Construction method and application of fingerprint spectrum of alpinia katsumadai medicinal material, standard decoction and traditional Chinese medicine formula granules thereof

Abstract

The invention provides a construction method of fingerprint spectrums of katsumadai medicinal material, standard decoction and formula granules thereof, which comprises the following steps: preparing a mixed reference solution from alpinetin, pinocembrin, cardamonin and alnudinone; preparing a test solution from the alpinia katsumadai medicinal material, the standard decoction or the formula granules; respectively detecting the reference solution and the test solution by adopting a high performance liquid chromatography, and constructing fingerprints of the alpinia katsumadai medicinal material, the standard decoction and the formula granules of the alpinia katsumadai medicinal material; wherein the conditions of the high performance liquid chromatography are as follows: a mixed solution of acetonitrile and methanol is taken as a mobile phase A, a phosphoric acid aqueous solution with the volume concentration of 0.08%-0.12% is taken as a mobile phase B, and a gradient elution procedure is adopted. The method has the advantages of being simple, stable and high in specificity, and a comprehensive, effective and rapid evaluation method is provided for quality control of alpinia katsumadai medicinal materials, standard decoction and formula granules of the alpinia katsumadai medicinal materials.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine identification and drug analysis, and specifically relates to a method for constructing an HPLC fingerprint of a cardamom herb, a standard decoction and its formula granules, and the identification application of confused products and plants of the same family. Background Art

[0002] Alpinia katsumadai Hayata is the dried nearly mature seeds of Alpinia katsumadai Hayata, a plant of the ginger family. Alpinia katsumadai Hayata is listed as cardamom in the "Ming Yi Bie Lu". Alpinia katsumadai Hayata tastes spicy and warm, and is in the spleen and stomach meridians. It has the functions of drying dampness, promoting qi and strengthening the spleen, warming the stomach and stopping vomiting. It is mainly used to treat symptoms such as internal obstruction of cold and dampness, abdominal distension, cold pain, belching, vomiting, and loss of appetite.

[0003] Market research has found that in recent years, some Chinese herbal medicine markets and Chinese herbal medicine companies have sold fake or adulterated cardamom. The most common adulterated product is Yunnan cardamom, which is the dried seeds of the ginger plant Alpinia blepharocalyx K. Schum., and its medicinal properties are similar to cardamom. Cardamom is the dried mature fruit of the ginger plant Amomurn kravanh Pierre ex Gagnep. or Java white cardamom Amomum comnpactum Soland ex Maton; red cardamom is the dried mature fruit of the ginger plant Alpinia galanga Willd.; nutmeg is the dried seed kernel of the Myristica fragrans Houtt. of the Myristica family.

[0004] The 2020 edition of the Chinese Pharmacopoeia records that the identification of cardamom is mainly through the identification of the appearance characteristics of the medicinal material and the identification of plant ecology. This method requires a high level of experience for the appraiser. In addition, Chinese medicine preparations such as standard decoctions and Chinese medicine formula granules lose the original properties of the medicinal materials after water extraction. In addition, ginger plants are rich in active ingredients such as flavonoids, volatile oils, and phenylheptanes, making it difficult to distinguish whether Chinese medicine preparations are derived from cardamom. At present, there are no reports on quality control methods for standard decoctions and formula granules of cardamom. Therefore, it is urgent to develop a method that can solve the problem of the inability to identify the common adulteration of standard decoctions and formula granules of cardamom on the market, so as to strengthen the identification of the specificity and overall quality control of standard decoctions and formula granules of cardamom, thereby conducting a comprehensive quality control method. Summary of the invention

[0005] In order to solve the above problems, this study provides a method for constructing HPLC fingerprints of cardamom medicinal materials, standard decoctions and their formula granules. The detection method is simple, stable and specific. The established HPLC fingerprint can be used to identify confused products and plants of the same family and evaluate the overall quality stability, providing a new scientific method for quality control of cardamom medicinal materials, standard decoctions and their formula granules.

[0006] The technical scheme of the present invention is as follows:

[0007] A method for constructing fingerprints of cardamom medicine, standard decoction and formula granules thereof comprises the following steps:

[0008] Take galangin, pine tincture, cardamomine and alnus ketone to prepare a mixed reference solution;

[0009] Prepare the test solution by taking the herbal medicine of cardamom, standard decoction or formula granules;

[0010] The reference solution and the test solution are respectively detected by high performance liquid chromatography to construct fingerprints of the cardamom herb, the standard decoction and the formula granules thereof;

[0011] The conditions of the high performance liquid chromatography method include: using a mixed solution of acetonitrile and methanol as mobile phase A, using a phosphoric acid aqueous solution with a volume concentration of 0.08% to 0.12% as mobile phase B, and using a gradient elution method;

[0012] The procedure of the gradient elution is:

[0013] From 0 to 10 min, the volume percentage of the mobile phase A increases from 28% to 32%;

[0014] 10min to 20min, the volume percentage of the mobile phase A increases from 32% to 50%;

[0015] 20min~28min, the volume percentage of the mobile phase A is 50%;

[0016] 28min-30min, the volume percentage of the mobile phase A increases from 50% to 65%;

[0017] 30min-37min, the volume percentage of the mobile phase A increases from 65% to 90%;

[0018] 37min-40min, the volume percentage of the mobile phase A is 90%.

[0019] In one embodiment, the volume ratio of acetonitrile to methanol in the mobile phase A is 4:1.

[0020] In one embodiment, the conditions of the high performance liquid chromatography method also include: using an Agilent Poroshell 120SB-C18 chromatographic column.

[0021] In one embodiment, the conditions of the high performance liquid chromatography method also include: a flow rate of 0.8 mL / min to 1.2 mL / min.

[0022] In one embodiment, the conditions of the high performance liquid chromatography method also include: a column temperature of 30°C to 40°C.

[0023] In one embodiment, the conditions of the high performance liquid chromatography method also include: a detection wavelength of 250nm to 270nm.

[0024] In one embodiment, the conditions of the high performance liquid chromatography method also include: the injection volume is 4 μL to 10 μL.

[0025] In one embodiment, 50% to 100% methanol by volume is used as a dissolving agent to prepare a mixed reference solution;

[0026] In the mixed reference solution, the concentration of galangin is 110 μg / mL to 130 μg / mL, the concentration of pine spatholobi is 80 μg / mL to 100 μg / mL, the concentration of cardamomum is 18 μg / mL to 22 μg / mL, and the concentration of alnus ketone is 70 μg / mL to 90 μg / mL.

[0027] In one embodiment, the preparation of the test solution comprises the following steps:

[0028] Extract the cardamom herb with water to obtain a residue, perform a first alcohol extraction on the residue, filter it, and take the filtrate to prepare a cardamom herb test solution;

[0029] Performing a second alcohol extraction on the powder of the standard decoction of cardamom, filtering, and taking the filtrate to prepare a test solution of the standard decoction of cardamom;

[0030] The powder of the cardamom formula granules is subjected to a third alcohol extraction, filtered, and the filtrate is taken to prepare a test solution of the cardamom formula granules.

[0031] In one of the embodiments, the water extraction is reflux extraction, and the volume ratio of the mass of the cardamom medicinal material test sample to water is 1g: (20-30)mL.

[0032] In one embodiment, the first alcohol extraction is to perform a first ultrasonic extraction on the residue using 50% to 100% methanol by volume as an extraction solvent.

[0033] In one embodiment, the second alcohol extraction is a second ultrasonic extraction of the cardamom standard decoction powder using 50% to 100% methanol as an extraction solvent, and the mass ratio of the cardamom standard decoction powder to the volume of the extraction solvent is 1g: (100 to 150) mL.

[0034] In one embodiment, the third alcohol extraction is to perform a third ultrasonic extraction on the cardamom formula granule powder using 50% to 100% methanol as an extraction solvent, and the mass ratio of the cardamom formula granule powder to the volume of the extraction solvent is 1g: (100 to 150) mL.

[0035] In one embodiment, the conditions of the first ultrasonic extraction, the second ultrasonic extraction and the second ultrasonic extraction are independently:

[0036] The power is 200W~300W, the frequency is 35kHz~45kHz, and the extraction time is 15min~45min.

[0037] In one embodiment, the fingerprint spectrum presents 10 characteristic peaks, of which 4 peaks correspond to the retention times of the corresponding reference peaks; the peak corresponding to the galangin reference peak is the S1 peak, and the relative retention times of peaks 1 to 2 and peak 4 with the S1 peak are calculated; the peak corresponding to the galangin reference peak is the S2 peak, and the relative retention times of peaks 6 to 7 and peak 9 with the S2 peak are calculated, and the relative retention times are within the range of ±10% of the specified value, and the specified values ​​are: peak 1 is 0.35, peak 2 is 0.68, peak 4 is 1.10, peak 6 is 1.20, peak 7 is 1.22, and peak 9 is 1.35.

[0038] The present invention also provides an application of a fingerprint spectrum constructed according to the method for constructing the fingerprint spectrum of cardamom medicinal materials, standard decoctions and formula granules thereof in identifying cardamom and Yunnan cardamom, or identifying cardamom and red cardamom, cardamom, nutmeg, or identifying cardamom and ginger, dried ginger, turmeric, zedoary zedoary, curcuma, and alkyl oxyphylla.

[0039] The present invention also provides a method for detecting the cardamom medicinal material, the standard decoction and the formula granules thereof, comprising the following steps:

[0040] Prepare the test solution by taking the herbal medicine of cardamom, standard decoction or formula granules;

[0041] The solution to be tested is detected by high performance liquid chromatography;

[0042] The conditions of the high performance liquid chromatography method include: using a mixed solution of acetonitrile and methanol as mobile phase A, using a phosphoric acid aqueous solution with a volume concentration of 0.08% to 0.12% as mobile phase B, and using a gradient elution method;

[0043] The procedure of the gradient elution is:

[0044] From 0 to 10 min, the volume percentage of the mobile phase A increases from 28% to 32%;

[0045] 10min to 20min, the volume percentage of the mobile phase A increases from 32% to 50%;

[0046] 20min~28min, the volume percentage of the mobile phase A is 50%;

[0047] 28min-30min, the volume percentage of the mobile phase A increases from 50% to 65%;

[0048] 30min-37min, the volume percentage of the mobile phase A increases from 65% to 90%;

[0049] 37min-40min, the volume percentage of the mobile phase A is 90%.

[0050] In one embodiment of the detection method, in the mobile phase A, the volume ratio of acetonitrile to methanol is 4:1.

[0051] In one embodiment of the detection method, the conditions of the high performance liquid chromatography method further include: using an Agilent Poroshell 120SB-C18 chromatographic column.

[0052] In one embodiment of the detection method, the conditions of the high performance liquid chromatography method also include: a flow rate of 0.8 mL / min to 1.2 mL / min.

[0053] In one embodiment of the detection method, the conditions of the high performance liquid chromatography method further include: a column temperature of 30°C to 40°C.

[0054] In one embodiment of the detection method, the conditions of the high performance liquid chromatography also include: the detection wavelength is 250nm to 270nm.

[0055] In one embodiment of the detection method, the conditions of the high performance liquid chromatography method also include: the injection volume is 4 μL to 10 μL.

[0056] The present invention also provides a method for constructing a high-resolution mass spectrometry component-based identification of cardamom medicinal materials, standard decoctions and formula granules thereof, comprising the following steps:

[0057] Take cardamom seeds, standard decoction or formula granules to prepare the test solution;

[0058] The test solution is subjected to high performance liquid chromatography-mass spectrometry analysis;

[0059] The conditions for high performance liquid chromatography analysis include elution according to the following mobile phase conditions: a mixed solution of acetonitrile and methanol as mobile phase A, an aqueous formic acid solution with a volume concentration of 0.08% to 0.12% as mobile phase B, and a gradient elution method;

[0060] The procedure of the gradient elution is:

[0061] From 0 to 10 min, the volume percentage of the mobile phase A increases from 28% to 32%;

[0062] 10min to 20min, the volume percentage of the mobile phase A increases from 32% to 50%;

[0063] 20min~28min, the volume percentage of the mobile phase A is 50%;

[0064] 28min-30min, the volume percentage of the mobile phase A increases from 50% to 65%;

[0065] 30min-37min, the volume percentage of the mobile phase A increases from 65% to 90%;

[0066] 37min-40min, the volume percentage of the mobile phase A is 90%.

[0067] In one embodiment of the construction method, in the mobile phase A, the volume ratio of acetonitrile to methanol is 4:1.

[0068] In one embodiment of the construction method, the conditions of the high performance liquid chromatography analysis also include: using an Agilent Poroshell 120SB-C18 chromatographic column.

[0069] In one embodiment of the construction method, the conditions of the high performance liquid chromatography analysis also include: a flow rate of 0.8 mL / min to 1.2 mL / min.

[0070] In one embodiment of the construction method, the conditions of the HPLC analysis also include: a column temperature of 30°C to 40°C.

[0071] In one embodiment of the construction method, the conditions of the HPLC analysis also include: the detection wavelength is 250nm to 270nm.

[0072] In one embodiment of the construction method, the conditions of the HPLC analysis also include: the injection volume is 4 μL to 10 μL.

[0073] In one embodiment of the construction method, the preparation of the test solution comprises the following steps:

[0074] Extract the cardamom herb with water to obtain a residue, perform a first alcohol extraction on the residue, filter it, and take the filtrate to prepare a cardamom herb test solution;

[0075] Performing a second alcohol extraction on the powder of the standard decoction of cardamom, filtering, and taking the filtrate to prepare a test solution of the standard decoction of cardamom;

[0076] The powder of the cardamom formula granules is subjected to a third alcohol extraction, filtered, and the filtrate is taken to prepare a test solution of the cardamom formula granules.

[0077] In one embodiment of the construction method, the water extraction is reflux extraction, and the mass ratio of the cardamom herb test sample to the volume of water is 1 g: (20-30) mL.

[0078] In one embodiment of the construction method, the first alcohol extraction is to perform a first ultrasonic extraction on the residue using 50% to 100% methanol by volume as an extraction solvent.

[0079] In one embodiment of the construction method, the second alcohol extraction is a second ultrasonic extraction of the cardamom standard decoction powder using 50% to 100% methanol as an extraction solvent, and the mass ratio of the cardamom standard decoction powder to the volume of the extraction solvent is 1g: (100 to 150) mL.

[0080] In one embodiment of the construction method, the third alcohol extraction is a third ultrasonic extraction of the cardamom formula granule powder using 50% to 100% methanol by volume as an extraction solvent, and the ratio of the mass of the cardamom formula granule powder to the volume of the extraction solvent is 1g: (100 to 150) mL.

[0081] In one embodiment of the construction method, the conditions of the first ultrasonic extraction, the second ultrasonic extraction and the third ultrasonic extraction are independently:

[0082] The power is 200W~300W, the frequency is 35kHz~45kHz, and the extraction time is 15min~45min.

[0083] In one embodiment of the construction method, the conditions for mass spectrometry analysis include: sheath gas flow rate of 30arb to 40arb; auxiliary gas flow rate of 8arb to 12arb; spray voltage of 3.5kV to 4.0kV; S-lens voltage of 45V to 55V; heating temperature of 320°C to 380°C; capillary temperature of 320°C to 380°C;

[0084] Positive ion mode and / or negative ion mode are used; the scanning range is 120m / z to 1200m / z; and the normalized collision energy is 30eV to 40eV.

[0085] The present invention has at least the following beneficial effects:

[0086] The invention adopts high performance liquid chromatography technology, uses a mixed solution of acetonitrile and methanol as mobile phase A, a phosphoric acid aqueous solution with a volume concentration of 0.08% to 0.12% as mobile phase B, adopts gradient elution and limits a specific elution program, and successfully constructs an HPLC fingerprint of the cardamom medicinal material, the standard decoction and the formula granules thereof. The established HPLC fingerprint can realize the identification of confused products and plants of the same family and the evaluation of the overall quality stability, and provides a new scientific method for the quality control of the cardamom medicinal material, the standard decoction and the formula granules thereof.

[0087] Furthermore, the fingerprint construction method of the cardamom medicinal material, standard decoction and formula granules thereof described in the present invention also has the advantages of being simple, stable and highly specific. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 Fingerprints of different wavelengths of cardamom medicine;

[0089] Figure 2 This is a 3D spectrum scan of the cardamom herb;

[0090] Figure 3 Fingerprints of different organic phases of cardamom herb;

[0091] Figure 4 Fingerprints of different aqueous phases of Amomum villosum medicinal material;

[0092] Figure 5 The chromatogram was investigated for the extraction solvent of the characteristic spectrum of the cardamom herb;

[0093] Figure 6 The chromatogram was investigated for the characteristic spectrum extraction method of cardamom herb;

[0094] Figure 7 The chromatogram was investigated for the extraction time of the characteristic spectrum of the cardamom herb;

[0095] Figure 8 To investigate the specificity of the fingerprint of cardamom;

[0096] Fig. 9 This is the fingerprint superposition of 20 batches of cardamom medicinal materials; among them, peak 3 (S1): galangin; peak 5 (S2): pine pine; peak 8: cardamomine; peak 10: alnus ketone;

[0097] Fig.10This is the fingerprint of the reference medicinal material of cardamom; among them, peak 3 (S1): galangin; peak 5 (S2): pine pine; peak 8: cardamomine; peak 10: alnus ketone;

[0098] Fig.11 This is the reference fingerprint of the medicinal material of cardamom; among them, peak 3 (S1): galangin; peak 5 (S2): pine pine; peak 8: cardamomine; peak 10: alnus ketone;

[0099] Fig.12 This is an overlay of fingerprints of 20 batches of standard decoctions of cardamom;

[0100] Fig.13 This is an overlay of fingerprints of three batches of cardamom formula granules;

[0101] Fig.14 This is the reference fingerprint of the standard decoction of cardamom; among them, peak 3 (S1): galangin; peak 5 (S2): pine pine; peak 8: cardamomine; peak 10: alnus ketone;

[0102] Fig.15 This is the reference fingerprint of the cardamom formula granules; among them, peak 3 (S1): galangin; peak 5 (S2): pine pine; peak 8: cardamomine; peak 10: alnus ketone;

[0103] Fig.16 The fingerprint comparison chromatograms of cardamom and Yunnan cardamom formula granules are shown;

[0104] Fig.17 This is the fingerprint overlay of the cardamom formula granules;

[0105] Fig.18 This is an overlay of fingerprints of cardamom formula granules;

[0106] Fig.19 This is the fingerprint overlay of the nutmeg formula granules;

[0107] Fig. 20 This is a comparison chart of fingerprints of red cardamom, cardamom and nutmeg formula granules;

[0108] Fig.21 This is the fingerprint overlay of the dried ginger formula granules;

[0109] Fig. 22 This is an overlay of the fingerprint of the turmeric formula granules;

[0110] Fig.23 This is the fingerprint overlay of ginger formula granules;

[0111] Fig.24 This is the fingerprint overlay of Alpinia oxyphylla formula granules;

[0112] Fig.25This is the fingerprint overlay of the Curcuma zedoaria formula granules;

[0113] Fig.26 This is the fingerprint overlay of Curcuma formula granules;

[0114] Fig. 27 This is a comparison chart of the fingerprint of the ginger formula granules;

[0115] Fig.28 This is the total ion flow diagram and UV absorption chromatogram of the test solution of the standard decoction of cardamom. DETAILED DESCRIPTION

[0116] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0117] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0118] The 2020 edition of the Chinese Pharmacopoeia stipulates under the item "Cardamom" that cardamom is the dried nearly mature seeds of the ginger plant Alpiniakatsumadai Hayata. There are counterfeits or adulterations on the market, and common confusing products are Yunnan cardamom, cardamom, red cardamom, nutmeg, etc. Identification and differentiation only by plant morphology or medicinal properties lacks intuitive differences. In addition, when the medicinal material of cardamom is made into standard decoctions, formula granules and other preparations, the original medicinal properties are lost, and it is impossible to identify whether the preparation is derived from cardamom, and ginger plants with similar active ingredients can be confused with it.

[0119] Therefore, in order to ensure the safety of medication, it is of great significance to establish a fast, accurate, objective and simple identification and differentiation method to study cardamom medicinal materials and their preparations.

[0120] The present invention provides a method for constructing a fingerprint spectrum of a cardamom medicinal material, a standard decoction and a formula granule thereof, comprising the following steps:

[0121] Take galangin, pine tincture, cardamomine and alnus ketone to prepare a mixed reference solution;

[0122] Prepare the test solution by taking the herbal medicine of cardamom, standard decoction or formula granules;

[0123] The reference solution and the test solution are respectively detected by high performance liquid chromatography to construct fingerprints of the cardamom herb, the standard decoction and the formula granules thereof;

[0124] The conditions of the high performance liquid chromatography method include elution according to the following mobile phase conditions: using a mixed solution of acetonitrile and methanol as mobile phase A, using a phosphoric acid aqueous solution with a volume concentration of 0.08% to 0.12% as mobile phase B, and using a gradient elution method;

[0125] The procedure of the gradient elution is:

[0126] From 0 to 10 min, the volume percentage of the mobile phase A increases from 28% to 32%;

[0127] 10min to 20min, the volume percentage of the mobile phase A increases from 32% to 50%;

[0128] 20min~28min, the volume percentage of the mobile phase A is 50%;

[0129] 28min-30min, the volume percentage of the mobile phase A increases from 50% to 65%;

[0130] 30min-37min, the volume percentage of the mobile phase A increases from 65% to 90%;

[0131] 37min-40min, the volume percentage of the mobile phase A is 90%.

[0132] In a specific example, in the mobile phase A, the volume ratio of acetonitrile to methanol is 4:1.

[0133] In a specific example, the conditions of the high performance liquid chromatography method also include: using an Agilent Poroshell 120SB-C18 chromatographic column.

[0134] More specifically, the specifications of the chromatographic column are: column length is 150 mm, inner diameter is 4.6 mm, and particle size of the internal filler is 2.7 μm.

[0135] In a specific example, the conditions of the high performance liquid chromatography also include: a flow rate of 0.8 mL / min to 1.2 mL / min, and the flow rate includes but is not limited to 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, and preferably, the flow rate is 1.0 mL / min.

[0136] In a specific example, the conditions of the high performance liquid chromatography method also include: a column temperature of 30°C to 40°C, including but not limited to 30°C, 32°C, 35°C, 38°C, and 40°C. Preferably, the column temperature is 35°C.

[0137] In a specific example, the conditions of the high performance liquid chromatography method also include: a detection wavelength of 250 nm to 270 nm, the detection wavelength includes but is not limited to 250 nm, 260 nm, and 270 nm, preferably, the detection wavelength is 260 nm.

[0138] In a specific example, the conditions of the high performance liquid chromatography also include: an injection volume of 4 μL to 10 μL, including but not limited to 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, and 10 μL. Preferably, the injection volume is 5 μL.

[0139] In a specific example, a mixed reference solution is prepared using 50% to 100% methanol by volume as a dissolving agent;

[0140] In the mixed reference solution, the concentration of galangin is 110 μg / mL to 130 μg / mL, the concentration of pine spatholobi is 80 μg / mL to 100 μg / mL, the concentration of cardamomum is 18 μg / mL to 22 μg / mL, and the concentration of alnus ketone is 70 μg / mL to 90 μg / mL.

[0141] Preferably, in the mixed reference solution, the concentration of galangin is 120 μg / mL, the concentration of pine tincture is 90 μg / mL, the concentration of cardamomum is 20 μg / mL, and the concentration of alnus ketone is 80 μg / mL.

[0142] In a specific example, the preparation of the test solution includes the following steps:

[0143] Extract the cardamom herb with water to obtain a residue, perform a first alcohol extraction on the residue, filter it, and take the filtrate to prepare a cardamom herb test solution;

[0144] Performing a second alcohol extraction on the powder of the standard decoction of cardamom, filtering, and taking the filtrate to prepare a test solution of the standard decoction of cardamom;

[0145] The powder of the cardamom formula granules is subjected to a third alcohol extraction, filtered, and the filtrate is taken to prepare a test solution of the cardamom formula granules.

[0146] It can be understood that before the cardamom medicinal material is hydrated, the cardamom medicinal material is crushed and passed through a No. 3 sieve to collect a powder sample with a particle size of about 50 mesh.

[0147] It can be understood that the residue is obtained by centrifuging the extract obtained after water extraction and concentrating the supernatant under reduced pressure.

[0148] Preferably, the concentration is carried out under reduced pressure at 60°C to 70°C, and more preferably, the concentration is carried out under reduced pressure at 65°C.

[0149] It can be understood that the preparation steps of the cardamom standard decoction powder are: grind the cardamom standard decoction freeze-dried powder, pass it through a No. 4 sieve, and collect the sample powder with a particle size of about 65 mesh.

[0150] It can be understood that the preparation steps of the cardamom formula granule powder are: grind the cardamom formula granules into fine powder, pass through a No. 4 sieve, and collect sample powder with a particle size of about 65 mesh.

[0151] In a specific example, the water extraction is reflux extraction, and the mass ratio of the cardamom herb sample to the volume of water is 1 g: (20-30) mL.

[0152] It can be understood that the "test sample" in the "mass ratio of the cardamom medicinal material test sample to the volume of water" refers to the cardamom medicinal material test sample powder sample.

[0153] Preferably, the reflux extraction time is 30 minutes, and after cooling, the lost weight is supplemented with water.

[0154] In a specific example, the first alcohol extraction is to perform a first ultrasonic extraction on the residue using methanol with a volume fraction of 50% to 100% as an extraction solvent.

[0155] In a specific example, the second alcohol extraction is to perform a second ultrasonic extraction on the standard decoction powder of cardamom using methanol with a volume fraction of 50% to 100% as an extraction solvent, and the mass ratio of the standard decoction powder of cardamom to the volume of the extraction solvent is 1g: (100 to 150) mL.

[0156] In a specific example, the third alcohol extraction is to perform a third ultrasonic extraction on the cardamom formula granule powder using 50% to 100% methanol as an extraction solvent, and the mass ratio of the cardamom formula granule powder to the volume of the extraction solvent is 1g: (100 to 150) mL.

[0157] In a specific example, the conditions of the first ultrasonic extraction, the second ultrasonic extraction and the third ultrasonic extraction are independently:

[0158] The power is 200W~300W, the frequency is 35kHz~45kHz, and the extraction time is 15min~45min.

[0159] Preferably, the conditions for ultrasonic extraction are: power of 250 W, frequency of 40 kHz, and extraction time of 30 min.

[0160] In a specific example, the fingerprint spectrum presents 10 characteristic peaks, of which 4 peaks correspond to the retention times of the corresponding reference peaks; the peak corresponding to the galangin reference peak is the S1 peak, and the relative retention times of peaks 1 to 2 and peak 4 with the S1 peak are calculated; the peak corresponding to the scutellaria baicalensis reference peak is the S2 peak, and the relative retention times of peaks 6 to 7 and peak 9 with the S2 peak are calculated, and the relative retention times are within the range of ±10% of the specified values, and the specified values ​​are: peak 1 is 0.35, peak 2 is 0.68, peak 4 is 1.10, peak 6 is 1.20, peak 7 is 1.22, and peak 9 is 1.35.

[0161] The present invention also provides an application of a fingerprint spectrum constructed according to the method for constructing the fingerprint spectrum of cardamom medicinal materials, standard decoctions and formula granules thereof in identifying cardamom and Yunnan cardamom, or identifying cardamom and red cardamom, cardamom, nutmeg, or identifying cardamom and ginger, dried ginger, turmeric, zedoary zedoary, curcuma, and alkyl oxyphylla.

[0162] The present invention also provides a method for detecting the cardamom medicinal material, the standard decoction and the formula granules thereof, comprising the following steps:

[0163] Prepare the test solution by taking the herbal medicine of cardamom, standard decoction or formula granules;

[0164] The solution to be tested is detected by high performance liquid chromatography;

[0165] The conditions of the high performance liquid chromatography method include elution according to the following mobile phase conditions: using a mixed solution of acetonitrile and methanol as mobile phase A, using a phosphoric acid aqueous solution with a volume concentration of 0.08% to 0.12% as mobile phase B, and using a gradient elution method;

[0166] The procedure of the gradient elution is:

[0167] From 0 to 10 min, the volume percentage of the mobile phase A increases from 28% to 32%;

[0168] 10min to 20min, the volume percentage of the mobile phase A increases from 32% to 50%;

[0169] 20min~28min, the volume percentage of the mobile phase A is 50%;

[0170] 28min-30min, the volume percentage of the mobile phase A increases from 50% to 65%;

[0171] 30min-37min, the volume percentage of the mobile phase A increases from 65% to 90%;

[0172] 37min-40min, the volume percentage of the mobile phase A is 90%.

[0173] In a specific example of the detection method, in the mobile phase A, the volume ratio of acetonitrile to methanol is 4:1.

[0174] In a specific example of the detection method, the conditions of the high performance liquid chromatography method also include: using an Agilent Poroshell 120SB-C18 chromatographic column.

[0175] In a specific example of the detection method, the conditions of the high performance liquid chromatography also include: a flow rate of 0.8 mL / min to 1.2 mL / min, and the flow rate includes but is not limited to 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, preferably, the flow rate is 1.0 mL / min.

[0176] In a specific example of the detection method, the conditions of the high performance liquid chromatography also include: a column temperature of 30°C to 40°C, including but not limited to 30°C, 32°C, 35°C, 38°C, and 40°C. Preferably, the column temperature is 35°C.

[0177] In a specific example of the detection method, the conditions of the high performance liquid chromatography also include: a detection wavelength of 250nm to 270nm, the detection wavelength includes but is not limited to 250nm, 260nm, 270nm, preferably, the detection wavelength is 260nm.

[0178] In a specific example of the detection method, the conditions of the high performance liquid chromatography also include: an injection volume of 4 μL to 10 μL, including but not limited to 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, and 10 μL. Preferably, the injection volume is 5 μL.

[0179] The present invention also provides a method for constructing a high-resolution mass spectrometry component-based identification of cardamom medicinal materials, standard decoctions and formula granules thereof, comprising the following steps:

[0180] Take cardamom seeds, standard decoction or formula granules to prepare the test solution;

[0181] The test solution is subjected to high performance liquid chromatography-mass spectrometry analysis;

[0182] The conditions for high performance liquid chromatography analysis include elution according to the following mobile phase conditions: using a mixed solution of acetonitrile and methanol as mobile phase A, using a formic acid aqueous solution with a volume concentration of 0.08% to 0.12% as mobile phase B, and using a gradient elution method;

[0183] The procedure of the gradient elution is:

[0184] From 0 to 10 min, the volume percentage of the mobile phase A increases from 28% to 32%;

[0185] 10min to 20min, the volume percentage of the mobile phase A increases from 32% to 50%;

[0186] 20min~28min, the volume percentage of the mobile phase A is 50%;

[0187] 28min-30min, the volume percentage of the mobile phase A increases from 50% to 65%;

[0188] 30min-37min, the volume percentage of the mobile phase A increases from 65% to 90%;

[0189] 37min-40min, the volume percentage of the mobile phase A is 90%.

[0190] In a specific example of the construction method, in the mobile phase A, the volume ratio of acetonitrile to methanol is 4:1.

[0191] In a specific example of the construction method, the conditions for the high performance liquid chromatography analysis also include: using an Agilent Poroshell 120SB-C18 chromatographic column.

[0192] In a specific example of the construction method, the conditions of the high performance liquid chromatography analysis also include: a flow rate of 0.8 mL / min to 1.2 mL / min, and the flow rate includes but is not limited to 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, and preferably, the flow rate is 1.0 mL / min.

[0193] In a specific example of the construction method, the conditions of the HPLC analysis also include: a column temperature of 30°C to 40°C, including but not limited to 30°C, 32°C, 35°C, 38°C, and 40°C. Preferably, the column temperature is 35°C.

[0194] In a specific example of the construction method, the conditions of the HPLC analysis also include: a detection wavelength of 250nm to 270nm, the detection wavelength includes but is not limited to 250nm, 260nm, 270nm, preferably, the detection wavelength is 260nm.

[0195] In a specific example of the construction method, the conditions of the HPLC analysis also include: an injection volume of 4 μL to 10 μL, including but not limited to 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, and 10 μL. Preferably, the injection volume is 5 μL.

[0196] In a specific example of the construction method, the preparation of the test solution includes the following steps:

[0197] Extract the cardamom herb with water to obtain a residue, perform a first alcohol extraction on the residue, filter it, and take the filtrate to prepare a cardamom herb test solution;

[0198] Performing a second alcohol extraction on the powder of the standard decoction of cardamom, filtering, and taking the filtrate to prepare a test solution of the standard decoction of cardamom;

[0199] The powder of the cardamom formula granules is subjected to a third alcohol extraction, filtered, and the filtrate is taken to prepare a test solution of the cardamom formula granules.

[0200] It can be understood that before the cardamom medicinal material is hydrated, the cardamom medicinal material is crushed and passed through a No. 3 sieve to collect a powder sample with a particle size of about 50 mesh.

[0201] It can be understood that the residue is obtained by centrifuging the extract obtained after water extraction and concentrating the supernatant under reduced pressure.

[0202] Preferably, the concentration is carried out under reduced pressure at 60°C to 70°C, and more preferably, the concentration is carried out under reduced pressure at 65°C.

[0203] It can be understood that the preparation steps of the cardamom standard decoction powder are: grind the cardamom standard decoction freeze-dried powder, pass it through a No. 4 sieve, and collect the sample powder with a particle size of about 65 mesh.

[0204] It can be understood that the preparation steps of the cardamom formula granule powder are: grind the cardamom formula granules into fine powder, pass through a No. 4 sieve, and collect sample powder with a particle size of about 65 mesh.

[0205] In a specific example, the water extraction is reflux extraction, and the mass ratio of the cardamom herb sample to the volume of water is 1 g: (20-30) mL.

[0206] It can be understood that the "test sample" in the "mass ratio of the cardamom medicinal material test sample to the volume of water" refers to the cardamom medicinal material test sample powder sample.

[0207] Preferably, the reflux extraction time is 30 minutes, and after cooling, the lost weight is supplemented with water.

[0208] In one embodiment of the construction method, the water extraction is reflux extraction, and the mass ratio of the test sample to the volume of water is 1 g: (20-30) mL.

[0209] In a specific example of the construction method, the first alcohol extraction is to perform a first ultrasonic extraction on the residue using methanol with a volume fraction of 50% to 100% as an extraction solvent.

[0210] In a specific example of the construction method, the second alcohol extraction is a second ultrasonic extraction of the cardamom standard decoction powder using 50% to 100% methanol as an extraction solvent, and the mass ratio of the cardamom standard decoction powder to the volume of the extraction solvent is 1g: (100 to 150) mL.

[0211] In a specific example of the construction method, the third alcohol extraction is a third ultrasonic extraction of the cardamom formula granule powder using 50% to 100% methanol as an extraction solvent, and the mass ratio of the cardamom formula granule powder to the volume of the extraction solvent is 1g: (100 to 150) mL.

[0212] In a specific example of the construction method, the conditions of the first ultrasonic extraction, the second ultrasonic extraction and the third ultrasonic extraction are independently:

[0213] The power is 200W~300W, the frequency is 35kHz~45kHz, and the extraction time is 15min~45min.

[0214] Preferably, the conditions for ultrasonic extraction are: power of 250 W, frequency of 40 kHz, and extraction time of 30 min.

[0215] In a specific example of the construction method, the conditions for mass spectrometry analysis include: sheath gas flow rate of 30arb to 40arb; auxiliary gas flow rate of 8arb to 12arb; spray voltage of 3.5kV to 4.0kV; S-lens voltage of 45V to 55V; heating temperature of 320℃ to 380℃; capillary temperature of 320℃ to 380℃;

[0216] Positive ion mode and / or negative ion mode are used; the scanning range is 120m / z to 1200m / z; and the normalized collision energy is 30eV to 40eV.

[0217] The present invention constructs HPLC fingerprints of cardamom medicinal materials, standard decoctions and formula particles thereof, which can fully reflect the characteristic peak information of the samples and demonstrate the chemical composition characteristics of cardamom.

[0218] The present invention simultaneously analyzes the fingerprints of cardamom medicinal materials, standard decoctions, formula granules, confused products, and preparations of plants of the same family, and can well distinguish confused products and plants of the same family, providing a new scientific method for quality control of cardamom medicinal materials, standard decoctions and formula granules thereof.

[0219] The following is a further detailed description of the fingerprint construction method of the cardamom medicinal material, standard decoction and its formula granules of the present invention in combination with specific examples. The raw materials used in the following examples are all commercially available products unless otherwise specified.

[0220] The instruments and reagents used in the embodiments of the present invention are as follows:

[0221] Example 1

[0222] This embodiment provides a method for constructing fingerprints of cardamom medicine, standard decoction and formula granules thereof, which is as follows:

[0223] 1. Instruments and test drugs

[0224] Instruments: Agilent high performance liquid chromatograph (1290, Agilent); Agilent Poroshell 120SB-C18 chromatographic column (4.6 mm × 150 mm, 2.7 μm); 1 / 10,000 analytical balance (ME204E, Mettler-Toledo); 1 / 10,000 analytical balance (XP26, Mettler-Toledo); CNC ultrasonic cleaner (KQ500DE, Kunshan Ultrasonic Instrument Co., Ltd.); constant temperature water bath (HWS28, Shanghai Yiheng Technology Co., Ltd.); ultrapure water system (Milli-QDirect, Merck & Co., Ltd.).

[0225] Reagents: ethanol (analytical grade, Tianjin Fuyu Fine Chemical Co., Ltd.); methanol (analytical grade, Tianjin Fuyu Fine Chemical Co., Ltd.); phosphoric acid (chromatographic grade, Tianjin Komiou Chemical Reagent Co., Ltd.); acetonitrile (chromatographic grade, Merck & Co., Ltd.); methanol (chromatographic grade, Merck & Co., Ltd.); ultrapure water (self-made in the laboratory).

[0226] Test drugs: Alpinia officinalis (batch number: 110762-201806, content: 99.2%, China Food and Drug Inspection Institute); Pinus schrenkiana (batch number: 111829-201703, content: 99.5%, China Food and Drug Inspection Institute); Cardamom (batch number: 110763-202105, content: 98.8%, China Food and Drug Inspection Institute); Alnus ketone (batch number: 111830-201703, content: 99.8%, China Food and Drug Inspection Institute); Cardamom Reference medicinal materials (batch number: 125007-201202A, calibrated by Guangdong Food and Drug Quality Research Institute); cardamom medicinal materials and their standard decoctions (20 batches each); cardamom formula granules (3 batches); red cardamom formula granules (3 batches); cardamom formula granules (3 batches); nutmeg formula granules (3 batches); dried ginger formula granules (3 batches); ginger formula granules (3 batches); turmeric formula granules (3 batches); curcuma formula granules (3 batches); zedoaria formula granules (3 batches); alkyl oxyphylla formula granules (3 batches), the origin and numbers are shown in Table 1.

[0227] Table 1 Sample information table

[0228]

[0229] 2. Chromatographic conditions and preparation of test solution

[0230] 2.1 Chromatographic conditions

[0231] An Agilent Poroshell 120SB-C18 (4.6 mm × 150 mm, 2.7 μm) chromatographic column was used; acetonitrile:methanol (4:1) was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 2 below; the flow rate was 1.0 ml per minute; the column temperature was 35°C; the detection wavelength was 260 nm; and the injection volume was 5 μl.

[0232] Table 2 Gradient elution program

[0233]

[0234] 2.2 Preparation of reference solution

[0235] Take appropriate amounts of shanpinin reference substance, pinocinol reference substance, cardamomum camphora reference substance, and alnus ketone reference substance, weigh them accurately, and add methanol to prepare mixed solutions containing 120 g shanpinin, 90 μg pinocinol, 20 μg cardamomum camphora and 80 μg alnus ketone per 1 mL.

[0236] 2.3 Preparation of standard decoction test solution

[0237] Take an appropriate amount of this product, grind it into powder, pass it through a No. 4 sieve, collect sample powder with a particle size of about 65 mesh, take about 0.2g, accurately weigh it, put it in a stoppered conical flask, accurately add 25mL of 70% methanol, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0238] 2.4 Preparation of test solution of formulated granules

[0239] Take an appropriate amount of this product, grind it into powder, pass it through a No. 4 sieve, collect sample powder with a particle size of about 65 mesh, take about 0.2g, accurately weigh it, put it in a stoppered conical flask, accurately add 25mL of 70% methanol, weigh it, ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0240] 2.5 Preparation of medicinal material test solution

[0241] Take about 2.0 g of the powder of this product (passed through No. 3 sieve), place it in a stoppered conical flask, add 50 mL of water, weigh it, heat and reflux for 30 minutes, cool, weigh it again, make up the lost weight with water, centrifuge, take 25 mL of the supernatant, concentrate under reduced pressure at 65°C, add 25 mL of 70% methanol to the residue, weigh it, treat it with ultrasound (power 250 W, frequency 40 kHz) for 30 minutes, cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0242] 2.6 Determination method

[0243] Accurately pipette 5 μL of the reference solution and the test solution respectively, inject into the high performance liquid chromatograph for determination.

[0244] 3. Optimization of chromatographic conditions

[0245] 3.1 Wavelength selection

[0246] Take about 2.0g of cardamom powder (CDK18), prepare the test solution according to the method in "2.5", inject and analyze according to the chromatographic conditions in "2.1", and record the absorption spectrum in the range of 190-400nm. Figure 2 The results show that by comparing the chromatograms of different detection wavelengths, it is found that at a wavelength of 260nm, the peak response and information are richer, the interference is small, and the separation is better. Therefore, 260nm is selected as the detection wavelength. Figure 1 .

[0247] 3.2 Selection of mobile phase

[0248] Take about 2.0g of cardamom powder (CDK18), prepare the test solution according to the method under "2.5", and analyze the sample according to the chromatographic conditions under "2.1". Compare the effects of different mobile phase combinations: acetonitrile-0.1% phosphoric acid, acetonitrile: methanol (4:1)-0.1% phosphoric acid, acetonitrile: methanol (4:1)-0.1% glacial acetic acid, acetonitrile: methanol (4:1)-0.1% formic acid. The results show that when the organic phase is acetonitrile-methanol (4:1) and the aqueous phase is 0.1% phosphoric acid, the characteristic peaks and separation effects are better. Therefore, the mobile phase is acetonitrile: methanol (4:1)-0.1% phosphoric acid. Figure 3-Figure 4 .

[0249] 3.3 Selection of different chromatographic columns

[0250] Take about 2.0g of cardamom powder (CDK18) and prepare the test solution according to the method under "2.5". Through a systematic investigation of different brands of chromatographic columns and different filling particle sizes (1.7, 1.8, 2.7, 3.0μm), it is found that different brands of chromatographic columns have a greater impact on the separation and peak shape of each peak. Among them, Agilent Poroshell 120SB-C18 (4.6mm×150mm, 2.7μm) chromatographic column has better characteristic peak shapes and separation effects. Agilent Poroshell 120SB-C18 (4.6mm×150mm, 2.7μm) was selected as the chromatographic column after comprehensive consideration.

[0251] 4. Investigation of the preparation method of test solution

[0252] 4.1 Investigation of extraction solvent

[0253] Take about 2.0g of cardamom (CDK18), accurately weigh, parallel 6 groups, 2 portions in each group, put in a stoppered conical bottle, add 50mL of water, weigh, heat and reflux for 30 minutes, cool, weigh again, make up the lost weight with water, centrifuge, take 25mL of supernatant, concentrate under reduced pressure at 65℃, accurately add 25mL of methanol, 70% methanol, 50% methanol, ethanol, 70% ethanol, and 50% ethanol to the residue, weigh, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and take the filtrate. Accurately aspirate 5μL of the above test solution, reference solution under "2.2" and blank solvent, and analyze according to the chromatographic conditions under "2.1". Record the peak area of ​​each characteristic peak and calculate "total peak area / sample weight". The results are shown in Table 3 below. Figure 5The results showed that the above six extraction solvents had little effect on the peak shape and separation effect of each characteristic peak, and the "total peak area / sample weight" was the largest when 70% methanol was used as the extraction solvent. Combined with the content determination results, 70% methanol was finally selected as the extraction solvent.

[0254] Table 3 Results of investigation on extraction solvents for the characteristic spectrum of cardamom

[0255]

[0256]

[0257] 4.2 Investigation of extraction methods

[0258] Take about 2.0g of cardamom (CDK18), accurately weigh, parallelize in 2 groups, 2 portions in each group, place in a stoppered conical flask, add 50mL of water, weigh, heat and reflux for 30 minutes, cool, weigh again, make up the lost weight with water, centrifuge, take 25mL of supernatant, concentrate under reduced pressure at 65℃, accurately add 25mL of 70% methanol to the residue, weigh, heat and reflux for 30 minutes, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate. Accurately aspirate 5μL of the above test solution, reference solution under "2.2" and blank solvent, analyze according to the chromatographic conditions under "2.1", record the peak area of ​​each characteristic peak, and calculate "total peak area / sample amount". The results are shown in Table 4 below. Figure 6 The results showed that there was no significant difference in the peak shape and separation effect of each characteristic peak between the two extraction methods. The effects of ultrasonic extraction and reflux extraction were equivalent. Considering the convenience of operation, ultrasonic extraction was selected as the extraction method for the characteristic spectrum of Amomum villosum.

[0259] Table 4 Results of investigation on the extraction method of characteristic spectrum of cardamom medicinal materials

[0260]

[0261] 4.3 Extraction time investigation

[0262] Take about 2.0g of cardamom powder (CDK18), and divide it into 3 parallel groups, 2 portions in each group, accurately weigh it, put it in a stoppered conical bottle, add 50mL of water, weigh it, heat and reflux for 30 minutes, let it cool, weigh it again, make up the lost weight with water, centrifuge, take 25mL of supernatant, concentrate it under reduced pressure at 65℃, accurately add 25mL of 70% methanol to the residue, weigh it, and treat it with ultrasound (power 250W, frequency 40kHz) for 15 minutes, 30 minutes, and 45 minutes respectively, 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 it. Accurately aspirate 5μL of the above test solution, the reference solution under "2.2" and the blank solvent, and analyze it according to the chromatographic conditions under "2.1", record the peak area of ​​each characteristic peak, and calculate the "total peak area / sample amount". The results are shown in Table 5 and Table 6 below. Figure 7 The results showed that different extraction times had little effect on the characteristic spectrum of Alpinia officinalis. To ensure complete extraction, ultrasonic treatment for 30 minutes was selected.

[0263] Table 5 Results of investigation on the extraction time of the characteristic spectrum of cardamom medicinal materials

[0264]

[0265]

[0266] 5. Methodological investigation

[0267] 5.1 Specificity inspection

[0268] Take about 2.0g of cardamom (CDK18), prepare the test solution according to the method in "2.5", accurately pipette 5μL of the test solution, the reference solution in "2.2" and the blank solvent, and analyze according to the chromatographic conditions in "2.1". The results show that the test and reference have the same chromatographic peaks at the corresponding retention times, and the blank solvent has no interference, indicating that the established method has good specificity. Figure 8 .

[0269] 5.2 Precision investigation

[0270] Take about 2.0g of the same batch of cardamom medicine (CDK18), prepare the test solution according to the method under "2.5", and repeat the injection and determination 6 times according to the chromatographic conditions under "2.1". Take the galangin peak as the reference peak S1, calculate the relative retention time of peaks 1 to 4 and S1; take the scutellariae peak as the reference peak S2, calculate the relative retention time of peaks 5 to 10 and S2, and calculate the RSD value. The results are shown in Table 6 below. The results show that the relative retention time RSD of each characteristic peak and S peak is less than 3%, indicating that the instrument has good precision.

[0271] Table 6 Precision investigation results of characteristic spectrum of cardamom (relative retention time)

[0272]

[0273] 5.3 Reproducibility study

[0274] Take about 2.0g of the same batch of cardamom medicine (CDK18), prepare 6 test solutions according to the method under "2.5", and inject and measure according to the chromatographic conditions under "2.1". Take the galangin peak as the reference peak S1, calculate the relative retention time of peaks 1 to 4 and S1; take the scutellariae peak as the reference peak S2, calculate the relative retention time of peaks 5 to 10 and S2, and calculate the RSD value. The results are shown in Table 7 below. The results show that the relative retention time RSD of each characteristic peak and S peak is less than 3%, indicating that the established method has good reproducibility.

[0275] Table 7 Results of reproducibility study of the characteristic spectrum of cardamom (relative retention time)

[0276]

[0277] 5.4 Stability investigation

[0278] Take about 2.0g of the same batch of cardamom medicine (CDK18), prepare the test solution according to the method under "2.5", and inject samples at 0, 2, 4, 6, 12, and 18 hours respectively according to the chromatographic conditions under "2.1". Take the shanjiangsu peak as the reference peak S1, calculate the relative retention time of peaks 1 to 4 and S1; take the scutellariae peak as the reference peak S2, calculate the relative retention time of peaks 5 to 10 and S2, and calculate the RSD value. The results are shown in Table 8 below. The results show that the RSD of the relative retention time of each characteristic peak is less than 3%, indicating that the test sample has good stability within 18 hours.

[0279] Table 8 Results of stability study of characteristic spectrum of cardamom medicinal materials (relative retention time)

[0280]

[0281] 5.5 Intermediate precision study

[0282] The operation was performed by different analysts in different laboratories and on instruments of different brands. About 2.0 g of the same batch of cardamom medicine (CDK18) was taken, and 6 test solutions were prepared according to the method under "2.5", and the samples were injected and measured according to the chromatographic conditions under "2.1". The relative retention time of peaks 1 to 4 and S1 was calculated using the peak of galangin as the reference peak S1; the relative retention time of peaks 5 to 10 and S2 was calculated using the peak of scutellaria baicalensis as the reference peak S2, and the RSD value was calculated. The results are shown in Table 9 below. The results show that the RSD of the relative retention time of each characteristic peak is less than 3%, indicating that the established method can be reproduced under different conditions such as different equipment and laboratories, and the intermediate precision of this method is good.

[0283] Table 9 Intermediate precision investigation results of the characteristic spectrum of cardamom (relative retention time)

[0284]

[0285]

[0286] 6. Establishment of the fingerprint of cardamom

[0287] 6.1 Results of fingerprint determination of cardamom

[0288] Take 20 batches of cardamom medicinal materials, according to the chromatographic conditions under "2.1" and the preparation method of the test solution under "2.5", take the galangin peak as the reference peak S1, calculate the relative retention time of peaks 1 to 4 and S1; take the pine pine peak as the reference peak S2, calculate the relative retention time of peaks 5 to 10 and S2, and calculate the RSD value. The experimental results are shown in Table 10. Combined with the established fingerprint analysis, the types of components of cardamom from different origins are basically the same. The fingerprint superposition of 20 batches of cardamom medicinal materials is shown in Fig. 9 .

[0289] Table 10 Fingerprints of 20 batches of cardamom (relative retention time)

[0290]

[0291] 6.2 Establishment of the common pattern of the herbal grain pattern of Amomum villosum

[0292] Take the cardamom reference medicinal material (batch number: 125007-201202A, calibrated by Guangdong Food and Drug Quality Institute) and 20 batches of cardamom medicinal materials, and inject and measure according to the chromatographic conditions under "2.1" and the test solution preparation method under "2.5", and obtain the fingerprint of the cardamom reference medicinal material ( Fig.10 ); the Chinese medicine chromatographic fingerprint similarity evaluation software was used to generate a reference map by the average method to establish a reference fingerprint of cardamom ( Fig.11 ).

[0293] It is determined that the fingerprint of cardamom medicine should present 10 characteristic peaks, and the retention times of the 10 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other; 4 peaks should correspond to the retention times of the corresponding reference peaks; the peak corresponding to the alpinia officinalis reference peak is the S1 peak, and the relative retention times of peaks 1-2, peak 4 and S1 are calculated; the peak corresponding to the pinecone reference peak is the S2 peak, and the relative retention times of peaks 6-7, peak 9 and S2 are calculated, and the relative retention times should be within the range of ±10% of the specified values, and the specified values ​​are: 0.35 (peak 1), 0.68 (peak 2), 1.10 (peak 4), 1.20 (peak 6), 1.22 (peak 7), and 1.35 (peak 9).

[0294] 6.3 Comparison of fingerprints of cardamom, standard decoction and its formula granules

[0295] Take 20 batches of standard decoction of cardamom and 3 batches of formula granules of cardamom, and according to the chromatographic conditions under "2.1" and the corresponding test solution preparation method under "2.3"-"2.4", sample injection and determination are performed. The peak of galangin is used as the reference peak S1, and the relative retention time of peaks 1 to 4 and S1 is calculated; the peak of scutellaria baicalensis is used as the reference peak S2, and the relative retention time of peaks 5 to 10 and S2 is calculated, and the RSD value is calculated (Table 11). The results showed that the standard decoction of cardamom and its formula granules all showed 10 characteristic peaks consistent with the cardamom medicinal material, and the relative retention times were within the specified range of the medicinal material, indicating that the established chromatographic conditions can be applied to cardamom medicinal materials, standard decoctions and their formula granules. Figure 12 to Figure 15 shown.

[0296] Table 11 Fingerprints of 20 batches of standard decoctions of cardamom and 3 batches of formula granules (relative retention time)

[0297]

[0298] 6.4 Identification of Mixed Products

[0299] 6.4.1 Comparison of counterfeit fingerprints

[0300] The preparation method and fingerprint detection method of the cardamom formula granules under "2.4" were used to study the fingerprints of three batches of Yunnan cardamom formula granules and compared with the fingerprints of the cardamom formula granules. The results are as follows: Fig.16 As shown in the figure, the fingerprints of cardamom and Yunnan cardamom formula granules are obviously different, and the common peaks are only peak 2 and peak 4, indicating that the chemical components of the two are quite different. Therefore, the established fingerprint method can well distinguish cardamom counterfeits.

[0301] 6.4.2 Comparison of fingerprints of cardamom

[0302] The preparation method of the cardamom formula granules and the fingerprint detection method were used to study the fingerprints of red cardamom, cardamom and nutmeg formula granules respectively. The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" was used to generate reference maps by the average method, and the reference fingerprints of red cardamom, nutmeg and cardamom formula granules were established and compared with the fingerprints of the cardamom formula granules. The results are as follows Figures 17-20 As shown. The results show that the fingerprints of red cardamom, cardamom, nutmeg and cardamom formula granules are obviously different. After retention time comparison analysis, the common peaks in the fingerprints of red cardamom and cardamom formula granules are only peak 1; cardamom and nutmeg and cardamom formula granules have no common peaks, indicating that the chemical components contained in red cardamom, cardamom and nutmeg are quite different. Therefore, the established fingerprint method can well distinguish cardamom and other cardamoms.

[0303] 6.4.3 Comparison of fingerprints of Zingiberaceae plants

[0304] The preparation method of cardamom formula granules and the fingerprint detection method were used to study the fingerprints of ginger, dried ginger, turmeric, zedoary turmeric, turmeric and alkyl oxyphylla formula granules from the ginger family. The "Chinese medicine chromatographic fingerprint similarity evaluation system" was used to generate reference maps according to the average method, and the reference fingerprints of the formula granules were established and compared with the fingerprints of the cardamom formula granules. The results are as follows: Figures 21 to 27 As shown. The results show that the fingerprints of the formula granules of ginger plants and the formula granules of cardamom are significantly different. After retention time comparison analysis, the common peaks in the fingerprints of dried ginger, ginger and cardamom formula granules are only peak 5; the common peaks in the fingerprints of turmeric and cardamom formula granules are only peaks 4 and 6; the common peaks in the fingerprints of zedoary and cardamom formula granules are only peaks 1 and 5; the fingerprints of Alpinia oxyphylla, turmeric and cardamom formula granules have no common peaks; this shows that the chemical components contained in the formula granules of ginger plants are quite different. Therefore, the established fingerprint method can well distinguish cardamom from other ginger plants.

[0305] Example 2

[0306] This embodiment provides a method for constructing a high-resolution mass spectrometry-based component identification of cardamom and its standard decoction, which is as follows:

[0307] 1. HPLC-MS conditions

[0308] An Agilent Poroshell 120SB-C18 (4.6 mm × 150 mm, 2.7 μm) chromatographic column was used; acetonitrile:methanol (4:1) was used as mobile phase A, 0.1% formic acid solution was used as mobile phase B, and gradient elution was performed according to the provisions of Table 12 below; the flow rate was 1.0 mL per minute; the column temperature was 35°C; the detection wavelength was 260 nm; and the injection volume was 5 μL.

[0309] Table 12 Gradient elution program

[0310]

[0311] The mass spectrometry conditions are shown in Table 13 below:

[0312] Table 13 Mass spectrometry parameters

[0313]

[0314] 2. Preparation of test solution

[0315] Take an appropriate amount of freeze-dried powder of standard decoction of cardamom, take about 0.2 g, and prepare the test solution according to the method under "2.3" of Example 1.

[0316] 3. Determination method

[0317] Accurately draw 5 μL of the test solution and inject it into the liquid chromatography-mass spectrometer for determination. Through the mass spectrometry accurate molecular weight and fragment ion comparison analysis, it is determined that peak 3 is galangin, peak 5 is pine pine, peak 8 is cardamomine, and peak 10 is alnus ketone. The total ion flow diagram and ultraviolet absorption chromatogram of the test solution are shown in Fig.28 , compound information is shown in Table 14.

[0318] Table 14 Mass spectrometry identification results of compounds in standard decoction of cardamom

[0319]

[0320]

[0321] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0322] The above-described embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solution of the present invention in detail, but it cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several modifications and improvements can be made, which all belong to the protection scope of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solution provided by the present invention are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing fingerprints of cardamom medicine, standard decoction and formula granules thereof, characterized in that: The steps include: Take galangin, pine tincture, cardamomine and alnus ketone to prepare a mixed reference solution; Prepare the test solution by taking the herbal medicine of cardamom, standard decoction or formula granules; The reference solution and the test solution are respectively detected by high performance liquid chromatography to construct fingerprints of the cardamom herb, the standard decoction and the formula granules thereof; The conditions of the high performance liquid chromatography method include: using a mixed solution of acetonitrile and methanol as mobile phase A, using a phosphoric acid aqueous solution with a volume concentration of 0.08% to 0.12% as mobile phase B, and using a gradient elution method; The procedure of the gradient elution is: From 0 to 10 min, the volume percentage of the mobile phase A increases from 28% to 32%; 10min to 20min, the volume percentage of the mobile phase A increases from 32% to 50%; 20min~28min, the volume percentage of the mobile phase A is 50%; 28min-30min, the volume percentage of the mobile phase A increases from 50% to 65%; 30min-37min, the volume percentage of the mobile phase A increases from 65% to 90%; 37min-40min, the volume percentage of the mobile phase A is 90%.

2. The construction method according to claim 1, characterized in that: In the mobile phase A, the volume ratio of acetonitrile to methanol is 4:

1.

3. The construction method according to claim 1, characterized in that: The conditions of the high performance liquid chromatography method also include at least one of the following conditions: (1) Using Agilent Poroshell 120SB-C18 column; (2) Flow rate: 0.8 mL / min to 1.2 mL / min; (3) Column temperature is 30℃~40℃; (4) The detection wavelength is 250nm~270nm; (5) The injection volume is 4 μL to 10 μL.

4. The construction method according to claim 1, characterized in that: Using 50% to 100% methanol by volume as a dissolving agent to prepare a mixed reference solution; In the mixed reference solution, the concentration of galangin is 110 μg / mL to 130 μg / mL, the concentration of pine spatholobi is 80 μg / mL to 100 μg / mL, the concentration of cardamomum is 18 μg / mL to 22 μg / mL, and the concentration of alnus ketone is 70 μg / mL to 90 μg / mL.

5. The construction method according to claim 1, characterized in that: The preparation of the test solution comprises the following steps: Extract the cardamom herb with water to obtain a residue, perform a first alcohol extraction on the residue, filter it, and take the filtrate to prepare a cardamom herb test solution; Performing a second alcohol extraction on the powder of the standard decoction of cardamom, filtering, and taking the filtrate to prepare a test solution of the standard decoction of cardamom; The powder of the cardamom formula granules is subjected to a third alcohol extraction, filtered, and the filtrate is taken to prepare a test solution of the cardamom formula granules.

6. The construction method according to claim 5, characterized in that: At least one of the following conditions is met: (1) The water extraction is a reflux extraction, and the mass ratio of the cardamom herb sample to the volume of water is 1 g: (20-30) mL; (2) the first alcohol extraction is to perform a first ultrasonic extraction on the residue using 50% to 100% methanol as an extraction solvent; (3) the second alcohol extraction is to perform a second ultrasonic extraction on the cardamom standard decoction powder using 50% to 100% methanol as an extraction solvent, and the mass ratio of the cardamom standard decoction powder to the volume ratio of the extraction solvent is 1 g: (100 to 150) mL; (4) The third alcohol extraction is to perform a third ultrasonic extraction on the cardamom formula granule powder using methanol with a volume fraction of 50% to 100% as an extraction solvent, and the mass ratio of the cardamom formula granule powder to the volume of the extraction solvent is 1g: (100 to 150) mL.

7. The construction method according to claim 6, characterized in that: The conditions of the first ultrasonic extraction, the second ultrasonic extraction and the third ultrasonic extraction are independently: The power is 200W~300W, the frequency is 35kHz~45kHz, and the extraction time is 15min~45min.

8. The construction method according to any one of claims 1 to 7, characterized in that: The fingerprint spectrum presents 10 characteristic peaks, of which 4 peaks correspond to the retention times of the corresponding reference substance peaks; the peak corresponding to the galangin reference substance peak is the S1 peak, and the relative retention times of peaks 1 to 2, peak 4 and S1 are calculated; the peak corresponding to the pyrocatechol reference substance peak is the S2 peak, and the relative retention times of peaks 6 to 7, peak 9 and S2 are calculated, and the relative retention times are within the range of ±10% of the specified values, and the specified values ​​are: peak 1 is 0.35, peak 2 is 0.68, peak 4 is 1.10, peak 6 is 1.20, peak 7 is 1.22, and peak 9 is 1.

35.

9. Application of the fingerprint obtained by the construction method according to any one of claims 1 to 8 in identifying Alpinia oxyphylla and Alpinia yunnanensis, or in identifying Alpinia oxyphylla and red cardamom, cardamom, nutmeg, or in identifying Alpinia oxyphylla and ginger, dried ginger, turmeric, zedoary zedoary, curcuma, and Alpinia oxyphylla.

10. A method for detecting the medicinal material of cardamom, standard decoction and formula granules thereof, characterized in that: The steps include: Prepare the test solution by taking the herbal medicine of cardamom, standard decoction or formula granules; The solution to be tested is detected by high performance liquid chromatography. The conditions of the high performance liquid chromatography method include: using a mixed solution of acetonitrile and methanol as mobile phase A, using a phosphoric acid aqueous solution with a volume concentration of 0.08% to 0.12% as mobile phase B, and using a gradient elution method; The procedure of the gradient elution is: From 0 to 10 min, the volume percentage of the mobile phase A increases from 28% to 32%; 10min to 20min, the volume percentage of the mobile phase A increases from 32% to 50%; 20min~28min, the volume percentage of the mobile phase A is 50%; 28min-30min, the volume percentage of the mobile phase A increases from 50% to 65%; 30min-37min, the volume percentage of the mobile phase A increases from 65% to 90%; 37min-40min, the volume percentage of the mobile phase A is 90%.

11. The detection method according to claim 10, characterized in that: In the mobile phase A, the volume ratio of acetonitrile to methanol is 4:

1.

12. The detection method according to claim 10, characterized in that: The conditions of the high performance liquid chromatography method also include at least one of the following conditions: (1) Using Agilent Poroshell 120SB-C18 column; (2) Flow rate: 0.8 mL / min to 1.2 mL / min; (3) Column temperature is 30℃~40℃; (4) The detection wavelength is 250nm~270nm; (5) The injection volume is 4 μL to 10 μL.

13. A method for constructing a high-resolution mass spectrometry-based component identification method for cardamom medicine, standard decoction and formula granules thereof, characterized in that: The steps include: Take cardamom seeds, standard decoction or formula granules to prepare the test solution; The test solution is subjected to high performance liquid chromatography-mass spectrometry analysis; The conditions for high performance liquid chromatography analysis include: using a mixed solution of acetonitrile and methanol as mobile phase A, using a formic acid aqueous solution with a volume concentration of 0.08% to 0.12% as mobile phase B, and using a gradient elution method; The procedure of the gradient elution is: From 0 to 10 min, the volume percentage of the mobile phase A increases from 28% to 32%; 10min to 20min, the volume percentage of the mobile phase A increases from 32% to 50%; 20min~28min, the volume percentage of the mobile phase A is 50%; 28min-30min, the volume percentage of the mobile phase A increases from 50% to 65%; 30min-37min, the volume percentage of the mobile phase A increases from 65% to 90%; 37min-40min, the volume percentage of the mobile phase A is 90%.

14. The construction method according to claim 13, characterized in that: In the mobile phase A, the volume ratio of acetonitrile to methanol is 4:

1.

15. The construction method according to claim 13, characterized in that: The conditions for the HPLC analysis also include at least one of the following conditions: (1) Using Agilent Poroshell 120SB-C18 column; (2) Flow rate: 0.8 mL / min to 1.2 mL / min; (3) Column temperature is 30℃~40℃; (4) The detection wavelength is 250nm~270nm; (5) The injection volume is 4 μL to 10 μL.

16. The construction method according to claim 13, characterized in that: The preparation of the test solution comprises the following steps: Extract the cardamom herb with water to obtain a residue, perform a first alcohol extraction on the residue, filter it, and take the filtrate to prepare a cardamom herb test solution; Performing a second alcohol extraction on the powder of the standard decoction of cardamom, filtering, and taking the filtrate to prepare a test solution of the standard decoction of cardamom; The powder of the cardamom formula granules is subjected to a third alcohol extraction, filtered, and the filtrate is taken to prepare a test solution of the cardamom formula granules.

17. The construction method according to claim 16, characterized in that: At least one of the following conditions is met: (1) The water extraction is a reflux extraction, and the mass ratio of the cardamom herb sample to the volume of water is 1 g: (20-30) mL; (2) the first alcohol extraction is to perform a first ultrasonic extraction on the residue using 50% to 100% methanol as an extraction solvent; (3) the second alcohol extraction is to perform a second ultrasonic extraction on the cardamom standard decoction powder using 50% to 100% methanol as an extraction solvent, and the mass ratio of the cardamom standard decoction powder to the volume ratio of the extraction solvent is 1 g: (100 to 150) mL; (4) The third alcohol extraction is to perform a third ultrasonic extraction on the cardamom formula granule powder using methanol with a volume fraction of 50% to 100% as an extraction solvent, and the mass ratio of the cardamom formula granule powder to the volume of the extraction solvent is 1g: (100 to 150) mL.

18. The construction method according to claim 17, characterized in that: The conditions of the first ultrasonic extraction, the second ultrasonic extraction and the third ultrasonic extraction are independently: The power is 200W~300W, the frequency is 35kHz~45kHz, and the extraction time is 15min~45min.

19. The method for constructing a solution according to any one of claims 13 to 18, characterized in that: The conditions for mass spectrometry analysis include: sheath gas flow rate of 30arb to 40arb; auxiliary gas flow rate of 8arb to 12arb; spray voltage of 3.5kV to 4.0kV; S-lens voltage of 45V to 55V; heating temperature of 320℃ to 380℃; capillary temperature of 320℃ to 380℃; Positive ion mode and / or negative ion mode are used; the scanning range is 120m / z to 1200m / z; and the normalized collision energy is 30eV to 40eV.

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