A method for constructing an antioxidant component fingerprint of Chaiyin granules and a method for detecting the content of index components

Abstract

The present application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for constructing an antioxidant component fingerprint of Chaiyin granules and a method for detecting the content of index components. The method for constructing the antioxidant component fingerprint of Chaiyin granules comprises the following steps: (1) uniformly mixing Chaiyin granules and a solvent to obtain a test sample solution; (2) feeding the test sample solution into an HPLC-ABTS-DAD-ESI-TOF / MS online detection system for detection to obtain antioxidant active components of Chaiyin granules; and (3) taking the antioxidant components in Chaiyin granules as common peaks to establish an antioxidant component fingerprint of Chaiyin granules. In the present application, HPLC-ABTS-DAD-ESI-TOF / MS is used to rapidly screen and identify the antioxidant components in Chaiyin granules, and the effectors that play a role in the antioxidant link of Chaiyin granules are preliminarily elucidated. The antioxidant components in Chaiyin granules are taken as common peaks to establish the antioxidant component fingerprint of Chaiyin granules.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection technology, specifically relating to a method for constructing a fingerprint spectrum of antioxidant components in Chaiyin granules and a method for detecting the content of its indicator components. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Chaiyin Granules originate from Chaige Jieji Decoction in *Shanghan Liushu* by Tao Hua of the Ming Dynasty and Yinqiao Powder in *Wenbing Tiaobian* by Wu Jutong, a famous physician of the Qing Dynasty. It is a compound preparation composed of 11 Chinese herbs: Bupleurum, Honeysuckle, Scutellaria, Kudzu Root, Schizonepeta, Artemisia, Forsythia, Platycodon, Bitter Almond, Mentha, and Houttuynia. In the formula, Bupleurum is the principal herb, used for its light and dispersing properties to clear heat from the exterior. Honeysuckle, Scutellaria, Forsythia, Kudzu Root, Artemisia, and Schizonepeta are the assistant herbs, used for clearing heat, relieving exterior symptoms, clearing lung heat, and reducing fever. Mentha, Houttuynia, and Bitter Almond are the adjuvant herbs, used for relieving sore throat, quenching thirst, clearing lung heat, resolving phlegm, and stopping cough. Platycodon, specifically entering the lung meridian, clears lung heat, resolves phlegm, relieves sore throat, and stops cough, acting as the guiding herb, allowing the other herbs in the formula to directly reach the lung system and achieve the effect of dispelling pathogens from the exterior. The combined effects of these herbs are heat-clearing and detoxifying, relieving sore throat and cough, and reducing fever and inflammation. Chaiyin granules are suitable for treating upper respiratory tract infections caused by exogenous wind-heat. They are widely used in traditional Chinese medicine for the treatment of influenza and have also played an important role in the prevention and treatment of the novel coronavirus.

[0004] However, to date, the Chinese Pharmacopoeia still does not include quality standards for Chaiyin granules. The 2020 edition of the Chinese Pharmacopoeia (Part I) includes a different dosage form of the same traditional Chinese medicine, Chaiyin oral liquid, but only uses HPLC to determine the baicalin content for its quality control, which cannot comprehensively reflect and evaluate the overall quality of the preparation.

[0005] Influenza virus, after invading host cells, triggers inflammation, leading to the production of a large number of free radicals in the body, causing oxidative damage to tissue cells, and further resulting in apoptosis. The imbalance of the redox environment is the basis of influenza virus infection and tissue damage; it can also increase the susceptibility of host epithelial cells to influenza virus and promote intercellular viral transmission. Inhibiting oxidative stress damage caused by viral infection is one of the effective strategies for treating influenza virus infection. Previous research in our group found that single herbs in Chaiyin granules, such as honeysuckle, contain antioxidant components such as caffeoylquinic acid, flavonoids, and iridoid glycosides. These components are also important active ingredients in its inhibition of neuraminidase, revealing its multi-target anti-influenza potential. To date, no research has been reported on the composition of antioxidant components in Chaiyin granules, and further research is needed to explore the use of the characteristic fingerprint of antioxidant components in Chaiyin granules for quality evaluation. Summary of the Invention

[0006] To overcome the above problems, this invention provides a method for constructing a fingerprint spectrum of antioxidant components in silver-containing biomass granules and a method for detecting the content of its indicator components. In this invention, HPLC-ABTS-DAD-ESI-TOF / MS is used for rapid screening and identification of antioxidant components in silver-containing biomass granules, preliminarily elucidating the effector substances that play a role in the antioxidant process of silver-containing biomass granules. Using the antioxidant components in silver-containing biomass granules as common peaks, a fingerprint spectrum of antioxidant components in silver-containing biomass granules is established.

[0007] The first aspect of this invention provides a method for constructing a fingerprint spectrum of antioxidant components in silver-containing granules, comprising the following steps:

[0008] (1) Mix the granules of cypermethrin and the solvent evenly to obtain the test solution;

[0009] (2) The test solution was injected into the HPLC-ABTS-DAD-ESI-TOF / MS online detection system for detection to obtain the antioxidant active ingredients of the Chaiyin granules;

[0010] (3) Using the antioxidant components in the Chaiyin particles as common peaks, a fingerprint spectrum of antioxidant components in Chaiyin particles was established.

[0011] In one or more embodiments, the solvent in step (1) is methanol, and the concentration of the test solution is 0.008 to 0.012 g / mL, preferably 0.01 g / mL.

[0012] In one or more embodiments, the HPLC system chromatographic conditions were as follows: Phenomenex Luna C18 column (250 mm × 4.6 mm, 5.0 μm), mobile phase A was 0.2% formic acid aqueous solution, mobile phase B was methanol, gradient elution, and flow rate was 0.8 mL / min. -1 The column temperature was 25℃, the detector wavelength was 280nm, and the injection volume was 5μL.

[0013] Preferred gradient elution conditions are as follows: 0–5 min, 7%–13% B; 5–20 min, 13%–22% B; 20–35 min, 22%–25% B; 35–41 min, 25%–28% B; 41–51 min, 28%–30% B; 51–57 min, 30%–32% B; 57–85 min, 32%–38% B; 85–113 min, 38%–52% B; 113–118 min, 52%–53% B; 118–130 min, 53%–70% B; 130–140 min, 70%–80% B; 140–145 min, 80%–85% B; 145–150 min, 85–100% B; 150–160 min, 100% B.

[0014] In one or more embodiments, the diode array detector (DAD) conditions are as follows: the ABTS solution is mixed with the column liner and introduced into the reaction coil via a three-way valve, with an ABTS flow rate of 0.6 mL / min. -1 The reaction tube has an inner diameter of 0.25 mm and a length of 10 m. The detector wavelength is 734 nm. Antioxidant components are screened by detecting the chromatographic peak corresponding to the inverted peak produced by ABTS at 734 nm.

[0015] In one or more embodiments, the mass spectrometry conditions are as follows: the mobile phase entering the high-resolution electrospray time-of-flight mass spectrometer (ESI-Q-TOF / MS) is split to 0.4 mL / min using a three-way split. -1 Electrospray ionization (ESI) source with positive and negative ion modes; full scan range m / z 100~2000; spray pressure 310.28kPa; drying gas volume flow rate 10.0L / min, temperature 325℃; capillary voltage 4.0kV; pyrolysis voltage 100V; cone voltage 60V.

[0016] In one or more embodiments, the antioxidant component fingerprint spectrum contains 44 chromatographic peaks, namely: peak 1 corresponds to ferulic acid, peak 4 corresponds to monoglucoside, peak 6 corresponds to 5-O-caffeoylquinic acid, peak 7 corresponds to loganic acid, peak 8 corresponds to forsythoside E, peak 9 corresponds to strychnoside hemiacetal lactone, peak 10 corresponds to amygdalin, peak 11 corresponds to 3′-hydroxypuerarin, peak 12 corresponds to chlorogenic acid, peak 13 corresponds to 4-O-caffeoylquinic acid, peak 15 corresponds to oxidized loganic acid, peak 16 corresponds to puerarin, peak 17 corresponds to 3-O-p-hydroxycinnamoylquinic acid or its isomer, peak 18 corresponds to 3-O-feruloylquinic acid, peak 19 corresponds to daidzein, peak 20 corresponds to forsythoside I or its isomer, peak 21 corresponds to oxidized strychnoside, peak 22 corresponds to... Peak 23 corresponds to hyperoside, peak 24 corresponds to hesperidin, peak 25 corresponds to (+)pinoresin-β-D-glucopyranoside, peak 26 corresponds to isoflavone glycoside A or its isomer, peak 27 corresponds to forsythoside A, peak 28 corresponds to isochlorogenic acid A, peak 29 corresponds to poplin 6-C-arabinoside 8-C-glucoside, peak 30 corresponds to luteolin, peak 32 corresponds to rutin, peak 33 corresponds to poplin 6-C-glucoside 8-C-arabinoside, peak 34 corresponds to isochlorogenic acid C, peak 35 corresponds to forsythoside, peak 37 corresponds to astragaloside, peak 38 corresponds to baicalin, peak 39 corresponds to poplin, peak 40 corresponds to apigenin 7-O-glucuronic acid, peak 41 corresponds to 6-O-methylbaicalin, peak 42 corresponds to wogonin, and peak 44 corresponds to baicalin.

[0017] In a second aspect, the present invention provides a method for detecting the content of indicator components in silver-containing granules, comprising the following steps:

[0018] The granules of cypermethrin and the solvent were mixed evenly to obtain the test solution; a gradient concentration reference solution was prepared, wherein the reference standard included one or more of chlorogenic acid, puerarin, forsythoside A, luteolin and baicalin;

[0019] The reference solution and the test solution were detected by high performance liquid chromatography (HPLC) to determine the peak area of ​​the chromatogram obtained by detecting the reference solution and the peak area of ​​the corresponding peak in the chromatogram obtained by detecting the test solution. The content of the index component corresponding to the reference in the test solution was calculated by the standard curve method.

[0020] In one or more embodiments, the high-performance liquid chromatography (HPLC) conditions were as follows: Phenomenex Luna C18 column (250 mm × 4.6 mm, 5.0 μm), mobile phase A was 0.2% formic acid aqueous solution, mobile phase B was methanol, gradient elution, and flow rate was 0.8 mL / min. -1 The column temperature was 25℃, the detector wavelength was 280nm, and the injection volume was 5μL.

[0021] Preferred gradient elution conditions are as follows: 0–5 min, 7%–13% B; 5–20 min, 13%–22% B; 20–35 min, 22%–25% B; 35–41 min, 25%–28% B; 41–51 min, 28%–30% B; 51–57 min, 30%–32% B; 57–85 min, 32%–38% B; 85–113 min, 38%–52% B; 113–118 min, 52%–53% B; 118–130 min, 53%–70% B; 130–140 min, 70%–80% B; 140–145 min, 80%–85% B; 145–150 min, 85–100% B; 150–160 min, 100% B.

[0022] The beneficial effects of this invention are as follows:

[0023] (1) In this invention, HPLC-ABTS-DAD-ESI-TOF / MS was used to rapidly screen and identify the antioxidant components in *Chaiyin* granules. Forty-four antioxidant components were screened from the *Chaiyin* granules, and 37 were preliminarily identified using DAD-ESI-Q-TOF / MS. A fingerprint spectrum of the antioxidant components in *Chaiyin* granules was established using the common peaks of these components. Simultaneously, activity verification showed that forsythoside A, luteolin, baicalin, chlorogenic acid, and puerarin all possessed good antioxidant activity. Using these five antioxidant components as indicator components, quantitative research and quality control of *Chaiyin* granules were achieved.

[0024] (2) This invention provides a method for constructing a fingerprint spectrum for qualitative analysis of silver-coated carbon dioxide particles, and also provides a method for detecting the content of index components that can be used for quantitative analysis. This provides a scientific experimental basis for the comprehensive quality evaluation of silver-coated carbon dioxide particles, and establishes a quality control method for silver-coated carbon dioxide particles that is highly specific, easy to operate and has good repeatability. Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 This is a graph showing the effect of using different packed columns to separate the test solution of *Cynanchum paniculatum* particles in Example 1.

[0027] Figure 2 This is the HPLC-DAD chromatogram of the *Chaiyin* granule extract detected using the optimized chromatographic conditions in Example 1.

[0028] Figure 3The HPLC-ABTS screening results for detecting the *Chaiyin* granule extract using optimized online screening conditions are shown in Example 2.

[0029] Figure 4 The fingerprint spectra of different batches of silver-coated particles from S1 to S10 in Experimental Example 1;

[0030] Figure 5 The HPLC-DAD chromatogram of the mixed reference standard and sample in Experimental Example 2; Detailed Implementation

[0031] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0033] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0034] 1. Instruments and reagents

[0035] The system includes: UltiMate 3000 high-performance liquid chromatograph, equipped with an autosampler, quaternary pump, column oven, and diode array detector (ThermoFisher, Germany); Bruker ImpactⅡ ESI-Q-TOF series high-resolution mass spectrometer (Bruker, Germany); 0.001% electronic analytical balance (Sartourius BSA, USA); Millipor Q-Plus ultrapure water treatment system (Millipore, USA); Tecan Infinite M20 series microplate reader (Tecan Group, Switzerland); ME54TE electronic balance (Mettler-Toledo, Mettler Toledo Instruments (China) Co., Ltd.); and SBL-10DT constant temperature ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.).

[0036] ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt) was purchased from Sigma-Aldrich; chlorogenic acid (batch number: B20782, purity ≥98%) and forsythoside A (batch number: B20727, purity ≥98%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; baicalin (batch number: 110715-200514, purity ≥98%) was purchased from the China Institute for Drug and Biological Products Testing; luteolin (batch number: MUST-16012405, purity 99.77%) was purchased from Chengdu Manster Biotechnology Co., Ltd.; puerarin (prepared in the laboratory, identified by spectroscopic data, purity ≥98%).

[0037] All reference medicinal materials used in the drug were purchased from Jinan Jianlian Pharmacy. Chaiyin Granules (light brownish-yellow to yellowish-brown granules, 8g x 10 sachets) were produced by Shandong Lunan Houpu Pharmaceutical Co., Ltd., with batch numbers 22201281, 22201281, 22201281, 22201281, 22220321, 22220321, 22220321, 22220881, 22220881, and 22220881, numbered S1 to S10, and purchased from various pharmacies in Jinan.

[0038] Methanol and acetonitrile were chromatographically pure (Merck, Germany); formic acid was chromatographically pure (Tianjin Kemeio Chemical Reagent Co., Ltd.); water was Millipore ultrapure water (18 MΩ); all other reagents were analytical grade.

[0039] 2. Solution preparation

[0040] Test solution: Accurately weigh 0.30 g of this product and place it in a stoppered Erlenmeyer flask. Add 30 mL of methanol and extract by ultrasonication for 30 min (300 W power, room temperature). Take the supernatant and filter it through a 0.22 μm microporous membrane to obtain the test solution.

[0041] Reference solutions: Accurately weigh appropriate amounts of chlorogenic acid, puerarin, forsythoside A, luteolin, and baicalin reference standards, place them in volumetric flasks, dissolve in methanol, and dilute to the mark to prepare solutions with mass concentrations of 1.000, 0.900, 0.900, 1.100, and 1.000 mg / mL. -1 The reference standard stock solution is obtained by passing it through a 0.22 μm microporous membrane.

[0042] Negative sample solution: Negative samples lacking honeysuckle, scutellaria, kudzu root, and forsythia were prepared according to the prescription ratio, the process specified in Part I of the 2020 Chinese Pharmacopoeia, and relevant literature. Accurately weigh 0.30 g of the negative sample, place it in a stoppered Erlenmeyer flask, add 30 mL of methanol, and extract ultrasonically for 30 min (300 W, room temperature). Collect the supernatant and filter it through a 0.22 μm microporous membrane to obtain the negative sample solution.

[0043] 3. HPLC-DAD / CAD-ESI-Q-TOF / MS analysis conditions

[0044] High-performance liquid chromatography (HPLC) conditions: Phenomenex Luna C18 column (250 mm × 4.6 mm, 5.0 μm); mobile phase A was 0.2% formic acid aqueous solution, mobile phase B was methanol, gradient elution program: 0–5 min, 7%–13% B; 5–20 min, 13%–22% B; 20–35 min, 22%–25% B; 35–41 min, 25%–28% B; 41–51 min, 28%–30% B; 51–57 min, 30%–32% B; 57–85 min, 32%–38% B; 85–113 min, 38%–52% B; 113–118 min, 52%–53% B; 118–130 min, 53%–70% B; 130–140 min, 70%–80% B. B, 140–145 min, 80%–85% B; 145–150 min, 85%–100% B; 150–160 min, 100% B. Injection volume 5 μL; detection wavelength 280 nm; column temperature 25 °C; flow rate 0.8 mL / min. -1 .

[0045] Diode array detector (DAD) conditions: ABTS solution is mixed with the column feedwater and introduced into the reaction coil via a three-way valve; ABTS flow rate is 0.6 mL / min. -1 The reaction tube has an inner diameter of 0.25 mm and a length of 10 m. The detector wavelength is 734 nm. Antioxidant components are screened by detecting the chromatographic peak corresponding to the inverted peak produced by ABTS at 734 nm.

[0046] Mass spectrometry conditions: The mobile phase entering the high-resolution electrospray time-of-flight mass spectrometer (ESI-Q-TOF / MS) was split to 0.4 mL / min using a three-way split. -1 Electrospray ionization (ESI) source with positive and negative ion modes; full scan range m / z 100~2000; spray pressure 310.28kPa; drying gas volume flow rate 10.0L / min, temperature 325℃; capillary voltage 4.0kV; pyrolysis voltage 100V; cone voltage 60V.

[0047] Example 1: Optimization of Chromatographic Conditions

[0048] Chaiyin granules are composed of 11 kinds of traditional Chinese medicine, and their composition is relatively complex. The chromatographic conditions, including column type, mobile phase system, gradient elution program, flow rate, column temperature, and detection wavelength, were investigated to obtain the best chromatographic separation effect.

[0049] Using a solution of *Chaiyin* granules as the test sample, the separation effects of several chromatographic columns with different packing materials (including: XSELECT™ HSS T3 column (150 mm × 3.0 mm, 3.5 μm), Agilent Zorbax SB-C18 column (250 mm × 4.6 mm, 5.0 μm), Phenomenex Luna C18 column (250 mm × 4.6 mm, 5.0 μm), Symmetry C18 column (250 mm × 4.6 mm, 5.0 μm), and Kromasil 100-5-C18 column (250 mm × 4.6 mm, 5.0 μm)) were investigated under the same gradient and mobile phase conditions. The results are as follows: Figure 1 As shown, the results indicate that the Phenomenex Luna C18 (250 mm × 4.6 mm, 5.0 μm) column provides better separation, more chromatographic peaks, higher column efficiency, and better peak symmetry, making it suitable for further analysis.

[0050] Different mobile phase compositions were compared under the same gradient elution mode, comparing the separation effects of acetonitrile-water and methanol-water mobile phase systems. The results showed that the methanol-water system resulted in better peak retention and separation, and a stable baseline in the chromatograms. To improve peak shape and reduce peak tailing, the separation effects of adding different concentrations of formic acid (0.2%, 0.4%, 0.6%, and 0.8%) to the aqueous phase were investigated. The results indicated that adding 0.2% formic acid to the aqueous phase significantly reduced tailing, resulting in clearer and more symmetrical peaks.

[0051] For different mobile phase flow rates (0.6, 0.8, 1.0 mL·min) -1 The column temperature (20, 25, 30℃) was also compared. The results showed that when the volumetric flow rate was 0.8 mL / min... -1The column efficiency is high, shortening the separation time without affecting the separation effect; therefore, it was chosen as the constant volumetric flow rate. It was found that the component chromatographic peak resolution and peak shape were better at 25℃. Under the optimization of the above conditions, the final liquid chromatography analysis conditions were obtained. Final HPLC conditions: Phenomenex Luna C18 column (250 mm × 4.6 mm, 5.0 μm); mobile phase A: 0.2% formic acid aqueous solution; mobile phase B: methanol; gradient elution program: 0–5 min, 7%–13% B; 5–20 min, 13%–22% B; 20–35 min, 22%–25% B; 35–41 min, 25%–28% B; 41–51 min, 28%–30% B; 51–57 min, 30%–32% B; 57–85 min, 32%–38% B; 85–113 min, 38%–52% B; 113–118 min, 52%–53% B; 118–130 min, 53%–70% B; 130–140 min, 70%–80% B. B, 140–145 min, 80%–85% B; 145–150 min, 85%–100% B; 150–160 min, 100% B. Injection volume 5 μL; detection wavelength 280 nm; column temperature 25 °C; flow rate 0.8 mL / min. -1 .

[0052] A diode array detector (DAD) was used to perform a full-wavelength scan (190-400 nm) of the test solution. The results showed that the sample had a large number of peaks at 280 nm, with good peak-to-peak resolution and a suitable ratio, and a relatively stable baseline. Therefore, 280 nm was chosen as the detection wavelength. The final DAD conditions were as follows: the ABTS solution was mixed with the solution at the column outlet and introduced into the reaction coil via a three-way valve; the ABTS flow rate was 0.6 mL / min. -1 The reaction tube has an inner diameter of 0.25 mm and a length of 10 m, and the detector wavelength is 734 nm.

[0053] Figure 2 The HPLC-DAD chromatogram shows the detection of *Chaiyin* granule extract using optimized chromatographic conditions.

[0054] Example 2: Optimization of Online Screening Conditions

[0055] This embodiment focuses on optimizing the ABTS flow rate and reaction tank specifications (inner diameter and length). The ABTS solution inflow rate was investigated at 0.2, 0.4, 0.6, 0.8, and 1.0 mL / min. -1The effects of the inverted peak intensity and baseline stability were investigated. Results showed that as the pump inflow rate increased within a certain range, the inverted peak height continuously increased; however, the increased flow rate also caused significant fluctuations in the signal baseline, thus affecting signal sensitivity. Ultimately, an ABTS flow rate of 0.6 mL / min was selected. -1 For use in subsequent screening studies.

[0056] The inner diameter and length of the reaction tube affect the reaction time and screening effect. This embodiment compares and optimizes different reaction tube inner diameters (0.18 mm, 0.25 mm) and lengths (5, 10, 15 m). The results show that a smaller inner diameter leads to a shorter reaction time and a decreased peak reversal response; a longer reaction tube increases the reaction time and peak reversal response, but a longer reaction time also causes peak broadening and decreased resolution. After comprehensive comparison, the optimal inner diameter of the reaction tube was determined to be 0.25 mm and the optimal length to be 10 m. Under the optimized screening conditions of this embodiment, the HPLC-ABTS screening results of the antioxidant components in the *Cinnamomum camphora* granule extract are as follows: Figure 3 As shown.

[0057] Example 1: Construction of the fingerprint spectrum of antioxidant active ingredients in Chaiyin granules

[0058] Test solutions of different batches of *Chaiyin* granules (S1–S10) were injected into an online HPLC-ABTS-DAD-ESI-TOF / MS system for detection to obtain the antioxidant active ingredients of the *Chaiyin* granules. The results are shown in Table 1. The fingerprint spectra of different batches of *Chaiyin* granules (S1–S10) are shown in Table 1. Figure 4 As shown.

[0059] Table 1 Antioxidant active ingredients in Chaiyin Granules

[0060]

[0061]

[0062]

[0063] In this invention, HPLC-ABTS-DAD-ESI-TOF / MS was used to rapidly screen and identify antioxidant components in the silver granules. Forty-four antioxidant components were screened out from the silver granules, and 37 of them were preliminarily identified by DAD-ESI-Q-TOF / MS. Peak 1 corresponds to ferulic acid, peak 4 to monoglucoside, peak 6 to 5-O-caffeoylquinic acid, peak 7 to loganic acid, peak 8 to forsythoside E, peak 9 to strychnos nuciferoside hemiacetal lactone, peak 10 to amygdalin, peak 11 to 3′-hydroxypuerarin, peak 12 to chlorogenic acid, peak 13 to 4-O-caffeoylquinic acid, peak 15 to oxidized loganic acid, peak 16 to puerarin, peak 17 to 3-O-p-hydroxycinnamoylquinic acid or its isomer, peak 18 to 3-O-feruloylquinic acid, peak 19 to daidzein, peak 20 to forsythoside I or its isomer, peak 21 to oxidized loganic acid, peak 22 to rhubarb glycoside, peak 23 to hyperoside, peak 2... Peak 4 corresponds to hesperidin, peak 25 corresponds to (+) pinoresinin-β-D-glucopyranoside, peak 26 corresponds to isoflavone glycoside A or its isomer, peak 27 corresponds to forsythoside A, peak 28 corresponds to isochlorogenic acid A, peak 29 corresponds to guarnin 6-C-arabinoside 8-C-glucoside, peak 30 corresponds to luteolin, peak 32 corresponds to rutin, peak 33 corresponds to guarnin 6-C-glucoside 8-C-arabinoside, peak 34 corresponds to isochlorogenic acid C, peak 35 corresponds to forsythoside, peak 37 corresponds to astragaloside, peak 38 corresponds to baicalin, peak 39 corresponds to guarnin, peak 40 corresponds to apigenin 7-O-glucuronic acid, peak 41 corresponds to 6-O-methylbaicalin, peak 42 corresponds to wogonin, and peak 44 corresponds to baicalin. Chromatographic peaks with potential antioxidant activity, good resolution, and stable occurrence in the samples were selected as common peaks in the fingerprint spectrum. Combining the analysis of multiple batches of samples, an antioxidant activity fingerprint spectrum for *Chaiyin* granules was constructed. Using the antioxidant components in *Chaiyin* granules as common peaks, an antioxidant component fingerprint spectrum for *Chaiyin* granules was established.

[0064] The similarity of fingerprint chromatograms of different batches of Chaiyin granules was analyzed using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) issued by the National Pharmacopoeia Commission. The chromatographic data were imported into the traditional Chinese medicine fingerprint similarity calculation software, and the aforementioned 44 common peaks were selected for peak matching. The common pattern was used as the reference fingerprint chromatogram for evaluating the similarity of different batches of Chaiyin granules. The cosine similarity method was used for the similarity calculation, and the results are shown in Table 2.

[0065] Table 2. Similarity evaluation results of HPLC fingerprint spectra of 10 batches of Chaiyin granule samples

[0066]

[0067] As can be seen from the table, the similarity of the 10 batches of Chaiyin granules is all above 0.99, which is quite close, indicating that the quality difference is small and the preparation process of different batches is relatively stable.

[0068] Experiment Example 2

[0069] Based on the antioxidant components screened in Chaiyin Granules in Experiment Example 1, and combined with the corresponding indicator components of each Chinese herb in the Chaiyin Granules compound in Part I of the 2020 edition of the Chinese Pharmacopoeia, five indicator components (chlorogenic acid, puerarin, forsythia suspensa, luteolin, and baicalin) from honeysuckle, scutellaria baicalensis, forsythia suspensa, and kudzu root were finally selected as the indicator components of Chaiyin Granules.

[0070] Specificity tests were conducted on the five identified index components (chlorogenic acid, puerarin, forsythoside A, luteolin, and baicalin).

[0071] Take the mixed reference solution, test solution, and negative sample solution, and inject them for analysis according to the optimized chromatographic conditions in Example 1. The HPLC-DAD chromatograms of the mixed reference solution and the sample are shown in [reference needed]. Figure 5 The peaks were well separated, and the test solution and the mixed reference solution had corresponding chromatographic peaks matching at the same retention time. However, the negative sample solution did not have any chromatographic peaks at the corresponding retention time positions of the mixed reference solution, indicating that there was no interference in the determination and the method had good specificity.

[0072] Methodological investigations were conducted on the five identified indicator components (chlorogenic acid, puerarin, forsythoside A, luteolin, and baicalin).

[0073] (1) Examination of linear relationships

[0074] The mass concentrations were 1.000, 0.900, 0.900, 1.100, and 1.000 mg·mL. -1 The reference stock solutions were analyzed under the optimized high-performance liquid chromatography (HPLC) conditions described in Example 1. Standard curves were plotted with the mass concentration of each reference standard as the abscissa (X) and the peak area A as the ordinate (Y). The limits of detection (LOD) and quantitation (LOQ) were set at signal-to-noise ratios of 3 and 10, respectively, to obtain the regression equations. The results are shown in Table 3. The correlation coefficients of the standard curves for the five index components were all greater than 0.9990, indicating that each component had a good linear relationship within its corresponding content range.

[0075] Table 3. Regression equations, limits of detection, and limits of quantitation for the five indicator components.

[0076]

[0077] (2) Precision test

[0078] The same batch of test solution (batch number: 22201281) was injected six times consecutively under the optimized high performance liquid chromatography conditions described in Example 1. The results showed that the peak area RSDs of chlorogenic acid, puerarin, forsythoside A, luteolin, and baicalin were 1.76%, 0.56%, 2.19%, 2.77%, and 0.69%, respectively, indicating good instrumental precision.

[0079] (3) Stability test

[0080] The same batch of test solution (batch number: 22201281) was tested at 0, 3, 6, 12, 18 and 24 h according to the optimized high performance liquid chromatography conditions in Example 1. The results showed that the peak areas RSD of chlorogenic acid, puerarin, forsythoside A, luteolin and baicalin were 1.94%, 0.63%, 0.90%, 3.90% and 1.23%, respectively, indicating that the test solution had good stability within 24 h.

[0081] (4) Repeatability test

[0082] Six test solutions were prepared in parallel from the same batch of samples (batch number: 22201281). The solutions were then analyzed under the optimized high-performance liquid chromatography conditions described in Example 1. Six parallel experiments were conducted. The peak areas RSD of chlorogenic acid, puerarin, forsythoside A, luteolin, and baicalin were 2.92%, 1.49%, 2.26%, 3.25%, and 1.14%, respectively, indicating that the method has good repeatability.

[0083] (5) Recovery test

[0084] Take the same batch of samples (batch number: 22201281), accurately weigh chlorogenic acid, puerarin, forsythoside A, luteolin, and baicalin reference standards respectively, and add them at the ratio of 80%, 100%, and 120% of the corresponding component content in the test sample. Detect the samples under the optimized high-performance liquid chromatography conditions in Example 1, determine the peak area of ​​each target compound, and calculate the spiked recovery rate (Table 4). The spiked recovery rates of the five index components are between 94.17% and 100.86%, indicating good method accuracy.

[0085] Table 4. Results of the recovery rate test for five indicator components (n=3)

[0086]

[0087] (6) Sample content determination

[0088] Ten batches of samples were taken and analyzed under the optimized high-performance liquid chromatography conditions described in Example 1. The contents of chlorogenic acid, puerarin, forsythoside A, luteolin, and baicalin in the samples were calculated, and the results are shown in Table 5. Among the components, baicalin had the highest content, with an average content of 9.880 mg / g; puerarin was the second highest, with a content of 3.678 mg / g; and luteolin had the lowest content among the five index components, with a content of 0.537 mg / g.

[0089] Table 5. Content of five key components in 10 batches of Chaiyin granules

[0090]

[0091] Experimental Example 3

[0092] The active components of the screened indicator components were validated using an in vitro ABTS free radical scavenging assay. The specific method included: reacting ABTS solution (7.0 mM, H2O) and potassium persulfate (4.9 mM, H2O) in the dark at 4°C for 12-16 h; diluting the ABTS·+ stock solution to an absorbance of 0.70 ± 0.02 at 734 nm to prepare the ABTS·+ working solution. The reference solution was diluted to a series of concentrations. 50 μL of the test solution and 100 μL of the ABTS·+ working solution were added to a 96-well plate and mixed thoroughly to form the sample group. After incubation at room temperature in the dark for 6 min, the absorbance (A) was measured at 734 nm using a microplate reader. Methanol was used as the blank group, and ascorbic acid (Vc) was used as the positive control group. The assay was repeated three times. The ABTS free radical scavenging rate was calculated using the formula. A standard curve was plotted with the scavenging rate as the ordinate (Y) and the mass concentration as the abscissa (X). The half-maximal scavenging concentration (IC50) of each reference ABTS free radical was calculated.

[0093]

[0094] A 样品 A 空白 The values ​​are A for the sample group and the blank group, respectively.

[0095] The results showed that all five indicator components possessed certain antioxidant activity and exhibited a good dose-response relationship within a certain range. Comparing the IC50 results and the inhibition rates at the same concentration, the order of antioxidant activity was: forsythoside A > luteolin > baicalin > chlorogenic acid > puerarin. Forsythoside A showed the highest activity in scavenging ABTS free radicals, with an IC50 of 11.08 ± 1.21 μmol·L⁻¹. -1 It was significantly better than the positive control ascorbic acid (22.99±1.35μmol·L⁻¹). -1 Despite its low content, luteolin-containing compounds exhibit strong activity, with an IC50 of 26.43 ± 2.12 μmol·L⁻¹. -1Its value was close to that of the positive control ascorbic acid. Chlorogenic acid and baicalin showed good activity, with IC50 values ​​of 31.65 ± 3.37 μmol·L⁻¹, respectively. -1 and 30.50±2.54μmol·L -1 Furthermore, the content determination results showed that forsythoside A, chlorogenic acid, and baicalin were present in high concentrations in the sample, suggesting that they may be the main components responsible for scavenging free radicals. The results also further indicate that the antioxidant activity of Chaiyin granules is the result of the synergistic effect of its multiple components.

[0096] Conclusion: This invention establishes a rapid screening method for antioxidant components in *Chaiyin* granules using HPLC-ABTS-DAD-ESI-TOF / MS technology. This method integrates chromatographic separation and activity screening, enabling rapid identification of antioxidant components in *Chaiyin* granule extract and screening out 44 antioxidant components. Further identification of 37 of these components using ESI-Q-TOF / MS technology was achieved. Based on this, an antioxidant fingerprint spectrum of *Chaiyin* granules was established. Combined with similarity analysis and quantitative determination of five active ingredients (chlorogenic acid, puerarin, forsythoside A, luteolin, and baicalin), the quality evaluation of *Chaiyin* granules was realized. This invention provides methodological and conceptual support for the rapid identification and overall quality evaluation of antioxidant components in traditional Chinese medicine preparations.

[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of antioxidant components in silver granules, characterized in that, Includes the following steps: (1) Mix the distillate particles and solvent evenly to obtain the test solution; the solvent is methanol; (2) The test solution was injected into an HPLC-ABTS-DAD-ESI-TOF / MS online detection system for detection to obtain the antioxidant active ingredients of the Chaiyin granules; HPLC system chromatographic conditions: Phenomenex Luna C18 column, mobile phase A was 0.2% formic acid aqueous solution, mobile phase B was methanol, gradient elution, detector wavelength was 280 nm; gradient elution conditions were: 0~5 min, 7%~13% B, 5~20 min, 13%~22% B, 20~35 min, 22%~25% B, 35~41 min, 25%~28% B, 41~51 min, 28%~30% B, 51~57 min, 30%~32% B, 57~85 min, 32%~38% B, 85~113 min, 38%~52% B, 113~118 min, 52%~53% B B, 118~130 min, 53%~70% B, 130~140 min, 70%~80% B, 140~145min, 80%~85% B, 145~150 min, 85~100% B, 150~160 min, 100% B; (3) Using the antioxidant components in the Chaiyin particles as common peaks, a fingerprint spectrum of the antioxidant components in Chaiyin particles was established.

2. The method for constructing the fingerprint spectrum of antioxidant components in *Chaiyin* particles as described in claim 1, characterized in that, In step (1), the concentration of the test solution is 0.008~0.012 g / mL.

3. The method for constructing the fingerprint spectrum of antioxidant components in *Chaiyin* particles as described in claim 1, characterized in that, The Phenomenex Luna C18 column has dimensions of 250 mm × 4.6 mm and a diameter of 5.0 μm.

4. The method for constructing the fingerprint spectrum of antioxidant components in *Chaiyin* particles as described in claim 1, characterized in that, The HPLC system chromatographic conditions also include: a flow rate of 0.8 mL / min. -1 The column temperature was 25 ºC; the injection volume was 5 μL.

5. The method for constructing the fingerprint spectrum of antioxidant components in *Chaiyin* particles as described in claim 1, characterized in that, Diode array detector conditions: The ABTS solution is mixed with the column solution via a three-way valve and introduced into the reaction coil; the ABTS flow rate is 0.6 mL / min. -1 The reaction tube has an inner diameter of 0.25 mm, a length of 10 m, and a detector wavelength of 734 nm.

6. The method for constructing the fingerprint spectrum of antioxidant components in *Chaiyin* particles as described in claim 1, characterized in that, Mass spectrometry conditions: The mobile phase entering the high-resolution electrospray time-of-flight mass spectrometer was split to 0.4 mL / min using a three-way valve. -1 Electrospray ionization (ESI) source with positive and negative ion modes; full scan range m / z 100~2000; spray pressure 310.28 kPa; drying gas volume flow rate 10.0 L / min, temperature 325 ºC; capillary voltage 4.0 kV; pyrolysis voltage 100 V; cone voltage 60 V.

7. The method for constructing the fingerprint spectrum of antioxidant components in *Chaiyin* particles as described in claim 1, characterized in that, The fingerprint spectrum of the antioxidant components contains 44 chromatographic peaks, namely: peak 1 corresponds to ferulic acid, peak 4 corresponds to monoglucoside, peak 6 corresponds to 5-O-caffeoylquinic acid, peak 7 corresponds to loganic acid, peak 8 corresponds to forsythoside E, peak 9 corresponds to strychnoside hemiacetal lactone, peak 10 corresponds to amygdalin, peak 11 corresponds to 3′-hydroxypuerarin, peak 12 corresponds to chlorogenic acid, peak 13 corresponds to 4-O-caffeoylquinic acid, peak 15 corresponds to oxidized loganic acid, peak 16 corresponds to puerarin, peak 17 corresponds to 3-O-p-hydroxycinnamoylquinic acid or its isomer, peak 18 corresponds to 3-O-feruloylquinic acid, peak 19 corresponds to daidzein, peak 20 corresponds to forsythoside I or its isomer, peak 21 corresponds to oxidized strychnoside, peak 22 corresponds to strychnoside, peak 13 corresponds to 4-O-caffeoylquinic acid, peak 15 corresponds to oxidized loganic acid, peak 21 corresponds to oxidized strychnoside, peak 22 corresponds to strychnoside, peak 23 corresponds to strychnoside, peak 24 corresponds to strychnoside, peak 25 corresponds to strychnoside, peak 26 corresponds to strychnoside, peak 27 corresponds to strychnoside, peak 28 corresponds to strychnoside, peak 29 corresponds to strychnoside, peak 20 corresponds to forsythoside I or its isomer, peak 21 corresponds to oxidized strychnoside, peak 22 corresponds to strychnoside, peak 23 corresponds to strychnoside, peak 24 corresponds to strychnoside, peak 25 corresponds Peak 23 corresponds to hyperoside, peak 24 corresponds to hesperidin, peak 25 corresponds to (+) pinoresinin-β-D-glucopyranoside, peak 26 corresponds to isoflavone glycoside A or its isomer, peak 27 corresponds to forsythoside A, peak 28 corresponds to isochlorogenic acid A, peak 29 corresponds to apigenin 6-C-arabinoside 8-C-glucoside, peak 30 corresponds to luteolin, peak 32 corresponds to rutin, peak 33 corresponds to apigenin 6-C-glucoside 8-C-arabinoside, peak 34 corresponds to isochlorogenic acid C, peak 35 corresponds to forsythoside, peak 37 corresponds to astragaloside, peak 38 corresponds to baicalin, peak 39 corresponds to apigenin, peak 40 corresponds to apigenin 7-O-glucuronic acid, peak 41 corresponds to 6-O-methylbaicalin, peak 42 corresponds to wogonin, and peak 44 corresponds to baicalin.

8. A method for detecting the content of indicator components in silver granules, characterized in that, Includes the following steps: The granules of cypermethrin and the solvent were mixed evenly to obtain the test solution; a gradient concentration reference solution was prepared, wherein the reference standard included chlorogenic acid, puerarin, forsythoside A, luteolin and baicalin; the solvent was methanol; The reference solution and the test solution were detected by high-performance liquid chromatography (HPLC) to determine the peak areas of the corresponding peaks in the chromatograms of the reference solution and the test solution. The content of the indicator component corresponding to the reference standard in the test solution was calculated using the standard curve method. The HPLC system chromatographic conditions were as follows: Phenomenex Luna C18 column; mobile phase A was 0.2% formic acid aqueous solution; mobile phase B was methanol; gradient elution was used; and the detector wavelength was 280 nm. The gradient elution conditions were: 0–5 min, 7%–13% B; 5–20 min, 13%–22% B; 20–35 min, 22%–25% B; 35–41 min, 25%–28% B; 41–51 min, 28%–30% B; 51–57 min, 30%–32% B; 57–85 min, 32%–38% B; 85–113 min, 38%–52% B; 113–118 min, 52%–53% B; 118–130 min, 53%–70% B; 130–140 min, 70%–80% B; 140–145 min. min, 80%~85% B, 145~150 min, 85~100% B, 150~160 min, 100% B.

9. The method for detecting the content of indicator components in silver-containing granules as described in claim 8, characterized in that, The Phenomenex Luna C18 column has dimensions of 250 mm × 4.6 mm and a diameter of 5.0 μm.

10. The method for detecting the content of indicator components in silver-containing granules as described in claim 8, characterized in that, The HPLC system chromatographic conditions also include: a flow rate of 0.8 mL / min. -1 The column temperature was 25 ºC; the injection volume was 5 μL.

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