Method for determining content of multiple flavonoid components in Sanwei Huangjing Tangsan

Abstract

The present application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for determining the content of multiple flavone components in Tibetan medicine Sanwei Huangjing Tangsan, which simultaneously determines the content of three flavone aglycone, i.e., quercetin, kaempferol and isorhamnetin in Sanwei Huangjing Tangsan by using high performance liquid chromatography (HPLC). The present application can accurately determine the content of main flavone effective components in Sanwei Huangjing Tangsan, has a good linear range, high accuracy, precision and repeatability, and is stable and feasible, and can be applied to the quality inspection of Sanwei Huangjing Tangsan.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection technology, specifically relating to a method for determining the content of multiple flavonoid components in Sanwei Huangjing Tang powder. Background Technology

[0002] The Sanwei Huangjing Tang San formula originates from the "Four Medical Classics". This formula consists of one part each of Tribulus terrestris (processed), Polygonatum sibiricum, and Himalayan Mirabilis jalapa. After being ground into coarse powder, the herbs are decocted with 1 part water and 16 parts water until the liquid reduces to 5 parts. One cup of water is taken at a time. It is used to treat various gynecological diseases such as lower abdominal distension and pain, menstrual disorders, dysmenorrhea, and abnormal leukorrhea.

[0003] From the perspective of Tibetan medicine, these symptoms are closely related to the imbalance between "Lung" and "Peigen". "Lung" is the driving force; its imbalance leads to disordered Qi, resulting in lower abdominal distension and menstrual irregularities. "Peigen" governs transportation and nourishment; its imbalance leads to internal cold and dampness, resulting in increased leukorrhea and dysmenorrhea. The formula uses Polygonatum to replenish menstrual blood and calm the imbalance of "Lung", addressing the root cause; Himalayan Mirabilis jalapa warms the kidneys and dispels cold, directly targeting the imbalance of "Peigen", addressing the symptoms; Tribulus terrestris is processed using the "stir-frying method", which enhances its effects of warming the kidneys, replenishing Qi, soothing the liver, and relieving depression, harmonizing the flow of "Lung" and acting as a pivot. These three herbs harmonize the liver, spleen, and kidneys, balancing Lung and Peigen, ultimately achieving harmony of Qi and blood, and unblocking the Chong and Ren meridians, thus healing various gynecological symptoms.

[0004] This invention establishes an HPLC method for determining the content of flavonoid index components quercetin, kaempferol, and isorhamnetin in Sanwei Huangjing Decoction Powder, providing a reference for the quality control of Tibetan medicine Sanwei Huangjing Decoction Powder and for improving and perfecting the quality standards included in the Chinese Pharmacopoeia. Summary of the Invention

[0005] The purpose of this invention is to provide a method for determining the content of various flavonoid components in Sanwei Huangjing Decoction Powder.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: The Three-Ingredient Polygonatum Decoction Powder of this invention is a medicinal powder composed of three medicinal materials: Tribulus terrestris (processed), Polygonatum sibiricum, and Himalayan Mirabilis jalapa in a weight ratio of 1:1:1.

[0007] The present invention discloses a method for determining the content of multiple flavonoid components in Sanwei Huangjing Decoction Powder. High-performance liquid chromatography (HPLC) was used to simultaneously determine the content of the main active ingredients, quercetin, kaempferol, and isorhamnetin. The main active ingredients determined by this invention are all flavonoids. Specifically, Tribulus terrestris contains quercetin, kaempferol, and isorhamnetin; Polygonatum sibiricum contains kaempferol and isorhamnetin; and Mirabilis jalapa contains kaempferol. All of these components possess anti-inflammatory, antioxidant, and immunomodulatory properties, and have good effects on treating lower abdominal distension, menstrual disorders, abnormal leukorrhea, and dysmenorrhea.

[0008] The chromatographic conditions of the high performance liquid chromatography method described in this invention are as follows: UV-Vis absorption detector, mobile phase is acetonitrile-0.05% phosphoric acid solution; gradient elution; flow rate is 1 mL / min; detection wavelength is 360 nm; column temperature is 30 °C.

[0009] In the chromatographic conditions described in this invention, the mobile phase system is acetonitrile-0.05% phosphoric acid water. Under these conditions, the methanol solutions of quercetin, kaempferol, and isorhamnetin can have good peak shapes, and the chromatographic peaks of the test samples have good resolution, which is beneficial for the accurate analysis of components.

[0010] The high-performance liquid chromatography method described in this invention uses an Ultimate XB-C column. 18 (250 mm × 4.6 mm, 5 μm) This chromatographic column is an octadecylsilane-bonded silica gel column, which is a commonly used column for the analysis of flavonoids. It can separate the components of the test solution, which is beneficial for the accurate analysis of the components.

[0011] The gradient elution program of the chromatographic conditions described in this invention includes the following steps: a) at 0 min, the acetonitrile ratio is 20%; b) at 25 min, the acetonitrile ratio is 31%; c) at 28 min, the acetonitrile ratio is 38%; d) at 45 min, the acetonitrile ratio is 38%; e) at 50 min, the acetonitrile ratio is 20%; f) at 60 min, the acetonitrile ratio is 20%.

[0012] Steps a) to b) are rinses performed to achieve complete separation of quercetin; steps c) to d) are rinses performed to achieve complete separation of kaempferol and quercetin; and steps e) to f) are to restore the gradient ratio before the procedure.

[0013] The chromatographic conditions described in this invention employ acetonitrile-0.05% phosphoric acid aqueous gradient elution. The sample solution contains a relatively complex composition, and good resolution is required to analyze the index components. Under these conditions, solvent peak tailing can be effectively reduced, and the signal-to-noise ratio, sensitivity, and theoretical plate number of the index component chromatographic peaks can be improved.

[0014] The method for preparing the test solution according to this invention is as follows: Polygonatum sibiricum, Tribulus terrestris (processed), and Mirabilis jalapa are placed in a 1:1:1 ratio in a grinder and pulverized into fine powder. 3 g of the fine powder is accurately weighed and placed in a 250 mL stoppered conical flask. 90 mL of a methanol-15% hydrochloric acid mixture (v:v, 4:1) is added, the mixture is shaken well, weighed, and heated under reflux in a 90 °C water bath for 4 h. After cooling to room temperature, the mixture is weighed again, and the weight is made up with the methanol-15% hydrochloric acid mixture (v:v, 4:1). The mixture is then filtered to obtain the test solution. The filtrate is filtered through a 0.45 μm organic microporous membrane and injected for analysis.

[0015] The Sanwei Huangjing Tang powder of this invention is a traditional powder made from pulverized raw herbs. This study investigated the effects of hydrochloric acid concentrations of 10%, 15%, 20%, 25%, and 30% (v / v) as hydrolysis solutions. The results showed that when the hydrochloric acid concentration was 10–15%, the content of flavonoid aglycones in the test solution was positively correlated. When the hydrochloric acid concentration was 15–30%, the change in flavonoid aglycone content in the test solution was not significant. However, when 20–30% hydrochloric acid was used as the hydrolysis solution, the prepared test solution was unstable within 60 minutes, and the flavonoid aglycone content decreased significantly. This was because the high concentration of hydrochloric acid further degraded the flavonoid aglycones. Therefore, a 15% hydrochloric acid solution was selected as the extraction solution. This study also investigated the effects of the material-to-liquid ratio and acid hydrolysis time on the flavonoid aglycone content in the test solution through single-factor experiments. The optimal material-to-liquid ratio was 1:30, and the optimal acid hydrolysis time was 4 hours.

[0016] The high-performance liquid chromatography method of this invention also includes a method for preparing the reference solution. Accurately weigh 10.74 mg of quercetin reference standard, 10.50 mg of kaempferol reference standard, and 7.75 mg of isorhamnetin reference standard. The quercetin and kaempferol reference standards are placed in separate 10 mL volumetric flasks, and the isorhamnetin reference standard is placed in a 50 mL volumetric flask. Methanol is added to dissolve and dilute to volume. After shaking well, the resulting solution has a mass concentration of 1060.04 μg·mL⁻¹. -1 1049.58 μg·mL -1 153.61 μg·mL -1 The reference standard stock solution was prepared by accurately pipetting 1 mL of kaempferol reference standard stock solution into a 10 mL volumetric flask, dissolving and diluting with methanol, and shaking well to prepare a solution with a mass concentration of 104.96 μg·mL⁻¹. -1 A diluted solution of kaempferol reference standard was prepared. Accurately pipette 0.6 mL of quercetin and 2.3 mL of isorhamnetin reference standard stock solution and 1.3 mL of kaempferol reference standard diluted solution into the same 50 mL volumetric flask, dissolve and dilute to volume with methanol, and shake well to prepare a solution containing quercetin, kaempferol, and isorhamnetin at mass concentrations of 12.72, 2.73, and 7.07 μg·mL, respectively. -1 A mixed reference solution.

[0017] The pure methanol solution described in this invention is methanol with a purity exceeding 99.9%.

[0018] The high performance liquid chromatography method of the present invention also includes a method for preparing a blank solution: the blank solution is prepared by omitting the addition of Sanwei Huangjing Decoction powder and performing the remaining operations in the same manner as the preparation of the test solution.

[0019] The beneficial effects of this invention are: (1) This invention establishes a high performance liquid chromatography method for the simultaneous quantitative determination of the index components quercetin, kaempferol and isorhamnetin in Sanwei Huangjing Decoction Powder and conducts methodological verification. The results show that the method is scientific and feasible. (2) Compared with separate analysis, the present invention can effectively reduce the number of sample injections and analysis time of the instrument, with short detection time, accurate detection results, and simple and easy operation; (3) The present invention has a wide linear range, good repeatability, stable and feasible sample processing method, and the obtained test solution has good stability at room temperature. (4) The method of the present invention is stable and accurate, and can be used for the quality evaluation of Sanwei Huangjing Decoction Powder, and can provide a reference for improving the quality standard of Sanwei Huangjing Decoction Powder. Attached Figure Description

[0020] Figure 1 The HPLC chromatogram is for the blank solution. Figure 2 This is the HPLC chromatogram of the mixed control solution; Figure 3 The HPLC chromatogram of the test solution is shown below. Figure 4 Linearity graph of quercetin reference standard; Figure 5 The linear relationship graph for kaempferol reference standard is shown. Figure 6 Linearity graph of isorhamnetin reference standard; Figure 7 Linear relationship graph of rutin reference standard; Detailed Implementation

[0021] The following experimental examples are used to further illustrate, but are not limited to, the present invention.

[0022] The Sanwei Huangjing Decoction Powder used in the following specific embodiments is prepared by weighing 30 g of Tribulus terrestris (processed), 30 g of Polygonatum sibiricum, and 30 g of Mirabilis jalapa, grinding them into fine powder, mixing them evenly, and packaging them in aluminum foil bags.

[0023] Kaempferol reference standard (batch number DSTDS005604, purity 99.96%) was purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.; quercetin reference standard (batch number 100081-202411, purity 98.7%) and isorhamnetin reference standard (batch number 110860-202012, purity 99.1%) were both purchased from the China National Institutes for Food and Drug Control; methanol (chromatographic grade, batch number DW55M121V02) and acetonitrile (chromatographic alcohol, batch number DW565121801) were both purchased from Hubei Futon Biochemical Co., Ltd. Technology Co., Ltd.; Phosphoric acid (analytical alcohol, batch number 20250603) and hydrochloric acid (analytical alcohol, batch number 20241015) were purchased from Sinopharm Chemical Reagent Co., Ltd.; Tribulus terrestris (batch number 20250702) and Polygonatum sibiricum (batch number 20250329) were from Bozhou Sihuai Traditional Chinese Medicine Planting Professional Cooperative; Himalayan Mirabilis jalapa (batch number 20231201) was from Tibet Shenhou Pharmaceutical Co., Ltd., and was tested as genuine by the CNAS testing laboratory of Jiangsu Shenhou Pharmaceutical Research Co., Ltd.; Ultrapure water was prepared by a pure water machine.

[0024] Example The contents of quercetin, kaempferol, and isorhamnetin in Sanwei Huangjing Tang powder were simultaneously determined by high performance liquid chromatography, including the following steps: (1) Preparation of the test solution Three medicinal herbs, Polygonatum sibiricum, Tribulus terrestris (processed), and Mirabilis jalapa, were ground into a fine powder using a grinder in a 1:1:1 ratio. 3 g of the fine powder was accurately weighed and placed in a 250 mL stoppered conical flask. 90 mL of a methanol-15% hydrochloric acid mixture (v:v, 4:1) was added, the mixture was shaken well, and the solution was weighed. The flask was heated under reflux in a 90 °C water bath for 4 h, cooled to room temperature, and weighed again. The weight was then made up with the methanol-15% hydrochloric acid mixture (v:v, 4:1). The solution was filtered to obtain the test solution. The filtrate was filtered through a 0.45 μm organic microporous membrane and injected for analysis.

[0025] (2) Preparation of mixed reference solution Accurately weigh 10.74 mg of quercetin reference standard, 10.50 mg of kaempferol reference standard, and 7.75 mg of isorhamnetin reference standard. Place the quercetin and kaempferol reference standards separately in 10 mL volumetric flasks, and the isorhamnetin reference standard in a 50 mL volumetric flask. Dissolve and dilute to volume with methanol, and shake well to obtain a mass concentration of 1060.04 μg·mL⁻¹. -1 1049.58 μg·mL -1 153.61 μg·mL -1 The reference standard stock solution was prepared by accurately pipetting 1 mL of kaempferol reference standard stock solution into a 10 mL volumetric flask, dissolving and diluting with methanol, and shaking well to prepare a solution with a mass concentration of 104.96 μg·mL⁻¹. -1A diluted solution of kaempferol reference standard was prepared. Accurately pipette 0.6 mL of quercetin and 2.3 mL of isorhamnetin reference standard stock solution and 1.3 mL of kaempferol reference standard diluted solution into the same 50 mL volumetric flask, dissolve and dilute to volume with methanol, and shake well to prepare a solution containing quercetin, kaempferol, and isorhamnetin at concentrations of 12.72, 2.73, and 7.07 μg / mL, respectively. -1 A mixed reference solution.

[0026] (3) Preparation of blank solution Without adding fine powder of medicinal materials, the blank solution is prepared by the same method as the preparation of the test solution in step (1).

[0027] (4) Chromatographic conditions The UV-Vis absorption detector was used with an acetonitrile-0.05% phosphoric acid solution as the mobile phase, gradient elution, a flow rate of 1 mL / min, a detection wavelength of 360 nm, a column temperature of 30 °C, and an injection volume of 10 μL.

[0028] (5) Methodological investigation System suitability test: Take the mixed reference solution, test solution, and blank solution, inject them according to the chromatographic conditions, record the chromatograms, and the results are shown in the figure. Figure 1 , Figure 2 , Figure 3 Using the extraction solvent as a blank, the resulting chromatogram showed no interfering peaks at the retention times of quercetin, kaempferol, and isorhamnetin. In the chromatogram of the test solution, the resolution of quercetin, kaempferol, and isorhamnetin peaks from adjacent peaks was greater than 1.5, and their retention times were consistent with those of the components in the reference solution. The suitability test of the chromatographic system met the requirements for content determination.

[0029] Linearity assessment: Accurately pipette 1.0, 2.0, 4.0, 5.0, 6.0, 8.0, and 10.0 mL of the mixed reference solution into separate 10 mL volumetric flasks, dilute to volume with methanol, and determine the concentration (µg·mL⁻¹). -1 (x) is the x-axis X Peak area as the ordinate Y Standard curves were plotted and regression analysis was performed. The results showed that quercetin concentrations ranged from 1.27 to 12.72 μg / mL. -1 The integral value of the inner area and the peak area show a good linear relationship, and the equation is as follows: Y =39.142 X -2.8852 ( r =0.999 9), kaempferol in the range of 0.27–2.73 μg·mL -1 The integral value of the inner area and the peak area show a good linear relationship, and the equation is as follows: Y =41.480 X -0.1243 (r =0.999 9), isorhamnetin in the range of 0.71–7.07 μg·mL -1 The integral value of the inner area and the peak area show a good linear relationship, and the equation is as follows: Y =37.577 X -0.6013 ( r =1.000 0), the result is as follows Figure 4 , Figure 5 , Figure 6 .

[0030] Precision test: Take the mixed reference solution, inject it according to the chromatographic conditions, and determine it 6 times consecutively. Measure the RSD of the peak areas of quercetin, kaempferol, and isorhamnetin. n =6) The values ​​of 0.15%, 0.38%, and 0.19% respectively indicate that the instrument has good precision.

[0031] Repeatability test: Prepare 6 parallel test solutions according to the test solution preparation method, inject and determine the RSD of the peak areas of quercetin, kaempferol, and isorhamnetin under chromatographic conditions. n =6) were 1.25%, 0.84%, and 1.12% respectively, indicating good repeatability.

[0032] Stability test: A fresh test solution was prepared, and measurements were taken at 2, 4, 6, 8, 10, and 12 h. The RSD of the peak areas of quercetin, kaempferol, and isorhamnetin was measured. n =6) were 1.88%, 0.27%, and 1.74% respectively, indicating that the solution was stable within 24 h.

[0033] Spiking recovery test: Accurately weigh 6 portions of Sanwei Huangjing Decoction Powder with known content, 3 g for each portion, and accurately add an appropriate amount of mixed reference solution to prepare the test solution. Inject the sample according to the chromatographic conditions and calculate the average recovery rate and RSD. The results are shown in Table 1.

[0034] Table 1 Results of the recovery rate test ( n =6)

[0035] (6) Determination of the content of various flavonoid components in Sanwei Huangjing Decoction Powder Six batches (S1~S6) of Sanwei Huangjing Decoction were taken, and two samples were taken from each sample. The samples were injected and analyzed according to the chromatographic conditions. The content of each component was determined and calculated. The results are shown in Table 2.

[0036] Table 2 Determination of the content of three flavonoid aglycones ( n =2)

[0037] This invention establishes a high-performance liquid chromatography (HPLC) method for the simultaneous quantitative determination of indicator components quercetin, kaempferol, and isorhamnetin in Sanwei Huangjing Tang powder. The method has been validated, and the results show that the method is scientific and feasible. The method exhibits a wide linear range, good repeatability, and stable and feasible sample preparation. The obtained test solution demonstrates good stability at room temperature. This method is stable and accurate, and can be used for the quality evaluation of Sanwei Huangjing Tang powder, providing a reference for improving the quality standards of this product.

[0038] The total flavonoid content in Sanwei Huangjing Tang powder was determined using ultraviolet detection, including the following steps: (1) Preparation of the test solution Mix Polygonatum sibiricum, Tribulus terrestris (processed), and Mirabilis jalapa in a 1:1:1 ratio, grind into powder, and sieve to obtain fine powder. Accurately weigh 1 g of the fine powder, add 30 mL of 60% ethanol solution, shake well, and weigh. After sonicating for 15 min, remove and cool to room temperature, weigh again, make up the weight with 60% ethanol solution, filter, and obtain the final product.

[0039] (2) Preparation of reference stock solution Preparation of reference stock solution: Accurately weigh 10.75 mg of rutin reference standard, dissolve it in 60% ethanol solution and dilute to 50 mL in a volumetric flask, shake well to obtain a rutin reference stock solution with a mass concentration of 0.215 mg·mL⁻¹.

[0040] (3) Methodological investigation Linearity assessment: 0, 1, 2, 3, 4, 5, and 6 mL of the reference solution were respectively placed in 20 mL volumetric flasks. 60% ethanol solution was added to a final volume of 6 mL. 1 mL of 5% NaNO₂ solution was added. After 6 min, 1 mL of 10% AlCl₃ solution was added. After 6 min, 5 mL of 10% NaOH solution was added. The mixture was shaken well, and after standing for 10 min, 60% ethanol solution was added to the mark for final dilution. The absorbance of the developed standard solution was measured at 510 nm. The concentration (μg·mL⁻¹) was expressed as [data missing]. -1 ) is the x-coordinate ( X ), absorbance ( Y A standard curve was plotted with the ordinate as the vertical axis, and regression analysis was performed. The resulting linear equation is: Y =0.0129 X +0.0001, r =0.999 2, the results showed that rutin was in the range of 10.54–63.21 μg·mL. -1 The linear relationship is good within the range, as shown in the results. Figure 7 .

[0041] Precision test: Take the Sanwei Huangjing Decoction powder sample, prepare the test solution according to the test solution preparation method, and measure the absorbance at a wavelength of 510 nm. The absorbance was measured 6 times in a row. The RSD of the absorbance was 0.58%, which indicates that the instrument has good precision.

[0042] Repeatability test: Prepare six parallel test solutions according to the same method as the test solution preparation procedure, measure the absorbance at a wavelength of 510 nm, and calculate the average concentration as 7.15 mg·g. -1 The RSD of the result content ( n =6) is 1.71%, indicating that the method has good repeatability.

[0043] Stability test: A new sample solution was prepared, and the absorbance was measured at 0, 10, 20, 30, 60, 90, and 120 min. The RSD was calculated to be 1.43%, indicating that the solution had good stability within 2 h.

[0044] Spiking recovery test: Six samples of Sanwei Huangjing Decoction Powder with known content were accurately weighed, each about 1.0 g, and rutin reference solution with known content was accurately added to prepare test solutions. The absorbance was measured at 510 nm, and the average recovery rate and RSD were calculated. The results are shown in Table 3.

[0045] Table 3. Experimental results of rutin recovery rate ( n = 6)

[0046] (4) Determination of rutin content in Sanwei Huangjing Decoction Powder Three batches of Sanwei Huangjing Decoction Powder (S1, S2, and S3) were prepared, and test solutions were prepared for each batch. The absorbance was measured at 510 nm. The results are shown in Table 4.

[0047] Table 4. Results of Rutin Content Determination in Sanwei Huangjing Decoction Powder

[0048] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for determining the content of multiple flavonoid components in Sanwei Huangjing Tang powder, characterized in that, The contents of quercetin, kaempferol and isorhamnetin in Sanwei Huangjing Decoction were determined simultaneously by high performance liquid chromatography.

2. The method for determining the content of multiple flavonoid components in Sanwei Huangjing Decoction Powder according to claim 1, characterized in that, The chromatographic conditions for the high performance liquid chromatography method are as follows: UV-Vis absorption detector, mobile phase is acetonitrile-0.05% phosphoric acid solution; gradient elution; flow rate is 1 mL / min; detection wavelength is 360 nm; column temperature is 30 ℃.

3. The content determination method according to claim 2, characterized in that, The gradient elution procedure under the chromatographic conditions includes the following steps: a) At 0 min, the acetonitrile ratio is 20%; b) At 25 min, the acetonitrile content was 31%; c) At 28 min, the acetonitrile content was 38%; d) At 45 min, the acetonitrile content was 38%; e) At 50 min, the acetonitrile ratio is 20%; f) At 60 min, the acetonitrile ratio is 20%.

4. The method for determining the content of multiple flavonoid components in Sanwei Huangjing Decoction Powder according to claim 2, characterized in that, The high-performance liquid chromatography column is an octadecylsilane-bonded silica column with dimensions of 250 mm × 4.6 mm and 5 μm.

5. The method for determining the content of multiple flavonoid components in Sanwei Huangjing Decoction Powder according to claim 1, characterized in that, The preparation method of the test solution in the high-performance liquid chromatography (HPLC) method is as follows: Polygonatum sibiricum, Tribulus terrestris (processed), and Mirabilis jalapa are placed in a 1:1:1 ratio in a grinder and pulverized into fine powder. 3 g of the fine powder is accurately weighed and placed in a 250 mL stoppered conical flask. 90 mL of a methanol-15% hydrochloric acid mixture (v:v, 4:1) is added, the mixture is shaken well, and the solution is weighed. The solution is heated under reflux in a 90 ℃ water bath for 4 h, cooled to room temperature, and weighed again. The weight is then made up with the methanol-15% hydrochloric acid mixture (v:v, 4:1), and the solution is filtered to obtain the test solution. The filtrate is filtered through a 0.45 μm organic microporous membrane and injected for analysis.

6. The method for determining the content of multiple flavonoid components in Sanwei Huangjing Decoction Powder according to claim 1, characterized in that, The method for preparing the reference solution in the high-performance liquid chromatography (HPLC) is as follows: Quercetin, kaempferol, and isorhamnetin are accurately weighed separately, dissolved in methanol and diluted to volume, then shaken well to prepare a reference stock solution. The quercetin, kaempferol, and isorhamnetin reference stock solutions are placed in the same volumetric flask, diluted to volume with methanol, and shaken well to prepare a mixed reference solution.

7. The method for determining the content of multiple flavonoid components in Sanwei Huangjing Decoction Powder according to claim 1, characterized in that, The injection volume in the high-performance liquid chromatography method is 10 μL.

8. The method for determining the content of multiple flavonoid components in Sanwei Huangjing Decoction Powder according to claim 1, characterized in that, The Sanwei Huangjing Decoction Powder is a drug composition consisting of three medicinal materials, Tribulus terrestris (processed), Polygonatum sibiricum, and Mirabilis jalapa, in a weight ratio of 1:1:1.

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