A method for efficiently extracting and purifying tubulosine

Abstract

This invention relates to the field of plant component extraction technology, specifically to a method for efficiently extracting and purifying berberine. The method includes: (1) selecting dried stems of *Radermachera sinensis*, crushing and sieving them to obtain *Radermachera sinensis* powder; (2) weighing the *Radermachera sinensis* powder and mixing it with a 20-30% (w / w) ammonium sulfate solution, then adding a compound enzyme preparation for enzymatic hydrolysis, adding isopropanol to obtain a mixture, extracting the mixture using an ultrasonic-microwave synergistic extraction method, centrifuging the extracted mixture for 10-13 min at 3500-4000 rpm, removing the isopropanol layer by distillation to obtain a crude extract; (3) dissolving the crude extract in water and loading it onto a polar macroporous resin for multiple elutions to obtain berberine. Compared with the prior art, this invention uses an enzyme preparation followed by an aqueous two-phase solvent combined with ultrasonic-microwave extraction technology to promote the maximum release of berberine from *Radermachera sinensis* powder, thereby increasing the yield. Finally, a gradient elution with macroporous resin is used to achieve efficient extraction and purification of berberine.

Description

Technical Field

[0001] This invention relates to the field of plant component extraction technology, specifically to a method for efficiently extracting and purifying berberine. Background Technology

[0002] Berberine is an alkaloid extracted from the dried stems of *Rhizoma Cirsium japonicum*, a plant in the Menispermaceae family. It possesses broad-spectrum antibacterial, antiviral, anti-inflammatory, and leukocyte-enhancing pharmacological effects, and has wide applications in clinical antibacterial drugs, chemotherapy drugs, pesticides, and veterinary drugs. Therefore, the extraction efficiency and purity of berberine significantly influence its application.

[0003] Traditional acid extraction and alkali precipitation methods have low extraction rates. For example, in the improvement of the process for extracting berberine from *Cephalotaxus fortunei*, *Cephalotaxus fortunei* is extracted by soaking in dilute sulfuric acid, adjusting the pH, allowing it to settle and precipitate, and then the filtrate is salted out, filtered, and dried to obtain crude berberine alkali. The crude alkali is then mixed with 80% alcohol at a ratio of 1:5, heated to boiling, and extracted. The pH is adjusted to 2, and the mixture is cooled to crystallize, yielding high-quality berberine. Using this method, the extraction rate of 500g of *Cephalotaxus fortunei* is only 2.25%, which is extremely low and the extraction effect is poor, failing to meet the requirements for berberine.

[0004] To improve the extraction rate of berberine, an acidic aqueous solution emulsification extraction method is used, as disclosed in CN201810765696.5. This method specifically discloses pulverizing *Gnaphalium affine* to obtain berberine powder, mixing the powder with an acetic acid solution of 0.1-0.5‰ at a solid-liquid mass ratio of 1:(8-10), emulsifying the mixture at 2500-3500 rpm and 25-30℃ for 15-30 minutes to obtain an emulsion, filtering the emulsion to obtain the filtrate, adding 6-8% sodium chloride (by weight of the filtrate) to the filtrate to adjust the pH to 9-10 to obtain an alkaline extract, allowing the alkaline extract to stand for 20-24 hours, filtering to obtain the precipitate, and drying the precipitate at 70-80℃ to obtain berberine. The crude product obtained using the above method was 1347g, which is a significant improvement compared to the 12738g crude product obtained by the traditional acid extraction process. However, this method does not involve purification, resulting in a high content of impurities and low purity of berberine in the product. Summary of the Invention

[0005] This invention provides a method for efficiently extracting and purifying berberine, aiming to solve the technical problems of low extraction efficiency or low purification yield in existing technologies.

[0006] To achieve the above objectives, the present invention provides a method for efficiently extracting and purifying berberine, the method comprising: (1) Select dried stems of rattan, crush and sieve them to obtain rattan powder; (2) Weigh out the powder of *Rhizoma Cimicifugae* and mix it with 20-30% (w / w) ammonium sulfate solution, then add a compound enzyme preparation for enzymatic hydrolysis, add isopropanol to obtain a mixture, the mixture is extracted by ultrasonic-microwave synergistic extraction, the extracted mixture is centrifuged for 10-13 min at 3500-4000 rpm, the isopropanol layer is taken and distilled to remove isopropanol to obtain crude extract; (3) Dissolve the crude extract in water and load it onto a polar macroporous resin for repeated elution to obtain berberine.

[0007] Preferably, in the above technical solution, the amount of the compound enzyme preparation is 0.1-0.5% of the mass of the tung oil powder.

[0008] Preferably, in the above technical solution, the compound enzyme preparation is composed of magnesium chloride, calcium chloride, cellulase, pectinase and citrate-sodium citrate buffer.

[0009] Preferably, in the above technical solution, the cellulase has an enzyme activity of 900-1100 U / g, and the pectinase has an enzyme activity of 700-900 U / g.

[0010] Preferably, in the above technical solution, the enzymatic hydrolysis conditions of the compound enzyme preparation are 1-1.5 h at a temperature of 45-50℃.

[0011] Preferably, in the above technical solution, the 28.6% (w / w) ammonium sulfate solution is prepared by dissolving 3.43 kg of ammonium sulfate in 8.57 L of deionized water and stirring to obtain 28.6% (w / w) ammonium sulfate.

[0012] Preferably, in the above technical solution, the extraction of the mixture using the ultrasonic-microwave synergistic extraction method specifically involves: the mixture first undergoing ultrasonic treatment for 10 minutes with an ultrasonic power of 300 W and a frequency of 25 kHz; then undergoing microwave extraction for 10 minutes with a microwave power of 500 W and a temperature setting of 75°C.

[0013] Preferably, in the above technical solution, in the step of dissolving the crude extract in water and loading it onto a polar macroporous resin for multiple elutions to obtain berberine, the multiple elutions refer to first eluting strongly polar impurities with water, then eluting weakly polar impurities with 20-30% ethanol, and then eluting berberine with 60-75% ethanol.

[0014] Preferably, in the above technical solution, the ethanol concentration for eluting weakly polar impurities is 30%, and the ethanol concentration for eluting berberine is 75%.

[0015] Compared with the prior art, the beneficial effects of this application are as follows: This application employs enzyme treatment followed by aqueous two-phase extraction as a solvent combined with ultrasonic-microwave extraction technology to maximize the release of berberine from the berberine powder, thereby increasing the yield. Gradient elution with macroporous resin is used to remove enzyme preparations, aqueous two-phase solvents, resin fragments, plant tissue particles, and lipid-soluble substances, achieving efficient separation of impurities from berberine.

[0016] The principle of compound enzyme preparation promoting the maximum release of berberine from berberine powder is as follows: using citrate buffer as a solvent provides the most suitable pH for cellulase and pectinase to maintain their enzyme activity and promote their specific hydrolysis of cellulose. Magnesium chloride and calcium chloride, as metal ion activators, can bind to enzyme proteins, promote the spatial structure stability of cellulase and pectinase, and enhance enzyme activity and enzyme effect. Detailed Implementation

[0017] The technical solutions in the embodiments of this invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Example 1

[0018] A method for efficiently extracting and purifying berberine, the method comprising: (1) Select dried stems of rattan, crush and sieve them to obtain rattan powder with a mesh size of 20; (2) Weigh 1 kg of *Rattania serratifolia* powder and place it in an extraction tank. Add 28.6% (w / w) ammonium sulfate solution to the extraction tank to mix the *Rattania serratifolia* powder and ammonium sulfate solution evenly. Then add the compound enzyme preparation to the extraction tank and stir the reaction at 50°C for 1.5 hours. Add 6 L of isopropanol to the extraction tank, tighten the tank lid, and place the extraction tank in an ultrasonic-microwave synergistic extraction instrument. Perform ultrasonic treatment for 10 min and microwave extraction for 10 min. Centrifuge the extracted mixture for 10 min at 4000 rpm. Remove the isopropanol layer by distillation to obtain the crude extract. The preparation method of the 28.6% (w / w) ammonium sulfate is as follows: Dissolve 3.43 kg of ammonium sulfate (analytical grade) in 8.57 L of deionized water and stir to dissolve to obtain 28.6%. (w / w)ammonium sulfate; the preparation method of the compound enzyme preparation is as follows: 0.2g magnesium chloride and 0.2g calcium chloride are added to 1L citrate-sodium citrate buffer, 33g cellulase (enzyme activity of about 1000U / g) is added to citrate-sodium citrate buffer and mixed evenly, and 16.5g pectinase (enzyme activity of about 800U / g) is added to the above solution to obtain the compound enzyme preparation; (3) Dissolve the crude extract in water and load it onto a polar macroporous resin. First, wash away the strong polar impurities with water, then wash away the weak polar impurities with 30% ethanol, and then wash away the target product with 75% ethanol to obtain berberine.

[0019] Example 2 A method for efficiently extracting and purifying berberine, the method comprising: (1) Select dried stems of rattan, crush and sieve them to obtain rattan powder with a mesh size of 20; (2) Weigh 1 kg of *Rattania serratifolia* powder and place it in an extraction tank. Add 28.6% (w / w) ammonium sulfate solution to the extraction tank to mix the *Rattania serratifolia* powder and ammonium sulfate solution evenly. Then add the compound enzyme preparation to the extraction tank and stir the reaction at 50°C for 1.5 hours. Add 6 L of isopropanol to the extraction tank, tighten the tank lid, and place the extraction tank in an ultrasonic-microwave synergistic extraction instrument. Perform ultrasonic treatment for 10 min and microwave extraction for 10 min. Centrifuge the extracted mixture for 10 min at 4000 rpm. Remove the isopropanol layer by distillation to obtain the crude extract. The preparation method of the 28.6% (w / w) ammonium sulfate is as follows: Dissolve 3.43 kg of ammonium sulfate (analytical grade) in 8.57 L of deionized water and stir to dissolve to obtain 28.6%. (w / w)ammonium sulfate; the preparation method of the compound enzyme preparation is as follows: 0.2g magnesium chloride and 0.2g calcium chloride are added to 1L citrate-sodium citrate buffer, 33g cellulase (enzyme activity of about 1000U / g) is added to citrate-sodium citrate buffer and mixed evenly, and 16.5g pectinase (enzyme activity of about 800U / g) is added to the above solution to obtain the compound enzyme preparation; (3) Dissolve the crude extract in water and load it onto a polar macroporous resin. First, wash away the strong polar impurities with water, then wash away the weak polar impurities with 30% ethanol, and then wash away the target product with 75% ethanol to obtain berberine. Example 3

[0020] A method for efficiently extracting and purifying berberine, the method comprising: (1) Select dried stems of rattan, crush and sieve them to obtain rattan powder with a mesh size of 20; (2) Weigh 1 kg of *Rattania serratifolia* powder and place it in an extraction tank. Add 28.6% (w / w) ammonium sulfate solution to the extraction tank to mix the *Rattania serratifolia* powder and ammonium sulfate solution evenly. Then add the compound enzyme preparation to the extraction tank and stir the reaction at 50°C for 1.5 hours. Add 6 L of isopropanol to the extraction tank, tighten the tank lid, and place the extraction tank in an ultrasonic-microwave synergistic extraction instrument. Perform ultrasonic treatment for 10 min and microwave extraction for 10 min. Centrifuge the extracted mixture for 10 min at 4000 rpm. Remove the isopropanol layer by distillation to obtain the crude extract. The preparation method of the 28.6% (w / w) ammonium sulfate is as follows: Dissolve 3.43 kg of ammonium sulfate (analytical grade) in 8.57 L of deionized water and stir to dissolve to obtain 28.6%. (w / w)ammonium sulfate; the preparation method of the compound enzyme preparation is as follows: 0.2g magnesium chloride and 0.2g calcium chloride are added to 1L citrate-sodium citrate buffer, 33g cellulase (enzyme activity of about 1000U / g) is added to citrate-sodium citrate buffer and mixed evenly, and 16.5g pectinase (enzyme activity of about 800U / g) is added to the above solution to obtain the compound enzyme preparation; (3) Dissolve the crude extract in water and load it onto a polar macroporous resin. First, wash away the strong polar impurities with water, then wash away the weak polar impurities with 30% ethanol, and then wash away the target product with 75% ethanol to obtain berberine.

[0021] Comparative Example 1 Weigh 500g of berberine powder and place it in an extraction vessel. Add 15%, 20%, 25%, 30%, and 35% (w / w) ammonium sulfate solutions to the extraction vessels respectively. Add 3L of isopropanol to each extraction vessel. Tighten the vessel caps and place the extraction vessels in an ultrasonic-microwave synergistic extraction instrument. Perform ultrasonic treatment for 10 minutes and microwave extraction for 10 minutes. Centrifuge at 4000 rpm. Remove the isopropanol layer by distillation to obtain the crude extract. Dissolve the crude extract in water and load it onto a polar macroporous resin. First, wash away the strongly polar impurities with water, then wash away the weakly polar impurities with 30% ethanol, and finally wash away the target product with 75% ethanol to obtain berberine.

[0022] Table 1. Effect of different ammonium sulfate solution concentrations (w / w) on the extraction of berberine. Ammonium sulfate solution concentration (w / w) 15% 20% 25% 30% 35% Berberine (g) 8.7 10.1 11.1 12.3 10.5 Extract yield (%) 1.74 2.02 2.22 2.46 2.10 As shown in Table 1, when the ammonium sulfate concentration is 30%, 12.3g of berberine can be extracted from 500g of *Coptis chinensis* powder, with an extract yield of 2.46%. However, when the ammonium sulfate concentration is 35%, only 10.5g of berberine can be extracted from 500g of *Coptis chinensis* powder, with an extract yield of 2.10%. The extracted berberine content decreases, and the extract yield decreases by 0.36%. This indicates that when the ammonium sulfate concentration is too high, it affects the solubility of berberine in isopropanol, causing it to saturate prematurely in isopropanol, resulting in insufficient precipitation of berberine from the *Coptis chinensis* powder.

[0023] Comparative Example 2 Weigh 500g of berberine powder and place it in an extraction vessel. Add 26%, 27%, 28%, and 29% (w / w) ammonium sulfate solutions to the extraction vessels respectively. Add 3L of isopropanol to each extraction vessel. Tighten the vessel caps and place the extraction vessels in an ultrasonic-microwave synergistic extraction instrument. Perform ultrasonic treatment for 10 minutes and microwave extraction for 10 minutes. Centrifuge at 4000 rpm. Remove the isopropanol layer by distillation to obtain the crude extract. Dissolve the crude extract in water and load it onto a polar macroporous resin. First, wash away the strongly polar impurities with water, then wash away the weakly polar impurities with 30% ethanol, and finally wash away the target product with 75% ethanol to obtain berberine.

[0024] Table 2. Effect of different ammonium sulfate solution concentrations (w / w) on berberine extraction. Ammonium sulfate solution concentration (w / w) 26% 27% 28% 29% Berberine (g) 11.4 11.8 12.5 12.9 Extract yield (%) 2.28 2.36 2.50 2.58 As can be seen from Table 2, when the concentration of ammonium sulfate solution is in the range of 28%-29% (w / w), the content of berberine obtained by extraction is 12.5-12.9g, and the extraction yield is 2.50-2.58%.

[0025] Comparative Example 3 Weigh 500g of berberine powder and place it in an extraction vessel. Add 28.2%, 28.4%, 28.6%, and 28.8% (w / w) ammonium sulfate solutions to the extraction vessels, respectively. Add 3L of isopropanol to each extraction vessel. Tighten the vessel caps and place the extraction vessels in an ultrasonic-microwave synergistic extraction instrument. Perform ultrasonic treatment for 10 minutes, followed by microwave extraction for 10 minutes. Centrifuge at 4000 rpm. Remove the isopropanol layer by distillation to obtain the crude extract. Dissolve the crude extract in water and load it onto a polar macroporous resin. First, wash away strongly polar impurities with water, then wash away weakly polar impurities with 30% ethanol, and finally wash away the target product with 75% ethanol to obtain berberine.

[0026] Table 3. Effect of different ammonium sulfate solution concentrations (w / w) on the extraction of berberine. Ammonium sulfate solution concentration (w / w) 28.2% 28.4% 28.6% 28.8% Berberine (g) 12.6 12.8 13.1 13.0 Extract yield (%) 2.52 2.56 2.62 2.60 As can be seen from Table 3, when the ammonium sulfate concentration is 28.6% (w / w), the extracted berberine is 13.1g, and the extract yield is 2.64%. Therefore, the best extraction effect of berberine powder is achieved by preparing an aqueous two-phase system with an ammonium sulfate solution of 28.6% (w / w) and isopropanol.

[0027] Comparative Example 4 Weigh 500g of berberine powder and place it in an extraction vessel. Add 28.6 (w / w) ammonium sulfate solution to the extraction vessel, add a compound enzyme preparation, and add 3L of isopropanol. Tighten the lid of the extraction vessel and place it in an ultrasonic-microwave synergistic extraction instrument. Perform ultrasonic treatment for 10 minutes and microwave extraction for 10 minutes. Centrifuge at 4000 rpm. Remove the isopropanol layer by distillation to obtain the crude extract. Dissolve the crude extract in water and load it onto a polar macroporous resin. First, wash away the strong polar impurities with water, then wash away the weak polar impurities with 30% ethanol, and finally wash away the target product with 75% ethanol to obtain berberine.

[0028] Table 4. Effects of different enzymes on the extraction of berberine

[0029] As shown in Table 4, the combined use of cellulase and pectinase is more effective than that of hemicellulase and pectinase. Furthermore, a cellulase to pectinase ratio of 2:1 yields the best extraction effect for berberine. This may be because: using citrate-sodium citrate buffer as the solvent for the compound enzyme preparation provides the optimal pH for both cellulase and pectinase; the addition of magnesium chloride and calcium chloride to the compound enzyme preparation allows them to bind to the proteins of either cellulase or pectinase, enhancing the spatial stability of the enzymes and thus improving their combined activity; and the use of cellulase to hydrolyze the cellulose component of berberine powder and pectinase to hydrolyze the pectin component, combined with cellulase, promotes a more porous structure in the berberine powder, facilitating the release of berberine or the dissolution of berberine by isopropanol.

[0030] Comparative Example 5 Weigh 500g of *Radermachera sinensis* powder and place it in an extraction vessel. Add 28.6g (w / w) ammonium sulfate solution to the extraction vessel. Add a compound enzyme preparation to the extraction vessel. The compound enzyme preparation is prepared by dissolving 20g of cellulase (enzyme activity approximately 1000U / g) and 10g of pectinase (enzyme activity approximately 800U / g) in 0.5L of water. Add 3L of isopropanol. Tighten the lid of the extraction vessel. Place the extraction vessel in an ultrasonic-microwave synergistic extraction instrument. Perform ultrasonic treatment for 10 minutes and then microwave extraction for 10 minutes. Centrifuge at 4000 rpm. Remove the isopropanol layer by distillation to obtain the crude extract. Dissolve the crude extract in water and load it onto a polar macroporous resin. First, wash away strong polar impurities with water, then wash away weak polar impurities with 30% ethanol, and finally wash away the target product with 75% ethanol to obtain rabinol.

[0031] Comparative Example 6 Weigh 500g of *Radermachera sinensis* powder and place it in an extraction vessel. Add 28.6g (w / w) ammonium sulfate solution to the extraction vessel. Add a compound enzyme preparation to the extraction vessel. The compound enzyme preparation is prepared by dissolving 20g of cellulase (enzyme activity approximately 1000U / g) and 10g of pectinase (enzyme activity approximately 800U / g) in 0.5L of citrate-sodium citrate buffer. Add 3L of isopropanol, tighten the lid of the extraction vessel, and place the extraction vessel in an ultrasonic-microwave synergistic extraction instrument. Perform ultrasonic treatment for 10 minutes and then microwave extraction for 10 minutes. Centrifuge the extracted mixture for 10 minutes at 4000 rpm. Remove the isopropanol layer by distillation to obtain the crude extract. Dissolve the crude extract in water and load it onto a polar macroporous resin. First, wash away strongly polar impurities with water, then wash away weakly polar impurities with 30% ethanol, and finally wash away the target product with 75% ethanol to obtain rabinol.

[0032] Comparative Example 7 Weigh 500g of *Radermachera sinensis* powder and place it in an extraction vessel. Add 28.6% (w / w) ammonium sulfate solution to the extraction vessel. Add a compound enzyme preparation to the extraction vessel. The compound enzyme preparation is prepared by dissolving 20g of cellulase (enzyme activity approximately 1000U / g) and 10g of pectinase (enzyme activity approximately 800U / g) in 0.5L of citrate-sodium citrate buffer. Add 3L of isopropanol, tighten the lid of the extraction vessel, and place the extraction vessel in an ultrasonic-microwave synergistic extraction instrument for ultrasonic extraction for only 10 minutes. Centrifuge the extracted mixture for 10 minutes at 4000 rpm. Remove the isopropanol layer by distillation to obtain the crude extract. Dissolve the crude extract in water and heat under reflux with 85% ethanol for 30 minutes. Filter and concentrate the filtrate to obtain the *Radermachera sinensis* product.

[0033] Comparative Example 8 Weigh 500g of *Radermachera sinensis* powder and place it in an extraction vessel. Add 28.6% (w / w) ammonium sulfate solution to the extraction vessel. Add a compound enzyme preparation to the extraction vessel. The compound enzyme preparation is prepared by dissolving 20g of cellulase (enzyme activity approximately 1000U / g) and 10g of pectinase (enzyme activity approximately 800U / g) in 0.5L of citrate-sodium citrate buffer. Add 3L of ethanol, tighten the lid of the extraction vessel, and place the extraction vessel in an ultrasonic-microwave synergistic extraction instrument for ultrasonic extraction for 10 minutes. Centrifuge the extracted mixture for 10 minutes at 4000 rpm. Remove the isopropanol layer by distillation to obtain the crude extract. Dissolve the crude extract in water and heat under reflux with 85% ethanol for 30 minutes. Filter and concentrate the filtrate to obtain the *Radermachera sinensis* product.

[0034] Table 5. Extraction of berberine and yield of berberine extract in different embodiments Example 1 Comparative Example 5 Comparative Example 6 Comparative Example 7 Comparative Example 8 Berberine (g) 28.9 13.4 13.9 12.7 10.3 Extract yield (%) 2.89 2.68 2.78 2.54 2.06 Table 5 shows that using citrate-sodium citrate buffer, calcium chloride, and magnesium chloride as components of the complex enzyme preparation resulted in a higher extract yield than using water. This indicates that citrate-sodium citrate buffer, calcium chloride, and magnesium chloride promote the release of berberine from the powder, and that isopropanol is more effective in dissolving berberine in the powder. A comparison of Example 1 with Comparative Examples 5-8 shows that the extraction solvent, extraction method, and purification method have a significant impact on the yield of berberine and the extract.

[0035] This invention can be implemented in various ways and is not limited to the embodiments described. Those skilled in the art will understand that the invention can be implemented in other specific ways without changing the technical concept or essential features. Therefore, it should be understood that the embodiments described above are exemplary and not intended to limit the invention.

Claims

1. A method for efficiently extracting and purifying berberine, characterized in that, The method includes: (1) Select dried stems of rattan, crush and sieve them to obtain rattan powder; (2) Weigh out the powder of *Rhizoma Cimicifugae* and mix it with 20-30% (w / w) ammonium sulfate solution, then add a compound enzyme preparation for enzymatic hydrolysis, add isopropanol to obtain a mixture, the mixture is extracted by ultrasonic-microwave synergistic extraction, the extracted mixture is centrifuged for 10-13 min at 3500-4000 rpm, the isopropanol layer is taken and distilled to remove isopropanol to obtain crude extract; (3) Dissolve the crude extract in water and load it onto a polar macroporous resin for repeated elution to obtain berberine.

2. The method for efficiently extracting and purifying berberine as described in claim 1, characterized in that, The amount of the compound enzyme preparation used is 0.1-0.5% of the mass of the tung oil powder.

3. The method for efficiently extracting and purifying berberine as described in claim 2, characterized in that, The compound enzyme preparation consists of magnesium chloride, calcium chloride, cellulase, pectinase, and citrate-sodium citrate buffer.

4. The method for efficiently extracting and purifying berberine as described in claim 3, characterized in that, The preparation method of the compound enzyme preparation is as follows: Take 0.1-0.2g of magnesium chloride and 0.1-0.2g of calcium chloride and add them to 0.5-3L of citrate-sodium citrate buffer solution. Take 20-35g of cellulase and add it to the citrate-sodium citrate buffer solution and mix well. Take 10-18g of pectinase and add it to the above solution and mix well to obtain the compound enzyme preparation.

5. The method for efficiently extracting and purifying berberine as described in claim 4, characterized in that, The cellulase has an enzyme activity of 900-1100 U / g, and the pectinase has an enzyme activity of 700-900 U / g.

6. The method for efficiently extracting and purifying berberine as described in claim 1, characterized in that, The enzymatic hydrolysis conditions for the compound enzyme preparation are: enzymatic hydrolysis reaction at a temperature of 45-50℃ for 1-1.5 hours.

7. The method for efficiently extracting and purifying berberine as described in claim 1, characterized in that, The 28.6% (w / w) ammonium sulfate solution was prepared by dissolving 3.43 kg of ammonium sulfate in 8.57 L of deionized water and stirring to obtain 28.6% (w / w) ammonium sulfate.

8. The method for efficiently extracting and purifying berberine as described in claim 1, characterized in that, The mixture was extracted using an ultrasonic-microwave synergistic extraction method as follows: the mixture was first subjected to ultrasonic treatment for 10 minutes at an ultrasonic power of 300 W and a frequency of 25 kHz; then it was subjected to microwave extraction for 10 minutes at a microwave power of 500 W and a temperature setting of 75°C.

9. The method for efficiently extracting and purifying berberine as described in claim 1, characterized in that, In the step of dissolving the crude extract in water and loading it onto a polar macroporous resin for multiple elutions to obtain berberine, the multiple elutions refer to first eluting strongly polar impurities with water, then eluting weakly polar impurities with 20-30% ethanol, and finally eluting berberine with 60-75% ethanol.

10. The method for efficiently extracting and purifying berberine as described in claim 9, characterized in that, The ethanol concentration for eluting weakly polar impurities was 30%, and the ethanol concentration for eluting berberine was 75%.

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