A method for extracting eleutheroside B by combining immobilized enzyme with high pressure

Abstract

The invention provides a method for extracting eleutheroside B by combining an immobilized enzyme with high pressure. Aiming at the problem that the immobilized enzyme is easy to absorb water and burst in the extract and has low utilization rate, polyvinyl alcohol, sodium alginate, calcium carbonate and chitosan are used as materials, and a composite enzyme (pectinase / cellulase) is loaded to prepare a novel CaCO3-CS-immobilized enzyme. The CaCO3-CS-immobilized enzyme has the advantages of high stability, easy separation and recovery, and can be reused. The enzymolysis of the composite enzyme on the cell wall is combined with the advantage of high pressure increasing the pressure difference inside and outside the cell, so as to improve the extraction efficiency of eleutheroside B. The non-heating extraction method can avoid the inactivation or denaturation of eleutheroside B due to high temperature. The operation is simple, the process parameters are easy to control, and it is suitable for being promoted from the laboratory to actual production.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction, and particularly relates to a method for extracting eleutheroside B by combining immobilized enzyme with high pressure. Background Art

[0002] Acanthopanax senticosus, also known as Siberian ginseng, is a perennial woody plant belonging to the Araliaceae family. According to the Compendium of Materia Medica, Acanthopanax senticosus has the effects of nourishing the kidney and calming the nerves. Acanthopanax senticosus contains a variety of active substances including eleutheroside B, eleutheroside E, flavonoids, polysaccharides, vitamins and minerals. Recent studies have found that eleutheroside B has anti-tumor, anti-depression, anti-viral and arrhythmia-improving pharmacodynamic effects. There are many methods for extracting eleutheroside B, among which the traditional heating reflux method is the most basic method for extracting glycoside components. Chinese patent CN112089741B uses this method to extract multiple components from Acanthopanax senticosus, but this method is time-consuming, uses a large amount of solvent, and has a low extraction rate. High-pressure assisted extraction is an efficient physical-assisted extraction technology developed in recent years. Under high-pressure conditions, the solvent can enter the interior of plant cells and fully dissolve the effective ingredients in the plant. At the same time, high pressure can also destroy the dense cell wall structure of the plant, disintegrate the cells, and make the effective ingredients quickly overflow with the solvent. It has the characteristics of short extraction time, low solvent consumption, easy control of process parameters and high extraction efficiency.

[0003] In addition, enzyme-assisted extraction technology has also been applied to the extraction of Acanthopanax glycosides. This method is green and environmentally friendly, and the highly specific enzyme catalysis can reduce the mass transfer resistance during the extraction of effective ingredients and improve the extraction rate of natural products in plants. There are abundant types of enzymes, but the decomposition effect of one enzyme on plant cell walls is often single, and the catalytic speed is slow; while the synergistic effect of multiple enzymes will accelerate the decomposition of cell walls, shorten the extraction time, and enhance the dissolution rate of effective ingredients in plants, which has great advantages in the extraction of plant-derived natural compounds. Chinese patent CN102977164A uses a composite enzyme-assisted method to extract hyperoside from Acanthopanax, and the extraction efficiency is significantly improved, but the invention does not give a specific method for how to remove the used composite enzyme, and the residual enzyme will increase the difficulty of subsequent separation and purification of active ingredients. In view of the above problems, immobilized enzyme technology is an effective solution. Enzyme immobilization is to bind or restrict the enzyme to a certain area with a solid material, carry out its unique catalytic reaction, and can be recycled and reused. Compared with free enzymes, immobilized enzymes not only maintain their high efficiency, specificity and mild enzyme catalytic reaction characteristics, but also overcome the shortcomings of free enzymes, showing the advantages of high stability, easy separation and recovery, and reusability.

[0004] Common immobilized enzymes are generally prepared with polyvinyl alcohol and sodium alginate. Polyvinyl alcohol is a substance rich in hydroxyl groups and has many enzyme binding sites, which can fully guarantee the enzyme fixation efficiency. Sodium alginate also has the characteristics of high elasticity, which makes the immobilized enzyme microspheres have good ball-forming properties. However, immobilized enzymes prepared with these two materials often have disadvantages such as weak mechanical strength and sensitivity to the outside world. The immobilized enzymes will absorb water and burst during use, causing the enzyme to leak out and become difficult to separate. This greatly limits the reusability of the immobilized enzymes and prevents them from being widely used in industrial production. In recent years, studies have found that: Ca 2+ It can form ionic bonds with the carboxyl groups on sodium alginate to increase the solidification strength of sodium alginate and improve the stability of immobilized enzymes; chitosan (CS), as a natural high molecular polymer, has certain bioadhesion properties, and its rich amino groups can be blended with the hydroxyl groups in polyvinyl alcohol to form hydrogen bonds, and the combined use can further increase the ductility of immobilized enzymes; at the same time, the amino groups on the CS molecules can also produce a good adsorption effect on the enzyme through complexation and ion exchange. Therefore, in order to improve the performance of immobilized enzymes, the present invention adds calcium carbonate and chitosan on the basis of commonly used materials to prepare a new type of calcium carbonate-chitosan-immobilized enzyme (CaCO3-CS-immobilized enzyme), and for the first time, the CaCO3-CS-immobilized enzyme is combined with high-pressure extraction technology for the extraction of eleutheroside B. The present invention establishes an efficient, green, energy-saving, and mild method for extracting eleutheroside B, which is easy to use in industrial production. Summary of the invention

[0005] Aiming at the shortcomings of the existing eleutheroside B extraction technology, such as large amount of solvent, low enzyme utilization, complicated operation and low extraction rate, the present invention provides a method for extracting eleutheroside B by combining immobilized enzyme with high pressure. Meanwhile, aiming at the problem that the immobilized enzyme is easy to absorb water and burst in the extract, a novel CaCO3-CS-immobilized enzyme is prepared. The CaCO3-CS-immobilized enzyme is used in coordination with the high pressure technology to improve the extraction efficiency of eleutheroside B, and the immobilized composite enzyme used in the extraction process can be directly filtered and separated, simplifying the subsequent purification steps. The separated immobilized composite enzyme can be recycled, reducing the production cost, and has a wide application prospect in industrial production.

[0006] The technical solution of the present invention is as follows:

[0007] A method for extracting eleutheroside B by using an immobilized enzyme combined with high pressure, the specific steps are as follows:

[0008] ① Take the dry powder of Acanthopanax senticosus rhizome, add deionized water at a solid-liquid ratio of 1:15-30 (w / v), and mix evenly; add homemade immobilized enzyme at a mass fraction of 0.5-2%, wrap it with a single layer of gauze and put it into the reaction system, maintain the temperature at 40°C, stir at 300rpm, and extract for 60-80min.

[0009] ② The extract of step ① is subjected to high-pressure extraction, and the high-pressure extraction conditions are: pressure 4.5-6MPa, temperature 45-55°C, and processing time 35-50min. The crude extract is filtered under reduced pressure to obtain a crude extract.

[0010] ③ Load the crude extract obtained in step ② onto an AB-8 macroporous resin column, with a loading concentration of 5 mg / mL, a loading volume of 0.2 BV, and a loading flow rate of 0.3 to 1 BV / h. After loading, elute with 70% ethanol at a concentration of 7 BV at a flow rate of 0.5 to 1.2 BV / h, collect the eluate, concentrate, and freeze-dry to obtain eleutheroside B.

[0011] Further, the preparation method of the CaCO3-CS-immobilized enzyme is as follows:

[0012] ①Put an appropriate amount of deionized water in a beaker, add 6-8% polyvinyl alcohol, 0.5-2% sodium alginate, 0.25-1% chitosan and 2-5% CaCO3 in proportion. After mixing evenly, stir magnetically at 95-100℃ and 200rpm for 10 minutes. After the reagents are fully dissolved, cool to 40℃ to obtain a gel solution.

[0013] ② Add pectin-cellulose complex enzyme to the solution in step ① at a mass fraction of 0.5-2%, maintain 40°C, 200 rpm, and stir magnetically for 10 minutes to obtain a complex enzyme gel solution. Take an appropriate amount of boric acid buffer solution with a pH of 6, add anhydrous calcium chloride at a mass fraction of 1.5-3.5%, and ultrasonicate at 60W for 30 minutes to prepare a cross-linking fixative.

[0014] ③ Use a 5mL syringe without a needle to absorb an appropriate amount of complex enzyme gel solution, and drop it into the cross-linking fixative at a rate of 2 seconds per drop, stirring with a glass rod while adding. After standing at 20℃ for 2 hours, take it out, rinse it twice with deionized water, and dry it at 50℃ to constant weight, and the preparation of immobilized enzyme microspheres is completed.

[0015] Furthermore, in the preparation step ① of the CaCO3-CS-immobilized enzyme, polyvinyl alcohol and sodium alginate are commonly used materials for preparing immobilized enzymes, and chitosan and CaCO3 are modified materials.

[0016] Furthermore, in step ② of preparing the CaCO3-CS-immobilized enzyme, the mass ratio of pectinase to cellulase in the composite enzyme is 0.5 to 2:1.

[0017] Furthermore, the CaCO3-CS-immobilized enzyme can be reused 3 to 5 times when combined with high pressure extraction of eleutheroside B.

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

[0019] 1. The present invention provides a method for preparing a novel immobilized complex enzyme. By loading calcium carbonate, the stability of the immobilized enzyme is increased, the problem of the immobilized enzyme easily absorbing water and bursting and introducing new impurities when extracting natural active ingredients is overcome, and the subsequent separation and purification steps of the extract are simplified.

[0020] 2. The activity of the immobilized composite enzyme prepared by the present invention can be maintained at more than 70% of the free enzyme, and the immobilized composite enzyme is more stable than the free enzyme, can be used repeatedly, and has a higher overall utilization rate than the free enzyme, thereby reducing the cost of enzyme use.

[0021] 3. The present invention discloses a method for extracting natural active ingredients by combining immobilized enzyme with high pressure and applies it to extracting eleutheroside B from Acanthopanax senticosus. The method combines the full enzymatic hydrolysis of the complex enzyme on the cell wall with the advantage of high pressure increasing the pressure difference between the inside and outside of the cell, promotes the dissolution of the intracellular active ingredients, and improves the extraction efficiency of eleutheroside B.

[0022] 4. The extraction technology disclosed in the present invention is a non-heating extraction technology, which can avoid the inactivation or denaturation of eleutheroside B due to high temperature, is simple to operate, and the process parameters are easy to control, and is suitable for promotion from the laboratory to actual production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The cross-section and internal scanning electron micrographs of the CaCO3-CS-immobilized enzyme (A: cross-section scanning, B: internal scanning)

[0024] Figure 2 The integrity of the immobilized enzyme after immersion in water (A: Example 1, B: Comparative Example 1)

[0025] Figure 3 is the average diameter of the immobilized enzyme after water absorption (①: Example 1, ②: Comparative Example 1)

[0026] Figure 4 For CaCO 3- Relative activity of enzymes in CS-immobilized enzymes (①: cellulase, ②: pectinase)

[0027] Figure 5 Effect of repeated utilization of CaCO3-CS-immobilized enzyme on the yield of eleutheroside B

[0028] Figure 6 Effects of different extraction methods on the yield of eleutheroside B DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0030] Example 1

[0031] Preparation of CaCO3-CS-immobilized enzyme

[0032] (1) Take 130 mL of deionized water and place it in a beaker. Add 9 g of polyvinyl alcohol, 2 g of sodium alginate, 3.9 g of calcium carbonate, and 0.65 g of chitosan. Mix well. Stir magnetically at 95°C and 200 rpm for 10 min. After the reagents are fully dissolved, cool to 40°C to obtain a gel solution.

[0033] (2) Take 0.78g pectinase and 0.52g cellulase, add them to the solution of step (1), maintain 40°C, 200rpm, and stir magnetically for 10min to obtain a composite enzyme gel solution. Take 100mL of pH=6 boric acid buffer solution, add 2g anhydrous calcium chloride, and ultrasonicate at 60W for 30min to prepare a cross-linking fixative.

[0034] (3) Use a 5 mL syringe without a needle to draw 3 mL of the complex enzyme gel solution and drip it into the cross-linking fixative at a rate of 2 seconds per drop, stirring with a glass rod while dripping. After standing at 20°C for 2 h for cross-linking, take it out, rinse it twice with deionized water, and dry it at 50°C to constant weight. The preparation of immobilized enzyme microspheres is completed.

[0035] Extraction of Acanthopanax Senticoside B

[0036] (1) Take the dry powder of Acanthopanax senticosus rhizomes, add deionized water at a solid-liquid ratio of 1:20 (w / v), and mix well; weigh 1% CaCO3-CS-immobilized enzyme, wrap it with a single layer of gauze and put it into the reaction system, maintain the temperature at 40°C, stir at 300 rpm, and extract for 60 minutes.

[0037] (2) The extract of step (1) was subjected to high-pressure extraction under the following conditions: pressure 5 MPa, temperature 50° C., and treatment time 45 min. The crude extract was filtered under reduced pressure to obtain a crude extract, and the immobilized enzyme microspheres were recovered.

[0038] (3) The crude extract obtained in step (2) was loaded onto an AB-8 macroporous resin column at a loading concentration of 5 mg / mL, a loading volume of 0.2 BV, a loading flow rate of 0.5 BV / h, and eluted with 7 BV of 70% ethanol at a flow rate of 1 BV / h. The eluate was collected, concentrated, and lyophilized to obtain eleutheroside B.

[0039] The yield of eleutheroside B was determined to be 1.685 mg / g. The yield determination steps of eleutheroside B in the present invention are as follows:

[0040] The elatoside B standard was prepared into different concentrations, and its absorbance was detected at 265 nm, and the absorbance was plotted against the elatoside B concentration to draw the elatoside B standard curve. The elatoside B extract was taken and the absorbance was detected at 265 nm. The elatoside B concentration was obtained by comparing the standard curve, and the elatoside B yield was calculated according to the following formula. The elatoside B yield was expressed as elatoside B equivalent (mg) / weight of elatoside powder (g).

[0041]

[0042] Where: m is the mass of Acanthopanax senticosus dry powder, g;

[0043] C is the concentration of eleutheroside B in Acanthopanax senticosus extract calculated from the standard curve, mg / mL;

[0044] V is the volume of the solution to be tested, mL;

[0045] Example 2

[0046] Preparation of CaCO3-CS-immobilized enzyme

[0047] (1) Take 100 mL of deionized water and place it in a beaker, add 8 g of polyvinyl alcohol, 2 g of sodium alginate, 3 g of calcium carbonate, and 0.5 g of chitosan, mix well, and stir magnetically at 100 ° C and 200 rpm for 10 min. After the reagents are fully dissolved, cool to 40 ° C to obtain a gel solution.

[0048] (2) Take 0.6g pectinase and 0.4g cellulase, add them to the solution of step (1), maintain 40°C, 200rpm, and stir magnetically for 10min to obtain a composite enzyme gel solution. Take 100mL of pH=6 boric acid buffer solution, add 3g anhydrous calcium chloride, and ultrasonicate at 60W for 30min to prepare a cross-linking fixative.

[0049] (3) Use a 5 mL syringe without a needle to draw 3 mL of the complex enzyme gel solution and drip it into the cross-linking fixative at a rate of 2 seconds per drop, stirring with a glass rod while dripping. After standing at 20°C for 2 h for cross-linking, take it out, rinse it twice with deionized water, and dry it at 50°C to constant weight. The preparation of immobilized enzyme microspheres is completed.

[0050] Extraction of Acanthopanax Senticoside B

[0051] (1) Take the dry powder of Acanthopanax senticosus rhizomes, add deionized water at a solid-liquid ratio of 1:30 (w / v), and mix well; weigh CaCO3-CS-immobilized enzyme at a mass fraction of 2%, wrap it with a single layer of gauze and put it into the reaction system, maintain the temperature at 40°C, stir at 300 rpm, and extract for 70 minutes.

[0052] (2) The extract of step (1) was subjected to high-pressure extraction under the following conditions: pressure 6 MPa, temperature 55° C., and treatment time 50 min. The crude extract was filtered under reduced pressure to obtain a crude extract, and the immobilized enzyme microspheres were recovered.

[0053] (3) The crude extract obtained in step (2) was loaded onto an AB-8 macroporous resin column at a loading concentration of 5 mg / mL, a loading volume of 0.2 BV, and a loading flow rate of 1 BV / h, and eluted with 7 BV of 70% ethanol at a flow rate of 0.5 BV / h, and the eluate was collected, concentrated, and freeze-dried to obtain eleutheroside B. The yield of eleutheroside B was determined according to the method of Example 1. The yield of eleutheroside B was determined to be 1.703 mg / g.

[0054] Comparative Example 1

[0055] Preparation of polyvinyl alcohol-sodium alginate-immobilized enzyme

[0056] (1) Take 130 mL of deionized water and place it in a beaker. Add 9 g of polyvinyl alcohol and 2 g of sodium alginate. Mix well. Stir magnetically at 95°C and 200 rpm for 10 min. After the reagents are fully dissolved, cool to 40°C to obtain a gel solution.

[0057] (2) Take 0.78g pectinase and 0.52g cellulase, add them to the solution of step (1), maintain 40°C, 200rpm, and stir magnetically for 10min to make the enzymes evenly dispersed in the solution to obtain a composite enzyme gel solution. Take 100mL of pH=6 boric acid buffer solution, add 2g anhydrous calcium chloride, and ultrasonicate at 60W for 30min to prepare a cross-linking fixative.

[0058] (3) Use a 5 mL syringe without a needle to draw 3 mL of the complex enzyme gel solution and drip it into the cross-linking fixative at a rate of 2 seconds per drop, stirring with a glass rod while dripping. After standing at 20°C for 2 h for cross-linking, take it out, rinse it twice with deionized water, and dry it at 50°C to constant weight. The immobilized enzyme preparation is completed.

[0059] Immobilized enzyme immobilization effect and performance analysis

[0060] 1. Immobilized enzyme structure

[0061] Figure 1 This is a SEM scan of the CaCO3-CS-immobilized enzyme microsphere structure prepared in Example 1. It can be seen from the figure that the microsphere has a complete sponge-like hollow structure. Further magnification shows that enzyme particles are attached to the inner wall of the sample (indicated by the white arrow), indicating that the immobilized enzyme microsphere prepared by the present invention has a good enzyme fixation effect.

[0062] 2. Mechanical strength

[0063] Take the immobilized enzymes prepared in Example 1 and Comparative Example 1. Take 10 pellets of each group of immobilized enzymes and place them in aqueous solutions stirred at 0 r / min and 300 r / min for 5 days, observe the integrity of each group of immobilized enzymes and calculate the integrity rate by the following formula.

[0064]

[0065] Where: n1: total number of immobilized enzymes tested (particles);

[0066] n2: Total number of undamaged immobilized enzymes (particles).

[0067] Depend on Figure 2 It can be seen that the immobilized enzyme prepared by the present invention remained intact after being stirred in water for 5 days, and the intact rate can reach 100%. However, the polyvinyl alcohol-sodium alginate-immobilized enzyme prepared by conventional wall materials in Comparative Example 1 swelled significantly after absorbing water, gradually became gel-like, and lost its original spherical shape, and the intact rate was 15% lower than that of the immobilized enzyme of the present invention.

[0068] 3. Volume expansion

[0069] Example 1 and Comparative Example 1 were used to prepare immobilized enzymes. Ten pellets of each group of immobilized enzymes were soaked in water for 5 consecutive days, and the diameter of each immobilized enzyme microsphere was measured at regular intervals every day. The results are shown in FIG. Figure 3 The immobilized enzyme prepared by the present invention has little volume increase (<20%) after 5 days due to the addition of calcium carbonate and chitosan, and has little water absorption and expansion. However, the volume of the polyvinyl alcohol-sodium alginate-immobilized enzyme prepared by conventional wall materials in Comparative Example 1 increased significantly (>45%) after 5 days, and had obvious water absorption and expansion.

[0070] 4. CaCO 3- Detection of enzyme activity in CS-immobilized enzyme

[0071] Determination of pectinase activity by DNS method

[0072] (1) Preparation of galacturonic acid standard curve: Prepare galacturonic acid standard solution of different concentrations, take 0.4 mL of each solution, add 0.3 mL of DNS reagent, boil in a water bath for 10 min, cool to room temperature, add 4.3 mL of water, measure the absorbance at 490 nm, and plot the absorbance against galacturonic acid concentration to obtain the galacturonic acid standard curve.

[0073] (2) Pectinase activity detection: Take 10 mL of 7.0 mg / mL pectin standard solution in two test tubes respectively, and preheat at 37°C for 10 min. Add 0.6 mg of free pectinase to one test tube, and add 1.0 g of the immobilized enzyme prepared in Example 1 to the other test tube. Take 0.4 mL of enzymatic solution from the test tube at different times, and add 0.3 mL of DNS reagent. After boiling in a water bath for 10 min, cool to room temperature, add 4.3 mL of deionized water, mix well, and detect the absorbance of each enzymatic solution at 490 nm. Compare with the standard curve to obtain the concentration of converted galacturonic acid.

[0074] The pectinase activity in the CaCO3-CS-immobilized enzyme prepared by the present invention is based on the concentration of galacturonic acid generated by enzymatic pectin conversion, and the free enzyme activity is set as 100%. The immobilized enzyme activity is divided by the free enzyme activity to obtain the relative activity of the immobilized enzyme. Figure 4 It can be seen that at 60 min, the pectinase activity in the CaCO3-CS-immobilized enzyme prepared by the present invention reached 76.6% of the free enzyme activity.

[0075] Determination of cellulase activity by DNS method

[0076] (1) Glucose standard curve drawing: Prepare glucose standard solution into different concentrations, take 2 mL of each solution, add 1 mL of DNS solution, boil in water for 10 min, cool to room temperature, and add 21.5 mL of deionized water. Measure the absorbance at 540 nm, and use the absorbance to glucose concentration to obtain the glucose standard curve.

[0077] (2) Cellulase activity detection: Take 20 mL of 7.0 mg / mL cellulose standard solution in two test tubes respectively, and preheat at 40°C for 10 min. Add 0.4 mg of free cellulase to one test tube, and add 2.0 g of immobilized enzyme prepared in Example 1 to the other test tube. Take 2.0 mL of enzymatic hydrolysate from the test tube at different times, and add 1.0 mL of DNS reagent. After boiling in a water bath for 10 min, cool to room temperature, add 21.5 mL of water, mix well, and detect the absorbance of each enzymatic hydrolysate at 540 nm. Compare with the standard curve to obtain the concentration of glucose generated by conversion.

[0078] The cellulase activity in the CaCO3-CS-immobilized enzyme prepared by the present invention is based on the glucose concentration generated by enzymatic cellulose hydrolysis, and the free enzyme activity is set as 100%. The relative activity of the immobilized enzyme is obtained by dividing the immobilized enzyme activity by the free enzyme activity. Figure 4 It can be seen that at 60 min, the cellulase activity in the CaCO3-CS-immobilized enzyme prepared by the present invention reached 71.3% of the free enzyme activity.

[0079] 5.CaCO3-CS-immobilized enzyme recycling times

[0080] The CaCO3-CS-immobilized enzyme recovered in Example 1 was used to extract eleutheroside B according to the steps in Example 1, and the operation was repeated 5 times. The yield of eleutheroside B was calculated according to the method in Example 1. The experimental results are shown in FIG. Figure 5 , 0 times represents the yield of eleutheroside B by simple high-pressure extraction. In the process of combined high-pressure extraction, the CaCO3-CS-immobilized enzyme can exert catalytic activity after 5 cycles. The yield of eleutheroside B is higher than that of single high-pressure extraction (1.378 mg / g), and the effect is better when it is used repeatedly for 3 times.

[0081] Comparative Example 2

[0082] Extraction of Acanthopanax Senticoside B

[0083] 1. Immobilized enzyme-assisted extraction of eleutheroside B

[0084] Take the dry powder of Acanthopanax rhizome, add deionized water at a solid-liquid ratio of 1:30 (w / v), and mix well; weigh the CaCO3-CS-immobilized enzyme prepared in Example 1 at a mass fraction of 1%, wrap it with a single layer of gauze and put it into the reaction system, maintain the temperature at 40°C, stir at 300 rpm, extract for 3 hours, and determine the yield of Acanthopanax glycoside B according to the method in Example 1.

[0085] 2. High-pressure assisted extraction of eleutheroside B

[0086] Take the dry powder of Acanthopanax rhizome, add deionized water at a solid-liquid ratio of 1:30 (w / v), mix well and then perform high-pressure extraction. The extraction conditions are: pressure 6 MPa, temperature 55° C., time 50 min. The yield of Acanthopanax glycoside B is determined according to the method in Example 1.

[0087] 3. Microwave-assisted extraction of eleutheroside B

[0088] Take the dry powder of Acanthopanax rhizome, add deionized water at a solid-liquid ratio of 1:30 (w / v), mix well and then perform microwave-assisted extraction. The extraction conditions are: power 600 W, temperature 70° C., time 20 min. The yield of Acanthopanax glycoside B is determined according to the method in Example 1.

[0089] 4. Immobilized enzyme combined with microwave extraction of eleutheroside B

[0090] (1) Take the dry powder of Acanthopanax senticosus rhizomes, add deionized water at a solid-liquid ratio of 1:30 (w / v), and mix well; weigh 1% of the CaCO3-CS-immobilized enzyme prepared in Example 1, wrap it with a single layer of gauze and put it into the reaction system, maintain the temperature at 40°C, stir at 300 rpm, and extract for 70 min.

[0091] (2) The extract of step (1) was subjected to microwave extraction under the following conditions: power 600 W, temperature 70° C., time 20 min, and the yield of eleutheroside B was determined according to the method of Example 1.

[0092] Depend on Figure 6 It can be seen that the combination of immobilized enzyme and modern extraction technology can increase the yield of eleutheroside B, among which high pressure combined with immobilized enzyme extraction has a better effect. Compared with the microwave combined with immobilized enzyme method, the extraction yield of eleutheroside B by high pressure combined with immobilized enzyme is increased by 7.8%.

Claims

1. A method for extracting eleutheroside B by using an immobilized enzyme combined with high pressure, characterized in that The specific steps are as follows: ① Take an appropriate amount of deionized water and place it in a beaker, add 6-8% polyvinyl alcohol, 0.5-2% sodium alginate, 0.25-1% chitosan and 2-5% CaCO3 in proportion, mix well, stir magnetically at 95-100°C and 200 rpm for 10 minutes, wait until the reagents are fully dissolved, cool to 40°C to obtain a gel solution; ② Add the complex enzyme to the solution in step ① at a mass fraction of 0.5-2%, maintain 40°C, 200 rpm, and magnetically stir for 10 minutes to obtain a complex enzyme gel solution, take an appropriate amount of boric acid buffer solution with a pH of 6, add anhydrous calcium chloride at a mass fraction of 1.5-3.5%, and ultrasonicate at 60W for 30 minutes to prepare a cross-linking fixative; ③ Use a 5mL syringe without a needle to draw an appropriate amount of complex enzyme gel solution, drop it into the cross-linking fixative at a rate of 2 seconds per drop, stir with a glass rod while adding, stand at 20℃ for 2h to cross-link, then take it out, rinse it twice with deionized water, and dry it at 50℃ to constant weight, and the preparation of immobilized enzyme microspheres is completed; ④ Take the dry powder of Acanthopanax senticosus rhizome, add deionized water at a solid-liquid ratio of 1:15-30 (w / v), mix well, add the homemade immobilized enzyme in step ③ at a mass fraction of 0.5-2%, wrap it with a single layer of gauze and put it into the reaction system, maintain the temperature at 40°C, stir at 300 rpm, and extract for 60-80 min; ⑤ subjecting the extract of step ④ to high-pressure extraction, the high-pressure extraction conditions being: pressure 4.5-6 MPa, temperature 45-55° C., treatment time 35-55 min, filtering the crude extract under reduced pressure to obtain a crude extract; ⑥ Load the crude extract obtained in step ⑤ onto an AB-8 macroporous resin column at a loading concentration of 5 mg / mL, a loading volume of 0.2 BV, a loading flow rate of 0.3 to 1 BV / h, and elute with 7 BV of 70% ethanol at a flow rate of 0.5 to 1.2 BV / h. Collect the eluate, concentrate it, and lyophilize it to obtain eleutheroside B.

2. The method for extracting eleutheroside B by combining immobilized enzyme with high pressure according to claim 1, characterized in that In the step ②, the mass ratio of pectinase to cellulase in the complex enzyme is 0.5-2:1.

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