Construction method and application of fingerprint spectrum of dampness-eliminating stomach-poria decoction

Abstract

The invention provides a construction method and application of a fingerprint spectrum of a dampness-removing stomach-poria cocos decoction, and belongs to the technical field of drug detection.The construction method comprises the steps that the dampness-removing stomach-poria cocos decoction is used for preparing a test solution, and then the fingerprint spectrum of the dampness-removing stomach-poria cocos decoction is obtained through high performance liquid chromatography detection with acetonitrile (A)-0.18-0.22 wt% formic acid aqueous solution as a mobile phase; a standard fingerprint spectrum is established by adopting a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system and is used for quality evaluation or control in the whole process of research / development / production / clinical application of the dampness-removing stomach-poria decoction. According to the construction method of the fingerprint spectrum of the dampness-eliminating stomach-poria cocos decoction, main medicinal material components in the dampness-eliminating stomach-poria cocos decoction can be detected only through high performance liquid chromatography, so that the quality condition of the dampness-eliminating stomach-poria cocos decoction can be reflected more comprehensively.

Description

Technical Field

[0001] The present invention relates to drug detection technology, and in particular to a method for constructing a fingerprint spectrum of Chushi Weiling Decoction and its application. Background Art

[0002] Chushi Weiling Decoction, from Volume 4 of "Surgery Authentic", has the effects of clearing away heat and dampness, strengthening the spleen and promoting diuresis. It is mainly used to treat damp-heat congestion in the spleen and lung meridians, which causes fire ulcers and causes pain. Waist fire ulcers (commonly known as snake sores) are damp, with yellow-white color, blisters of varying sizes, ulcers and water, and more pain than dry ones.

[0003] The main ingredients of the Dehumidifying Wei Ling Decoction are tangerine peel, gardenia, saposhnikovia divaricata, ginger and magnolia bark, stir-fried atractylodes, umbellate, oriental Chinese medicine, red tuckahoe, stir-fried white atractylodes, akebia, talcum powder, one qian each, licorice, cinnamon, three fen each, two cups of water, add twenty roots of rush (1.5m), boil for eight fen, and take before meals.

[0004] Since the prepared Chushi Weiling Decoction has lost the morphological characteristics of the original pieces, the simple qualitative identification and quantitative analysis of the index components are difficult to reflect its quality. As a standard reference for measuring whether the classic Chinese medicine prescription is basically consistent with the clinical decoction, its quality should strengthen the specific identification and multi-component and overall quality control.

[0005] The spectrum of Chinese medicine preparations refers to the spectrum that can reflect the information of multiple components and reflect its quality characteristics after the samples of Chinese medicine preparations are properly processed and analyzed by appropriate methods. The characteristic spectrum of Chinese medicine preparations is of great significance for identifying the key quality attributes of Chinese medicine preparations, studying the transfer of quantity values, evaluating the uniformity and stability of the quality of Chinese medicine preparations, and improving the overall quality control level of Chinese medicine preparations.

[0006] Therefore, by establishing the characteristic map, the overall quality of Chushi Weiling Decoction can be comprehensively controlled. Summary of the invention

[0007] In view of the above problems, the present invention provides a method for constructing a fingerprint of Chushi Weiling Decoction and its application.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is:

[0009] A method for constructing a fingerprint of Chushi Weiling Decoction, the method comprising the following steps:

[0010] S1. Prepare the test solution with Chushi Weiling Decoction;

[0011] S2, taking the test solution and performing high performance liquid chromatography to obtain the fingerprint of the Dehumidifying Weiling Decoction;

[0012] The mobile phase A of the high performance liquid chromatography detection is acetonitrile, and the mobile phase B is a formic acid aqueous solution with a concentration of 0.18-0.22wt%;

[0013] The elution method of the high performance liquid chromatography detection is gradient elution;

[0014] The elution conditions of the gradient elution are:

[0015] 0-70 min, 8% → 20% mobile phase A, 92% → 80% mobile phase B;

[0016] 70-105 min, 20% → 37% mobile phase A, 80% → 63% mobile phase B;

[0017] 105-120 min, 37% → 70% mobile phase A, 63% → 30% mobile phase B;

[0018] 120-130 min, 70% mobile phase A, 30% mobile phase B.

[0019] Furthermore, the detection wavelength of the high performance liquid chromatography detection is 260-270 nm.

[0020] Furthermore, the test solution is prepared by passing the dehumidified Wei Ling decoction liquid through a D101 macroporous adsorption resin column, washing with water until no obvious color is present, discarding the water, and continuing to elute with an ethanol aqueous solution, collecting the eluate, evaporating it in a water bath, adding a methanol aqueous solution to the residue to dissolve it, shaking it well, and filtering it.

[0021] Furthermore, the concentration of the ethanol aqueous solution used for elution is 90 to 98 vol%, and the amount used is 45 to 60 mL.

[0022] Furthermore, the concentration of the methanol aqueous solution used for dissolution is 40 to 60 vol%.

[0023] Furthermore, the dehumidifying Wei Ling Tang liquid is prepared by dissolving the dehumidifying Wei Ling Tang dry paste powder in water;

[0024] Alternatively, it can be prepared by diluting the concentrated solution of Chushi Weiling Decoction with water;

[0025] Alternatively, the decoction of Chushi Weiling Decoction is directly used as the liquid of Chushi Weiling Decoction;

[0026] When the dehumidified Weiling decoction dry paste powder is dissolved in water to prepare the dehumidified Weiling decoction liquid, the weight volume ratio of the dehumidified Weiling decoction dry paste powder to the prepared test solution is 0.064-0.096 g:1 mL;

[0027] When the Dehumidification Weiling Decoction concentrate is diluted with water to prepare the Dehumidification Weiling Decoction liquid, the volume ratio of the Dehumidification Weiling Decoction concentrate to the prepared test solution is 0.8-1.2 mL:1 mL;

[0028] When the Chushi Weiling Decoction decoction is used directly as the Chushi Weiling Decoction liquid, the volume ratio of the Chushi Weiling Decoction decoction to the prepared test sample solution is 4-6 mL:1 mL.

[0029] Furthermore, the flow rate of the HPLC detection is 0.8-1.2 mL / min, the column temperature is 40-50° C., and the injection volume is 5-20 μL;

[0030] The construction method also includes performing high performance liquid chromatography detection on a reference solution made of hesperidin.

[0031] An application of the standard fingerprint of Chushi Weiling Decoction obtained by the above-mentioned construction method in the quality evaluation or control of the whole process of research / development / production / clinical application of Chushi Weiling Decoction.

[0032] Furthermore, a total of 21 common peaks were identified in the standard fingerprint of Chushi Weiling Decoction;

[0033] Among them, peak 1 is genipin-1-gentiobioside, peak 2 is gardenia glycoside, peak 3 is magnolia glycoside B, peak 4 is glucosyl apigenin, peak 5 is cimicifuga glycoside, peak 6 is liquiritigenin, peak 7 is rutin, peak 8 is cimicifuga glycoside, peak 9 is naringin, peak 10 is 5-O-methylvisaminol glycoside, peak 11 (S) is hesperidin, peak 12 is ′-O-p-coumaroylquercetin gentiobioside, peak 13 is crocin I, peak 14 is citrus glycoside, peak 15 is chelidonol glycoside, peak 16 is glucosyl naringin, peak 17 is ammonium glycyrrhizate, peak 18 is 3,5,6,7,3',4',5'-heptamethoxyflavone, peak 19 is 5,6,7,8,4'-pentamethoxyflavone, peak 20 is honokiol, and peak 21 is magnolol.

[0034] Furthermore, the standard fingerprint is established using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System".

[0035] The beneficial effects of the construction method and application of the fingerprint spectrum of the Dehumidifying Weiling Decoction of the present invention are:

[0036] The method for constructing the fingerprint of Chushi Weiling Decoction of the present invention can detect the main medicinal ingredients in Chushi Weiling Decoction by high performance liquid chromatography, thereby more comprehensively reflecting the quality of Chushi Weiling Decoction;

[0037] The present invention combines the different physical and chemical properties of different effective ingredients in Chushi Weiling Decoction, and by adjusting the elution conditions, obtains a standard fingerprint spectrum with good separation, a relatively stable baseline, more peaks, and better reflection of the quality of Chushi Weiling Decoction; the elution conditions of the present invention are significantly better than other elution conditions;

[0038] The present invention obtains a standard fingerprint spectrum with good separation, relatively stable baseline, more peaks and better ability to reflect the quality of Chushi Weiling Decoction by adjusting the mobile phase;

[0039] The present invention achieves the purpose of separating and simultaneously detecting different effective components in the dehumidifying Weiling decoction by adjusting the detection wavelength; the detection wavelength of the present invention is significantly superior to other detection wavelengths;

[0040] The present invention can more fully extract the medicinal ingredients involved in the Chushi Weiling Decoction by adjusting the preparation method of the test solution, and the concentration of each medicinal ingredient in the obtained test solution can be separated and detected under the same high performance liquid chromatography conditions, and the peaks in the obtained chromatogram are good;

[0041] The present invention effectively optimizes the separation conditions by selecting appropriate process conditions such as chromatographic column, column temperature, flow rate, etc., thereby improving the peak stability of the standard fingerprint of Dehumidifying Weiling Decoction.

[0042] The construction method of the present invention also has good specificity and integrity, precision, stability, repeatability and durability;

[0043] The standard fingerprint of Chushi Weiling Decoction obtained by the present invention can provide effective guarantee for the quality monitoring of the subsequent Chushi Weiling Decoction in the whole process of research, development, production and clinical application.

[0044] By using the presence or absence and characteristics of the common characteristic peaks in the standard fingerprint of the Dehumidification Weiling Decoction obtained by the present invention, the product quality of the Dehumidification Weiling Decoction can be fully monitored. By comparing the similarity of the chromatographic fingerprint characteristics, the quality of the Dehumidification Weiling Decoction can be evaluated, and the stability and uniformity can be examined, which makes up for the shortcomings of the current quality control method. At the same time, the stability of the production process of the Dehumidification Weiling Decoction can also be monitored to ensure that its quality is stable, uniform and controllable.

[0045] The standard fingerprint of Chushi Weiling Decoction obtained by the present invention improves the quality monitoring standards of the finished and semi-finished products of Chushi Weiling Decoction, effectively prevents the occurrence of product counterfeiting incidents, and ensures the normal production and circulation order of Chushi Weiling Decoction; on the basis of the present invention, the correlation study between the fingerprint information and the pharmacodynamic activity information can also be carried out, so as to deeply clarify the correlation between the intrinsic chemical components of Chushi Weiling Decoction and the efficacy of the preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is the result of the investigation of the preparation method of the test solution in Example 1 of the present invention; wherein, from top to bottom, the reference numbers 1-3 are the characteristic spectrum of the test solution three, the characteristic spectrum of the test solution one, and the characteristic spectrum of the test solution two;

[0047] Figure 2It is a 3D fingerprint spectrum diagram in the detection wavelength investigation of Example 1 of the present invention;

[0048] Figure 3 It is the absorption spectrum such as the fingerprint spectrum in the detection wavelength investigation of Example 1 of the present invention;

[0049] Figure 4 It is the HPLC spectrum at a wavelength of 230 nm in the detection wavelength investigation of Example 1 of the present invention;

[0050] Figure 5 It is the HPLC spectrum at a wavelength of 240 nm in the detection wavelength investigation of Example 1 of the present invention;

[0051] Figure 6 It is the HPLC spectrum at a wavelength of 250 nm in the detection wavelength investigation of Example 1 of the present invention;

[0052] Figure 7 It is the HPLC spectrum at a wavelength of 260 nm in the detection wavelength investigation of Example 1 of the present invention;

[0053] Figure 8 It is the HPLC spectrum at a wavelength of 265 nm in the detection wavelength investigation of Example 1 of the present invention;

[0054] Fig. 9 It is the HPLC spectrum at a wavelength of 270 nm in the detection wavelength investigation of Example 1 of the present invention;

[0055] Fig.10 It is the HPLC spectrum at a wavelength of 280 nm in the detection wavelength investigation of Example 1 of the present invention;

[0056] Fig.11 It is the HPLC spectrum at a wavelength of 300 nm in the detection wavelength investigation of Example 1 of the present invention;

[0057] Fig.12 The peaks of each medicinal flavor in Example 1 of the present invention belong to the chromatogram Figure 1 ; Among them, from top to bottom, the numbers 1-6 are the characteristic spectrum of licorice, the characteristic spectrum of siler, the characteristic spectrum of ginger magnolia bark, the characteristic spectrum of gardenia, the characteristic spectrum of tangerine peel, and the characteristic spectrum of the test solution;

[0058] Fig.13 The peaks of each medicinal flavor in Example 1 of the present invention belong to the chromatogram Figure 2 ; Among them, from top to bottom, the labels 1-9 are the characteristic spectrum of earth-fried Atractylodes macrocephala, the characteristic spectrum of bran-fried Atractylodes lancea, the characteristic spectrum of red Poria cocos, the characteristic spectrum of Polyporus umbellatus, the characteristic spectrum of Akebia, the characteristic spectrum of Alisma orientalis, the characteristic spectrum of Juncus effusus, the characteristic spectrum of Cinnamomum cassia, and the characteristic spectrum of the test solution;

[0059] Fig.14The peak identification chromatogram of the reference substance in Example 1 of the present invention Figure 1 ; Wherein, from top to bottom, numbers 1-6 are the characteristic spectrum of liquiritin, the characteristic spectrum of ammonium glycyrrhizinate, the characteristic spectrum of magnolol, the characteristic spectrum of honokiol, the characteristic spectrum of the test solution, and the characteristic spectrum of the blank solution;

[0060] Fig.15 The peak identification chromatogram of the reference substance in Example 1 of the present invention Figure 2 ; Wherein, from top to bottom, numbers 1-6 are the characteristic spectrum of hesperidin, the characteristic spectrum of gardenia glycosides, the characteristic spectrum of cimicifuga glycosides, the characteristic spectrum of 5-O-methylvisaminol glycosides, the characteristic spectrum of the test solution, and the characteristic spectrum of the blank solution;

[0061] Fig.16 It is the UPLC-UV chromatogram and UPLC-MS total ion current diagram of the LC-MS determination in Example 1 of the present invention; wherein, from top to bottom, it is the positive ion mode TIC diagram, the negative ion mode TIC diagram, and the HPLC diagram at ultraviolet 265nm;

[0062] Fig.17 It is the fingerprint of Chushi Weiling Decoction and the reference in Example 2 of the present invention; wherein from top to bottom are the fingerprint of Chushi Weiling Decoction and the characteristic spectrum of the reference;

[0063] Fig.18 It is a common pattern diagram of the fingerprint of Chushi Weiling Decoction in Example 11 of the present invention; wherein from top to bottom are the control fingerprint and the fingerprints of two parallel samples of the first batch to two parallel samples of the fifteenth batch;

[0064] Fig.19 It is the standard fingerprint of Chushi Weiling Decoction in Example 11 of the present invention;

[0065] Fig. 20 It is a graph showing the results of the investigation of specificity and integrity in Example 12 of the present invention; from top to bottom, it is the characteristic spectrum of the test solution, the characteristic spectrum of the reference solution, and the characteristic spectrum of the blank solvent;

[0066] Fig.21 1 is a graph showing the results of the precision test in Example 12 of the present invention; wherein from top to bottom, numbers 1-6 are characteristic spectra of the test solution injected 6 times in succession;

[0067] Fig. 22 1 is a graph showing the results of the stability test in Example 12 of the present invention; wherein from top to bottom, numbers 1-7 are the characteristic spectrum at 0 h, the characteristic spectrum at 3 h, the characteristic spectrum at 6 h, the characteristic spectrum at 9 h, the characteristic spectrum at 12 h, the characteristic spectrum at 18 h, and the characteristic spectrum at 24 h;

[0068] Fig.23 1 is a diagram of the results of the repeatability test in Example 12 of the present invention; wherein the numbers 1-6 from top to bottom are characteristic spectra of 6 test sample solutions;

[0069] Fig.24 The fingerprints of different flow rates in Example 12 of the present invention are shown in FIG. 1 , wherein from top to bottom, they are the fingerprints at 0.8, 1.0, and 1.2 mL / min, respectively;

[0070] Fig.25 This is a similarity comparison diagram of different flow rates in Example 12 of the present invention; from top to bottom, they are the fingerprints and reference spectra at 0.8, 1.0, and 1.2 mL / min;

[0071] Fig.26 It is the fingerprint spectrum of different column temperatures in Example 12 of the present invention; wherein from top to bottom, they are the fingerprint spectrum at 40, 45, and 50°C;

[0072] Fig. 27 It is a similarity comparison diagram of different column temperatures in Example 12 of the present invention; wherein from top to bottom are the fingerprints and the reference spectra at 40, 45, and 50°C;

[0073] Fig.28 1 is the fingerprint of different chromatographic columns in Example 12 of the present invention; wherein from top to bottom, numbers 1-3 are respectively the fingerprint when using Pheromone Gemini 5u C18110A (4.6*250mm, 5μm), the fingerprint when using Welch Ultimate AQ-C18 (4.6*250mm, 5μm), and the fingerprint when using Shimadzu ODS-SP (4.6*250mm, 5μm);

[0074] Fig.29 1 is a similarity comparison diagram of different chromatographic columns in Example 12 of the present invention; from top to bottom, they are the fingerprint spectrum when using Pheromone Gemini 5u C18 110A (4.6*250mm, 5μm), the fingerprint spectrum when using Welch UltimateAQ-C18 (4.6*250mm, 5μm), and the fingerprint spectrum and the reference spectrum when using Shimadzu ODS-SP (4.6*250mm, 5μm);

[0075] Fig.30 The fingerprints of different instruments in Example 12 of the present invention are shown in FIG. 1 , wherein from top to bottom are the fingerprints of Shimadzu A20 and the fingerprints of Agilent 1260;

[0076] Fig.31This is a similarity comparison diagram of different instruments in Example 12 of the present invention; from top to bottom are the fingerprint spectrum when Shimadzu A20 is used, the fingerprint spectrum when Agilent 1260 is used, and the reference spectrum. DETAILED DESCRIPTION

[0077] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0078] Example 1 Screening of a method for constructing a fingerprint of Chushi Weiling Decoction

[0079] This embodiment uses the Dehumidification Weiling Decoction material standard as the Dehumidification Weiling Decoction sample, wherein the preparation method of the Dehumidification Weiling Decoction material standard is as follows:

[0080] Take one qian each of dried tangerine peel, gardenia, siler, ginger magnolia bark, stir-fried atractylodes, umbellate, oriental medicine, red tuckahoe, stir-fried atractylodes, akebia, talcum powder, three fen each of licorice and cinnamon, two cups of water, add twenty roots of rush (1.5m), boil for eight minutes, filter, and the decoction obtained is freeze-dried to obtain the dry paste powder of Dehumidifying Weiling Decoction, which is the material standard of Dehumidifying Weiling Decoction.

[0081] This example uses the Chushi Weiling Decoction material standard as the Chushi Weiling Decoction sample to screen the Chushi Weiling Decoction fingerprint construction method, as follows:

[0082] 1. Investigation of the preparation method of the test solution

[0083] Take 2.0 g of the standard substance of Dehumidifying Weiling Decoction, weigh it accurately, put it in a stoppered conical flask, accurately add 25 mL of methanol and 75 vol% ethanol aqueous solution respectively, ultrasonically treat for 30 min (power 800 W, frequency 40 kHz), take out, cool, shake well, filter, and obtain test solution 1 (solvent is methanol) and test solution 2 (solvent is 75 vol% ethanol aqueous solution).

[0084] Take 2.0 g of the standard substance of Dehumidification Weiling Decoction, accurately weigh it, add 25 mL of water, dissolve it with ultrasound, and pass it all through a D101 macroporous adsorption resin column (inner diameter 1.5 cm, column height 12 cm), wash it with water until there is no obvious color, discard the water, continue to elute with 50 mL of 95 vol% ethanol aqueous solution, collect the eluate, evaporate it in a water bath, dissolve the residue in 50 vol% methanol aqueous solution, transfer it to a 25 mL volumetric flask, add 50 vol% methanol aqueous solution to the scale, shake well, filter, and obtain the test solution three.

[0085] Accurately pipette 10 μL of each of the above test solution, inject it into the liquid chromatograph, measure, and record the chromatogram. The results are as follows: Figure 1 shown.

[0086] The HPLC conditions are as follows:

[0087] Octadecylsilane bonded silica gel is used as filler;

[0088] Flow rate: 1.0 mL / min;

[0089] The column temperature was 45°C;

[0090] The detection wavelength is 265nm;

[0091] Acetonitrile was used as mobile phase A, and 0.2 wt% formic acid aqueous solution was used as mobile phase B;

[0092] The elution method is gradient elution, and the elution conditions are:

[0093] 0-70 min, 8% → 20% mobile phase A, 92% → 80% mobile phase B;

[0094] 70-105 min, 20% → 37% mobile phase A, 80% → 63% mobile phase B;

[0095] 105-120 min, 37% → 70% mobile phase A, 63% → 30% mobile phase B;

[0096] 120-130 min, 70% mobile phase A, 30% mobile phase B.

[0097] The number of theoretical plates calculated based on the hesperidin peak should not be less than 2000.

[0098] Depend on Figure 1 It can be seen that through the investigation of the above-mentioned different methods for preparing the test solution, the method of eluting with a macroporous resin obtained more chromatographic peak information and a moderate response value, and after the test solution three was treated with impurities, the impurity peaks in the first 15 minutes of the chromatogram were improved. Finally, the preparation method of the test solution three was determined as the final test solution preparation method, that is, elution was performed using a macroporous adsorption resin column, and the eluent was collected by 95vol% ethanol aqueous solution, and the test solution was prepared by evaporating and re-dissolving.

[0099] The following test solution three was prepared by eluting with a macroporous adsorption resin column, collecting the eluate with 95vol% ethanol aqueous solution, evaporating and re-dissolving, and used as the test solution for construction method investigation.

[0100] 2. Detection wavelength investigation

[0101] Using a diode array detector, the test solution was scanned at a wavelength of 190-400 nm. The results were shown in 3D view and isoabsorption spectrum. Figure 2-Figure 3 .Depend on Figure 2 and Figure 3 It can be seen that within the wavelength range of 230 to 300 nm, the baseline is stable and the amount of information is large. The results of the analysis in this wavelength range are shown in Figure 4-Figure 11 By comparison Figure 4-Figure 11 It can be seen that the response values ​​of the chromatographic peaks at the wavelength of 265nm are the most uniform and have a large amount of information, so 265nm is preferred as the detection wavelength.

[0102] At the same time, since the chromatographic peak response values ​​at wavelengths of 260nm and 270nm are also relatively uniform, the final 21 characteristic peaks required can also be detected, so they can also be used as detection wavelengths.

[0103] 3. Peak attribution and reference selection

[0104] 1) Attribution of drug flavor peak

[0105] The material standard of Chushi Weiling Decoction is a material standard made by mixing 14 kinds of slices according to the prescribed drug flavor ratio and then decocting them in water. Its chemical components mainly include polysaccharides, saponins, flavonoids and other components. Considering the multi-component and overall quality control, the slices were decocted in water separately, and the decoction was prepared according to the fingerprint spectrum test sample preparation method. The chromatographic conditions in the test sample solution preparation method of step 1 were used to attribute the single drug peak. The results are shown in Figure 12-13 and Table 1. Among them, talc is a mineral drug, mainly inorganic, and no peak can be detected in the liquid phase, so its attribution study was not conducted.

[0106] Table 1 List of chromatographic peaks and their medicinal properties

[0107] Peak Attributed to medicinal taste 1 Gardenia 2 Gardenia 3 Magnolia officinalis 4 Tangerine peel 5 Windproof 6 Licorice 7 Gardenia 8 Windproof 9 Tangerine peel 10 Windproof 11 Tangerine peel 12 Gardenia 13 Gardenia 14 Tangerine peel 15 Windproof 16 Tangerine peel 17 Licorice 18 Tangerine peel 19 Tangerine peel 20 Magnolia officinalis 21 Magnolia officinalis

[0108] From Table 1 and Figure 12-13 It can be seen that the chromatographic peaks of Gardenia, Tangerine Peel, Saposhnikovia Divaricata, Licorice, and Magnolia officinalis can be attributed to the characteristic spectrum of Chusi Weiling Decoction, while the other medicinal ingredients have no specific components or the chromatographic peak response values ​​are low.

[0109] 2) Chromatographic peak identification

[0110] Through literature research, the main components related to each medicinal flavor were selected: gardenia glycoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, hesperidin, liquiritin, glycyrrhizic acid, honokiol and magnolol as reference substances, and the corresponding reference substance solutions were prepared. The chromatographic conditions in the preparation method of the test solution in step 1 were used for peak positioning research. At the same time, 50vol1% methanol aqueous solution was used as blank solution for determination. The results are as follows Figure 14-15The preparation method of each reference substance solution is to take an appropriate amount of the corresponding reference substance, accurately weigh it, and add methanol to make a solution containing 0.2 mg of the corresponding reference substance per 1 mL.

[0111] At the same time, the material standard of Chushi Weiling Decoction was detected by LC-MS to identify the chromatographic peaks in the fingerprint of Chushi Weiling Decoction. The obtained UPLC-UV chromatogram and total ion current (positive mode) are shown in Fig.16 Since the liquid phase part of the LC-MS instrument is UPLC, its chromatogram is somewhat different from the fingerprint spectrum obtained by high performance liquid chromatography in this embodiment. The specific peak correspondence is shown in Table 2.

[0112] In the LC-MS detection process, the chromatographic conditions are: acetonitrile as mobile phase A, 0.1wt% formic acid aqueous solution as mobile phase B, gradient elution; flow rate 0.4mL / min; column temperature 45°C; detection wavelength 265nm; injection volume 3μL; gradient elution conditions are:

[0113] 0-15 min, 8%→20% mobile phase A, 92%→80% mobile phase B;

[0114] 15-22 min, 20% → 37% mobile phase A, 80% → 63% mobile phase B;

[0115] 22-27 min, 37% → 70% mobile phase A, 63% → 30% mobile phase B;

[0116] 27-29min, 70%→8% mobile phase A, 30%→92% mobile phase B.

[0117] The mass spectrometry conditions were as follows: mass spectrometry detection was performed in positive and negative ion modes, respectively, with a drying gas temperature of 350°C, a drying gas flow rate of 10 L / min, a nebulizer gas pressure of 35 psi, a sheath gas temperature of 350°C, a sheath gas flow rate of 12 L / min, and a capillary voltage of 3500 V (positive mode) and 3500 V (negative mode). The primary mass spectrometry used the MS mode, with a mass scanning range of 100-1200 m / z. The secondary mass spectrometry used Auto MSMS, and the collision voltage was appropriately adjusted according to different compounds. The obtained LC-MS data were collected by Agilent MassHunter (B.08.00) software. Data processing was performed using Agilent software Qualitative Navigator (B.08.00) and Qualitative Workflows (B.08.00).

[0118] Table 2 Chromatographic peak identification results list

[0119]

[0120] By comparing with 8 reference substances with known ingredients (gardenia glycoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, hesperidin, liquiritigenin, glycyrrhizic acid, honokiol and magnolol), the corresponding compounds of the 8 chromatographic peaks were confirmed, and the analysis results were consistent with the mass spectrometry results.

[0121] Since the absorption intensity and retention time of the hesperidin chromatographic peak are moderate, the response is stable, and baseline separation is achieved among the identified chromatographic peaks, hesperidin is selected as the reference peak, and hesperidin is marked as peak S. At the same time, the hesperidin reference substance is used as a reference substance.

[0122] Example 2 A method for constructing a fingerprint of Dehumidifying Weiling Decoction

[0123] This embodiment uses the Dehumidification Weiling Decoction material standard as the Dehumidification Weiling Decoction sample, and the specific method for constructing the Dehumidification Weiling Decoction fingerprint is as follows:

[0124] S1. Solution preparation

[0125] S11. Preparation of reference solution

[0126] Take an appropriate amount of hesperidin reference substance, weigh it accurately, and add methanol to make a solution containing 0.2 mg per 1 mL to obtain the reference substance solution.

[0127] S12. Preparation of test solution

[0128] Take 2.0 g of the standard substance of Dehumidification Weiling Decoction (i.e., the dry paste powder of Dehumidification Weiling Decoction), accurately weigh it, add 25 mL of water, and dissolve it by ultrasonic treatment. The obtained Dehumidification Weiling Decoction liquid passes through a D101 macroporous adsorption resin column (inner diameter 1.5 cm, column height 12 cm), wash with water until there is no obvious color, discard the water, continue to elute with 50 mL of 95 vol 1% ethanol aqueous solution, collect the eluate, evaporate to dryness in a water bath, add 50 vol 1% methanol aqueous solution to dissolve the residue, transfer it to a 25 mL volumetric flask, add 50 vol 1% methanol aqueous solution to the scale, shake well, filter, and obtain the test solution.

[0129] S2. High performance liquid chromatography detection

[0130] Accurately pipette 10 μL of the reference solution and the test solution respectively, inject into the liquid chromatograph, measure under the chromatographic conditions in the test solution preparation method of step 1 of Example 1, record the chromatogram, and obtain the corresponding characteristic spectrum. Fig.17 ; Among them, the characteristic spectrum obtained by testing the test solution is the fingerprint spectrum of Chushi Weiling Decoction.

[0131] That is, the HPLC conditions are:

[0132] Octadecylsilane bonded silica gel is used as filler;

[0133] Flow rate: 1.0 mL / min;

[0134] The column temperature was 45°C;

[0135] The detection wavelength is 265nm;

[0136] Acetonitrile was used as mobile phase A, and 0.2 wt% formic acid aqueous solution was used as mobile phase B;

[0137] The elution method is gradient elution, and the elution conditions are:

[0138] 0-70 min, 8% → 20% mobile phase A, 92% → 80% mobile phase B;

[0139] 70-105 min, 20% → 37% mobile phase A, 80% → 63% mobile phase B;

[0140] 105-120 min, 37% → 70% mobile phase A, 63% → 30% mobile phase B;

[0141] 120-130 min, 70% mobile phase A, 30% mobile phase B.

[0142] The number of theoretical plates calculated based on the hesperidin peak should not be less than 2000.

[0143] Meanwhile, in the process of preparing the test solution in step S12, the Dehumidified Weiling Decoction concentrate can also be diluted with water to prepare the Dehumidified Weiling Decoction liquid to prepare the test solution; or, the Dehumidified Weiling Decoction decoction can be used as the Dehumidified Weiling Decoction liquid to prepare the test solution.

[0144] That is, take 5mL of Dehumidification Weiling Decoction concentrate and accurately weigh it, add 25mL of water, dissolve it by ultrasonic treatment, and then pass it through a D101 macroporous adsorption resin column (inner diameter 1.5cm, column height 12cm), wash it with water until there is no obvious color, discard the water, continue to elute with 50mL of 95vol1% ethanol aqueous solution, collect the eluate, evaporate it in a water bath, dissolve the residue in 50vol1% methanol aqueous solution, transfer it to a 5mL volumetric flask, add 50vol% methanol aqueous solution to the scale, shake well, filter, and prepare the test solution.

[0145] Alternatively, take 25 mL of the decoction of Chushi Weiling Decoction and directly pass it through a D101 macroporous adsorption resin column (inner diameter 1.5 cm, column height 12 cm), wash with water until there is no obvious color, discard the water, continue to elute with 50 mL of 95 vol% ethanol aqueous solution, collect the eluate, evaporate to dryness in a water bath, add 50 vol% methanol aqueous solution to dissolve the residue, transfer it to a 5 mL volumetric flask, add 50 vol% methanol aqueous solution to the scale, shake well, filter, and prepare the test solution.

[0146] Embodiments 3 to 10 Method for constructing fingerprints of Chushi Weiling Decoction

[0147] Examples 3 to 10 are respectively methods for constructing a fingerprint of Dehumidifying Weiling Decoction. According to the optimal construction conditions of the fingerprint of Dehumidifying Weiling Decoction given in Example 2, the range of construction conditions that can achieve a better fingerprint of Dehumidifying Weiling Decoction is further explored to obtain the construction methods in Examples 3 to 10. Among them, the construction methods of the fingerprint of Dehumidifying Weiling Decoction in Examples 3 to 10 are basically the same as those in Example 2, and the only difference is that some process parameters are different, as shown in Tables 3 and 4 for details:

[0148] Table 3 List of process parameters in Examples 3 to 6

[0149]

[0150] Table 4 List of process parameters in Examples 7 to 10

[0151]

[0152]

[0153] The contents and results of other parts of Examples 3 to 10 are the same as those of Example 2 and will not be repeated here (since other embodiments of the present invention have been able to fully prove that this method can better construct the fingerprint of Dehumidifying Weiling Decoction, no additional drawings will be added here).

[0154] Example 11 Standard fingerprint of Chushi Weiling Decoction and its application

[0155] 1) Common peak calibration

[0156] The fingerprint construction method in Example 2 was used to measure 15 batches of dehumidification Weiling decoction material benchmarks (two parallel samples were measured for each batch, a total of 30 copies), and the obtained fingerprints were analyzed. The chromatographic peaks with good stability and appropriate response values ​​in the fingerprints of 15 batches of dehumidification Weiling decoction material benchmarks (two parallel samples were measured for each batch) were selected as common peaks. A total of 21 common peaks were calibrated. The results are shown in FIG. Fig.18 .

[0157] 2) Establishment of standard fingerprint

[0158] The Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System 2012 Edition issued by the Chinese Pharmacopoeia Committee was used to automatically match the chromatographic peaks of the fingerprints of 15 batches of Dehumidification Weiling Decoction material standards (two parallel samples for each batch) to form a common pattern map and establish a standard fingerprint map. The results are shown in Fig.19 The established standard fingerprint was used as the control fingerprint for subsequent experiments.

[0159] At the same time, combined with the results of chromatographic peak identification, peak 1 is genipin-1-gentiobioside, peak 2 is gardenia glycoside, peak 3 is magnolin B, peak 4 is glucosylapigenin, peak 5 is cimicifuga glycoside, peak 6 is liquiritigenin, peak 7 is rutin, peak 8 is cimicifuga glycoside, peak 9 is naringin, peak 10 is 5-O-methylvisaminol glycoside, peak 11 (S) is hesperidin, peak 12 is ′-O-p-coumaroyl duquercetin Peak 13 is crocin I, peak 14 is citrus glycoside, peak 15 is chelidonol glycoside, peak 16 is glucosylnaringin, peak 17 is ammonium glycyrrhizate, peak 18 is 3,5,6,7,3',4',5'-heptamethoxyflavone, peak 19 is 5,6,7,8,4'-pentamethoxyflavone, peak 20 is magnolol, and peak 21 is magnolol.

[0160] 3) Calculation of similarity

[0161] The similarity evaluation software system of Chinese medicine chromatographic fingerprint was used to calculate the similarity of 15 batches of Chushi Weiling Decoction material benchmarks (two parallel samples were measured for each batch, a total of 30 copies) and the reference fingerprints based on the common peaks. According to the similarity calculated by the fingerprint, the similarity of 15 batches of Chushi Weiling Decoction material benchmarks and the reference fingerprints of Chushi Weiling Decoction was compared. The measured range was 0.986-1.000, all greater than 0.90, so the fingerprint was selected as the evaluation standard for the Chushi Weiling Decoction material benchmark. The fingerprint of Chushi Weiling Decoction should present a chromatographic peak with the same retention time as the reference chromatographic peak. The chromatographic spectrum of Chushi Weiling Decoction should be basically consistent with the reference fingerprint, with 21 corresponding common peaks. According to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the similarity is calculated based on the common peaks, and the similarity between the fingerprint of Chushi Weiling Decoction and the reference fingerprint shall not be less than 0.90.

[0162] 4) Application of standard fingerprint

[0163] The standard fingerprint of Chushi Weiling Decoction obtained in this example can be used for quality evaluation or control in the whole process of research / development / production / clinical application of Chushi Weiling Decoction.

[0164] Example 12 Fingerprint Methodology Study

[0165] This example conducts a methodological investigation on the construction method of the fingerprint of Dehumidifying Weiling Decoction in Example 2, mainly investigating the specificity and integrity, precision, stability, repeatability and durability of the construction method. The reference solution and the test solution in the methodological investigation process are prepared according to the method in Example 2, and the chromatographic conditions of the HPLC detection are also the chromatographic conditions in Example 2.

[0166] 1. Specificity and integrity

[0167] In order to investigate whether the blank solvent interferes with the reference fingerprint of the Dehumidifying Weiling Decoction, 50vol1% methanol aqueous solution was used as the blank solvent for investigation. 50vol1% methanol aqueous solution, reference solution and test solution were accurately aspirated for determination, and maintained at the final gradient of the elution procedure. The detection time was extended by 30min, and the chromatogram was recorded to investigate its integrity. The results are shown in Table 1. Fig. 20 .Depend on Fig. 20 It can be seen that through the specificity and integrity test, the blank solvent has no interference and the specificity is good; and there is no chromatographic peak within the extended 30 minutes, and the integrity is good.

[0168] 2. Precision Test

[0169] Take about 2.0g of the standard substance of Chushi Weiling Decoction, accurately weigh it, prepare a sample solution according to the method in Example 2, and measure it according to the method in Example 2, inject it 6 times continuously, and calculate the relative retention time of the remaining peaks with the S peak as a reference. And compare it with the reference spectrum R (using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System 2012 Edition" issued by the National Pharmacopoeia Committee, automatically match the chromatographic peaks of the repetitive characteristic spectrum to form a common pattern diagram, and establish the reference spectrum R, calculate the similarity, and the specific measurement results are shown in Fig.21 , Table 5 and Table 6.

[0170] Table 5 Precision similarity results list

[0171] Injection times R 1 2 3 4 5 6 R 1.000 0.998 0.998 0.998 0.998 0.998 0.998 1 0.998 1.000 1.000 1.000 1.000 1.000 1.000 2 0.998 1.000 1.000 1.000 1.000 1.000 1.000 3 0.998 1.000 1.000 1.000 1.000 1.000 1.000 4 0.998 1.000 1.000 1.000 1.000 1.000 1.000 5 0.998 1.000 1.000 1.000 1.000 1.000 1.000 6 0.998 1.000 1.000 1.000 1.000 1.000 1.000

[0172]

[0173] Depend on Figure 7 As can be seen from Tables 4 and 5, the fingerprint of Chushi Weiling Decoction presents chromatographic peaks with the same retention time as the chromatographic peaks of the reference, and presents 21 major chromatographic peaks, whose relative retention time RSD is less than 2%, and the similarity results are all greater than 0.95, indicating that the instrument has good precision.

[0174] 3. Stability test

[0175] Take about 2.0g of the standard substance of Chushi Weiling Decoction, accurately weigh it, prepare a sample solution according to the method in Example 2, and measure it according to the method in Example 2, respectively, inject and measure at 0, 3, 6, 9, 12, 18, and 24 hours, and use the S peak as a reference to calculate the relative retention time of the remaining peaks. And with the reference spectrum R (using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System 2012 Edition" promulgated by the State Pharmacopoeia Committee, the chromatographic peaks of the repetitive HPLC spectrum are automatically matched to form a common pattern diagram, and the reference spectrum R is established), calculate the similarity, and the specific measurement results are shown in Fig. 22 , Table 7 and Table 8.

[0176] Table 7 Precision similarity results list

[0177] R 0h 3h 6h 9h 12h 18h 24h R 1.000 0.998 0.998 0.998 0.998 0.998 0.998 0.998 0h 0.998 1.000 1.000 1.000 1.000 1.000 1.000 1.000 3h 0.998 1.000 1.000 1.000 1.000 1.000 1.000 1.000 6h 0.998 1.000 1.000 1.000 1.000 1.000 1.000 1.000 9h 0.998 1.000 1.000 1.000 1.000 1.000 1.000 1.000 12h 0.998 1.000 1.000 1.000 1.000 1.000 1.000 1.000 18h 0.998 1.000 1.000 1.000 1.000 1.000 1.000 1.000 24h 0.998 1.000 1.000 1.000 1.000 1.000 1.000 1.000

[0178]

[0179] Depend on Fig. 22 As can be seen from Table 7 and Table 8, the fingerprint of Chushi Weiling Decoction presents chromatographic peaks with the same retention time as the reference chromatographic peaks, and presents 21 main chromatographic peaks. The relative retention time RSD is less than 2%, and the similarity results are all greater than 0.95, indicating that the test solution prepared by the present invention has good stability within 24 hours.

[0180] 4. Repeatability test

[0181] Take about 2.0g (6 parts in total) of the material standard of Chushi Weiling Decoction respectively, accurately weigh it, prepare six test solutions according to the method in Example 2, and measure it according to the method in Example 2, with the S peak as a reference, calculate the relative retention time of the remaining peaks. And with the reference spectrum R (using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System 2012 Edition" promulgated by the National Pharmacopoeia Committee, automatically match the chromatographic peaks of the repetitive HPLC spectrum, form a common pattern diagram, and establish the reference spectrum R), calculate the similarity, and the specific measurement results are shown in Fig.23 , Table 9 and Table 10.

[0182] Table 9 Summary of repeatability similarity results

[0183] Sample No. R 1 2 3 4 5 6 R 1.000 0.999 0.999 1.000 1.000 1.000 1.000 1 0.999 1.000 1.000 0.999 0.998 0.997 0.998 2 0.999 1.000 1.000 0.999 0.999 0.998 0.999 3 1.000 0.999 0.999 1.000 1.000 0.999 1.000 4 1.000 0.998 0.999 1.000 1.000 1.000 1.000 5 1.000 0.997 0.998 0.999 1.000 1.000 1.000 6 1.000 0.998 0.999 1.000 1.000 1.000 1.000

[0184]

[0185] Depend on Fig.23 As can be seen from Table 9 and Table 10, the fingerprint of Chushi Weiling Decoction presents chromatographic peaks with the same retention time as the reference chromatographic peaks, and presents 21 main chromatographic peaks. The relative retention time RSD is less than 2%, and the similarity results are all greater than 0.95, indicating good repeatability.

[0186] 5. Durability test

[0187] The durability of the chromatographic conditions in Example 2 was investigated using different flow rates (0.8, 1.0, 1.2 mL / min), different column temperatures (40, 45, 50°C), different chromatographic columns (Shimadzu Inertsil ODS-SP, Pheromone Gemini C18) and different instruments (Shimadzu 20A, Agilent 1260), and the similarity was calculated with the reference spectrum R (using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System 2012 Edition" promulgated by the National Pharmacopoeia Committee, with repetitive HPLC spectrum chromatographic peaks automatically matched to form a common pattern diagram, and the reference spectrum R was established). Among them, the results of the different flow rate measurements are shown in Figure 24-25 and Table 11; the results of different column temperatures are shown in Figure 26-Figure 27 and Table 12; the results of different chromatographic columns are shown in Figure 28-Figure 29 and Table 13; the results of different instruments are shown in Figure 30-Figure 31 and Table 14.

[0188] Table 11 List of similarity results for different flow rates

[0189] R 1.2mL / min 1.0mL / min 0.8mL / min R 1.000 0.995 1.000 0.998 1.2mL / min 0.995 1.000 0.995 0.996 1.0mL / min 1.000 0.995 1.000 0.998 0.8mL / min 0.998 0.996 0.998 1.000

[0190] Table 12 Summary of similarity results at different column temperatures

[0191] R 50℃ 45℃ 40℃ R 1.000 0.997 0.999 0.998 50℃ 0.997 1.000 0.997 0.997 45℃ 0.999 0.997 1.000 0.996 40℃ 0.998 0.997 0.996 1.000

[0192] Table 13 Summary of similarity results of different chromatographic columns

[0193]

[0194] Table 14 Summary of similarity results of different instruments

[0195] R Agilent Shimadzu R 1.000 0.995 0.999 Agilent 0.995 1.000 0.993 Shimadzu 0.999 0.993 1.000

[0196] Depend on Figure 24-Figure 31 It can be seen from Tables 11 to 14 that the results under different column temperatures, different flow rates, different chromatographic columns, and different instrument conditions are basically consistent, and the peaks in the chromatograms are sharp, symmetrical, and have good separation, and the similarity is not less than 0.90. There are 21 main chromatographic peaks in the fingerprint of Chushi Weiling Decoction, which correspond to the characteristic peaks in the chromatographic peaks of the reference substance. It shows that the method of the present invention has good durability for different column temperatures, different flow rates, different chromatographic columns, and different instruments.

[0197] Obviously, the described embodiments 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 creative work are within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint of Chushi Weiling Decoction, characterized in that: The construction method comprises the following steps: S1. Prepare the test solution with Chushi Weiling Decoction; S2, taking the test solution and performing high performance liquid chromatography to obtain the fingerprint of the Dehumidifying Weiling Decoction; The mobile phase A of the HPLC detection is acetonitrile, and the mobile phase B is a formic acid aqueous solution with a concentration of 0.18-0.22wt%; The elution method of the high performance liquid chromatography detection is gradient elution; The elution conditions of the gradient elution are: 0-70 min, 8%→20% mobile phase A, 92%→80% mobile phase B; 70-105 min, 20%→37% mobile phase A, 80%→63% mobile phase B; 105-120 min, 37%→70% mobile phase A, 63%→30% mobile phase B; 120-130 min, 70% mobile phase A, 30% mobile phase B.

2. The method for constructing the fingerprint of Chushi Weiling Decoction according to claim 1, characterized in that: The detection wavelength of the high performance liquid chromatography detection is 260-270 nm.

3. The method for constructing the fingerprint of Chushi Weiling Decoction according to claim 1 or 2, characterized in that: The test solution is prepared by passing the dehumidified Wei Ling decoction liquid through a macroporous adsorption resin column, washing with water, discarding the water, eluting with an ethanol aqueous solution, collecting the eluate, evaporating it in a water bath, dissolving the residue with a methanol aqueous solution, shaking well, and filtering.

4. The method for constructing the fingerprint of Chushi Weiling Decoction according to claim 3, characterized in that: The concentration of the ethanol aqueous solution used for elution is 90-98 vol%, and the amount used is 45-60 mL.

5. The method for constructing the fingerprint of Chushi Weiling Decoction according to claim 3, characterized in that: The concentration of the methanol aqueous solution used for dissolution is 40 to 60 vol%.

6. The method for constructing the fingerprint of Chushi Weiling Decoction according to claim 3, characterized in that: The liquid of Chushi Weiling Decoction is prepared by dissolving the dry powder of Chushi Weiling Decoction in water; Alternatively, it can be prepared by diluting the concentrated solution of Chushi Weiling Decoction with water; Alternatively, the decoction of Chushi Weiling Decoction is directly used as the liquid of Chushi Weiling Decoction; When the dehumidified Weiling Decoction dry paste powder is dissolved in water to prepare the dehumidified Weiling Decoction liquid, the weight volume ratio of the dehumidified Weiling Decoction dry paste powder to the prepared test solution is 0.064-0.096 g:1 mL; When the Dehumidification Weiling Decoction concentrate is diluted with water to prepare the Dehumidification Weiling Decoction liquid, the volume ratio of the Dehumidification Weiling Decoction concentrate to the prepared test solution is 0.8~1.2mL:1mL; When the Chushi Weiling Decoction decoction is used directly as the Chushi Weiling Decoction liquid, the volume ratio of the Chushi Weiling Decoction decoction to the prepared test sample solution is 4~6mL:1mL.

7. The method for constructing the fingerprint of Dehumidifying Weiling Decoction according to claim 1, 2, 4, 5 or 6, characterized in that: The flow rate of the HPLC detection is 0.8-1.2 mL / min, the column temperature is 40-50°C, and the injection volume is 5-20 μL; The construction method also includes performing high performance liquid chromatography detection on a reference solution made of hesperidin.

8. Application of the standard fingerprint of Chushi Weiling Decoction obtained by the construction method described in any one of claims 1 to 7 in the quality evaluation or control of the whole process of research / development / production / clinical application of Chushi Weiling Decoction.

9. The use according to claim 8, characterized in that: A total of 21 common peaks were identified in the standard fingerprint of Chushi Weiling Decoction; Among them, peak 1 is genipin-1-gentiobioside, peak 2 is gardenia glycoside, peak 3 is magnolia glycoside B, peak 4 is glucosyl apigenin, peak 5 is cimicifuga glycoside, peak 6 is liquiritigenin, peak 7 is rutin, peak 8 is cimicifuga glycoside, peak 9 is naringin, peak 10 is 5-O-methylvisaminol glycoside, peak 11 (S) is hesperidin, peak 12 is 6'-O-p-coumaroylquercetin gentian Disaccharide glycoside, peak 13 is crocin I, peak 14 is citrus glycoside, peak 15 is chelidonol glycoside, peak 16 is glucosylnaringin, peak 17 is ammonium glycyrrhizate, peak 18 is 3,5,6,7,3',4',5'-heptamethoxyflavone, peak 19 is 5,6,7,8,4'-pentamethoxyflavone, peak 20 is magnolol, and peak 21 is magnolol.

10. The use according to claim 8 or 9, characterized in that: The standard fingerprint is established using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System".

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