A method for identifying the production area of psyllium based on UPLC-Q / TOF-MS

Abstract

The present application relates to the technical field of traditional Chinese medicine detection, and particularly relates to a plantago seed origin identification method based on UPLC-Q / TOF-MS. Based on the component information collected by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, combined with UNIFI spectral library retrieval and multivariate statistical analysis method, L-rhamnose, guaiacylglycerol-beta-ferulic acid ester, oleic acid, 5,7-dihydroxychromone, apigenin-7-O-glucoside and plantaginis are determined as key differential compounds for identifying plantago seeds from different origins, and an OPLS-DA identification model is established. The method can be effectively used for identifying and adulterating plantago seeds from different origins, and provides strong technical support for quality control of plantago seed medicinal materials.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine testing technology, specifically to a method for identifying the origin of Plantago asiatica based on UPLC-Q / TOF-MS. Background Technology

[0002] Plantaginis Semen is a plant belonging to the Plantaginaceae family. Plantago asiatica L. ) or flatcar ( Plantago depressa Willd. The dried, mature seeds of Plantago asiatica have the effects of clearing heat and promoting diuresis, eliminating dampness and stopping diarrhea, improving eyesight, and resolving phlegm. Plantago asiatica from Jiangxi Province (commonly known as "Phoenix Eye Plantago") is a traditional Chinese medicine with a long history of cultivation, accounting for over 70% of the national output. Its quality is superior to Plantago asiatica from Northeast China, Sichuan, and Hunan. Due to price differences, there are instances in the market where non-traditional medicinal materials are used to impersonate traditional medicinal materials, or where traditional medicinal materials are mixed with non-traditional medicinal materials.

[0003] Currently, the quality evaluation of Plantago asiatica mainly relies on its appearance and the content determination of indicator components (such as genipin and verbascoside) specified in the Chinese Pharmacopoeia. However, the appearance differences of Plantago asiatica from different origins are subtle, making the identification method based on appearance dependent on personal experience, highly subjective, and unable to identify the adulteration of non-authentic medicinal materials with authentic medicinal materials. Furthermore, Plantago asiatica has a wide geographical coverage, and the main difference in indicator components between different origins lies in their content. Moreover, there are no clear thresholds for the content of indicator components in Plantago asiatica from different origins. This makes it difficult to objectively determine the origin of Plantago asiatica by relying on the detection of a single or a few indicator components, and even more difficult to identify the adulteration of authentic medicinal materials with varying degrees of non-authentic medicinal materials. Existing technology CN116893227A discloses a method for constructing fingerprint spectra of Plantago asiatica and its processed products. This method mainly focuses on distinguishing between raw and processed products (salted and stir-fried), but it still cannot solve the problem of identifying Plantago asiatica from different origins or the adulteration of authentic medicinal materials with varying degrees of non-authentic medicinal materials.

[0004] Therefore, establishing a method that can comprehensively, quickly, and accurately identify the origin of Plantago asiatica from different regions and detect adulteration is of great significance for ensuring the safety of clinical drug use and maintaining market order. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for identifying the origin of psyllium based on UPLC-Q / TOF-MS, which can be effectively used for the identification and doping detection of psyllium from different origins.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for identifying the origin of psyllium husks based on UPLC-Q / TOF-MS, comprising the following steps: S1. Take plantain samples from different origins and prepare test solutions respectively. Prepare a mixed reference solution containing aucubin, genipin, plantainin, 7-hydroxycoumarin, luteolin, verbascoside and rhubarb glycoside. S2. The test solutions and mixed reference solutions of Plantago asiatica from different origins prepared in S1 were separated by ultra-high performance liquid chromatography and detected by high-resolution mass spectrometry using positive and negative ion MS. E The scanning mode was used to obtain total ion chromatograms in both positive and negative ion modes. The acquired UPLC-Q / TOF-MS data were used to extract compound information using UNIFI Portal software. Based on the retention time, accurate molecular ion peak, and secondary mass spectrometry information of the compounds, the components of the tested sample solution were analyzed and identified. S3. Import the mass spectrometry data of Plantago asiatica samples from different origins collected in positive and negative ion modes into Progenesis QI software, import all compound data into Ezinfo 3.0 software to obtain .txt files, and import the obtained files into SIMCA-P 14.1 software for principal component analysis and orthogonal partial least squares-discriminant analysis. S4. Using scatter plots and S-plot analysis, we obtained the first group of differential compounds between Plantago asiatica from Jiangxi and Plantago asiatica from Northeast China, and the second group of differential compounds between Plantago asiatica from Jiangxi and other producing areas. Based on the S-plot VIP>1 and the t-test P<0.05, we took the intersection of the two groups of differential analysis results between Plantago asiatica from Jiangxi and Northeast China, and between Plantago asiatica from Jiangxi and other producing areas, and identified L-rhamnose, guaiacylglycerol-β-ferulate, oleic acid, 5,7-dihydroxychromone, apigenin-7-O-glucoside, and plantagoside as the key differential compounds for identifying Plantago asiatica from different producing areas, and established an OPLS-DA identification model. S5. Prepare the test solution of the psyllium sample according to the method in S1, separate it by ultra-high performance liquid chromatography and detect it by high resolution mass spectrometry according to S2, obtain the mass spectrometry data of the psyllium sample in positive and negative ion modes, and import it into the OPLS-DA model established in S4 for identification.

[0007] Furthermore, the preparation method of the test solution in S1 is as follows: take plantain samples from different origins, crush them, sieve them, weigh them accurately, add 70% methanol aqueous solution, extract them by ultrasound, make up the lost weight after cooling, centrifuge them, take the supernatant and filter them to obtain the test solution.

[0008] Furthermore, ultrasonic extraction was performed at a power of 500W and a frequency of 40kHz for 30 minutes.

[0009] Furthermore, the psyllium samples in S1 include psyllium produced in Jiangxi, Heilongjiang, Liaoning, Sichuan, Hebei, Shaanxi, Anhui, and Jiangsu.

[0010] Furthermore, in S2, the chromatographic conditions used for ultra-high performance liquid chromatography separation are as follows: Column: Waters ACQUITY UPLC BEH C 18 Chromatographic column; Mobile phase A: Methanol; Mobile phase B: 0.1% formic acid aqueous solution; Gradient elution program: 0–1 min, 8% A; 1–10 min, 8%–50% A; 10–25 min, 50%–95% A; 25–28 min, 95% A; Column temperature: 40℃; Flow rate: 0.3 mL / min; Injection volume: 2 μL.

[0011] Furthermore, in S2, the mass spectrometry detection conditions used are as follows: the electrospray ionization source employs both positive and negative ion modes for MS. E scanning.

[0012] Furthermore, in positive ion mode, the capillary voltage is 3.0 kV, the ion source temperature is 110 °C, and the desolventizing temperature is 450 °C; in negative ion mode, the capillary voltage is 2.5 kV, the ion source temperature is 100 °C, and the desolventizing temperature is 350 °C; the source drift value is 80 V, the cone voltage is 40 V, and the sampling cone voltage is 40 V; the cone gas flow rate is 50 L / h, and the desolventizing gas flow rate is 800 L / h; the low collision energy is 6 V, and the high collision energy is set to 15 V–45 V; the mass scan range is 50–1200 m / z, and the scan time is 0.5 seconds; real-time mass correction is performed using leucine enkephalin, with the corrected ion in positive ion mode being m / z 556.2771 and the corrected ion in negative ion mode being m / z 554.2615.

[0013] Furthermore, the differential compounds in Group I of S4 are: L-rhamnose, aucubin, guaiacylglycerol-β-ferulate, oleic acid, 5,7-dihydroxychromone, 6-hydroxyluteolin, apigenin-7-O-glucoside, rhodioloside, senna-7-O-β-D-glucoside, ursolic acid, and psyllium glycoside.

[0014] Furthermore, the differential compounds in Group II of S4 are: L-rhamnose, guaiacylglycerol-β-ferulate, oleic acid, 5,7-dihydroxychromone, apigenin-7-O-glucoside, sorbitol, and psyllium glycoside.

[0015] Furthermore, the identification method in S5 is to use the cluster heatmap analysis in the OPLS-DA model to cluster the Jiangxi origin group, the Northeast origin group, and other origin groups into three categories respectively. The origin is directly determined based on the clustering results. When the sample to be tested deviates from the Jiangxi group clustering area, the Northeast group clustering area, and other other group clustering areas in the OPLS-DA model score map, it is determined that there is doping.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. This application provides a method for identifying the origin of Plantago asiatica based on UPLC-Q / TOF-MS, using methanol-water solution as the extraction solvent, preparing the test solution by ultrasonic-assisted extraction, and employing a Waters ACQUITY UPLC BEH C... 18 The chromatographic column was used for gradient elution with methanol / 0.1% formic acid in water, and MS was performed using both positive and negative ion modes. E The scanning process can simultaneously collect tens of thousands of component information. Using a self-built chemical component database of Plantago asiatica, a chemical component data table of Plantago asiatica is obtained. By grouping Plantago asiatica from different production areas and combining multivariate statistical methods to establish an OPLS-DA model, combined with S-polt plots and t-tests, two groups of differential compounds are screened out. The intersection of the analysis results of the two groups of differential compounds is then taken to finally obtain 6 key differential compounds with strong origin-specific characteristics, and an OPLS-DA identification model is established based on this.

[0017] 2. This method combines non-targeted metabolomics and multivariate statistical analysis, avoiding the subjectivity of manually selecting indicators. This identification method boasts high reliability and speed, effectively identifying Plantago asiatica from Jiangxi, Northeast China, and other non-authentic Plantago asiatica medicinal materials. It can also detect small amounts of Plantago asiatica from other origins mixed with Jiangxi Plantago asiatica; a mixing concentration of 10% or more is sufficient for the model to identify it. This solves the current challenge of detecting small amounts of Plantago asiatica from other origins mixed with authentic Jiangxi Plantago asiatica in the market. The method provided in this application can be effectively used for the identification and adulteration detection of Plantago asiatica from different origins, providing strong technical support for the quality control of Plantago asiatica medicinal materials. Attached Figure Description

[0018] Figure 1 This is the BPI chromatogram of the mixed reference solution in positive ion mode; where peak 1 (t) R Peak 2 (t = 2.29 min) is aucubin; peak 3 (t = 2.29 min) is aucubin. R =3.56min) Genipin glycosides; Peak 3 (t R =7.58min) is plantagoside; peak 4 (t R =8.42min) is 7-hydroxycoumarin; peak 5 (t R=8.60min) is luteolin; peak 6 (t R =9.24min) is verbascoside; peak 7 (t R =9.70min) is laccoside; Figure 2 The image shows the BPI chromatogram of the test solution; where A represents positive ion mode and B represents negative ion mode. Figure 3 PCA analysis chromatograms of Plantago asiatica from different origins; where A represents positive ion mode and B represents negative ion mode. Figure 4 A multivariate statistical analysis graph of Plantago asiatica from Jiangxi and Plantago asiatica from Northeast China; In the figure, A is the OPLS-DA analysis graph in positive ion mode; B is the OPLS-DA model cross-validation graph in positive ion mode; C is the S-plot in positive ion mode (components with VIP>1.0 are in red); D is the OPLS-DA analysis graph in negative ion mode; E is the OPLS-DA model cross-validation graph in negative ion mode; and F is the S-plot in negative ion mode (components with VIP>1.0 are in red). Figure 5 A multivariate statistical analysis graph of Plantago asiatica from Jiangxi and Plantago asiatica from other producing areas; In the figure, A is the OPLS-DA analysis graph in positive ion mode; B is the OPLS-DA model cross-validation graph in positive ion mode; C is the S-plot in positive ion mode (components with VIP>1.0 are in red); D is the OPLS-DA analysis graph in negative ion mode; E is the OPLS-DA model cross-validation graph in negative ion mode; and F is the S-plot in negative ion mode (components with VIP>1.0 are in red). Figure 6 Box plots showing the six key differentiating components of Plantago asiatica from different origins; Figure 7 Cluster heatmap of six key differential components of plantain seeds from different origins; among them, 1# is plantain glycoside; 2# is oleic acid; 3# is 5,7-dihydroxychromone; 4# is guaiacol glycerol-β-ferulate; 5# is L-rhamnose; and 6# is apigenin-7-O glucoside. Figure 8 Correlation analysis diagram of six key differential components of Plantago asiatica from different origins; Figure 9 OPLS-DA analysis chromatogram of a Jiangxi Plantago asiatica sample doped with Plantago asiatica from Northeast China; Figure 10 OPLS-DA analysis chromatogram of Plantago asiatica from Jiangxi Province mixed with Plantago asiatica from other producing areas; Figure 11OPLS-DA analysis chromatograms of Plantago asiatica samples from Jiangxi and Northeast China in the validation group; Figure 12 OPLS-DA analysis chromatograms of Plantago asiatica from Jiangxi and other Plantago asiatica samples from other producing areas in the validation group. Detailed Implementation

[0019] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Unless otherwise specified, all methods described herein are conventional methods, and all raw materials described herein are available from publicly available commercial sources.

[0021] Example 1: UPLC-Q / TOF-MS of chemical components of Psyllium husk E Qualitative identification 1. Instruments and Materials: The ultra-high performance liquid chromatography system included a Waters Acquity high performance liquid chromatograph, a SampleManager FTN autosampler, a Binary Solvent Manager binary pump, and a WAT-S4LV1467 column oven (Waters Corporation, USA); a Xevo G2-XS Q / TOF mass spectrometer equipped with Masslynx V4.2 analysis software, UNIFI Portal software, Progenesis QI software, and the TCM Chinese database (Waters Corporation, USA), and a KQ-500E CNC ultrasonic extractor (Kunshan Ultrasonic Instrument Co., Ltd.); an MS205DU electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); and a low-speed benchtop large-capacity multi-tube centrifuge (Shanghai Anting Scientific Instrument Factory).

[0022] Methanol was mass spectrometry grade (Merck, Germany); formic acid was mass spectrometry grade (Thermo Fisher Scientific (China) Co., Ltd.); purified water was (Hangzhou Wahaha Drinking Water Co., Ltd.).

[0023] 2. Source of reference standards: The reference standards required in the experiment, including aucubin (batch number AFCD1301), genipin (batch number AFCL2656), 7-hydroxycoumarin (batch number AFCI1953), luteolin (batch number AZCJ2517), verbascoside (batch number AZDF0707), and rutin (batch number AFEB2101), were purchased from Chengdu Efa Biotechnology Co., Ltd.; and psyllium glycoside (batch number CFS202601) was purchased from Wuhan Tianzhi Biotechnology Co., Ltd.

[0024] 3. Sample Source: The Plantaginis seeds used in the experiment were all directly purchased from the place of origin and identified by Professor Fu Xiaomei of the Department of Traditional Chinese Medicine Identification, School of Pharmacy, Jiangxi University of Traditional Chinese Medicine as Plantaginis Semen, a plant of the Plantaginaceae family. Plantago asiatica L. or flatcar front Plantago depressa Dried mature seeds of Willd. The specimens are preserved in the research laboratory of Jiangxi Provincial Institute for Drug Control. Information on 71 batches of Plantago asiatica samples used for modeling is shown in Table 1, and information on 17 batches of Plantago asiatica samples used for validation is shown in Table 2.

[0025] Table 1: Information Table of Modeled Carpenter's Forehead Samples

[0026] Table 2: Information on the Psyllium Fiber Samples

[0027] 3. Preparation of reference solutions: Accurately weigh 10 mg each of aucubin, genipin, plantago asiatica, 7-hydroxycoumarin, luteolin, verbascoside, and rutin, and place them in separate 10 mL volumetric flasks. Dissolve each in methanol and shake well to prepare a single reference solution with a mass concentration of approximately 1.00 mg / mL. Accurately pipette 100 μL of each of the above reference solutions into the same 10 mL volumetric flask, shake well, dilute to the mark with 70% methanol solution, and shake well to prepare a mixed reference solution with a mass concentration of approximately 10.00 μg / mL. Filter the solution through a 0.22 μm microporous membrane to obtain the final solution.

[0028] 4. Preparation of test solution: Accurately weigh 0.5 g of sample powder (passed through a No. 3 sieve), place it in an Erlenmeyer flask, add 20 mL of 70% methanol, extract by sonication (power: 500 W, frequency: 40 kHz) for 30 min, cool, replenish the lost weight with 70% methanol, centrifuge, and filter the supernatant through a 0.22 μm filter membrane to obtain the test solution.

[0029] 5. Chromatographic conditions: Waters ACQUITY UPLC BEH C was used. 18 The chromatographic column was used with mobile phase A being methanol and mobile phase B being 0.1% formic acid aqueous solution. The gradient elution program was as follows: 0–1 min, 8% A; 1–10 min, 8%–50% A; 10–25 min, 50%–95% A; 25–28 min, 95% A; column temperature 40℃; flow rate 0.3 mL / min; and injection volume 2 μL.

[0030] 6. Mass spectrometry conditions: Electrospray ionization (ESI) source, positive and negative ion MS. EScanning modes: In positive ion mode, the capillary voltage is 3.0 kV, the ion source temperature is 110℃, and the desolvation temperature is 450℃. In negative ion mode, the capillary voltage is 2.5 kV, the ion source temperature is 100℃, and the desolvation temperature is 350℃. Other main parameter settings are as follows: source drift value is 80V, orifice voltage is 40V, and sampling cone voltage is 40V. Orifice gas flow rate is 50 L / h, and desolvation gas flow rate is 800 L / h. Low collision energy is 6V, and high collision energy is set to 15~45V. The mass scan range is 50~1200 m / z, and the scan time is 0.5 seconds. Real-time mass correction is performed using leucine enkephalin; the correction ion is m / z 556.2771 in positive ion mode and m / z 554.2615 in negative ion mode. The search was conducted using the UNIFI database of chemical components of traditional Chinese medicine and the self-built database of plantago seed components (the self-built database of plantago seed components was established based on Waters' TCM (Chinese herbal medicine chemical components) database and publicly available literature data).

[0031] 7. Qualitative analysis of chemical components of Plantago asiatica by UPLC-Q / TOF-MS The mixed reference solution and the Plantago asiatica test solution were analyzed by positive and negative ion MS using a UPLC-Q / TOF-MS instrument. E Scanning was performed to obtain total ion chromatograms in both positive and negative ion modes, see [link / reference]. Figure 1 and Figure 2 Based on the Traditionall TCM database included in the UNIFI Portal software and the Plantago asiatica database built by our research group, preliminary mass spectrometry data analysis results were obtained. The possible compound list provided by the software was identified by examining the compound fragmentation patterns and related compound mass spectrometry data, ultimately identifying 87 compounds. Details of compound retention times, quasi-molecular ions, and fragment ions are shown in Table 3.

[0032] Table 3: Chemical Composition of Plantago asiatica identified by UPLC-Q / TOF-MS method

[0033] Note: For comparison with reference standards.

[0034] Example 2: Establishment of a multivariate statistical identification method for Psyllium husks from different origins using UPLC-Q / TOF MS. 1. Multivariate statistical analysis Data from 71 batches of Plantago asiatica samples from different origins, collected under both positive and negative ion modes, were imported into Progenesis QI software. All compound data were then imported into Ezinfo 3.0 software to obtain .txt files. The resulting files were then imported into SIMCA-P 14.1 software for multivariate statistical analysis, including PCA (principal component analysis) and OPLS-DA (orthogonal partial least squares-discriminant analysis).

[0035] PCA analysis results are as follows: Figure 3 The results showed that R²=0.575 and Q²=0.505 in the positive ion mode and R²=0.571 and Q²=0.504 in the negative ion mode. This indicates that the three groups of Plantago asiatica samples from Jiangxi, Northeast China and other production areas can be clearly distinguished.

[0036] OPLS-DA analysis results are as follows Figure 4 and Figure 5 As can be seen from the figure, the *Phyllanthus* samples from Jiangxi and Northeast China, as well as those from Jiangxi and other regions, are clearly distinguishable, and the model is reliable (both R² and Q² values ​​are high, and the replacement test is qualified). The two groups of *Phyllanthus* samples from Jiangxi and Northeast China show significant differences, especially under positive ion mode (…). Figure 4 (A)R 2 X=0.611, R 2 Y=0.984, Q 2 =0.975, in negative ion mode ( Figure 4 D)R 2 X=0.606, R 2 Y=0.949, Q 2 =0.94, indicating that the model has high reliability and strong predictive ability. (200 permutation test chart) Figure 4 China B and Figure 4 (E) shows that the R values ​​of the two groups of samples under positive and negative ion modes are... 2 and Q 2 The slopes of all of them are greater than 0, and the regression line Q 2 An intercept of <0 with the Y-axis indicates that the OPLS-DA model is reliable. Scatter score plot ( Figure 4 C and Figure 4 The S-plot (F) represents the differentially expressed components between groups. Compounds with significant differences (VIP > 1) can be screened out, resulting in differentially expressed compounds between Plantago asiatica from Jiangxi and Plantago asiatica from Northeast China. Eleven differentially expressed compounds were identified: L-rhamnose, aucubin, guaiacylglycerol-β-ferulate, oleic acid, 5,7-dihydroxychromone, 6-hydroxyluteolin, apigenin-7-O-glucoside, rhodioloside, senna-7-O-β-D-glucoside, ursolic acid, and psyllium glycoside.

[0037] There were significant differences between the two groups of samples of Plantago asiatica from Jiangxi and those from other regions, especially under positive ion mode. Figure 5 In equation A), R² = 0.452, R... 2 Y=0.897, Q²=0.869, ( Figure 5 In the negative ion mode (D), R² = 0.573, R 2 Y=0.827, Q²=0.796, indicating that the model has high reliability and strong predictive ability. The figure shows a significant difference between the Jiangxi-produced *Plantago asiatica* and other-produced *Plantago asiatica* samples, as shown in the 200-times permutation test figure (…). Figure 5 China B and Figure 5 (E) shows that the R values ​​of the two groups of samples under positive and negative ion modes are... 2 and Q 2 The slopes of all of them are greater than 0, and the regression line Q 2 An intercept of <0 with the Y-axis indicates that the OPLS-DA model is reliable. Scatter score plot ( Figure 5 C and Figure 5 The S-plot (F) indicates that compounds showing significant differences (VIP > 1) between groups were selected. Seven compounds were identified as differentially expressed: L-rhamnose, guaiacylglycerol-β-ferulate, oleic acid, 5,7-dihydroxychromone, apigenin-7-O-glucoside, sorbitol, and psyllium glycoside.

[0038] 2. Screening of key differentiating compounds By using S-plots (VIP>1) and combining them with t-tests (P<0.05), the intersection of the difference analysis results between Jiangxi and Northeast China, and between Jiangxi and other producing areas, yielded 6 common key differential compounds, as detailed in Table 4.

[0039] Table 4: Key Differentiating Compounds of Plantago asiatica from Different Origins

[0040] 3. Analysis of key differentiating compounds Box plot analysis ( Figure 6 The findings showed that L-rhamnose was found to be the lowest in products from Northeast China; guaiac glyceryl-β-ferulate was found to be the highest in other products; oleic acid, psyllium glycoside, and 5,7-dihydroxychromone were found to be the highest in products from Northeast China; and apigenin-7-O-glucoside was found to be the highest in products from Jiangxi Province.

[0041] Cluster heatmap analysis ( Figure 7 The study found that six key differentially expressed compounds clearly clustered samples from the three origins into three categories, validating their origin specificity. Furthermore, the differences in the content of these six key differentially expressed compounds further validated the results of the boxplot analysis.

[0042] Correlation analysis ( Figure 8 The study found that oleic acid, psyllium glycoside, and 5,7-dihydroxychromone showed a strong positive correlation.

[0043] Example 3: Application of methods for identifying plantain seeds from different origins 1. Sample source and preparation The sample information used in the verification experiment is the same as in Table 1. The preparation method of the reference solution is the same as that under "3. Preparation method of reference solution" in Example 1; the preparation method of the test solution is the same as that under "4. Preparation method of test solution" in Example 1.

[0044] Preparation of doped samples: Take the powder of Plantago asiatica from Jiangxi Province, and add 10%, 20%, 80% and 90% of the powder of Plantago asiatica from Northeast China, and 10%, 20%, 80% and 90% of the powder of Plantago asiatica from other places by mass, respectively. After mixing evenly, prepare the test solution according to the above method.

[0045] 2. Detection and Data Processing The chromatographic and mass spectrometry detection conditions were the same as those in "5. Chromatographic conditions" and "6. Mass spectrometry conditions" in Example 1. The OPLS-DA model established by the six key markers in Example 2 was used to analyze the psyllium husks from Jiangxi Province that were doped with small amounts (10%, 20%) and large amounts (80%, 90%) of psyllium husks from Northeast China, as well as psyllium husks from other origins.

[0046] in, Figure 9 This is an OPLS-DA analysis chromatogram of a Jiangxi plantago seed sample doped with plantago seed from Northeast China. Figure 10 The image shows the OPLS-DA analysis of Plantago asiatica samples from Jiangxi Province that are doped with Plantago asiatica from other producing areas. As can be seen from the image, the Jiangxi group can be clearly distinguished from the Northeast group and from other groups. Moreover, the doped samples can be significantly deviated from the clustering area of ​​pure Plantago asiatica in the score map, which proves that this method can be effectively used to identify Plantago asiatica from different producing areas and can detect doping of more than 10%.

[0047] 3. Verification of the identification of plantain samples from different origins The sample information used in the verification experiment is shown in Table 2. The preparation method of the reference solution is the same as that under "3. Preparation method of reference solution" in Example 1; the preparation method of the test solution is the same as that under "4. Preparation method of test solution" in Example 1.

[0048] The chromatographic and mass spectrometry detection conditions were the same as those in "5. Chromatographic conditions" and "6. Mass spectrometry conditions" in Example 1. The OPLS-DA model established by the six key biomarkers in Example 2 was used to analyze the Plantago asiatica samples from Jiangxi and Northeast China in the validation group, as well as the Plantago asiatica samples from Jiangxi and other origins in the validation group.

[0049] in, Figure 11 OPLS-DA analysis chromatograms of Plantago asiatica samples from Jiangxi and Northeast China were used for the validation group. Figure 12 The OPLS-DA analysis chromatograms of Plantago asiatica samples from Jiangxi and other production areas in the validation group are shown. As can be seen from the figure, Plantago asiatica from Jiangxi in the validation group can be clearly distinguished from Plantago asiatica from Northeast China, and Plantago asiatica from Jiangxi in the validation group can be distinguished from Plantago asiatica from other production areas.

[0050] Conclusion: The method of the present invention is simple to operate and accurate in yielding results. It can be effectively used for the identification and adulteration detection of Plantago asiatica from different origins, providing strong technical support for the quality control of Plantago asiatica medicinal materials.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and not to limit them; although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of this application or equivalent substitutions can be made to some technical features, all of which should be covered within the scope of the technical solutions claimed in this application.

Claims

1. A method for identifying the origin of psyllium husk based on UPLC-Q / TOF-MS, characterized in that, Includes the following steps: S1. Take plantain samples from different origins and prepare test solutions respectively. Prepare a mixed reference solution containing aucubin, genipin, plantainin, 7-hydroxycoumarin, luteolin, verbascoside and rhubarb glycoside. S2. The test solutions and mixed reference solutions of Plantago asiatica from different origins prepared in S1 were separated by ultra-high performance liquid chromatography and detected by high-resolution mass spectrometry using positive and negative ion MS. E The scanning mode was used to obtain total ion chromatograms in both positive and negative ion modes. The acquired UPLC-Q / TOF-MS data were used to extract compound information using UNIFI Portal software. Based on the retention time, accurate molecular ion peak, and secondary mass spectrometry information of the compounds, the components of the tested sample solution were analyzed and identified. S3. Import the mass spectrometry data of Plantago asiatica samples from different origins collected in positive and negative ion modes into ProgenesisQI software, import all compound data into Ezinfo 3.0 software to obtain .txt files, and import the obtained files into SIMCA-P 14.1 software for principal component analysis and orthogonal partial least squares-discriminant analysis. S4. Using scatter plots and S-plot analysis, we obtained the first group of differential compounds between Plantago asiatica from Jiangxi and Plantago asiatica from Northeast China, and the second group of differential compounds between Plantago asiatica from Jiangxi and other producing areas. Based on the S-plot VIP>1 and the t-test P<0.05, we took the intersection of the two groups of differential analysis results between Plantago asiatica from Jiangxi and Northeast China, and between Plantago asiatica from Jiangxi and other producing areas, and identified L-rhamnose, guaiacylglycerol-β-ferulate, oleic acid, 5,7-dihydroxychromone, apigenin-7-O-glucoside, and plantagoside as the key differential compounds for identifying Plantago asiatica from different producing areas, and established an OPLS-DA identification model. S5. Prepare the test solution of the psyllium sample according to the method in S1, separate it by ultra-high performance liquid chromatography and detect it by high resolution mass spectrometry according to S2, obtain the mass spectrometry data of the psyllium sample in positive and negative ion modes, and import it into the OPLS-DA model established in S4 for identification.

2. The method for identifying the origin of psyllium husk based on UPLC-Q / TOF-MS according to claim 1, characterized in that, The preparation method of the test solution in S1 is as follows: take Plantago asiatica samples from different origins, crush them, sieve them, weigh them accurately, add 70% methanol aqueous solution, extract them by ultrasound, make up the lost weight after cooling, centrifuge them, take the supernatant and filter them to obtain the test solution.

3. The method for identifying the origin of psyllium husk based on UPLC-Q / TOF-MS according to claim 1, characterized in that, The psyllium samples in S1 include those from Jiangxi, Heilongjiang, Liaoning, Sichuan, Hebei, Shaanxi, Anhui, and Jiangsu.

4. The method for identifying the origin of psyllium husk based on UPLC-Q / TOF-MS according to claim 1, characterized in that, In S2, the chromatographic conditions used for ultra-high performance liquid chromatography separation are as follows: Column: Waters ACQUITY UPLC BEH C 18 Chromatographic column; Mobile phase A: Methanol; Mobile phase B: 0.1% formic acid aqueous solution; Gradient elution program: 0–1 min, 8% A; 1–10 min, 8%–50% A; 10–25 min, 50%–95% A; 25–28 min, 95% A; Column temperature: 40℃; Flow rate: 0.3 mL / min; Injection volume: 2 μL.

5. The method for identifying the origin of psyllium husk based on UPLC-Q / TOF-MS according to claim 1, characterized in that, In S2, the mass spectrometry detection conditions employed include using both positive and negative ion modes for the electrospray ionization source. E scanning.

6. The method for identifying the origin of psyllium husk based on UPLC-Q / TOF-MS according to claim 5, characterized in that, In positive ion mode, the capillary voltage is 3.0 kV, the ion source temperature is 110℃, and the desolventizing temperature is 450℃; in negative ion mode, the capillary voltage is 2.5 kV, the ion source temperature is 100℃, and the desolventizing temperature is 350℃; the source drift value is 80 V, the cone voltage is 40 V, and the sampling cone voltage is 40 V; the cone gas flow rate is 50 L / h, and the desolventizing gas flow rate is 800 L / h; the low collision energy is 6 V, and the high collision energy is set to 15 V~45 V; the mass scan range is 50~1200 m / z, and the scan time is 0.5 seconds; real-time mass correction is performed using leucine enkephalin, with the corrected ion in positive ion mode being m / z 556.2771 and the corrected ion in negative ion mode being m / z 554.2615.

7. The method for identifying the origin of psyllium husk based on UPLC-Q / TOF-MS according to claim 1, characterized in that, The differential compounds in Group I of S4 are: L-rhamnose, aucubin, guaiacylglycerol-β-ferulate, oleic acid, 5,7-dihydroxychromone, 6-hydroxyluteolin, apigenin-7-O-glucoside, rhodioloside, senna-7-O-β-D-glucoside, ursolic acid, and psyllium glycoside.

8. The method for identifying the origin of psyllium husk based on UPLC-Q / TOF-MS according to claim 1, characterized in that, The differential compounds in Group II of S4 are: L-rhamnose, guaiacylglycerol-β-ferulate, oleic acid, 5,7-dihydroxychromone, apigenin-7-O-glucoside, sorbitol, and psyllium glycoside.

9. The method for identifying the origin of psyllium husk based on UPLC-Q / TOF-MS according to claim 1, characterized in that, The identification method in S5 is to use the cluster heatmap analysis in the OPLS-DA model to cluster the Jiangxi origin group, the Northeast origin group and other origin groups into three categories respectively. The origin is directly determined based on the clustering results. When the sample to be tested deviates from the Jiangxi group clustering area, the Northeast group clustering area and other other group clustering areas in the OPLS-DA model score map, it is determined that there is doping.

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