Fingerprint Detection Method for Freeze-dried Powder of Standard Decoction of Ginseng Schisandra Decoction and Its Fingerprint

Abstract

The present invention discloses a fingerprint detection method for freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction and its fingerprint, belonging to the technical field of detection of traditional Chinese medicine components. By taking the test solution of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction, using high performance liquid chromatography, with Hypersil C18 ODS (4.6×250 mm×5 μm) as the chromatographic column, using 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B) as the mobile phase for gradient elution, using an ultraviolet-visible light detector, and performing detection by switching multiple wavelengths. The detection results are analyzed by the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, a fingerprint of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction is established, and different batches of samples are analyzed to determine the standard fingerprint. Thirteen characteristic peaks are identified by mass spectrometry, which can comprehensively reflect the types and quantities of chemical components contained therein. This method has the characteristics of good stability, high precision, good reproducibility, etc., and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection of traditional Chinese medicine components, and particularly relates to a fingerprint detection method for freeze-dried powder of standard decoction of Ginseng and Schisandra Decoction and its fingerprint. Background Art

[0002] The original formula of Ginseng and Schisandra Decoction comes from the famous work "Youyou Jicheng" by Chen Fuzheng, a famous doctor in the Qing Dynasty. The whole formula consists of six medicinal materials: ginseng, atractylodes macrocephala, poria cocos, schisandra chinensis, ophiopogon japonicus, and licorice. It contains the two formulas of Sijunzi Decoction and Shengmai Powder, and has the effects of tonifying qi and strengthening the spleen, regulating qi and resolving phlegm, and consolidating the defense and nourishing the lungs. It is mainly used to treat diseases such as "chronic cough with spleen deficiency, weak middle qi, pale face and lips" and "cough that lasts for a long time without stopping". In the formula, ginseng greatly tonifies the primordial qi and "mainly tonifies the five zang-organs" to restore the root of qi deficiency; it is combined with poria cocos, atractylodes macrocephala, and licorice to invigorate the middle-jiao and strengthen the spleen, promote diuresis and assist transportation, and strengthen the earth qi in the middle-jiao; it is combined with schisandra chinensis to astringe the lung qi to consolidate the superficiality of qi; and ophiopogon japonicus is used to clear and moisten with its sweet and cold nature to clear the lung qi. Generally speaking, this formula tonifies the spleen with Sijunzi Decoction and protects the lungs with Shengmai Powder, containing the wonderful method of strengthening the earth to generate the metal. It has been confirmed by modern pharmacological research that it can not only enhance the phagocytic function of mononuclear phagocytes, induce interferon, enhance the body's immune function, but also has symptomatic treatment effects such as relieving cough, asthma, and expectoration. Among them, ginsenosides, glycyrrhizic acid, and polysaccharides are the main active ingredients. This formula has a mild property, good taste, high compliance, and has good clinical application value.

[0003] At present, due to the large variety of medicinal materials contained in this formula and the relatively complex traditional Chinese medicine components, the types and quantities of chemical components are not clear, and it is difficult to determine the components. There is no fingerprint detection method for Ginseng and Schisandra Decoction yet. Summary of the Invention

[0004] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a fingerprint detection method for freeze-dried powder of standard decoction of Ginseng and Schisandra Decoction and its fingerprint, so as to solve the problem of determining substances in Ginseng and Schisandra Decoction.

[0005] In order to achieve the above purpose, the present invention adopts the following technical solutions to implement:[[]]

[0006] The present invention discloses a fingerprint detection method for freeze-dried powder of standard decoction of Ginseng and Schisandra Decoction, including the following steps:

[0007] S1. Prepare test solutions of freeze-dried powder of standard decoction of Ginseng and Schisandra Decoction in different batches;

[0008] S2. Inject the test sample solutions of freeze-dried powder of different batches of standard decoction of ginseng and schisandra chinensis decoction in S1 into a high performance liquid chromatograph. The chromatographic conditions are as follows: chromatographic column: Hypersil C18 ODS, mobile phase A is 0.1% phosphoric acid aqueous solution, mobile phase B is acetonitrile, gradient elution, detection wavelength: 0-45min, 280nm; 45-50min, 254nm; 50-55min, 220nm; 55-110min, 254nm, record the chromatogram;

[0009] S3. According to the chromatograms of the test sample solutions of different batches of freeze-dried powder of standard decoction of ginseng and schisandra chinensis decoction obtained in S2, a fingerprint spectrum based on freeze-dried powder of standard decoction of ginseng and schisandra chinensis decoction was developed, similarity evaluation was performed, and characteristic peaks were identified by mass spectrometry.

[0010] Preferably, the specific steps of S1 are: weighing appropriate amounts of freeze-dried powder of standard decoction of Ginseng and Schisandra Decoction from different batches, placing them in stoppered conical flasks, adding methanol solution, ultrasonically extracting, and filtering the filtrate through a 0.45 μm microporous filter membrane to obtain a test solution.

[0011] More preferably, the ultrasonic time is 60 min.

[0012] Preferably, the chromatographic conditions in S2 are as follows: the elution procedure is as follows: 0-10 min 0.1% phosphoric acid aqueous solution, 85% by volume; 10-15 min 0.1% phosphoric acid aqueous solution, 85%-75% by volume; 15-25 min 0.1% phosphoric acid aqueous solution, 75%-70% by volume; 25-30 min 0.1% phosphoric acid aqueous solution, 70%-55% by volume; 30-45 min 0.1% phosphoric acid aqueous solution, 55%-50% by volume; 45-55 min 0.1% phosphoric acid aqueous solution, 50%-45% by volume; 55-75 min 0.1% phosphoric acid aqueous solution, 45%-40% by volume; 75-80 min 0.1% phosphoric acid aqueous solution, 40%-48% by volume; 80-90 min 0.1% phosphoric acid aqueous solution, 48%-70% by volume; 90-100 min 0.1% phosphoric acid aqueous solution, 70%-60% by volume; 100-110 min 0.1% phosphoric acid aqueous solution 60%-95% by volume.

[0013] Preferably, in S2, the column temperature is 30°C and the flow rate is 0.5 mL / min.

[0014] Preferably, the specific steps of S3 are as follows: Export the chromatograms of the test solution of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Soup obtained in S2, and generate a fingerprint through the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A; Select the chromatographic peaks that exist in the chromatograms of different batches as the common peaks; Generate a reference fingerprint by the average value calculation method, and export the similarity result table of the chromatograms of the test solution of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Soup in different batches; Calculate the relative retention time and relative peak area of each common peak; And identify the chemical components in the freeze-dried powder of the standard decoction of Ginseng and Schisandra Soup according to the mass spectrometry diagram; Confirm the reliability of the results based on the similarity result table and the chromatograms of the test solution of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Soup in different batches.

[0015] Preferably, 13 characteristic peaks in the constructed fingerprint are identified by mass spectrometry.

[0016] Preferably, the freeze-dried powder of the standard decoction of Ginseng and Schisandra Soup is prepared from the following six medicinal materials: ginseng, Atractylodes macrocephala, Poria cocos, Schisandra chinensis, Ophiopogon japonicus, and licorice. These materials are placed in a decocting pot, soaked in water, decocted twice, and the filtrate is dried.

[0017] More preferably, the ginseng is official selected ginseng, the Atractylodes macrocephala is bleached Atractylodes macrocephala, the Poria cocos is white Poria cocos, the Schisandra chinensis is northern Schisandra chinensis, the Ophiopogon japonicus is Hangzhou Ophiopogon japonicus, and the licorice is roasted licorice.

[0018] More preferably, the preparation method of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Soup is to make it by spray freeze-drying the standard decoction of Ginseng and Schisandra Soup.

[0019] The present invention also discloses the fingerprint of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Soup obtained by the above method. Peak 1 is liquiritigenin, Peak 2 is liquiritin, Peak 3 is apiosylisoliquiritigenin, Peak 4 is licorinchalcone B, Peak 5 is ginsenoside Rg1, Peak 6 is glycyrrhizic acid, Peak 7 is licorinchalcone A, Peak 8 is atractylenolide I, Peak 9 is schisantherin B, Peak 10 is schisandrin A, Peak 11 is schisandrin B, Peak 12 is schisandrin C, and Peak 13 is atractylenolide II.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The fingerprint detection method for the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction provided by the present invention uses high-performance liquid chromatography to establish the fingerprint detection method for the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction. The traditional Chinese medicine fingerprint can lay a foundation for the application of the quality standard of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction, and can comprehensively and objectively evaluate the quality of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction and the types and quantities of its contained chemical components, and then make an overall description and evaluation of the quality of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction. The traditional Chinese medicine fingerprint combined with high-performance liquid chromatography can comprehensively evaluate the authenticity, excellence and stability of the quality of traditional Chinese medicine by reflecting the chemical substance group that is the material basis of the efficacy of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction. In this method, when preparing the test solution, freeze-dried powders of the standard decoction of Ginseng and Schisandra Decoction from different batches are selected, which can make the experimental method have sufficient representativeness. In the chromatographic conditions, pure acetonitrile - 0.1% phosphoric acid aqueous solution is used as the mobile phase, which can ensure good separation effect of each component in the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction and the largest amount of chromatogram information. On this basis, the chromatographic conditions are optimized. The detection wavelength is investigated using an ultraviolet-visible absorption detector, and it is found that the detection wavelength conditions are: 0 - 45 min, 280 nm; 45 - 50 min, 254 nm; 50 - 55 min, 220 nm; 55 - 110 min, 254 nm. The chromatogram under this condition contains relatively comprehensive information and has a stable baseline, so this method is selected as the detection wavelength condition. By comparing the elution effects of different elution systems at different gradients, it is found that when pure acetonitrile - 0.1% phosphoric acid aqueous solution is used as the mobile phase, the separation effect of each component in the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction is better and the chromatogram information is the largest. Therefore, pure acetonitrile - 0.1% phosphoric acid aqueous solution is finally selected as the mobile phase. Based on the combination of high-performance liquid chromatography and traditional Chinese medicine fingerprint, this method optimizes the chromatographic conditions, has good adaptability, strong specificity, high precision, good stability and strong repeatability, meets the requirements for constructing fingerprints, can solve the problem of substance determination in Ginseng and Schisandra Decoction, is conducive to more comprehensively and effectively controlling the quality of clinical use of Ginseng and Schisandra Decoction, promoting the modernization of traditional Chinese medicine, and ensuring the safety and efficacy of clinical use more effectively.

[0022] Furthermore, through the investigation of the extraction solvent of the test sample, it is found that when pure methanol solution is used as the extraction solvent, the chromatogram information of the extract is relatively large and the component content is relatively high. Therefore, pure methanol solution is selected for extraction; through the investigation of the extraction method of the test sample, it is found that the chromatogram components obtained by ultrasonic extraction are relatively comprehensive and the resolution is good. Therefore, the ultrasonic extraction method is adopted.

[0023] Even further, through the investigation of the ultrasonic extraction time, it is found that the chromatogram components obtained by ultrasonic extraction with pure methanol for 60 min are relatively comprehensive, the resolution is good, and each chromatographic peak can be well separated. Therefore, 60 min of ultrasonic extraction is selected.

[0024] Furthermore, through the investigation of the elution procedure, it was found that when the gradient elution procedure was adopted, good resolution of each chromatographic peak in the fingerprint could be achieved.

[0025] Furthermore, through the investigation of different column temperatures, it was found that the peak emergence was optimal when the column temperature was maintained at 30°C, and the separation effect of each component was better. Therefore, the column temperature of 30°C was finally selected; through the investigation of different flow rates, it was found that the peak emergence was the best and the resolution was optimal when the flow rate was 0.5 mL / min. Therefore, the flow rate was maintained at 0.5 mL / min.

[0026] Furthermore, by optimizing the chromatographic conditions, including optimizing the composition of the mobile phase, detection wavelength, column temperature, flow rate, etc., the information content included in the chromatogram was the most comprehensive, the baseline was stable, and the separation effect was optimal.

[0027] Furthermore, based on the test results of multiple batches of samples, through the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints 2004A, the chromatographic peaks that existed in the chromatograms of the freeze-dried powders of the standard decoction of Ginseng and Schisandra Decoction in different batches were selected as common peaks; the control fingerprint was generated by the average value calculation method, and the similarity result table of the chromatograms of the test solutions of the freeze-dried powders of the standard decoction of Ginseng and Schisandra Decoction in different batches was derived; the relative retention time and relative peak area of each common peak were calculated; and the chemical components of the peaks in the fingerprint were identified according to the mass spectrometry diagram; according to the similarity result table and the chromatograms of the freeze-dried powders of the standard decoction of Ginseng and Schisandra Decoction in different batches, the reliability of the results was confirmed, and the standard fingerprint was obtained, which was used as the standard fingerprint of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction, so as to achieve the purpose of being able to control its quality more objectively, comprehensively, effectively and accurately.

[0028] The fingerprint of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction provided by the present invention can effectively characterize the quality of the Ginseng and Schisandra Decoction, objectively reflect the sequence and mutual relationship of the fingerprint characteristic peaks formed by each medicinal material, pay attention to the overall characteristics, and can avoid the one-sidedness of judging the quality of the Ginseng and Schisandra Decoction due to individual chemical components in the medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the control fingerprint of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction of the present invention;

[0030] Figure 2 is the mass spectrometry diagram of liquiritin of the present invention;

[0031] Figure 3 is the mass spectrometry diagram of liquiritin apioside and isoliquiritin apioside of the present invention;

[0032] Figure 4 is the mass spectrometry diagram of licorinchalcone B of the present invention;

[0033] Figure 5 Mass spectrum of ginsenoside Rg1 of the present invention;

[0034] Figure 6 Mass spectrum of glycyrrhizic acid of the present invention;

[0035] Figure 7 Mass spectrum of licorice chalcone A of the present invention;

[0036] Figure 8 Mass spectrum of atractylenolide I of the present invention;

[0037] Figure 9 Mass spectrum of schisantherin B of the present invention;

[0038] Figure 10 Mass spectrum of deoxyschizandrin of the present invention;

[0039] Figure 11 Mass spectrum of schisandrin B of the present invention;

[0040] Figure 12 Mass spectrum of schisandrin C of the present invention;

[0041] Figure 13 Mass spectrum of atractylenolide II of the present invention;

[0042] Figure 14 Chromatogram of the test solution of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction in 15 batches of the present invention. Detailed implementation manners

[0043] The present invention will be further described in detail below with reference to the accompanying drawings:

[0044] I. The fingerprint detection method for the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction provided by the present invention includes the following steps:

[0045] S1. Prepare the test solutions of the freeze-dried powders of the standard decoctions of Ginseng and Schisandra Decoction in different batches

[0046] Precisely weigh the freeze-dried powders of the standard decoctions of Ginseng and Schisandra Decoction in different batches respectively, place them in a stoppered conical flask, add methanol solution, extract by ultrasonic treatment, take the filtrate and filter it through a 0.45 μm microporous filter membrane to obtain the test solutions;

[0047] S2. Inject the test solutions of the freeze-dried powders of the standard decoctions of Ginseng and Schisandra Decoction in different batches in S1 into a high performance liquid chromatograph and record the chromatograms;

[0048] S3. Export the fingerprints of the test solutions of the freeze-dried powders of the standard decoctions of Ginseng and Schisandra Decoction in different batches obtained in S2, conduct similarity evaluation, and identify the characteristic peaks by mass spectrometry.

[0049] II. The fingerprint of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction obtained by the above method provided by the present invention is shown in Figures 1 to 13 . Thirteen characteristic peaks are identified by mass spectrometry. Peak 1 is liquiritin, Peak 2 is liquiritin apioside, Peak 3 is isoliquiritin apioside, Peak 4 is licorinchalcone B, Peak 5 is ginsenoside Rg1, Peak 6 is glycyrrhizic acid, Peak 7 is licorinchalcone A, Peak 8 is atractylenolide I, Peak 9 is schisantherin B, Peak 10 is deoxyschizandrin, Peak 11 is schizandrin B, Peak 12 is schizandrin C, and Peak 13 is atractylenolide II.

[0050] III. Optimization of the fingerprint establishment method:

[0051] S1. Optimization in the preparation of the test solution:

[0052] By experimentally investigating and comparing different extraction methods (ultrasonic extraction, reflux extraction, maceration), the present invention finds that the chromatogram components obtained by ultrasonic extraction are relatively comprehensive and the resolution is good. Therefore, the ultrasonic extraction method is adopted.

[0053] By experimentally investigating and comparing different extraction solvents (dichloromethane solution, chloroform solution, petroleum ether solution, pure methanol solution, 50% methanol aqueous solution, 75% methanol aqueous solution, pure aqueous solution), the present invention finds that when the pure methanol solution is used as the extraction solvent, the chromatogram of the extract has more information and higher component content. Therefore, the pure methanol solution is selected for extraction.

[0054] By experimentally investigating and comparing different ultrasonic extraction times (15 min, 30 min, 45 min, 60 min), the present invention finds that the chromatogram components obtained by ultrasonic extraction with pure methanol for 60 min are relatively comprehensive and the resolution is good. Therefore, the ultrasonic extraction method is adopted.

[0055] S2. Optimization in the chromatographic conditions:

[0056] The present invention uses an ultraviolet-visible absorption detector to investigate the detection wavelength, and extracts the chromatograms at 220 nm, 254 nm, and 280 nm. It is found that the detection wavelength conditions are: 0 - 45 min, 280 nm; 45 - 50 min, 254 nm; 50 - 55 min, 220 nm; 55 - 110 min, 254 nm. The chromatogram under this condition contains relatively comprehensive information and the baseline is stable. Therefore, this method is selected as the detection wavelength condition.

[0057] The present invention screens the flow rate (0.4 mL / min, 0.5 mL / min, 0.7 mL / min, 0.8 mL / min, 1.0 mL / min) and finds that the peak emergence is the best and the resolution is the best when the flow rate is 0.5 mL / min. Therefore, the flow rate is maintained at 0.5 mL / min.

[0058] The column temperature (25°C, 30°C, 35°C) was screened in the present invention. The results showed that the peak elution was optimal when the column temperature was maintained at 30°C, and the separation effect of each component was good. Therefore, the column temperature of 30°C was finally selected;

[0059] The present invention compared the elution effects of multiple different elution systems, namely pure methanol - water, pure acetonitrile - water, pure methanol - 0.1% phosphoric acid aqueous solution, pure acetonitrile - 0.1% phosphoric acid aqueous solution, pure methanol - 0.1% formic acid aqueous solution, pure methanol - 0.1% glacial acetic acid solution, and pure acetonitrile - 0.1% glacial acetic acid solution, at different gradients. The results showed that when the pure acetonitrile - 0.1% phosphoric acid aqueous solution was used as the mobile phase, the separation effect of each component in the freeze - dried powder of the standard decoction of Ginseng and Schisandra Decoction was good, and the chromatogram had the most information. Therefore, the pure acetonitrile - 0.1% phosphoric acid aqueous solution was finally selected as the mobile phase;

[0060] After determining the optimal mobile phase composition, the present invention screened the optimal gradient elution program through a large number of experiments. It was found that when the gradient elution program shown in Table 1 was adopted, good resolution of each chromatographic peak in the fingerprint could be achieved.

[0061] Table 1 Gradient elution program

[0062]

[0063] The implementation scheme of the present invention will be described in detail below in combination with examples. For those not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are adopted. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0064] Example 1 Fingerprint detection method for the freeze - dried powder of the standard decoction of Ginseng and Schisandra Decoction

[0065] The instruments and reagents used in the example are as follows:

[0066] Experimental equipment

[0067] 1. Instruments

[0068] The model and manufacturer of the instruments used in this experiment are shown in Table 2:

[0069] Table 2 Instruments

[0070]

[0071]

[0072] 2. Drugs and reagents

[0073] The batch numbers of the freeze - dried powder samples of the standard decoction of Ginseng and Schisandra Decoction in 15 batches are shown in Table 3:

[0074] Table 3 Batch numbers of 15 batches of lyophilized powder samples of Schisandrae Chinensis Decoction standard decoction

[0075]

[0076] The reagents used in the present invention are shown in Table 4:

[0077] Table 4 Reagents

[0078]

[0079]

[0080] 3. A fingerprint detection method for freeze-dried powder of standard decoction of ginseng and schisandra chinensis decoction, comprising the following steps:

[0081] S1. Preparation of test solution of freeze-dried powder of standard decoction of ginseng and schisandra chinensis decoction: accurately weigh 1.00 g of freeze-dried powder of standard decoction of ginseng and schisandra chinensis decoction from 15 batches respectively, put it into a 250 mL stoppered conical flask, add 100 mL of pure methanol solution, extract by ultrasonic for 60 min, and filter the filtrate through a 0.45 μm microporous filter membrane to obtain the test solution.

[0082] S2. Accurately pipette 15 batches of lyophilized powder of standard decoction of ginseng and schisandra chinensis decoction for testing respectively, inject into high performance liquid chromatograph, and record chromatogram; liquid chromatography conditions are as follows: chromatographic column: Hypersil C18 ODS (4.6×250mm×5μm); mobile phase: 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B), gradient elution; UV-visible absorption detector with multi-wavelength switching detection, detection wavelength: 0-45min, 280nm; 45-50min, 254nm; 50-55min, 220nm; 55-110min, 254nm; column temperature: 30℃; flow rate: 0.5mL / min; injection volume: 20μL; elution program see Table 5:

[0083] Table 5 Elution program

[0084]

[0085] S3. Export the fingerprint chromatograms of the freeze-dried powder test solutions of 15 batches of the standard decoction of Ginseng and Schisandra Decoction obtained in S2, and import them into the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines 2004A; generate a reference fingerprint chromatogram by the average value calculation method, and select the chromatographic peaks that exist in the chromatograms of the freeze-dried powders of 15 batches of the standard decoction of Ginseng and Schisandra Decoction as the common peaks; calculate the relative retention time and relative peak area of each common peak; perform mass spectrometry analysis on the freeze-dried powder test solutions of 15 batches of the standard decoction of Ginseng and Schisandra Decoction. Using an ultra-high performance liquid chromatography-mass spectrometer, the test conditions are electrospray ionization, spray voltage 3500V, sheath gas flow rate 40arb, auxiliary gas flow rate 10arb, auxiliary gas temperature 300°C, capillary temperature 300°C, scanning mode is full scan mode, and the mass-to-charge ratio scanning range m / z is 100 - 1300. Obtain the total ion current chromatogram and the mass spectrometry result chromatograms of 13 chemical components, import the detection data into Xcalibur software, enter the Qual Browser interface, and perform data analysis according to the elution situation of the chemical components.

[0086] After comparison, a total of 13 main components were identified, namely: liquiritigenin, liquiritin, apiosylisoliquiritigenin, licorinchalcone B, ginsenoside Rg1, glycyrrhizic acid, licorinchalcone A, atractylenolide I, schisantherin B, schisandrin A, schisandrin B, schisandrin C, atractylenolide II.

[0087] Chromatograms of the freeze-dried powders of 15 batches of the standard decoction of Ginseng and Schisandra Decoction were obtained through analysis and calculation. See Figure 14 , and there is good similarity among the common chromatographic peaks, indicating that the fingerprint chromatogram established by this method can well detect the quality of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction. The results are shown in Table 6.

[0088] Table 6 Similarity between each batch of samples and the common pattern

[0089]

[0090] Example 2 Methodology study on the fingerprint chromatogram construction method based on the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction

[0091] S1. Precision study

[0092] Take the freeze-dried powder test solution of the standard decoction of Ginseng and Schisandra Decoction prepared by the method of Example 1, analyze it according to the detection method of Example 1, inject samples in parallel 6 times, the injection volume is 20 μL, and use liquiritigenin, liquiritin, apiosylisoliquiritigenin, licorinchalcone B, ginsenoside Rg1, glycyrrhizic acid, licorinchalcone A, atractylenolide I, schisantherin B, schisandrin A, schisandrin B, schisandrin C, atractylenolide II as the reference peaks. By analyzing the peak area and retention time and calculating the RSD value, the results are shown in Table 7. The results show that the parallel injection precision of this equipment is good.

[0093] Table 7 Peak areas and retention times in precision study

[0094]

[0095]

[0096] S2. Stability study

[0097] Take the freeze-dried powder test solution of the standard decoction of Ginseng and Schisandra Decoction prepared by the method of Example 1, analyze it according to the detection method of Example 1, inject samples at different times of 0, 2, 6, 12, 18, and 24 h, with an injection volume of 20 μL. Using liquiritigenin, liquiritin, apiosylisoliquiritigenin, licorinchalcone B, ginsenoside Rg1, glycyrrhizic acid, licorinchalcone A, atractylenolide I, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, atractylenolide II as reference peaks, by analyzing the peak areas and retention times of the common peaks in the HPLC fingerprint of the sample and calculating the RSD values, the results are shown in Table 8, indicating that the chromatographic peaks of the freeze-dried powder test solution of the standard decoction of Ginseng and Schisandra Decoction hardly change within 24 h, and it has good stability.

[0098] Table 8 Peak areas and retention times in stability study

[0099]

[0100] S3. Repeatability study

[0101] Take the freeze-dried powder test solution of the standard decoction of Ginseng and Schisandra Decoction prepared by the method of Example 1, analyze it according to the detection method of Example 1, with an injection volume of 20 μL. Using liquiritigenin, liquiritin, apiosylisoliquiritigenin, licorinchalcone B, ginsenoside Rg1, glycyrrhizic acid, licorinchalcone A, atractylenolide I, schisantherin B, deoxyschizandrin, schisandrin B, schisandrin C, atractylenolide II as reference peaks, by analyzing the peak areas and retention times of the common peaks in the HPLC fingerprint of the sample and calculating the RSD values, the results are shown in Table 9, indicating that the chromatographic peaks of the sample have good reproducibility and the repeatability of this method is good.

[0102] Table 9 Peak areas and retention times in repeatability study

[0103]

[0104] The above experimental results show that the method for constructing the fingerprint of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction provided by the present invention has the characteristics of good stability, high precision, and good repeatability, and can provide quality assurance for the clinical efficacy of Ginseng and Schisandra Decoction.

[0105] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. Fingerprint detection method for freeze-dried powder of standard decoction of Ginseng Schisandra Decoction, characterized in that, The following steps are involved: S1. Using methanol solution as extraction solvent, prepare different batches of test sample solutions of freeze-dried powder of standard decoction of Ginseng and Schisandra Decoction; S2. Inject the test sample solutions of freeze-dried powder of standard decoction of Ginseng and Schisandra Decoction from different batches in S1 into a high performance liquid chromatograph. The chromatographic conditions are as follows: chromatographic column: Hypersil C18 ODS, mobile phase A is 0.1% phosphoric acid aqueous solution, mobile phase B is acetonitrile, gradient elution, detection wavelength: 0-45 min, 280 nm; 45-50 min, 254 nm; 50-55 min, 220 nm; 55-110 min, 254 nm, elution program: 0-10 min 0.1% phosphoric acid aqueous solution, volume 85%; 10-15 min 0.1% phosphoric acid aqueous solution, volume 85%-75%; 15-25 min 0.1% phosphoric acid aqueous solution, volume 75%-70%; 25-30 min 0.1% phosphoric acid aqueous solution, volume 70%-55%; 30-45 min 0.1% phosphoric acid aqueous solution, volume 55%-50%; 45-55 min 0.1% phosphoric acid aqueous solution, volume 50%-45%; 55-75 min 0.1% phosphoric acid aqueous solution, volume 45%-40%; 75-80 min 0.1% phosphoric acid aqueous solution, volume 40%-48%; 80-90 min 0.1% phosphoric acid aqueous solution, volume 48%-70%; 90-100 min 0.1% phosphoric acid aqueous solution, volume 70%-60%; 100-110 min 0.1% phosphoric acid aqueous solution, volume 60%-95%, record the chromatogram; S3. According to the chromatograms of the test solution of the freeze-dried powder of the standard decoction of ginseng and schisandra chinensis obtained in S2, the fingerprint of the freeze-dried powder of the standard decoction of ginseng and schisandra chinensis was constructed; with liquiritigenin, liquiritigenin, apigenin isoliquiritigenin, licorice chalcone B, ginsenoside Rg1, glycyrrhizic acid, licorice chalcone A, atractylodes lactone I, schisandrin B, schisandrin A, schisandrin B, schisandrin C and atractylodes lactone II as reference peaks, the similarity of the fingerprints of each batch was evaluated, and the characteristic peaks were identified by mass spectrometry; The ginseng and schisandra decoction standard decoction freeze-dried powder is prepared by placing six medicinal materials, namely ginseng, atractylodes, poria, schisandra, ophiopogon and licorice, in a decoction pot, soaking them in water, decocting them twice, and drying the filtrate.

2. The fingerprint detection method of the freeze-dried powder of the standard decoction of Ginseng Schisandra Decoction according to claim 1, characterized in that, The specific steps of S1 are: weigh appropriate amounts of freeze-dried powder of standard decoction of Ginseng and Schisandra Decoction from different batches, place them in stoppered conical bottles, add methanol solution, perform ultrasonic extraction, and filter the filtrate through a 0.45 μm microporous filter membrane to obtain the test solution.

3. The fingerprint detection method of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction according to claim 2, characterized in that, The ultrasonic time was 60 min.

4. The fingerprint detection method of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction according to claim 1, characterized in that, In S2, column temperature: 30°C; flow rate: 0.5 mL / min.

5. The fingerprint detection method of the freeze-dried powder of the standard decoction of Ginseng Schisandra Decoction according to claim 1, characterized in that, The specific steps of S3 are as follows: Export the chromatograms of the test solution of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction obtained in S2, and generate a fingerprint through the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A; Select the chromatographic peaks that exist in the chromatograms of different batches as common peaks; Generate a reference fingerprint by the average value calculation method, and export the similarity result table of the chromatograms of the test solution of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction in different batches; Calculate the relative retention time and relative peak area of each common peak; And identify the chemical components in the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction according to the mass spectrometry; Confirm the reliability of the results according to the similarity result table and the chromatograms of the test solution of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction in different batches.

6. The fingerprint detection method for the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction according to claim 1, wherein Thirteen characteristic peaks in the constructed fingerprint were identified by mass spectrometry.

7. The fingerprint detection method of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction according to claim 1, characterized in that, The ginseng is official selected ginseng, the atractylodes rhizome is bleached atractylodes rhizome, the poria is white poria, the schisandra fruit is northern schisandra fruit, the ophiopogon root is Hangzhou ophiopogon root, and the licorice is roasted licorice.

8. The fingerprint detection method of the freeze-dried powder of the standard decoction of Ginseng and Schisandra Decoction according to claim 1, characterized in that, Peak 1 is liquiritigenin, peak 2 is liquiritin, peak 3 is apiosylisoliquiritigenin, peak 4 is licorinchalcone B, peak 5 is ginsenoside Rg1, peak 6 is glycyrrhizic acid, peak 7 is licorinchalcone A, peak 8 is atractylenolide I, peak 9 is schisantherin B, peak 10 is schisandrin A, peak 11 is schisandrin B, peak 12 is schisandrin C, and peak 13 is atractylenolide II.

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