Preparation method of glycyrrhiza polysaccharide

Abstract

The invention relates to the field of preparation of pharmaceutical compositions, in particular to a preparation method of glycyrrhiza polysaccharide. The preparation method sequentially comprises the following steps: a solid state fermentation process, an alkali solution extraction process, a degreasing treatment process, a deproteinization treatment process, a decolorization treatment process, an ethanol graded extraction process, an amination modification process and a selenylation treatment process. The glycyrrhiza polysaccharide connected with selenoprotein obtained by the preparation method provided by the invention has the effects of resisting oxidation, improving immunity, resisting tumors and the like.

Description

Technical Field

[0001] The present disclosure relates to the field of pharmaceutical composition preparation, and in particular to a method for preparing glycyrrhiza polysaccharide. Background Art

[0002] Licorice is one of the world's most popular herbs, earning the title "King of Medicinal Herbs." Domestic and international research on the pharmacological effects of licorice has shown its efficacy in tonifying the spleen and replenishing qi, clearing away heat and detoxifying, relieving cough and removing phlegm, and harmonizing various medicinal herbs. It is primarily used for spleen and stomach deficiency, fatigue and weakness, palpitations and shortness of breath, cough and sputum, carbuncles and sores, and to alleviate drug toxicity. Licorice polysaccharides, one of its main components, possess antioxidant, antiviral, and anti-tumor biological activities. Currently, only the underground portion of licorice is used medicinally; its stems and leaves, as byproducts, also contain a wealth of active substances. However, due to the difficulty in fully extracting these active ingredients, their utilization rate is low.

[0003] Selenium, an essential trace element for the human body, is the primary active component of glutathione peroxidase. It can directly or indirectly scavenge oxygen free radicals in the body, inhibit lipid oxidation and 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, infection resistance, and stress resistance. Selenium is not only a key component of some enzymes, but also plays a crucial role in preventing tumorigenesis, treating cardiovascular and cerebrovascular diseases, and delaying aging. Selenium is widely present in nature and occurs in two main forms: inorganic selenium, including elemental selenium, metallic 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, and selenium disulfide; and organic selenium, primarily in the form of selenoproteins and selenopolysaccharides. Therefore, a preparation process for combining organic selenium with licorice extract is urgently needed. Summary of the Invention

[0004] The present invention provides a method for preparing glycyrrhiza polysaccharide to solve the deficiencies in the related art.

[0005] According to a first aspect of the embodiments of the present disclosure, a method for preparing glycyrrhiza polysaccharide is provided, wherein the glycyrrhiza polysaccharide is glycyrrhiza polysaccharide that has been selenized, and the preparation method comprises the following steps: Step 1: providing a licorice raw material, and subjecting the licorice raw material to solid-state fermentation to obtain the fermented licorice product of step 1; Step 2: drying the fermented licorice product, crushing it, sieving it, and then extracting it with an alkaline solution to obtain an aqueous solution of the crude licorice extract of step 2; Step 3: defatting the aqueous solution of the crude licorice extract to obtain the defatted crude licorice extract of step 3; Step 4: deproteinizing the defatted licorice crude extract to obtain the deproteinized licorice crude extract of step 4; Step 5: decolorizing the deproteinized licorice crude extract to obtain the decolorized licorice crude extract of step 5; Step 6: extracting the decolorized licorice crude extract with ethanol to obtain the licorice fractionated polysaccharides of step 6; Step 7: modifying the fractionated licorice polysaccharide by amino modification to obtain the amino-modified fractionated licorice polysaccharide of step 7; Step 8: subjecting the amino-modified licorice fractionated polysaccharide to selenization treatment to obtain the selenized licorice polysaccharide.

[0006] In one aspect of the embodiments of the present disclosure, step 1 includes the following steps: Step 1-1: providing a licorice raw material, mixing the licorice raw material with flour and soybean meal, then adding water and heating to 45° C.-55° C. for a heating time selected from 2-4 hours, and then adding a composite probiotic and cellulase to the water to obtain the mixed system of step 1-1; Step 1-2: fermenting the mixed system at 25° C.-35° C. for a fermentation time selected from 48-144 hours to obtain the fermented licorice product.

[0007] In one aspect of the embodiments of the present 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.

[0008] In one aspect of the embodiments of the present disclosure, in step 1-1, the composite probiotics include core bacteria and multiple compatible bacteria, the core bacteria are selected from Lactobacillus plantarum and / or Saccharomyces cerevisiae, and the compatible bacteria are selected from Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus reuteri, Lactobacillus paracasei, Bifidobacterium longum, Bacillus subtilis, Bifidobacterium infantis or Lactobacillus acidophilus.

[0009] In one aspect of the embodiments of the present disclosure, in step 1-1, preferably, the composite probiotic comprises Lactobacillus plantarum, Saccharomyces cerevisiae and Bacillus subtilis; specifically, the composite probiotic is a commercially available composite probiotic with a ratio of Lactobacillus plantarum: Saccharomyces cerevisiae: Bacillus subtilis = 1:1:1.

[0010] In one aspect of the embodiments of the present 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 the added water is selected from 40%-65%, the mass ratio of the added composite probiotics is selected from 0.5%-1.5%, and the mass ratio of the added cellulase is selected from 0.25%-0.5%.

[0011] In one aspect of the embodiments of the present disclosure, preferably, step 1-2 comprises: fermenting the mixed system at 30° C., and the fermentation time is selected from 96 hours.

[0012] In one aspect of the embodiments of the present disclosure, step 2 includes the following steps: Step 2-1: drying the fermented licorice product at 50° C.-70° C., and then passing through a 50-100 mesh sieve to obtain a powder of the fermented licorice product of step 2-1; Step 2-2: adding the fermented licorice product powder to a NaOH aqueous solution and heating to 70° C.-90° C. for a heating time selected from 1-2 hours, and then centrifuging the heated mixture to obtain the supernatant and solid of step 2-2; Step 2-3: adding the solid obtained in step 2-2 to a NaOH aqueous solution again and heating to 70° C.-90° C. for 1-2 h, and then centrifuging the heated mixture to obtain the supernatant and solid of step 2-3; Step 2-4: Combine the supernatant of step 2-2 and the supernatant of step 2-3 to obtain an aqueous solution of a crude licorice extract.

[0013] In one aspect of the embodiments of the present disclosure, in step 2-2 and step 2-3, the NaOH aqueous solution used is a NaOH aqueous solution with a concentration of 0.1-0.5 mol / L.

[0014] In one aspect of the embodiments 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.

[0015] In one aspect of the embodiments of the present 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.

[0016] In one aspect of the embodiments of the present 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 hours, and then centrifuging the heated mixture to obtain the supernatant and solid of step 2-2; wherein the mass of the NaOH aqueous solution is 16 times the mass of the fermented licorice product powder.

[0017] In one aspect of the embodiments of the present 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 hours, and 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.

[0018] In one aspect of the embodiments of the present disclosure, specifically, in step 2, the centrifugal speed is 3500 rpm.

[0019] In one aspect of the embodiments of the present disclosure, step 3 includes the following steps: Step 3-1: freeze-drying the aqueous solution of the crude licorice extract to obtain a solid of the crude licorice extract; Step 3-2: adding the solid of the crude licorice extract to n-hexane and soaking at room temperature for 5-10 hours to obtain the mixture of step 3-2; Step 3-3: The mixture of step 3-2 is filtered and dried to remove n-hexane, and then the obtained solid is added to a mixed solution of ethanol and acetone, and then heated to reflux at 65°C-75°C, and the heating reflux time is selected from 1-2.5 hours. The product after the reaction is concentrated and freeze-dried to obtain a defatted licorice crude extract.

[0020] In one aspect of the embodiments of the present disclosure, in step 3-2, the mass of the n-hexane is selected from 0.5 to 1.5 times the mass of the solid of the crude licorice extract.

[0021] In one aspect of the embodiments of the present disclosure, in the mixed solution of ethanol and acetone, the volume ratio of ethanol to acetone is selected from (2-4):1.

[0022] In one aspect of the embodiments of the present disclosure, specifically, step 3-2 includes: adding the solid of the crude licorice extract to n-hexane, the mass of which 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.

[0023] In one aspect of the embodiments of the present disclosure, specifically, step 3-3 includes: filtering and drying the mixture of step 3-2 to remove n-hexane, and then adding the obtained solid to a mixed solution of ethanol and acetone, wherein the volume ratio of ethanol and acetone in the mixed solution is selected from 3:1; then heating the mixed solution to reflux at 65°C-75°C for 1.5 hours, and concentrating and vacuum-drying the reaction product to obtain a defatted licorice crude extract.

[0024] In one aspect of the embodiments of the present disclosure, in step 4, the deproteinization step is completed by freeze-thaw cycle treatment; step 4 includes the following steps: Step 4-1: adding an appropriate amount of water to the defatted licorice crude extract to fully dissolve it, and then freezing it in an environment of -70°C to -50°C for 6-12 hours to obtain the frozen product of step 4-1; Step 4-2: The frozen product of step 4-1 is placed in a constant temperature environment of 5°C-15°C for natural thawing; Step 4-3: Repeat the process of step 4-1 and step 4-2 3-6 times to obtain a product after freeze-thaw cycles; Step 4-4: centrifuging the product after freeze-thaw cycles to obtain a supernatant and a solid, collecting the supernatant to obtain a deproteinized licorice crude extract.

[0025] In one aspect of the embodiment of the present disclosure, step 5 includes the following steps: Step 5-1: adding an appropriate amount of water and a first electrolyte solution to the deproteinized licorice crude extract to a final concentration of 0.02-0.08 mol / L to obtain the mixed solution of step 5-1; wherein the first electrolyte solution is a CaCl2 solution or a MgSO4 solution; Step 5-2: The mixed solution of step 5-1 is centrifuged to obtain a supernatant and a solid. The supernatant is collected and dialyzed to obtain the decolorized crude licorice extract.

[0026] In one aspect of an embodiment of the present disclosure, specifically, in step 5-1, a first electrolyte solution is added to a final concentration of 0.05 mol / L, and the first electrolyte solution is a CaCl2 solution.

[0027] In one aspect of the embodiment of the present disclosure, step 6 includes the following steps: Step 6-1: Concentrating the decolorized crude licorice extract to obtain a concentrated solution of step 6-1; Step 6-2: Add ethanol to the concentrated solution of step 6-1 until the concentration of ethanol in the final solution is 60%-70%. Collect and dry the precipitate obtained at this time to obtain licorice graded polysaccharides.

[0028] In one aspect of the embodiments of the present disclosure, in step 6-2, the ethanol is a 95% ethanol solution.

[0029] In one aspect of the disclosed embodiment, step 7 includes the following steps: Step 7-1: adding the fractionated licorice polysaccharide to a mixed solution of ethanol and propanol; Step 7-2: Under nitrogen protection, add a modified compound containing an amino group 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 modified compound containing an amino group is selected from propylenediamine, 3-methylaminopropylamine, tris(3-aminopropyl)amine, 3-diethylaminopropylamine or 3-dimethylaminopropylamine.

[0030] In one aspect of the embodiments of the present disclosure, in the mixed solution of step 7-1, the volume ratio of ethanol to propanol is selected from (0.5-1):1.

[0031] In one aspect of the embodiments of the present disclosure, preferably, the modifying compound containing an amino group is selected from 3-methylaminopropylamine.

[0032] In one aspect of the disclosed embodiment, step 8 includes the following steps: Step 8-1: dissolving the amino-modified licorice fractionated polysaccharide in water, adding a plant extract containing selenoprotein and a cross-linking agent thereto, and stirring at room temperature for 2-4 hours; Step 8-2: NaCl solution is added dropwise until a precipitate appears, and the NaCl solution is continued to be added dropwise until the precipitate no longer increases, and the precipitate is collected and dried to obtain selenized glycyrrhizic polysaccharide.

[0033] In one aspect of the embodiments of the present disclosure, in step 8-1, the cross-linking agent is selected from glutaraldehyde.

[0034] In one aspect of the embodiments of the present disclosure, the plant extract containing selenoprotein is selected from selenium-enriched Cardamine extract, selenium-enriched Lycium barbarum extract, selenium-enriched Salvia miltiorrhiza extract, selenium-enriched Honeysuckle extract or selenium-enriched Chlorella extract.

[0035] According to a second aspect of an embodiment of the present disclosure, a pharmaceutical composition is provided, comprising selenized glycyrrhiza polysaccharide obtained by the aforementioned preparation method.

[0036] According to a third aspect of the embodiments of the present disclosure, a health food is provided, comprising selenized licorice polysaccharide obtained by the aforementioned preparation method.

[0037] The technical solutions provided by the embodiments of the present disclosure may have the following beneficial effects: As can be seen from the above examples, the present disclosure provides a licorice polysaccharide connected to a selenoprotein, which has antioxidant, immunity-enhancing, anti-tumor and other effects.

[0038] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure. DETAILED DESCRIPTION

[0039] To make the purpose, technical solutions, and advantages of this application more clear, the technical solutions of this application will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of this application, rather than all the embodiments. The relevant embodiments described herein are illustrative and are used to provide a basic understanding of this application. The embodiments of this application should not be interpreted as limiting this application.

[0040] For the sake of clarity, only some numerical ranges are specifically disclosed herein. However, any lower limit may be combined with any upper limit to form an unspecified range; and any lower limit may be combined with other lower limits to form an unspecified range, and similarly, any upper limit may be combined with any other upper limit to form an unspecified range. In addition, each individually disclosed point or single value may itself serve as a lower limit or upper limit and be combined with any other point or single value, or with other lower limits or upper limits, to form an unspecified range.

[0041] As used herein, the terms "comprises," "comprising," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not preclude the presence of additional identical elements in the process, method, article, or apparatus that includes the element.

[0042] In the description herein, unless otherwise specified, “above” and “below” include the number itself.

[0043] Unless otherwise specified, the terms used in this disclosure have the commonly understood meanings commonly understood by those skilled in the art. Unless otherwise specified, the numerical values of the various parameters mentioned in this disclosure can be measured using various measurement methods commonly used in the art (for example, they can be tested according to the methods given in the examples of this disclosure).

[0044] The term "about" is used to describe and illustrate small changes. When used in conjunction with an event or situation, the term may refer to an example in which the event or situation occurs precisely and an example in which the event or situation occurs very approximately. For example, when used in conjunction with a numerical value, the term may refer to a variation range of 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%. In addition, amounts, ratios, and other numerical values are sometimes presented in this article in a range format. It should be understood that such range formats are for convenience and brevity, and should be flexibly understood to include not only numerical values explicitly designated as range limits, but also all individual numerical values or subranges encompassed within the range, as if each numerical value and subrange were explicitly designated.

[0045] A 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, the phrase "at least one of A and B" means only A; only B; or A and B. In another example, if items A, B, and C are listed, 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 can contain a single component or multiple components. Item B can contain a single component or multiple components. Item C can contain a single component or multiple components.

[0046] In the present disclosure, the raw materials of the selenium-enriched Cardamine extract, selenium-enriched Lycium barbarum extract, selenium-enriched Salvia miltiorrhiza extract, selenium-enriched honeysuckle extract or selenium-enriched Chlorella extract involved in the present disclosure are selenium-enriched Cardamine, selenium-enriched Lycium barbarum, selenium-enriched Salvia miltiorrhiza, selenium-enriched honeysuckle and selenium-enriched Chlorella, which can be obtained commercially.

[0047] In one aspect of the embodiments of the present disclosure, the selenium-enriched wolfberry extract is prepared by the following steps: Step 9-1: Grind the selenium-rich wolfberry fruit and pass it through an 80-mesh sieve to obtain selenium-rich wolfberry powder; Step 9-2: Add the selenium-rich wolfberry powder to a 0.5 mol / L NaHCO3 aqueous solution, ultrasonicate at 200 kHz for 45 minutes, and then heat to 75°C for 1 hour. Then, centrifuge the heated mixture at 4000 rpm for 5 minutes to obtain the supernatant and solid of step 9-2; collect the supernatant and concentrate it by rotary evaporation to obtain a selenium-rich wolfberry extract.

[0048] In one aspect of the embodiments of the present disclosure, the selenium-enriched Cardamine extract is prepared by the following steps: Step 10-1: Grind the stems and leaves of selenium-rich Cardamine and pass them through an 80-mesh sieve to obtain selenium-rich Cardamine powder; Step 10-2: Add the selenium-rich Cardamine powder to a 0.5 mol / L NaHCO3 aqueous solution, ultrasonicate at 200 kHz for 45 minutes, and then heat to 75°C for 1 hour. Then, centrifuge the heated mixture at 4000 rpm for 5 minutes to obtain the supernatant and solid of step 10-2; collect the supernatant and concentrate it by rotary evaporation to obtain a selenium-rich Cardamine extract.

[0049] In one aspect of the embodiments of the present disclosure, the selenium-enriched Salvia miltiorrhiza extract is prepared by the following steps: Step 11-1: Grind the selenium-rich Salvia miltiorrhiza and pass it through an 80-mesh sieve to obtain selenium-rich Salvia miltiorrhiza powder; Step 11-2: Add the selenium-rich Salvia miltiorrhiza powder to a 0.5 mol / L NaHCO3 aqueous solution, ultrasonicate at 200 kHz for 45 minutes, and then heat to 75°C for 2 hours. Then, centrifuge the heated mixture at 4000 rpm for 5 minutes to obtain the supernatant and solid of step 11-2; collect the supernatant and concentrate it by rotary evaporation to obtain a selenium-rich Salvia miltiorrhiza extract.

[0050] In one aspect of the embodiments of the present disclosure, the selenium-enriched Chlorella vulgaris extract is prepared by the following steps: Step 12-1: Add the dry matter of selenium-enriched Chlorella to water, soak for 50 minutes, and adjust the pH value to 4.0±0.2 with 30% phosphoric acid solution; Step 12-2: Add cellulase and pectinase to the water, raise the temperature to 65°C, and perform enzymatic hydrolysis for 2.5 hours; Step 12-3: The solution is heated to 95° C. to inactivate the enzyme, and then centrifuged at 4000 rpm for 5 minutes to obtain the supernatant and solid of step 12-3; the supernatant is collected and concentrated by rotary evaporation to obtain a selenium-enriched Chlorella vulgaris extract.

[0051] In one aspect of the embodiments of the present disclosure, the selenium-rich honeysuckle extract is prepared by the following steps: Step 13-1: drying the selenium-rich honeysuckle with hot air, crushing the mixture, and passing the mixture through a 100-mesh sieve to obtain selenium-rich honeysuckle powder; Step 13-2: Add the selenium-rich honeysuckle powder to a 0.5 mol / L NaHCO3 aqueous solution, ultrasonicate at 200 kHz for 45 minutes, and then heat to 75°C for 2 hours. Then, centrifuge the heated mixture at 4000 rpm for 5 minutes to obtain the supernatant and solid of step 13-2; collect the supernatant and concentrate it by rotary evaporation to obtain a selenium-rich honeysuckle extract.

[0052] In the present disclosure, the licorice raw materials are the stems and leaves of licorice.

[0053] In the present disclosure, the role of the cross-linking agent glutaraldehyde is to link the amino groups on proteins and polysaccharides.

[0054] The present disclosure is further described below with reference to the following examples. It should be understood that these examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure.

[0055] Examples and Comparative Examples: Example 1: Example 1 includes the following steps: Step 1: providing a licorice raw material (7 g each of stems and leaves), mixing the licorice raw material with flour (4 g) and soybean meal (2 g), then adding 12 mL of water and heating to 50° C. for 3 hours, then adding 240 mg of a commercially available composite probiotic (Lactobacillus plantarum: Saccharomyces cerevisiae: Bacillus subtilis = 1:1:1 composite probiotic) and 60 mg of cellulase to the water to obtain a mixed system; fermenting the mixed system at 30° C. for 96 hours to obtain a fermented licorice product; Step 2: Dry the fermented licorice product at 60° C. and then pass it through an 80-mesh sieve to obtain a fermented licorice product powder; add the fermented licorice product powder to a 0.3 mol / L NaOH aqueous solution and heat it to 85° C. for 1.5 hours, then centrifuge the heated mixture at 3500 rpm for 10 minutes to obtain a supernatant and a solid; wherein the mass of the NaOH aqueous solution is 16 times the mass of the fermented licorice product powder.

[0056] The resulting solid was added to a 0.3 mol / L aqueous NaOH solution and heated to 85°C for 1.5 hours. The heated mixture was then centrifuged at 3500 rpm for 10 minutes to obtain a supernatant and a solid. The mass of the aqueous NaOH solution was 8 times the mass of the fermented licorice powder. The supernatants were combined to obtain an aqueous solution of the crude licorice extract.

[0057] Step 3: Freeze-drying the aqueous solution of the crude licorice extract to obtain a solid crude licorice extract; adding the solid crude licorice extract to n-hexane, where the mass of n-hexane is 0.8 times the mass of the solid crude licorice extract, and soaking at room temperature for 8 hours to obtain a mixture. The mixture is filtered and dried to remove the n-hexane, and the resulting solid is then added to a mixed solution of ethanol and acetone, where the volume ratio of ethanol to acetone in the mixed solution is selected from 3:1; the mixed solution is then heated to reflux at 70°C for 1.5 hours. The reaction product is concentrated and vacuum-dried to obtain a defatted crude licorice extract.

[0058] Step 4: Add an appropriate amount of water to the defatted licorice crude extract to fully dissolve it, and then place it in a refrigerator at -60°C for 10 hours to obtain a frozen product; place the frozen product in a constant temperature environment of 8°C for natural thawing; repeat the above process 4 times (an appropriate amount of water can be added each time to replenish it) to obtain a product after freeze-thaw cycles; centrifuge the product after freeze-thaw cycles at 3500rpm for 10 minutes to obtain a supernatant and a solid, collect the supernatant, and obtain a deproteinized licorice crude extract.

[0059] Step 5: Add appropriate amounts of water and an electrolyte solution to the deproteinized licorice crude extract to a final concentration of 0.05 mol / L to obtain a mixed solution; wherein the electrolyte solution is a CaCl2 solution. The mixed solution is centrifuged to obtain a supernatant and a solid. The supernatant is collected and dialyzed to remove the CaCl2, thereby obtaining a decolorized licorice crude extract.

[0060] Step 6: The decolorized licorice crude extract was concentrated by 4 times its volume to obtain a concentrated solution, 95% ethanol was added to the concentrated solution until the ethanol concentration in the final solution was 60%, and the precipitate obtained at this time was collected and dried to obtain licorice graded polysaccharides.

[0061] Step 7: Add the fractionated 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 and stir for 2 h. After the reaction is completed, filter, wash with alcohol, and dry to obtain amino-modified fractionated licorice polysaccharide.

[0062] Step 8: The aforementioned amino-modified graded licorice polysaccharide was dissolved in 150 mL of water, and 2 g of selenium-enriched wolfberry extract (the extraction process was as described above) and 0.5 g of cross-linking agent glutaraldehyde were added thereto, and stirred at room temperature for 3 h; saturated NaCl solution was added dropwise until a precipitate appeared in the solution, and saturated NaCl solution was continued to be added dropwise until the precipitate no longer increased. The precipitate was collected and dried to obtain the selenized licorice polysaccharide of Example 1.

[0063] Example 2: Steps 1 to 6 of Example 2 are the same as those in Example 1; Step 7: Add the fractionated 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 and stir for 2 hours. After the reaction is completed, filter, wash with alcohol, and dry to obtain amino-modified fractionated licorice polysaccharide.

[0064] Step 8 of Example 2 is the same as that of Example 1, and the selenized licorice polysaccharide of Example 2 is obtained.

[0065] Example 3: Steps 1 to 6 of Example 3 are the same as those in Example 1; Step 7: Add the fractionated 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 and stir for 2 h. After the reaction is completed, filter, wash with alcohol, and dry to obtain amino-modified fractionated licorice polysaccharide.

[0066] Step 8 of Example 3 is the same as that of Example 1, and the selenized licorice polysaccharide of Example 3 is obtained.

[0067] Example 4: Steps 1 to 6 of Example 4 are the same as those in Example 1; Step 7: Add the fractionated 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 and stir for 2 hours. After the reaction is completed, filter, wash with alcohol, and dry to obtain amino-modified fractionated licorice polysaccharide.

[0068] Step 8 of Example 4 is the same as that of Example 1, and the selenized glycyrrhiza polysaccharide of Example 4 is obtained.

[0069] Example 5: Steps 1 to 6 of Example 5 are the same as those in Example 1; Step 7: Add the fractionated 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 and stir for 2 hours. After the reaction is completed, filter, wash with alcohol, and dry to obtain amino-modified fractionated licorice polysaccharide.

[0070] Step 8 of Example 5 is the same as that of Example 1, and the selenized licorice polysaccharide of Example 5 is obtained.

[0071] Comparative Example 1: Steps 1 to 6 of Comparative Example 1 are the same as those in Example 1; Step 7: Dissolve the graded licorice polysaccharides in 150 mL of water, and add 2 g of selenium-enriched wolfberry extract (the extraction process is as shown above) and 0.5 g of cross-linking agent glutaraldehyde, and stir at room temperature for 3 hours; 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.

[0072] The difference between Comparative Example 1 and Examples 1-5 is that the product of Comparative Example 1 has not been subjected to amination modification treatment.

[0073] Comparative Example 2: Steps 1 to 6 of Comparative Example 2 are the same as those in Example 1; Step 7: Add the licorice fractionated polysaccharide to ethanol (150 mL); under nitrogen protection, add 1 mol / L NaOH solution to the mixed solution to adjust the pH to 9.0, then add 1.8 mL of acetic anhydride and stir for 1 hour. After the reaction is completed, add 1 mol / L HCl solution to adjust the pH to 7.0, and then concentrate, precipitate with 95% ethanol, and dry to obtain acetylated modified licorice fractionated polysaccharide.

[0074] Step 8 of Comparative Example 2 is the same as that of Example 1, to obtain the selenized licorice polysaccharide of Comparative Example 2.

[0075] The difference between Comparative Example 2 and Examples 1-5 is that the product of Comparative Example 2 is not subjected to amination modification but is subjected to acetylation modification.

[0076] Comparative Example 3: Steps 1 to 6 of Comparative Example 3 are the same as those in Example 1; Step 7: Add the graded licorice polysaccharides to a mixed solution of ethanol (65 mL) and propanol (100 mL); under nitrogen protection, add 1 mol / L NaOH solution 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 hours, cool to room temperature after the reaction, and adjust the pH to 7.2 with glacial acetic acid; then concentrate, precipitate with 95% ethanol, and dry to obtain carboxymethyl-modified graded licorice polysaccharides.

[0077] The difference between Comparative Example 3 and Examples 1-5 is that the product of Comparative Example 3 is not subjected to an amination modification treatment but is subjected to a carboxymethylation modification treatment.

[0078] Comparative Example 4: The steps of Comparative Example 4 are the same as Step 1 to Step 7 in Example 1. The difference between Comparative Example 4 and Examples 1-5 is that Comparative Example 4 only prepares amino-modified glycyrrhiza polysaccharide.

[0079] Comparative Example 5: The steps of Comparative Example 5 are the same as Step 1 to Step 6 in Example 1. The difference between Comparative Example 5 and Examples 1-5 is that Comparative Example 5 only prepares the fractionated licorice polysaccharide.

[0080] DPPH radical scavenging rate test: The samples of Examples 1-5 and Comparative Examples 1-3 were respectively prepared into 0.75 mg / mL solutions, 0.0 mL of each of the above samples was taken, and 2 mL of mmol / L DPPH-ethanol solution was added to each of them. After thorough mixing, the mixture was kept away from light for 30 minutes, and then the absorbance value was measured at 517 nm and recorded as A1. The DPPH solution and sample were replaced by the same volume of ethanol and distilled water respectively for zero adjustment. The absorbance value measured by replacing the polysaccharide sample with the same volume of anhydrous ethanol was A0, and the absorbance value measured by replacing the DPPH-ethanol solution with the same volume of anhydrous ethanol was A2. Ascorbic acid was used as a positive control. The calculation formula for the DPPH radical scavenging rate was: DPPH radical scavenging rate (% = [1-(A1-A2) / A0] * 100%; the values of the DPPH radical scavenging rates of the samples of Examples 1-5 and Comparative Examples 1-3 are shown in Table 1 below: Table 1:

[0081] Comparing Examples 1-5, it can be seen that Example 1-2, in which licorice polysaccharide was modified by amino treatment with 3-methylaminopropylamine and propylenediamine, had better binding ability with the extracted plant selenoprotein. Comparing Examples 1-5 with Comparative Example 1-3, it can be seen that compared with licorice polysaccharides modified by other methods, the licorice polysaccharide modified by amino treatment had better binding ability with the extracted plant selenoprotein.

[0082] Those skilled in the art will readily appreciate other embodiments of the present disclosure after considering the specification and practicing the disclosure disclosed herein. This disclosure is intended to cover any variations, uses, or adaptations of the present disclosure that follow the general principles of the present 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 licorice polysaccharide that has been subjected to selenization treatment, and the preparation method comprises the following steps: Step 1: providing a licorice raw material, and subjecting the licorice raw material to solid-state fermentation to obtain the fermented licorice product of step 1; Step 2: drying the fermented licorice product, crushing it, sieving it, and then extracting it with an alkaline solution to obtain an aqueous solution of the crude licorice extract of step 2; Step 3: defatting the aqueous solution of the crude licorice extract to obtain the defatted aqueous solution of the crude licorice extract of step 3; Step 4: deproteinizing the aqueous solution of the defatted licorice crude extract to obtain the deproteinized licorice crude extract of step 4; Step 5: decolorizing the deproteinized licorice crude extract to obtain the decolorized licorice crude extract of step 5; Step 6: extracting the decolorized licorice crude extract with ethanol to obtain the licorice fractionated polysaccharides of step 6; Step 7: modifying the fractionated licorice polysaccharide by amino modification to obtain the amino-modified fractionated licorice polysaccharide of step 7; Step 8: subjecting the amino-modified licorice fractionated polysaccharide to selenization treatment to obtain the selenized licorice polysaccharide.

2. The preparation method according to claim 1, characterized in that Step 1 includes the following steps: Step 1-1: providing a licorice raw material, mixing the licorice raw material with flour and soybean meal, then adding water and heating to 45° C.-55° C. for a heating time selected from 2-4 hours, and then adding a composite probiotic and cellulase to the water to obtain the mixed system of step 1-1; Step 1-2: fermenting the mixed system at 25° C.-35° C. for a fermentation time selected from 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: drying the fermented licorice product at 50° C.-70° C., and then passing through a 50-100 mesh sieve to obtain a powder of the fermented licorice product of step 2-1; Step 2-2: adding the fermented licorice product powder to a NaOH aqueous solution and heating to 70° C.-90° C. for a heating time selected from 1-2 hours, and then centrifuging the heated mixture to obtain the supernatant and solid of step 2-2; Step 2-3: adding the solid obtained in step 2-2 to a NaOH aqueous solution again and heating to 70° C.-90° C. for 1-2 h, and then centrifuging the heated mixture to obtain the supernatant and solid of step 2-3; Step 2-4: Combine the supernatant of step 2-2 and the supernatant of step 2-3 to obtain an aqueous solution of a crude licorice extract.

4. The preparation method according to claim 1, characterized in that Step 3 includes the following steps: Step 3-1: freeze-drying the aqueous solution of the crude licorice extract to obtain a solid of the crude licorice extract; Step 3-2: adding the solid of the crude licorice extract to n-hexane and soaking at room temperature for 5-10 hours to obtain the mixture of step 3-2; Step 3-3: The mixture of step 3-2 is filtered and dried to remove n-hexane, and then the obtained solid is added to a mixed solution of ethanol and acetone, and then heated to reflux at 65°C-75°C, and the heating reflux time is selected from 1-2.5 hours. The product after the reaction is concentrated to obtain a defatted licorice crude extract.

5. The preparation method according to claim 1, characterized in that In step 4, deproteinization is completed by freeze-thaw cycle treatment; Step 4 includes the following steps: Step 4-1: adding an appropriate amount of water to the defatted licorice crude extract to fully dissolve it, and then freezing it in an environment of -70°C to -50°C for 6-12 hours to obtain the frozen product of step 4-1; Step 4-2: thawing the frozen product of step 4-1 naturally at a constant temperature of 5°C to 15°C; Step 4-3: Repeat the process of step 4-1 and step 4-2 3-6 times to obtain a product after freeze-thaw cycles; Step 4-4: centrifuging the product after freeze-thaw cycles to obtain a supernatant and a solid, collecting the supernatant to obtain a deproteinized licorice crude extract.

6. The preparation method according to claim 1, characterized in that Step 5 includes the following steps: Step 5-1: adding an appropriate amount of water and a first electrolyte solution to the deproteinized licorice crude extract to a final concentration of 0.02-0.08 mol / L to obtain the mixed solution of step 5-1; wherein the first electrolyte solution is a CaCl2 solution or a MgSO4 solution; Step 5-2: The mixed solution of step 5-1 is centrifuged to obtain a supernatant and a solid. 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: combining and concentrating the decolorized crude licorice extract to obtain the combined and concentrated product of step 6-1; Step 6-2: Add ethanol to the combined concentrated product of step 6-1 until the concentration of ethanol in the final solution is 60%-70%. Collect and dry the precipitate obtained at this time to obtain licorice fractionated polysaccharides.

8. The preparation method according to claim 1, characterized in that Step 7 includes the following steps: Step 7-1: adding the fractionated licorice polysaccharide to a mixed solution of ethanol and propanol; Step 7-2: Under nitrogen protection, add a modified compound containing an amino group 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 modified compound containing an amino group is selected from propylenediamine, 3-methylaminopropylamine, tris(3-aminopropyl)amine, 3-diethylaminopropylamine or 3-dimethylaminopropylamine.

9. The preparation method according to claim 1, characterized in that Step 8 includes the following steps: Step 8-1: dissolving the amino-modified licorice fractionated polysaccharide in water, adding a plant extract containing selenoprotein and a cross-linking agent thereto, and stirring at room temperature for 2-4 hours; Step 8-2: NaCl solution is added dropwise until a precipitate appears, and the NaCl solution is continued to be added dropwise until the precipitate no longer increases, and the precipitate is collected and dried to obtain selenized glycyrrhizic polysaccharide.

10. The preparation method according to claim 1, characterized in that The plant extract containing selenoprotein is selected from selenium-rich Cardamine extract, selenium-rich Lycium barbarum extract, selenium-rich Salvia miltiorrhiza extract, selenium-rich Honeysuckle extract or selenium-rich Chlorella extract.

Images