Use of proanthocyanidins in the preparation of a medicament for killing fish ectoparasitic ciliates in vitro

Abstract

The present application relates to the application of procyanidins in the preparation of a medicine for killing and inhibiting fish ectoparasitic ciliates, and belongs to the technical field of fish parasitic disease medicines. The application of procyanidins in the preparation of a medicine for killing and inhibiting fish ectoparasitic ciliates. The present application first applies procyanidins in the preparation of a medicine for killing and inhibiting marine fish ectoparasitic ciliates, especially for killing and inhibiting Ichthyophthirius multifiliis, which can completely kill and inhibit ectoparasites in an effective dose range, achieving the effect of preventing and treating Ichthyophthirius multifiliis disease in marine fish. The main drug component procyanidins used in the present application have a wide source, an industrialized preparation process, a low price, and a great potential for development into a new type of fish ectoparasitic ciliate fish medicine, and have a wide application prospect. Procyanidins have small toxic and side effects, are easy to degrade and eliminate, can effectively avoid negative effects such as environmental pollution and drug residues, and promote the sustainable, green and healthy development of the aquaculture industry.

Description

Technical Field

[0001] This invention relates to the application of proanthocyanidins in the preparation of drugs that inhibit and kill ectoparasitic ciliates in fish, and belongs to the field of fish parasitic disease drug technology. Background Technology

[0002] Ichthyophthirius multifiliis disease, also known as "white spot disease" in marine fish, is a parasitic disease caused by Cryptocaryon irritans Brown. It is one of the most threatening parasitic diseases in farmed bony fish, causing significant economic losses to the marine aquaculture industry. This parasite is mainly distributed in tropical and subtropical waters. Cryptocaryon irritans Brown has a direct, multimorphic life cycle, including a parasitic stage and a free-living stage, without an intermediate host. Its life cycle can be divided into four stages: trophonies, which are the stage after Cryptocaryon irritans Brown invades and infects the host fish, parasitizing the epithelial tissues of the host fish's gills, fins, and body surface, feeding on the host's body fluids, tissue fragments, and cells, and growing and developing; and protomontes, which are the stage after the trophonies mature, detach from the host tissue, enter the surrounding water, and before forming cysts. During the period when it lives freely in the water, swimming and crawling, the cyst precursor can detach naturally from the host from a mature trophozoite, or it can escape from a dead host and become a cyst precursor; it does not necessarily have to wait for the trophozoite to mature before it can detach from the host. The cyst (Tomont) is the period when the cyst precursor, after a brief period of free life in the water, adheres to the bottom wall and begins cell division and reproduction within the cyst. The larvae (Theronts) are the stage when the cysts, produced by cell division, are densely covered with cilia, oval or spindle-shaped, and can move rapidly in the water to find and infect hosts. Cryptocaryon irritans larvae spread with the flow of seawater. Upon encountering a host, they, as a histophilic parasite, invade and penetrate the basal layer of the host's epidermis, further differentiating and growing, thus infecting the fish. The larval infection temperature is 22–32℃, with the optimum water temperature being 25–27℃. Cryptocaryon irritans parasitizes wild hosts where the host's activity range is wide, movement is strong, and density is generally low, resulting in a relatively short parasitic period and minimal impact. However, in farmed hosts, due to the high density and limited activity range of farmed fish, Cryptocaryon irritans can rapidly infect and parasitize the entire host population, causing widespread disease and mass mortality, which is one of the main factors limiting the development of marine cage aquaculture. Currently, the prevention and control of "white spot disease" in marine fish mainly relies on physical and chemical methods, while immunotherapy is still under exploration and rarely used in actual production. Physical control methods include freshwater soaking, ozone or ultraviolet disinfection, rotation of aquaculture, and removal of cysts. These physical methods are cumbersome, costly, and labor-intensive, making them unsuitable for cage aquaculture. Chemical control is simple to operate and has significant effects, but the large-scale use of chemical drugs can easily cause negative impacts such as damage to the aquaculture environment, environmental pollution, and drug residues. There is an urgent need for a treatment method with good therapeutic effects and minimal negative impacts. In recent years, the scientific and safe use of drugs has become a focus of the development and use of fish medicines. my country has abundant resources of traditional Chinese medicinal herbs, and the use of traditional Chinese medicinal herbs to treat diseases of aquatic animals has a long history.Traditional Chinese medicine (TCM) is a green and environmentally friendly medicine, favored for its low environmental pollution and drug residues. However, due to the extreme complexity of its active ingredients, their precise identification is difficult, hindering accurate control in treatment. Natural compounds, the active ingredients of TCM, not only possess similar efficacy but also offer advantages such as controllable quality, clear mechanisms of action, stable efficacy, and rapid onset of action. Their safety and efficacy can be guaranteed, giving them significant advantages in production applications. Using natural compounds as medicines or feed additives for disease prevention and control in aquaculture fully meets the needs of developing pollution-free aquaculture and green aquatic products.

[0003] The proanthocyanidins proposed in this invention are derived from grape seeds, with a molecular weight of 594.52. They are slightly soluble in water. Proanthocyanidins, also known as condensed tannins, are polyphenolic compounds found in fruits, vegetables, nuts, legumes, grains, wine, and chocolate. They are composed of different amounts of catechins or epicatechins. Based on the number of polymers, they can be divided into oligomeric proanthocyanidins (2-4 polymers, abbreviated as OPC, easily soluble in water) and polymeric proanthocyanidins (≥5 polymers, abbreviated as PPC, poorly soluble in water). Proanthocyanidins have a wide range of pharmacological activities, including antioxidant, antitumor, anti-inflammatory, anti-allergic, and anti-edema effects. They also have anti-mutagenic, anti-diarrheal, antibacterial, antiviral, anti-caries, visual function improvement, prevention of Alzheimer's disease, and treatment of sports injuries. Studies have found that proanthocyanidins exert their anti-cancer activity through multiple mechanisms, including reducing intracellular ROS levels, inducing mitochondrial depolarization, inhibiting tumor cell proliferation, adhesion, and invasion potential, promoting apoptosis, and inhibiting cell cycle progression. They also have the potential to prevent and reduce dental caries by effectively inhibiting the growth, metabolism, and biofilm of Streptococcus mutans and suppressing virulence factors. Furthermore, they induce cell death in Staphylococcus aureus by disrupting cell permeability and reducing intracellular protein (enzyme) levels. Proanthocyanidins induce cell death through various mechanisms, thereby treating various diseases and demonstrating significant potential in disease prevention and control.

[0004]

[0005] Wang Yanjun. Effects of grape seed proanthocyanidins on the life cycle of *C. elegans* [D]. Beijing Forestry University, 2014. 1. Proanthocyanidins significantly prolong the lifespan of nematodes. 2. Proanthocyanidins significantly enhance the motility of nematodes. 3. Proanthocyanidins have no inhibitory effect on the growth of *Escherichia coli* OP50, indicating that the extension of nematode lifespan is not related to caloric restriction, but rather to the activity inherent in proanthocyanidins themselves. 4. Proanthocyanidins do not affect the reproductive capacity of nematodes.

[0006] There are currently no reports on the application of proanthocyanidins in inhibiting and killing ectoparasitic ciliates in fish. Summary of the Invention

[0007] The purpose of this invention is to provide the application of proanthocyanidins in the preparation of drugs that inhibit and kill fish ectoparasitic ciliates.

[0008] In one specific embodiment, the fish ectoparasitic ciliate is Cryptocaryon irritans Brown.

[0009] In one specific embodiment, the fish is the oval pomfret, Latin name Trachinotus ovatus.

[0010] In one specific embodiment, the concentration of proanthocyanidins in the drug is 10–200 μg / mL.

[0011] In one specific embodiment, the concentration of proanthocyanidins in the drug is 10–100 μg / mL.

[0012] In one specific embodiment, the dosage form of the drug is a mixed liquid, powder, emulsion, or a different dosage form prepared together with a pharmaceutically acceptable carrier and excipients.

[0013] Beneficial effects:

[0014] (1) This invention is the first to apply proanthocyanidins to the preparation of drugs that inhibit and kill external parasitic ciliates in marine fish, especially for inhibiting and killing Cryptocaryon irritans. Within the effective dosage range, it can completely inhibit and kill external parasites, thereby achieving the effect of preventing and treating Ichthyophthirius multifiliis disease in marine fish.

[0015] (2) The main drug component used in this invention, proanthocyanidins, has a wide range of sources, has an industrial preparation process, is inexpensive, and has great potential to be developed into a new type of fish medicine for the prevention and treatment of ectoparasitic ciliates in fish, with broad application prospects.

[0016] (3) Acute toxicity tests of proanthocyanidins on oval pomfret demonstrated that proanthocyanidins are highly safe for oval pomfret. At the same time, as a natural compound, proanthocyanidins have few toxic side effects, are easy to degrade and eliminate, and can effectively avoid negative impacts such as environmental pollution and drug residues, thus promoting the sustainable, green and healthy development of aquaculture. Attached Figure Description

[0017] Figure 1 This refers to the inhibitory effect of proanthocyanidins on Cryptocaryon larvae in Example 1.

[0018] Figure 2 In Example 2, proanthocyanidins reduced the mitochondrial membrane potential of Cryptocaryon larvae.

[0019] Figure 3 This refers to the inhibitory effect of proanthocyanidins on Cryptocaryon cysts in Example 3.

[0020] Figure 4 This refers to the inhibitory effect of proanthocyanidins on the precursor of Cryptocaryon cysts in Example 4.

[0021] Figure 5 The results of the acute toxicity test of proanthocyanidins administered by gavage to oval pomfret in Example 5 are shown. Detailed Implementation

[0022] Application of proanthocyanidins in the preparation of drugs that inhibit and kill fish ectoparasitic ciliates.

[0023] In one specific embodiment, the fish ectoparasitic ciliate is Cryptocaryon irritans Brown.

[0024] In one specific embodiment, the fish is the oval pomfret, Latin name Trachinotus ovatus.

[0025] In one specific embodiment, the concentration of proanthocyanidins in the drug is 10–200 μg / mL.

[0026] In one specific embodiment, the concentration of proanthocyanidins in the drug is 10–100 μg / mL.

[0027] In one specific embodiment, the dosage form of the drug is a mixed liquid, powder, emulsion, or a different dosage form prepared together with a pharmaceutically acceptable carrier and excipients.

[0028] The specific embodiments of the present invention will be further described below with reference to examples, but the present invention is not limited to the scope of the embodiments described herein.

[0029] The host model fish used in this embodiment was *Cryptocaryon ovatus*, sourced from a fish farm in Hainan Province. The parasite model was *Cryptocaryon irritans*, isolated from diseased *Cryptocaryon ovatus* in a net cage in Zhanjiang City, Guangdong Province, and identified as *Cryptocaryon irritans* by morphological and 18S rDNA molecular biology methods.

[0030] Example 1

[0031] Proanthocyanidins inhibit and kill Cryptocaryon larvae

[0032] (1) Preparation of the drug: Accurately weigh 12.50 mg of proanthocyanidins, dissolve them in 50% ethanol aqueous solution (V / V), and make up to 10 mL to prepare a proanthocyanidin solution with a concentration of 1.25 mg / mL. Dilute the solution with 50% ethanol aqueous solution to 25.0, 50.0, 75.0, 100.0, 250.0, 500.0, 750.0 and 1000.0 μg / mL proanthocyanidin solutions.

[0033] (2) Stimulate the hatching of Cryptocaryon cysts into larvae. Immediately collect the larvae using a centrifuge tube at 1000×g for 10 min. Collect the upper layer of viable larvae and discard the lower 100μL of impurities. Adjust the larval concentration to approximately 200 larvae / mL and use within 2 hours. Transfer 1mL of the larvae solution to a 24-well cell culture plate using a pipette for later use.

[0034] (3) 10 μL of proanthocyanidin solutions of different concentrations were added sequentially to 24-well cell culture plates containing larvae, resulting in final proanthocyanidin concentrations of 0.25, 0.50, 0.75, 1.00, 2.50, 5.00, 7.50, 10.00, and 12.50 μg / mL. 50% ethanol aqueous solution and formaldehyde solution with the same proanthocyanidin concentration were used as negative and positive controls, respectively. The cells were incubated at 27.0 ± 0.5℃ for 1 h. Larvae that did not move after 1 h were considered dead. The number of dead larvae (n1) was then counted under an inverted microscope. After counting, 200 μL of 4% paraformaldehyde was used to fix and stimulate the Cryptocaryon larvae for 10 min, and the total number of larvae in each well was counted (n2). The larval mortality rate (the percentage of the motionless larvae, PMT) was calculated as: n1 / n2 × 100%. The data on proanthocyanidin concentration and larval mortality were fitted using equation (1) in Origin Pro 8.5.1SR2 (OriginLabCorporation, USA) Sigmoidal fit gadget. The median effective dose (EC50) of proanthocyanidins for larvae was calculated using equation (1). 50 Each drug concentration group was tested in 5 parallel trials.

[0035] Fitting equation: Y=a / (1+exp(-k×(X-Xc))) (1)

[0036] Y and X represent the median mortality rate of larvae and the concentration of proanthocyanidins, respectively, while a, k, and Xc are constants.

[0037] Experimental results are as follows Figure 1 As shown, the mortality rate of Cryptocaryon larvae gradually increased with increasing proanthocyanidin concentration. When the proanthocyanidin concentration reached 5.00 μg / mL, the larval mortality rate reached 97.71%, and the EC50 of proanthocyanidins on larvae within 1 hour... 50 It is 1.84 μg / mL (R) 2 =0.996), lower than the EC50 of formaldehyde for larvae. 50 (5.98 μg / mL, R) 2 =0.992).

[0038] Note: Figure 1PMT: Larval mortality rate; * indicates a significant difference between the experimental group and the negative control group (p < 0.05); ** indicates an extremely significant difference between the experimental group and the negative control group (p < 0.01); A, Box plot of the inhibitory effect of proanthocyanidins on Cryptocaryon larvae; B, Box plot of the inhibitory effect of formaldehyde on Cryptocaryon larvae.

[0039] Example 2

[0040] Proanthocyanidins reduce stimulation of Cryptocaryon larvae mitochondria

[0041] (1) Preparation of the drug: Accurately measure 30 mg of proanthocyanidins, dissolve them in 50% ethanol aqueous solution, and make up to 10 mL to prepare a proanthocyanidin solution with a concentration of 3.0 mg / mL. Then, dilute with 50% ethanol aqueous solution to 0.1, 0.5, 1.0 and 2.0 mg / mL of proanthocyanidins.

[0042] (2) After the larvae of Cryptocaryon cysts hatched, the larvae were immediately collected in centrifuge tubes at 1000×g for 10 min. The upper layer of viable larvae was collected, while the lower 100 μL of weakly viable larvae and impurities was discarded. The larval concentration was adjusted to approximately 10,000 larvae / mL and used within 2 hours. The mitochondrial membrane potential of Cryptocaryon cysts was detected using an enhanced mitochondrial membrane potential detection kit (JC-1) (Beyotime Biotechnology, CAS: C2003S).

[0043] (2) Pipette 150 μL of larval suspension into a black 96-well microplate. Before proanthocyanidin treatment, add 50 μL of mitochondrial membrane potential staining working solution and incubate at 27.0 ± 0.5℃ for 20 min.

[0044] (3) Add 2 μL of proanthocyanidin solution of different concentrations to black 96-well microplates containing larvae in sequence to make the final concentration of proanthocyanidins 1.0, 5.0, 10.0, 20.0 and 30.0 μg / mL, and set up a negative control group (2 μL 50% ethanol aqueous solution).

[0045] Immediately afterwards, fluorescence intensity values ​​(excitation wavelength / emission wavelength = 490 / 530nm and 525 / 590nm) were detected using a fluorescence microplate reader (BioTek Synergy H1, BioTek Instruments, Winooski, VT, USA), with measurements taken every 5 minutes for a total of 60 minutes. Mitochondrial membrane potential was calculated based on the ratio of I490 / 530nm to I525 / 590nm, with 5 parallel experiments for each drug concentration group.

[0046] Experimental results are as follows Figure 2As shown, with increasing proanthocyanidin concentration, the mitochondrial membrane potential of Cryptocaryon stimuli larvae decreased significantly, and the mitochondrial membrane potential showed a decreasing trend over time. These results indicate that proanthocyanidins significantly reduce the mitochondrial membrane potential in Cryptocaryon stimuli larvae, potentially inducing larval apoptosis through this pathway, thereby inhibiting the activity of Cryptocaryon stimuli larvae.

[0047] Example 3

[0048] Proanthocyanidins inhibit and kill Cryptocaryon cysts

[0049] (1) Preparation of the drug: As in Case 1, accurately weigh 200 mg of proanthocyanidins, dissolve them in 50% ethanol aqueous solution, and make up to 10 mL to prepare a proanthocyanidin solution with a concentration of 20 mg / mL. Then, use 50% ethanol aqueous solution to dilute to 1.0, 2.5, 5.0, 7.5, 10.0 and 15.0 mg / mL proanthocyanidin solutions.

[0050] (2) Using the oval pomfret as a host, oval pomfret infected with Cryptocaryon irritans and exhibiting "white spots" were placed in a 300L funnel-shaped PVC collection tank, with glass dishes placed at the bottom for overnight collection. The next day, the glass dishes with cysts were removed, and the mucus on the surface of the cysts was cleaned with sterile filtered seawater. The cysts were then pipetted into 24-well cell culture plates, approximately 20 per well, and 2 mL of fresh sterile filtered seawater was added for later use.

[0051] (3) 20 μL of different concentrations of proanthocyanidins were added sequentially to 24-well cell culture plates containing cysts, so that the final concentrations of proanthocyanidins were 10.0, 25.0, 50.0, 75.0, 100.0, 150.0 and 200.0 μg / mL. 50% ethanol aqueous solution and formaldehyde solution with the same concentration as proanthocyanidins were used as negative control and positive control, respectively. The cells were cultured in a light incubator at 27.0±0.5℃ for 12 h. Afterwards, each well was washed with fresh seawater to remove proanthocyanidins and formaldehyde, and 2 mL of fresh seawater was added. The exact number of cysts was counted under a stereomicroscope.

[0052] (4) The cysts were cultured in a 27.0±0.5℃ light incubator for 5 days, with 1 mL of fresh seawater replaced daily. The hatching of the cysts was then observed under a stereomicroscope. The number of completely empty cysts in each well was counted as m2. The hatching rate (PTR) of the cysts that successfully released the ronts within 5 days was calculated. The PTR formula is (m2×100 / m1)%. The inhibition rate (ITRT) of the cysts was calculated using formula (2). OriginPro 8.5.1SR2 (OriginLabCorporation, USA) Sigmoidal fit was used. The fitting equation (Equation 1) in Gadget was used to fit the data on the inhibition rate of proanthocyanidin and formaldehyde concentrations on cyst hatching, where Y and X are the concentrations of ITRT and the experimental drugs proanthocyanidin and formaldehyde, respectively. The half-maximal effective dose (EC50) of proanthocyanidin and formaldehyde for inhibiting cyst hatching was calculated using the fitting equation (Equation 1). 50 Each drug concentration group was tested in parallel five times.

[0053] ITRT = ((PTR) nc -PTR t )×100 / PTR nc )% (2)

[0054] PTR nc and PTR t The values ​​represent the hatching rates of the negative control and reagent-treated groups, respectively.

[0055] Experimental results are as follows Figure 3 As shown, with increasing proanthocyanidin concentration, the encapsulation inhibition rate increases in a dose-dependent manner, indicating that proanthocyanidins have a significant effect on the EC50 inhibition rate of encapsulation. 50 =34.38 μg / mL (R) 2 =0.985), higher than the EC50 of formaldehyde on cysts. 50 (26.58 μg / mL, R) 2 =0.998).

[0056] Note: Figure 3 ITRT in the figure: Inhibition rate of cyst release of larvae; * indicates a significant difference between the experimental group and the negative control group (p < 0.05); ** indicates an extremely significant difference between the experimental group and the negative control group (p < 0.01); A, box plot of the inhibitory effect of proanthocyanidins on the stimulation of Cryptocaryon cyst hatching; B, box plot of the inhibitory effect of formaldehyde on the stimulation of Cryptocaryon cyst hatching.

[0057] Example 4

[0058] Proanthocyanidins inhibit and kill cryptocaryon cyst precursors

[0059] (1) Preparation of the drug: As in Case 1, accurately weigh 200 mg of proanthocyanidins, dissolve them in 50% ethanol aqueous solution, and make up to 10 mL to prepare a proanthocyanidin solution with a concentration of 20 mg / mL. Dilute the solution with 50% ethanol aqueous solution to 1.0, 2.5, 5.0, 10.0 and 15.0 mg / mL of proanthocyanidins.

[0060] (2) Collect mature Cryptocaryon stimuli trophozoites from the gill filaments of oval pomfret infected with Cryptocaryon stimuli for 2.5 days. Cut off the tail of oval pomfret with "small white spots" to bleed it. Cut off the gill filaments and place them on a plate containing filtered and sterilized seawater. The mature trophozoites fall off the gill filaments naturally. Rinse them 3 times with filtered and sterilized seawater and collect them. The size is about 200 μm. Place them in a 24-well cell culture plate, about 20 per well. Add 2 mL of fresh seawater. Incubate the trophozoites in a 27.0±0.5℃ light incubator for 1 h to transform them into cyst precursors for later use.

[0061] (3) 20 μL of proanthocyanidin solutions of different concentrations were added sequentially to 24-well cell culture plates containing cyst precursors, so that the final concentrations of proanthocyanidins were 10.0, 25.0, 50.0, 100.0, 150.0, and 200.0 μg / mL. 50% ethanol aqueous solution and formaldehyde solution with the same concentration as proanthocyanidins were used as negative and positive controls, respectively. The cyst precursors were incubated with different concentrations of reagents in a 27.0±0.5℃ light incubator for 12 h. Then, each well was washed with fresh seawater to remove proanthocyanidins and formaldehyde, and 2 mL of fresh seawater was added to replace the original solution. The exact number of cyst precursors in each well was counted under a stereomicroscope and recorded as m1.

[0062] (4) The protomontes were cultured in a 27.0±0.5℃ light incubator for 5 days, with 1 mL of fresh seawater replaced daily. The hatching of the protomontes was then observed under a stereomicroscope. Completely empty protomontes were used as the standard for evaluating whether the protomontes had successfully transformed into protomontes and released larvae. The number of completely empty protomontes in each well (m2) was counted, and the hatching rate (percentage of the alive protomonts, PAP) and inhibition rate (the inhibition of protomont transforming into protomont, IPTT) of the protomontes within 5 days were calculated. The hatching rate of the protomontes successfully transforming into larvae was calculated as (m2×100 / m1)%. The inhibition rate of the protomontes transforming into protomontes was calculated by formula (3). The fitting equation (equation 1) in Case 1 was used to fit the data on the inhibition rate of protocyanidin concentration on the transformation of protomontes. The half-maximal effective dose (EC50) of protocyanidin on the inhibition of protomonte transformation was calculated by fitting equation (1). 50 Five experiments were conducted in parallel for each drug concentration group.

[0063] IPTT = ((PAP) nc -PAP t )×100 / PAP nc )% (3)

[0064] PAP nc and PAP t The values ​​represent the hatching rates of the cyst precursors in the negative control and reagent-treated groups, respectively.

[0065] Experimental results are as follows Figure 4 As shown, with increasing proanthocyanidin concentration, the mortality rate of cysteine ​​precursors increases in a dose-dependent manner, and proanthocyanidins have a significant effect on the EC50 of cysteine ​​precursors. 50 =51.22 μg / mL (R) 2 =0.991), higher than the EC50 of formaldehyde for the encapsulation precursor. 50 (19.02 μg / mL, R) 2 =0.947).

[0066] Note: Figure 4 IPTT: Percentage of cyst precursors inhibited from converting to cysts and releasing larvae; * indicates a significant difference between the experimental group and the negative control group (p < 0.05); ** indicates an extremely significant difference between the experimental group and the negative control group (p < 0.01); A, Box plot of the inhibitory effect of proanthocyanidins on the hatching of Cryptocaryon cyst precursors; B, Box plot of the inhibitory effect of formaldehyde on the hatching of Cryptocaryon cyst precursors.

[0067] Example 5

[0068] Acute toxicity test of proanthocyanidins administered by gavage to pomfret

[0069] The oval pomfret used in the experiment were purchased from Chenhai Aquatic Products Company in Sanya City, Hainan Province. After being temporarily raised for 2 months to stabilize, 180 oval pomfrets weighing 27.15±2.36g were randomly selected and divided into 18 rearing tanks, forming 6 groups with 3 replicates per group. Different proanthocyanidin gavage doses were set at 0, 50, 100, 500, 1000, and 2000 mg / kg (proanthocyanidins / fish body weight). The rearing system was equipped with aeration equipment and a water pump. The water temperature was maintained at 26.0±1.0℃, pH 7±0.5, and DO above 6.5mg / L. The fish were fasted for 1 day before gavage.

[0070] Accurately weigh 0.068, 0.135, 0.675, 1.350, and 2.700 g of proanthocyanidins, dissolve them in PBS solution, and bring the volume to 5.0 mL to prepare proanthocyanidin solutions of 13.6, 27.0, 135.0, 270.0, and 540.0 mg / mL for later use. The safety of oral administration of different concentrations of proanthocyanidins to oval pomfret was tested by gavage. The gavage volume was 100 μL / fish, achieving drug concentration gradients of 0, 50, 100, 500, 1000, and 2000 mg / kg (proanthocyanidins / fish body weight) in the oval pomfret after oral administration. Observation was conducted for 14 days, monitoring the behavior of the experimental oval pomfret, recording their health status and mortality, and analyzing the data.

[0071] The survival rate of oval pomfret after gavage is as follows: Figure 5 As shown, in the 2000 mg / kg dosage group, 6 oval pomfret died within 14 days. Observation of the dead fish revealed damage to the gill covers, and dissection revealed ascites in the dead oval pomfret, while the internal organs were intact. Fish in other dosage groups and the control group did not experience any deaths or fatal symptoms; their behavior remained good, and they were able to eat on time. Careful examination of their appearance revealed no changes. Furthermore, during the dissection of all oval pomfret, no fluid accumulation or viscous material was found in the abdomen, and no organ swelling was observed. Proanthocyanidins have a high safe concentration of 1000 mg / kg (drug / fish body weight). Preliminary assessment indicates that proanthocyanidins have good safety, providing a safety guarantee for the development of feed additives for the treatment of "white spot disease".

[0072] Proanthocyanidins exhibit effective in vitro anti-irritant effects on Cryptocaryon larvae, cysts, and cyst precursors. They show significant larval killing effects. After incubation with larvae, proanthocyanidins can reduce the mitochondrial membrane potential of Cryptocaryon larvae, thereby reducing larval activity. Acute toxicity experiments via gavage show that this natural product has a high safe drug concentration, thus showing great potential for development as a feed additive or infusion solution.

Claims

1. Use of procyanidins for the preparation of a medicament for killing or inhibiting fish ectoparasitic ciliates, characterized in that, The fish ectoparasitic ciliate is Cryptocaryon irritans Brown in Latin. The structural formula of the procyanidin is: The molecular weight of the procyanidin is 594.

52.

2. The use of procyanidins according to claim 1 for the preparation of a medicament for the control of fish ectoparasitic ciliates in vitro, characterized in that, The fish is Trachinotus ovatus in Latin.

3. Use of procyanidins according to claim 1 or 2 for the preparation of a medicament for the control of fish ectoparasitic ciliates in vitro, characterized in that, The procyanidin concentration in the medicine is 10-200 μg / mL.

4. The use of procyanidins according to claim 3 for the preparation of a medicament for the control of fish ectoparasitic ciliates in vitro, characterized in that, The procyanidin concentration in the medicine is 10-100 μg / mL.

5. Use of procyanidins according to claim 1 or 2 for the preparation of a medicament for the control of fish ectoparasitic ciliates in vitro, characterized in that, The dosage form of the medicine is a medicine liquid for mixing, a powder or an emulsion.

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