Buddleja glycoside with URAT1 inhibitory activity and its preparation method and application

Abstract

The present invention discloses a budding glycoside having URAT1 inhibitory activity, and its preparation method and application. The preparation method comprises the following steps: washing and drying blackberry leaves, crushing and sieving to obtain blackberry leaf powder; subjecting the blackberry leaf powder to supercritical extraction and collecting the extract; concentrating the extract under reduced pressure to obtain a concentrate; purifying and eluting the concentrate and collecting the eluate; concentrating the eluate under reduced pressure and drying to obtain budding glycoside. The yield of budding glycoside is 0.84%, the HPLC purity is as high as 99.9%, and the preparation method is safe, environmentally friendly and low-cost. The present invention has also been experimentally verified to have budding glycoside with urate transporter 1 inhibitory activity, providing a new resource for the development of urate transporter 1 inhibitors, and also providing a new use for the high-value utilization of blackberry leaves.

Description

Technical Field

[0001] The present invention relates to the field of natural product development, and in particular to budding glycoside having URAT1 inhibitory activity, and a preparation method and application thereof. Background Art

[0002] Urate transporter 1 (URAT1), located in the apical membrane of the renal proximal tubule, is the primary transporter controlling renal urate reabsorption and a key target for the treatment of hyperuricemia. Drugs targeting URAT1 for the treatment of hyperuricemia, such as benzbromarone, inhibit URAT1's ability to transport urate, reducing urate reabsorption and thereby promoting uric acid excretion. However, benzbromarone exhibits significant hepatotoxicity and is unsuitable for use during gout flares, limiting its application. Other drugs that target URAT1 also have varying degrees of side effects and limitations. Therefore, the identification of URAT1 inhibitors from natural compounds has become a research hotspot.

[0003] Buddleja glycoside, with a molecular weight of 594.52, is a glycoside flavonoid with antithrombotic, hypoglycemic, anti-inflammatory, and antitumor activities. However, there are currently no reports on buddleja glycoside's inhibitory activity against URAT1 or its uric acid-lowering activity. Studies have reported that the main sources of buddleja glycoside include buddleja flower, Tilia amurensis flower, and raspberry, but some of these raw materials are relatively expensive. Blackberry leaves, on the other hand, contain a high content of buddleja glycoside. As a byproduct of the blackberry cultivation industry, blackberry leaves are relatively low-cost and can therefore be used as a raw material for the preparation of buddleja glycoside. The production process for buddleja glycoside primarily involves organic solvent extraction and reflux extraction, which have disadvantages such as high organic solvent consumption, long extraction time, and low extraction yield. Summary of the Invention

[0004] To address the shortcomings of the existing technology, the present invention provides a budding glycoside with URAT1 inhibitory activity, as well as its preparation method and application. The present invention utilizes supercritical carbon dioxide extraction technology combined with C18 reverse-phase silica gel column purification to efficiently extract and purify budding glycoside from blackberry leaves. The resulting budding glycoside has high yield and purity and exhibits strong urate transporter 1 inhibitory activity.

[0005] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for preparing budding glycoside having URAT1 inhibitory activity, comprising the following steps:

[0007] (1) Wash and dry the blackberry leaves, crush and sieve to obtain blackberry leaf powder;

[0008] (2) subjecting the blackberry leaf powder obtained in step (1) to supercritical extraction and collecting the extract;

[0009] (3) Concentrating the extract from step (2) under reduced pressure to obtain a concentrated solution;

[0010] (4) purifying and eluting the concentrated solution from step (3) and collecting the eluate;

[0011] (5) The eluate from step (4) is concentrated under reduced pressure and dried to obtain budding glycoside.

[0012] Furthermore, the mesh size of the sieving in step (1) is 40-100 meshes.

[0013] Furthermore, the supercritical extraction in step (2) adopts an extraction kettle, CO2 as the extraction agent, and 40%-80% ethanol as the entrainer; the conditions of the supercritical extraction are: extraction temperature of 30℃-50℃, extraction pressure of 35 Mpa-50 Mpa, extraction time of 40min-60 min, entrainer flow rate of 2 L / h-3 L / h, CO2 flow rate of 30 L / h-50 L / h, wherein the pressure of separation kettle 1 is 15 Mpa-20 Mpa, and the pressure of separation kettle 2 is 4 Mpa-8 Mpa.

[0014] Furthermore, the extract in step (3) and the eluent in step (5) are both concentrated under reduced pressure to 1 / 10-1 / 20 of their original volumes; the reduced pressure concentration conditions are: temperature of 40°C-55°C, and vacuum degree of -0.055 MPa ~-0.098 MPa.

[0015] Furthermore, the purification in step (4) is performed using a silica gel column, specifically a C18 reverse phase silica gel column with a particle size of 10-40 μm.

[0016] Furthermore, the elution conditions in step (4) are: elution with 40%-45% methanol solution for 2-3 BV, elution with 50%-60% methanol solution for 3 BV, thin layer chromatography for real-time detection, and collection of the components containing the target compound in the 50%-60% methanol eluate.

[0017] Furthermore, the drying method in step (5) is vacuum drying or freeze drying.

[0018] In summary, a method for preparing budding glycoside having URAT1 inhibitory activity comprises the following steps:

[0019] (1) Wash and dry the blackberry leaves, crush them, and pass them through a 40-100 mesh sieve to obtain blackberry leaf powder;

[0020] (2) Add blackberry leaf powder to the extraction kettle, use CO2 as the extractant and 40%-80% ethanol as the entrainer for supercritical extraction, set the extraction temperature to 30-50℃, the pressure to 35-50 MPa, the time to 40-60 min, the entrainer flow rate to 2-3 L / h, the CO2 flow rate to 30-50 L / h, the pressure of separation kettle 1 to 15-20 MPa, the pressure of separation kettle 2 to 4-8 MPa, and collect the extract in separation kettle 2;

[0021] (3) The extract of step (2) is concentrated under reduced pressure at a temperature of 40-55°C and a vacuum degree of -0.055 to -0.098 MPa to 1 / 10-1 / 20 of the original volume to obtain a concentrated solution;

[0022] (4) The concentrate of step (3) was purified using a C18 reverse phase silica gel column with a particle size of 10-40 μm. After loading, 2-3 BV of the concentrate was eluted with a 40%-45% methanol solution and 3 BV of the concentrate was eluted with a 50%-60% methanol solution. Thin layer chromatography was used for real-time detection, and the fraction containing the target compound in the 50%-60% methanol eluate was collected.

[0023] (5) The eluate from step (4) is concentrated under reduced pressure to 1 / 10-1 / 20 of the original volume at a temperature of 40-55° C. and a vacuum degree of -0.055 to -0.098 MPa, and vacuum dried or freeze-dried to obtain budding glycoside.

[0024] The present invention also provides budding glycoside, which is prepared by the preparation method and has a purity greater than 98%.

[0025] The present invention also provides the use of the budding glycoside in the preparation of a URAT1 inhibitor.

[0026] Furthermore, the budding glycoside inhibits the IC of URAT1 50 It is 7.77 µmol / L.

[0027] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0028] 1. The present invention provides a method for extracting and preparing budding glycoside from blackberry leaves. The method utilizes supercritical carbon dioxide extraction technology combined with C18 reverse-phase silica gel column purification to efficiently prepare high-purity budding glycoside with urate transporter 1 inhibitory activity. The yield of budding glycoside is 0.84%, and the HPLC purity is as high as 99.9%. In addition, the preparation method has the advantages of safety, environmental protection, and low cost.

[0029] 2. The budding glycosides derived from blackberry leaves provided by the present invention have been verified for the first time to have urate transporter 1 inhibitory activity, and the IC 50The activity of the urate transporter 1 inhibitor was 7.77 µmol / L, which was equivalent to that of benzbromarone, providing a new resource for the development of urate transporter 1 inhibitors and a new use for the high-value utilization of blackberry leaves.

[0030] 3. As a natural plant extract, budding glycoside from blackberry leaves has the advantages of less side effects on the human body and being green and safe compared to existing drugs. It has obvious advantages and broad application prospects in the preparation of functional products with urate transporter 1 inhibitory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the HPLC chromatogram of budding glycoside in Example 2, wherein the abscissa is time and the ordinate is electrical signal intensity.

[0032] Figure 2 The inhibitory activity of budding glycosides from different examples on URAT1 is shown in FIG. 1 , wherein the horizontal axis represents the budding glycosides prepared from different examples, and the vertical axis represents the inhibition rate.

[0033] Figure 3 The figure shows the inhibitory effects of benzbromarone and budding glycoside of Example 1 on URAT1, wherein the abscissa represents the concentration and the ordinate represents the inhibition rate. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with the examples, but the following examples are not intended to limit the present invention in any way, and any changes or substitutions made based on the present invention fall within the scope of protection of the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are all conventional reagents, methods and equipment in the art.

[0035] Purity determination of budding glycosides: HPLC was used for detection and the purity was calculated by the peak area normalization method. The chromatographic column was an Agilent ZORBAX SB-C18 column (5 μm, 4.6 mm×250 mm). The mobile phases were methanol (A) and 0.1% formic acid (B), with a ratio of 45% A and 55% B. The flow rate was 1.0 mL / min, the column temperature was 25°C, the detection wavelength was 365 nm, the injection volume was 20 μL, the sample concentration was 25 μg / mL, and the detection time was 15 min.

[0036] Calculation of budding glycoside yield: yield

[0037] Wherein, m1 is the mass of the obtained buddingoside solid powder, P is the purity of the buddingoside solid powder, and m2 is the mass of the blackberry leaf powder.

[0038] Example 1 Effect of process conditions on the preparation of budding glycosides

[0039] Wash, dry, crush, and sieve blackberry leaves to obtain 40-100 mesh blackberry leaf powder. Add 1 kg of blackberry leaf powder to an extraction kettle and perform supercritical extraction using CO₂ as the extractant and 60% ethanol as the entrainer. Set the extraction temperature to 40°C, the pressure to 40 MPa, the extraction time to 50 min, the entrainer flow rate to 3 L / h, the CO₂ flow rate to 35 L / h, the pressure in separator 1 to 16 MPa, and the pressure in separator 2 to 6 MPa. Collect the extract in separator 2 and concentrate under reduced pressure at 50°C and a vacuum of -0.090 MPa to 1 / 10 of the original volume to obtain a concentrate. The concentrate was purified using a C18 reverse-phase silica gel column with a particle size of 20 µm. After loading, 2 BV of the product was eluted with a 45% methanol solution and 3 BV of the product was eluted with a 55% methanol solution. Thin-layer chromatography was used for real-time detection. The eluate containing the target compound was collected and then concentrated under reduced pressure at 50°C and a vacuum degree of -0.090 MPa to 1 / 10 of the original volume. The product was then dried under vacuum to obtain budding glycoside.

[0040] (1) Effect of extraction temperature on the preparation of budding glycosides

[0041] On the basis of the above basic process, the extraction temperature of the extraction kettle was adjusted to 20, 30, 40, 50, 60, and 70°C respectively, and other process conditions remained unchanged to study the effects of different extraction temperatures on the preparation of budding glycoside.

[0042] Table 1 Effects of different extraction temperatures on the preparation of budding glycosides

[0043]

[0044] The results are shown in Table 1. When the temperature is 40°C, the yield and purity of buddingoside are the highest, and different extraction temperatures have little effect on the purity of buddingoside.

[0045] (2) Effect of extraction pressure on the preparation of budding glycosides

[0046] On the basis of the above basic process, the extraction pressures were set to 20, 25, 30, 35, 40, 45, and 50 MPa respectively, and other process conditions remained unchanged to study the effects of different extraction pressures on the preparation of buddlejac.

[0047] Table 2 Effect of different pressures on the preparation of budding glycosides

[0048]

[0049] The results are shown in Table 2. When the extraction pressure was 35 MPa, the yield of budding glycoside did not increase significantly when the pressure was increased, and when the extraction pressure was 35-45 MPa, the purity of budding glycoside was greater than 99.5%.

[0050] (3) Effect of extraction time on the preparation of budding glycosides

[0051] On the basis of the above basic process, the extraction time was set to 10, 20, 30, 40, 50, and 60 min respectively, and other process conditions remained unchanged to study the effect of different extraction times on the preparation of budding glycoside.

[0052] Table 3 Effect of extraction time on the preparation of budding glycosides

[0053]

[0054] The results are shown in Table 3. When the extraction time was 40 min, the purity and yield of budding glycoside were the highest.

[0055] (4) Effect of entrainer concentration on the preparation of budding glycosides

[0056] On the basis of the above basic process, the entrainer concentration was set to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% ethanol respectively, and other process conditions remained unchanged to study the effect of different entrainer concentrations on the preparation of budding glycoside.

[0057] Table 4 Effect of entrainer concentration on the preparation of budding glycosides

[0058]

[0059] The results are shown in Table 4. When the concentration of the entrainer ethanol is 50%, the yield and purity of budding glycoside are the highest.

[0060] (5) Effect of eluent methanol concentration on the preparation of budding glycosides

[0061] On the basis of the above basic process, the methanol concentration of the eluent was set to 40%, 45%, 50%, 55%, 60%, 65%, 70% and 75% respectively, and other process conditions remained unchanged to study the effects of different eluent concentrations on the preparation of budding glycosides.

[0062] Table 5 Effect of different methanol eluent concentrations on the preparation of budding glycosides

[0063]

[0064] The results are shown in Table 5. When the concentration of eluent methanol is 55%, the yield and purity of budding glycoside are the highest.

[0065] Example 2 Extraction and purification of buddingin from blackberry leaves

[0066] Wash and dry blackberry leaves, grind them in a grinder, and pass them through a 60-mesh sieve. Add 1 kg of blackberry leaf powder to an extraction kettle with 50% ethanol as the entrainer. Set the entrainer flow rate to 3 L / h and the CO2 flow rate to 40 L / h. Adjust the extraction kettle temperature to 40°C, the pressure to 35 MPa, and the extraction time to 40 min. Set the pressure in separation kettle 1 to 18 MPa and the pressure in separation kettle 2 to 6 MPa. The extract from separation kettle 2 was collected and concentrated under reduced pressure to 1 / 10 of the original volume. The concentrate was eluted with 45% methanol solution for 2 BV using a C18 reversed-phase silica gel column with a particle size of 10 μm, and then eluted with 55% methanol solution. A portion of 15 mL was collected and detected by thin-layer chromatography and the components containing a single point of budding glycoside were combined. A total of 3 BV were eluted. The collected budding glycoside solution was concentrated under reduced pressure and dried with a freeze dryer to obtain budding glycoside solid powder. The powder was dissolved and detected by HPLC. The calculated purity of the budding glycoside was 99.9% (see HPLC spectrum). Figure 1 ), the yield is 0.84%.

[0067] Example 3 Extraction and purification of buddingoside from blackberry leaves

[0068] Blackberry leaves were washed, dried, pulverized in a grinder, and passed through an 80-mesh sieve. 800 g of blackberry leaf powder was added to an extraction kettle with 75% ethanol as the entrainer. The entrainer flow rate was set at 2 L / h, the CO2 flow rate at 30 L / h, the extraction kettle temperature at 35°C, the pressure at 30 MPa, and the extraction time at 45 min. The pressure in separator 1 was set at 15 MPa, and the pressure in separator 2 was set at 5 MPa. The extract from separator 2 was collected and concentrated under reduced pressure at 55°C. The concentrate was then loaded onto a C18 reverse-phase silica gel column with a 20 μm particle size. Three batches of budding glycosides were eluted with 45% methanol, followed by 55% methanol. Thin-layer chromatography was used to detect and collect the fraction containing a single spot of budding glycosides, eluting for a total of three batches of glycosides. The fraction was then eluted with 100% methanol for another two batches of glycosides. The collected budding glycoside solution was concentrated under reduced pressure at 50°C and dried in a vacuum dessicator to obtain budding glycosides as a solid powder. The powder was dissolved and tested by HPLC, and the purity of the obtained budding glycoside was calculated to be 98.2% and the yield was 0.80%.

[0069] Example 4 Extraction and purification of buddingoside from blackberry leaves

[0070] 1 kg of 80-mesh blackberry leaf powder was added to an extraction kettle. The entrainer was 60% ethanol. The entrainer flow rate was set to 3 L / h, the CO2 flow rate was 35 L / h, the extraction kettle temperature was adjusted to 40°C, the pressure was 35 MPa, the extraction time was 40 min, and the pressure in separation kettle 1 was set to 20 MPa and the pressure in separation kettle 2 was set to 6 MPa. The extract from separation kettle 2 was collected and concentrated under reduced pressure at 50°C. The concentrate was loaded onto a C18 reverse-phase silica gel column and eluted with 45% methanol solution for 3 BV, then replaced with 55% methanol solution. A tube was collected every 20 mL. Thin-layer chromatography was performed and the fractions containing a single spot of budding glycoside were combined. A total of 3 BV of elution was obtained. Then, 100% methanol was replaced to elute 2 BV. The collected budding glycoside solution was concentrated under reduced pressure at 50°C and dried in a vacuum dessicator to obtain budding glycoside solid powder. The budding glycoside solid powder was dissolved and then tested by HPLC. The purity of the budding glycoside was calculated to be 99.0%, and the yield was 0.79%.

[0071] Example 5 Extraction and purification of buddingoside from blackberry leaves

[0072] Blackberry leaves were washed, dried, pulverized, and passed through a 60-mesh sieve. 600 g of blackberry leaf powder was added to an extraction kettle with 80% ethanol as the entrainer. The entrainer flow rate was set at 1.5 L / h and the CO2 flow rate was 35 L / h. The temperature of the extraction kettle was adjusted to 50°C, the pressure was 30 MPa, and the extraction time was 45 min. The pressure in separator 1 was set to 16 MPa, and the pressure in separator 2 was set to 5 MPa. The extract from separator 2 was collected and concentrated under reduced pressure at 55°C. The concentrate was purified using a C18 reverse-phase silica gel column. 3 BV of elution was performed with 45% methanol to remove impurities. The elution was then replaced with 55% methanol. Thin-layer chromatography was used to detect and collect a single fraction containing buddingin, eluting 3 BV in total. The collected buddingin solution was concentrated under reduced pressure and dried in a vacuum dessicator to obtain buddingin solid. The powder was dissolved and analyzed by HPLC. The purity of the buddingin was calculated to be 99.5%, and the yield was 0.76%.

[0073] Example 6 Extraction and purification of buddingoside from blackberry leaves

[0074] Blackberry leaves were washed, dried, pulverized, and passed through a 60-mesh sieve. 600 g of blackberry leaf powder was added to an extraction kettle with 70% ethanol as the entrainer. The entrainer flow rate was set at 1.5 L / h and the CO2 flow rate was 35 L / h. The temperature of the extraction kettle was adjusted to 50°C, the pressure was 30 MPa, and the extraction time was 45 min. The pressure in separator 1 was set to 16 MPa, and the pressure in separator 2 was set to 5 MPa. The extract from separator 2 was collected and concentrated under reduced pressure at 55°C. The concentrate was purified using a C18 reverse-phase silica gel column, eluting with 45% methanol for 3 BV, followed by 55% methanol for 3 BV. Thin-layer chromatography was used to detect and collect fractions containing a single spot of buddingin. The collected buddingin solution was concentrated under reduced pressure and then dried in a freeze dryer to obtain buddingin solid. The powder was dissolved and analyzed by HPLC. The purity of the buddingin was calculated to be 99.5%, and the yield was 0.76%.

[0075] Example 7 Inhibitory activity of budding glycosides on URAT1

[0076] In this example, the inhibitory activity of budding glycoside on the uptake of 6-carboxyfluorescein (6-CFL) by HEK-293T cells overexpressing URAT1 was determined by the following method:

[0077] (1) HEK-293T cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100 IU / mL), and streptomycin (100 μg / mL) in a 37°C, 5% CO2 incubator.

[0078] (2) URAT1-overexpressing HEK-293T-URAT1 cells and control HEK-293T-EGFP cells were constructed using lentiviral vectors. The cells were digested with trypsin and prepared into approximately 40×10 4 Cell suspension of 100 μL cells / mL was added to each well of a 96-well white cell culture plate. Experimental group, control group and blank group were set up, with 6 replicates in each group. 48 hours after plating, the culture medium was aspirated and washed once with HBSS solution. Then 100 μL of HBSS solution was added to each well and placed in an incubator for 10 minutes before adding the corresponding solution. Experimental group: 239.5 μmol / L 6-CFL was prepared with HBSS solution, and the corresponding concentrations of the test sample and positive drug were prepared with this solution; control group: 6-CFL was contained but no test sample was present; blank group: HBSS solution, 100 μL per well;

[0079] (3) After incubation in the incubator for 1 hour, remove the incubation solution and wash 3-5 times with 37°C PBS buffer. Aspirate the remaining PBS buffer and add 100 μL of 1mM NaOH solution to each well. Lyse at room temperature for 30 minutes in the dark. After shaking for 2 minutes to mix, read the result under the conditions of excitation and emission light at 490 and 525 nm, respectively. Calculate the inhibition rate according to the following formula:

[0080] Inhibition rate / %=(control group-experimental group) / (control group-blank group)×100%

[0081] (4) 2-fold gradient dilution of budding glycosides and positive drugs was performed, and HBSS was used as blank control. The inhibition rate of samples with different concentrations on URAT1 uptake of 6-CFL in cells was determined according to the above method, and the IC 50 value.

[0082] The URAT1 inhibitory activity of the budding glycosides prepared in Example 2, Example 3, Example 4, Example 5, and Example 6 was determined. Figure 2 The inhibition rates of budding glycosides at a concentration of 50 μmol / L on the absorption of 6-CFL by URAT1 in cells were 83.93%, 79.87%, 82.47%, 81.40% and 78.47%, respectively, indicating that budding glycosides can significantly inhibit the absorption of 6-CFL by URAT1.

[0083] The budding glycoside prepared in Example 1 was selected to study the inhibitory activity of budding glycoside at different concentrations on URAT1. Figure 3 The results showed that within the concentration range of 0~100 μmol / L, the inhibitory activity of budding glycoside on URAT1 gradually increased with the increase of budding glycoside concentration. When the concentration of budding glycoside was 25 μmol / L, its inhibition rate could reach 76.50%. As the concentration continued to increase, the inhibition rate growth trend tended to be slow. The IC 50 The activity of benzobromaron was 7.77 µmol / L, which was equivalent to that of benzbromarone.

[0084] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the above embodiments, or to replace some of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A method for preparing budding glycoside having URAT1 inhibitory activity, characterized in that: The preparation method comprises the following steps: (1) Wash and dry the blackberry leaves, crush and sieve to obtain blackberry leaf powder; (2) The blackberry leaf powder of step (1) is subjected to supercritical extraction, and the extract is collected; the supercritical extraction adopts an extraction kettle, CO2 is used as an extractant, and 40%-80% ethanol is used as an entrainer; the conditions of the supercritical extraction are: extraction temperature of 30°C-50°C, extraction pressure of 35 MPa-50 MPa, extraction time of 40 min-60 min, entrainer flow rate of 2 L / h-3 L / h, CO2 flow rate of 30 L / h-50 L / h, wherein the pressure of separation kettle 1 is 15 MPa-20 MPa, and the pressure of separation kettle 2 is 4 MPa-8 MPa; the extract is collected in separation kettle 2; (3) concentrating the extract from step (2) under reduced pressure to obtain a concentrated solution; (4) Purifying and eluting the concentrated solution of step (3) and collecting the eluate; the purification is performed using a silica gel column, specifically a C18 reverse phase silica gel column with a particle size of 10-40 µm; the elution conditions are: eluting with 40%-45% methanol solution for 2-3 BV, eluting with 50%-60% methanol solution for 3 BV, and performing real-time thin layer chromatography detection, collecting the components containing the target compound in the 50%-60% methanol eluate; (5) The eluate from step (4) is concentrated under reduced pressure and dried to obtain budding glycoside.

2. The preparation method according to claim 1, characterized in that The mesh size of the sieving in step (1) is 40-100 mesh.

3. The preparation method according to claim 1, wherein The extract in step (3) and the eluent in step (5) are both concentrated under reduced pressure to 1 / 10-1 / 20 of their original volumes; the reduced pressure concentration conditions are: temperature of 40°C-55°C, and vacuum degree of -0.055 MPa to -0.098 MPa.

4. The preparation method according to claim 1, characterized in that The drying method in step (5) is vacuum drying or freeze drying.

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