Preparation method of ganoderma ketonetriol and application of ganoderma ketonetriol in relieving cardiovascular hypoxia reoxygenation injury

Abstract

The invention relates to the field of active substance extraction, and provides a preparation method of ganoderma ketonetriol and application of ganoderma ketonetriol in relieving cardiovascular hypoxia reoxygenation injury. The preparation method comprises the following steps: (1) carrying out ultrasonic extraction on a lucid ganoderma raw material to obtain an extract, wherein the ultrasonic power is 50-100 W; purifying the extract with macroporous resin to obtain a crude component; (2) sequentially carrying out primary separation and secondary separation on the crude component to obtain ganoderma ketonetriol; a C18 column of 40-60 [mu] m is used for the first separation, and the flow velocity of a mobile phase is 50-100 mL / min; a C18 column of 5-10 [mu] m is used in the second separation, and the flow velocity of the mobile phase is 20-40 mL / min; a mobile phase A of two times of separation is a trifluoroacetic acid aqueous solution, and a mobile phase B is acetonitrile. According to the method, ultrasonic extraction and macroporous resin purification are firstly carried out, and then two times of liquid phase separation are carried out, so that the preparation yield and purity of ganoderma ketonetriol are remarkably improved. The prepared ganoderma lucidum ketonetriol can relieve cardiovascular hypoxia reoxygenation injury.

Description

Technical Field

[0001] The invention relates to the field of active substance extraction, in particular to a preparation method of ganoderma lucidum ketone triol and its application in alleviating cardiovascular hypoxia-reoxygenation damage. Background Art

[0002] The molecular formula of Ganodermanontriol (English name Ganodermanontriol, CAS.106518-63-2) is C 30 H 48 O4, molecular weight 472.7, structural formula as shown: Ganoderma lucidum ketone triol is mainly prepared by silica gel column chromatography (for example, Liu Chao. Study on chemical composition of Ganoderma lucidum and Ganoderma lucidum and determination of triterpenic acid content of Ganoderma lucidum [J]. Peking Union Medical College, 2009.), but this separation method takes a long time, has low efficiency, and obtains a small amount of target compounds, which restricts the in-depth research and effective development and application of Ganoderma lucidum ketone triol. Therefore, an ideal solution is needed. Summary of the invention

[0003] The invention provides a method for preparing ganoderma lucidum ketone triol and an application thereof for alleviating cardiovascular hypoxia-reoxygenation injury, wherein ultrasonic extraction and macroporous resin purification are first performed, and then liquid phase separation is performed twice, thereby significantly improving the preparation yield and purity of ganoderma lucidum ketone triol. The prepared ganoderma lucidum ketone triol can alleviate cardiovascular hypoxia-reoxygenation injury.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions: The preparation method of ganoderma lucidum ketone triol comprises the following steps: (1) extracting the raw material of Ganoderma lucidum by ultrasonic extraction, with the ultrasonic power being 50-100W; purifying the extract by using a macroporous resin to obtain a crude fraction; (2) The crude fraction is subjected to a first separation and a second separation in sequence to obtain ganoderma lucidum ketone triol; the first separation uses a 40-60 μm C18 column with a mobile phase flow rate of 50-100 mL / min; the second separation uses a 5-10 μm C18 column with a mobile phase flow rate of 20-40 mL / min; the mobile phase A for the two separations is trifluoroacetic acid aqueous solution, and the mobile phase B is acetonitrile.

[0005] Preferably, the ultrasonic extraction conditions in step (1) are as follows: adding ethanol 5-12 times the mass of the Ganoderma lucidum raw material, the ethanol volume concentration is ≥80%, and ultrasonication is performed for 20-30 minutes.

[0006] Preferably, the Ganoderma lucidum raw material is coarsely crushed Ganoderma lucidum fruiting bodies.

[0007] Preferably, the ultrasonic extraction is repeated 2-3 times.

[0008] Preferably, the extract is concentrated to 4-10 times the mass of the Ganoderma lucidum raw material.

[0009] As a preferred macroporous resin, D101 has a pore size of Specific surface area is 700-755m 2 / g.

[0010] Preferably, the steps of macroporous resin purification are: loading the column with ethanol having a volume concentration of 20-30vt% and loading the sample, the raw material feeding mass: macroporous resin column volume = 1g:1-2ml, eluting with 40-70vt% ethanol having a volume concentration of 3-6 times the volume of the macroporous resin column, and then eluting with 95vt% ethanol having a volume concentration of 5-8 times the volume of the macroporous resin column, receiving the 95vt% ethanol eluate, concentrating and drying to obtain a crude fraction rich in ganoderma lucidum ketone triol.

[0011] Preferably, the crude fraction is dissolved in DMSO before the first separation, and the first separation product is dissolved in DMSO before the second separation.

[0012] Preferably, the first separation step is: dissolving the crude fraction with DMSO and injecting it into a C18 column for liquid separation, collecting the preparation solution rich in ganoderma lucidum ketone triol, concentrating and drying it for the second separation.

[0013] Preferably, the concentration of the crude fraction after dissolution is 0.05-0.1 g / mL, and after ultrasonic dissolution, the supernatant is centrifuged and filtered to obtain the filtrate, which is injected into a C18 column for liquid separation.

[0014] Preferably, the second separation step is: dissolving the product of the first separation with DMSO and injecting it into a C18 column, collecting each single peak preparation liquid, and after high performance liquid phase analysis, finding the preparation liquid rich in ganoderma lucidum ketone triol, and concentrating and freeze-drying it to obtain the target isolate.

[0015] Preferably, the volume concentration of the trifluoroacetic acid aqueous solution is 0.05-0.2%, more preferably 0.1%.

[0016] The present invention also provides application of the ganoderma lucidum ketone triol obtained by the preparation method in alleviating cardiovascular hypoxia-reoxygenation damage.

[0017] Therefore, the beneficial effects of the present invention are as follows: (1) Ultrasonic extraction is superior to traditional extraction processes such as leaching and heating reflux in terms of content and yield, and has a short time, low energy consumption and a high safety factor.

[0018] (2) After purification, a large amount of impurities are removed, reducing the contamination of the C18 column; the effective ingredients are enriched, improving the monomer preparation rate and monomer purity.

[0019] (3) Two concentrations of ethanol were used for elution and purification to separate the medium polarity and low polarity triterpene acid components, thereby reducing the amount of organic phase used in the subsequent separation process and saving preparation time. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a QTOF qualitative analysis mass spectrum, in which 1 represents the peak of ganoderma lucidum ketone triol.

[0021] Figure 2 It is the high performance liquid chromatogram of the product of Example 1.

[0022] Figure 3 It is the high performance liquid chromatogram of the product of Example 4.

[0023] Figure 4 It is the high performance liquid chromatogram of the product of Comparative Example 6.

[0024] Figure 5 It is the high performance liquid chromatogram of the product of Comparative Example 7. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below through specific embodiments.

[0026] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the embodiments, unless otherwise specified, are all conventional methods in the art. Unless otherwise specified, parts are all weight parts, temperatures are expressed in ° C or at ambient temperature, and pressures are atmospheric pressure or near atmospheric pressure. There are many variations and combinations of reaction conditions (such as component concentrations, desired solvents, solvent mixtures, temperatures, pressures, and other reaction ranges) and conditions that can be used to optimize the purity and yield of the product obtained by the method, and only reasonable routine experiments will be required to optimize such method conditions.

[0027] Example The preparation method of ganoderma lucidum ketone triol comprises the following steps: Step 1: Ultrasonic extraction of Ganoderma lucidum raw materials to obtain extracts Take the roughly crushed Ganoderma lucidum raw material, add ethanol 5-12 times the mass of the Ganoderma lucidum raw material, the ethanol volume concentration is ≥80%, ultrasonic for 20-30 minutes, repeat the extraction 2-3 times, combine the ethanol extracts, and concentrate to 4-10 times the amount of the Ganoderma lucidum raw material.

[0028] Step 2: The extract is purified using a macroporous resin to obtain a crude fraction. Select macroporous resin D101 with a pore size of Specific surface area is 700-755m 2 / g. The column is loaded with ethanol having a volume concentration of 20-30vt% and the sample is loaded, the raw material input mass: the volume of the macroporous resin column = 1g: 1-2ml, after eluting with ethanol having a volume concentration of 40-70vt% which is 3-6 times the volume of the macroporous resin column, and then eluting with ethanol having a volume concentration of 95vt% which is 5-8 times the volume of the macroporous resin column, the 95vt% ethanol eluate is collected, concentrated and dried to obtain a crude fraction rich in ganoderma lucidum ketone triol.

[0029] Step 3: The crude fraction is subjected to two liquid phase separations to obtain high-purity ganoderma lucidum ketone triol S1. First liquid phase separation. Add dimethyl sulfoxide (DMSO) to the crude fraction, and the amount of DMSO is such that the concentration of the crude fraction after dissolution is 0.05-0.1 g / mL; after ultrasonic dissolution for 5-20 minutes, centrifuge and take the supernatant, filter the supernatant, take the filtrate, inject the filtrate into a 40-60 μm C18 preparative column, use 0.05-0.1 vt% trifluoroacetic acid as mobile phase A, acetonitrile as mobile phase B, adjust the flow rate to 50-100 mL / min, and collect the preparative liquid according to the preparative liquid phase spectrum. The preparative liquid is analyzed by high performance liquid chromatography, and the preparative liquid rich in ganoderma lucidum ketone triol is selected, and concentrated and dried for secondary separation.

[0030] S2. Second liquid phase separation. Use a 5-10 μm C18 preparative column, with 0.05-0.1vt% trifluoroacetic acid as mobile phase A, acetonitrile as mobile phase B, and adjust the flow rate to 20-40 mL / min. The product of the first liquid phase separation is ultrasonically dissolved in DMSO, injected into the preparative liquid phase, and each single peak preparative liquid is collected. After high performance liquid phase analysis, the preparative liquid rich in ganoderma lucidum ketone triol is found, and the target isolate ganoderma lucidum ketone triol is obtained after concentration and freeze drying.

[0031] UHPLC-QTOF was used to perform qualitative analysis and purity measurement on the target isolate, Ganoderma lucidum ketone triol, and the purity of Ganoderma lucidum ketone triol reached more than 90%.

[0032] Example 1 A method for preparing ganoderma lucidum ketone triol, comprising the following steps: Step 1: Ultrasonic extraction of Ganoderma lucidum raw materials to obtain extracts Take the roughly crushed Ganoderma lucidum fruiting body, add ethanol 12 times the mass of Ganoderma lucidum fruiting body, the ethanol volume concentration is 80vt%, ultrasonically perform 30min at 100W power, repeat ultrasonic extraction twice. Combine the ethanol extracts and concentrate to 10 times the amount of Ganoderma lucidum fruiting body feed to obtain the extract.

[0033] Step 2: The extract is purified using a macroporous resin to obtain a crude fraction. Select macroporous resin D101 (Cangzhou Maoxinxin Material Technology Co., Ltd., pore size Specific surface area is 700-755m 2 / g), use 25vt% ethanol to pack the column and load the sample. Use 4 times the volume of the extract and 50vt% ethanol to elute for the first elution, and use 6 times the volume of the extract and 95vt% ethanol to elute for the second elution. Collect the 95vt% ethanol eluate, concentrate and dry to obtain a crude fraction rich in ganoderma lucidum ketone triol.

[0034] Step 3: The crude fraction is subjected to two liquid phase separations to obtain high-purity ganoderma lucidum ketone triol S1. First liquid phase separation. Add 60 mL of dimethyl sulfoxide (DMSO) to 5 g of the crude fraction, dissolve it by ultrasonication for 10 min, centrifuge for 30 min, take the supernatant, filter the supernatant, take the filtrate, inject the filtrate into a 50 μm C18 preparative column, use 0.1 vt% trifluoroacetic acid as mobile phase A, acetonitrile as mobile phase B, adjust the flow rate to 80 mL / min, and collect the preparative liquid according to the preparative liquid phase spectrum. The preparative liquid is analyzed by high performance liquid chromatography, and the preparative liquid rich in ganoderma lucidum ketone triol is selected, which is concentrated and dried for secondary separation.

[0035] S2. Second liquid phase separation. Use a 5μm C18 preparative column, 0.1vt% trifluoroacetic acid as mobile phase A, acetonitrile as mobile phase B, and adjust the flow rate to 35mL / min. The product of the first liquid phase separation is ultrasonically dissolved in DMSO, injected into the preparative liquid phase, and each single peak preparative liquid is collected. After high-performance liquid phase analysis, the preparative liquid rich in ganoderma lucidum ketone triol is found, and the target isolate ganoderma lucidum ketone triol is obtained after concentration and freeze-drying.

[0036] UHPLC-QTOF was used to qualitatively analyze the target isolate Ganoderma lucidum triol obtained by S2. The mass spectrum of the QTOF qualitative analysis is shown in the figure below: Figure 1 The results are shown in Table 1.

[0037] Table 1. QTOF qualitative analysis Further cell experiments were carried out on the target isolate Ganoderma lucidum ketone triol obtained from S2.

[0038] The experimental method is as follows: S1. Take out the cells in the logarithmic growth phase from the CO2 incubator and remove the original culture medium. Add 2mL PBS to rinse the cells and discard the PBS. Add 2mL of trypsin to digest for 1min, add 6mL of complete culture medium to terminate the digestion, and use a pipette to blow the attached cells into a suspension. Mix 10μL of cell suspension + 10μL PBS in a new EP tube, pipette 10μL into the cell counting plate, insert the counting plate into the cell counter, click to capture the count, and record the number of live cells. Add culture medium according to the counting results and adjust the cell density to 7.5×10 4cells / mL, the cells were inoculated into 6-well plates, 2 mL per well, and cultured in a 37°C cell culture incubator with saturated humidity and 5% CO2.

[0039] S2. When the cultured cells grow to about 40% confluence, 2 mL of DMEM culture medium is added to the control group and the model group, and 2 mL of sample solution is added to the sample group, and the sample solution contains 25 μg / mL of ganoderma lucidum ketone triol. The cells are placed in a CO2 incubator and incubated for 6 hours. The cells are removed from the CO2 incubator. Except for the control group, the remaining groups are placed in an oxygen-deficient device, and nitrogen is passed into the device for 5 minutes to remove oxygen from the device. The device is sealed and placed in a CO2 incubator with the control group for 16 hours. After 16 hours, the cells in the oxygen-deficient device are removed and immediately placed in a CO2 incubator for reoxygenation for 4 hours.

[0040] S3. After 4 hours of cell treatment, the 6-well plate was taken out and rinsed twice with PBS. RNA of each group of cells was extracted. The RNA concentration and quality were determined by NanoDrop One ultra-micro UV-visible spectrophotometer. RNA was reverse transcribed into cDNA according to the operation method of RNA reverse transcription kit. The cDNA was amplified by SYBR Green I fluorescent dye method, and the relative expression of Bax and Bcl-2 mRNA was calculated according to the Ct value. The results are shown in Table 2.

[0041] Table 2. Bcl-2 / Bax values ​​of three parallel experiments Table 2 Data of three parallel experiments. The results show that after the H9C2 cells in the model group were treated with hypoxia and reoxygenation, the expression of Bcl-2 / Bax mRNA was significantly reduced, and ganoderma lucidum ketone triol can effectively increase the expression level of Bcl-2 / Bax mRNA. The activation rate calculation formula in the table is: activation rate (%) = (relative expression of sample group - relative expression of model group) ÷ relative expression of model group × 100%. The average activation rate of ganoderma lucidum ketone triol extracted by the present invention is 49.77%.

[0042] Example 2 The difference from Example 1 is that the volume concentration of ethanol in the second elution in step 2 is 80vt%. The details are as follows: A method for preparing ganoderma lucidum ketone triol, comprising the following steps: Step 1: Ultrasonic extraction of Ganoderma lucidum raw materials to obtain extracts Take the roughly crushed Ganoderma lucidum fruiting body, add ethanol 12 times the mass of Ganoderma lucidum fruiting body, the ethanol volume concentration is 80vt%, ultrasonically perform 30min at 100W power, repeat ultrasonic extraction twice. Combine the ethanol extracts and concentrate to 10 times the amount of Ganoderma lucidum fruiting body feed to obtain the extract.

[0043] Step 2: The extract is purified using a macroporous resin to obtain a crude fraction. Select macroporous resin D101, use ethanol with a volume concentration of 25vt% to pack the column and load the sample. Use ethanol with a volume concentration of 50vt% of 4 times the volume of the extract for the first elution, and use ethanol with a volume concentration of 80vt% of 6 times the volume of the extract for the second elution. Collect the 80vt% ethanol eluate, concentrate and dry to obtain a crude fraction rich in ganoderma lucidum ketone triol.

[0044] Step 3: The crude fraction is subjected to two liquid phase separations to obtain high-purity ganoderma lucidum ketone triol S1. First liquid phase separation. Add 60 mL of dimethyl sulfoxide (DMSO) to 5 g of the crude fraction, dissolve it by ultrasonication for 10 min, centrifuge for 30 min, take the supernatant, filter the supernatant, take the filtrate, inject the filtrate into a 50 μm C18 preparative column, use 0.1 vt% trifluoroacetic acid as mobile phase A, acetonitrile as mobile phase B, adjust the flow rate to 80 mL / min, and collect the preparative liquid according to the preparative liquid phase spectrum. The preparative liquid is analyzed by high performance liquid chromatography, and the preparative liquid rich in ganoderma lucidum ketone triol is selected, which is concentrated and dried for secondary separation.

[0045] S2. Second liquid phase separation. Use a 5μm C18 preparative column, 0.1vt% trifluoroacetic acid as mobile phase A, acetonitrile as mobile phase B, and adjust the flow rate to 35mL / min. The product of the first liquid phase separation is ultrasonically dissolved in DMSO, injected into the preparative liquid phase, and each single peak preparative liquid is collected. After high-performance liquid phase analysis, the preparative liquid rich in ganoderma lucidum ketone triol is found, and the target isolate ganoderma lucidum ketone triol is obtained after concentration and freeze-drying.

[0046] Example 3 The difference from Example 1 is that in step 2, macroporous resin D101 is selected from Bengbu Liaoyuan New Materials Co., Ltd., with a pore size of Specific surface area is 500-550m 2 / g.

[0047] Example 4 The difference from Example 1 is that the solvent used to dissolve the crude fraction and the first separated product in step 3 is methanol. A method for preparing ganoderma lucidum ketone triol, comprising the following steps: Step 1: Ultrasonic extraction of Ganoderma lucidum raw materials to obtain extracts Take the roughly crushed Ganoderma lucidum fruiting body, add ethanol 12 times the mass of Ganoderma lucidum fruiting body, the ethanol volume concentration is 80vt%, ultrasonically perform 30min at 100W power, repeat ultrasonic extraction twice. Combine the ethanol extracts and concentrate to 10 times the amount of Ganoderma lucidum fruiting body feed to obtain the extract.

[0048] Step 2: The extract is purified using a macroporous resin to obtain a crude fraction. Select macroporous resin D101, use ethanol with a volume concentration of 25vt% to pack the column and load the sample. Use ethanol with a volume concentration of 50vt% of 4 times the volume of the extract for the first elution, and use ethanol with a volume concentration of 95vt% of 6 times the volume of the extract for the second elution. Collect the 95vt% ethanol eluate, concentrate and dry to obtain a crude fraction rich in ganoderma lucidum ketone triol.

[0049] Step 3: The crude fraction is subjected to two liquid phase separations to obtain high-purity ganoderma lucidum ketone triol S1. First liquid phase separation. Add 60 mL of methanol to 5 g of crude fraction, dissolve by ultrasonic for 10 min, centrifuge for 30 min, take the supernatant, filter the supernatant, take the filtrate, inject the filtrate into a 50 μm C18 preparative column, use 0.1 vt% trifluoroacetic acid as mobile phase A, acetonitrile as mobile phase B, adjust the flow rate to 80 mL / min, and collect the preparative liquid according to the preparative liquid phase spectrum. The preparative liquid is analyzed by high performance liquid chromatography, and the preparative liquid rich in ganoderma lucidum ketone triol is selected, which is concentrated and dried for secondary separation.

[0050] S2. Second liquid phase separation. Use a 5μm C18 preparative column, 0.1vt% trifluoroacetic acid as mobile phase A, acetonitrile as mobile phase B, and adjust the flow rate to 35mL / min. The product of the first liquid phase separation is ultrasonically dissolved in methanol and injected into the preparative liquid phase. Each single peak preparative liquid is collected. After high performance liquid phase analysis, a preparative liquid rich in ganoderma lucidum ketone triol is found. After concentration and freeze drying, the target isolate ganoderma lucidum ketone triol is obtained.

[0051] Comparative Example 1 The difference from Example 1 is that step 1 adopts an extraction method, which is as follows: Take the roughly crushed Ganoderma lucidum fruiting body, add ethanol 12 times the mass of the Ganoderma lucidum fruiting body, the ethanol volume concentration is 80vt%, and extract twice, the first extraction time is 48h, and the second extraction time is 24h, filter, combine the filtrate, and concentrate to 10 times the amount of Ganoderma lucidum fruiting body feed.

[0052] Comparative Example 2 The difference from Example 1 is that step 1 adopts a heating reflux method, which is as follows: Take the roughly crushed Ganoderma lucidum fruiting body, add ethanol 12 times the mass of the Ganoderma lucidum fruiting body, the ethanol volume concentration is 80vt%, heat reflux extraction twice, the extraction time is 3h each time, filter, combine the filtrate, and concentrate to 10 times the amount of Ganoderma lucidum fruiting body feed.

[0053] Comparative Example 3 The difference from Example 1 is that the ultrasonic power in step 1 is 300 W, which exceeds the preferred range.

[0054] Comparative Example 4 The difference from Example 1 is that in step 2, macroporous resin NKA-II is used.

[0055] Comparative Example 5 The difference from Example 1 is that in step 2, the first elution is not performed, and the second elution is performed directly.

[0056] Comparative Example 6 The difference from Example 1 is that the mobile phase A in step 3 is an aqueous solution.

[0057] Comparative Example 7 The difference from Example 1 is that in step 3, the first liquid phase separation is not performed, and the second liquid phase separation is performed directly.

[0058] Results Analysis The results of the embodiments and comparative examples were analyzed and the results are as follows.

[0059] 1. Step 1: Effect of Ultrasonic Extraction Method on Ganoderma Lucidum Raw Materials Table 3. Effect of ultrasonic extraction method on Ganoderma lucidum raw materials Group Extraction method Content of ganoderma lucidum ketone triol in extract mg / ml Transfer rate % Extraction time Example 1 100W ultrasonic extraction 0.2186 91.0 1 Comparative Example 1 Extraction 0.1856 77.3 72 Comparative Example 2 Heating reflux 0.2079 86.6 6 Comparative Example 3 300W ultrasonic extraction 0.2093 91.4 1 The transfer rate in the table indicates the percentage of ganoderma lucidum ketone triol transferred from ganoderma lucidum to the extract after extraction, and the calculation formula is: transfer rate (%) = (volume of extract × content of extract) ÷ (raw material feed amount × raw material content) × 100%. As can be seen from Table 3, compared with traditional leaching or heating reflux method, the ultrasonic extraction method adopted by the present invention can improve the extraction rate under mild conditions and significantly shorten the extraction time. Comparative Example 3 increases the ultrasonic power, and the content of ganoderma lucidum ketone triol in the extract decreases instead, because too high ultrasonic power can cause the denaturation and degradation of ganoderma lucidum ketone triol. More importantly, the activation rate of Comparative Example 3 decreases to 34.47%. Therefore, the ultrasonic power needs to be controlled within a reasonable range.

[0060] 2. Effect of Step 2 Macroporous Resin Purification Table 4. Effect of macroporous resin purification Group Macroporous resin, elution process % of ganoderin in purified product Transfer rate % Example 1 D101, 50→95vt% ethanol 4.41 82.4 Example 2 D101, 50→80vt% ethanol 3.09 51.8 Example 3 Others D101, 50→95vt% ethanol 4.15 78.28 Comparative Example 4 NKA-II, 50→95vt% ethanol 0 0 Comparative Example 5 D101, 95vt% ethanol 0.62 80.5 The transfer rate in the table indicates the percentage of ganoderma lucidum ketone triol transferred from the ganoderma lucidum extract to the crude fraction after purification, and the calculation formula is: transfer rate (%) = (weight of purified product × content of purified product) ÷ (volume of extract × content of extract) × 100%. As can be seen from Table 4, the type of macroporous resin is very important for the purification of ganoderma lucidum ketone triol. Comparative Example 4 uses the macroporous resin NKA-II, which is unable to purify ganoderma lucidum ketone triol. Although Example 3 also uses D101, the performance of the same type of macroporous resin from different manufacturers is also different. The inventors found that controlling the pore size to The purification effect is better within the range. In addition, the elution procedure also affects the purification effect. Comparative Example 5 does not use gradient elution, and the amount of ganoderma lucidum ketone triol in the purified product decreases significantly. The ethanol concentration of the second elution of Example 2 is reduced from 95vt% in Example 1 to 80vt%, and the transfer rate decreases significantly. Therefore, the elution concentration is also very important.

[0061] 3. Impact of Liquid Phase Separation Figure 2-5 This is the HPLC chromatogram of the target isolate Ganoderma lucidum ketone triol. Figure 2 Corresponding to Example 1, the data are shown in Table 5, and ganoderma lucidum ketone triol with a purity of 100% was obtained.

[0062] Table 5. Figure 3 Corresponding to Example 4, the data are shown in Table 6. After the solvent was replaced by methanol from DMSO, the peak area of ​​the separated ganoderma lucidum ketone triol was only 11% of that in Example 1. The solubility of methanol was not as good as that of DMSO. Using methanol as a solvent, the ganoderma lucidum ketone triol in the sample could not be completely dissolved, so the yield was low and the purity was not high.

[0063] Table 6. Figure 4 Corresponding to Example 6, the data are shown in Table 7. The mobile phase A was replaced by a pure aqueous solution from a trifluoroacetic acid aqueous solution, the purity was only 85%, and the peak area was only 5.3% of that in Example 1. Under acidic conditions, ganoderma lucidum ketone triol was more easily eluted, and the separation effect was good.

[0064] Table 7. Figure 5 The data for the corresponding example 7 are shown in Table 8. Without the first liquid phase separation, the second liquid phase separation was directly performed, and the impurity peaks increased significantly, the peak response was poor, and the purity was low. Due to the complex components in the crude fraction, the small particle size C18 column was directly used, which caused the column to be blocked and the column efficiency to deteriorate, and it could not be effectively separated.

[0065] Table 8. Therefore, only under the preferred liquid phase conditions of the present invention can ganoderma lucidum ketone triol with a purity of 100% be obtained.

[0066] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing ganoderma lucidum ketone triol, characterized in that: The following steps are involved: (1) extracting the raw material of Ganoderma lucidum by ultrasonic extraction at an ultrasonic power of 50-100 W; purifying the extract by using a macroporous resin to obtain a crude fraction; (2) The crude fraction was subjected to a first separation and a second separation in sequence to obtain ganoderma lucidum ketone triol; the first separation was performed using a 40-60 μm C18 column with a mobile phase flow rate of 50-100 mL / min; the second separation was performed using a 5-10 μm C18 column with a mobile phase flow rate of 20-40 mL / min; the mobile phase A for both separations was trifluoroacetic acid aqueous solution, and the mobile phase B was acetonitrile.

2. The method for preparing ganoderma lucidum ketone triol according to claim 1, characterized in that: The conditions for ultrasonic extraction in step (1) are as follows: adding ethanol in an amount of 5-12 times the mass of the Ganoderma lucidum raw material, with the volume concentration of ethanol being ≥80%, and ultrasonicating for 20-30 min.

3. The method for preparing ganoderma lucidum ketone triol according to claim 1, characterized in that: The ultrasonic extraction was repeated 2-3 times.

4. The method for preparing ganoderma lucidum ketone triol according to claim 1, 2 or 3, characterized in that: The extract is concentrated to 4-10 times the mass of the Ganoderma lucidum raw material.

5. The method for preparing ganoderma lucidum ketone triol according to claim 1, characterized in that: The steps of macroporous resin purification are: pack the column with ethanol at a volume concentration of 20-30vt% and load the sample, elute with ethanol at a volume concentration of 40-70vt% which is 3-6 times the volume of the macroporous resin column, then elute with ethanol at a volume concentration of 95vt% which is 5-8 times the volume of the macroporous resin column, receive the 95vt% ethanol eluate, concentrate and dry to obtain a crude fraction rich in ganoderma lucidum ketone triol.

6. The method for preparing ganoderma lucidum ketone triol according to claim 1, characterized in that: The first separation step is: the crude fraction is dissolved in DMSO and then injected into a C18 column for liquid separation, and the preparation liquid rich in ganoderma lucidum ketone triol is collected, concentrated and dried for the second separation.

7. The method for preparing ganoderma lucidum ketone triol according to claim 6, characterized in that: The crude fraction was dissolved to a concentration of 0.05-0.1 g / mL. After ultrasonic dissolution, the supernatant was centrifuged and filtered to obtain the filtrate, which was injected into a C18 column for liquid separation.

8. The method for preparing ganoderma lucidum ketone triol according to claim 1, characterized in that: The steps of the second separation are: dissolving the product of the first separation with DMSO and injecting it into a C18 column, collecting each single peak preparation liquid, and after high performance liquid phase analysis, finding the preparation liquid rich in ganoderma lucidum ketone triol, and concentrating and freeze-drying it to obtain the target isolate.

9. The method for preparing ganoderma lucidum ketone triol according to claim 1 or 8, characterized in that: The volume concentration of the trifluoroacetic acid aqueous solution is 0.05-0.2%.

10. Use of ganoderma lucidum ketone triol prepared by the preparation method according to any one of claims 1 to 9 in alleviating cardiovascular hypoxia-reoxygenation damage.

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