Method for detecting contents of plumbagin and isovitexin in plumbago zeylanica
The detection of pterostilbene and isovitexin by liquid chromatography fills the gap in the detection of isovitexin in existing technologies, achieving efficient and accurate quality control of medicinal materials and ensuring the stability of medicinal material quality.
Patent Information
- Application Number
- CN202511286094.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-10
- Publication Date
- 2025-11-07
AI Technical Summary
Existing technologies lack a simple, easy-to-use, and highly accurate method for detecting the content of isovitexin in Plumbago salsa, making it impossible to effectively control the quality of the medicinal material.
The contents of plumbagoside and isovitexin were determined by liquid chromatography. The samples were dissolved in liquid chromatography, and the samples were extracted by ultrasonic extraction. After filtration, the samples were analyzed by liquid chromatography, and the contents were calculated based on the standard curve.
It achieves efficient and accurate detection of isovitexin content, has strong applicability, can comprehensively reflect the quality of medicinal materials, ensure their stability, and fill the gap in the quantitative control of isovitexin in medicinal materials.
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Figure CN120908362A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of drug detection, and particularly relates to a detection method for contents of leucodin and isofraxidin in leucodin. BACKGROUND
[0002] The dried roots, stems, leaves or whole plants of leucodin can be used as medicine. According to experimental research, the leucodin contains leucodin, isofraxidin, vanillic acid, zanthoxylin, americammi, leucodinone, trans-cinnamic acid, softwood ammi, thujaplicin, neoechinulin A, matsumtaquinone, 3-indole formaldehyde, mappianone, baizhuhuakone, 1,4-androstadiene-3,17-dione, 2,5-dimethyl-7-hydroxychromone and other components. The leucodin has the properties of pungent, bitter, astringent and warm, and is toxic. The leucodin has the effects of dissipating blood stasis, swelling, dispelling wind and relieving pain, and is used for treating rheumatoid arthritis, neuralgia, external injury and bleeding. The isofraxidin has the biological activities of anti-inflammatory, anticancer, antibacterial and antiviral, and is widely used in the research of tumor treatment. However, the existing literatures mainly record the quality control of leucodin, vanillic acid and other components, and rarely record the isofraxidin. Therefore, it is particularly important to develop a detection method for the quality control of isofraxidin, which is simple, accurate and reproducible. SUMMARY
[0003] The present application provides a detection method for the contents of leucodin and isofraxidin in leucodin, which is efficient, accurate, suitable and meets the requirements of method validation, and can be used for the quality control of the medicinal material. In addition, the detection method fills the blank of the quantitative control of the active component isofraxidin in leucodin, and can more comprehensively reflect the quality of the medicinal material and ensure the quality stability.
[0004] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:
[0005] The present application provides a detection method for the contents of leucodin and isofraxidin in leucodin, which includes the following steps:
[0006] (1) The leucodin standard and the isofraxidin standard are respectively dissolved in methanol to obtain a leucodin standard solution and an isofraxidin standard solution;
[0007] (2) The sample to be detected is crushed and sieved, and then is ultrasonically extracted with methanol to obtain an extraction solution after filtration;
[0008] (3) The leucodin standard solution and the isofraxidin standard solution are respectively detected by liquid chromatography to obtain a standard curve of leucodin and a standard curve of isofraxidin;
[0009] (4) detecting the extract obtained in step (2) by liquid chromatography, and obtaining the content of leucocyanidin and isofraxidin in the sample according to the standard curve of leucocyanidin and the standard curve of isofraxidin obtained in step (3).
[0010] Preferably, the content of leucocyanidin standard in the leucocyanidin standard solution in step (1) is 0.21-0.23 mg / mL, and the content of isofraxidin standard in the isofraxidin standard solution is 0.12-0.13 mg / mL.
[0011] Preferably, the mesh size of the sieve used in step (2) is 24-65 mesh.
[0012] Preferably, the ratio of the sample to be tested to methanol used in step (2) is 1 g:45-55 mL.
[0013] Preferably, the power of the ultrasonic wave in step (2) is 230-270 W, the frequency of the ultrasonic wave is 40-60 kHz, and the time of ultrasonic extraction is 50-70 min.
[0014] Preferably, the injection volume of the leucocyanidin standard solution and the isofraxidin standard solution in step (3) is independently 8-12 μL.
[0015] Preferably, the liquid chromatography in step (3) and step (4) uses a HALO 90A C18 (4.6 mm x 250 mm, 5 μm) column with octadecylsilane-bonded silica gel as the filler; acetonitrile is used as the mobile phase A, and 0.08-0.12% formic acid solution is used as the mobile phase B; the flow rate is 0.8-1.2 mL / min; the column temperature is 38-32°C, and the detection wavelength is 260-280 nm.
[0016] Preferably, the mobile phase A and the mobile phase B are eluted according to the following conditions:
[0017] 0-8 min: the proportion of the mobile phase A is uniformly increased from 12% to 15%, and the proportion of the mobile phase B is uniformly decreased from 88% to 85%;
[0018] 8-12 min: the proportion of the mobile phase A is uniformly increased from 15% to 20%, and the proportion of the mobile phase B is uniformly decreased from 85% to 80%;
[0019] 12-20 min: the proportion of the mobile phase A is 20%, and the proportion of the mobile phase B is 80%;
[0020] 20-25 min: the proportion of the mobile phase A is uniformly increased from 20% to 30%, and the proportion of the mobile phase B is uniformly decreased from 80% to 70%;
[0021] 25-30 min: the proportion of mobile phase A is uniformly increased from 30% to 50%, and the proportion of mobile phase B is uniformly decreased from 70% to 50%;
[0022] 30-35 min: the proportion of mobile phase A is uniformly increased from 50% to 68%, and the proportion of mobile phase B is uniformly decreased from 50% to 32%;
[0023] 35-40 min: the proportion of mobile phase A is uniformly increased from 68% to 80%, and the proportion of mobile phase B is uniformly decreased from 32% to 20%;
[0024] 40-60 min: the proportion of mobile phase A is 80%, and the proportion of mobile phase B is 20%.
[0025] Preferably, the injection amount of the extract solution in step (4) is 8-12 μL.
[0026] The application provides a detection method for the contents of plumbagin and isohamnetin in plumbago zeylanica, comprising the following steps: (1) dissolving plumbagin standard and isohamnetin standard in methanol to obtain plumbagin standard solution and isohamnetin standard solution; (2) crushing and sieving the sample to be detected, and then performing ultrasonic extraction with methanol, and filtering to obtain an extract solution; (3) performing liquid chromatography detection on the plumbagin standard solution and the isohamnetin standard solution respectively to obtain a standard curve of plumbagin and a standard curve of isohamnetin; and (4) performing liquid chromatography detection on the extract solution obtained in step (2), and obtaining the contents of plumbagin and isohamnetin in the sample according to the standard curve of plumbagin and the standard curve of isohamnetin obtained in step (3). The detection method meets the requirements of method validation, and the two components of plumbagin and isohamnetin can both be eluted within 35 min, the detection result is efficient and accurate, has strong applicability, and can be used for quality control of the medicinal material. In addition, the detection method fills the blank of quantitative control of the active component isohamnetin in plumbago zeylanica, can more comprehensively reflect the quality status of the medicinal material, and ensures the quality stability. BRIEF DESCRIPTION OF DRAWINGS
[0027] Fig. 1 Investigation with 0.1% formic acid-methanol mobile phase;
[0028] Fig. 2 Investigation with 0.1% formic acid-acetonitrile mobile phase;
[0029] Fig. 3 Investigation with 0.1% phosphoric acid-acetonitrile mobile phase;
[0030] Fig. 4 Investigation with elution gradient 1;
[0031] Fig. 5For elution gradient 2;
[0032] Fig. 6 For 250 nm detection wavelength;
[0033] Fig. 7 For 270 nm detection wavelength;
[0034] Fig. 8 For 320 nm detection wavelength;
[0035] Fig. 9 For plumbagin standard curve;
[0036] Fig. 10 For isovillose standard curve;
[0037] Fig. 11 For chromatogram of sample No. 2. DETAILED DESCRIPTION
[0038] The present application provides a detection method for the content of plumbagin and isovillose in plumbago, comprising the following steps:
[0039] (1) Dissolve plumbagin standard and isovillose standard in methanol respectively to obtain plumbagin standard solution and isovillose standard solution;
[0040] (2) Crush and sieve the sample to be detected, and then ultrasonically extract with methanol to obtain an extract after filtration;
[0041] (3) Detect the plumbagin standard solution and isovillose standard solution by liquid chromatography to obtain the standard curve of plumbagin and the standard curve of isovillose;
[0042] (4) Detect the extract obtained in step (2) by liquid chromatography, and obtain the content of plumbagin and isovillose in the sample according to the standard curve of plumbagin and the standard curve of isovillose obtained in step (3).
[0043] In the present application, the content of plumbagin standard in the plumbagin standard solution in step (1) is preferably 0.21-0.23 mg / mL, and further preferably 0.2226 mg / mL, and the content of isovillose standard in the isovillose standard solution is preferably 0.12-0.13 mg / mL, and further preferably 0.1254 mg / mL.
[0044] In the present application, the mesh size of the sieve used in step (2) is preferably 24-65 mesh, and further preferably 50 mesh.
[0045] In the present application, the ratio of the sample to be tested and methanol in step (2) is preferably 1 g: 45-55 mL, and more preferably 1 g: 50 mL.
[0046] In the present application, the power of the ultrasound in step (2) is preferably 230-270 W, and more preferably 250 W, the frequency of the ultrasound is preferably 40-60 kHz, and more preferably 50 kHz, and the time of the ultrasound extraction is preferably 50-70 min, and more preferably 60 min.
[0047] In the present application, the lost weight is preferably made up with methanol before filtration in step (2).
[0048] In the present application, the injection volume of the leucocyanidol standard solution and the isovitexin standard solution is independently preferably 8-12 μL, and more preferably 10 μL, when the liquid chromatography detection in step (3) is performed.
[0049] In the present application, the liquid chromatography detection in step (3) and step (4) is performed using a HALO 90A C18 (4.6 mm x 250 mm, 5 μm) column, preferably with octadecylsilane-bonded silica gel as the filler; preferably with acetonitrile as the mobile phase A and 0.08-0.12% formic acid solution as the mobile phase B, and more preferably with 0.1% formic acid solution as the mobile phase B; the flow rate is preferably 0.8-1.2 mL / min, and more preferably 1 mL / min; the column temperature is preferably 38-32°C, and more preferably 30°C, and the detection wavelength is preferably 260-280 nm, and more preferably 370 nm.
[0050] In the present application, the mobile phase A and the mobile phase B are preferably eluted according to the following conditions when the determination is performed:
[0051] 0-8 min: the proportion of the mobile phase A is uniformly increased from 12% to 15%, and the proportion of the mobile phase B is uniformly decreased from 88% to 85%;
[0052] 8-12 min: the proportion of the mobile phase A is uniformly increased from 15% to 20%, and the proportion of the mobile phase B is uniformly decreased from 85% to 80%;
[0053] 12-20 min: the proportion of the mobile phase A is 20%, and the proportion of the mobile phase B is 80%;
[0054] 20-25 min: the proportion of the mobile phase A is uniformly increased from 20% to 30%, and the proportion of the mobile phase B is uniformly decreased from 80% to 70%;
[0055] 25-30 min: the proportion of the mobile phase A is uniformly increased from 30% to 50%, and the proportion of the mobile phase B is uniformly decreased from 70% to 50%;
[0056] 30-35 min: the proportion of mobile phase A was uniformly increased from 50% to 68%, and the proportion of mobile phase B was uniformly decreased from 50% to 32%;
[0057] 35-40 min: the proportion of mobile phase A was uniformly increased from 68% to 80%, and the proportion of mobile phase B was uniformly decreased from 32% to 20%;
[0058] 40-60 min: the proportion of mobile phase A was 80%, and the proportion of mobile phase B was 20%.
[0059] In the present application, the injection amount of the extract solution in step (4) is preferably 8-12 μL, and further preferably 10 μL.
[0060] The technical solutions provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.
[0061] Instruments and reagents:
[0062] 1. The instruments used are shown in Table 1.
[0063] Table 1 Details of experimental instruments
[0064]
[0065]
[0066] 2. Reagents:
[0067] Formic acid, analytical pure (purchased from China National Pharmaceutical Group Chemical Reagent Co., Ltd.); phosphoric acid, analytical pure (purchased from Chongqing Chuandong Chemical Group Co., Ltd.); acetonitrile and methanol are both analytical pure; water is ultrapure water.
[0068] 3. Reference substance
[0069] Plumbagin (batch number: CFS202301, purity: ≥98%), purchased from Wuhan Tianzhi Biological Technology Co., Ltd.; Isofraxidin (batch number: 112098-202201, purity ≥100%), purchased from China Institute for Drug Control. The information of Plumbago zeylanica L. medicinal materials is shown in Table 2.
[0070] Table 2 Information of Plumbago zeylanica L. sample collection
[0071] No. Name Collection site Collection date 1 Plumbago zeylanica L. Hualan Township, Shangsi County, Fangchenggang City, Guangxi 2023 / 7 / 21 4 Plumbago zeylanica L. Sanying Village, Gongzheng Township, Shangsi County, Fangchenggang City, Guangxi 2023 / 9 / 11
[0072] Example 1
[0073] 1. Chromatographic conditions
[0074] The chromatographic column was HALO 90AC18 (4.6 mm x 250 mm, 5 μm); the mobile phase A was acetonitrile, the mobile phase B was 0.1% formic acid solution; the flow rate was 1 mL / min; the column temperature was 30°C; the injection volume was 10 μL; and the detection wavelength was 270 nm. Gradient elution was performed according to Table 3 below.
[0075] Table 3 Gradient elution sequence table
[0076]
[0077]
[0078] 2. Preparation of the reference solution
[0079] Precisely weigh 11.356 mg of plumbagin reference substance and 6.271 mg of isovitexin reference substance, respectively, into a 50 mL volumetric flask, add methanol to dissolve and dilute to the mark, shake well, to obtain the plumbagin reference solution with a concentration of 0.2226 mg / mL and the isovitexin reference solution with a concentration of 0.1254 mg / mL, respectively.
[0080] 3. Preparation of the test solution
[0081] Precisely weigh 0.5 g of plumbagin powder, add 50 mL of methanol, and treat under ultrasonic condition at 250 w and 50 kHz for 1 h, make up the weight loss with methanol, and filter, to obtain the test solution.
[0082] Investigation of the preparation of the test solution
[0083] 1. Investigation of the pretreatment method
[0084] Two different pretreatment methods were investigated, namely ultrasonic treatment and extraction after ultrasonic treatment.
[0085] Ultrasonic treatment: precisely weigh 1 g of the plumbagin sample powder No. 1 in Table 2, add 50 mL of methanol, treat under ultrasonic condition at 250 w and 50 kHz for 1 h, make up the weight loss with methanol, and filter, to obtain the test solution;
[0086] Extraction after ultrasonic treatment: precisely weigh 1 g of the plumbagin sample powder No. 1 in Table 2, add 50 mL of methanol, treat under ultrasonic condition at 250 w and 50 kHz for 1 h, make up the weight loss with methanol, filter, evaporate the filtrate to dryness, dissolve the residue in 20 mL of purified water, extract twice with 20 mL of ethyl acetate, combine the extract, evaporate to dryness, dissolve in methanol, transfer to a 5 mL volumetric flask, dilute to the mark, and filter, to obtain the test solution.
[0087] As shown by the results in Table 4 below, the pretreatment method of extraction after ultrasonic treatment results in the absence of the plumbagin peak, and the content of isovitexin component is lower than that of the ultrasonic treatment method, so the ultrasonic treatment method is selected as the pretreatment method.
[0088] Table 4 Pretreatment Investigation
[0089]
[0090] Note: "-" indicates that the peak of plumbagin was not found under this treatment.
[0091] 2. Extraction solvent investigation
[0092] Three extraction solvents were investigated, namely methanol, 50% methanol and water. 1 g of sample No. 2 in Table 2 was added with 50 mL of methanol, 50 mL of 50% methanol and 50 mL of water respectively, and then ultrasonically treated for 1 h under the condition of 250 w and 50 kHz. The lost weight was made up with methanol, and then filtered.
[0093] As shown in Table 5, when water was used as the extraction solvent, the contents of the two components to be tested were much lower than those of the other two extraction solvents. Compared with 50% methanol, the content of plumbagin was higher when methanol was used as the extraction solvent, while the content of isoflavanone did not differ much. Therefore, methanol was selected as the extraction solvent.
[0094] Table 5 Extraction solvent investigation
[0095]
[0096] 3. Ultrasonic time investigation
[0097] Different ultrasonic times were investigated, namely 0.5 h, 1 h and 1.5 h. 1 g of sample powder No. 2 in Table 2 was added with 50 mL of methanol, and then ultrasonically treated under the condition of 250 w and 50 kHz. The lost weight was made up with methanol, and then filtered.
[0098] As shown in Table 6, when the ultrasonic time was 0.5 h, the contents of the two components to be tested were both small. When the ultrasonic time was 1 h and 1.5 h, the contents of the two components did not differ much. Therefore, the ultrasonic extraction time was determined to be 1 h for fully extracting the two components in the sample and saving the sample preparation time.
[0099] Table 6 Ultrasonic time investigation
[0100]
[0101] 4. Sample amount investigation
[0102] Different sample amounts were investigated, namely 0.25 g, 0.5 g and 0.75 g. 1 g of sample powder No. 1 in Table 2 was added with 50 mL of methanol, and then ultrasonically treated under the condition of 250 w and 50 kHz. The lost weight was made up with methanol, and then filtered.
[0103] From Table 7, the contents of the two components are not much different under three different sampling amounts, and the sampling amount of 0.5 g is selected.
[0104] Table 7: Sampling amount investigation
[0105]
[0106] Example 3: Investigation of chromatographic conditions
[0107] 1. Investigation of mobile phase
[0108] Two different aqueous phases: 0.1% formic acid, 0.1% phosphoric acid; two different organic phases: methanol, acetonitrile are investigated respectively. Fig. 1 and Fig. 2 It can be known from the comparison that the peak response value and peak shape of acetonitrile are better than those of methanol; through Fig. 2 and Fig. 3 It can be known from the comparison that the peak shape and separation degree of 0.1% formic acid are slightly better than those of 0.1% phosphoric acid. Therefore, the aqueous phase is finally selected as 0.1% formic acid, and the organic phase is selected as acetonitrile.
[0109] 2. Investigation of elution gradient
[0110] With acetonitrile as mobile phase A and 0.1% formic acid as mobile phase B, two different gradients of mobile phase are investigated respectively (Table 8 and Table 9).
[0111] Elution gradient 1: acetonitrile as mobile phase A and 0.1% formic acid as mobile phase B, and the time program is shown in Table 8.
[0112] Table 8
[0113]
[0114]
[0115] Elution gradient 2: acetonitrile as mobile phase A and 0.1% formic acid as mobile phase B, and the time program is shown in Table 9.
[0116] Table 9
[0117] Time / min A / ％ B / ％ 0 12 88 8 15 85 12 20 80 20 20 80 25 30 70 30 50 50 35 68 32 40 80 20 60 80 20
[0118] It can be known from Fig. 4 , Fig. 5 that the elution gradient 1 has a long peak time and poor peak shape. The peak shape of each peak in the elution gradient 2 is good, and the separation degree and retention time are moderate, so the elution gradient 2 is finally selected.
[0119] 4. Investigation of detection wavelength
[0120] Three detection wavelengths 250 nm, 270 nm and 320 nm are investigated. From Fig. 6 ,Fig. 7 、 Fig. 8 It is known that isomiltirin and plumbagin have maximum absorption at 250 nm and 270 nm. In order to avoid interference of other impurities in the sample, 270 nm is finally selected as the detection wavelength.
[0121] Methodology investigation of Example 4
[0122] 1. Investigation of linear relationship
[0123] The plumbagin and isomiltirin standard solution (plumbagin: 0.2226 mg / mL; isomiltirin: 0.1254 mg / mL) is diluted 5 times, 10 times, 25 times, 50 times and 150 times, respectively, and then 5 μL and 10 μL of each injection solution is injected into the liquid chromatograph. The dilution and injection volume are shown in Table 10. The standard curve is plotted with the peak area (y) as the ordinate and the injection concentration (x) as the abscissa. The regression equation R2 is greater than 0.999, indicating that the plumbagin concentration in the range of 0.74 μg / mL to 111.29 μg / mL and the isomiltirin concentration in the range of 0.42 μg / mL to 62.71 μg / mL both present good linear relationship. The results are shown in Table 10. Fig. 9 、 Fig. 10 .
[0124] Table 10. Investigation of linear relationship
[0125]
[0126] 2. Investigation of precision
[0127] 10 μL of the plumbagin test sample solution is precisely taken and injected into the liquid chromatograph, and the chromatographic conditions in Table 3 are continuously injected for 6 times. The results are calculated according to the peak area of plumbagin and isomiltirin, and the RSD is 0.56% and 0.57%, respectively, indicating that the instrument precision is good. The results are shown in Table 11 and Table 12.
[0128] Table 11. Investigation results of precision-plumbagin (n=6)
[0129]
[0130]
[0131] Table 12. Investigation results of precision-isomiltirin (n=6)
[0132]
[0133] 3. Investigation of stability
[0134] Accurately pipette 10 μL of the test solution of V. agnus-castus, inject into the liquid chromatograph, and determine at 0 h, 4 h, 8 h, 12 h, 16 h, 18 h, and 24 h according to the chromatographic conditions in Table 3. The results are calculated based on the peak area of agnoside and isovitexin. The RSDs are 0.92% and 0.89%, respectively, indicating that the test solution has good stability, and the determination results are accurate and reliable within 24 h. The results are shown in Tables 13 and 14.
[0135] Table 13 Stability Investigation Results - Agnoside
[0136]
[0137] Table 14 Stability Investigation Results - Isovitexin
[0138]
[0139] 4. Reproducibility Investigation
[0140] Accurately pipette 6 portions of the same batch of V. agnus-castus powder, and prepare test solutions according to the method in Step 3 of Example 1. Inject and determine the peak area according to the chromatographic conditions in Table 3, and calculate the content and RSD of agnoside and isovitexin in the sample. The average content of agnoside and isovitexin in the batch sample is 0.4432 mg / g and 0.2497 mg / g, respectively, and the RSD is 1.62% and 2.22%, respectively, indicating that the method has good reproducibility. The results are shown in Tables 15 and 16.
[0141] Table 14 Reproducibility Investigation Results - Agnoside (n = 6)
[0142]
[0143] Table 16 Reproducibility Investigation Results - Isovitexin (n = 6)
[0144]
[0145]
[0146] 5. Sample Spiking Recovery Rate Investigation
[0147] Precisely weigh the same batch of known content of white flower Dan powder sample (white flower Danin: 0.4482 mg / g, isoflavone: 0.2498 mg / g), a total of 9, respectively, precisely add the appropriate volume of white flower Danin control solution with a concentration of 0.2226 mg / mL and isoflavone control solution with a concentration of 0.1254 mg / mL, prepare 9 test sample solutions according to the method of step 3 of example 1, determine the content of white flower Danin and isoflavone according to the chromatographic conditions of table 3, and calculate the recovery. The average recovery of white flower Danin is 103.43%, and the RSD is 1.68%; the average recovery of isoflavone is 103.66%, and the RSD is 1.97%, indicating that the determination method is accurate and feasible. The results are shown in tables 17 and 18.
[0148] Table 17 recovery rate of sample addition- white flower Danin (n = 9)
[0149]
[0150] Table 18 recovery rate of sample addition- isoflavone (n = 9)
[0151]
[0152]
[0153] 6. Durability investigation
[0154] The influence of different chromatographic columns on the determination method was investigated, and 90A column, Acclaim column, Kromail column and MGII column were used for test. The RSD of white flower Danin and isoflavone content determination results were 3.89% and 3.51% respectively, indicating that different chromatographic columns had little effect on the determination method. The results are shown in tables 19 and 20.
[0155] Table 19 durability investigation results- white flower Danin
[0156]
[0157] Table 20 durability investigation results- isoflavone
[0158]
[0159] According to the above methodological investigation results of the method, the designed method for detecting the content of white flower Danin and isoflavone in white flower Dan medicinal materials is effective and feasible.
[0160] Example 5 content determination results of white flower Dan
[0161] Take all batches of white flower Dan powder, according to the method of example 1 step 3, prepare test sample solution, two batches respectively, respectively, accurately suck white flower Dan element control solution, isoflavone control solution and test sample solution 10 μL, inject into the chromatograph, according to the chromatographic conditions of table 3, determine the peak area of control sample, and calculate the content of white flower Dan element and isoflavone in each batch of white flower Dan sample, the results are shown in table 21. The content of white flower Dan element in 24 batches of white flower Dan sample is in the range of 0.0745-2.0571 mg / g, and the content of isoflavone is in the range of 0.1366-7.5866 mg / g. By calculation, the 95% confidence interval of the content of white flower Dan element in the sample is 0.3108-0.7207 mg / g, and the 95% confidence interval of the content of isoflavone is 0.9598-2.2279 mg / g.
[0162] Table 21 white flower Dan sample content determination results
[0163]
[0164]
[0165] From the above examples, the present application provides a detection method for the content of white flower Dan element and isoflavone in white flower Dan, which comprises the following steps: (1) dissolving white flower Dan element standard and isoflavone standard in methanol respectively to obtain white flower Dan element standard solution and isoflavone standard solution; (2) crushing the sample to be tested, ultrasonic extraction with methanol, and filtering to obtain the extract; (3) detecting the white flower Dan element standard solution and isoflavone standard solution by liquid chromatography to obtain the standard curve of white flower Dan element and the standard curve of isoflavone; (4) detecting the extract obtained in step (2) by liquid chromatography, and obtaining the content of white flower Dan element and isoflavone in the sample according to the standard curve of white flower Dan element and the standard curve of isoflavone obtained in step (3). The detection method of the present application meets the requirements of method validation, and both white flower Dan element and isoflavone can be out of peak within 35 min, the detection result is efficient and accurate, has strong applicability, and can be used for quality control of the medicinal material. In addition, the detection method fills the blank of quantitative control of isoflavone, an active ingredient in white flower Dan medicinal material, can more comprehensively reflect the quality status of the medicinal material, and ensure the quality stability.
[0166] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.
Claims
1. A method for detecting the content of oleuropein and isohymenialdispin in olea europaea, characterized by, The method comprises the following steps: (1) dissolving the leucocyanidin standard and the isovitexin standard in methanol respectively to obtain leucocyanidin standard solution and isovitexin standard solution; (2) crushing and sieving the sample to be tested, and then extracting the sample with methanol under ultrasonic wave, and filtering to obtain an extract; (3) detecting the leucocyanidin standard solution and the isovitexin standard solution by liquid chromatography to obtain a standard curve of leucocyanidin and a standard curve of isovitexin; (4) detecting the extract obtained in step (2) by liquid chromatography, and obtaining the content of leucocyanidin and isovitexin in the sample according to the standard curve of leucocyanidin and the standard curve of isovitexin obtained in step (3).
2. The detection method according to claim 1, characterized in that, The content of the leucocyanidin standard in the leucocyanidin standard solution in step (1) is 0.21-0.23 mg / mL, and the content of the isovitexin standard in the isovitexin standard solution is 0.12-0.13 mg / mL.
3. The detection method according to claim 2, characterized in that, The mesh size of the sieve used in step (2) is 24-65.
4. The detection method according to claim 3, characterized in that, The ratio of the sample to be tested to methanol used in step (2) is 1 g:45-55 mL.
5. The detection method according to claim 4, characterized in that, The ultrasonic power in step (2) is 230-270 W, the ultrasonic frequency is 40-60 kHz, and the ultrasonic extraction time is 50-70 min.
6. The detection method according to claim 5, characterized in that, The lost weight is supplemented with methanol before filtering in step (2).
7. The detection method according to claim 6, characterized in that, The injection volume of the leucocyanidin standard solution and the isovitexin standard solution in step (3) is independently 8-12 μL.
8. The detection method according to claim 7, characterized in that, The liquid chromatography in steps (3) and (4) uses a HALO 90A C18 (4.6 mm×250 mm, 5 μm) chromatographic column, octadecylsilane-bonded silica gel as the filler, acetonitrile as the mobile phase A, and 0.08-0.12% formic acid solution as the mobile phase B, the flow rate is 0.8-1.2 mL / min, the column temperature is 38-32 ℃, and the detection wavelength is 260-280 nm.
9. The detection method according to claim 8, characterized in that, The mobile phase A and the mobile phase B are eluted according to the following conditions: 0-8 min: the proportion of the mobile phase A is uniformly increased from 12% to 15%, and the proportion of the mobile phase B is uniformly decreased from 88% to 85%; 8-12 min: the proportion of the mobile phase A is uniformly increased from 15% to 20%, and the proportion of the mobile phase B is uniformly decreased from 85% to 80%; 12-20 min: the proportion of the mobile phase A is 20%, and the proportion of the mobile phase B is 80%; 20-25 min: the proportion of the mobile phase A is uniformly increased from 20% to 30%, and the proportion of the mobile phase B is uniformly decreased from 80% to 70%; 25-30 min: the proportion of the mobile phase A is uniformly increased from 30% to 50%, and the proportion of the mobile phase B is uniformly decreased from 70% to 50%; 30-35 min: the proportion of the mobile phase A is uniformly increased from 50% to 68%, and the proportion of the mobile phase B is uniformly decreased from 50% to 32%; 35-40 min: the proportion of the mobile phase A is uniformly increased from 68% to 80%, and the proportion of the mobile phase B is uniformly decreased from 32% to 20%. 40-60 min: 80% of mobile phase A and 20% of mobile phase B.
10. The detection method according to claim 9, characterized in that, The injection volume of the extract solution in step (4) is 8-12 μL.