Method for separating and purifying ganoderma triterpenes from ganoderma lucidum sporocarp

Abstract

The invention discloses a method for separating and purifying ganoderma triterpenes from ganoderma lucidum sporocarp, and belongs to the technical field of natural product extraction. In order to solve the problems of high energy consumption, high cost, high environmental protection pressure, low ganoderma triterpene extraction rate, low ganoderma triterpene purity and the like in the prior art, the invention provides a method for separating and purifying ganoderma triterpene from ganoderma sporocarp. Microfiltration, ultrafiltration, nanofiltration, macroporous adsorption resin adsorption separation, concentration, precipitation and drying are performed to obtain the triterpenoid extract, the content of ganoderma triterpenoid is larger than or equal to 70%, and the yield is larger than or equal to 5.50%. According to the method, the extraction process flow is simplified, the product yield is greatly improved, and good separation efficiency is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction, and in particular relates to a method for separating and purifying ganoderma triterpenes from ganoderma fruiting bodies. Background Art

[0002] According to the differences in the physicochemical properties and biochemical reaction characteristics of triterpenes, triterpenoid compounds can be extracted by using solubility, adsorption, vapor pressure neutralization, saponification and enzymatic hydrolysis. At present, the organic solvent method is generally used to extract Ganoderma triterpenes, which usually requires a long extraction time and consumes a large amount of extraction solvent. At the same time, the use of a large amount of organic solvents will have adverse effects on the human body and the environment. CN102772448A uses enzymatic hydrolysis and ultrasonic extraction to extract the Ganoderma lucidum fruiting body to obtain a crude extract, and then uses an adsorption resin to purify the crude extract, and finally obtains a Ganoderma lucidum triterpene content of 30% to 50%, and the yield is 2% to 4%, but the process requires the use of 2 to 3 enzymes for enzymatic hydrolysis, resulting in high material costs; CN1560072A crushes the dried Ganoderma lucidum fruiting body and uses supercritical CO2 extraction technology to extract Ganoderma lucidum triterpene acid, and the Ganoderma lucidum triterpene acid content in the extract can reach up to 42.5%, but the supercritical CO2 extraction technology requires a large investment in equipment and high production costs, which is not suitable for industrialized life; CN109893547A uses sodium hydroxide solution to extract Ganoderma lucidum fruiting body under ultrasonic conditions, and then uses an adsorption resin to purify the extract, and finally obtains a Ganoderma lucidum triterpene content of 19% to 21%, but the yield is not mentioned. The above methods have high equipment costs for separating Ganoderma lucidum triterpene, and are difficult to industrialize. At the same time, the cumbersome production process will further reduce the product yield.

[0003] CN110664946A discloses a method for extracting triterpenoids from ganoderma lucidum, which uses a preparation process of first column chromatography and then dialysis to extract triterpenoids from ganoderma lucidum. The first column chromatography will increase the separation pressure of the column chromatography, and the process is difficult to implement and has high requirements on production equipment. Furthermore, the dialysis bag cannot be used in industrial production.

[0004] CN112138031A discloses a preparation method and application of a Ganoderma lucidum spore powder extract containing Ganoderma lucidum triterpenes and Ganoderma lucidum polysaccharides, and CN110330575B discloses a preparation method of a Ganoderma lucidum extract, both of which involve breaking the Ganoderma lucidum spore powder (ultrasonic crushing), ethanol extraction, and column extraction of Ganoderma lucidum triterpenes. Direct column extraction has poor separation effect, poor separation between triterpenoids and impurities, and great difficulty in process application; while ultrasonic treatment utilizes cavitation effect to increase the solubility of the target substance in the solvent, has high requirements on production equipment, but is rarely used in industrial production, which is not conducive to industrial production.

[0005] CN110144015B discloses a method for the simultaneous extraction and purification of ganoderma lucidum polysaccharide, ganoderma lucidum triterpenic acid and amino acid. In the preparation process, ionic resin is used in the column chromatography section, which involves ion exchange. This type of resin requires more acid and alkali for each regeneration, which puts great pressure on the environmental protection and cannot be reused.

[0006] CN114377039A discloses a method for extracting and separating multiple components of ganoderma lucidum, which comprises the following steps: crushing ganoderma lucidum and then extracting by breaking the wall, and separating the extract in multiple stages to obtain ganoderma lucidum polysaccharide enrichment and ganoderma lucidum triterpenoid enrichment, wherein the material-liquid (water) ratio of the extraction is 1:18-22, the extraction temperature is 60℃-80℃, and the multi-stage separation is to separate the extract by using a nanofiltration membrane with a molecular weight of 1000 to obtain ganoderma lucidum polysaccharide enrichment, and by using a nanofiltration membrane with a molecular weight of 500 to obtain ganoderma lucidum triterpenoid enrichment; before the multi-stage separation, the extract is first subjected to centrifugal microfiltration treatment, and the microfiltration clear liquid is then subjected to multi-stage separation by nanofiltration membrane. However, the content of ganoderma lucidum triterpenoid obtained by this method is low, reaching only about 2.3mg / g. Summary of the invention

[0007] The technical problem to be solved by the present invention is to address the problems existing in the prior art: the separation equipment cannot be industrialized, such as ultrasonic crushing and dialysis bag equipment technology cannot be industrialized for large-scale production; the equipment has high energy consumption, such as the working pressure of supercritical extraction equipment is generally 15 to 50 MPa, the energy consumption is high, and the separation cost is relatively high; environmental pressure, such as the use of ion exchange resins in the prior art will consume more acid and alkali, and the environmental pressure generated is relatively high; the extraction efficiency and purity of Ganoderma lucidum triterpenes are relatively low, etc.

[0008] In order to solve the problems of the prior art, the present invention provides a method for separating and purifying Ganoderma triterpenes from Ganoderma lucidum fruiting bodies. The method comprises crushing the Ganoderma lucidum fruiting bodies, and then using microfiltration, ultrafiltration, nanofiltration, and macroporous adsorption resin for adsorption separation, concentration, precipitation, and drying to obtain a Ganoderma triterpenoid product. The equipment used are all common equipment in current industrial production, and the energy consumption and equipment maintenance costs are relatively low. At the same time, the reagents used in the production process have little environmental pressure.

[0009] The present invention provides a method for separating and purifying triterpenes of Ganoderma lucidum from Ganoderma lucidum fruiting bodies, which comprises the following steps:

[0010] A. mixing the ganoderma lucidum fruiting body (the ganoderma lucidum fruiting body is dried before use) with an ethanol aqueous solution, and then grinding and refining the mixture under reflux to obtain a ganoderma lucidum fruiting body grinding liquid;

[0011] B. The Ganoderma lucidum fruiting body grinding liquid obtained in step A is first subjected to low-pressure crushing to a particle diameter of 1 to 5 μm, and then subjected to high-pressure crushing to a particle diameter of 0.1 to 1.0 μm to obtain a Ganoderma lucidum fruiting body homogenate;

[0012] C. Microfiltering the homogenate of the Ganoderma lucidum fruiting body obtained in step B through a microfiltration membrane with a pore size of 10 to 100 nm, and collecting the microfiltration permeate;

[0013] D. ultrafiltration the microfiltration permeate obtained in step C using an ultrafiltration membrane with a molecular weight cutoff of 800 to 2000 Da, and collecting the ultrafiltration permeate;

[0014] E. The ultrafiltration permeate obtained in step D is subjected to nanofiltration using a nanofiltration membrane with a molecular weight cutoff of 90 to 500 Da, and the nanofiltration retentate is collected; the nanofiltration retentate is loaded into a separation column equipped with a macroporous adsorption resin, and adsorbed, washed and eluted to obtain an eluate containing the Ganoderma lucidum triterpene extract;

[0015] F. Concentrate the eluate containing the triterpene extract of Ganoderma lucidum obtained in step E under reduced pressure, add a precipitant to the concentrate for precipitation, and dry the precipitate to obtain the triterpene extract of Ganoderma lucidum.

[0016] In the early stage of the present invention, the fruiting bodies of Ganoderma lucidum were directly immersed in a solvent and the target substance was extracted under ultrasonic conditions. However, the experiment found that the extraction effect and efficiency under ultrasonic treatment conditions were far lower than those of the current low-pressure and high-pressure crushing treatment methods used in the present invention.

[0017] The present invention successively adopts low-pressure crushing to a particle diameter of 1 to 5 μm, and then high-pressure crushing to prepare a homogenate of Ganoderma lucidum fruiting bodies that is conducive to subsequent microfiltration, ultrafiltration and nanofiltration multi-stage separation. If high-pressure crushing is directly used, it is easy to cause uneven crushing, which is not conducive to subsequent multi-stage filtration, and it will also affect the life of the high-pressure homogenizer, and it will also cause uneven crushing. At the same time, high-pressure crushing to a particle diameter of 0.1 to 1.0 μm can increase the specific surface area of ​​Ganoderma lucidum fruiting bodies to the greatest extent, so that the solvent can fully extract the target substance; if the high-pressure crushing diameter is too large, it will affect the microfiltration, ultrafiltration and nanofiltration multi-stage separation rate and separation effect. After testing, in the above method, in step A, it is preferred that low-pressure crushing to a particle diameter of 2 to 3 μm and high-pressure crushing to a particle diameter of 0.1 to 0.5 μm.

[0018] Wherein, in the above method, in step A, the volume concentration of the ethanol aqueous solution is 50% to 60%.

[0019] Wherein, in the above method, in step A, the mass volume ratio of the Ganoderma lucidum fruiting body to the ethanol aqueous solution is 1g:10-30mL.

[0020] Wherein, in the above method, in step A, the number of reflux grinding is 5 to 8 times.

[0021] Preferably, in the above method, in step A, the reflux grinding is performed 6 to 7 times.

[0022] Wherein, in the above method, in step A, a colloid mill is used for reflux grinding.

[0023] Wherein, in the above method, in step B, the working pressure of the low-pressure crushing is 400-600 bar.

[0024] Preferably, in the above method, in step B, the working pressure of the low-pressure crushing is 420-560 bar.

[0025] Wherein, in the above method, in step B, the working pressure of the high-pressure crushing is 1000-1300 bar.

[0026] Preferably, in the above method, in step B, the working pressure of the high-pressure crushing is 1100-1300 bar.

[0027] More preferably, in the above method, in step B, the working pressure of the high-pressure crushing is 1100-1250 bar.

[0028] Wherein, in the above method, in step B, the low-pressure crushing and the high-pressure crushing are both carried out in a homogenizer.

[0029] Wherein, in the above method, in step C, the pore size of the microfiltration membrane is 20 to 60 nm.

[0030] Preferably, in the above method, in step C, the pore size of the microfiltration membrane is 30 nm.

[0031] Wherein, in the above method, in step C, the working pressure during microfiltration is 0.2-1.0 MPa.

[0032] Preferably, in the above method, in step C, the working pressure during microfiltration is 0.4-0.8 MPa.

[0033] More preferably, in the above method, in step C, the working pressure during microfiltration is 0.5-0.6 MPa.

[0034] Wherein, in the above method, in step C, the microfiltration membrane is a ceramic membrane.

[0035] Preferably, in the above method, in step D, the molecular weight cutoff of the ultrafiltration membrane is 800 to 1000 Da.

[0036] Wherein, in the above method, in step D, the working pressure during ultrafiltration is 1.0 to 3.0 MPa.

[0037] Preferably, in the above method, in step D, the working pressure during ultrafiltration is 1.0 to 2.0 MPa.

[0038] More preferably, in the above method, in step D, the working pressure during ultrafiltration is 1.5 to 2.0 MPa.

[0039] Wherein, in the above method, in step D, the material of the ultrafiltration membrane is a material that does not react with the extract from the Ganoderma lucidum fruiting body, specifically, it can be polyethersulfone or polyolefin.

[0040] Preferably, in the above method, in step D, the material of the ultrafiltration membrane is polyolefin.

[0041] Wherein, in the above method, in step D, the ultrafiltration retentate is collected, dehydrated and dried to obtain the Ganoderma lucidum polysaccharide extract.

[0042] Preferably, in the above method, in step E, the molecular weight cutoff of the nanofiltration membrane is 150 to 300 Da.

[0043] Wherein, in the above method, in step E, the working pressure during nanofiltration is 1.0 to 3.0 MPa.

[0044] Preferably, in the above method, in step E, the working pressure during nanofiltration is 1.5-2.0 MPa.

[0045] Wherein, in the above method, in step E, the material of the nanofiltration membrane is a material that does not react with the extract from the Ganoderma lucidum fruiting body, specifically, it can be polyethersulfone or polyolefin.

[0046] Preferably, in the above method, in step E, the material of the nanofiltration membrane is polyolefin.

[0047] Wherein, in the above method, in step E, the macroporous adsorption resin is a non-polar macroporous resin, which is formed by condensation of styrene and divinylbenzene.

[0048] Wherein, in the above method, in step E, the particle size of the macroporous adsorption resin is 16-50 mesh, the particle size is 0.3-1.2 mm, the effective size is 0.4-0.6 mm, the humidity is 65±3%, and the specific surface area is ≥550m 2 / g.

[0049] Preferably, in the above method, in step E, the specific surface area of ​​the non-polar macroporous resin is preferably 550 to 700 m 2 / g.

[0050] Wherein, in the above method, in step E, the macroporous adsorption resin is first soaked in 95% ethanol for 24 hours before use to allow it to fully swell, and then the column bed is rinsed with 95% ethanol and then rinsed with distilled water until there is no alcohol smell before use.

[0051] Wherein, in the above method, in step E, the height-to-diameter ratio of the separation column is 5:1 to 7:1.

[0052] Preferably, in the above method, in step E, the height-to-diameter ratio of the separation column is 6:1 to 7:1.

[0053] Wherein, in the above method, in step E, the loading amount of the macroporous adsorption resin is calculated based on the mass of Ganoderma triterpenes in the nanofiltration retentate, and is loaded at 20 to 60 mg Ganoderma triterpenes / g macroporous adsorption resin.

[0054] Preferably, in the above method, in step E, the loading amount of the macroporous adsorption resin is 60 mg of Ganoderma triterpenes / g of macroporous adsorption resin.

[0055] Wherein, in the above method, in step E, water is used as the washing solvent during washing.

[0056] Wherein, in the above method, in step E, during elution, an ethanol aqueous solution with a volume concentration of 40% to 70% is used as the elution solvent.

[0057] Preferably, in the above method, in step E, during elution, an ethanol aqueous solution with a volume concentration of 60% is used as the elution solvent.

[0058] Wherein, in the above method, in step E, the loading flow rate of the nanofiltration retentate is 1.0 to 1.5 BV / h.

[0059] Preferably, in the above method, in step E, the loading flow rate of the nanofiltration retentate is 1.0 BV / h.

[0060] Wherein, in the above method, in step E, during washing, the loading flow rate of the washing solvent is 1.0 to 1.5 BV / h.

[0061] Preferably, in the above method, in step E, during washing, the loading flow rate of the washing solvent is 1.3 BV / h.

[0062] Wherein, in the above method, in step E, during elution, the loading flow rate of the elution solvent is 1.0 to 1.5 BV / h.

[0063] Preferably, in the above method, in step E, during elution, the loading flow rate of the elution solvent is 1.2 BV / h.

[0064] Wherein, in the above method, in step E, when washing (the purpose of washing is mainly to wash away pigment impurities), when the effluent becomes colorless, the washing is stopped.

[0065] Wherein, in the above method, in step E, the elution time is 5 to 8 hours.

[0066] Preferably, in the above method, in step E, the elution time is 6 to 7 hours.

[0067] Wherein, in the above method, in step F, the eluent is concentrated under reduced pressure to 1% to 2% of the volume of the eluent.

[0068] Wherein, in the above method, in step F, the temperature of the reduced pressure concentration is 40°C to 60°C, and the pressure is 20 to 35 mbar.

[0069] Preferably, in the above method, in step F, the temperature of the reduced pressure concentration is 48-55°C.

[0070] Wherein, in the above method, in step F, the precipitant is water.

[0071] Wherein, in the above method, in step F, the amount of the precipitant used is 2.0 to 6.0 times the volume of the concentrated solution.

[0072] Wherein, in the above method, in step F, the drying temperature is 40°C to 50°C.

[0073] Among them, in the above method, in step F, the specific operation of adding the precipitant to the concentrated solution for precipitation is: after the concentrated solution is cooled to room temperature, stirring at 100-200rpm for 20-30min, then adding the precipitant at one time (in the process of adding the precipitant to the concentrated solution for precipitation, the temperature of the precipitation system is maintained in the range of 10-25°C), stirring at 100-200rpm for 2.0-3.5h, stopping stirring, standing, solid-liquid separation, and obtaining a precipitate.

[0074] Beneficial effects of the present invention:

[0075] The colloid mill first used in the present invention utilizes a high-speed rotating grinding disc to cut and grind the material. It has a short crushing time for hard samples and can grind the samples into powder or particles evenly and quickly. The particle size after crushing reaches 0.1-0.5 μm; the homogenizer can achieve nano-level dispersion and homogenization of materials through high-pressure shearing, impact and cavitation, and has a large processing capacity to meet large-scale production needs.

[0076] The homogenate of the ganoderma lucidum fruiting body after low-pressure and high-pressure crushing is conducive to subsequent microfiltration, ultrafiltration and nanofiltration multi-stage separation, and filtration with different pore sizes or molecular weight cutoffs is adopted to preferentially remove impurities with large differences in molecular weight and triterpenoid substances, so as to reduce the pressure of the subsequent separation process (column chromatography); compared with the dialysis bag, the multi-stage filtration (microfiltration, ultrafiltration, nanofiltration) of the present invention has significantly improved efficiency and can be mass-produced; at the same time, the ganoderma lucidum polysaccharide in the ganoderma lucidum fruiting body is separated separately, and it can be produced as a by-product, thereby reducing the cost.

[0077] The present invention combines membrane separation technology with column chromatography technology to improve separation efficiency and target substance yield. The present invention uses non-polar resin for separation, does not involve ion exchange, and therefore does not need to use acid or alkali to regenerate the resin; the non-polar macroporous resin is an adsorption resin without any functional group obtained by polymerization of monomers with very small dipole moments, and has a strong hydrophobicity on the pore surface, which is suitable for adsorption of macromolecular substances, and the ethanol used in the resin regeneration process in the present invention can be recycled by reduced pressure concentration.

[0078] The equipment and instruments used in this application are all common equipment used by current industrial producers, with low cost and simple operation; the resin in the column chromatography section can be reused after regeneration, and the organic solvent can be repeatedly recovered, and the separation cost is low; the extraction process of the present invention simplifies the process flow, greatly improves the product yield, and has good separation efficiency. The content of Ganoderma triterpenoids in the Ganoderma triterpenoid extract is ≥70%, and the yield is ≥5.50%, which is more conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 The present invention is a schematic diagram of the process of separating and purifying triterpenes of Ganoderma lucidum.

[0080] Figure 2 This is a physical picture of the Ganoderma lucidum triterpene extract obtained in the example of the present invention. DETAILED DESCRIPTION

[0081] The present invention is further described in detail below by way of examples, but the protection scope of the present invention is not limited to the scope of the examples.

[0082] Example 1

[0083] (1) After the Ganoderma lucidum fruiting body is dried, 200 g of dry matter of the Ganoderma lucidum fruiting body is obtained, 2200 mL of 50% ethanol solution is added, and the mixture is repeatedly refluxed and ground for 6 times using a colloid mill to obtain a Ganoderma lucidum fruiting body grinding liquid.

[0084] (2) The working pressure of the homogenizer was set to low pressure 560 bar (low pressure crushing to particle diameter of 2-3 μm) and high pressure 1240 bar (high pressure crushing particle diameter of 0.1-0.5 μm), and the Ganoderma lucidum fruiting body grinding liquid was crushed and homogenized to obtain Ganoderma lucidum fruiting body homogenate liquid.

[0085] (3) A ceramic membrane with a pore size of 30 nm was used to microfilter the homogenate of Ganoderma lucidum fruiting bodies. The centrifugal pump pressure was 0.5 MPa, and the microfiltration permeate was collected.

[0086] (4) A polyolefin ultrafiltration membrane with a molecular weight cutoff of 800 to 1000 Da was used to filter the microfiltration permeate at a working pressure of 1.5 MPa, and the ultrafiltration permeate was collected; wherein the ultrafiltration retentate contained Ganoderma lucidum polysaccharide, and the ultrafiltration retentate was collected separately and the polysaccharide content was determined to be 3.52% using the phenol-sulfuric acid method.

[0087] (5) A polyolefin nanofiltration membrane with a molecular weight cutoff of 150 to 300 Da was used to filter the ultrafiltration permeate at a working pressure of 1.8 MPa. The nanofiltration retentate was collected to detect the content of Ganoderma triterpenoids. The macroporous adsorption resin was loaded onto a separation column chromatographic column according to a loading amount of 60 mg Ganoderma triterpenoids / g macroporous adsorption resin. The height-to-diameter ratio of the chromatographic column was 6:1, and the loading flow rate of the nanofiltration retentate was 1.0 BV / h. After the loading was completed, impurities were eluted with pure water at a flow rate of 1.3 BV / h. The elution was completed when the effluent was colorless. Ganoderma triterpenoids were eluted with 60% ethanol solution at a flow rate of 1.2 BV / h. After elution for 6.5 hours, the eluate containing Ganoderma triterpenoid extract was collected.

[0088] The macroporous adsorption resin is prepared by condensation of styrene and divinylbenzene; the particle size of the macroporous adsorption resin is 16-50 mesh, the particle size is 0.3-1.2 mm, the effective size is 0.4-0.6 mm, the humidity is 65±3%, and the specific surface area is 550-700 m 2 / g (this resin is used in the following examples).

[0089] (6) The eluate containing the triterpene extract of Ganoderma lucidum was concentrated by vacuum distillation at 48° C. and a vacuum degree of 23 mbar to obtain a concentrated solution (the volume of the concentrated solution is 1% to 2% of the volume of the eluate); when the concentrated solution was cooled to 25° C., it was stirred at a stirring speed of 150 rpm. After stirring for 30 minutes, the system was stable, and 2.5 times the volume of the concentrated solution was added with pure water at one time. The stirring was continued for 3 hours and then stopped. After suction filtration and drying at 40° C., a triterpene extract of Ganoderma lucidum with a content of 73.5% was obtained, and the yield was 5.51%.

[0090] The actual picture of the Ganoderma lucidum triterpene extract obtained is shown in Figure 2 .

[0091] Example 2

[0092] (1) After the Ganoderma lucidum fruiting body is dried, 350 g of dry matter of the Ganoderma lucidum fruiting body is obtained, 7000 mL of 50% ethanol solution is added, and the mixture is repeatedly refluxed and ground 7 times using a colloid mill to obtain a Ganoderma lucidum fruiting body grinding liquid.

[0093] (2) The working pressure of the homogenizer was set to 420 bar for low pressure (low pressure crushing to particle diameter of 2-3 μm) and 1100 bar for high pressure (high pressure crushing to particle diameter of 0.1-0.5 μm), and the Ganoderma lucidum fruiting body grinding liquid was crushed and homogenized to obtain Ganoderma lucidum fruiting body homogenate liquid.

[0094] (3) A ceramic membrane with a pore size of 30 nm was used to microfilter the homogenate of Ganoderma lucidum fruiting bodies. The centrifugal pump pressure was 0.6 MPa, and the microfiltration permeate was collected.

[0095] (4) A polyolefin ultrafiltration membrane with a molecular weight cutoff of 800 to 1000 Da was used to filter the microfiltration permeate at a working pressure of 1.5 MPa, and the ultrafiltration permeate was collected; wherein the ultrafiltration retentate contained Ganoderma lucidum polysaccharide, and the ultrafiltration retentate was collected separately and the polysaccharide content was determined to be 3.69% using the phenol-sulfuric acid method.

[0096] (5) A polyolefin nanofiltration membrane with a molecular weight cutoff of 150 to 300 Da was selected to filter the ultrafiltration permeate, and the working pressure was 2.0 MPa. The nanofiltration retentate was collected to detect the content of Ganoderma triterpenoids. The macroporous adsorption resin was loaded onto a separation column chromatographic column according to a loading amount of 60 mg Ganoderma triterpenoids / g macroporous adsorption resin. The height-to-diameter ratio of the chromatographic column was 6.5:1, and the loading flow rate of the nanofiltration retentate was 1.0 BV / h. After loading, impurities were eluted with pure water at a flow rate of 1.3 BV / h. When the effluent was colorless, the impurities were washed. The Ganoderma triterpenoids were eluted with a 60% ethanol solution at a flow rate of 1.2 BV / h. After elution for 7 hours, the eluate containing the Ganoderma triterpenoid extract was collected. The macroporous adsorption resin used was the same as that used in Example 1.

[0097] (6) The eluate containing the triterpene extract of Ganoderma lucidum was concentrated by vacuum distillation at 50° C. and a vacuum degree of 25 mbar to obtain a concentrated solution (the volume of the concentrated solution is 1% to 2% of the volume of the eluate); when the concentrated solution was cooled to 25° C., it was stirred at a stirring speed of 150 rpm. After stirring for 20 minutes, the system was stable, and 3.5 times the volume of the concentrated solution was added with pure water at one time. The stirring was continued for 2.5 hours and then stopped. After suction filtration and drying at 40° C., a triterpene extract of Ganoderma lucidum with a content of 71.2% was obtained, and the yield was 5.65%.

[0098] Example 3

[0099] (1) After the Ganoderma lucidum fruiting body is dried, 400 g of dry matter of the Ganoderma lucidum fruiting body is obtained, 10000 mL of 50% ethanol solution is added, and the mixture is repeatedly refluxed and ground for 6 times using a colloid mill to obtain a Ganoderma lucidum fruiting body grinding liquid.

[0100] (2) The working pressure of the homogenizer was set to 490 bar for low pressure (low pressure crushing to particle diameter of 2-3 μm) and 1220 bar for high pressure (high pressure crushing to particle diameter of 0.1-0.5 μm), and the Ganoderma lucidum fruiting body grinding liquid was crushed and homogenized to obtain Ganoderma lucidum fruiting body homogenate liquid.

[0101] (3) A ceramic membrane with a pore size of 30 nm was used to microfilter the homogenate of Ganoderma lucidum fruiting bodies. The centrifugal pump pressure was 0.6 MPa, and the microfiltration permeate was collected.

[0102] (4) A polyolefin ultrafiltration membrane with a molecular weight cutoff of 800 to 1000 Da was used to filter the microfiltration permeate at a working pressure of 1.8 MPa, and the ultrafiltration permeate was collected; wherein the ultrafiltration retentate contained Ganoderma lucidum polysaccharide, and the polysaccharide content of the ultrafiltration retentate was determined to be 2.91% by the phenol-sulfuric acid method.

[0103] (5) A polyolefin nanofiltration membrane with a molecular weight cutoff of 150 to 300 Da was selected to filter the ultrafiltration permeate, and the working pressure was 2.0 MPa. The nanofiltration retentate was collected to detect the content of Ganoderma triterpenoids. The macroporous adsorption resin was loaded onto a separation column chromatographic column according to a loading amount of 60 mg Ganoderma triterpenoids / g macroporous adsorption resin. The height-to-diameter ratio of the chromatographic column was 7:1, and the loading flow rate of the nanofiltration retentate was 1.0 BV / h. After loading, impurities were eluted with pure water at a flow rate of 1.3 BV / h. When the effluent was colorless, the impurities were washed. The Ganoderma triterpenoids were eluted with a 60% ethanol solution at a flow rate of 1.2 BV / h. After elution for 6 hours, the eluate containing the Ganoderma triterpenoid extract was collected. The macroporous adsorption resin used was the same as that used in Example 1.

[0104] (6) The eluate containing the triterpene extract of Ganoderma lucidum was concentrated by vacuum distillation at 50° C. and a vacuum degree of 20 mbar to obtain a concentrated solution (the volume of the concentrated solution is 1% to 2% of the volume of the eluate); when the concentrated solution was cooled to 25° C., it was stirred at a stirring speed of 150 rpm. After stirring for 25 minutes, the system was stable, and pure water 4 times the volume of the concentrated solution was added at one time. The stirring was continued for 3 hours and then stopped. After suction filtration and drying at 40° C., a triterpene extract of Ganoderma lucidum with a content of 70.5% was obtained, and the yield was 5.61%.

[0105] Example 4

[0106] (1) After the Ganoderma lucidum fruiting body is dried, 500 g of dry matter of the Ganoderma lucidum fruiting body is obtained, 14000 mL of 50% ethanol solution is added, and the mixture is repeatedly refluxed and ground for 6 times using a colloid mill to obtain a Ganoderma lucidum fruiting body grinding liquid.

[0107] (2) The working pressure of the homogenizer is set to a low pressure of 500 bar (low pressure crushing to a particle diameter of 2 to 3 μm) and a high pressure of 1250 bar (high pressure crushing to a particle diameter of 0.1 to 0.5 μm), and the Ganoderma lucidum fruiting body grinding liquid is crushed and homogenized to obtain a Ganoderma lucidum fruiting body homogenate liquid.

[0108] (3) A ceramic membrane with a pore size of 30 nm was used to microfilter the homogenate of Ganoderma lucidum fruiting bodies. The centrifugal pump pressure was 0.6 MPa, and the microfiltration permeate was collected.

[0109] (4) A polyolefin ultrafiltration membrane with a molecular weight cutoff of 800 to 1000 Da was used to filter the microfiltration permeate at a working pressure of 1.8 MPa, and the ultrafiltration permeate was collected; wherein the ultrafiltration retentate contained Ganoderma lucidum polysaccharide, and the polysaccharide content of the ultrafiltration retentate was determined to be 3.18% by the phenol-sulfuric acid method.

[0110] (5) A polyolefin nanofiltration membrane with a molecular weight cutoff of 150 to 300 Da was selected to filter the ultrafiltration permeate, and the working pressure was 2.0 MPa. The nanofiltration retentate was collected to detect the content of Ganoderma triterpenoids. The macroporous adsorption resin was loaded onto a separation column chromatographic column according to a loading amount of 60 mg Ganoderma triterpenoids / g macroporous adsorption resin. The height-to-diameter ratio of the chromatographic column was 7:1, and the loading flow rate of the nanofiltration retentate was 1.0 BV / h. After loading, impurities were eluted with pure water at a flow rate of 1.3 BV / h. When the effluent was colorless, the impurities were washed. The Ganoderma triterpenoids were eluted with a 60% ethanol solution at a flow rate of 1.2 BV / h. After elution for 6 hours, the eluate containing the Ganoderma triterpenoid extract was collected. The macroporous adsorption resin used was the same as that used in Example 1.

[0111] (6) The eluate containing the triterpene extract of Ganoderma lucidum was concentrated by vacuum distillation at 55° C. and a vacuum degree of 30 mbar to obtain a concentrated solution (the volume of the concentrated solution was 1% to 2% of the volume of the eluate); when the concentrated solution was cooled to 25° C., it was stirred at a stirring speed of 180 rpm. After stirring for 30 min, the system was stable, and pure water 6 times the volume of the concentrated solution was added at one time. The stirring was continued for 3.5 h and then stopped. After suction filtration and drying at 40° C., a triterpene extract of Ganoderma lucidum with a content of 73.5% was obtained, and the yield was 5.88%.

[0112] The present invention provides a method and idea for separating and purifying triterpenes from ganoderma lucidum. There are many methods and approaches to realize the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be realized by existing technologies.

Claims

1. A method for separating and purifying triterpenoids from ganoderma lucidum fruiting bodies, characterized in that: The following steps are involved: A. mixing the ganoderma lucidum fruiting body with an ethanol aqueous solution, and then grinding and refining the mixture under reflux to obtain a ganoderma lucidum fruiting body grinding liquid; B. The Ganoderma lucidum fruiting body grinding liquid obtained in step A is first subjected to low-pressure crushing to a particle diameter of 1 to 5 μm, and then subjected to high-pressure crushing to a particle diameter of 0.1 to 1.0 μm to obtain a Ganoderma lucidum fruiting body homogenate; C. Microfiltering the homogenate of the Ganoderma lucidum fruiting body obtained in step B through a microfiltration membrane with a pore size of 10 to 100 nm, and collecting the microfiltration permeate; D. ultrafiltration the microfiltration permeate obtained in step C using an ultrafiltration membrane with a molecular weight cutoff of 800 to 2000 Da, and collecting the ultrafiltration permeate; E. The ultrafiltration permeate obtained in step D is subjected to nanofiltration using a nanofiltration membrane with a molecular weight cutoff of 90 to 500 Da, and the nanofiltration retentate is collected; the nanofiltration retentate is loaded into a separation column equipped with a macroporous adsorption resin, and adsorbed, washed and eluted to obtain an eluate containing the Ganoderma lucidum triterpene extract; F. Concentrate the eluate containing the triterpene extract of Ganoderma lucidum obtained in step E under reduced pressure, add a precipitant to the concentrate for precipitation, and dry the precipitate to obtain the triterpene extract of Ganoderma lucidum.

2. The method for separating and purifying triterpenoids from ganoderma lucidum fruiting bodies according to claim 1, characterized in that: At least one of the following is met: In step A, the low pressure is crushed to a particle diameter of 2 to 3 μm, and the high pressure is crushed to a particle diameter of 0.1 to 0.5 μm; In step A, the volume concentration of the ethanol aqueous solution is 50% to 60%; In step A, the mass volume ratio of the Ganoderma lucidum fruiting body to the ethanol aqueous solution is 1 g: 10-30 mL; In step A, the reflux grinding is performed 5 to 8 times, preferably 6 to 7 times; In step A, reflux grinding is performed using a colloid mill.

3. The method for separating and purifying triterpenoids from ganoderma lucidum fruiting bodies according to claim 1, characterized in that: At least one of the following is met: In step B, the working pressure of the low-pressure crushing is 400-600 bar; preferably 420-560 bar; In step B, the working pressure of the high-pressure crushing is 1000-1300 bar; preferably 1100-1300 bar; more preferably 1100-1250 bar; In step B, the low-pressure crushing and the high-pressure crushing are both performed in a homogenizer.

4. The method for separating and purifying triterpenoids from ganoderma lucidum fruiting bodies according to claim 1, characterized in that: At least one of the following is met: In step C, the pore size of the microfiltration membrane is 20 to 60 nm, preferably 30 nm; In step C, the working pressure during microfiltration is 0.2-1.0 MPa; preferably 0.4-0.8 MPa; more preferably 0.5-0.6 MPa; In step C, the microfiltration membrane is a ceramic membrane.

5. The method for separating and purifying triterpenoids from ganoderma lucidum fruiting bodies according to claim 1, characterized in that: At least one of the following is met: In step D, the molecular weight cut-off of the ultrafiltration membrane is 800 to 1000 Da; In step D, the working pressure during ultrafiltration is 1.0 to 3.0 MPa; preferably 1.0 to 2.0 MPa; more preferably 1.5 to 2.0 MPa; In step D, the material of the ultrafiltration membrane is polyethersulfone or polyolefin, preferably polyolefin; In step D, the ultrafiltration retentate is collected, dehydrated and dried to obtain the Ganoderma lucidum polysaccharide extract.

6. The method for separating and purifying triterpenoids from ganoderma lucidum fruiting bodies according to claim 1, characterized in that: At least one of the following is met: In step E, the molecular weight cutoff of the nanofiltration membrane is 150 to 300 Da; In step E, the working pressure during nanofiltration is 1.0 to 3.0 MPa, preferably 1.5 to 2.0 MPa; In step E, the material of the nanofiltration membrane is polyethersulfone or polyolefin, preferably polyolefin.

7. The method for separating and purifying triterpenoids from ganoderma lucidum fruiting bodies according to claim 1, characterized in that: At least one of the following is met: In step E, the macroporous adsorption resin is a non-polar macroporous resin, which is formed by condensation of styrene and divinylbenzene; In step E, the macroporous adsorption resin has a particle size of 16-50 mesh, a particle diameter of 0.3-1.2 mm, an effective size of 0.4-0.6 mm, a humidity of 65±3%, and a specific surface area of ​​≥550 m 2 / g; In step E, the macroporous adsorption resin is soaked in 95% ethanol for 24 hours before use to fully swell the macroporous adsorption resin, and then the column bed is rinsed with 95% ethanol and then with distilled water until there is no alcohol smell before use; In step E, the height-to-diameter ratio of the separation column is 5:1 to 7:1; preferably 6:1 to 7:1; In step E, the loading amount of the macroporous adsorption resin is calculated based on the mass of the ganoderma triterpenes in the nanofiltration retentate, and is 20-60 mg of ganoderma triterpenes / g of macroporous adsorption resin; preferably 60 mg of ganoderma triterpenes / g of macroporous adsorption resin.

8. The method for separating and purifying triterpenoids from ganoderma lucidum fruiting bodies according to claim 1, characterized in that: At least one of the following is met: In step E, water is used as the washing solvent during washing; In step E, during elution, an ethanol aqueous solution with a volume concentration of 40% to 70% is used as the elution solvent; preferably, an ethanol aqueous solution with a volume concentration of 60% is used as the elution solvent; In step E, the loading flow rate of the nanofiltration retentate is 1.0 to 1.5 BV / h, preferably 1.0 BV / h; In step E, during the washing process, the washing solvent has a sample loading rate of 1.0 to 1.5 BV / h, preferably 1.3 BV / h; In step E, during elution, the loading flow rate of the elution solvent is 1.0 to 1.5 BV / h; preferably 1.2 BV / h; In step E, when washing impurities, when the effluent becomes colorless, the washing is stopped; In step E, the elution time is 5 to 8 hours, preferably 6 to 7 hours.

9. The method for separating and purifying triterpenoids from ganoderma lucidum fruiting bodies according to claim 1, characterized in that: At least one of the following is met: In step F, the mixture is concentrated under reduced pressure to 1% to 2% of the volume of the eluate; In step F, the temperature of the reduced pressure concentration is 40°C to 60°C, and the pressure is 20 to 35 mbar; preferably 48 to 55°C; In step F, the precipitant is water; In step F, the amount of the precipitant is 2.0 to 6.0 times the volume of the concentrated solution; In step F, the drying temperature is 40°C to 50°C.

10. The method for separating and purifying triterpenoids from ganoderma lucidum fruiting bodies according to any one of claims 1 to 9, characterized in that: In step F, the specific operation of adding the precipitant to the concentrated solution for precipitation is: after the concentrated solution is cooled to room temperature, stirring at 100-200 rpm for 20-30 min, then adding the precipitant all at once, stirring at 100-200 rpm for 2.0-3.5 h, stopping stirring, standing, solid-liquid separation, and obtaining a precipitate.

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