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CN-121991090-A - Method for extracting artemisinin by enzymolysis pretreatment

CN121991090ACN 121991090 ACN121991090 ACN 121991090ACN-121991090-A

Abstract

The invention relates to the technical field of plant active ingredient extraction, and discloses a method for extracting artemisinin by 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 refining; the composite enzyme system composed of cellulase, hemicellulase and pectase is adopted to carry out enzymolysis on the artemisia annua powder, the cell wall structure is destroyed to promote the release of artemisinin, the ultrasonic auxiliary extraction is combined to strengthen the extraction effect, and the purity of the product is improved through the impurity removal of chitosan, silica gel chromatography and twice crystallization. The method has the advantages of simple process, environmental protection and energy saving, effectively solves the problems of low extraction rate, insufficient purity and high cost of the traditional extraction method, is suitable for large-scale industrial production, and has higher practical value and industrialization prospect.

Inventors

  • ZHANG MEI
  • PENG XUEDONG
  • ZHAO JINZHAO
  • ZHOU LEI

Assignees

  • 湖南斯依康生物科技有限公司

Dates

Publication Date
20260508
Application Date
20260324

Claims (8)

  1. 1. The method for extracting artemisinin by enzymolysis pretreatment is characterized by comprising the following steps of: (1) Pretreating raw materials, namely selecting the dried aerial parts of the artemisia annua, removing impurities, crushing, and sieving with a 40-60-mesh sieve for later use; (2) The preparation method comprises the steps of (1) adding buffer solution into artemisia annua powder according to the feed-liquid ratio (1:10) - (1:20) (g/mL), regulating the pH value to 4.0-5.0, adding a compound enzyme preparation, carrying out enzymolysis for 1.5-3h at the constant temperature of 45-55 ℃, heating to 85-90 ℃ after enzymolysis, preserving heat for 15-20min, inactivating enzyme, and cooling to room temperature; (3) Ultrasonic assisted extraction, namely adding 90% -95% ethanol into enzymolysis materials, wherein the ethanol dosage is 8-12 times (mL/g) of the mass of the artemisia annua powder, performing ultrasonic extraction for 30-60min at the power of 200-300W and the temperature of 30-40 ℃, filtering and washing filter cakes, and combining filtrate and washing liquid to obtain crude extract; (4) Adding chitosan solution into the crude extract, adjusting chitosan concentration to 0.01-0.02g/mL, stirring, standing, filtering to remove impurities, collecting supernatant, concentrating under reduced pressure, and recovering ethanol to obtain artemisinin concentrate; (5) Purifying and crystallizing, namely dissolving the concentrate with 95% -99% ethanol, eluting by a chromatographic column, concentrating, refrigerating, crystallizing, washing and drying to obtain crude artemisinin; (6) Recrystallizing and refining, dissolving the crude product with 99% ethanol, filtering while hot, refrigerating for crystallization, and drying to obtain arteannuin product.
  2. 2. The method for extracting artemisinin by enzymolysis pretreatment according to claim 1, wherein in the step (1), the drying condition of the pretreatment of the raw materials is 60-70 ℃ oven drying for 2-3 hours.
  3. 3. The method for extracting artemisinin by enzymolysis pretreatment according to claim 1, wherein the buffer solution in the step (2) is a citric acid-sodium citrate buffer solution with the concentration of 0.05-0.1mol/L.
  4. 4. The method for extracting artemisinin by enzymolysis pretreatment according to claim 1, wherein the compound enzyme preparation in the step (2) is formed by mixing cellulase, hemicellulase and pectase according to the mass ratio of (2-3) (1-2) (1), wherein the enzyme activity of the cellulase is 100-150 mu/g, the enzyme activity of the hemicellulase is 80-120 mu/g, the enzyme activity of the pectase is 60-100 mu/g, and the adding amount of the compound enzyme preparation is 0.3% -0.8% of the mass of the artemisia annua powder.
  5. 5. The method for extracting artemisinin by enzymolysis pretreatment according to claim 1, wherein stirring is performed for 5-10min every 20-30min in the enzymolysis process in the step (2), and the stirring speed is 100-150r/min.
  6. 6. The method for extracting artemisinin by enzymolysis pretreatment according to claim 1, wherein in the step (4), chitosan solution is prepared by dissolving chitosan in 1% -2% acetic acid solution, the concentration is 0.1-0.2g/mL, the stirring time is 30-40min, the standing time is 1-2h, and the vacuum concentration condition is that the vacuum degree is-0.08-0.09 MPa and the temperature is 50-60 ℃.
  7. 7. The method for extracting artemisinin by enzymolysis pretreatment according to claim 1, wherein in the step (5), the chromatography medium of the chromatographic column is silica gel G, the column temperature is controlled to be 25-30 ℃, the eluent is petroleum ether-ethanol mixed solution, the volume ratio is 8-10:1, the elution flow rate is 1-2mL/min, the refrigeration crystallization temperature is 4-8 ℃, and the time is 8-12h.
  8. 8. The method for extracting artemisinin by enzymolysis pretreatment according to claim 1, wherein in the step (6), the solid-to-liquid ratio of recrystallization is 1:5-1:8 (g/mL), the dissolution temperature is 60-70 ℃, and the refrigerating crystallization time is 6-8h.

Description

Method for extracting artemisinin by enzymolysis pretreatment Technical Field The invention relates to the technical field of extraction of plant active ingredients, in particular to a method for extracting artemisinin by enzymolysis pretreatment. Background Artemisinin is a sesquiterpene lactone compound containing a peroxy bridge structure and extracted from artemisia annua, has high-efficiency, quick-acting and low-toxicity antimalarial activity, and has obvious curative effects on cerebral malaria and chloroquine malaria. In addition, the potential application value of artemisinin in the fields of anti-tumor, anti-schistosome, immunoregulation and the like further improves the market demand of artemisinin. At present, the industrialized extraction of artemisinin mainly depends on a natural product extraction method, and the traditional processes comprise a Soxhlet extraction method, a reflux extraction method, a cold leaching method and the like, and the problems of low extraction rate, large solvent consumption, long extraction time, insufficient product purity and the like generally exist in the methods. The core of the method is that the cell wall of the artemisia annua is mainly composed of cellulose, hemicellulose and the like, the artemisinin is wrapped in the cell wall, the cell wall structure is difficult to damage by the traditional extraction process, the mass transfer resistance of a solvent is high, the artemisinin is difficult to fully release, and the utilization rate of raw materials is low. In order to solve the problems, in the prior art, an attempt of enzymolysis auxiliary extraction appears, such as pretreatment by using cellulase alone, but the single enzyme system has limited action effect, unstable enzymolysis efficiency, unreasonable connection of subsequent extraction and purification steps, and the defects of insignificant improvement of extraction rate, complex process and high cost still exist. Part of the processes adopt strong acid environment or high-toxicity organic solvent, so that not only can the structural stability of artemisinin be destroyed, but also the environment is polluted, and the green production concept is not met. In addition, although the extraction rate of the high-efficiency methods such as supercritical CO 2 extraction is high, the equipment investment is huge, and the method is difficult to popularize and apply in small and medium enterprises. Therefore, the method for extracting the artemisinin by enzymolysis pretreatment has the advantages of simple process, high extraction rate, high product purity, environmental protection, energy conservation and controllable cost, and has important significance for promoting the industrialized development of the artemisinin. Disclosure of Invention In order to overcome the defects of the prior art, the invention aims to provide a method for extracting artemisinin by enzymolysis pretreatment, which realizes efficient and green extraction of artemisinin, improves the extraction rate and the product purity, reduces the production cost and meets the industrial production requirement. In order to achieve the purpose, the invention provides the following technical scheme that the method for extracting artemisinin by enzymolysis pretreatment comprises the following steps: (1) Raw material pretreatment, namely selecting the dried aerial parts of the artemisia annua, removing impurities, crushing, sieving with a 40-60 mesh sieve, and drying until the water content is less than or equal to 8 percent for later use; (2) The preparation method comprises the steps of (1) adding buffer solution into artemisia annua powder according to the feed-liquid ratio (1:10) - (1:20) (g/mL), regulating the pH value to 4.0-5.0, adding a compound enzyme preparation, carrying out enzymolysis for 1.5-3h at the constant temperature of 45-55 ℃, heating to 85-90 ℃ after enzymolysis, preserving heat for 15-20min, inactivating enzyme, and cooling to room temperature; (3) Ultrasonic assisted extraction, namely adding 90% -95% ethanol into enzymolysis materials, wherein the ethanol dosage is 8-12 times (mL/g) of the mass of the artemisia annua powder, performing ultrasonic extraction for 30-60min at the power of 200-300W and the temperature of 30-40 ℃, filtering and washing filter cakes, and combining filtrate and washing liquid to obtain crude extract; (4) Adding chitosan solution into the crude extract, adjusting chitosan concentration to 0.01-0.02g/mL, stirring, standing, filtering to remove impurities, collecting supernatant, concentrating under reduced pressure, and recovering ethanol to obtain artemisinin concentrate; (5) Purifying and crystallizing, namely dissolving the concentrate with 95% -99% ethanol, eluting by a chromatographic column, concentrating, refrigerating, crystallizing, washing and drying to obtain crude artemisinin; (6) Recrystallizing and refining, dissolving the crude product with 99% ethanol, filtering while hot, refrigerat