Method for determining content of flavone in pine pollen alcohol extract

Abstract

The invention belongs to the technical field of detection, and particularly relates to a method for determining the content of flavone in a pine pollen alcohol extract. According to the method, a NaNO-Al (NO)-NaOH color development method is adopted, rutin is taken as a reference substance, a pollen pini alcohol extract sample solution and a rutin reference substance solution are subjected to color development treatment, the absorbance is measured under a specific wavelength, and the content of the total flavonoids in the sample is calculated in combination with a standard curve. By optimizing the color development condition, the problems of insufficient optimization of the color development condition, large difference of measurement results, interference of matrix components and the like during measurement of flavone in the pollen pini alcohol extract in the prior art are solved, and the content measurement method which is stable, feasible, easy to operate and capable of meeting experimental requirements is provided. A basis is provided for measuring the content of flavone in the pollen pini alcohol extract, and the method has important significance on industrial development of pollen pini.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a method for determining the content of flavonoids in pine pollen alcohol extract. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Pine pollen is rich in active ingredients such as flavonoids, polyphenols, and polysaccharides. Among them, flavonoids have significant antioxidant, anti-inflammatory, and gut microbiota-regulating biological activities. Total flavonoid content is one of the core indicators for evaluating the quality of pine pollen and its processed products. Therefore, establishing accurate determination methods is of great significance for its industrial development.

[0004] Currently, the main methods for determining total flavonoids in plants are high-performance liquid chromatography (HPLC) and spectrophotometry. While HPLC can achieve the separation and quantification of monomeric flavonoids, its high instrument cost and complex operation make it difficult to meet the routine testing needs of grassroots production enterprises. The NaNO2-Al(NO3)3-NaOH colorimetric method in spectrophotometry is widely used due to its simplicity and low cost. Its principle is that flavonoids form colored complexes with metal salts under alkaline conditions, and the content is determined by colorimetric analysis.

[0005] However, existing technologies lack sufficient optimization of colorimetric conditions for pine pollen ethanol extracts, leading to significant differences in results across different laboratories. For example, the selection of parameters such as colorimetric reagent concentration and reaction time lacks systematic validation, potentially causing deviations in flavonoid content determination. Furthermore, pine pollen ethanol extracts contain matrix components such as organic acids and polysaccharides, which may interfere with the specificity of the colorimetric reaction. Therefore, there is an urgent need to optimize the flavonoid determination method for pine pollen ethanol extracts to improve the accuracy and reliability of the determination. Summary of the Invention

[0006] This invention provides a method for determining the content of flavonoids in pine pollen alcohol extract, aiming to solve the problems in the prior art such as insufficient optimization of color development conditions, large differences in test results between different laboratories, and measurement deviation caused by matrix component interference.

[0007] This invention employs the NaNO2-Al(NO3)3-NaOH colorimetric method, using rutin as a reference standard, and specifically includes the following steps: (1) Sample solution preparation: Accurately weigh the pine pollen alcohol extract, add ethanol solution for extraction, filter and collect the supernatant to obtain the pine pollen alcohol extract sample solution.

[0008] (2) Preparation of reference solution: Accurately weigh rutin reference standard, add methanol to dissolve and dilute to volume to obtain rutin standard solution.

[0009] (3) Color development treatment: Sodium nitrite solution, aluminum nitrate solution and sodium hydroxide solution were added to the sample solution and the reference solution in sequence and the reaction was carried out.

[0010] (4) Absorbance measurement: The absorbance of the solution after color development at a wavelength of 510 nm was measured.

[0011] (5) Standard curve plotting: By precisely aspirating different volumes of rutin standard solution, after color development, a standard curve is plotted with absorbance as the ordinate and rutin concentration as the abscissa. The curve shows a linear relationship between concentration and absorbance in the range of 8-48 μg / mL, and the linear regression equation is y=0.0066x+0.092 (R²=0.9994).

[0012] (6) Content calculation: The total flavonoid content in the sample is calculated according to the standard curve and the formula X=C×V×n / M, where X is the total flavonoid content in the sample (μg / g), C is the total flavonoid content calculated from the standard curve (μg / mL), n is the dilution factor of the filtrate, and M is the sample mass.

[0013] Meanwhile, the present invention also optimized the color development conditions, and determined the optimal color development conditions as follows: accurately pipette 2 mL of the sample solution to be tested into a 25 mL volumetric flask, titrate with distilled water to 6 mL, add 1 mL of 5% sodium nitrite solution, react for 6 min, add 1 mL of 10% aluminum nitrate solution, react for 6 min, add 10 mL of 4% sodium hydroxide solution, dilute to 25 mL, and react for 15 min.

[0014] Compared with the prior art, the technical advantages of the present invention are as follows: This invention establishes a method for determining the total flavonoid content in pine pollen. The method exhibits good precision, repeatability, and stability within 90 minutes, with a recovery rate of 90.26% and an RSD of 4.21%. This indicates that the method is stable, feasible, easy to operate, and meets experimental requirements in terms of precision and repeatability, providing a basis for subsequent determination of flavonoid content in pine pollen alcohol extracts.

[0015] Specifically, (1) High accuracy and reliability: By systematically optimizing the color development conditions, including the concentration, amount and reaction time of sodium nitrite solution, aluminum nitrate solution and sodium hydroxide solution, the measurement deviation caused by inconsistent color development conditions is effectively reduced. At the same time, the interference of matrix components such as organic acids and polysaccharides in pine pollen alcohol extract on the color development reaction is reduced, thus improving the accuracy and reliability of the measurement results.

[0016] (2) Good precision: According to the methodological investigation, the RSD of 6 consecutive measurements was 0.16%, which indicates that the instrument has good precision and ensures the stability of the measurement results.

[0017] (3) Good repeatability: In the repeatability experiment, the RSD of 6 parallel experiments was 0.12%, indicating that the method has good repeatability and can obtain relatively consistent results in multiple experiments.

[0018] (4) Strong stability: The RSD of the sample was 0.11% after multiple measurements within 90 min, indicating that the method has good stability within 90 min and is convenient for experimental operation and result determination.

[0019] (5) Ideal recovery rate: The average recovery rate was 90.26% and the RSD was 4.21%, which further verified the accuracy and feasibility of the method and met the requirements for the determination of flavonoid content in pine pollen alcohol extract.

[0020] (6) Simple operation and low cost: The NaNO2-Al(NO3)3-NaOH colorimetric method in spectrophotometry is adopted. Compared with high performance liquid chromatography, the instrument has low cost and simple operation, which is more suitable for the routine testing needs of grassroots production enterprises and is conducive to quality control in the industrialization development process of pine pollen and its processed products. Attached Figure Description

[0021] Figure 1 Full-wavelength scan of pine pollen.

[0022] Figure 2 : Rutin standard curve.

[0023] Figures 3-11 The results of the color development condition optimization include, among which Figure 3 To investigate the concentration of NaNO2 solution, Figure 4 To investigate the amount of NaNO2 solution used, Figure 5 To investigate the reaction time of NaNO2 solution, Figure 6 To investigate the concentration of Al(NO3)3 solution, Figure 7 To investigate the amount of Al(NO3)3 solution used, Figure 8 To investigate the reaction time of Al(NO3)3 solution, Figure 9 To investigate the concentration of NaOH solution, Figure 10 To investigate the amount of NaOH solution used, Figure 11 The reaction time of the NaOH solution was investigated. Detailed Implementation

[0024] To make the objectives and technical solutions of this invention clearer, the following embodiments are provided for further explanation. However, the scope of protection of this invention is not limited to these embodiments; the embodiments are merely for illustrative purposes. Those skilled in the art should understand that any changes or equivalent substitutions that do not depart from the concept of this invention are included within the scope of protection of this invention.

[0025] Method Principles Flavonoids possess a phenolic hydroxyl structure and can undergo a diazotization reaction with sodium nitrite under alkaline conditions. The resulting diazonium salt then forms a stable orange-red complex with aluminum nitrate, which exhibits a maximum absorption peak at 510 nm. By measuring the absorbance at this wavelength and comparing it with a rutin standard curve, the total flavonoid content in the sample can be calculated.

[0026] Reagents and Instruments Reagents: Rutin standard: purity ≥98% (purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.); Methanol, ethanol, sodium nitrite, aluminum nitrate, sodium hydroxide: all analytical grade (purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.); Experimental water: ultrapure water (prepared by Mingche™-D24UV pure water / ultrapure water system).

[0027] Instruments: Balance (AL204, METTLERTOLEDO International Trading (Shanghai) Co., Ltd.); Microplate reader (MaxPlus384, Molecular Devices, USA); Ultrasonic cleaner (KQ-300DE, Kunshan Ultrasonic Instruments Co., Ltd.); High-speed centrifuge (TDL-5A, Changzhou Wanhe Instrument Manufacturing Co., Ltd.); Rotary evaporator (R-210, BUCHI Labortechnik AG, Switzerland); Constant temperature water bath (HH-S4, Jintan Chengdong Xinrui Instrument Factory); Volumetric flasks (25mL, 50mL, 100mL, etc.); Pipettes (1mL, 2mL, 5mL, 10mL, etc.); 0.45μm organic filter membrane (Shanghai Anpu Experimental Technology Co., Ltd.).

[0028] Material source: The pine pollen used in this experiment was collected from Masson pine trees. Masson pine forests are distributed in Xiaba Town, Wudang District, Guiyang City, Guizhou Province. It was identified by Associate Professor Liu Shaohuan of Guizhou Medical University as Masson pine (Pinus massoniana Lamb.). When the flowers of Masson pine are just blooming, the flower spikes are picked, dried, and the pollen is collected. Impurities are removed.

[0029] Pine pollen ethanol extract: Fresh pine pollen was dried to remove impurities, washed, and dried. It was then extracted with 80% ethanol at 88℃ for 2 hours at a material-to-liquid ratio of 1:30 g / mL to obtain pine pollen ethanol extract. Determination method of total flavonoid content in pine pollen Preparation of pine pollen sample solution Accurately weigh 2g of pine pollen and place it in a 100mL Erlenmeyer flask. Add the pollen to the liquid at a ratio of 1:30 (g / mL). Add 60mL of 60% ethanol and sonicate three times for 30min each time. After cooling, add 60% ethanol to make up the weight, shake well, filter, and collect the supernatant.

[0030] Preparation of reference solution Preparation of rutin standard solution: Accurately weigh 20 mg of rutin reference standard (accurate to 0.01 mg), add an appropriate amount of methanol to dissolve, and transfer to a 100 mL volumetric flask. Dilute to the mark to obtain a rutin standard solution with a concentration of 0.2 mg / mL.

[0031] Preparation of 5% sodium nitrite solution: Accurately weigh 5g of sodium nitrite, dissolve it in water and transfer it to a 100mL volumetric flask, make up to 100mL, and shake well before use.

[0032] Preparation of 10% aluminum nitrate solution: Accurately weigh 17.60g of aluminum nitrate, dissolve it in water and transfer it to a 100mL volumetric flask, then dilute to 100mL and set aside.

[0033] Preparation of sodium hydroxide solution: Accurately weigh 4.30 g of sodium hydroxide, dissolve it in water, transfer it to a 100 mL volumetric flask, and dilute to 100 mL.

[0034] Selection of measurement wavelength Accurately pipette 2 mL of rutin reference solution and pine pollen sample solution into a 25 mL volumetric flask, titrate with distilled water to 6 mL, add 1 mL of 5% sodium nitrite solution, react for 6 min, add 1 mL of 10% aluminum nitrate solution, react for 6 min, add 10 mL of sodium hydroxide solution, dilute to the mark, react for 15 min, scan with an ELISA reader at all wavelengths (400-800 nm) to determine the maximum absorbance value A, and determine the maximum absorption wavelength.

[0035] Experimental results showed that the test solution of pine pollen and the rutin reference solution had the maximum absorbance at 510 nm, therefore 510 nm was determined as the measurement wavelength. The results are as follows... Figure 1 .

[0036] Construction of standard curves and examination of linear range Accurately pipette 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 mL of rutin standard solution into 25 mL volumetric flasks. Titrate with distilled water to 8 mL, add 1 mL of 5% sodium nitrite solution, react for 6 min, add 1 mL of 10% aluminum nitrate solution, react for 6 min, add 10 mL of sodium hydroxide solution, dilute to 25 mL, and react for 15 min to obtain a series of standard working solutions with rutin concentrations of 0-48 μg / mL. Using 0.0 mL of rutin standard solution as a blank sample, measure the absorbance (A) at 510 nm. Plot a standard curve with absorbance (A) on the ordinate and the concentration (C) of the rutin standard on the abscissa.

[0037] The linear regression equation for rutin reference solution in the range of 8-48 µg / mL was y = 0.0066x + 0.092, (R0...). 2 =0.9994), indicating a good linear relationship between concentration and absorbance within the range of 8-48 µg / mL. The rutin standard curve is shown below. Figure 2 As shown.

[0038] Determination of total flavonoid content in pine pollen This experiment used NaNO2-Al(NO3)3-NaOH

[79] The total flavonoid content in pine pollen was determined using a colorimetric method with rutin as a reference. 2 mL of sample was accurately pipetted into a 25 mL volumetric flask, titrated to 6 mL with distilled water, 1 mL of 5% sodium nitrite was added, and the mixture was shaken well and allowed to stand for 6 minutes. Then, 1 mL of 10% aluminum nitrate was added, shaken well, and allowed to stand for 6 minutes. Finally, 10 mL of 4% sodium hydroxide solution was added, and distilled water was added to the mark. The mixture was shaken well and allowed to stand for 15 minutes. This process was repeated three times. Using a blank reagent as a reference, the absorbance (A) was measured at 510 nm using a microplate reader.

[0039] The total flavonoid content is calculated using the formula: X = C × V × n / M In the formula: X—the total flavonoid content in the sample, in micrograms per gram (μg / g); C—the total flavonoid content calculated from the standard curve, in micrograms per milliliter (μg / mL); n—the dilution factor of the filtrate; M—the sample mass.

[0040] Optimization of colorimetric conditions for total flavonoids in pine pollen Investigation of NaNO2 solution concentration 2 mL of sample solution and rutin standard solution were transferred separately, and 1 mL of NaNO2 solution (1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%) was added. The absorbance value A was measured according to the procedure for "Determination of Total Flavonoid Content in Pine Pollen". Each experiment was repeated in triplicate. The results showed that the maximum absorbance value was obtained at a NaNO2 solution concentration of 5%. The experimental results are as follows... Figure 3 .

[0041] Investigation on the dosage of NaNO2 solution 2 mL of sample solution and rutin standard solution were transferred separately, and 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, and 1.75 mL of 5% NaNO2 solution were added. The absorbance value A was measured according to the procedure for "Determination of Total Flavonoids in Pine Pollen". Each experiment was repeated in triplicate. The results showed that the maximum absorbance value was obtained with 1 mL of NaNO2 solution. The experimental results are as follows... Figure 4 .

[0042] Investigation of the standing time after adding NaNO2 solution 2 mL of sample solution and rutin standard solution were transferred separately, and 1 mL of 5% NaNO2 solution was added. The solutions were then incubated for 0, 2, 4, 6, 8, 10, 12, and 14 min, respectively. The absorbance value (A) was measured according to the procedure for "Determination of Total Flavonoids in Pine Pollen." Each experiment was performed in triplicate. The results showed that the maximum absorbance value was observed 6 min after the addition of NaNO2 solution. The experimental results are as follows: Figure 5 .

[0043] Investigation of Al(NO3)3 solution concentration 2 mL of sample solution and rutin standard solution were transferred separately, and 1 mL of Al(NO3)3 solution (2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%) was added. The absorbance value A was measured according to the procedure for "Determination of Total Flavonoids in Pine Pollen". Each experiment was repeated in triplicate. The results showed that the 10% Al(NO3)3 solution concentration resulted in the highest absorbance value. The experimental results are as follows... Figure 6 .

[0044] Investigation on the dosage of Al(NO3)3 solution 2 mL of sample solution and rutin standard solution were transferred separately, and 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, and 1.75 mL of 10% Al(NO3)3 solution were added respectively. The procedure for "Determination of Total Flavonoid Content in Pinus massoniana Pollen Solution" was followed, and the absorbance value A was measured. Each experiment was repeated in triplicate. The results showed that the maximum absorbance value was achieved with 1 mL of Al(NO3)3 solution. The experimental results are as follows... Figure 7 .

[0045] Investigation of standing time after adding Al(NO3)3 solution 2 mL of sample solution and rutin standard solution were transferred separately, and 1 mL of 10% Al(NO3)3 solution was added. The solutions were then incubated for 0, 2, 4, 6, 8, 10, 12, and 14 min, respectively. The absorbance value (A) was measured according to the procedure for "Determination of Total Flavonoids in Pine Pollen." Each experiment was performed in triplicate. The results showed that the reaction time condition with the addition of Al(NO3)3 solution for 6 min resulted in the highest absorbance value. The experimental results are as follows: Figure 8 .

[0046] NaOH solution concentration investigation 2 mL of sample solution and rutin standard solution were transferred separately, and 10 mL of NaOH solution (3%, 4%, 5%, 6%, 8%, 10%, 12%, 14%) was added. The procedure for "Determination of Total Flavonoids in Pine Pollen" was followed, and the absorbance values ​​(A) were compared. Each experiment was repeated in triplicate. The results showed that the 4% NaOH solution concentration resulted in the highest absorbance value. The experimental results are as follows... Figure 9 .

[0047] Investigation on the dosage of NaOH solution 2 mL of sample solution and rutin standard solution were transferred separately, and (5, 6, 7, 8, 9, 10, 11, 12 mL) of NaOH solution were added respectively. The absorbance value A was measured according to the procedure for "Determination of Total Flavonoids in Pine Pollen". Each experiment was repeated in triplicate. The results showed that the maximum absorbance value was obtained with 10 mL of NaOH solution. The experimental results are as follows: Figure 10 .

[0048] Investigation of the standing time after adding NaOH solution 2 mL of sample solution and rutin standard solution were transferred separately, and 10 mL of 4% NaOH solution was added. The solutions were then allowed to stand for 10, 11, 12, 13, 14, 15, 16, and 17 min, respectively. The absorbance value (A) was measured according to the procedure for "Determination of Total Flavonoids in Pine Pollen". Each experiment was repeated in triplicate. The colorimetric results showed that the maximum absorbance value was achieved after 15 min of reaction following the addition of NaOH solution. The experimental results are as follows: Figure 11 .

[0049] Methodological investigation of the determination of total flavonoid content in pine pollen Precision test Accurately pipette 2 mL of pine pollen sample solution into a 25 mL volumetric flask, titrate with distilled water to 6 mL, and develop the color under optimal conditions and time. Measure the absorbance at the maximum absorption wavelength. Perform six consecutive measurements to calculate the total flavonoid content, average content, and RSD. The results are shown in Table 1. The RSD was 0.16% (n=6), indicating good instrument precision.

[0050] Table 1 Precision Experiment Results Repeated examination Accurately weigh 2g of pine pollen and extract it 6 times using the optimal extraction method to obtain the sample to be tested. Take 2mL of each sample and transfer it to a 25mL volumetric flask. Add distilled water and titrate to 6mL. Develop the color under the optimal colorimetric conditions and time, and measure the absorbance at 510nm. Repeat the measurement 6 times. Calculate the total flavonoid content, average content, and RSD. Table 2 shows that the RSD% is 0.12 (n=6), indicating good repeatability of the method.

[0051] Table 2 Results of repeatability experiments Stability test Accurately pipette 2 mL of the reference solution and sample solution into a 25 mL volumetric flask, titrate with distilled water to 6 mL, and develop the color under optimal conditions and time. Measure the absorbance at the maximum absorption wavelength, repeating the measurement six times at 0, 5, 15, 30, 60, and 90 min. Calculate the total flavonoid content, average content, and RSD. Table 3 shows that the RSD% is 0.11 (n=6), indicating that the method is stable within 90 min.

[0052] Table 3 Stability test results Recovery rate study Nine 2g portions of pine pollen were accurately weighed, divided into three groups of three. Rutin standard solution was added to each group at 0.5, 1, and 1.5 times the sample concentration, respectively. 2mL of each sample was transferred to a 25mL volumetric flask. Optimal colorimetric conditions and time were used for development, and the absorbance was measured at 510nm. The relative standard deviation (RSD) was calculated. Table 4 shows that the average recovery rate was 90.26%, with an RSD of 4.21% (n=9).

[0053] Table 4 Results of the recovery rate experiment This invention uses pine pollen as the research object, with flavonoid content as the indicator, and employs the NaNO2-Al(NO3)3-NaOH colorimetric method, with rutin as the reference standard. The maximum absorption wavelength of the pine pollen sample solution and the rutin reference standard was determined to be 510 nm. The experimental results show that the concentration and absorbance of the rutin reference standard solution are linearly related within the range of 8-48 µg / mL, indicating a good relationship. The linear regression equation is y = 0.0066x + 0.092 (R0). 2=0.9994). The optimal colorimetric conditions for the determination of total flavonoid content in pine pollen are as follows: Accurately pipette 2 mL of the sample solution to be tested into a 25 mL volumetric flask, titrate with distilled water to 6 mL, add 1 mL of 5% sodium nitrite solution, react for 6 min, add 1 mL of 10% aluminum nitrate solution, react for 6 min, add 10 mL of 4% sodium hydroxide solution, dilute to 25 mL, react for 15 min, and measure the absorbance A at 510 nm. Methodological evaluation results show that the instrument precision is good, the method repeatability is good, the experimental results are stable within 90 min, and the average recovery rate is 90.26% (RSD=9).

Claims

1. A method for determining the flavonoid content in pine pollen alcohol extract, characterized in that, The total flavonoid content in pine pollen alcohol extract was calculated by measuring absorbance and combining it with a standard curve using the NaNO2-Al(NO3)3-NaOH colorimetric method with rutin as a reference.

2. The method according to claim 1, characterized in that, Includes the following steps: (1) Preparation of pine pollen alcohol extract sample solution; (2) Preparation of rutin reference solution; (3) Perform color development treatment on the sample solution and the reference solution; (4) Measure the absorbance of the solution after color development at a specific wavelength; (5) Calculate the total flavonoid content in the sample based on the standard curve.

3. The method according to claim 2, characterized in that, The color development process involves sequentially adding sodium nitrite solution, aluminum nitrate solution, and sodium hydroxide solution and then reacting them.

4. The method according to claim 3, characterized in that, The concentration of the sodium nitrite solution is 1-8%, the amount used is 0.1-1.75 mL, and the reaction time after addition is 0-14 min.

5. The method according to claim 3, characterized in that, The concentration of the aluminum nitrate solution is 2-16%, the amount used is 0.1-1.75 mL, and the reaction time after addition is 0-14 min.

6. The method according to claim 3, characterized in that, The concentration of the sodium hydroxide solution is 3-14%, the amount used is 5-12 mL, and the reaction time after addition is 10-17 min.

7. The method according to claim 2, characterized in that, The specific wavelength is 510nm.

8. The method according to claim 2, characterized in that, The standard curve was plotted by precisely sampling different volumes of rutin standard solution, performing colorimetric treatment, and then plotting absorbance as the ordinate and rutin concentration as the abscissa.

9. The method according to claim 8, characterized in that, The concentration and absorbance of the rutin standard solution showed a linear relationship in the range of 8-48 μg / mL.

10. The method according to claim 1, characterized in that, The specific steps are as follows: (1) Preparation of pine pollen alcohol extract sample solution: Accurately weigh the pine pollen alcohol extract, add ethanol solution for extraction, filter and collect the supernatant to obtain the solution; (2) Preparation of rutin reference solution: Accurately weigh rutin reference standard, add methanol to dissolve and dilute to volume to obtain a rutin standard solution with a concentration of 0.2 mg / mL; (3) Color development treatment: Accurately pipette 2 mL of sample solution into a 25 mL volumetric flask, titrate with distilled water, add 1 mL of 5% sodium nitrite solution, react for 6 min, add 1 mL of 10% aluminum nitrate solution, react for 6 min, add 10 mL of 4% sodium hydroxide solution, dilute to 25 mL, and react for 15 min. (4) Absorbance measurement: The absorbance was measured at a wavelength of 510 nm; (5) Content calculation: The total flavonoid content was calculated based on the linear regression equation y=0.0066x+0.092 (R²=0.9994). The calculation formula is X=C×V×n / M, where X is the total flavonoid content in the sample (μg / g), C is the total flavonoid content calculated from the standard curve (μg / mL), n is the dilution factor of the filtrate, and M is the sample mass.

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