Construction method and application of HPLC characteristic fingerprint of walnut kernel medicinal material, decoction pieces, standard decoction and formula granules

Abstract

The present invention belongs to the field of detection technology, and particularly relates to a method for constructing HPLC characteristic fingerprints of walnut kernel medicinal materials, decoction pieces, standard decoctions, and formula granules, and its application. The HPLC characteristic fingerprint construction method provided by the present invention comprises the following steps: preparing a test sample, wherein the test sample is a walnut kernel medicinal material, walnut kernel decoction pieces, walnut kernel standard decoction, or walnut kernel formula granule; dissolving the test sample to obtain a test sample solution; measuring the test sample solution by high performance liquid chromatography to obtain the HPLC characteristic fingerprint of the corresponding test sample; the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, and mobile phase B is 0.1 wt% phosphoric acid solution, with gradient elution. The characteristic fingerprint construction method provided by the present invention can provide a more scientific basis for the identification of walnut kernel medicinal materials, decoction pieces, standard decoctions, and formula granules.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection, and particularly relates to a method for constructing HPLC characteristic fingerprints of walnut kernels as medicinal materials, cut pieces, standard decoctions, and formula granules, as well as its application. Background Art

[0002] Walnut kernel is the dried mature seed of Juglans regia L. of the Juglandaceae family, and has the effects of tonifying the kidney, warming the lungs, and moistening the intestines. It is used to treat or improve symptoms such as insufficient kidney yang, soreness and weakness of the waist and knees, impotence and spermatorrhea, deficiency-cold asthma, and intestinal dryness and constipation. In order to ensure the uniformity and stability of the quality of walnut kernel as medicinal materials, cut pieces, standard decoctions, and formula granules, it is very necessary to establish a new characteristic fingerprint method to control their quality. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a method for constructing HPLC characteristic fingerprints of walnut kernels as medicinal materials, cut pieces, standard decoctions, and formula granules, as well as its application. The construction method provided by the present invention can provide a more scientific basis for the identification of walnut kernels as medicinal materials, cut pieces, standard decoctions, and formula granules.

[0004] The present invention provides a method for constructing HPLC characteristic fingerprints of walnut kernels as medicinal materials, cut pieces, standard decoctions, and formula granules, comprising the following steps:

[0005] Prepare the test sample, and the test sample is walnut kernel as medicinal material, walnut kernel cut pieces, walnut kernel standard decoction or walnut kernel formula granule;

[0006] Dissolve the test sample to obtain a test sample solution;

[0007] Determine the test sample solution by high performance liquid chromatography to obtain the HPLC characteristic fingerprint of the corresponding test sample;

[0008] The chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is 0.1 wt% phosphoric acid solution, and gradient elution is performed.

[0009] In the construction method provided by the present invention, the walnut kernel cut pieces are the processed products of walnut kernel as medicinal materials; the walnut kernel standard decoction is the freeze-dried powder prepared from walnut kernel as medicinal materials after processing; the walnut kernel formula granule is the formula granule prepared by processing walnut kernel as medicinal materials according to the main quality indexes of the standard decoction.

[0010] In the construction method provided by the present invention, the solvent for dissolution is preferably 50 vol% methanol solution, and the chromatogram obtained under this solvent condition has a good peak shape and moderate resolution.

[0011] In the construction method provided by the present invention, when the test sample is walnut kernel medicinal material or walnut kernel decoction pieces, the specific process of dissolving and preparing the test sample solution preferably includes: decocting the test sample with water, cooling, filtering, evaporating the filtrate to dryness, mixing and dissolving the evaporated residue with a solvent, and filtering. The subsequent filtrate obtained is the test sample solution. Among them, the dosage ratio of the test sample to water is preferably (0.8 - 1.2) g: 30 mL, more preferably 1 g: 30 mL; the decocting time is preferably 25 - 35 min, more preferably 28 - 32 min, and still more preferably 30 min; the dosage ratio of the solvent to the test sample is preferably 10 mL: (0.8 - 1.2) g, more preferably 10 mL: 1 g.

[0012] In the construction method provided by the present invention, when the test sample is a standard decoction of walnut kernel, the specific process of dissolving and preparing the test sample solution preferably includes: mixing the test sample with a solvent, ultrasonically assisting in dissolution, cooling, shaking well, and filtering. The subsequent filtrate obtained is the test sample solution. Among them, the dosage ratio of the test sample to the solvent is preferably (0.2 - 0.4) g: 50 mL, more preferably 0.3 g: 50 mL; the power of the ultrasonic assistance is preferably 580 - 620 W, more preferably 600 W; the frequency of the ultrasonic assistance is preferably 35 - 45 kHz, more preferably 40 kHz; the time of the ultrasonic assistance is preferably 25 - 35 min, more preferably 30 min.

[0013] In the construction method provided by the present invention, when the test sample is walnut kernel formula granules, the specific process of dissolving and preparing the test sample solution preferably includes: grinding the test sample finely and then mixing it with a solvent, ultrasonically assisting in dissolution, cooling, shaking well, and filtering. The subsequent filtrate obtained is the test sample solution. Among them, the dosage ratio of the test sample to the solvent is preferably (0.2 - 0.4) g: 20 mL, more preferably 0.3 g: 20 mL; the power of the ultrasonic assistance is preferably 580 - 620 W, more preferably 600 W; the frequency of the ultrasonic assistance is preferably 35 - 45 kHz, more preferably 40 kHz; the time of the ultrasonic assistance is preferably 25 - 35 min, more preferably 30 min.

[0014] In the construction method provided by the present invention, when performing the determination by high performance liquid chromatography, the specific process of the gradient elution is preferably as follows:

[0015] 0 - 5 min, phase A: 2 vol%, phase B: 98 vol%;

[0016] 5 - 13 min, phase A: 2 - 10 vol%, phase B: 98 - 90 vol%;

[0017] 13 - 17 min, phase A: 10 - 13.5 vol%, phase B: 90 - 86.5 vol%;

[0018] 17 - 24 min, Phase A: 13.5 - 19.5 vol%, Phase B: 86.5 - 80.5 vol%;

[0019] 24 - 30 min, Phase A: 19.5 - 25 vol%, Phase B: 80.5 - 75 vol%.

[0020] In the construction method provided by the present invention, when performing the high - performance liquid chromatography determination, the flow rate of the mobile phase is preferably 0.3 mL / min. Under this flow rate condition, the peak shape of the chromatogram obtained is better and the resolution is moderate.

[0021] In the construction method provided by the present invention, when performing the high - performance liquid chromatography determination, the detection wavelength is preferably 254 nm. Under this detection wavelength condition, the chromatographic peaks of the chromatogram obtained have more information and the baseline is more stable.

[0022] In the construction method provided by the present invention, when performing the high - performance liquid chromatography determination, the injection volume is preferably 1 μL.

[0023] In the construction method provided by the present invention, when performing the high - performance liquid chromatography determination, the filler in the chromatographic column is octadecylsilyl bonded silica gel.

[0024] In the construction method provided by the present invention, when performing the high - performance liquid chromatography determination, the column temperature of the chromatographic column is preferably 25 °C. Under this column temperature condition, the peak shape of the chromatogram obtained is relatively symmetrical, the resolution is good, and the peak emergence is relatively complete.

[0025] In the construction method provided by the present invention, when performing the high - performance liquid chromatography determination, the theoretical plate number calculated based on the ellagic acid peak should be not less than 5000.

[0026] In the construction method provided by the present invention, it preferably further includes the following steps:

[0027] Dissolve the control medicinal material of walnut kernels to obtain a control medicinal material reference solution; dissolve gallic acid to obtain a gallic acid reference substance solution; dissolve ellagic acid to obtain an ellagic acid reference substance solution;

[0028] Determine the control medicinal material reference solution and the reference substance solution of the reference substance by high - performance liquid chromatography to obtain the chromatogram of the reference substance; and qualitatively analyze the components of the HPLC characteristic chromatogram of the test sample according to the chromatogram of the reference substance.

[0029] In the construction method provided by the present invention, the specific process of dissolving and preparing the reference solution of the control crude drug preferably includes: decocting the control crude drug of walnut kernels with water, cooling, filtering, evaporating the filtrate to dryness, mixing the evaporated residue with a solvent for dissolution, and filtering. The subsequent filtrate obtained is the reference solution of the control crude drug. Among them, the dosage ratio of the control crude drug of walnut kernels to water is preferably (0.8 - 1.2) g:30 mL, more preferably 1 g:30 mL; the decocting time is preferably 25 - 35 min, more preferably 28 - 32 min, and still more preferably 30 min; the solvent is preferably 50 vol% methanol solution; the dosage ratio of the solvent to the control crude drug of walnut kernels is preferably 10 mL:(0.8 - 1.2) g, more preferably 10 mL:1 g.

[0030] In the construction method provided by the present invention, the specific process of dissolving and preparing the reference solution of the gallic acid reference substance preferably includes: mixing gallic acid with a solvent to obtain the reference solution of the gallic acid reference substance. Among them, the solvent is preferably methanol; the dosage ratio of gallic acid to the solvent is preferably (10 - 30) μg:1 mL, more preferably 20 μg:1 mL.

[0031] In the construction method provided by the present invention, the specific process of dissolving and preparing the reference solution of the ellagic acid reference substance preferably includes: mixing ellagic acid with a solvent to obtain the reference solution of the ellagic acid reference substance. Among them, the solvent is preferably methanol; the dosage ratio of ellagic acid to the solvent is preferably (30 - 70) μg:1 mL, more preferably 50 μg:1 mL.

[0032] In the construction method provided by the present invention, the following steps are preferably further included:

[0033] Using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints to evaluate the similarity of the HPLC characteristic fingerprint of the walnut kernel crude drug, and obtaining the HPLC standard characteristic fingerprint of the walnut kernel crude drug composed of 6 characteristic peaks. In the HPLC standard characteristic fingerprint, the peak 1 corresponding to the gallic acid reference substance peak is the S1 peak. Calculate the relative retention times of peak 2 and peak 3 with respect to the S1 peak, and their relative retention times are within the range of ±10% of the specified values. The specified values are: 1.00 (peak 1), 2.23 (peak 2), 3.97 (peak 3). The peak 5 corresponding to the ellagic acid reference substance peak is the S2 peak. Calculate the relative retention times of peak 4 and peak 6 with respect to the S2 peak, and their relative retention times are within the range of ±10% of the specified values. The specified values are: 0.96 (peak 4), 1.00 (peak 5), 1.16 (peak 6).

[0034] In the construction method provided by the present invention, the following steps are preferably further included:

[0035] The similarity of the HPLC characteristic fingerprint of walnut kernel decoction pieces was evaluated using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprint. An HPLC standard characteristic fingerprint of walnut kernel decoction pieces consisting of 6 characteristic peaks was obtained. In the said HPLC standard characteristic fingerprint, peak 1 corresponding to the reference peak of gallic acid reference substance was S1 peak. The relative retention times of peak 2 and peak 3 with respect to S1 peak were calculated, and the relative retention times were within the range of ±10% of the specified values. The specified values were: 1.00 (peak 1), 2.23 (peak 2), 3.98 (peak 3). Peak 5 corresponding to the reference peak of ellagic acid reference substance was S2 peak. The relative retention times of peak 4 and peak 6 with respect to S2 peak were calculated, and the relative retention times were within the range of ±10% of the specified values. The specified values were: 0.96 (peak 4), 1.00 (peak 5), 1.16 (peak 6).

[0036] In the construction method provided by the present invention, preferably, the following steps are further included:

[0037] The similarity of the HPLC characteristic fingerprint of walnut kernel standard decoction was evaluated using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprint. An HPLC standard characteristic fingerprint of walnut kernel standard decoction consisting of 6 characteristic peaks was obtained. In the said HPLC standard characteristic fingerprint, peak 1 corresponding to the reference peak of gallic acid reference substance was S1 peak. The relative retention times of peak 2 and peak 3 with respect to S1 peak were calculated, and the relative retention times were within the range of ±10% of the specified values. The specified values were: 1.00 (peak 1), 2.22 (peak 2), 3.98 (peak 3). Peak 5 corresponding to the reference peak of ellagic acid reference substance was S2 peak. The relative retention times of peak 4 and peak 6 with respect to S2 peak were calculated, and the relative retention times were within the range of ±10% of the specified values. The specified values were: 0.96 (peak 4), 1.00 (peak 5), 1.16 (peak 6).

[0038] In the construction method provided by the present invention, preferably, the following steps are further included:

[0039] The similarity of the HPLC characteristic fingerprint of walnut kernel formula granules was evaluated using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprint. An HPLC standard characteristic fingerprint of walnut kernel formula granules consisting of 6 characteristic peaks was obtained. In the said HPLC standard characteristic fingerprint, peak 1 corresponding to the reference peak of gallic acid reference substance was S1 peak. The relative retention times of peak 2 and peak 3 with respect to S1 peak were calculated, and the relative retention times were within the range of ±10% of the specified values. The specified values were: 1.00 (peak 1), 2.23 (peak 2), 3.98 (peak 3). Peak 5 corresponding to the reference peak of ellagic acid reference substance was S2 peak. The relative retention times of peak 4 and peak 6 with respect to S2 peak were calculated, and the relative retention times were within the range of ±10% of the specified values. The specified values were: 0.96 (peak 4), 1.00 (peak 5), 1.16 (peak 6).

[0040] The present invention also provides a method for identifying walnut kernels as medicinal materials, cut pieces, standard decoctions, and formula granules. This method uses the HPLC characteristic chromatogram obtained by the construction method described in the above technical solution as the basis for identifying walnut kernels as medicinal materials, cut pieces, standard decoctions, and formula granules.

[0041] Compared with the prior art, the present invention provides a method for constructing an HPLC characteristic chromatogram of walnut kernels as medicinal materials, cut pieces, standard decoctions, and formula granules and its application. The method provided by the present invention can be used to establish the HPLC characteristic chromatogram of walnut kernels as medicinal materials, cut pieces, standard decoctions, and formula granules, and has good repeatability, precision, and stability, which can provide a more scientific basis for the identification of walnut kernels as medicinal materials, cut pieces, standard decoctions, and formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on the provided drawings without creative efforts.

[0043] Figure 1 It is the chromatogram of different wavelengths of the standard decoction of walnut kernels provided in Example 1 of the present invention;

[0044] Figure 2 It is the chromatogram for investigating the column temperature of the standard decoction of walnut kernels provided in Example 1 of the present invention;

[0045] Figure 3 It is the chromatogram for investigating the flow rate of the standard decoction of walnut kernels provided in Example 1 of the present invention;

[0046] Figure 4 It is the chromatogram for investigating the delay of the standard decoction of walnut kernels provided in Example 1 of the present invention;

[0047] Figure 5 It is the experimental result diagram for investigating the dissolution solvent during the preparation of the test solution of the standard decoction of walnut kernels provided in Example 1 of the present invention;

[0048] Figure 6 It is the experimental result diagram for investigating the dissolution method during the preparation of the test solution of the standard decoction of walnut kernels provided in Example 1 of the present invention;

[0049] Figure 7 It is the experimental result diagram for investigating the dissolution time during the preparation of the test solution of the standard decoction of walnut kernels provided in Example 1 of the present invention;

[0050] Figure 8It is the experimental result graph of the investigation on the solvent addition amount during the preparation of the test solution of the standard walnut kernel decoction provided in Example 1 of the present invention;

[0051] Figure 9 It is the chromatographic peak identification graph of the characteristic chromatogram of the standard walnut kernel decoction provided in Example 1 of the present invention;

[0052] Figure 10 It is the ultraviolet absorption spectrum graph of the gallic acid reference substance provided in Example 1 of the present invention;

[0053] Figure 11 It is the ultraviolet absorption spectrum graph of the gallic acid in the test sample provided in Example 1 of the present invention;

[0054] Figure 12 It is the ultraviolet absorption spectrum graph of the ellagic acid reference substance provided in Example 1 of the present invention;

[0055] Figure 13 It is the ultraviolet absorption spectrum graph of the ellagic acid in the test sample provided in Example 1 of the present invention;

[0056] Figure 14 It is the experimental result graph of the investigation on different instruments of the standard walnut kernel decoction provided in Example 1 of the present invention;

[0057] Figure 15 It is the experimental result graph of the investigation on different chromatographic columns of the standard walnut kernel decoction provided in Example 1 of the present invention;

[0058] Figure 16 It is the verification graph of the characteristic chromatogram of 16 batches of the standard walnut kernel decoction provided in Example 1 of the present invention;

[0059] Figure 17 It is the reference chromatogram of the characteristic chromatogram of the standard walnut kernel decoction provided in Example 1 of the present invention;

[0060] Figure 18 It is the chromatogram of the walnut kernel medicinal material at different wavelengths provided in Example 2 of the present invention;

[0061] Figure 19 It is the chromatogram of the investigation on the column temperature of the walnut kernel medicinal material provided in Example 2 of the present invention;

[0062] Figure 20 It is the chromatogram of the investigation on the flow rate of the walnut kernel medicinal material provided in Example 2 of the present invention;

[0063] Figure 21 It is the chromatogram of the investigation on the delay of the walnut kernel medicinal material provided in Example 2 of the present invention;

[0064] Figure 22 It is the experimental result graph of the investigation on the dissolution solvent during the preparation of the test solution of the walnut kernel medicinal material provided in Example 2 of the present invention;

[0065] Figure 23 This is a graph showing the experimental results of investigating the decoction time during the preparation of the walnut kernel medicinal material test solution provided in Example 2 of the present invention;

[0066] Figure 24 This is a graph showing the experimental results of investigating the amount of solvent added during the preparation of the walnut kernel medicinal material test sample solution provided in Example 2 of the present invention;

[0067] Figure 25 This is a graph showing the experimental results of investigating the dissolution mode during the preparation of the walnut kernel medicinal material test solution provided in Example 2 of the present invention;

[0068] Figure 26 This is a chromatographic peak identification diagram of the characteristic spectrum of walnut kernel medicinal material provided in Example 2 of the present invention;

[0069] Figure 27 This is a diagram showing the experimental results of different instruments investigating walnut kernel medicinal materials provided in Example 2 of the present invention;

[0070] Figure 28 This is a graph showing the experimental results of different chromatographic columns for investigating walnut kernel medicinal materials provided in Example 2 of the present invention;

[0071] Figure 29 This is a characteristic spectrum verification diagram of 16 batches of walnut kernel medicinal materials provided in Example 2 of the present invention;

[0072] Figure 30 This is a comparison of the characteristic atlas of walnut kernel medicinal materials provided in Example 1 of the present invention;

[0073] Figure 31 This is a characteristic spectrum verification diagram of 16 batches of walnut kernel slices provided in Example 2 of the present invention;

[0074] Figure 32 It is a comparison graph of the characteristic graph of the walnut kernel decoction piece provided in Example 2 of the present invention;

[0075] Figure 33 This is a chromatogram of different wavelengths of the walnut kernel formula granules provided in Example 3 of the present invention;

[0076] Figure 34 This is a column temperature chromatogram of the walnut kernel formula granules provided in Example 3 of the present invention;

[0077] Figure 35 This is a flow rate chromatogram of the walnut kernel formula granules provided in Example 3 of the present invention;

[0078] Figure 36 This is a chromatogram of the delayed investigation of the walnut kernel formula granules provided in Example 3 of the present invention;

[0079] Figure 37It is the experimental result diagram for investigating the dissolving solvent during the preparation of the test solution of walnut kernel formula granules provided in Example 3 of the present invention;

[0080] Figure 38 It is the experimental result diagram for investigating the dissolving method during the preparation of the test solution of walnut kernel formula granules provided in Example 3 of the present invention;

[0081] Figure 39 It is the experimental result diagram for investigating the dissolving time during the preparation of the test solution of walnut kernel formula granules provided in Example 3 of the present invention;

[0082] Figure 40 It is the experimental result diagram for investigating the amount of solvent added during the preparation of the test solution of walnut kernel formula granules provided in Example 3 of the present invention;

[0083] Figure 41 It is the chromatographic peak identification diagram of the characteristic chromatogram of walnut kernel formula granules provided in Example 3 of the present invention;

[0084] Figure 42 It is the experimental result diagram for investigating different instruments of walnut kernel formula granules provided in Example 3 of the present invention;

[0085] Figure 43 It is the experimental result diagram for investigating different chromatographic columns of walnut kernel formula granules provided in Example 3 of the present invention;

[0086] Figure 44 It is the verification diagram of the characteristic chromatogram of 3 batches of walnut kernel formula granules provided in Example 3 of the present invention;

[0087] Figure 45 It is the control chromatogram of the characteristic chromatogram of walnut kernel formula granules provided in Example 3 of the present invention. Detailed implementation manners

[0088] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without any creative efforts shall fall within the protection scope of the present invention.

[0089] Example 1

[0090] Construction of the HPLC characteristic chromatogram of the standard decoction of walnut kernel:

[0091] 1) Experimental instruments and materials

[0092] High performance liquid chromatograph: Agilent, Thermo Fisher, Waters type high performance liquid chromatographs. Unless otherwise specified, the Agilent high performance liquid chromatograph is used by default;

[0093] Electronic balance: ME204E / 02, MS205DU, XP26 (Mettler Toledo Instruments Co., Ltd.);

[0094] Ultra-pure water machine: Cell type 1810A (Shanghai Moller Scientific Instruments Co., Ltd.);

[0095] Ultrasonic cleaner: Model KQ600-DB (600W, 40KHz; Kunshan Ultrasonic Instruments Co., Ltd.);

[0096] Chromatographic column: Agilent C18-1, Agilent C18-2, Shimadzu C18 chromatographic column. Unless otherwise specified, Agilent C18-1 chromatographic column is used by default.

[0097] 2) Reagents and test drugs

[0098] Acetonitrile and phosphoric acid are of chromatographic purity, water is ultra-pure water, and the rest of the reagents are of analytical purity unless otherwise specified;

[0099] Gallic acid reference substance (National Institutes for Food and Drug Control, batch number: 110831-201906, purity: 91.5%);

[0100] Ellagic acid reference substance (National Institutes for Food and Drug Control, batch number: 111959-201903, purity: 88.8%)

[0101] Control crude drug of walnut kernel (Chengdu Deste Biotechnology Co., Ltd., batch number: DSTYH005301);

[0102] Standard decoction of walnut kernel (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: HTRBT01, HTRBT02, HTRBT03, HTRBT04, HTRBT05, HTRBT,06, HTRBT07, HTRBT08, HTRBT09, HTRBT10, HTRBT11, HTRBT12, HTRBT13, HTRBT14, HTRBT15, HTRBT16).

[0103] 3) Detection method for characteristic fingerprint

[0104] Chromatographic conditions and system suitability test: Using octadecylsilane chemically bonded silica as the filler; using acetonitrile as mobile phase A and 0.1wt% phosphoric acid solution as mobile phase B, gradient elution was carried out according to the regulations in Table 1; the flow rate was 0.3 mL per minute; the column temperature was 25°C; the detection wavelength was 254 nm; the number of theoretical plates calculated based on the ellagic acid peak should not be less than 5000.

[0105] Table 1 Gradient elution program

[0106] Time (min) Mobile Phase A (vol%) Mobile Phase B (vol%) 0～5 2 98 5～13 2→10 98→90 13～17 10→13.5 90→86.5 17～24 13.5→19.5 86.5→80.5 24～30 19.5→25 80.5→75

[0107] Preparation of reference solution: Take 1.0 g of the control medicinal material of walnut kernels, place it in a stoppered conical flask, add 30 mL of water, decoct for 30 minutes, cool, centrifuge (at a speed of 6000 revolutions per minute) for 4 minutes, take the supernatant, evaporate to dryness, dissolve the residue in 10 mL of 50 vol% methanol solution, filter, and take the subsequent filtrate as the reference solution of the control medicinal material; Separately take an appropriate amount of gallic acid reference substance, accurately weigh it, and prepare a solution containing 20 μg of gallic acid per 1 mL with methanol as the reference solution of gallic acid reference substance; Separately take an appropriate amount of ellagic acid reference substance, accurately weigh it, and prepare a solution containing 50 μg of ellagic acid per 1 mL with methanol as the reference solution of ellagic acid reference substance.

[0108] Preparation of test solution: Take 0.3 g of the powder of this product, place it in a stoppered conical flask, add 50 mL of 50 vol% methanol solution, tightly stopper, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate to obtain.

[0109] Determination method: Accurately pipette 1 μL each of the reference solution and the test solution, inject them into the liquid chromatograph, and determine to obtain.

[0110] 4) Investigation of chromatographic conditions

[0111] 4.1) Wavelength selection

[0112] On the basis of the above-determined experimental conditions, use a diode array detector to perform a full-band scan on the test solution, and separately extract the chromatograms of the test solution at wavelengths of 204 nm, 224 nm, 254 nm, 274 nm, 294 nm, and 314 nm. The results are shown in Figure 1 , Figure 1 which is the chromatograms of different wavelengths of the standard decoction of walnut kernels provided in Example 1 of the present invention. It can be seen from Figure 1 that when the detection wavelength is 254 nm, the chromatographic peak information is larger and the chromatogram baseline is more stable. Therefore, the detection wavelength is determined to be 254 nm.

[0113] 4.2) Column temperature investigation

[0114] On the basis of the above-determined experimental conditions, perform chromatographic detection on the test solution at column temperatures of 20 °C, 25 °C, and 30 °C respectively. The results are shown in Figure 2 , Figure 2 which is the chromatogram of column temperature investigation of the standard decoction of walnut kernels provided in Example 1 of the present invention. It can be seen from Figure 2 that when the column temperature is 25 °C, the chromatogram peak shape is relatively symmetrical, the resolution is better, and the peak emergence is more complete. Therefore, the column temperature is determined to be 25 °C.

[0115] 4.3) Flow rate investigation

[0116] On the basis of the above-determined experimental conditions, the test solution was chromatographically detected at flow rates of 0.25 mL / min, 0.3 mL / min, and 0.25 mL / min respectively. The results are shown in Figure 3 , Figure 3 which is the chromatogram of the flow rate investigation of the standard decoction of walnut kernels provided in Example 1 of the present invention. It can be seen from Figure 3 that when the flow rate is 0.3 mL / min, the peak shape of the chromatogram is good and the resolution is moderate. Therefore, the flow rate is determined to be 0.3 mL / min.

[0117] 4.4) Investigation of delay

[0118] On the basis of the above-determined experimental conditions, a delay test was carried out. The results are shown in Figure 4 , Figure 4 which is the chromatogram of the delay investigation of the standard decoction of walnut kernels provided in Example 1 of the present invention. It can be seen from Figure 4 that there are basically no useful chromatographic peaks in the sample after 30 minutes. Therefore, the sample detection time is set at 30 minutes.

[0119] 5) Investigation of the preparation of the test solution

[0120] 5.1) Investigation of the dissolution solvent

[0121] Take 0.3 g of the powder of this product, place it in a stoppered conical flask, add 20 mL of water, methanol, 70 vol% methanol, 50 vol% methanol, ethanol, and 50 vol% ethanol respectively, tightly stopper, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes respectively, let it cool, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0122] According to the above-determined experimental conditions, the test solutions prepared with different solvents were chromatographically determined respectively. The results are shown in Figure 5 , Figure 5 which is the experimental result diagram of the investigation of the dissolution solvent in the preparation process of the test solution of the standard decoction of walnut kernels provided in Example 1 of the present invention. It can be known from Figure 5 that when the solvent is 50 vol% methanol, the peak shapes of the characteristic peaks in the chromatogram are good and the resolution is moderate. The solvent is determined to be 50 vol% methanol.

[0123] 5.2) Investigation of the dissolution method

[0124] Take 0.3 g of the powder of this product, place it in a stoppered conical flask, add 20 mL of 50 vol% methanol, tightly stopper, ultrasonically treat (power 600 W, frequency 40 kHz) and heat under reflux for 30 minutes respectively, let it cool, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0125] Under the above - determined experimental conditions, chromatographic determinations were respectively carried out on the test solutions treated by ultrasonic treatment and heating under reflux. The results are shown in Figure 6 , Figure 6 which is the experimental result diagram for investigating the dissolution method during the preparation of the test solution of the standard decoction of walnut kernels provided in Example 1 of the present invention. Through Figure 6 it can be seen that there is little difference in the effects when the test sample is refluxed and ultrasonically treated. Since ultrasonic treatment is fast and simple, the dissolution method of the test sample is determined to be ultrasonic dissolution.

[0126] 5.3) Investigation of dissolution time

[0127] Take 0.3 g of the powder of this product, place it in a stoppered conical flask, add 20 mL of 50 vol% methanol, stopper it tightly, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 15 minutes, 30 minutes, and 45 minutes respectively. Let it cool, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0128] Under the above - determined experimental conditions, chromatographic determinations were respectively carried out on the test solutions ultrasonically treated for different times. The results are shown in Figure 7 , Figure 7 which is the experimental result diagram for investigating the dissolution time during the preparation of the test solution of the standard decoction of walnut kernels provided in Example 1 of the present invention. Through Figure 7 it can be seen that when the ultrasonic dissolution time is 30 min, the peak shape and resolution of the chromatogram are better. Therefore, the dissolution time is determined to be 30 min.

[0129] 5.4) Investigation of solvent addition amount

[0130] Take 0.3 g of the powder of this product, place it in a stoppered conical flask, and add 10 mL, 20 mL, and 50 mL of 50 vol% methanol respectively. Stopper it tightly, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0131] Under the above - determined experimental conditions, chromatographic determinations were respectively carried out on the test solutions with different solvent addition amounts. The results are shown in Figure 8 , Figure 8 [[ID=3S4]]which is the experimental result diagram for investigating the solvent addition amount during the preparation of the test solution of the standard decoction of walnut kernels provided in Example 1 of the present invention. Through Figure 8 it can be seen that when the solvent addition amount is 50 mL, the peak shapes and resolution of each chromatographic peak are better. Therefore, the solvent volume is selected as 50 mL.

[0132] 6) Methodology investigation

[0133] 6.1) Chromatographic peak identification

[0134] According to the above - determined experimental conditions, prepare the test solution of the standard decoction of walnut kernels, the reference solution of the reference medicinal material of walnut kernels, the reference solution of the reference substance of gallic acid, and the reference solution of the reference substance of ellagic acid. At the same time, referring to the above - determined experimental conditions, prepare the negative control solution (i.e., the blank solution) without the standard decoction of walnut kernels, and conduct chromatographic detection.

[0135] Locate the characteristic chromatogram peaks of the standard decoction of walnut kernels. The results are shown in Figures 9 - 13 , Figure 9 which is the chromatogram peak identification diagram of the characteristic chromatogram of the standard decoction of walnut kernels provided in Example 1 of the present invention, Figure 10 which is the ultraviolet absorption spectrum diagram of the reference substance of gallic acid provided in Example 1 of the present invention, Figure 11 which is the ultraviolet absorption spectrum diagram of gallic acid in the test sample provided in Example 1 of the present invention, Figure 12 which is the ultraviolet absorption spectrum diagram of the reference substance of ellagic acid provided in Example 1 of the present invention, Figure 13 which is the ultraviolet absorption spectrum diagram of ellagic acid in the test sample provided in Example 1 of the present invention. The results show that peak 1 is gallic acid and peak 5 is ellagic acid. In the following methodological investigations, the peak 1 corresponding to the gallic acid peak is set as S1 peak, and the relative retention times of peak 2, peak 3 and S1 peak are calculated. The peak 5 corresponding to the ellagic acid peak is set as S2 peak, and the relative retention times of peak 4, peak 6 and S2 peak are calculated to investigate the 6 characteristic peaks in the sample.

[0136] 6.2) Precision test

[0137] Take the test solution of the standard decoction of walnut kernels, inject samples continuously 6 times according to the determined experimental method, 1 μL each time, and calculate the retention times of each characteristic peak. The results are shown in Table 2.

[0138] Table 2 Precision investigation - retention time

[0139]

[0140] It can be seen from Table 2 that the RSD values of the retention times of each peak are 0.01% - 0.08%, indicating that the precision of the instrument is good.

[0141] 6.3) Repeatability investigation

[0142] Precisely weigh 6 portions of the standard decoction of walnut kernels, prepare the test solution and conduct chromatographic determination according to the determined experimental method. The results are shown in Table 3.

[0143] Table 3 Repeatability investigation - relative retention time of characteristic peaks

[0144]

[0145] As can be seen from Table 3, the RSD of the relative retention times of each characteristic peak is between 0.01% and 0.13%, indicating that the repeatability of this method is good.

[0146] 6.4) Intermediate precision investigation

[0147] 6.4.1) Investigation with different instruments

[0148] On the basis of the above-determined experimental conditions, accurately weigh 0.3 g of the standard decoction of walnut kernels (batch number: HTR-BT-230816) in three portions, prepare the test solution, and perform the determination on Waters, Agilent, and Thermo Fisher high-performance liquid chromatographs respectively. Calculate the relative retention times of each characteristic peak. The results are shown in Figure 14 and Table 4, Figure 14 which is the experimental result diagram of the investigation of the standard decoction of walnut kernels provided in Example 1 of the present invention with different instruments.

[0149] Table 4 Investigation with different instruments - Relative retention times of characteristic peaks

[0150]

[0151] As can be seen from Table 4, when the test solution is detected with the above three instruments, the RSD of the relative retention times of each characteristic peak is between 0.29% and 6.56%, indicating good instrument durability.

[0152] 6.4.2) Investigation with different personnel and times

[0153] On the basis of the above-determined experimental conditions, accurately weigh two portions of the standard decoction of walnut kernels by different personnel (A, B) at different times (T1, T2) respectively, prepare the test solution, and perform the determination. The results are shown in Table 5.

[0154] Table 5 Investigation with different personnel and times - Relative retention times of characteristic peaks

[0155]

[0156] As can be seen from Table 5, under the conditions of different sample preparation personnel and different sample preparation times, the RSD of the relative retention times of each characteristic peak is between 0.02% and 0.13%, indicating good method stability.

[0157] 6.5) Durability investigation

[0158] 6.5.1) Investigation on the durability of chromatographic columns

[0159] On the basis of the above-determined experimental conditions, investigate different brand chromatographic columns Agilent C18-1, Agilent C18-2, and Shimadzu C18 chromatographic columns respectively. The results are shown in Figure 15 and Table 6, Figure 15It is the experimental result diagram of the investigation on different chromatographic columns of the standard decoction of walnut kernels provided in Example 1 of the present invention.

[0160] Table 6 Investigation on the durability of chromatographic columns - relative retention time of characteristic peaks

[0161]

[0162]

[0163] It can be seen from Table 6 that the RSD of the relative retention time of each characteristic peak of different brands of chromatographic columns is between 0.10% and 8.70%, and the durability of the chromatographic column is good.

[0164] 6.5.2) Investigation on stability

[0165] On the basis of the above-determined experimental conditions, the same test solution was taken and determined at 0h, 4h, 8h, 12h, 16h, and 24h respectively. The results are shown in Table 7.

[0166] Table 7 Investigation on stability - retention time

[0167]

[0168] It can be seen from Table 7 that the RSD of the retention time of the corresponding characteristic peaks is between 0.02% and 0.27%, and the sample solution is relatively stable within 24 hours.

[0169] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in all the above investigations, and this method is good. The above 6 characteristic peaks are included in the subsequent investigations.

[0170] 7) Determination of characteristic peaks and establishment of reference chromatogram

[0171] 7.1) Verification results of 16 batches of standard decoctions of walnut kernels

[0172] Using the determined method, characteristic fingerprint analysis was carried out on 16 batches of samples, and the relative retention time was calculated. The results are shown in Figure 16 and Table 8. Figure 16 It is the verification diagram of the characteristic fingerprint of 16 batches of standard decoctions of walnut kernels provided in Example 1 of the present invention. Figure 16 Among them, S1 to S16 are respectively: HTRBT01, HTRBT02, HTRBT03, HTRBT04, HTRBT05, HTRBT06, HTRBT07, HTRBT08, HTRBT09, HTRBT10, HTRBT11, HTRBT12, HTRBT13, HTRBT14, HTRBT15, HTRBT16.

[0173] Table 8 Relative retention time of 16 batches of standard decoctions of walnut kernels

[0174]

[0175]

[0176] According to the principle of stable relative retention time, detectable in each batch of samples, and relatively high peaks, a total of 6 peaks with good durability were selected as characteristic peaks. According to the results of methodological investigation and the verification results of 16 batches of standard decoctions, it was determined that the theoretical plate number should not be less than 5000 calculated based on the ellagic acid peak.

[0177] 7.2) Establishment of the specified limit of relative retention time

[0178] The summary of the investigation items and verification results of the methodology is shown in Table 9:

[0179] Table 9 Summary of the results of each item of the methodology RSD(%) - Standard - Relative retention time (retention time)

[0180]

[0181] The retention time or relative retention time of each characteristic peak is stable and within the range of the average value ± 10%. Therefore, the specified range of the relative retention time of each peak was determined to be ± 10%.

[0182] Finally, it is specified that 6 characteristic peaks should be presented in the chromatogram of the test sample, and the retention times of the 6 characteristic peaks should correspond to those in the chromatogram of the reference medicinal material reference substance. Among them, Peak 1 and Peak 5 should correspond to the retention times of the gallic acid reference substance peak and the ellagic acid reference substance peak respectively. The peak corresponding to the gallic acid reference substance peak is the S1 peak. Calculate the relative retention times of Peak 2 and Peak 3 with respect to the S1 peak, and their relative retention times should be within the range of ± 10% of the specified value. The specified values are: 1.00 (Peak 1), 2.22 (Peak 2), 3.98 (Peak 3); the peak corresponding to the ellagic acid reference substance peak is the S2 peak. Calculate the relative retention times of Peak 4 and Peak 6 with respect to the S2 peak, and their relative retention times should be within the range of ± 10% of the specified value. The specified values are: 0.96 (Peak 4), 1.00 (Peak 5), 1.16 (Peak 6).

[0183] Using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition), 16 batches of standard decoctions of walnut kernels were synthesized to establish a reference chromatogram of the characteristic chromatogram of the standard decoction of walnut kernels, as Figure 17 shown, Figure 17 which is the reference chromatogram of the characteristic chromatogram of the standard decoction of walnut kernels provided in Example 1 of the present invention.

[0184] 8) Determination of the method for the characteristic chromatogram of the standard decoction of walnut kernels

[0185] Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica gel as the filler; use acetonitrile as mobile phase A and 0.1 wt% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in Table 1; the flow rate is 0.3 mL per minute; the column temperature is 25 °C; the detection wavelength is 254 nm; the number of theoretical plates calculated based on the ellagic acid peak should be not less than 5000.

[0186] Preparation of reference substance solution: Take 1.0 g of the control crude drug of walnut kernel, place it in a stoppered conical flask, add 30 mL of water, decoct for 30 minutes, cool, centrifuge (at a speed of 6000 revolutions per minute) for 4 minutes, take the supernatant, evaporate to dryness, dissolve the residue in 10 mL of 50 vol% methanol solution, filter, and take the subsequent filtrate as the reference substance solution of the control crude drug; separately weigh an appropriate amount of gallic acid reference substance accurately, dissolve it in methanol to make a solution containing 20 μg of gallic acid per 1 mL as the reference substance solution of gallic acid reference substance; separately weigh an appropriate amount of ellagic acid reference substance accurately, dissolve it in methanol to make a solution containing 50 μg of ellagic acid per 1 mL as the reference substance solution of ellagic acid reference substance.

[0187] Preparation of test solution: Take 0.3 g of the powder of this product, place it in a stoppered conical flask, add 50 mL of 50 vol% methanol solution, tightly stopper, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate, which is the test solution.

[0188] Determination method: Accurately pipette 1 μL each of the reference substance solution and the test solution, inject them into the liquid chromatograph for determination, and that's it.

[0189] Example 2

[0190] Construction of HPLC characteristic fingerprints of walnut kernel crude drug and cut crude drug:

[0191] 1) Experimental instruments and materials

[0192] Same as Example 1, not elaborated here.

[0193] 2) Reagents and test drugs

[0194] Walnut kernel crude drug (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: HTR01, HTR02, HTR03, HTR04, HTR05, HTR06, HTR07, HTR08, HTR09, HTR10, HTR11, HTR12, HTR13, HTR14, HTR15, HTR16);

[0195] Walnut kernel decoction pieces (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: HTR17, HTR18, HTR19, HTR20, HTR21, HTR22, HTR23, HTR24, HTR25, HTR26, HTR27, HTR28, HTR29, HTR30, HTR31, HTR32);

[0196] Other reagents and test drugs are the same as in Example 1 and will not be elaborated here.

[0197] 3) Feature mapping detection method

[0198] Chromatographic conditions and system suitability test: Using octadecylsilane-bonded silica gel as the filler; using acetonitrile as mobile phase A and 0.1 wt% phosphoric acid solution as mobile phase B, gradient elution is carried out according to the provisions in Table 1; the flow rate is 0.3 mL per minute; the column temperature is 25 °C; the detection wavelength is 254 nm; the number of theoretical plates calculated by the ellagic acid peak should be not less than 5000. [[ID=eleven]]

[0199] Preparation of reference solution: Take 1.0 g of walnut kernel reference medicinal material, place it in a stoppered conical flask, add 30 mL of water, decoct for 30 minutes, cool, centrifuge (at a speed of 6000 revolutions per minute) for 4 minutes, take the supernatant, evaporate to dryness, dissolve the residue in 10 mL of 50 vol% methanol solution, filter, and take the subsequent filtrate as the reference medicinal material reference solution; separately take an appropriate amount of gallic acid reference substance, accurately weigh it, dissolve it in methanol to make a solution containing 20 μg of gallic acid per 1 mL as the gallic acid reference substance reference solution; separately take an appropriate amount of ellagic acid reference substance, accurately weigh it, dissolve it in methanol to make a solution containing 50 μg of ellagic acid per 1 mL as the ellagic acid reference substance reference solution.

[0200] Preparation of test solution: Take 1.0 g of the powder of this product, accurately weigh it, place it in a stoppered conical flask, add 30 mL of water, decoct for 30 minutes, cool, centrifuge (at a speed of 6000 revolutions per minute) for 4 minutes, take the supernatant, evaporate to dryness, dissolve the residue in 10 mL of 50 vol% methanol solution, filter, and take the subsequent filtrate to obtain.

[0201] Determination method: Accurately pipette 1 μL each of the reference solution and the test solution, inject them into the liquid chromatograph for determination to obtain.

[0202] 4) Investigation of chromatographic conditions

[0203] 4.1) Wavelength selection

[0204] Based on the above-mentioned proposed experimental conditions, use a diode array detector to perform a full-band scan on the test solution, and separately extract the chromatograms of the test solution at wavelengths of 204 nm, 224 nm, 254 nm, 274 nm, 294 nm, and 314 nm. The results are shown in Figure 18 ,Figure 18 This is the chromatogram of walnut kernel medicinal materials at different wavelengths provided in Example 2 of the present invention. By Figure 18 it can be seen that when the detection wavelength is 254 nm, the chromatographic peak information is relatively large and the baseline of the chromatogram is more stable. Therefore, the detection wavelength is determined to be 254 nm.

[0205] 4.2) Column temperature investigation

[0206] On the basis of the above-determined experimental conditions, the test solution was chromatographically detected at column temperatures of 20 °C, 25 °C, and 30 °C respectively. The results are shown in Figure 19 , Figure 19 This is the chromatogram of the column temperature investigation of walnut kernel medicinal materials provided in Example 2 of the present invention. By Figure 19 it can be seen that when the column temperature is 25 °C, the chromatogram peak shape is relatively symmetric, the resolution is good, and the peak emergence is relatively complete. Therefore, the column temperature is determined to be 25 °C.

[0207] 4.3) Flow rate investigation

[0208] On the basis of the above-determined experimental conditions, the test solution was chromatographically detected at flow rates of 0.25 mL / min, 0.3 mL / min, and 0.25 mL / min respectively. The results are shown in Figure 20 , Figure 20 This is the chromatogram of the flow rate investigation of walnut kernel medicinal materials provided in Example 2 of the present invention. By Figure 20 it can be seen that when the flow rate is 0.3 mL / min, the chromatogram peak shape is good and the resolution is moderate. Therefore, the flow rate is determined to be 0.3 mL / min.

[0209] 4.4) Delay investigation

[0210] On the basis of the above-determined experimental conditions, a delay test was carried out. The results are shown in Figure 21 , Figure 21 This is the chromatogram of the delay investigation of walnut kernel medicinal materials provided in Example 2 of the present invention. By Figure 21 it can be seen that there are basically no useful chromatographic peaks in the sample after 30 min. Therefore, the sample detection time is set to 30 min.

[0211] 5) Preparation investigation of the test solution

[0212] 5.1) Investigation of the dissolution solvent

[0213] Take 1.0 g of the powder of this product, place it in a stoppered conical flask, add 30 ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, dissolve the residue in 10 mL each of methanol, 70 vol% methanol, 50 vol% methanol, 50 vol% ethanol, ethanol, and water respectively, filter, and take the subsequent filtrate to obtain the test solution.

[0214] Under the above - determined experimental conditions, chromatographic determinations were respectively carried out on the test solution prepared with different solvents. The results are shown in Figure 22 , Figure 22 which is the experimental result diagram for investigating the dissolution solvent during the preparation of the test solution of walnut kernel medicinal materials provided in Example 2 of the present invention. It can be seen from Figure 22 that when the solvent is 50 vol% methanol, the peak shapes of each characteristic peak in the chromatogram are good and the resolution is moderate. The solvent is determined to be 50 vol% methanol.

[0215] 5.2) Investigation of decocting time

[0216] Take 1.0 g of the powder of this product, place it in a stoppered conical flask, add 30 mL of water, decoct for 15 minutes, 30 minutes, and 45 minutes respectively, cool, filter, evaporate the filtrate to dryness, dissolve the residue with 10 mL of 50 vol% methanol, filter, and take the subsequent filtrate to obtain the test solution.

[0217] Under the above - determined experimental conditions, chromatographic determinations were respectively carried out on the test solutions decocted for different times. The results are shown in Figure 23 , Figure 23 which is the experimental result diagram for investigating the decocting time during the preparation of the test solution of walnut kernel medicinal materials provided in Example 2 of the present invention. It can be seen from Figure 23 that when the decocting time is 30 min, the peak shape and resolution of the chromatogram are better. Therefore, the decocting time is determined to be 30 min.

[0218] 5.3) Investigation of solvent addition amount

[0219] Take 1.0 g of the powder of this product, place it in a stoppered conical flask, add 30 mL of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, and dissolve the residue with 10 mL, 20 mL, and 50 mL of 50 vol% methanol respectively, filter, and take the subsequent filtrate to obtain the test solution.

[0220] Under the above - determined experimental conditions, chromatographic determinations were respectively carried out on the test solutions with different solvent addition amounts. The results are shown in Figure 24 , Figure 24 which is the experimental result diagram for investigating the solvent addition amount during the preparation of the test solution of walnut kernel medicinal materials provided in Example 2 of the present invention. It can be seen from Figure 24 that when the solvent addition amount is 10 mL, the peak shapes and resolution of each chromatographic peak are better. Therefore, the solvent volume is selected as 10 mL.

[0221] 5.4) Investigation of dissolution method

[0222] Take 1.0 g of the powder of this product, place it in a stoppered conical flask, add 30 mL of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, dissolve the residue in 10 mL of 50 vol% methanol, filter, and take the subsequent filtrate as the test solution; additionally, take about 1.0 g of this product, place it in a stoppered conical flask, add 10 mL of methanol, tightly stopper, weigh, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate as the test solution.

[0223] Under the above - determined experimental conditions, chromatographic determination was carried out on the test solutions prepared by decoction and ultrasonic treatment respectively, and the results are shown in Figure 25 , Figure 25 which is the experimental result diagram of the investigation on the dissolution method during the preparation of the test solution of walnut kernel medicinal materials provided in Example 2 of the present invention. Through Figure 25 it can be seen that the peaks of the test solution obtained by decoction are well - separated, so the dissolution method of the test product is determined to be decoction.

[0224] 6) Methodology investigation

[0225] 6.1) Chromatographic peak identification

[0226] Under the above - determined experimental conditions, prepare the test solution of walnut kernel medicinal materials, the reference solution of walnut kernel reference medicinal materials, the reference solution of gallic acid reference substance, and the reference solution of ellagic acid reference substance. At the same time, refer to the above - determined experimental conditions to prepare the negative control solution (i.e., blank solution) without walnut kernel medicinal materials, and conduct chromatographic detection.

[0227] Locate the peaks of the characteristic chromatogram of walnut kernel medicinal materials, and the results are shown in Figure 26 , Figure 26 which is the chromatographic peak identification diagram of the characteristic chromatogram of walnut kernel medicinal materials provided in Example 2 of the present invention. The results show that peak 1 is gallic acid and peak 5 is ellagic acid. In the following methodology investigation, the peak 1 corresponding to the gallic acid peak is set as S1 peak, calculate the relative retention times of peak 2 and peak 3 with respect to S1 peak, and the peak 5 corresponding to the ellagic acid peak is set as S2 peak, calculate the relative retention times of peak 4 and peak 6 with respect to S2 peak, and investigate the 6 characteristic peaks in the sample.

[0228] 6.2) Precision test

[0229] Take 1.0 g of walnut kernel medicinal materials to prepare the test solution, inject continuously 6 times according to the determined experimental method, 1 μL each time, and calculate the retention times of each characteristic peak. The results are shown in Table 10.

[0230] Table 10 Precision investigation - retention time

[0231]

[0232] As can be seen from Table 10, the RSD of the retention times of each characteristic peak in the precision investigation is between 0.00% and 0.06%, indicating good precision.

[0233] 6.3) Repeatability investigation

[0234] Take about 1.0 g of walnut kernel medicinal materials (passed through No. 3 sieve), 6 portions, prepare the test solution and perform chromatographic determination according to the proposed experimental method. The results are shown in Table 11.

[0235] Table 11 Repeatability investigation - relative retention time of characteristic peaks

[0236]

[0237] As can be seen from Table 11, the RSD of the relative retention times of characteristic peaks is between 0.08% and 1.50%, indicating good repeatability.

[0238] 6.4) Intermediate precision investigation

[0239] 6.4.1) Investigation of different personnel and time

[0240] Based on the above proposed experimental conditions, about 1.0 g of walnut kernel medicinal materials were weighed by different personnel (A, B) at different times (T1, T2), two portions each, to prepare the test samples and perform the determination. The results are shown in Table 12.

[0241] Table 12 Investigation of different personnel and time - relative retention time of characteristic peaks

[0242]

[0243] As can be seen from Table 12, under the conditions of different sample preparation personnel and different sample preparation times, the RSD of the relative retention times of each characteristic peak is between 0.01% and 0.08%, indicating good method stability.

[0244] 6.4.2) Investigation of different instruments

[0245] Based on the above proposed experimental conditions, about 1.0 g of walnut kernel medicinal materials were weighed respectively, three portions each, to prepare the test solution, and the determination was performed on Waters, Agilent, and Thermo Fisher high performance liquid chromatographs respectively. Calculate the relative retention time of each characteristic peak. The results are shown in Figure 27 and Table 13, Figure 27 which is the experimental result diagram of the investigation of different instruments for walnut kernel medicinal materials provided in Example 2 of the present invention.

[0246] Table 13 Investigation of different instruments - relative retention time of characteristic peaks

[0247]

[0248]

[0249] As can be seen from Table 13, when the test samples were detected with the above three instruments, the RSD of the relative retention time of each characteristic peak was between 0.30% and 6.32%, indicating good durability of the instruments.

[0250] 6.5) Durability investigation

[0251] 6.5.1) Chromatographic column durability investigation

[0252] On the basis of the above-determined experimental conditions, different brands of chromatographic columns, Agilent C18-1, Agilent C18-2, and Shimadzu C18 chromatographic columns, were investigated respectively, and the results are shown in Figure 28 and Table 14, Figure 28 which is the experimental result diagram of the investigation of different chromatographic columns of walnut kernel medicinal materials provided in Example 2 of the present invention.

[0253] Table 14 Chromatographic column durability investigation - relative retention time of characteristic peaks

[0254]

[0255] As can be seen from Table 14, when the samples were detected with the above three chromatographic columns, the RSD of the relative retention time of the characteristic peaks was between 0.10% and 8.78%, indicating good durability of the chromatographic columns.

[0256] 6.5.2) Stability investigation

[0257] On the basis of the above-determined experimental conditions, the same test sample solution was taken and measured at 0h, 4h, 8h, 12h, 16h, and 24h respectively, and the results are shown in Table 15.

[0258] Table 15 Stability investigation - retention time

[0259]

[0260] As can be seen from Table 15, the RSD of the corresponding retention time of the characteristic peaks was between 0.09% and 1.54%, and the sample solution was relatively stable within 24 hours.

[0261] In summary, the RSD of the relative retention time of each characteristic peak met the requirements in all the above investigations, and the method was good. The above 6 characteristic peaks were included in the subsequent investigations.

[0262] 7) Determination of characteristic peaks and establishment of reference chromatogram

[0263] 7.1) Verification results of 16 batches of walnut kernel medicinal materials

[0264] Using the proposed method, characteristic fingerprint analysis was performed on 16 batches of samples, and the relative retention time was calculated. The results are shown inFigure 29 and Table 16, Figure 29 which is the verification diagram of the characteristic chromatograms of 16 batches of walnut kernel medicinal materials provided in Example 2 of the present invention, Figure 29 where S1 to S16 are respectively: HTR01, HTR02, HTR03, HTR04, HTR05, HTR06, HTR07, HTR08, HTR09, HTR10, HTR11, HTR12, HTR13, HTR14, HTR15, HTR16.

[0265] Table 16 Relative retention times of 16 batches of walnut kernel medicinal materials

[0266]

[0267] According to the principle of stable relative retention time, all batches of samples can be detected and the peaks are relatively high, a total of 6 peaks with good repeatability were selected as characteristic peaks.

[0268] 7.2) Formulation of the specified value limit of relative retention time

[0269] Finally, it is specified that: there should be 6 characteristic peaks in the chromatogram of the test sample, and they should correspond to the retention times of the 6 characteristic peaks in the chromatogram of the reference medicinal material. Among them, Peak 1 and Peak 5 should correspond to the retention times of the reference peaks of gallic acid reference substance and ellagic acid reference substance respectively. The peak corresponding to the gallic acid reference substance reference peak is Peak S1. Calculate the relative retention times of Peak 2 and Peak 3 with respect to Peak S1, and their relative retention times should be within the range of ±10% of the specified value. The specified values are: 1.00 (Peak 1), 2.23 (Peak 2), 3.97 (Peak 3); the peak corresponding to the ellagic acid reference substance reference peak is Peak S2. Calculate the relative retention times of Peak 4 and Peak 6 with respect to Peak S2, and their relative retention times should be within the range of ±10% of the specified value. The specified values are: 0.96 (Peak 4), 1.00 (Peak 5), 1.16 (Peak 6).

[0270] Using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition) to synthesize 16 batches of walnut kernel medicinal materials, a reference chromatogram of the characteristic chromatograms of walnut kernel medicinal materials was established, as Figure 30 shown, Figure 30 which is the reference chromatogram of the characteristic chromatograms of walnut kernel medicinal materials provided in Example 1 of the present invention.

[0271] 7.3) Verification of the characteristic chromatograms of walnut kernel decoction pieces

[0272] Using the proposed method, perform characteristic chromatogram analysis on 16 batches of walnut kernel decoction piece samples, calculate the relative retention times, and the results are shown in Figure 31 and Table 17, Figure 31 which is the verification diagram of the characteristic chromatograms of 16 batches of walnut kernel decoction pieces provided in Example 2 of the present invention, Figure 29Among them, S1 to S21 are respectively: HTR17, HTR18, HTR19, HTR20, HTR21, HTR22, HTR23, HTR24, HTR25, HTR26, HTR27, HTR28, HTR29, HTR30, HTR31, HTR32.

[0273] Table 17 Relative retention times of 16 batches of walnut kernel decoction pieces

[0274]

[0275]

[0276] According to the principle of stable relative retention time, all batches of samples can be detected and the peaks are relatively high, a total of 6 peaks with good repeatability were selected as characteristic peaks.

[0277] Finally, it is stipulated that: 6 characteristic peaks should be presented in the chromatogram of the test sample, and the retention times of the 6 characteristic peaks should correspond to those in the chromatogram of the reference medicinal material reference substance. Among them, peak 1 and peak 5 should correspond to the retention times of the reference peaks of gallic acid reference substance and ellagic acid reference substance respectively. The peak corresponding to the gallic acid reference substance reference peak is the S1 peak. Calculate the relative retention times of peak 2 and peak 3 with respect to the S1 peak, and their relative retention times should be within the range of ±10% of the specified values, and the specified values are: 1.00 (peak 1), 2.23 (peak 2), 3.98 (peak 3); the peak corresponding to the ellagic acid reference substance reference peak is the S2 peak. Calculate the relative retention times of peak 4 and peak 6 with respect to the S2 peak, and their relative retention times should be within the range of ±10% of the specified values, and the specified values are: 0.96 (peak 4), 1.00 (peak 5), 1.16 (peak 6).

[0278] Using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition), 16 batches of walnut kernel decoction pieces were synthesized to establish a reference chromatogram of the characteristic chromatogram of walnut kernel decoction pieces, as Figure 32 shown, Figure 32 which is the reference chromatogram of the characteristic chromatogram of walnut kernel decoction pieces provided in Example 2 of the present invention.

[0279] 8) Determination of the characteristic chromatogram method for the standard decoction of walnut kernel

[0280] Chromatographic conditions and system suitability test: Octadecylsilane chemically bonded silica gel was used as the filler; acetonitrile was used as mobile phase A, and 0.1 wt% phosphoric acid solution was used as mobile phase B, and gradient elution was carried out according to the regulations in Table 1; the flow rate was 0.3 mL per minute; the column temperature was 25 °C; the detection wavelength was 254 nm; the number of theoretical plates calculated based on the ellagic acid peak should be not less than 5000.

[0281] Preparation of reference solution: Take 1.0 g of walnut kernel reference medicinal material, place it in a stoppered conical flask, add 30 mL of water, boil for 30 minutes, cool, centrifuge (speed is 6000 revolutions per minute) for 4 minutes, take the supernatant, evaporate to dryness, add 10 mL of 50 vol% methanol solution to dissolve the residue, filter, and take the filtrate as the reference medicinal material reference solution; take an appropriate amount of gallic acid reference substance, accurately weigh it, add methanol to make a solution containing 20 μg of gallic acid per 1 mL, as the gallic acid reference substance reference solution; take an appropriate amount of ellagic acid reference substance, accurately weigh it, add methanol to make a solution containing 50 μg of ellagic acid per 1 mL, as the ellagic acid reference substance reference solution.

[0282] Preparation of test solution: Take 1.0 g of the powder of this product, place it in a stoppered conical flask, add 30 mL of water, boil for 30 minutes, cool, centrifuge (speed is 6000 rpm) for 4 minutes, take the supernatant, evaporate to dryness, add 10 mL of 50 vol% methanol solution to dissolve the residue, filter, and take the filtrate.

[0283] Determination method: Accurately aspirate 1 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0284] Example 3

[0285] Construction of HPLC characteristic spectrum of walnut kernel formula granules:

[0286] 1) Experimental instruments and materials

[0287] Same as Example 1, no further details will be given.

[0288] 2) Reagents and test drugs

[0289] Walnut kernel formula granules (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: KL1, KL2, KL3);

[0290] Other reagents and drugs are the same as those in Example 1 and will not be described in detail.

[0291] 3) Feature spectrum detection method

[0292] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as mobile phase A, and 0.1 wt% phosphoric acid solution was used as mobile phase B, with gradient elution performed as specified in Table 1; the flow rate was 0.3 mL per minute; the column temperature was 25°C; the detection wavelength was 254 nm; and the number of theoretical plates calculated based on the ellagic acid peak was not less than 5000.

[0293] Preparation of reference solution: Take 1.0 g of the control medicinal material of walnut kernels, place it in a stoppered conical flask, add 30 mL of water, decoct for 30 minutes, cool, centrifuge (at a speed of 6000 revolutions per minute) for 4 minutes, take the supernatant, evaporate to dryness, dissolve the residue in 10 mL of 50 vol% methanol solution, filter, and take the consecutive filtrate as the reference solution of the control medicinal material; Take an appropriate amount of gallic acid reference substance, accurately weigh it, dissolve it in methanol to prepare a solution containing 20 μg of gallic acid per 1 mL as the reference solution of gallic acid reference substance; Take an appropriate amount of ellagic acid reference substance, accurately weigh it, dissolve it in methanol to prepare a solution containing 50 μg of ellagic acid per 1 mL as the reference solution of ellagic acid reference substance.

[0294] Preparation of test solution: Take an appropriate amount of this product, grind it finely, take about 0.3 g, place it in a stoppered conical flask, add 10 mL of 50 vol% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the consecutive filtrate, which is ready.

[0295] Determination method: Accurately pipette 1 μL each of the reference solution and the test solution, inject them into the liquid chromatograph for determination, and that's it.

[0296] 4) Investigation of chromatographic conditions

[0297] 4.1) Wavelength selection

[0298] Based on the above-mentioned established experimental conditions, use a diode array detector to perform a full-band scan on the test solution, and separately extract the chromatograms of the test solution at wavelengths of 204 nm, 224 nm, 254 nm, 274 nm, 294 nm, and 314 nm. The results are shown in Figure 33 , Figure 33 is the chromatograms of different wavelengths of the walnut kernel formula granules provided in Example 3 of the present invention. It can be seen from Figure 33 that when the detection wavelength is 254 nm, the chromatographic peak information is larger and the chromatogram baseline is more stable. Therefore, the detection wavelength is determined to be 254 nm.

[0299] 4.2) Column temperature investigation

[0300] Based on the above-mentioned established experimental conditions, perform chromatographic detection on the test solution at column temperatures of 20 °C, 25 °C, and 30 °C respectively. The results are shown in Figure 34 , Figure 34 is the chromatogram of column temperature investigation of the walnut kernel formula granules provided in Example 3 of the present invention. It can be seen from Figure 34 that when the column temperature is 25 °C, the chromatogram peak shape is relatively symmetrical, the resolution is better, and the peak emergence is more complete. Therefore, the column temperature is determined to be 25 °C.

[0301] 4.3) Flow rate investigation

[0302] On the basis of the above-mentioned experimental conditions, the test solution was chromatographically detected at the flow rates of 0.25 mL / min, 0.3 mL / min, and 0.25 mL / min respectively. The results are shown in Figure 35 , Figure 35 which is the chromatogram of the flow rate investigation of the walnut kernel formula granules provided in Example 3 of the present invention. It can be seen from Figure 35 that when the flow rate is 0.3 mL / min, the peak shape of the chromatogram is good and the resolution is moderate. Therefore, the flow rate is determined to be 0.3 mL / min.

[0303] 4.4) Delayed investigation

[0304] On the basis of the above-mentioned experimental conditions, the chromatogram acquisition time was extended to 60 min. The results are shown in Figure 36 , Figure 36 which is the chromatogram of the delayed investigation of the walnut kernel formula granules provided in Example 3 of the present invention. It can be seen from Figure 36 that when the chromatogram is acquired for 30 minutes, the chromatographic peaks have been completely acquired. Therefore, the chromatogram acquisition time is determined to be 30 min.

[0305] 5) Investigation on the preparation of the test solution

[0306] 5.1) Investigation on the dissolution solvent

[0307] Take an appropriate amount of this product, grind it finely, take 0.3 g, place it in a stoppered conical flask, add 10 mL of methanol, 70 vol% methanol, 50 vol% methanol, ethanol, 50 vol% ethanol, and water respectively, tightly stopper, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0308] According to the above-mentioned experimental conditions, the test solutions prepared with different solvents were chromatographically determined respectively. The results are shown in Figure 37 , Figure 37 which is the experimental result diagram of the investigation on the dissolution solvent in the preparation process of the test solution of the walnut kernel formula granules provided in Example 3 of the present invention. It can be known from Figure 22 that when the solvent is 50 vol% methanol, the peak shapes of each characteristic peak are good and the resolution is moderate. The solvent is determined to be 50 vol% methanol.

[0309] 5.2) Investigation on the dissolution method

[0310] Take an appropriate amount of this product, grind it finely, take 0.3 g, place it in a stoppered conical flask, add 10 mL of 50 vol% methanol, tightly stopper, and reflux and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes respectively, let it cool, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0311] Under the above - determined experimental conditions, chromatographic determinations were respectively carried out on the test solution prepared by reflux and ultrasonic treatment. The results are shown in Figure 38 , Figure 38 which is the experimental result diagram of the investigation of the dissolution method during the preparation of the test solution of the walnut kernel formula granules provided in Example 3 of the present invention. It can be seen from Figure 38 that the peaks of the test solution obtained by ultrasonic treatment are better separated. Therefore, the dissolution method of the test sample is determined to be ultrasonic.

[0312] 5.3) Investigation of dissolution time

[0313] Take an appropriate amount of this product, grind it finely, take 0.3 g, place it in a stoppered conical flask, add 10 mL of 50 vol% methanol, stopper it tightly, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 15 minutes, 30 minutes, and 45 minutes respectively. Let it cool, shake well, filter, and take the continuous filtrate to obtain the test solution.

[0314] Under the above - determined experimental conditions, chromatographic determinations were respectively carried out on the test solutions with different ultrasonic treatment times. The results are shown in Figure 39 , Figure 39 which is the experimental result diagram of the investigation of dissolution time during the preparation of the test solution of the walnut kernel formula granules provided in Example 3 of the present invention. It can be seen from Figure 39 that when the ultrasonic dissolution time is 30 min, the peak shape and resolution of the chromatogram are better. Therefore, the dissolution time is determined to be 30 min.

[0315] 5.4) Investigation of solvent addition amount

[0316] Take an appropriate amount of this product, grind it finely, take 0.3 g, place it in a stoppered conical flask, and add 10 mL, 20 mL, and 50 mL of 50 vol% methanol respectively, stopper it tightly, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the continuous filtrate to obtain the test solution.

[0317] Under the above - determined experimental conditions, chromatographic determinations were respectively carried out on the test solutions with different solvent addition amounts. The results are shown in Figure 40 , Figure 40 which is the experimental result diagram of the investigation of solvent addition amount during the preparation of the test solution of the walnut kernel formula granules provided in Example 3 of the present invention. It can be seen from Figure 40 that when the solvent addition amount is 20 mL, the peak shapes and resolution of each chromatographic peak are better. Therefore, the solvent volume is selected as 20 mL.

[0318] 6) Methodology investigation

[0319] 6.1) Chromatographic peak identification

[0320] Prepare the test solution of walnut kernel formula granules, the reference solution of walnut kernel reference crude drug, the reference solution of gallic acid reference substance, and the reference solution of ellagic acid reference substance according to the above-determined experimental conditions. At the same time, prepare the negative control solution (i.e., blank solution) without walnut kernel formula granules according to the above-determined experimental conditions for chromatographic detection.

[0321] Locate the characteristic peaks of the walnut kernel formula granules, and the results are shown in Figure 41 , Figure 41 which is the chromatographic peak identification diagram of the characteristic spectrum of the walnut kernel formula granules provided in Example 3 of the present invention. The results show that peak 1 is gallic acid and peak 5 is ellagic acid. In the following methodological investigations, the peak 1 corresponding to the gallic acid peak is set as S1 peak, and the relative retention times of peak 2 and peak 3 with respect to S1 peak are calculated. The peak 5 corresponding to the ellagic acid peak is set as S2 peak, and the relative retention times of peak 4 and peak 6 with respect to S2 peak are calculated to investigate the 6 characteristic peaks in the sample.

[0322] 6.2) Precision test

[0323] Take an appropriate amount of this product, grind it finely, take 0.3 g, and prepare the test solution of walnut kernel formula granules. Inject continuously for 6 times according to the determined experimental method, 1 μL each time, and calculate the retention times of each characteristic peak. The results are shown in Table 18.

[0324] Table 18 Precision investigation - Retention time

[0325]

[0326] It can be seen from Table 18 that the RSD of the retention times of each characteristic peak in the precision investigation is between 0.01% and 0.06%, indicating good precision.

[0327] 6.3) Repeatability investigation

[0328] Take an appropriate amount of this product, grind it finely, take 0.3 g, 6 portions, and prepare and determine according to the determined experimental method. The results are shown in Table 19.

[0329] Table 19 Repeatability investigation - Relative retention time of characteristic peaks

[0330]

[0331]

[0332] It can be seen from Table 19 that the RSD of the relative retention times of characteristic peaks is between 0.02% and 0.10%, indicating good repeatability.

[0333] 6.4) Intermediate precision investigation

[0334] 6.4.1) Investigation of different personnel and times

[0335] On the basis of the above - determined experimental conditions, different personnel (A, B) weighed the walnut kernel formula granules at different times (T1, T2), ground them finely, took 0.3 g, in two portions each, prepared the test samples, and carried out the determination. The results are shown in Table 20.

[0336] Table 20 Investigation of Different Personnel and Times - Relative Retention Time of Characteristic Peaks

[0337]

[0338] As can be seen from Table 20, under the conditions of different sample - preparing personnel and different sample - preparing times, the RSD of the relative retention time of each characteristic peak is between 0.01% and 0.11%, indicating good method stability.

[0339] 6.4.2) Investigation of Different Instruments

[0340] Take an appropriate amount of this product, grind it finely, take 0.3 g, prepare the test sample solution, and carry out the determination on Agilent, Waters, and Thermo Fisher high - performance liquid chromatographs respectively. The results are shown in Figure 42 and Table 21, Figure 42 which is the experimental result diagram of the investigation of different instruments for the walnut kernel formula granules provided in Example 3 of the present invention.

[0341] Table 21 Investigation of Different Instruments - Relative Retention Time of Characteristic Peaks

[0342]

[0343] As can be seen from Table 21, when the test samples are detected with the above three instruments, the RSD of the relative retention time of each characteristic peak is between 0.44% and 5.94%, indicating good instrument durability.

[0344] 6.5) Durability Investigation

[0345] 6.5.1) Investigation of Chromatographic Column Durability

[0346] On the basis of the above - determined experimental conditions, different brand chromatographic columns, namely Agilent C18 - 1, Agilent C18 - 2, and Shimadzu C18 chromatographic columns, were investigated respectively. The results are shown in Figure 43 and Table 22, Figure 43 which is the experimental result diagram of the investigation of different chromatographic columns for the walnut kernel formula granules provided in Example 3 of the present invention.

[0347] Table 22 Investigation of Chromatographic Column Durability - Relative Retention Time of Characteristic Peaks

[0348]

[0349]

[0350] As can be seen from Table 22, when the above three chromatographic columns were used to detect the samples, the RSD of the relative retention time of the characteristic peaks was between 0.05% and 8.44%, indicating good durability of the chromatographic columns.

[0351] 6.5.2) Stability investigation

[0352] Based on the above-determined experimental conditions, the same test solution was taken and measured at 0 h, 4 h, 8 h, 12 h, 16 h, and 24 h respectively. The results are shown in Table 15.

[0353] Table 23 Stability investigation - Retention time

[0354]

[0355] As can be seen from Table 23, the RSD of the retention time of the corresponding characteristic peaks was between 0.01% and 0.11%, and the sample solution was relatively stable within 24 hours.

[0356] In summary, the RSD of the relative retention time of each characteristic peak met the requirements in all the above investigations, and the method was good. The above 6 characteristic peaks were included in the subsequent investigations.

[0357] 7) Determination of characteristic peaks and establishment of reference chromatograms

[0358] 7.1) Verification results of 3 batches of walnut kernel formula granules

[0359] Using the proposed method, characteristic spectrum analysis was performed on 3 batches of samples, and the relative retention time was calculated. The results are shown in Figure 44 and Table 24, Figure 44 which is the verification diagram of the characteristic spectra of 3 batches of walnut kernel formula granules provided in Example 3 of the present invention.

[0360] Table 24 Relative retention times of 3 batches of walnut kernel formula granules

[0361]

[0362] According to the principle of stable relative retention time, all batches of samples can be detected and the peaks are relatively high, a total of 6 peaks with good repeatability were selected as characteristic peaks.

[0363] 7.2) Formulation of the specified limit of relative retention time

[0364] The summary of the investigation items and verification results of the methodology is shown in Table 25:

[0365] Table 25 Summary of the results of each item of the methodology RSD (%) - Relative retention time (retention time)

[0366]

[0367] Final Specification: Six characteristic peaks should be presented in the chromatogram of the test sample, and their retention times should correspond to those of the six characteristic peaks in the reference chromatogram of the reference medicinal material. Among them, Peak 1 and Peak 5 should respectively correspond to the retention times of the reference peaks of gallic acid reference substance and ellagic acid reference substance. The peak corresponding to the reference peak of gallic acid reference substance is Peak S1. Calculate the relative retention times of Peak 2 and Peak 3 with respect to Peak S1, and the relative retention times should be within the range of ±10% of the specified values. The specified values are: 1.00 (Peak 1), 2.23 (Peak 2), 3.98 (Peak 3). The peak corresponding to the reference peak of ellagic acid reference substance is Peak S2. Calculate the relative retention times of Peak 4 and Peak 6 with respect to Peak S2, and the relative retention times should be within the range of ±10% of the specified values. The specified values are: 0.96 (Peak 4), 1.00 (Peak 5), 1.16 (Peak 6).

[0368] The similarity evaluation system for traditional Chinese medicine chromatographic fingerprints (2012 edition) was used to synthesize the chromatograms of 3 batches of walnut kernel formula granules, and a reference chromatogram of the characteristic chromatogram of walnut kernel formula granules was established, as Figure 45 shown Figure 45 is the reference chromatogram of the characteristic chromatogram of walnut kernel formula granules provided in Example 3 of the present invention.

[0369] 8) Determination of the characteristic chromatogram method of walnut kernel formula granules

[0370] Chromatographic conditions and system suitability test: Octadecylsilane chemically bonded silica gel was used as the filler; acetonitrile was used as mobile phase A, and 0.1 wt% phosphoric acid solution was used as mobile phase B. Gradient elution was carried out according to the regulations in Table 1; the flow rate was 0.3 mL per minute; the column temperature was 25 °C; the detection wavelength was 254 nm; the number of theoretical plates calculated based on the ellagic acid peak should be not less than 5000.

[0371] Preparation of the reference substance solution: Take 1.0 g of the reference medicinal material of walnut kernel, place it in a stoppered conical flask, add 30 mL of water, decoct for 30 minutes, cool, centrifuge (at a speed of 6000 revolutions per minute) for 4 minutes, take the supernatant, evaporate to dryness, dissolve the residue in 10 mL of 50 vol% methanol solution, filter, and take the subsequent filtrate as the reference medicinal material reference substance solution; Take an appropriate amount of gallic acid reference substance, accurately weigh it, and prepare a solution containing 20 μg of gallic acid per 1 mL of methanol as the gallic acid reference substance solution; Take an appropriate amount of ellagic acid reference substance, accurately weigh it, and prepare a solution containing 50 μg of ellagic acid per 1 mL of methanol as the ellagic acid reference substance solution.

[0372] Preparation of the test solution: Take an appropriate amount of this product, grind it finely, take about 0.3 g, place it in a stoppered conical flask, accurately add 20 mL of 50 vol% methanol, stopper tightly, weigh, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the lost weight with 50 vol% methanol, shake well, filter, and take the subsequent filtrate, which is the test solution.

[0373] Determination method: Accurately pipette 1 μL each of the reference solution and the test solution, inject them into the liquid chromatograph, and conduct the determination to obtain the result.

[0374] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing HPLC characteristic patterns of walnut kernel medicinal materials, decoction pieces, standard decoctions, and formula granules, characterized in that: The following steps are involved: preparing a test sample, wherein the test sample is walnut kernel medicinal material, walnut kernel decoction slices, walnut kernel standard decoction or walnut kernel formula granules; Dissolve the test sample to obtain a test sample solution; the dissolving solvent is 50 vol% methanol solution; The test solution is measured by high performance liquid chromatography to obtain a HPLC characteristic spectrum of the corresponding test sample; The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the detection wavelength is 254 nm; the mobile phase A is acetonitrile, the mobile phase B is a 0.1 wt % phosphoric acid solution, and the gradient elution is performed; the gradient elution is specifically as follows: 0~5min, phase A: 2vol%, phase B: 98vol%; 5~13min, phase A: 2~10vol%, phase B: 98~90vol%; 13-17 min, phase A: 10-13.5 vol%, phase B: 90-86.5 vol%; 17~24min, phase A: 13.5~19.5vol%, phase B: 86.5~80.5vol%; 24~30min, phase A: 19.5~25vol%, phase B: 80.5~75vol%.

2. The construction method according to claim 1, characterized in that The chromatographic conditions of the high performance liquid chromatography method also include: a mobile phase flow rate of 0.3 mL / min; an injection volume of 1 μL; a column temperature of 25° C.; and a theoretical plate number of not less than 5000 calculated based on the ellagic acid peak.

3. The construction method according to claim 1, characterized in that The following steps are also included: Dissolve the walnut kernel reference medicinal material to obtain a reference medicinal material reference solution; dissolve gallic acid to obtain a gallic acid reference substance reference solution; dissolve ellagic acid to obtain an ellagic acid reference substance reference solution; The control medicinal material reference solution and the reference substance reference solution are measured by high performance liquid chromatography to obtain a chromatogram of the reference substance; and the components of the HPLC characteristic spectrum of the test sample are qualitatively analyzed based on the chromatogram of the reference substance.

4. The construction method according to claim 3, characterized in that The following steps are also included: The similarity of the HPLC characteristic spectrum of walnut kernel medicinal materials was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of walnut kernel medicinal materials consisting of 6 characteristic peaks was obtained; in the HPLC standard characteristic spectrum, peak 1 corresponding to the peak of the gallic acid reference substance was the S1 peak, and the relative retention times of peaks 2, 3, and S1 were calculated. The relative retention times were within the range of ±10% of the specified value, and the specified value was: 1.00 (peak 1), 2.23 (peak 2), 3.97 (peak 3). Peak 5 corresponding to the ellagic acid reference peak is the S2 peak. The relative retention times of peaks 4, 6 and S2 were calculated. The relative retention times were within ±10% of the specified values, which are: 0.96 (peak 4), 1.00 (peak 5), 1.16 (peak 6).

5. The construction method according to claim 3, characterized in that The following steps are also included: The similarity of the HPLC characteristic spectrum of the walnut kernel slices was evaluated using the Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of the walnut kernel slices consisting of 6 characteristic peaks was obtained; in the HPLC standard characteristic spectrum, peak 1 corresponding to the peak of the gallic acid reference substance was the S1 peak, and the relative retention times of peaks 2, 3 and S1 were calculated. The relative retention times were within the range of ±10% of the specified value, and the specified value was: 1.00 (peak 1), 2.23 (peak 2), 3.98 (peak 3). Peak 5 corresponding to the ellagic acid reference peak is the S2 peak. The relative retention times of peaks 4, 6 and S2 were calculated. The relative retention times were within ±10% of the specified values, which are: 0.96 (peak 4), 1.00 (peak 5), 1.16 (peak 6).

6. The construction method according to claim 3, characterized in that: The following steps are also included: The similarity of the HPLC characteristic spectrum of the walnut kernel standard decoction was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of the walnut kernel standard decoction consisting of 6 characteristic peaks was obtained; in the HPLC standard characteristic spectrum, peak 1 corresponding to the peak of the gallic acid reference substance was the S1 peak, and the relative retention times of peaks 2, 3, and S1 were calculated. The relative retention times were within the range of ±10% of the specified value, and the specified value was: 1.00 (peak 1), 2.22 (peak 2), 3.98 (peak 3). Peak 5 corresponding to the ellagic acid reference peak is the S2 peak. The relative retention times of peaks 4, 6 and S2 were calculated. The relative retention times were within ±10% of the specified values, which are: 0.96 (peak 4), 1.00 (peak 5), 1.16 (peak 6).

7. The construction method according to claim 3, characterized in that: The following steps are also included: The similarity of the HPLC characteristic spectrum of the walnut kernel formula granules was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of the walnut kernel formula granules consisting of 6 characteristic peaks was obtained; in the HPLC standard characteristic spectrum, peak 1 corresponding to the peak of the gallic acid reference substance was the S1 peak, and the relative retention times of peaks 2, 3, and S1 were calculated. The relative retention times were within the range of ±10% of the specified value, and the specified value was: 1.00 (peak 1), 2.23 (peak 2), 3.98 (peak 3). Peak 5 corresponding to the ellagic acid reference peak is the S2 peak. The relative retention times of peaks 4, 6 and S2 were calculated. The relative retention times were within ±10% of the specified values, which are: 0.96 (peak 4), 1.00 (peak 5), 1.16 (peak 6).

8. A method for identifying walnut kernel medicinal materials, decoction pieces, standard decoctions, and formula granules, characterized in that: The HPLC characteristic spectrum obtained by the construction method according to any one of claims 1 to 7 is used as the basis for identifying walnut kernel medicinal materials, decoction pieces, standard decoctions, and formula granules.

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