Quality detection method of ipomoea batatas medicinal material

Abstract

The invention discloses a method for detecting the quality of an operculous glorybower herb. In particular to a method for detecting the quality of a thoperculum turcicum medicinal material, which comprises a method a and / or a method b: the method a comprises the following steps: developing a thoperculum turcicum test solution on a thin-layer plate by using a developing solvent by adopting a thin-layer chromatography; and the method b comprises the following steps: carrying out gradient elution on the box fruit vine test sample solution on a chromatographic column by adopting a high performance liquid chromatography and using a mobile phase. The method for detecting the quality of the operculous turpethum provided by the invention is good in separation effect, high in sensitivity and good in reproducibility, complements thin-layer identification and content determination items lacked in the original quality standard, further improves the quality standard of the operculous turpethum, can better control the quality of the medicinal material of the operculous turpethum, and ensures the curative effect of clinical medication.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection of traditional Chinese medicines, and particularly relates to a quality detection method for a hamamelis chinensis medicinal material. Background Art

[0002] Convolvulaceae Convolvulaceae is a perennial herbaceous plant of the genus Convolvulaceae. Convolvulaceae Convolvulaceae is often used as a medicinal material. The commonly used medicinal parts are roots or root bark, which have the effects of expectoration, dampness, muscle relaxation and collateral activation, and can expel damp and cold substances and sticky, corrupt body fluids in the body. It is often used to treat edema, joint pain, sciatica, vitiligo and other diseases.

[0003] The "Xinjiang Uygur Autonomous Region Uygur Medicinal Materials Standard" (2010 edition) lists the medicinal part of the box fruit vine as the root or root bark, and the "Hubei Province Traditional Chinese Medicine Quality Standard" lists the medicinal part of the box fruit vine as the rhizome. The quality control of traditional Chinese medicines in the two standards consists of properties, microscopic identification and inspection items. The quality control of medicinal materials is not objective and accurate enough and cannot meet the current drug quality control requirements. The "Guangxi Zhuang Autonomous Region Zhuang Medicine Quality Standard" lists the medicinal part of the box fruit vine as the above-ground part. The overall level of this standard is not high, and it only makes provisions for the properties, cross-sections and thin-layer identification (using control medicinal materials as controls) of the medicinal materials.

[0005] In summary, the local standards for the three types of box fruit vines lack quality control research based on medicinal substances, and have not established qualitative and quantitative detection methods for effective substances or indicator components. They are unable to scientifically evaluate and control the quality of medicinal materials, and cannot ensure the safety and effectiveness of clinical medication and preparations.

[0006] Literature (C 21 steroids from the roots of Marsdenia tenacissima, J.-L. Liu et al. Phytochemistry 213 (2023) 113782) discloses compound P, which is an active ingredient found in the Convolvulaceae plant box fruit vine, belonging to C 21 Steroid compounds, C 21 Steroids have good anti-tumor, anti-oxidation, immune regulation, liver protection and other effects, especially in the anti-tumor research has been paid much attention. The literature also disclosed that compound P has good inhibitory activity against A549 cell line.

[0007]

[0008] Linolenic acid and linoleic acid are the main components of unsaturated fatty acids, and are important nutrients that are indispensable to the human body. They can participate in the development of brain cells and reduce blood lipids. They can synthesize arachidonic acid in the human body, help prevent cardiovascular and cerebrovascular diseases, and have certain antioxidant, anti-inflammatory, anti-tumor, and vision-improving effects. Linolenic acid and linoleic acid exist in many medicinal materials, such as red ginseng, hemp seeds, etc., but have not been reported in box fruit vine medicinal materials. The inventor of the present application also found that box fruit vine contains linolenic acid and linoleic acid.

[0009] It can be seen that compound P, linolenic acid and linoleic acid are all important active ingredients of drugs. However, there are very few studies on the ingredients and quality control of box fruit vine. Therefore, it is very important to study an accurate and stable quality detection method for the active ingredients in box fruit vine. Summary of the invention

[0010] Technical Problems to be Solved by the Invention In order to overcome the lack of quality control methods based on pharmacological substances in the quality detection methods of box fruit vine in the prior art, and the inability to scientifically evaluate and control the quality of medicinal materials, the present invention provides a quality detection method for box fruit vine medicinal materials. The method has good separation effect, high sensitivity, and good reproducibility, and can better control the quality of box fruit vine medicinal materials and ensure the clinical efficacy of medication.

[0011] The present invention provides a quality detection method for the medicinal material of box fruit vine, which comprises the following method a and / or method b:

[0012] Method a comprises the following steps:

[0013] Adopt thin layer chromatography method, use developing agent, and develop the box fruit vine test sample solution on the thin layer plate;

[0014] In terms of volume percentage, the developing agent includes 80-90% halogenated hydrocarbon solvent, 5-15% ester solvent and 3-8% alcohol solvent;

[0015] The halogenated hydrocarbon solvent is selected from one or both of dichloromethane and chloroform;

[0016] The ester solvent is selected from one or both of ethyl acetate and ethyl formate;

[0017] The alcohol solvent is selected from one or both of methanol and ethanol;

[0018] Method b comprises the following steps:

[0019] The high performance liquid chromatography method was adopted, and the mobile phase was used to gradiently elute the box fruit vine test sample solution on the chromatographic column;

[0020] The filler of the chromatographic column is octadecylsilane bonded silica gel;

[0021] The mobile phases are mobile phase A and mobile phase B;

[0022] The mobile phase A is a phosphoric acid aqueous solution, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.01-0.5%, and the mobile phase B is acetonitrile;

[0023] The gradient elution sequentially undergoes a first-stage gradient elution, a second-stage gradient elution, and a third-stage gradient elution;

[0024] The duration of the first stage gradient elution is 10-20 min. In the mobile phase, the volume percentage of mobile phase A changes from (15-25)% to (5-15)%; the volume percentage of mobile phase B changes from (75-85)% to (85-95)%;

[0025] The duration of the second stage gradient elution is 0.5-7 minutes. In the mobile phase, the volume percentage of mobile phase A changes from (5-15)% to (0-10)%; the volume percentage of mobile phase B changes from (85-95)% to (90-100)%;

[0026] The duration of the third stage gradient elution is 1-10 minutes. In the mobile phase, the volume percentage of mobile phase A is maintained at (0-10)%; and the volume percentage of mobile phase B is maintained at (90-100)%.

[0027] In the present invention, the duration of the gradient elution is the difference between the starting point and the end point of the elution time in the elution time period.

[0028] In a preferred embodiment, in the developing agent, the halogenated hydrocarbon solvent is dichloromethane or chloroform.

[0029] In a preferred embodiment, in the developing agent, the volume percentage of the halogenated hydrocarbon solvent is 80-85%, for example, 83% or 85%.

[0030] In a preferred embodiment, in the developing agent, the ester solvent is ethyl acetate.

[0031] In a preferred embodiment, in the developing agent, the volume percentage of the ester solvent is 6-10%, such as 8% or 8.5%.

[0032] In a preferred embodiment, in the developing agent, the alcohol solvent is methanol.

[0033] In a preferred embodiment, the volume percentage of the alcohol solvent in the developing agent is 5-8%, for example, 6.5% or 7%.

[0034] In a preferred embodiment, the developing agent further comprises an acid solvent, and the acid solvent is selected from one or both of formic acid and acetic acid, such as formic acid; the volume percentage of the acid solvent is 0.5-3%, such as 2%.

[0035] In a preferred embodiment, the developing agent consists of the halogenated hydrocarbon solvent, the ester solvent and the alcohol solvent; or consists of the halogenated hydrocarbon solvent, the ester solvent, the alcohol solvent and the acid solvent.

[0036] In a preferred embodiment, the developing agent is composed of dichloromethane, ethyl acetate and methanol, for example, the volume ratio of dichloromethane:ethyl acetate:methanol is 5:0.5:0.4.

[0037] In a preferred embodiment, the developing agent is composed of chloroform, ethyl acetate, methanol and formic acid, for example, the volume ratio of chloroform:ethyl acetate:methanol:formic acid is 5:0.5:0.4:0.1.

[0038] The test solution of the box fruit vine is loaded by a conventional loading method in the art, for example, a methanol solution of the box fruit vine extract.

[0039] The thin layer plate used in the thin layer chromatography method includes domestic silica gel GF 254 Board, high-efficiency Merk254 board, Yinlong high-efficiency silica gel prefabricated thin layer board, preferably domestic silica gel GF254 board and Yinlong high-efficiency silica gel prefabricated thin layer board.

[0040] The sample spotting volume for the thin layer chromatography method is conventional in the art, preferably 1 to 5 μL, such as 2 μL.

[0041] In a preferred embodiment, the thin layer chromatography detection method is a colorimetric method; the colorimetric method colorimetric agent is preferably a sulfuric acid ethanol solution, such as a 10% sulfuric acid ethanol solution.

[0042] In a preferred embodiment, the wavelength of the test is an ultraviolet light wavelength or a sunlight wavelength; the ultraviolet light wavelength is preferably 100-450nm, such as 366nm; the sunlight wavelength is preferably 400-760nm.

[0043] In a preferred embodiment, the development temperature is -4 to 35°C, for example 25°C.

[0044] In a preferred embodiment, the unfolding humidity is 15% to 70%, for example, 20%, 42% or 63%.

[0045] In a preferred embodiment, the method a further comprises the step of developing the reference solution of compound P on a thin layer plate using a developing agent (to determine whether compound P exists in the test solution of box fruit vine).

[0046] .

[0047] In a preferred embodiment, the reference solution of compound P is a methanol solution of compound P; in the reference solution of compound P, the mass volume ratio of compound P to methanol is (0.1-2) mg / ml, for example, 1 mg / ml.

[0048] In a preferred embodiment, the method a further comprises the step of developing the control medicinal material solution on a thin layer plate using a developing agent; the control medicinal material is preferably a Psoralea corylifolia control medicinal material produced in Zhaoqing, Guangdong.

[0049] In a preferred embodiment, the method a further comprises the step of developing the reference solution of compound P and the reference medicinal material solution on a thin layer plate using a developing agent.

[0050] In a preferred embodiment, the control medicinal material solution is a methanol solution of a control medicinal material; in the control medicinal material solution, the mass volume ratio of the control medicinal material to methanol is (0.01-2) g / ml.

[0051] In a preferred embodiment, in method a, the extract of Psoralea corylifolia is prepared by the following method:

[0052] Mixing and extracting the box fruit vine with a solvent to obtain the box fruit vine extract;

[0053] The solvent is selected from dichloromethane, methanol, an aqueous solution of an alcohol solvent, or a mixed solution consisting of petroleum ether and dichloromethane;

[0054] In the mixed solution composed of petroleum ether and dichloromethane, the volume ratio of petroleum ether to dichloromethane is 1: (0.5-2);

[0055] The alcohol solvent in the alcohol solvent aqueous solution is methanol or ethanol; the volume percentage of the alcohol solvent in the alcohol solvent aqueous solution is 40-98%, and the volume percentage is the volume percentage of the volume of the alcohol solvent to the volume of the alcohol solvent aqueous solution.

[0056] In a preferred embodiment, the box fruit vine is a box fruit vine root or root bark.

[0057] In a preferred embodiment, in the mixed solution of petroleum ether and dichloromethane, the volume ratio of petroleum ether to dichloromethane is 1:1.

[0058] In a preferred embodiment, the alcohol solvent in the alcohol solvent aqueous solution is methanol. Preferably, the volume percentage of methanol in the alcohol solvent aqueous solution is 40-60%, for example 50%.

[0059] In a preferred embodiment, the alcohol solvent in the alcohol solvent aqueous solution is ethanol. Preferably, the volume percentage of ethanol in the alcohol solvent aqueous solution is 80-98%, for example 95%.

[0060] In a preferred embodiment, the solvent is dichloromethane.

[0061] In a preferred embodiment, the mass volume ratio of the box fruit vine to the solvent is (0.01-2) g / ml; for example, 0.04 g / ml.

[0062] In a preferred embodiment, the extraction method is ultrasonic extraction.

[0063] In a preferred embodiment, the extraction time is 15 to 45 minutes, for example 30 minutes.

[0064] In a preferred embodiment, the extraction is performed once, twice or three times, preferably three times.

[0065] After the extraction, conventional post-processing methods in the art may be further performed, such as filtration and / or concentration (eg, rotary evaporation).

[0066] In a preferred embodiment, in method a, the extraction is performed three times, the solvent used in the first extraction is petroleum ether; the solvent used in the second extraction is a mixed solvent of petroleum ether and dichloromethane in a volume ratio of 1:1; and the solvent used in the third extraction is dichloromethane.

[0067] In a preferred embodiment, in the method b, the test solution of the Psoralea corylifolia is a methanol solution of the Psoralea corylifolia extract.

[0068] In a preferred embodiment, in the method b, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.2%, for example 0.1%.

[0069] In a preferred embodiment, in the method b, the duration of the first stage gradient elution is 12 to 18 min, for example, 15 min.

[0070] In a preferred embodiment, in the method b, the duration of the second stage gradient elution is 0.5 to 4 min, for example 1 min.

[0071] In a preferred embodiment, in the method b, the duration of the third stage gradient elution is 2 to 8 min, for example 4 min.

[0072] In a preferred embodiment, in the method b, in the first stage of gradient elution, in the mobile phase, the volume percentage of mobile phase A changes from (15~20)% to (5~10)%; the volume percentage of mobile phase B changes from (80~85)% to (90~95)%; for example, the volume percentage of mobile phase A changes from 20% to 10%; the volume percentage of mobile phase B changes from 80% to 90%.

[0073] In a preferred embodiment, in the method b, in the second stage gradient elution, in the mobile phase, the volume percentage of mobile phase A changes from (5~10)% to (0~5)%; the volume percentage of mobile phase B changes from (90~95)% to (95~100)%; for example, the volume percentage of mobile phase A changes from 10% to 5%; the volume percentage of mobile phase B changes from 90% to 95%.

[0074] In a preferred embodiment, in the method b, in the third stage gradient elution, in the mobile phase, the volume percentage of mobile phase A is maintained at (0~5)%; the volume percentage of mobile phase B is maintained at (95~100)%; for example, the volume percentage of mobile phase A is maintained at 5%; the volume percentage of mobile phase B is maintained at 95%.

[0075] In a preferred embodiment, in the method b, the gradient elution is:

[0076]

[0078] In a preferred embodiment, the chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a filler particle size of 5 μm, such as Agilent ZORBAX SB-C 18 (250 mm * 4.6 mm, 5 μm), Agilent Eclipse XDB-C 18 (250 mm* 4.6 mm, 5 μm) or SHISEIDO CAPCEll PAK C 18 (250 mm * 4.6 mm, 5 μm).

[0079] In a preferred embodiment, the filler of the chromatographic column is octadecylsilane bonded silica gel.

[0080] In a preferred embodiment, the column temperature of the chromatographic column is 25-35°C, for example, 30°C.

[0081] In a preferred embodiment, in the high performance liquid chromatography, the detection wavelength is 180-250 nm, for example 203 nm.

[0082] In a preferred embodiment, in the high performance liquid chromatography, the injection volume is 15-25 μl, for example 20 μl.

[0083] In a preferred embodiment, the flow rate of the mobile phase is 0.5-2 ml / min, preferably 0.8-1.2 ml / min, for example 1.0 ml / min.

[0084] In a preferred embodiment, the method b also includes the step of detecting the α-linolenic acid and / or linoleic acid reference solution by high performance liquid chromatography; the α-linolenic acid and / or linoleic acid reference solution is a methanol solution of α-linolenic acid and / or linoleic acid; in the α-linolenic acid and / or linoleic acid reference solution, the mass volume ratio of the α-linolenic acid and / or linoleic acid to the methanol solution is (0.2~3) mg / ml, for example 0.393 mg / ml or 1 mg / ml.

[0085] In a preferred embodiment, in the method b, the method for preparing the Psoralea corylifolia extract comprises the following steps: mixing and extracting the Psoralea corylifolia and an alcohol solvent to obtain the Psoralea corylifolia extract.

[0086] In a preferred embodiment, in the method b, the alcohol solvent is methanol.

[0087] In a preferred embodiment, in the method b, the mass volume ratio of the box fruit vine to the alcohol solvent is (0.005-0.02) g / ml, for example, 0.01 g / ml.

[0088] In a preferred embodiment, in the method b, the extraction is ultrasonic extraction, the power of the ultrasound is 100-400 W, for example, 250 W; the frequency of the ultrasound is 20-50 kHz, for example, 40 kHz.

[0089] In a preferred embodiment, in the method b, the extraction time is 10 to 60 minutes, for example 40 minutes.

[0090] In a preferred embodiment, in the method b, an alcohol solvent is used to make up for the lost weight after the extraction.

[0091] The extraction may be followed by a conventional post-processing method in the art, such as filtration, wherein the filtration is performed using a 0.2-0.5 µm filter membrane, such as a 0.45 µm filter membrane.

[0092] In a preferred embodiment, in the method b, the box fruit vine is the box fruit vine root bark.

[0093] In the quality inspection method for the medicinal material of Psoralea corylifolia, the quality inspection pass standard of the medicinal material of Psoralea corylifolia is: (1) compound P can be identified in thin layer chromatography; and / or (2) the content of α-linolenic acid and linoleic acid per gram of Psoralea corylifolia detected in high performance liquid chromatography is not less than 2 mg.

[0094] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0095] The reagents and raw materials used in the present invention are commercially available.

[0096] The positive progress of the present invention is that the quality detection method of box fruit vine provided by the present invention includes two aspects: thin layer chromatography identification and multi-component content detection. It makes up for the thin layer chromatography identification and high performance liquid chromatography content determination items missing in the original standard, and the method has good separation effect, high sensitivity and good reproducibility, and can more effectively reflect the quality of box fruit vine. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 :TLC images of 14 batches of Herba Hedyotis diffusae under white light (upper) and 366 nm (lower). 1: reference substance; 2: reference herbal medicine; 3-16: test herbal medicines 1-14, respectively.

[0098] Figure 2 : TLC graphs of reference substance P, sample 1, Psoralea corylifolia and Rhizoma tongguanyi from different origins under white light (upper) and 366 nm (lower). 1: reference substance P; 2: sample 1; 3: Psoralea corylifolia 2; 4: Psoralea corylifolia 3; 5: Psoralea corylifolia 4; 6: Psoralea corylifolia 5 (samples 1 and Psoralea corylifolia 2-5 correspond to test medicinal materials 15-19, collected in Nanning, Guangxi); 7: Rhizoma tongguanyi (purchased in Guangxi); 8: Rhizoma tongguanyi (Qiubei Badaoshao, Wenshan, Yunnan); 9: Rhizoma tongguanyi (Baicaoyuan Medicinal Materials Store, Kunming); 10: Rhizoma tongguanyi (Fushengtang Pharmacy, Kunming); 11: Rhizoma tongguanyi (Hengfeng Farmers' Market, Wenshan, Yunnan); 12: Rhizoma tongguanyi old root (Hengfeng Farmers' Market, Wenshan, Yunnan).

[0099] Figure 3 :TLC charts of the extraction solvents of the box fruit vine samples. 1: reference substance; 2: dichloromethane extraction; 3: methanol extraction; 4: 95% ethanol extraction; 5: 50% methanol extraction.

[0100] Figure 4 :TLC charts of the extraction methods of box fruit vine samples. 1: reference substance; 2: method ①; 3: method ②; 4: method ③.

[0101] Figure 5: TLC diagrams of the development system of box fruit vine samples. 1: reference substance; 2: sample; A: chloroform: acetone (4:1, secondary development); B: dichloromethane: ethyl acetate: methanol (5:0.5:0.4); C: chloroform: ethyl acetate: methanol: formic acid (5:0.5:0.4:0.1). The left diagram of A, B, and C is the TLC diagram under white light conditions, and the right diagram is the TLC diagram under 366 nm.

[0102] Figure 6 :TLC charts of box fruit vine sample spotting quantity under white light (left) and 366 nm (right). 1: reference substance; 2: 1 μl; 3: 2 μl; 4: 5 μl.

[0103] Figure 7 :TLC images of box fruit vine sample under white light (left) and 366 nm (right). 1: reference substance; 2: sample; A: 10% sulfuric acid ethanol colorimetric agent; B: vanillin concentrated sulfuric acid colorimetric agent.

[0104] Figure 8 :TLC graphs of box fruit vine samples under white light (left) and 366 nm (right). 1: reference; 2: sample; A: temperature -4 ℃; B: temperature 25 ℃.

[0105] Fig. 9 : White light (left) and 366 nm (right) TLC images of box fruit vine samples. 1: reference; 2: sample; A: humidity 20%; B: humidity 42%; C: humidity 63%. DETAILED DESCRIPTION

[0106] 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.

[0107] Example 1: Thin layer chromatography identification method of compound P in Psoralea corylifolia

[0108]

[0109] Experimental instruments and reagents

[0110] Analytical balance (Sartorius Scientific Instrument Co., Ltd.); TLC automatic spotter and TLC camera (CAMAG, Switzerland); KQ-250DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); methanol, chloroform, dichloromethane, ethyl acetate, formic acid, 95% ethanol were all analytical grade (Sinopharm Chemical Reagent Co., Ltd.), water was ultrapure water, silica gel GF254 plate (Yantai Jiangyou Silica Gel Development Reagent Factory), MN TLC plate, Merck TLC plate, MN HPTLC plate, size 10×20 cm; Compound P (Turpethum P) reference substance was prepared by the applicant according to the literature (C 21 Steroids from the roots of Marsdenia tenacissima, J.-L. Liu et al. Phytochemistry 213 (2023)113782) laboratory-made, (purity ≥98%); sample sources are shown in Table 1 below; Psoralea corylifolia root bark medicinal material (Xinjiang Uygur Pharmaceutical).

[0111] Table 1 Sample sources

[0112]

[0113] 1. Thin layer chromatography identification of compound P in Psoralea corylifolia

[0114] Preparation of test solution: Take 2.0 g of the test medicinal material (root bark powder of box fruit vine) in Table 1 above, add 50 ml of petroleum ether, ultrasonically treat for 30 min, and filter; evaporate the solvent from the filter residue, add 50 ml of a 1:1 mixed solvent of petroleum ether and dichloromethane, ultrasonically treat for 30 min, and filter; evaporate the solvent from the filter residue, add 50 ml of dichloromethane, ultrasonically treat for 30 min, dry the filtrate, and dissolve it in 1 ml of methanol to obtain the test solution.

[0115] Preparation of control medicinal material solution: Take 2.0 g of the control medicinal material powder of Heguoteng and prepare the control medicinal material solution according to the preparation method of the test sample solution.

[0116] Preparation of reference solution: Take an appropriate amount of compound P and dissolve it in methanol to prepare a 1 mg / ml reference solution.

[0117] Thin layer chromatography identification: 2 μl of reference medicinal material solution, 2 μl of test solution, and 5 μl of reference solution were taken and spotted on the same silica gel G thin layer plate, and chloroform-ethyl acetate-methanol-formic acid (5:0.5:0.4:0.1) was used as the development system. The development cylinder and thin layer plate were saturated for 5 min, developed, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105 °C for 3 min until the spots were clearly colored, and dried. Inspect under white light and 366 nm ultraviolet light.

[0118] The results are as follows Figure 1 As shown: in the chromatogram of the test sample of box fruit vine, the test sample medicinal material shows spots of the same color at the corresponding positions of the chromatograms of the control medicinal material and the control sample, and compound P can be detected.

[0119] 2. Detection of compound P, sample 1, box fruit vine and Tongguan vine:

[0120] Table 2 Sources of medicinal materials

[0121]

[0122] Tongguanteng is often used as a counterfeit of box fruit vine in the folk. Compound P can be used as a reference substance to distinguish box fruit vine from other counterfeits.

[0123] Figure 2 The sources of sample 1 and box fruit vine 2~5 are shown in Table 2. Sample 1 comes from the medicinal materials collected according to the characteristics of box fruit vine. The preparation method of the test solution of sample 1, box fruit vine 2~5 and Tongguanteng is the same as the preparation method of the test solution in "1. Thin layer chromatography identification method of compound P in box fruit vine", and the thin layer chromatography identification operation is the same as "1. Thin layer chromatography identification method of compound P in box fruit vine". The results are shown in Figure 2 As shown. Through experimental control, it was found that the medicinal material (root) of the box fruit vine and compound P showed the same fluorescent spots at the same position, while sample 1 and Tongguanteng did not.

[0124] In order to ensure that the thin layer identification method of the present invention is scientific, reasonable and feasible, the applicant conducted a series of experimental studies and investigations:

[0125] 3. Selection of extraction solvent

[0126] The test sample medicinal material (14) was ultrasonically extracted using four solvents, namely, dichloromethane, methanol, 95% ethanol, and 50% methanol water (the extraction method was the same as the preparation method of the test sample solution), and the test sample solution was prepared. The thin layer chromatography method in Part 1 of Example 1 was used for analysis, and the results (see Figure 3 ) showed that the samples extracted with dichloromethane were cleaner and free of interference spots at the same position of the reference sample than those extracted with the other three solvents.

[0127] 4. Selection of extraction method

[0128] ① Take 2.0 g of box fruit vine root peel powder, add 50 ml of petroleum ether, ultrasonically treat for 30 min, and filter; evaporate the solvent from the residue, add 50 ml of a 1:1 mixed solvent of petroleum ether and dichloromethane, ultrasonically treat for 30 min, and filter; evaporate the solvent from the residue, add 50 ml of dichloromethane, ultrasonically treat for 30 min, and evaporate the filtrate to dryness, and add 1 ml of methanol to dissolve.

[0129] ②Take 2.0 g of the root bark powder of the Chinese wolfberry vine, add 50 ml of dichloromethane, directly ultrasonicate for 30 min, evaporate the filtrate to dryness, and add 1 ml of methanol to dissolve it.

[0130] ③Take 2.0 g of the root bark powder of the fruit vine, add 50 ml of dichloromethane, reflux for 30 min, evaporate the filtrate to dryness, and add 1 ml of methanol to dissolve it.

[0131] The test solution (the sample is derived from the test medicinal material 14) prepared in methods ①-③ was analyzed by the thin layer chromatography method in Part 1 of Example 1. The results (see Figure 4 ) showed that the sample extracted by method ① had corresponding clear spots at the same position as the reference substance, with fewer interfering impurities.

[0132] 5. Selection of solvent

[0133] The same test solution (the sample comes from the test medicinal material 14) was developed with chloroform:acetone (4:1, secondary development), dichloromethane:ethyl acetate:methanol (5:0.5:0.4), chloroform:ethyl acetate:methanol:formic acid (5:0.5:0.4:0.1), and other operating conditions were the same as the thin layer chromatography method in Part 1 of Example 1. Results (see Figure 5 ) showed that the developing system of chloroform:ethyl acetate:methanol:formic acid (5:0.5:0.4:0.1) was better.

[0134] 6. Investigation of the amount of sample

[0135] The experiment investigated three sample volumes: 1 μl, 2 μl, and 5 μl. The same test solution (the sample was derived from the test herbal material 14) was taken, and other operating conditions were the same as the thin layer chromatography method in Part 1 of Example 1. The results showed (see Figure 6 ), the best effect is achieved when the sample volume is 2 μl.

[0136] 7. Investigation of colorimetric agents

[0137] The experiment investigated two color developing agents: vanillin solution and 10% sulfuric acid ethanol solution. The same test solution (the sample was derived from the test herbal material 14) was used, and other operating conditions were the same as the thin layer chromatography method in Part 1 of Example 1. Results (see Figure 7 ) showed that after color development with 10% sulfuric acid ethanol solution, the observation effect under white light and 366 ultraviolet light was better than that of vanillin solution.

[0138] 8. Temperature Investigation

[0139] The experiment observed the development at different temperatures of -4 ℃ and 25 ℃, taking the same test solution (the sample comes from the test medicinal material 14), and other operating conditions were the same as the thin layer chromatography method in Part 1 of Example 1. The results (see Figure 8 ) showed that the position and separation of spots were relatively good at 25 ℃.

[0140] 9. Humidity Investigation

[0141] The experiment observed the development at different humidity levels of 20%, 42%, and 63%, respectively. The same test solution (the sample was derived from the test medicinal material 14) was taken. Other operating conditions were the same as the thin layer chromatography method in Part 1 of Example 1. The results (see Fig. 9 ) showed that good results can be obtained in the humidity range of 20%~63%.

[0142] Example 2: Method for determining the contents of α-linolenic acid and linolenic acid in box fruit vine:

[0143] Experimental instruments and reagents

[0144] BSA323S electronic analytical balance (Beijing Sartorius Scientific Instrument Co., Ltd., China); Satorius BT25S electronic analytical balance (Beijing Sartorius Instrument System Co., Ltd., China); SCQ-5201 CNC ultrasonic cleaner (Shanghai Shengyan Ultrasonic Instrument Co., Ltd., China); water is ultrapure water, prepared in Milli-Q ultrapure water preparation instrument (Millipore, Bedford, MA); acetonitrile (Fisher Scientific, Canada). α-linolenic acid reference substance (batch number: S13011B127108, purity ≥98%) and linoleic acid reference substance (batch number: S29M11B113654, purity ≥98%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. The root bark sample of box fruit vine was provided by Xinjiang Uygur Pharmaceutical Co., Ltd. (the sample came from the test medicinal material 14).

[0145] 1. Determination of α-linolenic acid and linolenic acid in Psoralea corylifolia

[0146] Chromatographic conditions: octadecylsilane bonded silica gel as filler (column length 250 mm, inner diameter 4.6 µm, particle size 5 µm); 0.1% phosphoric acid water as mobile phase A, acetonitrile as mobile phase B, gradient elution order as shown in Table 3; flow rate 1.0 ml / min; column temperature 30 °C; detection wavelength 203 nm. The theoretical plate number calculated based on the linoleic acid peak should be no less than 6000.

[0147] Table 3 Gradient elution sequence

[0148]

[0149] Preparation of reference solution: Accurately weigh the α-linolenic acid reference substance and the linoleic acid reference substance, add the initial mobile phase to make solutions containing 393 μg of α-linolenic acid and 1000 μg of linoleic acid per 1 ml respectively.

[0150] Preparation of test solution: Take about 0.5 g of the root bark powder of the medicinal material, weigh it accurately, put it in a stoppered conical flask, add 50 ml of methanol accurately, stopper it tightly, weigh it, and treat it ultrasonically (power 250 W, frequency 40 kHz) for 40 minutes. Let it cool, weigh it again, make up the lost weight with methanol, filter it through a 0.45 µm filter membrane, and take the filtrate to obtain the test solution.

[0151] Determination method: Accurately pipette 20 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0152] Result: Calculated on the dry basis, the total amount of α-linolenic acid and linoleic acid contained in this product shall not be less than 0.20%.

[0153] In order to ensure that the content determination method of the present invention is scientific, reasonable and feasible, the applicant conducted a series of experimental studies and investigations:

[0154] 2. Selection of chromatographic conditions

[0155] 2.1 Establishment of test sample preparation method

[0156] 2.1.1 Investigation of extraction methods

[0157] The experiment used four extraction methods: methanol ultrasound, methanol reflux, dichloromethane ultrasound and petroleum ether ultrasound. Take about 1.0 g of the root bark of the box fruit vine, accurately weigh it, and each extraction method is parallel to 2 parts, placed in a stoppered conical flask, accurately add 25 ml of methanol, dichloromethane and petroleum ether respectively, weigh the weight, reflux (80 ℃) and ultrasonic treatment (250 W, 40 kHz) for 40 min, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and take the filtrate to obtain the test solution. Accurately draw 5 μl of each test solution, and analyze it according to the chromatographic conditions in Part 1 of Example 2. The results are shown in Table 4, and the methanol ultrasonic extraction method is the best.

[0158] Table 4 Investigation of extraction methods (n=2)

[0159]

[0160] 2.1.2 Extraction time investigation

[0161] Take about 1.0 g of the root bark of the box fruit vine, weigh it accurately, and place it in three parallel groups, with 2 portions in each group, in a stoppered conical bottle, accurately add 25 ml of the mobile phase solution, weigh it, and ultrasonically treat it (250 W, 40 kHz, 30 ℃) for 20 min, 40 min and 60 min respectively. The subsequent treatment steps are the same as 2.1.1. The results are shown in Table 5, and ultrasonic treatment for 40 min is the best.

[0162] Table 5 Extraction time investigation (n=2)

[0163]

[0164] 2.1.3 Investigation of extraction solvent dosage

[0165] Take about 1.0 g of the root bark of the box fruit vine, weigh it accurately, and place it in three parallel groups, with 2 portions in each group, in a stoppered conical bottle, and accurately add 25 ml, 50 ml and 100 ml of methanol solution, weigh it, and ultrasonically treat it (250 W, 40 kHz, 30 ℃) for 40 min. The subsequent treatment steps are the same as 2.1.1. The results are shown in Table 6. The best extraction volume of methanol is 100 ml (solid-liquid ratio is 1:100).

[0166] Table 6 Investigation of extraction solvent dosage (n=2)

[0167]

[0168] 2.2 Methodological validation

[0169] 2.2.1 Specificity test

[0170] The reference solution and the test solution in Part 1 of Example 2 were accurately aspirated, and the samples were analyzed according to the chromatographic conditions in Part 1 of Example 2. The results are shown in Table 7, and the peak conditions are consistent with those of the reference solution, indicating that the method is specific.

[0171] Table 7 Specificity of α-linolenic acid and linoleic acid

[0172]

[0173] 2.2.2 Linear relationship investigation

[0174] An appropriate amount of reference substance was accurately pipetted, and the initial mobile phase was added to prepare a mixed reference substance solution, and the sample was injected and analyzed according to the chromatographic conditions of Part 1 of Example 2. The results (Table 8) showed that the linear relationship was good.

[0175] Table 8 Linear range and detection limit of α-linolenic acid and linoleic acid

[0176]

[0177] 2.2.3 Precision test

[0178] 2.2.3.1 Instrument precision test

[0179] Take an appropriate amount of the reference solution in Part 1 of Example 2 and inject it continuously 6 times according to the chromatographic conditions in Part 1 of Example 2. The results are shown in Table 9. The results show that the instrument has good precision and meets the requirements of quantitative analysis.

[0180] Table 9 Precision test (n=6)

[0181]

[0182] 2.2.3.2 Inter-day precision test

[0183] The same reference solution (the reference solution in Part 1 of Example 2) was accurately pipetted and injected 6 times continuously for 3 consecutive days according to the chromatographic conditions in Part 1 of Example 2. The results are shown in Table 10, indicating that the instrument has good daytime precision.

[0184] Table 10 Precision test (n=6)

[0185]

[0186] 2.2.4 Stability test

[0187] The box fruit vine test solution in Part 1 of Example 2 was taken and placed at room temperature. Samples were injected and analyzed at 0, 2, 6, 12, 18, and 24 hours according to the chromatographic conditions in Part 1 of Example 2. The results are shown in Table 11, which show that α-linolenic acid and linoleic acid are stable within 24 hours.

[0188] Table 11 Stability test (n=6)

[0189]

[0190] 2.2.5 Repeatability test

[0191] Take the box fruit vine test solution in part 1 of Example 2, and make 6 parallel portions, and inject and analyze according to the chromatographic conditions in part 1 of Example 2. The results are shown in Table 12, which shows that the method has good repeatability.

[0192] Table 12 Repeatability test (n=6)

[0193]

[0194] 2.2.6 Sample recovery test

[0195] Take 0.5 g of the known content of the box fruit vine root bark sample, accurately weigh it, add the same amount of α-linolenic acid and linoleic acid reference substances as the known content, prepare the test solution according to the method in Part 1 of Example 2, and conduct 6 parallel injections according to the chromatographic conditions in Part 1 of Example 2 to calculate the recovery rate and its RSD value. The results are shown in Table 13, which shows that it meets the guidance principles for validation of analytical methods in Part IV 9101 of the Chinese Pharmacopoeia and meets the requirements for quantitative analysis.

[0196] Table 13 Sample recovery test (n=6)

[0197]

[0198] 2.2.7 Durability test

[0199] 2.2.7.1 Investigation of different column temperatures

[0200] The effects of different column temperatures of 25 ℃, 30 ℃ and 35 ℃ on the determination of Psoralea corylifolia content were compared.

[0201] Take 0.5 g of box fruit vine, and make two parallel portions. Prepare the test solution according to the method in Part 1 of Example 2. Except that the column temperature is 25 ℃, 30 ℃ and 35 ℃ respectively, the other chromatographic conditions are the same as those in Part 1 of Example 2. Inject and analyze, and record the peak area and retention time of α-linolenic acid and linoleic acid. The results are shown in Table 14. The results show that the analytical method has good durability within the column temperature range of 25-35 ℃.

[0202] Table 14 Durability column temperature investigation test (n=2)

[0203]

[0204] 2.2.7.2 Investigation of different flow rates

[0205] Compare the effects of different flow rates, 0.8 ml / min, 1.0 ml / min and 1.2 ml / min, on the determination of the content of box fruit vine. Take 0.5 g of box fruit vine and make two parallel portions. Prepare the test solution according to the method in Part 1 of Example 2. Except that the flow rates are 0.8 ml / min, 1.0 ml / min and 1.2 ml / min respectively, the other chromatographic conditions are the same as those in Part 1 of Example 2. Inject and analyze, and record the peak area and retention time of α-linolenic acid and linoleic acid. The results are shown in Table 15. The results show that the analytical method has good durability in the flow rate range of 0.8 ~1.2 ml / min.

[0206] Table 15 Durability flow rate test (n=2)

[0207]

[0208] 2.2.7.3 Investigation of different brands of chromatographic columns

[0209] Comparison of different brands of chromatographic columns, Agilent ZORBAX SB-C 18 (250 mm * 4.6 mm, 5 μm), Agilent Eclipse XDB-C 18 (250 mm * 4.6 mm, 5 μm) and SHISEIDO CAPCEll PAK C 18 (250mm * 4.6 mm, 5 μm) on the determination of box fruit vine content.

[0210] Take 0.5 g of box fruit vine, and make three parallel portions. Prepare the test solution according to the method in Part 1 of Example 2. Except for the different chromatographic columns, the other chromatographic conditions are the same as those in Part 1 of Example 2. Samples are injected for analysis, and the peak areas and retention times of α-linolenic acid and linoleic acid are recorded. The results are shown in Table 16. The results show that the analytical method has good durability under different chromatographic columns.

[0211] Table 16 Durability chromatographic column inspection test (n=2)

[0212]

[0213] 2.3 Content determination and content limit determination

[0214] Accurately weigh 15 batches of box fruit vines from different manufacturers and batches as shown in Table 1, prepare the test solution according to the method in Part 1 of Example 2, operate 2 portions in parallel, and analyze the samples according to the chromatographic conditions in Part 1 of Example 2, record the peak areas of α-linolenic acid and linoleic acid, and calculate their average contents. The results are shown in Table 17. The maximum value of the total content of α-linolenic acid and linoleic acid is 0.331%, and the minimum value is 0.199%. There is no regular difference in the content of medicinal materials. According to the measured data of 15 batches, the average value is reduced by 20%, and it is recommended that the lower limit of the total content of α-linolenic acid and linoleic acid in box fruit vine be not less than 0.20% (the average value is 0.267%).

[0215] Table 17 Contents of α-linolenic acid and linoleic acid in 15 batches of box fruit vines (%)

[0216]

Claims

1. A method for detecting the quality of a medicinal material of Psoralea corylifolia, characterized in that: It includes the following method a and / or method b: Method a comprises the following steps: Adopt thin layer chromatography method, use developing agent, and develop the box fruit vine test sample solution on the thin layer plate; In terms of volume percentage, the developing agent includes 80-90% halogenated hydrocarbon solvent, 5-15% ester solvent and 3-8% alcohol solvent; The halogenated hydrocarbon solvent is selected from one or both of dichloromethane and chloroform; The ester solvent is selected from one or both of ethyl acetate and ethyl formate; The alcohol solvent is selected from one or both of methanol and ethanol; Method b comprises the following steps: The high performance liquid chromatography method was adopted, and the mobile phase was used to gradiently elute the box fruit vine test sample solution on the chromatographic column; The filler of the chromatographic column is octadecylsilane bonded silica gel; The mobile phases are mobile phase A and mobile phase B; The mobile phase A is a phosphoric acid aqueous solution, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.01-0.5%, and the mobile phase B is acetonitrile; The gradient elution sequentially undergoes a first-stage gradient elution, a second-stage gradient elution, and a third-stage gradient elution; The duration of the first stage gradient elution is 10-20 min. In the mobile phase, the volume percentage of mobile phase A changes from (15-25)% to (5-15)%; the volume percentage of mobile phase B changes from (75-85)% to (85-95)%; The duration of the second stage gradient elution is 0.5-7 minutes. In the mobile phase, the volume percentage of mobile phase A changes from (5-15)% to (0-10)%; the volume percentage of mobile phase B changes from (85-95)% to (90-100)%; The duration of the third stage gradient elution is 1-10 minutes. In the mobile phase, the volume percentage of mobile phase A is maintained at (0-10)%; and the volume percentage of mobile phase B is maintained at (90-100)%.

2. The quality detection method of the medicinal material of box fruit vine as claimed in claim 1, characterized in that: Method a satisfies one or more of the following conditions: (1) In the developing agent, the halogenated hydrocarbon solvent is dichloromethane or chloroform; (2) In the developing agent, the volume percentage of the halogenated hydrocarbon solvent is 80-85%, for example, 83% or 85%; (3) In the developing agent, the ester solvent is ethyl acetate; (4) In the developing agent, the volume percentage of the ester solvent is 6-10%, for example 8% or 8.5%; (5) In the developing agent, the alcohol solvent is methanol; (6) In the developing agent, the volume percentage of the alcohol solvent is 5-8%, for example, 6.5% or 7%; (7) The developing agent further comprises an acid solvent; the acid solvent is selected from one or both of formic acid and acetic acid, and the volume percentage of the acid solvent is 0.5-3%; for example, the acid solvent is formic acid; the volume percentage of the acid solvent is preferably 2%; (8) The test solution of the Psoralea corylifolia is a methanol solution of the Psoralea corylifolia extract; (9) The sample spotting volume of the thin layer chromatography method is 1 to 5 μL, for example, 2 μL; (10) The thin layer chromatography method is a colorimetric method; the colorimetric agent of the colorimetric method is preferably a sulfuric acid ethanol solution, such as a 10% sulfuric acid ethanol solution; (11) The development temperature is -4 to 35°C, for example 25°C; (12) The unfolding humidity is 15% to 70%, for example, 20%, 42% or 63%; (13) The method a further comprises the step of developing the reference solution of compound P on the thin layer plate using a developing agent; ； (14) The method a also includes the step of developing the control medicinal material solution on the thin layer plate using a developing agent.

3. The quality detection method of the medicinal material of box fruit vine as claimed in claim 2, characterized in that: The method a satisfies one or more of the following conditions: (1) The developing agent is composed of the halogenated hydrocarbon solvent, the ester solvent and the alcohol solvent; or is composed of the halogenated hydrocarbon solvent, the ester solvent, the alcohol solvent and the acid solvent; Preferably, the developing solvent consists of dichloromethane, ethyl acetate and methanol; or consists of chloroform, ethyl acetate, methanol and formic acid; For example, the volume ratio of dichloromethane:ethyl acetate:methanol is 5:0.5:0.4; or the volume ratio of chloroform:ethyl acetate:methanol:formic acid is 5:0.5:0.4:0.1; (2) The reference solution of compound P is a methanol solution of compound P; in the reference solution of compound P, the mass volume ratio of compound P to methanol is (0.1-2) mg / ml, for example, 1 mg / ml; (3) The control medicinal material is a Herba Lycopodii fructus control medicinal material, produced in Zhaoqing, Guangdong; the control medicinal material solution is preferably a methanol solution of the control medicinal material; in the control medicinal material solution, the mass volume ratio of the control medicinal material to methanol is (0.01~2) g / ml.

4. The quality detection method of the medicinal material of box fruit vine as claimed in claim 2, characterized in that: In the method a, the box fruit vine extract is prepared by the following method: Mixing and extracting the box fruit vine with a solvent to obtain the box fruit vine extract; The solvent is selected from dichloromethane, methanol, an aqueous solution of an alcohol solvent, or a mixed solution consisting of petroleum ether and dichloromethane; In the mixed solution composed of petroleum ether and dichloromethane, the volume ratio of petroleum ether to dichloromethane is 1: (0.5-2); The alcohol solvent in the alcohol solvent aqueous solution is methanol or ethanol; the volume percentage of the alcohol solvent in the alcohol solvent aqueous solution is 40-98%.

5. The quality detection method of the medicinal material of box fruit vine as claimed in claim 4, characterized in that: The method a satisfies one or more of the following conditions: (1) The medicinal material of the Herba Lycopodii is the root or root bark of the Herba Lycopodii; (2) In the mixed solution of petroleum ether and dichloromethane, the volume ratio of petroleum ether to dichloromethane is 1:1; (3) The alcohol solvent in the alcohol solvent aqueous solution is methanol, preferably, in the alcohol solvent aqueous solution, the volume percentage of methanol is 40-60%, such as 50%; or the alcohol solvent in the alcohol solvent aqueous solution is ethanol, preferably, in the alcohol solvent aqueous solution, the volume percentage of ethanol is 80-98%, such as 95%; (4) The solvent is dichloromethane; (5) The mass volume ratio of the box fruit vine to the solvent is (0.01-2) g / ml; for example, 0.04 g / ml; (6) The extraction method is ultrasonic extraction; (7) The extraction time is 15 to 45 minutes, for example, 30 minutes; (8) The extraction is performed once, twice or three times, preferably three times; (9) After the extraction, the process also includes filtration and / or concentration.

6. The quality detection method of the medicinal material of box fruit vine as claimed in claim 5, characterized in that: In the method a, the extraction is performed three times, the solvent used in the first extraction is petroleum ether; the solvent used in the second extraction is a mixed solvent of petroleum ether and dichloromethane in a volume ratio of 1:1; and the solvent used in the third extraction is dichloromethane.

7. The quality detection method of the medicinal material of box fruit vine as claimed in claim 1, characterized in that: The method b satisfies one or more of the following conditions: (1) The test solution of the Herba Lycopodii is a methanol solution of the Herba Lycopodii extract; (2) The volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.05-0.2%, for example, 0.1%; (3) The duration of the first stage gradient elution is 12 to 18 minutes, for example, 15 minutes; (4) The duration of the second stage gradient elution is 0.5 to 4 min, for example, 1 min; (5) The duration of the third stage gradient elution is 2 to 8 minutes, for example, 4 minutes; (6) In the first stage of gradient elution, the volume percentage of mobile phase A in the mobile phase changes from (15-20)% to (5-10)%; the volume percentage of mobile phase B changes from (80-85)% to (90-95)%; for example, the volume percentage of mobile phase A changes from 20% to 10%; the volume percentage of mobile phase B changes from 80% to 90%; (7) In the second stage of gradient elution, the volume percentage of mobile phase A in the mobile phase changes from (5-10)% to (0-5)%; the volume percentage of mobile phase B changes from (90-95)% to (95-100)%; for example, the volume percentage of mobile phase A changes from 10% to 5%; the volume percentage of mobile phase B changes from 90% to 95%; (8) In the third stage of gradient elution, in the mobile phase, the volume percentage of mobile phase A is maintained at (0-5)%; and the volume percentage of mobile phase B is maintained at (95-100)%; for example, the volume percentage of mobile phase A is maintained at 5%; and the volume percentage of mobile phase B is maintained at 95%; (9) The column temperature of the chromatographic column is 25 to 35 °C, for example, 30 °C; (10) In the high performance liquid chromatography, the detection wavelength is 180-250 nm, for example, 203 nm; (11) In the high performance liquid chromatography, the injection volume is 15-25 μl, for example, 20 μl; (12) The flow rate of the mobile phase is 0.5 to 2 ml / min, preferably 0.8 to 1.2 ml / min, for example 1.0 ml / min; (13) The method b further comprises the step of detecting an α-linolenic acid and / or linoleic acid reference solution by high performance liquid chromatography; the α-linolenic acid and / or linoleic acid reference solution is a methanol solution of α-linolenic acid and / or linoleic acid; in the α-linolenic acid and / or linoleic acid reference solution, the mass volume ratio of the α-linolenic acid and / or linoleic acid to the methanol solution is (0.2-3) mg / ml, for example, 0.393 mg / ml or 1 mg / ml.

8. The quality detection method of the medicinal material of box fruit vine as claimed in claim 1, characterized in that: The method b satisfies one or both of the following conditions: (1) The gradient elution is: ; (2) The specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and filler particle size 5 µm, such as Agilent ZORBAXSB-C 18 (250 mm * 4.6 mm, 5 μm), Agilent Eclipse XDB-C 18 (250 mm * 4.6 mm, 5 μm) or SHISEIDO CAPCEll PAK C 18 (250 mm * 4.6 mm, 5 μm).

9. The quality detection method of the medicinal material of box fruit vine as claimed in claim 7, characterized in that: In the method b, the preparation method of the Psoralea corylifolia extract comprises the following steps: mixing and extracting the Psoralea corylifolia and an alcohol solvent to obtain the Psoralea corylifolia extract.

10. The quality detection method of the medicinal material of box fruit vine as claimed in claim 9, characterized in that: In the method b, the preparation method of the extract of Psoralea corylifolia also satisfies one or more of the following conditions: (1) The alcohol solvent is methanol; (2) The mass volume ratio of the box fruit vine to the alcohol solvent is (0.005-0.02) g / ml, for example, 0.01 g / ml; (3) The extraction is ultrasonic extraction, the power of the ultrasound is 100-400 W, for example, 250 W; the frequency of the ultrasound is 20-50 kHz, for example, 40 kHz; (4) The extraction time is 20 to 60 minutes, for example 40 minutes; (5) After the extraction, filtering is also included, and the filtration is preferably carried out using a 0.2-0.5 µm filter membrane, such as a 0.45 µm filter membrane; (6) The box fruit vine is the root or root bark of the box fruit vine.

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