Radish component functionalized magnetic microspheres, preparation method and application thereof

Abstract

The application belongs to the field of biological medicine, and particularly relates to a functionalized magnetic microsphere of Raphani Semen components, a preparation method and application thereof. Based on photo-crosslinking technology, the application constructs multi-component modified magnetic microspheres, solves the problems existing in traditional single-component probes, and simultaneously fixes multiple active components in a traditional Chinese medicine complex system on the surface of the magnetic microspheres. On this basis, the application integrates high-throughput identification technology of chemical proteomics and cell function verification experiments, systematically captures and identifies a target group acted on by a complex component system of traditional Chinese medicine, constructs an overall regulation network of components, targets and pathways, and analyzes the action mechanism of traditional Chinese medicine from the system biology level.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a functionalized magnetic microsphere of radish seed components, its preparation method, and its application. Background Technology

[0002] Flavonoids, alkaloids, phenolic acids, terpenes, and other active ingredients in traditional Chinese medicine (TCM) possess a variety of pharmacological activities, including anti-inflammatory, antioxidant, antitumor, and immunomodulatory effects, and have broad application prospects in the fields of oncology, cardiovascular and cerebrovascular diseases, and metabolic diseases. However, most TCM active ingredients suffer from poor water solubility, low stability, rapid in vivo metabolism, low bioavailability, and lack of targeting, resulting in limited efficacy and significant toxic side effects from traditional administration methods, which seriously restricts their clinical translation and modern application. Furthermore, current research methods that isolate and purify TCM active ingredients for single-target identification make it difficult to reproduce the overall efficacy of TCM compound formulas.

[0003] Magnetic microspheres, due to their advantages such as superparamagnetism, high specific surface area, rapid magnetic separation, easy surface modification, and good biocompatibility, have been widely introduced into the separation, enrichment, screening, and delivery of active ingredients in traditional Chinese medicine (TCM) in recent years. Under the guidance of an external magnetic field, magnetic microspheres can accumulate at the lesion site, achieving magnetic targeted delivery, increasing local drug concentration, and reducing systemic toxicity. Simultaneously, surface modification can enable intelligent drug release based on pH, temperature, and enzyme responses, prolonging the duration of drug action. Although functionalized magnetic microspheres for TCM active ingredients have made some progress in drug delivery, the diverse structures of TCM active ingredients and poor interfacial compatibility with magnetic microsphere carriers result in generally low loading rates. Furthermore, the loading process easily damages the structure of active ingredients, leading to rapid drug leakage and insufficient stability in the in vivo environment, affecting targeted enrichment and long-term release. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a functionalized magnetic microsphere of radish seed components. The microsphere has a magnetic core, a surface functionalized layer, and photoaffinity groups, successfully immobilizing the active ingredients in radish seed extract on the surface of the magnetic microsphere.

[0005] The present invention also provides a method for preparing the above-mentioned radish seed functionalized magnetic microspheres.

[0006] Another objective of this invention is to provide the application of radish seed functionalized magnetic microspheres.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention provides a method for preparing radish seed functionalized magnetic microspheres, comprising the following steps: (1) After heating the aqueous solution containing FeCl3·6H2O and FeCl2·4H2O, ammonia water was added under nitrogen protection and the reaction was stirred. After the reaction was completed, the product was washed to obtain Fe3O4 magnetic microspheres. (2) Disperse Fe3O4 magnetic microspheres in deoxygenated water, add oleic acid, and continue stirring the reaction; after the reaction is completed, wash and disperse them in n-hexane, then add the mixed solution to obtain a surface-modified Fe3O4 magnetic microsphere solution. (3) The surface-modified Fe3O4 magnetic microsphere solution was added to sodium periodate aqueous solution under stirring conditions and stirred to react. After the reaction was completed, the microspheres were washed and vacuum dried to obtain carboxylated magnetic microspheres. The obtained carboxylated magnetic microspheres were activated by adding EDC-HCl and NHS as activators to obtain activated carboxylated magnetic microspheres. The photoreaction intermediate was prepared by amidation reaction of p-azidobenzoic acid and 4,7,10-trioxo-1,13-tridecanediamine. (4) The activated carboxyl magnetic microspheres were dispersed in a dimethylformamide solution containing the photoreaction intermediate and stirred overnight at room temperature. After the reaction was completed, the product was thoroughly washed and the unreacted active sites were blocked with glycine. Finally, the magnetic microspheres modified with the photoreaction intermediate were obtained. (5) The magnetic microspheres modified with the photointermediate were dispersed in a methanol solution containing radish seed extract and irradiated with ultraviolet light to obtain radish seed functionalized magnetic microspheres.

[0008] Preferably, in step (1), the molar ratio of FeCl3·6H2O and FeCl2·4H2O in the aqueous solution is 1.5:1-2:1; the concentration of the ammonia water is 25%–28%; the volume ratio of the aqueous solution to the ammonia water is 12:1; and the reaction is carried out by stirring in an oil bath at 50 °C for 0.5 h.

[0009] Preferably, in step (2), the ratio of Fe3O4 magnetic microspheres to oleic acid is 1.0-1.2 g: 2 mL; the reaction is carried out at 50 °C for 1 h; the volume ratio of n-hexane to the mixed solution is 1:2; and the volume ratio of ethyl acetate to acetonitrile in the mixed solution is 1:1.

[0010] Preferably, in step (3), the volume ratio of the surface-modified Fe3O4 magnetic microsphere solution to the sodium periodate aqueous solution is 2:1; the concentration of the sodium periodate aqueous solution is 0.02-0.03 g / mL; and the stirring reaction is carried out at room temperature and 500 r·min. -1 The reaction was stirred for 30-35 minutes under the specified conditions; the molar ratio of the carboxylated magnetic microspheres, EDC-HCl and NHS was 1:2:2.

[0011] Preferably, in step (4), the mass ratio of the activated carboxyl magnetic microspheres to the photoreaction intermediate is 1.4-1.5:1; and the mass ratio of the glycine to the photoreaction intermediate is 1.0-1.1:1.

[0012] Preferably, in step (5), the concentration of the methanol solution of the radish seed extract is 100 mg·mL. -1 The mass ratio of the magnetic microspheres modified with the photointermediate to the radish seed extract is 2:1; the ultraviolet irradiation conditions are: irradiation at a wavelength of 365 nm for 1–3 h in an ultraviolet irradiator.

[0013] The present invention also provides a radish seed functionalized magnetic microsphere prepared by the above preparation method, wherein the radish seed functionalized magnetic microsphere is composed of an Fe3O4 magnetic nanolayer, an oleic acid modification layer, a photoreaction intermediate layer and a radish seed active small molecule layer from the inside out.

[0014] Preferably, the active small molecules of radish seeds are radish extract, sinapicine thiocyanate, adenosine, 3,4-dihydroxyphenylacetic acid, sinapic acid, ferulic acid, glucosaturttin, 4-methoxyglucobrassicin, glucobrassicin, 4-methylthio-3-butenyl-GSL, glucoraphenin, or 3-methylthionylpropylthioglycoside.

[0015] This invention also provides the application of the above-mentioned radish seed functionalized magnetic microspheres in the preparation of drugs for treating hypertension.

[0016] This invention utilizes photocrosslinking technology to construct multi-component modified magnetic microspheres, overcoming the limitations of traditional single-component probes. It simultaneously immobilizes multiple active ingredients from complex traditional Chinese medicine (TCM) systems onto the surface of the magnetic microspheres. Building upon this, it integrates high-throughput chemical proteomics identification technology with cell function verification experiments to systematically capture and identify target groups within the complex TCM component system. This constructs a comprehensive regulatory network of components, targets, and pathways, elucidating the mechanism of action of TCM from a systems biology perspective.

[0017] The beneficial effects of this invention are as follows: (1) The present invention successfully prepared Fe3O4 magnetic microspheres by coprecipitation method, and constructed functionalized magnetic microspheres with magnetic core, surface functionalized layer and photoaffinity group by oleic acid modification, sodium periodate oxidation, EDC / NHS activation and photoreaction intermediate grafting; and successfully immobilized the active ingredients in radish seed extract on the surface of magnetic microspheres under 365 nm ultraviolet light irradiation to obtain radish seed modified traditional Chinese medicine magnetic microspheres; the crystal structure of the magnetic core was not destroyed during the surface modification and photocrosslinking process, and the magnetic microspheres have good structural stability.

[0018] (2) The radish seed-modified magnetic microspheres constructed in this invention can specifically capture a variety of proteins related to oxidative stress, vascular function regulation and RAS, including ACE, ECE1, SOD1, SOD2, SOD3, NOS3, MME, CTSA and ALDH2, etc., indicating that radish seed may exert antihypertensive effects by regulating oxidative stress, improving endothelial function and synergistically acting on vasoactive peptide-related pathways.

[0019] (3) The method provided by the present invention realizes the transformation from a single component and a single target to a group of multiple components and multiple targets, providing a brand-new technical path for breaking the black box effect of traditional Chinese medicine and promoting the modernization of traditional Chinese medicine. Attached Figure Description

[0020] Figure 1 A schematic diagram of the synthesis route for functionalized magnetic microspheres; Figure 2 The mass spectrum of the photoreaction intermediate prepared in Example 1; Figure 3 A comparison of the total ion current before and after the photoreaction intermediate prepared in Example 1; Figure 4 Comparison of SEM morphology of blank magnetic microspheres and photocrosslinked magnetic microspheres; In the figure, A: Fe3O4 magnetic microspheres; B: magnetic microspheres after photocrosslinking and combination with plant extracts; Figure 5 The images show the FT-IR spectra of samples at different stages of the synthesis and surface functionalization of magnetic microspheres. In the figures, a: Fe3O4 magnetic microspheres; b: oleic acid-modified magnetic microspheres with carboxyl groups introduced by NaIO4 oxidation; c: functionalized magnetic microspheres grafted with photocrosslinking groups; d: magnetic microspheres after photocrosslinking is combined with plant extracts. Figure 6 XRD analysis of samples at different stages of the synthesis and surface functionalization of magnetic microspheres; in the figure, a: Fe3O4 magnetic microspheres; b: oleic acid modified and carboxyl groups introduced by oxidation; c: functionalized magnetic microspheres grafted with photocrosslinking groups; d: magnetic microspheres after photocrosslinking and combination with plant extracts. Figure 7 TIC images of radish seed extract before and after photocrosslinking fixation; Figure 8 SDS-PAGE electrophoresis images of histones from different treatments; Figure 9 Volcano plot of differentially expressed proteins; Figure 10 Bubble diagram for enrichment of GO biological processes; Figure 11 Bar chart for enrichment of GO biological processes. Detailed Implementation

[0021] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0022] Example 1 The experimental reagents and equipment used are shown in Tables 1 and 2.

[0023] 1. Experimental Materials 1.1 Reagents Table 1 Experimental drugs and reagents

[0024] 1.2 Instruments Table 2 Instrument Names and Manufacturers

[0025] 2. Experimental Methods 2.1 Preparation of photoreaction intermediate-modified magnetic microspheres (1) A 150 mL aqueous solution containing FeCl3·6H2O (11.2 mM, 454 mg) and FeCl2·4H2O (5.6 mM, 167 mg) was heated to 50 °C in an oil bath. Ammonia water (25%–28%, 12.5 mL) was added under nitrogen protection, and the mixture was stirred at 50 °C for 0.5 h. After the reaction was completed, the product was thoroughly washed to obtain Fe3O4 magnetic microspheres.

[0026] (2) 1100 mg of Fe3O4 magnetic microspheres were redispersed in 150 mL of deoxygenated water. The mixture was heated to 50 °C under nitrogen purging and continuous stirring. Then, 2 mL of oleic acid was added, and the reaction was continued for 1 h to achieve surface modification of the magnetic microspheres. After the reaction was completed, the magnetic particles were thoroughly washed with anhydrous ethanol and dispersed in 10 mL of n-hexane. Then, 20 mL of a 1:1 volume ratio ethyl acetate / acetonitrile mixed solution was added.

[0027] (3) Add sodium periodate aqueous solution (0.41 g, 15 mL) under stirring conditions, and incubate at room temperature and 500 r·min -1 The reaction was stirred for 30 min under the specified conditions. After the reaction was completed, the microspheres were washed and dried under vacuum to obtain carboxylated magnetic microspheres.

[0028] (4) Take the obtained carboxylated magnetic microspheres (513 mg), add 383 mg EDC-HCl and 230 mg NHS as activators, and then add 100 mL of mixed solvent (dichloromethane and DMF in a volume ratio of 95:5) to activate the carboxyl groups on the surface to obtain activated carboxylated magnetic microspheres.

[0029] (5) The photoreaction intermediate was prepared by amidation reaction of p-azidobenzoic acid and 4,7,10-trioxo-1,13-tridecanediamine. The specific process was as follows: 358.6 mg of p-azidobenzoic acid was dissolved in 100 mL of anhydrous dichloromethane / DMF mixed solvent, and 260 mg of NHS and 26.9 mg of DMAP were added. 460 mg of DCC was added under ice bath or 4 °C conditions, and the reaction was carried out for 4 h. After the reaction was completed, the dicyclohexylurea byproduct was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain crude p-azidobenzoic acid NHS active ester. 520 mg of the obtained crude active ester was dissolved in 100 mL of anhydrous dichloromethane. 5.1 g of 4,7,10-trioxo-1,13-tridecanediamine was dissolved in anhydrous dichloromethane, and the active ester solution was slowly added dropwise to the diamine solution under stirring conditions. After the addition was complete, the reaction was allowed to proceed at room temperature for 12 hours. After the reaction was complete, the mixture was filtered, and the filtrate was washed with water or saturated brine. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain the photoreaction intermediate.

[0030] (6) The washed activated carboxyl magnetic microspheres (the amount prepared in step (4)) were dispersed in 50 mL of dimethylformamide solution containing the photoreaction intermediate (the amount of the photoreaction intermediate was 10 mmol, 366 mg), and stirred overnight at room temperature. After the reaction was completed, the product was thoroughly washed, and 375.4 mg of glycine was added to block the unreacted active sites for 1 h. Finally, the magnetic microspheres modified with the photoreaction intermediate were obtained.

[0031] 2.2 Crosslinking of Radish Seed Extract on the Surface of Magnetic Microspheres Modified by Photointermediates 200 mg of magnetic microspheres modified with photointermediate were dispersed in a solution containing 1 mL of radish seed extract (100 mg·mL⁻¹). -1The sample was transferred to a methanol solution of radish seeds and irradiated at 365 nm for 1–3 h. During the irradiation, the azide groups (–N3) in the photointermediate molecules on the microsphere surface were photoactivated, thus successfully loading the radish seed components onto the surface of the magnetic microspheres. Methanol solutions of radish seeds before and after irradiation were collected for analysis. The specific analysis conditions were as follows: Chromatographic conditions: An ACQUITY UPLC® HSS T3 (2.1×100 mm, 1.8 µm) column was used. The mobile phase consisted of a gradient elution of 0.1% formic acid aqueous solution (A) and acetonitrile (B) (0–3 min, 0% B; 3–8 min, 0–5% B; 8–10 min, 5–5% B; 10–15 min, 5–15% B; 15–17 min, 15–15% B; 17–24 min, 15–30% B; 24–28 min, 30–70% B; 28–35 min, 70–100% B). The column temperature was 40 °C, the flow rate was 0.3 mL·min⁻¹, and the injection volume was 3 μL.

[0032] Mass spectrometry conditions: ESI ion source, positive and negative ion modes; capillary voltage 3500 V; sheath gas: 35.0 psi; auxiliary gas: 10.0 L·min -1 The capillary temperature was 350 °C; collision energies were 20.0, 40.0, and 60.0 eV; and the mass scan range was 80–1200 Da. After the reaction was complete, the microspheres were washed three times with methanol to remove unbound impurities, and then dried under nitrogen to obtain magnetic microspheres modified with traditional Chinese medicine components.

[0033] 2.3 Scanning Electron Microscopy (SEM) Characterization The morphology of the magnetic microspheres was observed using scanning electron microscopy. A suitable amount of sample was dried and uniformly dispersed on a conductive adhesive, then sputter-coated with gold before testing. The particle size distribution, morphological characteristics, and surface structure of the magnetic microspheres were analyzed using SEM images.

[0034] 2.4 FT-IR Infrared Spectroscopy Analysis Take appropriate amounts of dried samples, including Fe3O4 magnetic microspheres, carboxylated magnetic microspheres, photoreaction intermediate-modified magnetic microspheres, and radish seed-modified magnetic microspheres, and grind them evenly for later use. The potassium bromide pellet method is used for testing: weigh approximately 1-2 mg of sample, mix it with an appropriate amount of dried KBr, grind it finely, press it into a transparent thin film, and then place it in an infrared spectrometer for detection. The scanning range is set to 4000-400 cm⁻¹. -1 The resolution is 4 cm. -1 Subtraction was performed using blank KBr as the background.

[0035] 2.5 X-ray diffraction (XRD) analysis The crystal structure of the magnetic microsphere samples was analyzed using X-ray powder diffraction (XRD). Cu Kα rays with a wavelength of λ = 0.1541 nm were used as the radiation source. The instrument operating voltage was set to 40 kV and the operating current to 40 mA. The dried samples were uniformly spread in the sample cell, and diffraction tests were performed at room temperature. The data acquisition range was set to 2θ = 10°–80° with a step angle of 0.0262° to obtain the complete diffraction pattern of the samples. The obtained XRD patterns were analyzed to characterize the crystal phase composition and crystallization characteristics of the magnetic microspheres.

[0036] 2.6 Capture and Detection of Magnetic Microsphere Target Proteins from Traditional Chinese Medicine HUVECs were added to 1 mL of RIPA lysis buffer containing 1% PMSF and lysed on ice for 30 min. The mixture was then centrifuged at 4 ℃ and 12000 rpm for 10 min. The supernatant was collected and protein quantification was performed using standard methods. Equal amounts of total protein were divided into a control group, a binding group, and a competition group, with three replicates in each group. The control group consisted of 100 mg of blank magnetic microspheres without radish seed loading incubated with HUVECs cell lysis buffer at 4 ℃ for 12 h in a constant temperature shaker. The binding group consisted of 100 mg of magnetic microspheres modified with radish seed loading incubated with HUVECs cell lysis buffer at 4 ℃ for 12 h. The competition group consisted of HUVECs cell lysis buffer pre-incubated with radish seed extract solution, followed by the addition of 100 mg of magnetic microspheres modified with radish seed loading, and incubation continued at 4 ℃ for 12 h to reduce non-specific binding and verify the specificity of target protein binding.

[0037] After incubation, each group of samples was placed on a magnetic rack for magnetic separation. The supernatant was discarded, and the magnetic microspheres were washed 5–6 times with PBS buffer to remove non-specifically adsorbed proteins. Then, an appropriate amount of elution buffer was added to the magnetic bead-protein complex, and the mixture was gently pipetted to mix. After incubation at room temperature for 5–10 min, the mixture was placed back on the magnetic rack, and the supernatant was collected. The resulting supernatant was the eluted protein sample. The eluted protein was thoroughly mixed with an appropriate amount of 5×SDS loading buffer, heated at 95 °C for denaturation for 5 min, and cooled to room temperature for subsequent electrophoresis analysis.

[0038] Captured proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separating and stacking gels were prepared according to experimental requirements. After solidification, the electrophoresis apparatus was assembled, and the sample loading tanks were checked for sealing. Denatured protein samples (Marker) were added to each lane. Constant voltage electrophoresis was used, initially applying 80V to the stacking gel. Once the samples entered the separating gel, the voltage was increased to 120V, and electrophoresis was stopped when the bromophenol blue indicator migrated to the bottom of the gel. After electrophoresis, the gel was removed, rinsed with deionized water for 5 min, and stained with Coomassie Brilliant Blue at room temperature for 2 h. The staining solution was discarded, and the gel was washed again with deionized water for 5 min. Coomassie Brilliant Blue destaining solution was added for destaining, and the destaining solution was changed periodically according to the band development until the protein bands were clearly visible. After destaining, the gel was transferred to deionized water and washed for 5 min for subsequent development and mass spectrometry sample preparation.

[0039] Protein samples separated by SDS-PAGE were further identified and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS, Orbitrap Astral mass spectrometer coupled with Vanquish Neo UHPLC system, Thermo Fisher Scientific, USA). Mass spectrometry data were processed using label-free quantification (LFQ), and the differences in protein abundance among different experimental groups were visualized using a volcano plot. P <0.05 and Fold change (FC)>1.5 were used as differential protein screening criteria to identify potential target proteins captured by magnetic microspheres modified with radish seed components.

[0040] 2.7 UHPLC-Orbitrap Astral MS / MS Analytical Conditions Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was performed using an Orbitrap Astral mass spectrometer coupled with a Vanquish Neo ultra-high performance liquid chromatography (UHPLC) system (Thermo Fisher Scientific). Peptides for each sample were loaded onto a chromatographic column (50 cm Low-Load µPAC™ Neo HPLC Column, Thermo Scientific) at a flow rate of 2.2 μL / min. For reversed-phase high-performance liquid chromatography (RP-HPLC), mobile phase A consisted of an aqueous solution containing 0.1% formic acid, and mobile phase B consisted of an 80% acetonitrile solution containing 0.1% formic acid. The peptides eluted over 8 minutes at a flow rate of 1.25 μL / min through a linear gradient in mobile phase B. The linear gradient settings were as follows: 0–0.1 min, mobile phase B: 4%–6%; 0.1–1.1 min, B: 6%–12%; 1.1–4.3 min, B: 12%–25%; 4.3–6.1 min, B: 25%–45%; 6.1–6.5 min, B: 45%–99%; 6.5–8 min, B: 99%. The eluted peptides were detected on an Orbitrap Astral mass spectrometer. The data-independent acquisition (DIA) method included a single scan range of... m / z The precursor ion scan ranged from 380 to 980 ions, with a resolution of 240,000 ions, an automatic gain control (AGC) target of 500%, and an injection time of 5 ms. The DIA tandem mass spectrometry (MS / MS) scan was performed using an Astral mass analyzer. m / z Data collected within the range of 150-2000, with an isolation window of 2. m / z The AGC target value was 500%, and the injection time was 3 ms. The normalized collision energy was set to 25, and the cycle time was 0.6 s. Full-scan mass spectra and DIA scans were recorded in profile mode and centroid mode, respectively.

[0041] 3. Experimental Results 3.1 Analysis of the Synthesis Process of Magnetic Microspheres of Traditional Chinese Medicine From the inside out, it consists of a Fe3O4 magnetic nanolayer, an oleic acid modified layer, a photoreaction intermediate layer, and a radish seed active small molecule layer. Using Fe... 3+ :Fe 2+Fe3O4 magnetic nanospheres were prepared using a co-precipitation method with a ratio of 1.5:1 to 2:1 to enrich oleic acid protein. Then, sodium periodate was used to oxidize the C=C double bonds on the oleic acid surface to carboxyl groups, resulting in an oleic acid-modified layer. In the third layer, carboxyl magnetic beads were activated using a dual activator mixture of EDC HCl and NHS at a molar ratio of 1:1, and azide groups (–N3) from a photointermediate molecule were attached to the surface as photoaffinity groups. This structure serves as the core material for the herbal microspheres and can be used to connect different types of active small molecules. A certain amount of microspheres carrying photoaffinity groups and radish seed extract were irradiated at a wavelength of 365 nm to attach small molecules to the surface of the magnetic microspheres.

[0042] like Figure 1 As shown, the preparation process of TCM magnetic microspheres mainly includes three steps: magnetic core construction, surface functionalization, and photocrosslinking loading. First, Fe3O4 magnetic microspheres were prepared under alkaline conditions via a co-precipitation reaction of FeCl3·6H2O and FeCl2·4H2O. Then, oleic acid was used to hydrophobically modify the surface of the magnetic microspheres to improve their dispersibility and stability. After oxidation treatment with sodium periodate (NaIO4), carboxyl functional groups were successfully introduced onto the surface of the magnetic microspheres, providing reaction sites for subsequent coupling reactions. Under the action of an EDC / NHS dual activation system, the carboxyl groups underwent an amidation reaction with an amino-containing photoreaction intermediate, constructing functionalized magnetic microspheres with surface-modified photosensitive groups. Subsequently, under 365nm ultraviolet light irradiation, the photocrosslinking groups were activated, achieving covalent fixation of the active small molecules of TCM, ultimately obtaining TCM-MPs.

[0043] To further verify the correctness of the photocrosslinking group intermediate structure, mass spectrometry analysis was performed. The results are as follows: Figures 2-3 As shown, the main peak of the photoreaction intermediate m / z The value is 396.21, consistent with its theoretical molecular weight calculation, corresponding to the molecular formula C. 17 H 27 N5O4 indicates the successful synthesis of the target intermediate. The decrease in peak area before and after the reaction with carboxylated magnetic microspheres indicates that the photoreaction intermediate was bonded to the magnetic microspheres. These results, from a structural and quality analysis perspective, confirm the feasibility and reliability of the synthetic route for traditional Chinese medicine magnetic microspheres, providing a stable and functionally defined material basis for subsequent target protein fishing experiments.

[0044] 3.2 Scanning electron microscope images of blank magnetic microspheres and photocrosslinked magnetic microspheres The morphology of blank magnetic microspheres (Fe3O4 magnetic microspheres prepared in step (1) of Example 1) and photocrosslinked magnetic microspheres was observed using scanning electron microscopy. The results are as follows: Figure 4As shown in Figures A and B, the results are illustrated in the figure. The blank magnetic microspheres appear as approximately spherical or near-spherical particles with a relatively uniform particle size distribution. There is a certain degree of agglomeration between the particles, but the overall structure is relatively loose. The microsphere boundaries are relatively clear, and the surface is relatively flat, showing typical Fe3O4 magnetic microsphere morphology characteristics.

[0045] In contrast, the magnetic microspheres after photocrosslinking retained their overall spherical structure, but the surface roughness increased significantly, and the aggregation between microspheres was enhanced. In some areas, tighter interparticle connections were observed. Simultaneously, the photocrosslinked microspheres exhibited a more irregular structural feature, presumably related to the successful immobilization of the active ingredients and photosensitive groups of the traditional Chinese medicine onto the microsphere surface during the photocrosslinking process. This change in surface morphology may stem from the introduction of an organic molecular layer, transforming the microsphere surface from a relatively smooth state to a coated or modified state.

[0046] The SEM results above indicate that the photocrosslinking reaction did not destroy the overall structural integrity of the magnetic microspheres, but it did alter their surface morphology to some extent, reflecting the successful achievement of functionalization modification. This result corroborates the aforementioned mass spectrometry analysis and chromatographic peak area changes of the photocrosslinking intermediates, further demonstrating that the active ingredients of traditional Chinese medicine have been stably loaded onto the surface of the magnetic microspheres via photocrosslinking.

[0047] 3.3 FT-IR Spectroscopic Analysis of the Synthesis Process of Magnetic Microspheres To verify the changes in the surface chemical structure of the magnetic microspheres at different synthesis stages and to further substantiate the rationality of the synthesis route, Fourier transform infrared spectroscopy (FT-IR) analysis was performed on samples from each stage. The results are as follows: Figure 5 As shown. FT-IR spectroscopy results indicate that the samples exhibit corresponding characteristic absorption peaks at different functionalization stages. Group a shows a peak at approximately 580 cm⁻¹. -1 The presence of an absorption peak for the Fe-O stretching vibration at 2920 cm⁻¹ confirms the successful preparation of Fe₃O₄ magnetic microspheres. Compared to group a, group b showed an absorption peak at 2920 cm⁻¹. -1 and 2850cm -1 An absorption peak for the -CH2 stretching vibration of the fatty chain appears nearby, and also at approximately 1700 cm⁻¹. -1 The presence / enhanced carbonyl-related absorption nearby indicates that oleic acid was successfully modified onto the surface of the magnetic microspheres, and oxygen-containing functional groups were introduced after oxidation. Furthermore, group c showed absorption at approximately 1650 cm⁻¹. -1 and 1540 cm -1 A characteristic absorption peak of amide bonds appears nearby, along with a peak at 1100 cm⁻¹. -1 The enhanced absorption at the left and right ether bonds indicates that the photocrosslinking groups have been successfully grafted onto the surface of the magnetic microspheres. Group d shows absorption at 3400 cm⁻¹. -1 The nearby broad peaks are enhanced, and at 1600-1700 cm⁻¹-1 and 1000-1300 cm -1 The enhanced and more complex regional absorption indicates that the plant extracts were successfully incorporated into the surface of the functionalized magnetic microspheres. In summary, the FT-IR results confirm the stepwise surface modification and active molecule loading process of the magnetic microspheres.

[0048] 3.4 XRD Analysis of Magnetic Microspheres To verify whether the crystal structure of the magnetic microspheres changes at different functionalization stages, X-ray diffraction (XRD) analysis was performed on the samples. The results are as follows: Figure 6 As shown. In Figure 6 As shown in Figure a (Fe3O4 magnetic microspheres), characteristic diffraction peaks can be observed at 2θ = 30.1°, 35.4°, 43.1°, 53.4°, 56.9°, 62.5°, and 74.9°, corresponding to the (220), (311), (400), (422), (511), (440), and (533) crystal planes of Fe3O4, respectively. The positions of these diffraction peaks are consistent with the standard Fe3O4 magnetite crystal form, indicating the successful preparation of Fe3O4 magnetic microspheres with a good crystal structure. Figure 6 As shown in b (oleic acid-modified and NaIO4-oxidized carboxylated magnetic microspheres), the positions of each characteristic diffraction peak are related to... Figure 6 The values ​​of 'a' and 'a' are basically consistent, and no new impurity phase peaks appear, indicating that the oleic acid modification and oxidation treatment did not destroy the crystal structure of Fe3O4, but only introduced organic functional groups on the surface of the microspheres. Figure 6 The c (functionalized magnetic microspheres after grafting photocrosslinking groups) also retains the characteristic diffraction peaks of Fe3O4, with no significant shift in peak position, indicating that the grafting of photocrosslinking groups is a surface modification process and does not affect the crystal phase structure of the magnetic core. Figure 6 The d-type magnetic microspheres (after photocrosslinking and loading with plant extracts) still showed the same diffraction peak positions as Fe3O4, and no obvious new crystallization peaks were detected, indicating that the loading of plant extracts did not change the crystal form of the magnetic microspheres. Overall, the diffraction peak intensities of the samples at different stages varied slightly, which may be related to the attenuation of diffraction intensity caused by the surface organic layer coating. XRD results showed that the crystal structure of the magnetic microspheres remained unchanged throughout the entire process from the construction of the Fe3O4 magnetic core to the photocrosslinking and loading of plant extracts, and the magnetic core remained stable, providing structural assurance for its subsequent magnetic response separation and target protein fishing applications.

[0049] 3.5 Liquid chromatography-mass spectrometry analysis of radish seed extract before and after the reaction like Figure 7As shown, there are significant differences in the overall chemical composition spectrum of radish seed extract before and after the photocrosslinking reaction. After the reaction, the chromatographic peak response intensities of various components decreased significantly, indicating that some chemical components can bind to the photoreaction intermediate-modified magnetic microspheres and be fixed on the surface of the microspheres under UV activation conditions. Combined with the results in Table 3, it can be seen that the immobilization rates of different components vary significantly, reflecting that the binding ability between the various chemical components in radish seed extract and the functionalized magnetic microspheres is not the same. The immobilization rates of sinapic acid, 4-methylthio-3-butenylthioglycoside, and 3-methylthionylpropylthioglycoside were relatively high, at 91.62%, 90.59%, 90.34%, and 82.12%, respectively. Sinapic acid and sinapic acid thiocyanate, as representative phenolic acids and alkaloids in radish seeds, showed high immobilization rates, suggesting that they may be important material bases for subsequent target protein fishing. Meanwhile, ferulic acid, 3,4-dihydroxyphenylacetic acid, and brassinosteroids also showed moderate immobilization rates, indicating that they can be loaded onto the surface of magnetic microspheres to varying degrees. The immobilization rates of raphanin and raphanin were 47.25% and 43.56%, respectively, indicating that both have a certain immobilization capacity in this system. Raphanusinosteroids are important glucosinolates in radish seeds, while raphanin is an active isothiocyanate component derived from its enzymatic hydrolysis. The fact that both of these components showed a certain immobilization rate suggests that the antihypertensive active substances in radish seeds may not be limited to a single component, but rather involve multiple components working together. In contrast, the immobilization rates of adenosine and piracetamine were lower, at 10.32% and 13.56%, respectively, indicating that their binding ability to the surface of magnetic microspheres under the current photocrosslinking conditions is weaker, and they may remain more in the post-reaction solution.

[0050] Table 3. Fixation rate of photocrosslinking reaction of radish seeds

[0051] 3.6 Proteomic Analysis of Target Proteins in Magnetic Microspheres for Fishing from Traditional Chinese Medicine Based on the photoreaction intermediate modified magnetic microsphere technology, radish seed component-functionalized magnetic microspheres (TCM-MPs) were successfully constructed and used for the capture and identification of potential target proteins in HUVECs. SDS-PAGE results ( Figure 8 The results showed that the binding group exhibited specific protein bands, while the bands in the competition group were significantly weakened, indicating that the captured proteins had good specificity.

[0052] LC-MS / MS analysis results ( Figure 9 The results showed that a variety of significantly differentially expressed proteins were obtained through screening. P <0.05, FC>1.5). Functional protein analysis (Table 4) shows that these target proteins are mainly involved in RAS, oxidative stress regulation, and vasomotor processes.

[0053] Key enzymes such as ACE, MME, and CTSA are specifically captured and exhibit significant abundance differences. In terms of RAS system regulation, ACE participates in the classical ACE / Ang II pressor pathway, while MME and CTSA may participate in the ACE2 / Ang-(1–7) protective pathway and related bioactive peptide metabolism. Radish seed active components may exert a synergistic antihypertensive effect by inhibiting the pressor axis and enhancing the protective axis through a dual-pathway regulatory mechanism.

[0054] SOD1, SOD2, and SOD3 were significantly enriched, indicating that the active components of radish seeds have antioxidant effects by activating superoxide dismutase in the body. Changes in ALDH2 also suggest its potential role in aldehyde metabolism and the maintenance of mitochondrial redox homeostasis. Regarding vascular function regulation, NOS3 was significantly upregulated (FC=14.80), indicating that it enhances vasodilation and improves endothelial function by promoting NO production. Meanwhile, changes in ECE1, a key enzyme in endothelin production, suggest that radish seeds may participate in the regulation of the endothelin system, thereby affecting the balance between vasoconstriction and vasodilation.

[0055] like Figure 10 GO enrichment analysis showed that the differentially expressed proteins were significantly enriched in biological processes such as blood pressure regulation, reactive oxygen metabolism, systemic arterial blood pressure regulation, and vascular process regulation. Figure 11 Further analysis of the KEGG pathway revealed that differentially expressed proteins such as ACE, MME, and CTSA are involved in the renin-angiotensin system pathway, including Ang I / Ang II metabolism, Ang-(1-7) production, and vasomotor regulation. These results suggest that the active components of radish seeds may exert their antihypertensive effects by intervening in the RAS pathway, improving oxidative stress, and regulating vascular function.

[0056] In summary, radish seeds may regulate oxidative stress levels, improve endothelial function, and synergistically act on different branches of the RAS system to achieve comprehensive regulation of vascular tone and blood pressure.

[0057] Table 4. Functional table of 10 candidate proteins associated with hypertension

[0058] 4. Conclusion This invention focuses on the construction, characterization, and target protein fishing application of functionalized magnetic microspheres of radish seed components. The preparation, structural verification, and preliminary functional evaluation of the traditional Chinese medicine magnetic microspheres have been systematically completed.

[0059] Fe3O4 magnetic microspheres were successfully prepared using a co-precipitation method. Through oleic acid modification, sodium periodate oxidation, EDC / NHS activation, and grafting of photoreaction intermediates, functionalized magnetic microspheres with magnetic cores, surface functionalized layers, and photoaffinity groups were constructed. Under 365 nm UV irradiation, the active components of radish seed extract were successfully immobilized on the surface of the magnetic microspheres, resulting in radish seed-modified magnetic microspheres. Mass spectrometry results of the photoreaction intermediates showed that their molecular weight was consistent with the theoretical value, indicating successful synthesis of the target intermediates. Liquid chromatography-mass spectrometry (LC-MS) analysis before and after the reaction further demonstrated that multiple components of radish seed extract could be effectively immobilized on the surface of the magnetic microspheres, with differences in immobilization rates among different components, indicating that the constructed magnetic microspheres possess multi-component loading characteristics.

[0060] SEM results showed that the magnetic microspheres maintained a near-spherical structure before and after photocrosslinking. The surface roughness increased after functionalization, indicating that radish seed chemical components were successfully loaded onto the microsphere surface. FT-IR analysis showed that the magnetic microspheres exhibited corresponding characteristic absorption peak changes at each stage, including oleic acid modification, carboxylation, photocrosslinking group grafting, and radish seed loading, confirming the gradual realization of surface functionalization modification. XRD analysis showed that the samples at each stage retained the characteristic diffraction peaks of Fe3O4, indicating that the surface modification and photocrosslinking processes did not destroy the crystal structure of the magnetic core, and the magnetic microspheres possess good structural stability.

[0061] Based on the constructed magnetic microspheres modified with radish seed components, further proteomics analysis using SDS-PAGE and LC-MS / MS was conducted to screen potential target proteins in HUVECs. The results showed that this system could specifically capture various proteins related to oxidative stress, vascular function regulation, and RAS, including ACE, ECE1, SOD1, SOD2, SOD3, NOS3, MME, CTSA, and ALDH2. This indicates that radish seed may exert its antihypertensive effect by regulating oxidative stress, improving endothelial function, and synergistically acting on vasoactive peptide-related pathways.

[0062] Therefore, this invention successfully constructed and systematically characterized functionalized magnetic microspheres of radish seed components, verifying their feasibility and effectiveness in the fixation of multiple components of traditional Chinese medicine and the targeting of proteins, laying an experimental foundation for further in-depth analysis of the antihypertensive mechanism of radish seed.

Claims

1. A method for preparing radish seed-functionalized magnetic microspheres, characterized in that, Includes the following steps: (1) After heating the aqueous solution containing FeCl3·6H2O and FeCl2·4H2O, ammonia water was added under nitrogen protection and the reaction was stirred. After the reaction was completed, the product was washed to obtain Fe3O4 magnetic microspheres. (2) Disperse Fe3O4 magnetic microspheres in deoxygenated water, add oleic acid, and continue stirring the reaction; after the reaction is completed, wash and disperse them in n-hexane, then add the mixed solution to obtain a surface-modified Fe3O4 magnetic microsphere solution. (3) The surface-modified Fe3O4 magnetic microsphere solution was added to sodium periodate aqueous solution under stirring conditions and stirred to react. After the reaction was completed, the microspheres were washed and vacuum dried to obtain carboxylated magnetic microspheres. The obtained carboxylated magnetic microspheres were activated by adding EDC-HCl and NHS as activators to obtain activated carboxylated magnetic microspheres. The photoreaction intermediate was prepared by amidation reaction of p-azidobenzoic acid and 4,7,10-trioxo-1,13-tridecanediamine. (4) The activated carboxyl magnetic microspheres were dispersed in a dimethylformamide solution containing the photoreaction intermediate and stirred overnight at room temperature. After the reaction was completed, the product was thoroughly washed and unreacted active sites were blocked with glycine. Finally, magnetic microspheres modified with photoreaction intermediates were obtained; (5) The magnetic microspheres modified with the photointermediate were dispersed in a methanol solution containing radish seed extract and irradiated with ultraviolet light to obtain radish seed functionalized magnetic microspheres.

2. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of FeCl3·6H2O and FeCl2·4H2O in the aqueous solution is 1.5:1-2:1; the concentration of the ammonia water is 25%–28%; the volume ratio of the aqueous solution to the ammonia water is 12:1; and the reaction is carried out by stirring in an oil bath at 50°C for 0.5 h.

3. The preparation method according to claim 1 or 2, characterized in that, In step (2), the ratio of Fe3O4 magnetic microspheres to oleic acid is 1.0-1.2 g: 2 mL; the reaction is carried out at 50 °C for 1 h; the volume ratio of n-hexane to the mixed solution is 1:2; and the volume ratio of ethyl acetate to acetonitrile in the mixed solution is 1:

1.

4. The preparation method according to claim 3, characterized in that, In step (3), the volume ratio of the surface-modified Fe3O4 magnetic microsphere solution to the sodium periodate aqueous solution is 2:1; the concentration of the sodium periodate aqueous solution is 0.02-0.03 g / mL; and the stirring reaction is carried out at room temperature and 500 r·min. -1 The reaction was stirred for 30-35 minutes under the specified conditions; the molar ratio of the carboxylated magnetic microspheres, EDC-HCl and NHS was 1:2:

2.

5. The preparation method according to claim 1, characterized in that, In step (4), the mass ratio of the activated carboxyl magnetic microspheres to the photoreaction intermediate is 1.4-1.5:1; the mass ratio of the glycine to the photoreaction intermediate is 1.0-1.1:

1.

6. The preparation method according to claim 1, characterized in that, In step (5), the concentration of the methanol solution of the radish seed extract is 100 mg·mL. -1 The mass ratio of the magnetic microspheres modified with the photointermediate to the radish seed extract is 2:1; the ultraviolet irradiation conditions are: irradiation at a wavelength of 365 nm for 1–3 h in an ultraviolet irradiator.

7. A radish seed component-functionalized magnetic microsphere prepared by the preparation method according to any one of claims 1-6, characterized in that, The radish seed functionalized magnetic microspheres are composed of, from the inside out, an Fe3O4 magnetic nanolayer, an oleic acid modified layer, a photoreaction intermediate layer, and a radish seed active small molecule layer.

8. The radish seed functionalized magnetic microspheres according to claim 7, characterized in that, The active small molecules of radish seeds are radicin, sinigrin thiocyanate, adenosine, 3,4-dihydroxyphenylacetic acid, sinapic acid, ferulic acid, stigmasteric acid, indole-3-thioglucoside, brassinosteroidal glucoside, 4-methylthio-3-butenylthioglycoside, radicin, or 3-methylthionylpropylthioglycoside.

9. The use of radish seed functionalized magnetic microspheres as described in claim 7 in the preparation of drugs for treating hypertension.

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