A composite ionic liquid and its application

Abstract

The invention relates to the technical field of polysaccharide extraction, and discloses a composite ionic liquid and its application, wherein the composite ionic liquid is a mixture of trimethylamine hydrochloride and 1-ethyl-3-methylimidazolium acetate; the mass ratio of the trimethylamine hydrochloride and the 1-ethyl-3-methylimidazolium acetate is 1-1.5:2-3.5. The composite ionic liquid is used to extract polyporus polysaccharide. The invention uses the composite ionic liquid trimethylamine hydrochloride and 1-ethyl-3-methylimidazolium acetate to extract polyporus polysaccharide for the first time, and the extraction rate is 11.94%±0.36%, reaching 11.6-12.3mg / 100mg, which is 3-4 times the extraction rate of polyporus polysaccharide by the traditional water extraction method.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction, and in particular relates to a composite ionic liquid and application thereof. Background Art

[0002] Polyporus umbellatus (Pers.) Fries is the dried sclerotium of the Polyporaceae fungus. It tastes sweet and light, is neutral in nature, and enters the kidney and bladder meridians. It has the effect of promoting diuresis and removing dampness. It is mainly used to treat edema, dysuria, diarrhea, stranguria, leukorrhea, etc. It mainly contains chemical components such as polysaccharides, amino acids, ketones, and mineral elements. Polyporus polysaccharide is one of its main biologically active ingredients, which has anti-tumor, immunomodulatory, anti-radiation and liver protection effects.

[0003] The main extraction methods of polysaccharides include water extraction, ultrasonic extraction, microwave extraction, etc. The optimization of the extraction process involves orthogonal design, response surface optimization method, regression orthogonal experimental design method, etc. For example, Zhang Xiaoxia published a paper titled "Optimization of Extraction Process and Antioxidant Activity of Polyporus Polysaccharide" in the Journal of Chinese Traditional Medicine in 2016, in which a solvent-based ultrasonic extraction process was used to extract Polyporus polysaccharide, with an extraction rate of 1.773 mg / 100 mg; Zheng Yiqi published a paper titled "Response Surface Optimization of Ultrasonic-Assisted Extraction Process of Polyporus Sclerotium Polysaccharide and Analysis of Antioxidant Activity" in the Journal of Food Industry Science and Technology in 2023, in which ultrasonic-assisted technology was used to extract Polyporus polysaccharide. The effects of extraction time, ultrasonic power, extraction temperature and liquid-to-solid ratio on polysaccharide yield were investigated through single-factor experiments combined with response surface optimization, and the optimal extraction process was determined, with a maximum extraction rate of 2.50 mg / 100 mg. The extraction rates of Polyporus polysaccharides obtained by the above methods are all too low. Therefore, it is urgent to develop a substance for improving the extraction rate of Polyporus polysaccharide. Summary of the invention

[0004] The invention provides a composite ionic liquid and application thereof, which solves the problem of too low extraction rate when extracting polyporus polysaccharide in the prior art.

[0005] A composite ionic liquid is a mixture of trimethylamine hydrochloride and 1-ethyl-3-methylimidazolium acetate; the mass ratio of the trimethylamine hydrochloride to the 1-ethyl-3-methylimidazolium acetate is 1-1.5:2-3.5.

[0006] Too high or too low a mass ratio of trimethylamine hydrochloride and 1-ethyl-3-methylimidazolium acetate will reduce the extraction rate of Polyporus polysaccharide. In the early stage of extraction, 1-ethyl-3-methylimidazolium acetate is mainly responsible, but as the reaction proceeds, 1-ethyl-3-methylimidazolium acetate is difficult to enter the cells through the cell wall pores of Polyporus polysaccharide cells, making extraction difficult; at this time, the small molecule trimethylamine hydrochloride begins to work, breaking the macromolecular dissolution adhering to the outer surface of Polyporus polysaccharide particles under the action of ultrasound, opening up channels and entering the cells through the cell wall channels of Polyporus polysaccharide cells, continuing to dissolve polysaccharides, and further improving the extraction rate of Polyporus polysaccharide.

[0007] Preferably, the mass ratio of trimethylamine hydrochloride to 1-ethyl-3-methylimidazole acetate is 1 to 2:3.

[0008] The second purpose of the present invention is to protect the application of the composite ionic liquid in extracting Polyporus polysaccharide.

[0009] Preferably, the method for extracting Polyporus polysaccharide specifically comprises the following steps:

[0010] Adding the composite ionic liquid to deionized water to prepare a composite ionic liquid aqueous solution; wherein the concentration of the trimethylamine hydrochloride in the composite ionic liquid aqueous solution is 1.0 to 1.5 g / L, and the concentration of the 1-ethyl-3-methylimidazolium acetate is 2.0 to 3.5 g / L;

[0011] The umbelliferous powder and the composite ionic liquid aqueous solution are mixed at a material-liquid ratio of 1 g:20-30 mL, the pH of the mixed solution is adjusted to 6.5-7.0, the mixture is heated to 70-75° C., ultrasonicated, filtered, and the filtrate is purified to obtain the umbelliferous polysaccharide.

[0012] Preferably, the concentration of trimethylamine hydrochloride in the composite ionic liquid aqueous solution is 1.0 g / L, and the concentration of 1-ethyl-3-methylimidazolium acetate is 2.0 g / L.

[0013] Preferably, the ultrasonic condition is 50-70 min, 40-80 kHz. The purpose of the ultrasonic treatment is to promote the trimethylamine hydrochloride molecules to break up the macromolecular dissolution adhering to the outer surface of the Polyporus granules.

[0014] Preferably, HCl solution or NaOH solution is used to adjust the pH of the mixed solution.

[0015] Preferably, the concentration of the HCl solution or the NaOH solution is 0.1-0.2 mol / L.

[0016] Preferably, the purification method is: concentrating the filtrate to 1 / 3 of the original volume, adding ethanol to make the volume concentration of ethanol in the mixed solution 80%, standing for 6-8 hours, centrifuging and filtering, and the filter cake is crude polysaccharide. Ethanol is added to precipitate crude polysaccharide.

[0017] Preferably, the crude polysaccharide is purified with Sevage reagent to obtain a liquid with a protein content of less than or equal to 0.49%, and then freeze-dried to obtain refined Polyporus polysaccharide.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The present invention is the first to use composite ionic liquid trimethylamine hydrochloride and 1-ethyl-3-methylimidazolium acetate to extract Polyporus polysaccharide, with an extraction rate of 11.94%±0.36%, reaching 11.4-12.3 mg / 100 mg, which is 3-4 times the polysaccharide extraction rate of traditional water extraction.

[0020] 2. The present invention uses trimethylamine hydrochloride as the cationic liquid and 1-ethyl-3-methylimidazolium acetate as the anionic liquid. The extraction is carried out under the conditions of the optimal level of single-factor extraction: at the initial stage of extraction, the Polyporus umbellatus powder is heated at 70-75°C for a short time and is not softened. The extraction of Polyporus umbellatus polysaccharide is mainly caused by 1-ethyl-3-methylimidazolium acetate. However, as the extraction process proceeds, the Polyporus umbellatus powder gradually softens, and other water-soluble components in the cells are continuously released. Small molecules can diffuse rapidly into the extract, while macromolecular substances adhere to the outer surface of the Polyporus umbellatus particles and adsorb 1-ethyl-3-methylimidazolium acetate, making it difficult for 1-ethyl-3-methylimidazolium acetate to pass through. When the polysaccharide enters the cell wall pores of the Polyporus umbellatus cells, the dissolution rate of the polysaccharide will decrease. At this time, the small molecule trimethylamine hydrochloride begins to work. Because it is a cation and has a small molecular size, it itself has the ability to promote the dissolution of polysaccharides. In a hot environment, the trimethylamine hydrochloride molecules break the macromolecular dissolved substances adhering to the outer surface of the Polyporus umbellatus particles under the action of ultrasound, open up channels and enter the cells through the cell wall channels of the Polyporus umbellatus cells, continue to dissolve polysaccharides, and further improve the extraction rate of the polysaccharide, thereby avoiding the limitations of extracting polysaccharides with a single ionic liquid.

[0021] 3. Ionic liquids have the characteristics of good solubility, zero vapor pressure, high stability, non-volatility, low melting point, liquid at room temperature, wide solubility, good electrical conductivity, good thermal conductivity, green environmental protection, stable properties and high extraction rate.

[0022] 4. The refined polysaccharide obtained by the purification process of the crude polysaccharide extracted by the present invention is almost consistent with the refined polysaccharide extracted and purified by the traditional water extraction method in terms of antioxidant performance research, indicating that the composite ionic liquid adopted by the present invention only plays a role in improving the extraction rate in the extraction of Polyporus polysaccharide, and has no effect on the structure and performance of Polyporus polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention provides a process flow chart for extracting polyporus polysaccharide using a composite ionic liquid. DETAILED DESCRIPTION

[0024] The technical solutions in the present invention will be described clearly and completely below in conjunction with the specific implementation of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The methods described in the embodiments of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0026] The process flow chart of extracting Polyporus polysaccharide with composite ionic liquid provided in the embodiment of the present invention is shown in Figure 1 .

[0027] Example 1

[0028] A composite ionic liquid is a mixture of trimethylamine hydrochloride and 1-ethyl-3-methylimidazolium acetate; the mass ratio of the trimethylamine hydrochloride to the 1-ethyl-3-methylimidazolium acetate is 1:2.

[0029] A method for extracting polyporus polysaccharide is carried out according to the following steps:

[0030] Step 1, raw material pretreatment: wash the Polyporus umbellatus and cut it into 2 cm segments, put it into an oven and dry it at 80°C to constant weight, then crush it through a 60-mesh sieve to obtain raw material powder for later use.

[0031] Step 2: Add the composite ionic liquid to deionized water to prepare a composite ionic liquid aqueous solution as an extractant; wherein the concentration of trimethylamine hydrochloride is 1.0 g / L, and the concentration of 1-ethyl-3-methylimidazole acetate is 2.0 g / L.

[0032] Step 3, weigh 100g of raw material powder, add composite ionic liquid extractant according to 1g:20mL, mix and stir for 5min, adjust the pH of the mixed solution to 6.5 with 0.1mol / L HCl or 0.1mol / LNaOH solution, heat to 70℃ and maintain, and ultrasonically extract for 50min at 40kHz. After the extraction, the suspension is centrifuged at 4000r / min through centrifugal filter 1; the filtrate is concentrated under reduced pressure to 1 / 3 of the filtrate volume, and cooled to room temperature, 95% ethanol is added to the room temperature concentrate to make the ethanol volume concentration in the mixed solution 80%, and stand for 6h; the standing liquid is centrifuged for 15min through centrifugal filter 2 (centrifugal filter 1 and 2 are designed according to the process, and their types are the same to make the difference defined) and the filter cake is crude polysaccharide; the filtrate is sent to the distillation recovery device for recovery.

[0033] Step 4, purification: the crude polysaccharide obtained in step 3 was added to 50 mL of deionized water for redissolution, 1 / 5 volume of Sevage reagent was added to the solution, i.e., chloroform: n-butanol = 4:1, and the solution was vigorously shaken for 25 minutes and then centrifuged to separate the layers to remove the intermediate deformed protein and the lower organic reagent layer; the supernatant was repeated until the protein content in the supernatant was 0.48 mg / 100 mg; the protein content was determined by the Coomassie Brilliant Blue method.

[0034] Step 5, freeze drying: freeze-dry the supernatant with a protein content of 0.48 mg / 100 mg obtained in step 4 at -50°C for 14 h, and collect the supernatant to obtain refined Polyporus polysaccharide.

[0035] Example 2

[0036] A composite ionic liquid is a mixture of trimethylamine hydrochloride and 1-ethyl-3-methylimidazolium acetate; the mass ratio of the trimethylamine hydrochloride to the 1-ethyl-3-methylimidazolium acetate is 1:3.5.

[0037] A method for extracting polyporus polysaccharide is carried out according to the following steps:

[0038] Step 1, raw material pretreatment: wash the Polyporus umbellatus and cut it into 2 cm segments, put it into an oven and dry it at 80°C to constant weight, then crush it through a 60-mesh sieve to obtain raw material powder for later use.

[0039] Step 2: Add the composite ionic liquid to deionized water to prepare a composite ionic liquid aqueous solution as an extractant; wherein the concentration of trimethylamine hydrochloride is 1.0 g / L, and the concentration of 1-ethyl-3-methylimidazolium acetate is 3.5 g / L.

[0040] Step 3, weigh 100g of raw material powder, add composite ionic liquid extractant according to 1g:25mL, mix and stir for 8min, adjust the pH of the mixed solution to 6.5 with 0.2mol / L HCl solution, heat to 70℃ and maintain, and ultrasonically extract for 50min at 40kHz. After the extraction, the suspension is centrifuged at 4000r / min through centrifugal filter 1; the filtrate is concentrated under reduced pressure to 1 / 3 of the filtrate volume, and cooled to room temperature, 95% ethanol is added to the room temperature concentrate to make the ethanol volume concentration in the mixed solution 80%, and stand for 6h; the standing liquid is centrifuged through centrifugal filter 2 for 15min, and the filter cake is crude polysaccharide; the filtrate is sent to the distillation recovery device for recovery. Centrifugal filters 1 and 2 are designed according to the process, and their types are the same to define the difference.

[0041] Step 4, purification: the crude polysaccharide obtained in step 3 was added to 50 mL of deionized water for redissolution, 1 / 5 volume of Sevage reagent was added to the solution, i.e., chloroform: n-butanol = 4:1, and the solution was vigorously shaken for 25 minutes and then centrifuged to separate the layers to remove the intermediate deformed protein and the lower organic reagent layer; the supernatant was repeated until the protein content in the supernatant was 0.46 mg / 100 mg; the protein content was determined by the Coomassie Brilliant Blue method.

[0042] Step 5, freeze drying: freeze-dry the supernatant with a protein content of 0.46 mg / 100 mg obtained in step 4 at -50°C for 14 h, and collect the collected supernatant to obtain refined Polyporus polysaccharide.

[0043] Example 3

[0044] A composite ionic liquid is a mixture of trimethylamine hydrochloride and 1-ethyl-3-methylimidazolium acetate; the mass ratio of the trimethylamine hydrochloride to the 1-ethyl-3-methylimidazolium acetate is 1.5:2.

[0045] A method for extracting polyporus polysaccharide is carried out according to the following steps:

[0046] Step 1, raw material pretreatment: wash the Polyporus umbellatus and cut it into 3 cm segments, put it into an oven and dry it at 90°C to constant weight, then crush it through a 60-mesh sieve to obtain raw material powder for later use.

[0047] Step 2: Add the composite ionic liquid to deionized water to prepare a composite ionic liquid aqueous solution as an extractant; wherein the concentration of trimethylamine hydrochloride is 1.5 g / L, and the concentration of 1-ethyl-3-methylimidazolium acetate is 2.0 g / L.

[0048] Step 3, weigh 100g of raw material powder, add composite ionic liquid extractant according to 1g:30mL, mix and stir for 10min, adjust the pH of the mixed solution to 7.0 with 0.2mol / LNaOH solution, heat to 75℃ and maintain, and ultrasonically extract for 60min at 80kHz. After the extraction, the suspension is centrifuged at 4500r / min through centrifugal filter 1; the filtrate is concentrated under reduced pressure to 1 / 3 of the filtrate volume, and cooled to room temperature, 95% ethanol is added to the room temperature concentrate to make the ethanol volume concentration in the mixed solution 80%, and stand for 8h; the standing liquid is centrifuged through centrifugal filter 2 for 20min, and the filter cake is crude polysaccharide; the filtrate is sent to the distillation recovery device for recovery. Centrifugal filters 1 and 2 are designed according to the process, and their types are the same to define the difference.

[0049] Step 4, purification: the crude polysaccharide obtained in step 3 was re-dissolved in 50 mL of deionized water, 1 / 5 volume of Sevage reagent was added to the solution, i.e., chloroform: n-butanol = 4:1, and the solution was vigorously shaken for 25 minutes and then centrifuged to separate the layers to remove the intermediate deformed protein and the lower organic reagent layer; the supernatant was repeated until the protein content in the supernatant was 0.44 mg / 100 mg; the protein content was determined by the Coomassie Brilliant Blue method.

[0050] Step 5, freeze drying: freeze-dry the supernatant with a protein content of 0.44 mg / 100 mg obtained in step 4 at -50°C for 18 h, and collect the collected supernatant to obtain refined Polyporus polysaccharide.

[0051] Example 4

[0052] A composite ionic liquid is a mixture of trimethylamine hydrochloride and 1-ethyl-3-methylimidazolium acetate; the mass ratio of the trimethylamine hydrochloride to the 1-ethyl-3-methylimidazolium acetate is 1.5:3.5.

[0053] A method for extracting polyporus polysaccharide is carried out according to the following steps:

[0054] Step 1, raw material pretreatment: wash the Polyporus umbellatus and cut it into 3 cm segments, put it into an oven and dry it at 90°C to constant weight, then crush it through a 60-mesh sieve to obtain raw material powder for later use.

[0055] Step 2: Add the composite ionic liquid to deionized water to prepare a composite ionic liquid aqueous solution as an extractant; wherein the concentration of trimethylamine hydrochloride is 1.5 g / L, and the concentration of 1-ethyl-3-methylimidazolium acetate is 3.5 g / L.

[0056] Step 3, weigh 100g of raw material powder, add composite ionic liquid extractant according to 1g:30mL, mix and stir for 10min, adjust the pH of the mixed solution to 7.0 with 0.2mol / L HCl or 0.2mol / LNaOH solution, heat to 75℃ and maintain, and ultrasonically extract for 60min at 40kHz. After the extraction, the suspension is centrifuged at 4500r / min through centrifugal filter 1; the filtrate is concentrated under reduced pressure to 1 / 3 of the filtrate volume, and cooled to room temperature, 95% ethanol is added to the room temperature concentrate to make the ethanol volume concentration in the mixed solution 80%, and stand for 8h; the standing liquid is centrifuged through centrifugal filter 2 for 20min, and the filter cake is crude polysaccharide; the filtrate is sent to the distillation recovery device for recovery. Centrifugal filters 1 and 2 are designed according to the process, and their types are the same to define the difference.

[0057] Step 4, purification: the crude polysaccharide obtained in step 3 was added to 50 mL of deionized water for redissolution, 1 / 5 volume of Sevage reagent was added to the solution, i.e., chloroform: n-butanol = 4:1, and the solution was vigorously shaken for 25 minutes and then centrifuged to separate the layers to remove the intermediate deformed protein and the lower organic reagent layer; the supernatant was repeated until the protein content in the supernatant was 0.49 mg / 100 mg; the protein content was determined by the Coomassie Brilliant Blue method.

[0058] Step 5, freeze drying: freeze-dry the supernatant with a protein content of 0.49 mg / 100 mg obtained in step 4 at -50°C for 18 h, and collect the collected supernatant to obtain refined Polyporus polysaccharide.

[0059] Comparative Example 1

[0060] Step 1, raw material pretreatment: wash the Polyporus umbellatus and cut it into 2 cm segments, put it into an oven and dry it at 80°C to constant weight, then crush it through a 60-mesh sieve to obtain raw material powder for later use.

[0061] Step 2, weigh 100g of raw material powder, add deionized water as an extractant according to 1g:20mL, mix and stir for 5min, adjust the pH of the mixed solution to 7.0 with 0.1mol / LNaOH solution, heat to 75℃ and maintain, and extract ultrasonically at 40kHz for 60min. After the extraction, the suspension is centrifuged in a centrifugal filter 1 at a speed of 4500r / min; the filtrate is concentrated under reduced pressure to 1 / 3 of the filtrate volume, and cooled to room temperature, 95% ethanol is added to the room temperature concentrate to make the ethanol volume concentration in the mixed solution 80%, and stand for 8h; the standing liquid is centrifuged through a centrifugal filter 2, and the filter cake is crude polysaccharide.

[0062] Step 3, purification: the crude polysaccharide obtained in step 4 was added to 50 mL of deionized water for re-dissolution, 1 / 5 volume of Sevage reagent was added to the solution, i.e., chloroform: n-butanol = 4:1, and the solution was vigorously shaken for 25 minutes and then centrifuged to separate the layers to remove the intermediate deformed protein and the lower organic reagent layer; the supernatant was repeated until the protein content in the supernatant was 0.43 mg / 100 mg; the protein content was determined by the Coomassie Brilliant Blue method.

[0063] Step 4, freeze drying: freeze-dry the supernatant with a protein content of 0.43 mg / 100 mg obtained in step 4 at -50°C for 14 h, and collect the collected supernatant to obtain refined Polyporus polysaccharide.

[0064] Comparative Example 2

[0065] Step 1, raw material pretreatment: wash the Polyporus umbellatus and cut it into 2 cm segments, put it into an oven and dry it at 80°C to constant weight, then crush it through a 60-mesh sieve to obtain raw material powder for later use.

[0066] Step 2, preparing the ionic liquid solution: accurately prepare a solution of trimethylamine hydrochloride with a concentration of 1.5 g / L.

[0067] Step 2, weigh 100g of raw material powder, add trimethylamine hydrochloride ionic liquid solution as an extractant according to 1g:30mL, mix and stir for 10min, adjust the pH of the mixed solution to 7.0 with 0.1mol / LNaOH solution, heat to 75°C and maintain, and ultrasonically extract for 60min at 40kHz. After the extraction, the suspension is centrifuged in a centrifugal filter 1 at a speed of 4500r / min through a centrifuge; the filtrate is concentrated under reduced pressure to 1 / 3 of the filtrate volume, and cooled to room temperature, 95% ethanol is added to the room temperature concentrated solution to make the ethanol volume concentration in the mixed solution 80%, and stand for 8h; the standing liquid is centrifuged through a centrifugal filter 2, and the filter cake is a crude polysaccharide; the filtrate is sent to a distillation recovery device for recovery.

[0068] Step 3, purification: the crude polysaccharide obtained in step 4 was added to 50 mL of deionized water for redissolution, 1 / 5 volume of Sevage reagent was added to the solution, i.e., chloroform: n-butanol = 4:1, and the solution was vigorously shaken for 25 minutes and then centrifuged to separate the layers to remove the intermediate deformed protein and the lower organic reagent layer; the supernatant was repeated until the protein content in the supernatant was 0.47 mg / 100 mg; the protein content was determined by the Coomassie Brilliant Blue method.

[0069] Step 4, freeze drying: freeze-dry the supernatant with a protein content of 0.47 mg / 100 mg obtained in step 4 at -50°C for 18 h, and collect the collected supernatant to obtain refined Polyporus polysaccharide.

[0070] Comparative Example 3

[0071] Step 1, raw material pretreatment: wash the Polyporus umbellatus and cut it into 2 cm segments, put it into an oven and dry it at 80°C to constant weight, then crush it through a 60-mesh sieve to obtain raw material powder for later use.

[0072] Step 2, preparing ionic liquid solution: accurately prepare 1-ethyl-3-methylimidazolium acetate solution with a concentration of 3.5 g / L.

[0073] Step 2, weigh 100g of raw material powder, add 1-ethyl-3-methylimidazolium acetate ionic liquid as an extractant according to 1g:30mL, mix and stir for 10min, adjust the pH of the mixed solution to 7.0 with 0.1mol / LNaOH solution, heat to 75°C and maintain, and ultrasonically extract for 60min at 40kHz. After the extraction, the suspension is centrifuged in a centrifugal filter 1 at a speed of 4500r / min; the filtrate is concentrated under reduced pressure to 1 / 3 of the filtrate volume, and cooled to room temperature, 95% ethanol is added to the room temperature concentrated solution to make the ethanol volume concentration in the mixed solution 80%, and stand for 8h; the standing liquid is centrifuged through a centrifugal filter 2, and the filter cake is a crude polysaccharide; the filtrate is sent to a distillation recovery device for recovery.

[0074] Step 3, purification: the crude polysaccharide obtained in step 4 was added to 50 mL of deionized water for redissolution, 1 / 5 volume of Sevage reagent was added to the solution, i.e., chloroform: n-butanol = 4:1, and the solution was vigorously shaken for 25 minutes and then centrifuged to separate the layers to remove the intermediate deformed protein and the lower organic reagent layer; the supernatant was repeated until the protein content in the supernatant was 0.48 mg / 100 mg; the protein content was determined by the Coomassie Brilliant Blue method.

[0075] Step 4, freeze drying: freeze-dry the supernatant with a protein content of 0.48 mg / 100 mg obtained in step 4 at -50°C for 18 h, and collect the supernatant to obtain refined Polyporus polysaccharide.

[0076] Comparative Example 4

[0077] Step 1, raw material pretreatment: wash the Polyporus umbellatus and cut it into 2 cm segments, put it into an oven and dry it at 80°C to constant weight, then crush it through a 60-mesh sieve to obtain raw material powder for later use.

[0078] Step 2, preparing ionic liquid solution: accurately prepare 1-ethyl-3-methylimidazolium bromide solution with a concentration of 3.5 g / L.

[0079] Step 2, weigh 100g of raw material powder, add 1-ethyl-3-methylimidazolium bromide ionic liquid as an extractant according to 1g:30mL, mix and stir for 10min, adjust the pH of the mixed solution to 7.0 with 0.1mol / LNaOH solution, heat to 75°C and maintain, and ultrasonically extract for 60min at 40kHz. After the extraction, the suspension is centrifuged in a centrifugal filter 1 at a speed of 4500r / min; the filtrate is concentrated under reduced pressure to 1 / 3 of the filtrate volume, and cooled to room temperature, 95% ethanol is added to the room temperature concentrated solution to make the ethanol volume concentration in the mixed solution 80%, and stand for 8h; the standing liquid is centrifuged through a centrifugal filter 2, and the filter cake is a crude polysaccharide; the filtrate is sent to a distillation recovery device for recovery.

[0080] Step 3, purification: the crude polysaccharide obtained in step 4 was added to 50 mL of deionized water for redissolution, 1 / 5 volume of Sevage reagent was added to the solution, i.e., chloroform: n-butanol = 4:1, and the solution was vigorously shaken for 25 minutes and then centrifuged to separate the layers to remove the intermediate deformed protein and the lower organic reagent layer; the supernatant was repeated until the protein content in the supernatant was 0.45 mg / 100 mg; the protein content was determined by the Coomassie Brilliant Blue method.

[0081] Step 4, freeze drying: freeze-dry the supernatant with a protein content of 0.45 mg / 100 mg obtained in step 4 at -50°C for 18 h, and collect the collected supernatant to obtain refined Polyporus polysaccharide.

[0082] Comparative Example 5

[0083] Step 1, raw material pretreatment: wash the Polyporus umbellatus and cut it into 2 cm segments, put it into an oven and dry it at 80°C to constant weight, then crush it through a 60-mesh sieve to obtain raw material powder for later use.

[0084] Step 2, preparing the ionic liquid solution: accurately prepare a solution of 1-ethyl-3-methylimidazolium nitrate with a concentration of 3.5 g / L.

[0085] Step 2, weigh 100g of raw material powder, add 1-ethyl-3-methylimidazole nitrate ionic liquid as an extractant according to 1g:30mL, mix and stir for 10min, adjust the pH of the mixed solution to 7.0 with 0.1mol / LNaOH solution, heat to 75°C and maintain, and ultrasonically extract for 60min at 40kHz. After the extraction, the suspension is centrifuged in a centrifugal filter 1 at a speed of 4500r / min; the filtrate is concentrated under reduced pressure to 1 / 3 of the filtrate volume, and cooled to room temperature, 95% ethanol is added to the room temperature concentrated solution to make the ethanol volume concentration in the mixed solution 80%, and stand for 8h; the standing liquid is centrifuged through a centrifugal filter 2, and the filter cake is a crude polysaccharide; the filtrate is sent to a distillation recovery device for recovery.

[0086] Step 3, purification: the crude polysaccharide obtained in step 4 was added to 50 mL of deionized water for redissolution, 1 / 5 volume of Sevage reagent was added to the solution, i.e., chloroform: n-butanol = 4:1, and the solution was vigorously shaken for 25 minutes and then centrifuged to separate the layers to remove the intermediate deformed protein and the lower organic reagent layer; the supernatant was repeated until the protein content in the supernatant was 0.42 mg / 100 mg; the protein content was determined by the Coomassie Brilliant Blue method.

[0087] Step 4, freeze drying: freeze-dry the supernatant with a protein content of 0.42 mg / 100 mg obtained in step 4 at -50°C for 18 h, and collect the collected supernatant to obtain refined Polyporus polysaccharide.

[0088] Comparative Example 6

[0089] A composite ionic liquid is a mixture of tetramethylammonium chloride and 1-ethyl-3-methylimidazolium acetate; the mass ratio of the tetramethylammonium chloride to the 1-ethyl-3-methylimidazolium acetate is 1.5:3.5.

[0090] A method for extracting polyporus polysaccharide is carried out according to the following steps:

[0091] Step 1, raw material pretreatment: wash the Polyporus umbellatus and cut it into 2 cm segments, put it into an oven and dry it at 80°C to constant weight, then crush it through a 60-mesh sieve to obtain raw material powder for later use.

[0092] Step 2: configuring the composite ionic liquid into a composite ionic liquid solution; wherein the concentration of tetramethylammonium chloride is 1.5 g / L, and the concentration of 1-ethyl-3-methylimidazolium acetate is 3.5 g / L.

[0093] Step 3, weigh 100g of raw material powder, add composite ionic liquid extractant according to 1g:30mL, mix and stir for 10min, adjust the pH of the mixed solution to 7.0 with 0.1mol / LNaOH solution, heat to 75℃ and maintain, and ultrasonically extract for 60min at 40kHz. After the extraction, the suspension is centrifuged at 4500r / min through centrifugal filter 1; the filtrate is concentrated under reduced pressure to 1 / 3 of the filtrate volume, and cooled to room temperature, 95% ethanol is added to the room temperature concentrate to make the ethanol volume concentration in the mixed solution 80%, and stand for 8h; the standing liquid is centrifuged through centrifugal filter 2 for 20min, and the filter cake is crude polysaccharide; the filtrate is sent to the distillation recovery device for recovery. Centrifugal filters 1 and 2 are designed according to the process, and their types are the same to define the difference.

[0094] Step 4, purification: the crude polysaccharide obtained in step 3 was added to 50 mL of deionized water for redissolution, 1 / 5 volume of Sevage reagent was added to the solution, i.e., chloroform: n-butanol = 4:1, and the solution was vigorously shaken for 25 minutes and then centrifuged to separate the layers to remove the intermediate deformed protein and the lower organic reagent layer; the supernatant was repeated until the protein content in the supernatant was 0.43 mg / 100 mg; the protein content was determined by the Coomassie Brilliant Blue method.

[0095] Step 5, freeze drying: freeze-dry the supernatant with a protein content of 0.43 mg / 100 mg obtained in step 4 at -50°C for 18 h, and collect the supernatant to obtain refined Polyporus polysaccharide.

[0096] Results test:

[0097] The polysaccharide content in the concentrate was determined by the phenol-sulfuric acid method, and the polysaccharide extraction rate was calculated to be 11.6-12.3 mg / 100 mg of raw material.

[0098] Calculation method: extraction rate = obtained polysaccharide / Poria × 100%.

[0099] Table 1 Extraction rate of Polyporus polysaccharide extracted from different groups

[0100]

[0101] As shown in Table 1, the extraction rate of Polyporus polysaccharide in Examples 1 to 4 is 11.94% ± 0.36%, reaching 11.6 to 12.3 mg / 100 mg, which is higher than that of the conventional water extraction method, i.e., Comparative Example 1, and is 3-4 times the extraction rate of Comparative Example 1. Comparative Example 2 uses trimethylamine hydrochloride alone to extract Polyporus polysaccharide, which is only higher than Comparative Example 1. Comparative Example 3 uses 1-ethyl-3-methylimidazolium acetate alone to extract Polyporus polysaccharide, and the extraction rate of Polyporus polysaccharide is still low. Comparative Examples 2 and 3 show that the extraction rate of the embodiments of the present invention cannot be achieved by using any one of the composite ionic liquids alone. Comparative Examples 4 and 5 use other anionic liquids for extraction, but the effect is poor. Comparative Example 6 uses other cationic liquids and 1-ethyl-3-methylimidazolium acetate to form a composite ionic liquid for extraction, but the extraction rate is still much lower than that of Examples 1 to 5.

[0102] Reagent recovery: The filtrate obtained by centrifugal filtration in centrifugal filter 2 is subjected to reduced pressure distillation at an operating pressure of 60 kPa to recover ethanol. The concentration of the obtained ethanol solution can reach 90% and can be reused. The kettle liquid of the reduced pressure distillation mainly contains ionic liquid trimethylamine hydrochloride, 1-ethyl-3-methylimidazole acetate and water. The concentration of the ionic liquid therein is determined by liquid chromatography and can be reused.

[0103] DPPH free radical scavenging study: The method reported by Wang Hongliang et al. was adopted. References: Wang Hongliang, Hui Zhezhe, Liang Ben, et al. Study on the extraction process and antioxidant activity of polysaccharides from Gleditsia sinensis leaves [J]. Journal of the Chinese Cereals and Oils Association: 1-11 [2024-02-27. https: / / doi.org / 10.20048 / j.cnki.issn.1003-0174.000254. The DPPH free radical scavenging study of refined Polyporus polysaccharide was conducted. The results showed that the scavenging rate of Polyporus polysaccharide for DPPH free radicals was 94.3±0.63% at 4.0 mg / mL, which was equivalent to the scavenging rate of Vc at the same concentration.

[0104] ·OH free radical scavenging study: The method of Zhang et al. was adopted. Reference: AQ Zhang, NN Xiao, PF He, et al. Chemical analysis and antioxi-dant activity in vitro of polysaccharides extracted from Boletus edulis [J]. International Journal of Biological Macromolecules, 2011, 49 (5): 1092-1095. The ·OH free radical scavenging study of purified Polyporus umbellatus polysaccharide was conducted. The results showed that when Polyporus umbellatus polysaccharide was 3.6 mg / mL, the scavenging rate of ·OH free radical was 88.9 ± 0.63%, which was comparable to the scavenging rate of Vc at the same concentration.

[0105] Reducing ability determination: refer to the potassium ferrocyanide reduction method proposed by Ma Liyuan et al. References: Ma Liyuan, Huang Xueying, Shang Erkun, et al. Ultrasonic-assisted enzyme extraction of polysaccharides from corn husk and determination of their antioxidant activity [J]. Food and Machinery, 2023, 39(5): 186-192, 202. The reducing ability of refined Polyporus polysaccharide was determined. The results showed that as the concentration of Polyporus polysaccharide increased, its reducing ability gradually increased.

[0106] Table 2 Free radical scavenging and reducing abilities of polyporus polysaccharides extracted from different groups

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[0108]

[0109] As shown in Table 2, the antioxidant study shows that the reduction capacity of the Polyporus polysaccharides extracted by the composite ionic liquid provided in Examples 1 to 4 is similar to that of the Polyporus polysaccharides extracted by Comparative Examples 1 to 6. The data in Table 2 show that the performance of the extracted Polyporus refined polysaccharides is basically the same, whether in the examples or the comparative examples, indicating that the use of the composite ionic liquid provided by the present invention has no effect on the structure and performance of Polyporus polysaccharides, and the data difference is due to experimental errors.

[0110] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0111] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for extracting polyporus polysaccharide using composite ionic liquid, characterized in that: The specific steps include: The composite ionic liquid is a mixture of trimethylamine hydrochloride and 1-ethyl-3-methylimidazolium acetate; the mass ratio of trimethylamine hydrochloride to 1-ethyl-3-methylimidazolium acetate is 1-1.5:2-3.5; The composite ionic liquid is configured into a composite ionic liquid solution; wherein the concentration of the trimethylamine hydrochloride in the composite ionic liquid solution is 1.0-1.5 g / L, and the concentration of the 1-ethyl-3-methylimidazolium acetate is 2.0-3.5 g / L; The polyporus powder and the composite ionic liquid solution are mixed at a solid-liquid ratio of 1 g: 20-30 mL, the pH of the mixed solution is adjusted to 6.5-7.0, the temperature is raised to 70-75°C, ultrasonicated, filtered, and the filtrate is purified to obtain the polyporus polysaccharide.

2. The method according to claim 1, characterized in that The mass ratio of the trimethylamine hydrochloride to 1-ethyl-3-methylimidazole acetate is 1:2-3.

3. The method according to claim 1, characterized in that The concentration of trimethylamine hydrochloride in the composite ionic liquid is 1.0 g / L, and the concentration of 1-ethyl-3-methylimidazolium acetate is 2.0 g / L.

4. The method according to claim 1, characterized in that: The ultrasonic conditions are 50-70 min and 40-80 kHz.

5. The method according to claim 1, characterized in that When adjusting the pH of the mixed solution, use HCl solution or NaOH solution.

6. The method according to claim 5, characterized in that The concentration of the HCl solution or the NaOH solution is 0.1-0.2 mol / L.

7. The method according to claim 1, characterized in that The purification method is as follows: concentrating the filtrate to 1 / 3 of the original volume, adding ethanol to make the volume concentration of ethanol in the mixed solution 80%, standing for 6-8 hours, centrifuging and filtering, and the filter cake is crude polysaccharide.

8. The method according to claim 7, characterized in that The crude polysaccharide is purified with Sevage reagent to obtain a liquid with a protein content of less than or equal to 0.49%, and then freeze-dried to obtain refined Polyporus polysaccharide.

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