A method for constructing a feature map and a control map of broussonetia papyrifera and a method for distinguishing broussonetia papyrifera genuine and fake products

Abstract

The application discloses a kind of broussonetia papyrifera characteristic map and the construction method of contrast map and the method for distinguishing broussonetia papyrifera genuine fake.The broussonetia papyrifera test sample solution, reference substance solution is injected into ultra-high performance liquid chromatography respectively, and characteristic map is obtained;Characteristic map is introduced into traditional Chinese medicine chromatographic fingerprint similarity evaluation system to establish contrast map.The characteristic peak is compared with the peak of reference substance solution atlas, and the test sample is distinguished as broussonetia papyrifera or fake.The characteristic map of the application is rich in chromatographic information, high in specificity, short in time, good in chromatographic peak separation degree, and stable baseline.The technology has good reproducibility and high stability;It provides technical support for the authenticity identification of broussonetia papyrifera raw materials and preparations;The method is simple, accurate and reliable, easy to operate, compared with traditional crude drug identification method, less sample amount is needed, detection time is saved, and efficiency is higher;Solvent consumption is less, and the toxicity of solvent used is smaller, and the environmental pollution is small.

Description

Technical Field

[0001] This invention relates to a method for constructing characteristic and control spectra of Broussonetia papyrifera seeds and a method for distinguishing genuine and counterfeit Broussonetia papyrifera seeds, belonging to the field of drug quality control. Background Technology

[0002] Paper mulberry fruit (Broussonetia papyrifera (L.) Vent.), a plant in the Moraceae family, is the dried, ripe fruit of the paper mulberry tree. It is cold in nature, sweet in taste, and enters the liver and kidney meridians. It has the effects of tonifying the kidneys and clearing the liver, improving eyesight, and promoting diuresis. It is used for liver and kidney deficiency, soreness and weakness of the lower back and knees, consumptive fever, dizziness, blurred vision, pterygium, and edema. Paper mulberry fruit contains amino acids, flavonoids, alkaloids, and other chemical components, and has antioxidant, lipid-lowering, immune-enhancing, anti-tumor, and liver-protective effects.

[0003] Currently, the 2020 edition of the Chinese Pharmacopoeia only includes the characteristics, identification, inspection, and extracts of Broussonetia papyrifera fruit under the entry for "Broussonetia papyrifera fruit". Although these inspection items can reflect the quality of Broussonetia papyrifera fruit to a certain extent, their methods are relatively traditional and only applicable to raw materials and processed slices. In terms of the identification of genuine and counterfeit Broussonetia papyrifera fruit, Huang Baokang et al. used near-infrared diffuse reflectance spectroscopy (NIRDRS) combined with cluster analysis to compare and identify Broussonetia papyrifera fruit and two counterfeit products collected (Huang Baokang, Zhu Bin. Cluster analysis of near-infrared diffuse reflectance fingerprint spectra of Broussonetia papyrifera fruit and counterfeit products from different origins [J]. Chinese Medicinal Herbs, 2002(12):874-875.); Liu Linna et al. used characteristics, microscopy, thin layer chromatography and other methods to compare and identify Broussonetia papyrifera fruit and three kinds of mixed counterfeit products (Liu Linna, Zhou Xinbei, Ouyang Rong. Identification of adulterants in Broussonetia papyrifera fruit [J]. Journal of Hunan University of Traditional Chinese Medicine, 2004(3):16-18.). Spectroscopic methods combined with chemometrics require a large number of representative real samples to establish mature and accurate mathematical models. Furthermore, near-infrared spectroscopy has poor anti-interference capabilities, making the method difficult to generalize. Traditional pharmacognosy methods rely on human experience and judgment, are subjective, and are only applicable to raw materials and processed medicinal slices. For pharmaceutical preparations containing paper mulberry seeds, the inherent form of the seeds has been lost, making it difficult to evaluate their authenticity and quality using traditional identification methods, and thus difficult to guarantee that the raw material of the pharmaceutical preparation is indeed paper mulberry seeds.

[0004] Traditional Chinese medicine (TCM) characteristic chromatograms are a comprehensive and quantifiable multi-index quality control model that can fully reflect the types and quantities of chemical components in TCM, exhibiting higher specificity. By analyzing the number of characteristic peaks and the types of chemical components reflected in the characteristic chromatograms, the differences between Broussonetia papyrifera seeds and their adulterants can be identified more efficiently and concisely. This method is also applicable to drug preparations that have lost the inherent form of Broussonetia papyrifera seeds. Furthermore, the constructed TCM characteristic chromatogram method can be used simultaneously to evaluate the stability and consistency of the quality of TCM materials and their preparations, enabling holistic control, evaluation, and description of their quality. It can systematically, holistically, and specifically reflect and control the intrinsic quality of TCM. Therefore, there is an urgent need for a method that can accurately, rapidly, efficiently, and comprehensively control the quality of Broussonetia papyrifera seeds and their 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 paper mulberry fruit, and a method for distinguishing genuine and counterfeit paper mulberry fruit; the characteristic spectrum is highly specific, rich in information, efficient and accurate.

[0006] Technical Solution: To solve the above technical problems, this invention provides a method for constructing a feature map of Broussonetia papyrifera seeds, comprising the following steps:

[0007] (1) Take Broussonetia papyrifera seeds or their drug preparations, add extraction solvent, shake well, filter, and the filtrate is the test solution;

[0008] (2) Take the reference material of paper mulberry fruit, place it in a stoppered conical flask, add water, heat to reflux, concentrate to near dryness, add solvent, sonicate, shake well, filter, and take the filtrate as the reference solution.

[0009] (3) Inject the test solution and the reference solution into an ultra-high performance liquid chromatograph to obtain characteristic chromatograms. The characteristic chromatograms have 10 common peaks. Taking peak 6 as the S peak, calculate the relative retention time of each of the remaining characteristic peaks and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​for peaks 1 to 5 are 0.41, 0.45, 0.51, 0.57, and 0.80, respectively. The specified values ​​for peaks 7 to 10 are 1.07, 1.11, 1.19, and 1.33, respectively.

[0010] The Broussonetia papyrifera seeds or their pharmaceutical preparations include Broussonetia papyrifera raw materials, Broussonetia papyrifera slices, Broussonetia papyrifera standard decoction, Broussonetia papyrifera formula granules, Broussonetia papyrifera extract, or reference samples, intermediate products, or finished granules of classic prescriptions containing Broussonetia papyrifera seeds.

[0011] "Medicinal materials" refers to unprocessed or unfinished raw materials of traditional Chinese medicine. "Processed medicinal slices" refers to traditional Chinese medicine that has undergone processing according to needs, for use in prescriptions, or that can be directly used in clinical practice. "Standard decoctions" refers to single-herb decoctions prepared using standardized processes guided by traditional Chinese medicine theory and based on clinical application, referencing modern extraction methods. "Formulation granules" are granules prepared from single-herb processed medicinal slices according to traditional standards, extracted, and concentrated, for use in clinical prescriptions in traditional Chinese medicine. "Extracts" refer to extracts that meet certain quality standards, produced using standardized production processes, such as alcohol (e.g., methanol, ethanol) extracts, water extracts, etc. "Classic prescription reference samples" refer to medicinal substances prepared based on the preparation methods of ancient classic prescriptions recorded in ancient medical books; except for the molding process, the other preparation methods should be basically consistent with those recorded in ancient medical books. "Intermediate products of classic prescription preparations" or "finished granules of classic prescriptions" refer to granules prepared from ancient classic prescriptions recorded in ancient medical books, extracted, and concentrated, for use in clinical prescriptions in traditional Chinese medicine. The extraction solvent is an alcohol or an aqueous solution of an alcohol.

[0012] Preferably, the extraction solvent is methanol or an aqueous methanol solution.

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

[0014] The processing methods in step (1) include ultrasound, heating reflux, or shaking.

[0015] The processing time in step (1) is 30-120 minutes.

[0016] The processing time in step (1) is 15-90 minutes.

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

[0018] In step (3), the gradient elution of ultra-high performance liquid chromatography is as follows: 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] This invention also provides a method for establishing a reference map of paper mulberry seeds, comprising the following steps:

[0020] (1) Prepare multiple batches of Broussonetia papyrifera seed test solutions, multiple batches of Broussonetia papyrifera seed standard decoction test solutions, and multiple batches of Broussonetia papyrifera seed formula granule test solutions respectively;

[0021] (2) The test solution is tested according to the method of constructing the characteristic spectrum of Broussonetia papyrifera fruit to obtain the characteristic spectrum of Broussonetia papyrifera fruit, the characteristic spectrum of standard decoction and the characteristic spectrum of Broussonetia papyrifera fruit granules; the preparation of the test solution includes the following steps: take Broussonetia papyrifera fruit, standard decoction or its granules, add alcohol or alcohol aqueous solution, shake well, filter, and the filtrate is the test solution;

[0022] (3) Import the obtained feature chromatograms into the Chinese herbal chromatographic fingerprint chromatogram similarity evaluation system to establish a reference chromatogram of Broussonetia papyrifera seed, standard decoction or its formula granules.

[0023] Among them, there were 20 batches of Broussonetia papyrifera fruit samples, 20 batches of Broussonetia papyrifera fruit standard decoction samples, and 3 batches of Broussonetia papyrifera fruit formula granules samples.

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

[0025] (1) Take the paper mulberry fruit, standard decoction or its formula granules and counterfeit products respectively, add 10% methanol aqueous solution to treat them, shake well, filter, and the filtrate is the test solution;

[0026] (2) Take the reference material of paper mulberry fruit, place it in a stoppered conical flask, add water, heat to reflux, concentrate to near dryness, add solvent, sonicate, shake well, filter, and take the filtrate as the reference solution.

[0027] (3) Inject the test solution and the adulterant solution into the ultra-high performance liquid chromatograph for ultra-high performance liquid chromatography detection and record the chromatograms;

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

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

[0030] The processing methods in step (1) include ultrasonic or heating reflux.

[0031] The ultrasonic power is 250W to 600W and the frequency is 35kHz to 45kHz.

[0032] The ultra-high performance liquid chromatography (UHPLC) detection conditions were as follows: the chromatographic column was a CORTECS T3; mobile phase A was acetonitrile or methanol; mobile phase B was an aqueous solution of trifluoroacetic acid, an aqueous solution of acetic acid, an aqueous solution of phosphoric acid, or water; isocratic elution or gradient elution was performed using mobile phase A and mobile phase B; the flow rate was 0.95-1.05 mL / min; the column temperature was 28-32℃; and an ultraviolet detector was used with a wavelength of 200 nm-300 nm.

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

[0034] The counterfeit products include dodder seeds or perilla seeds.

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

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

[0037] In this invention, the paper mulberry fruit, its adulterant (perilla seed), and its adulterant (cuscutellaria seed) can be any one or more of the following: medicinal materials, processed slices, standard decoctions, extracts, and formula granules.

[0038] This invention provides a method for identifying the UPLC characteristic spectra of Broussonetia papyrifera seeds and their adulterants using UPLC analysis. This method can quickly and accurately distinguish and identify Broussonetia papyrifera seeds and their adulterants, providing a new analytical tool and approach for controlling the intrinsic quality of Broussonetia papyrifera seeds. Compared with existing technologies, this invention can be applied simultaneously to Broussonetia papyrifera seed raw materials, processed medicinal slices, and drug preparations containing Broussonetia papyrifera seeds that have lost the characteristics of processed medicinal slices. The method has strong specificity, good repeatability, excellent stability, requires a small sample volume, is simple to operate, and is more efficient and environmentally friendly. While detecting Broussonetia papyrifera seeds and their adulterants, it can accurately, quickly, efficiently, and comprehensively control the quality of Broussonetia papyrifera seeds and their preparations.

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

[0040] 1. This invention employs ultra-high performance liquid chromatography (UHPLC) and rationally controls chromatographic conditions to obtain characteristic chromatographic spectra with rich chromatographic information, high specificity, short processing time, good peak resolution, and stable baseline. This technique has good reproducibility and high stability.

[0041] 2. This invention establishes a high-performance liquid chromatography method for the detection and identification of Broussonetia papyrifera seeds and their adulterants. By using the difference in the number of characteristic peaks of Broussonetia papyrifera seeds and their adulterants as the identification point, Broussonetia papyrifera seeds and their adulterants Cuscuta chinensis seeds and Perilla frutescens seeds can be distinguished, which fully demonstrates the intrinsic quality differences of Broussonetia papyrifera seeds and their adulterants, and provides technical support for the identification of the authenticity of Broussonetia papyrifera seed raw materials and preparations.

[0042] 3. This invention can be effectively used for quality control and evaluation during the production process of paper mulberry fruit or its pharmaceutical preparations;

[0043] 4. The method of the present invention is simple, accurate and reliable, and easy to operate. Compared with traditional pharmacognosy identification methods, it requires less sample, saves detection time, and is more efficient; it consumes less solvent, the solvent used is less toxic, and causes less environmental pollution. Attached Figure Description

[0044] Figure 1 The image shows a 3D UPLC chromatogram of the Broussonetia papyrifera seed formulation particles under full wavelength scanning.

[0045] Figure 2 The UPLC chromatogram of Broussonetia papyrifera fruit particles under wavelength conditions of 220 nm to 280 nm is shown.

[0046] Figure 3 A characteristic atlas of Broussonetia papyrifera fruit as a reference medicinal material;

[0047] Figure 4 Comparative chromatograms of characteristic spectra of Broussonetia papyrifera fruit, standard decoction, and formulated granules;

[0048] Figure 5 A comparative chromatogram of the characteristic spectra of the standard decoction of Broussonetia papyrifera fruit, Perilla frutescens seed, and Cuscuta chinensis seed;

[0049] Figure 6 UPLC chromatograms of Broussonetia papyrifera seed formulation particles under different flow rates;

[0050] Figure 7 UPLC chromatograms of Broussonetia papyrifera seed formulation particles under different column temperatures;

[0051] Figure 8 The UPLC chromatogram for test1 in the comparative example;

[0052] Figure 9 The UPLC chromatogram for test2 in the comparative example;

[0053] Figure 10 The UPLC chromatogram for test3 in the comparative example is shown below.

[0054] Figure 11 The UPLC chromatogram for test4 in the comparative example;

[0055] Figure 12 The UPLC chromatogram for test5 in the comparative example;

[0056] Figure 13 This is the UPLC chromatogram of test6 under chromatographic conditions in the comparative example. Detailed Implementation

[0057] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0058] Instruments, reagents and samples:

[0059] Waters Acquity UPLC (Ultra-High Performance Liquid Chromatograph) (Waters Corporation); Empower 3 workstation (Waters Corporation); 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 Corporation); Agilent Mass Hunter Workstation data acquisition and qualitative analysis software (Agilent Technologies); electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); temperature-controlled water bath (Nantong Huatai Experimental Instruments Co., Ltd.); KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); pure water system (Sartorius); AS165W centrifuge (Assouwang (Shanghai) Trading Co., Ltd.). Acetonitrile (chromatographic grade, Thermo Fisher Scientific); water was ultrapure water; trifluoroacetic acid (chromatographic grade, Thermo Fisher Scientific); other reagents were all analytical grade.

[0060] The reference material of Broussonetia papyrifera fruit (batch number: 121720-201501) was purchased from the National Institutes for Food and Drug Control.

[0061] The raw materials of Broussonetia papyrifera (YC-C1~YC-C20), the standard decoction of Broussonetia papyrifera (DG-C1~DG-C20), the formula granules of Broussonetia papyrifera (KL1, KL2, KL3), and the counterfeit standard decoctions of Broussonetia papyrifera (standard decoctions of Perilla frutescens DG-Z1, DG-Z2, DG-Z3) and the counterfeit standard decoctions of Broussonetia papyrifera (standard decoctions of Cuscuta chinensis DG-T1, DG-T2, DG-T3) were all provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0062] Example 1: Construction of a UPLC characteristic spectrum identification method for paper mulberry fruit and its adulterants

[0063] 1. Preparation of reference solution

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

[0065] 2. Preparation of the test solution

[0066] Take 0.3g of Broussonetia papyrifera fruit granules, grind them into a fine powder, add 25ml of 10% methanol, weigh the powder, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the final product.

[0067] 3. Determination of chromatographic conditions

[0068] 3.1 Examination of Detection Wavelength

[0069] Take 2 μl of the above-mentioned test solution (formulation granule batch number: KL1) and record the absorption spectrum in the range of 200–400 nm. The results are as follows: Figure 1 , Figure 2 As shown, at wavelengths of 238 nm to 258 nm, the sample solution of Broussonetia papyrifera fruit formulation granules showed a large number of chromatographic peaks, with relatively richer chromatographic peak information, higher response values ​​for each chromatographic peak, and stable baselines. Among these, the preferred detection wavelength was 248 nm.

[0070] 3.2 Determination of chromatographic conditions

[0071] A CORTECS T3 column (150 mm in length, 4.6 mm in inner diameter, and 2.7 μm in particle size) was used; methanol was used as mobile phase A, and 0.2% trifluoroacetic acid solution was used as mobile phase B, with gradient elution 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 Maps of Broussonetia papyrifera Fruit, Standard Decoction, and Formula Granules

[0075] 1. Preparation of the test solution

[0076] Take 1g of Broussonetia papyrifera fruit, add 25ml of 10% methanol, heat under reflux for 60 minutes, cool, shake well, filter, and collect the filtrate to obtain the test solution of Broussonetia papyrifera fruit.

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

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

[0079] 2. Multiple batch measurements and establishment of characteristic spectra

[0080] According to the UPLC characteristic spectrum method constructed in Example 1 of this invention, multiple batches of reference solution, paper mulberry fruit, standard decoction, and formula granules were measured. 3 μL of each test solution was precisely pipetted. μl The samples were injected into an ultra-high performance liquid chromatograph (UPLC) for analysis, and the UPLC characteristic spectra of Broussonetia papyrifera fruit, standard decoction, and formulation granules were obtained. The retention time ratios of the characteristic peaks of each batch of samples were stable, and the RSDs were all less than 1%. The characteristic spectrum of the reference Broussonetia papyrifera fruit is shown below. Figure 3 The relative retention times of multiple batches of medicinal materials, standard decoctions, and formula granules are shown in Tables 2 to 4.

[0081] Table 2. Results of chromatographic determination of the medicinal characteristics of Broussonetia papyrifera seeds (relative retention time)

[0082]

[0083]

[0084] Table 3. Results of the characteristic chromatogram determination of the standard decoction of Broussonetia papyrifera seeds (relative retention time)

[0085]

[0086]

[0087] Table 4. Results of spectral analysis of the characteristic particles of Broussonetia papyrifera (relative retention time)

[0088]

[0089] Multiple batches of UPLC characteristic spectra of Broussonetia papyrifera fruit, standard decoction, and formula granules were analyzed using the "Similarity Evaluation System for Chromatographic Fingerprint Spectra of Traditional Chinese Medicine (2012 Edition)" recommended by the National Pharmacopoeia Commission. Comparison characteristic spectra of Broussonetia papyrifera fruit, standard decoction, and formula granules were established and compared. The results are shown below. Figure 4 The results show that the chromatograms of the raw paper mulberry fruit, standard decoction, and formula granules all exhibit 10 characteristic peaks, which correspond to the retention times of the 10 characteristic peaks in the reference chromatogram of the control herb. Taking peak 6 as the S peak, the relative retention times of the remaining characteristic peaks with respect to the S peak are calculated. These relative retention times should be within ±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 Broussonetia papyrifera seeds and their adulterants Perilla frutescens seeds and Cuscuta chinensis seeds

[0091] Take samples of Broussonetia papyrifera seeds (batch number: DG-C1), Broussonetia papyrifera adulterants (Perilla frutescens seeds, DG-Z1), and Broussonetia papyrifera adulterants (Cuscuta chinensis seeds, DG-T1), prepare test solutions according to the method described in Example 2, and determine them according to the method described in Example 1.

[0092] The characteristic chromatograms of the standard decoction of Broussonetia papyrifera fruit, Perilla frutescens seed, and Cuscuta chinensis seed were imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" recommended by the National Pharmacopoeia Commission for comparative analysis. The results are shown below. Figure 5 .

[0093] The results show that, compared with the 10 characteristic peaks in the characteristic spectrum of Broussonetia papyrifera seeds, the standard decoction of Cuscuta chinensis seeds has 5 shared characteristic peaks (peaks 1, 2, 3, 4, and 7), lacks 5 characteristic peaks (peaks 5, 6, 8, 9, and 10), and has 6 additional peaks (peaks T1, T2, T3, T4, T5, and T6); the standard decoction of Perilla frutescens seeds has 6 shared characteristic peaks (peaks 1, 2, 3, 4, 6, and 7), lacks 4 characteristic peaks (peaks 5, 8, 9, and 10), and has 1 additional peak (peak Z1). Therefore, the characteristic spectrum identification method established in this invention can be used to distinguish Broussonetia papyrifera seeds and their preparations from adulterants by the number of characteristic peaks.

[0094] Example 4: Methodological Validation of Feature Map Construction Method

[0095] 1. Precision test

[0096] Take 0.3g of Broussonetia papyrifera fruit granules (batch number: KL1), add 25ml of 10% methanol, weigh, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the test solution. Inject the sample six times consecutively to examine the consistency of the relative retention time and relative peak area of ​​the characteristic peaks. 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 precision of the characteristic spectral method is good.

[0097] Table 5. Precision Experiment Results (Relative Retention Time)

[0098]

[0099] Table 6. Precision Experiment Results (Relative Peak Area)

[0100]

[0101] 2 Stability

[0102] Take 0.3g of Broussonetia papyrifera fruit granules (batch number: KL1), add 25ml of 10% methanol, weigh, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the test solution. Inject the solution at 0, 4, 12, 16, 20, and 24 hours, 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] Six test solutions were prepared from the granulated Broussonetia papyrifera fruit (batch number: KL1) according to the test solution preparation method. The consistency of the relative retention time and relative peak area of ​​the characteristic peaks was examined. The results are shown in Tables 9 and 10. The relative retention time of each characteristic peak was less than 1%, and the RSD of the relative peak area was less than 3%, indicating good repeatability.

[0110] Table 9. Results of repeatability experiments (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 velocities

[0117] The effect of different flow rates on the robustness of the characteristic spectrum was investigated at flow rates of 0.28 ml / min, 0.30 ml / min, and 0.32 ml / min. Results are shown below. Figure 6 The separation effect of each characteristic peak was good in the flow rate range of 0.28 ml / min to 0.32 ml / min, indicating that the method has good robustness and can meet the system applicability requirements with small flow rate variations.

[0118] 4.2 Investigation at different column temperatures

[0119] The effect of different column temperatures on the robustness of the characteristic spectra was investigated at 28℃, 30℃, and 32℃. Results are shown below. Figure 7 The separation effect of each characteristic peak was good in the column temperature range of 28℃ to 32℃, indicating that the method has good robustness and can meet the system applicability requirements with small column temperature variations.

[0120] Comparative example:

[0121] This example examines the separation effect of Broussonetia papyrifera fruit samples under different mobile phase gradients.

[0122] A CORTECS T3 column (150 mm length, 4.6 mm inner diameter, 2.7 μm particle size) was used; acetonitrile or methanol was used as mobile phase A, and 0.2% formic acid solution, 0.2% phosphoric acid solution, or 0.2% trifluoroacetic acid solution was used as mobile phase B, with gradient elution performed according to the conditions in Tables 11–16. Results are shown in [Table data would be inserted here]. Figures 8 to 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] Results analysis: Figure 8 This corresponds to the UPLC characteristic spectrum of the paper mulberry fruit sample under chromatographic condition test1; Figure 9 This corresponds to the UPLC characteristic spectrum of the paper mulberry fruit sample under chromatographic condition test2; Figure 10This corresponds to the UPLC characteristic spectrum of the paper mulberry fruit sample under chromatographic condition test3; Figure 11 The corresponding UPLC characteristic spectrum of the Broussonetia papyrifera fruit sample under chromatographic conditions test4; Figure 12 The corresponding UPLC characteristic spectrum of the paper mulberry fruit sample under chromatographic conditions test5; Figure 13 The corresponding UPLC characteristic chromatograms of the *Broussonetia papyrifera* sample under chromatographic condition test6, i.e., the chromatographic conditions of this invention, are as follows: In the chromatograms obtained under chromatographic conditions test1 to test4 in this comparative example, the resolution of each chromatographic peak is poor. In the chromatogram under chromatographic condition test5, the resolution of each chromatographic peak is better, but the analysis time is longer and the baseline is not stable. The chromatogram obtained under chromatographic condition test6, i.e., the method of this invention, has rich chromatographic information, a shorter analysis time, good peak resolution, and a stable baseline.

Claims

1. A method for constructing a feature map of paper mulberry seeds, characterized by comprising the following steps: (1) Take the paper mulberry fruit, standard decoction or formula granules respectively, add methanol or methanol-water solution, shake well, filter, and the filtrate is the test solution; (2) Take the reference material of paper mulberry fruit, place it in a stoppered conical flask, add water, heat to reflux, concentrate to near dryness, add solvent, sonicate, shake well, filter, and take the filtrate as the reference material solution. (3) Inject the test solution and the reference medicinal material solution into an ultra-high performance liquid chromatograph to obtain characteristic chromatograms; the characteristic chromatograms have 10 common peaks, with 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 ​​for peaks 1 to 5 are 0.41, 0.45, 0.51, 0.57, and 0.80, respectively, and the specified values ​​for peaks 7 to 10 are: 1.07, 1.11, 1.19, 1.33; The ultra-high performance liquid chromatography column: CORTECS T3, specifications: 4.6×150mm, 2.7μm; detection wavelength: 238~258nm; Methanol as mobile phase A, 0.2% trifluoroacetic acid solution as mobile phase B for gradient elution; gradient elution: 0~5min, volume fraction of mobile phase A is 3%, volume fraction of mobile phase B is 97%; 5~13min, volume fraction of mobile phase A changes from 3→7%, volume fraction of mobile phase B changes from 97→93%; 13~28min, volume fraction of mobile phase A changes from 7→9%, volume fraction of mobile phase B changes from 93→81%.

2. The construction method according to claim 1, characterized in that, The processing methods in step (1) include ultrasonication, shaking, or heating reflux.

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

4. The construction method according to claim 1, characterized in that, In step (3), the injection volume of ultra-high performance liquid chromatography is 3µl; the flow rate is 0.95~1.05ml / min; and the column temperature is 28~32℃.

5. A method for establishing a reference map of Broussonetia papyrifera seeds, characterized in that, Includes the following steps: (1) Prepare multiple batches of Broussonetia papyrifera seed test solutions, multiple batches of Broussonetia papyrifera seed standard decoction test solutions, and multiple batches of Broussonetia papyrifera seed formula granule test solutions respectively; (2) The method for constructing the characteristic spectrum of Broussonetia papyrifera fruit according to any one of claims 1 to 4 is used to detect the test solution to obtain the characteristic spectrum of Broussonetia papyrifera fruit, the characteristic spectrum of standard decoction and the characteristic spectrum of Broussonetia papyrifera fruit granules; the preparation of the test solution includes the following steps: take Broussonetia papyrifera fruit, standard decoction or granules respectively, add methanol or methanol aqueous solution for treatment, shake well, filter, and the filtrate is the test solution; (3) Import the obtained feature chromatograms into the Chinese herbal chromatographic fingerprint chromatogram similarity evaluation system to establish a reference chromatogram of Broussonetia papyrifera seed, standard decoction or formula granules.

Images