Method for detecting content of isoquercitrin in chingma abutilon seed sample and application thereof

Abstract

The invention discloses a method for detecting the content of isoquercitrin in a chingma abutilon seed sample and application of the method. A test solution and a reference solution are prepared respectively and injected into an ultra-high performance liquid chromatograph, and specific chromatograms of the reference solution and the test solution are compared; and calculating the content of the isoquercitrin in the chingma abutilon seed sample by utilizing a regression equation of the sample size and the peak area of the isoquercitrin. The method disclosed by the invention is good in specificity and durability, and a new analysis means can be provided for quality detection of a chingma abutilon seed sample; the specific component of isoquercitrin is taken as an index, so that the method is more reasonable and accurate; the method is applicable to detection of chingma abutilon seed medicinal materials, standard decoction, formula granules and other products containing chingma abutilon seeds. The method is simple to operate, good in reproducibility and stability, reliable in recovery rate, low in detection cost, high in detection efficiency and small in environmental pollution.

Description

Technical Field

[0001] This invention relates to the detection of traditional Chinese medicine, and more particularly to a method for detecting the content of isoquercitrin in Abutilon theophrasti seeds and its application. Background Technology

[0002] Abutilon seeds are from the Malvaceae family plant Abutilon. Abutilon theophrasti Abutilon theophrasti seeds are dried, mature seeds. Mature fruits are harvested in autumn, dried, and the seeds are threshed and impurities removed. They are bitter and neutral in nature. They enter the large intestine, small intestine, and bladder meridians, and have the effects of clearing heat and detoxifying, promoting diuresis, and removing corneal opacity. They are used for dysentery (both bloody and white), painful urination, carbuncles and boils, and corneal opacity. Modern research shows that Abutilon theophrasti seeds contain fatty acids, amino acids, polysaccharides, and other chemical components. They have pharmacological effects such as diuresis, improving kidney function, and relieving edema. Abutilon theophrasti seeds are included in the Chinese Pharmacopoeia. The current 2025 edition of the Chinese Pharmacopoeia specifies linoleic acid as the key indicator for Abutilon theophrasti seeds. In addition, existing research on the quality of Abutilon theophrasti seeds includes: Wei Wei et al. determined the linoleic acid and oleic acid content of Abutilon theophrasti seeds from different origins; Shen Changhui et al. analyzed and identified its fatty acid composition; and Wang Linlin et al. used inductively coupled plasma atomic emission spectrometry to simultaneously determine multiple trace elements in Abutilon theophrasti seeds.

[0003] Abutilon seeds are mostly used in clinical practice by decoction. However, current technical methods for quality control of Abutilon seeds mainly involve fatty acids or other volatile components, which have low polarity and poor solubility in water. These methods are suitable for quality control of the raw materials, but their applicability to related preparations needs to be considered. The quality uniformity and effectiveness of the raw materials and related preparations are difficult to guarantee. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a method for detecting the isoquercitrin content in velvetleaf samples and its application, which solves the problem that existing velvetleaf quality control methods (such as those using fatty acids as indicators) are difficult to apply to the quality control of its water extracts (such as standard decoctions and formulation granules) due to the low polarity and poor water solubility of the indicator components.

[0005] Technical solution: The method for detecting isoquercitrin content in velvetleaf seed samples according to the present invention includes the following steps: (1) Preparation of test solutions for samples related to Abutilon theophrasti seeds; (2) Inject the test solution into the ultra-high performance liquid chromatograph, analyze and record the chromatogram; (3) Compare the chromatogram of the test solution with the chromatogram of isoquercitrin standard to calculate the content of isoquercitrin in the velvetleaf seed sample.

[0006] The jujube seed sample can be any one of the following: jujube seed medicinal material, standard decoction, extract, formula granules, or a reference sample, intermediate product, or finished product of a classic prescription containing jujube seed.

[0007] Preferably, the preparation of the test solution in step 1 includes: adding a methanol-water solution to the sample, extracting, filtering, and obtaining the test solution; more preferably, the methanol concentration in the methanol-water solution is not higher than 70%, and the amount used is 15-100 mL per gram of sample; more preferably, the extraction is ultrasonic extraction at 200-300 W power and 35-45 kHz frequency for 15-60 min.

[0008] Preferably, in step 2, the chromatographic conditions in the injection into the ultra-high performance liquid chromatograph include: a chromatographic column with octadecylsilane-bonded silica gel as the stationary phase; an eluent including mobile phase A and mobile phase B, wherein mobile phase A is methanol or acetonitrile, and mobile phase B is any one of water, formic acid aqueous solution, acetic acid aqueous solution, or phosphoric acid aqueous solution; an elution method with a mobile phase flow rate of 0.22-0.28 mL / min and gradient elution; and an elution column temperature of 28-32℃.

[0009] Preferably, the gradient elution procedure is as follows:

[0010] Preferably, the chromatographic column with octadecylsilane-bonded silica gel as the stationary phase is a CORTECS T3 column with dimensions of 2.1 × 100 mm and a particle size of 1.6 μm; the eluent includes mobile phase A, which is acetonitrile, and mobile phase B, which is an aqueous solution of acetic acid with a concentration of 0.1% by volume; the ultra-high performance liquid chromatograph is an ultra-high performance liquid chromatograph equipped with an ultraviolet detector; more preferably, the detection wavelength parameter of the ultra-high performance liquid chromatograph equipped with an ultraviolet detector is set to 250-400 nm.

[0011] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. This invention establishes a method for determining the content of isoquercitrin in Abutilon theophrasti seeds using ultra-high performance liquid chromatography (UHPLC) with reasonable chromatographic conditions. The method exhibits good specificity and robustness, providing a new analytical tool for the quality detection of Abutilon theophrasti seeds. 2. This invention uses isoquercitrin, a flavonoid component, as an indicator, rather than low-polarity components such as fatty acids, making it more suitable for samples using highly polar solvents as a matrix. 3. The scope of application of this invention is not limited to Abutilon theophrasti medicinal materials but also extends to the detection of standard decoctions, formulated granules, and other products containing Abutilon theophrasti seeds. It is the first to establish isoquercitrin as a key indicator component applicable to the entire process of quality control of Abutilon theophrasti from raw materials to water-extracted preparations. 4. This invention's method is simple to operate, has good reproducibility and stability, low detection cost, high detection efficiency, and minimal environmental pollution. Attached Figure Description

[0012] Figure 1 This is the UV absorption spectrum of isoquercitrin reference solution.

[0013] Figure 2 The image shows ultra-high performance liquid chromatograms of velvetleaf seed formulation granules extracted with different solvents.

[0014] Figure 3 This is a linear relationship between the injection amount and peak area of ​​isoquercitrin reference standard in ultra-high performance liquid chromatography.

[0015] Figure 4 Ultra-high performance liquid chromatograms showing the results of different flow rates.

[0016] Figure 5 Ultra-high performance liquid chromatograms of the results obtained at different temperatures.

[0017] Figure 6 Ultra-high performance liquid chromatograms showing the results of different S / N columns.

[0018] Figure 7 Ultra-high performance liquid chromatogram of the specificity test results for determining the content of Abutilon husk granules in the formula.

[0019] Figure 8 Ultra-high performance liquid chromatogram of the specificity test results for the determination of Abutilon theophrasti seed content in standard decoction.

[0020] Figure 9 Ultra-high performance liquid chromatogram of the results of the specificity test for the determination of Abutilon theophrasti seed content.

[0021] Figure 10 Ultra-high performance liquid chromatograms showing the results of different types of chromatographic columns. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Example 1: Construction of a method for determining the content of Abutilon husk granules in a formula 1. Preparation of reference solution Take an appropriate amount of isoquercitrin reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 5 μg per 1 ml.

[0024] 2. Chromatographic conditions (1) Determination of detection wavelength The isoquercitrin reference solution was scanned at wavelengths of 190–400 nm, and its ultraviolet absorption spectrum was recorded. The results are shown in the figure. Figure 1 The results showed that the isoquercitrin reference solution exhibited strong absorption in the range of 200 nm to 400 nm, making it suitable for detection. Considering the significant absorption peak around 255 nm, the optimal detection wavelength was 255 nm.

[0025] (2) Determination of chromatographic conditions A CORTECS T3 column (100 mm in length, 2.1 mm in inner diameter, and 1.6 μm in particle size) was used as the chromatographic column; acetonitrile was used as the mobile phase A, and 0.1% acetic acid solution was used as the mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 0.25 mL per minute; the column temperature was 30 °C; and the detection wavelength was 255 nm.

[0026]

[0027] 3. Preparation of the test solution (1) Investigation of extraction solvent Take an appropriate amount of Abutilon theophrasti seed granules (batch number: 21100109), grind them finely, and take approximately 0.9g (3 groups in total). Accurately weigh each group, and precisely add 25ml each of water, 70% methanol, and methanol. Seal the containers tightly and weigh them. Sonicate the mixture (250W power, 40kHz frequency) for 30 minutes, cool, and weigh again. Make up the lost weight with the corresponding solvent, shake well, filter, and collect the filtrate. Accurately pipette 2µl of each test solution and inject it into an ultra-high performance liquid chromatograph. Determine the content of isoquercitrin under the chromatographic conditions described above, record the peak area value, and calculate its content. The results are shown in Table 1. Figure 2 .

[0028] Table 1 Comparison of different extraction solvents

[0029] The results show that the highest content was obtained by extraction with 70% methanol, and 70% methanol is the preferred extraction solvent.

[0030] (2) Investigation of different extraction solvents Take an appropriate amount of Abutilon theophrasti seed granules (batch number: 21100109), grind them finely, and take about 0.9g in three groups. Accurately weigh each group and place them in a stoppered conical flask. Accurately add 25ml of 70% methanol, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, centrifuge, take 20ml of the supernatant, evaporate to dryness, add 25ml of water to dissolve, and extract three times with ethyl acetate or water-saturated n-butanol, 25ml each time. Combine the upper organic liquids, evaporate to dryness at 60℃, dissolve the residue in methanol, transfer to a 2ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate. Accurately pipette 2µl of each test solution and inject it into an ultra-high performance liquid chromatograph. Determine the content of isoquercitrin under the above chromatographic conditions, record the peak area value, and calculate its content. The results are shown in Table 2.

[0031] Table 2 Comparison of different extraction solvents

[0032] The results show that the content obtained after extraction is lower than that obtained by direct ultrasonic extraction, therefore extraction is not used.

[0033] Based on the above research results, the method for preparing the test solution is as follows: Take an appropriate amount of Abutilon theophrasti seed granules, grind them into a fine powder, take about 0.9g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, seal tightly, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake well, filter it, and take the filtrate to obtain the product.

[0034] (5) Detection of different batches of Abutilon husk granules Take appropriate amounts of three batches of Abutilon theophrasti seed formula granules, with two replicates for each batch. Prepare the test solution according to the method described in Example 1, inject it into an ultra-high performance liquid chromatograph, and determine the isoquercitrin content under the chromatographic conditions described above. The results are shown in Table 3.

[0035] Table 3. Determination of isoquercitrin content in Abutilon nut granules

[0036] The results showed that the RSD of the parallel samples in the same batch was less than 2%, indicating that the method for determining isoquercitrin in Abutilon husk samples is stable, feasible, reproducible, and reliable.

[0037] Example 2: Methodological validation of the content determination construction method 1. Linear relationship Accurately pipette 0.5 µl, 1.0 µl, 2.0 µl, 3.0 µl, and 4.0 µl of isoquercitrin (4.85 μg / ml) reference standard, respectively, and inject them into the ultra-high performance liquid chromatograph. Measure the values ​​under the chromatographic conditions described above. Plot a standard curve with peak area (µg) on ​​the ordinate and injection volume (µg) on ​​the abscissa. The regression equation obtained is: Y = 10894543.28X - 3100.33, R0 2 =1.0000, the results are shown in Table 4. Figure 3 .

[0038] Table 4. Relationship between peak area and injection volume of isoquercitrin reference standard

[0039] The results showed that within the injection range of 0.0024 μg to 0.0194 μg, the injection amount of isoquercitrin exhibited a good linear relationship with the peak area.

[0040] 2. Precision test Accurately pipette 2µl of the Abutilon husk test solution (batch number of Abutilon husk formula granules: 21100109) into the ultra-high performance liquid chromatograph, and determine it under the above chromatographic conditions. Perform 6 consecutive injections, record the peak area value of isoquercitrin, and calculate the relative standard deviation. The results are shown in Table 5.

[0041] Table 5 Instrument Precision Test

[0042] The results showed that the peak area RSD of isoquercitrin was 0.15%, indicating good instrument precision.

[0043] 3. Stability test Take one sample of Abutilon husk to prepare a test solution (batch number of Abutilon husk formula granules: 21100109). Inject 2µl every 4 hours, record the peak area of ​​isoquercitrin, calculate its RSD, and measure for a total of 24 hours. The results are shown in Table 6.

[0044] Table 6 Results of stability test

[0045] The results showed that the peak area RSD of isoquercitrin was 1.68%, and the test solution had good stability within 24 hours.

[0046] 4. Repeatability test Take an appropriate amount of Abutilon theophrasti seed sample (batch number of Abutilon theophrasti seed formula granules: 21100109), grind it into a fine powder, take about 0.9g, weigh it accurately, and make 6 parallel portions. Prepare the sample test solution according to the test solution preparation method. Inject 2µl of each sample, record the peak area value of isoquercitrin, calculate the content of isoquercitrin and the RSD value. The results are shown in Table 7.

[0047] Table 7 Repeatability Tests

[0048] The results showed that the peak area RSD of isoquercitrin was 0.51%, indicating good repeatability.

[0049] 5. Durability test (1) Investigation of different flow velocities One sample (batch number of Abutilon husk granules: 21100109) was taken and a test solution was prepared according to the preparation method of the test solution. The effect of three flow rates (0.22 ml / min, 0.25 ml / min, and 0.28 ml / min) on the content determination was investigated. The results are shown in Table 8. Figure 4The chromatographic column used was a CORTECS T3 (100 mm column length, 2.1 mm inner diameter, and 1.6 μm particle size); the mobile phase was acetonitrile-0.1% acetic acid solution; the detection wavelength was 255 nm; the column temperature was 30 °C; and gradient elution was used.

[0050] Table 8. Investigation at different flow velocities

[0051] The results showed that the separation of isoquercitrin chromatographic peaks met the requirements within the flow rate range of 0.22 ml / min to 0.28 ml / min, and the measured contents were basically consistent, indicating that small changes in flow rate had no significant effect on the determination of isoquercitrin content and that the flow rate was robust.

[0052] (2) Investigation at different column temperatures One sample (batch number of Abutilon husk granules: 21100109) was taken and a test solution was prepared according to the preparation method of the test solution. The effect of three temperatures (28℃, 30℃, and 32℃) on the content determination was investigated. The results are shown in Table 9. Figure 5 The chromatographic column used was a CORTECS T3 (100 mm column length, 2.1 mm inner diameter, and 1.6 μm particle size); the mobile phase was acetonitrile-0.1% acetic acid solution; the detection wavelength was 255 nm; the column temperature was 30 °C; and gradient elution was used.

[0053] Table 9. Investigation at different column temperatures

[0054] The results showed that the separation of isoquercitrin chromatographic peaks met the requirements within the column temperature range of 28℃ to 32℃, and the measured contents were basically consistent, indicating that small changes in column temperature had no significant effect on the determination of isoquercitrin content, and the column had good durability.

[0055] (3) Investigation of different chromatographic columns One sample (batch number of Abutilon theophrasti granules: 21100109) was taken and a test solution was prepared according to the preparation method for the test solution. The Abutilon theophrasti granule sample was analyzed using different S / N number CORTECS T3 columns (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm). Results are shown below. Figure 6 The mobile phase was acetonitrile-0.1% acetic acid solution; the detection wavelength was 255 nm; the column temperature was 30 °C; and gradient elution was used.

[0056] The results showed that the resolution of the isoquercitrin peak met the requirements in different S / N columns, indicating that different chromatographic columns had little impact on the determination of sample content and had good robustness.

[0057] 6. Accuracy Test Accurately weigh 0.45 g of Abutilon theophrasti granules (batch number: 21100109) with known content, add 10 ml of isoquercitrin (1.94 μg / ml) reference solution to each of the six portions, prepare the test solution according to the test solution preparation method, and determine the chromatographic conditions described above. Inject 2 µl of each solution, calculate the recovery rate and RSD value, and the results are shown in Table 10.

[0058] Table 10. Accuracy Test of Isoquercitrin

[0059] Results: The recovery rate of isoquercitrin in Abutilon julibrissin was 95.64%–104.80%, with an RSD of 2.75%, and the accuracy test showed good results.

[0060] Example 3: Specificity test for the determination of isoquercitrin content in Abutilon theophrasti seeds. Determination of isoquercitrin content in Abutilon husk granules Take approximately 0.9 g of Abutilon theophrasti seed formula granules (batch number: 23090109) and prepare the test solution according to the preparation method for the test solution. Inject 2 µl each of the Abutilon theophrasti seed formula granule test solution, isoquercitrin reference solution, and negative sample solution into the liquid chromatograph. The chromatographic conditions are as follows: column: CORTECS T3, 2.1 × 100 mm, particle size 1.6 μm; elution method: mobile phase flow rate 0.25 mL / min, gradient elution; elution column temperature: 30℃; detection wavelength: 255 nm; determine and record the chromatogram. The results are shown in the figure. Figure 7 The negative sample solution consisted of excipients (magnesium stearate, silicon dioxide, maltodextrin) and 70% methanol.

[0061] The results showed that the excipients and solvents did not interfere with the determination of isoquercitrin in Abutilon fibrosum granules. This method has strong specificity and is suitable for the content determination of Abutilon fibrosum granules.

[0062] Determination of isoquercitrin content in Abutilon theraegilate standard decoction Take approximately 0.9 g of Abutilon theophrasti standard decoction (batch number: DG2108337) and prepare the test solution according to the preparation method for the test solution. Inject 2 µl each of the Abutilon theophrasti granule test solution, isoquercitrin reference solution, and negative sample solution into the liquid chromatograph. The chromatographic conditions are as follows: column: CORTECS T3, 2.1 × 100 mm, particle size 1.6 μm; elution method: mobile phase flow rate 0.25 mL / min, gradient elution; column temperature: 30℃; detection wavelength: 255 nm; determine and record the chromatogram. The results are shown in the figure. Figure 8 .

[0063] Results: The solvent did not interfere with the determination of isoquercitrin in the standard decoction of Abutilon theophrasti seeds. This method has strong specificity and is suitable for the content determination of Abutilon theophrasti seeds in the standard decoction.

[0064] Determination of isoquercitrin content in Abutilon theophrasti seeds Take approximately 1g of Abutilon theophrasti seed (batch number: YC2108337) and prepare the test solution according to the preparation method for the test solution. Inject 2µl each of the Abutilon theophrasti granule test solution, isoquercitrin reference solution, and negative sample solution into the liquid chromatograph. The chromatographic conditions are as follows: column: CORTECS T3, 2.1×100mm, particle size 1.6μm; elution method: mobile phase flow rate 0.25mL / min, gradient elution; column temperature: 30℃; detection wavelength: 255nm; determine and record the chromatogram. The results are shown in the figure. Figure 9 .

[0065] Results: The solvent did not interfere with the determination of isoquercitrin in Abutilon theophrasti seeds. This method has strong specificity and is suitable for the content determination of Abutilon theophrasti seeds.

[0066] Comparative Example 1: Comparison of different chromatographic columns One sample (batch number of Abutilon theophrasti granules: 21100109) was taken, and a test solution was prepared according to the preparation method of the test solution. Five chromatographic columns were used to analyze the Abutilon theophrasti granule sample: CORTECS T3 (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm), HSS T3 (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm), BEH Sheild RP18 (column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm), EC-C18 (column length 100 mm, inner diameter 2.1 mm, particle size 1.9 μm), and EP-C18 (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm). The mobile phase was acetonitrile-0.1% acetic acid solution; the detection wavelength was 255 nm; the column temperature was 30 °C; and gradient elution was used. Results are shown below. Figure 10 In the chromatograms obtained from different columns in this comparative example, the BEH Sheild RP18 column showed an uneven baseline and small, unclear peaks; the HSS T3 column did not elute the isoquercitrin peak under gradient conditions; the EC-C18 column showed poor peak resolution and interference from other peaks; the EP-C18 column showed extremely low peaks that were difficult to identify; and the CORTECS T3 column showed a stable baseline with no other peaks, making it suitable for the detection of isoquercitrin in Abutilon theophrasti seeds.

Claims

1. A method for detecting the content of isoquercitrin in a sample of Abutilon theophrasti seeds, characterized in that, Includes the following steps: (1) Preparation of test solutions for samples related to Abutilon theophrasti seeds; (2) Inject the test solution into the ultra-high performance liquid chromatograph, analyze and record the chromatogram; (3) Compare the chromatogram of the test solution with the chromatogram of isoquercitrin standard to calculate the content of isoquercitrin in the velvetleaf seed sample.

2. The method according to claim 1, characterized in that, Step (2) The chromatographic conditions of the ultra-high performance liquid chromatograph include: the chromatographic column is an octadecylsilane-bonded silica gel column as the stationary phase; the eluent includes mobile phase A and mobile phase B, wherein mobile phase A is methanol or acetonitrile, and mobile phase B is any one of water, formic acid aqueous solution, acetic acid aqueous solution, or phosphoric acid aqueous solution; the elution method is gradient elution with a mobile phase flow rate of 0.22-0.28 mL / min; and the elution column temperature is 28-32℃.

3. The method according to claim 2, characterized in that, The gradient elution procedure is as follows: 。 4. The method according to claim 2, characterized in that, The chromatographic column used was a CORTECS T3.

5. The method according to claim 2, characterized in that, The ultra-high performance liquid chromatograph is an ultra-high performance liquid chromatograph equipped with an ultraviolet detector; the detection wavelength is 250-400nm.

6. The method according to claim 1, characterized in that, Step 1, the preparation of the test solution, includes: adding methanol-water solution to the sample, extraction, filtration, and obtaining the test solution.

7. The method according to claim 6, characterized in that, The methanol-water solution has a methanol concentration of 70% and is used at a rate of 15-100 mL per gram of sample.

8. The method according to claim 6, characterized in that, The extraction is performed using ultrasound at 200-300W power and 35-45kHz frequency for 15-60 minutes.

9. The method according to claim 1, characterized in that, The sample of Abutilon theophrasti seed can be any one of the following: Abutilon theophrasti raw material, standard decoction, extract, formula granules, or a reference sample, intermediate product, or finished product of a classic formula containing Abutilon theophrasti seed.

10. The application of the method according to any one of claims 1-9 in the quality control or quality testing of velvetleaf samples.

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