A fingerprint spectrum of Qihuzha granules and its establishment and detection method

Abstract

The invention relates to a fingerprint spectrum of Qihuzha granules and an establishment and detection method thereof. A method for establishing a fingerprint spectrum of Qihuzha granules is provided, and the steps include: S1, preparing a test solution of Qihuzha granules; S2, preparing a reference solution; S3, injecting the test solution and the reference solution into a high performance liquid chromatograph, and calculating and obtaining the fingerprint spectrum of Qihuzha granules according to the chromatogram of the test solution and the chromatogram of the reference solution. The established fingerprint spectrum detection method of Qihuzha granules is simple, has the advantages of high similarity, good stability, high precision, good reproducibility, etc., can comprehensively, objectively and accurately detect and evaluate the quality of Qihuzha products, and ensure clinical efficacy.

Description

Technical Field

[0001] The present invention relates to the field of fingerprint analysis methods for Chinese patent medicines, and in particular to a fingerprint of Qihuzha granules and an establishment and detection method thereof. Background Art

[0002] Astragalus, Hukou and Hawthorn Granules are composed of 11 Chinese medicinal herbs including astragalus, roasted licorice, Poria, Dendrobium, Hawthorn, Codonopsis, Tangerine peel, and Atractylodes. They have the effects of strengthening the spleen and stomach, replenishing qi and strengthening the exterior, and digesting food and relieving stagnation. They are mainly used to improve symptoms such as anorexia, picky eating, excessive sweating, irregular bowel movements, and susceptibility to colds caused by spleen and stomach qi deficiency and spleen dysfunction in children.

[0003] Clinical studies have found that Astragalus, Humulus and Hawthorn Granules are effective in assisting the treatment of acute upper respiratory tract infections in children, treating malnutrition in children with recurrent respiratory tract infections, treating spleen deficiency-type diarrhea in children, treating anorexia in children, and treating diarrhea in infants.

[0004] The current quality standard includes five thin-layer chromatography identifications and a single indicator (ammonium glycyrrhizate) content determination, but its quantitative quality control is relatively simple. The chemical composition of the 11 raw medicinal materials in the prescription is complex, and qualitative and quantitative analysis of individual components cannot fully reflect the types and quantities of the chemical components contained in Qihuzha granules. Fingerprints can more fully reflect the overall distribution of the various chemical components contained in the drug, improve the quality control of traditional Chinese medicine compounds, lay the foundation for research on the basic efficacy of preparations, and more effectively control the quality of preparations, ensuring their safe and effective clinical use.

[0005] Therefore, establishing a fingerprint of Qihuzha granules and establishing an identification method for Qihuzha products will be of great significance for the quality control of Qihuzha products, improving the quality standards of Qihuzha products and the safety of medication. Summary of the Invention

[0006] The purpose of the present invention is to provide a fingerprint spectrum of Qihuzha granules and a method for establishing and detecting the fingerprint spectrum of Qihuzha granules in response to the deficiencies in the prior art.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention is to provide a method for establishing a fingerprint spectrum of Qihuzha granules, comprising the steps of:

[0009] S1, preparing a test solution of the Qihuzha granules;

[0010] S2. Prepare a reference solution; the solute of the reference solution includes at least one of 5-hydroxymethylfurfural, chlorogenic acid, calycosin glucoside, liquiritin, naringin, hesperidin, codonopsis pilosula, formononetin, isoliquiritin, ammonium glycyrrhizate, dendrobium phenol, or atractylodes lactone III;

[0011] S3, injecting the test solution and the reference solution into a high performance liquid chromatograph respectively, and calculating the fingerprint of the Qihuzha granules according to the chromatogram of the test solution and the chromatogram of the reference solution;

[0012] The HPLC conditions include: a Capcell PAK ADME column, an inner diameter of 4 mm to 5 mm, a length of 200 mm to 300 mm, a filler particle diameter of 3 μm to 6 μm, a column temperature of 28° C. to 35° C., and a detection wavelength of 230 nm to 254 nm; an injection volume of 8 μL to 12 μL, a flow rate of 0.8 mL / min to 1.1 mL / min; mobile phase A is acetonitrile, mobile phase B is a 0.1% volume fraction of phosphoric acid aqueous solution, and the elution method is gradient elution;

[0013] The gradient elution comprises:

[0014]

[0015] Preferably, step S1 comprises: taking appropriate amounts of the Astragalus, Humulus and Hawthorn granules from several different batches of the Astragalus, Humulus and Hawthorn granules, grinding them into powder, and accurately weighing 2.7g-3.3g of the Astragalus, Humulus and Hawthorn granules, placing them in a stoppered conical flask, accurately adding 25mL of methanol, and sealing the flask, performing the first weighing, ultrasonic treatment, cooling treatment, and second weighing in sequence, supplementing the lost weight with methanol, and then shaking and filtering, taking the filtrate to obtain several different batches of the test solution.

[0016] More preferably, the ultrasonic treatment time is 20 min-40 min, the frequency is 30 KHz-40 KHz, and the power is 350 W-420 W.

[0017] Preferably, step S2 comprises: accurately weighing the solute, and quantitatively dissolving it in methanol to obtain the reference solution.

[0018] The second aspect of the present invention is to provide a fingerprint of Qihuzha granules obtained by the above-mentioned established method.

[0019] Preferably, the fingerprint of the Astragalus, Dendrobium and Hawthorn granules includes: peak 1 corresponding to the 5-hydroxymethylfurfural, peak 2 corresponding to the chlorogenic acid, peak 3 corresponding to the isoflavone glucoside, peak 7 corresponding to the liquiritin, peak 8 corresponding to the naringin, peak 9 corresponding to the hesperidin, peak 10 corresponding to the codonopsis pilosula, peak 11 corresponding to the formononetin, peak 12 corresponding to the isoliquiritin, peak 13 corresponding to ammonium glycyrrhizate, peak 14 corresponding to the dendrobium phenol and peak 15 corresponding to the atractylodes lactone III.

[0020] More preferably, taking peak No. 7 as the reference peak S, the retention time of peak No. 1 is 8.372, the retention time of peak No. 2 is 19.544, the retention time of peak No. 3 is 27.996, the retention time of peak No. 4 is 28.344, the retention time of peak No. 5 is 28.813, the retention time of peak No. 6 is 29.443, the retention time of peak No. 7 is 29.874, the retention time of peak No. 8 is 31.032, the retention time of peak No. 9 is 33.053, the retention time of peak No. 10 is 34.354, the retention time of peak No. 11 is 36.991, the retention time of peak No. 12 is 37.813, the retention time of peak No. 13 is 52.199, the retention time of peak No. 14 is 52.902, and the retention time of peak No. 15 is 60.388.

[0021] The third aspect of the present invention is to provide a quality detection method for the above-mentioned fingerprint spectrum of Astragalus, Humulus and Hawthorn Granules, the steps comprising: performing similarity evaluation, multi-point correction and Mark peak matching on the high-performance liquid chromatogram obtained after the Astragalus, Humulus and Hawthorn product to be tested is prepared into a solution and the fingerprint spectrum of the Astragalus, Humulus and Hawthorn Granules in turn, and determining the detection result based on the calculated similarity.

[0022] Preferably, if the similarity is 0.95-1.00, the test result indicates that the Astragalus, Dendrobium and Hawthorn product to be tested is qualified.

[0023] Preferably, the Astragalus, Dendrobium and Hawthorn products to be tested include at least one of Astragalus, Dendrobium and Hawthorn granules, Astragalus, Dendrobium and Hawthorn extracts and Astragalus, Dendrobium and Hawthorn intermediates.

[0024] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0025] The fingerprint of the Qihuzha granules of the present invention contains 15 common peaks, and the reference substance identifies 12 of the common peaks, including 5-hydroxymethylfurfural, chlorogenic acid, calycosin isoflavone glucoside, liquiritin, naringin, hesperidin, codonopsis pilosula, formononetin, isoliquiritin, glycyrrhizic acid, dendrobium phenol, and atractylodes lactone III. The common peaks are attributed to six medicinal materials, namely, Codonopsis pilosula, Astragalus membranaceus, Tangerine peel, Atractylodes macrocephala, Dendrobium officinale, and Licorice, thereby achieving the purpose of effectively controlling the quality of the Qihuzha granules. The present invention screens out the optimal detection wavelength, chromatographic column, mobile phase pH, column temperature, flow rate and other analytical conditions as well as the optimal test sample preparation conditions. The established fingerprint detection method for the Qihuzha granules is simple and has the advantages of high similarity, good stability, high precision, good reproducibility, etc., and can comprehensively, objectively and accurately detect and evaluate the quality of the Qihuzha products to ensure clinical efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the fingerprint of Qihuzha granules generated based on the chromatograms of 20 batches of test samples in the embodiment of the present invention;

[0027] Figure 2 This is an overlay of chromatograms of 20 batches of test samples in the embodiment of the present invention;

[0028] Figure 3 Comparison chart of the chromatograms of the test sample and reference sample in the embodiments of the present invention and the chromatograms of the negative preparation lacking Astragalus membranaceus in the embodiments;

[0029] Figure 4 It is a comparison chart of the chromatogram of the test sample, the chromatogram of the reference sample and the chromatogram of the negative preparation lacking licorice in the embodiment of the present invention;

[0030] Figure 5 It is a comparison diagram of the chromatogram of the test sample, the chromatogram of the reference sample and the chromatogram of the negative preparation lacking tangerine peel in the embodiment of the present invention;

[0031] Figure 6 It is a comparison chart of the chromatogram of the test sample and the reference sample in the embodiment of the present invention and the chromatogram of the negative preparation lacking Dendrobium in the embodiment;

[0032] Figure 7 It is a comparison chart of the chromatogram of the test sample, the chromatogram of the reference sample and the chromatogram of the negative preparation lacking Atractylodes macrocephala in the embodiment of the present invention;

[0033] Figure 8 The chromatograms of different wavelengths of the embodiment of the present invention and comparative example 1 are shown;

[0034] Figure 9 The chromatograms of different chromatographic columns of Example 2 of the present invention and Comparative Example 2 are shown;

[0035] Figure 10 The chromatograms of different mobile phases B of Example 3 of the present invention are shown;

[0036] Figure 11 The chromatograms of different column temperatures of Example 4 of the present invention and Comparative Example 4 are shown;

[0037] Figure 12 The chromatograms of different flow rates of Example 5 of the present invention and Comparative Example 5 are shown;

[0038] Figure 13 The following are chromatograms of solvent extraction of different test samples in Example 1 of the present invention and Comparative Example 6. DETAILED DESCRIPTION

[0039] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0040] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0042] Example

[0043] This embodiment provides a method for detecting the fingerprint of Qihuzha granules;

[0044] 1. Material Preparation

[0045] 1. Instruments and equipment

[0046] An Agilent 1260 high-performance liquid chromatograph equipped with a quaternary pump and a DAD detector was used (Agilent Technologies, USA); an XSE105DU electronic balance was used (Mettler-Toledo, Switzerland); an MS204TS / 02 electronic balance was used (Mettler-Toledo, Switzerland); a GmbH / P300H ultrasonic cleaner was used (Elma Schmidbauer, Germany); and a Master Touch-DUVF ultrapure water preparation apparatus was used (Hetai, Shanghai).

[0047] 2. Reagents

[0048] Acetonitrile, methanol (chromatographic grade, Damas-beta); methanol (analytical grade, General-Reagent); phosphoric acid (chromatographic grade, Fisher).

[0049] 3. Reference substances

[0050] 5-Hydroxymethylfurfural (Batch No.: 111626-202114, purity 98.7%), chlorogenic acid (Batch No.: 110753-202018, purity 96.1%), calycosin glucoside (Batch No.: 111920-201907, purity 96.8%), liquiritin (Batch No.: 111610-201908, purity 95.0%), hesperidin (Batch No.: 110721-202019, purity 95.3%), codonopsis pilosula (Batch No.: 111732-201908), ammonium glycyrrhizate (Batch No.: 1 10731-202021, purity 96.2%), dendrobium phenol (batch number: 111875-202003), atractylodes lactone III (batch number: 111978-201501, purity 99.9%), China Food and Drug Inspection Institutes; naringin (batch number: 10285, purity 98.7%), formononetin (batch number: 10240, purity 99.5%), Shanghai Shidande Biotechnology Co., Ltd.; isoliquiritigenin (batch number: PRF20040923, purity 98.2%), Chengdu Purifa Technology Development Co., Ltd.

[0051] 4. Samples

[0052] Astragalus, Huhu and Hawthorn Granules are provided by Kuihua Pharmaceutical Group (Guizhou) Hongqi Co., Ltd. The batch numbers of the 20 batches of drugs are 20200918, 20201007, 20210222, 20211211, 20211213, 20211214, 20211215, 20211217, 20220101, 20220102, 20220103, 20220104, 20220105, 20220106, 20220107, 20220108, 20220109, 20220110, 20220111, and 20220112.

[0053] 5. Negative preparations

[0054] Negative samples of various medicinal materials were missing and were provided by Kuihua Pharmaceutical Group (Guizhou) Hongqi Co., Ltd., and were extracted and prepared according to the prescription ratio and prescription process of each medicine.

[0055] 2. Establishment of the fingerprint of Qihuzha granules and sample testing

[0056] S1, take an appropriate amount of 20 batches of different batch numbers (batch numbers are 20200918, 20201007, 20210222, 20211211, 20211213, 20211214, 20211215, 20211217, 20220101, 20220102, 20220103, 20220104, 20220105, 20220106, 20220107, 20220108, 20220109, 2 0220110, 20220111, 20220112) of Astragalus, Hubula and Hawthorn granules were ground into powder and accurately weighed. The mixture was placed in a stoppered conical flask, and 25 mL of methanol was accurately added. After the flask was sealed, the first weighing, ultrasonic treatment, cooling and second weighing were carried out in sequence. The weight loss was supplemented with methanol, shaken and filtered, and the filtrate was taken to obtain 20 batches of test solution. The ultrasonic treatment time was 30 min, the frequency was 37 kHz, and the power was 380 W.

[0057] S2. Accurately weigh 1.0 mg of 5-hydroxymethylfurfural reference substance, 2.0 mg of chlorogenic acid reference substance, 1.0 mg of calycosin glucoside reference substance, 3.0 mg of liquiritin reference substance, 2.0 mg of naringin reference substance, 6.0 mg of hesperidin reference substance, 1.0 mg of codonopsis pilosula reference substance, 2.0 mg of formononetin reference substance, 1.0 mg of isoliquiritin reference substance, 14.0 mg of ammonium glycyrrhizate reference substance, 1.0 mg of dendrobium phenol reference substance, and 4.0 mg of atractylodes lactone III reference substance, place them in a 20 mL volumetric flask, add methanol to dissolve, and dilute to the mark. Shake well to obtain the reference solution.

[0058] S3, injecting 20 batches of test solution and reference solution into high performance liquid chromatograph to obtain 20 batches of test solution chromatograms and reference solution chromatograms; taking the common peaks of the 20 batches of test solution chromatograms, calculating the average to generate the fingerprint of Qihuzha granules ( Figure 1 ) and annotate the chemical components according to the retention time of the chromatogram of the reference solution;

[0059] The HPLC conditions included: a Capcell PAK ADME column with an inner diameter of 4.6 mm, a length of 250 mm, a filler particle diameter of 5 μm, a column temperature of 30° C., and a detection wavelength of 230 nm; an injection volume of 10 μL, a flow rate of 1.0 mL / min; mobile phase A was acetonitrile, mobile phase B was a 0.1% by volume aqueous phosphoric acid solution, and the elution method was gradient elution.

[0060] The gradient elution program includes:

[0061]

[0062] The retention times of the 15 common peaks are as follows:

[0063]

[0064]

[0065] S4, the chromatograms of the test solution of 20 batches and the fingerprint of Qihuzha granules were imported into the Chinese medicine chromatographic fingerprint similarity evaluation system (the superposition of the 20 batches of test solutions is as follows Figure 2 ), similarity evaluation, multi-point calibration and Mark peak matching were performed in sequence, and the test results of 20 batches of Qihuzha granules were determined according to the similarity;

[0066] The similarity results are shown in Table 1. The similarities between the fingerprint spectra of Qihuzha granules from different batches and the fingerprint spectra of Qihuzha granules are all above 0.95, and the similarity between different batches of Qihuzha granules is relatively high.

[0067] Table 1

[0068]

[0069] 3. Confirm the ownership of the common peak

[0070] Take the negative preparation lacking Astragalus, the negative preparation lacking Licorice, the negative preparation lacking Tangerine Peel, the negative preparation lacking Codonopsis, the negative preparation lacking Dendrobium and the negative preparation lacking Atractylodes, and prepare them according to the above test solution preparation method to obtain the negative sample solutions lacking each medicinal material.

[0071] According to the above chromatographic conditions, the chromatograms of the negative preparation lacking Astragalus, the negative preparation lacking Licorice, the negative preparation lacking Tangerine Peel, the negative preparation lacking Codonopsis, the negative preparation lacking Dendrobium, and the negative preparation lacking Atractylodes were measured respectively, and compared with the chromatograms of the test solution and the chromatogram of the reference solution. The retention times of the common peaks were compared, and finally the medicinal material identity of the common peaks in the fingerprint of Qihuzha granules was confirmed.

[0072] The comparison of the chromatogram of the negative preparation of Astragalus membranaceus with the chromatogram of the test solution and the chromatogram of the reference solution is shown in the figure below. Figure 3 As shown, Figure 3 In the figure a, the chromatogram of the reference substance of caerulea isoflavone glucoside is obtained, in the figure b, the chromatogram of the negative preparation lacking Astragalus, and in the figure c, the chromatogram of the test substance is obtained. It can be seen that the peak No. 3 (caerulea isoflavone glucoside) in the fingerprint is derived from the Astragalus medicinal material.

[0073] The comparison of the chromatogram of the negative preparation lacking licorice and the chromatogram of the test solution and the chromatogram of the reference solution is shown in the figure below. Figure 4 As shown, Figure 4Figure d is the chromatogram of the liquiritin reference substance, e is the chromatogram of the isoliquiritin reference substance, f is the chromatogram of the glycyrrhizic acid reference substance, g is the chromatogram of the negative preparation lacking licorice, and h is the chromatogram of the test sample. It can be seen that peaks 4, 5, 6, 7 (liquiritin), 12 (isoliquiritin), and 13 (glycyrrhizic acid) in the fingerprint are all derived from licorice herbs.

[0074] The comparison of the chromatogram of the negative preparation lacking tangerine peel with the chromatogram of the test solution and the chromatogram of the reference solution is shown in the figure below. Figure 5 As shown, Figure 5 In the figure, i is the chromatogram of the reference substance of naringin, j is the chromatogram of the reference substance of hesperidin, k is the chromatogram of the negative preparation lacking tangerine peel, and l is the chromatogram of the test sample. It can be seen that peak 8 (naringin) and peak 9 (hesperidin) in the fingerprint are both derived from tangerine peel.

[0075] The comparison of the chromatogram of the negative preparation of Dendrobium officinale and the chromatogram of the test solution and the chromatogram of the reference solution is shown in the figure below. Figure 6 As shown, Figure 6 In the figure, p is the chromatogram of the dendrobium phenol reference substance, q is the chromatogram of the negative preparation lacking dendrobium, and r is the chromatogram of the test sample. It can be seen that peak 14 (dendrobium phenol) in the fingerprint is derived from the dendrobium medicinal material.

[0076] The comparison of the chromatogram of the negative preparation lacking Atractylodes macrocephala and the chromatogram of the test solution and the chromatogram of the reference solution is shown in the figure below. Figure 7 As shown, Figure 7 In the figure, s is the chromatogram of the reference substance of Atractylodes lactone III, t is the chromatogram of the negative preparation lacking Atractylodes macrocephala, and u is the chromatogram of the test substance. It can be seen that peak 15 (Atractylodes lactone III) in the fingerprint spectrum comes from Atractylodes macrocephala.

[0077] 4. Precision Experiment

[0078] Take Qihuzha granules (batch number: 20220108) and prepare the test solution according to the above test solution preparation method to obtain the test solution;

[0079] According to the above chromatographic conditions, 6 injections were made continuously, and glycyrrhizin (peak 7) was used as the reference peak S. The relative retention time and relative peak area of each peak were calculated, and the relative standard deviation (RSD%) of each peak was calculated. The relative retention time RSD% values of each peak in the 6 injections of samples were between 0.00-0.17, and the relative peak area RSD% values were between 0.00-1.99, indicating that the instrument had good precision.

[0080] 5. Repeatability Experiment

[0081] Take Qihuzha Granules (Batch No.: 20220108), weigh 6 portions in parallel, and prepare the test solution according to the above method to obtain 6 test solution;

[0082] The samples were injected separately according to the above chromatographic conditions, and glycyrrhizin (peak 7) was used as the reference peak S to calculate the relative retention time and relative peak area of each peak. The relative standard deviation (RSD%) of each peak was also calculated. The relative retention time RSD% values of each peak in the 6-injection samples were between 0.05-0.17, and the relative peak area RSD% values were all between 2.46-4.27. The sample repeatability was good.

[0083] 6. Stability test

[0084] Take Qihuzha granules (batch number: 20220108) and prepare the test solution according to the above test solution preparation method to obtain the test solution;

[0085] According to the above chromatographic conditions, samples were injected at 0h, 2.5h, 5h, 10h, 17.5h, and 25h, respectively. Glycyrrhizin (peak 7) was used as the reference peak S. The relative retention time and relative peak area of each peak were calculated, and the relative standard deviation (RSD%) of each peak was calculated. The relative retention time RSD% values of each peak in the test solution were between 0.05-0.68, and the relative peak area RSD% values were all between 0.84-3.83, indicating that the test solution had good stability within 24h.

[0086] Comparative Example 1

[0087] This comparative example provides a method for establishing a fingerprint spectrum of Qihuzha granules;

[0088] Take Qihuzha granules (batch number: 20220108) and conduct the experiment according to the preparation method and chromatographic conditions of the test solution in the example, except that the detection wavelength of Comparative Example 1 is 254 nm;

[0089] The comparison of the chromatograms of the test sample in the embodiment and the chromatograms of the test sample at different wavelengths in Comparative Example 1 is shown in FIG. Figure 8 As shown, Figure 8 a is the chromatogram of the test sample at 230 nm, b is the chromatogram of the test sample at 254 nm, Figure 8 It can be seen that the chromatographic information at the detection wavelength of 230 nm is richer, the chromatographic peak response value is higher, and the baseline is more stable.

[0090] Comparative Example 2

[0091] This comparative example provides another method for establishing the fingerprint of Qihuzha granules;

[0092] Qihuzha granules (batch number: 20220108) were taken and the experiment was carried out according to the preparation method of the test solution and the chromatographic conditions in the example, except that the chromatographic columns Agilent Zorbax Eclipse Plus C18 (4.6×250 mm, 5 μm), Thermo Hypersil GOLD aQ (4.6×250 mm, 5 μm), and Capcell PAK MGLL C18 (4.6×250 mm, 5 μm) were used in Comparative Example 2;

[0093] The comparison diagram of the chromatogram of the test sample in the embodiment and the chromatogram of the test sample of different chromatographic columns in Comparative Example 2 is as follows Figure 9 As shown, Figure 9 In the figure, c is the chromatogram of the test sample on the Thermo Hypersil GOLD column, d is the chromatogram of the test sample on the Agilent Zorbax EclipsePlus C18 column, e is the chromatogram of the test sample on the Capcell PAK MGLL C18 column, and f is the chromatogram of the test sample on the Capcell PAK ADME column. Figure 9 It can be seen that the present invention sets the chromatographic column as CapcellPAK ADME (4.6×250 mm, 5 μm), and the separation effect of each chromatographic peak is better.

[0094] Comparative Example 3

[0095] This comparative example provides another method for establishing the fingerprint of Qihuzha granules;

[0096] Take Qihuzha granules (batch number: 20220108) and conduct the experiment according to the preparation method and chromatographic conditions of the test solution in the example, except that the mobile phase B in Comparative Example 3 is water, 0.05% phosphoric acid water, and 0.2% phosphoric acid water, respectively;

[0097] The comparison of the chromatogram of the test sample in the embodiment and the chromatogram of the test sample with different mobile phase B in comparative example 3 is shown in FIG. Figure 10 As shown, Figure 10 Where g is the chromatogram of the test sample with mobile phase B being water, h is the chromatogram of the test sample with mobile phase B being 0.05% phosphoric acid water, i is the chromatogram of the test sample with mobile phase B being 0.1% phosphoric acid water, and j is the chromatogram of the test sample with mobile phase B being 0.2% phosphoric acid water; Figure 10 It can be seen that in the present invention, the mobile phase B is set to 0.1% phosphoric acid water, and the separation effect of each chromatographic peak is better.

[0098] Comparative Example 4

[0099] This comparative example provides another method for establishing the fingerprint of Qihuzha granules;

[0100] Take Qihuzha granules (batch number: 20220108) and conduct the experiment according to the preparation method and chromatographic conditions of the test solution in the example, except that the column temperatures in comparative example 4 are 28°C, 32°C, and 35°C respectively;

[0101] The comparison of the chromatograms of the test samples in the embodiment and the chromatograms of the test samples at different column temperatures in Comparative Example 4 is shown in FIG. Figure 11 As shown, Figure 11 Where k is the chromatogram of the test sample at a column temperature of 28°C, l is the chromatogram of the test sample at a column temperature of 30°C, m is the chromatogram of the test sample at a column temperature of 32°C, and n is the chromatogram of the test sample at a column temperature of 35°C. Figure 11 It can be seen that the present invention sets the column temperature to 30° C., and the separation effect of each chromatographic peak is better.

[0102] Comparative Example 5

[0103] This comparative example provides another method for establishing the fingerprint of Qihuzha granules;

[0104] Take Qihuzha granules (batch number: 20220108) and conduct the experiment according to the preparation method and chromatographic conditions of the test solution in the example, except that the flow rates in comparative example 5 are 0.8 mL / min, 0.9 mL / min, and 1.1 mL / min respectively;

[0105] The comparison diagram of the chromatogram of the test sample in the embodiment and the chromatogram of the test sample at different flow rates in Comparative Example 5 is as follows: Figure 12 As shown, Figure 12 Where o is the chromatogram of the test sample at a flow rate of 0.8 mL / min, p is the chromatogram of the test sample at a flow rate of 0.9 mL / min, q is the chromatogram of the test sample at a flow rate of 1.0 mL / min, and r is the chromatogram of the test sample at a flow rate of 1.1 mL / min. Figure 12 It can be seen that the present invention sets the flow rate to 1.0 mL / min, and the separation effect of each chromatographic peak is better.

[0106] Comparative Example 6

[0107] This comparative example provides another method for establishing the fingerprint of Qihuzha granules;

[0108] Take Qihuzha granules (batch number: 20220108) and conduct the experiment according to the preparation method and chromatographic conditions of the test solution in the example, except that the extraction solvents of the test sample in Comparative Example 6 are water, 30% methanol, 50% methanol, and 70% methanol, respectively;

[0109] The chromatograms of the test samples in the embodiment and the chromatograms of the test samples with different extraction solvents in Comparative Example 6 are shown in FIG. Figure 13 As shown; Figure 13Where s is the chromatogram of the test sample when the extraction solvent is water, t is the chromatogram of the test sample when the extraction solvent is 30% methanol, u is the chromatogram of the test sample when the extraction solvent is 50% methanol, v is the chromatogram of the test sample when the extraction solvent is 70% methanol, and w is the chromatogram of the test sample when the extraction solvent is methanol. Figure 13 It can be seen that the present invention sets the sample extraction solvent to methanol, which has a higher extraction efficiency for each chromatographic peak with low polarity.

[0110] In summary, the fingerprint of Qihuzha granules provided by the present invention has 15 common peaks. Compared with the chromatograms of negative preparations lacking Astragalus, negative preparations lacking Licorice, negative preparations lacking Tangerine Peel, negative preparations lacking Codonopsis, negative preparations lacking Dendrobium, and negative preparations lacking Atractylodes, as well as the chromatograms of the reference substance, the common peak components are basically identified to determine the attribution of the common peak medicinal materials. The method is simple and has the advantages of high similarity, good stability, high precision, and good reproducibility.

[0111] The above description is only a preferred embodiment of the present invention and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for establishing a fingerprint of Qihuzha granules, characterized in that the steps include: S1, preparing a test solution of the Qihuzha granules, comprising: taking an appropriate amount of the Qihuzha granules, adding methanol and performing ultrasonic treatment; S2. Prepare a reference solution; the solutes of the reference solution include: 5-hydroxymethylfurfural, chlorogenic acid, calycosin glucoside, liquiritin, naringin, hesperidin, codonopsis pilosula, formononetin, isoliquiritin, ammonium glycyrrhizate, dendrobium phenol, and atractylodes lactone III; S3, injecting the test solution and the reference solution into a high performance liquid chromatograph respectively, and calculating the fingerprint of the Qihuzha granules according to the chromatogram of the test solution and the chromatogram of the reference solution; The high performance liquid chromatography conditions include: a Capcell PAK ADME column, an inner diameter of 4 mm to 5 mm, a length of 200 mm to 300 mm, a filler particle diameter of 3 μm to 6 μm, a column temperature of 28° C. to 35° C., and a detection wavelength of 230 nm to 254 nm; an injection volume of 8 μL to 12 μL, a flow rate of 0.8 mL / min to 1.1 mL / min; mobile phase A is acetonitrile, mobile phase B is a 0.1% volume fraction of phosphoric acid aqueous solution, and the elution method is gradient elution; The gradient elution comprises: ； The fingerprint of the Qihuzha granule includes: peak 1 corresponding to the 5-hydroxymethylfurfural, peak 2 corresponding to the chlorogenic acid, peak 3 corresponding to the calycosin isoflavone glucoside, peak 7 corresponding to the liquiritin, peak 8 corresponding to the naringin, peak 9 corresponding to the hesperidin, peak 10 corresponding to the codonopsis pilosula, peak 11 corresponding to the formononetin, peak 12 corresponding to the isoliquiritin, peak 13 corresponding to ammonium glycyrrhizate, peak 14 corresponding to the dendrobium phenol, and peak 15 corresponding to the atractylodes lactone III; Taking peak No. 7 as the reference peak S, the retention time of peak No. 1 is 8.372, the retention time of peak No. 2 is 19.544, the retention time of peak No. 3 is 27.996, the retention time of peak No. 4 is 28.344, the retention time of peak No. 5 is 28.813, the retention time of peak No. 6 is 29.443, the retention time of peak No. 7 is 29.874, the retention time of peak No. 8 is 31.032, the retention time of peak No. 9 is 33.053, the retention time of peak No. 10 is 34.354, the retention time of peak No. 11 is 36.991, the retention time of peak No. 12 is 37.813, the retention time of peak No. 13 is 52.199, the retention time of peak No. 14 is 52.902, and the retention time of peak No. 15 is 60.

388.

2. The establishment method according to claim 1, characterized in that Step S1 comprises: taking an appropriate amount of the Astragalus, Humulus and Hawthorn granules from several different batches of the Astragalus, Humulus and Hawthorn granules, grinding them, and accurately weighing 2.7g-3.3g of the Astragalus, Humulus and Hawthorn granules, placing them in a stoppered conical flask, accurately adding 25mL of methanol, and sealing them in turn by weighing the first weight, ultrasonic treatment, cooling, and weighing the second weight, supplementing the lost weight with methanol, and then shaking and filtering, taking the filtrate to obtain several different batches of the test sample solutions.

3. The establishment method according to claim 2, characterized in that: The ultrasonic treatment time is 20 min-40 min, the frequency is 30 KHz-40 KHz, and the power is 350 W-420 W.

4. The establishment method according to claim 1, characterized in that Step S2 comprises: accurately weighing the solute, and quantitatively dissolving it in methanol to obtain the reference solution.

5. A quality detection method for the fingerprint of Qihuzha granules obtained by the method according to any one of claims 1 to 4, characterized in that the steps include: The high performance liquid chromatogram obtained after the Astragalus, Dendrobium and Hawthorn product to be tested is prepared into a solution is subjected to similarity evaluation, multi-point correction and Mark peak matching with the Astragalus, Dendrobium and Hawthorn granule fingerprint spectrum in sequence, and the test result can be determined based on the calculated similarity.

6. The quality inspection method according to claim 5, characterized in that: If the similarity is 0.95-1.00, the test result shows that the Astragalus, Dendrobium and Hawthorn product to be tested is qualified.

7. The quality inspection method according to claim 5, characterized in that: The Astragalus, Dendrobium and Hawthorn products to be tested include at least one of Astragalus, Dendrobium and Hawthorn granules, Astragalus, Dendrobium and Hawthorn extracts, and Astragalus, Dendrobium and Hawthorn intermediates.

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