Method for constructing characteristic spectrum and contrast spectrum of fructus broussonetiae and method for distinguishing genuine and fake fructus broussonetiae

Abstract

The invention discloses a method for constructing a characteristic spectrum and a control spectrum of fructus broussonetiae and a method for distinguishing true and fake fructus broussonetiae. The method comprises the following steps: respectively injecting a fructus broussonetiae test solution and a reference substance solution into ultra-high performance liquid chromatography to obtain a characteristic chromatogram; and importing the characteristic chromatogram into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to establish a control chromatogram. And comparing the characteristic peak with the peak of the reference substance solution spectrum, and distinguishing the test sample as the fructus broussonetiae or the counterfeit product. The characteristic chromatogram is rich in chromatographic information, high in specificity, short in time, good in chromatographic peak separation degree and stable in base line, and the technology is good in reproducibility and high in stability; technical guarantee is provided for true and false identification of fructus broussonetiae raw materials and preparations; the method is simple, accurate, reliable and convenient to operate, and compared with a traditional biological identification method, the required sample amount is small, the detection time is saved, and the efficiency is high; and the solvent consumption is low, the toxicity of the used solvent is low, and the environmental pollution is small.

Description

Technical Field

[0001] The invention relates to a method for constructing a characteristic spectrum and a reference spectrum of Corydalis chinensis and a method for distinguishing genuine and fake Corydalis chinensis, and belongs to the field of drug quality control. Background Art

[0002] Cuscuta chinensis is the dried mature fruit of Broussonetia papyrifera (L.) Vent., a plant of the Moraceae family. It is cold in nature, sweet in taste, and enters the liver and kidney meridians. It has the effects of tonifying the kidney and clearing the liver, improving eyesight, and promoting urination. It is used for liver and kidney deficiency, soreness of the waist and knees, asthenia, dizziness, pannus, edema, and fullness. Cuscuta chinensis contains chemical components such as amino acids, flavonoids, and alkaloids, which have the effects of anti-oxidation, lowering blood lipids, enhancing immunity, anti-tumor, and protecting liver function.

[0003] At present, the 2020 edition of the Chinese Pharmacopoeia only includes properties, identification, inspection, and extracts under the item of "Curcuma bursa-pastor". Although these inspection items can reflect the quality of Curcuma bursa-pastor to a certain extent, their methods are relatively traditional and only applicable to medicinal materials and decoction pieces. At present, in terms of authenticity identification of Curcuma bursa-pastor, Huang Baokang et al. used near-infrared diffuse reflectance spectroscopy (NIRDRS) combined with cluster analysis to compare and identify Curcuma bursa-pastor and two counterfeits collected (Huang Baokang, Zhu Bin. Cluster analysis of near-infrared diffuse reflectance fingerprints of Curcuma bursa-pastor and counterfeits from different origins [J]. Chinese Medicinal Materials, 2002 (12): 874-875.); Liu Lina et al. used properties, microscopy, and thin layer analysis to compare and identify Curcuma bursa-pastor and three mixed counterfeits (Liu Lina, Zhou Xinbei, Ouyang Rong. Identification of adulterated products in Curcuma bursa-pastor [J]. Journal of Hunan University of Traditional Chinese Medicine, 2004 (3): 16-18.). Spectroscopy combined with chemometrics requires a large number of representative real samples to establish a mature and accurate mathematical model, and near-infrared spectroscopy has poor anti-interference ability, making the method difficult to promote. Traditional pharmacognosy methods rely on human experience and judgment, which is relatively subjective and only applicable to medicinal materials and decoction pieces. For pharmaceutical preparations containing Cuscuta australis, Cuscuta australis has lost its inherent morphology, making it difficult to evaluate its authenticity and quality through traditional identification methods, and it is difficult to ensure that the raw materials of pharmaceutical preparations are indeed Cuscuta australis.

[0004] The characteristic spectrum of traditional Chinese medicine is a comprehensive, quantifiable, multi-index quality control model that can comprehensively reflect the types and quantities of chemical components in traditional Chinese medicine and has higher specificity. The difference between Cuscuta australis and its counterfeits can be identified more efficiently and concisely through the number of characteristic peaks and types of chemical components reflected in the characteristic spectrum. It is also applicable to samples such as pharmaceutical preparations that have lost the inherent morphology of Cuscuta australis. In addition, the constructed characteristic spectrum method of traditional Chinese medicine can be used to evaluate the stability and consistency of the quality of traditional Chinese medicine and its preparations, and to control, evaluate and describe its quality as a whole, which can systematically, holistically and exclusively reflect and control the intrinsic quality of traditional Chinese medicine. Therefore, there is an urgent need for a method that can accurately, quickly, efficiently and comprehensively control the quality of Cuscuta australis and its preparations. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for constructing a characteristic spectrum and a reference spectrum of Corydalis chinensis and a method for distinguishing authentic and fake Corydalis chinensis; the characteristic spectrum has strong specificity, rich information, high efficiency and accuracy.

[0006] Technical solution: To solve the above technical problems, the present invention provides a method for constructing a characteristic spectrum of Corydalis chinensis seeds, comprising the following steps:

[0007] (1) Take the seeds of Cucurbitacin or its pharmaceutical preparation respectively, add the extraction solvent, shake well, filter, and the filtrate is the test solution;

[0008] (2) Take the reference medicinal material of Cuscuta australis, place it in a stoppered conical flask, add water, heat and reflux, concentrate to near dryness, add a solvent for ultrasonic treatment, shake well, filter, and take the filtrate as the reference solution;

[0009] (3) Take the test solution and the reference solution and inject them into the ultra performance liquid chromatography respectively to obtain a characteristic spectrum; the characteristic spectrum has 10 common peaks, peak 6 is taken as the S peak, and the relative retention time of the remaining characteristic peaks and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value. The specified values ​​of peaks 1 to 5 are: 0.41, 0.45, 0.51, 0.57, 0.80, respectively, and the specified values ​​of peaks 7 to 10 are: 1.07, 1.11, 1.19, 1.33, respectively.

[0010] Among them, the Cuscuta australis or its pharmaceutical preparation includes Cuscuta australis medicinal materials, Cuscuta australis decoction pieces, Cuscuta australis standard decoction, Cuscuta australis formula granules, Cuscuta australis extract or benchmark samples of classic prescriptions containing Cuscuta australis, preparation intermediates or finished granules.

[0011] "Medicinal materials" refer to raw materials of Chinese medicine that have not been processed or made into finished products. "Decoction pieces" refer to Chinese medicines that are processed according to needs and are used for prescriptions, or Chinese medicines that can be directly used in Chinese medicine clinics. "Standard decoctions" refer to single-flavor Chinese medicine decoction pieces water decoctions prepared by standardized processes under the guidance of Chinese medicine theory, based on clinical application, and with reference to modern extraction methods. "Formulated granules" are granules made from single-flavor Chinese medicine decoction pieces processed according to traditional standards and extracted and concentrated for use in Chinese medicine clinic prescriptions. "Extracts" refer to extracts that meet certain quality standards obtained by standardized production processes, such as alcohol (such as methanol, ethanol, etc.) extracts, water extracts, etc. "Classic prescription material benchmark samples" refer to Chinese medicine medicinal substances prepared based on the preparation methods of ancient classic prescriptions recorded in ancient medical books. Except for the molding process, the rest of the preparation methods should be basically consistent with the records in ancient medical books. "Classic prescription preparation intermediates" or "classic prescription finished granules" refer to granules made from ancient classic prescriptions recorded in ancient medical books through extraction and concentration for use in Chinese medicine clinic prescriptions. Wherein, the extraction solvent is alcohol or alcohol aqueous solution.

[0012] Preferably, the extraction solvent is methanol or a methanol-water solution.

[0013] More preferably, the concentration of the methanol aqueous solution is less than or equal to 30% by volume of methanol.

[0014] The treatment in step (1) includes ultrasound, heating reflux or shaking.

[0015] The treatment time in step (1) is 30-120 min.

[0016] Wherein, the treatment time in step (1) is 15-90 minutes.

[0017] The chromatographic conditions of the ultra-high performance liquid chromatography in step (3) are as follows: chromatographic column: CORTECS T3; injection volume: 3 μl; flow rate: 0.95-1.05 ml / min; column temperature: 28-32° C.; detection wavelength: 238-258 nm; methanol as mobile phase A, 0.2% trifluoroacetic acid solution as mobile phase B for gradient elution.

[0018] Wherein, the gradient elution of the ultra-high performance liquid chromatography in step (3) is: 0-5 min, the volume fraction of mobile phase A is 3%, and the volume fraction of mobile phase B is 97%; 5-13 min, the volume fraction of mobile phase A changes from 3 to 7%, and the volume fraction of mobile phase B changes from 97 to 93%; 13-28 min, the volume fraction of mobile phase A changes from 7 to 9%, and the volume fraction of mobile phase B changes from 93 to 81%.

[0019] The present invention also provides a method for establishing a comparison map of Corydalis chinensis seeds, comprising the following steps:

[0020] (1) preparing multiple batches of Cuscuta australis medicinal material test solution, multiple batches of Cuscuta australis standard decoction test solution, and multiple batches of Cuscuta australis formula granule test solution;

[0021] (2) According to the method for constructing the characteristic spectrum of Cuscuta australis, the test solution is tested respectively to obtain the characteristic spectrum of Cuscuta australis medicinal material, the characteristic spectrum of the standard decoction and the characteristic spectrum of Cuscuta australis formula granules; the preparation of the test solution comprises the following steps: taking Cuscuta australis medicinal material, the standard decoction or its formula granules respectively, adding alcohol or alcohol aqueous solution to treat, shaking well, filtering, and the filtrate is the test solution;

[0022] (3) The obtained characteristic spectrum is introduced into the traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system to establish a comparison spectrum of Cuscuta australis medicinal materials, standard decoctions or their formula particles.

[0023] Among them, there are 20 batches of Cuscuta australis medicinal materials test samples, 20 batches of Cuscuta australis standard decoction test samples, and 3 batches of Cuscuta australis formula granules test samples.

[0024] The present invention also provides a method for distinguishing paper mulberry seeds and their counterfeits, comprising the following steps:

[0025] (1) Take the Chinese Codonopsis pilosula, the standard decoction or its formula granules and the counterfeit respectively, add 10% methanol aqueous solution to treat, shake well, filter, and the filtrate is the test sample solution;

[0026] (2) Take the reference medicinal material of Cuscuta australis, place it in a stoppered conical flask, add water, heat and reflux, concentrate to near dryness, add a solvent for ultrasonic treatment, shake well, filter, and take the filtrate as the reference solution;

[0027] (3) Inject the test sample solution and the dummy sample solution into an ultra-high performance liquid chromatograph, perform ultra-high performance liquid chromatography detection, and record the chromatogram;

[0028] (4) Select peaks with stable relative retention times in the chromatogram as characteristic peaks, and then compare them with the peaks in the reference solution spectrum to determine 10 characteristic peaks; take peak 6 as the S peak, and calculate the relative retention times of the remaining characteristic peaks and the S peak. The relative retention times should be within ±10% of the specified values. The specified values ​​of peaks 1 to 5 are: 0.41, 0.45, 0.51, 0.57, 0.80, respectively; the specified values ​​of peaks 7 to 10 are: 1.07, 1.11, 1.19, 1.33, respectively;

[0029] (5) When the chromatogram of the test sample solution has 10 characteristic peaks, the test sample is Cuscuta australis, standard decoction or its formula granules; otherwise, the test sample is a counterfeit.

[0030] Wherein, the treatment method in step (1) includes ultrasound or heating reflux.

[0031] Wherein, the power of the ultrasound is 250W-600W, and the frequency is 35kHz-45kHz.

[0032] Among them, the ultra-high performance liquid chromatography detection conditions are: the chromatographic column is CORTECS T3; the mobile phase A is acetonitrile or methanol; the mobile phase B is trifluoroacetic acid aqueous solution, acetic acid aqueous solution, phosphoric acid aqueous solution or water; the mobile phase A and the mobile phase B are used for isocratic elution or gradient elution; the flow rate is 0.95-1.05mL / min; the column temperature is 28-32°C; and an ultraviolet detector is used with a wavelength of 200nm-300nm.

[0033] Wherein, the chromatographic conditions of the ultra-high performance liquid chromatography in step (3) are as follows: chromatographic column: CORTECS T3 (4.6×150 mm, 2.7 μm); injection volume: 3 μl; flow rate: 0.85-1.05 ml / min; column temperature: 28-32° C.; detection wavelength: 238-258 nm; methanol as mobile phase A, 0.2% trifluoroacetic acid solution as mobile phase B for gradient elution; gradient elution: 0-5 min, the volume fraction of mobile phase A is 3%, and the volume fraction of mobile phase B is 97%; 5-13 min, the volume fraction of mobile phase A changes from 3 to 7%, and the volume fraction of mobile phase B changes from 97 to 93%; 13-28 min, the volume fraction of mobile phase A changes from 7 to 9%, and the volume fraction of mobile phase B changes from 93 to 81%.

[0034] Among them, the counterfeit products include Cuscuta australis or Perilla australis.

[0035] Among them, when the chromatogram of the test sample solution has 5 characteristic peaks, the test sample is Cuscuta australis; the characteristic peaks are peak 1, peak 2, peak 3, peak 4, and peak 7.

[0036] Among them, when the chromatogram of the test sample solution has 6 characteristic peaks, the test sample is Perilla seed; the characteristic peaks are peak 1, peak 2, peak 3, peak 4, peak 6, and peak 7.

[0037] Among them, the paper mulberry seeds, paper mulberry seeds counterfeit products (perilla seeds), and paper mulberry seeds counterfeit products (cuscuta seeds) in the present invention can be any one or more of medicinal materials, decoction pieces, standard decoctions, extracts, and formula granules.

[0038] The present invention provides a method for identifying UPLC characteristic spectra of Cuscuta chinensis and its counterfeits using UPLC as an analytical means, which can quickly and accurately distinguish and identify Cuscuta chinensis and its counterfeits, and provide a new analytical means and idea for controlling the intrinsic quality of Cuscuta chinensis. Compared with the prior art, the present invention can be simultaneously applied to Cuscuta chinensis medicinal materials, decoction pieces, and Cuscuta chinensis-containing pharmaceutical preparations that have lost the properties of decoction pieces. The method has strong specificity, good repeatability, good stability, small sample quantity, simple operation, and is more efficient and environmentally friendly. While detecting Cuscuta chinensis and its counterfeits, the quality of Cuscuta chinensis and its preparations can be accurately, quickly, efficiently, and comprehensively controlled.

[0039] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0040] 1. The present invention adopts ultra-high performance liquid chromatography and reasonably controls the chromatographic conditions to obtain a characteristic spectrum with rich chromatographic information, high specificity, short time, good chromatographic peak separation, stable baseline, good reproducibility and high stability.

[0041] 2. The present invention establishes a high performance liquid chromatography method to detect and identify the seeds of Cuscuta australis and its counterfeits. The difference in the number of characteristic peaks of the seeds of Cuscuta australis and its counterfeits is used as a discrimination point, and the seeds of Cuscuta australis and its counterfeits, Cuscuta australis and Perilla australis, can be distinguished, which fully demonstrates the intrinsic quality difference between the seeds of Cuscuta australis and its counterfeits, and provides technical guarantee for the authenticity identification of the raw materials and preparations of Cuscuta australis;

[0042] 3. The present invention can be effectively used for quality control and evaluation in the production process of Cuscuta australis or its pharmaceutical preparations;

[0043] 4. The method of the present invention is simple, accurate, reliable, and easy to operate. Compared with the traditional pharmacognosy identification method, it requires less sample volume, saves detection time, and has higher efficiency; it consumes less solvent, the solvent used is less toxic, and has less pollution to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the 3D UPLC chromatogram of the pulp granules under full wavelength scanning;

[0045] Figure 2 This is the UPLC chromatogram of the formula granules of Cuscuta australis at a wavelength of 220nm to 280nm;

[0046] Figure 3 This is the characteristic spectrum of the reference medicinal material of Cuscuta australis;

[0047] Figure 4 This is a comparison chart of the characteristic spectra of Cuscuta australis medicinal materials, standard decoctions, and formula granules;

[0048] Figure 5 This is a comparison chart of the characteristic spectra of the standard decoction of Cuscuta australis, Perilla frutescens and Cuscuta australis.

[0049] Figure 6 The UPLC chromatograms of the granules of Codonopsis pilosula under different flow rate conditions;

[0050] Figure 7 The UPLC chromatograms of the formula granules of Cuscuta australis under different column temperature conditions;

[0051] Figure 8 It is the UPLC chromatogram of the chromatographic condition test1 in the comparative example;

[0052] Fig. 9 It is the UPLC chromatogram of the chromatographic condition test2 in the comparative example;

[0053] Fig.10 It is the UPLC chromatogram of the chromatographic condition test3 in the comparative example;

[0054] Fig.11 It is the UPLC chromatogram of the chromatographic condition test4 in the comparative example;

[0055] Fig.12 It is the UPLC chromatogram of the chromatographic condition test5 in the comparative example;

[0056] Fig.13 It is the UPLC chromatogram of the chromatographic condition test6 in the comparative example. DETAILED DESCRIPTION

[0057] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0058] Instruments, reagents and samples:

[0059] Waters Acquity UPLC ultra-high performance liquid chromatograph (Waters); Empower 3 workstation (Waters); Thermo Vanquish Flex ultra-high performance liquid chromatograph; Chromeleam 7.2SR4 Chameleon workstation; Agilent Technologies 1290 Infinity ultra-high performance liquid chromatograph; 1290 DAD diode array detector; 1290 MCT column oven; 1290 Vialsampler autosampler; 1290 Fiexible pump quaternary pump; Open LAB CDS2.3 chromatography workstation; Agilent G6530 Accurate-Mass Q-TOF mass spectrometer (Agilent); Agilent Mass Hunter Workstation data acquisition and qualitative analysis software (Agilent); electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); KQ-250B ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.); pure water system (Sartorius); AS165W centrifuge (Azov (Shanghai) Trading Co., Ltd.). Acetonitrile (chromatographic grade, Thermo Fisher); water is ultrapure water; trifluoroacetic acid (chromatographic grade, Thermo Fisher); other reagents are of analytical grade.

[0060] Cuscuta australis reference medicinal material (batch number: 121720-201501) was purchased from China Food and Drug Inspection Institutes.

[0061] Cucurbita chinensis medicinal materials (YC-C1~YC-C20), Cucurbita chinensis standard decoctions (DG-C1~DG-C20), Cucurbita chinensis formula granules (KL1, KL2, KL3), Cucurbita chinensis standard decoction counterfeits (Perilla seed standard decoction DG-Z1, DG-Z2, DG-Z3), and Cucurbita chinensis standard decoction counterfeits (Cuscuta seed standard decoction DG-T1, DG-T2, DG-T3) were all provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0062] Example 1: Construction of UPLC characteristic spectrum identification method for Corydalis chinensis and its counterfeit products

[0063] 1. Preparation of reference solution

[0064] Take 2 g of Cuscuta australis reference medicinal material, place it in a stoppered conical flask, add 50 ml of water, heat and reflux for 60 minutes, concentrate to near dryness, add 25 ml of 10% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate as the reference solution of the control medicinal material.

[0065] 2. Preparation of test solution

[0066] Take 0.3g of the formula granules of Cuscuta australis, grind them into powder, add 25ml of 10% methanol, weigh the weight, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0067] 3. Determination of chromatographic conditions

[0068] 3.1 Investigation of detection wavelength

[0069] Take 2 μl of the above test solution (formulated granule batch number: KL1) and record the absorption spectrum in the range of 200-400 nm. Figure 1 , Figure 2 As shown, at a wavelength of 238nm to 258nm, more chromatographic peaks were detected in the sample solution of the Cuscuta australis formula granules, the chromatographic peak information was relatively richer, the response value of each chromatographic peak was higher, and the baseline was stable. Among them, the preferred detection wavelength was 248nm.

[0070] 3.2 Determination of chromatographic conditions

[0071] CORTECS T3 was used as the chromatographic column (column length: 150 mm, inner diameter: 4.6 mm, particle size: 2.7 μm); methanol was used as mobile phase A, 0.2% trifluoroacetic acid solution was used as mobile phase B, and gradient elution was performed according to Table 1; the flow rate was 1.0 ml per minute; the column temperature was 30° C.; and the detection wavelength was 248 nm.

[0072] Table 1 Gradient elution table

[0073]

[0074] Example 2: Construction of characteristic profiles of Cuscuta australis medicinal materials, standard decoctions, and formula granules

[0075] 1 Preparation of test solution

[0076] Take 1 g of Cuscuta australis, add 25 ml of 10% methanol, heat and reflux for 60 minutes, cool, shake well, filter, and take the filtrate to obtain the Cuscuta australis test solution.

[0077] Take 0.3 g of the standard decoction of Cuscuta australis, add 25 ml of 10% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate to obtain the test solution of the standard decoction of Cuscuta australis.

[0078] Take 0.3 g of the Cuscuta chinensis formula granules, add 25 ml of 10% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate to obtain the Cuscuta chinensis formula granules test solution.

[0079] 2. Multi-batch determination and establishment of characteristic spectra

[0080] According to the UPLC characteristic spectrum method constructed in Example 1 of the present invention, multiple batches of reference solution, Cuscuta australis medicinal materials, standard decoction, and formula granules were measured, and 3 of each test solution was accurately taken. μl , injected into the ultra-high performance liquid chromatography for analysis, and the UPLC characteristic spectra of the Cuscuta chinensis, standard decoction, and formula granules were obtained. The retention time ratios of the characteristic peaks of the above batches of samples were stable, and the RSD was less than 1%. The characteristic spectrum of the Cuscuta chinensis control medicinal material is shown in Figure 3 The relative retention time results of multiple batches of medicinal materials, standard decoctions, and formula granules are shown in Tables 2 to 4.

[0081] Table 2 Determination results of characteristic spectrum of Cuscuta australis (relative retention time)

[0082]

[0083]

[0084] Table 3 Characteristic spectrum determination results of standard decoction of Cuscuta australis (relative retention time)

[0085]

[0086]

[0087] Table 4 Characteristic spectrum determination results of mulberry seed formula granules (relative retention time)

[0088]

[0089] The UPLC characteristic spectra of multiple batches of Cuscuta australis, standard decoction and formula granules were analyzed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" recommended by the Chinese Pharmacopoeia Committee. The comparative characteristic spectra of Cuscuta australis, standard decoction and formula granules were established and compared. The results are shown in Figure 4 . The results show that there are 10 characteristic peaks in the chromatograms of the test samples of Cuscuta australis, standard decoction, and formula granules, which correspond to the retention times of the 10 characteristic peaks in the chromatograms of the reference medicinal materials. Peak 6 is taken as the S peak, and the relative retention times of the remaining characteristic peaks and the S peak are calculated. The relative retention times should be within the range of ±10% of the specified values. The specified values ​​are: 0.41 (peak 1), 0.45 (peak 2), 0.51 (peak 3), 0.57 (peak 4), 0.80 (peak 5), 1.07 (peak 7), 1.11 (peak 8), 1.19 (peak 9), and 1.33 (peak 10).

[0090] Example 3: Identification of Cuscuta australis and its counterfeit products, Perilla frutescens and Cuscuta australis

[0091] Take samples of Cuscuta australis (batch number: DG-C1), Cuscuta australis fake (Perilla seed, DG-Z1), Cuscuta australis fake (Cuscuta australis, DG-T1), prepare test solutions according to the method described in Example 2, and measure them according to the method described in Example 1.

[0092] The characteristic spectra of the standard decoction of Cuscuta australis, Perilla frutescens and Cuscuta australis were imported into the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" recommended by the Chinese Pharmacopoeia Committee for comparative analysis. Figure 5 .

[0093] As can be seen from the results, compared with the 10 characteristic peaks in the characteristic spectrum of Cuscuta australis, the standard decoction of Cuscuta australis has 5 common characteristic peaks (peak 1, peak 2, peak 3, peak 4, peak 7), lacks 5 characteristic peaks (peak 5, peak 6, peak 8, peak 9, peak 10), and has 6 more peaks (peak T1, peak T2, peak T3, peak T4, peak T5, peak T6); the standard decoction of Perilla australis has 6 common characteristic peaks (peak 1, peak 2, peak 3, peak 4, peak 6, peak 7), lacks 4 characteristic peaks (peak 5, peak 8, peak 9, peak 10), and has 1 more peak (peak Z1). It can be seen that the characteristic spectrum identification method established by the present invention can be used to identify Cuscuta australis and its preparations and counterfeit products by the number of characteristic peaks.

[0094] Example 4: Methodological verification of the characteristic spectrum construction method

[0095] 1. Precision inspection

[0096] Take 0.3g of the formula granules of Cuscuta australis (batch number: KL1), add 25ml of 10% methanol, weigh the weight, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, cool, shake, filter, and take the filtrate to obtain the test solution. Inject the sample 6 times continuously to examine the consistency of the relative retention time and relative peak area of ​​the characteristic peak. The results are shown in Tables 5 and 6. The relative retention time of each characteristic peak is less than 1%, and the RSD value of the relative peak area is less than 3%, indicating that the characteristic spectrum method has good precision.

[0097] Table 5 Precision test results (relative retention time)

[0098]

[0099] Table 6 Precision test results (relative peak area)

[0100]

[0101] 2. Stability

[0102] Take 0.3g of the formula granules of Cuscuta australis (batch number: KL1), add 25ml of 10% methanol, weigh the weight, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, cool, shake, filter, take the filtrate, and get the test solution. Inject once at 0, 4, 12, 16, 20, and 24h, and measure for a total of 24 hours to examine the consistency of the relative retention time and relative peak area of ​​the characteristic peaks. The results are shown in Tables 7 and 8. The relative retention time of each characteristic peak is less than 1%, and the RSD of the relative peak area is less than 3%, indicating that the sample has good stability within 24 hours.

[0103] Table 7 Stability test results (relative retention time)

[0104]

[0105]

[0106] Table 8 Stability test results (relative peak area)

[0107]

[0108] 3. Repeatability

[0109] Take the formula granules of Cuscuta australis (batch number: KL1) and prepare 6 test solutions according to the test solution preparation method to examine the consistency of the relative retention time and relative peak area of ​​the characteristic peaks. The results are shown in Tables 9 and 10. The relative retention time of each characteristic peak is less than 1%, and the RSD of the relative peak area is less than 3%, with good repeatability.

[0110] Table 9 Repeatability test results (relative retention time)

[0111]

[0112] Table 10 Repeatability test results (relative peak area)

[0113]

[0114]

[0115] 4. Durability

[0116] 4.1 Investigation of different flow rates

[0117] The effect of different flow rates on the durability of the characteristic spectrum was investigated, and the flow rates were 0.28ml / min, 0.30ml / min, and 0.32ml / min respectively. Figure 6 The separation effect of each characteristic peak is good when the flow rate is in the range of 0.28ml / min to 0.32ml / min, which shows that the method has good durability and can meet the system applicability requirements when the flow rate changes slightly.

[0118] 4.2 Investigation of different column temperatures

[0119] The effect of different column temperatures on the durability of characteristic spectra was investigated, and the column temperatures were 28°C, 30°C, and 32°C respectively. Figure 7 The separation effect of each characteristic peak is good when the column temperature is in the range of 28℃~32℃, which shows that the method has good durability and can meet the system applicability requirements when the column temperature changes slightly.

[0120] Comparative Example:

[0121] This example investigates the separation effect of paper mulberry samples under different mobile phase gradients.

[0122] CORTECS T3 was used as the chromatographic column (column length: 150 mm, inner diameter: 4.6 mm, particle size: 2.7 μm); acetonitrile or methanol was used as the mobile phase A, 0.2% formic acid solution, 0.2% phosphoric acid solution or 0.2% trifluoroacetic acid solution was used as the mobile phase B, and gradient elution was performed according to the conditions in Tables 11 to 16, respectively. The results are shown in Figure 8 to Figure 13 .

[0123] Table 11 Chromatographic conditions test1

[0124]

[0125] Table 12 Chromatographic conditions test2

[0126]

[0127] Table 13 Chromatographic conditions test3

[0128]

[0129] Table 14 Chromatographic conditions test4

[0130]

[0131] Table 15 Chromatographic conditions test5

[0132]

[0133] Table 16 Chromatographic conditions test6

[0134]

[0135] Result analysis: Figure 8 The corresponding one is the UPLC characteristic spectrum of the paper mulberry seed sample under the chromatographic condition test1; Fig. 9 The corresponding one is the UPLC characteristic spectrum of the paper mulberry seed sample under the chromatographic condition test2; Fig.10The corresponding one is the UPLC characteristic spectrum of the paper mulberry seed sample under the chromatographic condition test3; Fig.11 The corresponding one is the UPLC characteristic spectrum of the paper mulberry seed sample under the chromatographic condition test4; Fig.12 The corresponding one is the UPLC characteristic spectrum of the paper mulberry seed sample under the chromatographic condition test5; Fig.13 The corresponding is the UPLC characteristic spectrum of the mulberry fruit sample under the chromatographic condition test6, i.e. the chromatographic condition of the present invention. In the chromatograms obtained under the chromatographic conditions test1 to test4 in this comparative example, the separation of each chromatographic peak is not good. In the chromatogram under the chromatographic condition test5, the separation of each chromatographic peak is good, but the analysis time is long and the baseline is not stable. The chromatographic condition test6, i.e. the chromatogram obtained by the method of the present invention, has rich chromatographic information, short time, good chromatographic peak separation, and a stable baseline.

Claims

1. A method for constructing a characteristic spectrum of Corydalis chinensis, characterized in that it comprises the following steps: (1) Take the Chinese Codonopsis pilosula, standard decoction or its formula granules respectively, add alcohol or alcohol aqueous solution to treat, shake well, filter, and the filtrate is the test solution; (2) Take the reference medicinal material of Cuscuta australis, place it in a stoppered conical flask, add water, heat and reflux, concentrate to near dryness, add a solvent for ultrasonic treatment, shake well, filter, and take the filtrate as the reference solution; (3) Take the test solution and the reference solution and inject them into the ultra-high performance liquid chromatography to obtain a characteristic spectrum; the characteristic spectrum has 10 common peaks, with peak 6 as the S peak, and calculate the relative retention time of the remaining characteristic peaks and the S peak. The relative retention time should be within the range of ±10% of the specified value. The specified values ​​of peaks 1 to 5 are: 0.41, 0.45, 0.51, 0.57, 0.80, respectively, and the specified values ​​of peaks 7 to 10 are: 1.07、1.11、1.19、1.33。 2. The construction method according to claim 1, characterized in that: The treatment in step (1) includes ultrasonication, shaking or heating under reflux.

3. The construction method according to claim 2, characterized in that: The power of the ultrasound is 250W-600W, and the frequency is 35kHz-45kHz.

4. The construction method according to claim 1, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography in step (3) are as follows: chromatographic column: CORTECS T3; injection volume: 3 μl; flow rate: 0.95-1.05 ml / min; column temperature: 28-32° C.; detection wavelength: 238-258 nm; methanol as mobile phase A, 0.2% trifluoroacetic acid solution as mobile phase B for gradient elution.

5. The construction method according to claim 1, characterized in that: The gradient elution of the ultra-high performance liquid chromatography in step (3) is as follows: from 0 to 5 min, the volume fraction of mobile phase A is 3%, and the volume fraction of mobile phase B is 97%; from 5 to 13 min, the volume fraction of mobile phase A changes from 3 to 7%, and the volume fraction of mobile phase B changes from 97 to 93%; from 13 to 28 min, the volume fraction of mobile phase A changes from 7 to 9%, and the volume fraction of mobile phase B changes from 93 to 81%.

6. A method for establishing a comparison map of Corydalis chinensis, characterized in that: The following steps are involved: (1) preparing multiple batches of Cuscuta australis medicinal material test solution, multiple batches of Cuscuta australis standard decoction test solution, and multiple batches of Cuscuta australis formula granule test solution; (2) According to the method for constructing the characteristic spectrum of Cuscuta australis fruit of claim 1, the test solution is tested respectively to obtain the characteristic spectrum of Cuscuta australis fruit medicinal material, the characteristic spectrum of the standard decoction and the characteristic spectrum of Cuscuta australis fruit formula granules; the preparation of the test solution comprises the following steps: taking Cuscuta australis fruit medicinal material, the standard decoction or its formula granules respectively, adding alcohol or alcohol aqueous solution to treat, shaking well, filtering, and the filtrate is the test solution; (3) The obtained characteristic spectrum is introduced into the traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system to establish a comparison spectrum of Cuscuta australis medicinal materials, standard decoctions or their formula particles.

7. A method for distinguishing paper mulberry seeds from their counterfeits, characterized in that: The following steps are involved: (1) Take Cuscuta australis medicinal materials, standard decoction or its formula granules and counterfeit products respectively, add alcohol or alcohol aqueous solution to treat them respectively, shake well, filter, and the filtrate is respectively the test sample solution; the counterfeit products include Cuscuta australis or Perilla australis; (2) Take the reference medicinal material of Cuscuta australis, place it in a stoppered conical flask, add water, heat and reflux, concentrate to near dryness, add a solvent for ultrasonic treatment, shake well, filter, and take the filtrate as the reference solution; (3) injecting the test solution and the dummy solution into an ultra-high performance liquid chromatograph respectively, performing ultra-high performance liquid chromatography detection, and recording the chromatogram; the chromatographic conditions of the ultra-high performance liquid chromatography are as follows: chromatographic column: CORTECS T3; injection volume: 3 μl; flow rate: 0.95-1.05 ml / min; column temperature: 28-32° C.; detection wavelength: 238-258 nm; methanol as mobile phase A, 0.2% trifluoroacetic acid solution as mobile phase B for gradient elution; gradient elution: 0-5 min, the volume fraction of mobile phase A is 3%, and the volume fraction of mobile phase B is 97%; 5-13 min, the volume fraction of mobile phase A changes from 3 to 7%, and the volume fraction of mobile phase B changes from 97 to 93%; 13-28 min, the volume fraction of mobile phase A changes from 7 to 9%, and the volume fraction of mobile phase B changes from 93 to 81%; (4) Select the peak with stable relative retention time in the chromatogram as the characteristic peak, and then compare it with the peak of the reference solution spectrum to determine 10 characteristic peaks; take peak 6 as the S peak, calculate the relative retention time of the remaining characteristic peaks and the S peak, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​of peaks 1 to 5 are: 0.41, 0.45, 0.51, 0.57, 0.80, respectively, and the specified values ​​of peaks 7 to 10 are: 1.07、1.11、1.19、1.33； (5) When the chromatogram of the test sample solution has 10 characteristic peaks, the test sample is Cuscuta australis, standard decoction or its formula granules; otherwise, the test sample is a counterfeit.

8. The method according to claim 7, characterized in that: The treatment in step (1) includes ultrasound or heating under reflux.

9. The method according to claim 7, characterized in that: When the chromatogram of the test sample solution has 5 characteristic peaks, the counterfeit product is Cuscuta australis; the characteristic peaks are peak 1, peak 2, peak 3, peak 4, and peak 7.

10. The method according to claim 7, characterized in that: When the chromatogram of the test sample solution has 6 characteristic peaks, the counterfeit product is Perilla seed; the characteristic peaks are peak 1, peak 2, peak 3, peak 4, peak 6, and peak 7.

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