Application of procyanidine in preparation of medicine for inhibiting and killing fish in-vitro parasitic ciliates

Abstract

The invention relates to application of procyanidine in preparation of a medicine for inhibiting and killing in-vitro parasitic ciliates of fishes, and belongs to the technical field of medicines for fish parasitic diseases. The procyanidine is applied to preparation of drugs for inhibiting and killing in-vitro parasitic ciliates of fishes. The procyanidine is applied to preparation of the medicine for inhibiting and killing the in-vitro parasitic ciliates of the marine fishes for the first time, especially for inhibiting and killing cryptocaryon irritans, the in-vitro parasites can be completely inhibited and killed within an effective dosage range, and the effect of preventing and treating the ichthyophthiriasis of the marine fishes is achieved. The procyanidine serving as a main medicine component is wide in source, has an industrial preparation process, is low in price, has a huge potential of being developed into a novel fish medicine for preventing and treating in-vitro parasitic ciliates of fishes, and is wide in application prospect. The procyanidine has small toxic and side effects, is easy to degrade and eliminate, can effectively avoid negative effects such as environmental pollution and drug residues, and promotes sustainable, green and healthy development of the aquaculture industry.

Description

Technical Field

[0001] The invention relates to application of proanthocyanidins in preparing a medicine for inhibiting and killing ectoparasitic ciliates of fish, and belongs to the technical field of medicines for fish parasitic diseases. Background Art

[0002] Ichthyophthirius scopariasis, also known as "white spot disease" of marine fish, is a parasitic disease caused by Cryptocaryon irritans Brown. It is one of the most threatening parasitic diseases of farmed bony fish and has caused huge economic losses to the marine aquaculture industry. The parasite is mainly distributed in tropical and subtropical waters. Cryptocaryon irritans has a direct polymorphic life history, including a parasitic stage and a free-living stage, without an intermediate host. Its life history can be divided into four stages. Trophozoites are the stage in which Cryptocaryon irritans invades and infects the host fish, parasitizes in the epithelial tissues of the host fish's gills, fins, and skin, feeds on the host's body fluids, tissue fragments and cells, and ingests nutrition and growth and development. Protomonts are the stage in which the trophozoites mature, detach from the host tissues, enter the surrounding water, and before forming cysts. During the period of free-living, swimming and crawling in water, the cyst precursor can be naturally separated from the host by a mature trophozoite, or it can escape from the dead host and become a cyst precursor. It is not necessary to wait for the trophozoite to mature before it can separate from the host; the cyst (Tomont) is the period when the cyst precursor adheres to the bottom wall of the water after a short period of free living in the water, and the cells in the cyst begin to divide and reproduce; the larvae (Theronts) are densely covered with cilia, oval or spindle-shaped, and can move quickly in the water to find a host and infect and invade. The larvae of Cryptocaryon irritans spread with the flow of seawater. When they encounter a host, they invade and penetrate the basal layer of the host epidermis as a tissue-tropic parasite and further differentiate and grow, thereby infecting the fish body. The infection temperature of the larvae is 22-32℃, and the optimum water temperature is 25-27℃. When Cryptocaryon irritans parasitize wild hosts, the hosts have a wide range of activities, strong movements, and generally low density. Its parasitic period is relatively short and the impact is relatively small. When parasitizing farmed hosts, due to the high density of farmed fish and the small range of activities, Cryptocaryon irritans can infect and parasitize the entire host population in a short period of time, causing large-scale infections and deaths, which is one of the main factors restricting the development of marine cage aquaculture. At present, the prevention and control methods for marine fish "white spot disease" are still mainly physical and drug prevention and control. Immune prevention and control is still under exploration and is rarely used in actual production. Physical prevention and control includes freshwater immersion, ozone or ultraviolet disinfection, rotational farming and cyst removal. These physical means are cumbersome, costly, and labor-intensive, and are not suitable for prevention and control measures in cage aquaculture models. Drug prevention and control is simple to operate and has significant effects, but the large-scale use of chemical drugs can easily cause negative effects such as damage to the aquaculture environment, environmental pollution, and drug residues. At present, there is an urgent need for a treatment method with good therapeutic effects and small negative effects. In recent years, scientific and safe drug use has become the focus of the development and use of fishery drugs. my country has rich resources of Chinese herbal medicine, and the use of Chinese herbal medicine to treat aquatic animal diseases has a long history.Chinese herbal medicine is a green and environmentally friendly medicine. It is favored by many people for its advantages such as low environmental pollution and low drug residues. However, the active ingredients of Chinese herbal medicine are extremely complex and difficult to determine, and cannot be accurately controlled in treatment. The natural compounds of the active ingredients of Chinese herbal medicine not only have similar functions to those of Chinese herbal medicine, but also have the advantages of controllable quality, clear mechanism of action, stable efficacy, and rapid onset compared to Chinese herbal medicine. Its safety and efficacy can be guaranteed, and it has obvious advantages in production application. Using natural compounds as drugs or feed additives for the prevention and control of aquaculture diseases fully meets the needs of developing pollution-free aquaculture and green aquatic products.

[0003] The proanthocyanidins mentioned in the present invention are derived from grape seeds, have a molecular weight of 594.52, and are slightly soluble in water. Proanthocyanidins are also called condensed tannins, are rich in polyphenolic compounds in fruits, vegetables, nuts, beans, cereals, wine and chocolate, and are composed of different amounts of catechins or epicatechins. According to the amount of polymers, they can be divided into oligomeric proanthocyanidins (2-4 polymers, referred to as OPC, easily soluble in water) and highly polymeric proanthocyanidins (≥5 polymers, referred to as PPC, poorly soluble in water). Proanthocyanidins have a wide range of pharmacological activities such as anti-oxidation, anti-tumor, anti-inflammatory, anti-allergic, and anti-edema, and have the effects of anti-mutation, anti-diarrhea, anti-bacterial and anti-viral, anti-caries, improving visual function, preventing senile dementia, and treating sports injuries. Studies have found that proanthocyanidins can exert their anti-cancer activity through multiple mechanisms, such as reducing intracellular ROS levels, inducing mitochondrial depolarization, inhibiting tumor cell proliferation, adhesion and invasion potential, promoting cell apoptosis, and hindering cell cycle progression; by effectively inhibiting the growth metabolism and biofilm of Streptococcus mutans and inhibiting virulence factors, they have the potential to prevent and reduce the occurrence of dental caries; by destroying cell permeability and reducing the amount of intracellular proteins (enzymes), Staphylococcus aureus loses its proliferation ability and causes death. Proanthocyanidins can cause cell death in a variety of ways, thereby treating various diseases, and have great potential in disease prevention and treatment.

[0004]

[0005] Wang Yanju. Effects of grape seed proanthocyanidins on the life cycle of Caenorhabditis elegans [D]. Beijing Forestry University, 2014. Public 1. Proanthocyanidins can significantly extend 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 calorie restriction, but the activity of proanthocyanidins themselves extends the lifespan of nematodes. 4. Proanthocyanidins do not affect the reproductive capacity of nematodes.

[0006] There is no report on the application of proanthocyanidins in inhibiting and killing ciliates parasitic in vitro in fish. Summary of the invention

[0007] The purpose of the present invention is to provide the use of proanthocyanidins in the preparation of drugs for inhibiting and killing parasitic ciliates in vitro of fish.

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

[0009] In a specific embodiment, the fish is Trachinotus ovatus.

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

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

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

[0013] Beneficial effects:

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

[0015] (2) The main drug ingredient proanthocyanidin used in the present invention has a wide source, an industrial preparation process, a low price, and has great potential for development into a new type of fishery drug for preventing and controlling external parasitic ciliates of fish, and has broad application prospects.

[0016] (3) The acute toxicity test of proanthocyanidins on oval pomfret proved that proanthocyanidins are highly safe for oval pomfret. At the same time, as a natural compound, proanthocyanidins have little toxic side effects and are easy to degrade and eliminate. They can effectively avoid negative effects such as environmental pollution and drug residues, and promote the sustainable, green and healthy development of aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Figure 2 The proanthocyanidins in Example 2 reduce the mitochondrial membrane potential of Cryptocaryon larvae.

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

[0020] Figure 4 This is the inhibitory effect of proanthocyanidins in Example 4 on the cyst precursors of Cryptocaryon irritans.

[0021] Figure 5 The results of the acute toxicity experiment on oval pomfret by gavage of proanthocyanidins in Example 5 are shown. DETAILED DESCRIPTION

[0022] Application of proanthocyanidins in the preparation of drugs for inhibiting and killing parasitic ciliates in fish ectoparasites.

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

[0024] In a specific embodiment, the fish is Trachinotus ovatus.

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

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

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

[0028] The specific implementation modes of the present invention are further described below in conjunction with embodiments, but the present invention is not limited to the scope of the embodiments.

[0029] The host model fish used in the embodiment is an oval pomfret, which comes from a farm in Hainan Province. The parasite model is Cryptocaryon irritans, which is isolated from an oval pomfret diseased in a cage in Zhanjiang City, Guangdong Province, and is identified as Cryptocaryon irritans by morphology and 18S rDNA molecular biology methods.

[0030] Example 1

[0031] Proanthocyanidins inhibit and kill Cryptocaryon irritans larvae

[0032] (1) Preparation of drugs: Accurately weigh 12.50 mg of proanthocyanidins, dissolve it in 50% ethanol aqueous solution (V / V), and dilute it to 10 mL to prepare a proanthocyanidin solution with a concentration of 1.25 mg / mL. The solution was then diluted 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 Cryptocaryon cysts to hatch larvae, and immediately collect the larvae using a centrifuge tube, centrifuge at 1000×g for 10 min; collect the upper layer of insect liquid with good vitality, discard 100μL of impurities in the lower layer, adjust the larvae concentration to about 200 / mL, and use within 2 hours. Use a pipette to transfer 1mL of insect liquid to a 24-well cell culture plate for later use;

[0034] (3) 10 μL of different concentrations of proanthocyanidin solution was added to the 24-well cell culture plate containing larvae in sequence, so that the final concentration of proanthocyanidin was 0.25, 0.50, 0.75, 1.00, 2.50, 5.00, 7.50, 10.00, and 12.50 μg / mL. In this experiment, 50% ethanol aqueous solution and formaldehyde solution with the same concentration of proanthocyanidin were used as negative and positive controls, respectively. The cells were cultured in a light incubator at 27.0±0.5℃ for 1 hour, and the larvae that did not move after 1 hour were used as the standard for dead insects. The number of dead larvae (n1) was then counted under an inverted microscope; after the counting was completed, 200 μL of 4% paraformaldehyde was used to fix and stimulate the Cryptocaryon larvae for 10 minutes, and the total number of larvae in each well was counted (n2); the percentage of the motionless theronts (PMT) formula was: n1 / n2×100%. The fitting equation (1) in the Sigmoidal fit gadget of Origin Pro 8.5.1SR2 (OriginLabCorporation, USA) was used to fit the data of the effect of proanthocyanidin concentration on larval mortality. The half effective dose (EC 50 ), each drug concentration group was repeated 5 times;

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

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

[0037] The experimental results are as follows Figure 1 As shown in Figure 2, with the increase of proanthocyanidin concentration, the mortality rate of Cryptocaryon irritans larvae gradually increased. When the proanthocyanidin concentration reached 5.00 μg / mL, the larval mortality rate reached 97.71%. The EC value of proanthocyanidin on larvae within 1 hour was 50 1.84 μg / mL (R 2 =0.996), which is lower than the EC value of formaldehyde for larvae. 50 (5.98 μg / mL, R 2 =0.992).

[0038] Note: Figure 1PMT: larval mortality rate; * indicates that the experimental group is significantly different from the negative control group (p < 0.05); ** indicates that the experimental group is extremely significantly different from the negative control group (p < 0.01); A, box plot of the inhibitory effect of proanthocyanidins on Cryptocaryon irritans larvae; B, box plot of the inhibitory effect of formaldehyde on Cryptocaryon irritans larvae.

[0039] Example 2

[0040] Proanthocyanidins reduce mitochondrial stimulation in Cryptocaryon larvae

[0041] (1) Preparation of drugs: Accurately weigh 30 mg of proanthocyanidins, dissolve it in 50% ethanol aqueous solution, and dilute it to 10 mL to prepare a proanthocyanidin solution with a concentration of 3.0 mg / mL. Subsequently, dilute it with 50% ethanol aqueous solution to 0.1, 0.5, 1.0, and 2.0 mg / mL of proanthocyanidins.

[0042] (2) When the larvae of Cryptocaryon cysts are hatched, the larvae are immediately collected using a centrifuge tube and centrifuged at 1000×g for 10 min. The upper layer of the liquid with good vitality is collected, and 100 μL of the larvae with weak vitality and impurities in the lower layer are discarded. The larvae concentration is adjusted to about 10,000 / mL and used within 2 hours. The enhanced mitochondrial membrane potential detection kit (JC-1) (Beyotime Biotechnology, CAS: C2003S) is used to detect the mitochondrial membrane potential of Cryptocaryon larvae.

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

[0044] (3) 2 μL of proanthocyanidin solutions of different concentrations were added to the black 96-well ELISA plate containing the larvae in sequence, so that the final concentration of proanthocyanidin was 1.0, 5.0, 10.0, 20.0, and 30.0 μg / mL, and a negative control group (2 μL of 50% ethanol aqueous solution) was set up.

[0045] Subsequently, the fluorescence intensity value (excitation wavelength / emission wavelength = 490 / 530 nm and 525 / 590 nm) was immediately detected using a fluorescence microplate reader (BioTek Synergy H1, BioTek Instruments, Winooski, VT, USA) every 5 minutes for a total of 60 minutes; the mitochondrial membrane potential was calculated based on the ratio of I490 / 530 nm and I525 / 590 nm, and 5 experiments were performed in parallel for each drug concentration group.

[0046] The experimental results are as follows Figure 2As shown in the figure, with the increase of proanthocyanidin concentration, the mitochondrial membrane potential of Cryptocaryon irritans larvae decreased significantly, and its mitochondrial membrane potential showed a decreasing trend over time. The results showed that proanthocyanidins significantly reduced the mitochondrial membrane potential of Cryptocaryon irritans larvae, and it is possible to induce larval apoptosis by reducing the mitochondrial membrane potential pathway, thereby inhibiting the activity of Cryptocaryon irritans larvae.

[0047] Example 3

[0048] Proanthocyanidins inhibit and kill Cryptocaryon cysts

[0049] (1) Preparation of drugs: As in Example 1, 200 mg of proanthocyanidins were accurately weighed, dissolved in 50% ethanol aqueous solution, and fixed to 10 mL to prepare a proanthocyanidin solution with a concentration of 20 mg / mL, and diluted with 50% ethanol aqueous solution to 1.0, 2.5, 5.0, 7.5, 10.0 and 15.0 mg / mL proanthocyanidin solutions.

[0050] (2) Using the ovate pomfret as the host, the ovate pomfret infected with Cryptocaryon irritans and with "white spots" was placed in a 300L funnel-shaped PVC collection bucket, and a glass dish was placed at the bottom for overnight collection. The next day, the glass dish with cysts was taken out, and the mucus on the surface of the cysts was cleaned with filtered and sterilized seawater. The cysts were pipetted into a 24-well cell culture plate with about 20 cysts per well, and 2 mL of fresh filtered and sterilized seawater was added for later use;

[0051] (3) 20 μL of different concentrations of proanthocyanidins were added to the 24-well cell culture plate containing cysts in sequence, so that the final concentration of proanthocyanidins was 10.0, 25.0, 50.0, 75.0, 100.0, 150.0 and 200.0 μg / mL. A 50% ethanol aqueous solution and a formaldehyde solution with the same concentration of proanthocyanidins were used as negative and positive controls, respectively. The cells were cultured in a light incubator at 27.0±0.5°C for 12 h. Each well was then 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 light incubator at 27.0±0.5℃ for 5 days, and 1 mL of fresh seawater was replaced every day. The cyst hatching was then observed under a stereomicroscope. The completely empty cyst shells in each well were used as the hatching standard, and the number of completely empty cysts in each well (m2) was counted. The percentage of the tomonts that successfully released theronts (PTR) within 5 days was calculated, and the PTR formula was (m2×100 / m1)%. The inhibition rate of tomont releasing theronts (ITRT) was calculated by formula (2). OriginPro 8.5.1SR2 (OriginLabCorporation, USA) was used for sigmoidal fitting. The fitting equation (Equation 1) in the gadget was used to fit the data of the inhibition rate of cyst hatching by proanthocyanidin concentration and formaldehyde concentration, where Y and X were the concentrations of ITRT and experimental drugs proanthocyanidin and formaldehyde, respectively. The half effective dose (EC 50 ), and each drug concentration group was subjected to 5 parallel experiments.

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

[0054] PTR nc and PTR t Represent the hatching rates of cysts in the negative control and reagent-treated groups, respectively.

[0055] The experimental results are as follows Figure 3 As shown in Figure 2, with the increase of proanthocyanidin concentration, the encapsulation inhibition rate increased in a dose-dependent manner. 50 =34.38 μg / mL (R 2 =0.985), which is higher than the EC of formaldehyde for encapsulation 50 (26.58 μg / mL, R 2 =0.998).

[0056] Note: Figure 3 ITRT: inhibition rate of larvae released from cysts; * indicates that the difference between the experimental group and the negative control group is significant (p < 0.05); ** indicates that the difference between the experimental group and the negative control group is extremely significant (p < 0.01); A, box plot of the inhibitory effect of proanthocyanidins on the hatching of Cryptocaryon cysts; B, box plot of the inhibitory effect of formaldehyde on the hatching of Cryptocaryon cysts.

[0057] Example 4

[0058] Proanthocyanidins inhibit and kill Cryptocaryon irritans cyst precursors

[0059] (1) Preparation of drugs: As in Example 1, 200 mg of proanthocyanidins were accurately weighed, dissolved in 50% ethanol aqueous solution, and the volume was fixed to 10 mL to prepare a proanthocyanidin solution with a concentration of 20 mg / mL, and the solution was diluted 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 irritans trophozoites on the gill filaments of ovate pomfret 2.5 days after infection with Cryptocaryon irritans. Cut off the tail of the ovate pomfret with "small white spots" and bleed it. Cut the gill filaments and place them on a plate filled with filtered sterilized seawater. The mature trophozoites fall off from the gill filaments naturally. Rinse them three times with filtered 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. The trophozoites are cultured in a light incubator at 27.0±0.5°C for 1 hour to transform them into cyst precursors for later use.

[0061] (3) 20 μL of proanthocyanidin solutions of different concentrations were sequentially added to the 24-well cell culture plate containing the cyst precursors, so that the final concentration of proanthocyanidin was 10.0, 25.0, 50.0, 100.0, 150.0, and 200.0 μg / mL. A 50% ethanol aqueous solution and a formaldehyde solution with the same concentration of proanthocyanidin were used as negative and positive controls, respectively. The cyst precursors were incubated with the reagent treatment groups of different concentrations in a 27.0±0.5°C light incubator for 12 h. Each well was then washed with fresh seawater to remove proanthocyanidin 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 cyst precursors were cultured in a 27.0±0.5℃ light incubator for 5 days, and 1 mL of fresh seawater was replaced every day. The cyst hatching was then observed under a stereomicroscope, and completely empty cysts were used as the standard for evaluating whether the cyst precursors had successfully transformed into cysts and released larvae. The number of completely empty cysts in each well was counted, and the hatching rate (the percentage of the alive protomonts, PAP) and inhibition rate (The inhibition of protomont transforming into tomont, IPTT) of cyst precursors within 5 days were calculated. The hatching rate of cyst precursors that successfully transformed into hatched larvae was calculated as: (m2×100 / m1)%. The inhibition rate of cyst precursor transformation to cyst was calculated by formula (3), and the fitting equation (Equation 1) in Case 1 was used to fit the data on the inhibition rate of cyst precursor transformation by proanthocyanidin concentration. The half effective dose (EC) of proanthocyanidins for inhibiting cyst precursor transformation was calculated by fitting equation (1). 50 ), and 5 experiments were performed in parallel for each drug concentration group.

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

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

[0065] The experimental results are as follows Figure 4 As shown in Figure 2, with the increase of proanthocyanidin concentration, the mortality rate of cyst precursors increased in a dose-dependent manner. 50 =51.22 μg / mL (R 2 =0.991), which is higher than the EC of formaldehyde for encapsulation precursors. 50 (19.02 μg / mL, R 2 =0.947).

[0066] Note: Figure 4 IPTT: percentage of inhibition of transformation of cyst precursors into cysts and release of larvae; * indicates significant difference between the experimental group and the negative control group (p < 0.05); ** indicates 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] Experiment on acute toxicity of proanthocyanidins to oval pomfret

[0069] The oval pomfret used in the experiment was purchased from Chenhai Aquatic Products Company in Sanya, Hainan. After being temporarily cultured for 2 months and stabilized, 180 oval pomfret weighing 27.15±2.36g were randomly selected and divided into 18 culture tanks, divided into 6 groups, 3 parallels in each group, and different proanthocyanidin gavage dose groups of 0, 50, 100, 500, 1000 and 2000 mg / kg (proanthocyanidin / fish body weight) were set. The culture system was equipped with an aeration device and a water pump, and the culture water was kept at a temperature of 26.0±1.0℃, pH 7±0.5, DO higher than 6.5mg / L, and fasted for 1d before gavage.

[0070] Weigh 0.068, 0.135, 0.675, 1.350 and 2.700 g of proanthocyanidins accurately, dissolve them in PBS solution, and make the volume to 5.0 mL to prepare 13.6, 27.0, 135.0, 270.0 and 540.0 mg / mL proanthocyanidin solutions for later use. The safety of oral administration of different concentrations of proanthocyanidins to the oval pomfret was tested by gavage administration. The gavage volume was 100 μL / tail, so that the oval pomfret reached a drug concentration gradient of 0, 50, 100, 500, 1000 and 2000 mg / kg (proanthocyanidins / fish body weight) after oral administration of different concentrations of proanthocyanidins. The oval pomfret was observed for 14 days, and the behavior of the experimental oval pomfret was noted, and their health status and death were recorded and analyzed.

[0071] The survival rate of oval pomfret after oral administration Figure 5 As shown in the data, 6 oval pomfret died in the 2000 mg / kg dose group within 14 days. The dead fish bodies were observed to have damaged gill covers. Dissection revealed ascites in the dead oval pomfret, but the internal organs were intact. No fish in other dose groups or the control group died or had any fatal symptoms. Their behavior remained good and they were able to take in feed on time. A careful examination of their appearance revealed no changes. In addition, when all oval pomfret were dissected, no water accumulation or sticky substances were found in the abdomen, and no swelling of the organs was found. Proanthocyanidins have a relatively high safety concentration of 1000 mg / kg (drug / fish weight). It can be preliminarily determined that proanthocyanidins have good safety, providing a safety guarantee for the development of feed additives for the treatment of "white spot disease".

[0072] Proanthocyanidins have good in vitro anti-Cryptocera larvae, cysts and cyst precursors, and have a significant killing effect on the larvae. After incubation with the larvae, proanthocyanidins can reduce the mitochondrial membrane potential of the larvae of Cryptocaryon irritans, thereby reducing the activity of the larvae. The acute toxicity experiment by oral administration shows that this natural product has a very high drug safety concentration, so it has a good development prospect in production as a feed additive or soaking solution.

Claims

1. Application of proanthocyanidins in the preparation of drugs for inhibiting and killing parasitic ciliates in fish ectoparasites.

2. The use of proanthocyanidins according to claim 1 in the preparation of a drug for inhibiting and killing parasitic ciliates in fish ectoparasites, characterized in that: The fish external parasitic ciliate is Cryptocaryon irritans Brown, with a Latin name of Cryptocaryon irritans.

3. Use of the proanthocyanidins according to claim 1 or 2 in the preparation of a drug for inhibiting and killing parasitic ciliates in fish ectoparasites, characterized in that: The fish is an oval pomfret, with the Latin name Trachinotus ovatus.

4. The use of proanthocyanidins according to claim 1 or 2 in the preparation of a drug for inhibiting and killing parasitic ciliates in fish ectoparasites, characterized in that: The concentration of proanthocyanidins in the medicine is 10 to 200 μg / mL.

5. The use of proanthocyanidins according to claim 4 in the preparation of a drug for inhibiting and killing parasitic ciliates in fish ectoparasites, characterized in that: The concentration of proanthocyanidins in the medicine is 10 to 100 μg / mL.

6. Use of the proanthocyanidins according to claim 1 or 2 in the preparation of a drug for inhibiting and killing parasitic ciliates in fish ectoparasites, characterized in that: The dosage form of the medicine is a mixed liquid, powder, pulverulent, emulsion or medicines of different dosage forms prepared together with pharmaceutically acceptable carriers and excipients.

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