Quality detection method of coptis chinensis-gardenia medicine pair

Abstract

The invention discloses a quality detection method of a coptis-gardenia medicine pair. According to the quality detection method, analysis conditions such as optimal mobile phase composition, elution procedure, mass spectrum, flow velocity and chromatographic column are screened through a large number of experiments. The chemical components of the coptis chinensis-gardenia medicine pair are analyzed and identified by adopting a UPLC-Q-TOF / MS technology, 99 components are identified, 41 coptis chinensis and 60 gardenia are common, and alkaloids, phenylpropanoids, iridoids, organic acids and flavonoids are taken as main components. On the basis, the UPLC-PDA (Ultra Performance Liquid Chromatography-Personal Digital Assistant) technology is used for carrying out content determination on the main effect components of the coptis-gardenia medicine pair, such as genipin 1-O-gentiobioside, geniposide, coptisine, epiberberine, cotetrandrine, jateorhizine, berberine hydrochloride and palmatine hydrochloride. Systematic methodology investigation and content determination prove that the method is good in precision, stability and repeatability, and reliable technical support is provided for quality control of coptis chinensis-gardenia medicine pairs and basic research of pharmacodynamic substances.

Description

Technical Field

[0001] This invention relates to a quality control method for traditional Chinese medicine, specifically a quality testing method for the Coptis chinensis-Gardenia jasminoides herb pair. Background Technology

[0002] Drug pairs are a common combination of drugs in traditional Chinese medicine clinical practice. They are a summary of the clinical experience of medical scientists throughout history. The combination of Coptis chinensis and Gardenia jasminoides comes from the Huanglian Jiedu Decoction (HLJDD) in "Waitai Miyao". It has the effect of clearing the intestines and purging fire. It is used clinically to treat enteritis, esophagitis, and oral ulcers.

[0003] Both Coptis chinensis and Gardenia jasminoides contain a variety of bioactive chemical components. Gardenia jasminoides contains genipin 1-O-gentiopicroside and genipin glycoside, while Coptis chinensis contains berberine, epiberberine, berberine hydrochloride, and palmatine hydrochloride, all of which have relatively high proportions of active ingredients. Berberine and genipin glycoside exhibit the most significant pharmacological effects and have been identified as quality control markers for HLJDD (Hormone-Liver-Distilled Drugs), considered core active ingredients in HLJDD for their anti-inflammatory effects. Furthermore, tetrandrine has been proven to have hypoglycemic activity. Accurate analysis and quantitative determination of the main effective components in medicinal materials are crucial for evaluating their efficacy.

[0004] Current research on the components of Coptis chinensis and Gardenia jasminoides as single herbs is relatively systematic, while research on the main effective components of the Coptis chinensis-Gardenia jasminoides combination is less common. To further elucidate the pharmacodynamic material basis and mechanism of action of the Coptis chinensis-Gardenia jasminoides combination, this invention employs UPLC technology to identify its effective components and performs quantitative analysis of the main active ingredients, providing experimental support for subsequent pharmacodynamic studies of the Coptis chinensis-Gardenia jasminoides combination. This technology not only provides a scientific basis for the quality control of Coptis chinensis-Gardenia jasminoides drugs but also helps ensure the stability and safety of their clinical use. Summary of the Invention

[0005] Purpose of the Invention: The purpose of this invention is to overcome the shortcomings of existing technologies. Through extensive experimental screening, this invention provides a method for identifying and determining the effective components of the Coptis chinensis-Gardenia jasminoides herb pair. This method has high detection sensitivity and good stability, and can objectively, comprehensively, and accurately evaluate the quality of the Coptis chinensis-Gardenia jasminoides herb pair, its extracts, and preparations. This is of great significance for quality control and ensuring efficacy.

[0006] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows:

[0007] A quality testing method for the Coptis chinensis-Gardenia jasminoides herb pair, including identification of active ingredients and content determination; the identification of active ingredients includes the following steps:

[0008] Step (1): Preparation of mixed reference solution

[0009] Take appropriate amounts of genipin, genipin 1-O-gentiopicroside, gentamicin, tetrandrine, berberine, berberine, epiberberine and palmatine hydrochloride reference standards, accurately weigh them, dissolve them in methanol, mix them well, and obtain a mixed reference standard solution of a certain concentration.

[0010] Step (2) Preparation of the test solution

[0011] Take Coptis chinensis and Gardenia jasminoides slices, soak them in a certain amount of water, then reflux to extract them. After extraction, filter them, combine the filtrates, concentrate them, and freeze dry them to obtain the Coptis chinensis-Gardenia jasminoides herbal pair sample.

[0012] Take a certain amount of Coptis chinensis-Gardenia jasminoides sample, mix them, dilute with ultrapure water, filter through a microporous membrane, and centrifuge to obtain the test solution;

[0013] Step (3) Sample injection analysis

[0014] The mixed reference standard from step (1) and the test solution from step (2) were injected into UPLC-Q-TOF / MS to analyze and identify the chemical components of the Coptis chinensis-Gardenia jasminoides drug pair.

[0015] As a preferred option, the quality testing method for the Coptis chinensis-Gardenia jasminoides pair described above, step (1) preparation of the mixed reference solution

[0016] Take appropriate amounts of genipin, genipin 1-O-gentiopicroside, gentamicin, tetrandrine, berberine, berberine, epiberberine, and palmatine hydrochloride reference standards, accurately weigh them, dissolve them in methanol, mix them well, and dilute them separately to prepare reference standard solutions with a concentration of 50 μg / mL.

[0017] As a preferred option, the method for quality testing of the Coptis chinensis-Gardenia jasminoides pair described above, step (2) of the preparation method of the test solution is as follows:

[0018] Extraction of medicinal materials: Take 250g of Coptis chinensis slices and 450g of Gardenia jasminoides slices, soak them in 8 times the amount of water for 2 hours, then reflux extract twice, 1.5 hours each time, filter through five layers of gauze, combine the two filtrates, concentrate and freeze dry to obtain Coptis chinensis-Gardenia jasminoides extract.

[0019] Take the extract of Coptis chinensis and Gardenia jasminoides, dilute it with ultrapure water to prepare a solution with a concentration of 1 mg / mL, filter it through a 0.22 μm microporous membrane, and centrifuge it at 13,000 rpm / min and 4℃ to obtain the test solution.

[0020] As a preferred option, the UPLC chromatographic conditions in step (3) of the above-described method for quality detection of the Coptis chinensis-Gardenia jasminoides pair are as follows:

[0021] The chromatographic column was an ACQUITY UPLC HSS T3 column, 2.1 mm × 100 mm, 1.8 μm, with a column temperature of 35 °C. The mobile phase was 0.1% formic acid-water (phase A) and acetonitrile (phase B), with a flow rate of 0.3 mL / min. The gradient elution program was: 0–3.0 min 95–85% A, 3–6 min 85–78% A, 6–13 min 78–5% A, 13–16 min 5–0% A, 16–17 min 0% A, 17–18 min 0–95% A, and 18–20 min 95% A. The injection volume was 3 μL.

[0022] As a preferred embodiment, the mass spectrometry parameters for the quality detection method of the Coptis chinensis-Gardenia jasminoides pair described above are set as follows: positive ion mode analysis, capillary voltage 3.0 kV, source temperature 100℃, desolvation temperature 350℃, cone gas flow rate 50 L / h, nitrogen nebulizer gas 600 L / h, full scan mode, scan range m / z 50–1200 Da; negative ion mode analysis, capillary voltage 2.5 kV, source temperature 100℃, desolvation temperature 280℃, cone gas flow rate 50 L / h, nitrogen nebulizer gas 600 L / h, full scan mode, scan range m / z 50–1200 Da; data are acquired and analyzed using MassLynx V4.2 software.

[0023] The quality detection method for the Coptis chinensis-Gardenia jasminoides herb pair provided by this invention includes the following steps for content determination:

[0024] (1) Preparation of reference solution

[0025] Accurately weigh genipin, genipin 1-O-gentiopicroside, gentamicin, tetrandrine, berberine, berberine hydrochloride, epiberberine, and palmatine hydrochloride, dissolve them in methanol, and prepare mixed reference solutions of genipin, genipin 1-O-gentiopicroside, gentamicin, tetrandrine, berberine, berberine hydrochloride, epiberberine, and palmatine hydrochloride at specific concentrations.

[0026] (2) Preparation of the test solution

[0027] Take Coptis chinensis and Gardenia jasminoides slices, soak them in a certain amount of water, then reflux to extract them. After extraction, filter them, combine the filtrates, concentrate them, and freeze dry them to obtain the Coptis chinensis-Gardenia jasminoides herbal pair sample.

[0028] Take a certain amount of Coptis chinensis-Gardenia jasminoides sample, dilute it with ultrapure water, filter it through a microporous membrane, and centrifuge it to obtain the test solution;

[0029] (3) Establishment of the linear regression equation

[0030] Take the mixed reference standard from step (1) and dilute it stepwise with methanol to obtain a series of reference standard solutions of different concentrations. Inject the solutions into UPLC sequentially, and use the mass concentration of the series of reference standards as the abscissa X and the corresponding peak area as the ordinate Y to perform linear regression analysis on each chemical component and calculate the linear regression equation.

[0031] (4) Content determination

[0032] The Coptis chinensis-Gardenia jasminoides drug pair test solution from step (2) was injected into UPLC for analysis. The content of each component in the test solution was calculated based on the linear regression equation from step (3).

[0033] As a preferred option, the quality testing method for the Coptis chinensis-Gardenia jasminoides pair described above includes step (1) preparation of the reference solution:

[0034] Accurately weigh genipin, genipin 1-O-gentiopicroside, gentamicin, tetrandrine, berberine, berberine hydrochloride, epiberberine, and palmatine hydrochloride, dissolve them in methanol, and prepare mixed reference solutions with concentrations of 2 mg / mL genipin, 1 mg / mL genipin 1-O-gentiopicroside, 1 mg / mL gentamicin, 1 mg / mL tetrandrine, 1 mg / mL berberine, 1 mg / mL berberine hydrochloride, 1 mg / mL epiberberine, and 1 mg / mL palmatine hydrochloride.

[0035] As a preferred option, in the quality testing method for the Coptis chinensis-Gardenia jasminoides pair described above, step (2) involves the preparation of the test solution:

[0036] Take 200g each of Coptis chinensis and Gardenia jasminoides slices, add 8 times the amount of water and soak for 2 hours, then reflux extract twice, 1.5 hours each time, filter through five layers of gauze, combine the two filtrates, concentrate and freeze dry to obtain Coptis chinensis-Gardenia jasminoides sample;

[0037] Take the Coptis chinensis-Gardenia jasminoides sample, dilute it with ultrapure water to prepare a solution with a concentration of 1 mg / mL, filter it through a 0.22 μm microporous membrane, and centrifuge it at 13,000 rpm / min and 4℃ to obtain the test solution.

[0038] As a preferred embodiment, the chromatographic conditions for steps (3) and (4) of the quality detection method for the Coptis chinensis-Gardenia jasminoides pair described above are as follows:

[0039] The chromatographic column was a Waters XSelect CSH C18, 2.1 mm × 150 mm, 2.5 μm. The mobile phase consisted of acetonitrile (phase A) and 0.1% formic acid (phase B). The gradient elution program was as follows: 0–1 min, 10% A; 1–3 min, 10%–15% A; 3–9 min, 15%–16% A; 9–17 min, 16%–18% A; 17–20 min, 18%–25% A; 20–25 min, 25%–91% A; 25–29 min, 91%–10% A; 29–31 min, 10% A. The flow rate was 0.2 mL / min. The detection wavelength was 256 nm. The injection volume was 1 μL. The column temperature was 30 °C.

[0040] Beneficial effects: The quality testing method for the Coptis chinensis-Gardenia jasminoides herb pair provided by this invention has the following advantages compared with the prior art:

[0041] This invention employs UPLC-Q-TOF / MS technology to analyze and identify the chemical components of the Coptis chinensis-Gardenia jasminoides herbal pair, identifying a total of 99 components: 41 from Coptis chinensis and 60 from Gardenia jasminoides. Two components are shared, primarily consisting of alkaloids, phenylpropanoids, iridoids, organic acids, and flavonoids. Furthermore, UPLC-PDA technology was used to quantitatively analyze the main effective components of the Coptis chinensis-Gardenia jasminoides herbal pair. Through systematic methodological investigation and content determination, the method was verified to have good precision, stability, and repeatability, providing reliable technical support for the quality control and pharmacodynamic material basis research of the Coptis chinensis-Gardenia jasminoides herbal pair. Attached Figure Description

[0042] Figure 1 This is a mass spectrum of Coptis chinensis and Gardenia jasminoides for component identification using UPLC-Q-TOF / MS technology.

[0043] Figure 2 Images showing the detection of components in the Coptis chinensis-Gardenia jasminoides herb pair using UPLC-Q-TOF / MS technology (A. Structural classification of compounds; B. Quantity of chemical components in each herb in the Coptis chinensis-Gardenia jasminoides herb pair; C. Structural formulas of chemical components in the Coptis chinensis-Gardenia jasminoides herb pair).

[0044] Figure 3 This is a graph showing the detection of components in the Coptis chinensis-Gardenia jasminoides herb pair using UPLC-Q-TOF / MS technology.

[0045] Figure 4 UPLC chromatograms of Coptis chinensis extract (A), Gardenia jasminoides extract (B), and Coptis chinensis-Gardenia jasminoides extract (C) solutions. Note: Peak 1: Genipin 1-O-gentiopicroside, Peak 2: Genipin glycoside, Peak 3: Coptisine, Peak 4: Epiberberine, Peak 5: Stephania tetrandra, Peak 6: Rhizophora alkaloid, Peak 7: Berberine hydrochloride, Peak 8: Palmatine hydrochloride.

[0046] Figure 5 This is the chromatogram for the first gradient elution method.

[0047] Figure 6 This is the chromatogram for the second gradient elution method. Detailed Implementation

[0048] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0049] Example 1

[0050] Analysis and identification of chemical components of Coptis chinensis-Gardenia jasminoides herb pair using UPLC-Q-TOF / MS technology

[0051] 1. Instruments and Reagents

[0052] Instruments and reagents Manufacturer SYNAPTG2-SiQ-TOF mass spectrometer Waters Technology (Shanghai) Co., Ltd. AcquityUPLCI-Class System Waters Technology (Shanghai) Co., Ltd. ACQUITYUPLCHSST3 column Waters Technology (Shanghai) Co., Ltd. Mettler M204 and Mettler MS105DU electronic analytical balances Mettler Toledo, Switzerland Milli-QIQ7000 Ultrapure Water System Millipore, USA Acetonitrile (mass spectrometry grade) Honeywell Burdick & Jackson Formic acid (mass spectrometry grade) Merck Darmstadt, Germany Berberine (batch number Yz110920), purity ≥98% Nanjing Yuanzhi Biotechnology Co., Ltd. Herba alkaloids (batch number Yz032022), purity ≥98%. Nanjing Yuanzhi Biotechnology Co., Ltd. Berberine (batch number Yz030723), purity ≥98%. Nanjing Yuanzhi Biotechnology Co., Ltd. Berberine (batch number Yz1227221), purity ≥98% Nanjing Yuanzhi Biotechnology Co., Ltd. Palmatine hydrochloride (batch number Yz070821), purity ≥98%. Nanjing Yuanzhi Biotechnology Co., Ltd. African tetrandrine (batch number Yz118221), purity ≥98%. Nanjing Yuanzhi Biotechnology Co., Ltd. Genipin (batch number 16052902), purity ≥98%. Nanjing Yuanzhi Biotechnology Co., Ltd. Genipin 1-O-gentiopicroside (batch number Yz1617222), purity ≥98%. Nanjing Yuanzhi Biotechnology Co., Ltd. Coptis chinensis (batch number: 20210101) Shaanxi Hanyi Shengcaotang Pharmaceutical Co., Ltd. Gardenia (Batch No.: 22050805) Anguo City Guangjitang Pharmaceutical Co., Ltd. .

[0053] 2. Method

[0054] 2.1 Preparation of the test solution

[0055] Extraction of medicinal materials: Take 250g of Coptis chinensis slices and 450g of Gardenia jasminoides slices, soak them in 8 times the amount of water for 2 hours, then reflux extract twice, 1.5 hours each time, filter through five layers of gauze, combine the two filtrates, concentrate and freeze dry to obtain Coptis chinensis-Gardenia jasminoides extract.

[0056] Take the extract of Coptis chinensis and Gardenia jasminoides, dilute it with ultrapure water to prepare a solution with a concentration of 1 mg / mL, filter it through a 0.22 μm microporous membrane, and centrifuge it at 13,000 rpm / min and 4℃ to obtain the test solution.

[0057] 2.2 Preparation of the reference solution:

[0058] Take appropriate amounts of genipin, genipin 1-O-gentiopicroside, gentamicin, tetrandrine, berberine, berberine, epiberberine, and palmatine hydrochloride reference standards, accurately weigh them, dissolve them in methanol, mix them well, and dilute them separately to prepare reference standard solutions with a concentration of 50 μg / mL.

[0059] 2.3 Sample Injection Analysis

[0060] The mixed reference solution from step 2.2 and the test solution from step 2.1 were injected into UPLC-Q-TOF / MS to analyze and identify the chemical components of the Coptis chinensis-Gardenia jasminoides drug pair.

[0061] The chromatographic conditions are as follows:

[0062] The chromatographic column was an ACQUITYUPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm), and the column temperature was 35 °C. The mobile phase was 0.1% formic acid-water (A) and acetonitrile (B), and the flow rate was 0.3 mL / min. The gradient elution program was: 0–3.0 min 95–85% A, 3–6 min 85–78% A, 6–13 min 78–5% A, 13–16 min 5–0% A, 16–17 min 0% A, 17–18 min 0–95% A, and 18–20 min 95% A. The injection volume was 3 μL.

[0063] Mass spectrometry conditions are:

[0064] The relevant parameters for mass spectrometry were set as follows: positive ion mode analysis, capillary voltage 3.0 kV, source temperature 100℃, desolvation temperature 350℃, cone gas flow rate 50 L / h, nitrogen nebulizer gas (600 L / h), full scan mode, scan range m / z 50–1200 Da; negative ion mode analysis, capillary voltage 2.5 kV, source temperature 100℃, desolvation temperature 280℃, cone gas flow rate 50 L / h, nitrogen nebulizer gas (600 L / h), full scan mode, scan range m / z 50–1200 Da. MassLynx V4.2 software was used for data acquisition and analysis.

[0065] 2.4 Data Processing and Result Analysis

[0066] A database of chemical components of the Coptis chinensis-Gardenia jasminoides herbal pair was constructed using databases such as TCMSP and Chemical Book, combined with literature. After importing the self-built database into the UNIFI platform, identification was performed based on mass spectrometry data, chromatographic elution sequence, and fragment ion characteristics, with a mass error of <10 ppm. Furthermore, for components not identified by the UNIFI platform, MassLynx 4.1 software was used for further identification, combining mass spectrometry fragmentation patterns with relevant literature.

[0067] 3. Experimental Results

[0068] Base peak chromatograms (BPI) of the Coptis chinensis-Gardenia jasminoides herb pair under positive and negative ion modes are shown in [reference needed]. Figure 1 A total of 99 components were identified, mainly alkaloids, phenylpropanoids, iridoids, organic acids, and flavonoids. Of these, 41 components came from Coptis chinensis and 60 from Gardenia jasminoides. Relevant information for the components is shown in Table 1, and their structural formulas are shown in [Table 1]. Figure 2 and Figure 3 .

[0069] Table 1. Qualitative Identification Information of the Coptis chinensis-Gardenia jasminoides Herb Pair

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076] The chemical constituents of the Coptis chinensis-Gardenia jasminoides herb pair were analyzed and identified using UPLC-Q-TOF / MS technology. Ninety-nine compounds were successfully identified, of which 41 were derived from Coptis chinensis, 60 from Gardenia jasminoides, and two were shared by both herbs. These components mainly include alkaloids, phenylpropanoids, iridoids, organic acids, flavonoids, fatty acids, and triterpenoids. This study provides a scientific basis for the quality control and standardization of traditional Chinese medicine, and has significant theoretical value and application prospects. Further research into the interactions of these chemical components may provide clues for the discovery of new pharmacologically active substances, laying the foundation for the component identification and application of the Coptis chinensis-Gardenia jasminoides herb pair.

[0077] Example 2: Quantitative analysis of the main effective components of the Coptis chinensis-Gardenia jasminoides herbal pair using UPLC-PDA technology.

[0078] 1. Materials

[0079] 1.1 Instruments and Reagents

[0080]

[0081]

[0082] 2 methods

[0083] 2.1 Solution Preparation

[0084] 2.1.1 Preparation of reference solution

[0085] Accurately weigh the reference standards of genipin, genipin 1-O-gentiopicroside, gentamicin, tetrandrine, berberine, berberine hydrochloride, epiberberine, and palmatine hydrochloride, dissolve them in methanol, and prepare reference solutions with concentrations of 2 mg / mL genipin, 1 mg / mL genipin 1-O-gentiopicroside, 1 mg / mL gentamicin, 1 mg / mL tetrandrine, 1 mg / mL berberine, 1 mg / mL berberine hydrochloride, 1 mg / mL epiberberine, and 1 mg / mL palmatine hydrochloride, respectively.

[0086] 2.1.2 Preparation of the test solution

[0087] Extraction of medicinal materials: Take 200g each of Coptis chinensis and Gardenia jasminoides slices, soak them in 8 times the amount of water for 2 hours, then reflux extract twice, 1.5 hours each time, filter through five layers of gauze, combine the two filtrates, concentrate and freeze dry to obtain Coptis chinensis and Gardenia jasminoides samples respectively.

[0088] Mix 5 mg each of Coptis chinensis and Gardenia jasminoides samples, dilute with ultrapure water to prepare a solution with a concentration of 1 mg / mL, filter through a 0.22 μm microporous membrane, and centrifuge (13,000 rpm / min, 4℃) to obtain the test solution of the Coptis chinensis-Gardenia jasminoides drug pair;

[0089] The preparation methods for the Coptis chinensis test solution and the Gardenia jasminoides test solution are the same as above.

[0090] 2.2 Chromatographic conditions

[0091] The chromatographic column was a Waters XSelect CSH C18 (2.1 mm × 150 mm, 2.5 μm); the mobile phase was acetonitrile (A)-0.1% formic acid water (B), with gradient elution (0-1 min, 10% A; 1-3 min, 10%-15% A; 3-9 min, 15%-16% A; 9-17 min, 16%-18% A; 17-20 min, 18%-25% A; 20-25 min, 25%-91% A; 25-29 min, 91%-10% A; 29-31 min, 10% A); the flow rate was 0.2 mL / min; the detection wavelength was 256 nm; the injection volume was 1 μL; and the column temperature was 30 °C.

[0092] 2.3 Methodological Validation

[0093] 2.3.1 Examination of Linear Range

[0094] Take an appropriate amount of the single reference solution under section “2.1.1”, dilute it stepwise with methanol, and inject it for detection according to the chromatographic conditions under section “2.2”.

[0095] 2.3.2 Precision Test

[0096] Prepare the reference solution according to the method in section "2.1.2", inject it continuously 6 times under the chromatographic conditions in section "2.2", and record the relative retention time and peak area of ​​each peak.

[0097] 2.3.3 Repeatability Test

[0098] Prepare the test solution according to the method in section “2.1.2”, and inject it continuously 6 times under the chromatographic conditions in section “2.2”, and record the relative retention time and peak area of ​​each peak.

[0099] 2.3.4 Stability Test

[0100] Prepare the test solution according to the method in section “2.1.2”. Take the same test solution and let it stand naturally at room temperature. Inject it at 0, 2, 4, 8, 12, 16, 24 and 48 h according to the chromatographic conditions in section “2.2”. Record the relative retention time and peak area of ​​each peak.

[0101] 2.3.5 Recovery Test

[0102] Take six samples of the Coptis chinensis-Gardenia jasminoides herbal pair with known content, each approximately 4 mg, and weigh accurately. Add a reference standard with a content equivalent to that of the analyte in the sample to each sample, and determine the analyte according to the chromatographic conditions under section "2.2". Record the corresponding peak area of ​​each analyte and calculate the recovery rate.

[0103] 2.3.6 Content Determination

[0104] Prepare the test solution according to the method in section "2.1.2", perform three parallel determinations, and calculate the content of each component in Coptis chinensis, Gardenia jasminoides, and the Coptis chinensis-Gardenia jasminoides pair according to the linear equation.

[0105] 3 Results and Discussion

[0106] 3.1 Optimization of chromatographic conditions

[0107] To achieve optimal separation of the eight main effective components within a short time, chromatographic conditions, including mobile phase, flow rate, column temperature, and elution gradient, were optimized in preliminary experiments. To improve peak shape, various mobile phase combinations were tested, including methanol-water, acetonitrile-water, methanol-0.1% formic acid solution, and acetonitrile-0.1% formic acid solution. Results showed that the acetonitrile-0.1% formic acid solution system provided the best separation for the eight components. After optimizing the flow rate and column temperature, a flow rate of 0.20 mL / min and a column temperature of 30℃ were ultimately determined. Analysis of the three-dimensional chromatograms of the samples in the 190–400 nm range and the maximum absorption wavelengths of each reference standard led to the selection of different detection wavelengths: 254, 280, and 256 nm. Comparison of the chromatograms detected at these three wavelengths revealed that the chromatogram detected at 256 nm had a flatter baseline and best reflected the chemical information of the two components in the Coptis chinensis-Gardenia jasminoides herb pair. Therefore, 256 nm was ultimately chosen as the detection wavelength.

[0108] This invention screened chromatograms with different elution gradients and selected the optimal gradient elution method:

[0109] The first gradient elution mode: 0-2.0 min 95-85% A, 2-5 min 85-78% A, 5-12 min 78-5% A, 12-15 min 5-0% A, 15-17 min 0% A, 17-18 min 0-95% A, 18-20 min 95% A. The chromatogram is shown below. Figure 5 As shown, the peak elution and separation are poor.

[0110] The second gradient elution method: 0-3.0 min 95-85% A, 3-6 min 85-75% A, 6-12 min 75-5% A, 12-17.5 min 5-2% A, 17.5-20 min 2% A, 20-22 min 2-95% A, 22-30 min 95% A. The chromatogram is shown below. Figure 6 As shown, the separation from several peaks is poor.

[0111] The third gradient elution method: 0-1 min, 10% A; 1-3 min, 10%-15% A; 3-9 min, 15%-16% A; 9-17 min, 16%-18% A; 17-20 min, 18%-25% A; 20-25 min, 25%-91% A; 25-29 min, 91%-10% A; 29-31 min, 10% A; flow rate: 0.2 mL / min; chromatogram as shown. Figure 4 As shown, the peaks are well separated. Therefore, this gradient elution method is preferred in this invention.

[0112] By comparing with standards, a total of 8 peaks were identified in the liquid chromatogram of the Coptis chinensis-Gardenia jasminoides pair, including 6 peaks from Coptis chinensis and 2 peaks from Gardenia jasminoides. These peaks were identified as genipin 1-O-gentiopicroside, genipin glycoside, berberine, epiberberine, tetrandrine, purslane, berberine hydrochloride, and palmatine hydrochloride. The liquid chromatograms of Coptis chinensis, Gardenia jasminoides, and the Coptis chinensis-Gardenia jasminoides pair are shown below. Figure 4 .

[0113] 3.2 Methodological Examination

[0114] 3.2.1 Examination of Linear Relationships

[0115] A standard curve was plotted with mass concentration (X) on the x-axis and peak area (Y) on the y-axis. The regression equation and linear range are shown in Table 2. The results show that each component has a good linear relationship within its respective range.

[0116] Table 2 Linear Relationships of Each Component

[0117] Element Regression equation r Linear range (μg / mL) Genipin 1-O-Gentianoid Disaccharide [Y = 3 x 10 6 X + 15729]] 0.9995 1.9531-250 Genipin [Y = 4 x 10 6 X + 48422]] 0.9998 7.8125-2000 Berberine [Y = 2 x 10 7 X-76735]]> 0.9998 7.8125-500 Root alkaloids <![CDATA[Y=2×10 7 X+41506]]> 0.9994 0.9766-125 African tetrandrine <![CDATA[Y=1×10 7 X+5475]]> 0.9998 1.9531-125 Berberine hydrochloride <![CDATA[Y=3×10 7 X+125802]]> 0.9998 15.6250-1000 Berberine <![CDATA[Y=2×10 7 X+5504]]> 0.9998 3.9063-250 Palmatine Hydrochloride <![CDATA[Y=2×10 7 X-33051]]> 0.9998 7.8125-500

[0118] 3.2.2 Precision Test

[0119] The RSDs of the peak areas of genipin, genipin 1-O-gentiopicroside, berberine, epiberberine, stephania tetrandra, purslane, berberine hydrochloride, and palmatine hydrochloride were 0.26%, 0.23%, 0.47%, 0.84%, 0.96%, 0.56%, 0.30%, and 0.57%, respectively (see Table 3). The results indicate that the instrument has good precision.

[0120] Table 3. Precision test results (n=6)

[0121]

[0122] 3.2.3 Repeatability Test

[0123] The RSDs of the peak areas of genipin, genipin 1-O-gentiopicroside, berberine, epiberberine, stephania tetrandra, purslane, berberine hydrochloride, and palmatine hydrochloride were 0.83%, 0.80%, 0.82%, 0.88%, 0.83%, 0.87%, 0.87%, and 1.25%, respectively (see Table 4). The results indicate that the method has good reproducibility.

[0124] Table 4. Results of repeatability testing (n=6)

[0125]

[0126] 3.2.4 Stability Test

[0127] The RSDs of the peak areas of genipin, genipin 1-O-gentiopicroside, berberine, epiberberine, stephania tetrandra, purslane, berberine hydrochloride, and palmatine hydrochloride were 0.49%, 1.17%, 0.36%, 0.44%, 0.52%, 1.02%, 0.48%, and 0.52%, respectively (see Table 5). The results indicate that the method has good stability.

[0128] 3.2.5 Recovery Test

[0129] Table 5. Results of the stability test

[0130]

[0131] The average recoveries of genipin, genipin 1-O-gentiopicroside, berberine, epiberberine, tetrandrine, purslane, berberine hydrochloride, and palmatine hydrochloride were 102.38%, 99.79%, 99.94%, 96.27%, 97.48%, 103.22%, 99.16%, and 96.13%, respectively (see Table 6). The results indicate that the recoveries meet the requirements.

[0132] Table 6. Recovery results of spiking (n=6)

[0133]

[0134]

[0135] 3.2.6 Content Determination

[0136] The results of the content determination of each component in Coptis chinensis, Gardenia jasminoides, Coptis chinensis-Gardenia jasminoides (5:9), and Coptis chinensis-Gardenia jasminoides (1:1) are shown in Table 7.

[0137] Table 7. Content of various components in Coptis chinensis, Gardenia jasminoides, and Coptis chinensis-Gardenia jasminoides medicinal materials (n=3)

[0138]

[0140] In this embodiment, UPLC-PDA technology was used to determine the content of the main effective components of the Coptis chinensis-Gardenia jasminoides herb pair. In particular, the extraction efficiency of Coptis chinensis-Gardenia jasminoides was significantly improved after decoction, while the extraction efficiency of the root alkaloids decreased after decoction. This also provides a scientific basis for the synergistic effect of the herb pair from the perspective of composition.

[0141] The present invention has been methodologically validated, demonstrating good precision, stability, and repeatability. The findings of this study not only provide reliable experimental evidence for in-depth research on the Coptis chinensis-Gardenia jasminoides herb pair, but also offer important reference for the improvement of its quality standards and its development and application.

[0142] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A quality testing method for a Coptis chinensis-Gardenia jasminoides herb pair, characterized in that, This includes the identification and content determination of active ingredients; the identification of active ingredients includes the following steps: Step (1): Preparation of mixed reference solution Take appropriate amounts of genipin, genipin 1-O-gentiopicroside, gentamicin, tetrandrine, berberine, berberine, epiberberine and palmatine hydrochloride reference standards, accurately weigh them, dissolve them in methanol, mix them well, and obtain a mixed reference standard solution of a certain concentration. Step (2) Preparation of the test solution Take Coptis chinensis and Gardenia jasminoides slices, soak them in a certain amount of water, then reflux to extract them. After extraction, filter them, combine the filtrates, concentrate them, and freeze dry them to obtain the Coptis chinensis-Gardenia jasminoides herbal pair sample. Take a certain amount of Coptis chinensis-Gardenia jasminoides sample, mix them, dilute with ultrapure water, filter through a microporous membrane, and centrifuge to obtain the test solution; Step (3) Sample injection analysis The mixed reference standard from step (1) and the test solution from step (2) were injected into UPLC-Q-TOF / MS to analyze and identify the chemical components of the Coptis chinensis-Gardenia jasminoides drug pair.

2. The quality testing method for the Coptis chinensis-Gardenia jasminoides herb pair according to claim 1, characterized in that, Step (1) Preparation of mixed reference solution Take appropriate amounts of genipin, genipin 1-O-gentiopicroside, gentamicin, tetrandrine, berberine, berberine, epiberberine, and palmatine hydrochloride reference standards, accurately weigh them, dissolve them in methanol, mix them well, and dilute them separately to prepare reference standard solutions with a concentration of 50 μg / mL.

3. The quality testing method for the Coptis chinensis-Gardenia jasminoides herb pair according to claim 1, characterized in that, The preparation method of the test solution in step (2) is as follows: Extraction of medicinal materials: Take 250g of Coptis chinensis slices and 450g of Gardenia jasminoides slices, soak them in 8 times the amount of water for 2 hours, then reflux extract twice, 1.5 hours each time, filter through five layers of gauze, combine the two filtrates, concentrate and freeze dry to obtain Coptis chinensis-Gardenia jasminoides extract. Take the extract of Coptis chinensis and Gardenia jasminoides, dilute it with ultrapure water to prepare a solution with a concentration of 1 mg / mL, filter it through a 0.22 μm microporous membrane, and centrifuge it at 13,000 rpm / min and 4℃ to obtain the test solution.

4. The quality testing method for the Coptis chinensis-Gardenia jasminoides herb pair according to claim 1, characterized in that, The chromatographic conditions for UPLC in step (3) are as follows: The chromatographic column was an ACQUITY UPLC HSS T3 column, 2.1 mm × 100 mm, 1.8 μm, with a column temperature of 35 °C. The mobile phase was 0.1% formic acid-water (phase A) and acetonitrile (phase B), with a flow rate of 0.3 mL / min. The gradient elution program was: 0–3.0 min 95–85% A, 3–6 min 85–78% A, 6–13 min 78–5% A, 13–16 min 5–0% A, 16–17 min 0% A, 17–18 min 0–95% A, 18–20 min 95% A, with an injection volume of 3 μL.

5. The quality testing method for the Coptis chinensis-Gardenia jasminoides herb pair according to claim 4, characterized in that, The mass spectrometry parameters were set as follows: positive ion mode analysis, capillary voltage 3.0 kV, source temperature 100℃, desolvation temperature 350℃, cone gas flow rate 50 L / h, nitrogen nebulizer gas 600 L / h, full scan mode, scan range m / z 50–1200 Da; negative ion mode analysis, capillary voltage 2.5 kV, source temperature 100℃, desolvation temperature 280℃, cone gas flow rate 50 L / h, nitrogen nebulizer gas 600 L / h, full scan mode, scan range m / z 50–1200 Da; data were acquired and analyzed using MassLynx V4.2 software.

6. The quality testing method for the Coptis chinensis-Gardenia jasminoides herb pair according to claim 1, characterized in that, Content determination includes the following steps: (1) Preparation of reference solution Accurately weigh genipin, genipin 1-O-gentiopicroside, gentamicin, tetrandrine, berberine, berberine hydrochloride, epiberberine, and palmatine hydrochloride, dissolve them in methanol, and prepare mixed reference solutions of genipin, genipin 1-O-gentiopicroside, gentamicin, tetrandrine, berberine, berberine hydrochloride, epiberberine, and palmatine hydrochloride at specific concentrations. (2) Preparation of the test solution Coptis chinensis and Gardenia jasminoides were taken separately, soaked in a certain amount of water, and then extracted by reflux. After extraction, the extracts were filtered, the filtrates were combined, concentrated and freeze-dried to obtain Coptis chinensis and Gardenia jasminoides samples. Take a certain amount of Coptis chinensis sample and Gardenia jasminoides sample, mix them, dilute with ultrapure water, filter through a microporous membrane, centrifuge, and obtain the test solution; (3) Establishment of the linear regression equation Take the mixed reference standard from step (1) and dilute it stepwise with methanol to obtain a series of reference standard solutions of different concentrations. Inject the solutions into UPLC sequentially, and use the mass concentration of the series of reference standards as the abscissa X and the corresponding peak area as the ordinate Y to perform linear regression analysis on each chemical component and calculate the linear regression equation. (4) Content determination The Coptis chinensis-Gardenia jasminoides drug pair test solution from step (2) was injected into UPLC for analysis. The content of each component in the test solution was calculated based on the linear regression equation from step (3).

7. The quality testing method for the Coptis chinensis-Gardenia jasminoides herb pair according to claim 6, characterized in that, Step (1) Preparation of the reference solution: Accurately weigh genipin, genipin 1-O-gentiopicroside, gentamicin, tetrandrine, berberine, berberine hydrochloride, epiberberine, and palmatine hydrochloride, dissolve them in methanol, and prepare mixed reference solutions with concentrations of 2 mg / mL genipin, 1 mg / mL genipin 1-O-gentiopicroside, 1 mg / mL gentamicin, 1 mg / mL tetrandrine, 1 mg / mL berberine, 1 mg / mL berberine hydrochloride, 1 mg / mL epiberberine, and 1 mg / mL palmatine hydrochloride.

8. The quality testing method for the Coptis chinensis-Gardenia jasminoides herb pair according to claim 6, characterized in that, Step (2) Preparation of the test solution: Take 200g each of Coptis chinensis and Gardenia jasminoides slices, add 8 times the amount of water and soak for 2 hours, then reflux extract twice, 1.5 hours each time, filter through five layers of gauze, combine the two filtrates, concentrate and freeze dry to obtain Coptis chinensis-Gardenia jasminoides sample; Take the Coptis chinensis-Gardenia jasminoides sample, dilute it with ultrapure water to prepare a solution with a concentration of 1 mg / mL, filter it through a 0.22 μm microporous membrane, and centrifuge it at 13,000 rpm / min and 4℃ to obtain the test solution.

9. The quality testing method for the Coptis chinensis-Gardenia jasminoides herb pair according to claim 6, characterized in that, The chromatographic conditions for steps (3) and (4) are as follows: Chromatographic column: Waters XSelect CSH C 18 The column has dimensions of 2.1 mm × 150 mm and a diameter of 2.5 μm. The mobile phase consists of acetonitrile (phase A) and 0.1% formic acid (phase B), with gradient elution: 0–1 min, 10% A; 1–3 min, 10%–15% A; 3–9 min, 15%–16% A; 9–17 min, 16%–18% A; 17–20 min, 18%–25% A; 20–25 min, 25%–91% A; 25–29 min, 91%–10% A; 29–31 min, 10% A. The flow rate is 0.2 mL / min. The detection wavelength is 256 nm. The injection volume is 1 μL. The column temperature is 30 °C.

10. The quality testing method for the Coptis chinensis-Gardenia jasminoides herb pair according to claim 6, characterized in that, The standard curve established in step (3) is as follows: 。

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