Fingerprint spectrum, construction method and detection method of leycesteria formosana medicinal material

Abstract

The invention relates to the field of medicine, in particular to a colepipe medicinal material fingerprint spectrum, a construction method and a detection method. According to the method disclosed by the invention, the coriolus formosanus medicinal material is taken as a research object, an acetonitrile-0. 1% phosphoric acid solution is taken as a mobile phase, the coriolus formosanus medicinal material fingerprint spectrum is constructed for the first time, nine common peaks are calibrated, and the characteristic component chlorogenic acid is identified; according to the construction method of the leylepipe fingerprint spectrum, the extraction time and the extraction method are optimized, so that the information amount of a leylepipe chromatographic peak is exposed to the maximum extent, the detection sensitivity is high, the specificity is high, the reproducibility and stability are good, the accuracy is high, and the quality of a leylepipe medicinal material can be rapidly and efficiently controlled. And by measuring the content of chlorogenic acid in the medicinal material of the leylepipe, the quality of the medicinal material of the leylepipe is more comprehensively and effectively controlled, and the curative effect and medication safety are ensured.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to the fingerprint spectrum, construction method and detection method of the medicinal material of Leycesteria formosa Wall. Background Art

[0002] Leycesteria formosa Wall. is a deciduous shrub or small shrub of the genus Leycesteria in the family Caprifoliaceae. There are about 8 species in the world, distributed in the Himalayan region and Myanmar. There are 6 species in China, and 4 species in Yunnan, which is the main distribution area of this genus of plants. Except for southern Yunnan, it is almost distributed throughout the province. Leycesteria formosa Wall. is also known as Fengchuixiao, Huopaozhutong, Guipaozhang, Kongxinmu, etc. It is widely distributed in Yunnan and grows in most areas at an altitude of 1400 - 3300m. It is rich in resources and is a commonly used medicinal plant of many ethnic groups. The Bai people call it "Xugai" and "Houshuangzhu", and the Yi people call it "Naitimei". It has the effects of promoting blood circulation to regulate menstruation, dispelling wind and dampness, resolving phlegm and relieving asthma, promoting diuresis and reducing swelling. It is mainly used to treat cystitis, edema, bronchial asthma, indigestion, abdominal distension, rheumatic pain, hemorrhoids, etc. Externally, it can treat fractures, and the leaves can be used as cooling medicine.

[0003] The characteristic spectrum of traditional Chinese medicine is a widely accepted quality evaluation model for traditional Chinese medicine at present. Its application reflects the trend of total quality management of traditional Chinese medicine and conforms to the characteristics of overall characterization analysis of traditional Chinese medicine quality control. At present, there is no clear quality standard system for the medicinal material of Leycesteria formosa Wall. Therefore, it is necessary to establish a characteristic spectrum or fingerprint spectrum of Leycesteria formosa Wall. to evaluate the consistency and stability of the quality of this medicinal material, which is of great significance in its production quality control. Summary of the Invention

[0004] In order to overcome the above technical defects, the present invention provides a fingerprint spectrum, construction method and detection method of the medicinal material of Leycesteria formosa Wall., providing a basis for quality detection and control of the medicinal material of Leycesteria formosa Wall.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] On the one hand, the present invention provides a construction method of a fingerprint spectrum of the medicinal material of Leycesteria formosa Wall., comprising the following steps:

[0007] S1. Prepare a reference substance solution by dissolving chlorogenic acid reference substance;

[0008] S2. Extract the extract of Leycesteria formosa Wall. to prepare a test sample solution:

[0009] S3. Respectively take the test sample solution and the reference substance solution and determine them by high performance liquid chromatography to obtain the fingerprint spectrum of the medicinal material of Leycesteria formosa Wall.;

[0010] The chromatographic conditions are as follows: The chromatographic column uses octadecylsilane-bonded silica gel as the filler, acetonitrile as mobile phase A, and 0.1 wt% phosphoric acid solution as mobile phase B for gradient elution. The flow rate is 0.5 mL / min - 1.5 mL / min, the column temperature is 25°C - 35°C, and the detection wavelength is 200 - 280 nm.

[0011] Furthermore, in S1, the reference solution is prepared by dissolving chlorogenic acid reference substance with an alcohol solution.

[0012] Furthermore, in S1, the alcohol solution is selected from one of methanol, ethanol, n-propanol, isopropanol, n-butanol, and isobutanol solutions.

[0013] Furthermore, in S1, the concentration of the alcohol solution is 50% - 100% wt.

[0014] Furthermore, in S1, the concentration of the reference solution is 200 - 300 μg / mL.

[0015] Furthermore, in S2, the Lepidagathis incurva extract is obtained by extracting Lepidagathis incurva with an extraction solvent; the extraction solvent is selected from one or more of water, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, methyl formate, ethyl formate, methyl acetate, ethyl acetate, dichloromethane, chloroform, carbon tetrachloride, dichloroethane, acetone, butanone, and pentanone.

[0016] Furthermore, in S2, the Lepidagathis incurva extract is the water extract or alcohol extract of Lepidagathis incurva.

[0017] Furthermore, in S2, the Lepidagathis incurva extract is prepared by the following steps: Take Lepidagathis incurva medicinal materials, crush them, add the extraction solvent, perform ultrasonic extraction, make up the volume, filter, and collect the filtrate.

[0018] Furthermore, in S2, the ultrasonic extraction frequency is 20 - 60 KHz;

[0019] and / or, the ultrasonic extraction time is 30 - 90 min.

[0020] Furthermore, in S3, the gradient elution conditions are as follows:

[0021] 0 - 15 min, the volume fraction of mobile phase A is 10%, and the volume fraction of mobile phase B is 90%;

[0022] 15 - 30 min, the volume fraction change of mobile phase A is 10% - 30%, and the volume fraction change of mobile phase B is 90% - 70%;

[0023] From 30 to 55 minutes, the volume fraction of mobile phase A changes from 30% to 80%, and the volume fraction of mobile phase B changes from 70% to 20%.

[0024] From 55 to 60 minutes, the volume fraction of mobile phase A changes from 80% to 100%, and the volume fraction of mobile phase B changes from 20% to 0%.

[0025] Furthermore, in S3, the test solution shows 9 common peaks. Among them, the 3rd peak corresponding to the reference substance peak is used as the reference peak. The relative retention time RSD value range of other common peaks relative to the reference peak is 0.01 - 0.38%, and the relative peak area RSD value range is 0.22 - 1.90%.

[0026] In the second aspect, the present invention provides a fingerprint spectrum of Lepidagathis incurva Wall. constructed by the above method. The test solution shows 9 common peaks. Taking the 3rd peak, the chlorogenic acid peak, as the reference peak, the relative retention time RSD value range of other common peaks relative to the reference peak is 0.01 - 0.38%, and the relative peak area RSD value range is 0.22 - 1.90%.

[0027] In the third aspect, the present invention provides a detection method for Lepidagathis incurva Wall. Comparing the fingerprint spectrum of Lepidagathis incurva Wall. constructed by the above method and the fingerprint spectrum of Lepidagathis incurva Wall. with the characteristic spectrum of the sample to be tested, the RSD value of the chlorogenic acid peak area should be within ±5% of the specified value for a qualified product; and / or, comparing the fingerprint spectrum of Lepidagathis incurva Wall. constructed by the above method and the fingerprint spectrum of Lepidagathis incurva Wall. with the characteristic spectrum of the sample to be tested for similarity evaluation, and a similarity greater than 0.90 is a qualified product.

[0028] Beneficial effects: The present invention takes Lepidagathis incurva Wall. as the research object, uses acetonitrile - 0.1% phosphoric acid solution as the mobile phase, and constructs the fingerprint spectrum of Lepidagathis incurva Wall. for the first time. A total of 9 common peaks are calibrated, and the characteristic component chlorogenic acid is identified. The construction method of the fingerprint spectrum of Lepidagathis incurva Wall. in the present invention maximally exposes the information volume of the chromatographic peaks of Lepidagathis incurva Wall. by optimizing the extraction time and extraction method, has high detection sensitivity, strong specificity, good reproducibility and stability, high accuracy, and can quickly and efficiently control the quality of Lepidagathis incurva Wall. By measuring the chlorogenic acid content in Lepidagathis incurva Wall., the quality of Lepidagathis incurva Wall. can be more comprehensively and effectively controlled to ensure the curative effect and medication safety. Description of the Drawings

[0029] Figure 1 It is the superposition chromatogram of the common pattern of 15 batches of Lepidagathis incurva Wall.

[0030] Figure 2 It is the HPLC control chromatogram of the reference substance solution and the test solution; among them, S1 is the test chromatogram, and S2 is the chlorogenic acid reference substance chromatogram.

[0031] Figure 3 This is the characteristic spectrum of the medicinal material of Guichuixiao under different ultrasonic conditions in Example 1.1;

[0032] Figure 4 This is the characteristic spectrum of the medicinal material of Guichuixiao under different extraction solvent conditions in Example 1.2;

[0033] Figure 5 This is a precision comparison chart in method validation;

[0034] Figure 6 This is a comparison chart of repeatability in method validation;

[0035] Figure 7 This is a stability comparison chart of the test solution in method validation. DETAILED DESCRIPTION

[0036] In order to make those skilled in the art better understand the technical scheme of the present invention, the present invention is described in detail below in conjunction with specific embodiments. The experimental methods for which specific conditions are not indicated in the following examples are usually carried out under normal conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar to or equal to the recorded content can all be applied to the present invention. The preferred implementation methods and materials described in the text are only for demonstration purposes.

[0037] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0038] The present invention aims to provide a fingerprint spectrum of a medicinal material of ghost blowing flute, and compare the characteristic spectrum of a sample to be tested with it. If the RSD value of the chlorogenic acid peak area is within ±5% of the specified value, it is a qualified product. If the similarity between the characteristic spectrum of the sample to be tested and the fingerprint spectrum of the medicinal material of ghost blowing flute is greater than 0.90, it is a qualified product. The fingerprint spectrum is included in the internal control quality standard of the enterprise, and the monitoring level of the quality of the medicinal material of ghost blowing flute by the enterprise is improved, so as to provide a reference basis for improving the quality control method of the medicinal material.

[0039] In some specific embodiments, a method for constructing a medicinal material fingerprint of Guichuixiao is provided, comprising the following steps:

[0040] S1. Prepare a reference solution by dissolving chlorogenic acid reference substance.

[0041] S2. Extract the extract of Leycesteria formosa Wall. to prepare a test solution:

[0042] S3. Respectively take the said test solution and the said reference solution and determine them by high performance liquid chromatography to obtain the fingerprint chromatogram of Leycesteria formosa Wall. medicinal materials;

[0043] The said chromatographic conditions are as follows: The chromatographic column is filled with octadecylsilane chemically bonded silica gel, acetonitrile is used as mobile phase A, 0.1wt% phosphoric acid solution is used as mobile phase B, gradient elution is carried out, the flow rate is 0.5mL / min - 1.5mL / min, the column temperature is 25℃ - 35℃, and the detection wavelength is 200 - 280nm.

[0044] In some specific embodiments, the said reference solution in S1 is prepared by dissolving chlorogenic acid reference substance with an alcohol solution;

[0045] In some specific embodiments, in S1, the said alcohol solution is selected from one of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol solutions; in some preferred embodiments, the said alcohol solution is selected from methanol;

[0046] In some specific embodiments, in S1, the concentration of the said alcohol solution is 50% - 100%wt; in actual operation, the concentration of the said alcohol solution can be selected as 50%wt, 60%wt, 70%wt, 80%wt, 90%wt, 100%wt;

[0047] In some specific embodiments, in S1, the concentration of the said reference solution is 200 - 300μg / mL; in actual operation, the concentration of the said reference solution can be selected as 200μg / mL, 220μg / mL, 240μg / mL, 260μg / mL, 280μg / mL, 300μg / mL;

[0048] In some specific embodiments, in S2, the said extract of Leycesteria formosa Wall. is obtained by extracting Leycesteria formosa Wall. with an extraction solvent; the said extraction solvent is selected from one or more of water, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, methyl formate, ethyl formate, methyl acetate, ethyl acetate, dichloromethane, chloroform, carbon tetrachloride, dichloroethane, acetone, butanone, pentanone; in some preferred embodiments, the said extraction solvent is selected from methanol;

[0049] In some specific embodiments, in S2, the said extract of Leycesteria formosa Wall. is prepared by the following steps: Take Leycesteria formosa Wall. medicinal materials, crush them, add an extraction solvent, carry out ultrasonic extraction, make up the volume, filter, and collect the filtrate.

[0050] In some specific embodiments, in S2, the ultrasonic extraction frequency is 20-60KHz; in actual operation, the ultrasonic extraction frequency can be selected to be 20KHz, 30KHz, 40KHz, 50KHz, 60KHz;

[0051] In some specific embodiments, the ultrasonic extraction time is 30-90 min; in actual operation, the ultrasonic extraction time can be selected to be 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min;

[0052] In some specific embodiments, in S3, the gradient elution conditions are:

[0053] 0-15min, the volume fraction of mobile phase A was 10%, and the volume fraction of mobile phase B was 90%;

[0054] 15-30min, the volume fraction of mobile phase A changes from 10% to 30%, and the volume fraction of mobile phase B changes from 90% to 70%;

[0055] 30-55min, the volume fraction of mobile phase A changes from 30% to 80%, and the volume fraction of mobile phase B changes from 70% to 20%;

[0056] From 55 to 60 min, the volume fraction of mobile phase A changes from 80% to 100%, and the volume fraction of mobile phase B changes from 20% to 0%.

[0057] In some specific embodiments, in S3, the test solution presents 9 common peaks, among which peak No. 3 corresponding to the reference peak is the reference peak, and the relative retention time RSD values ​​of the other common peaks and the reference peak range from 0.01 to 0.38%, and the relative peak area RSD values ​​range from 0.22 to 1.90%.

[0058] In a second aspect, the present invention provides a fingerprint of Guichuixiao medicinal materials constructed by the above method, wherein the test solution presents 9 common peaks, with the chlorogenic acid peak No. 3 as the reference peak (S peak), and the relative retention time RSD values ​​of the other common peaks and the S peak range from 0.01 to 0.38%, and the relative peak area RSD values ​​range from 0.22 to 1.90%.

[0059] The method for preparing the medicinal material fingerprint of Guichuixiao is also within the protection scope of the present invention.

[0060] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0061] Experimental instruments: Agilent 1260 (InfinityⅡ) high performance liquid chromatograph (Agilent Corporation, USA); SQP 1 / 100,000 electronic balance (Sartorius Scientific Instruments Co., Ltd., Germany); Milli-Q IQ7000 ultrapure water meter (Millipore Corporation, USA); XM-P101H ultrasonic cleaner (Kunshan Xiaomei Ultrasonic Instrument Co., Ltd.).

[0062] Reagents and materials: Chlorogenic acid (China Food and Drug Inspection Institute: 110753-201817, purity: 96.8%); acetonitrile and methanol were chromatographically pure and purchased from Merck KGaA; formic acid was analytically pure and purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.; water was ultrapure water.

[0063] Example 1 Construction of the fingerprint of medicinal materials of Guichuixiao

[0064] (1) Preparation of reference solution: Take an appropriate amount of chlorogenic acid reference substance, add 50 wt % methanol solution to the mark, shake well, and prepare a reference solution with a concentration of 266.3 μg / mL;

[0065] (2) Take 0.1 g of the ghost flute sample powder that has passed through a No. 3 sieve, weigh it accurately, put it in a stoppered conical flask, add 25 mL of a 50 wt% methanol solution, weigh it, and extract it ultrasonically at an ultrasonic frequency of 40 kHz for 30 min. Cool it to room temperature, add 50% methanol solution to make it heavier, shake it well, and filter it through a 0.22 μm microporous filter membrane to obtain the test solution;

[0066] (3) The test solution and the reference solution were respectively taken and measured by high performance liquid chromatography. The chromatographic conditions were as follows: the chromatographic column was a C18 chromatographic column, 250 mm×4.6 mm, 5 μm; acetonitrile was used as mobile phase A, and 0.1 wt% phosphoric acid solution was used as mobile phase B, and gradient elution was performed: 0-15 min, 10% acetonitrile; 15-30 min, 10-30% acetonitrile; 30-55 min, 30-8019% acetonitrile; 55-60 min, 80-100% acetonitrile; the flow rate was 1.0 mL / min; the column temperature was 30°C; the detection wavelength was 290 nm; and the injection volume was 10 μL.

[0067] According to the preparation method of the test solution, 15 batches of ghost flute test solution were prepared, and the ultra-high performance liquid chromatography was used for detection according to the above chromatographic conditions. The chromatograms were recorded, and the data were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". The chromatogram of sample S1 was used as the reference spectrum, and the median method was used with a time window width of 0.1 to perform multi-point correction to generate superimposed spectra and reference spectra ( Figure 1-2)。A total of 9 common peaks were calibrated. By comparing with the reference substance, 1 characteristic peak was identified, that is, peak 3 was chlorogenic acid. Since the 3rd chromatographic peak had a relatively large peak area and good resolution, it was used as the S peak (peak 3). The relative retention times and relative peak areas of the common peaks in the test solutions of different batches of *Leycesteria formosa* Wall. are shown in Table 1-2.

[0068] Table 1 Relative retention times of common peaks

[0069]

[0070]

[0071] Table 2 Relative peak areas of common peaks

[0072]

[0073] The average vector method was used to establish a common pattern for the fingerprints of 15 batches of *Leycesteria formosa* Wall.. Each fingerprint was compared with the reference fingerprint, and the cosine of the angle and the correlation coefficient between each sample and the reference sample were calculated to obtain the similarity between each fingerprint and the reference fingerprint. The analysis results are shown in Table 3.

[0074] Table 3

[0075]

[0076]

[0077] As can be seen from the above table, the similarities of the fingerprints of each component in the 15 batches of *Leycesteria formosa* Wall. samples

[0078] were all ≥ 0.994, meeting the requirements.

[0079] Example 1.1

[0080] The method of Example 1 was adopted, with the difference that the "ultrasonic extraction for 30 min at an ultrasonic frequency of 40 kHz" in S2 was replaced with "ultrasonic extraction for 90 min at an ultrasonic frequency of 20 kHz" and "ultrasonic extraction for 60 min at an ultrasonic frequency of 60 kHz" respectively to obtain the characteristic spectra under different ultrasonic conditions. As Figure 3 shown, under different ultrasonic extraction conditions, there were no obvious differences in the peak shapes and separation effects of the common peaks. Considering the durability of the method, ultrasonic extraction for 30 min at an ultrasonic frequency of 40 kHz was preferred.

[0081] Example 1.2

[0082] The method of Example 1 was adopted, with the difference that in S2, the "50 wt% methanol solution" was replaced with "100 wt% methanol solution", "50 wt% ethanol aqueous solution", and "50 wt% acetonitrile aqueous solution" to obtain the characteristic chromatograms of different extraction solvents, as Figure 4 shown. Methanol solution and acetonitrile aqueous solution as extraction solvents have better separation effects.

[0083] Example 2 Precision Experiment

[0084] Take the test solution of Lysimachia aspera Wall. ex DC. of Example 1, and inject samples continuously for 6 times under the chromatographic conditions of Example 1, and record the chromatogram (see Figure 5 ). Taking chlorogenic acid (peak No. 3) as the S peak, the RSD value range of the relative retention time of each common peak was calculated to be 0.01% - 0.52%, and the RSD value range of the relative peak area was 0.31 - 1.72%, indicating that the precision of the instrument is good.

[0085] Example 3 Repeatability Test

[0086] Take the same batch of Lysimachia aspera Wall. ex DC. samples, and prepare 6 test solutions in parallel according to the preparation method of the test solution of Lysimachia aspera Wall. ex DC. of Example 1, and inject samples under the chromatographic conditions of Example 1, and record the chromatogram (see Figure 6 ). Taking chlorogenic acid (peak No. 3) as the S peak, the RSD value range of the relative retention time of each common peak was calculated to be 0.13 - 0.27%, and the RSD value range of the relative peak area was 0.94% - 1.95%, indicating that the repeatability of this method is good.

[0087] Example 4 Stability Test

[0088] Take the test solution of Lysimachia aspera Wall. ex DC. of Example 1, and inject samples at 0, 4, 8, 12, 18, and 24 h respectively under the chromatographic conditions of Example 1, and record the chromatogram ( Figure 7 ). Taking chlorogenic acid (peak No. 3) as the S peak, the RSD value range of the relative retention time of each common peak was calculated to be 0.01 - 0.38%, and the RSD value range of the relative peak area was 0.22 - 1.90%, indicating that the test solution has good stability within 24 h.

[0089] Example 5 Spiked Recovery Test

[0090] Take 6 portions of Lysimachia aspera Wall. ex DC. test samples with known component contents, 0.05 g for each portion, and prepare 6 test solutions according to the preparation method of the test solution of Lysimachia aspera Wall. ex DC. of Example 1, add an appropriate amount of chlorogenic acid reference substance, and inject samples under the chromatographic conditions of Example 1, as shown in Table 4.

[0091] Table 4 Results of Spiked Recovery Test of Chlorogenic Acid (n = 6)

[0092]

[0093] The results in the above table show that the recovery rates are all between 95% and 105%, and the RSD of the recovery rate is 0.77%, indicating good accuracy.

[0094] Determination of Chlorogenic Acid Content in Leycesteria formosa Wall. in Example 6

[0095] Take the test solution S1 - S15 of Leycesteria formosa Wall. in Example 1, under the chromatographic conditions of Example 1, determine the peak area of chlorogenic acid, and calculate according to the external standard method to obtain the content of chlorogenic acid in Leycesteria formosa Wall. The results are shown in Table 5.

[0096] Table 5 Results of Chlorogenic Acid Determination in Leycesteria formosa Wall. (n = 3)

[0097]

[0098]

[0099] The above test results show that the method for constructing the fingerprint of Leycesteria formosa Wall. medicinal materials of the present invention has high precision, good stability and good reproducibility. The present invention also determines the active ingredient chlorogenic acid in Leycesteria formosa Wall. medicinal materials, which is of great significance for accurately controlling the quality of Leycesteria formosa Wall.

[0100] Finally, it should be noted that the above description is only the preferred embodiment of the present invention. Those skilled in the art can make various similar representations under the inspiration of the present invention without departing from the purpose and claims of the present invention. Such transformations all fall within the protection scope of the present invention.

Claims

1. A method for constructing a fingerprint of a medicinal material of Guichuixiao, characterized in that: The following steps are involved: S1. Prepare a reference solution by taking chlorogenic acid reference substance; S2. Extract the ghost flute extract to prepare the test solution: S3. Take the test solution and the reference solution respectively and measure them by high performance liquid chromatography to obtain the fingerprint of ghost flute medicinal materials; The chromatographic conditions are as follows: the chromatographic column uses octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A, and 0.1wt% phosphoric acid solution as a mobile phase B, with gradient elution, a flow rate of 0.5mL / min-1.5mL / min, a column temperature of 25°C-35°C, and a detection wavelength of 200-280nm.

2. The method for constructing the fingerprint of medicinal materials of Guichuixiao according to claim 1, characterized in that: In S1, the reference solution is prepared by dissolving the chlorogenic acid reference substance in an alcohol solution; And / or, the alcohol solution is selected from one of methanol, ethanol, n-propanol, isopropanol, n-butanol, and isobutanol solutions; and / or, the concentration of the alcohol solution is 50%-100%wt; And / or, the concentration of the reference solution is 200-300 μg / mL.

3. The method for constructing the fingerprint of Guichuixiao medicinal materials according to claim 1, characterized in that: In S2, the ghost flute extract is obtained by extracting the ghost flute with an extraction solvent; the extraction solvent is selected from one or more of water, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, methyl formate, ethyl formate, methyl acetate, ethyl acetate, dichloromethane, chloroform, carbon tetrachloride, dichloroethane, acetone, butanone, and pentanone.

4. The method for constructing the fingerprint of medicinal materials of Guichuixiao according to claim 1, characterized in that: In S2, the Herba Lycopodii extract is a Herba Lycopodii water extract or a Herba Lycopodii alcohol extract.

5. The method for constructing the fingerprint of medicinal materials of Guichuixiao according to claim 1, characterized in that: In S2, the ghost flute extract is prepared by the following steps: taking ghost flute medicinal materials, crushing, adding extraction solvent, ultrasonic extraction, constant volume, filtering, and collecting filtrate.

6. The method for constructing the fingerprint of Guichuixiao medicinal materials according to claim 5, characterized in that: In S2, the ultrasonic extraction frequency is 20-60KHz; And / or, the ultrasonic extraction time is 30-90 min.

7. The method for constructing the fingerprint of Guichuixiao medicinal materials according to claim 1, characterized in that: In S3, the gradient elution conditions are: 0-15min, the volume fraction of mobile phase A was 10%, and the volume fraction of mobile phase B was 90%; 15-30min, the volume fraction of mobile phase A changes from 10% to 30%, and the volume fraction of mobile phase B changes from 90% to 70%; 30-55min, the volume fraction of mobile phase A changes from 30% to 80%, and the volume fraction of mobile phase B changes from 70% to 20%; From 55 to 60 min, the volume fraction of mobile phase A changes from 80% to 100%, and the volume fraction of mobile phase B changes from 20% to 0%.

8. The method for constructing the fingerprint of the medicinal material of Guichuixiao according to any one of claims 1 to 7, characterized in that: The test solution presents 9 common peaks, among which peak No. 3 corresponding to the reference peak is the reference peak, and the relative retention time RSD values ​​of the other common peaks and the reference peak range from 0.01 to 0.38%, and the relative peak area RSD values ​​range from 0.22 to 1.90%.

9. A ghost flute medicinal material fingerprint constructed by the method described in any one of claims 1 to 7, characterized in that: The test solution showed 9 common peaks, with the No. 3 chlorogenic acid peak as the reference peak, and the relative retention time RSD values ​​of the other common peaks and the reference peaks ranged from 0.01 to 0.38%, and the relative peak area RSD values ​​ranged from 0.22 to 1.90%.

10. A method for detecting a medicinal material of Guichuxiao, characterized in that: Compare the ghost blowing flute medicinal material fingerprint constructed by the method described in claims 1-8 and the ghost blowing flute medicinal material fingerprint described in claim 9 with the characteristic spectrum of the sample to be tested, and the RSD value of the chlorogenic acid peak area should be within ±5% of the specified value to be a qualified product; and / or, evaluate the similarity of the ghost blowing flute medicinal material fingerprint constructed by the method described in claims 1-8 and the ghost blowing flute medicinal material fingerprint described in claim 9 with the characteristic spectrum of the sample to be tested, and the similarity greater than 0.90 is a qualified product.

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