Preparation method of negative bivalent active selenium
By using L-serine reaction with sodium methylselenoxide and N-acetyl-3-chloro-L-serine methyl ester, a high-purity negative divalent active selenium was prepared, which solved the problem of complex and high cost in the prior art, and achieved a simple, economical and suitable for large-scale production.
Patent Information
- Application Number
- CN202411808663.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-10
- Publication Date
- 2025-03-14
AI Technical Summary
When preparing negative divalent active selenium in the prior art, there are problems such as difficult to obtain reagents, expensive prices, harsh reaction conditions, and long reaction times, resulting in high production costs, complex processes, and difficult to produce on a large scale.
Using L-serine as raw material, the reaction with sodium methylselenoxide and N-acetyl-3-chloro-L-serine methyl ester is prepared through multiple steps of process processing, including step-by-step heating, extraction, hydrolysis and fermentation, etc.
It realizes the preparation of negative bivalent active selenium with simple process, high product purity, green and economical, and is suitable for large-scale industrial production.
Smart Images
Figure CN119613308A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of amino acid drug preparation, in particular to a method for preparing negative divalent active selenium. Background Art
[0002] Selenium is an essential trace element for the human body and an important component of many human enzymes. It has many functions such as anti-oxidation, anti-cancer, detoxification, growth promotion, and immunity enhancement. Once the body is deficient in selenium, it will directly lead to more than 40 diseases such as cardiovascular and cerebrovascular diseases, hypertension, metabolic syndrome, gastrointestinal diseases, diabetes, asthma, Parkinson's disease, liver disease, cancer, etc., and indirectly lead to the occurrence of more than 400 diseases. Therefore, the huge role of selenium in human health cannot be replaced by other substances. Data released by the World Health Organization show that more than 40 countries and regions in the world are suffering from selenium deficiency to varying degrees. Two-thirds of my country's regions are selenium-deficient areas, and 29.0% of the regions are severely selenium-deficient. Therefore, people living in these regions cannot meet the normal needs of the human body by relying on selenium in natural foods, and must rely on selenium supplements to make up for the insufficient intake of selenium in the diet. Currently, the selenium supplement products used are mainly inorganic selenium and organic selenium. Commonly used inorganic selenium is inorganic sodium selenite and sodium selenate-fortified foods and health products (such as: sodium selenite tablets, selenium-enriched salt, Selenium Baokang, etc.). Organic selenium (selenoprotein) refers to proteins rich in selenomethionine (SeMet) and selenocysteine (SeCys) that exist in animals and plants.
[0003] It has been proved that the toxicity and bioavailability of selenium are related to its chemical form: the absorption and utilization rate of inorganic selenium in vivo are low and toxic, and the poisoning dose is very close to the human body requirement, so its usage is strictly limited. Compared with inorganic selenium, the biggest feature of organic selenium is that it is absorbed by the human body and has a high utilization rate, strong biological activity, and little side effect, so the development and research of organic selenium products has received extensive attention. At present, the approach to obtain organic selenium mainly contains extraction method and chemical synthesis method, and the extraction method is subject to the restriction of natural organic selenium resources and low selenium content, and the extraction process is complicated, the yield is low, and the cost is high, which can not meet the needs of human nutrition, and the chemical synthesis method prepares organic selenium compounds, which has high selenium content, low production cost and can meet the nutritional needs of the human body, and has greater advantages.
[0004] Among them, negative divalent active selenium is the selenomethyl activated derivative of L-selenocysteine, the 21st essential amino acid for the human body. It is widely found in plants such as astragalus, garlic, onion and cabbage. It has clear chemical structure, low toxicity, high bioavailability, good selenium supplementation effect, strong anti-cancer activity and other characteristics. It has broad application prospects. In 2002, it was recognized by the US FDA as the latest generation of selenium source dietary supplements.
[0005] At present, the methods for obtaining negative divalent active selenium include direct chemical synthesis method preparation and indirect chemical splitting or enzyme splitting method preparation. The splitting methods all require additional consumption of chemical splitting reagents or enzyme splitting reagents, increase reaction steps and processing links, and the synthesis method is more complicated and the production cost is higher. The direct chemical synthesis method has a simple route, low production cost, and is easy to mass produce. However, the existing direct chemical synthesis method has problems such as reagents that are difficult to obtain, expensive, harsh reaction conditions, and long reaction time. For example, the chloroalanine diselenide sodium synthesis method (J.Med.Chem.1996,39,2040-2046) needs to be carried out under the condition of -70 ° C, and has strict requirements on equipment. It is difficult to put into large-scale production, such as the tert-butoxy acyl protection serine synthesis method, which has a long reaction time and involves high prices for raw materials and protective agents. Summary of the invention
[0006] 1. Technical problems solved:
[0007] In view of the shortcomings of the prior art, the present invention provides a method for preparing negative divalent active selenium. The method directly prepares negative divalent active selenium using L-serine, has a simple process, high product purity, is green and economical.
[0008] (II) Technical solution:
[0009] (1) Under stirring conditions, 7.8 g of methylselenol and 5 g of sodium carbonate were added with 50 ml of deionized water under nitrogen protection, and stirred at 60° C. for 2 h. A precipitate was generated. The mixture was cooled to room temperature and the precipitate was filtered out to obtain sodium methylselenol.
[0010] (2) Sodium methylselenol and N-acetyl-3-chloro-L-serine methyl ester in a molar ratio of (1-3):1 are added to a solvent and mixed, and reacted at -15-5°C for 1-5h, and then a step-by-step temperature increase method is adopted, and the reaction is stirred at 25-40°C for 1-3h, 40-65°C for 1-3h, and 65-75°C for 1-3h. After the reaction is completed, the temperature of the reaction system is cooled to -15-25°C, and the pH of the reaction system is slowly adjusted to 4.5-7 with 2-6 mol / L hydrochloric acid solution. The reaction is extracted with ethyl acetate for 3-5 times, the extracts are combined, washed with water until neutral, the organic phase is dried over anhydrous sodium sulfate, filtered, and the ethyl acetate is removed under reduced pressure to obtain N-acetyl-3-methylselenyl-L-serine methyl ester as a yellow oil.
[0011] (3) Mix a yellow oil of N-acetyl-3-methylseleno-L-serine methyl ester in a ratio of 1 g:(10-20) mL with a 2-6 mol / L hydrochloric acid solution, react at 80-95°C for 1.5-5 h, cool to room temperature 25-30°C, remove the hydrochloric acid solution under reduced pressure, collect the solid product, wash the solid product with 25-100°C water, collect the aqueous solution, add bentonite, stir and decolorize at 55-95°C for 0.5-1 h, filter out the bentonite after decolorization, wait until the temperature drops to -5-25°C, add pyridine to adjust the pH to 5.5-6.5, add anhydrous ethanol, then adjust the system temperature to -15-10°C, leave for 8-24 h, precipitate and dry to obtain a white solid hydrochloride.
[0012] (4) Put the hydrochloride into a special ceramic fermentation tank, add optimized culture medium, inoculate activated probiotic solution, lactic acid bacteria and bifidobacterium species, adjust the pH to 6.5, seal the tank, set the temperature to 6 degrees, take out the fermentation solution after two weeks, purify the active selenium solution, add hydrochloric acid solution to hydrolyze, filter out impurities, add solvent B to the filtrate, mix well, put it in a refrigerator for refrigeration, collect crystals, and obtain negative divalent active selenium white solid.
[0013] (5) Dissolving the negative divalent active selenium white solid in water at 25-100° C. to obtain a negative divalent active selenium solution, adding solvent A to the negative divalent active selenium solution, mixing evenly, then adding bentonite to decolorize, filtering, collecting the filtrate, adding solvent B to the filtrate, mixing evenly, then cooling to -10-25° C., standing for 2-12 hours, collecting white crystals, and obtaining negative divalent active selenium.
[0014] Preferably, the solvent in step (2) is at least one of methanol, ethanol, dioxane, tetrahydrofuran and toluene.
[0015] Preferably, in step (5), the ratio of negative divalent active selenium white solid to water is 1 g: (8-30) mL.
[0016] Preferably, the solvent A described in step (5) is one of dimethylformamide, methanol, ethanol, isopropanol, n-propanol, 2-butanol, isobutanol, n-butanol, glycerol, propylene glycol, cyclohexanol, pentanol, octanol, ethylene glycol, acetonitrile, diethylene glycol dimethyl ether and dimethylformamide.
[0017] Preferably, the solvent B described in step (5) is one of pyridine, acetone, triethylamine, tetrahydrofuran, methyl formate, tributylamine, dioxane, cyclohexanone, ethyl ether, methyl ethyl ketone, octanol, ethyl acetate, toluene, trioctylamine, isopropyl ether and n-butyl ether.
[0018] Preferably, the volume ratio of solvent A to water in step (5) is (1-40):50.
[0019] Preferably, the volume ratio of the solvent B to water in the step (5) is (1-40):50.
[0020] (III) Beneficial technical effects: The present invention selects N-acetyl-3-chloro-L-serine methyl ester as a synthetic raw material, undergoes a nucleophilic substitution reaction with sodium methylselenol to generate N-acetyl-3-methylseleno-L-serine methyl ester, and then undergoes a hydrolysis reaction under the action of hydrochloric acid to remove the N-acetyl group at the amino end and the methyl ester bond at the carboxylic acid end, respectively, to synthesize a hydrochloride with L-configuration maintained, and then activates it through bacterial fermentation, and further undergoes an impurity removal and refining process to obtain negative divalent active selenium with a purity of more than 98%.
[0021] The invention uses N-acetyl-3-chloro-L-serine methyl ester as a synthetic raw material to directly synthesize a hydrochloride with L-configuration maintained, thereby avoiding enzymatic splitting, chemical splitting and other splitting links, having a simple synthetic route, cheap and easily available raw materials, high yield and strong market competitiveness. Through a fermentation, refining and impurity removal process, negative divalent active selenium with a purity of more than 98% can be obtained. The invention has the characteristics of simple process, high product purity, greenness and environmental protection, and is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 This is the reaction formula for the preparation of negative divalent active selenium. DETAILED DESCRIPTION
[0023] Embodiment 1:
[0024] (1) Under stirring conditions, 7.8 g of methylselenol (obtained from Sichuan Xilaiwu Selenium-rich Agricultural Technology Research Institute) and 5 g of sodium carbonate were added to 50 ml of deionized water under nitrogen protection, and stirred at 60°C for 2 h. A precipitate was generated. The mixture was cooled to room temperature and the precipitate was filtered out to obtain sodium methylselenol.
[0025] (2) Sodium methylselenol, N-acetyl-3-chloro-L-serine methyl ester and anhydrous solvent methanol are mixed and reacted at -15°C for 2 hours, wherein the molar equivalent ratio of sodium methylselenol and N-acetyl-3-chloro-L-serine methyl ester is 1:1, and then a step-by-step temperature increase method is adopted, stirring and reacting at 25°C for 1 hour, stirring and reacting at 40°C for 1 hour, and stirring and reacting at 65°C for 1 hour. After the reaction is completed, the temperature of the reaction system is cooled to -15°C, and the pH of the reaction system is slowly adjusted to 7 with 2 mol / L hydrochloric acid solution. The reaction is extracted with ethyl acetate for 3 times, the extracts are combined, washed with water until neutral, the organic phase is dried over anhydrous sodium sulfate, filtered, and the ethyl acetate is removed under reduced pressure to obtain N-acetyl-3-methylselenol-L-serine methyl ester as a yellow oil.
[0026] (3) The yellow oil of N-acetyl-3-methylseleno-L-serine methyl ester obtained in step (2) was mixed with 2 mol / L hydrochloric acid solution in a mass volume ratio of 1 g:10 mL, reacted at 85° C. for 2 h, cooled to room temperature 30° C., removed the hydrochloric acid solution under reduced pressure, collected the solid product, washed the solid product with 25° C. water, collected the aqueous solution, added bentonite, stirred and decolorized at 60° C. for 0.5 h, filtered out the bentonite after decolorization, waited until the temperature dropped to -5° C., added pyridine to adjust the pH to 5.5, added anhydrous ethanol, and then adjusted the system temperature to -10° C., left for 8 h, precipitated and dried to obtain a hydrochloride white solid.
[0027] (4) The prepared hydrochloride is placed in a special ceramic fermentation tank, an optimized culture medium is added, activated probiotic solution, lactic acid bacteria and bifidobacterium are inoculated, the pH is adjusted to 6.5, the tank is sealed, and the temperature is set to 6 degrees. After two weeks, the fermentation solution is taken out, and the active selenium solution is purified, hydrochloric acid solution is added for hydrolysis, impurities are filtered out, tetrahydrofuran is added to the filtrate, mixed evenly, put into a refrigerator for refrigeration, and crystals are collected to obtain a negative divalent active selenium white solid.
[0028] (5) At 25° C., the negative divalent active selenium white solid obtained in step (4) is dissolved in water at a mass volume ratio of 1 g:8 mL to obtain a negative divalent active selenium solution, ether is added to the negative divalent active selenium solution at a volume ratio of 1:50 to water, the mixture is mixed evenly, bentonite is added for decolorization, the mixture is filtered, the filtrate is collected, tetrahydrofuran is added to the filtrate at a volume ratio of 1:50 to water, the mixture is mixed evenly, the mixture is cooled to -10° C., the mixture is allowed to stand for 2 h, white crystals are collected, and negative divalent active selenium is obtained.
[0029] Embodiment 2:
[0030] (1) Under stirring conditions, sodium methylselenol prepared from methylselenol and sodium carbonate, N-acetyl-3-chloro-L-serine methyl ester and anhydrous solvent ethanol are mixed and reacted at 15° C. for 5 hours, wherein the molar equivalent ratio of sodium methylselenol and N-acetyl-3-chloro-L-serine methyl ester is 2:1. Then, a step-by-step heating method is adopted, and the reaction is stirred at 40° C. for 3 hours, 65° C. for 3 hours, and 75° C. for 3 hours. After the reaction is completed, the temperature of the reaction system is cooled to 25° C., and the pH of the reaction system is slowly adjusted to 6 with 6 mol / L hydrochloric acid solution. The reaction is extracted with ethyl acetate for 5 times, the extracts are combined, washed with water until neutral, the organic phase is dried over anhydrous sodium sulfate, filtered, and the ethyl acetate is removed under reduced pressure to obtain N-acetyl-3-methylselenol-L-serine methyl ester as a yellow oil.
[0031] (2) The yellow oily substance of N-acetyl-3-methylseleno-L-serine methyl ester obtained in step (1) was mixed with 6 mol / L hydrochloric acid solution in a mass volume ratio of 1 g:20 mL, reacted at 95° C. for 5 h, cooled to room temperature (25° C.), the hydrochloric acid solution was removed under reduced pressure, the solid product was collected, the solid product was washed with 100° C. water, the aqueous solution was collected, bentonite was added, and the mixture was stirred at 95° C. for 1 h for decolorization. After decolorization, the bentonite was filtered out, and the temperature was lowered to 15° C., pyridine was added to adjust the pH to 6.5, anhydrous ethanol was added, and then the system temperature was adjusted to 10° C., and the mixture was allowed to stand for 12 h. The precipitation was dried to obtain a white solid hydrochloride.
[0032] (3) The prepared hydrochloride is placed in a special ceramic fermentation tank, an optimized culture medium is added, activated probiotic solution, lactic acid bacteria and bifidobacterium are inoculated, the pH is adjusted to 6.5, the tank is sealed, and the temperature is set to 6 degrees. After two weeks, the fermentation solution is taken out, and the active selenium solution is purified, hydrochloric acid solution is added for hydrolysis, impurities are filtered out, and dioxane is added to the filtrate, mixed evenly, put into a refrigerator for refrigeration, and crystals are collected to obtain a negative divalent active selenium white solid.
[0033] (4) At 100° C., the negative divalent active selenium white solid obtained in step (3) is dissolved in water at a mass volume ratio of 1 g:30 mL to obtain a negative divalent active selenium solution, methanol is added to the negative divalent active selenium solution in a volume ratio of 40:50 to water, the mixture is mixed evenly, bentonite is added for decolorization, the mixture is filtered, the filtrate is collected, dioxane is added to the filtrate in a volume ratio of 40:50 to water, the mixture is mixed evenly, the mixture is cooled to 25° C., the mixture is allowed to stand for 3 hours, white crystals are collected, and negative divalent active selenium is obtained.
[0034] Embodiment 3:
[0035] (1) Under stirring conditions, sodium methylselenol prepared from methylselenol and sodium carbonate, N-acetyl-3-chloro-L-serine methyl ester and anhydrous solvent tetrahydrofuran are mixed and reacted at 0°C for 3 hours, wherein the molar equivalent ratio of sodium methylselenol and N-acetyl-3-chloro-L-serine methyl ester is 3:1. Then, a step-by-step heating method is adopted, and the reaction is stirred at 30°C for 2 hours, 45°C for 2 hours, and 70°C for 2 hours. After the reaction is completed, the temperature of the reaction system is cooled to 0°C, and the pH of the reaction system is slowly adjusted to 5 with 4 mol / L hydrochloric acid solution. The reaction is extracted with ethyl acetate for 4 times, and the extracts are combined and washed with water until neutral. The organic phase is dried over anhydrous sodium sulfate, filtered, and the ethyl acetate is removed under reduced pressure to obtain N-acetyl-3-methylselenol-L-serine methyl ester as a yellow oil.
[0036] (2) The yellow oil of N-acetyl-3-methylseleno-L-serine methyl ester obtained in step (1) was mixed with 4 mol / L hydrochloric acid solution in a mass volume ratio of 1 g:15 mL, reacted at 90° C. for 3 h, cooled to 25° C., the hydrochloric acid solution was removed under reduced pressure, the solid product was collected, the solid product was washed with 80° C. water, the aqueous solution was collected, bentonite was added, and decolorization was carried out at 85° C. with stirring for 45 min. After decolorization, the bentonite was filtered out, and the temperature was reduced to 25° C., pyridine was added to adjust the pH to 6.0, anhydrous ethanol was added, and then the system temperature was adjusted to 0° C., left for 10 h, precipitated and dried to obtain a white solid hydrochloride.
[0037] (3) The prepared hydrochloride is placed in a special ceramic fermentation tank, an optimized culture medium is added, activated probiotic solution, lactic acid bacteria and bifidobacterium are inoculated, the pH is adjusted to 6.5, the tank is sealed, and the temperature is set to 6 degrees. After two weeks, the fermentation solution is taken out, and the active selenium solution is purified, hydrochloric acid solution is added for hydrolysis, impurities are filtered out, acetone is added to the filtrate, mixed evenly, put into a refrigerator for refrigeration, and crystals are collected to obtain a negative divalent active selenium white solid.
[0038] (4) At 80° C., the negative divalent active selenium white solid obtained in step (3) is dissolved in water at a mass volume ratio of 1 g:20 mL to obtain a negative divalent active selenium solution, isopropanol is added to the negative divalent active selenium solution in a volume ratio of 30:50 to water, the mixture is mixed evenly, bentonite is added for decolorization, the mixture is filtered, the filtrate is collected, acetone is added to the filtrate in a volume ratio of 20:50 to water, the mixture is mixed evenly, the mixture is cooled to 10° C., the mixture is allowed to stand for 4 hours, white crystals are collected, and negative divalent active selenium is obtained.
[0039] Embodiment 4:
[0040] (1) Under stirring conditions, sodium methylselenol prepared from methylselenol and sodium carbonate, N-acetyl-3-chloro-L-serine methyl ester and anhydrous solvent methanol are mixed and reacted at -5°C for 3 hours, wherein the molar equivalent ratio of sodium methylselenol and N-acetyl-3-chloro-L-serine methyl ester is 1.5:1, and then a step-by-step temperature increase method is adopted, stirring and reacting at 25°C for 2 hours, stirring and reacting at 45°C for 2 hours, and stirring and reacting at 75°C for 2 hours. After the reaction is completed, the temperature of the reaction system is cooled to 5°C, and the pH of the reaction system is slowly adjusted to 5 with 5 mol / L hydrochloric acid solution. The reaction is extracted with ethyl acetate for 3 times, the extracts are combined, washed with water until neutral, the organic phase is dried over anhydrous sodium sulfate, filtered, and the ethyl acetate is removed under reduced pressure to obtain N-acetyl-3-methylselenol-L-serine methyl ester as a yellow oil.
[0041] (2) The yellow oily substance of N-acetyl-3-methylseleno-L-serine methyl ester obtained in step (1) was mixed with 5 mol / L hydrochloric acid solution in a mass volume ratio of 1 g:16 mL, reacted at 85° C. for 2 h, cooled to 28° C., the hydrochloric acid solution was removed under reduced pressure, the solid product was collected, the solid product was washed with 90° C. water, the aqueous solution was collected, bentonite was added, and the mixture was stirred and decolorized at 95° C. for 30 min. After decolorization, the bentonite was filtered out, and the temperature was reduced to 5° C., pyridine was added to adjust the pH to 5.5, anhydrous ethanol was added, and then the system temperature was adjusted to -5° C., left for 12 h, precipitated and dried to obtain a white solid hydrochloride.
[0042] (3) The prepared hydrochloride is placed in a special ceramic fermentation tank, an optimized culture medium is added, activated probiotic solution, lactic acid bacteria and bifidobacterium are inoculated, the pH is adjusted to 6.5, the tank is sealed, and the temperature is set to 6 degrees. After two weeks, the fermentation solution is taken out, and the active selenium solution is purified, hydrochloric acid solution is added for hydrolysis, impurities are filtered out, solvent B is added to the filtrate, mixed evenly, put into a refrigerator for refrigeration, and crystals are collected to obtain a negative divalent active selenium white solid.
[0043] (4) At 60° C., the negative divalent active selenium white solid obtained in step (3) is dissolved in water at a mass volume ratio of 1 g:18 mL to obtain a negative divalent active selenium solution, dimethylformamide is added to the negative divalent active selenium solution in a volume ratio of 20:50 to water, the mixture is mixed evenly, bentonite is added for decolorization, the mixture is filtered, and the filtrate is collected, cyclohexanone is added to the filtrate in a volume ratio of 30:50 to water, the mixture is mixed evenly, and the mixture is cooled to 0° C. and allowed to stand for 6 hours, and white crystals are collected to obtain negative divalent active selenium.
[0044] Embodiment 5:
[0045] (1) Under stirring conditions, sodium methylselenol prepared from methylselenol and sodium carbonate, N-acetyl-3-chloro-L-serine methyl ester and anhydrous solvent dioxane are mixed and reacted at 0°C for 2 hours, wherein the molar equivalent ratio of sodium methylselenol and N-acetyl-3-chloro-L-serine methyl ester is 2.5:1. Then, a step-by-step heating method is adopted, and the reaction is stirred at 35°C for 1 hour, 55°C for 2 hours, and 75°C for 2 hours. After the reaction is completed, the temperature of the reaction system is cooled to 15°C, and the pH of the reaction system is slowly adjusted to 6.5 with 3 mol / L hydrochloric acid solution. The reaction is stirred evenly, extracted with ethyl acetate for 4 times, the extracts are combined, washed with water until neutral, the organic phase is dried over anhydrous sodium sulfate, filtered, and the ethyl acetate is removed under reduced pressure to obtain N-acetyl-3-methylselenol-L-serine methyl ester as a yellow oil.
[0046] (2) The yellow oil of N-acetyl-3-methylseleno-L-serine methyl ester prepared in step (1) was mixed with 3 mol / L hydrochloric acid solution in a mass volume ratio of 1 g:18 mL, reacted at 90° C. for 2.5 h, cooled to 25° C., the hydrochloric acid solution was removed under reduced pressure, the solid product was collected, the solid product was washed with 80° C. water, the aqueous solution was collected, bentonite was added, and decolorization was carried out at 65° C. with stirring for 40 min. After decolorization, the bentonite was filtered out, and the temperature was reduced to 20° C., pyridine was added to adjust the pH to 5.5, anhydrous ethanol was added, and then the system temperature was adjusted to -15° C., left for 24 h, precipitated and dried to obtain a white solid hydrochloride.
[0047] (3) The prepared hydrochloride is placed in a special ceramic fermentation tank, an optimized culture medium is added, activated probiotic solution, lactic acid bacteria and bifidobacterium are inoculated, the pH is adjusted to 6.5, the tank is sealed, and the temperature is set to 6 degrees. After two weeks, the fermentation solution is taken out, and the active selenium solution is purified, hydrochloric acid solution is added for hydrolysis, impurities are filtered out, toluene is added to the filtrate, mixed evenly, put into a refrigerator for refrigeration, and crystals are collected to obtain a negative divalent active selenium white solid.
[0048] (4) At 70° C., the negative divalent active selenium white solid obtained in step (3) is dissolved in water at a mass volume ratio of 1 g:14 mL to obtain a negative divalent active selenium solution, octanol is added to the negative divalent active selenium solution in a volume ratio of 10:50 to water, the mixture is mixed evenly, bentonite is added for decolorization, the mixture is filtered, the filtrate is collected, toluene is added to the filtrate in a volume ratio of 40:50 to water, the mixture is mixed evenly, the mixture is cooled to 5° C., the mixture is allowed to stand for 5 hours, white crystals are collected, and negative divalent active selenium is obtained.
[0049] Embodiment 6:
[0050] (1) Under stirring conditions, sodium methylselenol prepared from methylselenol and sodium carbonate, N-acetyl-3-chloro-L-serine methyl ester and anhydrous solvent toluene are mixed and reacted at 5°C for 1 hour, wherein the molar equivalent ratio of sodium methylselenol and N-acetyl-3-chloro-L-serine methyl ester is 1:1. Then, a step-by-step heating method is adopted, and the reaction is stirred at 35°C for 1 hour, 55°C for 1 hour, and 75°C for 3 hours. After the reaction is completed, the temperature of the reaction system is cooled to 10°C, and the pH of the reaction system is slowly adjusted to 5.5 with 4.5 mol / L hydrochloric acid solution. The reaction is stirred evenly, and extracted with ethyl acetate for 3 times. The extracts are combined, washed with water until neutral, and the organic phase is dried over anhydrous sodium sulfate, filtered, and the ethyl acetate is removed under reduced pressure to obtain N-acetyl-3-methylselenol-L-serine methyl ester as a yellow oil.
[0051] (2) The yellow oily substance of N-acetyl-3-methylseleno-L-serine methyl ester obtained in step (1) was mixed with 2.5 mol / L hydrochloric acid solution in a mass volume ratio of 1 g:15 mL, reacted at 80° C. for 1.5 h, cooled to 30° C., the hydrochloric acid solution was removed under reduced pressure, the solid product was collected, the solid product was washed with 60° C. water, the aqueous solution was collected, bentonite was added, and the mixture was stirred and decolorized at 55° C. for 30 min. After decolorization, the bentonite was filtered out, and the temperature was reduced to 10° C., pyridine was added to adjust the pH to 5.5, anhydrous ethanol was added, and then the system temperature was adjusted to -10° C., left for 24 h, and dried to obtain a white solid hydrochloride.
[0052] (3) The prepared hydrochloride is placed in a special ceramic fermentation tank, an optimized culture medium is added, activated probiotic solution, lactic acid bacteria and bifidobacterium are inoculated, the pH is adjusted to 6.5, the tank is sealed, and the temperature is set to 6 degrees. After two weeks, the fermentation solution is taken out, and the active selenium solution is purified, hydrochloric acid solution is added for hydrolysis, impurities are filtered out, ether is added to the filtrate, mixed evenly, put into a refrigerator for refrigeration, and crystals are collected to obtain a negative divalent active selenium white solid.
[0053] (4) At 60° C., the negative divalent active selenium white solid obtained in step (3) is dissolved in water at a mass volume ratio of 1 g:13 mL to obtain a negative divalent active selenium solution, acetonitrile is added to the negative divalent active selenium solution in a volume ratio of 40:50 to water, the mixture is mixed evenly, bentonite is added for decolorization, the mixture is filtered, and the filtrate is collected, and ether is added to the filtrate in a volume ratio of 10:50 to water, the mixture is mixed evenly, and the mixture is cooled to 5° C. and allowed to stand for 6 hours, and white crystals are collected to obtain negative divalent active selenium.
[0054] Embodiment 7:
[0055] (1) Under stirring conditions, sodium methylselenol prepared from methylselenol and sodium carbonate, N-acetyl-3-chloro-L-serine methyl ester and anhydrous solvent dioxane are mixed and reacted at -5°C for 1 hour, wherein the molar equivalent ratio of sodium methylselenol and N-acetyl-3-chloro-L-serine methyl ester is 3:1, and then a step-by-step heating method is adopted, stirring and reacting at 35°C for 2 hours, 55°C for 3 hours, and 75°C for 3 hours. After the reaction is completed, the temperature of the reaction system is cooled to 10°C, and the pH of the reaction system is slowly adjusted to 4.5 with 4.5 mol / L hydrochloric acid solution. The reaction is extracted with ethyl acetate for 3 times, the extracts are combined, washed with water until neutral, the organic phase is dried over anhydrous sodium sulfate, filtered, and the ethyl acetate is removed under reduced pressure to obtain N-acetyl-3-methylselenol-L-serine methyl ester as a yellow oil.
[0056] (2) The yellow oily substance of N-acetyl-3-methylseleno-L-serine methyl ester obtained in step (1) was mixed with 5 mol / L hydrochloric acid solution in a mass volume ratio of 1 g:18 mL, reacted at 80° C. for 3.5 h, cooled to 25° C., and the hydrochloric acid solution was removed under reduced pressure. The solid product was collected, and the solid product was washed with 60° C. water. The aqueous solution was collected, and bentonite was added. The mixture was stirred and decolorized at 55° C. for 30 min. After decolorization, the bentonite was filtered out, and the temperature was reduced to 5° C., pyridine was added to adjust the pH to 5.5, anhydrous ethanol was added, and then the system temperature was adjusted to -10° C., and the mixture was left for 24 h and dried to obtain a white solid hydrochloride.
[0057] (3) The prepared hydrochloride is placed in a special ceramic fermentation tank, an optimized culture medium is added, activated probiotic solution, lactic acid bacteria and bifidobacterium are inoculated, the pH is adjusted to 6.5, the tank is sealed, and the temperature is set to 6 degrees. After two weeks, the fermentation solution is taken out, and the active selenium solution is purified, hydrochloric acid solution is added for hydrolysis, impurities are filtered out, ethyl acetate is added to the filtrate, mixed evenly, put in a refrigerator for refrigeration, and crystals are collected to obtain a negative divalent active selenium white solid.
[0058] (4) At 60° C., the negative divalent active selenium white solid obtained in step (3) is dissolved in water at a mass volume ratio of 1 g:15 mL to obtain a negative divalent active selenium solution, and n-butanol is added to the negative divalent active methylselenocysteine solution at a volume ratio of 10:50 to water, and the mixture is mixed evenly. Bentonite is then added for decolorization, and the mixture is filtered and the filtrate is collected. Ethyl acetate is added to the filtrate at a volume ratio of 40:50 to water, and the mixture is mixed evenly. The mixture is then cooled to 5° C. and allowed to stand for 6 hours. White crystals are collected to obtain negative divalent active selenium.
[0059] Embodiment 8:
[0060] (1) Under stirring conditions, sodium methylselenol prepared from methylselenol and sodium carbonate, N-acetyl-3-chloro-L-serine methyl ester and anhydrous solvent methanol are mixed and reacted at 5°C for 2.5 hours, wherein the molar equivalent ratio of sodium methylselenol and N-acetyl-3-chloro-L-serine methyl ester is 1:1. Then, a step-by-step temperature increase method is adopted, and the reaction is stirred at 25°C for 2 hours, 45°C for 2 hours, and 70°C for 2 hours. After the reaction is completed, the temperature of the reaction system is cooled to 5°C, and the pH of the reaction system is slowly adjusted to 5 with 6 mol / L hydrochloric acid solution. The extract is extracted with ethyl acetate for 5 times, and the extract is washed with water until neutral. The organic phase is dried over anhydrous sodium sulfate, filtered, and the ethyl acetate is removed under reduced pressure to obtain N-acetyl-3-methylselenol-L-serine methyl ester as a yellow oil.
[0061] (2) The yellow oil of N-acetyl-3-methylseleno-L-serine methyl ester obtained in step (1) was mixed with 6 mol / L hydrochloric acid solution in a mass volume ratio of 1 g:12 mL, reacted at 95° C. for 5 h, cooled to 25° C., the hydrochloric acid aqueous solution was removed under reduced pressure, the solid product was collected, the solid product was washed with 60° C. water, the aqueous solution was collected, bentonite was added for decolorization, stirred at 75° C. for 1 h, the bentonite was filtered out, and when the temperature dropped to 0° C., pyridine was added to adjust the pH to 5.5, anhydrous ethanol was added, and then the system temperature was adjusted to 0° C., left for 8 h, and dried to obtain a white solid hydrochloride.
[0062] (3) The prepared hydrochloride is placed in a special ceramic fermentation tank, an optimized culture medium is added, activated probiotic solution, lactic acid bacteria and bifidobacterium are inoculated, the pH is adjusted to 6.5, the tank is sealed, and the temperature is set to 6 degrees. After two weeks, the fermentation solution is taken out, and the active selenium solution is purified, hydrochloric acid solution is added for hydrolysis, impurities are filtered out, toluene is added to the filtrate, mixed evenly, put into a refrigerator for refrigeration, and crystals are collected to obtain a negative divalent active selenium white solid.
[0063] (4) At 35° C., the negative divalent active selenium white solid obtained in step (3) is dissolved in water at a mass volume ratio of 1 g:30 mL to obtain a negative divalent methylselenocysteine solution, ethylene glycol is added to the negative divalent active selenium solution at a volume ratio of 45:50 to water, the mixture is mixed evenly, bentonite is added for decolorization, the mixture is filtered, the filtrate is collected, toluene is added to the filtrate at a volume ratio of 5:50 to water, the mixture is mixed evenly, the mixture is cooled to 10° C., the mixture is allowed to stand for 12 h, white crystals are collected, and negative divalent active selenium is obtained.
[0064] Table 1 shows the yield of the intermediate material and the purity of negative divalent active selenium in the embodiment.
[0065]
[0066]
[0067] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. A method for preparing negative divalent active selenium, characterized in that: The preparation method comprises: (1) adding methylselenol, sodium carbonate and deionized water under stirring and stirring at 60-65° C. for 2-2.5 hours until a precipitate is generated, cooling to room temperature, filtering out the precipitate, and obtaining sodium methylselenol; (2) Sodium methylselenol and N-acetyl-3-chloro-L-serine methyl ester in a molar ratio of (1-3):1 are added to a solvent and mixed, and reacted at -15-5°C for 1-5h, and then a step-by-step temperature increase method is adopted, and the reaction is stirred at 25-40°C for 1-3h, 40-65°C for 1-3h, and 65-75°C for 1-3h. After the reaction is completed, the temperature of the reaction system is cooled to -15-25°C, and the pH of the reaction system is slowly adjusted to 4.5-7 with a 2-6 mol / L hydrochloric acid solution. The solution is extracted with ethyl acetate for 3-5 times, the extracts are combined, washed with water until neutral, the organic phase is dried over anhydrous sodium sulfate, filtered, and the ethyl acetate is removed under reduced pressure to obtain N-acetyl-3-methylselenyl-L-serine methyl ester as a yellow oil; (3) Mix the yellow oil of N-acetyl-3-methylseleno-L-serine methyl ester with 2-6 mol / L hydrochloric acid solution, react at 80-95°C for 1.5-5h, cool to room temperature 25-30°C, remove the hydrochloric acid solution under reduced pressure, collect the solid product, wash the solid product with 25-100°C water, collect the aqueous solution, add bentonite, stir and decolorize at 55-95°C for 0.5-1h, filter out the bentonite after decolorization, wait until the temperature drops to -5-25°C, add pyridine to adjust the pH to 5.5-6.5, add anhydrous ethanol, then adjust the system temperature to -15-10°C, let stand for 8-24h, precipitate and dry to obtain a white solid hydrochloride; (4) placing the hydrochloride in a ceramic fermentation tank, adding optimized culture medium, inoculating activated probiotic solution, lactic acid bacteria and bifidobacterium species, adjusting the pH to 6.5, sealing the tank, setting the temperature to 6 degrees, taking out the fermentation solution after two weeks, purifying the active selenium solution, adding hydrochloric acid solution to hydrolyze, filtering out impurities, adding solvent B to the filtrate, mixing evenly, putting in a refrigerator for refrigeration, collecting crystals, and obtaining negative divalent active selenium white solid; (5) Dissolving the negative divalent active selenium white solid in water at 25-100° C. to obtain a negative divalent active selenium solution, adding solvent A to the negative divalent active selenium solution, mixing evenly, then adding bentonite to decolorize, filtering, collecting the filtrate, adding solvent B to the filtrate, mixing evenly, then cooling to -10-25° C., standing for 2-12 hours, collecting white crystals, and obtaining negative divalent active selenium.
2. The method for preparing negative divalent active selenium according to claim 1, characterized in that: The solvent in step (2) is at least one of methanol, ethanol, dioxane, tetrahydrofuran and toluene.
3. The method for preparing negative divalent active selenium according to claim 1, characterized in that: The ratio of N-acetyl-3-methylseleno-L-serine methyl ester to the hydrochloric acid solution in the step (3) is 1 g: (10-20) mL.
4. The method for preparing negative divalent active selenium according to claim 1, characterized in that: In the step (5), the ratio of the negative divalent active selenium white solid to water is 1 g: (8-30) mL.
5. The method for preparing negative divalent active selenium according to claim 1, characterized in that: The solvent A described in the step (5) is one of dimethylformamide, methanol, ethanol, isopropanol, n-propanol, 2-butanol, isobutanol, n-butanol, glycerol, propylene glycol, cyclohexanol, pentanol, octanol, ethylene glycol, acetonitrile, diethylene glycol dimethyl ether and dimethylformamide.
6. The method for preparing negative divalent active selenium according to claim 1, characterized in that: The solvent B described in step (5) is one of pyridine, acetone, triethylamine, tetrahydrofuran, methyl formate, tributylamine, dioxane, cyclohexanone, ethyl ether, methyl ethyl ketone, octanol, ethyl acetate, toluene, trioctylamine, isopropyl ether and n-butyl ether.
7. The method for preparing negative divalent active selenium according to claim 1, characterized in that: The volume ratio of solvent A to water in step (5) is (1-40):
50.
8. The method for preparing negative divalent active selenium according to claim 1, characterized in that: The volume ratio of the solvent B to water in the step (5) is (1-40):50.