Fingerprint spectrum construction method of prismatomeria tetrandra and application thereof

Abstract

The invention discloses a prismatomeria tetrandra fingerprint spectrum construction method and application thereof. Belongs to the technical field of medicine. The construction method comprises the following steps: taking scopoletin, coumarin, methyl isoalizarin-1-methyl ether and methyl isoalizarin reference substances, and dissolving the reference substances in a solvent to prepare a reference substance solution; taking a prismatomeris tetrandra sample, adding an extraction solvent for extraction, and filtering an extracting solution to prepare a test solution; respectively carrying out high performance liquid chromatography detection on the reference substance solution and the test solution, and constructing the fingerprint spectrum of the prismatomeria tetrandra. Compared with the prior art, the method has the beneficial effects that the fingerprint spectrum of the prismatomeria tetrandra is established by adopting an HPLC (High Performance Liquid Chromatography) method and is used for identifying and controlling the quality of the prismatomeria tetrandra medicinal material, so that a standard for comprehensively and effectively controlling the quality of the prismatomeria tetrandra medicinal material is expected to be established.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and more specifically to a method for constructing a fingerprint of Prismatomeris tetrandra roots and its application. Background Art

[0002] Prismatomeris tetrandra roots are the roots of Prismatomeris tetrandra (Roxb.) K. Schum of the Rubiaceae family, and have the effects of cooling blood to stop bleeding, strengthening tendons and bones, promoting diuresis to remove jaundice, and removing stasis to generate new tissues. They are mainly used for acute and chronic hepatitis with damp-heat accumulation and spleen deficiency and collateral stasis. Research shows that the extract of Prismatomeris tetrandra roots has significant effects on anti-silicosis fibrosis and anti-liver fibrosis.

[0003] There is little research on the quality control method of Prismatomeris tetrandra roots. Currently, there are only detection items such as character and microscopic identification. Its fingerprint has not been established yet, the content determination item is lacking, and a comprehensive and effective quality standard for controlling Prismatomeris tetrandra roots has not been formed. The fingerprint of traditional Chinese medicine is a comprehensive and quantifiable identification method. Establishing the fingerprint of Prismatomeris tetrandra roots can more comprehensively reflect the types and quantities of chemical components in Prismatomeris tetrandra roots, and then conduct an overall description and evaluation of the quality of Prismatomeris tetrandra root materials. The fingerprint should embody three basic principles: systematicness, characteristicness, and stability. The technical difficulty is how to comprehensively reflect the information of chemical components in Prismatomeris tetrandra roots and the identification of common peaks.

[0004] In summary, how to provide a fingerprint of Prismatomeris tetrandra roots and an HPLC research method for multi-component quantification is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method for constructing a fingerprint of Prismatomeris tetrandra roots and its application.

[0006] The present invention respectively uses different methods such as cold maceration, heat reflux, and ultrasound, as well as different solvents such as methanol and ethanol to optimize the extraction method of Prismatomeris tetrandra root samples. At the same time, the chemical components of Prismatomeris tetrandra roots are systematically studied, and components such as rubiadin-1-methyl ether and rubiadin are separated and identified, laying a foundation for the identification of common peaks.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] A method for constructing a fingerprint of Prismatomeris tetrandra roots, comprising the following steps:

[0009] (1) Take scopoletin, coumarin, rubiadin-1-methyl ether, and rubiadin reference substances, dissolve them with a solvent, and prepare a reference substance solution;

[0010] (2) Take Prismatomeris tetrandra root samples, add an extraction solvent for extraction, filter the extract, and prepare a test solution;

[0011] (3) Perform high performance liquid chromatography (HPLC) detection on the reference substance solution and the test sample solution respectively to construct the fingerprint of Xanthorrhizae Radix.

[0012] Further, in the step (1), the solvent is methanol.

[0013] Further, in the step (1), the mass concentrations of scopoletin, coumarin, rubiadin-1-methyl ether, and rubiadin reference substances in the reference substance solution are 0.0518, 0.0268, 10.5844, and 0.2610 mg / mL in sequence.

[0014] Further, in the step (2), methanol is used as the extraction solvent for ultrasonic extraction.

[0015] Further, the specific operation of the step (2) is: take the powder of Xanthorrhizae Radix, add methanol solution, the material-liquid ratio is 2:25 mg / mL, perform ultrasonic extraction for 40 min, and then filter through a 0.22 μm microporous filter membrane;

[0016] The ultrasonic conditions are: 100 W, 40 kHz.

[0017] Further, in the step (3),

[0018] The chromatographic column is a C18 chromatographic column;

[0019] The detection wavelength is 277 nm;

[0020] The column temperature is 30 °C,

[0021] The injection volume is 10 μL.

[0022] Further, in the step (3),

[0023] The mobile phase: acetonitrile is used as mobile phase A, and water is used as mobile phase B;

[0024] The mobile phase elution gradient is: 0 - 5 min, 20% A; 5 - 20 min, 20% - 42% A; 20 - 45 min, 42% A; 45 - 50 min, 42% - 60% A; 50 - 55 min, 60% - 95% A; 55 - 60 min, 95% A.

[0025] The fingerprint constructed by the above construction method.

[0026] The application of the above fingerprint in the identification and quality control of Xanthorrhizae Radix medicinal materials.

[0027] It can be seen from the above technical solutions that compared with the prior art, the beneficial effects obtained by the present invention are:

[0028] The present invention uses HPLC method to establish the fingerprint of Radix Xanthii, which is used for the identification and quality control of Radix Xanthii medicinal materials, in order to establish a comprehensive and effective standard for controlling the quality of Radix Xanthii medicinal materials. Description of the Drawings

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0030] Figure 1 It is the HPLC superposition chromatogram (S1 - S13) and the control chromatogram (R) of 13 batches of Radix Xanthii samples in Example 1 of the present invention;

[0031] Figure 2 It is the HPLC chromatogram of the mixed reference substance solution in Example 1 of the present invention. Among them, peak 2 represents scopoletin, peak 5 represents coumarin, peak 10 represents methylisocinchonain - 1 - methyl ether, and peak 12 represents methylisocinchonain;

[0032] Figure 3 It is the CA dendrogram of 13 batches of Radix Xanthii samples in Example 1 of the present invention;

[0033] Figure 4 It is the PCA score chart of 13 batches of Radix Xanthii samples in Example 1 of the present invention;

[0034] Figure 5 It is the OPLS - DA score chart of 13 batches of Radix Xanthii samples in Example 1 of the present invention;

[0035] Figure 6 It is the OPLS - DA VIP value of 13 batches of Radix Xanthii samples in Example 1 of the present invention. Detailed Embodiments

[0036] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0037] The medicaments required in the present invention are conventional experimental medicaments, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods, which will not be elaborated here one by one.

[0038] Example 1

[0039] (1) Preparation of the test solution

[0040] The 13 batches of medicinal materials were identified as Radix Xanthii, and the specific origin information is shown in Table 1.

[0041] Table 1 Information of Radix Xanthii samples

[0042]

[0043]

[0044] The Radix Xanthii samples of S1 - S13 were respectively ground into powders, passed through a No. 4 sieve. 2.0 g of the Radix Xanthii powder was precisely weighed and placed in a stoppered conical flask. 25 mL of methanol was precisely added, weighed, extracted by ultrasonic wave (100 W, 40 kHz) for 40 min, left to stand at room temperature, made up the weight with methanol, and filtered through a 0.22 μm microporous membrane to obtain the solution.

[0045] (2) Preparation of reference substance solution

[0046] Appropriately weighed scopoletin, coumarin, rubiadin - 1 - methyl ether, and rubiadin reference substances, precisely weighed, and made into a mixed reference substance solution containing 0.0518, 0.0268, 10.5844, and 0.2610 mg per 1 mL with methanol. Filtered and took the filtrate as the reference substance solution.

[0047] (3) Chromatographic conditions

[0048] The chromatographic column was a Techmate ST C18 column (4.6 mm × 250 mm, 5 μm); the mobile phase was acetonitrile (A) - water (B), with gradient elution (0 - 5 min, 20% A; 5 - 20 min, 20% - 42% A; 20 - 45 min, 42% A; 45 - 50 min, 42% - 60% A; 50 - 55 min, 60% - 95% A; 55 - 60 min, 95% A), the flow rate was 1.0 mL / min; the detection wavelength was 277 nm; the column temperature was 30 °C, and the injection volume was 10 μL.

[0049] (4) Fingerprint study

[0050] (41) Methodology investigation

[0051] Using rubiadin - 1 - methyl ether of peak 10 as the reference peak, taking the solution of the test sample of Radix Xanthii (S1), injecting continuously for 6 times, calculating the RSDs of the relative retention time and relative peak area of each common peak were 0.13% - 0.56% and 1.49% - 2.75% respectively, indicating good precision of the instrument.

[0052] Take the test solution of Huanggen (S1), inject samples for determination at 0, 2, 4, 8, 12, and 24 h after preparation, and calculate that the RSDs of the relative retention times and relative peak areas of each common peak are 0.11% - 0.48% and 1.28% - 2.61% respectively, indicating that the test solution is stable within 24 h.

[0053] Take 6 portions of the same batch of Huanggen test sample (S1), prepare the test solution according to the above method, inject samples for determination, and calculate that the RSDs of the relative retention times and relative peak areas of each common peak are 0.34% - 0.77% and 1.13% - 2.97% respectively, indicating that this method has good repeatability.

[0054] (42) Establishment of fingerprint and similarity evaluation

[0055] Using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition)", with the chromatogram of sample S1 as the reference chromatogram and the time window width of 0.01 min, generate the superimposed chromatogram and reference chromatogram through multi-point calibration and automatic matching, as shown in Figure 1 . A total of 12 common peaks were determined. By comparing with the reference substances ( Figure 2 ), 4 components were identified, namely scopoletin (12.395 min), coumarin (18.235 min), rubiadin-1-methyl ether (34.942 min), and rubiadin (53.987 min).

[0056] The similarity evaluation results are shown in Table 2. The similarities of 13 batches of Huanggen samples to the reference chromatogram are 0.937 - 0.996. From the perspective of similarity, the similarities of 13 batches of samples are relatively high, all above 0.900.

[0057] Table 2 Similarity evaluation results of 13 batches of Huanggen samples

[0058]

[0059] (43) Cluster analysis (CA)

[0060] Import the fingerprint data of 13 batches of Huanggen samples into SPSS 27.0 software, use the between-group linkage method and the average Euclidean distance method to perform cluster analysis on the samples, and the results are shown in Figure 3 . The 13 batches of Huanggen samples are divided into 3 categories. Samples S1 - S3, S5 - S8, and S11 - S13 are the first category, samples S9 - S10 are the second category, and sample S4 is the third category.

[0061] (44) Principal component analysis (PCA)

[0062] The fingerprint data of 13 batches of yellow root samples were imported into SIMCA14.1 software, and the peak areas of 12 common peaks were used as variables to perform PCA and draw PCA score graphs. The results are shown in Figure 4 The cumulative explanatory power parameter of the model is R 2 X is 0.835, and the prediction ability parameter Q 2 The value was 0.495, indicating that the model had good predictive power. The 13 batches of yellow root samples were divided into three categories, which was consistent with the CA results.

[0063] (45) Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA)

[0064] SIMCA27.0 software was used to perform OPLS-DA on 13 batches of yellow root samples. The score matrix is ​​shown in Figure 5 From the model verification parameters, we can see that the model has good stability (R 2 X=0.949,R 2 Y=0.611), cross-validation prediction ability Q 2 =0.00992. R of permutation test 2 The intercept of the fitting line on the Y axis is 0.226 (<0.3), Q 2 The intercept of the fitting line on the Y axis is -0.412 (<0.05), indicating that the constructed model does not have overfitting and can be used to analyze the differences between groups of samples.

[0065] The variable importance projection values ​​(VIP) of the 12 common peaks in the OPLS-DA model were extracted and screened with VIP>1 as the standard. Among them, VIP>1 of the three common peaks ( Figure 6 ), followed by peak 10 (methyl isorubiacin-1-methyl ether), peak 3, peak 2 (scopoletin), and peak 12 (methyl isorubiacin) with VIP=0.97.

[0066] In summary, combined with OPLS-DA VIP values, reference identification, and relevant pharmacodynamic literature, scopoletin, methyl isorubiacin-1-methyl ether, and methyl isorubiacin can be considered as differential markers of Xanthophyllum serrata.

[0067] (5) Content determination

[0068] (51) Linear relationship investigation

[0069] Take an appropriate amount of the mixed reference solution, dilute it with methanol and make up the volume, and measure it according to the above chromatographic conditions. Draw a standard curve with the concentration of the reference solution as the horizontal axis and the peak area as the vertical axis. The results are shown in Table 3.

[0070] Table 3 Results of linear relationship investigation of four components in yellow root

[0071]

[0072]

[0073] (52) Precision test

[0074] An appropriate amount of the mixed reference solution was taken and continuously injected for determination 6 times under the above chromatographic conditions. The RSDs of the peak areas of scopoletin, coumarin, rubiadin-1-methyl ether, and rubiadin were calculated to be 0.73%, 0.64%, 0.66%, and 0.59% respectively, indicating good precision of the instrument.

[0075] (53) Repeatability test

[0076] Six portions of the same batch of the test sample of Radix Xanthii (S1) were taken, and six portions of the test solution were prepared in parallel according to the above method. The test solution was injected for determination under the above chromatographic conditions. The average contents of scopoletin, coumarin, rubiadin, and rubiadin-1-methyl ether were calculated to be 0.0555, 0.0144, 5.5918, and 0.1202 mg / g respectively, and the RSDs were 1.72%, 1.57%, 1.13%, and 1.41% respectively, indicating good repeatability of the method.

[0077] (54) Stability test

[0078] The test solution of Radix Xanthii (S1) was taken and injected for determination at 0, 2, 4, 8, 16, and 24 h respectively. The RSDs of the peak areas of scopoletin, coumarin, rubiadin-1-methyl ether, and rubiadin were calculated to be 0.65%, 0.49%, 0.38%, and 0.46% respectively, indicating good stability of the test solution within 24 h.

[0079] (55) Recovery test

[0080] 1 g of the test sample of Radix Xanthii (S1) with known component content was accurately weighed, and a total of 6 portions were taken. Each reference substance was added at a ratio of 100%. The test solution was prepared according to the above method and injected for determination under the above chromatographic conditions. The average recoveries of the above 4 components were calculated to be 98.46% - 101.08%, and the RSDs were 0.71% - 1.89%.

[0081] (56) Determination of sample content

[0082] An appropriate amount of 13 batches of Radix Xanthii samples was taken, and the sample solution was prepared according to the above method and determined under the above chromatographic conditions. The contents of the 4 components in the samples were calculated, and the results are shown in Table 4.

[0083] Table 4 Results of content determination of 4 components in Radix Xanthii (mg / g, n = 3)

[0084]

[0085]

[0086] Comparative Example 1

[0087] Taking the contents of scopoletin, coumarin, rubiadin-1-methyl ether, and rubiadin as indicators, the present invention screened different methods such as cold maceration, heat reflux, and ultrasonic extraction, as well as different solvents such as methanol and ethanol to extract the yellow root samples.

[0088] Table 5 Comparison results of the contents of four components with different extraction methods and solvents (mg / g)

[0089]

[0090] As can be seen from Table 5, when methanol is used as the extraction solvent and ultrasonic extraction is carried out for 40 min, the overall extraction efficiency is relatively high. Therefore, it is selected as the extraction method and extraction solvent of the present invention.

[0091] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. The same or similar parts among the various embodiments can be referred to each other.

[0092] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing a fingerprint of Rhizoma Xanthophyllae, characterized in that: The steps include: (1) taking scopoletin, coumarin, methyl isorhizin-1-methyl ether, and methyl isorhizin reference substances, dissolving them in a solvent, and preparing a reference substance solution; (2) taking a yellow root sample, adding an extraction solvent to extract it, filtering the extract, and preparing a test solution; (3) The reference solution and the test solution are respectively subjected to high performance liquid chromatography to construct a fingerprint of the yellow root.

2. The construction method according to claim 1, characterized in that: In the step (1), the solvent is methanol.

3. The construction method according to claim 1, characterized in that: In the step (1), the mass concentrations of scopoletin, coumarin, methyl isorubiacin-1-methyl ether, and methyl isorubiacin reference substance in the reference substance solution are 0.0518, 0.0268, 10.5844, and 0.2610 mg / mL, respectively.

4. The construction method according to claim 1, characterized in that: In the step (2), methanol is used as the extraction solvent for ultrasonic extraction.

5. The construction method according to claim 1, characterized in that: The specific operation of step (2) is: taking the yellow root powder and adding it into the methanol solution, the solid-liquid ratio is 2:25 mg / mL, ultrasonic extraction for 40 minutes, and then filtering through a 0.22 μm microporous filter membrane; Ultrasonic conditions were: 100 W, 40 kHz.

6. The construction method according to claim 1, characterized in that: In the step (3), The chromatographic column is a C18 column; The detection wavelength is 277 mm; The column temperature was 30°C. The injection volume was 10 μL.

7. The construction method according to claim 1, characterized in that: In the step (3), Mobile phase: acetonitrile as mobile phase A, water as mobile phase B; The mobile phase elution gradient is: 0-5min, 20%A; 5-20min, 20%-42%A; 20-45min, 42%A; 45-50min, 42%-60%A; 50-55min, 60%-95%A; 55-60min, 95%A.

8. The fingerprint constructed by the construction method according to any one of claims 1 to 7.

9. Application of the fingerprint spectrum described in claim 8 in identification and quality control of Xanthoceras chinensis.

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