

[0031] Figure 2 The figure shows the changes in body weight of male mice during the toxicology experiment;

[0032] Figure 3 Figure 1 is a graph showing changes in body weight of female mice during the toxicology experiment;

[0033] Figure 4 This is the result of ALT and AST levels in serum of sexually mature white-feathered chickens after the experiment;

[0034] Figure 5 This is the result of ALP and γ-GT levels in serum of sexually mature white-feathered chickens after the experiment;

[0035] Figure 6This is the result of ALT and AST levels in serum of 2-month-old white-feathered chickens after the experiment;

[0036] Figure 7 This is the result of ALP and γ-GT levels in serum of 2-month-old white-feathered chickens after the experiment;

[0037] Figure 8 This is the result of the CAT, SOD and GPx content in the liver of sexually mature white-feathered chickens after the experiment;

[0038] Figure 9 This is the result of CAT, SOD and GPx content in the liver of 2-month-old white-feathered chickens after the experiment;

[0039] Figure 10 This is the result of MDA, PCO and AOPP content in chicken liver after the experiment of sexually mature white-feathered chickens;

[0040] Figure 11 This is the result of MDA, PCO and AOPP content in chicken liver after the experiment of 2-month-old white-feather chickens. DETAILED DESCRIPTION

[0041] In order to more clearly illustrate the overall concept of the present application, the following is a detailed description of the embodiments in conjunction with the accompanying drawings. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0042] Example 1: This example provides a method for preparing a liver protection complex, and the specific steps are as follows:

[0043] L1. Fresh spirulina was washed and blanched in 80°C water for 1 minute. The surface moisture was then air-dried to obtain a spirulina sample. Ginger charcoal was crushed and sieved to obtain ginger charcoal powder with a particle size of 80 μm. The ginger charcoal powder was dispersed in water to obtain a ginger charcoal suspension with a concentration of 4% by weight. The spirulina sample was completely immersed in the ginger charcoal suspension and allowed to stand at room temperature for 2 hours. After completion, the sample was washed, dried completely, and ground to obtain spirulina powder with a particle size of 10 μm.

[0044] L2. Weigh the spirulina powder obtained in step L1, a 3% xanthan gum aqueous solution, and epigallocatechin gallate in a mass ratio of 8:10:1. Mix the spirulina powder and xanthan gum aqueous solution and ultrasonically disperse until uniform. Then, add epigallocatechin gallate, adjust the pH to 7, and heat to 30°C for 4 hours to obtain Solution 1.

[0045] L3. The solution 1 obtained in step L2 was dialyzed at 4°C for 10 h using a dialysis bag with a molecular weight cutoff of 9 kDa. The dialysis medium (deionized water) was replaced twice during the dialysis. After the dialysis, the substance in the dialysis bag was pre-cooled at -60°C for 15 h and vacuum freeze-dried at a partition temperature of -50°C and a vacuum degree of 5 Pa to obtain a liver protection complex.

[0046] Example 2: This example provides a method for preparing a liver protection complex, and the specific steps are as follows:

[0047] L1. Fresh spirulina was washed and blanched in 80°C water for 1.5 minutes. The surface moisture was then air-dried to obtain a spirulina sample. Ginger charcoal was crushed and sieved to obtain ginger charcoal powder with a particle size of 90 μm. The ginger charcoal powder was dispersed in water to obtain a ginger charcoal suspension with a concentration of 5% by weight. The spirulina sample was completely immersed in the ginger charcoal suspension and allowed to stand at room temperature for 2.5 hours. After completion, the sample was washed, dried completely, and ground to obtain spirulina powder with a particle size of 15 μm.

[0048] L2. Weigh the spirulina powder obtained in step L1, a 4% xanthan gum aqueous solution, and epigallocatechin gallate in a mass ratio of 9:11:1. Mix the spirulina powder and xanthan gum aqueous solution and ultrasonically disperse until uniform. Then, add epigallocatechin gallate, adjust the pH to 7.5, and heat to 35°C for 6 hours to obtain Solution 1.

[0049] L3. The solution 1 obtained in step L2 was dialyzed at 4°C for 11 hours using a dialysis bag with a molecular weight cutoff of 10 kDa. The dialysis medium (deionized water) was replaced twice during the dialysis. After the dialysis, the substance in the dialysis bag was pre-cooled at -60°C for 12 hours and vacuum freeze-dried at a partition temperature of -50°C and a vacuum degree of 5 Pa to obtain a liver protection complex.

[0050] Example 3: This example provides a method for preparing a liver protection complex, and the specific steps are as follows:

[0051] L1. Fresh spirulina was washed and blanched in 80°C water for 2 minutes. The surface moisture was then air-dried to obtain a spirulina sample. Ginger charcoal was crushed and sieved to obtain a 100 μm ginger charcoal powder. The ginger charcoal powder was dispersed in water to obtain an 8% by weight ginger charcoal suspension. The spirulina sample was completely immersed in the ginger charcoal suspension and allowed to stand at room temperature for 3 hours. After washing, the sample was completely dried and ground to obtain a 20 μm spirulina powder.

[0052] L2. Weigh the spirulina powder obtained in step L1, a 5% xanthan gum aqueous solution, and epigallocatechin gallate in a mass ratio of 10:15:1. Mix the spirulina powder and xanthan gum aqueous solution and ultrasonically disperse until uniform. Then, add epigallocatechin gallate, adjust the pH to 8, and heat to 40°C for 8 hours to obtain Solution 1.

[0053] L3. The solution 1 obtained in step L2 was dialyzed at 4°C for 12 h using a dialysis bag with a molecular weight cutoff of 10 kDa. The dialysis medium (deionized water) was replaced three times during the dialysis. After the dialysis, the substance in the dialysis bag was pre-cooled at -60°C for 14 h and vacuum freeze-dried at a partition temperature of -50°C and a vacuum degree of 5 Pa to obtain a liver protection complex.

[0054] Example 4: This example provides a method for preparing a detoxification and degradation agent, and the specific steps are as follows:

[0055] S1. Weigh rosmarinic acid and 3 mol / L sodium hydroxide solution in a mass-to-volume ratio of 40 mg:6 mL, stir until uniformly dispersed, and adjust the pH to 7 with 1 mol / L hydrochloric acid solution to obtain Solution 2;

[0056] S2. Weigh EDC, NHS, and Solution 2 obtained in step S1 in a mass-to-volume ratio of 100 mg:60 mg:10 mL (EDC = 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, CAS No. 1892-57-5); NHS = N-hydroxysuccinimide, CAS No. 6066-82-6). Heat to 30°C, stir until uniformly dispersed, and then stand at room temperature for 20 minutes to obtain Solution 3.

[0057] S3. Laccase from Coriolus versicolor with an activity of 200 U / g and solution 3 obtained in step S2 were weighed at a mass-to-volume ratio of 80 mg:10 mL. The laccase was derived from Coriolus versicolor and had an enzyme activity of 200 U / g. The mixture was heated to 32°C and stirred at 100 rpm for 18 h. After completion, the mixture was centrifuged at 5000 g for 8 min. The supernatant was concentrated to 0.4 times its original volume and freeze-dried in vacuo to obtain a complex.

[0058] S4. Chitosan was dissolved in acetic acid solution with a mass percentage concentration of 2% to obtain a chitosan solution with a mass percentage concentration of 3%. The pH was adjusted to 5. The complex obtained in step S3 was added in an amount of 8 mg / mL. The mixture was stirred at 150 rpm for 20 min at room temperature. After the mixture was centrifuged at 8000 g for 10 min, the precipitate was dispersed in a trehalose solution with a mass percentage concentration of 6%. The mass volume concentration of the precipitate in the trehalose solution was 1 mg / mL. The mixture was stirred at an air flow rate of 1.5 m 3The detoxification degradation agent was obtained by spray drying at a temperature of 70°C, an inlet air temperature of 50°C and an outlet air temperature of 50°C.

[0059] Example 5: This example provides a method for preparing a detoxification and degradation agent, and the specific steps are as follows:

[0060] S1. Weigh rosmarinic acid and 3 mol / L sodium hydroxide solution in a mass-to-volume ratio of 45 mg:8 mL, stir until uniformly dispersed, and adjust the pH to 7.2 with 1 mol / L hydrochloric acid solution to obtain Solution 2;

[0061] S2. Weigh EDC, NHS, and Solution 2 obtained in step S1 in a mass-to-volume ratio of 110 mg:70 mg:12 mL (EDC = 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, CAS No. 1892-57-5); and NHS = N-hydroxysuccinimide, CAS No. 6066-82-6). Heat to 35°C, stir until uniformly dispersed, and then stand at room temperature for 24 minutes to obtain Solution 3.

[0062] S3. Laccase from Coriolus versicolor with an enzyme activity of 250 U / g and solution 3 obtained in step S2 were weighed at a mass-to-volume ratio of 90 mg:12 mL. The laccase was derived from Coriolus versicolor and had an enzyme activity of 250 U / g. The mixture was heated to 34°C and stirred at 120 rpm for 19 h. The mixture was then centrifuged at 5400 g for 9 min. The supernatant was concentrated to 0.45 times its original volume and freeze-dried in vacuo to obtain a complex.

[0063] S4. Chitosan was dissolved in acetic acid solution with a mass percentage concentration of 2% to obtain a chitosan solution with a mass percentage concentration of 4%. The pH was adjusted to 5.5. The complex obtained in step S3 was added in an amount of 9 mg / mL. The mixture was stirred at a speed of 180 rpm at room temperature for 24 minutes. After the mixture was centrifuged at a centrifugal force of 9000 g for 12 minutes, the precipitate was dispersed in a trehalose solution with a mass percentage concentration of 6%. The mass volume concentration of the precipitate in the trehalose solution was 2 mg / mL. The mixture was stirred at an air flow rate of 1.8 m 3 The detoxification degradation agent was obtained by spray drying at a temperature of 75°C, an air inlet temperature of 75°C and an air outlet temperature of 55°C.

[0064] Example 6: This example provides a method for preparing a detoxification and degradation agent, and the specific steps are as follows:

[0065] S1. Weigh rosmarinic acid and 3 mol / L sodium hydroxide solution in a mass-to-volume ratio of 50 mg:9 mL, stir until uniformly dispersed, and adjust the pH to 7.5 with 1 mol / L hydrochloric acid solution to obtain Solution 2;

[0066] S2. Weigh EDC, NHS, and Solution 2 obtained in step S1 in a mass-to-volume ratio of 120 mg:80 mg:15 mL (EDC = 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, CAS No. 1892-57-5; NHS = N-hydroxysuccinimide, CAS No. 6066-82-6). Heat to 40°C, stir until uniformly dispersed, and then stand at room temperature for 30 min to obtain Solution 3.

[0067] S3. Laccase from Coriolus versicolor with an enzyme activity of 300 U / g and solution 3 obtained in step S2 were weighed at a mass-to-volume ratio of 100 mg:15 mL. The laccase was derived from Coriolus versicolor and had an enzyme activity of 300 U / g. The mixture was heated to 36°C and stirred at 150 rpm for 20 h. Afterwards, the mixture was centrifuged at 6000 g for 10 min. The supernatant was concentrated to 0.5 times its original volume and freeze-dried in vacuo to obtain a complex.

[0068] S4. Chitosan was dissolved in acetic acid solution with a mass percentage concentration of 2% to obtain a chitosan solution with a mass percentage concentration of 5%, the pH was adjusted to 6, the complex obtained in step S3 was added, the amount of the complex added was 10 mg / mL, and the mixture was stirred at 200 rpm for 30 minutes at room temperature. After the end, the mixture was centrifuged at a centrifugal force of 10000 g for 15 minutes. The obtained precipitate was dispersed in a trehalose solution with a mass percentage concentration of 6%. The mass volume concentration of the precipitate in the trehalose solution was 3 mg / mL. The obtained mixture was heated at an air flow rate of 2m 3 The detoxification degradation agent was obtained by spray drying at a temperature of 80°C, an inlet air temperature of 80°C and an outlet air temperature of 60°C.

[0069] Example 7: This example provides a method for preparing a soluble powder for treating glyphosate-induced liver damage in livestock. The specific steps are as follows:

[0070] V1. Weigh 0.5 parts of sucrose, 2 parts of liver protection complex, and 3 parts of detoxification and degradation agent according to weight, wherein the liver protection complex is prepared by Example 1 and the detoxification and degradation agent is prepared by Example 4. Dissolve sucrose in water to obtain a sucrose solution with a mass percentage concentration of 5%. Mix the sucrose solution with the detoxification and degradation agent and simmer in an air flow of 2m 3 After spray drying at 50 °C / min, air inlet temperature 50 °C, and air outlet temperature 30 °C, the obtained micropowder was frozen at -20 °C for 48 h to obtain the core content;

[0071] V2. Dissolve the liver protection complex in water to obtain solution 4 with a mass percentage concentration of 10%. Add the core contents obtained in step V1 to solution 4, rapidly stir until uniformly dispersed, precool at -20°C for 15 hours, and vacuum freeze-dry at a partition temperature of -10°C and a vacuum degree of 8 Pa to obtain a soluble powder for treating glyphosate-induced liver damage in livestock.

[0072] Example 8: This example provides a method for preparing a soluble powder for treating glyphosate-induced liver damage in livestock. The specific steps are as follows:

[0073] V1. Weigh 1 part sucrose, 3 parts liver protection complex, and 4 parts detoxification and degradation agent by weight, wherein the liver protection complex is prepared by Example 2, and the detoxification and degradation agent is prepared by Example 5. Dissolve sucrose in water to obtain a sucrose solution with a mass percentage concentration of 6%. Mix the sucrose solution with the detoxification and degradation agent and simmer at an air flow rate of 2.5 m 3 After spray drying at 55°C / min, air inlet temperature 55°C, and air outlet temperature 35°C, the obtained micropowder was frozen at -20°C for 48h to obtain the core content;

[0074] V2. Dissolve the liver protection complex in water to obtain solution 4 with a mass percentage concentration of 12%. Add the core contents obtained in step V1 to solution 4, rapidly stir until uniformly dispersed, precool at -20°C for 18 hours, and vacuum freeze-dry at a shelf temperature of -10°C and a vacuum degree of 8 Pa to obtain a soluble powder for treating glyphosate-induced liver damage in livestock.

[0075] Example 9: This example provides a method for preparing a soluble powder for treating glyphosate-induced liver damage in livestock. The specific steps are as follows:

[0076] V1. Weigh 1.5 parts of sucrose, 5 parts of liver protection complex, and 5 parts of detoxification and degradation agent by weight, wherein the liver protection complex is prepared by Example 3 and the detoxification and degradation agent is prepared by Example 6. Dissolve sucrose in water to obtain a sucrose solution with a mass percentage concentration of 8%. Mix the sucrose solution with the detoxification and degradation agent and simmer in an air flow of 3m 3 After spray drying at 40°C, air inlet temperature 60°C, and air outlet temperature 40°C, the obtained micropowder was frozen at -20°C for 48h to obtain the core content;

[0077] V2. Dissolve the liver protection complex in water to obtain solution 4 with a mass percentage concentration of 15%. Add the core contents obtained in step V1 to solution 4, rapidly stir until uniformly dispersed, precool at -20°C for 20 hours, and vacuum freeze-dry at a partition temperature of -10°C and a vacuum degree of 8 Pa to obtain a soluble powder for treating glyphosate-induced liver damage in livestock.

[0078] Comparative Example 1: This comparative example provides a method for preparing a liquid preparation, and the specific steps are as follows:

[0079] 5 parts of the liver protection complex obtained in Example 3 and 1152 parts of water were weighed according to weight, mixed until uniformly dispersed, and stored at room temperature for 30 days to obtain the liquid preparation of this comparative example.

[0080] Comparative Example 2: This comparative example provides a method for preparing a liquid preparation, and the specific steps are as follows:

[0081] 5 parts of the detoxification and degradation agent obtained in Example 6 and 1152 parts of water were weighed by weight, mixed until uniformly dispersed, and stored at room temperature for 30 days to obtain the liquid preparation of this comparative example.

[0082] Comparative Example 3: This comparative example provides a method for preparing a liquid preparation, and the specific steps are as follows:

[0083] 5 parts of the soluble powder obtained in Example 9 and 500 parts of water were weighed according to weight, mixed until uniformly dispersed, and stored at room temperature for 30 days to obtain the liquid preparation of this comparative example.

[0084] Experimental test:

[0085] 1. Determination of dissolution time:

[0086] The dissolution time of the substances obtained in Examples 1-9 was tested. Taking Example 1 as an example, 25 g of the liver protective complex of Example 1 was weighed into a 500 mL beaker, 200 mL of cold boiled water (10° C.) was added, and the mixture was stirred. The time from the addition of cold boiled water to complete dissolution was calculated.

[0087] The dissolution time of the liver protection complex / detoxification degradation agent / soluble powder obtained in Examples 1-9 is as follows: Figure 1 As shown by Figure 1 It can be seen that all the above substances can be completely dissolved within 60s.

[0088] 2. Toxicology Experiments:

[0089] One hundred Kunming mice, half male and half female, were selected and acclimated for one week in an environment with a temperature of 20-26°C and a humidity of 40-60%. They were fed an SPF rat and mouse growth and breeding diet, produced by Beijing Keao Xieli Feed Co., Ltd., production license number: SCXK(Beijing)2012-0019. Sterile corncob bedding was used in the cages and changed twice weekly. The cages were also cleaned and disinfected. The use of experimental mice adhered to the regulations for the use and management of experimental animals and complied with animal welfare policies and relevant requirements.

[0090] After the adaptive feeding was completed, the experiment was started, and the mice were randomly divided into ten groups, which were recorded in order as the first to tenth groups. The first group was the control group, and the second to tenth groups corresponded to Examples 1-9, respectively. The dosage of the substance obtained in Examples 1-9 was 4 g / kg. The amount of the substance obtained in Examples 1-9 was calculated based on the weight of the mice, dissolved with water to a volume of 1.2 mL, and then gavage was performed. The control group was replaced with the same amount of normal saline for gavage, and the procedure was repeated once a day. The mice were observed for their daily feeding, drinking water, and activity, and the deaths were counted. If a mouse died, it was dissected in time to determine the cause of death, and the volume, color, texture, etc. of the main organs were observed. The experiment lasted for 21 days, and the results of the deaths of mice at the end of the experiment were as follows:

[0091] Table 1 Death of mice

[0092]

[0093] During the experiment, the weight changes of male and female mice were as follows: Figure 2 、 Figure 3 As shown by Figure 2 、 Figure 3 It can be seen that during the experiment, the mice in groups 2 to 10 lived normally and had no significant difference in weight from the mice in the control group, indicating that the liver protection complex / detoxification and degradation agent / soluble powder obtained in Examples 1-9 was non-toxic.

[0094] 3. Livestock Experiments

[0095] Taking into account the growth cycles of different livestock, chickens were selected to perform livestock experiments, and sexually mature white-feathered chickens and 2-month-old white-feathered chickens were selected for the experiments. After 10 days of adaptive feeding, the experiment began. Taking sexually mature white-feathered chickens as an example, they were randomly divided into 10 groups, with 15 chickens in each group. The first group was the control group, the second group was the drug group, and the third to tenth groups were all experimental groups, corresponding to Example 3, Example 6, Examples 7-9, and Comparative Examples 1-3, respectively. Example 3 and Example 6 were dissolved in the same proportion as Comparative Example 1 or Comparative Example 2, while Examples 7-9 were dissolved in the same proportion as Comparative Example 3. All examples were dissolved before use.

[0096] White-feathered chickens were not provided with drinking water from midnight to 7:00 AM daily. In the morning, before feeding, they were given specially prepared water. Both the control and drug groups received water without any drugs, while the experimental groups received water containing the liver protection complex, detoxification agent, and soluble powder, respectively. The amount of specially prepared water provided was 200 mL per bird. The chickens were then fed a normal diet. Both the drug and experimental groups received a diet containing 200 mg of glyphosate (analytical grade, Merck Chemicals (Shanghai) Co., Ltd.). The experiment was conducted for three months, after which the chickens were sacrificed and the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and γ-glutamyl transpeptidase (γ-GT) were measured (using kits purchased from Shanghai Enzyme Australia Biotechnology Co., Ltd., catalog numbers MO-J30625P, MO-J30626P, MO-J30660P, and MO-J31226P, respectively). Chicken livers were also harvested and the levels of catalase (CAT) (MO-J30715P), superoxide dismutase (SOD) (MO-J30494P), and glutathione peroxidase (GPx) (Nanjing Dulai Biotechnology Co., Ltd., E0117) were measured. Furthermore, the levels of malondialdehyde (MDA) (MO-J30490P), protein carbonyl compounds (PCO) (Nanjing Jiancheng Bioengineering Institute, A087), and advanced oxidation protein products (AOPP) (Jianglai Biotechnology, ELISA kit) were measured.

[0097] ALT, AST, ALP, and γ-GT are four common liver damage indicators. After consuming chicken feed containing glyphosate, the above indicators of sexually mature white-feathered chickens are as follows: Figure 4 、 Figure 5 As shown in the figure, the above index results of 2-month-old white-feathered chickens are as follows Figure 6 、 Figure 7 As shown. Figure 4-Figure 7 As can be seen, glyphosate significantly increases the levels of the above-mentioned indicators, showing significant differences from the control group. However, after drinking the water prepared according to Examples 7-9 beforehand, the above-mentioned indicators in Groups 5, 6, and 7 approached the control group values. A comparison of the values ​​in Groups 3 and 8 shows that the effect of the liver-protective complex after immediate dissolution is superior to that of the liquid preparation after 30 days of storage. A comparison of the values ​​in Groups 4 and 9 shows that the detoxification and degradation agent has similar properties, and the effect is also greater when immediately dissolved. A comparison of the values ​​in Groups 3, 4, and 7, as well as Groups 8, 9, and 10, shows that the special water obtained from either the liver-protective complex or the detoxification and degradation agent alone is less effective than the special water prepared from a soluble powder containing both of these substances. Thus, the comparison of the values ​​of the liver damage indicators shows that the soluble powder obtained by the present invention has a better liver-protective effect, and that immediate preparation is the optimal method of use.

[0098] CAT, SOD, and GPx characterize the antioxidant system of the liver. The relevant results of sexually mature white-feathered chickens and 2-month-old white-feathered chickens are as follows: Figure 8 、 Figure 9 As shown. Figure 8 、 Figure 9 It can be seen that after white-feathered chickens drink the freshly prepared special water made from soluble powder, their antioxidant system can be maintained at a good level, thereby continuously exerting the role of degrading and removing toxic substances.

[0099] MDA, PCO, and AOPP can reflect the degree of damage caused by liver tissue peroxidation, or be described as the degree of oxidative stress. Figure 10 、 Figure 11 The above index test results are respectively for sexually mature white-feathered chickens and 2-month-old white-feathered chickens. Figure 10 、 Figure 11 As can be seen, the second group of white-feathered chickens, which did not take any protective measures, experienced significant increases in MDA, PCO, and AOPP levels after consuming glyphosate-containing chicken feed. In contrast, groups five through seven maintained these indicators within acceptable ranges. Therefore, the soluble powder obtained by the present invention can effectively protect against oxidative stress caused by glyphosate, which is one of the manifestations of liver damage.

[0100] In addition, the above data show that early intervention in glyphosate resistance during the optimal growth and reproduction stage of white-feathered chickens will effectively avoid problems such as oxidative stress and liver damage caused by glyphosate, thereby helping white-feathered chickens gain weight and increase economic benefits.

[0101] The present invention also used aminomethylphosphonic acid with a purity of 99% purchased from Merck Chemical Technology (Shanghai) Co., Ltd. to conduct the above-mentioned livestock experiments, and obtained experimental results similar to those of glyphosate drugs, indicating that the soluble powder of the present application can more comprehensively deal with liver damage caused by glyphosate and its degradation products.

[0102] The above descriptions are merely several embodiments of the present invention and do not constitute any form of limitation to the present invention. Although the present invention is disclosed as above in terms of preferred embodiments, they are not intended to limit the present invention. Any technician familiar with the present profession who, without departing from the scope of the technical solution of the present invention, makes slight changes or modifications using the technical contents disclosed above are equivalent to equivalent implementation cases and fall within the scope of protection of the technical solution of the present invention.

Claims

1. A soluble powder for treating glyphosate-induced liver damage in livestock, characterized in that: Contains the following raw materials in parts by weight: 0.5-1.5 parts sucrose, 2-5 parts liver protection complex, 3-5 parts detoxification and degradation agent; The preparation method of the liver protection complex is as follows: L1. Fresh spirulina was washed, blanched, and air-dried to obtain a spirulina sample. Ginger charcoal was crushed and sieved to obtain ginger charcoal powder. The ginger charcoal powder was dispersed in water to obtain a ginger charcoal suspension with a concentration of 4-8% by weight. The spirulina sample was completely immersed in the ginger charcoal suspension and allowed to stand at room temperature. After completion, it was washed, completely dried, and ground to obtain spirulina powder with a particle size of 10-20 μm. L2. Weigh the spirulina powder, xanthan gum aqueous solution, and epigallocatechin gallate obtained in step L1 in a mass ratio of 8-10:10-15:

1. Mix the spirulina powder and xanthan gum aqueous solution and ultrasonically disperse them until uniform. Then, add epigallocatechin gallate, adjust the pH, and heat and maintain the mixture to obtain Solution 1. L3 The solution obtained in step L2 1 was dialyzed, and after completion the material in the dialysis bag was vacuum freeze-dried to obtain a liver protective complex; The preparation method of the detoxification degradation agent is as follows: S1. Weigh rosmarinic acid and sodium hydroxide solution in a mass-to-volume ratio of 40-50 mg: 6-9 mL, stir until uniformly dispersed, and adjust the pH to obtain Solution 2; S2. EDC, NHS and solution 2 obtained in step S1 were weighed in a mass volume ratio of 100-120 mg: 60-80 mg: 10-15 mL, heated, stirred until uniformly dispersed, and then allowed to stand at room temperature to obtain solution 3; S3. Laccase and solution 3 obtained in step S2 were weighed in a mass-to-volume ratio of 80-100 mg: 10-15 mL, heated, stirred, and centrifuged. The supernatant was concentrated to 0.4-0.5 times the original volume and freeze-dried in vacuo to obtain a complex. S4. Dissolve chitosan in acetic acid solution to obtain a chitosan solution with a mass percentage concentration of 3-5%, adjust the pH, add the complex obtained in step S3, and add 8-10 mg / mL of the complex. Stir at room temperature, centrifuge after completion, and disperse the obtained precipitate in trehalose solution to obtain a mixture. Spray dry the mixture to obtain a detoxifying degradation agent.

2. The soluble powder for treating glyphosate-induced liver damage in livestock according to claim 1, characterized in that: The mass percent concentration of the xanthan gum aqueous solution in step L2 is 3-5%.

3. The soluble powder for treating glyphosate-induced liver damage in livestock according to claim 2, characterized in that: The concentration of the sodium hydroxide solution in step S1 is 3 mol / L.

4. A method for preparing a soluble powder for treating glyphosate-induced liver damage in livestock according to any one of claims 1 to 3, characterized in that: The specific steps are as follows: V1. Dissolving sucrose in water to obtain a sucrose solution, mixing the sucrose solution with a detoxifying agent, spray drying, and freezing the resulting micropowder to obtain the core contents; V2. Dissolve the liver protection complex in water to obtain solution 4. Add the core contents obtained in step V1 to solution 4, quickly stir until uniformly dispersed, and vacuum freeze-dry to obtain a soluble powder for treating glyphosate-induced liver damage in livestock.

Images