A method for extracting alpinumisore from atractylodes rhizome

Abstract

The present application relates to the technical field of traditional Chinese medicine extract, and particularly relates to a method for extracting artabotrine from atractylodes lancea, comprising the following steps: S1, subjecting atractylodes lancea to enzymatic hydrolysis treatment to obtain an enzymatic hydrolysis mixture; S2, subjecting the enzymatic hydrolysis mixture to aqueous two-phase extraction treatment to obtain an extraction liquid; the aqueous two-phase extraction treatment adopts a mixed solution composed of ethanol with a mass fraction of 8-13% and sodium dihydrogen phosphate with a mass fraction of 25-30%; the solid-liquid ratio of the enzymatic hydrolysis mixture and the mixed solution is 1g:(16-24)mL; S3, subjecting the extraction liquid to concentration treatment to obtain an extract, then dissolving the extract with petroleum ether and performing gradient elution through liquid chromatography to obtain artabotrine. The extraction and purification method of the present application can effectively, mildly and efficiently extract artabotrine from atractylodes lancea, and has high extraction purity, high yield, simple process flow, short time consumption and low production cost.

Description

Technical Field

[0001] This invention relates to the technical field of extracts from traditional Chinese medicine, and specifically to a method for extracting atractylodes from Atractylodes lancea. Background Technology

[0002] Atractylodes lancea is the dried rhizome of Atractylodes lancea or Atractylodes dasycarpus, both belonging to the Asteraceae family. Its main active ingredient is volatile oil, which contains sesquiterpenoids (such as atractylodes alcohol, β-cineole, and atractylone) and polyvinylpyridines (such as atractylodes acetonide), as well as small amounts of phenols, organic acids, and flavonoids.

[0003] The 2015 edition of the Chinese Pharmacopoeia specifies atractylodes as an indicator component of Atractylodes lancea, with a content of approximately 0.3-0.6%. Atractylodes is a pale yellow needle-like crystal with the molecular formula C2. 13 H 10 O, possesses pharmacological effects such as stomachic, anti-inflammatory, lipid-lowering, antibacterial, and hepatoprotective properties. Due to the presence of unsaturated bonds in its chemical molecule, atractylodesin exhibits a certain degree of molecular instability. Currently, atractylodesin is typically extracted using high-temperature extraction; however, under high-temperature conditions, atractylodesin is prone to decomposition or transformation, leading to problems such as low extraction efficiency, significant loss of target components, and low extraction purity in conventional extraction methods.

[0004] Therefore, there is an urgent need for a method to extract atractylodes from Atractylodes lancea with high extraction efficiency and high purity. Summary of the Invention

[0005] The first aspect of this invention provides a method for extracting atractylodes from Atractylodes lancea, comprising the following steps: S1. Atractylodes lancea is subjected to enzymatic hydrolysis to obtain an enzymatic hydrolysate mixture; S2. The enzymatic hydrolysis mixture is subjected to aqueous two-phase extraction to obtain an extract; the aqueous two-phase extraction is performed using a mixed solution of 8-13% ethanol and 25-30% sodium dihydrogen phosphate; the ratio of the enzymatic hydrolysis mixture to the mixed solution is 1g:(16-24)mL. S3. The extract is concentrated to obtain an extract, which is then dissolved in petroleum ether and subjected to gradient elution by liquid chromatography to obtain atractylodesin.

[0006] The inventors discovered that using an ethanol / sodium dihydrogen phosphate aqueous two-phase extraction system exhibits excellent solubility selectivity and protective effect on atractylodesin, effectively improving its extraction purity and yield. However, the selection and dosage ratio of components in the aqueous two-phase extraction system require careful consideration. Improper component matching can affect yield and purity; improper dosage ratio control can lead to incomplete extraction and decreased yield, and may also result in the extraction of other substances simultaneously with atractylodesin, leading to decreased purity. Furthermore, the ratio of the enzymatic hydrolysis mixture to the extraction system also needs careful consideration; a ratio that is too low will result in incomplete extraction, while a ratio that is too high will increase preparation costs.

[0007] The aqueous two-phase extraction process uses a mixed solution of 8-13% ethanol and 25-30% sodium dihydrogen phosphate by mass. The mass fraction of ethanol can be, for example, but is not limited to, 8.0%, 8.2%, 8.4%, 8.6%, 8.8%, 9.0%, 9.2%, 9.4%, 9.6%, 9.8%, 10.0%, 10.2%, 10.4%, 10.6%, 10.8%, 11.0%, 11.2%, 11.4%, 11.6%, 11.8%, 12.0%, 12.2%, 12.4%, 12.6%, 12.8%, 13.0%, or a range of the above values. The mass fraction of sodium dihydrogen phosphate may be, for example, but not limited to, 25.0%, 25.2%, 25.4%, 25.6%, 25.8%, 26.0%, 26.2%, 26.4%, 26.6%, 26.8%, 27.0%, 27.2%, 27.4%, 27.6%, 27.8%, 28.0%, 28.2%, 28.4%, 28.6%, 28.8%, 29.0%, 29.2%, 29.4%, 29.6%, 29.8%, 30.0%, or a range of the above values.

[0008] In some preferred embodiments, the ratio of the enzymatic hydrolysis mixture to the mixed solution is 1g:20mL.

[0009] In some embodiments, in step S1, the enzymatic hydrolysis employs at least one of cellulase, pectinase, and papain.

[0010] In some preferred embodiments, the enzymatic hydrolysis employs a mixture of cellulase and pectinase, wherein the mass ratio of cellulase to pectinase is (1~3):1. The mass ratio of cellulase to pectinase can be, for example, but is not limited to, 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1, 2.2:1, 2.4:1, 2.6:1, 2.8:1, 3:1, or a range thereof.

[0011] In some preferred embodiments, the enzymatic hydrolysis employs a mixture of cellulase and pectinase, wherein the mass ratio of cellulase to pectinase is 2:1.

[0012] Enzymatic hydrolysis using cellulase and pectinase can specifically destroy the cell wall of Atractylodes lancea, enabling atractylodesin to be effectively extracted from Atractylodes lancea, thereby improving the yield of subsequent extraction and purification.

[0013] In some embodiments, in step S1, the enzymatic hydrolysis time is 1-3 hours, and the enzymatic hydrolysis temperature is 40-50°C. The enzymatic hydrolysis time can be, for example, but is not limited to, 1.0h, 1.2h, 1.4h, 1.6h, 1.8h, 2.0h, 2.2h, 2.4h, 2.6h, 2.8h, 3.0h, or a range thereof. The enzymatic hydrolysis temperature can be, for example, but is not limited to, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, or a range thereof.

[0014] In some preferred embodiments, in step S2, the aqueous two-phase extraction process uses a mixed solution consisting of 9% ethanol and 29% sodium dihydrogen phosphate by mass.

[0015] In some embodiments, in step S2, the extraction time is 0.5 to 1 hour, and the extraction temperature is 55 to 65°C. The extraction time can be, for example, but is not limited to, 0.5 hours, 0.6 hours, 0.7 hours, 0.8 hours, 0.9 hours, 1 hour, or a range thereof. The extraction temperature can be, for example, but is not limited to, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, or a range thereof.

[0016] In some embodiments, in step S3, the flow rate of the gradient elution is 250-350 mL / min. The flow rate of the gradient elution can be, for example, but is not limited to, 250 mL / min, 260 mL / min, 270 mL / min, 280 mL / min, 290 mL / min, 300 mL / min, 310 mL / min, 320 mL / min, 330 mL / min, 340 mL / min, 350 mL / min, or a range of the above values.

[0017] In some implementations, in step S3, the gradient elution flow rate is 300 mL / min.

[0018] In some implementations, the gradient elution procedure in step S3 is as follows: From 0 to 15 minutes, the volume fraction of petroleum ether in the mobile phase is 100%. After 15-25 minutes, the volume fraction of petroleum ether in the mobile phase is 92%. The volume fraction of petroleum ether in the mobile phase is 85% after 25-35 minutes. The mobile phase contains 80% petroleum ether at a volume fraction of 35-45 min.

[0019] For the extraction of atractylodes, gradient elution is superior to isocratic elution. This is because Atractylodes contains various active ingredients, such as sesquiterpenes, polyvinylacetylenes, phenolic acids, and flavonoids. These active ingredients exhibit significant differences in polarity. If isocratic elution is performed using a fixed proportion of mobile phase, highly polar impurities are difficult to elute or elute slowly, while less polar impurities may co-elute with atractylodes, ultimately leading to peak tailing, overlap, and reduced resolution of the atractylodes peak, resulting in poor product purity and yield. Gradient elution, particularly the gradient provided in this invention, effectively separates atractylodes from various impurities, improving both purity and yield.

[0020] In some implementations, the Atractylodes includes Atractylodes lancea and Atractylodes dasycarpus.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention employs enzymatic hydrolysis to extract atractylodes lancea, enabling better dissolution of atractylodes lancea. Based on the properties of atractylodes lancea, an aqueous two-phase extraction system of ethanol / sodium dihydrogen phosphate was developed, which is particularly suitable for the separation and purification of atractylodes lancea. Gradient elution is used to improve the extraction and purification efficiency. The method provided by this invention can gently and efficiently extract atractylodes lancea from Atractylodes lancea, achieving high purity and high yield. The process is simple, time-saving, and effectively reduces production costs. Attached Figure Description

[0022] Figure 1 Photographs of Atractylodes macrocephala used in the examples and comparative examples.

[0023] Figure 2 It is Atractylodes lancea powder.

[0024] Figure 3 It is an enzymatically hydrolyzed mixture.

[0025] Figure 4 It is a two-phase aqueous extraction.

[0026] Figure 5 It is Atractylodes lancea extract. Detailed Implementation

[0027] The following detailed embodiments further illustrate the content of the present invention. These embodiments do not constitute a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention. The raw materials, reagents, or devices used in the embodiments are all available from conventional commercial sources or can be obtained through existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0028] The Atractylodes lancea used in the examples and comparative examples were from the same batch, purchased from Qingping Medicinal Herbs Market. Figure 1 As shown.

[0029] Example 1 A method for extracting atractylodesin from Atractylodes lancea includes the following steps: S1. Pretreatment and enzymatic hydrolysis: Take 500g of Atractylodes lancea (… Figure 1 After washing with clean water, it is vacuum dried at 50℃ for 48 hours. Then, the Atractylodes lancea is pulverized and passed through a 40-mesh sieve to obtain Atractylodes lancea powder. Figure 2 A compound enzymatic hydrolysate was prepared, containing 0.5% (w / w) cellulase and 0.25% (w / w) pectinase, in an acetate buffer solution (pH=4.5). Atractylodes lancea powder was placed in the compound enzymatic hydrolysate and enzymatically hydrolyzed in a water bath at 45°C for 1.5 hours to obtain an enzymatic hydrolysate mixture. Figure 3 ).

[0030] S2, Aqueous Two-Phase Extraction: Aqueous two-phase extraction treatment of the enzymatic hydrolysis mixture ( Figure 4 The aqueous two-phase extraction process uses a mixed solution of 9% ethanol and 29% sodium dihydrogen phosphate. The enzymatic hydrolysate is mixed with the mixed solution at a ratio of 1 g:20 mL, and extraction is performed at 60°C for 40 minutes. After extraction, the upper phase rich in the target components is collected by centrifugation, and the solvent is recovered by vacuum concentration to obtain the extract.

[0031] S3. Gradient elution: The extract was dissolved in 10 times its volume of petroleum ether, filtered through 100 μm, 0.45 μm and 0.22 μm filter membranes, and then purified using a DAC dynamic axial compression preparative column system. The chromatographic packing material was 300-400 mesh silica gel, the mobile phase was petroleum ether and ethyl acetate, and the elution method was gradient elution. The elution program is shown in Table 1. The flow rate was 300 mL / min, and the UV detection wavelength was 340 nm.

[0032] Table 1 S4. Purification and Concentration: The collected target peak solution was concentrated to obtain 2.55g of pale yellow powder, with a yield of 0.51%. HPLC analysis showed that the purity of atractylodesin was 99.52%. Figure 5 ).

[0033] Example 2 The only difference from Example 1 is that in step S2, the aqueous two-phase extraction process uses a mixed solution of 9% ethanol and 25% sodium dihydrogen phosphate. The remaining steps are exactly the same as in Example 1.

[0034] S1. Pretreatment and Enzymatic Hydrolysis: Take 500g of Atractylodes lancea, wash it with clean water, and vacuum dry it at 50℃ for 48 hours. Then, pulverize the Atractylodes lancea and pass it through a 40-mesh sieve to obtain Atractylodes lancea powder. Prepare a compound enzymatic hydrolysate containing 0.5% (w / w) cellulase and 0.25% (w / w) pectinase, using acetate buffer (pH=4.5) as the solvent. Place the Atractylodes lancea powder in the compound enzymatic hydrolysate and enzymatically hydrolyze it in a water bath at 45℃ for 1.5 hours to obtain an enzymatic hydrolysate mixture.

[0035] S2. Aqueous Two-Phase Extraction: The enzymatic hydrolysate was subjected to aqueous two-phase extraction using a mixed solution of 9% ethanol and 25% sodium dihydrogen phosphate. The enzymatic hydrolysate and the mixed solution were mixed at a material-to-liquid ratio of 1 g:20 mL and extracted at 60°C for 40 minutes. After extraction, the upper phase rich in the target components was collected by centrifugation, and the solvent was recovered by vacuum concentration to obtain the extract.

[0036] S3. Gradient elution: The extract was dissolved in 10 times its volume of petroleum ether, filtered through 100 μm, 0.45 μm and 0.22 μm filter membranes, and then purified using a DAC dynamic axial compression preparative column system. The chromatographic packing material was 300-400 mesh silica gel, the mobile phase was petroleum ether and ethyl acetate, and the elution method was gradient elution. The elution program is shown in Table 1. The flow rate was 300 mL / min, and the UV detection wavelength was 340 nm.

[0037] In this example, 2.30 g of pale yellow powder was obtained, with a yield of 0.46%, and the purity of atractylodesin was 98.56% as determined by HPLC.

[0038] Example 3 The only difference from Example 1 is that in step S2, the aqueous two-phase extraction process uses a mixed solution of 12% ethanol and 26% sodium dihydrogen phosphate. The remaining steps are exactly the same as in Example 1.

[0039] S1. Pretreatment and Enzymatic Hydrolysis: Take 500g of Atractylodes lancea, wash it with clean water, and vacuum dry it at 50℃ for 48 hours. Then, pulverize the Atractylodes lancea and pass it through a 40-mesh sieve to obtain Atractylodes lancea powder. Prepare a compound enzymatic hydrolysate containing 0.5% (w / w) cellulase and 0.25% (w / w) pectinase, using acetate buffer (pH=4.5) as the solvent. Place the Atractylodes lancea powder in the compound enzymatic hydrolysate and enzymatically hydrolyze it in a water bath at 45℃ for 1.5 hours to obtain an enzymatic hydrolysate mixture.

[0040] S2. Aqueous Two-Phase Extraction: The enzymatic hydrolysate mixture is subjected to aqueous two-phase extraction using a mixed solution of 12% ethanol and 26% sodium dihydrogen phosphate. The enzymatic hydrolysate mixture and the mixed solution are mixed at a material-to-liquid ratio of 1g:20mL and extracted at 60°C for 40 minutes. After extraction, the upper phase rich in the target components is collected by centrifugation, and the solvent is recovered by vacuum concentration to obtain the extract.

[0041] S3. Gradient elution: The extract was dissolved in 10 times its volume of petroleum ether, filtered through 100 μm, 0.45 μm and 0.22 μm filter membranes, and then purified using a DAC dynamic axial compression preparative column system. The chromatographic packing material was 300-400 mesh silica gel, the mobile phase was petroleum ether and ethyl acetate, and the elution method was gradient elution. The elution program is shown in Table 1. The flow rate was 300 mL / min, and the UV detection wavelength was 340 nm.

[0042] In this example, 2.25g of pale yellow powder was obtained, with a yield of 0.45%, and the purity of atractylodesin was 98.99% as determined by HPLC.

[0043] Comparative Example 1 The only difference from Example 1 is that in step S2, the aqueous two-phase extraction process uses a mixed solution of 15% ethanol and 20% sodium dihydrogen phosphate by mass. The remaining steps are exactly the same as in Example 1.

[0044] S1. Pretreatment and Enzymatic Hydrolysis: Take 500g of Atractylodes lancea, wash it with clean water, and vacuum dry it at 50℃ for 48 hours. Then, pulverize the Atractylodes lancea and pass it through a 40-mesh sieve to obtain Atractylodes lancea powder. Prepare a compound enzymatic hydrolysate containing 0.5% (w / w) cellulase and 0.25% (w / w) pectinase, using acetate buffer (pH=4.5) as the solvent. Place the Atractylodes lancea powder in the compound enzymatic hydrolysate and enzymatically hydrolyze it in a water bath at 45℃ for 1.5 hours to obtain an enzymatic hydrolysate mixture.

[0045] S2. Aqueous Two-Phase Extraction: The enzymatic hydrolysate mixture is subjected to aqueous two-phase extraction using a mixed solution of 15% ethanol and 20% sodium dihydrogen phosphate. The enzymatic hydrolysate mixture and the mixed solution are mixed at a material-to-liquid ratio of 1g:20mL and extracted at 60°C for 40 minutes. After extraction, the upper phase rich in the target components is collected by centrifugation, and the solvent is recovered by vacuum concentration to obtain the extract.

[0046] S3. Gradient elution: The extract was dissolved in 10 times its volume of petroleum ether, filtered through 100 μm, 0.45 μm and 0.22 μm filter membranes, and then purified using a DAC dynamic axial compression preparative column system. The chromatographic packing material was 300-400 mesh silica gel, the mobile phase was petroleum ether and ethyl acetate, and the elution method was gradient elution. The elution program is shown in Table 1. The flow rate was 300 mL / min, and the UV detection wavelength was 340 nm.

[0047] This comparative example yielded 1.90 g of pale yellow powder, with a yield of 0.38%, and the purity of atractylodesin was 95.23% as determined by HPLC.

[0048] Comparative Example 2 The only difference from Example 1 is that in step S2, the aqueous two-phase extraction process uses a mixed solution consisting of 9% polyethylene glycol and 20% ammonium sulfate by mass. The remaining steps are exactly the same as in Example 1.

[0049] S1. Pretreatment and Enzymatic Hydrolysis: Take 500g of Atractylodes lancea, wash it with clean water, and vacuum dry it at 50℃ for 48 hours. Then, pulverize the Atractylodes lancea and pass it through a 40-mesh sieve to obtain Atractylodes lancea powder. Prepare a compound enzymatic hydrolysate containing 0.5% (w / w) cellulase and 0.25% (w / w) pectinase, using acetate buffer (pH=4.5) as the solvent. Place the Atractylodes lancea powder in the compound enzymatic hydrolysate and enzymatically hydrolyze it in a water bath at 45℃ for 1.5 hours to obtain an enzymatic hydrolysate mixture.

[0050] S2. Aqueous Two-Phase Extraction: The enzymatic hydrolysate is subjected to aqueous two-phase extraction using a mixed solution of 9% polyethylene glycol and 20% ammonium sulfate. The enzymatic hydrolysate and the mixed solution are mixed at a ratio of 1g:20mL, and extracted at 60°C for 40 minutes. After extraction, the upper phase rich in the target components is collected by centrifugation, and the solvent is recovered by vacuum concentration to obtain the extract.

[0051] S3. Gradient elution: The extract was dissolved in 10 times its volume of petroleum ether, filtered through 100 μm, 0.45 μm and 0.22 μm filter membranes, and then purified using a DAC dynamic axial compression preparative column system. The chromatographic packing material was 300-400 mesh silica gel, the mobile phase was petroleum ether and ethyl acetate, and the elution method was gradient elution. The elution program is shown in Table 1. The flow rate was 300 mL / min, and the UV detection wavelength was 340 nm.

[0052] This comparative example yielded 1.25 g of pale yellow powder, with a yield of 0.25%, and the purity of atractylodesin was 92.62% as determined by HPLC.

[0053] Comparative Example 3 The only difference from Example 1 is that in step S2, the enzymatic hydrolysis mixture and the mixed solution are mixed at a ratio of 1g:30mL, and the rest of the steps are exactly the same as in Example 1.

[0054] S1. Pretreatment and Enzymatic Hydrolysis: Take 500g of Atractylodes lancea, wash it with clean water, and vacuum dry it at 50℃ for 48 hours. Then, pulverize the Atractylodes lancea and pass it through a 40-mesh sieve to obtain Atractylodes lancea powder. Prepare a compound enzymatic hydrolysate containing 0.5% (w / w) cellulase and 0.25% (w / w) pectinase, using acetate buffer (pH=4.5) as the solvent. Place the Atractylodes lancea powder in the compound enzymatic hydrolysate and enzymatically hydrolyze it in a water bath at 45℃ for 1.5 hours to obtain an enzymatic hydrolysate mixture.

[0055] S2. Aqueous Two-Phase Extraction: The enzymatic hydrolysate mixture is subjected to aqueous two-phase extraction using a mixed solution of 9% ethanol and 29% sodium dihydrogen phosphate. The enzymatic hydrolysate mixture and the mixed solution are mixed at a material-to-liquid ratio of 1 g:30 mL and extracted at 60°C for 40 minutes. After extraction, the upper phase rich in the target components is collected by centrifugation, and the solvent is recovered by vacuum concentration to obtain the extract.

[0056] S3. Gradient elution: The extract was dissolved in 10 times its volume of petroleum ether, filtered through 100 μm, 0.45 μm and 0.22 μm filter membranes, and then purified using a DAC dynamic axial compression preparative column system. The chromatographic packing material was 300-400 mesh silica gel, the mobile phase was petroleum ether and ethyl acetate, and the elution method was gradient elution. The elution program is shown in Table 1. The flow rate was 300 mL / min, and the UV detection wavelength was 340 nm.

[0057] This comparative example yielded 2.05 g of pale yellow powder, with a yield of 0.41%, and the purity of atractylodesin was 96.25% as determined by HPLC.

[0058] Comparative Example 4 The only difference from Example 1 is that in step S2, the enzymatic hydrolysis mixture and the mixed solution are mixed at a ratio of 1g:10mL, and the rest of the steps are exactly the same as in Example 1.

[0059] S1. Pretreatment and Enzymatic Hydrolysis: Take 500g of Atractylodes lancea, wash it with clean water, and vacuum dry it at 50℃ for 48 hours. Then, pulverize the Atractylodes lancea and pass it through a 40-mesh sieve to obtain Atractylodes lancea powder. Prepare a compound enzymatic hydrolysate containing 0.5% (w / w) cellulase and 0.25% (w / w) pectinase, using acetate buffer (pH=4.5) as the solvent. Place the Atractylodes lancea powder in the compound enzymatic hydrolysate and enzymatically hydrolyze it in a water bath at 45℃ for 1.5 hours to obtain an enzymatic hydrolysate mixture.

[0060] S2. Aqueous Two-Phase Extraction: The enzymatic hydrolysate mixture is subjected to aqueous two-phase extraction using a mixed solution of 9% ethanol and 29% sodium dihydrogen phosphate. The enzymatic hydrolysate mixture and the mixed solution are mixed at a material-to-liquid ratio of 1 g:10 mL and extracted at 60°C for 40 minutes. After extraction, the upper phase rich in the target components is collected by centrifugation, and the solvent is recovered by vacuum concentration to obtain the extract.

[0061] S3. Gradient elution: The extract was dissolved in 10 times its volume of petroleum ether, filtered through 100 μm, 0.45 μm and 0.22 μm filter membranes, and then purified using a DAC dynamic axial compression preparative column system. The chromatographic packing material was 300-400 mesh silica gel, the mobile phase was petroleum ether and ethyl acetate, and the elution method was gradient elution. The elution program is shown in Table 1. The flow rate was 300 mL / min, and the UV detection wavelength was 340 nm.

[0062] This comparative example yielded 1.4 g of pale yellow powder with a yield of 0.28%, and the purity of atractylodesin was 89.36% as determined by HPLC.

[0063] Comparative Example 5 The only difference from Example 1 is that in step S3, isocratic elution is used instead of gradient elution, and the mobile phase is petroleum ether and ethyl acetate with a volume ratio of 8:2. The remaining steps are exactly the same as in Example 1.

[0064] S1. Pretreatment and Enzymatic Hydrolysis: Take 500g of Atractylodes lancea, wash it with clean water, and vacuum dry it at 50℃ for 48 hours. Then, pulverize the Atractylodes lancea and pass it through a 40-mesh sieve to obtain Atractylodes lancea powder. Prepare a compound enzymatic hydrolysate containing 0.5% (w / w) cellulase and 0.25% (w / w) pectinase, using acetate buffer (pH=4.5) as the solvent. Place the Atractylodes lancea powder in the compound enzymatic hydrolysate and enzymatically hydrolyze it in a water bath at 45℃ for 1.5 hours to obtain an enzymatic hydrolysate mixture.

[0065] S2. Aqueous Two-Phase Extraction: The enzymatic hydrolysate mixture is subjected to aqueous two-phase extraction using a mixed solution of 9% ethanol and 29% sodium dihydrogen phosphate. The enzymatic hydrolysate mixture and the mixed solution are mixed at a material-to-liquid ratio of 1 g: 20 mL and extracted at 60°C for 40 minutes. After extraction, the upper phase rich in the target components is collected by centrifugation, and the solvent is recovered by vacuum concentration to obtain the extract.

[0066] S3. Gradient elution: The extract was dissolved in 10 times its volume of petroleum ether, filtered through 100 μm, 0.45 μm and 0.22 μm filter membranes, and then purified using a DAC dynamic axial compression preparative column system. The chromatographic packing material was 300-400 mesh silica gel, the mobile phase was petroleum ether and ethyl acetate with a volume ratio of 8:2, the elution method was isocratic elution, the flow rate was 300 mL / min, and the UV detection wavelength was 340 nm.

[0067] This comparative example yielded 2g of pale yellow powder with a yield of 0.40%, and the purity of atractylodesin was 95.11% as determined by HPLC.

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for extracting atractylodesin from Atractylodes lancea, characterized in that, Includes the following steps: S1. Perform enzymatic hydrolysis on Atractylodes lancea to obtain an enzymatic hydrolysate mixture; S2. The enzymatic hydrolysis mixture is subjected to aqueous two-phase extraction to obtain an extract; the aqueous two-phase extraction is performed using a mixed solution of 8-13% ethanol and 25-30% sodium dihydrogen phosphate; the ratio of the enzymatic hydrolysis mixture to the mixed solution is 1g:(16-24)mL. S3. The extract is concentrated to obtain an extract, which is then dissolved in petroleum ether and subjected to gradient elution by liquid chromatography to obtain atractylodesin.

2. The method as described in claim 1, characterized in that, In step S1, the enzymatic hydrolysis uses at least one of cellulase, pectinase, and papain.

3. The method as described in claim 2, characterized in that, In step S1, the enzymatic hydrolysis uses a mixture of cellulase and pectinase, with the mass ratio of cellulase to pectinase being (1~3):

1.

4. The method as described in claim 1, characterized in that, In step S1, the enzymatic hydrolysis time is 1~3 hours and the enzymatic hydrolysis temperature is 40~50℃.

5. The method as described in claim 1, characterized in that, In step S2, the aqueous two-phase extraction process uses a mixed solution consisting of 9% ethanol and 29% sodium dihydrogen phosphate by mass.

6. The method as described in claim 1, characterized in that, In step S2, the extraction time is 0.5~1h and the extraction temperature is 55~65℃.

7. The method as described in claim 1, characterized in that, In step S3, the flow rate of the gradient elution is 250~350 mL / min.

8. The method as described in claim 1, characterized in that, In step S3, the gradient elution flow rate is 300 mL / min.

9. The method as described in claim 1, characterized in that, In step S3, the gradient elution procedure is as follows: From 0 to 15 minutes, the volume fraction of petroleum ether in the mobile phase is 100%. After 15-25 minutes, the volume fraction of petroleum ether in the mobile phase is 92%. The volume fraction of petroleum ether in the mobile phase is 85% after 25-35 minutes. The mobile phase contains 80% petroleum ether at a volume fraction of 35-45 min.

10. The method as described in claim 1, characterized in that, The Atractylodes lancea mentioned includes Atractylodes macrocephala and Atractylodes dasycarpus.

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