Rapid qualitative and quantitative detection method for coumarin compounds in angelica dahurica

Abstract

The application relates to a rapid qualitative and quantitative detection method of coumarin compounds in Angelica dahurica var. formosana. The application aims to solve the problem that the existing coumarin compound detection method is difficult to simultaneously qualitatively and quantitatively detect the coumarin compounds in Angelica dahurica var. formosana. The method comprises the following steps: one, extraction of the coumarin compounds in Angelica dahurica var. formosana; two, purification of the coumarin compounds in Angelica dahurica var. formosana; three, detection of the coumarin compounds in Angelica dahurica var. formosana; four, analysis and calculation of the detection data, and quantitative analysis is completed. The method can complete the detection of the coumarin compounds in Angelica dahurica var. formosana within 12 minutes, has the characteristics of high efficiency, sensitivity, accuracy and low requirement on equipment, and can be applied to the Angelica dahurica var. formosana food or medicine detection field. The application belongs to the field of qualitative and quantitative detection of coumarin compounds in Angelica dahurica var. formosana.

Description

Technical Field

[0001] This invention relates to a method for detecting various coumarin compounds in Angelica dahurica. Background Technology

[0002] *Heracleum dissectum* Ledeb., commonly known as Xing'an Angelica, is a perennial herb belonging to the Apiaceae family and the *Heracleum* genus. Its tender stems and leaves are edible, making it a popular wild vegetable, especially in the Greater Khingan Mountains region of Northeast China. As a traditional folk medicine, it is used to lower blood pressure, blood sugar, and cholesterol, dispel wind and dampness, and relieve pain. As a unique edible and medicinal plant resource in the Northeast forest region, it has significant development and utilization value. Coumarin compounds are important bioactive substances in *Heracleum dissectum*. Appropriate consumption or medicinal use has certain health benefits, but excessive consumption or medicinal use carries the risk of poisoning. Quality testing and safety analysis of *Heracleum dissectum* edible and medicinal products, as well as research on coumarin compounds in *Heracleum dissectum*, require qualitative or quantitative analysis of these compounds.

[0003] Angelica dahurica contains various coumarin compounds, including 7-hydroxycoumarin, scopolamine, psoralen, xanthotoxin, bergamot lactone, imperatorin, isopsoralen, isoapicin, and coumarins. The types and contents of these coumarin compounds vary significantly across different plant organs and developmental stages. Currently, there is a lack of rapid qualitative and quantitative detection methods for these coumarin compounds in Angelica dahurica, both domestically and internationally, which hinders the analysis and detection of coumarin compounds in Angelica dahurica and its processed products. Summary of the Invention

[0004] The purpose of this invention is to address the technical problem that existing methods for detecting coumarin compounds are difficult to simultaneously perform qualitative and quantitative detection of coumarin compounds in Angelica dahurica, and to provide a rapid qualitative and quantitative detection method for coumarin compounds in Angelica dahurica.

[0005] The rapid qualitative and quantitative detection method for coumarin compounds in Angelica pubescens is as follows:

[0006] I. Extraction of coumarin compounds from Angelica pubescens:

[0007] The dried, pulverized, or freshly crushed sample of Angelica dahurica was extracted with a 50%–100% methanol aqueous solution at a ratio of 1 g:(15–25) mL. The extraction was carried out at 28℃–38℃ with shaking or ultrasonication for 20–40 min. The sample was then centrifuged at 8500–11000 rpm, and the supernatant was collected. The precipitate was then extracted with the methanol aqueous solution at a ratio of 1 g:(15–25) mL at 28℃–38℃ with shaking or ultrasonication for 20–40 min. The sample was then centrifuged at 8500–11000 rpm, and the supernatant was collected. The extraction was repeated 2–4 times with shaking or ultrasonication. The supernatants were combined, filtered, and concentrated into a solid by centrifugation and vacuum extraction to obtain the extract.

[0008] II. Purification of coumarin compounds from Angelica pubescens:

[0009] The HC-C18 SPE columns were treated with 1 to 3 column volumes of methanol and deionized water, respectively, and then dried for later use.

[0010] The extract obtained in step one is dissolved in a 50%–100% methanol aqueous solution to obtain an extract solution. The extract solution is then passed through an HC-C18 SPE column. The total amount of coumarin compounds in the extract solution does not exceed 5% of the column packing material. After passing through the column for 10–15 min, the HC-C18 SPE column is eluted with an 80% methanol aqueous solution, and the eluent is collected. The eluent is concentrated by vacuum centrifugation and then dissolved in a 50%–100% methanol solution to obtain the extract solution of coumarin compounds from Angelica dahurica.

[0011] III. Detection of coumarin compounds in Angelica dahurica:

[0012] High-performance liquid chromatography (HPLC) and ultraviolet spectroscopy (UV spectroscopy) were performed using a high-performance liquid chromatography system equipped with a photodiode array as a liquid phase detector. The chromatographic column was a C18 column packed with octadecyl bonded silica gel. The sample injection volume was 10–20 μL. Mobile phase A was methanol and mobile phase B was water.

[0013] Mobile phase gradient elution program:

[0014] 0 min, 40%–60% A; 5 min, 50–70% A; 25 min, 70–90% A; 30 min, 80–100% A; flow rate 0.8–1 mL / min; column temperature 20–25℃; pressure 100–180 bar; quantitative detection wavelength 250–320 nm; UV spectroscopy detection wavelength 210–400 nm; the sum of the volume percentages of mobile phase A and mobile phase B at any time during the elution process is 100%; mobile phase flow rate: 0.8–1.2 mL / min;

[0015] Different concentrations of 7-hydroxycoumarin, scopolamine lactone, psoralen, xanthotoxin, bergamot lactone, imperatorin, isopsoralen, isofennelol, coumarin standard solutions, and extract solutions of coumarin compounds from Angelica dahurica were detected and analyzed.

[0016] IV. Analysis and Calculation of Test Data:

[0017] (1) Qualitative analysis of coumarin compounds in Angelica pubescens

[0018] The types of coumarin compounds in the *Angelica pubescens* sample were determined by high performance liquid chromatography retention time and ultraviolet spectra of the standard.

[0019] (2) Quantitative calculation of coumarin compounds in Angelica pubescens samples from Xing'an

[0020] The retention times of various standards and the peak areas corresponding to different concentrations of the standards were determined, and standard curves between the concentration and peak area of ​​each standard were plotted. The peak areas of coumarin compounds in the sample at different retention times were determined. The sample concentration was calculated based on the standard curves. The content of coumarin compounds in the sample of Angelica dahurica was calculated based on the sample concentration to complete the quantitative analysis.

[0021] The coumarin compounds are 7-hydroxycoumarin, scopolamine lactone, psoralen, xanthotoxin, bergamot lactone, imperatorin, isopsoralen, isoastatin, or coumarin.

[0022] The ultrasonic frequency for ultrasonic extraction in step one is 30KHz to 50KHz.

[0023] The methanol mentioned in step three is spectrally pure.

[0024] The C18 chromatographic column packing material with octadecyl bonded silica as the packing agent mentioned in step three is Hypersil BDSC18, with a size of 4.6 mm × 250 mm.

[0025] This method can complete the qualitative and quantitative detection and analysis of nine coumarin compounds in Angelica dahurica within 12 minutes, providing an effective method for the detection and analysis of various coumarin compounds in Angelica dahurica.

[0026] This invention solves the problem of existing methods being unable to simultaneously perform qualitative and quantitative detection of various coumarin compounds in Angelica dahurica, including 7-hydroxycoumarin, scopolamine, psoralen, xanthotoxin, bergamot lactone, imperatorin, isopsoralen, isoanethole, and coumarins. The invention includes the extraction, purification, chromatographic and spectroscopic detection, and data analysis and calculation of coumarin compounds in Angelica dahurica. This method has low requirements for detection equipment, is easy to implement and operate, and has a short detection time (less than 12 min), a wide linear detection range (0.122 mg / L to 500 mg / L), high sensitivity (limit of detection less than 0.17 mg / L, limit of quantitation less than 0.2 mg / L), good reproducibility (retention time deviation from standard within and between days less than 0.004%, peak area deviation from standard within and between days less than 0.026%), and high accuracy (average spiked recovery rate 97% to 125%, spiked recovery rate deviation from standard less than 4.6%). Attached Figure Description

[0027] Figure 1 This refers to the ultraviolet spectrum of scopolamine lactone involved in the data analysis and comparison of this invention;

[0028] Figure 2 This refers to the ultraviolet spectrum of 7-hydroxycoumarin involved in the analysis and comparison of detection data in this invention;

[0029] Figure 3 This refers to the coumarin ultraviolet spectrum involved in the analysis and comparison of detection data in this invention;

[0030] Figure 4 This refers to the ultraviolet spectrum of Sichuan pepper toxins involved in the data analysis and comparison of this invention;

[0031] Figure 5 This refers to the ultraviolet spectrum of psoralen involved in the data analysis and comparison of this invention;

[0032] Figure 6 This refers to the ultraviolet spectrum of isopsoralen involved in the data analysis and comparison of this invention;

[0033] Figure 7 This refers to the ultraviolet spectrum of isofennelolide involved in the data analysis and comparison of this invention;

[0034] Figure 8 This refers to the ultraviolet spectrum of bergamot lactone involved in the data analysis and comparison of this invention;

[0035] Figure 9 This refers to the ultraviolet spectrum of imperatorin involved in the analysis and comparison of detection data in this invention;

[0036] Figure 10This is a comparison of liquid chromatography (LC) chromatograms of the coumarin compound standard and the coumarin compound extract sample from Angelica dahurica in Example 1.

[0037] Figure 11 This is the ultraviolet spectrum of peak 1 in the extract of xanthotoxin and coumarin compounds from Angelica dahurica in Example 1;

[0038] Figure 12 This is the ultraviolet spectrum of peak 2 in the extracts of isofennelolide and coumarin compounds from Angelica dahurica in Example 1;

[0039] Figure 13 This is the ultraviolet spectrum of peak 3 in the extracts of bergamot lactone and coumarin compounds from Angelica dahurica in Example 1;

[0040] Figure 14 This is the ultraviolet spectrum of peak 4 in the extracts of imperatorin and coumarin compounds from Angelica dahurica in Example 1;

[0041] Figure 15 This is the standard curve for the quantitative analysis of zanthoxylate toxins in Example 1;

[0042] Figure 16 This is the standard curve for the quantitative analysis of bergamot lactones in Example 1;

[0043] Figure 17 This is the standard curve for the quantitative analysis of imperatorin in Example 1. Detailed Implementation

[0044] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0045] Specific Implementation Method 1: The rapid qualitative and quantitative detection method for coumarin compounds in Angelica pubescens in this implementation method is as follows:

[0046] I. Extraction of coumarin compounds from Angelica pubescens:

[0047] The dried, pulverized, or freshly crushed sample of Angelica dahurica was extracted with a 50%–100% methanol aqueous solution at a ratio of 1 g:(15–25) mL. The extraction was carried out at 28℃–38℃ with shaking or ultrasonication for 20–40 min. The sample was then centrifuged at 8500–11000 rpm, and the supernatant was collected. The precipitate was then extracted with the methanol aqueous solution at a ratio of 1 g:(15–25) mL at 28℃–38℃ with shaking or ultrasonication for 20–40 min. The sample was then centrifuged at 8500–11000 rpm, and the supernatant was collected. The extraction was repeated 2–4 times with shaking or ultrasonication. The supernatants were combined, filtered, and concentrated into a solid by centrifugation and vacuum extraction to obtain the extract.

[0048] II. Purification of coumarin compounds from Angelica pubescens:

[0049] The HC-C18 SPE columns were treated with 1 to 3 column volumes of methanol and deionized water, respectively, and then dried for later use.

[0050] The extract obtained in step one is dissolved in a 50%–100% methanol aqueous solution to obtain an extract solution. The extract solution is then passed through an HC-C18 SPE column. The total amount of coumarin compounds in the extract solution does not exceed 5% of the column packing material. After passing through the column for 10–15 min, the HC-C18 SPE column is eluted with an 80% methanol aqueous solution, and the eluent is collected. The eluent is concentrated by vacuum centrifugation and then dissolved in a 50%–100% methanol solution to obtain the extract solution of coumarin compounds from Angelica dahurica.

[0051] III. Detection of coumarin compounds in Angelica dahurica:

[0052] High-performance liquid chromatography (HPLC) and ultraviolet spectroscopy (UV spectroscopy) were performed using a high-performance liquid chromatography system equipped with a photodiode array as a liquid phase detector. The chromatographic column was a C18 column packed with octadecyl bonded silica gel. The sample injection volume was 10–20 μL. Mobile phase A was methanol and mobile phase B was water.

[0053] Mobile phase gradient elution program:

[0054] 0 min, 40%–60% A; 5 min, 50–70% A; 25 min, 70–90% A; 30 min, 80–100% A; flow rate 0.8–1 mL / min; column temperature 20–25℃; pressure 100–180 bar; quantitative detection wavelength 250–320 nm; UV spectroscopy detection wavelength 210–400 nm; the sum of the volume percentages of mobile phase A and mobile phase B at any time during the elution process is 100%; mobile phase flow rate: 0.8–1.2 mL / min;

[0055] Different concentrations of 7-hydroxycoumarin, scopolamine lactone, psoralen, xanthotoxin, bergamot lactone, imperatorin, isopsoralen, isofennelol, coumarin standard solutions, and extract solutions of coumarin compounds from Angelica dahurica were detected and analyzed.

[0056] IV. Analysis and Calculation of Test Data:

[0057] (1) Qualitative analysis of coumarin compounds in Angelica pubescens

[0058] The types of coumarin compounds in the *Angelica pubescens* sample were determined by high performance liquid chromatography retention time and ultraviolet spectra of the standard.

[0059] (2) Quantitative calculation of coumarin compounds in Angelica pubescens samples from Xing'an

[0060] The retention times of various standards and the peak areas corresponding to different concentrations of the standards were determined, and standard curves between the concentration and peak area of ​​each standard were plotted. The peak areas of coumarin compounds in the sample at different retention times were determined. The sample concentration was calculated based on the standard curves. The content of coumarin compounds in the sample of Angelica dahurica was calculated based on the sample concentration to complete the quantitative analysis.

[0061] The coumarin compounds are 7-hydroxycoumarin, scopolamine lactone, psoralen, xanthotoxin, bergamot lactone, imperatorin, isopsoralen, isoastatin, or coumarin.

[0062] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the ultrasonic frequency used for ultrasonic extraction in step one is 30kHz to 50kHz. Everything else is the same as in Specific Implementation Method One.

[0063] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One or Two in that the methanol used in step three is spectrally pure. Everything else is the same as in Specific Implementation Method One or Two.

[0064] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods One to Three in that the quantitative detection wavelength described in step three is 254 nm. Everything else is the same as in Specific Implementation Methods One to Three.

[0065] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in that the quantitative detection wavelength described in step three is 280nm. Everything else is the same as in Specific Implementation Methods One to Four.

[0066] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods One to Five in that the quantitative detection wavelength described in step three is 320nm. Everything else is the same as in Specific Implementation Methods One to Five.

[0067] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Methods One to Six in that the C18 chromatographic column packing material with octadecyl bonded silica as the packing material in step three is Hypersil BDS C18, with dimensions of 4.6 mm × 250 mm. Everything else is the same as in Specific Implementation Methods One to Six.

[0068] The effects of the present invention are verified using the following embodiments:

[0069] Example 1

[0070] The rapid qualitative and quantitative detection method for coumarin compounds in Angelica pubescens is as follows:

[0071] I. Extraction of coumarin compounds from Angelica pubescens:

[0072] 1g of dried Angelica dahurica sample powder was placed in a 50ml centrifuge tube and extracted with 15mL of 50% methanol aqueous solution. The extraction was carried out at 28℃ with shaking for 20min, and centrifuged at 11000rpm. The supernatant was collected. After centrifugation, the precipitate was mixed with methanol aqueous solution at a ratio of 1g:15mL and extracted at 28℃ with shaking for 20min. The extraction was carried out at 11000rpm and the supernatant was collected. The precipitate was extracted and centrifuged three times with shaking. The supernatants from the four extractions were combined, centrifuged, and vacuum concentrated into a solid to obtain the extract.

[0073] II. Purification of coumarin compounds from Angelica pubescens:

[0074] The HC-C18 SPE columns were treated with 1 to 3 column volumes of methanol and deionized water, respectively, and then dried for later use.

[0075] The extract obtained in step one was dissolved in a 50% (v / v) methanol aqueous solution to obtain an extract solution. The extract solution was passed through an HC-C18 SPE column. The total amount of coumarin compounds in the extract solution did not exceed 5% of the amount of packing material in the purification column. After passing through the column for 10 min, the HC-C18 SPE column was eluted with an 80% (v / v) methanol aqueous solution, and the eluent was collected. The eluent was concentrated by vacuum centrifugation and then dissolved in a 50% (v / v) methanol solution to obtain the extract solution of coumarin compounds from Angelica dahurica.

[0076] III. Detection of coumarin compounds in Angelica dahurica:

[0077] High-performance liquid chromatography (HPLC) and ultraviolet spectroscopy were performed using a high-performance liquid chromatography system equipped with a photodiode array (DAD) as a liquid phase detector. The chromatographic column was a C18 column packed with octadecyl bonded silica gel. The sample injection volume was 20 μL. Mobile phase A was methanol and mobile phase B was water.

[0078] Mobile phase gradient elution program:

[0079] 0 min, 40%–60% A; 5 min, 50–70% A; 25 min, 70–90% A; 30 min, 80–100% A; flow rate: 1 mL / min; column temperature: 23℃; pressure: 100–180 bar; quantitative detection wavelength: 320 nm; UV detection wavelength: 210–400 nm; the sum of the volume percentages of mobile phase A and mobile phase B at any time during the elution process is 100%; mobile phase flow rate: 1 mL / min.

[0080] Different concentrations of 7-hydroxycoumarin, scopolamine lactone, psoralen, xanthotoxin, bergamot lactone, imperatorin, isopsoralen, isofennelol, coumarin standard solutions, and extract solutions of coumarin compounds from Angelica dahurica were detected and analyzed.

[0081] The methanol mentioned in step three is spectrally pure.

[0082] The C18 chromatographic column packing material with octadecyl bonded silica as the packing agent mentioned in step three is Hypersil BDSC18, with a size of 4.6 mm × 250 mm.

[0083] IV. Analysis and Calculation of Test Data:

[0084] (1) Performance analysis of the detection method

[0085] Based on the above-mentioned optimized detection methods and conditions, the linear range, reproducibility, and accuracy of the detection methods were evaluated, and the detection analysis results are shown in Table 1.

[0086] Table 1. Linear range, limit of detection, limit of quantitation, relative standard deviation, and spiked recovery rate for the detection of coumarin compounds.

[0087]

[0088]

[0089] The results showed that the linear range of this method for the detection and analysis of the above nine coumarin compounds was as low as 0.122 mg / L and as high as 500 mg / L; the linearity was good. 2 The values ​​ranged from 0.9994 to 0.9999; the limit of detection was less than 0.17 mg / L and the limit of quantitation was less than 0.2 mg / L, indicating that the detection method had high sensitivity; the intra-day and inter-day relative standard deviations of retention time were less than 0.004%, and the intra-day and inter-day relative standard deviations of peak area were less than 0.026%, indicating that the method had good reproducibility and repeatability; the average spiked recovery rate was 97%–125%, and the spiked recovery rate RSD was less than 4.6%, indicating that the detection method had high accuracy.

[0090] (2) Qualitative analysis of coumarin compounds in Angelica dahurica 'Xingan'

[0091] The types of coumarin compounds in the *Angelica pubescens* sample were determined by high performance liquid chromatography retention time and ultraviolet spectra of the standard.

[0092] The high-performance liquid chromatography (HPLC) retention times and ultraviolet (UV) spectra of coumarin compound extracts from *Angelica pubescens* were compared with those of the standard samples to determine the types of coumarin compounds present in the *Angelica pubescens* samples. The HPLC retention times and UV spectra of the coumarin compound standard and the *Angelica pubescens* coumarin compound extract samples are shown below. Figure 10 As shown in the figure. The high-performance liquid chromatography retention times of the standard and the sample were compared. For example, the retention times of peaks 1, 2, 3 and 4 in the sample were 6.19, 7.11, 7.57 and 11.23, respectively, which are close to the retention times of the standard xanthotoxin, isoanisole, bergamot lactone and imperatorin, which were 6.21, 7.13, 7.59 and 11.26, respectively.

[0093] Further comparison of the UV spectra of substances with peaks 1, 2, 3, and 4 with the UV spectra of the standard was conducted, and the results are as follows: Figure 11 , Figure 12 , Figure 13 and Figure 14 As shown. Comparison of the UV spectra revealed that peak 2 did not match the UV spectrum of the standard isocarboxylidene. Therefore, although peak 2 and isocarboxylidene have similar high-performance liquid chromatography retention times, UV spectral analysis indicates that peak 2 is not isocarboxylidene. The UV spectra of peaks 1, 3, and 4 are consistent with those of xanthotoxin, bergamot lactone, and imperatorin, respectively. The trends and maximum and minimum absorption wavelengths of the UV spectra are the same. Therefore, qualitative analysis indicates that this sample of *Angelica dahurica* contains xanthotoxin, bergamot lactone, and imperatorin.

[0094] (3) Quantitative calculation of coumarin compounds in Angelica dahurica samples from Xing'an

[0095] The retention times of various standards and the peak areas corresponding to different concentrations of the standards were determined, and standard curves between the concentration and peak area of ​​each standard were plotted. The peak areas of coumarin compounds in the sample at different retention times were determined. The sample concentration was calculated based on the standard curves. The content of coumarin compounds in the sample of Angelica dahurica was calculated based on the sample concentration to complete the quantitative analysis.

[0096] The coumarin compounds are 7-hydroxycoumarin, scopolamine lactone, psoralen, xanthotoxin, bergamot lactone, imperatorin, isopsoralen, isoastatin, or coumarin.

[0097] Standard curve fitting: Based on the identification results of coumarin compounds in Angelica dahurica above, the contents of xanthotoxin, bergamot lactone, and imperatorin in the extract of coumarin compounds in Angelica dahurica were further quantitatively calculated. The peak areas of different concentrations of xanthotoxin, bergamot lactone, and imperatorin standards in liquid chromatography are shown in Table 2. The linear regression equations for xanthotoxin, bergamot lactone, and imperatorin were fitted as follows: Figure 15 , Figure 16 , Figure 17 As shown.

[0098] Table 2. Peak areas of liquid chromatography for different concentrations of xanthotoxin, bergamot lactone, and imperatorin standards.

[0099]

[0100] Sample Calculation: The peak areas of xanthotoxin, bergamot lactone, and imperatorin in the extract of coumarin compounds from *Angelica dahurica* were determined by liquid chromatography and are shown in Table 3. Based on the linear regression equation, the average concentrations of xanthotoxin, bergamot lactone, and imperatorin in the sample solution were calculated to be 0.003325 mg / mL, 0.03707 mg / mL, and 0.1188 mg / mL, respectively. The calculation formulas and results are shown in Table 3.

[0101] Table 3. Calculation of peak areas and concentrations of xanthotoxin, bergamot lactone, and imperatorin in extracts of coumarin compounds from Angelica dahurica.

[0102]

[0103]

[0104] Based on the concentration of coumarin compounds in the sample solution, calculate the content of coumarin compounds in the extract of Angelica dahurica using the following formulas.

[0105]

[0106]

[0107]

[0108] Calculate the content of coumarin compounds in the Xingan Angelica pubescens sample using the following formulas:

[0109]

[0110]

[0111]

[0112] The calculation results are shown in Table 4.

[0113]

[0114]

Claims

1. A rapid qualitative and quantitative detection method for coumarin compounds in Angelica dahurica, characterized in that... The rapid qualitative and quantitative detection method for coumarin compounds in Angelica dahurica is as follows: I. Extraction of coumarin compounds from Angelica pubescens: The dried, pulverized, or freshly crushed sample of Angelica dahurica was extracted with a 50%–100% methanol aqueous solution at a ratio of 1 g: (15–25) mL. The extraction was carried out at 28°C–38°C with shaking or ultrasonication for 20–40 min. The sample was then centrifuged at 8500–11000 rpm, and the supernatant was collected. The precipitate was then extracted with the methanol aqueous solution at a ratio of 1 g: (15–25) mL at 28°C–38°C with shaking or ultrasonication for 20–40 min. The sample was then centrifuged at 8500–11000 rpm, and the supernatant was collected. The extraction was repeated 2–4 times with shaking or ultrasonication. The supernatants were combined, filtered, and concentrated into a solid by centrifugation and vacuum extraction to obtain the extract. II. Purification of coumarin compounds from Angelica pubescens: The HC-C18 SPE columns were treated with 1 to 3 column volumes of methanol and deionized water, respectively, and then dried for later use. The extract obtained in step one is dissolved in a 50%~100% methanol aqueous solution to obtain an extract solution. The extract solution is passed through an HC-C18 SPE column. The total amount of coumarin compounds in the extract solution does not exceed 5% of the amount of packing material in the purification column. After passing through the column for 10~15 min, the HC-C18 SPE column is eluted with an 80% methanol aqueous solution and the eluent is collected. The eluent is concentrated by vacuum centrifugation and then dissolved in a 50%~100% methanol solution to obtain the extract solution of coumarin compounds from Angelica dahurica. III. Detection of coumarin compounds in Angelica dahurica: High-performance liquid chromatography (HPLC) and ultraviolet spectroscopy (UV spectroscopy) were performed using a high-performance liquid chromatography system equipped with a photodiode array as a liquid phase detector. The chromatographic column was a C18 column packed with octadecyl bonded silica gel. The sample injection volume was 10-20 μL. Mobile phase A was methanol and mobile phase B was water. Mobile phase gradient elution program: 0 min, 40%~60% A; 5 min, 50~70% A; 25 min, 70~90% A; 30 min, 80~100% A; column temperature 20~25 °C, pressure 100~180 bar, quantitative detection wavelength 250~320 nm; UV spectroscopy detection wavelength 210~400 nm; the sum of the volume percentages of mobile phase A and mobile phase B at any time during the elution process is 100%; mobile phase flow rate: 0.8~1.2 mL / min; Different concentrations of 7-hydroxycoumarin, scopolamine lactone, psoralen, xanthotoxin, bergamot lactone, imperatorin, isopsoralen, isofennelol, coumarin standard solutions, and extract solutions of coumarin compounds from Angelica dahurica were detected and analyzed. IV. Analysis and Calculation of Test Data: (1) Qualitative analysis of coumarin compounds in Angelica dahurica The types of coumarin compounds in the *Angelica pubescens* sample were determined by high performance liquid chromatography retention time and ultraviolet spectra of the standard. (2) Quantitative calculation of coumarin compounds in Angelica dahurica samples from Xing'an The retention times of various standards and the peak areas corresponding to different concentrations of the standards were determined, and standard curves between the concentration and peak area of ​​each standard were plotted. The peak areas of coumarin compounds in the sample at different retention times were determined. The sample concentration was calculated based on the standard curves. The content of coumarin compounds in the sample of Angelica dahurica was calculated based on the sample concentration to complete the quantitative analysis. The coumarin compounds are 7-hydroxycoumarin, scopolamine lactone, psoralen, xanthotoxin, bergamot lactone, imperatorin, isopsoralen, isoastatin, or coumarin.

2. The rapid qualitative and quantitative detection method for coumarin compounds in Angelica dahurica according to claim 1, characterized in that... The ultrasonic frequency for ultrasonic extraction in step one is 30 kHz to 50 kHz.

3. The rapid qualitative and quantitative detection method for coumarin compounds in Angelica dahurica according to claim 1, characterized in that... The methanol mentioned in step three is spectrally pure.

4. The rapid qualitative and quantitative detection method for coumarin compounds in Angelica dahurica according to claim 1, characterized in that... The quantitative detection wavelength described in step three is 254 nm.

5. The rapid qualitative and quantitative detection method for coumarin compounds in Angelica dahurica according to claim 1, characterized in that... The quantitative detection wavelength described in step three is 280 nm.

6. The rapid qualitative and quantitative detection method for coumarin compounds in Angelica dahurica according to claim 1, characterized in that... The quantitative detection wavelength described in step three is 320 nm.

7. The rapid qualitative and quantitative detection method for coumarin compounds in Angelica dahurica according to claim 1, characterized in that... The C18 chromatographic column packing material with octadecyl bonded silica as the packing material mentioned in step three is Hypersil BDS C18, with a size of 4.6 mm × 250 mm.

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