Method for determining content of isoorientin in pimpinella diversifolia fruit medicinal material

Abstract

The invention provides a method for determining the content of isoorientin in a pimpinella diversifolia fruit medicinal material. The invention provides a method for detecting isoorientin in a pimpinella diversifolia fruit medicinal material, which comprises the following steps: detecting a pimpinella diversifolia fruit medicinal material extract by adopting a high performance liquid chromatography method, and the chromatographic conditions are as follows: a chromatographic column is a C18 column; mobile phases are a mobile phase A and a mobile phase B; the mobile phase B is a phosphoric acid aqueous solution, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.01-0.3%, and the mobile phase A is methanol. The method is accurate in determination method, high in sensitivity, good in repeatability and reliable in result, and provides a basis for quality control and evaluation of the pimpinella diversifolia fruits.

Description

Technical Field

[0001] The invention relates to a method for determining the content of isoorientin in anisum fruit medicinal materials, and belongs to the technical field of medical detection. Background Art

[0002] Pineapple fruit is the dried mature fruit of Pimpinella anisum L., a plant of the Umbelliferae family. It has the effects of dispelling cold, promoting the body's natural composure, promoting diuresis and menstruation, moistening the intestines and relieving pain, and is used for dyspnea, cough and asthma. "Health Medicinal Garden": "Dispel cold and relieve pain, relieve cough and asthma"; "Annotated Medical Classic": "Mainly treat dyspnea; "Baidyi Medicinal Book": "Treat Naizilai cold". Pineapple fruit medicinal materials contain a large amount of flavonoids, sugars, volatile oils and other ingredients. The quality standard of this medicinal material will be included in the national drug standard in 2023, but only the source, properties, identification, inspection and other items are limited, and there is a lack of methods for determining the content of effective ingredients. Existing public literature (Tusongjiang Abdu Aini, Wang Yali, Salamati Aili. Research on the quality standard of fennel fruit [ J]. Chinese Journal of Ethnomedicine, 2013, 19(08): 51-52.) The method for determining the content of total flavonoids was published for the quality control of fennel fruit medicinal materials. Rutin was selected as the reference substance (index component), which belongs to the detection of large-category components. Since there are many types of flavonoid compounds and their components are complex, and rutin does not exist in fennel fruit, quantitative analysis of rutin alone may not reflect the actual content of total flavonoids in the medicinal materials. There are certain limitations, and the content of total flavonoids cannot distinguish the differences between different origins and different harvest periods.

[0003] Foreign studies have reported that isoorientin exists in fennel fruit, which is a natural flavonoid substance of luteolin glycoside. Studies have found that isoorientin has the effect of improving lung function and slowing down lung damage by reducing inflammatory factors and oxidative stress; isoorientin has anti-respiratory syncytial virus activity, has a protective effect on lipopolysaccharide-induced acute lung injury, and can reduce cellular inflammatory response. This effect is consistent with the efficacy of fennel fruit medicinal materials. The present invention uses isoorientin as an indicator component for the first time to establish a method for determining its content, providing a reference basis for further improving the quality standard of fennel fruit medicinal materials. The existing document "HPLC determination of total flavonoids and (iso) vitexin and (iso) orientin in cluster bamboo" discloses a method for determining the content of isoorientin in cluster bamboo, which uses an isocratic elution method to determine the content of isoorientin in cluster bamboo, a plant of the subfamily Bamboo in the Poaceae family. In the previous study, we used the isocratic elution method to try to determine the content of isoorientin in fennel fruit, but found that the separation effect was poor, the conditions were difficult to optimize, and the analysis time was long. Therefore, based on the existing public literature, a new method for determining the content of isoorientin in fennel fruit was established. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a reference basis for the lack of a method for determining the content of effective ingredients of anise fruit in the prior art and the lack of a perfect quality standard for anise fruit medicinal material. To this end, a method for determining the content of isoorientin in anise fruit medicinal material is provided. The method uses isoorientin as a determination index and adopts high performance liquid chromatography to establish a method for determining the content of anise fruit medicinal material, providing a basis for the research on the quality standard of anise fruit medicinal material. The method is accurate in determination, highly sensitive, and repeatable, and the result is reliable, providing a basis for quality control and evaluation of anise fruit.

[0005] The present invention provides a method for detecting isoorientin in anisum fruit medicinal material, which comprises the following steps:

[0006] The extract of fennel fruit medicinal material was tested by high performance liquid chromatography, and the chromatographic conditions included:

[0007] The chromatographic column is C 18 column;

[0008] The mobile phases are mobile phase A and mobile phase B;

[0009] The mobile phase B is a phosphoric acid aqueous solution, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.01-0.3%, and the mobile phase A is methanol.

[0010] In a certain embodiment, the preparation method of the anise fruit medicinal material extract comprises the following steps: mixing the anise fruit medicinal material and a solvent, extracting, cooling, making up for the weight loss, and filtering to obtain the anise fruit medicinal material extract;

[0011] The solvent is an alcohol solvent and water.

[0012] In a certain embodiment, the source of the anise fruit medicinal material may be Jiuquan, Gansu; the anise fruit medicinal material is the plant Anise of the Umbelliferae family.

[0013] In one embodiment, the mass volume ratio of the fennel fruit medicinal material to the solvent is 0.03 g / mL to 0.3 g / mL, such as 0.03 g / mL, 0.05 g / mL or 0.1 g / mL.

[0014] In one embodiment, the solvent is 50% to 100% methanol, preferably 50%, 70% or 100%; the percentage refers to the mass percentage of methanol in the solvent.

[0015] In a certain embodiment, the extraction method is ultrasonic treatment, reflux treatment or cold soaking method, preferably reflux treatment.

[0016] In a certain embodiment, the reflux treatment time is 15 to 40 minutes, preferably 20 minutes, 30 minutes or 40 minutes, for example 30 minutes.

[0017] In a certain embodiment, the cooling is natural cooling to room temperature.

[0018] In a certain embodiment, the lost weight is generally supplemented with a solvent in the solution obtained by extracting the anise fruit.

[0019] In one embodiment, the filtration is performed using an organic filter membrane, preferably a 0.45 μm organic filter membrane.

[0020] In a certain scheme, the preparation method of the anise fruit medicinal material extract preferably comprises the following steps: mixing 1 g of the anise fruit powder (passed through a No. 2 sieve) with 10 mL to 30 mL of 50% to 100% methanol, reflux extraction for 15 to 40 minutes, and after the mixed solution is cooled to room temperature, supplementing the lost weight with the 70% methanol, shaking well, filtering, and taking the filtrate to obtain the anise fruit medicinal material extract;

[0021] Further preferably, 1 g of the anise fruit powder (passed through a No. 2 sieve) is accurately weighed, placed in a 150 mL stoppered conical flask, 10 mL of 70% methanol is added, weighed, and refluxed for 30 minutes. After the sample is cooled to room temperature, 70% methanol is used to make up the lost weight, shaken well, filtered, and the filtrate is taken to obtain the anise fruit medicinal extract.

[0022] In a certain scheme, isoorientin is used as a reference substance, and the reference substance solution is prepared before detection: the isoorientin is dissolved in a methanol solution; the mass volume ratio of the isoorientin to the methanol solution is (3.2~64) μg / mL, for example, 12.8216 μg / mL.

[0023] In one embodiment, the methanol in the mobile phase A is chromatographically pure.

[0024] In a certain embodiment, the chromatographic column is a Waters C18 chromatographic column, an Agilent C18 chromatographic column or a GL C18 chromatographic column, for example, an Agilent C18 chromatographic column.

[0025] In a certain embodiment, in the detection method, the length of the chromatographic column is 250 mm;

[0026] The inner diameter of the chromatographic column is 4.6 mm;

[0027] The filler particle size of the chromatographic column is 5 μm.

[0028] In one embodiment, in the detection method, the chromatographic column is an Agilent C18 chromatographic column with a column length of 250 mm, an inner diameter of 4.6 mm, and a filler particle size of 5 µm.

[0029] In a certain embodiment, in the detection method, the high performance liquid chromatograph may be a Thermo Scientific Ultimate 3000, Thermo Scientific Vanquish, or Waters e2695 / 2998 high performance liquid chromatograph, such as Thermo Scientific Ultimate 3000.

[0030] In one embodiment, in the detection method, the detection wavelength is 345-350 nm, for example 347 nm.

[0031] In one embodiment, in the detection method, the injection volume is 5 to 20 μL, for example, 10 μL.

[0032] In one embodiment, in the detection method, the column temperature is 10-40°C, preferably 25-35°C, such as 25°C, 30°C or 35°C, preferably 35°C.

[0033] In one embodiment, the flow rate of the mobile phase is 0.1-2 mL / min, preferably 0.8-1.2 mL / min, for example 1.0 mL / min.

[0034] In a certain embodiment, in the detection method, the elution mode is gradient elution.

[0035] In a certain scheme, in the gradient elution, the total volume of the mobile phase A is 100%; at 0-36 min, the volume fraction of the mobile phase A is 28%, and the volume fraction of the mobile phase B is 72%; at 36-45 min, the volume fraction of the mobile phase A increases to 37%, and the volume fraction of the mobile phase B decreases to 63%; at 45-48 min, the volume fraction of the mobile phase A increases to 90%, and the volume fraction of the mobile phase B is 10%; at 48-51 min, the volume fraction of the mobile phase A is 90%, and the volume fraction of the mobile phase B is 10%; at 51-55 min, the volume fraction of the mobile phase A decreases to 28%, and the volume fraction of the mobile phase B increases to 72%.

[0036] In one embodiment, the chromatographic column is an Agilent C18 chromatographic column with a column length of 250 mm, an inner diameter of 4.6 mm, and a filler particle size of 5 µm;

[0037] The detection wavelength is 347nm;

[0038] The column temperature is 35°C;

[0039] The flow rate of the mobile phase is 1.0 mL / min;

[0040] The injection volume is 10 μL;

[0041] The mobile phase is methanol (A)-0.1% phosphoric acid solution (B);

[0042] The gradient elution is:

[0043]

[0044] Among them, the percentages in the table are the volume percentages of each component in the total volume of mobile phase A and mobile phase B;

[0045] The high performance liquid chromatograph is Thermo Scientific Ultimate3000.

[0046] In a certain embodiment, the method for detecting isoorientin in the anisum fruit medicinal material is:

[0047] The chromatographic conditions are as follows: Agilent C18 chromatographic column, 4.6 mm × 250 mm, 2.5 μm; mobile phase: mobile phase A is methanol, mobile phase B is 0.01-0.3% phosphoric acid aqueous solution, gradient elution; detection wavelength: 345-350 nm; column temperature: 10-40° C.; flow rate: 10-40° C.; injection volume: 5-20 μL;

[0048] The preparation of the reference solution before testing is as follows: accurately weigh the isoorientin reference substance, place it in a volumetric flask, and add methanol solution to make it (3.2~64) μg / mL;

[0049] The preparation method of the anise fruit medicinal material extract is as follows: 1 g of the anise fruit powder (passed through a No. 2 sieve) is mixed with 10 mL to 30 mL of 50% to 100% methanol, and reflux extraction is performed for 15 to 40 minutes. After the mixed solution is cooled to room temperature, the lost weight is supplemented with the 70% methanol, and the mixture is shaken and filtered to obtain the filtrate to obtain the anise fruit medicinal material extract.

[0050] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0051] The reagents and raw materials used in the present invention are commercially available.

[0052] The positive and progressive effects of the present invention are:

[0053] The invention uses isoorientin as a detection index and adopts high performance liquid chromatography to establish a content determination method for anisum fruit medicinal materials, and provides an experimental basis for the quality standard research of anisum fruit medicinal materials; the determination method is accurate, and the sample recovery RSD is less than 1.4%; the sensitivity is high, and the signal-to-noise ratio (S / N) is not less than 3 and can reach 10; the precision is less than 1.4%; the repeatability is good, and the result is reliable, which provides a basis for the quality control and evaluation of anisum fruit medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 is the HPLC chromatogram of the blank group;

[0055] Figure 2 is the HPLC chromatogram of the control group;

[0056] Figure 3 is the HPLC chromatogram of the sample group;

[0057] Figure 4 is the absorption curve of the control group of the example;

[0058] Figure 5 is the absorption curve of the example sample group. DETAILED DESCRIPTION

[0059] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0060] 1. Instruments and Materials

[0061] 1.1 Instrument

[0062] High performance liquid chromatograph (model: e2695, manufacturer: Waters Company); electronic balance (model: AL204, 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.).

[0063] 1.2 Materials

[0064] The methanol and phosphoric acid used in the mobile phase were chromatographic grade, the water was ultrapure water, and the other sample solvents were analytical grade. Isoorientin (111974-201401, China Food and Drug Inspection Institute, potency 94.0%); 3 batches of fennel fruit samples were collected from Jiuquan, Gansu, and were fennel (Pimpinlla anisum L.) of the Umbelliferae family.

[0065] In the present invention, % in 50% methanol and 70% methanol represents the mass percentage.

[0066] 2 Investigation of the preparation method of the test sample

[0067] 2.1 Investigation of different extraction methods

[0068] Example 1

[0069] Step 1: Preparation of reference solution: Take an appropriate amount of isoorientin reference substance (about 6 mg), weigh it accurately, place it in a 100mL stoppered brown volumetric flask, add 70% methanol to dissolve it, then add 70% methanol to the mark, and shake well. Accurately pipette 2mL of the above solution into a 10mL brown volumetric flask, add 70% methanol to dilute to the mark, mix well, filter with a 0.45μm organic filter membrane, and prepare a concentration of 12μg / mL as a reference solution for standby use.

[0070] Step 2: Preparation of test solution: Weigh 1 g of anise fruit powder (passed through No. 2 sieve), accurately weigh, place in a 150 mL stoppered conical flask, add 10 mL of 70% methanol, weigh, reflux and extract for 30 minutes, after the sample cools to room temperature, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0071] Step 3: Chromatographic conditions and system suitability Chromatographic column: Agilent ZORBAX-Eclipse-Plus-C18; methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B; detection wavelength: 347nm; flow rate: 1.0mL / min; column temperature: 30℃. Under these conditions, 10μL of reference solution and test solution were accurately drawn, injected into high performance liquid chromatograph (Waters e2695), eluted according to the elution gradient shown in Table 1, and determined.

[0072] Table 1 Gradient elution sequence

[0073]

[0074] The HPLC results of Example 1 are shown in Table 2. Figure 1 As shown, Figure 1-5 The blank, control, and sample chromatograms (red peaks) and the control and sample absorption curves are shown in Figure 1. Figure 4 and Figure 5 It can be seen that the isoorientin reference substance has a maximum absorption peak at 347nm, and the test solution has a maximum absorption peak at 347nm. It can be seen that both the reference substance and the test solution have maximum absorption at 347nm, so the detection wavelength is set to 347nm.

[0075] Table 2 Investigation of different wavelengths

[0076]

[0077] Example 2

[0078] The only difference from Example 1 is that the reflux treatment in step (2) is replaced by ultrasonic treatment.

[0079] The results of Example 1 and Example 2 are shown in Table 3. As shown in Table 3, the contents of isoorientin in the test solution obtained by ultrasonic treatment and reflux treatment are significantly different (RAD>4%), so reflux is selected as the extraction method.

[0080] Table 3 Investigation of different extraction methods

[0081]

[0082] 2.2 Investigation of different extraction solvents

[0083] Example 3

[0084] The difference from Example 1 is that the extraction solvent in step (2) is 50% methanol.

[0085] Example 4

[0086] The difference from Example 1 is that the extraction solvent in step (2) is 100% methanol.

[0087] The results of Example 1, Example 3 and Example 4 are shown in Table 4. As can be seen from Table 4, the contents of isoorientin in different extraction solvents are significantly different (RSD>4%), and through comprehensive consideration, it is determined that the extraction solvent is 70% methanol.

[0088] Table 4 Investigation of different extraction solvents

[0089]

[0090] 2.3 Investigation of solvent ratio (material-liquid ratio)

[0091] Example 5

[0092] The difference from Example 1 is that the mass volume ratio of the fennel fruit medicinal material and the extraction solvent in step (2) is 0.05 g / mL.

[0093] Example 6

[0094] The difference from Example 1 is that the mass volume ratio of the fennel fruit medicinal material and the extraction solvent in step (2) is 0.03 g / mL.

[0095] The results of Examples 5, 6, and 1 are shown in Table 5. As can be seen from Table 5, when the sample weight is 1 g and the extraction solvent volume is 10 mL and 20 mL, the content of isoorientin is relatively large, and there is no significant difference (relative average deviation RAD < 4%), so it is determined that the sample weight is 1 g and the solvent addition amount is 20 ml.

[0096] Table 5 Investigation of solvent ratio

[0097]

[0098] 2.4 Investigation of extraction time

[0099] Example 7

[0100] The difference from Example 5 is that the extraction time in step (2) is replaced with 20 min.

[0101] Example 8

[0102] The difference from Example 5 is that the extraction time in step (2) is replaced by 40 min.

[0103] The results of Examples 5, 7 and 8 are shown in Table 6. As shown in Table 6, there is no significant difference in the content of isoorientin with the change of extraction time (RSD<3%). Through comprehensive consideration, the extraction time is preliminarily determined to be 20 minutes.

[0104] Table 6 Investigation of different extraction times

[0105]

[0106] 3 Investigation of chromatographic conditions

[0107] 3.1 Investigation of column temperature

[0108] Example 9

[0109] The difference from Example 7 is that the column temperature in step (3) is changed to 25°C.

[0110] Example 10

[0111] The difference from Example 7 is that the column temperature in step (3) is changed to 35°C.

[0112] The results of Examples 9, 10 and 7 are shown in Table 7.

[0113] Table 7 Results of investigation at different column temperatures

[0114]

[0115] 3.2 Investigation of chromatographic columns

[0116] Embodiment 11

[0117] The only difference from Example 10 is that the chromatographic column in step (3) is Water SunFireC 18 5μm 4.6×250mm

[0118] Example 12

[0119] The only difference from Example 10 is that the chromatographic column in step (3) is GL Science Inertsustain C18 5μm 4.6×250mm

[0120] The results of Examples 10, 11, and 12 are shown in Table 8.

[0121] Table 8 Results of investigation on different chromatographic columns

[0122]

[0123] As shown in Table 8, different chromatographic columns have an impact on the content, separation and symmetry of isoorientin (RSD>4%). Considering the separation, symmetry and peak time, Agilent ZORBAX-Eclipse-Plus-C18 can be selected as the best chromatographic column for this experiment.

[0124] 3.3 Investigation of chromatograph

[0125] Embodiment 13

[0126] The only difference from Example 10 is that the chromatograph in step (3) is replaced by Thermo Scientific UltiMate 3000.

[0127] Embodiment 14

[0128] The only difference from Example 10 is that the chromatograph in step (3) is replaced by a Thermo Scientific Vanquish.

[0129] The results of Examples 10, 13 and 14 are shown in Table 9.

[0130] Table 9 Instrument investigation results

[0131]

[0132] 4. Validation of analytical method for content determination

[0133] Example 15 Linear Relationship Investigation

[0134] Accurately pipette 0.5mL, 1mL, 2mL, 4mL, 8mL, and 10mL of the reference solution (c=64.108μg / mL), place them in 10mL brown volumetric flasks, dilute to scale with 70% methanol, shake well, and filter with a 0.45μm organic filter membrane. Pipette 10μL respectively, and measure according to the chromatographic conditions of Example 10, with the peak area as the ordinate (Y) and the concentration of the isoorientin reference substance as the abscissa (X), draw a standard curve, and obtain the regression equation: Y=0.4880X+0.1001 (R 2 =0.9994), the results showed that when the concentration of isoorientin reference substance was in the range of 3.2054~64.108μg / mL, its concentration and peak area showed a good linear relationship.

[0135] Example 16 Precision Test

[0136] Take an appropriate amount (about 2 mL) of the reference solution (c=12.8216 μg / mL), filter it using an (organic) microporous membrane, and inject the sample 6 times continuously according to the test method of Example 10. The RSD value of the peak area of ​​isoorientin is 1.4% (n=6), see Table 10, indicating that the instrument has good precision.

[0137] Table 10 Precision test results (n=6)

[0138]

[0139] Example 17 Stability Test

[0140] According to the test method of Example 10, 10 μL of sample was injected at 0h, 4h, 9h, 14h, 24h, 31h, 38h, and 48h for determination. The results showed that the RSD value of the peak area of ​​isoorientin was 0.7% (n=9) (RSD<2%) as shown in Table 11, indicating that the test solution had good stability within the range of 48h.

[0141] Table 11 Stability test results (n=8)

[0142]

[0143] Example 18 Repeatability Test

[0144] Six portions of the same batch of anisum fruit medicinal materials produced in Gansu were taken, and the test solution was tested according to the test method of Example 10. The RSD value of the isoorientin content was 0.7% (n=6) (RSD<4%), as shown in Table 12, indicating good repeatability.

[0145] Table 12 Repeatability test results (n=6)

[0146]

[0147] Example 19 Sample recovery test

[0148] Take 9 samples with known isoorientin content, place them in a 150 mL conical flask, add the corresponding isoorientin reference solution at 50%, 100%, and 150%, measure the peak area according to the test method of Example 10, and calculate the sample recovery rate (%). The results are shown in Table 13.

[0149] Table 13 Sample recovery test results (n=9)

[0150]

[0151] Example 20 Sample Determination

[0152] Three batches of fennel fruit medicinal materials produced in Jiuquan, Gansu were taken, and the peak area was measured according to the test method of Example 10 to calculate the content of isoorientin in the fennel fruit medicinal materials in Jiuquan, Gansu. The results are shown in Table 14.

[0153] Table 14 Content determination results (n=3)

[0154]

[0155] in conclusion

[0156] The composition of traditional Chinese medicine is relatively complex, and there are fewer components that can be measured. When selecting content determination indicators, it is necessary to select active substances that have been clearly identified and have the same pharmacological activity as traditional Chinese medicine. Both anise fruit and isoorientin are effective in treating respiratory diseases and can be used for quality control of anise fruit. Taking isoorientin as the detection indicator and high performance liquid chromatography, a content determination method for anise fruit medicinal material was established, providing an experimental basis for the quality standard research of anise fruit medicinal material; the determination method is accurate, highly sensitive, and has good repeatability, and has good peak shape and separation, and the results are reliable.

Claims

1. A method for detecting isoorientin in anisum fruit medicinal material, comprising the following steps: The extract of fennel fruit medicinal material was tested by high performance liquid chromatography, and the chromatographic conditions included: The chromatographic column is C 18 column; The mobile phases are mobile phase A and mobile phase B; The mobile phase B is a phosphoric acid aqueous solution, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.01-0.3%, and the mobile phase A is methanol.

2. The method for detecting isoorientin in the medicinal material of Pimpinella foeniculum fruit as claimed in claim 1, characterized in that: The preparation method of the anise fruit medicinal material extract comprises the following steps: mixing the anise fruit medicinal material and a solvent, extracting, cooling, making up for weight loss, and filtering to obtain the anise fruit medicinal material extract, wherein the solvent is an alcohol solvent and water.

3. The method for detecting isoorientin in the medicinal material of Pimpinella foeniculum fruit as claimed in claim 2, characterized in that: The preparation method of the fennel fruit medicinal material extract meets one or more of the following conditions: (1) The mass volume ratio of the fennel fruit medicinal material to the solvent is 0.03 g / mL to 0.3 g / mL; (2) The solvent is 50% to 100% methanol, where the percentage refers to the mass percentage of methanol in the solvent; (3) The extraction method is ultrasonic treatment, reflux treatment or cold soaking; (4) The reflux treatment time is 15 to 40 minutes; (5) The cooling is natural cooling to room temperature; (6) When making up for the lost weight, the solvent in the solution obtained by extracting the fennel fruit is generally used; (7) The filtration is performed using an organic filter membrane.

4. The method for detecting isoorientin in the medicinal material of Pimpinella foeniculum fruit as claimed in claim 3, characterized in that: The preparation method of the fennel fruit medicinal material extract meets one or more of the following conditions: (1) The mass volume ratio of the fennel fruit medicinal material to the solvent is 0.03 g / mL, 0.05 g / mL or 0.1 g / mL; (2) The solvent is 50%, 70% or 100%; the percentage refers to the mass percentage of methanol in the solvent; (3) The extraction method is reflux treatment; (4) The reflux treatment time is 20 min, 30 min or 40 min; (5) The filtration is carried out using a 0.45 μm organic filter membrane.

5. The method for detecting isoorientin in the medicinal material of Pimpinella foeniculum fruit as claimed in claim 1, characterized in that: It meets one or more of the following conditions: (1) The anise fruit medicinal material is the plant Anise of the Umbelliferae family; (2) In the detection method, the length of the chromatographic column is 250 mm; (3) In the detection method, the inner diameter of the chromatographic column is 4.6 mm; (4) In the detection method, the particle size of the filler in the chromatographic column is 5 µm; (5) In the detection method, the detection wavelength is 345~350nm; (6) In the detection method, the injection volume is 5 to 20 μL; (7) In the detection method, the column temperature is 10~40℃; (8) In the detection method, the flow rate of the mobile phase is 0.1-2 mL / min; (9) In the detection method, the elution method is gradient elution.

6. The method for detecting isoorientin in the medicinal material of Pimpinella foeniculum fruit as claimed in claim 5, characterized in that: It meets one or more of the following conditions: (1) In the detection method, the detection wavelength is 347 nm; (2) In the detection method, the injection volume is 10 μL; (3) In the detection method, the column temperature is 25 to 35°C; (4) In the detection method, the flow rate of the mobile phase is 0.8-1.2 mL / min; (5) In the detection method, in the gradient elution, the total volume of the mobile phase A is 100%; at 0-36 min, the volume fraction of the mobile phase A is 28%, and the volume fraction of the mobile phase B is 72%; at 36-45 min, the volume fraction of the mobile phase A increases to 37%, and the volume fraction of the mobile phase B decreases to 63%; at 45-48 min, the volume fraction of the mobile phase A increases to 90%, and the volume fraction of the mobile phase B is 10%; at 48-51 min, the volume fraction of the mobile phase A is 90%, and the volume fraction of the mobile phase B is 10%; at 51-55 min, the volume fraction of the mobile phase A decreases to 28%, and the volume fraction of the mobile phase B increases to 72%.

7. The method for detecting isoorientin in the medicinal material of Pimpinella foeniculum fruit as claimed in claim 6, characterized in that: It meets one or more of the following conditions: (1) In the detection method, the column temperature is 25°C, 30°C or 35°C; (2) In the detection method, the flow rate of the mobile phase is 1.0 mL / min.

8. The method for detecting isoorientin in the medicinal material of Pimpinella foeniculum fruit as claimed in claim 1, characterized in that: Isoorientin is used as a reference substance, and the method also includes the preparation of a reference substance solution before detection: dissolving the isoorientin in a methanol solution; the mass volume ratio of the isoorientin to the methanol solution is (3.2-64) μg / mL.

9. The method for detecting isoorientin in the medicinal material of Pimpinella foeniculum fruit as claimed in claim 8, characterized in that: The mass volume ratio of the isoorientin to the methanol solution is 12.8216 μg / mL.

10. The method for detecting isoorientin in the medicinal material of Pimpinella foeniculum fruit as claimed in claim 1, characterized in that: The chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a filler particle size of 5 μm; The detection wavelength is 347nm; The injection volume was 10 μL; The column temperature was 35°C; The flow rate of the mobile phase is 1.0 mL / min; The mobile phase is methanol (A)-0.1% phosphoric acid solution (B); Gradient elution: The percentages in the table are the volume percentages of each component in the total volume of mobile phase A and mobile phase B.

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