Method for extracting artemisinin through enzymolysis pretreatment

Abstract

The invention relates to the technical field of plant effective component extraction, and discloses a method for extracting artemisinin through enzymolysis pretreatment, which comprises the steps of raw material pretreatment, composite enzymolysis pretreatment, ultrasonic-assisted extraction, impurity removal and concentration, purification and crystallization, and recrystallization and refining. A compound enzyme system composed of cellulase, hemicellulase and pectinase is adopted to conduct enzymolysis on artemisia annua powder, cell wall structures are destroyed to promote artemisinin release, ultrasonic-assisted extraction is combined to strengthen the extraction effect, and the product purity is improved through chitosan impurity removal, silica gel chromatography and two times of crystallization. The method is simple in process, environmentally friendly and energy-saving, effectively solves the problems that a traditional extraction method is low in extraction rate, insufficient in purity and high in cost, is suitable for large-scale industrial production, and has high practical value and industrialization prospects.

Description

Technical Field

[0001] This invention relates to the field of plant active ingredient extraction technology, specifically a method for extracting artemisinin through enzymatic pretreatment. Background Technology

[0002] Artemisinin is a sesquiterpene lactone compound with a peroxy-bridged structure extracted from Artemisia annua. It possesses highly effective, rapid-acting, and low-toxicity antimalarial activity, particularly showing significant efficacy against cerebral malaria and chloroquine-resistant malaria. Furthermore, the potential applications of artemisinin in antitumor, antischistosomiasis, and immunomodulatory fields have further increased its market demand.

[0003] Currently, the industrial extraction of artemisinin mainly relies on natural product extraction methods. Traditional processes include Soxhlet extraction, reflux extraction, and cold maceration extraction. These methods generally suffer from low extraction rates, high solvent consumption, long extraction times, and insufficient product purity. The core issue lies in the fact that the cell walls of Artemisia annua are primarily composed of cellulose and hemicellulose. Artemisinin is encapsulated within these cell walls, and traditional extraction processes struggle to disrupt this cell wall structure, resulting in high solvent mass transfer resistance, insufficient release of artemisinin, and low raw material utilization.

[0004] To address these issues, existing technologies have attempted enzymatic-assisted extraction, such as using cellulase alone for pretreatment. However, the effectiveness of single enzyme systems is limited, enzymatic hydrolysis efficiency is unstable, and the subsequent extraction and purification steps are not well-coordinated, resulting in drawbacks such as insignificant improvement in extraction rate, complex processes, and high costs. Some processes use strong acid environments or highly toxic organic solvents, which not only damage the structural stability of artemisinin but also cause environmental pollution, contradicting the concept of green production. Furthermore, while highly efficient methods such as supercritical CO2 extraction achieve high extraction rates, the huge investment required for equipment makes them difficult to widely apply in small and medium-sized enterprises.

[0005] Therefore, developing a simple, efficient, high-purity, environmentally friendly, energy-saving, and cost-controllable enzymatic pretreatment method for extracting artemisinin is of great significance for promoting the industrialization of artemisinin. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide a method for extracting artemisinin through enzymatic pretreatment, thereby achieving efficient and green extraction of artemisinin, improving the extraction rate and product purity, reducing production costs, and meeting the needs of industrial production.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for extracting artemisinin through enzymatic pretreatment, comprising the following steps: (1) Raw material pretreatment: Select the dried aerial parts of Artemisia annua, remove impurities, crush them, pass them through a 40-60 mesh sieve, and dry them until the moisture content is ≤8% for later use; (2) Pretreatment with compound enzyme hydrolysis: Add Artemisia annua powder to buffer solution at a material-to-liquid ratio of (1:10)-(1:20) (g / mL), adjust the pH value to 4.0-5.0, add compound enzyme preparation, and hydrolyze at a constant temperature of 45-55℃ for 1.5-3h. After hydrolysis, raise the temperature to 85-90℃ and keep it at 15-20min to inactivate the enzyme, and cool to room temperature. (3) Ultrasonic-assisted extraction: Add 90%-95% ethanol to the enzymatically hydrolyzed material. The amount of ethanol is 8-12 times (mL / g) of the mass of Artemisia annua powder. Ultrasonic extraction is performed for 30-60 min at a power of 200-300W and a temperature of 30-40℃. The filter cake is filtered and washed. The filtrate and washing liquid are combined to obtain the crude extract. (4) Impurity removal and concentration: Add chitosan solution to the crude extract, adjust the chitosan concentration to 0.01-0.02 g / mL, stir and let stand, filter to remove impurities, take the supernatant and concentrate under reduced pressure, recover ethanol, and obtain artemisinin concentrate; (5) Purification and crystallization: Dissolve the concentrate in 95%-99% ethanol, elute by chromatography, concentrate, crystallize under cold, wash and dry to obtain crude artemisinin; (6) Recrystallization purification: Dissolve the crude product in 99% ethanol, filter while hot, crystallize under cold, and dry to obtain artemisinin product.

[0008] Furthermore, in step (1), the drying conditions for the raw material pretreatment are drying in an oven at 60-70℃ for 2-3 hours.

[0009] Further, in step (2), the buffer solution is a citrate-sodium citrate buffer solution with a concentration of 0.05-0.1 mol / L.

[0010] Further, the compound enzyme preparation in step (2) is composed of cellulase, hemicellulase and pectinase mixed in a mass ratio of (2-3):(1-2):1; the cellulase activity is 1,000,000 to 1,500,000 u / g, the hemicellulase activity is 800,000 to 1,200,000 u / g, and the pectinase activity is 600,000 to 1,000,000 u / g; the amount of compound enzyme preparation added is 0.3% to 0.8% of the mass of Artemisia annua powder.

[0011] Furthermore, in step (2), the enzyme hydrolysis process is stirred for 5-10 minutes every 20-30 minutes, and the stirring speed is 100-150 r / min.

[0012] Further, in step (4), the chitosan solution is prepared by dissolving chitosan in 1%-2% acetic acid solution, with a concentration of 0.1-0.2 g / mL; the stirring time is 30-40 min, and the standing time is 1-2 h; the vacuum concentration conditions are vacuum degree -0.08~-0.09 MPa and temperature 50-60℃.

[0013] Further, in step (5), the chromatography medium of the chromatography column is silica gel G, the column temperature is controlled at 25-30℃, the eluent is a mixture of petroleum ether and ethanol with a volume ratio of 8-10:1, the elution flow rate is 1-2 mL / min, the refrigeration crystallization temperature is 4-8℃, and the time is 8-12 h.

[0014] Furthermore, in step (6), the solid-liquid ratio for recrystallization is 1:5-1:8 (g / mL), the dissolution temperature is 60-70℃, and the refrigerated crystallization time is 6-8h.

[0015] Furthermore, the extraction rate of the artemisinin product is ≥89%, and the purity is ≥98.5%.

[0016] Compared with the prior art, the present invention has the following beneficial technical effects: This invention employs a complex enzyme system composed of cellulase, hemicellulase, and pectinase. Each enzyme specifically degrades different components of the cell wall, achieving complete disruption of the cell wall structure and fully exposing the previously encapsulated artemisinin, thus removing obstacles for subsequent solvent extraction. Compared to single enzymatic hydrolysis, the synergistic effect of the complex enzyme system significantly enhances the cell wall disruption effect and substantially improves the release efficiency of artemisinin. Based on enzymatic cell wall disruption, ultrasonic-assisted extraction is combined. The cavitation effect of ultrasound creates localized high pressure and microjets, accelerating the penetration of the extraction solvent into the Artemisia annua tissue. Simultaneously, it promotes the rapid dissolution of released artemisinin into the solvent, significantly improving the contact efficiency and mass transfer rate between the solvent and artemisinin. Furthermore, processes such as enzyme inactivation and filter cake washing after enzymatic hydrolysis effectively avoid material loss during extraction, maximizing the extraction of artemisinin from the raw material and significantly improving the overall utilization rate of the raw material.

[0017] This invention utilizes the flocculation properties of chitosan to specifically remove water-soluble impurities such as polysaccharides, proteins, and pigments from the crude extract, achieving preliminary impurity removal without secondary pollution. Then, the adsorption-desorption difference of the chromatography column is used to separate artemisinin from trace impurities, achieving purification. Finally, a two-step crystallization process of purification crystallization and recrystallization is employed, utilizing the selectivity of crystal precipitation to remove residual trace organic solvents and small molecule impurities, allowing artemisinin to precipitate in a high-purity form. The solvents used in the entire extraction and dissolution process have high compatibility with the artemisinin structure, good solubility, and do not undergo chemical reactions. This ensures the complete dissolution of artemisinin while avoiding the formation of new contaminants from solvent-impurity combinations. Precise control of process parameters in each purification step ensures stable product purity, meeting the quality requirements of pharmaceutical-grade products.

[0018] The entire process operates at medium to low temperatures. Enzymatic hydrolysis and ultrasonic extraction do not require high temperature and high pressure conditions, and vacuum concentration is also carried out at low temperatures. Compared with traditional high-temperature reflux extraction and supercritical extraction, this significantly reduces heating energy consumption, achieving energy-saving production. Furthermore, the process does not use strong acids or alkalis, avoiding the generation of acidic or alkaline wastewater and simplifying subsequent wastewater treatment. In addition, the extracted Artemisia annua filter cake is a chemically free plant residue that can be reused as a resource, generating no harmful waste and aligning with the concept of a circular economy. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1 A method for extracting artemisinin through enzymatic pretreatment, comprising the following specific steps: Raw material pretreatment: Select dried Artemisia annua leaves, remove impurities, pulverize, and pass through a 40-mesh sieve to obtain Artemisia annua powder; place the powder in a 60℃ oven to dry for 3 hours, and cool to room temperature; Pretreatment with compound enzymatic hydrolysis: Weigh 100g of Artemisia annua powder and add it to the reaction vessel. Add 1500mL of 0.08mol / L citrate-sodium citrate buffer at a material-to-liquid ratio of 1:15 (g / mL) to adjust the pH of the system to 4.5. Add 0.5g of compound enzyme preparation (cellulase, hemicellulase, and pectinase in a mass ratio of 2:1:1, with cellulase activity of 1.2 million u / g, hemicellulase activity of 1 million u / g, and pectinase activity of 0.8 million u / g). After stirring evenly, enzymatic hydrolyze at a constant temperature of 50℃ for 2h. During the enzymatic hydrolysis, stir for 8min every 25min at a speed of 120r / min. After the enzymatic hydrolysis is completed, heat to 88℃ and incubate for 18min to inactivate the enzyme. Cool to room temperature to obtain the enzymatically hydrolyzed material. Ultrasonic-assisted extraction: Add 1000 mL of 92% ethanol (10 times the mass of Artemisia annua powder) to the enzymatic hydrolysate, stir well, and place in an ultrasonic device. Extract for 45 min using intermittent ultrasonic extraction (3 s for ultrasound, 2 s interval) at 250 W and 35 °C. After extraction, filter by pressure, and wash the filter cake twice with 50 mL of 92% ethanol each time. Combine the filtrate and washing liquid to obtain the crude extract. Impurity removal and concentration: Add 0.15 g / mL chitosan-acetic acid solution (chitosan is dissolved in 1.5% acetic acid solution) to the crude extract to adjust the chitosan concentration to 0.015 g / mL, stir for 35 min, let stand for 1.5 h, filter to remove precipitate; take the supernatant and place it in a rotary evaporator, concentrate under reduced pressure at a vacuum of -0.085 MPa and a temperature of 55 °C, recover ethanol, and obtain artemisinin concentrate; Purification and crystallization: The concentrate was dissolved in 98% ethanol to prepare a 0.15 g / mL solution, which was then added to a silica gel G column (column temperature 28℃). Elution was performed using a petroleum ether-ethanol mixture (volume ratio 9:1) at a flow rate of 1.5 mL / min. The eluent was collected. The eluent was concentrated under reduced pressure to 1 / 4 of its original volume, cooled to 6℃, and allowed to stand for 10 h to precipitate crystals. The crystals were collected by filtration, washed once with a small amount of ice-cold ethanol, and dried at 45℃ under a vacuum of -0.095 MPa for 2.5 h to obtain crude artemisinin. Recrystallization purification: Crude artemisinin was added to 99% ethanol at a solid-liquid ratio of 1:6 (g / mL), heated to 65℃ to dissolve completely, and filtered while hot; the filtrate was slowly cooled to room temperature and then refrigerated at 6℃ for 7 hours to precipitate crystals; after filtration, it was vacuum dried to constant weight to obtain the finished artemisinin product. The yield of artemisinin was calculated by weighing and material balance, and the extraction rate was 92.1%; the purity was 98.8% as determined by high performance liquid chromatography (HPLC).

[0021] Example 2 A method for extracting artemisinin through enzymatic pretreatment, comprising the following specific steps: Raw material pretreatment: Select dried Artemisia annua leaves and tender stems, remove impurities and crush them, pass them through a 50-mesh sieve to obtain Artemisia annua powder; place the powder in a 65℃ oven to dry for 2.5 hours, and cool to room temperature; Pretreatment with compound enzymatic hydrolysis: Weigh 100g of Artemisia annua powder and add it to the reaction vessel. Add 1200mL of 0.05mol / L citrate-sodium citrate buffer at a material-to-liquid ratio of 1:12 (g / mL) to adjust the pH of the system to 4.2. Add 0.3g of compound enzyme preparation (cellulase, hemicellulase, and pectinase in a mass ratio of 3:2:1, with cellulase activity of 1.5 million u / g, hemicellulase activity of 1.2 million u / g, and pectinase activity of 1 million u / g). After stirring evenly, enzymatically hydrolyze at a constant temperature of 48℃ for 1.5h. During the enzymatic hydrolysis, stir for 5min every 20min at a speed of 100r / min. After the enzymatic hydrolysis is completed, heat to 85℃ and incubate for 20min to inactivate the enzyme. Cool to room temperature to obtain the enzymatically hydrolyzed material. Ultrasonic-assisted extraction: Add 800 mL of 90% ethanol (8 times the mass of Artemisia annua powder) to the enzymatic hydrolysate, stir well, and place in an ultrasonic device. Perform intermittent ultrasonic extraction for 30 min at a power of 200 W and a temperature of 30 °C. After extraction, filter the filter cake and wash it three times with 40 mL of 90% ethanol each time. Combine the filtrate and washing liquid to obtain the crude extract. Impurity removal and concentration: Add 0.1 g / mL chitosan-acetic acid solution (chitosan is dissolved in 1% acetic acid solution) to the crude extract to adjust the chitosan concentration to 0.01 g / mL, stir for 30 min and let stand for 1 h, filter to remove precipitate; take the supernatant and concentrate under reduced pressure (vacuum degree -0.08 MPa, temperature 50℃), recover ethanol, and obtain artemisinin concentrate; Purification and crystallization: The concentrate was dissolved in 95% ethanol to prepare a 0.1 g / mL solution, which was then added to a silica gel G column (column temperature 25℃). The column was eluted with a petroleum ether-ethanol mixture (volume ratio 8:1) at a flow rate of 1 mL / min. The eluent was collected, concentrated to 1 / 5 of its original volume, cooled to 4℃, and allowed to stand for 8 h to precipitate crystals. After filtration, the crystals were washed with ice-cold ethanol and dried under vacuum at 40℃ for 2 h to obtain crude artemisinin. Recrystallization purification: The crude product was added to 99% ethanol at a solid-liquid ratio of 1:5 (g / mL), heated to 60℃ to dissolve, and filtered while hot; the filtrate was cooled and refrigerated for 6 hours to precipitate crystals, which were then dried to constant weight to obtain artemisinin product with an extraction rate of 89.5% and a purity of 98.6%.

[0022] Example 3 A method for extracting artemisinin through enzymatic pretreatment, comprising the following specific steps: Raw material pretreatment: Select dried Artemisia annua leaves, remove impurities, pulverize, and pass through a 60-mesh sieve to obtain Artemisia annua powder; dry in an oven at 70℃ for 2 hours, and cool to room temperature; Pretreatment with compound enzymatic hydrolysis: Weigh 100g of Artemisia annua powder and add it to the reaction vessel. Add 2000mL of 0.1mol / L citrate-sodium citrate buffer at a material-to-liquid ratio of 1:20 (g / mL) and adjust the pH to 4.8. Add 0.8g of compound enzyme preparation (cellulase, hemicellulase, and pectinase in a mass ratio of 2:2:1, with cellulase activity of 1 million u / g, hemicellulase activity of 800,000 u / g, and pectinase activity of 600,000 u / g). Hydrolyze at 55℃ for 3 hours, stirring for 10 minutes every 30 minutes at a speed of 150 r / min. After hydrolysis, incubate at 90℃ for 15 minutes to inactivate the enzyme and cool to room temperature. Ultrasonic-assisted extraction: Add 1200 mL of 95% ethanol (12 times the mass of Artemisia annua powder) to the enzymatically hydrolyzed material, stir well, and place it in an ultrasonic device. Under the conditions of 300 W power and 40 °C, use intermittent ultrasonic mode (3 s ultrasound, 2 s interval) for ultrasonic extraction for 60 min. After extraction, filter by pressure, collect the filtrate, and wash the filter cake three times with 60 mL of 90% ethanol each time. Combine the washing liquid and filtrate to obtain the crude extract. Impurity removal and concentration: Chitosan solution (preparation method: add chitosan to 2% acetic acid solution, stir until completely transparent, concentration is 0.2 g / mL) to crude extract, adjust chitosan concentration to 0.02 g / mL, stir for 40 min and let stand for 2 h to allow impurities to flocculate and precipitate; then filter to remove precipitate, take supernatant and place in rotary evaporator, concentrate under reduced pressure at vacuum degree -0.09 MPa and temperature 60℃, recover ethanol, concentrate until no ethanol distillation, to obtain artemisinin concentrate; Purification and crystallization: The artemisinin concentrate was dissolved in 99% ethanol to prepare a solution with a concentration of 0.2 g / mL. The solution was added to a chromatography column (chromatographic medium: silica gel G) for elution. The column temperature was controlled at 30℃, and the eluent was a petroleum ether-ethanol mixture (volume ratio 10:1). The elution flow rate was 2 mL / min, and the eluent was collected. The eluent was concentrated again under reduced pressure to 1 / 3 of its original volume, cooled to 8℃, and allowed to stand for 12 h to precipitate crystals. The crystals were collected by filtration, washed twice with a small amount of ice-cold ethanol, and dried to obtain crude artemisinin. Recrystallization purification: Crude artemisinin was added to 99% ethanol at a solid-liquid ratio of 1:8 (g / mL), heated to 70℃ to completely dissolve, and filtered while hot to remove insoluble impurities; the filtrate was slowly cooled to room temperature and then refrigerated for 8 hours to precipitate pure crystals; after filtration, it was vacuum dried to constant weight to obtain 9.32g of finished artemisinin product, with an extraction rate of 93.2%, and the purity was 99.0% as determined by high performance liquid chromatography (HPLC).

[0023] Comparative Example 1 The main difference between this comparative example and Example 3 is that the compound enzyme preparation only uses cellulase, without adding hemicellulase and pectinase, and the amount of cellulase added is the same as the total amount added in the original compound enzyme preparation.

[0024] The extraction rate was 76.5% and the purity was 95.2%. The single enzyme system could not completely degrade the cell wall, resulting in insufficient release of artemisinin and a significant decrease in the extraction rate. No additional impurities were introduced, but the purity was slightly reduced.

[0025] Comparative Example 2 The main difference between this comparative example and Example 3 is that the pH value of the compound enzymatic hydrolysis was adjusted to a neutral range of 7.0, while other enzymatic hydrolysis conditions remained unchanged. The extraction rate was 68.9%, and the purity was 94.5%. The neutral environment deviated from the optimal enzyme activity range of the compound enzyme system, resulting in a sharp drop in enzymatic hydrolysis efficiency, poor cell wall disruption, and a significant decrease in extraction rate. The pH setting did not introduce any impurities, leading to a slight decrease in purity.

[0026] Comparative Example 3 The main difference between this comparative example and Example 3 is that chitosan solution was not added in the impurity removal step, and the crude extract was directly concentrated under reduced pressure. The extraction rate was 90.5% and the purity was 86.3%. Without chitosan flocculation for impurity removal, polysaccharides, proteins, and other impurities remained in the crude extract, which could not be completely removed in subsequent purification, resulting in a significant decrease in purity. There was no material loss in the impurity removal step, but the extraction rate was slightly reduced.

[0027] Comparative Example 4 The main difference between this comparative example and Example 3 is that the silica gel G chromatography column elution step is omitted in the purification and crystallization process. The concentrate is directly dissolved in ethanol and then subjected to cold crystallization. The extraction rate is 91.8%, and the purity is 89.5%. Without the precise separation of the chromatography column, trace amounts of lipid-soluble impurities remain in the concentrate, resulting in a significant decrease in purity. Without the material loss from chromatography elution, the extraction rate is slightly lower.

[0028] Comparative Example 5 The main difference between this comparative example and Example 3 is that the temperature for the compound enzymatic hydrolysis was controlled at a low temperature of 30°C, while other enzymatic hydrolysis conditions remained unchanged. The extraction rate was 70.2%, and the purity was 94.3%. Low temperature inhibited the catalytic activity of the enzyme, making the enzymatic hydrolysis reaction difficult to proceed, resulting in incomplete cell wall degradation and a significant decrease in the extraction rate. Temperature did not affect the types of impurities, but the purity decreased slightly.

[0029] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0030] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0031] Those skilled in the art should understand that the above descriptions are merely several specific embodiments of the present invention, and not all embodiments.

Claims

1. A method for extracting artemisinin through enzymatic pretreatment, characterized in that, Includes the following steps: (1) Raw material pretreatment: Select the dried aerial parts of Artemisia annua, remove impurities, crush them, and pass them through a 40-60 mesh sieve for later use; (2) Pretreatment with compound enzyme hydrolysis: Add Artemisia annua powder to buffer solution at a material-to-liquid ratio of (1:10)-(1:20) (g / mL), adjust the pH value to 4.0-5.0, add compound enzyme preparation, and hydrolyze at a constant temperature of 45-55℃ for 1.5-3h. After hydrolysis, raise the temperature to 85-90℃ and keep it at 15-20min to inactivate the enzyme, and cool to room temperature. (3) Ultrasonic-assisted extraction: Add 90%-95% ethanol to the enzymatically hydrolyzed material. The amount of ethanol is 8-12 times (mL / g) of the mass of Artemisia annua powder. Ultrasonic extraction is performed for 30-60 min at a power of 200-300W and a temperature of 30-40℃. The filter cake is filtered and washed. The filtrate and washing liquid are combined to obtain the crude extract. (4) Impurity removal and concentration: Add chitosan solution to the crude extract, adjust the chitosan concentration to 0.01-0.02 g / mL, stir and let stand, filter to remove impurities, take the supernatant and concentrate under reduced pressure, recover ethanol, and obtain artemisinin concentrate; (5) Purification and crystallization: Dissolve the concentrate in 95%-99% ethanol, elute by chromatography, concentrate, crystallize under cold, wash and dry to obtain crude artemisinin; (6) Recrystallization purification: Dissolve the crude product in 99% ethanol, filter while hot, crystallize under cold, and dry to obtain artemisinin product.

2. The method for extracting artemisinin through enzymatic pretreatment according to claim 1, characterized in that, In step (1), the drying conditions for raw material pretreatment are drying in an oven at 60-70℃ for 2-3 hours.

3. The method for extracting artemisinin through enzymatic pretreatment according to claim 1, characterized in that, In step (2), the buffer solution is a citrate-sodium citrate buffer solution with a concentration of 0.05-0.1 mol / L.

4. The method for extracting artemisinin through enzymatic pretreatment according to claim 1, characterized in that, The compound enzyme preparation in step (2) is composed of cellulase, hemicellulase and pectinase mixed in a mass ratio of (2-3):(1-2):1; the cellulase activity is 1,000,000 to 1,500,000 u / g, the hemicellulase activity is 800,000 to 1,200,000 u / g, and the pectinase activity is 600,000 to 1,000,000 u / g; the amount of compound enzyme preparation added is 0.3% to 0.8% of the mass of Artemisia annua powder.

5. The method for extracting artemisinin through enzymatic pretreatment according to claim 1, characterized in that, In step (2), the enzyme hydrolysis process is stirred for 5-10 minutes every 20-30 minutes, and the stirring speed is 100-150 r / min.

6. The method for extracting artemisinin through enzymatic pretreatment according to claim 1, characterized in that, In step (4), the chitosan solution is prepared by dissolving chitosan in 1%-2% acetic acid solution, with a concentration of 0.1-0.2 g / mL; the stirring time is 30-40 min, and the standing time is 1-2 h; the vacuum concentration conditions are vacuum degree -0.08~-0.09 MPa and temperature 50-60℃.

7. The method for extracting artemisinin through enzymatic pretreatment according to claim 1, characterized in that, In step (5), the chromatography medium of the chromatography column is silica gel G, the column temperature is controlled at 25-30℃, the eluent is a mixture of petroleum ether and ethanol with a volume ratio of 8-10:1, the elution flow rate is 1-2 mL / min, the refrigeration crystallization temperature is 4-8℃, and the time is 8-12 h.

8. The method for extracting artemisinin through enzymatic pretreatment according to claim 1, characterized in that, In step (6), the solid-liquid ratio for recrystallization is 1:5-1:8 (g / mL), the dissolution temperature is 60-70℃, and the refrigerated crystallization time is 6-8h.