Method for distinguishing resveratrol and piceatannol

Abstract

A method for distinguishing resveratrol and piceatannol is characterized in that a 'Formaldehyde-Na2S2O5-Na2SO3-EDTA-D-gluconic acid-delta-lactone' pH clock reaction system is used as a distinguishing solution, and qualitative analysis of resveratrol and piceatannol is realized according to different influences of resveratrol and piceatannol on the highest pH value of the clock system and the time for reaching the highest pH value. The qualitative analysis method for resveratrol and piceatannol has the characteristics of high accuracy, easiness in operation, convenience, rapidness and the like.

Description

Technical Field

[0001] The present invention relates to a differentiation method, specifically, to establishing a "formaldehyde-Na2S2O5-Na2SO3-EDTA-D-glucono-δ-lactone" pH clock system. The method achieves differentiation of samples to be differentiated based on the different effects of the samples to be differentiated on the maximum pH value of the clock system and the time it takes to reach the maximum pH value. The method belongs to the field of analytical chemistry. Background Art

[0002] Piceatannol is a naturally occurring compound (structure shown in Structural Formula (I)) that plays a significant role in human health. It inhibits tumor cell proliferation and induces apoptosis by blocking tumor cell mitosis. Piceatannol is used to treat a variety of cancers, including ovarian cancer, breast cancer, and non-small cell lung cancer. It also has anti-leukemia properties, inhibits the activity of non-receptor-activated enzymes, spleen tyrosine kinase, and lymphocyte-specific protein tyrosine kinase, and helps delay the production of immature new adipocytes. Overall, piceatannol has significant research implications in human medicine.

[0003] Resveratrol, a non-flavonoid polyphenolic organic compound (structure shown in formula (Ill)), is an antitoxin produced by many plants when stimulated. Resveratrol can act as a potent activator of silent signaling factors, mimicking the anti-aging effects of caloric restriction and participating in the regulation of the average lifespan of organisms. Resveratrol has significant inhibitory effects on various tumor cells, including hepatocellular carcinoma, breast cancer, colon cancer, gastric cancer, and leukemia in mice. Resveratrol also exhibits antibacterial, antioxidant, immunomodulatory, and anti-asthmatic activities. These diverse biological activities have made resveratrol highly sought after.

[0004] Currently, common methods for distinguishing resveratrol from piceatannol include high-performance liquid chromatography (HPLC) and mass spectrometry (MS). While these analytical methods have their own advantages, they require expensive and sophisticated testing equipment, and the subsequent maintenance costs are also high, making them unsuitable for on-site testing. Therefore, it is imperative to find a detection and analysis method that is effective, simple to operate, and fast.

[0005]

[0006] Structural formula (Ⅰ) Piceatannol

[0007] Structural formula (ⅠⅠ) resveratrol Summary of the Invention

[0008] The present invention aims to provide a novel, convenient, and rapid method for distinguishing resveratrol from piceatannol. This method utilizes a "formaldehyde-Na2S2O5-Na2SO3-EDTA-D-glucono-δ-lactone" pH clock system as a distinguishing solution for qualitative detection of sample solutions. This method is based on the differential sensitivity of the pH clock system to the two substances. Specifically, the "formaldehyde-Na2S2O5-Na2SO3-EDTA-D-glucono-δ-lactone" pH clock reaction system is used as the distinguishing solution, and a pH profile of the time-dependent changes is recorded. Once the pH clock reaction begins, equal volumes of resveratrol and piceatannol sample solutions containing the same concentration are added to the two pH clock systems. Qualitative differentiation of the samples is achieved based on the varying effects of the samples on the clock systems' maximum pH values ​​and the time it takes to reach that peak.

[0009] The difference between the present qualitative differentiation method and the prior art is that the present invention uses the "formaldehyde-Na2S2O5-Na2SO3-EDTA-D-glucono-δ-lactone" pH clock system as a differentiation solution, and achieves differentiation of the samples to be differentiated according to the different effects of the samples to be differentiated on the maximum pH value of the clock system and the time to reach the maximum pH value: compared with the graph of pH value change over time without adding the solution to be differentiated, if the addition of the solution to be differentiated causes the maximum pH value of the clock system to drop significantly and the time to reach the maximum pH value to be significantly prolonged, then the added sample to be differentiated is a resveratrol sample; if the addition of the solution to be differentiated causes the maximum pH value of the clock system to drop slightly and the time to reach the maximum pH value to be slightly prolonged, then the added sample to be differentiated is a piceatannol sample; When the sample solution to be differentiated is detected in the differentiation solution (pH clock system), the temperature of the pH clock system is controlled at any specific temperature within the range of 25-35°C.

[0010] The concentration range of resveratrol and piceatannol in the differentiating solution (pH clock system) is 8×10 -5 -1.6×10 -4 mol / L.

[0011] The concentration range for differentiating the solutions described above represents the optimal concentration range determined experimentally. Within this concentration range, the effects of resveratrol and piceatannol on the differentiating solutions are distinct, making them easy to observe and analyze, and enabling easy differentiation. Furthermore, the concentration ranges for the components of the differentiating solutions (pH clock system) are shown in Table 1. The optimal solution for the differentiating solutions (pH clock system) obtained through multiple experiments is shown in Table 2: Table 1: Concentration of components in the pH clock system

[0012] Table 2: Optimal concentrations of components in the pH clock system

[0013] The specific experimental steps are as follows: 1. Prepare 40 mL of a differentiating 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 Start to monitor the pH of the solution. The computer records the pH change over time of the clock system, known as the pH clock spectrum. When testing a substance, add the substance to be tested immediately upon initiation of the pH clock system reaction, and record the pH change over time as a pH clock spectrum in the same manner.

[0014] The basic parameters of the pH clock spectrum include: The time for the clock system to reach the highest pH value: the time required from the start of the pH clock system reaction to the time when the pH of the clock system reaches the highest value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a graph showing the change in pH value of the differentiation solution (pH clock system) over time when no sample to be differentiated is added in Example 1.

[0016] Figure 2 In Example 1, 8×10 -5 mol / L resveratrol, and then the pH value of the solution (pH clock system) was distinguished as a inverse time spectrum.

[0017] Figure 3 In Example 1, 8×10 -5 mol / L piceatannol, and the pH value of the solution (pH clock system) was distinguished as a function of time.

[0018] Figure 4 This is a graph showing the change in pH value of the differentiation solution (pH clock system) over time when no sample to be differentiated is added in Example 2.

[0019] Figure 5 In Example 2, 1.2×10 -4 mol / L resveratrol, and then the pH value of the solution (pH clock system) was distinguished as a inverse time spectrum.

[0020] Figure 6 In Example 2, 1.2×10 -4 mol / L piceatannol, and the pH value of the solution (pH clock system) was distinguished as a function of time.

[0021] Figure 7 This is a graph showing the change in pH value of the differentiation solution (pH clock system) over time when no sample to be differentiated is added in Example 3.

[0022] Figure 8 In Example 3, 1.6×10 -4 mol / L resveratrol, and then the pH value of the solution (pH clock system) was distinguished as a function of time.

[0023] Figure 9 In Example 3, 1.6×10 -4 mol / L piceatannol, and the pH value of the solution (pH clock system) was distinguished as a function of time. DETAILED DESCRIPTION Example 1

[0024] This example verifies the feasibility of the method for distinguishing resveratrol from piceatannol according to the present invention by the following steps: (1) Preparation of differentiation 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 -3mol / L, the total volume was 40 mL, and the temperature was controlled at 27 °C.

[0025] At the same time, ethanol was used as a solvent to prepare a series of sample solutions containing resveratrol and piceatannol at different concentrations.

[0026] (2) Obtaining pH clock map The graph of the pH value of the prepared differentiation solution (pH clock system) changing with time is recorded by a computer equipped with a chemical signal acquisition and analysis program (without adding the sample to be tested). Figure 1 As shown in the figure, the time for the clock system to reach the highest pH value was 158s, and the highest pH value was about 9.52, which was used as a blank control. Two sets of differentiating solutions were prepared with the same concentration of each component as the above-mentioned differentiating solution. For one of the sets, 200 μL of 0.16 mol / L resveratrol sample solution was added to 40 mL of the pH clock system at the beginning of the reaction, so that its concentration in the differentiating solution was 8×10 -5 mol / L, the added resveratrol extended the time for the clock system to reach the highest pH value to 200 s, and the highest pH value was about 9.07. Figure 2 As shown; 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 its concentration in the differentiation solution was 8×10 -5 mol / L, the added piceatannol makes the time for the clock system to reach the highest pH value become 165 s, and the highest pH value is about 9.38. Figure 3 shown.

[0027] (3) Distinction Because resveratrol and piceatannol have different structures, their effects on the maximum pH value of the clock system and the time it takes to reach the maximum pH value are also different. Figure 1 、 Figure 2 、 Figure 3 Compared to the pH-time profile without the solution to be differentiated, the addition of resveratrol resulted in a significant decrease in the clock system's maximum pH and a significant extension of the time it took to reach the maximum pH. The addition of piceatannol resulted in a smaller decrease in the clock system's maximum pH and a smaller extension of the time it took to reach the maximum pH. These experiments demonstrate that resveratrol and piceatannol can be differentiated by comparing the clock system's maximum pH and the time it takes to reach the maximum pH.

[0028] Take two 0.16 mol / L solutions of the samples to be differentiated (one is a resveratrol solution and the other is a piceatannol solution, but the two have not been differentiated yet), mark one as sample 1 and the other as sample 2; prepare two sets of differentiation solutions with the same concentration of each component as above, add 200 μL of 0.16 mol / L sample 1 and sample 2 respectively, so that their concentrations in the differentiation solutions are 8×10 -5 mol / L.

[0029] Analysis and comparison show that: the addition of sample 1 makes the maximum pH value of the clock system drop significantly and the time to reach the maximum pH value is greatly extended (the maximum pH value and the time to reach the maximum pH value are different from the Figure 2 Corresponding to Figure 3 The addition of sample 2 resulted in a smaller decrease in the maximum pH value of the clock system and a smaller extension of the time to reach the maximum pH value (the maximum pH value and the time to reach the maximum pH value were not consistent with the Figure 3 Corresponding to Figure 2 Therefore, sample 1 is a resveratrol solution and sample 2 is a piceatannol solution, thereby achieving the distinction between resveratrol and piceatannol. Example 2

[0030] This example verifies the feasibility of the method for distinguishing resveratrol from piceatannol according to the present invention by the following steps: (1) Preparation of differentiation 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 -4mol / 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.

[0031] At the same time, ethanol was used as a solvent to prepare a series of sample solutions containing resveratrol and piceatannol at different concentrations.

[0032] (2) Obtaining pH clock map The graph of the pH value of the prepared differentiation solution (pH clock system) changing with time is recorded by a computer equipped with a chemical signal acquisition and analysis program (without adding the sample to be tested). Figure 4 As shown. The time for the clock system to reach the highest pH value was 157 s, and the highest pH value was about 9.50. A blank control was used as a blank control. Two sets of differentiating solutions were prepared with the same concentration of each component as the above-mentioned differentiating solution. For one of the sets, 200 μL of 0.24 mol / L resveratrol sample solution was added to 40 mL of the pH clock system at the beginning of the reaction, so that its concentration in the differentiating solution was 1.2×10 -4 mol / L, the added resveratrol extended the time for the clock system to reach the highest pH value to 237 s, and the highest pH value was about 8.51. Figure 5 For the other group, 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 resveratrol in the separation solution was 1.2×10 -4 mol / L, the addition of piceatannol made the time for the clock system to reach the highest pH value become 170 s, and the highest pH value was about 9.15. Figure 6 shown.

[0033] (3) Distinction Because resveratrol and piceatannol have different structures, their effects on the maximum pH value of the clock system and the time it takes to reach the maximum pH value are also different. Figure 4 、 Figure 5 、 Figure 6 It can be seen that the addition of resveratrol caused a significant decrease in the maximum pH value of the clock system and significantly prolonged the time it took to reach the maximum pH value; the addition of piceatannol caused a smaller decrease in the maximum pH value of the clock system and a smaller delay in the time it took to reach the maximum pH value. These experiments show that by comparing the maximum pH value and the time it takes to reach the maximum pH value of the clock system, resveratrol and piceatannol can be distinguished.

[0034] Take two 0.24 mol / L solutions of the samples to be differentiated (one is a resveratrol solution and the other is a piceatannol solution, but the two have not been differentiated yet), mark one as sample 1 and the other as sample 2; prepare two sets of differentiation solutions with the same concentration of each component as above, add 200 μL of 0.24 mol / L sample 1 and sample 2 respectively, so that their concentrations in the differentiation solutions are 1.2×10 -4 mol / L.

[0035] Analysis and comparison show that: the addition of sample 1 makes the maximum pH value of the clock system drop significantly and the time to reach the maximum pH value is greatly extended (the maximum pH value and the time to reach the maximum pH value are different from the Figure 5 Corresponding to Figure 6 The addition of sample 2 resulted in a smaller decrease in the maximum pH value of the clock system and a smaller extension of the time to reach the maximum pH value (the maximum pH value and the time to reach the maximum pH value were not consistent with the Figure 6 Corresponding to Figure 5 Therefore, sample 1 is a resveratrol solution and sample 2 is a piceatannol solution, thereby achieving the distinction between resveratrol and piceatannol. Example 3

[0036] This example verifies the feasibility of the method for distinguishing resveratrol from piceatannol according to the present invention by the following steps: (1) Preparation of differentiation 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 -4mol / 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.

[0037] At the same time, ethanol was used as the solvent to prepare a series of sample solutions of resveratrol and piceatannol with different concentrations.

[0038] (2) Obtaining pH clock map The graph of the pH value of the prepared differentiation solution (pH clock system) changing with time is recorded by a computer equipped with a chemical signal acquisition and analysis program (without adding the sample to be tested). Figure 7 As shown. The time for the clock system to reach the highest pH value was 158 s, and the highest pH value was about 9.51, which served as a blank control. Two sets of differentiating solutions were prepared with the same concentration of each component as the above-mentioned differentiating solution. For one of the sets, at the beginning of the reaction, 200 μL of 0.32 mol / L resveratrol sample solution was added to 40 mL of the pH clock system, so that its concentration in the differentiating solution was 1.6×10 -4 mol / L, the added resveratrol extended the time for the clock system to reach the highest pH value to 261 s, and the highest pH value was about 8.78. Figure 8 For the other group, at the beginning of the reaction, 200 μL of 0.32 mol / L piceatannol sample solution was added to the 40 mL pH clock system, so that the concentration of resveratrol in the separation solution was 1.6×10 -4 mol / L, the added piceatannol makes the time for the clock system to reach the highest pH value become 175 s, and the highest pH value is about 9.10. Figure 9 shown.

[0039] (3) Distinction Because resveratrol and piceatannol have different structures, their effects on the maximum pH value of the clock system and the time it takes to reach the maximum pH value are also different. Figure 7 、 Figure 8 、 Figure 9 It can be seen that the addition of resveratrol caused a significant decrease in the maximum pH value of the clock system and significantly prolonged the time it took to reach the maximum pH value; the addition of piceatannol caused a smaller decrease in the maximum pH value of the clock system and a smaller delay in the time it took to reach the maximum pH value. These experiments show that by comparing the maximum pH value and the time it takes to reach the maximum pH value of the clock system, resveratrol and piceatannol can be distinguished.

[0040] Take two 0.32 mol / L solutions of the samples to be differentiated (one is a resveratrol solution and the other is a piceatannol solution, but the two have not been differentiated yet), mark one as sample 1 and the other as sample 2; prepare two sets of differentiation solutions with the same concentration of each component as above, add 200 μL of 0.32 mol / L sample 1 and sample 2 respectively, so that their concentrations in the differentiation solutions are 1.6×10 -4 mol / L.

[0041] Analysis and comparison show that: the addition of sample 1 makes the maximum pH value of the clock system drop significantly and the time to reach the maximum pH value is greatly extended (the maximum pH value and the time to reach the maximum pH value are different from the Figure 8 Corresponding to Figure 9 The addition of sample 2 resulted in a smaller decrease in the maximum pH value of the clock system and a smaller extension of the time to reach the maximum pH value (the maximum pH value and the time to reach the maximum pH value were not consistent with the Figure 9 Corresponding to Figure 8 Therefore, sample 8 is a piceatannol solution and sample 9 is a resveratrol solution, thereby achieving the distinction between resveratrol and piceatannol.

[0042] It can be seen from the above examples that the concentration is 8×10 -5 -1.6×10 -4 Resveratrol and piceatannol within the mol / L range can be distinguished by the method of the present invention.

Claims

1. A method for distinguishing resveratrol from piceatannol, characterized in that: Using ethanol as solvent, prepare sample solutions of resveratrol and piceatannol to be differentiated; The pH clock reaction system of "formaldehyde-Na2S2O5-Na2SO3-EDTA-D-glucono-δ-lactone" was used as the differentiating solution, and the pH value of the clock system was recorded as a graph of changes over time. 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 begins, equal volumes of resveratrol and piceatannol sample solutions of the same concentration are added to the two groups of pH clock systems, respectively, and the samples to be differentiated are differentiated according to the different effects of the samples to be differentiated on the maximum pH value of the clock system and the time to reach the maximum pH value: compared with the pH value change over time graph without the addition of the solution to be differentiated, if the addition of the solution to be differentiated causes a significant decrease in the maximum pH value of the clock system and a significant extension in the time to reach the maximum pH value, then the added sample to be differentiated is the resveratrol sample; if the addition of the solution to be differentiated causes a slight decrease in the maximum pH value of the clock system and a slight extension in the time to reach the maximum pH value, then the added sample to be differentiated is the piceatannol sample; The molar concentration range of each component in the 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 solution is 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 concentration range of the sample to be differentiated in the differentiation solution is 8×10 -5 -1.6×10 -4 mol / L.

4. The method according to claim 1, wherein: The clock system temperature was controlled at 27°C.

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