A detection method suitable for Elsholtzia ciliata and its preparations

Abstract

The present invention provides a detection method applicable to Elsholtzia and its preparations, the detection method comprising: (1) preparing the Elsholtzia and its preparations to be tested into a test solution; (2) subjecting the test solution to high performance liquid chromatography detection to obtain a characteristic spectrum, and evaluating the quality of the Elsholtzia and its preparations according to the characteristic spectrum; the Elsholtzia and its preparations include Elsholtzia medicinal materials, Elsholtzia decoction pieces, Elsholtzia standard decoctions or Elsholtzia formula granules. The present invention establishes a detection method that can efficiently detect Elsholtzia and its preparations, such as Elsholtzia medicinal materials, Elsholtzia decoction pieces, Elsholtzia standard decoctions or Elsholtzia formula granules, and the analysis time is short, the elution gradient is simple, the characteristic peak separation in the characteristic spectrum is high, and the volatile and non-volatile components in the Elsholtzia and its preparations can be systematically characterized to comprehensively evaluate the quality of the Elsholtzia and its preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a detection method suitable for Elsholtzia ciliata and preparations thereof. Background Art

[0002] Elsholtzia is the dried aerial part of Mosla chinensis Maxim. or Mosla chinensis of the Lamiaceae family. It has a long history of medicinal use in my country. This variety is slightly warm and pungent in taste, and enters the lung and stomach meridians. It belongs to the category of pungent and warm antipyretic drugs. Its functions are to induce sweating and relieve the symptoms, eliminate dampness and harmonize the middle. It is mainly used for summer-heat cold, aversion to cold and fever, headache without sweating, abdominal pain, vomiting and diarrhea, edema, and urinary difficulty. The chemical components in Elsholtzia are mainly divided into two categories, one is volatile components, and the other is non-volatile components. Volatile components include aromatic compounds such as phenols, esters, olefins, terpenes, and alcohols, among which thymol and carvacrol are the main phenolic components; non-volatile components include baicalin-7-methyl ether, luteolin, quercetin, scutellaria baicalensis and apigenin, oroxylin, kaempferol, isorosmarinic acid glycosides, baicalin and other flavonoids, as well as phenolic acid components such as caffeic acid, rosmarinic acid, syringic acid, p-hydroxybenzoic acid, and oleanolic acid.

[0003] CN110824084A discloses the establishment of HPLC fingerprints of Elsholtzia ciliata from different origins and the method for determining its components, including the following steps: S1, preparation of reference solution: accurately weigh caffeic acid, rutin, apigenin, luteolin, luteolin, apigenin, baicalein-7-methyl ether and place them in a 25mL volumetric flask, add methanol to make up the volume, and prepare reference solutions respectively; S2, preparation of test solution; S3, methodological investigation; S4, establishment and analysis of fingerprints. This method uses a chromatographic column, a mobile phase of acetonitrile-0.2% phosphoric acid, a gradient elution, a column temperature of 30°C, a volume flow rate of 1.0 mL / min, and a detection wavelength of 210nm to establish HPLC fingerprints of Elsholtzia ciliata from different origins, and determine the content of 7 components, which can be used for quality control of Elsholtzia ciliata from different origins. However, this method is only disclosed for the detection and quality evaluation of Elsholtzia medicinal materials, and it is not disclosed whether it is applicable to the detection of various Elsholtzias and their preparations.

[0004] There are also reports that use HPLC effective benchmark characteristic maps to establish a liquid phase method that can comprehensively and accurately evaluate Elsholtzia ciliata slices. This method separates 15 characteristic peaks and 7 representative components of Elsholtzia ciliata (caffeic acid, isorosmarinic acid glycosides, baicalin, rosmarinic acid, baicalein-7-methyl ether, scutellaria baicalensis, and thymol), covering the three main chemical types of Elsholtzia ciliata: phenolic acids, flavonoids, and terpenes. The chromatographic conditions used were: Waters XBridge Shield RP18 column (4.6×250 mm, 5 μm), mobile phase: acetonitrile-0.2% phosphoric acid water, flow rate 0.8 mL / min, column temperature 20°C, detection wavelength 270nm, injection volume 20 μL, (“Study on Quality Evaluation of Elsholtzia jiangensis Pieces Based on Quality Characterization of Effective Benchmark Characteristic Spectra” Li Wenxia, ​​Kong Jing, Wu Qiuhong, etc., Global Chinese Medicine, 2021, Issue 14, Volume 05, Pages 789-798). However, this method is only publicly used for the detection and quality evaluation of Elsholtzia jiangensis pieces, and it is not disclosed whether it is applicable to the detection of various Elsholtzia jiangensis and its preparations.

[0005] CN113773907A discloses a method for detecting the chemical components of volatile oils from different parts of Elsholtzia ciliata, comprising the following steps: extracting volatile oils from Elsholtzia ciliata by steam distillation, and performing qualitative and quantitative analysis of volatile oils from Elsholtzia ciliata by GC-MS. This method uses a specific process to extract volatile oils from Elsholtzia ciliata, and effectively detects the components in volatile oils from Elsholtzia ciliata, but cannot fully detect volatile substances and non-volatile substances, and cannot be applied to the detection of various Elsholtzia ciliata and their preparations.

[0006] Therefore, developing a method for detecting various Elsholtzia and its preparations and detecting Elsholtzia from different origins is a research focus in this field. Summary of the invention

[0007] In view of the deficiencies in the prior art, the object of the present invention is to provide a detection method and a characteristic spectrum suitable for Elsholtzia and its preparations, which can be used to detect Elsholtzia medicinal materials, Elsholtzia decoction pieces, Elsholtzia standard decoction or Elsholtzia formula granules with high sensitivity and good accuracy.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a detection method applicable to Elsholtzia ciliata and its preparations, the detection method comprising:

[0010] (1) Prepare the Elsholtzia ciliata and its preparation to be tested into a test solution;

[0011] (2) subjecting the test solution to ultra-high performance liquid chromatography to obtain a characteristic spectrum, and conducting a quality evaluation of Elsholtzia ciliata and its preparations based on the characteristic spectrum;

[0012] The Elsholtzia and its preparations include Elsholtzia medicinal materials, Elsholtzia decoction pieces, Elsholtzia standard decoction or Elsholtzia formula granules.

[0013] In the prior art, the chemical components in the Elsholtzia medicinal materials or decoction pieces are characterized, but they are not related to other Elsholtzia products, such as the Elsholtzia standard decoction and the Elsholtzia formula granules. Therefore, it is particularly important to establish a method for identifying and characterizing the Elsholtzia medicinal materials and related products. In the prior art, there are two methods for characterizing the chemical components of Elsholtzia: ordinary high performance liquid chromatography and gas chromatography-mass spectrometry. The former has a long analysis time (85 min) and a complex gradient method (6 gradients); the latter has a short analysis time, but can only be used for volatile component analysis, not for non-volatile component analysis. Therefore, the present invention establishes a detection method that can efficiently detect the Elsholtzia medicinal materials, Elsholtzia decoction pieces, Elsholtzia standard decoction or Elsholtzia formula granules, which can comprehensively characterize various Elsholtzias and their preparations to evaluate the quality of Elsholtzias and their preparations.

[0014] Preferably, the test solution is prepared by the following method, which comprises: extracting the Elsholtzia ciliata and its preparation to obtain the test solution.

[0015] Preferably, the extraction comprises ultrasonic extraction and / or reflux extraction.

[0016] Preferably, the extraction time is 30-60 min, for example, 35 min, 40 min, 45 min or 50 min.

[0017] Preferably, the extractant used in the extraction is a methanol-water solution.

[0018] Preferably, the mass percentage of methanol in the methanol aqueous solution is 30-50%, for example, it can be 26%, 28%, 30%, 32% or 34%, etc., preferably 30%.

[0019] Preferably, the extraction further includes a filtering step.

[0020] Preferably, during the extraction, the solid-liquid ratio of the Elsholtzia ciliata medicinal material or Elsholtzia ciliata decoction pieces is 1:(20-30), for example, it can be 1:22, 1:24, 1:26 or 1:28.

[0021] Preferably, during the extraction, the solid-liquid ratio of the Elsholtzia standard decoction or the Elsholtzia formula granules is 1:(60-65), for example, it can be 1:61, 1:62, 1:63 or 1:64.

[0022] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0023] Preferably, the conditions for high performance liquid chromatography detection are as follows: the detection wavelength is 270-275 nm, for example, it can be 271 nm, 272 nm, 273 nm or 274 nm, etc., preferably 274 nm; the mobile phase A is acetonitrile, the mobile phase B is an aqueous phosphoric acid solution, and gradient elution is performed, and the flow rate is 0.2-0.4 mL / min, for example, it can be 0.25 mL / min, 0.3 mL / min or 0.35 mL / min, etc., preferably 0.3 mL / min; the column temperature is 33-37°C, for example, it can be 34°C, 35°C or 36°C, etc., preferably 35°C.

[0024] The mass percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.15%, for example, it can be 0.06%, 0.08%, 0.1%, 0.12% or 0.14%, etc., preferably 0.1%.

[0025] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0026] Preferably, the gradient elution process is: 0-14 min, for example, 1 min, 3 min, 5 min, 7 min, 9 min, 11 min, 13 min, etc., mobile phase A: 7%, mobile phase B: 93%; 14-55 min, for example, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, etc., mobile phase A: 7-46%, for example, 10%, 15%, 20%, 25%, 30%, 40%, 45%, etc., mobile phase B: 93-54%, for example, 92%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, etc.

[0027] Preferably, the detection method further comprises the step of preparing a control medicinal solution, and the step of detecting the control medicinal solution using the same detection method as the test solution to obtain a characteristic spectrum.

[0028] Preferably, the control medicinal material solution is prepared by the following method, which comprises: extracting Elsholtzia ciliata medicinal material to obtain the control medicinal material solution.

[0029] Preferably, the extraction time is 30-60 min, for example, 35 min, 40 min, 45 min or 50 min.

[0030] Preferably, the extractant used in the extraction is a methanol-water solution.

[0031] Preferably, the mass percentage of methanol in the methanol aqueous solution is 30-50%, for example, 32%, 35%, 40% or 45%.

[0032] Preferably, the extraction further includes a filtering step.

[0033] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0034] Preferably, the detection method further comprises the steps of preparing a reference solution and detecting the reference solution to obtain a characteristic spectrum using the same detection method as that for the test solution.

[0035] Preferably, the reference substance in the reference substance solution includes caffeic acid and rosmarinic acid.

[0036] Preferably, the preparation method of the reference substance solution is as follows, comprising: weighing a reference substance and dissolving it in a methanol aqueous solution to obtain the reference substance solution.

[0037] Preferably, the mass percentage of methanol in the methanol aqueous solution is 85-95%, for example, it can be 86%, 88%, 90%, 92% or 94%.

[0038] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0039] Preferably, the detection method comprises:

[0040] (1) Extract the Elsholtzia ciliata and its preparations to be tested with methanol-water solution for 30-60 min, and filter to obtain the test solution;

[0041] The extraction includes ultrasonic extraction and / or reflux extraction;

[0042] The mass percentage of methanol in the methanol aqueous solution is 30-50%;

[0043] The Elsholtzia and its preparations include Elsholtzia medicinal materials, Elsholtzia decoction pieces, Elsholtzia standard decoction or Elsholtzia formula granules;

[0044] During the extraction, the solid-liquid ratio of the Elsholtzia medicinal material or the Elsholtzia decoction pieces is 1:(20-30);

[0045] During the extraction, the solid-liquid ratio of the Elsholtzia standard decoction or the Elsholtzia formula granules is 1:(60-65);

[0046] (2) subjecting the test solution to ultra-high performance liquid chromatography to obtain a characteristic spectrum, and conducting a quality evaluation of Elsholtzia ciliata and its preparations based on the characteristic spectrum;

[0047] The conditions for the HPLC detection are as follows: the detection wavelength is 270-275 nm, the mobile phase A is acetonitrile, the mobile phase B is a phosphoric acid aqueous solution, the gradient elution is performed, the flow rate is 0.2-0.4 mL / min, and the column temperature is 33-37°C;

[0048] The mass percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.15%;

[0049] The gradient elution process is: 0-14 min, mobile phase A: 7%, mobile phase B: 93%; 14-55 min, mobile phase A: 7-46%, mobile phase B: 93-54%.

[0050] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0051] In a second aspect, the present invention provides a characteristic spectrum obtained by detection by the detection method provided in the first aspect, wherein the characteristic spectrum includes 11 characteristic peaks.

[0052] Preferably, taking the caffeic acid and rosmarinic acid as reference peaks, in the order of peak appearance, the relative retention time ranges of the characteristic peaks are: 0.56-0.58, for example, 0.565, 0.57 or 0.575, etc., 1.00, 1.06-1.08, for example, 1.065, 1.07 or 1.075, etc., 0.67-0.69, for example, 0.675, 0.68 or 0.685, etc., 0.88-0.90, for example, 0.885, 0.89 or 0.91. .895, etc., 0.91-0.93, for example, it can be 0.915, 0.92 or 0.925, etc., 1.00, 1.00-1.02, for example, it can be 1.005, 1.01 or 1.015, etc., 1.38-1.40, for example, it can be 1.385, 1.39 or 1.395, etc., 1.48-1.50, for example, it can be 1.485, 1.49 or 1.495, etc., 1.52-1.54, for example, it can be 1.525, 1.53 or 1.535, etc.

[0053] Preferably, in the order of peak elution, the relative retention times of the 11 characteristic peaks are 0.57, 1.00, 1.07, 0.68, 0.89, 0.92, 1.00, 1.01, 1.39, 1.49, and 1.53.

[0054] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] The present invention establishes a detection method for efficiently detecting Elsholtzia and its preparations, such as Elsholtzia medicinal materials, Elsholtzia slices, Elsholtzia standard decoction or Elsholtzia formula granules, and the analysis time is short, only 55 min, which can save 30 minutes compared with the prior art ("Study on Quality Evaluation of Elsholtzia Slices Based on Quality Characterization of Effective Benchmark Characteristic Spectra" Li Wenxia, ​​Kong Jing, Wu Qiuhong, etc., Global Chinese Medicine, 2021, 14, Volume 05, pp. 789-798), the elution gradient is simple (only 2 gradients, while the prior art is 6 gradients), the characteristic peak separation in the characteristic spectrum is high, and the characteristic peak separation in the present invention is greater than 3.0, and the separation of some chromatographic peaks in the prior art is poor. In addition, the present invention can realize the simultaneous detection of volatile and non-volatile components in Elsholtzia and its preparations, and systematically characterize its material basis to conduct a comprehensive quality evaluation of Elsholtzia and its preparations to ensure the effectiveness of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is the characteristic spectrum obtained by testing example 1;

[0058] Figure 2 This is the 3D full scan image obtained from the test case 4;

[0059] Figure 3 This is the characteristic spectrum obtained by detection in Example 1;

[0060] Figure 4 This is the characteristic spectrum obtained by detection in Example 6;

[0061] Figure 5 This is the characteristic spectrum obtained by detecting comparative example 1. DETAILED DESCRIPTION

[0062] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0063] As used herein, the terms "comprises," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0064] "Optional" or "any one" means that the subsequently described matter or event can or cannot occur, and that the description includes instances where the event occurs and instances where it does not.

[0065] The indefinite articles "a" and "an" before the elements or components of the present invention do not limit the quantity requirements (i.e. the number of occurrences) of the elements or components. Therefore, "a" or "an" should be interpreted as including one or at least one, and the elements or components in the singular form also include the plural form, unless the quantity obviously refers to the singular form only.

[0066] The terms "one embodiment", "some embodiments", "exemplarily", "specific examples" or "some examples" described in the present invention mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this document, the schematic representation of the above terms does not necessarily refer to the same embodiment or example.

[0067] The sources of reagents or instruments in the following examples are as follows:

[0068] Elsholtzia ciliata (Elsholtzia jiangensis) control drug: Chengdu Aifa Biotechnology Co., Ltd.;

[0069] Caffeic acid reference substance: batch number: 110885-201703, China Food and Drug Administration;

[0070] Rosmarinic acid reference substance: batch number: 111871-202007, China Food and Drug Administration;

[0071] Test sample: Elsholtzia ciliata (origin: Jiangxi, Henan, Hebei); other Elsholtzia ciliata-related preparations were made in the laboratory;

[0072] Column: ACQUITY UPLC® BEH Shield RP18 (1.7 µm, 2.1 × 150 mm);

[0073] Ultra-high performance liquid chromatograph: Agilent 1290 Infinity ultra-high performance liquid chromatograph (DAD detector).

[0074] Test Example 1

[0075] Investigation of different extraction solvents

[0076] Take 1 g of Elsholtzia ciliata slices, place in a stoppered conical flask, accurately add 25 mL of solvent (water, 30% methanol, 50% methanol, 70% methanol, 90% methanol, ethanol), seal, ultrasonicate (power 500 W, frequency 40 kHz) for 30 minutes, take out, cool, shake, filter, take the filtrate, and get 6 test solutions. Accurately pipette 3 μL of each test solution and inject it into the ultra-high performance liquid chromatography, with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the provisions in the following table; the flow rate is 0.3 mL per minute; the column temperature is 35°C; the detection wavelength is 274 nm. The number of theoretical plates calculated based on the rosmarinic acid peak should not be less than 10,000.

[0077]

[0078] The characteristic spectrum obtained by detection is as follows Figure 1 As shown in the figure, the extractants used in the figure are water, 30% methanol, 50% methanol, 70% methanol, 90% methanol, and ethanol from bottom to top. As can be seen from the figure, when water and ethanol are used as solvents, the chromatographic peak information is less; when 70% methanol and 90% methanol are used as solvents, the chromatographic peaks are interfered by solvent effects; 30% and 50% methanol have better effects, more spectral information, good peak shape and separation effects, and finally it is determined that the solvent for extracting Elsholtzia slices is 30%-50% methanol.

[0079] Test Example 2

[0080] Investigation of different extraction methods

[0081] Take 1 g of Elsholtzia ciliata, weigh accurately, place in a stoppered conical flask, accurately add 25 mL of 30% methanol, seal, and treat with ultrasound (power 500 W, frequency 40 kHz) or reflux for 30 minutes, take out, cool, shake, filter, and take the filtrate to obtain the test solution. Accurately pipette 3 μL of each test sample and inject it into the ultra-high performance liquid chromatography, with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the provisions in the following table; the flow rate is 0.3 mL per minute; the column temperature is 35 ° C; the detection wavelength is 274 nm. The number of theoretical plates calculated based on the rosmarinic acid peak should not be less than 10,000.

[0082]

[0083] The characteristic spectrum was detected, and the retention time and relative retention time of each characteristic peak were calculated as shown in Table 1; the corrected peak area results are shown in Table 2.

[0084] Table 1

[0085]

[0086] Table 2

[0087]

[0088] It can be seen from the table data that under the conditions of using the same test sample extraction solvent, the material information presented in the characteristic spectra obtained by different extraction methods is basically the same, and the difference in the peak area / sample weight ratio between the two is relatively small.

[0089] Test Example 3

[0090] Extraction time investigation

[0091] Take 1 g of Elsholtzia ciliata, weigh accurately, place in a stoppered conical flask, accurately add 25 mL of 30% methanol, seal, use ultrasonic treatment (power 500 W, frequency 40 kHz), extract for 30 min and 60 min respectively, take out, cool, shake well, filter, take the filtrate to obtain the test solution. Accurately draw 3 μL of each test sample and inject it into the ultra-high performance liquid chromatography, use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the provisions in the following table; the flow rate is 0.3 mL per minute; the column temperature is 35°C; the detection wavelength is 274 nm. The number of theoretical plates calculated based on the rosmarinic acid peak should not be less than 10,000.

[0092] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-14 7 93 14-55 7-46 93-54

[0093] The characteristic spectrum was detected, and the characteristic peak retention time and relative retention time corresponding to different extraction times are shown in Table 3; the corrected peak area results are shown in Table 4.

[0094] Table 3

[0095]

[0096] Table 4

[0097]

[0098] From the above results, it can be seen that under the same test sample concentration and extraction solvent conditions, the material information presented in the characteristic spectra obtained at different extraction times is basically the same, and the difference in the peak area / sample weight ratio of each characteristic peak is relatively small. Therefore, the extraction time for the test sample or control medicinal material in the present invention is preferably 30-60 min.

[0099] Preparation Example 1

[0100] This preparation example provides a sample solution of Elsholtzia ciliata medicinal material, and the preparation method is as follows:

[0101] Take 1 g of Elsholtzia ciliata powder, weigh it accurately, put it in a stoppered conical flask, accurately add 25 mL of 30% methanol, stopper it tightly, and treat it with ultrasound (power 500 W, frequency 40 kHz) for 30 minutes. Cool it, shake it well, filter it, and take the filtrate to obtain the Elsholtzia ciliata test solution.

[0102] Preparation Example 2

[0103] This preparation example provides a sample solution of Elsholtzia ciliata slices, and the preparation method is as follows:

[0104] Take 1 g of Elsholtzia slices, accurately weigh, place in a stoppered conical flask, accurately add 25 mL of 30% methanol, seal, and ultrasonically treat (power 500 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the Elsholtzia slice test solution.

[0105] Preparation Example 3

[0106] This preparation example provides a test solution of Elsholtzia standard decoction, and the preparation method is as follows:

[0107] Take 0.4 g of the standard decoction of Elsholtzia, weigh it accurately, put it in a stoppered conical flask, accurately add 25 mL of 30% methanol, stopper it tightly, and treat it with ultrasound (power 500 W, frequency 40 kHz) for 30 minutes. Cool it, shake it well, filter it, and take the filtrate to obtain the test solution of the standard decoction of Elsholtzia.

[0108] Preparation Example 4

[0109] This preparation example provides a test solution of Elsholtzia formula granules, and the preparation method is as follows:

[0110] Take 0.4 g of the Elsholtzia formula granules, accurately weigh, place in a stoppered conical flask, accurately add 25 mL of 30% methanol, seal, and ultrasonically treat (power 500 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the Elsholtzia formula granules test solution.

[0111] Preparation Example 5

[0112] This preparation example provides a control medicinal material solution of Elsholtzia ciliata (Elsholtzia jiangxiensis), and the preparation method is as follows:

[0113] Take 1 g of Elsholtzia ciliata (Elsholtzia jiangensis) reference medicinal material, accurately weigh it, put it in a stoppered conical flask, accurately add 25 mL of 30% methanol, seal it, and ultrasonically treat it (power 500 W, frequency 40 kHz) for 30 minutes. Cool it, shake it well, filter it, and take the filtrate to obtain the Elsholtzia ciliata (Elsholtzia jiangensis) reference medicinal material solution.

[0114] Preparation Example 6

[0115] This preparation example provides a caffeic acid reference solution, and the preparation method is as follows:

[0116] Weigh caffeic acid and add 90% methanol aqueous solution to prepare a reference solution containing 10 µg caffeic acid per 1 mL.

[0117] Preparation Example 7

[0118] This preparation example provides a caffeic acid reference solution, and the preparation method is as follows:

[0119] Weigh rosmarinic acid and add 90% methanol aqueous solution to prepare a reference solution containing 40 µg rosmarinic acid per 1 mL.

[0120] Preparation Example 8

[0121] This preparation example provides a sample solution of Elsholtzia ciliata slices, and the preparation method is as follows:

[0122] The only difference from Preparation Example 2 is that 19 batches of Elsholtzia ciliata slices are weighed separately, and the other reagents, dosages and preparation methods are the same as those in Preparation Example 2.

[0123] Test Example 4

[0124] Selection of detection wavelength

[0125] Accurately pipette 3 μL of the test solution obtained in Preparation Example 2 and inject it into the ultra-high performance liquid chromatograph. Use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. Perform gradient elution according to the requirements in the following table. The flow rate is 0.3 mL per minute. The column temperature is 35°C. Perform a 3D full scan. The test results are as follows: Figure 2 shown.

[0126]

[0127] As can be seen from the figure, the characteristic spectrum information at the detection wavelength of 210-230 nm or 260-285 nm is richer, the peak shape is better, and the interference of miscellaneous peaks is small.

[0128] Test Example 5

[0129] Column temperature selection

[0130] Accurately pipette 3 μL of the test solution obtained in Preparation Example 2 and inject it into an ultra-high performance liquid chromatograph, using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the provisions in the following table; the flow rate is 0.3 mL per minute; the detection wavelength is 274 nm, the column temperatures are 33°C, 35°C and 37°C, respectively, the theoretical plate number calculated based on the rosmarinic acid peak should not be less than 10,000, and a characteristic spectrum is obtained. The retention time and relative retention time of each characteristic peak are calculated, and the results are shown in Table 5.

[0131]

[0132] Table 5

[0133]

[0134] It can be seen from the table data that when different column temperatures (33°C, 35°C and 37°C) were examined, the RSD of the relative retention time of the characteristic peaks was less than 2%, indicating that the relative retention time of the characteristic peaks under this method has good durability to the column temperature.

[0135] Test Example 6

[0136] Different flow rate investigation

[0137] Accurately pipette 3 μL of the test solution obtained in Preparation Example 2 and inject it into an ultra-high performance liquid chromatograph, using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution as specified in the following table; the flow rate is 0.28 mL, 0.3 mL and 0.32 mL per minute; the detection wavelength is 274 nm, the column temperature is 35°C, the theoretical plate number is calculated based on the rosmarinic acid peak and should not be less than 10,000, and a characteristic spectrum is obtained. The retention time and relative retention time of each characteristic peak are calculated, and the results are shown in Table 6.

[0138]

[0139] Table 6

[0140]

[0141] It can be seen from the table data that the RSD of the relative retention time of each characteristic peak at different flow rates (0.28 mL / min, 0.30 mL / min and 0.32 mL / min) is less than 2%, indicating that the relative retention time of the characteristic peak under this method has good durability to the flow rate.

[0142] Test Example 7

[0143] Investigation of different chromatographic columns

[0144] 3 μL of the test solution obtained in Preparation Example 2 was accurately pipetted and injected into an ultra-high performance liquid chromatograph. Three ACQUITY UPLC® BEH Shield RP18 columns of the same model but different serial numbers were examined. 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 provisions in the following table; the flow rate was 0.3 mL per minute; the detection wavelength was 274 nm, the column temperature was 35°C, and the number of theoretical plates calculated based on the rosmarinic acid peak should not be less than 10,000. A characteristic spectrum was obtained, and the retention time and relative retention time of each characteristic peak were calculated. The results are shown in Table 7.

[0145]

[0146] Table 7

[0147]

[0148] It can be seen from the table data that when three ACQUITY UPLC® BEH Shield RP18 chromatographic columns of the same model but different serial numbers were investigated, the relative retention time RSD of each characteristic peak was less than 2%, indicating that the detection method provided by the present invention has good applicability when using this model of chromatographic column.

[0149] Example 1

[0150] This embodiment provides a method for detecting Elsholtzia ciliata and its preparations, and the method is as follows:

[0151] Accurately pipette 3 μL of the test solution obtained in Preparation Example 1 and inject it into an ultra-high performance liquid chromatograph, using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the provisions in the following table; the flow rate is 0.3 mL per minute; the detection wavelength is 274 nm, the column temperature is 35°C, and the theoretical plate number calculated based on the rosmarinic acid peak should be no less than 10,000. The test results are as follows: Figure 3 shown.

[0152]

[0153] As can be seen from the figure, there are 11 characteristic peaks in the chromatogram of the test sample, among which peak 2 and peak 7 correspond to the retention times of the corresponding reference solution peaks respectively; the peak corresponding to the caffeic acid reference peak is the S1 peak, and the relative retention times of peaks 1 and 3 and S1 are calculated, and their relative retention times should be within ±10% of the specified values, and the specified values ​​are: 0.57 (peak 1) and 1.07 (peak 3); the peak corresponding to the rosmarinic acid reference peak is the S2 peak, and the relative retention times of peaks 4-6, 8-11 and S2 are calculated, and their relative retention times should be within ±10% of the specified values, and the specified values ​​are: 0.68 (peak 4), 0.89 (peak 5), 0.92 (peak 6), 1.01 (peak 8), 1.39 (peak 9), 1.49 (peak 10), and 1.53 (peak 11).

[0154] In the characteristic spectrum, peak 2: caffeic acid (S1); peak 5: isosmarinic acid glycoside; peak 6: baicalin; peak 7: rosmarinic acid (S2); peak 9: baicalein-7-methyl ether; peak 10: cinnamaldehyde; peak 11: thymol.

[0155] Example 2

[0156] Repeatability experiment

[0157] The difference between this example and Example 1 is that 3 μL of the test solution obtained in Preparation 1 is precisely pipetted, and 6 portions are parallelly tested, and the rest of the reagents, dosages and detection methods are the same as those in Example 1. In the obtained characteristic spectrum, the retention time and relative retention time of each characteristic peak are calculated as shown in Table 8.

[0158] Table 8

[0159]

[0160] According to the repeatability test results, the relative retention time RSD of each characteristic peak is in the range of 0.00%-0.26%, indicating that the characteristic peak retention time of this characteristic spectrum has good repeatability.

[0161] Example 3

[0162] Intermediate precision test

[0163] The difference between this embodiment and embodiment 1 is that 6 different analysts accurately aspirate 3 μL of the test solution obtained in preparation 1 and perform detection respectively, and the remaining reagents, dosages and detection methods are the same as those in embodiment 1. The retention time and relative retention time in the obtained characteristic spectrum are shown in Table 9.

[0164] Table 9

[0165]

[0166] It can be seen from the table data that the RSD of the relative retention time of each characteristic peak is less than 2%, and the intermediate precision test meets the requirements.

[0167] Example 4

[0168] Stability test

[0169] The difference between this example and Example 1 is that 3 μL of the 6 test sample solutions obtained in Preparation 1 are precisely aspirated and tested at 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h, respectively, and the remaining reagents, dosages, and detection methods are the same as those in Example 1. The retention time and relative retention time in the obtained characteristic spectrum are shown in Table 10.

[0170] Table 10

[0171]

[0172] The results of the stability experiment show that the characteristic components in the sample solution are stable within 24 hours, and the relative retention time RSD of each characteristic peak is less than 2%.

[0173] Example 5

[0174] The difference between this example and Example 1 is that the test solution obtained in Preparation 1 is replaced with an equal amount of 19 batches of Elsholtzia scoparia decoction pieces test solution obtained in Preparation Example 8, and the tests are performed respectively, and the remaining reagents, dosages and detection methods are the same as those in Example 1. The relative retention times in the obtained characteristic spectra are shown in Table 11.

[0175] Table 11

[0176]

[0177] It can be seen from the table data that the test solutions obtained from 19 batches of Elsholtzia ciliata slices were tested, and the relative retention time RSD of each characteristic peak in the test results obtained was less than 0.15%, indicating that the detection method provided by the present invention has good applicability and stability.

[0178] Example 6

[0179] Study on the correlation between Elsholtzia ciliata medicinal materials, decoction pieces, standard decoction and formula granules

[0180] The difference between this embodiment and embodiment 1 is that the Elsholtzia medicinal material test solution obtained in Preparation 1, the Elsholtzia decoction piece test solution obtained in Preparation Example 2, the Elsholtzia standard decoction test solution obtained in Preparation Example 3, the Elsholtzia formula granule test solution obtained in Preparation Example 4 and the Elsholtzia (Elsholtzia jiangensis) control medicinal material solution obtained in Preparation Example 5 are respectively tested, and the remaining reagents, dosages and detection methods are the same as in Example 1.

[0181] The characteristic spectrum obtained is as follows Figure 4 As shown in the figure, it can be seen that by using the detection method provided by the present invention, in the preparation process of Elsholtzia granules, the spectral behaviors of Elsholtzia medicinal materials, Elsholtzia decoction pieces, Elsholtzia standard decoction and Elsholtzia formula granules are highly similar, with a similarity higher than 0.95, among which the similarity of Elsholtzia medicinal materials is 0.984, the similarity of Elsholtzia decoction pieces is 0.984, the similarity of Elsholtzia standard decoction is 0.986, and the similarity of Elsholtzia formula granules is 0.986, indicating that the characteristic spectrum method established by the present invention can perform comprehensive qualitative identification of Elsholtzia and its related products.

[0182] Comparative Example 1

[0183] This comparative example provides a method for detecting Elsholtzia ciliata and its preparation, and the method is as follows:

[0184] Refer to the detection method provided in "Study on Quality Evaluation of Elsholtzia jiangensis Pieces Based on Quality Characterization of Effective Benchmark Characteristic Spectra" by Li Wenxia, ​​Kong Jing, Wu Qiuhong, etc., Global Chinese Medicine, 2021, 14, Volume 05, Pages 789-798, to test Elsholtzia jiangensis medicinal materials, pieces, standard decoctions and formula granules. The characteristic spectra obtained by the test are as follows Figure 5As shown, S1-S4 are respectively Elsholtzia ciliata medicinal materials, decoction pieces, standard decoction, and formula granules. As shown in the figure, the analysis time is long when the existing technology is used for detection, and the chromatographic peak separation is poor.

[0185] The applicant declares that the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A detection method applicable to Elsholtzia ciliata medicinal materials and preparations thereof, characterized in that: The detection method comprises: (1) Prepare the Elsholtzia ciliata medicinal materials and preparations to be tested into a test solution; (2) subjecting the test solution to ultra-high performance liquid chromatography to obtain a characteristic spectrum, and conducting a quality evaluation of the Elsholtzia ciliata medicinal material and its preparation according to the characteristic spectrum; The Elsholtzia medicinal material and its preparations include Elsholtzia medicinal material, Elsholtzia decoction pieces, Elsholtzia standard decoction and Elsholtzia formula granules; The conditions for the ultra-high performance liquid chromatography detection are as follows: the detection wavelength is 270-275 nm, the mobile phase A is acetonitrile, the mobile phase B is a phosphoric acid aqueous solution, the gradient elution is performed, the flow rate is 0.2-0.4 mL / min, and the column temperature is 33-37°C; The mass percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.15%; The gradient elution process is: 0-14 min, mobile phase A: 7%, mobile phase B: 93%; 14-55 min, mobile phase A: 7-46%, mobile phase B: 93-54%; The chromatographic columns used for the ultra-high performance liquid chromatography detection are: ACQUITY UPLC® BEH Shield RP18, with specifications of 1.7 µm, 2.1×150 mm; The test solution is prepared by the following method, which comprises: extracting the Elsholtzia ciliata medicinal material and the preparation thereof to obtain the test solution; The extractant used in the extraction is a methanol-water solution, and the mass percentage of methanol in the methanol-water solution is 30-50%.

2. The detection method according to claim 1, characterized in that: The extraction includes ultrasonic extraction and / or reflux extraction.

3. The detection method according to claim 1, characterized in that: The extraction time is 30-60 min.

4. The detection method according to claim 1, characterized in that: The method further comprises a filtering step after the extraction.

5. The detection method according to claim 1, characterized in that: During the extraction, the solid-liquid ratio of the Elsholtzia medicinal material or the Elsholtzia decoction pieces is 1:(20-30) g / mL.

6. The detection method according to claim 1, characterized in that: During the extraction, the solid-liquid ratio of the Elsholtzia standard decoction or the Elsholtzia formula granules is 1:(60-65) g / mL.

7. The detection method according to claim 1, characterized in that: The detection method also includes the steps of preparing a control medicinal material solution and detecting the control medicinal material solution to obtain a characteristic spectrum using the same detection method as the test solution.

8. The detection method according to claim 7, characterized in that: The control medicinal material solution is prepared by the following method, which includes: extracting Elsholtzia ciliata medicinal material to obtain the control medicinal material solution.

9. The detection method according to claim 8, characterized in that: The extraction time of the control medicinal material solution is 30-60 min.

10. The detection method according to claim 8, characterized in that: The extractant used for extracting the control medicinal material solution is methanol-water solution.

11. The detection method according to claim 10, characterized in that: The weight percentage of methanol in the methanol-water solution used for extracting the control medicinal material solution is 30-50%.

12. The detection method according to claim 8, characterized in that: The control medicinal material solution further comprises a filtering step after extraction.

13. The detection method according to claim 1, characterized in that: The detection method also includes the steps of preparing a reference solution and detecting the reference solution using the same detection method as the test solution to obtain a characteristic spectrum.

14. The detection method according to claim 13, characterized in that: The reference substances in the reference substance solution include caffeic acid and rosmarinic acid.

15. The detection method according to claim 13, characterized in that: The preparation method of the reference substance solution is as follows, and the method comprises: weighing a reference substance and dissolving it in a methanol aqueous solution to obtain the reference substance solution.

16. The detection method according to claim 15, characterized in that: In the preparation method of the reference solution, the mass percentage of methanol in the methanol aqueous solution is 85-95%.

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