Wild chrysanthemum flower quality comprehensive evaluation method based on UHPLC (Ultra High Performance Liquid Chromatography) and application

Abstract

The invention discloses a wild chrysanthemum flower quality comprehensive evaluation method based on UHPLC (Ultra High Performance Liquid Chromatography) fingerprint spectrum and quantitative analysis, by optimizing chromatographic conditions, sample analysis of wild chrysanthemum flower medicinal materials or formula granules or extracts can be completed within 18 minutes, and compared with a wild chrysanthemum flower formula granule national standard method, the quality of the wild chrysanthemum flower is improved by 47% or above. The established fingerprint spectrum contains 17 common peaks, synchronous quantitative determination of eight index components of phenolic acids and flavonoids is realized, and a multi-index quality control standard is provided for wild chrysanthemum flower medicinal materials or formula granules or extracts. Through combination of the fingerprint spectrum and orthogonal partial least squares discriminant analysis (OPLS-DA), 100% accuracy rate of production place determination of chrysanthemum in three wild production places can be realized, and a component-based production place determination method is provided for wild chrysanthemum production place traceability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a comprehensive quality evaluation method of wild chrysanthemum based on ultra-high performance liquid chromatography (UHPLC) fingerprint, and application thereof in quality control of wild chrysanthemum medicinal materials, formula granules and extracts and origin discrimination of medicinal materials based on ingredients. BACKGROUND

[0002] Wild chrysanthemum (Chrysanthemum indicum L.) is a traditional Chinese medicinal material with the effects of clearing heat and detoxifying, calming the liver and improving eyesight, and is widely used in clinical and health care fields. The pharmacological activities of wild chrysanthemum mainly come from flavonoids, phenolic acids, volatile oils and other ingredients. However, due to the complex chemical composition and the influence of factors such as origin, harvesting and processing, the quality of medicinal materials is uneven, which seriously affects the clinical efficacy and product stability. The current quality control of wild chrysanthemum mainly relies on the determination of single indicators (such as geniposide and chlorogenic acid) or simple physicochemical identification, which is difficult to comprehensively reflect the overall quality. The traditional high performance liquid chromatography (HPLC) method has long analysis time and low separation efficiency, which is difficult to meet the needs of rapid detection of multiple components in wild chrysanthemum. At the same time, there is a lack of research on the quality control of deep processing products such as wild chrysanthemum formula granules and extracts, and there is a lack of unified scientific evaluation standard.

[0003] Geographical environmental differences may lead to different chemical composition characteristics, but in the origin discrimination of wild chrysanthemum, the existing technology is limited to sensory experience or analysis of a small number of markers, and there is a lack of objective origin discrimination means based on multi-component fingerprint. Although fingerprint technology has been applied in some traditional Chinese medicinal materials, the research on ultra-high performance liquid chromatography (UHPLC) fingerprint that can be used for efficient discrimination analysis of the origin of wild chrysanthemum has not been systematically carried out, especially in the aspects of efficient traceability and comprehensive quality evaluation of wild chrysanthemum origin combined with chemometrics, there is still a technical gap.

[0004] CN110579548A discloses a wild chrysanthemum medicinal material fingerprint and a quality evaluation method of one test multiple evaluation, which uses C-(18) as a filler and adopts one test multiple evaluation method to quantitatively determine 12 components in the wild chrysanthemum medicinal material fingerprint; however, the component determination time is as long as 58 minutes, which has a deficiency in detection efficiency, and the determination method fails to achieve origin discrimination analysis, and a more efficient and rapid analysis method needs to be developed.

[0005] Therefore, it is of great significance to develop an efficient, accurate and comprehensive wild chrysanthemum fingerprint analysis method based on UHPLC, establish a quality control system for wild chrysanthemum medicinal materials, formula granules and extracts, and realize scientific origin discrimination based on multiple components, so as to improve the quality standard level of the wild chrysanthemum industry chain. SUMMARY

[0006] The present application aims to provide a comprehensive quality evaluation method of Chrysanthemum indicum based on ultra-high performance liquid chromatography (UHPLC) fingerprint, so as to solve the problems of long time of traditional detection and analysis method, incomplete overall quality control, lack of unified quality evaluation standard for deep processing products such as formula granules and extracts, and dependence on subjective experience or a small number of markers for traditional origin judgment method, and lack of objective analysis means based on multi-component fingerprint.

[0007] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] A comprehensive quality evaluation method of Chrysanthemum indicum based on UHPLC fingerprint and quantitative analysis, comprising the following steps:

[0009] Step 1): accurately weigh a certain amount of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, and luteolin reference substance, add a solvent to prepare a mixed reference substance stock solution, and obtain six different concentrations of series mixed reference substance solutions by equal ratio dilution method;

[0010] Step 2): take 0.2-0.4 g of Chrysanthemum indicum medicinal materials or formula granules or extract powder of different origins, accurately weigh, place in a conical flask with a plug, accurately add 50 mL of solvent, weigh, reflux extract for 0.5-2 hours, weigh again, make up the weight loss with the above-mentioned solvent, shake well, filter, and take the filtrate to obtain a test sample solution;

[0011] Step 3): detect the mixed reference substance solution and the test sample solution by ultra-high performance liquid chromatography (UHPLC), and record the fingerprint of the mixed reference substance solution and the fingerprint of the test sample solution;

[0012] Step 4): import the fingerprint of the test sample solution in step 3) into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition to generate a control fingerprint, and calculate the similarity between the fingerprints of Chrysanthemum indicum medicinal materials of different origins and the similarity with the control fingerprint.

[0013] Preferably, the solvent in steps 1) and 2) is 30% methanol solution.

[0014] Preferably, the reflux in step 2) is water bath reflux.

[0015] Preferably, the concentrations of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, and luteolin in the mixed reference substance stock solution in step 1) are 15.89 μg, 91.33 μg, 13.33 μg, 4.40 μg, 36.60 μg, 287.12 μg, 35.14 μg, and 155.58 μg / mL, respectively.

[0016] Preferably, the detection condition of the ultra-high liquid chromatography in step 3) is that the chromatographic column is a C18 small particle size chromatographic column, the mobile phase is acetonitrile (A)-0.1% phosphoric acid (B), gradient elution is carried out, the gradient elution program is: 0-10 min, 6%→23% A; 10-12 min, 23%→28% A; 12-18 min, 28%→30% A; the flow rate of the mobile phase is 0.2-0.4 mL / min; the detection wavelength is 310 nm-350 nm; and the column temperature is 25-35 ℃.

[0017] Preferably, the fingerprint of the test sample solution in step 3) is calibrated by chromatographic peak matching, 17 common peaks are calibrated, and 8 chromatographic peaks are identified by comparison with the control sample, which are: neochlorogenic acid (peak 2), chlorogenic acid (peak 4), cryptochlorogenic acid (peak 5), caffeic acid (peak 6), isochlorogenic acid B (peak 12), isochlorogenic acid A (peak 13), isochlorogenic acid C (peak 15), and buddlejaflavone (peak 17).

[0018] Preferably, the comprehensive quality evaluation method of wild chrysanthemum based on UHPLC fingerprint in step 4) is specifically: taking the 17th buddlejaflavone chromatographic peak as a reference S peak, the relative retention time of other characteristic peaks is within ±10% of the specified value, the specified value of the relative retention time is: 0.37 (peak 1), 0.38 (peak 2), 0.44 (peak 3), 0.49 (peak 4), 0.51 (peak 5), 0.54 (peak 6), 0.58 (peak 7), 0.65 (peak 8), 0.73 (peak 9), 0.76 (peak 10), 0.77 (peak 11), 0.81 (peak 12), 0.83 (peak 13), 0.85 (peak 14), 0.87 (peak 15), 0.98 (peak 16), and the similarity with the control fingerprint is greater than 0.90.

[0019] Preferably, step 5) is further included: through the fingerprint combined with the orthogonal partial least squares discriminant analysis and the receiver operating characteristic curve (ROC) analysis method, OPLS-DA analysis is carried out on the wild chrysanthemum samples from different producing areas and the 17 common peak components, and then the producing area of the wild chrysanthemum is identified.

[0020] The established multi-component content determination method in the application is: the external standard one-point method is used to simultaneously determine the content of 8 index components, the linear relationship is good (R≥0.9998), the RSD of precision, repeatability and stability is less than 2%, and the sample addition recovery rate is between 96.97% and 101.70%;

[0021] The application provides a comprehensive quality evaluation method of wild chrysanthemum by using ultra-high performance liquid chromatography (UHPLC) method, which can realize characteristic spectrum and multi-index component content determination by one chromatographic condition, and shorten the detection cycle.

[0022] Compared with the prior art, the application has the following beneficial effects:

[0023] (1) Fast and efficient: 17 common peak analysis and 8 component quantification are completed within 18 minutes, which is more than 50% higher than the efficiency of traditional HPLC.

[0024] (2) Comprehensive quality control: simultaneous monitoring of phenolic acids, flavonoids and other active ingredients, suitable for quality control of wild chrysanthemum raw materials, extracts and formula granules.

[0025] (3) Accurate identification: 100% accurate identification of three origins is realized through chemometrics model, which provides scientific basis for wild chrysanthemum genuineness research and reflects the potential of the method in wild chrysanthemum origin traceability.

[0026] (4) Wide applicability: can be extended to the quality control of other chrysanthemum medicinal materials (such as Hangzhou white chrysanthemum and tribute chrysanthemum).

[0027] (5) Stable method: precision and repeatability RSD < 2%, meeting the international common analysis method verification requirements.

[0028] The application provides reliable technical support for the standardized production, quality control and origin identification of wild chrysanthemum and related products, and has moderate application value. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be considered as limiting the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.

[0030] Figure 1is the UHPLC fingerprint chromatogram of 22 batches of wild chrysanthemum from different producing areas in Example 3 of the present application and the control fingerprint chromatogram R, wherein peak 2 is neochlorogenic acid, peak 4 is chlorogenic acid, peak 5 is cryptochlorogenic acid, peak 6 is caffeic acid, peak 12 is isochlorogenic acid B, peak 13 is isochlorogenic acid A, peak 15 is isochlorogenic acid C, and peak 17 is bauhinia blue; S1-S22 are wild chrysanthemum 01 batch-22 batch, and R is the control fingerprint chromatogram;

[0031] Figure 2 is the chromatogram of the test sample solution and the mixed control solution in the content determination in Example 3 of the present application, wherein peak 1 is neochlorogenic acid, peak 2 is chlorogenic acid, peak 3 is cryptochlorogenic acid, peak 4 is caffeic acid, peak 5 is isochlorogenic acid B, peak 6 is isochlorogenic acid A, peak 7 is isochlorogenic acid C, and peak 8 is bauhinia blue; A is the chromatogram of the test sample solution, and B is the chromatogram of the mixed control solution;

[0032] Figure 3 is the OPLS-DA score plot of samples from different producing areas in Example 5 of the present application;

[0033] Figure 4 is the permutation validation plot of samples from different producing areas in Example 5 of the present application;

[0034] Figure 5 is the loading plot of samples from different producing areas in Example 5 of the present application;

[0035] Figure 6 is the ROC plot of samples from different producing areas in Example 5 of the present application;

[0036] Figure 7 is the chromatogram of different wild chrysanthemum samples (medicinal materials, formula granules, extracts) in Example 7 of the present application; S1 is wild chrysanthemum formula granules, S2 is wild chrysanthemum extract, and S3 is wild chrysanthemum medicinal materials;

[0037] Figure 8 is the UHPLC fingerprint chromatogram of Example 1 and Comparative Examples 1 and 2 of the present application. DETAILED DESCRIPTION

[0038] The embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0039] Example 1: UHPLC fingerprint chromatogram detection method of wild chrysanthemum medicinal materials

[0040] Take 0.4 g of wild chrysanthemum medicinal powder from different origins, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 30% methanol, stopper it, weigh it, heat and reflux it for 30 minutes, take it out, cool it to room temperature, make up the lost weight, filter it, and take the filtrate to obtain the test solution.

[0041] Accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, and monanoside reference substances, and add methanol to prepare mixed reference substance stock solutions with concentrations of 15.89 μg, 91.33 μg, 13.33 μg, 4.40 μg, 36.60 μg, 287.12 μg, 35.14 μg, and 155.58 μg / mL, respectively, for qualitative analysis.

[0042] The column was packed with octadecylsilane bonded silica gel; the mobile phase was acetonitrile (A)-0.1% aqueous phosphoric acid (B) with a gradient elution: 0-10 min, 6% → 23% A; 10-12 min, 23% → 28% A; 12-18 min, 28% → 30% A; the detection wavelength was 326 nm; the flow rate was 0.25 mL / min; and the column temperature was 30°C. The theoretical plate number, calculated based on the limonene peak, should be no less than 5000.

[0043] Accurately pipette 2 μl of reference solution and test solution respectively, inject into ultra-high performance liquid chromatography, measure, and record the chromatogram.

[0044] Example 2: Investigation of the fingerprint method of wild chrysanthemum

[0045] Precision: Take 0.4 g of powder from the same batch of wild chrysanthemum sample (S1), accurately weigh it, prepare the test solution according to the preparation method and chromatographic conditions and methods under Example 1, and continuously inject and measure 6 times.

[0046] Repeatability: Take 0.4 g of powder from the same batch of wild chrysanthemum sample (S1), accurately weigh it, and prepare 6 test solutions in parallel according to the preparation method of the test solution under Example 1, and measure it under the same chromatographic conditions as above.

[0047] Stability: Take 0.4 g of powder of the same batch of wild chrysanthemum sample (S1), accurately weigh it, and test it according to the preparation and chromatographic conditions of the test solution in Example 1. The samples are injected and measured at 0, 2, 6, 12, 18, and 24 hours after preparation.

[0048] The precision, repeatability and stability methodological test investigations were all represented by 17 common chromatographic peaks, with peak 17 (linarin) retention time and peak area as controls, and the RSD values of relative retention times (RRTs) and relative peak areas (RPAs) of other chromatographic peaks were used to evaluate the feasibility of methodological experiments. The test results showed that the RSD values of RRTs and RPAs of each chromatographic peak in precision test, repeatability test and stability test were all less than 2%. The above shows that the method is stable and feasible, and can be used for fingerprint analysis of the technical solution.

[0049] Example 3: Determination of Fingerprint of 22 Batches of Wild Chrysanthemum and Identification of Common Peaks

[0050] The powder of 22 batches of wild chrysanthemum was taken, and the sample preparation method and chromatographic conditions in Example 1 were used for UHPLC chromatograph injection detection, to obtain the fingerprint of different batches of wild chrysanthemum. The chromatograms of 22 batches of wild chrysanthemum were imported into the Similarity Evaluation System of Traditional Chinese Medicine Fingerprint 2012 version similarity software, and the UHPLC fingerprint was established by the median method to generate a control fingerprint (R) composed of 17 common peaks, as shown in Figures 1-2 Among the 17 common peaks, 8 were identified by comparison with the control, which were neochlorogenic acid (peak 2), chlorogenic acid (peak 4), cryptochlorogenic acid (peak 5), caffeic acid (peak 6), isochlorogenic acid B (peak 12), isochlorogenic acid A (peak 13), isochlorogenic acid C (peak 15), and linarin (peak 17). With peak 17 linarin chromatographic peak as a reference, the relative retention times of other characteristic peaks were within ±10% of the specified value, and the specified value of relative retention time was: 0.37 (peak 1), 0.38 (peak 2), 0.44 (peak 3), 0.49 (peak 4), 0.51 (peak 5), 0.54 (peak 6), 0.58 (peak 7), 0.65 (peak 8), 0.73 (peak 9), 0.76 (peak 10), 0.77 (peak 11), 0.81 (peak 12), 0.83 (peak 13), 0.85 (peak 14), 0.87 (peak 15), 0.98 (peak 16). The relative retention times are shown in Table 1 below, and the similarity degree matching results are shown in Table 2 below.

[0051]

[0052]

[0053] Example 4: Methodological investigation of 8 index ingredient content determination

[0054] Linear relationship: the mixed control stock solution in Example 1 was taken respectively, and 6 mixed control solutions with different concentrations were obtained by equal ratio dilution method. 2 μl of each mixed control solution with different concentration was injected into the UHPLC ultra-high liquid chromatograph, and the preparation of the test sample solution and the chromatographic condition method test were carried out according to the preparation method of the test sample solution and the chromatographic condition method test in Example 1. The injection concentration was taken as the abscissa (X), and the peak area integral value was taken as the ordinate (Y) to carry out linear regression analysis. The results showed that the linear relationship of the 8 components was good in the investigation range, and the correlation coefficient was above 0.999 8. The detailed results are shown in Table 3.

[0055] Precision: 0.4 g of the same batch of wild chrysanthemum sample (S1) powder was accurately weighed, and the preparation of the test sample solution and the chromatographic condition method test were carried out according to the preparation method of the test sample solution and the chromatographic condition method test in Example 1. The sample was continuously injected for determination for 6 times.

[0056] Repeatability: 0.4 g of the same batch of wild chrysanthemum sample (S1) powder was accurately weighed, and 6 test sample solutions were prepared in parallel according to the preparation method of the test sample solution in Example 1. The same chromatographic condition was used for determination.

[0057] Stability: 0.4 g of the same batch of wild chrysanthemum sample (S1) powder was accurately weighed, and the preparation of the test sample solution and the chromatographic condition method test were carried out according to the preparation method of the test sample solution and the chromatographic condition method test in Example 1. The sample was injected for determination at 0, 2, 6, 12, 18 and 24 h after preparation.

[0058] The RSD values of the peak areas of the 8 components were used to evaluate the feasibility of the precision, repeatability and stability methodological experiments. The test results showed that the method was stable and feasible, and could be used for the determination of the content of the 8 components in the technical scheme. The detailed test results are shown in Table 2.

[0059] Accuracy: 6 portions of wild chrysanthemum samples with known content (S1) were accurately weighed, and the mixed control solution of the 8 components was added according to the proportion of 1 of the content ratio of the control to the sample. The determination was carried out according to the ultra-high liquid chromatography condition, the average recovery rate and RSD of each component were calculated, and the determination results are shown in Table 3.

[0060] Table 3 Determination results of the content determination method of the 8 components

[0061]

[0062] Example 5 Determination of the content of the 8 components in 22 batches of samples

[0063] According to the established method, the content of the 8 components in 22 batches of samples was determined according to the preparation method of the test sample solution and the chromatographic condition method test in Example 1. The determination results are shown in Table 4.

[0064] Table 4 Determination results of the content of the 8 components in 22 batches of samples

[0065]

[0066] Example 6: A component-based Chrysanthemum indicum L. origin identification method

[0067] Orthogonal partial least squares discriminant analysis (OPLS-DA) was performed on 22 batches of Chrysanthemum indicum L. and 17 components, and supervised pattern recognition was performed on the sample origin. The results showed that through OPLS-DA, the samples from Hubei, Henan and Anhui could be effectively distinguished, the independent variable fitting index R2X and the dependent variable fitting index R2Y of the model were 0.961 and 0.948 respectively, and the model prediction index (Q2) was 0.862, both R2 and Q2 were more than 0.5 (as shown in Figures 3-4 The loading plot (as shown in Figure 5 ) showed that each component affected each sample from different origins. The contents of peaks 8 and 11 were relatively high in samples from Henan, the contents of peaks 3, 10, 14, 16 and luteolin were relatively rich in samples from Anhui, and the content of caffeic acid was relatively high in samples from Hubei. The differences in common peaks in different origins provided good data support for origin identification. It could be seen from 200 times of permutation test that the intersection point of Q2 regression line and vertical axis was less than 0, indicating that the model was acceptable and there was no overfitting (as shown in Figure 4 ), and could be used for Chrysanthemum indicum L. origin identification analysis. At the same time, the classification ability of the discriminant model was analyzed by receiver operating characteristic (ROC) curve, and the AUC of each origin of the OPLS-DA model was 1 (as shown in Figure 6 ), indicating that the model could accurately identify the origin of Chrysanthemum indicum L., and the accuracy of the discriminant model was 100%. It was shown that based on the common peak information of the fingerprint established in the present application, combined with the OPLS-DA model, the precise identification of Chrysanthemum indicum L. samples from three origins could be realized.

[0068] Example 7: Quality control of Chrysanthemum indicum L. samples, formula granules and extracts

[0069] Preparation of Chrysanthemum indicum L. formula granules: 50-100 g of Chrysanthemum indicum L. was decocted for 2-3 times, 8-10 times of water was added each time, and decocted for 20-30 minutes. The first soaking was for 30 minutes, then the filtrate was combined and concentrated. Appropriate amount of excipients was added, dried, and then appropriate amount of excipients was added for granulation, and Chrysanthemum indicum L. formula granules were obtained

[0070] Preparation of Chrysanthemum indicum L. extract: Chrysanthemum indicum L. was removed, 3-6 times of 30%-70% ethanol was added, and extraction was performed for 1-3 times, each time for 30-60 minutes. The filtrate was combined and concentrated, and dried to obtain Chrysanthemum indicum L. extract.

[0071] The fingerprint of wild chrysanthemum medicinal material, formula granules and extract was determined according to the preparation method of sample solution and the chromatographic condition method of Example 1. The results showed that the relative retention time of common peaks between wild chrysanthemum formula granules and extract and medicinal material was consistent, RSD≤1%, the separation degree of chromatographic peaks was all≥1.5, the methodological verification of fingerprint and quantitative analysis met the analysis requirements. It showed that the method had strong applicability in medicinal material and formula granules.

[0072] Comparative Example 1

[0073] The present application provides a wild chrysanthemum fingerprint and content establishment method.

[0074] The wild chrysanthemum medicinal material sample prepared in Example 1 was detected, the detection wavelength of ultraviolet detector was set to 326 nm, C18 high-pressure-resistant small particle size chromatographic column was selected; the chromatographic column temperature during detection was 30-40℃; the flow rate was selected; acetonitrile and 0.1% phosphoric acid were selected as mobile phase, the mobile phase was eluted in the following way, 0-5 min, 18%→24% A; 5-7 min, 24%→29% A; 7-13 min, 29%→30% A; 13-15 min, 30%→35% A; the detection wavelength was 326 nm; the flow rate was 0.25 mL / min; the column temperature was 30℃. The obtained spectrum is shown in Figure 7 .

[0075] Comparative Example 2

[0076] The present application provides a wild chrysanthemum medicinal material fingerprint establishment method.

[0077] The wild chrysanthemum medicinal material sample prepared in Example 1 was detected, the detection wavelength of ultraviolet detector was set to 326 nm, C18 high-pressure-resistant small particle size chromatographic column was selected; the chromatographic column temperature during detection was 30-40℃; the flow rate was selected; acetonitrile and 0.1% phosphoric acid were selected as mobile phase, the mobile phase was eluted in the following way, 0-5 min, 18%→24% A; 5-7 min, 24%→29% A; 7-15 min, 29%→30% A; 15-17 min, 30%→35% A; the detection wavelength was 326 nm; the flow rate was 0.25 mL / min; the column temperature was 30℃. The obtained spectrum is shown in Figure 7 .

[0078] Analysis comparison Figure 7Among the three fingerprints, the fingerprint obtained by Example 1 has obvious peaks of each component, high resolution and high signal-to-noise ratio. The fingerprint obtained by Comparative Example 1 cannot show the characteristic peaks of each component due to poor resolution of the sample. Although the resolution of the fingerprint of Comparative Example 2 is improved, the resolution, signal intensity and abundance are all poorer than those of Example 1. Comparative Examples 1 and 2 cannot realize quality control of the wild chrysanthemum samples.

[0079] In summary, the method for establishing the fingerprint of wild chrysanthemum provided in the present embodiment can accurately separate each component in the wild chrysanthemum sample and obtain a fingerprint with obvious and complete characteristics. The method can provide multi-index quality control standards for wild chrysanthemum medicinal materials or formula granules or extracts. Furthermore, the method can realize 100% accurate determination of the producing areas of wild chrysanthemum from three producing areas by combining the fingerprint with orthogonal partial least squares discriminant analysis (OPLS-DA), thereby providing a component-based method for determining the producing areas of wild chrysanthemum.

[0080] The above merely provides specific examples of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A comprehensive evaluation method for the quality of Chrysanthemum indicum based on UHPLC fingerprint and quantitative analysis, characterized in that: The steps include: Step 1): Accurately weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, and montanside reference substances, add solvent to prepare a mixed reference substance stock solution, and use the isoproportional dilution method to obtain 6 series of mixed reference substance solutions with different concentrations; Step 2): Take 0.2g to 0.4g of wild chrysanthemum medicinal materials or formulated granules or extract powder from different origins, accurately weigh, place in a stoppered conical flask, accurately add 50mL of solvent, weigh the weight, reflux extraction for 0.5 to 2 hours, weigh again, make up the loss with the above solvent, shake well, filter, and take the filtrate to obtain the test solution; Step 3) using ultra high performance liquid chromatography (UHPLC) to detect the mixed reference solution and the test solution, and record the fingerprint of the mixed reference solution and the fingerprint of the test solution; Step 4): The fingerprint of the test solution in step 3) is imported into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition to generate a reference fingerprint, and the similarity between the fingerprints of wild chrysanthemum medicinal materials from different origins and the similarity with the reference fingerprint are calculated.

2. The method according to claim 1, characterized in that The solvent in step 1) and step 2) is 30% methanol solution.

3. The method according to claim 1, characterized in that The reflux described in step 2) is a water bath reflux.

4. The method according to claim 1, wherein The concentrations of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, and monanoside in the mixed reference stock solution in step 1) were 15.89 μg, 91.33 μg, 13.33 μg, 4.40 μg, 36.60 μg, 287.12 μg, 35.14 μg, and 155.58 μg / mL, respectively.

5. The method according to claim 1, wherein The detection conditions of the ultrahigh performance liquid chromatography described in step 3) are as follows: the chromatographic column is a C18 small particle size chromatographic column, the mobile phase is acetonitrile (A)-0.1% phosphoric acid (B), and gradient elution is performed. The gradient elution program is: 0-10 min, 6%→23% A; 10-12 min, 23%→28% A; 12-18 min, 28%→30% A; the flow rate of the mobile phase is 0.2-0.4 mL / min; the detection wavelength is 310 nm to 350 nm; and the column temperature is 25-35 ° C.

6. The method according to claim 1, characterized in that In step 3), the fingerprint of the test solution was calibrated with 17 common peaks by chromatographic peak matching, and 8 chromatographic peaks were identified by comparison with the reference, namely: neochlorogenic acid (peak 2); chlorogenic acid (peak 4); cryptochlorogenic acid (peak 5); caffeic acid (peak 6); isochlorogenic acid B (peak 12); isochlorogenic acid A (peak 13); isochlorogenic acid C (peak 15); and chloroside (peak 17).

7. The method according to claim 1, characterized in that The comprehensive evaluation method for the quality of Chrysanthemum indicum based on UHPLC fingerprint in step 4) is as follows: using the chromatographic peak of No. 17 monanthoside as the reference S peak, the relative retention time of other characteristic peaks is specified to be within ±10% of the specified value, and the specified value of the relative retention time is: 0.37 (peak 1), 0.38 (peak 2), 0.44 (peak 3), 0.49 (peak 4), 0.51 (peak 5), 0.54 (peak 6), 0.58 (peak 7), 0.65 (peak 8), 0.73 (peak 9), 0.76 (peak 10), 0.77 (peak 11), 0.81 (peak 12), 0.83 (peak 13), 0.85 (peak 14), 0.87 (peak 15), and 0.98 (peak 16), and the similarity with the control fingerprint is greater than 0.

90.

8. The method according to claim 1, characterized in that The method also includes step 5): performing OPLS-DA analysis on wild chrysanthemum samples from different origins and 17 common peak components through fingerprint analysis combined with orthogonal partial least squares discriminant analysis and receiver operating characteristic curve (ROC) analysis, thereby identifying the origin of the wild chrysanthemum.

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