Enrichment Method of Ergosterol Peroxide from Sporoderm-Broken Ganoderma Lucidum Spore Powder

Abstract

The invention provides an enrichment method of Ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder, comprising the steps of preparing crude extract containing Ergosterol peroxide, purification by medium pressure preparative chromatography, purification by simulated moving-bed, and recrystallization. The operation of this invention is simple and stable, with higher yield and low cost, suiting for industrial continuous production.

Claims

1. An enrichment method of Ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder, comprising the following steps: (1) Preparing crude extract containing Ergosterol peroxide: taking the sporoderm-broken Ganoderma lucidum spore powder, performing extraction by percolation or reflux method, concentrating the extract liquid to dryness under reduced pressure, and obtaining the crude extract A containing Ergosterol peroxide; (2) Purification by medium pressure preparative chromatography: dissolving the crude extract A containing Ergosterol peroxide prepared in the step (1) with 60-95% methanol aqueous solution to obtain the sample solution for medium pressure preparative chromatography, performing purification by medium pressure preparative chromatography, collecting the fraction containing Ergosterol peroxide, concentrating it to dryness under reduced pressure to obtain the extractum B containing Ergosterol peroxide; (3) Purification by simulated moving-bed: dissolving the extractum B containing Ergosterol peroxide prepared in the step (2) with 60-95% methanol aqueous solution to obtain the sample solution, injecting the sample solution into the simulated moving-bed chromatography separation system for further purification, concentrating the extract liquor containing Ergosterol peroxide to dryness under reduced pressure, and obtaining the dry extract C rich in Ergosterol peroxide; (4) Dissolving the dry extract C rich in Ergosterol peroxide prepared in the step (3) with ethyl acetate-cyclohexane under heating conditions, transferring it into a environment at a temperature of 0-8° C. for recrystallization, filtering and washing the precipitate to obtain Ergosterol peroxide with high purity.

2. Enrichment method of Ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder according to claim 1, characterized in that the extraction solvent of percolation or reflux method said in step (1) is one of ethyl acetate, cyclohexane and petroleum, or a mixture of them with any ratio.

3. Enrichment method of Ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder according to claim 2, characterized in that the said percolation in step (1), wherein, the immersing time is 24-48 h, the immersing temperature is room temperature, and percolate is collected at approximate constant speed for 5-10 BV.

4. Enrichment method of Ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder according to claim 2, characterized in that the said reflux method in step (1), wherein, the liquid-solid ratio is 6-10, the extracting time at boiling condition is 1-2 h, the residue is repeatedly extracted for 1-2 times, and the filtrate is merged.

5. Enrichment method of Ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder according to claim 1, characterized in that the said step (2), wherein, the medium pressure preparative chromatography column is packed with 30-50 μm ODS silica gel, the height of column is 45 cm, the solid content of sample solution is 0.05-0.2 g/mL, the injection rate is 2-5 mL/min and the injection volume is 0.1 BV, performing isocratic elution with 60-95% methanol aqueous solution at the rate of 1.2-2.0 BV/h.

6-7. (canceled)

8. Enrichment method of Ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder according to claim 1, characterized in that the said step (3), wherein, the solid content of sample solution is 0.05-0.2 g/mL, the simulated moving-bed chromatography system uses ODS reversed silica gel with particle size of 30-50 μm as packing materials, the system, adopting 8-16 columns in series connection, is divided into four areas, each of which having 1-4 columns in series connection, the system parameters are set up as follows: room temperature, flow rate of injection pump is 3-6 mL/min, flow rate of elution pump is 16-38 mL/min, Extraction velocity is 10-23 mL/min, flow rate for raffinate is 9-21 mL/min, and switching time is 10-24 min.

9. Enrichment method of Ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder according to claim 2, characterized in that the said step (3), wherein, the solid content of sample solution is 0.05-0.2 g/mL, the simulated moving-bed chromatography system uses ODS reversed silica gel with particle size of 30-50 μm as packing materials, the system, adopting 8-16 columns in series connection, is divided into four areas, each of which having 1-4 columns in series connection, the system parameters are set up as follows: room temperature, flow rate of injection pump is 3-6 mL/min, flow rate of elution pump is 16-38 mL/min, Extraction velocity is 10-23 mL/min, flow rate for raffinate is 9-21 mL/min, and switching time is 10-24 min.

10. Enrichment method of Ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder according to claim 3, characterized in that the said step (3), wherein, the solid content of sample solution is 0.05-0.2 g/mL, the simulated moving-bed chromatography system uses ODS reversed silica gel with particle size of 30-50 μm as packing materials, the system, adopting 8-16 columns in series connection, is divided into four areas, each of which having 1-4 columns in series connection, the system parameters are set up as follows: room temperature, flow rate of injection pump is 3-6 mL/min, flow rate of elution pump is 16-38 mL/min, Extraction velocity is 10-23 mL/min, flow rate for raffinate is 9-21 mL/min, and switching time is 10-24 min.

11. Enrichment method of Ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder according to claim 4, characterized in that the said step (3), wherein, the solid content of sample solution is 0.05-0.2 g/mL, the simulated moving-bed chromatography system uses ODS reversed silica gel with particle size of 30-50 μm as packing materials, the system, adopting 8-16 columns in series connection, is divided into four areas, each of which having 1-4 columns in series connection, the system parameters are set up as follows: room temperature, flow rate of injection pump is 3-6 mL/min, flow rate of elution pump is 16-38 mL/min, Extraction velocity is 10-23 mL/min, flow rate for raffinate is 9-21 mL/min, and switching time is 10-24 min.

12. Enrichment method of Ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder according to claim 5, characterized in that the said step (3), wherein, the solid content of sample solution is 0.05-0.2 g/mL, the simulated moving-bed chromatography system uses ODS reversed silica gel with particle size of 30-50 μm as packing materials, the system, adopting 8-16 columns in series connection, is divided into four areas, each of which having 1-4 columns in series connection, the system parameters are set up as follows: room temperature, flow rate of injection pump is 3-6 mL/min, flow rate of elution pump is 16-38 mL/min, Extraction velocity is 10-23 mL/min, flow rate for raffinate is 9-21 mL/min, and switching time is 10-24 min.

13. Enrichment method of Ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder according to claim 1, characterized in that the said step (4), wherein, the dosage of ethyl acetate-cyclohexane is 10-20 times that of the dry extract C, the volume ratio of ethyl acetate to cyclohexane is 50:50-10:90, and the time of recrystallization is 36-48 h.

14. Enrichment method of Ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder according to claim 2, characterized in that the said step (4), wherein, the dosage of ethyl acetate-cyclohexane is 10-20 times that of the dry extract C, the volume ratio of ethyl acetate to cyclohexane is 50:50-10:90, and the time of recrystallization is 36-48 h.

15. Enrichment method of Ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder according to claim 3, characterized in that the said step (4), wherein, the dosage of ethyl acetate-cyclohexane is 10-20 times that of the dry extract C, the volume ratio of ethyl acetate to cyclohexane is 50:50-10:90, and the time of recrystallization is 36-48 h.

16. Enrichment method of Ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder according to claim 4, characterized in that the said step (4), wherein, the dosage of ethyl acetate-cyclohexane is 10-20 times that of the dry extract C, the volume ratio of ethyl acetate to cyclohexane is 50:50-10:90, and the time of recrystallization is 36-48 h.

17. Enrichment method of Ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder according to claim 5, characterized in that the said step (4), wherein, the dosage of ethyl acetate-cyclohexane is 10-20 times that of the dry extract C, the volume ratio of ethyl acetate to cyclohexane is 50:50-10:90, and the time of recrystallization is 36-48 h.

Description

EXAMPLES

[0023] The examples below further illustrate the invention, rather than limiting the scope thereof. The related methods and materials in this invention are separately the conventional methods and materials available in the market, unless specially stated otherwise.

Example 1

[0024] Acquiring ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder by following steps:

[0025] (1) Preparing crude extract containing Ergosterol peroxide: taking the sporoderm-broken Ganoderma lucidum spore powder, performing extraction by percolation, of which the extraction solvent is ethyl acetate, the immersing time is 40 h at room temperature, percolate is collected at approximate constant speed for 8 BV; concentrating the extract liquid to dryness under reduced pressure by rotary evaporator, the bathing temperature at 65° C., and the vacuum degree below −0.085, and obtaining the crude extract A with an ergosterol peroxide content of 0.62% revealed by the test results.

[0026] (2) Purification by medium pressure preparative chromatography: dissolving the crude extract A containing Ergosterol peroxide prepared in the step (1) with 60% methanol aqueous solution by heating and ultrasonic, filtrating the solution with 0.45 μm millipore filter to obtain the sample solution for medium pressure preparative chromatography, of which the solid content is 0.05 g/mL; performing purification by medium pressure preparative chromatography, of which the medium pressure preparative chromatography column is packed with 50 μm ODS silica gel, the height of column is 45 cm, the injection rate is 2 mL/min, and the injection volume is 0.1 BV, performing isocratic elution with 60% methanol aqueous solution at the rate of 1.2 BV/h; collecting the fraction containing Ergosterol peroxide, concentrating it to dryness under reduced pressure by rotary evaporator, the bathing temperature at 70° C., and the vacuum degree below −0.085, to obtain the extractum B with an Ergosterol peroxide content of 16.8%. In this step, the impurities with low polarity are removed almost, and the impurities with high polarity are partly removed.

[0027] (3) Purification by simulated moving-bed: simulated moving bed chromatography separation system includes elution pump, injection pump, extracting pump, chromatographic columns, electromagnetic valve, check valve, signal collector, system controller, and so on, thereinto, the flow rate of both elution pump and extracting pump are 0-250 mL/min, that of injection pump is 0-50 mL/min, the operating pressures and operating temperatures of the three pumps are respectively 0-10 MPa and 15-35° C. The system, using ODS reversed silica gel with particle size of 30 um as packing materials, adopting 16 columns in series connection, is divided into four areas, each of which having 4 columns in series connection. The positions of inlet and outlet can be changed at intervals by switchover according to the electromagnetic valve, thus simulated moving of the separated bed is achieved. Dissolving the extractum B containing Ergosterol peroxide prepared in the step (2) with 95% methanol aqueous solution by heating and ultrasonic, filtrating the solution with 0.45 μm millipore filter to obtain the sample solution, of which the solid content is 0.2 g/mL; injecting the sample solution into the simulated moving-bed chromatography separation system for further purification; The system parameters are set up as follows: room temperature, flow rate of injection pump is 3 mL/min, flow rate of elution pump is 16 mL/min, is 10 mL/min, flow rate of raffinate is 9 mL/min, and switching time is 24 min; concentrating the extract liquor containing Ergosterol peroxide to dryness under reduced pressure by rotary evaporator, the bathing temperature at 70° C., and the vacuum degree below −0.085, to obtain the dry extract C rich in Ergosterol peroxide.

[0028] (4) Dissolving the dry extract C rich in Ergosterol peroxide prepared in the step (3) with ethyl acetate-cyclohexane under heating conditions, transferring it into an environment at a temperature of 0° C. for recrystallization, filtering and washing the precipitate to obtain Ergosterol peroxide with a purity of 53.7%. In this step, the dosage of ethyl acetate-cyclohexane is 20 times that of the dry extract C, the volume ratio of ethyl acetate to cyclohexane is 10:90, and the time of recrystallization is 40 h.

[0029] In this example, the content of Ergosterol peroxide is tested by HPLC-ESLD, and the chromatographic conditions are as follows: Agilent 1200 Series High Performance Liquid Chromatography (HPLC) System, Chromachem evaporative light-scattering detector (ELSD) from ESA company in America, Waters Symmetry Shield RP18 columns (4.6×250 mm, 5 μm), 90% acetonitrile aqueous solution as the mobile phase, isocratic elution, flow rate: 0.8mL/min, column temperature: 30° C., temperature of drift tube: 80° C., temperature of atomizer: 60° C., nitrogen pressure: 25 psi, gain: 3, injection volume: 20 μL, purity calculation by external standard method.

Example 2

[0030] Acquiring ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder by following steps:

[0031] (1) Preparing crude extract containing Ergosterol peroxide: taking the sporoderm-broken Ganoderma lucidum spore powder, performing extraction by reflux method, of which the extraction solvent is cyclohexane-petroleum ether (1:1), the liquid-solid ratio is 10, the extracting time at boiling condition is 2 h, the residue is repeatedly extracted for once, and the filtrate is merged; concentrating the extract liquid to dryness under reduced pressure by rotary evaporator, the bathing temperature at 60° C., and the vacuum degree below −0.085, and obtaining the crude extract A with an ergosterol peroxide content of 0.98% revealed by the test results.

[0032] (2) Purification by medium pressure preparative chromatography: dissolving the crude extract A containing Ergosterol peroxide prepared in the step (1) with 95% methanol aqueous solution by heating and ultrasonic, filtrating the solution with 0.45 μm millipore filter to obtain the sample solution for medium pressure preparative chromatography, of which the solid content is 0.2 g/mL; performing purification by medium pressure preparative chromatography, of which the medium pressure preparative chromatography column is packed with 30 μm ODS silica gel, the height of column is 45 cm, the injection rate is 5 mL/min, and the injection volume is 0.1 BV, performing isocratic elution with 80% methanol aqueous solution at the rate of 2.0 BV/h; collecting the fraction containing Ergosterol peroxide, concentrating it to dryness under reduced pressure by rotary evaporator, the bathing temperature at 65° C., and the vacuum degree below −0.085, to obtain the extractum B with an Ergosterol peroxide content of 10.2%. In this step, the impurities with low polarity are removed almost, and the impurities with high polarity are partly removed.

[0033] (3) Purification by simulated moving-bed: simulated moving bed chromatography separation system includes elution pump, injection pump, extracting pump, chromatographic columns, electromagnetic valve, check valve, signal collector, system controller, and so on, thereinto, the flow rate of both elution pump and extracting pump are 0-250 mL/min, that of injection pump is 0-50 mL/min, the operating pressures and operating temperatures of the three pumps are respectively 0-10 MPa and 15-35° C. The system, using ODS reversed silica gel with particle size of 50 μm as packing materials, adopting 8 columns in series connection, is divided into four areas, each of which having 2 columns in series connection. The positions of inlet and outlet can be changed at intervals by switchover according to the electromagnetic valve, thus simulated moving of the separated bed is achieved. Dissolving the extractum B containing Ergosterol peroxide prepared in the step (2) with 60% methanol aqueous solution by heating and ultrasonic, filtrating the solution with 0.45 μm millipore filter to obtain the sample solution, of which the solid content is 0.05 g/mL; injecting the sample solution into the simulated moving-bed chromatography separation system for further purification; The system parameters are set up as follows: room temperature, flow rate of injection pump is 6 mL/min, flow rate of elution pump is 38 mL/min, Extraction velocity is 23 mL/min, flow rate of raffinate is 21 mL/min, and switching time is 10 min; concentrating the extract liquor containing Ergosterol peroxide to dryness under reduced pressure by rotary evaporator, the bathing temperature at 65° C., and the vacuum degree below −0.085, to obtain the dry extract C rich in Ergosterol peroxide.

[0034] (4) Dissolving the dry extract C rich in Ergosterol peroxide prepared in the step (3) with ethyl acetate-cyclohexane under heating conditions, transferring it into a environment at a temperature of 8° C. for recrystallization, filtering and washing the precipitate to obtain Ergosterol peroxide with a purity of 66.3%. In this step, the dosage of ethyl acetate-cyclohexane is 10 times that of the dry extract C, the volume ratio of ethyl acetate to cyclohexane is 50:50, and the time of recrystallization is 48 h.

[0035] In this example, the method testing the content of Ergosterol peroxide is the same as that in the example 1.

Example 3

[0036] Acquiring ergosterol peroxide from sporoderm-broken Ganoderma lucidum spore powder by following steps:

[0037] (1) Preparing crude extract containing Ergosterol peroxide: taking the sporoderm-broken Ganoderma lucidum spore powder, performing extraction by reflux method, of which the extraction solvent is ethyl acetate-cyclohexane (1:1), the liquid-solid ratio is 6, the extracting time at boiling condition is 1 h, the residue is repeatedly extracted for twice, and the filtrate is merged; concentrating the extract liquid to dryness under reduced pressure by rotary evaporator, the bathing temperature at 70° C., and the vacuum degree below −0.085, and obtaining the crude extract A with an ergosterol peroxide content of 1.23% revealed by the test results.

[0038] (2) Purification by medium pressure preparative chromatography: dissolving the crude extract A containing Ergosterol peroxide prepared in the step (1) with 80% methanol aqueous solution by heating and ultrasonic, filtrating the solution with 0.45 μm millipore filter to obtain the sample solution for medium pressure preparative chromatography, of which the solid content is 0.15 g/mL;

[0039] performing purification by medium pressure preparative chromatography, of which the medium pressure preparative chromatography column is packed with 40 μm ODS silica gel, the height of column is 45 cm, the injection rate is 4 mL/min, and the injection volume is 0.1 BV, performing isocratic elution with 80% methanol aqueous solution at the rate of 1.8 BV/h; collecting the fraction containing Ergosterol peroxide, concentrating it to dryness under reduced pressure by rotary evaporator, the bathing temperature at 60° C., and the vacuum degree below −0.085, to obtain the extractum B with an Ergosterol peroxide content of 20.4%. In this step, the impurities with low polarity are removed almost, and the impurities with high polarity are partly removed.

[0040] (3) Purification by simulated moving-bed: simulated moving bed chromatography separation system includes elution pump, injection pump, extracting pump, chromatographic columns, electromagnetic valve, check valve, signal collector, system controller, and so on, thereinto, the flow rate of both elution pump and extracting pump are 0-250 mL/min, that of injection pump is 0-50 mL/min, the operating pressures and operating temperatures of the three pumps are respectively 0-10 MPa and 15-35° C. The system, using ODS reversed silica gel with particle size of 40 μm as packing materials, adopting 12 columns in series connection, is divided into four areas, each of which having 3 columns in series connection. The positions of inlet and outlet can be changed at intervals by switchover according to the electromagnetic valve, thus simulated moving of the separated bed is achieved. Dissolving the extractum B containing Ergosterol peroxide prepared in the step (2) with 85% methanol aqueous solution by heating and ultrasonic, filtrating the solution with 0.45 μm millipore filter to obtain the sample solution, of which the solid content is 0.15 g/mL; injecting the sample solution into the simulated moving-bed chromatography separation system for further purification; The system parameters are set up as follows: room temperature, flow rate of injection pump is 5 mL/min, flow rate of elution pump is 33 mL/min, Extraction velocity is 20 mL/min, flow rate of raffinate is 18 mL/min, and switching time is 15 min; concentrating the extract liquor containing Ergosterol peroxide to dryness under reduced pressure by rotary evaporator, the bathing temperature at 60° C., and the vacuum degree below −0.085, to obtain the dry extract C rich in Ergosterol peroxide.

[0041] (4) Dissolving the dry extract C rich in Ergosterol peroxide prepared in the step (3) with ethyl acetate-cyclohexane under heating conditions, transferring it into a environment at a temperature of 4° C. for recrystallization, filtering and washing the precipitate to obtain Ergosterol peroxide with a purity of 79.63%. In this step, the dosage of ethyl acetate-cyclohexane is 15 times that of the dry extract C, the volume ratio of ethyl acetate to cyclohexane is 30:70, and the time of recrystallization is 36 h.

[0042] In this example, the method testing the content of Ergosterol peroxide is the same as that in the example 1.

[0043] The above are only preferred embodiments of the present disclosure and should not be used for limiting the present disclosure. For those skilled in the art, any modifications, equivalent replacements, improvements and the like within the spirit and principle of the present disclosure shall fall within the scope of protection of the present disclosure.