Method for detecting content of 4-coumaric acid and ferulic acid in polygonum and folium isatidis formula granules

Abstract

The invention provides a method for detecting the content of 4-coumaric acid and ferulic acid in polygonum-folium isatidis formula granules. The method comprises the following steps: acquiring a content detection map of a to-be-detected substance by adopting a high performance liquid chromatography; chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is used as a chromatographic column of a filler; the detection wavelength of 1, 4-cumaric acid is 290 to 330nm, and the detection wavelength of ferulic acid is 305 to 340nm; and acetonitrile and a 0.5%-1.5% glacial acetic acid solution in a ratio of (5-15): (85-95) are used as mobile phases. According to the method disclosed by the invention, organic acid components, namely 4-coumaric acid and ferulic acid, in an aqueous extract of the folium polygoni multiflori are taken as reference substances, and the high-performance liquid phase content determination and detection method for the coumaric acid and the ferulic acid in the folium polygoni multiflori formula granules is established, so that the contents of the 4-coumaric acid and the ferulic acid in the folium polygoni multiflori can be accurately and synchronously detected; the quality of a product can be better controlled on the whole, and effective guarantee is provided for production and clinical application of drugs.

Description

Technical Field

[0001] The present invention relates to the field of detection of traditional Chinese medicine components, and specifically relates to a method for detecting the contents of 4-coumaric acid and ferulic acid in Polygonum tinctorium Ait. formula granules. Background Art

[0002] Polygonum tinctorium Ait. is the dried leaf of the plant Polygonum tinctorium Ait. of the family Polygonaceae. It is harvested twice when the branches and leaves are lush in summer and autumn, the stems and branches and impurities are removed, and then dried. It is distributed in Liaoning, Hebei, Shaanxi, Shandong and other places. It has the effects of clearing heat and detoxifying, cooling blood and removing macules, and is mainly used for symptoms such as febrile disease with fever, macules and eruptions, cough and asthma due to lung heat, sore throat, mumps, erysipelas and carbuncle swelling.

[0003] Formula granules are obtained by taking water as the extraction medium, through extraction, concentration, spraying, drying and granulation. Substances with strong fat solubility are difficult to be completely extracted in the water extract, and there are great differences in the material basis. At present, the index components for the determination of the content in Polygonum tinctorium Ait. medicinal materials are usually substances with strong fat solubility such as indigo. Since the water solubility of such substances is poor, for Polygonum tinctorium Ait. formula granules, the measured range of indigo content is between 0.001% and 0.009%, less than two ten-thousandths, so it is not suitable as an index for its content determination.

[0004] As representative components of organic acids in Polygonum tinctorium Ait., 4-coumaric acid and ferulic acid mainly have pharmacological effects such as antibacterial, anti-inflammatory, antiviral, antioxidant and anti-tumor, and have activities consistent with the medicinal properties of Polygonum tinctorium Ait. At present, there is no relevant literature report on the synchronous detection of the contents of 4-coumaric acid and ferulic acid in the water extract of Polygonum tinctorium Ait. And when the currently disclosed method for synchronously detecting the contents of 4-coumaric acid and ferulic acid is applied to Polygonum tinctorium Ait., the peak shape and resolution of ferulic acid are both poor, there is interference of impurity peaks after the obvious peak, and the chromatographic peak purity cannot meet the detection requirements, so it cannot be stably detected. Summary of the Invention

[0005] The problem that the existing detection methods disclosed in the prior art cannot accurately synchronously detect the contents of 4-coumaric acid and ferulic acid in Polygonum tinctorium Ait. formula granules; the present invention provides a method for synchronously and accurately detecting the contents of 4-coumaric acid and ferulic acid in Polygonum tinctorium Ait. formula granules.

[0006] A method for detecting the contents of 4-coumaric acid and ferulic acid in Polygonum tinctorium Ait., using high performance liquid chromatography to obtain the chromatogram of the test substance; the chromatographic conditions of the high performance liquid chromatography are as follows:

[0007] A chromatographic column filled with octadecylsilane chemically bonded silica; the detection wavelength for 4-coumaric acid is 290 - 330 nm, and the detection wavelength for ferulic acid is 305 - 340 nm; the mobile phase is acetonitrile:(5 - 15):(85 - 95) 0.5% - 1.5% glacial acetic acid solution.

[0008] The above-mentioned content determination chromatogram is the chromatogram for content determination.

[0009] In the chromatographic conditions of the high performance liquid chromatography method described above, the column length of the chromatographic column is 250 mm, the column inner diameter is 4.6 mm, and the particle size is 5 μm;

[0010] and / or, the column temperature is 33 - 37 °C;

[0011] and / or, the flow rate is 0.8 - 1.2 ml / min;

[0012] and / or, the number of theoretical plates calculated based on the 4-coumaric acid peak should be not less than 5000.

[0013] In the high performance liquid chromatography method described above, the chromatographic column is thermo scientific TM Hypersil GOLD TM aQ, Waters Symmetry C18 or Zafex AQ-C18.

[0014] When the analyte is the test sample, the preparation process of the test sample solution is as follows: Take the water extract of Polygonum tinctorium Ait. or the preparation of the water extract of Polygonum tinctorium Ait., weigh accurately, add the solvent, tightly stopper, weigh, treat, take out, cool, weigh again, make up the lost weight with the solvent, shake well, filter, and take the subsequent filtrate to obtain;

[0015] When the analyte is the reference substance, the preparation process of the reference substance solution is as follows: Take the reference substance, weigh accurately, and make a reference substance solution with the solvent.

[0016] The solvents used in the test sample solution are methanol, water and aqueous alcohol solution;

[0017] and / or, the solvent used in the reference substance solution is methanol;

[0018] and / or, the reference substances include 4-coumaric acid reference substance and ferulic acid reference substance.

[0019] The mass concentration of the test sample in the test sample solution is 0.01 - 0.05 g / ml;

[0020] and / or, the mass concentration of the reference substance in the reference substance solution is 10 μg / ml.

[0021] In the preparation of the test sample solution, the treatment methods include heat treatment or ultrasonic treatment;

[0022] and / or, the treatment time is 10 - 30 min.

[0023] The power of the ultrasonic treatment is 250 W and the frequency is 40 kHz.

[0024] The test sample includes the preparation of Polygonum tinctorium Ait.

[0025] The preparation of Polygonum tinctorium Ait. includes the water extract paste of Polygonum tinctorium Ait., the dried powder of water extract of Polygonum tinctorium Ait., the formula granules of Polygonum tinctorium Ait. or the water extract preparation of Polygonum tinctorium Ait.

[0026] The technical solution of the present invention has the following advantages:

[0027] 1. The present invention provides a method for detecting the contents of 4-coumaric acid and ferulic acid in Polygonum tinctorium Ait. By using the organic acid components 4-coumaric acid and ferulic acid in the water extract of Polygonum tinctorium Ait. as reference substances, a high-performance liquid chromatography method for determining the contents of coumaric acid and ferulic acid in the formula granules of Polygonum tinctorium Ait. is established, which is beneficial to better controlling the quality of products as a whole and provides an effective guarantee for the production and clinical application of drugs.

[0028] 2. The method of the present invention has the advantages of simple operation, stability, high precision and good reproducibility, can quickly and accurately identify the quality of products, and provides a scientific basis for comprehensively establishing the quality control standard of the formula granules of Polygonum tinctorium Ait. Description of the Drawings

[0029] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0030] Figure 1 It is the control chromatogram for the specificity test in Example 2 of the present invention.

[0031] Figure 2 It is the standard curve of 4-coumaric acid in Example 2 of the present invention.

[0032] Figure 3 It is the standard curve of ferulic acid in Example 2 of the present invention.

[0033] Figure 4 It is the chromatogram for the content determination in Comparative Example 1 of the present invention. Detailed Embodiments

[0034] For those steps or conditions not specified in the examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments not specified for the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0035] Instruments: Waters ACQUITY UPLC H-Class ultra performance liquid chromatograph (Waters Corporation); ML204T electronic balance (Mettler Toledo Instruments (Shanghai) Co., Ltd.); JA1002 electronic balance (Shanghai Puchun Measuring Instruments Co., Ltd.); MSA6·6S-0CE-DM electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); KQ-100DE digital control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.);

[0036] Test drugs: 4-coumaric acid reference substance (batch number: 112037-202102, National Institutes for Food and Drug Control, purity 99.7%); ferulic acid reference substance (batch number: 110773-201915, National Institutes for Food and Drug Control, purity 99.4%);

[0037] Polygonum tinctorium Lour. formula granules: K478CP16, K47CP17, K478CP18.

[0038] Reagents: Methanol is of chromatographic grade, ethanol is of analytical grade, and water is Watsons distilled water.

[0039] Example 1

[0040] A method for detecting the contents of 4-coumaric acid and ferulic acid in Polygonum tinctorium Lour., the specific process is as follows:

[0041] 1. Preparation of test substance solution

[0042] Preparation of test solution: Take an appropriate amount of the test substance (Polygonum tinctorium Lour. formula granules), grind it finely, take about 0.5 g, place it in a stoppered conical flask, accurately add 20 ml of 30% methanol, stopper it tightly, weigh it accurately, ultrasonically treat it (power 250 W, frequency 40 kHz) for 20 minutes, take it out, let it cool, weigh it again, make up the lost weight with 30% methanol, shake well, filter, and take the subsequent filtrate, that is obtained.

[0043] Preparation of reference substance solution: Take appropriate amounts of 4-coumaric acid reference substance and ferulic acid reference substance, weigh them accurately, and make a reference substance solution containing 10 μg of each per 1 ml with methanol.

[0044] 2. Chromatographic method

[0045] The following chromatographic conditions are used for detection:

[0046] Using octadecylsilane chemically bonded silica as the filler (thermo scientific TM Hypersil GOLD TMaQ, column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); using acetonitrile - 1% glacial acetic acid solution (volume ratio 10:90) as the mobile phase; flow rate is 1.0 ml per minute; column temperature is 35 °C; detection wavelength for 4-coumaric acid is 310 nm; detection wavelength for ferulic acid is 322 nm. The number of theoretical plates calculated based on the 4-coumaric acid peak should be not less than 5000;

[0047] Precisely pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and measure to obtain the results.

[0048] 3. Inspection of Peak Purity

[0049] Take the test solution of Polygonum tinctorium Lour. granules, and use a diode array detector to conduct peak purity inspection on the 4-coumaric acid peak and ferulic acid peak. The results are shown in Table 1 below.

[0050] Table 1

[0051] Purity perspective Purity threshold 4-Coumaric acid peak 1.472 2.486 Ferulic acid 1.602 3.173

[0052] The results show that in the chromatogram of the test solution of Polygonum tinctorium Lour. granules, the peak purity angle of 4-coumaric acid is less than the purity threshold 1.472 < 2.486, and the peak purity angle of ferulic acid is less than the purity threshold 1.602 < 3.173, indicating that the peak purities of 4-coumaric acid and ferulic acid obtained by this method meet the analysis requirements.

[0053] 4. System Suitability Test

[0054] Using the 4-coumaric acid peak as a reference, conduct a system suitability test, and the results are shown in Table 2 below.

[0055] Table 2 Results of System Suitability Test

[0056]

[0057] The results show that the average value of the number of theoretical plates for 4-coumaric acid is 26969.6, the tailing factor is 0.98, and the repeatability RSD% is 1.56. The system suitability meets the analysis requirements.

[0058] 5. Accuracy Detection

[0059] 5.1. Accuracy of 4-coumaric acid

[0060] Accurately weigh 5.156 mg of 4-coumaric acid reference substance (purity 99.7%) and place it in a 100-ml volumetric flask. Add methanol to dissolve and make up to the mark, shake well, and use it as the mother liquor of the reference substance. Accurately measure 5 ml, 10 ml, and 15 ml of the mother liquor of the reference substance respectively and place them in 100-ml volumetric flasks. Add 30% methanol to make up to the mark, shake well, and use them as 4-coumaric acid reference solution 1, 4-coumaric acid reference solution 2, and 4-coumaric acid reference solution 3 respectively.

[0061] Take the formulated granules of Polygonum tinctorium Lour. leaves with known content (batch number: K478CP16, 4-coumaric acid 0.39 mg / g), grind them finely, take 9 portions, each portion is 0.25 g, accurately weigh them, and number them 1-9 respectively. For numbers 1-3, accurately add 20 ml of the above-mentioned 4-coumaric acid reference solution 1 respectively; for numbers 4-6, accurately add 20 ml of the above-mentioned 4-coumaric acid reference solution 2 respectively; for numbers 7-9, accurately add 20 ml of the above-mentioned 4-coumaric acid reference solution 3 respectively; the remaining operations are the same as the preparation method of the test solution. Calculate the recovery rate of 4-coumaric acid according to the following formula.

[0062]

[0063] 5.2 Accuracy of ferulic acid

[0064] Accurately weigh 5.267 mg of ferulic acid reference substance (purity 99.4%) and place it in a 100-ml volumetric flask. Add methanol to dissolve and make up to the mark, shake well, and use it as the mother liquor of the ferulic acid reference substance. Accurately measure 5 ml, 10 ml, and 15 ml of the mother liquor of the reference substance respectively and place them in 100-ml volumetric flasks. Add 30% methanol to make up to the mark, shake well, and use them as ferulic acid reference solution 1, ferulic acid reference solution 2, and ferulic acid reference solution 3 respectively.

[0065] Take the formulated granules of Polygonum tinctorium Lour. leaves with known content (batch number: K478CP16, ferulic acid 0.47 mg / g), grind them finely, take 9 portions, each portion is 0.25 g, accurately weigh them, and number them 1-9 respectively. For numbers 1-3, accurately add 20 ml of the above-mentioned ferulic acid reference solution 1 respectively; for numbers 4-6, accurately add 20 ml of the above-mentioned ferulic acid reference solution 2 respectively; for numbers 7-9, accurately add 20 ml of the above-mentioned ferulic acid reference solution 3 respectively; the remaining operations are the same as the preparation method of the test solution in the main text. Calculate the recovery rate of ferulic acid according to the following formula.

[0066]

[0067] The recovery rates of 4-coumaric acid and ferulic acid are as follows:

[0068] Table 3 Results of 4-coumaric acid recovery test

[0069]

[0070]

[0071] Table 4 Results of Ferulic Acid Recovery Test

[0072]

[0073] The results showed that the recovery rate of 4-coumaric acid measured in the recovery test ranged from 89.07% to 97.96%, and the RSD value was 2.77%; the recovery rate of ferulic acid ranged from 95.36% to 102.25%, and the RSD value was 2.48%. According to the regulations, when the content of the component to be measured is 0.01%, the spiked recovery rate range should be between 85% and 110%. Therefore, it meets the requirements of the recovery rate in the methodological verification, indicating that the results measured by this method are accurate.

[0074] Example 2

[0075] This example is used to conduct methodological verification on the chromatographic conditions in Example 1.

[0076] 1. Repeatability investigation

[0077] Take 6 portions of Polygonum tinctorium Lour. formula granules (batch number: K478CP16), grind them finely, and determine according to the method in Example 1. The content detection results of 4-coumaric acid and ferulic acid obtained are as follows.

[0078] Table 5 Results of 4-coumaric Acid Content in Repeatability Test

[0079]

[0081] Table 6 Results of Ferulic Acid Content in Repeatability Test

[0082]

[0083] The average value of 4-coumaric acid content in Polygonum tinctorium Lour. granules measured in the repeatability test was 0.40 mg / g, and the RSD was 0.86%; the average value of ferulic acid content was 0.46 mg / g, and the RSD was 1.66%; it meets the requirements of repeatability in the methodological verification.

[0084] 2. Intermediate precision investigation

[0085] Take 6 portions of Polygonum tinctorium Lour. formula granules (batch number: K478CP16), prepare samples according to the method in Example 1, and conduct intermediate precision tests using another high-performance liquid chromatograph by different analysts at different times. The content detection results of 4-coumaric acid and ferulic acid obtained are as follows.

[0086] Table 7 Results of 4-coumaric Acid Content in Intermediate Precision Test

[0087]

[0088] Table 8 Results Table of Ferulic Acid Content in Intermediate Precision Test

[0089]

[0090] The average content of 4 - Coumaric Acid in Polygonum tinctorium Lour. granules measured in the intermediate precision test was 0.40 mg / g, with an RSD of 1.14%, and the RSD of the detection results in the repeatability test was 1.00%; the average content of ferulic acid was 0.47 mg / g, with an RSD of 1.33%, and the RSD of the detection results in the repeatability test was 1.72%, meeting the requirements of methodological precision.

[0091] 3. Specificity Investigation

[0092] Take the test solution of Polygonum tinctorium Lour. formula granules, the mixed reference solution of 4 - Coumaric Acid and ferulic acid, the negative excipient control solution, and the blank solvent to obtain the chromatograms as shown in accordance with the method of Example 1. Figure 1 as shown

[0093] Through Figure 1 It can be seen that by investigating the specificity of the chromatographic conditions, no interference was found at the corresponding peak positions in the test solution chromatogram.

[0094] 4. Linearity

[0095] Take appropriate amounts of reference substances of 4 - Coumaric Acid and ferulic acid, accurately weigh them, dissolve them in an appropriate amount of methanol and make up to a certain volume to prepare a mixed reference solution containing 51.4053 μg of 4 - Coumaric Acid and 52.3540 μg of ferulic acid per 1 ml, as the linearity 5 solution; pipette 5 ml of the linearity 5 solution into a 10 - ml volumetric flask, add methanol to the scale and shake well, as the linearity 4; pipette 5 ml of the linearity 5 solution into a 20 - ml volumetric flask, add methanol to the scale and shake well, as the linearity 3; pipette 5 ml of the linearity 5 solution into a 50 - ml volumetric flask, add methanol to the scale and shake well, as the linearity 2; pipette 5 ml of the linearity 5 solution into a 100 - ml volumetric flask, add methanol to the scale and shake well, as the linearity 1.

[0096] Precisely pipette 10 μl of the continued filtrates of the above - mentioned solutions and inject them into the high - performance liquid chromatograph, and determine the peak areas of the 4 - Coumaric Acid and ferulic acid chromatographic peaks according to the method in Example 1. The results are as follows.

[0097] Table 9 Investigation of the Linear Relationship of 4 - Coumaric Acid

[0098]

[0099] Table 10 Investigation of the Linear Relationship of Ferulic Acid

[0100]

[0101]

[0102] Using the concentration of each component as the abscissa and the peak area of the chromatographic peak as the ordinate, a standard curve was plotted, as Figure 2 and Figure 3 shown. The regression equation of 4-coumaric acid was y = 78053.4366x - 3931.5912, R2 = 0.9999, and its linear range was 2.5703 μg / ml to 51.4053 μg / ml; the regression equation of ferulic acid was y = 55041.3235x - 5051.0108, R2 = 0.9999, and its linear range was 2.6177 μg / ml to 52.3540 μg / ml.

[0103] 5. Stability investigation

[0104] Take the formulated granules of Polygonum tinctorium Lour. (batch number: K478CP16) to prepare the test solution, and measure it at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h respectively. Calculate the contents of 4-coumaric acid and ferulic acid, and the test results are as follows.

[0105] Table 11 Stability results of 4-coumaric acid

[0106]

[0107] Table 12 Stability results of ferulic acid

[0108]

[0109] From the above data, it can be seen that the RSD value of the peak area of 4-coumaric acid within 24 hours was 1.71%, and the RSD value of the peak area of ferulic acid was 0.81%, indicating that the sample had good stability within 24 hours.

[0110] Example 3

[0111] A method for detecting the contents of 4-coumaric acid and ferulic acid in Polygonum tinctorium Lour., which is different from Example 1 in that the preparation of the test solution is different.

[0112] (1) Selection of extraction solvent

[0113] Take the formulated granules of Polygonum tinctorium Lour. leaves (batch number: K478CP16), grind them finely, take 9 portions respectively, each portion about 0.5 g, place them in a stoppered conical flask, accurately add 20 ml of the corresponding solvents (30% methanol, 50% methanol, 70% methanol, methanol, 30% ethanol, 50% ethanol, 70% ethanol, ethanol, water) respectively, perform ultrasonic treatment for 20 minutes (power 250 W, frequency 40 kHz), take out, let it cool, weigh again, make up the lost weight with the corresponding solvents respectively, shake well, filter, and obtain the filtrate. Calculate the contents of 4-coumaric acid and ferulic acid, and the results are as follows.

[0114] Table 13 Results of 4-coumaric acid contents with different extraction solvents

[0115]

[0116] Table 14 Results of ferulic acid contents with different extraction solvents

[0117]

[0118]

[0119] From the results of the content determination after investigating different extraction solvents, it can be seen that when ethanol is used as the extraction solvent, the contents of 4-coumaric acid and ferulic acid are extremely low; when the other solvents are used as the extraction solvents, the contents have little difference. To ensure complete extraction, 30% methanol is selected as the extraction solvent.

[0120] (2) Selection of solvent dosage

[0121] Take the formulated granules of Polygonum tinctorium Lour. leaves (batch number: K478CP16), grind them finely, take about 0.5 g, accurately weigh, place it in a stoppered conical flask, accurately add 10 ml, 20 ml, 30 ml of 30% methanol respectively, weigh, perform ultrasonic treatment (power 250 W, frequency 40 kHz) for 20 minutes, take out, let it cool, weigh again, make up the lost weight with 30% methanol, shake well, filter, take the continuous filtrate, and obtain the filtrate. Calculate the contents of 4-coumaric acid and ferulic acid, and the results are as follows.

[0122] Table 15 Results of 4-coumaric acid contents in the investigation of extraction solvent dosage

[0123]

[0124] Table 16 Results of ferulic acid contents in the investigation of extraction solvent dosage

[0125]

[0126]

[0127] As can be seen from the above table, there are no significant differences in the contents of 4-coumaric acid and ferulic acid with different amounts of extraction solvents. To ensure sufficient extraction of the samples, 20 ml was selected as the amount of extraction solvent.

[0128] (3) Selection of extraction time

[0129] Take the formulated granules of Polygonum tinctorium Lour. (batch number: K478CP16), grind them finely, take about 0.5 g, weigh accurately, place them in a stoppered conical flask, accurately add 20 ml of 30% methanol, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 10, 20, and 30 minutes respectively, take out, let cool, weigh again, make up the lost weight with 30% methanol, shake well, filter, and take the subsequent filtrate to obtain the solution. Calculate the contents of 4-coumaric acid and ferulic acid, and the results are as follows.

[0130] Table 17 Results of 4-coumaric acid content at different extraction times

[0131]

[0132] Table 18 Results of ferulic acid content at different extraction times

[0133]

[0134] It can be seen from the above results that 4-coumaric acid and ferulic acid can be completely extracted by ultrasonication for 20 minutes. Therefore, the extraction time was selected as 20 minutes.

[0135] (4) Selection of extraction method

[0136] Take the formulated granules of Polygonum tinctorium Lour. (batch number: K478CP16), grind them finely, take about 0.5 g, weigh accurately, place them in a stoppered conical flask, accurately add 20 ml of 30% methanol, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 20 minutes and reflux extract for 20 minutes respectively, take out, let cool, weigh, make up the lost weight with 30% methanol, shake well, filter, and take the subsequent filtrate to obtain the solution. Calculate the contents of 4-coumaric acid and ferulic acid, and the results are as follows.

[0137] Table 19 Results of 4-coumaric acid content with different extraction methods

[0138]

[0139] Table 20 Results of ferulic acid content with different extraction methods

[0140]

[0141] The above results show that there are no significant differences in the contents of 4-coumaric acid and ferulic acid in the test solution obtained by different extraction methods. Considering the convenience of experimental operation, ultrasonication was determined as the extraction method for the preparation of the test solution.

[0142] Example 4

[0143] A method for detecting the contents of 4-coumaric acid and ferulic acid in Polygonum tinctorium leaves, which is different from that in Example 1 in that the chromatographic conditions are different, specifically as follows:

[0144] (1) Investigation of different column temperatures

[0145] Take the Polygonum tinctorium leaves formula granules (batch number: K478CP16), prepare the test solution according to the preparation method of the test solution in Example 1, and measure according to the method in Example 1 at different column temperatures (33 °C, 35 °C and 37 °C) respectively to investigate the durability of the experimental method for the column temperature. The results are as follows:

[0146] Table 21 Results of determination of 4-coumaric acid content at different column temperatures

[0147]

[0148]

[0149] Table 22 Results of determination of ferulic acid content at different column temperatures

[0150]

[0151] The RSD values of the 4-coumaric acid content measured at different column temperatures are 0.65%, and the RSD values of the ferulic acid content are 2.75%, meeting the requirements of system suitability, indicating that the method has good durability for column temperature.

[0152] (2) Investigation of different flow rates

[0153] Take the Polygonum tinctorium leaves formula granules (batch number: K478CP16), prepare the test solution according to the preparation method of the test solution in Example 1, and measure according to the method in Example 1 at different flow rates (0.8 ml / min, 1.0 ml / min and 1.2 ml / min) respectively to investigate the durability of the experimental method for the flow rate. The results are as follows.

[0154] Table 23 Results of determination of 4-coumaric acid content at different flow rates

[0155]

[0156] Table 24 Results of determination of ferulic acid content at different flow rates

[0157]

[0159] The RSD values of the 4-coumaric acid content measured at different flow rates are 1.55%, and the RSD values of the ferulic acid content are 1.56%, indicating that the method has good durability for the flow rate.

[0160] (3) Investigation of different acid concentrations

[0161] Take the formula granules of Polygonum tinctorium leaves (batch number: K478CP16), prepare the test solution according to the preparation method of the test solution in Example 1, and measure it according to the method in Example 1 using glacial acetic acid solutions with different mass concentrations (0.5% glacial acetic acid, 1% glacial acetic acid, and 1.5% glacial acetic acid) respectively to investigate the durability of the experimental method for the flow rate. The results are as follows:

[0162] Table 25 Results of determination of 4-coumaric acid content at different acid concentrations

[0163]

[0164] Table 26 Results of determination of ferulic acid content at different acid concentrations

[0165]

[0166] The RSD values of the 4-coumaric acid content measured at different acid concentrations were 1.56%, and the RSD value of the ferulic acid content was 1.99%, indicating that the method has good durability for different acid concentrations.

[0167] (4) Investigation of different types of chromatographic columns

[0168] Take the formula granules of Polygonum tinctorium leaves (batch number: K478CP16), prepare the test solution according to the preparation method of the test solution in Example 1, and measure it according to the method in Example 1 using different types of chromatographic columns;

[0169] Chromatographic column 1: Waters C18 (4.6mm×250mm, 5μm),

[0170] Chromatographic column 2: thermo scientific TM Hypersil GOLD TM aQ (4.6mm×250mm, 5μm),

[0171] Chromatographic column 3: Zafex AQ-C18 (4.6mm×250mm, 5μm).

[0172] Investigate the durability of the experimental method for different chromatographic columns. The results are as follows:

[0173] Table 27 Results of determination of 4-coumaric acid content for different chromatographic columns

[0174]

[0175] Table 28 Results of determination of ferulic acid content for different chromatographic columns

[0176]

[0177] The RSD values of the 4-coumaric acid content measured by different chromatographic columns were 1.95%, and the RSD value of the ferulic acid content was 2.55%, meeting the system suitability requirements, indicating that this method has good durability for different chromatographic columns.

[0178] (5) Investigation of different detection wavelengths

[0179] Take the Herba Polygoni Tinctorii formula granules (batch number: K478CP16), prepare the test solution according to the preparation method of the test solution in Example 1, and perform the determination according to the method in Example 1 at different detection wavelengths to investigate the durability of the experimental method for different wavelengths. The results are as follows.

[0180] Table 29 Determination results of 4-coumaric acid content at different wavelengths

[0181]

[0182] Table 30 Determination results of ferulic acid content at different wavelengths

[0183]

[0184] The RSD values of the 4-coumaric acid content measured at different wavelengths were 1.73%, and the RSD value of the ferulic acid content was 1.88%, meeting the system suitability requirements, indicating that this method has good durability for different wavelengths.

[0185] (6) Investigation of different mobile phase ratios

[0186] Take the Herba Polygoni Tinctorii formula granules (batch number: K478CP16), prepare the test solution according to the preparation method of the test solution in Example 1, and perform the determination according to the method in Example 1 at different mobile phase ratios to investigate the durability of the experimental method for different mobile phase ratios. The results are as follows.

[0187] Table 31 Determination results of 4-coumaric acid content at different mobile phase ratios

[0188]

[0189] Table 32 Determination results of ferulic acid content at different mobile phase ratios

[0190]

[0191] The RSD values of the 4-coumaric acid content measured at different mobile phase ratios were 2.56%, and the RSD value of the ferulic acid content was 2.70%, meeting the system suitability requirements, indicating that this method has good durability for different mobile phase ratios.

[0192] Example 5

[0193] A method for detecting the contents of 4-coumaric acid and ferulic acid in Polygonum tinctorium leaves. Respectively take 24 batches of Polygonum tinctorium leaves decoction pieces and 3 batches of Polygonum tinctorium leaves formula granules. Use 24 batches of Polygonum tinctorium leaves decoction pieces to prepare the standard decoction of Polygonum tinctorium leaves. The preparation process of the standard decoction is as follows: Take Polygonum tinctorium leaves decoction pieces, add water for decoction, filter, concentrate the filtrate into a clear paste (the extraction rate of dry extract is 12.0%-16.9%), and freeze-dry to obtain.

[0194] Determine the contents of 4-coumaric acid and ferulic acid according to the method in Example 1; in the present invention, the production amount of Polygonum tinctorium leaves formula granules is 16.9% of the amount of decoction pieces. Combining with the extraction rate of the standard decoction, the contents of the corresponding granules can be calculated from the contents of each batch of standard decoctions. The results are as follows.

[0195] Table 33 Conversion table of the contents of 4-coumaric acid and ferulic acid in the finished product of Polygonum tinctorium leaves formula granules

[0196]

[0197]

[0198] Table 34 Determination results of the contents of three batches of granule samples

[0199]

[0200] According to the converted contents of 4-coumaric acid and ferulic acid in the finished product granules in Table 24, the range of the mean value of 4-coumaric acid ± 30% is 0.28 mg / g to 0.53 mg / g, and the range of the mean value ± 3SD is 0.21 mg / g to 0.60 mg / g; the range of the mean value of ferulic acid ± 30% is 0.29 mg / g to 0.55 mg / g, and the range of the mean value ± 3SD is 0.24 mg / g to 0.60 mg / g. Adopting the principle of the smaller range, it is recommended to use the range of the mean value ± 3SD as the granule content range, and round it off, that is, the content range of 4-coumaric acid is 0.20 mg / g to 0.60 mg / g, and the content range of ferulic acid is 0.20 mg / g to 0.60 mg / g. The following are the determination results of the contents of three batches of granules, all of which meet the regulations.

[0201] Comparative Example 1

[0202] The difference between this comparative example and Example 1 is that the chromatographic conditions are different, specifically as follows:

[0203] Chromatographic column thermo scientific TM Hypersil GOLD TM aQ (4.6×250 mm, 5 μm); flow rate 1.0 ml / min; column temperature 40 °C; detection wavelength 280 nm; use acetonitrile-0.1% phosphoric acid aqueous solution as the mobile phase for elution.

[0204] Precisely pipette 10 μl of each test solution respectively, inject into a liquid chromatograph, and determine to obtain Figure 4 the chromatogram as shown.

[0205] From Figure 4 the results, it can be seen that the peak shape and resolution of ferulic acid are both poor, and there are interfering peaks behind the obvious peak; the chromatographic peak purity cannot meet the detection requirements and cannot be stably detected. In the detection of 4-coumaric acid and ferulic acid in Polygonum tinctorium Lour. leaves, the mobile phase system of acetonitrile - 1% glacial acetic acid is superior to the mobile phase system of this comparative example (acetonitrile - 0.1% phosphoric acid).

[0206] Obviously, the above-mentioned embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or alterations derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for detecting the contents of 4-coumaric acid and ferulic acid in Polygonum tinctorium Lour. leaves, characterized in that, Obtain the chromatogram of the analyte to be tested by high performance liquid chromatography; the chromatographic conditions of the high performance liquid chromatography are as follows: A chromatographic column packed with octadecylsilyl silica gel; the detection wavelength for 4-coumaric acid is 290 - 330 nm, and the detection wavelength for ferulic acid is 305 - 340 nm; an acetonitrile: 0.5% - 1.5% glacial acetic acid solution with a volume ratio of (5 - 15):(85 - 95) is used as the mobile phase.

2. The method according to claim 1, wherein Among the chromatographic conditions of the high performance liquid chromatography, the column length of the chromatographic column is 250 mm, the column inner diameter is 4.6 mm, and the particle size is 5 μm; and / or, the column temperature is 33 - 37 °C; and / or, the flow rate is 0.8 - 1.2 ml / min; and / or, the number of theoretical plates calculated based on the 4-coumaric acid peak should be not less than 5000.

3. The method according to claim 1, wherein In the high performance liquid chromatography method, the chromatographic column is a thermo scientific TM Hypersil GOLD TM aQ, Waters Symmetry C18, or Zafex AQ-C18.

4. The method according to any one of claims 1 to 3, characterized in that When the analyte to be tested is the test sample, the preparation process of the test sample solution is as follows: Take the water extract of Polygonum tinctorium Lour. or the preparation of the water extract of Polygonum tinctorium Lour., weigh accurately, add the solvent, stopper tightly, weigh again, treat, take out, cool, weigh again, make up the lost weight with the solvent, shake well, filter, and take the subsequent filtrate to obtain it; When the analyte to be tested is the reference substance, the preparation process of the reference substance reference solution is as follows: Take the reference substance, weigh accurately, and prepare the reference substance reference solution with the solvent.

5. The method according to claim 4, characterized in that The solvents used in the test sample solution are methanol, water, and aqueous alcohol solution; and / or, the solvent used in the reference substance reference solution is methanol; and / or, the reference substances include 4-coumaric acid reference substance and ferulic acid reference substance.

6. The method according to claim 5, characterized in that, The mass concentration of the test sample in the test sample solution is 0.01 - 0.05 g / ml; and / or, the mass concentration of the reference substance in the reference substance reference solution is 10 μg / ml.

7. The method according to claim 4, characterized in that, In the preparation of the test sample solution, the treatment methods include heat treatment or ultrasonic treatment; and / or, the treatment time is 10 - 30 min.

8. The method according to claim 7, characterized in that, The power of the ultrasonic treatment is 250 W and the frequency is 40 kHz.

9. The method according to claim 5, characterized in that, The test sample includes the preparation of Polygonum tinctorium Lour.

10. The method according to claim 9, wherein The preparation of Polygonum tinctorium Lour. includes the water extract paste of Polygonum tinctorium Lour., the dried powder of the water extract of Polygonum tinctorium Lour., the formula granules of Polygonum tinctorium Lour., or the water extract preparation of Polygonum tinctorium Lour.

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