Method for detecting content of Jichuan decoction composition by quantitative analysis of multi-components by single marker

Abstract

The invention relates to a Jichuan decoction composition QAMS (quantitative analysis of multi-components by single marker) content detection method which comprises the following steps: extracting a Jichuan decoction composition by using an extraction solvent to prepare a test solution; dissolving the reference substance and the naringin by using a dissolving reagent to prepare a reference substance mixed solution and a naringin solution; respectively injecting the test solution, the reference substance mixed solution and the naringin solution, and carrying out ultra-high performance liquid chromatography detection; and by taking naringin as an internal reference substance, calculating the content of the component to be detected in the test solution according to a formula. According to the method, the content of eight components including caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, naringin, neohesperidin and chrysoidine in the Jichuan decoction composition is obtained by adopting a quantitative analysis of multi-components by single marker with naringin as an internal reference substance, the practicability is high, the operation is simple, the test is rapid and accurate, the cost is saved, the operation is simple and convenient, and the method is suitable for industrial production. And a basis is provided for establishing quality evaluation of the Jichuan decoction composition.

Description

Technical Field

[0001] The present application relates to the technical field of detection of traditional Chinese medicine preparations, and in particular to a one-test-multiple-evaluation content detection method for a Jichuan decoction composition. Background Art

[0002] Jichuan Decoction comes from the Complete Works of Jingyue written by Zhang Jingyue in the Ming Dynasty. It is composed of 6 herbs: Angelica sinensis, Achyranthes bidentata, Cistanche deserticola, Alisma orientalis, Cimicifuga heracleifolia, and Citrus aurantium. It has the effects of warming the kidney and benefiting the essence, moistening the intestines and relieving constipation. The book records that "for any disease involving deficiency and constipation, nitrate and yellow attack agents must not be used; if the bowel movement is inevitable, this is the main treatment. This is a tonic agent, the best and most wonderful." As an empirical prescription for the treatment of constipation, Jichuan Decoction has pharmacological effects such as promoting gastrointestinal motility, improving colon motility, regulating gastrointestinal hormones, and maintaining intestinal flora homeostasis. It is mainly used clinically for functional constipation in the elderly, slow-transmission constipation, functional outlet constipation, constipation in Parkinson's patients, constipation in stroke recovery patients, and constipation caused by opioids.

[0003] Jichuan Decoction is an ancient classic prescription and belongs to the "Catalogue of Ancient Classic Prescriptions (First Batch)" issued by the state. The research and development of classic prescriptions and corresponding Chinese medicine compound preparations are mainly divided into two stages: the development of "benchmark samples of ancient classic prescription Chinese medicine compound preparations (hereinafter referred to as material benchmarks, also known as standard decoctions)" and the development of "ancient classic prescription Chinese medicine compound preparations". Most of the research on Jichuan Decoction is clinical research and pharmacological research, and the quality control research mainly focuses on the content of index components and fingerprint analysis. The document "Simultaneous Determination of Four Index Components in Jichuan Decoction and Study on Its Material Benchmark Process" (Journal of Tianjin University of Traditional Chinese Medicine, Issue 4, 2023) established the HPLC fingerprint of Jichuan Decoction, but only simultaneously determined the contents of four index components, naringin, neohesperidin, β-ecdysterone, and echinacoside, and established its material benchmark preparation process. The document "Simultaneous Determination of Multiple Components in the Classic Recipe Jichuan Jian by HPLC" (Journal of Tianjin University of Traditional Chinese Medicine, Issue 2, 2022) only focuses on the six components that reflect the effects of regulating immunity, improving intestinal flora, and regulating gastrointestinal efficacy, namely, echinacoside, verbascoside, ligustilide, naringin, neohesperidin, and isoferulic acid, to establish a quality control method for the index components of Jichuan Jian.

[0004] Multi-index content determination has become a consensus for quality control of classic prescriptions. Classic prescriptions are complex with multiple medicinal flavors and components. The main drug Cistanche contains saponins, tannins, polysaccharides and other components; the auxiliary drug Angelica contains volatile oils, organic acids, polysaccharides and other components; the auxiliary drug Achyranthes contains saponins, alkaloids, polysaccharides and other components; the adjuvant drug Alisma contains terpenes, flavonoids and other components; the adjuvant drug Citrus aurantium contains volatile oils, flavonoids, coumarins, alkaloids and other components; the auxiliary drug Cimicifuga contains phenylpropanoids, alkaloids, saponins and other components. When using the external standard method for multi-index content determination, all components are often required as reference substances. Factors such as the difficulty of separation, unstable monomers, difficulty in supply, and high cost have limited the scientific research and development of the classic prescription Jichuanjian. The one-test-multiple-evaluation method utilizes the intrinsic functional relationship and proportional relationship of the effective ingredients of traditional Chinese medicine, takes cheap and easily available common ingredients as internal reference substances, and calculates the relative correction factors between the ingredients to be tested, achieving the advantages of strong practicality, simple operation, cost savings, and accurate and reliable test results. Summary of the invention

[0005] Based on this, it is necessary to provide a one-test, multi-evaluation content detection method for the Jichuan Jian combination, which can achieve accurate detection of multiple components in the Jichuan Jian combination, while saving costs, simplifying operations, and improving efficiency.

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

[0007] One aspect of the present invention provides a one-test, multiple-evaluation method for detecting the content of a Jichuanjian composition, comprising the following steps:

[0008] Extracting the Jichuanjian composition using an extraction solvent to prepare a test solution;

[0009] Using a dissolving reagent to dissolve a reference substance and naringin to prepare a reference substance mixed solution and a naringin solution; the reference substance comprises one or more of caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, neohesperidin and tangerine;

[0010] The test solution, the reference solution and the naringin solution were injected respectively for ultra-high performance liquid chromatography detection;

[0011] Taking naringin as the internal reference, the content of the component to be tested in the test solution was calculated according to the following formula:

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

[0013] Wherein, f is the relative correction factor of the component to be tested, 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 component to be tested in the test solution, and Ci is the concentration of the component to be tested in the test solution;

[0014] The raw materials for preparing the Jichuanjian composition include the following components by weight: 10-15 parts of angelica sinensis, 6-8 parts of Achyranthes bidentata, 8-10 parts of Cistanche deserticola, 4-6 parts of Orientalis alisma, 1-3 parts of Cimicifuga heracleifolia and 2-4 parts of Fructus aurantii.

[0015] In one embodiment, the components to be detected include one or more of caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, naringin, neohesperidin and tangerine.

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

[0017] The procedure of the gradient elution includes:

[0018] 0min~12.5min, the volume percentage of the mobile phase A increases from 12% to 13%;

[0019] From 12.5min to 20min, the volume percentage of the mobile phase A increases from 13% to 15%;

[0020] 20min to 30min, the volume percentage of the mobile phase A increases from 15% to 21%;

[0021] 30min-36min, the volume percentage of the mobile phase A increases from 21% to 30%;

[0022] From 36min to 43min, the volume percentage of the mobile phase A increases from 30% to 70%;

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

[0024] From 46 min to 50 min, the volume percentage of the mobile phase A was maintained at 12%.

[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) Column temperature is 28℃~32℃;

[0028] (2) Flow rate: 0.18 mL / min to 0.22 mL / min;

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

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

[0031] In one embodiment, the one-test-multiple-evaluation content detection method of the Jichuanjian composition further includes the following steps: using naringin as an internal reference, injecting a reference substance mixed solution of different concentrations, performing ultra-high performance liquid chromatography detection, recording the peak areas corresponding to the components to be tested in the reference substance mixed solution of different concentrations, and calculating the average relative 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 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 tested, C k is the mass concentration of the component to be measured.

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

[0035] Optionally, in the methanol aqueous solution, the volume percentage of methanol is 50% to 70%; in the acetonitrile aqueous solution, the volume percentage of acetonitrile is 50% to 70%.

[0036] In one embodiment, the dissolving agent includes methanol-water solution;

[0037] Optionally, in the methanol aqueous solution, the volume percentage of methanol is 50% to 70%.

[0038] In one embodiment, the raw materials for preparing the Jichuanjian composition include the following components in parts by mass: 14.92 parts of Angelica sinensis, 7.46 parts of Achyranthes bidentata, 9.33 parts of Cistanche deserticola, 5.60 parts of Alismatis, 2.61 parts of Cimicifuga heracleifolia and 3.73 parts of Fructus Aurantii Immaturus.

[0039] Another aspect of the present invention provides the use of the one-test-multiple-evaluation content detection method of the Jichuanjian composition as described above in the quality control of the Jichuanjian composition.

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

[0041] 1. This application adopts a one-test-multiple-evaluation method with naringin as an internal reference to obtain the contents of eight components in the Jichuanjian composition, including caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, naringin, neohesperidin, and tangerine. It is highly practical, simple to operate, fast and accurate in testing, and cost-effective.

[0042] 2. The method of the present application is simple and easy to operate, and the measurement results are accurate. The method has good repeatability, stability and durability, which provides a basis for establishing the quality evaluation of the Jichuanjian composition.

[0043] 3. The index components measured in this application are all chemical substances closely related to the functional properties of the Jichuan Jian composition, and have a unique biosynthetic pathway and chemical specificity. At the same time, they can reflect the clinical efficacy of the Jichuan Jian composition and should be used as important Q-marker components of the Jichuan Jian composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0045] Figure 1 This is the chromatogram of the mixed reference substance and the test substance in the investigation of the specificity of the content determination of the Jichuanjian composition in Example 1.

[0046] Figure 2 This is the chromatogram with missing negative in the investigation of specificity of content determination of Jichuanjian composition in Example 1.

[0047] Figure 3 This is a chromatogram of different column temperatures in the durability study of the content determination of the Jichuanjian composition in Example 1.

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

[0049] Figure 5 This is a chromatogram of different injection amounts in the durability study of the content determination of the Jichuanjian composition in Example 1.

[0050] Figure 6 This is a chromatogram of different gradient elution procedures in the durability investigation of the content determination of the Jichuanjian composition in Example 1.

[0051] Figure 7 This is a chromatogram of different mobile phases in the durability study of the content determination of the Jichuanjian composition in Example 1.

[0052] Figure 8 This is a chromatogram of different detection wavelengths in the durability investigation of the content determination of the Jichuanjian composition in Example 1.

[0053] In each figure, peak 1: caffeic acid; peak 2: echinacoside; peak 3: ferulic acid; peak 4: isoferulic acid; peak 5: verbascoside; peak 6: naringin; peak 7: neohesperidin; peak 8: tangeretin. DETAILED DESCRIPTION

[0054] The present application will be further described in detail below in conjunction with specific embodiments. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thoroughly understood.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0056] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0057] Herein, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0058] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0059] The percentage contents involved in this application, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.

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

[0061] The temperature parameters in this application, unless otherwise specified, allow both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±2°C, ±1°C, ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, and ±0.1°C are allowed. Normal temperature or room temperature in this application refers to no temperature control operation, generally 4°C to 35°C, preferably 20±5°C.

[0062] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0063] The existing detection of Jichuanjian composition mainly has the following problems: on the one hand, the existing liquid chromatography analysis is mainly aimed at the HPLC fingerprint of Jichuanjian and the simultaneous determination of the contents of multiple components, the detection time is long, and the quality control method of the index components is relatively insufficient; on the other hand, the existing liquid chromatography analysis mainly uses the external standard method to determine the content of Jichuanjian, the consumption of reference materials is large, and the detection cost is relatively expensive.

[0064] Based on this, the technical personnel of the present application have found through a large number of studies that a one-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, isoferulic acid, verbascoside, naringin, neohesperidin, and tangerine in the Jichuanjian composition, which meets the requirements of methodological verification. Multi-index content determination has become a consensus for quality control of classic prescriptions. The method of the present application is a multi-index synchronous quality control method, which determines the content of a representative component, calculates the content of the various components to be measured based on the relative correction factor, and controls the calculated value and the measured value to meet the requirements of quantitative methodology, overcoming the large consumption of reference substances and the high difficulty of detection operation during the implementation of the multi-index content determination method. The method has the characteristics of saving standard products and accurate and reliable results, and further promotes the promotion and application of quality evaluation research of Jichuanjian composition. The invention provides a method for detecting eight components in a Jichuanjian composition, namely, caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, naringin, neohesperidin and tangerine, which can save costs, simplify operations and improve efficiency.

[0065] One aspect of the present invention provides a one-test, multiple-evaluation method for detecting the content of a Jichuanjian composition, comprising the following steps:

[0066] Extracting the Jichuanjian composition using an extraction solvent to prepare a test solution;

[0067] Using a dissolving reagent to dissolve a reference substance and naringin to prepare a reference substance mixed solution and a naringin solution; the reference substance comprises one or more of caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, neohesperidin and tangerine;

[0068] The test solution, the reference solution and the naringin solution were injected respectively for ultra-high performance liquid chromatography detection;

[0069] Taking naringin as the internal reference, the content of the component to be tested in the test solution was calculated according to the following formula:

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

[0071] Wherein, f is the relative correction factor of the component to be tested, 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 component to be tested in the test solution, and Ci is the concentration of the component to be tested in the test solution;

[0072] The raw materials for preparing the Jichuanjian composition include the following components by weight: 10-15 parts of angelica sinensis, 6-8 parts of Achyranthes bidentata, 8-10 parts of Cistanche deserticola, 4-6 parts of Orientalis alisma, 1-3 parts of Cimicifuga heracleifolia and 2-4 parts of Fructus aurantii.

[0073] In some examples, the components to be tested include one or more of caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, naringin, neohesperidin and tangerine.

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

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

[0076] 0min~12.5min, the volume percentage of the mobile phase A increases from 12% to 13%;

[0077] From 12.5min to 20min, the volume percentage of the mobile phase A increases from 13% to 15%;

[0078] 20min to 30min, the volume percentage of the mobile phase A increases from 15% to 21%;

[0079] 30min-36min, the volume percentage of the mobile phase A increases from 21% to 30%;

[0080] From 36min to 43min, the volume percentage of the mobile phase A increases from 30% to 70%;

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

[0082] From 46 min to 50 min, the volume percentage of the mobile phase A was maintained at 12%.

[0083] In some examples, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.1% to 0.2%. It is understandable that the volume percentage of phosphoric acid in the phosphoric acid aqueous 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 ultra-high performance liquid chromatography detection conditions include: a column temperature of 28° C. to 32° C. It can be understood that the column temperature of the ultra-high performance liquid chromatography 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 the ultra-high performance liquid chromatography detection include: a flow rate of 0.18 mL / min to 0.22 mL / min. It is understandable that the flow rate for the ultra-high performance liquid chromatography detection includes but is 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 the ultra-high performance liquid chromatography detection include: the injection volume is 0.2 μL to 1.0 μL. It can be understood that the injection volume for the ultra-high performance liquid chromatography 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 detection conditions include: a wavelength of 286nm to 318nm. It is understandable that the wavelength of the ultra-high performance liquid chromatography detection includes but is not limited to 286nm, 290nm, 294nm, 298nm, 310nm, 312nm, and 318nm.

[0088] In some of the examples, the one-test-multiple-evaluation content detection method of the Jichuanjian composition also includes the following steps: using naringin as an internal reference, injecting a reference substance mixed solution of different concentrations, performing ultra-high performance liquid chromatography detection, recording the peak areas corresponding to the components to be tested in the reference substance mixed solution of different concentrations, and calculating the average relative 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 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 tested, C k is the mass concentration of the component to be measured.

[0091] In some examples, the extraction solvent includes a methanol aqueous solution or an acetonitrile aqueous solution. Further, in the methanol aqueous solution, the volume percentage of methanol is 50% to 70%; in the acetonitrile aqueous solution, the volume percentage of acetonitrile is 50% to 70%.

[0092] In some examples, the extraction is performed by ultrasound.

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

[0094] In some examples, the dissolving agent includes a methanol aqueous solution; further, the volume percentage of methanol in the methanol aqueous solution is 50% to 70%. 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 the examples, the raw materials for preparing the Jichuanjian composition include the following components in parts by mass: 14.92 parts of Angelica sinensis, 7.46 parts of Achyranthes bidentata, 9.33 parts of Cistanche deserticola, 5.60 parts of Alismatis orientalis, 2.61 parts of Cimicifuga heracleifolia and 3.73 parts of Fructus Aurantii Immaturus.

[0096] In one specific example, the one-test-multiple-evaluation content detection method of the Jichuanjian composition comprises the following steps:

[0097] Step 1: Chromatographic conditions, chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase: acetonitrile (A)-0.1% phosphoric acid aqueous solution (B); elution gradient (0-12.5min, 12%-13% A; 12.5-20min, 13%-15% A; 20-30min, 15%-21% A; 30-36min, 21%-30% A; 36-43min, 30%-70% A; 43-46min, 70%-12% A; 46-50min, 12% A); detection wavelength: 310nm; column temperature: 28-32°C; flow rate: 0.18-0.22mL / min; injection volume: 0.2-1.0μL; the number of theoretical plates 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, isoferulic acid, verbascoside, naringin, neohesperidin, and tangeretin, place them in a volumetric flask, mix them evenly, and then add 50% methanol aqueous solution to dissolve them to obtain the first mixed reference solution.

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

[0100] Step 4: Calculation of relative correction factor: Take the first mixed reference solution prepared in step 2, dilute it into a second mixed reference solution with a series of concentrations, inject it into the high performance liquid chromatograph, measure it according to the chromatographic conditions of step 1, record the peak area of ​​each component, and use naringin as the internal reference to calculate the relative correction factors (f k / s ), f k / s =f k / f s =(A k / C k ) / (A s / C s ), where A s is the peak area of ​​the internal reference, C s is the mass concentration of the internal reference substance, A k is the peak area of ​​the component to be measured, C k The relative correction factors f of caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, neohesperidin, tangerine and the internal reference naringin were calculated as the mass concentration of the tested components.

[0101] Step 5: Calculation of the content of the target compound. Take the test solution prepared in step 3 and inject it into the high performance liquid chromatograph for determination to obtain a chromatogram. Use the relative retention value in step 4 to locate the chromatographic peak of the component to be measured, and then calculate the content of caffeic acid, echinacoside, ferulic acid, isoferulic acid, 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 use of the one-test-multiple-evaluation content detection method of the Jichuanjian composition as described above in the quality control of the Jichuanjian composition.

[0103] The present application is further described in detail below in conjunction with specific examples. For experimental parameters not specified in the following specific examples, reference is made to the instructions given in the present application document, and reference may also be made to the experimental manual of the art or other experimental methods known in the art, or to the experimental conditions recommended by the manufacturer. It is understood that the instruments and raw materials used in the following examples are relatively specific, and may not be limited thereto in other specific examples.

[0104] Example 1

[0105] This embodiment provides a one-test, multiple-evaluation content detection method for the Jichuanjian composition, which is as follows:

[0106] 1. Instruments and test drugs

[0107] 1.1 Instrument

[0108] Waters H-class high performance liquid chromatograph (Waters, USA); Agilent 1290 high performance liquid chromatograph (Agilent, USA); ME204E one-tenth balance, XP26 one-millionth balance (METTLERTLEDO, 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 drugs

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

[0111] Trial drug: 9 batches of Jichuanjian combination were prepared in the laboratory, and the preparation method was as follows: take 14.92g of Angelica sinensis, 7.46g of Achyranthes bidentata, 9.33g of Cistanche deserticola (made with wine), 5.60g of Alismatis, 2.61g of Cimicifuga heracleifolia, and 3.73g of Fructus Aurantii Immaturus, a total of 43.65g, add 300mL of water, soak for 30min, boil to 150mL, filter, concentrate the filtrate and spray dry to obtain Jichuanjian 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%), isoferulic 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 tangerine (batch number 112054-202102, purity 99.7%) were all purchased from China Food and Drug Inspection Institutes.

[0113] 2. Experimental Methods

[0114] 2.1 Chromatographic conditions

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

[0116] Table 1 Mobile phase gradient elution conditions

[0117]

[0118] 2.2 Preparation of mixed reference solution

[0119] Accurately weigh appropriate amounts of caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, naringin, neohesperidin and tangeretin reference substances, and add 50% methanol aqueous solution to prepare mixed reference substance 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 test solution

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

[0122] 3. Methodological investigation

[0123] 3.1 Exclusivity Investigation

[0124] Accurately pipette appropriate amount of reference solution, test solution and negative sample solution, and perform the injection test according to the chromatographic conditions under "2.1". The results are shown in Figures 1-2 The results showed that the chromatographic peaks of each component had good separation, the theoretical plate number was not less than 5000, and the retention time was consistent with that of the chromatographic peak of the reference substance, indicating that the method had good specificity.

[0125] 3.2 Investigation of linear relationship

[0126] 0.1mL, 0.3mL, 0.5mL, 2.0mL, and 3.0mL of the mixed reference solution prepared under "2.2" were taken separately and placed in 5mL volumetric flasks, and 50% methanol aqueous solution was added to the scale. Shake well to prepare a series of mixed reference solutions of different concentrations. Samples were injected and measured according to the chromatographic conditions under "2.1", and the chromatogram was recorded. Linear regression was performed 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 Linear relationship investigation results (n = 6)

[0128]

[0129] 3.3 Precision investigation

[0130] Accurately pipette an appropriate amount of the mixed reference solution under item "2.2", and inject the sample according to the chromatographic conditions under item "2.1". Inject 6 injections continuously and record the peak area. The results are shown in Table 3. The RSDs of the peak areas of caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, naringin, neohesperidin, and tangerine were all less than 3%, indicating that the instrument had good precision.

[0131] Table 3 Precision investigation results

[0132]

[0133] 3.4 Repeatability study

[0134] Take an appropriate amount of the same Jichuanjian composition, prepare 6 test sample solutions in parallel according to the method under "2.3", and inject and measure according to the chromatographic conditions under "2.1". The results are shown in Table 4. The RSDs of the peak areas of caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, naringin, neohesperidin, and tangerine were all less than 3%, indicating that the method has good repeatability.

[0135] Table 4 Repeatability test results

[0136]

[0137] 3.5 Stability investigation

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

[0139] Table 5 Stability test results

[0140]

[0141] 3.6 Sample recovery test

[0142] Take 9 portions of the same Jichuanjian composition with known content, each portion is about 0.1g, accurately weighed, placed in a stoppered conical bottle, divided into 3 groups, accurately add appropriate amounts of mixed reference solution according to high, medium and low concentrations, prepare the test solution according to the method under "2.3", inject and measure according to the chromatographic conditions under "2.1", calculate the recovery rate, and the results are shown in Table 6. The calculated sample recovery rates of caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, naringin, neohesperidin, and tangerine are within 99.69% to 105.94%, the recovery rate limits specified in the pharmacopoeia, and the RSDs are all less than 3%, indicating that the method has good accuracy.

[0143] Table 6 Sample recovery results

[0144]

[0145]

[0146]

[0147] 4. Calculation of relative correction factor

[0148] Under the chromatographic conditions specified in the present invention, according to the linear relationship investigation results under item "3.2", naringin was used as the internal reference to calculate the relative correction factors (f k / s ), f k / s =f k / f s =(A k / C k ) / (A s / C s ), where A s is the peak area of ​​the internal reference, C s is the mass concentration of the internal reference substance, A k is the peak area of ​​the component to be measured, C k The relative correction factors f of caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, neohesperidin and tangerine were calculated based on the mass concentration of the tested components. The results are shown in Table 7.

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

[0150]

[0151] 5. System durability inspection

[0152] 5.1 Investigation of different instruments and chromatographic columns

[0153] Taking naringin as the internal reference, the relative correction factors of the other 7 components were investigated on two HPLC systems, Waters H-class and Agilent 1290, and two chromatographic columns, Waters ACQUITY UPLC HSS T3 (150mm×2.1mm, 1.8μm) and YMCTriart C18 (150mm×2.1mm, 1.9μm). The results are shown in Table 8. The RSD of each component was less than 3%, indicating that the replacement of instruments and chromatographic 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 of different column temperatures

[0157] Using naringin as the internal reference, a Waters H-class HPLC and a YMC Triart C18 column were used to investigate the effects of column temperatures of 28°C, 30°C, and 32°C on the relative correction factors of each component. The results are shown in Tables 9 and 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 rates

[0161] Naringin was used as the internal reference, and a Waters H-class HPLC was used with a YMC Triart C18 column at a flow rate of 0.18 mL min -1 , 0.20mL·min -1 、0.22mL·min -1 The results of the effects on the relative correction factors of 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 rates on relative correction factors

[0163]

[0164] 5.4 Investigation of different injection volumes

[0165] Using naringin as the internal reference, a Waters H-class HPLC and a YMC Triart C18 column were used to investigate 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. The results are shown in Tables 11 and 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. Positioning of chromatographic peaks of components to be measured

[0169] In the application of QAMS, the commonly used chromatographic peak positioning methods include relative retention value method, retention time difference method, time correction method, reference extract method, etc. The calculation formula of retention time difference method is: Δti / s=ti-ts. The calculation formula of relative retention value method is: ti / s=ti / ts. In this experiment, relative retention time was used to locate the chromatographic peak of the component to be tested. Naringin was used as the internal reference to investigate the relative retention time of the other 7 components under two high performance liquid chromatography systems, Waters H-class and Agilent 1290, and two chromatographic 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 locating the components to be tested.

[0170] Table 12 Relative retention time of each component

[0171]

[0172] 7. Comparison of the results of the QAMS and ESM

[0173] Take an appropriate amount of 9 batches of Jichuanjian composition samples, prepare the test solution according to the method under "2.3", sample injection and determination according to the chromatographic conditions under "2.1", and use the external standard method to determine the content of each component. The steps of the one-measurement-multiple-evaluation method of the present application are: take an appropriate amount of 9 batches of Jichuanjian composition samples, prepare the test solution according to the method under "2.3", sample injection and determination according to the chromatographic conditions under "2.1", substitute the relative correction factors of the 7 components under "4" into the formula to calculate the content of each component.

[0174] The results are shown in Table 13-1 and Table 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, isoferulic acid, verbascoside, neohesperidin, and tangerine are all less than 3%, indicating that there is no significant difference in the results calculated by the two methods.

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

[0176]

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

[0178]

[0179] In addition, the present application also investigates the conditions of the one-test-multiple-evaluation content detection method of the Jichuanjian composition, as follows:

[0180] 1. Selection of gradient elution program

[0181] As shown in Table 14-1 and Table 14-2, the elution gradient was optimized through comparative tests of various gradient elution conditions. Figure 6 ) It can be seen that when gradient elution program 1 is used, the chromatographic peak response is low, while when gradient elution program 2 is used, the chromatographic peak separation is good and the response is high. 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. Choice 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 when acetonitrile and methanol were selected, acetonitrile had a better separation effect and a higher chromatographic peak response. When methanol was used for gradient elution, the baseline noise was obvious and there was an obvious solvent peak. Because water has a larger polarity, fewer peak components and the peaks are stacked together, so it is considered to add acid. Under the condition of 0.1% phosphoric acid, the pH value of the solvent is closer to the pKa value of the index component to be measured, so that the separation effect is better, so the chromatographic peak shape is sharp and symmetrical. Therefore, acetonitrile-0.1% phosphoric acid was used for elution.

[0188] 3. Selection of detection wavelength

[0189] Through full wavelength scanning, it can be obtained that the maximum absorption wavelengths of ferulic acid and isoferulic acid in Angelica sinensis and Cimicifuga heracleifolia are around 270nm-320nm, the maximum absorption wavelengths of naringin, neohesperidin and tangerine in Fructus Aurantii Immaturus are around 280nm-320nm, and the maximum absorption wavelengths of echinacoside and verbascoside in Cistanche deserticola (wine-made) are 330nm. The maximum absorption wavelength of β-ecdysterone in Achyranthes bidentata is 270nm, the maximum absorption wavelengths of alismacol B and 23-acetyl alismacol B in Alisma orientalis are 208nm, and the maximum absorption wavelengths of alismacol C and 23-acetyl alismacol C are 246nm. The maximum absorption wavelengths of the two medicinal chemical components of Achyranthes bidentata and Alisma orientalis are quite different from those of other medicinal chemical components, so quantitative control of Achyranthes bidentata and Alisma orientalis is not performed in the present invention. Considering the stability of the overall baseline of the comprehensive spectrum, it was decided to select 286nm, 310nm, and 318nm as detection wavelengths for investigation. Figure 8 As shown in the figure, during the experiment, it was found that the wavelength was set to 310nm, which could detect caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, naringin, neohesperidin, and tangerine, and the baseline was stable at the corresponding wavelength, and the separation effect of each component was good. Therefore, 310nm was used as the detection wavelength.

[0190] 4. Preparation of test samples

[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 much different. 50% methanol and 50% acetonitrile ultrasonic extraction were used for experiments. As shown in Table 15, it was found that the extraction effect of 50% methanol 20mL ultrasonic extraction for 30 minutes was better, and the solubility of each component was relatively high. Therefore, 50% methanol 20mL ultrasonic extraction 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

[0195] Under the chromatographic conditions specified in this application, according to the linear relationship investigation results under "3.2", the 8 components were used as internal references to calculate the relative correction factors (f k / s ), f k / s =f k / f s =(A k / C k ) / (A s / C s ), where A s is the peak area of ​​the internal reference, C s is the mass concentration of the internal reference substance, A k is the peak area of ​​the component to be measured, Ck The results (see Table 16-1, Table 16-2, Table 16-3, Table 16-4, Table 16-5, Table 16-6, Table 16-7 and Table 16-8) show that the RSD of the relative correction factor of each component to be measured is less than 5% when the 8 components are used as internal references. Combined with the overall situation of the spectrum, naringin has a good peak shape, high response, and moderate retention time, so naringin is used as the internal reference.

[0196] Table 16-1 Relative correction factors of various components with caffeic acid as internal reference (n=6)

[0197]

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

[0199]

[0200] Table 16-3 Relative correction factors of various components with ferulic acid as internal reference (n=6)

[0201]

[0202] Table 16-4 Relative correction factors of each component with isoferulic acid as internal reference (n=6)

[0203]

[0204] Table 16-5 Relative correction factors of various components using verbascoside as internal reference (n=6)

[0205]

[0206] Table 16-6 Relative correction factors of various components using naringin as internal reference (n=6)

[0207]

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

[0209]

[0210] Table 16-8 Relative correction factors of various components with tangerine as internal reference (n=6)

[0211]

[0212] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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 above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A one-test, multiple-evaluation content detection method for Jichuanjian composition, characterized in that: The following steps are involved: Extracting the Jichuanjian composition using an extraction solvent to prepare a test solution; Using a dissolving reagent to dissolve a reference substance and naringin to prepare a reference substance mixed solution and a naringin solution; the reference substance comprises one or more of caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, neohesperidin and tangerine; The test solution, the reference solution and the naringin solution were injected respectively for ultra-high performance liquid chromatography detection; Taking naringin as the internal reference, the content of the component to be tested in the test solution was calculated according to the following formula: Ci = f*Cs*(Ai / As); Wherein, f is the relative correction factor of the component to be tested, 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 component to be tested in the test solution, and Ci is the concentration of the component to be tested in the test solution; The raw materials for preparing the Jichuanjian composition include the following components by weight: 10-15 parts of angelica sinensis, 6-8 parts of Achyranthes bidentata, 8-10 parts of Cistanche deserticola, 4-6 parts of Orientalis alisma, 1-3 parts of Cimicifuga heracleifolia and 2-4 parts of Fructus aurantii.

2. The one-test-multiple-evaluation content detection method of Jichuanjian composition according to claim 1, characterized in that: The components to be tested include one or more of caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, naringin, neohesperidin and tangerine.

3. The one-test-multiple-evaluation content detection method of Jichuanjian composition according to claim 1, characterized in that: The ultra-high performance liquid chromatography detection conditions include: mobile phase A is acetonitrile, mobile phase B is phosphoric acid aqueous solution, and gradient elution is used; The procedure of the gradient elution includes: 0min~12.5min, the volume percentage of the mobile phase A increases from 12% to 13%; From 12.5min to 20min, the volume percentage of the mobile phase A increases from 13% to 15%; 20min to 30min, the volume percentage of the mobile phase A increases from 15% to 21%; 30min-36min, the volume percentage of the mobile phase A increases from 21% to 30%; From 36min to 43min, the volume percentage of the mobile phase A increases from 30% to 70%; From 43 to 46 minutes, the volume percentage of the mobile phase A decreased from 70% to 12%; From 46 min to 50 min, the volume percentage of the mobile phase A was maintained at 12%.

4. The one-test-multiple-evaluation content detection method of the Jichuanjian composition according to claim 3, characterized in that: In the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.1% to 0.2%.

5. The one-test-multiple-evaluation content detection method of Jichuanjian composition according to claim 1, characterized in that: The ultra-high performance liquid chromatography detection also satisfies at least one of the following conditions: (1) Column temperature is 28℃~32℃; (2) Flow rate: 0.18 mL / min to 0.22 mL / min; (3) The injection volume is 0.2 μL to 1.0 μL; (4) The wavelength is 286nm~318nm.

6. The one-test-multiple-evaluation content detection method of the Jichuanjian composition according to any one of claims 1 to 5, characterized in that: The following steps are also included: Taking naringin as the internal reference, inject the reference solution of different concentrations, perform ultra-high performance liquid chromatography detection, record the peak area corresponding to the tested component in the reference solution of different concentrations, and calculate the average relative correction factor f according to the following formula: k / s : f k / s =f k / f s =(A k / C k ) / (A s / C s ); Among them, 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 tested, C k is the mass concentration of the component to be measured.

7. The one-test-multiple-evaluation content detection method of the Jichuanjian composition according to any one of claims 1 to 5, characterized in that: The extraction solvent includes methanol aqueous solution or acetonitrile aqueous solution; Optionally, in the methanol aqueous solution, the volume percentage of methanol is 50% to 70%; in the acetonitrile aqueous solution, the volume percentage of acetonitrile is 50% to 70%.

8. The one-test-multiple-evaluation content detection method of the Jichuanjian composition according to any one of claims 1 to 5, characterized in that: The dissolving agent includes methanol aqueous solution; Optionally, in the methanol aqueous solution, the volume percentage of methanol is 50% to 70%.

9. The one-test-multiple-evaluation content detection method of the Jichuanjian composition according to any one of claims 1 to 5, characterized in that: The raw materials for preparing the Jichuanjian composition include the following components by weight: 14.92 parts of Angelica sinensis, 7.46 parts of Achyranthes bidentata, 9.33 parts of Cistanche deserticola, 5.60 parts of Orientalis alisma, 2.61 parts of Cimicifuga heracleifolia and 3.73 parts of Fructus Aurantii Immaturus.

10. Use of the one-test, multiple-evaluation content detection method for the Jichuanjian composition according to any one of claims 1 to 9 in the quality control of the Jichuanjian composition.

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