A method for establishing HPLC characteristic patterns of cardamom medicinal materials, decoction pieces, standard decoctions and their formula granules

Abstract

The present invention provides a method for establishing an HPLC characteristic spectrum of cardamom medicinal materials, decoction pieces, standard decoctions, and formulated granules thereof, comprising the following steps: S1) dissolving the cardamom raw material in a solvent and extracting to obtain a test liquid; the cardamom raw material is a cardamom medicinal material, decoction piece, standard decoction, or formulated granules; S2) testing the test liquid using high-performance liquid chromatography to obtain an HPLC characteristic spectrum of the cardamom raw material. The method for detecting the characteristic spectrum of the cardamom medicinal material, decoction piece, standard preparation, and formulated granules thereof provided by the present invention can be used for quality testing of the cardamom medicinal material, decoction piece, standard decoction, and formulated granules, providing reliable results and providing a new method for quality control of cardamom and its preparations.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a method for establishing an HPLC characteristic spectrum of red cardamom medicinal materials, decoction pieces, standard decoctions and their formula particles. Background Art

[0002] Cardamom (Alpinia galanga (Linn.) Willd.) is the dried mature fruit of the ginger plant Alpinia galanga (Linn.) Willd. Harvested in autumn when the fruit turns red, it is then cleaned of impurities and dried in the shade. Cardamom has a pungent, warm nature, and is known to dispel cold and dampness, invigorate the spleen, and aid digestion. It is commonly used to treat cold and abdominal pain, food stagnation, vomiting, diarrhea, and excessive drinking.

[0003] Domestic and international literature studies have shown that the chemical composition of cardamom mainly includes volatile oils, flavonoids, saponins, and fatty acids. In recent years, no report has yet been published on the characteristic spectrum of cardamom. To effectively compare and analyze the differences and variations among medicinal materials, decoction pieces, standard decoctions, and formulated granules of cardamom, and to comprehensively evaluate and control the quality of these medicinal materials, decoction pieces, standard decoctions, and formulated granules, a unified method for determining the characteristic spectrum of these medicinal materials, decoction pieces, standard decoctions, and formulated granules is needed. This will facilitate the overall evaluation of the scientific and rationality of cardamom-related processes, better control the intrinsic quality of these medicinal materials, decoction pieces, standard decoctions, and formulated granules, and ensure the clinical efficacy of these formulated granules. Summary of the Invention

[0004] The technical problem solved by the present invention is to provide a method for establishing HPLC characteristic patterns of red cardamom medicinal materials, decoction pieces, standard decoctions and formula granules thereof, which can effectively analyze the chromatographic components of red cardamom medicinal materials, decoction pieces, standard decoctions and formula granules, and effectively evaluate the quality of red cardamom medicinal materials, decoction pieces, standard decoctions and formula granules.

[0005] In view of this, the present application provides a method for establishing HPLC characteristic profiles of cardamom medicinal materials, decoction pieces, standard decoctions and their formula particles, comprising the following steps:

[0006] S1) dissolving a cardamom raw material with a solvent, and extracting to obtain a test solution; the cardamom raw material is a cardamom medicinal material, a decoction piece, a standard decoction or a formula granule thereof;

[0007] S2) the liquid to be tested is detected by high performance liquid chromatography to obtain an HPLC characteristic spectrum of the cardamom raw material;

[0008] The conditions for the high performance liquid chromatography detection are: the chromatographic column is a C18 column, the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid solution, 0.1% acetic acid solution or 0.1% formic acid solution, and the elution is gradient.

[0009] Preferably, the method further comprises preparing a reference substance solution and a reference medicinal material solution:

[0010] The preparation of the reference substance solution is specifically as follows: dissolving 4-coumaric acid in 70% methanol to obtain a reference substance solution;

[0011] The preparation of the control medicinal material reference solution is specifically as follows: decocting the red cardamom reference medicinal material with water, evaporating the filtered filtrate to dryness, dissolving it in 70% methanol, and ultrasonically treating it to obtain the control medicinal material reference solution;

[0012] The reference substance solution and the reference medicinal material solution are measured by high performance liquid chromatography to obtain chromatograms of the reference substance and the reference medicinal material, respectively;

[0013] The components of the HPLC characteristic spectra of red cardamom medicinal materials, decoction pieces, standard decoctions and their formula granules are qualitatively determined based on the chromatograms of the reference substance and the reference medicinal materials.

[0014] Preferably, the solvent is 70% methanol;

[0015] The cardamom raw material is cardamom medicinal material or decoction pieces, the extraction method is heating reflux, and the ratio of the cardamom raw material to the solvent is 1g: (20-30)ml;

[0016] The cardamom raw material is a standard cardamom decoction or its formula particles, the extraction method is heating reflux or ultrasonic extraction, the ratio of the cardamom raw material to the solvent is 0.5g: (20-30)ml, the power of the ultrasonic extraction is 600W, the frequency is 40kHz; and the extraction time is 15-45min.

[0017] Preferably, the gradient elution is specifically:

[0018] 0-7 min, phase A: 5→10%, phase B: 95→90%;

[0019] 7-30 min, phase A: 10→18%, phase B: 90→82%;

[0020] 30~42min, phase A: 18→25%, phase B: 82→75%;

[0021] 42-44 min, phase A: 25→95%, phase B: 75→5%;

[0022] 44-50min, phase A: 95%, phase B: 5%.

[0023] Preferably, the flow rate of the mobile phase is 0.4-0.6 ml / min, and the detection wavelength is 310 nm from 0 to 28 min and 260 nm from 28 to 50 min.

[0024] Preferably, the chromatographic column has a length of 150 mm, an inner diameter of 3.0 mm, a particle size of 2.7 μm, and a column temperature of 25°C.

[0025] Preferably, the similarity of the HPLC characteristic spectra of red cardamom medicinal materials, decoction pieces, standard decoctions and their formula granules is evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to obtain HPLC standard characteristic spectra of red cardamom medicinal materials, decoction pieces, standard decoctions and their formula granules consisting of 9 characteristic peaks, wherein peak 1: p-hydroxybenzaldehyde, peak 5 (S): 4-coumaric acid.

[0026] Preferably, in the characteristic spectrum of the standard decoction of red cardamom, the 4-coumaric acid reference peak is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±10% of the specified value, and the specified values ​​are: 0.67 (peak 1), 0.78 (peak 2), 0.86 (peak 3), 0.93 (peak 4), 1.26 (peak 6), 1.85 (peak 7), 2.00 (peak 8), and 2.04 (peak 9).

[0027] Preferably, in the medicinal material characteristics of red cardamom or the atlas of red cardamom slices, the 4-coumaric acid reference peak is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified value is: 0.68 (peak 1), 0.78 (peak 2), 0.86 (peak 3), 0.93 (peak 4), 1.26 (peak 6), 1.86 (peak 7), 2.03 (peak 8), 2.07 (peak 9).

[0028] Preferably, in the characteristic spectrum of the red cardamom formula granules, the 4-coumaric acid reference peak is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified value is: 0.67 (peak 1), 0.78 (peak 2), 0.85 (peak 3), 0.93 (peak 4), 1.26 (peak 6), 1.85 (peak 7), 1.99 (peak 8), 2.03 (peak 9).

[0029] The present application provides a method for establishing an HPLC characteristic spectrum of red cardamom medicinal materials, decoction pieces, standard decoctions and their formula granules, which comprises the following steps: first, dissolving the red cardamom raw material in a solvent, extracting to obtain a test liquid, and then detecting the test liquid by high performance liquid chromatography, thereby obtaining an HPLC characteristic spectrum of the red cardamom raw material; the method for establishing an HPLC characteristic spectrum of the red cardamom medicinal materials, decoction pieces, standard decoctions and their formula granules provided in the present application establishes an HPLC characteristic spectrum of the red cardamom medicinal materials, decoction pieces, standard decoctions and their formula granules by adopting high performance liquid chromatography, using acetonitrile-0.1% phosphoric acid solution as the mobile phase and 4-coumaric acid as a reference substance, and the method has the characteristics of good repeatability, high accuracy and good stability, thereby effectively evaluating the quality of the red cardamom medicinal materials, decoction pieces, standard decoctions and formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The chromatograms of the red cardamom standard decoction under different mobile phases of the present invention are as follows;

[0031] Figure 2 The chromatograms of the red cardamom standard decoction of the present invention at different wavelengths are shown;

[0032] Figure 3 The chromatograms of the red cardamom standard decoction under different flow rates of the present invention are as follows;

[0033] Figure 4 The chromatograms of the red cardamom standard decoction of the present invention at different column temperatures are shown;

[0034] Figure 5 This is a chromatogram showing the delayed properties of the standard decoction of red cardamom according to the present invention;

[0035] Figure 6 The chromatograms of the red cardamom standard decoction under different extraction solvents of the present invention are as follows;

[0036] Figure 7 The chromatograms of the red cardamom standard decoction under different extraction methods of the present invention are as follows;

[0037] Figure 8 The chromatograms of the red cardamom standard decoction at different extraction times are shown in FIG.

[0038] Figure 9 This is a chromatogram of the red cardamom standard decoction of the present invention with different solvent addition amounts;

[0039] Figure 10 This is the chromatographic peak identification spectrum of the characteristic spectrum of the standard decoction of red cardamom of the present invention;

[0040] Figure 11 4-Coumaric acid spectrum of the reference substance of the present invention;

[0041] Figure 124-Coumaric acid spectrum of the standard decoction of red cardamom of the present invention;

[0042] Figure 13 It is the p-hydroxybenzaldehyde spectrogram of the reference substance of the present invention;

[0043] Figure 14 This is the p-hydroxybenzaldehyde spectrum of the standard decoction of red cardamom of the present invention;

[0044] Figure 15 The chromatograms of the red cardamom standard decoction under different instruments of the present invention are as follows;

[0045] Figure 16 The chromatograms of the red cardamom standard decoction under different chromatographic columns of the present invention are as follows;

[0046] Figure 17 Characteristic spectra of red cardamom standard decoction under different batch numbers of the present invention;

[0047] Figure 18 This is a comparison chart of the standard decoction of red cardamom of the present invention;

[0048] Figure 19 Characteristic spectra of red cardamom medicinal materials under different batches of the present invention;

[0049] Figure 20 This is a comparative characteristic spectrum of the cardamom medicinal material of the present invention;

[0050] Figure 21 Characteristic spectra of red cardamom formula granules under different batches of the present invention;

[0051] Figure 22 This is a comparative characteristic spectrum of the cardamom formula granules of the present invention;

[0052] Figure 23 This is a comparative characteristic spectrum of the cardamom medicinal material of the present invention;

[0053] Figure 24 This is a comparison characteristic spectrum of the standard decoction of red cardamom of the present invention;

[0054] Figure 25 This is a comparative characteristic spectrum of the cardamom formula granules of the present invention;

[0055] Figure 26 This is a comparative characteristic spectrum diagram of the red cardamom medicinal material, standard decoction and formula granules of the present invention. DETAILED DESCRIPTION

[0056] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0057] In view of the differences and changes in the existing technology of red cardamom medicinal materials, decoction pieces, standard decoctions, and formula granules, and the overall evaluation and control of the quality requirements of red cardamom medicinal materials, decoction pieces, standard decoctions, and formula granules, the present application provides a method for establishing an HPLC characteristic spectrum of red cardamom medicinal materials, decoction pieces, standard decoctions, and formula granules, which can accurately and reliably detect the characteristic spectrum of red cardamom and its preparations; the authenticity and quality consistency and stability of red cardamom and its preparations can be effectively detected and controlled, providing a basis for effectively controlling and more comprehensively evaluating the quality of red cardamom medicinal materials, decoction pieces, standard decoctions, and formula granules, so as to ensure the stability of the quality of red cardamom and its preparations. Specifically, an embodiment of the present invention discloses a method for establishing an HPLC characteristic spectrum of red cardamom medicinal materials, decoction pieces, standard decoctions, and formula granules, comprising the following steps:

[0058] S1) dissolving a cardamom raw material with a solvent, and extracting to obtain a test solution; the cardamom raw material is a cardamom medicinal material, a decoction piece, a standard decoction or a formula granule;

[0059] S2) the liquid to be tested is detected by high performance liquid chromatography to obtain an HPLC characteristic spectrum of the cardamom raw material;

[0060] The conditions for the high performance liquid chromatography detection are as follows: the chromatographic column is a C18 column, the mobile phase A is acetonitrile, the mobile phase B is a 0.1% phosphoric acid solution, and the elution is gradient.

[0061] In the process of establishing HPLC characteristic profiles of cardamom medicinal materials, decoction pieces, standard decoctions, and formulated granules, the present application first extracts the cardamom raw material with a solvent to obtain a test solution; the cardamom raw material is a cardamom medicinal material, decoction piece, standard decoction, or formulated granules. The solvent is 70% methanol. Specifically, for the cardamom standard decoction, cardamom decoction pieces, cardamom medicinal materials, and cardamom formulated granules, the solvent is selected from 70% methanol. Using this extraction solvent, each characteristic peak has a good peak shape.

[0062] The cardamom raw material is cardamom medicinal material or decoction pieces, the extraction method is heating reflux, the ratio of the cardamom raw material to the solvent is 1g: (20-30)ml, specifically, the ratio of the cardamom raw material to the solvent is 1g: 25ml; the cardamom raw material is a cardamom standard decoction or its formula granules, the extraction method is heating reflux or ultrasonic extraction, the ratio of the cardamom raw material to the solvent is 0.5g: (20-30)ml, specifically, the ratio of the cardamom raw material to the solvent is 0.5g: 25ml; the power of the ultrasonic extraction is 600W, and the frequency is 40kHz; the extraction time is 15-45min.

[0063] The extraction time is 10 to 45 minutes, specifically, 30 minutes. The peak shape of the chromatogram during the extraction time is moderate.

[0064] During this process, a reference substance solution and a reference medicinal material reference solution were also prepared. Specifically, the reference medicinal material reference solution was prepared by decocting red cardamom reference medicinal material with water, evaporating the filtered filtrate to dryness, dissolving it in 70% methanol, and ultrasonically treating it to obtain the reference medicinal material reference solution;

[0065] The preparation of the reference substance solution is specifically as follows: 4-coumaric acid is dissolved in 70% methanol to obtain a reference substance solution.

[0066] In the above-mentioned control medicinal material reference solution, the ratio of red cardamom control medicinal material and methanol is 1g:25ml, and in the above-mentioned control substance reference solution, the ratio of 4-coumaric acid and 70% methanol is 40μg:1ml.

[0067] The present application then uses high performance liquid chromatography to detect the test liquid to obtain the HPLC characteristic spectrum of the cardamom raw material. Specifically, when the cardamom raw material is cardamom medicinal material or cardamom decoction slices, the HPLC characteristic spectrum of the cardamom medicinal material can be obtained; when the cardamom raw material is cardamom standard decoction, the HPLC characteristic spectrum of cardamom standard decoction can be obtained; when the cardamom raw material is cardamom formula granules, the HPLC characteristic spectrum of cardamom formula granules can be obtained.

[0068] In the present application, the conditions of the HPLC method are as follows: the chromatographic column is a C18 column, the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid solution, 0.1% acetic acid solution or 0.1% formic acid solution, and the elution is gradient.

[0069] Specifically, the gradient elution is:

[0070] 0-7 min, phase A: 5→10%, phase B: 95→90%;

[0071] 7-30 min, phase A: 10→18%, phase B: 90→82%;

[0072] 30~42min, phase A: 18→25%, phase B: 82→75%;

[0073] 42-44 min, phase A: 25→95%, phase B: 75→5%;

[0074] 44-50min, phase A: 95%, phase B: 5%.

[0075] Under the above mobile phase selection conditions, phosphoric acid is relatively stable, so 0.1% phosphoric acid is used as mobile phase B.

[0076] The column length of the chromatographic column is 150 mm, the inner diameter is 3.0 mm, the particle size is 2.7 μm, and the column temperature is 25° C. When the column temperature is 25° C., the peak shape of the chromatogram is relatively symmetrical, the separation is good, and the peak is more complete.

[0077] In high performance liquid chromatography, the mobile phase flow rate is 0.4-0.6 ml / min, preferably 0.5 ml / min; the detection wavelength is 310 nm from 0 to 28 min and 260 nm from 28 to 50 min. The amount of information of the secondary chromatographic peaks is larger and the chromatogram baseline is more stable; the injection volume is 2 μL.

[0078] After the above conditions are determined, while the cardamom and its preparation are measured by high performance liquid chromatography, the reference substance solution and the reference medicinal material reference substance solution are measured by high performance liquid chromatography to obtain chromatograms of the reference substance and the reference medicinal material, respectively;

[0079] The components of the HPLC characteristic spectra of red cardamom medicinal materials, decoction pieces, standard decoctions and their formula granules are qualitatively determined based on the chromatograms of the reference substance and the reference medicinal materials.

[0080] Based on the research of the above method, this application determined the HPLC characteristic spectrum method of red cardamom raw material:

[0081] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 edition General Chapter 0512):

[0082] Chromatographic conditions and system suitability tests were conducted using octadecylsilane bonded silica gel as the filler (column length: 150 mm, inner diameter: 3.0 mm, particle size: 2.7 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 21; flow rate: 0.5 ml / min; column temperature: 25°C; detection wavelength: 310 nm from 0 to 28 minutes, 260 nm from 28 to 50 minutes; the number of theoretical plates calculated based on the 4-coumaric acid peak should be no less than 5000.

[0083] Preparation of reference solution: Take 1 g of red cardamom reference medicinal material, place it in a stoppered conical flask, add 25 ml of water, boil for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 10 ml of 70% methanol to the residue, ultrasonically treat for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material solution;

[0084] Take appropriate amount of 4-coumaric acid reference substance and p-hydroxybenzaldehyde reference substance, add 70% methanol to make reference substance solutions containing 40 μg of each per 1 ml, and use them as reference substance solutions;

[0085] Preparation of test solution: Take 0.5 g of the product, place it in a stoppered conical flask, add 25 ml of 70% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate.

[0086] Determination method: Accurately aspirate 2 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0087] For cardamom medicinal materials or decoction pieces, the final regulations are: there should be 9 characteristic peaks in the chromatogram of the test sample, and the retention times should correspond to the 9 characteristic peaks in the chromatogram of the control medicinal material reference material, among which Peak 5 should correspond to the retention time of the 4-coumaric acid reference material peak; the peak corresponding to the 4-coumaric acid reference material peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value; the specified values ​​are: 0.68 (peak 1), 0.78 (peak 2), 0.86 (peak 3), 0.93 (peak 4), 1.26 (peak 6), 1.86 (peak 7), 2.03 (peak 8), and 2.07 (peak 9).

[0088] For the standard decoction of red cardamom, the final regulations are as follows: 9 characteristic peaks should be present in the chromatogram of the test sample, and the retention times should correspond to the 9 characteristic peaks in the chromatogram of the reference medicinal material. Among them, Peak 5 should correspond to the retention time of the corresponding reference peak of 4-coumaric acid; the peak corresponding to the 4-coumaric acid reference is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value; the specified values ​​are: 0.67 (peak 1), 0.78 (peak 2), 0.86 (peak 3), 0.93 (peak 4), 1.26 (peak 6), 1.85 (peak 7), 2.00 (peak 8), and 2.04 (peak 9).

[0089] For red cardamom formula granules, the final regulations are: 9 characteristic peaks should appear in the chromatogram of the test sample, and the retention times should correspond to the 9 characteristic peaks in the chromatogram of the control medicinal material reference substance, among which Peak 5 should correspond to the retention time of the 4-coumaric acid reference substance peak; the peak corresponding to the 4-coumaric acid reference substance peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value; the specified values ​​are: 0.67 (peak 1), 0.78 (peak 2), 0.85 (peak 3), 0.93 (peak 4), 1.26 (peak 6), 1.85 (peak 7), 1.99 (peak 8), and 2.03 (peak 9).

[0090] The HPLC characteristic spectrum establishment method provided by the present invention uses the traditional Chinese medicine chromatographic fingerprint to control the substance groups of red cardamom medicinal materials, decoction pieces, standard decoctions and their formula granules under liquid chromatography conditions, and locates the fingerprint spectrum with 4-coumaric acid; it can greatly reduce the cost of detection and realize qualitative detection.

[0091] The similarity of the HPLC characteristic spectra of red cardamom medicinal materials, decoction pieces, standard decoctions and their formula granules was evaluated using the Chinese medicine chromatographic fingerprint similarity evaluation system. The HPLC standard characteristic spectra of red cardamom medicinal materials, decoction pieces, standard decoctions and their formula granules were obtained, which consisted of 9 characteristic peaks, including peak 1: p-hydroxybenzaldehyde and peak 5 (S): 4-coumaric acid.

[0092] This invention, for the first time, uses ultra-high performance liquid chromatography to establish a method for simultaneously determining the characteristic profiles of cardamom medicinal materials, decoction pieces, standard decoctions, and formulated granules. This method also offers the advantages of simple sample preparation and a wide selection range. The method achieves effective separation even when the extraction solvent is 70% methanol and the extraction method is ultrasonic and reflux conditions.

[0093] The present invention adopts ultra-high performance liquid chromatography for the first time to establish a characteristic spectrum method for the simultaneous determination of red cardamom medicinal materials, decoction pieces, standard soups and formula granules; and successfully identifies p-hydroxybenzaldehyde and 4-coumaric acid, among which 4-coumaric acid is identified for the first time.

[0094] The invention has the characteristics of strong operability, convenient analysis and stability, and uses high-performance liquid fingerprint technology to control the quality of red cardamom medicinal materials, decoction pieces, standard decoctions and formula granules, so that product quality is effectively controlled.

[0095] The present invention adopts high performance liquid chromatography for the first time to establish a method for simultaneously determining the characteristic spectrum of red cardamom medicinal materials, decoction pieces, red cardamom standard soup, and red cardamom formula granules. The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) is used to combine the reference spectrum of red cardamom medicinal materials, red cardamom decoction pieces, red cardamom standard soup, and red cardamom formula granules on one graph for comparison. Figure 26 shown.

[0096] The present invention can analyze the differences and changes of red cardamom medicinal materials, decoction pieces, standard decoctions, and formula granules, which is conducive to the overall evaluation of the scientificity and rationality of red cardamom-related process, and can better control the intrinsic quality of red cardamom medicinal materials, decoction pieces, standard decoctions, and formula granules as a whole, thereby ensuring the clinical efficacy of red cardamom formula granules.

[0097] In order to further understand the present invention, the method for establishing the HPLC characteristic spectrum of the red cardamom standard decoction, decoction pieces, medicinal materials, and formula granules provided by the present invention is described in detail below in conjunction with the examples. The scope of protection of the present invention is not limited by the following examples.

[0098] Example 1 Red cardamom standard decoction

[0099] An ultra-high performance liquid chromatography characteristic spectrum of red cardamom standard decoction was established, which can be used as an overall quality control method for red cardamom standard decoction.

[0100] 1.1 Materials, reagents, and instruments

[0101] High performance liquid chromatograph: Agilent inifity1260 high performance liquid chromatograph, Thermo Fisher high performance liquid chromatograph;

[0102] Electronic balance: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instrument Co., Ltd.);

[0103] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0104] Ultrasonic cleaner: KQ600-DB (600 W, 40 kHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0105] Chromatographic columns: Agilent C18 3.0 mm × 150 mm, 2.7 μm; Waters C18 3 mm × 150 mm, 2.7 μm;

[0106] Acetonitrile and phosphoric acid were of chromatographic grade, water was ultrapure water, and the remaining reagents were of analytical grade;

[0107] 4-Coumaric acid (China Food and Drug Control Institutes, batch number: 112037-202102, purity: 99.7%);

[0108] p-Hydroxybenzaldehyde (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTDD12901);

[0109] Cardamom reference medicinal material (China Food and Drug Inspection Institute, batch number: 121012-201704);

[0110] Cardamom standard decoction freeze-dried powder (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd.).

[0111] 1.2 Chromatographic conditions and system suitability test

[0112] Octadecylsilane bonded silica gel was used as the filler (column length: 150 mm, inner diameter: 3.0 mm, particle size: 2.7 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution as specified in Table 1 below; the flow rate was 0.5 ml / min; the column temperature was 25°C; the detection wavelengths were 310 nm from 0 to 28 minutes and 260 nm from 28 to 50 minutes; the number of theoretical plates calculated based on the 4-coumaric acid peak should be no less than 5000;

[0113] Table 1 Gradient elution data of red cardamom standard decoction

[0114] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～7 5→10 95→90 7～30 10→18 90→82 30～42 18→25 82→75 42～44 25→95 75→5 44～50 95 5

[0115] 1.3 Preparation of reference solution

[0116] Take 1 g of red cardamom reference medicinal material, place it in a stoppered conical flask, add 25 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 10 ml of 70% methanol to the residue, ultrasonically treat for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the control medicinal material;

[0117] Take an appropriate amount of 4-coumaric acid reference substance, weigh accurately, and add 70% methanol to make a solution containing 40 μg per 1 ml, which serves as the reference substance solution.

[0118] 1.4 Preparation of test solution

[0119] Take 0.5 g of the powder of this product, place it in a stoppered conical flask, add 25 ml of 70% methanol, and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake well, filter, and take the filtrate to obtain the product.

[0120] 1.5 Determination method

[0121] Determination method: Accurately aspirate 2μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine. 1.6 Chromatographic conditions and system suitability test

[0122] 1.6.1 Mobile phase selection

[0123] Based on the experimental conditions proposed above, the separation effects of three different mobile phases were investigated: acetonitrile-0.1% acetic acid, acetonitrile-0.1% phosphoric acid, and acetonitrile-0.1% formic acid. Figure 1 As shown by Figure 1 It can be seen that the chromatographic peaks under different mobile phase conditions are not much different, but the properties of phosphoric acid are relatively stable. Therefore, acetonitrile-0.1% phosphoric acid solution was used as the mobile phase for the characteristic spectrum determination method of red cardamom standard decoction.

[0124] 1.6.2 Wavelength Selection

[0125] Based on the experimental conditions proposed above, the diode array detector was used to scan the entire wavelength range of the test solution, and the chromatograms of the test solution at wavelengths of 260 nm, 280 nm, 310 nm, 330 nm, and 360 nm were extracted respectively. Figure 2 As shown by Figure 2It can be seen that when the detection wavelength is 310 nm from 0 to 28 minutes and 260 nm from 28 to 50 minutes, the chromatographic peak information volume is larger and the chromatogram baseline is more stable. Therefore, the detection wavelength is determined to be 310 nm from 0 to 28 minutes and 260 nm from 28 to 50 minutes.

[0126] 1.6.3 Flow rate investigation

[0127] Based on the experimental conditions proposed above, the flow rates of 0.4 mL / min, 0.5 mL / min, and 0.6 mL / min were investigated. Figure 3 , as shown in Table 2 and Table 3,

[0128] Table 2 Flow rate investigation-retention time characteristic peak data table

[0129]

[0130] Table 3 Flow rate investigation-relative retention time characteristic peak data table

[0131]

[0132] The results showed that the separation effects of different flow rates on each chromatographic peak were not much different, and the flow rate was temporarily set at 0.5 ml / min for subsequent investigation.

[0133] 1.6.4 Column temperature investigation

[0134] Based on the experimental conditions proposed above, the column temperatures of 20℃, 25℃ and 30℃ were investigated respectively. Figure 4 , as shown in Table 4 and Table 5;

[0135] Table 4 Column temperature investigation-retention time characteristic data table

[0136]

[0137] Table 5 Column temperature investigation-relative retention time characteristic peak data table

[0138]

[0139] The results showed that when the column temperature was 25℃, the chromatogram peak shape was more symmetrical, the separation was better, and the peaks were more complete; therefore, the column temperature was determined to be 25℃.

[0140] 1.6.5 Delay test

[0141] Based on the experimental conditions proposed above, a delay test was conducted. The results are shown in Figure 5 .

[0142] The results showed that the sample had basically no chromatographic peak after 50 minutes, so the sample detection time was set to 50 minutes.

[0143] 1.6.6 System suitability test results

[0144] Octadecylsilane bonded silica gel was used as the filler (column length: 150 mm, inner diameter: 3.0 mm, particle size: 2.7 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution as specified in Table 6; the flow rate was 0.5 ml / min; the column temperature was 25°C; the detection wavelengths were 310 nm from 0 to 28 minutes and 260 nm from 28 to 50 minutes; the number of theoretical plates calculated based on the 4-coumaric acid peak should be no less than 5000;

[0145] Table 6 Gradient elution data of red cardamom standard decoction

[0146] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～7 5→10 95→90 7～30 10→18 90→82 30～42 18→25 82→75 42～44 25→95 75→5 44～50 95 5

[0147] 1.7 Preparation of test solution

[0148] 1.7.1 Extraction solvent investigation

[0149] Take 0.5g of this product and place it in a stoppered conical flask. The extraction solvents for the test sample are methanol, 30% methanol, 70% methanol, 50% ethanol, ethanol, and water, 25ml each. Seal the flask tightly and treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate. The results are as follows: Figure 6 As shown by Figure 6 It can be seen that when the extraction solvent is 70% methanol, the peak shapes of the characteristic peaks are good, and the extraction solvent is temporarily set to 70% methanol.

[0150] 1.7.2 Investigation of extraction methods

[0151] Take 0.5g of this product, place it in a stoppered conical flask, add 25ml of 70% methanol, seal it tightly, and examine the extraction method of the test sample when it is refluxed and ultrasonically. The extraction time is 30 minutes, let it cool, shake it well, filter it, and take the filtrate to obtain, such as Figure 7 ,Depend on Figure 7 It can be seen that there is little difference in the effects of reflux and ultrasonic extraction of the test sample. The ultrasonic method is fast and simple, so the extraction method of the test sample was determined to be ultrasonic extraction.

[0152] 1.7.3 Extraction time investigation

[0153] Take 0.5g of this product, place it in a stoppered conical flask, add 25ml of 70% methanol, seal it tightly, and treat it with ultrasound (power 600W, frequency 40kHz). The extraction time of the test product is 15 minutes, 30 minutes, and 45 minutes respectively. Let it cool, shake it well, filter it, and take the filtrate to obtain the product; if Figure 8 As shown by Figure 8It can be seen that when the extraction time is 30 minutes, the chromatogram peak shape is moderate. Therefore, the extraction time is determined to be 30 minutes.

[0154] 1.7.4 Investigation of solvent addition amount

[0155] Take 0.5g of this product and place it in a stoppered conical flask. Add 10ml, 25ml and 50ml of 70% methanol respectively. Seal the flask tightly and treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes. Let it cool, shake well, filter and take the filtrate to obtain the following: Figure 9 As shown by Figure 9 It can be seen that when the amount of extraction solvent added is 25 ml, the peak shapes of each chromatographic peak are moderate, so the solvent amount is selected as 25 ml.

[0156] In summary, the preparation method of the test solution of the characteristic spectrum of red cardamom soup is determined as follows: take 0.5 g of the product, place it in a stoppered conical flask, add 25 ml of 70% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0157] 1.8 Methodological Review

[0158] 1.8.1 Chromatographic peak identification

[0159] Preparation of test solution: Prepare the test solution of red cardamom standard decoction according to the experimental conditions proposed above.

[0160] Preparation of reference solution: Take 1 g of red cardamom reference medicinal material, place it in a stoppered conical flask, add 25 ml of water, boil for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 10 ml of 70% methanol to the residue, ultrasonically treat for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material solution.

[0161] Take appropriate amounts of 4-coumaric acid reference substance and p-hydroxybenzaldehyde reference substance, add 70% methanol to prepare reference substance solutions containing 40 μg of each per 1 ml.

[0162] Preparation of negative control solution: According to the experimental conditions proposed above, prepare a negative control solution of the standard decoction lacking red cardamom.

[0163] Locate the characteristic spectrum peaks of the standard decoction of red cardamom, such as Figures 10-14 As shown, the results show that peak 1 is p-hydroxybenzaldehyde and peak 5 is 4-coumaric acid. In the following methodological investigation, 9 characteristic peaks in the sample were investigated.

[0164] 1.8.2 Precision test

[0165] Take the test solution of red cardamom standard decoction and inject 2 μl of it 6 times continuously according to the proposed experimental method. Calculate the retention time and peak area of ​​each characteristic peak, as shown in Table 7 and Table 8.

[0166] Table 7 Precision Investigation-Retention Time Characteristic Data Table

[0167]

[0168]

[0169] Table 8 Precision Investigation-Peak Area Characteristic Peak Data Table

[0170]

[0171] As can be seen from the above table, the RSD of the retention time of each characteristic peak is 0.02% to 1.31%, and the RSD of the peak area of ​​each characteristic peak is 0.09% to 1.59%, indicating that the instrument has good precision.

[0172] 1.8.3 Repeatability Study

[0173] Accurately weigh 6 portions of freeze-dried powder of red cardamom standard decoction, prepare and measure according to the proposed experimental method, and calculate the relative retention time and relative peak area of ​​each characteristic peak; as shown in Tables 9 and 10;

[0174] Table 9 Repeatability study - relative retention time characteristic peak data table

[0175]

[0176] Table 10 Repeatability study - relative peak area characteristic peak data table

[0177]

[0178]

[0179] The results showed that the RSD of relative retention time of 6 samples was 0.00%-0.03%, and the RSD of relative peak area was 0.00%-2.64%, indicating that the method had good repeatability.

[0180] 1.8.4 Intermediate precision study

[0181] 1.8.4.1 Inspection of different instruments

[0182] Based on the experimental conditions proposed above, the freeze-dried powder of red cardamom standard decoction was accurately weighed to prepare the test solution, which was measured on Agilent 1260 and Thermo Fisher ultra-high performance liquid chromatographs and the relative retention time was calculated; Figure 15 , as shown in Table 11;

[0183] Table 11 Instrument durability investigation - relative retention time characteristic peak data table

[0184]

[0185] The results showed that when the two instruments were used to detect the test samples, the RSDs of the relative retention times of the characteristic peaks were 0.17% to 6.21%, indicating that the instruments had good durability.

[0186] 1.8.4.2 Inspection by different personnel and time

[0187] Based on the experimental conditions proposed above, different personnel (A, B) accurately weighed the red cardamom standard decoction at different times (T1, T2) to prepare the test sample, and measured and calculated the relative retention time and relative peak area, as shown in Tables 12 and 13;

[0188] Table 12 Personnel and time investigation - relative retention time characteristic peak data table

[0189]

[0190] Table 13 Personnel and time investigation - relative peak area characteristic peak data table

[0191]

[0192]

[0193] The results showed that when different personnel measured the same sample at different times, the RSDs of the relative retention times of the characteristic peaks were 0.00% to 0.05%, and the RSDs of the relative peak areas were 0.00% to 0.94%, indicating that the method was stable.

[0194] 1.8.5 Durability Assessment

[0195] 1.8.5.1 Column Durability Assessment

[0196] Based on the experimental conditions proposed above, the chromatographic columns Agilent 1 (Agilent 1, LNB17453) C18 3.0mm×150mm, 2.7μm; Agilent 2 (Agilent 2, LNB18153) C18 3.0mm×150mm, 2.7μm; Waters C18 3mm×150mm 2.7μm were used for analysis and investigation, and the relative retention time of each characteristic peak was calculated, as shown in the following figure: Figure 16 , as shown in Table 14;

[0197] Table 14 Chromatographic column durability investigation - relative retention time characteristic data table

[0198]

[0199] The results showed that the RSDs of the relative retention times of the characteristic peaks were between 0.10% and 1.47% when the three chromatographic columns were used to detect the samples, indicating that the chromatographic columns had good durability.

[0200] 1.8.5.2 Stability Study

[0201] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h, and 24h, as shown in Table 15 and Table 16;

[0202] Table 15 Stability Study - Retention Time Characteristic Peak Data

[0203]

[0204] Table 16 Stability Study - Peak Area Characteristic Peak Data

[0205]

[0206]

[0207] The results showed that the RSD of the characteristic peak retention time was 0.02% to 0.05%, and the RSD of the characteristic peak area was 0.11% to 0.59%. The sample solution was stable within 24 hours.

[0208] In summary, the RSDs of the relative retention times and peak areas of each characteristic peak met the requirements in all the above investigations, indicating that the method was good. The above 9 characteristic peaks were included in the subsequent investigation.

[0209] 1.9 Determination of characteristic peaks and establishment of reference spectrum

[0210] The proposed method was used to determine the characteristic spectra of 16 batches of samples of this product, and the relative retention time and relative peak area were calculated, such as Figure 17 , as shown in Tables 17 and 18; Figure 17 Among them, S1 to S16 are HDK-BT-230801 to HDK-BT-230816 respectively;

[0211] Table 17 Relative retention time data of red cardamom standard decoction

[0212]

[0213] Table 18 Relative peak area data of cardamom standard decoction

[0214]

[0215]

[0216] Nine robust peaks were selected as characteristic peaks based on their stability in relative retention time, consistent detection across all sample batches, and relatively high peak height. Based on the methodological review and validation results from 16 batches of standard broth, the number of theoretical plates, calculated based on 4-coumaric acid, was tentatively set to be no less than 5,000.

[0217] Final regulations: There should be 9 characteristic peaks in the chromatogram of the test sample, and the retention times should correspond to the 9 characteristic peaks in the chromatogram of the control medicinal material reference substance, among which Peak 5 should correspond to the retention time of the corresponding reference substance peak of 4-coumaric acid; the peak corresponding to the 4-coumaric acid reference substance is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value; the specified values ​​are: 0.67 (peak 1), 0.78 (peak 2), 0.86 (peak 3), 0.93 (peak 4), 1.26 (peak 6), 1.85 (peak 7), 2.00 (peak 8), and 2.04 (peak 9).

[0218] Sixteen batches of red cardamom standard decoctions were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a comparison spectrum of the characteristic spectrum of red cardamom standard decoctions was established, such as Figure 18 As shown in the figure, peak 1: p-hydroxybenzaldehyde; peak 5 (S): 4-coumaric acid; chromatographic column: Agilent C18 150mm×3.0mm, 2.7μm.

[0219] Example 2 Red cardamom medicinal material

[0220] With reference to the investigation results of the characteristic spectrum of the standard decoction of red cardamom, an ultra-high performance liquid chromatography characteristic spectrum of red cardamom was established, which can be used as an overall quality control method for red cardamom.

[0221] 2.1 Materials, reagents, and instruments

[0222] 2.1.1 Experimental instruments and materials

[0223] Electronic balances: ME-04E, ME204E, XPE26 (Mettler-Toledo Instruments Co., Ltd.);

[0224] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0225] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0226] Electronic constant temperature water bath: DK-98-Ⅱ (Beijing Zhongxing Weiye Instrument Co., Ltd.);

[0227] Electronic constant temperature water bath: DZKW-4 (Beijing Zhongxing Weiye Instrument Co., Ltd.);

[0228] Chromatographic column: Agilent C18 (150 mm × 3.0 mm, 2.7 μm).

[0229] 2.1.2 Reagents and test drugs

[0230] Methanol (Sigma-Aldrich, chromatographic grade); phosphoric acid, acetic acid, and formic acid (chromatographic grade); ultrapure water; all other reagents were of analytical grade.

[0231] Isoquercetin (China Food and Drug Administration, batch number: 111809-201804, purity: 97.2%);

[0232] Cardamom reference medicinal material (Sichuan Institute of Drug Control, batch number: No. SCZD008-202006).

[0233] 2.2 Chromatographic conditions and system suitability test

[0234] Same as 1.2 Chromatographic conditions and system suitability test.

[0235] 2.3 Preparation of reference solution

[0236] Prepare the reference solution in the same way as in 1.3.

[0237] 2.4 Preparation of test solution

[0238] Take 1 g of the powder (passed through No. 3 sieve) of this product, place it in a stoppered conical flask, add 25 ml of 70% methanol, stopper it tightly, weigh it, heat it under 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 to obtain the product.

[0239] 2.5 Assay

[0240] Same as 1.5 Chromatographic conditions and system suitability test.

[0241] 2.6 Determination of characteristic peaks and establishment of reference spectrum

[0242] The proposed method was used to determine the characteristic spectra of 16 batches of samples of this product and calculate the relative retention time. Figure 19 , as shown in Table 19; Figure 19 Among them, S1 to S15 are XXLS2023071154-XXLS2023071168 respectively; S16 is 010201-2308001.

[0243] Table 19 Relative retention time data of characteristic spectrum of cardamom medicinal materials

[0244] Serial number batch number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5(S) Peak 6 Peak 7 Peak 8 Peak 9 1 XXLS2023071154 0.680 0.781 0.861 0.931 1.000 1.262 1.861 2.025 2.068 2 XXLS2023071155 0.679 0.780 0.861 0.931 1.000 1.262 1.860 2.024 2.066 3 XXLS2023071156 0.679 0.781 0.861 0.932 1.000 1.263 1.863 2.028 2.070 4 XXLS2023071157 0.680 0.781 0.862 0.933 1.000 1.263 1.863 2.029 2.071 5 XXLS2023071158 0.680 0.781 0.862 0.933 1.000 1.264 1.867 2.033 2.075 6 XXLS2023071159 0.680 0.781 0.862 0.934 1.000 1.263 1.866 2.033 2.075 7 XXLS2023071160 0.680 0.781 0.862 0.933 1.000 1.263 1.864 2.030 2.073 8 XXLS2023071161 0.680 0.781 0.861 0.932 1.000 1.263 1.863 2.029 2.071 9 XXLS2023071162 0.680 0.781 0.861 0.932 1.000 1.263 1.863 2.029 2.071 10 XXLS2023071163 0.679 0.781 0.861 0.932 1.000 1.263 1.862 2.027 2.070 11 XXLS2023071164 0.679 0.781 0.861 0.933 1.000 1.263 1.862 2.028 2.070 12 XXLS2023071165 0.679 0.781 0.861 0.933 1.000 1.263 1.864 2.029 2.071 13 XXLS2023071166 0.680 0.781 0.861 0.932 1.000 1.263 1.864 2.031 2.073 14 XXLS2023071167 0.680 0.781 0.862 0.933 1.000 1.263 1.864 2.030 2.072 15 XXLS2023071168 0.680 0.781 0.862 0.933 1.000 1.263 1.863 2.030 2.072 16 010201-2308001 0.680 0.781 0.862 0.933 1.000 1.263 1.865 2.031 2.074 RSD (%) 0.07 0.03 0.06 0.09 0.00 0.04 0.09 0.12 0.12 average value 0.680 0.781 0.861 0.933 1.000 1.263 1.863 2.029 2.071 ±10％ 0.612 0.703 0.775 0.840 0.900 1.137 1.677 1.826 1.864 ±10％ 0.748 0.859 0.947 1.026 1.100 1.389 2.049 2.232 2.278

[0245] Based on the principle of stable relative retention times and the fact that peaks can be detected in all batches of samples at relatively high levels, a total of nine peaks with good reproducibility were selected as characteristic peaks. The relative retention time RSDs of the nine characteristic peaks in 16 batches of red cardamom medicinal materials were all less than 1.0%.

[0246] Final regulations: There should be 9 characteristic peaks in the chromatogram of the test sample, and the retention times should correspond to the 9 characteristic peaks in the chromatogram of the control medicinal material reference substance, among which Peak 5 should correspond to the retention time of the 4-coumaric acid reference substance peak; the peak corresponding to the 4-coumaric acid reference substance peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value; the specified values ​​are: 0.68 (peak 1), 0.78 (peak 2), 0.86 (peak 3), 0.93 (peak 4), 1.26 (peak 6), 1.86 (peak 7), 2.03 (peak 8), and 2.07 (peak 9).

[0247] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 16 batches of red cardamom medicinal materials, and a reference characteristic spectrum of red cardamom medicinal materials was established, such as Figure 20 As shown in the figure, peak 1: p-hydroxybenzaldehyde; peak 5 (S): 4-coumaric acid; chromatographic column: Agilent C18, 3.0 mm × 150 mm, 2.7 μm.

[0248] Example 3 Red Cardamom Formula Granules

[0249] An HPLC characteristic spectrum of red cardamom formula granules was established, which can be used as an overall quality control method for red cardamom formula granules.

[0250] 3.1 Materials, reagents, and instruments

[0251] Same as 1.1. Materials, reagents, and instruments.

[0252] Cardamom formula granules (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd.).

[0253] 3.2 Chromatographic conditions and system suitability test

[0254] Same as 1.2 Chromatographic conditions and system suitability test.

[0255] 3.3 Preparation of reference solution

[0256] Same as 1.3 for preparation of reference solution.

[0257] 3.4 Preparation of test solution

[0258] Take 0.5g of this product, grind it into powder, put it into a stoppered conical flask, add 25ml of 70% methanol, and ultrasonically treat it (power 600W, frequency 40kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0259] 3.5 Determination method

[0260] Same as 1.5 Chromatographic conditions and system suitability test.

[0261] 3.6 Determination of characteristic peaks and establishment of reference maps

[0262] The proposed method was used to determine the characteristic spectra of three batches of samples of this product and calculate the relative retention time, such as Figure 21 As shown in Table 20, Figure 21 Characteristic spectrum of red cardamom formula granules, in which S1 to S3 are: 2308095, 2308096, 2308097;

[0263] Table 20 Relative retention time data of cardamom formula granules

[0264]

[0265] Based on the principles of stable relative retention times, consistent detection across all batches of samples, and relatively high peak heights, nine robust peaks were selected as characteristic peaks: Peak 1, Peak 2, Peak 3, Peak 4, Peak 5, Peak 6, Peak 7, Peak 8, and Peak 9. Based on the methodological review results and the validation results of three batches of particles, the theoretical plate number calculated based on the 4-coumaric acid peak was tentatively set to be no less than 5,000.

[0266] Final regulations: The test sample chromatogram should show nine characteristic peaks, and their retention times should correspond to those of the nine characteristic peaks in the chromatogram of the reference medicinal material. Peak 5 should correspond to the retention time of the 4-coumaric acid reference peak. The peak corresponding to the 4-coumaric acid reference peak is the S peak. The relative retention times of each characteristic peak and the S peak should be calculated, and the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.67 (peak 1), 0.78 (peak 2), 0.85 (peak 3), 0.93 (peak 4), 1.26 (peak 6), 1.85 (peak 7), 1.99 (peak 8), and 2.03 (peak 9).

[0267] Three batches of red cardamom formula granules were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a comparison map of the characteristic spectrum of red cardamom formula granules was established, such as Figure 22 As shown in the figure, peak 1: p-hydroxybenzaldehyde; peak 5 (S): 4-coumaric acid; chromatographic column: Agilent C18 150mm×3.0mm, 2.7μm.

[0268] Example 4

[0269] The process of establishing the HPLC characteristic spectrum of cardamom slices is the same as that of cardamom medicinal materials.

[0270] The present invention adopts high performance liquid chromatography for the first time to establish a characteristic spectrum method for simultaneously determining red cardamom medicinal materials, red cardamom decoction pieces, red cardamom standard soup and red cardamom formula granules.

[0271] The chromatographic conditions for this method are as follows: octadecylsilane bonded silica gel as the filler (column length: 150 mm, inner diameter: 3.0 mm, particle size: 2.7 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 21 below; flow rate: 0.5 ml / min; column temperature: 25°C; detection wavelength: 310 nm from 0 to 28 minutes, 260 nm from 28 to 50 minutes. The theoretical plate number calculated based on the 4-coumaric acid peak should be no less than 5000.

[0272] Table 21 Gradient elution data of cardamom raw material

[0273]

[0274]

[0275] This method can effectively separate the components of the chromatographic peak of red cardamom and successfully identify p-hydroxybenzaldehyde and 4-coumaric acid. The characteristic spectra of red cardamom and red cardamom formula granules were synthesized into a reference spectrum using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version); a reference spectrum of the characteristic spectra of red cardamom medicinal material, standard decoction, and red cardamom granules was established. Figures 23-25 .

[0276] For cardamom medicinal materials or cardamom decoction pieces, the final regulations are: 9 characteristic peaks should be present in the chromatogram of the test sample, and the retention times should correspond to the 9 characteristic peaks in the chromatogram of the control medicinal material reference material, among which Peak 5 should correspond to the retention time of the 4-coumaric acid reference material peak; the peak corresponding to the 4-coumaric acid reference material peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value; the specified values ​​are: 0.68 (peak 1), 0.78 (peak 2), 0.86 (peak 3), 0.93 (peak 4), 1.26 (peak 6), 1.86 (peak 7), 2.03 (peak 8), and 2.07 (peak 9).

[0277] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 16 batches of red cardamom medicinal materials, and a reference characteristic spectrum of red cardamom medicinal materials was established; Figure 23 As shown in the figure, peak 1: p-hydroxybenzaldehyde; peak 5 (S): 4-coumaric acid; chromatographic column: Agilent C18, 3.0 mm × 150 mm, 2.7 μm.

[0278] For the standard decoction of red cardamom, the final regulations are as follows: 9 characteristic peaks should be present in the chromatogram of the test sample, and the retention times should correspond to the 9 characteristic peaks in the chromatogram of the reference medicinal material. Among them, Peak 5 should correspond to the retention time of the corresponding reference peak of 4-coumaric acid; the peak corresponding to the 4-coumaric acid reference is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value; the specified values ​​are: 0.67 (peak 1), 0.78 (peak 2), 0.86 (peak 3), 0.93 (peak 4), 1.26 (peak 6), 1.85 (peak 7), 2.00 (peak 8), and 2.04 (peak 9).

[0279] Sixteen batches of red cardamom standard decoctions were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a comparison of the characteristic spectra of red cardamom standard decoctions was established. Figure 24 In the figure, peak 1: p-hydroxybenzaldehyde; peak 5 (S): 4-coumaric acid; chromatographic column: Agilent C18 150mm×3.0mm, 2.7μm.

[0280] For red cardamom formula granules, the final regulations are: 9 characteristic peaks should appear in the chromatogram of the test sample, and the retention times should correspond to the 9 characteristic peaks in the chromatogram of the control medicinal material reference substance, among which Peak 5 should correspond to the retention time of the 4-coumaric acid reference substance peak; the peak corresponding to the 4-coumaric acid reference substance peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value; the specified values ​​are: 0.67 (peak 1), 0.78 (peak 2), 0.85 (peak 3), 0.93 (peak 4), 1.26 (peak 6), 1.85 (peak 7), 1.99 (peak 8), and 2.03 (peak 9).

[0281] Three batches of red cardamom formula granules were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a comparison map of the characteristic spectrum of red cardamom formula granules was established, such as Figure 25 In the figure, peak 1: p-hydroxybenzaldehyde; peak 5 (S): 4-coumaric acid; chromatographic column: Agilent C18 150mm×3.0mm, 2.7μm.

[0282] The above embodiments are only intended to help understand the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by those skilled in the art, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0283] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for establishing HPLC characteristic patterns of cardamom medicinal materials, decoction pieces, standard decoctions and their formula granules, comprising the following steps: S1) dissolving a cardamom raw material in a solvent and extracting it to obtain a test solution; the cardamom raw material is cardamom medicinal material, decoction pieces, standard decoction or its formula granules; the solvent is 70% methanol; S2) detecting the test solution by high performance liquid chromatography to obtain an HPLC characteristic spectrum of the cardamom raw material; The conditions for the HPLC detection are as follows: a C18 column, mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid solution, 0.1% acetic acid solution or 0.1% formic acid solution, and gradient elution; the mobile phase flow rate is 0.4-0.6 ml / min, the detection wavelength is 310 nm from 0 to 28 min, and 260 nm from 28 to 50 min; the column length is 150 mm, the inner diameter is 3.0 mm, and the particle size is 2.7 μm; The gradient elution is specifically as follows: 0-7 min, phase A: 5→10%, phase B: 95→90%; 7-30 min, phase A: 10→18%, phase B: 90→82%; 30~42min, phase A: 18→25%, phase B: 82→75%; 42-44 min, phase A: 25→95%, phase B: 75→5%; 44~50min, phase A: 95%, phase B: 5%.

2. The establishment method according to claim 1, characterized in that It also includes the preparation of reference substance solutions and reference medicinal material solutions: The preparation of the reference substance solution is specifically as follows: dissolving 4-coumaric acid in 70% methanol to obtain a reference substance solution; The preparation of the control medicinal material reference solution is specifically as follows: decocting the red cardamom reference medicinal material with water, evaporating the filtered filtrate to dryness, dissolving it in 70% methanol, and ultrasonically treating it to obtain the control medicinal material reference solution; The reference substance solution and the reference medicinal material solution are measured by high performance liquid chromatography to obtain chromatograms of the reference substance and the reference medicinal material, respectively; The components of the HPLC characteristic spectra of red cardamom medicinal materials, decoction pieces, standard decoctions and their formula granules are qualitatively determined based on the chromatograms of the reference substance and the reference medicinal materials.

3. The establishment method according to claim 1 or 2, characterized in that: The cardamom raw material is cardamom medicinal material or decoction pieces, the extraction method is heating reflux, and the ratio of the cardamom raw material to the solvent is 1g: (20-30)ml; The cardamom raw material is a standard cardamom decoction or its formula granules, the extraction method is heating reflux or ultrasonic extraction, the ratio of the cardamom raw material to the solvent is 0.5g: (20~30)ml, the power of the ultrasonic extraction is 600W, the frequency is 40kHz; and the extraction time is 15~45min.

4. The establishment method according to claim 1 or 2, characterized in that: The column temperature of the chromatographic column is 25°C.

5. The establishment method according to claim 1 or 2, characterized in that: The similarity of the HPLC characteristic spectra of red cardamom medicinal materials, decoction pieces, standard decoctions and their formula granules was evaluated using the Chinese medicine chromatographic fingerprint similarity evaluation system. The HPLC standard characteristic spectra of red cardamom medicinal materials, decoction pieces, standard decoctions and their formula granules were obtained, which consisted of 9 characteristic peaks, including peak 1: p-hydroxybenzaldehyde and peak 5: 4-coumaric acid.

6. The establishment method according to claim 5, characterized in that: In the characteristic spectrum of the standard decoction of red cardamom, the 4-coumaric acid reference peak is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±10% of the specified value, and the specified values ​​are: 0.67 (peak 1), 0.78 (peak 2), 0.86 (peak 3), 0.93 (peak 4), 1.26 (peak 6), 1.85 (peak 7), 2.00 (peak 8), and 2.04 (peak 9).

7. The establishment method according to claim 5, characterized in that: In the characteristic spectrum of red cardamom medicinal materials or red cardamom decoction slices, the 4-coumaric acid reference peak is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±10% of the specified value, and the specified value is: 0.68 (peak 1), 0.78 (peak 2), 0.86 (peak 3), 0.93 (peak 4), 1.26 (peak 6), 1.86 (peak 7), 2.03 (peak 8), and 2.07 (peak 9).

8. The establishment method according to claim 5, characterized in that: In the characteristic spectrum of red cardamom formula granules, the 4-coumaric acid reference peak is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value. The specified value is: 0.67 (peak 1), 0.78 (peak 2), 0.85 (peak 3), 0.93 (peak 4), 1.26 (peak 6), 1.85 (peak 7), 1.99 (peak 8), and 2.03 (peak 9).

Images