Application of the bark of Aopsis pilosula in preparing products for preventing and / or treating marine fish scutellaria disease

Abstract

The present invention discloses the use of conophora japonica in the preparation of products for preventing and / or treating shield ciliate disease in marine fish. The conophora japonica may be a conophora japonica preparation. The preparation comprises an active ingredient, and the active ingredient is conophora japonica, an alcohol extract of conophora japonica, or an aqueous extract of conophora japonica. The present invention confirms through in vitro insecticide tests and animal infection tests that the addition of conophora japonica can kill shield ciliates, and can significantly reduce the infection rate and mortality rate of shield ciliates in turbot, thus having a good effect in preventing and treating shield ciliate disease. Conophora japonica has the advantages of low toxicity to turbot and environmental protection, and can reduce the pollution to the environment caused by formaldehyde, copper sulfate, etc. emitted during the turbot farming process for the treatment of shield ciliate disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquaculture disease prevention and control, and in particular relates to the application of Acanthopanax serrata in preparing products for preventing and / or treating marine fish clade ciliate disease. Background Art

[0002] The turbot (Scophthalmus maximus), commonly known as the turbot, originally ranged from the eastern Atlantic Ocean to the northeastern European coast, living a benthic life on the seafloor. Since its introduction to my country, turbot has generated considerable economic benefits due to its unique advantages, and its cultivation has become a crucial pillar of my country's marine aquaculture sector. However, with the continuous expansion of turbot aquaculture and increasing stocking densities, coupled with slow progress in disease control, various aquaculture diseases have emerged one after another. Among these aquaculture diseases, parasitic diseases caused by scutellum, cryptorchids, trichodina, and flagellates have caused significant losses to the turbot aquaculture industry. The most serious of these is scutellum disease, a rapidly fatal systemic infection. Turbot diseases caused by scutellum are highly contagious and devastating. An outbreak of these diseases, especially in seedlings, often results in significant economic losses for aquaculture farmers. The primary pathogen is Heterozoan cerebri, a parasite that can enter the aquaculture system through various pathways, causing disease in turbot. Shield ciliates are not only found in large numbers in the affected areas, body surfaces, and gills of turbot, but also in the liver, spleen, kidneys, brain, heart, and other organs. Significant quantities have also been found in the ascites. In the early stages of infection, white spots appear on the fish's body surface and mucus secretion increases. As the disease progresses, the affected tissues become red and swollen, feeling soft and swollen to the touch. In severe cases, lesions may also appear on the fish's trunk, sometimes developing ulcers and bleeding. Affected turbot typically darken in color, exhibit significantly reduced vitality, decrease feed intake, and slow growth. They no longer cluster in the aquaculture pond, but instead disperse, often exhibiting abnormal swimming behavior such as circling. Furthermore, ciliates can infect most internal organs, including the brain.

[0003] Currently, there are few effective treatments for scutellaria diseases in China. In actual aquaculture production, antibiotics and chemical disinfectants are often used to treat fish diseases to prevent and control disease. Therefore, the search for new, safe, reliable, and environmentally friendly insecticides has become an urgent issue.

[0004] Aopikopi belongs to the genus Liriodendron of the Magnoliaceae family, also known as horse laurel bark. It is warm in nature and spicy in taste, and has the effects of dispelling wind and dampness, dispersing cold and relieving cough. It is generally used to treat rheumatic pain, wind-cold cough and other diseases. There is currently no report that Aopikopi can kill shield ciliates. Summary of the Invention

[0005] The present invention aims to provide the use of the cortex ophiopogonis in the preparation of a product for preventing and / or treating scutellariasis in marine fish. This product has the advantages of low toxicity to turbot and environmental friendliness, reducing environmental pollution caused by formaldehyde, copper sulfate, and other substances emitted during turbot aquaculture to treat scutellariasis.

[0006] In a first aspect, the present invention provides the use of Psoralea corylifolia or its preparation in the preparation of a product for preventing and / or treating chelonian disease in marine fish.

[0007] In at least one embodiment of the present invention, the marine fish is turbot.

[0008] In at least one embodiment of the present invention, the Cortex Phellodendri or its preparation is used as the sole active ingredient;

[0009] The preparation includes an active ingredient, which is the cortex ophiopogonis, the cortex ophiopogonis alcohol extract or the cortex ophiopogonis water extract. It is understandable that, in order to facilitate the preparation of various dosage forms, the preparation may also include excipients acceptable in traditional Chinese medicine preparations.

[0010] In at least one embodiment of the present invention, the product is a medicine or feed. Depending on the mode of administration (e.g., oral administration or medicinal bath), the dosage form of the preparation includes but is not limited to tablets, powders, granules, decoctions, and wines.

[0011] As an example, the preparation is a powder of Aopikopi (Aopikopi) root, and its preparation method includes the following steps: taking Aopikopi (Aopikopi) root, pulverizing it, and passing it through a 100-mesh sieve. Alternatively, the dosage is as follows: adding 100 to 1000 g (e.g., 200 g) of Aopikopi (Aopikopi) powder to every 100 kg of feed, and feeding it to sick turbot.

[0012] In an alternative embodiment, the preparation is an alcohol extract of the conophora bark. The preparation method of the alcohol extract of the conophora bark comprises the following steps: extracting the conophora bark and adding ethanol for maceration extraction. Furthermore, the ratio of conophora bark to ethanol is 1000g: (8000-12000)mL, including but not limited to 1000g:10000mL; the mass concentration of the ethanol is 95%-100%, including but not limited to food-grade 95% ethanol (aqueous solution); it is understood that 100% refers to anhydrous ethanol; during the maceration extraction step, the temperature is 25°C-50°C, including but not limited to room temperature of 25°C; during the maceration extraction step, each extraction lasts 48-96 hours, including but not limited to 48 hours; during the maceration extraction step, the extraction is repeated 2-3 times, and the extracts are combined. Furthermore, after extraction, the extract is concentrated to 1g / mL based on the crude drug amount. Optionally, the usage and dosage are as follows: take 1000g of conophora bark, extract it with ethanol, and then sprinkle the extract into 2000kg of water (breeding density 30 to 40 fish / m³) to perform a medicinal bath on the diseased turbot.

[0013] In an alternative embodiment, the method for preparing the aqueous extract of the Cortex Phellodendri includes the following steps: The method for preparing the aqueous extract of the Cortex Phellodendri comprises: taking the Cortex Phellodendri, adding water, and decocting the Cortex Phellodendri, and concentrating the extract after decocting. Furthermore, the ratio of the Cortex Phellodendri to water is 1000 g: (8000-12000 mL), including but not limited to 1000 g: 10000 mL; the decocting step is performed 2-3 times (e.g., 2 times), each time for 1 hour; and the concentration step is performed to a crude drug content of 1 g / ml.

[0014] In the above application of the present invention, the therapeutic effect is specifically reflected in reducing the number of turbot shield ciliates parasites and / or reducing the mortality rate of turbot.

[0015] In a second aspect, the present invention provides a marine fish feed comprising a basic fish feed and a feed additive, wherein the feed additive is Psoralea corylifolia or a preparation thereof.

[0016] In at least one embodiment of the present invention, the marine fish is turbot. For example, the composition of the basic fish feed, by weight, is as follows: 45%-50% fish meal, 20%-25% soybean meal, 15%-20% wheat flour, 5%-8% fish oil, 2%-3% soy lecithin, 1%-2% monocalcium phosphate, 1%-1.5% vitamin premix, and 1%-1.5% mineral premix. The specific composition can be adjusted based on the needs of the turbot at different growth stages.

[0017] In at least one embodiment of the present invention, the Cortex Phellodendri or its preparation is used as the sole active ingredient;

[0018] The preparation includes an active ingredient, which is the cortex phellodendri, an alcohol extract of the cortex phellodendri or a water extract of the cortex phellodendri. It can be understood that in order to facilitate the preparation of various dosage forms, the preparation may also include excipients acceptable in each preparation. The dosage form of the preparation can be reasonably selected according to the type of feed.

[0019] The mass percentage of the feed additive in the marine fish feed is 0.1% to 1% based on the scutellaria baicalensis. As an example, the preparation is a scutellaria baicalensis powder, and the mass percentage of the scutellaria baicalensis powder in the marine fish feed is 0.2%.

[0020] In a third aspect, the present invention provides the use of Psoralea corylifolia or its preparation in any of the following:

[0021] A1) Use in the preparation of a product for killing shield ciliates;

[0022] A2) Killing of shield ciliates in vitro.

[0023] In the above applications, the in vitro killing of shield ciliates is for non-therapeutic purposes and does not include wounds and infected areas.

[0024] In at least one embodiment of the present invention, the Cortex Phellodendri or its preparation is used as the sole active ingredient.

[0025] The formulation includes an active ingredient, which is the cortex ophiopogonis, an alcohol extract of the cortex ophiopogonis, or an aqueous extract of the cortex ophiopogonis. It is understood that, to facilitate preparation into various dosage forms, the formulation may also include excipients acceptable in various formulations. The formulation form of the formulation can be appropriately selected based on its application scenario. Optionally, the product is an insecticide.

[0026] In a preferred embodiment of the present invention, the active ingredient is an alcohol extract of the tangerine peel. The preparation method of the alcohol extract of the tangerine peel comprises the following steps: extracting the tangerine peel and adding ethanol for maceration extraction. Furthermore, the ratio of the tangerine peel to ethanol is 1000g: (8000-12000)mL, including but not limited to 1000g:10000mL; the mass concentration of the ethanol is 95%-100%, including but not limited to food-grade 95% ethanol; it is understood that 100% refers to anhydrous ethanol; the maceration extraction step is performed at a temperature of 25°C-50°C, including but not limited to room temperature of 25°C; each extraction step lasts 48-96 hours, including but not limited to 48 hours; the extraction step is performed two to three times, and the extracts are combined. Furthermore, after extraction, the extract is concentrated to 1g / mL based on the crude drug amount.

[0027] In an alternative embodiment, the method for preparing the aqueous extract of the Cortex Phellodendri includes the following steps: The method for preparing the aqueous extract of the Cortex Phellodendri comprises: taking the Cortex Phellodendri, adding water, and decocting the Cortex Phellodendri, and concentrating the extract after decocting. Furthermore, the ratio of the Cortex Phellodendri to water is 1000 g: (8000-12000 mL), including but not limited to 1000 g: 10000 mL; the decocting step is performed 2-3 times (e.g., 2 times), each time for 1 hour; and the concentration step is performed to a concentration level of 1 g / mL based on the crude drug amount.

[0028] Optionally, based on the conopsis bark, the addition concentration (based on the amount of crude drug) of the conopsis bark or its preparation in the water body (such as seawater) infected with shield ciliates is ≥0.25g / L, such as 0.25g / L.

[0029] The present invention has the following beneficial effects:

[0030] The invention proves through in vitro insecticide test and animal infection test that adding the conophaga bark can kill the shield ciliates, can significantly reduce the infection rate and mortality rate of the shield ciliates of turbot, and has a good effect of preventing and treating the shield ciliates disease.

[0031] The present invention discovers for the first time that the cortex phellodendri has the effect of killing shield ciliates, can significantly reduce the mortality rate of turbot shield ciliate disease, and reduces the parasitic number of turbot shield ciliates. It has the advantages of low toxicity to turbot and environmental protection, and can reduce the pollution to the environment caused by formaldehyde, copper sulfate, etc. emitted during the turbot breeding process due to the treatment of shield ciliate disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a photo of the diseased turbot temporarily reared in Example 1 of the present invention.

[0033] Figure 2 These are photos of the group experiment in Example 1 of the present invention. DETAILED DESCRIPTION

[0034] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0035] Unless otherwise specified, the methods used in the following examples are all conventional methods and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0036] The Aopsis pilosula medicinal materials in the following embodiments are Chinese herbal medicine slices purchased from Chengdu Yuhetang E-Commerce Co., Ltd.

[0037] Preparation Example 1: Preparation of Aopuki Powder

[0038] Take the Aopikopi medicinal material, grind it, and pass it through a 100-mesh sieve to obtain the Aopikopi powder.

[0039] Dosage and administration: Take 0.2 kg of the above powder, mix it into 100 kg of feed, and feed according to the daily intake.

[0040] Preparation Example 2: Preparation of Alcohol Extract of Psoralea corylifolia

[0041] Take 1000 g of the bark of Psoralea corylifolia, add 10000 mL of food-grade 95% ethanol, soak and extract for 48 hours, extract twice, combine the extracts, and concentrate under reduced pressure to 1000 mL.

[0042] Dosage and administration: Take 1L of the above extract and sprinkle it into 2000kg of seawater (breeding density 30-40 fish / m³) for medicated bathing of turbot infected with scutellaria disease. Bath for 30 minutes every day for 5-7 consecutive days.

[0043] Preparation Example 3: Preparation of Aqueous Extract of Psoralea corylifolia

[0044] Take 1000g of Aopsis pilosula, add 10000mL of water, decoct twice, each time for 1 hour, combine the decoctions, and concentrate under reduced pressure to 1000mL.

[0045] Example 1, Preparation Example 1 Pharmacological Experiment

[0046] This example is used to observe the efficacy of Preparation Example 1 in preventing and treating turbot shield ciliate disease.

[0047] The following is an explanation of the anti-shield ciliate test of Aopikopi officinalis with reference to specific experiments.

[0048] A turbot farm with shield ciliate disease was selected. The infected turbot were confirmed by microscopic observation of gills with shield ciliates. The infected turbot were selected and transferred to a temporary holding pond ( Figure 1 ), 30 turbot (200g-300g / piece) infected with Shield Ciliate Disease were selected and randomly divided into 2 groups ( Figure 2 After acclimation, one group was administered the drug from Preparation Example 1. 0.2 kg of Preparation Example 1 was mixed into 100 kg of feed (see Table 1 for the recipe) and fed at a rate of 3%. The other group served as the model control group and received no medication. The maintenance temperature was 18°C. During the experiment, diseased gills from the test and model control groups were gently scraped with an inoculating loop and smeared onto a glass slide. The viability and number of ciliates were observed under a microscope. The experiment was conducted for 5 days. The results are shown in Table 2.

[0049]

[0050] Evaluation criteria:

[0051] Abundant: At 100x magnification, there are few spaces between the insects, and they are almost packed together, filling the entire field of view. This is scored as 6 points.

[0052] Many: More than 20 insects are within the field of view at 100x magnification. Score 5 points.

[0053] Medium: No more than 20 insects are visible in the field of view at 100x magnification. Score: 4 points.

[0054] Few: Under a 100x lens, there are no more than 10 insects in the field of view. Score: 3 points.

[0055] Very few: No more than 3 insects are visible in the field of view at 100x magnification. Score 1 point.

[0056] No parasites: No parasites are found in the field of view at 100x magnification, and no parasites are found after changing the field of view. The score is 0.

[0057]

[0058] "*" indicates that the evaluation score of the Example group after 5 days of medication was significantly different from that of the model group.

[0059] The experimental results showed that Aopicoides can significantly reduce the number of shield ciliates parasites in turbot and reduce the mortality rate, while the number of shield ciliates parasites in the model group is still increasing. Aopicoides has a significant effect compared with the model control group, indicating that Aopicoides has a significant effect in resisting turbot shield ciliate disease.

[0060] Pharmacological efficacy experiments of Example 2, Preparation Example 2 and Preparation Example 3

[0061] In vitro insecticidal test: Take a 96-well deep-well plate, first add 0.6 mL of seawater to each well, then add 0.6 mL of Preparation Example 2 to the first well, dilute in series, and dilute to the 12th well. The drug concentrations are 1 / 2-1 / 4096 (for example, 0.6 mL of drug solution + 0.6 mL of seawater corresponds to a drug concentration of 1:2, and then a double dilution corresponds to a drug concentration of 1:4). According to this method, Preparation Example 3 and food-grade 95% ethanol are diluted, 120 μL of each diluted sample is pipetted, and added to a 96-well cell culture plate, and then 120 μL of shield ciliate solution is added to each well (shield ciliates are derived from clinically diseased turbot, cultured and propagated in vitro, and can be used for in vitro insecticidal tests when there are more than 10 shield ciliates in the 100x microscope field of view). Gently shake the cell culture plate for 1 minute, then let it stand for 2 hours, and observe under an inverted microscope to see if the ciliates are dead to determine the drug effect. At the same time, a blank control group was set up. The concentration of shield ciliates in the blank control group was the same as that in the experimental group, and only an equal amount of seawater or food-grade 95% ethanol was added.

[0062] The minimum insecticidal concentration was defined as the lowest concentration that killed all shield ciliates under microscopic examination. The results showed that the minimum insecticidal concentration for the 95% food-grade ethanol solvent control was 1:8, the in vitro minimum insecticidal concentration for Preparation 2 was 1:4096, and the minimum insecticidal concentration for Preparation 3 was 1:512 (0.000244 ml / ml). This indicates that the alcohol extract of the cortex ophiopogonis has a strong in vitro insecticidal effect on shield ciliates, and the water extract of the cortex ophiopogonis also has in vitro insecticidal effects on shield ciliates.

[0063] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, can implement the present invention in a wider range under equivalent parameters, concentration and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any variation, purposes or improvements of the present invention, including departing from the disclosed scope in the application and the changes made with conventional techniques known in the art.

Claims

1. Use of the Cortex Aconiti Lateralis Preparata or its preparation in the preparation of a product for preventing and / or treating Shield ciliate disease in marine fish; The marine fish is turbot.

2. The use according to claim 1, characterized in that: The preparation comprises an active ingredient, and the active ingredient is the cortex ophiopogonis, the cortex ophiopogonis alcohol extract or the cortex ophiopogonis water extract.

3. The use according to claim 1, characterized in that: The product is a medicine or feed.

4. Use of Aopsoriasis bark or its preparation in any of the following: A1) Preparation of products for killing turbot shield ciliates; A2) In vitro killing of turbot shield ciliates.

5. The use according to claim 4, characterized in that: The preparation comprises an active ingredient, and the active ingredient is the cortex ophiopogonis, the cortex ophiopogonis alcohol extract or the cortex ophiopogonis water extract.

6. The use according to claim 5, characterized in that: The preparation method of the alcohol extract of the Atractylodes lancea bark comprises the following steps: taking the Atractylodes lancea bark, adding ethanol and performing immersion extraction.

7. The use according to claim 6, characterized in that: The ratio of the scutellaria baicalensis bark to ethanol is 1000 g: (8000-12000) mL; The mass concentration of the ethanol is 95% to 100%; In the maceration extraction step, the temperature is 25°C to 50°C; In the maceration extraction step, each extraction takes 48 to 96 hours; In the immersion extraction step, the extraction times are 2 to 3 times, and the extracts are combined.

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