Method for evaluating quality of chrysanthemum by quantitative analysis of multi-components by single marker and application of method

Abstract

The invention discloses a method for evaluating the quality of chrysanthemum through a single-marker multi-evaluation method and application of the method, and belongs to the technical field of medicinal material quality control. Chlorogenic acid and galuteolin are used as internal reference substances, and high performance liquid chromatography is adopted. Chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, and luteoloside, luteolin-7-O-beta-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-beta-D-(6 ''-O-malonyl)-glucoside, and apigenin 7-O-(6''-O-malonyl) and calculating the contents of the 17 components in the chrysanthemum according to the relative retention time and correction factor of-beta-D-glucoside, diosmetin, linarin, luteolin and acacetin. The method disclosed by the invention is simple, convenient, efficient, low in cost, high in sensitivity, good in repeatability and accurate and reliable in result, and realizes quantitative analysis of multiple components by one marker on the quality control of the chrysanthemum.

Description

Technical Field

[0001] The present invention belongs to the technical field of quality control of medicinal materials, and particularly relates to a method for evaluating the quality of Chrysanthemi Flos by quantitative analysis of multicomponents by single marker (QAMS) and its application. Background Art

[0002] The components of traditional Chinese medicine are complex and variable, and their pharmacological activities and clinical efficacy are often the result of the combined action of multiple components. Using the content of a single component or a certain type of component as an evaluation index usually cannot comprehensively and fully reflect the quality of traditional Chinese medicine. However, controlling the quality of traditional Chinese medicine by measuring the content of multiple components has many problems such as scarce, unstable, and high-cost reference substances, which cause great difficulties in detection.

[0003] Quantitative analysis of multicomponents by single marker (QAMS) is a new model for simultaneous quality control of multiple components in the field of traditional Chinese medicine. By using the internal functional relationship and proportional relationship of the active components of traditional Chinese medicine, the simultaneous determination of multiple components (for which reference substances are difficult to obtain or supply) can be achieved by only measuring one stable, inexpensive, and easily available component, which can effectively solve the problems such as the large number of reference substances used, high cost, and difficult supply in the process of multi-index quality control of traditional Chinese medicine. This method will be the development direction of simultaneous quantitative determination of multiple components in traditional Chinese medicine.

[0004] Chrysanthemi Flos is the capitulum of the plant Chrysanthemum morifolium Ramat of the Compositae family and is an important bulk medicinal material. Chrysanthemi Flos contains active components such as organic acids, flavonoids, vitamin E, ellagic acid, and amino acids, and has the effects of dispelling wind and heat, calming the liver fire, improving eyesight, and expelling toxins. Chrysanthemi Flos has a wide range of uses. In addition to being used in medical prescriptions, it is also an important raw material for more than 40 kinds of traditional Chinese medicine preparations.

[0005] At present, most of the research on the quality standard of chrysanthemum is the quantitative analysis of multiple components by external standard method. Not only does it require high purity of reference materials and expensive ingredients, but it is also difficult to reflect the intrinsic quality of traditional Chinese medicine. In the provisions of the Chinese Pharmacopoeia on chrysanthemum, the high performance liquid external standard method is used to determine the content of chlorogenic acid, luteolin and isochlorogenic acid A in chrysanthemum. Patent CN111537653B discloses a method for determining the content of chemical components in chrysanthemum, which uses ultra-high performance liquid chromatography-mass spectrometry and external standard method to determine the content of 13 chemical components in chrysanthemum; the 13 chemical components include: isochlorogenic acid C, hesperidin, cosmosin, quercetin, acacia, hyperoside, chlorogenic acid, luteolin-7-O-glucuronide, luteolin, apigenin, diosmetin, isoquercitrin, cryptochlorogenic acid; ... 0133153A discloses a method for simultaneously determining the contents of five chemical components in chrysanthemum medicinal materials by high performance liquid chromatography, and the contents of five components, namely, chlorogenic acid, luteolin-7-O-β-D-glucoside, luteolin-7-O-β-D-glucuronide, 3,5-dicaffeoylquinic acid and apigenin-7-O-β-D-glucoside, are determined by external standard method; the methods for determining the effective components in chrysanthemum reported in the existing literature are as follows: (1) using Shim-pack C18 (250mm×4.6mm, 5μm) chromatographic column; the mobile phase is acetonitrile (A)-0.1% phosphoric acid solution (B), gradient elution; volume flow rate is 1.0mL / min; column temperature is 30℃; injection volume is 10μL; detection wavelength is 348nm. Chlorogenic acid is used as the internal reference, and its correction factor with luteolin, quercetin and acacetin is established, and the content of each component is calculated. (Wang Fucheng, Gong Daofeng, Shi Weijing, et al. Simultaneous determination of the contents of four active ingredients in chrysanthemum by a one-test-multiple-evaluation method [J]. Chinese Medicinal Materials, 2016, 39(05): 1086-1089.); (2) Using 3-caffeoylquinic acid as the internal standard, the correction factors of this component and 5-caffeoylquinic acid, 4-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid were established, and the contents of the six caffeoylquinic acids were calculated using the correction factors. (Deng Xiuqing, Mao Yinchun, Wu Yichao, et al. Determination of six caffeoylquinic acids in chrysanthemum by a one-test-multiple-evaluation method [J]. West China Journal of Pharmacy, 2016, 31(06): 640-644.).

[0006] The evaluation methods of chrysanthemum quality reported so far have problems such as few test components, many standard products, complicated operation steps, long test time and high cost. There is no research report on the determination of 7 organic acids and 10 flavonoids in chrysanthemum by high performance liquid chromatography and establishing correction factors for organic acids and flavonoids. Therefore, it is of great significance to establish a stable, controllable, low-cost, accurate and efficient method for chrysanthemum quality control. Summary of the invention

[0007] In order to solve the technical problems existing in the prior art, the present invention provides an HPLC method for determining the contents of 17 organic acids and flavonoids, namely chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteoloside, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucoside, apigenin, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, diosmetin, buddleoside, luteolin and acacetin, in chrysanthemum. This method uses chlorogenic acid and luteoloside as internal references, and calculates the contents of neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, diosmetin, buddleoside, luteolin and acacetin with correction factors, thereby establishing a comprehensive and controllable quality evaluation method for chrysanthemum, greatly reducing the detection cost, and ensuring that the detection results are stable and controllable.

[0008] The present invention is achieved through the following technical solutions:

[0009] The first aspect of the present invention relates to a method for evaluating the quality of chrysanthemum by the multiple-component determination with a single reference method. In this method, chlorogenic acid and luteoloside are used as internal references, and high performance liquid chromatography is adopted. The contents of 17 components in chrysanthemum are calculated based on the relative retention times and correction factors of chlorogenic acid with neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C, and the relative retention times and correction factors of luteoloside with luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, diosmetin, buddleoside, luteolin and acacetin.

[0010] Preferably, the specific steps of the method are as follows:

[0011] (1) Preparation of the test solution: Powder the chrysanthemum, take the chrysanthemum powder, extract it with an extraction solvent by ultrasonic extraction, and filter to obtain the test solution;

[0012] (2) Preparation of the mixed reference standard solution: Weigh accurately appropriate amounts of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteoloside, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, diosmetin, buddleoside, luteolin, and acacetin, dissolve them to obtain single reference standard stock solutions, and then mix them to obtain the mixed reference standard stock solution, from which the mixed reference standard solution is obtained;

[0013] (3) Precisely pipette the reference standard solution and the test sample solution, inject them into a high-performance liquid chromatograph for HPLC determination. Using chlorogenic acid and luteoloside in the reference standard solution as internal standards, calculate the correction factors of neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, diosmetin, buddleoside, luteolin, and acacetin. Calculate the contents of 7 organic acid compounds, namely chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C in the test sample by comparing with the correction factor of chlorogenic acid, and calculate the contents of 10 flavonoid compounds, namely luteoloside, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, diosmetin, buddleoside, luteolin, and acacetin in the test sample by comparing with the correction factor of luteoloside;

[0014] The specific conditions for HPLC determination in step (3) are as follows: Chromatographic column: C18 chromatographic column; injection volume: 1 - 10 μL; detection wavelength: 322 - 332 nm; column temperature: 20 - 30 °C; flow rate: 0.8 - 1.0 mL / min; mobile phase: 0.1% phosphoric acid water as mobile phase A and acetonitrile as mobile phase B; gradient elution program: 0 - 15 min, 90% A → 80% A; 15 - 25 min, 80% A → 78% A; 25 - 35 min, 78% A → 65% A.

[0015] Preferably, the C18 chromatographic column is selected from one of the Waters CORTECS T3 chromatographic column with a specification of 4.6×150mm, 2.7μm, the Waters XSelect Premier T3 chromatographic column with a specification of 4.6×150mm, 2.7μm, and the Agilent ZorbaxEclipse Pluse C18 chromatographic column with a specification of 2.1×100mm, 2.7μm.

[0016] More preferably, the specific conditions for HPLC determination are as follows:

[0017] Chromatographic column: Waters CORTECS T3 chromatographic column with a specification of 4.6×150mm, 2.7μm; detection wavelength: 327nm; injection volume: 5μL; flow rate: 1.0mL / min; column temperature: 25°C; mobile phase: 0.1% phosphoric acid water as mobile phase A, acetonitrile as mobile phase B; gradient elution program: 0 - 15min, 90%A → 80%A; 15 - 25min, 80%A → 78%A; 25 - 35min, 78%A → 65%A.

[0018] Preferably, the specific steps for preparing the test solution in step (1) are as follows: Weigh accurately 0.25g of chrysanthemum powder and place it in a stoppered conical flask, add an appropriate amount of extraction solvent, weigh, perform ultrasonic extraction, make up the weight, shake well and filter, and filter the filtrate through a 0.45μm microporous filter membrane to obtain the solution.

[0019] More preferably, the extraction solvent in step (1) is selected from 70% methanol, methanol or ethanol; 70% methanol is preferred.

[0020] More preferably, the extraction time in step (1) is 15min, 30min, 40min or 60min; 40min is preferred.

[0021] More preferably, the mass - to - volume ratio of the material to the liquid during extraction in step (1) is 0.25g:10 - 50mL, preferably 0.25g:25mL.

[0022] Preferably, the preparation of the mixed standard solution in step (2) includes the following steps:

[0023] B1: Accurately weigh about 5 - 10 mg of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteoloside, luteolin - 7 - O - β - D - glucuronide, apigenin - 7 - O - glucuronide, apigenin, luteolin - 7 - O - β - D - (6'' - O - malonyl) - glucoside, apigenin 7 - O - (6'' - O - malonyl) - β - D - glucoside, diosmetin, buddleoside, luteolin, acacetin respectively, and place them in 10 mL or 50 mL volumetric flasks;

[0024] B2: Add an appropriate amount of 70% methanol solution to dissolve and make up to the mark to prepare a single standard stock solution;

[0025] B3: Accurately pipette an appropriate volume of the 17 standard stock solutions in step B2 into a 20 mL volumetric flask, dilute with 70% methanol to the mark, and prepare a mixed reference stock solution with different concentrations;

[0026] B4: Accurately pipette 0.2 mL, 0.4 mL, 1 mL, 1.6 mL of the mixed reference stock solution in step B3 into 2 mL volumetric flasks respectively, dilute with 70% methanol to the mark to obtain a series of mixed reference working solutions.

[0027] Preferably, the correction factors between neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C and the internal reference substance chlorogenic acid in step (3), and the correction factors between luteolin - 7 - O - β - D - glucuronide, apigenin - 7 - O - glucuronide, apigenin, luteolin - 7 - O - Β - D - (6'' - O - malonyl) - glucoside, apigenin 7 - O - (6'' - O - malonyl) - β - D - glucoside, diosmetin, buddleoside, luteolin, acacetin and the internal reference substance luteoloside are calculated according to the following method:

[0028] a) Accurately pipette 5 μL of the mixed reference working solution and inject it into a high - performance liquid chromatograph;

[0029] b) Determine the peak areas of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteoloside, luteolin - 7 - O - β - D - glucuronide, apigenin - 7 - O - glucuronide, apigenin, luteolin - 7 - O - Β - D - (6'' - O - malonyl) - glucoside, apigenin 7 - O - (6'' - O - malonyl) - β - D - glucoside, diosmetin, buddleoside, luteolin, acacetin by HPLC;

[0030] c) Calculate the correction factors of chlorogenic acid with neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C, and the correction factors of luteoloside with luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, diosmetin, buddleoside, luteolin, and acacetin according to the correction factor calculation formula f = (As / Cs) / (Ai / Ci), where Cs is the mass concentration of the internal reference substance, As is the chromatographic peak area of the internal reference substance, Ci is the mass concentration of other components, and Ai is the chromatographic peak area of other components.

[0031] The second aspect of the present invention relates to the application of the method for evaluating the quality of chrysanthemum by the multi-component quantification method in the quality standard of chrysanthemum extract or related preparations containing chrysanthemum.

[0032] Compared with the prior art, the beneficial effects that the present invention can achieve are:

[0033] The beneficial effects of the present invention are: (1) The method of the present invention is based on high performance liquid chromatography technology, and the chromatographic conditions are optimized. While ensuring that the resolution meets the requirements, the detection time is greatly shortened, the cost is reduced, the operation is simple, easy to master, and convenient for further popularization; (2) The present invention uses chlorogenic acid and luteoloside as internal reference substances at the same time, which can more accurately calculate the contents of organic acid components and flavonoid components in chrysanthemum. Chlorogenic acid and luteoloside are cheap and easy to obtain, thus greatly reducing the detection cost. Moreover, this method is rapid, accurate, and can more comprehensively control the quality of chrysanthemum, which is of great significance for the quality control of the large-scale medicinal material chrysanthemum and ensuring its clinical efficacy. Description of the Drawings

[0034] Figure 1 : HPLC chromatograms of the mixed standard solution and the test solution. In the figure, A is the mixed standard solution, B is the test solution, and the numbers 1-17 are in sequence: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, luteoloside, luteolin-7-O-β-D-glucuronide, isochlorogenic acid B, isochlorogenic acid A, apigenin-7-O-glucuronide, apigenin, isochlorogenic acid C, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, diosmetin, buddleoside, luteolin, and acacetin; It should be noted that: the peak height of the 8th peak of isochlorogenic acid A in B is too high to provide a complete chromatogram, but it does not affect the determination of this peak and the calculation of the corresponding values;

[0035] Figure 2 : Effects of different extraction conditions on the contents of organic acid components and flavonoid components in chrysanthemum. Detailed implementation mode

[0036] The present invention will be described below through embodiments to make the technical solution of the present invention easier to understand and master, but the present invention is not limited thereto. The experimental methods described in the following embodiments are all conventional methods unless otherwise specified; the medicinal materials and reagents can be obtained from commercial channels unless otherwise specified; and the performance of products from different sources has no significant impact.

[0037] The equipment used in the present invention includes: Agilent 1260 Infinity II HPLC; Waters ACQUITY HPLC; 5430R high-speed refrigerated centrifuge; KQ52000E ultrasonic cleaner; XSR105 electronic balance; ME204 electronic balance; IQ7000 ultrapure water instrument.

[0038] The reagent materials selected in the present invention are: chrysanthemum medicinal materials (purchased externally), neochlorogenic acid (product number: B21396), chlorogenic acid (product number: A10016), cryptochlorogenic acid (product number: B21587), caffeic acid (product number: B20660), luteoloside (product number: B20887), luteolin-7-O-β-D-glucuronide (product number: A10216), isochlorogenic acid B (product number: B21540), isochlorogenic acid A (product number: B21539), apigenin-7-O-glucuronide (product number: B20985), isochlorogenic acid C (product number: B21541), apigenin 7-O-(6”-O-malonyl)-β-D-glucoside (product number: B26437), buddleoside (product number: PS000691), apigenin (product number: B20981), luteolin-7-O-β-D-(6”-O-malonyl)-glucoside (product number: B28125), diosmetin (product number: B21369), luteolin (product number: B20888), acacetin (product number: B20627) are all purchased from Shanghai Yeyuan Technology Co., Ltd., acetonitrile (chromatographic grade, Merck), water (self-made, first-grade water), methanol (analytical grade, Sinopharm Chemical Reagent Co., Ltd.), phosphoric acid (analytical grade, Sinopharm Chemical Reagent Co., Ltd.).

[0039] Example 1:

[0040] Determination of the contents of organic acid components (chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C) and flavonoid components (luteoloside, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, diosmetin, buddleoside, luteolin and acacetin) in Chrysanthemi Flos by multi-component assay with single internal standard method, the specific steps are as follows:

[0041] (1) Preparation of mixed standard solution: Accurately weigh about 10 mg of reference substances of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin-7-O-β-D-glucuronide and apigenin 7-O-(6”-O-malonyl)-β-D-glucoside respectively, and place them in 10 mL volumetric flasks; accurately weigh about 5 mg of reference substances of luteoloside and buddleoside respectively, and place them in 50 mL volumetric flasks; accurately weigh about 10 mg of reference substances of apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, diosmetin, luteolin and acacetin, place them in 50 mL volumetric flasks, add appropriate amount of 70% methanol to dissolve respectively, and dilute to the scale, shake well, and prepare single standard solutions with concentrations of 1.034 mg / mL, 1.095 mg / mL, 1.061 mg / mL, 0.981 mg / mL, 0.995 mg / mL, 1.079 mg / mL, 1.023 mg / mL, 1.034 mg / mL, 0.880 mg / mL, 0.077 mg / mL, 0.099 mg / mL, 0.251 mg / mL, 0.214 mg / mL, 0.201 mg / mL, 0.233 mg / mL, 0.204 mg / mL and 0.218 mg / mL respectively. Accurately pipette 0.5, 1, 0.5, 1, 1, 1, 1, 1, 0.4, 1.5, 1, 1, 0.6, 1, 1, 1, 1 mL of the above 17 standard solutions into a 20 mL volumetric flask, dilute to the scale with methanol, shake well, and use it as the mixed standard solution for standby;

[0042] (2) Preparation of test solution: Take about 0.25 g of Chrysanthemi Flos powder (passed through No. 1 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 70% methanol, stopper it, weigh it, ultrasonically treat it for 40 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake well, filter, take the continuous filtrate, filter it through a 0.45 μm microporous filter membrane to obtain the test solution for standby;

[0043] (3) Chromatographic conditions:

[0044] Chromatographic column: Chromatographic column: Waters CORTECS T3 chromatographic column (4.6mm×150mm, 2.7μm), injection volume: 5μL, detection wavelength: 327nm; flow rate: 1.0mL / min; column temperature: 25°C; mobile phase: 0.1% phosphoric acid (A) acetonitrile (B), wherein the gradient elution process is as follows: 0-15min, 90%A→80%A; 15-25min, 80%A→78%A; 25-35min, 78%A→65%A;

[0045] (4) Determination of relative retention time and correction factor: Accurately pipette 5 μL of the series of mixed standard working solutions in step (1) and inject them into the high performance liquid chromatograph respectively. Perform analysis and determination according to the chromatographic conditions in step (3), record the chromatographic peak area and retention time, and calculate the relative retention time of chlorogenic acid and neochlorogenic acid according to the correction factor calculation formula fs / i=(As / Cs) / (Ai / Ci) (where Cs is the internal reference concentration, As is the internal reference chromatographic peak area, Ai is the analyte concentration, and Ci is the analyte chromatographic peak area). , cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6"-O-malonyl)-glucoside, apigenin 7-O-(6"-O-malonyl)-β-D-glucoside, dioscorea glycoside, montanol, luteolin and acacetin correction factors, determine the relative retention time and correction factor; the results are shown in the following table:

[0046] Table 1: Relative retention time and correction factor of organic acid components to be tested

[0047]

[0048] Table 2: Relative retention time and correction factor of flavonoid components to be tested

[0049]

[0050]

[0051] (5) Content determination of the sample: Precisely pipette 5 μL each of the chlorogenic acid and luteoloside reference substance solutions and the test solution, inject them into the high-performance liquid chromatograph, and determine under the chromatographic conditions of system suitability in step (3). According to the correction factor in step (4), calculate the contents of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteoloside, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, diosmetin, buddleoside, luteolin, and acacetin by the calculation formula of multi-component quantification by single standard, and the calculation formula of multi-component quantification by single standard is as follows:

[0052]

[0053] Among them, w is the content of each component to be determined, fs / i is the correction factor of each component to be determined, As is the chromatographic peak area of the remaining components to be determined, Cr is the concentration of the chlorogenic acid or luteoloside reference substance, V is the total volume, Ar is the peak area of chlorogenic acid or luteoloside, and m is the weighed amount of the sample.

[0054] The results showed that the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteoloside, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, diosmetin, buddleoside, luteolin, and acacetin in the chrysanthemum sample were 0.034%, 0.884%, 0.054%, 0.009%, 0.158%, 2.077%, 0.627%, 0.127%, 0.034%, 0.624%, 0.106%, 0.015%, 0.367%, 0.085%, 0.028%, 0.101%, and 0.127% respectively.

[0055] Example 2: Effects of extraction solvents on the contents and stabilities of organic acid components and flavonoid components in chrysanthemum

[0056] Respectively change the extraction solvents in step (2) of Example 1 to 70% methanol, methanol, and ethanol, and keep other operation methods unchanged to obtain the test solutions of the 70% methanol solvent group, the ethanol solvent group, and the methanol solvent group. The results showed that compared with the 70% methanol solvent group, the contents of organic acid components in the test solutions prepared from the methanol solvent group and the ethanol solvent group were lower, as shown in Figure 2 .

[0057] Example 3: Effect of ultrasonic extraction time on the contents of organic acid components and flavonoid components in chrysanthemum

[0058] Based on Example 1, the effect of ultrasonic extraction time on the contents of organic acid components and flavonoid components in chrysanthemum in the preparation method of the test solution of the present invention was studied:

[0059] The ultrasonic extraction time in step (2) of Example 1 was respectively changed to 15 min, 30 min, 40 min and 60 min, and other operation methods remained unchanged, obtaining the 15-min ultrasonic group, 30-min ultrasonic group, 40-min ultrasonic group and 60-min ultrasonic group. The results showed that the contents of organic acid components and flavonoid components in the test solutions prepared by the 40-min ultrasonic group and 60-min ultrasonic group were relatively high, as shown in Figure 2 .

[0060] Example 4: Effect of solid-liquid ratio on the contents of organic acid components and flavonoid components in chrysanthemum

[0061] Based on Example 1, the effect of solid-liquid ratio on the contents of organic acid components and flavonoid components in chrysanthemum in the preparation method of the test solution of the present invention was studied:

[0062] The volume of the extraction solvent in step (2) of Example 1 was respectively changed to 10 mL, 25 mL and 50 mL, and other operation methods remained unchanged, obtaining the 10-mL volume group, 25-mL volume group and 50-mL volume group. The results showed that the contents of organic acid components and flavonoid components in the test solutions prepared by the 25-mL volume group and 50-mL volume group were relatively high, as shown in Figure 2 .

[0063] Example 5: Effect of extraction method on the contents of organic acid components and flavonoid components in chrysanthemum

[0064] Based on Example 1, the effect of extraction method on the contents of organic acid components and flavonoid components in chrysanthemum in the preparation method of the test solution of the present invention was studied:

[0065] The extraction method in step (2) of Example 1 was respectively changed to heating reflux extraction and ultrasonic extraction, and other operation methods remained unchanged, obtaining the heating reflux extraction group and the ultrasonic extraction group. The results showed that the contents of organic acid components and flavonoid components in the test solution prepared by the ultrasonic extraction group were relatively high, as shown in Figure 2 .

[0066] Example 6: Methodology investigation

[0067] Based on Example 1, the methodology of the method of the present invention was verified from the following aspects:

[0068] 6.1 Investigation of linear relationship

[0069] Precisely pipette 5 μL of the series of mixed standard working solutions in step (1) and inject them into the high-performance liquid chromatograph respectively. Analyze and determine according to the chromatographic conditions in step (3), record the corresponding peak areas. Using the mass concentration (μg / mL) as the abscissa (x) and the peak area as the ordinate (y), plot the standard curve to obtain the linear relationships of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteoloside, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, diosmetin, buddleoside, luteolin and acacetin as shown in the table.

[0070] Table 3: Linear relationships of each component

[0071]

[0072]

[0073] 6.2 Precision test

[0074] Precisely pipette the test solution in step (2) respectively and analyze and determine according to the chromatographic conditions in step (3). Inject samples continuously for 6 times, record the corresponding peak areas, and calculate the RSD values of the peak areas of each analyte. See Table 4. The results show that the precision of the instrument is good.

[0075] 6.3 Stability test

[0076] Analyze and determine the test solution in step (2) according to the chromatographic conditions in step (3). Inject samples at 0, 3, 6, 9, 12, and 24 h respectively, record the corresponding peak areas, and calculate the RSD of the peak areas of each analyte. See Table 4. The results show that the test solution is stable within 24 h.

[0077] 6.4 Repeatability test

[0078] Precisely weigh 6 portions of chrysanthemum powder, each portion being 0.25 g, prepare 6 portions of test solutions according to the method in step (2), analyze and determine under the chromatographic conditions in step (3), record the corresponding peak areas, and calculate the RSD of the contents of each component in the 6 samples. See Table 4. The results show that the method of the present invention has good repeatability.

[0079] 6.5 Spiking recovery test

[0080] Accurately weigh 0.10 g, 0.125 g, and 0.25 g of chrysanthemum powder with known content, three portions each. Precisely add an equal mass of the mixed standard solution to each portion, prepare the test solution according to the method in step (2), and conduct analysis and determination under the chromatographic conditions in step (3). The results are shown in Table 4, indicating that the method of the present invention has good accuracy.

[0081] Table 4: Investigation of precision, repeatability, stability, and accuracy

[0082]

[0083]

[0084] Example 7: Investigation of the durability of the correction factor

[0085] Based on Example 1, the effects of different detection wavelengths, chromatographic columns, instruments, column temperatures, and flow rates on the correction factor in the method of the present invention were investigated respectively.

[0086] 7.1 Effect of different detection wavelengths on the correction factor

[0087] Take the mixed standard solution in step (3), and investigate the effects of different detection wavelengths at 322 nm, 327 nm, and 332 nm on the correction factor. Using chlorogenic acid and luteoloside as the internal standard peaks, calculate the correction factors of other organic acid components to be measured and the correction factors of flavonoid components to be measured respectively. The results show that the RSD of the correction factors of each component is < 2.0%. The results indicate that there is no significant difference in the correction factors measured at different detection wavelengths. See Tables 5 and 6.

[0088] 7.2 Effect of different chromatographic columns on the correction factor

[0089] Using a Waters high-performance liquid chromatograph, investigate the effects of three different chromatographic columns, namely Waters CORTECS T3 (chromatographic column 1), Waters XSelect Premier T3 (chromatographic column 2), and Agilent Zorbax Eclipse Pluse C18 (chromatographic column 3), on the correction factors of each component. Using chlorogenic acid and luteoloside as the internal standard peaks, calculate the correction factors of other organic acid components to be measured and the correction factors of flavonoid components to be measured respectively. The results show that the RSD of the correction factors of each component is < 2.0%. The results indicate that there is no significant difference in the correction factors measured with different chromatographic columns. See Tables 5 and 6.

[0090] 7.3 Effect of different instruments on the correction factor

[0091] Using Waters CORTECS T3 chromatographic column, the effects of two different instruments, Waters Arc HPLC and Agilent 1260 Infinity II HPLC, on the correction factors were investigated. Chlorogenic acid and luteoloside were used as internal standard peaks, and the correction factors of other organic acid components to be measured and the correction factors of flavonoid components to be measured were calculated respectively. The results showed that the RSD of the correction factors of each component was <2.0%. The results indicated that there was no significant difference in the correction factors measured by different detection instruments, as shown in Table 5 and Table 6.

[0092] 7.4 Effects of Different Column Temperatures and Flow Rates on the Correction Factors

[0093] The effects of different column temperatures (20, 25, 30 °C) and different flow rates (0.8, 1.0, 1.2 mL / min) on the correction factors were investigated. Chlorogenic acid and luteoloside were used as internal standard peaks, and the correction factors of other organic acid components to be measured and the correction factors of flavonoid components to be measured were calculated respectively. The results showed that the RSD of the correction factors of each component was <2.0%. The results indicated that there was no significant difference in the correction factors measured at different column temperatures and flow rates, as shown in Table 5 and Table 6.

[0094] Table 5: Effects of Different Detection Wavelengths, Chromatographic Columns, Instruments, Column Temperatures and Flow Rates on the Correction Factors of Organic Acid Components

[0095]

[0096]

[0097] Table 6: Effects of Different Detection Wavelengths, Chromatographic Columns, Instruments, Column Temperatures and Flow Rates on the Correction Factors of Flavonoid Components

[0098]

[0099]

[0100] 7.5 Localization of Chromatographic Peaks

[0101] The accurate positioning of chromatographic peaks is a prerequisite for ensuring the application of the QAMS method. Generally, parameters such as retention time difference and relative retention time can be used for positioning. In this method, the reproducibility of the relative retention time of each component to be measured was investigated on two high-performance liquid chromatographs, Agilent 1290 infinity Ⅱ and Waters Acquity, and on three different specifications of chromatographic columns, Waters CORTECS T3 (column 1), Agilent Zorbax Eclipse Pluse C18 (column 2), and Waters CORTECS C18 (column 3). The results showed that the RSD values of the relative retention times of 17 components under different instruments and chromatographic columns were all less than 2.0%, indicating that this method can accurately locate the target peaks. Therefore, it is feasible to use the relative retention time as a method for positioning the target chromatographic peaks of chrysanthemum, as shown in Tables 7 and 8.

[0102] Table 7: Relative retention times of organic acid components measured on different chromatographic columns and different instruments

[0103]

[0104] Table 8: Relative retention times of flavonoid components measured on different chromatographic columns and different instruments

[0105]

[0106] Example 8: Comparison between the Quantitative Analysis of Multiple Components by Single Marker (QAMS) method and the External Standard Method (ESM)

[0107] Based on Example 1, 10 batches of chrysanthemum samples were respectively prepared into chrysanthemum powders according to step (2), and the test solutions were prepared according to step (3). The contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteoloside, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, diosmetin, buddleoside, luteolin, and acacetin in the samples were determined by the External Standard Method (ESM). Then, they were calculated by the Quantitative Analysis of Multiple Components by Single Marker (QAMS) method established in the present invention. The results showed that the P values were all 0.99, indicating that there was no significant difference between the two methods, suggesting that this method can be used for the quality evaluation of chrysanthemum, as shown in Table 9.

[0108] Table 9-1: Comparison of contents between the External Standard Method and the Quantitative Analysis of Multiple Components by Single Marker (QAMS) method

[0109]

[0110]

[0111] Table 9-2: Comparison of Contents between External Standard Single-Point Method and QAMS

[0112]

[0113] Example 9: Comparison with the Literature "Simultaneous Determination of 12 Components in Flos Chrysanthemi Indici by QAMS"

[0114] The selection of internal reference substance is a prerequisite for ensuring the accuracy of correction factor and relative retention time. Generally, components with relatively high stability, high content, low price and easy availability are used as internal reference substances. Based on Example 1, according to the correction factor calculation formula in step (4), taking chlorogenic acid as the internal reference substance (Method 1), luteoloside as the internal reference substance (Method 2), and chlorogenic acid and luteoloside as the internal reference substances simultaneously (Method 3), the correction factors and relative retention times of organic acid components and flavonoid components are calculated respectively, as shown in Table 10.

[0115] Table 10: Correction Factors and Relative Retention Times Obtained with Different Internal Reference Substances

[0116]

[0117]

[0118] The contents are calculated respectively with the correction factors obtained by the three methods, and at the same time, the contents of components in the samples determined by the external standard single-point method (ESM) are compared, as shown in Table 11.

[0119] Table 11: Results of Content Comparison (%)

[0120]

[0121]

[0122] It can be found from the content comparison results in Table 11 that there is no significant difference between the content calculated with the correction factor of organic acid components obtained with chlorogenic acid as the internal reference substance and the content calculated by the external standard single-point method, while there is a significant difference between the content of flavonoid components and the content calculated by the external standard single-point method. There is no significant difference between the content of flavonoid components calculated with luteoloside as the internal reference substance and the content calculated by the external standard single-point method, while there is a significant difference between the content of organic acid components and the content calculated by the external standard single-point method. Therefore, choosing chlorogenic acid as the internal reference substance for organic acid components and luteoloside as the internal reference substance for flavonoid components, and calculating the correction factors respectively, can more truly reflect the quality of chrysanthemum samples.

[0123] Example 10: Detection Cost Analysis

[0124] As can be seen from Table 12, the total cost of purchasing 17 reference substances on the market exceeds 20,100 yuan. The method of the present invention uses the multiple components by single marker method, and only needs to use chlorogenic acid and luteoloside reference substances (a total of 640 yuan) to detect the contents of 7 organic acid components and 10 flavonoid components, greatly reducing the detection cost.

[0125] Table 12: Price Details of 17 Reference Substances (Specification: 20 mg)

[0126]

[0127]

[0128] The above test results show that the detection method of the present invention has high practicability, short time consumption, simple operation, low cost, accurate results and good repeatability. It can make up for the deficiencies of existing single-component and multi-index content determination methods, and can effectively detect the contents of 17 compounds including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteoloside, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, diosmetin, buddleoside, luteolin and acacetin in chrysanthemum, and can more comprehensively reflect the quality status of chrysanthemum, providing a basis for establishing quality evaluation standards for different varieties of chrysanthemum medicinal materials. At the same time, it is of great significance for the quality control of bulk medicinal materials chrysanthemum and ensuring its clinical efficacy.

[0129] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art does not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for evaluating chrysanthemum quality by one-test-multiple-evaluation method, characterized in that: The method uses chlorogenic acid and luteolin as internal reference substances, adopts high performance liquid chromatography, and calculates the contents of the above 17 components in chrysanthemum through the relative retention time and correction factor of chlorogenic acid and neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C, and the relative retention time and correction factor of luteolin and luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6"-O-malonyl)-glucoside, apigenin 7-O-(6"-O-malonyl)-β-D-glucoside, dioscorea glycoside, montanol, luteolin and acacetin.

2. The method for evaluating chrysanthemum quality by one-test-multiple-evaluation method according to claim 1, characterized in that: The specific steps of the method are: (1) Preparation of the test solution: grind chrysanthemum into powder, extract the chrysanthemum powder with an ultrasonic extraction solvent, and filter to obtain the test solution; (2) Preparation of mixed standard solution: accurately weigh appropriate amounts of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6"-O-malonyl)-glucoside, apigenin 7-O-(6"-O-malonyl)-β-D-glucoside, dioscorea glycoside, montanol, luteolin, and acacetin, dissolve them to obtain single standard stock solutions, and mix the standard stock solutions to obtain mixed standard solutions; (3) Accurately pipette the standard mixed solution and the test solution, inject into the high performance liquid chromatograph, and perform HPLC determination. Using chlorogenic acid and luteolin in the standard solution as internal standards, calculate the correction factors for neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, dioscorea glycoside, montanol, luteolin, and acaciadin. The contents of 7 kinds of organic acid compounds, including chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C, in the test samples were calculated by the correction factor with chlorogenic acid. The contents of 10 kinds of flavonoid compounds, including luteolin, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6”-O-malonyl)-glucoside, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside, dioscorea glycoside, montanol, luteolin and acacetin, in the test samples were calculated by the correction factor with luteolin. The specific conditions of the HPLC determination in step (3) are as follows: chromatographic column: C18 chromatographic column, injection volume: 1-10 μL, detection wavelength: 322-332 nm, column temperature: 20-30° C., flow rate: 0.8-1.0 mL / min, mobile phase: 0.1% phosphoric acid water as mobile phase A, acetonitrile as mobile phase B, gradient elution program: 0-15 min, 90% A→80% A; 15-25 min, 80% A→78% A; 25-35 min, 78% A→65% A.

3. The method for evaluating chrysanthemum quality by one-test-multiple-evaluation method according to claim 2, characterized in that: The C18 chromatographic column is selected from a Waters CORTECS T3 chromatographic column with a specification of 4.6×150 mm and 2.7 μm, a Waters XSelect Premier T3 chromatographic column with a specification of 4.6×150 mm and 2.7 μm, and an Agilent Zorbax Eclipse Pluse C18 chromatographic column with a specification of 2.1×100 mm and 2.7 μm.

4. The method for evaluating chrysanthemum quality by one-test-multiple-evaluation method according to claim 2, characterized in that: The specific conditions of the HPLC determination are: Chromatographic column: Waters CORTECS T3, 4.6×150 mm, 2.7 μm column, detection wavelength: 327 nm; injection volume: 5 μL, flow rate: 1.0 mL / min; column temperature: 25°C; mobile phase: 0.1% phosphoric acid water as mobile phase A, acetonitrile as mobile phase B, gradient elution program: 0-15 min, 90% A→80% A; 15-25 min, 80% A→78% A; 25-35 min, 78% A→65% A.

5. The method for evaluating chrysanthemum quality by one-test-multiple-evaluation method according to claim 2, characterized in that: The specific steps of preparing the test solution in step (1) are: accurately weighing 0.25 g of chrysanthemum powder and placing it in a stoppered conical flask, adding an appropriate amount of extraction solvent, measuring the weight, ultrasonic extraction, replenishing the weight, shaking and filtering, and filtering the filtrate with a 0.45 μm microporous filter membrane to obtain the solution.

6. The method for evaluating chrysanthemum quality by one-test-multiple-evaluation method according to claim 2, characterized in that The preparation of the mixed standard solution in step (2) comprises the following steps: B1: Accurately weigh about 5-10 mg of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-β-D-(6"-O-malonyl)-glucoside, apigenin 7-O-(6"-O-malonyl)-β-D-glucoside, dioscorea glycoside, montanol, luteolin, and acaciadin, and place them in 10 mL or 50 mL volumetric flasks respectively; B2: Add appropriate amount of 70% methanol solution to dissolve and dilute to scale to prepare a single standard stock solution; B3: Accurately pipette an appropriate volume of the 17 standard stock solutions in step B2, place them in a 20 mL volumetric flask, add 70% methanol to dilute to the mark, and prepare mixed reference stock solutions of different concentrations; B4: Accurately pipette 0.2mL, 0.4mL, 1mL, and 1.6mL of the mixed standard stock solution in step B3 into a 2mL volumetric flask, add 70% methanol to dilute to the scale, and obtain a series of mixed standard working solutions.

7. The method for evaluating chrysanthemum quality by one-test-multiple-evaluation method according to claim 2, characterized in that: The correction factors between the neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C and the internal reference chlorogenic acid in step (3), and the correction factors between luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-Β-D-(6"-O-malonyl)-glucoside, apigenin 7-O-(6"-O-malonyl)-β-D-glucoside, dioscorea glycoside, montanol, luteolin, acacetin and the internal reference luteolin are calculated according to the following method: a) Accurately pipette 5 μL of the mixed standard working solution and inject it into the HPLC; b) HPLC determination of the peak areas of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, luteolin, luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-Β-D-(6"-O-malonyl)-glucoside, apigenin 7-O-(6"-O-malonyl)-β-D-glucoside, dioscorea glycoside, montanol, luteolin, and acacetin; c) According to the correction factor calculation formula f = (As / Cs) / (Ai / Ci), the correction factors of chlorogenic acid and neochlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C, and the correction factors of luteolin and luteolin-7-O-β-D-glucuronide, apigenin-7-O-glucuronide, apigenin, luteolin-7-O-Β-D-(6"-O-malonyl)-glucoside, apigenin 7-O-(6"-O-malonic acid monoacyl)-β-D-glucoside, dioscorea glycoside, montanol, luteolin, and acacetin were calculated, wherein Cs is the mass concentration of the internal reference, As is the chromatographic peak area of ​​the internal reference, Ci is the mass concentration of other components, and Ai is the chromatographic peak area of ​​other components.

8. The method for evaluating chrysanthemum quality by one-test-multiple-evaluation method according to claim 2, characterized in that: The extraction solvent in step (1) is selected from 70% methanol, methanol or ethanol, the extraction time is 15min, 30min, 40min or 60min, and the mass volume ratio of the feed liquid is 0.25g:10-50mL.

9. Application of the method for evaluating chrysanthemum quality by the one-test-multiple-evaluation method according to any one of claims 1 to 8 in the quality standardization of chrysanthemum extracts or related preparations containing chrysanthemum.

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