Method for constructing specific chromatogram of traditional chinese medicine compound preparation and method for measuring content of five marker ingredients thereof

Abstract

The present application relates to the technical field of traditional Chinese medicine testing, and in particular to a method for constructing a specific chromatogram of a traditional Chinese medicine compound preparation and a method for measuring the content of five marker ingredients thereof. Conditions for liquid chromatography detection in the technical solution of the present application comprise: mobile phase A being acetonitrile, and mobile phase B being water; 0 min-10 min, keeping the volume percentage of mobile phase A at 12%; 10 min-11 min, increasing the volume percentage of mobile phase A from 12% to 24%; 11 min-30 min, increasing the volume percentage of mobile phase A from 24% to 26%; 30 min-31 min, increasing the volume percentage of mobile phase A from 26% to 45%; and 31 min-45 min, increasing the volume percentage of mobile phase A from 45% to 47%. The technical solution of the present application can simultaneously and accurately measure five marker ingredients of a traditional Chinese medicine compound preparation in one chromatographic detection system, is easy to operate, and achieves convenient calculation.

Description

Methods for constructing characteristic spectra of traditional Chinese medicine compound preparations and methods for determining the content of five indicator components.

[0001] Related applications

[0002] This application claims priority to Chinese patent application filed on January 9, 2025, with application number 2025100344182, entitled "Method for constructing characteristic spectra of traditional Chinese medicine compound preparations and method for determining the content of five index components", the entire text of which is incorporated herein by reference. Technical Field

[0003] This application relates to the field of traditional Chinese medicine detection technology, and in particular to a method for constructing characteristic spectra of a traditional Chinese medicine compound preparation and a method for determining the content of its five indicator components. Background Technology

[0004] Shen Ge Bu Shen Capsules are a traditional Chinese medicine compound preparation. The main ingredients, kudzu root, epimedium, and codonopsis, are used in traditional Chinese medicine based on their effective components: total flavonoids from kudzu root and epimedium, and polysaccharides from codonopsis. They possess the effects of tonifying the kidneys and replenishing essence, and warming and tonifying kidney yang. They are mainly used to treat mild to moderate depression caused by deficiency of both qi and yin, and insufficiency of kidney qi. The existing quality standards for this formula mainly control puerarin and icariin, and do not measure other active ingredients. To better control the quantitative and qualitative changes of active ingredients during the production process, it is necessary to develop a method for simultaneously measuring the content of multiple active ingredients.

[0005] Kudzu root is the dried root of *Pueraria lobata* or *Pueraria truncata*, both belonging to the legume family. It is a traditional Chinese medicine that is both food and medicine. Its main pharmacologically active components are isoflavones, with puerarin being the most abundant. It possesses various pharmacological effects, including antioxidant, anti-inflammatory, cardioprotective, vasodilatory, anticancer, and neuroprotective properties, and can be used to treat diseases such as Alzheimer's and Parkinson's. Codonopsis pilosula is the dried tuberous root of *Codonopsis pilosula*, a plant in the Caryophyllaceae family. It has the effects of invigorating qi and strengthening the spleen, promoting body fluid production and moistening the lungs. Polysaccharides are one of its main active components, possessing various biological activities such as hypoglycemic, anti-inflammatory, and immunomodulatory effects. Epimedium, the dried leaves of *Epimedium brevicornu*, *Epimedium sagittatum*, *Epimedium pubescens*, and *Epimedium koreanum* (all belonging to the Berberidaceae family), possesses the effects of tonifying kidney yang, strengthening tendons and bones, and dispelling wind and dampness. Flavonoids such as icariin A, icariin B, icariin C, and icariin are its main active ingredients, exhibiting pharmacological activities including antibacterial, antioxidant, antitumor, immune-enhancing, cardiovascular-cerebrovascular-improving, endocrine-regulating, anti-osteoporosis, anti-inflammatory, and learning and memory-enhancing effects. Therefore, using icariin, icariin A, icariin B, icariin C, and puerarin as indicator components in the liquid chromatography analysis of Shen Ge Bushen Capsules helps to better control the quantitative and qualitative changes of the effective components during the production process.

[0006] Numerous methods have been reported for the detection of the aforementioned components. For example, Gao Junqing et al. were able to determine puerarin in Naomaitai capsules using a gradient elution of acetonitrile and 1.0 g / L phosphate; Li Jing was able to determine puerarin in Getong Tongluo capsules using isocratic elution of formic acid and water; Yang Ru and Teng Xichao et al. were able to determine icariin, cyproheptadine A, cyproheptadine B, cyproheptadine C, and cyproheptadine I using a gradient elution of acetonitrile and water; and Feng Liang et al. were able to determine icariin in Xianling Gubao capsules using a gradient elution of acetonitrile and 0.5% formic acid combined with a three-wavelength switching method. However, the above elution procedures were not specifically designed for the determination of traditional Chinese medicine compound preparations composed of pueraria, epimedium, and codonopsis pilosula. When used in this formula, their applicability was poor, and the separation effect and peak shape of the chromatographic peaks were unsatisfactory. One method discloses an HPLC method for the determination of icariin, cuscutaidine A, cuscutaidine B, and cuscutaidine C in Shenge Bushen Capsules. This method uses high-performance liquid chromatography (HPLC) with a Waters ACQUITY UPLC HSS T3 C18 column (2.1 × 100 mm, 1.8 μm) and a gradient elution system of acetonitrile-0.05% phosphoric acid aqueous solution. However, this method does not take puerarin into account and cannot effectively separate it. Additionally, other methods have been reported for the chromatographic detection of puerarin, but these also cannot simultaneously determine puerarin and total flavonoid glycosides in epimedium.

[0007] Therefore, it is necessary to develop a method that can simultaneously determine icariin, chopradin A, chopradin B, chopradin C and puerarin in Shenge Bushen Capsules. Summary of the Invention

[0008] Based on this, one or more embodiments of this application provide a method for constructing characteristic chromatograms of traditional Chinese medicine compound preparations and a method for determining the content of five indicator components, wherein each indicator component has high separation and good chromatographic peak shape, and can be simultaneously determined under one chromatographic condition. Furthermore, the determination method has passed the specificity, precision, repeatability and robustness tests through methodological validation.

[0009] The technical solution of this application includes the following:

[0010] 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 kudzu root, codonopsis root and epimedium;

[0011] The method for constructing the feature map includes the following steps:

[0012] Take the tested traditional Chinese medicine compound preparation, add an extraction solvent to extract it, and obtain the test solution;

[0013] The test solution was subjected to liquid chromatography to obtain the characteristic chromatogram of the traditional Chinese medicine compound preparation.

[0014] The conditions for the liquid chromatography detection include:

[0015] (1) Acetonitrile is used as mobile phase A and water is used as mobile phase B;

[0016] (2) Gradient elution is employed, wherein the gradient elution procedure includes:

[0017] From 0 min to 10 min, the volume percentage of the mobile phase A is maintained at 12%.

[0018] Within 10 to 11 minutes, the volume percentage of the mobile phase A increased from 12% to 24%.

[0019] From 11 min to 30 min, the volume percentage of the mobile phase A increased from 24% to 26%.

[0020] Between 30 and 31 minutes, the volume percentage of the mobile phase A increased from 26% to 45%.

[0021] Between 31 and 45 minutes, the volume percentage of the mobile phase A increased from 45% to 47%.

[0022] (3) The detection wavelength is 265nm~275nm.

[0023] In some embodiments, the conditions for liquid chromatography detection further include at least one of the following:

[0024] (1) The chromatographic column packing material is octadecylsilane bonded silica gel;

[0025] (2) The column temperature is 28℃~32℃;

[0026] (3) The injection volume is 3 μL to 7 μL; and,

[0027] (4) The flow rate is 0.8 mL / min to 1.2 mL / min.

[0028] 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 packing material is 5 μm.

[0029] In some embodiments, the extraction solvent is an aqueous alcohol-water solution with a volume concentration of 70% to 90%, optionally, the alcohol includes ethanol; and / or,

[0030] The extraction method includes ultrasound, optionally with a power of 240W–260W, a frequency of 30kHz–50kHz, and a duration of 20min–40min; and / or,

[0031] The mass-to-volume ratio of the tested traditional Chinese medicine compound preparation to the extraction solvent is 0.2–0.6 g: 100 mL.

[0032] In some embodiments, the characteristic spectrum of the traditional Chinese medicine compound preparation includes five characteristic peaks, namely peak 1, peak 2, peak 3, peak 4, and peak 5.

[0033] Among them, peak 1 is puerarin, peak 2 is ascorbic acid A, peak 3 is ascorbic acid B, peak 4 is ascorbic acid C, and peak 5 is icariin.

[0034] Using 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%.

[0035] A method for determining the content of five indicator components in a traditional Chinese medicine compound preparation, wherein the traditional Chinese medicine compound preparation is a mixture of water extracts of kudzu root, codonopsis root, and epimedium; the five indicator components are, in order, puerarin, cuscutaidine A, cuscutaidine B, cuscutaidine C, and epimedium glycoside.

[0036] The content determination method includes the following steps:

[0037] A mixture of reference standards for the five indicator components was taken, and liquid chromatography was performed with different injection volumes of reference standard solution to detect them and construct standard curves for the mass and peak area of ​​puerarin, cytosine A, cytosine B, cytosine C and icariin.

[0038] Take the traditional Chinese medicine compound preparation to be tested, add an extraction solvent to extract it, and obtain the test solution;

[0039] The test solution was subjected to liquid chromatography to obtain the peak areas of puerarin, astragaloside A, astragaloside B, astragaloside C and icariin in the test solution. The peak areas were then substituted into the standard curves of the concentrations and peak areas of the puerarin, astragaloside A, astragaloside B, astragaloside C and icariin, respectively, for calculation.

[0040] The conditions for the liquid chromatography detection include:

[0041] (1) Acetonitrile is used as mobile phase A and water is used as mobile phase B;

[0042] (2) Gradient elution is employed, wherein the gradient elution procedure includes:

[0043] From 0 min to 10 min, the volume percentage of the mobile phase A is maintained at 12%.

[0044] Within 10 to 11 minutes, the volume percentage of the mobile phase A increased from 12% to 24%.

[0045] From 11 min to 30 min, the volume percentage of the mobile phase A increased from 24% to 26%.

[0046] Between 30 and 31 minutes, the volume percentage of the mobile phase A increased from 26% to 45%.

[0047] Between 31 and 45 minutes, the volume percentage of the mobile phase A increased from 45% to 47%.

[0048] (3) The detection wavelength is 265nm~275nm.

[0049] In some embodiments, the conditions for liquid chromatography detection further include at least one of the following:

[0050] (1) The chromatographic column packing material is octadecylsilane bonded silica gel;

[0051] (2) The column temperature is 28℃~32℃;

[0052] (3) The injection volume is 3 μL to 7 μL; and

[0053] (4) The flow rate is 0.8 mL / min to 1.2 mL / min.

[0054] 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 packing material is 4 μm to 6 μm.

[0055] In some embodiments, the extraction solvent is an aqueous alcohol-water solution with a volume concentration of 70% to 90%, optionally, the alcohol includes ethanol; and / or,

[0056] The extraction method includes ultrasound, optionally with a power of 240W–260W, a frequency of 30kHz–50kHz, and a duration of 20min–40min; and / or,

[0057] The mass-to-volume ratio of the traditional Chinese medicine compound preparation to be tested to the extraction solvent is 0.2–0.6 g: 100 mL.

[0058] In some embodiments, the dissolving solvent is an aqueous alcohol solution with a volume concentration of 70% to 90%, and optionally, the alcohol includes ethanol.

[0059] The technical solution of this application establishes a characteristic chromatogram of traditional Chinese medicine compound preparations based on a suitable liquid chromatography analysis method, which enables the characterization of five indicator components of traditional Chinese medicine compound preparations—puerarin, cuscutaidine A, cuscutaidine B, cuscutaidine C, and icariin. The chromatographic peaks of each indicator component have good separation and are not mutually interfered with, and the content can be determined in the same chromatographic detection system, thus providing a reference for the quality control of traditional Chinese medicine compound preparations.

[0060] The technical solution of this application enables the simultaneous determination of the content of five indicator components in traditional Chinese medicine compound preparations, reducing the complexity of existing standards that require separate liquid chromatography conditions for their detection, improving testing efficiency, and reducing the amount of organic reagents used. The external standard method is selected, which is simple to operate and convenient to calculate. Attached Figure Description

[0061] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0062] Figure 1 is a chromatogram of the test solution obtained under the chromatographic detection conditions of Scheme 1 as described in Section 1.4.6 of this application;

[0063] Figure 2 is a chromatogram of the test solution obtained under the chromatographic detection conditions of Scheme 2 as described in Section 1.4.6 of this application;

[0064] Figure 3 is a chromatogram of the test solution obtained using the chromatographic detection conditions of Scheme 3 as described in Section 1.4.6 of this application;

[0065] Figure 4 is a chromatogram of the test solution obtained under the chromatographic detection conditions of Scheme 4 as described in Section 1.4.6 of this application;

[0066] Figure 5 is a chromatogram of the test solution obtained using the chromatographic detection conditions of Scheme 5 as described in Section 1.4.6 of this application;

[0067] Figure 6 is a chromatogram of the test solution obtained under the chromatographic detection conditions of Scheme 6 as described in Section 1.4.6 of this application;

[0068] Figure 7 is a chromatogram of the test solution obtained using the chromatographic detection conditions of Scheme 7 as described in Section 1.4.6 of this application;

[0069] Figure 8 is a chromatogram of the test solution obtained using the chromatographic detection conditions of Scheme 8 as described in Section 1.4.6 of this application;

[0070] Figure 9 shows the chromatograms of blank excipients, negative samples, and test solutions obtained using the chromatographic detection conditions of Scheme 8 in this application.

[0071] Figure 10 is a chromatogram of the mixed reference standard obtained using the chromatographic detection conditions of Scheme 8 in this application. Detailed Implementation

[0072] The present application is further described below with reference to embodiments and examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the protection scope of the appended claims.

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0074] the term

[0075] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0076] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations encompass 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 using 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 connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: 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, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0077] In this document, terms such as "preferred," "better," and "more preferred" are merely descriptions of implementation methods or examples that achieve better results, and should be understood as not constituting a limitation on the scope of protection of this application.

[0078] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0079] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0080] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0081] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, optional numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0082] The technical solution of this application, based on a suitable liquid chromatography analysis method and a characteristic spectrum established by a dual-wavelength detection system, enables the characterization of five indicator components of traditional Chinese medicine compound preparations—icariin, cuscutaidine A, cuscutaidine B, cuscutaidine C, and puerarin. The chromatographic peaks of each indicator component have good separation and are not mutually interfered with, and their content can be determined in the same chromatographic detection system, thus providing a reference for the quality control of traditional Chinese medicine compound preparations.

[0083] In this article, the traditional Chinese medicine compound preparation is a mixture of extracts from kudzu root, codonopsis root, and epimedium.

[0084] In this article, the five indicator components in the traditional Chinese medicine compound preparation are, in order, puerarin, chopradoxime A, chopradoxime B, chopradoxime C, and icariin.

[0085] The method for constructing the characteristic spectrum of a traditional Chinese medicine compound preparation according to an embodiment of this application includes the following steps:

[0086] Take the tested traditional Chinese medicine compound preparation, add an extraction solvent to extract it, and obtain the test solution;

[0087] The test solution was subjected to liquid chromatography to obtain the characteristic chromatogram of the traditional Chinese medicine compound preparation.

[0088] The conditions for liquid chromatography detection include:

[0089] (1) Acetonitrile is used as mobile phase A and water is used as mobile phase B;

[0090] (2) Gradient elution is used. The gradient elution procedure includes:

[0091] From 0 min to 10 min, maintain the volume percentage of mobile phase A at 12%;

[0092] Between 10 and 11 minutes, the volume percentage of mobile phase A increased from 12% to 24%.

[0093] From 11 min to 30 min, the volume percentage of mobile phase A increased from 24% to 26%.

[0094] Between 30 and 31 minutes, the volume percentage of mobile phase A increased from 26% to 45%.

[0095] Between 31 and 45 minutes, the volume percentage of mobile phase A increased from 45% to 47%.

[0096] (3) Detection wavelength 265nm~275nm.

[0097] In some embodiments, the conditions for liquid chromatography detection further include at least one of the following:

[0098] (1) The chromatographic column packing material is octadecylsilane bonded silica gel;

[0099] (2) The column temperature is 28℃~32℃;

[0100] (3) The injection volume is 3 μL to 7 μL; and,

[0101] (4) The flow rate is 0.8 mL / min to 1.2 mL / min.

[0102] 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 packing material is 4 μm to 6 μm.

[0103] Optionally, the length of the chromatographic column can be 240mm, 250mm, 260mm, etc., the diameter can be 4.4mm, 4.5mm, 4.6mm, etc., and the particle size of the packing material can be 4μm, 5μm, 6μm, etc.

[0104] In some embodiments, the extraction solvent is an aqueous alcohol solution with a volume concentration of 70% to 90%, and optionally, the alcohol includes ethanol.

[0105] In this embodiment, the volume concentration ratio of alcohol to aqueous solution is 70% to 90%, for example, 70%, 75%, 80%, 85%, 90%, etc.

[0106] In some embodiments, the extraction method includes ultrasound, optionally with a power of 240W to 260W, a frequency of 30kHz to 50kHz, and a duration of 20min to 40min.

[0107] Optionally, the ultrasonic power can be 240W, 250W, 260W, etc., the frequency can be 30kHz, 40kHz, 50kHz, etc., and the time can be 20min, 30min, 40min, etc.

[0108] In some embodiments, the mass-to-volume ratio of the tested traditional Chinese medicine compound preparation to the extraction solvent is 0.2–0.6 g:100 mL. For example, the mass-to-volume ratio of the tested traditional Chinese medicine compound preparation to the extraction solvent can be 0.2 g:100 mL, 0.3 g:100 mL, 0.4 g:100 mL, 0.5 g:100 mL, 0.6 g:100 mL, etc.

[0109] In some embodiments, the characteristic spectrum of the traditional Chinese medicine compound preparation includes five characteristic peaks, namely peak 1, peak 2, peak 3, peak 4, and peak 5.

[0110] Among them, peak 1 is puerarin, peak 2 is ascorbic acid A, peak 3 is ascorbic acid B, peak 4 is ascorbic acid C, and peak 5 is icariin;

[0111] Using 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%.

[0112] The method for determining the content of five indicator components in a traditional Chinese medicine compound preparation according to an embodiment of this application includes the following steps:

[0113] A mixture of reference standards for five indicator components was taken, and liquid chromatography was performed with different injection volumes of reference standard solution to detect them. Standard curves for the mass and peak area of ​​puerarin, cytosine A, cytosine B, cytosine C and icariin were constructed.

[0114] Take the traditional Chinese medicine compound preparation to be tested, add an extraction solvent to extract it, and obtain the test solution;

[0115] The test solution was subjected to liquid chromatography to obtain the peak areas of puerarin, ascorbic acid A, ascorbic acid B, ascorbic acid C and icariin in the test solution. The peak areas were then substituted into the standard curves of the concentrations and peak areas of puerarin, ascorbic acid A, ascorbic acid B, ascorbic acid C and icariin to calculate the peak areas.

[0116] The conditions for liquid chromatography detection include:

[0117] (1) Acetonitrile is used as mobile phase A and water is used as mobile phase B;

[0118] (2) Gradient elution is used. The gradient elution procedure includes:

[0119] From 0 min to 10 min, maintain the volume percentage of mobile phase A at 12%;

[0120] Between 10 and 11 minutes, the volume percentage of mobile phase A increased from 12% to 24%.

[0121] From 11 min to 30 min, the volume percentage of mobile phase A increased from 24% to 26%.

[0122] Between 30 and 31 minutes, the volume percentage of mobile phase A increased from 26% to 45%.

[0123] Between 31 and 45 minutes, the volume percentage of mobile phase A increased from 45% to 47%.

[0124] (3) The detection wavelength is 265nm~275nm.

[0125] In some embodiments, the conditions for liquid chromatography detection further include at least one of the following:

[0126] (1) The chromatographic column packing material is octadecylsilane bonded silica gel;

[0127] (2) The column temperature is 28℃~32℃;

[0128] (3) The injection volume is 3 μL to 7 μL; and

[0129] (4) The flow rate is 0.8 mL / min to 1.2 mL / min.

[0130] In the method for determining the content of five indicator components in the traditional Chinese medicine compound preparation of this application embodiment, the chromatographic conditions used can be referenced from the limitations on the corresponding chromatographic conditions in the characteristic chromatogram of the traditional Chinese medicine compound preparation of this application embodiment.

[0131] In the method for determining the content of five indicator components in a traditional Chinese medicine compound preparation in this application embodiment, in the step of taking the traditional Chinese medicine compound preparation to be tested, adding an extraction solvent for extraction, and obtaining the solution to be tested, the extraction solvent and extraction method used can be referred to the extraction solvent and extraction method used for the extraction of the traditional Chinese medicine compound preparation in the characteristic spectrum of the traditional Chinese medicine compound preparation in this application embodiment.

[0132] In some embodiments, the dissolving solvent is an aqueous alcohol solution with a volume concentration of 70% to 90%, and optionally, the alcohol includes ethanol.

[0133] In this embodiment, the volume concentration ratio of alcohol to aqueous solution is 70% to 90%, for example, 70%, 75%, 80%, 85%, 90%, etc.

[0134] In some embodiments, the mass concentration of each indicator component in the reference solution is 1 μg / mL to 120 μg / mL.

[0135] In this embodiment, the mass concentrations of puerarin, icariin, trichosamine A, trichosamine B, and trichosamine C in the reference solution were 80 μg / mL to 120 μg / mL, 1 μg / mL to 5 μg / mL, 2 μg / mL to 8 μg / mL, 5 μg / mL to 15 μg / mL, and 15 μg / mL to 25 μg / mL, respectively.

[0136] The technical solution of this application enables the simultaneous determination of the content of five indicator components in traditional Chinese medicine compound preparations, reducing the complexity of existing standards that require separate liquid chromatography conditions for their detection, improving testing efficiency, and reducing the amount of organic reagents used. The external standard method is selected, which is simple to operate and convenient to calculate.

[0137] The following are some specific examples.

[0138] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0139] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0140] Example 1

[0141] This embodiment provides a method for constructing the characteristic spectrum of the traditional Chinese medicine compound preparation of this application.

[0142] 1.1. Experimental medicinal materials

[0143] Ginseng and Pueraria Kidney-Nourishing Capsules

[0144] 1.2. Experimental Reagents and Materials

[0145] Methanol, ultrapure water, ethanol, phosphoric acid, acetonitrile; puerarin reference standard, icariin reference standard, chopradoxime A reference standard, chopradoxime B reference standard, chopradoxime C reference standard, silicon dioxide.

[0146] 1.3. Instruments and Equipment

[0147] Analytical balance; high performance liquid chromatograph; ultrasonic instrument.

[0148] 1.4. Experimental Procedure

[0149] 1.4.1. Preparation of reference stock solution

[0150] Accurately weigh appropriate amounts of puerarin reference standard, icariin reference standard, chopradoxime A reference standard, chopradoxime B reference standard, and chopradoxime C reference standard. Dissolve them in ethanol, then dilute to the mark with 50% ethanol 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, chopradoxime A, chopradoxime B, and chopradoxime C, respectively, as stock solutions.

[0151] 1.4.2. Preparation of mixed reference solution

[0152] Accurately measure the stock solution from 1.4.1 and dilute it to the mark with 50% ethanol solution to prepare a solution containing puerarin, icariin, chopradoxime A, chopradoxime B, and chopradoxime C at concentrations of 100 μg / mL, 2.0 μg / mL, 5.0 μg / mL, 10 μg / mL, and 20 μg / mL, respectively, as a mixed reference solution.

[0153] 1.4.3. Preparation of the test solution

[0154] Test solution: Weigh approximately 10 mg of the contents of Shenge Bushen Capsules accurately into a 25 mL volumetric flask, add an appropriate amount of 80% ethanol, sonicate for 30 minutes, cool, dilute to the mark with 80% ethanol, shake well, filter through a microporous membrane (0.45 μm), and collect the filtrate.

[0155] 1.4.4. Negative Solution

[0156] Use an 80% ethanol solution as the negative sample solution.

[0157] 1.4.5. Blank excipient solution

[0158] Take an appropriate amount of blank excipient (silica), about 0.25g, place it in a 50mL volumetric flask, add an appropriate amount of 80% ethanol solution, sonicate for 30 minutes, cool, dissolve and dilute to the mark with 80% ethanol solution, shake well, filter through a microporous membrane (0.45μm), and collect the filtrate to obtain the product.

[0159] 1.4.6. Chromatographic conditions

[0160] Using octadecylsilane-bonded silica gel as the packing material (250 mm × 4.6 mm), gradient elution was performed according to the specifications in the table below; column temperature 30℃; injection volume 5 μL; detection wavelength 270 nm; theoretical plate number calculated based on the icariin peak was not less than 8000.

[0161] Table 1 Eight Elution Protocols

[0162] The chromatographic detection results of the above eight schemes are shown in Figures 1 to 8, respectively. Peaks 1, 2, 3, 4, and 5 are puerarin, ascorbic acid A, ascorbic acid B, ascorbic acid C, and icariin, respectively. The comparison shows that scheme eight yields more characteristic peaks, with high resolution, moderate response, minimal impurity interference, and a stable baseline.

[0163] Scheme 8 was used to detect the test solution, negative solution, and blank excipient, and the corresponding chromatograms were obtained. The comparison results of the chromatograms of the test solution, negative solution, and blank excipient solution are shown in Figure 9. The mixed reference solution was detected, and the chromatogram shown in Figure 10 was obtained.

[0164] Comparison of Figures 9 and 10 revealed that Scheme 8 could detect 5 characteristic peaks of the reference standard, demonstrating good separation.

[0165] 2. Methodological Examination

[0166] 2.1 Specificity: Take 5 μL each 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 8 under section 1.4.6 to confirm whether there is any interference (the detection results can be seen in Figures 9 and 10).

[0167] 2.2 Precision: Take the test solution and inject it 6 times consecutively under the chromatographic conditions of Scheme 8 in section 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 solutions in parallel according to the method in section 1.4.3. Determine and record the peak areas according to the chromatographic conditions in scheme 8 of section 1.4.6 and calculate the relative standard deviation (RSD) of each peak area.

[0169] 2.4 Robustness: 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 8 under section 1.4.6. Measure and record the peak areas of the five components and calculate the relative standard deviation (RSD) of the peak areas of each component within 48h.

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

[0171] This application determined 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 the eight elution programs in Table 1) in chromatographic detection. The determined mobile phase to be acetonitrile (A)-water (B) and the elution program to be: 0-10 min: 12% A; 10-11 min: 12%-24% A; 11-30 min: 24%-26% A; 30-31 min: 26%-45% A; 31-45 min: 45%-47% A. This method showed the best separation effect for puerarin and icariin and is most suitable for their analysis and detection.

[0172] 3. Establishment of the standard curve

[0173] The mixed reference solution from section 1.4.2 was injected at chromatographic conditions (Scheme 8) under section 1.4.6 at concentrations of 1 μL, 2 μL, 4 μL, 8 μL, 10 μL, 20 μL, and 40 μL, respectively. The peak areas of the five components were measured and recorded. Linear regression was performed with the injection volume (μg) as the abscissa (x) and the peak area as the ordinate (y). The linear regression equations and correlation coefficients for puerarin, astragalin A, astragalin B, astragalin C, and icariin were calculated. Table 3 shows that the five components exhibited good linear relationships.

[0174] Table 3 Linear Regression Equations

[0175] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0176] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments 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.

[0177] The embodiments described above merely illustrate several implementation methods of this application and should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

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

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

3. The construction method according to claim 2, characterized in that, The chromatographic column has a length of 240 mm to 260 mm, a diameter of 4.4 mm to 4.8 mm, and a particle size of 4 μm to 6 μm for the packing material.

4. The construction method according to any one of claims 1 to 3, characterized in that, At least one of the following conditions must be met: (1) The extraction solvent is an aqueous alcohol solution with a volume concentration of 70% to 90%, and optionally, the alcohol includes ethanol; (2) The extraction method includes ultrasound, optionally with a power of 240W–260W, a frequency of 30kHz–50kHz, and a duration of 20min–40min; and (3) The mass-volume ratio of the tested traditional Chinese medicine compound preparation and the extraction solvent is 0.2-0.6 g: 100 mL.

5. The construction method according to any one of claims 1 to 4, characterized in that, The characteristic spectrum of the traditional 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 ascorbic acid A, peak 3 is ascorbic acid B, peak 4 is ascorbic acid C, and peak 5 is icariin; Using 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 content of five indicator components in a traditional Chinese medicine compound preparation, characterized in that, The traditional Chinese medicine compound preparation is a mixture of water extracts of kudzu root, codonopsis root, and epimedium; the five indicator components are, in order, puerarin, cuscutaidine A, cuscutaidine B, cuscutaidine C, and epimedium glycoside. The content determination method includes the following steps: A mixture of reference standards for the five indicator components was taken, and liquid chromatography was performed with different injection volumes of reference standard solution to detect them and construct standard curves for the mass and peak area of ​​puerarin, cytosine A, cytosine B, cytosine C and icariin. Take the traditional Chinese medicine compound preparation to be tested, add an extraction solvent to extract it, and obtain the test solution; The test solution was subjected to liquid chromatography to obtain the peak areas of puerarin, astragaloside A, astragaloside B, astragaloside C and icariin in the test solution. The peak areas were then substituted into the standard curves of the mass and peak area of ​​puerarin, astragaloside A, astragaloside B, astragaloside C and icariin for calculation. The conditions for the liquid chromatography detection include: (1) Acetonitrile is used as mobile phase A and water is used as mobile phase B; (2) Gradient elution is employed, wherein the gradient elution procedure includes: From 0 min to 10 min, the volume percentage of the mobile phase A is maintained at 12%. Within 10 to 11 minutes, the volume percentage of the mobile phase A increased from 12% to 24%. From 11 min to 30 min, the volume percentage of the mobile phase A increased from 24% to 26%. Between 30 and 31 minutes, the volume percentage of the mobile phase A increased from 26% to 45%. From 31 min to 45 min, the volume percentage of mobile phase A increased from 45% to 47%; and (3) Detection wavelength 265nm~275nm.

7. The content determination method according to claim 6, characterized in that, The conditions for liquid chromatography detection also include at least one of the following: (1) The chromatographic column packing material is octadecylsilane bonded silica gel; (2) The column temperature is 28℃~32℃; (3) The injection volume is 3 μL to 7 μL; and, (4) The flow rate is 0.8 mL / min to 1.2 mL / min.

8. The content determination method according to claim 7, characterized in that, The chromatographic column has a length of 240 mm to 260 mm, a diameter of 4.4 mm to 4.8 mm, and a particle size of 4 μm to 6 μm for the packing material.

9. The method for content determination according to any one of claims 6 to 8, characterized in that, At least one of the following conditions must be met: (1) The extraction solvent is an aqueous alcohol solution with a volume concentration of 70% to 90%, and optionally, the alcohol includes ethanol; (2) The extraction method includes ultrasound, optionally with a power of 240W to 260W, a frequency of 30kHz to 50kHz, and a duration of 20min to 40min; and (3) The mass-volume ratio of the Chinese herbal compound preparation to be tested to the extraction solvent is 0.2-0.6 g: 100 mL.

10. The method for content determination according to any one of claims 6 to 9, characterized in that, The dissolving solvent is an aqueous alcohol solution with a volume concentration of 70% to 90%, and optionally, the alcohol includes ethanol.

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