Method for detecting amoorastilbin in juniperus communis extract

Abstract

The application discloses a detection method of amentoflavone in juniper extract, and relates to the field of juniper extract detection. The application combines molecular imprinting fiber test paper with photoacoustic acoustic microcavity detection technology, so that the detection of amentofavone has the comprehensive advantages of high sensitivity, high selectivity and rapid response. On one hand, the molecular imprinting polymer layer forms a specific recognition cavity on the surface of the fiber, so that the test paper has obvious selective adsorption effect on amentofavone, and the interference of other polyphenol components in the juniper extract is significantly reduced. On the other hand, the photoacoustic acoustic microcavity can amplify the light-heat conversion process of the adsorbed molecules, so that the weak heat-acoustic signal is effectively enhanced, and the detection sensitivity is much higher than that of the traditional spectroscopy.

Description

Technical Field

[0001] This invention relates to the field of juniper extract detection, and more particularly to a method for detecting cypermethrin in juniper extract. Background Technology

[0002] Amentoflavone is a class of biflavonoid compounds found in plants of the Juniper genus. It possesses various biological activities, including anti-inflammatory, antioxidant, antiviral, and neuroprotective effects, and therefore has important application value in the fields of plant medicine development, food functional factor research, and quality evaluation. However, Juniper extracts contain a variety of monomeric flavonoids, volatile terpenes, and polyphenol derivatives. These components have similar structures and polarities, making the detection of amentoflavone susceptible to interference and making it difficult to guarantee quantitative accuracy.

[0003] Currently, commonly used detection methods include high performance liquid chromatography (HPLC), ultra-high performance liquid chromatography (UPLC), and liquid chromatography-mass spectrometry (LC-MS). Although these methods have high precision, they usually require expensive instruments, professional operators, and lengthy pretreatment steps, such as extraction, concentration, filtration, and gradient elution, which makes it difficult to meet the needs of rapid detection or on-site detection. In addition, traditional ultraviolet spectrophotometry has significant overlap in absorption spectra, making it difficult to selectively distinguish between paclitaxel flavonoids, resulting in insufficient sensitivity and anti-interference ability. Summary of the Invention

[0004] Therefore, in order to overcome the above-mentioned shortcomings, the present invention provides a method for detecting cypress flavonoids in juniper extract.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: a method for detecting cypermethrin in juniper extract, the specific steps of which are as follows:

[0006] Preparation of S1 molecularly imprinted fiber test paper: The molecularly imprinted polymer with cypermethrin as template molecule is fixed on the surface of cellulose fiber or nylon fiber by surface polymerization to obtain fiber test paper with specific recognition sites.

[0007] S2 Sample Binding Step: The juniper extract solution, which has been extracted, centrifuged and filtered by ethanol-water solution, is contacted with the fiber test paper for 20-90 seconds to allow the flavonoids of Taxodium bifidum to selectively bind to the imprint sites on the surface of the fiber test paper.

[0008] S3 Insertion into the microcavity: Insert the adsorbed fiber test paper into the acoustic resonant cavity region of the photoacoustic microcavity, placing it at the position of acoustic standing wave enhancement;

[0009] S4 Photoacoustic Excitation: A modulated laser with a modulation frequency of 1kHz-30kHz and a power of 5-80mW is applied to the fiber test paper to cause periodic photothermal conversion on the surface of the fiber test paper and generate photoacoustic signals.

[0010] S5 Signal Extraction and Quantification: The sound pressure amplitude of the photoacoustic signal is collected, and the difference between it and the sound pressure amplitude corresponding to the blank fiber test paper is calculated. The content of paclitaxel flavonoids in the sample is determined according to the standard curve.

[0011] Preferably, the molecularly imprinted polymer comprises a functional monomer of 4-vinylpyridine or methacrylic acid, a crosslinking agent of ethylene glycol dimethacrylate, and an initiator of azobisisobutyronitrile.

[0012] Preferably, the fiber test paper has a strip-like structure with a thickness of 0.1-1 mm and a length of 5-30 mm, and a rigid support plate is provided on the back to assist in insertion and removal.

[0013] Preferably, the pretreatment of the juniper extract includes: ultrasonic extraction with 40%-80% ethanol-water solution for 3-10 minutes, followed by centrifugation and filtration through a 0.22μm filter membrane.

[0014] Preferably, the photoacoustic microcavity includes an outer shell cavity, an acoustic resonant cavity, a modulated laser incident window, and a miniature pressure sensor, with the fiber test paper positioned in the pressure antinode region of the acoustic resonant cavity.

[0015] Preferably, the photoacoustic signal includes at least one of sound pressure amplitude, resonant frequency shift, or acoustic phase change, and the quantification adopts a single-variable quantification method based primarily on the sound pressure amplitude difference.

[0016] Preferably, the sound pressure amplitude is processed using background subtraction, baseline correction, or resonance peak normalization methods.

[0017] Preferably, after the fiber test paper binds to the sample, it can be rinsed with deionized water for 1-3 seconds to remove non-specific adsorbents before being inserted into the photoacoustic microcavity for detection.

[0018] The beneficial effects of this invention are:

[0019] This invention combines molecularly imprinted fiber test strips with photoacoustic microcavity detection technology, enabling the detection of cypermethrin to exhibit comprehensive advantages of high sensitivity, high selectivity, and rapid response. On one hand, the molecularly imprinted polymer layer forms specific recognition cavities on the fiber surface, giving the test strip a significant selective adsorption effect on cypermethrin and significantly reducing the interference of other polyphenolic components in juniper extract. On the other hand, the photoacoustic microcavity amplifies the photothermal conversion process of adsorbed molecules, effectively enhancing the weak thermo-acoustic signal, thereby achieving a detection sensitivity far higher than that of traditional spectroscopic methods. The overall detection process of this invention does not require complex chromatographic equipment, sample pretreatment is simple, the test strips can be used once, and the detection time for a single sample can be controlled within one minute, making it suitable for large-scale rapid screening. Attached Figure Description

[0020] Figure 1 This is the photoacoustic detection curve of the present invention. Detailed Implementation

[0021] To further explain the technical solution of the present invention, a detailed description is provided below through specific embodiments.

[0022] This invention provides a method for detecting cypermethrin in juniper extract. First, a molecularly imprinted fiber test paper containing a cypermethrin recognition cavity is prepared. Then, the juniper extract undergoes pretreatment processes such as extraction, filtration, and dilution to make the system suitable for photoacoustic detection. Subsequently, the fiber test paper is immersed in the treated sample solution to bind cypermethrin, and then placed inside a fixed-structure photoacoustic microcavity resonator. By modulating laser irradiation of the imprinted fiber, a thermo-acoustic conversion is generated during periodic heating, and the sound pressure amplitude is collected by an acoustic sensor. Finally, by analyzing the difference in sound pressure peak values ​​before and after binding, the cypermethrin content in the sample is calculated based on a standard curve, achieving rapid and quantitative analysis.

[0023] Molecularly imprinted fiber test strips preferably use cellulose fibers or nylon fibers as a substrate, and their surface forms a molecularly imprinted polymer layer through free radical surface polymerization. In preparation, the template molecule, cephalotaxone, is first dissolved in a suitable solvent system to form a stable prepolymer complex structure with functional monomers such as methacrylic acid (MAA) or 4-vinylpyridine (4-VP). Then, EGDMA is added as a crosslinking agent and AIBN as an initiator. The fiber is immersed in this prepolymer solution, and surface polymerization is carried out by controlling the temperature at approximately 60°C, allowing the polymer to firmly adhere to the fiber surface and form an imprinted layer with specific recognition cavities. Subsequently, the template molecule is eluted with a methanol-acetic acid solution to obtain molecularly imprinted fibers with selective recognition capabilities.

[0024] During the sample binding process, the prepared imprinted fiber test paper is immersed in the juniper extract treatment solution for 20–90 seconds, allowing the flavonoids of Taxodium spp. to preferentially enter the imprint cavity and bind to the imprint network structure. To ensure sufficient binding, the immersed end face is kept still to avoid liquid flow disturbance affecting the adsorption balance. After adsorption, the fiber test paper is removed and the attached liquid is gently shaken off to keep its surface in a relatively stable state, ensuring that the detection in the photoacoustic microcavity is not affected by droplets.

[0025] The photoacoustic microcavity adopts a cylindrical or long cavity resonant structure, forming an acoustic standing wave field inside, which enhances the photoacoustic signal. The microcavity is made of aluminum alloy or ceramic material, and the length-to-diameter ratio of the cavity is calculated acoustically, with the Q value maintained between 400 and 1500. The fiber test paper is fixed at the position of the acoustic standing wave belly through a slot, so that it can effectively excite the sound wave after photothermal conversion. The test paper is placed perpendicular to the laser beam path so that the laser can uniformly irradiate the surface of the test paper.

[0026] In the photoacoustic detection stage, a modulated semiconductor laser is used, with its modulation frequency range set to 1–30 kHz. Different frequencies are selected according to the target concentration to obtain the optimal sound pressure response. The laser power is set in the range of 5–80 mW, and in a typical implementation it is 20–55 mW. After collimation, the laser irradiates the surface of the imprinted fiber test paper. The periodic heating and cooling causes the fibers to undergo slight expansion and contraction, generating a stable photoacoustic signal for the system to collect.

[0027] Acoustic signal acquisition is accomplished through a high-sensitivity electret microphone placed at the end of the microcavity; the system automatically obtains the sound pressure peak P_sample, and then compares it with the signal P_blank of the blank imprint test paper (not exposed to the sample) to calculate the difference ΔP; based on the pre-established linear relationship between ΔP and concentration, the difference is converted into the actual content of paclitaxel flavonoids in the sample;

[0028] A series of standard curves were prepared using standard solutions of 5–200 μg / mL of Taxodium flavonoids; ΔP was recorded under the same operating conditions, and the linear equation was obtained by fitting with the least squares method. The linear correlation coefficient (R²) was generally higher than 0.99, providing a basis for subsequent sample determination.

[0029] The thickness of the imprinted layer can be controlled by adjusting the monomer ratio, polymerization temperature, and polymerization time. For example, shortening the polymerization time or reducing the crosslinking agent ratio can form a thinner layer of 100–150 nm, which improves photothermal response efficiency; while a thicker layer of 250–300 nm can improve selectivity. The present invention preferably uses the 100–300 nm range to obtain the best balance between recognition performance and photoacoustic response.

[0030] Juniper fruit powder was mixed with 60% ethanol at a mass-volume ratio of 1:10 and heated under reflux for 1 hour to fully dissolve the flavonoids of Taxodium spp. Solid particles were removed by passing the mixture through a 0.45μm filter membrane and then diluted to the required concentration to prepare a test solution. This pretreatment ensured that the sample was transparent and free of particles, thus avoiding photoacoustic noise caused by particles absorbing light energy.

[0031] To simplify testing, an integrated testing device can be fabricated, which integrates a photoacoustic microcavity, laser, drive modulator, acoustic sensor, and signal processing module into the same device. The device includes a slot for fixing the test strip, a laser collimation module, an acoustic cavity, and a data processing unit. Users only need to insert the test strip and press the test button to complete the analysis, reducing the difficulty of operation.

[0032] Example 1

[0033] Preparation of imprinted fibers: fiber: cellulose, length 30 mm; template: 0.05 mmol of cypermethrin, 0.3 mmol of MAA, 2 mmol of EGDMA; solvent: acetonitrile; imprint layer thickness approximately 150 nm.

[0034] Sample preparation: Juniper extract diluted to 50 μg / mL; binding time: 60 seconds.

[0035] Photoacoustic detection conditions: Laser: 30mW, Frequency: 12kHz, Q value: 980;

[0036] Sound pressure results: blank: 22 Pa, sample: 78 Pa, ΔP=56 Pa, calculated concentration: 48 μg / mL (error 4%).

[0037] Example 2

[0038] Imprint preparation: nylon fiber, template: 0.08 mmol, 4-VP: 0.4 mmol, EGDMA: 2.5 mmol, imprint layer thickness: 260 nm

[0039] Sample, concentration: 120 μg / mL, binding time: 60 seconds;

[0040] Photoacoustic conditions: Laser: 55mW, Frequency: 10kHz;

[0041] Test results: Blank: 30 Pa, Sample: 158 Pa, ΔP = 128 Pa;

[0042] Calculated concentration: 118 μg / mL (error 2%).

[0043] Example 3

[0044] Imprinting, MAA system but with half the template concentration, improves the accuracy of structure recognition;

[0045] Sample concentration: 0.5 μg / mL, binding time: 60 seconds;

[0046] Photoacoustic detection, laser: 40mW, frequency: 15kHz, Q value: 1500;

[0047] Sound pressure results: blank: 5 Pa, sample: 17 Pa, ΔP = 12 Pa, calculated concentration: 0.48 μg / mL (error 4%).

[0048] In the above embodiments, the Q value refers to the acoustic quality factor of the photoacoustic microcavity, which is used to characterize the microcavity's ability to enhance photoacoustic sound pressure signals. It is a core physical parameter that supports the differences in sensitivity and embodiments, and is a recognized and necessary technical feature in the field.

[0049] Appendix Figure 1 The diagram shows the photoacoustic pressure difference (ΔP) response curves of Examples 1 to 3 under different concentrations of paclitaxel flavonoids. The horizontal axis represents the concentration of the paclitaxel flavonoid standard solution, ranging from 20 to 120 μg / mL; the vertical axis represents the sound pressure difference ΔP (Pa) obtained by photoacoustic detection. The three curves correspond to three groups of molecularly imprinted fiber test strips prepared under different conditions. Among them, the curve of Example 2 is located at the highest position of the three curves, indicating that it produces a larger photoacoustic response value under the same concentration conditions, indicating that its imprint layer recognition performance and photothermal conversion efficiency are the best, and it has higher sensitivity. The curve of Example 1 is the second best, and its photoacoustic response increases linearly with the increase of concentration, but the response amplitude is slightly lower. The curve of Example 3 is at the lowest position, indicating that its imprint structure or polymerization parameters result in insufficient photoacoustic signal generation and relatively low sensitivity. From the slope and linearity characteristics of the three sets of curves, it can be seen that all three show a good linear relationship, but Example 2 has a higher response amplitude and a steeper slope, reflecting its superior performance in the detection of paclitaxel flavonoids. Therefore, in the embodiments of the present invention, Example 2 can be regarded as the best embodiment.

[0050] Example 2 utilizes the surface activity of nylon fibers and the strong π–π interaction of 4-VP to significantly enhance the binding ability between the molecular imprint layer and cephalotaxine, resulting in a higher number of imprinted sites and a more stable structure. This allows the test strip to exhibit extremely high acoustic pressure response intensity (ΔP up to 128 Pa) under photoacoustic excitation. At the same time, the combination of a moderate imprint layer thickness and a high Q-value microcavity structure enables the detection repeatability to reach an industry-leading level (RSD of only 0.8%). Ultimately, this achieves rapid, accurate, and sensitive quantitative detection of cephalotaxine in a high concentration range, with overall performance superior to existing photoacoustic detection methods and conventional HPLC pretreatment systems.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting amentoflavone in a juniper extract, characterized by: The specific steps are as follows: Preparation of S1 molecularly imprinted fiber test paper: A molecularly imprinted polymer with cypermethrin as a template molecule is fixed on the surface of cellulose fiber or nylon fiber by surface polymerization to obtain fiber test paper with specific recognition sites; the molecularly imprinted polymer includes functional monomers of 4-vinylpyridine or methacrylic acid, crosslinking agent of ethylene glycol dimethacrylate and initiator of azobisisobutyronitrile. S2 Sample Binding Step: The juniper extract solution, which has been extracted, centrifuged and filtered by ethanol-water solution, is contacted with the fiber test paper for 20-90 seconds to allow the flavonoids of Taxodium bifidum to selectively bind to the imprint sites on the surface of the fiber test paper. S3 Insertion into the microcavity: The adsorbed fiber test paper is inserted into the acoustic resonant cavity region of the photoacoustic microcavity, placing it at the position of acoustic standing wave enhancement; the photoacoustic microcavity includes an outer shell cavity, an acoustic resonant cavity, a modulated laser incident window, and a miniature pressure sensor, and the fiber test paper is placed in the pressure antinode region of the acoustic resonant cavity. The photoacoustic microcavity adopts a cylindrical or long cavity resonant structure, forming an acoustic standing wave field inside. The ratio of cavity length to diameter is calculated acoustically, and the Q value is maintained between 400 and 1500. S4 photoacoustic excitation: A modulated laser with a modulation frequency of 1kHz-30kHz and a power of 5-80mW is applied to the adsorbed fiber test paper to cause periodic photothermal conversion on the surface of the fiber test paper and generate photoacoustic signals. S5 Signal Extraction and Quantification: The sound pressure amplitude of the photoacoustic signal is collected, and the difference ΔP is calculated between it and the sound pressure amplitude corresponding to the blank molecularly imprinted fiber test paper not exposed to the sample. The content of paclitaxel flavonoids in the sample is determined according to the standard curve of ΔP versus concentration.

2. The method for detecting cypress flavonoids in juniper extract according to claim 1, characterized in that: The fiber test paper has a strip-like structure with a thickness of 0.1-1 mm and a length of 5-30 mm, and a rigid support plate is provided on the back to assist in insertion and removal.

3. The method for detecting cypress flavonoids in juniper extract according to claim 1, characterized in that: The pretreatment of the juniper extract includes: ultrasonic extraction with 40%-80% ethanol-water solution for 3-10 minutes, followed by centrifugation and filtration through a 0.22μm filter membrane.

4. The method according to claim 1, wherein the extract of Du- song is extracted from Ilicium verum Hook. f. The quantitative method used is a univariate quantitative method based primarily on the difference in sound pressure amplitude.

5. The method according to claim 4, wherein the extract of Du- song is extracted from Ilex asprella. The sound pressure amplitude is processed using background subtraction, baseline correction, or resonance peak normalization methods.

6. The method according to claim 1, wherein the extract of Du- song is extracted from Ilicium verum Hook. f. After the fiber test paper binds to the sample, it can be rinsed with deionized water for 1-3 seconds to remove non-specific adsorbates before being inserted into the photoacoustic microcavity for detection.

Images