Construction method and application of ginseng extract fingerprint spectrum

Abstract

The invention provides a construction method and application of a ginseng extract fingerprint spectrum, and relates to the technical field of pharmaceutical analysis. The construction method comprises the following steps: analyzing a sample to be detected by adopting high performance liquid chromatography to obtain a ginseng extract fingerprint spectrum; analysis conditions of the high performance liquid chromatography are as follows: a filler of a chromatographic column is octadecylsilane chemically bonded silica; a mobile phase A is acetonitrile, a mobile phase B is water, and a gradient elution procedure is adopted. The fingerprint spectrum obtained by the construction method contains chromatographic peaks of rare ginsenosides 20 (R)-Rh2, 20 (S)-Rg3 and 20 (R)-Rg3, and the problem that the rare ginsenosides cannot be evaluated in quality detection of the ginseng extract in the prior art is solved.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for constructing a ginseng extract fingerprint spectrum and an application thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Ginseng is a perennial herb belonging to the Araliaceae family. It tastes sweet, slightly bitter, and is neutral in nature. It enters the spleen, lung, and heart meridians. It has the effects of greatly replenishing vital energy, restoring pulse and consolidating collapse, replenishing spleen and lungs, promoting body fluid, and calming the mind. It is often used to treat physical weakness and collapse, cold limbs and weak pulse, spleen deficiency and poor appetite, lung deficiency and cough, thirst due to loss of body fluid, internal heat and thirst, long-term illness and weakness, palpitations and insomnia, etc. Ginseng Chinese medicine slices are extracted with water, concentrated, dried, and granulated to obtain ginseng formula granules, which have many advantages such as retaining the original decoction efficacy, flexible use according to symptoms, and convenient use. The main active ingredient is ginsenoside.

[0004] At present, the quality inspection of ginseng or ginseng products mostly uses ginsenosides as the inspection standard. The 2020 edition of the Chinese Pharmacopoeia stipulates the content requirements of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 under ginseng, but does not include other types of ginsenosides, such as rare ginsenoside 20(R)-Rh2, rare ginsenoside 20(S)-Rg3 and rare ginsenoside 20(R)-Rg3, etc. It is difficult to comprehensively reflect the mass fraction of rare saponins in ginseng extracts, which is far from meeting the requirements of product quality control. Ginsenosides are a mixture of sterols contained in ginseng. These rare saponins are all active ingredients in ginseng, and they each have unique effects and functions, such as enhancing human immune function, anti-tumor, anti-fatigue and improving cardiovascular function. Therefore, it is very necessary to establish a quality evaluation method that can comprehensively reflect the quality of the main active components in ginseng extracts.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing a ginseng extract fingerprint and its application, so as to alleviate the problem that the prior art cannot evaluate rare ginsenosides in the quality detection of ginseng extract.

[0007] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0008] In a first aspect, a method for constructing a ginseng extract fingerprint is provided, the method comprising analyzing a sample to be tested by high performance liquid chromatography to obtain a ginseng extract fingerprint;

[0009] The analysis conditions of HPLC were:

[0010] The filler of the chromatographic column is octadecylsilane bonded silica gel;

[0011] Mobile phase: Mobile phase A is acetonitrile, mobile phase B is water;

[0012] The gradient elution procedure is as follows, based on volume percentage:

[0013] time Mobile phase Av / v% Mobile phase B v / v% 0min 15％ 85％ 20min 30％ 70％ 40min 35％ 65％ 60min 45％ 55％ 65min 45％ 55 90min 75％ 25％

[0014] The detection wavelength is 200~400nm.

[0015] In a second aspect, a ginseng extract quality control method is provided, comprising using the construction method described in the first aspect to obtain a fingerprint of the ginseng extract to be evaluated, comparing the fingerprint of the ginseng extract to be evaluated with a control fingerprint, and calculating the similarity between the fingerprint of the ginseng extract to be evaluated and the control fingerprint, and if the similarity is ≥90%, the quality of the ginseng extract to be evaluated is determined to be qualified.

[0016] In the third aspect, a method for constructing the fingerprint of the ginseng extract described in the first aspect, or the use of the ginseng extract quality control method described in the second aspect in the preparation of a drug containing ginseng extract is provided.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention establishes a method for constructing a fingerprint of ginseng extract, which provides an important basis for the quality control and evaluation of products containing ginseng extract. The present invention establishes an HPLC fingerprint, which can better separate the components in the ginseng extract, and determines 11 common fingerprint peaks and 5 confirmed components according to the test results, including two common saponins and three rare saponins, specifically ginsenoside Re, ginsenoside Rb1, rare ginsenoside 20 (R) -Rh2, rare ginsenoside 20 (S) -Rg3 and rare ginsenoside 20 (R) -Rg3. The construction method provided by the present invention is simple, and the fingerprint of the ginseng extract obtained has good stability and repeatability, which lays a foundation for the quality evaluation of ginseng extract and its products. The fingerprint of the ginseng extract obtained by the construction method provides a reference for the quality evaluation of ginseng extract and products containing it. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The chromatogram of the test solution of Example 1;

[0021] Figure 2 This is a qualitative analysis diagram of the standard product of Example 1;

[0022] Figure 3 HPLC fingerprints of 8 batches (S1-S8) of ginseng extract samples and their control (R) in Example 2;

[0023] Figure 4 The reference fingerprint of ginseng extract constructed in Example 2;

[0024] Figure 5 The fingerprints under different mobile phase conditions in Example 3;

[0025] Figure 6 The fingerprints under different elution gradient conditions in Example 4, S1 to S7 represent elution condition gradient 1 to gradient 7 respectively;

[0026] Figure 7 The fingerprints of different chromatographic columns in Example 5, S1 is Diamonsil C18 (250 mm × 4.6 mm, 5 μm), S2 is Agilent ZORBAX SB-C18 (250 mm × 4.6 mm, 5 μm), and S3 is Platisil ODS (250 mm × 4.6 mm, 5 μm);

[0027] Figure 8 is the chromatogram of 6 injections in the precision test experiment in Example 6, S1 to S6 are the chromatograms of 6 injections, and R(11) is the control chromatogram generated by 6 precision samples;

[0028] Fig. 9 is the chromatogram of 6 injections in the repeatability test in Example 6, S1 to S6 are the chromatograms of 6 injections, and R(11) is the chromatogram generated for 6 repeatability samples;

[0029] Fig.10 These are the chromatograms of the stability test in Example 6 injected at 0, 3, 6, 9, 12, and 24 hours, respectively. S1 to S6 are the chromatograms of the stability test injected at 0, 3, 6, 9, 12, and 24 hours, respectively. The control chromatogram is generated by the 6 stability samples of R(11). DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. 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.

[0031] It should be noted that:

[0032] In the present invention, unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined with each other to form a new technical solution.

[0033] In the present invention, unless otherwise specified, all technical features and preferred features mentioned herein can be combined with each other to form a new technical solution.

[0034] In the present invention, unless otherwise specified, the components or preferred components involved can be combined with each other to form a new technical solution.

[0035] In the present invention, unless otherwise specified, the numerical range "a-b" represents an abbreviation of any real number combination between a and b, where a and b are real numbers. For example, the numerical range "6-22" means that all real numbers between "6-22" have been listed in this document, and "6-22" is just an abbreviation of these numerical combinations.

[0036] The “range” disclosed in the present invention is in the form of a lower limit and an upper limit, which can be one or more lower limits, and one or more upper limits, respectively.

[0037] In the present invention, unless otherwise specified, each reaction or operation step can be carried out in sequence or not. Preferably, the reaction method herein is carried out in sequence.

[0038] Unless otherwise specified, the professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content may also be applied to the present invention.

[0039] In a first aspect, a method for constructing a ginseng extract fingerprint is provided, the method comprising analyzing a sample to be tested by high performance liquid chromatography to obtain a ginseng extract fingerprint.

[0040] In an optional embodiment, the ginseng extract is a ginseng extract produced by Guangzhou Zeli Pharmaceutical Technology Co., Ltd.'s high-efficiency, high-pressure difference, low-temperature continuous extraction, concentration and separation technology (HHLSE) and complete set of equipment (Heli Intelligent Low-temperature and High-efficiency Extraction Complete Set of Equipment).

[0041] The analysis conditions of HPLC were:

[0042] Chromatographic column: The filler of the chromatographic column is octadecylsilane bonded silica gel. In an optional embodiment, the chromatographic column is Platisil ODS, with a specification of 250 mm×4.6 mm and a 5 μm chromatographic column.

[0043] In an optional embodiment, the column temperature of the chromatographic column is 25-35°C, preferably 30°C.

[0044] Mobile phase: Mobile phase A is acetonitrile and mobile phase B is water.

[0045] In an optional embodiment, the flow rate is 0.8 to 1.2 mL / min, preferably 1 mL / min.

[0046] Elution program: Calculated by volume percentage, the gradient elution program is:

[0047] Table 1

[0048] time Mobile phase Av / v% Mobile phase B v / v% 0min 15％ 85％ 20min 30％ 70％ 40min 35％ 65％ 60min 45％ 55％ 65min 45％ 55 90min 75％ 25％

[0049] The detection wavelength is 200-400 nm, for example, but not limited to 200, 210, 220, 250, 280, 300, 350 or 400 nm. Optionally, the detection wavelength is 203-210 nm.

[0050] In an optional embodiment, the sample to be tested includes a test solution, and the test solution is an aqueous solution of ginseng extract.

[0051] In an optional embodiment, the test solution is an aqueous solution of ginseng extract, and the concentration of ginseng extract is 0.02 g / mL.

[0052] In an optional embodiment, the sample to be tested also includes a reference solution, which is a methanol solution, and the reference solution contains at least one of the following components: ginsenoside Re, ginsenoside Rb1, ginsenoside 20(R)-Rh2, ginsenoside 20(S)-Rg2 and ginsenoside 20(R)-Rg3.

[0053] In an optional embodiment, each 1 mL of the reference solution contains 122.9 μg of ginsenoside Re.

[0054] In an optional embodiment, each 1 mL of the reference solution contains 94.3 μg of ginsenoside Rb1.

[0055] In an optional embodiment, each 1 mL of the reference solution contains 219.5 μg of ginsenoside 20(R)-Rh2.

[0056] In an optional embodiment, each 1 mL of the reference solution contains 105.8 μg of ginsenoside 20(S)-Rg2.

[0057] In an optional embodiment, each 1 mL of the reference solution contains 145 μg of ginsenoside 20(R)-Rg3.

[0058] In an optional embodiment, the construction method further comprises providing fingerprints of different batches of test samples, calibrating common peaks and synthesizing a control fingerprint.

[0059] In an optional embodiment, the control fingerprint has a total of 11 common peaks, peak 1 is identified as ginsenoside Re, peak 2 is identified as ginsenoside Rb1, peak 7 is identified as ginsenoside 20(S)-Rg3, peak 9 is identified as ginsenoside 20(R)-Rg3, and peak 10 is identified as ginsenoside 20(R)-Rh2, with the ginsenoside Rb1 peak in the fingerprint being used as the reference peak.

[0060] In a specific embodiment, the method for constructing the fingerprint of ginseng extract comprises:

[0061] (a) Preparation of test solution: Take about 0.5 g of ginseng extract, weigh accurately, dissolve it in pure water and make up to volume in a 25 mL volumetric flask, shake well, and filter through a 0.45 μm microporous filter membrane to obtain the solution.

[0062] (b) Preparation of reference solution: Accurately weigh appropriate amounts of ginsenoside Re, ginsenoside Rb1, rare ginsenoside 20(R)-Rh2, rare ginsenoside 20(S)-Rg2 and rare ginsenoside 20(R)-Rg3 standards, and use methanol as solvent to prepare 1 mL of a solution containing 122.9 μg, 94.3 μg, 219.5 μg, 105.8 μg and 145 μg of ginsenoside Re, ginsenoside Rb1, ginsenoside 20(R)-Rh2, ginsenoside 20(S)-Rg3 and 20(R)-Rg3, respectively. This is the reference solution.

[0063] (c) Accurately pipette 20 μL of the reference solution and the test solution, respectively, inject them into the high performance liquid chromatograph, record the spectrum, and process the spectrum with the fingerprint spectrum software to obtain the fingerprint spectrum of the ginseng extract. The chromatographic conditions are as follows: the chromatographic column is: Platisil ODS (250 mm × 4.6 mm, 5 μm); acetonitrile is used as the mobile phase A, water is used as the mobile phase B, gradient elution, flow rate is 1.0 mL / min, column temperature is 30°C, and detection wavelength is 203 nm. The gradient elution program is shown in Table 1 above.

[0064] In a second aspect, a ginseng extract quality control method is also provided, the method comprising using the construction method described in the first aspect to obtain a fingerprint of the ginseng extract to be evaluated, comparing the fingerprint of the ginseng extract to be evaluated with a control fingerprint, calculating the similarity between the fingerprint of the ginseng extract to be evaluated and the control fingerprint, and if the similarity is ≥90%, the quality of the ginseng extract to be evaluated is determined to be qualified.

[0065] In an optional embodiment, if the similarity between the fingerprint of the ginseng extract to be evaluated and the control fingerprint is at least 90%, at least 92%, at least 95%, at least 97% or at least 99%, the ginseng extract to be evaluated is judged to be of qualified quality.

[0066] In an optional embodiment, the reference fingerprint is at least one fingerprint of a ginseng extract of known qualified quality obtained using the construction method described in the first aspect.

[0067] In an optional embodiment, the reference fingerprint is a reference fingerprint synthesized from multiple fingerprints of ginseng extracts of known qualified quality obtained using the construction method described in the first aspect.

[0068] In an optional embodiment, the control fingerprint has 11 common peaks, peak 1 is identified as ginsenoside Re, peak 2 is identified as ginsenoside Rb1, peak 7 is identified as ginsenoside 20(S)-Rg3, peak 9 is identified as ginsenoside 20(R)-Rg3, and peak 10 is identified as ginsenoside 20(R)-Rh2;

[0069] Taking the ginsenoside Rb1 peak in the control fingerprint as the reference peak, the relative retention times of the remaining peaks are as follows:

[0070] Peak 1: 0.530~0.550, peak 3: 1.070~1.075, peak 4: 1.130~1.145, peak 5: 1.250~1.280, peak 6: 1.530~1.580, peak 7: 1.735~1.785, peak 8: 1.750~1.805, peak 9: 1.780~1.835, peak 10: 2.025~2.085, peak 11: 2.105~2.165.

[0071] In an optional embodiment, the ginsenoside Rb1 peak in the control fingerprint is used as the reference peak, and the relative retention times of the remaining peaks are as follows: Peak 1: 0.534-0.548, Peak 3: 1.070-1.076, Peak 4: 1.130-1.143, Peak 5: 1.252-1.276, Peak 6: 1.539-1.579, Peak 7: 1.737-1.784, Peak 8: 1.753-1.804, Peak 9: 1.782-1.833, Peak 10: 2.029-2.084, Peak 11: 2.108-2.165.

[0072] In an optional embodiment, in the control fingerprint, the ginsenoside Rb1 peak in the control fingerprint is used as the reference peak, and the relative peak areas of the remaining peaks are as follows:

[0073] Peak 1: 0.860~0.890, peak 3: 0.545~0.585, peak 4: 0.270~0.285, peak 5: 0.355~0.380, peak 6: 0.053~0.057, peak 7: 0.078~0.082, peak 8: 0.040~0.045, peak 9: 0.029~0.030, peak 10: 0.075~0.084, peak 11: 0.045~0.048.

[0074] In an optional embodiment, in the control fingerprint, the ginsenoside Rb1 peak in the control fingerprint is used as the reference peak, and the relative peak areas of the remaining peaks are as follows:

[0075] Peak 1: 0.835~0.887, peak 3: 0.540~0.585, peak 4: 0.272~0.291, peak 5: 0.355~0.380, peak 6: 0.051~0.057, peak 7: 0.077~0.082, peak 8: 0.041~0.044, peak 9: 0.029~0.030, peak 10: 0.075~0.084, peak 11: 0.044~0.048.

[0076] In a third aspect, a method for constructing a fingerprint of a ginseng extract according to the first aspect, or an application of the quality control method of a ginseng extract according to the second aspect in the preparation of a drug containing a ginseng extract is provided.

[0077] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0078] The test sample in the following embodiment is a ginseng extract produced by the high-efficiency high-pressure difference low-temperature continuous extraction concentration separation technology (HHLSE) and complete equipment (Heli Intelligent Low-temperature High-efficiency Extraction Complete Equipment) of Guangzhou Zeli Pharmaceutical Technology Co., Ltd. The specific extraction method is: the raw material is crushed and 11 times 60% ethanol is added and mixed evenly, and then extracted (cyclone extraction, not less than 12h), ethanol recovery, membrane filtration, vacuum concentration (concentrated to a soluble solid content of 31-35%) to prepare a concentrated solution, and the alcohol extraction centrifugal residue is added 9 times purified water is added for extraction (extraction pressure 30-40Mpa, temperature 20-25°C, extraction time is nearly instantaneous, less than 0.2 seconds), membrane filtration, membrane concentration, vacuum concentration (concentrated to a soluble solid content of 16-20%) to prepare a concentrated solution, the alcohol-extracted concentrated solution and the water-extracted concentrated solution are mixed and sterilized and filtered, and an appropriate amount of maltodextrin is added at a ratio of 6:4 of filtrate to dryness and maltodextrin, and spray-dried slurry adjustment, spray drying, powder screening and total mixing are performed to prepare a spray-dried powder (input: output = 1: 0.4-0.6).

[0079] Example 1

[0080] 1. Preparation conditions of test solution:

[0081] (1) Preparation of test solution: Take about 0.5 g of ginseng extract, weigh accurately, dissolve it in pure water and dilute it to 25 mL volumetric flask, shake well, filter through a 0.45 μm microporous filter membrane, and accurately pipette 20 μL into a high performance liquid chromatograph for determination.

[0082] (2) Preparation of standards: Ginsenoside Re, ginsenoside Rb1, rare ginsenoside 20(R)-Rh2, rare ginsenoside 20(S)-Rg3 and rare ginsenoside 20(R)-Rg3 standards were dissolved in methanol to obtain ginsenoside standards. Each 1 mL of ginsenoside Re reference solution contains 122.9 μg of ginsenoside Re, each 1 mL of ginsenoside Rb1 reference solution contains 94.3 μg of ginsenoside Rb1, each 1 mL of ginsenoside 20(R)-Rh2 reference solution contains 219.5 μg of ginsenoside 20(R)-Rh2, each 1 mL of ginsenoside 20(S)-Rg2 reference solution contains 105.8 μg of ginsenoside 20(S)-Rg2, and each 1 mL of reference solution contains 145 μg of ginsenoside 20(R)-Rg3.

[0083] 2. Chromatographic conditions:

[0084] (1) Chromatographic column: Agilent 1100 chromatographic column: Platisil ODS (250 mm × 4.6 mm, 5 μm);

[0085] (2) Mobile phase: Mobile phase A: acetonitrile; Mobile phase B: water;

[0086] (3) Elution conditions:

[0087] Table 1 Mobile phase gradient elution conditions

[0088] time Acetonitrile (A) v / v% Water (B) v / v% 0min 15％ 85％ 20min 30％ 70％ 40min 35％ 65％ 60min 45％ 55％ 65min 45％ 55 90min 75％ 25％

[0089] (4) Detection wavelength: 203 nm;

[0090] (5) Column temperature: 30°C;

[0091] (6) Flow rate: 1 mL / min;

[0092] The above chromatographic conditions were used to detect the test solution, and the chromatogram was as follows: Figure 1 The above chromatographic conditions were used to detect the standard solution, and the chromatogram was as shown in Figure 2 As shown, it was found that the ginseng extract contained ginsenoside Re, ginsenoside Rb1, rare ginsenoside 20(R)-Rh2, rare ginsenoside 20(S)-Rg3 and rare ginsenoside 20(R)-Rg3.

[0093] Example 2 Establishment of control fingerprint

[0094] In order to detect the specificity of the fingerprint of ginseng extract, samples from different batches were collected. The sample data are as follows:

[0095] S1(2020101), S2(20201101), S3(20210101), S4(20211201), S5(20220101), S6(20221101), S7(20230101), S8(20231201)

[0096] Weigh 0.5 g of each S1-S8 ginseng extract respectively, prepare the test solution according to the method of Example 1, and detect according to the liquid phase conditions under Example 1 to obtain the common peak pattern (11 common peaks) of 8 batches of samples ( Figure 3 ) and ginseng extract reference fingerprint ( Figure 4 ).

[0097] Example 3 Comparison of different mobile phases

[0098] 1. Preparation conditions of test solution:

[0099] (1) Preparation of test solution: Take about 0.5 g of ginseng extract, weigh accurately, dissolve it in pure water and dilute it to 25 mL volumetric flask, shake well, filter through a 0.45 μm microporous filter membrane, and accurately pipette 20 μL into a high performance liquid chromatograph for determination.

[0100] 2. Chromatographic conditions:

[0101] Different mobile phases: mobile phase: S1 (mobile phase A: acetonitrile; mobile phase B: 0.01% phosphoric acid), S2 (mobile phase A: acetonitrile; mobile phase B: 0.1% phosphoric acid), S3 (mobile phase A: acetonitrile; mobile phase B: water), other chromatographic conditions are the same as in Example 1.

[0102] Test results such as Figure 5 As shown, acetonitrile (mobile phase A)-water (mobile phase B) as the elution mobile phase has a large number of chromatographic peaks and good separation, and the baseline is relatively stable. Under the chromatographic conditions of Example 1, acetonitrile (mobile phase A)-water (mobile phase B) has the best effect.

[0103] Example 4 Comparison of different elution gradient conditions

[0104] 1. Preparation conditions of test solution:

[0105] (1) Preparation of test solution: Take about 0.5 g of ginseng extract, weigh accurately, dissolve it in pure water and dilute it to 25 mL volumetric flask, shake well, filter through a 0.45 μm microporous filter membrane, and accurately pipette 20 μL into a high performance liquid chromatograph for determination.

[0106] 2. Chromatographic conditions:

[0107] (1) Chromatographic column: Platisil ODS (250 mm × 4.6 mm, 5 μm); mobile phase: mobile phase A: acetonitrile; mobile phase B: water; injection volume 20 μL; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 203 nm;

[0108] (2) Elution was performed according to the following elution conditions, where the gradient was calculated as a percentage of mobile phase A, and the percentage of mobile phase A and mobile phase B was 100%:

[0109] Gradient 1: 0-20 min, 15-35% A, 20-30 min, 35-35% A, 30-45 min, 35-45% A, 45-50 min, 45-45% A, 50-80 min, 45-75% A;

[0110] Gradient 2: 0-25min, 15-35%A, 25-35min, 35-35%A, 35-55min, 35-45%A, 55-60min, 45-45%A, 60-80min, 45-75%A;

[0111] Gradient 3: 0-25 min, 15-35% A, 25-65 min, 35-45% A, 60-90 min, 45-75% A;

[0112] Gradient 4: 0-25min, 15-35%A, 25-55min, 35-40%A, 55-65min, 40-45%A, 65-70min, 45-45%A, 70-90min, 45-75%A;

[0113] Gradient 5: 0-25min, 15-35%A, 25-35min, 35-35%A, 35-65min, 35-45%A, 65-70min, 45-45%A, 70-90min, 45-75%A;

[0114] Gradient 6: 0-5min, 15-20%A, 5-20min, 20-35%A, 20-30min, 35-35%A, 30-50min, 35-45%A, 50-55min, 45-45%A, 55-80min, 45-75%A;

[0115] Gradient 7: 0-20min, 15-30%A, 20-40min, 30-35%A, 40-60min, 35-45%A, 60-65min, 45-45%A, 65-90min, 45-75%A.

[0116] Gradient 7 is the elution condition of Example 1.

[0117] 3. Test results: Fingerprints under different elution gradient conditions are as follows Figure 6 As shown, from Figure 6 It can be seen that the number of chromatographic peaks eluted by gradient 7 is the largest, the chromatographic peak separation is better, and the baseline is relatively stable. Therefore, under the chromatographic conditions of Example 1, gradient 7 has the best effect.

[0118] Example 5 Comparison of different chromatographic columns

[0119] 1. Preparation conditions of test solution:

[0120] (1) Preparation of test solution: Take about 0.5 g of ginseng extract, weigh accurately, dissolve it in pure water and dilute it to 25 mL volumetric flask, shake well, filter through a 0.45 μm microporous filter membrane, and accurately pipette 20 μL into a high performance liquid chromatograph for determination.

[0121] 2. Chromatographic conditions:

[0122] (1) Different chromatographic columns: S1: Diamonsil C18 (250 mm × 4.6 mm, 5 μm), S2: Agilent ZORBAXSB-C18 (250 mm × 4.6 mm, 5 μm), S3: Platisil ODS (250 mm × 4.6 mm, 5 μm);

[0123] (2) Injection volume 20 μL; flow rate 1.0 mL / min; column temperature 30°C; detection wavelength 203 nm; gradient as follows:

[0124] Gradient 7: 0-20min, 15-30%A, 20-40min, 30-35%A, 40-60min, 35-45%A, 60-65min, 45-45%A, 65-90min, 45-75%A;

[0125] Test results such as Figure 7 As shown: In the chromatogram of S3 (Platisil ODS), the chromatographic peak intensity is relatively high and the chromatographic peak separation is optimal. Therefore, under the chromatographic conditions of Example 1, the Platisil ODS chromatographic column is the best.

[0126] Example 6 Methodological Investigation

[0127] The following methodological investigations all adopted the chromatographic conditions of Example 1.

[0128] 1. Precision test

[0129] Take ginseng extract, prepare the test solution according to the method of Example 1, inject the sample 6 times continuously, and record each chromatogram. Figure 8 As shown. The retention time and peak area of ​​each common peak were investigated, and ginsenoside Rb1 (peak 2) was used as the reference peak to calculate the relative peak area RSD and relative retention time RSD of each common peak. The similarity between each spectrum was calculated using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software. The results showed that the RSD of the relative retention time of each common peak was ≤0.4% (as shown in Table 2), the RSD of the relative peak area was ≤3% (as shown in Table 3), and the similarity of each chromatogram was above 0.99, indicating that the instrument had good precision and met the technical requirements of the fingerprint spectrum.

[0130] Table 2 Relative retention time of common peaks in precision test (n=6)

[0131]

[0132]

[0133] Table 3 Precision test common peak relative peak area (n = 6)

[0134]

[0135] 2. Repeatability test

[0136] Take ginseng extract, prepare the test solution according to the method of Example 1, inject the sample 6 times continuously, and record each chromatogram. Fig. 9, the retention time and peak area of ​​each common peak were investigated, and ginsenoside Rb1 (peak 2) was used as the reference peak to calculate the relative peak area RSD and relative retention time RSD of each common peak. The similarity between each spectrum was calculated using the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software. The results showed that the relative retention time RSD of each common peak was ≤1.5% (as shown in Table 4), the relative peak area RSD was ≤3% (as shown in Table 5), and the similarity of each chromatogram was above 0.99, indicating that the method had good repeatability and met the technical requirements of fingerprint.

[0137] Table 4 Relative retention time of common peaks in repeated experiments (n=6)

[0138]

[0139] Table 5 Relative peak areas of common peaks in repeatability test (n=6)

[0140]

[0141]

[0142] 3. Stability test

[0143] Take ginseng extract and prepare the test solution according to the method of Example 1. Inject the sample at 0, 3, 6, 9, 12, and 24 hours respectively, and record each chromatogram. Fig.10 , the retention time and peak area of ​​each common peak were investigated, and ginsenoside Rb1 (peak 2) was used as the reference peak to calculate the relative peak area RSD and relative retention time RSD of each common peak. The similarity between each spectrum was calculated using the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software. The results showed that the relative retention time RSD of each common peak was ≤0.5% (as shown in Table 6), the relative peak area RSD was ≤3% (as shown in Table 7), and the similarity of each chromatogram was above 0.99, indicating that the sample stability was good within 24 hours and met the technical requirements of the fingerprint spectrum.

[0144] Table 6 Relative retention time of common peaks in stability test (n=6)

[0145]

[0146] Table 7 Relative peak areas of common peaks in stability test (n=6)

[0147]

[0148]

[0149] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a fingerprint of a ginseng extract, characterized in that: The method comprises adopting high performance liquid chromatography to analyze the sample to be tested to obtain a fingerprint spectrum of the ginseng extract; The analytical conditions for HPLC include: The filler of the chromatographic column is octadecylsilane bonded silica gel; Mobile phase: Mobile phase A is acetonitrile, mobile phase B is water; The gradient elution procedure is as follows, based on volume percentage: The detection wavelength is 200~400nm.

2. The construction method according to claim 1, characterized in that: The chromatographic column is Platisil ODS, with specifications of 250 mm × 4.6 mm, and a 5 μm chromatographic column.

3. The construction method according to claim 1, characterized in that: The column temperature is 25-35°C, preferably 30°C; Optionally, the mobile phase flow rate is 0.8 to 1.2 mL / min, preferably 1 mL / min; Optionally, the detection wavelength is 203-210 nm.

4. The construction method according to claim 1, characterized in that: The sample to be tested includes a test solution, and the test solution is an aqueous solution of ginseng extract; Optionally, the test solution is an aqueous solution of ginseng extract, and the concentration of ginseng extract is 0.02 g / mL; Optionally, the sample to be tested also includes a reference solution, which is a methanol solution and contains at least one of the following components: ginsenoside Re, ginsenoside Rb1, ginsenoside 20(R)-Rh2, ginsenoside 20(S)-Rg2 and ginsenoside 20(R)-Rg3.

5. The construction method according to any one of claims 1 to 4, characterized in that: The construction method also includes providing fingerprints of different batches of test samples, calibrating common peaks and synthesizing control fingerprints.

6. The construction method according to claim 5, characterized in that: The control fingerprint has a total of 11 common peaks, peak 1 is identified as ginsenoside Re, peak 2 is identified as ginsenoside Rb1, peak 7 is identified as ginsenoside 20(S)-Rg3, peak 9 is identified as ginsenoside 20(R)-Rg3, and peak 10 is identified as ginsenoside 20(R)-Rh2, with the ginsenoside Rb1 peak in the fingerprint being used as the reference peak.

7. A method for controlling the quality of ginseng extract, characterized in that: The method comprises obtaining a fingerprint of the ginseng extract to be evaluated by using the construction method according to any one of claims 1 to 6, comparing the fingerprint of the ginseng extract to be evaluated with a reference fingerprint, calculating the similarity between the fingerprint of the ginseng extract to be evaluated and the reference fingerprint, and if the similarity is ≥90%, determining that the ginseng extract to be evaluated is of qualified quality; Optionally, the reference fingerprint is at least one fingerprint of a ginseng extract of known qualified quality obtained using the construction method described in any one of claims 1 to 6.

8. The method for controlling the quality of ginseng extract according to claim 7, characterized in that: The control fingerprint has 11 common peaks, peak 1 is identified as ginsenoside Re, peak 2 is identified as ginsenoside Rb1, peak 7 is identified as ginsenoside 20(S)-Rg3, peak 9 is identified as ginsenoside 20(R)-Rg3, and peak 10 is identified as ginsenoside 20(R)-Rh2; Taking the ginsenoside Rb1 peak in the control fingerprint as the reference peak, the relative retention times of the remaining peaks are as follows: Peak 1: 0.530~0.550, peak 3: 1.070~1.075, peak 4: 1.125~1.145, peak 5: 1.240~1.280, peak 6: 1.530~1.580, peak 7: 1.735~1.785, peak 8: 1.750~1.805, peak 9: 1.780~1.835, peak 10: 2.025~2.085, peak 11: 2.105~2.

165.

9. The method for quality control of ginseng extract according to claim 8, characterized in that: Taking the ginsenoside Rb1 peak in the control fingerprint as the reference peak, the relative peak areas of the remaining peaks are as follows: Peak 1: 0.845~0.890, peak 3: 0.545~0.585, peak 4: 0.280~0.290, peak 5: 0.355~0.380, peak 6: 0.053~0.057, peak 7: 0.078~0.082, peak 8: 0.040~0.045, peak 9: 0.029~0.030, peak 10: 0.075~0.084, peak 11: 0.045~0.

048.

10. Use of the method for constructing the fingerprint of ginseng extract according to any one of claims 1 to 6, or the method for quality control of ginseng extract according to any one of claims 7 to 9 in the preparation of a drug containing ginseng extract.

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