Characteristic spectrum detection method, origin identification method and quality control method of Amomum villosum products

Abstract

The present invention relates to the technical field of traditional Chinese medicine detection, and discloses a characteristic spectrum detection method, a base identification method and a quality control method for a fructus amomi product. The characteristic spectrum detection method of the fructus amomi product provided by the present invention uses octadecylsilane bonded silica gel as a filler, acetonitrile-phosphoric acid aqueous solution as a mobile phase for gradient elution, selects a specific elution program, and can detect a characteristic spectrum containing 11 characteristic peaks, not only significantly improves the number of common characteristic peaks, but also achieves good separation of these common characteristic peaks, the elution program of the characteristic spectrum detection method is simple, the obtained characteristic spectrum baseline is stable, each characteristic peak has a good peak shape, and the separation between each characteristic peak is high, and the peak positions of 7 characteristic peaks including vanillic acid, epicatechin, isoquercetin and quercetin can be accurately located, providing a basis for quality detection and control of the fructus amomi product.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine detection, and in particular to a characteristic spectrum detection method, a base identification method and a quality control method for an Amomum villosum product. Background Art

[0002] Amomum villosum is pungent and warm in nature, and enters the spleen, stomach, and kidney meridians. It has the effects of removing dampness and stimulating appetite, warming the spleen and stopping diarrhea, regulating qi and calming the fetus. It is often used for dampness and turbidity blocking the middle part of the body, abdominal distension without hunger, spleen and stomach deficiency and cold, vomiting and diarrhea, nausea and vomiting during pregnancy, and fetal movement disorder, and has broad market prospects.

[0003] Amomum villosum formula granules are products made from Amomum villosum slices under the guidance of traditional Chinese medicine theory. They not only retain the medicinal properties and efficacy of Chinese herbal medicine slices, but also have the advantages of no decoction, easy to take, easy to carry, and can be added or subtracted according to symptoms. They meet the needs of modern consumption and life and are an important supplement to Chinese herbal medicine slices.

[0004] There are various relevant testing standards for Amomum villosum granules in the related art. However, during the process of implementing the present invention, the inventors found that the high-performance liquid chromatography method used in the relevant testing standards for Amomum villosum granules suffers from low separation and a small number of shared characteristic peaks. Summary of the Invention

[0005] In view of this, the present invention provides a characteristic spectrum detection method, origin identification method and quality control method of Amomum villosum product, so as to solve the problems of low separation and small number of common characteristic peaks when using high performance liquid chromatography in relevant detection standards to detect Amomum villosum formula granules.

[0006] In a first aspect, the present invention provides a method for detecting a characteristic spectrum of an Amomum villosum product, comprising the following steps:

[0007] The test solution was taken and tested by high performance liquid chromatography; wherein the chromatographic conditions of the high performance liquid chromatography method include:

[0008] Octadecylsilane bonded silica gel was used as a filler, acetonitrile was used as mobile phase A, and 0.08% to 0.12% by volume phosphoric acid aqueous solution was used as mobile phase B. Gradient elution was performed. The gradient elution procedure included:

[0009] From 0 to 13 min, the volume percentage of acetonitrile in the mobile phase was 2% → 7%;

[0010] From 13 to 36 minutes, the volume percentage of acetonitrile in the mobile phase is 7% → 18%;

[0011] 36-40 min, the volume percentage of acetonitrile in the mobile phase is 18%.

[0012] The characteristic spectrum detection method of the above-mentioned Amomum villosum product provided by the present invention uses octadecylsilane bonded silica gel as a filler, acetonitrile-phosphoric acid aqueous solution as a mobile phase for gradient elution, and selects a specific elution procedure to detect and obtain a characteristic spectrum containing 11 characteristic peaks, which not only significantly increases the number of common characteristic peaks, but also achieves good separation of these common characteristic peaks. The elution procedure of this characteristic spectrum detection method is simple, the baseline of the obtained characteristic spectrum is stable, the peak shape of each characteristic peak is good, the separation between the characteristic peaks is high, and the peak positions of 7 characteristic peaks including vanillic acid, epicatechin, isoquercetin and quercetin can be accurately located.

[0013] By using the above-mentioned characteristic spectrum detection method, characteristic spectra of Amomum villosum products with different origins can be constructed, and the obtained characteristic spectra are highly characteristic and rich in chromatographic information, which can fully display the chemical composition characteristics of Amomum villosum products with different origins. In addition, the above-mentioned detection method has high precision and good stability, providing a basis for quality inspection and control of Amomum villosum products and identification and differentiation of Amomum villosum origins.

[0014] In an optional embodiment, the chromatographic conditions of the high performance liquid chromatography method further include at least one of the following conditions:

[0015] 1) The detection wavelength is 258nm~262nm;

[0016] 2) Column temperature is 28°C to 32°C;

[0017] 3) Flow rate: 0.30 mL / min to 0.37 mL / min;

[0018] 4) The injection volume is 1 μL to 3 μL;

[0019] 5) The chromatographic column was a CORTECS UPLC T3 column with an inner diameter of 2.1 mm, a column length of 100 mm, and a particle size of 1.6 μm.

[0020] In an optional embodiment, the preparation method of the test solution is as follows:

[0021] Take the test sample, add solvent, extract, separate solid and liquid, and take liquid to obtain the test sample solution;

[0022] Optionally, the solvent is a methanol aqueous solution, and the volume percentage of methanol in the methanol aqueous solution is 50% to 70%;

[0023] Optionally, relative to 0.5 g of the test sample, the amount of the solvent added is 20 mL to 50 mL;

[0024] Optionally, the extraction is ultrasonic extraction, and the ultrasonic time is 45 minutes to 60 minutes.

[0025] In an optional embodiment, the characteristic spectrum detection method further comprises the steps of preparing a reference solution using a vanillic acid reference substance, an epicatechin reference substance, an isoquercetin reference substance, and a quercetin reference substance, and detecting the reference solution using the high performance liquid chromatography method to obtain a reference spectrum of the reference substances;

[0026] Optionally, the solvent for preparing the reference solution is selected from a methanol aqueous solution with a volume percentage of 50% to 100%, and each 1 mL of the reference solution contains 10 μg to 50 μg of vanillic acid, epicatechin, isoquercetin and quercetin.

[0027] In an optional embodiment, the characteristic spectrum detection method further comprises the steps of preparing a control medicinal material solution using Amomum villosum as a control medicinal material, and detecting the control medicinal material solution using high performance liquid chromatography in the characteristic spectrum detection method to obtain a control medicinal material reference spectrum;

[0028] Optionally, the preparation process of the control medicinal material solution includes: taking Amomum villosum control medicinal material, adding water and heating to reflux, separating the solid and liquid and taking the liquid, evaporating to dryness, adding a solvent to the obtained residue, extracting and separating the solid and liquid, and taking the liquid to obtain the control medicinal material solution.

[0029] In an optional embodiment, the test sample in the test solution includes an Amomum villosum aqueous extract or an Amomum villosum preparation;

[0030] Optionally, the Amomum villosum preparation includes Amomum villosum formula granules.

[0031] In a second aspect, the present invention further provides a method for identifying the origin of an Amomum villosum product, comprising the following steps:

[0032] Obtaining a characteristic spectrum of the Amomum villosum product to be tested according to the characteristic spectrum detection method described above, and identifying the vanillic acid peak, isoquercetin peak, and quercetin peak in the characteristic spectrum;

[0033] respectively determining the relative peak areas of the vanillic acid peak and the isoquercetin peak, and the relative peak areas of the vanillic acid peak and the quercetin peak;

[0034] When the relative peak area between the vanillic acid peak and the isoquercetin peak is 28.6 to 34.1, it is determined that the Amomum villosum base corresponding to the Amomum villosum product is green shell sand;

[0035] and / or, when the relative peak area between the vanillic acid peak and the isoquercetin peak is not less than 34.1, determining that the Amomum villosum base corresponding to the Amomum villosum product is Amomum hainanensis;

[0036] And / or, when the relative peak area between the vanillic acid peak and the quercetin peak is not less than 4.4, it is determined that the Amomum villosum base corresponding to the Amomum villosum product is Amomum villosum.

[0037] The origin identification method of the above-mentioned Amomum villosum products provided by the present invention, based on the characteristic spectrum obtained in this application, can accurately identify and distinguish related products of Amomum villosum with different origins, namely, Amomum villosum yangchunensis, Amomum villosum green shell and Amomum villosum hainanensis, providing a rapid and reliable detection method for interspecies identification of Amomum villosum products.

[0038] In a third aspect, the present invention also provides the application of the above-mentioned characteristic spectrum detection method and / or origin identification method in the quality control of Amomum villosum products.

[0039] In a fourth aspect, the present invention further provides a method for quality control of an Amomum villosum product, comprising the steps of obtaining a characteristic spectrum of the Amomum villosum product to be tested according to the characteristic spectrum detection method described above, and comparing the characteristic spectrum with a control characteristic spectrum;

[0040] Wherein, the control characteristic spectrum is obtained by fitting the characteristic spectrum obtained by using at least one batch of standard products of Amomum villosum according to the characteristic spectrum detection method described above by the average value or median method;

[0041] And / or, it includes the step of identifying the Amomum villosum origin corresponding to the Amomum villosum product to be tested according to the origin identification method described above.

[0042] In an optional embodiment, the control characteristic spectrum includes 11 characteristic peaks, wherein peak 1 is a protocatechuic acid peak, peak 4 is a vanillic acid peak, peak 5 is a catechin peak, peak 7 is a procyanidin B2 peak, peak 8 is an epicatechin peak, peak 10 is an isoquercetin peak, and peak 11 is a quercetin peak;

[0043] Taking the epicatechin peak as the S peak, the relative retention time of each characteristic peak and the S peak is within ±10% of the specified value, among which the specified value corresponding to peak 1 is 0.21, the specified value corresponding to peak 2 is 0.39, the specified value corresponding to peak 3 is 0.42, the specified value corresponding to peak 5 is 0.65, the specified value corresponding to peak 6 is 0.90, the specified value corresponding to peak 7 is 0.94, and the specified value corresponding to peak 9 is 1.25. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0045] Figure 1 This is the chromatogram containing 12 chromatographic peaks confirmed in Example 2 of the present invention;

[0046] Figure 2 This is a chromatogram containing 11 chromatographic peaks confirmed in Example 2 of the present invention;

[0047] Figure 3 is the control characteristic spectrum determined in Example 2 of the present invention;

[0048] Figure 4 This is a chromatogram obtained by using methanol-0.1% phosphoric acid as the mobile phase in Example 3 of the present invention;

[0049] Figure 5 This is a chromatogram obtained by using acetonitrile-0.1% phosphoric acid as the mobile phase in Example 3 of the present invention;

[0050] Figure 6 is a chromatogram detected at a wavelength of 260 nm in Example 3 of the present invention;

[0051] Figure 7 is a chromatogram detected at a wavelength of 280 nm in Example 3 of the present invention;

[0052] Figure 8 This is the negative control chromatogram during the specificity verification in Example 4 of the present invention;

[0053] Figure 9 This is the chromatogram of the test sample during the specificity verification in Example 4 of the present invention;

[0054] Figure 10 is a chromatogram of integrity verification in Example 4 of the present invention;

[0055] Figure 11 This is a chromatogram of the precision verification in Example 4 of the present invention;

[0056] Figure 12 This is a chromatogram of the repeatability verification in Example 4 of the present invention;

[0057] Figure 13 This is a chromatogram of the intermediate precision verification in Example 4 of the present invention;

[0058] Figure 14 is a chromatogram of stability verification in Example 4 of the present invention;

[0059] Figure 15 1. It is the liquid phase characteristic spectrum of 3 batches of Amomum villosum (Amomum villosum) formula granules in Example 9 of the present invention. DETAILED DESCRIPTION

[0060] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0061] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0062] In order to solve the problems existing in the above-mentioned related technologies, according to the first aspect of the present invention, a characteristic spectrum detection method, an origin identification method and a quality control method of Amomum villosum product are provided to solve the problems of low separation and small number of common characteristic peaks when using the high performance liquid chromatography method in the relevant detection standards to detect Amomum villosum formula granules.

[0063] In a first aspect, the present invention provides a method for detecting a characteristic spectrum of an Amomum villosum product, comprising the following steps:

[0064] The test solution was taken and tested by high performance liquid chromatography; wherein the chromatographic conditions of the high performance liquid chromatography method include:

[0065] Octadecylsilane bonded silica gel was used as a filler, acetonitrile was used as mobile phase A, and 0.08% to 0.12% by volume phosphoric acid aqueous solution was used as mobile phase B. Gradient elution was performed. The gradient elution procedure included:

[0066] From 0 to 13 min, the volume percentage of acetonitrile in the mobile phase was 2% → 7%;

[0067] From 13 to 36 minutes, the volume percentage of acetonitrile in the mobile phase is 7% → 18%;

[0068] 36-40 min, the volume percentage of acetonitrile in the mobile phase is 18%.

[0069] The characteristic spectrum detection method of the above-mentioned Amomum villosum product provided by the present invention uses octadecylsilane bonded silica gel as a filler, acetonitrile-phosphoric acid aqueous solution as a mobile phase for gradient elution, and selects a specific elution procedure to detect and obtain a characteristic spectrum containing 11 characteristic peaks, which not only significantly increases the number of common characteristic peaks, but also achieves good separation of these common characteristic peaks. The elution procedure of this characteristic spectrum detection method is simple, the baseline of the obtained characteristic spectrum is stable, the peak shape of each characteristic peak is good, the separation between the characteristic peaks is high, and the peak positions of 7 characteristic peaks including vanillic acid, epicatechin, isoquercetin and quercetin can be accurately located.

[0070] By using the above-mentioned characteristic spectrum detection method, characteristic spectra of Amomum villosum products with different origins can be constructed, and the obtained characteristic spectra are highly characteristic and rich in chromatographic information, which can fully display the chemical composition characteristics of Amomum villosum products with different origins. In addition, the above-mentioned detection method has high precision and good stability, providing a basis for quality inspection and control of Amomum villosum products and identification and differentiation of Amomum villosum origins.

[0071] In an optional embodiment, the chromatographic conditions of the high performance liquid chromatography method further include at least one of the following conditions:

[0072] 1) The detection wavelength is 258nm~262nm;

[0073] 2) Column temperature is 28°C to 32°C;

[0074] 3) Flow rate: 0.30 mL / min to 0.37 mL / min;

[0075] 4) The injection volume is 1 μL to 3 μL;

[0076] 5) The chromatographic column was a CORTECS UPLC T3 column with an inner diameter of 2.1 mm, a column length of 100 mm, and a particle size of 1.6 μm.

[0077] Illustratively, mobile phase B can be an aqueous phosphoric acid solution with a volume percentage of 0.08%, 0.10% or 0.12%; the detection wavelength can be 258 nm, 260 nm or 262 nm; the column temperature can be 28°C, 30°C or 32°C; the flow rate can be 0.30 mL / min, 0.35 mL / min or 0.37 mL / min; and the injection volume can be 1 μL, 2 μL or 3 μL.

[0078] In an optional embodiment, the preparation method of the test solution is as follows:

[0079] Take the test sample, add solvent, extract, separate solid and liquid, and take liquid to obtain the test sample solution;

[0080] Optionally, the solvent is a methanol aqueous solution, and the volume percentage of methanol in the methanol aqueous solution is 50% to 70%;

[0081] Optionally, relative to 0.5 g of the test sample, the amount of the solvent added is 20 mL to 50 mL;

[0082] Optionally, the extraction is ultrasonic extraction, and the ultrasonic time is 45 minutes to 60 minutes.

[0083] Illustratively, the preparation method of the test solution is as follows: take an appropriate amount of the product, grind it into powder, accurately weigh it, accurately add the solvent, weigh it, ultrasonically treat it, make up the lost weight with the solvent, shake it well, filter it, and take the filtrate to obtain it.

[0084] In a particularly preferred embodiment, the preparation method of the test solution is as follows: take an appropriate amount of the product, grind it, take about 0.5 g, accurately weigh it, accurately add 25 mL of 70% methanol, weigh it, and ultrasonically treat it (power 250 W, frequency 40 KHz) for 45 minutes, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0085] In an optional embodiment, the characteristic spectrum detection method further comprises the steps of preparing a reference solution using a vanillic acid reference substance, an epicatechin reference substance, an isoquercetin reference substance, and a quercetin reference substance, and detecting the reference solution using the high performance liquid chromatography method to obtain a reference spectrum of the reference substances;

[0086] Optionally, the solvent for preparing the reference solution is selected from a methanol aqueous solution with a volume percentage of 50% to 100%, and each 1 mL of the reference solution contains 10 μg to 50 μg of vanillic acid, epicatechin, isoquercetin and quercetin.

[0087] Illustratively, in the reference solution, the concentration of the vanillic acid reference substance may be 10 μg / mL to 50 μg / mL, for example, 12 μg / mL; the concentration of the epicatechin reference substance may be 10 μg / mL to 50 μg / mL, for example, 40 μg / mL; the concentration of the isoquercetin reference substance may be 10 μg / mL to 50 μg / mL, for example, 12 μg / mL; the concentration of the quercetin reference substance may be 10 μg / mL to 50 μg / mL, for example, 12 μg / mL.

[0088] In an optional embodiment, the characteristic spectrum detection method further comprises the steps of preparing a control medicinal material solution using Amomum villosum as a control medicinal material, and detecting the control medicinal material solution using high performance liquid chromatography in the characteristic spectrum detection method to obtain a control medicinal material reference spectrum;

[0089] Optionally, the preparation process of the control medicinal material solution includes: taking Amomum villosum control medicinal material, adding water and heating to reflux, separating the solid and liquid and taking the liquid, evaporating to dryness, adding a solvent to the obtained residue, extracting and separating the solid and liquid, and taking the liquid to obtain the control medicinal material solution.

[0090] Illustratively, the preparation process of the control medicinal material solution includes: taking Amomum villosum (Amomum villosum) control medicinal material, adding water, heating and refluxing, filtering, evaporating the filtrate to dryness, adding a solvent to the residue, ultrasonically treating, shaking, filtering, and obtaining the control medicinal material solution.

[0091] In a particularly preferred embodiment, the preparation process of the control medicinal material solution includes: taking 2.5 g of Amomum villosum (Amomum villosum) control medicinal material, adding 50 mL of water, heating and reflux for 45 minutes, filtering, evaporating the filtrate to dryness, adding 20 mL of 70% methanol to the residue, ultrasonically treating (power 250 W, frequency 40 KHz) for 45 minutes, shaking, filtering, and using as the control medicinal material solution.

[0092] In an optional embodiment, the test sample in the test solution includes an Amomum villosum aqueous extract or an Amomum villosum preparation;

[0093] Optionally, the Amomum villosum preparation includes Amomum villosum formula granules.

[0094] In a second aspect, the present invention further provides a method for identifying the origin of an Amomum villosum product, comprising the following steps:

[0095] Obtaining a characteristic spectrum of the Amomum villosum product to be tested according to the characteristic spectrum detection method described above, and identifying the vanillic acid peak, isoquercetin peak, and quercetin peak in the characteristic spectrum;

[0096] respectively determining the relative peak areas of the vanillic acid peak and the isoquercetin peak, and the relative peak areas of the vanillic acid peak and the quercetin peak;

[0097] When the relative peak area between the vanillic acid peak and the isoquercetin peak is 28.6 to 34.1, it is determined that the Amomum villosum base corresponding to the Amomum villosum product is green shell sand;

[0098] and / or, when the relative peak area between the vanillic acid peak and the isoquercetin peak is not less than 34.1, determining that the Amomum villosum base corresponding to the Amomum villosum product is Amomum hainanensis;

[0099] And / or, when the relative peak area between the vanillic acid peak and the quercetin peak is not less than 4.4, it is determined that the Amomum villosum base corresponding to the Amomum villosum product is Amomum villosum.

[0100] The origin identification method of the above-mentioned Amomum villosum products provided by the present invention, based on the characteristic spectrum obtained in this application, can accurately identify and distinguish related products of Amomum villosum with different origins, namely, Amomum villosum yangchunensis, Amomum villosum green shell and Amomum villosum hainanensis, providing a rapid and reliable detection method for interspecies identification of Amomum villosum products.

[0101] In a third aspect, the present invention also provides the application of the above-mentioned characteristic spectrum detection method and / or origin identification method in the quality control of Amomum villosum products.

[0102] In a fourth aspect, the present invention further provides a method for quality control of an Amomum villosum product, comprising the steps of obtaining a characteristic spectrum of the Amomum villosum product to be tested according to the characteristic spectrum detection method described above, and comparing the characteristic spectrum with a control characteristic spectrum;

[0103] Wherein, the control characteristic spectrum is obtained by fitting the characteristic spectrum obtained by using at least one batch of standard products of Amomum villosum according to the characteristic spectrum detection method described above by the average value or median method;

[0104] And / or, it includes the step of identifying the Amomum villosum origin corresponding to the Amomum villosum product to be tested according to the origin identification method described above.

[0105] In an optional embodiment, the control characteristic spectrum includes 11 characteristic peaks, wherein peak 1 is a protocatechuic acid peak, peak 4 is a vanillic acid peak, peak 5 is a catechin peak, peak 7 is a procyanidin B2 peak, peak 8 is an epicatechin peak, peak 10 is an isoquercetin peak, and peak 11 is a quercetin peak;

[0106] Taking the epicatechin peak as the S peak, the relative retention time of each characteristic peak and the S peak is within ±10% of the specified value, among which the specified value corresponding to peak 1 is 0.21, the specified value corresponding to peak 2 is 0.39, the specified value corresponding to peak 3 is 0.42, the specified value corresponding to peak 5 is 0.65, the specified value corresponding to peak 6 is 0.90, the specified value corresponding to peak 7 is 0.94, and the specified value corresponding to peak 9 is 1.25.

[0107] The present invention is further described in detail below with reference to specific examples. These examples should not be construed as limiting the scope of protection claimed in the present invention.

[0108] The instruments, reagents and reagents involved in the examples are as follows:

[0109] 1. Preparation process of freeze-dried powder of Amomum villosum decoction standard decoction:

[0110] Take the medicinal material of Amomum villosum and prepare it into a medicinal piece that meets the requirements according to the relevant provisions of the 2020 edition of the Chinese Pharmacopoeia. Take the medicinal piece, crush it, soak it in room temperature water, add boiling water and simmer it over low heat, filter it, add room temperature water to the residue, boil it over high heat, then simmer it over low heat, filter it, and combine the two filtrates to obtain the standard decoction of Amomum villosum medicinal piece. Take the medicinal solution and concentrate it under reduced pressure to a concentrated extract. Place the concentrated extract in a freeze dryer and freeze-dry it. Take it out, grind it into powder and divide it into vials to obtain the freeze-dried powder of the standard decoction of Amomum villosum medicinal piece.

[0111] Preparation process of Amomum villosum (Amomum villosum) formula granules:

[0112] Take the medicinal material of Amomum villosum (Amomum villosum), and prepare it into a medicinal piece that meets the requirements according to the relevant provisions of the 2020 edition of the Chinese Pharmacopoeia. Take the medicinal piece, crush it before processing, extract the volatile oil, and encapsulate the volatile oil for later use. Add boiling water to the first decoction for boiling extraction, filter, add room temperature water to the second decoction, heat to boiling, extract, filter, combine the filtrate, and decompress and concentrate it to a concentrated extract. Add the volatile oil inclusion compound and drying excipients to mix, spray dry, add the adjusted amount of excipients to mix, and dry granulate.

[0113] 2. Instruments and test drugs

[0114] (1) Instruments and equipment

[0115] Electronic analytical balance: METTLER TOLEDO XA205DV; Sartovins B24s;

[0116] Water bath: HH-8 (Changzhou Aohua Instrument Co., Ltd.);

[0117] Rotary evaporator: R-300 (BUCHI);

[0118] Ultrasonic instrument: KQ-500DE (Kunshan Ultrasonic Instrument Co., Ltd.);

[0119] Chromatograph: Waters ACQUITY UPLC H-Class PLUS.

[0120] (2) Chromatographic column

[0121] Columns 1, 2, and 3: Waters CORTECS T3, 2.1 × 100 mm, 1.6 μm;

[0122] Column 4: Thermo Hypersil GOLD, 2.1*100 mm, 1.9 μm;

[0123] Column 5: Agilent SB-C18, 2.1*100 mm, 1.8 μm.

[0124] (3) Reagents

[0125] Acetonitrile and methanol were of chromatographic grade, water was ultrapurified water, and other reagents were of analytical grade.

[0126] (4) Drug testing

[0127] Protocatechuic acid reference substance (batch number: 110809-201906, for content determination, calculated as 97.7%) was purchased from the China Food and Drug Inspection Institutes;

[0128] Vanillic acid reference substance (batch number: 110776-201503, for content determination, calculated as 99.8%) was purchased from the China Food and Drug Inspection Institutes;

[0129] Catechin reference substance (batch number: 110877-202005, for content determination, calculated as 95.1%) was purchased from the China Food and Drug Inspection Institutes;

[0130] Epicatechin reference substance (batch number: 110878-201703, for content determination, calculated as 99.7%) was purchased from the China Food and Drug Inspection Institute;

[0131] Proanthocyanidin B2 reference substance (batch number: ST13510105, for content determination, calculated as 98.0%) was purchased from Shanghai Shidande Biotechnology Co., Ltd.

[0132] Isoquercetin reference substance (batch number: 111809-201804, for content determination, calculated as 97.2%) was purchased from the China Food and Drug Inspection Institutes;

[0133] Quercetin reference substance (batch number: 111538-202007, for content determination, calculated as 93.5%) was purchased from the China Food and Drug Inspection Institutes;

[0134] Amomum villosum (Amomum villosum) control medicinal material (batch number: 120985-201406, for thin layer chromatography identification) was purchased from the China Food and Drug Inspection Institute;

[0135] Freeze-dried powder of standard decoction of Amomum villosum (Amomum villosum) slices (Batch No.: 2102001Y, 2102002Y, 2102003Y, 2102004Y, 2102005Y, 2102006Y, 2102007Y, 2102008Y, 2102009Y, 2102011Y, 2102014Y, 2102016Y, 2102020Y, 2102021Y, 2102024Y, 2109025Y, 2109026Y);

[0136] Freeze-dried powder of standard decoction of Amomum villosum (Hainan Amomum villosum) slices (Batch numbers: 2102018Y, 2102019Y, 2102022Y, 2111030Y, 2111031Y, 2111032Y);

[0137] Freeze-dried powder of standard decoction of Amomum villosum (Green Shell Sand) slices (Batch No.: 2207033Y);

[0138] Amomum villosum (Amomum villosum) formula granules (Batch numbers: 2112001Y, 2112002Y, 2112003Y).

[0139] Example 1

[0140] This embodiment provides a characteristic spectrum detection method for a freeze-dried powder of a standard decoction of Amomum villosum (Amomum villosum) (Batch No.: 2102001Y):

[0141] (1) Solution preparation:

[0142] Preparation of control medicinal material solution: Take 2.5 g of Amomum villosum (Amomum villosum) control medicinal material, add 50 mL of water, heat and reflux for 45 minutes, filter, evaporate the filtrate to dryness, add 20 mL of 70% methanol to the residue, ultrasonically treat (power 250 W, frequency 40 kHz) for 45 minutes, shake well, filter, and use as the control medicinal material solution.

[0143] Preparation of reference solution: Take appropriate amounts of vanillic acid reference, epicatechin reference, isoquercetin reference, and quercetin reference, accurately weigh them, and add methanol to make a mixed solution containing 12 μg each of vanillic acid, isoquercetin, and quercetin and 40 μg of epicatechin per 1 mL, which is used as the reference solution.

[0144] Preparation of test solution: Take an appropriate amount of this product, grind it into powder, take about 0.5g, accurately weigh it, accurately add 25mL of 70% methanol, weigh it, ultrasonically treat it (power 250W, frequency 40KHz) for 45 minutes, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate.

[0145] (2) Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 General Chapter 0512):

[0146] Chromatographic conditions: A CORTECS UPLC T3 column (100 mm length, 2.1 mm inner diameter, 1.6 μm particle size) was used; mobile phase A was acetonitrile, and mobile phase B was 0.1% (v / v) phosphoric acid in water, with gradient elution as specified in Table 1; the flow rate was 0.35 mL / min; the column temperature was 30°C; and the detection wavelength was 260 nm. The number of theoretical plates, calculated based on the vanillic acid peak, should be no less than 5000.

[0147] Table 1 Gradient elution table

[0148]

[0149] Assay: Accurately pipette 1 μl of each of the reference medicinal material solution, reference substance solution, and test solution into a high-performance liquid chromatograph for determination. The basic information of each chromatographic peak in the resulting chromatogram is shown in Table 2.

[0150] Table 2 Test results of reference substances and test substances

[0151]

[0152] As shown in Table 2, the test sample chromatogram should exhibit 11 characteristic peaks, and their retention times should correspond to those of the 11 characteristic peaks in the control herb chromatogram. Four of these peaks should correspond to the retention times of the corresponding reference peaks. Of these 11 characteristic peaks, Peak 1 is protocatechuic acid, Peak 4 is vanillic acid, Peak 5 is catechin, Peak 7 is procyanidin B2, Peak 8 is epicatechin, Peak 10 is isoquercetin, and Peak 11 is quercetin. The characteristic peak corresponding to the epicatechin peak is designated as the S peak. The relative retention times of each characteristic peak and the S peak are calculated. These relative retention times should be within ±10% of the specified values, which are: 0.21 (Peak 1), 0.39 (Peak 2), 0.42 (Peak 3), 0.65 (Peak 5), 0.90 (Peak 6), 0.94 (Peak 7), and 1.25 (Peak 9).

[0153] (3) Origin identification:

[0154] Identification of freeze-dried powder of standard decoction of Amomum villosum (Amomum villosum): Calculate the relative peak area of ​​Peak 4 and Peak 11. The relative peak area should be within the specified range, which is: should not be less than 4.4 (Peak 4).

[0155] Identification of freeze-dried powder of standard decoction of Amomum villosum (green shell sand) slices: calculate the relative peak area of ​​peak 4 and peak 10, and the relative peak area should be within the specified range, which is: 28.6-34.1 (peak 4).

[0156] Identification of freeze-dried powder of standard decoction of Amomum villosum (Hainan Amomum) slices: calculate the relative peak area of ​​Peak 4 and Peak 10. The relative peak area should be within the specified range, which is: not less than 34.1 (Peak 4).

[0157] Example 2

[0158] This example is used to illustrate the establishment of a comparative characteristic spectrum of Amomum villosum products:

[0159] The fingerprint similarity evaluation software "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used. The liquid phase characteristic spectra of multiple batches of representative standard decoctions (lyophilized powders) of Amomum villosum (Amomum villosum) were used to generate a reference liquid phase characteristic spectrum. By consulting the literature, the chemical properties of each characteristic peak of the standard decoction (lyophilized powder) of Amomum villosum (Amomum villosum) were analyzed, and protocatechuic acid, vanillic acid, catechin, epicatechin, procyanidin B2, isoquercetin, and quercetin reference substances were selected to locate and identify them.

[0160] (1) Selection of characteristic peaks of characteristic spectra

[0161] Basis for developing characteristic spectrum category components: According to literature reports, the main chemical components of Amomum villosum include volatile oils such as bornyl acetate, camphor, camphor, borneol, limonene, and α-pinene; phenolic acids such as vanillic acid, protocatechuic acid, stearic acid, and palmitic acid; polyphenolic hydroxyl flavonoids such as epicatechin and catechin; flavonoids such as quercetin, isoquercetin, and quercitrin; and polysaccharides. Since the standard decoction of Amomum villosum slices is extracted after boiling in water, the water solubility of the chemical components should be considered. Substances with lower content may not be present after water dissolution. Among the above categories, phenolic acids, flavanols, and flavonoids are more water-soluble and easily present in the standard decoction of Amomum villosum slices. Therefore, they are used as the main chemical components of the characteristic spectrum of the standard decoction of Amomum villosum slices.

[0162] The fingerprint test results of 17 batches of standard decoctions (lyophilized powder) of Amomum villosum (Amomum villosum) slices were analyzed. The fingerprint similarity evaluation software "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to generate a reference characteristic spectrum. The characteristic peaks were identified and pointed out by HPLC and LC / MS / MS, and 12 chromatographic peaks with good separation were confirmed, such as Figure 1 As shown in the figure, peak 2 has a smaller peak shape in the characteristic spectrum of some batches. Considering the applicability of the characteristic spectrum method to different batches of chromatographic columns and different laboratories, it is not used as the characteristic peak. Peaks 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 are selected as characteristic peaks, and the chromatographic peak numbers 1 to 11 are rearranged according to the order of the chromatographic peaks, as shown in the figure. Figure 2 shown.

[0163] According to the results of peak identification and reference substance positioning, peak 1 was determined to be protocatechuic acid, peak 4 to be vanillic acid, peak 5 to be catechin, peak 7 to be procyanidin B2, peak 8 to be epicatechin, peak 10 to be isoquercetin, and peak 11 to be quercetin. The above identified components all belong to organic acids, flavanols, and flavonoids. The characteristic spectrum contains the main chemical components of Amomum villosum, which is consistent with the information reported in the literature.

[0164] (2) Reference Peak S Peak Selection Criteria

[0165] According to the peak identification and peak selection results of the characteristic peaks in the liquid phase characteristic spectrum of the standard decoction (lyophilized powder) of Amomum villosum (Amomum villosum) medicinal slices, the chromatographic peaks that are easily obtained by the reference substance are protocatechuic acid, vanillic acid, catechin, epicatechin, isoquercetin, and quercetin. Among them, in the characteristic spectrum of the standard decoction (lyophilized powder) of Amomum villosum (Amomum villosum) medicinal slices, the peak time of epicatechin is in the middle of the entire spectrum, and it is also the indicator component for content determination. The relative retention time of the characteristic peak is calculated by taking epicatechin as the S peak of the liquid phase characteristic spectrum; isoquercetin and quercetin have good thermal stability and moderate response, and the relative peak area of ​​the characteristic peak is calculated. The final control characteristic spectrum is as follows: Figure 3shown.

[0166] (3) Determination of characteristic peaks of characteristic spectra

[0167] The characteristic peaks were identified and assigned by HPLC and LC / MS / MS, and 7 characteristic peaks were located and assigned by reference substances, and it was determined that peak 1 was protocatechuic acid, peak 4 was vanillic acid, peak 5 was catechin, peak 7 was procyanidin B2, peak 8 was epicatechin, peak 10 was isoquercetin, and peak 11 was quercetin.

[0168] The above peaks were analyzed by LC / MS / MS, and it was confirmed that their molecular structures were consistent with those of protocatechuic acid, vanillic acid, catechin, procyanidin B2, epicatechin, isoquercetin, and quercetin reference substances, respectively. The results are shown in Table 3.

[0169] Table 3 Figure 2 LC / MS / MS analysis results of the liquid phase characteristic spectrum shown

[0170]

[0171] Example 3 Chromatographic Conditions

[0172] (1) Selection of mobile phase

[0173] Methanol and acetonitrile were used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. The elution effects of the two mobile phases on the same freeze-dried powder of Amomum villosum (Amomum villosum) decoction standard decoction (batch number: 2102001Y) were investigated, and the injection volume was 1 μL.

[0174] Test results such as Figure 4 and Figure 5 As shown, Figure 4 This is the chromatogram obtained when methanol-0.1% phosphoric acid was used as the mobile phase. Figure 5 The chromatogram obtained when acetonitrile-0.1% phosphoric acid was used as the mobile phase. Figure 4 and Figure 5 It can be seen that compared with the case of using methanol-0.1% phosphoric acid as the mobile phase, the time for peak elution to be complete is earlier, the detection time is shorter, and more components are eluted when using acetonitrile-0.1% phosphoric acid as the mobile phase.

[0175] (2) Optimization of gradient elution program

[0176] Using a gradient elution method, the same sample solution (Batch No. 2102001Y) was analyzed using multiple different elution gradients. By comparing the chromatograms and data obtained from different elution procedures, the gradient that provided richer chromatographic information, better peak separation, more stable baselines, and more reasonable analysis time was selected. Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Elution was performed according to the elution gradients shown in Tables 4-7, respectively. The detection wavelength was 260 nm, the column temperature was 30°C, and the flow rate was 0.35 mL / min. The chromatographic data corresponding to the four elution gradients are shown in Tables 8-11.

[0177] Table 4 Condition 1 Elution Gradient

[0178]

[0179] Table 5 Condition 2 Elution Gradient

[0180]

[0181] Table 6 Condition 3 elution gradient

[0182]

[0183] Table 7 Condition 4 Elution Gradient

[0184]

[0185] Table 8 Chromatographic data corresponding to elution gradient of condition 1

[0186]

[0187]

[0188] Table 9 Chromatographic data corresponding to elution gradient of condition 2

[0189]

[0190] Table 10 Chromatographic data corresponding to elution gradient of condition 3

[0191]

[0192] Table 11 Chromatographic data corresponding to elution gradient of condition 4

[0193]

[0194] It can be seen from Tables 8-11 that under condition 4 elution gradient, the peaks are better separated and symmetrical.

[0195] (3) Selection of detection wavelength

[0196] Take the same sample solution (batch number: 2102001Y), use 3D full wavelength scanning, select the chromatograms of the two wavelengths with more peak information (260nm, 280nm) for comparison, and determine the detection wavelength by comparing the number of chromatographic peaks and peak heights. Among them, the chromatogram detected at a wavelength of 260nm is as follows Figure 6 As shown, the chromatogram detected at a wavelength of 280 nm is as follows Figure 7 shown.

[0197] Depend on Figure 6 and Figure 7 It can be seen that at a wavelength of 260-280 nm, as the wavelength increases, the total peak area of ​​the liquid phase characteristic spectrum continues to increase, and at a wavelength of 280 nm, the peak information volume of the liquid phase characteristic spectrum is relatively large, and the response value of each peak is relatively high, but the baseline is not stable enough. The chromatographic peak has a stable baseline at a wavelength of 260 nm, and the peak symmetry is relatively good.

[0198] (4) Selection of mobile phase acid

[0199] The same sample solution (batch number: 2102001Y) was used to examine the elution effects of acetonitrile with 0.1% formic acid, 0.1% acetic acid, and 0.1% phosphoric acid as the mobile phase. The information content of the chromatographic peak and the system suitability parameters were used as evaluation indicators to determine the appropriate mobile phase acid type. The results are shown in Tables 12-14.

[0200] Table 12 System suitability parameters obtained from the 0.1% formic acid-acetonitrile system test

[0201]

[0202] Table 13 System suitability parameters obtained from the 0.1% acetic acid-acetonitrile system test

[0203]

[0204] Table 14 System suitability parameters obtained from the 0.1% phosphoric acid-acetonitrile system test

[0205]

[0206]

[0207] It can be seen from Tables 12-14 that when 0.1% phosphoric acid is used as mobile phase B, the symmetry of the chromatographic peaks is better than that of acetic acid and formic acid at the same concentration.

[0208] (5) Investigation of phosphoric acid concentration in mobile phase B

[0209] The same sample solution (batch number: 2102001Y) was used to examine the elution effects of acetonitrile with 0.2% phosphoric acid and 0.1% phosphoric acid as the mobile phase. The information content of the chromatographic peak and the system suitability parameters were used as evaluation indicators to determine the appropriate mobile phase acid concentration. The specific results are shown in Tables 15 and 16.

[0210] Table 15 System suitability parameters obtained from the 0.2% phosphoric acid-acetonitrile system test

[0211]

[0212] Table 16 System suitability parameters obtained from the 0.1% phosphoric acid-acetonitrile system test

[0213]

[0214] As can be seen from Tables 15 and 16, there is no significant difference in the system suitability parameters of the chromatographic peaks using different concentrations of phosphoric acid-acetonitrile systems.

[0215] (6) Selection of column temperature

[0216] The same sample solution (batch number: 2102001Y) was taken, and the information content of the detected chromatographic peaks and the system suitability parameters were used as evaluation indicators to examine the chromatographic peak separation effect at different column temperatures (25°C, 30°C, 35°C) and determine the appropriate chromatographic column temperature. The specific results are shown in Tables 17-19.

[0217] Table 17 System suitability parameters obtained at a column temperature of 25°C

[0218]

[0219]

[0220] Table 18 System suitability parameters obtained at a column temperature of 30°C

[0221]

[0222] Table 19 System suitability parameters obtained at a column temperature of 35°C

[0223]

[0224] As shown in Tables 17-19, within the range of 25-30°C, column temperature changes have minimal impact on the chromatographic system adaptability parameters. In particular, at a column temperature of 30°C, characteristic peaks exhibited improved symmetry, with each peak experiencing minimal interference from surrounding smaller peaks.

[0225] (7) Selection of flow rate

[0226] The same test solution (batch number: 2102001Y) was taken, and the information content of the detected chromatographic peaks and the system suitability parameters were used as evaluation indicators to examine the chromatographic peak separation effect at different flow rates (0.3, 0.35, and 0.4 mL / min) and determine the appropriate flow rate. The specific results are shown in Tables 20-22.

[0227] Table 20 System suitability parameters obtained at a flow rate of 0.30 mL / min

[0228]

[0229]

[0230] Table 21 System suitability parameters obtained at a flow rate of 0.35 mL / min

[0231]

[0232] Table 22 System suitability parameters obtained at a flow rate of 0.40 mL / min

[0233]

[0234] As can be seen from Tables 20-22, different flow rates not only affect the retention time and peak area of ​​each peak, but also the separation of each characteristic peak from surrounding minor peaks. In particular, within the flow rate range of 0.30-0.40 mL / min, the separation of peaks 1 and 7 from surrounding minor peaks is better at a flow rate of 0.35 mL / min.

[0235] Example 4

[0236] This example is used to perform methodological verification on the detection method in Example 1:

[0237] (1) Specificity verification

[0238] Use 70% methanol as negative control solution. Accurately pipette 1 μl of the test solution (batch number: 2102001Y) and negative control solution of Amomum villosum (Amomum villosum) slice standard decoction (lyophilized powder) and inject them into the high performance liquid chromatography instrument. Figure 8 and 9 As shown, Figure 8 is the negative control chromatogram, Figure 9 is the chromatogram of the test product. Figure 8 and Figure 9 It can be seen that the negative has no interference.

[0239] (2) Integrity Verification

[0240] Take the test sample solution (batch number: 2102001Y) and measure it according to the chromatographic conditions of the liquid phase characteristic spectrum of the standard decoction of Amomum villosum (Amomum yangchunsha) medicinal slices (lyophilized powder). The measurement is still carried out after the peak appears, and the measurement time is twice the time of the liquid phase characteristic spectrum of the standard decoction of Amomum villosum (Amomum villosum) medicinal slices (lyophilized powder).

[0241] The results are as follows Figure 10 As shown, it can be seen that after 80 minutes, there is basically no chromatographic peak in the liquid phase characteristic spectrum of the standard decoction (lyophilized powder) of Amomum villosum (Amomum yangchunsha) medicinal slices, indicating that the method can already determine the main chromatographic peaks of the liquid phase characteristic spectrum of the standard decoction (lyophilized powder) of Amomum villosum (Amomum villosum) medicinal slices, indicating that the method has good integrity.

[0242] (3) Precision verification

[0243] The same sample solution (batch number: 2102001Y) was injected 6 times (S1-S6) to measure the relative retention time and relative peak area of ​​11 characteristic peaks. The RSDs were all within the qualified range, indicating that the method and instrument precision were good. Figure 11 , Table 23 and Table 24.

[0244] Table 23 Precision relative retention time (t / ts) test results

[0245]

[0246] Table 24 Precision relative peak area (S / Ss) test results

[0247]

[0248] (4) Repeatability verification

[0249] Take the same portion of Amomum villosum (Amomum villosum) decoction standard decoction (lyophilized powder) (batch number: 2102001Y) to prepare 6 test solutions, and measure the relative retention time and relative peak area of ​​11 characteristic peaks. The RSDs are all within the qualified range, indicating that the method has good repeatability. Figure 12 , Table 25 and Table 26.

[0250] Table 25 Method repeatability relative retention time (t / ts) test results (n=6)

[0251]

[0252]

[0253] Table 26 Method repeatability relative peak area (S / Ss) test results (n=6)

[0254]

[0255] (5) Intermediate precision verification

[0256] The same standard decoction of Amomum villosum (Amomum villosum) slices (lyophilized powder) (batch number: 2102001Y) was taken and prepared by three experimenters. Each experimenter prepared two samples and measured the relative retention time and relative peak area of ​​11 characteristic peaks. The RSDs were all within the qualified range, indicating that the intermediate precision of the method was good. Figure 13 , Table 27 and Table 28.

[0257] Table 27 Intermediate precision relative retention time (t / ts) test results (different operators)

[0258]

[0259] Table 28 Intermediate precision relative peak area (S / Ss) test results (different operators)

[0260]

[0261]

[0262] (6) Stability verification

[0263] The same sample solution (batch number: 2102001Y) was injected at 0, 4, 8, 12, 18, and 24 hours after preparation. The relative retention time and relative peak area of ​​11 characteristic peaks were measured. The RSDs were all within the qualified range, indicating that the method was stable within 24 hours and met the determination requirements. Figure 14 , Table 29 and Table 30.

[0264] Table 29 Stability relative retention time (t / ts) test results

[0265]

[0266] Table 30 Stability relative peak area (S / Ss) test results

[0267]

[0268] (7) Durability verification

[0269] ①Verification at different flow rates

[0270] The same sample solution (batch number: 2102001Y) was taken and the flow rates were set to 0.33 mL / min, 0.35 mL / min, and 0.37 mL / min respectively. The changes in the relative retention time and relative peak area of ​​the characteristic peaks of this product when the flow rate changed slightly were investigated. The results are shown in Tables 31 and 32.

[0271] Table 31 Relative retention time results at different flow rates (mL / min)

[0272]

[0273] Table 32 Relative peak area results at different flow rates (mL / min)

[0274]

[0275] ②Verification at different column temperatures

[0276] Take the same test solution (batch number: 2102001Y) and set the column temperature to 28℃, 30℃, and 32℃ respectively. The changes in the relative retention time and relative peak area of ​​the characteristic peaks when the column temperature changes are investigated. The results are shown in Tables 33 and 34.

[0277] Table 33 Relative retention time results at different column temperatures

[0278]

[0279] Table 34 Relative peak area results at different column temperatures

[0280]

[0281]

[0282] ③Verification at different phosphoric acid concentrations

[0283] The same sample solution (lot number: 2102001Y) was used to compare the separation performance of 0.08% phosphoric acid-acetonitrile, 0.1% phosphoric acid-acetonitrile, and 0.12% phosphoric acid-acetonitrile to determine the most suitable mobile phase system. Specific results are shown in Tables 35-37.

[0284] Table 35 Liquid phase characteristic spectrum system adaptability parameters obtained using 0.08% phosphoric acid detection

[0285]

[0286] Table 36 Liquid phase characteristic spectrum system adaptability parameters obtained using 0.1% phosphoric acid detection

[0287]

[0288] Table 37 Liquid phase characteristic spectrum system adaptability parameters obtained using 0.12% phosphoric acid detection

[0289]

[0290] ④ Validation of different types of chromatographic columns

[0291] Take the same sample solution (batch number: 2102001Y) and examine the separation effect of different types of chromatographic columns in the laboratory on the chromatographic peaks to determine whether a fixed chromatographic column is needed:

[0292] Column a: Waters CORTECS T3, 2.1*100 mm, 1.6 μm;

[0293] Column b: Thermo Hypersil GOLD, 2.1*100 mm, 1.9 μm;

[0294] Chromatographic column c: Agilent SB-C18, 2.1*100 mm, 1.8 μm.

[0295] The test results are shown in Tables 38-40.

[0296] Table 38 System suitability parameters of the liquid phase characteristic spectrum obtained by detection using chromatographic column a

[0297]

[0298] Table 39 System suitability parameters of the liquid phase characteristic spectrum obtained by detection using chromatographic column b

[0299]

[0300] Table 40 System suitability parameters of the liquid phase characteristic spectrum obtained by detection using chromatographic column c

[0301]

[0302] The results show that when using the Waters CORTECS T3 column, the peaks are less interfered with by the surrounding small chromatographic peaks and the symmetry of the characteristic peaks is better. When using other types of chromatographic columns, there are missing characteristic peaks or the characteristic peaks are more interfered with by the surrounding small chromatographic peaks.

[0303] Example 5

[0304] According to the method of Example 1, the liquid phase characteristic spectra of 17 batches of freeze-dried powder of standard decoction of Amomum villosum (Amomum villosum) slices were measured respectively, and the measurement results are shown in Tables 41-43.

[0305] Table 41 Relative retention time determination results of liquid phase characteristic spectra of 17 batches of standard decoction of Amomum villosum (Amomum villosum) slices (lyophilized powder)

[0306]

[0307]

[0308] Table 42 Results of relative peak area determination of liquid phase characteristic spectra of 17 batches of standard decoction of Amomum villosum (Amomum villosum) slices (lyophilized powder)

[0309]

[0310]

[0311] Table 43 Results of relative peak area determination of liquid phase characteristic spectra of 17 batches of standard decoction of Amomum villosum (Amomum villosum) slices (lyophilized powder)

[0312]

[0313] Example 6

[0314] According to the method of Example 1, the liquid phase characteristic spectra of 6 batches of freeze-dried powder of standard decoction of Amomum villosum (Hainan Amomum) slices were measured respectively, and the measurement results are shown in Tables 44-46.

[0315] Table 44 Relative retention time determination results of liquid phase characteristic spectra of 6 batches of standard decoction of Amomum villosum (Hainan Amomum villosum) slices (lyophilized powder)

[0316]

[0317]

[0318] Table 45 Results of relative peak area determination of liquid phase characteristic spectra of 6 batches of standard decoction of Amomum villosum (Hainan Amomum villosum) slices (lyophilized powder)

[0319]

[0320] Table 46 Results of relative peak area determination of liquid phase characteristic spectra of 6 batches of standard decoction of Amomum villosum (Hainan Amomum villosum) slices (lyophilized powder)

[0321]

[0322] Example 7

[0323] According to the method of Example 1, the liquid phase characteristic spectrum of a batch of freeze-dried powder of standard decoction of Amomum villosum (green shell sand) slices was determined, and the determination results are shown in Tables 47-49.

[0324] Table 47 Relative retention time determination results of liquid phase characteristic spectrum of 1 batch of Amomum villosum (green shell sand) decoction standard decoction (lyophilized powder)

[0325]

[0326] Table 48 Relative peak area determination results of liquid phase characteristic spectrum of 1 batch of Amomum villosum (green shell sand) decoction standard decoction (lyophilized powder)

[0327]

[0328] Table 49 Results of relative peak area determination of liquid phase characteristic spectrum of 1 batch of Amomum villosum (green shell sand) decoction standard decoction (lyophilized powder)

[0329]

[0330] The results of Examples 5-7 show that the liquid phase characteristic chromatograms of multiple batches of standard decoctions (lyophilized powders) of Amomum villosum slices exhibit 11 characteristic peaks, which correspond to the retention times of the 11 characteristic peaks in the chromatograms of the reference medicinal material. Four of these peaks correspond to the retention times of the corresponding reference peaks. The peak corresponding to the epicatechin reference peak is the S peak. The relative retention times of each characteristic peak and the S peak were calculated, and the relative retention times were within ±10% of the specified values, which are: 0.21 (peak 1), 0.39 (peak 2), 0.42 (peak 3), 0.65 (peak 5), 0.90 (peak 6), 0.94 (peak 7), and 1.25 (peak 9).

[0331] There are obvious differences in the relative peak areas of peak 4 (vanillic acid) compared with peak 10 (isoquercetin) and peak 11 (quercetin) in the liquid phase characteristic spectra of the standard decoctions (lyophilized powder) of Amomum villosum (Yangchunsha), Amomum villosum (Hainansha) and Amomum villosum (Green Shell Sand) medicinal pieces.

[0332] Example 8

[0333] According to the method of Example 1, the liquid phase characteristic spectrum of a batch of Amomum villosum (Amomum villosum) formula granules (batch number: 2112001Y) was measured, and the measurement results are shown in Table 50.

[0334] Table 50 Chromatographic peak system adaptability parameters of Amomum villosum (Amomum villosum) formula granules

[0335]

[0336]

[0337] It can be seen from Table 50 that when the chromatographic conditions of the liquid phase characteristic spectrum of the standard decoction (lyophilized powder) of Amomum villosum (Yangchunsha) medicinal slices are used for Amomum villosum (Yangchunsha) formula granules, the liquid phase characteristic spectrum method system of Amomum villosum (Yangchunsha) formula granules has good adaptability and specificity. Therefore, the chromatographic conditions of the liquid phase characteristic spectrum of the standard decoction (lyophilized powder) of Amomum villosum (Yangchunsha) medicinal slices can be used as the chromatographic conditions of the liquid phase characteristic spectrum of Amomum villosum (Yangchunsha) formula granules.

[0338] The chromatogram of the Amomum villosum (Amomum villosum) formula granules should show 11 characteristic peaks, and their retention times should correspond to those of the 11 characteristic peaks in the chromatogram of the reference medicinal material. Four of these peaks should correspond to the retention times of the corresponding reference material peaks. The peak corresponding to the epicatechin reference peak is the S peak. Calculate the relative retention time of each characteristic peak to the S peak. The relative retention time should be within ±10% of the specified value: 0.21 (peak 1), 0.39 (peak 2), 0.42 (peak 3), 0.65 (peak 5), 0.90 (peak 6), 0.94 (peak 7), and 1.25 (peak 9). Calculate the relative peak area of ​​Peak 4 to Peak 11. The relative peak area should be within the specified range: no less than 4.4 (peak 4).

[0339] Example 9

[0340] Determination of liquid phase characteristic spectra of multiple batches of Amomum villosum (Amomum villosum) formula granules:

[0341] The liquid phase characteristic spectrum of three batches of Amomum villosum (Yangchunsha) formula granules (batch number: 2112001Y, 2112002Y, 2112003Y) was determined. The results showed that the chromatograms of the three batches of Amomum villosum (Yangchunsha) formula granules showed 11 characteristic peaks, and the retention times should correspond to the 11 characteristic peaks in the chromatogram of the reference medicinal material reference. Among them, 4 peaks should correspond to the retention times of the corresponding reference peaks, and the relative retention time and relative peak area are within the required range. The liquid phase characteristic spectrum is qualified. Among them, the liquid phase characteristic spectrum is shown in Figure 15 , relative retention times are shown in Table 51, and relative peak areas are shown in Table 52. Figure 15 Among them, the batch number corresponding to S1 is: 2112001Y, the batch number corresponding to S2 is: 2112002Y, and the batch number corresponding to S3 is: 2112003Y.

[0342] Table 51 Relative retention time of liquid phase characteristic spectra of three batches of Amomum villosum (Amomum villosum) formula granules

[0343]

[0344] Table 52 Relative peak areas of liquid phase characteristic spectra of three batches of Amomum villosum (Amomum villosum) formula granules

[0345]

[0346] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A characteristic spectrum detection method for Amomum villosum products, characterized in that: The steps include: The test solution was taken and tested by ultra-high performance liquid chromatography; wherein the chromatographic conditions of the ultra-high performance liquid chromatography include: Acetonitrile was used as mobile phase A and 0.08% to 0.12% by volume phosphoric acid aqueous solution was used as mobile phase B for gradient elution. The gradient elution procedure included: From 0 to 13 min, the volume percentage of acetonitrile in the mobile phase is 2% → 7%; From 13 to 36 minutes, the volume percentage of acetonitrile in the mobile phase is 7% → 18%; 36-40 min, the volume percentage of acetonitrile in the mobile phase was 18%; The detection wavelength is 258nm~262nm; The chromatographic column was a CORTECS UPLC T3 column with an inner diameter of 2.1 mm, a column length of 100 mm, and a particle size of 1.6 μm; The preparation method of the test solution is as follows: taking a test sample, adding a solvent, extracting and separating the solid and liquid, and taking the liquid to obtain the test solution; the solvent is a methanol aqueous solution; The method also includes the steps of preparing a reference solution using a protocatechuic acid reference substance, a vanillic acid reference substance, a catechin reference substance, a procyanidin B2 reference substance, an epicatechin reference substance, an isoquercetin reference substance, and a quercetin reference substance, and the step of detecting the reference solution using the ultra-high performance liquid chromatography method to obtain a reference spectrum of the reference substances.

2. The characteristic spectrum detection method according to claim 1, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography method further include at least one of the following conditions: 1) Column temperature is 28℃~32℃; 2) Flow rate: 0.30 mL / min to 0.37 mL / min; 3) The injection volume is 1µL to 3µL.

3. The characteristic spectrum detection method according to claim 1, characterized in that: The volume percentage of methanol in the methanol aqueous solution is 50% to 70%.

4. The characteristic spectrum detection method according to claim 1, characterized in that: The amount of the solvent added is 20 mL to 50 mL relative to 0.5 g of the test sample.

5. The characteristic spectrum detection method according to claim 1, wherein: The extraction is ultrasonic extraction, and the ultrasonic time is 45 minutes to 60 minutes.

6. The characteristic spectrum detection method according to claim 1, characterized in that: The solvent for preparing the reference solution is selected from a methanol aqueous solution with a volume percentage of 50% to 100%, and each 1 mL of the reference solution contains 10 μg to 50 μg of vanillic acid, epicatechin, isoquercetin and quercetin.

7. The characteristic spectrum detection method according to claim 1, characterized in that: The characteristic spectrum detection method further includes the steps of preparing a control medicinal material solution using Amomum villosum as a control medicinal material, and detecting the control medicinal material solution using the ultra-high performance liquid chromatography method to obtain a control medicinal material reference spectrum.

8. The characteristic spectrum detection method according to claim 7, characterized in that: The preparation process of the control medicinal material solution includes: taking Amomum villosum control medicinal material, adding water and heating to reflux, separating the solid and liquid and taking the liquid, evaporating to dryness, adding a solvent to the obtained residue, extracting and separating the solid and liquid, and taking the liquid to obtain the control medicinal material solution.

9. The characteristic spectrum detection method according to any one of claims 1 to 8, characterized in that: The test sample in the test solution includes an Amomum villosum water extract or an Amomum villosum preparation.

10. The characteristic spectrum detection method according to claim 9, characterized in that: The Amomum villosum preparation includes Amomum villosum formula granules.

11. Use of the characteristic spectrum detection method according to any one of claims 1 to 10 in quality control of Amomum villosum products.

12. A quality control method for Amomum villosum products, characterized in that: The method comprises the steps of obtaining a characteristic spectrum of the Amomum villosum product to be tested according to the characteristic spectrum detection method according to any one of claims 1 to 10, and comparing the characteristic spectrum with a control characteristic spectrum; The control characteristic spectrum is obtained by fitting the characteristic spectrum obtained by the characteristic spectrum detection method according to any one of claims 1 to 10 using at least one batch of standard products of Amomum villosum products through the average value or median method.

13. The quality control method according to claim 12, characterized in that: The control characteristic spectrum includes 11 characteristic peaks, wherein peak 1 is a protocatechuic acid peak, peak 4 is a vanillic acid peak, peak 5 is a catechin peak, peak 7 is a procyanidin B2 peak, peak 8 is an epicatechin peak, peak 10 is an isoquercetin peak, and peak 11 is a quercetin peak; Taking the epicatechin peak as the S peak, the relative retention time of each characteristic peak and the S peak is within ±10% of the specified value, among which the specified value corresponding to peak 1 is 0.21, the specified value corresponding to peak 2 is 0.39, the specified value corresponding to peak 3 is 0.42, the specified value corresponding to peak 5 is 0.65, the specified value corresponding to peak 6 is 0.90, the specified value corresponding to peak 7 is 0.94, and the specified value corresponding to peak 9 is 1.25.

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