Application of ecarin in preparation of jellyfish repellent and ecarin-copper composite repellent system

Abstract

The application discloses application of ecarizine in preparation of jellyfish repellent and an ecarizine-copper composite repellent system. The application first discovers that ecarizine has significant repellent activity on jellyfish through systematic behavior screening. Then, aiming at the defects of low solubility of ecarizine in seawater and high ecological toxicity of copper ions, an ecarizine-loaded copper-cyclodextrin metal organic framework composite (ICA@Cu-MOF) is further constructed. The loading amount of ecarizine in the composite is 18.55%±2%, and the apparent solubility in artificial seawater is increased by 2.08 times; the cumulative release rate of copper ions is reduced from 93.83% to 71.42%, and the burst release is effectively slowed down. The behavior experiment shows that the repellent index of ICA@Cu-MOF reaches 100%, which is significantly better than 66.67% of the physical mixture. The application first applies ecarizine to the field of jellyfish repellent, and through the synergistic composite with copper ions, the repellent effect is significantly improved, the preparation performance is improved, and the ecological risk is reduced, thereby providing a new technical scheme for the preventive protection of jellyfish stings.

Description

Technical Field

[0001] This invention relates to the fields of marine biological protection and materials science, specifically to the novel use of icocaridine in the preparation of jellyfish repellents, as well as the composite repellent system based on icocaridine and copper ions, its preparation method, and its application. Background Technology

[0002] Jellyfish stings are one of the most common marine injuries sustained during coastal activities, affecting approximately 150 million people globally each year. The nematocysts on jellyfish tentacles can inject toxins into the body, causing severe pain, skin inflammation, blisters, and in severe cases, systemic poisoning and even death. Currently, there are no specific antiseptic drugs for jellyfish stings; therefore, preventative protection by avoiding jellyfish has significant clinical and public health implications.

[0003] Reports on jellyfish repellents are currently extremely limited. A few studies have attempted to use substances such as copper ions, but the direct application of copper ions poses a high risk of ecotoxicity. Most organic repellents have low solubility in seawater, making it difficult to rapidly diffuse and form effective repellent concentrations. More importantly, to date, there are no reports of using systematic behavioral screening to identify jellyfish repellent active substances.

[0004] Icaridin is a known insect repellent widely used to repel arthropods such as mosquitoes, ticks, and fleas. Its mechanism of action is mainly related to interfering with insect olfactory receptors and regulating voltage-gated sodium channels. However, jellyfish belong to the phylum Cnidaria and have completely different nervous system structures and physiological characteristics. Whether icacaridin can produce a repellent effect on jellyfish has not been reported in any previous studies or provided any technical indications. Summary of the Invention

[0005] This invention addresses the aforementioned problems. Given the lack of reports on the discovery of jellyfish-repelling active substances using systematic behavioral screening, this invention employs a self-designed three-chamber behavioral screening system to systematically evaluate 16 candidate substances, discovering for the first time that icacridin possesses significant jellyfish-repelling activity. Building upon this, to further address the practical problems of low seawater solubility of icacridin and excessively rapid release of copper ions, this invention further designs an icacridin-copper composite repellent system, specifically by loading icacridin into a copper-cyclodextrin metal-organic framework (Cu-MOF), forming a solid composite repellent with synergistic effects.

[0006] Experimental results showed that the loading of icolinergic acid in the ICA@Cu-MOF complex was 18.55% ± 2%, and the apparent solubility in artificial seawater increased by 2.08 times. The cumulative release rate of copper ions decreased from 93.83% to 71.42%, effectively mitigating the sudden release. Behavioral experiments showed that the repellency index of ICA@Cu-MOF reached 100%, significantly better than the 66.67% of the physical mixture. ICA@Cu-MOF treatment significantly altered the movement pattern of jellyfish in the experimental setup, causing them to gradually move away from the administration area and gather in the opposite area.

[0007] Based on the above research, the technical solution to be protected by this invention is as follows: In a first aspect, the present invention provides a novel use of icocaridine in the preparation of jellyfish repellents.

[0008] This invention uses systematic behavioral screening to add 16 candidate substances (including metal salts, insect repellents, plant essential oils, surfactants, etc.) to one side of a three-chamber water tank, record the distribution and movement changes of jellyfish in each area, and calculate the repellency index. The results show that among the 6 organic compounds tested, only icacridin (ICA) showed significant repellency activity, while widely used insect repellents such as DEET, pyrethroids, and menthol had no repellency effect (Table 1). This discovery has the following important significance: (1) It is the first time that icacridin has repellency activity against jellyfish, filling the gap in the field of jellyfish protection; (2) It suggests that the avoidance perception mechanism of jellyfish may have different molecular targets than that of insects, and the result that DEET is ineffective while ICA is effective has important scientific implications.

[0009] Therefore, icocaridine can be used as a single active ingredient in the preparation of jellyfish repellents, or it can be used in combination with other substances that have jellyfish repellent activity, such as copper, which also has jellyfish repellent effects, to obtain better repellent effects.

[0010] In a second aspect, the present invention explores the formulation of icocaridine and provides a composite repellency system comprising icocaridine and copper ions.

[0011] Although icacridin alone possesses jellyfish repellent activity, this invention has found that its solubility in seawater is low (only 6.75 mg / mL at 25°C), affecting its rapid release and diffusion. Meanwhile, while copper ions have a strong repellent effect, high-concentration copper exposure poses an ecotoxicity risk. To address these issues, this invention combines icacridin with copper ions to mutually improve formulation performance.

[0012] Preferably, the present invention provides an icocaredin-supported copper-cyclodextrin metal-organic framework complex (ICA@Cu-MOF). The metal-organic framework is formed by the coordination of copper ions and cyclodextrin, and icocaredin is loaded onto the pores or surface of the framework through physical adsorption or coordination.

[0013] Experiments show that ICA@Cu-MOF possesses the following excellent properties: (1) The apparent solubility of icocaridine in artificial seawater increased to 14.06 mg / mL, which is 2.08 times that of free icocaridine; (2) The cumulative release rate of copper ions decreased from 93.83% in Cu-MOF to 71.42%, which effectively slowed down the sudden release of copper ions and reduced the risk of ecotoxicity. (3) In jellyfish behavior experiments, the repellency index of ICA@Cu-MOF reached 100%, which was significantly better than the 66.67% of the physical mixture of icostigma and Cu-MOF. p (< 0.01), which proves that structural integration brings about synergistic effects.

[0014] Furthermore, this invention provides a method for preparing ICA@Cu-MOF composites, comprising the following steps: (1) Synthesis of copper-cyclodextrin metal-organic framework (Cu-MOF): copper salt and β-cyclodextrin or its analogues were mixed in deionized water at a molar ratio of (3-8):1. A first proton polar solvent was added, and the mixture was reacted at 30-60℃ for 1-4 h. A second proton polar solvent was added to precipitate the mixture, and the mixture was washed and dried to obtain Cu-MOF.

[0015] (2) Preparation of ICA@Cu-MOF: Icaridin is dissolved in anhydrous methanol and mixed with Cu-MOF in a certain ratio (e.g., Icaridin:Cu-MOF=25:100, w / w). The mixture is then sealed and stirred at 30-50℃ for 1-3 h and dried to obtain the final product.

[0016] In a preferred embodiment of the present invention, in step (1), the copper salt is selected from any one of copper chloride, copper sulfate, copper nitrate, copper acetate, and copper phosphate, preferably copper chloride; The β-cyclodextrin analogue is selected from any one of α-cyclodextrin, γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin, and methyl-β-cyclodextrin; Further preferred, the molar ratio between copper chloride and β-cyclodextrin is 5:1.

[0017] Preferably, the copper salt is mixed with β-cyclodextrin or its analogue by stirring and adding a first proton polar solvent dropwise under stirring until the solution is clear; The proton polar solvent is selected from ethanol, methanol, isopropanol, n-butanol, ethylene glycol, and glycerol; wherein, the first proton polar solvent is preferably ethanol, and the second proton polar solvent is preferably methanol.

[0018] Furthermore, β-cyclodextrin (β-CD) can be dissolved in deionized water first, and anhydrous ethanol can be added dropwise under stirring until the solution is clear. Then, CuCl2 solution can be slowly added dropwise to the β-CD solution, and the reaction can be stirred at 40°C for 2 h.

[0019] The precipitate obtained was vacuum filtered, washed three times with methanol, and dried at 40~60℃ (preferably 50℃) to obtain a light blue solid Cu-MOF.

[0020] In a third aspect, the present invention provides a jellyfish repellent comprising the aforementioned icostigma or ICA@Cu-MOF complex, and an acceptable carrier or substrate. It can be a spray (applied directly to skin or clothing), a coating (applied to fishing nets, boat hulls, lifesaving equipment), or an impregnating agent (impregnating fabrics such as swimsuits, diving suits, and life jackets), etc.

[0021] In a fourth aspect, the present invention provides a jellyfish repellent product, comprising a carrier and a jellyfish repellent agent coated on the carrier, wherein the carrier may be a fabric or a fishing net, a boat hull, a life-saving device, or the like.

[0022] Compared with the prior art, the beneficial effects of this application are as follows: Through systematic behavioral screening, this invention demonstrates for the first time that icacridin has a significant repellent effect on jellyfish, filling a technological gap in the field of jellyfish protection. This discovery provides a novel active ingredient option for the development of jellyfish repellents. Combining icacridin with copper ions for jellyfish repellency achieves synergistic effects. It significantly improves the seawater solubility and release behavior of icacridin: by loading it onto Cu-MOF, the apparent solubility of icacridin increases by 2.08 times, facilitating rapid diffusion and effectiveness in seawater. It reduces the ecotoxicity risk of copper ions: the cumulative release rate of copper ions in the complex decreases from 93.83% to 71.42%, mitigating the potential impact on the marine environment. The repellent effect is significant; the highest repellency index of ICA@Cu-MOF reaches 100%, superior to physical mixtures (66.67%). The preparation process is simple: using a mild solvothermal method and solution loading method, the conditions are mild, the operation is simple, and it is easy to scale up for production. Attached Figure Description

[0023] Figure 1 A schematic diagram of the behavioral screening experimental process of the present invention is shown.

[0024] Figure 2The screening results of seven major candidate compounds are shown, indicating that icacridin (ICA) and copper salts have significant repellent activity, while DEET and others are ineffective. The changes in jellyfish spatial distribution and activity status over time under different treatment conditions are shown, including: (A) blank control; (B) ICA; (C) DEET; (D) CuCl2 (copper chloride); (E) CuSO4 (copper sulfate); (F) Cu(OAc)2 (copper acetate); (G) NaAc (sodium acetate); (H) LaCl3 (lanthanum chloride). Stacked bar charts represent the proportion of motile jellyfish distributed in the non-treated area (orange), central area (pink), and treated area (green) at each time point; while dashed lines represent the number of inactive jellyfish. Motile and inactive individuals were counted separately in the experiment, and the sum of the two equals the total number of jellyfish in each experiment. (I) The comparison of the number of jellyfish remaining in the treated area 3 minutes after treatment between the blank control group and the positive control group reflects its immediate repellent effect. Compared with the control group, *p <0.05, **p < 0.01.

[0025] Figure 3 Characterization results of Cu-MOF are shown: (A) Representative scanning electron microscope (SEM) image of Cu-MOF, showing its typical microstructure. Scale bar: 20 μm. (B) Energy dispersive X-ray spectroscopy (EDS) elemental surface scan of Cu-MOF, showing the uniform elemental distribution within the particles. (C) Powder X-ray diffraction (PXRD) patterns of Cu-MOF and a reference sample used to analyze crystal structure. (D) Fourier transform infrared (FTIR) spectra of β-cyclodextrin (β-CD) and Cu-MOF, showing their characteristic functional groups and coordination characteristics. (E) and (F) are thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) curves of β-CD and Cu-MOF, respectively, used to compare their thermal behavior.

[0026] Figure 4Characterization and comparison of solubility and release curves of ICA@Cu-MOF are shown: (A) Schematic diagram of ICA incorporation into Cu-MOF to form an integrated composite material. (B) Typical scanning electron microscope (SEM) image of ICA@Cu-MOF, showing its unique microstructure. Scale bar: 30 μm. (C) Fourier transform infrared (FTIR) spectra of ICA, Cu-MOF, and ICA@Cu-MOF. (D) Comparison of solubility of free ICA and ICA@Cu-MOF in distilled water and artificial seawater. (E) Cumulative release curves of ICA, ICA / Cu-MOF (physical mixture PM), and ICA@Cu-MOF in artificial seawater. (F) Copper (Cu) release curves of ICA@Cu-MOF and Cu-MOF in artificial seawater. Data are expressed as mean ± standard deviation (SD) (n = 3).

[0027] Figure 5 The graph shows a comparison of the repulsion index (RI) over time between ICA@Cu-MOF and the physical mixture: the spatial distribution and activity status of jellyfish under different treatment conditions, including (A) ICA@Cu-MOF and (B) ICA / Cu-MOF (physical mixture PM). Stacked bar charts represent the proportion of jellyfish distributed in the untreated area (orange), the central area (pink), and the treated area (green) at each observation time point; while dashed lines represent the number of jellyfish that have lost their ability to move. (C) Comparison of repulsion index over time between ICA@Cu-MOF and the physical mixture (PM). Data are expressed as mean ± standard deviation (SD) (n = 3). Statistical differences between the two groups at each time point were analyzed using two-way ANOVA and multiple comparisons, and are marked as follows: *p < 0.05, **p < 0.01, ***p <0.001, ****p < 0.0001, ns represents no significant difference.

[0028] Figure 6 The analysis of jellyfish movement trajectories is shown: Figure (A) displays the individual trajectories of five jellyfish, with the origin of the coordinate system set at the corner of the untreated area. The start and end points of each trajectory are marked with circles and arrows, respectively. Figure (F) shows the superimposed trajectories of all five jellyfish. It can be seen that the jellyfish have a wider range of activity and are more dispersed in the untreated and central areas; while in the treated area, their movement is significantly restricted, and their spatial distribution is more limited. This analysis is based on video recordings of an in vitro jellyfish repellency experiment. Detailed Implementation

[0029] To make the inventive objectives, technical solutions, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. It should be understood that the embodiments described are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this description.

[0030] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0031] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques from the fields of biomedical engineering, biophysics, pharmaceutics, pharmaceutical analysis, medicinal chemistry, analytical chemistry, molecular biology, biochemistry, and related areas. These techniques have been well described in existing literature.

[0032] The present invention is further illustrated below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. The processes, conditions, experimental methods, etc., for implementing the present invention, except as specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0033] I. Experimental Methods 1.1 Screening of jellyfish repellent active ingredients

[0034] The red moon jellyfish were purchased from Mengzhan (Shanghai) Information Technology Co., Ltd., and were reared in artificial seawater (salinity 25‰, temperature 20 ± 1 ℃) for 48 h before use in the experiment. The red moon jellyfish used in this study are invertebrates, and according to relevant ethical management regulations, no ethics committee approval was required. All experimental procedures were conducted in accordance with the principle of minimizing animal stress and harm. The experimental apparatus and methods used for the jellyfish repulsion experiment are described in [link to relevant documentation]. Figure 1Preliminary screening of different categories of candidate substances was conducted (Table 1). To systematically screen candidate substances with potential jellyfish repellent activity, a candidate substance screening strategy centered on behavioral evaluation was established, combining the ecological and behavioral characteristics of jellyfish and existing research experience on repellents. Chemical substances reported to have repellent or stimulating effects on other species were given priority as candidates. Repellents typically act on the sensory systems of animals, thereby inducing avoidance behavior, and are currently widely used in insects and other animals. Furthermore, there is often some overlap in the behavioral response mechanisms among different species; that is, a chemical substance that can repel one type of organism may also have similar behavioral effects on other species under certain conditions. Therefore, this study, referring to research experience on repellents for terrestrial and marine organisms, selected some common mosquito repellents, shark repellents, and related organic compounds as candidate substances to explore the potential role of repellents from other species in repelling jellyfish.

[0035] Based on preliminary literature review and analysis of existing market components, the following three types of compounds were selected as candidate active ingredients for repellents: (1) Metal salts: including copper acetate, copper sulfate, lanthanum chloride, etc., which are commonly used in marine antifouling, algae control and shark repellent products, and are speculated to have repellent or paralyzing effects on invertebrates; (2) Conventional mosquito repellent ingredients: including ICA, DEET, and natural source ingredients such as clove oil and menthol, which are commonly used as mosquito repellents. Therefore, considering that they may interfere with the sensory system, these substances are an important source for screening jellyfish repellents; (3) Other chemical stimulants: such as sodium dodecyl sulfate (SDS), benzyl isothiocyanate, dyes and nonsteroidal anti-inflammatory drugs, etc. These substances cover surfactants, irritant compounds and drug molecules, aiming to broadly explore the effects of different chemical signals on jellyfish movement behavior and avoid missing potential active ingredients.

[0036] 1.2. Synthesis of copper-cyclodextrin metal-organic frameworks (Cu-MOF)

[0037] Anhydrous copper chloride (CuCl2, 5 mmol) was dissolved in 5 mL of deionized water. Separately, β-cyclodextrin (β-CD, 1 mmol) was dissolved in 15 mL of deionized water. Anhydrous ethanol was added dropwise with stirring until the solution became clear. The CuCl2 solution was slowly added dropwise to the β-CD solution, and the mixture was stirred at 40 °C for 2 h. After the reaction was complete, methanol was added as an antisolvent to precipitate the solid. The precipitate was filtered under vacuum, washed three times with methanol, and dried at 50 °C to obtain a light blue solid, Cu-MOF.

[0038] 1.3. Preparation of ICA@Cu-MOF composite

[0039] Icaridin (ICA) was dissolved in anhydrous methanol, and Cu-MOF prepared in step 1.2 was added at a ratio of ICA:Cu-MOF=25:100 (w / w). The sealed system was magnetically stirred at 40℃ (350 rpm) for 2 h and dried at 50℃ to obtain ICA@Cu-MOF.

[0040] 1.4. Solubility Test

[0041] Excess analyte (ICA or ICA@Cu-MOF) was placed in 1 mL of pure water and artificial seawater, respectively. After thorough mixing, the mixture was placed in a constant-temperature shaking incubator at 25℃ and 150 rpm for 0.5 h to allow the system to reach dissolution equilibrium. The supernatant was then filtered through a 0.22 μm microporous membrane, and 0.5 mL of the filtrate was placed in a 10 mL volumetric flask and diluted to the mark with the mobile phase. The ICA content in the solution was determined by high-performance liquid chromatography (HPLC) to evaluate the apparent solubility of ICA and ICA@Cu-MOF in water (n = 3).

[0042] 1.5. Release Behavior Test

[0043] The in vitro release behavior of ICA@Cu-MOF was determined according to Method II (paddle method) of General Chapter 0931, Dissolution and Release Determination, Part IV, 2025 Edition of the Chinese Pharmacopoeia. To simulate the actual seawater environment, artificial seawater (salinity 25‰) was used as the release medium. Approximately 600 mg of ICA@Cu-MOF sample was accurately weighed and placed in 500 mL of ultrasonically degassed artificial seawater. The release experiment was conducted at 25°C and 75 rpm. Samples were taken at predetermined time intervals, and an equal volume of fresh medium at the same temperature was added to maintain a constant system volume. After sampling, the samples were processed accordingly, and ICA and Cu were determined by high-performance liquid chromatography (HPLC) and flame atomic absorption spectrometry (FAS), respectively. 2+ The release amount. To compare the release characteristics of the active ingredient in different systems, the ICA release experiment simultaneously included an ICA raw material group and an ICA / Cu-MOF physical mixture group. For Cu... 2+ The release experiment included a Cu-MOF group as a control, and comparisons were made under the same conditions. All experiments were performed in triplicate, and the cumulative release amount was calculated and release curves were plotted based on the measurement results.

[0044] 1.6. Evaluation of the repellent effect of ICA@Cu-MOF on venomous jellyfish

[0045] To systematically evaluate the repellency effect of the ICA@Cu-MOF composite system, this study set up an experimental group and a control group for comparative analysis. The experimental group was the ICA@Cu-MOF group, used to evaluate the repellency performance of ICA loaded with Cu-MOF. For each experiment, 4 g of ICA@Cu-MOF was weighed and evenly sprinkled in the drug addition area of ​​the water tank, so that ICA and Cu... 2+ A local concentration gradient is formed to exert a repellent effect. The control groups included the ICA / Cu-MOF (PM) group and a blank control group. The ICA / Cu-MOF (PM) group was prepared as follows: ICA raw material and blank Cu-MOF carrier were accurately weighed and placed in a centrifuge tube at the same mass ratio as ICA@Cu-MOF, vortexed for 5 min to ensure uniform dispersion, resulting in a physical mixture. This group was used to compare the repellent effect between the physical mixture system and the drug-loaded system, thereby analyzing the influence of carrier structure on the repellent effect. The blank control group was a treatment without any added repellent substance, used to observe the distribution behavior of jellyfish under natural conditions. Each treatment group had three parallel experiments (n=3). All experiments were conducted under the same environmental conditions and by the same operator to reduce human error. The experimental sequence and apparatus location were randomized to avoid the influence of time and space factors on the experimental results.

[0046] The repellency behavior experiment was conducted based on an established jellyfish repellency behavior evaluation model. During the experiment, a treatment zone was set up on one side of the repellency experimental apparatus, and the other side served as a control zone. The test samples were placed in the treatment zone. Subsequently, a certain number of jellyfish were placed in the central area of ​​the apparatus, and the baffle was removed to observe their movement behavior and spatial distribution changes within the experimental apparatus. The distribution of jellyfish in each zone was recorded within a specified time, and the repellency rate was calculated to quantitatively characterize the repellency effect under different treatment conditions. The repellency activity of the ICA@Cu-MOF system was comprehensively evaluated by comparing the differences between the ICA@Cu-MOF, ICA / Cu-MOF (PM) group, and the blank control group.

[0047] 1.7. Jellyfish Trajectory Analysis

[0048] The motion trajectories of jellyfish were extracted from behavioral measurement videos using the open-source motion analysis software Tracker (V6.3.2). The videos were filmed from a fixed side view to capture the jellyfish's two-dimensional motion within the vertical plane of the tank. During tracking, an automatic tracking module based on a template matching algorithm was used to track individual jellyfish, with the geometric center of the jellyfish's bell defined as a point mass. Five jellyfish were tracked independently under each experimental condition. To accommodate the periodic contraction and deformation of the jellyfish during swimming, the parameters of the automatic tracking were optimized and manually corrected as needed to ensure trajectory continuity. Furthermore, using the known tank length (50 cm) as a reference scale, pixel coordinates were converted to physical distances through spatial calibration. Finally, the horizontal (x-axis) and vertical (y-axis) position data over time were exported for subsequent trajectory visualization and qualitative comparison.

[0049] 1.8. Statistical Analysis

[0050] Statistical analyses were performed using GraphPad Prism version 6.02. For comparisons of three or more groups, one-way ANOVA was used, followed by multiple group comparisons. Statistically significant differences were defined as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. The abbreviation 'ns' indicates no significant difference between the relevant treatment groups.

[0051] II. Experimental Results 2.1. Repellent effects of different types of substances

[0052] Among the metal salts, the jellyfish in the copper acetate, copper sulfate, and copper chloride treatment groups showed significant spatial distribution changes during the experiment, namely, the number of jellyfish decreased in the treatment area and the number of jellyfish relatively increased in the non-treatment area. Figure 2 D-2F). In the lanthanum chloride treatment group, the distribution of jellyfish in each region did not change significantly ( Figure 2 H). In mosquito repellent formulations, the number of jellyfish in the ICA-treated group gradually decreased within the treatment area and migrated to the non-treatment area. Figure 2 B); DEET ( Figure 2 In the C), clove oil, and menthol treatment groups, the distribution of jellyfish varied little across regions. Among other categories of substances, sodium acetate ( Figure 2 In the treatment groups containing sodium dodecyl sulfate, anhydrous ethanol, active black KN-B, benzyl isothiocyanate, permethrin, diclofenac sodium, and magnesium chloride, the jellyfish did not show significant regional distribution changes during the experiment. See Table 1 for details. Table 1. Repellent effects of different candidate substances on jellyfish

[0053] (+): Repellent activity observed; (-): No repellent activity observed. The comparison of the number of jellyfish remaining in the treatment area 3 minutes after drug administration between the blank control group and the positive control group reflects its immediate repellency effect. Figure 2 I).

[0054] 2.2 Characterization of Cu-MOF and ICA@Cu-MOF Representative scanning electron microscope (SEM) images of Cu-MOFs show their typical microstructure. Figure 3 A); Energy dispersive X-ray spectroscopy (EDS) elemental surface scans of Cu-MOF show that the elemental distribution within the particles is uniform ( Figure 3 B). Characterization techniques, including PXRD, FTIR, TGA, and DSC, confirmed the successful construction of the Cu-MOF support, whose structure and physicochemical properties differed significantly from the original β-CD. Figure 3 The solvothermal method yields products with more uniform particle morphology, clearer crystal structure, and higher reaction efficiency, making it a superior synthesis method for Cu-MOF.

[0055] See the schematic diagram of the ICA@Cu-MOF composite system. Figure 4 A, SEM images reveal its unique microstructure ( Figure 4 B), FTIR detection results showed characteristic differences among ICA, Cu-MOF, and ICA@Cu-MOF ( Figure 4 C).

[0056] 2.3. Solubility Evaluation of ICA@Cu-MOF

[0057] After 0.5 h of equilibration, the solubility of ICA in water was measured to be 10.40 mg / mL, while the solubility of ICA@Cu-MOF under the same conditions was 18.99 mg / mL, approximately 1.83 times that of the original ICA. In the artificial seawater system, the solubility of ICA was 6.76 mg / mL, while the solubility of ICA@Cu-MOF increased to 14.06 mg / mL, approximately 2.08 times that of the original ICA. Figure 4 D). The results showed that the apparent solubility of ICA@Cu-MOF in both water and artificial seawater was improved compared with that of free ICA, indicating that Cu-MOF as a carrier can improve the dispersion of ICA in the aqueous system to a certain extent, thus exhibiting a certain solubilizing effect.

[0058] 2.4. Research on Release Behavior

[0059] To evaluate the release behavior of ICA in the ICA@Cu-MOF system, in vitro release experiments were conducted in artificial seawater (salinity 25‰, 25℃), and compared with ICA active pharmaceutical ingredient (API) and a physical mixture (PM) of ICA and Cu-MOF. All three systems exhibited a rapid release trend in the initial stage, with the release amount increasing rapidly within 0.5 h. In contrast, the ICA@Cu-MOF system showed a faster release rate throughout the entire process, reaching approximately 90% cumulative release at 0.5 h, then gradually stabilizing, and reaching near-complete release (approximately 99%) at 2 h. The release rate of ICA / Cu-MOF (PM) was slightly lower than that of the ICA@Cu-MOF system, with a cumulative release of approximately 90% at 2 h, while the release rate of the ICA API was relatively slower, with a final cumulative release of approximately 82%. The above results indicate that, compared with the active pharmaceutical ingredient (API), the ICA@Cu-MOF system can achieve faster ICA release in an artificial seawater environment. This may be related to the dispersion effect of the Cu-MOF carrier on ICA, which improves the diffusion efficiency of ICA in the release medium. Meanwhile, the release curve of the ICA@Cu-MOF system is relatively stable, indicating that the system can release most of the drug in a shorter time, providing a basis for its application in aquatic environments. Figure 4 E).

[0060] To investigate Cu 2+ Release behavior in artificial seawater was investigated using in vitro release experiments on ICA@Cu-MOF and Cu-MOF. Both materials exhibited a rapid release trend in the initial stage, with Cu showing a rapid release rate within a short period. 2+ The release rate increases rapidly, then gradually slows down and stabilizes after about 1 hour. Specifically, in the Cu-MOF system, Cu... 2+ The release rate is relatively fast, with a cumulative release of nearly 93% after 2 hours; while in the ICA@Cu-MOF system, Cu... 2+ The release is relatively small, with a cumulative release of approximately 71% over the same period of time. Figure 4 F). This result indicates that ICA loading enhances the effect of Cu on Cu in Cu-MOF. 2+ The release behavior had a certain impact, causing Cu 2+ The release rate of Cu was reduced. This may be because ICA molecules occupy part of the space in the material structure or interact with Cu-MOF, thus hindering the release of Cu to some extent. 2+ The release process. Overall, Cu 2 The system exhibited rapid release behavior in artificial seawater, and the loading of ICA had a certain regulatory effect on its release, providing a reference for further research on the behavior of this system in practical applications.

[0061] 2.5. Evaluation of Avoidance Effect

[0062] To compare the repellency effects of ICA@Cu-MOF and ICA / Cu-MOF (PM), the spatial distribution and repellency rate of jellyfish in the experimental setup were analyzed. Under ICA@Cu-MOF treatment conditions, jellyfish rapidly left the treatment area after the experiment began. Figure 5 A). As time progressed, the number of jellyfish in the treatment area continued to decrease and gradually stabilized at a low level. Simultaneously, the number of jellyfish that lost their mobility gradually increased over time, indicating that this group had a significant impact on jellyfish behavior. In contrast, in the ICA / Cu-MOF (PM) treatment group, the spatial distribution of jellyfish in the apparatus was relatively unstable, the number of individuals in the treatment area fluctuated less, and almost no loss of mobility in jellyfish was observed during the experiment. Figure 5 B). Further comparison of the avoidance index over time under the two treatment conditions showed that the avoidance index rapidly increased within 1 minute after ICA@Cu-MOF treatment and remained at a high level throughout the subsequent experiment. Figure 5 C); while the aversion index of the ICA / Cu-MOF (PM) group was generally lower and more volatile. Figure 5 D). The above results indicate that, compared with simple physical mixing systems, ICA@Cu-MOF composites exhibit stronger and more stable repellency effects.

[0063] 2.6. Extraction and Quantitative Analysis of Jellyfish Movement Trajectories

[0064] To reveal the effects of ICA@Cu-MOF on jellyfish behavior patterns more precisely and objectively, this study, based on the avoidance rate assessment, further extracted and quantified the motion trajectories in the experimentally recorded videos using computer vision, thereby providing more concrete behavioral evidence. The results are as follows: Figure 6 The analysis of jellyfish movement trajectories revealed distinctly different kinematic characteristics between the ICA@Cu-MOF treated group and the control group. The trajectories of the ICA@Cu-MOF treated jellyfish exhibited strong directional and spatial repulsion. A typical trajectory characteristic was the initial acceleration of movement in the jellyfish during the early stages of drug diffusion, possibly due to the initial Cu... 2+ At higher concentrations, the jellyfish's trajectories quickly turned away from the drug source and remained on the side furthest from the drug zone. Many trajectories exhibited a characteristic of initially approaching but then rapidly turning back, reflecting the jellyfish's detection and active avoidance behavior towards the repellent's diffusion front. In contrast, the jellyfish trajectories in the control group were relatively evenly and continuously distributed within the device.

[0065] To further analyze the impact of ICA@Cu-MOF on jellyfish behavior patterns, based on the aforementioned trajectory extraction and quantitative analysis, the jellyfish's movement trajectory within the experimental setup was visualized using two-dimensional coordinates. Figure 6 As observed in the AE (Anaerobic Observation) results, different individuals exhibited a relatively consistent movement trend under ICA@Cu-MOF treatment. After moving around near the initial experimental position for a period of time, the jellyfish gradually moved away from the drug administration area and eventually stopped at the far end of the device. Some individuals showed obvious turning behavior when approaching the boundary of the drug administration area, and then continued to swim away from the drug administration area. At the same time, in some tracks, the phenomenon of jellyfish gradually stopping movement (non-motile) after moving away from the drug administration area could be observed. The superimposed map of the movement trajectories of multiple individuals can more intuitively reflect the overall movement trend. The results show that most tracks are concentrated on the side away from the drug administration area, while the number of tracks in the area closer to the drug administration area is significantly reduced, indicating that jellyfish exhibit a significant spatial avoidance trend after ICA@Cu-MOF treatment. In summary, ICA@Cu-MOF treatment can significantly change the movement pattern of jellyfish in the experimental device, causing them to gradually move away from the drug administration area and gather in the opposite area. This result is consistent with the aforementioned avoidance rate statistics, further verifying from a kinetic perspective that ICA@Cu-MOF has a significant avoidance effect on jellyfish.

[0066] III. Results Analysis This study screened 16 candidate substances based on a previously constructed jellyfish repellency experimental setup and method, and the results showed that Cu... 2+ Both CuCl2 and ICA exhibited significant repellent activity and can serve as key active ingredients for the subsequent construction of composite repellent systems. Cu-MOFs were prepared from CuCl2 and β-CD using vapor diffusion and solvothermal methods, respectively. Characterization techniques including SEM, EDS, PXRD, FTIR, TGA, and DSC confirmed the successful construction of the Cu-MOF support, whose structure and physicochemical properties differed significantly from the original β-CD. Comparison of the two preparation methods revealed that the solvothermal method yielded products with more uniform particle morphology, clearer crystal structures, and higher reaction efficiency, making it a superior method for the synthesis of Cu-MOFs. An ICA@Cu-MOF composite system was constructed, and optimal drug loading conditions were determined. By loading ICA onto Cu-MOFs, the ICA@Cu-MOF composite system was successfully constructed.

[0067] Drug loading experiments showed that the feed ratio, loading time, and loading temperature all affected the drug loading performance of the system. Specifically, the system exhibited the best drug loading effect with an average loading rate of approximately 18.55% when the ICA:Cu-MOF feed ratio was 1:4 (w / w), the loading time was 120 min, and the loading temperature was 40℃. Characterization results further confirmed that ICA was successfully loaded into the Cu-MOF system. The ICA@Cu-MOF system improved the solubility of ICA and had a certain regulatory effect on the release behavior of the active ingredient. Solubility studies showed that the apparent solubility of ICA@Cu-MOF in water and artificial seawater was higher than that of the ICA raw material, suggesting that the Cu-MOF carrier can improve the dispersion and solubility of ICA in aqueous systems to a certain extent.

[0068] In vitro release experiments showed that ICA@Cu-MOF exhibited relatively stable release behavior in artificial seawater, with ICA release being relatively gradual. While increasing the ICA release rate, it also limited the release of Cu. 2+ The large-scale release indicates that the system has a certain regulatory effect on the release of the two active ingredients.

[0069] Behavioral evaluation results showed that after treatment with ICA@Cu-MOF, jellyfish were able to quickly move away from the drug administration area, and the repellency rate increased rapidly over time, reaching a maximum of 100%. In contrast, the repellency rate of the ICA / Cu-MOF (PM) group was lower and fluctuated more significantly. Further analysis of movement trajectories revealed that the movement behavior of jellyfish in the ICA@Cu-MOF treatment group changed from random swimming to directional movement away or decreased mobility, indicating that the constructed composite system can effectively induce stable avoidance behavior in jellyfish, demonstrating good repellency activity.

[0070] In summary, this study successfully constructed an ICA@Cu-MOF composite system, realizing Cu 2+ Effective integration with ICA. This system combines drug loading capacity, solubility improvement, and release regulation characteristics, and demonstrated good application effects in the evaluation of jellyfish repellency behavior. The results indicate that constructing an active ingredient delivery system based on a metal-organic framework is feasible for repelling toxic jellyfish, providing an experimental basis for the further design, preparation, and application evaluation of related repellent agents. Although this study has made some progress in jellyfish repellent screening, carrier construction, and repellency behavior evaluation, some issues still require further in-depth research. Future research can further optimize the material structure and drug loading method, systematically examine the release behavior and stability under different environmental conditions, and extend the application to more jellyfish species or actual seawater environments to verify the repellency effect, thereby further enhancing the application potential of this system in the fields of marine ecological security and jellyfish control.

[0071] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above specific embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. Application of icocaridine in the preparation of jellyfish repellent.

2. The application according to claim 1, characterized in that, The jellyfish repellent uses icocaridine as the sole active ingredient, or icocaridine is combined with other substances with jellyfish repellent activity as active components.

3. The application according to claim 2, characterized in that, The other substance with jellyfish-repelling activity is copper ions.

4. A jellyfish repellent composition, characterized in that, The system is an icocaredin-copper complex repellent system comprising icocaredin and copper ions, wherein the copper ions are provided in the form of a soluble copper salt or a copper-cyclodextrin metal-organic framework Cu-MOF; The copper-cyclodextrin metal-organic framework is formed by the coordination of copper ions and cyclodextrin, and the icocaridine is loaded in the framework to form an ICA@Cu-MOF complex.

5. The jellyfish repellent composition according to claim 4, characterized in that, The preparation method is as follows: (1) Synthesis of copper-cyclodextrin metal-organic framework: copper salt and cyclodextrin were mixed in deionized water at a molar ratio of 3 to 8:1, a first proton polar solvent was added, and the mixture was reacted at 30 to 60 °C for 1 to 4 h. A second proton polar solvent was added to precipitate the mixture, and the mixture was washed and dried to obtain Cu-MOF. (2) Preparation of ICA@Cu-MOF: Icaridin was dissolved in anhydrous methanol and mixed with Cu-MOF in a certain proportion. The mixture was then sealed and stirred at 30-50℃ for 1-3 h and dried to obtain the final product.

6. The jellyfish repellent composition according to claim 5, characterized in that: In step (1), the copper salt is selected from any one of copper chloride, copper sulfate, copper nitrate, copper acetate, and copper phosphate; The cyclodextrin is selected from any one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfonyl-β-cyclodextrin, and methyl-β-cyclodextrin; The copper salt and cyclodextrin were mixed by stirring, and the first proton polar solvent was added dropwise under stirring until the solution became clear. The first proton polar solvent and the second proton polar solvent are each selected from any one of ethanol, methanol, isopropanol, n-butanol, ethylene glycol, and glycerol; In step (2), the mass ratio between icostigmine and Cu-MOF is 25:100; Icaridin was mixed with Cu-MOF and then magnetically stirred at 350 rpm.

7. The jellyfish repellent composition according to claim 6, characterized in that, The copper salt is selected from copper chloride, the cyclodextrin is selected from β-cyclodextrin, the first proton polar solvent is selected from ethanol, the second proton polar solvent is selected from methanol, and the molar ratio between copper chloride and β-cyclodextrin is 5:

1.

8. A jellyfish repellent, characterized in that, The jellyfish repellent composition according to any one of claims 4 to 7 is used as the active component, and also includes excipients for preparing different product forms.

9. The jellyfish repellent according to claim 8, characterized in that, Selected from sprays, coatings, or impregnation agents.

10. A product with jellyfish-repelling function, characterized in that, Includes a carrier, the surface of which is coated with the jellyfish repellent of claim 8 or 9.

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