Method for detecting chlorogenic acid and application thereof

Abstract

The application relates to the field of biological monitoring and particularly belongs to a detection method of chlorogenic acid and application thereof. The method comprises the following steps: taking palladium nitrate, iron nitrate nonahydrate and glutathione as raw materials to prepare FePd2S4 nano-enzyme particles; mixing different concentrations of chlorogenic acid standard solution with acetic acid-sodium acetate buffer solution, H2O2, 3,3',5,5'-tetramethylbenzidine and FePd2S4 nano-enzyme suspension, incubating, measuring the absorbance value at 652 nm, taking the concentration of chlorogenic acid as the abscissa and the absorbance difference as the ordinate, drawing a standard curve and obtaining a linear equation; processing a sample to be measured, extracting chlorogenic acid in the sample to be measured, and preparing a sample solution; taking the sample solution to replace the chlorogenic acid standard solution to repeat the above steps and measure the absorbance value; and calculating the content of chlorogenic acid in the sample to be measured according to the linear equation of S.2. The method has the advantages of simplicity, low cost, high stability, environmental friendliness and the like and is suitable for large-scale preparation.

Description

Technical Field

[0001] This invention relates to the field of biological monitoring, specifically to a method for detecting chlorogenic acid and its application. Background Technology

[0002] Chlorogenic acid is a naturally occurring polyphenolic compound widely found in various plants, including coffee, tea, and honeysuckle. The 2010 edition of the Chinese Pharmacopoeia uses chlorogenic acid content as one of the quality evaluation indicators for traditional Chinese medicinal materials such as honeysuckle, wild honeysuckle, and eucommia leaves. Its unique molecular structure, containing caffeoyl and quinic acid groups, endows it with significant redox activity and various biological functions. Numerous studies have demonstrated that chlorogenic acid possesses powerful antioxidant, anti-inflammatory, and hypoglycemic activities, making it an important component in functional foods, pharmaceuticals, and health supplements. Therefore, accurate determination of chlorogenic acid content is crucial for product quality control, pharmacological research, and clinical applications.

[0003] Currently, the main analytical methods for chlorogenic acid include high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS). While these techniques offer advantages such as high precision and selectivity, they also have limitations, such as expensive equipment, complex sample pretreatment requirements, and lengthy analytical procedures. These limitations significantly restrict their application in rapid on-site detection and real-time monitoring. In this context, nanozyme-based colorimetric sensing technology has emerged as a promising alternative due to its advantages of ease of operation, rapid response, low cost, and potential for miniaturization.

[0004] Traditional enzyme-based colorimetric sensors typically utilize enzymes such as peroxidase or laccase, benefiting from the inherent specificity of biorecognition elements. However, these sensors face challenges including poor operational stability, sensitivity to environmental conditions, limited lifespan due to enzyme denaturation, and stringent requirements for pH and temperature. In contrast, enzyme-like sensors, based on advanced functional materials, offer greater stability, a wider range of operating conditions, and longer lifespans, although they often fall short of achieving the same level of specificity as enzyme-based sensors. The unique physicochemical properties of nanozymes make them ideal sensing elements for interactions with non-specific analytes; therefore, the use of nanozymes to construct array-based sensing methods holds great promise for applications in analytical detection.

[0005] Patent CN105758956A proposes a method for detecting chlorogenic acid in mulberries and their products. This application uses high-performance liquid chromatography (HPLC) to detect chlorogenic acid content. This process is stable, reliable, environmentally friendly, and has a high extraction yield. However, the equipment used in this method is expensive, the sample pretreatment requirements are complex, and the analytical process is lengthy, making it unsuitable for widespread application in production and daily life.

[0006] Patent CN112980434A proposes a silicon quantum dot method for detecting chlorogenic acid and a method for detecting chlorogenic acid. The chlorogenic acid detection method established using silicon quantum dots in this application is characterized by high efficiency, sensitivity, high specificity, and fast detection speed, providing sensitive and accurate detection results for the specific detection of chlorogenic acid. However, this method requires a long detection time and has a slow reaction speed, making it unsuitable for rapid detection.

[0007] Patent CN116087357A discloses a method for determining chlorogenic acid compounds in beverages. This application can effectively separate seven chlorogenic acids, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and 1,5-dicaffeoylquinic acid, and can detect all seven chlorogenic acids in a beverage simultaneously. This method has advantages such as high sensitivity and accurate qualitative and quantitative analysis. However, this method still employs chromatography, which is costly, involves complex pretreatment steps, requires a long time, and demands strict detection conditions, making it inconvenient for widespread use in production testing. Summary of the Invention

[0008] In view of the problems existing in the prior art, the purpose of this invention is to design and provide a method for detecting chlorogenic acid and its application.

[0009] The present invention is specifically implemented using the following technical solutions: The first aspect of this invention provides a method for detecting chlorogenic acid, comprising the following steps: S.1. FePd2S4 nanozyme particles were prepared using palladium nitrate, ferric nitrate nonahydrate, and glutathione as raw materials.

[0010] S.2. Chlorogenic acid standard solutions of different concentrations were mixed with acetate-sodium acetate buffer, H2O2, 3,3',5,5'-tetramethylbenzidine and FePd2S4 nanozyme suspension, and incubated. The absorbance at 652 nm was measured. A standard curve was plotted with chlorogenic acid concentration on the x-axis and absorbance difference on the y-axis, and the linear equation was obtained.

[0011] S.3. Process the sample to be tested, extract chlorogenic acid from the sample to be tested, and prepare the test solution.

[0012] S.4. Replace the chlorogenic acid standard solution with the test solution and repeat step S.2 to determine the absorbance value.

[0013] S.5. The content of chlorogenic acid in the sample to be tested is calculated based on the linear equation in S.2.

[0014] Furthermore, the specific preparation of FePd2S4 nanozyme particles in step S.1 includes the following steps: Y.1. Dissolve Fe(NO3)3·9H2O, Pd(NO3)2·xH2O and L-cysteine ​​in water, add ethylene glycol, and stir until homogeneous.

[0015] Y.2. Heat the solution and maintain the temperature for a period of time, then cool it to room temperature.

[0016] Y.3. The precipitate was washed and dried after cooling to obtain FePd2S4 nanozyme particles.

[0017] Further, the molar ratio in step Y.1 is Fe(NO3)3·9H2O:Pd(NO3)2·xH2O:L-cysteine ​​= 1:2-4:7-9, with 25-100 mL of water and 25-100 mL of ethylene glycol.

[0018] Furthermore, in step Y.2, the heating is carried out in a stainless steel autoclave lined with polytetrafluoroethylene, and the temperature is raised to 150-180℃ and maintained for 7-9 hours.

[0019] Furthermore, in step Y.3, the washing is performed 3-5 times each with deionized water and ethanol, and the drying method is vacuum drying at 60-80°C.

[0020] Further, in step S.2, the pH of the acetate-sodium acetate buffer solution is 3.0-5.0, and the concentration is 0.1-0.3M; the final concentration of H2O2 is 0.05-0.2mM; the final concentration of 3,3',5,5'-tetramethylbenzidine is 100-200μM; and the final concentration of the FePd2S4 nanozyme suspension is 10-50μg / mL. -1 .

[0021] Furthermore, the incubation temperature in step S.2 is 25-37℃, and the incubation time is 2-10 minutes.

[0022] Furthermore, the standard curve equation in step S.2 is: A - A0 = 0.026 × C 绿原酸 -0.018; Absorbance value A, absorbance of blank A0, C 绿原酸 This represents the concentration of chlorogenic acid, in μg / mL. -1 .

[0023] Furthermore, the method for processing the sample to be tested in step S.3 is as follows: weigh the sample powder to be tested, add 75% methanol solution at a material-to-liquid ratio of 1:100 (g:mL), mix well, place in an ultrasonic extractor, set the ultrasonic power to 500W and the frequency to 40kHz, and ultrasonically extract for 40min. After extraction, filter through filter paper under reduced pressure, collect the filtrate, and store it in a refrigerator at 4℃ for later use.

[0024] A second aspect of the present invention provides an application of the method described above in the rapid detection of food or Chinese medicinal materials.

[0025] The present invention has the following beneficial effects: (1) The detection method of this application does not require expensive and complicated instruments such as chromatography-mass spectrometry instruments, and can realize rapid and efficient detection of chlorogenic acid in Chinese medicinal materials or food. At the same time, the preparation method of FePd2S4 nanozyme particles is simple, low cost, high stability and environmentally friendly, and is suitable for large-scale preparation.

[0026] (2) The amount of nanozyme required in the detection process of this application is small, which greatly reduces the detection cost. The detection sensitivity is high and the response time is short. The color change can be observed within a few minutes, which greatly shortens the detection time.

[0027] (3) This application has the feature of visualization, which can realize on-site quantitative or qualitative detection, which is fast and convenient, and has great application value and market prospects. Attached Figure Description

[0028] Figure 1 This is a scanning electron microscope image of the FePd2S4 nanozyme prepared in Example 1 of the present invention; Figure 2 EDS image of the FePd2S4 nanozyme prepared in Example 1 of this invention; Figure 3 This is a graph showing the peroxidase-like activity of FePd2S4 in Example 2 of the present invention. Figure 4 This is the chlorogenic acid standard curve established in Example 3 of the present invention; Figure 5 The images show the 300-800nm ​​ultraviolet scans of samples with different concentrations in this detection system in Example 4 of the present invention. Detailed Implementation

[0029] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0030] Example 1: Preparation of FePd2S4 nanozymes (1) Raw material weighing: In a molar ratio of Fe∶Pd∶L-cysteine ​​= 1∶2∶8, accurately weigh 0.4040 g of ferric nitrate nonahydrate, 0.4609 g of palladium nitrate hydrate, and 0.96938 g of L-cysteine, and place the above raw materials together in a 100 mL beaker.

[0031] (2) Solution preparation and stirring: Add 25 mL of deionized water and 25 mL of ethylene glycol to a beaker and stir continuously for 30 min at room temperature to fully dissolve and mix the raw materials.

[0032] (3) Hydrothermal reaction: Transfer the well-mixed solution to a 100 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene. After sealing the reactor, place it in a heating device, raise the temperature to 160 °C and keep it at a constant temperature for 8 h.

[0033] (4) Post-processing: After the reaction is completed, turn off the heating equipment and let the reactor cool naturally to room temperature. Take out the product from the reactor. Wash the product alternately with deionized water and anhydrous ethanol, three times each, to remove residual impurity ions and unreacted raw materials on the surface of the product. Place the washed product in a vacuum drying oven, set the temperature to 60 °C, and dry to constant weight to obtain the target product FePd2S4 powder.

[0034] The FePd2S4 nanoparticles prepared above were analyzed by scanning electron microscopy (SEM) and EDS, as follows: Figure 1 and Figure 2 As shown, the FePd2S4 surface exhibits a rough granular surface. EDS analysis shows that Fe, Pd, and S are uniformly distributed in the material, indicating that FePd2S4 was successfully synthesized on the surface.

[0035] Example 2: Study on the properties of FePd2S4 nanozymes Using colorless TMB as a chromogenic substrate, the peroxidase-like properties of FePd2S4 were evaluated by the amount of blue oxidized TMB (oxTMB) generated. The experimental procedure was as follows: FePd2S4 (final concentration: 5 mg / L) was added to acetate-sodium acetate buffer (0.2 M, pH 3.5). –1 The substrates ABTS (final concentration: 0.25 mM, characteristic absorption peak at 420 nm) and OPD (final concentration: 0.5 mM, characteristic absorption peak at 450 nm) were incubated in a 37 ℃ water bath for 20 min, and the UV-Vis absorption spectra of the solutions in the wavelength range of 300–800 nm were recorded.

[0036] like Figure 3As shown, under weakly acidic conditions, FePd2S4 catalyzes the oxidation of TMB to a blue oxidized state (oxTMB, which has a characteristic UV-Vis absorption peak at 652 nm), while TMB itself does not show obvious color change, indicating that FePd2S4 has good peroxidase-like activity.

[0037] Example 3: Application of FePd2S4 nanozyme in the detection of chlorogenic acid in Hangzhou white chrysanthemum (1) Establishing a standard curve for chlorogenic acid 1) Add chlorogenic acid standard solution in different volumes to acetate-sodium acetate buffer (pH 3.5, 0.2 M); H2O2 (final concentration 0.1 mM); 3,3',5,5'-tetramethylbenzidine (final concentration 150 μM); and FePd2S4 (final concentration 20 μg / mL). -1 .

[0038] 2) After incubating at 25°C for 5 minutes, terminate the reaction with an ice bath.

[0039] 3) Take 200 μL of each mixture and measure its absorbance A at 652 nm using a full-wavelength scanning microplate reader. Simultaneously record the absorbance A0 of the blank without standard. Calculate the standard curve equation for the difference between chlorogenic acid concentration and absorbance ΔA: The equation of the standard curve is ΔA = 0.026 × C. 绿原酸 -0.018, such as Figure 3 .

[0040] Where ΔA is the absorbance difference, and C 绿原酸 This represents the concentration of chlorogenic acid, in μg / mL. -1 Correlation coefficient R 2 =0.982, the detection linear range is 0-40 μg / mL -1 The detection limit is 1 μg / mL. -1 .

[0041] (2) Treatment of Hangzhou white chrysanthemum / bamboo leaves 1) Raw material weighing: Weigh 0.5020g of Hangzhou white chrysanthemum powder accurately according to the material-liquid ratio of 1:100 (g:mL) and place it in two 100mL beakers, labeled as beaker A (Hangzhou white chrysanthemum) and beaker B (bamboo leaf).

[0042] 2) Addition and mixing of extraction solvent: Add 50 mL of 75% methanol solution to beakers A and B respectively, and stir thoroughly with a glass rod to mix the medicinal powder with the methanol solution evenly.

[0043] 3) Ultrasonic extraction: Place the three beakers in an ultrasonic extractor, set the ultrasonic extraction power to 500W and the ultrasonic frequency to 40kHz, and perform ultrasonic extraction for 40 minutes at room temperature.

[0044] 4) Filtration and Preservation: After ultrasonic extraction, the mixture in each beaker was filtered using quantitative filter paper, and the filtrates were collected to obtain the Hangzhou white chrysanthemum extract and bamboo leaf extract. The two extracts were then transferred to sealed containers and refrigerated at 4°C for later use. When ready for analysis, the filtrates were concentrated or diluted to ensure that chlorogenic acid remained within the linear detection range.

[0045] (3) Determination of chlorogenic acid in the Hangzhou white chrysanthemum to be tested Replace the chlorogenic acid standard solution in step (1) with the chrysanthemum sample treated in step (2), repeat the operation in step (1), and measure its absorbance value at 652 nm. The absorbance value was 0.425.

[0046] (4) Calculation of chlorogenic acid concentration in Hangzhou white chrysanthemum Substituting the absorbance value measured in step (3) into the linear equation obtained in step (1), the concentration of chlorogenic acid in the tested Hangzhou white chrysanthemum was calculated to be 17.038 μg / mL. -1 .

[0047] Example 4: Application of FePd2S4 nanozyme in the detection of chlorogenic acid in bamboo leaves 1) Prepare bamboo leaf extract according to step (2) in Example 2, and add standard chlorogenic acid solution to the bamboo leaf extract at concentrations ranging from 5-50 μg / mL. -1 .

[0048] 2) Mixed acetate-sodium acetate buffer (pH 3.5, 0.2M), FePd2S4 nanozyme (final concentration 20 μg / mL) -1 TMB (final concentration 150 μM) and H2O2 (final concentration 100 μM) were added to the bamboo leaf samples treated in step 1).

[0049] 3) Incubate at 25℃ for 5 minutes.

[0050] 4) Scanning with ultraviolet wavelengths (300-800nm) Figure 5 Bamboo leaf samples containing chlorogenic acid standards showed an enhanced absorption peak at 652 nm, thus the presence of chlorogenic acid can be preliminarily identified by colorimetric reaction and ultraviolet wavelength scanning.

[0051] 5) Determination of chlorogenic acid in bamboo leaves Replace the chlorogenic acid standard solution in step (1) of Example 3 with the bamboo leaf sample treated in step 1), repeat the operation of step (1), and measure its absorbance value at 652 nm. The absorbance value was 0.225.

[0052] 6) Calculation of chlorogenic acid concentration in bamboo leaves Substituting the absorbance value measured in step 5) into the linear equation obtained in step (1) of Example 3, the concentration of chlorogenic acid in the bamboo leaves to be tested was calculated to be 9.346 μg / mL. -1 .

Claims

1. A method for detecting chlorogenic acid, characterized in that, Includes the following steps: S.

1. FePd2S4 nanozyme particles were prepared using palladium nitrate, ferric nitrate nonahydrate and glutathione as raw materials; S.

2. Chlorogenic acid standard solutions of different concentrations were mixed with acetate-sodium acetate buffer, H2O2, 3,3',5,5'-tetramethylbenzidine and FePd2S4 nanozyme suspension, and incubated. The absorbance value at 652 nm was measured. A standard curve was plotted with chlorogenic acid concentration as the abscissa and absorbance difference as the ordinate, and the linear equation was obtained. S.

3. Process the sample to be tested, extract chlorogenic acid from the sample to be tested, and prepare the test solution; S.

4. Replace the chlorogenic acid standard solution with the test solution and repeat step S.2 to determine the absorbance value; S.

5. The content of chlorogenic acid in the sample to be tested is calculated based on the linear equation in S.

2.

2. The method for detecting chlorogenic acid as described in claim 1, characterized in that, The preparation of FePd2S4 nanozyme particles in step S.1 specifically includes the following steps: Y.

1. Dissolve Fe(NO3)3·9H2O, Pd(NO3)2·xH2O and L-cysteine ​​in water, add ethylene glycol, and stir until homogeneous; Y.

2. Heat the solution and maintain this temperature for a period of time, then cool to room temperature; Y.

3. Wash the precipitate with the cooled solution and dry it to obtain FePd2S4 nanozyme particles.

3. The method for detecting chlorogenic acid as described in claim 2, characterized in that, The molar ratio in step Y.1 is Fe(NO3)3·9H2O:Pd(NO3)2·xH2O:L-cysteine ​​= 1:2-4:7-9, with 25-100 mL of water and 25-100 mL of ethylene glycol.

4. The method for detecting chlorogenic acid as described in claim 2, characterized in that, In step Y.2, the heating process involves placing the product in a stainless steel autoclave lined with polytetrafluoroethylene (PTFE) and heating it to 150-180°C for 7-9 hours.

5. The method for detecting chlorogenic acid as described in claim 2, characterized in that, In step Y.3, the washing is performed 3-5 times each with deionized water and ethanol, and the drying method is vacuum drying at 60-80℃.

6. The method for detecting chlorogenic acid as described in claim 1, characterized in that, In step S.2, the pH of the acetate-sodium acetate buffer solution is 3.0-5.0, and the concentration is 0.1-0.3M; the final concentration of H2O2 is 0.05-0.2mM; the final concentration of 3,3',5,5'-tetramethylbenzidine is 100-200μM; and the final concentration of the FePd2S4 nanozyme suspension is 10-50μg / mL. -1 .

7. The method for detecting chlorogenic acid as described in claim 1, characterized in that, The incubation temperature in step S.2 is 25-37℃, and the incubation time is 2-10 minutes.

8. The method for detecting chlorogenic acid as described in claim 1, characterized in that, The equation of the standard curve in step S.2 is: A - A0 = 0.026 × C 绿原酸 -0.018; Absorbance value A, absorbance of blank A0, C 绿原酸 This represents the concentration of chlorogenic acid, in μg / mL. -1 .

9. The method for detecting chlorogenic acid as described in claim 1, characterized in that, The method for processing the sample to be tested in step S.3 is as follows: weigh the sample powder to be tested, add 75% methanol solution at a material-to-liquid ratio of 1:100 (g:mL), mix well, place in an ultrasonic extractor, set the ultrasonic power to 500W and the frequency to 40kHz, and extract ultrasonically for 40min. After extraction, filter through filter paper under reduced pressure, collect the filtrate, and store it in a refrigerator at 4℃ for later use.

10. The application of the method as described in any one of claims 1-9 in the rapid detection of food or Chinese medicinal materials.

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