Detection method of traditional Chinese medicine composition

Abstract

The invention discloses a method for detecting the content of a traditional Chinese medicine composition, the traditional Chinese medicine composition comprises caulis sinomenii, radix sophorae flavescentis, rhizoma anemarrhenae, herba siegesbeckiae, rhizoma corydalis, ajuga ciliata, ovateleaf holly bark, yam rhizome and acanthopanax senticosus, the method comprises the following steps: taking a traditional Chinese medicine composition test sample solution for HPLC detection, acetonitrile is used as a mobile phase A, a phosphate buffer solution (pH 5.8) is used as a mobile phase B, and elution in chromatographic conditions of HPLC detection is gradient elution. Systematic methodology investigation and verification show that the method is high in specificity and good in repeatability and accuracy. The content of the contained chemical components is detected by utilizing the method disclosed by the invention, so that the component information of the particles or a method for determining the component content information is established, the quality of the particles can be comprehensively reflected, and the quality of the preparation can be integrally described and evaluated.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry, and particularly relates to a method for detecting a traditional Chinese medicine composition. Background Art

[0002] A prescription composed of nine herbs, namely Sinomenium acutum, Sophora flavescens, Anemarrhena asphodeloides, Siegesbeckia orientalis, Corydalis yanhusuo, Ajuga ciliata, Ilex rotunda, Dioscorea septemloba, and Acanthopanax senticosus, has the effects of clearing collaterals and relaxing tendons, dissipating binds and relieving pain, dispelling wind and removing dampness. It is mainly used for treating the recurrence of arthralgia syndrome with symptoms such as muscle spasms, limited flexion and extension, and subcutaneous nodules; or the acute attack period of arthralgia syndrome with obvious symptoms of joint swelling, redness, heat and pain, accompanied by fever and thirst. Pharmacological studies have shown that it has good clinical efficacy in the treatment of active rheumatoid arthritis (RA). By regulating the inflammatory response and targeting the angiogenesis target of RA, and by inhibiting angiogenesis, it can achieve the purpose of improving symptoms and treating rheumatic diseases. At present, the relevant research still stays in the study of pharmacodynamics, and the research on the pharmacodynamic material basis is relatively weak.

[0003] Traditional Chinese medicine compounds have complex components and the characteristics of multiple targets and multiple effects. They follow the compatibility rules of monarch, minister, assistant and envoy in medication, with the monarch drug playing the main therapeutic role and the minister drug enhancing the treatment of the main disease. The monarch drugs in this formula, Sinomenium acutum and Sophora flavescens, are both commonly used traditional Chinese medicines for treating rheumatoid arthritis and have good curative effects. The alkaloids in Sinomenium acutum are the main active ingredients for relieving wind and pain, and sinomenine is its main active ingredient and also the main index component for the quality control and evaluation of Sinomenium acutum. The previous research by the inventor has shown that various components such as matrine and sophocarpine in Sophora flavescens have anti-tumor activity and anti-inflammatory and analgesic effects; the main chemical component of the minister drug Corydalis yanhusuo, alkaloids, has analgesic, sedative, anti-inflammatory, anti-tumor and other effects, and the total alkaloids of Corydalis yanhusuo can effectively inhibit central and peripheral pain. Therefore, in this paper, an HPLC method is established for simultaneously determining the contents of 6 components, namely matrine, sophocarpine, sinomenine, protopine, tetrahydropalmatine, and corydaline, to provide a reference for the research on the pharmacodynamic material basis, quality control and subsequent preparation development of the traditional Chinese medicine composition. Summary of the Invention

[0004] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] A method for detecting the content of a traditional Chinese medicine composition, characterized in that a test solution of the traditional Chinese medicine composition is taken for HPLC detection, and the chromatographic conditions for HPLC detection include:

[0006] An Agilent ZORBAX Eclipse XDB-C18 column (5 μm, 4.6×250 mm) was used; acetonitrile was used as mobile phase A, and phosphate buffer solution (pH 5.8) was used as mobile phase B. The elution in the HPLC detection chromatographic conditions was gradient elution, and the gradient elution program could be: 0 - 25 min, 5% A; 25 - 35 min, 5% - 7% A; 35 - 50 min, 7% - 15% A; 50 - 60 min, 15% - 19% A; 60 - 80 min, 19% - 20% A; 80 - 90 min, 20% - 35% A; 90 - 115 min, 35% A; 115 - 120 min, 35% - 44% A; 120 - 145 min, 44% - 48% A.

[0007] Specifically, the traditional Chinese medicine composition comprises nine herbs, namely Sinomenium acutum, Sophora flavescens, Anemarrhena asphodeloides, Siegesbeckia orientalis, Corydalis yanhusuo, Ajuga ciliata, Ilex rotunda, Dioscorea septemloba, and Acanthopanax senticosus.

[0008] Further, the chromatographic conditions include: the flow rate is 0.8 - 1.0 mL / min, the column temperature is 25 - 35 °C, and the detection wavelength is 208 - 212 nm.

[0009] More specifically, the chromatographic conditions include: the flow rate is 0.9 mL / min, the column temperature is 30 °C, and the detection wavelength is 210 nm.

[0010] Specifically, the C18 chromatographic column is selected from Phenomenex Gemini C18 chromatographic column or Phenomenex Luna C18 chromatographic column.

[0011] Specifically, the traditional Chinese medicine composition can be a solid, liquid or external preparation; for example: decoction, granule, capsule, tablet, pill, oral liquid, tincture, syrup, suppository, gel, spray, injection, etc.

[0012] Further, when the traditional Chinese medicine composition is a granule, its preparation method includes: 10 parts of Sinomenium acutum, 10 parts of Sophora flavescens, 15 parts of Siegesbeckia orientalis, 15 parts of Corydalis yanhusuo, 15 parts of Ajuga ciliata, 15 parts of Ilex rotunda, 10 parts of Dioscorea septemloba, 12 parts of Acanthopanax senticosus, and 10 parts of Anemarrhena asphodeloides. Add 10 times the amount of water and decoct twice, each time for 1.5 hours. Concentrate under reduced pressure to an extract with a relative density of 1.08 - 1.12 (60 °C - 70 °C), spray dry, pulverize, add excipients, and granulate by dry granulation to obtain the product.

[0013] Optionally, the preparation of the test sample solution is as follows: Weigh accurately 0.64 g of the extract of this product, place it in a stoppered conical flask, add 30 mL of purified water, and ultrasonically treat for 15 minutes to dissolve it (power 500 W, frequency 40 kHz). Then transfer it to a separating funnel, extract with chloroform 5 times, 30 mL each time. Combine the chloroform layers, evaporate to dryness, dissolve the residue in methanol, and make up the volume to 10 mL in a volumetric flask to obtain the solution.

[0014] Optionally, the preparation of the test sample solution is as follows: Weigh accurately 0.3 g of the granule powder of this product, place it in a stoppered conical flask, add 30 mL of purified water, and ultrasonically treat for 15 minutes to dissolve it (power 500 W, frequency 40 kHz). Then transfer it to a separating funnel, extract with chloroform 5 times, 30 mL each time. Combine the chloroform layers, evaporate to dryness, dissolve the residue in methanol, and make up the volume to 10 mL in a volumetric flask to obtain the solution.

[0015] Furthermore, the above detection method also includes the detection of reference substances, and the reference substances include one or more of the reference standards of matrine, sophocarpine, sinomenine, protopine, tetrahydropalmatine, and tetrahydropalmatine.

[0016] Specifically, the method for preparing the reference substances into a solution for detection is as follows: Take the reference standards of matrine, sophocarpine, sinomenine, protopine, tetrahydropalmatine, and tetrahydropalmatine, and add methanol to make a single-standard or mixed-standard solution containing 7.98 - 319.37 μg of matrine, 3.21 - 320.75 μg of sophocarpine, 9.15 - 366.03 μg of sinomenine, 0.27 - 27.44 μg of protopine, 0.47 - 46.96 μg of tetrahydropalmatine, and 0.42 - 42.00 μg of tetrahydropalmatine per 1 mL.

[0017] By establishing a standard curve for one or more reference substances, the information on the composition or the content of the components of the composition is finally obtained.

[0018] The present invention provides a method for detecting the component content of a composition including Sinomenium acutum, Sophora flavescens, Anemarrhena asphodeloides, Siegesbeckia orientalis, Corydalis yanhusuo, Ajuga ciliata, Ilex rotunda, Dioscorea hypoglauca, and Eleutherococcus senticosus. After systematic methodological investigation and verification, it shows that this method has strong specificity, and its durability, repeatability, and accuracy are all good. Using the method of the present invention to detect the chemical components or the content of the components in the composition, and accordingly establishing a method for determining the content of the granules can comprehensively reflect its quality and provide an overall description and evaluation of the quality of the preparation. Description of the Drawings

[0019] Figure 1 Chromatogram of the investigation of different mobile phases for the detection of the component content of the traditional Chinese medicine composition;

[0020] Figure 2 Chromatogram of the investigation of the gradient elution program for the detection of the component content of the traditional Chinese medicine composition;

[0021] Figure 3 Chromatogram for the specificity study of the component content determination of the traditional Chinese medicine composition. Among them, 1 - matrine; 2 - sophocarpine; 3 - sinomenine; 4 - protopine; 5 - tetrahydropalmatine; 6 - corydaline. Detailed implementation manners

[0022] The present invention discloses a detection method for a traditional Chinese medicine composition comprising Sinomenium acutum, Sophora flavescens, Anemarrhena asphodeloides, Siegesbeckia orientalis, Corydalis yanhusuo, Ajuga decumbens, Ilex rotunda, Dioscorea hypoglauca, and Acanthopanax senticosus. Sinomenium acutum is the dried vine stem of Sinomenium acutum (Thunb.) Rehd. et Wils. and Sinomenium acutum (Thunb.) Rehd. et Wils. var. cinereum Rehd. et Wils. of the family Menispermaceae; Sophora flavescens is the dried root of Sophora flavescens Ait. of the family Leguminosae; Siegesbeckia orientalis is the dried above-ground part of Siegesbeckia orientalis L., Siegesbeckia pubescens Makino or Siegesbeckia glabrescens Makino of the family Compositae; Corydalis yanhusuo (Yuanhu) is the dried tuber of Corydalis yanhusuo W.T. Wang of the family Papaveraceae; Ajuga decumbens is the dried whole herb of Ajuga decumbens Thunb. of the family Labiatae; Ilex rotunda is the dried bark of Ilex rotunda Thunb. of the family Aquifoliaceae; Dioscorea hypoglauca is the dried rhizome of Dioscorea hypoglauca Palibin of the family Dioscoreaceae; Acanthopanax senticosus is the dried root, rhizome or stem of Acanthopanax senticosus (Rupr. et Maxim.) Harms of the family Araliaceae; Anemarrhena asphodeloides is the dried rhizome of Anemarrhena asphodeloides Bge. of the family Liliaceae.

[0023] Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all such substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0024] Instruments and reagents

[0025] Mettler XP6 Electronic Analytical Balance (Mettler Company);

[0026] Mettler Toledo AL204 Electronic Analytical Balance (Mettler Company);

[0027] Mettler Toledo MS204 Electronic Analytical Balance (Mettler Company);

[0028] Mettler Toledo MS-105DU Electronic Analytical Balance (Mettler Company);

[0029] KQ5200DA CNC Ultrasonic Cleaner (Kunshan Ultrasonic Instruments Co., Ltd.);

[0030] HH Digital Display Constant Temperature Water Bath (Changzhou Guoyu Instrument Manufacturing Co., Ltd.);

[0031] Mettler Toledo Seven Compact plus S120 pH Meter (Mettler Company);

[0032] Milli-Q Ultra-pure Water Instrument (Millipore Corporation, USA).

[0033] High Performance Liquid Chromatograph:

[0034] Waters Arc High Performance Liquid Chromatograph, 2998PDA Detector;

[0035] Agilent 1260 High Performance Liquid Chromatograph, DAD UV Detector;

[0036] Agilent 1200 High Performance Liquid Chromatograph, MWD UV Detector;

[0037] Sample: Traditional Chinese Medicine Composition Preparation (Jiangsu Kanion Pharmaceutical Co., Ltd., Batch No.: 230501)

[0038] Reference Substance: Matrine Reference Substance (National Institutes for Food and Drug Control, Batch No.: 110805-202010, for content determination, purity 98.7%);

[0039] Sinomenine Reference Substance (National Institutes for Food and Drug Control, Batch No.: 110774-201808, for content determination, purity 94.6%);

[0040] Sophocarpine Reference Substance (National Institutes for Food and Drug Control, Batch No.: 112052-202001, for content determination, purity 91.8%);

[0041] Protopine reference substance (National Institutes for Food and Drug Control, batch number: 110853-201805, for content determination, purity 99.6%)

[0042] Tetrahydropalmatine reference substance (National Institutes for Food and Drug Control, batch number: 110726-202020, for content determination, purity 99.3%)

[0043] Corydaline reference substance (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 250152-202204, for content determination, purity 98%)

[0044] Reagents: Acetonitrile (Merck Millipore, USA, chromatographic grade); Potassium dihydrogen phosphate (Shanghai Macklin Biochemical Co., Ltd., chromatographic grade); Dipotassium hydrogen phosphate (Shanghai Macklin Biochemical Co., Ltd., chromatographic grade); Other reagents are all of analytical grade.

[0045] Example 1

[0046] 1 Chromatographic conditions

[0047] 1.1 Selection of mobile phase

[0048] Acetonitrile: [0.01 mol / L ammonium acetate methanol solution (adjusted to pH 8.1 with concentrated ammonia solution)](3:2) - 0.01 mol / L ammonium acetate aqueous solution (adjusted to pH 8.1 with concentrated ammonia solution), acetonitrile - 0.033 mol / L potassium dihydrogen phosphate (0.05% triethylamine), and acetonitrile - phosphate buffer salt (pH 5.8) were used as the elution systems. Through experiments, matrine, sophocarpine, sinomenine, protopine, tetrahydropalmatine, and corydaline were detected. In the systems of acetonitrile: [0.01 mol / L ammonium acetate methanol solution (adjusted to pH 8.1 with concentrated ammonia solution)](3:2) - 0.01 mol / L ammonium acetate aqueous solution (adjusted to pH 8.1 with concentrated ammonia solution) and acetonitrile - 0.033 mol / L potassium dihydrogen phosphate (0.05% triethylamine), the resolution of the 6 target peaks in the sample was poor, and the peak shape of the reference substance was poor; in the system of acetonitrile - phosphate buffer salt (pH 5.8), matrine, sophocarpine, sinomenine, protopine, tetrahydropalmatine, and corydaline were detected, and the peak shapes and resolutions of each chromatographic peak were good, the retention time was stable, the preparation steps of the mobile phase solution were simple, the test solution (extraction method refer to 1.2.2) was stable and the extraction of the target components reached the standard. Therefore, acetonitrile - phosphate buffer salt (pH 5.8) was selected as the elution system. Other chromatographic conditions: The flow rate of the HPLC detection was 0.9 mL / min, a Phenomenex Gemini C18 chromatographic column was used, the column temperature was 30 °C, the injection volume was 10 μL, and the detection wavelength was 210 nm. The results are shown in Tables 1 - 2, attached Figure 1 。

[0049] Table 1 Gradient elution program 1

[0050]

[0051] Table 2 Gradient Elution Program 2

[0052]

[0053] By investigating different elution gradients, acetonitrile-phosphate buffer solution (pH 5.8) was selected as the mobile phase for gradient elution. The results of different elution programs are shown in Tables 3 - 5 and the appendix Figure 2 。

[0054] Table 3 Gradient Elution Program 3

[0055]

[0056] Table 4 Gradient Elution Program 4

[0057]

[0058] Table 5 Gradient Elution Program 5

[0059]

[0060]

[0061] By investigating different elution gradients, under the elution conditions of Gradient Elution Programs 3 and 4, the resolution of the target peak in the sample was less than 1.5, and baseline separation was not achieved. Further optimization is required in the follow-up; under the elution conditions of Gradient Elution Program 5, the elution program had the best effect. In the test sample chromatogram, the resolution between matrine, sophocarpine, sinomenine, protopine, tetrahydropalmatine, and palmatine and adjacent chromatographic peaks was greater than 1.5, achieving baseline separation, and the symmetry factor was 1.0. Therefore, Gradient Elution Program 5 of acetonitrile-phosphate buffer (pH 5.8) in Table 5 was selected, as shown in Table 6 for details.

[0062] Table 6 Resolution Results of Different Elution Programs

[0063]

[0064] The finally established detection conditions are:

[0065] The elution in the chromatographic conditions for HPLC detection is gradient elution, and the gradient elution program is as follows: 0 - 25 min, 5% A; 25 - 35 min, 5% - 7% A; 35 - 50 min, 7% - 15% A; 50 - 60 min, 15% - 19% A; 60 - 80 min, 19% - 20% A; 80 - 90 min, 20% - 35% A; 90 - 115 min, 35% A; 115 - 120 min, 35% - 44% A; 120 - 145 min, 44% - 48% A. The flow rate for HPLC detection is 0.9 mL / min, using a Phenomenex Gemini C18 chromatographic column, with a column temperature of 30 °C, an injection volume of 10 μL, and a detection wavelength of 210 nm.

[0066] 1.2 System suitability test

[0067] 1.2.1 Preparation of reference solution

[0068] Appropriately weigh an appropriate amount of reference substances of matrine, sophocarpine, sinomenine, protopine, tetrahydropalmatine, and palmatine, and accurately weigh them. Dissolve them in methanol to prepare a mixed reference solution containing 31.94 μg, 12.83 μg, 36.60 μg, 1.10 μg, 1.88 μg, and 1.68 μg of matrine, sophocarpine, sinomenine, protopine, tetrahydropalmatine, and palmatine per 1 mL respectively.

[0069] 1.2.2 Preparation of test solution

[0070] Take about 0.3 g of the granules of this product, accurately weigh it, place it in a stoppered conical flask, add 30 mL of purified water, and ultrasonically treat it for 15 minutes to dissolve (power 500 W, frequency 40 kHz). Then transfer it to a separating funnel, add chloroform and extract 5 times, 30 mL each time. Combine the chloroform layers, evaporate to dryness, dissolve the residue in methanol, and make up the volume to 10 mL in a volumetric flask to obtain the solution.

[0071] 1.2.3 Determination

[0072] Precisely pipette 10 μL of the reference solution, inject it into the liquid chromatograph, continuously inject samples repeatedly 6 times, perform the determination, and calculate the relative standard deviation of the measured peak area values; precisely pipette 10 μL of the test solution, inject it into the high-performance liquid chromatograph, detect according to the chromatographic conditions screened above, continuously inject samples repeatedly 6 times, and calculate the peak area, theoretical plate number, resolution, and tailing factor of the components to be measured.

[0073] 1.2.4 Results

[0074] Under the above chromatographic conditions, the components to be measured in the test sample chromatogram are baseline-separated from the adjacent chromatographic peaks, and the symmetry factor, relative standard deviation of the chromatographic system repeatability, and theoretical plate number all meet the analysis requirements, indicating good system suitability.

[0075] 1.3 Investigation on the preparation method of the test sample solution

[0076] To optimize a reasonable and simple preparation method for the test sample solution, the sample amount, extraction times, and extraction volume were investigated respectively. The results are shown in Tables 7 - 9.

[0077] Table 7 Results of the sample amount test

[0078]

[0079] Table 8 Results of the extraction times test

[0080]

[0081] Table 9 Results of the extraction volume test

[0082]

[0083]

[0084] According to the above test results, the preparation method of the test sample solution was determined as follows: Take about 0.3 g of the granules of this product, accurately weigh, place in a stoppered conical flask, add 30 mL of purified water, ultrasonically treat for 15 minutes to dissolve (power 500 W, frequency 40 kHz), then transfer to a separatory funnel, add chloroform for extraction 5 times, 30 mL each time, combine the chloroform layers, evaporate to dryness, dissolve the residue in methanol, and dilute to 10 mL in a volumetric flask, that is obtained.

[0085] 1.4 Specificity test

[0086] 1.4.1 Preparation of the reference solution

[0087] Take appropriate amounts of matrine, sophocarpine, sinomenine, protopine, tetrahydropalmatine, and palmatine reference substances, accurately weigh, and dissolve in methanol to prepare a mixed reference solution containing 31.94 μg, 12.83 μg, 36.60 μg, 1.10 μg, 1.88 μg, and 1.68 μg of matrine, sophocarpine, sinomenine, protopine, tetrahydropalmatine, and palmatine respectively per 1 mL.

[0088] 1.4.2 Preparation of the test sample solution and the negative test sample solution

[0089] Take about 0.3 g of the granules of this product, accurately weigh, place in a stoppered conical flask, add 30 mL of purified water, ultrasonically treat for 15 minutes to dissolve (power 500 W, frequency 40 kHz), then transfer to a separating funnel, add chloroform and extract 5 times, 30 mL each time. Combine the chloroform layers, evaporate to dryness, dissolve the residue in methanol, and make up the volume to 10 mL in a volumetric flask to obtain the solution. Prepare the negative control sample solution without Sinomenium acutum, Sophora flavescens, and Corydalis yanhusuo in the same way.

[0090] 1.4.3 Determination

[0091] Precisely pipette 10 μL each of the reference substance solution, the test sample solution, and the negative control sample solution, inject into a high performance liquid chromatograph for determination. The results show that the negative control sample has no interference in the determination of matrine, sophocarpine, sinomenine, protopine, tetrahydropalmatine, and tetrahydropalmatine. The results are shown in the appendix Figure 3 .

[0092] 1.5 Investigation of linear relationship

[0093] Weigh appropriate amounts of matrine, sophocarpine, sinomenine, protopine, tetrahydropalmatine, and palmatine reference substances accurately, and dissolve them separately in methanol to prepare solutions with concentrations of 0.7988 mg / mL for matrine, 0.3450 mg / mL for sophocarpine, 0.9113 mg / mL for sinomenine, 0.0263 mg / mL for protopine, 0.045 mg / mL for tetrahydropalmatine, and 0.0413 mg / mL for palmatine, which are the stock solutions of matrine, sophocarpine, sinomenine, protopine, tetrahydropalmatine, and palmatine reference substances. Take 0.1 mL, 0.2 mL, 0.4 mL, 1.0 mL, 2.0 mL, 4.0 mL, and 10.0 mL of this stock solution and place them in 10-mL volumetric flasks respectively, then add methanol to prepare mixed reference substance solutions 1 - 7 with a series of standard curve concentrations. Precisely pipette 10 μL of each of the above solutions and inject them into the high-performance liquid chromatograph, and record the peak areas of the chromatographic peaks. Using the concentration (μg / mL) as the abscissa (X) and the peak area as the ordinate (Y), plot the standard curve and calculate the regression equation and correlation coefficient as follows: For matrine: Y = 19188.83X - 4777.92; r = 0.9999; for sophocarpine: Y = 27119.81X + 5174.56; r = 0.9999; for sinomenine: Y = 44623.79X - 24674.28; r = 1.0000; for protopine: Y = 87252.11X + 886.14; r = 0.9999; for tetrahydropalmatine: Y = 91187.27X - 1172.67; r = 1.0000; for palmatine: Y = 86913.70X - 966.72; r = 1.0000. As a result, matrine and sinomenine showed good linear relationships in the concentration range of 1 - 6 (matrine 8.0 - 319.4 μg / mL, sinomenine 9.2 - 366.0 μg / mL); sophocarpine, protopine, tetrahydropalmatine, and palmatine showed good linear relationships in the concentration range of 1 - 7 (sophocarpine 3.2 - 320.7 μg / mL, protopine 0.3 - 27.4 μg / mL, tetrahydropalmatine 0.5 - 47.0 μg / mL, palmatine 0.4 - 42.0 μg / mL), and r was greater than or equal to 0.9999. The results are shown in Tables 10 - 15.

[0094] Table 10 Results of the investigation on the linear relationship of matrine

[0095]

[0096] Table 11 Results of the investigation on the linear relationship of sophocarpine

[0097]

[0098]

[0099] Table 12 Results of the investigation on the linear relationship of sinomenine

[0100]

[0101] Results of the investigation on the linear relationship of protopine

[0102]

[0103] Results of the investigation on the linear relationship of tetrahydropalmatine

[0104]

[0105] Results of the investigation on the linear relationship of corydaline

[0106]

[0107] 1.6 Precision test

[0108] Precisely pipette 10 μL of the reference substance solutions at three different concentrations (matrine concentrations are 7.98, 31.94, 319.37 μg / mL respectively; sophocarpine concentrations are 3.21, 12.83, 320.75 μg / mL respectively; sinomenine concentrations are 9.15, 36.60, 366.03 μg / mL respectively; protopine concentrations are 0.27, 1.10, 27.44 μg / mL respectively; tetrahydropalmatine concentrations are 0.47, 1.88, 46.96 μg / mL respectively; corydaline concentrations are 0.42, 1.68, 42.00 μg / mL respectively) and the test solution, inject them repeatedly for 6 times, measure, and calculate the injection precision. The results show that the instrument precision is good. The results are shown in Tables 16 and 17.

[0109] Table 16 Results of the precision test of the reference substance solution

[0110]

[0111]

[0112] Table 17 Results of the precision test of the test solution

[0113]

[0114] 1.7 Stability test

[0115] Take the same reference solution (matrine concentration: 79.84 μg / mL; sophocarpine concentration: 32.07 μg / mL; sinomenine concentration: 91.51 μg / mL; protopine concentration: 2.74 μg / mL; tetrahydropalmatine concentration: 4.70 μg / mL; corydalis A concentration: 4.20 μg / mL) and the test solution, inject samples at 0 h, 5 h, 10 h, 15 h, 20 h, 25 h, 30 h, 35 h, 40 h, and 45 h respectively, with each injection volume of 10 μL, and conduct the determination. Calculate the RSD of the peak areas of the reference solution, which are 0.95% for matrine, 1.07% for sophocarpine, 0.94% for sinomenine, 1.42% for protopine, 0.85% for tetrahydropalmatine, and 0.74% for corydalis A; the RSD of the peak areas of the test solution are 0.64% for matrine, 0.60% for sophocarpine, 0.55% for sinomenine, 0.79% for protopine, 0.38% for tetrahydropalmatine, and 1.14% for corydalis A. The results show that the reference solution and the test solution are basically stable when placed at room temperature for 45 h. The results are shown in Tables 18 and 19.

[0116] Table 18 Results of the stability test of the reference substance

[0117]

[0118] Table 19 Results of the stability test of the test substance

[0119]

[0120] 1.8 Repeatability test

[0121] Take about 0.3 g of the traditional Chinese medicine composition granules, prepare 6 portions of the test solution, conduct the determination, calculate the content. The content of matrine in the sample is measured to be 1.83 mg / g, with an RSD of 2.82%; the content of sophocarpine is 0.78 mg / g, with an RSD of 2.86%; the content of sinomenine is 2.30 mg / g, with an RSD of 2.77%; the content of protopine is 0.07 mg / g, with an RSD of 1.57%; the content of tetrahydropalmatine is 0.10 mg / g, with an RSD of 2.70%; the content of corydalis A is 0.07 mg / g, with an RSD of 3.57%. The results show that the method has good repeatability. The results are shown in Table 20.

[0122] Table 20 Results of the repeatability test

[0123]

[0124] 1.9 Recovery test

[0125] Take about 0.15 g of a sample with known content (matrine content is 1.83 mg / g, sophocarpine content is 0.78 mg / g, sinomenine content is 2.30 mg / g, protopine content is 0.07 mg / g, tetrahydropalmatine content is 0.10 mg / g, and palmatine content is 0.07 mg / g), accurately weigh it, weigh 9 portions in parallel, 3 portions as a group, place them in a stoppered conical flask. To each group, add appropriate amounts of reference substance solutions of matrine, sophocarpine, sinomenine, protopine, tetrahydropalmatine, and palmatine (matrine 319.37 mg / mL, sophocarpine 128.30 mg / mL, sinomenine 366.03 mg / mL, protopine 10.98 mg / mL, tetrahydropalmatine 18.78 mg / mL, palmatine 16.80 mg / mL), and then prepare the test solution according to the method. Determine the content, and calculate that the average recovery rate of matrine is 94.32%, RSD = 3.24%; the average recovery rate of sophocarpine is 96.89%, RSD = 4.99%; the average recovery rate of sinomenine is 97.83%, RSD = 4.95%; the average recovery rate of protopine is 109.53%, RSD = 4.85%; the average recovery rate of tetrahydropalmatine is 99.43%, RSD = 2.52%; the average recovery rate of palmatine is 87.13%, RSD = 5.95%. The test results show that this method has good accuracy. The results are shown in Tables 21 - 26.

[0126] Table 21 Results of the Recovery Test of Matrine

[0127]

[0128] Table 22 Results of the Recovery Test of Sophocarpine

[0129]

[0130] Table 23 Results of the Recovery Test of Sinomenine

[0131]

[0132] Table 24 Results of the Recovery Test of Protopine

[0133]

[0134]

[0135] Table 25 Results of the Recovery Test of Tetrahydropalmatine

[0136]

[0137] Table 26 Results of the Recovery Test of Palmatine

[0138]

[0139] Preparation Example: Take 328 g of Sinomenium acutum, 328 g of Sophora flavescens, 492 g of Siegesbeckia orientalis, 492 g of Corydalis yanhusuo processed with vinegar, 492 g of Ajuga ciliata, 492 g of Ilex rotunda, 328 g of Dioscorea hypoglauca, 393.6 g of Eleutherococcus senticosus, and 328 g of Anemarrhena asphodeloides. Add 10 times the amount of water and decoct twice, each time for 1.5 hours. Concentrate under reduced pressure to an extract with a relative density of 1.08 - 1.12 (60°C - 70°C), spray dry, pulverize, add excipients such as dextrin, stevioside, sucralose, aspartame, mannitol, thaumatin, etc., and granulate by dry granulation to obtain the product.

[0140] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art of this technology, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for detecting the content of a Chinese medicine composition, wherein the Chinese medicine composition comprises Caulis Sinomenii, Sophora flavescens, Rhizoma Anemarrhenae, Herba Siegesbeckiae, Corydalis, Herba Ajugae, Herba Scutellariae, Herba Dioscoreae, Herba Acanthopanacis senticosus; characterized in that, The test solution of the Chinese medicine composition was taken for HPLC detection. The chromatographic conditions of HPLC detection included: A C18 chromatographic column (5 μm, 4.6×250 mm) is used; acetonitrile is used as mobile phase A, and phosphate buffer solution (pH 5.8) is used as mobile phase B. The elution in the chromatographic conditions of the HPLC detection is gradient elution, and the gradient elution program is: 0-25 min, 5% A; 25-35 min, 5%-7% A; 35-50 min, 7%-15% A; 50-60 min, 15%-19% A; 60-80 min, 19%-20% A; 80-90 min, 20%-35% A; 90-115 min, 35% A; 115-120 min, 35%-44% A; 120-145 min, 44%-48% A.

2. The method according to claim 1, characterized in that The preparation of the test solution includes taking the product, adding 30 mL of pure water, ultrasonically treating for 15 minutes to dissolve (power 500 W, frequency 40 kHz), then transferring to a separatory funnel, adding chloroform to extract 5 times, 30 mL each time, combining the chloroform layers, evaporating to dryness, adding methanol to dissolve the residue, and diluting to a 10 mL volumetric flask to obtain the product.

3. The method according to claim 1, characterized in that The chromatographic conditions include: a flow rate of 0.8 to 1.0 mL / min, a column temperature of 25 to 35° C., and a detection wavelength of 208 to 212 nm.

4. The method according to claim 1, characterized in that: The chromatographic conditions include: the flow rate is 0.9 mL / min, the column temperature is 30° C., and the detection wavelength is 210 nm.

5. The method according to claim 1, characterized in that The C18 chromatographic column is selected from a Phenomenex Gemini C18 chromatographic column or a Phenomenex Luna C18 chromatographic column.

6. The method according to claim 1, characterized in that The method further comprises the detection of a reference substance, wherein the reference substance comprises one or more of matrine, sophocarpine, sinomenine, protopine, tetrahydropalmatine, and tetrahydropalmatine standard substances.

7. The method according to claim 6, characterized in that The method for preparing the reference substance into a solution comprises: taking one or more of matrine, sophocarpine, sinomenine, protopine, tetrahydropalmatine, and tetrahydropalmatine standard substances, and adding methanol to prepare a single standard or mixed standard solution containing 7.98-319.37 μg of matrine, 3.21-320.75 μg of sophocarpine, 9.15-366.03 μg of sinomenine, 0.27-27.44 μg of protopine, 0.47-46.96 μg of tetrahydropalmatine, and 0.42-42.00 μg of tetrahydropalmatine per 1 mL.

8. The method according to claim 1, characterized in that: The Chinese medicine composition includes solid, liquid or external preparation.

9. The method according to claim 1, characterized in that When the Chinese medicine composition is in the form of granules, its preparation method comprises: adding 10 times the amount of water to nine medicinal materials including Caulis Sinomenii, Sophora flavescens, Siegesbeckia ulmoides, Corydalis yanhusuo, Ajuga chinensis, Rhizoma Cibotii, Radix Dioscoreae, Acanthopanax senticosus and Rhizoma Anemarrhenae, boiling and decocting for 2 times, each time for 1.5 hours, concentrating under reduced pressure to an extract with a relative density of 1.08-1.12 (60° C.-70° C.), spray drying, crushing, adding auxiliary materials, and dry granulating to obtain the granules.

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