Fingerprint of Zhishu Granules and Detection Method of Content Determination

Abstract

The present application provides a method for establishing a fingerprint of Zhishu granules and a method for determining the content of its components. The fingerprint of Zhishu granules is established by high performance liquid chromatography and by reasonably selecting chromatographic conditions. The method has the advantages of simplicity, high sensitivity, fast analysis speed, and strong specificity, so it can be used for quality control of Zhishu granules, and also provides a basis for further studying the chemical composition and quality standards of Zhishu granules. The present application realizes the determination of the contents of 7 chemical components in Zhishu granules, introduces the multi-wavelength method into the determination of Zhishu granules for the first time, and realizes the simultaneous content determination of rutin, naringin, hesperidin, neohesperidin, naringenin, nobiletin, and atractylodes lactone III in Zhishu granules. The method is simple, fast, and highly specific, and has excellent stability, reproducibility, and accuracy. It is beneficial to the content determination and quality control of Zhishu granules of the present invention, and simultaneously realizes the quality monitoring of the three-flavor prescription medicine in the preparation.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine analysis, and in particular to a detection method for fingerprint spectrum and content determination of a traditional Chinese medicine Zhishu granule. Background Art

[0002] Traditional Chinese medicine and its preparations are complex multi-component systems. Therefore, their quality should be evaluated by appropriate detection methods that can provide rich identification information. The establishment of traditional Chinese medicine fingerprints and the detection of characteristic component contents will be able to more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine and its preparations, and thus provide an overall description and evaluation of the quality of the medicines.

[0003] Strict quality control of Zhishu granules is of great significance to ensure the safety, effectiveness and stability of the preparations. Zhishu granules are composed of three herbs: fried Zhishi, fried Atractylodes macrocephala and lotus leaf. This method establishes a method for content determination while conducting fingerprint research, and simultaneously realizes the quality monitoring of the three prescription drugs in the preparation, thus improving the preparation quality control method.

[0004] Zhishu Granule is an exclusive product of Nanjing Zhongshan Pharmaceutical Co., Ltd. It is composed of three herbs: fried Zhishi, fried Atractylodes macrocephala, and lotus leaf. It has the effects of strengthening the spleen and digestion, promoting qi and removing dampness. It has a good effect on spleen and stomach weakness, poor digestion, abdominal distension and other symptoms. Zhishu Granule is a national Class IV innovative Chinese medicine that uses modern extraction methods to modify the dosage form of Zhishu Pills. It has the characteristics of clear efficacy and convenient administration.

[0005] In the prior art, there are few research reports on Zhishu granules, and there is no report on simultaneous fingerprint and content determination to comprehensively detect the quality of the three-ingredient prescription. The existing methods for detection cannot achieve effective separation and accurate quantification of multiple components. Based on the prior art, the present invention mainly studies the fingerprint and content determination of its main components, which can better control the product quality. Summary of the invention

[0006] The purpose of this application is to provide a method for establishing a fingerprint of Zhishu Granules and a method for determining the content of its components, which can obtain the fingerprint of Zhishu Granules, separate 25 common peaks, identify 11 chromatographic peaks, and simultaneously determine the content of at least 7 chemical components in Zhishu Granules, which can be used for quality control of Zhishu Granules. The specific technical solution is as follows:

[0007] The first object of the present invention is to provide a method for establishing a fingerprint of Zhishu granules, the method comprising:

[0008] (1) Preparation of the test solution of Zhishu granules: Take Zhishu granules with a mass of M1, and extract them with methanol with a volume fraction of 50-100% and a volume of V1 as a solvent to obtain a test solution; wherein M1:V1=1g:(10-50)mL; when the preparation method of the test solution of the present application is adopted, combined with the chromatographic conditions of the high performance liquid chromatography method of the present application, it is beneficial to obtain more characteristic peaks in a shorter analysis time, and the fingerprint of Zhishu granules can be comprehensively established from the perspective of chemical composition, so that the fingerprint of Zhishu granules can be established quickly, accurately, comprehensively and reliably, which can be used for the quality control of Zhishu granules.

[0009] (2) Preparation of mixed reference solution: Accurately weigh reference substances of naringin, nuciferine, naringin, hesperidin, neohesperidin, naringenin, hesperidin, nobiletin, tangeretin, atractylodes lactone III, and atractylodes lactone II, place them in a volumetric flask, dilute to the mark with 50-100% methanol, shake well, and prepare a reference solution; in this application, the methanol with a volume fraction of 50-100% refers to a methanol aqueous solution with a volume fraction of 50-100%.

[0010] (3) using high performance liquid chromatography to detect the test solution to obtain a chromatogram of the Zhishu granules;

[0011] Chromatographic conditions included:

[0012] Chromatographic column: octadecylsilane bonded silica gel chromatographic column;

[0013] Mobile phase: Phase A is 0.05-0.15% phosphoric acid aqueous solution, and phase B is acetonitrile;

[0014] Elution procedure: gradient elution with a volume fraction of 25-90% phase A and 10-75% phase B;

[0015] Flow rate: 0.8-1.2mL / min;

[0016] Column temperature: 30-40°C;

[0017] Injection volume: 10-20 μL;

[0018] Detection wavelength: 220±5nm;

[0019] By adopting the chromatographic conditions of the present application, the chemical components in the Zhishu granules can be better separated.

[0020] (4) The chromatogram of the Zhishu Granules obtained in step (3) is analyzed using a traditional Chinese medicine chromatographic fingerprint similarity evaluation software to obtain a fingerprint of the Zhishu Granules.

[0021] Furthermore, in step (1), the extraction is ultrasonic extraction, the extraction time is 20-60 min, the extraction power is 150-200 W, and the extraction frequency is 30-50 kHz.

[0022] Further, step (2) preparation of a mixed reference solution: accurately weigh naringin, nuciferine, naringin, hesperidin, neohesperidin, naringenin, hesperidin, nobiletin, tangeretin, atractylodes lactone III, and atractylodes lactone II reference substances, and add 100% methanol to prepare a reference solution containing 50±5 μg / mL of naringin, 50±5 μg / mL of nuciferine, 100±5 μg / mL of naringin, 50±5 μg / mL of hesperidin, 100±5 μg / mL of neohesperidin, 50±5 μg / mL of naringenin, 50±5 μg / mL of hesperidin, 50±5 μg / mL of nobiletin, 50±5 μg / mL of tangeretin, 50±5 μg / mL of atractylodes lactone III, and 50±5 μg / mL of atractylodes lactone II;

[0023] Preferably, a reference solution containing 50 μg / mL of naringin, 50 μg / mL of nuciferine, 100 μg / mL of naringin, 50 μg / mL of hesperidin, 100 μg / mL of neohesperidin, 50 μg / mL of naringenin, 50 μg / mL of hesperidin, 50 μg / mL of nobiletin, 50 μg / mL of tangeretin, 50 μg / mL of atractylodes lactone III, and 50 μg / mL of atractylodes lactone II is prepared.

[0024] Furthermore, the elution procedure in step (3) is:

[0025]

[0026] The second object of the present invention is to provide a method for establishing a comparative fingerprint of Zhishu granules, the method comprising:

[0027] Take R batches of Zhishu Granules, and obtain the fingerprints of each batch of Zhishu Granules according to the aforementioned method for establishing the fingerprint of Zhishu Granules; analyze the fingerprints of the R batches of Zhishu Granules using the Chinese medicine chromatographic fingerprint similarity evaluation software to obtain the Zhishu Granule control fingerprint; the R is ≥15.

[0028] This application does not limit the parameter settings of the Chinese medicine chromatographic fingerprint similarity evaluation software when analyzing, as long as the purpose of this application can be achieved. The fingerprint of the R batch of Zhishu granules is analyzed by the Chinese medicine chromatographic fingerprint similarity evaluation software, and the control fingerprint of Zhishu granules can be obtained according to the mean method or the median method.

[0029] The third object of the present invention is to provide a control fingerprint of Zhishu Granules obtained by the aforementioned establishment method.

[0030] Furthermore, the control fingerprint of the Zhishu granules includes 25 common peaks, identifying 11 chromatographic peaks, of which peak 10 is naringin with a retention time of 31.796 min, peak 11 is nuciferine with a retention time of 32.878 min, peak 12 is naringin with a retention time of 35,722 min, peak 13 is hesperidin with a retention time of 37.937 min, and peak 14 is neohesperidin with a retention time of 40.8 77min, Peak 18 is naringenin with a retention time of 51.926min, Peak 19 is hesperetin with a retention time of 53.147min, Peak 22 is nobiletin with a retention time of 57.817min, Peak 23 is tangeretin with a retention time of 60.025min, Peak 24 is atractylodes lactone III with a retention time of 60.482min, Peak 25 is atractylodes lactone II with a retention time of 65.342min.

[0031] In the present application, the "common peak" refers to the chromatographic peak with the same retention time and good separation that exists in the chromatograms of R batches of Zhishu granules, that is, the common peak in the chromatograms of each Zhishu granule.

[0032] A fourth object of the present invention is to provide a method for controlling the quality of Zhishu granules, the method comprising:

[0033] a. Take the Zhishu granules to be tested, and according to the aforementioned establishment method, a chromatogram of the Zhishu granules to be tested is obtained;

[0034] b. Evaluate the similarity of the chromatogram obtained in step a with the aforementioned Zhishu granules control fingerprint. If the similarity is ≥0.90, the quality of the Zhishu granules to be tested is determined to be qualified.

[0035] Specifically, the present application can perform quality control by the following method: the fingerprint spectrum or control fingerprint spectrum of Zhishu Granules is obtained by the method of the present application; for Zhishu Granules of unknown mass, a test solution is prepared according to the method of the present application; the test solution is detected according to the chromatographic conditions of the present application to obtain a chromatogram of Zhishu Granules of unknown mass; the chromatogram of the unknown mass of Zhishu Granules is evaluated for similarity with the fingerprint spectrum of Zhishu Granules or the control fingerprint spectrum of Zhishu Granules, and a similarity of ≥0.90 is obtained, thereby judging that the quality of Zhishu Granules is qualified.

[0036] The fifth object of the present invention is to provide a method for determining the content of chemical components in Zhishu granules, using high performance liquid chromatography to determine the content of chemical components in Zhishu granules, wherein the chemical components include: naringin, naringin, hesperidin, neohesperidin, naringenin, nobiletin, and atractylodes lactone III;

[0037] The method comprises:

[0038] Z1. Establish standard curves for each chemical component:

[0039] Using 50-100% methanol as solvent, 5-12 mixed reference solutions of various chemical components with concentration gradients are prepared within the concentration range;

[0040] Under the same chromatographic conditions, the mixed reference substance solutions of each concentration gradient are respectively injected into the high performance liquid chromatograph, the chromatographic peaks of each chemical component are determined according to the retention time of each chemical component, and the chromatographic peak area of ​​each chemical component is obtained;

[0041] The chromatographic conditions include:

[0042] Chromatographic column: octadecylsilane bonded silica gel chromatographic column;

[0043] Mobile phase: Phase A is 0.05-0.15% phosphoric acid aqueous solution, and phase B is acetonitrile;

[0044] Elution procedure: gradient elution with a volume fraction of 25-90% phase A and 10-75% phase B;

[0045] Flow rate: 0.8-1.2mL / min;

[0046] Column temperature: 30-40°C;

[0047] Injection volume: 10-20 μL; Detection wavelength: 190-900 nm full wavelength scan, including 220±5 nm wavelength for detecting Atractylodes lactone III, 280 nm±5 nm wavelength for detecting naringin, naringin, hesperidin, neohesperidin, naringenin, and 332 nm±5 nm wavelength for detecting nobiletin;

[0048] The peak area of ​​each chemical component chromatographic peak is used as the ordinate, and the concentration of each chemical component is used as the abscissa to establish the standard curve of each chemical component;

[0049] Z2. Obtain the chromatographic peak area of ​​each chemical component in the Zhishu granules to be tested:

[0050] Take the Zhishu granules to be tested with a mass of M1, extract them with a volume of V1 and a volume fraction of 50-100% methanol as a solvent to obtain a test solution; wherein M1:V1=1g:(10-50)mL;

[0051] Under the same chromatographic conditions as step Z1, the test solution is injected into a high performance liquid chromatograph to obtain a chromatogram of the Zhishu granules to be tested, the chromatographic peaks of each chemical component are determined according to the retention time of each chemical component, and the chromatographic peak area of ​​each chemical component is obtained;

[0052] Z3. Determine the content of each chemical component in the Zhishu granules to be tested:

[0053] According to the standard curves of each chemical component established by Z1, the concentration C1 of each chemical component was obtained from the chromatographic peak area of ​​each chemical component in the Zhishu Granules to be tested by Z2, and the content of each chemical component in the Zhishu Granules to be tested was calculated according to the formula C=C1×V1 / M1.

[0054] Further, in the mixed reference solution of Z1, the concentration range of naringin is 0.0240-0.3993 mg / ml, the concentration range of naringin is 0.1807-3.0123 mg / ml, the concentration range of hesperidin is 0.0121-0.2011 mg / ml, the concentration range of neohesperidin is 0.1502-2.5032 mg / ml, the concentration range of naringenin is 0.0020-0.0325 mg / ml, the concentration range of nobiletin is 0.0027-0.0442 mg / ml, and the concentration range of atractylodes lactone III is 0.0019-0.0313 mg / ml;

[0055] Further, the elution procedure of Z1 is specifically as follows:

[0056]

[0057] Furthermore, in step Z2, the extraction is ultrasonic extraction, the extraction time is 20-60 min, the extraction power is 150-200 W, and the extraction frequency is 30-50 kHz.

[0058] The present application does not have any particular limitation on the preparation method of the mixed reference solution, as long as the purpose of the present application can be achieved. For example, a mixed reference stock solution can be prepared first, in which the concentration of each component is greater than or equal to the concentration of each chemical component in the mixed reference solution, and then the mixed reference solution is obtained by dilution.

[0059] In the present application, the mixed reference solution used to establish a standard curve may include the mixed reference stock solution; the present application has no particular limitation on the preparation of the mixed reference stock solution, as long as the purpose of the present application can be achieved. For example, the mixed reference stock solution can be prepared by preferentially preparing the stock solution of each chemical component separately, and then taking the stock solution of each chemical component separately.

[0060] By adopting the preparation method of the test solution of the present application, a test solution including 7 chemical components is obtained, making the detection results of the chemical component contents in the Zhishu granules more comprehensive, accurate and reliable.

[0061] The beneficial effects of the technical solution of the present invention are:

[0062] The present application provides a method for establishing a fingerprint of Zhishu Granules, which adopts high performance liquid chromatography (HPLC) and reasonably selects chromatographic conditions to achieve the establishment of the fingerprint of Zhishu Granules. The method has the advantages of simplicity, high sensitivity, fast analysis speed, and strong specificity, and can be used for the quality control of Zhishu Granules, and also provides a basis for further research on the chemical composition and quality standards of Zhishu Granules.

[0063] The present application also provides a method for establishing a control fingerprint of Zhishu granules, a control fingerprint of Zhishu granules, and use thereof in quality control. By analyzing the fingerprints of multiple batches of Zhishu granules, a control fingerprint of Zhishu granules can be established more accurately, so that the quality of Zhishu granules can be evaluated more accurately and reliably. By comparing the similarity between the Zhishu granules to be tested and the established control fingerprint of Zhishu granules, it is possible to accurately, efficiently, and simply determine whether the quality of the Zhishu granules to be tested is qualified.

[0064] The present application further provides a method for determining the content of chemical components in Zhishu granules, using the same chromatographic conditions as in the method for establishing the fingerprint of Zhishu granules to achieve the determination of the content of at least 7 chemical components in Zhishu granules. The present invention introduces a multi-wavelength method for determining Zhishu granules for the first time, and for the first time realizes the simultaneous determination of naringin, naringin, hesperidin, neohesperidin, naringenin, nobiletin, and atractylodes lactone III in Zhishu granules. The method is simple to operate, rapid, highly specific, and has excellent stability, reproducibility, and accuracy. It is conducive to the content determination and quality control of Zhishu granules of the present invention, and simultaneously realizes the quality monitoring of the three-flavor prescription medicine in the preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 It is the chromatogram of the mixed reference substance in Example 1, wherein the chemical components corresponding to the serial numbers are: peak 10 is naringin, peak 11 is nuciferine, peak 12 is naringin, peak 13 is hesperidin, peak 14 is neohesperidin, peak 18 is naringenin, peak 19 is hesperetin, peak 22 is nobiletin, peak 23 is hesperetin, peak 24 is atractylodes lactone III, and peak 25 is atractylodes lactone II.

[0066] Figure 2 It is a superimposed spectrum of the fingerprints of the Zhishu Granules numbered S1-S24 in Examples 1-24 and the reference fingerprint R of the Zhishu Granules in Example 25;

[0067] Figure 3It is the control fingerprint spectrum R of the Zhishu granules in Example 25; among the chemical components corresponding to each serial number, the known chemical components are: peak 10 is naringin, peak 11 is nuciferine, peak 12 is naringin, peak 13 is hesperidin, peak 14 is neohesperidin, peak 18 is naringenin, peak 19 is hesperidin, peak 22 is nobiletin, peak 23 is hesperidin, peak 24 is atractylodes lactone III, and peak 25 is atractylodes lactone II.

[0068] Figure 4 It is the chromatogram of the mixed reference solution in Example 26; wherein the chemical components corresponding to the serial numbers are: peak 10 is naringin, peak 12 is naringin, peak 13 is hesperidin, peak 14 is neohesperidin, peak 18 is naringenin, peak 22 is nobiletin, and peak 24 is atractylodes lactone III.

[0069] Figure 5 This is the chromatogram of the test solution of the Zhishu Granules numbered S1 in Example 26. DETAILED DESCRIPTION

[0070] The instruments, reagents and materials used in this application are described below.

[0071] Instruments: Shimadzu LC-40D liquid chromatograph; RODI-220A1 pure water machine (Rui Sijie Water Purification Technology Co., Ltd.); ML104 / 02 1 / 10,000 electronic balance, 1 / 10,000 electronic balance (Mettler-Toledo, Switzerland); KH-500E ultrasonic cleaner (Kunshan Hechuang Ultrasonic Instrument Co., Ltd.); Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition): State Pharmacopoeia Committee.

[0072] Reagents: acetonitrile chromatography grade (batch number: 22075301; manufacturer: Anhui Tiandi High Purity Solvent Co., Ltd.); phosphoric acid chromatography grade (batch number: C14276009; manufacturer: Shanghai McLean Biochemical Technology Co., Ltd.); methanol analytical grade (batch number: 20220624; manufacturer: Sinopharm Chemical Reagent Co., Ltd.); the experimental water was RODI-220A1 ultrapure water.

[0073] Test drugs: reference substances: naringin (purity 99.27%, Chengdu Maiske Pharmaceutical Technology Co., Ltd.), nuciferine (purity 99.8%, China Food and Drug Control Institute), naringin (purity 93.4%, China Food and Drug Control Institute), hesperidin (purity 95.3%, China Food and Drug Control Institute), neohesperidin (purity 99.4%, China Food and Drug Control Institute), naringenin (purity 98%, Shanghai Ronghe Pharmaceutical Technology Co., Ltd.), hesperidin (purity 98%, Dester Biological), nobiletin (purity 100%, China Food and Drug Control Institute), hesperidin (purity 98%, Shanghai Ronghe Pharmaceutical Technology Co., Ltd.), atractylodes lactone III (purity 99.9%, China Food and Drug Control Institute), atractylodes lactone II (purity 99.9%, China Food and Drug Control Institute). The 24 batches of Zhishu granules samples were from Nanjing Zhongshan Pharmaceutical Co., Ltd., and all of them have passed the inspection. The sample information is shown in Table 1.

[0074] Table 1 Information of Zhishu granules samples

[0075]

[0076] Example 1

[0077] Grind the contents of the Zhishu granules numbered S1, accurately weigh 1 g of mass M1, accurately add 10 mL of 100% methanol solution, seal, weigh, and ultrasonicate for 30 min with an extraction power of 180 W and an extraction frequency of 40 kHz; cool, weigh again, make up the lost amount with methanol solution, shake well, filter through a 0.22 μm microporous filter membrane, and take the filtrate to obtain.

[0078] Accurately weigh naringin, nuciferine, naringin, hesperidin, neohesperidin, naringenin, hesperidin, nobiletin, tangeretin, atractylodes lactone III, and atractylodes lactone II reference substances, and add 100% methanol to prepare 49.54 μg / mL, 51.35 μg / mL, 102.07 μg / mL, 50.45 μg / mL, 102.76 μg / mL, 49.92 μg / mL, 50.50 μg / mL, 51.52 μg / mL, 51.98 μg / mL, 51.52 μg / mL, and 50.02 μg / mL mixed reference substance solutions.

[0079] The mixed reference solution and the test solution were detected by high performance liquid chromatography to obtain a mixed reference solution chromatogram ( Figure 1 ) and the chromatogram of Zhishu granules numbered S1 ( Figure 2 ), wherein the chromatographic conditions include:

[0080] Chromatographic column: Waters Symmetry C18 (4.6*250mm, 5μm);

[0081] Mobile phase: 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B),

[0082] Elution gradient: 0-7min, 90%-85%A; 7-20min, 85%-82%A; 20-30min, 82%-81%A; 30-45min, 81%-6 5%A; 45-55min, 65%-40%A; 55-65min, 40%-25%A; 65-70min, 25%-90%A; 70-80min, 90% A;

[0083] Flow rate: 1.0 mL / min;

[0084] Column temperature: 30°C;

[0085] The detection wavelength fingerprint spectrum is 220nm,

[0086] Injection volume: 10 μL.

[0087] The chromatogram of Zhishu Granules No. S1 was introduced into the similarity evaluation system of Chinese medicine chromatographic fingerprints to establish the fingerprint of Zhishu Granules No. S1, such as Figure 2 As shown in S1.

[0088] Example 2-24

[0089] Except that the Zhishu granules numbered S2 to S24 were used to replace the Zhishu granules numbered S1 in Example 1, the rest was the same as Example 1, and the fingerprints of the Zhishu granules numbered S2 to S24 were obtained respectively; wherein the fingerprints of the Zhishu granules numbered S2 to S24 were respectively as follows: Figure 2 As shown in S2-S24 in .

[0090] Embodiment 25

[0091] (1) The fingerprints of the Zhishu Granules No. S1-S24 obtained in Examples 1-24 were analyzed using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, the fingerprint of the Zhishu Granule No. S20 was set as a reference, the time window width was set to 0.1 min, and the average method was used to perform multi-point correction and automatic matching of the chromatographic peaks to obtain a control fingerprint of the Zhishu Granules, and the similarity was calculated; the control fingerprint of the Zhishu Granules obtained was as follows: Figure 2 and Figure 3 As shown in R.

[0092] After analysis, 25 common peaks were identified, that is, Zhishu Granules included 25 common chromatographic peaks, which were numbered in sequence. Among them, 11 chromatographic peaks were identified by comparison with the reference substance, among which peak No. 10 was naringin with a retention time of 31.796 min, peak No. 11 was nuciferine with a retention time of 32.878 min, peak No. 12 was naringin with a retention time of 35,722 min, peak No. 13 was hesperidin with a retention time of 37.937 min, peak No. 14 was neohesperidin with a retention time of 40.877 min, peak No. 18 was naringenin with a retention time of 51.926 min, and peak No. 19 was naringenin with a retention time of 52. Peak No. 21 is hesperidin with a retention time of 53.147 min, Peak No. 22 is nobiletin with a retention time of 57.817 min, Peak No. 23 is hesperidin with a retention time of 60.025 min, Peak No. 24 is atractylodes lactone III with a retention time of 60.482 min, and Peak No. 25 is atractylodes lactone II with a retention time of 65.342 min. The similarity evaluation results are shown in Table 2, and it can be seen that the similarities are all above 0.90, indicating that the established material benchmark of the fingerprint of Zhishu granules has good stability and can reflect its fingerprint characteristics. The fingerprints of each batch of Zhishu granules have high consistency.

[0093] Table 2 Similarity results

[0094]

[0095]

[0096]

[0097]

[0098] (2) Methodological investigation of the establishment of the fingerprint of Zhishu granules

[0099] (2-1) Precision test

[0100] Take the Zhishu granules numbered S6, prepare the test solution according to the method of Example 1, continuously inject 6 times according to the chromatographic conditions of Example 1, and record the HPLC chromatogram; introduce the obtained chromatograms into the Chinese medicine chromatographic fingerprint similarity evaluation system, and the measured similarities are all 1.000, indicating that the instrument has good precision.

[0101] (2-2) Stability test

[0102] Take the Zhishu granules numbered S6, prepare the test solution according to the method of Example 1, and according to the chromatographic conditions of Example 1, sample injection and detection are performed at 0, 2, 4, 6, 8, 10, 12, and 24 h, and the HPLC chromatograms are recorded; each chromatogram obtained is introduced into the Chinese medicine chromatographic fingerprint similarity evaluation system, and the measured similarities are all 1.000, indicating that the test solution is stable within 24 h.

[0103] (2-3) Repeatability test

[0104] Take the Zhishu granules numbered S6, and prepare 6 test solutions in parallel according to the method of Example 1. The samples were injected and tested according to the chromatographic conditions of Example 1, and the HPLC chromatograms were recorded; the obtained chromatograms were introduced into the Chinese medicine chromatographic fingerprint similarity evaluation system, and the measured similarities were all 1.000, indicating that the method had good repeatability.

[0105] Embodiment 26

[0106] Z1. Detection wavelength

[0107] Scan at full wavelength of 190-900nm, with a wavelength of 220nm for detecting atractylodes lactone III; a wavelength of 280nm for detecting naringin, naringin, hesperidin, neohesperidin, and naringenin; and a wavelength of 332nm for detecting nobiletin;

[0108] Z2. Establish standard curves for each chemical component

[0109] Accurately weigh 4, 30, 2, and 25 mg of naringin, naringin, hesperidin, and neohesperidin reference substances into a 10 ml volumetric flask 1; accurately weigh 1.5, 2, and 1.5 mg of naringenin, nobiletin, and atractylodes lactone III into a 10 ml volumetric flask 2, and dilute to volume with methanol; take 2 ml of the solution in volumetric flask 2 into volumetric flask 1, and dilute to volume with methanol to obtain a mixed reference substance stock solution. Take 0.3, 0.5, 1.0, 1.5, 2.0, and 2.5 of the mixed reference substance stock solution, dilute to a 5 ml volumetric flask with methanol, and obtain 7 mixed reference substance solutions containing different known concentrations of each chemical component, including the mixed reference substance stock solution, as standard curve solutions.

[0110] The concentration range of naringin in standard curve solution 1 is 0.0240 mg / ml, naringin is 0.1807 mg / ml, hesperidin is 0.0121 mg / ml, neohesperidin is 0.1502 mg / ml, naringenin is 0.0020 mg / ml, nobiletin is 0.0027 mg / ml, and atractylodes lactone III is 0.0019 mg / ml;

[0111] The concentration range of naringin in standard curve solution 2 was 0.0399 mg / ml, naringin was 0.3012 mg / ml, hesperidin was 0.0201 mg / ml, neohesperidin was 0.2503 mg / ml, naringenin was 0.0033 mg / ml, nobiletin was 0.0044 mg / ml, and atractylodes lactone III was 0.0031 mg / ml;

[0112] In the standard curve solution 3, the concentration range of naringin was 0.0799 mg / ml, the concentration range of naringin was 0.6025 mg / ml, the concentration range of hesperidin was 0.0402 mg / ml, the concentration range of neohesperidin was 0.5006 mg / ml, the concentration range of naringenin was 0.0065 mg / ml, the concentration range of nobiletin was 0.0088 mg / ml, and the concentration range of atractylodes lactone III was 0.0063 mg / ml;

[0113] In the standard curve solution 4, the concentration range of naringin was 0.1198 mg / ml, the concentration range of naringin was 0.9037 mg / ml, the concentration range of hesperidin was 0.0603 mg / ml, the concentration range of neohesperidin was 0.7510 mg / ml, the concentration range of naringenin was 0.0098 mg / ml, the concentration range of nobiletin was 0.0133 mg / ml, and the concentration range of atractylodes lactone III was 0.0094 mg / ml;

[0114] In the standard curve solution 5, the concentration range of naringin was 0.1597 mg / ml, the concentration range of naringin was 1.2049 mg / ml, the concentration range of hesperidin was 0.0804 mg / ml, the concentration range of neohesperidin was 1.0013 mg / ml, the concentration range of naringenin was 0.0130 mg / ml, the concentration range of nobiletin was 0.0177 mg / ml, and the concentration range of atractylodes lactone III was 0.0125 mg / ml;

[0115] In the standard curve solution 6, the concentration range of naringin was 0.1997 mg / ml, the concentration range of naringin was 1.5062 mg / ml, the concentration range of hesperidin was 0.1006 mg / ml, the concentration range of neohesperidin was 1.2516 mg / ml, the concentration range of naringenin was 0.0163 mg / ml, the concentration range of nobiletin was 0.0221 mg / ml, and the concentration range of atractylodes lactone III was 0.0157 mg / ml;

[0116] The concentration range of naringin in standard curve solution 7 (i.e., mixed reference stock solution) was 0.3993 mg / ml, naringin was 3.0123 mg / ml, hesperidin was 0.2011 mg / ml, neohesperidin was 2.5032 mg / ml, naringenin was 0.0325 mg / ml, nobiletin was 0.0442 mg / ml, and atractylodes lactone III was 0.0313 mg / ml;

[0117] Under the same chromatographic conditions, each mixed reference solution is injected into a high performance liquid chromatograph, the chromatographic peak of each chemical component is determined according to the retention time of each chemical component, and the chromatographic peak area of ​​each chemical component is obtained;

[0118] Wherein, the chromatographic conditions include:

[0119] Chromatographic column: Waters Symmetry C18 (4.6*250mm, 5μm);

[0120] Mobile phase: 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B),

[0121] Elution gradient: 0-7min, 90%-85%A; 7-20min, 85%-82%A; 20-30min, 82%-81%A; 30-45min, 81%-6 5%A; 45-55min, 65%-40%A; 55-65min, 40%-25%A; 65-70min, 25%-90%A; 70-80min, 90% A;

[0122] Flow rate: 1.0 mL / min;

[0123] Column temperature: 30°C;

[0124] The detection wavelength fingerprint is 220nm, 280nm, 332nm,

[0125] Injection volume: 10 μL.

[0126] Regression analysis was performed with the peak area (Y) of the chromatographic peak of each chemical component as the ordinate and the concentration (X) of each chemical component as the abscissa, and the standard curve of each chemical component was established to obtain the linear regression equation and correlation coefficient r of each chemical component. The standard curve results of the 7 chemical components are shown in Table 3. The results showed that the correlation coefficient r of the 7 chemical components within their respective linear ranges was ≥ 0.999, indicating that the 7 chemical components had a good linear relationship within their linear ranges.

[0127] Table 3 Standard curves of 7 chemical components

[0128]

[0129] Z3. Obtain the chromatographic peak area of ​​each chemical component in the Zhishu granules to be tested:

[0130] Grind the contents of Zhishu Granule No. S1, accurately weigh 1g of M1, accurately add 10mL of methanol solution, seal, weigh, ultrasonicate for 30min, extract with 180W power and 40kHz frequency; cool, weigh again, add methanol solution to make up the lost amount, shake well, filter with 0.22μm microporous membrane, and take the filtrate. Repeat to prepare 3 portions of the test solution of Zhishu Granule No. S1.

[0131] Under the same chromatographic conditions as step Z2, the test solution is injected into a high performance liquid chromatograph to obtain a chromatogram of the Zhishu granules, the chromatographic peaks of each chemical component are determined according to the retention time of each chemical component, and the chromatographic peak area of ​​each chemical component in the Zhishu granules numbered S1 is obtained;

[0132] Z4. Determine the content of each chemical component in the Zhishu granules to be tested:

[0133] According to the standard curves of each chemical component established by Z2, the concentration C1 of each chemical component was obtained from the chromatographic peak area of ​​each chemical component in the Zhishu Granules No. S1, and the content C of each chemical component in the Zhishu Granules No. S1 was calculated according to the formula C=C1×V1 / M1=C1×10mL / 1g. The results are shown in Table 4, and the unit of the content of each chemical component in the Zhishu Granules is expressed in mg / g.

[0134] Examples 27-49

[0135] Except that the Zhishu granules numbered S2-S24 were used to replace the Zhishu granules numbered S1 in Example 26, the rest was the same as Example 26. The contents of each chemical component in the Zhishu granules numbered S2-S24 were obtained, and the results are shown in Table 4.

[0136] Table 4 Content of each chemical component in Zhishu granules No. S1-S24 (mg / g)

[0137]

[0138] Example 50 Methodological Investigation of the Determination Method of Chemical Components in Zhishu Granules

[0139] (1) Specificity inspection

[0140] Take the fourth mixed reference solution (i.e., standard curve solution 4) in Example 26, and obtain its chromatogram as shown in the following figure: Figure 4 As shown; Take the test solution of Zhishu granules numbered S1 in Example 26, and obtain its chromatogram as shown in Figure 5 shown; according to Figure 4 and Figure 5 It can be seen that at the corresponding positions of the reference substances of naringin, naringin, hesperidin, neohesperidin, naringenin, nobiletin, and atractylodes lactone III, chromatographic peaks with the same retention time in the test solution appeared, and the separation of each chemical component was good, indicating that the method has good specificity.

[0141] (2) Precision test

[0142] The fourth mixed reference solution in Example 26 (i.e., standard curve solution 4) was taken, and the sample was continuously injected and measured 6 times according to the chromatographic conditions in Example 26. The HPLC chromatogram was recorded. The retention time RSD values ​​of naringin, naringin, hesperidin, neohesperidin, naringenin, nobiletin, and atractylodes lactone III were calculated to be 0.05%, 0.05%, 0.03%, 0.02%, 0.01%, 0.01%, and 0.01%, respectively, and the peak area RSD values ​​were 0.24%, 0.24%, 0.33%, 0.25%, 0.24%, 0.24%, and 0.11%, respectively. The results showed that the instrument had good precision.

[0143] (3) Stability test

[0144] Take the Zhishu granules numbered S6, prepare a test solution according to the method of Example 26, and according to the chromatographic conditions of Example 26, sample injection and detection are performed at 0, 2, 4, 6, 8, 10, 12, and 24 h, respectively, and record the HPLC chromatogram. The retention time RSD values ​​of naringin, naringin, hesperidin, neohesperidin, naringenin, nobiletin, and atractylodes lactone III are calculated to be 0.03%, 0.04%, 0.02%, 0.02%, 0.01%, 0.01%, and 0.01%, respectively, and the peak area RSD values ​​are 4.70%, 1.04%, 1.10%, 1.10%, 0.94%, 0.91%, and 0.80%, respectively, indicating that the test solution is stable within 24 h.

[0145] (4) Repeatability

[0146] Take the Zhishu granules numbered S6, and prepare 6 test solutions in parallel according to the method of Example 26. The samples were injected and measured according to the chromatographic conditions of Example 26, and the HPLC chromatogram was recorded. The RSD values ​​of naringin, naringin, hesperidin, neohesperidin, naringenin, nobiletin, and atractylodes lactone III were calculated to be 2.38%, 2.42%, 2.38%, 2.48%, 2.55%, 2.18%, and 2.10%, respectively. The results showed that the method had good repeatability.

[0147] (5) Sample recovery rate

[0148] 9 portions of Zhishu granules numbered S6 with known contents of 7 chemical components were accurately weighed, and mixed reference substances or mixed reference substance solutions containing 80%, 100%, and 120% of the 7 chemical components were accurately added, and spiked test solutions were prepared in parallel (n=3) according to the method in Example 26, and the contents of each chemical component were calculated according to the chromatographic conditions in Example 26; wherein, the original amount, added amount, and measured amount of each chemical component were respectively shown in Table 5. According to the formula: sample recovery rate (%) = (measured amount - original amount) / added amount × 100%, the sample recovery rate of each chemical component was calculated, and the average recovery rate and RSD value were calculated. The results are shown in Table 5, and the average recovery rates of naringin, naringin, hesperidin, neohesperidin, naringenin, nobiletin, and atractylodes lactone III were 87.6%-114.3%, and the RSD value was 0.66%-3.21%. The results show that the method has good accuracy.

[0149] Table 5

[0150]

[0151]

[0152]

[0153] In summary, the method for establishing the fingerprint of Zhishu Granules and the method for determining the content of its components provided in this application adopt the same chromatographic conditions to achieve the establishment of the fingerprint of Zhishu Granules, and can determine the contents of at least 7 chemical components in Zhishu Granules. The method is accurate, has good repeatability and specificity, and has the advantages of simplicity, high efficiency, high sensitivity, fast analysis speed, strong specificity, etc. It can comprehensively, accurately and reliably evaluate the quality of Zhishu Granules, and provides a good reference basis for the material basis of the efficacy of Zhishu Granules and comprehensive quality control.

[0154] The above description is only a preferred embodiment of the present application and is not intended to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application are included in the protection scope of the present application.

Claims

1. A method for establishing a fingerprint of Zhishu granules, characterized in that: The method comprises: (1) Preparation of Zhishu Granule Test Solution: Take Zhishu Granule with a mass of M1, extract it with a volume of V1 and a volume fraction of 50-100% methanol as a solvent to obtain a test solution; wherein M1:V1=1g:(10-50)mL; (2) Preparation of mixed reference solution: accurately weigh naringin, nuciferine, naringin, hesperidin, neohesperidin, naringenin, hesperidin, nobiletin, tangeretin, atractylodes lactone III, and atractylodes lactone II reference substances, place them in a volumetric flask, dilute to the mark with 50-100% methanol, and shake well to prepare a reference solution; (3) using high performance liquid chromatography to detect the test solution to obtain a chromatogram of the Zhishu granules; Chromatographic conditions included: Chromatographic column: octadecylsilane bonded silica gel chromatographic column; Mobile phase: Phase A is 0.05-0.15% phosphoric acid aqueous solution, and phase B is acetonitrile; Elution procedure: gradient elution with a volume fraction of 25-90% phase A and 10-75% phase B; Flow rate: 0.8-1.2mL / min; Column temperature: 30-40°C; Injection volume: 10-20 μL; Detection wavelength: 220±5nm; (4) analyzing the chromatogram of the Zhishu Granules obtained in step (3) using a traditional Chinese medicine chromatographic fingerprint similarity evaluation software to obtain a fingerprint of the Zhishu Granules; The elution procedure in step (3) is:

2. The establishment method according to claim 1, characterized in that: In step (1), the extraction is ultrasonic extraction, the extraction time is 20-60 min, the extraction power is 150-200 W, and the extraction frequency is 30-50 kHz.

3. The establishment method according to claim 1, characterized in that: Step (2) Preparation of mixed reference solution: Accurately weigh reference substances of naringin, nuciferine, naringin, hesperidin, neohesperidin, naringenin, hesperidin, nobiletin, tangeretin, atractylodes lactone III, and atractylodes lactone II, and add 100% methanol to prepare a reference solution containing 50±5 μg / mL of naringin, 50±5 μg / mL of nuciferine, 100±5 μg / mL of naringin, 50±5 μg / mL of hesperidin, 100±5 μg / mL of neohesperidin, 50±5 μg / mL of naringenin, 50±5 μg / mL of hesperidin, 50±5 μg / mL of nobiletin, 50±5 μg / mL of tangeretin, 50±5 μg / mL of atractylodes lactone III, and 50±5 μg / mL of atractylodes lactone II.

4. The establishment method according to claim 3, characterized in that: A reference solution containing 50 μg / mL of naringin, 50 μg / mL of nuciferine, 100 μg / mL of naringin, 50 μg / mL of hesperidin, 100 μg / mL of neohesperidin, 50 μg / mL of naringenin, 50 μg / mL of hesperidin, 50 μg / mL of nobiletin, 50 μg / mL of tangeretin, 50 μg / mL of atractylodes lactone III, and 50 μg / mL of atractylodes lactone II was prepared.

5. A method for establishing a comparative fingerprint of Zhishu granules, characterized in that: The method comprises: Take R batches of Zhishu Granules, and obtain the fingerprints of each batch of Zhishu Granules according to the method for establishing the fingerprint of Zhishu Granules according to any one of claims 1 to 4; analyze the fingerprints of the R batches of Zhishu Granules using the Chinese medicine chromatographic fingerprint similarity evaluation software to obtain a control fingerprint of Zhishu Granules; the R is ≥15.

6. The establishment method according to claim 5, characterized in that: The control fingerprint of Zhishu granules obtained by the method includes 25 common peaks, and 11 chromatographic peaks are identified, wherein peak 10 is naringin with a retention time of 31.796 min, peak 11 is nuciferine with a retention time of 32.878 min, peak 12 is naringin with a retention time of 35.722 min, peak 13 is hesperidin with a retention time of 37.937 min, peak 14 is neohesperidin with a retention time of 40.8 77min, Peak 18 is naringenin with a retention time of 51.926min, Peak 19 is hesperetin with a retention time of 53.147min, Peak 22 is nobiletin with a retention time of 57.817min, Peak 23 is tangeretin with a retention time of 60.025min, Peak 24 is atractylodes lactone III with a retention time of 60.482min, Peak 25 is atractylodes lactone II with a retention time of 65.342min.

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