Construction method of fingerprint spectrum of paeonia veitchii granules and fingerprint spectrum

Abstract

The invention provides a construction method of a fingerprint spectrum of Xiangshao granules and the fingerprint spectrum. According to the fingerprint spectrum of the Xiangshao granules provided by the invention, neohesperidin is taken as an internal reference substance, and the relative retention time of the neohesperidin is 1.00, so that the relative retention time of other 26 common peaks in the fingerprint spectrum is determined to be 0.10, 0.11, 0.13, 0.15, 0.35, 0.42, 0.44, 0.48, 0.58, 0.64, 0.71, 0.73, 0.77, 0.79, 0.82, 0.83, 0.91, 0.95, 1.04, 1.18, 1.28, 1.39, 1.44, 1.47, 1.48 and 1.56. Therefore, the obtained fingerprint spectrum can be used for comprehensively and effectively evaluating the quality of the paeonia veitchii granules.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine detection technology, specifically to a method for constructing a fingerprint spectrum of Xiangshao granules and the fingerprint spectrum thereof. Background Technology

[0002] Xiangshao Granules were launched as a new drug in 2005 (National Drug Approval Number Z20050425). This drug is made from 10 kinds of pure natural plant medicines, including Cyperus rotundus, Paeonia lactiflora, Melia toosendan, Bupleurum chinense, Ligusticum chuanxiong, Citrus aurantium, and Amomum villosum. It is mainly used to treat menopausal syndrome and has significant effects on a series of symptoms such as hot flashes, sweating, irritability, insomnia, palpitations, rib pain, dizziness, tinnitus, anxiety, and depression. In addition, it is also effective for symptoms such as premenstrual irritability, headache, insomnia, lower abdominal distension and pain, stomach distension and pain, nausea and vomiting.

[0003] However, to date, the Chinese Pharmacopoeia still does not include a quality standard for Xiangshao granules. Traditional Chinese medicine fingerprinting refers to the chromatographic or spectral representation of one or more characteristic components common to a particular Chinese medicinal material or prepared Chinese medicine. At present, given that the effective components in most Chinese medicinal materials are not clearly identified, traditional Chinese medicine fingerprinting is of great significance for effectively controlling and evaluating the quality of Chinese medicinal materials or prepared Chinese medicines. Summary of the Invention

[0004] To overcome the above problems, the present invention provides a method for constructing a fingerprint spectrum of peony granules and a fingerprint spectrum thereof.

[0005] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for constructing a fingerprint spectrum of peony granules, comprising: The test solution was analyzed by high performance liquid chromatography (HPLC), and neohesperidin was used as an internal reference to obtain the fingerprint spectrum of Xiangshao granules. The preparation method of the test solution includes: dispersing peony granules in a methanol-water solution and then ultrasonically treating the solution; the volume fraction of methanol in the methanol-water solution is 50%~70%. In the high performance liquid chromatography (HPLC) detection process, acetonitrile was used as mobile phase A, 0.1% (v / v) phosphoric acid solution was used as mobile phase B, gradient elution was performed, and the detection wavelength was 210 nm to 260 nm. The conditions for gradient elution include: .

[0006] In one or more embodiments, the volume fraction of methanol in the methanol-water solution is 70%.

[0007] In one or more embodiments, the concentration of peony granules in the test solution is 0.4~0.6 mg / mL, preferably 0.5 mg / mL.

[0008] In one or more embodiments, the ultrasonic treatment power is 200~300 W, preferably 250 W; the ultrasonic treatment time is 20~40 min, preferably 30 min.

[0009] In one or more embodiments, during high-performance liquid chromatography (HPLC) detection, the mobile phase flow rate is 0.9~1.1 mL / min, preferably 1.0 mL / min.

[0010] In one or more embodiments, the detection wavelength during high-performance liquid chromatography is 210 nm.

[0011] In one or more embodiments, during high-performance liquid chromatography (HPLC) detection, the column temperature is 25~35℃, preferably 30℃.

[0012] In a second aspect, the present invention provides a fingerprint spectrum of peony granules, which is constructed by the construction method described in the first aspect; using neohesperidin as an internal reference, its relative retention time is 1.00, and the relative retention times of other common peaks in the fingerprint spectrum are 0.10, 0.11, 0.13, 0.15, 0.35, 0.42, 0.44, 0.48, 0.58, 0.64, 0.71, 0.73, 0.77, 0.79, 0.82, 0.83, 0.91, 0.95, 1.04, 1.18, 1.28, 1.39, 1.44, 1.47, 1.48 and 1.56. The deviation in relative retention time is within 8%.

[0013] In one or more embodiments, when the relative retention times are 0.64, 0.77, 0.79, 0.95, 1.47, 1.48 and 1.56, the corresponding compounds are, in order, paeoniflorin, ferulic acid, glycyrrhizin, naringin, glycyrrhizic acid, saikosaponin A and dehydroauracetin.

[0014] A third aspect of the present invention provides the application of the fingerprint spectrum of Xiangshao granules described in the second aspect in the quality evaluation or control of the entire process of research, development, production or clinical application of Xiangshao granules.

[0015] The beneficial effects of this invention are as follows: This invention provides a method for constructing a fingerprint spectrum of Xiangshao granules and the fingerprint spectrum thereof. The fingerprint spectrum of Xiangshao granules provided by this invention uses neohesperidin as an internal reference, with a relative retention time of 1.00. The relative retention times of the other 26 common peaks in the fingerprint spectrum are determined to be 0.10, 0.11, 0.13, 0.15, 0.35, 0.42, 0.44, 0.48, 0.58, 0.64, 0.71, 0.73, 0.77, 0.79, 0.82, 0.83, 0.91, 0.95, 1.04, 1.18, 1.28, 1.39, 1.44, 1.47, 1.48, and 1.56. The fingerprint spectrum provided by this invention can comprehensively and effectively evaluate the quality of Xiangshao granules. The method for constructing a fingerprint spectrum of *Prunus armeniaca* granules provided by this invention uses acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, employing gradient elution. By optimizing the gradient elution program and the detection wavelength, a fingerprint spectrum of *Prunus armeniaca* granules with good peak separation, peak symmetry, and high response values ​​is obtained. The fingerprint spectrum established by this method can be used for quality control and evaluation of *Prunus armeniaca* granules. Specificity, stability, repeatability, and robustness tests were conducted on the method provided by this invention. The test results show that the method provided by this invention exhibits good specificity, stability, repeatability, and robustness. Attached Figure Description

[0016] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0017] Figure 1 This is the chromatogram obtained in Example 2 from the detection under the provisional fingerprint chromatogram detection conditions described in Example 1; Figure 2 The chromatograms are those obtained by detecting the gradient elution conditions shown in Tables 5 and 7 in Example 3, where elution condition 1 is the gradient elution condition shown in Table 5 and elution condition 2 is the gradient elution condition shown in Table 7. Figure 3 The chromatograms of the reference solutions of glycyrrhizin, saikosaponin A and ferulic acid in Example 4 are shown below. Figure 4 The chromatograms of the paeoniflorin, dehydroauracetam, and glycyrrhizic acid reference solutions in Example 4 are shown below. Figure 5 The chromatograms of the reference solutions of naringin and neohesperidin in Example 4 are shown below. Figure 6 The chromatograms are for detection wavelengths of 210 nm, 230 nm, and 260 nm in Example 5; where a~h are paeoniflorin, ferulic acid, glycyrrhizin, naringin, neohesperidin, glycyrrhizic acid, saikosaponin A, and dehydroausolic acid lactone, respectively. Figure 7 The chromatograms are those of the mixed reference solution and the test solution in Example 7; wherein, a~h are paeoniflorin, ferulic acid, glycyrrhizin, naringin, neohesperidin, glycyrrhizic acid, saikosaponin A and dehydroauracetin lactone, respectively. Figure 8 Chromatograms of six batches of test sample solutions; Figure 9 The standard fingerprint spectrum of Xiangshao granules; Figure 10 Typical liquid chromatogram of paeoniflorin reference standard specificity; Figure 11 This is a typical liquid chromatogram of the specificity of ferulic acid reference standard; Figure 12 Typical liquid chromatogram of glycyrrhizin reference standard specificity; Figure 13 Typical liquid chromatogram of the specificity of naringin reference standard; Figure 14 Typical liquid chromatogram of the specificity of the new hesperidin reference standard; Figure 15 This is a typical liquid chromatogram of the specificity of glycyrrhizic acid reference standard; Figure 16 Typical liquid chromatogram of the specificity of saikosaponin A reference standard; Figure 17 This is a typical liquid chromatogram for the specificity of dehydroauracetam reference standard. Detailed Implementation

[0018] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0019] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0020] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0021] The instruments used in the following examples are shown in Table 1, the reagents used in the experiments are shown in Table 2, the samples used in the experiments are shown in Table 3, and the reference standards used in the experiments are shown in Table 4.

[0022] Table 1. Experimental Instruments

[0023] Table 2 Reagents used in the experiment

[0024] Table 3 Samples used in the experiment

[0025] Table 4. Controls used in the experiment

[0026] Preparation of the test solution: Take the contents of the peony granule sample, grind it into a fine powder, take 1.5 g of the fine powder, weigh it accurately, put it in a stoppered conical flask, add 50 mL of 70% methanol aqueous solution, weigh it, sonicate (250 W, 40 kHz) for 30 min, cool it to room temperature, weigh it again, make up the weight loss with 70% methanol aqueous solution, shake well, filter, and take the filtrate to obtain the test solution.

[0027] Preparation of reference solution: Take appropriate amounts of paeoniflorin, ferulic acid, glycyrrhizin, naringin, neohesperidin, glycyrrhizic acid, saikosaponin A and dehydroauracetin lactone reference standards, accurately weigh them, and dissolve them in methanol to prepare a solution with a concentration of 0.5 mg / mL.

[0028] Example 1 Based on a comprehensive analysis of the conditions for determining the fingerprint chromatograms of the medicinal materials in the prescription and Xiangshao granules, the following HPLC conditions were determined: Column: Welch Xtimate@ C18 (4.6 mm × 250 mm, 5 μm); Mobile phase: Acetonitrile was used as mobile phase A, and 0.1% (v / v) phosphoric acid water was used as mobile phase B, eluted according to the gradient shown in Table 5. Flow rate: 1.0 mL / min; column temperature: 30 ℃; detection wavelength: 210 nm.

[0029] Table 5 Gradient elution conditions

[0030] Example 2 Xiangshao granules are an oral preparation. The selection of the extraction solvent mainly considers the consistency with the solvent and extraction method used in the process preparation. The following test sample solution preparation scheme is designed, and the details are shown in Table 6: Table 6. Preparation schemes for experimental sample solutions

[0031] Assay: Accurately measure 20 μL of each of the test solutions prepared in methanol aqueous solutions of different concentrations as shown in Table 6, inject them into the liquid chromatograph, and detect them according to the provisional fingerprint chromatogram determination conditions in Example 1. The obtained chromatograms are shown below. Figure 1 As shown.

[0032] from Figure 1 As can be seen, when 50% and 70% methanol aqueous solutions were selected as extraction solvents, the number of chromatographic peaks in the sample solution and the baseline separation between each peak could be basically achieved, and there was no significant difference in the extraction results between the two solvents. Considering the actual preparation method of the extract of Xiangshao granules, 70% methanol aqueous solution was used as the extraction solvent for the contents of Xiangshao granules.

[0033] Example 3 Because the Xiangshao granules prescription contains many medicinal ingredients and has a complex composition, in order to effectively identify the characteristic fingerprint peaks or common peaks of the material basis of Xiangshao granules, it is necessary to optimize the elution method in order to separate or elute as many material basis components as possible. The specific experimental design is as follows: the test solution is detected according to the chromatographic conditions described below.

[0034] Chromatographic conditions include: Column: Octadecyl bonded silica gel (4.6 mm × 250 mm, 5 μm); Mobile phase A: Acetonitrile; Mobile phase B: 0.1% (v / v) phosphoric acid solution; Flow rate: 1.0 mL / min; Column temperature: 30 ℃; Detection wavelength: 210 nm; The gradient elution conditions are shown in Table 5 of Example 1 and in Table 7.

[0035] Table 7 Gradient elution conditions

[0036] Assay: Accurately measure 20 μL of the above test solution and inject it into the liquid chromatograph. Elute according to the gradient elution conditions shown in Tables 5 and 7. The chromatograms are shown below. Figure 2 As shown. From Figure 2 As can be seen from the table, when the test is performed according to the gradient elution conditions shown in Table 5, the fingerprint spectrum of the test sample solution shows good separation of each peak, and the peak response is better than that shown in Table 7. The analysis time is also moderate. Therefore, the elution conditions shown in Table 5 are used as the determination conditions for the fingerprint spectrum of Xiangshao granules.

[0037] Example 4 The main material basis of Xiangshao granules is as follows: Assay: 20 μL of the reference solution was injected into the liquid chromatograph, and the determination was performed under the optimized chromatographic conditions described in Example 3. The results are as follows: Figures 3-5 As shown. From Figures 3-5 As can be seen, all reference standards responded appropriately and can be used as identifiers to distinguish the fingerprint characteristic peaks of Xiangshao granules.

[0038] Example 5 To more effectively reflect the material properties of Xiangshao granules, it is necessary to examine the characteristic peak (S peak) of the fingerprint as the substance that responds well at a specific wavelength. Therefore, the selection of the specific wavelength needs to be further optimized.

[0039] The ultraviolet absorption of the peony granules is mainly concentrated around wavelengths of 210 nm, 230 nm, and 260 nm. To ensure the comprehensiveness of the material basis detection, characteristic localization was used to examine the characteristics of chromatograms at different wavelengths, confirming the wavelengths required for fingerprint detection. The test solution and reference solution were injected into the liquid chromatograph, and chromatograms at detection wavelengths of 210 nm, 230 nm, and 260 nm were recorded. The experimental results are as follows: Figure 6 As shown. From Figure 6 As can be seen, when the detection wavelengths are 210 nm, 230 nm and 260 nm, the characteristic indicators of the drugs composed of Xiangshao granules, such as paeoniflorin, ferulic acid, glycyrrhizin, naringin, neohesperidin, glycyrrhizic acid, saikosaponin A and dehydroausendone, all respond; while under the wavelength of 210 nm, the ultraviolet characteristics of each substance in the sample respond better; thus confirming that the detection wavelength is 210 nm.

[0040] Example 6 Based on the experimental results in Examples 1-5 above, the fingerprint spectrum determination conditions for Xiangshao granules are summarized as follows: Octadecyl-bonded silica gel (4.6 mm × 250 mm, 5 μm) was used as the packing material; acetonitrile was used as mobile phase A and 0.1% (v / v) phosphoric acid was used as mobile phase B. Gradient elution was performed according to the conditions shown in Table 5, with a flow rate of 1.0 mL per minute; column temperature of 30 ℃; and detection wavelength of 210 nm.

[0041] Preparation of the test solution: Take the contents of the peony granule sample, grind it into a fine powder, take 1.5 g of the fine powder, weigh it accurately, put it in a stoppered conical flask, add 50 mL of 70% methanol aqueous solution, weigh it, sonicate (250 W, 40 kHz) for 30 min, cool it to room temperature, weigh it again, make up the weight loss with 70% methanol aqueous solution, shake well, filter, and take the filtrate to obtain the test solution.

[0042] Assay: Accurately pipette 20 µL each of the reference solution and the test solution into the liquid chromatograph, measure and record the chromatogram to obtain the result.

[0043] Example 7 The known chemical components in Xiangshao granules mainly include paeoniflorin, ferulic acid, glycyrrhizin, naringin, neohesperidin, glycyrrhizic acid, saikosaponin A, and dehydrocostunolide, etc. The known components are located and identified in the chromatogram of the test solution.

[0044] Assay: The mixed reference solution and the test solution were injected and analyzed under the chromatographic conditions determined in Example 6. The peaks in the chromatograms of the test solution and each reference solution were compared, and the characteristic peaks were assigned. The results are as follows: Figure 7 As shown.

[0045] from Figure 7 As can be seen from the chromatogram, the characteristic peaks in the test sample solution chromatogram identify eight known components. Among them, peaks a to h are, in order: paeoniflorin, ferulic acid, glycyrrhizin, naringin, neohesperidin, glycyrrhizic acid, saikosaponin A, and dehydroausendone. The peaks of paeoniflorin, ferulic acid, glycyrrhizin, naringin, neohesperidin, glycyrrhizic acid, saikosaponin A, and dehydroausendone are relatively prominent and can be used as characteristic peaks. The neohesperidin peak has a high response and can be used as the reference main S peak.

[0046] Example 8 Take an appropriate amount of one batch of Xiangshao granules for testing, and prepare 6 test solutions (s1~s6) according to the preparation method of the test solution determined in Example 6. Under the chromatographic conditions specified in Example 6, accurately measure 20 μL and inject them into the liquid chromatograph for determination according to law, and record the chromatogram.

[0047] Calibration of common characteristic fingerprint peaks: The chromatograms of six test solutions from one batch of Xiangshao granules were analyzed and compared to determine their common peaks.

[0048] The chromatograms of six test solutions from one batch of Xiangshao granules all showed 27 chromatographic peaks, of which eight were common characteristic peaks, accounting for more than 90% of the total peak area. Using neohesperidin as a reference peak, the relative retention times were calculated, and the results are as follows: Figure 8 As shown in Table 8.

[0049] Table 8 Characteristic Peak Parameters

[0050] The standard fingerprint spectrum of Xiangshao granules is as follows: Figure 9As shown, the fingerprint spectrum should exhibit 27 characteristic peaks. The relative retention times of each characteristic peak and the neohesperidin peak are as follows: 0.10 (peak 1), 0.11 (peak 2), 0.13 (peak 3), 0.15 (peak 4), 0.35 (peak 5), 0.42 (peak 6), 0.44 (peak 7), 0.48 (peak 8), 0.58 (peak 9), 0.64 (peak 10), 0.71 (peak 11), 0.73 (peak 12), 0.77 (peak 13), 0.79 (peak 14), 0.82 (peak 15), and 0.83 (peak 16). 0.91 (peak 17), 0.95 (peak 18), 1.00 (S peak 19), 1.04 (peak 20), 1.18 (peak 21), 1.28 (peak 22), 1.39 (peak 23), 1.44 (peak 24), 1.47 (peak 25), 1.48 (peak 26), 1.56 (peak 27); among which peaks 10, 13, 14, 18, 19 (S), 25, 26, and 27 are paeoniflorin, ferulic acid, glycyrrhizin, naringin, neohesperidin, glycyrrhizic acid, saikosaponin A, and dehydroausolic acid lactone, respectively.

[0051] Characteristic peak limit range: The characteristic spectrum of the test sample should show 27 characteristic peaks, among which peaks 10, 13, 14, 18, 19 (S), 25, 26, and 27 are paeoniflorin, ferulic acid, glycyrrhizin, naringin, neohesperidin, glycyrrhizic acid, saikosaponin A, and dehydroausolic acid lactone, respectively; the peak corresponding to the retention time of the reference neohesperidin peak is designated as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The deviation of the relative retention time should be within 8%.

[0052] Example 9 Validation of fingerprinting method: (1) Specificity test: Take 20 μL of the test solution and the reference solution respectively, inject them into the liquid chromatograph, and determine them under the chromatographic conditions determined in Example 6. Record the chromatograms. Typical specific liquid chromatograms are shown below. Figures 10-17 As shown, the method-specific characteristic peaks and common peak information are the same as those shown in Table 8.

[0053] The experimental data above showed that 27 characteristic peaks were identified in the sample solution, including those of paeoniflorin, ferulic acid, glycyrrhizin, naringin, neohesperidin, glycyrrhizic acid, saikosaponin A, and dehydroauscinactone. The chromatographic peaks were well separated from other peaks, the peaks were symmetrical, and the response values ​​were high, indicating that the specificity under this condition was strong. The blank solvent did not interfere with the determination of characteristic peaks and common peaks, indicating that the method has good specificity.

[0054] (2) Stability test: Because Xiangshao granules contain many medicinal ingredients and have complex components, in order not to affect fingerprint research during the experiment, it is necessary to examine the stability of the test solution at room temperature to ensure that the test solution does not affect the evaluation of changes in the material basis of the sample.

[0055] Assay: Accurately measure 20 μL of the test solution and incubate it at room temperature in the dark for 0 h, 3 h, 6 h, 9 h, 12 h, and 15 h, respectively. Inject the sample under the chromatographic conditions determined in Example 6 and calculate the ratio of the relative retention time to the peak area of ​​the common characteristic peak in the fingerprint chromatogram. Limitations: The relative standard deviation of the relative peak area of ​​the common characteristic peak in the fingerprint chromatogram should not exceed 5.0%, and the relative standard deviation of the relative retention time of the characteristic peak should not exceed 2.0%, indicating that the test solution is relatively stable. The sample stability results are shown in Tables 9-11.

[0056] Table 9 Sample stability - relative peak area

[0057] Table 10 Sample stability - relative retention time

[0058] Table 11 Stability Similarity Results

[0059] Results: As shown in Tables 9-11, at a wavelength of 210 nm, the relative standard deviation of the relative retention time of each chromatographic peak in the test solution compared with the reference peak of neohesperidin was the largest at 0.228% and the relative standard deviation of the relative peak area was the largest at 4.790% within 15 h. The similarity between the fingerprint spectra of the sample solution after 15 h was greater than 0.9, indicating that the sample has good stability.

[0060] (3) Repeatability test: Six test solutions were prepared in parallel. 20 μL of each of the six test solutions was injected into the liquid chromatograph, and the chromatograms were determined and recorded according to the established chromatographic conditions. The repeatability results are shown in Table 12.

[0061] Table 12 Repeatability test data

[0062] Experimental results: The experimental data above show that the sample similarity is greater than 0.9, which indicates that the sample has good stability.

[0063] (4) Durability test: The effects of fine-tuning column temperature (25 ℃, 35 ℃) and different flow rates (0.9 mL / min, 1.1 mL / min) on the determination results of common peaks were investigated, and the relative standard deviation of the relative retention time was used to judge the robustness of the sample.

[0064] Assay: Accurately measure 20 μL of the test solution, inject it under the chromatographic conditions determined in Example 6, and record the chromatogram. The robustness results are shown in Tables 13 and 14.

[0065] Table 13 Sample durability similarity results

[0066] Table 14 Results of relative retention time for sample durability

[0067] Analysis of the measured data shows that, at a detection wavelength of 210 nm, with fine-tuning of column temperature (25 ℃, 30 ℃) and different flow rates (0.9 mL / min, 1.1 mL / min), the sample similarity is greater than 0.9, and the maximum relative standard deviation of relative retention time is 6.566%, indicating that the determined chromatographic conditions are robust and the samples are robust.

[0068] (5) Similarity evaluation: The fingerprint spectrum test data of 6 sample solutions of 1 batch of Xiangshao granules (batch number: 2310050) were imported into the Chinese medicine fingerprint spectrum similarity software. The software was used to calculate the control fingerprint spectrum of Xiangshao granules. The test data of each fingerprint spectrum were calculated based on the similarity of characteristic peaks. The results are shown in Table 15.

[0069] Table 15 Similarity Results

[0070] Analysis of the data showed that the fingerprint spectrum and characteristic peak similarity of the six Xiangshao granule samples were all no less than 0.90.

[0071] This invention, through different test solution preparations and chromatographic conditions, determined the test solution preparation method and detection conditions for the fingerprint spectrum of Xiangshao granules, and identified eight characteristic peaks: paeoniflorin, ferulic acid, glycyrrhizin, naringin, neohesperidin, glycyrrhizic acid, saikosaponin A, and dehydroausendone. These peaks effectively reflect the material basis of Xiangshao granules and can serve as a basis for quality control of Xiangshao granules, used to evaluate the authenticity, quality, and stability of the preparation.

[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of peony granules, characterized in that, include: The test solution was detected by high performance liquid chromatography, and neohesperidin was used as an internal reference to obtain the fingerprint spectrum of Xiangshao granules. The preparation method of the test solution includes: dispersing peony granules in a methanol-water solution and then ultrasonically treating the solution; the volume fraction of methanol in the methanol-water solution is 50%~70%. In the high performance liquid chromatography (HPLC) detection process, acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, gradient elution was performed, and the detection wavelength was 210 nm to 260 nm. The conditions for gradient elution include: 。 2. The construction method as described in claim 1, characterized in that, The volume fraction of methanol in the methanol-water solution is 70%.

3. The construction method as described in claim 1, characterized in that, The concentration of peony granules in the test solution is 0.4~0.6 mg / mL.

4. The construction method as described in claim 1, characterized in that, The ultrasonic treatment power is 200~300 W; the ultrasonic treatment time is 20~40 min.

5. The construction method as described in claim 1, characterized in that, During high-performance liquid chromatography (HPLC) detection, the mobile phase flow rate is 0.9~1.1 mL / min.

6. The construction method as described in claim 1, characterized in that, During high-performance liquid chromatography (HPLC) detection, the detection wavelength is 210 nm.

7. The construction method as described in claim 1, characterized in that, During high-performance liquid chromatography (HPLC) detection, the column temperature is 25~35 ℃.

8. A fingerprint spectrum of peony granules, characterized in that, It is constructed by the construction method according to any one of claims 1 to 7; using neohesperidin as an internal reference, its relative retention time is 1.00, and the relative retention times of other common peaks in the fingerprint spectrum are 0.10, 0.11, 0.13, 0.15, 0.35, 0.42, 0.44, 0.48, 0.58, 0.64, 0.71, 0.73, 0.77, 0.79, 0.82, 0.83, 0.91, 0.95, 1.04, 1.18, 1.28, 1.39, 1.44, 1.47, 1.48 and 1.56 respectively; The deviation in relative retention time is within 8%.

9. The fingerprint spectrum of peony granules as described in claim 8, characterized in that, When the relative retention times were 0.64, 0.77, 0.79, 0.95, 1.47, 1.48 and 1.56, the corresponding compounds were paeoniflorin, ferulic acid, glycyrrhizin, naringin, glycyrrhizic acid, saikosaponin A and dehydroausolic acid lactone, respectively.

10. The application of the fingerprint spectrum of Xiangshao granules as described in claim 8 or 9 in the quality evaluation or control of the entire process of research, development, production or clinical application of Xiangshao granules.

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