Qinba selenium mushroom polysaccharide, extraction and purification method and application

Abstract

The invention relates to the technical field of medicine, in particular to Qinba selenium mushroom polysaccharide, an extraction and purification method and application. The method comprises the steps of water extraction and alcohol precipitation, microfiltration and ultrafiltration combined filtration, ion purification and gel purification. The microfiltration and ultrafiltration combined filtration comprises the following steps: repeatedly freezing and thawing the Qinba selenium mushroom crude polysaccharide prepared by water extraction and alcohol precipitation, dissolving and freezing, centrifuging the thawed solution, and reserving the supernate; and sequentially carrying out microfiltration and ultrafiltration on the supernate, collecting filtrate, and drying to obtain the Qinba selenium mushroom extracellular crude polysaccharide. According to the method, the Qinba selenium mushroom polysaccharide can be effectively extracted, and the Qinba selenium mushroom polysaccharide can be widely applied due to good purity; the method is simple in steps, easy to operate and low in extraction and purification cost, and can be effectively applied to various fields.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a Qinba selenium mushroom polysaccharide, an extraction and purification method and an application thereof. Background Art

[0002] Qinba selenium mushroom, also known as selenium-rich Agaricus blazei, is rich in various active ingredients. It can be used as a source of immunomodulators, anticancer agents, antimutagents and antibacterial substances, and has received increasing attention in recent years.

[0003] In the prior art, the polysaccharide components are generally extracted by boiling in water. Although the yield of this extraction method is relatively high, what is obtained is mainly crude polysaccharide of Qinba selenium mushroom with low purity. The effects in anti-tumor, immune enhancement, antiviral and blood sugar lowering are relatively poor, which makes it difficult to effectively apply Qinba selenium mushroom.

[0004] In order to improve the purity, ion purification or gel purification can usually be used, and these technical means have become mature. However, ion purification or gel purification based on water boiling will result in a very low yield, which may often be reduced by less than one percent, which leads to a serious reduction in the extraction efficiency of Qinba selenium mushroom polysaccharide, and is not conducive to the promotion and application of Qinba selenium mushroom polysaccharide.

[0005] In addition, some ion purification or gel purification conditions are not compatible with Qinba selenium mushroom polysaccharides, and the steps are cumbersome and costly, resulting in a lot of waste of Qinba selenium mushroom raw materials.

[0006] Therefore, in order to meet market demand and improve the medicinal and nutritional value of Qinba selenium mushroom, it is urgent to study an extraction and purification process for crude polysaccharides of Qinba selenium mushroom. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a Qinba selenium mushroom polysaccharide, an extraction and purification method and an application thereof.

[0008] The technical solution of the present invention to solve the above technical problems is as follows:

[0009] The present invention provides a method for extracting and purifying Qinba selenium mushroom polysaccharide, which comprises water extraction and alcohol precipitation, microfiltration and ultrafiltration combined filtration, ion purification and gel purification; the microfiltration and ultrafiltration combined filtration comprises the following steps:

[0010] The crude polysaccharide of Qinba selenium mushroom prepared by the water extraction and alcohol precipitation is dissolved and frozen after repeated freezing and thawing, the thawed solution is centrifuged and the supernatant is retained; the supernatant is microfiltered and ultrafiltered in sequence, the filtrate is collected and dried to obtain the crude extracellular polysaccharide of Qinba selenium mushroom.

[0011] Based on the above technical solution, the present invention can also be improved as follows.

[0012] Further, the microfiltration and ultrafiltration combined filtration comprises the following steps:

[0013] S2-1, centrifuging the thawed solution at 7000-7500 rpm for multiple times, with each centrifugation time being 10-20 min, to obtain a supernatant;

[0014] S2-2, sequentially performing microfiltration on the supernatant obtained in step S2-1 using at least two microfiltration membranes to obtain a microfiltrate;

[0015] S2-3, firstly ultrafiltering the microfiltrate using an ultrafiltration membrane, and then filtering using a microfiltration membrane to obtain an ultrafiltrate;

[0016] S2-4, detecting the ionic strength of the ultrafiltrate.

[0017] Further, when the ionic strength of the ultrafiltrate is lower than 10Μs / cm, the microfiltration-ultrafiltration combined filtration is completed; when the ionic strength of the ultrafiltrate is higher than 10Μs / cm, the step S2-2 is repeated until the ionic strength of the ultrafiltrate is lower than 10Μs / cm.

[0018] Furthermore, in step S2-2, there are three types of microfiltration membranes, namely, 0.8 μm microfiltration membrane, 0.45 μm microfiltration membrane and 0.22 μm microfiltration membrane.

[0019] Furthermore, in step S2-3, the ultrafiltration membrane is an ultrafiltration membrane with a molecular weight of 100 kDa, and the microfiltration membrane is a 0.22 μm microfiltration membrane.

[0020] Further, the water extraction and alcohol precipitation comprises the following steps:

[0021] S1-1, obtaining Qinba selenium mushroom powder;

[0022] S1-2, mixing the Qinba selenium mushroom powder with water and heating, maintaining at 100° C. for 2.5 to 3.5 hours, and concentrating the filtrate after repeated filtration to obtain a concentrated solution;

[0023] S1-3, after centrifuging the concentrated solution, adding 95% by volume ethanol, centrifuging and collecting the precipitate, and drying to obtain the Qinba selenium mushroom crude polysaccharide; the volume ratio of the concentrated solution to the ethanol is 1:4-5.

[0024] Furthermore, the crude extracellular polysaccharide of Qinba selenium mushroom was prepared into a solution with a mass concentration of 20 mg / mL, and then a diethylaminoethyl cellulose ion exchange chromatography column was used for the ion purification; the eluents for the ion purification were deionized water, 0.1 mol / L NaCl solution and 0.2 mol / L NaCl solution respectively; the loading amount for the ion purification was 1 mL, and the flow rate was 0.25 mL / min.

[0025] Furthermore, the eluate obtained from the ion purification is filtered and then subjected to the gel purification; the filler of the gel column for the gel purification is SephacryS-200, with a specification of 16mm×100cm; the loading amount of the gel purification is 1mL / each time, the eluent is deionized water, and the elution flow rate is 1mL / min.

[0026] The present invention also provides a kind of Qinba selenium mushroom polysaccharide, which is prepared by the above method.

[0027] The present invention also provides an application of the Qinba selenium mushroom polysaccharide as described above, which can be used to prepare a preparation for intervening in acute myeloid leukemia.

[0028] The beneficial effects of the present invention are:

[0029] (1) The extraction and purification method of Qinba selenium mushroom polysaccharide of the present invention adopts a method combining water extraction and alcohol precipitation with ion purification and gel purification, so that Qinba selenium mushroom polysaccharide can be effectively extracted and has good purity, so that it can be widely used;

[0030] (2) The extraction and purification method of Qinba selenium mushroom polysaccharide of the present invention first performs microfiltration and ultrafiltration combined filtration before ion purification and gel purification to achieve impurity removal, thereby effectively improving the effect of extraction and purification;

[0031] (3) The extraction and purification method of Qinba selenium mushroom polysaccharide of the present invention has simple steps, is easy to operate, and has low extraction and purification costs;

[0032] (4) The Qinba selenium mushroom polysaccharide of the present invention has good purity and can be effectively applied in various fields, especially in the preparation of preparations for intervening in acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a flow chart of the method for extracting and purifying Qinba selenium mushroom polysaccharide of the present invention;

[0034] Figure 2 This is the standard curve diagram of Example 1 of the extraction and purification method of Qinba selenium mushroom polysaccharide of the present invention;

[0035] Figure 3This is the standard curve diagram of Example 2 of the method for extracting and purifying Qinba selenium mushroom polysaccharide of the present invention;

[0036] Figure 4 The method for extracting and purifying Qinba selenium mushroom polysaccharide of the present invention, in the ion purification of Example 3, is a curve diagram of the tube number, polysaccharide concentration and NaCl concentration of each tube;

[0037] Figure 5 The standard curve diagram of Example 3 is the extraction and purification method of Qinba selenium mushroom polysaccharide of the present invention;

[0038] Figure 6 The method for extracting and purifying the polysaccharide of Qinba selenium mushroom of the present invention is a curve diagram of the tube number and polysaccharide concentration of each tube in the gel purification of Example 4;

[0039] Figure 7 The standard curve diagram of Example 4 is the extraction and purification method of Qinba selenium mushroom polysaccharide of the present invention;

[0040] Figure 8 The present invention provides a method for extracting and purifying the polysaccharide of Qinba selenium mushroom. In Example 5, the network pharmacology diagram of Qinba selenium mushroom fructose intervening in acute myeloid leukemia is shown. Figure 8 A in the middle is the potential active ingredient-target network; Figure 8 Middle B is the protein interaction network diagram;

[0041] Fig. 9 The extraction and purification method of Qinba selenium mushroom polysaccharide of the present invention, in Example 5, Qinba selenium mushroom polysaccharide intervenes in the biological process of inflammatory factors regulating the immune microenvironment-related targets of acute myeloid leukemia, enrichment analysis and pathway analysis diagram; Fig. 9 A in the middle is a GO enrichment analysis diagram. Fig. 9 Middle B is a bar graph of enrichment analysis of biological processes, cellular components, and molecular functions; Fig. 9 Middle C is a KEGG enrichment analysis pathway bar chart, Fig. 9 Middle D is the KEGG enrichment analysis pathway bubble diagram. DETAILED DESCRIPTION

[0042] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0043] The extraction and purification method of Qinba selenium mushroom polysaccharide of the present invention comprises water extraction and alcohol precipitation, microfiltration and ultrafiltration combined filtration, ion purification and gel purification; the microfiltration and ultrafiltration combined filtration comprises the following steps:

[0044] The crude polysaccharide of Qinba selenium mushroom prepared by water extraction and alcohol precipitation is dissolved and frozen after repeated freezing and thawing, centrifuged after thawing and the supernatant is retained; the supernatant is microfiltered and ultrafiltered in sequence, and the filtrate is collected and dried to obtain the crude extracellular polysaccharide of Qinba selenium mushroom.

[0045] The extraction and purification method of Qinba selenium mushroom polysaccharide of the present invention adopts a method combining water extraction and alcohol precipitation with ion purification and gel purification, so that Qinba selenium mushroom polysaccharide can be effectively extracted, and through having good purity, it can be widely used. At the same time, in the extraction and purification method of the present invention, before ion purification and gel purification, microfiltration and ultrafiltration combined filtration are first performed to achieve impurity removal, thereby effectively improving the effect of extraction and purification.

[0046] like Figure 1 As shown, the extraction and purification method of the present invention has the following steps:

[0047] S1. Water extraction and alcohol precipitation: The water extraction and alcohol precipitation method can utilize the thermal effect of hot water to diffuse the polysaccharide from the cells of Qinba selenium mushroom into the external solution. The specific steps are as follows:

[0048] S1-1. Obtain Qinba selenium mushroom powder: Grind the Qinba selenium mushroom raw material into powder for later use.

[0049] S1-2, mixing Qinba selenium mushroom powder with water and heating, maintaining at 100° C. for 2.5 to 3.5 hours, filtering repeatedly and concentrating the filtrate to obtain a concentrated solution.

[0050] Preferably, the mass volume ratio of Qinba selenium mushroom powder to water is 2-3g:300-400ml.

[0051] Specifically, in one embodiment of the present invention, 4000 ml of water is added to every 30 g of Qinba selenium mushroom polysaccharide powder, and the mixture is extracted with boiling water for 3 hours. The extract is filtered with gauze, and the filter residue is subjected to the above operation for 3 times, and impurities are removed. The filtrate is then concentrated to obtain a polysaccharide concentrate, which is stored overnight in a refrigerator at 4°C.

[0052] S1-3. After centrifuging the concentrated solution, add 95% by volume ethanol, centrifuge and collect the precipitate, and dry to obtain the crude polysaccharide of Qinba selenium mushroom; the volume ratio of the concentrated solution to ethanol is 1:4-5.

[0053] Preferably, in one embodiment of the present invention, the concentrate is centrifuged at 4500 rpm for 10 min to remove the bottom precipitate, then 4 times the volume of 95% ethanol is added while vigorously stirring, and then after flocs appear, centrifugation is performed at 7500 rpm for 15 min, the precipitate is collected and dried at 60°C to precipitate the polysaccharide, and then centrifuged and dried to obtain crude polysaccharide.

[0054] S2, microfiltration and ultrafiltration combined filtration; this step can effectively remove impurities in the crude polysaccharide, so that the final extraction and purification effect is greatly improved.

[0055] S2-1. Centrifuge the thawed solution several times at 7000-7500 rpm, with each centrifugation time being 10-20 min, to obtain the supernatant.

[0056] Preferably, the thawing solution is prepared from crude polysaccharide of Qinba selenium mushroom and water, and the mass volume ratio of the two is 1g:3-5ml.

[0057] In one embodiment of the present invention, the Qinba selenium mushroom crude polysaccharide was repeatedly frozen and thawed, 30g of Qinba selenium mushroom polysaccharide crude particles were weighed into a beaker, 120mL of pure water was added, stirred to dissolve, and frozen at -20°C, taken out after being completely frozen, thawed at room temperature, centrifuged at 7500rpm for 15min, and the supernatant was separated, and the previous operation was repeated until no precipitation was precipitated.

[0058] S2-2, sequentially using at least two microfiltration membranes to microfilter the supernatant obtained in step S2-1 to obtain a microfiltrate.

[0059] Preferably, there are three types of microfiltration membranes, namely 0.8 μm microfiltration membrane, 0.45 μm microfiltration membrane and 0.22 μm microfiltration membrane.

[0060] S2-3, firstly ultrafilter the microfiltrate with an ultrafiltration membrane, and then filter with a microfiltration membrane to obtain an ultrafiltrate.

[0061] Preferably, the ultrafiltration membrane is an ultrafiltration membrane with a molecular weight of 100 kDa, and the microfiltration membrane is a 0.22 μm microfiltration membrane.

[0062] S2-3. Detect the ionic strength of the ultrafiltrate.

[0063] Preferably, when the ionic strength of the ultrafiltrate is lower than 10Μs / cm, the microfiltration-ultrafiltration combined filtration is completed; when the ionic strength of the ultrafiltrate is higher than 10Μs / cm, the step S2-2 is repeated until the ionic strength of the ultrafiltrate is lower than 10Μs / cm.

[0064] S3, ion purification:

[0065] The crude extracellular polysaccharide of Qinba selenium mushroom is prepared into a solution with a mass concentration of 20 mg / mL, and then ion purification is performed using a diethylaminoethyl cellulose ion exchange chromatography column; the eluents for ion purification are deionized water, 0.1 mol / L NaCl solution and 0.2 mol / L NaCl solution in sequence; the loading amount of the ion purification is 1 mL, and the flow rate is 0.25 mL / min. The above-mentioned concentration gradient eluent can improve the polysaccharide extraction rate, reduce energy consumption, and shorten the extraction time.

[0066] S4. Gel purification:

[0067] The eluate obtained from ion purification was filtered and then subjected to gel purification; the filler of the gel column for gel purification was SephacryS-200, with a specification of 16mm×100cm; the loading amount of gel purification was 1mL / each time, the eluent was deionized water, and the elution flow rate was 1mL / min.

[0068] Preferably, the method of the present invention further comprises a step of determining the polysaccharide content by phenol-sulfuric acid method. In order to ensure the effect of the extraction and purification method, a determination can be performed after each extraction step to detect the effect of the extraction and purification process.

[0069] The Qinba selenium mushroom polysaccharide of the present invention is specifically a polysaccharide-β-glucan, so glucose can be used as a standard when quantifying it. In the method of the present invention, in order to quantitatively detect the content of Qinba selenium mushroom polysaccharide in the obtained extract, a standard curve of its absorbance value and concentration can be drawn using multiple concentration gradient glucose standard solutions in each step, and the extract is also subjected to absorbance detection, so that the content of the polysaccharide component in the extract can be calculated through the standard curve.

[0070] The present invention also provides a kind of Qinba selenium mushroom polysaccharide, which is prepared by adopting the above method.

[0071] The present invention also provides an application of the Qinba selenium mushroom polysaccharide as described above, which can be used to prepare a preparation for intervening in acute myeloid leukemia.

[0072] The present invention is described below by specific embodiments:

[0073] Example 1 Water extraction and alcohol precipitation

[0074] This example uses water extraction and alcohol precipitation to prepare crude polysaccharides, and the specific preparation steps are as follows:

[0075] (1) Pretreatment of Qinba selenium mushroom powder: Take Qinba selenium mushroom raw material and place it in a grinder for grinding to obtain 30g of Qinba selenium mushroom fine powder for later use.

[0076] (2) Water extraction: Add 4000 ml of water to every 30 g of Qinba selenium mushroom polysaccharide powder, extract with boiling water for 3 hours, filter the extract with gauze, repeat the above operation on the residue, repeat the operation 3 times, remove impurities, and then concentrate the filtrate to obtain 30 ml of polysaccharide concentrate. After standing overnight, store it in a refrigerator at 4°C.

[0077] (3) Alcohol precipitation and drying: The concentrate was centrifuged at 4500 rpm for 10 min to remove the bottom precipitate, and then 4 times the volume of 95% ethanol was added while vigorously stirring. After flocs appeared, the concentrate was centrifuged at 7500 rpm for 15 min. The precipitate was collected and dried at 60°C to precipitate the polysaccharide. After centrifugation and drying, the crude polysaccharide was obtained.

[0078] The polysaccharide content was determined by the phenol-sulfuric acid method, and the standard curve was drawn: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, and 1.0 mL of 100 μg / mL glucose standard solution were respectively drawn into stoppered glass test tubes, and pure water was added to 1.0 mL. After vortex mixing, 0.5 mL of 5% phenol solution was added, and after mixing, 2.5 mL of concentrated sulfuric acid was added, and the mixture was quickly mixed. The mixture was placed in a 100°C water bath for 15 min, and after cooling, 200 μL of the sample was drawn and placed in an enzyme labeling plate strip, and the absorbance value at a wavelength of 490 nm was measured. The standard curve was drawn with the glucose concentration and the A490 absorbance value as the ordinate, as shown in FIG. Figure 2 shown.

[0079] The method for determining the crude polysaccharide content is to weigh 2.55 mg of the sample, dissolve it in pure water and dilute it to 100 μg / mL, take 1 mL of the diluted crude polysaccharide, and analyze the absorbance value at a wavelength of 490 nm using the same method as the above-mentioned standard, and calculate the polysaccharide content in the sample using the standard curve.

[0080] For comparative verification, this example is recorded as JXY crude polysaccharide (without impurities removed) and the sugar content therein is measured, and after the measurement, the steps of Example 2 are continued.

[0081] Example 2 Microfiltration and ultrafiltration combined filtration

[0082] In this example, the crude polysaccharide obtained in Example 1 was subjected to microfiltration and ultrafiltration to remove impurities, thereby obtaining the Qinba selenium mushroom extracellular crude polysaccharide.

[0083] The polysaccharide content was determined by the phenol-sulfuric acid method, and the standard curve was drawn: 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of 100 μg / mL glucose standard solution were respectively drawn into stoppered glass test tubes, and pure water was added to 1.0 mL. After vortex mixing, 0.5 mL of 5% phenol solution was added, and after mixing, 2.5 mL of concentrated sulfuric acid was added, and the mixture was quickly mixed, and a 100°C water bath was placed for 15 min. After cooling, the mixture was mixed and 200 μL of the sample was drawn and placed in an enzyme labeling plate strip, and the absorbance value at a wavelength of 490 nm was measured. The standard curve was drawn with the glucose concentration and the A490 absorbance value as the ordinate, as shown in FIG. Figure 3 shown.

[0084] The method for determining the polysaccharide content in the crude polysaccharide of Qinba selenium mushroom is to weigh 2.55 mg of the sample, dissolve it in pure water and dilute it to 100 μg / mL, take 1 mL of the diluted crude polysaccharide of Qinba selenium mushroom, and analyze the absorbance value at a wavelength of 490 nm in the same way as the above-mentioned standard, and calculate the polysaccharide content in the crude polysaccharide of Qinba selenium mushroom through the standard curve.

[0085] (1) After repeated freezing and thawing, weigh 30 g of crude granules of Qinba selenium mushroom polysaccharide into a beaker, add 120 mL of pure water, stir to dissolve, freeze at -20°C, take out after being completely frozen, thaw at room temperature, centrifuge at 7500 rpm for 15 min, separate the supernatant, and repeat the previous operation until no precipitate is precipitated.

[0086] (2) Microfiltration and ultrafiltration: The supernatant was filtered through 0.8 μm, 0.45 μm, and 0.22 μm filter membranes, and pure water was added. The filtrate was further purified by an ultrafiltration membrane with a molecular weight of 100 kDa, and then filtered through a 0.22 μm filter membrane and stored in a refrigerator at 4°C. The above operation was repeated until the ionic strength of the ultrafiltration effluent reached below 10 Μs / cm, and the filtrate was collected and freeze-dried to obtain the crude extracellular polysaccharide of Qinba selenium mushroom, which was frozen for later use.

[0087] For comparison, the crude extracellular polysaccharide of Oyster mushroom obtained in this example was recorded as JXY crude polysaccharide (impurities removed), and the purity was measured, followed by further ion purification.

[0088] Example 3 Ion Purification

[0089] In this example, the crude extracellular polysaccharide of Selenium qinbaensis obtained in Example 2 after removing impurities was used for ion purification, and the specific process was as follows:

[0090] The crude extracellular polysaccharide of Qinba selenium mushroom was prepared into a solution with a concentration of 10 mg / mL, and the crude extracellular polysaccharide solution of Qinba selenium mushroom was purified using a diethylaminoethyl cellulose ion exchange chromatography column (0.45 μm, 26 mm×100 cm). The specific purification process was as follows:

[0091] The crude extracellular polysaccharide solution of Qinba selenium mushroom was centrifuged at 10000 rpm for 20 min, and the supernatant was taken. 40 mL was added to the sample loop for loading, and ion exchange chromatography was performed, and 5 mL was injected into the sample loop each time.

[0092] The chromatographic column was subjected to linear gradient elution, and the eluted solutions were numbered respectively, and the numbers were all odd. Among them, deionized water was used for tubes 1-29, 0.1 mol / L NaCl solution was used for tubes 31-51, 0.2 mol / L NaCl solution was used for tubes 53-69, and 0.3 mol / L NaCl solution was used for tubes 71-87. The elution mode was automatic injection, and the concentrate was prepared into a solution with a concentration of 20 mg / mL, the sample volume was 1 mL, the flow rate was 0.25 mL / min, and 2 mL was collected in each tube.

[0093] The phenol-sulfuric acid method was used to detect the sugar-containing test tubes. The test method was to add 200 μL of 5% phenol solution and mix well, then add 0.5 mL of concentrated sulfuric acid and mix well, place at room temperature for 15 minutes, and filter the Qinba selenium mushroom polysaccharide filtrate using a Sephadex G100 gel filtration chromatography column (0.45 μm). The absorbance value of each tube solution was measured at 490 nm, and the polysaccharide ion purification curve was drawn with the absorbance value as the ordinate and the number of tubes as the abscissa, as shown in the figure. Figure 4 The same process as the sample treatment was performed using glucose solutions with multiple concentration gradients, and the obtained results were plotted as the glucose standard curve of this embodiment, as shown in FIG. Figure 5 shown.

[0094] according to Figure 4 It can be seen that the curve contains three peaks, which appear when eluting with deionized water, 0.1 mol / L NaCl solution and 0.2 mol / L NaCl solution, respectively. This shows that a large amount of polysaccharides are eluted. However, when eluting with 0.3 mol / L NaCl solution, there is almost no peak, indicating that the elution of polysaccharides by NaCl solution of this concentration is not obvious.

[0095] The solutions in the tubes corresponding to each peak were collected and combined. Specifically, the 37th to 43rd tubes were combined, the 9th to 21st tubes were combined, the 59th to 63rd tubes were combined, and the above three parts were further combined, which was recorded as JXY-D.

[0096] The polysaccharide content in JXY-D was calculated using the glucose standard curve of this example, and then the gel purification was continued.

[0097] Example 4 Gel Purification

[0098] The Qinba selenium mushroom polysaccharide sample obtained by ion exchange chromatography was made into a 20 mg / mL solution with 0.22 μm water film deionized water, and separated and purified using the FPLC chromatography system of Amersham bioscience. The filler was SephacryS-200 gel column (16 mm × 100 cm), the sample volume was 1 mL each time, and eluted with 0.22 μm water film deionized water. The elution flow rate was 1 mL / min, and the samples were connected by tubes, and 2.5 mL was collected in each tube. Specifically, the eluted solutions were renumbered, and the numbers were all odd numbers from 1 to 57. The eluent was detected by a RID-10A differential refractometer, and the absorbance value of each tube solution was measured at 490 nm. The polysaccharide ion purification curve was drawn with the absorbance value as the vertical coordinate and the number of tubes as the horizontal coordinate, as shown in FIG. Figure 6 The same process as the sample treatment was performed using glucose solutions with multiple concentration gradients, and the obtained results were plotted as the glucose standard curve of this embodiment, as shown in FIG. Figure 7shown.

[0099] according to Figure 6 It can be seen that the curve contains a peak, and the solutions in the corresponding tubes are collected and merged. The specific merging method is to merge tubes 35 to 41, and the merged solution is recorded as JXY-N. JXY-N is concentrated by vacuum decompression, dialyzed for desalination, freeze-dried, and then diluted 50 times. The absorbance value of the diluted solution is further measured, and the content of Qinba selenium mushroom polysaccharide is calculated according to the standard curve. The measurement result is, and then the elution results of Qinba selenium mushroom polysaccharide by gel chromatography column chromatography are obtained.

[0100] The measurement results of Examples 1 to 4 are shown in Tables 1 and 2.

[0101] Table 1 Purity and quality of polysaccharides

[0102]

[0103] Table 2 Polysaccharide extraction and purification yield

[0104]

[0105] By comparing Table 1 and Table 2, it can be seen that the purity of crude polysaccharide extraction without impurity removal is only 18.4%, while after impurity removal, the purity of polysaccharide can be increased to 73.4%, which is significantly improved.

[0106] On the basis of the above, ion purification and gel purification were continued to be used to further improve the extraction effect of the Brazilian polysaccharide. Finally, 1.406g of polysaccharide can be extracted using 30g of Qinba selenium mushroom, and the yield can reach 4.69%.

[0107] Example 5 Application of Qinba Selenium Mushroom Polysaccharide in Intervention of Acute Myeloid Leukemia

[0108] This example verifies the application of the Qinba selenium mushroom polysaccharide prepared in Example 4.

[0109] Qinba selenium mushroom polysaccharide intervenes in acute myeloid leukemia network pharmacology Figure 8 As shown, Figure 8 A in the middle is the potential active ingredient-target network; Figure 8 B is a protein interaction network diagram. Qinba selenium mushroom polysaccharide intervenes in the biological process of inflammatory factors regulating the immune microenvironment-related targets of acute myeloid leukemia, enrichment analysis and pathway analysis are shown in Figure 1. Fig. 9 As shown, Fig. 9 A and B in the middle are GO enrichment analysis bar graphs and BP (biological process), CC (cellular component), and MF (molecular function); Fig. 9C and D in the middle are KEGG enrichment analysis pathway bubble diagrams. The size of the circle represents the gene data enriched in the corresponding pathway, and the change from green to red represents a gradually decreasing P value.

[0110] The prediction results show that Qinba selenium mushroom polysaccharide can cause pyroptosis of leukemia cells and exert an anti-tumor effect.

[0111] It has been found that the Chinese medicine Qinba selenium mushroom monomer and compound preparations have anti-tumor effects with diverse mechanisms. They not only kill leukemia cells, but also have multiple functions such as regulating immunity, anti-oxidation, free radical scavenging, and enhancing the sensitivity of chemotherapy drugs. Therefore, better extraction and preparation of Qinba selenium mushroom polysaccharides have good application prospects in clinical applications.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting and purifying polysaccharides from Qinba selenium mushroom, characterized in that: The method comprises water extraction and alcohol precipitation, microfiltration and ultrafiltration combined filtration, ion purification and gel purification to obtain the Qinba selenium mushroom polysaccharide; the microfiltration and ultrafiltration combined filtration comprises the following steps: The crude polysaccharide of Qinba selenium mushroom prepared by the water extraction and alcohol precipitation is dissolved and frozen after repeated freezing and thawing, the thawed solution is centrifuged and the supernatant is retained; the supernatant is microfiltered and ultrafiltered in sequence, the filtrate is collected and dried to obtain the crude extracellular polysaccharide of Qinba selenium mushroom.

2. The method for extracting and purifying polysaccharides from Selenium chinense mushroom according to claim 1, characterized in that: The microfiltration and ultrafiltration combined filtration comprises the following steps: S2-1, centrifuging the thawed solution at 7000-7500 rpm for multiple times, each centrifugation time being 10-20 min, and obtaining the supernatant after each centrifugation for the next centrifugation; S2-2, sequentially performing microfiltration on the supernatant obtained in step S2-1 using at least two microfiltration membranes to obtain a microfiltrate; S2-3, firstly ultrafiltering the microfiltrate using an ultrafiltration membrane, and then filtering using a microfiltration membrane to obtain an ultrafiltrate; S2-4, detecting the ionic strength of the ultrafiltrate.

3. The method for extracting and purifying Qinba selenium mushroom polysaccharide according to claim 2, characterized in that: When the ionic strength of the ultrafiltrate is lower than 10Μs / cm, the microfiltration-ultrafiltration combined filtration is completed; when the ionic strength of the ultrafiltrate is higher than 10Μs / cm, the step S2-2 is repeated until the ionic strength of the ultrafiltrate is lower than 10Μs / cm.

4. The method for extracting and purifying Qinba selenium mushroom polysaccharide according to claim 2, characterized in that: In the step S2-2, there are three types of microfiltration membranes, namely, 0.8 μm microfiltration membrane, 0.45 μm microfiltration membrane and 0.22 μm microfiltration membrane.

5. The method for extracting and purifying polysaccharide from Qinba selenium mushroom according to claim 2, characterized in that: In the step S2-3, the ultrafiltration membrane is an ultrafiltration membrane with a molecular weight of 100 kDa, and the microfiltration membrane is a 0.22 μm microfiltration membrane.

6. The method for extracting and purifying Qinba selenium mushroom polysaccharide according to any one of claims 1 to 5, characterized in that: The water extraction and alcohol precipitation comprises the following steps: S1-1, obtaining Qinba selenium mushroom powder; S1-2, mixing the Qinba selenium mushroom powder with water and heating, maintaining at 100° C. for 2.5 to 3.5 hours, and concentrating the filtrate after repeated filtration to obtain a concentrated solution; S1-3, after centrifuging the concentrated solution, adding 95% by volume ethanol, centrifuging and collecting the precipitate, and drying to obtain the Qinba selenium mushroom crude polysaccharide; the volume ratio of the concentrated solution to the ethanol is 1:4-5.

7. The method for extracting and purifying Qinba selenium mushroom polysaccharide according to claim 6, characterized in that: The crude extracellular polysaccharide of Qinba selenium mushroom is prepared into a solution with a mass concentration of 20 mg / mL, and then a diethylaminoethyl cellulose ion exchange chromatography column is used for the ion purification; the eluents for the ion purification are deionized water, 0.1 mol / L NaCl solution and 0.2 mol / L NaCl solution, respectively; the loading amount for the ion purification is 1 mL, and the flow rate is 0.25 mL / min.

8. The method for extracting and purifying Qinba selenium mushroom polysaccharide according to claim 7, characterized in that: The eluate obtained from the ion purification is filtered and then subjected to the gel purification; the filler of the gel column for the gel purification is SephacryS-200, with a specification of 16mm×100cm; the loading amount of the gel purification is 1mL / each time, the eluent is deionized water, and the elution flow rate is 1mL / min.

9. A Qinba selenium mushroom polysaccharide, characterized in that: The method is prepared by any one of claims 1 to 8.

10. An application of Qinba selenium mushroom polysaccharide as claimed in claim 9, characterized in that: The invention can be used for preparing preparations for intervening in acute myeloid leukemia.

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