Method for determining content of active substances in clerodendrum japonicum based on high performance liquid chromatography

Abstract

The invention relates to the technical field of component detection of clerodendrum japonicum, in particular to a method for determining the content of active substances in clerodendrum japonicum based on a high performance liquid chromatography method. The invention provides a method for determining the content of active substances in clerodendrum japonicum based on high performance liquid chromatography, which comprises the following steps: (1) mixing the clerodendrum japonicum medicinal material with ethanol, and carrying out ultrasonic treatment to obtain a test solution; the method comprises the following steps: respectively taking a chlorogenic acid solution, an apigenin-7-O-diglucuronic acid solution, a verbascoside solution and an acacetin-7-O-[beta-D-glucopyranuronyl (1-> 2)-O-D-glucopyranuronide] solution as reference substance solutions; and (2) performing high performance liquid chromatography detection on the test solution and the reference solution obtained in the step (1). The method can be used for simultaneously detecting the contents of four active components in clerodendrum japonicum, is low in detection limit and wide in linear range, and ensures the accuracy and reliability of a detection result.

Description

Technical Field

[0001] This invention relates to the field of lacquerware component detection technology, and in particular to a method for determining the content of active substances in lacquerware based on high performance liquid chromatography. Background Technology

[0002] Clerodendrum japonicum is a traditional medicinal plant with properties of clearing heat and detoxifying, promoting blood circulation and reducing swelling. It is widely used in folk medicine to treat inflammation, rheumatism, and skin diseases. Modern research shows that its pharmacological activity is closely related to flavonoids, phenylethanoid glycosides, and triterpenoids. Components such as apigenin, luteolin, and acteoside possess antioxidant, anti-inflammatory, and anti-tumor effects. Therefore, establishing accurate and efficient methods for determining the content of active ingredients is of great significance for the quality control and further development and utilization of Clerodendrum japonicum.

[0003] Currently, the determination of active components in *Cornus arvense* mainly employs ultraviolet spectrophotometry or thin-layer chromatography, but these methods suffer from low sensitivity and poor selectivity, making it difficult to achieve simultaneous analysis of multiple components. Furthermore, some studies have used conventional liquid chromatography, but it faces challenges such as insufficient separation efficiency and long analysis times, particularly with poor separation of structurally similar flavonoids and phenylethanoid glycosides. Therefore, developing an efficient and accurate quantitative analytical method to overcome the shortcomings of traditional techniques has become a key research focus.

[0004] High-performance liquid chromatography (HPLC) has become a commonly used technique for the analysis of natural products due to its high resolution, high sensitivity, and good reproducibility. In recent years, by optimizing the chromatographic column, mobile phase, and detection conditions, HPLC can achieve rapid separation and quantification of multiple components in complex matrices. For example, using a C18 reversed-phase column combined with a gradient elution program can effectively separate active components with significant polarity differences in *Clerodendrum trichotomum*; while diode array detectors (DAD) or high-performance liquid chromatography-mass spectrometry (HPLC-MS) can further improve the accuracy and specificity of detection. Therefore, establishing an HPLC-based method for the determination of active components in *Clerodendrum trichotomum* is expected to provide reliable technical support for its quality standardization and pharmacological research. Summary of the Invention

[0005] The purpose of this invention is to provide a method for determining the content of active substances in *Corydalis yanhusuo* based on high performance liquid chromatography.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for determining the content of active substances in *Cortex Cercidiphyllum* based on high performance liquid chromatography, comprising the following steps:

[0008] (1) Mix the medicinal material of *Corydalis yanhusuo* with ethanol and sonicate to obtain the test solution;

[0009] Chlorogenic acid solution, apigenin-7-O-diglucuronic acid solution, verbascoside solution, and farnesin-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] solution were used as reference solutions, respectively.

[0010] (2) The test solution and reference solution obtained in step (1) are subjected to high performance liquid chromatography for detection: acetonitrile is used as mobile phase A and formic acid solution is used as mobile phase B, and elution is performed according to the gradient elution program in Table 1; the flow rate is 0.8 to 1.2 mL / min, the column temperature is 28 to 32℃, and the detection wavelength is 310 to 330 nm.

[0011] Table 1

[0012]

[0013] Preferably, in step (2), the chromatographic column is a Thermo column with a size of 5 μm and 4.6 × 250 mm; and octadecylsilane-bonded silica gel is used as the packing material.

[0014] Preferably, in step (2), the injection volume is 8-12 μL; the volume concentration of the formic acid solution is 0.08-0.12%.

[0015] Preferably, the ratio of the *Pterocarya stenoptera* medicinal material to ethanol in step (1) is 0.5–1.0 g: 50 mL; and the concentration of the ethanol is 45–55%.

[0016] Preferably, the power of the ultrasound in step (1) is 450-550W and the time is 25-35min.

[0017] Preferably, the chlorogenic acid solution, apigenin-7-O-diglucuronic acid solution, verbascoside solution, and acaciain-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] solution in step (1) are all in methanol as solvent.

[0018] Preferably, the concentration of the chlorogenic acid solution in step (1) is 50-60 μg / mL; and the concentration of the apigenin-7-O-diglucuronic acid solution is 350-400 μg / mL.

[0019] Preferably, the concentration of the verbascoside solution in step (1) is 400-430 μg / mL; and the concentration of the farnesin-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] solution is 2.0-2.4 mg / mL.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention can simultaneously detect the content of four active ingredients in tung oil, with a low detection limit and a wide linear range, ensuring the accuracy and reliability of the measurement results.

[0022] This invention uses acetonitrile-0.1% formic acid as the mobile phase and combines it with a gradient elution program to effectively solve the problem of separating active components with large polarity differences in cinnamon. The peaks of each component are symmetrical and highly separated, avoiding the overlapping peak problem commonly found in traditional methods.

[0023] This invention utilizes ultrasonic extraction to prepare the test solution, which significantly shortens the pretreatment time compared to traditional methods such as reflux extraction. It also boasts high extraction efficiency, making it suitable for batch sample analysis. Furthermore, method validation demonstrates good stability of the test solution within 24 hours, facilitating practical application.

[0024] This invention not only overcomes the shortcomings of traditional ultraviolet spectrophotometry and thin-layer chromatography in terms of low sensitivity and poor selectivity, but also significantly improves analytical efficiency and accuracy, providing reliable technical support for the quality standardization, pharmacological research and industrial application of *Corydalis yanhusuo* medicinal materials. Attached Figure Description

[0025] Figure 1 Chromatograms of different absorption wavelengths.

[0026] Figure 2 Chromatograms of different mobile phases.

[0027] Figure 3 Chromatograms for different flow rates.

[0028] Figure 4 Specific chromatograms; 1: chlorogenic acid; 2: apigenin-7-O-diglucuronide; 3: verbascoside; 4: farnesin-7-O-[β-D-glucuronide (1→2)-OD-glucuronide].

[0029] Figure 5 The graph shows the linearity of the four active substances. Detailed Implementation

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] The medicinal material Clerodendrum japonicum (Thunb.) Sweet. was identified by Engineer Huang Qingquan of the Guangxi Zhuang Autonomous Region Institute for Drug Control as the dried aerial part of Clerodendrum japonicum (Thunb.) Sweet., a plant of the Verbenaceae family. Specific sample information is shown in Table 2.

[0032] Table 2 Information on samples of *Corydalis yanhusuo* medicinal materials

[0033]

[0034]

[0035] Note: "-" indicates that there is no collection number.

[0036] Chlorogenic acid purity 96.3%, from the National Institutes for Food and Drug Control; verbascoside purity 95.2%, from the National Institutes for Food and Drug Control; apigenin-7-O-diglucuronide purity 98.5%, from Shanghai Shidander Standards Technical Service Co., Ltd.; acaciain-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] purity 98.5%, from Shanghai Shidander Standards Technical Service Co., Ltd.

[0037] Example 1

[0038] A method for determining the content of active substances in *Cortex rubrum* based on high performance liquid chromatography, comprising the following steps:

[0039] Preparation of the test solution: Take about 0.75g of *Pterocarya stenoptera* powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50mL of 50% ethanol, weigh it, sonicate (500W, 40Kz) for 30 minutes, cool it, weigh it, make up the weight loss with 50% ethanol, shake it well, filter it, and filter it through a 0.45μm filter membrane to obtain the test solution.

[0040] Preparation of reference solution: Weigh an appropriate amount of chlorogenic acid reference standard accurately and add methanol to prepare a solution containing 57.42 μg per 1 mL.

[0041] Take an appropriate amount of apigenin-7-O-diglucuronic acid reference standard, accurately weigh it, and add methanol to prepare a solution containing 374 μg per 1 mL.

[0042] Take an appropriate amount of verbascoside reference standard, accurately weigh it, and add methanol to prepare a solution containing 417 μg per 1 mL.

[0043] Take an appropriate amount of acacia-7-O-[β-D-glucuronylpyranoside (1→2)-OD-glucuronylpyranoside] reference standard, accurately weigh it, and add methanol to prepare a solution containing 2.16 mg per 1 mL.

[0044] Chromatographic conditions were determined as follows: A Thermo column (5 μm, 4.6 × 250 mm) was used, with octadecylsilane-bonded silica gel as the packing material. Acetonitrile was used as mobile phase A, and 0.1% formic acid aqueous solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1. The column temperature was 30 °C; the flow rate was 1.0 mL per minute; the detection wavelength was 320 nm; and the injection volume was 10 μL.

[0045] Example 2

[0046] 1. Optimization of chromatographic conditions (based on the detection method in Example 1, the same applies below)

[0047] (1) Wavelength selection: The chromatograms of *Clerodendrum trichotomum* at different wavelengths were investigated. 320 nm was found to be the wavelength with the largest absorption for the four components requiring content determination, showing good separation and baseline stability. Therefore, 320 nm was ultimately selected as the detection wavelength for content determination. (See below) Figure 1 .

[0048] (2) Investigation of mobile phases: Two different mobile phases, acetonitrile-0.1% glacial acetic acid and acetonitrile-0.1% formic acid, were investigated respectively. See Figure 2 When the mobile phase was acetonitrile-0.1% formic acid, the chromatographic peak shape was better and the resolution was higher. Therefore, acetonitrile-0.1% formic acid was finally selected as the mobile phase.

[0049] (3) Flow rate investigation: Three different flow rates, 0.8 mL / min, 0.9 mL / min, and 1.0 mL / min, were investigated. The results showed that different flow rates had little effect on peak shape. Finally, a flow rate of 1.0 mL / min was selected. See [link to relevant documentation]. Figure 3 .

[0050] 2. Optimization of extraction method

[0051] (1) Selection of extraction solvent: The effect of different extraction solvents on the content of *Corydalis yanhusuo* (No.: *Corydalis yanhusuo-4) was investigated. 0.75 g of the sample was accurately weighed and added to 50 mL of anhydrous methanol, 30% methanol, 50% methanol, anhydrous ethanol, 30% ethanol, and 50% ethanol, respectively. The samples were then sonicated for 30 minutes. The results showed that the contents of the four components were higher when using 50% methanol and 50% ethanol as solvents. However, ethanol has lower toxicity, so 50% ethanol was chosen as the extraction solvent. See Table 3 for details.

[0052] (2) Selection of extraction method: The effect of different extraction methods on the content of *Corydalis yanhusuo* (No.: *Corydalis yanhusuo*-4) was investigated. 0.75 g of sample was accurately weighed and 50 mL of 50% ethanol was added. The sample was treated with ultrasound and reflux for 30 minutes respectively. The results are shown in Table 4. The content of different extraction methods did not differ much. Considering that ultrasound treatment is fast and convenient, ultrasound was finally selected as the extraction method.

[0053] Table 3 Comparison of extraction solvents

[0054]

[0055]

[0056] Note: "-" indicates that the chromatographic peak was not extracted.

[0057] Table 4 Comparison of Extraction Methods

[0058]

[0059] Selection of extraction time: The effect of different ultrasonic times on the extraction content of *Corydalis yanhusuo* (No.: *Corydalis yanhusuo*-4) was investigated. 0.75 g of sample was accurately weighed and 50 mL of 50% ethanol was accurately added. The samples were ultrasonically treated for 30 minutes and 45 minutes, respectively. The results showed that the components to be tested were basically completely extracted after ultrasonic treatment for 30 minutes. See Table 5 for details.

[0060] Table 5 Comparison of extraction times

[0061]

[0062]

[0063] (4) Selection of sampling amount: The effect of different sampling amounts on the extraction of four analytes from *Corydalis yanhusuo* (No.: *Corydalis yanhusuo*-4) was investigated. 0.375 g, 0.75 g, and 1.125 g of the sample were weighed accurately, and 50 mL of 50% ethanol was added. The sample was then sonicated for 30 minutes. The results showed that the extraction of the analytes was basically consistent when the sampling amounts were the above three. After comprehensive consideration, the sampling amount of 0.75 g was selected. See Table 6 for details.

[0064] Table 6 Selection of Sampling Size

[0065]

[0066] Example 3 Methodological Investigation

[0067] 1. Exclusivity

[0068] Chromatis chinensis (sample number: Chromatis-20) was tested according to the method described in Example 1. Chromatogenic acid, apigenin-7-O-diglucuronide, verbascoside, and acaciain-7-O-[β-D-glucuronylpyranoside (1→2)-OD-glucuronylpyranoside] reference standards, Chromatis chinensis test sample, and blank (50% ethanol) chromatograms were plotted. The results are shown in the figure. Figure 4 In the chromatogram of the test sample, chromatographic peaks with retention times consistent with those of the reference standards appeared, and no interference occurred in the blank control. The experiment demonstrates that this method has good specificity.

[0069] 2. Linearity and Range

[0070] Accurately weigh an appropriate amount of chlorogenic acid reference standard, and prepare a solution containing 25.09 μg / mL with methanol. Use this as the stock solution. Accurately pipette 1 mL of the stock solution into a 10 mL volumetric flask, dilute to the mark with methanol, and mix well to obtain a solution with a concentration of 5.017 μg / mL. Accurately pipette 1 mL of the 5.017 μg / mL solution into a 10 mL volumetric flask, dilute to the mark with methanol, and mix well to obtain a solution with a concentration of 0.502 μg / mL.

[0071] Accurately weigh an appropriate amount of apigenin-7-O-diglucuronide reference standard, and prepare a solution containing 100.88 μg / mL in methanol. Use this as the stock solution. Accurately pipette 5 mL and 1 mL of the stock solution into 10 mL volumetric flasks, dilute to the mark with methanol, and mix well to obtain solutions with concentrations of 50.441 μg / mL and 10.088 μg / mL. Accurately pipette 1 mL of the 10.088 μg / mL solution into a 5 mL volumetric flask, dilute to the mark with methanol, and mix well to obtain a solution with a concentration of 2.018 μg / mL.

[0072] Accurately weigh an appropriate amount of verbascoside reference standard and prepare a solution containing 218.24 μg / mL in methanol. Use this as the stock solution. Accurately pipette 5 mL and 1 mL of the stock solution into 10 mL volumetric flasks, dilute to the mark with methanol, and mix well to obtain solutions with concentrations of 109.118 μg / mL and 21.824 μg / mL. Accurately pipette 1 mL of the 21.824 μg / mL solution into a 10 mL volumetric flask, dilute to the mark with methanol, and mix well to obtain a solution with a concentration of 2.182 μg / mL.

[0073] Accurately weigh an appropriate amount of farnesin-7-O-[β-D-glucuronylpyranoside (1→2)-OD-glucuronylpyranoside] reference standard, and prepare a solution containing 432.102 μg / mL in methanol. Use this as the stock solution. Accurately pipette 5 mL and 1 mL of the stock solution into 10 mL and 5 mL volumetric flasks, respectively. Dilute to the mark with methanol and mix well to obtain solutions with concentrations of 216.051 μg / mL and 86.420 μg / mL. Accurately pipette 1 mL of the 86.420 μg / mL solution into a 10 mL volumetric flask, dilute to the mark with methanol, and mix well to obtain a solution with a concentration of 8.642 μg / mL.

[0074] The diluted reference standard was injected and analyzed. A standard curve was plotted with the peak area integral value on the ordinate and the injection volume on the abscissa. The results are shown in [Figure number missing]. Figure 5 .

[0075] The results showed that chlorogenic acid reference standard had good linearity in the range of 0.0025 μg to 0.2509 μg, apigenin-7-O-diglucuronide reference standard in the range of 0.0101 μg to 1.5132 μg, verbascoside reference standard in the range of 0.0109 μg to 3.2735 μg, and farnesin-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] in the range of 0.0432 μg to 3.2408 μg (see Table 7).

[0076] Table 7 Linear Relationship Table

[0077]

[0078] 3 Repeatability

[0079] Six parallel test samples were prepared from the same batch of *Corydalis yanhusuo* medicinal material (number: *Corydalis yanhusuo*-20) according to the preparation method of the test sample. The contents of each component to be tested were determined and calculated according to the method. The results are shown in Table 8. The RSD value of the contents is less than 3%. The experiment shows that the repeatability of this method is good.

[0080] Table 8 Repeatability

[0081]

[0082] 4 Precision

[0083] One sample of *Corydalis yanhusuo* (number: *Corydalis yanhusuo*-20) was taken, and a test solution was prepared. The sample was injected six times consecutively, and the peak area was determined according to the method. The RSD was calculated. The results are shown in Tables 3-7. The peak area RSDs of chlorogenic acid, apigenin-7-O-diglucuronide, verbascoside, and farnesin-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] were all less than 1%, which met the method requirements and indicated that the method had good precision. See Table 9.

[0084] Table 9 Precision

[0085]

[0086] 5. Stability

[0087] One sample of *Clerodendrum trichotomum* (number: *Clerodendrum trichotomum*-20) was taken, and a test solution was prepared. The sample was injected at different time points within 24 hours, and the determination was carried out according to the method. The chromatograms were recorded and integrated, and the peak areas were measured. The results are shown in Table 10. The peak area RSDs of chlorogenic acid, apigenin-7-O-diglucuronide, verbascoside, and farnesin-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] were all less than 3%. The experiment showed that the peak areas of the test solution were basically stable within 24 hours.

[0088] Table 10 Stability

[0089]

[0090] 6. Durability

[0091] Based on the above chromatographic conditions, Agilent 1200 and Watres E2695 were used to determine the chromatographic properties of *Corydalis yanhusuo* (No.: *Corydalis yanhusuo*-20), and different brands of chromatographic columns were used to investigate the durability of the instruments and columns. The brands are shown in Table 11, and the results are shown in Tables 12 and 13.

[0092] The RSDs of chlorogenic acid, apigenin-7-O-diglucuronide, verbascoside, and acaciain-7-O-[β-D-glucuronylpyranoside (1→2)-OD-glucuronylpyranoside] were all less than 3%. Experiments showed that different instruments and column brands had virtually no impact on the determination of the content of *Clerodendrum trichotomum* medicinal materials, and this method exhibited good robustness.

[0093] Table 11 Chromatographic Column Brands

[0094]

[0095]

[0096] Table 12 Agilent - Results of determination using different chromatographic columns

[0097]

[0098] Table 13 Results of Waters measurements using different chromatographic columns

[0099]

[0100] 7. Accuracy

[0101] Accurately weigh three portions each of *Clerodendrum trichotomum* (No.: *Clerodendrum trichotomum-20*) with known content: 0.1875g, 0.375g, and 0.5625g, for a total of nine portions. Accurately weigh 3.975mg of chlorogenic acid, place it in a 50mL volumetric flask, add 50% ethanol to dissolve it, and dilute to the mark. Shake well to obtain a chlorogenic acid stock solution (1) with a concentration of 0.0766mg / mL. Accurately measure 10mL of chlorogenic acid stock solution (1), place it in a 20mL volumetric flask, add 50% ethanol to the mark, and shake well to obtain a chlorogenic acid stock solution (2) with a concentration of 0.0383mg / mL. Accurately weigh 10.186 mg of apigenin-7-O-diglucuronide, place it in a 20 mL volumetric flask, add 50% ethanol to dissolve it and dilute to the mark, shake well, and you will get a stock solution of apigenin-7-O-diglucuronide with a concentration of 0.4991 mg / mL (1).

[0102] Accurately measure 10 mL of apigenin-7-O-diglucuronide stock solution (1), place it in a 20 mL volumetric flask, add 50% ethanol to the mark, and shake well to obtain apigenin-7-O-diglucuronide stock solution (2) with a concentration of 0.2496 mg / mL. Accurately weigh 11.685 mg of verbascoside, place it in a 20 mL volumetric flask, add 50% ethanol to dissolve it, and dilute to the mark. Shake well to obtain verbascoside stock solution (1) with a concentration of 0.5562 mg / mL.

[0103] Accurately measure 10 mL of verbascoside stock solution (1), place it in a 20 mL volumetric flask, add 50% ethanol to the mark, and shake well to obtain verbascoside stock solution (2) with a concentration of 0.2781 mg / mL. Accurately weigh 11.806 mg of farnesin-7-O-[β-D-glucuronide (1→2)-OD-glucuronide], place it in a 200 mL volumetric flask, add 50% ethanol to dissolve it, and dilute to the mark. Shake well to obtain farnesin-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] stock solution (1) with a concentration of 0.0578 mg / mL.

[0104] Accurately weigh 8.826 mg of farnesin-7-O-[β-D-glucuronide (1→2)-OD-glucuronide], place it in a 50 mL volumetric flask, add 50% ethanol to dissolve it and dilute to the mark, shake well, and you will get a stock solution (2) of farnesin-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] with a concentration of 0.1730 mg / mL.

[0105] Accurately measure 0.5 mL each of chlorogenic acid stock solution (1), apigenin-7-O-diglucuronide stock solution (1), and verbascoside stock solution (1). Then accurately measure 25 mL of acaciain-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] stock solution (1). Place the solutions in a 200 mL volumetric flask, add 50% ethanol to the mark, and shake well to obtain 0.1875 g of solvent for sample recovery. Accurately measure 50 mL of each solution and add it to the three weighed samples. Process the solutions according to the test solution preparation method under the content determination section to obtain sample recovery solutions 1-1 to 1-3. Accurately measure 1 mL each of chlorogenic acid stock solution (1), apigenin-7-O-diglucuronide stock solution (1), and verbascoside stock solution (1). Then accurately measure 25 mL of acaciain-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] stock solution (1) and 50 mL of the solution. Place them in a 200 mL volumetric flask, add 50% ethanol to the mark, and shake well to obtain 0.375 g of solvent recovered by spiking. Accurately measure 50 mL of the solution and add it to the three weighed samples. Process the solution according to the test solution preparation method under the content determination section to obtain sample solutions 2-1 to 2-3. Accurately measure 3 mL each of chlorogenic acid stock solution (2), apigenin-7-O-diglucuronide stock solution (2), and verbascoside stock solution (2). Then accurately measure 25 mL of acaciain-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] stock solution (2), place them in a 200 mL volumetric flask, add 50% ethanol to the mark, and shake well to obtain 0.5625 g of solvent recovered by spiking. Accurately measure 50 mL and add it to the three weighed samples, and process them according to the test solution preparation method under the content determination section to obtain sample solutions 3-1 to 3-3.

[0106] Accurately pipette 10 μL of each of the above solutions, determine according to the method, record the chromatogram and integrate. The recovery rate is within the specified range of 99.63% to 108.44%, and the RSD is less than 3% (see Table 14).

[0107] Table 14 Recovery Rate

[0108]

[0109]

[0110] 8. The contents of chlorogenic acid, apigenin-7-O-diglucuronide, verbascoside and acaciain-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] in 20 batches of *Clerodendrum trichotomum* from different origins were determined using the established method described above. The results are shown in Table 15.

[0111] Table 15 Results of the determination of the content of *Corydalis yanhusuo* medicinal material

[0112]

[0113] The measured chlorogenic acid content in 20 batches of medicinal materials ranged from 0.07 mg / g to 1.79 mg / g, with a 95% confidence interval of [0.3528, 0.8112]. The lower limit for content determination was set down by 20% from the average of the 95% confidence intervals, meaning the chlorogenic acid content in the medicinal materials should not be less than 0.0484%. The measured apigenin-7-O-diglucuronide content ranged from 0.28 mg / g to 3.88 mg / g, with a 95% confidence interval of [0.7998, 1.5972]. The lower limit for content determination was set down by 20% from the average of the 95% confidence intervals, meaning the apigenin-7-O-diglucuronide content in the medicinal materials should not be less than 0.1028%. The measured verbascoside content ranged from 0.43 mg / g to 9.7 mg / g. The content of 7 mg / g, with a 95% confidence interval of [2.0872, 4.4178], is determined by lowering the average value of the data in the 95% confidence interval by 20%, meaning that the content of verbascoside in the medicinal material shall not be less than 0.2637%. The measured range of the content of farnesin-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] is 1.53 mg / g to 8.55 mg / g, with a 95% confidence interval of [3.4267, 5.2133]. The content of farnesin-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] in the medicinal material is determined by lowering the average value of the data in the 95% confidence interval by 20%, meaning that the content of farnesin-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] in the medicinal material shall not be less than 0.3631%.

[0114] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for determining the content of active substances in *Corydalis yanhusuo* based on high performance liquid chromatography, characterized in that, Includes the following steps: (1) Mix the medicinal material of *Corydalis yanhusuo* with ethanol and sonicate to obtain the test solution; Chlorogenic acid solution, apigenin-7-O-diglucuronic acid solution, verbascoside solution, and farnesin-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] solution were used as reference solutions, respectively. (2) The test solution and reference solution obtained in step (1) are subjected to high performance liquid chromatography for detection: acetonitrile is used as mobile phase A and formic acid solution is used as mobile phase B, and elution is performed according to the gradient elution program in Table 1; the flow rate is 0.8 to 1.2 mL / min, the column temperature is 28 to 32℃, and the detection wavelength is 310 to 330 nm. Table 1 2. The method according to claim 1, characterized in that, In step (2), the chromatographic column is a Thermo column with a size of 5 μm and 4.6 × 250 mm; the packing material is octadecylsilane bonded silica gel.

3. The method according to claim 1, characterized in that, In step (2), the injection volume is 8-12 μL; the volume concentration of the formic acid solution is 0.08-0.12%.

4. The method according to claim 1, characterized in that, In step (1), the ratio of the medicinal material *Corydalis yanhusuo* to ethanol is 0.5–1.0 g: 50 mL; the concentration of the ethanol is 45–55%.

5. The method according to claim 1, characterized in that, The ultrasonic power in step (1) is 450-550W and the time is 25-35min.

6. The method according to claim 1, characterized in that, In step (1), the chlorogenic acid solution, apigenin-7-O-diglucuronic acid solution, verbascoside solution, and acaciain-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] solution are all in methanol as solvent.

7. The method according to claim 1, characterized in that, The concentration of the chlorogenic acid solution in step (1) is 50-60 μg / mL; the concentration of the apigenin-7-O-diglucuronic acid solution is 350-400 μg / mL.

8. The method according to claim 1 or 7, characterized in that, The concentration of the verbascoside solution in step (1) is 400-430 μg / mL; the concentration of the farnesin-7-O-[β-D-glucuronide (1→2)-OD-glucuronide] solution is 2.0-2.4 mg / mL.

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