Method for detecting content of active ingredients of traditional Chinese medicine

Abstract

The invention discloses a traditional Chinese medicine active ingredient content detection method which comprises the following steps: preparing standard substances of traditional Chinese medicine active ingredients to obtain standard substance solutions with different concentrations; respectively carrying out chromatographic analysis on a sample containing the traditional Chinese medicine active ingredients and the standard substance solution, and carrying out linear regression by taking the sample injection concentration of the standard substance solution as a horizontal coordinate and the peak area as a vertical coordinate to obtain a linear regression equation; determining the peak area of the traditional Chinese medicine active ingredients in the sample according to the chromatographic analysis result, and substituting the peak area into a linear regression equation to obtain the content of the traditional Chinese medicine active ingredients, according to the detection method, the content of the traditional Chinese medicine active ingredients in the sample can be detected by limiting a specific process, the detection is rapid, the sensitivity is high, and a technical basis is provided for traditional Chinese medicine research.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and particularly relates to a method for detecting the content of active ingredients in traditional Chinese medicine. Background Art

[0002] The active ingredients of traditional Chinese medicine are the material basis for treating diseases and an important source for finding new lead compounds in innovative drugs. However, due to the complexity of the composition of traditional Chinese medicine itself, the integrity of its effects, and the unclear mechanism of action, clarifying the active ingredients and their mechanism of action responsible for the efficacy of traditional Chinese medicine remains a key issue in the research of traditional Chinese medicine.

[0003] In addition, traditional Chinese medicine generally needs to be decocted or extracted during the taking process to extract the active ingredients in the traditional Chinese medicine. However, the extraction content of the active ingredients in traditional Chinese medicine directly affects the overall efficacy. Therefore, it is particularly crucial to detect the content of the active ingredients in the extracted samples of traditional Chinese medicine.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting the content of active ingredients in traditional Chinese medicine. The detection method of the present invention can detect the content of active ingredients in the sample, with rapid detection and high sensitivity, providing a technical basis for the research of traditional Chinese medicine.

[0006] In order to achieve the above object of the present invention, the following technical solutions are specifically adopted: The first aspect of the present invention provides a method for detecting the content of active ingredients in traditional Chinese medicine, and the detection method includes the following steps: (a) Prepare standard products of active ingredients in traditional Chinese medicine to obtain standard product solutions with different concentrations; (b) Perform chromatographic analysis on the sample containing the active ingredients in traditional Chinese medicine and the standard product solution respectively, and perform linear regression with the injection concentration of the standard product solution as the abscissa and the peak area as the ordinate to obtain a linear regression equation; (c) According to the chromatographic analysis results, determine the peak area of the active ingredients in traditional Chinese medicine in the sample, and then substitute the peak area into the linear regression equation to obtain the content of the active ingredients in traditional Chinese medicine.

[0007] Preferably, the active ingredients in traditional Chinese medicine include ligustilide, α - cyperone, and atractylodin.

[0008] Preferably, when the active ingredient of traditional Chinese medicine is ligustilide, standard solutions with concentrations of 0.001 - 0.004 mg / mL, 0.004 - 0.007 mg / mL, 0.01 - 0.02 mg / mL, 0.02 - 0.03 mg / mL, 0.04 - 0.05 mg / mL, 0.07 - 0.1 mg / mL, and 0.15 - 0.2 mg / mL are prepared; When the active ingredient of traditional Chinese medicine is α - cyperone, standard solutions with concentrations of 0.0002 - 0.0003 mg / mL, 0.0004 - 0.0006 mg / mL, 0.001 - 0.002 mg / mL, 0.002 - 0.003 mg / mL, 0.004 - 0.005 mg / mL, 0.006 - 0.009 mg / mL, and 0.01 - 0.02 mg / mL are prepared; When the active ingredient of traditional Chinese medicine is atractylodin, standard solutions with concentrations of 0.0004 - 0.0006 mg / mL, 0.001 - 0.002 mg / mL, 0.002 - 0.003 mg / mL, 0.004 - 0.005 mg / mL, 0.007 - 0.009 mg / mL, 0.01 - 0.02 mg / mL, and 0.03 - 0.04 mg / mL are prepared.

[0009] Preferably, the chromatographic column used in the chromatographic analysis process is an Elite commercial C18 column.

[0010] Preferably, the length of the chromatographic column is 40 - 60 mm, the inner diameter is 4 - 5 mm, and the particle size of the packing is 4 - 6 μm.

[0011] Preferably, in the chromatographic analysis process: Mobile phase A is acetonitrile, and mobile phase B is water; the gradient elution program is 60% mobile phase A from 0 - 24 min, 60% - 45% mobile phase A from 24 - 34 min, 45% - 60% mobile phase A from 34 - 45 min, and 60% mobile phase A from 45 - 55 min; the detection wavelengths are 254 nm, 330 nm, and 340 nm; the flow rate is 0.8 - 1.2 mL / min; the column temperature is 22 - 28 °C.

[0012] Preferably, the sample containing the active ingredient of traditional Chinese medicine is a traditional Chinese medicine extract.

[0013] Preferably, before chromatographic analysis, a 0.4 - 0.5 µm microporous filter membrane is used to filter the sample containing the active ingredient of traditional Chinese medicine and the standard solution.

[0014] Compared with the prior art, the beneficial effects of the present invention at least include: The detection method of the present invention can detect the content of active ingredients in traditional Chinese medicine in samples through the limitation of specific processes, with fast detection and high sensitivity. Specifically, by setting chromatographic conditions, such as mobile phase, gradient elution program, chromatographic column, etc., it can achieve the detection of the contents of ligustilide, α-cyperone, and atractylodin in samples. The lowest detectable limit of ligustilide can reach 2.7 μg / mL, that of α-cyperone can reach 0.266 μg / mL, and that of atractylodin can reach 0.499 μg / mL. It can be seen that the detection method of this application has high detection sensitivity and provides a technical basis for traditional Chinese medicine research. Description of the Drawings

[0015] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0016] Figure 1 It is the liquid chromatogram of the mixed standard solution in the embodiment of the present invention; Figure 2 It is the liquid chromatogram of the extract of Xiangzhi Jieyu Prescription in the embodiment of the present invention. Detailed Embodiments

[0017] The following will describe in detail the embodiments of the technical solutions of the present invention in combination with the embodiments. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, so they are only examples and cannot be used to limit the protection scope of the present invention.

[0018] It should be noted that unless otherwise specified, the technical terms or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art to which the present invention belongs.

[0019] The embodiment of the present invention provides a method for detecting the content of active ingredients in traditional Chinese medicine, and the detection method includes the following steps: (a) Prepare standard products of active ingredients in traditional Chinese medicine to obtain standard solutions with different concentrations; (b) Perform chromatographic analysis on the samples containing active ingredients in traditional Chinese medicine and the standard solutions respectively, perform linear regression with the injection concentration of the standard solution as the abscissa and the peak area as the ordinate to obtain a linear regression equation; (c) According to the chromatographic analysis results, determine the peak area of the active ingredients in traditional Chinese medicine in the sample, and then substitute the peak area into the linear regression equation to obtain the content of the active ingredients in traditional Chinese medicine.

[0020] The detection method of the present invention can detect the content of traditional Chinese medicine active ingredients in a sample through the limitation of specific processes, with fast detection speed and high sensitivity. Specifically, through the setting of chromatographic conditions, such as mobile phase, gradient elution program, chromatographic column, etc., the content detection of ligustilide, α-cyperone and atractylodin in the sample can be realized. The lowest detectable amount of ligustilide can reach 2.7 μg / mL, the lowest detectable amount of α-cyperone can reach 0.266 μg / mL, and the lowest detectable amount of atractylodin can reach 0.499 μg / mL. It can be seen that the detection method of this application has high detection sensitivity and provides a technical basis for traditional Chinese medicine research.

[0021] In some embodiments, the traditional Chinese medicine active ingredients include ligustilide, α-cyperone and atractylodin.

[0022] In some embodiments, when the traditional Chinese medicine active ingredient is ligustilide, standard solutions with concentrations of 0.001 - 0.004 mg / mL, 0.004 - 0.007 mg / mL, 0.01 - 0.02 mg / mL, 0.02 - 0.03 mg / mL, 0.04 - 0.05 mg / mL, 0.07 - 0.1 mg / mL and 0.15 - 0.2 mg / mL are prepared; When the traditional Chinese medicine active ingredient is α-cyperone, standard solutions with concentrations of 0.0002 - 0.0003 mg / mL, 0.0004 - 0.0006 mg / mL, 0.001 - 0.002 mg / mL, 0.002 - 0.003 mg / mL, 0.004 - 0.005 mg / mL, 0.006 - 0.009 mg / mL and 0.01 - 0.02 mg / mL are prepared; When the traditional Chinese medicine active ingredient is atractylodin, standard solutions with concentrations of 0.0004 - 0.0006 mg / mL, 0.001 - 0.002 mg / mL, 0.002 - 0.003 mg / mL, 0.004 - 0.005 mg / mL, 0.007 - 0.009 mg / mL, 0.01 - 0.02 mg / mL and 0.03 - 0.04 mg / mL are prepared.

[0023] In one embodiment, the chromatographic column used in the chromatographic analysis process is an Elite commercial C18 column.

[0024] In one embodiment, the length of the chromatographic column is 40 - 60 mm, the inner diameter is 4 - 5 mm, and the filler particle size is 4 - 6 μm.

[0025] In one embodiment, during the chromatographic analysis process: Mobile phase A is acetonitrile and mobile phase B is water; the gradient elution program is as follows: 0 - 24 min, 60% mobile phase A; 24 - 34 min, 60% - 45% mobile phase A; 34 - 45 min, 45% - 60% mobile phase A; 45 - 55 min, 60% mobile phase A; the detection wavelengths are 254 nm, 330 nm, and 340 nm; the flow rate is 0.8 - 1.2 mL / min; the column temperature is 22 - 28 °C.

[0026] In one embodiment, the sample containing the active ingredients of traditional Chinese medicine is an extract of traditional Chinese medicine, such as the extract of the raw materials of Xiangzhi Jieyu Pills.

[0027] In one embodiment, before chromatographic analysis, the sample containing the active ingredients of traditional Chinese medicine and the standard solution are filtered through a 0.4 - 0.5 µm microporous filter membrane.

[0028] The technical solution of the present invention will be further described in detail through specific examples below.

[0029] Example This example is a method for detecting the contents of three active ingredients of traditional Chinese medicine (ligustilide, α - cyperone, and atractylodin) in Xiangzhi Jieyu Pills. The detection method includes the following steps: 1. Experimental method 1.1 Chromatographic conditions Chromatographic conditions: The chromatographic column is an Elite commercial C 18 Column (5 μm, 4.6 mm × 25 cm); using acetonitrile (A) - water (B) as the mobile phase; the gradient elution program is: 0 - 24 min, 60% A; 24 - 34 min, 60% - 45% A; 34 - 45 min, 45% - 60% A; 45 - 55 min, 60% A, the detection wavelengths are 254 nm, 330 nm, and 340 nm; the flow rate is 1 mL / min; the column temperature is 25 °C.

[0030] The gradient elution program is shown in Table 1: Table 1 Time (min) Mobile phase A (%) Mobile phase B (%) 0 60 40 24 60 40 34 45 55 45 60 40 55 60 40 1.2 Preparation of the standard solution: Preparation of reference substances and linear series working solutions: ①Accurately weigh 3.642 mg of ligustilide, place it in a 10 mL volumetric flask, dissolve it with methanol and dilute to the mark, shake well to obtain a reference substance stock solution with a concentration of 0.3642 mg / mL. Respectively take the reference substance mother liquor and dilute it to a series of working solutions with concentrations of 0.0027, 0.0054, 0.0108, 0.0216, 0.0433, 0.0866, and 0.173 mg / mL. Filter them respectively through a 0.45 μm microporous filter membrane, and the filtrate is used as the linear series working solution of the reference substance. ②Accurately weigh 0.34 mg of α-cyperone, place it in a 10 mL volumetric flask, dissolve it with methanol and dilute to the mark, shake well to obtain a reference substance stock solution with a concentration of 0.034 mg / mL. Respectively take the reference substance mother liquor and dilute it to a series of working solutions with concentrations of 0.00027, 0.00053, 0.00107, 0.00214, 0.0043, 0.0085, and 0.0171 mg / mL. Filter them respectively through a 0.45 μm microporous filter membrane, and the filtrate is used as the linear series working solution of the reference substance. ③Accurately weigh 0.639 mg of atractylenolide, place it in a 10 mL volumetric flask, dissolve it with methanol and dilute to the mark, shake well to obtain a reference substance stock solution with a concentration of 0.0639 mg / mL. Respectively take the reference substance mother liquor and dilute it to a series of working solutions with concentrations of 0.0005, 0.001, 0.002, 0.004, 0.008, 0.016, and 0.032 mg / mL. Filter them respectively through a 0.45 μm microporous filter membrane, and the filtrate is used as the linear series working solution of the reference substance.

[0031] 1.3 Preparation of sample solutions: Precisely measure 1 mL of the sample solution of the Xiangzhi Jieyu formula extract, place it in a 25 mL volumetric flask, make up the volume with methanol, shake well, filter it through a 0.45 µm microporous filter membrane, and take the filtrate to obtain the sample solution.

[0032] 2. Experimental results The chromatograms of the extract of the Xiangzhi Jieyu formula and the mixed reference substance solution are as Figure 2 , Figure 1 shown. The mixed reference substance solution is obtained by mixing equal volumes of the ligustilide reference substance mother liquor with a concentration of 0.3642 mg / mL, the α-cyperone reference substance mother liquor with a concentration of 0.034 mg / mL, and the reference substance mother liquor with a concentration of 0.0639 mg / mL. Take the reference substance solution and determine it under the above chromatographic conditions. Using the injection concentration (X) of the reference substance as the abscissa and the peak area (Y) as the ordinate, perform linear regression to obtain the linear regression equation. The results are shown in Table 2.

[0033] Table 2 Results of the investigation on the linear relationship of three active monomers Component Regression equation r Linear range Ligustilide y = 2E+07x + 26640 0.9987 2.70 μg / mL - 346.2 μg / mL α-Cyperone y = 4E+07x + 6396 0.9988 0.266 μg / mL - 34.17 μg / mL Atractylodin y = 9E+07x + 14040 0.9989 0.499 μg / mL - 63.9 μg / mL According to the obtained linear equation, the contents of ligustilide, α-cyperone and atractylodin in the extract of Xiangzhi Jieyu Prescription were calculated to be 11.42 mg / g, 0.78 mg / g and 2.7 mg / g respectively.

Claims

1. A method for detecting the content of active ingredients in traditional Chinese medicine, characterized in that, The detection method includes the following steps: (a) Prepare standard substances of traditional Chinese medicine active ingredients to obtain standard substance solutions with different concentrations; (b) Perform chromatographic analysis on the sample containing traditional Chinese medicine active ingredients and the standard substance solutions respectively. Take the injection concentration of the standard substance solution as the abscissa and the peak area as the ordinate to perform linear regression to obtain a linear regression equation; (c) According to the chromatographic analysis results, determine the peak area of the traditional Chinese medicine active ingredient in the sample, and then substitute the peak area into the linear regression equation to obtain the content of the traditional Chinese medicine active ingredient; The traditional Chinese medicine active ingredients include ligustilide, α-cyperone, and atractylodin; The chromatographic column used in the chromatographic analysis process is an Elite commercial C 18 column; the length of the chromatographic column is 40-60 mm, the inner diameter is 4-5 mm, and the filler particle size is 4-6 μm; During the chromatographic analysis process: mobile phase A is acetonitrile, and mobile phase B is water; the gradient elution program is 0 - 24 min, 60% mobile phase A; 24 - 34 min, 60% - 45% mobile phase A, 34 - 45 min, 45% - 60% mobile phase A, 45 - 55 min, 60% mobile phase A; the detection wavelengths are 254 nm, 330 nm, and 340 nm; the flow rate is 0.8 - 1.2 mL / min; the column temperature is 22 - 28 °C.

2. The detection method according to claim 1, wherein When the traditional Chinese medicine active ingredient is ligustilide, prepare standard substance solutions with concentrations of 0.001 - 0.004 mg / mL, 0.004 - 0.007 mg / mL, 0.01 - 0.02 mg / mL, 0.02 - 0.03 mg / mL, 0.04 - 0.05 mg / mL, 0.07 - 0.1 mg / mL, and 0.15 - 0.2 mg / mL; When the traditional Chinese medicine active ingredient is α-cyperone, prepare standard substance solutions with concentrations of 0.0002 - 0.0003 mg / mL, 0.0004 - 0.0006 mg / mL, 0.001 - 0.002 mg / mL, 0.002 - 0.003 mg / mL, 0.004 - 0.005 mg / mL, 0.006 - 0.009 mg / mL, and 0.01 - 0.02 mg / mL; When the traditional Chinese medicine active ingredient is atractylodin, prepare standard substance solutions with concentrations of 0.0004 - 0.0006 mg / mL, 0.001 - 0.002 mg / mL, 0.002 - 0.003 mg / mL, 0.004 - 0.005 mg / mL, 0.007 - 0.009 mg / mL, 0.01 - 0.02 mg / mL, and 0.03 - 0.04 mg / mL.

3. The detection method according to claim 1, characterized in that, The sample containing traditional Chinese medicine active ingredients is a traditional Chinese medicine extract.

4. The detection method according to claim 1, characterized in that, Before chromatographic analysis, filter the sample containing traditional Chinese medicine active ingredients and the standard substance solutions with a 0.4 - 0.5 µm microporous filter membrane.

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