A method for constructing a fingerprint of Chuanxiong Cha Tiao Powder based on high performance liquid chromatography and its application

Abstract

The present invention provides a method for constructing a Chuanxiong Tea Tiao Powder fingerprint based on high-performance liquid chromatography and its application, relating to the technical field of traditional Chinese medicine. The method comprises the following steps: A. preparing a Chuanxiong Tea Tiao Powder sample solution; B. determining the Chuanxiong Tea Tiao Powder sample solution using high-performance liquid chromatography to obtain a Chuanxiong Tea Tiao Powder HPLC spectrum; and C. constructing a Chuanxiong Tea Tiao Powder fingerprint based on the Chuanxiong Tea Tiao Powder HPLC spectrum. This method enables simultaneous detection of multiple components of Chuanxiong Tea Tiao Powder, improving the comprehensiveness and accuracy of the constructed Chuanxiong Tea Tiao Powder fingerprint. The method can be used to evaluate the quality of Chuanxiong Tea Tiao Powder, quantitatively detect the contents of multiple chemical components in Chuanxiong Tea Tiao Powder, and lay the foundation for subsequent research on Chuanxiong Tea Tiao Powder.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a method for constructing a fingerprint spectrum of Chuanxiong Cha Tiao Powder based on high performance liquid chromatography and its application. Background Art

[0002] Chuanxiong Cha Tiao San, first recorded in the Taiping Huimin Hejiju Fang, is a classic TCM prescription for treating exogenous wind-induced headaches. It is composed of eight Chinese herbs: Chuanxiong, Qianghuo, Baizhi, Fangfeng, Xixin, Mentha, Jingjie, and Gancao. It is mainly used to treat headaches caused by exogenous wind-induced headaches, or with chills, fever, and nasal congestion. Studies have shown that Chuanxiong Cha Tiao San has anti-inflammatory and analgesic effects. It is currently widely used for various vascular headaches, especially for the treatment of migraines. The 2020 edition of the Chinese Pharmacopoeia includes both Chuanxiong Cha Tiao San and Chuanxiong Cha Tiao Granules. The two have the same formula and efficacy, but their quality control indicators are different. In the 2020 edition of the Chinese Pharmacopoeia, Chuanxiong Cha Tiao San only uses ferulic acid as a quality control indicator, while Chuanxiong Cha Tiao Granules use ferulic acid and glycyrrhizic acid as quality control indicators. However, Chuanxiong Cha Tiao Powder contains multiple ingredients, including Z-ligustilide, ferulic acid, notopterygol, imperatorin, cimicifugoside, and asarone. Using only ferulic acid and glycyrrhizic acid as indicators neither comprehensively controls its quality nor fully demonstrates its efficacy. Chuanxiong Cha Tiao Powder, a powder made from eight medicinal herbs, including Chuanxiong rhizome, has wind-dispelling and analgesic properties and is used to treat wind-induced headaches, as well as aversion to cold, fever, and nasal congestion. Its therapeutic efficacy has been clinically validated. However, ensuring the quality of Chuanxiong Cha Tiao Powder is fundamental to its effectiveness. Describing the intrinsic quality of Chuanxiong Cha Tiao Powder solely based on one or two active ingredients presents a one-sided problem in traditional Chinese medicine quality control, let alone focusing on inactive ingredients. Therefore, characterizing and controlling only one or two chemical components is insufficient for quality control of Chuanxiong Cha Tiao Powder; the entire substance group must be macroscopically controlled. A traditional Chinese medicine fingerprint is a chromatographic or spectral profile of one or more characteristic components common to a particular Chinese herbal medicine or Chinese patent medicine. Given that the active ingredients of most Chinese herbal medicines are currently unknown, fingerprinting plays a crucial role in effectively controlling the quality of these materials and Chinese patent medicines. Fingerprinting has gained international recognition as a quality control method for Chinese herbal medicines and their extracts. Studies have examined the fingerprint of Chuanxiong Cha Tiao San (Chuanxiong Cha Tiao San) granules. For example, Mei Jixiong et al. conducted an HPLC fingerprint study of Chuanxiong Cha Tiao San granules in 2012, using an acetonitrile-0.05% formic acid solution as the mobile phase, gradient elution, and a detection wavelength of 254 nm. However, the fingerprint developed resulted in a long elution time of 135 minutes, limited detection parameters, and only identified ammonium glycyrrhizate. CN116678978B establishes a fingerprint spectrum and chemical component content determination method for Chuanxiong Tea Powder. The method uses methanol-acetonitrile-water as the mobile phase, gradient elution, and a detection wavelength of 270-280 nm. However, the fingerprint spectrum established can only identify seven peaks, namely, cimicifugoside, liquiritin, cimicifugoside, 5-O-methylasvimethol glycoside, ligusticolide I, chelidonol glycoside, and ligusticolide A. This is not sufficient to fully evaluate the quality control of Chuanxiong Tea Powder. Summary of the Invention

[0003] One of the purposes of the present invention is to provide a method for constructing a fingerprint of Chuanxiong Tea Tiao Powder based on high performance liquid chromatography, so as to solve the problem that the fingerprint established in the prior art can only identify 7 peaks, namely, cimicifugoside, liquiritin, cimicifugoside, 5-O-methylaswimilol glycoside, ligusticum lactone I, chelidonol glycoside, and ligusticum lactone A, which is not sufficient to comprehensively evaluate the quality control of Chuanxiong Tea Tiao Powder.

[0004] The second object of the present invention is to provide the application of the aforementioned method for constructing the fingerprint of Chuanxiong Tea Powder in qualitative analysis of the active ingredients of Chuanxiong Tea Powder, quantitative analysis of the active ingredients of Chuanxiong Tea Powder or evaluation of the quality of Chuanxiong Tea Powder.

[0005] The third object of the present invention is to provide a method for evaluating the quality of Chuanxiong tea powder.

[0006] In a first aspect, the present invention provides a method for constructing a fingerprint of Chuanxiongcha Tiaosan based on high performance liquid chromatography, comprising the following steps:

[0007] A. placing the Chuanxiong Tea Powder sample in methanol for ultrasonic treatment and then filtering to obtain a Chuanxiong Tea Powder sample solution;

[0008] B. Using high performance liquid chromatography to determine the Chuanxiong Tea Adjusting Powder sample solution to obtain the Chuanxiong Tea Adjusting Powder HPLC spectrum;

[0009] C. Constructing the fingerprint of Chuanxiongcha Tiaosan based on the HPLC spectrum of Chuanxiongcha Tiaosan;

[0010] The mobile phase A in the high performance liquid chromatography determination is acetonitrile, and the mobile phase B is formic acid with a mass concentration of 0.01-0.10%.

[0011] Furthermore, the amount of the Chuanxiong tea powder sample added to methanol is 0.5-1.5 g of the Chuanxiong tea powder sample per 100 ml of methanol.

[0012] Furthermore, the amount of the Chuanxiong tea powder sample added is 1 g of the Chuanxiong tea powder sample per 100 ml of methanol;

[0013] Preferably, the ultrasonic treatment time is 20 to 40 minutes, preferably 30 minutes;

[0014] Preferably, the power of the ultrasound is 200-400 W, and the frequency is 40-50 kHz, preferably the power is 300 W, and the frequency is 45 kHz;

[0015] Preferably, after the ultrasonic treatment, the method further comprises cooling and weighing the mass, and supplementing the lost mass with methanol;

[0016] Preferably, the filtration comprises coarse filtration followed by filtration of the filtrate through a microporous membrane of 0.22-0.3 μm, preferably 0.22 μm.

[0017] Furthermore, the fingerprint of the Chuanxiong tea powder sample solution was constructed using a Cl8 chromatographic column with a length of 25 cm, a particle size of 5 μm, and an inner diameter of 4.6 mm; gradient elution, a flow rate of 1.0 mL / min; a column temperature of 30°C; an injection volume of 10 μL; and a detection wavelength of 280 nm.

[0018] Furthermore, the gradient elution conditions include:

[0019] 0~15min, 9%A, 91%B;

[0020] 15~18min, 9%~15%A, 91%~85%B;

[0021] 18-60 min, 15% A, 85% B;

[0022] 60~85min, 15%~27%A, 85%~73%B;

[0023] 85~115min, 27%~40%A, 73%~60%B;

[0024] 115~130min, 40%~45%A, 60%~55%B;

[0025] 130~131min, 45%~55%A, 55%~45%B;

[0026] 131~145min, 55%A, 45%B.

[0027] Furthermore, the step C comprises:

[0028] C1. Extract characteristic peaks from the HPLC spectrum of Chuanxiongchatiao powder;

[0029] C2. Perform similarity analysis on the characteristic peaks to construct a fingerprint of Chuanxiong Cha Tiao Powder that represents the overall characteristics of Chuanxiong Cha Tiao Powder.

[0030] Furthermore, after step C, the method further comprises verifying the fingerprint of Chuanxiongchatiaosan using the fingerprint of a standard product;

[0031] If the characteristic peaks of the fingerprint of Chuanxiong Cha Tiao Powder are consistent with those of the standard product fingerprint, the construction is successful, otherwise it is reconstructed.

[0032] Furthermore, the HPLC spectrum of the Chuanxiong tea powder includes at least one HPLC spectrum of Chuanxiong tea powder of the same specification but different batches.

[0033] In a second aspect, the present invention provides the application of the aforementioned method for constructing a fingerprint of Chuanxiong Tea Regulating Powder in any of the following:

[0034] D1) Qualitative analysis of the active ingredients in Chuanxiongchatiaosan;

[0035] D2) Quantitative analysis of the active ingredients in Chuanxiongchatiaosan;

[0036] D3) Evaluate the quality of Chuanxiong Tea Powder.

[0037] In a third aspect, the present invention provides a method for evaluating the quality of Chuanxiong Tea Powder, comprising using the aforementioned Chuanxiong Tea Powder fingerprint as a reference fingerprint, comparing the characteristic peaks of the Chuanxiong Tea Powder fingerprint to be evaluated with the characteristic peaks of the reference fingerprint, and judging the quality of the Chuanxiong Tea Powder to be evaluated;

[0038] Preferably, judging the quality of the Chuanxiong Tea Powder to be evaluated includes: if the number of peaks and the peak time of the fingerprint of the Chuanxiong Tea Powder to be evaluated are consistent with those of the control fingerprint, it indicates that the quality of the Chuanxiong Tea Powder to be evaluated is consistent with the Chuanxiong Tea Powder used to construct the above-mentioned Chuanxiong Tea Powder fingerprint, otherwise the quality does not meet the requirements.

[0039] The present invention provides a method for constructing a Chuanxiong Tea Tiao Powder fingerprint based on high-performance liquid chromatography. The use of methanol during the preparation of a Chuanxiong Tea Tiao Powder sample solution maximizes the determination of low-polarity components in the sample, resulting in the greatest number of characteristic peaks in the test solution's fingerprint, laying the foundation for further multi-component detection and analysis. High-performance liquid chromatography enables simultaneous detection of multiple components of Chuanxiong Tea Tiao Powder. Improving the mobile phase to acetonitrile (A)-0.05% formic acid (B) addresses the technical issues of uneven baselines and suboptimal separation of characteristic peaks in the Chuanxiong Tea Tiao Powder HPLC spectrum. This improves the comprehensiveness and accuracy of the constructed Chuanxiong Tea Tiao Powder fingerprint, enabling its use in evaluating the quality of Chuanxiong Tea Tiao Powder and laying the foundation for quantitative detection of the contents of multiple chemical components in Chuanxiong Tea Tiao Powder and subsequent research on Chuanxiong Tea Tiao Powder. The invention solves the problem that the fingerprint established in the prior art can only identify seven peaks, namely, cimicifuga glycoside, liquiritin, cimicifuga, 5-O-methylasvimethicone glycoside, ligusticum lactone I, chelidonol glycoside, and ligusticum lactone A, which is not enough to comprehensively evaluate the quality control of Chuanxiong Tea Powder. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0041] Figure 1 HPLC spectrum of Chuanxiongcha Tiaosan provided in Example 1 of the present invention;

[0042] Figure 2 HPLC color spectrum of the standard product provided in Example 1 of the present invention;

[0043] Figure 3 HPLC spectrum of Chuanxiongcha Tiaosan provided in Example 2 of the present invention;

[0044] Figure 4 HPLC spectrum of Chuanxiongcha Tiaosan provided in Example 3 of the present invention;

[0045] Figure 5 HPLC spectrum of Chuanxiongcha Tiaosan provided in Comparative Example 1 of the present invention;

[0046] Figure 6 HPLC spectrum of Chuanxiongcha Tiaosan provided in Comparative Example 2 of the present invention;

[0047] Figure 7 HPLC spectrum of Chuanxiongcha Tiaosan obtained in process 1 of optimizing the mobile phase and gradient elution program provided in experiment 1 of the present invention;

[0048] Figure 8 HPLC spectrum of Chuanxiongcha Tiaosan obtained in process 2 of optimizing the mobile phase and gradient elution program provided in experiment 1 of the present invention;

[0049] Figure 9 HPLC spectrum of Chuanxiongcha Tiaosan obtained in process 3 of optimizing the mobile phase and gradient elution program provided in experiment 1 of the present invention;

[0050] Figure 10 This is the fingerprint of the sample to be tested provided in Application Example 1 of the present invention. DETAILED DESCRIPTION

[0051] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms is non-limiting.

[0052] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0053] On the one hand, the present invention provides a method for constructing a fingerprint of Chuanxiongcha Tiaosan based on high performance liquid chromatography, comprising the following steps:

[0054] A. placing the Chuanxiong Tea Powder sample in methanol for ultrasonic treatment and then filtering to obtain a Chuanxiong Tea Powder sample solution;

[0055] B. Using high performance liquid chromatography to determine the Chuanxiong Tea Adjusting Powder sample solution to obtain the Chuanxiong Tea Adjusting Powder HPLC spectrum;

[0056] C. Constructing the fingerprint of Chuanxiongcha Tiaosan based on the HPLC spectrum of Chuanxiongcha Tiaosan;

[0057] The mobile phase A in the high performance liquid chromatography determination is acetonitrile, and the mobile phase B is formic acid with a mass concentration of 0.01-0.10%.

[0058] The use of methanol in the preparation of the Chuanxiong Tea Powder sample solution maximizes the determination of low-polarity components in the sample, maximizing the number of characteristic peaks in the sample's fingerprint, paving the way for further multi-component analysis. High-performance liquid chromatography (HPLC) enabled simultaneous detection of multiple components in Chuanxiong Tea Powder. Improving the mobile phase to acetonitrile (A)-0.05% formic acid (B) addressed the technical issues of uneven baselines and suboptimal separation of characteristic peaks in the HPLC chromatogram of Chuanxiong Tea Powder. This improved the comprehensiveness and accuracy of the constructed Chuanxiong Tea Powder fingerprint, enabling its use in evaluating its quality and laying the foundation for quantitative determination of the various chemical components in Chuanxiong Tea Powder and subsequent research on the substance. The invention solves the problem that the fingerprint established in the prior art can only identify seven peaks, namely, cimicifuga glycoside, liquiritin, cimicifuga, 5-O-methylasvimethicone glycoside, ligusticum lactone I, chelidonol glycoside, and ligusticum lactone A, which is not enough to comprehensively evaluate the quality control of Chuanxiong Tea Powder.

[0059] The mass concentration of formic acid as mobile phase B can be, but is not limited to, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1%, preferably 0.05%.

[0060] In some specific embodiments, the amount of the Chuanxiong tea powder sample added to methanol is 0.5-1.5 g of the Chuanxiong tea powder sample per 100 ml of methanol.

[0061] The amount of Chuanxiong tea powder sample added to every 100 ml of methanol can be, but is not limited to, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1.0 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g or 1.5 g, and can also be any value between 0.5 and 1.5 g.

[0062] In some specific embodiments, the added amount of the Chuanxiong tea powder sample is 1 g of Chuanxiong tea powder sample per 100 ml of methanol.

[0063] In some specific embodiments, the ultrasonic treatment time is 20 to 40 minutes.

[0064] The ultrasonic treatment time may be, but is not limited to, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min or 40 min, and may be any value between 20 and 40 min, preferably 30 min.

[0065] In some specific embodiments, the ultrasound has a power of 200-400 W and a frequency of 40-50 kHz, preferably a power of 300 W and a frequency of 45 kHz.

[0066] In some specific embodiments, the ultrasonic treatment further comprises cooling and then weighing the mass, and supplementing the lost mass with methanol.

[0067] In some specific embodiments, the filtration includes coarse filtration followed by filtering the filtrate through a 0.22-0.3 μm microporous membrane.

[0068] The pore size of the microporous membrane may be, but is not limited to, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm, 0.29 μm or 0.3 μm, or any value between 0.22 and 0.3 μm, preferably 0.22 μm.

[0069] In some specific embodiments, the fingerprint of the Chuanxiong tea powder sample solution is constructed using a Cl8 chromatographic column with a length of 25 cm, a particle size of 5 μm, and an inner diameter of 4.6 mm; gradient elution, a flow rate of 1.0 mL / min; a column temperature of 30°C; an injection volume of 10 μL; and a detection wavelength of 280 nm.

[0070] In some specific embodiments, the gradient elution conditions include:

[0071] 0~15min, 9%A, 91%B;

[0072] 15~18min, 9%~15%A, 91%~85%B;

[0073] 18-60 min, 15% A, 85% B;

[0074] 60~85min, 15%~27%A, 85%~73%B;

[0075] 85~115min, 27%~40%A, 73%~60%B;

[0076] 115~130min, 40%~45%A, 60%~55%B;

[0077] 130~131min, 45%~55%A, 55%~45%B;

[0078] 131~145min, 55%A, 45%B.

[0079] The aforementioned gradient elution conditions can further enhance the separation and detection of characteristic peaks of the various components of Chuanxiong Tea Powder. Experimental results indicate that 26 characteristic peaks can be identified in Chuanxiong Tea Powder, 16 of which are as follows: chlorogenic acid (peak 1), caffeic acid (peak 2), vanillin (peak 4), ferulic acid (peak 5), liquiritin (peak 6), cimicifugoside (peak 9), hesperidin (peak 10), rosmarinic acid (peak 11), montanol (peak 13), pulegone (peak 15), coniferyl ferulate (peak 18), glycyrrhizic acid (peak 20), Z-ligustilide (peak 22), notopterygium wilfordii alcohol (peak 24), imperatorin (peak 25), and isoimperatorin (peak 26). This allows for the simultaneous detection of multiple components of Chuanxiong Tea Powder, enabling a more comprehensive evaluation of its quality while also improving detection efficiency and reducing costs. This method can quantify the contents of various chemical components in Chuanxiong Cha Tiao Powder, laying the foundation for subsequent research on Chuanxiong Cha Tiao Powder.

[0080] Where A is acetonitrile as mobile phase A, and B is formic acid with a mass concentration of 0.01~0.10% of mobile phase B.

[0081] In some specific embodiments, the step C comprises:

[0082] C1. Extract characteristic peaks from the HPLC spectrum of Chuanxiongchatiao powder;

[0083] C2. Perform similarity analysis on the characteristic peaks to construct a fingerprint of Chuanxiong Cha Tiao Powder that represents the overall characteristics of Chuanxiong Cha Tiao Powder.

[0084] The similarity analysis involves using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software to generate a common pattern spectrum for characteristic peaks, which serves as the reference fingerprint for Chuanxiong Tea Tiao Powder. Specifically, the Chuanxiong Tea Tiao Powder sample chromatogram is imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System," where multi-point correction and peak matching are performed, followed by the median method to generate the reference fingerprint. This ultimately forms the Chuanxiong Tea Tiao Powder fingerprint.

[0085] According to a specific implementation method, a total of 26 characteristic peaks of different components of Chuanxiong Cha Tiao Powder can be separated, and the components represented by 16 peaks can be identified through mixed comparison.

[0086] In some specific embodiments, the step C further includes verifying the fingerprint of Chuanxiong Cha Tiao Powder using a standard fingerprint;

[0087] If the characteristic peaks of the fingerprint of Chuanxiong Cha Tiao Powder are consistent with those of the standard product fingerprint, the construction is successful, otherwise it is reconstructed.

[0088] In some specific embodiments, the HPLC spectrum of the Chuanxiong Tea Powder includes at least one HPLC spectrum of Chuanxiong Tea Powder of the same specification but different batches.

[0089] By using one or more different batches, the chemical composition and characteristics of the sample can be more accurately reflected, reducing the accidental errors caused by a single batch of samples; sufficient sample batches can also help verify the repeatability and consistency of the fingerprint spectrum, ensuring the stability and reliability of the results; by analyzing multiple batches of samples, the variables in the experimental process can be better controlled, and the repeatability of the experiment can be improved. By collecting 20 different batches of samples, the embodiment of the present invention found that different batches of Chuanxiong Tea Powder all had the same fingerprint spectrum. In subsequent applications, there is no need to use 20 batches of Chuanxiong Tea Powder to synthesize the fingerprint spectrum. It is only necessary to test the samples according to the patented method to directly obtain the fingerprint spectrum of Chuanxiong Tea Powder.

[0090] According to another aspect of the present invention, there is also provided the use of the aforementioned Chuanxiong Tea Powder pre-treatment method or the aforementioned Chuanxiong Tea Powder fingerprint construction method in any of the following:

[0091] D1) Qualitative analysis of the active ingredients in Chuanxiongchatiaosan;

[0092] D2) Quantitative analysis of the active ingredients in Chuanxiongchatiaosan;

[0093] D3) Evaluate the quality of Chuanxiong Tea Powder.

[0094] According to another aspect of the present invention, a method for evaluating the quality of Chuanxiong Tea Powder is provided, comprising using the aforementioned Chuanxiong Tea Powder fingerprint as a reference fingerprint, comparing the characteristic peaks of the Chuanxiong Tea Powder fingerprint to be evaluated with the characteristic peaks of the reference fingerprint, and judging the quality of the Chuanxiong Tea Powder to be evaluated;

[0095] Preferably, judging the quality of the Chuanxiong Tea Powder to be evaluated includes: if the number of peaks and the peak time of the fingerprint of the Chuanxiong Tea Powder to be evaluated are consistent with those of the control fingerprint, it indicates that the quality of the Chuanxiong Tea Powder to be evaluated is consistent with the Chuanxiong Tea Powder used to construct the above-mentioned Chuanxiong Tea Powder fingerprint; otherwise, they are inconsistent and are deemed to be of unqualified quality.

[0096] Among them, when judging whether the number of peaks and the peak time of the fingerprint spectrum of the Chuanxiong Tea Powder to be evaluated are consistent with those of the control fingerprint spectrum, the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system can also be used. The fingerprint spectrum of the test sample and the control fingerprint spectrum are calculated after similarity, and they are consistent when the similarity is not less than 0.90.

[0097] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0098] Preparation of standard solution: Accurately weigh 1.224 mg of chlorogenic acid, 2.098 mg of caffeic acid, 1.053 mg of vanillin, 3.029 mg of ferulic acid, 2.019 mg of ammonium glycyrrhizate, 1.9823 mg of cimicifugoside, 1.023 mg of hesperidin, 1.654 mg of rosmarinic acid, 2.319 mg of mongoside, 2.081 mg of pulegone, 1.983 mg of coniferyl ferulate, 1.082 mg of liquiritin, 1.723 mg of Z-ligustilide, 1.892 mg of notopterygium wilfordii alcohol, 1.439 mg of imperatorin, and 1.782 mg of isoimperatorin into a brown volumetric flask. Add methanol to make up to 100 mL to obtain the mixed reference solution (store at 4°C).

[0099] Example 1

[0100] A method for constructing a fingerprint of Chuanxiong Cha Tiao Powder based on high performance liquid chromatography, specifically following the steps below:

[0101] (1) Test solution: Take about 0.5 g of Chuanxiong tea powder, weigh it accurately, place it in a stoppered conical flask, add 50 mL of methanol accurately, stopper it tightly, weigh it, and treat it ultrasonically (power 300 W, frequency 45 kHz) for 30 min. Let it cool, weigh it again, make up the lost mass with methanol, shake it well, filter it, take the filtrate, filter it with a 0.22 μm microporous membrane, and collect the filtrate as the test solution.

[0102] (2) Chromatographic conditions: The chromatographic column was Agilent Eclipse Plus Cl8 (4.6 mm × 250 mm, 5 μm); the mobile phase was acetonitrile (A)–0.05% formic acid (B), with gradient elution; the flow rate was 1.0 mL / min; the column temperature was 30 °C; the injection volume was 10 μL; and the detection wavelength was 280 nm.

[0103] (3) Mobile phase and gradient elution procedure: Acetonitrile was used as mobile phase A and 0.05% formic acid solution was used as mobile phase B for gradient elution. The gradient elution procedure was as follows:

[0104] 0~15min, 9%A, 91%B;

[0105] 15~18min, 9%~15%A, 91%~85%B;

[0106] 18-60 min, 15% A, 85% B;

[0107] 60~85min, 15%~27%A, 85%~73%B;

[0108] 85~115min, 27%~40%A, 73%~60%B;

[0109] 115~130min, 40%~45%A, 60%~55%B;

[0110] 130~131min, 45%~55%A, 55%~45%B;

[0111] 131~145min, 55%A, 45%B.

[0112] (4) Sample determination: Under the above chromatographic conditions, accurately aspirate 10 μl of the standard solution and the test solution, and perform high performance liquid chromatography analysis. Obtain the HPLC spectrum of the standard solution (such as Figure 2 ) and the HPLC spectrum of Chuanxiongcha Tiaosan (as shown in Figure 1 20 batches were tested, respectively recorded as S-1 to S-20. The chromatograms of Chuanxiong Cha Tiao Powder samples were sequentially imported into the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System". With S-20 as the reference spectrum, multi-point correction and chromatographic peak matching were performed in sequence, and the control fingerprint spectrum was generated using the median method. The final Chuanxiong Cha Tiao Powder fingerprint spectrum is shown in the figure below. Figure 1 , the standard chromatogram is as follows Figure 2 . ) 26 common peaks (peaks 1 to 26) were calibrated for all 20 samples of Chuanxiong Tea Powder. Figure 1 As shown in the figure, 16 peaks were identified by comparison with the standard: chlorogenic acid (peak 1), caffeic acid (peak 2), vanillin (peak 4), ferulic acid (peak 5), liquiritin (peak 6), cimicifugoside (peak 9), hesperidin (peak 10), rosmarinic acid (peak 11), montanol (peak 13), pulegone (peak 15), coniferyl ferulate (peak 18), glycyrrhizic acid (peak 20), Z-ligustilide (peak 22), notopterygium wilfordii alcohol (peak 24), imperatorin (peak 25), and isoimperatorin (peak 26).

[0113] Example 2

[0114] A method for constructing a fingerprint of Chuanxiong Cha Tiao Powder based on high performance liquid chromatography, specifically following the steps below:

[0115] (1) Test solution: Take about 0.75 g of Chuanxiong tea powder, weigh it accurately, place it in a stoppered conical flask, add 50 mL of methanol accurately, stopper it tightly, weigh it, and treat it ultrasonically (power 300 W, frequency 45 kHz) for 20 min. Let it cool, weigh it again, make up the lost mass with methanol, shake it well, filter it, take the filtrate, filter it with a 0.3 μm microporous membrane, and collect the filtrate as the test solution.

[0116] (2) Chromatographic conditions: The chromatographic column was Agilent Eclipse Plus Cl8 (4.6 mm × 250 mm, 5 μm); the mobile phase was acetonitrile (A)–0.01% formic acid (B), with gradient elution; the flow rate was 1.0 mL / min; the column temperature was 30 °C; the injection volume was 10 μL; and the detection wavelength was 280 nm.

[0117] (3) Mobile phase and gradient elution procedure: Acetonitrile was used as mobile phase A and 0.01% formic acid solution was used as mobile phase B for gradient elution. The gradient elution procedure was as follows:

[0118] 0~15min, 9%A, 91%B;

[0119] 15~18min, 9%~15%A, 91%~85%B;

[0120] 18-60 min, 15% A, 85% B;

[0121] 60~85min, 15%~27%A, 85%~73%B;

[0122] 85~115min, 27%~40%A, 73%~60%B;

[0123] 115~130min, 40%~45%A, 60%~55%B;

[0124] 130~131min, 45%~55%A, 55%~45%B;

[0125] 131~145min, 55%A, 45%B.

[0126] (4) Sample determination: Under the above chromatographic conditions, accurately draw 10 μl of the sample solution and perform high performance liquid chromatography analysis to obtain the fingerprint of Chuanxiong Cha Tiao Powder, as shown in the following figure: Figure 3As shown in the figure, there are 26 characteristic peaks in total, of which 16 characteristic peaks are identified as follows: chlorogenic acid (peak 1), caffeic acid (peak 2), vanillin (peak 4), ferulic acid (peak 5), liquiritin (peak 6), cimicifugoside (peak 9), hesperidin (peak 10), rosmarinic acid (peak 11), montanol (peak 13), pulegone (peak 15), coniferyl ferulate (peak 18), glycyrrhizic acid (peak 20), Z-ligustilide (peak 22), notopterygium wilfordii alcohol (peak 24), imperatorin (peak 25), and isoimperatorin (peak 26).

[0127] Example 3

[0128] A method for constructing a fingerprint of Chuanxiong Cha Tiao Powder based on high performance liquid chromatography, specifically following the steps below:

[0129] (1) Test solution: Take about 0.25 g of Chuanxiong tea powder, weigh it accurately, place it in a stoppered conical flask, add 50 mL of methanol accurately, stopper it tightly, weigh it, and treat it ultrasonically (power 300 W, frequency 45 kHz) for 40 min. Let it cool, weigh it again, make up the lost mass with methanol, shake it well, filter it, take the filtrate, filter it with a 0.25 μm microporous membrane, and collect the filtrate as the test solution.

[0130] (2) Chromatographic conditions: The chromatographic column was Agilent Eclipse Plus Cl8 (4.6 mm × 250 mm, 5 μm); the mobile phase was acetonitrile (A)–0.1% formic acid (B), with gradient elution; the flow rate was 1.0 mL / min; the column temperature was 30 °C; the injection volume was 10 μL; and the detection wavelength was 280 nm.

[0131] (3) Mobile phase and gradient elution procedure: Acetonitrile was used as mobile phase A and 0.1% formic acid solution was used as mobile phase B for gradient elution. The gradient elution procedure was as follows:

[0132] 0~15min, 9%A, 91%B;

[0133] 15~18min, 9%~15%A, 91%~85%B;

[0134] 18-60 min, 15% A, 85% B;

[0135] 60~85min, 15%~27%A, 85%~73%B;

[0136] 85~115min, 27%~40%A, 73%~60%B;

[0137] 115~130min, 40%~45%A, 60%~55%B;

[0138] 130~131min, 45%~55%A, 55%~45%B;

[0139] 131~145min, 55%A, 45%B.

[0140] (4) Sample determination: Under the above chromatographic conditions, accurately draw 10 μl of each test solution and perform high performance liquid chromatography analysis to obtain the HPLC spectrum of Chuanxiong Tea Powder. Figure 4 As shown in the figure, there are 26 characteristic peaks in total, of which 16 characteristic peaks are identified as follows: chlorogenic acid (peak 1), caffeic acid (peak 2), vanillin (peak 4), ferulic acid (peak 5), liquiritin (peak 6), cimicifugoside (peak 9), hesperidin (peak 10), rosmarinic acid (peak 11), montanol (peak 13), pulegone (peak 15), coniferyl ferulate (peak 18), glycyrrhizic acid (peak 20), Z-ligustilide (peak 22), notopterygium wilfordii alcohol (peak 24), imperatorin (peak 25), and isoimperatorin (peak 26).

[0141] Comparative Example 1

[0142] The difference from Example 1 is that (1) the test solution: take about 0.5 g of Chuanxiong tea powder, accurately weigh it, accurately add 50 mL of pure water, weigh the mass, boil it over high heat, continue to simmer for 30 minutes, seal it, let it cool, weigh the mass again, make up the lost mass with pure water, shake it well, filter it, take the filtrate, filter it with a 0.22 μm microporous membrane, and collect the filtrate as the test solution.

[0143] The results are as follows Figure 5 As shown in the figure, there are 11 characteristic peaks in total, of which only 5 characteristic peaks can be identified: caffeic acid (peak 2), cimicifugoside (peak 9), hesperidin (peak 10), rosmarinic acid (peak 11), and mongoside (peak 13). Compared with the examples, it is obvious that most of the components cannot be effectively separated.

[0144] Comparative Example 2

[0145] The difference from Example 1 is that (2) the chromatographic conditions are: the chromatographic column is Agilent Eclipse Plus Cl8 (4.6 mm × 250 mm, 5 μm); the mobile phase is acetonitrile-methanol-water, and gradient elution is performed; the flow rate is 1.0 mL / min; the column temperature is 30°C; the injection volume is 10 μL; and the detection wavelength is 280 nm.

[0146] (3) Mobile phase and gradient elution procedure: Use methanol-acetonitrile in a volume ratio of 1:1, recorded as mobile phase A, and water as mobile phase B for gradient elution. The gradient elution procedure is as follows:

[0147] 0~15min, 9%A, 91%B;

[0148] 15~18min, 9%~15%A, 91%~85%B;

[0149] 18-60 min, 15% A, 85% B;

[0150] 60~85min, 15%~27%A, 85%~73%B;

[0151] 85~115min, 27%~40%A, 73%~60%B;

[0152] 115~130min, 40%~45%A, 60%~55%B;

[0153] 130~131min, 45%~55%A, 55%~45%B;

[0154] 131~145min, 55%A, 45%B.

[0155] The results are as follows Figure 6 As shown, the characteristic peaks could not be effectively separated.

[0156] Experiment 1 Optimization of mobile phase and gradient elution program

[0157] Optimization process 1

[0158] The difference from Example 1 is that (3) the mobile phase and gradient elution procedure is: acetonitrile is used as mobile phase A and 0.05% formic acid solution is used as mobile phase B for gradient elution. The gradient elution procedure is as follows in the following volume ratio:

[0159] 0-30min, 20%A, 80%B;

[0160] 30~40min, 20%~40%A, 80%~60%B;

[0161] 40~60min, 40%~75%A, 60%~25%B;

[0162] 60~65min, 75%~100%A, 25%~0B;

[0163] 65~80min, 100%~10%A, 0~90%B.

[0164] The results are as follows Figure 7 As shown, most of the characteristic peaks were not effectively separated by this method, and the gradient elution procedure needs to be further optimized.

[0165] Optimization process 2

[0166] The difference from Example 1 is that (3) the mobile phase and gradient elution procedure is: acetonitrile is used as mobile phase A and 0.05% formic acid solution is used as mobile phase B for gradient elution. The gradient elution procedure is as follows in the following volume ratio:

[0167] 0~10min, 20%A, 80%B;

[0168] 10~15min, 20%~30%A, 80%~70%B;

[0169] 15~20min, 30%~40%A, 70%~60%B;

[0170] 20~25min, 40%~45%A, 60%~55%B;

[0171] 25~45min, 45%~60%A, 55%~40B;

[0172] 45~80min, 60%~100%A, 40%~0B.

[0173] The results are as follows Figure 8 As shown, most of the characteristic peaks could not be effectively separated by this method, so the gradient elution procedure needs to be further optimized.

[0174] Optimization process 3

[0175] The difference from Example 1 is that (3) the mobile phase and gradient elution procedure is: acetonitrile is used as mobile phase A and 0.05% formic acid solution is used as mobile phase B for gradient elution. The gradient elution procedure is as follows in the following volume ratio:

[0176] 0-10min, 10%A, 90%B;

[0177] 10-20min, 10%~18%A, 90%~82%B;

[0178] 20-50min, 18%A, 82%B;

[0179] 50-70min, 18%~27%A, 82%~73%B;

[0180] 70-100min, 27%~42%A, 73%~58%B;

[0181] 100-120min, 42%~45%A, 58%~55%B;

[0182] 120-130 min, 45% A, 55% B;

[0183] 130-140min, 45%~55%A, 55%~45%B;

[0184] 140-150min, 55%~100%A, 45%~0B.

[0185] The results are as follows Figure 9 As shown, this method produces more characteristic peaks than the first two methods, but the separation effect of the components is not perfect enough, so the gradient elution program was further optimized to obtain the gradient elution program used in Examples 1 to 3.

[0186] Experiment 2 Methodological investigation of the fingerprint of Chuanxiong Cha Tiao Powder RSD value

[0187] Using the fingerprint of Chuanxiongchatiao powder constructed in Example 1, with ferulic acid (peak 5) as the reference peak, the following steps were performed:

[0188] 1. Precision test

[0189] 0.5 g of Chuanxiong Tea Powder was used to prepare a test solution according to the method in Example 1. The sample was injected according to the chromatographic conditions in Example 1, and the chromatographic peak No. 5 of ferulic acid was used as the reference peak (S). The relative retention time and relative peak area RSD of each common peak were calculated. The results are shown in Table 1. It can be seen that the relative retention time is within 0.5%, and the relative peak area RSD is within 2.5%, indicating that the precision of the instrument is good.

[0190] 2. Reproducibility test

[0191] 0.5 g of Chuanxiong Tea Powder was used to prepare a test solution according to the method in Example 1. Continuous measurements were performed under the chromatographic conditions of Example 1, using the No. 5 ferulic acid peak as the reference peak (S). The relative retention times and relative peak area RSDs of the common peaks were calculated. The results are shown in Table 1. The relative retention times were within 0.5% and the relative peak area RSDs were within 2%, indicating good reproducibility of the method.

[0192] 3. Stability test

[0193] Chuanxiong tea powder was used to prepare a test solution according to the method in Example 1. The test solution was assayed at 0, 3, 6, 9, and 12 hours after preparation using the chromatographic conditions in Example 1. The solution was stored in a refrigerator at 4°C before assay. Using the ferulic acid peak No. 5 as the reference peak (S), the relative retention times of the common peaks were calculated to be within 1%, and the relative peak area RSDs were within 2%, indicating good stability of the test solution within 12 hours.

[0194] Table 1 RSD values ​​of the methodological investigation of the fingerprint of Chuanxiong Cha Tiao Powder

[0195]

[0196] The relative peak areas are: 0.474-0.771 (#1), 0.179-1.117 (#2), 0.106-0.306 (#3), 0.054-0.209 (#4), 1.000 (#5 reference peak), 0.325-1.540 (#6), 0.556-1.776 (#7), 0.174-0.368 (#8), 0.367-1.361 (#9), 3.573-6.904 (#10), 2.071-5.160 (#11), 0.316-1.141 (#12), 0.184-0.327 (#13), 0.476-1.129 (#14), 0.109-0.235 (#15), 0.110-0. 227 (#16), 0.131~0.330 (#17), 0.322~1.310 (#18), 0.192~0.594 (#19), 0.135~0.276 (#20), 0.066~0.382 (#21), 2.262~6.143 (#22), 0.160~0.334 (#23), 0.183~0.431 (#24), 0.259~0.599 (#25), 0.198~0.635 (#26).

[0197] Application Example 1: Detection of Chuanxiong Tea Powder

[0198] 1. Quantitative detection

[0199] The fingerprint of Chuanxiong Tea Powder constructed in Example 1 was used as the control fingerprint. 0.5 g of Chuanxiong Tea Powder powder was taken and a test solution was prepared according to the method of Example 1. The main active ingredients in Chuanxiong Tea Powder were quantitatively detected according to the HPLC conditions of Example 1 to obtain the fingerprint of the sample to be tested (as shown in Figure 1). Figure 10 The content of each component was calculated using HPLC, and the content of Chuanxiong Cha Tiao Powder was evaluated based on the amount of each component. The results of the 16 identified peaks are shown in Table 2.

[0200] Table 2

[0201]

[0202] 2. Quality evaluation

[0203] The fingerprint of the Chuanxiong Tea Modulation Powder constructed in Example 1 was used as the control fingerprint, and the fingerprint of the sample to be detected obtained by quantitative detection in Example 1 (such as Figure 10The number of characteristic peaks and the time of their appearance (as shown) are compared with those of the reference fingerprint. If they are consistent, it is considered that the quality of the reference fingerprint has been achieved and the quality is good. If some chromatographic peaks are missing or extra, it is considered that there is a possibility that the Chuanxiong Cha Tiao Powder sample is missing drug components or is a counterfeit.

[0204] Specific results such as Figure 10 As shown, the spectrum was compared with the control fingerprint spectrum ( Figure 1 ) were compared, and it was found that since the characteristic chromatographic peak of Chuanxiong Tea Powder in Application Example 2 was consistent with the peak shape and peak retention time of the control fingerprint, it was shown that the ingredients contained were consistent, indicating that the quality of the Chuanxiong Tea Powder to be evaluated was good and there was no adulteration.

[0205] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a fingerprint of Chuanxiong Tea Flowing Powder based on high performance liquid chromatography, characterized in that: The following steps are involved: A. placing the Chuanxiong Tea Powder sample in methanol for ultrasonic treatment and then filtering to obtain a Chuanxiong Tea Powder sample solution; B. Using high performance liquid chromatography to determine the Chuanxiong Tea Adjusting Powder sample solution to obtain the Chuanxiong Tea Adjusting Powder HPLC spectrum; C. Constructing the fingerprint of Chuanxiongcha Tiaosan based on the HPLC spectrum of Chuanxiongcha Tiaosan; A Cl8 column with a length of 25 cm, a particle size of 5 μm, and an inner diameter of 4.6 mm was used; gradient elution was performed at a flow rate of 1.0 mL / min; the column temperature was 30°C; the injection volume was 10 μL; and the detection wavelength was 280 nm. The mobile phase A in the HPLC assay is acetonitrile, and the mobile phase B is formic acid with a mass concentration of 0.01-0.10%; The gradient elution conditions include: 0~15min, 9%A, 91%B; 15~18min, 9%~15%A, 91%~85%B; 18-60 min, 15% A, 85% B; 60~85min, 15%~27%A, 85%~73%B; 85~115min, 27%~40%A, 73%~60%B; 115~130min, 40%~45%A, 60%~55%B; 130~131min, 45%~55%A, 55%~45%B; 131-145 min, 55% A, 45% B; The fingerprint of Chuanxiong tea powder contains 26 characteristic peaks, among which peak 1 is chlorogenic acid, peak 2 is caffeic acid, peak 4 is vanillin, peak 5 is ferulic acid, peak 6 is liquiritin, peak 9 is cimicifugoside, peak 10 is hesperidin, peak 11 is rosmarinic acid, peak 13 is mongoside, peak 15 is pulegone, peak 18 is coniferyl ferulate, peak 20 is glycyrrhizic acid, peak 22 is Z-ligustilide, peak 24 is notopterygium wilfordii alcohol, peak 25 is imperatorin, and peak 26 is isoimperatorin.

2. The construction method according to claim 1, characterized in that The added amount of the Chuanxiong tea powder sample is 0.5-1.5g of Chuanxiong tea powder sample per 100ml of methanol.

3. The construction method according to claim 2, characterized in that The amount of the Chuanxiong tea powder sample added to methanol is 1 g of the Chuanxiong tea powder sample per 100 ml of methanol.

4. The construction method according to claim 2, characterized in that The ultrasonic treatment time is 20 to 40 minutes.

5. The construction method according to claim 4, characterized in that: The ultrasonic treatment time is 30 min.

6. The construction method according to claim 2, characterized in that The power of the ultrasound is 200-400W, and the frequency is 40-50kHz.

7. The construction method according to claim 6, characterized in that The power of the ultrasound is 300W and the frequency is 45kHz.

8. The construction method according to claim 2, characterized in that: After the ultrasonic treatment, the method further includes cooling and weighing the mass, and using methanol to make up for the lost mass.

9. The construction method according to claim 2, characterized in that: The filtration includes coarse filtration followed by filtration of the filtrate through a 0.22-0.3 μm microporous membrane.

10. The construction method according to claim 9, characterized in that: The filtration includes coarse filtration followed by filtration of the filtrate through a 0.22 μm microporous filter membrane.

11. The construction method according to claim 1, characterized in that: The step C comprises: C1. Extract characteristic peaks from the HPLC spectrum of Chuanxiongchatiao powder; C2. Perform similarity analysis on the characteristic peaks to construct a fingerprint of Chuanxiong Cha Tiao Powder that represents the overall characteristics of Chuanxiong Cha Tiao Powder.

12. The construction method according to claim 1, characterized in that: After step C, the fingerprint of Chuanxiongchatiao powder is verified using the fingerprint of the standard; If the characteristic peaks of the fingerprint of Chuanxiong Cha Tiao Powder are consistent with those of the standard product fingerprint, the construction is successful, otherwise it is reconstructed.

13. Use of the method for constructing the fingerprint of Chuanxiong Cha Tiao Powder according to any one of claims 1 to 12 in any of the following: D1) Qualitative analysis of the active ingredients in Chuanxiongchatiaosan; D2) Quantitative analysis of the active ingredients in Chuanxiongchatiaosan; D3) Evaluate the quality of Chuanxiong Tea Powder.

Images