Method for determining content of astragaloside IV in kernel-bighead atractylodes rhizome stomach-invigorating granules
By improving extraction and chromatographic conditions, the accuracy problem of astragaloside A content determination in Renshu Jianwei Granules, a traditional Chinese medicine preparation, was solved, achieving rapid and accurate detection and ensuring consistency between quality control and efficacy.
Patent Information
- Application Number
- CN202512021301.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-30
- Publication Date
- 2026-03-13
AI Technical Summary
Existing technologies are insufficient for accurately determining the content of astragaloside A in the complex traditional Chinese medicine preparation Renshu Jianwei Granules. There are many interfering factors, the detection methods are unstable, and the consistency of quality control and efficacy is affected.
By employing improved extraction methods and chromatographic conditions, astragaloside A was extracted by heating and reflux. Combined with an octadecylsilane-bonded silica gel column and an evaporative light scattering detector, a more efficient and stable method for determining the content of astragaloside A was established, reducing interference from impurities and improving resolution and system applicability.
This method enables rapid and accurate determination of astragaloside A content, reduces the detection limit, improves reproducibility and stability, and ensures the quality consistency and clinical efficacy of Renshu Jianwei Granules.
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Figure CN121656446A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for determining the content of active ingredients in traditional Chinese medicine preparations, specifically a method for determining the content of astragaloside A in Renshu Jianwei granules. Background Technology
[0002] Ren Shu Jian Wei Granules were developed under the leadership of Professor Shan Zhaowei, a nationally renowned traditional Chinese medicine doctor. As the academic leader of the Department of Gastroenterology at Jiangsu Provincial Hospital of Traditional Chinese Medicine (National Clinical Research Base for Gastroenterology), Professor Shan, through over forty years of clinical and research experience, systematically proposed that the core pathogenesis of atrophic gastritis and precancerous lesions is "qi deficiency, blood stasis, and heat stagnation," and established "tonifying qi, activating blood circulation, and clearing heat" as the fundamental treatment principle. Based on this, he created this preparation. The entire formula consists of roasted astragalus, stir-fried atractylodes macrocephala, coix seed, and wine-processed scutellaria baicalensis, among other herbs.
[0003] Astragalus membranaceus (processed with honey) is the principal ingredient in this formula, primarily used to invigorate qi and strengthen the spleen to address the root cause of the illness. Modern pharmacological research indicates that astragaloside A (a triterpenoid saponin compound) contained in processed astragalus membranaceus is one of its key active ingredients and the main material basis for the efficacy of Renshu Jianwei Granules. Therefore, accurate determination of the astragaloside A content is crucial for controlling the quality of this product. However, due to the complexity of the chemical components in the compound formula and the presence of many interfering factors, establishing a precise and stable detection method that can eliminate interference is particularly necessary. Summary of the Invention
[0004] To improve the accuracy and reliability of quality control, this invention makes significant improvements to the method for determining astragaloside A content, mainly achieving breakthroughs in extraction method and chromatographic conditions. The new method, by changing the extraction method, more efficiently enriches the target component and reduces interference from impurities; simultaneously, it employs more stable chromatographic conditions, resulting in significantly improved peak shape and resolution of astragaloside A, and good system applicability. The new method demonstrates superior performance in terms of limit of detection, limit of quantitation, repeatability, accuracy, and robustness, and can be more reliably used for the quality evaluation of Renshu Jianwei Granules, thus providing a solid guarantee for the consistency of its clinical efficacy.
[0005] The technical solution of this invention is as follows: A method for determining the astragaloside A content in Renshu Jianwei Granules includes the following steps: Step 1, Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, and dissolve it in 80% methanol to obtain the solution; Step 2, Preparation of the test solution: Take Renshu Jianwei Granules and add them to a 50% methanol solution containing 4% concentrated ammonia test solution. Heat under reflux, centrifuge, take the supernatant, evaporate to dryness, add 80% methanol to the residue to make up to volume, filter, and take the filtrate to obtain the test solution. Step 3: Accurately pipette the reference solution and the test solution separately and inject them into the liquid chromatograph. Measure using an evaporative light scattering detector. Plot the natural logarithm of the peak area on the ordinate and the natural logarithm of the injected volume on the abscissa using the external standard two-point method to obtain the measurement results. The liquid chromatography conditions are as follows: Column packing material: octadecylsilane-bonded silica gel; Mobile phase: Acetonitrile is used as mobile phase A and water is used as mobile phase B. Gradient elution is performed according to the specifications in the table below. The detector is an evaporative light scattering detector.
[0006]
[0007] Furthermore, the concentration of astragaloside A reference standard in the reference solution described in step 1 is 0.5 mg / ml.
[0008] Furthermore, the concentration of Renshu Jianwei granules in step 2 in a 50% methanol solution containing 4% concentrated ammonia solution is 40-80 mg / ml.
[0009] Furthermore, the heating reflux time in step 2 is 60 min to 90 min, preferably 60 min.
[0010] Furthermore, the centrifugation in step 2 is performed at 4000 r / min for 5 minutes.
[0011] Furthermore, the residue described in step 2 is diluted to 5 ml with 80% methanol.
[0012] Furthermore, the liquid chromatography conditions described in step 3 are a flow rate of 1.0 ml / min and a column temperature of 30 °C.
[0013] Furthermore, the evaporative light scattering detector conditions were: gas flow rate 1.80 SLM, evaporator temperature 70°C, atomizer temperature 60°C, and PMT gain 1.0.
[0014] The beneficial effects of this invention are as follows: The sample detection method established in this invention is rapid and accurate, with low detection and quantitation limits, no interference from negative samples, strong specificity, high reproducibility and stability, and good peak shape of the obtained chromatograms, which can more effectively control the astragaloside A content of Renshu Jianwei Granules. Attached Figure Description
[0015] Figure 1 The figure shows the experimental results of the specificity of astragaloside A.
[0016] Figure 2 The liquid chromatogram of Renshu Jianwei Granules sample. Detailed Implementation
[0017] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.
[0018] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.
[0019] The instruments and reagents used in the examples are as follows: Instruments: Agilent Technologies 1260II, including the quaternary pump system 1260 Infinity II, the fully automated injection system 1260 Infinity, the automatic temperature-controlled column oven 1260 Infinity; evaporative light scattering detector 1260 Infinity II ELSD; ML104 / 02 electronic analytical balance (Mettler Toledo); XPR2 electronic analytical balance (Mettler Toledo); DK-98-IIA electric thermostatic water bath (Tianjin Tester Instrument Co., Ltd.); KH-500E ultrasonic instrument (Kunshan Hechuang Ultrasonic Instrument Co., Ltd.).
[0020] Drug trials: Renshu Jianwei Granules Sample (220702, Nanjing Zhongshan Pharmaceutical Co., Ltd.); Negative samples were prepared according to the prescription ingredients, excluding the processed Astragalus membranaceus. Astragaloside A reference standard was purchased from the National Institutes for Food and Drug Control. Methanol (analytical grade); concentrated ammonia (analytical grade); acetonitrile (chromatographic grade); ultrapure water.
[0021] Example 1: Determination of Astragaloside A Content in Renshu Jianwei Granules Step 1: Preparation of the reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, and add 80% methanol to prepare a solution containing 0.5 mg per ml.
[0022] Step 2, Preparation of the test solution: Take an appropriate amount of Renshu Jianwei Granules and a negative sample, grind them into a fine powder, take about 2g, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 50% methanol solution containing 4% concentrated ammonia test solution (take 4ml of concentrated ammonia test solution, add 50% methanol to 100ml, shake well), stopper tightly, weigh, heat under reflux for 1h, cool, weigh again, use 50% methanol solution containing 4% concentrated ammonia test solution to make up the lost weight, shake well, centrifuge at 4000r / min for 5 minutes, accurately measure 25ml of supernatant, evaporate to dryness, add 80% methanol to dissolve the residue, transfer to a 5ml volumetric flask, add 80% methanol to the mark, shake well, filter, and take the filtrate to obtain the product.
[0023] Step 3: Accurately pipette 2 μl, 5 μl (or 10 μl) of the reference solution from Step 1 and 20 μl of the test solution from Step 2, respectively, and inject them into the liquid chromatograph. Measure using an evaporative light scattering detector. Plot the natural logarithm of the peak area on the ordinate and the natural logarithm of the injection volume on the abscissa, and calculate using the external standard two-point method to obtain the determination result.
[0024] The liquid chromatography conditions were as follows: column packing material: octadecylsilane-bonded silica gel (YMC-pack ODS-A, 5μm*4.6mm*250mm); flow rate: 1.0ml / min; column temperature: 30℃.
[0025] Mobile phase: Acetonitrile is used as mobile phase A and water is used as mobile phase B. Gradient elution is performed according to the specifications in the table below.
[0026] The evaporation light scattering conditions were: gas flow rate 1.80 SLM, evaporator temperature 70℃, atomizer temperature 60℃, and PMT gain 1.0.
[0027]
[0028] The chromatogram of the test sample is shown below. Figure 1 The results showed that under the sample processing method and chromatographic conditions, the target peak of astragaloside A was well separated, with few impurities, no interfering peaks before and after the target peak, and the chromatographic peak was located at a relatively flat baseline, which could accurately achieve the quantitative detection of astragaloside A.
[0029] Example 2: Sample Pretreatment Investigation 1. Examination of extraction methods The extraction methods of the test samples were investigated, and the effects of different extraction methods (ultrasound, heating and reflux) on the extraction efficiency of astragaloside A were compared to determine the optimal extraction method.
[0030] Preparation of the test solution: Take Renzhu Jianwei Granules, grind them into a fine powder, take about 2g, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 50% methanol solution containing 4% concentrated ammonia test solution (take 4ml of concentrated ammonia test solution, add 50% methanol to 100ml, shake well), stopper tightly, weigh, and then treat with ultrasound (power 500W, frequency 40kHz) and heat under reflux for 1h, cool, weigh again, and make up the lost weight with 50% methanol solution containing 4% concentrated ammonia test solution, shake well, centrifuge at 4000r / min for 5 minutes, accurately measure 25ml of the supernatant, evaporate to dryness, dissolve the residue in 80% methanol, transfer it to a 5ml volumetric flask, add 80% methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.
[0031] The reference solution was prepared according to the method of Example 1, and the detection was performed according to the detection conditions of Example 1.
[0032] Table 1. Effects of different extraction methods on astragaloside A content (n=3)
[0033] The results showed that different extraction methods had a significant impact on the extraction efficiency of astragaloside A, and heating reflux was selected as the extraction method.
[0034] 2. Examination of sample quantity The sample weight was investigated to compare the effects of different sample weights (2g, 4g) on the extraction efficiency of astragaloside A, and the sample weight was determined.
[0035] Preparation of the test solution: Take Renzhu Jianwei granules, grind them finely, and accurately weigh 2g and 4g respectively. Place them in a stoppered conical flask, and accurately add 50ml of 50% methanol solution containing 4% concentrated ammonia test solution (take 4ml of concentrated ammonia test solution, add 50% methanol to 100ml, and shake well). Stopper tightly, weigh, heat under reflux for 1h, cool, weigh again, and replenish the lost weight with 50% methanol solution containing 4% concentrated ammonia test solution. Shake well, centrifuge at 4000r / min for 5 minutes, accurately measure 25ml of the supernatant, evaporate to dryness, dissolve the residue in 80% methanol, transfer to a 5ml volumetric flask, add 80% methanol to the mark, shake well, filter, and collect the filtrate to obtain the test solution. Prepare the reference solution according to the method of Example 1, and perform detection according to the detection conditions of Example 1.
[0036] Table 2. Effect of different sample weights on astragaloside A content (n=3)
[0037] The results showed that different sample weights had little effect on the extraction efficiency of astragaloside A. Considering the simplicity of the experiment, 2g was chosen as the sample weight.
[0038] 3. Examination of extraction time The treatment methods of the test samples were investigated, and the effects of different extraction times (30 min, 60 min, 90 min) on the extraction efficiency of astragaloside A were compared to determine the optimal extraction method.
[0039] Preparation of the test solution: Take Renzhu Jianwei granules, grind them finely, take about 2g, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 50% methanol solution containing 4% concentrated ammonia test solution (take 4ml of concentrated ammonia test solution, add 50% methanol to 100ml, shake well), stopper tightly, weigh, heat under reflux for 30min, 60min, and 90min respectively, cool, weigh again, replenish the lost weight with 50% methanol solution containing 4% concentrated ammonia test solution, shake well, centrifuge at 4000r / min for 5 minutes, accurately measure 25ml of supernatant, evaporate to dryness, dissolve the residue in 80% methanol, transfer to a 5ml volumetric flask, add 80% methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution. Prepare the reference solution according to the method of Example 1, and perform detection according to the detection conditions of Example 1.
[0040] Table 3. Effect of different extraction times on astragaloside A content (n=3)
[0041] The results showed that an extraction time of 30 min resulted in a lower extraction efficiency of astragaloside A than extraction times of 60 min and 90 min. The extraction times of 60 min and 90 min had little effect on the extraction efficiency of astragaloside A. Considering the need to save time, the study selected an extraction time of 60 min.
[0042] 4. Investigation of extraction solvent The treatment methods for the test samples were investigated, and the effects of different extraction solvents (methanol solution containing 4% concentrated ammonia, 80% methanol solution containing 4% concentrated ammonia, 50% methanol solution containing 4% concentrated ammonia, and 25% methanol solution containing 4% concentrated ammonia) on the extraction efficiency of astragaloside A were compared to determine the optimal extraction method.
[0043] Preparation of the test solution: Take Renzhu Jianwei granules, grind them finely, take about 2g, accurately weigh it, and place it in a stoppered conical flask. Accurately add 50ml each of the following solutions: methanol solution containing 4% concentrated ammonia test solution, 80% methanol solution containing 4% concentrated ammonia test solution, 50% methanol solution containing 4% concentrated ammonia test solution, and 25% methanol solution containing 4% concentrated ammonia test solution. Seal tightly, weigh, heat under reflux for 60min, cool, and weigh again. Make up the weight loss with the extraction solvent, shake well, centrifuge at 4000r / min for 5 minutes, accurately measure 25ml of the supernatant, evaporate to dryness, dissolve the residue in 80% methanol, transfer to a 5ml volumetric flask, add 80% methanol to the mark, shake well, filter, and collect the filtrate to obtain the test solution. Prepare the reference solution according to the method of Example 1, and perform detection according to the detection conditions of Example 1.
[0044] Table 4. Effects of different extraction solvents on astragaloside A content (n=3)
[0045] The results showed that different extraction solvents had a significant impact on the extraction efficiency of astragaloside A. Among them, 50% methanol containing 4% concentrated ammonia solution had the highest extraction efficiency, so it was selected as the extraction solvent.
[0046] Example 3 Methodological Investigation: Limit of detection and limit of quantitation The limits of detection (LOD) and quantitation (LOQ) of the method were determined by a signal-to-noise ratio (S / N) of 3 and 10, respectively. The LOD of astragaloside A was 0.025 μg and the LOQ was 0.084 μg.
[0047] 1. Specificity test The test solution and negative sample solution were prepared according to the method in Example 1. 20 μl of the negative sample solution, 2 μl of the control solution, and 20 μl of the test solution were respectively taken and tested according to the detection conditions in Example 1.
[0048] The results showed that, according to the method of this invention, no corresponding chromatographic peak appeared in the negative sample at the corresponding retention time of astragaloside A, and the negative sample did not interfere with the determination. Figure 2 .
[0049] 2. Linear relationship Accurately pipette 2 μl, 3 μl, 4 μl, 5 μl, 7 μl, 10 μl, and 15 μl of a 0.5001 mg / mL reference solution and inject them into the liquid chromatograph for determination. The result is expressed as IgA. 峰面积 Let lgm be the vertical axis. 进样量 Let x be the x-axis, and construct a linear equation. The linear equation is y = 1.7567x + 1.9829, R ∈ R. 2 =0.9996, indicating that astragaloside A has a good linear relationship in the range of 1.0002ug to 7.5015ug.
[0050] 3. Precision The reference solution prepared according to the method in Example 1 was injected six times consecutively with an injection volume of 7 μl. The RSD value of the peak area was 0.50%, indicating that the instrument has good parallel injection precision.
[0051] Table 5 Precision test results
[0052] 4. Repeatability Six samples were accurately weighed in parallel and prepared into six test solutions according to the method in Example 1. The results were determined according to the above chromatographic conditions. The RSD of astragaloside A was 3.44%, indicating that the method has good repeatability.
[0053] Table 6 Repeatability Test Results
[0054] 5. Stability The test solution was prepared according to the method in Example 1, and its stability was determined by injection at different time points of 0, 2, 4, 6, 8, 10, 12, 18, and 24 hours. The RSD of the peak area of astragaloside A was 2.23%, indicating that the method has good stability.
[0055] Table 7 Stability Test Results
[0056] 6. Recovery rate Nine 2g portions of sample were accurately weighed, and 1.6ml, 2.0ml, and 2.4ml of a 0.3577mg / ml reference solution were added respectively. The samples were prepared according to the test sample preparation method and determined under the chromatographic conditions described above. The recovery rates were calculated. The RSD of the recovery rate of astragaloside A was 97.09%-107.85%, and the RSD of the recovery rate was 3.24%, indicating that the method has high accuracy. The results are shown in Table 8.
[0057] Table 8 Results of the recovery test
[0058] The above experimental results show that the method for determining the astragaloside A content of Renshu Jianwei Granules provided by the present invention has good stability, high precision, and good repeatability. It can objectively evaluate the quality of Renshu Jianwei Granules and is of great significance for ensuring clinical efficacy.
[0059] 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 astragaloside A in Renshu Jianwei Granules, characterized in that, Includes the following steps: Step 1, Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, and dissolve it in 80% methanol to obtain the solution; Step 2, Preparation of the test solution: Take Renshu Jianwei Granules and add them to a 50% methanol solution containing 4% concentrated ammonia test solution. Heat under reflux, centrifuge, take the supernatant, evaporate to dryness, add 80% methanol to the residue to make up to volume, filter, and take the filtrate to obtain the test solution. Step 3: Accurately pipette the reference solution and the test solution separately and inject them into the liquid chromatograph. Measure using an evaporative light scattering detector. Plot the natural logarithm of the peak area on the ordinate and the natural logarithm of the injected volume on the abscissa using the external standard two-point method to obtain the measurement results. The liquid chromatography conditions were as follows: column packing material: octadecylsilane-bonded silica gel; Mobile phase: Using acetonitrile as mobile phase A and water as mobile phase B, gradient elution is performed according to the specifications in the table below: The detector is an evaporative light scattering detector. 。 2. The determination method according to claim 1, characterized in that, The concentration of astragaloside A reference standard in the reference solution described in step 1 is 0.5 mg / ml.
3. The determination method according to claim 1, characterized in that, The concentration of Renshu Jianwei granules in step 2 in a 50% methanol solution containing 4% concentrated ammonia solution is 40-80 mg / ml.
4. The determination method according to claim 1, characterized in that, The heating and reflux time in step 2 is 60 min to 90 min, preferably 60 min.
5. The determination method according to claim 1, characterized in that, The centrifugation in step 2 is performed at 4000 r / min for 5 minutes.
6. The determination method according to claim 1, characterized in that, The volume adjustment in step 2 is 5 ml.
7. The determination method according to claim 1, characterized in that, The liquid chromatography conditions described in step 3 are a flow rate of 1.0 ml / min and a column temperature of 30 °C.
8. The determination method according to claim 1, characterized in that, The evaporative light scattering detector conditions were: gas flow rate 1.80 SLM, evaporator temperature 70°C, atomizer temperature 60°C, and PMT gain 1.0.