A preparation method and application of American ginseng saponin components

Abstract

This invention discloses a method for preparing and applying ginsenosides from American ginseng. The method involves extracting crude American ginseng powder with ethanol to obtain total ginsenosides. The total ginsenosides are then separated and purified using gradient countercurrent chromatography, followed by further separation and purification using liquid chromatography. This yields nine monomers with individual sample purity exceeding 95%, including ginsenoside Rg1, acetylginsenoside Rg1, ginsenoside Rb1, Rc, Rg2, Rb3, American ginsenoside R1, ginsenoside Rd, gypenosides XVII, and notoginsenoside Fd. This application can effectively extract ginsenosides from American ginseng and separate each component, resulting in high purity of each monomer.

Description

Technical Field

[0001] The invention relates to a preparation method and application of American ginseng saponin components. Background Art

[0002] Panax quinquefolius Panax quinquefolium The dried root of L. has the functions of replenishing qi and nourishing yin, clearing away heat and promoting body fluid. It is used for qi deficiency and yin deficiency, deficiency heat and fatigue, cough and asthma, sputum and blood, internal heat and thirst, dry mouth and throat, etc., and is widely distributed in Shandong, Hebei, Jilin and other places in my country. Modern pharmacological studies have shown that American ginseng has multiple pharmacological activities such as lowering blood sugar, regulating immunity, anti-fatigue, and neuroprotection. In addition, American ginseng has been included in the pilot material management of both food and Chinese medicinal materials, and is about to enter the food field as a raw material. It has been widely used in functional foods, daily chemicals, etc. Saponin components in American ginseng are its main active substances. For example, the Chinese Pharmacopoeia stipulates that the content of ginsenosides Rg1, Re and Rb1 should not be less than 2.0%. At the same time, the content of ginsenosides Rc, Rg2, Rb3 and other components in American ginseng is relatively high, and has special pharmacological effects. Therefore, how to efficiently prepare high-purity ginsenoside monomers is of great significance for activity screening, product development, and quality control. The preparation of saponin components in American ginseng usually adopts traditional preparation methods such as polyamide chromatography, silica gel column chromatography and Sephadex LH-20, which have the disadvantages of long separation time, poor reproducibility, severe dead adsorption, and large solvent consumption. Preparative liquid chromatography has the advantages of high separation efficiency and good reproducibility, but for samples with more complex components, this method has low separation efficiency and strict sample pretreatment.

[0003] Currently, there is no method that can separate and purify the saponin components contained in American ginseng. Summary of the invention

[0004] The invention provides a preparation method and application of American ginseng saponin components, and solves the technical problem of separating and purifying the saponin components contained in American ginseng.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A method for preparing American ginsenoside components is carried out according to the following steps:

[0007] 1) Preparation of total saponins of American ginseng: Weigh 1 part of American ginseng crude powder, add ethanol 8 times the mass of American ginseng crude powder, heat to 100℃ and reflux extract twice, each time for 2 hours, combine the two extracts, recover the ethanol under reduced pressure, evaporate the sample to extract, add water 1 times the mass of American ginseng crude powder to disperse, extract 3 times with petroleum ether equal to the volume of water, then extract 3 times with n-butanol equal to the volume of water, concentrate the n-butanol extract under reduced pressure, and freeze-dry to obtain total saponins of American ginseng;

[0008] 2) Gradient countercurrent chromatography to separate and purify total saponins of American ginseng: the stationary phase was water saturated with ethyl acetate, the mobile phase A was ethyl acetate saturated with water, and the mobile phase B was n-butanol saturated with water. The gradient flow rate was set to 0 min, 90%A; 180 min, 75%A; 190 min, 60%A; 300 min, 60%A. The column volume of the high-speed countercurrent chromatography was 200-400, the sample load was 50-400 mg, the rotation speed was 600-1000 rpm, the flow rate was 1-10 mL / min, and the detector was an evaporative light detector. Two monomers of ginsenoside Re and Rb1, and four mixed components were separated at one time.

[0009] Liquid chromatography separation: The four mixed components were separated and purified by liquid chromatography. The separation and purification conditions were as follows: acetonitrile-water as the mobile phase, flow rate of 3.0 mL / min, 50 min, detection wavelength of 203 nm, and ginsenoside Rg1, acetyl ginsenoside Rg1, ginsenosides Rb1, Rc, Rg2, Rb3, American ginsenoside R1, ginsenoside Rd, gypenosides X VII and notoginsenoside Fd9 were obtained;

[0010] The fineness of the American ginseng powder is 50-200 mesh;

[0011] The ethanol is a 95-98% ethanol aqueous solution;

[0012] The volume ratio of acetonitrile to water is 36:64.

[0013] The column volume of the high-speed countercurrent chromatograph is 300 mL; the sample loading amount is 150 mg; the rotation speed is 800 rpm; and the flow rate is 2.0 mL / min.

[0014] A method for analyzing saponins of American ginseng, liquid phase conditions: C 18 column, UV detection wavelength 203 nm, column temperature: 25°C, flow rate: 1.0 mL / min, injection volume: 10 μL, elution solvents are water (A) and acetonitrile (B), gradient conditions are: 0 min, 22% acetonitrile; 15 min, 22% acetonitrile; 25 min, 60% acetonitrile; 50 min, 60% acetonitrile;

[0015] The C 18 The columns were 250 mm × 4.6 mm id, 2.5 μm.

[0016] An application of American ginsenoside components, wherein the prepared American ginsenoside is applied to anti-oxidation or lowering blood sugar.

[0017] The invention has the following beneficial technical effects:

[0018] 1. This patent fully considers countercurrent chromatography as an enrichment method, and innovatively adopts gradient countercurrent chromatography mode, which greatly improves the separation efficiency. At the same time, combined with preparative liquid chromatography technology, the rapid preparation of the main saponin components in American ginseng is achieved. At the same time, the prepared saponin components are screened for activity, providing technical support for their development in functional foods.

[0019] 2. The preparation method of the present invention utilizes linear gradient countercurrent chromatography for wide K D The valuable compounds are separated and purified, and finally 11 monomer compounds are separated by preparative liquid chromatography. The cost is lower than the existing technology, the operation is simple, the efficiency is high, and a large number of American ginseng saponin monomer compounds with a purity greater than 98% can be separated and prepared. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the process flow chart of Example 1;

[0021] Figure 2 This is the linear gradient countercurrent chromatogram of total saponins of American ginseng in Example 1;

[0022] Figure 3 This is the liquid phase diagram after linear gradient countercurrent separation of total saponins of American ginseng in Example 1;

[0023] Figure 4 This is the liquid phase diagram of the total saponin monomer of American ginseng in Example 1;

[0024] Figure 5 This is a structural diagram of the isolated monomer of American ginsenoside in Example 1;

[0025] Figure 6 This is the antioxidant result diagram of the monomer in Example 1;

[0026] Figure 7 This is a graph showing the hypoglycemic results of the monomer in Example 1;

[0027] Figure 8 The linear gradient countercurrent chromatogram in Comparative Example 1;

[0028] Fig. 9 It is the linear gradient countercurrent chromatogram in Comparative Example 2. DETAILED DESCRIPTION

[0029] The present invention is further described below with reference to specific examples.

[0030] like Figure 1 A preparation method flow chart of an efficient preparation method for high-purity saponin components of American ginseng.

[0031] 1. Preparation of Total Saponins from American Ginseng

[0032] Preparation of total saponins of American ginseng: Weigh an appropriate amount of American ginseng coarse powder, and extract it twice with 8 times the amount of 95% ethanol under heating and reflux, each time for 2 hours. Combine the two extracts, recover the ethanol under reduced pressure until there is no alcohol taste, add an appropriate amount of water to disperse, and extract it three times with equal volumes of petroleum ether and n-butanol respectively. Concentrate the n-butanol extract under reduced pressure and freeze-dry to obtain total saponins of American ginseng.

[0033] 2. Separation and purification of total saponins from American ginseng using gradient countercurrent chromatography

[0034] The stationary phase was water saturated with ethyl acetate, the mobile phase A was ethyl acetate saturated with water, the mobile phase B was n-butanol saturated with water, the column volume of the high-speed countercurrent chromatography was 300 mL, the sample load was 150 mg, the rotation speed was 800 rpm, the water phase was the stationary phase, the flow rate was 2.0 mL / min, and the detector was an evaporative light detector.

[0035] The specific operation steps are as follows: put 800 mL of ethyl acetate and 400 mL of water into a separation funnel, shake vigorously, let the solution stand for a few minutes, separate into two phases, the upper organic phase is mobile phase A, and the lower aqueous phase is stationary phase. Mix 800 mL of n-butanol and 100 mL of water in a separation funnel and separate into two phases, the upper organic phase is mobile phase B. The sample solution is 150 mg of total saponins of American ginseng dissolved in 5 mL of mobile phase A and 5 mL of stationary phase for standby use. A high-speed countercurrent chromatograph is used, which is composed of a plunger pump, an injection valve, an evaporative light detector, and a chromatographic separation column (a spiral column formed by winding multiple layers of polytetrafluoroethylene tubes, with a capacity of 300 mL). First, put the injection valve in the injection state, fill the chromatographic separation column with a pump at a certain flow rate, and stop the pump. Turn on the speed controller to rotate the chromatographic separation column of the high-speed flow chromatograph forward and reverse. When the speed reaches 800 rpm, pump in mobile phase A at a flow rate of 2.0 mL / min. After equilibrium, inject it into the liquid storage tube of the injection valve of the countercurrent chromatograph with a syringe, rotate the injection valve to the column connection state, and allow the sample to enter the chromatographic separation column. Set the pump gradient flow rate to 0 min, 90%A; 180 min, 75%A; 190 min, 60%A; 300 min, 60%A, and perform elution. According to the evaporative light detector ( Figure 2 ) receives the target component and obtains two monomers of ginsenoside Re and Rb1 and four mixed components.

[0036] 3. Preparative HPLC Separation and Purification and HPLC Analysis

[0037] The above four mixed components were subjected to secondary preparation by preparative liquid chromatography, and the mobile phase was acetonitrile-water (36:64, v / v), flow rate of 3.0 mL / min, 50 min, detection wavelength of 203 nm, and 11 monomers including ginsenoside Rg1, ginsenoside Re, acetyl ginsenoside Rg1, ginsenoside Rb1, Rc, Rg2, Rb3, American ginsenoside R1, ginsenoside Rd, gypenosyl saponin X VII, and notoginsenoside Fd were obtained, and the purity of the monomers was higher than 95%.

[0038] The separated substances were analyzed by high performance liquid chromatography. The liquid phase conditions were: C 18 Column (250 mm × 4.6 mm id, 2.5 μm), UV detection wavelength 203 nm, column temperature: 25℃, flow rate: 1.0 mL / min, injection volume: 10 μL, elution solvents are water (A) and acetonitrile (B), gradient conditions are: 0 min, 22% acetonitrile; 15 min, 22% acetonitrile; 25 min, 60% acetonitrile; 50 min, 60% acetonitrile.

[0039] 4. Structural analysis

[0040] The separated ginsenosides were detected and identified by ESI-MS and MS-MS. The mass spectrometry conditions were as follows: m / z 100-1500, ion source temperature 300℃, spray voltage 4 kV, cone voltage 40 V, carrier gas flow rate 6 L / min, positive and negative ions were detected, and the measured data are as follows:

[0041] Compound 1: retention time 11.87 min, [M - H] - m / z 799.4029, [M + HCOOH - H] - m / z 845.4017, molecular formula C 42 H 72 O 14 MS / MS result: 637.3699 [M - Glc - H] - , 619.3579 [M - Glc -H2O - H] - , 475.3307 [M - 2Glc - H] - , which was identified as ginsenoside Rg1.

[0042] Compound 2: retention time 11.90 min, [M - H] - m / z 945.4473, [M + HCOOH - H]- m / z 991.4480, molecular formula C 48 H 82 O 18 .MS / MS results: m / z 991.4480 [M + HCOOH - H] - , 945.4473 [M -H] - , 799.4137 [M - Rha - H] - , 783.4070 [M - Glc - H] - , 637.3470 [M-Glc-Rha-H] - , 475.2196 [M-2Glc-Rha-H] - , which was identified as ginsenoside Re.

[0043] Compound 3: retention time 23.31 min, [M - H] - m / z 841.4098, [M + HCOOH - H] - 887.4125, molecular formula C 44 H 74 O 15 .MS / MS results: m / z 799.4037 [M - Ac - H] - ,781.3962 [M - Ac-H2 O - H] - , 799.4037 [M - Ac - H] - , 637.3688 [M-Glc-Ac-H] - , 618.3597 [M -Glc - Ac - H2O - H] - , which was identified as acetylginsenoside Rg1.

[0044] Compound 4: retention time 31.51 min, [M - H] - m / z 1107.4875, molecular formula C 54 H 92 O 23 .MS / MS results: m / z 1077.4875 [M - H]- , 945.4491 [M - Glc - H] - , 783.4108 [M - 2Glc - H] - , 765.4026 [M-2Glc-H2O-H] - , 621.3747 [M - 3Glc - H] - , which was identified as ginsenoside Rb1.

[0045] Compound 5: retention time 32.49 min, [M - H] - m / z 1077.4784, molecular formula C 54 H 90 O 22 .MS / MS results: m / z 945.4459 [M - Araf - H] - , 783.4114 [M-Glc-Araf-H] - , 765.4021 [M-Glc-Araf-H2O-H] - , 621.3747 [M-2Glc-Araf-H] - , which was identified as ginsenoside Rc.

[0046] Compound 6: retention time 32.68 min, [M - H] - m / z 783.4121, [M + HCOOH - H] - 829.2932, molecular formula C 42 H 72 O 13 .MS / MS results: m / z 637.3689 [M - Rha - H] - , 619.3593 [M -Rha- H2 O - H] - , 475.3319 [M-Glc-Rha-H] - , which was identified as ginsenoside Rg2.

[0047] Compound 7: retention time 33.90 min, [M - H] - m / z1077.4790, molecular formula C 53 H 90 O 22 .MS / MS results: m / z 945.4475 [M - Xyl - H] - , 783.4122 [M-Glc-Xyl-H] - , 765.4042 [M-Glc-Xyl-H2O-H] - , 621.3756 [M-2Glc-Xyl-H] - , 459.2348 [M-3Glc-Xyl-H] - , which was identified as ginsenoside Rb3.

[0048] Compound 8: retention time 34.85 min, [M - H] - m / z 1149.4921, molecular formula C 53 H 90 O 22 .MS / MS results: m / z 1149.4921 [M - H] - , 1107.4853 [M - Ac - H] - ,1089.4769 [M - Ac - H 2 O -H] - ,945.4479 [M-Glc-Ac-H] - , 783.4115 [M-2Glc-Ac-H] - , 765.4019 [M -3Glc - Ac - H2O - H] - , 621.3733 [M-3Glc-Ac-H] - , 459.2178 [M-4Glc-Ac-H] - , which was identified as American ginsenoside R1.

[0049] Compound 9: retention time 36.26 min, [M - H] - m / z 945.4481, [M + HCOOH - H] - 991.4487, molecular formula C 56 H94 O 24 .MS / MS results: m / z 783.4118 [M - Glc - H] - ,621.3744 [M-2Glc-H] - , 459.3369 [M - 3Glc - H] - , which was identified as ginsenoside Rd.

[0050] Compound 10: retention time 38.35 min, [M - H] - m / z 945.4481, [M + HCOOH - H] - 991.4487, molecular formula C 48 H 82 O 18 .MS / MS results: m / z 783.4124 [M - Glc - H] - , 621.3766 [M-2Glc-H] - , 459.3405 [M - 3Glc - H] - , which was identified as gypenoside X VII.

[0051] Compound 11: retention time 40.20 min, [M - H] - m / z 915.4396, [M + HCOOH - H] - 961.4414, molecular formula C 48 H 82 O 18 .MS / MS results: m / z 783.4097 [M - Xyl - H] - , 621.3753 [M-Glc-Xyl-H] - , 459.3389 [M-2Glc-Xyl-H] - , which was identified as notoginsenoside Fd.

[0052] 5. Antioxidant evaluation

[0053] Weigh 96 mg ABTS and 16.6 mg potassium persulfate in two 25 mL volumetric flasks, dilute to volume with water, mix ABTS solution and potassium persulfate solution in a ratio of 1:1, store in the dark for 12 h, and then dilute with PBS to an absorbance of 0.70 ± 0.02 at 734 nm to prepare ABTS working solution. Add 100 μL of 0.8 mM saponin sample solution or vitamin E solution or water to each well of a 48-well plate, then take 900 μL of ABTS working solution, and measure the absorbance at 734 nm using an enzyme reader after 6 min. The ABTS clearance rate represents the ABTS clearance ability, and the calculation formula is as follows:

[0054] ABTS clearance rate (%) = [1 - A 1 / A 0 ] × 100%

[0055] The antioxidant results showed that ( Figure 6 ), the clearance rates of ginsenosides Rb1 and Rd were 58% and 55% respectively, showing good antioxidant activity. This indicates that it has further application prospects in the field of auxiliary antioxidant functional products.

[0056] 6. Rating of hypoglycemic activity

[0057] The hypoglycemic activity was evaluated by the inhibition experiment of α-glucosidase. 10 mg of α-glucosidase was weighed and dissolved in 100 mL of 0.02 mol / L PBS to prepare a 1 U / mL α-glucosidase solution for use. First, 40 µL of sample (0.5 mM), acarbose or PBS were added to a 48-well plate and mixed with 80 µL of α-glucosidase (1 U / mL). After incubation at 37°C for 15 min, 80 µL of 2.5 mmol / L p -NPG substrate was used to start the reaction. The reaction was continued at 37°C for 15 min, and 320 µL of 1 mol / L sodium carbonate solution was added to terminate the reaction. The absorbance of p-nitrobenzene produced was immediately measured at a wavelength of 405 nm, and the inhibition rate of α-glucosidase was calculated using the following formula:

[0058] Inhibition rate (%) = [1 - A 1 / A 0 ] × 100%

[0059] in, A 0 is the absorbance of the blank control, A 1 is the absorbance of the sample or positive control. Using SPSS analysis software, IC was calculated based on the sample concentration and the inhibition rate of α-glucosidase activity. 50 Value, sample IC 50The lower the value, the stronger its ability to inhibit the enzyme, that is, the stronger its hypoglycemic activity.

[0060] The results of hypoglycemic treatment showed that ( Figure 7 ), the clearance rates of ginsenosides Rb1 and Rd were 64% and 61% respectively, showing good hypoglycemic activity. This indicates that it has further application prospects in the field of auxiliary hypoglycemic functional products.

[0061] Example 2

[0062] 1. Preparation of Total Saponins from American Ginseng

[0063] Weigh an appropriate amount of American ginseng powder, and extract it twice with 8 times the amount of 95% ethanol under heating and reflux, each time for 2 hours. Combine the two extracts, recover the ethanol under reduced pressure until there is no alcohol taste, add an appropriate amount of water to disperse, and extract it three times with equal volumes of petroleum ether and n-butanol respectively. Concentrate the n-butanol extract under reduced pressure and freeze-dry to obtain the total saponins of American ginseng.

[0064] 2. Separation and purification of saponin monomers from American ginseng using high-speed countercurrent chromatography

[0065] The stationary phase was water saturated with ethyl acetate, the mobile phase A was ethyl acetate saturated with water, the mobile phase B was n-butanol saturated with water, the column volume of the high-speed countercurrent chromatography was 300 mL, the sample load was 180 mg, the rotation speed was 700 rpm, the water phase was the stationary phase, the flow rate was 3.0 mL / min, 50 min, and the detector was an evaporative light detector.

[0066] The specific operation steps are as follows: put 800 mL of ethyl acetate and 400 mL of water into a separation funnel, shake vigorously, let the solution stand for a few minutes, separate into two phases, the upper organic phase is mobile phase A, and the lower aqueous phase is stationary phase. Mix 800 mL of n-butanol and 100 mL of water in a separation funnel and separate into two phases, the upper organic phase is mobile phase B. The sample solution is 180 mg of total saponins of American ginseng dissolved in 5 mL of mobile phase A and 5 mL of stationary phase for standby use. A high-speed countercurrent chromatograph is used, which is composed of a plunger pump, an injection valve, an evaporative light detector, and a chromatographic separation column (a spiral column formed by winding multiple layers of polytetrafluoroethylene tubes, with a capacity of 300 mL). First, put the injection valve in the injection state, fill the chromatographic separation column with a pump at a certain flow rate, and stop the pump. Turn on the speed controller to rotate the chromatographic separation column of the high-speed flow chromatograph forward and reverse. When the speed reaches 800 rpm, pump in the mobile phase A at a flow rate of 3.0 mL / min. After equilibrium, inject it into the liquid storage tube of the injection valve of the countercurrent chromatograph with a syringe, and rotate the injection valve to the column connection state to allow the sample to enter the chromatographic separation column. Set the pump gradient flow rate to 0 min, 90%A; 180 min, 75%A; 190 min, 60%A; 300 min, 60%A, and perform elution for a total of 50 min. According to the target component received by the evaporative light detector, two monomers containing ginsenoside Re and Rb1, and four mixed components were obtained.

[0067] 3. Preparative Liquid Chromatography Separation and Purification

[0068] The mixture was subjected to secondary preparation by preparative liquid chromatography, and the mobile phase was acetonitrile-water (40:60, v / v ), flow rate of 3.5 mL / min, detection wavelength of 203 nm, and 11 monomers including ginsenoside Rg1, ginsenoside Re, acetyl ginsenoside Rg1, ginsenoside Rb1, Rc, Rg2, Rb3, American ginsenoside R1, ginsenoside Rd, gypenosaphenosate X VII and notoginsenoside Fd were obtained, and the purity of the monomers was higher than 95%.

[0069] Comparative Example 1:

[0070] Conditions: The stationary phase is water saturated with ethyl acetate, the mobile phase A is ethyl acetate saturated with water, the mobile phase B is n-butanol saturated with water, the column volume of the high-speed countercurrent chromatography is 300 mL, the sample load is 100 mg, the speed is 800 rpm, the water phase is the stationary phase, the flow rate is 2.0 mL / min, and the detector is an evaporative light detector. The mobile phase ratio of the countercurrent chromatography is 0 min, 90% A; 240 min, 0% A; 360 min, 0% A, and the stationary phase retention rate is only 22%. Figure 8 .

[0071] Comparative Example 2:

[0072] Conditions: The stationary phase is water saturated with ethyl acetate, the mobile phase A is water saturated with ethyl acetate, the mobile phase B is water saturated with n-butanol, the column volume of the high-speed countercurrent chromatography is 300 mL, the sample load is 150 mg, the speed is 800 rpm, the water phase is the stationary phase, the flow rate is 2.0 mL / min, and the detector is an evaporative light detector. The mobile phase ratio of the countercurrent chromatography is 0 min, 90%A; 30 min, 80%A; 150 min, 75%A; 180 min, 55%A; 240 min, 40%A; 300 min, 40%A, the injection volume is 100 mg, 2.0 mL / min, and the stationary phase retention rate is only 16%. Fig. 9 .

[0073] Depend on Figure 8 and Fig. 9 It can be seen that the comparative examples (1 and 2) and Example 1 change the proportion of the solvent system in the solvent system, which results in a too fast peak elution time, resulting in the compounds not being effectively separated.

[0074] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without creative work are still within the scope of protection of the present invention.

Claims

1. A method for preparing American ginsenoside components, characterized in that: Follow these steps: 1) Preparation of total saponins of American ginseng: Weigh 1 part of American ginseng crude powder, add ethanol 8 times the mass of American ginseng crude powder, heat to 100℃ and reflux extract twice, each time for 2 hours, combine the two extracts, recover the ethanol under reduced pressure, evaporate the sample to extract, add water 1 times the mass of American ginseng crude powder to disperse, extract 3 times with petroleum ether equal to the volume of water, then extract 3 times with n-butanol equal to the volume of water, concentrate the n-butanol extract under reduced pressure, and freeze-dry to obtain total saponins of American ginseng; 2) Gradient countercurrent chromatography to separate and purify total saponins of American ginseng: the stationary phase was water saturated with ethyl acetate, the mobile phase A was ethyl acetate saturated with water, and the mobile phase B was n-butanol saturated with water. The gradient flow rate was set to 0 min, 90% A; 180 min, 75% A; 190 min, 60% A; 300 min, 60% A. The column volume of the high-speed countercurrent chromatography was 200-400, the sample load was 50-400 mg, the rotation speed was 600-1000 rpm, the flow rate was 1-10 mL / min, and the detector was an evaporative light detector. Two monomers of ginsenoside Re and Rb1, and four mixed components were separated at one time. Liquid chromatography separation: The four mixed components were separated and purified by liquid chromatography. The separation and purification conditions were as follows: acetonitrile-water as the mobile phase, flow rate of 3.0 mL / min, 50 min, detection wavelength of 203 nm, and ginsenoside Rg1, acetyl ginsenoside Rg1, ginsenosides Rb1, Rc, Rg2, Rb3, American ginsenoside R1, ginsenoside Rd, gypenosyl saponin X VII and notoginsenoside Fd9 were obtained; The fineness of the American ginseng powder is 50-200 mesh; The ethanol is a 95-98% ethanol aqueous solution; The volume ratio of acetonitrile to water is 36:64; The column volume of the high-speed countercurrent chromatograph is 300 mL; the sample loading amount is 150 mg; the rotation speed is 800 rpm; and the flow rate is 2.0 mL / min; Liquid phase conditions: C 18 column, UV detection wavelength 203 nm, column temperature: 25 °C, flow rate: 1.0 mL / min, injection volume: 10 μL, elution solvents: water (A) and acetonitrile (B), gradient conditions: 0 min, 22% acetonitrile; 15 min, 22% acetonitrile; 25 min, 60% acetonitrile; 50 min, 60% acetonitrile; The C 18 The columns were 250 mm × 4.6 mm id, 2.5 μm.

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