Method for quantitatively detecting piceatannol

Abstract

The invention relates to a method for quantitatively detecting piceatannol, which is characterized in that a 'Formaldehyde-Na2S2O5-Na2SO3-EDTA (Ethylene Diamine Tetraacetic Acid)-D-gluconic acid-delta-lactone' pH (Potential of Hydrogen) clock reaction system is used as a detection solution, and quantitative analysis on piceatannol is realized according to different responses of the system to piceatannol with different concentrations, namely different times for reaching the highest pH value. The quantitative analysis method for piceatannol has the characteristics of high accuracy, easiness in operation, convenience, rapidness and the like.

Description

Technical Field

[0001] The present invention relates to an analytical detection method, specifically, establishing a "formaldehyde-Na2S2O5-Na2SO3-EDTA-D-glucono-δ-lactone" pH clock reaction system. Based on the different responses of the system to different concentrations of piceatannol, that is, the different times when the maximum pH value is reached, a quantitative analysis method for piceatannol is achieved, belonging to the field of analytical chemistry. Background Art

[0002] The molecular formula of piceatannol is C 14 H 12 O4 (structure shown in formula (I)) is a natural small molecule compound with anti-leukemia activity and important pharmaceutical applications. Piceatannol is a diterpenoid compound with multiple chiral centers, one of which is most important because it enables piceatannol to exhibit anti-tumor activity.

[0003] It inhibits tumor cell proliferation and induces apoptosis by blocking the process of tumor cell mitosis. Piceatannol can be used to treat a variety of cancers, such as ovarian cancer, breast cancer, and non-small cell lung cancer.

[0004] The determination of piceatannol primarily relies on instrumental analysis methods, such as high-performance liquid chromatography (HPLC) and high-performance liquid chromatography with ultraviolet detection (HPLC-UV). Fluorescence and spectrophotometry have also been reported. Chromatographic methods offer the advantages of high sensitivity and accuracy, but they can reduce analytical sensitivity for samples with complex matrices. Therefore, it is crucial to develop a rapid, simple, and effective detection and analysis method.

[0005]

[0006] Structural formula (Ⅰ) Piceatannol Summary of the Invention

[0007] This invention aims to provide a novel quantitative detection method for piceatannol using a "formaldehyde-Na2S2O5-Na2SO3-EDTA-D-glucono-δ-lactone" pH clock system as the detection solution. This method utilizes a standard curve (working curve) method developed based on the sensitive response of this pH clock system to piceatannol. Specifically, the "formaldehyde-Na2S2O5-Na2SO3-EDTA-D-glucono-δ-lactone" pH clock reaction system is used as the detection solution, and a pH profile is recorded as a function of time. When the pH clock reaction begins, equal volumes of a series of piceatannol sample solutions of varying concentrations are added to the pH clock system. Quantitative detection of the piceatannol sample is achieved based on the varying effects of the sample concentrations on the time it takes for the clock system to reach its maximum pH value.

[0008] A working curve was established based on the relationship between the concentration of piceatannol in the pH clock system and the time to reach the highest pH value, where the horizontal axis is the concentration of piceatannol in the pH clock system and the vertical axis is the time t to reach the highest pH value; when the concentration of piceatannol in the system was 4×10 -5 mol / L to 2×10 -4 mol / L, the time t to reach the highest pH value is in a linear relationship with the concentration of piceatannol, based on which the quantitative detection of piceatannol in the sample can be achieved.

[0009] This quantitative detection method differs from the prior art in that it uses a "formaldehyde-Na2S2O5-Na2SO3-EDTA-D-glucono-δ-lactone" pH clock system as the detection solution, and that the system responds differently to different concentrations of piceatannol, i.e., it takes different times for the system to reach the highest pH value, thereby achieving quantitative analysis of piceatannol.

[0010] The concentration range of piceatannol in the test solution (pH clock system) was 4×10 -5 mol / L to 2×10 -4 mol / L.

[0011] When piceatannol is detected in a detection solution (pH clock system), the temperature of the pH clock system is controlled at any specific temperature within the range of 25-35°C.

[0012] Using this pH clock system, the concentration range within which piceatannol can be detected falls within the optimal experimentally determined concentration range. Within this concentration range, the time to reach the maximum pH value responds well to changes in piceatannol concentration, with a large linear correlation coefficient. Furthermore, the concentration ranges of the components in the detection solution (pH clock system) are shown in Table 1. The optimal concentrations of the detection solution (pH clock system) obtained through multiple experiments are shown in Table 2: Table 1: Concentration of components in the pH clock system

[0013] Table 2: Optimal concentrations of components in the pH clock system

[0014] The specific experimental steps are as follows: 1. Prepare a test solution (pH clock system) within the concentration range specified in Table 1. Keep its temperature constant at a specific value between 25°C and 35°C. Insert the prepared working electrode (pH combination electrode, Leici, E-331) into the solution. Connect the other end of the working electrode to a computer via a potential / temperature / pH integrated tester (Jiaxing Disheng Electronic Technology Co., Ltd., ZHFX-595). Open the chemical signal acquisition and analysis program on the computer, set the acquisition time and sampling rate, and quickly click the start button to monitor the pH of the solution. The computer records the pH change curve over time, which is the pH clock spectrum. When a substance is required for testing, the substance to be tested is added immediately upon the start of the pH clock system reaction. The pH change over time is recorded in the same manner as the pH clock spectrum.

[0015] The basic parameters of the pH clock spectrum include: Time to reach the maximum pH value: the time from the start of the pH clock system reaction to the time the pH of the clock system reaches the maximum value.

[0016] 2. Establish a working curve for the relationship between the concentration of piceatannol in the test solution and the time when the pH reaches the highest pH value Using ethanol as solvent, 0.008 mol / L, 0.016 mol / L, 0.024 mol / L, 0.032 mol / L, and 0.040 mol / L piceatannol solutions were prepared as sample solutions. At the start of the pH clock system reaction, 200 μL of the sample solutions of different concentrations were added to 40 mL of the pH clock system using a pipette to make the piceatannol concentration in the system 4×10 -5 mol / L to 2×10 -4mol / L; the change in the pH clock system response is the time to reach the highest pH value, recorded as t; when the concentration of piceatannol in the system is different, the time t when the pH clock system reaches the highest pH value is also different; the graph is drawn with the concentration of piceatannol in the system as the horizontal axis and t as the vertical axis; when the concentration of piceatannol in the system is between 4×10 -5 mol / L to 2×10 -4 mol / L, the time t when the pH clock system reaches the highest pH value is in a linear relationship with the concentration of piceatannol, and a working curve is obtained.

[0017] Quantitative detection of piceatannol By adding a test sample of unknown concentration to the detection solution pH clock system at the beginning of the pH clock system reaction, the time (t) for the corresponding pH clock system to reach the highest pH value can be measured. Based on the corresponding relationship between t and concentration on the working curve, the concentration of piceatannol in the detection system can be obtained, and then the concentration of piceatannol in the test sample can be calculated. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the change in pH value of the test solution (pH clock system) over time when no sample to be tested is added in Example 1.

[0019] Figure 2 In Example 1, 4×10 -5 mol / L piceatannol, and the pH value of the detection solution (pH clock system) changes with time.

[0020] Figure 3 In Example 1, 8×10 -5 mol / L piceatannol, and the pH value of the detection solution (pH clock system) changes with time.

[0021] Figure 4 This is a working curve of the time t at which the pH reaches the highest pH value and the concentration of piceatannol in Example 1.

[0022] Figure 5 This is a graph showing the change in pH value of the test solution (pH clock system) over time when no sample to be tested is added in Example 2.

[0023] Figure 6 In Example 2, 1.2×10 -4 mol / L piceatannol, and the pH value of the detection solution (pH clock system) changes with time.

[0024] Figure 7 In Example 2, 1.6×10 -4mol / L piceatannol, and the pH value of the detection solution (pH clock system) changes with time.

[0025] Figure 8 This is the working curve between the time t at which the pH reaches the highest pH value and the piceatannol concentration in Example 2.

[0026] Figure 9 This is a graph showing the change in pH value of the test solution (pH clock system) over time when no sample to be tested is added in Example 3.

[0027] Figure 10 In Example 3, 1.6×10 -4 mol / L piceatannol, and the pH value of the detection solution (pH clock system) changes with time.

[0028] Figure 11 In Example 3, 2×10 -4 mol / L piceatannol, and the pH value of the detection solution (pH clock system) changes with time.

[0029] Figure 12 This is the working curve between the time t at which the pH reaches the highest pH value and the concentration of piceatannol in Example 3. DETAILED DESCRIPTION Example 1

[0030] A pH clock system consisting of formaldehyde, Na₂S₂O₅, Na₂SO₃, EDTA, D-glucono-δ-lactone was used as the detection solution for quantitative analysis of piceatannol. Equal volumes of piceatannol sample solutions of varying concentrations were added to the pH clock system. A working curve (e.g., a linear relationship) was established, correlating the piceatannol concentration in the detection system with the time it took to reach the maximum pH value. This allowed the piceatannol concentration in the pH clock system to be measured, allowing the piceatannol concentration in the test sample to be calculated.

[0031] (1) Prepare the test solution First, prepare 0.256 mol / L formaldehyde solution, a mixed solution of 0.025 mol / L Na2S2O5, 0.00625 mol / L Na2SO3 and 0.001 mol / L EDTA, and 0.00748 mol / L D-glucono-δ-lactone solution in distilled water. Add 20 mL of 0.025 mol / L Na2S2O5, 0.00625 mol / L Na2SO3 and 0.001 mol / L EDTA solution to a 50 mL beaker. mol / L EDTA solution, 15 mL 0.00748 mol / L D-glucono-δ-lactone solution, and 5 mL 0.256 mol / L formaldehyde solution were added to ensure that the concentration of each component in the "formaldehyde-Na2S2O5-Na2SO3-EDTA-D-glucono-δ-lactone" pH clock system was 3.20×10 -2 mol / L, Na2S2O5 1.25×10 -2 mol / L, Na2SO3 3.13×10 -3 mol / L, EDTA 5.00×10 -4 mol / L, D-glucono-δ-lactone 2.81×10 -3 mol / L, the total volume was 40 mL, and the temperature was controlled at 27 °C.

[0032] At the same time, a series of piceatannol sample solutions with different concentrations were prepared using ethanol as solvent.

[0033] (2) Obtaining pH clock map The graph of the pH value of the prepared test solution changing with time is recorded by a computer equipped with a chemical signal acquisition and analysis program (without adding the test sample), such as Figure 1 As shown. The time for pH to reach the highest pH value was 158 s, which was used as a blank control. Two other test solutions were prepared with the same concentration of each component as the above test solution. For one of the groups, at the beginning of the reaction, 200 μL of 0.08 mol / L piceatannol sample solution was added to 40 mL of the pH clock system, so that the concentration of piceatannol in the test solution was 4×10 -5 mol / L, the addition of piceatannol prolonged the time to reach the highest pH value to 160s. Figure 2 For the other group, at the beginning of the reaction, 200 μL of 0.16 mol / L piceatannol sample solution was added to 40 mL of the pH clock system, so that the concentration of piceatannol in the test solution was 8×10 -5 mol / L, the added piceatannol makes the time to reach the highest pH value become 165s, such as Figure 3 shown. Figure 2 、 Figure 3 It was confirmed that different concentrations of piceatannol in the test solution resulted in different times when the pH clock system reached the highest pH value. -5 mol / L to 2×10 -4 When the concentration is between 100 mol / L, the different results of the pH clock system reaching the highest pH value due to different concentrations can be observed.

[0034] (3) Quantitative detection A working curve was established based on the relationship between the concentration of piceatannol in the test system and the time to reach the highest pH value, such as Figure 4 As shown in the figure, the horizontal axis is the concentration of piceatannol in the pH clock system, and the vertical axis is the time t when the maximum pH value is reached. When the concentration of piceatannol in the detection system is 4×10 -5 mol / L to 2×10 -4 mol / L, the time to reach the highest pH value is linearly related to the concentration of piceatannol, and the linear equation is t=1.25×10 5 c+155.4, R 2 = 0.9952. Based on this, the quantitative detection of piceatannol in the sample can be achieved. Example 2

[0035] (1) Prepare the test solution First, 0.256 mol / L formaldehyde solution, a mixed solution of 0.025 mol / L Na2S2O5, 0.00625 mol / L Na2SO3 and 0.001 mol / L EDTA, and 0.00748 mol / L D-glucono-δ-lactone solution were prepared using distilled water. To a 50 mL beaker, add 20.1 mL of a mixed solution of 0.025 mol / L Na2S2O5, 0.00625 mol / L Na2SO3, and 0.001 mol / L EDTA, 14.7 mL of 0.00748 mol / L D-glucono-δ-lactone solution, and 5.2 mL of 0.256 mol / L formaldehyde solution in order to ensure that the concentration of each component in the "formaldehyde-Na2S2O5-Na2SO3-EDTA-D-glucono-δ-lactone" pH clock system is 3.33×10 -2 mol / L, Na2S2O5 1.26×10 -2 mol / L, Na2SO3 3.14×10 -3 mol / L, EDTA 5.03×10 -4 mol / L, D-glucono-δ-lactone 2.75×10 -3 mol / L, the total volume was 40 mL, and the temperature was controlled at 27 °C.

[0036] At the same time, a series of piceatannol sample solutions with different concentrations were prepared using ethanol as solvent.

[0037] (2) Obtaining pH clock map The graph of the pH value of the prepared test solution changing with time is recorded by a computer equipped with a chemical signal acquisition and analysis program (without adding the test sample), such as Figure 5 As shown. The time for pH to reach the highest pH value was 158 s, which was used as a blank control. Two other test solutions with the same concentration of each component as the above test solution were prepared. For one of the groups, at the beginning of the reaction, 200 μL of 0.24 mol / L piceatannol sample solution was added to 40 mL of the pH clock system, so that the concentration of piceatannol in the test solution was 1.2×10 -4 mol / L, the addition of piceatannol prolonged the time to reach the highest pH value to 170s. Figure 6 For the other group, at the beginning of the reaction, 200 μL of 0.32 mol / L piceatannol sample solution was added to 40 mL of the pH clock system, so that the concentration of piceatannol in the test solution was 1.6×10 -4 mol / L, the added piceatannol makes the time to reach the highest pH value become 175 s. Figure 7 shown. Figure 6 、 Figure 7 It was confirmed that different concentrations of piceatannol in the test solution resulted in different times when the pH clock system reached the highest pH value. -5 mol / L to 2×10 -4 When the concentration is between 100 mol / L, the different results of the pH clock system reaching the highest pH value due to different concentrations can be observed.

[0038] (3) Quantitative detection A working curve was established based on the relationship between the concentration of piceatannol in the test system and the time to reach the highest pH value, such as Figure 8 As shown in the figure, the horizontal axis is the concentration of piceatannol in the pH clock system, and the vertical axis is the time t when the maximum pH value is reached. When the concentration of piceatannol in the detection system is 4×10 -5 mol / L to 2×10 -4 mol / L, the time to reach the highest pH value is linearly related to the concentration of piceatannol, and the linear equation is t=1.3×10 5 c+154.6, R 2 =0.9985. Based on this, the quantitative detection of piceatannol in the sample can be achieved. Example 3

[0039] (1) Prepare the test solution First, 0.256 mol / L formaldehyde solution, a mixed solution of 0.025 mol / L Na2S2O5, 0.00625 mol / L Na2SO3 and 0.001 mol / L EDTA, and 0.00748 mol / L D-glucono-δ-lactone solution were prepared using distilled water. To a 50 mL beaker, add 19.8 mL of a mixed solution of 0.025 mol / L Na2S2O5, 0.00625 mol / L Na2SO3, and 0.001 mol / L EDTA, 15 mL of 0.00748 mol / L D-glucono-δ-lactone solution, and 5.2 mL of 0.256 mol / L formaldehyde solution in order to ensure that the concentration of each component in the "formaldehyde-Na2S2O5-Na2SO3-EDTA-D-glucono-δ-lactone" pH clock system is 3.33×10 -1 mol / L, Na2S2O5 1.24×10 -2 mol / L, Na2SO3 3.09×10 -3 mol / L, EDTA 4.95×10 -4 mol / L, D-glucono-δ-lactone 2.81×10 -3 mol / L, the total volume was 40 mL, and the temperature was controlled at 27 °C.

[0040] At the same time, a series of piceatannol sample solutions with different concentrations were prepared using ethanol as solvent.

[0041] (2) Obtaining pH clock map The graph of the pH value of the prepared test solution changing with time is recorded by a computer equipped with a chemical signal acquisition and analysis program (without adding the test sample), such as Figure 9 As shown. The time for pH to reach the highest pH value was 156 s, which was used as a blank control. Two other test solutions were prepared with the same concentration of each component as the above test solution. For one of the groups, at the beginning of the reaction, 200 μL of 0.32 mol / L piceatannol sample solution was added to 40 mL of the pH clock system, so that the concentration of piceatannol in the test solution was 1.6×10 -4 mol / L, the addition of piceatannol prolonged the time to reach the highest pH value to 175s. Figure 10 As shown; for the other group, at the beginning of the reaction, 200 μL of 0.40 mol / L piceatannol sample solution was added to 40 mL of the pH clock system, so that the concentration of piceatannol in the test solution was 2×10 -4 mol / L, the added piceatannol makes the time to reach the highest pH value become 181 s, such as Figure 11 shown. Figure 10 、 Figure 11 It was confirmed that different concentrations of piceatannol in the test solution resulted in different times when the pH clock system reached the highest pH value. -5 mol / L to 2×10 -4 When the concentration is between 100 mol / L, the different results of the pH clock system reaching the highest pH value due to different concentrations can be observed.

[0042] (3) Quantitative detection A working curve was established based on the relationship between the concentration of piceatannol in the test system and the time to reach the highest pH value, such as Figure 12 As shown in the figure, the horizontal axis is the concentration of piceatannol in the pH clock system, and the vertical axis is the time t when the maximum pH value is reached. When the concentration of piceatannol in the detection system is 4×10 -5 mol / L to 2×10 -4 mol / L, the time to reach the highest pH value is linearly related to the concentration of piceatannol, and the linear equation is t=1.2×10 5 c+155.8, R 2 =0.9982, thus the quantitative detection of piceatannol in the sample can be achieved.

Claims

1. A method for quantitatively detecting piceatannol, characterized in that: Using ethanol as solvent, a solution of the sample piceatannol to be tested was prepared; The pH clock reaction system of "formaldehyde-Na2S2O5-Na2SO3-EDTA-D-glucono-δ-lactone" was used as the detection solution, and the pH change over time was recorded. The temperature of the pH clock system is controlled at any specific temperature within the range of 25-35°C. When the pH clock reaction starts, equal volumes of a series of piceatannol sample solutions with different concentrations are added to the pH clock system. According to the different effects of different concentrations of the sample on the time when the clock system reaches the highest pH value, quantitative detection of the sample is achieved: a working curve is established based on the relationship between the concentration of piceatannol in the pH clock system and the time when the highest pH value is reached, in which the horizontal axis is the concentration of piceatannol in the pH clock system and the vertical axis is the time t when the highest pH value is reached. When the piceatannol concentration in the system is 4×10 -5 mol / L to 2×10 -4 When the pH value is between 0.1 and 0.2 mol / L, there is a linear relationship between the time t to reach the highest pH value and the concentration of piceatannol, thereby achieving the quantitative detection of piceatannol in the sample; The molar concentration range of each component in the test solution is: formaldehyde 2.88×10 -2 -3.58×10 -2 mol / L, Na2S2O51.21×10 -2 -1.32×10 -2 mol / L、Na2SO33.08×10 -3 -3.28×10 -3 mol / L, EDTA 4.82×10 -4 -5.08×10 -4 mol / L, D-glucono-δ-lactone 2.67×10 -3 -2.87×10 -3 mol / L.

2. The method according to claim 1, wherein: The molar concentration of each component in the test solution is formaldehyde 3.20×10 -2 mol / L, Na2S2O5 1.25×10 -2 mol / L, Na2SO3 3.13×10 -3 mol / L, EDTA 5.00×10 -4 mol / L, D-glucono-δ-lactone 2.81×10 -3 mol / L.

3. The method according to claim 1, wherein: The temperature of the pH clock system was controlled at 27°C when measuring the piceatannol solution.

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