A method for constructing a characteristic spectrum of tsaoko kernels and their preparations and a method for distinguishing them from counterfeits

Abstract

The present invention belongs to the field of traditional Chinese medicine detection technology, and specifically relates to a method for constructing a characteristic spectrum of a tsaoko kernel and its preparation and a method for distinguishing the same from counterfeit products, comprising the following steps: (1) preparing a test solution; (2) taking the test solution and detecting it by high performance liquid chromatography, with a wavelength of 238-242 nm, octadecylsilane bonded silica gel as a filler, a mobile phase comprising an aqueous solution containing phosphoric acid and acetonitrile, and a gradient elution program comprising: 0 → 20 min → 30 min → 50 min, and the volume percentage of acetonitrile in the mobile phase being: 4% → 19% → 34% → 45%. The characteristic spectrum obtained by the method has a stable baseline, good characteristic peak shape, high separation, and can accurately locate the peak positions of protocatechuic acid, proanthocyanidin B2, and citral (neral, geranial), fully reflecting the integrity and characteristic properties of the tsaoko kernel and its preparation, and providing a basis for quality detection and control of the tsaoko kernel and its preparation.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for constructing a characteristic spectrum of tsaoko kernels and their preparations and a method for distinguishing them from counterfeits. Background Art

[0002] Tsao-ko (Amomum tsao-ko Crevost et Lemaire), a plant of the ginger family, is the dried, mature fruit of the tsao-ko plant. Its seeds are the tsao-ko kernels, which are often used as medicine in Traditional Chinese Medicine. The fruit is harvested in autumn when ripe, freed of impurities, and sun-dried or low-temperature dried. It is oblong, with three blunt ridges, 2 to 4 cm long, and 1 to 2.5 cm in diameter. Its surface is grayish-brown to reddish-brown, with longitudinal grooves and ridges. It has a rounded, protruding column at the apex, and a pedicel or pedicel scar at the base. The cortex is tough and easily torn longitudinally. Peeling the outer skin reveals a yellowish-brown septum in the middle, dividing the seed mass into three lobes, each containing 8 to 11 seeds. The seeds are conical, polyhedral, approximately 5 mm in diameter. They have a reddish-brown surface, covered with an off-white, membranous aril. The ridge is a longitudinal groove, with a concave hilum at the apex. They are hard, with an off-white endosperm. They have a distinctive aroma and a pungent, slightly bitter taste. Their main functions include drying dampness, warming the middle-Jiangsu region, and relieving malaria and expectoration. It is used for internal obstruction of cold and dampness, abdominal distension and pain, fullness and vomiting, chills and fever due to malaria, and fever due to plague.

[0003] Amomum villosum kernel is a botanical medicine, and its components are mostly volatile oils, terpenes, phenols, flavonoids, etc. The 2020 edition of the Chinese Pharmacopoeia only includes properties, microscopic identification, thin layer identification, and volatile oil content determination under the "Amomum villosum kernel" item.

[0004] Existing research on the quality standards of Amomum villosum kernels or their identification from common counterfeits has primarily focused on properties, identification, content determination, and fingerprint analysis. Previously, GC-MS chromatography was the most common method for fingerprint identification in the study of these components. Another study employed HPLC, using protocatechuic aldehyde and parahydroxybenzoic acid as reference substances, to analyze the HPLC fingerprints of 15 batches of Amomum villosum kernels from different origins. The results identified 21 common characteristic peaks and a common fingerprint pattern for Amomum villosum kernels. However, the separation of the characteristic peaks was poor, and the distinctness was unclear. Especially for Tsaoko kernel preparations, since the effective ingredients of preparations such as Chinese medicine formula granules prepared from Tsaoko kernel water extracts are obviously different from the medicinal materials themselves after extraction, grinding, concentration, drying and other processes, the characteristic spectrum constructed by this method has fewer characteristic peaks and the separation effect is worse. In addition, the use of high-performance liquid chromatography to distinguish Tsaoko kernels and their preparations from common counterfeits is still a blank. How to improve the detection standards of Tsaoko kernels and their preparations and comprehensively control the quality of the finished products is a technical problem that the present invention needs to solve. Summary of the Invention

[0005] Therefore, the first purpose of the present invention is to provide a method for constructing a characteristic spectrum of cardamom kernels and their preparations. This method strengthens specific identification and multi-component and holistic quality control based on the characteristics of cardamom kernels and their preparations, and establishes a characteristic spectrum of this variety. The method has a large number of characteristic peaks with high separation and obvious characteristic, which can be used for comprehensive quality control of cardamom kernels and their preparations.

[0006] Specifically, the present invention discloses a method for constructing a characteristic spectrum of tsaoko kernel and its preparation, comprising the following steps:

[0007] (1) Preparation of test solution;

[0008] (2) The sample solution was detected by high performance liquid chromatography at a wavelength of 238-242 nm, using octadecylsilane bonded silica gel as the filler, the mobile phase comprising an aqueous solution containing phosphoric acid and acetonitrile, and the gradient elution program comprising: 0 → 20 min → 30 min → 50 min, and the volume percentage of acetonitrile in the mobile phase was: 4% → 19% → 34% → 45%.

[0009] In the present invention, the tsaoko kernel and its preparation can be tsaoko kernel medicinal materials, tsaoko kernel decoction pieces, or preparations made from tsaoko kernel water extract, such as powders, granules, tablets, etc.

[0010] In certain preferred embodiments, in step (2), the flow rate is 0.25-0.32 mL / min, the column temperature is 28-32° C., and the injection volume is 1-10 μL.

[0011] Furthermore, the volume percentage of phosphoric acid in the phosphoric acid-containing aqueous solution is 0.08-0.12%.

[0012] Furthermore, the gradient elution program also includes 50 min→51 min→56 min→56.01 min→62 min, and the volume percentage of acetonitrile in the mobile phase is: 45%→95%→95%→4%→4%.

[0013] In certain preferred embodiments, step (1) comprises weighing a test sample of tsaoko kernel, extracting it with a solvent to obtain an extract, separating the solid from the liquid, and obtaining the liquid, which is the test sample solution;

[0014] In certain preferred embodiments, the step (1) further satisfies any one or more of the following AEs:

[0015] A. The mass to volume ratio of the tsaoko kernel sample to the solvent is 0.1-0.4:15-50; the mass to volume ratio is g / mL;

[0016] B. The extraction method is reflux extraction or ultrasonic extraction;

[0017] C. Extraction time is ≥30 min, preferably 30-60 min;

[0018] D. The solid-liquid separation is selected from centrifugation or filtration;

[0019] E. The solvent is selected from at least one of methanol, water and ethanol; preferably a methanol-water solution with a volume percentage of 30-70%.

[0020] In certain preferred embodiments, the construction method further includes the steps of preparing a reference solution using at least one solubilizing agent selected from protocatechuic acid, proanthocyanidin B2, and citral (including neral and geranial), and detecting the reference solution by high performance liquid chromatography according to any of the construction methods described in the present invention to obtain a reference spectrum; and / or, using tsaoko kernel as a reference material, preparing a reference solution according to step (1) of any of the construction methods described in the present invention, and detecting the reference solution by high performance liquid chromatography according to any of the construction methods described in the present invention to obtain a reference spectrum.

[0021] Preferably, each 1 mL of the reference substance solution contains 5-50 μg of each reference substance; and / or, the solvent used in the preparation of the reference substance solution is selected from methanol or a methanol aqueous solution with a volume fraction of not less than 50%.

[0022] Certain preferred embodiments further include constructing a comparative characteristic spectrum of tsaoko kernels and their preparations. Characteristic spectra obtained from testing multiple batches of tsaoko kernels and their preparations are generated using a traditional Chinese medicine chromatographic characteristic spectrum similarity evaluation system. At least two batches of tsaoko kernel granules are used, for example, 4 batches, 7 batches, 15 batches, and 17 batches of tsaoko kernel granules.

[0023] In certain preferred embodiments, after generating the control characteristic spectrum of Tsaoko kernel and its preparation using the Chinese medicine chromatographic characteristic spectrum similarity evaluation software, the step of marking the common characteristic peaks is also included.

[0024] The second purpose of the present invention is to provide a method for identifying cardamom kernels and their preparations from counterfeits. The method can identify and distinguish cardamom kernels from a variety of common counterfeits and their preparations, such as Amomum villosum, Amomum villosum longifolia, Amomum villosum niuginosa, Amomum villosum fragrant, wild cardamom, and pseudo cardamom, so as to better monitor the quality of cardamom kernel preparations.

[0025] To this end, the present invention also provides a method for distinguishing tsaoko kernels and their preparations from counterfeits, comprising the step of comparing a characteristic spectrum of the product to be identified with a control characteristic spectrum of the tsaoko kernels and their preparations; the characteristic spectrum of the product to be identified is constructed using the product to be identified according to any of the above-mentioned construction methods, and the control characteristic spectrum of the tsaoko kernels and their preparations is selected from any one of the following (1)-(4):

[0026] (1) It has 15 common characteristic peaks, the peak corresponding to the reference peak of proanthocyanidin B2 is the S1 peak, and the relative retention times of peaks 1 to 3 and peaks 5 to 10 with respect to the S1 peak are within the range of ±10% of the specified value; the specified values ​​of peaks 1 to 3 and peaks 5 to 10 are: 0.38, 0.60, 0.80, 1.11, 1.27, 1.31, 1.40, 1.72 and 1.88, respectively; Peak 15 is the S2 peak, and the relative retention times of peaks 11 to 14 with respect to the S2 peak are within the range of ±10% of the specified value, and the specified values ​​of peaks 11 to 14 are: 0.77, 0.87, 0.94, 0.95, respectively;

[0027] (2) It has 15 common characteristic peaks, of which 4 peaks correspond to the retention times of the reference peaks of protocatechuic acid, proanthocyanidin B2, neral, and geranyl aldehyde, respectively. The peak corresponding to the reference peak of proanthocyanidin B2 is the S1 peak. The relative retention times of peaks 2 to 3 and peaks 5 to 10 with respect to the S1 peak are within the range of ±10% of the specified value. Peak 15 is the S2 peak, and the relative retention times of peaks 11 to 13 with respect to the S2 peak are within the range of ±10% of the specified value. The specified values ​​are: 0.60 (peak 2), 0.80 (peak 3), 1.11 (peak 5), 1.27 (peak 6), 1.31 (peak 7), 1.40 (peak 8), 1.72 (peak 9), 1.88 (peak 10), 0.77 (peak 11), 0.87 (peak 12), and 0.94 (peak 13).

[0028] (3) Characteristic spectra of the tsaoko kernels and / or their preparations obtained by any of the above-mentioned construction methods using a single batch or multiple batches of tsaoko kernels and / or their preparations as test samples;

[0029] (4) Using multiple batches of tsaoko kernels and / or their preparations as test samples, the characteristic spectra obtained according to any of the construction methods described above are used to prepare a control characteristic spectra by using the average value or median method.

[0030] Furthermore, in the control characteristic spectrum of the tsaoko kernel and its preparation, the relative peak area of ​​peak 7 and peak 1 is within the specified value range, and the specified value is: not less than 5.9 (peak 7).

[0031] Furthermore, the counterfeit products include one or more of Amomum villosum and its preparations, Alpinia fasciata seeds and its preparations, Amomum villosum and its preparations, and Amomum villosum and its preparations; preferably, the Amomum villosum includes one or more of Amomum villosum yangchunensis, Amomum villosum longifolia, Amomum villosum niugu, and Amomum villosum fragrant.

[0032] If the characteristic spectrum of the product to be identified shows 15 characteristic peaks corresponding to the control characteristic spectrum of the cardamom kernel formula granules, and / or the relative peak area of ​​peak 7 and peak 1 is higher than 5.9, then it is cardamom kernel or its preparation, otherwise it is not.

[0033] The present invention also provides a method for detecting the quality of grass kernels and their preparations, comprising the step of comparing a characteristic spectrum of the grass kernel product to be tested with a control characteristic spectrum of the grass kernel and their preparations; the characteristic spectrum of the grass kernel product to be tested is obtained by using the grass kernel product to be tested according to any of the construction methods described above, and the control characteristic spectrum of the grass kernel and their preparations is selected from any one of the following (1) to (4):

[0034] (1) It has 15 common characteristic peaks, the peak corresponding to the reference peak of proanthocyanidin B2 is the S1 peak, and the relative retention times of peaks 1 to 3 and peaks 5 to 10 with respect to the S1 peak are within the range of ±10% of the specified value; the specified values ​​of peaks 1 to 3 and peaks 5 to 10 are: 0.38, 0.60, 0.80, 1.11, 1.27, 1.31, 1.40, 1.72 and 1.88, respectively; Peak 15 is the S2 peak, and the relative retention times of peaks 11 to 14 with respect to the S2 peak are within the range of ±10% of the specified value, and the specified values ​​of peaks 11 to 14 are: 0.77, 0.87, 0.94, 0.95, respectively;

[0035] (2) It has 15 common characteristic peaks, of which 4 peaks correspond to the retention times of the reference peaks of protocatechuic acid, proanthocyanidin B2, neral, and geranyl aldehyde, respectively. The peak corresponding to the reference peak of proanthocyanidin B2 is the S1 peak. The relative retention times of peaks 2 to 3 and peaks 5 to 10 with respect to the S1 peak are within the range of ±10% of the specified value. Peak 15 is the S2 peak, and the relative retention times of peaks 11 to 13 with respect to the S2 peak are within the range of ±10% of the specified value. The specified values ​​are: 0.60 (peak 2), 0.80 (peak 3), 1.11 (peak 5), 1.27 (peak 6), 1.31 (peak 7), 1.40 (peak 8), 1.72 (peak 9), 1.88 (peak 10), 0.77 (peak 11), 0.87 (peak 12), and 0.94 (peak 13).

[0036] (3) Characteristic spectra of the tsaoko kernels and / or their preparations obtained by any of the above-mentioned construction methods using a single batch or multiple batches of tsaoko kernels and / or their preparations as test samples;

[0037] (4) Using multiple batches of tsaoko kernels and / or their preparations as test samples, the characteristic spectra obtained according to any of the construction methods described above are used to prepare a control characteristic spectra by using the average value or median method.

[0038] The tsaoko kernel product to be tested in the present invention can be tsaoko kernel medicinal material, tsaoko kernel decoction pieces, or a preparation made from tsaoko kernel water extract, such as powder, granules, tablets, etc.

[0039] The relative peak area is used to evaluate the quality of the tested grass kernel formula granule product. If the relative peak area between peak 1 and peak 7 of the characteristic spectrum of the tested grass kernel formula granule product is not less than 5.9, the quality is qualified; if it is less than 5.9, it is unqualified.

[0040] The technical solution of the present invention has the following advantages:

[0041] 1. The method for constructing the characteristic spectrum of the tsaoko kernel and its preparation described in the present invention has a wavelength of 238-242 nm, uses octadecylsilane bonded silica gel as a filler, and the mobile phase includes an aqueous solution containing phosphoric acid and acetonitrile. Through a specific gradient elution program, 15 common characteristic peaks are obtained, and the separation of common characteristic peaks including protocatechuic acid, proanthocyanidin B2, neral, and geranial is achieved. The elution program is simple, the obtained characteristic spectrum has a stable baseline, good characteristic peak shape, high separation, and can accurately locate the peak positions of protocatechuic acid, proanthocyanidin B2, and citral (neral, geranial), fully reflecting the integrity and characteristic of the tsaoko kernel and its preparation, and providing a basis for quality detection and control of the tsaoko kernel and its preparation.

[0042] 2. The method for constructing the characteristic spectrum of the tsaoko kernel and its preparation described in the present invention is to investigate the extraction conditions such as the optimization of chromatographic conditions, detection wavelength, extraction solvent, extraction time, etc., and determine the optimal extraction process, so that the peak area is higher, and the quality of the tsaoko kernel and its preparation can be more comprehensively monitored.

[0043] 3. The method for distinguishing the tsaoko kernels and their preparations from counterfeits of the present invention is to construct a characteristic spectrum using the above-mentioned method of the present invention and compare it with the control characteristic spectrum of the tsaoko kernels and their preparations, so as to distinguish the tsaoko kernels and their preparations from common counterfeits of Amomum villosum, including Amomum villosum yangchunensis, Amomum villosum longifolia, Amomum villosum niuginosa, and Amomum villosum fragrant, and their preparations, wild tsaoko and their preparations, pseudo-tsaoko and their preparations, and cardamom kernels and their preparations.

[0044] 4. The quality detection method of the grass kernel and its preparation described in the present invention can comprehensively, clearly and effectively detect the quality of the grass kernel and its preparation by comparing the characteristic spectrum of the grass kernel and its preparation product to be tested with the control characteristic spectrum of the grass kernel and its preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0046] Figure 1 The chromatogram is at 200 nm under the conditions of acetonitrile-0.1% phosphoric acid water;

[0047] Figure 2 Chromatogram at 210 nm under acetonitrile-0.1% phosphoric acid water conditions;

[0048] Figure 3 Chromatogram at 220 nm under acetonitrile-0.1% phosphoric acid water conditions;

[0049] Figure 4 Chromatogram at 230 nm under acetonitrile-0.1% phosphoric acid water conditions;

[0050] Figure 5 Chromatogram at 240 nm under acetonitrile-0.1% phosphoric acid water conditions;

[0051] Figure 6 The chromatogram is at 250 nm under the conditions of acetonitrile-0.1% phosphoric acid water;

[0052] Figure 7 Chromatogram at 260 nm under acetonitrile-0.1% phosphoric acid water conditions;

[0053] Figure 8 Chromatogram at 270 nm under acetonitrile-0.1% phosphoric acid water conditions;

[0054] Figure 9 Chromatogram at 280 nm under acetonitrile-0.1% phosphoric acid water conditions;

[0055] Figure 10 The chromatogram is at 300 nm under the conditions of acetonitrile-0.1% phosphoric acid water;

[0056] Figure 11 This is the chromatogram under the conditions of methanol-0.1% phosphoric acid water;

[0057] Figure 12 is the chromatogram of elution gradient 1;

[0058] Figure 13 is the chromatogram of elution gradient 2;

[0059] Figure 14 is the chromatogram of elution gradient 3;

[0060] Figure 15 is the chromatogram of elution gradient 4;

[0061] Figure 16 is the chromatogram of elution gradient 5;

[0062] Figure 17 is the chromatogram of elution gradient 6;

[0063] Figure 18 is the chromatogram of elution gradient 7;

[0064] Figure 19 is the chromatogram of elution gradient 8;

[0065] Figure 20 is the chromatogram of elution gradient 9;

[0066] Figure 21 This is the reference characteristic spectrum of the standard decoction of Tsaoko kernel slices; Peak 1: protocatechuic acid; Peak 4: proanthocyanidin B2; Peak 14 and Peak 15: citral (Peak 14: neral; Peak 15: geranial);

[0067] Figure 22 This is the characteristic spectrum of the reference medicinal material Amomum villosum;

[0068] Figure 23 Characteristic spectra of three batches of tsaoko kernel formula granules;

[0069] Figure 24 is the chromatogram of negative blank solution;

[0070] Figure 25 is the instrument precision chromatogram;

[0071] Figure 26 is a reproducible chromatogram;

[0072] Figure 27 Chromatograms for different personnel;

[0073] Figure 28 is the stability chromatogram;

[0074] Figure 29 The chromatogram was examined for a phosphoric acid concentration of 0.08%;

[0075] Figure 30 The chromatogram is for 0.10% phosphoric acid concentration;

[0076] Figure 31 The chromatogram is for 0.12% phosphoric acid concentration;

[0077] Figure 32 This is the protocatechuic acid reference substance spectrum;

[0078] Figure 33This is the reference sample of proanthocyanidin B2;

[0079] Figure 34 This is the citral reference substance spectrum;

[0080] Figure 35 This is the sample profile of the tsaoko kernel formula granules;

[0081] Figure 36 Comparison of the characteristic spectrum of Amomum villosum kernel and Amomum villosum; S1: Amomum villosum; S2: Amomum villosum;

[0082] Figure 37 Comparison of the characteristic spectra of Amomum villosum kernel and Amomum villosum; S1: Amomum villosum; S2-4: Amomum villosum;

[0083] Figure 38 Comparison of the characteristic spectrum of Amomum villosum and Amomum villosum; S1: Amomum villosum; S2: Amomum villosum;

[0084] Figure 39 Comparison of the characteristic spectra of Amomum villosum and Amomum villosum; S1: Amomum villosum; S2: Amomum villosum;

[0085] Figure 40 Comparison of the characteristic spectrum of Tsaoko kernel and Fragrant sand; S1: Tsaoko kernel; S2: Fragrant sand;

[0086] Figure 41 Comparison of the characteristic spectrum of tsaoko kernel and wild tsaoko; S1: tsaoko; S2-4: wild tsaoko;

[0087] Figure 42 Comparison of the characteristic spectrum of Amomum villosum kernel and Amomum villosum pseudo-Amomum villosum; S1: Amomum villosum; S2-4: Amomum villosum pseudo-Amomum villosum. DETAILED DESCRIPTION

[0088] The following examples are provided to further understand the present invention better, are not limited to the best mode of implementation, and do not limit the content and scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts falls within the scope of protection of the present invention. If specific experimental steps or conditions are not specified in the examples, the operation or conditions of the conventional experimental steps described in the literature in this area can be carried out. If the manufacturer of the reagents or instruments used is not specified, they are all conventional reagent products that can be obtained commercially.

[0089] Experimental Example 1 Investigation of Construction Method

[0090] 1. Instruments, reagents and test drugs

[0091] Chromatograph: Waters ACQUITY UPLC I-Class chromatography system, including quaternary solvent manager (ACQ-QSM), automatic sample injector (ACQ-FTN), original imported chromatography column oven (ACQ-CM), diode array ultraviolet detector (ACQ-TUV), and Empower chromatography management system.

[0092] Column: ACQUITY BEH C18 (column length: 150 mm, inner diameter: 2.1 mm, particle size: 1.7 μm); Waters symmetry column (250 mm×4.6 mm, 5 μm).

[0093] Electronic analytical balance: Mettler Toledo NewClassic MS 100,000th balance, Jing TianFA2044A 10,000th balance.

[0094] Test drug: The tsaoko kernel formula granules can be prepared by conventional methods in the art. For example, the present invention is prepared according to the following steps: 6800 g of tsaoko kernel slices are taken, crushed, and extracted once by boiling (100° C.). Water is added with a mass of 18 times the mass of the slices, and extracted for 1.5 hours to obtain an extract and collect a sufficient amount of volatile oil. The volatile oil is taken by grinding, and 7 times the mass of β-cyclodextrin is added to prepare an inclusion compound. The extract is filtered while hot through a 200-mesh filter cloth, and the filtrate is concentrated under reduced pressure at 70° C. to a relative density of 1.03 to 1.10 (65±5° C.). The inclusion compound is added to the concentrate, mixed, spray-dried, crushed, dry-granulated, packaged, and sealed for storage.

[0095] The freeze-dried powder of the standard decoction of tsaoko kernel slices can be prepared by conventional methods in the field. For example, in the present invention, it is prepared according to the following steps: take the tsaoko kernel slices, crush them, add 2 times the amount of water to soak for 30 minutes, add 6 times the amount of water, boil with boiling water and simmer (200W) for 5 minutes, filter with 200 mesh filter cloth while hot, add 6 times the amount of water to the residue, boil with high heat (500W), then turn to low heat (200W) and boil for 25 minutes, filter with 200 mesh filter cloth while hot, combine the two filtrates, and the filtrate is the standard decoction of tsaoko kernel slices.

[0096] Take the standard decoction of Tsaoko kernel slices and concentrate it at 50-65°C to a concentrate with a relative density of 1.02mg / ml-1.06mg / ml, freeze-dry it to obtain the freeze-dried powder of the standard decoction of Tsaoko kernel slices, and weigh it. The standard decoction of Tsaoko kernel slices was used as a test sample, and its volatile oil content was determined according to the volatile oil determination method (General Rules 2204 of the 2020 edition of the Chinese Pharmacopoeia). Calculate the volume of volatile oil contained in the standard decoction of Tsaoko kernel slices of equal mass and the mass of freeze-dried powder. According to this result, the freeze-dried powder and volatile oil equivalent to the same amount of slices were mixed, and the mixture of freeze-dried powder of the standard decoction of Tsaoko kernel slices and volatile oil was referred to as freeze-dried powder of the standard decoction of Tsaoko kernel slices (mixture containing volatile oil) hereinafter.

[0097] Protocatechuic acid reference substance (batch number: 110809-202207, purchased from the China Food and Drug Administration); eucalyptol reference substance (batch number: 110788-202108, purchased from the China Food and Drug Administration); proanthocyanidin B2 (batch number: wkq21031002, purchased from Sichuan Weikeqi Biotechnology Co., Ltd.); citral (batch number: 190178-201701, purchased from the China Food and Drug Administration); and Amomum villosum reference medicinal material (batch number 121550-201602, purchased from the China Food and Drug Administration). Methanol and acetonitrile were chromatographically pure, and water was ultrapure; all other reagents were of analytical grade.

[0098] 2. Optimization of chromatographic conditions

[0099] Take 0.2 g of freeze-dried powder (containing a mixture of volatile oils) of the standard decoction of Tsaoko kernel slices and place it in a stoppered conical flask. Add 25 ml of 50% methanol and weigh the weight. Ultrasonic extraction is performed for 30 minutes. The weight loss is made up with 50% methanol. Filter and collect the filtrate to obtain the test solution. The test solution is examined as follows.

[0100] (1) Investigation of absorption wavelength and organic phase

[0101] The test solutions were analyzed by high-performance liquid chromatography using either methanol-0.1% phosphoric acid aqueous solution or acetonitrile-0.1% phosphoric acid aqueous solution, respectively, under the following gradient conditions. The chromatographic column was a Waters symmetry column (250 mm × 4.6 mm, 5 μm), the column temperature was 30°C, the flow rate was 1 ml / min, and the injection volume was 10 μL. The wavelengths were 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, and 300 nm, respectively.

[0102] Table 1 Gradient elution program

[0103]

[0104] By the attached Figure 1-11 As can be seen, the 240 nm absorption wavelength exhibits more chromatographic peaks and a more moderate response compared to other wavelengths, so 240 nm was selected as the absorption wavelength for characteristic chromatographic analysis. Methanol as the mobile phase results in an unstable baseline, and acetonitrile has a stronger elution ability than methanol, so acetonitrile was selected as the mobile phase.

[0105] (2) Mobile phase gradient investigation

[0106] The above test solution was detected by high performance liquid chromatography. The chromatographic column was a Waters symmetry column (250 mm × 4.6 mm, 5 μm), the mobile phase was acetonitrile and 0.1% phosphoric acid water, and the following elution gradients 1 to 4 and flow rates were used for elution, the detection wavelength was 240 nm; the column temperature was 30°C, and 10 μL was injected.

[0107] Elution gradient 4 did not achieve good separation results and the analysis time was long. Therefore, our company continued to optimize the mobile phase gradient on the UPLC. The chromatographic column was a Waters ACQUITY BEH C18 column (150 mm × 2.1 mm, 1.7 μm), and the mobile phase was acetonitrile and 0.1% phosphoric acid water. The following elution gradients 5 to 9 and flow rates were used for elution, respectively. The detection wavelength was 240 nm; the column temperature was 30°C, and 1 μL was injected.

[0108] Table 2 Elution gradient 1 to 3

[0109]

[0110]

[0111] Table 3 Elution gradient 4 to 6

[0112]

[0113] Table 4 Elution gradient 7 to 9

[0114]

[0115] By the attached Figure 12-20 It can be seen that compared with other elution gradients, the characteristic spectrum peak shape and separation degree obtained by elution gradient 9 are better. The spectrum shown has a large amount of information, a stable baseline, a good peak shape, and a good separation degree (the separation degrees of the 15 characteristic peaks are all greater than 1.2). Therefore, this method is determined to be a characteristic spectrum construction method.

[0116] 3. Preparation of test solution

[0117] (1) Selection of extraction solvent

[0118] Take 0.2 g of the powder of the standard decoction of Tsaoko kernel slices (containing a mixture of volatile oils), accurately weigh it, place it in a stoppered conical flask, accurately add 20 ml each of 30% methanol, 50% methanol, 70% methanol, and methanol, stopper it, weigh it, and ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, accurately draw 1 μL of the filtrate, inject it into a high performance liquid chromatograph, and determine it according to the chromatographic conditions of an elution gradient of 9.

[0119] Table 5 Comparison of extraction solvent chromatographic peak system adaptability parameters

[0120]

[0121]

[0122]

[0123] Summary: From the above results, it can be seen that the peak shapes of the peaks are better and the peak areas are larger when 70% methanol is extracted, so 70% methanol is preferred as the extraction solvent for this experiment.

[0124] (2) Selection of extraction method

[0125] Take 0.5 g of two portions of standard decoction powder of Tsaoko kernel slices (containing a mixture of volatile oils), accurately weigh them, place them in stoppered conical flasks, accurately add 25 ml of 70% methanol to each, stopper them, weigh them, ultrasonically treat them (power 250 W, frequency 40 kHz), reflux them for 30 minutes, take them out, cool them, weigh them again, make up the lost weight with 70% methanol, shake them well, filter them, accurately draw 1 μL of the filtrate, inject them into a high performance liquid chromatograph, and determine them according to the chromatographic conditions of an elution gradient of 9.

[0126] Table 6 Comparison of chromatographic peak system adaptability parameters of standard decoction of tsaoko kernel slices (lyophilized powder) extraction methods

[0127]

[0128] Conclusion: When different extraction methods were used for the standard decoction of tsaoko kernel slices, the system adaptability parameters of the chromatographic peaks were better, with better separation and symmetry. The reflux method would lead to the loss of volatile oil. Therefore, ultrasound was the preferred extraction method for the characteristic spectrum of the standard decoction of tsaoko kernel slices.

[0129] (3) Selection of sampling volume

[0130] Take 0.1, 0.2, and 0.4 g of the standard decoction powder of Tsaoko kernel slices (containing a mixture of volatile oils) respectively, accurately weigh them, place them in a stoppered conical flask, accurately add 25 ml of 70% methanol respectively, stopper them, weigh them, and ultrasonically treat them (power 250 W, frequency 40 kHz) for 30 minutes. Take them out, let them cool, weigh them again, make up the lost weight with 70% methanol, shake them well, filter them, take 1 μL of the filtrate, inject it into a high performance liquid chromatograph, and determine it according to the chromatographic conditions of an elution gradient of 9.

[0131] Table 7 Comparison of sampling volume chromatographic peak system adaptability parameters

[0132]

[0133] The test results show that when the sampling amount is between 0.1g and 0.4g, the peak area of ​​the standard decoction of the tsaoko kernel slices basically increases proportionally, and 0.2g is preferably used as the sampling amount of the standard decoction of the tsaoko kernel slices.

[0134] Experimental Example 2 Determination of characteristic peaks and establishment of reference spectrum

[0135] (1) Construction method

[0136] The control medicinal material reference solution and the reference substance reference solution were prepared according to the method of Example 1. 17 batches of lyophilized powder of standard decoction of Tsaoko kernel slices (containing a mixture of volatile oils) (Batch Nos.: 2103001Y, 2103002Y, 2103003Y, 2103004Y, 2103005Y, 2103006Y, 2103007Y, 2103008Y, 2103009Y, 2103010Y, 2103011Y, 2103013Y, 2103014Y, 2103015Y, 230301Y, 230302Y, 230303Y) were taken as test samples, and the test sample solutions were prepared according to the method of Example 1. The above-mentioned reference solution and 17 batches of test sample solutions were subjected to high performance liquid chromatography detection, and the chromatographic conditions were the same as those in Example 1. The characteristic spectrum of the reference medicinal material of Amomum villosum can be found in Figure 22 shown.

[0137] According to the research results, it was determined that 15 characteristic peaks should be present in the characteristic spectrum of the standard decoction of Tsaoko kernel slices, and the retention times of the 15 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other, of which 4 peaks should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the reference peak of proanthocyanidin B2 is the S1 peak, and the relative retention times of peaks 2 to 3 and peaks 5 to 10 with the S1 peak are calculated. Peak 15 is the S2 peak, and the relative retention times of peaks 11 to 13 with the S2 peak are calculated. The relative retention times should be within the range of ±10% of the specified values, which are: 0.60 (peak 2), 0.80 (peak 3), 1.11 (peak 5), 1.27 (peak 6), 1.31 (peak 7), 1.40 (peak 8), 1.72 (peak 9), 1.88 (peak 10), 0.77 (peak 11), 0.87 (peak 12), and 0.94 (peak 13). Calculate the relative peak area of ​​Peak 7 to Peak 1. The relative peak area should be within the specified range, which is not less than 5.9 (Peak 7). The characteristic spectra of 17 batches of standard decoctions of Tsaoko kernel slices and the comparative characteristic spectra of standard decoctions of Tsaoko kernel slices are shown in the table below.

[0138] Table 8 Relative retention time results of characteristic spectra determination of 17 batches of standard decoctions of Amomum villosum slices

[0139]

[0140]

[0141] Table 9 Relative peak area results of characteristic spectrum determination of 17 batches of standard decoctions of Amomum villosum slices

[0142]

[0143]

[0144]

[0145] (2) The characteristic spectra of 17 batches of standard decoctions of Tsaoko kernel slices were synthesized by using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a reference spectrum of standard decoctions of Tsaoko kernel slices was established. Figure 21 shown.

[0146] The reference spectrum was fitted using Mark peak fitting.

[0147] Table 10 Relative retention time of the comparison chart of standard decoction of tsaoko kernel

[0148]

[0149] Table 11 Relative peak areas of the comparison chart of standard decoction of tsaoko kernel

[0150]

[0151] Table 12 Relative retention time of characteristic spectra of Tsaoko control medicinal materials

[0152]

[0153] Table 13 Relative peak areas of characteristic spectrum of Tsaoko control medicinal materials

[0154]

[0155] (3) Formula granules

[0156] Take 3 batches of tsaoko kernel formula granules (2303001Y, 2303002Y, 2303003Y), prepare the test solution according to the method of Example 1 and measure it, and construct the characteristic spectrum of 3 batches of tsaoko kernel formula granules, see Figure 23 The test results show that the relative retention time and relative peak area of ​​each characteristic peak of the three batches of tsaoko kernel formula granules are within the specified value range.

[0157] Table 14 Relative retention time results of characteristic spectra of three batches of tsaoko kernel formula granules

[0158]

[0159] Table 15 Relative peak area results of characteristic spectra of three batches of tsaoko kernel formula granules

[0160]

[0161] Experimental Example 3 Methodology Verification

[0162] 1. Exclusivity

[0163] Accurately pipette the test solution and negative blank sample solution obtained in Example 1 (mix 0.5 g maltodextrin and 0.5 g beta-cyclodextrin, prepared according to the test solution preparation method), inject them into the high performance liquid chromatograph, and test according to the chromatographic conditions of Example 1. Figure 24 and 35 As shown, the results showed that the negative had no interference.

[0164] 2. Precision

[0165] Take the same sample solution of the Tsaoko kernel formula granules prepared according to the method of Example 1, repeat the injection 6 times, record the spectrum, and measure the relative retention time and relative peak area of ​​15 characteristic peaks. The results are shown in Figure 2. Figure 25 The relative retention times of the 15 characteristic peaks were all less than or equal to 0.06%, indicating good precision.

[0166] 3. Repeatability

[0167] Take the same sample of the Tsaoko kernel formula granules and repeat the sample 6 times according to the method of Example 1. Follow the chromatographic conditions of Example 1 for sample analysis and record the chromatogram. The results are shown in Figure 26 , the relative retention times of the 15 characteristic peaks were all less than or equal to 2.0%, indicating good repeatability.

[0168] 4. Intermediate precision (different operators)

[0169] Two inspectors used the same equipment at different times to measure the same batch of Tsaoko kernel formula granules according to the test solution preparation method and chromatographic conditions described in Example 1. The relative retention time and relative peak area of ​​15 common peaks were measured and analyzed. The results are shown in Table 1. Figure 27 The relative retention times of the 15 characteristic peaks were all less than or equal to 0.05%, indicating that the intermediate precision of this method was good.

[0170] 5. Stability inspection

[0171] Take the same sample solution prepared in Example 1 and analyze it at 0, 4, 8, 12, 18, and 24 hours according to the chromatographic conditions described in Example 1. Record the chromatogram and measure the relative retention time and relative peak area of ​​15 characteristic peaks. Figure 28 The relative retention times of the 15 characteristic peaks were all less than or equal to 0.08%, indicating that the method had good stability and could meet the needs of determination.

[0172] 6. Investigation of different mobile phase concentrations

[0173] Take the test solution prepared in Example 1 and analyze it under different phosphoric acid concentrations according to the chromatographic conditions described in Example 1. Record the chromatograms. The results are shown in Figures 29-31 , analyzed the separation effect of each characteristic peak, and the results showed that when the phosphoric acid concentration changed, the separation effect of each characteristic peak was less affected, and the relative retention time of the 15 characteristic peaks was less than or equal to 0.08%. This method has good durability for different phosphoric acid concentrations.

[0174] According to the results of the above-mentioned methodological investigation, among the 15 characteristic peaks of the characteristic spectrum of the cardamom kernel formula granules established in Example 1, each chromatographic peak is affected to a certain extent by different column temperatures and flow rates, and the relative retention time value of each characteristic peak is in the range of -10% to 10%. In order to adapt to its durability, it is recommended to control the relative retention time range of each characteristic peak within ±10%.

[0175] Example 1

[0176] This embodiment provides a method for constructing a characteristic spectrum of tsaoko kernel formula particles, comprising the following steps:

[0177] Preparation of test solution: Take an appropriate amount of the test sample, grind it into powder, take about 0.2 g, accurately weigh it, place it in a stoppered conical flask, add 25 ml of 70% methanol, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, cool it, shake it well, filter it, and take the filtrate to obtain the product.

[0178] Preparation of Reference Substance Solution: Accurately weigh an appropriate amount of protocatechuic acid and proanthocyanidin B2 reference substances, add 70% methanol to prepare a mixed solution containing 20 μg of each per 1 ml, which will serve as the reference substance solution. Accurately weigh an appropriate amount of citral reference substance, add methanol to prepare a solution containing 30 μg of the reference substance per 1 ml, which will serve as the reference substance solution.

[0179] Preparation of control medicinal material reference solution: Take about 3 g of Amomum villosum control medicinal material, weigh accurately, place in a stoppered conical flask, add 25 ml of methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0180] Assay: Inject 1 μL each of the test solution, reference solution, and reference medicinal material solution of the aforementioned tsaoko kernel granules into an ultra-high performance liquid chromatograph using the following chromatographic conditions: ACQUITY BEH C18 column, 2.1 × 150 mm, 1.7 μm; acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, gradient elution as specified in the table below; flow rate, 0.3 mL / min; column temperature, 30°C; detection wavelength, 240 nm. The number of theoretical plates, calculated based on the protocatechuic acid peak, should be no less than 5000.

[0181]

[0182]

[0183] Table 16 Peak results of the test samples

[0184]

[0185] Table 17 Peak results of citral reference substance

[0186]

[0187] Table 18 Peak results of proanthocyanidin B2 reference substance

[0188]

[0189] Table 19 Peak results of protocatechuic acid reference substance

[0190]

[0191] See the results Figures 32-35The tsaoko kernel formula granules showed 15 characteristic peaks in the chromatogram, and the retention times of the 15 characteristic peaks in the chromatogram of the reference medicinal material corresponded to each other. Four of these peaks corresponded to the retention times of the corresponding reference material peaks. Furthermore, the baseline was stable, the characteristic peaks had good peak shapes, and the resolution was high. Furthermore, the peak positions of protocatechuic acid, proanthocyanidin B2, and citral (neral, geranial) could be accurately located.

[0192] The peak corresponding to the reference peak of proanthocyanidin B2 is designated as peak S1. The relative retention times of peaks 2 to 3 and peaks 5 to 10 relative to peak S1 are calculated. Peak 15 is designated as peak S2, and the relative retention times of peaks 11 to 13 relative to peak S2 are calculated. The relative retention times of the above peaks are: 0.63 (peak 2), 0.80 (peak 3), 1.12 (peak 5), 1.29 (peak 6), 1.36 (peak 7), 1.47 (peak 8), 1.77 (peak 9), 2.01 (peak 10), 0.78 (peak 11), 0.88 (peak 12), and 0.94 (peak 13). The relative peak area of ​​peak 7 relative to peak 1 is 39.3, which meets the specified value of not less than 5.9.

[0193] Example 2

[0194] According to relevant information, the main counterfeit products of Amomum villosum are Amomum villosum, ... Wild Amomum villosum, and Pseudo Amomum villosum (Guangxi Amomum villosum), etc.

[0195] The present embodiment provides a method for distinguishing tsaoko kernel formula granules from counterfeits, using tsaoko kernel medicinal materials, Amomum villosum Lour. (scientific name: dried mature fruit of Zingiberaceae plant Amomum villosum Lour.), Amomum gagnepainii (scientific name: dried mature fruit of Zingiberaceae plant Amomum gagnepainii TLWu et al.), Amomum zerumbet (scientific name: dried mature fruit of Zingiberaceae plant Alpinia zerumbet (Pers.) Burtt. & Smith), Amomum muricarpum (scientific name: dried mature fruit of Zingiberaceae plant Amomum muricarpum Elm.), Alpinia katsumadai Hayata (scientific name: dried nearly mature seeds of Zingiberaceae plant Alpinia katsumadai Hayata), Amomum koenigii (scientific name: dried mature fruit of Zingiberaceae plant Amomum koenigii JFGmelin), and Amomum tsaoko (scientific name: dried mature fruit of Zingiberaceae plant Amomum tsaoko). The dried mature fruit of SQTong & Y.M.Xia was used as the test sample, and characteristic spectra were prepared according to the method of Example 1. The characteristic spectra of each counterfeit product were compared with the characteristic spectra of the tsaoko kernel medicinal material, and the results are shown in FIG. Figures 36-42 shown.

[0196] Compared with the characteristic spectrum of Amomum villosum, peaks 4, 6, 8, 12, 14, and 15 are absent from the Amomum villosum characteristic spectrum, and the relative peak area of ​​peak 7 relative to peak 1 is far less than 5.9. Furthermore, unique peaks of Amomum villosum are present at 30-32 minutes, 43-45 minutes, 47 minutes, and 48 minutes. Peaks 11 and 12 are absent from the Amomum villosum characteristic spectrum, and peaks 13-15 are essentially undetectable. Furthermore, the relative peak area of ​​peak 7 relative to peak 1 is far less than 5.9. Peaks 9 and 11 are absent from the Amomum villosum characteristic spectrum, and peaks 12-15 are essentially undetectable. Furthermore, the relative peak area of ​​peak 7 relative to peak 1 is far less than 5.9. Peaks 7, 11, 12, and 13 are absent from the Amomum villosum characteristic spectrum, and peaks 14 and 15 are essentially undetectable. Peaks 13-15 are absent from the characteristic spectrum of Xiangsha, and the relative peak area of ​​Peak 7 to Peak 1 is much lower than 5.9. Peaks at 26, 50, and 36 minutes are all unique to Xiangsha. Peaks 5-7 and 9-12 are absent from the characteristic spectrum of Y. tsaoko, and Y. tsaoko-specific peaks appear around 37, 45, and 50 minutes. Peaks 4, 7, 9, and 10 are absent from the characteristic spectrum of Pseudo-tsaoko, but Pseudo-tsaoko-specific peaks appear in the latter half of the characteristic spectrum, around 31 and 36 minutes.

[0197] It can be seen from this that the identification method of the present invention can be used to identify grass kernels and their preparations from counterfeits. If the characteristic spectrum of the product to be identified shows 15 characteristic peaks corresponding to the control characteristic spectrum of the grass kernel formula granules, and / or the relative peak area of ​​peak 7 and peak 1 is higher than 5.9, then it is grass kernels or their preparations, otherwise it is not.

[0198] The examples provided herein are intended to illustrate, not to limit, the methods of implementation of the present invention. Those skilled in the art will readily appreciate that other variations or modifications based on the above description are possible. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic spectrum of tsaoko kernel and its preparation, characterized in that: The following steps are included: (1) Preparation of the test solution. The preparation of the test solution includes: weighing the tsaoko kernel test sample, adding a solvent for extraction, obtaining an extract, separating the solid and the liquid, and obtaining the liquid, which is the test solution; the solvent is selected from methanol, methanol-water solution or ethanol; (2) The test solution and the reference solution were respectively detected by ultra-high performance liquid chromatography at a wavelength of 238-242 nm using a Waters ACQUITY BEH C18 column with the following specifications: column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm, mobile phases of phosphoric acid-containing aqueous solution and acetonitrile, gradient elution program including: 0→20 min→30 min→50 min, the volume percentage of acetonitrile in the mobile phase including: 4%→19%→34%→45%; the volume percentage of phosphoric acid in the phosphoric acid-containing aqueous solution including 0.08-0.12%; the preparation method of the reference solution including using protocatechuic acid, proanthocyanidin B2, neral, geranial reference substances and solvent to prepare the reference solution.

2. The construction method according to claim 1, characterized in that In step (2), the flow rate was 0.25-0.32 mL / min, the column temperature was 28-32 °C, and the injection volume was 1-10 µL.

3. The construction method according to claim 1, characterized in that The step (1) also satisfies any one or more of the following AEs: A. The mass to volume ratio of the tsaoko kernel sample to the solvent is 0.1-0.4:15-50; the mass to volume ratio is g / mL; B. The extraction method is reflux extraction or ultrasonic extraction; C. Extraction time is ≥30min; D. The solid-liquid separation is selected from centrifugation or filtration; E. The volume percentage of methanol in the methanol aqueous solution is 30-70%.

4. The construction method according to claim 3, characterized in that In the step (1), the extraction time is 30-60 minutes.

5. The construction method according to any one of claims 1 to 4, characterized in that: and the step of detecting the reference solution by ultra-high performance liquid chromatography in accordance with the construction method described in any one of claims 1 to 4 to obtain a reference spectrum; and / or, using the Amomum villosum kernel reference medicinal material as a reference substance, preparing the reference solution by step (1) in the construction method described in any one of claims 1 to 4, and detecting the reference solution by ultra-high performance liquid chromatography in accordance with the construction method described in any one of claims 1 to 4 to obtain a reference spectrum.

6. The construction method according to claim 1, characterized in that Each 1 mL of the reference substance solution contains 5-50 μg of each reference substance; and / or, the solvent used in the preparation of the reference substance solution is selected from methanol or a methanol-water solution with a volume fraction of not less than 50%.

7. A method for distinguishing grass kernels and their preparations from counterfeits, characterized in that: The method comprises the steps of comparing the characteristic spectrum of the product to be identified with the control characteristic spectrum of Amomum villosum kernel and its preparation; the counterfeit product comprises one or more of Amomum villosum and its preparation, Amomum villosum kernel and its preparation, Amomum villosum and its preparation, and Amomum villosum pseudo-Amomum villosum and its preparation; the characteristic spectrum of the product to be identified is constructed using the product to be identified according to the construction method described in any one of claims 1 to 6, and the control characteristic spectrum of Amomum villosum kernel and its preparation is selected from any one of the following (1) to (4): (1) It has 15 common characteristic peaks. The peak corresponding to the reference peak of proanthocyanidin B2 is the S1 peak. The relative retention times of peaks 1 to 3 and peaks 5 to 10 with respect to the S1 peak are within the range of ±10% of the specified value. The specified values ​​of peaks 1 to 3 and peaks 5 to 10 are 0.38, 0.60, 0.80, 1.11, 1.27, 1.31, 1.40, 1.72 and 1.88, respectively. Peak 15 is the S2 peak. The relative retention times of peaks 11 to 14 with respect to the S2 peak are within the range of ±10% of the specified value. The specified values ​​of peaks 11 to 14 are 0.77, 0.87, 0.94 and 0.95, respectively. (2) It has 15 common characteristic peaks, of which 4 peaks correspond to the retention times of protocatechuic acid, proanthocyanidin B2, nerol and geranyl aldehyde reference peaks respectively. The peak corresponding to the proanthocyanidin B2 reference peak is the S1 peak. The relative retention times of peaks 2 to 3 and peaks 5 to 10 with the S1 peak are within the range of ±10% of the specified value. Peak 15 is the S2 peak, and the relative retention times of peaks 11 to 13 with the S2 peak are within the range of ±10% of the specified value. The specified values ​​of peaks 2 to 3 and peaks 5 to 13 are: 0.60, 0.80, 1.11, 1.27, 1.31, 1.40, 1.72, 1.88, 0.77, 0.87 and 0.94 respectively. (3) Characteristic spectrum of the tsaoko kernels and / or their preparations obtained by the construction method according to any one of claims 1 to 6 using a single batch or multiple batches of tsaoko kernels and / or their preparations as test samples; (4) Using multiple batches of tsaoko kernels and / or their preparations as test samples, the characteristic profiles obtained according to the construction method described in any one of claims 1 to 6 are used to prepare a control characteristic profile by using the average value or median method.

8. The method for distinguishing between tsaoko kernels and their preparations and counterfeits according to claim 7, characterized in that: The Amomum villosum includes one or more of Amomum villosum yangchunensis, Amomum villosum longifolia, Amomum villosum niuginosa, and Amomum villosum fragrant.

9. A quality detection method for tsaoko kernels and their preparations, characterized in that: The method comprises the steps of comparing the characteristic spectrum of the tested grass kernel product with the control characteristic spectrum of the grass kernel and its preparation; the characteristic spectrum of the tested grass kernel product is obtained by using the tested grass kernel product according to the construction method described in any one of claims 1 to 6, and the control characteristic spectrum of the grass kernel and its preparation is selected from any one of the following (1) to (4): (1) It has 15 common characteristic peaks. The peak corresponding to the reference peak of proanthocyanidin B2 is the S1 peak. The relative retention times of peaks 1 to 3 and peaks 5 to 10 with respect to the S1 peak are within the range of ±10% of the specified value. The specified values ​​of peaks 1 to 3 and peaks 5 to 10 are 0.38, 0.60, 0.80, 1.11, 1.27, 1.31, 1.40, 1.72 and 1.88, respectively. Peak 15 is the S2 peak. The relative retention times of peaks 11 to 14 with respect to the S2 peak are within the range of ±10% of the specified value. The specified values ​​of peaks 11 to 14 are 0.77, 0.87, 0.94 and 0.95, respectively. (2) It has 15 common characteristic peaks, of which 4 peaks correspond to the retention times of protocatechuic acid, proanthocyanidin B2, nerol and geranyl aldehyde reference peaks respectively. The peak corresponding to the proanthocyanidin B2 reference peak is the S1 peak. The relative retention times of peaks 2 to 3 and peaks 5 to 10 with the S1 peak are within the range of ±10% of the specified value. Peak 15 is the S2 peak, and the relative retention times of peaks 11 to 13 with the S2 peak are within the range of ±10% of the specified value. The specified values ​​of peaks 2 to 3 and peaks 5 to 13 are: 0.60, 0.80, 1.11, 1.27, 1.31, 1.40, 1.72, 1.88, 0.77, 0.87 and 0.94 respectively. (3) Characteristic spectrum of the tsaoko kernels and / or their preparations obtained by the construction method according to any one of claims 1 to 6 using a single batch or multiple batches of tsaoko kernels and / or their preparations as test samples; (4) Using multiple batches of tsaoko kernels and / or their preparations as test samples, the characteristic spectra obtained according to the construction method described in any one of claims 1 to 6 are used to prepare a control characteristic spectra by using the average value or median method.

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