Method for establishing fingerprint of gypsophila gracile preparation, fingerprint and application thereof

Abstract

The present invention provides a method for establishing a fingerprint of a gypsophila preparation, comprising the following steps: S1, preparation of a test solution: weighing the gypsophila preparation, adding methanol, ultrasonically treating, filtering, and obtaining a test solution; S2, preparation of a reference solution: weighing eight reference substances, including cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, polydatin, astilbin, oxidized resveratrol, quercetin, and resveratrol, mixing, adding methanol, and obtaining a reference solution; S3, establishment of a fingerprint: respectively measuring the test solution and the reference solution prepared above by HPLC, recording the chromatograms and comparing them, identifying the common peaks, and obtaining a fingerprint of the gypsophila preparation. The invention successfully established a fingerprint of the gypsophila preparation by optimizing the extraction conditions and HPLC chromatographic conditions of the gypsophila preparation. The fingerprint establishment method is accurate, reliable, and simple to operate, and the detection method has high precision, good specificity, stability, and repeatability.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine fingerprint analysis, and particularly relates to a method for establishing a fingerprint of a gypsophila chinensis preparation and the fingerprint thereof. Background Art

[0002] Jingangteng preparation is a single preparation made from Jingangteng of the Liliaceae family through water extraction and alcohol precipitation. It has the effects of clearing away heat and detoxifying, reducing swelling and dispersing nodules. The main ones include Jingangteng syrup and Jingangteng capsule.

[0003] Currently, quality control for Jingangteng syrup is still primarily based on ministerial standards, primarily employing TLC comparative analysis of the raw medicinal material and foam testing for qualitative identification. These methods are overly simplistic and difficult to achieve the desired quality control objectives. The existing statutory quality standards for Jingangteng capsules are contained in "National Drug Standards for New Drug Conversion, Volume 35." The content determination item uses thin-layer scanning to determine diosgenin, a method with poor reproducibility. Furthermore, Traditional Chinese Medicine (TCM) ingredients are complex, and their pharmacological effects are often the result of the combined action of multiple components. Determination of a single component cannot comprehensively and scientifically evaluate the quality of TCMs.

[0004] Fingerprinting is an important method for evaluating the batch uniformity of traditional Chinese medicine preparations. Multi-component content determination has been a major content determination method promoted in recent years. The combination of these two methods plays a crucial role in effectively controlling batch uniformity and efficacy. Therefore, it is necessary to establish a fingerprint method for simultaneously determining the content of the main components of jingangteng preparations. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for establishing a fingerprint of a gypsophila preparation, which can at least solve some of the defects in the prior art.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for establishing a fingerprint of a gypsophila preparation comprises the following steps:

[0008] S1. Preparation of test solution: Weigh the Knotweed preparation, add methanol, sonicate, and filter to obtain the test solution;

[0009] S2. Preparation of reference solution: Weigh 8 reference substances, including cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, polydatin, astilbin, oxidized resveratrol, quercetin, and resveratrol, mix them, and add methanol to prepare a reference solution;

[0010] S3. Establishment of fingerprint: The test solution and reference solution prepared above were measured by HPLC (high performance liquid chromatography), the chromatograms were recorded and compared, and the common peaks were identified to obtain the fingerprint of the Kantow vine preparation.

[0011] Furthermore, in step S1, the methanol concentration is 90%, the ultrasonic treatment power is 200 W, the frequency is 40 kHz, and the ultrasonic treatment time is 15 min.

[0012] Furthermore, in the reference solution, the concentration of cryptochlorogenic acid was 2.190 μg / mL, the concentration of neochlorogenic acid was 18.914 μg / mL, the concentration of chlorogenic acid was 46.320 μg / mL, the concentration of polydatin was 1.661 μg / mL, the concentration of astilbin was 46.379 μg / mL, the concentration of oxidized resveratrol was 12.524 μg / mL, the concentration of quercetin was 46.411 μg / mL, and the concentration of resveratrol was 2.5967 μg / mL.

[0013] Furthermore, in step S3, the HPLC chromatographic conditions are as follows: the chromatographic column is CAPCELL PAK C18, 250 mm × 4.6 mm, 5 μm; the mobile phase is 0.1% phosphoric acid solution-acetonitrile, with gradient elution; the detection wavelength is 200 nm to 400 nm; the flow rate is 1 mL / min; the column temperature is 25°C to 40°C; the injection volume is 10 μL; and the number of theoretical plates calculated based on the chlorogenic acid peak should be no less than 10,000.

[0014] Furthermore, the HPLC chromatographic conditions are as follows: the chromatographic column is CAPCELLPAK C18, 250 mm × 4.6 mm, 5 μm; the mobile phase is 0.1% phosphoric acid solution-acetonitrile, with gradient elution; the detection wavelength is 303 nm; the flow rate is 1 mL / min; the column temperature is 35°C; the injection volume is 10 μL; and the number of theoretical plates calculated based on the chlorogenic acid peak should be no less than 10,000.

[0015] Furthermore, the gradient elution program is as follows by volume percentage: 0-5 min, 6% acetonitrile, 94% 0.1% phosphoric acid solution; 5-15 min, 6%-10% acetonitrile, 94%-90% 0.1% phosphoric acid solution; 15-60 min, 10%-29% acetonitrile, 90%-71% 0.1% phosphoric acid solution; 60-65 min, 29%-6% acetonitrile, 71%-94% 0.1% phosphoric acid solution; 65-70 min, 6% acetonitrile, 94% 0.1% phosphoric acid solution.

[0016] Furthermore, in step S3, the chromatogram of the test solution is imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System Software" for similarity evaluation with the chromatogram of the reference solution to determine the similarity and identify the common peaks.

[0017] Furthermore, the gypsophila gracile preparation is gypsophila gracile syrup or gypsophila gracile capsule.

[0018] In addition, the present invention also provides a fingerprint spectrum obtained according to the above-mentioned method for establishing the fingerprint spectrum of the gypsophila preparation.

[0019] The present invention also provides the application of the method for establishing the fingerprint of the above-mentioned scutellaria baicalensis preparation in the determination of the content of ingredients in the scutellaria baicalensis preparation and the quality inspection of the scutellaria baicalensis preparation.

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

[0021] (1) The present invention successfully established the fingerprint of the Vinegar Thunb preparation by optimizing the extraction conditions and HPLC chromatography conditions of the Vinegar Thunb preparation. The fingerprint establishment method is accurate, reliable, and simple to operate. The detection method is highly precise, specific, stable, and reproducible.

[0022] (2) The present invention can simultaneously detect the contents of eight active ingredients in the Kung Paeonia lactiflora preparation, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, oxidized resveratrol, quercetin, and resveratrol, providing a basis for the quality control of the Kung Paeonia lactiflora preparation.

[0023] The present invention will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of HPLC superimposed patterns and reference pattern R of 15 batches of Jingangteng syrup in Example 1;

[0025] Figure 2 is the HPLC chromatogram of the reference substance solution in Example 1;

[0026] Figure 3 This is the HPLC chromatogram of the Jingangteng syrup in Example 1;

[0027] Figure 4 is the HPLC chromatogram of the blank sample solution in Example 1;

[0028] Figure 5 HPLC chromatograms of Jingangteng syrup in Example 2 under different extraction solvents; wherein (a) methanol, (b) 90% methanol, and (c) ethanol;

[0029] Figure 6 HPLC chromatograms of Jingangteng syrup in Example 2 using different extraction methods; (a) ultrasonic extraction, (b) reflux extraction;

[0030] Figure 7 HPLC chromatograms of Jingangteng syrup at different extraction times in Example 2, wherein (a) 15 min, (b) 30 min, and (c) 60 min;

[0031] Figure 8The HPLC chromatograms of the Jingangteng syrup in Example 2 using different mobile phases are shown; wherein (a) 0.1% phosphoric acid (A)-acetonitrile (B), (b) 0.1% glacial acetic acid (A)-acetonitrile (B), and (c) 0.1% formic acid (A)-acetonitrile (B);

[0032] Figure 9 HPLC chromatograms of Jingangteng syrup in Example 2 at different detection wavelengths; (a) 203 nm, (b) 290 nm, (c) 303 nm;

[0033] Figure 10 The HPLC chromatograms of the Jingangteng syrup in Example 2 at different column temperatures are shown, wherein (a) is 25°C, (b) is 30°C, (c) is 35°C, and (d) is 40°C.

[0034] Figure 11 The HPLC chromatograms of Jingangteng syrup in Example 2 using different chromatographic columns are shown; (a) WondaSil C18, (b) Nano Chrom C18, (c) SHIMADZU C18, (d) Thermo scientific C18, (e) Agilent 5TC-C18 (2) C18, (f) GL Sciences Inc Inertsil ODS-3 C18, (g) Capcellpak C18, and (f) Waters C18.

[0035] Figure 12 HPLC chromatograms of Jingangteng syrup at different flow rates in Example 2, wherein (a) 0.8 mL / min, (b) 1.0 mL / min, and (c) 1.2 mL / min;

[0036] Figure 13 HPLC chromatograms of Jingangteng syrup at different running times in Example 2, wherein (a) 65 min, (b) 70 min, and (c) 75 min;

[0037] Figure 14 Schematic diagram of HPLC superimposed patterns and reference pattern R of 15 batches of Jingangteng Capsules in Example 4;

[0038] Figure 15 is the HPLC chromatogram of the reference substance solution in Example 4;

[0039] Figure 16 This is the HPLC chromatogram of Jingangteng Capsule in Example 4;

[0040] Figure 17 is the HPLC chromatogram of the blank sample solution in Example 4;

[0041] Figure 18 HPLC chromatograms of Jingangteng capsule in Example 4 under different extraction solvents; wherein (a) methanol, (b) 90% methanol, and (c) 60% methanol;

[0042] Figure 19 HPLC chromatograms of Jingangteng Capsules in Example 4 using different extraction methods; (a) ultrasonic extraction, (b) reflux extraction;

[0043] Figure 20 HPLC chromatograms of Jingangteng Capsules at different extraction times in Example 4, wherein (a) 15 min, (b) 30 min, and (c) 60 min;

[0044] Figure 21 HPLC chromatograms of Jingangteng Capsules in Example 4 using different mobile phases; (a) 0.1% phosphoric acid (A)-acetonitrile (B), (b) 0.1% glacial acetic acid (A)-acetonitrile (B), and (c) 0.1% formic acid (A)-acetonitrile (B);

[0045] Figure 22 HPLC chromatograms of Jingangteng Capsules in Example 4 at different detection wavelengths; (a) 280 nm, (b) 303 nm, and (c) 326 nm;

[0046] Figure 23 HPLC chromatograms of Jingangteng Capsules in Example 4 at different column temperatures, wherein (a) is 25°C, (b) is 30°C, and (c) is 35°C.

[0047] Figure 24 The HPLC chromatograms of Jingangteng Capsules in Example 4 using different chromatographic columns are shown; (a) CAPCELLPAK C18, (b) GL Science WondaSil C18, (c) NanoChrom ChromCoreC18, and (d) WatersSunFire C18. DETAILED DESCRIPTION

[0048] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0049] Experimental instruments and reagents:

[0050] 1. Instruments

[0051] Electronic balance [Mettler Toledo MS105, Mettler-Toledo Instruments (Shanghai) Co., Ltd.]; ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.); ultrapure water machine (PGJ-20-YS, Wuhan Pinguan Instrument Equipment Co., Ltd.); electric constant temperature water bath (DK-900-II, Tianjin Test Instrument Co., Ltd.); high performance liquid chromatograph [UltiMate3000, Thermo Fisher Scientific (China) Co., Ltd.], including DAD detector (DAD-300) and chromatography workstation (Chromelenon 7).

[0052] 2. Drug reagents

[0053] Cryptochlorogenic acid (purity 98.0%, CAS No. 905-99-7); neochlorogenic acid (purity 98.0%, CAS No. 906-33-2); and oxidized resveratrol (purity 98.0%, CAS No. 29700-22-9) were purchased from Shanghai Topu Biopharmaceutical Technology Co., Ltd.; polydatin (purity 87.3%, batch No. 111575-201603), astilbin (purity 95.0%, batch No. 111798-202306), quercetin (purity 95.3%, batch number 111538-202308), chlorogenic acid [purity 96.3%, batch number 110753-202119], quercetin (purity 99.1%, batch number 100081-201610), and resveratrol (purity 99.4%, batch number 111535-201703) were purchased from the China Food and Drug Inspection Institutes.

[0054] There were 15 batches of Jingangteng syrup (numbered S1 to S15), with batch numbers 20220101, 20220605, 20220901, 20220902, 20220903, 20220904, 20221101, 20230101, 20230102, 20240101, 20240102, 20240103, 20240104, 20240105, and 20240106, all provided by Hubei Furen Pharmaceutical Co., Ltd.

[0055] There are 15 batches of Jingangteng Capsules (numbered S1 to S15), batch numbers 20210101, 20210103, 20210105, 20210108, 20220402, 20231217, 20231218, 20231219, 20231220, 20240105, 20240106, 20240107, 20240108, 20240109, and 20240110, all provided by Hubei Furen Pharmaceutical Co., Ltd.

[0056] Phosphoric acid (superior grade, Tianjin Komiou Chemical Reagent Co., Ltd.), water was ultrapure water prepared by a Piguan ultrapure water machine, acetonitrile and methanol (chromatographic grade, Shanghai Sarao Industrial Co., Ltd.).

[0057] Example 1: Establishment of fingerprint of jingangteng syrup

[0058] 1. Solution preparation

[0059] 1.1 Reference substance solution: Accurately weigh appropriate amounts of 8 reference substances including cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, polydatin, astilbin, oxidized resveratrol, quercetin, and resveratrol, and add 90% methanol to prepare a mixed solution containing 2.190 μg / mL of cryptochlorogenic acid, 18.914 μg / mL of neochlorogenic acid, 46.320 μg / mL of chlorogenic acid, 1.661 μg / mL of polydatin, 46.379 μg / mL of astilbin, 12.524 μg / mL of oxidized resveratrol, 46.411 μg / mL of quercetin, and 2.597 μg / mL of resveratrol.

[0060] 1.2 Test solution: Accurately take 10 mL of Vinegar syrup and place it in a 50 mL volumetric flask. Add appropriate amount of 90% methanol and treat it with ultrasound (200W, 40 kHz) for 15 minutes. Take it out and let it stand to room temperature. Add 90% methanol to the scale, shake well, pass it through a microporous filter membrane (0.22 μm), and take the filtrate.

[0061] 2. Chromatographic conditions

[0062] The chromatographic column was Capcell Pak C18, 4.6 mm × 250 mm, 5.0 μm. The mobile phase was 0.1% phosphoric acid solution (A)-acetonitrile (B). The gradient elution program shown in Table 1 was used for elution. The column temperature was 35°C, the flow rate was 1.0 mL / min, the detection wavelength was 303 nm, and the injection volume was 10 μL.

[0063] Table 1: Gradient elution program

[0064]

[0065] 3. Investigation of fingerprint methodology

[0066] 3.1 Precision Test: Take Jingangteng syrup (S10) and prepare a test solution according to the method in "1.2 Test Solution". Measure six times according to "2. Chromatographic Conditions". Record the peak areas of each common peak. The RSDs of the peak areas of the eight components (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, oxyresveratrol, quercetin, and resveratrol) were calculated to be 0.55%, 0.52%, 0.55%, 1.11%, 0.88%, 0.55%, 1.42%, and 0.77%, respectively. The RSDs of the relative peak areas of each common peak were all <1.5%, indicating good instrument precision.

[0067] 3.2 Stability Test: Take Jingangteng Syrup (S10) and prepare a test solution according to the method in "1.2 Test Solution". Measure according to "2. Chromatographic Conditions". Record the relative peak areas of the common peaks within 0, 4, 8, 12, 16, and 24 hours. The RSDs of the peak areas of the eight components (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, oxidized resveratrol, quercetin, and resveratrol) within 24 hours were calculated to be 0.68%, 0.43%, 0.70%, 0.94%, 0.43%, 0.83%, 1.80%, and 0.99%, respectively. The RSDs of the relative peak areas of the common peaks were all less than 2.0%, indicating that Jingangteng Syrup had good stability within 24 hours.

[0068] 3.3 Repeatability Test: Six test solutions were prepared from the syrup of Rhizoma Coptidis (S10) according to the method in "1.2 Test Solution". The peak areas of the common peaks were recorded according to "2. Chromatographic Conditions". The RSDs of the peak areas of the eight components (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, oxyresveratrol, quercetin, and resveratrol) were calculated to be 0.55%, 0.10%, 0.18%, 0.74%, 0.20%, 0.72%, 0.52%, and 0.70%, respectively. The RSDs of the relative peak areas of the common peaks were all <1.0%, indicating good repeatability of the method.

[0069] 3.4 Establishment of fingerprints and identification of chromatographic peaks: Take 15 batches of Kungfu Teng syrup, prepare the test solution according to the method of "1.2 Test solution", inject the sample according to the chromatographic conditions under "2. Chromatographic conditions", record the chromatogram, set the integration parameters on the Dion Chromeleon chromatography workstation (version 7.2.10), and integrate the main chromatographic peaks. Export the CDF files of the spectra one by one in the form of CDF data files. Import the exported CDF files of the above 15 batches of chromatograms into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System Software (2012 Edition)", cut off the solvent peak of the first 3 minutes, and establish the overlay map and control fingerprint of 15 batches of samples through the evaluation system, as shown below: Figure 1 As shown. By comparing with the chromatogram of mixed reference solution (such as Figure 2(as shown) comparison, Figure 1 The eight common peaks were identified, in order: peak 3 neochlorogenic acid, peak 5 chlorogenic acid, peak 6 cryptochlorogenic acid, peak 12 polydatin, peak 14 astilbin, peak 16 oxidized resveratrol, peak 18 quercetin, and peak 21 resveratrol; the fingerprint of Jingangteng syrup was obtained as follows Figure 3 As shown in the figure, the eight common peaks were renumbered from 1 to 8, and peaks 1 to 8 represent neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, oxidized resveratrol, quercetin, and resveratrol, respectively.

[0070] 3.5 Similarity Evaluation: Using the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System Software (2012 Edition)," the similarity between the chromatograms of 15 batches of Jingangteng Teng syrup and the control fingerprint was calculated. The similarity results are shown in Table 2. As can be seen from Table 2, the similarities were all > 0.90, indicating that the chemical composition of different batches of Jingangteng Teng syrup was highly uniform and stable, with similar contents of major components and no significant quality differences. Furthermore, their fingerprint characteristics were effectively and accurately reflected in the fingerprints.

[0071] Table 2: Similarity calculation results of 15 batches of Jingangteng syrup

[0072]

[0073] 4. Blank sample test

[0074] Take the auxiliary materials of Jingangteng Syrup to prepare a negative blank solution, inject 10 μL of it using the chromatographic conditions under "2. Chromatographic Conditions", and record the chromatogram, such as Figure 4 The results show that the negative blank solution does not have a chromatographic peak corresponding to the test solution in the chromatogram, and there is no interference.

[0075] Example 2: Preparation method of Jingangteng syrup test solution and optimization of chromatographic conditions

[0076] 1. Selection of extraction solvent

[0077] Take three portions of Jingangteng Syrup S10 (batch number: 20240101), accurately add 50 mL of methanol, 90% methanol solution and ethanol solution respectively, weigh the weight, ultrasonically treat at power 200W and frequency 40kHz for 15 minutes, let cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and take the filtrate as the test solution. Determine under the same method, the chromatograms are as follows Figure 5 (a) Figure 5 (b) Figure 5As shown in Figure (c), the optimal extraction solvent was determined by comparison. The results showed that ethanol extraction resulted in fewer and smaller chromatographic peaks, indicating incomplete extraction. However, extraction with 90% methanol produced higher peak heights, higher extraction efficiency, and better peak shape. Therefore, 90% methanol was selected as the extraction solvent for preparing the Jingangteng Teng syrup sample.

[0078] 2. Selection of extraction method

[0079] Take two portions of Jingangteng syrup S10 (batch number: 20240101), accurately add 50mL of 90% methanol solution, weigh the weight, and treat with ultrasound (power 200W, frequency 40kHz) for 15 minutes and reflux extraction for 15 minutes respectively. Let cool, weigh the weight again, make up the lost weight with 90% methanol, shake well, filter, and take the filtrate as the test solution. The chromatograms are as follows when measured under the same method. Figure 6 (a) and Figure 6 (b) The optimal extraction method was determined by comparison. The results showed that the main chromatographic peak height in the chromatogram was higher and the peak shape was better when ultrasonic extraction was used; therefore, ultrasonic treatment was selected as the extraction method for preparing the Jingangteng syrup sample.

[0080] 3. Selection of extraction time

[0081] Take three portions of Jingangteng syrup S10 (batch number: 20240101), accurately add 50mL of 90% methanol solution, weigh the weight, and treat with ultrasound (power 200W, frequency 40kHz) for 15min, 30min, and 60min respectively. Let cool, weigh the weight again, make up the lost weight with 90% methanol, shake well, filter, and take the filtrate as the test solution. Determine under the same method, the chromatograms are as follows Figure 7 (a) Figure 7 (b) and Figure 7 As shown in Figure (c), the optimal extraction time was determined by comparison. The results showed that there was no significant difference in the chromatograms after ultrasonic extraction for 15 minutes and 30 minutes, and the peak shape was good. However, after 60 minutes of ultrasonic treatment, the peak areas of multiple chromatographic peaks decreased. In order to ensure good extraction efficiency and prevent the degradation of chemical components during the extraction process, 15 minutes of ultrasonic treatment was selected as the optimal extraction time for preparing the Jingangteng Teng syrup sample.

[0082] 4. Selection of mobile phase

[0083] An appropriate amount of Jingangteng syrup S10 (batch number: 20240101) was taken and prepared according to the test solution preparation method in Example 1. The following mobile phases were compared using a C18 chromatographic column: ① 0.1% phosphoric acid (A)-acetonitrile (B), ② 0.1% glacial acetic acid (A)-acetonitrile (B), and ③ 0.1% formic acid (A)-acetonitrile (B). Gradient elution was performed using the gradient elution program in Example 1. The chromatograms were as follows: Figure 8 (a) Figure 8 (b) Figure 8 As shown in (c), although the three mobile phases mentioned above all achieved separation, mobile phase ① had the best separation effect, while mobile phase ② had poor separation effect on peaks 2 and 3; when mobile phase ③ was used for separation, the response of most peaks was lower than that of mobile phase ①, and peaks 7 and 8 could not be completely separated; therefore, mobile phase ① was selected as the mobile phase for the test sample of Jingangteng Teng Syrup.

[0084] 5. Selection of detection wavelength

[0085] Take an appropriate amount of Jingangteng syrup S10 (batch number: 20240101), prepare it according to the test solution preparation method of Example 1, inject it into the high performance liquid chromatography for analysis, and follow the chromatographic conditions of Example 1, wherein the detection wavelengths are 203nm, 290nm, and 303nm for comparison. The chromatograms are as follows: Figure 9 (a) Figure 9 (b) Figure 9 The results show that the detection wavelength of 303 nm provides richer chromatographic information, a more stable baseline, and more balanced responses across all peaks, which comprehensively reflects the components in the sample. In particular, the peak areas of Peaks 2 and 6, which have lower responses, are larger than those at other wavelengths. Therefore, 303 nm was selected as the detection wavelength for the Jingangteng Teng Syrup sample.

[0086] 6. Selection of column temperature

[0087] Take 4 portions of Jingangteng syrup S10 (batch number: 20240101), prepare the test solution according to the preparation method of Example 1, and inject it into the high performance liquid chromatography for analysis, wherein the column temperature is 25℃, 30℃, 35℃, and 40℃ respectively. The chromatograms are as follows: Figure 10 (a) Figure 10 (b) Figure 10 (c) Figure 10 (d) The results showed that when the column temperature was 30°C, the chromatographic peak separation effect was poor, especially at 30°C, peaks 4 and 5 included nearby impurity peaks, affecting the accuracy of the results; when the column temperature was 25°C, peak 8 may have included impurity peaks, while when the column temperature was 35°C, the HPLC chromatogram separation effect of Jingangteng syrup was the best; therefore, 35°C was selected as the column temperature for the Jingangteng syrup fingerprint.

[0088] 7. Selection of chromatographic columns

[0089] An appropriate amount of Jingangteng syrup S10 (batch number: 20240101) was taken and prepared according to the test solution preparation method of Example 1, and injected into a high performance liquid chromatograph for analysis. The analysis was performed using chromatographic columns WondaSil C18, Nano Chrom C18, SHIMADZU C18, Thermo scientific C18, Agilent 5TC-C18(2)C18, GL Sciences Inc Inertsil ODS-3C18, Capcell pak C18 and Waters C18, respectively. The chromatograms were as shown below: Figure 11 (a) Figure 11 (b) Figure 11 (c) Figure 11 (d), 11(e), Figure 11 (f) Figure 11 (g) Figure 11 (h) As shown. The results showed that, using WondaSil C18 column, peak 2 and peak 3 could not be completely separated, and peak 6 could not be separated from impurities; using Nano Chrom C18 column, peak 5 could not be separated from impurities, and all peaks had obvious tailing; using SHIMADZU C18 column, peak 7 and peak 8 were not separated from impurities, and the impurity peak on the left of peak 2 disappeared; using Thermo scientific C18 column, peak 7 and peak 8 were not separated from impurities; using Agilent 5TC-C18(2)C18, peak 7 and peak 8 were not separated from impurities, and the impurity peak on the left of peak 2 disappeared; using GL Sciences Inc Inertsil ODS-3C18 column, peak 6 was not separated from impurities, and the impurity peak on the left of peak 2 disappeared; using Waters C18 column, the chromatographic peaks had serious tailing; therefore, Capcellpak C18 column was selected as the chromatographic column for the determination of the fingerprint of King Kong Teng Syrup.

[0090] 8. Selection of column flow rate

[0091] Take an appropriate amount of Jingangteng syrup S10 (batch number: 20240101), prepare the test solution according to the preparation method of Example 1, and inject it into the high performance liquid chromatography for analysis, wherein the flow rates are 0.8mL / min, 1.0mL / min, and 1.2mL / min, respectively. The chromatograms are as follows: Figure 12 (a) Figure 12 (b) Figure 12(c) The results show that at a flow rate of 0.8 mL / min, Peak 2 could not be separated from the impurity peak on the left; at a flow rate of 1.2 mL / min, Peak 6 appeared as a double peak. A flow rate of 1.0 mL / min demonstrated superior resolution, peak shape, and system pressure control in the HPLC analysis of Jingangteng Teng Syrup S10. Therefore, 1.0 mL / min was selected as the optimal flow rate.

[0092] 9. Selection of column running time

[0093] Take an appropriate amount of Jingangteng syrup S10 (batch number: 20240101), prepare it according to the test solution preparation method of Example 1, and inject it into the high performance liquid chromatography for analysis. The running time is 65min, 70min, and 75min respectively. The chromatograms are as follows: Figure 13 (a) Figure 13 (b) Figure 13 (c) The results show that when the run time is 65 minutes, Peak 8 may contain impurities; when the run time is 75 minutes, Peak 6 has a double peak; and when the run time is 70 minutes, the chromatographic peaks are clear and well resolved, so 70 minutes is selected as the optimal run time.

[0094] Example 3: Determination of the contents of 8 components in Jingangteng syrup

[0095] 1. Preparation of solution: same as in Example 1 above.

[0096] 2. Chromatographic conditions: same as in Example 1 above.

[0097] 3. Determination of ingredient content and methodology investigation

[0098] 3.1 Linearity Assessment: Each reference substance was accurately weighed and dissolved in 90% methanol to prepare a mixed solution containing 1623.860 μg / mL of cryptochlorogenic acid, 840.840 μg / mL of neochlorogenic acid, 2010.744 μg / mL of chlorogenic acid, 521.181 μg / mL of polydatin, 951.900 μg / mL of astilbin, 530.180 μg / mL of oxyresveratrol, 428.850 μg / mL of quercetin, and 381.828 μg / mL of resveratrol. The solution was serially diluted and measured according to "2. Chromatographic Conditions." Chromatograms were recorded, with the abscissa (x, μg / mL) representing mass concentration and the ordinate (y) representing peak area. Linearity analysis was performed for each component. The results are shown in Table 3. As can be seen from Table 3, the r values ​​were all greater than 0.9999, indicating that each component exhibited good linearity within its respective range.

[0099] Table 3: Linear equations and linear ranges of 8 components

[0100]

[0101] 3.2 Precision test: Same as “3.1 Precision test” in Example 1 above.

[0102] 3.3 Stability test: Same as “3.2 Stability test” in Example 1 above.

[0103] 3.4 Repeatability test: Same as “3.3 Repeatability test” in Example 1 above.

[0104] 3.5 Sample Addition Recovery Test: Six portions of the same Jingangteng Teng syrup sample (S10) were taken, 5 ml of each portion was accurately measured, and approximately equal amounts of each single reference substance were added. The test solution was prepared according to the method in "1.2 Test Solution". The determination was carried out according to "2. Chromatographic Conditions". The average sample addition recoveries of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, oxidized resveratrol, quercetin, and resveratrol were calculated. The results are shown in Table 4. The results showed that the average sample addition recoveries of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, oxidized resveratrol, quercetin, and resveratrol were 102.3%, 96.5%, 97.4%, 103.4%, 98.7%, 95.6%, 108.4%, and 100.6%, respectively (n=6), indicating that the method had a good recovery rate.

[0105] Table 4: Recovery rates of various components in syrup of Jingangteng

[0106]

[0107]

[0108] 3.6 Sample content determination: Take the syrup of King Kong Teng (S1~S15), prepare the test solution according to the method of "1.2 Test solution", and perform sample injection and determination according to "2. Chromatographic conditions". Calculate the contents of the 8 components in the sample. The results are shown in Table 5.

[0109] Table 5: Determination results of 8 components in 15 batches of Jingangteng syrup (μg / mL)

[0110]

[0111] Example 4: Establishment of fingerprint of Jingangteng capsule

[0112] 1. Solution preparation

[0113] 1.1 Reference solution: same as in Example 1 above.

[0114] 1.2 Test solution: Same as Example 1, except that the test sample is Jingangteng capsule.

[0115] 2. Chromatographic conditions: same as in Example 1 above.

[0116] 3. Investigation of fingerprint methodology

[0117] 3.1 Precision Test: Take the Jingangteng Capsule (S10) test solution and inject it six times continuously according to the chromatographic conditions under "2. Chromatographic Conditions". Record the retention time and peak area of ​​each common peak. Among them, the peak area RSD values ​​of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, oxidized resveratrol, quercetin, and resveratrol are 1.25%, 0.89%, 0.92%, 0.98%, 1.07%, 1.01%, 1.32%, and 1.23%, respectively. The RSD of the peak area of ​​each component is less than 1.26%. The calculated RSD of the retention time of each common peak is less than 0.12%, indicating good instrument precision.

[0118] 3.2 Stability Test: Jingangteng Capsules (S10) were used to prepare a test solution according to the method under "1.2 Test Solution". Samples were injected and measured at 0, 4, 8, 12, 16, 20, and 24 h according to the chromatographic conditions under "2. Chromatographic Conditions". The peak areas of the components at the seven time points are shown in Table 6. As shown in Table 6, the RSDs of the retention times of the common peaks were all <0.06%, and the RSDs of the peak areas were all <1.85%, indicating that the test solution had good stability within 24 h.

[0119] Table 6: Peak area results of stability test

[0120]

[0121]

[0122] 3.3 Repeatability Test: Six test solutions were prepared from the same Jingangteng capsule sample (S10) according to the method described in "1.2 Test Solution." The solutions were injected and tested under the chromatographic conditions described in "2. Chromatographic Conditions." The RSD values ​​for the retention times and peak areas of the components were calculated. The results are shown in Table 7. As shown in Table 7, the RSDs for the peak areas of the common peaks were all <2.43%, and the RSDs for the retention times were all <0.14%, indicating good repeatability of the method.

[0123] Table 7: Peak area results of repeatability test

[0124]

[0125] 3.4 Establishment of fingerprints and identification of chromatographic peaks: Take 15 batches of Jingangteng capsules, prepare the test solution according to the method under "1.2 Test solution", inject the sample according to the chromatographic conditions under "2. Chromatographic conditions", record the chromatogram, and integrate the main chromatographic peaks. Export the CDF files of the spectrum one by one in the form of CDF data files, and import the exported CDF files of the above 15 batches of chromatograms into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System Software (2012 Edition)". Through the evaluation system, the overlay map and reference fingerprint of 15 batches of samples were established, as shown in the following figure: Figure 14 As shown. A total of 8 common peaks were identified and compared with the chromatogram of the mixed reference substance (such as Figure 15 The eight common peaks were identified and renumbered in order: peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is cryptochlorogenic acid, peak 4 is polydatin, peak 5 is astilbin, peak 6 is oxidized resveratrol, peak 7 is quercetin, and peak 8 is resveratrol. Figure 16 shown.

[0126] 3.5 Similarity Evaluation: The "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System Software (2012 Edition)" was used to calculate the similarity between the chromatograms of 15 batches of Jingangteng Capsules and the control fingerprint. The similarity results are shown in Table 8. As can be seen from Table 8, the similarities ranged from 0.860 to 0.991, indicating good batch uniformity of Jingangteng Capsules.

[0127] Table 8: Similarity calculation results of 15 batches of Jingangteng capsules

[0128]

[0129] 4. Blank sample test

[0130] Take the excipients of Jingangteng Capsule to prepare a negative blank solution, inject 10 μL of the solution using the chromatographic conditions under “2. Chromatographic conditions”, and record the chromatogram. Figure 17 The results show that the negative blank solution does not have a chromatographic peak corresponding to the test solution in the chromatogram, and there is no interference.

[0131] Example 5: Preparation method of Jingangteng capsule test solution and optimization of chromatographic conditions

[0132] 1. Selection of extraction solvent

[0133] Take three portions of Jingangteng Capsule S10 (batch number: 20240105), each about 2g, and accurately add 50mL of methanol, 90% methanol, and 60% methanol respectively, weigh the weight, and ultrasonically treat at a power of 500W and a frequency of 40kHz for 15min. Let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate as the test solution. Determine it by comparison under the same method and determine the best extraction solvent. The chromatograms are as follows: Figure 18 (a) Figure 18 (b) Figure 18 As shown in (c), the chromatographic peaks of the 60% methanol and methanol extractions were small, indicating inadequate extraction. However, the 90% methanol extraction resulted in a higher main chromatographic peak height, higher extraction efficiency, and better peak shape. Therefore, 90% methanol was selected as the extraction solvent for the Jingangteng Teng capsule sample.

[0134] 2. Selection of extraction method

[0135] Take two portions of Jingangteng Capsule S10 (batch number: 20240105), each about 2 g, place them in a stoppered conical flask, accurately add 90% methanol 50 mL, weigh the weight, respectively, use ultrasonic treatment (power 200 W, frequency 40 kHz) for 15 minutes and reflux extraction for 15 minutes, let cool, weigh the weight again, make up the lost weight with 90% methanol, shake well, filter, take the filtrate as the test solution, and determine it under the same method. The best extraction method is determined by comparison. The chromatograms are as follows: Figure 19 (a) Figure 19 The main chromatographic peak height in the chromatogram obtained by ultrasonic extraction for 15 min was higher than that obtained by reflux extraction for 15 min. To ensure good extraction efficiency and prevent degradation of chemical components during heating, ultrasonic treatment for 15 min was selected as the extraction method for preparing Jingangteng Capsules.

[0136] 3. Selection of extraction time

[0137] Take three portions of Jingangteng Capsule S10 (batch number: 20240105), each about 2g, and place them in a stoppered conical flask. Add 50mL of 90% methanol and weigh the weight. Ultrasonicate (power 500W, frequency 40kHz) for 15min, 30min, and 60min, respectively. Let cool, weigh the weight again, make up the lost weight with 90% methanol, shake well, filter, and take the filtrate as the test solution. Determine the optimal extraction time by comparison under the same method. The chromatograms are as follows: Figure 20 (a) Figure 20 (b) Figure 20(c) Among them, the chromatograms of ultrasonic extraction for 15 min and 30 min showed no significant difference, and the peak shape was good. However, after 60 min of ultrasonic treatment, the area of ​​some chromatographic peaks decreased. Therefore, 15 min of ultrasonic treatment was selected as the optimal extraction time for preparing Jingangteng capsule test samples.

[0138] 4. Selection of mobile phase

[0139] An appropriate amount of Jingangteng Capsule S10 (batch number: 20240105) was taken and prepared according to the test solution preparation method of Example 1. The following mobile phases were compared using a C18 chromatographic column: ① 0.1% phosphoric acid (A)-acetonitrile (B), ② 0.1% glacial acetic acid (A)-acetonitrile (B), and ③ 0.1% formic acid (A)-acetonitrile (B). Gradient elution was performed using the gradient elution program in Example 1. The chromatograms were as follows: Figure 21 (a) Figure 21 (b) Figure 21 As shown in (c), while all three mobile phases achieved separation, mobile phase ① provided the best separation. Mobile phase ② did not separate Peak 2 from the preceding peaks well. Mobile phase ③ also had lower response than mobile phase ① for most peaks. Therefore, mobile phase ① was selected as the mobile phase for the Jingangteng capsule sample.

[0140] 5. Selection of detection wavelength

[0141] Take an appropriate amount of Jingangteng Capsule S10 (batch number: 20240105), prepare the test solution according to the preparation method of Example 1, inject it into the high performance liquid chromatography for analysis, and follow the chromatographic conditions of Example 1, wherein the detection wavelengths are 280nm, 303nm, and 326nm for comparison. The chromatograms are as follows: Figure 22 (a) Figure 22 (b) Figure 22 (c) Among them, using 303 nm as the detection wavelength, the chromatographic information is richer, the baseline is more stable, and the response values ​​of each peak are more balanced, which can relatively comprehensively reflect the component information in the sample. In particular, the response of Peak 5 is larger at other wavelengths. Therefore, 303 nm was selected as the detection wavelength for the Jingangteng Teng capsule sample.

[0142] 6. Selection of column temperature

[0143] Take an appropriate amount of Jingangteng Capsule S10 (batch number: 20240105), prepare the test solution according to the preparation method of Example 1, and inject it into the high performance liquid chromatography for analysis, wherein the column temperature is 25℃, 30℃, and 35℃ respectively. The separation of the chromatogram is as follows: Figure 23 (a) Figure 23 (b) Figure 23(c) At a column temperature of 25°C, the chromatographic peak separation was poor, especially for peaks 2 and 4, which were not separated from nearby impurity peaks, affecting the accuracy of the results. At a column temperature of 30°C, the response values ​​of the peaks were not as high as those at 35°C. Therefore, 35°C was selected as the column temperature for the fingerprint of Jingangteng Capsule.

[0144] 7. Selection of chromatographic columns

[0145] Take an appropriate amount of Jingangteng Capsule S10 (batch number: 20240105), prepare the test solution according to the preparation method of Example 1, inject it into the high performance liquid chromatography for analysis, and analyze it with chromatograms ①CAPCELL PAK C18, ②GL Science WondaSil C18, ③NanoChrom ChromCoreC18, and ④Waters SunFire C18, respectively. The chromatograms are as follows: Figure 24 (a) Figure 24 (b) Figure 24 (c) Figure 24 As shown in (d), it can be seen that using the GL Science WondaSil C18 column (②), peaks 2 and 6 were not separated from the preceding and following impurity peaks. However, using columns (①), (③), and (④), the target components were all well separated, indicating that this method is well adapted to different brands of columns.

[0146] Example 6: Determination of the contents of 8 ingredients in Jingangteng Capsules

[0147] 1. Preparation of solution: same as in Example 1.

[0148] 2. Chromatographic conditions: same as in Example 1.

[0149] 3. Determination of ingredient content and methodology investigation

[0150] 3.1 Linear relationship investigation: Take appropriate amount of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, oxidized resveratrol, quercetin, and resveratrol reference substances, accurately weigh them, add 90% methanol to dissolve them and prepare reference substance stock solutions containing 841 μg / mL of neochlorogenic acid, 2011 μg / mL of chlorogenic acid, 1624 μg / mL of cryptochlorogenic acid, polydatin, astilbin, oxidized resveratrol, quercetin, and resveratrol, respectively. Dilute the above reference substance stock solutions stepwise with 90% methanol to prepare series of mixed standard solutions No. 1 to No. 8. Determine according to the chromatographic conditions under "2. Chromatographic Conditions" and record the chromatograms. A linear regression equation was drawn with the concentration of the linear solution series as the abscissa (x) and the corresponding peak area as the ordinate (y). The linear equations and linear ranges of each component are shown in Table 9. As can be seen from Table 9, r is greater than 0.999, and each component has a good linear relationship within its respective range.

[0151] Table 9: Linear equations and linear ranges of 8 components

[0152]

[0153] 3.2 Precision test: Same as “3.1 Precision test” in Example 4 above.

[0154] 3.3 Stability test: Same as “3.2 Stability test” in Example 4 above.

[0155] 3.4 Repeatability test: Same as “3.3 Repeatability test” in Example 4 above.

[0156] 3.5 Sample recovery test: 1 g of Jingangteng Capsule (S10) with known component contents was accurately weighed. The contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, oxidized resveratrol, quercetin, and resveratrol were 1.08 mg / g, 2.58 mg / g, 1.23 mg / g, 0.17 mg / g, 0.18 mg / g, 0.68 mg / g, 0.23 mg / g, and 0.47 mg / g, respectively. Each reference solution was accurately added in a 1:1 ratio of the components in the sample. Six replicates were prepared. The test solution was prepared according to the method under "1.2 Test Solution". The sample was injected and determined according to the chromatographic conditions under "2. Chromatographic Conditions". The average recovery of each component was calculated. The results are shown in Table 10. The results showed that the average recoveries of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, polydatin, astilbin, oxidized resveratrol, quercetin and resveratrol were 103.15%, 94.55%, 96.79%, 92.08%, 99.87%, 92.77%, 96.44% and 92.23% (n=6), respectively. The recovery rate of this method was good.

[0157] Table 10: Recovery rates of 8 components in 15 batches of Jingangteng capsules

[0158]

[0159] 3.6 Sample content determination: Take the test sample solution of Jingangteng Capsules (S1~S15), and inject and determine the sample according to the chromatographic conditions under "2. Chromatographic conditions". Calculate the contents of the 8 components in the sample. The results are shown in Table 11.

[0160] Table 11: Content determination results of 8 components of 15 batches of Jingangteng capsules (mg / g)

[0161]

[0162] In summary, by optimizing the HPLC chromatographic conditions, the HPLC fingerprint of the Vajra chinensis preparation was successfully established, and a total of 8 main common peaks were identified. These 8 common peaks were attributed, and 8 active ingredients could be determined simultaneously. The similarity between the fingerprints of 15 batches of Vajra chinensis preparations and the control was greater than 0.9, indicating that the types of chemical components contained in each batch of samples were basically consistent and there was good uniformity between the samples. This detection method is accurate, reliable, and simple to operate, and can provide a basis for the quality control of the preparation.

[0163] The above examples are merely illustrative of the present invention and do not limit the scope of protection of the present invention. Any design that is identical or similar to the present invention falls within the scope of protection of the present invention.

Claims

1. A method for establishing a fingerprint of a gypsophila preparation, characterized in that: The steps include: S1. Preparation of test solution: Weigh the gypsophila preparation, add methanol, sonicate, and filter to obtain a test solution; wherein the methanol concentration is 90%, the sonication power is 200 W, the frequency is 40 kHz, and the sonication time is 15 min; S2. Preparation of reference solution: Weigh 8 reference substances, including cryptochlorogenic acid, neochlorogenic acid, chlorogenic acid, polydatin, astilbin, oxidized resveratrol, quercetin, and resveratrol, mix them, and add methanol to prepare a reference solution; S3. Establishment of fingerprint: The test solution and reference solution prepared above were measured by HPLC, the chromatograms were recorded and compared, and the common peaks were identified to obtain the fingerprint of the scutellaria preparation; The HPLC chromatographic conditions are as follows: CAPCELL PAK C18 column, 250 mm × 4.6 mm, 5 μm; mobile phase: 0.1% phosphoric acid solution-acetonitrile, gradient elution; detection wavelength: 200 nm to 400 nm; flow rate: 1 mL / min; column temperature: 25°C to 40°C; injection volume: 10 μL; the theoretical plate number calculated based on the chlorogenic acid peak should be no less than 10,000; The gradient elution program is as follows by volume percentage: 0-5 min, 6% acetonitrile, 94% 0.1% phosphoric acid solution; 5-15 min, 6%-10% acetonitrile, 94%-90% 0.1% phosphoric acid solution; 15-60 min, 10%-29% acetonitrile, 90%-71% 0.1% phosphoric acid solution; 60-65 min, 29%-6% acetonitrile, 71%-94% 0.1% phosphoric acid solution; 65-70 min, 6% acetonitrile, 94% 0.1% phosphoric acid solution.

2. The method for establishing the fingerprint of the scutellaria baicalensis preparation according to claim 1, wherein: The concentration of cryptochlorogenic acid in the reference solution was 2.190 μg / mL, the concentration of neochlorogenic acid was 18.914 μg / mL, the concentration of chlorogenic acid was 46.320 μg / mL, the concentration of polydatin was 1.661 μg / mL, the concentration of astilbin was 46.379 μg / mL, the concentration of oxidized resveratrol was 12.524 μg / mL, the concentration of quercetin was 46.411 μg / mL, and the concentration of resveratrol was 2.5967 μg / mL.

3. The method for establishing the fingerprint of the scutellaria baicalensis preparation according to claim 1, wherein: The HPLC chromatographic conditions are as follows: the chromatographic column is CAPCELL PAK C18, 250 mm × 4.6 mm, 5 μm; the mobile phase is 0.1% phosphoric acid solution-acetonitrile, with gradient elution; the detection wavelength is 303 nm; the flow rate is 1 mL / min; the column temperature is 35°C; the injection volume is 10 μL; and the theoretical plate number calculated based on the chlorogenic acid peak should be no less than 10,000.

4. The method for establishing the fingerprint of the scutellaria baicalensis preparation according to claim 1, wherein: In step S3, the chromatogram of the test solution is imported into the "Chinese medicine chromatographic fingerprint similarity evaluation system software" to perform similarity evaluation with the chromatogram of the reference solution, determine the similarity, and identify the common peaks.

5. The method for establishing the fingerprint of the scutellaria baicalensis preparation according to claim 1, wherein: The scutellaria baicalensis preparation is scutellaria baicalensis syrup or scutellaria baicalensis capsule.

6. A fingerprint obtained by the method for establishing the fingerprint of the scutellaria baicalensis preparation according to any one of claims 1 to 5.

7. Application of the method for establishing the fingerprint of the scutellaria baicalensis preparation as described in any one of claims 1 to 5 in the determination of the content of ingredients in the scutellaria baicalensis preparation and the quality inspection of the scutellaria baicalensis preparation.

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