Method for establishing fingerprint spectrum of aqueous extract in Xinnaoning capsule

Abstract

The invention belongs to the technical field of quality detection of traditional Chinese medicine preparations, and provides an establishment method and application of a fingerprint spectrum of a Xinnaoning capsule aqueous extract. The method comprises the following steps: preparing Xinnaoning capsule test sample solutions, collecting an HPLC (High Performance Liquid Chromatography) chromatogram of each test sample solution by adopting a high performance liquid chromatography, and carrying out similarity evaluation on the obtained chromatograms. The standard fingerprint spectrum of the Xinnaoning capsule HPLC aqueous extract obtained by the method has the characteristics of high similarity, small spectrum difference and high accuracy, meets the analysis requirements of the fingerprint spectrum, and reflects the integrity and uniformity of a sample. Therefore, the aqueous extract fingerprint spectrum established by the invention is stable and reliable, has important significance on component analysis and identification and quality control of the Xinnaoning capsule preparation, and provides technical support for realizing more comprehensive and effective product quality control.

Description

Technical Field

[0001] The present invention relates to the technical field of quality detection of traditional Chinese medicine preparations, and in particular to a method for establishing a fingerprint spectrum of a water extract of Xinnaoning capsules. Background Art

[0002] Xinnaoning Capsule is a product of Guizhou Jingcheng Pharmaceutical Co., Ltd. and is included in the 2020 edition of the "Chinese Pharmacopoeia". It is composed of ginkgo leaves, boxwood, salvia miltiorrhiza, Litsea cubeba and Allium macrostemon. It has the effects of promoting blood circulation, promoting qi, unblocking meridians and relieving pain. It is clinically used for chest pain, headache, and dizziness caused by qi stagnation and blood stasis, with symptoms such as chest tightness and stabbing pain, palpitations, dizziness, etc., as well as patients with coronary heart disease and cerebral arteriosclerosis with the above symptoms.

[0003] A traditional Chinese medicine (TCM) fingerprint refers to a chromatogram or spectrum that characterizes the chemical properties of certain TCM materials or preparations after appropriate processing and analytical methods. It comprehensively reflects the types and quantities of chemical constituents within the medicinal material, effectively demonstrating the integrity and combined effects of TCM components. Its rapidity and accuracy have led to its widespread application in TCM analysis, identification, and quality control. This facilitates the establishment of more precise, comprehensive, and batch-by-batch quality control methods for commercially available TCMs. Currently, no studies have examined the fingerprint of the aqueous extract of Xinnaoning Capsules. Therefore, establishing a fingerprint for the aqueous extract of Xinnaoning Capsules is crucial for component analysis, identification, and quality control. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for establishing a fingerprint of a water extract in Xinnaoning capsule.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The method for establishing the fingerprint of the water extract of Xinnaoning Capsule of the present invention comprises the following steps:

[0007] S1. Preparation of Xinnaoning Capsule test solution:

[0008] S11. Take the contents of Xinnaoning capsule, add 5-20 times the amount of water, weigh, heat and reflux to extract for 30-120 minutes, make up the weight with water, shake well, centrifuge, and take the supernatant to obtain Xinnaoning aqueous extract;

[0009] S12. Accurately pipette 200 μl of the Xinnaoning aqueous extract into a stoppered centrifuge tube, add 200 μl of 0.3 mol / L NaOH, mix thoroughly, add 160 μl of 0.5 mol / L PMP methanol solution, mix thoroughly, and heat in a 70°C water bath for 30 min. Cool to room temperature, neutralize the alkali solution with 200 μl of 0.3 mol / L HCl solution, add 1240 μl of purified water and 2 ml of chloroform, shake well, centrifuge for 5 min, discard the chloroform layer, and repeat the extraction three times; filter the aqueous layer through a 0.45 μm filter membrane, and use the filtrate as the Xinnaoning capsule test solution;

[0010] S2. Chromatographic conditions:

[0011] The chromatographic column was Kromasil C18, 4.6 × 250 mm, 5 μm; mobile phase A was 0.1 mol / L potassium dihydrogen phosphate buffer, mobile phase B was acetonitrile, gradient elution, flow rate: 0.8 ml / min; column temperature: 35°C; injection volume: 10 μl; detection wavelength: 250 nm; gradient elution program:

[0012]

[0013] S3. Detection:

[0014] According to the above chromatographic conditions, 10 μl of the above test solution was accurately aspirated and tested by HPLC to obtain a chromatogram of the Xinnaoning Capsule test solution.

[0015] Preferably, the amount of water added in step S11 of the present invention is 5-10 times.

[0016] Further preferably, the amount of water added in step S11 of the present invention is 10 times.

[0017] Preferably, the extraction time in step S11 of the present invention is 90-120 minutes.

[0018] Further preferably, the extraction time in step S11 of the present invention is 90 minutes.

[0019] The heating reflux extraction method in step S11 of the present invention can also be replaced by ultrasonic extraction.

[0020] The potassium dihydrogen phosphate buffer in step S2 of the present invention needs to be adjusted to pH 6.7 using 40% NaOH.

[0021] The method for establishing the fingerprint of the water extract of Xinnaoning Capsule of the present invention also includes: importing the obtained chromatogram of the Xinnaoning Capsule test solution into the traditional Chinese medicine fingerprint similarity evaluation software, using the latest batch of samples as the reference spectrum, selecting the average method, setting the time window width to 0.2-0.5, multi-point correction, and full spectrum peak matching to generate the reference fingerprint of the Xinnaoning Capsule substance benchmark water extract, and using the produced control fingerprint as the benchmark to calculate the similarity between the fingerprint of each batch of Xinnaoning Capsule substance benchmark water extract and the control spectrum, and the similarity is not less than 0.90.

[0022] The invention relates to an application of the fingerprint of the water extract of Xinnaoning Capsules obtained by the method for establishing the fingerprint of the water extract of Xinnaoning Capsules in characterizing the components of the water extract of Xinnaoning Capsules or detecting the quality of the water extract of Xinnaoning Capsules.

[0023] The invention relates to an application of the fingerprint of the water extract of Xinnaoning Capsules obtained by the method for establishing the fingerprint of the water extract of Xinnaoning Capsules in the quality control of Xinnaoning Capsules.

[0024] Beneficial effects of the present invention:

[0025] The present invention provides the construction of a method for determining the fingerprint spectrum of the components of the water extract of Xinnaoning Capsules, which has the advantages of high stability, high precision and good repeatability. The water extract of Xinnaoning Capsules was evaluated for similarity, and the samples showed high similarity and small differences in the spectra. This shows that the quality of the water extracts of each batch of Xinnaoning Capsules is relatively stable, and the overall characteristics of the components of the water extracts are slightly different, which meets the analysis requirements of the fingerprint spectrum and reflects the integrity and uniformity of the sample. This shows that the fingerprint spectrum of the water extract established by the present invention is stable and reliable, which is of great significance to the analysis and identification of the components and quality control of the Xinnaoning Capsules preparation, and provides technical support for achieving more comprehensive and effective control of product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 :Fingerprint of Xinnaoning Capsule water extract (ultrasonic extraction)

[0027] Figure 2 :Fingerprint of Xinnaoning Capsule Water Extract (Heating Reflux Extraction)

[0028] Figure 3 :Overlay of fingerprints of Xinnaoning capsule water extract (extraction method investigation) (from bottom to top: heating reflux extraction, ultrasonic extraction)

[0029] Figure 4 :Fingerprint of Xinnaoning Capsule water extract (5 times solvent)

[0030] Figure 5 :Fingerprint of Xinnaoning Capsule water extract (10 times solvent)

[0031] Figure 6:Fingerprint of Xinnaoning Capsule water extract (20 times solvent)

[0032] Figure 7 :Superposition of fingerprints of Xinnaoning capsule water extract (investigation of extraction solvent multiples) (from bottom to top: 5 times solvent, 10 times solvent, 20 times solvent)

[0033] Figure 8 :Fingerprint of Xinnaoning Capsule water extract (extraction 30min)

[0034] Figure 9 :Fingerprint of Xinnaoning Capsule water extract (extraction 60min)

[0035] Figure 10 :Fingerprint of Xinnaoning Capsule water extract (extraction 90min)

[0036] Figure 11 :Fingerprint of Xinnaoning Capsule water extract (extraction 120min)

[0037] Figure 12 :Superposition of fingerprints of Xinnaoning capsule water extract (extraction time investigation) (from bottom to top: 30min, 60min, 90min, 120min)

[0038] Figure 13 : Fingerprint of Xinnaoning Capsule water extract under the optimal determination method

[0039] Figure 14 :Precision investigation (from bottom to top: precision 1 to precision 6)

[0040] Figure 15 :Repeatability investigation (from bottom to top: repeatability 1 to repeatability 6)

[0041] Figure 16 :Stability investigation (from bottom to top: stability 1 to stability 6)

[0042] Figure 17 :HPLC fingerprint comparison of water extract (26 batches)

[0043] Figure 18 :Overlay of HPLC fingerprints of water extracts (26 batches)

[0044] Figure 19 :Comparison of HPLC fingerprints of water extracts (2017 batch)

[0045] Figure 20 : Overlay of HPLC fingerprints of water extracts (2017 batch)

[0046] Figure 21 :Comparison of HPLC fingerprints of water extracts (2018)

[0047] Figure 22 :Overlay of HPLC fingerprints of water extracts (2018 batch)

[0048] Figure 23 :Comparison of HPLC fingerprints of water extracts (2019)

[0049] Figure 24 :Overlay of HPLC fingerprints of water extracts (2019 batch)

[0050] Figure 25 :Overlay of HPLC fingerprints of water extract (4-year batch)

[0051] Figure 26 : Overlay of HPLC fingerprints of water extracts (4 years) (S1: Xinnaoning 20160916; S2-S4: control spectra of 2017, 2018, and 2019 respectively) DETAILED DESCRIPTION

[0052] The following is a detailed description of the technical solution of the present invention in conjunction with specific embodiments. The following embodiments are only for explanation and illustration, and do not constitute a limitation of the technical solution of the present invention.

[0053] Example 1 Establishment of a method for determining the fingerprint of the water extract of Xinnaoning capsule

[0054] S1. Preparation of Xinnaoning Capsule test solution:

[0055] S11. Accurately weigh 1 g of the contents of Xinnaoning capsules, add 10 ml of water, weigh, heat under reflux for 90 min, make up the weight, shake well, centrifuge, and collect the supernatant to obtain the Xinnaoning aqueous extract;

[0056] S12. Accurately pipette 200 μl of the Xinnaoning aqueous extract into a stoppered centrifuge tube, add 200 μl of 0.3 mol / L NaOH, mix thoroughly, add 160 μl of 0.5 mol / L PMP methanol solution, mix thoroughly, and heat in a 70°C water bath for 30 min. Cool to room temperature, neutralize the alkali solution with 200 μl of 0.3 mol / L HCl solution, add 1240 μl of purified water, add 2 ml of chloroform, shake well, centrifuge at 3500 rpm for 5 min, discard the chloroform layer, and repeat the extraction three times; filter the aqueous layer through a 0.45 μm filter membrane, and take the filtrate as the Xinnaoning capsule test solution;

[0057] S2. Chromatographic conditions:

[0058] The chromatographic column was a Kromasil C18 column, 4.6 × 250 mm, 5 μm; mobile phase A was 0.1 mol / L potassium dihydrogen phosphate buffer (adjusted to pH 6.7 with 40% NaOH), and mobile phase B was acetonitrile. Gradient elution was performed at a flow rate of 0.8 ml / min. The column temperature was 35°C; the injection volume was 10 μl; and the detection wavelength was 250 nm. The gradient elution program was as follows:

[0059]

[0060] S3. Detection:

[0061] According to the above chromatographic conditions, 10 μl of the above test solution was accurately aspirated and detected by HPLC to obtain the chromatogram of Xinnaoning Capsules.

[0062] Example 2 Establishment of the fingerprint determination method of Xinnaoning capsule water extract

[0063] S1. Preparation of Xinnaoning Capsule test solution:

[0064] S11. Accurately weigh 2 g of the contents of Xinnaoning capsules, add 10 ml of water, weigh, heat under reflux for 60 min, make up the weight, shake well, centrifuge, and collect the supernatant to obtain the Xinnaoning aqueous extract;

[0065] S12. Accurately pipette 200 μl of the Xinnaoning aqueous extract into a stoppered centrifuge tube, add 200 μl of 0.3 mol / L NaOH, mix thoroughly, add 160 μl of 0.5 mol / L PMP methanol solution, mix thoroughly, and heat in a 70°C water bath for 30 min. Cool to room temperature, neutralize the alkali solution with 200 μl of 0.3 mol / L HCl solution, add 1240 μl of purified water, add 2 ml of chloroform, shake well, centrifuge at 3500 rpm for 5 min, discard the chloroform layer, and repeat the extraction three times; filter the aqueous layer through a 0.45 μm filter membrane, and take the filtrate as the Xinnaoning capsule test solution;

[0066] S2. Chromatographic conditions:

[0067] The chromatographic column was a Kromasil C18 column, 4.6 × 250 mm, 5 μm; mobile phase A was 0.1 mol / L potassium dihydrogen phosphate buffer (adjusted to pH 6.7 with 40% NaOH), and mobile phase B was acetonitrile. Gradient elution was performed at a flow rate of 0.8 ml / min. The column temperature was 35°C; the injection volume was 10 μl; and the detection wavelength was 250 nm. The gradient elution program was as follows:

[0068]

[0069] S3. Detection:

[0070] According to the above chromatographic conditions, 10 μl of the above test solution was accurately aspirated and detected by HPLC to obtain the chromatogram of Xinnaoning Capsules.

[0071] Example 3 Establishment of the fingerprint determination method of Xinnaoning capsule water extract

[0072] S1. Preparation of Xinnaoning Capsule test solution:

[0073] S11. Accurately weigh 0.5 g of the contents of Xinnaoning capsules, add 10 ml of water, weigh, heat under reflux for 120 min, make up the weight, shake well, centrifuge, and collect the supernatant to obtain the Xinnaoning aqueous extract;

[0074] S12. Accurately pipette 200 μl of the Xinnaoning aqueous extract into a stoppered centrifuge tube, add 200 μl of 0.3 mol / L NaOH, mix thoroughly, add 160 μl of 0.5 mol / L PMP methanol solution, mix thoroughly, and heat in a 70°C water bath for 30 min. Cool to room temperature, neutralize the alkali solution with 200 μl of 0.3 mol / L HCl solution, add 1240 μl of purified water, add 2 ml of chloroform, shake well, centrifuge at 3500 rpm for 5 min, discard the chloroform layer, and repeat the extraction three times; filter the aqueous layer through a 0.45 μm filter membrane, and take the filtrate as the Xinnaoning capsule test solution;

[0075] S2. Chromatographic conditions:

[0076] The chromatographic column was a Kromasil C18 column, 4.6 × 250 mm, 5 μm; mobile phase A was 0.1 mol / L potassium dihydrogen phosphate buffer (adjusted to pH 6.7 with 40% NaOH), and mobile phase B was acetonitrile. Gradient elution was performed at a flow rate of 0.8 ml / min. The column temperature was 35°C; the injection volume was 10 μl; and the detection wavelength was 250 nm. The gradient elution program was as follows:

[0077]

[0078] S3. Detection:

[0079] According to the above chromatographic conditions, 10 μl of the above test solution was accurately aspirated and detected by HPLC to obtain the chromatogram of Xinnaoning Capsules.

[0080] Example 4 Establishment of the comparative fingerprint of Xinnaoning capsule water extract

[0081] The chromatogram of Xinnaoning Capsule obtained in Example 1 was imported into the traditional Chinese medicine fingerprint similarity evaluation software. The latest batch of samples was used as the reference spectrum. The average method was selected, the time window width was set to 0.5, multi-point correction was performed, and the full spectrum peak was matched to generate the reference fingerprint spectrum of the Xinnaoning Capsule substance benchmark water extract. The similarity between the fingerprint spectrum of each batch of Xinnaoning Capsule substance benchmark water extract and the reference spectrum was calculated based on the production control fingerprint spectrum. The similarity was not less than 0.90.

[0082] Example 5 Establishment of the comparative fingerprint of Xinnaoning capsule water extract

[0083] The chromatogram of Xinnaoning Capsule obtained in Example 2 was imported into the Chinese medicine fingerprint similarity evaluation software. The latest batch of samples was used as the reference spectrum. The average method was selected, the time window width was set to 0.4, multi-point correction was performed, and the full spectrum peak was matched to generate the reference fingerprint spectrum of the Xinnaoning Capsule substance benchmark water extract. The similarity between the fingerprint spectrum of each batch of Xinnaoning Capsule substance benchmark water extract and the reference spectrum was calculated based on the production control fingerprint spectrum. The similarity was not less than 0.90.

[0084] Example 6 Establishment of the comparative fingerprint of Xinnaoning capsule water extract

[0085] The chromatogram of Xinnaoning Capsule obtained in Example 3 was imported into the traditional Chinese medicine fingerprint similarity evaluation software, and the latest batch of samples was used as the reference spectrum. The average method was selected, the time window width was set to 0.2, multi-point correction, and full spectrum peak matching were performed to generate the monosaccharide reference fingerprint spectrum of the Xinnaoning Capsule substance benchmark water extract; the similarity between the fingerprint spectrum of each batch of Xinnaoning Capsule substance benchmark water extract and the reference spectrum was calculated based on the production control fingerprint spectrum, and the similarity was not less than 0.90.

[0086] In order to further verify the reliability of the present invention, the inventors conducted a series of experiments, as follows:

[0087] 1. Establishment of fingerprint determination method for Xinnaoning capsule water extract

[0088] 1 Experimental instruments and materials

[0089] 1.1 Experimental instruments: Agilent 1200 high-performance liquid chromatograph (including online vacuum degasser G1322A, quaternary pump G1311A, autosampler G1329A, column oven G1316A, diode array detector G1315A, and chromatography workstation). UPT-C-20 ultrapure water machine and HH-4 digital constant-temperature water bath (Bangsi Instrument Technology (Shanghai) Co., Ltd.); FA2204 electronic analytical balance (Lichen Technology (Ningbo Yinzhou Huafeng Electronic Instrument Factory)); PS-60 ultrasonic cleaner (Dongguan Jiekang Ultrasonic Equipment Co., Ltd.); FE28 pH meter (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); TDL-80-2B centrifuge (Shanghai Anting Scientific Instrument Factory).

[0090] 1.2 Experimental reagents: acetonitrile (chromatographic grade, Grace Company); methanol, sodium hydroxide, potassium dihydrogen phosphate (analytical grade, Tianjin Zhiyuan Chemical Reagent Co., Ltd.); 3-methyl-1-phenyl-5-pyrazolone (PMP) (analytical grade, Aladdin Holding Group Co., Ltd.); hydrochloric acid (analytical grade, Guangzhou Chemical Reagent Factory).

[0091] 1.3 Experimental materials: Xinnaoning Capsules (batch number: 20190336; provided by Guizhou Jingcheng Pharmaceutical Co., Ltd.).

[0092] 2 Experimental content and results

[0093] 2.1 Investigation of the extraction method of the test solution

[0094] 2.1.1 Chromatographic conditions

[0095] Chromatographic column: Kromasil C18 (4.6*250mm, 5μm); mobile phase: 0.1mol / L potassium dihydrogen phosphate buffer (adjusted to pH 6.7 with 40% NaOH) (A) - acetonitrile (B), gradient elution, flow rate: 0.8ml / min; column temperature: 35℃; injection volume: 10μl; detection wavelength: 250nm.

[0096] Table 1 Gradient elution program

[0097]

[0098] Note: The column was equilibrated with 13% acetonitrile for 10 min.

[0099] 2.1.2 Preparation of test solution

[0100] Extraction method 1: ultrasonic extraction

[0101] Accurately weigh 1g of Xinnaoning capsule contents, add 10ml of water, weigh, and ultrasonically extract for 60 minutes. Make up the weight, shake well, centrifuge, and collect the supernatant to obtain the Xinnaoning aqueous extract. Accurately pipette 200μl of the Xinnaoning aqueous extract into a stoppered centrifuge tube, add 200μl of 0.3mol / L NaOH, mix thoroughly, add 160μl of 0.5mol / L PMP methanol solution, mix well, and heat in a 70°C water bath for 30 minutes. Cool to room temperature, neutralize the alkaline solution with 200μl of 0.3mol / L HCl solution, add 1240μl of purified water and 2ml of chloroform, shake well, centrifuge at 3500 rpm for 5 minutes, discard the chloroform layer, and repeat the extraction three times. Pass the aqueous layer through a 0.45μm filter, and the filtrate is used as test solution 1.

[0102] Extraction method 2: Heating reflux extraction

[0103] Accurately weigh 1g of Xinnaoning capsule contents, add 10ml of water, weigh, and heat under reflux for 60 minutes. Make up the weight, shake well, centrifuge, and collect the supernatant to obtain the Xinnaoning aqueous extract. Accurately pipette 200μl of the Xinnaoning aqueous extract into a stoppered centrifuge tube, add 200μl of 0.3mol / L NaOH, mix thoroughly, add 160μl of 0.5mol / L PMP methanol solution, mix well, and heat in a 70°C water bath for 30 minutes. Cool to room temperature, neutralize the alkali solution with 200μl of 0.3mol / L HCl solution, add 1240μl of purified water and 2ml of chloroform, shake well, centrifuge at 3500 rpm for 5 minutes, discard the chloroform layer, and repeat the extraction three times. Pass the aqueous layer through a 0.45μm filter, and the filtrate is used as test solution 2.

[0104] 2.1.3 Determination method

[0105] According to the chromatographic conditions, 10 μl of each of the test solutions 1 and 2 were accurately aspirated, and the results were compared by HPLC to determine the best extraction method.

[0106] 2.1.4 Measurement results

[0107] The experimental results are shown in Figures 1 to 3 According to the experimental results, the chromatographic peaks at 18 to 28 minutes were better separated by heating reflux extraction and worse separated by ultrasonic extraction. According to the total peak area of ​​the main chromatographic peaks, the total peak area of ​​heating reflux extraction was larger, so heating reflux extraction was selected as the extraction method for the test sample.

[0108] 2.2 Investigation of the extraction solvent multiples of the test solution

[0109] 2.2.1 Chromatographic conditions: Same as “2.1.1 Chromatographic conditions”.

[0110] 2.2.2 Preparation of test solution

[0111] Test Solution 1 (5x solvent): Accurately weigh 2g of Xinnaoning capsule contents, add 10ml of water, weigh, and heat under reflux for 60min. Make up to the required weight, shake well, centrifuge, and remove the supernatant to obtain the Xinnaoning aqueous extract. Accurately pipette 200μl of the Xinnaoning aqueous extract into a stoppered centrifuge tube, add 200μl of 0.3mol / L NaOH, mix thoroughly, add 160μl of 0.5mol / L PMP methanol solution, mix well, and heat in a 70°C water bath for 30min. Cool to room temperature, neutralize the alkali solution with 200μl of 0.3mol / L HCl solution, add 1240μl of purified water, add 2ml of chloroform, shake well, centrifuge at 3500 rpm for 5min, discard the chloroform layer, and repeat the extraction three times. Pass the aqueous layer through a 0.45μm filter, and the filtrate is used as Test Solution 1.

[0112] Test Solution 2 (10x solvent): Accurately weigh 1g of Xinnaoning capsule contents, add 10ml of water, weigh, and heat under reflux for 60 minutes. Make up to the required weight, shake well, centrifuge, and remove the supernatant to obtain the Xinnaoning aqueous extract. Accurately pipette 200μl of the Xinnaoning aqueous extract into a stoppered centrifuge tube. Add 200μl of 0.3mol / L NaOH and mix thoroughly. Add 160μl of 0.5mol / L PMP methanol solution. Mix well, then heat in a 70°C water bath for 30 minutes. Cool to room temperature, neutralize the alkali solution with 200μl of 0.3mol / L HCl solution, add 1240μl of purified water, add 2ml of chloroform, shake well, centrifuge at 3500 rpm for 5 minutes, discard the chloroform layer, and repeat the extraction three times. Pass the aqueous layer through a 0.45μm filter, and the filtrate is used as Test Solution 2.

[0113] Test Solution 3 (20x solvent): Accurately weigh 0.5g of Xinnaoning capsule contents, add 10ml of water, weigh, and heat under reflux for 60min. Make up to the required weight, shake well, centrifuge, and collect the supernatant to obtain the Xinnaoning aqueous extract. Accurately pipette 200μl of the Xinnaoning aqueous extract into a stoppered centrifuge tube. Add 200μl of 0.3mol / L NaOH and mix thoroughly. Add 160μl of 0.5mol / L PMP methanol solution. Mix well, then heat in a 70°C water bath for 30min. Cool to room temperature, neutralize the alkali solution with 200μl of 0.3mol / L HCl solution, add 1240μl of purified water and 2ml of chloroform, shake well, centrifuge at 3500 rpm for 5min, discard the chloroform layer, and repeat the extraction three times. Pass the aqueous layer through a 0.45μm filter, and the filtrate is used as Test Solution 3.

[0114] 2.2.3 Determination method

[0115] According to the chromatographic conditions, 10 μl of each of the test solutions 1, 2, and 3 were accurately aspirated, and the results were compared by HPLC to determine the optimal extraction solvent multiple.

[0116] 2.2.4 Measurement results

[0117] The experimental results are shown in Figures 4 to 7 According to the experimental results, the 18-28 min chromatographic peaks were better separated by 5 times solvent and 10 times solvent, but poorly separated by 20 times solvent. According to the total peak area of ​​the main chromatographic peaks, the total peak area of ​​the 10 times solvent spectrum was the largest, so the 10 times solvent was selected as the extraction solvent multiple of the test sample.

[0118] 2.3 Investigation of the extraction time of the test solution

[0119] 2.3.1 Chromatographic conditions: Same as “2.1.1 Chromatographic conditions”.

[0120] 2.3.2 Preparation of test solution

[0121] Test Solution 1 (30 min extraction): Accurately weigh 1 g of Xinnaoning capsule contents, add 10 ml of water, weigh, heat under reflux for 30 min, make up to the required weight, shake well, centrifuge, and collect the supernatant to obtain the Xinnaoning aqueous extract. Accurately pipette 200 μl of the Xinnaoning aqueous extract into a stoppered centrifuge tube, add 200 μl of 0.3 mol / L NaOH, mix thoroughly, add 160 μl of 0.5 mol / L PMP methanol solution, mix well, and heat in a 70°C water bath for 30 min. Cool to room temperature, neutralize the alkaline solution with 200 μl of 0.3 mol / L HCl solution, add 1240 μl of purified water, add 2 ml of chloroform, shake well, centrifuge at 3500 rpm for 5 min, discard the chloroform layer, and repeat the extraction three times. Pass the aqueous layer through a 0.45 μm filter, and the filtrate is used as Test Solution 1.

[0122] Test Solution 2 (60 min extraction): Accurately weigh 1 g of Xinnaoning capsule contents, add 10 ml of water, weigh, heat under reflux for 60 min, make up to the required weight, shake well, centrifuge, and collect the supernatant to obtain the Xinnaoning aqueous extract. Accurately pipette 200 μl of the Xinnaoning aqueous extract into a stoppered centrifuge tube, add 200 μl of 0.3 mol / L NaOH, mix thoroughly, add 160 μl of 0.5 mol / L PMP methanol solution, mix well, and heat in a 70°C water bath for 30 min. Cool to room temperature, neutralize the alkaline solution with 200 μl of 0.3 mol / L HCl solution, add 1240 μl of purified water, add 2 ml of chloroform, shake well, centrifuge at 3500 rpm for 5 min, discard the chloroform layer, and repeat the extraction three times. Pass the aqueous layer through a 0.45 μm filter, and the filtrate is used as Test Solution 2.

[0123] Test Solution 3 (90 min extraction): Accurately weigh 1 g of Xinnaoning capsule contents, add 10 ml of water, weigh, and extract under reflux for 90 min. Make up to the required weight, shake well, centrifuge, and collect the supernatant to obtain the Xinnaoning aqueous extract. Accurately pipette 200 μl of the Xinnaoning aqueous extract into a stoppered centrifuge tube. Add 200 μl of 0.3 mol / L NaOH and mix thoroughly. Add 160 μl of 0.5 mol / L PMP methanol solution. Mix thoroughly, then heat in a 70°C water bath for 30 min. Cool to room temperature, neutralize the alkali solution with 200 μl of 0.3 mol / L HCl solution, add 1240 μl of purified water and 2 ml of chloroform, shake well, centrifuge at 3500 rpm for 5 min, discard the chloroform layer, and repeat the extraction three times. Pass the aqueous layer through a 0.45 μm filter, and the filtrate is used as Test Solution 3.

[0124] Test Solution 4 (120 min extraction): Accurately weigh 1 g of Xinnaoning capsule contents, add 10 ml of water, weigh, and heat under reflux for 120 min. Make up to the required weight, shake well, centrifuge, and collect the supernatant to obtain the Xinnaoning aqueous extract. Accurately pipette 200 μl of the Xinnaoning aqueous extract into a stoppered centrifuge tube. Add 200 μl of 0.3 mol / L NaOH and mix thoroughly. Add 160 μl of 0.5 mol / L PMP methanol solution. Mix well, then heat in a 70°C water bath for 30 min. Cool to room temperature, neutralize the alkaline solution with 200 μl of 0.3 mol / L HCl solution, add 1240 μl of purified water and 2 ml of chloroform, shake well, centrifuge at 3500 rpm for 5 min, discard the chloroform layer, and repeat the extraction three times. Pass the aqueous layer through a 0.45 μm filter, and the filtrate is used as Test Solution 4.

[0125] 2.3.3 Determination method

[0126] According to the chromatographic conditions, 10 μl of each of the test solutions 1, 2, 3, and 4 were accurately aspirated, and the results were compared by HPLC to determine the optimal extraction time.

[0127] 2.3.4 Measurement results

[0128] The experimental results are shown in Figures 8 to 12 According to the experimental results, the chromatographic peaks at 18-28 min, 90 min and 120 min were better separated; according to the total peak area of ​​the main chromatographic peaks, the total peak area of ​​the 90 min spectrum was larger, and the total peak area of ​​the 120 min spectrum did not increase significantly, so the extraction time of the test sample was selected as 90 min.

[0129] 3 Conclusion

[0130] By optimizing the preparation of the test solution and the chromatographic conditions for determination, the optimal determination method for Xinnaoning Capsules was obtained, as follows:

[0131] (1) Chromatographic conditions

[0132] Chromatographic column: Kromasil C18 (4.6*250mm, 5μm); mobile phase: 0.1mol / L potassium dihydrogen phosphate buffer (adjusted to pH 6.7 with 40% NaOH) (A) - acetonitrile (B), gradient elution, flow rate: 0.8ml / min; column temperature: 35℃; injection volume: 10μl; detection wavelength: 250nm.

[0133] Gradient elution program

[0134]

[0135] Note: The column was equilibrated with 13% acetonitrile for 10 min.

[0136] (2) Preparation of test solution

[0137] Accurately weigh 1g of Xinnaoning capsule contents, add 10ml of water, weigh, and heat under reflux for 90 minutes. Make up the weight, shake well, centrifuge, and collect the supernatant to obtain the Xinnaoning aqueous extract. Accurately pipette 200μl of the Xinnaoning aqueous extract into a stoppered centrifuge tube, add 200μl of 0.3mol / L NaOH, mix thoroughly, add 160μl of 0.5mol / L PMP methanol solution, mix well, and heat in a 70°C water bath for 30 minutes. Cool to room temperature, neutralize the alkali solution with 200μl of 0.3mol / L HCl solution, add 1240μl of purified water and 2ml of chloroform, shake well, centrifuge at 3500 rpm for 5 minutes, discard the chloroform layer, and repeat the extraction three times. Pass the aqueous layer through a 0.45μm filter, and the filtrate is used as the test solution.

[0138] (3) Measurement method

[0139] According to the chromatographic conditions, accurately draw 10 μl of the above test solution and analyze the results by HPLC.

[0140] 2. Methodological Investigation of the Fingerprint of Xinnaoning Capsule Water Extract

[0141] 2.1 Precision test

[0142] Take Xinnaoning Capsule (batch number: 20190336), prepare the test solution according to the preparation method of the test solution, and perform the test according to the chromatographic conditions. The injection volume is 10 μl, and the sample is injected 6 times continuously. The fingerprint spectrum is measured and the retention time and peak area of ​​each common chromatographic peak are recorded. With the retention time and peak area of ​​chromatographic peak No. 9 as a reference, the relative retention time and relative peak area of ​​the main chromatographic peaks in the sample are calculated, and the corresponding average value and relative standard deviation (RSD%) are calculated. The results are shown in Figure 14 and Tables 2 to 5.

[0143] Table 2 Precision investigation-retention time

[0144]

[0145] Table 3 Precision investigation-peak area

[0146]

[0147] Table 4 Precision investigation - relative retention time

[0148]

[0149] Table 5 Precision investigation - relative retention peak area

[0150]

[0151] Through analysis Figure 14 According to the above precision test results table, the RSD value of retention time in the precision test is less than

[0152] The RSD values ​​of the relative retention time and the relative peak area were less than 1.00%, less than 4.00%, and less than 5.00%, respectively. This shows that the instrument has good precision.

[0153] 2.2 Repeatability test

[0154] Take Xinnaoning Capsule (batch number: 20190336), prepare 6 test solutions according to the preparation method of the test solution, and measure according to the chromatographic conditions. The injection volume is 10μl, measure the fingerprint, and record the retention time and peak area of ​​each common chromatographic peak. Using the retention time and peak area of ​​chromatographic peak No. 9 as a reference, calculate the relative retention time and relative peak area of ​​the main chromatographic peaks in the sample, and calculate the corresponding average value and relative standard deviation (RSD%). The results are shown in Figure 15 and Tables 6 to 9.

[0155] Table 6 Repeatability Study - Retention Time

[0156]

[0157] Table 7 Repeatability Study-Peak Area

[0158]

[0159] Table 8 Repeatability study - relative retention time

[0160]

[0161] Table 9 Repeatability study - relative peak area

[0162]

[0163] Through analysis Figure 15 According to the above repeatability test results, the RSD values ​​of retention time in the repeatability test were less than

[0164] 1.00%, the RSD value of the chromatographic peak area is less than 4.00%, the RSD value of the relative retention time is less than 1.00%, and the RSD value of the relative peak area is less than 5.00%. It can be seen that the RSD values ​​are all less than 5.00%, indicating that the experiment has good repeatability.

[0165] 2.3 Stability test

[0166] Take Xinnaoning Capsule (batch number: 20190336), prepare the test solution according to the preparation method of the test solution, and measure it according to the chromatographic conditions. The injection volume is 10μl, and the sample is injected and measured at 0h, 2h, 4h, 8h, and 12h respectively. Measure the fingerprint spectrum and record the retention time and peak area of ​​each common chromatographic peak. Using the retention time and peak area of ​​chromatographic peak No. 9 as a reference, calculate the relative retention time and relative peak area of ​​the main chromatographic peaks in the sample, and calculate the corresponding average value and relative standard deviation (RSD%). The results are shown in Figure 16 and Tables 10 to 13.

[0167] Table 10 Stability Study-Retention Time

[0168]

[0169] Table 11 Stability Study-Peak Area

[0170]

[0171] Table 12 Stability Study-Relative Retention Time

[0172]

[0173] Table 13 Stability Study - Relative Peak Area

[0174]

[0175] By analyzing the above Figure 16 From the stability investigation result table, it is concluded that in the stability test, the RSD values ​​of the retention time are all less than 1.00%, the RSD values ​​of the peak area are all less than 5.00%, the RSD values ​​of the relative retention time are less than 1.00%, and the RSD values ​​of the relative peak area of ​​the chromatographic peak are all less than 5.00%. It can be seen that the RSD values ​​are all less than 5%, indicating that the samples prepared by the present invention have good stability within 12 hours.

[0176] 2.4 Conclusion

[0177] Using the established chromatographic conditions for Xinnaoning Capsules, the precision, repeatability and stability of the methodological investigation were studied. It was found that in each investigation item, the RSD values ​​of retention time, peak area, relative retention time and relative peak area were all less than 5.00%, proving that the instrument used has good precision, the determination method has good repeatability, and the prepared samples have good stability within 12 hours.

[0178] 3. Study on the Similarity of Fingerprints of Xinnaoning Capsules Water Extract

[0179] Twenty-six batches of Xinnaoning capsules were used for similarity study and were provided by Guizhou Jingcheng Pharmaceutical Co., Ltd. The batch numbers and serial numbers are shown in Table 14 ;

[0180] Table 14 Xinnaoning Capsule serial number and batch number

[0181]

[0182] 3.1 Experimental methods

[0183] The determination was carried out according to the optimal determination method of Xinnaoning Capsule determined under the aforementioned “3 Conclusions”.

[0184] (1) Analysis of all 26 batches of samples

[0185] 26 batches of samples were tested by HPLC to obtain fingerprints. The obtained fingerprints were analyzed using the software "Chinese Herbal Chromatographic Fingerprint Similarity Software Evaluation System (2012 Edition)" of the National Pharmacopoeia Committee. The latest batch (20190722) of sample S25 was used as the reference spectrum. The average method was used to generate the reference spectrum, the time window width was set to 0.3, multi-point correction was performed, and full spectrum peak matching was performed to generate the reference spectrum. Figures 17 and 18 , calculate the similarity, and the results are shown in Table 15 below.

[0186] Table 15 HPLC fingerprint similarity results of Xinnaoning capsule water extract (26 batches)

[0187]

[0188] According to the experimental results, the similarity of the 26 batches of samples was greater than 0.950, and the average similarity was 0.976, indicating that the samples had high similarity and small chromatographic differences.

[0189] (2) 2017 sample analysis

[0190] In 2017, 9 batches of samples were tested by HPLC, and the HPLC fingerprint of the aqueous extract of Xinnaoning Capsules was obtained. The obtained fingerprint was analyzed using the software "Chinese Medicine Chromatographic Fingerprint Similarity Software Evaluation System (2012 Edition)" of the National Pharmacopoeia Committee. The latest batch (20171111) sample S9 was used as the reference spectrum. The average method was used to generate the reference spectrum, the time window width was set to 0.2, multi-point correction was performed, and full spectrum peak matching was performed to generate the reference spectrum. See Figures 19 to 20 , calculate the similarity, and the results are shown in Table 16.

[0191] Table 16 HPLC fingerprint similarity results of Xinnaoning capsule water extract (2017 batch)

[0192]

[0193] According to the experimental results, the similarities of the nine batches of samples in 2017 were all greater than 0.970, with an average similarity of 0.986, indicating that the samples were highly similar, the chromatographic differences were effective, and the production process was stable.

[0194] (3) 2018 sample analysis

[0195] In 2018, five batches of samples were tested by HPLC and fingerprints were obtained. The obtained fingerprints were analyzed using the software "Chinese Herbal Chromatographic Fingerprint Similarity Software Evaluation System (2012 Edition)" of the National Pharmacopoeia Committee. The latest batch (20181133) of sample S4 was used as the reference spectrum. The average method was used to generate the reference spectrum, the time window width was set to 0.2, multi-point correction was performed, and full spectrum peak matching was performed to generate the reference spectrum. Figures 21 to 22 , calculate the similarity, and the results are shown in Table 17.

[0196] Table 17 HPLC fingerprint similarity results of Xinnaoning capsule water extract (2018 batch)

[0197]

[0198] According to the experimental results, the similarities of the five batches of samples in 2018 were all greater than 0.970, with an average similarity of 0.987, indicating that the samples were highly similar and the chromatographic differences were small.

[0199] (4) 2019 sample analysis

[0200] In 2019, 11 batches of samples were tested by HPLC and fingerprints were obtained. The obtained fingerprints were analyzed using the software "Chinese Herbal Chromatographic Fingerprint Similarity Software Evaluation System (2012 Edition)" of the National Pharmacopoeia Committee. The latest batch (20190722) sample S11 was used as the reference spectrum. The average method was used to generate the reference spectrum, the time window width was set to 0.3, multi-point correction was performed, and full spectrum peak matching was performed to generate the reference spectrum. Figures 23 to 24, calculate the similarity, and the results are shown in Table 18.

[0201] Table 18 HPLC fingerprint similarity results of Xinnaoning capsule water extract (2019 batch)

[0202]

[0203] According to the experimental results, the similarities of the 11 batches of samples in 2019 were all greater than 0.970, with an average similarity of 0.981, indicating that the samples were highly similar and the chromatographic differences were small.

[0204] (5) 4-year comparative analysis of Xinnaoning capsules

[0205] Since there was only one batch of samples in 2016, the 20160916 batch of samples was used to represent the 2016 samples, and the similarity of the samples in the four years was analyzed by comparing them with the chromatograms of 2017, 2018, and 2019. The obtained spectra were analyzed using the software "Chinese Medicine Chromatographic Fingerprint Similarity Software Evaluation System (2012 Edition)" of the National Pharmacopoeia Committee. The latest year (2019 reference spectrum) sample S4 was used as the reference spectrum. The average method was used to generate the reference spectrum, the time window width was set to 0.2, multi-point correction was performed, and full spectrum peak matching was performed to generate the reference spectrum. Figures 25 to 26 , calculate the similarity, and the results are shown in Table 19.

[0206] Table 19 HPLC fingerprint similarity results of Xinnaoning capsule water extract (4-year batch)

[0207]

[0208] According to the experimental results, the similarity of the control maps of the four years was greater than 0.980, and the average similarity was 0.991, indicating that the samples were highly similar and the chromatographic differences were small.

[0209] 3.2 Conclusion

[0210] 1) From 2016 to 2019, a total of 26 batches of samples had similarities greater than 0.950, with an average similarity of 0.976. Analysis of similarity of samples from the same year showed that the similarities of 9 batches of samples in 2017 were all greater than 0.970, with an average similarity of 0.986; the similarities of 5 batches of samples in 2018 were all greater than 0.970, with an average similarity of 0.986; and the similarities of 11 batches of samples in 2019 were all greater than 0.970, with an average similarity of 0.981.

[0211] 2) The similarity of samples from a batch in 2016 was analyzed with the reference maps in 2017, 2018, and 2019. The similarity of the reference maps of the four years was greater than 0.980, and the average similarity was 0.991.

[0212] 3) According to the experimental results, among the 26 batches of samples over the four years, all batches and samples between years were highly similar, with small chromatographic differences.

[0213] The above experimental conclusions indicate that the fingerprints of Xinnaoning capsule samples exhibit high similarity and minimal variability, indicating that the overall characteristics of the water extract components in each batch of Xinnaoning capsule are similar, the quality of the water extract is relatively stable, and meets the analytical requirements of the fingerprint spectrum, reflecting the integrity, stability, and uniformity of the samples. This demonstrates that the water extract fingerprint determination method established in this invention is stable and reliable, and is of great significance for the analysis and identification of the components and quality control of Xinnaoning capsule preparations.

[0214] Although the present invention has been described in detail above using general descriptions, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of the present invention.

Claims

1. A method for establishing a fingerprint of the water extract of Xinnaoning capsule, characterized in that: The following steps are involved: S1. Preparation of Xinnaoning Capsule test solution: S11. Take the contents of Xinnaoning capsule, add 5-20 times the amount of water, weigh, heat and reflux to extract for 30-120 minutes, make up the weight with water, shake well, centrifuge, and take the supernatant to obtain Xinnaoning aqueous extract; S12. Accurately pipette 200 μl of the Xinnaoning aqueous extract into a stoppered centrifuge tube, add 200 μl of 0.3 mol / L NaOH, mix thoroughly, add 160 μl of 0.5 mol / L PMP methanol solution, mix thoroughly, and heat in a 70°C water bath for 30 min. Cool to room temperature, neutralize the alkali solution with 200 μl of 0.3 mol / L HCl solution, add 1240 μl of purified water and 2 ml of chloroform, shake well, centrifuge for 5 min, discard the chloroform layer, and repeat the extraction three times; filter the aqueous layer through a 0.45 μm filter membrane, and use the filtrate as the Xinnaoning capsule test solution; S2. Chromatographic conditions: The chromatographic column was Kromasil C18, 4.6 × 250 mm, 5 μm; mobile phase A was 0.1 mol / L potassium dihydrogen phosphate buffer, mobile phase B was acetonitrile, gradient elution, flow rate: 0.8 ml / min; column temperature: 35°C; injection volume: 10 μl; detection wavelength: 250 nm; gradient elution program: S3. Detection: According to the above chromatographic conditions, 10 μl of the above test solution was accurately aspirated and tested by HPLC to obtain a chromatogram of the Xinnaoning Capsule test solution.

2. The method for establishing the fingerprint of the water extract of Xinnaoning Capsule according to claim 1, characterized in that: The amount of water added in step S11 is 5-10 times.

3. The method for establishing the fingerprint of the water extract of Xinnaoning Capsule according to claim 2, characterized in that: The amount of water added in step S11 is 10 times.

4. The method for establishing the fingerprint of the water extract of Xinnaoning Capsule according to claim 1, characterized in that: The extraction time in step S11 is 90-120 min.

5. The method for establishing the fingerprint of the water extract of Xinnaoning Capsule according to claim 4, characterized in that: The extraction time in step S11 is 90 minutes.

6. The method for establishing the fingerprint of the water extract of Xinnaoning Capsule according to claim 1, characterized in that: The heating reflux extraction in step S11 can also be replaced by ultrasonic extraction.

7. The method for establishing the fingerprint of the water extract of Xinnaoning Capsule according to claim 1, characterized in that: The potassium dihydrogen phosphate buffer in step S2 needs to be adjusted to pH 6.7 using 40% NaOH.

8. The method for establishing the fingerprint of the water extract of Xinnaoning Capsule according to claim 1, characterized in that: The method further comprises: importing the obtained chromatogram of the Xinnaoning capsule test solution into a traditional Chinese medicine fingerprint similarity evaluation software, using the latest batch of samples as a reference spectrum, selecting the average method, setting the time window width to 0.2-0.5, multi-point correction, and full spectrum peak matching to generate a reference fingerprint spectrum of the Xinnaoning capsule substance benchmark water extract, and using the production reference fingerprint spectrum as a benchmark to calculate the similarity between the fingerprint spectrum of each batch of Xinnaoning capsule substance benchmark water extract and the reference spectrum, wherein the similarity is not less than 0.

90.

9. Application of the fingerprint of the water extract of Xinnaoning Capsules obtained by the method for establishing the fingerprint of the water extract of Xinnaoning Capsules as claimed in claim 1 in characterizing the components of the water extract of Xinnaoning Capsules or detecting its quality.

10. Application of the fingerprint of Xinnaoning Capsules water extract obtained by the method for establishing the fingerprint of Xinnaoning Capsules water extract as claimed in claim 1 in the quality control of Xinnaoning Capsules.

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