Jichuan decoction composition one-sample multi-evaluation content detection method

Abstract

The application relates to a one-measurement-multiple-evaluation content detection method for Jichuan decoction composition, which comprises the following steps: using an extraction solvent to extract the Jichuan decoction composition to prepare a test sample solution; using a dissolving reagent to dissolve a control sample and naringin to prepare a control sample mixed solution and a naringin solution; respectively sampling the test sample solution, the control sample mixed solution and the naringin solution to perform ultrahigh performance liquid chromatography detection; taking naringin as an internal reference, and calculating the content of the to-be-detected component in the test sample solution according to a formula. The method adopts the one-measurement-multiple-evaluation method taking naringin as the internal reference, obtains the contents of eight components, i.e. caffeic acid, pinitol, ferulic acid, isoferulic acid, verbascoside, naringin, neohesperidin and hesperidin in the Jichuan decoction composition, and has the advantages of high practicability, simple operation, rapid testing, accuracy, cost saving, simple and convenient operation, and the like, and provides a basis for establishing the quality evaluation of the Jichuan decoction composition.

Description

Technical Field

[0001] This application relates to the field of traditional Chinese medicine preparation testing technology, and in particular to a method for detecting the content of Jichuan Decoction composition using a single test and multiple evaluations. Background Technology

[0002] Jichuan Decoction originates from *Jingyue Quanshu*, written by Zhang Jingyue in the Ming Dynasty. It consists of six herbs: Angelica sinensis, Achyranthes bidentata, Cistanche deserticola, Alisma plantago-aquatica, Cimicifuga foetida, and Citrus aurantium. It has the effects of warming the kidneys and replenishing essence, moistening the intestines and promoting bowel movements. The book states, "For diseases involving deficiency and constipation, drastic remedies such as Glauber's salt and rhubarb should not be used; if there is no other way to relieve constipation, this decoction is appropriate. This is a remedy that combines tonification and purgation, truly wonderful." As an experienced formula for treating constipation, Jichuan Decoction has pharmacological effects such as promoting gastrointestinal motility, improving colonic motility, regulating gastrointestinal hormones, and maintaining intestinal flora homeostasis. Clinically, it is mainly used for functional constipation in the elderly, slow transit constipation, functional outlet constipation, constipation in Parkinson's disease patients, constipation in stroke recovery patients, and constipation caused by opioids.

[0003] Jichuan Decoction is a classic ancient Chinese medicine formula, included in the "List of Classic Ancient Chinese Medicine Formulas (First Batch)" published by the state. The development of classic formulas and corresponding traditional Chinese medicine compound preparations mainly involves two stages: the development of "reference samples for traditional Chinese medicine compound preparations based on classic ancient Chinese medicine formulas (hereinafter referred to as material references, also known as standard decoctions)" and the development of "compound preparations based on classic ancient Chinese medicine formulas." Research on Jichuan Decoction primarily focuses on clinical and pharmacological studies, while quality control research mainly concentrates on the content of indicator components and fingerprint analysis. The literature "Simultaneous Determination of Four Indicator Components in Jichuan Decoction and Research on its Material Reference Process" (Journal of Tianjin University of Traditional Chinese Medicine, 2023, No. 4) established an HPLC fingerprint of Jichuan Decoction, but only simultaneously determined the content of four indicator components—naringin, neohesperidin, β-ecdysterone, and echinacoside—and established their material reference preparation process. The literature "Simultaneous Determination of Multiple Components in the Classic Formula Jichuan Decoction by HPLC" (Journal of Tianjin University of Traditional Chinese Medicine, Vol. 2, 2022) focuses on six components that demonstrate the effects of regulating immunity, improving intestinal flora, and regulating gastrointestinal efficacy: echinacoside, verbascoside, ligustilide, naringin, neohesperidin, and isoflavonic acid. It establishes a quality control method for the indicative components of Jichuan Decoction.

[0004] Multi-index content determination has become a consensus in the quality control of classic prescriptions. Classic prescriptions are complex entities composed of multiple herbs and components. The principal herb, Cistanche deserticola, contains saponins, tannins, and polysaccharides; the assistant herb, Angelica sinensis, contains volatile oils, organic acids, and polysaccharides; the assistant herb, Achyranthes bidentata, contains saponins, alkaloids, and polysaccharides; the adjuvant herb, Alisma plantago-aquatica, contains terpenes and flavonoids; the adjuvant herb, Citrus aurantium, contains volatile oils, flavonoids, coumarins, and alkaloids; and the guiding herb, Cimicifuga foetida, contains phenylpropanoids, alkaloids, and saponins. When using the external standard method for multi-index content determination, all components are often required as reference standards. The difficulty in separation, the instability of monomers, the scarcity of available materials, and the high cost limit the scientific research and development of the classic prescription, Jichuan Decoction. The one-test-multiple-evaluation method utilizes the intrinsic functional and proportional relationships of the effective components of traditional Chinese medicine, using inexpensive and readily available common components as internal references to calculate the relative correction factors between the test components. It has the advantages of strong practicality, simple operation, cost-saving, and accurate and reliable test results. Summary of the Invention

[0005] Therefore, it is necessary to provide a method for detecting the content of multiple components in Jichuan Decoction in a single test. This method can accurately detect multiple components in Jichuan Decoction while saving costs, simplifying operation, and improving efficiency.

[0006] The technical solution of this application is as follows:

[0007] One aspect of the present invention provides a method for detecting the content of Jichuan Decoction composition using a single test and multiple evaluation methods, comprising the following steps:

[0008] The Jichuan Decoction composition was extracted using an extraction solvent to prepare a test solution;

[0009] The reference standard and naringin were dissolved using a dissolving agent to prepare a mixed solution of the reference standard and a solution of naringin; the reference standard included one or more of caffeic acid, echinacoside, ferulic acid, isoflavone, verbascoside, neohesperidin and citrus red.

[0010] The test solution, the mixed solution of reference standard and the naringin solution were injected separately and detected by ultra-high performance liquid chromatography.

[0011] Using naringin as an internal control, the content of the analyte in the test solution was calculated according to the following formula:

[0012] Ci = f * Cs * (Ai / As);

[0013] In the formula, f is the relative correction factor of the analyte, As is the peak area of ​​the internal reference naringin, Cs is the concentration of the internal reference naringin in the reference solution, Ai is the peak area of ​​the analyte in the test solution, and Ci is the concentration of the analyte in the test solution.

[0014] The raw materials for preparing the Jichuan Decoction composition, by mass parts, include the following components: 10-15 parts Angelica sinensis, 6-8 parts Achyranthes bidentata, 8-10 parts Cistanche deserticola, 4-6 parts Alisma plantago-aquatica, 1-3 parts Cimicifuga foetida, and 2-4 parts Citrus aurantium.

[0015] In one embodiment, the test ingredient includes one or more of caffeic acid, echinacoside, ferulic acid, isoflavone, verbascoside, naringin, neohesperidin, and citrus red.

[0016] In one embodiment, the conditions for ultra-high performance liquid chromatography detection include: mobile phase A is acetonitrile, mobile phase B is an aqueous phosphoric acid solution, and gradient elution is used;

[0017] The gradient elution procedure includes:

[0018] From 0 min to 12.5 min, the volume percentage of the mobile phase A increased from 12% to 13%.

[0019] Between 12.5 and 20 minutes, the volume percentage of the mobile phase A increased from 13% to 15%.

[0020] Between 20 and 30 minutes, the volume percentage of the mobile phase A increased from 15% to 21%.

[0021] Between 30 and 36 minutes, the volume percentage of the mobile phase A increased from 21% to 30%.

[0022] Between 36 and 43 minutes, the volume percentage of the mobile phase A increased from 30% to 70%.

[0023] Between 43 and 46 minutes, the volume percentage of the mobile phase A decreased from 70% to 12%.

[0024] The volume percentage of the mobile phase A is maintained at 12% for 46 to 50 minutes.

[0025] In one embodiment, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.1% to 0.2%.

[0026] In one embodiment, the ultra-high performance liquid chromatography detection also satisfies at least one of the following conditions:

[0027] (1) The column temperature is 28℃~32℃;

[0028] (2) The flow rate is 0.18 mL / min to 0.22 mL / min;

[0029] (3) The injection volume is 0.2 μL to 1.0 μL;

[0030] (4) Wavelength is 286nm~318nm.

[0031] In one embodiment, the method for detecting the content of the Jichuan Decoction composition using a single test and multiple evaluations further includes the following steps: using naringin as an internal reference, injecting mixed solutions of reference standards at different concentrations, performing ultra-high performance liquid chromatography (UHPLC) detection, recording the peak areas corresponding to the analyte in the mixed solutions of reference standards at different concentrations, and calculating the average value relative to the correction factor f according to the following formula. k / s :

[0032] f k / s =f k / f s =(A k / C k ) / (A s / C s );

[0033] Among them, A s C represents the peak area of ​​the internal reference. s A represents the mass concentration of the internal reference. k C represents the peak area of ​​the component to be measured. k This represents the mass concentration of the component to be measured.

[0034] In one embodiment, the extraction solvent includes an aqueous methanol solution or an aqueous acetonitrile solution;

[0035] Optionally, the methanol aqueous solution contains 50% to 70% methanol by volume; the acetonitrile aqueous solution contains 50% to 70% acetonitrile by volume.

[0036] In one embodiment, the dissolving agent comprises an aqueous methanol solution;

[0037] Optionally, the methanol aqueous solution contains 50% to 70% methanol by volume.

[0038] In one embodiment, the raw materials for preparing the Jichuan Decoction composition, by mass parts, include the following components: 14.92 parts Angelica sinensis, 7.46 parts Achyranthes bidentata, 9.33 parts Cistanche deserticola, 5.60 parts Alisma plantago-aquatica, 2.61 parts Cimicifuga foetida, and 3.73 parts Citrus aurantium.

[0039] Another aspect of the present invention provides the application of the above-described method for detecting the content of Jichuan Decoction composition in the quality control of Jichuan Decoction composition.

[0040] Compared with the prior art, this application has the following beneficial effects:

[0041] 1. This application uses a one-test-multiple-evaluation method with naringin as an internal reference to obtain the content of eight components in the Jichuan Decoction composition, namely caffeic acid, echinacoside, ferulic acid, isoflavonic acid, verbascoside, naringin, neohesperidin and tangerine. It is practical, simple to operate, fast, accurate and cost-effective.

[0042] 2. The method of this application is simple and easy to operate, and the test results are accurate. The method has good repeatability, stability and durability, providing a basis for establishing the quality evaluation of Jichuan Decoction composition.

[0043] 3. The index components measured in this application are all chemical substances closely related to the functional properties of the Jichuan Decoction composition, and have a unique biosynthetic pathway, possessing chemical specificity. At the same time, they can reflect the clinical efficacy of the Jichuan Decoction composition and should be regarded as important Q-marker components of the Jichuan Decoction composition. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0045] Figure 1 This is a chromatogram of the mixed reference standard and the test sample in the specificity study of the content determination of the Jichuan Decoction composition in Example 1.

[0046] Figure 2 The chromatogram for the absence of a negative result in the specificity study of the Jichuan Decoction composition in Example 1 is shown.

[0047] Figure 3 The chromatograms are obtained from different column temperatures during the durability study of the Jichuan Decoction composition in Example 1.

[0048] Figure 4 This is a chromatogram of different flow rates used in the durability study of the Jichuan Decoction composition content determination in Example 1.

[0049] Figure 5 This is a chromatogram of different injection volumes during the durability study of the Jichuan Decoction composition content determination in Example 1.

[0050] Figure 6 This is a chromatogram of different gradient elution procedures used in the durability study of the Jichuan Decoction composition content determination in Example 1.

[0051] Figure 7 This is a chromatogram of different mobile phases used in the durability study of the Jichuan Decoction composition in Example 1.

[0052] Figure 8 This is a chromatogram of different detection wavelengths used in the durability study of the Jichuan Decoction composition content determination in Example 1.

[0053] In each figure, peak 1: caffeic acid; peak 2: echinacoside; peak 3: ferulic acid; peak 4: isoflavonic acid; peak 5: verbascoside; peak 6: naringin; peak 7: neohesperidin; peak 8: tangerine. Detailed Implementation

[0054] The present application will be further described in detail below with reference to specific embodiments. The present application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0056] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0057] In this document, terms such as “further,” “even further,” and “especially” are used to describe the purpose and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0058] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0059] Unless otherwise specified, the percentage content mentioned in this application refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0060] Unless otherwise specified, all percentage concentrations mentioned in this application refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0061] Unless otherwise specified, the temperature parameters in this application allow for both isothermal processing and processing within a certain temperature range. The isothermal processing allows temperature fluctuations within the precision range of instrument control, such as ±5℃, ±2℃, ±1℃, ±0.5℃, ±0.4℃, ±0.3℃, ±0.2℃, and ±0.1℃. In this application, room temperature or ambient temperature refers to the absence of temperature control operations, generally meaning 4℃ to 35℃, and preferably 20±5℃.

[0062] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0063] The existing methods for detecting the composition of Jichuan Decoction mainly have the following problems: On the one hand, existing liquid chromatography analysis mainly targets the HPLC fingerprint of Jichuan Decoction and the simultaneous determination of the content of multiple components, which takes a long time and the quality control methods for indicator components are relatively insufficient; on the other hand, existing liquid chromatography analysis mainly uses the external standard method to determine the content of Jichuan Decoction, which consumes a large amount of reference standards and the detection cost is relatively expensive.

[0064] Based on this, the technical personnel of this application, addressing the shortcomings of existing multi-index content determination methods, have discovered through extensive research that a single-measurement-multiple-evaluation method (i.e., QAMS method) can be used to establish a UPLC method for determining the content of caffeic acid, echinacoside, ferulic acid, isoflavone, verbascoside, naringin, neohesperidin, and tangerine in the Jichuan Decoction composition, which meets the requirements of methodological validation. Multi-index content determination has become a consensus in the quality control of classic prescriptions. The method of this application is a multi-index simultaneous quality control method. By determining the content of a representative component, the content of multiple analytes is calculated based on a relative correction factor, and the calculated values ​​are controlled to meet the quantitative methodological requirements, overcoming the problems of high consumption of reference standards and high operational difficulty in the implementation of multi-index content determination methods. This method has the characteristics of saving standard standards and providing accurate and reliable results, further promoting the popularization and application of quality evaluation research on the Jichuan Decoction composition. This invention provides a cost-effective, simplified, and efficient method for detecting eight components in the Jichuan Decoction composition: caffeic acid, echinacoside, ferulic acid, isoflavonic acid, verbascoside, naringin, neohesperidin, and tangerine.

[0065] One aspect of the present invention provides a method for detecting the content of Jichuan Decoction composition using a single test and multiple evaluation methods, comprising the following steps:

[0066] The Jichuan Decoction composition was extracted using an extraction solvent to prepare a test solution;

[0067] The reference standard and naringin were dissolved using a dissolving agent to prepare a mixed solution of the reference standard and a solution of naringin; the reference standard included one or more of caffeic acid, echinacoside, ferulic acid, isoflavone, verbascoside, neohesperidin and citrus red.

[0068] The test solution, the mixed solution of reference standard and the naringin solution were injected separately and detected by ultra-high performance liquid chromatography.

[0069] Using naringin as an internal control, the content of the analyte in the test solution was calculated according to the following formula:

[0070] Ci = f * Cs * (Ai / As);

[0071] In the formula, f is the relative correction factor of the analyte, As is the peak area of ​​the internal reference naringin, Cs is the concentration of the internal reference naringin in the reference solution, Ai is the peak area of ​​the analyte in the test solution, and Ci is the concentration of the analyte in the test solution.

[0072] The raw materials for preparing the Jichuan Decoction composition, by mass parts, include the following components: 10-15 parts Angelica sinensis, 6-8 parts Achyranthes bidentata, 8-10 parts Cistanche deserticola, 4-6 parts Alisma plantago-aquatica, 1-3 parts Cimicifuga foetida, and 2-4 parts Citrus aurantium.

[0073] In some of these examples, the analyte includes one or more of caffeic acid, echinacoside, ferulic acid, isoflavone, verbascoside, naringin, neohesperidin, and citrus.

[0074] In some of these examples, the conditions for ultra-high performance liquid chromatography detection include: mobile phase A being acetonitrile, mobile phase B being an aqueous phosphoric acid solution, and gradient elution.

[0075] In some of these examples, the gradient elution procedure includes:

[0076] From 0 min to 12.5 min, the volume percentage of the mobile phase A increased from 12% to 13%.

[0077] Between 12.5 and 20 minutes, the volume percentage of the mobile phase A increased from 13% to 15%.

[0078] Between 20 and 30 minutes, the volume percentage of the mobile phase A increased from 15% to 21%.

[0079] Between 30 and 36 minutes, the volume percentage of the mobile phase A increased from 21% to 30%.

[0080] Between 36 and 43 minutes, the volume percentage of the mobile phase A increased from 30% to 70%.

[0081] Between 43 and 46 minutes, the volume percentage of the mobile phase A decreased from 70% to 12%.

[0082] The volume percentage of the mobile phase A is maintained at 12% for 46 to 50 minutes.

[0083] In some of these examples, the volume percentage of phosphoric acid in the aqueous phosphoric acid solution is 0.1% to 0.2%. It will be understood that the volume percentage of phosphoric acid in the aqueous phosphoric acid solution includes, but is not limited to, 0.1%, 0.12%, 0.14%, 0.16%, 0.18%, and 0.2%.

[0084] In some examples, the conditions for ultra-high performance liquid chromatography (UHPLC) detection include a column temperature of 28°C to 32°C. It is understood that the column temperature for UHPLC detection includes, but is not limited to, 28°C, 29°C, 30°C, 31°C, and 32°C.

[0085] In some examples, the conditions for ultra-high performance liquid chromatography (UHPLC) detection include a flow rate of 0.18 mL / min to 0.22 mL / min. It is understood that the flow rates for UHPLC detection include, but are not limited to, 0.18 mL / min, 0.19 mL / min, 0.20 mL / min, 0.21 mL / min, and 0.22 mL / min.

[0086] In some examples, the conditions for ultra-high performance liquid chromatography (UHPLC) detection include an injection volume of 0.2 μL to 1.0 μL. It is understood that the injection volume for UHPLC detection includes, but is not limited to, 0.2 μL, 0.4 μL, 0.6 μL, 0.8 μL, and 1.0 μL.

[0087] In some examples, the ultra-high performance liquid chromatography (UHPLC) detection conditions include wavelengths of 286 nm to 318 nm. It is understood that the wavelengths for UHPLC detection include, but are not limited to, 286 nm, 290 nm, 294 nm, 298 nm, 310 nm, 312 nm, and 318 nm.

[0088] In some examples, the method for detecting the content of the Jichuan Decoction composition using a single test and multiple evaluations further includes the following steps: using naringin as an internal reference, injecting mixed solutions of reference standards at different concentrations, performing ultra-high performance liquid chromatography (UHPLC) detection, recording the peak areas corresponding to the analytes in the mixed solutions of reference standards at different concentrations, and calculating the average value relative to the correction factor f according to the following formula. k / s :

[0089] f k / s =f k / f s =(A k / C k ) / (A s / C s );

[0090] Among them, A s C represents the peak area of ​​the internal reference. s A represents the mass concentration of the internal reference. k C represents the peak area of ​​the component to be measured. k This represents the mass concentration of the component to be measured.

[0091] In some examples, the extraction solvent comprises an aqueous methanol solution or an aqueous acetonitrile solution. Further, the aqueous methanol solution contains 50% to 70% methanol by volume; the aqueous acetonitrile solution contains 50% to 70% acetonitrile by volume.

[0092] In some of these examples, the extraction is performed using ultrasound.

[0093] In some examples, during the extraction step, the mass-to-volume ratio of the Jichuan Decoction composition to the extraction solvent is 0.2 g: (20–50) mL. It is understood that the mass-to-volume ratio of the Jichuan Decoction composition to the extraction solvent during the extraction step includes, but is not limited to, 0.2 g: 20 mL, 0.2 g: 30 mL, 0.2 g: 40 mL, and 0.2 g: 50 mL.

[0094] In some examples, the dissolving agent comprises an aqueous methanol solution; further, the methanol solution contains 50% to 70% methanol by volume. It is understood that the volume percentage of methanol in the dissolving agent includes, but is not limited to, 50%, 55%, 60%, 65%, and 70%.

[0095] In some of these examples, the raw materials for preparing the Jichuan Decoction composition, by mass parts, include the following components: 14.92 parts Angelica sinensis, 7.46 parts Achyranthes bidentata, 9.33 parts Cistanche deserticola, 5.60 parts Alisma plantago-aquatica, 2.61 parts Cimicifuga foetida, and 3.73 parts Citrus aurantium.

[0096] In one specific example, the method for detecting the content of the Jichuan Decoction composition using a single test and multiple evaluation methods includes the following steps:

[0097] Step 1: Chromatographic conditions: Column: Octadecylsilane-bonded silica gel as packing material; Mobile phase: Acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B); Elution gradient (0–12.5 min, 12%–13% A; 12.5–20 min, 13%–15% A; 20–30 min, 15%–21% A; 30–36 min, 21%–30% A; 36–43 min, 30%–70% A; 43–46 min, 70%–12% A; 46–50 min, 12% A); Detection wavelength: 310 nm; Column temperature: 28–32℃; Flow rate: 0.18–0.22 mL / min; Injection volume: 0.2–1.0 μL; The theoretical plate number, calculated based on the naringin peak, should be no less than 5000.

[0098] Step 2: Preparation of mixed reference solution. Accurately weigh appropriate amounts of caffeic acid, echinacoside, ferulic acid, isoflavonic acid, verbascoside, naringin, neohesperidin, and citrus red pigment, place them in a volumetric flask, mix them evenly, and then dissolve them in 50% methanol aqueous solution to obtain the first mixed reference solution.

[0099] Step 3: Preparation of the test solution. Accurately weigh an appropriate amount of the Jichuan Decoction composition, accurately add a methanol aqueous solution with a volume concentration of 50% to 70%, perform ultrasonic extraction, filter, and collect the filtrate to obtain the test solution.

[0100] Step 4: Calculation of relative correction factors. Take the first mixed reference solution prepared in Step 2, dilute it to prepare a series of second mixed reference solutions, inject them into the high-performance liquid chromatograph, and determine them under the chromatographic conditions of Step 1. Record the peak area of ​​each component. Using naringin as an internal reference, calculate the relative correction factors (f) of the other 7 components. k / s ), f k / s =f k / f s =(A k / C k ) / (A s / C s ), where A s C represents the peak area of ​​the internal reference material. s A represents the mass concentration of the internal reference substance. k C represents the peak area of ​​the component to be measured. k Given the mass concentration of the analyte, calculate the relative correction factor f for caffeic acid, echinacoside, ferulic acid, isoferrulic acid, verbascoside, neohesperidin, citrus rubigin, and the internal reference naringin.

[0101] Step 5: Calculation of the content of the target compound. Inject the test solution prepared in Step 3 into a high-performance liquid chromatograph for determination and obtain a chromatogram. Locate the chromatographic peak of the analyte using the relative retention value used in Step 4. Then, calculate the contents of caffeic acid, echinacoside, ferulic acid, isoflavone, verbascoside, neohesperidin, and tangerine according to the relative correction factor f of each component calculated in Step 4.

[0102] Another aspect of the present invention provides the application of the above-described method for detecting the content of Jichuan Decoction composition in the quality control of Jichuan Decoction composition.

[0103] The present application will be further described in detail below with reference to specific embodiments. Experimental parameters not specified in the following embodiments should first be referred to the guidelines given in this application, and may also be referred to experimental manuals or other experimental methods known in the art, or the experimental conditions recommended by the manufacturer. It is understood that the instruments and materials used in the following embodiments are relatively specific, and may not be limited to these in other embodiments.

[0104] Example 1

[0105] This embodiment provides a method for detecting the content of Jichuan Decoction composition using a single test and multiple evaluation methods, as detailed below:

[0106] 1. Instruments and reagents

[0107] 1.1 Instruments

[0108] Waters H-class high-performance liquid chromatograph (Waters Corporation, USA); Agilent 1290 high-performance liquid chromatograph (Agilent Technologies, USA); ME204E part per ten thousand balance and XP26 part per million balance (METTLERTDLEDO, Switzerland); KQ-500DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Milli-Q Direct ultrapure water system (Merck, Germany).

[0109] 1.2 Reagents and Test Chemicals

[0110] Reagents: Methanol and acetonitrile were of chromatographic grade (Merck, Germany); phosphoric acid was of chromatographic grade (Tianjin Kemeio Chemical Reagent Co., Ltd.); water was ultrapure water; and other reagents were of analytical grade.

[0111] Test drug: Nine batches of Jichuan Decoction composition were prepared in the laboratory. The preparation method was as follows: 14.92g of Angelica sinensis, 7.46g of Achyranthes bidentata, 9.33g of Cistanche deserticola (processed with wine), 5.60g of Alisma plantago-aquatica, 2.61g of Cimicifuga foetida, and 3.73g of Citrus aurantium, totaling 43.65g, were added to 300mL of water, soaked for 30min, decocted to 150mL, filtered, the filtrate was concentrated and spray-dried to obtain Jichuan Decoction spray-dried powder.

[0112] Caffeic acid (batch number 110885-201703, purity 99.7%), echinacoside (batch number 111670-201907, purity 91.8%), ferulic acid (batch number 110773-202316, purity 99.3%), isoferrulic acid (batch number 111698-201904, purity 99.3%), verbascoside (batch number 111530-202315, purity 97.6%), naringin (batch number 110722-202417, purity 96.1%), neohesperidin (batch number 111857-202305, purity 99.6%), and citrus red (batch number 112054-202102, purity 99.7%) were all purchased from the National Institutes for Food and Drug Control.

[0113] 2. Experimental Methods

[0114] 2.1 Chromatographic conditions

[0115] Chromatographic column: YMC Triart C18 Column (120mm×2.1mm, 1.9μm); Mobile phase: Acetonitrile as mobile phase A, and 0.1% phosphoric acid aqueous solution as mobile phase B, with gradient elution as specified in Table 1; Detection wavelength: 310nm; Column temperature: 30℃; Flow rate: 0.2mL / min; Injection volume: 1μL.

[0116] Table 1 Gradient elution conditions of the mobile phase

[0117]

[0118] 2.2 Preparation of mixed reference solution

[0119] Accurately weigh appropriate amounts of caffeic acid, echinacoside, ferulic acid, isoflavone, verbascoside, naringin, neohesperidin, and hesperidin reference standards, and add them to 50% methanol aqueous solution to prepare mixed reference standard solutions with mass concentrations of 99.1018 μg / mL, 283.1112 μg / mL, 408.8322 μg / mL, 101.9811 μg / mL, 101.1507 μg / mL, 119.9520 μg / mL, 375.1220 μg / mL, 302.2860 μg / mL, and 118.0448 μg / mL, respectively.

[0120] 2.3 Preparation of the test solution

[0121] Take an appropriate amount of Jichuan Decoction composition, about 0.2g, accurately weigh it, add 25mL of 50% methanol aqueous solution, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol aqueous solution, shake well, filter it, and take the filtrate to obtain the test solution.

[0122] 3. Methodological Examination

[0123] 3.1 Specificity Examination

[0124] Accurately pipette appropriate amounts of the reference solution, test solution, and negative sample solution, and inject them according to the chromatographic conditions described in section "2.1". The results are shown in the table below. Figures 1-2 The results showed that the chromatographic peaks of each component had good resolution, with theoretical plate numbers not less than 5000, and retention times consistent with those of the reference standard peaks, indicating that the method has good specificity.

[0125] 3.2 Examination of Linear Relationships

[0126] Take 0.1 mL, 0.3 mL, 0.5 mL, 2.0 mL, and 3.0 mL of the mixed reference solution prepared under section "2.2" respectively, and place them in 5 mL volumetric flasks. Add 50% methanol aqueous solution to the mark and shake well to prepare a series of mixed reference solutions of different concentrations. Inject and determine the solutions according to the chromatographic conditions under section "2.1" and record the chromatograms. Perform linear regression with the mass concentration of the reference solution as the abscissa (x, μg / mL) and the peak area as the ordinate (y). The results are shown in Table 2. The linear relationship of each component is good within its respective concentration range.

[0127] Table 2 Results of linear relationship examination (n=6)

[0128]

[0129] 3.3 Precision Examination

[0130] Accurately pipette an appropriate amount of the mixed reference solution from section "2.2" and inject it according to the chromatographic conditions from section "2.1". Inject six times consecutively and record the peak areas. The results are shown in Table 3. The RSDs of the peak areas of caffeic acid, echinacoside, ferulic acid, isoflavone, verbascoside, naringin, neohesperidin, and tangerine were all less than 3%, indicating good instrument precision.

[0131] Table 3. Precision test results

[0132]

[0133] 3.4 Repeatability Test

[0134] Take an appropriate amount of the same Jichuan Decoction composition and prepare 6 test solutions in parallel according to the method in section "2.3". Inject and determine the solutions according to the chromatographic conditions in section "2.1". The results are shown in Table 4. The RSD of the peak areas of caffeic acid, echinacoside, ferulic acid, isoflavonic acid, verbascoside, naringin, neohesperidin, and citrus red pigment were all less than 3%, indicating that the method has good repeatability.

[0135] Table 4 Results of Repeatability Testing

[0136]

[0137] 3.5 Stability Test

[0138] Accurately pipette the test solution from section "2.3" and inject it at 0, 2, 4, 8, 12, and 24 hours after preparation, according to the chromatographic conditions from section "2.1". The results are shown in Table 5. The RSDs of the peak areas of caffeic acid, echinacoside, ferulic acid, isoflavone, verbascoside, naringin, neohesperidin, and tangerine were all less than 3%, indicating that the sample was stable within 24 hours.

[0139] Table 5. Results of the stability test

[0140]

[0141] 3.6 Recovery Test

[0142] Nine portions of the same known concentration of Jichuan Decoction composition, each approximately 0.1 g, were accurately weighed and placed in stoppered conical flasks, divided into three groups. Appropriate amounts of the mixed reference solution were accurately added to each group according to high, medium, and low concentrations. The test solutions were prepared according to the method described in section "2.3," and the samples were injected and analyzed under the chromatographic conditions described in section "2.1." The recoveries were calculated, and the results are shown in Table 6. The recoveries of caffeic acid, echinacoside, ferulic acid, isoferrulic acid, verbascoside, naringin, neohesperidin, and citrus red were within 99.69%–105.94%, all within the recovery limits specified in the pharmacopoeia, with RSDs less than 3%, indicating good accuracy of the method.

[0143] Table 6. Recovery Rate Results

[0144]

[0145]

[0146]

[0147] 4. Calculation of relative correction factor

[0148] Under the chromatographic conditions defined in this invention, based on the linearity study results under section "3.2", using naringin as an internal reference, the relative correction factors (f) of the other seven components were calculated. k / s ), f k / s =f k / f s =(A k / C k ) / (A s / C s ), where A s C represents the peak area of ​​the internal reference material. s A represents the mass concentration of the internal reference substance. k C represents the peak area of ​​the component to be measured. k Given the mass concentration of the components to be measured, the relative correction factors f for caffeic acid, echinacoside, ferulic acid, isoflavonic acid, verbascoside, neohesperidin, and citrus red were calculated, and the results are shown in Table 7.

[0149] Table 7. Relative correction factors for each component (n=6)

[0150]

[0151] 5. System durability assessment

[0152] 5.1 Investigation using different instruments and chromatographic columns

[0153] Using naringin as an internal reference, the relative correction factors of the other seven components were investigated using two high-performance liquid chromatography (HPLC) systems (Waters H-class and Agilent 1290) and two columns (Waters ACQUITY UPLC HSS T3 (150 mm × 2.1 mm, 1.8 μm) and YMCTriart C18 (150 mm × 2.1 mm, 1.9 μm). The results are shown in Table 8. The RSD of each component was less than 3%, indicating that the replacement of instruments and columns had no significant effect on the relative correction factors of each component.

[0154] Table 8. Effects of different instruments and chromatographic columns on relative correction factors.

[0155]

[0156] 5.2 Investigation at different column temperatures

[0157] Using naringin as an internal reference, the effects of column temperatures of 28℃, 30℃, and 32℃ on the relative correction factors of each component were investigated using a Waters H-class high-performance liquid chromatograph and a YMC Triart C18 column. The results are shown in Table 9. Figure 3 The RSD of each component was less than 3%, indicating that the fluctuation of column temperature had no significant effect on the relative correction factor of each component.

[0158] Table 9. Effect of different column temperatures on relative correction factors

[0159]

[0160] 5.3 Investigation of different flow velocities

[0161] Using naringin as an internal control, a Waters H-class high-performance liquid chromatograph and a YMC Triart C18 column were used to investigate the flow rate at 0.18 mL / min. -1 0.20 mL·min -1 0.22 mL·min -1 The effects of the relative correction factors on each component are shown in Table 10 and... Figure 4 The RSD of each component was less than 3%, indicating that different flow rates had no significant effect on the relative correction factors of each component.

[0162] Table 10. Effect of different flow velocities on relative correction factor

[0163]

[0164] 5.4 Investigation of different injection volumes

[0165] Using naringin as an internal control, the effects of injection volumes of 0.2 μL, 0.5 μL, 0.8 μL, and 1.0 μL on the relative correction factors of each component were investigated using a Waters H-class high-performance liquid chromatograph and a YMC Triart C18 column. The results are shown in Table 11. Figure 5 The RSD of each component was less than 3%, indicating that different injection volumes had no significant effect on the relative correction factors of each component.

[0166] Table 11 Effect of different injection volumes on relative correction factors

[0167]

[0168] 6. Localization of the chromatographic peaks of the analyte

[0169] In QAMS applications, commonly used chromatographic peak localization methods include relative retention value method, retention time difference method, time correction method, and reference extract method. The retention time difference method calculation formula is: Δti / s = ti - ts. The relative retention value method calculation formula is: ti / s = ti / ts. This experiment used relative retention time to locate the chromatographic peaks of the analytes. Naringin was used as an internal reference. The relative retention times of the other seven components were investigated using two high-performance liquid chromatography (HPLC) systems: Waters H-class and Agilent 1290, and two columns: Waters ACQUITY UPLC HSS T3 (100mm × 2.1mm, 1.8μm) and YMC Triart C18 (150mm × 2.1mm, 1.9μm). The results are shown in Table 12. The RSD of each component using the relative retention value method was less than 3%, indicating that the relative retention value method is feasible for analyte localization.

[0170] Table 12 Relative retention times of each component

[0171]

[0172] 7. Comparison of results from the Quality Assurance System (QAMS) and the External Standard Method (ESM)

[0173] Take appropriate amounts of samples from 9 batches of Jichuan Decoction composition, prepare test solutions according to the method in section "2.3", and inject and determine the content of each component under the chromatographic conditions in section "2.1". The content of each component is determined by the external standard method. The steps of the one-test-multiple-evaluation method of this application are as follows: take appropriate amounts of samples from 9 batches of Jichuan Decoction composition, prepare test solutions according to the method in section "2.3", inject and determine the content of each component under the chromatographic conditions in section "2.1", and calculate the content of each component by substituting the relative correction factors of the 7 components in section "4" into the formula.

[0174] The results are shown in Tables 13-1 and 13-2. The content results calculated by the two methods are basically consistent. The relative standard deviation (RSD) and relative error (RE) of caffeic acid, echinacoside, ferulic acid, isoflavone, verbascoside, neohesperidin, and citrus red are all less than 3%, indicating that there is no significant difference between the results calculated by the two methods.

[0175] Table 13-1 Comparison of results from external standard method and one-test-multiple-evaluation method

[0176]

[0177] Table 13-2 Comparison of results from external standard method and one-test-multiple-evaluation method

[0178]

[0179] In addition, this application also examines the conditions for the one-test-multiple-evaluation content detection method of Jichuan Decoction composition, as follows:

[0180] 1. Selection of gradient elution program

[0181] As shown in Tables 14-1 and 14-2, the elution gradient was optimized through comparative experiments with various gradient elution conditions. The results ( Figure 6 As can be seen, gradient elution program 1 resulted in a lower chromatographic peak response, while gradient elution program 2 resulted in good peak resolution and a higher response. Therefore, gradient elution program 2 is the optimal gradient elution program.

[0182] Table 14-1 Gradient elution program 1

[0183]

[0184] Table 14-2 Gradient elution program 2

[0185]

[0186] 2. Selection of mobile phase

[0187] Acetonitrile-0.1% phosphoric acid, methanol-0.1% phosphoric acid, acetonitrile-0.2% phosphoric acid, and methanol-0.2% phosphoric acid were selected as mobile phases for investigation. Figure 7 As shown in the figure, during the experiment, it was found that acetonitrile had better separation effect and higher chromatographic peak response when using acetonitrile and methanol. Methanol gradient elution resulted in significant baseline noise and obvious solvent peaks. Due to the high polarity of water, fewer peak components were emitted, and the peaks clustered together. Therefore, acid was considered. Under 0.1% phosphoric acid conditions, the solvent pH was closer to the pKa value of the analyte, resulting in better separation and sharper, symmetrical peaks. Therefore, acetonitrile-0.1% phosphoric acid elution was used.

[0188] 3. Selection of detection wavelength

[0189] Full-wavelength scanning revealed that the maximum absorption wavelengths of ferulic acid and isoferrulic acid in Angelica sinensis and Cimicifuga foetida are approximately 270 nm to 320 nm; the maximum absorption wavelengths of naringin, neohesperidin, and citrus jujuba var. spinosa in Citrus aurantium are approximately 280 nm to 320 nm; and the maximum absorption wavelength of echinacoside and verbascoside in Cistanche deserticola (processed with wine) is 330 nm. The maximum absorption wavelength of β-ecdysterone in Achyranthes bidentata is 270 nm; the maximum absorption wavelengths of alisol B and 23-acetylalisol B in Alisma plantago-aquatica are 208 nm; and the maximum absorption wavelengths of alisol C and 23-acetylalisol C are 246 nm. The maximum absorption wavelengths of these two medicinal components, Achyranthes bidentata and Alisma plantago-aquatica, differ significantly from those of other medicinal components; therefore, quantitative control of Achyranthes bidentata and Alisma plantago-aquatica was not performed in this invention. Considering the overall stability of the chromatogram baseline, 286 nm, 310 nm, and 318 nm were selected as detection wavelengths for investigation. Figure 8 As shown in the experiment, it was found that a wavelength of 310 nm could detect caffeic acid, echinacoside, ferulic acid, isoflavone, verbascoside, naringin, neohesperidin, and tangerine, and the baseline was stable at the corresponding wavelength, indicating good separation of each component. Therefore, 310 nm was chosen as the detection wavelength.

[0190] 4. Preparation of the test sample

[0191] The preparation of the test sample was determined by examining the extraction conditions. The peak areas of ultrasonic extraction and reflux extraction were not significantly different, so ultrasonic extraction with 50% methanol and 50% acetonitrile was used in the experiments. As shown in Table 15, the experiment found that ultrasonic extraction with 20 mL of 50% methanol for 30 minutes yielded better results, with relatively higher dissolution rates of each component. Therefore, ultrasonic extraction with 20 mL of 50% methanol for 30 minutes was used.

[0192] Table 15 Comparison of extraction effects of 50% methanol and 50% acetonitrile (n=3)

[0193]

[0194] 5. Selection of internal reference materials

[0195] Under the chromatographic conditions specified in this application, based on the linearity study results under section "3.2", the relative correction factors (f) for the other 7 components were calculated using 8 components as internal references. k / s ), f k / s =f k / f s =(A k / C k ) / (A s / C s ), where A s C represents the peak area of ​​the internal reference material. s A represents the mass concentration of the internal reference substance. k C represents the peak area of ​​the component to be measured.k The values ​​represent the mass concentrations of the analytes. The results (see Tables 16-1, 16-2, 16-3, 16-4, 16-5, 16-6, 16-7, and 16-8) show that, using the eight analytes as internal controls, the RSD of the relative correction factor for each analyte was less than 5%. Considering the overall chromatogram, naringin showed a good peak shape, high response, and moderate retention time; therefore, naringin was chosen as the internal control.

[0196] Table 16-1 Relative correction factors for each component using caffeic acid as an internal reference (n=6)

[0197]

[0198] Table 16-2 Relative correction factors for each component using echinacoside as an internal reference (n=6)

[0199]

[0200] Table 16-3 Relative correction factors for each component using ferulic acid as an internal reference (n=6)

[0201]

[0202] Table 16-4 Relative correction factors for each component using isoflavonic acid as an internal control (n=6)

[0203]

[0204] Table 16-5 Relative correction factors for each component using verbascoside as an internal reference (n=6)

[0205]

[0206] Table 16-6 Relative correction factors for each component using naringin as an internal reference (n=6)

[0207]

[0208] Table 16-7 Relative correction factors for each component using neohesperidin as an internal reference (n=6)

[0209]

[0210] Table 16-8 Relative correction factors for each component using hesperidin as an internal reference (n=6)

[0211]

[0212] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0213] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for determining the content of a Jichuan decoction composition by quantitative analysis of multiple components, characterized in that, The method comprises the following steps: extracting the Jichuan decoction composition to prepare a test sample solution, wherein the extraction solvent comprises a methanol aqueous solution or an acetonitrile aqueous solution; dissolving the control sample and naringin to prepare a control sample mixed solution and a naringin solution, wherein the control sample comprises caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, neohesperidin, and red orange; respectively injecting the test sample solution, the control sample mixed solution, and the naringin solution to perform ultra-high performance liquid chromatography detection; taking naringin as an internal reference, and calculating the content of the to-be-tested component in the test sample solution according to the following formula: Ci = f * Cs * (Ai / As); in the formula, f is a relative correction factor of the to-be-tested component, As is the peak area of the internal reference naringin, Cs is the concentration of the internal reference naringin in the control sample mixed solution, Ai is the peak area of the to-be-tested component in the test sample solution, and Ci is the concentration of the to-be-tested component in the test sample solution; the conditions of the ultra-high performance liquid chromatography detection comprise: the mobile phase A is acetonitrile, the mobile phase B is a phosphoric acid aqueous solution, and gradient elution is adopted; the program of the gradient elution comprises: 0 min~12.5 min, the volume percentage of the mobile phase A is increased from 12% to 13%; 12.5 min~20 min, the volume percentage of the mobile phase A is increased from 13% to 15%; 20 min~30 min, the volume percentage of the mobile phase A is increased from 15% to 21%; 30 min~36 min, the volume percentage of the mobile phase A is increased from 21% to 30%; 36 min~43 min, the volume percentage of the mobile phase A is increased from 30% to 70%; 43 min~46 min, the volume percentage of the mobile phase A is decreased from 70% to 12%; 46 min~50 min, the volume percentage of the mobile phase A is maintained at 12%; the chromatographic column is filled with octadecylsilane-bonded silica gel; the preparation raw material of the Jichuan decoction composition comprises, in mass fraction, the following components: 10-15 parts of angelica, 6-8 parts of radix cyathulae, 8-10 parts of cistanche, 4-6 parts of alisma, 1-3 parts of radix clematidis, and 2-4 parts of fructus aurantii.

2. The method according to claim 1, wherein the Jichuan decoction composition is a ratin method. The to-be-tested component further comprises naringin.

3. The method according to claim 1, wherein the Jichuan decoction composition is a ratin method. In the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.1%-0.2%.

4. The method according to claim 1, wherein the Jichuan decoction composition is a quality control method for multiple assays of one sample. The ultra-high performance liquid chromatography detection further satisfies at least one of the following conditions: (1) the column temperature is 28°C-32°C; (2) the flow rate is 0.18 mL / min-0.22 mL / min; (3) the injection amount is 0.2 μL-1.0 μL; (4) the wavelength is 286 nm-318 nm.

5. The method for detecting the content of Jichuan Decoction composition by one test and multiple evaluations according to any one of claims 1 to 4, characterized in that, The method further comprises the following steps: With naringin as the internal reference, the sample of different concentrations of the control product mixed solution was detected by ultra-high performance liquid chromatography, the peak area of the test component in the control product mixed solution of different concentrations was recorded, and the relative correction factor f was calculated according to the following formula and the average value was taken k / s : f k / s =f k / f s =(A k / C k ) / (A s / C s ); Wherein, A s is the peak area of the internal reference, C s is the mass concentration of the internal reference, A k is the peak area of the component to be measured, C k is the mass concentration of the component to be measured.

6. The method according to any one of claims 1 to 4, wherein the Jichuan Decoction is prepared according to the method of claim 5. In the methanol aqueous solution, the volume percentage of methanol is 50%-70%; in the acetonitrile aqueous solution, the volume percentage of acetonitrile is 50%-70%.

7. The method according to any one of claims 1 to 4, wherein the Jichuan decoction composition is a quality control method for the determination of the content of one or more components in the Jichuan decoction composition. The dissolving reagent comprises a methanol aqueous solution.

8. The method according to any one of claims 1-4, wherein the Jichuan decoction composition is a quality control method for the determination of the contents of multiple components by one test. In the methanol aqueous solution, the volume percentage of methanol is 50%-70%.

9. The method according to any one of claims 1-4, wherein the Jichuan decoction composition is a quality control method for the determination of the contents of multiple components by one test. The preparation raw materials of the Jichuan decoction composition comprise the following components in mass fraction: 14.92 parts of angelica, 7.46 parts of radix cyathulae, 9.33 parts of cistanche, 5.60 parts of alisma, 2.61 parts of cimicifuga and 3.73 parts of fructus aurantii.

10. Application of the QAMS content detection method of the Jichuan decoction composition in the quality control of the Jichuan decoction composition.

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