Methods for constructing and detecting characteristic spectra of Malva nut medicinal material, standard decoction, and traditional Chinese medicine granules.

Abstract

This application relates to the field of traditional Chinese medicine analysis technology, specifically to a method for constructing and detecting characteristic chromatograms of Malva nut (Sterculia lychnophora) raw material, standard decoction of Malva nut, and Malva nut traditional Chinese medicine formula granules. This application constructs characteristic chromatograms by establishing suitable chromatographic conditions for liquid chromatography analysis of the standard decoction of Malva nut. The obtained characteristic chromatograms have good specificity, good separation of each characteristic peak, and rich chemical information, effectively characterizing the mass transfer from raw material to decoction. This fully demonstrates the material basis characteristics of Malva nut raw material to standard decoction and granules, providing a rapid and comprehensive detection method for the quality of Malva nut raw material, standard decoction, and granules.

Description

Technical Field

[0001] This application relates to the field of traditional Chinese medicine detection technology, and in particular to the construction and detection methods of characteristic spectra of Malva nut, standard decoction and traditional Chinese medicine formula granules. Background Technology

[0002] Malva nut, also known as Pangdahai, is the dried, mature seed of *Sterculia lychnophora* Hance, a plant in the Sterculiaceae family. It is a well-known imported fruit-based traditional Chinese medicine, used both as food and medicine, and has a high market penetration. Malva nut is sweet and cold in nature, and enters the lung and large intestine meridians. It has the effects of clearing heat and moistening the lungs, relieving sore throat and hoarseness, and moistening the intestines and promoting bowel movements. The chemical components of malva nut include polysaccharides, reducing sugars, phenols, tannins, amino acids, peptides, proteins, oils, and volatile oils. Round-grained stellaria media, belonging to the same genus as malva nut, is often used as a substitute. Dried mature olive fruits and unripe olives are also easily mixed in.

[0003] Malva nut (Sterculia lychnophora) grows in tropical regions and is distributed in Vietnam, Indonesia, Malaysia, and Thailand. The seeds are oval, resembling olives, 2-3 cm long and 1-1.8 cm in diameter. The surface is yellowish-brown or brown with irregular fine wrinkles. The outer seed coat is extremely thin, brittle, and easily falls off. The middle seed coat is a dark brown parenchyma that rapidly swells into a spongy state upon contact with water. The inner seed coat is slightly leathery, reddish-brown, with a faint odor and a slightly sweet taste. The best quality Malva nuts are large, firm, brown, and have fine wrinkles and a glossy surface. Malva nuts are available in China in two main phases: from February to May, mainly from Vietnam, with rounder fruits, commonly known as round-fruited Malva nuts; and in July, mainly from Thailand, with longer fruits, commonly known as long-fruited Malva nuts. Long-fruited Malva nuts are generally more expensive than round-fruited ones in the market, and these two types of slices are often sold together.

[0004] Currently, there are few documented methods for the quality analysis of Malva nut (Sterculia lychnophora). Reports mostly focus on the composition of Malva nut, with only a few studies on quality standard evaluation. Thin-layer chromatography (TLC) and liquid chromatography are generally used for the detection and identification of Malva nut. Furthermore, research on the specific components of Malva nut is lacking, and there is no differentiation between different grades of Malva nut. Although some literature reports the use of characteristic chromatographic methods for the quality evaluation of Malva nut, these methods have certain applicability issues and low resolution. Therefore, there is an urgent need to provide a more efficient and reproducible detection method applicable to Malva nut.

[0005] In view of this, the technical content of this application is hereby presented. Summary of the Invention

[0006] The purpose of this application is to provide a method for constructing and detecting characteristic spectra of Malva nut medicinal material, Malva nut standard decoction, and Malva nut traditional Chinese medicine formula granules. This detection method can be used to identify Malva nut medicinal material, its standard decoction, and traditional Chinese medicine formula granules from different sources, providing a reference for the formulation of Malva nut quality standards.

[0007] The first aspect of this application provides a method for constructing a characteristic spectrum of Sterculia lychnophora herbal material, Sterculia lychnophora standard decoction, or Sterculia lychnophora traditional Chinese medicine formula granules, comprising the following steps:

[0008] Take the test sample, add an extraction solvent to extract it, and prepare a test sample solution. The test sample is selected from the medicinal material of Malva nut, the standard decoction of Malva nut, or the traditional Chinese medicine formula granules of Malva nut.

[0009] The test solution was subjected to liquid chromatography analysis to establish characteristic chromatograms of Malva nut medicinal material, Malva nut standard decoction or Malva nut Chinese medicine formula granules;

[0010] The conditions for the liquid chromatography analysis include:

[0011] (1) Mobile phase A is methanol, and mobile phase B is a 0.04%~0.06% acid solution;

[0012] (2) Gradient elution is employed, wherein the gradient elution procedure includes:

[0013] Within 0-5 minutes, the volume percentage of mobile phase A increased from 13% to 14%.

[0014] For 5 to 22 minutes, maintain the volume percentage of mobile phase A at 14%.

[0015] Between 22 and 37 minutes, the volume percentage of mobile phase A increased from 14% to 31%.

[0016] Between 37 and 50 minutes, the volume percentage of mobile phase A increased from 31% to 50%.

[0017] Between 50 and 60 minutes, the volume percentage of the mobile phase B increased from 50% to 85%.

[0018] A second aspect of this application provides a method for detecting *Sterculia lychnophora* medicinal material, *Sterculia lychnophora* standard decoction, or *Sterculia lychnophora* traditional Chinese medicine formula granules, comprising the following steps:

[0019] Take the analyte and extract it with an extraction solvent to prepare the analyte solution;

[0020] The test sample solution was subjected to liquid chromatography analysis to obtain the characteristic chromatogram of the test sample.

[0021] The conditions for the liquid chromatography analysis include:

[0022] 1) Mobile phase A is methanol, and mobile phase B is a 0.04%~0.06% aqueous solution of phosphoric acid;

[0023] 2) Gradient elution is employed, and the gradient elution procedure includes:

[0024] Within 0-5 minutes, the volume percentage of mobile phase A increased from 13% to 14%.

[0025] For 5 to 22 minutes, maintain the volume percentage of mobile phase A at 14%.

[0026] Between 22 and 37 minutes, the volume percentage of mobile phase A increased from 14% to 31%.

[0027] Between 37 and 50 minutes, the volume percentage of mobile phase A increased from 31% to 50%.

[0028] Between 50 and 60 minutes, the volume percentage of the mobile phase B increased from 50% to 85%.

[0029] The third aspect of this application provides the application of the feature map obtained by the feature map construction method of the first aspect of this application and / or the detection method of the second aspect of this application in any of the following (1) to (3):

[0030] (1) Differentiate between long-fruited and round-fruited Malva nut medicinal materials;

[0031] (2) Differentiate between the standard decoction of long-fruited and round-fruited Malva nut;

[0032] (3) Differentiate between long-fruited and round-fruited Malva nut Chinese medicine formula granules.

[0033] This application establishes suitable chromatographic conditions for liquid chromatography analysis of the standard decoction of Malva nut, constructs its characteristic chromatogram, and obtains a characteristic chromatogram with good specificity, good separation of each characteristic peak, and rich chemical information. It can well characterize the mass transfer from raw materials to decoction, and fully demonstrate the material basis characteristics of Malva nut medicinal materials to Malva nut standard decoction and Malva nut traditional Chinese medicine formula granules. It provides a rapid and comprehensive detection method for the quality of Malva nut medicinal materials, Malva nut standard decoction and Malva nut traditional Chinese medicine formula granules.

[0034] This application conducts extensive research on the characteristic components of Malva nut, its standard decoction, and traditional Chinese medicine granules. By establishing suitable chromatographic conditions, nine common characteristic peaks were obtained: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, and peak 9. The application also identifies characteristic components of multiple herbs, including protocatechuic acid, protocatechuic aldehyde, catechin, caffeic acid, 4-coumaric acid, and ferulic acid (peak 1 is protocatechuic acid, peak 2 is protocatechuic aldehyde, peak 5 is catechin, peak 6 is caffeic acid, peak 7 is 4-coumaric acid, and peak 9 is ferulic acid). This clarifies the intrinsic chemical information of Malva nut and can provide important reference for its quality testing, identification, and clinical use. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 The 3D chromatogram of the standard decoction of Malva nut in Example 1 is shown below.

[0037] Figure 2 This is a comparison of the chromatograms obtained from the standard decoction of Pangdahai (Sterculia lychnophora) in Example 1 under both wavelength switching and single wavelength conditions.

[0038] Figure 3 This is a comparison of chromatograms obtained from the standard decoction of Malva nut under different elution procedures in Example 1;

[0039] Figure 4 This is a comparison of chromatograms obtained from the standard decoction of Malva nut in Example 1 under different mobile phase conditions;

[0040] Figure 5 This is a comparison of chromatograms obtained from the standard decoction of Malva nut in Example 1 under different concentrations of phosphoric acid eluent conditions;

[0041] Figure 6 This is a superimposed image of the characteristic maps of 15 batches of round-fruited Malva nut obtained in Example 1;

[0042] Figure 7 This is an examination of the specificity of the detection method for Malva nut in Example 1;

[0043] Figure 8 This is a superimposed image of the characteristic maps of 10 batches of long-fruited Malva nut obtained in Example 2;

[0044] Figure 9This is a control characteristic spectrum of the long-fruited Malva nut prepared in Example 2;

[0045] Figure 10 This is a control characteristic spectrum of the round-fruited Malva nut prepared in Example 2;

[0046] Figure 11 This is a comparison chart of the test results for long-fruited and round-fruited Malva nut in Example 2. Detailed Implementation

[0047] The present application is further described below with reference to the embodiments, examples, and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to the application, and these equivalent forms also fall within the protection scope of the appended claims.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0049] the term

[0050] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0051] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations encompass any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that, in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0052] In this document, terms such as "preferred," "better," and "more preferred" are merely descriptions of implementation methods or examples that achieve better results, and should be understood as not constituting a limitation on the scope of protection of this application.

[0053] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0054] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0055] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, optional numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0056] In this application, weight can be a well-known unit of mass in the chemical industry, such as μg, mg, g, or kg.

[0057] In this application, "%" represents a percentage, referring to the proportion by weight; however, the percentage of a solution, unless otherwise specified, refers to the amount of solute contained in 100 mL of the solution; the percentage of alcohol refers to the proportion by volume at 20°C. Furthermore, the following symbols may be used as needed: %(g / g) indicates the amount of solute contained in 100 g of solution; %(mL / mL) indicates the amount of solute contained in 100 mL of solution; %(mL / g) indicates the amount of solute contained in 100 g of solution; %(g / mL) indicates the amount of solute contained in 100 mL of solution.

[0058] Malva nut has the effects of clearing heat and moistening the lungs, relieving sore throat and hoarseness, and moistening the intestines and relieving constipation. It is generally taken by steeping in boiling water or decocting. Its substance transfer is based on decoction. However, the current detection methods for malva nut are limited to morphological identification and internal chemical composition identification, which cannot reflect the internal chemical information of malva nut and limit its application in modern medicine.

[0059] One aspect of this application relates to a method for constructing a characteristic spectrum of Malva nut medicinal material, Malva nut standard decoction, or Malva nut traditional Chinese medicine formula granules. The characteristic spectrum prepared by this method can comprehensively reflect the intrinsic chemical information of Malva nut medicinal material, Malva nut standard decoction, and Malva nut traditional Chinese medicine formula granules, providing an effective reference for the quality control, identification, and clinical use of Malva nut medicinal material.

[0060] In some embodiments of this application, the method for constructing the index spectrum of Malva nut medicinal material, Malva nut standard decoction, or Malva nut traditional Chinese medicine formula granules includes the following steps:

[0061] Take the test sample, add extraction solvent to extract it, and prepare the test sample solution. The test sample is selected from the medicinal material of Malva nut, the standard decoction of Malva nut, or the traditional Chinese medicine formula granules of Malva nut.

[0062] Take the test solution and perform liquid chromatography analysis to establish characteristic chromatograms of Malva nut medicinal material, Malva nut standard decoction or Malva nut Chinese medicine formula granules;

[0063] The conditions for liquid chromatography analysis include:

[0064] 1) Mobile phase A is methanol, and mobile phase B is a 0.04%~0.06% acid solution;

[0065] 2) Gradient elution is used. The gradient elution procedure includes:

[0066] From 0 to 5 minutes, the volume percentage of mobile phase A increased from 13% to 14%.

[0067] From 5 min to 22 min, maintain the volume percentage of mobile phase A at 14%.

[0068] Between 22 and 37 minutes, the volume percentage of mobile phase A increased from 14% to 31%.

[0069] Between 37 and 50 minutes, the volume percentage of mobile phase A increased from 31% to 50%.

[0070] Between 50 and 60 minutes, the volume percentage of mobile phase B increased from 50% to 85%.

[0071] In some embodiments, the conditions for liquid chromatography analysis further include at least one of the following:

[0072] (1) The liquid chromatography analysis was ultra-high performance liquid chromatography analysis;

[0073] (2) The flow rate for gradient elution is 0.8 mL / min to 1.2 mL / min;

[0074] (3) The column temperature of the chromatographic column is 32℃~38℃;

[0075] (4) The injection volume is 2μL~4μL.

[0076] In some embodiments, the acid solution of mobile phase B is selected from aqueous solutions of phosphoric acid, formic acid, and acetic acid, and is further selected from aqueous solutions of phosphoric acid, which can obtain characteristic peaks with better separation and clearer peak shapes, while also having suitable response signal values.

[0077] Furthermore, a phosphoric acid concentration of 0.04% to 0.06% in an aqueous phosphoric acid solution can achieve better chromatographic peak separation while also providing suitable response signal values ​​and peak shapes.

[0078] In some implementations, in liquid chromatography analysis, the detection wavelength is 265nm~275nm for 0~23min and 320nm~330nm for 23min~60min, which can obtain richer chromatographic peak information, more suitable response signal values, and at the same time, the baseline is stable and interference is small.

[0079] In some embodiments, the chromatographic column is a T3 column; further, the column type is WatersAtlantis T3.

[0080] In some implementations, the liquid chromatography analysis is ultra-high performance liquid chromatography analysis.

[0081] In some implementations, the gradient elution flow rate is 0.8 mL / min to 1.2 mL / min.

[0082] In some implementations, the column temperature is 32°C to 38°C.

[0083] In some implementations, the injection volume is 2 μL to 4 μL.

[0084] In some embodiments, the extraction solvent for the test sample is an aqueous alcohol solution with an alcohol volume ratio of 60% to 80%;

[0085] Furthermore, the alcohol in the alcohol-water solution is methanol;

[0086] Furthermore, the mass-to-volume ratio of the test sample to the extraction solvent is 1 g:(15~25) mL.

[0087] In some implementations, the extraction method includes ultrasound;

[0088] Furthermore, the ultrasonic power is 200W~400W and the frequency is 30kHz~50kHz.

[0089] In some preferred embodiments, the chromatographic conditions include: using an Atlantis T3 column (4.6 mm × 150 mm, 3 μm); using methanol as mobile phase A and 0.05% phosphoric acid solution as mobile phase B, employing gradient elution. The gradient elution program includes: 0–5 min, increasing the volume percentage of mobile phase A from 13% to 14%; 5 min–22 min, maintaining the volume percentage of mobile phase A at 14%; 22 min–37 min, increasing the volume percentage of mobile phase A from 14% to 31%; 37 min–50 min, increasing the volume percentage of mobile phase A from 31% to 50%; 50 min–60 min, increasing the volume percentage of mobile phase B from 50% to 85%; a flow rate of 1.0 mL per minute; a column temperature of 35 °C; a detection wavelength of 280 nm from 0 min–23 min and a detection wavelength of 325 nm from 23 min–60 min; and an injection volume of 3 μL.

[0090] In some implementations, the method for constructing feature maps further includes the following steps:

[0091] A reference solution is provided, and the reference solution is subjected to liquid chromatography analysis, wherein the liquid chromatography analysis conditions are as described in any of the above technical solutions for the liquid chromatography analysis of the test sample;

[0092] The reference solution included protocatechuic acid, protocatechuic aldehyde, catechin, caffeic acid, ferulic acid, and 4-coumaric acid.

[0093] In some embodiments, the preparation steps of the reference solution include: taking appropriate amounts of protocatechuic acid, protocatechuic aldehyde, catechin, caffeic acid, ferulic acid, and 4-coumaric acid, and dissolving them in a solvent;

[0094] The preferred solvent is an aqueous alcohol solution with an alcohol volume ratio of 60% to 80%.

[0095] Furthermore, the alcohol in the alcohol-water solution is methanol;

[0096] Furthermore, the total mass of each reference substance and the mass-to-volume ratio of the solvent were (10~40) μg:1mL.

[0097] In some embodiments, the characteristic spectrum of the Malva nut herb, the standard Malva nut decoction, or the Malva nut traditional Chinese medicine formula granules includes the following 9 common peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, and peak 9, wherein peak 1 is protocatechuic acid, peak 2 is protocatechuic aldehyde, peak 5 is catechin, peak 6 is caffeic acid, peak 7 is 4-coumaric acid, and peak 9 is ferulic acid.

[0098] This application also relates to a method for detecting Sterculia lychnophora herbal material, Sterculia lychnophora standard decoction, or Sterculia lychnophora traditional Chinese medicine formula granules, which includes the following steps:

[0099] Take the analyte and extract it with an extraction solvent to prepare the analyte solution;

[0100] The sample solution was subjected to liquid chromatography analysis to obtain the characteristic chromatogram of the sample.

[0101] The conditions for liquid chromatography analysis include:

[0102] 1) Mobile phase A is methanol, and mobile phase B is a 0.04%~0.06% aqueous solution of phosphoric acid;

[0103] 2) Gradient elution is used. The gradient elution procedure includes:

[0104] From 0 to 5 minutes, the volume percentage of mobile phase A increased from 13% to 14%.

[0105] From 5 min to 22 min, maintain the volume percentage of mobile phase A at 14%.

[0106] Between 22 and 37 minutes, the volume percentage of mobile phase A increased from 14% to 31%.

[0107] Between 37 and 50 minutes, the volume percentage of mobile phase A increased from 31% to 50%.

[0108] Between 50 and 60 minutes, the volume percentage of mobile phase B increased from 50% to 85%.

[0109] In some embodiments, the liquid chromatography analysis conditions in the detection method for Malva nut medicinal material, Malva nut standard decoction, or Malva nut traditional Chinese medicine formula granules further include at least one of the following:

[0110] (1) The liquid chromatography analysis is ultra-high performance liquid chromatography analysis;

[0111] (2) The flow rate of the gradient elution is 0.8 mL / min to 1.2 mL / min;

[0112] (3) The column temperature of the chromatographic column is 32℃~38℃;

[0113] (4) The injection volume is 2μL~4μL.

[0114] In some implementation methods, the chromatographic column used in the detection methods of Malva nut medicinal material, Malva nut standard decoction or Malva nut Chinese medicine formula granules is a T3 chromatographic column.

[0115] Furthermore, the chromatographic column is a Waters Atlantis T3.

[0116] In some implementation methods, the extraction solvent in the detection methods of Malva nut medicinal material, Malva nut standard decoction or Malva nut Chinese medicine formula granules is an alcohol-water solution with an alcohol volume ratio of 60% to 80%.

[0117] Furthermore, the alcohol in the alcohol-water solution is methanol.

[0118] In some implementation methods, the extraction method in the detection of Malva nut medicinal material, Malva nut standard decoction or Malva nut Chinese medicine formula granules includes ultrasound.

[0119] Furthermore, the ultrasonic power is 200W~400W and the frequency is 30kHz~50kHz.

[0120] In another aspect, this application also provides the application of the feature map constructed by the feature map construction method described in any of the above technical solutions and / or the detection method described in any of the above technical solutions in any of the following (1) to (3):

[0121] (1) Differentiate between long-fruited and round-fruited Malva nut medicinal materials;

[0122] (2) Differentiate between the standard decoction of long-fruited and round-fruited Malva nut;

[0123] (3) Differentiate between long-fruited and round-fruited Malva nut Chinese medicine formula granules.

[0124] Furthermore, the above identification steps include:

[0125] Take the sample to be tested, add an extraction solvent to extract it, and prepare the sample solution.

[0126] Perform liquid chromatography analysis on the sample solution to establish a characteristic chromatogram of the sample;

[0127] The characteristic spectrum of the sample to be tested is compared with the characteristic spectrum of the Malva nut medicinal material, Malva nut standard decoction or Malva nut Chinese medicine formula granules constructed by the characteristic spectrum construction method described in any of the above technical solutions, so as to achieve the identification of the sample to be tested.

[0128] The conditions for liquid chromatography analysis include:

[0129] (1) Mobile phase A is methanol, and mobile phase B is a 0.04%~0.06% acid solution;

[0130] (2) Gradient elution is employed, wherein the gradient elution procedure includes:

[0131] From 0 to 5 minutes, the volume percentage of mobile phase A increased from 13% to 14%.

[0132] From 5 min to 22 min, maintain the volume percentage of mobile phase A at 14%.

[0133] Between 22 and 37 minutes, the volume percentage of mobile phase A increased from 14% to 31%.

[0134] Between 37 and 50 minutes, the volume percentage of mobile phase A increased from 31% to 50%.

[0135] Between 50 and 60 minutes, the volume percentage of mobile phase B increased from 50% to 85%.

[0136] In the above identification methods, the conditions of liquid chromatography analysis (including the instrument used for analysis, flow rate, column temperature, injection volume, column type, etc.) and the extraction solvent and extraction method used in the preparation of the test solution are as described in the relevant content of the first or second aspect of this application, and will not be repeated here.

[0137] In some embodiments, the characteristic chromatograms of the *Sterculia lychnophora* herbal material, *Sterculia lychnophora* standard decoction, or *Sterculia lychnophora* herbal formula granules established by the construction method described in any of the above technical solutions include:

[0138] In the characteristic chromatograms of *Sterculia lychnophora*, standard decoctions, or granules of its traditional Chinese medicine formula, with peak S as a reference, the relative peak area of ​​peak 4 is 0~0.15.

[0139] In the characteristic chromatograms of round-fruited Malva nut, standard decoction, or herbal formula granules, with peak S as a reference, the relative peak area of ​​peak 4 is 0.3~2.

[0140] The characteristic spectrum of the sample to be tested is compared with the characteristic spectrum of the Malva nut medicinal material, Malva nut standard decoction, or Malva nut Chinese medicine formula granules constructed by the construction method described in any of the above technical solutions:

[0141] In the characteristic spectrum of the sample to be tested, with peak S as a reference, if the relative peak area of ​​peak 4 is 0~0.15, then the sample to be tested is selected from long-fruited Malva nut, standard decoction or its traditional Chinese medicine formula granules.

[0142] In the characteristic spectrum of the sample to be tested, with peak S as a reference, if the relative peak area of ​​peak 4 is 0.3~2, then the sample to be tested is selected from round-fruited Malva nut, standard decoction, or Chinese herbal formula granules.

[0143] The following are some specific examples.

[0144] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0145] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0146] Instruments and reagents

[0147] Instruments: Waters ACQUITY H-class ultra-high performance liquid chromatograph (Waters Technologies Inc.); Thermo UHPLC (Vanquish, Thermo Fisher Scientific); Waters Atlantis T3 column (4.6mm × 150mm, 3μm); 0.0001 g balance (ME204E, Mettler Toledo); 0.00001 g balance (ABT220-5DM, Guangzhou Dexiang Technology Co., Ltd.); 0.00001 g balance (XP26, Mettler Toledo); Electronic balance (JJ600, Changshu Shuangjie Test Instrument Factory); CNC ultrasonic cleaner (KQ500DE, Kunshan Ultrasonic Instrument Co., Ltd.); Constant temperature water bath (HWS28, Shanghai Yiheng Technology Co., Ltd.); Ultrapure water system (Villi-Q Direct, Merck KGaA).

[0148] Reagents: Ethanol (Tianjin Fuyu Fine Chemical Co., Ltd.) and methanol (Tianjin Fuyu Fine Chemical Co., Ltd.) were analytical grade; phosphoric acid (Tianjin Kemeio Chemical Reagent Co., Ltd.) and methanol (Merck, Inc.) were chromatographic grade; water was ultrapure water (prepared in the laboratory). Protocatechuic acid (batch number: 110809-201906, content: 97.7%, China National Institutes for Food and Drug Control); Protocatechuic aldehyde (batch number: 110810-201909, content: 99.6%, China National Institutes for Food and Drug Control); Catechin (batch number: 110877-802005, content: 95.1%, China National Institutes for Food and Drug Control); Caffeic acid (batch number: 110773-201915, content: 99.4%, China National Institutes for Food and Drug Control); Ferulic acid (batch number: 110885-201703, content: 99.7%, China National Institutes for Food and Drug Control); 4-Coumaric acid (batch number: 112037-202102, content: 99.7%, China National Institutes for Food and Drug Control); Malva nut reference material (batch number: 121287-201704, China National Institutes for Food and Drug Control).

[0149] The medicinal materials used in this application were identified by the Quality Center of Guangdong Yifang Pharmaceutical Co., Ltd. as dried, mature seeds of the plant *Sterculia lychnophora* Hance, belonging to the Sterculiaceae family. Specific information on the medicinal materials is shown in Table 1.

[0150] Table 1. Information on Malva nut medicinal materials

[0151]

[0152] Example 1: Establishment of the Feature Map Construction Method

[0153] 1.1 Determine chromatographic conditions

[0154] 1.1.1 Determination of the optimal absorption wavelength

[0155] Take an appropriate amount of standard decoction of Pangdahai (Sterculia lychnophora), grind it into a fine powder, and accurately weigh about 0.5g. Prepare the test solution according to the method for preparing the test solution under section "1.3". Perform gradient elution under the chromatographic conditions under section "1.1.4", and use full-wavelength scanning as the detection method. The detection results are as follows: Figure 1 ,according to Figure 1 The standard decoction of Malva nut showed the highest absorbance at wavelengths of 280 nm and 325 nm. The detection method was adjusted to 280 nm, with wavelengths of 280 nm for 0 min to 23 min and 325 nm for 23 min to 60 min. The detection results are as follows. Figure 2 ,according to Figure 2 The wavelength-switching operation (detection wavelength of 280nm from 0min to 23min and detection wavelength of 325nm from 23min to 60min) yielded more chromatographic peak information and larger peaks, while the baseline was stable and interference was less. Therefore, considering all factors, the wavelength-switching operation was performed.

[0156] 1.1.2 Determination of the elution gradient

[0157] Based on the separation effect of each chromatographic peak, the chromatographic conditions were optimized to determine the optimal chromatographic conditions. A Waters Atlantis T3 (4.6 mm × 150 mm, 3 μm) column was used, with methanol as mobile phase A and 0.05% phosphoric acid solution as mobile phase B, and gradient elution was performed according to the specifications in Table 2; the flow rate was 1.0 mL per minute; the column temperature was 35℃; the detection wavelength was 280 nm from 0 min to 23 min and 325 nm from 23 min to 60 min; the injection volume was 3 μL.

[0158] Table 2 Gradient Elution Table

[0159]

[0160] See results Figure 3 As shown ( Figure 3 The question mark icon in the upper left corner indicates that the sample type is "unknown". The results show that by optimizing the chromatographic conditions, the separation of each characteristic peak is better and the running time is shorter when the chromatographic conditions are Method 4. Therefore, Method 4 was determined as the final chromatographic condition.

[0161] 1.1.3 Determination of the mobile phase

[0162] The effects of different water phases on each characteristic peak were investigated to determine the optimal mobile phase. The results are shown in [Figure number missing]. Figure 4 and Figure 5 As shown ( Figure 5 The question mark icon in the upper left corner indicates that the sample type is "unknown". The results showed that the separation of each chromatographic peak was best when 0.05% phosphoric acid was used as the aqueous phase. Therefore, methanol-0.05% phosphoric acid was finally selected as the final mobile phase.

[0163] 1.1.4 Determined chromatographic conditions

[0164] Based on the above experimental results, the chromatographic conditions for the standard decoction of Malva nut can be determined as follows: an Atlantis T3 column (4.6 mm × 150 mm, 3 μm) is used; methanol is used as mobile phase A, and 0.05% phosphoric acid solution is used as mobile phase B, with gradient elution performed according to the specifications in Table 3; the flow rate is 1.0 mL per minute; the column temperature is 35℃; the detection wavelength is 280 nm from 0 min to 23 min, and 325 nm from 23 min to 60 min; the injection volume is 3 μL.

[0165] Table 3 Gradient elution table

[0166] 1.2 Preparation of reference solution

[0167] Take appropriate amounts of protocatechuic acid reference standard, protocatechuic aldehyde reference standard, catechin reference standard, caffeic acid reference standard, ferulic acid reference standard, and 4-coumaric acid reference standard, accurately weigh them, and add 70% methanol to prepare a mixed solution containing 40 μg of protocatechuic acid, 10 μg of protocatechuic aldehyde, 30 μg of catechin, 30 μg of caffeic acid, 20 μg of ferulic acid, and 10 μg of 4-coumaric acid per 1 mL, as the reference solution.

[0168] 1.3 Preparation of the test solution

[0169] Medicinal material: Take about 1.0g of sample powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 70% methanol, sonicate for 30 minutes (power 300W, frequency 40kHz), centrifuge, take the supernatant and evaporate to dryness, add 20mL of water to dissolve the residue, extract twice with ethyl acetate, 60mL each time, combine the ethyl acetate layers, evaporate to dryness, add 70% methanol to dissolve the residue, transfer to a 2mL volumetric flask, add 70% methanol to the mark, shake well, filter, and take the filtrate to obtain the final product.

[0170] Standard decoction: Take an appropriate amount of this product, grind it into a fine powder, accurately weigh about 1.0 g, place it in a stoppered conical flask, accurately add 25 mL of 70% methanol, sonicate for 30 minutes (power 300W, frequency 40kHz), centrifuge, take the supernatant and evaporate to dryness, add 20 mL of water to dissolve the residue, extract twice with ethyl acetate, 60 mL each time, combine the ethyl acetate layers, evaporate to dryness, add 70% methanol to dissolve the residue, transfer to a 2 mL volumetric flask, add 70% methanol to the mark, shake well, filter, and take the filtrate to obtain the decoction.

[0171] Formula granules: Take an appropriate amount of this product, grind it into a fine powder, accurately weigh about 1.0 g, place it in a stoppered conical flask, accurately add 25 mL of 70% methanol, sonicate for 30 minutes (power 300W, frequency 40kHz), centrifuge, take the supernatant and evaporate to dryness, add 20 mL of water to dissolve the residue, extract twice with ethyl acetate, 60 mL each time, combine the ethyl acetate layers, evaporate to dryness, add 70% methanol to dissolve the residue, transfer to a 2 mL volumetric flask, add 70% methanol to the mark, shake well, filter, and take the filtrate to obtain the product.

[0172] 1.4 Determination Method

[0173] Accurately pipette 3 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0174] 1.5 Determination of Malva nut samples

[0175] Fifteen batches of *Sterculia lychnophora* (numbered PY01~PY15) were collected. Test solutions were prepared according to the method described in section "1.3". Chromatographic analysis was performed under the conditions specified in section "1.1". The resulting superimposed characteristic chromatograms of the 15 batches of *Sterculia lychnophora* are shown below. Figure 6 Using peak 6 as the reference peak S, the relative retention times and relative peak areas of other chromatographic peaks and peak S were calculated, and the results are shown in Tables 4 and 5.

[0176] Table 4. Relative retention times of characteristic chromatograms of 15 batches of *Sterculia lychnophora* (round-fruited sterculia lychnophora) medicinal materials.

[0177]

[0178] Table 5. Relative peak areas of characteristic spectra of 15 batches of round-fruited Malva nut medicinal materials

[0179]

[0180] 1.6 Characteristic Peak Identification

[0181] The test solution was subjected to ultraviolet spectral analysis. By comparing the ultraviolet spectra with those of the reference standard, the components contained in Malva nut were finally determined to be as follows: peak 1 is protocatechuic acid, peak 2 is protocatechuic aldehyde, peak 5 is catechin, peak 6 is caffeic acid, peak 7 is 4-coumaric acid, and peak 9 is ferulic acid.

[0182] 1.7 Specificity Examination

[0183] See results Figure 7 The characteristic peaks in the chromatogram of the test sample have the same chromatographic peaks at the corresponding retention times as those in the chromatogram of the reference solution, and there is no negative interference, indicating that the method has good specificity.

[0184] Example 2: Application of Differentiation between Round and Long Fruits of Malva Nutmeg

[0185] 2.1 Chromatographic conditions for characteristic spectra

[0186] The chromatographic column was a Waters Atlantis T3 column (4.6 mm × 150 mm, 3 μm), and the flow rate was 1.0 mL / min. Methanol was used as mobile phase A, and 0.05% phosphoric acid was used as mobile phase B. Gradient elution was performed as follows: 0 min–5 min, 13% A–14% A; 5 min–22 min, 14% A; 22 min–37 min, 14% A–31% A; 37 min–50 min, 31% A–50% A; 50 min–60 min, 50% A–85% A. The detection wavelength was 280 nm from 0 min to 23 min and 325 nm from 23 min to 60 min. The column temperature was 35 °C. The injection volume was 3 μL.

[0187] 2.2 Preparation of reference solution

[0188] Take appropriate amounts of protocatechuic acid reference standard, protocatechuic aldehyde reference standard, catechin reference standard, caffeic acid reference standard, ferulic acid reference standard and 4-coumaric acid reference standard, accurately weigh them, add 70% methanol to prepare a mixed solution containing 40 μg of protocatechuic acid, 10 μg of protocatechuic aldehyde, 30 μg of catechin, 30 μg of caffeic acid, 20 μg of ferulic acid and 10 μg of 4-coumaric acid per 1 mL, and use it as the reference solution.

[0189] 2.3 Preparation of control herbal solution

[0190] Take about 1g of the reference herb *Sterculia lychnophora*, place it in a stoppered conical flask, add 50mL of water, heat under reflux for 30min, filter, evaporate to dryness, add 25mL of 70% methanol to the residue, heat under reflux for 30min, centrifuge, take the supernatant and evaporate to dryness, add 20mL of water to dissolve the residue, extract twice with 60mL of ethyl acetate each time, combine the ethyl acetate layers, evaporate to dryness, add 70% methanol to dissolve the residue, transfer to a 2mL volumetric flask, add 70% methanol to the mark, shake well, filter, and take the filtrate as the reference solution for the reference herb.

[0191] 2.4 Preparation of the test solution

[0192] Accurately weigh approximately 1.0 g of sample powder (passed through a No. 3 sieve), place it in a stoppered conical flask, accurately add 25 mL of 70% methanol, sonicate for 30 minutes (300 W power, 40 kHz frequency), centrifuge, take the supernatant and evaporate to dryness, add 20 mL of water to dissolve the residue, extract twice with ethyl acetate, 60 mL each time, combine the ethyl acetate layers, evaporate to dryness, add 70% methanol to dissolve the residue, transfer to a 2 mL volumetric flask, add 70% methanol to the mark, shake well, filter, and take the filtrate to obtain the sample.

[0193] 2.5 Comparison of characteristic images of round-fruited and long-fruited Malva nut

[0194] Ten batches of *Sterculia lychnophora* (numbered PC01~PC10) were also collected, and the chromatographic conditions under section “2.1” were used to determine the characteristics of these ten batches. The results are shown in Tables 6 and 7. The overlay of the characteristic chromatograms obtained from the ten batches of *Sterculia lychnophora* is shown in the figure below. Figure 8 .

[0195] The "Similarity Evaluation System for Chromatographic Characteristic Magnetism of Traditional Chinese Medicine (Version 2012.0)" recommended by the National Pharmacopoeia Commission was used to process the characteristic chromatograms of 10 batches of long-fruited Malva nut and 15 batches of round-fruited Malva nut samples from "Example 1". Common peaks were identified, and a control characteristic chromatogram of long-fruited Malva nut was established. Figure 9 ) and the comparative characteristic atlas of round-fruited Malva nut medicinal materials ( Figure 10 ).

[0196] A comparison chart of the test results for long-fruited and round-fruited Malva nut ( Figure 11 As can be seen, all batches of round-fruited Malva nut have 10 characteristic peaks, while most batches of long-fruited Malva nut lack peak 4, and some batches detect peak 4 but with low signal intensity. According to the detection results of this application, in the chromatogram of long-fruited Malva nut, with peak S as a reference, the relative peak area of ​​peak 4 is 0~0.15, while in the chromatogram of round-fruited Malva nut, with peak S as a reference, the relative peak area of ​​peak 4 is 0.3~2. The peak area of ​​peak 4 relative to peak S can be used as the basis for distinguishing between long-fruited and round-fruited Malva nut.

[0197] ① Results of the determination of the characteristic spectrogram of Sterculia lychnophora (long-fruited Malva nut)

[0198] Table 6. Relative retention times of characteristic chromatograms of 10 batches of *Sterculia lychnophora* (long-fruited sterculia lychnophora) medicinal materials.

[0199]

[0200] Table 7. Relative peak areas of characteristic spectra of 10 batches of *Sterculia lychnophora* (long-fruited sterculia lychnophora) medicinal materials.

[0201] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0202] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples 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.

[0203] The embodiments described above merely illustrate several implementation methods of this application and should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing characteristic chromatograms of Malva nut medicinal material, Malva nut standard decoction, or Malva nut traditional Chinese medicine formula granules, characterized in that, Includes the following steps: Take the test sample, add an extraction solvent for extraction, take the supernatant and evaporate to dryness, dissolve the residue in water, extract with ethyl acetate to obtain the test sample solution, wherein the test sample is selected from Malva nut medicinal material, Malva nut standard decoction or Malva nut Chinese medicine formula granules; the extraction solvent is an alcohol-water solution with an alcohol volume ratio of 60%~80%, wherein the alcohol in the alcohol-water solution is methanol; A reference solution is prepared, wherein the reference solution comprises protocatechuic acid, protocatechuic aldehyde, catechin, caffeic acid, ferulic acid and 4-coumaric acid; The test solution and the reference solution were subjected to liquid chromatography analysis to establish characteristic chromatograms of Malva nut medicinal material, Malva nut standard decoction or Malva nut Chinese medicine formula granules; The conditions for the liquid chromatography analysis include: (1) Mobile phase A is methanol, and mobile phase B is a 0.04%~0.06% acid solution; (2) Gradient elution is employed, wherein the gradient elution procedure includes: Within 0-5 minutes, the volume percentage of mobile phase A increased from 13% to 14%. For 5 to 22 minutes, maintain the volume percentage of mobile phase A at 14%. Between 22 and 37 minutes, the volume percentage of mobile phase A increased from 14% to 31%. Between 37 and 50 minutes, the volume percentage of mobile phase A increased from 31% to 50%. Over 50-60 minutes, the volume percentage of the mobile phase B increased from 50% to 85%. The detection wavelength for 0 min to 23 min is 280 nm, and the detection wavelength for 23 min to 60 min is 325 nm. (3) The flow rate of the gradient elution is 0.8 mL / min to 1.0 mL / min; (4) The chromatographic column is a T3 column with a length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 3 μm for the packing material.

2. The method for constructing the characteristic spectrum of the *Sterculia lychnophora* herbal material, *Sterculia lychnophora* standard decoction, or *Sterculia lychnophora* herbal formula granules as described in claim 1, characterized in that, The conditions for the liquid chromatography analysis also include at least one of the following: (1) The column temperature of the chromatographic column is 32℃~38℃; (2) The injection volume is 2μL~4μL.

3. The method for constructing the characteristic spectrum of the *Sterculia lychnophora* herbal material, *Sterculia lychnophora* standard decoction, or *Sterculia lychnophora* herbal formula granules as described in claim 1, characterized in that, The extraction method includes ultrasound.

4. The method for constructing the characteristic spectrum of the *Sterculia lychnophora* herbal material, *Sterculia lychnophora* standard decoction, or *Sterculia lychnophora* herbal formula granules as described in claim 3, characterized in that, The power of ultrasound is 200W~400W and the frequency is 30kHz~50kHz.

5. The method for constructing the characteristic spectrum of the *Sterculia lychnophora* herbal material, *Sterculia lychnophora* standard decoction, or *Sterculia lychnophora* herbal formula granules as described in any one of claims 1 to 4, characterized in that, The characteristic spectrum of the Malva nut medicinal material, Malva nut standard decoction or Malva nut Chinese medicine formula granules includes the following 9 common peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8 and peak 9, wherein peak 1 is protocatechuic acid, peak 2 is protocatechuic aldehyde, peak 5 is catechin, peak 6 is caffeic acid, peak 7 is 4-coumaric acid and peak 9 is ferulic acid.

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