Quality evaluation method and application of dandelion medicinal material and extract thereof

Abstract

The invention discloses construction and application of a dandelion fingerprint spectrum and a multi-component quantitative analysis method. According to the method, the UHPLC fingerprint spectrum of the phenol components and the triterpenoid components of the dandelion is constructed for the first time, quantitative analysis of the six phenol components and the three triterpenoid components can be achieved at the same time, and the method can be used for comprehensive evaluation and quality control of dandelion raw materials and extracts of the dandelion raw materials.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis technology, specifically to a method and application for evaluating the quality of dandelion based on fingerprinting and multi-component quantification. Background Technology

[0002] Dandelion is a commonly used medicinal and edible herb in my country, possessing the effects of clearing heat and detoxifying, reducing swelling and dissipating nodules, and promoting diuresis. It mainly contains phenolic compounds and triterpenes. Modern pharmacological studies have shown that the phenolic components (phenolic acids and flavonoids) in dandelion have anti-tumor, antioxidant, and anti-inflammatory activities; while the triterpenoid components in dandelion have anti-inflammatory, liver-damage-treating, antioxidant, and anti-tumor activities.

[0003] The quantitative index for dandelion under the content determination section of the Chinese Pharmacopoeia is chicoric acid, which is a relatively simple component. In recent years, there have been some studies on HPLC and UPLC fingerprinting of dandelion, but these studies mainly focus on the phenolic components in dandelion. There are also a few quantitative analysis studies on the triterpenoid components in dandelion. However, in terms of fingerprinting and quantitative analysis of the whole components of dandelion, there is a lack of methods to simultaneously achieve fingerprinting and quantitative analysis of both phenolic and triterpenoid components, and there is a lack of methods and means to comprehensively evaluate the whole components of dandelion.

[0004] In order to better achieve the quality evaluation of dandelion medicinal materials, extracts and downstream preparations, it is urgent to develop a method that can simultaneously achieve quantitative analysis of phenolic and triterpenoid components and determination of full component fingerprint spectrum. Summary of the Invention

[0005] The purpose of this invention is to provide a method for the simultaneous quantitative analysis and fingerprinting of six phenolic acid components and three triterpenoid components from dandelion. The fingerprint constructed by this method simultaneously contains both phenolic and triterpenoid components, enabling quality control of both components in a single detection. This method can be used for the quality evaluation of dandelion medicinal materials, dandelion extracts, and their preparations.

[0006] The technical solution provided by this invention is as follows: A method for quality evaluation of dandelion medicinal material and its extract includes the following steps: (1) Preparation of test solution: Take the pulverized dandelion medicinal material or extract, add the extraction solvent to the test sample for extraction, filter, take the filtrate to obtain the test solution.

[0007] Preferably, the extraction solvent in step (1) is at least one of water, methanol and ethanol.

[0008] Preferably, the extraction solvent is 70% ethanol.

[0009] Preferably, the extraction step employs reflux extraction, with an extraction time of 120 minutes.

[0010] (2) Preparation of reference solution: Take monocaffeoyl tartaric acid, chlorogenic acid, caffeic acid, chicoric acid, isochlorogenic acid A, luteolin, lupeol, taraxasterol and taraxerol reference standards and prepare a mixed reference solution; Preferably, methanol is used as the solvent for preparing the reference standard.

[0011] Each 1 mL of the reference solution contains 50–200 μg of monocaffeoyl tartaric acid; and / or, Each 1 mL of the reference solution contains 10–100 μg of chlorogenic acid; and / or, Each 1 mL of the reference solution contains 10–100 μg of caffeic acid; and / or, Each 1 mL of the reference solution contains 50–250 μg of chicoric acid; and / or, Each 1 mL of the reference solution contains 5–50 μg of isochlorogenic acid A; and / or Each 1 mL of the reference solution contains 2–20 μg of luteolin; and / or, Each 1 mL of the reference solution contains 2–50 μg of lupeol; and / or, Each 1 mL of the reference solution contains 2–50 μg of taraxasterol; and / or, Each 1 mL of the reference solution contains 2–50 μg of taraxerol.

[0012] (3) Chromatographic analysis conditions: The column was packed with octadecylsilane-bonded silica gel, and gradient elution was performed with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B. The detection wavelength was dynamically switched, the injection volume was 1 μL, and the detector was a DAD or VWD detector.

[0013] This invention investigated the mobile phase system. When acetic acid and formic acid were added, the baseline drift of the triterpenoid components in the chromatogram was severe. The results showed that the separation effect of each target component in the chromatogram was better when acetonitrile-phosphoric acid aqueous solution was used as the mobile phase system. Furthermore, acetonitrile was selected as mobile phase A and 0.1% phosphoric acid aqueous solution was selected as mobile phase B for gradient elution.

[0014] Preferably, the gradient elution procedure is as follows:

[0015] During the research process of this invention, the characteristics of phenolic and triterpenoid components were combined to further determine the dynamic switching wavelength range of the DAD detector. Preferably, the specific method for dynamic wavelength switching is: 0–35 min, 327 nm; 35–50 min, 210 nm; this method can also be applied to the VWD detector.

[0016] This invention also investigated and screened different models and brands of chromatographic columns. Considering the characteristics of each target component and the instrument, the separation performance of Shim-pack Scepter C18-120, Waters ACQUITY UPLC HSS T3, and Excsep UPLC SCB-C18 columns for the target components was investigated.

[0017] Preferably, the chromatographic column is a Shim-pack Scepter C18-120.

[0018] Preferably, the chromatographic column has a specification of 2.1×100mm and the packing particle size is 1.9μm.

[0019] This invention also investigated the effects of different column temperatures (20℃, 25℃, 30℃) and different flow rates (0.1 mL / min, 0.2 mL / min, 0.3 mL / min) on the separation degree of each target component.

[0020] Preferably, the flow rate is 0.3 mL / min and the column temperature is 30 °C.

[0021] (4) Establishment of fingerprint spectrum: The test solution was injected and measured, the chromatogram was recorded and imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Version" for processing to obtain the dandelion fingerprint spectrum and calculate the similarity.

[0022] This invention conducts a methodological investigation on the fingerprint spectroscopy determination method, including investigations on precision, repeatability, and stability. The results show that the method has good precision, repeatability, and stability.

[0023] The nine chromatographic peaks identified were: monocaffeoyl tartaric acid (peak 1), chlorogenic acid (peak 2), caffeic acid (peak 3, S peak), chicoric acid (peak 4), isochlorogenic acid A (peak 5), luteolin (peak 6), lupeol (peak 7), taraxasterol (peak 8), and taraxerol (peak 9). The retention times of the nine common peaks were: peak 1 (0.40), peak 2 (0.52), peak 3 (0.56), peak 4 (1.00), peak 5 (1.04), peak 6 (1.27), peak 7 (2.08), peak 8 (2.15), and peak 9 (2.19). The similarity to the reference chromatogram was calculated to be no less than 0.94.

[0024] (5) Quantitative analysis: Following steps (1) to (4), quantitative analysis was performed on nine phenolic and triterpenoid components in the sample, including monocaffeoyl tartaric acid, chlorogenic acid, caffeic acid, chicoric acid, isochlorogenic acid A, luteolin, lupeol, taraxasterol and taraxerol, for the quality control of medicinal materials and extracts.

[0025] Methodological studies have shown that this method has good precision, repeatability, and stability. All nine components showed good separation, good linearity, good limit of quantitation, and high accuracy, making it suitable for quantitative analysis of components.

[0026] The method for determining the content of nine index components in step (5) of this invention has a good linear relationship (R ≥ 0.99982), and the RSD of precision, repeatability and stability are all less than 2%, with the recovery rate between 97.55% and 102.69%.

[0027] Compared with the prior art, the present invention has the following advantages: 1. This invention is the first to achieve quantitative analysis of 6 phenolic components and 3 triterpenoid components in dandelion. The established fingerprint spectrum and the quality information characterized by the quantitative analysis components are more comprehensive, and the dandelion products can be comprehensively evaluated through phenols and triterpenoids.

[0028] 2. The analytical method for phenolic and triterpenoid components provided by this invention is convenient and efficient, and can achieve the determination of two types of components at one time, providing a more comprehensive evaluation of the types of components. While achieving the same analytical efficiency, it can avoid two determinations for phenolic and triterpenoid components. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 UHPLC chromatograms of dandelion test solution, mixed reference solution, and blank solvent; Figure 2 Overlay pattern of dandelion sample and control fingerprint (R); Figure 3 UHPLC chromatograms of dandelion medicinal materials, extracts, and granules; Figure 4 UHPLC chromatograms corresponding to different chromatographic columns; Figure 5 UHPLC spectra corresponding to different solvent concentrations; Figure 6 UHPLC chromatograms corresponding to different extraction times; Figure 7 UHPLC spectra corresponding to different phosphoric acid concentrations. Detailed Implementation

[0031] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this application. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. All reagents or instruments used are commercially available conventional products.

[0032] Example 1: Determination of Dandelion Samples (1) Take dandelion medicinal material, crush it and sieve it. Take about 0.5 g, weigh it accurately, put it in a stoppered conical flask, add 25 mL of 70% ethanol accurately, stopper tightly, reflux for 120 min, take it out, let it cool, weigh it again, make up the weight loss with 70% ethanol, shake well, filter, and take the filtrate to obtain the test solution.

[0033] Take appropriate amounts of monocaffeoyl tartaric acid, chlorogenic acid, caffeic acid, chicoric acid, isochlorogenic acid A, luteolin, lupeol, taraxasterol, and taraxerol reference standards respectively, accurately weigh them, and add methanol to prepare mixed reference standard solutions containing 79.66, 19.70, 14.36, 168.37, 7.04, 8.84, 18.47, 20.42, and 32.50 μg of monocaffeoyl tartaric acid, chlorogenic acid, caffeic acid, chicoric acid, isochlorogenic acid A, luteolin, lupeol, taraxasterol, and taraxerol per mL, respectively.

[0034] An octadecylsilane-bonded silica gel column was used as the packed column; gradient elution was performed using acetonitrile (A)-0.1% phosphoric acid aqueous solution (B) as the mobile phase: 0–12 min, 5%→15%A; 12–20 min, 15%→28%A; 20–25 min, 28%→35%A; 25–27 min, 35%→95%A; 27–35 min, 95%→97%A; 35–50 min, 97%A; the mobile phase flow rate was 0.3 mL / min; dynamic wavelength switching was used: 0–35 min, 327 nm; 35–50 min, 210 nm; the column temperature was 30℃. The theoretical plate number, calculated based on chicoric acid, should not be less than 10,000.

[0035] Accurately pipette 1 μL each of the mixed reference solution and the test solution into the ultra-high performance liquid chromatograph, measure and record the chromatograms.

[0036] The UHPLC chromatograms of dandelion test solution, mixed reference solution, and blank solvent are shown below. Figure 1 As shown, the results indicate that the resolution (Ri) of each chromatographic peak is greater than 1.5, there is no interference from the blank solution, and the method has good specificity. It can be used for the fingerprinting and quantitative analysis of dandelion.

[0037] Example 2: Construction of Dandelion Fingerprint Spectrum The raw material for dandelion is dandelion grown in alkaline soil. Taraxacum borealisinense Kitam. Samples S1-5 were collected from Gansu, S6-10 from Henan, and S11-15 from Shanxi. Fingerprint analysis was conducted using samples S1-15.

[0038] Precision: 0.5 g of dandelion sample (S1) was accurately weighed and the test solution was prepared according to the method in Example 1. The sample was injected and measured 6 times consecutively. Peak 4 (chicoric acid) was used as the reference peak (S). The results showed that the RSD values ​​of the relative retention time and relative peak area of ​​each common peak were 0.02% to 0.18% and 0.17% to 0.86%, respectively, both less than 2.0%.

[0039] Repeatability: Take dandelion sample (S1), accurately weigh it, and prepare 6 test solutions in parallel according to the test solution preparation method in Example 1. Inject and determine the solutions. Using peak 4 (chicoric acid) as the reference peak (S), the RSD values ​​of the relative retention time and relative peak area of ​​each common peak were calculated to be 0.03% to 0.22% and 0.41% to 1.76%, respectively, both less than 2.0%.

[0040] Stability: The same batch of dandelion medicinal material samples (S1) were accurately weighed and the test solution was prepared according to the test solution method in Example 1. The samples were placed for 0, 4, 8, 12, 16 and 24 h and then injected for determination. The RSD values ​​of the relative retention time and relative peak area of ​​each common peak were calculated using peak 4 (chicoric acid) as the reference peak (S). The values ​​were 0.08% to 0.47% and 0.37% to 1.87%, respectively, both less than 2.0%.

[0041] Fingerprint chromatogram establishment and similarity evaluation: Fifteen batches of dandelion samples were analyzed according to the method in Example 1. The chromatographic data were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". After median method and multi-point correction, chromatographic peak matching was performed, and a total of 9 common peaks were identified. Superimposed chromatograms of dandelion samples and control fingerprint chromatograms were generated respectively. Figure 2By comparing with the reference standard, all nine chromatographic peaks were identified as follows: monocaffeoyl tartaric acid (peak 1), chlorogenic acid (peak 2), caffeic acid (peak 3), chicoric acid (peak 4, S peak), isochlorogenic acid A (peak 5), luteolin (peak 6), lupeol (peak 7), taraxasterol (peak 8), and taraxerol (peak 9). The relative retention times (RSD) of the common peaks in each batch of samples ranged from 0.11% to 0.46%. The established dandelion fingerprint chromatogram comprehensively reflects the component characteristics of dandelion. The similarity (R) between the fingerprint chromatograms of 15 batches of dandelion samples and the reference fingerprint chromatogram was all 0.940 or higher (see Table 1), indicating good consistency among the batches of medicinal materials.

[0042] Table 1. Fingerprint similarity matching data of 15 batches of dandelion medicinal materials

[0043] Example 3: Quantitative Analysis of Nine Components from Dandelion Linearity assessment: A mixed reference stock solution containing nine components from Example 1 was used, and six reference solutions of different concentrations were obtained through serial dilution. 1 μL of each of the six different concentration reference solutions was injected into a UHPLC ultra-high performance liquid chromatograph, and the analysis was performed under the chromatographic conditions described in Example 1. The x-axis was plotted as the injection concentration (…). X ), with the peak area integral value as the ordinate ( Y Linear regression analysis was performed, and the results showed that all nine components had a good linear relationship within the linear range, with correlation coefficients of [missing information]. R All values ​​were above 0.99982, as shown in Table 2.

[0044] Table 2 Results of the methodological study on the quantitative analysis of nine components

[0045] Precision: Take 0.5g of the same batch of dandelion medicinal material sample, accurately weigh it, prepare the test solution according to the method in Example 1, and continuously inject and determine it 6 times under the same chromatographic conditions to examine the RSD value of the peak area of ​​9 components.

[0046] Repeatability: Take the same batch of dandelion samples, weigh them accurately, prepare 6 test solutions in parallel according to the method in Example 1, and determine them under the same chromatographic conditions. Calculate the RSD of the mass fraction of each component.

[0047] Stability: Take the same batch of dandelion medicinal material samples, weigh them accurately, and prepare the test solution according to the method in Example 1. After preparation, the samples were placed for 0, 4, 8, 12, 16 and 24 h and the RSD of the mass fraction of each component was calculated.

[0048] The results showed that the precision, repeatability, and stability RSD values ​​of monocaffeoyl tartaric acid, chlorogenic acid, caffeic acid, chicoric acid, isochlorogenic acid A, luteolin, lupeol, taraxasterol, and taraxerol were between 0.18% and 0.68%, 0.63% and 1.88%, and 0.47% and 1.25%, respectively, indicating that the instrument had good precision, and the method had good repeatability and stability.

[0049] Recovery test: Six dandelion samples with measured content, each approximately 0.25 g, were accurately weighed. Appropriate amounts of monocaffeoyl tartaric acid, chlorogenic acid, caffeic acid, chicoric acid, isochlorogenic acid A, luteolin, lupeol, taraxasterol, and taraxerol reference standards were added at a 1:1 ratio to the sample content. Six test solutions were prepared in parallel according to the method described in Example 1, and analyzed. The average recovery rates of monocaffeoyl tartaric acid, chlorogenic acid, caffeic acid, chicoric acid, isochlorogenic acid A, luteolin, lupeol, taraxasterol, and taraxerol were calculated. The results are shown in Table 2. The recoveries of the nine components ranged from 97.55% to 102.69%, with RSDs ranging from 0.79% to 1.93%, indicating good recovery of the method.

[0050] Example 4: Determination of the content of nine components in 15 batches of dandelion samples According to the method and chromatographic conditions described in Example 1, the contents of nine components—caffeoyl tartaric acid, chlorogenic acid, caffeic acid, chicoric acid, isochlorogenic acid A, luteolin, lupeol, taraxasterol, and taraxerol—in 15 batches of dandelion samples were determined. The results of the content determination for the 15 batches of dandelion are shown in Table 3. The average contents of the components, in descending order, are chicoric acid, monocaffeoyl tartaric acid, chlorogenic acid, caffeic acid, isochlorogenic acid A, taraxerol, taraxasterol, lupeol, and luteolin.

[0051] Table 3. Results of content determination of 9 components in 15 batches of dandelion medicinal materials ( n =2)

[0052] Example 5: UHPLC determination of dandelion extract and single-ingredient preparations Weigh 0.15 g of dandelion extract and 0.2 g of dandelion granules into separate Erlenmeyer flasks, add 50 mL of 70% ethanol, sonicate for 30 min, replenish the weight loss with 70% ethanol, shake well, filter, and collect the filtrate. Inject and determine the fingerprints of the three batches of dandelion extract, three batches of granules, and three batches of medicinal materials according to the chromatographic conditions described in Example 1. The fingerprint chromatograms obtained are as follows: Figure 3As shown, the results indicate that the relative retention times of the medicinal material, extract, and granules are consistent, and the similarity of the chromatograms of the nine batches is greater than 0.996, indicating good peak resolution and meeting the quantitative requirements. Methodological studies also demonstrate the applicability of this method for the quality analysis of extracts and granules. The UHPLC chromatograms of dandelion medicinal material, extract, and granules are shown below. Figure 3 As shown, this method can be used for the quality control of medicinal materials, extracts, and granules prepared from extracts.

[0053] Example 6: Effect of different chromatographic columns on UHPLC separation efficiency Different chromatographic columns have a significant impact on the separation of dandelion components, especially phenolic components. The following are the chromatograms of three columns (Shim-pack scepter C18 120, ACQUITY HSS T3, and Excsep SCB-C18) at 35 minutes (see...). Figure 4 As can be seen, the Shim-pack scepter C18 120 is more effective than the other two chromatographic columns for phenolic components, and this column was ultimately selected for screening other chromatographic conditions.

[0054] Example 7: Investigation of Different Extraction Solvent Concentrations Approximately 0.5 g of pulverized and sieved dandelion material was accurately weighed and prepared into test solutions according to the test sample preparation procedure in Example 1. The difference between the samples lay in the extraction solvent: the solvents were water, 30% ethanol, 50% ethanol, 70% ethanol, and 95% ethanol, respectively. The chromatograms comparing extraction with different solvents are shown below. Figure 5 As shown, the results indicate that 70% ethanol and 70% methanol can fully extract phenolic and triterpenoid components. Considering environmental factors, 70% ethanol was further selected as the extraction solvent.

[0055] Example 8: Investigation of different extraction times Approximately 0.5 g of pulverized and sieved dandelion material was accurately weighed and prepared into test solutions according to the test sample preparation procedure in Example 1. The difference between the samples was the extraction time: 30, 60, 90, 120, and 150 min, respectively. Chromatograms comparing different extraction times are shown below. Figure 6 As shown, the results indicate that after 120 min of extraction, the area of ​​each chromatographic peak did not change significantly. Therefore, 120 min is the optimal extraction time in terms of extraction efficiency.

[0056] Example 9 Effect of different phosphoric acid concentrations on UHPLC separation efficiency Organic acids such as formic acid and acetic acid have a significant impact on the triterpenoid components of dandelion. Therefore, acetonitrile-water and acetonitrile-phosphoric acid aqueous solutions were selected to investigate the effect of phosphoric acid concentration, corresponding to phosphoric acid concentrations of 0%, 0.05%, and 0.1%, respectively. The chromatograms show that the introduction of phosphoric acid improved the chromatogram and the separation of various phenolic components, especially the highly polar components (see...). Figure 7 After comprehensive comparison, acetonitrile-0.1% phosphoric acid was finally selected for gradient elution.

Claims

1. A method for quality evaluation of dandelion medicinal material and its extract, characterized in that, Includes the following steps: (1) Preparation of test solution: Take the pulverized dandelion medicinal material or extract, add the extraction solvent to the test sample for extraction, filter, take the filtrate to obtain the test solution; (2) Preparation of reference solution: Take monocaffeoyl tartaric acid, chlorogenic acid, caffeic acid, chicoric acid, isochlorogenic acid A, luteolin, lupeol, taraxasterol and taraxerol reference standards and prepare a mixed reference solution; (3) Chromatographic analysis conditions: The column packed with octadecylsilane bonded silica gel was used with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B for gradient elution. The detection wavelength was dynamically switched. The injection volume was 1 μL. The detector was a DAD or VWD detector. The gradient elution procedure is as follows: ； (4) Establishment of fingerprint spectrum: The test solution was injected and measured, the chromatogram was recorded and imported into the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine 2012 Version" for processing to obtain the fingerprint spectrum of dandelion and calculate the similarity; (5) Quantitative analysis: Following steps (1) to (4), quantitative analysis was performed on nine phenolic and triterpenoid components in the sample, including monocaffeoyl tartaric acid, chlorogenic acid, caffeic acid, chicoric acid, isochlorogenic acid A, luteolin, lupeol, taraxasterol and taraxerol, for the quality control of medicinal materials and extracts.

2. The method according to claim 1, characterized in that, The extraction solvent in step (1) is at least one of water, methanol and ethanol.

3. The method according to claim 2, characterized in that, The extraction solvent is 70% ethanol.

4. The method according to claim 1, characterized in that, The extraction step in step (1) uses reflux extraction, and the extraction time is 120 min.

5. The method according to claim 1, characterized in that, In step (2), the solvent for preparing the reference standard is methanol; each 1 mL of the reference standard solution contains 50–200 μg of monocaffeoyl tartaric acid; each 1 mL of the reference standard solution contains 10–100 μg of chlorogenic acid; each 1 mL of the reference standard solution contains 10–100 μg of caffeic acid; each 1 mL of the reference standard solution contains 50–250 μg of chicoric acid; each 1 mL of the reference standard solution contains 5–50 μg of isochlorogenic acid A; each 1 mL of the reference standard solution contains 2–20 μg of luteolin; each 1 mL of the reference standard solution contains 2–50 μg of lupeol; each 1 mL of the reference standard solution contains 2–50 μg of taraxasterol; each 1 mL of the reference standard solution contains 2–50 μg of taraxerol.

6. The method according to claim 1, characterized in that, The specific method for dynamically switching wavelengths in step (3) is as follows: 0–35 min, 327 nm; 35–50 min, 210 nm.

7. The method according to claim 1, characterized in that, In step (3), the chromatographic column model is Shim-pack Scepter C18-120, the column size is 2.1×100 mm, the packing particle size is 1.9 μm, the flow rate is 0.3 mL / min, and the column temperature is 30℃.

8. The method according to claim 1, characterized in that, The nine common peaks identified in step (4) are monocaffeoyl tartaric acid (peak 1), chlorogenic acid (peak 2), caffeic acid (peak 3, S peak), chicoric acid (peak 4), isochlorogenic acid A (peak 5), luteolin (peak 6), lupeol (peak 7), taraxasterol (peak 8), and taraxerol (peak 9); the relative retention times of the nine common peaks are peak 1 (0.40), peak 2 (0.52), peak 3 (0.56), peak 4 (1.00), peak 5 (1.04), peak 6 (1.27), peak 7 (2.08), peak 8 (2.15), and peak 9 (2.19); the similarity is calculated and the similarity with the reference spectrum is not less than 0.

94.

9. The method according to claim 1, characterized in that, In step (5), the content determination methods of the nine index components showed a linear relationship with R≥0.99982, and the RSD of precision, repeatability and stability were all less than 2%, with the recovery rate between 97.55% and 102.69%.

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