Construction method and application of characteristic chromatograms of Java amomum cardamomum medicinal material, standard decoction and traditional Chinese medicine formula granules

Abstract

The invention relates to a construction method and a detection method of characteristic chromatograms of a Java amomum cardamomum medicinal material, a standard decoction and a traditional Chinese medicine formula granule. According to the method, proper ultra-high performance liquid chromatography conditions are selected, protocatechuic acid, (4R, 6R)-6-hydroxypiperidone, (4R, 5R)-4-Hydroxy-5-isopropyl-2-methylaminohex-2-enone, vanillic acid and (4S, 5R)-4-Hydroxy-5-isopropyl-2-methylaminohex-2-enone are used for preparing a reference substance solution, a Java amomum cardamomum medicinal material, a standard decoction or / and formula granules are used as a test sample for detection, a characteristic spectrum is constructed, and the characteristic spectrum is applied to the detection of the Java amomum cardamomum medicinal material, the standard decoction or / and the formula granules. The characteristic chromatogram contains 9 characteristic peaks, is rich in chromatogram information, can reflect the quality of the Java amomum cardamomum medicinal material, can reflect the quality of standard Java amomum cardamomum decoction and traditional Chinese medicine formula granules, can be used for detecting the quality of the Java amomum cardamomum medicinal material, the standard Java amomum cardamomum decoction and the traditional Chinese medicine formula granules, and has a wide application prospect. The invention provides an effective method for quality detection of the Java amomum cardamomum and the modern preparation thereof, and also provides a scientific experimental basis for research on comprehensive quality evaluation of the Java amomum cardamomum and the modern preparation thereof.

Description

Technical Field

[0001] The present invention relates to the research field of quality standards of traditional Chinese medicines, and in particular to a method for constructing characteristic spectra of Javanese white cardamom medicines, standard decoctions and traditional Chinese medicine formula granules and applications thereof. Background Art

[0002] Javanese white cardamom (Amomum compactum Soland ex Maton) is a tropical medicinal plant of the ginger family, native to Java Island, Indonesia. It is a traditional and important imported southern medicine and pharmacopoeia Chinese medicine variety in my country. Its dried mature fruit is used as medicine, which has the medicinal functions of removing dampness and eliminating abdominal distension, promoting qi and warming the middle, stimulating appetite and digestion. It is mainly used to treat dampness and turbidity blocking the middle part of the body, loss of appetite, initial onset of damp-heat, chest tightness without hunger, vomiting and regurgitation due to cold and dampness, chest and abdominal distension and pain, and indigestion. It is also widely used as a food seasoning, with a large market demand, high economic value and broad development prospects.

[0003] The main chemical components of Java white cardamom are volatile oil and flavonoids, among which the main components of volatile oil are eucalyptol, p-cymene, beta-pinene, limonene, alpha-terpineol, camphor, etc. Other literatures have found that Java white cardamom also contains phenolic acid components and trace elements such as Zn, Cu, Fe, Mn, Co, Ni, Cr, and Mo. Modern pharmacological studies have confirmed that Java white cardamom has pharmacological effects such as antioxidant, anti-tumor, anti-inflammatory, and analgesic. The overall composition and content of the above chemical components are the basis for the clinical efficacy of Java white cardamom. Therefore, to ensure its clinical efficacy, it is necessary to conduct overall quality control of its active ingredients.

[0004] The 2020 edition of the Chinese Pharmacopoeia includes cardamom, which is defined as the dried mature fruit of Amomum kravanh Pierre ex Gagnep. or Amomum compactum Soland ex Maton, a plant of the ginger family. It is divided into "original cardamom" and "Indonesian white cardamom" according to its origin. However, the 2020 edition of the Chinese Pharmacopoeia only records its properties and inspection items, as well as the method of determining the volatile oil content by gas chromatography, and has not yet conducted its characteristic spectrum control.

[0005] Some other literature studies have reported on the detection of white cardamom, such as Zeng Zhi, Fu Lin, et al. Comparison of volatile oil components of white cardamom, red cardamom, cardamom and nutmeg [J]. Applied Chemistry, 2012, 29(11); Wu Huiqin, Huang Xiaolan, et al. Study on GC-MS fingerprint of volatile oil of white cardamom [J]. Chinese Medicinal Materials, 2006, 29(8). However, the above studies are all about the use of GC-MS to determine the volatile components in medicinal materials, and there are no reports on the characteristic spectrum research of white cardamom standard decoction and formula granules.

[0006] Therefore, how to provide a detection method that can not only detect Javanese white cardamom medicinal materials, but also is suitable for the detection of Javanese white cardamom standard decoctions and Chinese medicine formula granules is crucial to the quality detection of modern Javanese white cardamom preparations. Summary of the invention

[0007] To solve the above problems, the first purpose of the present invention is to provide a method for constructing a characteristic spectrum of Java white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules; the second purpose of the present invention is to provide a method for detecting Java white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules. The construction method provided by the present invention can not only reflect the quality of Java white cardamom medicinal materials, but also reflect the quality of Java white cardamom standard decoctions and Chinese medicine formula granules, and can be simultaneously applied to the quality detection of Java white cardamom medicinal materials, standard decoctions and their formula granules.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] A method for constructing characteristic spectra of Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules, the construction method comprising the following steps:

[0010] Prepare reference substance solutions by taking protocatechuic acid, (4R,6R)-6-hydroxypiperidinone, (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, vanillic acid, and (4S,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone;

[0011] Extracting a Javanese white cardamom sample with an extraction solvent to prepare a sample solution, wherein the Javanese white cardamom sample is selected from one or more of Javanese white cardamom medicinal materials, Javanese white cardamom standard decoctions, and Javanese white cardamom formula granules;

[0012] The reference substance solution and the test substance solution are detected by ultra-high performance liquid chromatography to construct a characteristic spectrum;

[0013] The conditions of the ultra-high performance liquid chromatography method include: using a mixed solution of methanol and acetonitrile as mobile phase A, using a phosphoric acid aqueous solution as mobile phase B, and performing gradient elution.

[0014] In one embodiment, the procedure of the gradient elution is:

[0015] From 0 to 10 min, the volume percentage of the mobile phase A changes from 6% to 7%;

[0016] 10min-20min, the volume percentage of the mobile phase A changes from 7% to 10%;

[0017] 20min-25min, the volume percentage of the mobile phase A changes from 10% to 13%;

[0018] 25min~42min, the volume percentage of the mobile phase A is 13%;

[0019] From 42min to 50min, the volume percentage of the mobile phase A changes from 13% to 30%;

[0020] From 50 min to 60 min, the volume percentage of the mobile phase A changes from 30% to 40%.

[0021] In one of the embodiments, in the mixed solution of methanol and acetonitrile, the volume ratio of methanol to acetonitrile is 1:1 to 1:3.

[0022] In one of the embodiments, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.15%.

[0023] In one embodiment, the conditions of the ultra-high performance liquid chromatography method also include: using a WatersCortecs T3 chromatography column.

[0024] In one embodiment, the conditions of the ultra-high performance liquid chromatography also include: detection wavelength: 0-26 min, 290 nm-310 nm; 27 min-60 min, 230 nm-250 nm.

[0025] In one embodiment, the conditions of the ultra-high performance liquid chromatography also include: a flow rate of 0.23 mL / min to 0.27 mL / min.

[0026] In one embodiment, the conditions of the ultra-high performance liquid chromatography method also include: a column temperature of 36°C to 40°C.

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

[0028] Protocatechuic acid, (4R,6R)-6-hydroxypiperidinone, (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, vanillic acid, (4S,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone and methanol with a volume fraction of 80% to 100% are mixed.

[0029] In one embodiment, in the reference substance solution, each 1 mL of the reference substance solution contains 20 μg to 30 μg of the protocatechuic acid, 20 μg to 30 μg of the (4R, 6R)-6-hydroxypiperidinone, 20 μg to 30 μg of the (4R, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, 20 μg to 30 μg of the vanillic acid, and 20 μg to 30 μg of the (4S, 5R)-4-Hy droxy-5-isopropyl-2-methylcyclohex-2-enone.

[0030] In one embodiment, the Javanese white cardamom test sample is a Javanese white cardamom medicinal material, the extraction solvent is 80% to 100% methanol by volume, the Javanese white cardamom medicinal material is crushed, sieved, mixed with 80% to 100% methanol by volume, ultrasonically extracted, filtered, and the filtrate is collected;

[0031] The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

[0032] In one embodiment, the Javanese white cardamom test sample is a Javanese white cardamom standard decoction, the extraction solvent is 80% to 100% methanol by volume, the Javanese white cardamom standard decoction is ground, mixed with 80% to 100% methanol by volume, ultrasonically extracted, filtered, and the filtrate is collected;

[0033] The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

[0034] In one embodiment, the Javanese white cardamom test sample is a Javanese white cardamom formula granule, the extraction solvent is 80% to 100% methanol by volume, the Javanese white cardamom formula granule is ground, mixed with 80% to 100% methanol by volume, ultrasonically extracted, filtered, and the filtrate is collected;

[0035] The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

[0036] In one embodiment, the characteristic spectrum of the Javanese white cardamom medicinal material, standard decoction, and formula granules includes 9 characteristic peaks, wherein peak 1 is protocatechuic acid, peak 3 is vanillic acid, peak 6 is (4R, 6R)-6-hydroxypiperidinone, peak 7 is (4R, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, and peak 9 is (4S, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone; the peak corresponding to the (4R, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone reference peak is the S peak, and the relative retention times of peaks 2, 5, and 8 with respect to the S peak are calculated, and the relative retention times are within the range of ±10% of the specified value, and the specified value is: 0.25 for peak 2, 0.80 for peak 5, and 1.06 for peak 8.

[0037] A method for detecting Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules, the detection method comprising the following steps:

[0038] Extracting the sample to be tested with an extraction solvent to prepare a solution of the sample to be tested;

[0039] The test solution is tested by ultra-high performance liquid chromatography, and the test spectrum obtained is compared with the characteristic spectrum constructed by the above construction method;

[0040] The conditions of the ultra-high performance liquid chromatography method include: using a mixed solution of methanol and acetonitrile as mobile phase A, using a phosphoric acid aqueous solution as mobile phase B, and performing gradient elution.

[0041] In one embodiment of the detection method, the procedure of the gradient elution is:

[0042] From 0 to 10 min, the volume percentage of the mobile phase A changes from 6% to 7%;

[0043] 10min-20min, the volume percentage of the mobile phase A changes from 7% to 10%;

[0044] 20min-25min, the volume percentage of the mobile phase A changes from 10% to 13%;

[0045] 25min~42min, the volume percentage of the mobile phase A is 13%;

[0046] From 42min to 50min, the volume percentage of the mobile phase A changes from 13% to 30%;

[0047] From 50 min to 60 min, the volume percentage of the mobile phase A changes from 30% to 40%.

[0048] In one embodiment of the detection method, in the mixed solution of methanol and acetonitrile, the volume ratio of methanol to acetonitrile is 1:1 to 1:3.

[0049] In one embodiment of the detection method, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.15%.

[0050] In one embodiment of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography also include: using a Waters Cortecs T3 chromatographic column.

[0051] In one embodiment of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography also include: detection wavelength: 0-26 min, 290 nm-310 nm; 27 min-60 min, 230 nm-250 nm.

[0052] In one embodiment of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a flow rate of 0.23 mL / min to 0.27 mL / min.

[0053] In one embodiment of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a column temperature of 36°C to 40°C.

[0054] In one embodiment of the detection method, the sample to be tested is Java white cardamom medicinal material, the extraction solvent is methanol with a volume fraction of 80% to 100%, the Java white cardamom medicinal material is crushed, sieved, mixed with methanol with a volume fraction of 80% to 100%, ultrasonically extracted, filtered, and the filtrate is collected;

[0055] The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

[0056] In one embodiment of the detection method, the sample to be tested is a Javanese white cardamom standard decoction, the extraction solvent is 80% to 100% methanol by volume, the Javanese white cardamom standard decoction is ground, mixed with 80% to 100% methanol by volume, ultrasonically extracted, filtered, and the filtrate is collected;

[0057] The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

[0058] In one embodiment of the detection method, the sample to be tested is Java white cardamom formula granules, the extraction solvent is methanol with a volume fraction of 80% to 100%, the Java white cardamom formula granules are ground, mixed with methanol with a volume fraction of 80% to 100%, ultrasonic extraction is performed, and filtration is performed to obtain a filtrate;

[0059] The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] The invention selects ultra-high performance liquid chromatography conditions which are simultaneously applicable to Java white cardamom medicinal materials, standard decoctions thereof and Chinese medicine formula granules, uses the medicinal materials, standard decoctions or / and Chinese medicine formula granules as test samples for detection, and constructs a characteristic spectrum. The characteristic spectrum comprises 9 characteristic peaks and is rich in spectrum information. The characteristic spectrum can not only reflect the quality of the Java white cardamom medicinal materials, but also reflect the quality of the Java white cardamom standard decoctions and Chinese medicine formula granules. The characteristic spectrum can be simultaneously used for quality detection of the Java white cardamom medicinal materials, standard decoctions thereof and Chinese medicine formula granules, provides an effective method for quality detection of the Java white cardamom and its modern preparations, and also provides a scientific experimental basis for research on comprehensive quality evaluation of the Java white cardamom and its modern preparations.

[0062] In addition, the characteristic spectrum constructed by the present invention can display the characteristic peak information of protocatechuic acid, (4R, 6R)-6-hydroxypiperidinone, (4R, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, vanillic acid, and (4S, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, which is an expansion of the prior art that only uses volatile oil components as intrinsic quality indicators, and further enriches the research on comprehensive quality evaluation of Javanese white cardamom.

[0063] The present invention applies ultra-high performance liquid chromatography to the quality inspection of Javanese white cardamom medicinal materials and their modern preparations for the first time, filling the gap in the quality inspection of Javanese white cardamom medicinal materials and their modern preparations, realizing the quality control and specificity identification of Javanese white cardamom from medicinal materials to Chinese medicine formula granules in terms of intrinsic quality, further ensuring the quality of Javanese white cardamom medicinal materials, standard decoctions, and formula granules, and being beneficial to ensuring the effectiveness of clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 This is the characteristic spectrum of Javanese white cardamom formula granules under different gradient elution conditions;

[0065] Figure 2 This is the characteristic spectrum of organic phase in different proportions of Javanese white cardamom formula granules;

[0066] Figure 3 This is the characteristic spectrum of different acids in Javanese white cardamom formula granules;

[0067] Figure 4 This is a specific property diagram of Javanese white cardamom formula granules; wherein, peak 1: protocatechuic acid, peak 3: vanillic acid, peak 6: (4R, 6R)-6-hydroxypiperidinone, peak 7: (4R, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, peak 9: (4S, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone;

[0068] Figure 5 This is the durability test diagram of the characteristic spectrum of Javanese white cardamom formula granules at different column temperatures;

[0069] Figure 6 This is the characteristic spectrum of Javanese white cardamom formula granules at different flow rates for durability investigation;

[0070] Figure 7 This is an overlay of characteristic spectra of three batches of Javanese white cardamom formula granules;

[0071] Figure 8 This is an overlay of characteristic spectra of 10 batches of Javanese white cardamom medicinal materials;

[0072] Fig. 9 This is an overlay of the characteristic spectra of 10 batches of standard decoctions of Javanese white cardamom. DETAILED DESCRIPTION

[0073] 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.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally 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 optional scope of the term "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of related listed items, and the combinations include any two related listed items, any more related listed items, or all related listed items.

[0075] In the present invention, "first aspect", "second aspect" and the like are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features.

[0076] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0077] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0078] Existing detection methods are difficult to effectively compare and analyze the differences and changes in the characteristic spectra of white cardamom medicinal materials, standard decoctions, and formula granules. Therefore, establishing a method that can be used to simultaneously determine the characteristic spectra of Java white cardamom medicinal materials, standard decoctions, and formula granules is conducive to the overall evaluation of the scientificity and rationality of the relevant process of Java white cardamom, and can better control the intrinsic quality of Java white cardamom medicinal materials, standard decoctions, and formula granules as a whole, and ensure the clinical efficacy of Java white cardamom.

[0079] The present invention provides a method for constructing a characteristic spectrum of Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules, the construction method comprising the following steps:

[0080] Prepare reference substance solutions by taking protocatechuic acid, (4R,6R)-6-hydroxypiperidinone, (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, vanillic acid, and (4S,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone;

[0081] Extracting a Javanese white cardamom sample with an extraction solvent to prepare a sample solution, wherein the Javanese white cardamom sample is selected from one or more of Javanese white cardamom medicinal materials, Javanese white cardamom standard decoctions, and Javanese white cardamom formula granules;

[0082] The reference substance solution and the test substance solution are detected by ultra-high performance liquid chromatography to construct a characteristic spectrum;

[0083] The conditions of the ultra-high performance liquid chromatography method include: using a mixed solution of methanol and acetonitrile as mobile phase A, using a phosphoric acid aqueous solution as mobile phase B, and performing gradient elution.

[0084] In one example, the gradient elution program is:

[0085] From 0 to 10 min, the volume percentage of the mobile phase A changes from 6% to 7%;

[0086] 10min-20min, the volume percentage of the mobile phase A changes from 7% to 10%;

[0087] 20min-25min, the volume percentage of the mobile phase A changes from 10% to 13%;

[0088] 25min~42min, the volume percentage of the mobile phase A is 13%;

[0089] From 42min to 50min, the volume percentage of the mobile phase A changes from 13% to 30%;

[0090] From 50 min to 60 min, the volume percentage of the mobile phase A changes from 30% to 40%.

[0091] In one example, in the mixed solution of methanol and acetonitrile, the volume ratio of methanol to acetonitrile is 1:1 to 1:3.

[0092] It can be understood that the volume ratio of methanol to acetonitrile includes but is not limited to 1:1, 1:1.5, 1:2, 1:3; preferably, the volume ratio of methanol to acetonitrile is 1:2.

[0093] In one example, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.15%.

[0094] It can be understood that in the phosphoric acid aqueous solution, the volume fraction of the phosphoric acid includes but is not limited to 0.05%, 0.08%, 0.1%, 0.12%, and 0.15%; preferably, the volume fraction of the phosphoric acid is 0.1%.

[0095] In one example, the conditions of the ultra-high performance liquid chromatography method also include: using a Waters CortecsT3 chromatography column.

[0096] Specifically, the specifications of the chromatographic column are: column length of 150 mm, inner diameter of 2.1 mm, and particle size of 1.6 μm.

[0097] In one example, the conditions of the ultra-high performance liquid chromatography also include: detection wavelength: 0-26 min, 290 nm-310 nm; 27 min-60 min, 230 nm-250 nm.

[0098] Preferably, the detection wavelength is: 0-26 min, 300 nm; 27 min-60 min, 240 nm.

[0099] In one example, the conditions of the ultra-high performance liquid chromatography also include: a flow rate of 0.23 mL / min to 0.27 mL / min.

[0100] It can be understood that the flow rate includes but is not limited to 0.23 mL / min, 0.25 mL / min, 0.27 mL / min; preferably, the flow rate is 0.25 mL / min.

[0101] In one example, the conditions of the ultra-high performance liquid chromatography also include: a column temperature of 36°C to 40°C.

[0102] It can be understood that the column temperature includes but is not limited to 36°C, 37°C, 38°C, 39°C, and 40°C; preferably, the column temperature is 38°C.

[0103] In one example, the injection volume was 1 μL to 3 μL.

[0104] In one example, the preparation of the reference substance solution comprises the following steps:

[0105] Protocatechuic acid, (4R,6R)-6-hydroxypiperidinone, (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, vanillic acid, (4S,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone and methanol with a volume fraction of 80% to 100% are mixed.

[0106] It can be understood that in the preparation of the reference substance solution, the volume fraction of methanol includes but is not limited to 80%, 90%, and 100%; preferably, the volume fraction of methanol is 100%.

[0107] In one example, in the reference substance solution, each 1 mL of the reference substance solution contains 20 μg to 30 μg of protocatechuic acid, 20 μg to 30 μg of (4R, 6R)-6-hydroxypiperidinone, 20 μg to 30 μg of (4R, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, 20 μg to 30 μg of vanillic acid, and 20 μg to 30 μg of (4S, 5R)-4-Hydr oxy-5-isopropyl-2-methylcyclohex-2-enone.

[0108] In one example, the Javanese white cardamom test sample is a Javanese white cardamom medicinal material, the extraction solvent is 80% to 100% methanol by volume, the Javanese white cardamom medicinal material is crushed, sieved, mixed with 80% to 100% methanol by volume, ultrasonically extracted, filtered, and the filtrate is collected;

[0109] The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

[0110] Preferably, the Javanese white cardamom medicinal material is crushed and sieved, which means that the Javanese white cardamom medicinal material is made into powder, and passed through a No. 3 sieve to collect medicinal material powder of about 50 mesh.

[0111] In one example, the ratio of Javanese white cardamom powder to methanol is (0.5-2.0) g:10 mL, preferably 1.0 g:10 mL.

[0112] It can be understood that the volume fraction of the methanol includes but is not limited to 80%, 90%, and 100%; preferably, the volume fraction of the methanol is 100%.

[0113] It can be understood that the filtration includes but is not limited to filtration using filter paper; the filtrate is preferably the subsequent filtrate collected after removing the initial filtrate.

[0114] It can be understood that the time for the ultrasonic extraction includes but is not limited to 25 min, 30 min, 35 min; preferably, the time is 30 min.

[0115] It can be understood that the power includes but is not limited to 250W, 280W, 300W, 320W, and 350W; preferably, the power is 300W.

[0116] It can be understood that the frequency includes but is not limited to 35kHZ, 40kHZ, 45kHZ; preferably, the frequency is 40kHZ.

[0117] In one example, the Javanese white cardamom test sample is a Javanese white cardamom standard decoction, the extraction solvent is 80% to 100% methanol by volume, the Javanese white cardamom standard decoction is ground, mixed with 80% to 100% methanol by volume, ultrasonically extracted, filtered, and the filtrate is collected;

[0118] The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

[0119] It can be understood that the Javanese white cardamom standard decoction in the present invention is in the form of freeze-dried powder.

[0120] Preferably, the grinding of the Javanese white cardamom standard decoction refers to grinding the freeze-dried powder of the Javanese white cardamom standard decoction into a powdery state.

[0121] In one example, the ratio of Javanese white cardamom standard decoction sample powder to methanol is (0.2-1.0) g:10 mL, preferably 0.5 g:10 mL.

[0122] It can be understood that the volume fraction of the methanol includes but is not limited to 80%, 90%, and 100%; preferably, the volume fraction of the methanol is 100%.

[0123] It can be understood that the filtration includes but is not limited to filtration using filter paper; the filtrate is preferably the subsequent filtrate collected after removing the initial filtrate.

[0124] It can be understood that the time for the ultrasonic extraction includes but is not limited to 25 min, 30 min, 35 min; preferably, the time is 30 min.

[0125] It can be understood that the power includes but is not limited to 250W, 280W, 300W, 320W, and 350W; preferably, the power is 300W.

[0126] It can be understood that the frequency includes but is not limited to 35kHZ, 40kHZ, 45kHZ; preferably, the frequency is 40kHZ.

[0127] In one example, the Javanese white cardamom test sample is a Javanese white cardamom formula granule, the extraction solvent is 80% to 100% methanol by volume, the Javanese white cardamom formula granule is ground, mixed with 80% to 100% methanol by volume, ultrasonically extracted, filtered, and the filtrate is collected;

[0128] The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

[0129] Preferably, grinding the Javanese white cardamom formula particles means grinding the Javanese white cardamom formula particles into powder.

[0130] In one example, the ratio of Javanese white cardamom formula granule sample powder to methanol is (0.2-1.0) g:10 mL, preferably 0.5 g:10 mL.

[0131] It can be understood that the volume fraction of the methanol includes but is not limited to 80%, 90%, and 100%; preferably, the volume fraction of the methanol is 100%.

[0132] It can be understood that the time for the ultrasonic extraction includes but is not limited to 25 min, 30 min, 35 min; preferably, the time is 30 min.

[0133] It can be understood that the power includes but is not limited to 250W, 280W, 300W, 320W, and 350W; preferably, the power is 300W.

[0134] It can be understood that the frequency includes but is not limited to 35kHZ, 40kHZ, 45kHZ; preferably, the frequency is 40kHZ.

[0135] In one example, the characteristic spectrum of the Javanese white cardamom medicinal material, standard decoction, and formula granules includes 9 characteristic peaks, among which peak 1 is protocatechuic acid, peak 3 is vanillic acid, peak 6 is (4R, 6R)-6-hydroxypiperidinone, peak 7 is (4R, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, and peak 9 is (4S, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone; the peak corresponding to the (4R, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone reference peak is the S peak, and the relative retention times of peaks 2, 5, and 8 with respect to the S peak are calculated, and the relative retention times are within ±10% of the specified values, and the specified values ​​are: 0.25 for peak 2, 0.80 for peak 5, and 1.06 for peak 8.

[0136] The Javanese white cardamom characteristic spectrum method established by the present invention through reasonable selection of ultra-high performance liquid chromatography conditions can be applicable to Javanese white cardamom medicinal materials, standard decoctions and traditional Chinese medicine formula granules. The characteristic spectrum contains 9 characteristic peaks and is rich in spectrum information. It can not only reflect the quality of Javanese white cardamom medicinal materials, but also reflect the quality of Javanese white cardamom standard decoctions and traditional Chinese medicine formula granules.

[0137] The present invention also provides a method for detecting Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules, comprising the following steps:

[0138] Extracting the sample to be tested with an extraction solvent to prepare a solution of the sample to be tested;

[0139] The test solution is tested by ultra-high performance liquid chromatography, and the test spectrum obtained is compared with the characteristic spectrum constructed by the above construction method;

[0140] The conditions of the ultra-high performance liquid chromatography method include: using a mixed solution of methanol and acetonitrile as mobile phase A, using a phosphoric acid aqueous solution as mobile phase B, and performing gradient elution.

[0141] In one example of the detection method, the gradient elution procedure is:

[0142] From 0 to 10 min, the volume percentage of the mobile phase A changes from 6% to 7%;

[0143] 10min-20min, the volume percentage of the mobile phase A changes from 7% to 10%;

[0144] 20min-25min, the volume percentage of the mobile phase A changes from 10% to 13%;

[0145] 25min~42min, the volume percentage of the mobile phase A is 13%;

[0146] From 42min to 50min, the volume percentage of the mobile phase A changes from 13% to 30%;

[0147] From 50 min to 60 min, the volume percentage of the mobile phase A changes from 30% to 40%.

[0148] In one example of the detection method, in the mixed solution of methanol and acetonitrile, the volume ratio of methanol to acetonitrile is 1:1 to 1:3.

[0149] It can be understood that the volume ratio of methanol to acetonitrile includes but is not limited to 1:1, 1:1.5, 1:2, 1:3; preferably, the volume ratio of methanol to acetonitrile is 1:2.

[0150] In one example of the detection method, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.15%.

[0151] It can be understood that the volume fraction of the phosphoric acid includes but is not limited to 0.05%, 0.08%, 0.1%, 0.12%, and 0.15%; preferably, the volume fraction of the phosphoric acid is 0.1%.

[0152] In one example of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography also include: using a Waters Cortecs T3 chromatographic column.

[0153] Specifically, the specifications of the chromatographic column are: column length of 150 mm, inner diameter of 2.1 mm, and particle size of 1.6 μm.

[0154] In one example of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography also include: detection wavelength: 0-26 min, 290 nm-310 nm; 27 min-60 min, 230 nm-250 nm.

[0155] Preferably, the detection wavelength is: 0-26 min, 300 nm; 27 min-60 min, 240 nm.

[0156] In one example of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a flow rate of 0.23 mL / min to 0.27 mL / min.

[0157] It can be understood that the flow rate includes but is not limited to 0.23 mL / min, 0.25 mL / min, 0.27 mL / min; preferably, the flow rate is 0.25 mL / min.

[0158] In one example of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a column temperature of 36°C to 40°C.

[0159] It can be understood that the column temperature includes but is not limited to 36°C, 37°C, 38°C, 39°C, and 40°C; preferably, the column temperature is 38°C.

[0160] In one example, the injection volume was 1 μL to 3 μL.

[0161] In one example of the detection method, the sample to be tested is Java white cardamom medicinal material, the extraction solvent is methanol with a volume fraction of 80% to 100%, the Java white cardamom medicinal material is crushed, sieved, mixed with methanol with a volume fraction of 80% to 100%, ultrasonically extracted, filtered, and the filtrate is collected;

[0162] The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

[0163] Preferably, the Javanese white cardamom medicinal material is crushed and sieved, which means that the Javanese white cardamom medicinal material is made into powder, and passed through a No. 3 sieve to collect medicinal material powder of about 50 mesh.

[0164] In one example of the detection method, the ratio of Javanese white cardamom powder to methanol is (0.5-2.0) g:10 mL, preferably 1.0 g:10 mL.

[0165] It can be understood that the volume fraction of the methanol includes but is not limited to 80%, 90%, and 100%; preferably, the volume fraction of the methanol is 100%.

[0166] It can be understood that the filtration includes but is not limited to filtration using filter paper; the filtrate is preferably the subsequent filtrate collected after removing the initial filtrate.

[0167] It can be understood that the time for the ultrasonic extraction includes but is not limited to 25 min, 30 min, 35 min; preferably, the time is 30 min.

[0168] It can be understood that the power includes but is not limited to 250W, 280W, 300W, 320W, and 350W; preferably, the power is 300W.

[0169] It can be understood that the frequency includes but is not limited to 35kHZ, 40kHZ, 45kHZ; preferably, the frequency is 40kHZ.

[0170] In one example of the detection method, the sample to be tested is a Javanese white cardamom standard decoction, the extraction solvent is 80% to 100% methanol by volume, the Javanese white cardamom standard decoction is ground, mixed with 80% to 100% methanol by volume, ultrasonically extracted, filtered, and the filtrate is collected;

[0171] The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

[0172] It can be understood that the Javanese white cardamom standard decoction in the present invention is in the form of freeze-dried powder.

[0173] Preferably, the grinding of the Javanese white cardamom standard decoction refers to grinding the freeze-dried powder of the Javanese white cardamom standard decoction into a powdery state.

[0174] In one example of the detection method, the ratio of Javanese white cardamom standard decoction sample powder to methanol is (0.2-1.0) g:10 mL, preferably 0.5 g:10 mL.

[0175] It is understood that the volume fraction of the methanol includes but is not limited to 80%, 90%, 100%; preferably, the volume fraction of the methanol is 100%. It is understood that the filtration includes but is not limited to filtration using filter paper; the filtrate is preferably the subsequent filtrate collected after removing the primary filtrate.

[0176] It can be understood that the time for the ultrasonic extraction includes but is not limited to 25 min, 30 min, 35 min; preferably, the time is 30 min.

[0177] It can be understood that the power includes but is not limited to 250W, 280W, 300W, 320W, and 350W; preferably, the power is 300W.

[0178] It can be understood that the frequency includes but is not limited to 35kHZ, 40kHZ, 45kHZ; preferably, the frequency is 40kHZ.

[0179] In one embodiment of the detection method, the sample to be tested is Java white cardamom formula granules, the extraction solvent is methanol with a volume fraction of 80% to 100%, the Java white cardamom formula granules are ground, mixed with methanol with a volume fraction of 80% to 100%, ultrasonic extraction is performed, and filtration is performed to obtain a filtrate;

[0180] The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

[0181] Preferably, grinding the Javanese white cardamom formula particles means grinding the Javanese white cardamom formula particles into powder.

[0182] In one example, the ratio of Javanese white cardamom formula granule sample powder to methanol is (0.2-1.0) g:10 mL, preferably 0.5 g:10 mL.

[0183] It is understood that the volume fraction of methanol includes but is not limited to 80%, 90%, 100%; preferably, the volume fraction of methanol is 100%. It is understood that the time of ultrasonic extraction includes but is not limited to 25min, 30min, 35min; preferably, the time is 30min.

[0184] It can be understood that the power includes but is not limited to 250W, 280W, 300W, 320W, and 350W; preferably, the power is 300W.

[0185] It can be understood that the frequency includes but is not limited to 35kHZ, 40kHZ, 45kHZ; preferably, the frequency is 40kHZ.

[0186] The present invention applies ultra-high performance liquid chromatography to the quality inspection of Javanese white cardamom medicinal materials and their modern preparations for the first time, filling the gap in the quality inspection of Javanese white cardamom medicinal materials and their modern preparations, realizing the quality control and specificity identification of Javanese white cardamom from medicinal materials to Chinese medicine formula granules in terms of intrinsic quality, further ensuring the quality of Javanese white cardamom medicinal materials, standard decoctions, and formula granules, and being beneficial to ensuring the effectiveness of clinical medication.

[0187] The characteristic spectrum construction method of Java white cardamom medicinal material, standard decoction and Chinese medicine formula granules of the present invention is further described in detail below in conjunction with specific examples. The raw materials used in the following examples, unless otherwise specified, are all commercially available products.

[0188] Example 1: Method for establishing UPLC characteristic spectrum of Javanese white cardamom formula granules

[0189] 1. Instruments, reagents and test drugs

[0190] Instruments: Waters H-class ultra-high performance liquid chromatograph (Waters Corporation), Thermo Vanquish ultra-high performance liquid chromatograph (Thermo Fisher Scientific Inc.), Waters Cortecs T3 (2.1 mm × 100 mm, 1.6 μm) chromatographic column, one-ten-thousandth balance (ME204E, Mettler-Toledo), one-millionth balance (XP26, Mettler-Toledo), CNC ultrasonic cleaner (KQ500D, Kunshan Ultrasonic Instrument Co., Ltd.), electric constant temperature blast drying oven (DHG-9146A, Jinghong Instrument Co., Ltd.), ultrapure water system (Milli-Q Direct, Merck & Co., Ltd.).

[0191] Reagents: Methanol (Xilong Scientific Co., Ltd.) was of analytical grade; phosphoric acid (Tianjin Komiou Chemical Reagent Co., Ltd.), methanol (Merck Co., Ltd.), and acetonitrile (Merck Co., Ltd.) were of UPLC chromatography grade; and water was ultrapure water (self-made in the laboratory).

[0192] Test drugs: protocatechuic acid (batch number: 110809-202207, content: 97.5%, China Food and Drug Inspection Institute); vanillic acid (batch number: 110776-201503, content: 99.8%, China Food and Drug Inspection Institute); (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone (source: Guangdong Yifang Pharmaceutical Co., Ltd.), (4S,5R)-4-Hydr oxy-5-isopropyl-2-methylcyclohex-2-enone (source: Guangdong Yifang Pharmaceutical Co., Ltd.), (4R,6R)-6-hydroxypiperidinone (source: Guangdong Yifang Pharmaceutical Co., Ltd.) are all homemade in the laboratory; Java white cardamom formula granules (batch numbers: KL 01, KL02, KL03; source: Guangdong Yifang Pharmaceutical Co., Ltd.).

[0193] 2. Methods and Results

[0194] 2.1 Chromatographic conditions

[0195] A Waters Cortecs T3 (150 mm × 2.1 mm, 1.6 μm) column was used; methanol-acetonitrile (1:2) 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 1 below; the flow rate was 0.25 mL per minute; the column temperature was 38°C; the detection wavelength was 300 nm from 0 to 26 min, and the detection wavelength was 240 nm from 27 to 60 min. The injection volume was 1 μL.

[0196] Table 1 Gradient elution table

[0197]

[0198] 2.2 Preparation of test solution

[0199] Take an appropriate amount of this product, grind it into powder, take about 0.5g, weigh it accurately, put it in a stoppered conical flask, add 10mL of methanol accurately, weigh it, treat it with ultrasound (power 300W, frequency 40kHz) for 30 minutes, cool it, shake it well, filter it, and take the filtrate to obtain it.

[0200] 2.3 Preparation of reference substance solution

[0201] Take appropriate amounts of protocatechuic acid reference substance, (4R,6R)-6-hydroxypiperidinone reference substance, (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone reference substance, vanillic acid reference substance, and (4S,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone reference substance, accurately weigh them, and add methanol to make a mixed solution containing 25 μg of each per 1 mL, which serves as the reference substance solution.

[0202] 2.4 Determination method

[0203] Accurately pipette 1 μL of the reference substance solution and the test substance solution respectively, inject into the liquid chromatograph, and measure to obtain the characteristic spectrum of the Javanese white cardamom formula granules.

[0204] 3. Chromatographic conditions

[0205] 3.1 Optimization of gradient conditions

[0206] Chromatographic conditions: Waters CORTECS T3 (2.1mm×150mm, 1.6μm) chromatographic column; methanol-acetonitrile (1:1) as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution according to the provisions in Tables 2 and 3 below; flow rate of 0.25mL per minute, 0-26min, detection wavelength of 300nm; 27min-60min, detection wavelength of 240nm. Injection volume of 1μl. Figure 1 .

[0207] Table 2 Gradient elution program 1

[0208]

[0209] Table 3 Gradient elution program 2

[0210]

[0211] The results showed that by comparing two different gradient conditions, it was found that the chromatogram obtained according to the "Gradient Elution Program 2" in Table 3 was more evenly distributed, the chromatographic peaks were better separated, the peak shape was good, and the baseline was stable. Therefore, the gradient elution program 2 in Table 3 was selected as the gradient elution program condition for the characteristic spectrum analysis of Javanese white cardamom formula granules.

[0212] 3.2 Optimization of organic phase ratio

[0213] In view of the change in the ratio of the organic phase, the best organic phase is selected, and the specific conditions are as follows:

[0214] Chromatographic conditions: Waters CORTECS T3 (2.1mm×150mm, 1.6μm) chromatographic column; methanol-acetonitrile (1:1), methanol-acetonitrile (1:1.5), methanol-acetonitrile (1:2), methanol-acetonitrile (1:3) as mobile phase A, 0.2% phosphoric acid solution as mobile phase B, gradient elution according to the provisions in Table 3 above; flow rate of 0.25mL per minute, 0-26min, detection wavelength of 300nm; 27min-60min, detection wavelength of 240nm. Injection volume of 1μL. Figure 2 .

[0215] The results showed that when methanol-acetonitrile (1:2) was used as mobile phase A, the chromatogram obtained was more evenly distributed, the chromatographic peaks had better separation, the peak shape was good, and the baseline was stable. Therefore, methanol-acetonitrile (1:2) was used as the mobile phase A as the analysis condition for the characteristic spectrum of Javanese white cardamom formula granules.

[0216] 3.3 Optimization of different acids

[0217] According to the changes of different acids, the optimal acid concentration is selected, and the specific conditions are as follows:

[0218] Chromatographic conditions: Waters CORTECS T3 (2.1mm×150mm, 1.6μm) chromatographic column; methanol-acetonitrile (1:2) as mobile phase A, 0.2% phosphoric acid, 0.1% phosphoric acid, and 0.1% formic acid solution as mobile phase B, respectively, and gradient elution according to the provisions in Table 3; flow rate of 0.25mL per minute, 0-26min, detection wavelength of 300nm; 27min-60min, detection wavelength of 240nm. Injection volume of 1μL. Figure 3 .

[0219] The results showed that when 0.1% phosphoric acid by volume was used as mobile phase B, the chromatogram obtained was more evenly distributed, the chromatographic peaks had better separation, the peak shape was good, and the baseline was stable. Therefore, 0.1% phosphoric acid was used as the mobile phase B as the analysis condition for the characteristic spectrum of Javanese white cardamom formula granules.

[0220] 3.4 Determination of chromatographic conditions

[0221] A Waters Cortecs T3 (150 mm × 2.1 mm, 1.6 μm) chromatographic column was used; methanol-acetonitrile (1:2) 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 of "Gradient Elution Program 2" in Table 3; the flow rate was 0.25 mL per minute; the column temperature was 38°C; the detection wavelength was 300 nm from 0 to 26 min; the detection wavelength was 240 nm from 27 to 60 min. The injection volume was 1 μL.

[0222] 4. Methodological investigation

[0223] 4.1 Specificity Investigation

[0224] Each characteristic peak in the chromatogram of the test sample has the same chromatographic peak at the corresponding retention time of the chromatogram of the reference solution, and there is no negative interference, such as Figure 4 This indicates that this method has good specificity.

[0225] 4.2 Durability test

[0226] (1) Investigation of different column temperatures

[0227] Take a sample of Javanese white cardamom formula granules (KL01) and prepare the test solution according to the test solution preparation method specified in "2.2". Except that the column temperature is 36℃, 38℃ and 40℃ respectively, other chromatographic conditions are the same as those specified in "2.1". Sample injection and analysis are performed. Peak 7 is used as the reference peak S. The relative retention time and relative peak area of ​​each characteristic peak and the S peak are calculated, and the RSD value is calculated. The results are as follows Figure 5 It was shown that the RSD values ​​of relative retention time and relative peak area were less than 5.0% at different column temperatures, indicating that the method had good durability for different column temperatures.

[0228] (2) Investigation of different flow rates

[0229] Take a sample of Javanese white cardamom formula granules (KL01) and prepare the test solution according to the test solution preparation method determined in "2.2". Except that the flow rates are 0.23mL / min, 0.25mL / min and 0.27mL / min respectively, other chromatographic conditions are the same as those specified in "2.1". Sample injection analysis is performed, and peak 7 is used as the reference peak S. The relative retention time and relative peak area of ​​each characteristic peak and peak S are calculated, and the RSD value is calculated. The results are as follows: Figure 6 It was shown that the RSD values ​​of relative retention time and relative peak area were less than 3.0% at different flow rates, indicating that the method had good durability for different flow rates.

[0230] 4.3 Identification of characteristic peaks

[0231] Take Javanese white cardamom formula granules, prepare the test solution according to the test solution preparation method determined in "2.2", and according to the chromatographic conditions in "2.1", respectively take the test solution and the reference solution for analysis, compare the retention time and spectrum of the characteristic peaks, and determine that peak 1 is protocatechuic acid, peak 3 is vanillic acid, peak 6 is (4R, 6R)-6-hydroxypiperidinone, peak 7 is (4R, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, and peak 9 is (4S, 5R)-4-Hydroxy-5-isopr opyl-2-methylcyclohex-2-enone.

[0232] 4.4 Determination of formula granule samples

[0233] Take 3 batches of Javanese white cardamom formula granules (batch numbers: KL01, KL02, KL03), prepare the test solution according to the test solution preparation method determined in "2.2", accurately pipette the above test solution and Javanese white cardamom reference medicinal material reference solution, respectively, and inject and measure according to the chromatographic conditions in "2.1". The characteristic spectrum overlay of the 3 batches of samples is shown in Figure 7 According to the characteristic spectrum, 9 common peaks were identified. Taking peak 7 (4R, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone as the reference peak S, the relative retention time and relative peak area of ​​each characteristic peak and the S peak were calculated and shown in Tables 4 and 5.

[0234] Table 4 Characteristic spectra of three batches of Javanese white cardamom formula granules (relative retention time)

[0235]

[0236] Table 5 Characteristic spectra (relative peak areas) of three batches of Javanese white cardamom formula granules

[0237]

[0238] According to Tables 4 and 5, 9 peaks can be detected in all three batches of Javanese white cardamom formula granules.

[0239] Example 2 Determination of the characteristic spectrum of Javanese white cardamom medicinal materials and standard decoctions

[0240] (1) Preparation method of test sample:

[0241] Javanese white cardamom medicinal material: Take about 1.0g of the powder of this product (passed through No. 3 sieve), weigh it accurately, put it in a stoppered conical flask, add 10ml of methanol accurately, weigh it, ultrasonically treat it (power 300W, frequency 40kHz) for 30 minutes, cool it, shake it well, filter it, and take the filtrate to obtain it.

[0242] Standard decoction of Javanese white cardamom: Take an appropriate amount of this product, grind it into powder, take about 0.5g, weigh it accurately, put it in a stoppered conical flask, add 10ml of methanol accurately, weigh it, ultrasonically treat it (power 300W, frequency 40kHz) for 30 minutes, cool it, shake it well, filter it, and take the filtrate to obtain it.

[0243] Take 10 batches of Java white cardamom medicinal material powder (passed through No. 3 sieve) and corresponding 10 batches of Java white cardamom standard decoction powder, prepare test solution according to the above method, accurately pipette the above test solution, and refer to the chromatographic conditions under "2.1" in Example 1 for sample injection and determination. The characteristic spectrum superposition diagram of 10 batches of Java white cardamom medicinal materials is as follows Figure 8 As shown in the figure, the characteristic spectrum overlay of 10 batches of Javanese white cardamom standard decoction is as follows Fig. 9 As shown in Figure 1, according to the characteristic spectrum, the Javanese white cardamom medicinal material chromatogram and the Javanese white cardamom standard decoction chromatogram both have 9 common peaks. Taking peak 7 as the reference peak S, the relative retention time, relative peak area and RSD value of each characteristic peak and peak S were calculated, and the calculation results are shown in Tables 6 to 9.

[0244] Table 6 Characteristic spectra of 10 batches of Javanese white cardamom (relative retention time)

[0245]

[0246] Table 7 Characteristic spectra of 10 batches of Javanese white cardamom medicinal materials (relative peak area)

[0247]

[0248] Table 8 Characteristic spectra of 10 batches of Javanese white cardamom standard decoctions (relative retention time)

[0249]

[0250] Table 9 Characteristic spectra of 10 batches of Javanese white cardamom standard decoction (relative peak area)

[0251]

[0252] According to Tables 6 to 9, it can be seen that 9 characteristic peaks can be detected in different batches of Javanese white cardamom medicinal materials and their standard decoctions.

[0253] The results of the above examples show that the established method is stable and reliable with good reproducibility. The method constructed by the present invention can be simultaneously applied to the determination of Javanese white cardamom medicinal materials, standard decoctions and formula granules thereof.

[0254] The characteristic spectrum of Java white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules constructed by the present invention through reasonable control of ultra-high performance liquid chromatography conditions can be used for quality detection of Java white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules, which fills the gap in quality detection of Java white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules, provides an effective method for identification of modern Java white cardamom preparations, and also provides a scientific experimental basis for research on comprehensive quality evaluation of Java white cardamom medicinal materials, standard decoctions and formula granules, further ensures the quality of Java white cardamom medicinal materials, standard decoctions and formula granules, and is conducive to ensuring the effectiveness of clinical medication.

[0255] The invention establishes a simple, reliable and practical detection method for Javanese white cardamom medicinal materials, standard decoctions and formula granules. The established ultra-high performance liquid phase method is simple and efficient, and the results are objective, precise and reliable.

[0256] 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.

[0257] The above-described embodiments only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in detail, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, 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 solutions 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 contents of the attached claims, and the description and drawings can be used to interpret the contents of the claims.

Claims

1. A method for constructing characteristic spectra of Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules, characterized in that: The construction method comprises the following steps: Prepare reference substance solutions by taking protocatechuic acid, (4R,6R)-6-hydroxypiperidinone, (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, vanillic acid, and (4S,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone; Extracting a Javanese white cardamom sample with an extraction solvent to prepare a sample solution, wherein the Javanese white cardamom sample is selected from one or more of Javanese white cardamom medicinal materials, Javanese white cardamom standard decoctions, and Javanese white cardamom formula granules; The reference substance solution and the test substance solution are detected by ultra-high performance liquid chromatography to construct a characteristic spectrum; The conditions of the ultra-high performance liquid chromatography method include: using a mixed solution of methanol and acetonitrile as mobile phase A, using a phosphoric acid aqueous solution as mobile phase B, and performing gradient elution.

2. The method for constructing characteristic spectra of Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules according to claim 1, characterized in that: The procedure of the gradient elution is: From 0 to 10 min, the volume percentage of the mobile phase A changes from 6% to 7%; 10min-20min, the volume percentage of the mobile phase A changes from 7% to 10%; 20min-25min, the volume percentage of the mobile phase A changes from 10% to 13%; 25min~42min, the volume percentage of the mobile phase A is 13%; From 42min to 50min, the volume percentage of the mobile phase A changes from 13% to 30%; From 50 min to 60 min, the volume percentage of the mobile phase A changes from 30% to 40%.

3. The method for constructing characteristic spectra of Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules according to claim 1, characterized in that: In the mixed solution of methanol and acetonitrile, the volume ratio of methanol to acetonitrile is 1:1 to 1:

3.

4. The method for constructing characteristic spectra of Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules according to claim 1, characterized in that: In the phosphoric acid aqueous solution, the volume fraction of phosphoric acid is 0.05% to 0.15%.

5. The method for constructing the characteristic spectrum of Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules according to any one of claims 1 to 4, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography satisfy at least one of the following (1) to (4): (1) Using Waters Cortecs T3 chromatographic column; (2) Detection wavelength: 0-26 min, 290 nm-310 nm; 27 min-60 min, 230 nm-250 nm; (3) Flow rate: 0.23 mL / min to 0.27 mL / min; (4) The column temperature is 36℃~40℃.

6. The method for constructing the characteristic spectrum of Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules according to any one of claims 1 to 4, characterized in that: The preparation of the reference substance solution comprises the following steps: Protocatechuic acid, (4R,6R)-6-hydroxypiperidinone, (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, vanillic acid, (4S,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone and methanol with a volume fraction of 80% to 100% are mixed.

7. The method for constructing characteristic spectra of Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules according to claim 6, characterized in that: In the reference substance solution, each 1 mL of the reference substance solution contains 20 μg to 30 μg of protocatechuic acid, 20 μg to 30 μg of (4R, 6R)-6-hydroxypiperidinone, 20 μg to 30 μg of (4R, 5R)-4-Hydroxy-5-isopr opyl-2-methylcyclohex-2-enone, 20 μg to 30 μg of vanillic acid, and 20 μg to 30 μg of (4S, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone.

8. The method for constructing characteristic spectra of Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules according to any one of claims 1 to 4, characterized in that: The Javanese white cardamom test sample is a Javanese white cardamom medicinal material, and the extraction solvent is 80% to 100% methanol by volume. The Javanese white cardamom medicinal material is crushed, sieved, mixed with 80% to 100% methanol by volume, subjected to ultrasonic extraction, filtered, and a filtrate is collected; The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

9. The method for constructing characteristic spectra of Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules according to any one of claims 1 to 4, characterized in that: The Javanese white cardamom test sample is a Javanese white cardamom standard decoction, and the extraction solvent is 80% to 100% methanol by volume. The Javanese white cardamom standard decoction is ground, mixed with 80% to 100% methanol by volume, subjected to ultrasonic extraction, filtered, and a filtrate is obtained; The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

10. The method for constructing characteristic spectra of Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules according to any one of claims 1 to 4, characterized in that: The Javanese white cardamom test sample is a Javanese white cardamom formula granule, and the extraction solvent is 80% to 100% methanol by volume. The Javanese white cardamom formula granule is ground, mixed with 80% to 100% methanol by volume, subjected to ultrasonic extraction, filtered, and a filtrate is collected; The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

11. The method for constructing characteristic spectra of Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules according to any one of claims 1 to 4, characterized in that: The characteristic spectrum of the Javanese white cardamom medicinal material, standard decoction, and formula granules includes 9 characteristic peaks, wherein peak 1 is protocatechuic acid, peak 3 is vanillic acid, peak 6 is (4R, 6R)-6-hydroxypiperidinone, peak 7 is (4R, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, and peak 9 is (4S, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone; the peak corresponding to the (4R, 5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone reference peak is the S peak, and the relative retention times of peaks 2, 5, and 8 with respect to the S peak are calculated, and the relative retention times are within the range of ±10% of the specified values, and the specified values ​​are: 0.25 for peak 2, 0.80 for peak 5, and 1.06 for peak 8.

12. A method for detecting Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules, characterized in that: The detection method comprises the following steps: Extracting the sample to be tested with an extraction solvent to prepare a solution of the sample to be tested; The test solution is tested by ultra-high performance liquid chromatography, and the test spectrum obtained is compared with the characteristic spectrum constructed by the construction method according to any one of claims 1 to 11; The conditions of the ultra-high performance liquid chromatography method include: using a mixed solution of methanol and acetonitrile as mobile phase A, using a phosphoric acid aqueous solution as mobile phase B, and performing gradient elution.

13. The method for detecting Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules according to claim 12, characterized in that: The procedure of the gradient elution is: From 0 to 10 min, the volume percentage of the mobile phase A changes from 6% to 7%; 10min-20min, the volume percentage of the mobile phase A changes from 7% to 10%; 20min-25min, the volume percentage of the mobile phase A changes from 10% to 13%; 25min~42min, the volume percentage of the mobile phase A is 13%; From 42min to 50min, the volume percentage of the mobile phase A changes from 13% to 30%; From 50 min to 60 min, the volume percentage of the mobile phase A changes from 30% to 40%.

14. The method for detecting Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules according to claim 12, characterized in that: In the mixed solution of methanol and acetonitrile, the volume ratio of methanol to acetonitrile is 1:1 to 1:

3.

15. The method for detecting Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules according to claim 12, characterized in that: In the phosphoric acid aqueous solution, the volume fraction of phosphoric acid is 0.05% to 0.15%.

16. The method for detecting Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine granules according to any one of claims 12 to 15, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography satisfy at least one of the following (1) to (4): (1) Using Waters Cortecs T3 chromatographic column; (2) Detection wavelength: 0-26 min, 290 nm-310 nm; 27 min-60 min, 230 nm-250 nm; (3) Flow rate: 0.23 mL / min to 0.27 mL / min; (4) The column temperature is 36℃~40℃.

17. The method for detecting Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine granules according to any one of claims 12 to 15, characterized in that: The sample to be tested is Java white cardamom medicinal material, and the extraction solvent is methanol with a volume fraction of 80% to 100%. The Java white cardamom medicinal material is crushed, sieved, mixed with methanol with a volume fraction of 80% to 100%, ultrasonically extracted, filtered, and the filtrate is collected; The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

18. The method for detecting Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine formula granules according to any one of claims 12 to 15, characterized in that: The sample to be tested is a Javanese white cardamom standard decoction, and the extraction solvent is 80% to 100% methanol by volume. The Javanese white cardamom standard decoction is ground, mixed with 80% to 100% methanol by volume, subjected to ultrasonic extraction, filtered, and a filtrate is collected; The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

19. The method for detecting Javanese white cardamom medicinal materials, standard decoctions and Chinese medicine granules according to any one of claims 12 to 15, characterized in that: The sample to be tested is Java white cardamom formula granules, and the extraction solvent is methanol with a volume fraction of 80% to 100%. The Java white cardamom formula granules are ground, mixed with methanol with a volume fraction of 80% to 100%, ultrasonically extracted, filtered, and the filtrate is collected; The ultrasonic extraction time is 25 min to 35 min, the power is 250 W to 350 W, and the frequency is 35 kHz to 45 kHz.

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