A preparation method of ginsenoside Rc reference substance

Abstract

The present invention discloses a method for preparing a ginsenoside Rc reference standard, comprising: a) adding an ethanol solution to a sample of ginsenoside components, stirring and ultrasonicating the sample until completely dissolved, filtering the sample through a membrane, loading the sample onto a reverse-phase chromatography column with a large-particle packing, performing linear gradient elution with an ethanol solution, and analyzing and combining the target fractions containing ginsenoside Rc to obtain a crude fraction; b) loading the crude fraction onto a reverse-phase chromatography column with a small-particle packing, performing isocratic elution with an ethanol solution, and analyzing and combining the target fractions containing ginsenoside Rc to obtain a crude ginsenoside Rc solution; c) loading the crude product solution onto a hydrophilic chromatography column with a small-particle packing, performing isocratic elution with an ethanol-water solution to obtain a pure ginsenoside Rc solution; and d) concentrating and removing alcohol from the pure product solution, and freeze-drying the solution to obtain a ginsenoside Rc reference standard. The ginsenoside Rc monomer obtained by the present invention has a purity exceeding 98%, and the process is simple, easy to operate, and low-cost. No toxic or hazardous solvents are used in the preparation process.

Description

Technical Field

[0001] The invention belongs to the technical field of modernization of traditional Chinese medicine, and particularly relates to a method for preparing a ginsenoside Rc reference substance. Background Art

[0002] Ginseng (Panax ginseng CA Meyer) is a perennial herbaceous plant in the Araliaceae family and genus Panax. Its fleshy root is a tonic, suitable for regulating blood pressure, restoring heart function, and treating neurasthenia and physical weakness. It also has stomachic, diuretic, and stimulant properties. Ginsenosides, a class of steroid compounds considered the active ingredient in ginseng, have become a research target. They have been shown to enhance immunity, combat fatigue, and improve memory and learning abilities.

[0003] In addition to its anti-inflammatory and antioxidant effects, ginsenoside Rc also has the effects of preventing diabetes and lowering lipids, especially the inhibitory effect on cancer cells.

[0004] The molecular formula of ginsenoside Rc is C 53 H 90 O 22 , the structural formula is as follows:

[0005]

[0006] Ginsenoside Rc is a ginsenoside diol type and is one of the main saponins in ginseng. Existing saponin monomer separation and purification methods, such as macroporous resin and silica gel column chromatography, are difficult to isolate high-purity monomer compounds. In addition, toxic solvents are easily introduced during the separation process, and the operation is cumbersome and not conducive to scale-up. Summary of the Invention

[0007] In view of this, the present invention aims to provide a method for preparing a ginsenoside Rc reference substance, which is used to solve the problems of easy introduction of toxic and harmful reagents, complex operation, low yield and purity during the preparation process of the existing method.

[0008] To achieve the above object, the present invention adopts a method for preparing a ginsenoside Rc reference substance, comprising the following steps:

[0009] a) Add 25%-35% ethanol solution to a sample of ginsenosides and sonicate until completely dissolved. Filter through a 0.45 μm organic filter membrane and pass through a reversed-phase column packed with 30-60 μm particles. Elute using a linear gradient of ethanol solution concentration from 25% to 90%. Finely cut and collect fractions for UPLC analysis. Combine the target fractions containing ginsenoside Rc to form the crude fraction.

[0010] b) connecting a reverse phase chromatography column filled with 5-10 μm particle size filler to a preparative chromatography system, allowing the crude separated solution to flow through the chromatography column for purification via a preparative chromatography system pump, isocratically eluting with 50%-60% ethanol, collecting fractions according to chromatographic peaks, performing UPLC analysis, and combining the target fractions containing ginsenoside Rc to obtain a crude ginsenoside Rc solution;

[0011] c) connecting a hydrophilic chromatographic column with a 5-10 μm particle size filler to a preparative chromatographic system, passing the crude product solution through the chromatographic column for purification via a preparative chromatographic system pump, and isocratically eluting with an 80%-70% ethanol aqueous solution to obtain a pure ginsenoside Rc solution;

[0012] d) The pure product solution is concentrated to remove alcohol and freeze-dried to obtain ginsenoside Rc reference substance.

[0013] Preferably, the ginsenoside total saponin fraction sample is prepared by the following steps:

[0014] 1) Soak ginseng slices in 50% to 80% ethanol for 0.5 to 1 hour, decoct for 2 to 3 hours, and filter the liquid; decoct the residue 2 to 3 times, each time for 2 to 3 hours, filter the liquid, and combine and concentrate the liquids to obtain a ginseng extract solution;

[0015] 2) The ginseng extract solution was filtered through a membrane and passed through a reverse phase chromatography column. The elution was then performed with 2 to 4 column volumes of water and 4 to 6 column volumes of a 50 to 70% ethanol solution, and then with 2 to 5 column volumes of a 70 to 95% ethanol solution. The eluate was collected, concentrated, and dried to obtain a ginsenoside total saponin component sample.

[0016] Preferably, the reverse phase chromatography column filler matrix in step a) is silica gel or a high molecular polymer.

[0017] Preferably, the reverse phase chromatography column filler matrix in step b) is silica gel.

[0018] Preferably, the hydrophilic chromatography column filler in step c) is a filler with polar groups bonded to the surface of silica gel, and the bonded polar groups are one or more of diol groups, amides, amino groups, carboxyl groups, sugar groups, and zwitterions.

[0019] The beneficial effects of the present invention are as follows:

[0020] 1. The method of the present invention combines high performance liquid chromatography separation technology and can effectively separate the target saponin from other saponins, impurities, etc. through chromatographic fillers with different particle sizes and different separation selectivities, thereby achieving efficient separation, purification and preparation of saponin monomer compounds, and improving the purity of saponin monomer compounds.

[0021] 2. The solvents used in the preparation process of the present invention are ethanol and purified water, and no toxic or harmful reagents are required, thus achieving green preparation and production.

[0022] 3. The entire experimental operation process of the method of the present invention is simple, and both the eluent and the regeneration agent can be recycled and reused, saving solvent costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the UPLC analysis result of the total saponin fraction sample of ginseng;

[0024] Figure 2 This is the UPLC analysis result of the crude fraction of the total ginsenosides sample;

[0025] Figure 3 This is the UPLC analysis result of crude ginsenoside Rc solution;

[0026] Figure 4 This is the UPLC analysis result of pure ginsenoside Rc solution;

[0027] Figure 5 This is a flow chart for the preparation of ginsenoside Rc reference substance. DETAILED DESCRIPTION

[0028] In order to more fully understand the features and technical contents of the present invention, the implementation of the present invention is described in detail below.

[0029] The preparation process of a ginsenoside Rc reference substance of the present invention is as follows: Figure 5 As shown, including the following:

[0030] Step 101: Add a 25% to 35% ethanol solution to a sample of ginsenoside components, stir and sonicate until completely dissolved, filter through a 0.45 μm organic filter membrane, and then pass through a reversed-phase chromatography column packed with 30 to 60 μm particles. Elute using a linear gradient of ethanol solution concentration from 25% to 90%, then finely cut and collect the fractions for UPLC analysis. Combine the target fractions containing ginsenoside Rc to form the crude fraction.

[0031] Preferably, the reverse phase chromatography column filler matrix is silica gel or a high molecular polymer;

[0032] Preferably, the ginsenoside total saponin fraction sample is prepared by the following steps:

[0033] 1) Soak ginseng slices in 50% to 80% ethanol for 0.5 to 1 hour, decoct for 2 to 3 hours, and filter the liquid; decoct the residue 2 to 3 times, each time for 2 to 3 hours, filter the liquid, and combine and concentrate the liquids to obtain a ginseng extract solution;

[0034] 2) filtering the ginseng extract solution through a membrane and then passing it through a reverse phase chromatography column, eluting it with 2 to 4 column volumes of water and 4 to 6 column volumes of a 50 to 70% ethanol solution, and then eluting it with 2 to 5 column volumes of a 70 to 95% ethanol solution, collecting the eluate, concentrating it, and drying it to obtain a ginsenoside total saponin component sample;

[0035] Step 102: Connect a reverse phase chromatography column filled with 5-10 μm particle size filler to a preparative chromatography system, allow the crude separated solution to flow through the chromatography column for purification via a preparative chromatography system pump, and isocratically elute with 50%-60% ethanol. Collect fractions according to chromatographic peaks for UPLC analysis, and combine the target fractions containing ginsenoside Rc to obtain a crude ginsenoside Rc solution.

[0036] Preferably, the reverse phase chromatography column filler matrix is silica gel;

[0037] Step 103: Connect a hydrophilic chromatography column with 5-10 μm particle size filler to a preparative chromatography system, and purify the crude product solution by flowing it through the chromatography column through a preparative chromatography system pump. Then, the crude product solution is isocratically eluted with 80%-70% ethanol aqueous solution to obtain a pure ginsenoside Rc solution.

[0038] Preferably, the hydrophilic chromatographic column filler is a filler with polar groups bonded to the surface of silica gel, and the bonded polar groups are one or more of diol groups, amide groups, amino groups, carboxyl groups, sugar groups, and zwitterions;

[0039] Step 104: The pure product solution is concentrated to remove alcohol and freeze-dried to obtain ginsenoside Rc reference substance.

[0040] At this point, the preparation process of the ginsenoside Rc reference substance of the present invention is completed.

[0041] The preparation process of ginsenoside Rc reference substance is described below by listing specific examples.

[0042] Example 1

[0043] Step 101: Take 75 g of ginsenosides sample and add it to 1.5 L of 33% ethanol solution, stir and sonicate until completely dissolved. Filter through a 0.45 μm organic filter membrane to obtain the reconstituted clear solution, and perform UPLC (Waters ACQUITY UPLC I-Class system) analysis. The chromatographic analysis results are as follows: Figure 1 As shown;

[0044] A DAC100 column (silica-based reverse-phase C18 packing, mass 1500 g, particle size 30 μm) was connected to a preparative chromatography system (Hanbang preparative chromatography system, including NP7000 preparative pump, NC3000 detector), the flow rate was set at 150 mL / min, and the column was flushed with 33% ethanol equilibration buffer for 3 BV. The above-mentioned reconstituted clarified liquid was passed through a C18 reverse-phase column for purification through the preparative chromatography system pump. The first half was eluted with a 33-50% ethanol gradient, and the second half was eluted with a 50-70% ethanol gradient. Each fraction was then finely cut and fractions were collected for UPLC (Waters ACQUITY UPLC I-Class system) analysis. The target fractions containing ginsenoside Rc were combined to form the crude separation solution. The UPLC analysis results of the crude separation solution are shown in Figure 2. Figure 2 As shown;

[0045] Step 102: Connect a DAC50 column (silica-based reverse-phase C18 packing, mass 300 g, particle size 7 μm) to a preparative chromatography system (Hanbang preparative chromatography system, including NP7000 preparative pump, NC3000 detector), set the flow rate to 35 mL / min, flush the column with 55% ethanol equilibration buffer for 3 BV, and allow the crude separation solution to flow through a C18 reverse-phase column for purification via the preparative chromatography system pump. Elute isocratically with 55% ethanol, collect fractions according to chromatographic peaks, and perform UPLC analysis (Waters ACQUITY UPLC I-Class system). The target fractions containing ginsenoside Rc are combined to form a crude ginsenoside Rc solution. The UPLC analysis results of the crude solution are shown in FIG. Figure 3 As shown;

[0046] Step 103: Connect a DAC50 column (zwitterionic hydrophilic chromatography packing, mass 300 g, particle size 10 μm) to a preparative chromatography system (Hanbang preparative chromatography system, including NP7000 preparative pump, NC3000 detector), set the flow rate to 35 mL / min, and flush the column with 72% ethanol (i.e., 28% aqueous solution) equilibration buffer for 3 BV. The crude product solution is passed through the zwitterionic hydrophilic chromatography column through the preparative chromatography system pump for purification. The pure ginsenoside Rc solution is obtained by isocratic elution with 72% ethanol aqueous solution. Fractions are collected according to the chromatographic peaks and analyzed by UPLC (Waters ACQUITY UPLC I-Class system). The UPLC analysis results of the pure solution are shown in FIG. Figure 4 As shown, the purity is 98.74%;

[0047] Step 104: The pure product solution was concentrated, de-alcoholized, and freeze-dried to obtain 1.44 g of ginsenoside Rc reference substance with a yield of 1.92% and a recovery rate of 90.5%.

[0048] Example 2

[0049] Step 101: 85 g of a total ginsenoside fraction sample was added to 1.5 L of a 35% ethanol solution, stirred and ultrasonicated until completely dissolved, and filtered through a 0.45 μm organic filter membrane to obtain a reconstituted clear solution;

[0050] A DAC100 column (silica-based reverse-phase C18 packing, mass 1500 g, particle size 30 μm) was connected to a preparative chromatography system (Hanbang preparative chromatography system, including NP7000 preparative pump, NC3000 detector), the flow rate was set at 120 mL / min, and the column was flushed with 35% ethanol equilibration buffer for 3 BV. The above-mentioned reconstituted clarified liquid was passed through a C18 reverse-phase column for purification through the preparative chromatography system pump. The first half was eluted with a 35-52% ethanol gradient, and the second half was eluted with a 52-80% ethanol gradient. Each fraction was then finely cut and collected for UPLC analysis (Waters ACQUITY UPLC I-Class system). The target fractions containing ginsenoside Rc were combined to form the crude separation solution;

[0051] Step 102: Connect a DAC50 column (silica-based reverse-phase C18 packing, mass 300 g, particle size 7 μm) to a preparative chromatography system (Hanbang preparative chromatography system, including an NP7000 preparative pump and an NC3000 detector), set the flow rate to 30 mL / min, flush the column with 3 BV of 50% ethanol equilibration buffer, and allow the crude fraction to flow through a C18 reverse-phase column for purification via the preparative chromatography system pump. Elute isocratically with 50% ethanol, and collect fractions according to chromatographic peaks for UPLC analysis (Waters ACQUITY UPLC I-Class system). Combine the target fractions containing ginsenoside Rc to obtain the crude ginsenoside Rc solution.

[0052] Step 103: Connect a DAC50 column (amide-based hydrophilic packing, mass 300 g, particle size 10 μm) to a preparative chromatography system (Hanbang preparative chromatography system, including an NP7000 preparative pump and an NC3000 detector). Set the flow rate to 30 mL / min and flush the column with 3 BV of 75% ethanol (i.e., 25% aqueous solution) equilibration buffer. Pass the crude product solution through a C18 reverse-phase column for purification via the preparative chromatography system pump. Elute isocratically with 75% ethanol-water solution to obtain a pure solution of ginsenoside Rc. Fractions were collected according to chromatographic peaks and analyzed by UPLC (Waters ACQUITY UPLC I-Class system). The purity was 98.32%.

[0053] Step 104: The pure product solution was concentrated to remove alcohol and then freeze-dried to obtain 1.52 g of ginsenoside Rc reference substance with a yield of 1.79% and a recovery rate of 91.2%.

[0054] Example 3

[0055] Step 101: 100 g of a total ginsenoside sample was added to 1.5 L of a 35% ethanol solution, stirred and ultrasonicated until completely dissolved, and filtered through a 0.45 μm organic filter membrane to obtain a reconstituted clear solution;

[0056] A DAC100 column (silica-based reverse-phase C18 packing, mass 1500 g, particle size 30 μm) was connected to a preparative chromatography system (Hanbang preparative chromatography system, including NP7000 preparative pump, NC3000 detector), the flow rate was set at 100 mL / min, and the column was flushed with 35% ethanol equilibration buffer for 3 BV. The above-mentioned reconstituted clarified liquid was passed through a C18 reverse-phase column for purification through the preparative chromatography system pump. The first half was eluted with a 35-55% ethanol gradient, and the second half was eluted with a 55-80% ethanol gradient. Each fraction was then finely cut and collected for UPLC analysis (Waters ACQUITY UPLC I-Class system). The target fractions containing ginsenoside Rc were combined to form the crude separation solution;

[0057] Step 102: Connect a DAC50 column (silica-based reverse-phase C18 packing, 300 g, 7 μm particle size) to a preparative chromatography system (Hanbang preparative chromatography system, including an NP7000 preparative pump and an NC3000 detector). Set the flow rate to 25 mL / min, flush the column with 3 BV of 57% ethanol equilibration buffer, and allow the crude fraction to flow through a C18 reverse-phase column for purification via the preparative chromatography system pump. Elute isocratically with 57% ethanol, collect fractions according to chromatographic peaks, and analyze them by UPLC (Waters ACQUITY UPLC I-Class system). Combine the target fractions containing ginsenoside Rc to obtain the crude ginsenoside Rc solution.

[0058] Step 103: Connect a DAC50 column (300 g of diol-based hydrophilic packing material, 10 μm particle size) to a preparative chromatography system (Hanbang preparative chromatography system, including an NP7000 preparative pump and an NC3000 detector). Set the flow rate to 25 mL / min and flush the column with 3 BV of 78% ethanol (i.e., a 22% aqueous solution) equilibration buffer. Pass the crude product solution through a C18 reverse-phase column for purification via the preparative chromatography system pump. Elute isocratically with 78% ethanol in water to obtain a pure solution of ginsenoside Rc. Fractions were collected according to chromatographic peaks and analyzed by UPLC (Waters ACQUITY UPLC I-Class system). The purity was 98.66%.

[0059] Step 104: The pure product solution was concentrated to remove alcohol and then freeze-dried to obtain 1.68 g of ginsenoside Rc reference substance with a yield of 1.68% and a recovery rate of 92.3%.

[0060] In the above examples, the UPLC analysis conditions are as follows:

[0061] (1) Analytical column: BEH C18 (2.1 × 100 mm, 1.7 μm);

[0062] (2) Mobile phase A: acetonitrile, B: 0.1% phosphoric acid water;

[0063] (3) Flow rate: 0.4 mL / min;

[0064] (4) Gradient conditions: 0-7 min: 19% phase A, 7-11 min: 19-29% phase A, 11-14.5 min: 29% phase A, 14.5-20 min: 29-40% phase A, 20-25 min: 40-90% phase A.

[0065] (5) Column temperature: 40°C;

[0066] (6) Detection wavelength: 203 nm;

[0067] (7) Injection volume: 5 μL.

[0068] The separation of ginsenoside Rc monomer compounds is difficult, but after optimization by the present invention, the entire experimental operation process is simplified, efficient, and low in preparation cost, and finally a high-purity and high-yield ginsenoside Rc reference substance can be obtained.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Therefore, any equivalent changes or modifications made in accordance with the scope of the patent of the present invention shall fall within the scope of the patent claims of the present invention.

Claims

1. A method for preparing a ginsenoside Rc reference substance, characterized in that: The following steps are involved: a) soaking ginseng slices in 50% to 80% ethanol for 0.5 to 1 hour, decocting for 2 to 3 hours, and filtering the liquid; decocting the residue 2 to 3 times, each time for 2 to 3 hours, filtering the liquid, and combining and concentrating the liquids to obtain a ginseng extract solution; b) filtering the ginseng extract solution through a membrane and then passing it through a reverse phase chromatography column, eluting it with 2 to 4 column volumes of water and 4 to 6 column volumes of a 50 to 70% ethanol solution, and then eluting it with 2 to 5 column volumes of a 70 to 95% ethanol solution, collecting the eluate, concentrating it, and drying it to obtain a ginsenoside total saponin component sample; c) Add 25%-35% ethanol solution to a sample of ginsenoside components and stir with sonication until completely dissolved. Filter through a 0.45 μm organic filter membrane and pass through a reversed-phase column packed with 30-60 μm particles. Elute using a linear gradient of ethanol solution concentration from 25% to 90%. Finely cut and collect fractions for UPLC analysis. Combine the target fractions containing ginsenoside Rc to form the crude fraction. d) connecting a reverse phase chromatography column filled with 5-10 μm particle size filler to a preparative chromatography system, passing the crude fraction through the chromatography column for purification via a preparative chromatography system pump, isocratically eluting with 50%-60% ethanol, collecting fractions according to chromatographic peaks, performing UPLC analysis, and combining the target fractions containing ginsenoside Rc to obtain the crude ginsenoside Rc solution; e) connecting a hydrophilic chromatographic column with 5-10 μm particle size filler to a preparative chromatographic system, passing the crude product solution through the chromatographic column through a preparative chromatographic system pump for purification, and isocratically eluting with 80%-70% ethanol aqueous solution to obtain a pure ginsenoside Rc solution; f) concentrating the pure product solution to remove alcohol and freeze-drying to obtain ginsenoside Rc reference substance; The reverse phase chromatography column filler in step c) and step d) is a silica gel reverse phase C18 filler, and the hydrophilic chromatography column filler in step e) is a filler with polar groups bonded to the silica gel surface, wherein the bonded polar groups are diol groups, amides or zwitterions.

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