Construction Method and Application of Characteristic Maps of Javanese White Cardamom, Standard Decoction, and Traditional Chinese Medicine Granules

Abstract

This invention relates to a method for constructing and detecting characteristic spectra of Java white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules. This invention selects suitable ultra-high performance liquid chromatography (UHPLC) conditions to prepare reference solutions using protocatechuic acid, (4R,6R)-6-hydroxypiperone, (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, vanillic acid, and (4S,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone. Java cardamom, standard decoctions, and / or formulation granules are used as test samples for detection. A characteristic chromatogram is constructed, containing nine characteristic peaks and rich spectral information. This chromatogram reflects not only the quality of Java cardamom but also the quality of Java cardamom standard decoctions and traditional Chinese medicine formulation granules. It can be used simultaneously for the quality detection of Java cardamom, its standard decoctions, and traditional Chinese medicine formulation granules, providing an effective method for the quality detection of Java cardamom and its modern preparations. It also provides a scientific experimental basis for the comprehensive quality evaluation of Java cardamom and its modern preparations.

Description

Technical Field

[0001] This invention relates to the field of research on quality standards for Chinese medicinal materials, specifically to a method for constructing characteristic spectra of Java white cardamom, standard decoctions, and Chinese medicine formula granules, and their application. Background Technology

[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 Chinese medicinal herb and a variety listed in the Chinese Pharmacopoeia. Its dried, mature fruit is used medicinally and has functions such as resolving dampness and stagnation, regulating qi and warming the middle jiao, and stimulating appetite and digestion. It is mainly used to treat symptoms such as dampness and turbidity obstructing the middle jiao, loss of appetite, early stage of damp-heat syndrome, chest tightness and lack of hunger, cold-dampness vomiting, chest and abdominal distension and pain, and indigestion. It is also widely used as a food flavoring agent. It has a large market demand, high economic value, and broad development prospects.

[0003] Javanese white cardamom's main chemical components include volatile oils and flavonoids. The volatile oils primarily contain eucalyptol, p-cymene, β-pinene, limonene, alpha-terpineol, and camphor. Other literature has found that Javanese white cardamom also contains phenolic acids and trace elements such as Zn, Cu, Fe, Mn, Co, Ni, Cr, and Mo. Modern pharmacological studies have confirmed that Javanese white cardamom possesses antioxidant, antitumor, anti-inflammatory, and analgesic effects. The overall composition and content of these chemical components are fundamental to the clinical efficacy of Javanese white cardamom; therefore, to ensure its clinical efficacy, comprehensive quality control of its active ingredients is necessary.

[0004] The 2020 edition of the Chinese Pharmacopoeia includes cardamom in Part I, defining it as the dried, mature fruit of *Amomum kravanh Pierre ex Gagnep.* or *Amomum compactum Soland ex Maton*, belonging to the ginger family. It is classified as "original cardamom" and "Indonesian cardamom" depending on the place of origin. However, the 2020 edition only records its properties and testing items, as well as the method for determining the volatile oil content using gas chromatography; it does not yet provide characteristic chromatographic control.

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

[0006] Therefore, providing a detection method that can detect not only Java white cardamom but also standard decoctions and granules of Java white cardamom is crucial for the quality testing of modern Java white cardamom preparations. Summary of the Invention

[0007] To address the aforementioned problems, the first objective of this invention is to provide a method for constructing characteristic chromatograms of Java white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules; the second objective of this invention is to provide a method for detecting Java white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules. The construction method provided by this invention can reflect not only the quality of Java white cardamom medicinal material, but also the quality of Java white cardamom standard decoction and traditional Chinese medicine formula granules, and can be simultaneously applied to the quality detection of Java white cardamom medicinal material, standard decoction, and their formula granules.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] A method for constructing characteristic spectra of Java white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules, the method comprising the following steps:

[0010] A reference solution of protocatechuic acid, (4R,6R)-6-hydroxypiperone, (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, vanillic acid, and (4S,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone was prepared.

[0011] The test sample solution is prepared by extracting the Java white cardamom sample with an extraction solvent. The Java white cardamom sample is selected from one or more of the following: Java white cardamom medicinal material, Java white cardamom standard decoction, and Java white cardamom formula granules.

[0012] The reference solution and the test solution were detected by ultra-high performance liquid chromatography (UHPLC) to construct characteristic chromatograms.

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

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

[0015] From 0 to 10 minutes, the volume percentage of the mobile phase A changed from 6% to 7%.

[0016] Over a period of 10 to 20 minutes, the volume percentage of the mobile phase A changed from 7% to 10%.

[0017] Over 20 to 25 minutes, the volume percentage of the mobile phase A changed from 10% to 13%.

[0018] During the period of 25 min to 42 min, the volume percentage of the mobile phase A was 13%.

[0019] Over a period of 42 to 50 minutes, the volume percentage of the mobile phase A changed from 13% to 30%.

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

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

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

[0023] In one embodiment, the conditions for the ultra-high performance liquid chromatography further include the use of a WatersCortecs T3 column.

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

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

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

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

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

[0029] In one embodiment, each 1 mL of the reference solution contains 20 μg to 30 μg of protocatechuic acid, 20 μg to 30 μg of (4R,6R)-6-hydroxypiperone, 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-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone.

[0030] In one embodiment, the Java white cardamom test sample 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 pulverized, sieved, mixed with methanol with a volume fraction of 80% to 100%, 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 Java white cardamom test sample is a standard decoction of Java white cardamom, and the extraction solvent is methanol with a volume fraction of 80% to 100%. The standard decoction of Java white cardamom is ground, mixed with methanol with a volume fraction of 80% to 100%, and subjected to ultrasonic extraction, 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 Java white cardamom sample 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%, and subjected to ultrasonic extraction, 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 chromatograms of the Javanese white cardamom medicinal material, standard decoction, and formulation granules include nine characteristic peaks, wherein peak 1 is protocatechuic acid, peak 3 is vanillic acid, peak 6 is (4R,6R)-6-hydroxypiperone, 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 reference peak of (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone is the S peak. The relative retention times of peaks 2, 5, and 8 with peak S are calculated, and their relative retention times are within ±10% of a specified value, wherein the specified values ​​are: 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 material, standard decoction, and traditional Chinese medicine granules, the method comprising the following steps:

[0038] The sample to be tested is extracted using an extraction solvent to prepare a test solution;

[0039] The test solution was detected by ultra-high performance liquid chromatography, and the detected chromatogram was compared with the characteristic chromatogram constructed by the above construction method.

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

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

[0042] From 0 to 10 minutes, the volume percentage of the mobile phase A changed from 6% to 7%.

[0043] Over a period of 10 to 20 minutes, the volume percentage of the mobile phase A changed from 7% to 10%.

[0044] Over 20 to 25 minutes, the volume percentage of the mobile phase A changed from 10% to 13%.

[0045] During the period of 25 min to 42 min, the volume percentage of the mobile phase A was 13%.

[0046] Over a period of 42 to 50 minutes, the volume percentage of the mobile phase A changed from 13% to 30%.

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

[0048] In one embodiment of the detection method, the volume ratio of methanol to acetonitrile in the mixed solution of methanol and 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 further include: using a Waters Cortecs T3 column.

[0051] In one embodiment of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography further 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 further 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 further 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, and the extraction solvent is methanol with a volume fraction of 80% to 100%. The Java white cardamom is pulverized, 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 standard decoction of Java white cardamom, and the extraction solvent is methanol with a volume fraction of 80% to 100%. The standard decoction of Java white cardamom is ground, mixed with methanol with a volume fraction of 80% to 100%, 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%, ultrasonically extracted, filtered, and the filtrate is collected.

[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] This invention selects ultra-high performance liquid chromatography (UHPLC) conditions suitable for both Javanese white cardamom medicinal material and its standard decoction and traditional Chinese medicine formula granules. Using the medicinal material, standard decoction, and / or traditional Chinese medicine formula granules as test samples, a characteristic chromatogram is constructed. This chromatogram contains nine characteristic peaks and is rich in information, reflecting not only the quality of Javanese white cardamom medicinal material but also the quality of Javanese white cardamom standard decoction and traditional Chinese medicine formula granules. It can be used simultaneously for the quality detection of Javanese white cardamom medicinal material, its standard decoction, and traditional Chinese medicine formula granules, providing an effective method for the quality detection of Javanese white cardamom and its modern preparations. It also provides a scientific experimental basis for the comprehensive quality evaluation of Javanese white cardamom and its modern preparations.

[0062] Furthermore, the characteristic spectrum constructed in this invention can display characteristic peak information of protocatechuic acid, (4R,6R)-6-hydroxypiperone, (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, vanillic acid, and (4S,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone. This expands upon the existing technology that only uses volatile oil components as intrinsic quality indicators, further enriching the research on the comprehensive quality evaluation of Java white cardamom.

[0063] This invention is the first to apply ultra-high performance liquid chromatography to the quality testing of Java white cardamom and its modern preparations, filling a gap in the quality testing of Java white cardamom and its modern preparations. It achieves quality control and specific identification of Java white cardamom from raw materials to traditional Chinese medicine formula granules from the perspective of intrinsic quality, further ensuring the quality of Java white cardamom herbal materials, standard decoctions, and formula granules, which is conducive to ensuring the effectiveness of clinical medication. Attached Figure Description

[0064] Figure 1 Characteristic spectra of different gradient elution conditions for Java white cardamom formulation granules;

[0065] Figure 2 Characteristic spectra of organic phases in different proportions of Java white cardamom formulation granules;

[0066] Figure 3 Characteristic spectra of different acids in Java white cardamom formulation granules;

[0067] Figure 4 This is a specificity diagram for Java white cardamom granules; among them, peak 1: protocatechuic acid, peak 3: vanillic acid, peak 6: (4R,6R)-6-hydroxypiperone, peak 7: (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, and peak 9: (4S,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone;

[0068] Figure 5 A graph showing the characteristic granules of Java white cardamom formulation and its durability at different column temperatures;

[0069] Figure 6 A graph showing the characteristic granules of Java white cardamom formulation and its durability at different flow rates;

[0070] Figure 7 Overlay of characteristic chromatograms of three batches of Java white cardamom granules;

[0071] Figure 8 This is a superimposed image of the characteristic atlases of 10 batches of Java white cardamom medicinal materials;

[0072] Figure 9 This is a superimposed image of the characteristic spectra of 10 batches of standard decoction of Java white cardamom. Detailed Implementation

[0073] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The optional range of the term "and / or" as used herein includes any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all related listed items.

[0075] In this invention, terms such as "first aspect" and "second aspect" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features.

[0076] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0077] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows temperature fluctuations within the precision range controlled by the instrument.

[0078] Existing detection methods are insufficient for effectively comparing and analyzing the differences and changes in the characteristic spectra of Java white cardamom raw materials, standard decoctions, and formulated granules. Therefore, establishing a method that can be applied simultaneously to determine the characteristic spectra of Java white cardamom raw materials, standard decoctions, and formulated granules is beneficial for comprehensively evaluating the scientific validity and rationality of related processes, and can better control the intrinsic quality of Java white cardamom raw materials, standard decoctions, and formulated granules, thus ensuring the clinical efficacy of Java white cardamom.

[0079] This invention provides a method for constructing characteristic spectra of Java white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules. The construction method includes the following steps:

[0080] A reference solution of protocatechuic acid, (4R,6R)-6-hydroxypiperone, (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, vanillic acid, and (4S,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone was prepared.

[0081] The test sample solution is prepared by extracting the Java white cardamom sample with an extraction solvent. The Java white cardamom sample is selected from one or more of the following: Java white cardamom medicinal material, Java white cardamom standard decoction, and Java white cardamom formula granules.

[0082] The reference solution and the test solution were detected by ultra-high performance liquid chromatography (UHPLC) to construct characteristic chromatograms.

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

[0084] In one example, the gradient elution procedure is as follows:

[0085] From 0 to 10 minutes, the volume percentage of the mobile phase A changed from 6% to 7%.

[0086] Over a period of 10 to 20 minutes, the volume percentage of the mobile phase A changed from 7% to 10%.

[0087] Over 20 to 25 minutes, the volume percentage of the mobile phase A changed from 10% to 13%.

[0088] During the period of 25 min to 42 min, the volume percentage of the mobile phase A was 13%.

[0089] Over a period of 42 to 50 minutes, the volume percentage of the mobile phase A changed from 13% to 30%.

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

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

[0092] Understandably, the volume ratio of methanol to acetonitrile includes, but is not limited to, 1:1, 1:1.5, 1:2, and 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 aqueous phosphoric acid solution is 0.05% to 0.15%.

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

[0095] In one example, the conditions for the ultra-high performance liquid chromatography also include the use of a Waters Cortecs T3 column.

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

[0097] In one example, the conditions for 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 for the ultra-high performance liquid chromatography also include a flow rate of 0.23 mL / min to 0.27 mL / min.

[0100] Understandably, the flow rate includes, but is not limited to, 0.23 mL / min, 0.25 mL / min, and 0.27 mL / min; preferably, the flow rate is 0.25 mL / min.

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

[0102] Understandably, 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 solution includes the following steps:

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

[0106] Understandably, in the preparation of the reference 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, each 1 mL of the reference solution contains 20 μg to 30 μg of protocatechuic acid, 20 μg to 30 μg of (4R,6R)-6-hydroxypiperone, 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-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone.

[0108] In one example, the Java white cardamom test sample 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 pulverized, sieved, mixed with methanol with a volume fraction of 80% to 100%, 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 pulverization and sieving of Java white cardamom refers to making Java white cardamom into powder and passing it through a No. 3 sieve to collect approximately 50-mesh powder.

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

[0112] Understandably, the volume fraction of methanol includes, but is not limited to, 80%, 90%, and 100%; preferably, the volume fraction of methanol is 100%.

[0113] Understandably, the filtration includes, but is not limited to, filtration using filter paper; the filtrate is preferably the subsequent filtrate collected after the initial filtrate has been removed.

[0114] Understandably, the ultrasonic extraction time includes, but is not limited to, 25 min, 30 min, and 35 min; preferably, the time is 30 min.

[0115] Understandably, the power includes, but is not limited to, 250W, 280W, 300W, 320W, and 350W; preferably, the power is 300W.

[0116] Understandably, the frequency includes, but is not limited to, 35 kHz, 40 kHz, and 45 kHz; preferably, the frequency is 40 kHz.

[0117] In one example, the Java white cardamom test sample is a standard decoction of Java white cardamom, and the extraction solvent is methanol with a volume fraction of 80% to 100%. The standard decoction of Java white cardamom is ground, mixed with methanol with a volume fraction of 80% to 100%, 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] Understandably, the Javanese white cardamom standard decoction described in this invention is in freeze-dried powder form.

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

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

[0122] Understandably, the volume fraction of methanol includes, but is not limited to, 80%, 90%, and 100%; preferably, the volume fraction of methanol is 100%.

[0123] Understandably, the filtration includes, but is not limited to, filtration using filter paper; the filtrate is preferably the subsequent filtrate collected after the initial filtrate has been removed.

[0124] Understandably, the ultrasonic extraction time includes, but is not limited to, 25 min, 30 min, and 35 min; preferably, the time is 30 min.

[0125] Understandably, the power includes, but is not limited to, 250W, 280W, 300W, 320W, and 350W; preferably, the power is 300W.

[0126] Understandably, the frequency includes, but is not limited to, 35 kHz, 40 kHz, and 45 kHz; preferably, the frequency is 40 kHz.

[0127] In one example, the Java white cardamom sample 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.

[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 Java white cardamom formula granules means grinding the Java white cardamom formula granules into a fine powder.

[0130] In one example, the ratio of Java white cardamom granules to methanol was (0.2–1.0) g:10 mL, preferably 0.5 g:10 mL.

[0131] Understandably, the volume fraction of methanol includes, but is not limited to, 80%, 90%, and 100%; preferably, the volume fraction of methanol is 100%.

[0132] Understandably, the ultrasonic extraction time includes, but is not limited to, 25 min, 30 min, and 35 min; preferably, the time is 30 min.

[0133] Understandably, the power includes, but is not limited to, 250W, 280W, 300W, 320W, and 350W; preferably, the power is 300W.

[0134] Understandably, the frequency includes, but is not limited to, 35 kHz, 40 kHz, and 45 kHz; preferably, the frequency is 40 kHz.

[0135] In one example, the characteristic chromatograms of the Javanese cardamom medicinal material, standard decoction, and formula granules include nine characteristic peaks, where peak 1 is protocatechuic acid, peak 3 is vanillic acid, peak 6 is (4R,6R)-6-hydroxypiperone, 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 reference peak of (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone is the S peak. The relative retention times of peaks 2, 5, and 8 with peak S are calculated, and their relative retention times are within ±10% of a specified value, which is: 0.25 for peak 2, 0.80 for peak 5, and 1.06 for peak 8.

[0136] The present invention establishes a characteristic chromatogram method for Java white cardamom by rationally selecting ultra-high performance liquid chromatography conditions. This method is applicable to Java white cardamom medicinal materials, standard decoctions, and traditional Chinese medicine granules. The characteristic chromatogram contains nine characteristic peaks and is rich in chromatographic information. It can reflect not only the quality of Java white cardamom medicinal materials, but also the quality of Java white cardamom standard decoctions and traditional Chinese medicine granules.

[0137] This invention also provides a method for detecting Javanese white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules, comprising the following steps:

[0138] The sample to be tested is extracted using an extraction solvent to prepare a test solution;

[0139] The test solution was detected by ultra-high performance liquid chromatography, and the detected chromatogram was compared with the characteristic chromatogram constructed by the above construction method.

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

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

[0142] From 0 to 10 minutes, the volume percentage of the mobile phase A changed from 6% to 7%.

[0143] Over a period of 10 to 20 minutes, the volume percentage of the mobile phase A changed from 7% to 10%.

[0144] Over 20 to 25 minutes, the volume percentage of the mobile phase A changed from 10% to 13%.

[0145] During the period of 25 min to 42 min, the volume percentage of the mobile phase A was 13%.

[0146] Over a period of 42 to 50 minutes, the volume percentage of the mobile phase A changed from 13% to 30%.

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

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

[0149] Understandably, the volume ratio of methanol to acetonitrile includes, but is not limited to, 1:1, 1:1.5, 1:2, and 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] Understandably, 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 the use of a Waters Cortecs T3 column.

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

[0154] In one example of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography further 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 further include a flow rate of 0.23 mL / min to 0.27 mL / min.

[0157] Understandably, the flow rate includes, but is not limited to, 0.23 mL / min, 0.25 mL / min, and 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] Understandably, 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, and the extraction solvent is methanol with a volume fraction of 80% to 100%. The Java white cardamom is pulverized, 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 pulverization and sieving of Java white cardamom refers to making Java white cardamom into powder and passing it through a No. 3 sieve to collect approximately 50-mesh powder.

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

[0165] Understandably, the volume fraction of methanol includes, but is not limited to, 80%, 90%, and 100%; preferably, the volume fraction of methanol is 100%.

[0166] Understandably, the filtration includes, but is not limited to, filtration using filter paper; the filtrate is preferably the subsequent filtrate collected after the initial filtrate has been removed.

[0167] Understandably, the ultrasonic extraction time includes, but is not limited to, 25 min, 30 min, and 35 min; preferably, the time is 30 min.

[0168] Understandably, the power includes, but is not limited to, 250W, 280W, 300W, 320W, and 350W; preferably, the power is 300W.

[0169] Understandably, the frequency includes, but is not limited to, 35 kHz, 40 kHz, and 45 kHz; preferably, the frequency is 40 kHz.

[0170] In one example of the detection method, the sample to be tested is a standard decoction of Java white cardamom, and the extraction solvent is methanol with a volume fraction of 80% to 100%. The standard decoction of Java white cardamom is ground, mixed with methanol with a volume fraction of 80% to 100%, 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] Understandably, the Javanese white cardamom standard decoction described in this invention is in freeze-dried powder form.

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

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

[0175] Understandably, the volume fraction of methanol includes, but is not limited to, 80%, 90%, and 100%; preferably, the volume fraction of methanol is 100%. Understandably, 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.

[0176] Understandably, the ultrasonic extraction time includes, but is not limited to, 25 min, 30 min, and 35 min; preferably, the time is 30 min.

[0177] Understandably, the power includes, but is not limited to, 250W, 280W, 300W, 320W, and 350W; preferably, the power is 300W.

[0178] Understandably, the frequency includes, but is not limited to, 35 kHz, 40 kHz, and 45 kHz; preferably, the frequency is 40 kHz.

[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%, ultrasonically extracted, filtered, and the filtrate is collected.

[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 Java white cardamom formula granules means grinding the Java white cardamom formula granules into a fine powder.

[0182] In one example, the ratio of Java white cardamom granules to methanol was (0.2–1.0) g:10 mL, preferably 0.5 g:10 mL.

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

[0184] Understandably, the power includes, but is not limited to, 250W, 280W, 300W, 320W, and 350W; preferably, the power is 300W.

[0185] Understandably, the frequency includes, but is not limited to, 35 kHz, 40 kHz, and 45 kHz; preferably, the frequency is 40 kHz.

[0186] This invention is the first to apply ultra-high performance liquid chromatography to the quality detection of Java white cardamom and its modern preparations, filling the gap in the quality detection of Java white cardamom and its modern preparations. It realizes quality control and specific identification of Java white cardamom from raw materials to traditional Chinese medicine formula granules from the perspective of intrinsic quality, further ensuring the quality of Java white cardamom, standard decoctions, and formula granules, which is conducive to ensuring the effectiveness of clinical medication.

[0187] The following detailed description, with reference to specific embodiments, illustrates the method for constructing characteristic spectra of Java white cardamom, standard decoction, and traditional Chinese medicine granules according to the present invention. Unless otherwise specified, all raw materials used in the following embodiments are commercially available products.

[0188] Example 1: Method for establishing UPLC characteristic maps of Java white cardamom formulation granules

[0189] 1. Instruments, reagents and reagents

[0190] Instruments: Waters H-class ultra-high performance liquid chromatograph (Waters Corporation), Thermo Vanquish ultra-high performance liquid chromatograph (Thermo Fisher Scientific), Waters Cortecs T3 (2.1mm×100mm, 1.6μm) column, 0.001% balance (ME204E, Mettler Toledo), 0.1% balance (XP26, Mettler Toledo), CNC ultrasonic cleaner (KQ500D, Kunshan Ultrasonic Instrument Co., Ltd.), electric thermostatic drying oven (DHG-9146A, Jinghong Instrument Co., Ltd.), ultrapure water system (Milli-Q Direct, Merck KE).

[0191] Reagents: Methanol (Xilong Scientific Co., Ltd.) was analytical grade; phosphoric acid (Tianjin Kemeio Chemical Reagent Co., Ltd.), methanol (Merck Co., Ltd.), and acetonitrile (Merck Co., Ltd.) were UPLC chromatographic grade; water was ultrapure water (prepared in the laboratory).

[0192] Test reagents: Protocatechuic acid (batch number: 110809-202207, content: 97.5%, China National Institutes for Food and Drug Control); Vanillic acid (batch number: 110776-201503, content: 99.8%, China National Institutes for Food and Drug Control); (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone (source: Guangdong Yifang Pharmaceutical Co., Ltd.), (4S,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone (source: Guangdong Yifang Pharmaceutical Co., Ltd.), and (4R,6R)-6-hydroxypiperone (source: Guangdong Yifang Pharmaceutical Co., Ltd.) were all prepared in the laboratory; Javanese cardamom 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, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the flow rate was 0.25 mL / min; the column temperature was 38 °C; the detection wavelength was 300 nm from 0 to 26 min, and 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 the test solution

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

[0200] 2.3 Preparation of reference solution

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

[0202] 2.4 Determination Method

[0203] Accurately pipette 1 μL each of the reference solution and the test solution, inject them into the liquid chromatograph, and determine the characteristic chromatogram of the Java white cardamom formula granules.

[0204] 3. Chromatographic conditions

[0205] 3.1 Optimization of Gradient Conditions

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

[0207] Table 2 Gradient elution program 1

[0208]

[0209] Table 3 Gradient elution program 2

[0210]

[0211] The results showed that, comparing two different gradient conditions, the chromatogram obtained according to "gradient elution program 2" in Table 3 had a more uniform distribution, better resolution of each chromatographic peak, good peak shape, and stable baseline. Therefore, gradient elution program 2 in Table 3 was selected as the gradient elution program condition for the characteristic chromatographic analysis of Java white cardamom formula granules.

[0212] 3.2 Optimization of the organic phase ratio

[0213] To optimize the organic phase ratio, the following conditions are specified:

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

[0215] The results showed that when the mobile phase was methanol-acetonitrile (1:2) as the mobile phase A, the chromatograms were more uniformly distributed, the peaks were better separated, the peak shapes were good, and the baselines were stable. Therefore, methanol-acetonitrile (1:2) was used as the analytical condition for the characteristic chromatograms of Java white cardamom granules.

[0216] 3.3 Optimization of different acids

[0217] To determine the optimal acid concentration based on variations in different acids, the specific conditions are as follows:

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

[0219] The results showed that, when comparing three different acids, using 0.1% phosphoric acid as mobile phase B resulted in a more uniform chromatogram distribution, better peak resolution, better peak shape, and a stable baseline. Therefore, 0.1% phosphoric acid was chosen as the analytical condition for the characteristic chromatogram of Java white cardamom granules.

[0220] 3.4 Determination of chromatographic conditions

[0221] 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, and 0.1% phosphoric acid solution was used as mobile phase B, following the gradient elution procedure 2 in Table 3; the flow rate was 0.25 mL / min; 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.

[0222] 4. Methodological Examination

[0223] 4.1 Specificity Examination

[0224] The characteristic peaks in the chromatogram of the test sample have the same chromatographic peaks at the corresponding retention times as those in the chromatogram of the reference solution, and there is no negative interference. Figure 4 As shown, this demonstrates the good specificity of the method.

[0225] 4.2 Durability Test

[0226] (1) Investigation at different column temperatures

[0227] Take the Javanese white cardamom granules (KL01) sample and prepare the test solution according to the method specified in section "2.2". Except for the column temperatures of 36℃, 38℃, and 40℃, all other chromatographic conditions are the same as specified in section "2.1". Inject and analyze, using peak 7 as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak and peak S, and calculate the RSD value. The results are as follows... Figure 5 The results show that the RSD values ​​of relative retention time and relative peak area are all less than 5.0% at different column temperatures, indicating that the method has good robustness to different column temperatures.

[0228] (2) Investigation of different flow velocities

[0229] Take the Javanese white cardamom granules (KL01) sample and prepare the test solution according to the method specified in section "2.2". Except for the flow rates of 0.23 mL / min, 0.25 mL / min, and 0.27 mL / min, all other chromatographic conditions are the same as specified in section "2.1". Inject the sample for analysis, using peak 7 as the reference peak S. Calculate the relative retention time and relative peak area of ​​each characteristic peak and peak S, and calculate the RSD value. The results are as follows: Figure 6 The results show that the RSD values ​​of relative retention time and relative peak area are all less than 3.0% at different flow rates, indicating that the method has good robustness to different flow rates.

[0230] 4.3 Identification of Characteristic Peaks

[0231] Take Java white cardamom granules and prepare the test solution according to the test solution preparation method determined in section "2.2". Under the chromatographic conditions in section "2.1", inject the test solution and the reference solution into the sample for analysis. Compare the retention time and spectrum of the characteristic peaks to determine that peak 1 is protocatechuic acid, peak 3 is vanillic acid, peak 6 is (4R,6R)-6-hydroxypiperone, 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.

[0232] 4.4 Determination of Formulation Granule Samples

[0233] Three batches of Javanese white cardamom granules (batch numbers: KL01, KL02, and KL03) were taken. Test solutions were prepared according to the method specified in section "2.2". The test solutions and the Javanese white cardamom reference material solution were accurately pipetted and injected according to the chromatographic conditions specified in section "2.1". The superimposed characteristic chromatograms of the three batches are shown in [Figure number missing]. Figure 7 Based on the characteristic spectrum, nine common peaks were identified. Using peak 7(4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone as reference peak S, the relative retention times and relative peak areas of each characteristic peak and peak S were calculated and are shown in Tables 4 and 5.

[0234] Table 4. Characteristic chromatograms (relative retention times) of three batches of Javanese white cardamom formulation granules.

[0235]

[0236] Table 5. Characteristic spectral data (relative peak area) of three batches of Javanese white cardamom formulation granules.

[0237]

[0238] According to Tables 4 and 5, nine peaks were detected in all three batches of Javanese white cardamom granules.

[0239] Example 2: Determination of the characteristic chromatograms of Java white cardamom and its standard decoction

[0240] (1) Method for preparing the test sample:

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

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

[0243] Ten batches of Javanese white cardamom medicinal material powder (passed through a No. 3 sieve) and ten corresponding batches of Javanese white cardamom standard decoction powder were taken respectively. Test solutions were prepared according to the above method. The test solutions were precisely pipetted and injected under the chromatographic conditions described in "2.1" of Example 1 for determination. The superimposed characteristic chromatograms of the ten batches of Javanese white cardamom medicinal material are shown below. Figure 8 As shown in the overlay image, the characteristic spectra of 10 batches of Javanese white cardamom standard decoction are as follows: Figure 9 As shown in the characteristic chromatograms, both the chromatogram of Java white cardamom and the chromatogram of Java white cardamom standard decoction have 9 common peaks. Using 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. The calculation results are shown in Tables 6 to 9.

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

[0245]

[0246] Table 7. Characteristic chromatograms (relative peak areas) of 10 batches of Javanese white cardamom medicinal materials

[0247]

[0248] Table 8. Characteristic chromatograms (relative retention times) of 10 batches of standard decoctions of Javanese white cardamom.

[0249]

[0250] Table 9. Characteristic chromatograms (relative peak areas) of 10 batches of Javanese white cardamom standard decoction

[0251]

[0252] According to Tables 6 to 9, nine characteristic peaks can be detected in different batches of Javanese white cardamom and its standard decoction.

[0253] The results of the above embodiments show that the established method is stable and reliable with good reproducibility. The method constructed in this invention can be applied to the determination of Java white cardamom, standard decoction and its formulation granules.

[0254] This invention provides characteristic chromatograms of Java white cardamom medicinal materials, standard decoctions, and traditional Chinese medicine formula granules constructed by rationally controlling ultra-high performance liquid chromatography conditions. These chromatograms can be used for quality detection of Java white cardamom medicinal materials, standard decoctions, and traditional Chinese medicine formula granules, filling a gap in the quality detection of these materials. This provides an effective method for the identification of modern Java white cardamom preparations and offers a scientific experimental basis for its comprehensive quality evaluation, further ensuring the quality of Java white cardamom medicinal materials, standard decoctions, and formula granules, and contributing to the effectiveness of clinical medication.

[0255] This invention establishes a simple, reliable, and practical method for detecting Java white cardamom medicinal materials, standard decoctions, and formulation granules. The established ultra-high performance liquid chromatography method is simple, efficient, and produces objective, precise, and reliable results.

[0256] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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 embodiments described above are merely illustrative of several implementations of the present invention, designed to facilitate a detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this invention patent should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing characteristic chromatograms of Javanese white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules, characterized in that, The construction method includes the following steps: A reference solution of protocatechuic acid, (4R,6R)-6-hydroxypiperone, (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, vanillic acid, and (4S,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone was prepared. A test solution is prepared by extracting a sample of Java white cardamom using an extraction solvent. The Java white cardamom sample is selected from one or more of the following: Java white cardamom medicinal material, Java white cardamom standard decoction, and Java white cardamom formula granules. The Java white cardamom sample is either Java white cardamom medicinal material or a standard decoction of Java white cardamom, in which case the extraction solvent is 80%–100% methanol (by volume). Alternatively, the Java white cardamom sample may be a standard decoction of Java white cardamom, in which case the extraction solvent is 80%–100% methanol (by volume). Finally, the Java white cardamom sample may be a formula granule of Java white cardamom, in which case the extraction solvent is 80%–100% methanol (by volume). The reference solution and the test solution were detected by ultra-high performance liquid chromatography (UHPLC) to construct characteristic chromatograms. The conditions for the ultra-high performance liquid chromatography method include: A gradient elution was performed using a mixed solution of methanol and acetonitrile with a volume ratio of 1:2 as mobile phase A and an aqueous solution of phosphoric acid with a volume fraction of 0.05% to 0.15% as mobile phase B. The gradient elution procedure is as follows: From 0 to 10 minutes, the volume percentage of the mobile phase A changed from 6% to 7%. Over a period of 10 to 20 minutes, the volume percentage of the mobile phase A changed from 7% to 10%. Over 20 to 25 minutes, the volume percentage of the mobile phase A changed from 10% to 13%. During the period of 25 min to 42 min, the volume percentage of the mobile phase A was 13%. Over a period of 42 to 50 minutes, the volume percentage of the mobile phase A changed from 13% to 30%. Over a period of 50 to 60 minutes, the volume percentage of the mobile phase A changed from 30% to 40%. A Waters Cortecs T3 column was used, with the following specifications: column length 150 mm, inner diameter 2.1 mm, and particle size 1.6 μm. Detection wavelengths: 0–26 min, 290 nm–310 nm; 26 min–60 min, 230 nm–250 nm.

2. The method for constructing characteristic spectra of Javanese white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules according to claim 1, characterized in that, The chromatographic conditions of the ultra-high performance liquid chromatography satisfy at least one of the following (1) to (2): (1) The flow rate is 0.23 mL / min to 0.27 mL / min; (2) The column temperature is 36℃~40℃.

3. The method for constructing characteristic spectra of Javanese white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules according to claim 1, characterized in that, The preparation of the reference solution includes the following steps: Protocatechuic acid, (4R,6R)-6-hydroxypiperone, (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, vanillic acid, and (4S,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone were mixed with methanol at a volume fraction of 80%–100%.

4. The method for constructing characteristic spectra of Javanese white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules according to claim 3, characterized in that, The reference solution contains, per 1 mL, 20 μg to 30 μg of protocatechuic acid, 20 μg to 30 μg of (4R,6R)-6-hydroxypiperone, 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-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone.

5. The method for constructing characteristic spectra of Javanese white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules according to claim 1, characterized in that, The Javanese white cardamom medicinal material was pulverized, sieved, mixed with methanol of 80% to 100% by volume, ultrasonically extracted, filtered, and the filtrate was 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.

6. The method for constructing characteristic spectra of Javanese white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules according to claim 1, characterized in that, The Java white cardamom standard decoction was ground, mixed with methanol at a volume fraction of 80% to 100%, and subjected to ultrasonic extraction. The mixture was then filtered, and the filtrate was 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.

7. The method for constructing characteristic spectra of Javanese white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules according to claim 1, characterized in that, The Java white cardamom granules were ground, mixed with methanol at a volume fraction of 80% to 100%, and subjected to ultrasonic extraction. The mixture was then filtered, and the filtrate was 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.

8. The method for constructing characteristic spectra of Javanese white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules according to claim 1, characterized in that, The characteristic chromatograms of the Javanese white cardamom medicinal material, standard decoction, and formula granules include nine characteristic peaks, of which peak 1 is protocatechuic acid, peak 3 is vanillic acid, peak 6 is (4R,6R)-6-hydroxypiperone, peak 7 is (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone, and peak 9 is (4S,5R)-4-Hydroxy-5-isopropyl-2-methylcycl ohex-2-enone; the peak corresponding to the reference peak of (4R,5R)-4-Hydroxy-5-isopropyl-2-methylcyclohex-2-enone is the S peak. The relative retention times of peaks 2, 5, and 8 with peak S are calculated, and their relative retention times are within ±10% of the specified values, which are: 0.25 for peak 2, 0.80 for peak 5, and 1.06 for peak 8.

9. A method for detecting Javanese white cardamom medicinal material, standard decoction, and traditional Chinese medicine granules, characterized in that, The detection method includes the following steps: The sample to be tested is extracted with an extraction solvent to prepare a sample solution; the sample to be tested is selected from one or more of Java white cardamom medicinal material, Java white cardamom standard decoction, and Java white cardamom formula granules; 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 sample to be tested is Java white cardamom standard decoction, and the extraction solvent is methanol with a volume fraction of 80% to 100%; 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 sample solution to be tested was detected by ultra-high performance liquid chromatography, and the detected chromatogram was compared with the characteristic chromatogram constructed by the construction method according to any one of claims 1 to 8; The conditions for the ultra-high performance liquid chromatography method include: A gradient elution was performed using a mixed solution of methanol and acetonitrile with a volume ratio of 1:2 as mobile phase A and an aqueous solution of phosphoric acid with a volume fraction of 0.05% to 0.15% as mobile phase B. The gradient elution procedure is as follows: From 0 to 10 minutes, the volume percentage of the mobile phase A changed from 6% to 7%. Over a period of 10 to 20 minutes, the volume percentage of the mobile phase A changed from 7% to 10%. Over 20 to 25 minutes, the volume percentage of the mobile phase A changed from 10% to 13%. During the period of 25 min to 42 min, the volume percentage of the mobile phase A was 13%. Over a period of 42 to 50 minutes, the volume percentage of the mobile phase A changed from 13% to 30%. Over a period of 50 to 60 minutes, the volume percentage of the mobile phase A changed from 30% to 40%. A Waters Cortecs T3 column was used, with the following specifications: column length 150 mm, inner diameter 2.1 mm, and particle size 1.6 μm. Detection wavelengths: 0–26 min, 290 nm–310 nm; 26 min–60 min, 230 nm–250 nm.

10. The method for detecting Javanese white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules according to claim 9, characterized in that, The chromatographic conditions of the ultra-high performance liquid chromatography satisfy at least one of the following (1) to (2): (1) The flow rate is 0.23 mL / min to 0.27 mL / min; (2) The column temperature is 36℃~40℃.

11. The detection method for Javanese white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules according to claim 9, characterized in that, The Javanese white cardamom medicinal material was pulverized, sieved, mixed with methanol of 80% to 100% by volume, ultrasonically extracted, filtered, and the filtrate was 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.

12. The detection method for Javanese white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules according to claim 9, characterized in that, The Java white cardamom standard decoction was ground, mixed with methanol at a volume fraction of 80% to 100%, and subjected to ultrasonic extraction. The mixture was then filtered, and the filtrate was 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.

13. The detection method for Javanese white cardamom medicinal material, standard decoction, and traditional Chinese medicine formula granules according to claim 9, characterized in that, The Java white cardamom granules were ground, mixed with methanol at a volume fraction of 80% to 100%, and subjected to ultrasonic extraction. The mixture was then filtered, and the filtrate was 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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