Application of copper sulfate in preparation of jellyfish stings protection product with jellyfish repelling and nematocyst launching inhibition dual functions

Abstract

The invention provides an application of copper sulfate in preparation of a jellyfish stings protection product with double functions of repelling jellyfish and inhibiting nematocyst emission. The result shows that the low-concentration copper sulfate can inhibit the emission of the nematocyst; and it is proved that the influence of copper sulfate on the jellyfish is acute and instant, the jellyfish can make a large reaction just when making contact with copper sulfate, and the repelling effect on the jellyfish is good. The invention provides a new scheme and thought for repelling the jellyfish and inhibiting the emission of the nematocyst.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to a new application of copper sulfate in jellyfish sting protection and jellyfish sting prevention. BACKGROUND

[0002] There are various marine organisms in China's sea area, among which there are more than thousands of toxic and harmful marine organisms. Marine organism injury and poisoning have become an important part of marine special environment medicine. Among them, jellyfish have a large number of species, wide distribution, large total biomass, strong toxin activity and great harm, and their stings have become the most common marine organism injury (Med Sci Monit 2024, 30:e944265). Generally, jellyfish sting causes local symptoms such as pain, whip mark redness or itching; when the toxin enters the blood, it may cause systemic symptoms such as nausea, vomiting or headache; and severe cases may even lead to death due to respiratory, circulatory and liver and kidney function failure. Current treatment of jellyfish stings mainly focuses on local treatment and systemic symptomatic treatment, and there is also a lack of scientific and effective preventive measures for jellyfish outbreaks and jellyfish stings. Due to the unknown composition and mechanism of jellyfish toxins, there is still a lack of specific antitoxic drugs for jellyfish stings. The Chironex fleckeri antivenom developed by Australia is the only commercialized jellyfish antivenom (J Venom Anim Toxins Incl Trop Dis 2014, 20: 34). However, for jellyfish and other toxic organisms with weak self-swimming ability, effective preventive measures are a better choice to prevent their harm. Therefore, the avoidance and sting protection of jellyfish have a better application prospect than antivenom.

[0003] Jellyfish belong to the phylum Cnidaria, and their tentacles are covered with stinging cells that can rapidly fire barbed nematocysts into the human or animal body and inject venom, causing stings. Nematocyst is the most important functional organelle in stinging cells, with good stability and few exogenous regulation limitations. Therefore, researchers often use nematocysts as experimental objects to study their structural properties and emission control mechanisms under laboratory conditions. Nematocysts remain active after being separated from the organism, so researchers have tried various methods to inhibit the emission of isolated nematocysts in an attempt to provide a theoretical basis for preventing jellyfish stings. However, due to the diversity of jellyfish nematocyst morphology, structure and function, the experimental results obtained in previous studies are not consistent, and even contradictory. For example, literature reports that vinegar can inhibit nematocyst emission, but other studies have shown that vinegar can stimulate nematocysts to further emit (Reg Environ Change 2023, 23: 114); the effect of urine on nematocyst emission rate is also not conclusive. Different species of jellyfish, different handling methods in different laboratories, and different evaluation systems for nematocyst emission have led to no consensus on effective active components for inhibiting nematocyst emission.

[0004] At present, some patents have reported on jellyfish sting protection products that inhibit the launch of nematocysts: For example, patent EP2363135 mentioned that La 3+ When mixed with plant extracts such as oats and tea, it has a good inhibitory effect on the emission of nematocysts; Patent EP2380577 mentions that the appropriate concentration of Zn 2+ and La 3+ The composition has a good inhibitory effect on the emission of nematocysts; Patent US6338837 describes the use of antihistamine diphenhydramine and metal cations such as Ca 2+ , K + , Mg 2+ etc. can effectively inhibit the emission of nematocysts of jellyfish; Zhang Zhi et al. (Shanghai: Second Military Medical University, 2010) determined that a mixed solution of 500 mmol / L LaCl3, 25 mmol / L MgCl2, and 25 mmol / L CaCl2 has the best inhibitory effect on the emission of nematocysts of the hair-bearing jellyfish.

[0005] In summary, while there has been considerable research on the screening of active substances for inhibiting nematocyst emission, relatively few have combined this with jellyfish repellency to validate the jellyfish repellency of these active substances. Therefore, based on a comprehensive review of these previous studies, the present invention screened and identified active substances that exhibit both jellyfish repellency and nematocyst emission inhibition, achieving both jellyfish repellency and sting protection. Summary of the Invention

[0006] The present invention addresses the above-mentioned challenges. A first objective is to provide a new application for copper sulfate, specifically its use in the preparation of a jellyfish sting protection product that has the dual functions of repelling jellyfish and inhibiting the release of nematocysts. A second objective is to provide a jellyfish sting protection product containing copper sulfate as an active ingredient.

[0007] The research process of this invention is as follows: First, substances that inhibit the activity of jellyfish nematocysts were screened. Sand jellyfish, the most common species in my country's waters, were selected as experimental subjects. Based on literature analysis and the research team's previous experimental results, active substances that affect nematocyst emission were collected and organized, and relevant chemical substances and drugs were selected, including magnesium chloride, lanthanum chloride, sulfide, isopropyl alcohol, urea, and copper sulfate. Then, equal amounts of fresh freeze-dried nematocysts were added to solutions of different active substances to form nematocyst suspensions. The inhibitory performance of the different substances was compared under the same pressure stimulation, and the results showed that copper sulfate had the best inhibitory effect. Next, the efficiency of copper sulfate in inhibiting nematocyst emission was evaluated. Equal amounts of pure nematocyst suspensions were mixed with copper sulfate solutions of different concentrations (0.1mM, 0.5mM, and 1mM). The results showed that the inhibitory effect was positively correlated with the copper sulfate concentration.

[0008] Second, the effect of copper sulfate on jellyfish avoidance was explored: the red moon jellyfish was used as the research object, which has autonomy in movement compared to other species of jellyfish, and can move autonomously without water flow assistance or when the water flow is small. Different concentrations of copper sulfate (50, 40, 30 mg / mL) were added to the jellyfish glass water tank containing uniform upward water flow, and then the number of jellyfish in each area was observed and counted for 5 minutes. The effective avoidance concentration of copper sulfate was determined by the percentage of the number of jellyfish in different distance areas from the slow-release drug, and finally the reduction percentage of jellyfish in the first two areas near the drug was used as the avoidance standard of the active substance. The results showed that there were no jellyfish in the drug-added area within the first 4 minutes, and the trend of the increase of the number of jellyfish that could not move was most obvious between 0-1 minutes, which proved that the jellyfish would react greatly when they first contacted copper sulfate, and the effect of copper sulfate on jellyfish was acute and instantaneous; the avoidance effect of jellyfish increased with the increase of the concentration of copper sulfate, and compared with high concentration, the avoidance effect decreased with the decrease of the concentration; when the effective concentration of copper sulfate pentahydrate in water was greater than 0.3 mg / mL, it had the effect of avoiding jellyfish.

[0009] Based on the above research, the specific technical solutions of the present application are as follows:

[0010] In a first aspect of the present application, the application of copper sulfate in the preparation of a jellyfish sting protection product is provided.

[0011] Preferably, the protection product has the dual functions of jellyfish avoidance and inhibition of nematocyst emission.

[0012] Further, copper sulfate is one of the active components in the protection product.

[0013] Further, the concentration (effective concentration) of copper sulfate released in water in the protection product is not less than 0.3 mg / mL. Considering safety and protection effect, it is preferably 0.3-0.5 mg / mL.

[0014] In a second aspect of the present application, a jellyfish sting protection product is provided, and the active component of the product is copper sulfate.

[0015] Preferably, the jellyfish sting protection product includes a coating and a portable jellyfish sting protection product. The coating is preferably a coating arranged on a wearable marine protection equipment or a net cage for aquaculture. The coating arranged on the wearable marine protection equipment can effectively avoid and inhibit the emission of nematocyst of jellyfish, thereby protecting the safety of marine workers; the coating on the net cage for aquaculture can effectively prevent jellyfish from climbing on the net cage, which not only prevents the weight of the net cage from increasing and the damage to the net cage, but also protects the marine breeders and the breeding products to a certain extent.

[0016] The portable jellyfish sting protection product is a water-soluble capsule, a solution preparation or a spray, and the concentration of copper sulfate in the preparation is preferably 30-50 mg / mL. When performing activities in water where jellyfish may appear, the capsule or solution preparation can be placed in the water in advance, or a certain dose of the spray can be sprayed on the water surface to form a repellent area in a small area of water around the body to prevent jellyfish from approaching and protect the body from danger in a short time.

[0017] Effects of the application

[0018] The application provides application of copper sulfate in preparation of a jellyfish sting protection product with double functions of jellyfish repellence and inhibition of discharge of nematocysts. Results show that low-concentration copper sulfate can inhibit discharge of nematocysts; and it is proved that the influence of copper sulfate on jellyfish is acute and instantaneous, and jellyfish will make a great response when just contacting copper sulfate, and the repellence effect on jellyfish is better. The application provides a new scheme and idea for jellyfish repellence and inhibition of discharge of nematocysts. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The jellyfish repellence effects of different concentrations of copper sulfate are shown: A, standard curve of copper sulfate pentahydrate; B, 50 mg / mL copper sulfate treatment group; C, 40 mg / mL copper sulfate treatment group; D, 30 mg / mL copper sulfate treatment group; E, 25 mg / mL copper sulfate treatment group; F, actual distribution of jellyfish in the model (0 min, 1 min, 2 min) of the 50 mg / mL copper sulfate treatment group (n=3). DETAILED DESCRIPTION

[0020] The application will be described in detail below with reference to the embodiments and drawings. However, the following embodiments should not be regarded as limiting the scope of the application.

[0021] Example 1: Inhibition of discharge of nematocysts of jellyfish by copper sulfate

[0022] I. Screening of active substances for inhibiting discharge of nematocysts of jellyfish

[0023] Active substances affecting discharge of nematocysts are collected and sorted, and related chemical substances and drugs are selected, including magnesium chloride, lanthanum chloride, sulfide, isopropyl alcohol, urea and copper sulfate.

[0024] Different kinds of intervention active substance solutions are prepared, and equal amounts of fresh freeze-dried nematocysts are taken, and the nematocyst suspension is prepared after adding the intervention active substance solution. 20 μL of the nematocyst suspension is taken and placed on a glass slide, and the cover glass is slowly covered and given pressure stimulation, and then the total number and the number of discharged nematocysts are counted. The discharge rate of the jellyfish nematocyst suspension is calculated according to the formula: nematocyst discharge rate = discharged nematocyst / nematocyst total number x 100%.

[0025] The results showed that only copper sulfate had the effect of inhibiting the emission of nematocysts.

[0026] 2. Evaluation of the efficiency of copper sulfate in inhibiting nematocyst emission

[0027] 1. Preparation of Nematocysts: Frozen jellyfish tentacles were autolyzed by adding an equal volume of high-magnesium artificial seawater to the sample at 4°C for 4 days. The sample was sieved twice, and the filtrate was centrifuged at 2000 × g for 3 min at 4°C. The pellet was reconstituted with artificial seawater. Different concentration gradients of Percoll solutions were prepared using high-magnesium artificial seawater. Tubes were stacked in descending order of concentration. The reconstituted sample was mixed with 60% Percoll in a 1:1 ratio and layered on top of the medium. The solution was centrifuged at 1000 × g for 20 min at 4°C in a swing-out rotor. Cellular debris and ejected nematocysts were removed from the upper portion based on the color and turbidity of the separation system. The remaining nematocyst Percoll suspension was centrifuged at 10,000 × g for 20 min at 4°C, washed once with high-magnesium artificial seawater, and centrifuged at 10,000 × g for 20 min at 4°C. The pellet represented the unfired nematocysts of the jellyfish.

[0028] 2. Identification of Nematocyst Emissivity: Prepare a nematocyst suspension by adding an appropriate amount of freeze-dried nematocysts to an appropriate amount of artificial seawater. Pipette 50 μL of the nematocyst suspension and mix thoroughly with 50 μL of copper sulfate solution of varying concentrations (0.1 mM, 0.5 mM, and 1 mM) in an EP tube. Incubate at 4°C for 30 min. Pipette 10 μL of the mixed solution onto a glass slide, cover with a coverslip, and remove excess artificial seawater. Observe and count the nematocysts under a standard light microscope. Place a 150g weight on the coverslip to apply pressure to the nematocysts, and observe and count them under a standard light microscope. Count three slides for each solution, counting the total number of nematocysts and the number of nematocysts emitted. An equal amount of nematocysts was added to artificial seawater as a control for analysis. Calculate the emissivity of the jellyfish nematocyst suspension using the following formula: Nematocyst Emissivity = Emitted Nematocysts / Total Nematocysts × 100%. The nematocyst emission rate of the model group was used as the benchmark (100%) to adjust the nematocyst emission rate of the experimental group. The results are shown in Table 1 below:

[0029] Table 1 Emission suppression efficiency of copper sulfate nematocysts

[0030]

[0031] From the results in Table 1, it can be seen that compared with the model group, copper sulfate intervention can significantly reduce the emission of nematocysts in a dose-dependent manner.

[0032] Example 2 Copper sulfate can effectively repel jellyfish

[0033] 1. Establishment of jellyfish repellent model

[0034] 1. Jellyfish breeding: The jellyfish culture tank used in the jellyfish repellent model is a glass tank with a length of 1 m, a width of 30 cm, and a height of 30 cm. A hole is left every 20 cm at the bottom of the tank to connect the rubber pipe of the water pump. The power of the water pump is adjusted to 0.5 L / min to provide a uniform upward flow of water in the tank, ensuring that the jellyfish can move freely and preventing them from sinking to the bottom. According to the interval of the two water pumps, 1-5 activity areas are divided. To inject seawater into the tank and add 50 small jellyfish with a size of 2 cm, observe their random and uniform distribution in the water and whether they will gather to the left or right without being affected by the water flow, and determine the objective random distribution of the jellyfish.

[0035] 2. Effective jellyfish repellent concentration: Different concentrations of active substances to be tested are placed on the tank wall at the far right end of the water body, and then the number of jellyfish distributed in each area is observed and counted for 5 minutes. The effective repellent concentration of the active substance is determined according to the percentage of the number of jellyfish in different distance areas from the active substance, and finally the reduction percentage of jellyfish in the first two areas near the drug is taken as the repellent standard of the drug.

[0036] II. Determination of the jellyfish repellent effect of copper sulfate

[0037] Seawater is injected into the tank and 50 small jellyfish with a size of 2 cm are added, and their random and uniform distribution in the water is observed without being affected by the water flow, and whether they will gather to the left or right is determined, and the objective random distribution of the jellyfish is determined. Different concentrations of copper sulfate (50, 40, 30 mg / mL) are placed on the tank wall at the far right end of the water body, and then the number of jellyfish distributed in each area is observed and counted for 5 minutes.

[0038] The repellent effect of copper sulfate on jellyfish is determined according to the percentage of the number of jellyfish in different distance areas from the copper sulfate, and finally the reduction percentage of jellyfish in the first two areas near the copper sulfate is taken as the repellent effect of copper sulfate.

[0039] The results are shown in Figure 1 As shown in the figure, copper sulfate is an effective jellyfish repellent active substance ( Figure 1 A); After treatment with 50 mg / mL of copper sulfate pentahydrate, no jellyfish were active in the drug-added area within the first 4 minutes, while the number of jellyfish that lost the ability to move increased with time, with the most obvious trend between 0-1 minutes, indicating that jellyfish will react greatly when they first come into contact with copper sulfate ( Figure 1 B); The distribution of active jellyfish in the 40 mg / mL copper sulfate treatment group was below 25% all the time. Similarly, the trend of jellyfish losing the ability to move was most obvious between 0-1 minutes, so it can be inferred that the effect of copper sulfate on jellyfish is acute and instantaneous ( Figure 1 C); Figure 1D is the distribution rate statistics of 30 mg / mL copper sulfate treatment group, Figure 1 E is the 25 mg / mL copper sulfate treatment group, compared with high concentration, the concentration is reduced, the repellent effect is reduced. The effective concentration of 50 mg / mL copper sulfate pentahydrate in water is 0.5 mg / mL.

[0040] In summary, the screened copper sulfate has the dual functions of jellyfish repellent and nematocyst inhibition, and can be used as a high-efficiency active substance to prepare jellyfish sting protection products.

[0041] The basic principles, main features and significant advantages of the present application have been described in detail. The purpose of the description in the examples and the specification is only to further illustrate the operation principle of the present application. Without violating the core idea and the legal protection range of the present application, the present application allows and covers all kinds of reasonable changes and improvements. These changes and improvements will be considered as belonging to the scope of protection requested by the present application. Specifically, the protection scope of the present application will be clearly and strictly defined by the appended claims and their equivalent legal effects.

Claims

1. Application of copper sulfate in the preparation of jellyfish sting protection products.

2. The use according to claim 1, characterized in that The protective product has the dual functions of repelling jellyfish and inhibiting the emission of nematocysts.

3. The use according to claim 2, characterized in that The concentration of copper sulfate released into water by the protective product is not less than 0.3 mg / mL.

4. A jellyfish sting protection product, characterized in that: Its active ingredients include copper sulfate.

5. The jellyfish sting protection product according to claim 4, characterized in that: The jellyfish sting protection product includes a coating and a portable jellyfish sting protection product.

6. The jellyfish sting protection product according to claim 5, characterized in that: The coating is a coating provided on wearable marine protective equipment or an aquaculture cage.

7. The jellyfish sting protection product according to claim 8, characterized in that: The portable jellyfish sting protection product is a water-soluble capsule, solution preparation or spray.

8. The jellyfish sting protection product according to claim 7, characterized in that: The concentration of copper sulfate in portable jellyfish sting protection products shall not be less than 30 mg / mL.

9. The jellyfish sting protection product according to claim 8, characterized in that: The concentration of copper sulfate in portable jellyfish sting protection products is 30~50mg / mL.

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