Construction and quality control of lung-tonifying and detoxifying granule fingerprint spectrum

Abstract

The invention relates to construction and quality control of a lung-tonifying and detoxifying granule fingerprint spectrum. An HPLC (High Performance Liquid Chromatography) fingerprint spectrum of the lung-tonifying and detoxifying granules has 17 characteristic fingerprint chromatographic peaks, wherein the peak 1 is guanosine, the peak 3 is protocatechualdehyde, the peak 4 is quinic acid, the peak 6 is chlorogenic acid, the peak 10 is loganin, the peak 14 is forsythiaside A, and the peak 17 is forsythin.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine analysis, and particularly relates to the construction of a fingerprint spectrum of Yifei Jiedu granules and its quality control. Background Art

[0002] Yifei Jiedu Granules (YFJDG) are the first specialty medical institution preparation approved by the Shaanxi Provincial Drug Administration for the prevention and treatment of COVID-19 [Shaanxi Drug Administration Letter (2020) No. 59]. It is a recommended prescription in the Second Edition of the Traditional Chinese Medicine Treatment Plan for the Prevention and Treatment of Pneumonia Caused by the Novel Coronavirus. Developed by renowned TCM practitioner Ma Zhanping of Shaanxi Provincial Hospital of Traditional Chinese Medicine, this prescription is based on the classic formulas "Yupingfeng Powder" and "Yinqiao Jiedu Powder." It includes 11 herbs: honeysuckle, astragalus, stir-fried white atractylodes, saposhnikovia divaricata, forsythia suspensa, roasted lily bulb, dendrobium, platycodon, reed root, imperata root, and licorice. All 11 herbs in the prescription are primarily designed for "light, clear, and penetrating properties." Their combination of herbs enhances the efficacy of "tonifying qi, strengthening the exterior, detoxifying, and promoting fluid production." In addition to treating pneumonia caused by the novel coronavirus, YFJDG is also widely used in clinical practice to treat conditions such as colds caused by wind and toxins, the common cold, winter influenza, and viral pneumonia.

[0003] At present, there are few research reports on YFJDG, which mainly focus on the quality control of single medicinal materials. The traditional quality evaluation model is to evaluate specific indicators, but a single evaluation method cannot comprehensively evaluate the quality of the compound. As an important means of quality control of traditional Chinese medicine, HPLC characteristic spectrum method uses spectroscopy, chromatography and other technologies to conduct an overall study of chemical components, which can comprehensively reflect the chemical information and content information contained in the compound, as well as the relationship between different batches, etc., and is now a widely used quality evaluation method. Therefore, the present invention establishes a YFJDG fingerprint based on HPLC chromatography, and adopts an external standard method to determine the content of 7 components including guanosine, protocatechuic aldehyde, quinic acid, chlorogenic acid, strychnine, forsythiaside A, and forsythiaside, so as to provide strong data support for comprehensively controlling the quality of the prescription and ensuring clinical safety. Summary of the Invention

[0004] The present invention provides an HPLC fingerprint of Yifei Jiedu Granules (or a method for constructing the same), characterized in that when the HPLC chromatographic conditions are as follows, the HPLC fingerprint of the Yifei Jiedu Granules is substantially the same as that of Figure 2 consistent;

[0005] HPLC chromatographic conditions are as follows:

[0006] The chromatographic column is Agilent 5TC-C 18Chromatographic column, specifications: 250mm×4.6mm, 5μm; mobile phase: methanol as phase A, 0.1% phosphoric acid aqueous solution as phase B; gradient elution conditions: 0-20min, 5%-15% A; 20-40min, 15%-28% A; 40-70min, 28%-38% A; 70-80min, 38%-48% A; 80-90min, 48%-5% A; 90-100min, 5% A, flow rate: 1ml / min, column temperature: 30℃, detection wavelength: 230nm, injection volume: 10μl.

[0007] Another embodiment of the present invention provides the HPLC fingerprint of the Yifei Jiedu Granules (or its construction method), characterized in that the HPLC fingerprint of the Yifei Jiedu Granules is substantially the same as Figure 2 The results were consistent with those of the other two samples, with 17 characteristic fingerprint chromatographic peaks, of which peak 1 was guanosine, peak 3 was protocatechuic aldehyde, peak 4 was quinic acid, peak 6 was chlorogenic acid, peak 10 was strychnine, peak 14 was forsythiaside A, and peak 17 was forsythiaside.

[0008] Another embodiment of the present invention provides the use of the HPLC fingerprint of the Yifei Jiedu Granules (or its construction method) in the quality control and component analysis of the Yifei Jiedu Granules.

[0009] Another embodiment of the present invention provides an application of the HPLC fingerprint of the Yifei Jiedu Granules (or a method for constructing the same) in quality control of the Yifei Jiedu Granules, characterized in that the application comprises the following steps:

[0010] (1) Prepare a methanol solution of a certain concentration by preparing Yifei Jiedu granules to obtain a test solution;

[0011] (2) Take the test solution obtained in step (1) and test it by HPLC to obtain an HPLC chart of the test sample, wherein the chromatographic conditions are as follows:

[0012] The chromatographic column is Agilent 5TC-C 18 Chromatographic column, specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: methanol as phase A, 0.1% phosphoric acid aqueous solution as phase B; gradient elution conditions: 0-20 min, 5%-15% A; 20-40 min, 15%-28% A; 40-70 min, 28%-38% A; 70-80 min, 38%-48% A; 80-90 min, 48%-5% A; 90-100 min, 5% A; flow rate: 1 ml / min; column temperature: 30°C; detection wavelength: 230 nm; injection volume: 10 μl;

[0013] (3) Compare the HPLC chromatogram of the test sample obtained in step (2) with the HPLC fingerprint of the Yifei Jiedu Granules described in the present invention. The Yifei Jiedu Granules with a similarity of more than 0.90 (preferably 0.99) are qualified products.

[0014] The concentration of the test solution in step (1) is preferably a methanol solution of 20-60 mg / mL of Yifei Jiedu Granules, and more preferably 30-50 mg / mL; when preparing the test solution, it is preferred to first ultrasonically treat the Yifei Jiedu Granules with an appropriate volume fraction of 50%-60% methanol solution for 30-40 minutes, then adjust the volume and filter to obtain the test solution.

[0015] Another embodiment of the present invention provides a method for analyzing the components of Yifei Jiedu Granules, characterized in that the method comprises the following steps:

[0016] (1) Preparation of mixed reference solution:

[0017] Accurately weigh appropriate amounts of guanosine, protocatechuic aldehyde, quinic acid, chlorogenic acid, loganin, forsythiaside A, and forsythiaside reference substances, dissolve them in 50% methanol aqueous solution to prepare mixed reference substance solutions with mass concentrations of 387.712 μg / ml, 125.552 μg / ml, 68.780 μg / ml, 49.774 μg / ml, 42.771 μg / ml, 261.366 μg / ml, and 44.436 μg / ml, respectively;

[0018] (2) Preparation of standard curves for each reference substance in step (1):

[0019] The mixed reference solution prepared in step (1) was diluted stepwise to 0.8, 0.6, 0.4, 0.2, and 0.1 times the concentration of the original solution, and HPLC was performed according to the chromatographic conditions of the present invention. The peak area Y was used as the ordinate and the reference concentration X was used as the abscissa to obtain the regression equation. The results were as follows: guanosine y = 5877.8x-3.3902, r = 0.9999, linear range 38.771 ~ 387.712 μg / ml; protocatechuic aldehyde y = 5011x-1.0258, r = 0.9999, linear range 12.555 ~ 125.552 μg / ml; quinic acid y = 14784x-1.4509, r = 0.9 999, linear range 6.878~68.780μg / ml; chlorogenic acid y=15695x-0.036, r=0.9999, linear range 4.977~49.774μg / ml; strychnine y=14092x+0.1358, r=0.9999, linear range 4.277~42.771μg / ml; forsythiaside Ay=10076x+17.682, r=0.9999, linear range 26.137~261.366μg / ml; forsythiaside y=18336x+0.154, r=0.9999, linear range 4.444~44.436μg / ml;

[0020] (3) Preparation of test solution: Yifei Jiedu granules were prepared into a methanol solution of a certain concentration to obtain the test solution;

[0021] (4) taking the test solution obtained in step (3), detecting it by HPLC, and calculating the contents of guanosine, protocatechuic aldehyde, quinic acid, chlorogenic acid, loganin, forsythiaside A, and forsythiaside reference substances according to their concentrations and retention times in the fingerprint and the linear equation in step (2);

[0022] The chromatographic conditions for HPLC detection in steps (2) and (4) are as follows:

[0023] The chromatographic column is Agilent 5TC-C 18 Chromatographic column, specifications: 250mm×4.6mm, 5μm; mobile phase: methanol as phase A, 0.1% phosphoric acid aqueous solution as phase B; gradient elution conditions: 0-20min, 5%-15% A; 20-40min, 15%-28% A; 40-70min, 28%-38% A; 70-80min, 38%-48% A; 80-90min, 48%-5% A; 90-100min, 5% A, flow rate: 1ml / min, column temperature: 30℃, detection wavelength: 230nm, injection volume: 10μl.

[0024] The concentration of the test solution in step (3) is preferably 20-60 mg / mL of methanol solution of Yifei Jiedu Granules, and more preferably 30-50 mg / ml; when preparing the test solution, it is preferred to first ultrasonically treat Yifei Jiedu Granules with an appropriate volume fraction of 50%-60% methanol solution for 30-40 minutes, then adjust the volume and filter to obtain the test solution.

[0025] Unless otherwise specified, the ratio of the mobile phase in the mixed solution and chromatographic conditions in the present invention is by volume; 0.1% phosphoric acid aqueous solution refers to a phosphoric acid aqueous solution with a mass fraction of 0.1%.

[0026] Compared with the prior art, the advantages of the present invention are: (1) the present invention makes up for the deficiencies of the prior art, establishes a high performance liquid chromatography (HPLC) fingerprint of Yifei Jiedu Granules, and provides an effective method for quality control of Yifei Jiedu Granules; (2) the present invention matches the chromatograms of 10 batches of Yifei Jiedu Granule samples to obtain a control spectrum. In the 10 batches of samples, a total of 17 chromatographic peaks are identified as common peaks in the fingerprint of Yifei Jiedu Granules, and the common pattern of the HPLC fingerprint of Yifei Jiedu Granules is determined; (3) the present invention establishes a method for simultaneously determining the fingerprint and content determination of guanosine, protocatechuic aldehyde, quinic acid, chlorogenic acid, strychnine, forsythiaside A, and forsythiaside in YFJDG, and comprehensively evaluates the quality of YFJDG from the perspective of integrity and quantification. The method is simple, rapid, accurate, and feasible, and can provide a reference and solution for the quality standard of YFJDG. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 These are the HPLC fingerprints of 10 batches of Yifei Jiedu Granules (S1 to S10) and the control fingerprint (R);

[0028] Figure 2 The fingerprints of 10 batches of Yifei Jiedu Granules (S1-S10) of the present invention are fitted to generate the control fingerprints;

[0029] Figure 3 It is the HPLC spectrum of the mixed reference solution, wherein peak 1 is guanosine, peak 3 is protocatechuic aldehyde, peak 4 is quinic acid, peak 6 is chlorogenic acid, peak 10 is strychnine, peak 14 is forsythiaside A, and peak 17 is forsythiaside. DETAILED DESCRIPTION

[0030] 1. Instruments and reagents

[0031] 1.1 Instruments

[0032] Agilent 1260 high performance liquid chromatograph (Agilent Technologies), analytical balance (Sartorius Scientific Instruments Co., Ltd.); water bath (Beijing Kewei Yongxing Instruments Co., Ltd.); ultrasonic cleaner (Ningbo Xinyi Ultrasonic Equipment Co., Ltd.); ultrapure water system (Beijing Puxi General Instruments Co., Ltd.).

[0033] 1.2 Drug testing

[0034] The reference substance guanosine batch number: (B20905, HPLC ≥ 98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., the reference substance protocatechuic aldehyde batch number: (CHB230627, HPLC ≥ 98%), the reference substance quinic acid batch number: (CHB240224, HPLC ≥ 98%), the reference substance chlorogenic acid batch number: (CHB231010, HPLC ≥ 98%), the reference substance strychnine batch number: (CHB231021, HPLC ≥ 98%), the reference substance forsythiaside A batch number: (CHB231017, HPLC ≥ 98%), and the reference substance forsythiaside batch number: (CHB231121, HPLC ≥ 98%) were purchased from China Food and Drug Identification Institute. Analytical grade methanol and chromatographic grade phosphoric acid were purchased from Tianjin Tianli Chemical Reagent Co., Ltd., and chromatographic grade methanol was purchased from Thermo Fisher Scientific Co., Ltd. The experimental water was homemade ultrapure water. YFJDG (batch numbers are: 20211201, 20211218, 20221122, 20221201, 20221203, 20221205, 20221206, 20230102, 2023013, 20230104, Shaanxi Provincial Hospital of Traditional Chinese Medicine).

[0035] 2 Methods and Results

[0036] 2.1 Chromatographic conditions

[0037] An Agilent 5TC-C18 (250 mm × 4.6 mm, 5 μm) column was used with a mobile phase of methanol (A)-0.1% phosphoric acid solution (B). The elution sequence was as follows: 0-20 min, 5%-15% A; 20-40 min, 15%-28% A; 40-70 min, 28%-38% A; 70-80 min, 38%-48% A; 80-90 min, 48%-5% A; 90-100 min, 5% A. The flow rate was 1 ml / min, the column temperature was 30°C, the detection wavelength was 230 nm, and the injection volume was 10 μl.

[0038] 2.2 Solution preparation

[0039] 2.2.1 Mixed reference solution

[0040] Accurately weigh appropriate amounts of the reference substances guanosine, protocatechuic aldehyde, quinic acid, chlorogenic acid, loganin, forsythiaside A, and forsythin, dissolve them in 50% methanol and water, dilute to the mark on the volumetric flask, and shake well. Prepare reference solutions with mixed standard concentrations of 387.712 μg / ml, 125.552 μg / ml, 68.780 μg / ml, 49.774 μg / ml, 42.771 μg / ml, 261.366 μg / ml, and 44.436 μg / ml for each of the seven components. Dilute the above reference substances to 0.8, 0.6, 0.4, 0.2, and 0.1 times the original solution concentrations, respectively, and set aside.

[0041] 2.2.2 Preparation of test solution

[0042] Accurately weigh 0.500 g of YFJDG sample and place it in a conical flask. Add 10 ml of 50% methanol solution by volume, sonicate for 30 min, cool and then adjust to volume. Filter with a 0.45 μm filter membrane to obtain the YFJDG test solution.

[0043] 2.3 Linear relationship investigation

[0044] Mixed standard solutions of guanosine (387.712 μg / ml), protocatechuic aldehyde (125.552 μg / ml), quinic acid (68.780 μg / ml), chlorogenic acid (49.774 μg / ml), strychnine (42.771 μg / ml), forsythiaside A (261.366 μg / ml), and forsythiaside (44.436 μg / ml) were prepared and diluted to 0.8, 0.6, 0.4, 0.2, and 0.1 times the concentration of the mother solution, respectively. Standard curves were established with the peak area Y as the ordinate and the reference substance concentration X as the abscissa, and the regression equations were calculated. As shown in Table 1, the components had a good relationship within their respective linear ranges.

[0045] Table 1 Regression equations and linear relationships of 7 components

[0046]

[0047] 2.4 Methodological validation

[0048] 2.4.1 Precision experiment

[0049] Accurately aspirate 10 μl of the test solution and inject the sample six times continuously according to the chromatographic conditions under "2.1". The peak area of ​​each component is determined. The results show that the RSDs (n=6) of guanosine, protocatechuic aldehyde, quinic acid, chlorogenic acid, strychnine, forsythiaside A, and forsythiaside are 1.42%, 0.19%, 0.15%, 0.31%, 0.22%, 0.38%, and 1.67%, respectively, indicating that the precision of the instrument is good.

[0050] 2.4.2 Stability test

[0051] The test solution of batch number 20230102 was injected at 0, 2, 4, 6, 8, and 12 h, and the peak area of ​​each component was measured according to the chromatographic conditions under "2.1". The results showed that the RSDs (n=6) of guanosine, protocatechuic aldehyde, quinic acid, chlorogenic acid, strychnine, forsythiaside A, and forsythin were 1.42%, 0.21%, 0.15%, 0.73%, 0.65%, 0.45%, and 3.31%, respectively, indicating good stability of the sample over time.

[0052] 2.4.3 Repeatability Experiment

[0053] An appropriate amount of sample from batch number 20230102 was taken and prepared in parallel using the method described in "2.1." Six test solutions were injected (10 μl) and the peak areas were determined. The results showed that the RSDs (n=6) for guanosine, protocatechuic aldehyde, quinic acid, chlorogenic acid, strychnine, forsythiaside A, and forsythin were 2.44%, 1.86%, 1.86%, 2.49%, 1.95%, 3.37%, and 1.86%, respectively, all within 5%, indicating good reproducibility among samples within the same batch.

[0054] 2.5 Establishment of HPLC fingerprint and common peak attribution

[0055] Ten different batches of samples were prepared into test solutions according to the preparation method under "2.2.2". The solutions were then assayed according to the method under "2.1" and the chromatograms recorded. The 10 batches of samples were sequentially imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) in AIA format. Using the S2 sample map as a reference, the median method was used. After multi-point correction, peaks with high response values ​​and good peak shapes were selected for recording. A total of 17 common peaks were calibrated. The calculated similarities between the sample and standard chromatograms were 0.993, 0.998, 0.998, 0.999, 0.997, 0.997, 0.996, 0.998, and 0.996, respectively, all greater than 0.990. The results indicate good similarity, indicating that the chemical composition and quality of the 10 batches of YFJDG are relatively stable.

[0056] 2.6 Identification of chromatographic peaks

[0057] Ten batches of YFJDG samples were taken, and mixed reference and test solutions were prepared according to the method under "2.2" for injection, analysis and comparison. A total of seven components were calibrated, namely guanosine, protocatechuic aldehyde, quinic acid, chlorogenic acid, strychnine, forsythiaside A, and forsythin.

[0058] 2.7 Sample recovery experiment

[0059] Six samples of Yifei Jiedu Granules (batch number 20230102) with known content were accurately weighed, and each test solution was prepared according to the "2.2.2" method. The reference solution was accurately added at a ratio of 1:1, and the recovery rate was calculated. The average sample recovery rates of guanosine, protocatechuic aldehyde, quinic acid, chlorogenic acid, strychnine, forsythiaside A, and forsythiaside were 100.35%, 99.21%, 99.39%, 100.61%, 100.20%, 101.38%, and 100.35%, respectively, and the RSDs were 2.11%, 2.50%, 1.75%, 2.32%, 3.10%, 2.52%, and 2.11%, respectively. This shows that the sample recovery rate and RSD values ​​of this test are within the required range, indicating that the sample recovery rate of this method is good.

[0060] Table 2 Recovery results of 7 components in YFJDG (n=6)

[0061]

[0062]

[0063]

[0064] 2.8 Sample content determination

[0065] Ten batches of YFJDG samples were prepared according to the method in "2.2.2." The samples were then injected into the instrument for analysis according to the method in "2.1," and the peak areas were recorded. The contents of guanosine, protocatechuic aldehyde, quinic acid, chlorogenic acid, strychnine, forsythiaside A, and forsythin were calculated using the external standard method. The results are shown in Table 3.

[0066] Table 3 Determination results of the content of each component

[0067]

[0068] 3 Discussions

[0069] 3.1 Selection of YFJDG mobile phase

[0070] The present invention first extracted the YFJDG sample with a methanol solution with a volume fraction of 80%, and investigated two mobile phase systems: methanol-0.1% phosphoric acid and acetonitrile-0.1% phosphoric acid. The results showed that when the mobile phase was acetonitrile-0.1% phosphoric acid, the chromatogram showed no chromatographic peak after 30 minutes; when eluted with methanol-0.1% phosphoric acid, the sample had a larger number of peaks throughout the entire time period. Comparing the two mobile phase conditions of methanol-0.1% phosphoric acid and methanol-0.1% formic acid, when using methanol-0.1% phosphoric acid, the chromatographic peak baseline was more stable, there were fewer impurity peaks, the characteristic peak response was higher, and the separation was better. Therefore, the present invention ultimately selected methanol-0.1% phosphoric acid solution as the mobile phase.

[0071] 3.2 Selection of extraction conditions

[0072] The samples were extracted using methanol solutions with a volume fraction of 20%, 50%, and 80%, respectively, and ultrasonicated for 30 minutes. The results showed that the chromatogram peak response value of the 50% methanol solution extraction was higher and the peak shape was symmetrical. The ultrasonication time of the samples (20 minutes, 30 minutes, and 40 minutes) was also investigated, and the results showed that there was no significant difference in the chromatograms of 30 minutes and 40 minutes. Therefore, the present invention uses 30 minutes of ultrasonication as the YFJDG sample preparation time.

[0073] 3.3 Investigation of chromatographic conditions

[0074] Full wavelength scanning was used to investigate the peaks at different wavelengths (230nm, 240nm, 255nm, 260nm, 270nm, 290nm, 300nm, 320nm, 330nm, 350nm, and 370nm). The results showed that the peak response at 230nm was higher, the baseline was stable, and the number of peaks was larger; the peak response at 270nm was smaller; no peak appeared at 290nm after 60 minutes; and no peak appeared at 350nm after 50 minutes. The present invention also combined the types of ingredients in the medicinal materials to find that chlorogenic acid, strychnine A, forsythiaside A, and forsythin had better absorption at 230-250nm. Taking all the above into consideration, 230nm was selected as the wavelength of the present invention.

[0075] Subsequently, the present invention further investigated the changes in the peaks of the YFJDG test sample under gradient conditions at different time periods (80 min, 90 min, 100 min, 120 min, 130 min, and 150 min). The results showed that the peaks at 130 min and 150 min were more dispersed, while the peaks were more concentrated after the time was shortened. In summary, 100 min was used as the final investigation time.

[0076] Next, three commonly used column temperatures (25℃, 30℃, and 35℃) were investigated. When the temperature was 25℃, the baseline of the spectrum was unstable, the peak area was small, and the characteristic peak response was poor. When the temperature was 35℃, the separation of the chromatographic peaks was insufficient, and peaks with larger peak areas would produce tailing and front extension. Therefore, 30℃ was selected as the optimal column temperature.

[0077] 3.4 Selection of indicator components

[0078] The present invention establishes a YFJDG fingerprint map, and the selected index components are all associated with the clinical indications of YFJDG. At the same time, the seven components of YGJDG all have anti-inflammatory, antioxidant and antiviral effects. Chlorogenic acid, as a natural polyphenol compound that accounts for a relatively large proportion, can destroy the permeability of bacterial cell membranes, inhibit the activity of influenza virus neuraminidase, and improve insulin sensitivity. Forsythiaside and forsythiaside A are the main active ingredients in Forsythia, with a wide range of pharmacological activities. They can inhibit the Toll-Like Receptor 4 (TLR4) / mitogen-activated protein kinase (MAPK) / nuclear transcription factor-κB (NF-κB) pathway and alleviate acute lung injury; they can also block the replication of SARS-CoV-2 and downregulate the expression of inflammatory factors mediated by NF-κB. Guanosine, a purine nucleotide metabolite in Astragalus, plays a crucial role in the repair of pulmonary vascular injury through the nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway. This pathway regulates the physiological and biochemical metabolism of the pulmonary vasculature and is crucial for the repair of pulmonary vascular injury. sGC, which converts into guanosine, also plays a crucial role in lung diseases. Studies have shown that activation of sGC can reduce inflammation and neutrophil infiltration, improve lung mechanics, and thus alleviate lung injury. In patients with chronic obstructive pulmonary disease, sGC expression is significantly reduced, correlating with decreased lung function. By activating sGC, it can alleviate airway hyperreactivity caused by acute smoke exposure. Protocatechuic aldehyde can reduce the production of nitric oxide (NO) in macrophages, exerting a protective effect in animal models of lipopolysaccharide-induced acute lung injury. It not only activates the Nrf2-PPARγ-HO-1 signaling pathway to reduce excessive inflammatory mediators and cell death, but also prevents fatal lung injury caused by influenza A virus (H1N1). Quinic acid improves alveolar-capillary permeability by inhibiting alveolar macrophage infiltration and the release of proinflammatory cytokines. In a sepsis model, it reduces lung tissue damage by inhibiting caspase-1-mediated pyroptosis. Strychnosin has significant anti-inflammatory and anti-shock effects, whose mechanisms include activating NF-κB and alleviating acute lung injury and its complications.Lei Ming et al. were able to dose-dependently inhibit nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome-mediated Caspase-1 activation and mature interleukin-1β (IL-1β) secretion in a mouse model of sepsis, thereby alleviating acute lung injury. The pharmacological activities of these seven components have potential therapeutic effects in pneumonia caused by novel coronavirus infection, so the simultaneous determination of the content of these seven components is of great significance for the quality control of YFJDG.

Claims

1. An HPLC fingerprint of Yifei Jiedu Granules, characterized in that When the HPLC chromatographic conditions are as follows, the HPLC fingerprint of the Yifei Jiedu Granules is substantially consistent with FIG2 ; HPLC chromatographic conditions are as follows: The chromatographic column is Agilent 5TC-C 18 Chromatographic column, specifications: 250mm×4.6mm, 5μm; mobile phase: methanol as phase A, 0.1% phosphoric acid aqueous solution as phase B; gradient elution conditions: 0-20min, 5%-15% A; 20-40min, 15%-28% A; 40-70min, 28%-38% A; 70-80min, 38%-48% A; 80-90min, 48%-5% A; 90-100min, 5% A, flow rate is 1ml / min, column temperature is 30℃, detection wavelength is 230nm, injection volume is 10μl.

2. The HPLC fingerprint of the Yifei Jiedu granules according to claim 1 is characterized in that The HPLC fingerprint of the Yifei Jiedu granules has 17 characteristic fingerprint chromatographic peaks, wherein peak 1 is guanosine, peak 3 is protocatechuic aldehyde, peak 4 is quinic acid, peak 6 is chlorogenic acid, peak 10 is strychnine, peak 14 is forsythiaside A, and peak 17 is forsythiaside.

3. The method for constructing the HPLC fingerprint of the Yifei Jiedu Granule according to any one of claims 1 to 2, characterized in that When the HPLC chromatographic conditions are as follows, the HPLC fingerprint of the Yifei Jiedu Granules has 17 characteristic fingerprint chromatographic peaks, wherein peak 1 is guanosine, peak 3 is protocatechuic aldehyde, peak 4 is quinic acid, peak 6 is chlorogenic acid, peak 10 is strychnine, peak 14 is forsythiaside A, and peak 17 is forsythiaside.

4. Application of the HPLC fingerprint of Yifei Jiedu Granules according to any one of claims 1 to 3 or the method for constructing the same in quality control and component analysis of Yifei Jiedu Granules.

5. Application of the HPLC fingerprint of Yifei Jiedu Granules or the method for constructing the same according to any one of claims 1 to 3 in quality control of Yifei Jiedu Granules, characterized in that The application comprises the following steps: (1) Prepare a methanol solution of a certain concentration by preparing Yifei Jiedu granules to obtain a test solution; (2) Take the test solution obtained in step (1) and test it by HPLC to obtain an HPLC chart of the test sample, wherein the chromatographic conditions are as follows: The chromatographic column is Agilent 5TC-C 18 Chromatographic column, specifications: 250 mm × 4.6 mm, 5 μm; mobile phase: methanol as phase A, 0.1% phosphoric acid aqueous solution as phase B; gradient elution conditions: 0-20 min, 5%-15% A; 20-40 min, 15%-28% A; 40-70 min, 28%-38% A; 70-80 min, 38%-48% A; 80-90 min, 48%-5% A; 90-100 min, 5% A; flow rate: 1 ml / min; column temperature: 30°C; detection wavelength: 230 nm; injection volume: 10 μl; (3) Compare the HPLC chromatogram of the test sample obtained in step (2) with the HPLC fingerprint of the Yifei Jiedu Granules according to any one of claims 1 to 3. The Yifei Jiedu Granules with a similarity of more than 0.90 (preferably 0.99) are qualified products.

6. The use according to claim 5, characterized in that The concentration of the test solution in step (1) is preferably 20-60 mg / ml of methanol solution of Yifei Jiedu Granules, and more preferably 30-50 mg / ml; when preparing the test solution, it is preferred to first ultrasonically treat Yifei Jiedu Granules with an appropriate volume fraction of 50%-60% methanol solution for 30-40 minutes, then adjust the volume and filter to obtain the test solution.

7. A method for analyzing the ingredients of Yifei Jiedu Granules, characterized in that The method comprises the following steps: (1) Preparation of mixed reference solution: Accurately weigh appropriate amounts of guanosine, protocatechuic aldehyde, quinic acid, chlorogenic acid, loganin, forsythiaside A, and forsythiaside reference substances, dissolve them in 50% methanol aqueous solution to prepare mixed reference substance solutions with mass concentrations of 387.712 μg / ml, 125.552 μg / ml, 68.780 μg / ml, 49.774 μg / ml, 42.771 μg / ml, 261.366 μg / ml, and 44.436 μg / ml, respectively; (2) Preparation of standard curves for each reference substance in step (1): The mixed reference solution prepared in step (1) was diluted stepwise to 0.8, 0.6, 0.4, 0.2, and 0.1 times the concentration of the original solution, and HPLC was performed according to the chromatographic conditions of the present invention. The peak area Y was used as the ordinate and the reference concentration X was used as the abscissa to obtain the regression equation. The results were as follows: guanosine y = 5877.8x-3.3902, r = 0.9999, linear range 38.771 ~ 387.712 μg / ml; protocatechuic aldehyde y = 5011x-1.0258, r = 0.9999, linear range 12.555 ~ 125.552 μg / ml; quinic acid y = 14784x-1.4509, r = 0.9 999, linear range 6.878~68.780μg / ml; chlorogenic acid y=15695x-0.036, r=0.9999, linear range 4.977~49.774μg / ml; strychnine y=14092x+0.1358, r=0.9999, linear range 4.277~42.771μg / ml; forsythiaside Ay=10076x+17.682, r=0.9999, linear range 26.137~261.366μg / ml; forsythiaside y=18336x+0.154, r=0.9999, linear range 4.444~44.436μg / ml; (3) Preparation of test solution: Yifei Jiedu granules were prepared into a methanol solution of a certain concentration to obtain the test solution; (4) The test solution obtained in step (3) was taken and tested by HPLC. The contents of guanosine, protocatechuic aldehyde, quinic acid, chlorogenic acid, loganin, forsythiaside A and forsythiaside reference substances and their retention times in the fingerprint and the linear equation in step (2) were calculated.

8. The method according to claim 7, characterized in that The chromatographic conditions for HPLC detection in steps (2) and (4) are as follows: The chromatographic column is Agilent 5TC-C 18 Chromatographic column, specifications: 250mm×4.6mm, 5μm; mobile phase: methanol as phase A, 0.1% phosphoric acid aqueous solution as phase B; gradient elution conditions: 0-20min, 5%-15% A; 20-40min, 15%-28% A; 40-70min, 28%-38% A; 70-80min, 38%-48% A; 80-90min, 48%-5% A; 90-100min, 5% A, flow rate is 1ml / min, column temperature is 30℃, detection wavelength is 230nm, injection volume is 10μl.

9. The method according to any one of claims 7 to 8, characterized in that The concentration of the test solution described in step (3) is preferably 20-60 mg / ml of methanol solution of Yifei Jiedu Granules, and more preferably 30-50 mg / ml; when preparing the test solution, it is preferred to first ultrasonically treat the Yifei Jiedu Granules with an appropriate volume fraction of 50%-60% methanol solution for 30-40 minutes, then adjust the volume and filter to obtain the test solution.

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