A method for efficiently extracting plant polysaccharides

Abstract

The invention relates to the technical field of plant polysaccharide extraction, and is used to solve the problem that impurities such as inorganic salts, proteins, pigments and small molecules are often present in the existing eucommia plant polysaccharide extracts, which have an adverse effect on subsequent polysaccharide purification and refining steps. The invention specifically relates to a method for efficiently extracting plant polysaccharides. In the method, polysaccharides in eucommia leaf dry powder are extracted by ultrasonic treatment, and the extraction efficiency is high and the effect is good, so as to obtain crude plant polysaccharides. Then, the crude plant polysaccharides are dissolved, and a purification adsorbent is used to adsorb the crude plant polysaccharide solution. The purification adsorbent has excellent adsorption and impurity removal effect, is easy to use, improves the efficiency of extracting plant polysaccharides, fully removes impurities in the crude plant polysaccharides, and has almost no loss of polysaccharides, and improves the quality of the extracted plant polysaccharides.

Description

Technical Field

[0001] The invention relates to the technical field of plant polysaccharide extraction, and in particular to a method for efficiently extracting plant polysaccharides. Background Art

[0002] Polysaccharides are one of the main active and index components of plants, and are widely present in various parts and organs of plants. The development of efficient plant polysaccharide extraction technology is one of the hot spots in the research and development of natural products. At present, the main method of plant polysaccharide extraction is water extraction or combined with microwave, ultrasound, subcritical, high-speed shear, high-pressure pulse and other means to assist water extraction. Eucommia ulmoides, belonging to the Eucommia genus of the Eucommia family, is a tonic medicinal material with a wide range of applications in functional foods and medicine. A large number of studies have shown that Eucommia leaves contain a variety of medicinal ingredients and nutrients, mainly chemical components such as flavonoids and polysaccharide compounds. Existing studies have shown that Eucommia leaf polysaccharides have antihypertensive, lipid-lowering and blood sugar-lowering effects, anti-tumor effects, and anti-osteoporosis effects.

[0003] In plant polysaccharide extracts, there are often impurities such as inorganic salts, proteins, pigments and small molecules, which will have an adverse effect on the subsequent polysaccharide purification and refining steps. Among them, the molecular weight of protein is similar to that of polysaccharides and is difficult to separate. Therefore, it is the main impurity in polysaccharide extracts and also the technical difficulty of polysaccharide purification. Currently, the commonly used protein removal methods mainly include Sevage method and enzyme method, but the Sevage method uses toxic solvents such as chloroform, and the enzyme method (mostly using imported pectinase) is costly and not suitable for industrial production. The protein removal rate is generally less than 80%. Even if it exceeds 80%, the polysaccharide loss rate is also high, which is usually above 30%. In addition, there are disadvantages such as cumbersome operation and long time consumption, and some methods have harmful solvent residues.

[0004] How to improve the impurities such as inorganic salts, proteins, pigments and small molecules in the polysaccharide extracts of Eucommia ulmoides, which often have an adverse effect on the subsequent polysaccharide purification and refining steps, is the key to the present invention. Therefore, a method for efficiently extracting plant polysaccharides is urgently needed to solve the above problems. Summary of the invention

[0005] In order to overcome the above-mentioned technical problems, the purpose of the present invention is to provide a method for efficiently extracting plant polysaccharides, which solves the problem that the existing Eucommia plant polysaccharide extracts often contain impurities such as inorganic salts, proteins, pigments and small molecules, which will have an adverse effect on the subsequent polysaccharide purification and refining steps.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A method for efficiently extracting plant polysaccharides comprises the following steps:

[0008] Step 1: add eucommia leaf dry powder and deionized water that have passed through a 40-50 mesh sieve into a single-necked flask, perform ultrasonic treatment for 25-50 minutes at a temperature of 50-60°C and an ultrasonic power of 350-400W, centrifuge the reaction product at a centrifugal speed of 4500-6000r / min after the reaction, take the supernatant and add it to an ethanol solution for alcohol precipitation, wash the precipitate with anhydrous ethanol and acetone for 2-3 times in sequence, and then place it in a vacuum drying oven and dry it at a temperature of 30-35°C for 3-5 hours to obtain crude plant polysaccharides;

[0009] Step 2: Add crude plant polysaccharides and deionized water to a three-necked flask equipped with a stirrer and a thermometer, stir and mix for 15-20 minutes at a temperature of 60-70°C and a stirring rate of 400-500r / min, then adjust the pH to 5.5-6.5 with a hydrochloric acid solution, then add a purification adsorbent and continue stirring and mixing for 30-40 minutes, and after the reaction is completed, cool the reaction product to room temperature, then use a magnet to remove the purification adsorbent, take the supernatant and add it to an ethanol solution for alcohol precipitation, wash the precipitate with anhydrous ethanol for 2-3 times, and then place it in a vacuum drying oven and dry it at a temperature of 25-35°C for 6-8 hours to obtain plant polysaccharides.

[0010] As a further solution of the present invention: the dosage ratio of the Eucommia leaf dry powder and deionized water in step 1 is 1g:15-30mL, and the volume fraction of the ethanol solution is 95%.

[0011] As a further solution of the present invention: the usage ratio of the crude plant polysaccharide, deionized water and purification adsorbent in step 2 is 10g:500-1000mL:0.1-1.0g, the mass fraction of the hydrochloric acid solution is 15-20%, and the volume fraction of the ethanol solution is 95%.

[0012] As a further solution of the present invention: the purification adsorbent is prepared by the following steps:

[0013] A1: Wash the diatomaceous earth with distilled water for 3-5 times, then place it in a vacuum drying oven, dry it at a temperature of 100-105°C for 2-3 hours, then grind it and pass it through a 60-100 mesh sieve to obtain pretreated diatomaceous earth;

[0014] A2: adding the pretreated diatomaceous earth into deionized water, and then ultrasonically treating it for 10-15 minutes at a temperature of 25-30°C and an ultrasonic frequency of 45-55kHz to disperse it evenly, thereby obtaining a diatomaceous earth dispersion;

[0015] A3: Add the polysaccharide clarifier and acetic acid solution into a three-necked flask equipped with a stirrer, a thermometer and a constant pressure dropping funnel, stir and mix for 20-30 minutes at a temperature of 45-50°C and a stirring rate of 400-500 r / min to obtain a clarifier solution;

[0016] A4: Add ferroferric oxide to deionized water, and then ultrasonically treat for 15-20 minutes at a temperature of 25-30°C and an ultrasonic frequency of 45-55kHz to obtain a magnetic fluid.

[0017] A5: Add diatomaceous earth dispersion, clarifier solution and magnetic fluid into a three-necked flask equipped with a stirrer and a thermometer, stir and react for 3-5 hours at a temperature of 60-70°C and a stirring rate of 400-500r / min. After the reaction, cool the reaction product to room temperature, then centrifuge at a centrifugal rate of 4500-6000r / min, wash the precipitate with distilled water for 2-3 times, then place it in a vacuum drying oven, dry it at a temperature of 60-65°C for 3-5 hours, then grind it and pass it through a 60-100 mesh sieve to obtain a purified adsorbent.

[0018] As a further solution of the present invention: the dosage ratio of the pretreated diatomaceous earth and deionized water in step A2 is 1 g: 10-15 mL.

[0019] As a further solution of the present invention: the usage ratio of the polysaccharide clarifier and the acetic acid solution in step A3 is 3-5 g:40-50 mL, and the mass fraction of the acetic acid solution is 2-3%.

[0020] As a further solution of the present invention: the dosage ratio of the ferrosoferric oxide and deionized water in step A4 is 1 g: 10-15 mL.

[0021] As a further solution of the present invention: the usage ratio of the diatomaceous earth dispersion, the clarifier solution and the magnetic fluid in step A5 is 10-15 mL: 5-25 mL: 10-15 mL.

[0022] As a further solution of the present invention: the polysaccharide clarifier is prepared by the following steps:

[0023] B1: o-chloroaniline and sulfuric acid solution are added to a three-necked flask equipped with a stirrer and a thermometer, and stirred for reaction at a temperature of 90-100°C and a stirring rate of 400-500r / min for 30-40min, then cooled to 25-30°C, and then added with paraformaldehyde and continued to stir for reaction for 1-2h, and then heated to 80-85°C while stirring, and the heating rate was controlled to be 1-2°C / min, and then stirred for reaction at a constant temperature for 2-3h. After the reaction, deionized water is added to the reaction product, and the temperature is maintained at 75-80°C. Then, the pH is adjusted to 5-6 with sodium hydroxide solution, and a precipitate is precipitated, and then vacuum filtered, and the filter cake is washed with distilled water for 2-3 times, and then placed in a vacuum drying oven, and dried at a temperature of 60-65°C for 2-3h to obtain intermediate 1;

[0024] The reaction principle is as follows:

[0025]

[0026] B2: Add intermediate 1, epichlorohydrin, methyl isobutyl ketone and deionized water to a three-necked flask equipped with a stirrer, a thermometer and a constant pressure dropping funnel, stir and react for 8-10 hours at a temperature of 75-80°C and a stirring rate of 400-500r / min, then cool to 30-40°C, then add benzyltriethylammonium chloride solution dropwise while stirring, and control the dropping rate to 1-2 drops / s, then add sodium hydroxide solution dropwise while stirring, and control the dropping rate to 1-2 drops / s, and continue to stir and react for 1-2 hours under the condition of heating to 80-85°C after the addition is complete. After the reaction is completed, cool the reaction product to room temperature, let it stand for stratification, introduce carbon dioxide into the organic phase until saturated, then wash with sodium chloride solution 2-3 times, then rotary evaporate to remove the solvent, then filter while hot, collect the filtrate to obtain intermediate 2;

[0027] The reaction principle is as follows:

[0028]

[0029] B3: Chitosan and acetic acid solution were stirred and mixed to obtain chitosan solution, sodium hydroxide solution was added to a three-necked flask equipped with a stirrer, a thermometer and a constant pressure dropping funnel, and the chitosan solution was added dropwise to the three-necked flask while stirring at a temperature of 25-30°C and a stirring rate of 400-500r / min, and the dropping rate was controlled to be 1-2 drops / s. After the dropwise addition was completed, the stirring reaction was continued for 15-20 minutes. After the reaction was completed, the reaction product was vacuum filtered, and the filter cake was added to a copper sulfate solution, and then stirred for 20-30 minutes, and then allowed to stand for 1-2 hours, and then vacuum filtered. The filter cake was washed with distilled water and anhydrous ethanol for 2-3 times in sequence, and then placed in a vacuum drying oven, and dried at a temperature of 50-55°C for 1-2 hours to obtain intermediate 3;

[0030] The reaction principle is as follows:

[0031]

[0032] B4: Add intermediate 2, intermediate 3, deionized water, isopropanol and sodium hydroxide solution into a three-necked flask equipped with a stirrer and a thermometer, stir and react for 80-100 min at a temperature of 45-50°C and a stirring rate of 400-500 r / min. After the reaction is completed, vacuum filter the reaction product, wash the filter cake with distilled water, anhydrous ethanol and acetone for 2-3 times in sequence, and then place it in a vacuum drying oven and dry it at a temperature of 50-55°C for 1-2 h to obtain intermediate 4;

[0033] The reaction principle is as follows:

[0034]

[0035] B5: Add intermediate 4 and hydrochloric acid solution into a single-necked flask, perform ultrasonic treatment for 10-15 min at a temperature of 45-50°C and an ultrasonic frequency of 45-55kHz. After the reaction is completed, vacuum filter the reaction product, wash the filter cake with distilled water until it is neutral, and then place it in a vacuum drying oven and dry it at a temperature of 50-55°C for 1-2 hours to obtain a polysaccharide clarifier.

[0036] The reaction principle is as follows:

[0037]

[0038] As a further scheme of the present invention: the dosage ratio of the o-chloroaniline, sulfuric acid solution, paraformaldehyde and deionized water in step B1 is 0.1 mol: 50-60 mL: 0.05-0.055 mol: 300-500 mL, the mass fraction of the sulfuric acid solution is 25-30%, the molecular weight of the paraformaldehyde is 90.08, and the mass fraction of the sodium hydroxide solution is 25-30%.

[0039] As a further scheme of the present invention: the dosage ratio of the intermediate 1, epichlorohydrin, methyl isobutyl ketone, deionized water, benzyltriethylammonium chloride solution and sodium hydroxide solution in step B2 is 10mmol:40-50mmol:80-100mL:25-35mL:18-26mL:25-30mL, the mass fraction of the benzyltriethylammonium chloride solution is 50-55%, the mass fraction of the sodium hydroxide solution is 35-40%, and the mass fraction of the sodium chloride solution is 5-8%.

[0040] As a further solution of the present invention: the amount ratio of the chitosan, acetic acid solution, sodium hydroxide solution and copper sulfate solution in step B3 is 3g:12-15mL:150-200mL:20-30mL, the mass fraction of the acetic acid solution is 2-3%, the mass fraction of the sodium hydroxide solution is 10-15%, and the mass fraction of the copper sulfate solution is 8-10%.

[0041] As a further solution of the present invention: the usage ratio of the intermediate 2, intermediate 3, deionized water, isopropanol and sodium hydroxide solution in step B4 is 1-5mL: 1g: 20-25mL: 3-5mL: 3-5mL, and the mass fraction of the sodium hydroxide solution is 5-10%.

[0042] As a further solution of the present invention: the usage ratio of the intermediate 4 and the hydrochloric acid solution in step B5 is 1 g: 15-20 mL, and the molar concentration of the hydrochloric acid solution is 1-1.2 mol / L.

[0043] Beneficial effects of the present invention:

[0044] The invention discloses a method for efficiently extracting plant polysaccharides. The method comprises the following steps: adding eucommia leaf dry powder and deionized water to a single-necked flask for ultrasonic treatment, centrifuging the reaction product, taking the supernatant for alcohol precipitation, washing the precipitate, and drying to obtain plant crude polysaccharides; adding the plant crude polysaccharides and deionized water to a three-necked flask for stirring and mixing, adjusting the pH value with a hydrochloric acid solution, adding a purification adsorbent, and continuing to stir and mix; cooling the reaction product to room temperature after the reaction is completed, removing the purification adsorbent with a magnet, taking the supernatant for alcohol precipitation, washing the precipitate, and drying to obtain plant polysaccharides; in the method, the polysaccharides in the eucommia leaf dry powder are extracted by ultrasonic treatment, the extraction efficiency is high and the effect is good, the plant crude polysaccharides are obtained, the plant crude polysaccharides are dissolved, the plant crude polysaccharide solution is adsorbed by the purification adsorbent, impurities in the plant crude polysaccharides are removed, and there is almost no loss of polysaccharides, the polysaccharide retention rate is more than 98%, the protein removal rate is more than 87%, and the solid removal rate is more than 31%;

[0045] In the process of extracting plant polysaccharides, a purification adsorbent is first prepared, o-chloroaniline is reacted with polyformaldehyde to generate intermediate 1 containing amino groups and chlorine atoms, and then the chlorine atoms on epichlorohydrin are used to undergo affinity substitution reaction with the amino groups on intermediate 1 to introduce epoxy groups to obtain intermediate 2, and chitosan is dissolved and added to a copper sulfate solution so that the amino groups on chitosan form a complex with copper ions to obtain intermediate 3, and since C6-OH on intermediate 3 is more active than C3-OH, it preferentially undergoes nucleophilic substitution reaction with the chlorine atoms on intermediate 2 to obtain intermediate 4, and then intermediate 4 removes copper ions under acidic conditions to obtain a polysaccharide clarifier, and then the polysaccharide clarifier, diatomaceous earth and ferrous oxide form a loading agent. The magnetic diatomaceous earth of the polysaccharide clarifier is used to obtain a purification adsorbent. The chitosan molecules of the polysaccharide clarifier in the purification adsorbent contain a large amount of -OH and -NH2, which can adsorb substances such as protein, gelatin, and polyphenols. In addition, a large amount of epoxy groups on the chitosan molecules can undergo SN2 ring-opening reactions with nucleophilic groups such as amino, hydroxyl, and thiol groups on protein molecules, thereby being able to fully adsorb impurities in the polysaccharide. The diatomaceous earth in the purification adsorbent has excellent adsorption and decolorization effects, which further enhances its decolorization and impurity removal effects. The ferroferric oxide in the purification adsorbent makes it magnetic, which is convenient for separation, recovery, and recycling. The purification adsorbent has excellent adsorption and impurity removal effects and is easy to use, thereby improving the efficiency of extracting plant polysaccharides and the quality of the extracted plant polysaccharides. DETAILED DESCRIPTION

[0046] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0047] Embodiment 1:

[0048] This embodiment is a method for preparing a polysaccharide clarifier, comprising the following steps:

[0049] B1: 0.1 mol o-chloroaniline and 50 mL of 25% sulfuric acid solution were added to a three-necked flask equipped with a stirrer and a thermometer, and stirred for 30 min at 90°C and 400 r / min, then cooled to 25°C, and then 0.05 mol of polyformaldehyde with a molecular weight of 90.08 was added and stirred for 1 h, and then heated to 80°C while stirring, and the heating rate was controlled to be 1°C / min, and then stirred for 2 h at a constant temperature. After the reaction, 300 mL of deionized water was added to the reaction product, and the temperature was maintained at 75°C. Then, the pH was adjusted to 5 with 25% sodium hydroxide solution to precipitate, and then vacuum filtered. The filter cake was washed twice with distilled water, and then placed in a vacuum drying oven and dried at 60°C for 2 h to obtain intermediate 1;

[0050] B2: 10 mmol of intermediate 1, 40 mmol of epichlorohydrin, 80 mL of methyl isobutyl ketone and 25 mL of deionized water were added to a three-necked flask equipped with a stirrer, a thermometer and a constant pressure dropping funnel, and stirred for 8 h at a temperature of 75 ° C and a stirring rate of 400 r / min, then cooled to 30 ° C, and then 18 mL of 50% benzyltriethylammonium chloride solution was added dropwise while stirring, and the dropping rate was controlled to 1 drop / s, and then 25 mL of 35% sodium hydroxide solution was added dropwise while stirring, and the dropping rate was controlled to 1 drop / s. After the addition was completed, the temperature was raised to 80 ° C and the stirring reaction was continued for 1 h. After the reaction was completed, the reaction product was cooled to room temperature, allowed to stand and stratified, carbon dioxide was introduced into the organic phase until saturated, and then washed twice with 5% sodium chloride solution, and then the solvent was removed by rotary evaporation, and then filtered while hot, and the filtrate was collected to obtain intermediate 2;

[0051] B3: 3 g of chitosan and 12 mL of 2% acetic acid solution were stirred and mixed to obtain a chitosan solution. 150 mL of 10% sodium hydroxide solution was added to a three-necked flask equipped with a stirrer, a thermometer and a constant pressure dropping funnel. The chitosan solution was added dropwise to the three-necked flask while stirring at a temperature of 25°C and a stirring rate of 400 r / min. The dropping rate was controlled to be 1 drop / s. After the addition was completed, the stirring reaction was continued for 15 minutes. After the reaction was completed, the reaction product was vacuum filtered, and the filter cake was added to 20 mL of 8% copper sulfate solution, then stirred for 20 minutes, then allowed to stand for 1 hour, and then vacuum filtered. The filter cake was washed twice with distilled water and anhydrous ethanol in turn, and then placed in a vacuum drying oven and dried at a temperature of 50°C for 1 hour to obtain intermediate 3;

[0052] B4: 1 mL of intermediate 2, 1 g of intermediate 3, 20 mL of deionized water, 3 mL of isopropanol and 3 mL of 5% sodium hydroxide solution were added to a three-necked flask equipped with a stirrer and a thermometer, and stirred at 45°C and 400 r / min for 80 min. After the reaction, the reaction product was vacuum filtered, and the filter cake was washed twice with distilled water, anhydrous ethanol and acetone in sequence, and then placed in a vacuum drying oven and dried at 50°C for 1 h to obtain intermediate 4;

[0053] B5: Add 1 g of intermediate 4 and 15 mL of 1 mol / L hydrochloric acid solution into a single-necked flask, and perform ultrasonic treatment at 45°C and 45 kHz for 10 min. After the reaction is completed, vacuum filter the reaction product, wash the filter cake with distilled water until it is neutral, and then place it in a vacuum drying oven and dry it at 50°C for 1 h to obtain a polysaccharide clarifier.

[0054] Embodiment 2:

[0055] This embodiment is a method for preparing a polysaccharide clarifier, comprising the following steps:

[0056] B1: 0.1 mol o-chloroaniline and 60 mL of 30% sulfuric acid solution were added to a three-necked flask equipped with a stirrer and a thermometer, and stirred for 40 min at 100°C and 500 r / min, then cooled to 30°C, and then 0.055 mol of polyformaldehyde with a molecular weight of 90.08 was added and stirred for 2 h, and then heated to 85°C while stirring, and the heating rate was controlled to 2°C / min, and then stirred for 3 h at a constant temperature. After the reaction, 500 mL of deionized water was added to the reaction product, and the temperature was maintained at 80°C. Then, the pH was adjusted to 6 with 30% sodium hydroxide solution to precipitate, and then vacuum filtered. The filter cake was washed with distilled water 3 times, and then placed in a vacuum drying oven and dried at 65°C for 3 h to obtain intermediate 1;

[0057] B2: 10 mmol of intermediate 1, 50 mmol of epichlorohydrin, 100 mL of methyl isobutyl ketone and 35 mL of deionized water were added to a three-necked flask equipped with a stirrer, a thermometer and a constant pressure dropping funnel, and stirred for 10 h at a temperature of 80 ° C and a stirring rate of 500 r / min, then cooled to 40 ° C, and then 26 mL of 55% benzyltriethylammonium chloride solution was added dropwise while stirring, and the dropping rate was controlled to 2 drops / s, and then 30 mL of 40% sodium hydroxide solution was added dropwise while stirring, and the dropping rate was controlled to 2 drops / s. After the addition was completed, the temperature was raised to 85 ° C and the stirring reaction was continued for 2 h. After the reaction was completed, the reaction product was cooled to room temperature, allowed to stand and stratified, carbon dioxide was introduced into the organic phase until saturated, and then washed 3 times with 8% sodium chloride solution, and then the solvent was removed by rotary evaporation, and then filtered while hot, and the filtrate was collected to obtain intermediate 2;

[0058] B3: 3 g of chitosan and 15 mL of 3% acetic acid solution were stirred and mixed to obtain a chitosan solution. 200 mL of 15% sodium hydroxide solution was added to a three-necked flask equipped with a stirrer, a thermometer and a constant pressure dropping funnel. The chitosan solution was added dropwise to the three-necked flask while stirring at a temperature of 30°C and a stirring rate of 500 r / min. The dropping rate was controlled to be 2 drops / s. After the addition was completed, the stirring reaction was continued for 20 minutes. After the reaction was completed, the reaction product was vacuum filtered, and the filter cake was added to 30 mL of 10% copper sulfate solution, and then stirred for 30 minutes, and then allowed to stand for 2 hours, and then vacuum filtered. The filter cake was washed three times with distilled water and anhydrous ethanol in sequence, and then placed in a vacuum drying oven and dried at a temperature of 55°C for 2 hours to obtain intermediate 3.

[0059] B4: 5 mL of intermediate 2, 1 g of intermediate 3, 25 mL of deionized water, 5 mL of isopropanol and 5 mL of 10% sodium hydroxide solution were added to a three-necked flask equipped with a stirrer and a thermometer, and stirred for 100 min at a temperature of 50°C and a stirring rate of 500 r / min. After the reaction was completed, the reaction product was vacuum filtered, and the filter cake was washed three times with distilled water, anhydrous ethanol and acetone in sequence, and then placed in a vacuum drying oven and dried at a temperature of 55°C for 2 h to obtain intermediate 4;

[0060] B5: Add 1 g of intermediate 4 and 20 mL of 1.2 mol / L hydrochloric acid solution into a single-necked flask, and perform ultrasonic treatment at 50°C and 55 kHz for 15 min. After the reaction is completed, vacuum filter the reaction product, wash the filter cake with distilled water until it is neutral, and then place it in a vacuum drying oven and dry it at 55°C for 2 h to obtain a polysaccharide clarifier.

[0061] Embodiment 3:

[0062] This embodiment is a method for preparing a purification adsorbent, comprising the following steps:

[0063] A1: The diatomaceous earth was washed with distilled water for 3 times, and then placed in a vacuum drying oven, dried at 100°C for 2 hours, and then ground and passed through a 60-mesh sieve to obtain pretreated diatomaceous earth;

[0064] A2: 1 g of pretreated diatomaceous earth was added to 10 mL of deionized water, and then ultrasonically treated for 10 min at a temperature of 25°C and an ultrasonic frequency of 45 kHz to obtain a diatomaceous earth dispersion;

[0065] A3: 3 g of the polysaccharide clarifier from Example 1 and 40 mL of 2% acetic acid solution were added to a three-necked flask equipped with a stirrer, a thermometer and a constant pressure dropping funnel, and stirred and mixed for 20 min at a temperature of 45° C. and a stirring rate of 400 r / min to obtain a clarifier solution;

[0066] A4: 1 g of ferroferric oxide was added to 10 mL of deionized water, and then ultrasonically treated for 15 min at a temperature of 25°C and an ultrasonic frequency of 45 kHz to obtain a magnetic fluid.

[0067] A5: Add 10 mL of diatomaceous earth dispersion, 5 mL of clarifier solution and 10 mL of magnetic fluid into a three-necked flask equipped with a stirrer and a thermometer, and stir the reaction at 60°C and 400 r / min for 3 hours. After the reaction, cool the reaction product to room temperature and centrifuge it at 4500 r / min. Wash the precipitate twice with distilled water, place it in a vacuum drying oven, dry it at 60°C for 3 hours, grind it, and pass it through a 60-mesh sieve to obtain a purified adsorbent.

[0068] Embodiment 4:

[0069] This embodiment is a method for preparing a purification adsorbent, comprising the following steps:

[0070] A1: The diatomaceous earth was washed with distilled water for 5 times, and then placed in a vacuum drying oven, dried at 105°C for 3 hours, and then ground and passed through a 100-mesh sieve to obtain pretreated diatomaceous earth;

[0071] A2: 1 g of pretreated diatomaceous earth was added to 15 mL of deionized water, and then ultrasonically treated for 15 min at a temperature of 30°C and an ultrasonic frequency of 55 kHz to obtain a diatomaceous earth dispersion;

[0072] A3: 5 g of the polysaccharide clarifier from Example 2 and 50 mL of 3% acetic acid solution were added to a three-necked flask equipped with a stirrer, a thermometer and a constant pressure dropping funnel, and stirred and mixed for 30 min at a temperature of 50° C. and a stirring rate of 500 r / min to obtain a clarifier solution;

[0073] A4: 1 g of ferroferric oxide was added to 15 mL of deionized water, and then ultrasonically treated for 20 min at a temperature of 30°C and an ultrasonic frequency of 55 kHz to obtain a magnetic fluid.

[0074] A5: Add 15 mL of diatomaceous earth dispersion, 25 mL of clarifier solution and 15 mL of magnetic fluid into a three-necked flask equipped with a stirrer and a thermometer, and stir the reaction at 70°C and 500 r / min for 5 hours. After the reaction, cool the reaction product to room temperature and centrifuge it at 6000 r / min. Wash the precipitate with distilled water for 3 times, place it in a vacuum drying oven, dry it at 65°C for 5 hours, grind it, and pass it through a 100-mesh sieve to obtain a purified adsorbent.

[0075] Embodiment 5:

[0076] This embodiment is a method for efficiently extracting plant polysaccharides, comprising the following steps:

[0077] Step 1: Add 1 g of Eucommia leaf dry powder passed through a 40-mesh sieve and 15 mL of deionized water into a single-necked flask, perform ultrasonic treatment for 25 min at a temperature of 50°C and an ultrasonic power of 350 W, centrifuge the reaction product at a centrifugal speed of 4500 r / min after the reaction, take the supernatant and add it to a 95% ethanol solution by volume for alcohol precipitation, wash the precipitate with anhydrous ethanol and acetone twice in sequence, then place it in a vacuum drying oven and dry it at a temperature of 30°C for 3 h to obtain crude plant polysaccharides;

[0078] Step 2: Add 10g of crude plant polysaccharides and 500mL of deionized water to a three-necked flask equipped with a stirrer and a thermometer, stir and mix for 15min at a temperature of 60°C and a stirring rate of 400r / min, then adjust the pH to 5.5 with a 15% hydrochloric acid solution, then add 0.1g of the purified adsorbent from Example 3 and continue stirring and mixing for 30min. After the reaction is completed, the reaction product is cooled to room temperature, and then the purified adsorbent is removed with a magnet, and the supernatant is added to a 95% ethanol solution by volume for alcohol precipitation. The precipitate is washed twice with anhydrous ethanol, and then placed in a vacuum drying oven and dried at 25°C for 6h to obtain plant polysaccharides.

[0079] Embodiment 6:

[0080] This embodiment is a method for efficiently extracting plant polysaccharides, comprising the following steps:

[0081] Step 1: Add 1 g of Eucommia leaf dry powder passed through a 50-mesh sieve and 30 mL of deionized water into a single-necked flask, perform ultrasonic treatment for 50 min at a temperature of 60°C and an ultrasonic power of 400 W. After the reaction is completed, centrifuge the reaction product at a centrifugal speed of 6000 r / min, take the supernatant and add it to a 95% ethanol solution by volume for alcohol precipitation, wash the precipitate with anhydrous ethanol and acetone three times in sequence, then place it in a vacuum drying oven and dry it at a temperature of 35°C for 5 hours to obtain crude plant polysaccharides;

[0082] Step 2: Add 10g of crude plant polysaccharides and 1000mL of deionized water to a three-necked flask equipped with a stirrer and a thermometer, stir and mix for 20min at a temperature of 70°C and a stirring rate of 500r / min, then adjust the pH to 6.5 with a 20% hydrochloric acid solution, then add 1.0g of the purified adsorbent from Example 4 and continue stirring and mixing for 40min. After the reaction is completed, the reaction product is cooled to room temperature, and then the purified adsorbent is removed with a magnet, and the supernatant is added to a 95% ethanol solution by volume for alcohol precipitation. The precipitate is washed 3 times with anhydrous ethanol, and then placed in a vacuum drying oven and dried at 35°C for 8h to obtain plant polysaccharides.

[0083] Comparative Example 1:

[0084] The difference between Comparative Example 1 and Example 6 is that no purification adsorbent is added.

[0085] Comparative Example 2:

[0086] The difference between Comparative Example 2 and Example 6 is that diatomaceous earth is added instead of the purification adsorbent.

[0087] Comparative Example 3:

[0088] The difference between Comparative Example 3 and Example 6 is that chitosan is added instead of the purification adsorbent.

[0089] The plant polysaccharides of Examples 5-6 and Comparative Examples 1-3 were tested, and the test results are shown in the following table:

[0090] sample Polysaccharide retention rate, % Protein clearance, % Solid removal rate, % Example 5 98.9 87.4 31.6 Example 6 99.3 89.7 32.8 Comparative Example 1 98.0 20.3 8.1 Comparative Example 2 98.6 62.9 21.5 Comparative Example 3 98.8 79.6 29.5

[0091] Referring to the data in the above table, according to Examples 5-6, it can be seen that the polysaccharide retention rate of the plant polysaccharide of the present invention is more than 98%, the protein removal rate is more than 87%, and the solid removal rate is more than 31%. According to Example 6, compared with Comparative Examples 1-3, it can be seen that adding diatomaceous earth, chitosan, and purification adsorbent can effectively remove proteins and solids in polysaccharides, improve the purity of plant polysaccharides, and thus improve their quality, among which the purification adsorbent has the best improvement effect.

[0092] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0093] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the invention or exceed the scope defined by the claims, they shall all fall within the protection scope of the present invention.

Claims

1. A method for efficiently extracting plant polysaccharides, characterized in that: The following steps are involved: Step 1: adding eucommia leaf powder and deionized water into a single-necked flask for ultrasonic treatment, then centrifuging the reaction product, taking the supernatant for alcohol precipitation, washing the precipitate, and then drying to obtain crude plant polysaccharides; Step 2: Add crude plant polysaccharides and deionized water into a three-necked flask and stir to mix, then adjust the pH with hydrochloric acid solution, then add a purification adsorbent and continue to stir and mix, after the reaction is completed, cool the reaction product to room temperature, then use a magnet to remove the purification adsorbent, take the supernatant for alcohol precipitation, wash the precipitate, and then dry to obtain plant polysaccharides; The purified adsorbent is prepared by the following steps: A1: Wash and dry the diatomaceous earth, then grind and sieve to obtain pretreated diatomaceous earth; A2: adding the pretreated diatomaceous earth into deionized water for ultrasonic treatment to obtain a diatomaceous earth dispersion; A3: Add the polysaccharide clarifier and acetic acid solution into a three-necked flask and stir to mix to obtain a clarifier solution; A4: Add ferroferric oxide to deionized water for ultrasonic treatment to obtain a magnetic fluid; A5: Add the diatomite dispersion, the clarifier solution and the magnetic fluid into a three-necked flask and stir for reaction. After the reaction is completed, cool the reaction product to room temperature, then centrifuge it, wash and dry the precipitate, grind it, and sieve it to obtain a purified adsorbent; The polysaccharide clarifier is prepared by the following steps: B1: o-chloroaniline and sulfuric acid solution were added to a three-necked flask and stirred for reaction, then paraformaldehyde was added and the reaction was continued with stirring, after the reaction was completed, deionized water was added to the reaction product, then the pH was adjusted with sodium hydroxide solution to precipitate, then vacuum filtered, the filter cake was washed and dried to obtain intermediate 1; B2: Add intermediate 1, epichlorohydrin, methyl isobutyl ketone and deionized water into a three-necked flask and stir to react, then add benzyltriethylammonium chloride solution and sodium hydroxide solution dropwise while stirring, continue stirring to react after the addition is complete, and after the reaction is completed, cool the reaction product to room temperature, stand and separate, introduce carbon dioxide into the organic phase until saturated, then wash, rotary evaporate, filter while hot, collect the filtrate to obtain intermediate 2; B3: Chitosan and acetic acid solution are stirred and mixed to obtain chitosan solution, sodium hydroxide solution is added to a three-necked flask, and the chitosan solution is added dropwise to the three-necked flask while stirring, and the stirring reaction is continued after the dropwise addition is completed. After the reaction is completed, the reaction product is vacuum filtered, and the filter cake is added to a copper sulfate solution and stirred, and then allowed to stand, vacuum filtered, and the filter cake is washed and dried to obtain intermediate 3; B4: Add intermediate 2, intermediate 3, deionized water, isopropanol and sodium hydroxide solution into a three-necked flask and stir to react. After the reaction is completed, vacuum filter the reaction product, wash and dry the filter cake to obtain intermediate 4; B5: Add the intermediate 4 and hydrochloric acid solution into a single-necked flask for ultrasonic treatment. After the reaction is completed, vacuum filter the reaction product, wash and dry the filter cake to obtain a polysaccharide clarifier.

2. A method for efficiently extracting plant polysaccharides according to claim 1, characterized in that: The dosage ratio of the Eucommia leaf dry powder and deionized water in step one is 1g:15-30mL; the dosage ratio of the crude plant polysaccharide, deionized water and purification adsorbent in step two is 10g:500-1000mL:0.1-1.0g, and the mass fraction of the hydrochloric acid solution is 15-20%.

3. The method for efficiently extracting plant polysaccharides according to claim 1, characterized in that: The dosage ratio of the pretreated diatomaceous earth and deionized water in step A2 is 1 g: 10-15 mL; the dosage ratio of the polysaccharide clarifier and acetic acid solution in step A3 is 3-5 g: 40-50 mL, and the mass fraction of the acetic acid solution is 2-3%.

4. A method for efficiently extracting plant polysaccharides according to claim 1, characterized in that: The usage ratio of the ferroferric oxide and deionized water in step A4 is 1 g: 10-15 mL; the usage ratio of the diatomaceous earth dispersion, the clarifier solution and the magnetic fluid in step A5 is 10-15 mL: 5-25 mL: 10-15 mL.

5. The method for efficiently extracting plant polysaccharides according to claim 1, characterized in that: The amount ratio of the o-chloroaniline, sulfuric acid solution, paraformaldehyde and deionized water in step B1 is 0.1 mol: 50-60 mL: 0.05-0.055 mol: 300-500 mL, the mass fraction of the sulfuric acid solution is 25-30%, and the mass fraction of the sodium hydroxide solution is 25-30%; the amount ratio of the intermediate 1, epichlorohydrin, methyl isobutyl ketone, deionized water, benzyltriethylammonium chloride solution and sodium hydroxide solution in step B2 is 10 mmol: 40-50 mmol: 80-100 mL: 25-35 mL: 18-26 mL: 25-30 mL, the mass fraction of the benzyltriethylammonium chloride solution is 50-55%, and the mass fraction of the sodium hydroxide solution is 35-40%.

6. The method for efficiently extracting plant polysaccharides according to claim 1, characterized in that: The amount ratio of the chitosan, acetic acid solution, sodium hydroxide solution and copper sulfate solution in step B3 is 3g:12-15mL:150-200mL:20-30mL, the mass fraction of the acetic acid solution is 2-3%, the mass fraction of the sodium hydroxide solution is 10-15%, and the mass fraction of the copper sulfate solution is 8-10%; the amount ratio of the intermediate 2, intermediate 3, deionized water, isopropanol and sodium hydroxide solution in step B4 is 1-5mL:1g:20-25mL:3-5mL:3-5mL, and the mass fraction of the sodium hydroxide solution is 5-10%; the amount ratio of the intermediate 4 and hydrochloric acid solution in step B5 is 1g:15-20mL, and the molar concentration of the hydrochloric acid solution is 1-1.2mol / L.

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