Construction method of characteristic chromatogram of traditional Chinese medicine compound preparation and content determination method of five index components of traditional Chinese medicine compound preparation

Abstract

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a construction method of a characteristic chromatogram of a traditional Chinese medicine compound preparation and a content determination method of five index components of the traditional Chinese medicine compound preparation. According to the technical scheme, the conditions of liquid chromatography detection are as follows: a mobile phase A is acetonitrile, and a mobile phase B is water; the volume percentage of the mobile phase A is maintained to be 12% in 0-10 min; the volume percentage of the mobile phase A is increased to 24% from 12% in 10-11 min; the volume percentage of the mobile phase A is increased from 24% to 26% in 11-30 min; the volume percentage of the mobile phase A is increased from 26% to 45% in 30-31 min; the volume percentage of the mobile phase A is increased to 47% from 45% in 31-45 min. According to the technical scheme, the five index components of the traditional Chinese medicine compound preparation can be accurately measured in one chromatographic detection system at the same time, operation is easy, and calculation is convenient.

Description

Technical Field

[0001] The present application relates to the technical field of traditional Chinese medicine detection, and in particular to a method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation and a method for determining the contents of its five index components. Background Art

[0002] Shen Ge Bushen Capsule is a traditional Chinese medicine compound preparation. The main raw materials are Pueraria root, Epimedium and Radix Pseudostellariae, which are used as medicine with their effective ingredients Pueraria root, Epimedium flavonoids and Radix Pseudostellariae polysaccharides respectively. It has the effects of nourishing the kidney and replenishing essence, warming and nourishing kidney yang, and is mainly used to treat mild to moderate depression caused by deficiency of both qi and yin and deficiency of kidney qi. The existing quality standards of this prescription mainly control puerarin and icariin, and do not measure other active ingredients. In order to better control the changes in the quantity and quality transfer of effective ingredients during the production process, it is necessary to develop a content determination method that can simultaneously determine multiple active ingredients.

[0003] Pueraria root is the dried root of Pueraria lobata or Pueraria lobata, a plant of the Leguminosae family. It is a traditional Chinese medicinal material that is both a medicine and food. Its main pharmacologically active ingredients are isoflavone compounds, among which puerarin accounts for the largest proportion. It has multiple pharmacological effects such as antioxidant, anti-inflammatory, cardioprotective, vasodilation, anti-cancer, and neuroprotective effects, and can be used to treat diseases such as Alzheimer's disease and Parkinson's disease. Pseudostellaria heterophylla is the dried root of the Caryophyllaceae plant Herba Glehniae, which has the effects of invigorating qi and strengthening the spleen, promoting fluid production and moistening the lungs. Polysaccharides are one of its main active ingredients, and have multiple biological activities such as hypoglycemic, anti-inflammatory, and immunomodulatory. Epimedium is the dried leaves of the plants Epimedium, Epimedium sagittatum, Epimedium pubescens and Epimedium koreana of the Berberidaceae family. It has the effects of nourishing kidney yang, strengthening tendons and bones, and removing rheumatism. Flavonoids such as chondrin A, chondrin B, chondrin C and icariin are its main active ingredients, which have antibacterial, antioxidant, anti-tumor, immune enhancement, cardiovascular and cerebrovascular function improvement, endocrine regulation, osteoporosis resistance, anti-inflammatory, and learning and memory enhancement. Therefore, using icariin, chondrin A, chondrin B, chondrin C, and puerarin as indicator components in the liquid phase detection of Shen Ge Bushen Capsules helps to better control the changes in the quantity and quality transfer of effective ingredients during the production process.

[0004] There have been many reports on the detection methods for the above-mentioned index components. For example, Gao Junqing et al. used acetonitrile and 1.0 g / L phosphoric acid gradient elution to determine puerarin in Naomai Tai Capsules, Li Jing used formic acid and water isocratic elution to determine puerarin in Ge Ke Tong Luo Capsules, Yang Ru and Teng Xichao et al. used acetonitrile and water gradient elution to determine icariin, epimedin A, epimedin B, epimedin C and baohuoside I, Feng Liang et al. used acetonitrile and 0.5% formic acid gradient elution combined with the three-wavelength switching method to determine icariin in Xianling Gubao Capsules. However, the above elution procedure was not used for the determination of the traditional Chinese medicine compound preparation composed of Pueraria lobata, epimedium and Pseudostellaria heterophylla, and its applicability was poor when used for this prescription, and the separation effect and peak shape of the chromatographic peaks were poor. A method disclosed a HPLC determination method for the content of icariin, epimedin A, epimedin B, and epimedin C in Shengge Bushen Capsule, using high performance liquid chromatography, the chromatographic column was Waters ACQUITY UPLC HSS T3 C18 (2.1×100 mm, 1.8 μm), and the mobile phase was acetonitrile-0.05% phosphoric acid aqueous solution system gradient elution. However, this method did not take puerarin into account and could not effectively separate it. In addition, there are methods that report the development of a chromatographic detection method for puerarin, which also cannot achieve the simultaneous determination of puerarin and total flavonoid glycosides in epimedium.

[0005] Based on this, it is necessary to develop a method for the simultaneous determination of icariin, chamotidine A, chamotidine B, chamotidine C and puerarin in Shengge Bushen Capsule. Summary of the invention

[0006] Based on this, one or more embodiments of the present application provide a method for constructing a characteristic spectrum of a Chinese medicine compound preparation and a method for determining the contents of its five index components, wherein the separation degree of each index component is high, the chromatographic peak shape is good, and simultaneous determination can be achieved under one chromatographic condition, and the determination method has been methodologically validated and passed the specificity, precision, repeatability and durability tests.

[0007] The technical solution of this application includes the following contents:

[0008] A method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation, wherein the traditional Chinese medicine compound preparation is a mixture of extracts of Pueraria root, Radix Pseudostellariae and Herba Epimedii;

[0009] The method for constructing the characteristic map comprises the following steps:

[0010] Take the Chinese herbal compound preparation to be tested, add the extraction solvent to extract it, and obtain the test solution;

[0011] The test solution is subjected to liquid chromatography detection to obtain a characteristic spectrum of the Chinese medicine compound preparation;

[0012] The conditions for the liquid chromatography detection include:

[0013] (1) Acetonitrile was used as mobile phase A and water was used as mobile phase B;

[0014] (2) Gradient elution is used, and the procedure of gradient elution includes:

[0015] 0min~10min, maintaining the volume percentage of the mobile phase A at 12%;

[0016] From 10min to 11min, the volume percentage of the mobile phase A increased from 12% to 24%;

[0017] From 11min to 30min, the volume percentage of the mobile phase A increased from 24% to 26%;

[0018] From 30min to 31min, the volume percentage of the mobile phase A increased from 26% to 45%;

[0019] From 31min to 45min, the volume percentage of the mobile phase A increased from 45% to 47%;

[0020] (3) The detection wavelength is 265nm~275nm.

[0021] In some embodiments, the conditions for liquid chromatography detection further include at least one of the following:

[0022] (1) The filler of the chromatographic column is octadecylsilane bonded silica gel;

[0023] (2) Column temperature is 28°C~32°C;

[0024] (3) The injection volume is 3 μL to 7 μL; and

[0025] (4) The flow rate is 0.8 mL / min~1.2 mL / min.

[0026] In some embodiments, the length of the chromatographic column is 240 mm to 260 mm, the diameter is 4.4 mm to 4.8 mm, and the particle size of the filler is 5 μm.

[0027] In some embodiments, the extraction solvent is an alcohol aqueous solution with a volume concentration ratio of 70% to 90%, and optionally, the alcohol includes ethanol; and / or,

[0028] The extraction method includes ultrasound, and optionally, the frequency of ultrasound is 240W-260W, 30kHz-50kHz, and the time is 20min-40min; and / or,

[0029] The mass volume ratio of the tested Chinese herbal compound preparation and the extraction solvent is 0.2~0.6g:100mL.

[0030] In some embodiments, the characteristic spectrum of the Chinese medicine compound preparation includes 5 characteristic peaks, namely peak 1, peak 2, peak 3, peak 4, and peak 5;

[0031] Among them, peak 1 is puerarin, peak 2 is epimedin A, peak 3 is epimedin B, peak 4 is epimedin C, and peak 5 is icariin.

[0032] Taking peak 1 as the reference peak, the relative retention times of the other characteristic peaks are as follows: peak 2: 3.11±10%; peak 3: 3.26±10%; peak 4: 3.45±10%; peak 5: 3.66±10%.

[0033] A method for determining the contents of five index components in a traditional Chinese medicine compound preparation, wherein the traditional Chinese medicine compound preparation is a mixture of water extracts of Pueraria lobata, Radix Pseudostellariae and Epimedium; the five index components are puerarin, epimedin A, epimedin B, epimedin C and icariin in order;

[0034] The content determination method comprises the following steps:

[0035] A mixture of the reference substances of the five index components is taken, and liquid chromatography is performed with different injection volumes of the reference substance solution to construct a standard curve of the mass and peak area of ​​puerarin, epimedin A, epimedin B, epimedin C and icariin;

[0036] Take the Chinese herbal compound preparation to be tested, add an extraction solvent to extract it, and obtain a test solution;

[0037] Taking the test solution for liquid chromatography detection, obtaining the peak areas of puerarin, epimedin A, epimedin B, epimedin C and icariin in the test solution, and substituting them into the standard curves of the concentration and peak area of ​​puerarin, epimedin A, epimedin B, epimedin C and icariin for calculation;

[0038] Wherein, the conditions for the liquid chromatography detection include:

[0039] (1) Acetonitrile was used as mobile phase A and water was used as mobile phase B;

[0040] (2) Gradient elution is used, and the procedure of gradient elution includes:

[0041] 0min~10min, maintaining the volume percentage of the mobile phase A at 12%;

[0042] From 10min to 11min, the volume percentage of the mobile phase A increased from 12% to 24%;

[0043] From 11min to 30min, the volume percentage of the mobile phase A increased from 24% to 26%;

[0044] From 30min to 31min, the volume percentage of the mobile phase A increased from 26% to 45%;

[0045] From 31min to 45min, the volume percentage of the mobile phase A increased from 45% to 47%;

[0046] (3) The detection wavelength is 265nm~275nm.

[0047] In some embodiments, the conditions for liquid chromatography detection further include at least one of the following:

[0048] (1) The filler of the chromatographic column is octadecylsilane bonded silica gel;

[0049] (2) Column temperature is 28°C~32°C;

[0050] (3) The injection volume is 3 μL to 7 μL; and

[0051] (4) The flow rate is 0.8 mL / min~1.2 mL / min.

[0052] In some embodiments, the length of the chromatographic column is 240 mm to 260 mm, the diameter is 4.4 mm to 4.8 mm, and the particle size of the filler is 4 μm to 6 μm.

[0053] In some embodiments, the extraction solvent is an alcohol aqueous solution with a volume concentration ratio of 70% to 90%, and optionally, the alcohol includes ethanol; and / or,

[0054] The extraction method includes ultrasound, and optionally, the frequency of ultrasound is 240W-260W, 30kHz-50kHz, and the time is 20min-40min; and / or,

[0055] The mass volume ratio of the Chinese herbal compound preparation to be tested and the extraction solvent is 0.2-0.6 g:100 mL.

[0056] In some embodiments, the dissolving solvent is an alcohol aqueous solution with a volume concentration ratio of 70% to 90%, and optionally, the alcohol includes ethanol.

[0057] The technical solution of the present application realizes the characterization of the five index components of the Chinese medicine compound preparation, namely puerarin, epimedin A, epimedin B, epimedin C and icariin, based on the characteristic spectrum of the Chinese medicine compound preparation established based on a suitable liquid chromatography analysis method. The chromatographic peaks of each index component have good separation and are not interfered with each other. The content can be determined in the same chromatographic detection system, thereby providing a reference for the quality control of the Chinese medicine compound preparation.

[0058] The technical solution of the present application realizes the simultaneous determination of the contents of the five index components in the traditional Chinese medicine compound preparation, reduces the complex operation of the existing standard for their detection using separate liquid phase conditions, improves the inspection efficiency, and reduces the amount of organic reagents used. The external standard method is selected, which is simple to operate and convenient to calculate. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] 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.

[0060] Figure 1 The chromatogram of the test solution obtained using the chromatographic detection conditions of Scheme 1 in Section 1.4.6 of this application;

[0061] Figure 2 The chromatogram of the test solution obtained using the chromatographic detection conditions of Scheme 2 in Section 1.4.6 of this application;

[0062] Figure 3 The chromatogram of the test solution obtained using the chromatographic detection conditions of Scheme 3 in Section 1.4.6 of this application;

[0063] Figure 4 The chromatogram of the test solution obtained using the chromatographic detection conditions of Scheme 4 in Section 1.4.6 of this application;

[0064] Figure 5 The chromatogram of the test solution obtained using the chromatographic detection conditions of Scheme 5 in Section 1.4.6 of this application;

[0065] Figure 6 The chromatogram of the test solution obtained using the chromatographic detection conditions of Scheme 6 in Section 1.4.6 of this application;

[0066] Figure 7 The chromatogram of the test solution obtained using the chromatographic detection conditions of Scheme 7 in Section 1.4.6 of this application;

[0067] Figure 8 The chromatogram of the test solution obtained using the chromatographic detection conditions of Scheme 8 in Section 1.4.6 of this application;

[0068] Fig. 9 The chromatograms of blank excipients, negative samples and test solution obtained using the chromatographic detection conditions of Scheme 8 for this application;

[0069] Fig.10This is the chromatogram of the mixed reference substance obtained using the chromatographic detection conditions of Scheme 8 in this application. DETAILED DESCRIPTION

[0070] The present application will be further described below in conjunction with the embodiments and examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. In addition, it should be understood that after reading the content taught in the present application, those skilled in the art may make various changes or modifications to the present application, and these equivalent forms also fall within the protection scope of the claims attached to the present application.

[0071] 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.

[0072] the term

[0073] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0074] The terms "and / or", "or / and", and "and / or" used in this article have a selection range that includes any one of two or more related listed items, and also includes any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0075] Herein, "preferred", "preferable", "better", etc. are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0076] In the present application, "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 the present application.

[0077] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0078] 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.

[0079] In this application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0080] The technical solution of the present application is based on a suitable liquid chromatography analysis method and a characteristic spectrum of a Chinese medicine compound preparation established based on a dual-wavelength detection system to achieve the characterization of five index components of the Chinese medicine compound preparation - icariin, epimedin A, epimedin B, epimedin C, and puerarin. The chromatographic peaks of each index component have good separation and are not mutually interfering. The content determination can be achieved in the same chromatographic detection system, thereby providing a reference for the quality control of the Chinese medicine compound preparation.

[0081] Herein, the Chinese herbal compound preparation is a mixture of extracts of Pueraria lobata, Pseudostellaria heterophylla and Epimedium brevicornum.

[0082] In this article, the five index ingredients in the Chinese herbal compound preparation are puerarin, epimedin A, epimedin B, epimedin C and icariin.

[0083] The method for constructing a characteristic spectrum of a Chinese medicine compound preparation according to an embodiment of the present application comprises the following steps:

[0084] Take the Chinese herbal compound preparation to be tested, add the extraction solvent to extract it, and obtain the test solution;

[0085] The test solution is subjected to liquid chromatography to obtain a characteristic spectrum of the Chinese medicine compound preparation;

[0086] The conditions for liquid chromatography detection include:

[0087] (1) Acetonitrile was used as mobile phase A and water was used as mobile phase B;

[0088] (2) Gradient elution is used. The procedure of gradient elution includes:

[0089] 0min~10min, maintain the volume percentage of mobile phase A at 12%;

[0090] From 10 to 11 minutes, the volume percentage of mobile phase A increased from 12% to 24%;

[0091] From 11min to 30min, the volume percentage of mobile phase A increased from 24% to 26%;

[0092] From 30 to 31 minutes, the volume percentage of mobile phase A increased from 26% to 45%;

[0093] From 31 to 45 minutes, the volume percentage of mobile phase A increased from 45% to 47%;

[0094] (3) Detection wavelength: 265nm~275nm.

[0095] In some embodiments, the conditions for liquid chromatography detection further include at least one of the following:

[0096] (1) The filler of the chromatographic column is octadecylsilane bonded silica gel;

[0097] (2) Column temperature is 28°C~32°C;

[0098] (3) The injection volume is 3 μL to 7 μL; and

[0099] (4) The flow rate is 0.8 mL / min~1.2 mL / min.

[0100] In some embodiments, the length of the chromatographic column is 240 mm to 260 mm, the diameter is 4.4 mm to 4.8 mm, and the particle size of the filler is 4 μm to 6 μm.

[0101] Optionally, the length of the chromatographic column can be 240 mm, 250 mm, 260 mm, etc., the diameter can be 4.4 mm, 4.5 mm, 4.6 mm, etc., and the particle size of the filler can be 4 μm, 5 μm, 6 μm, etc.

[0102] In some embodiments, the extraction solvent is an alcohol aqueous solution with a volume concentration ratio of 70% to 90%, and optionally, the alcohol includes ethanol.

[0103] In this embodiment, the volume concentration ratio of the alcohol aqueous solution is 70% to 90%, for example 70%, 75%, 80%, 85%, 90%, etc.

[0104] In some embodiments, the extraction method includes ultrasound, and optionally, the power of the ultrasound is 240W~260W, the frequency is 30kHz~50kHz, and the time is 20min~40min.

[0105] Optionally, the power of ultrasound can be 240W, 250W, 260W, etc., the frequency can be 30kHz, 40kHz, 50kHz, etc., and the time can be 20min, 30min, 40min, etc.

[0106] In some embodiments, the mass volume ratio of the tested Chinese herbal compound preparation and the extraction solvent is 0.2~0.6g:100mL. For example, the mass volume ratio of the tested Chinese herbal compound preparation and the extraction solvent can be 0.2g:100mL, 0.3g:100mL, 0.4g:100mL, 0.5g:100mL, 0.6g:100mL, etc.

[0107] In some embodiments, the characteristic spectrum of the Chinese medicine compound preparation includes 5 characteristic peaks, namely peak 1, peak 2, peak 3, peak 4, and peak 5;

[0108] Among them, peak 1 is puerarin, peak 2 is epimedin A, peak 3 is epimedin B, peak 4 is epimedin C, and peak 5 is icariin;

[0109] Taking peak 1 as the reference peak, the relative retention times of the other characteristic peaks are as follows: peak 2: 3.11±10%; peak 3: 3.26±10%; peak 4: 3.45±10%; peak 5: 3.66±10%.

[0110] The method for determining the contents of five index components in a Chinese medicine compound preparation according to an embodiment of the present application comprises the following steps:

[0111] A mixture of reference substances of the five index components was taken, and liquid chromatography was performed with different injection volumes of reference substance solutions to construct standard curves of the mass and peak area of ​​puerarin, epimedin A, epimedin B, epimedin C and icariin;

[0112] Take the Chinese herbal compound preparation to be tested, add an extraction solvent to extract it, and obtain a test solution;

[0113] Taking the test solution for liquid chromatography detection, obtaining the peak areas of puerarin, epimedin A, epimedin B, epimedin C and icariin in the test solution, and substituting them into the standard curves of concentration and peak area of ​​puerarin, epimedin A, epimedin B, epimedin C and icariin for calculation;

[0114] Among them, the conditions for liquid chromatography detection include:

[0115] (1) Acetonitrile was used as mobile phase A and water was used as mobile phase B;

[0116] (2) Gradient elution is used. The procedure of gradient elution includes:

[0117] 0min~10min, maintain the volume percentage of mobile phase A at 12%;

[0118] From 10 to 11 minutes, the volume percentage of mobile phase A increased from 12% to 24%;

[0119] From 11min to 30min, the volume percentage of mobile phase A increased from 24% to 26%;

[0120] From 30 to 31 minutes, the volume percentage of mobile phase A increased from 26% to 45%;

[0121] From 31 to 45 minutes, the volume percentage of mobile phase A increased from 45% to 47%;

[0122] (3) The detection wavelength is 265nm~275nm.

[0123] In some embodiments, the conditions for liquid chromatography detection further include at least one of the following:

[0124] (1) The filler of the chromatographic column is octadecylsilane bonded silica gel;

[0125] (2) Column temperature is 28°C~32°C;

[0126] (3) The injection volume is 3 μL~7 μL; and

[0127] (4) The flow rate is 0.8 mL / min~1.2 mL / min.

[0128] In the method for determining the contents of the five index components in the traditional Chinese medicine compound preparation of the embodiment of the present application, the various chromatographic conditions used can refer to the definition of the corresponding chromatographic conditions in the characteristic spectrum of the traditional Chinese medicine compound preparation of the embodiment of the present application.

[0129] In the method for determining the contents of five index components in the Chinese herbal compound preparation of the embodiment of the present application, the Chinese herbal compound preparation to be tested is taken, an extraction solvent is added for extraction, and in the step of obtaining a test solution, the extraction solvent and extraction method used can refer to the extraction solvent and extraction method used for extracting the test Chinese herbal compound preparation in the characteristic spectrum of the Chinese herbal compound preparation of the embodiment of the present application.

[0130] In some embodiments, the dissolving solvent is an alcohol aqueous solution with a volume concentration ratio of 70% to 90%, and optionally, the alcohol includes ethanol.

[0131] In this embodiment, the volume concentration ratio of the alcohol aqueous solution is 70% to 90%, for example 70%, 75%, 80%, 85%, 90%, etc.

[0132] In some embodiments, the mass concentration of each indicator component in the reference solution is 1 μg / mL to 120 μg / mL.

[0133] In this embodiment, in the reference solution, the mass concentration of puerarin is 80 μg / mL~120 μg / mL, the mass concentration of icariin is 1 μg / mL~5 μg / mL, the mass concentration of chondrin A is 2 μg / mL~8 μg / mL, the mass concentration of chondrin B is 5 μg / mL~15 μg / mL, and the mass concentration of chondrin C is 15 μg / mL~25 μg / mL.

[0134] The technical solution of the present application realizes the simultaneous determination of the contents of the five index components in the traditional Chinese medicine compound preparation, reduces the complex operation of the existing standard for their detection using separate liquid phase conditions, improves the inspection efficiency, and reduces the amount of organic reagents used. The external standard method is selected, which is simple to operate and convenient to calculate.

[0135] The following are some specific embodiments.

[0136] For experimental parameters not specified in the following specific embodiments, reference is made to the instructions given in the present application document, and reference may also be made to experimental manuals in the art or other experimental methods known in the art, or to experimental conditions recommended by manufacturers.

[0137] The raw materials and reagents involved in the following specific examples can be obtained from commercial sources, or can be prepared by those skilled in the art according to known methods.

[0138] Example 1

[0139] This embodiment provides a method for constructing a characteristic spectrum of the Chinese medicine compound preparation of the present application.

[0140] 1.1. Experimental herbs

[0141] Shen Ge Bu Shen Capsule

[0142] 1.2. Experimental reagents and materials

[0143] Methanol, ultrapure water, ethanol, phosphoric acid, acetonitrile; puerarin reference substance, icariin reference substance, chondrin A reference substance, chondrin B reference substance, chondrin C reference substance, silicon dioxide.

[0144] 1.3. Instruments and Equipment

[0145] Analytical balance; high performance liquid chromatograph; ultrasonic instrument.

[0146] 1.4 Experimental procedures

[0147] 1.4.1. Preparation of reference stock solution

[0148] Accurately weigh appropriate amounts of puerarin reference substance, icariin reference substance, choledoyl A reference substance, choledoyl B reference substance and choledoyl C reference substance, add ethanol to dissolve them, and then add 50% ethanol to dilute to the scale to prepare solutions with concentrations of 1000 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL for puerarin, icariin, choledoyl A, choledoyl B and choledoyl C, respectively, which are used as stock solutions.

[0149] 1.4.2. Preparation of mixed reference solution

[0150] Accurately measure the stock solution in 1.4.1 and add 50% ethanol solution to dilute to the scale to prepare solutions containing 100 μg / mL, 2.0 μg / mL, 5.0 μg / mL, 10 μg / mL, and 20 μg / mL of puerarin, icariin, choledinol A, choledinol B, and choledinol C, respectively, as mixed reference solutions.

[0151] 1.4.3. Preparation of test solution

[0152] Test solution: Take about 10 mg of the contents of Shen Ge Bushen Capsule, accurately weigh it, put it into a 25 mL volumetric flask, add an appropriate amount of 80% ethanol, ultrasonically treat it for 30 minutes, cool it, add 80% ethanol to dilute it to the scale, shake it well, filter it with a microporous filter membrane (0.45 μm), and take the filtrate to obtain the test solution.

[0153] 1.4.4. Negative solution

[0154] Take 80% ethanol solution as the negative sample solution.

[0155] 1.4.5. Blank excipient solution

[0156] Take an appropriate amount of blank auxiliary material (silicon dioxide), about 0.25 g, put it in a 50 mL volumetric flask, add an appropriate amount of 80% ethanol solution, ultrasonicate for 30 minutes, cool, dissolve and dilute to the scale with 80% ethanol solution, shake well, filter with a microporous filter membrane (0.45μm), and take the filtrate to obtain.

[0157] 1.4.6. Chromatographic conditions

[0158] Octadecylsilane bonded silica gel was used as the filler (250 mm × 4.6 mm) and gradient elution was performed according to the following table; the column temperature was 30°C; the injection volume was 5 μL and the detection wavelength was 270 nm; the theoretical plate number was not less than 8000 calculated based on the icariin peak.

[0159] Table 1 Eight elution schemes

[0160]

[0161]

[0162] The chromatographic test results of the above eight schemes are shown in Figures 1 to 8 , where peak 1, peak 2, peak 3, peak 4 and peak 5 are puerarin, epimedin A, epimedin B, epimedin C and icariin, respectively. Comparison shows that Scheme 8 can obtain more characteristic peaks, and each characteristic peak has high separation, moderate response, small impurity interference and stable baseline.

[0163] Scheme 8 was used to detect the test solution, negative solution, and blank auxiliary material to obtain the corresponding chromatograms. The chromatogram comparison results of the test solution, negative solution, and blank auxiliary material solution are as follows: Fig. 9 . Test the mixed reference solution and obtain Fig.10 Chromatogram shown.

[0164] Comparison Fig. 9 and Fig.10 It was found that five characteristic peaks of the reference substance could be detected using Scheme 8 with good separation.

[0165] 2. Methodological investigation

[0166] 2.1 Specificity: Take 5 µL of the test solution, mixed reference solution, negative sample solution and blank excipient solution and inject them into the HPLC chromatograph according to the chromatographic conditions of Scheme VIII under 1.4.6 to confirm whether there is any interference (the test results can be found in Fig. 9 and Fig.10 ).

[0167] 2.2 Precision: Take the test solution and inject it 6 times continuously according to the chromatographic conditions of Scheme VIII under 1.4.6, record the peak area and calculate the relative standard deviation (RSD) of each peak area.

[0168] 2.3 Repeatability: Take samples from the same batch number and prepare 6 test sample solutions in parallel according to the method in 1.4.3. Determine and record the peak areas according to the chromatographic conditions of Scheme VIII in 1.4.6 and calculate the relative standard deviation (RSD) of each peak area.

[0169] 2.4 Durability: Take the same test solution and inject 5 µL at 0h, 4h, 8h, 12h, 18h, 24h, 36h and 48h according to the chromatographic conditions of Scheme VIII in 1.4.6 to measure and record the peak areas of the five components. Calculate the relative standard deviation (RSD) of the peak area corresponding to each component within 48 h.

[0170] Table 2 Relative standard deviation of peak area corresponding to each component of method precision, repeatability and robustness

[0171]

[0172] The present application determines the contents of puerarin and icariin by changing the type of mobile phase (methanol-water, acetonitrile-water and acetonitrile-water-phosphoric acid) and elution program (see eight elution programs in Table 1) in the chromatographic detection, and determines that the mobile phase is acetonitrile (A)-water (B), and the elution program is: 0-10min: 12% A; 10-11min: 12%-24% A; 11-30min: 24%-26% A; 30-31min: 26%-45% A; 31-45min: 45%-47% A. The separation effect of puerarin and icariin is the best, and it is most suitable for the analysis and detection of puerarin and icariin.

[0173] 3. Establishment of standard curve

[0174] Take the mixed reference solution in 1.4.2 and inject 1µL, 2µL, 4µL, 8µL, 10µL, 20µL and 40µL respectively according to the chromatographic conditions of Scheme VIII under 1.4.6 to measure and record the peak areas of the five components. Perform linear regression with the injection volume (μg) as the abscissa (x) and the peak area as the ordinate (y) to calculate the linear regression equation and correlation coefficient of puerarin, chondrin A, chondrin B, chondrin C and icariin. Table 3 shows that the five components have a good linear relationship.

[0175] Table 3 Linear regression equation

[0176]

[0177] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0178] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples 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.

[0179] The above-described embodiments only express several implementation methods of the present application, but they cannot be understood as limiting the scope of the patent application. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present application, several variations and improvements can also be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solutions provided in the present application through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for constructing a characteristic spectrum of a Chinese medicine compound preparation, characterized in that: The Chinese medicine compound preparation is a mixture of extracts of Pueraria root, Radix Pseudostellariae and Herba Epimedii; The method for constructing the characteristic map comprises the following steps: Take the Chinese herbal compound preparation to be tested, add the extraction solvent to extract it, and obtain the test solution; The test solution is subjected to liquid chromatography detection to obtain a characteristic spectrum of the Chinese medicine compound preparation; The conditions for the liquid chromatography detection include: (1) Acetonitrile was used as mobile phase A and water was used as mobile phase B; (2) Gradient elution is used, and the procedure of gradient elution includes: 0min~10min, maintaining the volume percentage of the mobile phase A at 12%; From 10min to 11min, the volume percentage of the mobile phase A increased from 12% to 24%; From 11min to 30min, the volume percentage of the mobile phase A increased from 24% to 26%; From 30min to 31min, the volume percentage of the mobile phase A increased from 26% to 45%; From 31min to 45min, the volume percentage of the mobile phase A increased from 45% to 47%; (3) Detection wavelength: 265nm~275nm.

2. The construction method according to claim 1, characterized in that: The conditions for the liquid chromatography detection also include at least one of the following: (1) The filler of the chromatographic column is octadecylsilane bonded silica gel; (2) Column temperature is 28°C~32°C; (3) The injection volume is 3 μL to 7 μL; and (4) The flow rate is 0.8 mL / min~1.2 mL / min.

3. The construction method according to claim 2, characterized in that: The length of the chromatographic column is 240 mm to 260 mm, the diameter is 4.4 mm to 4.8 mm, and the particle size of the filler is 4 μm to 6 μm.

4. The construction method according to any one of claims 1 to 3, characterized in that: The extraction solvent is an alcohol aqueous solution with a volume concentration ratio of 70% to 90%, and optionally, the alcohol includes ethanol; and / or, The extraction method includes ultrasound, optionally, the power of ultrasound is 240W~260W, the frequency is 30kHz~50kHz, and the time is 20min~40min; and / or, The mass volume ratio of the tested Chinese herbal compound preparation and the extraction solvent is 0.2~0.6g:100mL.

5. The construction method according to any one of claims 1 to 3, characterized in that: The characteristic spectrum of the Chinese medicine compound preparation includes 5 characteristic peaks, namely peak 1, peak 2, peak 3, peak 4, and peak 5; Among them, peak 1 is puerarin, peak 2 is epimedin A, peak 3 is epimedin B, peak 4 is epimedin C, and peak 5 is icariin; Taking peak 1 as the reference peak, the relative retention times of the other characteristic peaks are as follows: peak 2: 3.11±10%; peak 3: 3.26±10%; peak 4: 3.45±10%; peak 5: 3.66±10%.

6. A method for determining the contents of five index components in a Chinese medicine compound preparation, characterized in that: The Chinese medicine compound preparation is a mixture of water extracts of Pueraria lobata, Radix Pseudostellariae and Epimedium; the five index components are puerarin, epimedin A, epimedin B, epimedin C and icariin in order; The content determination method comprises the following steps: A mixture of the reference substances of the five index components is taken, and liquid chromatography is performed with different injection volumes of the reference substance solution to construct a standard curve of the mass and peak area of ​​puerarin, epimedin A, epimedin B, epimedin C and icariin; Take the Chinese herbal compound preparation to be tested, add an extraction solvent to extract it, and obtain a test solution; Taking the test solution for liquid chromatography detection, obtaining the peak areas of puerarin, epimedin A, epimedin B, epimedin C and icariin in the test solution, and substituting them into the standard curves of mass and peak area of ​​puerarin, epimedin A, epimedin B, epimedin C and icariin for calculation; Wherein, the conditions for the liquid chromatography detection include: (1) Acetonitrile was used as mobile phase A and water was used as mobile phase B; (2) Gradient elution is used, and the procedure of gradient elution includes: 0min~10min, maintaining the volume percentage of the mobile phase A at 12%; From 10min to 11min, the volume percentage of the mobile phase A increased from 12% to 24%; From 11min to 30min, the volume percentage of the mobile phase A increased from 24% to 26%; From 30min to 31min, the volume percentage of the mobile phase A increased from 26% to 45%; From 31min to 45min, the volume percentage of the mobile phase A increased from 45% to 47%; (3) Detection wavelength: 265nm~275nm.

7. The content determination method according to claim 6, characterized in that: The conditions for the liquid chromatography detection also include at least one of the following: (1) The filler of the chromatographic column is octadecylsilane bonded silica gel; (2) Column temperature is 28°C~32°C; (3) The injection volume is 3 μL to 7 μL; and (5) The flow rate is 0.8 mL / min~1.2 mL / min.

8. The content determination method according to claim 7, characterized in that: The length of the chromatographic column is 240 mm to 260 mm, the diameter is 4.4 mm to 4.8 mm, and the particle size of the filler is 4 μm to 6 μm.

9. The content determination method according to any one of claims 6 to 8, characterized in that: The extraction solvent is an alcohol aqueous solution with a volume concentration ratio of 70% to 90%, and optionally, the alcohol includes ethanol; and / or, The extraction method includes ultrasound, and optionally, the frequency of ultrasound is 240W-260W, 30kHz-50kHz, and the time is 20min-40min; and / or, The mass volume ratio of the Chinese herbal compound preparation to be tested and the extraction solvent is 0.2-0.6 g:100 mL.

10. The content determination method according to any one of claims 6 to 8, characterized in that: The dissolving solvent is an alcohol aqueous solution with a volume concentration ratio of 70% to 90%, and optionally, the alcohol includes ethanol.

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