Construction method and application of fingerprint spectrum of Golden Premna Herb capsule

Abstract

The invention provides a construction method and application of a fingerprint spectrum of a golden-firewood capsule, and belongs to the technical field of medicine detection.The construction method comprises the steps that a test solution is prepared firstly, then acetonitrile (A)-0.05-0.2 wt% phosphoric acid aqueous solution (B) serves as a mobile phase, the test solution is detected through high performance liquid chromatography, and the fingerprint spectrum of the golden-firewood capsule is obtained; a standard fingerprint spectrum is established by adopting 'Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System' in 2012 edition; the method is used for quality evaluation or control in the whole process of research / development / production / clinical application of the golden firewood capsules. According to the construction method of the fingerprint spectrum of the golden leaf capsule, main medicinal material components in the golden leaf capsule can be detected only through high performance liquid chromatography, and the quality condition of the golden leaf capsule is comprehensively reflected.

Description

Technical Field

[0001] The invention relates to drug detection technology, and in particular to a construction method and application of a Jinchai capsule fingerprint spectrum. Background Art

[0002] Jinchai Capsule is an antiviral drug composition, which is composed of honeysuckle, forsythia, bupleurum, scutellaria, pinellia, American ginseng, and cyperus rotundus. It has the functions of expelling pathogens and detoxifying, clearing the lungs and relieving heat. It is used for upper respiratory tract infection of wind-heat syndrome, with symptoms of fever, aversion to wind, dry throat and sore throat, nasal congestion and runny nose, headache, cough, etc. It is different from the traditional Chinese medicines used in Jinchai Kanggan Capsule, which is composed of seven traditional Chinese medicines: honeysuckle, isatis indigo leaf, forsythia, bupleurum, scutellaria, platycodon and menthol, and Jinchaihuang Granule, which is composed of honeysuckle extract, scutellaria extract and bupleurum extract.

[0003] Chinese invention patent CN200610081518.8 discloses the specific formula of Jinchai Capsules and its preparation method. Chinese invention patent application CN201410354037.4 discloses the quality control method of Jinchai Capsules, including the determination of chlorogenic acid and baicalin content, as well as the determination of baicalin, chlorogenic acid, forsythiaside, and ginsenoside Rb. 1 and Rg 1 Thin layer identification.

[0004] The spectrum of Chinese medicine preparations refers to the spectrum that can reflect the information of multiple components and reflect its quality characteristics after the samples of Chinese medicine preparations are properly processed and analyzed by appropriate methods. The characteristic spectrum of Chinese medicine preparations is of great significance for identifying the key quality attributes of Chinese medicine preparations, studying the transfer of quantity values, evaluating the uniformity and stability of the quality of Chinese medicine preparations, and improving the overall quality control level of Chinese medicine preparations.

[0005] Therefore, in order to strengthen specific identification and multi-component and overall quality control, it is necessary to control the overall quality of Jinchai Capsules by establishing a map. Summary of the invention

[0006] In view of the above problems, the present invention provides a method for constructing a fingerprint spectrum of Jinchai Capsules and its application.

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

[0008] A method for constructing a fingerprint spectrum of Jinchai capsules, the method comprising the following steps:

[0009] S1. Prepare the test solution by taking Jinchai capsules;

[0010] S2, taking the test solution and performing high performance liquid chromatography to obtain the fingerprint of the Jinchai capsule;

[0011] The mobile phase A of the HPLC detection is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution with a concentration of 0.05-0.2 wt%;

[0012] The elution method of the high performance liquid chromatography detection is gradient elution;

[0013] The elution conditions of the gradient elution are:

[0014] 0-10 min, 10%→15% mobile phase A, 90%→85% mobile phase B;

[0015] 10-50 min, 15% → 25% mobile phase A, 85% → 75% mobile phase B;

[0016] 50-60 min, 25% mobile phase A, 75% mobile phase B;

[0017] 60-90 min, 25% → 50% mobile phase A, 75% → 50% mobile phase B;

[0018] 90-100 min, 50% → 90% mobile phase A, 50% → 10% mobile phase B;

[0019] 100-100.1 min, 90% → 10% mobile phase A, 10% → 90% mobile phase B;

[0020] 100.1~110min, 10% mobile phase A, 90% mobile phase B.

[0021] Furthermore, the detection wavelength of the high performance liquid chromatography detection is 200-210 nm, and the chromatographic column is Agilent ZORBAX SB-C18 (250x4.6mm, 5um).

[0022] Furthermore, the test solution is prepared by extracting the contents of Jinchai capsules with methanol aqueous solution / dilute ethanol and filtering.

[0023] Further, the concentration of the methanol aqueous solution is 25 to 100 vol%;

[0024] The weight-to-volume ratio of the contents of Jinchai capsules to methanol aqueous solution / dilute ethanol is 0.8 g:20-40 mL.

[0025] Furthermore, the extraction time is 15 to 45 minutes, and the extraction method is ultrasound or reflux.

[0026] Furthermore, the flow rate of the HPLC detection is 0.8-1.2 mL / min, the column temperature is 20-30° C., and the injection volume is 5-20 μL.

[0027] Furthermore, the construction method further comprises performing high performance liquid chromatography detection on at least one of the single medicinal material solution, the negative sample solution lacking the single medicinal material, and the mixed reference solution;

[0028] The single herbal medicine solution is prepared by taking the single herbal medicines used in Jinchai Capsules respectively;

[0029] The negative sample solution lacking a single medicinal material is prepared by using the method for preparing Jinchai capsules to prepare the contents of capsules lacking a single medicinal material, and then extracting to prepare the negative sample solution lacking a single medicinal material;

[0030] The mixed reference solution is made of chlorogenic acid, baicalin, forsythiaside, ginsenoside Rb 1 , made by adding methanol.

[0031] An application of the standard fingerprint spectrum of Jinchai Capsule obtained by the above construction method in the quality evaluation or control of the whole process of research / development / production / clinical application of Jinchai Capsule.

[0032] Furthermore, a total of 13 common peaks were identified in the standard fingerprint of the Jinchai capsule;

[0033] Among them, peak 3 is chlorogenic acid, peak 4 is cryptochlorogenic acid, peak 7 is forsythiaside A, peak 9 is baicalin, peak 10 is forsythiaside, peak 11 is wogonin, peak 12 is ginsenoside Rb 1 , Peak 13 is wogonin.

[0034] Furthermore, in the standard fingerprint of Jinchai Capsule, peak 3 is S1 peak, and the relative retention time of peaks 1 to 11 and S1 peak is calculated; peak 12 is S2 peak, and the relative retention time of peak 13 and S2 peak is calculated;

[0035] Among them, the specified value of peak 1 is 0.60, the specified value of peak 2 is 0.66, the specified value of peak 4 is 1.06, the specified value of peak 5 is 2.12, the specified value of peak 6 is 2.36, the specified value of peak 7 is 2.43, the specified value of peak 8 is 2.79, the specified value of peak 9 is 4.39, the specified value of peak 10 is 4.55, the specified value of peak 11 is 5.90, and the specified value of peak 13 is 1.16.

[0036] The beneficial effects of the construction method and application of the Jinchai capsule fingerprint spectrum of the present invention are:

[0037] The method for constructing the fingerprint of Jinchai Capsule of the present invention can detect the main medicinal ingredients in Jinchai Capsule by high performance liquid chromatography, and comprehensively reflect the quality status of Jinchai Capsule;

[0038] The present invention combines the different physical and chemical properties of different effective ingredients in Jinchai Capsules and adjusts the elution conditions to obtain a standard fingerprint spectrum with good separation, a relatively stable baseline, more peaks and better reflection of the quality of Jinchai Capsules; the elution conditions of the present invention are significantly better than other elution conditions;

[0039] The present invention achieves the purpose of separating and simultaneously detecting different effective components in Jinchai Capsules by adjusting the detection wavelength; the detection wavelength of the present invention is significantly better than other detection wavelengths;

[0040] The present invention obtains a standard fingerprint spectrum with good separation, relatively stable baseline, more peaks and better ability to reflect the quality of Jinchai Capsules by adjusting the mobile phase.

[0041] The present invention can more fully extract the medicinal ingredients involved in the Jinchai capsule by adjusting the extraction solvent and the extraction volume, and the concentration of each medicinal ingredient in the obtained test solution can be separated and detected under the same high performance liquid chromatography conditions, and the peaks in the obtained chromatogram are good;

[0042] The present invention effectively optimizes the separation conditions by selecting appropriate process conditions such as chromatographic column, column temperature, flow rate, etc., thereby improving the peak stability of the standard fingerprint spectrum of Jinchai capsules;

[0043] The construction method of the present invention also has good specificity, precision, repeatability, stability and durability;

[0044] The standard fingerprint spectrum of Jinchai Capsule obtained by the present invention can provide effective guarantee for the quality monitoring of Jinchai Capsule in the whole process of research, development, production and clinical application.

[0045] By using the presence or absence and characteristics of the common characteristic peaks in the standard fingerprint spectrum of Jinchai Capsules obtained by the present invention, the product quality of Jinchai Capsules can be fully monitored. By comparing the similarity of the chromatographic fingerprint characteristics, the quality of Jinchai Capsules can be evaluated, and the stability and uniformity of Jinchai Capsules can be examined, which makes up for the shortcomings of the current quality control method. At the same time, the stability of the production process of Jinchai Capsules can also be monitored to ensure the stability, uniformity and controllability of its quality.

[0046] The standard fingerprint of Jinchai Capsule obtained by the present invention improves the quality monitoring standard of finished and semi-finished products of Jinchai Capsule, effectively prevents the occurrence of product counterfeiting incidents, and ensures the normal production and circulation order of Jinchai Capsule; on the basis of the present invention, the correlation study between fingerprint information and pharmacodynamic activity information can also be carried out, so as to deeply clarify the correlation between the intrinsic chemical composition of Jinchai Capsule and the efficacy of the preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1It is the attribution chromatogram of the chromatographic peaks of honeysuckle in Example 1 of the present invention, wherein, from top to bottom, they are the fingerprint of the negative sample solution lacking honeysuckle, the fingerprint of the single medicinal material solution of honeysuckle, and the fingerprint of the Jinchai capsule sample;

[0048] Figure 2 It is the chromatogram of the chromatographic peak attribution of Forsythia suspensa in Example 1 of the present invention, wherein, from top to bottom, they are the fingerprint of the negative sample solution lacking Forsythia suspensa, the fingerprint of the Forsythia suspensa single medicinal material solution, and the fingerprint of the Jinchai capsule sample;

[0049] Figure 3 It is the chromatogram of the chromatographic peak attribution of Scutellaria baicalensis in Example 1 of the present invention, wherein, from top to bottom, they are the fingerprint of the negative sample solution lacking Scutellaria baicalensis, the fingerprint of the single medicinal material solution of Scutellaria baicalensis, and the fingerprint of the Jinchai capsule sample;

[0050] Figure 4 It is the attribution chromatogram of the chromatographic peaks of American ginseng in Example 1 of the present invention, wherein, from top to bottom, they are the fingerprint of the negative sample solution lacking American ginseng, the fingerprint of the American ginseng single medicinal material solution, and the fingerprint of the Jinchai capsule sample;

[0051] Figure 5 It is the chromatogram of the chromatographic peak attribution of Bupleurum in Example 1 of the present invention, wherein, from top to bottom, they are the fingerprint of the negative sample solution lacking Bupleurum, the fingerprint of the Bupleurum single medicinal material solution, and the fingerprint of the Jinchai capsule sample;

[0052] Figure 6 It is the attribution chromatogram of the chromatographic peaks of the Mianma Guanzhong in Example 1 of the present invention, wherein, from top to bottom, they are the fingerprint of the negative sample solution lacking Mianma Guanzhong, the fingerprint of the Mianma Guanzhong single medicinal material solution, and the fingerprint of the Jinchai capsule sample;

[0053] Figure 7 It is the attribution chromatogram of the chromatographic peaks of Qing Banxia in Example 1 of the present invention, wherein, from top to bottom, they are the fingerprint of the negative sample solution lacking Qing Banxia, ​​the fingerprint of the Qing Banxia single medicinal material solution, and the fingerprint of the Jinchai capsule sample;

[0054] Figure 8 It is the chromatographic peak identification spectrum in Example 1 of the present invention, wherein, from top to bottom, they are the fingerprint spectrum of reference solution 1, the fingerprint spectrum of reference solution 2, the fingerprint spectrum of the mixed reference solution, the fingerprint spectrum of reference solution 3, the fingerprint spectrum of reference solution 4, and the fingerprint spectrum of the Jinchai capsule sample;

[0055] Fig. 9 It is the standard fingerprint of Jinchai Capsule in Example 7 of the present invention;

[0056] Fig.10It is a comparison map generated by the fingerprint similarity evaluation software of 10 batches of Jinchai capsule samples in Example 7 of the present invention;

[0057] Fig.11 It is a comparative spectrum of the specificity test in Example 8 of the present invention, wherein, from top to bottom, it is the fingerprint spectrum of the blank solvent, the fingerprint spectrum of dextrin, the fingerprint spectrum of the mixed reference substance and the fingerprint spectrum of the Jinchai capsule sample;

[0058] Fig.12 It is a comparative spectrum of durability tests at different column temperatures in Example 8 of the present invention, wherein, from top to bottom, it is the fingerprint spectrum of the Jinchai capsule sample when the column temperature is 20°C, the fingerprint spectrum of the Jinchai capsule sample when the column temperature is 25°C, and the fingerprint spectrum of the Jinchai capsule sample when the column temperature is 30°C;

[0059] Fig.13 It is a comparative graph of durability tests at different flow rates in Example 8 of the present invention, wherein, from top to bottom, they are the fingerprint of the Jinchai capsule sample at a flow rate of 0.8 mL / min, the fingerprint of the Jinchai capsule sample at a flow rate of 0.9 mL / min, the fingerprint of the Jinchai capsule sample at a flow rate of 1.0 mL / min, the fingerprint of the Jinchai capsule sample at a flow rate of 1.1 mL / min, and the fingerprint of the Jinchai capsule sample at a flow rate of 1.2 mL / min;

[0060] Fig.14 It is a comparative spectrum of durability tests of different chromatographic columns in Example 8 of the present invention, wherein, from top to bottom, they are the fingerprint spectrum of the Jinchai capsule sample when the chromatographic column is Agilent ZORBAX SB-C18 (250x4.6mm, 5um), the fingerprint spectrum of the Jinchai capsule sample when the chromatographic column is Agilent ZORBAX SB-C18 (250x4.6mm, 5um), the fingerprint spectrum of the Jinchai capsule sample when the chromatographic column is Agilent ZORBAX SB-Aq (250x4.6mm, 5um), and the fingerprint spectrum of the Jinchai capsule sample when the chromatographic column is COSMOSIL5C18-MS-II (250x4.6mm, 5um);

[0061] Fig.15 This is a comparative spectrum 1 of the investigation test of different extraction solvents in Example 9 of the present invention, wherein, from top to bottom, it is the fingerprint spectrum of the Jinchai capsule sample when the solvent is ethanol, the fingerprint spectrum of the Jinchai capsule sample when the solvent is water, and the fingerprint spectrum of the Jinchai capsule sample when the solvent is methanol;

[0062] Fig.16This is the second comparative spectrum of the investigation test of different extraction solvents in Example 9 of the present invention, wherein, from top to bottom, it is the fingerprint spectrum of the Jinchai capsule sample when the solvent is dilute ethanol, the fingerprint spectrum of the Jinchai capsule sample when the solvent is 75 vol% methanol aqueous solution, the fingerprint spectrum of the Jinchai capsule sample when the solvent is 25 vol% methanol aqueous solution, and the fingerprint spectrum of the Jinchai capsule sample when the solvent is 50 vol% methanol aqueous solution;

[0063] Fig.17 It is a comparative spectrum of the investigation test of different extraction methods in Example 9 of the present invention, wherein the upper figure is the fingerprint spectrum of the Jinchai capsule sample during ultrasonic extraction, and the lower figure is the fingerprint spectrum of the Jinchai capsule sample during reflux extraction;

[0064] Fig.18 It is a comparative spectrum of the investigation test of different extraction volumes in Example 9 of the present invention, wherein, from top to bottom, it is the fingerprint spectrum of the Jinchai capsule sample when the extraction volume is 20mL, the fingerprint spectrum of the Jinchai capsule sample when the extraction volume is 30mL, and the fingerprint spectrum of the Jinchai capsule sample when the extraction volume is 40mL;

[0065] Fig.19 It is a comparative spectrum of the investigation test of different extraction times in Example 9 of the present invention, wherein, from top to bottom, it is the fingerprint spectrum of the Jinchai capsule sample when the extraction time is 15 minutes, the fingerprint spectrum of the Jinchai capsule sample when the extraction time is 30 minutes, and the fingerprint spectrum of the Jinchai capsule sample when the extraction time is 45 minutes;

[0066] Fig. 20 It is a comparative spectrum of the investigation test of different detection wavelengths in Example 9 of the present invention, wherein, from top to bottom, it is the fingerprint spectrum of the Jinchai capsule sample when the detection wavelength is 203nm, the fingerprint spectrum of the Jinchai capsule sample when the detection wavelength is 327nm, the fingerprint spectrum of the Jinchai capsule sample when the detection wavelength is 240nm, and the fingerprint spectrum of the Jinchai capsule sample when the detection wavelength is 280nm;

[0067] Fig.21 It is a comparative spectrum of the investigation test of different elution conditions in Example 9 of the present invention, wherein, from top to bottom, it is the fingerprint spectrum of the Jinchai capsule sample using the elution condition one, the fingerprint spectrum of the Jinchai capsule sample using the elution condition two, the fingerprint spectrum of the Jinchai capsule sample using the elution condition three, and the fingerprint spectrum of the Jinchai capsule sample using the elution condition four;

[0068] Fig. 22It is a comparative spectrum of the different mobile phase investigation tests in Example 9 of the present invention, wherein, from top to bottom, they are the fingerprint spectrum of the Jinchai capsule sample when acetonitrile-0.1wt% phosphoric acid aqueous solution is used as the mobile phase, the fingerprint spectrum of the Jinchai capsule sample when acetonitrile-0.2wt% phosphoric acid aqueous solution is used as the mobile phase, the fingerprint spectrum of the Jinchai capsule sample when acetonitrile-0.05wt% phosphoric acid aqueous solution is used as the mobile phase, the fingerprint spectrum of the Jinchai capsule sample when acetonitrile-0.1wt% formic acid aqueous solution is used as the mobile phase, and the fingerprint spectrum of the Jinchai capsule sample when acetonitrile-0.1wt% acetic acid aqueous solution is used as the mobile phase. DETAILED DESCRIPTION

[0069] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0070] Example 1 A method for constructing a fingerprint of Jinchai capsule

[0071] In this embodiment, the contents of Jinchai capsules are used as Jinchai capsule samples, and the specific method for constructing the fingerprint of Jinchai capsules is as follows:

[0072] S1. Solution preparation

[0073] S11. Preparation of test solution

[0074] Take 0.8 g of Jinchai capsule sample, place it in a stoppered conical flask, accurately add 30 mL of 50 vol% methanol aqueous solution, ultrasonically treat for 30 min (power 300 W, frequency 40 kHz), cool, shake well, filter, and take the filtrate to obtain the test solution.

[0075] S12. Preparation of single herbal medicine solution

[0076] Honeysuckle, forsythia, bupleurum, scutellaria, pinellia tuber, American ginseng, and rhizoma cyrtomii are taken to prepare the single medicinal material dry paste powder corresponding to each single medicinal material according to the method disclosed in Chinese invention patent CN200610081518.8 (the medicinal materials corresponding to each single medicinal material dry paste powder are honeysuckle, forsythia, bupleurum, pinellia tuber, and rhizoma cyrtomii), scutellaria extract powder, and American ginseng powder;

[0077] Take the dry paste powder of each single medicinal material, the powder of Scutellaria baicalensis extract, and the powder of American ginseng to replace the contents of Jinchai Capsules, and prepare each single medicinal material solution according to the method for preparing the test sample solution.

[0078] S13. Preparation of negative sample solution lacking a single medicinal material

[0079] According to the method for preparing Jinchai capsules disclosed in Chinese invention patent CN200610081518.8, the contents of each capsule lacking a single medicinal material were prepared respectively, and then the contents of each capsule lacking a single medicinal material were used to replace the contents of Jinchai capsules respectively, and negative sample solutions lacking a single medicinal material were prepared according to the method for preparing test sample solutions.

[0080] S14. Preparation of reference solution

[0081] Accurately weigh an appropriate amount of wogonin reference substance and add methanol to prepare a reference substance solution containing 1.0 mg of wogonin per 1 mL.

[0082] Accurately weigh an appropriate amount of chlorogenic acid reference substance and add methanol to prepare a reference substance solution II containing 1.0 mg chlorogenic acid per 1 mL.

[0083] Accurately weigh an appropriate amount of wogonin reference substance and add methanol to prepare a reference substance solution containing 1.0 mg of wogonin per 1 mL.

[0084] Accurately weigh an appropriate amount of forsythiaside A reference substance and add methanol to prepare a reference substance solution containing 1.0 mg forsythiaside A per 1 mL.

[0085] Accurately weigh chlorogenic acid reference substance, baicalin reference substance, forsythiaside reference substance, ginsenoside Rb 1 An appropriate amount of reference substance was added with methanol to prepare a solution containing 0.3 mg chlorogenic acid, 1.0 mg baicalin, 70 μg forsythiaside, and 0.2 mg ginsenoside Rb per 1 mL. 1 The mixed solution was used as the mixed reference solution.

[0086] S2. Fingerprint Detection

[0087] S21. Test solution detection

[0088] Accurately pipette the test sample solutions respectively, inject them into the high performance liquid chromatograph, and perform high performance liquid chromatography detection to obtain the Jinchai Capsule spectrum (i.e. the fingerprint spectrum of the Jinchai Capsule sample).

[0089] Among them, the chromatographic conditions for high performance liquid chromatography detection are:

[0090] The chromatographic column used octadecylsilane bonded silica as filler (Agilent ZORBAX SB-C18, 250x4.6mm, 5μm);

[0091] The column temperature was 25°C;

[0092] The detection wavelength is 203nm;

[0093] The flow rate was 1.0 mL / min;

[0094] The injection volume was 10 μL;

[0095] Mobile phase A is acetonitrile, and mobile phase B is 0.1 wt % phosphoric acid aqueous solution, namely acetonitrile-0.1 wt % phosphoric acid aqueous solution.

[0096] The elution method is gradient elution, and the elution conditions are:

[0097] 0-10 min, 10%→15% mobile phase A, 90%→85% mobile phase B;

[0098] 10-50 min, 15% → 25% mobile phase A, 85% → 75% mobile phase B;

[0099] 50-60 min, 25% mobile phase A, 75% mobile phase B;

[0100] 60-90 min, 25% → 50% mobile phase A, 75% → 50% mobile phase B;

[0101] 90-100 min, 50% → 90% mobile phase A, 50% → 10% mobile phase B;

[0102] 100-100.1 min, 90% → 10% mobile phase A, 10% → 90% mobile phase B;

[0103] 100.1-110 min, 10% mobile phase A, 90% mobile phase B;

[0104] The theoretical plate number calculated based on the chlorogenic acid peak should not be less than 3000.

[0105] S22, Chromatographic Peaks and Taste Attribution

[0106] The test sample solution, each single herb solution and the corresponding negative sample solution lacking a single herb were taken separately and tested under the above-mentioned HPLC conditions to attribute the herb source of the chromatographic peaks in the Jinchai Capsule spectrum and determine the herb attribution of each chromatographic peak in the sample spectrum. The specific results are shown in Tables 1 and Figure 1 to Figure 7 .

[0107] Table 1 Chromatographic peaks and medicinal properties

[0108] Peak Attributed to medicinal taste 1 Forsythia 2 honeysuckle 3(S1) honeysuckle 4 honeysuckle 5 Forsythia 6 Forsythia 7 Forsythia 8 Forsythia 9 Scutellaria baicalensis 10 Forsythia 11 Scutellaria baicalensis 12(S2) American Ginseng 13 Scutellaria baicalensis

[0109] S23, Chromatographic Peak Identification

[0110] Take reference solution 1 to reference solution 7, mix the reference solution and the test solution, and test them under the above HPLC conditions. The results are shown in Figure 8 .

[0111] Depend on Figure 8It can be seen that by comparing with different reference substances, 8 ingredients in Jinchai Capsules were identified, as follows:

[0112] Table 2 List of reference peak identification results

[0113]

[0114]

[0115] Due to baicalin, chlorogenic acid, forsythiaside, ginsenoside Rb 1 They are the main active ingredients of Scutellaria baicalensis, honeysuckle, forsythia suspensa, and American ginseng, which are relatively stable. Therefore, only baicalin, chlorogenic acid, forsythia suspensa, and ginsenoside Rb were selected. 1 As a reference substance, chlorogenic acid and ginsenoside Rb 1 The retention time of the chromatographic peak is moderate and the baseline separation is achieved, so chlorogenic acid and ginsenoside Rb are selected. 1 The reference peaks used as the control are marked as Peak S1 and Peak S2.

[0116] Example 2-6 Method for constructing Jinchai capsule fingerprint

[0117] Examples 2 to 6 are respectively a method for constructing a Jinchai capsule fingerprint. Based on the optimal Jinchai capsule fingerprint construction conditions given in Example 1, the range of construction conditions that can achieve a better Jinchai capsule fingerprint is further explored to obtain the construction methods in Examples 2 to 6. Among them, the construction method of the Jinchai capsule fingerprint in Examples 2 to 5 is basically the same as that in Example 1, and the only difference is that some process parameters are different, as shown in Table 3 for details:

[0118] Table 3 List of process parameters in Examples 2 to 5

[0119]

[0120] The method for constructing the fingerprint of Jinchai Capsule in Example 6 is basically the same as that in Example 1, except that Example 6 uses dilute ethanol instead of 50 vol% methanol aqueous solution as the extraction solvent;

[0121] The dilute ethanol is prepared according to the test solution in Appendix XVB of the Chinese Pharmacopoeia Volume I. Take 529 mL of ethanol and dilute it to 1000 mL with water.

[0122] The contents and results of other parts of Examples 2 to 6 are the same as those of Example 1 and will not be repeated here (since other embodiments of the present invention have been able to fully prove that this method can better construct the fingerprint spectrum of Jinchai Capsules, no additional drawings will be added here).

[0123] Example 7 Standard fingerprint of Jinchai capsule and its application

[0124] 1) Establishment of standard fingerprint

[0125] The chromatographic peaks in the fingerprints of 10 batches of Jinchai capsule samples were matched to form a common pattern map, and the standard fingerprint of Jinchai capsule was established using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" of the Chinese Pharmacopoeia Committee (see Fig. 9 ), and combined with the peak identification results of the reference substance, the 8 common peaks were identified, peak 3 was chlorogenic acid, peak 4 was cryptochlorogenic acid, peak 7 was forsythiaside A, peak 9 was baicalin, peak 10 was forsythiaside, peak 11 was wogonin, peak 12 was ginsenoside Rb 1 , Peak 13 is wogonin.

[0126] 2) Common peak calibration and similarity calculation

[0127] The method for constructing the fingerprint of Jinchai Capsule in Example 1 was used to measure 10 batches of Jinchai Capsule samples, and the obtained liquid phase spectrum was analyzed by the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) of the State Pharmacopoeia Committee". The characteristic peaks with relatively stable, high response value and good peak shape were selected for multi-point correction, and the similarity was calculated. The results are shown in Table 4. The similarity of the 10 batches of samples is greater than 0.99. The spectrum is shown in Table 4. Fig.10 .

[0128] Table 4 Similarity calculation results of 10 batches of Jinchai capsule samples

[0129]

[0130] 3) Selection of reference peaks and determination of relative retention times

[0131] Peak 3, which corresponds to the peak of the chlorogenic acid reference substance, is the S1 peak. The relative retention time of peaks 1 to 11 and S1 is calculated. 1 The peak 12 corresponding to the reference peak is the S2 peak, and the relative retention time of peak 13 and S2 peak is calculated;

[0132] The specified value of relative retention time is determined according to the relative retention time of the standard fingerprint of Jinchai Capsule. The relative retention times of the chromatographic peaks in the fingerprints of 10 batches of Jinchai Capsule samples should be within the range of ±10% of the specified value. The specified values ​​are: 0.60 (peak 1), 0.66 (peak 2), 1.06 (peak 4), 2.12 (peak 5), 2.36 (peak 6), 2.43 (peak 7), 2.79 (peak 8), 4.39 (peak 9), 4.55 (peak 10), 5.90 (peak 11), and 1.16 (peak 13).

[0133] The standard fingerprint of Jinchai Capsule obtained in this example can be used for quality evaluation or control in the whole process of research / development / production / clinical application of Jinchai Capsule.

[0134] Example 8 Fingerprint Methodology Study

[0135] This example conducts a methodological investigation on the construction method of the Jinchai Capsule fingerprint in Example 1, mainly investigating the specificity, precision, repeatability, stability and durability of the construction method. The mixed reference solution and the test solution in the methodological investigation process are prepared according to the method in Example 1, and the chromatographic conditions of the HPLC detection are also the chromatographic conditions in Example 1.

[0136] 1. Exclusivity

[0137] Weigh 0.8 g of dextrin, add 30 mL of 50 vol% methanol aqueous solution, ultrasonicate for 30 min, cool to room temperature, shake well, filter, and take the filtrate to obtain a dextrin solution.

[0138] In order to investigate whether the blank solvent and the auxiliary material dextrin have any interference, the blank solvent (50% methanol), dextrin solution, mixed reference solution and test solution were injected into the high performance liquid chromatograph for determination and the chromatogram was recorded. The results are shown in Fig.11 As shown, it can be seen that the blank solvent and the auxiliary material dextrin have no interference, indicating that the construction method of the present invention has good specificity.

[0139] 2. Precision Test

[0140] Take a sample of Jinchai capsules, prepare a test solution, inject it 6 times continuously, record the corresponding fingerprint, and calculate the RSD of the relative retention time of each characteristic peak. The results are shown in the following table:

[0141] Table 5 List of precision inspection results

[0142]

[0143] As can be seen from Table 5, the RSD of the relative retention time of each characteristic peak in the obtained fingerprint spectrum was less than 1% after 6 consecutive injections using the construction method of the present invention, indicating that the construction method of the present invention has good precision.

[0144] 3. Repeatability test

[0145] Take the same batch of Jinchai capsule samples, prepare 6 test solutions, inject them separately, record the corresponding fingerprints, and calculate the RSD of the relative retention time of each characteristic peak. The results are shown in the following table:

[0146] Table 6 Summary of repeatability test results

[0147]

[0148]

[0149] As can be seen from Table 6, when the construction method of the present invention was used to detect 6 test sample solutions, the relative retention time RSD of each characteristic peak in the obtained fingerprint spectrum was less than 1%, indicating that the construction method of the present invention has good repeatability.

[0150] 4. Stability test

[0151] Take a sample of Jinchai capsules and prepare a test solution. Inject it into the liquid chromatograph at 0h, 4h, 9h, 16h, 24h, 30h, 37h, 47h, and 54h, record the corresponding fingerprint, and calculate the RSD of the relative retention time of each characteristic peak. The results are shown in the table below:

[0152] Table 7 Summary of stability investigation results

[0153]

[0154] As can be seen from Table 7, the relative retention time RSD of each characteristic peak in the fingerprint spectrum obtained by using the construction method of the present invention to detect the test solution placed for different time periods is less than 1%, indicating that the test solution has good stability within 54 hours.

[0155] 5. Durability test

[0156] The durability of the chromatographic conditions of the present invention with different chromatographic columns, different mobile phase systems, different column temperatures and different flow rates was investigated, and the peak types of their common peaks were evaluated. The relative retention time of the chromatographic peaks of the repeatable fingerprint spectra was used to determine the determined value of the relative retention time and investigate the durability.

[0157] (1) Effect of different column temperatures

[0158] The same batch of Jinchai capsule samples were taken to prepare the test solution. Under the chromatographic conditions of Example 1, the effects of different column temperatures such as 20°C, 25°C, and 30°C were investigated (i.e., only the column temperature in the chromatographic conditions in Example 1 was changed, and other conditions remained unchanged). The results are shown in Table 1. Fig.12 .Depend on Fig.12 It can be seen that as the column temperature increases, the retention time of each characteristic peak is slightly advanced, but the relative retention time meets the proposed requirements. In summary, the construction method of the present invention has good durability within the column temperature range of 20 to 30°C.

[0159] (2) Effect of different flow rates

[0160] Take the same batch of Jinchai capsule samples to prepare the test solution. Under the chromatographic conditions of Example 1, the effects of different flow rates such as 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, and 1.2 mL / min were investigated (i.e., only the flow rate in the chromatographic conditions in Example 1 was changed, and other conditions remained unchanged). The results are shown in Table 1. Fig.13 .Depend on Fig.13 It can be seen that with the increase of flow rate, the retention time of each characteristic peak is slightly advanced, but the relative retention time meets the proposed requirements. In summary, the construction method of the present invention has good durability within the flow rate range of 0.8 to 1.2 mL / min.

[0161] (3) Influence of different chromatographic columns

[0162] The same batch of Jinchai capsule samples were taken to prepare the test solution. Under the chromatographic conditions of Example 1, the influence of the same type of chromatographic columns of different brands was investigated (i.e., only the chromatographic columns in the chromatographic conditions in Example 1 were changed, and other conditions remained unchanged). The results are shown in FIG. Fig.14 .Depend on Fig.14 It can be seen that the separation degree of characteristic peak 3 and characteristic peak 4 in the fingerprint spectrum obtained by using the Agilent ZORBAX SB-Aq chromatographic column is not good, and the peak order changes; the separation degree of characteristic peak 6 and characteristic peak 7 in the fingerprint spectrum obtained by using the COSMOSIL 5C18-MS-II chromatographic column is not good; and the use of different batches of Agilent ZORBAX SB-C18 (250x4.6mm, 5um) chromatographic columns has no significant effect on the fingerprint spectrum of the Jinchai capsule sample, and the relative retention time and separation degree meet the requirements. It shows that for the chromatographic columns of the same manufacturer, different batches are relatively stable. Therefore, it is determined that the chromatographic column used in the method of the present invention is Agilent ZORBAX SB-C18 (250x4.6mm, 5um).

[0163] Example 9 Investigation of various process conditions in the construction method

[0164] In this example, various process conditions in the method for constructing the fingerprint of Jinchai Capsules in Example 1 are investigated. During the investigation, the mixed reference solution and the test solution are prepared according to the method in Example 1, and the chromatographic conditions of the HPLC detection also adopt the chromatographic conditions in Example 1.

[0165] 1. Investigation of the preparation method of the test solution

[0166] 1) Investigation of extraction solvent

[0167] The same batch of Jinchai capsule samples were extracted using ethanol, water, methanol, dilute ethanol, 75 vol% methanol aqueous solution, 25 vol% methanol aqueous solution, and 50 vol% methanol aqueous solution as different extraction solvents (i.e., only the extraction solvent in the preparation process of the test solution in Example 1 was changed, and other conditions remained unchanged). The results are shown in Table 1. Figure 15-16 .

[0168] Depend on Fig.15 It can be seen that when methanol is used as the extraction solvent, the response and uniformity of the chromatographic peak are better than those of water and pure ethanol. Fig.16 It can be seen that the extraction effect is poor when 25 vol% methanol aqueous solution is used as the extraction solvent; while when 75 vol% methanol aqueous solution, 50 vol% methanol aqueous solution and dilute ethanol are used as the three extraction solvents, there is no obvious difference in the response and uniformity of the chromatographic peak. Therefore, 50 vol% methanol aqueous solution is used as the extraction solvent in the best construction method of the present invention.

[0169] 2) Investigation of extraction methods

[0170] The same batch of Jinchai capsule samples were extracted by ultrasound and reflux respectively (i.e., only the extraction method in the preparation process of the test solution in Example 1 was changed, and other conditions remained unchanged). The results are shown in Fig.17 .

[0171] Depend on Fig.17 It can be seen that from the perspective of the number of chromatographic peaks and the response, there is no significant difference in the extraction effect between the two extraction methods. However, since the ultrasonic extraction method is simple and easy, the ultrasonic extraction method is used as the extraction method in the best construction method of the present invention.

[0172] 3) Extraction volume investigation

[0173] Take 0.8g of the same batch of Jinchai capsule samples and extract them using three extraction volumes of 20mL, 30mL and 40mL respectively (that is, only the amount of 50vol% methanol aqueous solution in the preparation process of the test solution in Example 1 is changed, and other conditions remain unchanged). The results are shown in Fig.18 .

[0174] Depend on Fig.18 It can be seen that when 20 mL of 50 vol% methanol aqueous solution was used to extract ginsenoside Rb 1 The peak response was the largest, but the baicalin peak was overloaded at this time. The other two extraction volumes had no significant effect, so 30 mL was used as the extraction volume in the optimal construction method of the present invention.

[0175] 4) Extraction time investigation

[0176] The same batch of Jinchai capsule samples were extracted using three extraction times of 15min, 30min, and 45min (i.e., only the extraction time in the preparation process of the test solution in Example 1 was changed, and other conditions remained unchanged). The results are shown in Table 1. Fig.19 .

[0177] Depend on Fig.19 It can be seen that there is no significant difference in the chromatographic peak response and uniformity of the three extraction times in the obtained fingerprint. In the optimal construction method of the present invention, 30 minutes is used as the extraction time.

[0178] 2. Investigation of Chromatographic Conditions

[0179] 1) Investigation of detection wavelength

[0180] Since the main active ingredients in the Jinchai capsule samples include phenolic acids, saponins and flavonoids, etc. Therefore, four detection wavelengths of 203nm, 240nm, 280nm and 327nm were investigated. The same batch of Jinchai capsule samples were taken to prepare the test solution. Under the chromatographic conditions of Example 1, the effects of the four detection wavelengths of 203nm, 240nm, 280nm and 327nm were investigated (that is, only the detection wavelength in the chromatographic conditions in Example 1 was changed, and other conditions remained unchanged). The results are shown in Table 1. Fig. 20 .

[0181] Depend on Fig. 20 It can be seen that when the detection wavelength is 203 nm, the number of chromatographic peaks in the obtained fingerprint is the largest, the peak shape and response value are both good, and the baseline is stable. Therefore, 203 nm is used as the optimal detection wavelength in the construction method of the present invention.

[0182] 2) Investigation of elution conditions

[0183] The same batch of Jinchai capsule samples were taken to prepare the test solution. Under the chromatographic conditions of Example 1, the influence of different elution conditions was investigated (i.e., only the elution conditions in the chromatographic conditions in Example 1 were changed, and other conditions remained unchanged). The results are shown in Fig.21 .

[0184] Among them, different elution conditions are as follows:

[0185] Acetonitrile was used as mobile phase A, and 0.1 wt% phosphoric acid aqueous solution was used as mobile phase B.

[0186] Elution condition one is as follows: 0-5 min, 5% mobile phase A, 95% mobile phase B; 5-7 min, 5%→15% mobile phase A, 95%→85% mobile phase B; 7-20 min, 15% mobile phase A, 85% mobile phase B; 20-60 min, 15%→25% mobile phase A, 85%→75% mobile phase B; 60-90 min, 25%→55% mobile phase A, 75%→45% mobile phase B; 90-91 min, 55%→5% mobile phase A, 45%→95% mobile phase B; 91-100 min, 5% mobile phase A, 95% mobile phase B.

[0187] Elution condition two is as follows: 0-20 min, 15% mobile phase A, 85% mobile phase B; 20-60 min, 15%→25% mobile phase A, 85%→75% mobile phase B; 60-90 min, 25%→55% mobile phase A, 75%→45% mobile phase B; 90-100 min, 55%→90% mobile phase A, 45%→10% mobile phase B; 100-101 min, 90%→15% mobile phase A, 10%→85% mobile phase B; 101-110 min, 15% mobile phase A, 85% mobile phase B.

[0188] Elution condition three is 0-5 min, 10% mobile phase A, 90% mobile phase B; 5-15 min, 10%→15% mobile phase A, 90%→85% mobile phase B; 15-55 min, 15%→25% mobile phase A, 85%→75% mobile phase B; 55-60 min, 25% mobile phase A, 75% mobile phase B; 60-90 min, 25%→50% mobile phase A, 75%→50% mobile phase B; 90-100 min, 50%→90% mobile phase A, 50%→10% mobile phase B; 100-100.1 min, 90%→10% mobile phase A, 10%→90% mobile phase B; 100.1-110 min, 10% mobile phase A, 90% mobile phase B;

[0189] Elution condition 4 is the elution condition of Example 1.

[0190] Depend on Fig.21 It can be seen that on the same chromatographic column, the number of chromatographic peaks and the degree of separation in the fingerprint obtained using elution condition 4 are significantly better than those of the other three elution conditions. Therefore, elution condition 4 is used as the elution condition in the construction method of the present invention.

[0191] 3) Investigation of mobile phase system

[0192] Take the same batch of Jinchai capsule samples to prepare test solutions. Under the chromatographic conditions of Example 1, the effects of five different mobile phase systems, namely, acetonitrile-0.1wt% phosphoric acid aqueous solution, acetonitrile-0.2wt% phosphoric acid aqueous solution, acetonitrile-0.05wt% phosphoric acid aqueous solution, acetonitrile-0.1wt% formic acid aqueous solution, and acetonitrile-0.1wt% acetic acid aqueous solution, were investigated (i.e., only the mobile phase system in the chromatographic conditions in Example 1 was changed, and other conditions remained unchanged, wherein mobile phase A was acetonitrile). The results are shown in Table 1. Fig. 22 .

[0193] Depend on Fig. 22 It can be seen that the elution ability of the mobile phase B using 0.1wt% formic acid aqueous solution or 0.1wt% acetic acid aqueous solution is weaker than the elution ability of the mobile phase B using 0.05wt% phosphoric acid aqueous solution, and the baseline fluctuation in the corresponding fingerprint spectrum obtained when the mobile phase B uses 0.1wt% formic acid aqueous solution or 0.1wt% acetic acid aqueous solution is large, while the mobile phase B uses different concentrations of phosphoric acid aqueous solution to the separation, response and uniformity of the chromatographic peaks in the obtained fingerprint spectrum. Therefore, acetonitrile-0.1% phosphoric acid aqueous solution is used as the mobile phase system in the optimal construction method of the present invention.

[0194] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint of Jinchai Capsule, characterized in that: The construction method comprises the following steps: S1. Prepare the test solution by taking Jinchai capsules; S2, taking the test solution and performing high performance liquid chromatography to obtain the fingerprint of the Jinchai capsule; The mobile phase A of the HPLC detection is acetonitrile, and the mobile phase B is a phosphoric acid aqueous solution with a concentration of 0.05-0.2 wt%; The elution method of the high performance liquid chromatography detection is gradient elution; The elution conditions of the gradient elution are: 0-10 min, 10%→15% mobile phase A, 90%→85% mobile phase B; 10-50 min, 15% → 25% mobile phase A, 85% → 75% mobile phase B; 50-60 min, 25% mobile phase A, 75% mobile phase B; 60-90 min, 25%→50% mobile phase A, 75%→50% mobile phase B; 90-100 min, 50% → 90% mobile phase A, 50% → 10% mobile phase B; 100-100.1 min, 90% → 10% mobile phase A, 10% → 90% mobile phase B; 100.1~110min, 10% mobile phase A, 90% mobile phase B.

2. The method for constructing the fingerprint of Jinchai Capsule according to claim 1, characterized in that: The detection wavelength of the high performance liquid chromatography detection is 200-210 nm, and the chromatographic column is Agilent ZORBAX SB-C18.

3. The method for constructing the fingerprint of Jinchai Capsule according to claim 1 or 2, characterized in that: The test solution is prepared by extracting the contents of Jinchai capsules with methanol aqueous solution / dilute ethanol and filtering.

4. The method for constructing the fingerprint of Jinchai Capsule according to claim 3, characterized in that: The concentration of the methanol aqueous solution is 25 to 100 vol%; The weight-to-volume ratio of the contents of Jinchai capsules to methanol aqueous solution / dilute ethanol is 0.8 g:20-40 mL.

5. The method for constructing the fingerprint of Jinchai Capsule according to claim 3, characterized in that: The extraction time is 15 to 45 minutes, and the extraction method is ultrasound or reflux.

6. The method for constructing the fingerprint of Jinchai Capsule according to claim 1, 2, 4 or 5, characterized in that: The flow rate of the high performance liquid chromatography detection is 0.8-1.2 mL / min, the column temperature is 20-30° C., and the injection volume is 5-20 μL.

7. The method for constructing the fingerprint of Jinchai Capsule according to claim 1, 2, 4 or 5, characterized in that: The construction method also includes performing high performance liquid chromatography on at least one of the single medicinal material solution, the negative sample solution lacking the single medicinal material, and the mixed reference solution; The single herbal medicine solution is prepared by taking the single herbal medicines used in Jinchai Capsules respectively; The negative sample solution lacking a single medicinal material is prepared by using the method for preparing Jinchai capsules to prepare the contents of capsules lacking a single medicinal material, and then extracting to prepare the negative sample solution lacking a single medicinal material; The mixed reference solution is prepared by adding methanol to chlorogenic acid, baicalin, forsythiaside, and ginsenoside Rb1.

8. Application of the standard fingerprint of Jinchai Capsule obtained by the construction method described in any one of claims 1 to 7 in the quality evaluation or control of the whole process of research / development / production / clinical application of Jinchai Capsule.

9. The use according to claim 8, characterized in that: A total of 13 common peaks were identified in the standard fingerprint of Jinchai Capsule; Among them, peak 3 is chlorogenic acid, peak 4 is cryptochlorogenic acid, peak 7 is forsythiaside A, peak 9 is baicalin, peak 10 is forsythiaside, peak 11 is baicalin, peak 12 is ginsenoside Rb1, and peak 13 is wogonin.

10. The use according to claim 8 or 9, characterized in that: In the standard fingerprint of Jinchai Capsule, peak 3 is S1 peak, and the relative retention time of peaks 1 to 11 and S1 peak is calculated; peak 12 is S2 peak, and the relative retention time of peak 13 and S2 peak is calculated; Among them, the specified value of peak 1 is 0.60, the specified value of peak 2 is 0.66, the specified value of peak 4 is 1.06, the specified value of peak 5 is 2.12, the specified value of peak 6 is 2.36, the specified value of peak 7 is 2.43, the specified value of peak 8 is 2.79, the specified value of peak 9 is 4.39, the specified value of peak 10 is 4.55, the specified value of peak 11 is 5.90, and the specified value of peak 13 is 1.16.

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