Color developing agent for rapidly measuring lignan content and preparation method of color developing agent

Abstract

The invention relates to the technical field of analytical chemistry detection, in particular to a color developing agent for rapidly measuring the content of lignan and a preparation method thereof.The color developing agent is composed of a liquid component and a solid component dispersed in the liquid component, and the liquid component comprises the following components by volume: a color developing agent, a 2, 4-dihydroxy-2, 4-dihydroxy-2, 4-dihydroxy-2, 4-dihydroxy-2, 4-dihydroxy-2, 4-dihydroxy-2, 4-dihydroxy-2, 4-dihydroxy-2, 4- adding an acidic ethanol solution of 2, 4-dinitrophenylhydrazine; the nano signal enhancer is surface modified gold nanorod sol with the concentration of 3 to 8 nM; the catalytic oxidant is iron (III) porphyrin with the concentration of 0.05 to 0.15 mmol / L; a dispersion stabilizer: polyethylene glycol 400 with a volume fraction of 1-3%; the solvent system is a mixed solvent of a buffer solution with the pH value of 2.0-3.0 and ethanol; the solid component is lignan molecularly imprinted polymer microspheres, and the concentration of the lignan molecularly imprinted polymer microspheres in the color developing agent is 0.5-2.0 g / L. According to the method, lignans can be conveniently detected, the sensitivity is high, the molecularly imprinted polymer provides specific recognition capability, and rapid, high-sensitivity and high-selectivity colorimetric detection on lignan components is realized.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry detection technology, and in particular to a colorimetric reagent for rapid determination of lignan content and its preparation method. Background Technology

[0002] Lignans are a class of important bioactive components widely found in plants. In the fields of pharmaceuticals, health products, and food, the rapid and accurate determination of lignan content in raw materials or products is a key step in quality control and efficacy evaluation.

[0003] Currently, the main methods for determination include chromatography and spectrometry. Chromatographic methods, such as high-performance liquid chromatography (HPLC), and spectroscopic methods, such as ultraviolet-visible spectrophotometry, offer good accuracy and resolution, but rely on expensive instruments and are complex and time-consuming, making them unsuitable for rapid on-site or large-volume sample screening. Traditional spectrophotometric methods, such as the vanillin-concentrated sulfuric acid method, are relatively simple to operate, but have significant drawbacks: Firstly, colorimetric reactions usually require prolonged heating, such as more than 30 minutes in a boiling water bath, which is inefficient. Secondly, the reaction selectivity is poor, and common components in the sample such as polyphenols, flavonoids, and pigments can easily cause interference, leading to falsely high results. Third, the concentrated sulfuric acid used is highly corrosive and oxidizing, posing a safety hazard, and the colorimetric solution has poor stability, requiring strict control of the measurement time.

[0004] Therefore, developing a dedicated colorimetric reagent that is fast-responding, highly selective, safe to operate, and stable is of great significance for achieving high-throughput and rapid quantitative analysis of lignans. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a colorimetric reagent for the rapid determination of lignan content and its preparation method. This colorimetric reagent combines specific chemical reactions, nano-plasma resonance enhancement effects, and molecular recognition technology. Under the action of a biomimetic catalyst, the colorimetric reaction is rapid, facilitating the detection of lignans with high sensitivity. Furthermore, the introduction of molecularly imprinted polymers provides specific recognition capabilities, enabling rapid, highly sensitive, and highly selective colorimetric detection of lignan components.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions: In a first aspect, the present invention provides a colorimetric reagent for the rapid determination of lignan content, comprising a liquid component and a solid component dispersed therein, wherein the liquid component comprises, by volume, the following components: Colorimetric reagent: an acidic ethanol solution of 2,4-dinitrophenylhydrazine with a concentration of 5-10 mmol / L; Nano-signal enhancer: Surface-modified gold nanorod sol with a concentration of 3-8 nM; Catalytic oxidant: Iron(III) porphyrin at a concentration of 0.05-0.15 mmol / L; Dispersant stabilizer: 1-3% by volume of polyethylene glycol 400; Solvent system: a mixture of buffer solution with pH 2.0-3.0 and ethanol; The solid component is lignan molecularly imprinted polymer microspheres, and its concentration in the colorimetric agent is 0.5-2.0 g / L.

[0007] As a further embodiment of the present invention, the surface-modified gold nanorods are gold nanorods whose surfaces are modified with polyvinylpyrrolidone and have an aspect ratio of 3.5-4.5.

[0008] As a further embodiment of the present invention, the lignan molecularly imprinted polymer microspheres are prepared by precipitation polymerization using schisandrin A as a template molecule, methacrylic acid as a functional monomer, and ethylene glycol dimethacrylate as a crosslinking agent.

[0009] As a further embodiment of the present invention, the buffer solution is citrate-disodium hydrogen phosphate buffer or citrate-sodium citrate buffer.

[0010] As a further aspect of the present invention, in the acidic ethanol solution, the volume ratio of ethanol to buffer solution is 1:3 to 1:5.

[0011] Secondly, the present invention provides a method for preparing a colorimetric reagent for rapid determination of lignan content, comprising the following steps: a) Preparation of liquid phase base solution: Under light-protected conditions, 2,4-dinitrophenylhydrazine is dissolved in an acidic ethanol solution composed of buffer solution with pH value of 2.0-3.0 and anhydrous ethanol at a volume ratio of (3-5):1, and stirred until completely dissolved to obtain the colorimetric reagent solution; the colorimetric reagent solution, polyethylene glycol 400, and the remaining buffer solution and anhydrous ethanol are stirred and mixed to obtain the liquid phase base solution; b) Constructing a nano-reinforced liquid phase: Under stirring conditions, surface-modified gold nanorod sol is added to the liquid phase base liquid obtained in step S1, and then ultrasonic treatment is performed to obtain mixture A; c) Adding catalytic oxidant: Under stirring conditions, iron(III) porphyrin is added to the mixture A obtained in step S2 and mixed evenly to obtain mixture B; d) Solid-phase dispersion and curing: Under stirring conditions of 50-200 rpm, lignan molecularly imprinted polymer microspheres are added to the mixture B obtained in step S3, and the mixture is continuously stirred to disperse it evenly. Then, it is allowed to stand and cure at 2-8℃ for 20-28 hours to obtain the color developer.

[0012] As a further aspect of the present invention, in step a), the stirring and dissolving conditions are to stir at 300-500 rpm for 20-40 minutes at 20-30°C.

[0013] As a further aspect of the present invention, in step b), the power of the ultrasonic treatment is 200-400 W, and the treatment time is 10-20 minutes.

[0014] As a further aspect of the present invention, in step b), the surface-modified gold nanorod sol is premixed with a portion of the calculated amount of polyethylene glycol 400 before being added, and then ultrasonically treated at a power of 100-200 W for 5-10 minutes.

[0015] As a further embodiment of the present invention, step c) is carried out under ice bath or cold water bath conditions, the stirring speed during mixing is 200-400 rpm, and the mixing time is 10-20 minutes.

[0016] As a further aspect of the present invention, in step d), the lignan molecularly imprinted polymer microspheres are pre-dried under vacuum at 40-60°C for 2-4 hours before being added.

[0017] As a further embodiment of the present invention, in step d), after adding the lignan molecularly imprinted polymer microspheres, the mixture is stirred at 100-200 rpm for 45-75 minutes to ensure uniform dispersion.

[0018] Thirdly, the present invention also provides a method for rapidly determining the lignan content in a sample using a colorimetric reagent, comprising the following steps: S1: Sample pretreatment: Take the sample solution to be tested, add the colorimetric reagent, and vortex mix. S2: Heat the mixture in a water bath at 60-85℃ for 3-8 minutes, then remove and cool to room temperature; S3: Centrifuge the reaction mixture, take the supernatant, and use the colorimetric reagent without the sample solution as a reference after the same treatment. Measure the absorbance at a wavelength of 520-550 nm on a UV-Vis spectrophotometer. S4: Calculate the lignan content in the sample to be tested based on the standard curve plotted using lignan standards.

[0019] As a further aspect of the present invention, in step S1, the volume ratio of the sample solution to the colorimetric reagent is 1:2 to 1:4.

[0020] As a further embodiment of the present invention, in step S2, the heating reaction temperature is 80°C and the reaction time is 5 minutes.

[0021] As a further embodiment of the present invention, in step S3, the centrifugation conditions are 3000-5000 rpm for 2-5 minutes.

[0022] As a further aspect of the present invention, in step S4, the lignan standard is schisandrin A.

[0023] Compared with existing technologies, the colorimetric reagent and its preparation method for rapid determination of lignan content provided by the present invention have the following beneficial effects: 1. This invention introduces iron(III)porphyrin as a biomimetic catalytic oxidant, which can mildly and efficiently catalyze the oxidation of lignan characteristic structures, thereby rapidly and specifically condensing with the chromogenic main agent 2,4-dinitrophenylhydrazine (DNPH). This significantly reduces the water bath heating time of over 30 minutes required by traditional colorimetric methods, greatly meeting the needs of high-throughput rapid screening.

[0024] 2. This invention also utilizes the localized surface plasmon resonance effect of gold nanorods to strongly enhance the light absorption signal of the colorimetric product, thereby improving the detection sensitivity of the colorimetric reagent compared to the traditional vanillin-sulfuric acid method, enabling accurate detection of trace amounts of lignans in the sample. Furthermore, the molecularly imprinted polymer microspheres prepared using schisandrin A as a template can specifically recognize and enrich lignans and their structural analogs from complex sample matrices, effectively eliminating interference from common impurities such as polysaccharides, flavonoids, and simple phenols. The anti-interference ability against polyphenols is improved compared to non-specific colorimetric methods, resulting in more accurate and reliable measurement results.

[0025] 3. This invention eliminates the use of highly corrosive and hazardous reagents such as concentrated sulfuric acid and perchloric acid in traditional methods, and adopts a mild acidic buffer system, which significantly reduces the safety risks of experimental operations and environmental pollution, and is more in line with the requirements of modern green analytical chemistry.

[0026] These or other aspects of the invention will become more apparent from the following description of embodiments. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only, and are not intended to limit the invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0028] The technical solutions in the exemplary embodiments of the present invention will be clearly and completely described below with reference to exemplary embodiments of the present invention. Obviously, the described exemplary embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Specifically, the embodiments of this application will be further described below. Example 1

[0030] This application provides a method for preparing a colorimetric reagent for the rapid determination of lignan content. The reagents involved in this preparation method include: 2,4-dinitrophenylhydrazine, polyvinylpyrrolidone-modified gold nanorod sol (aspect ratio 4.0), iron(III)tetraphenylporphyrin, polyethylene glycol 400, schisandrin A standard, methacrylic acid, ethylene glycol dimethacrylate, azobisisobutyronitrile, methanol, anhydrous ethanol, citric acid, and disodium hydrogen phosphate. All reagents used are of analytical grade.

[0031] The samples used in this preparation method were commercially available dried fruit powder of Schisandra chinensis and root powder of Kadsura coccinea. The instruments used included: an electronic analytical balance, an ultrasonic cell disruptor, a magnetic stirrer, a constant temperature water bath, a high-speed centrifuge, a UV-Vis spectrophotometer, a vortex mixer, and a vacuum drying oven.

[0032] In the preparation method of this colorimetric agent, the preparation method of lignan molecularly imprinted polymer (MIP) microspheres is as follows: The template molecule schisandrin A (1.0 mmol) and the functional monomer methacrylic acid (4.0 mmol) were dissolved in acetonitrile (50 mL) and pre-assembled at 4 °C for 12 hours.

[0033] Add the crosslinking agent ethylene glycol dimethacrylate (20 mmol) and the initiator azobisisobutyronitrile (0.1 mmol) to the above solution, and purge with nitrogen gas to remove oxygen for 20 minutes.

[0034] The polymerization reaction was carried out in an oil bath at 60°C for 24 hours. After the reaction was completed, the product was collected by centrifugation, and the template molecules were eluted by Soxhlet extraction with a methanol-acetic acid (9:1, v / v) mixture for 48 hours, followed by washing with methanol until neutral.

[0035] The obtained polymer microspheres were vacuum dried at 50°C to constant weight, ground and passed through a 200-mesh sieve for later use.

[0036] The preparation method of this colorimetric reagent includes the following steps: (1) Preparation of the base solution for liquid chromatography: Under light-protected conditions, 0.097 g (0.5 mmol) of 2,4-dinitrophenylhydrazine was dissolved in 10 mL of an acidic ethanol solution consisting of a pH 2.5 citrate-disodium hydrogen phosphate buffer and anhydrous ethanol at a volume ratio of 4:1. The solution was stirred at 25 °C and 400 rpm for 30 minutes until completely dissolved to obtain the colorimetric reagent solution. This solution, 1.5 mL of polyethylene glycol 400, and the remaining pH 2.5 buffer and anhydrous ethanol mixture (total volume made up to approximately 85 mL) were mixed and stirred until homogeneous to obtain the base solution for liquid chromatography.

[0037] (2) Construction of nano-reinforced liquid phase: Take 5 mL of 5 nM PVP-modified gold nanorod sol and premix it with 0.5 mL of polyethylene glycol 400. Sonicate the mixture at 150 W for 8 minutes. With stirring, slowly add the mixture to the liquid phase base solution in step (1). Then sonicate the whole mixture at 350 W for 15 minutes to obtain mixture A.

[0038] (3) Adding catalytic oxidant: Under ice bath and stirring at 300 rpm, 1.0 mL of 5 mmol / L iron(III) porphyrin methanol solution was slowly added dropwise to mixture A, and stirring was continued for 15 minutes to obtain mixture B.

[0039] (4) Solid-phase dispersion and curing: 0.1 g of MIPs microspheres, which had been pre-dried under vacuum at 50°C for 3 hours, was slowly added to mixture B under stirring at 150 rpm. After the addition was complete, the stirring was continued at this speed for 60 minutes. The resulting suspension was transferred to a brown reagent bottle and allowed to stand and cure for 24 hours at 4°C to obtain the final colorimetric reagent. In this colorimetric reagent, the concentration of 2,4-dinitrophenylhydrazine was approximately 5 mmol / L, the concentration of gold nanorods was approximately 5 nM, the concentration of iron(III)porphyrin was approximately 0.05 mmol / L, the volume fraction of polyethylene glycol 400 was approximately 2%, and the concentration of MIPs microspheres was approximately 1.0 g / L.

[0040] For the standard curve preparation, accurately weigh 5.0 mg of schisandrin A standard, dissolve it in methanol, and dilute to 50 mL to obtain a stock solution of 100 μg / mL. The stock solution was serially diluted with methanol to prepare a series of standard solutions with concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL. Accurately pipette 1.0 mL of each standard solution into a 5 mL stoppered centrifuge tube, add 3.0 mL of the colorimetric reagent prepared in the example, and vortex for 10 seconds. Place each centrifuge tube in an 80°C constant temperature water bath and heat for 5 minutes. Immediately after removal, cool to room temperature in an ice-water bath for 2 minutes. Centrifuge at 4500 rpm for 3 minutes, carefully aspirate the supernatant, and use a system prepared in the same manner but with 1.0 mL of methanol instead of the sample solution as a reference. Measure the absorbance (A) at 538 nm using a UV-Vis spectrophotometer with a 1 cm path length cuvette. Linear regression was performed with schisandrin A concentration (C, μg / mL) on the x-axis and absorbance (A) on the y-axis. The standard curve equation was obtained as: A = 0.0198C + 0.0052, and the correlation coefficient R0 was [value missing]. 2 =0.9993. This indicates that schisandrin A has a good linear relationship in the concentration range of 5-50 μg / mL. Example 2

[0041] This application provides a method for preparing a colorimetric reagent for the rapid determination of lignan content. The reagents involved in this preparation method include: 2,4-dinitrophenylhydrazine, polyvinylpyrrolidone-modified gold nanorod sol (aspect ratio 3.8), iron(III)tetraphenylporphyrin, polyethylene glycol 400, schisandrin A standard, magnolol standard, and honokiol standard, methacrylic acid, ethylene glycol dimethacrylate, azobisisobutyronitrile, acetonitrile, methanol, anhydrous ethanol, citric acid, and sodium citrate. Unless otherwise specified, all reagents used are of analytical grade.

[0042] The sample used in this preparation method is commercially available dried bark powder of Magnolia officinalis. The instruments used include: a high-performance liquid chromatograph equipped with a diode array detector, an electronic analytical balance, an ultrasonic cleaner, a magnetic stirrer, a constant temperature water bath, a high-speed centrifuge, a UV-Vis spectrophotometer, a vortex mixer, and a vacuum drying oven.

[0043] In the preparation method of this colorimetric agent, the preparation method of lignan molecularly imprinted polymer (MIPs) microspheres is the same as in Example 1, and MIPs microspheres with schisandrin A as template are prepared.

[0044] The preparation method of this colorimetric reagent includes the following steps: (1) Preparation of the base solution for liquid chromatography: Under light-protected conditions, 0.198 g (1.0 mmol) of 2,4-dinitrophenylhydrazine was dissolved in 10 mL of an acidic ethanol solution consisting of a pH 3.0 citrate-sodium citrate buffer and anhydrous ethanol in a volume ratio of 3:1. The solution was stirred at 30 °C and 500 rpm for 20 minutes until completely dissolved to obtain the colorimetric reagent solution. This solution, 3.0 mL of polyethylene glycol 400, and the remaining pH 3.0 buffer and anhydrous ethanol mixed solvent (total volume made up to approximately 85 mL) were mixed and stirred until homogeneous to obtain the base solution for liquid chromatography.

[0045] (2) Construction of nano-reinforced liquid phase: Take 8 mL of PVP-modified gold nanorod sol with a concentration of 8 nM, and premix it with 1.0 mL of polyethylene glycol 400. Sonicate it at 200 W for 5 minutes. With stirring, slowly add this mixture to the liquid phase base solution in step (1), and then sonicate the whole mixture at 400 W for 10 minutes to obtain mixture A.

[0046] (3) Adding catalytic oxidant: Under the conditions of cold water bath (~10℃) and stirring at 400 rpm, 3.0 mL of 5 mmol / L iron(III) porphyrin methanol solution was slowly added dropwise to mixture A, and stirring was continued for 10 minutes to obtain mixture B.

[0047] (4) Solid-phase dispersion and curing: 0.2 g of MIPs microspheres, which had been pre-dried under vacuum at 60℃ for 2 hours, was slowly added to mixture B under stirring at 200 rpm. After the addition was complete, the stirring speed was adjusted to 100 rpm and stirring was continued for 75 minutes. The resulting suspension was transferred to a brown reagent bottle and allowed to stand and cure for 28 hours in a refrigerator at 2℃ to obtain the final colorimetric reagent. In this colorimetric reagent, the concentration of 2,4-dinitrophenylhydrazine was approximately 10 mmol / L, the concentration of gold nanorods was approximately 8 nM, the concentration of iron(III)porphyrin was approximately 0.15 mmol / L, the volume fraction of polyethylene glycol 400 was approximately 3%, and the concentration of MIPs microspheres was approximately 2.0 g / L.

[0048] When plotting the standard curve, the same procedure as in step 4 of Example 1 was followed, using schisandrin A as the standard. The resulting standard curve equation was: A = 0.0205C + 0.0038, with a correlation coefficient R0. 2 =0.9991. This indicates a good linear relationship within the concentration range of 5-50 μg / mL. Example 3

[0049] This application provides a method for preparing a colorimetric reagent for the rapid determination of lignan content. The reagents involved in this preparation method include: 2,4-dinitrophenylhydrazine, polyvinylpyrrolidone-modified gold nanorod sol (aspect ratio 3.5), iron(III)tetraphenylporphyrin, polyethylene glycol 400, schisandrin A standard, glucose, bovine serum albumin, and tannic acid. All reagents used are of analytical grade.

[0050] In this preparation method, complex sample A was prepared using commercially available "Anshen Bunao Liquid" (containing Schisandra chinensis and processed Polygonum multiflorum); complex sample B was prepared using laboratory-made Schisandra chinensis-Magnolia officinalis mixed extract powder (Schisandra chinensis:Magnolia officinalis = 1:1, w / w). The instruments used were the same as in Example 1.

[0051] In the preparation method of this colorimetric agent, the preparation method of lignan molecularly imprinted polymer (MIP) microspheres is the same as in Example 1.

[0052] The preparation method of this colorimetric reagent includes the following steps: (1) Preparation of the base solution for liquid chromatography: Under light-protected conditions, 0.097 g (0.5 mmol) of 2,4-dinitrophenylhydrazine was dissolved in 10 mL of an acidic ethanol solution consisting of a pH 2.0 citrate-disodium hydrogen phosphate buffer and anhydrous ethanol at a volume ratio of 5:1. The solution was stirred at 20 °C and 300 rpm for 40 minutes until completely dissolved to obtain the colorimetric reagent solution. This solution, 1.0 mL of polyethylene glycol 400, and the remaining pH 2.0 buffer and anhydrous ethanol mixed solvent (total volume made up to approximately 90 mL) were mixed and stirred until homogeneous to obtain the base solution for liquid chromatography.

[0053] (2) Construction of nano-reinforced liquid phase: Take 3 mL of PVP-modified gold nanorod sol with a concentration of 3 nM, and premix it with 0.3 mL of polyethylene glycol 400. Sonicate it at 100 W for 10 minutes. With stirring, slowly add this mixture to the liquid phase base solution in step (1), and then sonicate the whole mixture at 200 W for 20 minutes to obtain mixture A.

[0054] (3) Adding catalytic oxidant: Under ice bath (0-4℃) and stirring at 200 rpm, 1.0 mL of 5 mmol / L iron(III) porphyrin methanol solution was slowly added dropwise to mixture A, and stirring was continued for 20 minutes to obtain mixture B.

[0055] (4) Solid-phase dispersion and curing: 0.05 g of MIPs microspheres, which had been pre-dried under vacuum at 40℃ for 4 hours, was slowly added to mixture B under stirring at 50 rpm. After the addition was complete, the stirring speed was increased to 100 rpm and stirring was continued for 45 minutes. The resulting suspension was transferred to a brown reagent bottle and allowed to stand at 8℃ for 20 hours to obtain the final colorimetric reagent. In this colorimetric reagent, the concentration of 2,4-dinitrophenylhydrazine was approximately 5 mmol / L, the concentration of gold nanorods was approximately 3 nM, the concentration of iron(III)porphyrin was approximately 0.05 mmol / L, the volume fraction of polyethylene glycol 400 was approximately 1%, and the concentration of MIPs microspheres was approximately 0.5 g / L.

[0056] When plotting the standard curve, schisandrin A was used as the standard, as in Example 1. The resulting standard curve equation was: A = 0.0191C + 0.0060, with a correlation coefficient R0. 2 =0.9989. This indicates a good linear relationship within the concentration range of 5-50 μg / mL, proving that the system still possesses excellent analytical performance even when the lower limit of the parameter is used.

[0057] Performance testing: The performance of the colorimetric reagents used for the rapid determination of lignan content in Examples 1-3 was tested. During the tests, linear range, sensitivity, and precision were performed based on the colorimetric reagents prepared in Examples 1-3. First, schisandrin A standard was accurately weighed and prepared into a 100 μg / mL stock solution with methanol. Then, the stock solution was diluted to prepare a series of standard solutions with concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL. 1.0 mL of each concentration standard solution was accurately pipetted and 3.0 mL of the colorimetric reagent was added, followed by vortexing to mix. The mixture was reacted in an 80°C water bath for 5 minutes, immediately followed by ice bath cooling for 2 minutes. After centrifugation at 4500 rpm for 3 minutes, the supernatant was collected. Using a blank reagent as a reference, the mixture was centrifuged at 538 °C. The absorbance (A) was measured at a wavelength of nm; linear regression was performed with concentration (C) on the x-axis and absorbance (A) on the y-axis; the same Schisandra chinensis test solution was measured 6 times consecutively, and the relative standard deviation (RSD) was calculated to examine the intraday precision.

[0058] The accuracy was tested using a spiked recovery experiment. The procedure was as follows: six portions of Schisandra chinensis test solution with known lignan content were taken; schisandrin A standard solution with low, medium, and high concentrations (10 μg / mL, 15 μg / mL, and 20 μg / mL) was added to each sample, with two replicates for each level; 1.0 mL of each concentration standard solution was precisely pipetted into each sample, and 3.0 mL of colorimetric reagent (prepared in Examples 1-3) was added, and the mixture was vortexed; the mixture was reacted in an 80°C water bath for 5 minutes, and immediately cooled in an ice bath for 2 minutes; the mixture was centrifuged at 4500 rpm for 3 minutes, and the supernatant was collected. The absorbance (A) was measured at a wavelength of 538 nm using a blank reagent as a reference. Colorimetric development, centrifugation, and measurement were performed; the spiked recovery rate was calculated (average recovery rate % = (total measured amount - background amount) / spiked amount × 100%).

[0059] The performance test results are shown in the table below: Table 1. Performance test results of the colorimetric reagents used for rapid determination of lignan content in Examples 1-3. All performance test data consistently show that the composite colorimetric reagent and its detection method provided by this invention exhibit significant advantages in terms of speed, sensitivity, accuracy, anti-interference, and stability. Its comprehensive performance far exceeds that of existing conventional colorimetric technologies, achieving a technological breakthrough and possessing significant practical application value and market prospects.

[0060] This invention introduces iron(III)porphyrin as a biomimetic catalytic oxidant, which can mildly and efficiently catalyze the oxidation of lignan characteristic structures, thereby rapidly and specifically condensing with the chromogenic main agent 2,4-dinitrophenylhydrazine (DNPH). This shortens the water bath heating time of over 30 minutes required by traditional colorimetric methods, greatly meeting the needs of high-throughput rapid screening. Furthermore, this invention utilizes the localized surface plasmon resonance effect of gold nanorods to strongly enhance the absorbance signal of the chromogenic product, improving the detection sensitivity of the chromogenic agent compared to the traditional vanillin-sulfuric acid method, enabling accurate detection of trace amounts of lignans in samples. Moreover, the molecularly imprinted polymer microspheres prepared using schisandrin A as a template can specifically recognize and enrich lignans and their structural analogs from complex sample matrices, effectively eliminating interference from common impurities such as polysaccharides, flavonoids, and simple phenols. The anti-interference ability against polyphenols is improved compared to non-specific colorimetric methods, resulting in more accurate and reliable measurement results. This invention eliminates the use of highly corrosive and hazardous reagents such as concentrated sulfuric acid and perchloric acid in traditional methods, and adopts a mild acidic buffer system, which significantly reduces the safety risks of experimental operations and environmental pollution, and is more in line with the requirements of modern green analytical chemistry.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A colorimetric reagent for the rapid determination of lignan content, characterized in that, It consists of a liquid component and a solid component dispersed therein, wherein the liquid component comprises the following components by volume: Colorimetric reagent: an acidic ethanol solution of 2,4-dinitrophenylhydrazine with a concentration of 5-10 mmol / L; Nano-signal enhancer: Surface-modified gold nanorod sol with a concentration of 3-8 nM; Catalytic oxidant: Iron(III) porphyrin at a concentration of 0.05-0.15 mmol / L; Dispersant stabilizer: 1-3% by volume of polyethylene glycol 400; Solvent system: a mixture of buffer solution with pH 2.0-3.0 and ethanol; The solid component is lignan molecularly imprinted polymer microspheres, and its concentration in the colorimetric agent is 0.5-2.0 g / L.

2. The colorimetric reagent for rapid determination of lignan content as described in claim 1, characterized in that, The surface-modified gold nanorods are gold nanorods whose surfaces are modified with polyvinylpyrrolidone, and have an aspect ratio of 3.5-4.

5.

3. The colorimetric reagent for rapid determination of lignan content as described in claim 2, characterized in that, The lignan molecularly imprinted polymer microspheres were prepared by precipitation polymerization using schisandrin A as a template molecule, methacrylic acid as a functional monomer, and ethylene glycol dimethacrylate as a crosslinking agent.

4. The colorimetric reagent for rapid determination of lignan content as described in claim 1, characterized in that, The buffer solution is either citrate-disodium hydrogen phosphate buffer or citrate-sodium citrate buffer.

5. The colorimetric reagent for rapid determination of lignan content as described in claim 1, characterized in that, In the acidic ethanol solution, the volume ratio of ethanol to buffer solution is 1:3 to 1:

5.

6. A method for preparing a colorimetric reagent for rapid determination of lignan content, used to prepare the colorimetric reagent according to any one of claims 1-5; characterized in that, The preparation method includes the following steps: a) Preparation of liquid phase base solution: Under light-protected conditions, 2,4-dinitrophenylhydrazine is dissolved in an acidic ethanol solution composed of buffer solution with pH value of 2.0-3.0 and anhydrous ethanol at a volume ratio of (3-5):1, and stirred until completely dissolved to obtain the colorimetric reagent solution; the colorimetric reagent solution, polyethylene glycol 400, and the remaining buffer solution and anhydrous ethanol are stirred and mixed to obtain the liquid phase base solution; b) Constructing a nano-reinforced liquid phase: Under stirring conditions, surface-modified gold nanorod sol is added to the liquid phase base liquid obtained in step S1, and then ultrasonic treatment is performed to obtain mixture A; c) Adding catalytic oxidant: Under stirring conditions, iron(III) porphyrin is added to the mixture A obtained in step S2 and mixed evenly to obtain mixture B; d) Solid-phase dispersion and curing: Under stirring conditions of 50-200 rpm, lignan molecularly imprinted polymer microspheres are added to the mixture B obtained in step S3, and the mixture is continuously stirred to disperse it evenly. Then, it is allowed to stand and cure at 2-8℃ for 20-28 hours to obtain the color developer.

7. The method for preparing the colorimetric reagent for rapid determination of lignan content as described in claim 6, characterized in that, In step a), the stirring and dissolving conditions are: stirring at 300-500 rpm for 20-40 minutes at 20-30°C.

8. The method for preparing the colorimetric reagent for rapid determination of lignan content as described in claim 6, characterized in that, In step b), the power of the ultrasonic treatment is 200-400 W, and the treatment time is 10-20 minutes; Before being added, the surface-modified gold nanorod sol is premixed with a portion of the calculated amount of polyethylene glycol 400 and ultrasonically treated at 100-200 W power for 5-10 minutes.

9. The method for preparing the colorimetric reagent for rapid determination of lignan content as described in claim 6, characterized in that, In step c), the mixing is carried out under ice bath or cold water bath conditions, with a stirring speed of 200-400 rpm and a mixing time of 10-20 minutes.

10. The method for preparing the colorimetric reagent for rapid determination of lignan content as described in claim 6, characterized in that, In step d), the lignan molecularly imprinted polymer microspheres are pre-dried under vacuum at 40-60°C for 2-4 hours before being added; after adding the lignan molecularly imprinted polymer microspheres, they are stirred at 100-200 rpm for 45-75 minutes to ensure uniform dispersion.

Images