Preparation method of calipteroxin and application of calipteroxin as marker for identifying peucedanum praeruptorum
By preparing and detecting calipteryxin in Peucedanum praeruptorum, the problem of identifying counterfeit Peucedanum praeruptorum was solved, the accuracy of medicinal material quality testing and the safety of clinical use were improved, and pharmacopoeia standard support was provided.
Patent Information
- Application Number
- CN202511748129.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-26
- Publication Date
- 2026-02-03
AI Technical Summary
Existing technologies cannot effectively distinguish and detect adulterated Peucedanum praeruptorum, which affects the quality and efficacy of the medicinal material, and there is a lack of corresponding testing standards.
The marker calipteryxin in Angelica dahurica was prepared, and the content of calipteryxin in mixed powders of Angelica dahurica and Angelica dahurica with different mass ratios was detected by ultra-high performance liquid chromatography (UPLC). The detection method and limit standard were established.
This method enables accurate identification of adulterated Angelica dahurica (hairy Angelica dahurica), improves the pass rate of medicinal material quality testing, ensures the safety and effectiveness of clinical drug use, and provides a basis for relevant pharmacopoeia standards.
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Figure CN121453967A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of identification technology of Chinese medicinal materials, specifically involving the preparation method of calipteryxin in Peucedanum praeruptorum and the identification of adulterated Peucedanum praeruptorum, as well as the detection method and limit standard of calipteryxin. Background Technology
[0002] Qianhu Peucedanum praeruptorum Dunn is the dried root of *Peucedanum praeruptorum* (white-flowered angelica), belonging to the genus *Peucedanum* of the Apiaceae family. It is a commonly used traditional Chinese medicine, bitter and pungent in taste, slightly cold in nature, and enters the lung meridian. It has the effects of lowering qi and resolving phlegm, dispelling wind and clearing heat. It is used for phlegm-heat wheezing, thick yellow phlegm, and wind-heat cough with copious phlegm. Mao Qianhu (hairy angelica), on the other hand, is *Ligusticum striatum* (short-sliced ligusticum), belonging to the genus *Ligusticum* of the Apiaceae family. Ligusticopsis brachyloba The dried root of *Franch.* Leuteum jasminoides* is sometimes used as a substitute for *Angelica pubescens* in folk medicine because of the similarity in appearance between *Angelica pubescens* and *Angelica pubescens*. However, its main components do not meet the main testing requirements of the 2025 edition of the Pharmacopoeia of the People's Republic of China. Adding *Angelica pubescens* to *Angelica pubescens* will also affect the pass rate of *Angelica pubescens* testing, and its efficacy also needs to be verified.
[0003] To improve the quality inspection pass rate of Peucedanum praeruptorum and ensure the safety of its clinical use, this invention prepares and identifies the unknown main components of Peucedanum praeruptorum, extracts them from mixed powders of Peucedanum praeruptorum and Peucedanum praeruptorum in different mass ratios, detects the components, and sets limits of quantitation. This provides technical support for the quality of Peucedanum praeruptorum and the safety of its clinical use, and can also provide a basis for relevant standards to limit the detection of impurities in Peucedanum praeruptorum, which is of great significance. Summary of the Invention
[0004] Purpose of the invention: The purpose of this invention is to screen out the detection markers for adulterated *Peucedanum praeruptorum* (also known as *Gentiana macrophylla*). It also provides a method for preparing calipteryxin, a marker for adulterated *Peucedanum praeruptorum*, and a method for limiting the detection of calipteryxin in *Peucedanum praeruptorum*.
[0005] This invention provides technical support for the quality, efficacy, and safety of the traditional Chinese medicine Peucedanum praeruptorum. The invention utilizes phytochemical methods to prepare calipteryxin and employs UPLC to determine the calipteryxin content in mixed powders of Peucedanum praeruptorum and Angelica pubescens in different mass ratios. This method demonstrates high feasibility and practicality in identifying adulteration in Peucedanum praeruptorum varieties.
[0006] To achieve the above objectives, the technical steps employed in this invention are as follows: A method for preparing a calipteryxin compound, comprising the following steps: (1) Take the dried root of *Ligusticum striatum*, a plant of the genus *Ligusticum* in the family Apiaceae, crush it, extract it with methanol, and combine the extracts; concentrate it until alcohol-free, extract it with dichloromethane, combine the dichloromethane phases, concentrate it to dryness, and obtain the extract. (2) The compound calipteryxin was obtained by silica gel column chromatography with a gradient elution of petroleum ether: ethyl acetate in different volume ratios. The eluent was collected, concentrated to dryness, dissolved in methanol, separated by preparative liquid chromatography, and the preparative solution was concentrated, extracted with dichloromethane, and concentrated under reduced pressure.
[0007] As a preferred embodiment, the preparation method described above includes the following steps: Take the dried root of *Ligusticum striatum*, a plant of the genus *Ligusticum* in the family Apiaceae, pulverize it, add 10-90% methanol at a mass-to-volume ratio of 1:5-20, and extract in a water bath at 50-70℃ 1-3 times, 0.5-2 hours each time. Combine the extracts; concentrate until alcohol-free, extract with dichloromethane, combine the dichloromethane phases, concentrate to dryness, and obtain the extract. (3) Separate the extract from step (1) by silica gel column chromatography, using a gradient elution of petroleum ether:ethyl acetate with volume ratios of 100:0; 75:25; 50:50; 25:75; 0:100. Take the eluent with a petroleum ether:ethyl acetate ratio of 75:25, concentrate it to dryness, dissolve it in methanol, and use preparative liquid chromatography. Under the conditions of a flow rate of 20 mL / min, a detection wavelength of 321 nm, and a column temperature of 25 °C, first use acetonitrile:water with a mobile phase of 75:25, C 18 The fraction with a retention time of 8 minutes was collected by column separation; then, the fraction with a retention time of 13 minutes was collected by C8 column separation with a mobile phase of methanol:water = 72:28. The fraction was concentrated to a small volume at 45°C, extracted with dichloromethane, and concentrated under reduced pressure in dichloromethane phase to obtain the compound calipteryxin.
[0008] The compound calipteryxin prepared by the above method can be used as a marker for detecting Peucedanum praeruptorum, as a component in the detection of adulterated Peucedanum praeruptorum, or as a marker for identifying adulterated Peucedanum praeruptorum in Peucedanum praeruptorum.
[0009] This invention detects calipteryxin in Peucedanum praeruptorum, Peucedanum pubescens, and mixtures of Peucedanum praeruptorum and Peucedanum pubescens for identification purposes, and includes the following steps: (1) Preparation of Calipteryxin standard solution and establishment of standard curve: Preparation of Calipteryxin standard stock solution: Accurately weigh calipteryxin, dissolve it in methanol and make up to volume, seal it in a brown bottle and store it away from light; Preparation of Calipteryxin standard solution: Take calipteryxin standard stock solution and dilute it with methanol to obtain calipteryxin standard solutions of different concentrations. Inject calipteryxin standard solutions of different concentrations into an ultra-high performance liquid chromatograph. Perform linear regression analysis with peak area as the ordinate (y) and calipteryxin reference solution concentration as the abscissa (x) to plot the calipteryxin standard curve equation. (2) Preparation of test solutions of Peucedanum praeruptorum, Peucedanum pubescens, and Peucedanum praeruptorum mixed with Peucedanum pubescens: Take whole, mold-free and rot-free Peucedanum root and Peucedanum hairy root, and air-dry, bake, crush and sieve to obtain Peucedanum powder and Peucedanum hairy powder; mix Peucedanum powder and Peucedanum hairy powder in a certain mass ratio. Then, take the powder of Peucedanum praeruptorum, the powder of Peucedanum praeruptorum pubescens and the mixture of Peucedanum praeruptorum pubescens and the mixture of Peucedanum praeruptorum pubescens, respectively, add 8 to 15 times the volume of methanol, extract by ultrasonication for 30 to 60 minutes, filter the extract, dilute the filtrate with methanol and filter it through a microporous membrane, and use the filtrate from the microporous membrane to obtain the test solution of Peucedanum praeruptorum pubescens, the mixture of Peucedanum praeruptorum pubescens and ... (3) Determination of calipteryxin in samples of Peucedanum praeruptorum, Peucedanum pubescens, and Peucedanum praeruptorum mixed with Peucedanum pubescens: Take the test solutions of Peucedanum praeruptorum, Peucedanum praeruptorum var. pubescens and Peucedanum praeruptorum var. pubescens mixed with Peucedanum praeruptorum from step (2) and inject them into an ultra-high performance liquid chromatograph for analysis; then, substitute the peak area of calipteryxin into the standard curve equation of step (1) and calculate the content of calipteryxin in each test sample.
[0010] As a preferred embodiment, the chromatographic conditions for steps (1) and (3) above are as follows: the mobile phase consists of ultrapure water as phase A and acetonitrile as phase B, with gradient elution; the detection wavelength is 321 nm; the flow rate is 0.2 mL / min; the injection volume is 2 µL; and the retention time is 60 min. The gradient elution conditions are shown in the table below: Gradient elution conditions and time (min) Mobile phase B (%) Mobile phase A (%) 0~20 40~40 60~60 20~30 40~50 60~50 30~50 50~50 50~50 50~55 50~40 50~60 55~60 40~40 60~60 As a preferred embodiment, in the application described above, the preparation of the sample solution of *Peucedanum praeruptorum* mixed with *Peucedanum praeruptorum* in step (2) is as follows: Weigh *Peucedanum praeruptorum* powder and *Peucedanum praeruptorum* powder S1~S8 (where the mass ratio of *Peucedanum praeruptorum* is S1 50%: 1.0 g-1.0 g, S2 30%: 1.4 g-0.6 g, S3 20%: 1.6 g-0.4 g, S4 10%: 1.8 g-0.2 g, S5 5%: 1.9 g-0.1 g, S6 3%: 1.94 g-0.06 g, S7 2%: 1.96 g-0.04 g, S8 1%: 1.98 g-0.02 g), place them in a 250 mL stoppered conical flask, add 20 mL of methanol, weigh, sonicate for 30 min, cool, weigh to make up the weight, filter, take 2 mL of the filtrate and add 10 mL of methanol. Dilute to volume with methanol in a volumetric flask, filter through a 0.45 µm microporous membrane to obtain the test solution of Peucedanum praeruptorum mixed with Angelica dahurica.
[0011] As a preferred option, the above-described application involves step (1) establishing the calipteryxin standard stock solution and standard curve: Calipteryxin standard stock solution: Accurately weigh 4.487 mg calipteryxin, dissolve it in methanol, and dilute to 10 mL to obtain a calipteryxin standard stock solution with a concentration of 448.7 μg / mL; Calipteryxin Standard Solution: Accurately pipette 5.00 mL of calipteryxin stock solution (448.7 μg / mL) into a 10 mL volumetric flask, dilute to the mark with methanol to obtain a calipteryxin standard solution of 224.35 μg / mL; pipette 5.00 mL of the calipteryxin standard solution (224.35 μg / mL) into a 10 mL volumetric flask, dilute to the mark with methanol to obtain a calipteryxin standard solution of 112.75 μg / mL; repeat the above steps to obtain calipteryxin standard solutions of 56.09 μg / mL, 28.04 μg / mL, and 14.02 µg / mL respectively; inject the six different concentrations of calipteryxin standard solutions into an ultra-high performance liquid chromatograph (UHPLC), and perform linear regression analysis with concentration as the x-axis and peak area as the y-axis. The Calipteryxin standard curve is plotted as y = 0.2123x - 0.1675, R0. 2 = 1, linear range: 14.02~448.70 µg / mL. Based on the signal-to-noise ratio, the limit of detection (DL) of calipteryxin is calculated to be 0.3719 µg / mL, and the limit of quantitation (QL) is 1.2396 µg / mL.
[0012] Methodological Investigation on the Analytical Detection of Calipteryxin 1. Precision, Repeatability and Stability Tests Precision test: Appropriately pipette a calipteryxin reference substance solution, prepare a reference substance solution with a concentration of 112.75 μg / mL, inject samples continuously 6 times under chromatographic conditions, record the peak area of calipteryxin and calculate the relative standard deviation (RSD). The results show that the RSD of the peak area of calipteryxin is 0.91%, indicating good precision of the instrument.
[0013] Repeatability test: According to the preparation method of the test solution of Peucedanum praeruptorum adulterated with P. pubescens, take 6 parallel groups of the test solution of P. pubescens, conduct detection and analysis, record the peak area of calipteryxin and calculate the RSD. The results show that the RSD of the peak area of calipteryxin is 1.31%, indicating good repeatability of this method.
[0014] Stability test: Take one portion of the test solution of P. pubescens in the repeatability test, determine the peak area of calipteryxin at 0, 4, 8, 12, and 24 h respectively, and calculate its RSD. The results show that the RSD of the peak area is 1.37%, indicating good stability of P. pubescens within 24 h.
[0015] 2. Spiked Recovery Test Take 0.5 g of P. praeruptorum with the determined calipteryxin content, add calipteryxin reference substance at 3 levels of 80%, 100%, and 120% of the known content respectively to prepare a mixed test solution, determine at 321 nm, and calculate the spiked recovery rate. The results show that the average recovery rate of calipteryxin is 114.69% and the RSD is 3.66%, indicating good accuracy of this method. Beneficial Effects
[0016] The existing Chinese Pharmacopoeia standards do not stipulate the quality detection of Peucedanum praeruptorum adulterated with P. pubescens. In the market, P. pubescens is used to adulterate P. praeruptorum for medicinal use, reducing the qualified rate of P. praeruptorum detection and affecting the medicinal efficacy. The present invention qualitatively and quantitatively determines calipteryxin extracted and separated from P. pubescens to identify whether P. praeruptorum is adulterated with P. pubescens and the content of the adulteration, so as to provide a scientific basis for ensuring the safety and effectiveness of P. praeruptorum in clinical use. At the same time, the present invention can also provide a basis for the limit detection of impurities in P. praeruptorum and provide a basis for relevant regulations of the Pharmacopoeia to add impurity detection items.
[0017] This method was tested through precision tests, repeatability tests, stability tests, and sample recovery tests. The results show that this method is stable, reliable, and accurate in determining calipteryxin in the mixed solution of Peucedanum praeruptorum and Angelica dahurica. Attached Figure Description
[0018] Figure 1 This is the structural formula for calipteryxin.
[0019] Figure 2 The 1H NMR spectrum of calipteryxin dissolved in deuterated chloroform.
[0020] Figure 3 The carbon NMR spectrum of calipteryxin dissolved in deuterated chloroform.
[0021] Figure 4 Chromatograms of the test solutions of reference standards calipteryxin, Peucedanum praeruptorum, and Angelica pubescens. Figure 5 Chromatograms of test solutions containing 50%, 30%, and 20% *Peucedanum praeruptorum*.
[0022] Figure 6 Chromatograms of test solutions containing 10%, 5%, and 3% *Peucedanum praeruptorum*.
[0023] Figure 7 The chromatogram is of a mixed test solution of 2% and 1% *Peucedanum praeruptorum*. Detailed Implementation
[0024] The following examples further illustrate the present invention. These are merely embodiments of the present experiment. Any person skilled in the art can use the above-described technical solutions to improve the present invention or modify it into an equivalent technical solution.
[0025] Example 1 The preparation method of calipteryxin from Angelica dahurica includes the following steps: (1) Take the dried root of Ligusticum sepium (common name: Angiotensinus pubescens), a plant of the genus Ligusticum in the family Apiaceae, crush it, add 90% methanol at a mass-to-volume ratio of 1:5, and extract three times in a water bath at 70°C for 2 hours each time. Combine the extracts. Concentrate until alcohol is removed, extract twice with dichloromethane, combine the dichloromethane phases, and concentrate to dryness to obtain the extract.
[0026] The extract was subjected to silica gel column chromatography, eluting with a gradient of petroleum ether:ethyl acetate at volume ratios of 100:0; 75:25; 50:50; 25:75; 0:100. The petroleum ether:ethyl acetate eluent at 75:25 was concentrated to dryness, dissolved in methanol, and then separated by preparative liquid chromatography. Specific chromatographic conditions were: 321 nm, 25 °C, 20 mL / min. The mobile phase was initially acetonitrile:water = 75:25. 18 The fraction with a retention time of 8 minutes was collected by column chromatography, followed by further separation using a C8 column with a mobile phase of methanol:water (72:28) and collection of the fraction with a retention time of 13 minutes. The qualified preparation solution was concentrated to a small volume at 45°C, extracted with dichloromethane, and concentrated under reduced pressure in the dichloromethane phase to obtain white, blocky crystals with a purity of over 95%. 1 H-NMR (400 MHz, CDCl3) showed δ 7.58(d, J = 9.6 Hz, 1H) and 6.20 (d, J = 9.6 Hz, 1H) H-4 and H-3 signals for a pair of olefin protons in coumarin. δ 7.35(d, J =8.8 Hz, 1H) and 6.80 (d, J = 8.8 Hz, 1H) represents the proton signal coupled between H-5 and H-6 at the ortho positions of the benzene ring. δ 5.99 (m, 1H) and 1.97 (m, 3H), 1.83 (p, J= 1.6 Hz, 3H) indicates the presence of angeloyl, while the olefin proton δ 5.65 (p, J = 1.6 Hz, 1H) and the two allyl methyl signals δ 2.14 (3H, d, J = 1.2 Hz) and 1.88 (3H, d, J = 1.6 Hz) belong to senecioyl. Combined with the literature (Nielsen BE, SoineTO. Coumarins. IV. Coumarins of Pteryxia terebinthina. Structures of two newcoumarins, isopteryxin and calipteryxin. J Pharm Sci, 1967, 56(2):184-187.). Based on the carbon spectral signal and the literature (Tim M. Swager, John H. Cardellina II. Coumarins from Musineon divaricatum [J]. Phytochemistry, 1985, 24(4):805-813 and Buendía-Trujillo AI, Torrres-Valencia JM, Joseph-Nathan P, Burgueño-Tapia E. Absolute Configuration Assignment of 3',4'-di-O-acylkhellactones Using Vibrational Circular Dichroism Exciton Chirality. Nat Prod Commun, 2015, 10(6):1027-1032.), the compound was identified as calipteryxin, and its structural formula is as follows. Figure 1 As shown. Its 1H-NMR and 13C-NMR spectra are as follows. Figure 2 and 3 .
[0027] Example 2 The method for determining calipteryxin in Peucedanum praeruptorum, Peucedanum praeruptorum var. pubescens, and Peucedanum praeruptorum var. pubescens mixed with Peucedanum praeruptorum var. pubescens includes the following steps: (1) Preparation of Calipteryxin standard solution and establishment of standard curve: Calipteryxin standard stock solution: Accurately weigh 4.487 mg of Calipteryxin, dissolve it in methanol, and dilute to 10 mL to obtain a Calipteryxin standard stock solution with a concentration of 448.7 μg / mL; Calipteryxin standard solution: Accurately pipette 5.00 mL of calipteryxin standard stock solution with a concentration of 448.7 μg / mL into a 10 mL volumetric flask, and dilute to the mark with methanol to obtain a calipteryxin standard solution with a concentration of 224.35 μg / mL; take 5.00 mL of the calipteryxin standard solution with a concentration of 224.35 μg / mL into a 10 mL volumetric flask, and dilute to the mark with methanol to obtain a calipteryxin standard solution with a concentration of 112.18 μg / mL; repeat the above steps to obtain calipteryxin standard solutions with concentrations of 56.09 μg / mL, 28.05 μg / mL, and 14.03 µg / mL respectively; Six calipteryxin standard solutions of different concentrations were injected into an ultra-high performance liquid chromatograph. Linear regression analysis was performed with concentration as the x-axis and peak area as the y-axis. The calipteryxin standard curve was plotted as y = 0.2123x - 0.1675. R 2 = 1, linear range: 14.02~448.70 µg / mL. Based on the signal-to-noise ratio, the limit of detection (DL) of calipteryxin is calculated to be 0.3719 µg / mL, and the limit of quantitation (QL) is 1.2396 µg / mL.
[0028] The chromatographic conditions were as follows: a Waters ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 × 100 mm); ultrapure water (A) and acetonitrile (B) as the mobile phase, with gradient elution; detection wavelength of 321 nm; flow rate of 0.2 mL / min; column temperature of 35℃; injection volume of 2 µL; and retention time of 60 min. The theoretical plate number, calculated according to calipteryxin, should not be less than 3000.
[0029] The gradient elution conditions are shown in the table below: Gradient elution conditions and time (min) Mobile phase B (%) Mobile phase A (%) 0~20 40~40 60~60 20~30 40~50 60~50 30~50 50~50 50~50 50~55 50~40 50~60 55~60 40~40 60~60 (2) Preparation of the test solution of Peucedanum praeruptorum mixed with Peucedanum praeruptorum: Take whole, mold-free, and rot-free Peucedanum praeruptorum roots and hairy Peucedanum praeruptorum roots, air-dry them naturally, bake them, pulverize them, and pass them through a No. 3 sieve. Accurately weigh the Peucedanum praeruptorum powder - hairy Peucedanum praeruptorum powder S1~S8 (S1 50%: 1.0023g-1.0006g, S2 30%: 1.4021g-0.6002g, S3 20%: 1.6013g-0.4016g, S4 10%: 1.8078g-0.2011g, S5 5%: 1.9015g-0.1007g, S6 3%: 1.9424g-0.0613g, S7 2%: 1.9604g-0.0410g, S8 1%: 1.9855g-0.0210g) into a 250 mL stoppered conical flask, add 20 Weigh the sample in mL of methanol, sonicate at 80 kHz for 30 min, cool, weigh again to make up the weight, filter, add 2 mL of the filtrate to a 10 mL volumetric flask, dilute to volume with methanol, and filter through a 0.45 µm microporous membrane to obtain the sample solution of Peucedanum praeruptorum mixed with Angelica dahurica.
[0030] (3) Preparation of test solution for genuine Chinese herbal medicine Peucedanum praeruptorum: Take whole, mold-free, and rot-free Peucedanum root, air-dry, oven-dry, pulverize, and pass through a No. 3 sieve. Accurately weigh Peucedanum powder P1~P4 (P1 2.0059g; P2 2.0083g; P3 2.0098g; P4 2.0024g) into a 250 mL stoppered conical flask, add 20 mL of methanol, weigh, sonicate at 80 kHz for 30 min, cool, weigh to make up the weight, filter, take 2 mL of the filtrate and add it to a 10 mL volumetric flask, dilute to volume with methanol, filter through a 0.45 µm microporous membrane to obtain the Peucedanum praeruptorum test solution.
[0031] (4) Determination of calipteryxin in Peucedanum praeruptorum adulterated with virgin Peucedanum praeruptorum and in virgin Peucedanum praeruptorum test samples: Take the test solution of the adulterated Peucedanum praeruptorum in step (2) and the test solution of the genuine Peucedanum praeruptorum in step (3) respectively, and inject them into the ultra-high performance liquid chromatograph for analysis under the same chromatographic conditions as in step (1); then substitute the peak area of calipteryxin into the standard curve equation in step (1) to calculate the content of calipteryxin in the adulterated Peucedanum praeruptorum test sample and the genuine Peucedanum praeruptorum test sample.
[0032] 3. The calipteryxin content in each batch of Peucedanum praeruptorum adulterated with Peucedanum praeruptorum was as follows: S1 35.4855 µg / mL; S2 22.9382 µg / mL; S3 16.1713 µg / mL; S4 9.7379 µg / mL; S5 6.0515 µg / mL; S6 4.5985 µg / mL; S7 4.1185 µg / mL; S8 3.6499 µg / mL.
[0033] 4. Based on chromatographic data, the lowest detectable calipteryxin concentration is determined by mixing 1% of Peucedanum praeruptorum with 1% of Peucedanum praeruptorum. That is, the lowest limit of quantitation for calipteryxin in Peucedanum praeruptorum mixed with 1% of Peucedanum praeruptorum is 3.6499 µg / mL, which is equivalent to a mass fraction of 0.1819 mg / g.
[0034] 5. Calipterine content was not detected in any batch of genuine Peucedanum praeruptorum products.
[0035] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing a calipteryxin compound, characterized in that, Includes the following steps: (1) Take the dried root of *Ligusticum striatum*, a plant of the genus *Ligusticum* in the family Apiaceae, crush it, extract it with methanol, and combine the extracts; concentrate it until alcohol-free, extract it with dichloromethane, combine the dichloromethane phases, concentrate it to dryness, and obtain the extract. (2) The compound calipteryxin was obtained by silica gel column chromatography with a gradient elution of petroleum ether: ethyl acetate in different volume ratios. The eluent was collected, concentrated to dryness, dissolved in methanol, separated by preparative liquid chromatography, and the preparative solution was concentrated, extracted with dichloromethane, and concentrated under reduced pressure.
2. The preparation method according to claim 1, characterized in that, Includes the following steps: Take the dried root of *Ligusticum striatum*, a plant of the genus *Ligusticum* in the family Apiaceae, pulverize it, add 10-90% methanol at a mass-to-volume ratio of 1:5-20, and extract in a water bath at 50-70℃ 1-3 times, 0.5-2 hours each time. Combine the extracts; concentrate until alcohol-free, extract with dichloromethane, combine the dichloromethane phases, concentrate to dryness, and obtain the extract. (2) Separate the extract from step (1) by silica gel column chromatography, using a gradient elution of petroleum ether:ethyl acetate with volume ratios of 100:0; 75:25; 50:50; 25:75; 0:
100. Take the eluent with a petroleum ether:ethyl acetate ratio of 75:25, concentrate it to dryness, dissolve it in methanol, and use preparative liquid chromatography. Under the conditions of a flow rate of 20 mL / min, a detection wavelength of 321 nm, and a column temperature of 25 °C, first use acetonitrile:water with a mobile phase of 75:25, C 18 The solution was separated by column chromatography, followed by further separation using a C8 column with a mobile phase of methanol:water = 72:
28. The qualified preparation solution was concentrated to a small volume at 45°C, extracted with dichloromethane, and concentrated under reduced pressure in the dichloromethane phase to obtain the compound calipteryxin.
3. The application of the compound calipteryxin prepared by the preparation method according to claim 1 or 2 as a marker for the detection of Angelica pubescens.
4. The application of the compound calipteryxin prepared by the preparation method according to claim 1 or 2 as a component in the detection of adulterants of Angelica dahurica.
5. The application of the compound calipteryxin prepared by the preparation method according to claim 1 or 2 as a marker for identifying adulteration of Angelica dahurica with hairy Angelica.
6. The application according to any one of claims 3 to 5, for detecting calipteryxin in Peucedanum praeruptorum, Peucedanum pubescens, and mixtures of Peucedanum praeruptorum and Peucedanum pubescens, characterized in that, Includes the following steps: (1) Preparation of Calipteryxin standard solution and establishment of standard curve: Preparation of Calipteryxin standard stock solution: Accurately weigh calipteryxin, dissolve it in methanol and make up to volume, seal it in a brown bottle and store it away from light; Preparation of Calipteryxin standard solution: Take calipteryxin standard stock solution and dilute it with methanol to obtain calipteryxin standard solutions of different concentrations. Inject calipteryxin standard solutions of different concentrations into an ultra-high performance liquid chromatograph. Perform linear regression analysis with peak area as the ordinate (y) and calipteryxin reference solution concentration as the abscissa (x) to plot the calipteryxin standard curve equation. (2) Preparation of test solutions of Peucedanum praeruptorum, Peucedanum pubescens, and Peucedanum praeruptorum mixed with Peucedanum pubescens: Take whole, mold-free and rot-free Peucedanum root and Peucedanum hairy root, and air-dry, bake, crush and sieve to obtain Peucedanum powder and Peucedanum hairy powder; mix Peucedanum powder and Peucedanum hairy powder in a certain mass ratio. Then, take the powder of Peucedanum praeruptorum, the powder of Peucedanum praeruptorum pubescens and the mixture of Peucedanum praeruptorum pubescens and the mixture of Peucedanum praeruptorum pubescens, respectively, add 8 to 15 times the volume of methanol, extract by ultrasonication for 30 to 60 minutes, filter the extract, dilute the filtrate with methanol and filter it through a microporous membrane, and use the filtrate from the microporous membrane to obtain the test solution of Peucedanum praeruptorum pubescens, the mixture of Peucedanum praeruptorum pubescens and ... (3) Determination of calipteryxin in samples of Peucedanum praeruptorum, Peucedanum pubescens, and Peucedanum praeruptorum mixed with Peucedanum pubescens: Take the test solutions of Peucedanum praeruptorum, Peucedanum praeruptorum var. pubescens and Peucedanum praeruptorum var. pubescens mixed with Peucedanum praeruptorum from step (2) and inject them into an ultra-high performance liquid chromatograph for analysis; then, substitute the peak area of calipteryxin into the standard curve equation of step (1) and calculate the content of calipteryxin in each test sample.
7. The application according to claim 6, characterized in that, The chromatographic conditions for steps (1) and (3) are as follows: the mobile phase is ultrapure water as phase A and acetonitrile as phase B, with gradient elution; the detection wavelength is 321 nm; the flow rate is 0.2 mL / min; the injection volume is 2 µL; and the retention time is 60 min.
8. The application according to claim 6, characterized in that, Preparation of the test solution of Peucedanum praeruptorum mixed with Angelica dahurica in step (2): Weigh Peucedanum praeruptorum powder and Angelica dahurica powder S1~S8, wherein the mass ratio of Angelica dahurica is S1 50%, S2 30%, S3 20%, S4 10%, S5 5%, S6 3%, S7 2%, and S8 1%, place them in a stoppered conical flask, add 20 mL of methanol, weigh, sonicate for 30 min, cool, weigh to make up the weight, filter, take 2 mL of the filtrate and add it to a 10 mL volumetric flask, make up to volume with methanol, filter with a 0.45 µm microporous membrane to obtain the test solution of Peucedanum praeruptorum mixed with Angelica dahurica.
9. The application according to claim 7, characterized in that, The gradient elution conditions are shown in the table below: 。 10. The application according to claim 4 or 5, characterized in that, The limit of quantification for adulteration of Peucedanum praeruptorum with calipteryxin shall not exceed 0.1819 mg / g.