Separation and detection method for flavonoid components in chrysanthemum

Abstract

The invention relates to the technical field of analysis and detection, and discloses a method for separating and detecting flavonoid components in chrysanthemum, which comprises the following steps: extracting a chrysanthemum sample to be detected by using a methanol aqueous solution to obtain an extracting solution; octadecylsilane chemically bonded silica is adopted as a chromatographic column filler, high performance liquid chromatography detection is performed on the extracting solution according to a specific elution procedure, and a chromatogram is obtained; and performing qualitative and / or quantitative analysis on the flavonoid components in the chrysanthemum sample to be detected according to the chromatogram. The method is used; according to the present invention, a variety of flavonoid components such as chlorogenic acid luteolin-7-O-beta-D-glucoside, quercetin-3-O-B-D-galactoside, isochlorogenic acid B, kaempferol-3-O-rutinoside, isochlorogenic acid A, isochlorogenic acid C, diosmetin-7-O-B-D-glucoside, luteolin and apigenin can be effectively separated from chrysanthemum, synchronous detection of the components is realized, interference of other components in the chrysanthemum can be prevented, and the method has relatively high detection accuracy.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to a method for separating and detecting flavonoids in chrysanthemum. Background Technology

[0002] Flavonoids are a class of natural polyphenolic compounds widely found in plants, possessing various biological activities such as antioxidant, anti-inflammatory, and immunomodulatory effects. They are also an important active ingredient in chrysanthemum. Their basic structure consists of two benzene rings (ring A and ring B) with phenolic hydroxyl groups linked by a central three-carbon atom, with a 2-phenylchromone nucleus as the parent nucleus. Flavonoids are generally poorly soluble in water, but in chrysanthemum, they often exist in combination with sugars or glycosides, increasing their water solubility and facilitating their pharmacological effects in the human body.

[0003] Detecting the flavonoids in chrysanthemums to determine their types and contents is beneficial for quality control. However, existing methods for detecting chrysanthemum flavonoids are not comprehensive enough, resulting in poor separation and a limited number of detectable flavonoid species. For example, the patent with publication number CN109374786A uses ultra-high performance liquid chromatography (UHPLC) to detect the active ingredients in Hangzhou chrysanthemum, but it can only achieve qualitative and quantitative detection of chlorogenic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, luteolin, and buddleoside. Summary of the Invention

[0004] To address the shortcomings of existing liquid chromatography methods in detecting chrysanthemum flavonoids, such as insufficient comprehensiveness, poor separation efficiency, and a limited range of detectable flavonoid species, this invention provides a method for the separation and detection of flavonoid components in chrysanthemum. Using this method, multiple common flavonoid components in chrysanthemum can be detected simultaneously, preventing interference from other components and achieving high detection accuracy.

[0005] The specific technical solution of this invention is as follows: A method for separating and detecting flavonoids in chrysanthemum, comprising the following steps: S1: Extract the chrysanthemum sample to be tested with methanol-water solution to obtain the extract; S2: Using octadecylsilane-bonded silica gel as the column packing material, the extract was analyzed by high-performance liquid chromatography (HPLC) to obtain a chromatogram. The mobile phase used in the elution program consisted of aqueous acetic acid and acetonitrile. The change in the proportion of aqueous acetic acid was as follows: The initial concentration was 90-91% v / v from the start to 4.5-5.5 min; gradually decreased to 85-86% v / v from 9.5-10.5 min; gradually decreased to 83-83.5% v / v from 39.0-40.5 min; gradually decreased to 82-82.5% v / v from 44.5-45.5 min; gradually decreased to 81-81.5% v / v from 49.5-50.5 min; gradually decreased to 68-71% v / v from 54.5-55.5 min; gradually decreased to 47-49% v / v from 64.0-65.0 min; gradually decreased to 4.5-5.5% v / v from 64.8-65.3 min, maintained until 69.5-70.0 min; gradually decreased to 90-91% v / v from 70.1-70.3 min. S3: Perform qualitative and / or quantitative analysis of flavonoid components in the chrysanthemum sample to be tested based on the chromatogram.

[0006] The elution procedure used in this invention can be represented as follows:

[0007] The elution procedures listed in the table above use the conventional representation of elution procedures in liquid chromatography, meaning that the elution procedure consists of the following stages: 1) The initial mobile phase, by volume percentage, consists of the following components: 90-91% v / v aqueous acetic acid solution, with the balance being acetonitrile; 2) The mobile phase formulation remained unchanged until 4.5-5.5 min, and then the partition ratio of each group gradually changed until 9.5-10.5 min, when it changed to: 85-86% v / v acetic acid aqueous solution, with the remainder being acetonitrile; 3) The distribution ratio of each group of mobile phase continued to change gradually until it changed to 83-83.5% v / v acetic acid aqueous solution, with the remainder being acetonitrile, at 39.0-40.5 min. 4) The distribution ratio of each group of mobile phase continued to change gradually until it changed to 82-82.5% v / v acetic acid aqueous solution, with the remainder being acetonitrile, at 44.5-45.5 min. 5) The distribution ratio of each group of mobile phase continued to change gradually until it changed to 81-81.5% v / v acetic acid aqueous solution, with the remainder being acetonitrile, at 49.5-50.5 min. 6) The distribution ratio of each component of the mobile phase continued to change gradually until it changed to 68-71% v / v acetic acid aqueous solution at 54.5-55.5, with the remainder being acetonitrile; 7) The distribution ratio of each group of mobile phase continued to change gradually until it changed to 47-49% v / v acetic acid aqueous solution at 64.0-65.0 min, with the remainder being acetonitrile; 8) The distribution ratio of each group of mobile phase continued to change gradually until it changed to 4.5-5.5% v / v acetic acid aqueous solution, with the remainder being acetonitrile, at 64.8-65.3 min. 9) The mobile phase formulation remained unchanged until 69.5~70.0 min, and then the partition ratio of each group gradually changed until 70.1~70.3 min, when it changed to: 90~91% v / v acetic acid aqueous solution, with the remainder being acetonitrile; 10) The mobile phase formulation remains unchanged and the duration is unlimited.

[0008] This invention uses methanol-water extraction to extract chrysanthemum samples, followed by a specific chromatographic column packing and gradient elution program. This allows for the effective separation of various common flavonoids in chrysanthemums (including chlorogenic acid, luteolin-7-O-β-D-glucoside, quercetin-3-OBD-galactoside, isochlorogenic acid B, kaempferol-3-O-rutinoside, isochlorogenic acid A, isochlorogenic acid C, geraniol-7-OBD-glucoside, luteolin, and apigenin). This method avoids interference from other components in chrysanthemums in the detection of these flavonoids and ensures good separation between these flavonoids, preventing peak overlap. Therefore, it enables simultaneous detection of various flavonoids while ensuring high detection accuracy and sensitivity.

[0009] Flavonoids have similar structures and properties, making them difficult to separate during high-performance liquid chromatography (HPLC) detection. This presents significant challenges to designing methods for the simultaneous detection of multiple flavonoids. The choice of column packing material, the selection of the mobile phase in the gradient elution program, and variations in the mobile phase formulation at different stages all affect the separation efficiency of flavonoids, thus impacting detection accuracy. For example, this invention uses acetic acid, which has relatively weak dissociation capabilities. The low ionic strength of the acetic acid aqueous solution weakens the shielding effect on the silanol groups (-SiOH) on the column (stationary phase) surface. Residual silanol groups can form weak hydrogen bonds with the glycosyl or hydroxyl groups of flavonoid glycosides, enhancing their retention specificity. Conversely, if a more dissociative acid is used, the higher ionic strength of its aqueous solution will shield the secondary action sites of the silanol groups. Simultaneously, acid radicals may compete for binding to the stationary phase surface, leading to reduced retention specificity of flavonoid glycosides and overlapping chromatographic peaks of some flavonoids, preventing the simultaneous detection of these components.

[0010] Preferably, the flavonoid components include at least one of chlorogenic acid, luteolin-7-O-β-D-glucoside, quercetin-3-OBD-galactoside, isochlorogenic acid B, kaempferol-3-O-rutin, isochlorogenic acid A, isochlorogenic acid C, geraniol-7-OBD-glucoside, luteolin, and apigenin.

[0011] Using the method of the present invention, common flavonoid components in the above 10 chrysanthemums can be effectively separated, and the simultaneous detection of these 10 chrysanthemum flavonoids can be achieved with high detection accuracy and sensitivity.

[0012] Preferably, in step S2, the high-performance liquid chromatography (HPLC) detection conditions are as follows: the chromatographic column is an Agilent Eclipse XDB-C18, the injection volume is 5~20 µL, the flow rate is 0.5~2.0 mL / min, the column temperature is 20~35℃, and the detection wavelength is 280~560 nm.

[0013] The Agilent Eclipse XDB-C18 column is one of the chromatographic columns that uses octadecylsilane-bonded silica gel as the packing material. It employs high-density bonded C18 chains and double-end sealing technology, which can largely shield the active sites of silanol groups, reduce secondary interactions of flavonoid glycosides (such as hydrogen bonding), thereby reducing tailing and improving column efficiency. In addition, the column has hydrophobic and moderately polar intercalating groups, which is beneficial for distinguishing flavonoid glycosides by differences in glycosyl substitution positions (such as luteolin-7-O-glucoside and quercetin-3-O-galactoside) and isomers (such as isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C). Furthermore, it also has good resolution for flavonoid components with small polarity differences (such as isochlorogenic acid A and kaempferol-3-O-rutin glycoside).

[0014] Preferably, in step S1, the chrysanthemum sample to be tested is chrysanthemum powder or chrysanthemum extract, and the particle size of the chrysanthemum powder is no greater than 360 µm.

[0015] Preferably, the volume fraction of methanol in the methanol aqueous solution is 70-75%.

[0016] Preferably, in step S1, the extraction method is ultrasonic extraction for 40-50 min.

[0017] Preferably, in step S2, the volume fraction of acetic acid in the aqueous acetic acid solution is 0.01~0.5%.

[0018] Preferably, in step S3, the qualitative analysis involves determining the correspondence between the types of flavonoids and the retention times of chromatographic peaks based on the detection results of the target flavonoid reference standard under the same conditions, and determining the types of flavonoids based on the retention times of chromatographic peaks in the chromatogram of the chrysanthemum sample to be tested; the quantitative analysis involves determining the content of the corresponding flavonoids by substituting the chromatographic peak area into the standard curve.

[0019] Furthermore, the correspondence between the types of flavonoid components and the retention times of chromatographic peaks is as follows: Chlorogenic acid: 9~10 min; Luteolin-7-O-β-D-glucoside: 26~28 min; Quercetin-3-OBD-galactoside: 27~29 min; Isochlorogenic acid B: 32~34 min; Kaempferol-3-O-rutin: 36~38 min; Isochlorogenic acid A: 38~40 min; Isochlorogenic acid C: 41~48 min; Geraniol-7-OBD-glucoside: 50~55min; Luteolin: 55~61min; Apigenin: 61~55 min.

[0020] Furthermore, the method for constructing the standard curve is as follows: the target flavonoid reference standard is dissolved in methanol to prepare a series of reference standard solutions with different concentration gradients. The reference standard solutions are then detected by high performance liquid chromatography according to step S2. Based on the concentration of the target flavonoid in the reference standard solution and its peak area in the chromatogram, a standard curve is plotted.

[0021] Compared with the prior art, the present invention has the following advantages: (1) The method of the present invention can effectively separate a variety of flavonoids in chrysanthemum, including chlorogenic acid luteolin-7-O-β-D-glucoside, quercetin-3-OBD-galactoside, isochlorogenic acid B, kaempferol-3-O-rutin, isochlorogenic acid A, isochlorogenic acid C, geraniol-7-OBD-glucoside, luteolin and apigenin, so as to realize the simultaneous detection of these components, improve the comprehensiveness of chrysanthemum flavonoid detection, and prevent interference from other components in chrysanthemum, and has high detection accuracy and sensitivity.

[0022] (2) This invention can separate various common flavonoids in chrysanthemum from interfering substances and separate these flavonoids from each other through simple pretreatment steps and high performance liquid chromatography, thereby reducing the difficulty of operation and sources of error and improving detection efficiency. Attached Figure Description

[0023] Figure 1 The chromatogram obtained in Example 1 is shown.

[0024] Figure 2 This is the chromatogram obtained in Example 2.

[0025] Figure 3 This is the chromatogram obtained in Example 3.

[0026] Figure 4 The chromatogram obtained in Comparative Example 1 is shown.

[0027] Figure 5 The chromatogram obtained in Comparative Example 2 is shown.

[0028] Figure 6 The chromatogram obtained in Comparative Example 3 is shown.

[0029] Figure 7 This is the chromatogram obtained in Example 4. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments.

[0031] General Implementation Examples A method for separating and detecting flavonoids in chrysanthemum, comprising the following steps: S1: Extract the chrysanthemum sample to be tested with methanol-water solution to obtain the extract; S2: Using octadecylsilane-bonded silica gel as the column packing material, the extract was subjected to high-performance liquid chromatography (HPLC) detection according to the elution program in the table below, and the chromatogram was obtained:

[0032] S3: Perform qualitative and / or quantitative analysis of flavonoid components in the chrysanthemum sample to be tested based on the chromatogram.

[0033] In some specific embodiments, the flavonoid components include at least one of chlorogenic acid, luteolin-7-O-β-D-glucoside, quercetin-3-OBD-galactoside, isochlorogenic acid B, kaempferol-3-O-rutinoside, isochlorogenic acid A, isochlorogenic acid C, geraniol-7-OBD-glucoside, luteolin, and apigenin.

[0034] In some specific embodiments, in step S1, the chrysanthemum sample to be tested is chrysanthemum powder or chrysanthemum extract, and the particle size of the chrysanthemum powder is no greater than 360 µm.

[0035] In some specific embodiments, the volume fraction of methanol in the methanol-water solution is 70-75%.

[0036] In some specific embodiments, in step S1, the extraction method is ultrasonic extraction for 40-50 min.

[0037] In some specific embodiments, in step S2, the volume fraction of acetic acid in the aqueous acetic acid solution is 0.01~0.5%.

[0038] In some specific embodiments, the high-performance liquid chromatography (HPLC) detection conditions in step S2 are as follows: the chromatographic column is an Agilent Eclipse XDB-C18, the injection volume is 5~20 µL, the flow rate is 0.5~2.0 mL / min, the column temperature is 20~35℃, and the detection wavelength is 280~560 nm.

[0039] In some specific embodiments, in step S3, the qualitative analysis is to determine the correspondence between the types of flavonoids and the retention times of chromatographic peaks based on the detection results of the target flavonoid reference standard under the same conditions, and to determine the types of flavonoids based on the retention times of chromatographic peaks in the chromatogram of the chrysanthemum sample to be tested; the quantitative analysis is to determine the content of the corresponding flavonoids by substituting the chromatographic peak area into the standard curve.

[0040] In the above specific implementation method: Optionally or preferably, the correspondence between the types of flavonoid components and the retention times of chromatographic peaks is as follows:

[0041] Optionally or preferably, the standard curve is constructed as follows: the target flavonoid reference standard is dissolved in methanol to prepare a series of reference standard solutions with different concentration gradients. The reference standard solutions are then detected by high performance liquid chromatography according to step S2. The standard curve is plotted based on the concentration of the target flavonoid in the reference standard solution and its peak area in the chromatogram. Specific Implementation The present invention will now be described through specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0043] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Unless otherwise specified, the raw materials and equipment used in this invention are conventional in the art and can be obtained through conventional commercial means; unless otherwise specified, the methods used in this invention are conventional methods in the art.

[0044] The tables relating to elution procedures in the following examples and comparative examples all use the conventional representation of elution procedures in liquid chromatography.

[0045] Example 1: The separation and detection effect of the method of the present invention on 10 kinds of chrysanthemum flavonoids The following steps were used to prepare a mixed reference solution, which was then analyzed by high-performance liquid chromatography: S1: Preparation of mixed reference solution Accurately weigh the following 10 reference standards: chlorogenic acid, luteolin-7-O-β-D-glucoside, quercetin-3-OBD-galactoside, isochlorogenic acid B, kaempferol-3-O-rutin, isochlorogenic acid A, isochlorogenic acid C, geraniol-7-OBD-glucoside, luteolin, and apigenin. Ten reference standards were placed in volumetric flasks, dissolved by sonication with methanol, and then diluted to the mark with methanol. The solutions were shaken well to obtain a mixed reference solution containing 0.1011 mg / mL chlorogenic acid, 0.0925 mg / mL luteolin-7-O-β-D-glucoside, 0.1011 mg / mL quercetin-3-OBD-galactoside, 0.1051 mg / mL isochlorogenic acid B, 0.1113 mg / mL kaempferol-3-O-rutin, 0.1022 mg / mL isochlorogenic acid, 0.1096 mg / mL isochlorogenic acid C, 0.0558 mg / mL geraniol-7-OBD-glucoside, 0.1044 mg / mL luteolin, and 0.1054 mg / mL apigenin.

[0046] S2: High-performance liquid chromatography detection The mixed reference solution was injected into a high-performance liquid chromatograph (HPLC) for detection. An Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm) was used as the column. The injection volume was 10 µL, the flow rate was 1 mL / min, the column temperature was 30 °C, and the detection wavelength was 348 nm. During elution, a 0.1% v / v aqueous acetic acid solution was used as mobile phase A, and acetonitrile was used as mobile phase B. The gradient elution program was as follows:

[0047] The chromatographic peaks obtained in this embodiment are found in Figure 1 Each substance in Figure 1 The retention periods are as follows:

[0048] from Figure 1 It can be seen that the chromatographic peaks of the 10 flavonoids are relatively independent, indicating that the method of this embodiment can effectively separate these 10 flavonoids.

[0049] Example 2: The separation and detection effect of the method of the present invention on 10 kinds of chrysanthemum flavonoids The following steps were used to prepare a mixed reference solution, which was then analyzed by high-performance liquid chromatography: S1: Preparation of mixed reference solution Accurately weigh the following 10 reference standards: chlorogenic acid, luteolin-7-O-β-D-glucoside, quercetin-3-OBD-galactoside, isochlorogenic acid B, kaempferol-3-O-rutin, isochlorogenic acid A, isochlorogenic acid C, geraniol-7-OBD-glucoside, luteolin, and apigenin. Ten reference standards were placed in volumetric flasks, dissolved by sonication with methanol, and then diluted to the mark with methanol. The solutions were shaken well to obtain a mixed reference solution containing 0.1011 mg / mL chlorogenic acid, 0.0925 mg / mL luteolin-7-O-β-D-glucoside, 0.1011 mg / mL quercetin-3-OBD-galactoside, 0.1051 mg / mL isochlorogenic acid B, 0.1113 mg / mL kaempferol-3-O-rutin, 0.1022 mg / mL isochlorogenic acid, 0.1096 mg / mL isochlorogenic acid C, 0.0558 mg / mL geraniol-7-OBD-glucoside, 0.1044 mg / mL luteolin, and 0.1054 mg / mL apigenin.

[0050] S2: High-performance liquid chromatography detection The mixed reference solution was injected into a high-performance liquid chromatograph (HPLC) for detection. An Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm) was used as the column. The injection volume was 10 µL, the flow rate was 1 mL / min, the column temperature was 30 °C, and the detection wavelength was 348 nm. During elution, a 0.5% v / v aqueous acetic acid solution was used as mobile phase A, and acetonitrile was used as mobile phase B. The gradient elution program was as follows:

[0051] The chromatographic peaks obtained in this embodiment are found in Figure 2 .from Figure 2 It can be seen that the chromatographic peaks of the 10 flavonoids are relatively independent, indicating that the method of this embodiment can effectively separate these 10 flavonoids.

[0052] Example 3: The separation and detection effect of the method of the present invention on 10 kinds of chrysanthemum flavonoids The following steps were used to prepare a mixed reference solution, which was then analyzed by high-performance liquid chromatography: S1: Preparation of mixed reference solution Accurately weigh the following 10 reference standards: chlorogenic acid, luteolin-7-O-β-D-glucoside, quercetin-3-OBD-galactoside, isochlorogenic acid B, kaempferol-3-O-rutin, isochlorogenic acid A, isochlorogenic acid C, geraniol-7-OBD-glucoside, luteolin, and apigenin. Ten reference standards were placed in volumetric flasks, dissolved by sonication with methanol, and then diluted to the mark with methanol. The solutions were shaken well to obtain a mixed reference solution containing 0.1011 mg / mL chlorogenic acid, 0.0925 mg / mL luteolin-7-O-β-D-glucoside, 0.1011 mg / mL quercetin-3-OBD-galactoside, 0.1051 mg / mL isochlorogenic acid B, 0.1113 mg / mL kaempferol-3-O-rutin, 0.1022 mg / mL isochlorogenic acid, 0.1096 mg / mL isochlorogenic acid C, 0.0558 mg / mL geraniol-7-OBD-glucoside, 0.1044 mg / mL luteolin, and 0.1054 mg / mL apigenin.

[0053] S2: High-performance liquid chromatography detection The mixed reference solution was injected into a high-performance liquid chromatograph (HPLC) for detection. An Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm) was used as the column. The injection volume was 10 µL, the flow rate was 1 mL / min, the column temperature was 30 °C, and the detection wavelength was 348 nm. During elution, a 0.01% v / v aqueous acetic acid solution was used as mobile phase A, and acetonitrile was used as mobile phase B. The gradient elution program was as follows:

[0054] The chromatographic peaks obtained in this embodiment are found in Figure 3 .from Figure 3 It can be seen that the chromatographic peaks of the 10 flavonoids are relatively independent, indicating that the method of this embodiment can effectively separate these 10 flavonoids.

[0055] Comparative Example 1: Effect of gradient elution program on the separation and detection of 10 chrysanthemum flavonoids The following steps were used to prepare a mixed reference solution, which was then analyzed by high-performance liquid chromatography: S1: Preparation of mixed reference solution Accurately weigh the following 10 reference standards: chlorogenic acid, luteolin-7-O-β-D-glucoside, quercetin-3-OBD-galactoside, isochlorogenic acid B, kaempferol-3-O-rutin, isochlorogenic acid A, isochlorogenic acid C, geraniol-7-OBD-glucoside, luteolin, and apigenin. Ten reference standards were placed in volumetric flasks, dissolved by sonication with methanol, and then diluted to the mark with methanol. The solutions were shaken well to obtain a mixed reference solution containing 0.1011 mg / mL chlorogenic acid, 0.0925 mg / mL luteolin-7-O-β-D-glucoside, 0.1011 mg / mL quercetin-3-OBD-galactoside, 0.1051 mg / mL isochlorogenic acid B, 0.1113 mg / mL kaempferol-3-O-rutin, 0.1022 mg / mL isochlorogenic acid, 0.1096 mg / mL isochlorogenic acid C, 0.0558 mg / mL geraniol-7-OBD-glucoside, 0.1044 mg / mL luteolin, and 0.1054 mg / mL apigenin.

[0056] S2: High-performance liquid chromatography detection The mixed reference solution was injected into a high-performance liquid chromatograph (HPLC) for detection. An Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm) was used as the column. The injection volume was 10 µL, the flow rate was 1 mL / min, the column temperature was 30 °C, and the detection wavelength was 348 nm. During elution, a 0.1% v / v aqueous acetic acid solution was used as mobile phase A, and acetonitrile was used as mobile phase B. The gradient elution program was as follows:

[0057] The chromatographic peaks obtained in this comparative example are found in Figure 4 .from Figure 4 As can be seen, there is a certain degree of overlap among many chromatographic peaks, including peak 3 (quercetin-3-OBD-galactoside), peak 4 (isochlorogenic acid B), peak 5 (kaempferol-3-O-rutinoside), and peak 6 (isochlorogenic acid A), indicating that the method in this comparative example is not very effective in separating these flavonoids. The reason for this is that the gradient elution program used in this comparative example has an excessively high proportion of organic phase and an excessively steep gradient slope, causing the above five flavonoids to be eluted within relatively close time windows, resulting in ineffective separation.

[0058] Comparative Example 2: The effect of mobile phase selection on the separation and detection of 10 chrysanthemum flavonoids The following steps were used to prepare a mixed reference solution, which was then analyzed by high-performance liquid chromatography: S1: Preparation of mixed reference solution Accurately weigh the following 10 reference standards: chlorogenic acid, luteolin-7-O-β-D-glucoside, quercetin-3-OBD-galactoside, isochlorogenic acid B, kaempferol-3-O-rutin, isochlorogenic acid A, isochlorogenic acid C, geraniol-7-OBD-glucoside, luteolin, and apigenin. Ten reference standards were placed in volumetric flasks, dissolved by sonication with methanol, and then diluted to the mark with methanol. The solutions were shaken well to obtain a mixed reference solution containing 0.1011 mg / mL chlorogenic acid, 0.0925 mg / mL luteolin-7-O-β-D-glucoside, 0.1011 mg / mL quercetin-3-OBD-galactoside, 0.1051 mg / mL isochlorogenic acid B, 0.1113 mg / mL kaempferol-3-O-rutin, 0.1022 mg / mL isochlorogenic acid, 0.1096 mg / mL isochlorogenic acid C, 0.0558 mg / mL geraniol-7-OBD-glucoside, 0.1044 mg / mL luteolin, and 0.1054 mg / mL apigenin.

[0059] S2: High-performance liquid chromatography detection The mixed reference solution was injected into a high-performance liquid chromatograph (HPLC) for detection. An Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm) was used as the column. The injection volume was 10 µL, the flow rate was 1 mL / min, the column temperature was 30 °C, and the detection wavelength was 348 nm. During elution, a 0.1% v / v aqueous acetic acid solution was used as mobile phase A, and acetonitrile was used as mobile phase B. The gradient elution program was as follows:

[0060] The chromatographic peaks obtained in this comparative example are found in Figure 5 .from Figure 5 As can be seen, peaks 3 (quercetin-3-OBD-galactoside), 4 (isochlorogenic acid B), 5 (kaempferol-3-O-rutinoside), and 6 (isochlorogenic acid A) overlap significantly and cannot be separated. The reason for this lies in the combined effect of the differences in the properties of the two acids on the mobile phase pH, the dissociation state of the compounds, and the stationary phase. Specifically, acetic acid (weak acid, pKa≈4.76) and phosphoric acid (strong acid, pKa1≈2.15) have significantly different dissociation abilities, leading to large differences in the actual pH and ionic strength of the mobile phase; the glacial acetic acid system has low ionic strength (mainly containing CH3COO). - and H + The shielding effect of residual silanol groups (-SiOH) on the surface of the C18 stationary phase is relatively weak. These residual silanol groups can form weak hydrogen bonds with the glycosyl or hydroxyl groups of flavonoid glycosides, enhancing their retention specificity. In contrast, the phosphate ions (PO4) in the phosphoric acid system...3- / HPO4 2- High concentrations and strong ionic strength can shield the secondary action sites of silanol groups. At the same time, phosphate groups may compete for binding to the surface of the stationary phase, resulting in a decrease in the retention specificity of flavonoid glycosides (such as a decrease in the separation degree between quercetin glycoside and kaempferol glycoside). In addition, the retention behavior of isochlorogenic acid A is dominated by hydrophobicity and tends to be similar to the retention time of flavonoid glycosides.

[0061] Comparative Example 3: The effect of column selection on the separation and detection of 10 chrysanthemum flavonoids The following steps were used to prepare a mixed reference solution, which was then analyzed by high-performance liquid chromatography: S1: Preparation of mixed reference solution Accurately weigh the following 10 reference standards: chlorogenic acid, luteolin-7-O-β-D-glucoside, quercetin-3-OBD-galactoside, isochlorogenic acid B, kaempferol-3-O-rutin, isochlorogenic acid A, isochlorogenic acid C, geraniol-7-OBD-glucoside, luteolin, and apigenin. Ten reference standards were placed in volumetric flasks, dissolved by sonication with methanol, and then diluted to the mark with methanol. The solutions were shaken well to obtain a mixed reference solution containing 0.1011 mg / mL chlorogenic acid, 0.0925 mg / mL luteolin-7-O-β-D-glucoside, 0.1011 mg / mL quercetin-3-OBD-galactoside, 0.1051 mg / mL isochlorogenic acid B, 0.1113 mg / mL kaempferol-3-O-rutin, 0.1022 mg / mL isochlorogenic acid, 0.1096 mg / mL isochlorogenic acid C, 0.0558 mg / mL geraniol-7-OBD-glucoside, 0.1044 mg / mL luteolin, and 0.1054 mg / mL apigenin.

[0062] S2: High-performance liquid chromatography detection The mixed reference solution was injected into a high-performance liquid chromatograph (HPLC) for detection. An Agilent ZORBAX SB-AQ column (4.6 mm × 150 mm, 3.5 μm) was used as the column. The injection volume was 10 µL, the flow rate was 1 mL / min, the column temperature was 30 °C, and the detection wavelength was 348 nm. During elution, a 0.1% v / v aqueous acetic acid solution was used as mobile phase A, and acetonitrile was used as mobile phase B. The gradient elution program was as follows:

[0063] The chromatographic peaks obtained in this comparative example are found in Figure 6 .from Figure 6As observed in this comparative example, when detecting 10 types of chrysanthemum flavonoids using this method, some flavonoid components failed to elute normally. The reason for this is that the ZORBAX SB-AQ is a highly water-compatible C18 column with hydrophilic groups modified on its surface, suitable for high-aqueous mobile phases (e.g., >95% water). However, when detecting chrysanthemum flavonoids, its hydrophilic properties cause flavonoid glycosides to be repelled by the strongly hydrophilic stationary phase due to the hydrophilicity of their glycosyl groups, resulting in excessively short retention times. Furthermore, isochlorogenic acid (highly hydrophobic) may suddenly elute in the later stages of the gradient, causing peak broadening or loss.

[0064] Example 4: Actual Sample Testing The following steps were used to detect the flavonoids in chrysanthemums: S1: Preparation of mixed reference solution Accurately weigh the following 10 reference standards: chlorogenic acid, luteolin-7-O-β-D-glucoside, quercetin-3-OBD-galactoside, isochlorogenic acid B, kaempferol-3-O-rutin, isochlorogenic acid A, isochlorogenic acid C, geraniol-7-OBD-glucoside, luteolin, and apigenin. Ten reference standards were placed in volumetric flasks, dissolved by sonication with methanol, and then diluted to the mark with methanol. The mixture was shaken well to obtain a mixed reference standard stock solution containing chlorogenic acid 1.011 mg / mL, luteolin-7-O-β-D-glucoside 0.925 mg / mL, quercetin-3-OBD-galactoside 1.011 mg / mL, isochlorogenic acid B 1.051 mg / mL, kaempferol-3-O-rutin 1.113 mg / mL, isochlorogenic acid 1.022 mg / mL, isochlorogenic acid C 1.096 mg / mL, geraniol-7-OBD-glucoside 0.558 mg / mL, luteolin 1.044 mg / mL, and apigenin 1.054 mg / mL. Then, 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, and 1 mL of the mixed reference standard stock solution were taken and diluted to 10 mL volumetric flasks to obtain a series of mixed reference standard solutions with different concentration gradients.

[0065] S2: Plot the standard curve Each mixed reference solution was injected separately into a high-performance liquid chromatograph (HPLC) for detection. An Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm) was used as the column, with an injection volume of 10 µL, a flow rate of 1 mL / min, a column temperature of 30 °C, and a detection wavelength of 348 nm. During elution, a 0.1% v / v aqueous acetic acid solution was used as mobile phase A, and acetonitrile was used as mobile phase B. The gradient elution program was as follows:

[0066] Based on the concentrations of each flavonoid component in the mixed reference solution and the corresponding peak areas, a standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The results are as follows:

[0067] Based on the chromatograms obtained from each mixed reference solution, the correspondence between the types of flavonoid components and the retention times of the chromatographic peaks was established as follows:

[0068] S3: Sample pretreatment After passing the chrysanthemum powder through a No. 3 sieve, accurately weigh 0.5 g and place it in a stoppered conical flask. Add 50 mL of 70% v / v methanol aqueous solution, weigh, and extract by ultrasonication at 60℃ for 40 min. After cooling, replenish the weight of the solution lost during ultrasonication, filter through a membrane, collect the filtrate, and obtain the extract.

[0069] S4: Sample Detection The extract was injected into a high-performance liquid chromatograph (HPLC) for detection. An Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm) was used as the column. The injection volume was 10 µL, the flow rate was 1 mL / min, the column temperature was 30℃, and the detection wavelength was 348 nm. During elution, a 0.1% v / v aqueous acetic acid solution was used as mobile phase A, and acetonitrile was used as mobile phase B. The gradient elution program was as follows:

[0070] The chromatogram of the extract obtained in this embodiment is shown in [reference needed]. Figure 7 Based on the retention time of the chromatographic peaks, and combined with the correspondence between the types of flavonoids and the retention times of the chromatographic peaks established in step S2, the types of flavonoids contained in the chrysanthemum sample to be tested are determined.

[0071] Detection Figure 7 The peak areas of each characteristic peak were substituted into the standard curve obtained in step S2 to calculate the content of each flavonoid component in the chrysanthemum sample to be tested. The results are as follows:

Claims

1. A method for separating and detecting flavonoids in chrysanthemum, characterized in that, Includes the following steps: S1: Extract the chrysanthemum sample to be tested with methanol-water solution to obtain the extract; S2: Using octadecylsilane-bonded silica gel as the column packing material, the extract was analyzed by high-performance liquid chromatography (HPLC) to obtain a chromatogram. The mobile phase used in the elution program consisted of aqueous acetic acid and acetonitrile. The change in the proportion of aqueous acetic acid was as follows: The initial concentration was 90-91% v / v from the start to 4.5-5.5 min; gradually decreased to 85-86% v / v from 9.5-10.5 min; gradually decreased to 83-83.5% v / v from 39.0-40.5 min; gradually decreased to 82-82.5% v / v from 44.5-45.5 min; gradually decreased to 81-81.5% v / v from 49.5-50.5 min; gradually decreased to 68-71% v / v from 54.5-55.5 min; gradually decreased to 47-49% v / v from 64.0-65.0 min; gradually decreased to 4.5-5.5% v / v from 64.8-65.3 min, maintained until 69.5-70.0 min; gradually decreased to 90-91% v / v from 70.1-70.3 min. S3: Perform qualitative and / or quantitative analysis of flavonoid components in the chrysanthemum sample to be tested based on the chromatogram.

2. The separation and detection method according to claim 1, characterized in that, The flavonoid components include at least one of chlorogenic acid, luteolin-7-O-β-D-glucoside, quercetin-3-OBD-galactoside, isochlorogenic acid B, kaempferol-3-O-rutinoside, isochlorogenic acid A, isochlorogenic acid C, geraniol-7-OBD-glucoside, luteolin, and apigenin.

3. The separation and detection method according to claim 1, characterized in that, In step S2, the conditions for high performance liquid chromatography detection are as follows: the chromatographic column is an Agilent Eclipse XDB-C18, the injection volume is 5~20 µL, the flow rate is 0.5~2.0 mL / min, the column temperature is 20~35℃, and the detection wavelength is 280~560 nm.

4. The separation and detection method according to claim 1, characterized in that, In step S1, the chrysanthemum sample to be tested is chrysanthemum powder or chrysanthemum extract, and the particle size of the chrysanthemum powder is no greater than 360 µm.

5. The separation and detection method according to claim 1, characterized in that, In step S1, the volume fraction of methanol in the methanol aqueous solution is 70-75%.

6. The separation and detection method according to claim 1 or 5, characterized in that, In step S1, the extraction method is ultrasonic extraction for 40-50 min.

7. The separation and detection method according to claim 1, characterized in that, In step S2, the volume fraction of acetic acid in the acetic acid aqueous solution is 0.01~0.5%.

8. The separation and detection method according to claim 1, characterized in that, In step S3, the qualitative analysis is to determine the correspondence between the types of flavonoids and the retention times of chromatographic peaks based on the detection results of the target flavonoid reference standard under the same conditions, and to determine the types of flavonoids based on the retention times of chromatographic peaks in the chromatogram of the chrysanthemum sample to be tested. Quantitative analysis involves determining the content of the corresponding flavonoid components by substituting the chromatographic peak area into the standard curve.

9. The separation and detection method according to claim 8, characterized in that, The correspondence between the types of flavonoids and the retention times of chromatographic peaks is as follows: Chlorogenic acid: 9~10 min; Luteolin-7-O-β-D-glucoside: 26~28 min; Quercetin-3-OBD-galactoside: 27~29 min; Isochlorogenic acid B: 32~34 min; Kaempferol-3-O-rutin: 36~38 min; Isochlorogenic acid A: 38~40 min; Isochlorogenic acid C: 41~48 min; Geraniol-7-OBD-glucoside: 50~55min; Luteolin: 55~61min; Apigenin: 61~55 min.

10. The separation and detection method according to claim 1, characterized in that, The standard curve is constructed as follows: the target flavonoid reference standard is dissolved in methanol to prepare a series of reference standard solutions with different concentration gradients. The reference standard solutions are then detected by high performance liquid chromatography according to step S2. The standard curve is plotted based on the concentration of the target flavonoid in the reference standard solution and its peak area in the chromatogram.

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