Method for quantitatively detecting ellagic acid
By using a nonlinear oscillation system catalyzed by tetraazacetetracyclodiene nickel complex, a working curve of ellagic acid concentration and inhibition time was established, and the complex and cost-effective ellagic acid detection in the prior art was solved, and a simple and accurate quantitative analysis of ellagic acid was achieved.
Patent Information
- Application Number
- CN202510690020.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-27
- Publication Date
- 2025-08-01
AI Technical Summary
The prior art methods for detecting ellagic acid are complex and costly, making it difficult to achieve efficient and accurate quantitative analysis.
A nonlinear oscillation system catalyzed by tetraazacetetracyclodiene nickel complex [NiL](ClO4)2 was used to establish a working curve for quantitative analysis by different concentrations of ellagic acid.
It provides a quantitative detection method for ellagic acid that is easy to operate, low cost and high accuracy, which can quickly detect ellagic acid content in food and medicine without being disturbed by other substances.
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Figure CN120404866A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for quantitatively analyzing fisetin, which is characterized in that: the nonlinear chemical oscillation system of "H2SO4-KIO3-[NiL](ClO4)2-malonic acid-H2O2" is used as a detection solution to quantitatively analyze fisetin. In [NiL](ClO4)2, the ligand L is 5, 7, 7, 12, 14, 14-hexamethyl-1, 4, 8, 11-tetraazacyclotetradeca-4, 11-diene. By using the different inhibition times of fisetin with different concentrations on the oscillation pattern, a working curve establishing the correlation between the concentration of the analyte and the inhibition time is established to achieve the purpose of detecting the content of fisetin in the sample. The present invention belongs to the field of analytical chemistry. Background Art
[0002] The structural formula of fisetin is shown as structural formula (I). Fisetin is a polyphenol dilactone compound and a dimer derivative of gallic acid. Fisetin is a natural polyphenol component widely present in various plant tissues such as soft fruits and nuts. Fisetin shows obvious inhibitory effects on chemically induced carcinogenesis and other various carcinogens, especially has good inhibitory effects on colon cancer, esophageal cancer, liver cancer, lung cancer, tongue and skin tumors, etc. Fisetin also has various biological activity functions, such as antioxidant function, antimutagenic property, and inhibitory effect on human immunodeficiency virus. In addition, fisetin is also an effective coagulant, has good inhibitory effects on various bacteria and viruses, can protect the wound surface from the invasion of bacteria, prevent infection, and inhibit ulcers. At the same time, it has been found through research that fisetin also has blood pressure lowering and sedative effects.
[0003] At present, the main method for detecting fisetin in China is high performance liquid chromatography (HPLC) method, combined with evaporative light scattering detector (ELSD) or DAD detector, which can efficiently and accurately detect the content change of fisetin in the sample. However, this method has the disadvantages of complex operation and high cost. The quantitative analysis method of fisetin involved in the present invention has the characteristics of high accuracy, easy operation, convenience and rapidity, etc.
[0004]
[0005] Structural formula (I) Fisetin (Ellagic acid) Summary of the Invention
[0006] The present invention aims to provide a new detection method for ellagic acid, namely a detection method for ellagic acid based on a nonlinear oscillation system catalyzed by nickel tetraazacyclotetradecadiene complex [NiL](ClO4)2. This method is an electrochemical oscillation system method developed based on the sensitive response of the nonlinear system (i.e., the oscillation system) catalyzed by this complex to ellagic acid. Specifically, different concentrations of ellagic acid sample solutions are added to the oscillation system, resulting in different inhibition times, and a working curve (such as a linear relationship diagram) establishing the correlation between the concentration of the analyte and the inhibition time is established, thereby realizing the quantitative analysis of ellagic acid.
[0007] The nickel tetraazacyclotetradecadiene complex referred to in the present invention is a nickel tetraazamacrocyclic complex with 5, 7, 7, 12, 14, 14-hexamethyl-1, 4, 8, 11-tetraazacyclotetradeca-4, 11-diene as a ligand, and its chemical formula is [NiL](ClO4)2, and its structure is shown in formula (II).
[0008] Structural formula (II) [NiL](ClO4)2 The structure of this complex is very similar to the key structure porphyrin ring of myoglobin, hemoglobin, chlorophyll and some metalloenzymes in vivo. The chemical oscillation reaction catalyzed by [NiL](ClO4)2 is similar to the biochemical oscillation in plant and animal cells. Therefore, this system has a stable amplitude, a long oscillation lifetime and a sensitive response to ellagic acid.
[0009] [NiL](ClO4)2 is synthesized according to the reference: 1) Prepare L·2HClO4; 2) Prepare [NiL](ClO4)2 from L·2HClO4.
[0010] (1) Prepare L·2HClO4: References: 1. Curtis, N. F. and Hay, R. W., J. Chem. Soc., Chem. Commun., 1966, p.534. 2. Gang Hu, Panpan Chen, Wei Wang, Lin Hu, Jimei Song, Lingguang Qiu, Juan Song, Electrochimica Acta, 2007, Vol. 52, pp.7996 - 8002. 3. Lin Hu, Gang Hu, Han - Hong Xu, J. Anal. Chem., 20060 Vol. 61, NO.10, pp.1021 - 1025. 4. Hu Gang, Doctoral Dissertation of University of Science and Technology of China, p25 - 27, Hefei, 2005.
[0011] (2) Preparation of [NiL](ClO4)2 from L·2HClO4: References: 1. N. F. Curtis, J. Chem. Soc. Dolton Tran., 1972, Vol.13, 1357. 2. Hu Gang, Doctoral Dissertation of University of Science and Technology of China, p42 - 43, Hefei, 2005.
[0012] It was confirmed as the complex by elemental analysis and infrared characterization.
[0013] The difference between this detection method and the existing technologies is that the H2SO4 - KIO3 - [NiL](ClO4)2 - malonic acid - H2O2 Briggs - Rauscher non - linear oscillation system is used as the detection solution. By using the different inhibition times of ellagic acid with different concentrations on the oscillation pattern, a working curve correlating the concentration of the analyte with the inhibition time is established, and then the quantitative analysis of ellagic acid is realized. The concentrations of each component in the detection solution are shown in Table 1: Table 1: Concentration ranges of each component in the chemical oscillation system <![CDATA[H2SO4 (mol / L)]]> <![CDATA[KIO3 (mol / L)]]> <2 (mol / L)]]> Malonic acid (mol / L) <![CDATA[H2O2 (mol / L)]]> 0.02445-0.02693 0.024369-0.02458 0.000846-0.0008947 0.1715-0.1783 1.2727-1.3236 The specific operation is as follows: 1. Prepare the detection solution according to Table 1, and record the curve of the change of the potential of this solution with time (Potential - Time curve), namely the chemical potential oscillation pattern.
[0014] Put the prepared detection solution into a 50 mL small beaker and add a magnetic stirrer of appropriate size. Place it on a constant - temperature magnetic heating stirrer, keep the stirring speed at 800 revolutions per minute, and maintain the temperature in the beaker at - 4 ~ 2℃ under ice - bath conditions. Then, insert the prepared working electrode (platinum electrode) and reference electrode (double - salt - bridge calomel electrode) into the solution to prepare for potential monitoring of the solution. The other ends of the working electrode and the reference electrode are connected to a data collector (Go! LINK) through an amplifier (Instrument Amplifier) and then to a computer. After setting the acquisition time and sampling speed in the logger lite program in the computer, quickly click the start button to monitor the potential of the solution. The computer records the curve of the collected potential values changing with time, namely the chemical potential oscillation pattern. When it is necessary to detect the analyte ellagic acid, when the oscillation pattern reaches the stability of the oscillation system, quickly add ellagic acid to the oscillation system, usually when the potential is at the lowest potential during the 4th to 7th oscillations.
[0015] The basic parameters of the oscillation spectrum include: Induction time: The time required from adding the last substance to the start of oscillation of the solution.
[0016] Oscillation amplitude: The potential difference between the lowest potential and the next highest potential during the oscillation process.
[0017] Oscillation period: The time required from one lowest (highest) potential to the next lowest (highest) potential during the oscillation process.
[0018] Highest potential: The highest potential point that appears in the system during stable oscillation.
[0019] Lowest potential: The lowest potential point that appears in the system during stable oscillation.
[0020] Oscillation lifetime: The time required from the start to the end of oscillation.
[0021] Equilibrium potential: The potential when the system reaches the thermodynamic equilibrium state. At this moment, the potential does not change with time.
[0022] Inhibition time (T in ): The time required from the start of inhibition of oscillation after adding the ellagic acid solution to the restoration of oscillation.
[0023] 2. Establish a working curve for the relationship between the ellagic acid sample concentration and the oscillation characteristic response parameter (inhibition time) Prepare a series of ellagic acid solutions with different concentrations as sample solutions. Add the prepared sample solutions to the stable oscillation system and add them when the potential is at the lowest potential during the 4th to 7th oscillations. The added ellagic acid will temporarily inhibit the oscillation for a period of time, and then the oscillation resumes, that is, the inhibition time T in is generated. Adding ellagic acid with different concentrations will result in different inhibition times. The oscillation characteristic response parameter is the inhibition time (T in ).
[0024] Using the inhibition time T in as the ordinate and the concentration c of the ellagic acid sample solution as the abscissa to plot a graph, a working curve is obtained for the ellagic acid concentration range from 5.00×10 -6 mol / L to 1.50×10 -5 mol / L in the system.
[0025] 3. Quantitative analysis of ellagic acid Add the sample to be tested to the stable oscillation system (all samples to be tested are added when the potential is at the lowest potential during the 4th oscillation). The oscillation response is the generation of an inhibition time, and T inThe value, according to the working curve, can be used to obtain the concentration of ellagic acid in the sample to be tested.
[0026] This method can conveniently and quickly detect the content of ellagic acid in food or medicine. Experiments show that other substances in the sample have no interference with the detection. Description of the Drawings
[0027] Figure 1 It is the chemical potential oscillation spectrum of the detection solution (without adding samples) in Example 1.
[0028] Figure 2 It is the oscillation response spectrum of the oscillation system after adding 5.00×10 -6 mol / L ellagic acid in Example 1.
[0029] Figure 3 It is the oscillation response spectrum of the oscillation system after adding 7.50×10 -6 mol / L ellagic acid in Example 1.
[0030] Figure 4 It is the working curve of the inhibition time T of ellagic acid concentration c in Example 1 in
[0031] Figure 5 It is the chemical potential oscillation spectrum of the detection solution (without adding samples) in Example 2.
[0032] Figure 6 It is the oscillation response spectrum of the oscillation system after adding 1.00×10 -5 mol / L ellagic acid in Example 2.
[0033] Figure 7 It is the oscillation response spectrum of the oscillation system after adding 1.25×10 -5 mol / L ellagic acid in Example 2.
[0034] Figure 8 It is the working curve of ellagic acid concentration c and inhibition time T in Example 2 in
[0035] Figure 9 It is the chemical potential oscillation spectrum of the detection solution (without adding samples) in Example 3.
[0036] Figure 10 It is the oscillation response spectrum of the oscillation system after adding 1.25×10 -5 mol / L ellagic acid in Example 3.
[0037] Figure 11 It is the oscillation response spectrum of the oscillation system after adding 1.50×10 -5 mol / L ellagic acid in Example 3.
[0038] Figure 12 is the working curve of ellagic acid concentration c and inhibition time T in Example 3 in of the working curve graph. Detailed implementation manners Example 1
[0039] Apply the H2SO4 - KIO3 - [NiL](ClO4)2 - malonic acid - H2O2 Briggs - Rauscher oscillation system as the detection solution. Utilize the different inhibition times of ellagic acid with different concentrations on the oscillation pattern to establish a working curve that correlates the concentration of the analyte with the inhibition time, and then achieve the quantitative analysis of.
[0040] (1) Prepare the H2SO4 - KIO3 - [NiL](ClO4)2 - malonic acid - H2O2 detection solution Firstly, prepare a 0.025 mol / L H2SO4 solution as the solvent with 98% H2SO4; then use the 0.025 mol / L H2SO4 solvent to prepare a 0.14 mol / L KIO3 solution, a 2 mol / L malonic acid solution, a 0.0173 mol / L [NiL](ClO4)2 solution, and a 4.0 mol / L H2O2 solution respectively. Then, successively add 14.5 mL of 0.025 mol / L H2SO4 solution; 7 mL of 0.14 mol / L KIO3 solution; 2 mL of 0.0173 mol / L [NiL](ClO4)2; 3.5 mL of 2 mol / L malonic acid solution; 13 mL of 4.0 mol / L H2O2 solution into an open system of a 50 mL beaker. Finally, the concentration of H2SO4 in the system is 0.025 mol / L, the concentration of KIO3 is 0.02445 mol / L, the concentration of [NiL](ClO4)2 is 0.0008673 mol / L, the concentration of malonic acid is 0.174 mol / L, and the concentration of H2O2 is 1.293 mol / L.
[0041] Use ethanol as the solvent to prepare a series of ellagic acid sample solutions with different concentrations.
[0042] (2) Obtain the oscillation pattern The potential oscillation pattern of the chemical oscillation system is recorded by a computer equipped with the logger lite program, Figure 1At typical concentrations (H2SO4 0.025 mol / L, KIO3 0.02445 mol / L, [NiL](ClO4)2 0.0008673 mol / L, malonic acid 0.174 mol / L, H2O2 1.293 mol / L), the oscillation spectrum of the above-mentioned differentiated solution without adding the sample to be tested was used as a blank control. 20 μL of 0.01 mol / L ellagic acid sample solution was added to the prepared oscillation solution, so that its concentration in the system was 5.00×10 -6 mol / L. The added ellagic acid produced a characteristic response to the oscillation spectrum ( Figure 2 ), where the inhibition time T in was 143 seconds. For another group, 30 μL of 0.01 mol / L ellagic acid sample solution was added to the prepared oscillation solution, so that its concentration in the system was 7.50×10 -6 mol / L. The added one produced a characteristic response to the oscillation spectrum ( Figure 3 ), where the inhibition time T in was 389 seconds. The addition time for each time was at the start of the 4th oscillation when the potential was at the lowest potential. From Figure 2 , Figure 3 it can be seen that ellagic acid at different concentrations would produce different inhibition times.
[0043] (3) Analysis According to the relationship between the concentration c of ellagic acid in the system and the inhibition time T in , a working curve was established. As Figure 4 shown, where the abscissa is the concentration c of ellagic acid added to the oscillation solution, and the ordinate is the inhibition time T in . When the concentration of ellagic acid in the system was between 5.00×10 -6 mol / L and 1.50×10 -5 mol / L, the inhibition time T in had a linear relationship with the concentration c of the ellagic acid solution. The linear equation was T in =6.7×10 7 c - 169.6, R 2 =0.98212. Based on this, quantitative analysis of ellagic acid can be achieved. Example 2
[0044] (1) Preparation of H2SO4 - KIO3 - [NiL](ClO4)2 - malonic acid - H2O2 detection solution First, prepare a 0.025 mol / L H2SO4 solution using 98% H2SO4 as the solvent. Then, use the 0.025 mol / L H2SO4 solvent to prepare a 0.14 mol / L KIO3 solution, a 2 mol / L malonic acid solution, a 0.0173 mol / L [NiL](ClO4)2 solution, and a 4.0 mol / L H2O2 solution. Then, successively add 14.5 mL of the 0.025 mol / L H2SO4 solution, 7 mL of the 0.14 mol / L KIO3 solution, 2 mL of the 0.0173 mol / L [NiL](ClO4)2, 3.5 mL of the 2 mol / L malonic acid solution, and 13 mL of the 4.0 mol / L H2O2 solution to an open system in a 50 mL beaker. Finally, the concentration of H2SO4 in the system is 0.025 mol / L, the concentration of KIO3 is 0.02439 mol / L, the concentration of [NiL](ClO4)2 is 0.0008765 mol / L, the concentration of malonic acid is 0.1732 mol / L, and the concentration of H2O2 is 1.301 mol / L.
[0045] Prepare a series of ellagic acid sample solutions with different concentrations using ethanol as the solvent.
[0046] (2) Obtain the oscillation spectrum The potential oscillation spectrum of the chemical oscillation system is recorded by a computer equipped with the logger lite program. Figure 5 It is the oscillation spectrum of the above-mentioned differentiated solution without adding the test sample at typical concentrations (H2SO4 0.025 mol / L, KIO3 0.02439 mol / L, [NiL](ClO4)2 0.0008765 mol / L, malonic acid 0.1732 mol / L, H2O2 1.301 mol / L) for blank control. Add 40 μL of the 0.01 mol / L ellagic acid sample solution to the prepared oscillation solution so that its concentration in the system is 1.00×10 -5 mol / L. The added ellagic acid produces a characteristic response to the oscillation spectrum ( Figure 6 ), where the inhibition time T in is 470 seconds. For another group, add 50 μL of the 0.01 mol / L ellagic acid sample solution to the prepared oscillation solution so that its concentration in the system is 1.25×10 -5 mol / L. The added ellagic acid produces a characteristic response to the oscillation spectrum ( Figure 7 ), where the inhibition time T in is 652 seconds. The addition time for each time is at the start of the 4th oscillation when the potential is at the lowest potential. From Figure 6 、Figure 7 It can be seen that ellagic acid at different concentrations produces different inhibition times.
[0047] (3) Analysis Based on the concentration c of ellagic acid and the inhibition time T in the system in to establish a working curve, as Figure 8 shown, where the abscissa is the concentration c of ellagic acid added to the oscillating solution, and the ordinate is the inhibition time T in , when the concentration of ellagic acid in the system is between 5.00×10 -6 mol / L and 1.50×10 -5 mol / L, the inhibition time T in has a linear relationship with the concentration c of the ellagic acid solution, and the linear equation is T in =6.7×10 7 c - 168.8, R 2 =0.98427. Based on this, quantitative analysis of ellagic acid can be achieved. Example 3
[0048] (1) Prepare the H2SO4 - KIO3 - [NiL](ClO4)2 - malonic acid - H2O2 detection solution First, prepare a 0.025 mol / L H2SO4 solution with 98% H2SO4 as the solvent; then use the 0.025 mol / L H2SO4 solvent to prepare a 0.14 mol / L KIO3 solution, a 2 mol / L malonic acid solution, a 0.0173 mol / L [NiL](ClO4)2 solution, and a 4.0 mol / L H2O2 solution respectively. Then, successively add 14.5 mL of 0.025 mol / L H2SO4 solution; 7 mL of 0.14 mol / L KIO3 solution; 2 mL of 0.0173 mol / L [NiL](ClO4)2; 3.5 mL of 2 mol / L malonic acid solution; 13 mL of 4.0 mol / L H2O2 solution to the open system of a 50 mL beaker. Finally, the concentration of H2SO4 in the system is 0.025 mol / L, the concentration of KIO3 is 0.02457 mol / L, the concentration of [NiL](ClO4)2 is 0.0008821 mol / L, the concentration of malonic acid is 0.1756 mol / L, and the concentration of H2O2 is 1.3151 mol / L.
[0049] Using ethanol as the solvent, prepare a series of ellagic acid sample solutions with different concentrations.
[0050] (2) Obtain the oscillation spectrum The potential oscillation spectra of the chemical oscillation system were recorded by a computer equipped with the logger lite program. Figure 9 Under typical concentrations (H2SO4 0.025 mol / L, KIO3 0.02457 mol / L, [NiL](ClO4)2 0.0008821 mol / L, malonic acid 0.1756 mol / L, H2O2 1.3151 mol / L), the oscillation spectra of the above-mentioned differentiated solution without adding the test sample were used as a blank control. 50 μL of 0.01 mol / L ellagic acid sample solution was added to the prepared oscillation solution, so that its concentration in the system was 1.25×10 -5 mol / L. The added ellagic acid produced a characteristic response to the oscillation spectra ( Figure 10 ), where the inhibition time T in was 645 seconds. For another group, 60 μL of 0.02 mol / L ellagic acid sample solution was added to the prepared oscillation solution, so that its concentration in the system was 1.50×10 -5 mol / L. The added ellagic acid produced a characteristic response to the oscillation spectra ( Figure 11 ), where the inhibition time T in was 851 seconds. The addition time for each time was at the start of the 4th oscillation when the potential was at the lowest potential. From Figure 10 , Figure 11 it can be seen that ellagic acid at different concentrations would produce different inhibition times.
[0051] (3) Analysis According to the relationship between the concentration c of ellagic acid in the system and the inhibition time T in , a working curve was established. As Figure 12 shown, where the abscissa is the concentration c of ellagic acid added to the oscillation solution, and the ordinate is the inhibition time T in . When the concentration of ellagic acid in the system was between 5.00×10 -6 mol / L and 1.50×10 -5 mol / L, the inhibition time T in had a linear relationship with the concentration c of the ellagic acid solution. The linear equation was T in =6.7×10 7 c - 170.6, R 2 =0.98235. Based on this, quantitative analysis of ellagic acid can be achieved.
Claims
1. A method for detecting ellagic acid, characterized in that : Using the H2SO4 - KIO3 - [NiL](ClO4)2 - malonic acid - H2O2 Briggs - Rauscher oscillating system as the detection solution, record the inhibition time of ellagic acid with different concentrations on the oscillating system, establish a working curve correlating the concentration of the analyte with the inhibition time, and achieve the quantitative analysis of ellagic acid; in [NiL](ClO4)2, L is 5, 7, 7, 12, 14, 14 - hexamethyl - 1, 4, 8, 11 - tetraazacyclotetradeca - 4, 11 - diene; The detection steps are as follows: Prepare the H2SO4 - KIO3 - [NiL](ClO4)2 - malonic acid - H2O2 detection solution. In the detection solution, the molar concentrations of each component are H2SO4 0.02445 - 0.02693 mol / L, KIO3 0.024369 - 0.02458 mol / L, [NiL](ClO4)2 0.000846 - 0.0008947 mol / L, malonic acid 0.1715 - 0.1783 mol / L, and H2O2 1.2727 - 1.3236 mol / L; Using ethanol as the solvent, prepare a series of ellagic acid solutions with different concentrations.
2. Obtain the oscillation pattern The oscillogram of the detection solution is recorded by a computer; different concentrations of ellagic acid solution are added to the prepared oscillating solution, and the addition time each time is when the oscillation starts for the 4th time and the potential is at the lowest potential; the added ellagic acid will temporarily inhibit the oscillation for a period of time, and then the oscillation resumes, that is, the inhibition time T is generated. in , different concentrations of ellagic acid will produce different inhibition times.
3. Analyze Based on the relationship between the concentration c of ellagic acid and the inhibition time T in the system in a working curve is established; where the abscissa is the concentration c of ellagic acid added to the oscillating solution, and the ordinate is the inhibition time T in , when the concentration of ellagic acid in the system is between 5.00×10 -6 mol / L and 1.50×10 -5 mol / L, the inhibition time T in has a linear relationship with the concentration c of the ellagic acid solution, and based on this, quantitative analysis of ellagic acid in the sample can be achieved.