Method for constructing fingerprint of Toosendan fruit preparation and method for identifying the same

Abstract

The present application relates to a method for constructing a fingerprint of a Toosendan preparation and a method for identifying the same. The method for constructing a fingerprint of the Toosendan preparation is a Toosendan standard decoction or a stir-fried Toosendan standard decoction; the method comprises the following steps: preparation of a reference solution: mixing a 5-hydroxymethylfurfural reference substance and a rutin reference substance with a first solvent to prepare the reference solution; preparation of a test solution; mixing the Toosendan preparation with a second solution, centrifuging, taking the supernatant for column chromatography, and collecting the eluate; then evaporating the eluate to dryness, dissolving it with a third solution, and preparing the test solution; the reference solution and the test solution are subjected to ultra-high performance liquid chromatography. The above-mentioned method has high separation and response values ​​of the characteristic peaks in the UPLC characteristic spectrum of Toosendan or stir-fried Toosendan standard decoction, good accuracy, specificity and reproducibility, and high detection efficiency.

Description

Technical Field

[0001] The present application relates to the technical field of traditional Chinese medicine analysis, and in particular to a method for constructing a fingerprint spectrum of a Toosendan fruit preparation and a method for identifying the same. Background Art

[0002] Toosendan fruit is the dried, mature fruit of the Meliaceae plant, Melia toosendan Sieb. et Zucc., of the Meliaceae family. It is bitter, cold, and slightly toxic, and has the effects of soothing the liver and relieving heat, promoting qi circulation and relieving pain, and killing insects. Stir-fried Toosendan fruit, which has reduced toxicity, is more suitable for clinical use and improves medication safety. Current efforts to distinguish and identify raw and stir-fried Toosendan fruit focus on the differences in properties and chemical composition between raw and stir-fried Toosendan fruit.

[0003] In addition, a method has been used to study the fingerprints of Toosendan Fructus and stir-fried Toosendan Fructus by high performance liquid chromatography (HPLC). The fingerprints identified 10 common characteristic peaks, suggesting that the processing mechanism of stir-fried Toosendan Fructus may be related to the changes in the content of chemical components. However, this method:

[0004] 1) Focus on comparative studies of decoction pieces, while traditional Chinese medicine is mainly used in the form of decoctions. Decoctions are the material basis for the clinical efficacy of Chinese medicine. Standard decoctions of Chinese medicine are single-ingredient decoction pieces prepared by standardized processes under the guidance of traditional Chinese medicine theory and based on clinical application, with reference to modern extraction methods. As a standard substance or standard system, they are often used to standardize the clinical use of Chinese medicine, standardize the clinical use of new Chinese medicine pieces such as Chinese medicine formula granules, and ensure the accuracy of clinical use of Chinese medicine. Standard decoctions use water as a solvent, indicating that water-soluble components should be an important material basis for the decoction to exert its efficacy. In addition, the chemical composition of Chinese medicine may also undergo certain changes during the decoction process. Therefore, it is difficult to reflect the effective components of standard decoctions in research on decoction pieces, especially the characteristics of water-soluble components that are more instructive for clinical use;

[0005] 2) The fingerprint spectrum identified a total of 10 common characteristic peaks, but this method had a long analysis time and poor separation, low response values ​​of the main chromatographic peaks, and a lack of identification of common peaks, making it difficult to reflect the intrinsic quality characteristics of Toosendan fruit and stir-fried Toosendan fruit.

[0006] In addition, this method does not clearly define the criteria for distinguishing between Toosendan fruit, stir-fried Toosendan fruit, and its counterfeit product, Melia toosendan fruit. Summary of the Invention

[0007] Based on this, the present application provides a method for constructing a fingerprint spectrum of Toosendan preparations and an identification method with more accurate detection results and shorter detection time.

[0008] In a first aspect of the present application, a method for constructing a fingerprint of a Toosendan fruit preparation is provided, wherein the Toosendan fruit preparation is a Toosendan fruit standard decoction, a stir-fried Toosendan fruit standard decoction, a Toosendan fruit traditional Chinese medicine formula granule, or a stir-fried Toosendan fruit traditional Chinese medicine formula granule; the construction method comprises the following steps:

[0009] Preparation of a reference solution: mixing a 5-hydroxymethylfurfural reference substance and a rutin reference substance with a first solvent to prepare the reference solution;

[0010] Preparation of a test solution: mixing the Toosendan preparation with the second solution, centrifuging, taking the supernatant for column chromatography, and collecting the eluate; then evaporating the eluate to dryness and dissolving it with the third solution to prepare the test solution;

[0011] The reference solution and the test solution are subjected to ultra-high performance liquid chromatography (UPLC) detection, wherein the conditions for the UPLC detection include the following items (1) to (3):

[0012] (1) Mobile phase A is acetonitrile and methanol in a volume ratio of (3-5):1, and mobile phase B is a 0.2% to 0.4% phosphoric acid aqueous solution. The elution gradient is as follows:

[0013] From 0 min to 8 min, the volume percentage of the mobile phase A changes from 3% to 8%;

[0014] From 8 min to 16 min, the volume percentage of the mobile phase A was changed from 8% to 11%;

[0015] From 16 min to 19 min, the volume percentage of the mobile phase A was changed from 11% to 19%;

[0016] From 19 min to 27 min, the volume percentage of the mobile phase A was changed from 19% to 20%;

[0017] From 27 min to 31 min, the volume percentage of the mobile phase A was changed from 20% to 36%;

[0018] From 31 to 38 minutes, the volume percentage of the mobile phase A was changed from 36% to 45%;

[0019] (2) Detection wavelength: 300nm-320nm from 0min to 19min; 350nm-370nm from 19min to 38min;

[0020] (3) Chromatographic column: Waters Cortecs T3 C 18 Chromatographic column.

[0021] In some embodiments, the ultra-high performance liquid chromatography detection conditions further include at least one of the following (1) to (3):

[0022] (1) Flow rate: 0.2–0.4 mL / min;

[0023] (2) Column temperature is 28°C to 32°C;

[0024] (3) The injection volume is 1 μL to 5 μL.

[0025] In some embodiments, the column chromatography conditions include: using a macroporous adsorption resin column as a chromatography column, performing elution and washing in sequence, the elution solution is water, the elution solution is an ethanol solution with a volume fraction of 50% to 100%, and collecting the solution of the elution process to obtain the eluate.

[0026] In some embodiments, the volume of the eluted solution is 30 mL to 70 mL; and / or,

[0027] The loading volume of the supernatant is 3 mL to 7 mL.

[0028] In some embodiments, the first solvent is a methanol solution with a volume fraction of 45% to 55%; and / or,

[0029] The second solution is water; and / or,

[0030] The third solution is a methanol solution with a volume fraction of 45% to 55%.

[0031] In some embodiments, the fingerprint spectrum includes 10 characteristic peaks, including the characteristic peaks of 5-hydroxymethylfurfural, vanillin, coniferaldehyde, rutin, threo-guaiac-β-coniferaldehyde ether and erythro-guaiac-β-coniferaldehyde ether.

[0032] In a second aspect of the present application, a method for identifying a Toosendan fruit preparation is provided, wherein the Toosendan fruit preparation includes at least two of Toosendan fruit standard decoction, stir-fried Toosendan fruit standard decoction, and Melia azedarach fruit standard decoction, or the Toosendan fruit preparation includes at least two of Toosendan fruit traditional Chinese medicine formula granules, stir-fried Toosendan fruit traditional Chinese medicine formula granules, and Melia azedarach fruit traditional Chinese medicine formula granules; the identification method comprises the following steps:

[0033] Preparation of a sample solution to be tested; mixing the Toosendan preparation with the second solution, centrifuging, taking the supernatant for column chromatography, and collecting the eluate; then evaporating the eluate to dryness and dissolving it with the third solution to prepare the sample solution to be tested;

[0034] The sample solution to be tested is subjected to ultra-high performance liquid chromatography to obtain a characteristic spectrum of the Toosendan fruit preparation and compare the spectrum;

[0035] The conditions for the ultra-high performance liquid chromatography detection include the following (1) to (3):

[0036] (1) Mobile phase A is acetonitrile and methanol in a volume ratio of (3-5):1, and mobile phase B is a 0.2% to 0.4% phosphoric acid aqueous solution. The elution gradient is as follows:

[0037] From 0 min to 8 min, the volume percentage of the mobile phase A changes from 3% to 8%;

[0038] From 8 min to 16 min, the volume percentage of the mobile phase A was changed from 8% to 11%;

[0039] From 16 min to 19 min, the volume percentage of the mobile phase A was changed from 11% to 19%;

[0040] From 19 min to 27 min, the volume percentage of the mobile phase A was changed from 19% to 20%;

[0041] From 27 min to 31 min, the volume percentage of the mobile phase A was changed from 20% to 36%;

[0042] From 31 to 38 minutes, the volume percentage of the mobile phase A was changed from 36% to 45%;

[0043] (2) Detection wavelength: 300nm-320nm from 0min to 19min; 350nm-370nm from 19min to 38min;

[0044] (3) Chromatographic column: Waters Cortecs T3 C 18 Chromatographic column.

[0045] In some embodiments, the ultra-high performance liquid chromatography detection conditions further include at least one of the following (1) to (3):

[0046] (1) Flow rate: 0.2–0.4 mL / min;

[0047] (2) Column temperature is 28°C to 32°C;

[0048] (3) The injection volume is 1 μL to 5 μL.

[0049] In some embodiments, the column chromatography conditions include: using a macroporous adsorption resin column as a chromatography column, performing elution and washing in sequence, the elution solution is water, the elution solution is an ethanol solution with a volume fraction of 50% to 100%, and collecting the solution of the elution process to obtain the eluate.

[0050] In some embodiments, the volume of the eluted solution is 30 mL to 70 mL; and / or,

[0051] The loading volume of the supernatant is 3 mL to 7 mL.

[0052] In some embodiments, the second solution is water; and / or,

[0053] The third solution is a methanol solution with a volume fraction of 45% to 55%.

[0054] In some embodiments, the comparison includes at least one of the following items (1) to (3):

[0055] (1) If the relative peak area ratio of Peak 1 to Peak S2 is less than 0.91, and the relative peak area ratio of the sum of Peaks 8 and 9 to the relative peak area of ​​Peak S2 is greater than or equal to 0.44, the Toosendan Fructus preparation is determined to be Toosendan Fructus standard decoction;

[0056] (2) If the relative peak area ratio of Peak 1 to Peak S2 is ≥0.91, and the relative peak area ratio of the sum of Peaks 8 and 9 to the relative peak area of ​​Peak S2 is ≥0.39, the Toosendan Fructus preparation is determined to be a stir-fried Toosendan Fructus standard decoction;

[0057] (3) The ratio of the sum of the relative peak areas of Peak 8 and Peak 9 to the relative peak area of ​​Peak S2 is less than 0.39, and the Toosendan Fructus preparation is determined to be a standard decoction of Toosendan Fructus;

[0058] Among them, peak 1 is a characteristic peak with a relative retention time of 0.71±10%, peak 8 is a characteristic peak of threo-guaiac-β-coniferaldehyde ether, peak 9 is a characteristic peak of erythro-guaiac-β-coniferaldehyde ether, and peak S2 is a characteristic peak of rutin.

[0059] The above-mentioned method for constructing a fingerprint of a standard decoction of Toosendan fruit or stir-fried Toosendan fruit is constructed by rationally controlling the chromatographic conditions of UPLC to obtain a UPLC fingerprint of the standard decoction of Toosendan fruit or stir-fried Toosendan fruit, and the fingerprint has high resolution and response values ​​for each characteristic peak, good accuracy, specificity, and reproducibility, and can better reflect the intrinsic quality characteristics of the standard decoction of Toosendan fruit or stir-fried Toosendan fruit (water-soluble components), meet the quality control needs of the standard decoction of Toosendan fruit or stir-fried Toosendan fruit, and provide an important reference for the quality control of Toosendan fruit, stir-fried Toosendan fruit formula granules, and related traditional Chinese medicine preparations. At the same time, the construction method is short in time and has high detection efficiency.

[0060] In addition, the above UPLC chromatographic conditions are also applicable to the identification of any two of Toosendan fruit standard decoction, stir-fried Toosendan fruit standard decoction, and Melia azedarach standard decoction, and are particularly applicable to the identification and differentiation of Melia azedarach standard decoction from counterfeit Melia azedarach standard decoction. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is the ultraviolet absorption spectrum of the Toosendan Fructus standard decoction sample in the range of 190-400nm;

[0062] Figure 2 This is the chromatogram of Toosendan Fructus standard decoction sample at 220 nm;

[0063] Figure 3 This is the chromatogram of Toosendan Fructus standard decoction sample at 260nm;

[0064] Figure 4 This is the chromatogram of Toosendan Fructus standard decoction sample at 310 nm;

[0065] Figure 5 This is the chromatogram of Toosendan Fructus standard decoction sample at 360nm;

[0066] Figure 6 To investigate the effects of different chromatographic columns on the fingerprints of Toosendan Fructus standard decoction;

[0067] Figure 7 To investigate the effects of acid solutions of different concentrations on the fingerprints of Toosendan Fructus standard decoction;

[0068] Figure 8 To investigate the effect of different sample volumes on the fingerprint of Toosendan Fructus standard decoction;

[0069] Figure 9 To investigate the effects of different elution solvents on the fingerprint of Toosendan Fructus standard decoction;

[0070] Figure 10 To investigate the effects of different elution solvent dosages on the fingerprint of Toosendan Fructus standard decoction;

[0071] Figure 11 To investigate the specificity of the fingerprint of Toosendan Fructus standard decoction;

[0072] Figure 12 This is an overlay of fingerprints of 20 batches of Toosendan Fructus standard decoctions;

[0073] Figure 13 This is an overlay of fingerprints of 20 batches of stir-fried Toosendan fruit standard decoctions;

[0074] Figure 14 This is the reference fingerprint of the standard decoction of Toosendan fruit, in which, Peak 2 (S1): 5-hydroxymethylfurfural; Peak 4: vanillin; Peak 5: coniferaldehyde; Peak 7 (S2): rutin; Peak 8: threo-guaiac-β-coniferaldehyde ether; Peak 9: erythro-guaiac-β-coniferaldehyde ether;

[0075] Figure 15This is the reference fingerprint of the standard decoction of stir-fried Toosendan fruit, in which Peak 2 (S1): 5-hydroxymethylfurfural; Peak 4: vanillin; Peak 5: coniferaldehyde; Peak 7 (S2): rutin; Peak 8: threo-guaiac-β-coniferaldehyde ether; Peak 9: erythro-guaiac-β-coniferaldehyde ether;

[0076] Figure 16 This is the total ion current and ultraviolet absorption chromatogram of the test solution of Toosendan Fructus standard decoction;

[0077] Figure 17 This is an overlay of characteristic spectra of 7 batches of standard decoctions of Melia azedarach. DETAILED DESCRIPTION

[0078] The following is a further detailed description of the fingerprint construction method and identification method of the Toosendan Fructus preparation of the present application in conjunction with specific examples. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0080] The optional scope of the terms "and / or", "or / and", and "and / or" used in this document includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, and the said any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items.

[0081] Herein, "at least one" refers to any one, any two, or any two or more of the listed items.

[0082] In this application, terms such as "first aspect," "second aspect," "third aspect," and "fourth aspect" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, terms such as "first," "second," "third," and "fourth" serve only as non-exhaustive enumeration and description, and should not constitute a closed-ended limitation on quantity.

[0083] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0084] In this application, when referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0085] Unless otherwise specified, percentages in this application refer to mass percentages for solid-liquid mixtures and solid-solid mixtures, and volume percentages for liquid-liquid mixtures. For solutions in this application, unless otherwise specified, the solvent is water.

[0086] The percentage concentrations mentioned in this application, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0087] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range of instrument control.

[0088] The room temperature in this application generally refers to 4°C to 30°C, preferably 20±5°C.

[0089] Some examples of the present application provide a method for constructing a fingerprint of a Toosendan Fructus preparation, wherein the Toosendan Fructus preparation is a Toosendan Fructus standard decoction, a stir-fried Toosendan Fructus standard decoction, a Toosendan Fructus traditional Chinese medicine formula granule, or a stir-fried Toosendan Fructus traditional Chinese medicine formula granule; the construction method comprises the following steps:

[0090] Preparation of a reference solution: mixing a 5-hydroxymethylfurfural reference substance and a rutin reference substance with a first solvent to prepare the reference solution;

[0091] Preparation of a test solution: mixing the Toosendan preparation with the second solution, centrifuging, taking the supernatant for column chromatography, and collecting the eluate; then evaporating the eluate to dryness and dissolving it with the third solution to prepare the test solution;

[0092] The reference solution and the test solution are subjected to ultra-high performance liquid chromatography (UPLC) detection, wherein the conditions for the UPLC detection include the following items (1) to (3):

[0093] (1) Mobile phase A is acetonitrile and methanol in a volume ratio of (3-5):1, and mobile phase B is a 0.2% to 0.4% phosphoric acid aqueous solution. The elution gradient is as follows:

[0094] From 0 min to 8 min, the volume percentage of the mobile phase A changes from 3% to 8%;

[0095] From 8 min to 16 min, the volume percentage of the mobile phase A was changed from 8% to 11%;

[0096] From 16 min to 19 min, the volume percentage of the mobile phase A was changed from 11% to 19%;

[0097] From 19 min to 27 min, the volume percentage of the mobile phase A was changed from 19% to 20%;

[0098] From 27 min to 31 min, the volume percentage of the mobile phase A was changed from 20% to 36%;

[0099] From 31 to 38 minutes, the volume percentage of the mobile phase A was changed from 36% to 45%;

[0100] (2) Detection wavelength: 300nm-320nm from 0min to 19min; 350nm-370nm from 19min to 38min;

[0101] (3) Chromatographic column: Waters Cortecs T3 C 18 Chromatographic column.

[0102] Understandably, in theory, the efficacy and composition of standard decoctions and TCM granules must be consistent. Quality control of standard decoctions also provides guidance for the development of quality standards for TCM granules, and the standards for TCM granules are determined with reference to standard decoctions. Therefore, the above technical solution is applicable to the construction of fingerprints for both standard decoctions and TCM granules.

[0103] Specifically, in mobile phase A, the volume ratio of acetonitrile to methanol includes but is not limited to: 3:1, 3.5:1, 4:1, 4.5:1, 5:1 or any two of the foregoing.

[0104] Specifically, the volume fraction of the phosphoric acid aqueous solution in the mobile phase B includes, but is not limited to, 0.2%, 0.25%, 0.3%, 0.35%, 0.4% or any two of the foregoing.

[0105] Specifically, the detection wavelength is: 310 nm from 0 min to 19 min; 360 nm from 19 min to 38 min.

[0106] Specifically, the specifications of the chromatographic column are: diameter of 2 mm to 2.3 mm, length of 140 mm to 160 mm, and filler particle size of 1.5 μm to 2 μm. More specifically, the filler particle size includes, but is not limited to, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2 μm, or any combination thereof.

[0107] In some examples, the flow rate of the ultra-high performance liquid chromatography detection is 0.2-0.4 mL / min. Specifically, the flow rate of the ultra-high performance liquid chromatography detection includes but is not limited to: 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, or any two of the foregoing.

[0108] In some examples, the column temperature of the ultra-high performance liquid chromatography detection is 28° C. to 32° C. Specifically, the column temperature of the ultra-high performance liquid chromatography detection includes but is not limited to: 28° C., 29° C., 30° C., 31° C., 32° C., or any two of the foregoing.

[0109] In some examples, the injection volume of the ultra-high performance liquid chromatography detection is 1 μL to 5 μL. Specifically, the injection volume of the ultra-high performance liquid chromatography detection includes but is not limited to: 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, or any two of the foregoing.

[0110] In some examples, the column chromatography conditions include: using a macroporous adsorption resin column as a chromatography column, performing elution and washing in sequence, the elution solution is water, the elution solution is an ethanol solution with a volume fraction of 50% to 100%, and collecting the solution of the elution process to obtain the eluate.

[0111] Specifically, the elution solution includes but is not limited to pure ethanol or an ethanol aqueous solution with a volume fraction of 50%, an ethanol aqueous solution with a volume fraction of 60%, an ethanol aqueous solution with a volume fraction of 70%, an ethanol aqueous solution with a volume fraction of 80%, an ethanol aqueous solution with a volume fraction of 90% or a range of any two of the foregoing.

[0112] Specifically, the macroporous adsorption resin column is a D101 macroporous adsorption resin column.

[0113] Specifically, the inner diameter of the macroporous adsorption resin column is 0.8 cm to 1.2 cm, and the column length is 13 cm to 17 cm.

[0114] In some examples, the volume of the eluted solution is 30 mL to 70 mL. Specifically, the volume of the eluted solution includes but is not limited to: 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, or any two of the foregoing.

[0115] In some examples, the loading volume of the supernatant is 3 mL to 7 mL. Specifically, the loading volume of the supernatant includes but is not limited to: 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, or any two of the foregoing.

[0116] In some examples, the first solvent is a methanol aqueous solution with a volume fraction of 45% to 55%.

[0117] In some examples, the second solution is water.

[0118] In some examples, the third solution is a methanol aqueous solution with a volume fraction of 45% to 55%.

[0119] In some examples, the fingerprint spectrum includes 10 characteristic peaks, including characteristic peaks of 5-hydroxymethylfurfural, vanillin, coniferaldehyde, rutin, threo-guaiac-β-coniferaldehyde ether and erythro-guaiac-β-coniferaldehyde ether.

[0120] Some other examples of the present application provide a method for identifying a Toosendan fruit preparation, wherein the Toosendan fruit preparation includes at least two of Toosendan fruit standard decoction, stir-fried Toosendan fruit standard decoction, and Melia azedarach standard decoction, or the Toosendan fruit preparation includes at least two of Toosendan fruit traditional Chinese medicine formula granules, stir-fried Toosendan fruit traditional Chinese medicine formula granules, and Melia azedarach traditional Chinese medicine formula granules; the identification method comprises the following steps:

[0121] Preparation of a sample solution to be tested; mixing the Toosendan preparation with the second solution, centrifuging, taking the supernatant for column chromatography, and collecting the eluate; then evaporating the eluate to dryness and dissolving it with the third solution to prepare the sample solution to be tested;

[0122] The sample solution to be tested is subjected to ultra-high performance liquid chromatography to obtain a characteristic spectrum of the Toosendan fruit preparation and compare the spectrum;

[0123] The conditions for the ultra-high performance liquid chromatography detection include the following (1) to (3):

[0124] (1) Mobile phase A is acetonitrile and methanol in a volume ratio of (3-5):1, and mobile phase B is a 0.2% to 0.4% phosphoric acid aqueous solution. The elution gradient is as follows:

[0125] From 0 min to 8 min, the volume percentage of the mobile phase A changes from 3% to 8%;

[0126] From 8 min to 16 min, the volume percentage of the mobile phase A was changed from 8% to 11%;

[0127] From 16 min to 19 min, the volume percentage of the mobile phase A was changed from 11% to 19%;

[0128] From 19 min to 27 min, the volume percentage of the mobile phase A was changed from 19% to 20%;

[0129] From 27 min to 31 min, the volume percentage of the mobile phase A was changed from 20% to 36%;

[0130] From 31 to 38 minutes, the volume percentage of the mobile phase A was changed from 36% to 45%;

[0131] (2) Detection wavelength: 300nm-320nm from 0min to 19min; 350nm-370nm from 19min to 38min;

[0132] (3) Chromatographic column: Waters Cortecs T3 C 18 Chromatographic column.

[0133] It can be understood that the preparation of the sample solution to be tested and the conditions for ultra-high performance liquid chromatography detection in this identification method are similar to the preparation of the test solution and the conditions for ultra-high performance liquid chromatography detection in the above-mentioned method for constructing the fingerprint spectrum of the Toosendan preparation, and will not be repeated here.

[0134] Understandably, the efficacy and composition of standard decoctions and TCM granules must theoretically be consistent. Quality control of standard decoctions also provides guidance for the development of quality standards for TCM granules, and the standards for TCM granules are determined with reference to standard decoctions. Therefore, the above technical solution is applicable to the identification of both standard decoctions and TCM granules.

[0135] In some examples, the comparison includes at least one of the following items (1) to (3):

[0136] (1) If the relative peak area ratio of Peak 1 to Peak S2 is less than 0.91, and the relative peak area ratio of the sum of Peaks 8 and 9 to the relative peak area of ​​Peak S2 is greater than or equal to 0.44, the Toosendan Fructus preparation is determined to be Toosendan Fructus standard decoction;

[0137] (2) If the relative peak area ratio of Peak 1 to Peak S2 is ≥0.91, and the relative peak area ratio of the sum of Peaks 8 and 9 to the relative peak area of ​​Peak S2 is ≥0.39, the Toosendan Fructus preparation is determined to be a stir-fried Toosendan Fructus standard decoction;

[0138] (3) The ratio of the sum of the relative peak areas of Peak 8 and Peak 9 to the relative peak area of ​​Peak S2 is less than 0.39, and the Toosendan Fructus preparation is determined to be a standard decoction of Toosendan Fructus;

[0139] Among them, peak 1 is a characteristic peak with a relative retention time of 0.71±10%, peak 8 is a characteristic peak of threo-guaiac-β-coniferaldehyde ether, peak 9 is a characteristic peak of erythro-guaiac-β-coniferaldehyde ether, and peak S2 is a characteristic peak of rutin.

[0140] The following are specific examples. Unless otherwise specified, the reagents used in the examples are all commercially available products.

[0141] Example 1

[0142] This embodiment is a method for constructing the fingerprint of Toosendan Fructus standard decoction or stir-fried Toosendan Fructus standard decoction.

[0143] 1. Instruments, reagents and test drugs

[0144] Instruments: Waters ultra-high performance liquid chromatograph (H-class, Waters); Waters Cortecs T3 C 18 Chromatographic column (2.1 mm × 150 mm, 1.6 μm), one-ten-thousandth balance (ME204E, Mettler-Toledo), one-millionth balance (XP26, Mettler-Toledo), JJ600 electronic balance (ZG-0191, Changshu Shuangjie Testing Instrument Factory), CNC ultrasonic cleaner (KQ500DE, Kunshan Ultrasonic Instrument Co., Ltd.), and ultrapure water system (Milli-Q Direct, Merck & Co., Ltd.) were used.

[0145] Reagents: ethanol (analytical grade, Xilong Scientific Co., Ltd.); phosphoric acid (Tianjin Komiou Chemical Reagent Co., Ltd.), acetonitrile (Merck Co., Ltd.), and methanol (Merck Co., Ltd.) for liquid phase chromatography were of chromatographic grade; and water was ultrapure water (prepared in the laboratory).

[0146] Test drugs: 5-hydroxymethylfurfural (batch number: 111626-202215, content: 99.5%, China Food and Drug Inspection Institute), rutin (batch number: 100080-202012, content: 91.6%, China Food and Drug Inspection Institute); the numbers of 20 batches of Toosendan fruit standard decoction and 20 batches of stir-fried Toosendan fruit standard decoction and the origin information of the medicinal materials used are shown in Table 1.

[0147] Table 1 Sample information table

[0148]

[0149]

[0150] 2 Preparation of standard decoction

[0151] Take Toosendan fruit medicinal material, remove impurities, and crush into irregular pieces with a diameter of 1.0 cm to 2.0 cm to prepare Toosendan fruit decoction slices. Take Toosendan fruit decoction slices, stir-fry at 135° C. for 15 minutes until the surface turns brown, and prepare stir-fried Toosendan fruit decoction slices.

[0152] 100g each of Toosendan Fructus slices and stir-fried Toosendan Fructus slices were decocted twice with water. For the first decoction, 8 times the amount of water was added, the mixture was soaked for 30 minutes, and then the mixture was boiled over high heat, then kept slightly boiling over low heat for 30 minutes. The decoction was filtered through a 350-mesh sieve, and the filtrate was quickly cooled with cold water. For the second decoction, 6 times the amount of water was added, the mixture was boiled over high heat, then kept slightly boiling over low heat for 25 minutes, and the decoction was filtered through a 350-mesh sieve. The filtrate was quickly cooled with cold water, and the two decoctions were combined. The mixture was concentrated under reduced pressure and freeze-dried in a vacuum to obtain freeze-dried powders of standard decoctions of Toosendan Fructus and stir-fried Toosendan Fructus.

[0153] 3. Fingerprint chromatographic conditions and preparation method of test solution

[0154] 3.1 Chromatographic conditions

[0155] Using Waters Cortecs T3 C 18 Chromatographic column (2.1 mm × 150 mm, 1.6 μm); acetonitrile:methanol (volume ratio 4:1) as mobile phase A, 0.3% volume fraction of phosphoric acid aqueous solution as mobile phase B, gradient elution according to the regulations in Table 2; flow rate, 0.30 mL / min; column temperature, 30°C; detection wavelength, 310 nm from 0 to 19 minutes, and 360 nm from 19 to 38 minutes; injection volume, 2 μL.

[0156] Table 2 Gradient elution table

[0157]

[0158] 3.2 Preparation of reference solution

[0159] Take appropriate amount of 5-hydroxymethylfurfural reference substance and rutin reference substance, weigh accurately, add 50% methanol aqueous solution by volume to make a mixed solution containing 15 μg of each per 1 mL, which is used as the reference substance solution.

[0160] 3.3 Preparation of standard decoction test solution

[0161] Take an appropriate amount of freeze-dried powder of Toosendan fruit standard decoction or freeze-dried powder of stir-fried Toosendan fruit standard decoction, grind it finely, and take approximately 0.5 g, accurately weigh it, and place it in a stoppered conical flask. Add 10 mL of water to dissolve it. Centrifuge it, and pass 5 mL of the supernatant through a D101 macroporous adsorption resin column (1 cm inner diameter, 15 cm length). Elute it with 50 mL of water, discard the water, and then elute it with 50 mL of ethanol. Collect the eluate, evaporate it to dryness, and dissolve the residue in 50% (v / v) methanol aqueous solution. Transfer it to a 5 mL volumetric flask, add 50% (v / v) methanol aqueous solution to the mark, shake it well, filter it, and collect the filtrate to obtain the product.

[0162] 3.4 Determination method

[0163] Accurately pipette 2 μL of reference solution and test solution respectively, inject them into ultra-high performance liquid chromatography instrument, and measure to obtain the result.

[0164] 4 Optimization of chromatographic conditions

[0165] 4.1 Determination of the optimal absorption wavelength

[0166] Take an appropriate amount of Toosendan standard decoction, grind it into powder, take about 0.5g, prepare the test solution according to the method under "3.3", and analyze the sample according to the chromatographic conditions under "3.1". Record the ultraviolet absorption spectrum of Toosendan standard decoction sample in the range of 190-400nm ( Figure 1 ) and compare the chromatograms of the samples at detection wavelengths of 220 nm, 260 nm, 310 nm and 360 nm ( Figures 2 to 5 ), experiments showed that when 220nm and 260nm were selected as detection wavelengths, the response of each chromatographic peak was poor and the baseline was unstable. When 310nm was selected as the detection wavelength, the response values ​​of each chromatographic peak from 0 to 19 minutes were large, the baseline was stable, and the interference was small, but the baseline of the chromatographic peak after 19 minutes was unstable. At the detection wavelength of 360nm, the response values ​​of each chromatographic peak after 19 minutes were large, the baseline was stable, and the interference was small. Therefore, based on comprehensive considerations, the detection wavelengths of 310nm were selected from 0 to 19 minutes, and 360nm were selected from 19 to 34 minutes.

[0167] 4.2 Investigation of different chromatographic columns

[0168] Take an appropriate amount of Toosendan fruit standard decoction, grind it into powder, take about 0.5g, and prepare the test solution according to the method under "3.3". The chromatographic conditions are Waters BEH C 18 (2.1mm×150mm,1.7μm), SHIMADZU Shim-packScepter C 18 (2.1mm×150mm, 1.9μm), Waters HSS T3 C 18(2.1mm×150mm, 1.8μm) and WatersCORTECS T3 C 18 Except for the column diameter (2.1 mm × 150 mm, 1.6 μm), the rest are the same as in item 3.1. The effects of different chromatographic columns on the fingerprint of Toosendan standard decoction are investigated (see Figure 6 The results showed that the Waters CORTECS T3 C 18 (2.1mm×150mm, 1.6μm) chromatographic column, the overall separation and distribution of each characteristic peak are good, so the Waters CORTECS T3 C 18 (2.1 mm × 150 mm, 1.6 μm) column was used as the chromatographic column.

[0169] 4.3 Investigation of different acid concentrations

[0170] Take an appropriate amount of Toosendan standard decoction, grind it into powder, take about 0.5 g, and prepare the test solution according to the method under "3.3". The chromatographic conditions are the same as those under "3.1", except that the mobile phase B is 0.05% phosphoric acid aqueous solution, 0.2% phosphoric acid aqueous solution and 0.3% phosphoric acid aqueous solution, respectively. The effects of different concentrations of acid solution on the fingerprint of Toosendan standard decoction are investigated (see Figure 7 The results show that, by comparing the elution effects of three different mobile phases, the acetonitrile:methanol (4:1)-0.3% phosphoric acid solution provides better peak separation and a more stable baseline. Therefore, the acetonitrile:methanol (4:1)-0.3% phosphoric acid solution was selected as the mobile phase.

[0171] 5 Investigation of the preparation method of test solution

[0172] 5.1 Investigation of the sample volume of the test solution

[0173] Take an appropriate amount of Toosendan fruit standard decoction (No. BT13), grind it into powder, take about 0.5 g, accurately weigh it, and place it in 3 parallel groups in stoppered conical flasks. Add 10 mL of water to dissolve it, centrifuge it, take 3 mL, 5 mL, and 7 mL of the supernatant, pass it through a D101 macroporous adsorption resin column (with an inner diameter of 1 cm and a column length of 15 cm), elute it with 50 mL of water, discard the water, and then elute it with 50 mL of ethanol. Collect the eluate, evaporate it to dryness, dissolve the residue in 50% methanol aqueous solution by volume, transfer it to a 5 mL volumetric flask, add 50% methanol aqueous solution by volume to the scale, shake it well, filter it, take the filtrate, and analyze it according to the chromatographic conditions under "3.1". The results are as follows: Figure 8As shown in the figure, the results show that, using "total peak area / sample weight / sample volume" as an indicator, when the sample volume is 3-5 mL, the adsorption of each chemical component is good and basically reaches adsorption equilibrium. However, when the sample volume is 7 mL, the "total peak area / sample weight / sample volume" of each characteristic peak decreases. Therefore, the sample volume of 5 mL was selected based on the response of each characteristic peak.

[0174] 5.2 Investigation of elution solvent

[0175] Take an appropriate amount of Toosendan fruit standard decoction (No. BT13), grind it into powder, take about 0.5g, weigh it accurately, and place it in 3 parallel groups in stoppered conical flasks. Add 10mL of water to each to dissolve it, centrifuge it, take 5mL of the supernatant and pass it through a D101 macroporous adsorption resin column (inner diameter 1cm, column length 15cm), elute it with 50mL of water, and discard the water. Elute it with 50% ethanol aqueous solution, 70% ethanol aqueous solution, and 50mL of ethanol respectively, collect the eluate, evaporate it to dryness, dissolve the residue with 50% methanol aqueous solution, transfer it to a 5mL volumetric flask, add 50% methanol aqueous solution to the scale, shake it well, filter it, take the filtrate, and analyze it according to the chromatographic conditions under "3.1". The results are as follows: Figure 9 As shown in the figure, the results show that there is no obvious difference in the peak shape and separation effect of each characteristic peak when using different extraction and elution solvents, but the "total peak area / sample amount" is the largest when ethanol is used as the elution solvent. In order to ensure complete elution, ethanol is selected as the elution solvent.

[0176] 5.3 Investigation of elution solvent dosage

[0177] Take an appropriate amount of Toosendan fruit standard decoction (No. BT13), grind it into powder, take about 0.5g, weigh it accurately, and place it in 3 parallel groups in stoppered conical flasks. Add 10mL of water to each to dissolve it. Centrifuge, take 5mL of the supernatant and pass it through a D101 macroporous adsorption resin column (inner diameter 1cm, column length 15cm), elute with 50mL of water, and discard the water. Elute with 30mL, 50mL and 70mL of ethanol respectively, collect the eluate, evaporate to dryness, dissolve the residue in 50% methanol aqueous solution by volume, transfer it to a 5mL volumetric flask, add 50% methanol aqueous solution by volume to the scale, shake well, filter, take the filtrate, and analyze it according to the chromatographic conditions under "3.1". The results are as follows Figure 10 As shown in the figure, the results show that there is no obvious difference in the "total peak area / sample amount" of the 10 characteristic peaks with different elution solvent amounts, indicating that 30 mL can be completely eluted. Considering the influence of the experimental environment, in order to ensure the durability of the method, the elution solvent amount was selected as 50 mL.

[0178] 6 Methodological Validation

[0179] 6.1 Specificity Investigation

[0180] Take an appropriate amount of Toosendan Fructus Standard Decoction (No. BT13), grind it into powder, take about 0.5 g, prepare the test solution according to the method under "3.3", take 2 μL each of the test solution, reference solution and blank solvent, inject them into the ultra-high performance liquid chromatography, and analyze the samples according to the chromatographic conditions under "3.1". The results are as follows: Figure 11 As shown in the figure, the results show that the test sample and the reference sample have the same chromatographic peaks at the corresponding retention times, and there is no interference from the blank solvent, indicating that the established method has good specificity.

[0181] 6.2 Precision investigation

[0182] Take an appropriate amount of Toosendan standard decoction (No.: BT13), grind it into powder, take about 0.5 g, prepare the test solution according to the method under "3.3", repeat the injection 6 times according to the chromatographic conditions under "3.1", use the 5-hydroxymethylfurfural chromatographic peak as the reference peak S1, calculate the relative retention time and relative peak area RSD value of peak 1, peak 3, peak 4 and peak S1, use the rutin chromatographic peak as the reference peak S2, calculate the relative retention time and relative peak area RSD value of peak 5, peak 6, peak 8, peak 9, peak 10 and peak S2, all of which are less than 3.0%, indicating good instrument precision.

[0183] 6.3 Repeatability Study

[0184] About 0.5 g of the same batch of Toosendan Fructus standard decoction (No.: BT13) was taken, accurately weighed, and 6 parallel portions were made. 6 test solutions were prepared according to the method under "3.3". The samples were injected and analyzed according to the chromatographic conditions under "3.1". The chromatographic peak of 5-hydroxymethylfurfural was used as the reference peak S1, and the relative retention time and relative peak area RSD values ​​of peak 1, peak 3, peak 4 and peak S1 were calculated; the chromatographic peak of rutin was used as the reference peak S2, and the relative retention time and relative peak area RSD values ​​of peak 5, peak 6, peak 8, peak 9, peak 10 and peak S2 were calculated. The results were all less than 3.0%, indicating that the method had good repeatability.

[0185] 6.4 Stability investigation

[0186] Take an appropriate amount of Toosendan standard decoction (No.: BT13), grind it into powder, take about 0.5 g, prepare the test solution according to the method under "3.3", and according to the chromatographic conditions determined under "3.1", sample analysis was performed at 0 hour, 2 hours, 4 hours, 6 hours, 10 hours, 12 hours, and 24 hours, respectively. The 5-hydroxymethylfurfural chromatographic peak was used as the reference peak S1, and the relative retention time and relative peak area RSD values ​​of peak 1, peak 3, peak 4 and peak S1 were calculated; the rutin chromatographic peak was used as the reference peak S2, and the relative retention time and relative peak area RSD values ​​of peak 5, peak 6, peak 8, peak 9, peak 10 and peak S2 were calculated. The results were all less than 3.0%, indicating that the test solution had good stability within 24 hours.

[0187] 7 Establishment of fingerprints of standard decoctions of Toosendan fruit and stir-fried Toosendan fruit

[0188] According to the test solution preparation method under "3.3" and the chromatographic conditions under "3.1", 20 batches of Toosendan Fructus and stir-fried Toosendan Fructus standard decoction samples were fingerprinted and 10 common peaks with good peak shape, high separation and relatively pure chromatographic peaks were selected as the characteristic peaks of Toosendan Fructus and stir-fried Toosendan Fructus standard decoction. The results are shown in Figure 2. Figures 12-13 As shown. Taking the chromatographic peak of peak 2 5-hydroxymethylfurfural as the reference peak S1, the average relative retention times of peak 1, peak 3, and peak 4 of the fingerprints of 20 batches of Toosendan fruit and stir-fried Toosendan fruit standard decoction samples with respect to peak S1 were calculated as follows: 0.71 (peak 1), 1.32 (peak 3), and 3.90 (peak 4); taking the chromatographic peak of peak 7 rutin as the reference peak S2, the average relative retention times of peak 5, peak 6, peak 8, peak 9, and peak 10 with respect to peak S2 were calculated as 0.96 (peak 5), 0.98 (peak 6), 1.07 (peak 8), 1.10 (peak 9), and 1.35 (peak 10). The UPLC fingerprints of 20 batches of Toosendan fruit standard decoction and stir-fried Toosendan fruit standard decoction were matched using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines", and the control fingerprints of Toosendan fruit and stir-fried Toosendan fruit standard decoction were generated by the average method. The results are shown in Figures 14-15 shown.

[0189] 8 Identification of characteristic peaks

[0190] 8.1 Ultra-high performance liquid chromatography conditions

[0191] Except that mobile phase B is 0.1% by volume formic acid aqueous solution, the other chromatographic conditions are the same as those in item "3.1".

[0192] 8.2 Mass spectrometry conditions

[0193] The mass spectrometry parameters are shown in Table 3.

[0194] Table 3 Mass spectrometry parameters

[0195]

[0196] 8.3 Preparation of test solution

[0197] Take an appropriate amount of Toosendan Fructus Standard Decoction (No. BT13), grind it into powder, take about 0.5 g, and prepare the test solution according to the method under "3.3".

[0198] 8.4 Determination method

[0199] Accurately pipette 2 μL of the test solution, inject it into the liquid chromatography-mass spectrometer, and measure it.

[0200] Through mass spectrometry accurate molecular weight and fragment ion comparison analysis, it was confirmed that the characteristic peak 2 in the fingerprint of Toosendan decoction was 5-hydroxymethylfurfural, the characteristic peak 4 was vanillin, the characteristic peak 5 was coniferaldehyde, the characteristic peak 7 was rutin, the characteristic peak 8 was threonine-guaiacine-β-coniferaldehyde ether, and the characteristic peak 9 was erythroguaiacine-β-coniferaldehyde ether. The total ion current and ultraviolet absorption chromatogram of the test solution are shown in Figure 16 , compound information is shown in Table 4.

[0201] Table 4 Mass spectrometry identification results of compounds in Toosendan Fructus standard decoction

[0202]

[0203]

[0204] Example 2

[0205] This example is the identification of Toosendan Fructus and stir-fried Toosendan Fructus standard decoction by UPLC characteristic patterns.

[0206] A comparative analysis of the fingerprints (characteristic) of 20 batches of Toosendan Fructus standard decoctions and stir-fried Toosendan Fructus standard decoctions revealed 10 characteristic peaks in both the pre- and post-processing standard decoctions. Furthermore, the peak area of ​​Peak 1 in the stir-fried Toosendan Fructus standard decoction increased significantly after processing, while the peak area of ​​Peak S2 (Peak 7) remained relatively stable. Therefore, the relative peak areas of Peak 1 and S2 were used to distinguish Toosendan Fructus and stir-fried Toosendan Fructus standard decoctions. The relative peak area ratios of Peak 1 to S2 for the 20 batches of Toosendan Fructus and stir-fried Toosendan Fructus standard decoctions are shown in Table 5. The results showed that the relative peak area ratios of peak 1 to peak S2 in the characteristic spectra of 20 batches of Toosendan Fructus standard decoctions ranged from 0.195 to 0.814, with an average of 0.401. The relative peak area ratios of peak 1 to peak S2 in the characteristic spectra of the stir-fried Toosendan Fructus standard decoctions ranged from 0.914 to 5.048, with an average of 2.184. Based on the relative peak area determination results of peak 1 to peak S2 in 20 batches of Toosendan Fructus and stir-fried Toosendan Fructus standard decoctions, the minimum relative peak area of ​​peak 1 to peak S2 in the characteristic spectra of the 20 batches of stir-fried Toosendan Fructus standard decoctions was taken as the lower limit, and the relative peak area of ​​peak 1 / S2 in stir-fried Toosendan Fructus standard decoction should not be less than 0.91, in order to distinguish and identify Toosendan Fructus and stir-fried Toosendan Fructus standard decoctions.

[0207] Table 5 Comparison of relative peak areas of peak 1 and peak S2 of standard decoction of Toosendan fruit and stir-fried Toosendan fruit

[0208]

[0209]

[0210] Example 3

[0211] This example is the identification of Toosendan fruit, stir-fried Toosendan fruit and Melia azedarach fruit standard decoctions by UPLC characteristic patterns.

[0212] Melia azedarach L. is the dried mature fruit of the Meliaceae plant. However, in modern pharmacological studies, Melia azedarach L. is more toxic than Toosendan L. In addition, Toosendan L. and Melia azedarach L. are often mixed in the market. Therefore, they should be distinguished when used.

[0213] In this study, 7 batches of Melia azedarach fruit medicinal materials were collected, impurities removed, and crushed. According to the method under "2", 7 batches of Melia azedarach fruit standard decoctions were prepared, numbered: KBT1~KBT7. According to the test solution preparation method under "3.3" and the chromatographic conditions under "3.1", the characteristic spectra of the 7 batches of Melia azedarach fruit standard decoctions were determined. The results are shown in Figure 17 The results show that the characteristic spectra of the standard decoctions of Toosendan Fructus, stir-fried Toosendan Fructus, and Meliae Arnebiae all have 10 common characteristic peaks with consistent retention times. The peak area ratios of the sum of peaks 8 and 9 in the characteristic spectra of 20 batches of Toosendan Fructus and stir-fried Toosendan Fructus to the peak S2 (peak 7) were calculated, and the results are shown in Table 6. The peak area ratios of the sum of peaks 8 and 9 in the characteristic spectra of 20 batches of Toosendan Fructus to the peak S2 ranged from 0.44 to 2.09, and the peak area ratios of the sum of peaks 8 and 9 in the characteristic spectra of 20 batches of stir-fried Toosendan Fructus to the peak S2 ranged from 0.39 to 2.20. The peak area ratios of the sum of peaks 8 and 9 in the characteristic spectra of 7 batches of Meliae Arnebiae standard decoction to the peak S2 ranged from 0.24 to 0.29 (see Table 7). The minimum value of the ratio of the sum of the peak areas of peak 8 and peak 9 in the characteristic spectra of 20 batches of Toosendan fruit and stir-fried Toosendan fruit standard decoction to the peak area of ​​S2 peak was taken as the lower limit, and it was stipulated that the relative peak area of ​​the sum of the peak areas of peak 8 and peak 9 in the characteristic spectra of Toosendan fruit standard decoction to the S2 peak should not be less than 0.44, and the relative peak area of ​​the sum of the peak areas of peak 8 and peak 9 in the characteristic spectra of stir-fried Toosendan fruit standard decoction to the S2 peak should not be less than 0.39. In this way, Toosendan fruit standard decoction, stir-fried Toosendan fruit standard decoction and Melia azedarach standard decoction can be distinguished and identified.

[0214] Table 6 Relative peak areas of peaks 8+9 / S2 in characteristic spectra of Toosendan fruit and stir-fried Toosendan fruit standard decoction

[0215]

[0216] Table 7 Relative peak areas of peaks 8+9 / S2 in characteristic spectra of 7 batches of standard decoctions of Melia azedarach

[0217]

[0218]

[0219] In addition, based on the above research results, the fingerprint (characteristic) spectrum standard of Toosendan fruit standard decoction was determined as follows: 10 characteristic peaks should be present in the chromatogram of the test sample, among which peak 2 and peak 7 should correspond to the retention time of the corresponding reference peak of the control sample respectively; the peak corresponding to the 5-hydroxymethylfurfural reference peak is taken as the S1 peak, and the relative retention times of peak 1, peak 3, peak 4 and S1 peak are calculated, and the relative retention times should be within the range of ±10% of the specified value, and the specified values ​​are: 0.71 (peak 1), 1.32 (peak 3), 3.90 (peak 4); the peak corresponding to the rutin reference peak is taken as the S2 peak, and the relative retention times of peak 5, peak 6, peak 8, peak 9, peak 10 and S2 peak are calculated, and the relative retention times should be within the range of ±10% of the specified value, and the specified values ​​are: 0.96 (peak 5), 0.98 (peak 6), 1.07 (peak 8), 1.10 (peak 9), 1.35 (peak 10). The ratio of the sum of the peak areas of Peak 8 and Peak 9 to the peak area of ​​Peak S2 should not be less than 0.44.

[0220] The fingerprint (characteristic) spectrum standard of the stir-fried Toosendan fruit decoction is as follows: the test sample should show 10 characteristic peaks in the chromatogram, of which peaks 2 and 7 should correspond to the retention times of the corresponding reference peaks; the peak corresponding to the 5-hydroxymethylfurfural reference peak is the S1 peak, and the relative retention times of peaks 1, 3, and 4 to the S1 peak are calculated. The relative retention times should be within the range of ±10% of the specified values, which are: 0.71 (peak 1), 1.32 (peak 3), and 3.90 (peak 4); the peak corresponding to the 5-hydroxymethylfurfural reference peak is the S1 peak. The peak corresponding to the substance peak is the S2 peak. The relative retention time of Peak 5, Peak 6, Peak 8, Peak 9, Peak 10 and Peak S2 is calculated. The relative retention time should be within the range of ±10% of the specified value. The specified value is: 0.96 (Peak 5), 0.98 (Peak 6), 1.07 (Peak 8), 1.10 (Peak 9), 1.35 (Peak 10); the relative peak area of ​​Peak 1 and Peak S2 should not be less than 0.91, and the ratio of the sum of the peak areas of Peak 8 and Peak 9 to the peak area of ​​Peak S2 should not be less than 0.39.

[0221] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0222] The embodiments described above only express several implementation methods of the present application, which are convenient for understanding the technical solutions of the present application in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of this application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing a fingerprint of a Toosendan fruit preparation, characterized in that: The Toosendan fruit preparation is Toosendan fruit standard decoction, stir-fried Toosendan fruit standard decoction, Toosendan fruit traditional Chinese medicine formula granules or stir-fried Toosendan fruit traditional Chinese medicine formula granules; the construction method comprises the following steps: Preparation of a reference solution: mixing a 5-hydroxymethylfurfural reference substance and a rutin reference substance with a first solvent to prepare the reference solution; Preparation of the test solution: mixing the Toosendan preparation with the second solution, centrifuging, taking the supernatant for column chromatography, and collecting the eluate; Then, the eluate is evaporated to dryness and dissolved in the third solution to prepare the test solution; The reference solution and the test solution are subjected to ultra-high performance liquid chromatography (UPLC) detection. The conditions for the UPLC detection include the following (1) to (3): (1) Mobile phase A is acetonitrile and methanol in a volume ratio of (3-5):1, and mobile phase B is a 0.2%-0.4% volume fraction of phosphoric acid aqueous solution. The elution gradient is as follows: From 0 min to 8 min, the volume percentage of the mobile phase A was changed from 3% to 8%; From 8 min to 16 min, the volume percentage of the mobile phase A was changed from 8% to 11%; From 16 min to 19 min, the volume percentage of the mobile phase A was changed from 11% to 19%; From 19 min to 27 min, the volume percentage of the mobile phase A was changed from 19% to 20%; From 27 min to 31 min, the volume percentage of the mobile phase A was changed from 20% to 36%; From 31 min to 38 min, the volume percentage of the mobile phase A changed from 36% to 45%; (2) Detection wavelength: 300nm~320nm from 0min to 19min; 350nm~370nm from 19min to 38min; (3) Chromatographic column: Waters Cortecs T3 C 18 Chromatographic columns; The column chromatography conditions include: using a macroporous adsorption resin column as a chromatography column, sequentially performing elution and washing, wherein the elution solution is water, the elution solution is an ethanol solution with a volume fraction of 50% to 100%, and collecting the solution in the elution process to obtain the eluate; The first solvent is a methanol solution with a volume fraction of 45% to 55%; The second solution is water; The third solution is a methanol solution with a volume fraction of 45% to 55%.

2. The method for constructing the fingerprint of Toosendan Fructus preparation according to claim 1, characterized in that: The conditions for ultra-high performance liquid chromatography detection also include at least one of the following (1) to (3): (1) Flow rate: 0.2~0.4mL / min; (2) Column temperature is 28°C~32°C; (3) The injection volume is 1μL~5μL.

3. The method for constructing the fingerprint of Toosendan Fructus preparation according to claim 1, characterized in that: The volume of the eluted solution is 30 mL to 70 mL.

4. The method for constructing the fingerprint of Toosendan Fructus preparation according to claim 1, characterized in that: The loading volume of the supernatant is 3 mL to 7 mL.

5. The method for constructing the fingerprint of the Toosendan Fructus preparation according to any one of claims 1 to 4, characterized in that: The fingerprint spectrum includes 10 characteristic peaks, including 5-hydroxymethylfurfural, vanillin, coniferyl aldehyde, rutin, threonine-guaiac- β -Piniferaldehyde and red-guaiac- β -Characteristic peaks of coniferaldehyde ether.

6. A method for identifying Toosendan fruit preparations, characterized in that: The Toosendan fruit preparation includes at least two of Toosendan fruit standard decoction, stir-fried Toosendan fruit standard decoction, and Melia azedarach fruit standard decoction, or the Toosendan fruit preparation includes at least two of Toosendan fruit traditional Chinese medicine formula granules, stir-fried Toosendan fruit traditional Chinese medicine formula granules, and Melia azedarach fruit traditional Chinese medicine formula granules; the identification method includes the following steps: Preparation of a sample solution to be tested; mixing the Toosendan preparation with the second solution, centrifuging, taking the supernatant for column chromatography, and collecting the eluate; then evaporating the eluate to dryness and dissolving it with the third solution to prepare the sample solution to be tested; The sample solution to be tested is subjected to ultra-high performance liquid chromatography to obtain a characteristic spectrum of the Toosendan fruit preparation and compare the spectrum; The conditions for ultra-high performance liquid chromatography detection include the following (1) to (3): (1) Mobile phase A is acetonitrile and methanol in a volume ratio of (3-5):1, and mobile phase B is a 0.2%-0.4% volume fraction of phosphoric acid aqueous solution. The elution gradient is as follows: From 0 min to 8 min, the volume percentage of the mobile phase A was changed from 3% to 8%; From 8 min to 16 min, the volume percentage of the mobile phase A was changed from 8% to 11%; From 16 min to 19 min, the volume percentage of the mobile phase A was changed from 11% to 19%; From 19 min to 27 min, the volume percentage of the mobile phase A was changed from 19% to 20%; From 27 min to 31 min, the volume percentage of the mobile phase A was changed from 20% to 36%; From 31 min to 38 min, the volume percentage of the mobile phase A changed from 36% to 45%; (2) Detection wavelength: 300nm~320nm from 0min to 19min; 350nm~370nm from 19min to 38min; (3) Chromatographic column: Waters Cortecs T3 C 18 Chromatographic columns; The column chromatography conditions include: using a macroporous adsorption resin column as a chromatography column, sequentially performing elution and washing, wherein the elution solution is water, the elution solution is an ethanol solution with a volume fraction of 50% to 100%, and collecting the solution in the elution process to obtain the eluate; The second solution is water; The third solution is a methanol solution with a volume fraction of 45% to 55%.

7. The identification method of Toosendan Fructus preparation according to claim 6, characterized in that: The conditions for ultra-high performance liquid chromatography detection also include at least one of the following (1) to (3): (1) Flow rate: 0.2~0.4mL / min; (2) Column temperature is 28°C~32°C; (3) The injection volume is 1μL~5μL.

8. The identification method of Toosendan Fructus preparation according to claim 6, characterized in that: The volume of the eluted solution is 30 mL to 70 mL.

9. The identification method of Toosendan Fructus preparation according to claim 6, characterized in that: The loading volume of the supernatant is 3 mL to 7 mL.

10. The identification method of Toosendan Fructus preparation according to any one of claims 6 to 9, characterized in that: The comparison includes at least one of the following items (1) to (3): (1) If the relative peak area ratio of Peak 1 to Peak S2 is less than 0.91, and the relative peak area ratio of the sum of Peak 8 and Peak 9 to the relative peak area of ​​Peak S2 is greater than or equal to 0.44, the Toosendan Fructus preparation is determined to be Toosendan Fructus standard decoction; (2) If the relative peak area ratio of Peak 1 to Peak S2 is ≥0.91, and the relative peak area ratio of the sum of Peak 8 and Peak 9 to Peak S2 is ≥0.39, the Toosendan Fructus preparation is determined to be a stir-fried Toosendan Fructus standard decoction; (3) The ratio of the sum of the relative peak areas of peaks 8 and 9 to the relative peak area of ​​peak S2 is less than 0.39, and the Toosendan preparation is determined to be a standard decoction of Melia azedarach; Among them, peak 1 is a characteristic peak with a relative retention time of 0.71±10%, peak 8 is a characteristic peak of thio-guaiac- β -The characteristic peak of coniferaldehyde ether, peak 9 is red-guaiac- β -The characteristic peak of coniferaldehyde, S2 peak is the characteristic peak of rutin.

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