Fingerprint spectrum of guihuichang medicinal material, construction method and detection method

Abstract

The present application relates to the field of medicine, and more particularly to a fingerprint spectrum of a medicine of a ghost flute, a method for constructing the fingerprint spectrum and a method for detecting the fingerprint spectrum, wherein the ghost flute medicine is taken as a research object, acetonitrile-0.1% phosphoric acid solution is taken as a mobile phase, a fingerprint spectrum of the ghost flute medicine is constructed for the first time, nine common peaks are calibrated, and a characteristic component chlorogenic acid is identified; the method for constructing the fingerprint spectrum of the ghost flute optimizes extraction time and extraction methods, so that information of chromatographic peaks of the ghost flute is exposed to the maximum extent, has high detection sensitivity, has strong specificity, good reproducibility and stability, high accuracy, and can quickly and efficiently control the quality of the ghost flute medicine. The content of chlorogenic acid in the ghost flute medicine is determined, so that the quality of the ghost flute medicine is more comprehensively and effectively controlled, and the efficacy and safety of the medicine are ensured.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of pharmaceuticals, and in particular relates to a fingerprint of Leycesteria formosa Wall. medicinal material, a construction method and a detection method thereof. BACKGROUND ART

[0002] Leycesteria formosa Wall. is a deciduous or small shrub plant of the genus *Leycesteria* in the family Caprifoliaceae. There are about 8 species worldwide, distributed in the Himalayan region and Myanmar. There are 6 species in China and 4 species in Yunnan Province, which is the main distribution area of plants of this genus. Except for southern Yunnan, it is found almost throughout the province. Leycesteria formosa Wall., also known as Fengchui Xiao, Huopao Tong, Guipaozhang, Kongxin Mu, etc., is widely distributed in Yunnan, grows in most areas at an altitude of 1400 to 3300m, and has abundant resources. It is a commonly used medicinal plant for multiple ethnic groups. It is called "Xugai" and "Houshuangzhu" by the Bai people, and "Naitimo" by the Yi people. It has the effects of breaking blood and regulating menstruation, dispelling wind and removing dampness, resolving phlegm and relieving asthma, and promoting diuresis to reduce swelling. It is mainly used for treating cystitis, edema, bronchial asthma, food stagnation, abdominal distension, rheumatalgia, hemorrhoids, etc. It can be used externally to treat fractures, and its leaves can be used as a cooling medicine.

[0003] Traditional Chinese medicine characteristic chromatogram is a currently widely accepted quality evaluation mode for traditional Chinese medicine. Its application reflects the trend of comprehensive quality management of traditional Chinese medicine and conforms to the characteristics of overall characterization analysis for traditional Chinese medicine quality control. At present, there is no clear quality standard system for Leycesteria formosa Wall. medicinal materials, so it is necessary to establish a characteristic chromatogram or fingerprint of Leycesteria formosa Wall. to evaluate the consistency and stability of the quality of the medicinal material, which is of great significance for its production quality control. SUMMARY OF THE INVENTION

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

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

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

[0007] S1. taking chlorogenic acid reference substance to prepare a reference substance solution;

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

[0009] S3. taking the test sample solution and the reference substance solution respectively and performing measurement by high performance liquid chromatography to obtain the fingerprint of Leycesteria formosa Wall. medicinal material;

[0010] The chromatographic conditions were as follows: the column was packed with octadecylsilane-bonded silica gel, acetonitrile was used as mobile phase A, and 0.1 wt% phosphoric acid solution was used as mobile phase B, gradient elution was performed, the flow rate was 0.5 mL / min-1.5 mL / min, the column temperature was 25℃-35℃, and the detection wavelength was 200-280 nm.

[0011] Furthermore, in step S1, the reference solution is prepared by dissolving chlorogenic acid reference standard in an alcohol solution;

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

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

[0014] Further, in step S1, the concentration of the reference solution is 200-300 μg / mL;

[0015] Further, in step S2, the extract of *Ghost Blowing Flute* is obtained by extracting *Ghost Blowing Flute* using 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 step S2, the extract of *Ghost Blowing Flute* is either a water extract or an alcohol extract of *Ghost Blowing Flute*.

[0017] Further, in S2, the extract of the Ghostly Flute is prepared by the following steps: take the Ghostly Flute medicinal material, crush it, add the extraction solvent, extract it by ultrasound, make up the volume, filter it, and collect the filtrate.

[0018] Furthermore, in step 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] From 0 to 15 minutes, the volume fraction of mobile phase A was 10%, and the volume fraction of mobile phase B was 90%.

[0022] Over 15-30 minutes, the volume fraction of mobile phase A changes by 10%-30%, and the volume fraction of mobile phase B changes by 90%-70%.

[0023] Over 30-55 minutes, the volume fraction of mobile phase A changes by 30%-80%, and the volume fraction of mobile phase B changes by 70%-20%.

[0024] Over 55-60 minutes, the volume fraction of mobile phase A changes by 80%-100%, while the volume fraction of mobile phase B changes by 20%-0%.

[0025] Furthermore, in step S3, the test solution exhibits nine common peaks, among which peak No. 3, corresponding to the reference peak, is the reference peak. 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%.

[0026] Secondly, the present invention provides a fingerprint spectrum of the medicinal material *Ghost Blowing Flute* constructed using the above method. The test solution exhibits 9 common peaks, with peak 3 (chlorogenic acid peak) as the reference peak. 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%.

[0027] Thirdly, the present invention provides a method for detecting the medicinal herb *Ghost Flute*, wherein the fingerprint spectrum of *Ghost Flute* constructed by the above method and the fingerprint spectrum of *Ghost Flute* are compared 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 for the product to be qualified; and / or, the similarity evaluation is performed on the fingerprint spectrum of *Ghost Flute* constructed by the above method and the fingerprint spectrum of *Ghost Flute* and the characteristic spectrum of the sample to be tested, and the similarity is greater than 0.90 for the product to be qualified.

[0028] Beneficial Effects: This invention uses the medicinal herb *Gnaphalium affine* as the research object and acetonitrile-0.1% phosphoric acid solution as the mobile phase to construct, for the first time, a fingerprint chromatogram of *Gnaphalium affine*, identifying nine common peaks and pinpointing the characteristic component chlorogenic acid. The fingerprint chromatogram construction method of this invention optimizes extraction time and method, maximizing the exposure of information from the chromatographic peaks, resulting in high detection sensitivity, strong specificity, good reproducibility and stability, and high accuracy. This allows for rapid and efficient quality control of *Gnaphalium affine*. Furthermore, by determining the chlorogenic acid content in *Gnaphalium affine*, the quality of the herb can be controlled more comprehensively and effectively, ensuring efficacy and medication safety. Attached Figure Description

[0029] Figure 1 The chromatograms of the common pattern of 15 batches of the medicinal material "Ghost Blowing Flute" are superimposed.

[0030] Figure 2 These are HPLC reference chromatograms of the reference solution and the test solution; where S1 is the chromatogram of the test solution and S2 is the chromatogram of the chlorogenic acid reference standard.

[0031] Figure 3 is characteristic chromatograms of *Leycesteria formosa* Wall. medicinal material under different ultrasonic conditions in Example 1.1;

[0032] Figure 4 is characteristic chromatograms of *Leycesteria formosa* Wall. medicinal material under different extraction solvent conditions in Example 1.2;

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

[0034] Figure 6 is a repeatability comparison diagram in method validation;

[0035] Figure 7 is a test solution stability comparison diagram in method validation. DETAILED EMBODIMENTS

[0036] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is described in detail below with reference to specific embodiments. For experimental methods without specific conditions indicated in the following examples, conventional conditions or conditions recommended by the manufacturer are generally followed. Unless otherwise specified, all test materials used in the following examples are purchased from conventional biochemical reagent stores. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only.

[0037] The endpoints and any values of the ranges disclosed herein 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, one or more new numerical ranges can be obtained by combining the endpoints of each range, combining the endpoints of each range with an individual point value, and combining individual point values with each other, and these numerical ranges shall be regarded as specifically disclosed herein.

[0038] The present invention aims to provide a fingerprint of *Leycesteria formosa* Wall. medicinal material. The characteristic chromatogram of a sample to be tested is compared with the fingerprint; a product is qualified if the RSD of the chlorogenic acid peak area is within ±5% of the specified value, and a product is qualified if the similarity between the characteristic chromatogram of the sample to be tested and the fingerprint of *Leycesteria formosa* Wall. medicinal material is greater than 0.90. The fingerprint is incorporated into the enterprise internal control quality standard to improve the enterprise's monitoring level of the quality of *Leycesteria formosa* Wall. medicinal material, so as to provide a reference for improving the quality control method of this medicinal material.

[0039] In some specific embodiments, a method for constructing a fingerprint of *Leycesteria formosa* Wall. medicinal material is provided, comprising the following steps:

[0040] S1. Taking chlorogenic acid reference substance to prepare a reference solution;

[0041] S2. Extracting Leycesteria formosa extract to prepare a test solution:

[0042] S3. Taking the test solution and the reference solution respectively for measurement by high performance liquid chromatography to obtain the fingerprint of Leycesteria formosa medicinal material;

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

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

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

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

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

[0048] In some specific embodiments, in said S2, the Leycesteria formosa extract is obtained by extracting Leycesteria formosa 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; in some preferred embodiments, the extraction solvent is selected from methanol;

[0049] In some specific embodiments, in said S2, the Leycesteria formosa extract is prepared by the following steps: taking Leycesteria formosa medicinal material, pulverizing, adding an extraction solvent, performing ultrasonic extraction, adjusting the volume to a constant volume, filtering, and collecting the filtrate.

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

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

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

[0053] From 0 to 15 minutes, the volume fraction of mobile phase A was 10%, and the volume fraction of mobile phase B was 90%.

[0054] Over 15-30 minutes, the volume fraction of mobile phase A changes by 10%-30%, and the volume fraction of mobile phase B changes by 90%-70%.

[0055] Over 30-55 minutes, the volume fraction of mobile phase A changes by 30%-80%, and the volume fraction of mobile phase B changes by 70%-20%.

[0056] Over 55-60 minutes, the volume fraction of mobile phase A changes by 80%-100%, while the volume fraction of mobile phase B changes by 20%-0%.

[0057] In some specific embodiments, in step S3, the test solution presents 9 common peaks, of which peak 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] Secondly, the present invention provides a fingerprint spectrum of the medicinal material *Ghost Blowing Flute* constructed using the above method. The test solution exhibits 9 common peaks, with peak 3 (chlorogenic acid peak) as the reference peak (S peak). The relative retention time RSD values ​​of the other common peaks and 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 fingerprint spectrum of the medicinal material "Ghost Blowing Flute" obtained by the above method also falls within the protection scope of this invention.

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

[0061] Experimental instruments: Agilent 1260 (Infinity II) high performance liquid chromatograph (Agilent Technologies, USA); SQP electronic balance (Sartorius Scientific Instruments GmbH, Germany); Milli-Q IQ7000 ultrapure water system (Millipore, USA); XM-P101H ultrasonic cleaner (Kunshan Xiaomei Ultrasonic Instrument Co., Ltd.).

[0062] Reagents and materials: Chlorogenic acid (China National Institutes for Food and Drug Control: 110753-201817, purity: 96.8%); Acetonitrile and methanol were chromatographic grade and purchased from Merck & Co. Ltd.; Formic acid was analytical grade and purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.; Water was ultrapure water.

[0063] Example 1: Construction of fingerprint spectrum of the medicinal herb "Ghost Blowing Flute"

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

[0065] (2) Take 0.1g of the Ghost Blowing Flute sample powder that has passed through No. 3 sieve, weigh it accurately, place it in a stoppered conical flask, add 25mL of 50wt% methanol solution, weigh it, extract it by ultrasonication at 40kHz for 30min, cool it to room temperature, add 50% methanol solution to make up the weight, shake it well, filter it through a 0.22μm microporous membrane to obtain the test solution;

[0066] (3) The test solution and the reference solution were determined by high performance liquid chromatography. The chromatographic conditions were as follows: the column was a C18 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, 30-80% acetonitrile; 55-60 min, 80-100% acetonitrile; the flow rate was 1.0 mL / min; the column temperature was 30 ℃; the detection wavelength was 290 nm; and the injection volume was 10 μL.

[0067] Fifteen batches of *Ghost Blowing Flute* test solutions were prepared according to the preparation method of the test solution. Ultra-high performance liquid chromatography (UHPLC) was used for detection under the above chromatographic conditions, and chromatograms were recorded. The data were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". Using the chromatogram of sample S1 as the reference chromatogram, the median method was used with a time window width of 0.1 for multi-point correction, generating overlay chromatograms and reference chromatograms. Figure 1-2Nine common peaks were identified. By comparing with the reference standard, one characteristic peak, peak 3, was identified as chlorogenic acid. Because peak 3 has a large peak area and good resolution, it was designated as peak S (peak 3). The relative retention times and relative peak areas of the common peaks in each batch of the *Ghost Flute* test solution are shown in Table 1-2.

[0068] Table 1. Relative retention times of common peaks

[0069]

[0070]

[0071] Table 2 Relative Peak Area of ​​Common Peaks

[0072]

[0073] The average vector method was used to establish a common pattern in the fingerprint images of 15 batches of "ghost blowing flute" (a type of Chinese flute). Each fingerprint image was compared with a control fingerprint image, and the cosine of the angle between each sample and the baseline sample and the correlation coefficient were calculated to obtain the similarity between each fingerprint image and the control fingerprint image. The analysis results are shown in Table 3.

[0074] Table 3

[0075]

[0076]

[0077] As can be seen in the table above, the similarity of the fingerprint spectra of each component in the 15 batches of "Ghost Blowing Flute" samples is...

[0078] ≥0.994, meets the requirements.

[0079] Example 1.1

[0080] The method of Example 1 was used, except that "ultrasonic extraction at 40 kHz for 30 min" in S2 was replaced with "ultrasonic extraction at 20 kHz for 90 min" and "ultrasonic extraction at 60 kHz for 60 min" respectively, to obtain characteristic spectra under different ultrasonic conditions, such as... Figure 3 As shown, under different ultrasonic extraction conditions, there was no significant difference in the peak shape and separation effect of each common peak. Considering the robustness of the method, the ultrasonic frequency of 40kHz and ultrasonic extraction for 30min are preferred.

[0081] Example 1.2

[0082] The method of Example 1 is used, with the difference that "50wt% methanol solution" in step S2 is replaced with "100wt% methanol solution", "50wt% ethanol aqueous solution" and "50wt% acetonitrile aqueous solution", to obtain characteristic chromatograms of different extraction solvents, as shown in Figure 4 , when methanol solution and acetonitrile aqueous solution are used as extraction solvents, the separation effect is better.

[0083] Example 2 Precision Experiment

[0084] Take the test solution of *Leycesteria formosa* Wall. prepared in Example 1, inject and measure continuously for 6 times according to the chromatographic conditions of Example 1, and record the chromatograms (see Figure 5 ), with (peak 3) chlorogenic acid as the S peak, the RSD range of relative retention time of each common peak is calculated to be 0.01% to 0.52%, and the RSD range of relative peak area is 0.31% to 1.72%, indicating that the precision of the instrument is good.

[0085] Example 3 Repeatability Test

[0086] Take *Leycesteria formosa* Wall. samples of the same batch, prepare 6 test solutions in parallel according to the preparation method of *Leycesteria formosa* Wall. test solution in Example 1, inject and measure according to the chromatographic conditions of Example 1, and record the chromatograms (see Figure 6 ), with (peak 3) chlorogenic acid as the S peak, the RSD range of relative retention time of each common peak is calculated to be 0.13% to 0.27%, and the RSD range of relative peak area is 0.94% to 1.95%, indicating that the method has good repeatability.

[0087] Example 4 Stability Test

[0088] Take the *Leycesteria formosa* Wall. test solution of Example 1, inject and measure at 0, 4, 8, 12, 18 and 24h respectively according to the chromatographic conditions of Example 1, and record the chromatograms (see Figure 7 ), with (peak 3) chlorogenic acid as the S peak, the RSD range of relative retention time of each common peak is calculated to be 0.01% to 0.38%, and the RSD range of relative peak area is 0.22% to 1.90%, indicating that the test solution has good stability within 24h.

[0089] Example 5 Sample Recovery Test

[0090] Take 6 portions of *Leycesteria formosa* Wall. samples with known component content, 0.05g per portion, prepare 6 test solutions according to the preparation method of *Leycesteria formosa* Wall. test solution in Example 1, add an appropriate amount of chlorogenic acid reference substance, inject and measure according to the chromatographic conditions of Example 1, the results are shown in Table 4.

[0091] Table 4 Results of chlorogenic acid sample recovery test (n=6)

[0092]

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

[0094] Example 6: Determination of chlorogenic acid content in *Ghost Blowing Flute*

[0095] Take the test solution S1-S15 of the Ghostly Flute from Example 1, and determine the peak area of ​​chlorogenic acid under the chromatographic conditions of Example 1. Calculate the content of chlorogenic acid in the Ghostly Flute using the external standard method. The results are shown in Table 5.

[0096] Table 5. Results of chlorogenic acid determination in *Ghost Blowing Flute* (n=3)

[0097]

[0098]

[0099] The above test results demonstrate that the fingerprinting method for the medicinal herb *Ghostly Flute* of this invention exhibits high precision, good stability, and good reproducibility. This invention also determined the active ingredient chlorogenic acid in *Ghostly Flute*, which is of great significance for accurately controlling the quality of *Ghostly Flute*.

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

Claims

1. A method for constructing a fingerprint spectrum of a medicinal herb called "Ghost Blowing Flute," characterized in that, Includes the following steps: S1. Prepare a reference solution by dissolving chlorogenic acid reference standard in a methanol solution with a concentration of 50%-100%wt to prepare a reference solution with a concentration of 200-300µg / mL. S2. Extracting the extract of *Ghost Blowing Flute* and preparing a test solution; wherein, the extract of *Ghost Blowing Flute* is prepared by the following steps: taking *Ghost Blowing Flute* medicinal material, crushing it, adding methanol, ultrasonically extracting it, making up to a certain volume, filtering it, and collecting the filtrate; S3. Take the test solution and the reference solution respectively and determine them by high performance liquid chromatography to obtain the fingerprint spectrum of the *Ghost Blowing Flute* medicinal material; The chromatographic conditions were as follows: the column was packed with octadecylsilane-bonded silica gel, acetonitrile was used as mobile phase A, and 0.1 wt% phosphoric acid solution was used as mobile phase B, with gradient elution at a flow rate of 0.5 mL / min. 1.5 mL / min, column temperature 25℃ 35℃, detection wavelength 200~280 nm; The gradient elution conditions are as follows: 0 After 15 minutes, the volume fraction of mobile phase A was 10%, and the volume fraction of mobile phase B was 90%. 15 After 30 minutes, the volume fraction of mobile phase A changed by 10%. The volume fraction of mobile phase B changes by 30% to 90%. 70%; 30 After 55 minutes, the volume fraction of mobile phase A changed by 30%. 80%, the volume fraction of mobile phase B changes by 70%. 20%; 55 After 60 minutes, the volume fraction of mobile phase A changed by 80%. 100%, the volume fraction of mobile phase B changes by 20%. 0%.

2. The method for constructing the fingerprint spectrum of the medicinal herb "Ghost Blowing Flute" according to claim 1, characterized in that, In S2, the ultrasonic extraction frequency is 20-60KHz.

3. The method for constructing the fingerprint spectrum of the medicinal herb "Ghost Blowing Flute" according to claim 1, characterized in that, In S2, the ultrasonic extraction time is 30-90 min.

4. The method for constructing the fingerprint spectrum of the medicinal material "Ghost Blowing Flute" according to any one of claims 1-3, characterized in that, The test solution exhibits 9 common peaks. Among them, peak 3, corresponding to the chlorogenic acid peak, is used as the reference peak. 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%.

5. A method for detecting the medicinal material of *Leycesteria formosa* Wall., characterized in that The fingerprint spectrum of the medicinal material "Ghost Blowing Flute" constructed by the method described in any one of claims 1-4 is compared 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 the product to be considered qualified.

6. A method for detecting the medicinal herb *Ghostly Flute*, characterized in that, The similarity evaluation is performed between the fingerprint spectrum of the medicinal material "Ghost Blowing Flute" constructed by the method described in any one of claims 1-4 and the feature spectrum of the sample to be tested. If the similarity is greater than 0.90, it is considered a qualified product.

7. A method for detecting the medicinal herb "Ghostly Flute," characterized in that, The fingerprint spectrum of the medicinal material *Ghost Blowing Flute* constructed by the method described in any one of claims 1-4 is evaluated 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, and the spectrum similarity should be greater than 0.90 to be considered a qualified product.

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