A method for preparing licorice polysaccharide

Abstract

This disclosure relates to the field of pharmaceutical composition preparation, and more particularly to a method for preparing licorice polysaccharide; the preparation method includes the following steps: solid-state fermentation, alkaline solution extraction, defatting, deproteinization, decolorization, ethanol fractionation extraction, amination modification, and selenization. The licorice polysaccharide linked to selenoproteins obtained by the preparation method provided in this disclosure has antioxidant, immune-enhancing, and anti-tumor effects.

Description

Technical Field

[0001] This disclosure relates to the field of pharmaceutical composition preparation, and more particularly to a method for preparing licorice polysaccharide. Background Technology

[0002] Licorice is one of the world's most popular herbs, often referred to as the "King of Herbs." Pharmacological studies of licorice both domestically and internationally have shown that it tonifies the spleen and replenishes qi, clears heat and detoxifies, relieves cough and phlegm, and harmonizes the effects of other herbs. It is mainly used for spleen and stomach weakness, fatigue, palpitations, shortness of breath, cough with excessive phlegm, carbuncles, and boils, and can also alleviate drug toxicity. Polysaccharides, one of the main components of licorice, possess antioxidant, antiviral, and antitumor biological activities. Currently, only the lower part of licorice is used medicinally; the stems and leaves, as byproducts, also contain abundant active substances, but their utilization rate is low due to the difficulty in fully extracting their effective components.

[0003] Selenium, an essential trace element for the human body, is the main active component of glutathione peroxidase. It can directly or indirectly scavenge oxygen free radicals in the body, inhibit lipid oxidation or peroxidation, block the pathogenic effects of reactive oxygen species and free radicals, protect cells from peroxide damage, and maintain cell membrane stability. It plays a vital role in animal growth, reproduction, immunity, anti-infection, and anti-stress. Selenium is not only an important component of some enzymes, but it also plays a significant role in preventing tumors, treating cardiovascular and cerebrovascular diseases, and delaying aging. Selenium is widely distributed in nature, mainly in two forms: inorganic selenium, including elemental selenium, metal selenium compounds, selenites (sodium selenite, calcium selenite, zinc selenite, etc.), selenates (sodium selenate, etc.), selenides (hydrogen selenide, zinc selenide, sodium selenide, etc.), selenium sulfide, selenium dioxide, selenium disulfide, etc.; and organic selenium, mainly existing as selenium-containing proteins and selenium polysaccharides. Therefore, there is an urgent need for a preparation process that can combine organic selenium with licorice extract. Summary of the Invention

[0004] This disclosure provides a method for preparing licorice polysaccharide to address the shortcomings of related technologies.

[0005] According to a first aspect of the present disclosure, a method for preparing licorice polysaccharide is provided, wherein the licorice polysaccharide is licorice polysaccharide after selenization treatment, and the preparation method includes the following steps:

[0006] Step 1: Provide licorice raw material, and ferment the licorice raw material in a solid state to obtain the fermented licorice product of Step 1;

[0007] Step 2: After drying the fermented licorice product, pulverize it and sieve it. Then, extract it with an alkaline solution to obtain an aqueous solution of the crude licorice extract from Step 2.

[0008] Step 3: The aqueous solution of the crude licorice extract is defatted to obtain the defatted crude licorice extract of Step 3;

[0009] Step 4: The defatted licorice crude extract is subjected to a deproteinization process to obtain the deproteinized licorice crude extract of Step 4.

[0010] Step 5: The deproteinized crude licorice extract is subjected to a decolorization treatment to obtain the decolorized crude licorice extract of Step 5.

[0011] Step 6: Extract the decolorized crude licorice extract with ethanol to obtain the graded licorice polysaccharide from Step 6;

[0012] Step 7: The licorice graded polysaccharide is modified by amylation to obtain the amylated licorice graded polysaccharide of Step 7.

[0013] Step 8: The aminated and modified licorice graded polysaccharide is subjected to selenization treatment to obtain the selenized licorice polysaccharide.

[0014] In one aspect of this disclosure, step 1 includes the following steps:

[0015] Step 1-1: Provide licorice raw material, mix the licorice raw material with flour and soybean meal, then add water and heat to 45℃-55℃, the heating time is selected from 2-4h, then add compound probiotics and cellulase to the water to obtain the mixture system of Step 1-1;

[0016] Steps 1-2: Ferment the mixture at 25℃-35℃ for 48-144 hours to obtain the fermented licorice product.

[0017] In one aspect of the embodiments of this disclosure, in step 1-1, the mass ratio of the licorice raw material, flour and soybean meal is selected from (6-8):(1-3):1; preferably, the mass ratio of the licorice raw material, flour and soybean meal is selected from 7:2:1.

[0018] In one aspect of the embodiments of this disclosure, in step 1-1, the compound probiotics include core bacteria and a variety of complementary bacteria. The core bacteria are selected from Lactobacillus plantarum and / or Saccharomyces cerevisiae, and the complementary bacteria are selected from Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus reuteri, Lactobacillus paracasei, Bifidobacterium longum, Bacillus subtilis, Bifidobacterium infantis, or Lactobacillus acidophilus.

[0019] In one aspect of the embodiments of this disclosure, in step 1-1, preferably, the compound probiotics include Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus subtilis; specifically, the compound probiotics are commercially available compound probiotics with Lactobacillus plantarum: Saccharomyces cerevisiae: Bacillus subtilis = 1:1:1.

[0020] In one aspect of the embodiments of this disclosure, in the mixing system of step 1-1, based on the total mass of the solid mixture (i.e., licorice raw material, flour and soybean meal), the mass ratio of water added is selected from 40% to 65%, the mass ratio of the added compound probiotics is selected from 0.5% to 1.5%, and the mass ratio of the added cellulase is selected from 0.25% to 0.5%.

[0021] In one aspect of the present disclosure, preferably, steps 1-2 include: fermenting the mixture at 30°C for a time of 96 hours.

[0022] In one aspect of this disclosure, step 2 includes the following steps:

[0023] Step 2-1: Dry the fermented licorice product at 50℃-70℃, and then pass it through a 50-100 mesh sieve to obtain the fermented licorice product powder of Step 2-1.

[0024] Step 2-2: Add the fermented licorice product powder to NaOH aqueous solution and heat to 70℃-90℃ for 1-2 hours. Then, centrifuge the heated mixture to obtain the supernatant and solid from Step 2-2.

[0025] Step 2-3: Add the solid obtained in step 2-2 back into the NaOH aqueous solution and heat to 70℃-90℃ for 1-2 hours. Then, centrifuge the heated mixture to obtain the supernatant and solid from step 2-3.

[0026] Step 2-4: Combine the supernatant from Step 2-2 and the supernatant from Step 2-3 to obtain an aqueous solution of crude licorice extract.

[0027] In one aspect of the embodiments of this disclosure, in steps 2-2 and 2-3, the NaOH aqueous solution used is a NaOH aqueous solution with a concentration of 0.1-0.5 mol / L.

[0028] In one aspect of the present disclosure, preferably, in step 2-2, the mass of the NaOH aqueous solution used is 15-20 times the mass of the fermented licorice product powder.

[0029] In one aspect of the embodiments of this disclosure, preferably, in step 2-3, the mass of the NaOH aqueous solution used is 5-10 times the mass of the solid obtained in step 2-2.

[0030] In one aspect of this disclosure, specifically, step 2-2 includes: adding the fermented licorice product powder to a 0.3 mol / L NaOH aqueous solution and heating it to 85°C for 1.5 h; then centrifuging the heated mixture to obtain the supernatant and solid from step 2-2; wherein the mass of the NaOH aqueous solution is 16 times the mass of the fermented licorice product powder.

[0031] In one aspect of the embodiments of this disclosure, specifically, step 2-3 includes: adding the solid obtained in step 2-2 to a 0.3 mol / L NaOH aqueous solution and heating it to 85°C for 1.5 h; then centrifuging the heated mixture to obtain the supernatant and solid of step 2-3; wherein the mass of the NaOH aqueous solution is 8 times the mass of the fermented licorice product powder.

[0032] In one aspect of this disclosure, specifically in step 2, the centrifugation rate is 3500 rpm.

[0033] In one aspect of this disclosure, step 3 includes the following steps:

[0034] Step 3-1: Freeze-dry the aqueous solution of crude licorice extract to obtain a solid crude licorice extract;

[0035] Step 3-2: Add the solid of the crude licorice extract to n-hexane and soak at room temperature for 5-10 hours to obtain the mixture from step 3-2;

[0036] Step 3-3: The mixture from Step 3-2 is filtered and dried to remove n-hexane. The resulting solid is then added to a mixed solution of ethanol and acetone and heated under reflux at 65℃-75℃ for 1-2.5 hours. The product after reaction is concentrated and freeze-dried to obtain defatted crude licorice extract.

[0037] In one aspect of this disclosure, in step 3-2, the mass of the n-hexane is selected from 0.5-1.5 times the mass of the solid of the crude licorice extract.

[0038] In one aspect of this disclosure, in the mixed solution of ethanol and acetone, the volume ratio of ethanol to acetone is selected from (2-4):1.

[0039] In one aspect of the embodiments of this disclosure, specifically, step 3-2 includes: adding the solid of the crude licorice extract to n-hexane, wherein the mass of the n-hexane is 0.8 times the mass of the solid of the crude licorice extract, and soaking at room temperature for 8 hours to obtain the mixture of step 3-2.

[0040] In one aspect of the embodiments of this disclosure, specifically, step 3-3 includes: filtering and drying the mixture from step 3-2 to remove n-hexane, then adding the resulting solid to a mixed solution of ethanol and acetone, wherein the volume ratio of ethanol to acetone in the mixed solution is selected as 3:1; then heating the mixed solution under reflux at 65°C-75°C for 1.5 hours, and concentrating and vacuum drying the product after the reaction to obtain defatted crude licorice extract.

[0041] In one aspect of this disclosure, in step 4, the deproteinization step is completed by a freeze-thaw cycle; step 4 includes the following steps:

[0042] Step 4-1: Add an appropriate amount of water to the defatted crude licorice extract and dissolve it completely. Then freeze it in an environment of -70℃ to -50℃ for 6-12 hours to obtain the frozen product of Step 4-1.

[0043] Step 4-2: Place the frozen product from Step 4-1 in a constant temperature environment of 5℃-15℃ to thaw naturally;

[0044] Step 4-3: Repeat steps 4-1 and 4-2 3-6 times to obtain the product after freeze-thaw cycles;

[0045] Step 4-4: After centrifuging the product after freeze-thaw cycle, obtain supernatant and solid. Collect the supernatant to obtain deproteinized crude licorice extract.

[0046] In one aspect of this disclosure, step 5 includes the following steps:

[0047] Step 5-1: Add an appropriate amount of water and a first electrolyte solution to the deproteinized crude licorice extract, and add the first electrolyte solution until the final concentration is 0.02-0.08 mol / L to obtain the mixed solution of Step 5-1; wherein, the first electrolyte solution is CaCl2 solution or MgSO4 solution;

[0048] Step 5-2: After centrifuging the mixed solution from Step 5-1, a supernatant and a solid are obtained. The supernatant is collected and dialyzed to obtain the decolorized crude licorice extract.

[0049] In one aspect of the embodiments of this disclosure, specifically, in step 5-1, a first electrolyte solution is added to a final concentration of 0.05 mol / L, wherein the first electrolyte solution is a CaCl2 solution.

[0050] In one aspect of this disclosure, step 6 includes the following steps:

[0051] Step 6-1: The decolorized crude licorice extract is concentrated to obtain the concentrated solution of Step 6-1;

[0052] Step 6-2: Add ethanol to the concentrated solution from Step 6-1 until the concentration of ethanol in the final solution is 60%-70%. Collect and dry the precipitate obtained at this point to obtain graded licorice polysaccharides.

[0053] In one aspect of this disclosure, in step 6-2, the ethanol is a 95% ethanol solution.

[0054] In one aspect of this disclosure, step 7 includes the following steps:

[0055] Step 7-1: Add the graded licorice polysaccharide to a mixed solution of ethanol and propanol;

[0056] Step 7-2: Under nitrogen protection, add the amino-containing modified compound to the mixed solution, stir for 1-3 hours, and after the reaction is completed, filter, wash and dry to obtain amino-modified licorice graded polysaccharides; wherein the amino-containing modified compound is selected from propylenediamine, 3-methylaminopropylamine, tris(3-aminopropyl)amine, 3-diethylaminopropylamine or 3-dimethylaminopropylamine.

[0057] In one aspect of this disclosure, in the mixed solution of step 7-1, the volume ratio of ethanol to propanol is selected from (0.5-1):1.

[0058] In one aspect of the embodiments of this disclosure, preferably, the amino-containing modified compound is selected from 3-methylaminopropylamine.

[0059] In one aspect of this disclosure, step 8 includes the following steps:

[0060] Step 8-1: Dissolve the aminated licorice graded polysaccharide in water, and add plant extract containing selenoprotein and cross-linking agent to it, and stir at room temperature for 2-4 hours;

[0061] Step 8-2: Add NaCl solution dropwise until a precipitate appears, continue adding NaCl solution dropwise until the precipitate no longer increases, collect the precipitate and dry it to obtain selenized licorice polysaccharide.

[0062] In one aspect of this disclosure, in step 8-1, the crosslinking agent is selected from glutaraldehyde.

[0063] In one aspect of the embodiments of this disclosure, the plant extract containing selenoprotein is selected from selenium-enriched barley grass extract, selenium-enriched wolfberry extract, selenium-enriched salvia miltiorrhiza extract, selenium-enriched honeysuckle extract, or selenium-enriched chlorella extract.

[0064] According to a second aspect of the present disclosure, a pharmaceutical composition is provided comprising selenized licorice polysaccharide obtained by the aforementioned preparation method.

[0065] According to a third aspect of the present disclosure, a health food is provided, the health food comprising selenized licorice polysaccharide obtained by the aforementioned preparation method.

[0066] The technical solutions provided by the embodiments of this disclosure may include the following beneficial effects:

[0067] As can be seen from the above embodiments, this disclosure provides a licorice polysaccharide linked to selenoprotein, which has antioxidant, immune-enhancing, and anti-tumor effects.

[0068] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit this disclosure. Detailed Implementation

[0069] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with embodiments. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. The embodiments described herein are illustrative in nature and are used to provide a basic understanding of this application. The embodiments of this application should not be construed as limiting this application.

[0070] For the sake of brevity, this article only discloses a few specific numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, just as any upper limit can be combined with any other upper limit to form an unspecified range. Furthermore, each individually disclosed point or single value can itself serve as a lower or upper limit and be combined with any other point or single value or with other lower or upper limits to form an unspecified range.

[0071] In this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0072] In this description, unless otherwise stated, "above" and "below" include the stated number.

[0073] Unless otherwise stated, the terms used in this disclosure have their common meanings as commonly understood by those skilled in the art. Unless otherwise stated, the values ​​of the parameters mentioned in this disclosure can be measured using various measurement methods commonly used in the art (e.g., they can be tested according to the methods given in the embodiments of this disclosure).

[0074] The term "about" is used to describe and indicate small variations. When used in conjunction with an event or situation, the term may refer to examples in which the event or situation occurred precisely or in examples in which the event or situation occurred very approximately. For example, when used in conjunction with numerical values, the term may refer to a range of variation less than or equal to ±10% of the numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%. Additionally, quantities, ratios, and other numerical values ​​are sometimes presented in range format herein. It should be understood that such range format is for convenience and brevity and should be interpreted flexibly to include not only numerical values ​​explicitly specified as range limits but also all individual numerical values ​​or subranges covered within the range, as if each numerical value and subrange were explicitly specified.

[0075] The list of items connected by the terms "at least one of," "at least one of," "at least one of," or other similar terms can mean any combination of the listed items. For example, if items A and B are listed, then the phrase "at least one of A and B" means only A; only B; or A and B. In another instance, if items A, B, and C are listed, then the phrase "at least one of A, B, and C" means only A; or only B; only C; A and B (excluding C); A and C (excluding B); B and C (excluding A); or all of A, B, and C. Item A may contain a single component or multiple components. Item B may contain a single component or multiple components. Item C may contain a single component or multiple components.

[0076] In this disclosure, the raw materials for the selenium-enriched *Cephalotaxus fortunei* extract, selenium-enriched *Lycium barbarum* extract, selenium-enriched *Salvia miltiorrhiza* extract, selenium-enriched *Lonicera japonica* extract, or selenium-enriched *Chlorella* extract mentioned in this disclosure are selenium-enriched *Cephalotaxus fortunei*, selenium-enriched *Lycium barbarum*, selenium-enriched *Salvia miltiorrhiza*, selenium-enriched *Lonicera japonica*, and selenium-enriched *Chlorella*, which are all commercially available.

[0077] In one aspect of this disclosure, the selenium-enriched wolfberry extract is prepared by the following steps:

[0078] Step 9-1: After crushing the selenium-enriched goji berries, pass them through an 80-mesh sieve to obtain selenium-enriched goji berry powder.

[0079] Step 9-2: Add the selenium-enriched wolfberry powder to a 0.5 mol / L NaHCO3 aqueous solution, sonicate at 200 kHz for 45 min, then heat to 75 °C for 1 h, and then centrifuge the heated mixture at 4000 rpm for 5 min to obtain the supernatant and solid from Step 9-2; collect the supernatant and concentrate it by rotary evaporation to obtain the selenium-enriched wolfberry extract.

[0080] In one aspect of this disclosure, the selenium-enriched rice barley extract is prepared by the following steps:

[0081] Step 10-1: After crushing the stems and leaves of selenium-enriched rice straw, pass them through an 80-mesh sieve to obtain selenium-enriched rice straw powder.

[0082] Step 10-2: Add the selenium-enriched crushed rice straw powder to a 0.5 mol / L NaHCO3 aqueous solution, sonicate at 200 kHz for 45 min, then heat to 75 °C for 1 h, and then centrifuge the heated mixture at 4000 rpm for 5 min to obtain the supernatant and solid from Step 10-2; collect the supernatant and concentrate it by rotary evaporation to obtain the selenium-enriched crushed rice straw extract.

[0083] In one aspect of this disclosure, the selenium-enriched Salvia miltiorrhiza extract is prepared by the following steps:

[0084] Step 11-1: Crush the selenium-enriched Salvia miltiorrhiza into powder and pass it through an 80-mesh sieve to obtain selenium-enriched Salvia miltiorrhiza powder;

[0085] Step 11-2: Add the selenium-enriched Salvia miltiorrhiza powder to a 0.5 mol / L NaHCO3 aqueous solution, sonicate at 200 kHz for 45 min, then heat to 75 °C for 2 h, and then centrifuge the heated mixture at 4000 rpm for 5 min to obtain the supernatant and solid from Step 11-2; collect the supernatant and concentrate it by rotary evaporation to obtain the selenium-enriched Salvia miltiorrhiza extract.

[0086] In one aspect of this disclosure, the selenium-enriched Chlorella extract is prepared by the following steps:

[0087] Step 12-1: Add the dry matter of selenium-enriched Chlorella to water, soak for 50 minutes, and adjust the pH to 4.0±0.2 with a 30% phosphoric acid solution;

[0088] Step 12-2: Add cellulase and pectinase to the water, heat to 65℃, and enzymatically hydrolyze for 2.5 hours;

[0089] Step 12-3: Heat the solution to 95°C to inactivate the enzyme, and then centrifuge at 4000 rpm for 5 min to obtain the supernatant and solid from step 12-3; collect the supernatant and concentrate it by rotary evaporation to obtain selenium-enriched Chlorella extract.

[0090] In one aspect of this disclosure, the selenium-enriched honeysuckle extract is prepared by the following steps:

[0091] Step 13-1: After drying the selenium-enriched honeysuckle with hot air, pulverize it and pass it through a 100-mesh sieve to obtain selenium-enriched honeysuckle powder.

[0092] Step 13-2: Add the selenium-enriched honeysuckle powder to a 0.5 mol / L NaHCO3 aqueous solution, sonicate at 200 kHz for 45 min, then heat to 75 °C for 2 h, and then centrifuge the heated mixture at 4000 rpm for 5 min to obtain the supernatant and solid from Step 13-2; collect the supernatant and concentrate it by rotary evaporation to obtain the selenium-enriched honeysuckle extract.

[0093] In this disclosure, the licorice raw material refers to the stems and leaves of licorice.

[0094] In this disclosure, the cross-linking agent glutaraldehyde serves to link amino groups on proteins and polysaccharides.

[0095] The present disclosure is further illustrated below with reference to embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure.

[0096] Examples and comparative examples:

[0097] Example 1: Example 1 includes the following steps:

[0098] Step 1: Provide licorice raw materials (7g each of stems and leaves). Mix the licorice raw materials with flour (4g) and soybean meal (2g), then add 12mL of water and heat to 50℃ for 3 hours. Then add 240mg of compound probiotics (commercially available, a compound probiotic mixture of Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus subtilis in a 1:1:1 ratio) and 60mg of cellulase to the water to obtain a mixed system. Ferment the mixed system at 30℃ for 96 hours to obtain fermented licorice product.

[0099] Step 2: Dry the fermented licorice product at 60℃, then pass it through an 80-mesh sieve to obtain fermented licorice product powder; add the fermented licorice product powder to a 0.3 mol / L NaOH aqueous solution and heat to 85℃ for 1.5 h; then centrifuge the heated mixture at 3500 rpm for 10 min to obtain the supernatant and solid; wherein the mass of the NaOH aqueous solution is 16 times the mass of the fermented licorice product powder.

[0100] The obtained solid was added to a 0.3 mol / L NaOH aqueous solution and heated to 85°C for 1.5 h. The heated mixture was then centrifuged at 3500 rpm for 10 min to obtain a supernatant and a solid. The mass of the NaOH aqueous solution was 8 times the mass of the fermented licorice product powder. The supernatants were combined to obtain an aqueous solution of crude licorice extract.

[0101] Step 3: The aqueous solution of crude licorice extract was freeze-dried to obtain a solid crude licorice extract. The solid crude licorice extract was added to n-hexane, with the mass of n-hexane being 0.8 times the mass of the solid crude licorice extract. The mixture was soaked at room temperature for 8 hours to obtain a mixture. The mixture was filtered and dried to remove the n-hexane. The resulting solid was then added to a mixed solution of ethanol and acetone, with a volume ratio of ethanol to acetone of 3:1. The mixed solution was then refluxed at 70°C for 1.5 hours. The product after the reaction was concentrated and vacuum dried to obtain a defatted crude licorice extract.

[0102] Step 4: Add an appropriate amount of water to the defatted crude licorice extract and dissolve it completely. Then freeze it in a freezer at -60°C for 10 hours to obtain the frozen product. Thaw the frozen product naturally in a constant temperature environment at 8°C. Repeat the above process 4 times (add an appropriate amount of water each time to replenish the product) to obtain the product after freeze-thaw cycle. Centrifuge the product after freeze-thaw cycle at 3500 rpm for 10 minutes to obtain the supernatant and solid. Collect the supernatant to obtain the deproteinized crude licorice extract.

[0103] Step 5: Add an appropriate amount of water and electrolyte solution to the deproteinized crude licorice extract, and add the electrolyte solution to a final concentration of 0.05 mol / L to obtain a mixed solution; wherein the electrolyte solution is CaCl2 solution. After centrifuging the above mixed solution, a supernatant and a solid are obtained. Collect the supernatant and remove CaCl2 by dialysis to obtain the decolorized crude licorice extract.

[0104] Step 6: After the decolorized crude licorice extract is concentrated by 4 times its volume, a concentrated solution is obtained. 95% ethanol is added to the concentrated solution until the concentration of ethanol in the final solution is 60%. The precipitate obtained at this time is collected and dried to obtain graded licorice polysaccharides.

[0105] Step 7: Add the graded licorice polysaccharide to a mixed solution of ethanol (65 mL) and propanol (100 mL); under nitrogen protection, add 0.75 g of 3-methylaminopropylamine (910 μL) to the mixed solution, stir for 2 h, and after the reaction is completed, filter, wash with alcohol and dry to obtain aminated graded licorice polysaccharide.

[0106] Step 8: Dissolve the aforementioned aminated licorice graded polysaccharide in 150 mL of water, and add 2 g of selenium-enriched wolfberry extract (extraction process as shown above) and 0.5 g of cross-linking agent glutaraldehyde. Stir at room temperature for 3 h; add saturated NaCl solution dropwise until a precipitate appears in the solution, and continue to add saturated NaCl solution dropwise until the precipitate no longer increases. Collect the precipitate and dry it to obtain the selenized licorice polysaccharide of Example 1.

[0107] Example 2: Steps 1 to 6 of Example 2 are the same as those in Example 1;

[0108] Step 7: Add the graded licorice polysaccharide to a mixed solution of ethanol (65 mL) and propanol (100 mL); under nitrogen protection, add 0.63 g of propylenediamine (730 μL) to the mixed solution, stir for 2 h, and after the reaction is completed, filter, wash with alcohol and dry to obtain aminated graded licorice polysaccharide.

[0109] Step 8 of Example 2 is the same as that in Example 1, and the selenized licorice polysaccharide of Example 2 is obtained.

[0110] Example 3: Steps 1 to 6 of Example 3 are the same as those in Example 1;

[0111] Step 7: Add the graded licorice polysaccharide to a mixed solution of ethanol (65 mL) and propanol (100 mL); under nitrogen protection, add 1.60 g of tris(3-aminopropyl)amine (1690 μL) to the mixed solution, stir for 2 h, and after the reaction is completed, filter, wash with alcohol and dry to obtain aminated graded licorice polysaccharide.

[0112] Step 8 of Example 3 is the same as that in Example 1, and the selenized licorice polysaccharide of Example 3 is obtained.

[0113] Example 4: Steps 1 to 6 of Example 4 are the same as those in Example 1;

[0114] Step 7: Add the graded licorice polysaccharide to a mixed solution of ethanol (65 mL) and propanol (100 mL); under nitrogen protection, add 1.11 g of 3-diethylaminopropylamine (1350 μL) to the mixed solution, stir for 2 h, and after the reaction is completed, filter, wash with alcohol and dry to obtain aminated graded licorice polysaccharide.

[0115] Step 8 of Example 4 is the same as that in Example 1, and the selenized licorice polysaccharide of Example 4 is obtained.

[0116] Example 5: Steps 1 to 6 of Example 5 are the same as those in Example 1;

[0117] Step 7: Add the graded licorice polysaccharide to a mixed solution of ethanol (65 mL) and propanol (100 mL); under nitrogen protection, add 0.869 g of 3-dimethylaminopropylamine (1070 μL) to the mixed solution, stir for 2 h, and after the reaction is completed, filter, wash with alcohol and dry to obtain aminated graded licorice polysaccharide.

[0118] Step 8 of Example 5 is the same as that in Example 1, and the selenized licorice polysaccharide of Example 5 is obtained.

[0119] Comparative Example 1: Steps 1 to 6 of Comparative Example 1 are the same as those in Example 1;

[0120] Step 7: Dissolve the graded licorice polysaccharide in 150 mL of water, add 2 g of selenium-enriched wolfberry extract (extraction process as shown above) and 0.5 g of cross-linking agent glutaraldehyde, and stir at room temperature for 3 h; add saturated NaCl solution dropwise until a precipitate appears in the solution, continue to add saturated NaCl solution dropwise until the precipitate no longer increases, collect the precipitate and dry it to obtain the selenized licorice polysaccharide of Comparative Example 1.

[0121] The difference between Comparative Example 1 and Examples 1-5 is that the product of Comparative Example 1 was not subjected to amination modification treatment.

[0122] Comparative Example 2: Steps 1 to 6 of Comparative Example 2 are the same as those in Example 1;

[0123] Step 7: Add the graded licorice polysaccharide to ethanol (150 mL); under nitrogen protection, add 1 mol / L NaOH solution dropwise to the mixed solution to adjust the pH to 9.0, then add 1.8 mL of acetic anhydride, stir for 1 h, and after the reaction is complete, add 1 mol / L HCl solution dropwise to adjust the pH to 7.0. Then, after concentration, precipitation with 95% ethanol, and drying, the acetylated graded licorice polysaccharide is obtained.

[0124] Step 8 of Comparative Example 2 is the same as that in Example 1, resulting in the selenized licorice polysaccharide of Comparative Example 2.

[0125] The difference between Comparative Example 2 and Examples 1-5 is that the product of Comparative Example 2 was not modified by amination but by acetylation.

[0126] Comparative Example 3: Steps 1 to 6 of Comparative Example 3 are the same as those in Example 1;

[0127] Step 7: Add the graded licorice polysaccharide to a mixed solution of ethanol (65 mL) and propanol (100 mL); under nitrogen protection, add 1 mol / L NaOH solution dropwise to the mixed solution to adjust the pH to 10.0, then add 3.0 mL of chloroacetic acid, heat to 70 °C, stir for 3 h, cool to room temperature after the reaction is complete, and adjust the pH to 7.2 with glacial acetic acid; then concentrate, precipitate with 95% ethanol, and dry to obtain carboxymethylated graded licorice polysaccharide.

[0128] The difference between Comparative Example 3 and Examples 1-5 is that the product of Comparative Example 3 was modified by carboxymethylation instead of amination.

[0129] Comparative Example 4: The steps in Comparative Example 4 are the same as steps 1 to 7 in Example 1. The difference between Comparative Example 4 and Examples 1-5 is that Comparative Example 4 only prepared glycyrrhizin with aminated modification.

[0130] Comparative Example 5: The steps in Comparative Example 5 are the same as steps 1 to 6 in Example 1. The difference between Comparative Example 5 and Examples 1-5 is that Comparative Example 5 only prepared graded licorice polysaccharides.

[0131] DPPH free radical scavenging rate test: Samples from Examples 1-5 and Comparative Examples 1-3 were prepared as 0.75 mg / mL solutions. 0.0 mL of each sample was taken, and 2 mL of mmol / L DPPH-ethanol solution was added to each. After thorough mixing, the samples were stored in the dark for 30 min, and the absorbance was measured at 517 nm, recorded as A1. The same volume of ethanol and distilled water were used to replace the DPPH solution and sample, respectively, for zeroing. The absorbance value was A0 when the same volume of anhydrous ethanol was used instead of the polysaccharide sample, and A2 when the same volume of anhydrous ethanol was used instead of the DPPH-ethanol solution. Ascorbic acid was used as a positive control. The formula for calculating the DPPH free radical scavenging rate is: DPPH free radical scavenging rate (% = [1 - (A1 - A2) / A0] * 100%); The DPPH free radical scavenging rates of the samples from Examples 1-5 and Comparative Examples 1-3 are shown in Table 1 below.

[0132] Table 1:

[0133] Comparing Examples 1-5, it can be seen that Examples 1-2, which use 3-methylaminopropylamine and propylenediamine to modify licorice polysaccharides through amination, exhibit better binding ability with the extracted plant selenoproteins. Comparing Examples 1-5 and Comparative Examples 1-3, it can be seen that, compared to licorice polysaccharides modified using other methods, amination-modified licorice polysaccharides exhibit better binding ability with the extracted plant selenoproteins.

[0134] Other embodiments of this disclosure will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This disclosure is intended to cover any variations, uses, or adaptations of this disclosure that follow the general principles of this disclosure and include common knowledge or customary techniques in the art not disclosed herein.

Claims

1. A method for preparing licorice polysaccharide, characterized in that, The licorice polysaccharide is a selenized licorice polysaccharide, and the preparation method includes the following steps: Step 1: Provide licorice raw material, and ferment the licorice raw material in a solid state to obtain the fermented licorice product of Step 1; Step 2: After drying the fermented licorice product, pulverize it and sieve it. Then, extract it with an alkaline solution to obtain an aqueous solution of the crude licorice extract from Step 2. Step 3: The aqueous solution of the crude licorice extract is defatted to obtain the defatted aqueous solution of crude licorice extract from Step 3; Step 4: The aqueous solution of the defatted crude licorice extract is subjected to a deproteinization treatment to obtain the deproteinized crude licorice extract of Step 4. Step 5: The deproteinized crude licorice extract is subjected to a decolorization treatment to obtain the decolorized crude licorice extract of Step 5. Step 6: Extract the decolorized crude licorice extract with ethanol to obtain the graded licorice polysaccharide from Step 6; Step 7: The licorice graded polysaccharide is modified by amylation to obtain the amylated licorice graded polysaccharide of Step 7. Step 8: The aminated and modified licorice graded polysaccharide is subjected to selenization treatment to obtain the selenized licorice polysaccharide; Step 7 includes the following steps: Step 7-1: Add the graded licorice polysaccharide to a mixed solution of ethanol and propanol; Step 7-2: Under nitrogen protection, add an amino-containing modified compound to the mixed solution, stir for 1-3 hours, and after the reaction is completed, filter, wash and dry to obtain amino-modified licorice graded polysaccharides; wherein, the amino-containing modified compound is selected from propylenediamine, 3-methylaminopropylamine, tris(3-aminopropyl)amine, 3-diethylaminopropylamine or 3-dimethylaminopropylamine; Step 8 includes the following steps: Step 8-1: Dissolve the aminated licorice graded polysaccharide in water, and add plant extract containing selenoprotein and cross-linking agent to it, and stir at room temperature for 2-4 hours; Step 8-2: Add NaCl solution dropwise until a precipitate appears, continue adding NaCl solution dropwise until the precipitate no longer increases, collect the precipitate and dry it to obtain selenized licorice polysaccharide; In step 8-1, the crosslinking agent is selected from glutaraldehyde.

2. The preparation method according to claim 1, characterized in that, Step 1 includes the following steps: Step 1-1: Provide licorice raw material, mix the licorice raw material with flour and soybean meal, then add water and heat to 45℃-55℃, the heating time is selected from 2-4h, then add compound probiotics and cellulase to the water to obtain the mixture system of Step 1-1; Steps 1-2: Ferment the mixture at 25℃-35℃ for 48-144 hours to obtain the fermented licorice product.

3. The preparation method according to claim 1, characterized in that, Step 2 includes the following steps: Step 2-1: Dry the fermented licorice product at 50℃-70℃, and then pass it through a 50-100 mesh sieve to obtain the fermented licorice product powder of Step 2-1; Step 2-2: Add the fermented licorice product powder to NaOH aqueous solution and heat to 70℃-90℃ for 1-2 hours. Then, centrifuge the heated mixture to obtain the supernatant and solid from Step 2-2. Step 2-3: Add the solid obtained in step 2-2 back into the NaOH aqueous solution and heat to 70℃-90℃ for 1-2 hours. Then, centrifuge the heated mixture to obtain the supernatant and solid from step 2-3. Step 2-4: Combine the supernatant from Step 2-2 and the supernatant from Step 2-3 to obtain an aqueous solution of crude licorice extract.

4. The preparation method according to claim 1, characterized in that, Step 3 includes the following steps: Step 3-1: Freeze-dry the aqueous solution of the crude licorice extract to obtain a solid crude licorice extract; Step 3-2: Add the solid of the crude licorice extract to n-hexane and soak at room temperature for 5-10 hours to obtain the mixture from step 3-2; Step 3-3: The mixture from Step 3-2 is filtered and dried to remove n-hexane. The resulting solid is then added to a mixed solution of ethanol and acetone and heated under reflux at 65℃-75℃ for 1-2.5 hours. The product after the reaction is concentrated to obtain defatted crude licorice extract.

5. The preparation method according to claim 1, characterized in that, In step 4, the deproteinization process is completed through freeze-thaw cycles; step 4 includes the following steps: Step 4-1: Add an appropriate amount of water to the defatted crude licorice extract and dissolve it completely. Then freeze it in an environment of -70℃ to -50℃ for 6-12 hours to obtain the frozen product of Step 4-1. Step 4-2: Place the frozen product from Step 4-1 in a constant temperature environment of 5℃-15℃ to thaw naturally; Step 4-3: Repeat steps 4-1 and 4-2 3-6 times to obtain the product after freeze-thaw cycles; Step 4-4: After centrifuging the product after freeze-thaw cycle, obtain supernatant and solid. Collect the supernatant to obtain deproteinized crude licorice extract.

6. The preparation method according to claim 1, characterized in that, Step 5 includes the following steps: Step 5-1: Add an appropriate amount of water and a first electrolyte solution to the deproteinized crude licorice extract, and add the first electrolyte solution until the final concentration is 0.02-0.08 mol / L to obtain the mixed solution of Step 5-1; wherein, the first electrolyte solution is CaCl2 solution or MgSO4 solution; Step 5-2: After centrifuging the mixed solution from Step 5-1, a supernatant and a solid are obtained. The supernatant is collected and dialyzed to obtain the decolorized crude licorice extract.

7. The preparation method according to claim 1, characterized in that, Step 6 includes the following steps: Step 6-1: The decolorized crude licorice extract is combined and concentrated to obtain the combined and concentrated product of Step 6-1; Step 6-2: Add ethanol to the combined and concentrated product from Step 6-1 until the concentration of ethanol in the final solution is 60%-70%. Collect and dry the precipitate obtained at this point to obtain graded licorice polysaccharides.

8. The preparation method according to claim 1, characterized in that, The plant extract containing selenoprotein is selected from selenium-enriched barley grass extract, selenium-enriched wolfberry extract, selenium-enriched salvia miltiorrhiza extract, selenium-enriched honeysuckle extract, or selenium-enriched chlorella extract.

Images