A method for constructing a characteristic spectrum of Amomum villosum or Amomum villosum kernel or a derivative product thereof, and a method for identifying Amomum villosum kernel or a derivative product thereof from counterfeit products, Amomum villosum kernel or a derivative product thereof with shell

Abstract

The application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a construction method of a characteristic spectrum of Amomum tsao-ko or Amomum tsao-ko kernel or a derivative product thereof and a method for identifying Amomum tsao-ko kernel or a derivative product thereof and pseudo products, Amomum tsao-ko with a shell or a derivative product thereof. The construction method of the characteristic spectrum of Amomum tsao-ko or Amomum tsao-ko kernel or a derivative product thereof comprises high performance liquid chromatography detection, and the chromatographic conditions comprise: using octadecylsilane bonded silica gel as a filler, using a phosphoric acid-containing aqueous solution as a mobile phase B, using acetonitrile as a mobile phase A, and gradient elution. The construction method of the characteristic spectrum of Amomum tsao-ko or Amomum tsao-ko kernel or a derivative product thereof has more characteristic peaks, better baseline separation, and simultaneously displays water-soluble components and volatile components in the same chromatogram, and is simple and easy to operate, and thus is more suitable for quality detection of Amomum tsao-ko or Amomum tsao-ko kernel or a derivative product thereof.

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 or tsaoko kernels or derivatives thereof, and a method for distinguishing tsaoko kernels or derivatives thereof from counterfeits, tsaoko with shells or derivatives thereof. Background Art

[0002] Amomum villosum has multiple pharmacological effects such as gastrointestinal regulation, antibacterial, anti-inflammatory, antioxidant, anti-tumor, hypoglycemic and lipid-regulating effects. Its main components include volatile oils, phenols, diphenylheptanes and bicyclononanes; when regulating the gastrointestinal system, the active ingredients are volatile oils and polyphenols.

[0003] Amomum villosum is the dried mature fruit of the ginger family plant Amomum tsao-ko Crevost et Lemaire. Its seeds are amomum villosum kernels, which are often used as medicine in traditional Chinese medicine.

[0004] There are studies on the identification of cardamom kernels or their derivatives from common counterfeits in the prior art, but the baseline separation is poor, there are many noise peaks, there is a large slope in the characteristic spectrum and some characteristic peaks are high, which is not conducive to the overall identification and judgment of cardamom, cardamom kernels and their derivatives, and the detection time is long; in addition, when using cardamom kernels or their derivatives clinically, the cardamom shell in the cardamom needs to be removed and only the cardamom kernels are used, but how to use high-performance liquid chromatography to identify cardamom kernels or their derivatives from cardamom with shells is still a blank. How to improve the quality detection method of cardamom or cardamom kernels or their derivatives and effectively control the quality of the finished product is a technical problem that the present invention needs to solve. Summary of the Invention

[0005] Therefore, the first object of the present invention is to provide a method for constructing a characteristic map of cardamom or cardamom kernels or their derivatives. This method establishes a characteristic map of the variety based on the characteristics of cardamom or cardamom kernels or their derivatives. The good baseline stability further suppresses the noise peak, controls the overall height balance of the characteristic peak, eliminates the intermediate slope, and facilitates overall identification and judgment, so as to perform comprehensive quality control of cardamom or cardamom kernels or their derivatives.

[0006] The second purpose of the present invention is to provide a method for identifying tsaoko and its derivatives or tsaoko kernels and its derivatives from counterfeits. The method can identify and distinguish tsaoko, tsaoko kernels and their derivatives from a variety of common counterfeits and their preparations, including tsaoko, tsaoko, tsaoko, and tsaoko, so as to better control the quality of tsaoko, tsaoko kernels and their derivatives.

[0007] The third object of the present invention is to provide a method for identifying cardamom kernels and their derivatives from cardamom in shells and their derivatives. The method can identify and distinguish cardamom kernels and their derivatives from cardamom in shells and their derivatives, facilitate further detection of whether cardamom kernels and their derivatives contain cardamom shells, and facilitate clinical use without using cardamom kernels and their derivatives mixed with cardamom shells.

[0008] To this end, the present invention provides the following technical solutions.

[0009] The present invention provides a method for constructing a characteristic spectrum of tsaoko or tsaoko kernel or its derivatives, comprising detecting by high performance liquid chromatography, wherein the chromatographic conditions include:

[0010] Octadecylsilane bonded silica gel was used as the filler, aqueous solution containing phosphoric acid was used as the mobile phase B, acetonitrile was used as the mobile phase A, and gradient elution was performed. The gradient elution procedure included:

[0011] From 0 to 12 minutes, the volume percentage of mobile phase A was 9% → 16%, and the volume percentage of mobile phase B was 91% → 84%;

[0012] 12-18 minutes, the volume percentage of mobile phase A is 16% → 24%, and the volume percentage of mobile phase B is 84% ​​→ 76%;

[0013] 18-24 minutes, the volume percentage of mobile phase A is 24% → 30%, and the volume percentage of mobile phase B is 76% → 70%;

[0014] 24-35 minutes, the volume percentage of mobile phase A is 30% → 52%, and the volume percentage of mobile phase B is 70% → 48%;

[0015] From 35 to 45 minutes, the volume percentage of mobile phase A is 52%→55%, and the volume percentage of mobile phase B is 48%→45%.

[0016] Optionally, the chromatographic conditions include at least one of the following:

[0017] (1) The detection wavelength is 230-240 nm; optionally, 230 nm for 0-30 minutes and 240 nm for 30-45 minutes;

[0018] (2) The flow rate is 0.45-0.55 ml / min; optionally, the flow rate is 0.5 ml / min;

[0019] (3) Column temperature 25-35°C; optionally, column temperature 30°C;

[0020] (4) The injection volume is 1-5 μL; optionally, the injection volume is 1-3 μL; further optionally, the injection volume is 2 μL;

[0021] (5) The phosphoric acid concentration in the aqueous solution containing phosphoric acid in mobile phase B is 0.05 to 0.2% v / v; optionally, the phosphoric acid concentration is 0.1% v / v;

[0022] (6) The specifications of the chromatographic column are: column length 100-150 mm, inner diameter 3.0-4.6 mm, and particle size 1.8-2.7 μm; optionally, the column length is 150 mm, the inner diameter is 3.0 mm, and the particle size is 1.8 μm.

[0023] Optionally, the method further includes the preparation of a test sample solution, including: weighing the test sample, adding a solvent for extraction, solid-liquid separation, and taking a liquid, which is the test sample solution.

[0024] Optionally, the preparation of the test solution satisfies at least one of the following:

[0025] A. The ratio of the mass of the test sample to the volume of the solvent is (0.2-1.0):25; the relationship between mass and volume is g / mL;

[0026] B. The extraction method is ultrasonic extraction;

[0027] C. Extraction time is ≥15 minutes, preferably 30 minutes;

[0028] D. The solid-liquid separation is selected from centrifugation or membrane filtration;

[0029] E. The solvent is selected from one or more of methanol, ethanol, and water; preferably, a methanol-water solution with a volume percentage of 50-70%.

[0030] Optionally, the construction method further comprises the steps of preparing a reference solution using at least one of catechin, epicatechin, and citral, and detecting the reference solution by high performance liquid chromatography according to the construction method to obtain a reference spectrum of the reference substance;

[0031] Preferably, each 1 mL of catechin reference solution contains 10-30 μg of catechin; preferably, 20 μg;

[0032] Preferably, each 1 mL of epicatechin reference solution contains 20-40 μg of epicatechin; preferably, 30 μg;

[0033] Preferably, each 1 mL of citral reference solution contains 20-40 μg of citral; preferably, 30 μg;

[0034] Preferably, the solvent used in the preparation of the reference solution is selected from methanol or a methanol aqueous solution with a volume fraction of not less than 50-70%; more preferably, the solvent used in the preparation of the reference solution is selected from a 70% methanol aqueous solution;

[0035] Preferably, the construction method further comprises the steps of preparing a control medicinal material solution using the Amomum villosum control medicinal material, and detecting the control medicinal material solution by the high performance liquid chromatography method in the construction method to obtain a control medicinal material reference atlas;

[0036] Preferably, the preparation method of the control medicinal material solution is the same as the preparation method of the test solution;

[0037] Preferably, the tsaoko or its derivative products include at least one of tsaoko medicinal materials, tsaoko decoction pieces or tsaoko preparations; the tsaoko kernels or its derivative products include at least one of tsaoko kernel decoction pieces or tsaoko kernel preparations.

[0038] The present invention also provides a method for determining the content of tsaoko or tsaoko kernel or its derivatives, comprising:

[0039] Taking the test solution and the reference solution, respectively, and detecting them by the high performance liquid chromatography method in the method for constructing the characteristic spectrum of the tsaoko or tsaoko kernel or its derivatives;

[0040] Wherein, the reference substance includes at least one of catechin, epicatechin and citral.

[0041] The present invention also provides a method for detecting the quality of tsaoko or tsaoko kernels or derivatives thereof, comprising the steps of comparing a characteristic spectrum of the product to be detected with a control characteristic spectrum of the tsaoko or tsaoko kernels or derivatives thereof;

[0042] The characteristic spectrum of the product to be tested is constructed according to the construction method;

[0043] The control characteristic spectrum of the tsaoko or tsaoko kernel or its derivative products is selected from any one of the following (1)-(4):

[0044] (1) It has 10 common characteristic peaks. The peak corresponding to the reference peak of epicatechin is the S peak. The relative retention times of peaks 1-2, 4, 6-8 and S peak are calculated. The relative retention times should be within the range of ±10% of the specified values. The specified values ​​of peaks 1-2, 4 and 6-8 are: 0.41, 0.61, 0.87, 1.15, 1.31 and 1.78 respectively.

[0045] (2) It has 10 common characteristic peaks, of which 4 peaks correspond to the retention times of the reference peaks of catechin, epicatechin, and citral, respectively. The peak corresponding to the reference peak of epicatechin is the S peak. The relative retention times of peaks 1-2, 4, and 6-8 to the S peak are calculated. The relative retention times should be within the range of ±10% of the specified values. The specified values ​​of peaks 1-2, 4, and 6-8 are: 0.41, 0.61, 0.87, 1.15, 1.31, and 1.78, respectively; optionally, peak 1 corresponds to protocatechuic acid; peak 2 corresponds to protocatechuic aldehyde; peak 3 corresponds to catechin; peak 4 corresponds to proanthocyanidin B2; peak 5 (S) corresponds to epicatechin; peaks 9 and 10 correspond to citral;

[0046] (3) Characteristic spectra of tsaoko kernels and / or their derivatives obtained by the construction method using a single batch or multiple batches of tsaoko kernels and / or their derivatives as test samples;

[0047] (4) Using multiple batches of tsaoko and / or tsaoko kernels and / or their derivatives as test samples, the characteristic spectra obtained according to the construction method are prepared into a control characteristic spectra by using the average value or median method.

[0048] The present invention also provides a method for distinguishing between tsaoko or tsaoko kernels or derivatives thereof and counterfeits, comprising:

[0049] Test according to the above-mentioned quality test method for tsaoko or tsaoko kernels or their derivatives; wherein, for tsaoko or tsaoko kernels or their derivatives, the relative peak area of ​​peak 7 and peak S should be within the specified range, which is not less than 0.31;

[0050] Optionally, the counterfeit product includes one or more of Amomum villosum or its derivatives, Alpinia fasciata seeds or its derivatives, Amomum villosum or its derivatives, Amomum villosum or its derivatives; preferably, the Amomum villosum includes one or more of Amomum villosum yangchunensis, Amomum villosum longifolia, Amomum villosum niugu, and Amomum villosum fragrant.

[0051] The present invention also provides a method for identifying tsaoko kernels or derivatives thereof and tsaoko in shell or derivatives thereof, comprising:

[0052] The product to be tested is subjected to the step of detecting the characteristic spectrum by high performance liquid chromatography in the construction method;

[0053] The chromatographic conditions of the high performance liquid chromatography method also include a detection wavelength of 192-194 nm;

[0054] The reference solution also includes a reference solution containing 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol and / or meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol;

[0055] For tsaoko kernel or its derivative products, the peak area of ​​the chromatographic peak of 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol and / or meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol in the characteristic spectrum is ≤ the peak area of ​​the chromatographic peak of the corresponding reference solution of 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol and / or meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol;

[0056] For shelled Amomum villosum or its derivative products, the peak area of ​​the chromatographic peak of 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol and / or meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol in the characteristic spectrum is greater than the peak area of ​​the chromatographic peak of the corresponding reference solution of 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol and / or meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol.

[0057] Optionally, each 1 mL of the reference solution contains 1.6-2.5 μg of meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol or 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol;

[0058] Preferably, for the shelled tsaoko or its derivative products, the chromatographic peak area of ​​the meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol is detected to be greater than 155239 μAU·S; for the tsaoko kernel or its derivative products, the chromatographic peak area of ​​the meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol is detected to be ≤155239 μAU·S.

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

[0060] 1. The method for constructing a characteristic spectrum of tsaoko or tsaoko kernel or its derivative products provided by the present invention 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, 10 common characteristic peaks are obtained, and effective separation of the common characteristic peaks is achieved. Moreover, the obtained characteristic spectrum has more characteristic peaks and better baseline separation, and the water-soluble components and volatile components are simultaneously displayed in the same chromatogram. The method is simple, easy to operate, and has a short detection time. It can accurately locate the peak positions including protocatechuic acid, protocatechuic aldehyde, proanthocyanidin B2, catechin, protocatechuin, and citral, fully reflecting the integrity and characteristic of tsaoko or tsaoko kernel or its derivative products (such as tsaoko kernel formula granules and other derivative products), and providing a basis for quality detection and content determination of tsaoko or tsaoko kernel or its derivative products.

[0061] 2. The method for constructing the characteristic spectrum of the tsaoko or tsaoko kernel or its derivative products provided by the present invention is to determine the optimal chromatographic conditions by optimizing the chromatographic conditions, so that the peak area is higher and the separation effect is better, and the quality of the tsaoko, tsaoko kernel or its derivative products can be more comprehensively monitored.

[0062] 3. The method for identifying tsaoko or tsaoko kernels or their derivatives from counterfeits provided by the present invention can effectively identify tsaoko or tsaoko kernels or their derivatives from counterfeits by comparing the characteristic spectrum of the product to be identified with the control characteristic spectrum of tsaoko or tsaoko kernels or their derivatives. The counterfeits include Amomum villosum and its preparations, Amomum cardamomum seeds and its preparations, wild tsaoko and its preparations, and pseudo-tsaoko and its preparations; the Amomum villosum includes Amomum villosum, Amomum villosum longifolium, Amomum villosum niugu, and Amomum villosum fragrant.

[0063] 4. The present invention provides a method for identifying tsaoko kernels or their derivatives from tsaoko shells or their derivatives. The method comprises the following steps: detecting whether the product to be tested contains 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol and / or meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol, preparing a meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol reference solution, and comparing the peak area of ​​the meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol reference solution at a concentration of 1.6-2.5 μg / mL with the peak area of ​​meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol in the product to be tested. This method can effectively detect whether the product to be tested contains tsaoko shells, thereby ensuring that tsaoko kernels or their derivatives mixed with tsaoko shells are not used clinically.

[0064] 5. The quality detection method of the cardamom or cardamom kernel or its derivative products provided by the present invention compares the characteristic spectrum of the cardamom or cardamom kernel product to be tested with the control characteristic spectrum of the cardamom or cardamom kernel or its derivative products, thereby more comprehensively detecting the quality of the cardamom or cardamom kernel or its derivative products, thereby proving that the quality of the product is effective and controllable. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] 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.

[0066] Figure 1 is a characteristic spectrum of the tsaoko kernel formula granules; specifically, Figure 1-(1) is the test sample; Figure 1-(2) is catechin; Figure 1-(3) is epicatechin; Figure 1-(4) is citral; Figure 1-(5) is the tsaoko control medicinal material; Figure 1-(6) is the tsaoko kernel control medicinal material;

[0067] Figure 2 is the chromatogram of gradient condition 1 in Experimental Example 1;

[0068] Figure 3 is the chromatogram of gradient condition 2 in Experimental Example 1;

[0069] Figure 4 is the chromatogram of gradient condition 3 in Experimental Example 1;

[0070] Figure 5 is the chromatogram of gradient condition 4 in Experimental Example 1;

[0071] Figure 6 is the chromatogram of gradient condition 5 in Experimental Example 1;

[0072] Figure 7 is the chromatogram of gradient condition 6 in Experimental Example 1;

[0073] Figure 8 is the chromatogram of gradient condition 7 in Experimental Example 1;

[0074] Figure 9 is the chromatogram of gradient condition 8 in Experimental Example 1;

[0075] Figure 10 is the chromatogram of gradient condition 9 in Experimental Example 1;

[0076] Figure 11 is the chromatogram of gradient condition 10 in Experimental Example 1;

[0077] Figure 12 This is the chromatogram at a flow rate of 0.45 ml / min in the flow rate investigation in Experimental Example 1;

[0078] Figure 13 This is the chromatogram at a flow rate of 0.50 ml / min in the flow rate investigation in Experimental Example 1;

[0079] Figure 14 This is the chromatogram at a flow rate of 0.55 ml / min in the flow rate investigation in Experimental Example 1;

[0080] Figure 15 This is the chromatogram when the column temperature is 25°C in the investigation of column temperature in Experimental Example 1;

[0081] Figure 16 This is the chromatogram when the column temperature is 30°C in the investigation of column temperature in Experimental Example 1;

[0082] Figure 17 This is the chromatogram when the column temperature is 35°C in the investigation of column temperature in Experimental Example 1;

[0083] Figure 18This is a chromatogram of the mobile phase of acetonitrile-water in the investigation of the mobile phase in Experimental Example 1;

[0084] Figure 19 This is a chromatogram of the mobile phase of acetonitrile-0.1% formic acid aqueous solution in the investigation of the mobile phase in Experimental Example 1;

[0085] Figure 20 This is a chromatogram of the mobile phase of acetonitrile-0.1% glacial acetic acid aqueous solution in the investigation of the mobile phase in Experimental Example 1;

[0086] Figure 21 This is a chromatogram of the mobile phase of acetonitrile-0.05% phosphoric acid aqueous solution in the investigation of the mobile phase in Experimental Example 1;

[0087] Figure 22 This is a chromatogram of the mobile phase of acetonitrile-0.1% phosphoric acid aqueous solution in the investigation of the mobile phase in Experimental Example 1;

[0088] Figure 23 This is a chromatogram of the mobile phase of acetonitrile-0.2% phosphoric acid aqueous solution in the investigation of the mobile phase in Experimental Example 1;

[0089] Figure 24 This is a chromatogram with a detection wavelength of 230 nm in the investigation of the detection wavelength in Experimental Example 1;

[0090] Figure 25 This is a chromatogram with a detection wavelength of 240 nm in the investigation of the detection wavelength in Experimental Example 1;

[0091] Figure 26 This is a chromatogram with a detection wavelength of 254 nm in the investigation of the detection wavelength in Experimental Example 1;

[0092] Figure 27 The chromatogram is a chromatogram with a detection wavelength of 230 nm for 0-30 minutes and 240 nm for 30-45 minutes in the investigation of the detection wavelength in Experimental Example 1;

[0093] Figure 28 This is the chromatogram of the injection volume of 1 μL in the injection volume investigation in Experimental Example 1;

[0094] Figure 29 This is the chromatogram of the injection volume of 2 μL in the injection volume investigation in Experimental Example 1;

[0095] Figure 30 This is the chromatogram of the injection volume of 3 μL in the injection volume investigation in Experimental Example 1;

[0096] Figure 31 This is the chromatogram of the injection volume of 4 μL in the injection volume investigation in Experimental Example 1;

[0097] Figure 32 This is the chromatogram of the injection volume of 5 μL in the injection volume investigation in Experimental Example 1;

[0098] Figure 33 Characteristic spectra of three batches of tsaoko kernel formula granules in Experimental Example 2;

[0099] Figure 34 This is the characteristic spectrum of the reference medicinal material Amomum villosum in Experimental Example 3;

[0100] Figure 35 Characteristic spectra of 17 batches of standard decoction of tsaoko kernel slices (lyophilized powder) in Experimental Example 3 (batch numbers from bottom to top are: 2103001Y, 2103002Y, 2103003Y, 2103004Y, 2103005Y, 2103006Y, 2103007Y, 2103008Y, 2103009Y, 2103010Y, 2103011Y, 2103013Y, 2103014Y, 2103015Y, 230301Y, 230302Y, 230303Y);

[0101] Figure 36 The characteristic spectrum of the control is shown in Figure 1. Peak 1: protocatechuic acid; Peak 2: protocatechuic aldehyde; Peak 3: catechin; Peak 4: procyanidin B2; Peak 5: epicatechin; Peak 9 and Peak 10: citral;

[0102] Figure 37 This is the reference sample positioning map; from bottom to top in the figure: Tsaoko kernel formula granules (batch number: 2303001Y); protocatechuic acid reference; protocatechuic aldehyde reference; catechin reference; procyanidin B2 reference; epicatechin reference; citral reference;

[0103] Figure 38 is the chromatogram of negative blank solution;

[0104] Figure 39 Comparison of the characteristic spectra of Amomum villosum kernel and Amomum villosum pseudo-Amomum villosum; S1-3: Amomum villosum pseudo-Amomum villosum; S4: Amomum villosum kernel;

[0105] Figure 40 Comparison of the characteristic spectra of tsaoko kernel and wild tsaoko; S1-3: wild tsaoko; S4: tsaoko kernel;

[0106] Figure 41 Comparison of the characteristic spectra of Amomum villosum and Amomum villosum; S1-3: Amomum villosum; S4: Amomum villosum; S5: Amomum villosum; S6: Amomum villosum; S7: Amomum villosum; S8: Amomum villosum;

[0107] Figure 42 This is the UV spectrum of 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol;

[0108] Figure 43This is the UV spectrum of meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol;

[0109] Figure 44 These are the chromatograms of tsaoko-related samples and tsaoko kernel-related samples at a wavelength of 192 nm; S1: tsaoko medicinal material; S2: tsaoko kernel decoction pieces; S3: tsaoko freeze-dried powder; S4: tsaoko kernel freeze-dried powder; S5: 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol reference substance; S6: meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol reference substance. DETAILED DESCRIPTION

[0110] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection 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 shall fall within the scope of protection of the present invention.

[0111] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0112] The test sample in the following embodiments and experimental examples is a formula granule of tsaoko kernel. The specific preparation method of the formula granule of tsaoko kernel is as follows: 6800g of tsaoko kernel slices are crushed, boiled and extracted, 18 times the weight of water is added, extracted for 1.5 hours, and an extract is obtained while collecting a sufficient amount of volatile oil. The volatile oil is taken by grinding, 7 times the weight of volatile oil is added to β-cyclodextrin to prepare an inclusion compound, the extract is filtered while hot through a 200-mesh filter cloth, the filtrate is concentrated under reduced pressure at 70°C to a relative density of 1.03 to 1.10 (65±5°C), the concentrate is added to the inclusion compound, mixed, spray-dried, crushed, dry-granulated, packaged, and sealed for storage.

[0113] The standard decoction of tsaoko kernel slices of the present invention is prepared by the following method: taking tsaoko kernel slices, crushing them, adding 2 times the amount of water to soak for 30 minutes, adding 6 times the amount of water, boiling with boiling water and simmering over low heat (200W) for 5 minutes, filtering with a 200-mesh filter cloth while hot, adding 6 times the amount of water to the residue, boiling with high heat (500W), then switching to low heat (200W) and boiling for 25 minutes, filtering with a 200-mesh filter cloth while hot, and combining the two filtrates to obtain the standard decoction of tsaoko kernel slices.

[0114] The main instruments and reagents involved in the present invention are as follows:

[0115] 1. Instruments, reagents and test drugs

[0116] Chromatograph 1: Waters ACQUITY UPLC H-Class PLUS chromatography system, including four-element solvent manager (ACQ-QSM), sample manager (ACQ-FTN), imported original chromatographic column oven (ACQ-CM), diode array ultraviolet detector (ACQ-PDA), Empower chromatography management system;

[0117] Chromatograph 2: Agilent 1290 Infinity II chromatography system, including four-element solvent manager (1290 Flexible Pump), sample manager (1290 Vial sampler), imported original chromatographic column oven (1290 MCT), diode array ultraviolet detector (1290 PDA FS), OpenLAB CDS chromatography management system;

[0118] Chromatograph 3: Thermo Vanquish chromatography system, including four-element solvent manager (Pump), sample manager (Autosampler), imported original chromatographic column oven (Column-Compartment), diode array ultraviolet detector (Detector), Chromeleon 7.2 chromatography management system.

[0119] Chromatographic column: Waters HSST3 (column length 150 mm, inner diameter 3.0 mm, particle size 1.8 μm); CAPCELL CORE C18 (column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm); Welch core-shell (column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm).

[0120] Ultrasonic instrument: KQ-500DE (Kunshan Ultrasonic Instrument Co., Ltd.)

[0121] 2, reagents and reagents:

[0122] Protocatechuic acid reference substance (batch number: 110809-202207, purchased from China Food and Drug Inspection Institute, purity 97.5%); protocatechuic aldehyde reference substance (batch number: 110810-202210, purchased from China Food and Drug Inspection Institute, purity 99.9%); citral reference substance (batch number: 190178-201701, purchased from China Food and Drug Inspection Institute); catechin reference substance (batch number: 110877-202005, purchased from China Food and Drug Inspection Institute, purity 95.1%); epicatechin reference substance (batch number: 110878-201703, purchased from the China Food and Drug Administration, purity 99.7%); proanthocyanidin B2 reference substance (batch number: 000897-202006, purchased from Jiangxi Baicaoyuan Biotechnology Co., Ltd.); tsaoko reference medicinal material (batch number: 121550-201602, purchased from the China Food and Drug Administration); tsaoko kernel reference medicinal material (batch number: 230138-202401, Shanghai Hongyong Biotechnology Co., Ltd.). Acetonitrile was chromatographically pure (Thermo Fisher Scientific), water was ultrapure water, and all other reagents were of analytical grade.

[0123] Tsaoko kernel formula granules (batch numbers: 2303001Y, 2303002Y, 2303003Y).

[0124] As used herein, the amomum villosum refers to the amomum villosum shell and amomum villosum kernel, and is commonly used for the amomum villosum in the shell.

[0125] Example 1

[0126] This embodiment provides a method for constructing a characteristic spectrum of tsaoko or tsaoko kernel or its derivatives, comprising:

[0127] (1) Preparation of test solution: Using the tsaoko kernel granules as the test sample, take approximately 0.2 g of the test sample powder, accurately weigh it, place it in a stoppered conical flask, add 25 ml of 70% v / v methanol aqueous solution, weigh it, and sonicate (power 250 W, frequency 40 kHz) for 30 minutes. Remove it, let it cool, and make up the lost weight with 70% v / v methanol aqueous solution. Shake well, filter, and take the filtrate to obtain the solution.

[0128] Preparation of reference medicinal material solution: Take 1g of Tsaoko control medicinal material and 1g of Tsaoko kernel control medicinal material, and prepare Tsaoko control medicinal material reference solution and Tsaoko kernel control medicinal material reference solution according to the above "Preparation of test solution";

[0129] Preparation of reference substance solutions: Take an appropriate amount of epicatechin and citral reference substances, accurately weigh them, and add methanol to make a solution containing 30 μg per 1 ml to prepare epicatechin reference substance solution and citral reference substance solution. Take an appropriate amount of catechin reference substance, accurately weigh it, and add methanol to make a solution containing 20 μg per 1 ml to prepare catechin reference substance solution.

[0130] (2) Take 2 μL of each of the test solution, reference medicinal material solution and reference substance solution and use high performance liquid chromatography to determine the content of the sample. The chromatographic conditions are as follows: octadecylsilane bonded silica gel as the filler (Waters HSST3 column (150 mm length, 3.0 mm inner diameter, 1.8 μm particle size); acetonitrile as mobile phase A, 0.1% (v / v) phosphoric acid solution as mobile phase B, gradient elution as specified in the table below; flow rate, 0.5 ml / min; column temperature, 30°C; detection wavelengths, 230 nm for 0-30 minutes, and 240 nm for 30-45 minutes. The number of theoretical plates, calculated based on the epicatechin peak, should be no less than 5000.

[0131] Table 1 Gradient elution table

[0132]

[0133] The results are shown in Table 2 and Figure 1. Figure 1-(1) to Figure 1-(6) It can be seen that the characteristic spectrum of the tsaoko kernel formula granules corresponds to the chromatographic peak of the characteristic spectrum of the reference medicinal material. The characteristic spectrum of the tsaoko kernel formula granules has 10 characteristic peaks, and all 10 characteristic peaks are effectively separated. Each characteristic peak has a good peak shape, a stable baseline, a short detection time, and a uniform peak height or peak area. Among them, peak 3 and peak 5 correspond to the retention time of the catechin reference substance peak and the epicatechin reference substance peak, respectively, and peak 9 and peak 10 correspond to the retention time of the citral reference substance peak; the peak corresponding to the retention time of the epicatechin reference substance peak is the S peak. The relative retention times of peaks 1-2, peak 4 and peaks 6-8 with the S peak are calculated, and their relative retention times are within the range of ±10% of the specified value, and the specified values ​​are: 0.41 (peak 1), 0.61 (peak 2), 0.87 (peak 4), 1.15 (peak 6), 1.31 (peak 7), and 1.78 (peak 8). Among them, peak 1 corresponds to protocatechuic acid; peak 2 corresponds to protocatechuic aldehyde; peak 3 corresponds to catechin; peak 4 corresponds to procyanidin B2; peak 5 (S) corresponds to epicatechin; peaks 9 and 10 correspond to citral (peaks 9 and 10 correspond to the two isomers of citral, neral and geranial).

[0134] Both the cardamom and cardamom kernel control medicinal materials show 10 characteristic peaks, and the chromatographic peaks are not much different. Considering that the current control supplied by the China National Institute for Food and Drug Control is the cardamom control medicinal material, and the cardamom kernel control medicinal material is provided by a third-party reference material company, the supply quality of the cardamom control medicinal material is more guaranteed. Therefore, the cardamom control medicinal material is used as the control, and the cardamom kernel control medicinal material is no longer used as the control.

[0135] Table 2 System adaptability results

[0136]

[0137]

[0138] Experimental Example 1

[0139] 1. Investigation of chromatographic conditions

[0140] 1. Selection of mobile phase gradient

[0141] Take the same sample solution prepared in Example 1, and inject 2 μL of it; use octadecylsilane bonded silica gel as filler (Waters HSST3) column length: 150 mm, inner diameter: 3.0 mm, particle size: 1.8 μm; acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution (v / v) was used as mobile phase B, and gradient elution was performed according to the requirements in Table 3; the flow rate was 0.3 ml / min; the column temperature was 30°C; the detection wavelength was 240 nm, and the results are shown in Table 3. Figure 2 .

[0142] Table 3 Gradient conditions 1

[0143]

[0144] Depend on Figure 2 It can be seen that gradient elution was performed using gradient condition 1. The water-soluble components with greater polarity were eluted in 0-20 minutes, and the non-polar components such as volatile oils were eluted in 30-40 minutes. There was basically no chromatographic peak after 40 minutes. However, the chromatographic peak separation was poor. Therefore, the mobile phase ratio was further optimized and gradient elution was performed according to the regulations in Table 4. The results are shown in FIG. Figure 3 ;

[0145] Table 4 Gradient Condition 2

[0146]

[0147] Depend on Figure 3 It can be seen that the elution effect of the water-soluble component chromatographic peak is poor at 10-20 minutes when gradient condition 2 is used for elution. Further optimization is carried out according to the provisions in Table 5. The results are shown in Table 5. Figure 4 ;

[0148] Table 5 Gradient conditions 3

[0149]

[0150] Depend on Figure 4 It can be seen that the separation of each chromatographic peak is improved by using gradient condition 3 for elution, but the analysis time is longer. Further optimization is carried out and gradient elution is carried out according to the provisions in Table 6. The results are shown in Figure 5 ;

[0151] Table 6 Gradient Condition 4

[0152]

[0153] Depend on Figure 5 It can be seen that the analysis time is shortened after the elution flow rate and elution gradient are adjusted by using gradient condition 4, but the separation of the chromatographic peaks from 0 to 7 minutes is poor. Further optimization is carried out according to the provisions in Table 7. The results are shown in Table 7. Figure 6 ;

[0154] Table 7 Gradient Condition 5

[0155]

[0156]

[0157] Depend on Figure 6 It can be seen that the elution was performed using gradient condition 5, and the chromatographic peaks were well separated in the period 0-20 minutes. However, the baseline was not stable in the period 20-30 minutes due to the rapid gradient change. Further optimization was performed and the gradient elution was performed according to the provisions in Table 8. The results are shown in FIG. Figure 7 ;

[0158] Table 8 Gradient Condition 6

[0159]

[0160] Depend on Figure 7 It can be seen that the problem of unstable baseline can be improved by slowing down the gradient change when using gradient condition 6 for elution, but the analysis time is increased. Further optimization is carried out according to the provisions in Table 9 for gradient elution. The results are shown in Figure 8 ;

[0161] Table 9 Gradient Conditions 7

[0162]

[0163] Depend on Figure 8 It can be seen that the chromatographic peak separation between 6 and 10 minutes was poor when elution was performed using gradient condition 7. The elution gradient was adjusted and the analysis time was shortened. Further optimization was performed and gradient elution was performed according to the regulations in Table 10. The results are shown in FIG. Figure 9 ;

[0164] Table 10 Gradient Conditions 8

[0165]

[0166]

[0167] Depend on Figure 9 It can be seen that the separation effect of each characteristic peak is good when the gradient condition 8 is used for elution. The elution gradient is slightly adjusted to further shorten the analysis time. The gradient elution is performed according to the regulations in Table 11. The results are shown in FIG. Figure 10 ;

[0168] Table 11 Gradient Conditions 9

[0169]

[0170] Depend on Figure 10 It can be seen that when gradient condition 9 is used for elution, the chromatographic peak separation is poor due to the rapid change of the elution gradient from 13 to 25 minutes. Further optimization is carried out according to the provisions in Table 12. The results are shown in Figure 11 ;

[0171] Table 12 Gradient conditions 10

[0172]

[0173] Depend on Figure 11 It can be seen that the separation effect of each characteristic peak is better and the analysis time is shorter when gradient condition 10 is used for elution. Therefore, gradient condition 10 is temporarily set as the elution gradient, and the flow rate, column temperature and detection wavelength are further investigated.

[0174] 2. Investigation of flow rate

[0175] In addition to the flow rate, the chromatographic conditions determined by the selection of the mobile phase gradient in 1 were used, with the flow rate as the variable. The same sample solution prepared in Example 1 was taken and measured at flow rates of 0.45 ml / min, 0.50 ml / min, and 0.55 ml / min, respectively. The resulting chromatograms are shown in FIG. Figure 12-14 As shown. Figure 12-14 It can be seen that when the elution flow rate changes slightly, the separation differences of the characteristic peaks are not obvious, so the flow rate is temporarily set at 0.50 ml / min for subsequent condition screening.

[0176] 3. Investigation of column temperature

[0177] In addition to the column temperature, according to the chromatographic conditions determined by the investigation of 2 and flow rate, with column temperature as the variable, take the same sample solution prepared in Example 1 and measure it at column temperatures of 25°C, 30°C, and 35°C, respectively. The resulting chromatogram is as follows Figure 15-17 As shown. Figure 15-17It can be seen that the change of column temperature has a great influence on the separation of chromatographic peaks in 10-25 minutes. The chromatographic effect obtained by the column temperature of 30℃ is relatively better. Therefore, the column temperature is temporarily set at 30℃ for subsequent condition screening.

[0178] 4. Investigation of mobile phase

[0179] In addition to the mobile phase, according to the chromatographic conditions determined by the investigation of 3, column temperature, with the mobile phase as the variable, the same test solution prepared according to Example 1 was taken and measured under different mobile phases (acetonitrile-water, acetonitrile-0.1% v / v formic acid aqueous solution, acetonitrile-0.1% v / v glacial acetic acid aqueous solution, acetonitrile-0.05% v / v phosphoric acid aqueous solution, acetonitrile-0.1% v / v phosphoric acid aqueous solution, acetonitrile-0.2% v / v phosphoric acid aqueous solution). The resulting chromatograms are shown in FIG. Figure 18-23 As shown. Figure 18-23 It can be seen that different mobile phases have a great influence on the characteristic spectrum. When formic acid or glacial acetic acid is selected as the mobile phase, the baseline is not stable. When 0.1% phosphoric acid aqueous solution is used as the mobile phase, the chromatographic peak separation and peak shape are better. Therefore, acetonitrile-0.1% phosphoric acid aqueous solution is temporarily selected as the mobile phase for subsequent condition screening.

[0180] 5. Investigation of detection wavelength

[0181] In addition to the detection wavelength, according to the chromatographic conditions determined in 4. Investigation of the mobile phase, with the detection wavelength as a variable, the same sample solution prepared in Example 1 was taken and measured at different detection wavelengths of 230 nm, 240 nm, 254 nm (230 nm for 0-30 minutes and 240 nm for 30-45 minutes). The resulting chromatogram is shown in FIG. Figures 24-27 As shown. Figures 24-27 It can be seen that different detection wavelengths have a great influence on the chromatographic peak response. At a wavelength of 230nm, the chromatographic peak response of the water-soluble components in the first half of the characteristic spectrum is better. At a wavelength of 240nm, the chromatographic peak response of the volatile components in the second half of the characteristic spectrum is better. At a wavelength of 254nm, the overall chromatographic peak response is lower. Therefore, it is chosen to switch the wavelength from 230nm to 240nm in 30 minutes to ensure that there is enough time to switch the wavelength without affecting the detection of volatile oil components. Therefore, the detection wavelength is temporarily set to 230nm from 0 to 30 minutes and 240nm from 30 to 45 minutes for subsequent condition screening and investigation.

[0182] 6. Sample volume inspection

[0183] According to the chromatographic conditions determined in 5. Detection wavelength, with the injection volume as the variable, take the same test solution prepared in Example 1, and accurately aspirate 1 μL, 2 μL, 3 μL, 4 μL, and 5 μL of the test solution for measurement. The resulting chromatogram is shown in FIG. Figure 28-32 As shown. Figure 28-32It can be seen that different injection volumes have a greater impact on the chromatographic peak response. When the injection volume is 4μL or 5μL, the chromatographic peak is deformed. When the injection volume is 2μL, the chromatographic peak shape is better and the peak response is moderate. Therefore, the injection volume is tentatively set to 2μL.

[0184] 7. Determination of optimal chromatographic conditions

[0185] Octadecylsilane bonded silica gel as filler (Waters HSST3 column (150 mm length, 3.0 mm inner diameter, 1.8 μm particle size); acetonitrile as mobile phase A, 0.1% (v / v) phosphoric acid solution as mobile phase B, gradient elution as specified in Table 13; flow rate, 0.5 ml / min; column temperature, 30°C; detection wavelengths: 230 nm from 0 to 30 minutes, 240 nm from 30 to 45 minutes. Injection volume: 2 μL.

[0186] Table 13 Gradient elution table

[0187]

[0188] Experimental Example 2 Construction of characteristic maps of three batches of tsaoko kernel formula granules

[0189] Three batches of tsaoko kernel formula granule samples were taken as test samples, and the test sample solution was prepared according to the method of Example 1; the tsaoko control medicinal material was taken according to the method of Example 1 to prepare the tsaoko control medicinal material reference solution; the catechin, epicatechin, and citral reference substances were taken according to the method of Example 1 to prepare catechin reference substance reference solution, epicatechin reference substance reference solution, and citral reference substance reference solution.

[0190] The above solution was tested by high performance liquid chromatography in Example 1 to obtain characteristic spectra of three batches of tsaoko kernel formula granules. The results are shown in Tables 14-15 (Note: In Table 14, since the wavelength is switched to 240 nm at 30-45 minutes, Peak 9 is taken as the S peak, and the relative retention time of Peak 10 and Peak 9 is calculated) and Figure 33 The results show that the relative retention times and relative peak areas of the three batches of tsaoko kernel formula granules are within the required range.

[0191] Table 14 Relative retention time determination results of three batches of tsaoko kernel formula granules

[0192]

[0193] Table 15 Relative peak area determination results of three batches of tsaoko kernel formula granules

[0194]

[0195] Experimental Example 3: Establishment of control characteristic spectrum

[0196] (1) Generation of control feature maps

[0197] 17 batches of standard decoction of Tsaoko kernel slices (lyophilized powder) (2103001Y, 2103002Y, 2103003Y, 2103004Y, 2103005Y, 2103006Y, 2103007Y, 2103008Y, 2103009Y, 2103010Y, 2103011Y, 2103013Y, 2103014Y, 2103015 Y, 230301Y, 230302Y, 230303Y) were used as test samples, and a test sample solution was prepared according to the method of Example 1; the tsaoko control medicinal material was taken and the tsaoko control medicinal material reference solution was prepared according to the method of Example 1; the catechin, epicatechin, and citral reference substances were taken and the catechin reference substance reference solution, epicatechin reference substance reference solution, and citral reference substance reference solution were prepared according to the method of Example 1.

[0198] The above solution was tested by the high performance liquid chromatography method of Example 1. The test results are shown in Tables 16-21 below, and Figures 34-35 The measurement results show that the characteristic spectra of 17 batches of standard decoctions of Tsaoko kernel slices have 10 characteristic peaks in total, and the retention times should correspond to the 10 characteristic peaks in the chromatograms of the reference medicinal materials. The fingerprint similarity evaluation software "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to generate a control characteristic spectrum for the characteristic spectra of 17 batches of standard decoctions of Tsaoko kernel slices. The Mark peak fitting method was used for fitting the control spectrum, and the common characteristic peaks were identified. A total of 10 common characteristic peaks were identified, such as Figure 36 (Note: in Table 16, since the wavelength was switched to 240 nm from 30 to 45 minutes, Peak 9 was taken as the S peak, and the relative retention time of Peak 10 and Peak 9 was calculated).

[0199] Table 16 Relative retention time of reference spectrum

[0200] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5(S) Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 min 4.904 7.315 8.473 10.4 12.013 13.848 15.687 21.405 37.711 38.566 t / ts 0.41 0.61 0.71 0.87 1.00 1.15 1.31 1.78 1.00 1.02

[0201] Table 17 Comparative chromatogram relative peak area

[0202] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5(S) Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 A 62.985 58.328 398.577 379.004 981.677 235.453 602.445 87.41 157.189 260.211 A / As 0.064 0.059 0.406 0.386 1.000 0.240 0.614 0.089 1.000 1.655

[0203] Table 18 Relative retention time of characteristic spectrum of Tsaoko control medicinal materials

[0204] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5(S) Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 min 4.806 7.219 8.34 10.292 11.935 13.806 15.676 21.344 37.68 38.542 t / ts 0.40 0.60 0.70 0.86 1.00 1.16 1.31 1.79 1.00 1.02

[0205] Table 19 Relative peak areas of characteristic spectrum of Tsaoko control medicinal materials

[0206] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5(S) Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 A 10028 10634 48979 152348 467607 149880 382517 54502 30042 52049 A / As 0.021 0.023 0.105 0.326 1.000 0.321 0.818 0.117 1.000 1.733

[0207] Table 20 Relative retention time determination results of 17 batches of standard decoction of tsaoko kernel slices (lyophilized powder)

[0208]

[0209]

[0210] Table 21 Relative peak area determination results of 17 batches of standard decoction of tsaoko kernel slices (lyophilized powder)

[0211]

[0212]

[0213] (3) Identification of characteristic peaks in the reference spectrum

[0214] Take appropriate amounts of protocatechuic acid, protocatechuic aldehyde, epicatechin, proanthocyanidin B2, and citral reference substances, accurately weigh them, and add methanol to make solutions containing 30 μg per 1 ml to prepare protocatechuic acid reference substance solution, protocatechuic aldehyde reference substance solution, epicatechin reference substance solution, proanthocyanidin B2 reference substance solution, and citral reference substance solution. Take appropriate amount of catechin reference substance, accurately weigh it, and add methanol to make a solution containing 20 μg per 1 ml to prepare catechin reference substance solution.

[0215] The above solution was detected by high performance liquid chromatography method of Example 1 to obtain the reference chromatogram, which was compared with the reference characteristic spectrum. The results are shown in FIG. Figure 37 , 10 characteristic peaks were located and identified by reference substances. The retention times of peaks 1, 2, 3, 4, and 5 corresponded to the retention times of protocatechuic acid, protocatechuic aldehyde, catechin, procyanidin B2, and epicatechin reference substances, respectively. The retention times of peaks 9-10 corresponded to the retention times of citral reference substances. Peak 1 was determined to be protocatechuic acid, peak 2 to protocatechuic aldehyde, peak 3 to catechin, peak 4 to procyanidin B2, peak 5 to epicatechin, and peaks 9 and 10 to citral (peaks 9 and 10 correspond to neral and geranial, two isomers of citral).

[0216] Using reference substances, the first half of the spectrum was primarily composed of polyphenolic components such as protocatechuic acid, protocatechuic aldehyde, catechin, procyanidin B2, and epicatechin, while the second half contained volatile components such as citral. The same method can be used to determine the main water-soluble and volatile active ingredients in tsaoko kernel granules. Among the water-soluble components in the first part of the characteristic spectrum, epicatechin showed a high response, so epicatechin was selected as the S peak for calculating the relative retention times of the characteristic peaks.

[0217] In summary, the test sample chromatogram should show 10 characteristic peaks, and the retention times should correspond to the 10 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 3, 5, 9, and 10 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the epicatechin reference peak is the S peak. The relative retention times of peaks 1-4, 6-10 and the S peak are calculated. The relative retention times should be within ±10% of the specified values: 0.41 (peak 1), 0.61 (peak 2), 0.87 (peak 4), 1.15 (peak 6), 1.31 (peak 7), and 1.78 (peak 8). Peak 1 is protocatechuic acid, peak 2 is protocatechuic aldehyde, peak 3 is catechin, peak 4 is procyanidin B2, peak 5 is epicatechin, and peaks 9 and 10 correspond to citral.

[0218] Experimental Example 4 Methodology Verification

[0219] 1. Precision

[0220] The same sample solution of the tsaoko kernel formula granules prepared according to the method of Example 1 was taken and injected 6 times. The spectrum was recorded and the relative retention time and relative peak area of ​​10 characteristic peaks were measured. The results are shown in Tables 22-23. The RSDs of the relative retention times of the 10 characteristic peaks were all less than 2%; the differences in the relative peak areas of the characteristic peaks were small, indicating good precision.

[0221] Table 22 Precision test relative retention time results

[0222]

[0223] Table 23 Precision test relative peak area results

[0224]

[0225]

[0226] 2. Repeatability test

[0227] The same sample of the tsaoko kernel formula granules was taken for testing and the sample was repeated 6 times according to the method of Example 1. The sample was injected and analyzed according to the chromatographic conditions described in Example 1, and the chromatogram was recorded. The results are shown in Tables 24-25. The RSDs of the relative retention times of the 10 characteristic peaks were all less than 2%, and the differences in the relative peak areas of the characteristic peaks were small, indicating good repeatability.

[0228] Table 24 Relative retention time of repeatability test

[0229]

[0230] Table 25 Precision test relative peak area results

[0231]

[0232]

[0233] 3. Intermediate precision (different operators)

[0234] Three 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 ​​10 common peaks were measured and analyzed. The results are shown in Tables 26-27. The RSDs of the relative retention times of the 10 characteristic peaks were all less than 2%, indicating that the method had good intermediate precision.

[0235] Table 26 Intermediate precision relative retention time

[0236]

[0237] Table 27 Intermediate precision relative retention area

[0238]

[0239] 4. Stability test

[0240] The same test solution prepared in Example 1 was taken, and the sample was injected and analyzed at 0, 4, 8, 12, 18, and 24 h according to the chromatographic conditions described in Example 1. The chromatogram was recorded, and the relative retention time and relative peak area of ​​10 characteristic peaks were measured and analyzed. The results are shown in Tables 28-29. The RSD of the relative retention time of the 10 characteristic peaks was less than 2%, indicating that the method has good stability and can meet the needs of the determination.

[0241] Table 28 Relative retention time of stability test

[0242]

[0243] Table 29 Relative retention area of ​​stability test

[0244]

[0245] 5. Exclusivity

[0246] Preparation of negative blank solvent: Take the negative particles prepared with maltodextrin, grind them into powder, take about 0.2 g, accurately weigh, place in a stoppered conical flask, add 25 ml of 70% v / v methanol aqueous solution, weigh the weight, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, let cool, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate.

[0247] Accurately pipette the test solution (2 μL) and negative blank solvent (2 μL) obtained in Example 1 and inject them into the high performance liquid chromatograph respectively. Test according to the chromatographic conditions of Example 1. Figure 38 As shown in Figure 1, the results show that the negative test has no interference.

[0248] 6. Durability inspection

[0249] 6.1. Investigation of different flow rates

[0250] The same sample solution prepared in Example 1 was taken and measured according to the chromatographic conditions described in Example 1 at flow rates of 0.45 ml / min, 0.50 ml / min, and 0.55 ml / min, respectively. The chromatograms were recorded, and the relative retention times and relative peak areas at different flow rates were measured and analyzed. The results are shown in Tables 30-31.

[0251] Table 30 Results of investigation on relative retention time at different flow rates

[0252]

[0253] Table 31 Results of relative peak area investigation at different flow rates

[0254]

[0255] Different flow rates have a certain influence on the information content of the chromatographic peaks and the system adaptability parameters. After comparative analysis of the above results, when the flow rates are 0.45 ml / min and 0.55 ml / min, the separation of peak 2 and peak 6 is affected. Therefore, the flow rate of 0.50 ml / min is selected as the best in the present invention.

[0256] 6.2. Investigation of different column temperatures

[0257] The same sample solution prepared in Example 1 was taken and measured according to the chromatographic conditions described in Example 1 at column temperatures of 25°C, 30°C, and 35°C, respectively. The chromatograms were recorded, and the relative retention times and relative peak areas at different column temperatures were measured and analyzed. The results are shown in Tables 32-33.

[0258] Table 32 Results of relative retention time at different column temperatures

[0259]

[0260] Table 33 Results of relative peak area investigation at different column temperatures

[0261]

[0262] Different column temperatures have a certain impact on the information content of the chromatographic peaks and the system adaptability parameters. After comparative analysis of the above results, when the column temperature is 25°C and 35°C, the separation of peaks 2 and 3 is affected. Therefore, the present invention selects a column temperature of 30°C as the best.

[0263] 6.3. Inspection of different brands of chromatographic columns

[0264] The same sample solution prepared in Example 1 was taken and measured using different chromatographic columns (chromatographic column 1: Waters ACQUITY HSS T3; chromatographic column 2: Welch core-shell; chromatographic column 3: CAPCELLCORE C18) according to the chromatographic conditions described in Example 1. The chromatograms were recorded, and the relative retention times and relative peak areas of the different chromatographic columns were measured and analyzed. The results are shown in Tables 34-35.

[0265] Table 34 Results of relative retention time investigation on different chromatographic columns

[0266]

[0267] Table 35 Results of relative peak area investigation on different chromatographic columns

[0268]

[0269] Different chromatographic columns have a certain impact on the information content of chromatographic peaks and system suitability parameters. After comparative analysis of the above results, the chromatographic separation obtained with the Waters ACQUITY HSS T3 chromatographic column was good. Therefore, the present invention selected the Waters ACQUITY HSS T3 (1.8 μm, 3.0×150 mm) as the optimal chromatographic column.

[0270] 6.4. Investigation of different chromatographs

[0271] The same sample solution prepared in Example 1 was taken and measured using different chromatographs (Chromatograph 1: Waters ACQUITY UPLC H-Class PLUS chromatographic system; Chromatograph 2: Agilent 1290 Infinity II chromatographic system; Chromatograph 3: Thermo Vanquish chromatographic system) according to the chromatographic conditions described in Example 1. The chromatograms were recorded, and the relative retention times and relative peak areas of the different instruments were measured and analyzed. The results are shown in Tables 36-37.

[0272] Table 36 Results of relative retention time investigation on different chromatographic columns

[0273]

[0274]

[0275] Table 37 Results of relative peak area investigation on different chromatographic columns

[0276]

[0277] After comparative analysis of the above results, the reproducibility of the chromatographic peaks on different chromatographs was good, indicating that the method provided by the present invention has good durability.

[0278] Example 2

[0279] 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.

[0280] The present embodiment provides a method for distinguishing tsaoko or tsaoko kernels or their derivatives from counterfeits, using tsaoko 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 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 Tsaoko, and the results are shown in FIG. Figures 39-41 shown.

[0281] Compared with the control characteristic spectrum of Amomum villosum, the characteristic spectrum of Amomum villosum, Amomum villosum, Amomum villosum, Amomum villosum, Amomum villosum, Amomum villosum, and Amomum villosum has basically no chromatographic peaks at peaks 7 and 8. Considering that peak 8 in the control characteristic spectrum of Amomum villosum is smaller, the relative peak area of ​​peak 8 and peak S is also smaller (0.046-0.174). Therefore, the minimum value of the relative peak of peak 7 and peak S is specified (0.390×0.8=0.312) to distinguish Amomum villosum from related counterfeits.

[0282] It can be seen from this that the identification method of the present invention can be used to identify tsaoko or tsaoko kernels or their derivatives and counterfeits. If peak 7 appears in the characteristic spectrum of the product to be identified and the relative peak area of ​​peak 7 and peak S is ≥0.31, it is tsaoko or its derivatives, otherwise it is not.

[0283] Example 3

[0284] According to relevant information, the shell of Amomum villosum should be removed when used clinically.

[0285] This embodiment provides a method for identifying tsaoko kernels or their derivatives from tsaoko in shells or their derivatives, comprising the following steps:

[0286] (1) Using Tsaoko medicinal materials, Tsaoko kernel slices, Tsaoko kernel freeze-dried powder (standard decoction), and Tsaoko (standard decoction) as test samples, respectively, prepare test sample solutions according to the method of Example 1; taking appropriate amounts of 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol and meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol reference substances, respectively, prepare 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol reference substance solutions and meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol reference substance solutions containing 2.5 μg per 1 ml according to the method of Example 1.

[0287] (2) According to Figures 42-43 As shown in FIG, the optimal absorption wavelength of meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol and 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol is 192 nm, so the wavelength of 192 nm was selected for the HPLC determination.

[0288] Take 2 μL of the test solution and reference solution and use high performance liquid chromatography to determine the content of the sample. The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as the filler (Waters HSST3) column length: 150 mm, inner diameter: 3.0 mm, particle size: 1.8 μm; acetonitrile as mobile phase A, 0.1% v / v phosphoric acid solution as mobile phase B, gradient elution as specified in Table 1; flow rate: 0.5 ml / min; column temperature: 30°C; detection wavelength: 192 nm. Results are shown in Tables 38-39 and Figure 44 shown.

[0289] Table 38 Comparison of calculated concentration results of 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol

[0290]

[0291] Table 39 Comparison of calculated concentration results of meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol

[0292]

[0293]

[0294] according to Figure 44 As shown in the results, the detection peaks of meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol and 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol in tsaoko and its derivatives are relatively high; the above two components are basically not detected in tsaoko kernels and their derivatives. Therefore, meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol and 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol can be used to identify and distinguish tsaoko kernels and their derivatives from shelled tsaoko and its derivatives.

[0295] It can be seen from Tables 38-39 that the concentration of meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol is more different from that of 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol in Tsaoko and its derivatives and Tsaoko kernels and their derivatives. Based on cost considerations, only the peak area of ​​a certain concentration of meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol reference solution is specified to identify and differentiate the shelled Tsaoko.

[0296] The minimum concentration of meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol in samples related to tsaoko was 2.72 μg / ml, and the maximum concentration in samples related to tsaoko kernel was 1.057 μg / ml. Therefore, the concentration of the meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol reference substance was determined to be 2.5 μg / ml. At a wavelength of 192 nm, the peak area of ​​the chromatographic peak of meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol in the sample to be tested must not be greater than the peak area of ​​the meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol reference substance. The results are shown in Table 40.

[0297] Table 40 Comparison of peak area results of meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol

[0298]

[0299]

[0300] The peak area of ​​the meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol reference substance at a concentration of 2.5 μg / ml is 155239 μAU·S. According to Table 40, the peak areas of the tsaoko-related samples are 181611-377171 μAU·S, the peak areas of the tsaoko kernel-related samples are 10574-70568 μAU·S, and the peak areas of the tsaoko kernel-related derivatives are 44865-55420 μAU·S. It can be seen that the peak area of ​​the meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol reference substance at a concentration of 2.5 μg / ml can effectively distinguish the tsaoko kernel and its derivatives from the tsaoko shell.

[0301] It can be seen from this that the identification method of the present invention can be used to identify tsaoko kernels or their derivatives and shelled tsaoko kernels or their derivatives. For shelled tsaoko kernels or their derivatives, the chromatographic peak area of ​​the meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol is detected to be greater than 155239 μAU·S; for tsaoko kernels or their derivatives, the chromatographic peak area of ​​the meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol is detected to be ≤155239 μAU·S.

[0302] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. 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 or tsaoko kernel or its derivatives, characterized in that: The method includes high performance liquid chromatography detection, and the chromatographic conditions include: Octadecylsilane bonded silica gel was used as the filler, an aqueous solution containing phosphoric acid was used as the mobile phase B, and acetonitrile was used as the mobile phase A. The phosphoric acid concentration in the aqueous solution containing phosphoric acid in the mobile phase B was 0.05-0.2% v / v. The detection wavelength was 230-240 nm. The chromatographic column specifications were: column length 100-150 mm, inner diameter 3.0-4.6 mm, particle size 1.8-2.7 μm. Gradient elution was performed, and the gradient elution procedure included: From 0 to 12 minutes, the volume percentage of mobile phase A was 9%→16%, and the volume percentage of mobile phase B was 91%→84%; From 12 to 18 minutes, the volume percentage of mobile phase A was 16% → 24%, and the volume percentage of mobile phase B was 84% ​​→ 76%; From 18 to 24 minutes, the volume percentage of mobile phase A was 24% → 30%, and the volume percentage of mobile phase B was 76% → 70%; From 24 to 35 minutes, the volume percentage of mobile phase A is 30% → 52%, and the volume percentage of mobile phase B is 70% → 48%; From 35 to 45 minutes, the volume percentage of mobile phase A is 52% → 55%, and the volume percentage of mobile phase B is 48% → 45%; The preparation of the test solution includes: weighing the test sample, adding a solvent for extraction, separating the solid and the liquid, and taking the liquid to obtain the test solution; the solvent is selected from one or more of methanol, ethanol, and water; The method also includes the step of preparing a reference solution by using catechin, epicatechin and citral.

2. The construction method according to claim 1, characterized in that The chromatographic conditions include at least one of the following: (1) The detection wavelength is 230-240nm; (2) Flow rate is 0.45-0.55 ml / min; (3) Column temperature 25-35℃; (4) The injection volume is 1-5 μL; (5) The phosphoric acid concentration in the aqueous solution containing phosphoric acid in mobile phase B is 0.1% v / v; (6) The specifications of the chromatographic column are: column length 150 mm, inner diameter 3.0 mm, and particle size 1.8 μm.

3. The construction method according to claim 2, characterized in that The chromatographic conditions include at least one of the following: (1) Flow rate is 0.5 ml / min; (2) Column temperature is 30°C; (3) The injection volume is 1-3 μL.

4. The construction method according to claim 3, characterized in that The chromatographic conditions include: an injection volume of 2 μL.

5. The construction method according to claim 4, characterized in that The preparation of the test solution satisfies at least one of the following: A. The ratio of the mass of the test sample to the volume of the solvent is (0.2-1.0):25; the relationship between mass and volume is g / mL; B. The extraction method is ultrasonic extraction; C. Extraction time is ≥15 minutes; D. The solid-liquid separation is selected from centrifugation or membrane filtration; E. preferably a methanol aqueous solution with a volume percentage of 50-70%.

6. The construction method according to claim 5, characterized in that: The extraction time was 30 minutes.

7. The construction method according to any one of claims 1 to 6, characterized in that: The construction method further comprises the step of detecting the reference substance solution according to the high performance liquid chromatography method in the construction method to obtain a reference spectrum of the reference substance.

8. The construction method according to claim 7, characterized in that: Each 1 mL of catechin reference solution contains 10-30 μg of catechin.

9. The construction method according to claim 8, characterized in that: Each 1 mL of catechin reference solution contains 20 μg of catechin.

10. The construction method according to claim 9, characterized in that: Each 1 mL of epicatechin reference solution contains 20-40 μg of epicatechin.

11. The construction method according to claim 10, characterized in that: Each 1 mL of epicatechin reference solution contains 30 μg of epicatechin.

12. The construction method according to claim 11, characterized in that: Each 1 mL of citral reference solution contains 20-40 μg of citral.

13. The construction method according to claim 12, characterized in that: Each 1 mL of citral reference solution contains 30 μg of citral.

14. The construction method according to claim 13, characterized in that: The solvent used in the preparation of the reference solution is selected from methanol or a methanol aqueous solution with a volume fraction of not less than 50-70%.

15. The construction method according to claim 14, characterized in that: The solvent used in the preparation of the reference solution was selected from 70% methanol aqueous solution.

16. The construction method according to claim 15, characterized in that: The construction method also includes the steps of preparing a control medicinal material solution using the Amomum villosum control medicinal material, and the step of detecting the control medicinal material solution using the high performance liquid chromatography method in the construction method to obtain a control medicinal material reference atlas.

17. The construction method according to claim 16, characterized in that: The tsaoko or its derivative products include at least one of tsaoko medicinal materials, tsaoko decoction pieces or tsaoko preparations; the tsaoko kernels or its derivative products include at least one of tsaoko kernel decoction pieces or tsaoko kernel preparations.

18. A method for determining the content of tsaoko or tsaoko kernel or its derivatives, characterized in that: include: Take the test solution and the reference solution, and respectively detect them by the high performance liquid chromatography method in the method for constructing the characteristic spectrum of the tsaoko or tsaoko kernel or its derivative products according to any one of claims 1 to 17; Among them, the reference substances include catechin, epicatechin and citral.

19. A method for detecting the quality of tsaoko or tsaoko kernels or their derivatives, characterized in that: The method comprises the steps of comparing the characteristic spectrum of the product to be tested with the control characteristic spectrum of the tsaoko or tsaoko kernel or its derivative products; The characteristic spectrum of the product to be tested is constructed according to the construction method according to any one of claims 1 to 17; The control characteristic spectrum of the tsaoko or tsaoko kernel or its derivative products is selected from any one of the following (1) to (4): (1) It has 10 common characteristic peaks. The peak corresponding to the reference peak of epicatechin is the S peak. The relative retention times of peaks 1-2, 4, 6-8 and S peak are calculated. The relative retention times should be within the range of ±10% of the specified values. The specified values ​​of peaks 1-2, 4 and 6-8 are 0.41, 0.61, 0.87, 1.15, 1.31 and 1.78 respectively. (2) It has 10 common characteristic peaks, of which 4 peaks correspond to the retention times of the reference peaks of catechin, epicatechin, and citral, respectively. The peak corresponding to the reference peak of epicatechin is the S peak. The relative retention times of peaks 1-2, 4, 6-8 and the S peak are calculated. The relative retention times should be within the range of ±10% of the specified values. The specified values ​​of peaks 1-2, 4, and 6-8 are 0.41, 0.61, 0.87, 1.15, 1.31, and 1.78, respectively. (3) Characteristic spectra of tsaoko and / or tsaoko kernels and / or their derivatives obtained by the construction method according to any one of claims 1 to 17 using a single batch or multiple batches of tsaoko and / or tsaoko kernels and / or their derivatives as test samples; (4) Using multiple batches of tsaoko and / or tsaoko kernels and / or their derivatives as test samples, the characteristic spectrum obtained according to the construction method described in any one of claims 1 to 17 is used to prepare a control characteristic spectrum by using the average value or median method.

20. The quality inspection method of tsaoko or tsaoko kernel or its derivative products according to claim 19, characterized in that: Peak 1 corresponds to protocatechuic acid; peak 2 corresponds to protocatechuic aldehyde; peak 3 corresponds to catechin; peak 4 corresponds to procyanidin B2; peak 5 (S) corresponds to epicatechin; peaks 9 and 10 correspond to citral.

21. A method for distinguishing between tsaoko or tsaoko kernels or their derivatives and counterfeits, characterized in that: include: The test is carried out according to the quality detection method of the tsaoko or tsaoko kernel or its derivative products as described in claim 20; wherein, for the tsaoko or tsaoko kernel or its derivative products, the relative peak area of ​​peak 7 and peak S should be within the specified range, and the specified value is: not less than 0.

31.

22. The method for distinguishing between tsaoko or tsaoko kernels or derivatives thereof and counterfeits according to claim 21, characterized in that: The counterfeit products include one or more of Amomum villosum or its derivatives, Alpinia fasciata seeds or its derivatives, Amomum villosum or its derivatives, and Amomum villosum or its derivatives.

23. The method for distinguishing between tsaoko or tsaoko kernels or derivatives thereof and counterfeits according to claim 22, characterized in that: The Amomum villosum includes one or more of Amomum villosum yangchunensis, Amomum villosum longifolia, Amomum villosum niuginosa, and Amomum villosum fragrant.

24. A method for distinguishing between tsaoko kernels or their derivatives and tsaoko in shell or their derivatives, characterized in that: include: The step of detecting the characteristic spectrum of the product to be tested by high performance liquid chromatography in the construction method according to any one of claims 1 to 17; The chromatographic conditions of the high performance liquid chromatography method also include a detection wavelength of 192-194 nm; The reference solution also includes a reference solution containing 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol and / or meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol; For tsaoko kernel or its derivative products, the peak area of ​​the chromatographic peak of 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol and / or meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol in the characteristic spectrum is ≤ the peak area of ​​the chromatographic peak of the corresponding reference solution of 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol and / or meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol; For shelled Amomum villosum or its derivative products, the peak area of ​​the chromatographic peak of 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol and / or meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol in the characteristic spectrum is greater than the peak area of ​​the chromatographic peak of the corresponding reference solution of 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol and / or meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol.

25. The method for identifying tsaoko kernels or their derivatives and tsaoko in shell or their derivatives according to claim 24, characterized in that: Each 1 mL of the reference solution contains 1.6-2.5 μg of meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol or 1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol.

26. The method for identifying tsaoko kernels or their derivatives and tsaoko in shell or their derivatives according to claim 25, characterized in that: For shelled tsaoko or its derivatives, the chromatographic peak area of ​​the meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol is detected to be greater than 155239 μAU·S; for tsaoko kernel or its derivatives, the chromatographic peak area of ​​the meso-1,7-bis-(4-hydroxyphenyl)-3,5-heptanediol is detected to be ≤155239 μAU·S.

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