Method for simultaneously detecting contents of strychnine and ginsenosides Rg1, Rb1 and Re in ginseng

Abstract

The invention provides a method for simultaneously detecting the contents of strychnine and ginsenosides Rg1, Rb1 and Re in ginseng, and belongs to the technical field of high performance liquid chromatography detection. The ethanol aqueous solution is used as an extraction solvent, strychnine and ginsenoside can be extracted at the same time, and the problem that strychnine cannot be effectively extracted through Soxhlet extraction is avoided; through selection of a mobile phase and adjustment of gradient elution, effective separation of strychnine and ginsenoside is realized, interfering substances are reduced, and accurate detection of contents of strychnine and ginsenoside Rg1, Rb1 and Re is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of high performance liquid chromatography detection, and in particular relates to a method for simultaneously detecting the contents of strychnine and ginsenosides Rg1, Rb1 and Re in ginseng. Background Art

[0002] Ginseng is a special medicinal material produced in Changbai Mountain. It has the effects of replenishing qi and blood, nourishing yin and promoting body fluid, strengthening physical fitness, and delaying aging. It can also be used for anti-tumor, anti-fatigue, regulating immunity, protecting the nervous system, and antibacterial treatment. Ginsenosides are the main active ingredients in ginseng, among which ginsenosides Rg1, Rb1, and Re are the main components of ginsenosides. Therefore, the determination of ginsenosides Rg1, Rb1, and Re plays a vital role in evaluating the content of ginsenosides and controlling the quality of ginseng.

[0003] Ginseng is widely used in clinical applications and health foods, and ginseng also contains the toxic ingredient strychnine. Strychnine has a central nervous system stimulant effect and is used as a central nervous system stimulant, which restricts its use by athletes. Moreover, the safety window of strychnine is narrow, and the adult toxic dose is 5 to 10 mg. The determination of its content is related to the safety of ginseng use. Due to the complex composition of ginseng and the relatively small content of strychnine in ginseng powder, the detection is difficult and inaccurate. In some detection attempts, various components with similar chemical structures in ginseng will produce chromatographic peaks similar to strychnine during detection, making it difficult to accurately distinguish the signal of strychnine. The detection method recorded in the "Chinese Pharmacopoeia" (Volume 1 2019 edition) cannot achieve the purpose of accurate detection, which is not conducive to the quality evaluation and use control of ginseng in actual production. Summary of the invention

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

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

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

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

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

[0009] (3) subjecting the test solution obtained in step (2) to high performance liquid chromatography to obtain the contents of strychnine and ginsenosides Rg1, Rb1, and Re in ginseng;

[0010] The mobile phases for the HPLC detection are 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%;

[0011] The elution condition of the high performance liquid chromatography detection is gradient elution, and the gradient elution is:

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

[0015] Preferably, the volume content of ethanol in the ethanol aqueous 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: the chromatographic column is a C18 chromatographic column, the flow rate is 0.8-1.2 mL / min, the column temperature is 30-40° C., the detection wavelength is 203 nm, and the injection volume is 8-12 μL.

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

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

[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, and the power of the ultrasonic extraction is 200-300W, the frequency is 50kHz, and the time is 1-2h.

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

[0023] The present invention provides a method for simultaneously detecting the contents of strychnine and ginsenosides Rg1, Rb1 and Re in ginseng, comprising the following steps: (1) mixing ginseng powder with an extraction solvent for leaching, and then drying the obtained liquid phase to obtain a ginseng extract; the extraction solvent is an ethanol aqueous solution; (2) mixing the ginseng extract obtained in the step (1) with methanol to obtain a test solution; (3) subjecting the test solution obtained in the step (2) to high performance liquid chromatography detection to obtain strychnine and ginsenosides Rg1, Rb1 and Re in ginseng. e content; the mobile phase for the high performance liquid chromatography 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 condition for the high performance liquid chromatography detection is gradient elution, and the gradient elution is: 0-35min, 80% mobile phase A, 20% mobile phase B; 35-60min, 80-20% mobile phase A, 20-80% mobile phase B; 60-70min, 20-80% mobile phase A, 80-20% mobile phase B. The present invention uses ethanol aqueous solution as the extraction solvent, can extract strychnine and ginsenosides at the same time, avoids the inability of Soxhlet extraction to effectively extract strychnine; through the selection of the mobile phase and the adjustment of the gradient elution, the effective separation of strychnine and ginsenosides is achieved, the interfering substances are reduced, and the accurate detection of the contents of strychnine and ginsenosides Rg1, Rb1, and Re is achieved. The test results show that, according to the method provided by the present invention, the linear range of strychnine is 0.004-0.030 μg / mL, the average recovery rate is 97.95%, and the RSD is 1.46%; the linear range of ginsenoside Rg1 is 0.08-0.60 μg / mL, the average recovery rate is 98.09%, and the RSD is 1.26%; the linear range of ginsenoside Rb1 is 0.032-0.24 μg / mL, the average recovery rate is 98.92%, and the RSD is 1.17%; the linear range of ginsenoside Re is 0.20-1.50 μg / mL, the average recovery rate is 98.91%, and the RSD is 1.57%; the RSD of repeatability detection is less than 1%. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0026] Figure 3 This is the HPLC chromatogram of detecting the second transplanted ginseng in Example 2 of the present invention. DETAILED DESCRIPTION

[0027] All raw materials of the present invention have no particular limitation on their sources and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0028] There is no particular limitation on the purity of all raw materials in the present invention. The present invention preferably uses chromatographically pure raw materials or raw material purities commonly used in the field of high performance liquid chromatography detection.

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

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

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

[0032] (3) subjecting the test solution obtained in step (2) to high performance liquid chromatography to obtain the contents of strychnine and ginsenosides Rg1, Rb1, and Re in ginseng; the mobile phases for the high performance liquid chromatography detection are 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 the high performance liquid chromatography detection are gradient elution, and the gradient elution is:

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

[0036] The invention mixes ginseng powder with an extraction solvent for leaching, and then dries the obtained liquid phase to obtain a ginseng extract.

[0037] The present invention has no special requirements on the specifications of the ginseng powder, and the specifications of ginseng powder well known to those skilled in the art can be used. As an embodiment of the present invention, the ginseng powder can be obtained by crushing ginseng with a grinder and then passing it through an 80-mesh sieve.

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

[0039] In the present invention, the mass ratio of the ginseng powder to the extraction solvent is preferably 1: (25-35), more preferably 1: (27-32); as an embodiment of the present invention, the mass ratio of the ginseng powder to the extraction solvent can be 1: 26, 1: 27, 1: 28, 1: 29, 1: 30, 1: 31, 1: 32, 1: 33 or 1: 34. The mass ratio of ginseng powder to the extraction solvent within the above range is conducive to further improving the extraction efficiency of ginseng extract.

[0040] In the present invention, the extraction is preferably ultrasonic extraction, and the power of the ultrasonic extraction is preferably 200-300W, more preferably 230-270W; the frequency of the ultrasonic extraction is preferably 50kHz; the time of the ultrasonic extraction is preferably 1-2h, more preferably 1.3-1.8h. In an embodiment of the present invention, the power of the ultrasonic extraction is 250W, the frequency is 50kHz, and the time is 1.5h. Using ultrasonic extraction within the above parameter range is conducive to further improving the extraction efficiency of ginseng extract.

[0041] In the present invention, after the extraction is completed, solid-liquid separation is preferably performed, and the solid-liquid separation method is preferably centrifugal separation. The present invention has no special requirements on the parameters of the centrifugal separation, and the conventional centrifugal separation parameters in the art can be used. Centrifugal separation can simply and efficiently separate the extract from the residue, which is conducive to further improving the extraction efficiency of the ginseng extract.

[0042] The present invention has no 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 a test product solution.

[0044] In the present invention, the amount of methanol to ginseng powder is preferably 100 mL: (1-3) g, more preferably 100 mL: 1 g. The amount of methanol to ginseng powder is within the above range, which is conducive to the separation of strychnine and ginsenosides Rg1, Rb1, Re during gradient elution. As an embodiment of the present invention, the methanol extract can be placed in a volumetric flask and fixed 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 contents of strychnine and ginsenosides Rg1, Rb1, and Re in ginseng.

[0046] In the present invention, the mobile phases for the HPLC detection are 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%, preferably 0.05%. The present invention uses the above mobile phases, which is conducive to separating strychnine and ginsenosides Rg1, Rb1, and Re from ginseng extract during gradient elution.

[0047] In the present invention, the elution condition of the high performance liquid chromatography detection is gradient elution, and the gradient elution is:

[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] 60-70 min, 20-80% mobile phase A, 80-20% mobile phase B. The present invention uses the above elution conditions to separate strychnine and ginsenosides Rg1, Rb1, and Re from ginseng extract, thereby improving the accuracy of detection.

[0051] In the present invention, the conditions for the HPLC detection preferably also include: the chromatographic column is a C18 chromatographic column, the flow rate is 0.8-1.2 mL / min, the column temperature is 30-40°C, the detection wavelength is 203 nm, and the injection volume is 8-12 μL; more preferably: the chromatographic column is a C18 chromatographic column, the flow rate is 1.0 mL / min, the column temperature is 35°C, the detection wavelength is 203 nm, and the injection volume is 10 μL. Using the above detection conditions for HPLC detection is conducive to further improving the accuracy of the detection.

[0052] In the present invention, the external standard method is preferably used in 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] The present invention uses ethanol aqueous solution as an extraction solvent, and can simultaneously extract strychnine and ginsenosides, thereby avoiding the inability of Soxhlet extraction to effectively extract strychnine; through the selection of mobile phase and the adjustment of 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.

[0054] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] Example 1

[0056] instrument

[0057] Liquid chromatograph (Shimadzu LC--20AT); electronic balance (Sartorius Scientific Instruments Co., Ltd.); ultrapure water machine (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 substance (Desit Biological Co., Ltd., HPLC ≥ 98%), Rg1, Rb1, Re reference substances (Shanghai Baoman Biotechnology Co., Ltd., HPLC ≥ 98%).

[0060] A method for simultaneously detecting the contents of strychnine and ginsenosides Rg1, Rb1, and Re in ginseng, comprising the following specific steps:

[0061] Preparation of test solution

[0062] Take four-year straight ginseng from Changbai Mountain, crush the ginseng with a grinder, pass it through an 80-mesh sieve, take 1g of ginseng powder, accurately weigh it, add 30g of 80% ethanol aqueous solution by volume, and put it into ultrasonic extraction (power 250W, frequency 50kHz, 1.5h); centrifuge to take the supernatant, dry it to constant weight, dissolve the residue with methanol, transfer it to a 100mL volumetric flask, add methanol to make up to volume, shake it well and use it as the test solution.

[0063] Standard product testing

[0064] A standard mixed solution was prepared, in which the concentration of strychnine was 0.4 μg / mL, the concentration of ginsenoside Rg1 was 8.0 μg / mL, the concentration of ginsenoside Rb1 was 3.2 μg / mL, and the concentration of ginsenoside Re was 20.0 μg / mL; the standard solution was tested according to the above chromatographic conditions to obtain an HPLC chromatogram of the standard, as shown in FIG. Figure 1 As shown. Figure 1 It can be seen that the peaks of strychnine and ginsenosides Rg1, Rb1, and Re (see the markings in the figure) provide a reference for subsequent detection.

[0065] Preparation of mixed standard solution

[0066] Accurately weigh 1 mg of strychnine, dissolve it with an appropriate amount of methanol and dilute it to a 50 mL volumetric flask, shake well; weigh 14 mg of ginsenoside Rg, 11.6 mg of ginsenoside Rb, and 10 mg of ginsenoside Re, dissolve them with an appropriate amount of methanol and dilute them to a 10 mL volumetric flask, shake well, and prepare standard solutions of strychnine and ginsenosides Rg1, Rb1, and Re. Accurately transfer the same volume of each of the above standard solutions to the same volumetric flask, add an appropriate amount of methanol to dilute to 10 mL, shake well, and the volume of each standard solution transferred each time is 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] Chromatographic column: XBridge C18 5μm (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 °C;

[0076] Detection wavelength: 203nm;

[0077] Injection volume: 10 μL.

[0078] Linear relationship investigation

[0079] The series of mixed standard solutions were filtered through a 0.22 μm filter membrane, and 10 μL was injected into the HPLC. Each concentration of mixed standard solution was injected three times in parallel, and the average peak area S was calculated. 均 The relationship between strychnine, ginsenoside Rg1, Rb1 and Re was obtained by the relationship between the injection volume and the linear relationship between strychnine, ginsenoside Rg1, Rb1 and Re. The experimental data and results are shown in Tables 1 to 5.

[0080] Table 1 Linear relationship data table of strychnine

[0081] Solution concentration (μg / mL) Injection volume (μg) <![CDATA[Strychnine S 均 (mV·s)]]> 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 Linear data of ginsenoside Rg1

[0083] Solution concentration (μg / mL) Injection volume (μg) <![CDATA[Rg1S 均 (mV s)]]> 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 Linear data of ginsenoside Rb1

[0085]

[0086]

[0087] Table 4 Linear data table of ginsenoside Re

[0088] Solution concentration (μg / mL) Injection volume (μg) <![CDATA[ReS 均 (mV s)]]> 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 Linear relationship investigation record of strychnine, ginsenoside Rg1, Rb1, Re

[0090] Compound Linear equations Linear range (μg / mL) <![CDATA[R 2 ]]> Strychnine 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] Through the above-mentioned linear relationship investigation, we can clearly see the corresponding peak areas of each component at different concentrations and the good linear law, which lays the foundation for the subsequent accurate determination of the content.

[0092] Precision inspection

[0093] Take the mixed standard solution, filter it through a 0.22μm filter membrane, take 10μL and inject it into the high performance liquid chromatograph, inject it in parallel 6 times a day, record the peak areas S1~S6 of strychnine, ginsenosides Rg1, Rb1, and Re, and calculate their RSD values. According to the above method, inject once a day for 6 consecutive days, record the peak areas S1~S6 of strychnine, ginsenosides Rg1, Rb1, and Re, and calculate their RSD values. The RSD values ​​of the peak areas of strychnine, ginsenosides Rg1, Rb1, and Re are all less than 2%, indicating that the instrument has good precision. The experimental results are shown in Tables 6~7.

[0094] Table 6 Intra-day precision inspection record

[0095] serial number S1(mV·s) S2(mV·s) S3(mV·s) S4(mV·s) S5(mV·s) S6(mV·s) RSD% Strychnine 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 Daily precision inspection record

[0097]

[0098]

[0099] It can be seen from the precision test results that the instrument used in the present invention can stably give reliable data under this detection method, ensuring the accuracy and repeatability of the detection.

[0100] Repeatability test

[0101] Take 6 portions of the same concentration of mixed standard solution, filter through a 0.22 μm filter membrane, inject each portion three times, 10 μL each time, and measure the peak area S of strychnine, ginsenoside Rg1, Rb1, and Re 均 , the content of each compound was calculated by external standard method, and the RSD value was calculated. The RSD values ​​of strychnine, ginsenoside Rg1, Rb1, and Re were all less than 2%, indicating that the method had good repeatability. The experimental results are shown in Table 8.

[0102] Table 8 Repeatability test record of strychnine, ginsenoside Rg1, Rb1, Re

[0103] serial number <![CDATA[Strychnine S 均 (mV·s)]]> <![CDATA[Rg1S 均 (mV s)]]> <![CDATA[Rb1S 均 (mV s)]]> <![CDATA[ReS 均 (mV s)]]> 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] The repeatability test further verified that the method of the present invention can stably determine the content of each component when preparing different samples during multiple operations, and has good repeatability.

[0105] Stability test

[0106] Take 6 mixed standard solutions of the same concentration, filter through a 0.22μm filter membrane, and inject 10μL every 2h for 12h. Record the retention time t and peak area S of strychnine, ginsenoside Rg1, Rb1, and Re, and calculate the RSD value. The results show that the RSD values ​​of the retention time t and peak area S of strychnine, ginsenoside Rg1, Rb1, and Re are all less than 2%, indicating that the sample solution has good stability within 12h. The experimental results are shown in Tables 9 and 10.

[0107] Table 9 Stability test record of strychnine and ginsenoside Rg1

[0108]

[0109]

[0110] Table 10 Stability test record of ginsenoside Rb1 and Re

[0111] serial number Rb1S(mV·s) Rb1t(s) ReS(mV·s) Ret(s) 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 results of the stability test prove that the prepared sample solution is stable within a certain time range, which can ensure the reliability of the test data and will not fluctuate greatly due to time factors.

[0113] Sample recovery rate

[0114] Take 6 portions of ginseng powder (pass through 80 mesh sieve), prepare 6 sample solutions according to the preparation method of the test solution, pass through 0.22μm filter membrane, inject each portion three times, inject 10μL each time, determine the peak area of ​​strychnine, ginsenoside Rg1, Rb1, Re, calculate the content, recovery rate, average recovery rate and RSD value. The results show that the RSD value of the recovery rate is less than 2%, indicating that the recovery rate of this method is good. The experimental results are shown in Tables 11 to 14.

[0115] Table 11 Strychnine sample recovery rate inspection record

[0116]

[0117] Table 12 Recovery rate of ginsenoside Rg1

[0118]

[0119] Table 13 Recovery rate of ginsenoside Rb1

[0120]

[0121] Table 14 Recovery rate of ginsenoside Re

[0122]

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

[0124] Content determination

[0125] Take three samples of the test solution, filter them through a 0.22 μm filter membrane, and inject them into a high performance liquid chromatograph, injecting 10 μL each time, to obtain a HPLC chromatogram, as shown in the figure below: Figure 2 As shown. Figure 2 The peak areas of strychnine, ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Re at their elution positions (marked in the figure) were recorded, and the contents of strychnine, ginsenoside Rg1, Rb1 and Re were calculated by the external standard method, and the average value of the three samples was taken.

[0126] Example 2

[0127] Take two transplanted ginsengs and prepare the test solution according to the method of Example 1, and perform high performance liquid chromatography to obtain an HPLC chromatogram. Figure 3 As shown. Figure 3The peak areas of strychnine, ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Re were recorded at the peak positions (marked in the figure), and the contents of strychnine, ginsenoside Rg1, Rb1 and Re were calculated by external standard method, and the average values ​​of three samples were taken. The results are shown in Table 15.

[0128] Table 15 Record of the contents of strychnine and ginsenosides Rg1, Rb1, and Re in different embodiments

[0129] / Strychnine average (mg / g) Rg1 average (mg / g) Rb1 average (mg / g) Re average (mg / g) 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 medicinal material content in the Chinese Pharmacopoeia (Volume 1 2019 Edition):

[0132] 1. Chromatographic conditions

[0133] Mobile phase: acetonitrile (A)-water (B);

[0134] Gradient elution conditions:

[0135] 0-35min, 19% A;

[0136] 35-55min, 19-29% A;

[0137] 55-70min, 29% A;

[0138] 70-100min, 29-40%A;

[0139] Detection wavelength: 203nm;

[0140] Flow rate: 1 mL / min;

[0141] Column temperature: 35 °C;

[0142] Injection volume: 10 μL.

[0143] 2. Preparation of reference solution

[0144] Accurately weigh ginsenoside Rg1 reference substance, ginsenoside Re reference substance and ginsenoside Rb1 reference substance, add methanol to make a mixed solution containing 0.2 mg of each substance per 1 mL, shake well, and obtain.

[0145] 3. Preparation of test solution

[0146] Take 1 g of the powder of this product (passed through a No. 4 sieve), accurately weigh it, place it in a Soxhlet extractor, add chloroform and heat to reflux for 3 hours, discard the chloroform solution, evaporate the solvent from the residue, move it into a 100 mL conical flask together with the filter paper tube, accurately add 50 mL of saturated n-butanol with water, plug it tightly, leave it overnight, treat it with ultrasound (power 250 W, frequency 50 kHz) for 30 minutes, filter, discard the initial filtrate, accurately measure 25 mL of the subsequent filtrate, place it in an evaporating dish and evaporate it to dryness, dissolve the residue in methanol and transfer it to a 5 mL volumetric flask, add methanol to dilute to the scale, shake well, filter, and take the subsequent filtrate.

[0147] 4. Content Determination

[0148] Take three portions of the test solution, filter through a 0.22 μm filter membrane, and inject into a high performance liquid chromatograph, inject 10 μL each time, record the peak area, and calculate the content of strychnine, ginsenoside Rg1, Rb1, and Re by the external standard method. The results are shown in Table 16.

[0149] Table 16 Content record of strychnine, ginsenoside Rg1, Rb1 and Re in pharmacopoeia method

[0150] Strychnine average (mg / g) Rg1 average (mg / g) Rb1 average (mg / g) Re average (mg / g) Four-year-old ginseng Not detected 3.283 1.294 4.442 Transplanting ginseng Not detected 2.631 1.934 3.309

[0151] By comparing Table 15 and Table 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 result is closer to the true value. The deviations of the two methods meet 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 is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for simultaneously detecting the contents of strychnine and 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 a ginseng extract; the extraction solvent is an ethanol aqueous solution; (2) mixing the ginseng extract obtained in step (1) with methanol to obtain a test solution; (3) subjecting the test solution obtained in step (2) to high performance liquid chromatography to obtain the contents of strychnine and ginsenosides Rg1, Rb1, and Re in ginseng; The mobile phases for the HPLC detection are 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 condition of the high performance liquid chromatography detection is gradient elution, and 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; 60-70min, 20-80% mobile phase A, 80-20% mobile phase B.

2. The method according to claim 1, characterized in that: The volume content of ethanol in the ethanol aqueous solution in step (1) is 70-80%.

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

4. The method according to claim 1, characterized in that The conditions for high performance liquid chromatography detection in step (3) include: a C18 chromatographic 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.

5. The method according to claim 4, characterized in that The conditions for high performance liquid chromatography detection in step (3) include: a C18 chromatographic 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.

6. The method according to claim 1, characterized in that The amount of methanol in step (2) and the amount of ginseng powder in step (1) are in the order of 100 mL: (1-3) g.

7. The method according to claim 1, characterized in that In the step (3), the external standard method is used for high performance liquid chromatography detection.

8. The method according to claim 7, characterized in that The mixed standard solution used in the external standard method is a methanol solution of strychnine, ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Re.

9. The method according to claim 1, characterized in that: The extraction in step (1) is ultrasonic extraction, and the power of the ultrasonic extraction is 200-300W, the frequency is 50kHz, and the time is 1-2h.

10. The method according to claim 1 or 9, characterized in that: After the extraction in step (1) is completed, solid-liquid separation is performed, and the solid-liquid separation method is centrifugal separation.

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