A method for simultaneously detecting the contents of ginsenoside Rg1, Rb1 and Re and sinesinine in ginseng

Abstract

The application provides a method for simultaneously detecting the contents of strophanthin and ginsenosides Rg1, Rb1 and Re in ginseng, and belongs to the technical field of high performance liquid chromatography detection. The method uses an ethanol aqueous solution as an extraction solvent, can simultaneously extract strophanthin and ginsenosides, avoids the problem that strophanthin cannot be effectively extracted by a soxhlet extraction, realizes effective separation of strophanthin and ginsenosides by selecting a mobile phase and adjusting gradient elution, reduces interfering substances, and realizes accurate detection of the contents of strophanthin and ginsenosides Rg1, Rb1 and Re.

Description

Technical Field

[0001] This invention belongs to the field of high performance liquid chromatography detection technology, and particularly relates to a method for simultaneously detecting the contents of ginsenosides Rg1, Rb1 and Re in ginseng. Background Technology

[0002] Ginseng is a medicinal herb native to the Changbai Mountain region. It possesses the effects of invigorating qi and blood, nourishing yin and promoting body fluids, strengthening the body, and delaying aging. It can also be used for various treatments, including anti-tumor, anti-fatigue, immune regulation, nervous system protection, and antibacterial properties. Ginsenosides are the main active ingredients in ginseng, with ginsenosides Rg1, Rb1, and Re being the main components. Therefore, the determination of ginsenosides Rg1, Rb1, and Re plays a crucial role in evaluating ginsenoside content and controlling ginseng quality.

[0003] Ginseng is widely used in clinical applications and health foods, but it also contains the toxic component strychnine. Strychnine has a central nervous system stimulant effect and is used as a central nervous system stimulant, limiting its use in athletes. Furthermore, strychnine has a narrow safety window; the toxic dose for adults is 5–10 mg, making its content determination crucial for the safe use of ginseng. Due to the complex composition of ginseng and the relatively low content of strychnine in ginseng powder, detection is difficult and inaccurate. In some testing attempts, various components with similar chemical structures in ginseng produce chromatographic peaks similar to strychnine during detection, making it difficult to accurately distinguish the strychnine signal. The detection methods described in the Chinese Pharmacopoeia (Part I, 2019 edition) cannot achieve accurate detection, hindering the quality evaluation and use control of ginseng in actual production. Summary of the Invention

[0004] The purpose of this invention is to provide a method for simultaneously detecting the content of strychnine and ginsenosides Rg1, Rb1, and Re in ginseng. The method provided by this invention can simultaneously detect the content of strychnine and ginsenosides Rg1, Rb1, and Re in ginseng, and has high detection accuracy.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for simultaneously detecting the contents of ginsenosides Rg1, Rb1, and Re in ginseng, comprising the following steps:

[0007] (1) Ginseng powder is mixed with an extraction solvent for extraction, and then the resulting liquid phase is dried to obtain ginseng extract; the extraction solvent is an aqueous ethanol solution;

[0008] (2) Mix the ginseng extract obtained in step (1) with methanol to obtain the test solution;

[0009] (3) The test solution obtained in step (2) is subjected to high performance liquid chromatography to detect the content of strychnine and ginsenosides Rg1, Rb1 and Re in ginseng;

[0010] The mobile phases used in the high-performance liquid chromatography (HPLC) detection are mobile phase A: an aqueous solution of phosphoric acid, and mobile phase B: acetonitrile; the mass concentration of phosphoric acid in the aqueous solution of phosphoric acid is 0.05–0.1%.

[0011] The elution conditions for the high-performance liquid chromatography detection are gradient elution, and the gradient elution is as follows:

[0012] 0–35 min, 80% mobile phase A, 20% mobile phase B;

[0013] 35–60 min, 80–20% mobile phase A, 20–80% mobile phase B;

[0014] 60–70 min, 20–80% mobile phase A, 80–20% mobile phase B.

[0015] Preferably, the volume content of ethanol in the aqueous ethanol solution in step (1) is 70-80%.

[0016] Preferably, in step (1), the mass ratio of ginseng powder to extraction solvent is 1:(25-35).

[0017] Preferably, the conditions for high performance liquid chromatography detection in step (3) include: a C18 column, a flow rate of 0.8 to 1.2 mL / min, a column temperature of 30 to 40 °C, a detection wavelength of 203 nm, and an injection volume of 8 to 12 μL.

[0018] Preferably, the conditions for high performance liquid chromatography detection in step (3) include: a C18 column, a flow rate of 1.0 mL / min, a column temperature of 35 °C, a detection wavelength of 203 nm, and an injection volume of 10 μL.

[0019] Preferably, the external standard method is used for high performance liquid chromatography detection in step (3).

[0020] Preferably, the mixed standard solution used in the external standard method is a methanol solution of strychnine, ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Re.

[0021] Preferably, the extraction in step (1) is ultrasonic extraction, wherein the ultrasonic extraction power is 200-300W, the frequency is 50kHz, and the time is 1-2h.

[0022] Preferably, after the extraction is completed in step (1), solid-liquid separation is performed, and the solid-liquid separation method is centrifugation.

[0023] This invention provides a method for simultaneously detecting the content of ginsenosides Rg1, Rb1, and Re in ginseng, comprising the following steps: (1) mixing ginseng powder with an extraction solvent for extraction, and then drying the resulting liquid phase to obtain ginseng extract; the extraction solvent is an aqueous ethanol solution; (2) mixing the ginseng extract obtained in step (1) with methanol to obtain a test solution; (3) performing high performance liquid chromatography on the test solution obtained in step (2) to obtain the content of ginsenosides Rg1, Rb1, and Re in ginseng. The content of e; the mobile phase for high-performance liquid chromatography (HPLC) detection is mobile phase A: phosphoric acid aqueous solution, and mobile phase B: acetonitrile; the mass concentration of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.1%; the elution conditions for HPLC detection are gradient elution, wherein the gradient elution is: 0-35 min, 80% mobile phase A, 20% mobile phase B; 35-60 min, 80-20% mobile phase A, 20-80% mobile phase B; 60-70 min, 20-80% mobile phase A, 80-20% mobile phase B. This invention uses an ethanol aqueous solution as the extraction solvent, which can simultaneously extract strychnine and ginsenosides, avoiding the ineffective extraction of strychnine by Soxhlet extraction; by selecting the mobile phase and adjusting the gradient elution, effective separation of strychnine and ginsenosides is achieved, interfering substances are reduced, and accurate detection of the contents of strychnine and ginsenosides Rg1, Rb1, and Re is achieved. The experimental results show that the method provided by this invention has the following linear ranges: strychnine (0.004–0.030 μg / mL), average recovery rate 97.95%, RSD 1.46%; ginsenoside Rg1 (0.08–0.60 μg / mL), average recovery rate 98.09%, RSD 1.26%; ginsenoside Rb1 (0.032–0.24 μg / mL), average recovery rate 98.92%, RSD 1.17%; and ginsenoside Re (0.20–1.50 μg / mL), average recovery rate 98.91%, RSD 1.57%. The repeatability RSD is less than 1%. Attached Figure Description

[0024] Figure 1 HPLC chromatograms of strychnine and ginsenosides Rg1, Rb1, and Re standards;

[0025] Figure 2 This is the HPLC chromatogram of four-year-old straight-grown ginseng in Example 1 of the present invention;

[0026] Figure 3 This is the HPLC chromatogram of ginseng transplanted from two different plants in Example 2 of the present invention. Detailed Implementation

[0027] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0028] The purity of all raw materials used in this invention is not particularly limited. However, this invention preferably uses chromatographically pure raw materials or raw materials with purity commonly used in the field of high-performance liquid chromatography.

[0029] This invention provides a method for simultaneously detecting the contents of ginsenosides Rg1, Rb1, and Re in ginseng, comprising the following steps:

[0030] (1) Ginseng powder is mixed with an extraction solvent for extraction, and then the resulting liquid phase is dried to obtain ginseng extract; the extraction solvent is an aqueous ethanol solution;

[0031] (2) Mix the ginseng extract obtained in step (1) with methanol to obtain the test solution;

[0032] (3) The test solution obtained in step (2) is subjected to high performance liquid chromatography (HPLC) to detect the content of strychnine and ginsenosides Rg1, Rb1, and Re in ginseng; the mobile phase for HPLC detection is mobile phase A: phosphoric acid aqueous solution and mobile phase B: acetonitrile; the mass concentration of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.1%; the elution conditions for HPLC detection are gradient elution, and the gradient elution is as follows:

[0033] 0–35 min, 80% mobile phase A, 20% mobile phase B;

[0034] 35–60 min, 80–20% mobile phase A, 20–80% mobile phase B;

[0035] 60–70 min, 20–80% mobile phase A, 80–20% mobile phase B.

[0036] This invention involves mixing ginseng powder with an extraction solvent for extraction, and then drying the resulting liquid phase to obtain ginseng extract.

[0037] This invention does not have any special requirements on the specifications of the ginseng powder; specifications of ginseng powder well known to those skilled in the art can be used. In one embodiment of this invention, the ginseng powder can be obtained by pulverizing ginseng using a pulverizer and then passing it through an 80-mesh sieve.

[0038] In this invention, the extraction solvent is an aqueous ethanol solution, and the volume content of ethanol in the aqueous ethanol solution is preferably 70-80%, more preferably 75-80%. As one embodiment of this invention, the volume content of ethanol in the aqueous ethanol solution can be 71%, 72%, 73%, 74%, 76%, 77%, 78%, 79%, or 80%. An ethanol volume content within the above range is beneficial for further improving the extraction efficiency of ginseng extract.

[0039] In this invention, the preferred mass ratio of ginseng powder to extraction solvent is 1:(25-35), more preferably 1:(27-32). As one embodiment of this invention, the mass ratio of ginseng powder to extraction solvent can be 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, or 1:34. A mass ratio of ginseng powder to extraction solvent within the above ranges is beneficial for further improving the extraction efficiency of ginseng extract.

[0040] In this invention, the extraction is preferably performed using ultrasonic extraction. The power of the ultrasonic extraction is preferably 200-300W, more preferably 230-270W; the frequency of the ultrasonic extraction is preferably 50kHz; and the extraction time is preferably 1-2 hours, more preferably 1.3-1.8 hours. In an embodiment of this invention, the ultrasonic extraction power is 250W, the frequency is 50kHz, and the time is 1.5 hours. Using ultrasonic extraction with the above parameter range is beneficial for further improving the extraction efficiency of ginseng extract.

[0041] In this invention, solid-liquid separation is preferably performed after the extraction is completed, and the preferred method for solid-liquid separation is centrifugation. This invention does not have special requirements for the centrifugation parameters; conventional centrifugation parameters in the art can be used. Centrifugation can simply and efficiently separate the extract from the residue, which is beneficial for further improving the extraction efficiency of ginseng extract.

[0042] The present invention does not have any particular requirements for the specific drying method, as long as the solvent in the liquid phase is removed.

[0043] After obtaining the ginseng extract, the present invention mixes the ginseng extract with methanol to obtain the test solution.

[0044] In this invention, the preferred ratio of methanol to ginseng powder is 100 mL:(1-3) g, more preferably 100 mL:1 g. A methanol-ginseng powder ratio within this range is beneficial for the separation of strychnine and ginsenosides Rg1, Rb1, and Re during gradient elution. As one embodiment of this invention, the methanol extract can be placed in a volumetric flask and diluted to volume with methanol.

[0045] After obtaining the test solution, the present invention performs high performance liquid chromatography on the test solution to obtain the content of ginsenosides and ginsenosides Rg1, Rb1 and Re in ginseng.

[0046] In this invention, the mobile phase for high-performance liquid chromatography (HPLC) detection is mobile phase A: an aqueous solution of phosphoric acid, and mobile phase B: acetonitrile; the mass concentration of phosphoric acid in the aqueous solution of phosphoric acid is 0.05-0.1%, preferably 0.05%. The use of the above-mentioned mobile phases in this invention facilitates the separation of strychnine and ginsenosides Rg1, Rb1, and Re from ginseng extract during gradient elution.

[0047] In this invention, the elution conditions for high-performance liquid chromatography detection are gradient elution, wherein the gradient elution is as follows:

[0048] 0–35 min, 80% mobile phase A, 20% mobile phase B;

[0049] 35–60 min, 80–20% mobile phase A, 20–80% mobile phase B;

[0050] Elution time: 60–70 min, 20–80% mobile phase A, 80–20% mobile phase B. Using the above elution conditions, this invention can separate strychnine and ginsenosides Rg1, Rb1, and Re from ginseng extract, improving the accuracy of detection.

[0051] In this invention, the preferred conditions for high-performance liquid chromatography (HPLC) detection include: a C18 column, a flow rate of 0.8–1.2 mL / min, a column temperature of 30–40 °C, a detection wavelength of 203 nm, and an injection volume of 8–12 μL; more preferably: a C18 column, a flow rate of 1.0 mL / min, a column temperature of 35 °C, a detection wavelength of 203 nm, and an injection volume of 10 μL. Using the above detection conditions for HPLC detection helps to further improve the accuracy of detection.

[0052] In this invention, the external standard method is preferably used for the high performance liquid chromatography detection; the mixed standard solution used in the external standard method is preferably a methanol solution of strychnine, ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Re.

[0053] This invention uses an ethanol-water solution as the extraction solvent, which can simultaneously extract strychnine and ginsenosides, avoiding the ineffective extraction of strychnine by Soxhlet extraction. By selecting the mobile phase and adjusting the gradient elution, effective separation of strychnine and ginsenosides is achieved, interfering substances are reduced, and the contents of strychnine and ginsenosides Rg1, Rb1, and Re are accurately detected.

[0054] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0055] Example 1

[0056] instrument

[0057] Liquid chromatograph (Shimadzu LC-20AT); electronic balance (Sartorius Scientific Instruments Co., Ltd.); ultrapure water system (Shanghai Gaosen Instrument Equipment Co., Ltd.).

[0058] Drug testing

[0059] Acetonitrile and methanol (chromatographic grade, Liaoning Quanrui Reagent Co., Ltd.), phosphoric acid (analytical grade, Tianjin Damao Chemical Reagent Factory), strychnine reference standard (Dest Biotechnology Co., Ltd., HPLC ≥ 98%), Rg1, Rb1, Re reference standards (Shanghai Baoman Biotechnology Co., Ltd., HPLC ≥ 98%).

[0060] A method for simultaneously detecting the contents of ginsenosides Rg1, Rb1, and Re in ginseng, the specific steps of which are as follows:

[0061] Preparation of the test solution

[0062] Four-year-old ginseng from the Changbai Mountain region was pulverized using a pulverizer and passed through an 80-mesh sieve. 1g of ginseng powder was accurately weighed and then 30g of 80% ethanol aqueous solution was added. The mixture was subjected to ultrasonic extraction (power 250W, frequency 50kHz, 1.5h). The supernatant was collected by centrifugation, dried to constant weight, and the residue was dissolved in methanol. The residue was transferred to a 100mL volumetric flask, diluted to volume with methanol, and shaken well to obtain the test solution.

[0063] Standard product testing

[0064] Prepare a mixed solution of standards with the following concentrations: strychnine 0.4 μg / mL, ginsenoside Rg1 8.0 μg / mL, ginsenoside Rb1 3.2 μg / mL, and ginsenoside Re 20.0 μg / mL. Perform chromatographic analysis on the standard solution under the above conditions to obtain the HPLC chromatogram of the standards, as shown below. Figure 1 As shown. From Figure 1 The peak elution patterns of strychnine and ginsenosides Rg1, Rb1, and Re can be observed (see the annotations in the figure), providing a reference for subsequent detection.

[0065] Preparation of mixed standard solutions

[0066] Accurately weigh 1 mg of strychnine, dissolve it in an appropriate amount of methanol, and dilute to 10 mL in a volumetric flask. Shake well. Weigh 4 mg of ginsenoside Rg1, 1.6 mg of ginsenoside Rb1, and 10 mg of ginsenoside Re, dissolve them separately in an appropriate amount of methanol, and dilute to 10 mL in a volumetric flask. Shake well to prepare standard solutions of strychnine and ginsenosides Rg1, Rb1, and Re. Accurately transfer the same volume of each standard solution to the same volumetric flask, add an appropriate amount of methanol, and dilute to 10 mL. Shake well. The volumes of each standard solution transferred each time are 0.2 mL, 0.5 mL, 0.8 mL, 1 mL, 1.2 mL, and 1.5 mL, respectively, to obtain a series of mixed standard solutions.

[0067] Chromatographic conditions:

[0068] Column: XBridge C 185μm (4.6×250nm);

[0069] Mobile phase: Acetonitrile (B) - 0.05% phosphoric acid (A);

[0070] Gradient elution conditions:

[0071] 0–35 min, 80% mobile phase A, 20% mobile phase B;

[0072] 35–60 min, 80–20% mobile phase A, 20–80% mobile phase B;

[0073] 60–70 min, 20–80% mobile phase A, 80–20% mobile phase B;

[0074] Flow rate: 1.0 mL / min;

[0075] Column temperature: 35℃;

[0076] Detection wavelength: 203nm;

[0077] Injection volume: 10 μL.

[0078] Examining linear relationships

[0079] The series of mixed standard solutions were filtered through a 0.22 μm filter membrane, and 10 μL of each solution was injected into the high-performance liquid chromatograph. Each concentration of the mixed standard solution was injected in triplicate, and the average peak area S was calculated. 均 The relationship between the injection volume and the linear relationship of strychnine, ginsenosides Rg1, Rb1, and Re was obtained. The experimental data and results are shown in Tables 1-5.

[0080] Table 1. Linear Relationship Data of Shi Ning

[0081] 0.4 0.004 24546 1.0 0.010 79337 1.6 0.016 131240 2.0 0.020 164970 2.4 0.024 200324 3.0 0.030 298437

[0082] Table 2. Linearity data for ginsenoside Rg1

[0083] 8 0.08 25922 20 0.20 41986 32 0.32 59814 40 0.40 71586 48 0.48 83539 60 0.60 101154

[0084] Table 3. Linearity data for ginsenoside Rb1

[0085]

[0086]

[0087] Table 4. Linearity data of ginsenoside Re

[0088] 20 0.20 35350 50 0.50 66417 80 0.80 96961 100 1.00 119074 120 1.20 137743 150 1.50 168629

[0089] Table 5. Record of linear relationship investigation of strychnine, ginsenosides Rg1, Rb1, and Re.

[0090] Shi Ning y = 170988x - 6929.7 0.004～0.030 0.9995 Ginsenoside Rg1 y = 1454.9x + 13564 0.08～0.60 0.9997 Ginsenoside Rb1 y = 1156.8x - 243.52 0.032～0.24 0.9996 Ginsenoside Re y = 1025.2x + 15159 0.20～1.50 0.9998

[0091] The above linear relationship examination clearly shows the peak area of ​​each component at different concentrations and the good linearity, laying the foundation for accurate content determination in the future.

[0092] Precision test

[0093] Take the mixed standard solution, filter it through a 0.22 μm filter membrane, and inject 10 μL into the high-performance liquid chromatograph. Perform six parallel injections per day, recording the peak areas S1–S6 of strychnine, ginsenosides Rg1, Rb1, and Re, and calculating their RSD values. Continue this method, injecting once daily for six consecutive days, recording the peak areas S1–S6 of strychnine, ginsenosides Rg1, Rb1, and Re, and calculating their RSD values. The RSD values ​​of the peak areas of strychnine, ginsenosides Rg1, Rb1, and Re are all less than 2%, indicating good instrument precision. The experimental results are shown in Tables 6 and 7.

[0094] Table 6 Precision Assessment Record Sheet within 6 Days

[0095] Shi Ning 53337 53456 53534 53227 53478 53456 0.21 Rg1 59824 59523 58532 59761 59338 59712 0.81 Rb1 14665 14683 14713 14801 14633 14654 0.41 Re 96963 96872 96793 96981 96321 96478 0.28

[0096] Table 7. Daytime Precision Examination Record Sheet

[0097]

[0098]

[0099] The results of the precision test show that the instrument used in this invention can stably provide reliable data under this detection method, ensuring the accuracy and repeatability of the detection.

[0100] Repeated examination

[0101] Six mixed standard solutions of the same concentration were taken, filtered through a 0.22 μm filter membrane, and each solution was injected three times, with 10 μL injected each time. The peak areas S of strychnine, ginsenosides Rg1, Rb1, and Re were measured. 均 The content of each compound was calculated using the external standard method, and the RSD values ​​were calculated. The RSD values ​​of strychnine, ginsenosides Rg1, Rb1, and Re were all less than 2%, indicating good reproducibility of the method. The experimental results are shown in Table 8.

[0102] Table 8. Record of repeatability studies of ginsenosides, ginsenosides Rg1, Rb1, and Re.

[0103] 1 82078 83535 21933 137746 2 82834 83312 21845 137658 3 82535 83478 22019 137749 4 82034 83557 21722 137321 5 82678 83678 21843 137901 6 82176 83456 21698 137589 RSD% 0.41 0.15 0.56 0.14

[0104] Repeatability tests further verified that the method of the present invention can stably determine the content of each component in multiple operations for different sample preparations, and has good repeatability.

[0105] Stability test

[0106] Six mixed standard solutions of the same concentration were filtered through a 0.22 μm filter membrane and injected every 2 hours for 12 consecutive hours, with each injection being 10 μL. The retention times (t) and peak areas (S) of strychnine, ginsenosides Rg1, Rb1, and Re were recorded, and the RSD values ​​were calculated. The results showed that the RSD values ​​of the retention times (t) and peak areas (S) of strychnine, ginsenosides Rg1, Rb1, and Re were all less than 2%, indicating good stability of the sample solutions within 12 hours. The experimental results are shown in Tables 9 and 10.

[0107] Table 9. Record of Stability Study of Ginsenoside Rg1 and Ginsenoside Rg1

[0108]

[0109]

[0110] Table 10: Record of Stability Study of Ginsenosides Rb1 and Re

[0111] 1 9188 51.330 66417 33.523 2 9156 51.567 66314 33.345 3 9233 51.459 66123 33.149 4 9245 51.743 65789 33.713 5 9102 51.298 67251 33.534 6 9244 51.345 66934 33.632 RSD% 0.63 0.33 0.81 0.61

[0112] The stability test results proved that the prepared sample solution is stable within a certain time range, which can ensure the reliability of the detection data and prevent large fluctuations due to time factors.

[0113] Recovery rate of spiking

[0114] Six portions of ginseng powder (passed through an 80-mesh sieve) were used to prepare six sample solutions according to the method for preparing the test solution. Each solution was filtered through a 0.22 μm filter membrane, and each sample was injected three times, with 10 μL injected each time. The peak areas of strychnine, ginsenosides Rg1, Rb1, and Re were measured, and the contents, recoveries, average recoveries, and RSD values ​​were calculated. The results showed that the RSD values ​​of the recoveries were all less than 2%, indicating that the method had good recovery. The experimental results are shown in Tables 11–14.

[0115] Table 11 Record of Recovery Rate of Strychnine by Spiking

[0116]

[0117] Table 12 Record of Recovery Rate of Ginsenoside Rg1

[0118]

[0119] Table 13 Record of Recovery Rate of Ginsenoside Rb1

[0120]

[0121] Table 14. Record of Recovery Rate of Ginsenoside Re

[0122]

[0123] The spiking recovery test demonstrated that the method of the present invention can accurately recover and determine the content after adding standard substances to the sample, which further verified the accuracy and reliability of the method.

[0124] Content determination

[0125] Take three aliquots of the test solution, filter them through a 0.22 μm filter membrane, and inject them separately into a high-performance liquid chromatograph (HPLC), injecting 10 μL each time. Obtain the HPLC chromatograms, as shown below. Figure 2 As shown. According to Figure 2 Record the peak positions (marked in the figure) and peak areas of ginsenoside Rg1, ginsenoside Rb1, and ginsenoside Re. Calculate the contents of ginsenoside Rg1, Rb1, and Re using the external standard method, and take the average value of the three test samples.

[0126] Example 2

[0127] Two transplanted ginseng plants were used to prepare the test solution according to the method in Example 1, and the solution was detected by high-performance liquid chromatography (HPLC). The HPLC chromatogram was obtained as follows: Figure 3 As shown. According to Figure 3The peak positions (marked in the figure) and peak areas of strychnine, ginsenoside Rg1, ginsenoside Rb1, and ginsenoside Re were recorded. The contents of strychnine, ginsenoside Rg1, Rb1, and Re were calculated using the external standard method, and the average value of the three test samples was taken. The results are shown in Table 15.

[0128] Table 15. Content records of ginsenosides Rg1, Rb1, and Re in different embodiments.

[0129] Example 1 0.135 3.487 1.388 4.642 Example 2 0.064 2.751 2.005 3.419

[0130] Comparative Example 1

[0131] According to the determination of ginseng content in the Chinese Pharmacopoeia (Part I, 2019 edition):

[0132] 1. Chromatographic conditions

[0133] Mobile phase: Acetonitrile (A) - Water (B);

[0134] Gradient elution conditions:

[0135] 0–35 min, 19% A;

[0136] 35–55 min, 19–29% A;

[0137] 55–70 min, 29% A;

[0138] 70–100 min, 29–40% A;

[0139] Detection wavelength: 203nm;

[0140] Flow rate: 1 mL / min;

[0141] Column temperature: 35℃;

[0142] Injection volume: 10 μL.

[0143] 2. Preparation of reference solution

[0144] Accurately weigh ginsenoside Rg1 reference standard, ginsenoside Re reference standard and ginsenoside Rb1 reference standard, add methanol to prepare a mixed solution containing 0.2 mg of each per 1 mL, shake well and the solution is obtained.

[0145] 3. Preparation of the test solution

[0146] Accurately weigh 1g of the powder (passed through a No. 4 sieve), place it in a Soxhlet extractor, add chloroform and heat under reflux for 3 hours. Discard the chloroform solution, evaporate the solvent from the residue, and transfer it along with the filter paper tube into a 100mL Erlenmeyer flask. Accurately add 50mL of water-saturated n-butanol, seal tightly, and let stand overnight. Sonicate (power 250W, frequency 50kHz) for 30 minutes, filter, discard the initial filtrate, accurately measure 25mL of the subsequent filtrate, evaporate it to dryness in an evaporating dish, dissolve the residue in methanol and transfer it to a 5mL volumetric flask, dilute with methanol to the mark, shake well, filter, and collect the subsequent filtrate to obtain the final product.

[0147] 4. Content determination

[0148] Three test solutions were taken, filtered through a 0.22 μm filter membrane, and injected into a high-performance liquid chromatograph (HPLC) at a rate of 10 μL per injection. The peak areas were recorded, and the contents of strychnine, ginsenosides Rg1, Rb1, and Re were calculated using the external standard method. The results are shown in Table 16.

[0149] Table 16. Content records of ginsenosides Rg1, Rb1, and Re according to pharmacopoeia methods.

[0150] Four-year straight-grown ginseng Not detected 3.283 1.294 4.442 Second transplant ginseng Not detected 2.631 1.934 3.309

[0151] Comparing Tables 15 and 16, it can be seen that the method of the present invention can detect the content of strychnine, while the pharmacopoeia standard method cannot. At the same time, the ginsenoside content detected by the method of the present invention is higher than that detected by the pharmacopoeia standard method, and the results are closer to the true value. Moreover, the deviation between the two methods meets the requirements, which fully demonstrates the advantages of the method of the present invention in the determination of the content of ginseng-related components.

[0152] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for simultaneously detecting the contents of ginsenosides Rg1, Rb1, and Re in ginseng, comprising the following steps: (1) Ginseng powder is mixed with an extraction solvent for extraction, and then the resulting liquid phase is dried to obtain ginseng extract; the extraction solvent is an aqueous ethanol solution; (2) Mix the ginseng extract obtained in step (1) with methanol to obtain the test solution; (3) The test solution obtained in step (2) is subjected to high performance liquid chromatography to detect the content of strychnine and ginsenosides Rg1, Rb1 and Re in ginseng; The mobile phases used in the high-performance liquid chromatography (HPLC) detection are mobile phase A: aqueous phosphoric acid solution, and mobile phase B: acetonitrile; the mass concentration of phosphoric acid in the aqueous phosphoric acid solution is 0.05~0.1%. The elution conditions for the high-performance liquid chromatography detection are gradient elution, and the gradient elution is as follows: 0~35min, 80% mobile phase A, 20% mobile phase B; 35~60 min, 80~20% mobile phase A, 20~80% mobile phase B; 60-70 min, 20-80% mobile phase A, 80-20% mobile phase B; In step (1), the volume content of ethanol in the aqueous ethanol solution is 70-80%. The extraction in step (1) is ultrasonic extraction, with a power of 200~300W, a frequency of 50kHz, and a time of 1~2h. The conditions for high performance liquid chromatography detection in step (3) include: a C18 column, a flow rate of 0.8~1.2 mL / min, a column temperature of 30~40℃, a detection wavelength of 203 nm, and an injection volume of 8~12 μL; In step (3), the external standard method is used for high performance liquid chromatography detection.

2. The method according to claim 1, characterized in that, In step (1), the mass ratio of ginseng powder to extraction solvent is 1:(25~35).

3. The method according to claim 1, characterized in that, The conditions for high performance liquid chromatography detection in step (3) include: a C18 column, a flow rate of 1.0 mL / min, a column temperature of 35 °C, a detection wavelength of 203 nm, and an injection volume of 10 μL.

4. The method according to claim 1, characterized in that, The ratio of methanol to ginseng powder in step (1) in step (2) is 100mL:(1~3)g.

5. The method according to claim 1, characterized in that, The external standard method uses a mixed standard solution consisting of strychnine, ginsenoside Rg1, ginsenoside Rb1, and ginsenoside Re in methanol.

6. The method according to claim 1, characterized in that, After the extraction is completed in step (1), solid-liquid separation is performed, and the solid-liquid separation method is centrifugation.

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