Establishment of fingerprint of water extract in Xinuanling capsule

Abstract

The application belongs to the technical field of traditional Chinese medicine preparation quality detection, and provides a method for establishing a water extract fingerprint of Xinnaoning capsules and application of the method. The method comprises preparation of a test solution of the Xinnaoning capsules, collection of HPLC chromatograms of each test solution by high performance liquid chromatography (HPLC), and similarity evaluation of the obtained chromatograms. The method of the application obtains a HPLC water extract standard fingerprint of the Xinnaoning capsules, and has the characteristics of high similarity, small difference between the fingerprints, and high accuracy, and meets the analysis requirements of the fingerprint, and reflects the integrity and uniformity of the sample. It is indicated that the water extract fingerprint established by the application is stable and reliable, and has important significance for component analysis, identification and quality control of the Xinnaoning capsule preparation, and provides technical support for more comprehensive and effective control of product quality.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of quality detection of traditional Chinese medicine preparation, and particularly relates to a method for establishing a water extract fingerprint of Xinnaoning capsules. BACKGROUND

[0002] Xinnaoning capsules are products of Guizhou Jingcheng Pharmaceutical Co., Ltd., and are recorded in the Chinese Pharmacopoeia 2020 edition. Xinnaoning capsules are composed of Ginkgo leaves, Potoptelea stenophylla, Salvia miltiorrhiza, Litsea chenii and Allium bakeri, have the effects of activating blood and promoting qi, dredging collaterals and relieving pain, and are clinically used for treating chest pain, headache and dizziness caused by qi stagnation and blood stasis, and are used for treating chest pain, headache and dizziness caused by qi stagnation and blood stasis, and are used for treating chest pain, headache and dizziness caused by qi stagnation and blood stasis.

[0003] Traditional Chinese medicine fingerprint refers to a chromatogram or a spectrum obtained by adopting a certain analysis method after some traditional Chinese medicinal materials or traditional Chinese medicine preparation is properly treated, which can indicate the chemical characteristics. The traditional Chinese medicine fingerprint can comprehensively reflect the types and quantities of chemical components contained in medicinal materials, effectively embodies the overallity and comprehensive action of traditional Chinese medicine components, and has been widely applied in traditional Chinese medicine analysis and quality control due to its rapidness and accuracy. The traditional Chinese medicine fingerprint is beneficial to establishing a more accurate, comprehensive and cross-batch quality control method for traditional Chinese medicine on the market. At present, there is no research on the fingerprint of water extract of Xinnaoning capsules. Therefore, it is of great significance to establish the fingerprint of water extract of Xinnaoning capsules for component analysis and quality control. SUMMARY

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

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

[0006] The method for establishing a water extract fingerprint of Xinnaoning capsules provided by the present application comprises the following steps:

[0007] S1, preparation of a test solution of Xinnaoning capsules:

[0008] S11, taking the contents of Xinnaoning capsules, adding 5-20 times the amount of water, weighing, heating and refluxing for 30-120 minutes, supplementing the weight with water, shaking uniformly, centrifuging, and taking the supernatant, to obtain a water extract of Xinnaoning capsules;

[0009] S12, precisely suck 200ul of Xinnaoling water extract solution in a centrifuge tube with a plug, add 200ul of 0.3mol / L NaOH, mix thoroughly, add 160ul of 0.5mol / L PMP methanol solution, mix thoroughly, and then place in a 70℃ water bath for 30min, cool to room temperature, neutralize the alkali with 200ul of 0.3mol / L HCl solution, add 1240ul of purified water, add 2ml of chloroform, shake well, centrifuge for 5min, discard the chloroform layer, repeat the extraction for 3 times; pass the water layer through a 0.45um filter membrane, take the subsequent filtrate as the Xinnaoling capsule test sample solution;

[0010] S2, chromatographic conditions:

[0011] The chromatographic column is Kromasil C18, with a specification of 4.6*250mm, 5um; the mobile phase A is 0.1mol / L potassium dihydrogen phosphate buffer salt, the mobile phase B is acetonitrile, gradient elution, flow rate: 0.8ml / min; column temperature: 35℃; injection volume 10ul; detection wavelength: 250nm; the gradient elution program is as follows:

[0012]

[0013] S3, detection:

[0014] According to the above chromatographic conditions, 10ul of the above test sample solution is precisely sucked and detected by HPLC to obtain the chromatogram of the Xinnaoling capsule test sample solution.

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

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

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

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

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

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

[0021] The method for establishing the water extract fingerprint of Xinnaoning capsules further comprises: importing the chromatogram of the obtained Xinnaoning capsule test product solution into traditional Chinese medicine fingerprint similarity evaluation software, taking the latest batch sample as a reference spectrum, selecting the average method, setting the time window width to 0.2-0.5, performing multi-point correction, and performing full spectrum peak matching to generate a control fingerprint spectrum of the water extract of the material basis of Xinnaoning capsules, taking the produced control fingerprint spectrum as a basis, calculating the similarity of the water extract fingerprint spectrum of each batch of the material basis of Xinnaoning capsules and the control spectrum, and the similarity is not less than 0.90.

[0022] The water extract fingerprint of Xinnaoning capsules obtained by the method for establishing the water extract fingerprint of Xinnaoning capsules has the application in characterizing the water extract composition or quality detection of Xinnaoning capsules.

[0023] The water extract fingerprint of Xinnaoning capsules obtained by the method for establishing the water extract fingerprint of Xinnaoning capsules has the application in quality control of Xinnaoning capsules.

[0024] The present application has the following advantages:

[0025] The present application provides a method for establishing the water extract composition fingerprint determination method of Xinnaoning capsules, which has the advantages of high stability, high precision and good repeatability. The similarity of the water extract of Xinnaoning capsules is evaluated, and the result sample has high similarity and small spectrum difference. It is shown that the quality of the water extract of each batch of Xinnaoning capsules is stable, the overall characteristic difference of the water extract composition is small, the analysis requirement of the fingerprint spectrum is met, and the overallity and uniformity of the sample are reflected. It is shown that the water extract fingerprint established by the present application is stable and reliable, and has important significance for the composition analysis and identification and quality control of Xinnaoning capsule preparation, and provides technical support for realizing more comprehensive and effective control of product quality. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 : Water extract fingerprint spectrum of Xinnaoning capsules (ultrasonic extraction)

[0027] Figure 2 : Water extract fingerprint spectrum of Xinnaoning capsules (heating reflux extraction)

[0028] Figure 3 : Water extract fingerprint spectrum superimposition of Xinnaoning capsules (extraction method investigation) (the spectrum from bottom to top is: heating reflux extraction, ultrasonic extraction spectrum)

[0029] Figure 4 : Water extract fingerprint spectrum of Xinnaoning capsules (5 times solvent)

[0030] Figure 5 : Water extract fingerprint spectrum of Xinnaoning capsules (10 times solvent)

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

[0032] Figure 7 Superimposed fingerprint of Xinnaoning Capsule water extract (investigation of extraction solvent times) (from bottom to top, the spectra are 5 times solvent, 10 times solvent, 20 times solvent)

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

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

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

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

[0037] Figure 12 Superimposed fingerprint of Xinnaoning Capsule water extract (investigation of extraction time) (from bottom to top, the spectra are 30 min, 60 min, 90 min, 120 min)

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

[0039] Figure 14 Investigation of precision (from bottom to top, the spectra are precision 1 to precision 6)

[0040] Figure 15 Investigation of repeatability (from bottom to top, the spectra are repeatability 1 to repeatability 6)

[0041] Figure 16 Investigation of stability (from bottom to top, the spectra are stability 1 to stability 6)

[0042] Figure 17 Reference spectrum of water extract HPLC fingerprint (26 batches)

[0043] Figure 18 Superimposed spectrum of water extract HPLC fingerprint (26 batches)

[0044] Figure 19 Reference spectrum of water extract HPLC fingerprint (2017 batches)

[0045] Figure 20 Superimposed spectrum of water extract HPLC fingerprint (2017 batches)

[0046] Figure 21 : Water extract HPLC fingerprint chromatogram control map (2018)

[0047] Figure 22 : Water extract HPLC fingerprint chromatogram overlay (2018 batch)

[0048] Figure 23 : Water extract HPLC fingerprint chromatogram control map (2019)

[0049] Figure 24 : Water extract HPLC fingerprint chromatogram overlay (2019 batch)

[0050] Figure 25 : Water extract HPLC fingerprint chromatogram overlay (4-year batch)

[0051] Figure 26 : Water extract HPLC fingerprint chromatogram overlay (4-year batch) (S1: Xinnaoling 20160916; S2-S4: 2017, 2018, and 2019 control maps, respectively) DETAILED DESCRIPTION

[0052] The technical solutions of the present application are described in detail below in combination with specific embodiments. The following embodiments are merely used for explanation and illustration, and do not constitute a limitation on the technical solutions of the present application.

[0053] Example 1: Establishment of a method for determining the water extract fingerprint chromatogram of Xinnaoling capsules

[0054] S1, Preparation of the test solution of Xinnaoling capsules:

[0055] S11, 1 g of the content of Xinnaoling capsules was precisely weighed, 10 ml of water was added, the weight was made up, and the mixture was heated to reflux for 90 min, the weight was made up, and the mixture was shaken, centrifuged, and the supernatant was taken, thereby obtaining the water extract of Xinnaoling;

[0056] S12, 200 μl of the water extract solution of Xinnaoling was precisely pipetted into a centrifuge tube with a stopper, 200 μl of 0.3 mol / L NaOH was added, the mixture was thoroughly mixed, 160 μl of 0.5 mol / L PMP methanol solution was added, the mixture was mixed and then placed in a 70℃ water bath for heating for 30 min, the mixture was cooled to room temperature, 200 μl of 0.3 mol / L HCl solution was added to neutralize the alkali, 1240 μl of purified water was added, 2 ml of chloroform was added, the mixture was shaken and centrifuged at 3500 rpm for 5 min, the chloroform layer was discarded, and the extraction was repeated for 3 times; the water layer was filtered through a 0.45 μm filter membrane, and the filtrate was taken as the test solution of Xinnaoling capsules;

[0057] S2, Chromatographic conditions:

[0058] The chromatographic column was a Kromasil C18, with dimensions of 4.6 × 250 mm and a diameter of 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 used at a flow rate of 0.8 mL / min, a column temperature of 35 °C, an injection volume of 10 μL, and a detection wavelength of 250 nm. The gradient elution program was as follows:

[0059]

[0060] S3, Detection:

[0061] Under the above chromatographic conditions, 10 μl of the above test solution was accurately pipetted and analyzed by HPLC to obtain the chromatogram of Xinnaoning capsules.

[0062] Example 2: Establishment of a fingerprint spectroscopy method for the aqueous extract of Xinnaoning capsules

[0063] S1. Preparation of the test solution for Xinnaoning capsules:

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

[0065] S12. Accurately pipette 200 μl of the aqueous extract of Xinnaoning 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℃ 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 3 times; filter the aqueous layer through a 0.45 μm filter membrane, and take the filtrate as the test solution for Xinnaoning capsules;

[0066] S2, Chromatographic conditions:

[0067] The chromatographic column was a Kromasil C18, with dimensions of 4.6 × 250 mm and a diameter of 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 used at a flow rate of 0.8 mL / min, a column temperature of 35 °C, an injection volume of 10 μL, and a detection wavelength of 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 precisely taken and detected by HPLC to obtain the chromatogram of the Xinnaoling capsule.

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

[0072] S1, Preparation of the test solution of the Xinnaoling capsule:

[0073] S11, 0.5 g of the content of the Xinnaoling capsule was precisely weighed, 10 ml of water was added, the weight was supplemented, and the mixture was heated to reflux for 120 min, the weight was supplemented, the mixture was shaken, centrifuged, and the supernatant was taken to obtain the water extract of the Xinnaoling capsule;

[0074] S12, 200 μl of the water extract solution of the Xinnaoling capsule was precisely taken into a centrifuge tube with a plug, 200 μl of 0.3 mol / L NaOH was added, the mixture was thoroughly mixed, 160 μl of 0.5 mol / L PMP methanol solution was added, the mixture was mixed and then placed in a 70℃ water bath for heating for 30 min, the mixture was cooled to room temperature, 200 μl of 0.3 mol / L HCl solution was added to neutralize the alkali, 1240 μl of purified water was added, 2 ml of chloroform was added, the mixture was shaken and centrifuged at 3500 rpm for 5 min, the chloroform layer was discarded, and the extraction was repeated for 3 times; the water layer was filtered through a 0.45 μm filter membrane, and the filtrate was taken as the test solution of the Xinnaoling capsule;

[0075] S2, Chromatographic conditions:

[0076] The chromatographic column was Kromasil C18 with a specification of 4.6 x 250 mm and 5 μm; the mobile phase A was 0.1 mol / L potassium dihydrogen phosphate buffer salt (40% NaOH was added to adjust the pH to 6.7), the mobile phase B was acetonitrile, gradient elution, the flow rate was 0.8 ml / min; the column temperature was 35℃; the injection volume was 10 μl; the detection wavelength was 250 nm; and 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 precisely taken and detected by HPLC to obtain the chromatogram of the Xinnaoling capsule.

[0080] Example 4: Establishment of the control fingerprint of the water extract of the Xinnaoling capsule

[0081] The chromatogram of the Xinnaoning capsule obtained in Example 1 is introduced into the traditional Chinese medicine fingerprint similarity evaluation software, the latest batch sample is used as a reference spectrum, the average method is selected, the time window width is set to 0.5, the multi-point correction is used, the full spectrum peak matching is used, the Xinnaoning capsule material standard water extract control fingerprint spectrum is generated, the production control fingerprint spectrum is used as a reference, the similarity of each batch of Xinnaoning capsule material standard water extract fingerprint spectrum and the control spectrum is calculated, and the similarity is not less than 0.90.

[0082] Example 5: Establishment of Xinnaoning capsule water extract control fingerprint spectrum

[0083] The chromatogram of the Xinnaoning capsule obtained in Example 2 is introduced into the traditional Chinese medicine fingerprint similarity evaluation software, the latest batch sample is used as a reference spectrum, the average method is selected, the time window width is set to 0.4, the multi-point correction is used, the full spectrum peak matching is used, the Xinnaoning capsule material standard water extract control fingerprint spectrum is generated, the production control fingerprint spectrum is used as a reference, the similarity of each batch of Xinnaoning capsule material standard water extract fingerprint spectrum and the control spectrum is calculated, and the similarity is not less than 0.90.

[0084] Example 6: Establishment of Xinnaoning capsule water extract control fingerprint spectrum

[0085] The chromatogram of the Xinnaoning capsule obtained in Example 3 is introduced into the traditional Chinese medicine fingerprint similarity evaluation software, the latest batch sample is used as a reference spectrum, the average method is selected, the time window width is set to 0.2, the multi-point correction is used, the full spectrum peak matching is used, the Xinnaoning capsule material standard water extract monosaccharide control fingerprint spectrum is generated; the production control fingerprint spectrum is used as a reference, the similarity of each batch of Xinnaoning capsule material standard water extract fingerprint spectrum and the control spectrum is calculated, and the similarity is not less than 0.90.

[0086] In order to further verify the reliability of the present application, the inventors have carried out a series of tests, as follows:

[0087] I. Establishment of Xinnaoning capsule water extract fingerprint spectrum determination method

[0088] 1. Experimental instruments and materials

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

[0090] 1.2 Experimental reagents: acetonitrile (chromatographic pure, Grace Company); methanol, sodium hydroxide, potassium dihydrogen phosphate (analytical pure, Tianjin Chuyuan Chemical Reagent Co., Ltd.); 3-methyl-1-phenyl-5-pyrazolone (PMP) (analytical pure, Aladdin Holding Group Co., Ltd.); hydrochloric acid (analytical pure, 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 test sample solution extraction method

[0094] 2.1.1 Chromatographic conditions

[0095] Chromatographic column Kromasil C18 (4.6*250mm, 5μm); mobile phase: 0.1mol / L potassium dihydrogen phosphate buffer salt (40% NaOH to pH 6.7) (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: Equilibrate with 13% acetonitrile for 10min before column.

[0099] 2.1.2 Preparation of test sample solution

[0100] Extraction method one: ultrasonic extraction

[0101] Accurately weigh 1 g of Xinnaoning capsule contents, add 10 ml of water, weigh, ultrasonic extraction for 60 min, make up the weight, shake well, centrifuge, take the supernatant, i.e. Xinnaoning water extract. Accurately pipette 200 μl of Xinnaoning water extract solution into a centrifuge tube with a stopper, add 0.3 mol / L NaOH 200 μl, mix well, add 0.5 mol / L PMP methanol solution 160 μl, mix well, and then place in a 70°C water bath for heating for 30 min, cool to room temperature, neutralize the alkali with 0.3 mol / L HCl solution 200 μl, add purified water 1240 μl, add chloroform 2 ml, shake well, centrifuge at 3500 rpm for 5 min, discard the chloroform layer, repeat the extraction for 3 times. Pass the water layer through a 0.45 μm filter membrane, take the filtrate as the test sample solution 1.

[0102] Extraction method two: heating reflux extraction

[0103] Accurately weigh 1 g of Xinnaoning capsule contents, add 10 ml of water, weigh, heating reflux extraction for 60 min, make up the weight, shake well, centrifuge, take the supernatant, i.e. Xinnaoning water extract. Accurately pipette 200 μl of Xinnaoning water extract solution into a centrifuge tube with a stopper, add 0.3 mol / L NaOH 200 μl, mix well, add 0.5 mol / L PMP methanol solution 160 μl, mix well, and then place in a 70°C water bath for heating for 30 min, cool to room temperature, neutralize the alkali with 0.3 mol / L HCl solution 200 μl, add purified water 1240 μl, add chloroform 2 ml, shake well, centrifuge at 3500 rpm for 5 min, discard the chloroform layer, repeat the extraction for 3 times. Pass the water layer through a 0.45 μm filter membrane, take the filtrate as the test sample solution 2.

[0104] 2.1.3 Determination method

[0105] According to the chromatographic conditions, accurately pipette 10 μl of each of the above test sample solutions 1 and 2, respectively, and detect by HPLC, compare the determination results, and preferably select the best extraction method.

[0106] 2.1.4 Determination results

[0107] The experimental results are shown in Figures 1 to 3 According to the experimental results, the chromatographic peaks at 18-28 min are better separated by heating reflux extraction, and are poorer separated by ultrasonic extraction; according to the total peak area of the main chromatographic peaks, the total peak area is larger by heating reflux extraction, and the extraction method of the test sample is selected as heating reflux extraction.

[0108] 2.2 Investigation of the extraction solvent multiple of the test sample solution

[0109] 2.2.1 Chromatographic conditions: same as "2.1.1 Chromatographic conditions".

[0110] 2.2.2 Preparation of test sample solution

[0111] Test solution one (5 times solvent): precisely weigh 2 g of Xinnualing capsule content, add 10 ml of water, weigh, heat reflux extraction for 60 min, make up the weight, shake well, centrifuge, take the supernatant, and Xinnualing water extract is obtained. Accurately pipette 200 μl of Xinnualing water extract solution into a centrifuge tube with a plug, add 0.3 mol / L NaOH 200 μl, mix well, add 0.5 mol / L PMP methanol solution 160 μl, mix well, and then place in a 70°C water bath for heating for 30 min, cool to room temperature, neutralize the alkali with 0.3 mol / L HCl solution 200 μl, add purified water 1240 μl, add chloroform 2 ml, shake well, centrifuge at 3500 rpm for 5 min, discard the chloroform layer, repeat extraction for 3 times. The water layer is filtered through a 0.45 μm filter membrane, and the filtrate is used as test solution 1.

[0112] Test solution two (10 times solvent): precisely weigh 1 g of Xinnualing capsule content, add 10 ml of water, weigh, heat reflux extraction for 60 min, make up the weight, shake well, centrifuge, take the supernatant, and Xinnualing water extract is obtained. Accurately pipette 200 μl of Xinnualing water extract solution into a centrifuge tube with a plug, add 0.3 mol / L NaOH 200 μl, mix well, add 0.5 mol / L PMP methanol solution 160 μl, mix well, and then place in a 70°C water bath for heating for 30 min, cool to room temperature, neutralize the alkali with 0.3 mol / L HCl solution 200 μl, add purified water 1240 μl, add chloroform 2 ml, shake well, centrifuge at 3500 rpm for 5 min, discard the chloroform layer, repeat extraction for 3 times. The water layer is filtered through a 0.45 μm filter membrane, and the filtrate is used as test solution 2.

[0113] Test solution three (20 times solvent): precisely weigh 0.5 g of Xinnualing capsule content, add 10 ml of water, weigh, heat reflux extraction for 60 min, make up the weight, shake well, centrifuge, take the supernatant, and Xinnualing water extract is obtained. Accurately pipette 200 μl of Xinnualing water extract solution into a centrifuge tube with a plug, add 0.3 mol / L NaOH 200 μl, mix well, add 0.5 mol / L PMP methanol solution 160 μl, mix well, and then place in a 70°C water bath for heating for 30 min, cool to room temperature, neutralize the alkali with 0.3 mol / L HCl solution 200 μl, add purified water 1240 μl, add chloroform 2 ml, shake well, centrifuge at 3500 rpm for 5 min, discard the chloroform layer, repeat extraction for 3 times. The water layer is filtered through a 0.45 μm filter membrane, 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 above test sample solutions 1, 2 and 3 was precisely taken and subjected to HPLC detection, and the detection results were compared to select the best extraction solvent multiple.

[0116] 2.2.4 Detection results

[0117] The experimental results are shown in Table 2.2.4. Figures 4 to 7 According to the experimental results, the separation of the 5-fold solvent and 10-fold solvent was better for the chromatographic peaks at 18-28 min, and the separation of the 20-fold solvent was poorer. According to the total peak area of the main chromatographic peaks, the total peak area of the 10-fold solvent spectrum was the largest, and the extraction solvent multiple of the test sample was selected as 10-fold solvent.

[0118] 2.3 Investigation of extraction solvent time of test sample solution

[0119] 2.3.1 Chromatographic conditions: same as "2.1.1 Chromatographic conditions".

[0120] 2.3.2 Preparation of test sample solution

[0121] Test sample solution one (extraction for 30 min): 1 g of the contents of Xinnangning capsules was precisely weighed, 10 ml of water was added, the weight was adjusted, and the mixture was extracted by heating reflux for 30 min. The weight was adjusted, the mixture was shaken and centrifuged, and the supernatant was obtained, which was Xinnangning water extract. 200 μl of the Xinnangning water extract solution was precisely taken into a centrifuge tube with a stopper, 200 μl of 0.3 mol / L NaOH was added, the mixture was thoroughly mixed, 160 μl of 0.5 mol / L PMP methanol solution was added, the mixture was mixed and then placed in a 70°C water bath for heating for 30 min, the mixture was cooled to room temperature, 200 μl of 0.3 mol / L HCl solution was added to neutralize the alkali, 1240 μl of purified water was added, 2 ml of chloroform was added, the mixture was shaken and centrifuged at 3500 rpm for 5 min, the chloroform layer was discarded, and the extraction was repeated for 3 times. The water layer was filtered through a 0.45 μm filter membrane, and the filtrate was used as test sample solution 1.

[0122] Test sample solution two (extraction for 60 min): 1 g of the contents of Xinnangning capsules was precisely weighed, 10 ml of water was added, the weight was adjusted, and the mixture was extracted by heating reflux for 60 min. The weight was adjusted, the mixture was shaken and centrifuged, and the supernatant was obtained, which was Xinnangning water extract. 200 μl of the Xinnangning water extract solution was precisely taken into a centrifuge tube with a stopper, 200 μl of 0.3 mol / L NaOH was added, the mixture was thoroughly mixed, 160 μl of 0.5 mol / L PMP methanol solution was added, the mixture was mixed and then placed in a 70°C water bath for heating for 30 min, the mixture was cooled to room temperature, 200 μl of 0.3 mol / L HCl solution was added to neutralize the alkali, 1240 μl of purified water was added, 2 ml of chloroform was added, the mixture was shaken and centrifuged at 3500 rpm for 5 min, the chloroform layer was discarded, and the extraction was repeated for 3 times. The water layer was filtered through a 0.45 μm filter membrane, and the filtrate was used as test sample solution 2.

[0123] Test solution three (extraction for 90 min): 1 g of the contents of the Xinnaoning capsules was precisely weighed, 10 ml of water was added, the weight was adjusted, and the mixture was extracted by reflux for 90 min. The weight was adjusted, the mixture was shaken, and centrifuged. The supernatant was obtained, and was used as the water extract of the Xinnaoning capsules. 200 μl of the water extract solution was precisely pipetted into a centrifuge tube with a stopper, 200 μl of 0.3 mol / L NaOH was added, the mixture was thoroughly mixed, 160 μl of 0.5 mol / L PMP methanol solution was added, the mixture was mixed, and was placed in a water bath at 70°C for 30 min. The mixture was cooled to room temperature, 200 μl of 0.3 mol / L HCl solution was added to neutralize the alkali, 1240 μl of purified water was added, 2 ml of chloroform was added, the mixture was shaken, and was centrifuged at 3500 rpm for 5 min. The chloroform layer was discarded, and the extraction was repeated for 3 times. The water layer was filtered through a 0.45 μm filter membrane, and the filtrate was used as test solution 3.

[0124] Test solution four (extraction for 120 min): 1 g of the contents of the Xinnaoning capsules was precisely weighed, 10 ml of water was added, the weight was adjusted, and the mixture was extracted by reflux for 120 min. The weight was adjusted, the mixture was shaken, and centrifuged. The supernatant was obtained, and was used as the water extract of the Xinnaoning capsules. 200 μl of the water extract solution was precisely pipetted into a centrifuge tube with a stopper, 200 μl of 0.3 mol / L NaOH was added, the mixture was thoroughly mixed, 160 μl of 0.5 mol / L PMP methanol solution was added, the mixture was mixed, and was placed in a water bath at 70°C for 30 min. The mixture was cooled to room temperature, 200 μl of 0.3 mol / L HCl solution was added to neutralize the alkali, 1240 μl of purified water was added, 2 ml of chloroform was added, the mixture was shaken, and was centrifuged at 3500 rpm for 5 min. The chloroform layer was discarded, and the extraction was repeated for 3 times. The water layer was filtered through a 0.45 μm filter membrane, and the filtrate was used as test solution 4.

[0125] 2.3.3 Determination method

[0126] According to the chromatographic conditions, 10 μl of each of the above test solutions 1, 2, 3, and 4 was precisely pipetted, and was detected by HPLC. The determination results were compared, and the optimal extraction time was selected.

[0127] 2.3.4 Determination results

[0128] The experimental results are shown in Table 1. Figures 8 to 12 According to the experimental results, the chromatographic peaks at 18-28 min were separated well, and the separation was better at 90 min and 120 min. According to the total peak area of the main chromatographic peaks, the total peak area of the chromatogram at 90 min was larger, and the total peak area at 120 min did not increase obviously. Therefore, the extraction time of the test sample was selected as 90 min.

[0129] 3. Conclusion

[0130] Through the optimization of the preparation of the test sample solution and the chromatographic conditions of the determination, the optimal determination method of the Xinnaoning capsules was obtained, and was as follows:

[0131] (1) Chromatographic conditions

[0132] Chromatographic column Kromasil C18 (4.6*250mm, 5μm); mobile phase: 0.1mol / L potassium dihydrogen phosphate buffer (pH 6.7 adjusted 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: Equilibrate with 13% acetonitrile for 10 min before column.

[0136] (2) Preparation of test solution

[0137] Precisely weigh 1g of the contents of Xinnangling capsules, add 10ml of water, weigh, heat and reflux extract for 90min, make up the weight, shake well, centrifuge, and take the supernatant, which is the Xinnangling water extract. Accurately pipette 200μl of the Xinnangling water extract solution into a centrifuge tube with a stopper, add 200μl of 0.3mol / L NaOH, mix thoroughly, add 160μl of 0.5mol / L PMP methanol solution, mix well, and then place in a 70℃ water bath for heating for 30min, cool to room temperature, neutralize the alkali with 200μl of 0.3mol / L HCl solution, add 1240μl of purified water, add 2ml of chloroform, shake well, centrifuge at 3500rpm for 5min, discard the chloroform layer, and repeat the extraction for 3 times. Pass the water layer through a 0.45μm filter membrane, and take the filtrate as the test solution.

[0138] (3) Determination method

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

[0140] II. Methodology investigation of Xinnangling capsule water extract fingerprint

[0141] 2.1 Precision test

[0142] Take Xinnangling capsules (batch number: 20190336), prepare the test solution according to the preparation method of test solution, determine according to the chromatographic conditions, the injection volume is 10μl, continuously inject 6 times, determine the fingerprint, and record the retention time and peak area of each common chromatographic peak. Take the retention time and peak area of peak No. 9 as the 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 14 and Tables 2 to 5.

[0143] Table 2 Precision study - retention time

[0144]

[0145] Table 3 Precision study - peak area

[0146]

[0147] Table 4 Precision study - relative retention time

[0148]

[0149] Table 5 Precision study - relative peak area

[0150]

[0151] By analyzing Figure 14 and the precision test results table, it is concluded that in the precision test, the RSD value of the retention time is less than

[0152] 1.00%, the RSD value of the peak area is less than 5.00%, 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 4.00%, all less than 5.00%. It can be seen that the precision of the instrument is good.

[0153] 2.2 Reproducibility test

[0154] Take the Xinnuanling capsule (batch number: 20190336), according to the preparation method of the test solution, prepare 6 test solutions, and measure according to the chromatographic conditions, the injection amount is 10 μl, measure the fingerprint, record the retention time and peak area of each common chromatographic peak. Take the retention time and peak area of No. 9 chromatographic peak as the 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 Reproducibility study - retention time

[0156]

[0157] Table 7 Reproducibility study - peak area

[0158]

[0159] Table 8 Reproducibility study - relative retention time

[0160]

[0161] Table 9 Reproducibility study - relative peak area

[0162]

[0163] By analyzing the above Figure 15 and the repeatability test results table, it is concluded that in the repeatability test, the RSD values of the retention time are less than 1.00%, the RSD values of the chromatographic peak peak area are less than 4.00%, the RSD values of the relative retention time are less than 1.00%, the RSD values of the relative peak area are less than 5.00%, and the RSD values are less than 5.00%, which indicates that the experiment has good repeatability.

[0164] 1.00%, the RSD values of the chromatographic peak peak area are less than 4.00%, the RSD values of the relative retention time are less than 1.00%, the RSD values of the relative peak area are less than 5.00%, and the RSD values are less than 5.00%, which indicates that the experiment has good repeatability.

[0165] 2.3 Stability test

[0166] Take Xinnangning capsules (batch number: 20190336), prepare the test sample solution according to the preparation method of the test sample solution, and determine according to the chromatographic conditions, the injection amount is 10 μl, and the sample is injected and determined at 0 h, 2 h, 4 h, 8 h and 12 h, the fingerprint spectrum is determined, and the retention time and peak area of each common chromatographic peak are recorded. With the retention time and peak area of No. 9 chromatographic peak as the 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 obtained. The results are shown in Figure 16 and Tables 10 to 13.

[0167] Table 10 Stability test-retention time

[0168]

[0169] Table 11 Stability test-peak area

[0170]

[0171] Table 12 Stability test-relative retention time

[0172]

[0173] Table 13 Stability test-relative peak area

[0174]

[0175] By analyzing the above Figure 16 and the stability test results table, it is concluded that in the stability test, the RSD values of the retention time are less than 1.00%, the RSD values of the peak area are less than 5.00%, the RSD values of the relative retention time are less than 1.00%, the RSD values of the relative peak area of the chromatographic peak are less than 5.00%, and the RSD values are less than 5%, which indicates that the prepared sample has good stability within 12 h.

[0176] 2.4 Conclusion

[0177] Using the established chromatographic conditions of Xinnaoning Capsules, the precision, repeatability and stability in the methodology study were researched. The RSD values of retention time, peak area, relative retention time and relative peak area in each investigation item were less than 5.00%, which proved that the instrument had good precision, the determination method had good repeatability, and the prepared sample had good stability within 12 h.

[0178] III. Similarity Study of Water Extract Fingerprint of Xinnaoning Capsules

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

[0180] Table 14 Codes and Batch Numbers of Xinnaoning Capsules

[0181]

[0182] 3.1 Experimental Method

[0183] The determination was performed according to the optimal determination method of Xinnaoning Capsules determined in the above "3 Conclusion" item.

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

[0185] The 26 batches of samples were detected by HPLC to obtain the fingerprint. The obtained spectra were analyzed by the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software (2012 Edition)" software of the State Pharmacopoeia Commission. The latest batch (20190722) sample S25 was used as the reference spectrum. The average method was used for the control spectrum generation method. The time window width was set to 0.3. The multi-point correction was used for full spectrum peak matching to generate the control spectrum, as shown in Figures 17 to 18 . The similarity was calculated, and the results are shown in Table 15 below.

[0186] Table 15 Similarity Results of HPLC Fingerprint of Water Extract of Xinnaoning Capsules (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. This indicated that the sample similarity was high, and the spectrum difference was small.

[0189] (2) Analysis of 2017 samples

[0190] The HPLC fingerprint of the water extract of the Xinnaoling capsules was obtained by HPLC detection of the 2017 samples. The obtained chromatograms were analyzed by the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software of the State Pharmacopoeia Commission, with the latest batch (20171111) sample S9 as the reference chromatogram. The average method was used for the generation of the reference chromatogram, the time window width was set to 0.2, the multi-point correction was used, and the full spectrum peak matching was used to generate the reference chromatogram, as shown in Figures 19 to 20 The similarity was calculated, and the results are shown in Table 16.

[0191] Table 16 Similarity results of HPLC fingerprint of water extract of Xinnaoling capsules (2017 batch)

[0192]

[0193] According to the experimental results, the similarity of the 2017 samples was greater than 0.970, and the average similarity was 0.986, indicating that the sample similarity was high, and the difference between the chromatograms was small.

[0194] (3) Sample analysis in 2018

[0195] The HPLC fingerprint of the water extract of the Xinnaoling capsules was obtained by HPLC detection of the 2017 samples. The obtained chromatograms were analyzed by the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software of the State Pharmacopoeia Commission, with the latest batch (20181133) sample S4 as the reference chromatogram. The average method was used for the generation of the reference chromatogram, the time window width was set to 0.2, the multi-point correction was used, and the full spectrum peak matching was used to generate the reference chromatogram, as shown in Figures 21 to 22 The similarity was calculated, and the results are shown in Table 17.

[0196] Table 17 Similarity results of HPLC fingerprint of water extract of Xinnaoling capsules (2018 batch)

[0197]

[0198] According to the experimental results, the similarity of the 2018 samples was greater than 0.970, and the average similarity was 0.987, indicating that the sample similarity was high, and the difference between the chromatograms was small.

[0199] (4) Sample analysis in 2019

[0200] The HPLC fingerprint of the water extract of the Xinnaoling capsules was obtained by HPLC detection of the 2017 samples. The obtained chromatograms were analyzed by the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software of the State Pharmacopoeia Commission, with the latest batch (20190722) sample S11 as the reference chromatogram. The average method was used for the generation of the reference chromatogram, the time window width was set to 0.3, the multi-point correction was used, and the full spectrum peak matching was used to generate the reference chromatogram, as shown in Figures 23 to 24, the results are shown in Table 18.

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

[0202]

[0203] According to the experimental results, the similarity of 11 batches of samples in 2019 is greater than 0.970, and the average similarity is 0.981, indicating that the sample similarity is high and the spectrum difference is small.

[0204] (5) Analysis of 4-year control spectrum of Xinnaoning Capsule

[0205] Because there is only one batch of sample in 2016, the sample of 20160916 represents the sample of 2016, and the control spectrum of 2017, 2018 and 2019 is analyzed to analyze the similarity of samples in 4 years. The obtained spectrum was analyzed by using the software of "Chinese medicine chromatographic fingerprint similarity software evaluation system (2012 edition)" of the State Pharmacopoeia Committee, taking the sample S4 of the latest year (2019 control spectrum) as the reference spectrum, the control spectrum generation method was selected as the average method, the time window width was set to 0.2, the multi-point correction was used, and the control spectrum was generated by full spectrum peak matching, see Figures 25 to 26 , the results are shown in Table 19.

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

[0207]

[0208] According to the experimental results, the similarity of 4-year control spectrum is greater than 0.980, and the average similarity is 0.991, indicating that the sample similarity is high and the spectrum difference is small.

[0209] 3.2 Conclusion

[0210] 1) The similarity of 26 batches of samples from 2016 to 2019 is greater than 0.950, and the average similarity is 0.976. The similarity of samples in the same year is analyzed, the similarity of 9 batches of samples in 2017 is greater than 0.970, and the average similarity is 0.986, the similarity of 5 batches of samples in 2018 is greater than 0.970, and the average similarity is 0.986, the similarity of 11 batches of samples in 2019 is greater than 0.970, and the average similarity is 0.981.

[0211] 2) The similarity of samples in 4 years is analyzed by taking 1 batch of sample in 2016 as the control spectrum of 2017, 2018 and 2019, and the similarity of 4-year control spectrum is greater than 0.980, and the average similarity is 0.991.

[0212] 3) According to the experimental results, 26 batches of samples in 4 years, all batches and annual samples have high similarity and small spectrum difference.

[0213] From the above experimental conclusions, the fingerprint similarity between each sample of Xinnaoning Capsule is high, and the difference is small, which indicates that the overall characteristic difference of the water extract components in each batch of Xinnaoning Capsule is small, the quality of the water extract is stable, meets the analysis requirements of the fingerprint spectrum, and reflects the overall stability and uniformity of the sample. It is shown that the water extract fingerprint spectrum determination method established by the present application is stable and reliable, and has important significance for the component analysis and identification and quality control of Xinnaoning Capsule preparation.

[0214] Although the present application has been described in detail in the foregoing description with general description, specific embodiments and experiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application.

Claims

1. A method for establishing the fingerprint spectrum of Xinnualing capsule water extract, characterized in that, It comprises the following steps: S1, preparation of Xinnaoning capsule test solution: S11, take the content of Xinnaoning capsule, add 10 times the amount of water, weigh, heat reflux extraction for 90 min, add water to make up the weight, shake well, centrifuge, take the supernatant, namely Xinnaoning water extract; S12, accurately pipette 200 μl of Xinnaoning water extract solution into a centrifuge tube with a plug, add 200 μl of 0.3 mol / L NaOH, mix well, add 160 μl of 0.5 mol / L PMP methanol solution, the PMP is 1-phenyl-3-methyl-5-pyrazolone, mix well, place in a 70°C water bath for 30 min, cool to room temperature, neutralize the alkali with 200 μl of 0.3 mol / L HCl solution, add 1240 μl of purified water, add 2 ml of chloroform, shake well, centrifuge for 5 min, discard the chloroform layer, repeat the extraction for 3 times; pass the water layer through a 0.45 μm filter membrane, take the filtrate as the test solution of Xinnaoning capsule; S2, chromatographic conditions: The chromatographic column is Kromasil C18, with a specification of 4.6x250 mm, 5 μm; the mobile phase A is 0.1 mol / L potassium dihydrogen phosphate buffer salt with pH adjusted to 6.7 by 40% NaOH, the mobile phase B is acetonitrile, gradient elution, flow rate: 0.8 ml / min; column temperature: 35°C; injection volume 10 μl; detection wavelength: 250 nm; the gradient elution program is as follows: ； S3, detection: According to the above chromatographic conditions, accurately pipette 10 μl of the above test solution, detect by HPLC, obtain the chromatogram of Xinnaoning capsule test solution; S4, fingerprint generation: Import the obtained chromatogram of Xinnaoning capsule test solution into traditional Chinese medicine fingerprint similarity evaluation software, take the latest batch sample as the reference spectrum, select the average method, set the time window width to 0.2-0.5, multi-point correction, full spectrum peak matching, generate the control fingerprint spectrum of Xinnaoning capsule material standard water extract, take the generated control fingerprint spectrum as the reference, calculate the similarity of each batch of Xinnaoning capsule material standard water extract fingerprint spectrum and the control spectrum, the similarity is not less than 0.

90.

2. The application of Xinnaoning capsule water extract fingerprint spectrum obtained by the method of claim 1 in characterizing the composition of Xinnaoning capsule water extract.

3. The application of Xinnaoning capsule water extract fingerprint spectrum obtained by the method of claim 1 in the quality control of Xinnaoning capsule.

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