A method for constructing a fingerprint spectrum of shentong zhuyu decoction and application thereof

Abstract

The application discloses a kind of shentong zhuyu decoction fingerprint construction method and application, belong to the technical field of drug detection.The application is detected by high performance liquid chromatography to several batches of shentong zhuyu decoction by the selection of mobile phase and the cooperation of chromatographic column, the high performance liquid chromatography of different batches of shentong zhuyu decoction is compared, obtains the common peak containing loganic acid, amygdalin, hydroxyl safflower yellow pigment A, aniseed acid, qianghuo alcohol, isoeucommarin, ammonium glycyrrhizinate, kaempferol characteristic peak, and the fingerprint of shentong zhuyu decoction is constructed according to the common peak.The fingerprint constructed by the application can better detect 12 medicinal materials, shorten the detection time of single sample to 125 min or less, improve efficiency, and improve the quality control level of product.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical testing technology, specifically to a method for constructing and applying the fingerprint spectrum of Shentong Zhuyu Decoction. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Shen Tong Zhu Yu Tang (Body Pain Relief Decoction) is a classic Chinese medicine formula composed of 12 herbs: Gentiana macrophylla, Ligusticum chuanxiong, Prunus persica, Carthamus tinctorius, Glycyrrhiza uralensis, Notopterygium incisum, Commiphora myrrha, Angelica sinensis, Trogopterus xanthipes, Cyperus rotundus, Achyranthes bidentata, and Pheretima aspergillum. Its functions are to invigorate blood circulation, promote qi circulation, resolve phlegm, unblock meridians, and relieve pain. It is mainly used for syndromes of blood stasis obstructing meridians, such as shoulder pain, arm pain, back pain, leg pain, or general body pain, sharp, stabbing pain that is persistent, accompanied by a dark tongue or ecchymosis, and a choppy pulse. According to the inventor's research, although there are currently detection methods for the characteristic chromatograms of Shen Tong Zhu Yu Tang, liquid chromatography detection takes more than 3 hours and cannot fully represent all 12 herbs, making it difficult to effectively control its intrinsic quality during production. Furthermore, even with shorter detection times, the peak shapes of the constructed characteristic chromatograms of Shen Tong Zhu Yu Tang are poor, and the separation of each peak is insufficient, making it impossible to simultaneously detect the content of various active ingredients in Shen Tong Zhu Yu Tang, thus hindering precise quality control. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for constructing and applying a fingerprint spectrum of Shentong Zhuyu Decoction. The fingerprint spectrum constructed by this invention contains common peaks of loganic acid, amygdalin, hydroxysafflower yellow A, ferulic acid, notopterygium alcohol, isoimperatorin, ammonium glycyrrhizate, and kaempferol, which can better detect the 12 medicinal materials in Shentong Zhuyu Decoction, shorten the detection time, and simultaneously detect the content of the above 8 components, thereby improving the quality control level of the product.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] The first aspect of this invention provides a method for constructing the fingerprint spectrum of Shentong Zhuyu Decoction, comprising the following steps:

[0007] The test solution and the reference solution were taken separately and determined by high performance liquid chromatography. Common peaks containing the characteristic peaks of loganic acid, amygdalin, hydroxysafflower yellow A, ferulic acid, notopterygol, isoimperatorin, glycyrrhizic acid ammonium, and kaempferol were obtained, and a fingerprint spectrum of Shentong Zhuyu Decoction was established.

[0008] The detection conditions for the high-performance liquid chromatography method include:

[0009] Mobile phase A is acetonitrile, and mobile phase B is a 0.03–0.08 mol / L potassium dihydrogen phosphate buffer solution with pH = 3.0–4.0. Gradient elution is used, and the detection wavelength is 200–250 nm.

[0010] The inventors discovered that current methods for detecting the characteristic chromatograms of Shentong Zhuyu Decoction mostly use acetonitrile and phosphoric acid aqueous solution as the mobile phase. While these methods can successfully establish characteristic chromatograms for Shentong Zhuyu Decoction, the peak shapes are poor and the separation between peaks is insufficient. This prevents the simultaneous detection of the content of various components in Shentong Zhuyu Decoction, requiring different elution procedures to detect the content of each component. To address this, the inventors conducted extensive research and discovered that using acetonitrile and potassium dihydrogen phosphate buffer solution as the mobile phase, and increasing the proportion of the organic phase from 5% to 68%, allows most components of Shentong Zhuyu Decoction to be displayed in the chromatogram. This enables comprehensive detection of the 12 medicinal herbs in Shentong Zhuyu Decoction and allows for multiple evaluations in one measurement, simultaneously detecting the content of eight components in Shentong Zhuyu Decoction: loganic acid, amygdalin, hydroxysaffron yellow A, ferulic acid, notopterygium alcohol, isoimperatorin, ammonium glycyrrhizate, and kaempferol. The method of this invention simultaneously detects 12 medicinal materials in Shentong Zhuyu Decoction from both qualitative and quantitative perspectives, and the detection results are accurate, thereby improving the quality control level of Shentong Zhuyu Decoction products.

[0011] This invention selects 200-250 nm as the detection wavelength for the fingerprint spectrum of Shentong Zhuyu Decoction. This wavelength is rich in chemical composition information, and 27 common peaks can be obtained. The positions of loganic acid, amygdalin, hydroxysafflower yellow A, ferulic acid, notopterygium alcohol, isoimperatorin, ammonium glycyrrhizate, and kaempferol are identified by reference standards.

[0012] In some embodiments of the present invention, the method for preparing the test solution includes the following steps:

[0013] Methanol was added to the Shen Tong Zhu Yu Decoction to be tested, the volume was adjusted, and the mixture was sonicated, cooled, and the weight loss was replenished with methanol. The mixture was then shaken well and filtered through a membrane to obtain the test solution. Sonication helps to evenly disperse the Shen Tong Zhu Yu Decoction in the methanol; however, the increased temperature during sonication causes methanol to evaporate and its concentration to rise, leading to higher errors. Therefore, adding methanol after cooling reduces the impact of methanol evaporation and minimizes measurement errors. Furthermore, the sonication time is 20–40 minutes.

[0014] Preferably, the concentration of Shentong Zhuyu Decoction in the test solution is 48-52% by volume.

[0015] In some embodiments of the present invention, the method for preparing the reference solution includes the following steps:

[0016] Loganic acid, amygdalin, hydroxysafflower yellow A, ferulic acid, gentianol, isoimperatorin, ammonium glycyrrhizate, and kaempferol were dissolved in solvents to prepare reference solutions.

[0017] Preferably, the solvent is methanol. Methanol has better solubility for loganic acid, amygdalin, hydroxysaffron yellow A, ferulic acid, citronellol, isoimperatorin, ammonium glycyrrhizate, and kaempferol, which facilitates the dispersion of each compound, improves the detector's detection efficiency, and enhances detection stability. Furthermore, using the same solvent for the test solution and the reference solution reduces matrix effects and improves the accuracy of the fingerprint spectrum.

[0018] Preferably, the concentrations of loganic acid, amygdalin, hydroxysaffron yellow A, ferulic acid, gentianol, isoimperatorin, ammonium glycyrrhizate, and kaempferol in the reference solution are all 0.08–0.12 mg / mL.

[0019] In some embodiments of the present invention, in the high-performance liquid chromatography method, the column packing material is 4-6 μm octadecylsilane-bonded silica gel, the column length is 200-300 mm, the column diameter is 4.5-4.7 mm, the column temperature is 25-35 °C, the mobile phase flow rate is 0.8-1.2 mL / min, and the injection volume is 4-10 μL.

[0020] Preferably, the chromatographic column is an Agilent XDB-C18 (250*4.6mm, 5μm).

[0021] In some embodiments of the present invention, the gradient elution procedure is as follows:

[0022] 0–14 min, 5%–11% mobile phase A, balance mobile phase B;

[0023] 14–26 min, 11% mobile phase A, balance mobile phase B;

[0024] 26–42 min, 11%–18% mobile phase A, balance mobile phase B;

[0025] 42–47 min, 18% mobile phase A, balance mobile phase B;

[0026] 47–66 min, 18%–26% mobile phase A, balance mobile phase B;

[0027] 66–85 min, 26%–50% mobile phase A, balance mobile phase B;

[0028] 85–12 5 min, 50%–68% mobile phase A, balance mobile phase B.

[0029] In some embodiments of the present invention, the process of establishing the feature map is as follows:

[0030] High-performance liquid chromatography (HPLC) was used to detect different batches of Shentong Zhuyu Decoction. The HPLC chromatograms of different batches of Shentong Zhuyu Decoction and the reference solution were compared to obtain common peaks containing the characteristic peaks of loganic acid, amygdalin, hydroxysaffron yellow A, ferulic acid, notopterygium alcohol, isoimperatorin, ammonium glycyrrhizate, and kaempferol. The fingerprint spectrum of Shentong Zhuyu Decoction was established based on the common peaks.

[0031] In some embodiments of the present invention, the detection wavelength is 210–230 nm.

[0032] In some embodiments of the present invention, the fingerprint spectrum of the Body Pain Relief Decoction includes 27 characteristic peaks, wherein peaks 5, 12, and 20 are from Gentiana macrophylla; peaks 24 and 27 are from Ligusticum chuanxiong; peaks 4, 10, and 20 are from Prunus persica; peaks 11, 17, and 18 are from Carthamus tinctorius; peaks 14, 15, 20, and 23 are from Glycyrrhiza uralensis; peaks 3, 4, and 7 are from Notopterygium incisum; peaks 20 and 27 are from Commiphora myrrha; peaks 16 and 19 are from Trogopterus xanthipes; and peak 22 is from Pheretima aspergillum.

[0033] Using peak 13 as reference peak S, the relative retention times of each characteristic peak and peak S are within ±10% of the specified values; the specified values ​​are: 0.168 (peak 1), 0.231 (peak 2), 0.253 (peak 3), 0.365 (peak 4), 0.393 (peak 5), 0.403 (peak 6), 0.419 (peak 7), 0.447 (peak 8), 0.481 (peak 9), 0.510 (peak 10), 0.595 (peak 11), 0.634 (peak 12), 1.000 (peak S), 1.024 (peak 14), 1.046 (peak 15), 1.113 Peak 16, 1.209, Peak 17, 1.236, Peak 18, 1.286, Peak 19, 1.389, Peak 20, 1.731, Peak 21, 1.746, Peak 22, 1.768, Peak 23, 1.975, Peak 24, 2.074, Peak 25, 2.136, Peak 26, 2.215; among which, Peak 5 is loganic acid, Peak 10 is amygdalin, Peak 11 is hydroxysaffron yellow A, Peak 13 is ferulic acid, Peak 21 is kaempferol, Peak 23 is ammonium glycyrrhizate, Peak 25 is notopterygium alcohol, and Peak 27 is isoimperatorin.

[0034] In a second aspect, the present invention provides an application of the above-mentioned method for constructing the fingerprint spectrum of Shentong Zhuyu Decoction in the quality testing of Shentong Zhuyu Decoction in industrial production. The fingerprint spectrum of Shentong Zhuyu Decoction is obtained according to the above-mentioned construction method; a high-performance liquid chromatography (HPLC) of the sample to be tested is obtained according to the above-mentioned construction method; a similarity evaluation is performed between the HPLC of the sample to be tested and the fingerprint spectrum of Shentong Zhuyu Decoction, and the sample to be tested with a relative retention time within ±10% of a specified value is considered a qualified product.

[0035] A second aspect of the present invention provides a method for detecting eight chemical components in a body pain relief and blood stasis removal decoction, comprising the following steps:

[0036] The test solution was analyzed by high performance liquid chromatography to obtain the content of eight chemical components, which include loganic acid, amygdalin, hydroxysafflower yellow A, ferulic acid, gentianol, isoimperatorin, ammonium glycyrrhizate and kaempferol.

[0037] The conditions for detection by ultra-high performance liquid chromatography include:

[0038] Mobile phase A is acetonitrile, and mobile phase B is a 0.03–0.08 mol / L potassium dihydrogen phosphate buffer solution with pH = 3.0–4.0. Gradient elution is used, and the detection wavelength is 200–250 nm.

[0039] The gradient elution procedure is as follows:

[0040] 0–14 min, 5%–11% mobile phase A, balance mobile phase B;

[0041] 14–26 min, 11% mobile phase A, balance mobile phase B;

[0042] 26–42 min, 11%–18% mobile phase A, balance mobile phase B;

[0043] 42–47 min, 18% mobile phase A, balance mobile phase B;

[0044] 47–66 min, 18%–26% mobile phase A, balance mobile phase B;

[0045] 66–85 min, 26%–50% mobile phase A, balance mobile phase B;

[0046] 85–12 5 min, 50%–68% mobile phase A, balance mobile phase B;

[0047] The chromatographic column packing is octadecylsilyl-bonded silica gel with a particle size of 4 - 6 μm, and the chromatographic column is Agilent XDB-C18 (250 * 4.6 mm, 5 μm); the column temperature is 25 - 35 °C; the flow rate of the mobile phase is 0.8 - 1.2 mL / min, and the injection volume is 4 - 10 μL;

[0048] The preparation method of the test solution comprises the following steps:

[0049] Methanol is added to the Shentong Zhuyu Decoction to be tested, fixed volume, ultrasonic treatment is carried out, cooled, the lost weight is made up with methanol, shaken well, and filtered through a filter membrane to obtain the test solution; in the test solution, the concentration of the Shentong Zhuyu Decoction is 48 - 52% (v / v).

[0050] The beneficial effects of the present invention are as follows:

[0051] The present invention provides a method for constructing a fingerprint of Shentong Zhuyu Decoction, which comprises the following steps: taking the test solution and the reference solution respectively, and determining by high performance liquid chromatography to obtain the common peaks containing the characteristic peaks of loganin acid, amygdalin, hydroxysafflor yellow A, ferulic acid, notopterol, isopimpinellin, ammonium glycyrrhizinate and kaempferol, and establishing the fingerprint of Shentong Zhuyu Decoction; the detection conditions of the high performance liquid chromatography include: mobile phase A is acetonitrile, mobile phase B is a potassium dihydrogen phosphate buffer solution with a pH of 3.0 - 4.0 and a concentration of 0.03 - 0.08 mol / L, gradient elution, and the detection wavelength is 200 - 250 nm. Through the selection of the mobile phase and the cooperation of the chromatographic column, more chemical components of Shentong Zhuyu Decoction can be well separated. Specifically, the present invention selects acetonitrile and potassium dihydrogen phosphate buffer solution as the mobile phase, and the proportion of the organic phase increases from 5% to 68% during the elution process, so that most components of Shentong Zhuyu Decoction are shown in the chromatogram, and 12 herbs in Shentong Zhuyu Decoction can be comprehensively detected; by selecting acetonitrile and potassium dihydrogen phosphate buffer solution as the mobile phase, the peak shapes of 8 components such as loganin acid, amygdalin, hydroxysafflor yellow A, ferulic acid, notopterol, isopimpinellin, ammonium glycyrrhizinate and kaempferol in Shentong Zhuyu Decoction are more excellent, and the resolution of each peak reaches the separation standard. Therefore, the separation of the above 8 components can be achieved at one time, and the simultaneous detection of the contents of the 8 components can be realized, the detection efficiency is improved, and the detection time of a single sample is shortened to within 125 min. The method of the present invention simultaneously detects 12 herbs in Shentong Zhuyu Decoction from both qualitative and quantitative aspects, and the detection result is accurate, which improves the quality control level of Shentong Zhuyu Decoction products. Description of the Drawings

[0052] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0053] Figure 1 High-performance liquid chromatography of the reference solution in Example 10 of this invention;

[0054] Figure 2 This is the high-performance liquid chromatography (HPLC) spectrum of Example 1 of the present invention;

[0055] Figure 3 This is the high-performance liquid chromatography (HPLC) of Example 2 of the present invention.

[0056] Figure 4 This is the high-performance liquid chromatography (HPLC) of Example 3 of the present invention;

[0057] Figure 5 This is the high-performance liquid chromatography (HPLC) of Example 5 of the present invention;

[0058] Figure 6 The fingerprint spectrum generated by measuring the spectrum under the scheme of Example 10 of the present invention contains 5, loganic acid, 10, amygdalin, 11, hydroxysafflower yellow pigment A, 13, ferulic acid, 21, kaempferol, 23, ammonium glycyrrhizate, 25, notopterygium alcohol, 27, and isoimperatorin.

[0059] Figure 7 High performance liquid chromatography for negative samples of Gentiana macrophylla and Shentong Zhuyu Decoction;

[0060] Figure 8 High-performance liquid chromatography for negative samples of Ligusticum chuanxiong and Shentong Zhuyu Decoction;

[0061] Figure 9 High-performance liquid chromatography for peach kernel negative samples and Shentong Zhuyu Decoction;

[0062] Figure 10 High-performance liquid chromatography for safflower negative samples and Shentong Zhuyu Decoction;

[0063] Figure 11 High-performance liquid chromatography of licorice negative samples and Shentong Zhuyu Decoction;

[0064] Figure 12 High-performance liquid chromatography for the negative sample of Qianghuo and Shentong Zhuyu Decoction;

[0065] Figure 13 High-performance liquid chromatography for myrrh-negative samples and Shentong Zhuyu Decoction;

[0066] Figure 14 High-performance liquid chromatography of Angelica sinensis negative sample and Shentong Zhuyu Decoction;

[0067] Figure 15High-performance liquid chromatography (HPLC) of the negative sample of Wulingzhi and Shentong Zhuyu Decoction;

[0068] Figure 16 High-performance liquid chromatography for the negative sample of Cyperus rotundus and Shentong Zhuyu Decoction;

[0069] Figure 17 High-performance liquid chromatography for negative samples of Achyranthes bidentata and Shentong Zhuyu Decoction;

[0070] Figure 18 High-performance liquid chromatography of earthworm negative samples and Shentong Zhuyu Decoction. Detailed Implementation

[0071] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0072] The relevant information regarding the equipment, reagents, test solutions, and reference solutions used in the following examples is shown below:

[0073] Instruments: Agilent 1260 high performance liquid chromatograph; 0.0001 g electronic balance, Mettler Toledo Instruments (Shanghai) Co., Ltd.; KQ-300DE CNC ultrasonic cleaner.

[0074] Test reagents: Loganilic acid, amygdalin, hydroxysafflower yellow A, ferulic acid, notopterygium alcohol, isoimperatorin, ammonium glycyrrhizate, kaempferol; reference standards were all purchased from the China National Institutes for Food and Drug Control. The Shen Tong Zhu Yu Decoction was prepared by Jinan Tonglu Pharmaceutical Technology Development Co., Ltd., batch number 20231013. Methanol was chromatographically pure; acetonitrile was chromatographically pure; phosphoric acid was chromatographically pure; water was ultrapure water.

[0075] Preparation of test solution:

[0076] Accurately measure 5 mL of the Shen Tong Zhu Yu Tang preparation and place it in a 10 mL volumetric flask. Add an appropriate amount of methanol, sonicate for 20 min, and dilute to the mark with methanol. Shake well, filter the supernatant through a 0.45 μm filter membrane, and collect the filtrate to obtain the test solution.

[0077] Preparation of reference solution:

[0078] Accurately weigh appropriate amounts of loganic acid, amygdalin, hydroxysaffron yellow A, ferulic acid, notopterygium alcohol, isoimperatorin, ammonium glycyrrhizate, and kaempferol, and add methanol to prepare a reference solution containing 0.1 mg / mL loganic acid, 0.1 mg / mL amygdalin, 0.1 mg / mL hydroxysaffron yellow A, 0.1 mg / mL ferulic acid, 0.1 mg / mL notopterygium alcohol, 0.1 mg / mL isoimperatorin, 0.1 mg / mL kaempferol, and 0.1 mg / mL ammonium glycyrrhizate.

[0079] Experimental Example 1

[0080] Accurately pipette 5 μL each of the test solution and the reference solution and inject them into the high-performance liquid chromatograph for detection.

[0081] Chromatographic conditions: Agilent XDB-C18 column (250*4.6mm, 5μm); mobile phase acetonitrile (A)-0.1% phosphoric acid (B), gradient elution sequence: 0–5 min, 5% A; 5–19 min, 5%–11% A; 19–31 min, 11% A; 31–61 min, 11%–26% A; 61–80 min, 26%–50% A; 80–120 min, 50%–68% A; column temperature 30℃; flow rate 1.0 mL / min; detection wavelength 220 nm.

[0082] Test results as follows Figure 2 As shown, many peaks failed to achieve baseline separation, thus failing to achieve the goal of rapid fingerprint detection.

[0083] Experimental Example 2

[0084] Accurately pipette 10 μL each of the test sample and reference solution and inject them into the high-performance liquid chromatograph for detection.

[0085] Chromatographic conditions: Agilent XDB-C18 column (250*4.6mm, 5μm), mobile phase acetonitrile (A)-0.1% phosphoric acid (B), gradient elution sequence: 0–14 min, 5%–11% A; 14–26 min, 11% A; 26–61 min, 11%–26% A; 61–80 min, 26%–50% A; 80–120 min, 50%–68% A; column temperature 30℃, injection volume 10μL, flow rate 1.0 mL / min, detection wavelength 220 nm.

[0086] Test results as follows Figure 3 As shown, the separation is insufficient, and some components cannot be separated from the baseline.

[0087] Experimental Example 3

[0088] Accurately pipette 10 μL each of the test sample and reference solution and inject them into the high-performance liquid chromatograph for detection.

[0089] Chromatographic conditions: Agilent SB-C18 column (250*4.6mm, 5μm), mobile phase acetonitrile (A)-0.1% phosphoric acid (B), gradient elution sequence: 0–14 min, 5%–11% A; 14–26 min, 11% A; 26–61 min, 11%–26% A; 61–80 min, 26%–50% A; 80–120 min, 50%–68% A; column temperature 30℃, injection volume 10μL, flow rate 1.0 mL / min, detection wavelength 220 nm.

[0090] The results are as follows Figure 4 As shown, the common characteristic peaks are not as well separated as those on the XDB column, and baseline separation is not achieved.

[0091] Experiment Example 4

[0092] Accurately pipette 10 μL each of the test sample and reference solution and inject them into the high-performance liquid chromatograph for detection.

[0093] Chromatographic conditions: Agilent SB-C18 column (250*4.6mm, 5μm); mobile phase: acetonitrile (A) - 0.05mol / L potassium dihydrogen phosphate buffer solution (pH=3.0) (B); gradient elution sequence: 0–14 min, 5%–11% A; 14–26 min, 11% A; 26–61 min, 11%–26% A; 61–80 min, 26%–50% A; 80–120 min, 50%–68% A; column temperature: 30℃; injection volume: 10μL; flow rate: 1.0mL / min; detection wavelength: 220nm.

[0094] The column with common characteristic peaks did not achieve high resolution and baseline separation, unlike the XDB column.

[0095] Experimental Example 5

[0096] Accurately pipette 10 μL each of the test sample and reference solution and inject them into the high-performance liquid chromatograph for detection.

[0097] Chromatographic conditions: Agilent XDB-C18 column (250*4.6mm, 5μm), mobile phase acetonitrile (A)-0.1% phosphoric acid (B), gradient elution sequence: 0–14 min, 5%–11% A; 14–26 min, 11% A; 26–42 min, 11%–18% A; 42–47 min, 18% A; 47–66 min, 18%–26% A; 66–85 min, 26%–50% A; 85–125 min, 50%–68% A; column temperature 30℃, injection volume 10μL, flow rate 1.0mL / min, detection wavelength 220nm.

[0098] Test results as follows Figure 5 As shown, this indicates that the separation of some components is poor.

[0099] Experimental Example 6

[0100] Accurately pipette 10 μL each of the test solution and the reference solution and inject them into the high-performance liquid chromatograph for detection.

[0101] Chromatographic conditions: Agilent XDB-C18 column (250*4.6mm, 5μm); mobile phase: acetonitrile (A) - 0.08mol / L potassium dihydrogen phosphate buffer solution (pH=3.5) (B); gradient elution sequence: 0–14 min, 5%–11% A; 14–26 min, 11% A; 26–42 min, 11%–18% A; 42–47 min, 18% A; 47–66 min, 18%–26% A; 66–85 min, 26%–50% A; 85–125 min, 50%–68% A; column temperature: 30℃; injection volume: 10μL; flow rate: 1.0mL / min; detection wavelength: 250nm.

[0102] The test results showed that the separation of some components was poor.

[0103] Experimental Example 7

[0104] Accurately pipette 10 μL each of the test solution and the reference solution and inject them into the high-performance liquid chromatograph for detection.

[0105] Chromatographic conditions: Agilent XDB-C18 column (250*4.6mm, 5μm); mobile phase: acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B); gradient elution sequence: 0–14 min, 5%–11% A, flow rate 0.8 mL / min; 14–26 min, 11% A, flow rate 0.8 mL / min; 26–42 min, 11%–18% A, flow rate 0.8 mL / min; 42–47 min, 18% A, flow rate 0.8 mL / min; 47–66 min, 18%–26% A, flow rate 0.8–1.0 mL / min; 66–85 min, 26%–50% A, flow rate 1.0 mL / min; 85–125 min, 50%–68% A, flow rate 1.0 mL / min; column temperature 30℃; injection volume 10 μL; detection wavelength 220 nm.

[0106] The test results show that a good characteristic spectrum can be obtained by using a 0.1% phosphoric acid aqueous solution, but the peak shape and separation of individual peaks are poor.

[0107] Experimental Example 8

[0108] Accurately pipette 10 μL each of the test sample and reference solution and inject them into the high-performance liquid chromatograph for detection.

[0109] Chromatographic conditions: Agilent XDB-C18 column (250*4.6mm, 5μm); mobile phase: acetonitrile (A) - 0.03mol / L potassium dihydrogen phosphate buffer solution (pH=3.0) (B); gradient elution sequence: 0–14 min, 5%–11% A, flow rate 0.8 mL / min; 14–26 min, 11% A, flow rate 0.8 mL / min; 26–42 min, 11%–18% A, flow rate 0.8 mL / min. L / min; 42–47 min, 18% A, flow rate 0.8 mL / min; 47–66 min, 18%–26% A, flow rate 0.8–1.0 mL / min; 66–85 min, 26%–50% A, flow rate 1.0 mL / min; 85–125 min, 50%–68% A, flow rate 1.0 mL / min; column temperature 30℃, injection volume 10 μL, detection wavelength 200 nm.

[0110] The test results show that the method yields a good feature map, but the peak shape and separation of individual peaks are poor.

[0111] Experimental Example 9

[0112] Accurately pipette 10 μL each of the test sample and reference solution and inject them into the high-performance liquid chromatograph for detection.

[0113] Chromatographic conditions: Agilent XDB-C18 column (250*4.6mm, 5μm); mobile phase: acetonitrile (A) - 0.08mol / L potassium dihydrogen phosphate buffer solution (pH=4.0) (B); gradient elution sequence: 0–14 min, 5%–11% A, flow rate 0.8 mL / min; 14–26 min, 11% A, flow rate 0.8 mL / min; 26–42 min, 11%–18% A, flow rate 0.8 mL / min. L / min; 42–47 min, 18% A, flow rate 0.8 mL / min; 47–66 min, 18%–26% A, flow rate 0.8–1.0 mL / min; 66–85 min, 26%–50% A, flow rate 1.0 mL / min; 85–125 min, 50%–68% A, flow rate 1.0 mL / min; column temperature 30℃, injection volume 10 μL, detection wavelength 220 nm.

[0114] The test results show that the feature spectrum obtained by this method is worse than that of Example 8, with poorer peak shape and separation of individual peaks.

[0115] Example 10

[0116] Best implementation method:

[0117] Precisely aspirate 10 μL each of the test sample and reference solution, and inject them into a high-performance liquid chromatograph for detection.

[0118] Chromatographic conditions: Chromatographic column: Agilent XDB-C18 (250 * 4.6 mm, 5 μm), mobile phase: acetonitrile (A) - 0.05 mol / L potassium dihydrogen phosphate buffer solution (pH = 3.5) (B), gradient elution sequence: 0 - 14 min, 5% - 11% A, flow rate 0.8 mL / min; 14 - 26 min, 11% A, flow rate 0.8 mL / min; 26 - 42 min, 11% - 18% A, flow rate 0.8 mL / min; 42 - 47 min, 18% A, flow rate 0.8 mL / min; 47 - 66 min, 18% - 26% A, flow rate 0.8 - 1.0 mL / min; 66 - 85 min, 26% - 50% A, flow rate 1.0 mL / min; 85 - 125 min, 50% - 68% A, flow rate 1.0 mL / min; column temperature 30 °C, injection volume 10 μL, detection wavelength 220 nm.

[0119] The test results are as Figure 6 shown. Using 0.1% phosphoric acid aqueous solution is not as good as using 0.05 mol / L potassium dihydrogen phosphate buffer solution. The latter gives a better peak shape in the chromatogram because the potassium dihydrogen phosphate buffer solution has the functions of salt balance and can control the pH of the system. Therefore, under this system, the resolution of each peak has better separation and improvement.

[0120] Example 11

[0121] Establishment of the fingerprint of Shentong Zhuyu Decoction:

[0122] Calibration of common peaks: Ten batches of samples (S1 - S10) of Shentong Zhuyu Decoction with batch numbers 231101, 231102, 231103, 231201, 231202, 231301, 231302, 231303, 231401, and 231402 were determined according to the chromatographic conditions of Example 10. The chromatogram measured at 220 nm was imported into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" to generate the common mode of the fingerprint. Set the S1 sample with more chemical composition information as the reference chromatogram. As Figure 6 shown, 27 common peaks were calibrated. The 13th peak with a large peak area and good resolution was used as the reference peak S, and the relative retention times of other common peaks and peak S were calculated, as shown in Table 1.

[0123] Table 1 Relative retention times

[0124]

[0125]

[0126] Similarity evaluation: The Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines was used to evaluate the similarity of the above 10 batches of Shentong Zhuyu Decoction. The similarity evaluations of S1 - S10 are shown in Table 3. From the similarity results, it can be seen that the similarities of the 10 batches of Shentong Zhuyu Decoction with the reference chromatogram are all above 0.9 (see Table 2), indicating that the 10 batches of samples all have good similarities.

[0127] Table 2 Similarity evaluations of S1 - S10

[0128] S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Comparison S1 1 S2 0.979 1 S3 0.948 0.968 1 S4 0.954 0.934 0.958 1 S5 0.95 0.955 0.967 0.943 1 S6 0.952 0.956 0.997 0.994 0.996 1 S7 0.925 0.953 0.967 0.979 0.958 0.972 1 S8 0.935 0.941 0.963 0.989 0.962 0.985 0.963 1 S9 0.829 0.933 0.943 0.985 0.961 0.972 0.946 0.976 1 S10 0.867 0.943 0.979 0.986 0.963 0.945 0.929 0.989 0.985 1 Comparison 0.984 0.972 0.983 0.973 0.981 0.992 0.981 0.986 0.977 0.976 1

[0129] Correlation between Shentong Zhuyu Decoction and each medicinal material:

[0130] Fingerprint detections were carried out on the Shentong Zhuyu Decoction negative samples lacking certain flavors, single - herb samples of Gentiana macrophylla, Ligusticum chuanxiong, Prunus persica, Carthamus tinctorius, Glycyrrhiza uralensis, Notopterygium incisum, Myrrha, Angelica sinensis, Trogopterus dung, Cyperus rotundus, Achyranthes bidentata, and Pheretima aspergillum, as well as Shentong Zhuyu Decoction. It was found that common peaks 5, 12, and 20 came from Gentiana macrophylla; peaks 24 and 27 came from Ligusticum chuanxiong; peaks 4, 10, and 20 came from Prunus persica; peaks 11, 17, and 18 came from Carthamus tinctorius; peaks 14, 15, 20, and 23 came from Glycyrrhiza uralensis; peaks 3, 4, and 7 came from Notopterygium incisum; peaks 20 and 27 came from Myrrha; peaks 16 and 19 came from Trogopterus dung; peak 22 came from Pheretima aspergillum. The chromatogram of the negative sample is shown in Figures 7-18 。By comparing with the reference substance solution, peak 5 was identified as loganic acid; peak 10 was identified as amygdalin; peak 11 was identified as hydroxysafflor yellow A; peak 13 was identified as ferulic acid; peak 21 was identified as kaempferol; peak 23 was identified as ammonium glycyrrhizinate; peak 25 was identified as notopterol; peak 27 was identified as isopimpinellin.

[0131] Determination of sample content:

[0132] Take 3 batches of Shentong Zhuyu Decoction, and determine the contents (%) of 6 components including loganic acid, ferulic acid, kaempferol, ammonium glycyrrhizinate, notopterol, and isopimpinellin according to the above method. The results of content determination are shown in Table 4.

[0133] Table 4 Results of sample content determination (mg / mL)

[0134] batch number Strychnos nuciferic acid ferulic acid Kaempferol ammonium glycyrrhizate Qianghuo alcohol Euphorbia pekinensis 231101 0.02782 0.01869 0.002501 0.03826 0.001761 0.0002253 231102 0.02738 0.01742 0.002487 0.03807 0.001802 0.0002087 231103 0.02796 0.01825 0.002515 0.03879 0.001784 0.0002154

[0135] Methodological investigation:

[0136] Precision of fingerprint:

[0137] Take 5 mL of Shentong Zhuyu Decoction, and determine it according to the above method. Inject samples continuously for 6 times, record the fingerprint, and calculate the relative retention times of each common peak with the retention time of peak 13 as the reference (see Table 3). The relative retention times of each common peak are less than 3%, indicating good precision.

[0138] Table 3 Relative Retention Time

[0139]

[0140]

[0141]

[0142] Fingerprint repeatability:

[0143] Take 5 mL of Shen Tong Zhu Yu Decoction and prepare 6 samples in parallel according to the above method. Record the fingerprint spectrum and calculate the relative retention time of each common peak using the retention time of peak 13 as a reference (see Table 4 below). The relative retention time of each common peak is less than 3%, indicating good repeatability.

[0144] Table 4 Relative Retention Time

[0145]

[0146]

[0147] Fingerprint stability:

[0148] Take 5 mL of Shen Tong Zhu Yu Decoction and prepare it according to the above method. Inject the sample at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h after sample preparation and record the fingerprint spectrum. Using the retention time of peak 13 as a reference, calculate the relative retention time of each common peak (see Table 5). The relative retention time of each common peak is less than 3%, indicating that the test solution has good stability within 24 h.

[0149] Table 5 Relative Retention Time

[0150]

[0151]

[0152]

[0153] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing the fingerprint spectrum of a body pain relief and blood stasis-removing decoction, characterized in that, Includes the following steps: The test solution and the reference solution were taken separately and determined by high performance liquid chromatography. Common peaks containing the characteristic peaks of loganic acid, amygdalin, hydroxysafflower yellow A, ferulic acid, notopterygol, isoimperatorin, glycyrrhizic acid ammonium, and kaempferol were obtained, and a fingerprint spectrum of Shentong Zhuyu Decoction was established. The detection conditions for the high-performance liquid chromatography method include: Mobile phase A is acetonitrile, and mobile phase B is a 0.03-0.08 mol / L potassium dihydrogen phosphate buffer solution with pH=3.0-4.

0. Gradient elution is used, and the detection wavelength is 200-250 nm. In the high-performance liquid chromatography method, the chromatographic column is an Agilent XDB-C18, 250*4.6mm, 5μm; the column temperature is 25~35℃; the flow rate of the mobile phase is 0.8~1.2 mL / min; and the injection volume is 4~10 μL. The gradient elution procedure is as follows: 0 ~ 14 min, 5% ~ 11% mobile phase A, balance mobile phase B; 14 ~ 26 min, 11% mobile phase A, balance mobile phase B; 26 ~ 42 min, 11% ~ 18% mobile phase A, balance mobile phase B; 42 ~ 47 min, 18% mobile phase A, balance mobile phase B; 47 ~ 66 min, 18% ~ 26% mobile phase A, balance mobile phase B; 66 ~ 85 min, 26% ~ 50% mobile phase A, balance mobile phase B; 85 ~ 12 5 min, 50% ~ 68% mobile phase A, balance mobile phase B.

2. The method for constructing the fingerprint spectrum of the Body Pain Relief Decoction as described in claim 1, characterized in that, The preparation method of the test solution includes the following steps: Add methanol to the body pain relief decoction to be tested, make up to volume, sonicate, cool, make up the weight loss with methanol, shake well, and filter with a filter membrane to obtain the test solution.

3. The method for constructing the fingerprint spectrum of the Body Pain Relief Decoction as described in claim 2, characterized in that, The concentration of Shen Tong Zhu Yu Tang in the test solution was 48-52% by volume.

4. The method for constructing the fingerprint spectrum of the Body Pain Relief Decoction as described in claim 1, characterized in that, The preparation method of the reference solution includes the following steps: Loganic acid, amygdalin, hydroxysafflower yellow A, ferulic acid, gentianol, isoimperatorin, ammonium glycyrrhizate, and kaempferol were dissolved in solvents to prepare reference solutions.

5. The method for constructing the fingerprint spectrum of the Body Pain Relief Decoction as described in claim 4, characterized in that, The solvent is methanol.

6. The method for constructing the fingerprint spectrum of the Body Pain Relief Decoction as described in claim 4, characterized in that, The concentrations of loganic acid, amygdalin, hydroxysaffron yellow A, ferulic acid, gentianol, isoimperatorin, ammonium glycyrrhizate, and kaempferol in the reference solution were all 0.08–0.12 mg / mL.

7. The method for constructing the fingerprint spectrum of the Body Pain Relief Decoction as described in claim 1, characterized in that, The process of establishing the fingerprint map is as follows: High-performance liquid chromatography (HPLC) was used to detect different batches of Shentong Zhuyu Decoction. The HPLC chromatograms of different batches of Shentong Zhuyu Decoction and the reference solution were compared to obtain common peaks containing the characteristic peaks of loganic acid, amygdalin, hydroxysaffron yellow A, ferulic acid, notopterygium alcohol, isoimperatorin, ammonium glycyrrhizate, and kaempferol. The fingerprint spectrum of Shentong Zhuyu Decoction was established based on the common peaks.

8. The method for constructing the fingerprint spectrum of the Body Pain Relief Decoction as described in claim 1, characterized in that, The detection wavelength is 210~230nm.

9. The method for constructing the fingerprint spectrum of the Body Pain Relief Decoction as described in claim 1, characterized in that, The fingerprint spectrum of the Shen Tong Zhu Yu Decoction includes 27 characteristic peaks; Using peak 13 as the reference peak S, the relative retention times of each characteristic peak are within ±10% of the specified values; the specified values ​​are: peak 1 0.168, peak 2 0.231, peak 3 0.253, peak 4 0.365, peak 5 0.393, peak 6 0.403, peak 7 0.419, peak 8 0.447, peak 9 0.481, peak 10 0.510, peak 11 0.595, peak 12 0.634, peak 13 1.000, peak 14 1.024, peak 15 1.046, and peak 16 1.

11.

3. Peak 17 is 1.209, peak 18 is 1.236, peak 19 is 1.286, peak 20 is 1.389, peak 21 is 1.731, peak 22 is 1.746, peak 23 is 1.768, peak 24 is 1.975, peak 25 is 2.074, peak 26 is 2.136, and peak 27 is 2.

215. Among them, peak 5 is loganic acid, peak 10 is amygdalin, peak 11 is hydroxysafflower yellow A, peak 13 is ferulic acid, peak 21 is kaempferol, peak 23 is ammonium glycyrrhizate, peak 25 is notopterygium alcohol, and peak 27 is isoimperatorin.

10. The application of a fingerprint spectrum construction method for Shentong Zhuyu Decoction according to any one of claims 1 to 9 in the quality testing of Shentong Zhuyu Decoction in industrial production, characterized in that, The fingerprint spectrum of Shentong Zhuyu Decoction is obtained according to the construction method of any one of claims 1 to 9; the high performance liquid chromatogram of the sample to be tested is obtained according to the construction method of any one of claims 1 to 9; the similarity evaluation between the high performance liquid chromatogram of the sample to be tested and the fingerprint spectrum of Shentong Zhuyu Decoction is performed, and the sample to be tested with a relative retention time within ±10% of the specified value is a qualified product.

11. A method for detecting eight chemical components in Shentong Zhuyu Decoction, characterized in that, Includes the following steps: The test solution was analyzed by high performance liquid chromatography to obtain the content of eight chemical components, which include loganic acid, amygdalin, hydroxysafflower yellow A, ferulic acid, gentianol, isoimperatorin, ammonium glycyrrhizate and kaempferol. The conditions for detection by high performance liquid chromatography include: Mobile phase A is acetonitrile, and mobile phase B is a 0.03-0.08 mol / L potassium dihydrogen phosphate buffer solution with pH=3.0-4.

0. Gradient elution is used, and the detection wavelength is 200-250 nm. The gradient elution procedure is as follows: 0 ~ 14 min, 5% ~ 11% mobile phase A, balance mobile phase B; 14 ~ 26 min, 11% mobile phase A, balance mobile phase B; 26 ~ 42 min, 11% ~ 18% mobile phase A, balance mobile phase B; 42 ~ 47 min, 18% mobile phase A, balance mobile phase B; 47 ~ 66 min, 18% ~ 26% mobile phase A, balance mobile phase B; 66 ~ 85 min, 26% ~ 50% mobile phase A, balance mobile phase B; 85 ~ 12 5 min, 50% ~ 68% mobile phase A, balance mobile phase B; The chromatographic column was an Agilent XDB-C18, 250*4.6mm, 5μm; the column temperature was 25~35℃; the flow rate of the mobile phase was 0.8~1.2mL / min, and the injection volume was 4~10 μL. The preparation method of the test solution comprises the following steps: Add methanol to the Shhentong Zhuyu Decoction to be tested, make up the volume, perform ultrasonic treatment, cool down, supplement the lost weight with methanol, shake well, and filter through a filter membrane to obtain the test solution; in the test solution, the concentration of the Shhentong Zhuyu Decoction is 48-52% (v / v).

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