TWI882319B - A method of purifying triterpenes - Google Patents

Abstract

A method of purifying triterpenes comprising providing a crude extract of Ganoderma lucidum, performing a preceding treatment to the crude extract of Ganoderma lucidumand obtaining an extract of Ganoderma lucidum, and operating a simulated moving bed chromatography to separate and purify compounds of 1st type triterpene from the extract and compounds of 2nd type of triterpene from the extract.

Description

Method for purifying triterpenoid compounds

The present invention relates to a purification method, in particular to a purification method for triterpenoid compounds.

The traditional Chinese medicinal material Ganoderma lucidum has high medicinal value and has become one of the main raw materials for health food. Studies have found that Ganoderma lucidum has many functions such as regulating the immune system and cardiovascular system. The main active ingredients in Ganoderma lucidum include triterpenoid compounds such as ganoderic acid and ganoderic alcohol. Currently, there are methods to separate and purify triterpenoid compounds from Ganoderma lucidum, but due to the complex composition of Ganoderma lucidum and other factors, the purity and yield need to be improved.

The present invention provides a method for purifying triterpenoid compounds, which can separate and purify high-purity triterpenoid compounds and is suitable for the scale-up production of high-purity triterpenoid compounds. The triterpenoid compounds separated and purified by the method of the present invention are suitable for biotransformation, thereby facilitating bioavailability.

The present invention also provides a method for glycosylation of triterpenoid compounds, which can produce triterpenoid compound derivatives with high water solubility and facilitate bioavailability.

The method for purifying triterpenoid compounds provided by the present invention comprises the steps of: providing a crude extract of Ganoderma lucidum, which contains a first triterpenoid compound and a second triterpenoid compound, wherein the polarity of the first triterpenoid compound is higher than the polarity of the second triterpenoid compound; and dissolving the crude extract of Ganoderma lucidum and performing a pretreatment to obtain a Ganoderma lucidum extract; the Ganoderma lucidum extract contains the first triterpenoid compound and the second triterpenoid compound; and using a simulated moving bed chromatography method to separate the first triterpenoid compound from the Ganoderma lucidum extract. The method comprises: (i) providing a first simulated moving bed, which is composed of a first mobile phase and a stationary phase, and includes a flushing end, a feed inlet, an extraction end, a raffinate end, and a first section, a second section and a third section arranged in sequence, wherein the first mobile phase flows from the flushing end through the first section, the second section and the third section in the same direction relative to the first simulated moving bed, and the stationary phase simulates moving in the opposite direction relative to the first mobile phase; (ii) injecting the ganoderma lucidum extract from the feed inlet into the space between the second section and the third section of the first simulated moving bed, and allowing the first triterpenoid compound to move with the stationary phase to the extraction end between the first section and the second section, and allowing the second triterpenoid compound to move with the first mobile phase to the extraction end of the third section, so as to separate and purify the first triterpenoid compound and the second triterpenoid compound.

The method for purifying triterpenoid compounds provided by the present invention comprises the steps of: purifying triterpenoid compounds according to the aforementioned method; and providing enzymes and glycosyl donors, mixing the enzymes, glycosyl donors and purified triterpenoid compounds, so that the enzymes can perform in vitro biotransformation. The purified triterpenoid compounds include first-class triterpenoid compounds and second-class triterpenoid compounds.

The present invention adopts a step of pre-treating the crude extract of Ganoderma lucidum before the simulated moving bed chromatography, thereby improving the separation and purification efficiency of the simulated moving bed chromatography, and increasing the yield and purity.

In order to make the above and other purposes, features and advantages of the present invention more clearly understood, embodiments are specifically cited below and described in detail with reference to the accompanying drawings.

The triterpenoid compounds herein are preferably triterpenoid compounds contained in Ganoderma lucidum, or Ganoderma triterpenoids. Ganoderma triterpenoids may exist in Ganoderma lucidum and its extracts. In a broad sense, Ganoderma lucidum extracts include any Ganoderma lucidum extracts such as extraction stage, separation stage, and purification stage. For example, Ganoderma lucidum crude extracts, purified Ganoderma lucidum extracts, etc. can all be regarded as Ganoderma lucidum extracts. Ganoderma lucidum extracts may be in liquid or solid form. The Ganoderma lucidum triterpenoids herein mainly include Ganoderic acid A, Ganoderic acid F, and Ganoderin B.

The present invention provides a method for purifying triterpenoid compounds. In an embodiment of the present invention, the triterpenoid compounds are further ganoderma triterpenoid compounds, also known as ganoderma triterpenes, and include a first type of triterpenoid compounds and a second type of triterpenoid compounds. As shown in FIG1 , the purification method of the present invention includes step S810: providing a ganoderma crude extract, which includes a first type of triterpenoid compounds and a second type of triterpenoid compounds, wherein the polarity of the first type of triterpenoid compounds is higher than the polarity of the second type of triterpenoid compounds; step S820: dissolving the ganoderma crude extract and performing a pretreatment to obtain a ganoderma extract; the ganoderma extract contains the first type of triterpenoid compounds and the second type of triterpenoid compounds; and step S830: using a simulated moving bed chromatography to separate the first type of triterpenoid compounds from the ganoderma extract; The triterpenoid compounds are separated from the second triterpenoid compounds, and the simulated moving bed chromatography method comprises: (i) providing a first simulated moving bed, which is composed of a first mobile phase and a stationary phase, and includes a flushing end, a feed inlet, an extraction end, a raffinate end, and a first section, a second section and a third section arranged in sequence, wherein the first mobile phase flows from the flushing end through the first section, the second section and the third section in the same direction relative to the first simulated moving bed, and the stationary phase simulates moving in the opposite direction relative to the first mobile phase; (ii) injecting the ganoderma lucidum extract from the feed inlet into the space between the second section and the third section of the first simulated moving bed, and allowing the first triterpenoid compound to move with the stationary phase to the extraction end between the first section and the second section, and allowing the second triterpenoid compound to move with the first mobile phase to the extraction end of the third section, so as to separate and purify the first triterpenoid compound and the second triterpenoid compound.

[Preparation of Ganoderma Lucidum Crude Extract]

Step S810 further includes step S811: extracting Ganoderma lucidum using a supercritical fluid to obtain a crude extract of Ganoderma lucidum. The example is shown in Example 1. <Step S811: Example 1> This example uses commercially available Ganoderma lucidum fruiting bodies, such as commercially available diced Ganoderma lucidum fruiting bodies (about 1~3 mm) or commercially available Ganoderma lucidum fruiting bodies diced for standby use. This example uses an extraction device with an extraction tank volume of 1 L (ID. 10.5 cm, L. 11.1 cm), and the extraction conditions are: extraction pressure 350 bar, extraction temperature 60°C. About 100 g of diced Ganoderma lucidum fruiting bodies are loaded each time, and extraction is performed at a carbon dioxide flow rate of 60 g/min. The cosolvent (10 wt% anhydrous ethanol (99.5%)) is pre-mixed with _CO2 at a flow rate of 8.45 mL/min before entering the extraction tank inlet and then heated. Samples are taken from the separation tank every half hour, and the extraction is carried out for a total of 2.5 to 3.5 hours. During the extraction, anhydrous ethanol is pumped in from the extraction tank outlet at a flow rate of 8.45 mL/min to avoid blockage of the extract. The collected extract can also be mixed, concentrated, and filtered to obtain a crude extract of Ganoderma lucidum. The extraction curves of the total extract and Ganoderma lucidum acid A, Ganoderma alcohol B, and Ganoderma lucidum acid F are shown in Figures 2A-2B. In addition, based on Example 1, the present invention implements an embodiment and tests whether the extraction conditions have the ability to be scaled up. The example is shown in Example 2. <Example 2: Scale-up production of crude Ganoderma lucidum extract> This example uses an extraction device with an extraction tank volume of 25 L. The collected extract contains ethanol as a cosolvent, and an extract is obtained after the ethanol evaporates. Figures 3A-3B are the extraction curves of the total extract and ganoderic acid A, ganoderic alcohol B, and ganoderic acid F in Example 2. As shown in Figures 3A-3B, after 240 minutes of extraction, 18.93 g/kg of Ganoderma lucidum extract can be obtained. This result is better than the test result of the 1 L extraction device. It is speculated that the possible reason is that the diameter of the 25 L extraction tank is 15 cm, which is similar to the 11 cm diameter of the 1 L extraction tank, but the lengths of the extraction tanks are 142 cm and 11.5 cm, respectively, and the L/D ratios are 9.5 and 1, respectively. When the extraction is performed in a 25 L extraction tank, the contact time between the same amount of supercritical fluid and the raw material is longer, so a larger amount of extract can be obtained. In addition, in the Ganoderma lucidum raw material used in this example, the Ganoderma lucidum triterpenoids are mainly ganoderic acid A, and the content of ganoderic acid F and ganoderic alcohol B is relatively small, so the total extraction amount of ganoderic acid A can reach 986 mg/kg-loaded, which is much higher than the extraction amount of ganoderic acid A in Example 1 of 814 mg/kg-loaded, while the final extraction amount of ganoderic alcohol B and ganoderic acid F has no significant difference. The results show that when the operating conditions of Example 1 are scaled up to an extraction tank 25 times larger, similar results can still be obtained.

[Analysis method]

In the embodiment of the present invention, an analysis method for Ganoderma triterpenes and glycosylated Ganoderma triterpenes is established using HPLC/UV equipment, which is used to perform quantitative analysis of Ganoderma triterpenes in Ganoderma crude extracts and Ganoderma extracts, and to confirm the effect of glycosylation of Ganoderma triterpenes, as shown in Examples 3 and 4, respectively. <Example 3: Establishment of Ganoderma triterpenoid analysis method> This example uses HPLC/UV (Pump: 2130, Hitachi; UV: L-2455, Hitachi) to analyze the content of various Ganoderma triterpenoid components in Ganoderma extracts. Agilent Eclipse XDB-C18 (250 mm × 4.6 mm, 5 μm) was used as the analysis column; the analysis wavelength was set to 252 nm; and the mobile phase of 1 mL/min was eluted with the gradient shown in Table 1. Table 1: Mobile phase gradient design Time(min) 0 25 50 60 85 100 101 110 Acetonitrile ratio 28 30 39 60 100 100 28 28 0.1% acetic acid water ratio 72 70 61 40 0 0 72 72 In the present embodiment, the triterpenoid compounds in the ganoderma extract include ganoderic acid A, ganoderic acid F and ganoderic alcohol B. In this example, a series of standard solutions with different concentrations are prepared to prepare the calibration curve, wherein the concentrations of ganoderic acid A are 140.9, 281.6, 563.2 and 1126.5 mg/L, the concentrations of ganoderic acid F are 153.7, 307.4, 614.8 and 1229.6 mg/L, and the concentrations of ganoderic alcohol B are 27.6, 55.1 and 110.3 mg/L. By setting the horizontal coordinate to the injection concentration (mg/L) and the vertical coordinate to the signal peak area of the HPLC spectrum, a standard curve can be prepared and the regression equations are A = 8216.2 × C (ganoderic acid A), A = 7400.5 × C (ganoderic acid F), and A = 18939 × C (ganoderic alcohol B). FIG4 is an HPLC/UV analysis spectrum of the ganoderma lucidum extract of an embodiment of the present invention, wherein the retention times of ganoderic acid A, ganoderic acid F, and ganoderic alcohol B are 36.5 minutes, 55.2 minutes, and 87.7 minutes, respectively. For example, using the above calibration curves to estimate the content of ganoderic acid A, ganoderic acid F, and ganoderic alcohol B in the Ganoderma lucidum extract, the concentrations can be calculated to be 115.8, 182.48, and 69.4 mg/L, respectively. <Example 4: Establishment of the analytical method for glycosylated Ganoderma lucidum triterpenes> This example uses an Agilent 1100 HPLC system (Santa Clara, CA, USA) and a gradient pump (Waters 600, Waters, Milford, MA, USA) to analyze the content of various glycosylated Ganoderma lucidum triterpenes. Sharpsil H-C18 (5 μm, 4.6 ^ID × 250 mm) was used as the analytical column; the analytical wavelength was set to 252 nm; and the mobile phase of 1 mL/min was eluted with the gradient shown in Table 2. Table 2: Mobile phase gradient design Time(min) 0 20 25 28 35 Methanol ratio 20 50 50 20 20 1% acetic acid water ratio 80 50 50 80 80 FIG5 is a graph of various glycosylated derivatives of ganoderic acid A according to an embodiment of the present invention, wherein the retention times of GAA, GAA-15-G, GAA-G2, GAA-G3 and GAA-G4 are 24.4 minutes, 21.3 minutes, 20.8 minutes, 20.1 minutes and 19.3 minutes, respectively.

[Pre-treatment of Ganoderma lucidum extract]

The polarity distribution of various components in the Ganoderma lucidum extract, such as the aforementioned Ganoderma lucidum crude extract, is relatively broad, and the solubility in different concentrations of ethanol is different. If the Ganoderma lucidum crude extract is directly subjected to simulated moving bed chromatography, precipitation may occur, resulting in column blockage, thereby reducing the separation efficiency of the first triterpenoid compound and the second triterpenoid compound. Step S820 can be used to separate the low-polarity impurities in the Ganoderma lucidum crude extract of step S810 in advance, so as to avoid precipitation within the concentration range of the mobile phase containing ethanol in the simulated moving bed chromatography, thereby preventing the column from being blocked.

As shown in FIG6A , step S820 further includes step S821: dissolving the crude extract of Ganoderma lucidum in an alcohol/water system, and step S822: centrifuging the alcohol/water system to separate a supernatant, wherein the supernatant contains the first triterpenoid compound and the second triterpenoid compound. Step S821 preferably further includes a step of treating the alcohol/water system in which the crude extract of Ganoderma lucidum is dissolved at a low temperature (hereinafter referred to as winterization method). The operations of step S821 and step S822 are respectively illustrated in Example 5 and Example 6. <Step S821: Example 5> 5.1: The crude extract of Ganoderma lucidum is first completely dissolved in anhydrous ethanol (purity 99.5%), and the volume and weight are recorded. 5.2: Add a preset amount of pure water to the completely dissolved Ganoderma lucidum crude extract to prepare an ethanol/water system (or ethanol-water solution) of a specific weight ratio, as shown in Table 3, and record the volume and weight. At this time, a precipitate will be found. <Step S822: Example 6> Separate the supernatant from the precipitate by centrifugation, extract the supernatant, and record the volume, weight, and dry weight of the supernatant. Among them, Example 5.2 further includes a winterization method: refrigerate the configured ethanol/water system of a specific weight ratio for several hours, for example, at 4°C for more than 12 hours, and Example 6 further includes: separate the supernatant from the precipitate by centrifugation at a low temperature, extract the supernatant, and record the volume, weight, and dry weight of the supernatant. In addition, the precipitate can be dissolved by adding an appropriate volume of anhydrous ethanol. Table 3: Ethanol/water addition amount planning for Example 5 Group Ethanol/water weight ratio (wt %) Sample weight (g) Anhydrous ethanol addition amount (g) Pure water addition amount (g) 1 80% 0.5 10 2.50 2 75% 0.5 10 3.33 3 60% 0.5 10 6.67 4 55% 0.5 10 8.12 5 50% 0.5 10 10.00 The present invention also uses different weight ratios of ethanol/water systems and analyzes the ratios of ganoderin B, ganoderic acid A and ganoderic acid F in the supernatant and the precipitate. The results are shown in Tables 4 and 5. Table 4: Changes in the ratio of ganoderic triterpenes in the supernatant and the precipitate in Examples 5 to 6 Group Ethanol/water ratio (wt%) Sample status Total dry weight (mg) Total amount of triterpenoids in Ganoderma lucidum (mg) Ganoderma lucidum triterpenoid ratio (%) Ganoderic acid A ratio (%) Ganoderic acid F ratio (%) Lingzhi alcohol B ratio (%) Recovery rate% Original solution 99.5% Supernatant 187.05 13.90 7.43% 4.48% 2.02% 0.93% - 1 80% Supernatant 163.45 13.74 8.40% 5.18% 2.21% 1.02% 87.38 Sediment re-dissolution 23.60 0.37 1.59% 0.99% 0.43% 0.17% 12.62 2 75% Supernatant 148.85 13.71 9.21% 5.68% 2.43% 1.10% 79.58 Sediment re-dissolution 38.20 0.54 1.40% 0.71% 0.32% 0.38% 20.42 3 60% Supernatant 103.55 12.64 12.21% 7.57% 3.30% 1.34% 55.36 Sediment re-dissolution 83.50 1.49 1.78% 0.98% 0.44% 0.36% 44.64 4 55% Supernatant 86.65 12.68 14.64% 9.74% 3.64% 1.26% 46.32 Sediment re-dissolution 100.40 2.14 2.14% 1.07% 0.46% 0.60% 53.68 5 50% Supernatant 76.95 12.59 16.36% 10.51% 4.68% 1.17% 41.14 Sediment re-dissolution 110.10 2.19 1.99% 0.87% 0.40% 0.72% 58.86 Table 5: Changes in the ratio of Ganoderma triterpenes in the supernatant and sediment after winterization Group Ethanol/water ratio (wt%) Sample status Total dry weight (mg) Total amount of triterpenoids in Ganoderma lucidum (mg) Ganoderma lucidum triterpenoid ratio (%) Ganoderic acid A ratio (%) Ganoderic acid F ratio (%) Lingzhi alcohol B ratio (%) Recovery rate% Original solution 99.5% Supernatant 187.05 13.90 7.43% 4.48% 2.02% 0.93% - 1 80% Supernatant 151.05 12.94 8.57% 5.21% 2.36% 1.00% 80.75 Sediment re-dissolution 36.00 0.76 2.11% 1.28% 0.56% 0.27% 19.25 2 75% Supernatant 139.35 12.66 9.08% 5.50% 2.40% 1.19% 74.50 Sediment re-dissolution 47.70 0.78 1.64% 0.82% 0.36% 0.46% 25.50 3 60% Supernatant 91.95 12.52 13.62% 8.38% 3.66% 1.57% 49.16 Sediment re-dissolution 95.10 2.78 2.92% 1.55% 0.66% 0.71% 50.84 4 55% Supernatant 71.75 12.55 17.49% 11.15% 4.76% 1.58% 38.36 Sediment re-dissolution 115.30 3.21 2.78% 1.44% 0.65% 0.68% 61.64 5 50% Supernatant 53.25 12.57 23.61% 15.27% 6.71% 1.64% 28.47 Sediment re-dissolution 133.80 3.05 2.28% 1.11% 0.50% 0.67% 71.53 Tables 4 and 5 respectively summarize the changes in the ratio of Ganoderma triterpenes in the supernatant and precipitate of different ratios of ethanol/water systems dissolved with Ganoderma lucidum crude extracts before and after winterization. The results show that more precipitates will be produced after refrigeration, which is due to the decrease in the solubility of Ganoderma lucidum extracts in low temperature environments. In a lower concentration ethanol/water environment, the content of Ganoderma triterpenes in the supernatant increased significantly after winterization, indicating that the solubility of low-polarity substances is lower than that of Ganoderma triterpenes. Compared with the ratio of Ganoderma triterpenes in the original solution, when the Ganoderma triterpenes are removed from the precipitate at 75% ethanol, the ratio of Ganoderma triterpenes can be increased to more than 9.08%, and when the precipitate is removed from the ethanol at a lower concentration (less than 60%), it can be increased to more than 13.62%. The supernatant can be used as the Ganoderma extract and used in step S830.

As shown in FIG6B, step S820 may further include step S823: providing an adsorbent to be added to the supernatant for adsorption, and step S824: filtering to separate the adsorbent and the filtrate, and step S825: desorbing the substance in the adsorbent and obtaining a desorbed liquid. The filtrate and the desorbed liquid contain the first triterpenoid compound, the second triterpenoid compound or a combination thereof. The operations of step S823, step S824 and step S825 are respectively illustrated in Example 7, Example 8 and Example 9. : <Step S823: Example 7> 7.1: Measure the ethanol/water system supernatant of the specific weight ratio of step S822, and add a specific weight of the adsorbent per milliliter of the supernatant, as shown in Table 6. 7.2: Stir evenly for 2 hours at room temperature to perform adsorption. <Step S824: Example 8> Use 0.45 μm filter paper for vacuum filtration and collect the filtrate. During the process, ethanol/water of the same weight concentration can be used for rinsing to avoid sample precipitation or re-dissolution of the adsorbed substance. Record the volume, weight and dry weight of the filtrate. <Step S825: Example 9> 9.1: Use anhydrous ethanol to desorb the substance in the adsorbent. After adding an appropriate volume of anhydrous ethanol, stir evenly for 2 hours to perform the desorption procedure. 9.2: Use 0.45 μm filter paper for vacuum filtration and collect the filtrate. Anhydrous ethanol can be used for rinsing during the process. Record the volume, weight and dry weight of the filtrate. Collect the adsorbent and repeat the desorption. The adsorbent in Example 9.1 can be, for example, SP70 or UBK555, and preferably SP70. The amount of SP70 can be, for example, 0.1-0.2 g/mL. Table 6: Ethanol/water ratio and adsorbent addition amount planning Group Adsorbent Name Sample ethanol/water ratio (wt %) Adsorbent addition amount (g/mL supernatant) 1 SP70 75% 0.1 2 75% 0.2 3 60% 0.1 4 60% 0.2 5 UBK555 75% 0.1 6 75% 0.2 7 60% 0.1 8 60% 0.2 The filtrate and the desorption liquid can be used as the Ganoderma lucidum extract and used in step S830. FIG7 shows the HPLC analysis charts of the stock solution (ethanol/water system in which the Ganoderma lucidum crude extract is dissolved, A), the supernatant after winterization in 60% ethanol/water system (B), the precipitate after winterization in 60% ethanol/water system (C), the filtrate after SP70 adsorption of the supernatant after winterization in 60% ethanol/water system (D), and the desorption liquid after SP70 adsorption of the supernatant after winterization in 60% ethanol/water system (E).

[Separation of triterpenoids]

In step S830, the substances in the supernatant and/or filtrate and desorption liquid of step S820, including the first triterpenoid compound and the second triterpenoid compound, are separated into extract and raffinate by using a suitable mobile phase, a stationary phase and a column system of a simulated moving bed. In the embodiment of the present invention, the first mobile phase of the first simulated moving bed (SMB) contains an ethanol aqueous solution, and the stationary phase thereof contains a surface-modified silica filler.

Step S830 may further include selecting the first mobile phase and stationary phase of the first simulated moving bed. The stationary phase of the embodiment of the present invention may be selected from prepared grade fillers such as Welch Ultisil AQ-C18 (40-70 μm, 100Å, referred to as Welch AQ), Welch Ultisil Polar RP (40-70 μm, 100Å, referred to as Polar) and YMC ODS-AQ-HG (12 nm S-50μm, referred to as YMC AQ). Welch AQ, Polar, and YMC AQ fillers may be filled in a packed column of, for example, 1 cm ^ID × 25 cm ^L , and installed in an HPLC device to screen the mobile phase. The first mobile phase of the embodiment of the present invention may be selected from ethanol aqueous solutions of different proportions prepared with anhydrous ethanol (99.5%). The screening may include performing single column chromatography tests with different fillers such as Welch AQ, Polar, and YMC AQ in combination with different proportions of ethanol-water solutions to obtain information on the retention behavior of ganoderin B, ganoderin acid A, and ganoderin acid F.

From the single column analysis test results, information such as whether the highly polar and less polar substances in the ganoderma lucidum crude extract can be effectively separated and the length of their retention time can be obtained. Among them, the one that can effectively separate the highly polar and less polar substances and has a short retention time is preferred, that is, it is more conducive to the separation operation of the simulated moving bed analysis. In a preferred embodiment of the present invention, the first simulated moving bed uses a first mobile phase of ethanol: water at a ratio of 90:10 and a stationary phase of polar filler. In different embodiments, a first mobile phase of ethanol: water at a ratio of 60:40 can also be used to effectively separate ganoderic acid A and ganoderic acid F.

FIG8 shows a single column chromatogram of the supernatant after the winterization method in the stationary phase of the Polar filler and the first mobile phase of ethanol: water of 90:10. As shown in FIG8, the retention time of ganoderic acid A and ganoderic acid F are similar, 6.10 minutes and 6.11 minutes respectively, while the retention time of ganoderic alcohol B is 10.67 minutes. The retention time can be used to design the operating conditions of the first simulated moving bed in step S830 (described in detail below).

Step S830 may also include a configuration design of the first simulated moving bed. As shown in FIG9 , the first simulated moving bed 100 includes a distillation end D, a feed port F, an extraction end E, a raffinate end R, and a first section 110A, a second section 110B, and a third section 110C arranged in sequence, wherein the first mobile phase flows from the distillation end D through the first section 110A, the second section 110B, and the third section 110C in the same direction relative to the first simulated moving bed, and the stationary phase simulates moving in the opposite direction relative to the first mobile phase.

In the embodiment of the present invention, the first simulated moving bed 100 may include eight packed columns C1-C8. The packed columns C1-C8 may be, for example, packed columns of 1 cm ^ID × 25 cm ^L , but are not limited thereto. The eight packed columns are connected in series, and the packed columns C1-C2 form a first section 110A, the packed columns C3-C5 form a second section 110B, and the packed columns C6-C8 form a third section 110C, wherein the flushing end D is located in the first section 110A and is used to allow the first mobile phase to flow through the first section 110A, the second section 110B, and the third section 110C in the direction X1. The feed port F is located between the second section 110B and the third section 110C, and is used to allow feed such as the supernatant and/or the filtrate and the desorption liquid of step S820 to be injected therein. The extraction end E is located between the first section 110A and the second section 110B, and the raffinate end R is located in the third section 110C. Through the operation of the first simulated moving bed 100, the lower polar substances in the feed, such as the first triterpenoid compounds, can move to the extraction end E along with the stationary phase, and the higher polar substances, such as the second triterpenoid compounds, can move to the raffinate end R along with the first mobile phase, thereby separating and purifying the first triterpenoid compounds and the second triterpenoid compounds.

In a preferred embodiment of the present invention, the filler used in the columns C1 to C8 is Welch Ultisil Polar RP (40-70 μm, 100Å), the first mobile phase is a 90/10 ethanol-water solution, and the feed is preferably the supernatant after the winterization in the 60% ethanol/water system in step S820. The operation of the first simulated moving bed 100 is shown in Example 10. <Step S830: Example 10> 10.1: Based on the triangle theory and the retention time of ganoderic acid A, ganoderic acid F and ganoderic alcohol B in FIG8 , as well as the operating range of the pump flow rate, a series of experiments with different switching times were conducted at a fixed flow rate; wherein the flow rates were set as flushing end flow rate (QD) = 6.5 mL/min, extraction end flow rate (QE) = 3.5 mL/min, feed inlet flow rate (QF) = 0.3 mL/min, raffinate end flow rate (QR) = 3.3 mL/min, and the valve switching time (tsw) was 4.0 minutes, 4.5 minutes, 5.0 minutes, 5.5 minutes and 6.5 minutes in five experiments. After the operation of the first simulated moving bed 100 reaches four cycles of steady-state operation, the raffinate product and the extraction product are collected at the raffinate end R and the extraction end E, and HPLC analysis is performed. Figure 10 shows the analysis spectrum of HPLC/UV. 10.2: Part of the samples collected at each switching time are further dried to calculate the weight percentage (wt) of ganoderic acid A, ganoderic acid F and ganoderic alcohol B in each product. The concentrations and weight percentages of ganoderic acid A, ganoderic acid F and ganoderic alcohol B obtained at the raffinate end R and the extraction end E are shown in Table 7. Table 7: Ganoderma triterpene concentration and weight percentage results of Example 10 Feed Concentration(g/L) Weight percentage wt (%) C _A C _F C _B wt _A w _F wt _B 731.87 233.15 113.15 5.71 1.82 0.88 t _sw (min) Residue Extraction product Residue Extraction product C _A C _F C _B C _A C _F C _B wt _A w _F Total wt _B 4.0 1.07 1.74 ND 15.75 5.10 4.25 - - - - 4.5 32.93 10.49 ND ND ND 4.21 7.35 2.34 9.69 2.24 5.0 33.33 10.79 ND ND ND 4.90 7.18 2.33 9.51 2.15 5.5 35.05 11.31 ND ND ND 4.99 6.00 1.94 7.94 3.37 6.5 38.45 12.40 ND ND ND 3.81 6.41 2.07 8.47 3.52 ND: below the detection limit, _CA : Ganoderic acid A concentration, _CF : Ganoderic acid F concentration, _CB : Ganoderic alcohol B concentration, _wtA : Ganoderic acid A weight percentage, _wtF : Ganoderic acid F weight percentage, _wtB : Ganoderic alcohol B weight percentage, “-”: close to 0 As shown in Figure 10, when the switching time is 4.0 minutes, most of the triterpenes of Ganoderma lucidum are collected at the extraction end E (4.0-E), while only a small amount of ganoderic acid A and ganoderic acid F are collected at the residual end R (4.0-R); when the switching time is increased to 4.5 minutes, most of the ganoderic acid A and ganoderic acid F are collected at the residual end R (4.5-R), while the extraction end E (4.5-E) only collects substances with a retention time greater than 70 minutes, including ganoderic alcohol B. As shown in Table 7, when the switching time is 4.5 minutes, the weight percentage of ganoderic acid A in the residual end R is 7.35%, ganoderic acid F is 2.34 wt%, and the total is 9.69 wt%, while the ganoderic alcohol B in the extraction end E is 2.24 wt%. It can also be observed from Figure 10 and Table 7 that the number of peaks at the extraction end E in the spectrum is small, but the weight percentage of Ganoderma lucidum alcohol B is only 2~4%, indicating that there may be a large amount of substances in the product of the extraction end E that cannot be measured by UV, resulting in a low weight percentage of Ganoderma lucidum triterpenes. Similarly, the residue end R may also have this situation. Therefore, if a second separation and purification is performed, the total content of Ganoderma lucidum triterpenes should be increased.

In a preferred embodiment of the present invention, as shown in FIG. 11A , step S830 may further include step S831: injecting the raffinate of step S830 from the feed port into the space between the second section and the third section of the first simulated moving bed, and allowing the first triterpenoid compound to move with the stationary phase to the extraction end between the first section and the second section, and allowing the second triterpenoid compound to move with the first mobile phase to the raffinate end of the third section, so as to further separate and purify the first triterpenoid compound and the second triterpenoid compound. In some embodiments of the present invention, the operation of step S831 may be performed as described above and may be performed on the basis of, for example, Example 10. In a preferred embodiment of the present invention, step S831 is further designed with a valve switching time (tsw) of 4 minutes and 2 seconds, and the feed used is the raffinate (4.5-R) with a switching time of 4.5 minutes in step S830. Step S831 can obtain, for example, 13.34 wt% of ganoderic acid A and 3.22 wt% of ganoderic acid F, and can also obtain ganoderic alcohol B with higher purity.

In a preferred embodiment of the present invention, as shown in FIG. 11B , step S830 further includes step S832: (iii) providing a second simulated moving bed, which is composed of a second mobile phase and a stationary phase, and includes a bicarbonate end, a feed inlet, an extraction end, a raffinate end, and a first section, a second section, and a third section arranged in sequence, wherein the second mobile phase flows from the bicarbonate end through the first section, the second section, and the third section in the same direction relative to the second simulated moving bed, and the stationary phase is relative to the second mobile phase. Simulate the movement in the reverse direction; (iv) inject the raffinate of step S830 or step S831 from the feed port of the second simulated moving bed into the space between the second section and the third section, and make the ganoderic acid A in the second triterpenoid compound move with the stationary phase to the extraction end between the first section and the second section, and make the ganoderic acid F in the second triterpenoid compound move with the second mobile phase to the raffinate end of the third section, so as to separate and purify ganoderic acid A and ganoderic acid F; wherein the second mobile phase is different from the first mobile phase. In this embodiment of the present invention, the second mobile phase of the second simulated moving bed is different from the first mobile phase and contains a supercritical carbon dioxide fluid. Since the second moving phase of the second simulated moving bed contains supercritical fluid, it is also called SF-SMB (Supercritical Fluid Simulated Moving Bed) in this article. The stationary phase of the second simulated moving bed contains surface-modified silica filler.

Preferably, step S832 can further effectively separate ganoderic acid A and ganoderic acid F, and separate other substances with properties similar to ganoderic acid triterpenes. Step S832 can include selecting a second mobile phase and a stationary phase of a second simulated moving bed, wherein a second mobile phase and a column system with lower polarity are preferably selected to effectively improve the purity of ganoderic acid triterpenes. In the embodiment of the present invention, a single column chromatography test is performed using Welch Ultisil 2-EP (40-70 μm, 100Å) filler as the stationary phase and supercritical carbon dioxide with a cosolvent as the second mobile phase to obtain information about the retention time of ganoderic acid A and ganoderic acid F.

FIG12 shows the results of single column chromatography of 4.5-R raffinate in a Welch Ultisil 2-EP (2-ethylpyridine) stationary phase and a second mobile phase of supercritical carbon dioxide with 27 wt% cosolvent (95% ethanol), wherein the operating parameters are 140 bar and 50°C. As shown in FIG12 , the retention time of ganoderic acid A is 8.38 minutes, the retention time of ganoderic acid F is 4.49 minutes, and the 4.5-R raffinate has peaks of different sizes between minutes, indicating that in addition to ganoderic acid A and ganoderic acid F, there are other components in the product that can be separated by the 2-EP packed column. The retention time can be used to design the operating conditions of the second simulated moving bed in step S832 (described below).

Step S832 may also include the configuration design of the second simulated moving bed. As shown in FIG13 , the second simulated moving bed 200 includes a distillation end D’, a feed port F’, an extraction end E’, a raffinate end R’, and a first section 220A, a second section 220B, and a third section 220C arranged in sequence, wherein the second mobile phase flows from the distillation end D’ through the first section 220A, the second section 220B, and the third section 220C in the same direction X1 relative to the second simulated moving bed, and the stationary phase simulates moving in the opposite direction X2 relative to the second mobile phase.

In the embodiment of the present invention, the second simulated moving bed 100 may include six packing columns C1'~C6'. The packing columns C1'~C6' may be, for example, 1 cm ^ID × 15 cm ^L packing columns, but are not limited thereto. The six packing columns are connected in series, and the packing columns C1'~C2' form the first section 220A, the packing columns C3'~C4' form the second section 220B, and the packing columns C5'~C6' form the third section 220C, wherein the flushing end D' is located in the first section 220A and is used to make the second moving phase flow through the first section 220A, the second section 220B and the third section 220C in the direction X1. The feed port F' is located between the second section 220B and the third section 220C, and is used to inject feed such as the raffinate of steps S830 and S831. The extraction end E' is located between the first section 220A and the second section 220B, and the raffinate end R' is located in the third section 220C. Through the operation of the second simulated moving bed 200, the ganoderic acid A and substances with similar properties in the feed can be moved to the extraction end E' along with the stationary phase, while the ganoderic acid F and substances with similar properties can be moved to the raffinate end R along with the second mobile phase for separation.

In a preferred embodiment of the present invention, the filler used in the columns C1'~C8' is Welch Ultisil 2-EP (40-70 μm, 100Å), and the second mobile phase is supercritical carbon dioxide with 27% cosolvent (95% ethanol), and the feed is preferably the raffinate of step S831, or the raffinate of step S830, such as 4.5-R raffinate. The operation of the second simulated moving bed 200 is shown in Example 11. <Step S832: Example 11> Through the triangle theory and the retention time of ganoderic acid A, ganoderic acid F and ganoderic alcohol B in Figure 12, as well as the operating range of the flowmeter and pump flow, three different switching time tests were conducted at a fixed flow rate. The flow rate settings are shown in Table 8. The valve switching time (tsw) has three tests of 11.0 minutes, 10.0 minutes, and 9.0 minutes. After the second simulated moving bed 200 has reached five cycles of steady-state operation, the raffinate and the extract product are collected at the raffinate end R' and the extraction end E', and HPLC analysis is performed. Figures 14A-14B show the HPLC analysis spectrum. Table 8: Flow rate settings for Example 11 Supercritical CO ₂ flow Punch end (D) (g/min) Extraction end (E) (g/min) Feed (feed port F) (g/min) Residue end (R) (g/min) 5.0 2.42 0.313 2.894 Cosolvent (ethanol) flow rate Flushing end (D) (mL/min) Extraction end (E) (mL/min) Feed (feed port F) (mL/min) Residue end (R) (mL/min) 2.300 1.112 0.144 1.331 Supercritical fluid flow Flushing end (D) (mL/min) Extraction end (E) (mL/min) Feed (feed port F) (mL/min) Residue end (R) (mL/min) 7.955 3.848 0.497 4.604 As shown in FIG. 14A , when the switching time is 9.0 minutes, the material with a retention time of 0 to 60 minutes can be collected at the extraction end E' (9-E), and the material with a retention time of 45 to 110 minutes can be collected at the raffinate end R' (9-R). FIG. 14B shows that when the switching time is increased to 10.0 minutes, the material with a retention time of 0 to 36 minutes can be collected at the extraction end E', and the material with a retention time of 37 to 110 minutes can be collected at the raffinate end R'. Because the retention time of ganoderic acid A is 35.5 minutes, the maximum proportion of ganoderic acid A can be obtained, while ganoderic acid F appears at the raffinate end R'. The weight percentage of ganoderic acid A is calculated to be 27.99%.

In a preferred embodiment of the present invention, after step S830, steps S831 and S832 may be performed in sequence. Steps S830 to S832 may also be used to scale up the production of Ganoderma triterpenes, as shown in Example 12. <Example 12: Scale-up production of Ganoderma triterpenes using the first simulated moving bed 100 and the second simulated moving bed 200> 12.1: First separation and purification ( ^1st SMB) using the first simulated moving bed 100 12.1 can be performed on the basis of Example 10, with the difference that the filling columns C1~C8 are preferably, for example, 3 cm ^ID × 25 cm ^L , and the flow rates are set as flushing end flow rate (QD) = 97.5 mL/min, extraction end flow rate (QE) = 52.5 mL/min, feed inlet flow rate (QF) = 4.5 mL/min, and raffinate end flow rate (QR) = 49.5 mL/min. 12.2: Perform the second separation and purification ( ^2nd SMB) with the first simulated moving bed 100. The operating conditions can refer to 12.1. 12.3: Perform the separation and purification with the second simulated moving bed 200 12.3 can be performed on the basis of Example 11, and the difference mainly lies in the flow setting, as shown in Table 9. Table 9: Flow setting of Example 12.3 Supercritical CO ₂ flow Punch end (D) (g/min) Extraction end (E) (g/min) Feed (feed port F) (g/min) Residue end (R) (g/min) 75.0 36.3 4.96 43.66 Cosolvent (ethanol) flow rate Flushing end (D) (mL/min) Extraction end (E) (mL/min) Feed (feed port F) (mL/min) Residue end (R) (mL/min) 34.5 16.68 2.16 19.98 Supercritical fluid flow Flushing end (D) (mL/min) Extraction end (E) (mL/min) Feed (feed port F) (mL/min) Residue end (R) (mL/min) 119.32 57.72 7.46 69.06 The results of separation and purification by two first simulated moving beds 100 and one second simulated moving bed 200 are summarized in Tables 10 and 11. The results show that after the first separation by the first simulated moving bed 100, the concentrations of ganoderic acid A and ganoderic acid F increased from 5.82% and 1.85% to 7.35% and 2.34%, respectively. After the second separation by the first simulated moving bed 100, the concentrations of ganoderic acid A and ganoderic acid F increased to 13.47% and 3.18%, respectively. Finally, after the separation by the second simulated moving bed 200, a product containing 28.08% ganoderic acid A was obtained. Table 10: Results of concentration and weight percentage of ganoderic acid triterpenes in Examples 12.1-12.2 Feed Concentration (g/L) wt (%) C _A C _F C _B wt _A w _F wt _B 731.87 233.15 113.15 5.82 1.85 0.91 t _sw (min) Residue Extraction product Residue Extraction product C _A C _F C _B C _A C _F C _B wt _A w _F Total wt _B 1st SMB 4.6 32.93 10.49 ND ND ND 4.21 7.35 2.34 9.69 4.5 ^2nd SMB 4.1 26.99 6.51 ND ND ND ND 13.47 3.18 16.65 ND Table 11: Ganoderma lucidum triterpenoid concentration and weight percentage results of Example 12 Feed Concentration (g/L) wt (%) C _A C _F C _B wt _A w _F wt _B 731.87 233.15 113.15 5.82 1.85 0.91 t _sw (min) Residue Extraction product Residue Extraction product C _A C _F C _B C _A C _F C _B wt _A w _F wt _A SF-SMB 10 ND 5.41 ND 22.42 ND ND ND 5.05 28.08

[Glycosylation of triterpenoids]

The present invention also provides a method for glycosylation of triterpenoid compounds. Glycosylated triterpenoid compounds can have higher water solubility, which is conducive to absorption and utilization by organisms. As shown in Figure 15, the method for glycosylation of triterpenoid compounds includes step S910: purifying triterpenoid compounds according to the method including steps S810~830, and step S920: providing enzymes and glycosyl donors, mixing enzymes, glycosyl donors and purified triterpenoid compounds, so that the enzymes can perform in vitro biotransformation. The purified triterpenoid compounds include first-class triterpenoid compounds and second-class triterpenoid compounds.

In the embodiment of the present invention, step S920 further includes step S921: providing a recombinant Escherichia coli having a recombinant gene, and the recombinant gene can produce an enzyme. The recombinant gene can include a glycosyltransferase family 1 (GT1) gene such as uridine diphosphate-dependent glycosyltransferase (UGT) genes BsUGT398 and BsUGT489 , and an amylosucrase gene such as amylosucrase DgAS of Deinococcus geothermalis . The recombinant gene can be further located on a vector, and the recombinant Escherichia coli carries the vector having the recombinant gene and can express a glycosyltransferase or DgAS. In some embodiments of the present invention, the provided genetic recombinant Escherichia coli carries a pETDuet-BsUGT489 plasmid and can be induced to express uridine diphosphate-dependent glycosyltransferase (hereinafter referred to as BsUGT489). In other embodiments, step S921 can provide a genetic recombinant Escherichia coli carrying a pETDuet-DgAS plasmid and can be induced to express DgAS. For the cultivation and induction of recombinant E. coli and the purification of the protein products BsUGT489 or DgAS, please refer to the references of TS Chang et al. [1][2]: [1] Uridine diphosphate-dependent glycosyltransferases from Bacillus subtilis ATCC 6633 catalyze the 15- O -glycosylation of ganoderic acid A, International Journal of Molecular Sciences 19(11) (2018) 3469; [2] Potential industrial production of a well-soluble, alkaline-stable, and anti-inflammatory isoflavone glucoside from 8-hydroxydaidzein glucosylated by recombinant amylosucrase of Deinococcus geothermalis , Molecules 24(12) (2019) 2236. In some embodiments of the present invention, the enzyme in step S920 may also be a cyclodextrin glucosyltransferase. In addition, the enzyme in step S920 may also be a commercially available enzyme such as ^Toruzyme® . The final purified protein product may be prepared into a 50% glycerol storage solution and stored at -80°C for future use.

The operation of mixing the enzyme, the glycosyl donor and the purified triterpenoid compound in step S920 to allow the enzyme to perform in vitro bioconversion is exemplified in Example 13. <Example 13: In vitro bioconversion> 13.1: Preparation of purified triterpenoid compound The residues and extracts of steps S830, S831 and S832 can be used for in vitro bioconversion. In this example, the raffinate (4.5-R, 5.0-R and 6.5-R raffinate) and extract (4.5-E, 5.0-E and 6.5-E extract) of step S830 such as example 10, the raffinate (AFII405-R) and extract (402E) of steps S830~S831, and multiple raffinate products (5CR) are mixed to perform in vitro biotransformation. Unpurified triterpenoid compounds such as the crude extract of Ganoderma lucidum in step S810 are used as a control group and in vitro biotransformation is performed. The raffinate, extract and unpurified triterpenoid compounds are concentrated and freeze-dried, and then resuspended in dimethyl sulfoxide (DMSO) for standby use. 13.2: This example uses purified BsUGT489 for in vitro bioconversion. The in vitro bioconversion is performed in 0.1 mL of a reaction mixture containing 0.2 µg of purified BsUGT489, 5% (v/v) of the sample of Example 13.1, 2 mM uridine diphosphate-glucose (UDP-G), 10 mM MgCl ₂ , and 50 mM Tris (pH 8.0). The reaction is carried out at a temperature above room temperature for several hours, for example, at 40°C for 24 hours. After the reaction is completed, an equal volume of methanol is added to terminate the reaction and the mixture is analyzed by HPLC as in Example 14. <Example 14: HPLC analysis> This example was performed using an Agilent 1100 series HPLC system (Santa Clara, CA, USA) equipped with a gradient pump (Waters 600, Waters, Milford, MA, USA). The stationary phase was a C18 column (5 μm, 4.6 ^ID × 250 mm ^L ; Sharpsil H-C18), and the mobile phase was 1% acetic acid in water (A) and methanol (B). The washing conditions were a linear gradient from 20% (B) to 50% (B) from 0 to 20 minutes, an isocratic washing containing 50% (B) from 20 to 25 minutes, a linear gradient from 50% (B) to 20% (B) from 25 to 28 minutes, and an isocratic washing of 20% (B) from 28 to 35 minutes. All washing solutions were washed at a flow rate of 1 mL/min. The injection volume was 10 μL. The detection wavelength was 254 nm. The HPLC/UV analysis chromatograms of the sample of Example 13.1 before and after glycosylation are shown in Figures 16A to 25B. The efficiency of in vitro bioconversion was calculated based on the HPLC area according to the present invention, as shown in Table 12. In this example, the obtained in vitro bioconversion rate can reach 59.9%. Table 12: Glycosylation of triterpenoids by BsUGT489 Sample In vitro bioconversion rate Ganoderma Lucidum Extract ⎼ ¹ Extract 4.5-R +++ Extraction product 4.5-E ⎼ Extract 402-E + Extract 5.0-R + Extraction product 5.0-E ⎼ Extract 5CR ++ Raffinate 6.5-R + Extraction product 6.5-E ⎼ Extraction product AFII 405-R ++ “⎼” indicates no significant product; “+” indicates the conversion is significantly lower than 50%; “+++” indicates the conversion is significantly higher than 50%; “++” indicates the conversion is higher than 50%. Conversion was determined by HPLC peak area.

[Purification of Ganoderma Lucidum Glycoside]

In the embodiment of the present invention, the glycosylated triterpenoid compounds include ganoderin B, ganoderin acid A and ganoderin acid F. The embodiment of the present invention further purifies ganoderin glycosides and tests the water solubility of the purified ganoderin glycosides, as shown in Examples 15 and 16, respectively. <Example 15: Purification of ganoderin glycosides> 15.1: Amplification of bioconversion reaction First, a bioconversion reaction is amplified based on Example 13. A reaction mixture containing purified BsUGT489, the sample of Example 13.1, uridine diphosphate-glucose (UDP-G), etc. is amplified to 20 mL (1 mL per tube, 20 tubes in total), and shaken at 40°C at 180 rpm for 24 hours. After the reaction, an equal volume of methanol is added to terminate the bioconversion. 15.2: HPLC purification The reaction mixture of Example 15.1 was filtered through a 0.45 μm nylon membrane and the filtrate was injected into a preparative YongLin HPLC system equipped with a preparative C18 reverse phase column (10 μm, 20.0 ^ID × 250 ^L mm, ODS 3; Inertsil, GL Sciences, Eindhoven, The Netherlands). The operating conditions were the same as those for analytical HPLC. The eluate corresponding to the metabolite peak in the HPLC analysis was collected, concentrated and lyophilized. <Example 16: Water solubility test> Take 2 mg of the product purified by HPLC and dissolve it in 100 μl of double deionized water, shake it at 180 rpm for 1 hour at 25°C, and then centrifuge it at 10,000 g for 30 minutes at 25°C. The supernatant is filtered through a 0.2 μm nylon membrane and analyzed by HPLC. During the analysis, the supernatant is diluted 10 times with 50% methanol. The content is calculated by calibrating the calibration curve with a glucoside standard solution. The calibration curve is diluted with 50% methanol, and the prepared concentrations are 30, 90, 120, 150 or 210 mg/L. The measured solubility may vary depending on the sample in Example 13.1. Taking the raffinate 4.5-R of Example 10 as an example, the concentration may be, for example, 1812.5±41.8 mg/L.

In summary, the purification method of the embodiment of the present invention can separate and purify more than 2 wt% of ganoderic alcohol B, more than 5 wt% of ganoderic acid A, and more than 2 wt% of ganoderic acid F, wherein the content of ganoderic acid A can be further increased to more than 7 wt%. After two times of SMB separation and purification, the content of ganoderic acid A can be further increased to more than 10 wt%, such as 13.34 wt%, and the total content of ganoderic alcohol B, ganoderic acid A and ganoderic acid F can be more than 15 wt%, such as 16.56 wt%. Under the preparation-grade specification, the embodiment of the present invention can be scaled up to produce ganoderic acid A up to 28 wt%. The product produced by the purification method of the embodiment of the present invention exhibits a higher in vitro bioconversion rate than the crude extract; on the one hand, it reflects the higher amount of Ganoderma triterpenes in the product, as well as the high purity of Ganoderic acid A, Ganoderic acid F, and Ganoderic alcohol B; on the other hand, it indicates that the product produced by the embodiment of the present invention can be derived into water-soluble derivatives and has a higher bioavailability.

Although the present invention has been disclosed as above by way of embodiments, they are not intended to limit the present invention. A person having ordinary knowledge in the technical field to which the present invention belongs may make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined in the attached patent application.

100: First simulated moving bed 200: Second simulated moving bed 110A, 220A: First section 110B, 220B: Second section 110C, 220C: Third section C1~C8, C1’~C6’: Packing column D, D’: Flushing end F, F’: Feed inlet E, E’: Extraction end R, R’: Residue end

FIG1 is a schematic flow chart of a method for purifying triterpenoid compounds in an embodiment of the present invention. FIG2A and FIG2B are extraction curves of an embodiment of the present invention, respectively. FIG3A and FIG3B are extraction curves of another embodiment of the present invention, respectively. FIG4 is an HPLC/UV analysis chart of a Ganoderma lucidum extract in an embodiment of the present invention. FIG5 is an HPLC/UV analysis chart of a derivative of a Ganoderma lucidum extract in an embodiment of the present invention. FIG6A-6B are schematic diagrams of the pretreatment steps for purifying triterpenoid compounds in an embodiment of the present invention. FIG7 is an HPLC analysis chart of a Ganoderma lucidum extract in another embodiment of the present invention. FIG8 is a single column chromatography chart of a Ganoderma lucidum extract in an embodiment of the present invention. FIG. 9 is a schematic diagram of the configuration of the first simulated moving bed of an embodiment of the present invention. FIG. 10 is an HPLC/UV analysis spectrum of the first simulated moving bed product of an embodiment of the present invention. FIG. 11A is a flow diagram of a method for purifying triterpenoid compounds of another embodiment of the present invention. FIG. 11B is a flow diagram of a method for purifying triterpenoid compounds of yet another embodiment of the present invention. FIG. 12 is a single column chromatography spectrum of a Ganoderma lucidum extract of another embodiment of the present invention. FIG. 13 is a schematic diagram of the configuration of the second simulated moving bed of an embodiment of the present invention. FIG. 14A is an HPLC analysis spectrum of the second simulated moving bed product of an embodiment of the present invention. FIG. 14B is an HPLC analysis spectrum of the second simulated moving bed product of another embodiment of the present invention. FIG. 15 is a schematic flow chart of a method for glycosylation of triterpenoid compounds according to an embodiment of the present invention. FIG. 16A-16B to FIG. 25A-25B are HPLC/UV analysis spectra of Ganoderma lucidum extracts and their glycosylated derivatives according to different embodiments of the present invention.

S810, S820, S830: Steps

Claims (20)

A method for purifying triterpenoid compounds, comprising: Providing a crude extract of Ganoderma lucidum; the crude extract of Ganoderma lucidum contains a first triterpenoid compound and a second triterpenoid compound, wherein the polarity of the first triterpenoid compound is higher than the polarity of the second triterpenoid compound; Dissolving the crude extract of Ganoderma lucidum and performing a pretreatment to obtain a Ganoderma lucidum extract; the Ganoderma lucidum extract contains the first triterpenoid compound and the second triterpenoid compound; wherein the pretreatment comprises: Dissolving the crude extract of Ganoderma lucidum in an alcohol/water system; Low-temperature treatment of the alcohol/water system in which the crude extract of Ganoderma lucidum is dissolved; and Centrifuging the alcohol/water system to separate a supernatant, wherein the supernatant contains the first triterpenoid compound and the second triterpenoid compound; and The first triterpenoid compound and the second triterpenoid compound in the ganoderma lucidum extract are separated by simulated moving bed chromatography; the simulated moving bed chromatography comprises: (i) providing a first simulated moving bed; the first simulated moving bed is composed of a first mobile phase and a stationary phase, and includes a flushing end, a feed inlet, an extraction end, a raffinate end, and a first section, a second section and a third section arranged in sequence, wherein the first mobile phase flows from the flushing end through the first section, the second section and the third section in the same direction relative to the first simulated moving bed, and the stationary phase simulates moving in the opposite direction relative to the first mobile phase; (ii) injecting the ganoderma lucidum extract from the feed port into the space between the second section and the third section of the first simulated moving bed, and allowing the first triterpenoid compound to move with the stationary phase to the extraction end between the first section and the second section, and allowing the second triterpenoid compound to move with the first mobile phase to the raffinate end of the third section, so as to separate and purify the first triterpenoid compound and the second triterpenoid compound; wherein a raffinate product is released from the raffinate end; (iii) providing a second simulated moving bed; the second simulated moving bed is composed of a second mobile phase and a stationary phase, and includes a flushing end, a feed inlet, an extraction end, a raffinate end, and a first section, a second section and a third section arranged in sequence, wherein the second mobile phase flows from the flushing end through the first section, the second section and the third section in the same direction relative to the second simulated moving bed, and the stationary phase simulates moving in the opposite direction relative to the second mobile phase; and (iv) injecting the raffinate from the feed port of the second simulated moving bed into the space between the second section and the third section, and allowing the ganoderic acid A in the second triterpenoid compound to move with the stationary phase to the extraction end between the first section and the second section, and allowing the ganoderic acid F in the second triterpenoid compound to move with the second mobile phase to the raffinate end of the third section, so as to separate and purify ganoderic acid A and ganoderic acid F; wherein the second mobile phase is different from the first mobile phase, and the second mobile phase contains a supercritical carbon dioxide fluid; and the stationary phase contains a surface-modified silica filler. The method for purifying triterpenoid compounds as described in claim 1, wherein the step (ii) further comprises obtaining a raffinate from the raffinate end, and the simulated moving bed chromatography method further comprises: Injecting the raffinate from the feed inlet into the space between the second section and the third section of the first simulated moving bed, and allowing the first triterpenoid compounds to move with the stationary phase to the extraction end between the first section and the second section, and allowing the second triterpenoid compounds to move with the first mobile phase to the raffinate end of the third section, so as to separate and purify the first triterpenoid compounds and the second triterpenoid compounds. The method for purifying triterpenoid compounds as described in claim 1, wherein the step of dissolving the crude extract of Ganoderma lucidum and performing the pretreatment further comprises: treating the alcohol/water system in which the crude extract of Ganoderma lucidum is dissolved at a low temperature. The method for purifying triterpenoid compounds as described in claim 3, wherein the alcohol/water system further comprises 60-75 wt% of an alcohol aqueous solution; the low temperature treatment step further comprises refrigerating the alcohol/water system in which the crude extract of Ganoderma lucidum is dissolved, and centrifuging the alcohol/water system at a low temperature. The method for purifying triterpenoid compounds as described in claim 1, wherein the step of dissolving the crude extract of Ganoderma lucidum and performing the pretreatment further comprises: providing an adsorbent to be added to the supernatant for adsorption; and performing filtration to separate the adsorbent and a filtrate, wherein the filtrate contains the first triterpenoid compound, the second triterpenoid compound or a combination thereof. The method for purifying triterpenoid compounds as described in claim 5, wherein the step of dissolving the crude extract of Ganoderma lucidum and performing the pretreatment further comprises: desorbing the substance in the adsorbent and obtaining a desorption liquid, wherein the desorption liquid contains the first triterpenoid compound, the second triterpenoid compound or a combination thereof. The method for purifying triterpenoid compounds as described in claim 1, wherein the first mobile phase contains an ethanol aqueous solution and the stationary phase contains a surface-modified silica filler. The method for purifying triterpenoid compounds as described in claim 7, wherein the ratio of ethanol/water in the ethanol aqueous solution is 90/10, and the separation conditions of the first simulated moving bed are: The flow rate of the ethanol aqueous solution is 6.5 mL/min at the flushing end, 0.3 mL/min at the feed inlet, 3.5 mL/min at the extraction end, and 3.3 mL/min at the raffinate end, and the switching time of the first simulated moving bed is 4 minutes to 5 minutes. The method for purifying triterpenoid compounds as described in claim 7, wherein the ratio of ethanol/water in the ethanol aqueous solution is 90/10, and the separation conditions of the first simulated moving bed are: The flow rate of the ethanol aqueous solution is 97.5 mL/min at the flushing end, 4.5 mL/min at the feed inlet, 52.5 mL/min at the extraction end, and 49.5 mL/min at the raffinate end, and the switching time of the first simulated moving bed is 4 minutes to 5 minutes. A method for purifying triterpenoid compounds as described in claim 1, wherein the second mobile phase further contains a cosolvent, the cosolvent contains 95% ethanol, and the separation conditions of the second simulated moving bed are: The flow rate of the supercritical carbon dioxide fluid is 5.0 g/min at the distillation end, 0.313 g/min at the feed port, 2.42 g/min at the extraction end, and 2.894 g/min at the raffinate end; The flow rate of the cosolvent is 2.300 mL/min at the distillation end, 0.144 mL/min at the feed port, 1.112 mL/min at the extraction end, and 1.331 mL/min at the raffinate end; and The flow rate of the second mobile phase is 7.955 mL/min at the flushing end, 0.497 mL/min at the feed port, 3.848 mL/min at the extraction end, and 4.604 mL/min at the raffinate end, and the switching time of the second simulated moving bed is 9 minutes to 10 minutes. A method for purifying triterpenoid compounds as described in claim 1, wherein the second mobile phase further contains a cosolvent, the cosolvent contains 95% ethanol, and the separation conditions of the second simulated moving bed are: The flow rate of the supercritical carbon dioxide fluid is 75.0 g/min at the flushing end, 4.96 g/min at the feed inlet, 36.3 g/min at the extraction end, and 43.66 g/min at the raffinate end; The flow rate of the cosolvent is 34.5 mL/min at the flushing end, 2.16 mL/min at the feed inlet, 16.68 mL/min at the extraction end, and 19.98 mL/min at the raffinate end; and The flow rate of the second mobile phase is 119.32 mL/min at the flushing end, 7.46 mL/min at the feed port, 57.72 mL/min at the extraction end, and 69.06 mL/min at the raffinate end, and the switching time of the second simulated moving bed is 9 minutes to 10 minutes. The method for purifying triterpenoid compounds as described in claim 1, wherein the preparation method of the crude extract of Ganoderma lucidum comprises: extracting Ganoderma lucidum using a supercritical fluid to obtain the crude extract of Ganoderma lucidum. A method for purifying triterpenoid compounds as described in claim 1, wherein the content of ganoderin B in the separated and purified first-type triterpenoid compounds is greater than 2 wt%, the content of ganoderic acid A in the separated and purified second-type triterpenoid compounds is greater than 5 wt%, and the content of ganoderic acid F in the separated and purified second-type triterpenoid compounds is greater than 2 wt%. The method for purifying triterpenoid compounds as described in claim 13, wherein the content of the separated and purified ganoderic acid A is greater than 10 wt%, and the total content of ganoderic alcohol B, ganoderic acid A and ganoderic acid F is greater than 15 wt%. A method for glycosylation of triterpenoid compounds, comprising: Purifying triterpenoid compounds according to the method of any one of claims 1 to 14; and Providing an enzyme and a glycosyl donor, mixing the enzyme, the glycosyl donor and the purified triterpenoid compounds, so that the enzyme performs in vitro bioconversion; Wherein the purified triterpenoid compounds include a first triterpenoid compound and a second triterpenoid compound. The method for glycosylation of triterpenoid compounds as described in claim 15, wherein the step of providing the enzyme further comprises providing a genetically recombinant Escherichia coli; the recombinant Escherichia coli has a recombinant gene, and the recombinant gene can produce the enzyme. A method for glycosylation of triterpenoid compounds as described in claim 16, wherein the recombinant gene includes a glycosyltransferase family 1 (GT1) gene. A method for glycosylation of triterpenoid compounds as described in claim 17, wherein the recombinant gene is a uridine diphosphate-dependent glycosyltransferase (UGT) gene. The method for glycosylation of triterpenoid compounds as described in claim 15, wherein the sugar donor comprises uridine diphosphate-glucose (UDP-G). A method for glycosylation of triterpenoid compounds as described in claim 15, wherein the first type of triterpenoid compounds includes ganoderin B, and the second type of triterpenoid compounds includes ganoderin acid A and ganoderin acid F.