Use of epigallocatechin gallate in preparation of medicine for preventing and treating ichthyophthiriasis
By extracting epicatechin gallate (ECG) from *Sargentodoxa cuneata*, the problems of high toxicity and environmental pollution of existing drugs have been solved, achieving highly efficient killing of Ichthyophthirius multifiliis and safe prevention and control of fish diseases, providing a safe, effective, and green drug solution.
Patent Information
- Application Number
- CN202310270335.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-03-20
- Publication Date
- 2025-11-18
- Estimated Expiration
- 2043-03-20
AI Technical Summary
Existing chemical drugs used to control Ichthyophthirius multifiliis disease in fish have problems such as high toxicity, high residue, and environmental pollution, and there is a lack of safe, effective, and green alternative drugs.
epicatechin gallate (ECG), isolated and purified from *Sargentodoxa cuneata*, was used to verify its killing and control effects on *Ichthyophthirius multifiliis* through in vitro and in vivo experiments. It was found that ECG has a killing effect on predators, cyst precursors and cysts of *Ichthyophthirius multifiliis*, and is safe and non-toxic to fish at low concentrations.
ECG can rapidly and efficiently kill Ichthyophthirius multifiliis at low doses. In vitro tests show that the EC50 is 0.14-5.78 mg/L, and in vivo tests show that a concentration of 2-4 mg/L can cure Ichthyophthirius multifiliis in goldfish. It is also safe and non-toxic to fish, and has high safety.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of fishery disease prevention and control technology, and specifically relates to the application of epicatechin gallate in the preparation of drugs for the prevention and treatment of Ichthyophthirius multifiliis disease in fish. Background Technology
[0002] Ichthyophthirius multifiliis Fouquet (1876) (hereinafter referred to as Ichthyophthirius) is a pathogenic protozoan that parasitizes the body surface and gills of various freshwater fish, causing Ichthyophthiriasis and resulting in huge economic losses to the global aquaculture industry. Malachite green and mercury salt preparations, which are effective compounds for controlling Ichthyophthiriasis, have been banned due to their carcinogenic, teratogenic, and mutagenic properties. Other chemical substances, such as formalin and copper sulfate, also face problems such as high residues, high toxicity, and environmental pollution. Therefore, developing safe, effective, and green alternative drugs is imperative.
[0003] Traditional Chinese medicine (TCM) herbs, characterized by low toxicity to non-target organisms, easy degradation, and environmental friendliness, are an important source for new drug development. Existing studies have found that chili-ginger mixtures, garlic, and magnolia bark extracts exhibit significant inhibitory activity against ichthyophthirius multifiliis (white spot disease) in its isolated stage. Furthermore, monomeric compounds purified from crude extracts, such as 10-gingerol and emodin, have shown stronger insecticidal activity and efficacy in controlling Ichthyophthirius multifiliis in vivo. However, most of these compounds have complex structures, are difficult to synthesize, and are highly toxic, currently remaining in the laboratory research stage. Therefore, further research and development of TCM herbs for the control of Ichthyophthirius multifiliis is needed, along with the discovery and screening of more insecticidal compounds from plant resources, and the identification of more lead compounds with excellent drug-like properties, simple chemical structures, and ease of artificial synthesis and modification, to promote the development of TCM herbs for the control of Ichthyophthirius multifiliis.
[0004] In the early stages of the experiment, we found that the alcoholic extract of *Sargentodoxa cuneata* exhibited significant insecticidal activity, with a median effective concentration (EC50) against *Ichthyophthirius multifiliis* predators. 50 The concentration was only 0.1 mg / L, compared to previously reported plant extracts. 50 The value is lower and it is relatively safe for the host goldfish (LC). 50 The concentration was 70.3 mg / L, indicating its potential for development into a novel anti-Ichthyophthirius multifiliis drug. Furthermore, guided by activity tracking and combined with various chromatographic and spectroscopic methods, the insecticidal compound epicatechin gallate (ECG) was isolated and identified from the alcoholic extract of *Sargentodoxa cuneata*, with the following structural formula:
[0005]
[0006] epicatechin gallate, with the molecular formula C0 22 H18 O 10 The compound, with a molecular weight of 442, is a white powder, readily soluble in water and organic solvents such as ethanol. Literature review revealed that ECG can effectively inhibit grouper iridovirus infection. Considering the significant differences between viruses and parasites in terms of structure, reproduction, and invasion, we believe that using ECG to prepare drugs for the prevention and treatment of ichthyophthirius multifiliis disease in fish has broad application prospects. Summary of the Invention
[0007] The purpose of this invention is to provide the application of epicatechin gallate in killing Ichthyophthirius multifiliis and in the preparation of drugs for the prevention and treatment of Ichthyophthirius multifiliis disease in fish.
[0008] The objective of this invention is achieved through the following solution:
[0009] The insecticidal activity of different concentrations of ECG against different life stages of *Ichthyophthirius multifiliis* was investigated by culturing *Ichthyophthirius multifiliis* in vitro. The results showed that ECG had a killing effect on the predator, cyst precursor, and cyst of *Ichthyophthirius multifiliis*, with a dose-response relationship, and could kill up to 100% of *Ichthyophthirius multifiliis*. Specifically, ECG had a significant effect on the ECG of the predator *Ichthyophthirius multifiliis*. 50 The median lethal concentration (LD50) was 0.14 mg / L, with a 95% confidence interval of 0.13–0.15 mg / L; for the EC50 precursor of the cyst... 50 The concentration was 0.52 mg / L, with a 95% confidence interval of 0.46–0.60 mg / L; for encapsulated EC... 50 The concentration was 5.78 mg / L, with a 95% confidence interval of 4.38–7.65 mg / L. EC 50 It is an important parameter for guiding the dosage of insecticides. This invention can kill insects faster and more efficiently with a lower dosage.
[0010] Next, the acute toxicity of ECG to aquatic fish was investigated. The results showed that the LC50 of ECG on goldfish was [not specified]. 50 The concentration was 63.66 mg / L, with a 95% confidence interval of 60.93-66.38 mg / L, which is 6.3-212.3 times the effective in vitro insecticidal concentration (0.3-10.0 mg / L). This demonstrates that ECG is safe and non-toxic to fish within its effective insecticidal range.
[0011] Further investigation was conducted into the in vivo efficacy of ECG in preventing Ichthyophthirius multifiliis (white spot disease). Results showed that when the ECG concentration was ≥2 mg / L, after 12 days of treatment in a mixed-species system of diseased and healthy goldfish, the infection rate and intensity of all goldfish were 0. Therefore, treatment with 2 mg / L for 12 days could cure Ichthyophthirius multifiliis in goldfish and protect healthy goldfish from infection. The survival rates of healthy and diseased goldfish were 96.7% and 86.7%, respectively, significantly higher than the untreated group. However, at a concentration of 8 mg / L, the survival rate of diseased goldfish was only 20%, a significant decrease compared to the healthy group. Therefore, the recommended concentration (final drug concentration) for treating Ichthyophthirius multifiliis in goldfish is 2-4 mg / L of aquaculture water.
[0012] Further experiments revealed that, at the same dosage concentration, other catechin compounds besides ECG (such as epicatechin and catechin) were ineffective against Ichthyophthirius multifiliis, indicating that catechin compounds exhibit a certain degree of selectivity in their efficacy against Ichthyophthirius multifiliis. Meanwhile, ECG showed low insecticidal activity against other common freshwater fish parasites, with a mortality rate of only 13.6% against Tetrahymena, and was ineffective against Trichodina, Cyclostome, and Dactylogyrus, suggesting that ECG has a certain degree of specificity in its efficacy against freshwater fish parasites.
[0013] In summary, ECG can be used for in vitro insecticidal treatment of Ichthyophthirius multifiliis and for in vivo prevention and treatment of Ichthyophthirius multifiliis in freshwater fish. When used in vivo or in vitro, ECG can be used alone as an active ingredient in combination with a drug carrier to prepare insecticides, or it can be combined with other active ingredients in combination with a drug carrier to prepare insecticides.
[0014] The beneficial effects of this invention are:
[0015] (1) This invention is the first to apply ECG to kill Ichthyophthirius multifiliis and control Ichthyophthirius multifiliis disease, and can achieve 100% killing of insects within the effective dosage range.
[0016] (2) The ECG of the present invention is obtained by separating and purifying the natural plant Sargentodoxa cuneata. It is non-toxic and harmless to humans and animals and has a high safety profile. Detailed Implementation
[0017] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.
[0018] The materials used in the following examples were as follows: The host model was goldfish, with a total length of 7.1-10.0 (8.2±0.7) cm, a body length of 4.6-5.8 (5.1±0.3) cm, and a weight of 3.1-7.0 (4.7±1.0) g, purchased from an aquaculture farm in Shanghai. The parasite model was *Ichthyophthirius multifiliis*, obtained from the Hubei Provincial Collaborative Innovation Center for Freshwater Aquaculture Health at Huazhong Agricultural University. Epicatechin gallate was extracted in the inventor's laboratory.
[0019] Extraction process of epicatechin gallate:
[0020] The stems of *Sargentodoxa cuneata* were dried to constant weight in an oven at 55℃, then pulverized and filtered through a 60-mesh sieve to obtain 10 kg of powder. 10 kg of powder was extracted twice with 100 L of anhydrous ethanol at a volume ratio of 1:10 (g:mL) in a 45℃ water bath, each extraction lasting 2 hours. The extracts were combined and filtered to obtain a filtrate containing residual powder. The filtrate was concentrated under reduced pressure in a rotary evaporator at 55℃. The concentrated product was dried in an oven at 55℃ to remove residual ethanol until constant weight was obtained, yielding 1.08 kg of *Sargentodoxa cuneata* extract. 1 kg of the extract was suspended in 11 L of distilled water and extracted sequentially with petroleum ether (4 × 11 L), ethyl acetate (6 × 11 L), and n-butanol (6 × 11 L). The n-butanol extract (230 g) of the active fraction was subjected to primary silica gel column chromatography with gradient elution using ethyl acetate:methanol (1:0, 4:1, 2:1, 1:1, 1:2, 1:4, 0:1) as the elution system. The ethyl acetate:methanol = 2:1 extract (14.3 g) of the active fraction was subjected to secondary silica gel column chromatography with gradient elution using ethyl acetate:methanol (6:1, 4:1, 3:1, 2:1, 1:1, 0:1) as the elution system. The ethyl acetate:methanol = 2:1 extract (2.12 g) of the active fraction was subjected to semi-preparative high-performance liquid chromatography (mobile phase A: acetonitrile, mobile phase B: 0.2% aqueous acetic acid; gradient elution: 0-15 min: 15% A; 15-20 min: 15%-20% A; 20-30 min: 20% A; 30-35 min: 20%-25% A). R (min) = 25.8 min, and finally epicatechin gallate monomer was obtained.
[0021] Example 1: Killing effect of epicatechin gallate on infecting Ichthyophthirius multifiliis larvae (predators)
[0022] (1) Preparation of the drug: Accurately weigh 0.2 g of epicatechin gallate, dissolve it in 10.0 mL of dimethyl sulfoxide to prepare a 20 mg / mL solution, and then dilute it to the concentration required for the test. Using the content of dimethyl sulfoxide (0.02%) in the highest concentration solution used in the test as the standard, prepare a control treatment solution containing 0.02% dimethyl sulfoxide without the compound.
[0023] (2) Add 100 μL of a solution containing approximately 300 predators to each well of a 96-well plate, followed by 100 μL of drug solutions of different concentrations, making the drug concentrations 0.5, 0.4, 0.3, 0.2, 0.1, and 0.05 mg / L. Groups with only distilled water and groups with only 0.02% dimethyl sulfoxide were set as negative controls. Each group was tested in triplicate. The experimental temperature was 24 ± 0.5℃. The 100% lethal time of predators in different treatment groups was recorded. After 4 hours, the number of surviving predators in each well was counted, and the predator mortality rate and median effective concentration (EC50) were calculated. 50 The calculation formula is as follows:
[0024] Mortality rate (%) = (1 - number of surviving predators in the experimental group / number of predators before drug administration) × 100%
[0025] The results showed that the killing effect of epicatechin gallate on Ichthyophthirius multifiliis (white spot disease) predators was dose-dependent; the higher the concentration, the shorter the lethal time. At a concentration of 0.3 mg / L, 100% of the predators were killed within 30.6 minutes; while at 0.2 mg / L, the lethality of epicatechin gallate against the predators was as high as 91.9% within 4 hours. The EC50 of epicatechin gallate against Ichthyophthirius multifiliis predators was calculated using SPSS 23.0. 50 The concentration was 0.14 mg / L (95% confidence interval: 0.13–0.15 mg / L).
[0026] Table 1. Killing effect of catechin gallate on Ichthyophthirius multifiliis predator.
[0027] Concentration (mg / L) Lethal time (min) mortality rate(%) 0.5 10.2±0.4 100.0±0.0 0.4 18.6±1.4 100.0±0.0 0.3 30.6±0.4 100.0±0.0 0.2 ﹣ 91.9±5.9 0.1 ﹣ 18.3±8.7 0.05 ﹣ 5.0±4.4 <![CDATA[0(H2O)]]> ﹣ 0.0±0.0 0 (0.02% DMSO) ﹣ 0.0±0.0
[0028] Example 2: Killing effect of epicatechin gallate on adult Ichthyophthirius multifiliis (cyst precursor)
[0029] (1) The drug preparation is the same as in Example 1.
[0030] (2) Add 1 mL of insect solution containing approximately 30 cyst precursors to each well of a 12-well plate and accurately count them. Then add 1 mL of drug solution of different concentrations to make the drug concentrations 2.0, 1.0, 0.5, 0.25, 0.13, and 0.06 mg / L, respectively. The groups with only distilled water and those with only 0.05% dimethyl sulfoxide were set as negative controls. Each group had three replicates. After incubating in a 24℃ incubator for 6 hours, record the number of cysts in each treatment group. Then place the 12-well plate in a 24℃ incubator and allow it to hatch for 12 hours. Count the number of predators in each well and calculate the average number of predators hatched per cyst in each treatment group. The calculation formula is as follows:
[0031] Mortality rate (%) = (Number of dead cyst precursors in the experimental group / Number of cyst precursors before drug administration) × 100%
[0032] Average number of predators hatched per cyst = Total number of predators per pore / Number of cysts per pore
[0033] The results showed that when the concentration of epicatechin gallate was greater than 2.0 mg / L, it could kill 100% of the cyst precursors within 6 hours; when the concentrations were 1.0 mg / L and 0.5 mg / L, the lethality rates against the cyst precursors were 92.2% and 56.7%, respectively, and the number of predators released after the surviving cyst precursors formed cysts was significantly different from that of the control group (p < 0.01). The EC50 of epicatechin gallate against Ichthyophthirius multifiliis cyst precursors was calculated using SPSS 23.0. 50 The value was 0.52 mg / L (95% confidence interval: 0.46–0.60 mg / L).
[0034] Table 2. Killing effect of catechin gallate on Ichthyophthirius multifiliis cyst precursor.
[0035] Concentration (mg / L) mortality rate(%) Number of hatches 2.0 100.0±0.0 0.0±0.0 1.0 92.2±6.9 113.3±98.7 0.5 56.7±3.3 183.5±20.9 0.25 21.1±10.2 318.7±117.2 0.13 4.4±1.9 387.0±118.5 0.06 2.2±1.9 468.8±71.5 <![CDATA[0(H2O)]]> 0.0±0.0 454.2±61.3 0 (0.05% DMSO) 0.0±0.0 439.1±31.1
[0036] Example 3: Killing effect of epicatechin gallate on Ichthyophthirius multifiliis cysts
[0037] (1) The drug preparation is the same as in Example 1.
[0038] (2) Add 1 mL of insect solution containing approximately 30 cyst precursors to each well of a 12-well plate and accurately count them. Incubate the 12-well plate in a 24℃ incubator for 4 hours. After the cyst precursors develop into cysts, accurately count the number of cysts in each well. Then add 1 mL of drug solution of different concentrations to make the final drug concentrations 10.0, 8.0, 6.0, 4.0, 2.0, 1.0, 0.5, and 0.25 mg / L, respectively. The groups with only distilled water and those with only 0.05% dimethyl sulfoxide were set as negative controls. Each group was set up in triplicate. Incubate the 12-well plate in a 24℃ incubator for 12 hours, record the mortality rate of cysts in each treatment group, count the number of predators in each well, and calculate the average number of predators hatched per cyst in each treatment group. The calculation formula is as follows:
[0039] Mortality rate (%) = (Number of dead cysts in the experimental group / Number of cysts formed in the experimental group) × 100%
[0040] Average number of predators hatched per cyst = Total number of predators per pore / Number of cysts per pore
[0041] The results showed that epicatechin gallate concentration of 10.0 mg / L completely inhibited cyst hatching; while at 2.0 mg / L it could not kill 100% of the cysts, it significantly inhibited cyst reproduction compared to the control group (p < 0.01). The EC50 of epicatechin gallate against Ichthyophthirius multifiliis cysts was calculated using SPSS 23.0. 50 The concentration was 5.78 mg / L (95% confidence interval: 4.38–7.65 mg / L).
[0042] Table 3. Killing effect of catechin gallate on Ichthyophthirius multifiliis cysts.
[0043] Concentration (mg / L) mortality rate(%) Number of hatches 10.0 100.0±0.0 0.0±0.0 8.0 69.7±6.1 9.1±4.3 6.0 44.5±6.8 16.4±9.1 4.0 34.8±3.5 15.3±9.4 2.0 27.9±9.1 29.5±5.2 1.0 0.0±0.0 366.1±23.6 0.5 0.0±0.0 335.1±20.6 <![CDATA[0(H2O)]]> 0.0±0.0 357.5±23.8 0 (0.05% DMSO) 0.0±0.0 366.7±26.5
[0044] Example 4: Acute toxicity test of epicatechin gallate on goldfish
[0045] (1) Preparation of the medicine solution: The method is the same as in Example 1.
[0046] (2) Preliminary experiment: The concentration of epicatechin gallate that caused all goldfish to die within 24 hours was 90 mg / L, and the concentration that did not cause death within 96 hours was 40 mg / L.
[0047] (3) 5L of aerated tap water and 10 goldfish were added to each of 24 aquariums. The experimental temperature was maintained at 23±0.5℃, and oxygen was supplied by an oxygen pump. Based on the preliminary experiment, six concentration groups of 90, 80, 70, 60, 50, and 40 mg / L were set up. Groups with only distilled water and groups with only 0.1% dimethyl sulfoxide were set as negative controls. Each concentration was replicated in triplicate. During the experiment, the poisoning status of the fish at each concentration was observed continuously. If any goldfish died from poisoning, they were removed promptly to avoid water quality contamination and affecting the experimental results. The experimental results were statistically analyzed after 96 hours, and the median lethal concentration (LC50) was calculated. 50 The results are shown in Table 4, which calculate the 95% confidence interval (using the SPSS software probit program).
[0048] Table 4. Acute toxicity of catechin gallate to goldfish
[0049]
[0050] The acute toxicity test results of epicatechin gallate on goldfish showed that epicatechin gallate had a 96-hour LC50 value for goldfish. 50 The concentration was 63.66 mg / L (95% confidence interval: 60.93-66.38 mg / L), which is 6.3-212.3 times the effective in vitro insecticidal concentration (0.3-10.0 mg / L). This indicates that epicatechin gallate is safe for fish within its effective insecticidal range.
[0051] Example 5: The preventive and therapeutic effects of epicatechin gallate on Ichthyophthirius multifiliis (white spot disease).
[0052] (1) Preparation of the medicine solution: The method is the same as in Example 1.
[0053] (2) Healthy goldfish were quantitatively infected with predator at a ratio of 10000:1 and cultured at 25℃ for 4-5 days to obtain 75 goldfish severely infected with Ichthyophthirius multifiliis (502.5±41.2 white spots per fish). These goldfish were randomly divided into 15 rearing tanks (32cm×22cm×19cm) containing 5L of aerated water, with 5 fish per tank. 150 healthy goldfish were randomly selected and randomly divided into various rearing tanks, with 10 healthy fish per tank, and kept together with the infected goldfish. A porous partition (0.6cm×0.6cm mesh) was used to separate the infected and healthy goldfish. Different volumes of epicatechin gallate stock solution were added to each rearing tank to achieve final concentrations of 8, 4, 2, 1, and 0 mg / L, with three replicates per group. The same volume of aerated water was replaced and medication was added daily to maintain the same drug concentration in each experimental group as on the first day. Medication was continued for 12 days. The number of dead goldfish was observed and recorded daily, and dead fish were promptly removed to prevent water pollution. During the experiment, an oxygen pump was used to increase oxygen levels, and no food was provided. On the 12th day of drug treatment, the infection rate, infection intensity (only the number of trophozoites on the tail fin was counted), and survival rate of the goldfish were recorded. The results are shown in Table 5.
[0054] Table 5. Evaluation of the control effect of catechin gallate on Ichthyophthirius multifiliis disease.
[0055]
[0056] “I” indicates a goldfish that was sick before the experiment; “N” indicates a goldfish that was healthy before the experiment.
[0057] Infection rate = (Number of fish with white spots on their body surface / Number of surviving fish) × 100%;
[0058] Infection intensity = Total number of white spots on the tail fin / Number of fish with white spots on their body surface;
[0059] Survival rate = (Number of surviving fish after the experiment / Total number of goldfish before the experiment) × 100%;
[0060] The results showed that when epicatechin gallate concentration ≥2 mg / L was used to treat a mixed-species system of diseased and healthy goldfish for 12 days, the infection rate and infection intensity of all goldfish were 0. Therefore, immersion treatment with 2 mg / L drug for 12 days can cure Ichthyophthirius multifiliis disease in goldfish and protect healthy goldfish from infection. The survival rates of healthy and diseased goldfish were 96.7% and 86.7%, respectively. At a concentration of 1 mg / L, the infection rate and infection intensity of healthy goldfish were 44.4% and 39.4 Ich / fish, respectively, while those of diseased goldfish were 31.7% and 16.3 Ich / fish, respectively. Therefore, we consider this concentration to be ineffective. When the drug concentration was 8 mg / L, the survival rate of diseased goldfish was only 20%. Therefore, a drug concentration of 2-4 mg / L per liter of aquaculture water is recommended.
[0061] Example 6: The in vitro killing effect of tea polyphenols on Ichthyophthirius multifiliis
[0062] (1) The drug preparation is the same as in Example 1.
[0063] (2) The test method was the same as in Example 2, and the results are shown in Table 6.
[0064] Table 6. Killing effect of tea polyphenols on Ichthyophthirius multifiliis cyst precursors.
[0065] Compound Name Concentration (mg / L) mortality rate(%) Number of hatches Epicatechin gallate 2.0 100.0±0.0 0.0±0.0 Catechins 2.0 0.0±0.0 462.2±56.2 Catechin 2.0 0.0±0.0 431.8±49.4
[0066] The results showed that, at the same drug concentration of 2.0 mg / L, neither epicatechin nor catechins could effectively kill the precursor of Ichthyophthirius multifiliis cysts, indicating that catechin compounds have a certain selectivity in their efficacy against Ichthyophthirius multifiliis.
[0067] Example 7: Insecticidal activity of epicatechin gallate against parasites in other freshwater fish
[0068] (1) The drug preparation is the same as in Example 1.
[0069] (2) ECG killing test on Trichodina / Dactylogyrus: 100 μL of a solution containing approximately 30 Trichodina / 1600 Tetrahymena was added to each well of a 96-well plate, followed by 100 μL of 4.0 mg / L ECG solution to achieve a final drug concentration of 2.0 mg / L. After 4 hours of treatment, the number of surviving parasites in each well was counted. ECG killing test on Dactylogyrus: The tail fins (10 parasites / tail) of goldfish with mild Dactylogyrus infection were cut off and transferred to a 12-well plate. 4.0 mg / L ECG solution was added to achieve a final drug concentration of 2.0 mg / L. After 4 hours of treatment, the number of surviving Dactylogyrus on the tail fins in each well was counted. ECG killing test on Trichodina / Dactylogyrus: 10 parasites / tail of goldfish with mild Dactylogyrus infection were cut off and transferred to a 12-well plate. 4.0 mg / L ECG solution was added to achieve a final drug concentration of 2.0 mg / L. After 4 hours of treatment, the number of surviving Dactylogyrus on the tail fins in each well was counted. ECG killing test on Trichodina / Dactylogyrus: 100 μL of a solution containing approximately 30 Trichodina / 1600 Tetrahymena was added to each well of a 96-well plate, followed by 100 μL of 4.0 mg / L ECG solution to achieve a final drug concentration of 2.0 mg / L. After 4 hours of treatment, the number of surviving Dactylogyrus on the tail fins in each well was counted. ECG killing test on Trichodina / Dactylogyrus: 100 μL of a solution containing approximately 30 Trichodina / 1600 Tetrahymena was added to each well of a Killing test of *Cyclocarya paliurus*: The gills of yellow catfish infected with *Cyclocarya paliurus* were cut off and transferred into 6-well plates. 4.0 mg / L ECG solution was added to make the final drug concentration 2.0 mg / L. After treatment for 4 h, the number of surviving *Cyclocarya paliurus* in each well was counted. Each treatment group was set up in 3 replicates. The groups with only distilled water and only 0.02% dimethyl sulfoxide were set as negative controls. The experiment was carried out at 24±0.5℃. The results are shown in Table 7.
[0070] Table 7. Killing effect of catechin gallate on parasites in freshwater fish.
[0071] Parasite name Concentration (mg / L) mortality rate(%) Wheelworm 2.0 0.0±0.0 Tetrahymena 2.0 13.6±4.8 twigworm 2.0 0.0±0.0 Dactylogyrus 2.0 0.0±0.0 Ichthyophthirius multifiliis 2.0 100.0±0.0
[0072] The results showed that the insecticidal activity of ECG against other common freshwater fish parasites (Trichodina, Tetrahymena, Cytomegalovirus, and Dactylogyrus) was investigated under the same drug concentration and conditions. The results showed that 2.0 mg / L ECG had a mortality rate of only 13.6% against Tetrahymena within 4 hours, and had no killing effect on Trichodina, Cytomegalovirus, and Dactylogyrus. However, 2.0 mg / L ECG had a 100% killing effect on the predator and cyst precursor stages of Ichthyophthirius multifiliis, indicating that ECG has a certain degree of specificity in its efficacy against Ichthyophthirius multifiliis.
[0073] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. The application of epicatechin gallate in the preparation of drugs for the prevention and treatment of Ichthyophthirius multifiliis disease in goldfish, characterized in that: The final concentration of epicatechin gallate in the drug is 2-4 mg / L.