Preparation method of granules of amomum villosum lour formula, quality control method of amomum villosum lour and determination method of effective component content

Abstract

The application belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a preparation method of a formula granule of Amomum villosum, a quality control method of Amomum villosum and a determination method of effective component content. The identification method comprises preparation of a test sample solution and a control medicinal material solution, and development by using a first developing agent or a second developing agent. The first developing agent system comprises methanol, glacial acetic acid and water, and the second developing agent system comprises ethyl acetate, methanol and water. The chromatogram obtained by the identification method has good spot separation effect, clear spots, high accuracy, good durability to temperature and humidity, and can be used as a thin layer identification method of Amomum villosum.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine analysis technology, specifically relating to a method for preparing Amomum villosum formula granules, a method for quality control of Amomum villosum, and a method for determining the content of effective components. Background Technology

[0002] Amomum villosum, a perennial herb belonging to the genus Amomum in the ginger family, has the effects of resolving dampness and promoting qi circulation, warming the middle jiao and stopping diarrhea, and calming the fetus. It is mainly used to treat stagnation of spleen and stomach qi, dampness obstructing the middle jiao, spleen and stomach deficiency and cold, vomiting and diarrhea, etc., and has certain medicinal value.

[0003] Existing technologies for studying Amomum villosum only establish methods for content determination and characteristic chromatographic methods, but do not establish quality control methods for related preparations, nor are there any reports on the analysis of phenolic acid components, flavonoid components, etc., which cannot guarantee the accuracy and effectiveness of quality control of Amomum villosum preparations. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defects in the prior art, such as the inability to determine the content of effective components in cardamom and the poor accuracy of the quality control method, so as to provide a method for preparing cardamom formula granules, a method for cardamom quality control, and a method for determining the content of effective components.

[0005] To this end, the present invention provides the following technical solution.

[0006] This invention provides a thin-layer chromatography method for identifying Amomum villosum, characterized by comprising the following steps:

[0007] Test solution: Prepare a test solution from the test sample;

[0008] Reference herb solution: Prepare a reference herb solution from the reference herb.

[0009] Take the test solution and the reference medicinal material solution, spot them, develop them with the first or second developing solvent system, remove them and examine them;

[0010] The first developing agent system includes methanol, glacial acetic acid and water;

[0011] The second developing solvent system comprises ethyl acetate, methanol, and water.

[0012] The first developing solvent system comprises methanol, glacial acetic acid, and water in a volume ratio of (6-12):(0.8-1.2):(0.8-1.2);

[0013] Preferably, the first developing agent system comprises methanol, glacial acetic acid, and water in a volume ratio of 9:1:1;

[0014] Preferably, the second developing solvent system comprises ethyl acetate, methanol, and water in a volume ratio of (6-10):(3-5):(0.4-0.6);

[0015] Preferably, the second developing solvent system comprises ethyl acetate, methanol, and water in a volume ratio of 8:4:0.5.

[0016] In the thin-layer identification method, the test sample is Amomum villosum medicinal material, Amomum villosum slices, Amomum villosum formula granules, or freeze-dried powder of Amomum villosum standard decoction;

[0017] Preferably, the sample volume is 5ul to 20ul;

[0018] Preferably, the origin of the cardamom is Yangchun cardamom, green-shelled cardamom, or Hainan cardamom.

[0019] In the thin-layer identification method, the test solution is prepared by extraction.

[0020] Preferably, the extraction solvent is ethyl acetate and / or n-butanol;

[0021] Preferably, the reference medicinal material solution is prepared by extraction.

[0022] Preferably, the solvent for extraction is ethyl acetate and / or n-butanol.

[0023] When performing thin-layer chromatography identification, if the test sample is a medicinal material or decoction piece, the preparation method of the test sample solution includes: taking the test sample, adding water, heating under reflux, filtering, adding extraction solvent, extracting 2-4 times, combining the filtrates, evaporating to dryness, adding methanol to the residue, dissolving, and obtaining the test sample solution.

[0024] In addition, the present invention also provides a method for determining the content of vanillic acid and epicatechin in Amomum villosum, comprising the following steps:

[0025] Test solution: Prepare a test solution from the test sample;

[0026] Reference solution: Prepare a reference solution from the reference standard;

[0027] Determination: Determined by high performance liquid chromatography; Chromatographic conditions: Acetonitrile as mobile phase A, phosphoric acid aqueous solution as mobile phase B, gradient elution;

[0028] The gradient elution program is as follows: 0-5 min, 6-8% → 13-18% mobile phase A, 92-94% → 82-87% mobile phase B; 5-6 min, 13-18% → 28-35% mobile phase A, 82-87% → 65%-72% mobile phase B.

[0029] The chromatographic conditions also included: using octadecylsilane-bonded silica gel as the packing material, with a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.6 µm;

[0030] Preferably, the gradient elution program is as follows: 0-5 min, 7%→15% mobile phase A, 93%→85% mobile phase B; 5-6 min, 15%→30% mobile phase A, 85%→70% mobile phase B; or, the gradient elution program is as follows: 0-5 min, 7%→14.5% mobile phase A, 93%→85.5% mobile phase B; 5-6 min, 14.5%→30% mobile phase A, 85.5%→70% mobile phase B;

[0031] Preferably, the flow rate is 0.28–0.32 ml / min;

[0032] Preferably, the column temperature is 28–32°C;

[0033] Preferably, the wavelength is 255–265 nm;

[0034] Preferably, the injection volume is 1–3 μL.

[0035] Furthermore, each 1 ml of the reference solution contains 5–20 μg of vanillic acid;

[0036] Preferably, each 1 ml of the reference solution contains 20–80 μg of epicatechin;

[0037] Preferably, the detection limit for vanillic acid in the cardamom is 0.5~2.7 mg / g;

[0038] Preferably, the detection limit for epicatechin in the cardamom is 1.1~3.8 mg / g.

[0039] Furthermore, this invention also provides a method for determining the content of bornyl acetate in Amomum villosum, comprising the following steps:

[0040] Test solution: Prepare a test solution from the test sample;

[0041] Reference solution: Prepare a reference solution from the reference standard;

[0042] Determination: Gas chromatography was used for determination. Chromatographic conditions: The column temperature was programmed as follows: the initial temperature was 95-110℃, held for 18-22 min, and then increased to 220-245℃ at a rate of 15-25℃ / min.

[0043] Chromatographic conditions also include: a capillary column with 100% dimethylpolysiloxane as the stationary phase, specifications: column length 30 m, inner diameter 0.25 mm, and membrane thickness 0.25 μm; and / or,

[0044] The injection port temperature is 220–245℃; and / or,

[0045] Detector temperature 250–260°C; and / or,

[0046] 10:1 split ratio; and / or,

[0047] The injection volume is 1–3 μL; and / or,

[0048] Each 1 ml of the reference solution contains 0.1–0.3 mg of bornyl acetate; and / or,

[0049] The detection limit for bornyl acetate in the cardamom is 2.6-10.3 mg / g.

[0050] When determining vanillic acid, epicatechin, or borneol acetate in Amomum villosum, the original source of Amomum villosum is Amomum villosum var. yangchunense, Amomum villosum var. yunnanense, or Amomum villosum var. yunnanense.

[0051] Furthermore, the present invention also provides a method for preparing Amomum villosum granules, comprising the following steps:

[0052] (1) Decoction of Amomum villosum, collection of volatile oil, inclusion complex to obtain volatile oil inclusion complex;

[0053] (2) The decoction was filtered and concentrated under reduced pressure to obtain a paste;

[0054] (3) Mix the extract with the inclusion complex, spray dry, add excipients, and granulate;

[0055] Preferably, during the inclusion process, the mass ratio of volatile oil, β-cyclodextrin, and water is 1:7:21;

[0056] Preferably, the inlet air temperature of the spray dryer is 150-210℃, and the outlet air temperature is 70-115℃.

[0057] The preparation method for the test solution in determining borneol acetate in Amomum villosum includes: accurately weighing 20-50 g of the granulated test sample, and conducting the test according to Method A of the Determination of Volatile Oils (General Chapter 2204, Chinese Pharmacopoeia 2020 Edition), placing it in a round-bottom flask, adding 300 ml of water and several glass beads, shaking to mix, connecting the volatile oil analyzer, adding water from the top of the analyzer until it fills the graduated section and overflows into the flask, then adding 5 ml of n-hexane, connecting the reflux condenser, heating and maintaining a gentle boil for 3 hours, cooling, and after the solution has clearly separated into layers, separating the n-hexane solution and placing it in a 10 ml volumetric flask, washing the inner wall of the volatile oil analyzer with a small amount of n-hexane several times, combining the n-hexane washings with the same volumetric flask, adding n-hexane to the mark, shaking well, then accurately measuring 1 ml into a 20 ml volumetric flask, adding n-hexane to the mark, shaking well to obtain the test solution.

[0058] The method for preparing Amomum villosum granules provided by this invention is applicable to Amomum villosum of any origin.

[0059] The technical solution of this invention has the following advantages:

[0060] 1. The present invention provides a thin-layer chromatography identification method for Amomum villosum, comprising the preparation of a test solution and a reference herbal solution, and developing the sample using a first or second developing solvent. The first developing solvent system comprises methanol, glacial acetic acid, and water, and the second developing solvent system comprises ethyl acetate, methanol, and water. This identification method yields chromatographic spots with good separation, clear spots, high accuracy, and good durability against temperature and humidity variations, and can be used as a thin-layer chromatography identification method for Amomum villosum.

[0061] 2. The method for determining the content of vanillic acid and epicatechin in Amomum villosum provided by the present invention has a short analysis time and high accuracy in determining the content of vanillic acid and epicatechin.

[0062] 3. The method for determining the content of bornyl acetate in Amomum villosum provided by the present invention has high accuracy in determining the content of bornyl acetate. Attached Figure Description

[0063] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0064] Figure 1 These are thin-layer chromatograms of different spotting amounts observed in Experimental Example 1 of this invention;

[0065] Figure 2 These are thin-layer chromatograms of Experiment 1 of the present invention at different temperatures, where a is a thin-layer identification chromatogram of Amomum villosum formula particles at room temperature, and b is a thin-layer identification chromatogram of Amomum villosum formula particles at 4.3℃.

[0066] Figure 3 These are thin-layer chromatograms of experimental example 1 of the present invention under different humidity conditions, where a is the thin-layer identification chromatogram of Amomum villosum formula particles under 88% humidity, and b is the thin-layer identification chromatogram of Amomum villosum (Yangchun Amomum) formula particles under 35% humidity.

[0067] Figure 4 These are the thin-layer chromatograms of different manufacturers' thin-layer plates examined in Experiment 1 of this invention, where a is the thin-layer identification chromatogram of Amomum villosum formula particles (Shanghai polyamide film plate), and b is the thin-layer identification chromatogram of Amomum villosum formula particles (Zhejiang Taizhou plate).

[0068] Figure 5 This is a thin-layer chromatogram of Experimental Example 2 of the present invention, showing the results of different sample amounts.

[0069] Figure 6 These are thin-layer chromatograms of experimental example 2 of the present invention at different temperatures, where a is a thin-layer identification chromatogram of Amomum villosum formula particles at 24℃ and RH 58%, and b is a thin-layer identification chromatogram of Amomum villosum formula particles at 4℃ and RH 65%.

[0070] Figure 7 These are thin-layer chromatograms of experimental example 2 of the present invention under different humidity conditions, where a is a thin-layer identification chromatogram of Amomum villosum formula particles under 24℃ and RH88% conditions, and b is a thin-layer identification chromatogram of Amomum villosum formula particles under 54℃ and RH32% conditions.

[0071] Figure 8 These are the thin-layer chromatograms of different manufacturers' thin-layer plates examined in Experimental Example 2 of this invention. Among them, a is the thin-layer identification chromatogram of Shanghai Jinsui Amomum villosum formula granules at 24℃ and RH 5%, b is the thin-layer identification chromatogram of Qingdao Bangkai Amomum villosum formula granules at 24℃ and RH 5%, and c is the thin-layer identification chromatogram of Sipute Amomum villosum formula granules at 24℃ and RH 58%.

[0072] Figure 9 This is a thin-layer chromatogram of Amomum villosum slices from Example 3 of the present invention;

[0073] Figure 10 This is the chromatogram obtained in Example 4 of the present invention;

[0074] Figure 11 This is the chromatogram obtained in Example 5 of the present invention. Detailed Implementation

[0075] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0076] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0077] instrument:

[0078] KQ-700DE Ultrasonic Extractor: Kunshan Ultrasonic Instruments Co., Ltd.; BS224S, Sartorius 0.001g electronic analytical balance: Sartorius GmbH, Germany; XR205SM-DR 0.001g electronic analytical balance: Precisa GmbH, Switzerland; Thin-layer chromatography imager: CAMAG GmbH, Switzerland; Polyamide film plates: Luqiao Sijia Biochemical Plastics Factory, Taizhou City, Zhejiang Province; Shanghai Jinsui Biotechnology Co., Ltd.; Qingdao Bangkai; Sipute;

[0079] METTLER TOLEDO XS204 0.0001 g balance (METTLER, Switzerland); KQ-500DE ultrasonic instrument (Kunshan Ultrasonic Instrument Co., Ltd.); HH-8 water bath (Changzhou Aohua Instrument Co., Ltd.); ATS4 (CAMAG) fully automatic spotting device; developing tank; YOKO-PN electric sprayer (Wuhan Pharmaceutical New Technology Development Co., Ltd.); electronic analytical balance: METTLERTOLEDO XA205DV; Sartivins BS224s; ultrasonic instrument: KQ-500DE (Kunshan Ultrasonic Instrument Co., Ltd.); chromatograph: Waters ACQUITY UPLC H-Class PLUS, column: Waters CORTECS T3, 2.1×100mm, 1.6µm; chromatograph: Shimadzu GC2010 PLUS; column: Agilent, DB-1, column length 30m, inner diameter 0.25mm, membrane thickness 0.25μm.

[0080] Reagents and reagents:

[0081] Ethyl acetate, methanol, ethanol, and n-hexane were all of analytical grade. Acetonitrile was of chromatographic grade.

[0082] Test drug: Amomum villosum (Yangchun Amomum villosum) reference material, batch number: 120985-201406, purchased from China National Institutes for Food and Drug Control;

[0083] Amomum villosum granules (originating from Amomum villosum var. yangchunense) batch numbers: K1, K2, K3;

[0084] Amomum villosum slices (originating from Amomum villosum var. yangchunense) batch number: YP01-YP03;

[0085] Amomum villosum slices (origin of green-shelled amomum villosum) batch number: YP04-YP06;

[0086] Amomum villosum slices (originating from Hainan Amomum villosum) batch number: YP07-YP09;

[0087] Freeze-dried powder of Amomum villosum standard decoction (origin of Amomum villosum var. yangchunense) batch number: BZTJ01-BZTJ17;

[0088] Negative blank particles, using maltodextrin as negative blank particles;

[0089] Vanillic acid (batch number: 110776-201503, for content determination, calculated at 99.8%) was purchased from the National Institutes for Food and Drug Control; epicatechin (batch number: 110878-201703, for content determination, calculated at 99.7%) was purchased from the National Institutes for Food and Drug Control; bornyl acetate (batch number: 110759-202007, for content determination, calculated at 99.3%) was purchased from the National Institutes for Food and Drug Control.

[0090] Example 1

[0091] This embodiment provides a thin-layer chromatography identification method for Amomum villosum granules (originating from Amomum villosum var. yangchunense), including the following steps:

[0092] Test solution: Take 0.5g of Amomum villosum granules as test sample, add 30ml of water to dissolve it, extract with ethyl acetate 3 times, 20ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue to obtain the test solution.

[0093] Reference herb solution: Take 1g of Amomum villosum reference herb (originating from Amomum villosum var. yangchunense), add 30ml of water, heat under reflux for 45min, filter, extract with ethyl acetate 3 times, 20ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and prepare the reference herb solution.

[0094] Preparation of negative control solution: Take negative blank particles, grind them finely, add 30 ml of water, extract with ethyl acetate 3 times, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1 ml of methanol to the residue, dissolve, filter, and use as negative control solution.

[0095] According to the thin-layer chromatography method (General Chapter 0502 of the Chinese Pharmacopoeia 2020), 10 μL of the test solution, 15 μL of the reference medicinal material solution, and 10 μL of the negative control solution were spotted separately onto the same polyamide film plate. A methanol, glacial acetic acid, and water solution (volume ratio 9:1:1) was used as the developing solvent. After development, the plate was removed, air-dried, sprayed with 3% aluminum trichloride solution, dried with hot air, and examined under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions as in the chromatogram of the reference medicinal material.

[0096] Experimental Example 1

[0097] This experimental example provides a methodological verification of the thin-layer chromatography identification method for Amomum villosum granules (originating from Amomum yangchunense) in Example 1.

[0098] (1) Investigation of sample size

[0099] Five solutions of Amomum villosum granules and five solutions of control medicinal materials were prepared according to Example 1. The sample amount was used as a variable, and the results were determined according to Example 1. (See attached table for details.) Figure 1 The sample loading volumes were 5µL, 8µL, 10µL, 15µL, and 20µL, respectively. Figure 1 Starting from the left, the first five (1-5) are chromatograms of the test sample solution obtained at different spotting volumes, from left to right: 5µL, 8µL, 10µL, 15µL, and 20µL; the last five (6-10) are chromatograms of the reference medicinal material solution obtained at different spotting volumes, from left to right: 5µL, 8µL, 10µL, 15µL, and 20µL.

[0100] from Figure 1 As can be seen, the spots in the chromatogram become clearer with increasing sample volume. However, when the sample volume increases to a certain extent, the test solution becomes slightly blurred. Overall, however, the chromatograms of the Amomum villosum granules test solution and the reference herb show spots of the same color at the corresponding positions. Based on the separation and clarity of the spots, the preferred sample volume for the test solution is 10 µL, and the preferred sample volume for the reference herb solution is 15 µL.

[0101] (2) Investigation at different temperatures

[0102] According to Example 1, a test solution of Amomum villosum granules, a reference herb solution of Amomum villosum, and a negative control solution were prepared. The sample volume of the test solution was 10 µL, the sample volume of the reference herb solution was 15 µL, and the sample volume of the negative control solution was 10 µL. All samples were spotted onto the same polyamide film plate and measured under room temperature and low temperature conditions. The results are shown in [Figure 1]. Figure 2 .in, Figure 2 'a' represents normal temperature conditions, which is 25.2℃. Figure 2 b represents a low-temperature condition, where low temperature refers to 4.3℃; Figure 2 a and Figure 2 In b, counting from the left, the first three (1-3) are test solutions obtained from different batches of Amomum villosum formula granules, corresponding to batch numbers K1-K3, the fourth (4) is the control medicinal material solution, and the last one (5) is the negative control solution.

[0103] from Figure 2 It can be seen that under normal temperature and low temperature conditions, the thin-film chromatography of Amomum villosum formula granules and the thin-layer chromatography of Amomum villosum reference material show spots of the same color at the corresponding positions, with no negative interference and good separation of chromatographic spots. This indicates that temperature has little effect on the thin-layer identification of Amomum villosum formula granules, and this thin-layer identification method has good temperature resistance.

[0104] (3) Investigation of different humidity levels

[0105] According to Example 1, a test solution of Amomum villosum granules, a reference herb solution of Amomum villosum, and a negative control solution were prepared. The sample volume of the test solution was 10 µL, the sample volume of the reference herb solution was 15 µL, and the sample volume of the negative control solution was 10 µL. All samples were spotted onto the same polyamide film plate and measured under high humidity and low humidity conditions, respectively. The results are shown in [Figure 1]. Figure 3 .in, Figure 3 'a' represents a high humidity condition, where high humidity is 88%. Figure 3 b represents a low humidity condition, where low humidity is 35%. Figure 3 Counting from the left, the first three (1-3) are test solutions obtained from different batches of Amomum villosum formula granules, corresponding to batch numbers K1-K3; the fourth (4) is the control herb solution; and the last (5) is the negative control solution.

[0106] from Figure 3 It can be seen that under both high and low humidity conditions, the chromatograms of the Amomum villosum formula granules and the Amomum villosum reference material show spots of the same color at the corresponding positions, and the chromatogram spots are well separated with no interference from negative samples. The experimental results indicate that humidity has little effect on the thin-layer identification of Amomum villosum formula granules and that the material exhibits good tolerance to humidity.

[0107] (4) Investigation of thin-layer boards from different manufacturers

[0108] According to Example 1, a test solution of Amomum villosum granules, a reference herb solution of Amomum villosum, and a negative control solution were prepared. The sample volume of the test solution was 10 µL, the sample volume of the reference herb solution was 15 µL, and the sample volume of the negative control solution was 10 µL. These solutions were spotted onto polyamide film plates provided by different manufacturers and measured according to the method in Example 1. The results are shown in [Figure 1]. Figure 4 Among them, the manufacturers are Shanghai Polyamide Film Board (…). Figure 4 a) Zhejiang Taizhou board ( Figure 4 (b) Figure 4 Counting from the left, the first three (1-3) are test solutions obtained from different batches of Amomum villosum formula granules, corresponding to batch numbers K1-K3; the fourth (4) is the control herb solution; and the last (5) is the negative control solution.

[0109] from Figure 4 It can be seen that the thin-layer plates of Amomum villosum granules and Amomum villosum reference material from different manufacturers show spots of the same color at the corresponding positions in the polyamide film plates. The spots are well separated and clear, with no interference from negative samples. This identification method has good adaptability to polyamide film plates from different manufacturers, and the chromatographic bands are clear.

[0110] The above methodological studies demonstrate that the thin-layer chromatography method using methanol-glacial acetic acid-water as the developing solvent system exhibits good robustness, excellent separation effect, and clear spots, making it suitable as a thin-layer chromatography method for identifying Amomum villosum.

[0111] Example 2

[0112] This embodiment provides a thin-layer chromatography identification method for Amomum villosum granules (originating from Amomum villosum var. yangchunense), including the following steps:

[0113] Test solution: Take 0.5g of Amomum villosum granules as test sample, add 30ml of water to dissolve it, extract with ethyl acetate 3 times, 20ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue to obtain the test solution.

[0114] Reference herb solution: Take 1g of Amomum villosum reference herb, add 30ml of water, heat under reflux for 45min, filter, extract with ethyl acetate 3 times, 20ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and obtain the reference herb solution.

[0115] Preparation of negative control solution: Take negative blank particles (maltodextrin), grind them finely, add 30 ml of water, extract with ethyl acetate 3 times, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1 ml of methanol to the residue, dissolve, filter, and use as negative control solution.

[0116] According to the thin-layer chromatography method (General Chapter 0502 of the Chinese Pharmacopoeia 2020), 10 μL each of the test solution, the reference medicinal material solution, and the negative control solution were spotted onto the same polyamide film. Developed using ethyl acetate, methanol, and water (volume ratio 8:4:0.5), the film was removed, air-dried, sprayed with 3% aluminum trichloride solution, dried with hot air, and examined under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions as in the chromatogram of the reference medicinal material.

[0117] Experimental Example 2

[0118] This experimental example provides a methodological verification of the thin-layer chromatography identification method for Amomum villosum granules (originating from Amomum yangchunense) in Example 2.

[0119] (1) Sampling quantity investigation

[0120] Five solutions of Amomum villosum granules, a control herb solution, and a negative control solution were prepared according to Example 2. The sample amount was used as a variable, and the results were determined according to Example 2. The results are shown in [Figure 2]. Figure 5 The sample volumes for the test solutions were 1 µL, 5 µL, 8 µL, 10 µL, and 15 µL, respectively; the sample volumes for the control medicinal materials solutions were 1 µL, 5 µL, 10 µL, and 15 µL, respectively; and the sample volume for the negative control solution was 10 µL. Figure 5Starting from the left, the first five (1-5) are chromatograms of the test sample solution obtained under different spotting volumes, from left to right: 1µL, 5µL, 8µL, 10µL, 15µL; the last four (6-9) are chromatograms of the reference medicinal material solution obtained under different spotting volumes, from left to right: 1µL, 5µL, 10µL, 15µL; the last one (10) is the negative control solution.

[0121] from Figure 5 It can be seen that the chromatograms of the Amomum villosum granules test sample and the Amomum villosum reference material show spots of the same color at the corresponding positions, and there is no interference from the negative control. Based on the separation and clarity of the spots, when using a second developing solvent system, the spotting volume of the test sample solution and the reference material solution is preferably 10 µL.

[0122] (2) Investigation at different temperatures

[0123] According to Example 2, a test solution of Amomum villosum granules and a reference solution of Amomum villosum were prepared. The sample volume was 10 µL, and both were spotted onto the same polyamide film plate. Measurements were performed at room temperature and low temperature. The results are shown below. Figure 6 .in, Figure 6 'a' represents normal temperature conditions, which is 24.0℃. Figure 6 b represents a low-temperature condition, where low temperature refers to 4.0℃; Figure 6 Counting from the left, the first three (1-3) are test solutions obtained from different batches of Amomum villosum formula granules, corresponding to batch numbers K1-K3, and the last one (4) is the control herbal solution.

[0124] from Figure 6 It can be seen that under normal and low temperature conditions, the chromatograms of the test solution of Amomum villosum granules and the chromatogram of Amomum villosum reference material show spots of the same color at the corresponding positions, and the separation effect of the chromatographic spots is good. The experimental results show that temperature has no significant effect on the thin-layer identification of Amomum villosum granules, and this thin-layer identification method has good temperature resistance.

[0125] (3) Investigation of different humidity levels

[0126] According to Example 2, a test solution of Amomum villosum granules and a reference solution of Amomum villosum were prepared. The sample volume was 10 µL, and both were spotted onto the same polyamide film plate. Measurements were performed under high and low humidity conditions, respectively. The results are shown in [Figure 1]. Figure 7 .in, Figure 7 'a' represents a high humidity condition, where high humidity is 88%. Figure 7 b represents a low humidity condition, where low humidity is 32%. Figure 7 Counting from the left, the first three (1-3) are test solutions obtained from different batches of Amomum villosum formula granules, corresponding to batch numbers K1-K3, and the last one (4) is the control herbal solution. Figure 7The results show that under high humidity and low temperature conditions, the chromatograms of the Amomum villosum granules and the Amomum villosum reference material show spots of the same color at the corresponding positions, and the spot separation is good. The experimental results indicate that humidity has no significant effect on the thin-layer chromatography identification of Amomum villosum granules, and this thin-layer chromatography identification method has good durability against humidity.

[0127] (4) Investigation of thin-layer boards from different manufacturers

[0128] The test solution of Amomum villosum granules and the reference medicinal material solution of Amomum villosum were prepared according to Example 2. The sample volume was 10 µL, and the samples were spotted onto polyamide film plates provided by different manufacturers. The results were determined according to the method in Example 2. The results are shown below. Figure 8 .in, Figure 8 a, b, and c correspond to the manufacturers Shanghai Jinsui, Qingdao Bangkai, and Siput, respectively. Figure 8 Counting from the left, the first three (1-3) are test solutions obtained from different batches of Amomum villosum formula granules, corresponding to batch numbers K1-K3, and the last one (4) is the control herbal solution.

[0129] Figure 8 The results show that the chromatograms of the Amomum villosum (Yangchun Amomum) formula granules obtained from polyamide films from different manufacturers show spots of the same color at corresponding positions as the Amomum villosum reference material, and the chromatogram spots are well separated. The experimental results indicate that polyamide films from different manufacturers have no significant impact on the thin-layer identification of Amomum villosum (Yangchun Amomum) formula granules, and this thin-layer identification method has good durability with polyamide films from different manufacturers.

[0130] In summary, the thin-layer chromatography method using the second developing solvent system demonstrates good robustness, effective separation, and clear spots, making it suitable for the thin-layer identification of Amomum villosum granules (derived from Amomum tsao-ko). The thin-layer identification results for all three batches of Amomum villosum granules met the requirements.

[0131] Example 3

[0132] This embodiment provides a thin-layer chromatography identification method for Amomum villosum slices (derived from Yangchun Amomum villosum, Green Shell Amomum villosum, and Hainan Amomum villosum), comprising the following steps:

[0133] Test solution: Take 1g of Amomum villosum slices, add 30ml of water, heat under reflux for 45min, filter, extract with ethyl acetate 3 times, 20ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and obtain the test solution.

[0134] Reference herb solution: Take 1g of Amomum villosum reference herb, add 30ml of water, heat under reflux for 45min, filter, extract with ethyl acetate 3 times, 20ml each time, combine the ethyl acetate solutions, evaporate to dryness, add 1ml of methanol to dissolve the residue, and obtain the reference herb solution.

[0135] According to the thin-layer chromatography method (General Chapter 0502 of the Chinese Pharmacopoeia 2020), 10 μL each of the test solution and the reference medicinal material solution were spotted separately onto the same polyamide film. A mixture of methanol, glacial acetic acid, and water (volume ratio 9:1:1) was used as the developing solvent. After development, the film was removed, air-dried, sprayed with 3% aluminum trichloride solution, dried with hot air, and examined under ultraviolet light (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions as in the chromatogram of the reference medicinal material.

[0136] The thin-layer chromatography of Amomum villosum slices with batch numbers YP01-YP09 was determined as described above, and the results are shown below. Figure 9 , Figure 9 1 represents the reference medicinal material of Amomum villosum (Yangchun Amomum villosum), 2-4 correspond to batch numbers YP01-YP03 Yangchun Amomum villosum slices, 5-7 correspond to batch numbers YP04-YP06 Green Shell Amomum villosum slices, and 8-10 correspond to batch numbers YP07-YP09 Hainan Amomum villosum slices.

[0137] Example 4

[0138] This embodiment provides a method for determining the content of vanillic acid and epicatechin in Amomum villosum granules (based on Amomum villosum var. yangchunense), including the following steps:

[0139] Test solution: Take an appropriate amount of Amomum villosum granules, grind it into a fine powder, take about 0.5g, weigh it accurately, add 25ml of 70% methanol accurately, weigh it, sonicate it (power 250W, frequency 40KHz) for 45min, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0140] Reference solution: Take appropriate amounts of vanillic acid and epicatechin reference standards, accurately weigh them, and add methanol to prepare a mixed reference solution containing 12 μg vanillic acid and 40 μg epicatechin per 1 ml of reference solution.

[0141] Determination: The determination was performed according to high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition). Octadecylsilane-bonded silica gel was used as the packing material. The chromatographic column was a Waters CORTECS T3, 2.1 × 100 mm, 1.6 µm. Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B. Gradient elution was performed at a flow rate of 0.3 ml / min, a column temperature of 30℃, and a detection wavelength of 260 nm. The gradient elution program was as follows: 0-5 min, 7% → 15% mobile phase A, 93% → 85% mobile phase B; 5-6 min, 15% → 30% mobile phase A, 85% → 70% mobile phase B. The theoretical plate number, calculated based on the vanillic acid peak, should be no less than 5000.

[0142] Take 1 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine them.

[0143] Adaptability assessment:

[0144] Injection was performed under the chromatographic conditions described above, and the results are shown in Table 1 and... Figure 10 The vanillic acid and epicatechin chromatographic peaks in the Amomum villosum formula granules were not interfered with by other chromatographic peaks, and the separation was good, meeting the requirements for content determination. The theoretical plate number was greater than 5000, which met the requirements.

[0145] Table 1. Adaptability parameters of chromatographic peak system

[0146]

[0147] The contents of vanillic acid and epicatechin in Amomum villosum granules with batch numbers K1-K3 were calculated, and the results are shown in Table 2. The specific calculation method was as follows: by measuring the peak areas of vanillic acid and epicatechin in the reference solution and the test solution, the contents of vanillic acid and epicatechin in the Amomum villosum granules were calculated using the external standard method. The results in Table 2 show that the contents of vanillic acid and epicatechin in the Amomum villosum granules meet the requirements.

[0148] Table 2. Results of Granule Content Determination in Three Batches of Amomum villosum Formula

[0149]

[0150] The upper and lower limits of the specified values ​​in Table 2 were obtained from 17 batches of freeze-dried Amomum villosum standard decoction powder. Table 3 shows the vanillic acid and epicatechin content in the 17 batches of freeze-dried Amomum villosum standard decoction powder, and the upper and lower limits were obtained based on the vanillic acid and epicatechin content of the 17 batches of freeze-dried Amomum villosum standard decoction powder. Among them, the detection limit for vanillic acid content is 0.5-2.8 mg / g, and the detection limit for epicatechin content is 1.1-3.8 mg / g.

[0151] Table 317 shows the vanillic acid and epicatechin content in freeze-dried powder of Amomum villosum standard decoction.

[0152]

[0153] Example 5

[0154] This embodiment provides a method for determining the content of bornyl acetate in Amomum villosum granules (based on Amomum villosum var. yangchunense), including the following steps:

[0155] Test solution: Accurately weigh 30g of the formula granules for testing, and conduct the test according to Method A of the Determination of Volatile Oil (General Chapter 2204, Chinese Pharmacopoeia 2020 Edition). Place the mixture in a round-bottom flask, add 300ml of water and several glass beads, shake to mix, connect the volatile oil tester, add water from the top of the tester until it fills the mark and overflows into the flask, then add 5ml of n-hexane, connect the reflux condenser, heat and maintain a gentle boil for 3 hours, cool, and after the solution has clearly separated into layers, separate the n-hexane solution and place it in a 10ml volumetric flask. Wash the inner wall of the volatile oil tester with a small amount of n-hexane several times, and combine the n-hexane washings with the same volumetric flask. Add n-hexane to the mark, shake well, then accurately measure 1ml into a 20ml volumetric flask, add n-hexane to the mark, shake well, and obtain the test solution.

[0156] Reference solution: Weigh an appropriate amount of bornyl acetate reference standard accurately, add n-hexane to prepare a reference solution containing 0.2 mg of bornyl acetate per 1 ml of solution.

[0157] Determination: Gas chromatography (General Chapter 0521, Chinese Pharmacopoeia 2020 Edition) was used for determination. Chromatographic conditions: Chromatograph: Shimadzu GC2010 PLUS; Capillary column with 100% dimethylpolysiloxane as the stationary phase; Column: Agilent DB-1, specifications: column length 30m, inner diameter 0.25mm, film thickness 0.25μm; Column temperature program: initial temperature 100℃, increased to 230℃ at a rate of 20℃ / min, held for 20 minutes; Injector temperature 230℃; Detector (FID) temperature 250℃; Split ratio 10:1; The theoretical plate number calculated based on the bornyl acetate peak should not be less than 10000.

[0158] Accurately pipette 1 μL each of the reference solution and the test solution into the gas chromatograph and determine their properties.

[0159] Adaptability assessment

[0160] Injection was performed under the chromatographic conditions described above for the content determination method. The results are shown in Table 4 and... Figure 11 The test results show that there are no other chromatographic peaks around the chromatographic peak of borneol acetate in the Amomum villosum formula granules, the separation is good, the theoretical plate number is greater than 10,000, which meets the requirements for content determination.

[0161] Table 4. Adaptability parameters of chromatographic peak system

[0162]

[0163] The content of bornyl acetate in Amomum villosum granules with batch numbers K1-K3 was calculated, and the results are shown in Table 5. Specific calculation method: The content of bornyl acetate in Amomum villosum granules was calculated using the external standard method by measuring the peak area of ​​bornyl acetate in the reference solution and the test solution. The results in Table 5 show that the content of bornyl acetate in Amomum villosum granules meets the requirements.

[0164] Table 5. Results of Granule Content Determination in Three Batches of Amomum villosum (Yangchun Amomum) Formula

[0165]

[0166] The upper and lower limits of the specified values ​​in Table 5 were obtained from 17 batches of freeze-dried Amomum villosum standard decoction powder. Table 6 shows the content of borneol acetate in the 17 batches of freeze-dried Amomum villosum standard decoction powder, and the upper and lower limits of the specified values ​​were obtained based on the content of borneol acetate in the 17 batches of freeze-dried Amomum villosum standard decoction powder. Among them, the limit of borneol acetate content is 4.1-13.0 mg / g.

[0167] Table 6. Content of Borneol Acetate in Freeze-Dried Powder of Amomum villosum Standard Decoction in Batch 617

[0168]

[0169] Example 6

[0170] This embodiment provides a method for preparing Amomum villosum granules (based on Amomum villosum var. yangchunense), where 1g of granules corresponds to 4.5g of medicinal slices, including the following steps:

[0171] (1) Take the Amomum villosum medicinal material, remove impurities, crush it, soak it in water for 30 minutes, decoct it, and collect the volatile oil at the same time. Add β-cyclodextrin and water to the volatile oil, grind it in a colloid mill for 30 minutes, and encapsulate the volatile oil to obtain the volatile oil inclusion complex. The mass ratio of volatile oil, β-cyclodextrin and water is 1:7:21. The time for collecting the volatile oil is 30 minutes, and the decoction time is 30 minutes.

[0172] (2) The decoction was filtered through a 200-mesh filter and concentrated under reduced pressure at 70°C to a liquid with a relative density of 1.06-1.14 g / ml (60±5°C);

[0173] (3) The extract and volatile oil inclusion complex are mixed evenly, spray-dried, maltodextrin is added, mixed evenly, and granulated. The parameters set for the spray drying instrument are: inlet air temperature 180℃, outlet air temperature 95℃, and induced draft frequency 40Hz.

[0174] Research on the Granulation Process of Amomum villosum Formula

[0175] (1) Investigate the effects of different extraction times on the content of volatile oil and the content of bornyl acetate in volatile oil.

[0176] Take Amomum villosum slices and extract volatile oil according to Example 6. The difference is that the volatile oil was extracted at different time periods and different extraction times according to the table below. The results are shown in the table below.

[0177] Table 7. Content of volatile oil and bornyl acetate obtained at different time periods and extraction times.

[0178]

[0179] The results above show that the total amount of volatile oil reached a high level in the first 10 minutes of extraction. After 30 minutes, the oil extraction rate of volatile oil decreased significantly, indicating that the volatile oil can be basically extracted after 30 minutes of extraction. The content of bornyl acetate per unit of volatile oil obtained at different time periods was basically the same. Therefore, the optimal extraction time for volatile oil is 30 minutes.

[0180] (2) Take 100g of Amomum villosum slices, use an electric heating mantle to heat and reflux for decoction, add water to soak for 30min, after soaking, add different amounts of water (14 times, 18 times, 22 times) and decoct for 30min, filter with 200 mesh filter cloth, and obtain the result. Two parallel experiments were conducted and the results are shown in the table below.

[0181] Table 8. Content of vanillic acid and epicatechin obtained by decoction with different amounts of water.

[0182]

[0183] The results above show that the yield, vanillic acid content, and epicatechin content of extracts obtained by adding different amounts of water are relatively similar. Therefore, the preferred amount of water added is 14-22 times.

[0184] (3) Extraction time

[0185] Take slices of Amomum villosum, soak them in water for 30 minutes, then decoct them for 30 minutes, 45 minutes, and 60 minutes respectively. Filter the decoction through a 200-mesh filter cloth to obtain the final product. Two parallel experiments were conducted, and the results are shown in the table below.

[0186] Table 9. Content of vanillic acid and epicatechin obtained at different decoction times

[0187]

[0188] The results above show that the extraction time has little effect on the yield and content of key components. Therefore, the optimal extraction time is 30-60 min (i.e., the decoction time of the medicinal materials).

[0189] (4) Encapsulation process

[0190] ① Ratio of volatile oil to packaging excipients

[0191] When encapsulating volatile oils, the ratio of volatile oil to water is 1:21. Taking the ratio of volatile oil to β-cyclodextrin as a variable, the mass ratio of volatile oil to β-cyclodextrin and the corresponding inclusion rate of volatile oil inclusion compounds are shown in the table below.

[0192] Table 10 Inclusion rates of volatile oil inclusion compounds obtained with different inclusion excipients

[0193]

[0194] Note: Each group consists of 2 parallel experiments.

[0195] The results show that when the ratio of volatile oil to β-cyclodextrin is 1:7, the effect reaches a high level. Therefore, 1:7 is preferred as the mass ratio of volatile oil to β-cyclodextrin.

[0196] ②Ratio of volatile oil to water

[0197] When the volatile oil was included, the ratio of volatile oil to β-cyclodextrin was 1:7. The mass ratio of volatile oil to water and the corresponding inclusion rate of the volatile oil inclusion complex are shown in the table below, with the ratio of volatile oil to water as the variable.

[0198] Table 11 Inclusion rates of volatile oil inclusion compounds obtained with different inclusion excipients

[0199]

[0200] Note: Each group consists of 2 parallel experiments.

[0201] The results show that, taking into account both the inclusion rate of the volatile oil inclusion complex and the amount of raw materials, the optimal mass ratio of volatile oil to water is 1:21.

[0202] ③ Enclosure time

[0203] When encapsulating the volatile oil, the mass ratio of volatile oil, β-cyclodextrin, and water was 1:7:21. The colloid milling time and encapsulation rate are shown in the table below.

[0204] Table 12 Inclusion rates obtained at different grinding times

[0205]

[0206] Note: Each group consists of 2 parallel experiments.

[0207] The above results indicate that, taking into account both grinding time and inclusion rate, the optimal grinding time for the colloid is 30 min.

[0208] Based on the above experimental results, the preferred volatile oil encapsulation process is as follows: the preferred mass ratio of volatile oil, encapsulation additives, and water is 1:7:21, and the colloid grinding time is 30 min.

[0209] (4) Concentration process

[0210] According to the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules," the concentration temperature and concentration time of Amomum villosum (Yangchun Amomum) decoction were investigated to clarify the impact of each factor on the loss rate of vanillic acid and epicatechin content. Finally, the vacuum degree for concentrating Amomum villosum (Yangchun Amomum) formula granules was set to -0.08 MPa to -0.04 MPa, the density of the fluid extract was set to 1.06 to 1.14 g / mL (60℃±5℃), and the concentration temperature was controlled to not exceed 70℃.

[0211] (5) Drying process

[0212] Spray drying is chosen as the drying method because it has a fast drying speed, short material heating time, suitable vacuum degree, and less loss of volatile oil. Finally, the spray drying parameters were determined (extract density 1.04-1.12 g / mL (60℃±5℃), inlet air temperature 150-210℃, outlet air temperature 70-115℃, induced draft frequency 30Hz~50Hz) to further study the influence of spray drying method on the index components of Amomum villosum (Yangchun Amomum).

[0213] (6) Amount of excipients

[0214] Take the same amount of Amomum villosum granules extract, add excipients equivalent to 0%, 3%, and 5% of the amount of medicinal slices, spray dry, and determine the yield, moisture, solubility, and content of the extract. The results are shown in the table below.

[0215] Table 13 Effects of different excipients on the formulation granules

[0216]

[0217] The above results show that the drying process of Amomum villosum granules can be smooth and with minimal loss without the addition of excipients. The resulting granule extract powder has good solubility and is suitable for granulation.

[0218] Based on the above results, the drying process for Amomum villosum (Yangchun Amomum) formula granules is tentatively set as follows: the density of the extract is controlled at 1.06~1.14g / mL (60℃±5℃), volatile oil inclusion complex is added, mixed evenly, and spray dried. The spray drying parameters are: inlet air temperature 150-210℃, outlet air temperature 70-115℃, and induced draft frequency 30Hz~50Hz.

[0219] (7) Molding process research

[0220] ① Study on the powder characteristics of the formulated granule extract

[0221] Determination of the angle of repose

[0222] The angle of repose is the maximum angle formed between the free slope of a powder accumulation layer and the horizontal plane.

[0223] Evaluation criteria: Angle of repose ≤ 30° indicates that the material has excellent flowability; angle of repose ≤ 40° indicates that the material's flowability meets the requirements of the production process; angle of repose > 40° indicates that the material has poor flowability and cannot meet the requirements of the production process, requiring the addition of an appropriate amount of lubricant to change its flowability before granulation.

[0224] Table 14 Results of Angle of Repose Measurement

[0225]

[0226] Note: T1 is an extract of Amomum villosum obtained without the addition of excipients before granulation, without the granulation step.

[0227] Conclusion: The angle of repose of the Amomum villosum (Yangchun Amomum) formula granule extract is all <40°, indicating that the flow properties of the Amomum villosum formula granule extract meet the flowability requirements required in the production process.

[0228] Determination of bulk density

[0229] Bulk density refers to the density obtained by dividing the mass of a powder by the volume of the container occupied by the powder. It is divided into loose density and compaction.

[0230] Evaluation criteria: The lower the bulk density, the larger the air gaps in the powder, and the less powder can be packed per unit volume; conversely, the smaller the air gaps in the powder, the more powder can be packed per unit volume. From a production perspective, the higher the bulk density, the better, but excessively high bulk density can lead to poor powder flowability.

[0231] Table 15 Results of extract bulk density determination

[0232]

[0233] Conclusion: The bulk density of the Amomum villosum granule extract is 0.35 g·cm³. -3 ~0.43 g·cm -3 Within this range, it indicates that the interstitial spaces in the Amomum villosum formula granule extract powder are relatively large, and the mass per unit volume is relatively small.

[0234] ② Formulation process investigation

[0235] Dry granulation is a method of uniformly mixing drugs and excipients, compressing them into large flakes or strips, and then pulverizing them into granules of the desired size. Compared with traditional wet granulation, it eliminates the processes of preparing soft materials, drying, and granulation, simplifying the process and overcoming the disadvantage of large excipient usage, thus increasing drug loading. Based on the characteristics of the Amomum villosum granulated extract, dry granulation was chosen as the formulation process.

[0236] The effect of 0% and 40% of the additives on the granulation effect was investigated. The granulation parameters were: roller speed 4~7 rpm; extrusion pressure 5~9 MPa; and feeding speed 20~30 rpm.

[0237] Table 16 Results of Process Investigation with Different Amounts of Additives

[0238]

[0239] Note: The extracts in the table are in powder form; Particle 1 is the particle obtained by dry granulation of extract T1 without the addition of excipients; Particle 2 is the particle obtained by dry granulation of extract T1 with 40% excipients.

[0240] Conclusion: Under the same granulation process parameters, different amounts of excipients can be added to prepare Amomum villosum granules well, and all evaluation indicators meet the production requirements. This indicates that the addition of different amounts of excipients has little impact on the granulation process parameters of Amomum villosum granules. In other words, the same granulation process parameters can meet the formulation requirements of Amomum villosum granules with different excipient ratios.

[0241] (8) Performance study of the Amomum villosum granules prepared by the above method

[0242] ① For the Amomum villosum granules with batch numbers K1-K3, the particle size distribution was determined by the double sieve method, Method II (General Chapter 0982 of the Chinese Pharmacopoeia 2020). The total particle size distribution of the granules that could not pass through sieve No. 1 and that could pass through sieve No. 5 should not exceed 15%. The particle size results of the three batches of granules were 5.8%, 3.8%, and 3.1%, respectively, all of which met the requirements.

[0243] ②The moisture content of the Amomum villosum granules in batches K1-K3, determined by the toluene method (General Chapter 0832, Chinese Pharmacopoeia 2020 Edition), shall not exceed 8.0%. The moisture content of the three batches of granules were 3.3%, 3.9%, and 3.9%, respectively, which meet the requirements.

[0244] ③ For the Amomum villosum granules with batch numbers K1-K3, the solubility test for granules was performed according to (General Chapter 0104 of the Chinese Pharmacopoeia 2020 Edition). 1g of the packaged weight of the Amomum villosum granules was added to 200ml of hot water and stirred for 5 minutes. Immediate observation was required; the granules should completely dissolve without any charred residue or other foreign matter. The solubility results for all three batches of granules were: completely dissolved, with no charred residue, meeting the requirements.

[0245] ④ For the Amomum villosum granules with batch numbers K1-K3, the content variation of granules was determined according to (General Chapter 0104 of the Chinese Pharmacopoeia 2020 Edition). The content variation limit for this product was 1g ± 10%. The content variation of all three batches of granules met the requirements.

[0246] ⑤ Take an appropriate amount of this product, grind it into a fine powder, accurately weigh 2g, accurately add 100ml of ethanol, and determine the soluble extract by the hot extraction method under the "Determination of Alcohol-Soluble Extractives" section (General Chapter 2201, Chinese Pharmacopoeia 2020 Edition). The batch numbers are 2102001Y, 2102002Y, 2102003Y, 2102004Y, 2102005Y, 2102006Y, 2102007Y, 2102008Y, 2102009Y, 2102011Y, and 2102014. The measured range of extractives from the freeze-dried powder of Amomum villosum standard decoction (Y, 2102016Y, 2102020Y, 2102021Y, 2102024Y, 2109025Y, and 2109026Y) was 13.5%-27.0%. The extractives from Amomum villosum formulation granules (K1-K3) were tested in parallel with two groups per batch. The extractives were 19.5%, 13.9%, and 19.8%, with RSDs of 0.7%, 0.5%, and 1.4%, respectively, all meeting the requirements. The extractives from all three batches of granules were within the measured range of the freeze-dried powder extractives from the Amomum villosum standard decoction.

[0247] ⑥ Take 30g of this product, add 500ml of water and follow the method for determination of volatile oil (General Chapter 2204 of Chinese Pharmacopoeia 2020). The volatile oil content of the freeze-dried powder of Amomum villosum standard decoction with batch numbers 2102001Y, 2102002Y, 2102003Y, 2102004Y, 2102005Y, 2102006Y, 2102007Y, 2102008Y, 2102009Y, 2102011Y, 2102014Y, 2102016Y, 2102020Y, 2102021Y, 2102024Y, 2109025Y, and 2109026Y ranges from 1.0% to 3.2%, in ml / g. The volatile oil content of the granules in the formula is specified to be 1.0% to 3.2%, in ml / g. The volatile oil content of the K1-K3 Amomum villosum granules was tested, with two parallel experiments for each batch of granules. The results were 2.3%, 2.2%, and 2.3%, with RSDs of 0%, 0.1%, and 0.9%, respectively, all meeting the requirements. The extracts from the three batches of granules were within the measured range of the extracts from the freeze-dried powder of the standard Amomum villosum decoction.

[0248] The above results demonstrate that the properties of the Amomum villosum granules prepared by this invention meet the production requirements.

[0249] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for determining the content of vanillic acid and epicatechin in Amomum villosum, characterized in that, Includes the following steps: Test solution: The test solution is prepared by extracting the test sample with 70% methanol; the test sample is Amomum villosum medicinal material, Amomum villosum slices, Amomum villosum formula granules or freeze-dried powder of Amomum villosum standard decoction; Reference solution: Prepare a reference solution by mixing vanillic acid and epicatechin reference standards; Determination: Determined by ultra-high performance liquid chromatography; Chromatographic conditions: acetonitrile as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution; The gradient elution program is as follows: 0-5 min, 7% → 15% mobile phase A, 93% → 85% mobile phase B; 5-6 min, 15% → 30% mobile phase A, 85% → 70% mobile phase B; The chromatographic column used for the determination was a Waters CORTECS T3, 2.1 × 100 mm, 1.6 µm; The measurement wavelength is 255–265 nm.

2. The determination method according to claim 1, characterized in that, Chromatographic conditions also include: The flow rate was 0.28–0.32 ml / min; The column temperature is 28–32℃; The injection volume is 1–3 μL.

3. The determination method according to claim 1 or 2, characterized in that, Each 1 ml of the reference solution contains 5–20 μg of vanillic acid; Each 1 ml of the reference solution contains 20–80 μg of epicatechin; The detection limit for vanillic acid in the cardamom is 0.5–2.7 mg / g; The detection limit for epicatechin in the cardamom is 1.1–3.8 mg / g.

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