Preparation and quality detection method of pimpinella diversifolia fruit standard decoction

Abstract

The invention discloses a preparation and quality detection method of a Pimpinella diversifolia fruit standard decoction, and particularly provides a detection method of the Pimpinella diversifolia fruit standard decoction, which comprises the following steps: detecting the Pimpinella diversifolia fruit standard decoction by high performance liquid chromatography; the pimpinella diversifolia fruit standard decoction is prepared from chlorogenic acid, isoorientin, isovitexin, luteolin and apigenin. Chromatographic conditions of the high performance liquid chromatography are as follows: a chromatographic column is an octadecyl bonded silica gel chromatographic column; the mobile phase A is acetonitrile, and the mobile phase B is 0.05-0.1% acetic acid aqueous solution; and gradient elution. The method is good in stability, high in precision and good in reproducibility. The fingerprint spectrum of the standard decoction of the pimpinella diversifolia fruit decoction pieces has seven common peaks, five components are determined through identification and are all main effective components of the pimpinella diversifolia fruit, a basis can be provided for establishment and quality control of the fingerprint spectrum of the formula granules, and the clinical effectiveness of the pimpinella diversifolia fruit formula granules is ensured.

Description

Technical Field

[0001] The invention relates to a preparation method and quality detection method of a standard decoction of fennel fruit. Background Art

[0002] The safety and effectiveness of Chinese medicine are the result of thousands of years of precipitation and clinical practice. In the long history of Chinese medicine, decoction has always been the main form of clinical application, and its unique efficacy and safety have been widely recognized and applied. This traditional treatment method not only contains a profound cultural heritage, but also reflects the wisdom and experience of Chinese medicine. In August 2016, the State Pharmacopoeia Committee issued the "Technical Requirements for Quality Control and Standardization of Chinese Medicine Formula Granules (Draft for Comments)", which indicates that a new model of quality control of Chinese medicine formula granules may be opened. The new standard applies characteristic spectrum quality control technology, strengthens the overall quality control level of Chinese medicine formula granules, especially the introduction of the concept of "standard decoction" in the process of standard formulation, which not only provides a basis for measuring the rationality of the formulation of Chinese medicine formula granules and quality control, but also promotes the innovation and development of Chinese medicine quality control mode.

[0003] Anise fruit is the dried mature fruit of Pimpinella anisum L., a plant of the Umbelliferae family. It has the pharmacological effects of dispelling cold, promoting the body's natural composure, promoting diuresis and menstruation, and moistening the intestines and relieving pain. Its medicinal value has been confirmed by medical literature. For example, "Health Medicinal Garden" records that it has the effects of "dispelling cold and relieving pain, relieving cough and relieving asthma"; "Notes on Medical Classics" describes that it can: "clear the blockage, relieve pain, and eliminate dizziness". Modern scientific research has further revealed the chemical composition of anise fruit, and found that it is rich in bioactive ingredients such as flavonoids, flavonoid glycosides and phenolic acids. These ingredients give anise fruit multiple pharmacological effects such as antioxidant, anti-tumor, anti-depression and anxiety relief, antibacterial, antispasmodic, analgesic and anti-inflammatory.

[0004] There are few research reports on the quality control of anise fruit at home and abroad. The original standards only include properties and microscopic identification, and the quality control is relatively simple. In 2013, the document "Research on Quality Standards of Anise Fruit" disclosed its thin-layer identification of anisole. On this basis, the "Xinjiang Uygur Autonomous Region Traditional Chinese Medicine Uyghur Medicinal Pieces Processing Specifications" (2020 edition) included anisole thin-layer identification in the standard. In the newly revised national drug standards in 2023, only the properties and microscopic and thin-layer identification of anise fruit are used as quality control indicators. These methods are not enough to fully control the quality of medicinal materials. There are low efficiency of thin-layer identification, lack of content determination and fingerprint spectrum, which are not enough to fully control the quality of anise fruit. At present, there is no research and preparation method for standard decoction of anise fruit.

[0005] These problems lead to the inaccuracy of the quality control of the standard decoction of fennel fruit slices, which affects the safety and effectiveness of its clinical application. The present invention establishes a method for preparing a standard decoction of fennel fruit and uses thin layer chromatography technology and high performance liquid chromatography technology to establish a thin layer identification and characteristic spectrum method to improve the quality control level of the standard decoction of fennel fruit slices, further ensure the quality and stability of fennel fruit formula particles, and ensure its consistency and reliability in production and clinical application. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the defect that the quality control of anise fruit in the prior art is not accurate enough, and to provide a standard decoction of anise fruit and a preparation method and a detection method thereof, so as to better control the quality and particle stability of anise fruit formula granules.

[0007] The present invention solves the above technical problems through the following technical solutions:

[0008] The present invention provides a method for detecting a standard decoction of fennel fruit, which comprises the following steps:

[0009] The sample to be tested is detected by high performance liquid chromatography; the sample to be tested contains one or more of chlorogenic acid, isorientin, isovitexin, luteolin and apigenin;

[0010] The chromatographic conditions of the high performance liquid chromatography are:

[0011] The chromatographic column is an octadecyl bonded silica gel chromatographic column;

[0012] Mobile phase A was acetonitrile, and mobile phase B was 0.05% to 0.1% acetic acid in water;

[0013] Gradient elution, the gradient method is: the initial volume percentage of mobile phase A is 10-15%, changes to 40% within 40-50 minutes, changes to 90% within 5-10 minutes after rising to 40%; 5-10 minutes after rising to 90%, it decreases to 10-15%; the volume percentage of mobile phase B is always 1-the volume percentage of mobile phase A; during the change process of the gradient elution, the volume percentage of mobile phase A keeps rising or flat. In some embodiments, the chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0014] In some embodiments, the chromatography column is an Agilent ZORBAX Eclipse Plus C18.

[0015] In some embodiments, in the chromatographic conditions of the high performance liquid chromatography, the column temperature is 25-35°C; such as 28-35°C; preferably 30°C.

[0016] In some embodiments, in the chromatographic conditions of the high performance liquid chromatography, the flow rate is 0.9-1.1 ml / min; such as 1-1.1 ml / min, preferably 1 ml / min.

[0017] In some embodiments, in the chromatographic conditions of the high performance liquid chromatography, the detection wavelength is 300-360 nm; such as 320-350 nm; for example 320 nm, 335 nm or 350 nm.

[0018] In some embodiments, the mobile phase B is 0.05% acetic acid in water;

[0019] In some embodiments, the standard decoction of anise fruit is tested in the form of a test solution, and the test solution is prepared by ultrasonicating or refluxing a 50%-100% methanol aqueous solution of the standard decoction of anise fruit.

[0020] In some embodiments, in the test solution, the 50%-100% methanol aqueous solution is preferably 70%-100% methanol aqueous solution; for example, 100% methanol.

[0021] In some embodiments, in the test solution, the ultrasound or reflux is preferably ultrasound.

[0022] In some embodiments, in the test solution, the mass-to-volume ratio of the standard decoction of fennel fruit and the solvent is (0.01-0.1) g / ml; for example, 0.025 g / ml, 0.05 g / ml or 0.075 g / ml.

[0023] In some embodiments, in the test solution, the extract is obtained by ultrasonic or reflux extraction for 15 to 60 minutes; for example, the extract is obtained by ultrasonic or reflux extraction for 15 minutes, 30 minutes or 45 minutes.

[0024] In some embodiments, the detection method further comprises detecting a reference solution; the reference solution is a solution comprising one or more of chlorogenic acid, isoorientin, isovitexin, luteolin and apigenin.

[0025] In some embodiments, the solvent of the reference solution is aqueous methanol; such as 50-100% aqueous methanol; for example 70% aqueous methanol.

[0026] In some embodiments, the concentration of chlorogenic acid, isoorientin, isovitexin, luteolin or apigenin in the reference solution is 20-50 μg / ml, for example 30 μg / ml.

[0027] In some embodiments, the gradient elution method is any of the following schemes:

[0028] Gradient elution method 1

[0029]

[0030] Gradient elution method 2

[0031]

[0032] Gradient elution method 3

[0033]

[0034] For example, "0→10 minutes, the volume of mobile phase A is 13%" means that within the range of 0 to 10 minutes, the mobile phase A (the volume of mobile phase A as a percentage of the total volume of mobile phase) is fixed at 13%; "15→25 minutes, the volume of mobile phase A is 13%→16%" means that within the range of 10 to 25 minutes, the mobile phase A (the volume of mobile phase A as a percentage of the total volume of mobile phase) changes linearly from 13% to 16%;

[0035] Preferably, the gradient elution method is gradient elution method 3.

[0036] In some embodiments, the high performance liquid chromatography is detected using a diode array detector.

[0037] In some embodiments, the detection method of the standard decoction of fennel fruit constructs a characteristic spectrum; the number of characteristic peaks in the characteristic spectrum is not less than 5.

[0038] In some embodiments, the characteristic spectrum has no less than 7 characteristic peaks.

[0039] In some embodiments, the characteristic spectrum has five characteristic peaks identified as chlorogenic acid peak, isoorientin peak, isovitexin peak, luteolin peak and apigenin peak.

[0040] In some embodiments, the standard decoction of anise fruit is prepared by the following method for preparing the standard decoction of anise fruit, which comprises the following steps:

[0041] The crushed fennel fruit slices are decocted with water for the first time and then filtered, and the filter residue is decocted with water for the second time and then filtered, the two filtrates are combined, concentrated, and freeze-dried.

[0042] In some embodiments, in the preparation of the standard decoction of anise fruit, the anise fruit is soaked in water for 50 to 70 minutes before decocting; for example, soaked in water for 60 minutes.

[0043] In some embodiments, in the preparation of the standard decoction of anise fruit, the decocting step is to boil over high heat and then keep the decocting step at a slight boiling state, for example, keep the decocting step at a slight boiling state for 20 or 30 minutes.

[0044] In some embodiments, in the preparation of the standard decoction of anise fruit, during the first decoction, the mass ratio of the anise fruit slices to the water is 1:(6-8); for example, 1:7.

[0045] In some embodiments, in the preparation of the standard decoction of fennel fruit, during the second decoction, the mass ratio of the fennel fruit slices to the water is 1:(5-7); for example, 1:6.

[0046] In some embodiments, the concentration is vacuum concentration at ≤ 60°C.

[0047] In some embodiments, the concentration refers to concentration until the mass to volume ratio of the fennel fruit slices and the concentrated solution is (0.8~1.2) g / ml; for example, 1 g / ml.

[0048] In some embodiments, the freeze-drying temperature is -30 to -10°C; for example -20°C.

[0049] In some embodiments, the freeze drying includes a pre-freezing stage and a drying stage; the pre-freezing stage lasts for 10 to 14 hours (e.g., 12 hours); the drying stage lasts for 30 to 42 hours (e.g., 36 hours). The present invention also provides a method for detecting a standard decoction of anise fruit, which includes the following steps:

[0050] Spot the test solution and reference solution on the TLC plate respectively, develop with a developing agent, develop the color, and inspect visually under ultraviolet light;

[0051] The developing solvent is a mixture of methanol, water and formic acid in a volume ratio of 4:1:(0.25-1);

[0052] The color development is performed by spraying aluminum chloride ethanol solution and then heating;

[0053] The test sample is a standard decoction of fennel fruit;

[0054] The reference substance is isoorientin.

[0055] In some embodiments, the thin layer chromatography plate is a polyamide plate.

[0056] In some embodiments, the aluminum chloride ethanol solution is a 1-10% aluminum chloride ethanol solution; for example, a 2-5% aluminum chloride ethanol solution; such as a 2% aluminum chloride ethanol solution or a 5% aluminum chloride ethanol solution.

[0057] In some embodiments, the heating temperature is 95-115°C; for example 105°C.

[0058] In some embodiments, the heating time is 2-8 minutes; for example, 5 minutes.

[0059] In some embodiments, the wavelength of the ultraviolet lamp is 365 nm.

[0060] In some embodiments, the test solution is a methanol-water solution of the standard decoction of anise fruit; for example, a 70% methanol-water solution of the standard decoction of anise fruit.

[0061] In some embodiments, the developing agent is a mixture of methanol, water and formic acid in a volume ratio of 4:1:(0.5-0.75); for example, a mixture of methanol, water and formic acid in a volume ratio of 4:1:0.5 or a mixture of methanol, water and formic acid in a volume ratio of 4:1:0.75.

[0062] In some embodiments, the test solution is prepared by the following method: mixing the standard decoction of fennel fruit with methanol aqueous solution and dissolving by ultrasonication. The ultrasonic dissolution time can be 10-30 minutes; for example, 20 minutes.

[0063] In some embodiments, the reference solution is a methanol aqueous solution of isoorientin; for example, a 70% methanol aqueous solution of isoorientin.

[0064] In some embodiments, the mass-to-volume ratio of the standard decoction of anise fruit to the methanol-water solution is 1 g / (10-30) ml; for example, 1 g / 20 ml.

[0065] In some embodiments, the standard decoction of anise fruit is prepared by any of the aforementioned methods for preparing the standard decoction of anise fruit.

[0066] The present invention also provides a method for preparing a standard decoction of fennel fruit, which comprises the following steps:

[0067] The crushed fennel fruit slices are decocted with water for the first time and then filtered, and the filter residue is decocted with water for the second time and then filtered, the two filtrates are combined, concentrated, and freeze-dried.

[0068] In some embodiments, the conditions and operations of the method for preparing the standard decoction of anise fruit are as described in any of the preceding items.

[0069] In the present invention, the term "X% AB solution" refers to a solution composed of A and solvent B; when A is liquid at room temperature (such as methanol aqueous solution), X% refers to the percentage of the volume of A to the total volume of the solution; when A is solid at room temperature (such as aluminum chloride ethanol solution), X% refers to the percentage of the mass of A to the total volume of the solution, and the unit is g / ml; for example, in a 5% aluminum chloride ethanol solution, the mass of aluminum chloride in 100 ml of the solution is 5g.

[0070] The positive progressive effect of the present invention is that the present invention provides a thin layer identification and fingerprint method for a standard decoction of anise fruit slices. It overcomes the one-sidedness of the existing thin layer identification of anise fruit to determine the overall quality, and provides a new method and means for complete and accurate analysis and evaluation of the quality of the standard decoction of anise fruit slices. The method of the present invention has good stability, high precision, good reproducibility, and is convenient and easy to master. The fingerprint of the standard decoction of anise fruit slices has a total of 7 common peaks, and 5 components were identified by identification, all of which are the main active ingredients of anise fruit, which can provide a basis for the establishment of the fingerprint spectrum and quality control of the formula granules, and ensure the clinical effectiveness of the anise fruit formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 It is a thin layer chromatogram of Example 2; (from left to right, it corresponds to the isoorientin reference substance, 3 batches of standard decoctions HQ240201, HQ240202, and HQ240203 respectively).

[0072] Figure 2 This is the thin layer chromatogram of Example 3; (from left to right, they correspond to the isoorientin reference substance, and three batches of standard decoctions HQ240201, HQ240202, and HQ240203, respectively).

[0073] Figure 3 It is a thin layer chromatogram of Example 4; (from left to right, it corresponds to the isoorientin reference substance, and three batches of standard decoctions HQ240201, HQ240202, and HQ240203, respectively).

[0074] Figure 4 The thin layer chromatogram of Example 5; (from left to right, they correspond to the isoorientin reference substance, 3 batches of anisum fruit medicinal materials Y2304051, Y2308025, and Y2311046)

[0075] Figure 5 Thin layer chromatogram of comparative example 1; (from left to right correspond to anisole reference substance, 3 batches of standard decoctions HQ240201, HQ240202, HQ240203 respectively).

[0076] Figure 6 This is the thin layer chromatogram of comparative example 2; the left side is the isoorientin reference substance, and the right side is the standard decoction HQG240201.

[0077] Figure 7 High performance liquid chromatography spectra at different detection wavelengths.

[0078] Figure 8 High performance liquid chromatograms under different elution gradients.

[0079] Fig. 9 High performance liquid chromatograms at different column temperatures.

[0080] Fig.10 High performance liquid chromatograms at different flow rates.

[0081] Fig.11 High performance liquid chromatograms of different extraction methods.

[0082] Fig.12 HPLC chromatograms of different extraction solvents.

[0083] Fig.13 High performance liquid chromatograms of different material ratios.

[0084] Fig.14 The high performance liquid chromatograms at different extraction times.

[0085] Fig.15 This is the HPLC chromatogram of the blank group in the specificity investigation.

[0086] Fig.16 This is the HPLC chromatogram of the control group in the specificity investigation.

[0087] Fig.17 This is the HPLC chromatogram of the test sample group in the specificity investigation.

[0088] Fig.18 These are the fingerprints of 15 batches of samples of standard decoction of anise fruit and the reference fingerprints of standard decoction of anise fruit.

[0089] Fig.19 This is the high performance liquid chromatogram of the medicinal material Pimpinella foeniculum. DETAILED DESCRIPTION

[0090] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0091] Instruments and reagents

[0092] High performance liquid chromatograph (model: e2695, manufacturer: Waters); electronic balance (model: AL204, manufacturer: Mettler-Toledo Instrument (Shanghai) Co., Ltd.); electronic balance (model: MS205DU, manufacturer: Mettler-Toledo Instrument (Shanghai) Co., Ltd.); electronic balance (model: STX-1202ZH, manufacturer: Ohaus Instrument (Changzhou) Co., Ltd.); electric constant temperature water bath (model: HWS-26, manufacturer: Shanghai Qixin Scientific Instrument Co., Ltd.).

[0093] Chlorogenic acid reference substance (batch number 110753-202119, content 96.3%), isoorientin reference substance (batch number 111974-201401, content 94.0%), anisole reference substance (batch number 111556-200101, content 98.0%), luteolin reference substance (batch number 111520-202107, content 96.3%), apigenin reference substance (111901-202205, content 98.4%) were purchased from China Food and Drug Inspection Institute, isovitexin reference substance (batch number 250215-202308, content 98.0%). Acetonitrile, methanol, phosphoric acid and glacial acetic acid used in liquid phase were all chromatographically pure, and other reagents were analytically pure. Pineapple fruit medicinal material (Batch number: Y2304051, Y2308025, Y2311046).

[0094] The HPLC spectrum of the embodiment of the present application is calibrated with 7 common characteristic peaks, marked as peaks 1-7; 5 peaks are identified by the reference substance, and peaks 1, 3, 5, 6, and 7 are chlorogenic acid, isoorientin, isovitexin, luteolin, and apigenin, respectively.

[0095] Example 1

[0096] A method for preparing a standard decoction of fennel fruit comprises the following steps:

[0097] a. (Take fennel fruit medicinal material and process it into medicinal slices according to the "remove impurities and sieve out dust and soil" under the fennel fruit item in the "Xinjiang Uygur Autonomous Region Traditional Chinese Medicine Uygur Medicinal Slices Processing Specifications") Take 100g of fennel fruit medicinal slices, crush them, add purified water (mass ratio of medicinal slices: purified water = 1:7) and soak for 60 minutes, boil over high heat, keep it slightly boiling and continue to boil for 30 minutes, filter it while hot with a 200-mesh standard sieve; add purified water (mass ratio of medicinal slices: purified water = 1:6) to the residue, boil over high heat, keep it slightly boiling and continue to boil for 20 minutes, and collect the filtrate.

[0098] b. Combine the two filtrates and concentrate at low temperature (≤60℃, -0.08MPa) on a rotary evaporator to 100mL.

[0099] c. Take the above concentrated solution and put it into freeze dryer for freeze drying (pre-freezing at -20℃ for 12h, drying for 36h) to obtain the standard decoction powder of anise fruit.

[0100] 18 batches of standard decoctions, HQ240201, HQ240202, HQ240203 and HQ01-15, were obtained by the above preparation method; among them, standard decoctions HQ240201, HQ240202 and HQ240203 were prepared from fennel fruit medicinal material batches Y2304051, Y2308025 and Y2311046, respectively. The corresponding relationship between HQ01-15 and medicinal material batch numbers is shown in the following table.

[0101]

[0102] Example 2 TLC Qualitative Identification

[0103] Take 0.5g of the standard decoction powder, add 10mL of 70% methanol, and ultrasonically dissolve for 20 minutes. Take another isoorientin reference substance, add 70% methanol to make a solution containing 10μg per 1mL as the reference substance solution; according to the thin layer chromatography method (Appendix VI B of the 2020 edition of the "Chinese Pharmacopoeia"), take 1.5μL of the above solution and spot it on the same polyamide plate, use methanol-water-formic acid (4:1:0.5) as the developing agent, develop, take out, dry, spray with 5% aluminum chloride ethanol solution, heat at 105℃ for 5min, and inspect at 365nm. If Figure 1 As shown, in the chromatogram of the test sample, a spot of the same color appears at the corresponding position of the chromatogram of the reference sample, R f is 0.6.

[0104] Example 3 TLC Qualitative Identification

[0105] Take 0.5g of the standard decoction powder, add 10mL of 70% methanol, and ultrasonically dissolve for 20 minutes. Take another isoorientin reference substance, add 70% methanol to make a solution containing 10μg per 1mL as the reference substance solution; according to the thin layer chromatography method (Appendix VI B of the 2020 edition of the "Chinese Pharmacopoeia"), take 2μL of the above solution and spot it on the same polyamide plate, use methanol-water-formic acid (4:1:0.75) as the developing agent, develop, take out, dry, spray with 5% aluminum chloride ethanol solution, heat at 105℃ for 5min, and inspect at 365nm. If Figure 2 As shown, in the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the reference sample.

[0106] Example 4 TLC Qualitative Identification

[0107] Take 0.5g of the standard decoction powder, add 10mL of 70% methanol, and ultrasonically dissolve for 20 minutes. Take another isoorientin reference substance, add 70% methanol to make a solution containing 10μg per 1mL as the reference substance solution; according to the thin layer chromatography method (Appendix VI B of the 2020 edition of the "Chinese Pharmacopoeia"), take 2μL of the above solution and spot it on the same polyamide plate, use methanol-water-formic acid (4:1:0.5) as the developing agent, develop, take out, dry, spray with 2% aluminum chloride ethanol solution, heat at 105℃ for 5min, and inspect at 365nm. If Figure 3 As shown, in the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the reference sample.

[0108] Example 5 Thin layer chromatography transport test of standard decoction

[0109] Take 0.5 g of fennel fruit powder (obtained by crushing fennel fruit), add 10 mL of 70% methanol, and ultrasonically dissolve for 20 minutes. Take another isoorientin reference substance, add 70% methanol to make a solution containing 10 μg per 1 mL as the reference substance solution; according to the thin layer chromatography method (Appendix VI B of the 2020 edition of the "Chinese Pharmacopoeia"), take 1.5 μL of the above solution and spot it on the same polyamide plate, use methanol-water-formic acid (4:1:0.5) as the developing agent, develop, take out, dry, spray with 5% aluminum chloride ethanol solution, heat at 105°C for 5 minutes, and inspect at 365 nm. If Figure 4 As shown, in the chromatogram of the test sample, a spot of the same color appears at the corresponding position of the chromatogram of the reference sample, R f is 0.6; Figure 1 By comparison, the detection results of the medicinal materials were consistent with those of the standard decoction, and the detection of isoorientin was also achieved in the fennel fruit medicinal material and the fennel fruit standard decoction.

[0110] Using the TLC conditions of Examples 3 and 4, only the powder of the standard decoction of fennel fruit was replaced with the fennel fruit medicinal material for TLC detection, which also enabled the detection of isoorientin, thus achieving the transfer from the medicinal material to the standard decoction.

[0111] Comparative Example 1 TLC Qualitative Identification

[0112] The test was conducted with reference to the public release of the revised draft of the national drug standards for fennel fruit medicinal materials.

[0113] Take 1g of anise fruit powder, add 20ml of ether, ultrasonically treat for 10 minutes, filter, evaporate the filtrate, add 1ml of anhydrous ethanol to the residue to dissolve it, and use it as the test solution. Take another anisole reference substance, add anhydrous ethanol to make a solution containing 1μl per 1ml, which is used as the reference solution. According to the thin layer chromatography method (General Rules 0502 of the 2015 edition of the Chinese Pharmacopoeia), 2μl of each of the above two solutions were taken and spotted on the same silica gel G thin layer plate, and petroleum ether (60-90℃)-ethyl acetate (17:2.5) was used as the developing solvent. Develop, take out, dry, and spray with 5% phosphomolybdic acid ethanol test solution. The results are as follows. Figure 5 As shown in the figure, in the chromatogram of the test sample, a spot of the same color appears at the corresponding position of the chromatogram of the reference sample. f is 0.8.

[0114] Will Figure 5 and Figure 1-4 By comparison, the results show that the thin layer chromatography detection method provided by the present application obtains far more spots than the existing methods. A large number of spots means that more sample spots can be separated, which helps to improve separation efficiency and shorten analysis data. By increasing the number of spots, a multi-stage expansion technology can be used to overcome the tailing phenomenon caused by small adsorbent particles, so that the circular or elliptical spots are concentrated in one line, thereby increasing the sample concentration per unit area of ​​the spots and improving the detection sensitivity. At the same time, more spots mean more data points, which helps to enhance the reliability and repeatability of the experimental results. Each spot is an independent experimental result, which increases the diversity and accuracy of the data.

[0115] Comparative Example 2

[0116] Take 0.5g of the standard decoction powder, add 10mL of 70% methanol, and ultrasonically dissolve for 20 minutes. Take another isoorientin reference substance, add 70% methanol to make a solution containing 10μg per 1mL as the reference substance solution; according to the thin layer chromatography method (Appendix VI B of the 2020 edition of the "Chinese Pharmacopoeia"), take 1.5μL of the above solution and spot it on the same polyamide plate, use methanol-water (4:1) as the developing agent, develop, take out, dry, spray with 5% aluminum chloride ethanol solution, heat at 105℃ for 5min, and inspect at 365nm. If Figure 6 As shown, in the chromatogram of the test sample, tailing is serious and the fuzzy separation effect is poor.

[0117] Example 6 Establishment of HPLC Characteristic Spectrum Method for Standard Decoction

[0118] 1 Chromatographic conditions and system suitability test

[0119] 1.1 Proposed chromatographic conditions

[0120] Octadecyl bonded silica gel was used as the filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); (Agilent ZORBAX Eclipse Plus C18, 250 mm × 4.6 mm, 5 μm) mobile phase A was acetonitrile, mobile phase B was 0.05% glacial acetic acid, gradient elution method was shown in Table 1, column temperature was 30 °C; flow rate was 1 mL / min; injection volume was 10 μL; detection wavelength was 350 nm.

[0121] Table 1 Gradient elution method

[0122]

[0123] The percentage sign in the table means volume percentage.

[0124] 1.2 Solution preparation

[0125] (1) Preparation of reference solution

[0126] Take appropriate amount of chlorogenic acid, isoorientin, isovitexin, luteolin, and apigenin reference substances, accurately weigh them, and add 70% methanol to prepare a mixed reference substance solution containing 30 μg of each substance per 1 mL.

[0127] (2) Preparation of test solution

[0128] Accurately weigh 1 g of standard decoction powder, place it in a 150 mL conical flask, add 20 mL of methanol, weigh it, extract it by ultrasonic for 45 min, make up the weight with methanol, filter, and take the filtrate to obtain the product.

[0129] 1.3 Determination method

[0130] Take 10 μL of the reference solution and test solution respectively, inject them into the high performance liquid chromatograph, and measure to obtain the result.

[0131] 2. Investigation of chromatographic conditions

[0132] 2.1 Investigation of the optimal absorption wavelength

[0133] Based on the proposed experimental conditions, the diode array detector was used to scan the full wavelength of the test solution of the standard decoction of anise fruit, and the chromatogram of the test solution at wavelengths of 320 mm, 335 mm, and 350 mm was extracted. The results are as follows: Figure 7 shown.

[0134] 2.2 Elution gradient investigation

[0135] The chromatograms of anise fruit under three different gradients were investigated. The gradient elution is shown in the table below. The other chromatographic conditions were determined in the same way as in "1.1". The results are as follows: Figure 8 shown.

[0136] Table 2 Gradient elution method 1

[0137]

[0138] Table 3 Gradient elution method 2

[0139]

[0140] Table 4 Gradient elution method 3

[0141]

[0142] The results showed that the separation was better when gradient elution 3 was selected, so gradient elution 3 was finally selected.

[0143] 2.3 Column temperature investigation

[0144] The column temperature was set to 25℃, 30℃, and 35℃ respectively. The other chromatographic conditions were the same as those in "1.1". The results were as follows: Fig. 9 ,The experimental results show that, under the above temperature, 30℃ has the best separation effect, and the column temperature is selected to be 30℃.

[0145] 2.4 Flow rate investigation

[0146] The flow rates were set to 0.9 mL / min, 1 mL / min, and 1.1 mL / min respectively. The other chromatographic conditions were the same as those in "1.1". The results were as follows: Fig.10 ,The experimental results show that the separation effect is best when the flow rate is 1mL / min, and the flow rate is 1mL / min.

[0147] In summary, the chromatographic conditions and system suitability test for the characteristic spectrum of the standard decoction of anise fruit were determined as follows: octadecyl bonded silica gel was used as the filler (column length was 250 mm, inner diameter was 4.6 mm, and particle size was 5 μm); (Agilent ZORBAX Eclipse Plus C18, 250 mm × 4.6 mm, 5 μm) the mobile phase A was acetonitrile, the mobile phase B was 0.05% glacial acetic acid, the gradient elution method was as shown in the following table, the column temperature was 30 °C; the flow rate was 1 mL / min; the injection volume was 10 μL; and the detection wavelength was 350 nm.

[0148] Table 5 Gradient elution method

[0149]

[0150] 3. Investigation of the preparation method of test solution

[0151] 3.1 Investigation of extraction methods

[0152] Compare the two extraction methods of heating reflux and ultrasonic extraction. Take an appropriate amount of the powder sample of the standard decoction of anise fruit, accurately weigh two portions, each 0.5g, put them in a stoppered conical bottle, accurately add 20mL of 70% methanol, weigh the weight, extract one portion under reflux for 30 minutes, and extract the other portion under ultrasonic extraction for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake well, filter, and get it. Determine according to the above-determined chromatographic conditions, such as Fig.11 The experimental results show that the two extraction methods have the same separation effect. Due to the higher extraction efficiency and wide adaptability of ultrasonic extraction, ultrasonic extraction was selected as the extraction method for the characteristic spectrum of the standard decoction of anise fruit.

[0153] 3.2 Investigation of extraction solvent

[0154] The extraction solvents for the preparation of the characteristic spectrum of the sample of the standard decoction of anise fruit were investigated, and 50% methanol, 70% methanol, and 100% methanol were used as extraction solvents. Take an appropriate amount of the powder sample of the standard decoction of anise fruit, accurately weigh 3 portions, 0.5g each, put them in a stoppered conical flask, accurately add 20mL of 50% methanol to one portion, 20mL of 70% methanol to one portion, and 20mL of 100% methanol to one portion, weigh the weight, extract with ultrasound for 30 minutes, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and obtain. Determine according to the chromatographic conditions determined above, such as Fig.12 As shown in the figure, the experimental results show that when 70% and 100% methanol are used as extraction solvents, there is only a difference in peak area, and the number of peaks is the same, but the chromatogram extracted with 100% methanol has a better peak shape and a stable baseline. After comprehensive consideration, 100% methanol was selected as the extraction solvent for the characteristic spectrum of the standard decoction of fennel fruit.

[0155] 3.3 Material ratio investigation

[0156] Take an appropriate amount of the powder sample of the standard decoction of anise fruit, accurately weigh three portions, 0.5g, 1g, and 1.5g respectively, place in a stoppered conical flask, accurately add 20mL of 100% methanol, weigh the weight, ultrasonically extract for 30 minutes, cool, weigh again, make up the lost weight with 100% methanol, shake well, filter, and obtain. Determine according to the above-determined chromatographic conditions, such as Fig.13 The experimental results show that by comparing the chromatograms of the test sample of 0.5g, 1g, and 1.5g, it is found that the peak shape and separation of the chromatographic peaks of 0.5 and 1g are better. Considering comprehensively, 0.5g is selected as the material amount of the test sample for the characteristic spectrum of the standard decoction of anise fruit.

[0157] 3.4 Extraction time investigation

[0158] Take an appropriate amount of the powder sample of the standard decoction of anise fruit, accurately weigh three portions, 0.5 g each, place in a stoppered conical flask, accurately add 20 mL of 100% methanol, weigh the weight, ultrasonically extract for 15 minutes, 30 minutes, and 45 minutes, cool, weigh again, make up the lost weight with 100% methanol, shake well, filter, and obtain. Determine according to the above-determined chromatographic conditions, such as Fig.14 The experimental results show that the number of characteristic peaks is consistent when comparing the chromatograms of the test sample at 15 minutes, 30 minutes, and 45 minutes. However, the peak shape of the chromatogram at 45 minutes is better and the sample powder is fully dissolved. Considering comprehensively, 45 minutes is selected as the extraction time for the characteristic spectrum of the test sample of the standard decoction of anise fruit.

[0159] In summary, the preparation method of the test sample and reference solution for detecting the characteristic spectrum of the standard decoction of fennel fruit is as follows: Preparation of solution

[0160] (1) Preparation of reference solution

[0161] Take appropriate amount of chlorogenic acid, isoorientin, isovitexin, luteolin, and apigenin reference substances, accurately weigh them, and add 70% methanol to prepare a mixed reference substance solution containing 30 μg of each substance per 1 mL.

[0162] (2) Preparation of test solution

[0163] Accurately weigh 0.5 g of standard decoction powder, place it in a 150 mL conical flask, add 20 mL of methanol, weigh it, extract it by ultrasonic for 45 min, make up the weight with methanol, filter, and take the filtrate to obtain the product.

[0164] 1.3 Determination method

[0165] Take 10 μL of the reference solution and the test solution respectively, inject them into the high performance liquid chromatograph, and measure to obtain the result.

[0166] 4. Validation of feature map methodology

[0167] 4.1 Specificity Investigation

[0168] Take appropriate amount of the above-mentioned reference solution, test solution and blank solvent (100% methanol), inject and measure according to the chromatographic conditions under "1.1", and record the chromatogram. The results show that the baselines of the reference and test chromatograms are stable, the chromatographic peak separation and symmetry are good, the retention time of the characteristic peaks in the test sample is basically the same as that of the reference, and there is no interference from the blank solvent. See the results. Figure 15-17 .

[0169] 4.2 Precision test

[0170] Take 1 g of the standard decoction powder of fennel fruit (No. HQ01) from the same batch, weigh it accurately, prepare it according to the test sample method, and repeat the measurement 6 times according to the chromatographic conditions under "1.1". Take the chromatographic peak of isoorientin as the reference peak S, calculate the relative retention time and relative peak area of ​​each fingerprint peak and the S peak, the relative retention time RSD of each fingerprint peak is 0.03%~0.11%, and the relative peak area RSD is 0.36%~1.24%, indicating that the instrument has good precision.

[0171] 4.3 Repeatability test

[0172] Take about 1 g of the same batch of standard decoction powder of fennel fruit (No. HQ01), accurately weigh it, and make 6 parallel portions. Prepare 6 test sample solutions according to the test sample method, and inject and measure according to the chromatographic conditions under "1.1". Take the chromatographic peak of isoorientin as the reference peak S, calculate the relative retention time and relative peak area of ​​each fingerprint peak and the S peak, the relative retention time RSD of each fingerprint peak is 0.01%~0.16%, and the relative peak area RSD is 0.48%~1.42%, indicating that the method has good repeatability.

[0173] 4.4 Stability test

[0174] Take the test solution under "4.3" for parallel experiment. According to the chromatographic conditions under "1.1", the samples were injected and measured at 0h, 4h, 9h, 19h, 28h, 40h and 48h respectively. The chromatographic peak of isoorientin was used as the reference peak S. The relative retention time and relative peak area of ​​each fingerprint peak and the S peak were calculated. The RSD of the relative retention time of each fingerprint peak was 0.01%~0.44%, and the RSD of the relative peak area was 0.30%~1.85%, indicating that the test solution had good stability within 48h.

[0175] 4.5 Establishment and analysis of feature maps

[0176] According to the sample preparation method, 15 batches of standard decoction of fennel fruit were prepared as test samples. According to the chromatographic conditions under "1.1", the samples were injected respectively and the chromatograms were recorded. The "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 edition) of the State Pharmacopoeia Committee was used, and the HQ01 fingerprint spectrum was used as the reference spectrum to identify common peaks. A total of 7 common fingerprint peaks were calibrated, and 5 peaks were identified through the reference substance. Peaks 1, 3, 5, 6, and 7 are chlorogenic acid, isoorientin, isovitexin, luteolin, and apigenin, respectively. Multi-point retention time correction and peak matching were performed to generate the reference fingerprint spectrum of the standard decoction of fennel fruit (R) (see Fig.18 ). The similarity between the fingerprints of 15 batches of standard decoctions of fennel fruit and the reference fingerprints is 0.957~0.998 (see the table below), indicating that all 15 batches of samples have high similarity.

[0177] Table 6 Similarity evaluation of 15 batches of standard decoctions of fennel fruit

[0178]

[0179] 5. HPLC delivery test of standard decoction

[0180] Take 0.5 g of fennel fruit powder (obtained by crushing fennel fruit), put it in a 150 mL conical flask, add 20 mL of methanol, weigh it, extract it by ultrasonic for 45 min, make up the weight with methanol, filter it, take the filtrate and obtain the test solution.

[0181] Take the test solution, inject it according to the chromatographic conditions under "1.1", and record the chromatogram. Fig.19 shown.

[0182] The results showed that the detection method of the present application can also be used to obtain seven characteristic peaks, 1-7, of the fennel fruit medicinal material (the 6th, 14th, 15th, 19th, 21st, 35th and 36th peaks in the figure correspond to the aforementioned peaks 1-7, respectively), realizing the transfer from medicinal materials to standard decoctions.

Claims

1. A method for detecting a standard decoction of fennel fruit, characterized in that: It includes the following steps: The standard decoction of anise fruit is detected by high performance liquid chromatography; the standard decoction of anise fruit contains one or more of chlorogenic acid, isorientin, isovitexin, luteolin and apigenin; The chromatographic conditions of the high performance liquid chromatography are: The chromatographic column is an octadecyl bonded silica gel chromatographic column; Mobile phase A was acetonitrile, and mobile phase B was 0.05% to 0.1% acetic acid in water; Gradient elution, the gradient elution method is: the initial volume percentage of mobile phase A is 10-15%, changes to 40% within 40-50 minutes, changes to 90% within 5-10 minutes after rising to 40%; and decreases to 10-15% 5-10 minutes after rising to 90%; the volume percentage of mobile phase B is always 1-the volume percentage of mobile phase A; during the change process of the gradient elution, the volume percentage of mobile phase A keeps rising or remains the same.

2. The detection method of the standard decoction of fennel fruit as claimed in claim 1, characterized in that: It meets one or more of the following conditions: (1) The chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; (2) The chromatographic column is Agilent ZORBAX Eclipse Plus C18; (3) In the chromatographic conditions of the high performance liquid chromatography, the column temperature is 25-35°C; such as 28-35°C; preferably 30°C; (4) In the chromatographic conditions of the high performance liquid chromatography, the flow rate is 0.9-1.1 ml / min; such as 1-1.1 ml / min, preferably 1 ml / min; (5) In the chromatographic conditions of the high performance liquid chromatography, the detection wavelength is 300-360 nm; such as 320-350 nm; for example 320 nm, 335 nm or 350 nm; (6) The mobile phase B is 0.05% acetic acid aqueous solution; (7) The standard decoction of fennel fruit is tested in the form of a test solution, and the test solution is prepared by ultrasonication or reflux of a 50%-100% methanol aqueous solution of the standard decoction of fennel fruit; (8) The detection method further comprises detecting a reference solution; the reference solution is a solution containing one or more of chlorogenic acid, isorientin, isovitexin, luteolin and apigenin; (9) The gradient elution method is any one of the following schemes: Gradient elution method 1 ； Gradient elution method 2 ； Gradient elution method 3 ； Preferably, the gradient elution method is gradient elution method 3; (10) the high performance liquid chromatography method is detected using a diode array detector; and, (11) The detection method of the standard decoction of fennel fruit constructs a characteristic spectrum; the number of characteristic peaks in the characteristic spectrum is not less than 5; for example, the number of characteristic peaks in the characteristic spectrum is not less than 7; preferably, there are 5 characteristic peaks in the characteristic spectrum, namely, chlorogenic acid peak, isorientin peak, isovitexin peak, luteolin peak and apigenin peak.

3. The detection method of the standard decoction of fennel fruit as claimed in claim 2, characterized in that, It meets one or more of the following conditions: (1) In the preparation of the test solution, the 50%-100% methanol aqueous solution is a 70%-100% methanol aqueous solution; for example, 100% methanol; (2) In the preparation of the test solution, the ultrasound or reflux is ultrasound; for example, ultrasound for 15 to 60 minutes, or for example, ultrasound for 45 minutes; (3) In the preparation of the test solution, the concentration of the 50%-100% methanol aqueous solution of the standard decoction of fennel fruit is (0.01-0.1) g / ml; for example, 0.025 g / ml, 0.05 g / ml or 0.075 g / ml; (4) The solvent of the reference solution is methanol-water solution; such as 50-100% methanol-water solution; for example, 70% methanol-water solution; and, (5) In the reference solution, the concentration of chlorogenic acid, isorientin, isovitexin, luteolin or apigenin is 20-50 μg / ml, for example 30 μg / ml.

4. The detection method of the standard decoction of fennel fruit as claimed in claim 1, characterized in that: The standard decoction of fennel fruit is prepared by the following preparation method of the standard decoction of fennel fruit, which comprises the following steps: The crushed fennel fruit slices are decocted with water for the first time and then filtered, and the filter residue is decocted with water for the second time and then filtered, the two filtrates are combined, concentrated, and freeze-dried.

5. The detection method of the standard decoction of fennel fruit as claimed in claim 4, characterized in that: The preparation method of the standard decoction of fennel fruit meets one or more of the following conditions: (1) In the preparation of the standard decoction of fennel fruit, the fennel fruit slices are soaked in water for 50 to 70 minutes before decocting; for example, soaked in water for 60 minutes; (2) In the preparation of the standard decoction of anise fruit, the decoction is boiled over high heat and then kept at a slight boil, for example, kept at a slight boil for 20 or 30 minutes; (3) In the preparation of the standard decoction of fennel fruit, during the first decoction, the mass ratio of the fennel fruit slices to the water is 1:(6-8); for example, 1:7; (4) In the preparation of the standard decoction of fennel fruit, during the second decoction, the mass ratio of the fennel fruit slices to the water is 1:(5-7); for example, 1:6; (5) The concentration is vacuum concentration at ≤60°C; (6) The concentration refers to concentration until the mass to volume ratio of the fennel fruit slices and the concentrated solution is (0.8-1.2) g / ml; for example, 1 g / ml; (7) The freeze-drying temperature is -30 to -10°C; for example -20°C; and, (8) The freeze-drying process includes a pre-freezing stage and a drying stage; the pre-freezing stage lasts for 10 to 14 hours (e.g., 12 hours); the drying stage lasts for 30 to 42 hours (e.g., 36 hours).

6. A method for detecting a standard decoction of Pimpinella foeniculum, characterized in that: It includes the following steps: Spot the test solution and reference solution on the TLC plate respectively, develop with a developing agent, develop the color, and inspect visually under ultraviolet light; The developing solvent is a mixture of methanol, water and formic acid in a volume ratio of 4:1:(0.25-1); The color development is performed by spraying aluminum chloride ethanol solution and then heating; The test solution is a solution of standard decoction of fennel fruit; The reference solution is a solution of isoorientin.

7. The detection method of the standard decoction of fennel fruit as claimed in claim 6, characterized in that: It meets one or more of the following conditions: (1) The thin layer chromatography plate is a polyamide plate; (2) The aluminum chloride ethanol solution is a 1-10% aluminum chloride ethanol solution; for example, a 2-5% aluminum chloride ethanol solution; such as a 2% aluminum chloride ethanol solution or a 5% aluminum chloride ethanol solution; (3) The heating temperature is 95-115°C, for example 105°C; (4) The heating time is 2-8 minutes, for example 5 minutes; (5) The wavelength of the ultraviolet lamp is 365nm; (6) The test solution is a methanol-water solution of the standard decoction of fennel fruit; for example, a 70% methanol-water solution of the standard decoction of fennel fruit; (7) The reference solution is a methanol aqueous solution of isoorientin; for example, a 70% methanol aqueous solution of isoorientin; and, (8) The developing solvent is a mixture of methanol, water and formic acid in a volume ratio of 4:1:(0.5-0.75); for example, a mixture of methanol, water and formic acid in a volume ratio of 4:1:0.5 or a mixture of methanol, water and formic acid in a volume ratio of 4:1:0.

75.

8. The detection method of the standard decoction of fennel fruit as claimed in claim 7, characterized in that: It meets one or more of the following conditions: (1) The test solution is prepared by the following method: mixing the standard decoction of fennel fruit with methanol aqueous solution and dissolving by ultrasonication; the ultrasonic dissolution time can be 10-30 minutes, for example, 20 minutes; (2) In the test solution, the mass-to-volume ratio of the standard decoction of fennel fruit to the methanol aqueous solution is 1 g / (10-30) ml; for example, 1 g / 20 ml; and, (3) The standard decoction of anise fruit is prepared by the preparation method of the standard decoction of anise fruit according to claim 4 or 5.

9. A method for preparing a standard decoction of fennel fruit, characterized in that: It includes the following steps: The crushed fennel fruit slices are decocted with water for the first time and then filtered, and the filter residue is decocted with water for the second time and then filtered, the two filtrates are combined, concentrated, and freeze-dried.

10. The method for preparing the standard decoction of fennel fruit as claimed in claim 9, characterized in that: The standard decoction of anise fruit is prepared by the preparation method of the standard decoction of anise fruit according to claim 5.

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