Method for preparing high-purity hypericin through counter-current chromatography

Abstract

Aiming at the three technical bottlenecks of low separation efficiency, high product purity and high solvent toxicity in the existing hypericin extraction and purification process, the mass transfer efficiency is improved through a steam explosion directional wall breaking technology, and the release efficiency of intracellular active ingredients is improved through multienzyme-ultrasonic coupling; multistage countercurrent chromatography separation is adopted to break through the selective limitation of traditional column chromatography on naphthdianthrone components, and the hypericin with the purity larger than or equal to 98% is finally obtained. The steam explosion technology is adopted, the dissolution rate of hypericin and other effective components is greatly increased, meanwhile, the extraction rate is increased, the yield is increased, and the cost is saved. According to the method, multienzyme-ultrasonic coupling and counter-current chromatography are combined, the problems of high energy consumption, high pollution and the like caused by strong acid (alkali) in a traditional plant extraction technology are integrally solved, oxidative deterioration caused by long-time heating of active ingredients is improved, meanwhile, the product purity is remarkably improved, and the method is suitable for industrial production. And a technical support is provided for construction of extraction production of clean, stable and high-quality natural products.

Description

1. Technical Field

[0001] The invention belongs to the technical field of natural product extraction and purification, and particularly relates to a method for preparing high-purity hypericin by countercurrent chromatography. 2. Background Technology

[0002] Hypericin is the core component of Hypericum perforatum with antiviral and antidepressant activities. Its molecular formula is C 30 H 16 O8 has poor thermal stability and is easily oxidized. Existing extraction processes, such as the invention patent with publication number CN 105693494A, "A process for extracting and purifying hypericin from Hypericum perforatum," have the following drawbacks: ① The use of organic solvents such as chloroform and ethyl acetate causes significant environmental pollution; ② Silica gel column chromatography has low separation efficiency, resulting in a product purity of only 8.5-9.0%; ③ Multiple extractions lead to loss of active ingredients, resulting in a yield of less than 60%. Another example is the invention patent with publication number CN 105596387A, "A method for producing a Hypericum perforatum extract," which has the following drawbacks: ① The use of large amounts of alkaline and organic solvents for extraction results in a long production cycle, severe pollution, and increased production costs; ② The use of traditional extraction methods results in low extraction yields, resulting in a significant waste of Hypericum perforatum resources; ③ The extraction and concentration processes in this production process require extended heating time, causing some active ingredients to oxidize and deteriorate due to prolonged heat, resulting in low product quality.

[0003] Research has shown that steam explosion pretreatment effectively disrupts plant cell wall structure, increasing the degradation rate of lignin in Hypericum perforatum. Countercurrent chromatography, compared to traditional column chromatography, offers advantages such as the absence of a solid support, high separation efficiency, and ease of industrial scale-up. However, existing technologies have yet to achieve the synergistic application of these integrated techniques for the extraction and preparation of hypericin from Hypericum perforatum. 3. Summary of the Invention

[0004] The present invention addresses the three major technical bottlenecks of low separation efficiency, product purity, and high solvent toxicity in existing hypericin extraction and purification processes. The invention provides a method for preparing high-purity hypericin by countercurrent chromatography. The method has the advantages of simple production process, low production cost, safe operation, low energy consumption, and high efficiency. The hypericin prepared by the present invention has the advantages of high product purity and stable performance.

[0005] The technical solution of the present invention is based on the following mechanism: the steam explosion cell wall disruption mechanism: under the action of 1.5-2.0MPa saturated steam, the lignin in the cell wall of Hypericum perforatum undergoes a glass transition. When the pressure is instantly released, the shear force generated by the water vaporization causes micron-scale cracks in the cell wall (the porosity is increased by more than 80%), thereby significantly improving the accessibility of hypericin during the subsequent enzymatic hydrolysis process. Multi-enzyme-ultrasound coupled depolymerization: laccase, cellulase and pectinase are formed into a complex enzyme system. Through the synergistic action of the complex enzyme system's specific catalysis and the ultrasonic cavitation effect, ultrasound destroys the dense structure of the substrate and increases the enzyme contact area. The two work together to completely expose the glandular hair structure (the main storage site of hypericin), improving the dissolution efficiency of hypericin. Countercurrent chromatography distribution enhancement mechanism: A quaternary solvent system of n-hexane-ethyl acetate-methanol-water (5:5:4:3) is used, and the difference in the distribution coefficient of hypericin between the two phases (K=1.2~1.5) is utilized. The constant centrifugal force field generated by high-speed planetary centrifugation is used to achieve dynamic separation of the target component based on polarity gradient in the spiral column, thereby overcoming the adsorption interference of the silica gel column on non-polar impurities.

[0006] The present invention achieves its objectives by providing a method for preparing high-purity hypericin by countercurrent chromatography. This method utilizes steam explosion directional cell wall disruption technology to improve mass transfer efficiency, utilizes multi-enzyme-ultrasound coupling to enhance the release efficiency of intracellular active ingredients, and employs multi-stage countercurrent chromatography to overcome the selectivity limitations of conventional column chromatography for naphthodianthrone components, ultimately obtaining hypericin with a purity of ≥98%. The specific steps of the method are as follows:

[0007] (1) Preparation of Hypericum perforatum powder

[0008] The commercially available dry whole herb of Hypericum perforatum is used as raw material, which is first crushed into 20-80 meshes by a high-speed grinder to ensure a sieving rate of ≥95%, and the crushed Hypericum perforatum coarse powder is collected; the coarse powder is then put into a steam explosion tank, and 1.5-2.0 MPa saturated steam is introduced, and the pressure is maintained for 3-5 minutes, with an instantaneous pressure relief rate of ≥0.8 MPa / s. After the explosion, the pore diameter of the powder is 5-30 μm, and the exploded Hypericum perforatum powder is collected for later use.

[0009] (2) Preparation of Hypericum perforatum enzymatic extract

[0010] After step (1) is completed, the Hypericum perforatum powder is added to the phosphate buffer solution at a ratio of 1:15-30 of the mass of Hypericum perforatum powder to the volume of pH 4-5-5.5 phosphate buffer. Laccase (400-600 U / g): cellulase (800-1200 U / g): pectinase (1200-1800 U / g) are uniformly mixed at a ratio of 1:2:3 of their enzyme activities to obtain a composite enzyme system. The composite enzyme system is then added to the Hypericum perforatum powder mixture at a ratio of 1:0.03-0.08 of the volume of the Hypericum perforatum powder mixture to the mass of the composite enzyme system. The mixture is stirred evenly to obtain a mixed extract. The mixed extract was transferred to an ultrasonic reactor and treated at 45-50°C with low-frequency ultrasound at 38-42 kHz for 15-30 minutes to ensure adequate contact between the enzyme and substrate. This was followed by high-frequency ultrasound at 50 kHz for 15-45 minutes to promote the dissolution of intracellular components. Following the multi-enzyme-ultrasound coupled treatment, the enzymes were inactivated at 80-90°C for 5-10 minutes. The resulting mixture, the enzymatic hydrolyzed extract of Hypericum perforatum, was collected and used for the next step.

[0011] (3) Preparation of hypericin crude extract

[0012] After step (2) is completed, an extraction solvent is prepared according to a volume ratio of n-butanol to ethyl acetate of 1:3-6. The enzymatic hydrolyzed extract of Hypericum perforatum collected in step (2) and the extraction solvent are then injected into a secondary countercurrent centrifugal extractor at a volume ratio of 1:0.8-1.2. The temperature of the centrifugal extractor system is controlled at 13-17°C, and countercurrent centrifugal extraction is performed at a speed of 3500-4000 rpm. After the centrifugal extraction is completed, the organic phase and the aqueous phase are collected separately. Anhydrous magnesium sulfate is then added to the organic phase at a ratio of organic phase volume (mL) to anhydrous magnesium sulfate mass (g) of 1:0.05-0.15 for dehydration for 10-20 minutes. After impurities are removed through a 0.22-0.45 μm filter membrane, the extract is concentrated by rotary evaporation at 40-45°C and -0.08-0.06 MPa to 1 / 8-1 / 10 of the original volume to obtain a crude hypericin extract, which is sealed and nitrogen-filled and stored at -20°C for later use.

[0013] (4) Preparation of high-purity hypericin

[0014] After step (3) is completed, a two-phase solvent is prepared in a ratio of n-hexane: ethanol: water of 1:5-7:3-5 by volume. Ultrasonic degassing is carried out for 20-30 minutes, and the mixture is allowed to stand for 10-12 hours to separate into layers. The upper layer is the stationary phase, and the lower layer is the mobile phase. The crude hypericin extract is dissolved in the stationary phase in a ratio of 1:10-50 by volume of the crude hypericin extract: the stationary phase to obtain the injection solution. The stationary phase is then pumped into the chromatographic column, and the main engine speed is adjusted to 800 rpm. The mobile phase is pumped in at a flow rate of 1-5 mL / min until the system is balanced. The injection solution is then injected through the injection valve, and the target fraction is received according to the detector spectrum. The collected target fraction is first filtered through a 0.22 μm microporous filter membrane, then pre-frozen at -21°C for 2 to 4 hours, placed in a freeze-drying device, and freeze-dried at 10 to 20 Pa and -50 to -60°C for 24 to 36 hours to prepare high-purity hypericin with a purity of 98.2 to 98.7%.

[0015] After the present invention adopts the above technical solution, it has the following main effects:

[0016] 1. In the production process of the present invention, the method steps are simple, the product yield is high, the comprehensive utilization rate of resources is high, and it is very convenient for industrial production.

[0017] 2. The present invention adopts steam explosion technology in the production process, which greatly improves the dissolution rate of hypericin and other effective ingredients, and at the same time improves the extraction rate, thereby increasing the output and saving costs.

[0018] 3. The combination of multi-enzyme-ultrasound coupling and countercurrent chromatography in the present invention solves the problems of high energy consumption and high pollution caused by strong acids (alkalis) in traditional plant extraction technology, and improves the oxidative deterioration of active ingredients caused by long-term heating. At the same time, it significantly improves the product purity, providing technical support for the construction of clean, stable, high-quality natural product extraction production.

[0019] 4. The present invention avoids the use of toxic and harmful solvents in the production process, uses non-toxic substances such as ethanol and purified water, and recycles them.

[0020] 5. The hypericin product obtained by the present invention has high purity and good stability.

[0021] The products prepared by the method of the present invention can be widely used in medicine, health care products, food, daily chemicals and other industries. 4. Specific Implementation Methods

[0022] The present invention will be further described below with reference to specific embodiments.

[0023] Example 1

[0024] A method for preparing high-purity hypericin by countercurrent chromatography, the specific steps of the method are as follows:

[0025] (1) Preparation of Hypericum perforatum powder

[0026] The commercially available dry whole herb of Hypericum perforatum is used as raw material, which is first crushed to 20 mesh by a high-speed grinder to make a screening rate ≥95%, and the crushed Hypericum perforatum coarse powder is collected; the coarse powder is then placed in a steam explosion tank, and 1.5 MPa saturated steam is introduced, and the pressure is maintained for 3 minutes, with an instantaneous pressure relief rate of ≥0.8 MPa / s. After the explosion, the pore diameter of the powder is 5 μm, and the exploded Hypericum perforatum powder is collected for later use.

[0027] (2) Preparation of Hypericum perforatum enzymatic extract

[0028] After step (1) is completed, the Hypericum perforatum powder is added to the phosphate buffer solution at a ratio of 1:15 of the mass of Hypericum perforatum powder to the volume of pH 4.5 phosphate buffer solution. The three enzymes are evenly mixed at a ratio of 1:2:3 of the enzyme activity of laccase (400U / g): cellulase (800U / g): pectinase (1200U / g) to obtain a composite enzyme system. The composite enzyme system is then added to the Hypericum perforatum powder mixture at a ratio of 1:0.03 of the volume of the Hypericum perforatum powder mixture to the mass of the composite enzyme system (g), and stirred evenly to obtain a mixed extract. The mixed extract is transferred to an ultrasonic reactor and treated at a low frequency of 38kHz for 15 minutes at 45°C to allow the enzyme to fully contact the substrate, and then switched to a high frequency of 50kHz for 15 minutes to promote the dissolution of intracellular components. After the multi-enzyme-ultrasound coupling treatment, the enzyme was inactivated at 80°C for 5 minutes, and the mixture was collected as the Hypericum perforatum enzymatic extract for the next step of treatment.

[0029] (3) Preparation of hypericin crude extract

[0030] After step (2) is completed, an extraction solvent is prepared according to a volume ratio of n-butanol to ethyl acetate of 1:3. The enzymatic hydrolyzed extract of Hypericum perforatum collected in step (2) and the extraction solvent are then injected into a secondary countercurrent centrifugal extractor at a volume ratio of 1:0.8. The temperature of the centrifugal extractor system is controlled at 13°C, and countercurrent centrifugal extraction is performed at a speed of 3500 rpm. After the centrifugal extraction is completed, the organic phase and the aqueous phase are collected separately. Anhydrous magnesium sulfate is then added to the organic phase at a ratio of organic phase volume (mL): anhydrous magnesium sulfate mass (g) of 1:0.05 for dehydration for 10 minutes. After impurities are removed through a 0.22 μm filter membrane, the extract is concentrated by rotary evaporation at 40°C and -0.08 MPa to 1 / 8 of the original volume to obtain a crude hypericin extract, which is then sealed and filled with nitrogen and stored at -20°C for later use.

[0031] (4) Preparation of high-purity hypericin

[0032] After step (3), a two-phase solvent was prepared in a ratio of 1:5:3 of n-hexane: ethanol: water by volume, ultrasonically degassed for 20 minutes, and allowed to stand for 10 hours. The upper layer was the stationary phase and the lower layer was the mobile phase. The crude hypericin extract was dissolved in the stationary phase in a ratio of 1:10 of the mass of the crude hypericin extract to the volume of the stationary phase to obtain the injection solution. The stationary phase was then pumped into the chromatographic column, and the main engine speed was adjusted to 800 rpm. The mobile phase was pumped in at a flow rate of 1 mL / min until the system was balanced. The injection solution was then injected through the injection valve and the target fraction was received according to the detector spectrum. The collected target fraction was first filtered through a 0.22 μm microporous filter membrane, then pre-frozen at -21°C for 2 hours, placed in a freeze-drying device, and freeze-dried at 10 Pa and -50°C for 24 hours to prepare high-purity hypericin with a purity of 98.2%.

[0033] Example 2

[0034] A method for preparing high-purity hypericin by countercurrent chromatography, the specific steps of the method are as follows:

[0035] (1) Preparation of Hypericum perforatum powder

[0036] The commercially available dry whole herb of Hypericum perforatum was used as the raw material, which was first crushed to 60 meshes by a high-speed grinder to make the screening rate ≥95%, and the crushed Hypericum perforatum coarse powder was collected; the coarse powder was then placed in a steam explosion tank, and 1.8MPa saturated steam was introduced, and the pressure was maintained for 4 minutes, with an instantaneous pressure relief rate of ≥0.8MPa / s. After the explosion, the powder pore diameter was 15μm, and the Hypericum perforatum powder after the explosion was collected for use. (2) Preparation of Hypericum perforatum enzymatic extract

[0037] After step (1) is completed, the Hypericum perforatum powder is added to the phosphate buffer solution at a ratio of 1:20 of the mass of Hypericum perforatum powder to the volume of pH 5.0 phosphate buffer solution to obtain a Hypericum perforatum powder mixture. Laccase (500U / g): cellulase (1000U / g): pectinase (1600U / g) are uniformly mixed at a ratio of 1:2:3 of enzyme activity to obtain a composite enzyme system. The composite enzyme system is then added to the Hypericum perforatum powder mixture at a ratio of 1:0.05 of the volume of the Hypericum perforatum powder mixture to the mass of the composite enzyme system (g), and stirred to obtain a mixed extract. The mixed extract is transferred to an ultrasonic reactor and treated at a low frequency of 40kHz for 20min at 48°C to allow the enzyme to fully contact the substrate, and then switched to a high frequency of 50kHz for 30min to promote the dissolution of intracellular components. After the multi-enzyme-ultrasound coupling treatment, the enzyme was inactivated at 85°C for 8 minutes, and the mixture was collected as the Hypericum perforatum enzymatic extract for the next step of treatment.

[0038] (3) Preparation of hypericin crude extract

[0039] After step (2) is completed, an extraction solvent is prepared according to a volume ratio of n-butanol to ethyl acetate of 1:4. The enzymatic hydrolyzed extract of Hypericum perforatum collected in step (2) and the extraction solvent are then injected into a secondary countercurrent centrifugal extractor at a volume ratio of 1:1.0. The temperature of the centrifugal extractor system is controlled at 15°C, and countercurrent centrifugal extraction is performed at a speed of 3800 rpm. After the centrifugal extraction is completed, the organic phase and the aqueous phase are collected separately. Anhydrous magnesium sulfate is then added to the organic phase at a ratio of organic phase volume (mL): anhydrous magnesium sulfate mass (g) of 1:0.10 for dehydration for 15 minutes. After impurities are removed through a 0.22 μm filter membrane, the extract is concentrated by rotary evaporation at 42°C and -0.07 MPa to 1 / 9 of the original volume to obtain a crude hypericin extract, which is then sealed and filled with nitrogen and stored at -20°C for later use.

[0040] (4) Preparation of high-purity hypericin

[0041] After step (3), a two-phase solvent was prepared in a ratio of n-hexane: ethanol: water of 1:6:4 by volume, ultrasonically degassed for 25 minutes, and allowed to stand for 11 hours. The upper layer was the stationary phase and the lower layer was the mobile phase. The crude hypericin extract was dissolved in the stationary phase in a ratio of 1:25 by volume of the crude hypericin extract: the stationary phase to obtain the injection solution. The stationary phase was then pumped into the chromatographic column, and the main engine speed was adjusted to 800 rpm. The mobile phase was pumped in at a flow rate of 3 mL / min until the system was balanced. The injection solution was then injected through the injection valve and the target fraction was received according to the detector spectrum. The collected target fraction was first filtered through a 0.22 μm microporous filter membrane, then pre-frozen at -21°C for 3 hours, placed in a freeze-drying device, and freeze-dried at 15 Pa and -55°C for 30 hours to prepare high-purity hypericin with a purity of 98.5%.

[0042] Example 3

[0043] A method for preparing high-purity hypericin by countercurrent chromatography, the specific steps of the method are as follows:

[0044] (1) Preparation of Hypericum perforatum powder

[0045] The commercially available dry whole herb of Hypericum perforatum was used as the raw material, which was first crushed to 80 meshes by a high-speed grinder to make the screening rate ≥95%, and the crushed Hypericum perforatum coarse powder was collected; the coarse powder was then placed in a steam explosion tank, and 2.0 MPa saturated steam was introduced, and the pressure was maintained for 5 minutes, with an instantaneous pressure relief rate of ≥0.8 MPa / s. After the explosion, the powder pore diameter was 30 μm, and the exploded Hypericum perforatum powder was collected for later use. (2) Preparation of Hypericum perforatum enzymatic extract

[0046] After step (1) is completed, the Hypericum perforatum powder is added to the phosphate buffer solution at a ratio of 1:30 of the mass of Hypericum perforatum powder to the volume of pH 5.5 phosphate buffer solution. Laccase (600U / g): cellulase (1200U / g): pectinase (1800U / g) are uniformly mixed at a ratio of 1:2:3 of enzyme activity to obtain a composite enzyme system. The composite enzyme system is then added to the Hypericum perforatum powder mixture at a ratio of 1:0.08 of the volume of the Hypericum perforatum powder mixture to the mass of the composite enzyme system (g), and the mixture is stirred to obtain a mixed extract. The mixed extract is transferred to an ultrasonic reactor and treated at a low frequency of 42kHz for 30min at 50℃ to allow the enzyme to fully contact the substrate, and then switched to a high frequency of 50kHz for 45min to promote the dissolution of intracellular components. After the multi-enzyme-ultrasound coupling treatment, the enzyme was inactivated at 90°C for 10 minutes, and the mixture was collected as the Hypericum perforatum enzymatic extract for the next step of treatment.

[0047] (3) Preparation of hypericin crude extract

[0048] After step (2) is completed, an extraction solvent is prepared according to a volume ratio of n-butanol to ethyl acetate of 1:6. The enzymatic hydrolyzed extract of Hypericum perforatum collected in step (2) and the extraction solvent are then injected into a secondary countercurrent centrifugal extractor at a volume ratio of 1:1.2. The temperature of the centrifugal extractor system is controlled at 17°C, and countercurrent centrifugal extraction is performed at a speed of 4000 rpm. After the centrifugal extraction is completed, the organic phase and the aqueous phase are collected separately. Anhydrous magnesium sulfate is then added to the organic phase at a ratio of organic phase volume (mL): anhydrous magnesium sulfate mass (g) of 1:0.15 for dehydration for 20 minutes. After impurities are removed through a 0.45 μm filter membrane, the extract is concentrated by rotary evaporation at 45°C and -0.06 MPa to 1 / 10 of the original volume to obtain a crude hypericin extract, which is then sealed and filled with nitrogen and stored at -20°C for later use.

[0049] (4) Preparation of high-purity hypericin

[0050] After step (3), a two-phase solvent was prepared in a ratio of n-hexane: ethanol: water of 1:7:5 by volume, ultrasonically degassed for 30 minutes, and allowed to stand for 12 hours. The upper layer was the stationary phase and the lower layer was the mobile phase. The crude hypericin extract was dissolved in the stationary phase in a ratio of 1:50 by volume of the crude hypericin extract: the stationary phase to obtain the injection solution. The stationary phase was then pumped full into the chromatographic column, and the main engine speed was adjusted to 800 rpm. The mobile phase was pumped in at a flow rate of 5 mL / min until the system was balanced. The injection solution was then injected through the injection valve and the target fraction was received according to the detector spectrum. The collected target fraction was first filtered through a 0.22 μxm microporous filter membrane, then pre-frozen at -21°C for 4 hours, placed in a freeze-drying device, and freeze-dried at 20 Pa and -60°C for 36 hours to prepare high-purity hypericin with a purity of 98.7%.

Claims

1. A method for preparing high-purity hypericin by countercurrent chromatography, the specific steps of the method are as follows: (1) Preparation of Hypericum perforatum powder The whole herb of Hypericum perforatum available on the market is used as raw material, which is first crushed to 20-80 meshes by a high-speed grinder to ensure a sieving rate of ≥95%, and the crushed Hypericum perforatum coarse powder is collected; the coarse powder is then placed in a steam explosion tank, and saturated steam of 1.5-2.0 MPa is introduced for 3-5 minutes, with an instantaneous pressure relief rate of ≥0.8 MPa / s, so that the pore diameter of the powder after explosion is 5-30 μm, and the exploded Hypericum perforatum powder is collected for later use; (2) Preparation of Hypericum perforatum enzymatic extract After the completion of step (1), the Hypericum perforatum powder is added to the phosphate buffer solution at a ratio of 1 g: 15-30 mL of the mass of the Hypericum perforatum powder: the volume of the pH 4.5-5.5 phosphate buffer solution to obtain a Hypericum perforatum powder mixture; the three are uniformly mixed at a ratio of 1:2:3 of the enzyme activity of laccase 400-600 U / g: cellulase 800-1200 U / g: pectinase 1200-1800 U / g to obtain a composite enzyme system; and the composite enzyme system is added at a ratio of 1 mL: 0.03-0.08 g of the volume of the Hypericum perforatum powder mixture: the mass of the composite enzyme system. The mixture is added to the mixed solution of Hypericum perforatum powder and stirred evenly to obtain a mixed extract; the mixed extract is transferred to an ultrasonic reactor, and a low-frequency ultrasonic treatment of 38 to 42 kHz is initiated at 45 to 50° C. for 15 to 30 minutes to allow sufficient contact between the enzyme and the substrate, and then switched to a high-frequency ultrasonic treatment of 50 kHz for 15 to 45 minutes to promote the dissolution of intracellular components; after the multi-enzyme-ultrasound coupling treatment is completed, the enzyme is inactivated at a temperature of 80 to 90° C. for 5 to 10 minutes, and the mixture is collected to obtain the Hypericum perforatum enzymatic extract for use in the next step; (3) Preparation of hypericin crude extract After step (2) is completed, an extraction solvent is prepared according to a volume ratio of n-butanol to ethyl acetate of 1:3-6; the enzymatic hydrolyzed extract of Hypericum perforatum collected in step (2) and the extraction solvent are injected into a secondary countercurrent centrifugal extractor at a volume ratio of 1:0.8-1.2, the temperature of the centrifugal extractor system is controlled at 13-17°C, and countercurrent centrifugal extraction is performed at a speed of 3500-4000 rpm. After the centrifugal extraction is completed, the organic phase and the aqueous phase are collected respectively; anhydrous magnesium sulfate is added to the organic phase at a ratio of organic phase volume to anhydrous magnesium sulfate mass of 1 mL: 0.05-0.15 g for dehydration for 10-20 minutes, and after impurities are removed through a 0.22-0.45 μm filter membrane, the extract is concentrated by rotary evaporation at 40-45°C and -0.08--0.06 MPa to 1 / 8-1 / 10 of the original volume to obtain a crude hypericin extract, which is sealed and nitrogen-filled and stored at -20°C for later use; (4) Preparation of high-purity hypericin After step (3) is completed, prepare a two-phase solvent in a ratio of n-hexane: ethanol: water of 1:5-7:3-5 by volume, perform ultrasonic degassing for 20-30 minutes, let it stand for 10-12 hours to separate into layers, the upper layer is the stationary phase, and the lower layer is the mobile phase; dissolve the crude hypericin extract in the stationary phase in a ratio of 1:10-50 by volume of the crude hypericin extract to the stationary phase to obtain the injection solution; then pump the stationary phase to fill the chromatographic column, and then adjust the main engine speed to 800 rpm, and use 1 After the mobile phase is pumped in at a flow rate of 5 mL / min until the system is balanced, the injection liquid is injected through the injection valve, and the target fraction is received according to the detector spectrum; the collected target fraction is first filtered through a 0.22 μm microporous filter membrane, and then pre-frozen at -21°C for 2 to 4 hours, placed in a freeze-drying device, and freeze-dried at 10 to 20 Pa and -50 to -60°C for 24 to 36 hours to prepare high-purity hypericin with a purity of 98.2 to 98.7%.