A method for constructing a characteristic map of ginseng leaves and its application

Abstract

The present invention belongs to the field of drug analysis technology, and specifically relates to a method for constructing and applying a ginseng leaf characteristic spectrum. The construction method comprises: preparing a test solution, preparing a reference solution, and determining the solution according to high performance liquid chromatography, and defining acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B and a specific gradient elution program. The characteristic spectrum obtained by this construction method has good separation of the characteristic peaks, strong spectrum specificity, and is easy to implement, providing a basis for effectively controlling and scientifically evaluating the quality of ginseng leaf medicinal materials and their preparations. The method provided by the present invention is simple to operate, provides accurate results, and has good reproducibility of the results.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis, and in particular relates to a method for constructing a ginseng leaf characteristic spectrum and its application. Background Art

[0002] Ginseng leaf is the dried leaf of Panax ginseng (Camey), a plant of the Araliaceae family. Ginseng has numerous medicinal parts, including ginseng, ginseng leaves, and ginseng flowers. The chemical composition of different ginseng parts is similar, primarily composed of ginsenosides. The chemical composition of American ginseng leaves, a plant of the same family, is also primarily composed of ginsenosides. Due to the similarity of chemical composition within the Panax genus, it is difficult to distinguish ginseng leaf from other plants of the genus according to pharmacopoeial standards. Traditional Chinese medicine granules are produced by extracting Chinese herbal medicine slices with water and using extraction, drying, and granulation processes. These granules lack the identifying characteristics of the original medicinal material, making it impossible to inspect and identify the medicinal material based on its shape, size, texture, and other aspects. Therefore, a method for identifying ginseng leaf is needed to provide a basis for effectively controlling and scientifically evaluating its quality. The characteristic spectra of ginseng leaf obtained by existing technologies suffer from problems such as poor separation between characteristic peaks and insufficient spectra specificity. Summary of the Invention

[0003] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the existing technology in evaluating the quality of ginseng leaves, such as poor separation of characteristic peaks and weak specificity in the constructed spectrum, thereby providing a method and application for constructing a ginseng leaf characteristic spectrum.

[0004] To this end, the present invention provides the following technical solutions.

[0005] The present invention provides a method for constructing a ginseng leaf characteristic map, comprising the following steps:

[0006] Preparation of test solution: Prepare the test sample into test solution;

[0007] Preparation of reference solution: Prepare reference solution from control medicinal materials;

[0008] Determination: Determined by high performance liquid chromatography;

[0009] The chromatographic conditions of the high performance liquid chromatography method include: acetonitrile as mobile phase A, phosphoric acid aqueous solution as mobile phase B, and gradient elution;

[0010] The gradient elution program included: 0-10 min, 5→20% mobile phase A, 95→80% mobile phase B; 10-18 min, 20→22% mobile phase A, 80→78% mobile phase B; 18-20 min, 22→31% mobile phase A, 78→69% mobile phase B; 20-30 min, 31→33% mobile phase A, 69→67% mobile phase B;

[0011] The gradient elution program also includes: 30-40 min, 33→60% mobile phase A, 67→40% mobile phase B; 40-45 min, 60→90% mobile phase A, 40→10% mobile phase B; or, 30-50 min, 33→80% mobile phase A, 67→20% mobile phase B.

[0012] The chromatographic conditions of the high performance liquid chromatography method further include: using an octadecylsilane bonded silica gel column as a filler, the chromatographic column specifications are: inner diameter 4.6 mm, column length 150 mm, particle size 2.7 μm; and / or,

[0013] The column temperature is 23-27°C; and / or,

[0014] A wavelength of 201-205 nm; and / or

[0015] The injection volume is 5-15 μL; and / or,

[0016] The flow rate is 0.9-1.1 ml / min; and / or,

[0017] Use 0.08-0.12% phosphoric acid aqueous solution as mobile phase B.

[0018] The test sample is at least one of ginseng leaf slices, ginseng leaf formula granules, ginseng leaf standard decoction freeze-dried powder, ginseng leaf extract powder and ginseng leaf medicinal materials.

[0019] The method for preparing the test solution comprises: taking the test sample, adding an extraction solvent, extracting, and filtering;

[0020] Preferably, when preparing the test solution, the extraction solvent is at least one of water, methanol and ethanol;

[0021] Preferably, the extraction solvent is methanol;

[0022] Preferably, when preparing the test solution, the extraction method is reflux extraction or ultrasonic extraction.

[0023] When the test sample is at least one of ginseng leaf formula granules, ginseng leaf standard decoction freeze-dried powder and ginseng leaf extract powder, when preparing the test sample solution, the ratio of the test sample mass to the extraction solvent volume is 1g:(45-55)ml.

[0024] The preparation method of the reference solution comprises: taking a reference medicinal material, extracting it with water, filtering it and then evaporating it to dryness to obtain a water extract; adding an extraction solvent to the water extract, extracting it, and filtering it;

[0025] Preferably, when preparing the reference solution, the extraction solvent is at least one of water, methanol and ethanol;

[0026] Preferably, the extraction solvent is methanol;

[0027] Preferably, the ratio of the control medicinal material mass to the extraction solvent volume is 1 g: (15-19) ml;

[0028] Preferably, when preparing the reference solution, the extraction method is reflux extraction or ultrasonic extraction.

[0029] The construction method also includes the preparation of a reference substance solution;

[0030] The reference substance is at least one of a kaempferol-3-O-sophoroside reference substance, a ginsenoside Rg1 reference substance, a ginsenoside Re reference substance, and a ginsenoside Rd reference substance.

[0031] The characteristic spectrum obtained by the construction method includes 14 characteristic peaks;

[0032] Taking Peak 2 as reference peak 1, the specified value of the relative retention time of Peak 1 is: 0.58;

[0033] Taking peak 4 as reference peak 2, the specified values ​​of the relative retention times of peaks 5, 6, 7, 8, 9, 10, 12, 13, and 14 are 1.11, 1.25, 1.29, 1.35, 1.43, 1.48, 1.72, 1.76, and 1.79, respectively;

[0034] Among them, the relative retention time of the characteristic peak is within ±10% of the specified value.

[0035] The present invention also provides a method for identifying ginseng leaves, which uses the above-mentioned construction method to obtain a characteristic spectrum of a test sample, and uses the areas of peak 3 and peak 11 to identify whether the test sample is ginseng leaf.

[0036] In the identification method, when a is 0.43-1.5, the sample to be tested is ginseng leaf;

[0037] in,

[0038] S 11号峰 is the peak area of ​​peak 11, S 3号峰 is the peak area of ​​Peak 3.

[0039] The technical solution of the present invention has the following advantages:

[0040] 1. The present invention provides a method for constructing a ginseng leaf characteristic spectrum, comprising: preparing a test solution, preparing a reference solution, and performing a high-performance liquid chromatography (HPLC) assay using acetonitrile as mobile phase A and aqueous phosphoric acid as mobile phase B, along with a specific gradient elution procedure. This method yields a characteristic spectrum with good separation of characteristic peaks, strong chromatographic specificity, and ease of implementation, providing a basis for effective control and scientific evaluation of the quality of ginseng leaf medicinal materials and their preparations. The method provided by the present invention is simple to operate, produces accurate results, and offers highly reproducible results.

[0041] 2. The identification method of ginseng leaves provided by the present invention can distinguish ginseng leaves from other medicinal parts of ginseng and distinguish ginseng leaves from plants of the same family. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0043] Figure 1 This is the characteristic spectrum of 18 batches of ginseng leaf standard decoction freeze-dried powder and 3 batches of ginseng leaf formula granules in Example 1 of the present invention;

[0044] Figure 2 This is the reference atlas of ginseng leaves in Example 1 of the present invention;

[0045] Figure 3 This is the characteristic spectrum of the ginseng leaf formula granules obtained by different gradient elution procedures in Experimental Example 1 of the present invention;

[0046] Figure 4 This is the characteristic spectrum of the ginseng leaf formula granules obtained at different flow rates in Experimental Example 1 of the present invention;

[0047] Figure 5 This is the characteristic spectrum of the ginseng leaf formula granules obtained at different column temperatures in Experimental Example 1 of the present invention;

[0048] Figure 6 This is the characteristic spectrum of the ginseng leaf formula granules obtained with different mobile phases B in Experimental Example 1 of the present invention;

[0049] Figure 7 This is the characteristic spectrum of the ginseng leaf formula granules obtained with different mobile phase B concentrations in Experimental Example 1 of the present invention;

[0050] Figure 8 This is the characteristic spectrum of the ginseng leaf formula granules obtained by different chromatographic columns in Experimental Example 1 of the present invention;

[0051] Figure 9 This is the characteristic spectrum of the ginseng leaf formula granules obtained from different batches of chromatographic columns in Experimental Example 1 of the present invention;

[0052] Figure 10 This is the characteristic spectrum of the test sample obtained by different extraction methods in Experimental Example 2 of the present invention;

[0053] Figure 11 This is the characteristic spectrum of the test sample obtained at different extraction times in Experimental Example 2 of the present invention;

[0054] Figure 12 This is the characteristic spectrum of the test sample obtained with different extraction solvents in Experimental Example 2 of the present invention;

[0055] Figure 13 This is the characteristic spectrum of the test sample obtained with different concentrations of extraction solvent in Experimental Example 2 of the present invention;

[0056] Figure 14 This is the characteristic spectrum of the chromatographic condition system adaptability of Experimental Example 3 of the present invention;

[0057] Figure 15 is the spectrum of the negative sample of Experimental Example 3 of the present invention;

[0058] Figure 16 This is the characteristic spectrum of different samples to be tested in Example 2 of the present invention. DETAILED DESCRIPTION

[0059] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0060] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0061] The instruments and reagents used in the following examples are:

[0062] Instruments: Chromatograph 1: Waters e2695 chromatography system, including a quaternary gradient infusion pump (Alliance2695), a 120-position high-performance autosampler, an imported column oven, a Waters 2998 diode array UV detector, and an Empower chromatography management system; Chromatograph 2: Shimadzu chromatography system, including an LC-20AT quaternary pump, a SIL-20AC autosampler, an SPD-M20A PDA diode array detector, a CTO-20AC column oven, a CBM-20A system controller, and an Empower chromatography management system; Chromatograph 3: Dionex UltiMate 3000 chromatography system, including an LPG-3400A quaternary pump, a WPS-3000TSL autosampler, a PDA diode array detector, and a chromatography workstation.

[0063] Chromatographic columns: (1) Agilent ZORBAX SB-Aq, 4.6 mm × 150 mm, 3.5 μm; (2) CAPCELL COREC18, 4.6 mm × 150 mm, 2.7 μm; (3) CORTECS T3, 4.6 mm × 150 mm, 2.7 μm.

[0064] Reagents: Acetonitrile was of chromatographic grade, water was ultrapure water; other reagents were of analytical grade.

[0065] Test drugs: Kaempferol-3-O-sophoroside (batch number: 19895-95-5, for content determination, calculated at 90.8%) was purchased from Shanghai Hongyong Biotechnology Co., Ltd.; ginsenoside Rg1 (batch number: 110703-201933, for content determination, calculated at 93.4%) was purchased from the China Food and Drug Inspection Institute; ginsenoside Re (batch number: 110754-202028, for content determination, calculated at 93.9%) was purchased from the China Food and Drug Inspection Institute; ginsenoside Rd (batch number: 111818-201603, for content determination, calculated at 92.1%) was purchased from the China Food and Drug Inspection Institute; ginseng leaf control medicinal material (batch number: 370006-202101; purchased from Shanghai Hongyong Biotechnology Co., Ltd.).

[0066] Ginseng leaf formula granules (batch numbers: 2110001Y, 2110002Y, 2110003Y).

[0067] Ginseng leaf slices (batch numbers: 2010001Y, 2010002Y, 2010003Y, 2010004Y, 2010005Y, 2010006Y, 2010008Y, 2010009Y, 2010010Y, 2010012Y, 2010013Y, 2010017Y, 2010018Y, 2010019Y, 2010020Y, 210801Y, 210802Y, 210803Y).

[0068] Ginseng leaf standard decoction freeze-dried powder: The above 18 batches of ginseng leaf slices were used as raw materials and prepared by the following method: take an appropriate amount of ginseng leaf slices, soak for 30 minutes, decoct twice, add 12 times the amount of slices of water for the first decoction, boil over high heat (500W) and then simmer for 30 minutes, add 10 times the amount of slices of water for the second decoction, boil over high heat (500W) and then simmer for 25 minutes, combine the filtrate, concentrate at 65°C to a material-liquid ratio of about 1:1 (relative density of 1.06-1.12,°C), and freeze-dry to obtain There are 18 batches of ginseng leaf standard decoction freeze-dried powder, and the batch numbers correspond to 2010001D, 2010002D, 2010003D, 2010004D, 2010005D, 2010006D, 2010008D, 2010009D, 2010010D, 2010012D, 2010013D, 2010017D, 2010018D, 2010019D, 2010020D, 210801D, 210802D, and 210803D respectively.

[0069] Example 1

[0070] This embodiment provides a method for constructing a ginseng leaf characteristic map, comprising the following steps:

[0071] Preparation of the test solution: take the test sample, add the extraction solvent, extract, filter, and obtain the test solution. Specifically, when the test sample is a decoction piece or medicinal material, the preparation of the test solution is: take about 1.5g of the test sample, place it in a stoppered conical flask, add 50ml of water, heat and reflux for 1h, filter, evaporate the filtrate to dryness, add 25ml of 80% methanol to the residue, seal it tightly, and ultrasonically treat it for 30min with a power of 250W and a frequency of 40kHz, let it cool, shake it well, filter it, and take the filtrate to obtain the test solution. When the test sample is a formula granule, a standard decoction freeze-dried powder, or an extract powder, the preparation of the test solution is: take an appropriate amount of the test sample, grind it finely, take about 0.5g, place it in a stoppered conical flask, add 25ml of 80% methanol, seal it tightly, and ultrasonically treat it for 30min with an ultrasonic power of 250W and a frequency of 40kHz, let it cool, filter it, and take the filtrate to obtain the test solution.

[0072] Preparation of reference solution: Take about 1.5 g of ginseng leaf reference medicinal material, place it in a stoppered conical flask, add 5 ml of water, heat and reflux for 1 hour, filter, evaporate the filtrate to dryness, add 25 ml of 80% methanol to the residue, seal it tightly, and ultrasonically treat it for 30 minutes at an ultrasonic power of 250 W and a frequency of 40 kHz. Cool, shake well, filter, and take the filtrate to obtain the reference solution.

[0073] Preparation of reference solution: Take appropriate amounts of kaempferol-3-O-sophoroside reference, ginsenoside Rg1 reference, ginsenoside Re reference, and ginsenoside Rd reference, accurately weigh them, and add methanol to make mixed solutions containing 0.1 mg of kaempferol-3-O-sophoroside, 0.25 mg of ginsenoside Rg1, 0.5 mg of ginsenoside Re, and 0.2 mg of ginsenoside Rd per 1 ml to obtain reference solution.

[0074] Determination: Determined according to the high performance liquid chromatography method (General Chapter 0512 of the 2020 edition of the Chinese Pharmacopoeia). Chromatographic conditions: Octadecylsilane bonded silica gel column as the filler, column specifications: inner diameter 4.6mm, column length 150mm, particle size 2.7μm; flow rate 1.0ml / min; column temperature 25℃; detection wavelength 203nm; acetonitrile as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution, gradient elution program: 0-10min, 5→20% mobile phase A, 95→80% mobile phase B; 10-18min, 20→22% Mobile phase A, 80→78% mobile phase B; 18-20 min, 22→31% mobile phase A, 78→69% mobile phase B; 20-30 min, 31→33% mobile phase A, 69→67% mobile phase B; 30-40 min, 33→60% mobile phase A, 67→40% mobile phase B; 40-45 min, 60→90% mobile phase A, 40→10% mobile phase B; the theoretical plate number calculated based on ginsenoside Re should be no less than 1500.

[0075] Accurately pipette 10 μL of test solution, reference solution and reference solution respectively, inject into liquid chromatograph and measure to obtain the result.

[0076] Confirmation of characteristic peaks

[0077] Take 18 batches of ginseng leaf slices standard decoction freeze-dried powder and 3 batches of ginseng leaf formula granules, prepare ginseng leaf slices test solution and ginseng leaf formula granule test solution according to the above steps, and measure according to the above chromatographic conditions to obtain the characteristic spectrum of each test solution. The results are shown in Figure 1 .in, Figure 1 S1-S18 correspond to the characteristic spectra of 18 batches of ginseng leaf standard decoction freeze-dried powder, and S19-S21 correspond to the characteristic spectra of 3 batches of ginseng leaf formula granules.

[0078] The fingerprint similarity evaluation software "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to fit and generate the reference characteristic spectrum using the "multi-point correction, MARK peak matching" mode. Figure 2 As shown in Table 1, there are 14 characteristic chromatographic peaks in the spectrum. The relative retention time of the characteristic spectrum is determined according to the research results: There are 14 characteristic peaks in the characteristic spectrum of ginseng leaves, namely peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13 and peak 14, among which peak 2, peak 3, peak 4 and peak 11 correspond to the retention time of the reference substance peaks of kaempferol-3-O-sophoroside, ginsenoside Rg1, ginsenoside Re and ginsenoside Rd, respectively. It is confirmed that the components of peak 2, peak 3, peak 4 and peak 11 are kaempferol-3-O-sophoroside, ginsenoside Rg1, ginsenoside Re and ginsenoside Rd, respectively. Ginsenoside Rg1, ginsenoside Re and ginsenoside Rd; using double reference peaks, the relative retention time of each characteristic peak was calculated segmentally, with the characteristic peak of kaempferol-3-O-sophoroside as the reference peak 1 (S1), and the relative retention time of peak 1 was specified to be 0.58; using the characteristic peak of ginsenoside Re as the reference peak 2 (S2), the relative retention times of peaks 5, 6, 7, 8, 9, 10, 12, 13 and 14 were specified to be 1.11, 1.25, 1.29, 1.35, 1.43, 1.48, 1.72, 1.76 and 1.79, respectively.

[0079] Table 1 Retention time of each characteristic peak of ginseng leaf standard decoction freeze-dried powder and formula granules

[0080]

[0081]

[0082] Note: t / min is the retention time, t / ts is the relative retention time.

[0083] Identification of characteristic peaks

[0084] Ginseng leaves have 14 characteristic peaks, which were identified and assigned using HPLC and LC / MS / MS. Peak 1 is L-tryptophan, peak 2 is kaempferol-3-O-sophoroside, peak 3 is ginsenoside Rg1, peak 4 is ginsenoside Re, peak 5 is Vietnamese ginsenoside R4, peak 6 is ginsenoside F5, peak 7 is ginsenoside Rb1, peak 8 is ginsenoside Rc, peak 9 is ginsenoside Rb2, peak 10 is ginsenoside F1, peak 11 is ginsenoside Rd, peak 12 is ginsenoside F4, peak 13 is 20(S)-ginsenoside F2, and peak 14 is ginsenoside Rh4. The results are shown in the table below.

[0085] Table 2 Components corresponding to each characteristic peak

[0086]

[0087] Experimental Example 1 Determination of chromatographic conditions

[0088] 1.1 Gradient elution procedure

[0089] Take the same batch of ginseng leaf formula granules and prepare 4 test solutions according to the "Preparation of test solution" in Example 1. Except for the different gradient elution procedures, the other procedures are the same as Example 1. The gradient elution procedures 1-3 are as follows. The results are shown in the table below. Figure 3 :

[0090] Table 3 Gradient elution program 1

[0091] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0～10 1 5→20 95→80 10～20 1 20→25 80→75 20～35 1 25→35 75→65 35～45 1 35→80 65→20 45～50 1 80→90 20→10 40～45 1 90 10

[0092] Table 4 Gradient elution program 2

[0093] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0～10 1.0 5→20 95→80 10～18 1.0 20→22 80→78 18～20 1.0 22→31 78→69 20～30 1.0 31→33 69→67 30～50 1.0 33→80 67→20

[0094] Table 5 Gradient elution program 3

[0095] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0～10 1.0 5→20 95→80 10～18 1.0 20→22 80→78 18～20 1.0 22→31 78→69 20～30 1.0 31→33 69→67 30～40 1.0 33→60 67→40 40～45 1.0 60→90 40→10

[0096] The above results show that gradient elution procedure 1 has poor resolution between characteristic peaks between 20 and 30 minutes and cannot effectively separate the characteristic peaks. Gradient elution procedures 2 and 3 have good resolution between characteristic peaks and can both be used to construct a characteristic spectrum for ginseng leaves. However, compared with gradient elution procedures 2 and 3, gradient elution procedure 3 has a shorter time and an earlier peak elution time, making it superior.

[0097] 1.2 Flow rate

[0098] The same batch of ginseng leaf formula granules was used to prepare three test solutions according to the "Preparation of Test Solution" in Example 1. The characteristic spectra of different test solutions were obtained according to the "Determination" item in Example 1, with the flow rate as the variable; the flow rates were 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min, respectively. The separation of the chromatographic peaks and the system suitability parameters were used as the evaluation indicators. The results are shown in the following table and Figure 4 .

[0099] Table 6 Spectra of test products obtained at various flow rates

[0100]

[0101]

[0102] From the above results, it can be seen that different flow rates affect the retention time and peak area of ​​each characteristic peak. When the flow rate is 1.0 ml / min, the separation effect of peak 7, peak 11 and the surrounding small peaks is better. The flow rate of 1.0 ml / min is the optimal flow rate.

[0103] 1.3 Column temperature

[0104] Take the same batch of ginseng leaf formula granules and prepare 3 test solutions according to the "Preparation of Test Solution" in Example 1. With column temperature as the variable, according to the "Determination" item in Example 1, characteristic spectra of different test solutions are obtained; the column temperatures are 20°C, 25°C and 30°C respectively, and the separation of chromatographic peaks and system suitability parameters are used as evaluation indicators. The results are shown in the following tables and Figure 5 .

[0105] Table 7 Spectra of the test samples obtained at different column temperatures

[0106]

[0107]

[0108] The above results show that when the column temperature is 30°C, Peak 7 is subject to greater interference. When the column temperature is 25°C, the separation of Peaks 7 and 12 is better, and the separation of characteristic peaks and system adaptability parameters are also better. Therefore, 25°C is the optimal column temperature.

[0109] 1.4 Mobile phase B

[0110] The same batch of ginseng leaf formula granules was used to prepare three test solutions according to the "Preparation of Test Solution" in Example 1. The characteristic spectra of different test solutions were obtained according to the "Determination" item in Example 1, with mobile phase B as the variable; the mobile phases were acetonitrile-0.1% phosphoric acid aqueous solution, acetonitrile-0.1% formic acid aqueous solution, and acetonitrile-0.1% acetic acid aqueous solution, respectively. The separation of the chromatographic peaks and the system suitability parameters were used as evaluation indicators. The results are shown in the following table and Figure 6 The results show that using phosphoric acid as mobile phase B results in a relatively stable baseline and excellent separation between the characteristic peaks of Peaks 5, 11, and 13 (acetic acid and formic acid). Therefore, acetonitrile was used as mobile phase A and aqueous phosphoric acid solution was used as mobile phase B.

[0111] Table 8 Spectra of the test products obtained with different mobile phases B

[0112]

[0113]

[0114] 1.5 Mobile phase B concentration

[0115] The same batch of ginseng leaf formula granules was used to prepare three test solutions according to the "Preparation of Test Solution" in Example 1. The concentration of mobile phase B was used as a variable, and the characteristic spectra of different test solutions were obtained according to the "Determination" item in Example 1; the concentrations of mobile phase B were 0.05%, 0.1%, and 0.2%, respectively. The separation of the chromatographic peaks and the system suitability parameters were used as evaluation indicators. The results are shown in the following table and Figure 7 The results show that when 0.1% phosphoric acid aqueous solution is used as mobile phase B, the symmetry of the obtained spectrum is better, and 0.1% phosphoric acid aqueous solution is preferably used as mobile phase B.

[0116] Table 9 Spectra of the test products obtained with different concentrations of mobile phase B

[0117]

[0118]

[0119] 1.6 Chromatographic column

[0120] The same batch of ginseng leaf formula granules was used to prepare three test solutions according to the "Preparation of Test Solution" in Example 1. The characteristic spectra of different test solutions were obtained according to the "Assay" item in Example 1, using the chromatographic column as a variable. The chromatographic columns were ① Agilent ZORBAX SB-Aq, 4.6 mm × 150 mm, 3.5 μm; ② CAPCELL CORE C18, 4.6 mm × 150 mm, 2.7 μm; and ③ CORTECS T3, 4.6 mm × 150 mm, 2.7 μm. The results are shown in the following table and Figure 8 .

[0121] Table 10 Retention time of the spectra of the test samples obtained by different chromatographic columns

[0122]

[0123] Table 11 Retention time relative peak area of ​​the samples obtained by different chromatographic columns

[0124]

[0125]

[0126] From the above results, it can be seen that the chromatographic separation effect obtained by using CORTECS T3, 4.6 mm × 150 mm, 2.7 μm as the chromatographic column is the best. Therefore, this chromatographic column is preferred in the present invention.

[0127] The effect of different batches of chromatographic columns (CORTECS T3, 4.6 mm × 150 mm, 2.7 μm) on the chromatogram was further investigated. Three test solutions were prepared from the same batch of ginseng leaf granules according to the "Preparation of Test Solution" in Example 1. Three chromatographic columns of different batches (denoted as Column 1, Column 2, and Column 3) were used. Following the "Assay" step in Example 1, characteristic spectra of the different test solutions were obtained. The results are shown in the following tables and Figure 9 .

[0128] Table 12 Comparison of relative retention time results of different batches of chromatographic columns

[0129]

[0130] Table 13 Comparison of relative peak area results of different batches of chromatographic columns

[0131]

[0132]

[0133] The above results show that the deviation of the relative retention time of each characteristic peak is less than 2%. The characteristic spectrum results will not change with the chromatographic column batch number, and the retention time of each characteristic chromatographic peak does not change much. Therefore, it is recommended that the chromatographic column CORTECS T3, 4.6mm×150mm, 2.7μm be preferred as the chromatographic column for subsequent research and investigation.

[0134] Based on the above, the optimal chromatographic conditions were determined as follows: octadecylsilane bonded silica gel was used as the filler (column length was 150 mm, inner diameter was 4.6 mm, particle size was 2.7 μm); acetonitrile was used as the mobile phase A, 0.1% phosphoric acid was used as the mobile phase B, gradient elution was performed, and the gradient elution program was as follows: 0-10 min, 5→20% mobile phase A, 95→80% mobile phase B; 10-18 min, 20→22% mobile phase A, 80→78% mobile phase B; 18 -20 min, 22→31% mobile phase A, 78→69% mobile phase B; 20-30 min, 31→33% mobile phase A, 69→67% mobile phase B; 30-40 min, 33→60% mobile phase A, 67→40% mobile phase B; 40-45 min, 60→90% mobile phase A, 40→10% mobile phase B; detection wavelength is 203 nm; the number of theoretical plates calculated based on ginsenoside Re should be no less than 1500.

[0135] Experimental Example 2 Determination of the preparation method of the test solution

[0136] 2.1 Extraction method of test solution

[0137] The same batch of ginseng leaf formula granules were used to extract the test sample using different methods, and the characteristic spectra of different test sample solutions were obtained according to the "Determination" item in Example 1; the first method was the same as the "Preparation of Test Sample Solution" in Example 1; the second method used reflux extraction, specifically comprising: taking an appropriate amount of the product, grinding it, taking about 0.5g, placing it in a stoppered conical flask, adding 25ml of 80% methanol, heating and refluxing for 30min, cooling, filtering, and taking the filtrate to obtain the test sample solution. The results are shown in the following table and Figure 10 .

[0138] Table 14 Spectra obtained by different extraction methods of test solution

[0139]

[0140]

[0141] Judging from the above results, ultrasonic extraction and reflux extraction have little effect on the characteristic spectrum. Considering the simplicity of the experimental method, ultrasonic extraction is preferred.

[0142] 2.2 Extraction time

[0143] Take the same batch of ginseng leaf formula granules, and use the ultrasonic treatment time as a variable. Prepare 4 test solutions according to the "Preparation of Test Solution" in Example 1, and obtain characteristic spectra of different test solutions according to the "Determination" item in Example 1; the ultrasonic treatment time is 15min, 30min, 45min and 60min respectively. Figure 11 The experimental results show that, from 15 to 60 min, as the extraction time increases, the total peak area of ​​the characteristic peaks does not differ much. Within this range, all characteristic components are completely extracted. Considering the extraction efficiency, 30 min is the preferred extraction time.

[0144] 2.3 Ultrasonic power

[0145] Using the same batch of ginseng leaf granules, three test solutions were prepared according to the "Preparation of Test Solutions" section in Example 1. Characteristic spectra of the different test solutions were obtained according to the "Measurement" section in Example 1, using ultrasonic power as a variable; the ultrasonic powers were 200 W, 250 W, and 300 W, respectively. During the experiment, it was found that ultrasonic power had little effect on the experimental results.

[0146] 2.4 Extraction solvent

[0147] The same batch of ginseng leaf formula granules was taken, and 3 test solutions were prepared according to the "Preparation of Test Solution" in Example 1, with the extraction solvent as the variable. The characteristic spectra of different test solutions were obtained according to the "Determination" item in Example 1. When preparing the test solution, the extraction solvents were water, methanol and ethanol, respectively. The results are shown in the following table and Figure 12 .from Figure 12 It can be seen that when methanol is used as the extraction solvent, the baseline of the characteristic spectrum obtained is relatively stable and there is less interference from impurity peaks. Methanol is preferably used as the extraction solvent.

[0148] Table 15 Spectra of the test samples obtained with different extraction solvents

[0149]

[0150]

[0151] 2.5 Extraction solvent concentration

[0152] The same batch of ginseng leaf formula granules was used, and 4 test solutions were prepared according to the "Preparation of Test Solution" in Example 1, with the concentration of the extraction solvent as a variable. The characteristic spectra of different test solutions were obtained according to the "Determination" item in Example 1. When preparing the test solution, the extraction solvent concentrations were 40% methanol aqueous solution, 60% methanol aqueous solution, 80% methanol aqueous solution and methanol, respectively. The results are shown in the following table and Figure 13 .

[0153] Table 16 Retention time and peak area of ​​the test solution chromatograms obtained at different extraction solvent concentrations

[0154]

[0155]

[0156] From the above results and Figure 13 It can be seen that there is no obvious difference in the peak area of ​​the characteristic peaks in the characteristic spectra obtained with methanol aqueous solutions of different concentrations. Combined with the dissolution effect of ginseng leaf formula granules, 80% methanol is preferably used as the extraction solvent for the test solution.

[0157] 2.6 Extraction solvent volume

[0158] The same batch of ginseng leaf formula granules was taken, and the extraction solvent volume was used as a variable. Three test solutions were prepared according to the "Preparation of Test Solution" in Example 1, and characteristic spectra of different test solutions were obtained according to the "Determination" item in Example 1. When preparing the test solutions, the extraction solvent volumes were 10 ml, 25 ml and 50 ml, respectively. The results showed that the response values ​​of each chromatographic peak presented by about 0.5 g of the test formula granules corresponding to 25 ml of 80% methanol extraction were relatively good. Therefore, the extraction solvent volume of 25 ml was selected in this experiment for subsequent investigation.

[0159] 2.7 Sample volume of test product

[0160] Take the same batch of ginseng leaf formula granules, take the sample sampling amount as a variable, prepare 5 test solutions according to the "Preparation of Test Solution" in Example 1, and obtain the characteristic spectra of different test solutions according to the "Determination" item in Example 1; when preparing the test solutions, the sample sampling amounts are 0.20g, 0.40g, 0.50g, 0.60g and 0.80g respectively. It can be seen from the results that with the increase of the sample sampling amount, the cover level of each characteristic peak increases by a corresponding multiple, indicating that each characteristic peak is completely extracted. When the sampling amount is 0.5g, the peak height and peak width of the characteristic peak are relatively moderate, and 0.5g is preferably used as the sample sampling amount.

[0161] In summary, the optimal preparation method for the test solution is determined. Specifically, when the test sample is a formula granule or a standard decoction freeze-dried powder, the preparation method is as follows: take an appropriate amount of the product, grind it into powder, take about 0.5g, place it in a stoppered conical flask, add 25ml of 80% methanol, seal it tightly, ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes, let it cool, filter it, and take the filtrate to obtain the product. When the test sample is a medicinal material or a piece of decoction, the preparation method is as follows: take about 1.5g of the test sample, place it in a stoppered conical flask, add 50ml of water, heat and reflux for 1h, filter it, evaporate the filtrate to dryness, add 25ml of 80wt% methanol to the residue, seal it tightly, ultrasonically treat it for 30min with a power of 250W and a frequency of 40kHz, let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0162] Experimental Example 3 Chromatographic Conditions and System Suitability

[0163] (1) HPLC analysis was performed according to the preparation method of the test solution determined in Experimental Example 2 and the chromatographic conditions determined in Experimental Example 1 to verify the chromatographic conditions and system adaptability of the ginseng leaf formula granules, and the chromatograms were recorded as shown in the following table and Figure 14 shown.

[0164] Table 17 System adaptability characteristic spectrum

[0165] Serial number Retention time area %area high width Separation s / n Symmetry Factor USP theoretical plate number 1 6.819 644723 5.49 135937 14 31.42 1.6 49552 2 11.701 3638637 30.97 1063977 9.8 46.89 245.96 1.3 274628 3 20.546 1061897 9.04 166729 29 71.38 38.54 1.1 256212 4 20.914 2221159 18.9 494197 17.9 2.72 114.24 1.15 541605 5 23.187 150318 1.28 38339 14.1 22.15 8.86 1.55 899827 6 26.196 250188 2.13 41948 28 25.22 9.7 1.39 515684 7 26.926 646493 5.5 95148 15 4.38 22 0.98 334842 8 28.303 338909 2.88 44535 29.4 7.19 10.3 1 316606 9 30.03 424758 3.61 47736 29.9 8.15 11.03 0.91 246521 10 30.912 377049 3.21 44063 28.1 3.91 10.19 1.18 307452 11 33.474 1228445 10.45 171306 31.5 12.79 39.6 0.85 453028 12 35.924 86882 0.74 25193 13.8 18.62 5.82 1.37 2807559 13 36.867 520585 4.43 132926 20 10.49 30.73 1.26 2271068 14 37.289 160746 1.37 44392 16.2 4.6 10.26 1.91 2834581

[0166] From the above results, it can be seen that the separation degree of each characteristic peak is greater than 1.5, and the theoretical plate number of the characteristic peak is greater than 1500, indicating that the method of the present invention has good system adaptability and can be used as a detection method for the characteristic spectrum of ginseng leaf formula granules.

[0167] (2) A negative sample was prepared according to the test solution preparation method determined in Experimental Example 2, and HPLC analysis was performed according to the chromatographic conditions determined in Experimental Example 1. The results are shown in Figure 15. The above results show that the negative sample has no interference with the spectrum. The method of the present invention has good system adaptability and specificity, and can be used as a detection method for the characteristic spectrum of ginseng leaf formula granules. The preparation method of the negative sample: the auxiliary material maltodextrin dry preparation obtains a negative sample, grinds it into powder, takes about 0.5g, puts it into a stoppered conical flask, adds 25ml of 80% methanol, seals it, and ultrasonically treats it for 30min at a power of 250W and a frequency of 40kHz. Let it cool, filter, and take the filtrate to obtain a negative sample.

[0168] Experimental Example 4: Verification of analytical method

[0169] 4.1 Instrument precision

[0170] The test solution was prepared according to the "Preparation of Test Solution" in Example 1. The sample was injected six times according to the "Determination" in Example 1. The relative retention time and relative peak area of ​​each characteristic peak were measured. The results showed that the relative retention time RSD of each characteristic peak was less than 2%. Except for Peak 14, the relative peak area RSD of each characteristic peak was less than 2%, indicating good instrument precision.

[0171] 4.2 Repeatability Experiment

[0172] Six test solutions were prepared from the same batch of ginseng leaf granules according to the "Preparation of Test Solution" procedure in Example 1. Characteristic spectra of the different test solutions were obtained according to the "Assay" procedure in Example 1. The relative retention times and relative peak areas of the characteristic peaks were measured. The results showed that the relative retention times of the characteristic peaks were less than 2%, and the relative peak areas of the characteristic peaks, except for Peak 14, were less than 2%. These results demonstrate the good reproducibility of the method.

[0173] 4.3 Precision of different operators

[0174] Three different examiners, each at different times, sampled the same batch of ginseng leaf granules and prepared test solutions according to the "Preparation of Test Solution" procedure in Example 1. Using the same equipment, they obtained characteristic spectra of the different test solutions, following the "Assay" procedure in Example 1. The relative retention times and relative peak areas of the characteristic peaks were measured. The results showed that the relative retention time deviations for each characteristic peak were less than 2%. With the exception of Peak 14, the relative peak area deviations for all characteristic peaks were less than 2%, indicating good intermediate precision.

[0175] 4.4 Intermediate precision of different HPLC instruments

[0176] The same examiner, at different times, prepared three test solutions from the same batch of ginseng leaf granules according to the "Test Solution Preparation" procedure in Example 1. Characteristic spectra of the different test solutions were obtained according to the "Assay" section in Example 1, using the HPLC instrument as the variable. The relative retention times and peak areas of the characteristic peaks were measured using three HPLC instruments: a Waters e2695, a Shimadzu, and a Dionex UltiMate 3000. The results show that the relative retention times of the characteristic peaks varied by less than 3%, but the relative peak areas of the characteristic peaks were significantly affected by the instrument. This suggests that the influence of different instruments on the relative retention times of the characteristic peaks is minimal.

[0177] 4.5 Stability investigation

[0178] Take the same batch of ginseng leaf formula granules, and prepare 6 test solutions according to the "Preparation of Test Solution" in Example 1. The samples were placed at room temperature for 0 h, 4 h, 8 h, 12 h, 18 h, and 24 h, respectively. Then, according to the "Determination" item in Example 1, the characteristic spectrum of the test solution was obtained, and the relative peak area and relative retention time were calculated, and the RSD was calculated. The results showed that the relative retention time RSD of each characteristic peak was less than 2%, and except for peak 14, the relative peak area RSD of each characteristic peak was less than 2%, indicating that the test solution was stable within 24 h and met the determination requirements.

[0179] Example 2

[0180] This embodiment provides a method for identifying ginseng leaves, comprising the following steps:

[0181] Prepare the test sample solution according to the method provided in Example 1;

[0182] The test solution was tested according to the "Determination" item of Example 1 to obtain a characteristic spectrum of the test solution. First, observe whether Peak 3 and Peak 14 appear. If they do, use the following formula for identification. When a is 0.43-1.5, the test sample is ginseng leaf. If it does not meet the following formula, it means that the test sample is not ginseng leaf.

[0183]

[0184] S 11号峰 is the peak area of ​​peak 11, S 3号峰 is the peak area of ​​Peak 3.

[0185] verify

[0186] Ginseng, ginseng flower, ginseng stem and American ginseng leaf were prepared into test sample solutions according to Example 1, and then tested according to the "Determination" item of Example 1 to obtain the characteristic spectra of ginseng, ginseng flower, ginseng stem and American ginseng leaf. Figure 16 .

[0187] from Figure 16 It can be seen that there are no peaks 12 and 14 in the characteristic spectrum of ginseng flower, and the response values ​​of peaks 1, 2 and 3 are lower than those of ginseng leaf; the peak area ratio of peak 11 to peak 3 is 1.54-7.61.

[0188] The characteristic spectrum of ginseng stems lacks peaks 6, 8, 9, 10, and 12. Furthermore, the response values ​​for peaks 1, 2, 3, 4, 5, 7, 11, 13, and 14 are lower than those in ginseng leaves, indicating that the contents of the components corresponding to these peaks are low in ginseng stems. The peak area ratio of peak 11 to peak 3 ranges from 0.12 to 0.40.

[0189] The ginseng characteristic spectrum lacks peaks 2, 13, and 14, and the response values ​​of each characteristic peak are lower than those of the ginseng leaf sample. The peak area ratio of peak 11 to peak 3 is 0.15-0.20.

[0190] The characteristic spectrum of American ginseng leaves does not contain peaks 5, 6, and 13. The peak area ratio of peak 11 to peak 3 is 3.36-6.67.

[0191] The peak area ratio of Peak 11 to Peak 3 in the characteristic spectrum of ginseng leaves is 0.57-1.16, which has no intersection with the range of relative peak area ratios of ginseng flowers, ginseng stems, ginseng and American ginseng leaves, and there is a significant difference.

[0192] The upper limit of the ginseng leaf range of 0.57-1.16 is multiplied by the coefficient 1.3, that is, 1.16×1.3, and the upper limit of the range is set to 1.5; the lower limit of the ginseng leaf range of 0.57-1.16 is multiplied by the coefficient 0.75, that is, 0.57×0.75. Therefore, the peak area ratio of Peak 11 and Peak 3 in the ginseng leaf characteristic spectrum is set to 0.43-1.5, which is used to distinguish ginseng leaves from other parts, American ginseng leaves, etc.

[0193] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic spectrum of ginseng leaves, characterized in that: The following steps are involved: Preparation of the test solution: preparing the test sample into a test solution; when preparing the test solution, the extraction solvent is at least one of water, methanol and ethanol; Preparation of reference solution: Prepare reference solution from control medicinal materials; Determination: Determined by high performance liquid chromatography; The chromatographic conditions of the high performance liquid chromatography method include: acetonitrile as mobile phase A, 0.08-0.12% aqueous phosphoric acid as mobile phase B, gradient elution; chromatographic column: CORTECS T3, 4.6 mm × 150 mm, 2.7 µm; column temperature: 25°C; wavelength: 201-205 nm; The gradient elution program included: 0-10 min, 5→20% mobile phase A, 95→80% mobile phase B; 10-18 min, 20→22% mobile phase A, 80→78% mobile phase B; 18-20 min, 22→31% mobile phase A, 78→69% mobile phase B; 20-30 min, 31→33% mobile phase A, 69→67% mobile phase B; 30-40 min, 33→60% mobile phase A, 67→40% mobile phase B; 40-45 min, 60→90% mobile phase A, 40→10% mobile phase B; Or, 0-10 min, 5→20% mobile phase A, 95→80% mobile phase B; 10-18 min, 20→22% mobile phase A, 80→78% mobile phase B; 18-20 min, 22→31% mobile phase A, 78→69% mobile phase B; 20-30 min, 31→33% mobile phase A, 69→67% mobile phase B; 30-50 min, 33→80% mobile phase A, 67→20% mobile phase B; The characteristic spectrum of ginseng leaves has a total of 14 characteristic peaks, peak 1 is L-tryptophan, peak 2 is kaempferol-3-O-sophoroside, peak 3 is ginsenoside Rg1, peak 4 is ginsenoside Re, peak 5 is Vietnamese ginsenoside R4, peak 6 is ginsenoside F5, peak 7 is ginsenoside Rb1, peak 8 is ginsenoside Rc, peak 9 is ginsenoside Rb2, peak 10 is ginsenoside F1, peak 11 is ginsenoside Rd, peak 12 is ginsenoside F4, peak 13 is 20(S)-ginsenoside F2, and peak 14 is ginsenoside Rh4.

2. The construction method according to claim 1, characterized in that The chromatographic conditions of the high performance liquid chromatography method further include: an injection volume of 5-15 μL; and / or, The flow rate is 0.9-1.1 ml / min.

3. The construction method according to claim 1 or 2, characterized in that The test sample is at least one of ginseng leaf slices, ginseng leaf formula granules, ginseng leaf standard decoction freeze-dried powder, ginseng leaf extract powder and ginseng leaf medicinal materials.

4. The construction method according to claim 1 or 2, characterized in that The preparation method of the test solution comprises: taking the test sample, adding an extraction solvent, extracting, and filtering.

5. The construction method according to claim 4, characterized in that The extraction solvent is methanol.

6. The construction method according to claim 4, characterized in that When preparing the test solution, the extraction method is reflux extraction or ultrasonic extraction.

7. The construction method according to claim 4, characterized in that: When the test sample is at least one of ginseng leaf formula granules, ginseng leaf standard decoction freeze-dried powder and ginseng leaf extract powder, when preparing the test sample solution, the ratio of the test sample mass to the extraction solvent volume is 1g:(45-55)ml.

8. The construction method according to claim 1, characterized in that The preparation method of the reference solution comprises: taking a reference medicinal material, adding water to extract, filtering and then evaporating to dryness to obtain a water extract; adding an extraction solvent to the water extract, extracting, and filtering.

9. The construction method according to claim 8, characterized in that: When preparing the reference solution, the extraction solvent is at least one of water, methanol and ethanol.

10. The construction method according to claim 9, characterized in that: The extraction solvent is methanol.

11. The construction method according to claim 9, characterized in that: The ratio of the control medicinal material mass to the extraction solvent volume is 1 g: (15-19) ml.

12. The construction method according to claim 9, characterized in that: When preparing the reference solution, the extraction method is reflux extraction or ultrasonic extraction.

13. The construction method according to claim 1 or 2, characterized in that: It also includes the preparation of reference substance solutions; The reference substance is at least one of a kaempferol-3-O-sophoroside reference substance, a ginsenoside Rg1 reference substance, a ginsenoside Re reference substance, and a ginsenoside Rd reference substance.

14. The construction method according to claim 1, characterized in that: The characteristic spectrum obtained by the construction method includes 14 characteristic peaks; Taking Peak 2 as reference peak 1, the specified value of the relative retention time of Peak 1 is: 0.58; Taking peak 4 as reference peak 2, the specified values ​​of the relative retention times of peaks 5, 6, 7, 8, 9, 10, 12, 13, and 14 are 1.11, 1.25, 1.29, 1.35, 1.43, 1.48, 1.72, 1.76, and 1.79, respectively; Among them, the relative retention time of the characteristic peak is within ±10% of the specified value.

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