Method for constructing characteristic maps and detection methods of Glechoma longituba medicinal materials, standard decoctions or traditional Chinese medicine formula granules and their applications

Abstract

The present invention provides a method for constructing a characteristic spectrum of a Glechoma galbana medicinal material, a standard decoction, or a traditional Chinese medicine formula granule, comprising the following steps: preparing a chlorogenic acid reference substance and a chlorogenic acid reference substance solution; preparing a mixed reference substance solution and a neochlorogenic acid and cryptochlorogenic acid reference substance solution; preparing a test solution and subjecting the chlorogenic acid reference substance solution, the mixed reference substance solution, and the test solution to ultra-high performance liquid chromatography analysis, respectively; wherein the ultra-high performance liquid chromatography analysis conditions include: mobile phase A is acetonitrile, mobile phase B is aqueous phosphoric acid, and a gradient elution procedure is employed. The characteristic spectrum constructed by the method described in the present invention is rich in information, highly characteristic, and has good reproducibility.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine quality control, and in particular to a method for constructing and detecting a characteristic spectrum of a cleavers medicinal material, a standard decoction or a traditional Chinese medicine formula granule, and applications thereof. Background Art

[0002] Galium aparine L., a plant of the Rubiaceae family, was included in the 1977 edition of the Chinese Pharmacopoeia. It is the dried whole herb of Galium aparine L., a plant of the Rubiaceae family. It has pungent, bitter, cooling, and slightly cold properties, and has the effects of clearing heat and detoxifying, as well as promoting diuresis and reducing swelling. It is commonly used clinically to treat tumors, dysentery, mastitis, urinary tract infections, amenorrhea, traumatic swelling, gallbladder disorders, bone diseases, and nocturnal emission.

[0003] In recent years, research on Glechoma longituba has primarily focused on evaluating its quality or establishing quality standards through content determination, thin-layer chromatography (TLC) identification, and ultraviolet spectrophotometry. The 2018 edition of the Hubei Provincial Standard for Traditional Chinese Medicine, the 2014 edition of the Sichuan Provincial Standard for Tibetan Medicinal Materials, and the 1994 edition of the Shanghai Municipal Standard for Traditional Chinese Medicine all include Glechoma longituba. The 2014 edition of the Sichuan Provincial Standard for Tibetan Medicinal Materials added TLC identification and content determination methods for Glechoma longituba. Content determination in Glechoma longituba primarily uses chlorogenic acid, a phenylpropanoid active ingredient, as a research indicator. However, Glechoma longituba also contains flavonoids and iridoids. Chlorogenic acid content alone lacks specificity and cannot objectively reflect the changes in Glechoma longituba composition, thus failing to accurately assess the intrinsic quality of the sample. TLC qualitative identification methods often use a reference standard, which can only reflect the changes in a single spot. The lack of a reference standard for identifying other spots prevents comprehensive information from being captured by TLC. The above two analysis methods are not comprehensive enough and cannot reflect as much sample information as possible. In addition, the preparation method of the test samples is complicated. When analyzing a large amount of sample data, it requires a lot of manpower and material resources, which reduces the detection efficiency.

[0004] Characteristic spectra are images, spectra, and data obtained using modern information collection technology and quality analysis methods that reveal the properties of traditional Chinese medicines or other dosage forms. They comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicines, thereby comprehensively evaluating their quality. However, research on the characteristic spectra of Glechoma longituba is almost nonexistent. Summary of the Invention

[0005] Based on this, the present invention provides a method for constructing a characteristic spectrum of a Glechoma longituba medicinal material, a standard decoction or a Chinese medicine formula granule. The constructed characteristic spectrum is rich in information, highly characteristic and has good reproducibility.

[0006] The present invention is achieved through the following technical solutions.

[0007] A method for constructing a characteristic spectrum of a Chinese medicinal material, a standard decoction, or a Chinese medicine formula granule comprises the following steps:

[0008] Take chlorogenic acid reference substance to prepare chlorogenic acid reference substance solution; take neochlorogenic acid and cryptochlorogenic acid reference substances to prepare mixed reference substance solution;

[0009] Prepare the test solution by taking the medicinal material of Glehnia littoralis, standard decoction or Chinese medicine granules;

[0010] The chlorogenic acid reference solution, the mixed reference solution, and the test solution are subjected to ultra-performance liquid chromatography analysis respectively;

[0011] The conditions for ultra-high performance liquid chromatography analysis include: mobile phase A is acetonitrile, mobile phase B is aqueous phosphoric acid; a gradient elution program is used; the gradient elution program includes: from 0 min to 5 min, the volume percentage of mobile phase A is 6%; from 5 min to 20 min, the volume percentage of mobile phase A changes from 6% to 21%; from 20 min to 20.1 min, the volume percentage of mobile phase A changes from 21% to 90%; and from 20.1 min to 25 min, the volume percentage of mobile phase A is maintained at 90%.

[0012] In one embodiment, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.2%.

[0013] In one embodiment, the conditions for ultra-high performance liquid chromatography analysis also include: the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 28°C to 32°C; the flow rate is 0.28mL / min to 0.32mL / min; and the wavelength is 310nm to 350nm.

[0014] In one embodiment, the dimensions of the chromatographic column used in ultra-high performance liquid chromatography analysis include: a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm ± 0.2 μm.

[0015] In one embodiment, preparing the test solution comprises the following steps:

[0016] Mixing the galangal medicinal material with a methanol aqueous solution with a volume fraction of 60% to 80%, heating and refluxing for extraction, and then filtering to obtain a filtrate; or

[0017] Mixing the standard decoction of galangal with 60% to 80% ethanol aqueous solution, performing ultrasonic extraction, and then filtering to obtain a filtrate; or

[0018] The Chinese medicinal formula granules of Glechoma longituba are mixed with an ethanol aqueous solution with a volume fraction of 60% to 80%, subjected to ultrasonic extraction, and then filtered to obtain a filtrate.

[0019] In one embodiment, the following steps are also included:

[0020] Mixing the control medicinal material of Galium japonicum with water, heating and refluxing for extraction, then filtering, evaporating the filtrate, taking the residue and mixing it with ethanol and water solution, ultrasonically treating it, then filtering, taking the filtrate, and preparing the control medicinal material reference solution;

[0021] The control medicinal material reference solution is subjected to ultra-high performance liquid chromatography analysis.

[0022] In one embodiment, preparing a chlorogenic acid reference solution comprises the following steps: mixing the chlorogenic acid reference solution with an ethanol aqueous solution;

[0023] The preparation of the mixed reference solution comprises the following steps: mixing the neochlorogenic acid reference substance, the cryptochlorogenic acid reference substance and an ethanol aqueous solution.

[0024] In one embodiment, the characteristic spectrum of the Glechoma longituba medicinal material, standard decoction or Chinese medicine formula granules has a total of 6 characteristic peaks; among them, peak 2 is the chromatographic peak of chlorogenic acid, peak 1 is the chromatographic peak of neochlorogenic acid, and peak 3 is the chromatographic peak of cryptochlorogenic acid.

[0025] The present invention also provides the use of the method for constructing the characteristic spectrum of the above-mentioned Glechoma longituba medicinal material, standard decoction or traditional Chinese medicine formula granule in identifying the authenticity of Glechoma longituba.

[0026] The present invention also provides a method for detecting a medicinal material, a standard decoction or a Chinese medicine granule, comprising the following steps:

[0027] Take the product to be tested and prepare a solution of the product to be tested;

[0028] The test solution is subjected to ultra-high performance liquid chromatography determination;

[0029] The conditions for ultra-high performance liquid chromatography analysis include: mobile phase A is acetonitrile, mobile phase B is aqueous phosphoric acid; a gradient elution program is used; the gradient elution program includes: from 0 min to 5 min, the volume percentage of mobile phase A is 6%; from 5 min to 20 min, the volume percentage of mobile phase A changes from 6% to 21%; from 20 min to 20.1 min, the volume percentage of mobile phase A changes from 21% to 90%; and from 20.1 min to 25 min, the volume percentage of mobile phase A is maintained at 90%.

[0030] In one embodiment, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.2%.

[0031] In one embodiment, the conditions for ultra-high performance liquid chromatography analysis also include: the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 28°C to 32°C; the flow rate is 0.28mL / min to 0.32mL / min; and the wavelength is 310nm to 350nm.

[0032] In one embodiment, the dimensions of the chromatographic column used in ultra-high performance liquid chromatography analysis include: a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm ± 0.2 μm.

[0033] Compared with the prior art, the method for constructing a characteristic spectrum of a Glechoma longituba medicinal material, a standard decoction or a Chinese medicine formula granule of the present invention has the following beneficial effects:

[0034] The present invention establishes a fingerprint of the Chinese galium medicinal material, standard decoction or traditional Chinese medicine formula granules by using UPLC, and limits the mobile phase solvent and gradient elution program, thereby achieving the goals of rich characteristic spectrum information, strong characteristic and good reproducibility. It can comprehensively reflect the intrinsic quality of the Chinese galium sample, can be used for the quality control of the Chinese galium medicinal material, standard decoction and traditional Chinese medicine formula granules, and can also be effectively used for the identification of authentic and counterfeit Chinese galium, thereby ensuring the safety and effectiveness of clinical medication.

[0035] Furthermore, the method for constructing the fingerprint of the Glechoma longituba medicinal material, standard decoction or traditional Chinese medicine formula granule described in the present invention is simple to operate and has a short analysis time, thus saving detection costs and improving analysis efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 A comparison chart of solvents used to extract the Chinese medicinal formula granules of Glechoma longituba provided by the present invention;

[0037] Figure 2 A comparative diagram of the extraction methods of the Chinese medicinal formula granules of the galangal provided by the present invention;

[0038] Figure 3 A comparison chart of the extraction time of the Chinese medicinal formula granules of the galangal provided by the present invention;

[0039] Figure 4 Investigation on the specificity of the characteristic spectrum of the Chinese medicinal formula granules of Glechoma longituba provided by the present invention;

[0040] Figure 5 Characteristic spectra of the Chinese medicinal formula granules of Glechoma longituba under different instruments provided by the present invention;

[0041] Figure 6 Characteristic spectra of the Chinese medicinal formula granules of Glechoma longituba at different column temperatures provided by the present invention;

[0042] Figure 7Characteristic spectra of the Chinese medicinal formula granules of Glechoma longituba at different flow rates provided by the present invention;

[0043] Figure 8 Characteristic spectra of the Chinese medicinal formula granules of Glechoma longituba under different brands of chromatographic columns provided by the present invention;

[0044] Figure 9 The characteristic spectrum of the Chinese medicinal formula granules of Glechoma longituba under the same brand chromatographic column provided by the present invention;

[0045] Figure 10 Overlapping chromatograms of 15 batches of Glechoma longituba medicinal materials provided by the present invention;

[0046] Figure 11 Overlapping chromatograms of 15 batches of standard decoctions of Glechoma longituba provided by the present invention;

[0047] Figure 12 Overlapping chromatograms of three batches of Glechoma longituba Chinese medicinal formula granules provided by the present invention;

[0048] Figure 13 Characteristic spectra of the standard decoction of Glechoma longituba under different gradient elution programs provided by the present invention;

[0049] Figure 14 The wavelength scanning 3D diagram provided by the present invention;

[0050] Figure 15 Characteristic spectra of the standard decoction of Glechoma longituba at different wavelengths provided by the present invention;

[0051] Figure 16 A comparative diagram of Glechoma longituba and Glechoma longituba provided by the present invention. DETAILED DESCRIPTION

[0052] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0053] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. In the description of the present invention, the meaning of "several" is at least one, such as one, two, etc., unless otherwise clearly and specifically defined.

[0054] The terms "preferably," "more preferably," and the like, used herein refer to embodiments of the present invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the present invention.

[0055] When a numerical range is disclosed herein, the range is considered continuous and includes 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 indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0056] Unless otherwise indicated, all percentages, fractions, and ratios are calculated based on the total weight of the compositions of the present invention. Unless otherwise indicated, all masses relating to listed ingredients are given as active ingredients and therefore do not include solvents or byproducts that may be present in commercially available materials. The term "mass percentage content" may be expressed herein with the symbol "%." All molecular weights herein are weight-average molecular weights expressed in Daltons, unless otherwise indicated. All formulations and testing herein took place at 25°C, unless otherwise indicated. The terms "comprise," "include," "contain," "have," "have," and other variations herein are intended to encompass non-exclusive inclusions, and do not distinguish between these terms. The term "comprising" means that additional steps and ingredients that do not affect the end result may be added. The compositions and methods / processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein. The terms "efficacy," "performance," "effect," and "efficacy" are not used herein to distinguish between.

[0057] The weights of the relevant components mentioned in the description of the embodiments of the present invention may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally increased or decreased according to the description of the embodiments of the present invention, it is within the scope disclosed in the description of the embodiments of the present invention. Specifically, the weights mentioned in the description of the embodiments of the present invention may be mass units known in the chemical industry, such as ug, mg, g, and kg.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0059] The present invention provides a method for constructing a characteristic spectrum of a Chinese medicinal material, a standard decoction or a Chinese medicine formula granule, comprising the following steps:

[0060] Take chlorogenic acid reference substance to prepare chlorogenic acid reference substance solution; take neochlorogenic acid and cryptochlorogenic acid reference substances to prepare mixed reference substance solution;

[0061] Prepare the test solution by taking the medicinal material of Glehnia littoralis, standard decoction or Chinese medicine granules;

[0062] The chlorogenic acid reference solution, mixed reference solution and test solution were subjected to ultra-performance liquid chromatography analysis respectively;

[0063] Among them, the conditions for ultra-high performance liquid chromatography analysis include: mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution; a gradient elution program is adopted; the gradient elution program includes: 0 min to 5 min, the volume percentage of mobile phase A is 6%; 5 min to 20 min, the volume percentage of mobile phase A changes from 6% to 21%; 20 min to 20.1 min, the volume percentage of mobile phase A changes from 21% to 90%; 20.1 min to 25 min, the volume percentage of mobile phase A is maintained at 90%.

[0064] In a specific example, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.2%.

[0065] It can be understood that in the present invention, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution includes but is not limited to 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, and 0.2%.

[0066] In a specific example, the conditions for ultra-high performance liquid chromatography analysis also include: the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 28°C to 32°C; the flow rate is 0.28mL / min to 0.32mL / min; and the wavelength is 310nm to 350nm.

[0067] It is understood that in the present invention, column temperatures include but are not limited to 28°C, 29°C, 30°C, 31°C, and 32°C;

[0068] Flow rates include but are not limited to 0.28 mL / min, 0.29 mL / min, 0.30 mL / min, 0.31 mL / min, 0.32 mL / min;

[0069] Wavelengths include but are not limited to 310 nm, 315 nm, 320 nm, 325 nm, 326 nm, 327 nm, 328 nm, 329 nm, 330 nm, 331 nm, 332 nm, 333 nm, 334 nm, 335 nm, 340 nm, 345 nm, and 350 nm.

[0070] In a specific example, the dimensions of the chromatographic column used in ultra-high performance liquid chromatography analysis include: a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm±0.2 μm.

[0071] Preferably, the dimensions of the chromatographic column used in ultra-high performance liquid chromatography analysis include: column length of 100 mm, inner diameter of 2.1 mm, and particle size of 1.8 μm.

[0072] More preferably, the chromatographic column model used in the ultra-performance liquid chromatography analysis is Waters HSS T3 (2.1 mm×100 mm, 1.8 μm).

[0073] In a specific example, the injection volume is 1 μL.

[0074] In a specific example, preparing the test solution includes the following steps:

[0075] Mixing the medicinal material of Galium japonicum with a methanol aqueous solution with a volume fraction of 60% to 80%, heating and refluxing for extraction, and then filtering to obtain a filtrate; or

[0076] Mixing the standard decoction of Glechoma longituba with 60% to 80% ethanol aqueous solution, performing ultrasonic extraction, and then filtering to obtain the filtrate; or

[0077] The Chinese medicinal formula granules of Glechoma longituba are mixed with an ethanol aqueous solution with a volume fraction of 60% to 80%, subjected to ultrasonic extraction, and then filtered to obtain a filtrate.

[0078] It can be understood that in the present invention, the volume fraction of methanol in the methanol aqueous solution includes but is not limited to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, and 80%; the volume fraction of ethanol in the ethanol aqueous solution includes but is not limited to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, and 80%.

[0079] In a specific example, the heating extraction time is 30 minutes to 45 minutes. Preferably, the heating extraction time is 30 minutes.

[0080] In a specific example, the ultrasonic extraction time is 30 minutes to 45 minutes. Preferably, the ultrasonic extraction time is 30 minutes.

[0081] In a specific example, the conditions for ultrasonic extraction include: power of 300 W and frequency of 40 kHz.

[0082] More specifically, preparing the test solution comprises the following steps:

[0083] Take the powder of the medicinal material of the Chinese galangal that has passed through a No. 4 sieve, add 70% methanol, heat and reflux for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate; or

[0084] Take the standard decoction of Galium salsa, grind it into powder, add 70% ethanol, ultrasonicate it for 30 minutes, let it cool, shake it well, filter it, and take the filtrate; or

[0085] Take the Chinese medicinal formula granules of Glechoma longituba, grind them into powder, add 70% ethanol, ultrasonically treat them for 30 minutes, cool them, shake them well, filter them, and take the filtrate.

[0086] In a specific example, the following steps are also included:

[0087] Mixing the control medicinal material of Galium japonicum with water, heating and refluxing for extraction, then filtering, evaporating the filtrate, taking the residue and mixing it with ethanol and water solution, ultrasonically treating it, then filtering, taking the filtrate, and preparing the control medicinal material reference solution;

[0088] The control medicinal material reference solution was subjected to ultra-performance liquid chromatography analysis.

[0089] More specifically, preparing the control medicinal material reference solution includes the following steps:

[0090] Take the control medicinal material of Galium salviae var. amara, add water, heat and reflux for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 70% ethanol to the residue, ultrasonically treat for 30 minutes, shake well, filter, and take the filtrate.

[0091] In a specific example, preparing a chlorogenic acid reference solution comprises the following steps: mixing a chlorogenic acid reference solution with an ethanol aqueous solution;

[0092] The preparation of the mixed reference solution comprises the following steps: mixing the neochlorogenic acid reference substance, the cryptochlorogenic acid reference substance and the ethanol aqueous solution.

[0093] In a specific example, the number of characteristic peaks in the characteristic spectrum of the medicinal material Glechoma longituba, standard decoction or Chinese medicine formula granules is 6; among them, peak 2 is the chromatographic peak of chlorogenic acid, peak 1 is the chromatographic peak of neochlorogenic acid, and peak 3 is the chromatographic peak of cryptochlorogenic acid.

[0094] The present invention also provides an application of the method for constructing a characteristic spectrum of the above-mentioned Glechoma longituba medicinal material, standard decoction or traditional Chinese medicine formula granule in identifying the authenticity of Glechoma longituba.

[0095] In one specific example, counterfeit hogweed includes, but is not limited to, hogweed.

[0096] The present invention also provides a method for detecting a medicinal material, a standard decoction or a Chinese medicine granule, comprising the following steps:

[0097] Take the product to be tested and prepare a solution of the product to be tested;

[0098] The test solution is subjected to ultra-high performance liquid chromatography;

[0099] Among them, the conditions for ultra-high performance liquid chromatography analysis include: mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution; a gradient elution program is adopted; the gradient elution program includes: 0 min to 5 min, the volume percentage of mobile phase A is 6%; 5 min to 20 min, the volume percentage of mobile phase A changes from 6% to 21%; 20 min to 20.1 min, the volume percentage of mobile phase A changes from 21% to 90%; 20.1 min to 25 min, the volume percentage of mobile phase A is maintained at 90%.

[0100] In a specific example, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.2%.

[0101] In a specific example, the conditions for ultra-high performance liquid chromatography analysis also include: the chromatographic column is an octadecylsilane bonded silica gel column; the column temperature is 28°C to 32°C; the flow rate is 0.28mL / min to 0.32mL / min; and the wavelength is 310nm to 350nm.

[0102] In a specific example, the dimensions of the chromatographic column used in ultra-high performance liquid chromatography analysis include: a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm±0.2 μm.

[0103] The following is a detailed description of the construction method and detection method of the characteristic spectrum of the Chinese medicinal material, standard decoction or Chinese medicinal granules of the present invention in conjunction with specific examples. The raw materials used in the following examples, unless otherwise specified, are all commercially available products.

[0104] Example 1

[0105] This embodiment provides a method for constructing a characteristic spectrum of a Chinese medicinal formula granule of Glechoma longituba, which is as follows:

[0106] 1. Instruments and Materials

[0107] Instruments: Thermo high-performance liquid chromatograph (Thermo Vanquish, Thermo Fisher Scientific); Shimadzu high-performance liquid chromatograph (LC40 Dxs, Shimadzu Corporation), Waters high-performance liquid chromatograph (Waters H-Class, Waters Corporation); Waters HSS T3 chromatographic column (2.1 mm × 100 mm, 1.8 μm), Waters CORTECS T3 chromatographic column (2.1 mm × 100 mm, 1.6 μm), Agilent SB C18 chromatographic column (2.1 mm × 100 mm, 1.8 μm); one hundredth of a day balance (JJ600, Changshu Shuangjie Testing Instrument Factory); one ten-thousandth of a day balance (ME204E, Mettler-Toledo); one millionth of a day balance (XP26, Mettler-Toledo); a digitally controlled ultrasonic cleaner (KQ500DE, Kunshan Ultrasonic Instrument Co., Ltd.); an electric constant temperature water bath (HWS28, Shanghai Yiheng Technology Co., Ltd.); and an ultrapure water system (Milli-Q Direct, Merck KGaA).

[0108] Reagents: acetonitrile (Merck Co., Ltd., chromatographic grade), phosphoric acid (Tianjin Komiou Chemical Reagent Co., Ltd., chromatographic grade), ethanol (Xilong Scientific Co., Ltd., analytical grade), methanol (Xilong Scientific Co., Ltd., analytical grade); water was ultrapure water (self-made in the laboratory).

[0109] Test drugs: Chinese herbal formula granules of Glechoma longituba (batch number: CG01, CG02, CG03); neochlorogenic acid (batch number: wkq18030107; content: 98%; manufacturer: Sichuan Weikeqi Biotechnology Co., Ltd.), chlorogenic acid (batch number: 110753-202018; content: 96.1%; manufacturer: China Food and Drug Inspection Institute), cryptochlorogenic acid (batch number: DST210427-035; content: 98.24%; manufacturer: Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.), Glechoma longituba control medicinal material (batch number: 121513-200501; manufacturer: China Food and Drug Inspection Institute); maltodextrin (batch number: FF2106006, FH2107001-A, manufacturer: Weifang Shengtai Pharmaceutical Co., Ltd.).

[0110] 2 Methods and Results

[0111] 2.1 Chromatographic conditions

[0112] A Waters HSS T3 (2.1 mm × 100 mm, 1.8 μm) column was used, acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the requirements in Table 1; the column temperature was 30°C, the flow rate was 0.3 ml per minute, the detection wavelength was 330 nm, and the injection volume was 1 μl.

[0113] Table 1 Gradient elution table

[0114]

[0115] 2.2 Preparation of reference solution

[0116] Take appropriate amount of neochlorogenic acid reference substance and cryptochlorogenic acid reference substance, accurately weigh them, and add 70% ethanol to make a mixed solution containing 20 μg of each per 1 ml as the mixed reference solution.

[0117] Take an appropriate amount of chlorogenic acid reference substance, accurately weigh it, and add 70% ethanol to make a solution containing 20 μg per 1 ml as the chlorogenic acid reference solution.

[0118] 2.3 Reference solution of medicinal materials

[0119] Take 1 g of Glechoma longituba reference medicinal material, place it in a stoppered conical flask, add 25 ml of water, heat and reflux for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25 ml of 70% ethanol to the residue, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate as the reference solution of the control medicinal material.

[0120] 2.4 Preparation of test solution

[0121] Take an appropriate amount of Chinese medicinal formula granules of Glechoma longituba, grind them into powder, take 0.2g, place it in a stoppered conical flask, add 25ml of 70% ethanol, ultrasonically treat (power 300W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0122] 2.5 Establishment of feature maps

[0123] Accurately pipette 1 μl each of the above-mentioned reference solution, reference medicinal material reference solution, and test solution, inject them into the liquid chromatograph, and measure according to the chromatographic conditions under "2.1" in Example 1 to obtain the product.

[0124] Six common characteristic peaks were determined, and by comparing the retention times with those of the chromatographic peaks of the reference substance, the chromatographic peaks of some components were identified: Peak 2 was the chromatographic peak of chlorogenic acid, Peak 1 was the chromatographic peak of neochlorogenic acid, and Peak 3 was the chromatographic peak of cryptochlorogenic acid.

[0125] 3. Investigation of the preparation method of test solution

[0126] 3.1 Extraction solvent

[0127] This experiment investigated the effects of different extraction solvents on the characteristic spectra of the Chinese herbal formula granules of Glechoma elegans. 50% methanol, 70% methanol, methanol, 50% ethanol, 70% ethanol, and ethanol were used as extraction solvents. The peak shapes and separations of the six characteristic peaks consistent with the retention times of the characteristic peaks of the standard decoction were observed, and the "total peak area / sample weight" of the six characteristic peaks was calculated to compare the effects of different extraction solvents on the characteristic spectra of the Chinese herbal formula granules of Glechoma elegans, and the optimal extraction solvent was selected.

[0128] Take an appropriate amount of Chinese medicinal formula granules of Glechoma longituba (CG01), grind it into powder, take about 0.2g, and divide it into 6 parallel groups, 2 portions in each group, and place them in a stoppered conical flask. Accurately add 50% methanol, 70% methanol, methanol, 50% ethanol, 70% ethanol, and 25ml of ethanol respectively, weigh the weight, and ultrasonicate (power 300W, frequency 40kHz) for 30 minutes. Let it cool, weigh the weight again, make up the lost weight with the corresponding solvent, shake well, filter, and take the filtrate. According to the chromatographic conditions under "2.1", the results are as follows: Figure 1 As shown in Table 2.

[0129] The results showed that when different extraction solvents were used, the "total peak area / sample weight" of each characteristic peak was the largest when 70% ethanol was used as the extraction solvent. Therefore, 70% ethanol was preferably used as the extraction solvent.

[0130] Table 2 Results of investigation on extraction solvents for characteristic spectrum of Chinese medicinal formula granules of Glechoma longituba

[0131]

[0132] 3.2 Extraction method

[0133] This experiment investigated the effects of different extraction methods on the characteristic spectra of the Chinese herbal formula granules of Glechoma elegans, mainly investigating the two extraction methods of ultrasound and reflux. The peak shape and separation degree of the six characteristic peaks consistent with the retention time of the characteristic peaks of the standard decoction were observed, and the "total peak area / sample amount" of the six characteristic peaks were calculated to compare the effects of different extraction methods on the characteristic spectra of the Chinese herbal formula granules of Glechoma elegans.

[0134] Take an appropriate amount of Chinese medicinal formula granules of Glechoma longituba (CG01), grind it into powder, take about 0.2g, accurately weigh it, and place it in two parallel groups, 2 portions in each group, place it in a stoppered conical flask, accurately add 25ml of 70% ethanol, weigh it, and ultrasonically treat it (power 300W, frequency 40kHz) for 30 minutes, and heat it under reflux for 30 minutes, remove it, let it cool, weigh it again, make up the lost weight with 70% ethanol, shake it well, filter it, and take the filtrate. Sample analysis is carried out according to the chromatographic conditions under "2.1". The experimental results are as follows Figure 2 As shown in Table 3.

[0135] The results showed that there was no significant difference in the peak shape, separation effect, and "total peak area / sample weight" of the characteristic peaks of the Chinese herbal formula granules of Glechoma longituba using different extraction methods. When ultrasonic extraction was used, the "total peak area / sample weight" of each characteristic peak was the largest, so ultrasonic extraction was chosen.

[0136] Table 3 Results of investigation on the extraction method of characteristic spectrum of Chinese medicinal formula granules of Glechoma longituba

[0137]

[0138] 3.3 Extraction time

[0139] This experiment investigated the effect of ultrasound time on the characteristic spectrum of the Chinese medicine formula granules of Glechoma salsa. The effects of different ultrasound times on the characteristic spectrum of the Chinese medicine formula granules of Glechoma salsa were compared by calculating the "total peak area / sample amount" of the six characteristic peaks that were consistent with the retention time of the characteristic peaks of the standard decoction.

[0140] Take an appropriate amount of Chinese medicinal formula granules of Glechoma longituba (CG01), grind it into powder, take about 0.2g, accurately weigh it, and place it in three parallel groups, 2 portions in each group, into a stoppered conical flask, accurately add 25ml of 70% ethanol, weigh it, and sonicate it (power 300W, frequency 40kHz) for 15 minutes, 30 minutes, and 45 minutes respectively. Remove it, let it cool, weigh it again, make up the lost weight with 70% ethanol, shake it well, filter it, and take the filtrate. Sample analysis is carried out according to the chromatographic conditions under "2.1". The experimental results are as follows Figure 3 As shown in Table 4.

[0141] The results showed that there was no significant difference in the peak shape, separation effect and "total peak area / sample weight" of the six characteristic peaks after ultrasonic treatment for 30 minutes and 45 minutes, respectively. This indicates that ultrasonic treatment for 30 minutes can completely extract the sample. In order to save time, the ultrasonic extraction time was selected as 30 minutes.

[0142] Table 4 Results of the investigation on the extraction time of characteristic spectrum of Chinese medicinal formula granules of Glechoma longituba

[0143]

[0144] 4 Methodological Investigation

[0145] 4.1 Exclusivity

[0146] According to the research data on the process of preparing Chinese medicinal formula granules of Gleam, maltodextrin is added as an excipient during the preparation of this variety. Therefore, this experiment investigated the effect of the negative excipients lacking Gleam on the characteristic spectrum of Chinese medicinal formula granules of Gleam.

[0147] Take maltodextrin as a negative sample and prepare a negative sample solution according to the test solution preparation method specified in "2.4". Take an appropriate amount of Chinese medicinal formula granules of Glehnia littoralis (CG01) and prepare a test solution according to the test solution preparation method specified in "2.4".

[0148] Take the test sample solution, negative sample solution, reference solution, and reference medicinal material solution, and analyze them according to the chromatographic conditions under "2.1". Record the chromatogram, such as Figure 4 As shown in the figure, the experimental results show that the test sample chromatogram has the same chromatographic peak at the corresponding retention time as the reference sample chromatogram, and there is no interference from the negative sample, indicating that the method has good specificity.

[0149] 4.2 Precision

[0150] Grind an appropriate amount of Glechoma longituba TCM granules (CG01) to a fine powder. Accurately weigh approximately 0.2 g of the granules. Prepare a test solution according to the test solution preparation method specified in "2.4." Inject and analyze the sample according to the chromatographic conditions in "2.1." Inject the same sample solution six times consecutively. Using Peak 2 as the reference peak, S, calculate the relative retention time and peak area of ​​each characteristic peak relative to Peak S, and calculate the RSD. The experimental results are shown in Tables 5 and 6.

[0151] The results showed that the same sample solution was injected six times continuously, with the chromatographic peak of peak 2 as the reference peak S. The relative retention time RSDs of each characteristic peak and the S peak were in the range of 0.05% to 0.14%, and the relative peak area RSDs were in the range of 0.14% to 1.25%, all less than 3.0%, indicating that the instrument had good precision.

[0152] Table 5 Precision investigation results of characteristic spectrum of Chinese medicinal formula granules of Glechoma longituba (relative retention time)

[0153]

[0154] Table 6 Precision investigation results of characteristic spectrum of Chinese medicinal formula granules of Glechoma longituba (relative peak area)

[0155]

[0156] 4.3 Repeatability

[0157] Grind an appropriate amount of Glechoma longituba (CG01) granules, taking approximately 0.2 g and accurately weighing six replicates. Prepare six test solutions according to the test solution preparation method specified in "2.4." Inject and analyze according to the chromatographic conditions in "2.1." Using Peak 2 as the reference peak, calculate the relative retention time and peak area of ​​each characteristic peak relative to Peak S, and calculate the RSD. The experimental results are shown in Tables 7 and 8.

[0158] The results showed that the same batch of samples were measured six times, with the chromatographic peak of peak 2 as the reference peak S. The relative retention time RSDs of the characteristic peaks and the S peak were in the range of 0.09% to 0.21%, and the relative peak area RSDs were in the range of 0.30% to 2.75%, all less than 3.0%, indicating that the method had good repeatability.

[0159] Table 7 Repeatability test results of characteristic spectrum of Chinese medicinal formula granules of Glechoma longituba (relative retention time)

[0160]

[0161] Table 8 Repeatability test results of characteristic spectrum of Chinese medicinal formula granules of Glechoma longituba (relative peak area)

[0162]

[0163] 4.4 Stability

[0164] An appropriate amount of Glechoma longituba TCM granules (CG01) was ground into powder. Approximately 0.2 g was accurately weighed and the test solution was prepared according to the test solution preparation method specified in "2.4". Samples were injected and analyzed at 0, 2, 3, 4, 10, and 12 hours using the chromatographic conditions in "2.1". Peak 2 was used as the reference peak, S. The relative retention times and peak areas of each characteristic peak relative to peak S were calculated, and the RSD values ​​were calculated. The experimental results are shown in Tables 9 and 10.

[0165] The results showed that the same sample solution was sampled and analyzed at 0, 2, 3, 4, 10 and 12 hours respectively. Taking peak 2 as the reference peak S, the relative retention time RSDs of each characteristic peak and the S peak were in the range of 0.04% to 0.09%, and the relative peak area RSDs were in the range of 0.10% to 2.35%, all less than 3.0%, indicating that the sample solution was relatively stable within 12 hours.

[0166] Table 9 Characteristic spectrum stability test results of Chinese medicinal formula granules of Glechoma longituba (relative retention time)

[0167]

[0168] Table 10 Stability test results of characteristic spectrum of Chinese medicinal formula granules of Glechoma longituba (relative peak area)

[0169]

[0170] 4.5 Intermediate precision

[0171] Different analysts operated on different instruments on different days. An appropriate amount of Chinese medicinal formula granules of Glehnia littoralis (CG01) was taken and ground. About 0.2 g was taken and 6 replicates were accurately weighed. Six replicates of the test solution were prepared according to the test solution preparation method specified in "2.4". The samples were injected and analyzed according to the chromatographic conditions in "2.1". Peak No. 2 was used as the reference peak S. The relative retention time and relative peak area of ​​each characteristic peak and the S peak were recorded, and the RSD values ​​were calculated. The experimental results are shown in Tables 11 and 12. The intermediate precision inspection chromatogram is shown in Tables 11 and 12. Figure 5 shown.

[0172] The results showed no significant differences in peak shape or separation between the characteristic peaks, performed by different analysts on different instruments on different days. Six replicates of the same sample batch were performed, using Peak 2 as the reference peak S. The relative retention time (RSD) and relative peak area (RSD) values ​​for each characteristic peak relative to Peak S were all less than 3%. The RSDs for the relative retention times and the six repeatability data ranged from 1.85% to 6.33%, and the RSDs for the relative peak areas and the six repeatability data ranged from 0.74% to 3.04%. The relative peak areas showed minimal variability, but the relative retention times showed significant variability. However, the relative retention time RSDs for Peaks 4, 5, and 6, while exceeding 3%, remained within ±10% of the specified values. The relative retention time RSD for Peak 1 was larger, allowing for accurate localization using a reference standard. Overall, this method demonstrated good intermediate precision.

[0173] Table 11 Intermediate precision test results of Chinese medicinal formula granules of Glechoma longituba (relative retention time)

[0174]

[0175]

[0176] Table 12 Intermediate precision investigation results of Chinese medicinal formula granules of Glechoma longituba (relative peak area)

[0177]

[0178] Experimenter 1; Instrument model: Shimadzu LC40 Dxs

[0179] Experimenter 2; Instrument model: Thermo Fisher Vanquish

[0180] 4.6 Durability

[0181] 4.6.1 Column temperature

[0182] The effects of different column temperatures (28℃, 30℃ and 32℃) on the characteristic spectra of the Chinese medicine formula granules of Glechoma longituba were compared.

[0183] Take an appropriate amount of Chinese medicinal formula granules of Glechoma longituba (CG01), grind it into powder, take about 0.2g, weigh it accurately, and prepare the test solution according to the test solution preparation method determined in "2.4". Except that the column temperature is 28℃, 30℃ and 32℃ respectively, other chromatographic conditions are the same as those specified in "2.1". Sample analysis is carried out, and the relative retention time and relative peak area of ​​each characteristic peak and the S peak are calculated with the peak 2 as the reference peak S, and the RSD value is calculated. The experimental results are shown in Table 13, Table 14 and Figure 6 shown.

[0184] The results showed that at different column temperatures, the RSDs of the relative retention times of each characteristic peak and the S peak were in the range of 1.27% to 4.16%, all less than 5%, and the variation range of the relative retention times of each characteristic peak was within ±10%; the RSDs of the relative peak areas were in the range of 1.13% to 2.91%, and the RSD values ​​were less than 3.0%, indicating that the method had good durability when the column temperature was ±2°C.

[0185] Table 13 Column temperature investigation results of characteristic spectrum of Chinese medicinal formula granules of Glechoma longituba (relative retention time)

[0186]

[0187] Table 14 Column temperature investigation results of characteristic spectrum of Chinese medicinal formula granules of Glechoma longituba (relative peak area)

[0188]

[0189] 4.6.2 Flow rate

[0190] The effects of different flow rates, 0.28ml / min, 0.30ml / min and 0.32ml / min, on the characteristic spectrum of the Chinese medicine formula granules of Glechoma longituba were compared.

[0191] Take the test solution under the "Different column temperature investigation" item, except that the flow rates are 0.28ml / min, 0.30ml / min and 0.32ml / min respectively, the other chromatographic conditions are the same as those specified under "2.1", and inject the sample for analysis. Take the peak 2 chromatographic peak as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the RSD value. The experimental results are shown in Table 15, Table 16 and Figure 7 shown.

[0192] The results showed that at different flow rates, the RSD of the relative retention time of each characteristic peak and the S peak was in the range of 0.38% to 1.68%. Except for Peak 5, the RSD of the relative peak area of ​​each characteristic peak was in the range of 0.15% to 2.81%, all of which were less than 3.0%. Combined with the spectrum, an increase of 0.02 ml / min in flow rate had a greater impact on the peak shape of Peak 5. Except for Peak 5, the analytical method had good durability within the flow rate range of 0.28 ml / min to 0.30 ml / min.

[0193] Table 15 Flow rate investigation results of characteristic spectrum of Chinese medicinal formula granules of Glechoma longituba (relative retention time)

[0194]

[0195] Table 16 Flow rate investigation results of characteristic spectrum of Chinese medicinal formula granules of Glechoma longituba (relative peak area)

[0196]

[0197] 4.6.3 Chromatographic columns

[0198] (1) Chromatographic columns of different brands

[0199] Take an appropriate amount of Chinese medicinal formula granules of Glechoma longituba (CG01), grind it into powder, take about 0.2g, accurately weigh it, and prepare the test solution according to the test solution preparation method specified in "2.4". Except that the chromatographic columns are Waters HSS T3 (2.1mm×100mm, 1.8μm), Waters CORTECS T3 (2.1mm×100mm, 1.6μm), and Agilent SB C18 (2.1mm×100mm, 1.8μm), the other chromatographic conditions are the same as those specified in "2.1". Samples were injected and analyzed, with peak No. 2 as the reference peak S. The relative retention time and relative peak area of ​​each characteristic peak and peak S were calculated, and the RSD value was calculated. The experimental results are shown in Tables 17 and 18. Figure 8 shown.

[0200] The results showed that the characteristic peaks were well separated across different brands of columns. The relative retention time RSDs of the characteristic peaks and the S peak ranged from 1.68% to 15.54%, and the relative peak area RSDs of the characteristic peaks ranged from 1.40% to 19.47%. The peak elution times were unstable, and there was some fluctuation in peak area between different columns. The peak elution time was more moderate when using the Waters HSS T3 column. Therefore, the Waters HSS T3 (2.1 mm × 100 mm, 1.8 μm) column is recommended.

[0201] Table 17 Characteristic chromatographic column investigation results of the Chinese medicinal formula granules of the Chinese herbal medicine (relative retention time)

[0202]

[0203] Table 18 Characteristic spectrum of Chinese medicinal formula granules of Glechoma longituba chromatographic column investigation results (relative peak area)

[0204]

[0205] (2) Chromatographic columns of the same brand

[0206] Take an appropriate amount of Chinese medicinal formula granules of Glechoma longituba (CG01), grind it into powder, take about 0.2g, weigh it accurately, and prepare the test solution according to the test solution preparation method specified in "2.4". Except that the chromatographic columns are Waters HSS T3 with different numbers (2.1mm×100mm, 1.8μm, numbers: HSS T3-1, HSS T3-2, HSS T3-3), other chromatographic conditions are the same as those specified in "2.1". Samples are injected and analyzed, with peak No. 2 as the reference peak S, and the relative retention time and relative peak area of ​​each characteristic peak and peak S are calculated, and the RSD value is calculated. The experimental results are shown in Tables 19 and 20. Figure 9 shown.

[0207] The results showed that the characteristic peaks could be well separated using the same type of chromatographic columns with different numbers. The RSDs of the relative retention times of the characteristic peaks and the S peak were in the range of 0.42% to 0.84%, and the RSDs of the relative peak areas of the characteristic peaks were in the range of 0.86% to 2.14%. The RSDs were all less than 3.0%. Therefore, it was recommended that the chromatographic column be Waters HSS T3 (2.1mm×100mm, 1.8μm).

[0208] Table 19 Characteristic chromatogram of Chinese medicinal formula granules of Glehnia littoralis with the same brand of chromatographic columns (relative retention time)

[0209]

[0210] Table 20 Characteristic Spectra of Chinese Herbal Formula Granules of Glehnia littoralis Result of Chromatographic Column of the Same Brand (Relative Peak Area)

[0211]

[0212] The results of the durability investigation showed that the characteristic spectrum method used Peak 2 as the reference peak. The relative retention time and relative peak area of ​​each characteristic peak and the S peak were less affected by column temperature, flow rate and chromatographic columns of the same type with different numbers. Therefore, the analytical method had good durability.

[0213] Example 2

[0214] This embodiment provides a method for detecting the medicinal material of Glechoma longituba, which is as follows:

[0215] Preparation of the test solution of Glechoma longituba medicinal material: take about 0.5g of Glechoma longituba medicinal material powder (passed through No. 4 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, weigh the weight, heat and reflux for 30 minutes, cool, weigh the weight again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain.

[0216] Accurately pipette 1 μl of the test solution and inject it into the liquid chromatograph. Determine according to the chromatographic conditions under "2.1" in Example 1 to obtain the product.

[0217] According to the above method, the characteristic spectra of 15 batches of Glechoma longituba were analyzed. Peak 2 was used as the reference peak S. The relative retention time and relative peak area of ​​each characteristic peak and peak S were calculated, and the RSD value was calculated. The medicinal material batch number and the results are shown in Table 21, Table 22 and Table 23. Figure 10 shown.

[0218] Table 21 Characteristic spectra of 15 batches of Glechoma longituba medicinal materials (relative retention time)

[0219]

[0220] Table 22 Characteristic spectra of 15 batches of Glechoma longituba medicinal materials (relative peak area)

[0221]

[0222] Example 3

[0223] This embodiment provides a method for detecting a standard decoction of Glechoma longituba, which is as follows:

[0224] Preparation of the test solution of standard decoction of Galium: Take an appropriate amount of standard decoction of Galium, grind it into powder, take 0.2 g, place it in a stoppered conical flask, add 25 ml of 70% ethanol, ultrasonically treat it (power 300 W, frequency 40 kHz) for 30 minutes, cool it, shake it well, filter it, and take the filtrate to obtain the product.

[0225] Accurately pipette 1 μl of the test solution and inject it into the liquid chromatograph. Determine according to the chromatographic conditions under "2.1" in Example 1 to obtain the product.

[0226] According to the above method, the characteristic spectra of 15 batches of standard decoction of Glechoma salsa were analyzed. Peak 2 was used as the reference peak S. The relative retention time and relative peak area of ​​each characteristic peak and peak S were calculated, and the RSD value was calculated. The standard decoction batch number and the results are shown in Table 23, Table 24 and Table 25. Figure 11 shown.

[0227] Table 23 Characteristic spectra of 15 batches of standard decoction of Glehnia littoralis (relative retention time)

[0228]

[0229] Table 24 Characteristic spectra of 15 batches of standard decoction of Glehnia littoralis (relative peak area)

[0230]

[0231] Example 4

[0232] This embodiment provides a method for detecting Chinese medicinal formula granules of Glechoma longituba, which is as follows:

[0233] Prepare the test solution according to the method for preparing the test solution of the Chinese medicinal formula granules of Glechoma longituba determined under "2.4" in Example 1, accurately draw 1 μl of the test solution, inject it into the liquid chromatograph, and measure it according to the chromatographic conditions under "2.1" in Example 1 to obtain the product.

[0234] According to the above method, three batches of Chinese medicinal formula granules of Glechoma longituba were measured, and peak 2 was used as the reference peak S. The relative retention time and relative peak area of ​​each characteristic peak and peak S were calculated, and the RSD value was calculated. The batch number of Chinese medicinal formula granules of Glechoma longituba and the results are shown in Table 25, Table 26 and Table 27. Figure 12 shown.

[0235] Table 25 Characteristic spectra of three batches of Chinese medicinal formula granules of Glehnia littoralis (relative retention time)

[0236]

[0237] Table 26 Characteristic spectra (relative peak area) of three batches of Chinese medicinal formula granules of Glehnia littoralis

[0238]

[0239] Example 5

[0240] This example investigates the ultra-high performance liquid chromatography conditions, which are as follows:

[0241] 1. Elution procedure

[0242] Take an appropriate amount of standard decoction of Glechoma longituba, grind it into powder, take about 0.2g, place it in a stoppered conical flask, accurately add 25ml of 70% ethanol, weigh it, and treat it with ultrasound (power 300W, frequency 40kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate.

[0243] A Waters HSS T3 (100 mm × 2.1 mm, 1.8 μm) column was used; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the requirements in Table 27; the column temperature was 30°C; the flow rate was 0.3 ml per minute; the detection wavelength was 330 nm; and the injection volume was 1 μl. Figure 13 shown.

[0244] The results showed that the chromatogram obtained by gradient condition 3 was more evenly distributed, and the peak shapes and separation of each chromatographic peak were good. It was selected as the analysis condition for the characteristic spectrum of the standard decoction of Glehnia littoralis.

[0245] Table 27 Gradient elution table

[0246]

[0247] 2. Wavelength

[0248] Take the test solution of the standard decoction of Glechoma longituba and scan the whole wavelength at 190nm~400nm to determine the best absorption wavelength. The results are as follows: Figures 14 and 15 As shown in the figure, it can be seen that under various inspection wavelength conditions, the chromatographic peak response value obtained at a wavelength of 330nm is the highest, with the most chromatographic peak information, the least interference, and a stable baseline, so 330nm is selected as the detection wavelength.

[0249] Example 6

[0250] This embodiment provides a method for distinguishing the medicinal material Glechoma longituba from its common counterfeit medicinal material Glechoma longituba, as follows:

[0251] The medicinal sources of the counterfeit pig sprouts are shown in Table 28.

[0252] Table 28 Information of medicinal materials of pigtooth herb

[0253]

[0254] Preparation of the test solution of Glechoma longituba medicinal material: take about 0.5g of Glechoma longituba medicinal material powder (passed through No. 4 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, weigh the weight, heat and reflux for 30 minutes, cool, weigh the weight again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain.

[0255] Preparation of the test solution of Gnaphalium wilfordii medicinal material: take about 0.5g of Gnaphalium wilfordii medicinal material powder (passed through No. 4 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, weigh the weight, heat and reflux for 30 minutes, cool, weigh the weight again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain.

[0256] Accurately draw 1 μl of the test solution and inject it into the liquid chromatograph. Measure according to the chromatographic conditions specified in item "2.1" of Example 1. Figure 16 shown.

[0257] The results showed that the chromatogram of the medicinal material of pig's tooth did not show the same chromatographic peak at the corresponding retention time with the chromatogram of the medicinal material of Geum-sinensis, indicating that this method can effectively distinguish Geum-sinensis from the counterfeit pig's tooth.

[0258] 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.

[0259] The embodiments described above only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all fall within the scope of protection of the present invention. 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 by the present invention are all within the scope of protection of the claims attached to the present invention. Therefore, the scope of protection of the patent of the present invention 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 characteristic spectrum of a medicinal material, a standard decoction or a Chinese medicine granule, characterized in that: The steps include: Take chlorogenic acid reference substance to prepare chlorogenic acid reference substance solution; take neochlorogenic acid and cryptochlorogenic acid reference substances to prepare mixed reference substance solution; The preparation of a test solution from a medicinal material of Glechoma longituba, a standard decoction or a Chinese medicine granule comprises the following steps: mixing the medicinal material of Glechoma longituba with a methanol aqueous solution having a volume fraction of 60% to 80%, heating and refluxing for extraction, and then filtering to obtain a filtrate; or The standard decoction of cleavers is mixed with an ethanol aqueous solution with a volume fraction of 60% to 80%, subjected to ultrasonic extraction, and then filtered to obtain a filtrate; or The Chinese medicinal formula granules of Glechoma longituba are mixed with an ethanol aqueous solution with a volume fraction of 60% to 80%, subjected to ultrasonic extraction, and then filtered to obtain a filtrate; The chlorogenic acid reference solution, the mixed reference solution, and the test solution are subjected to ultra-performance liquid chromatography analysis respectively; The conditions for ultra-high performance liquid chromatography analysis include: mobile phase A is acetonitrile, mobile phase B is aqueous phosphoric acid, and the volume fraction of phosphoric acid in the aqueous phosphoric acid is 0.05% to 0.2%; a gradient elution program is used; the gradient elution program includes: from 0 min to 5 min, the volume percentage of mobile phase A is 6%; from 5 min to 20 min, the volume percentage of mobile phase A changes from 6% to 21%; from 20 min to 20.1 min, the volume percentage of mobile phase A changes from 21% to 90%; and from 20.1 min to 25 min, the volume percentage of mobile phase A remains at 90%; the chromatographic column is an octadecylsilane bonded silica gel column, model Waters HSST3, with a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm; and a wavelength of 310 nm to 350 nm.

2. The method for constructing a characteristic spectrum of the medicinal material, standard decoction or traditional Chinese medicine granules of Glehnia littoralis according to claim 1, characterized in that: The conditions for ultra-high performance liquid chromatography analysis also include: column temperature of 28° C. to 32° C.; and flow rate of 0.28 mL / min to 0.32 mL / min.

3. The method for constructing a characteristic spectrum of the medicinal material, standard decoction or traditional Chinese medicine granules of Glechoma longituba according to claim 1, characterized in that: The following steps are also included: Mixing the control medicinal material of Galium japonicum with water, heating and refluxing for extraction, then filtering, evaporating the filtrate, taking the residue and mixing it with ethanol and water solution, ultrasonically treating it, then filtering, taking the filtrate, and preparing the control medicinal material reference solution; The control medicinal material reference solution is subjected to ultra-high performance liquid chromatography analysis.

4. The method for constructing a characteristic spectrum of the medicinal material, standard decoction or traditional Chinese medicine granules of Glehnia littoralis according to claim 1, characterized in that: The preparation of the chlorogenic acid reference solution comprises the following steps: mixing the chlorogenic acid reference solution with an ethanol aqueous solution; The preparation of the mixed reference solution comprises the following steps: mixing the neochlorogenic acid reference substance, the cryptochlorogenic acid reference substance and an ethanol aqueous solution.

5. The method for constructing a characteristic spectrum of the medicinal material, standard decoction or traditional Chinese medicine granules of Glechoma longituba according to any one of claims 1 to 4, characterized in that: There are 6 characteristic peaks in the characteristic spectrum of the Glechoma longituba medicinal material, standard decoction or Chinese medicine formula granules; among them, peak 2 is the chromatographic peak of chlorogenic acid, peak 1 is the chromatographic peak of neochlorogenic acid, and peak 3 is the chromatographic peak of cryptochlorogenic acid.

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