Magnolol compound and application thereof

Abstract

The invention discloses a magnolol compound and application thereof, and belongs to the technical field of aquatic animal disease control. The magnolol compound is a derivative of magnolol, comprises compounds C1-C4, and has an excellent effect of resisting fish parasites. The magnolol compound kills the fish parasites such as ichthyophthirius multifilis and cryptocaryon irritans through soaking administration or oral administration, the killing effect is better than that of magnolol, the ichthyophthirius multifilis and / or cryptocaryon irritans grazing bodies can be killed, and breeding of ichthyophthirius multifilis and / or cryptocaryon irritans cysts is effectively inhibited. On the basis of keeping the original green and pollution-free advantages of magnolol, the killing activity of magnolol on fish parasites is enhanced, and a green, safe and high-activity medicament is provided for prevention and control of aquatic diseases.

Description

Technical Field

[0001] This invention belongs to the field of aquatic animal disease prevention and control technology, and relates to a magnolol compound and its application. Background Technology

[0002] In recent years, with the rapid development of aquaculture, diseases have frequently occurred in both freshwater and marine aquaculture, seriously restricting the healthy development of the industry and becoming one of the key issues that urgently need to be addressed to achieve high efficiency and high yield in aquaculture. Currently, the incidence of fish parasitic diseases is relatively high, and the epidemic period is relatively long, posing a serious threat to aquaculture. Among them, Ichthyophthirius multifiliis (Ichthyophthirius multifiliis) is particularly prevalent. Ichthyophthirius multifiliis ) and stimulating cryptocaryon ( Cryptocaryon irritans Brown These are two types of ciliated ectoparasites that cause significant damage, serving as pathogens of "white spot disease" in freshwater and marine fish, respectively. They have a wide host range and low host specificity, infecting most freshwater or marine fish species and causing similar pathological changes, often leading to large-scale mortality of farmed fish.

[0003] Currently, the treatment of white spot disease mainly involves chemical drugs such as copper sulfate, quinine, methylene blue, malachite green, benzalkonium chloride, phenoxyethanol, and formalin. While chemical drugs can provide good therapeutic effects, they easily cause environmental pollution, and drug residues in fish can endanger the safety of edible fish, thus jeopardizing human health. Furthermore, long-term use of chemical drugs increases parasite resistance, and some relatively safe drugs, such as medium-chain fatty acids, do not meet efficacy standards. Therefore, finding environmentally friendly drugs with highly effective antiparasitic activity is crucial for the prevention and control of Ichthyophthirius multifiliis and Cryptocaryon irritans.

[0004] Patent CN105726522A discloses that magnolol, a natural product extracted and isolated from the dried bark and root bark of *Magnolia officinalis* (a plant in the Magnoliaceae family), possesses excellent insecticidal activity, and that magnolol and honokiol exhibit significant activity against *Cryptocaryon irritans*. However, while magnolol and honokiol show significant insecticidal effects, they still suffer from drawbacks such as poor physicochemical and pharmacokinetic properties and relatively low therapeutic index. Therefore, based on the basic skeletal structure of the natural active molecules of magnolol and honokiol, this paper proposes to design and synthesize magnolol derivatives. This aims to improve their pharmacological properties, enhance their insecticidal activity, reduce toxicity, and increase oral bioavailability while maintaining their green and pollution-free advantages. This is of great significance for further enhancing the application of this type of compound in the prevention and control of aquatic diseases. Summary of the Invention

[0005] To solve the above technical problems, the present application provides a magnolol compound and its application. The magnolol compound is a derivative of magnolol, including compounds C1-C4, and has excellent anti-fish parasite effect. The magnolol compound of the present application can kill Ichthyophthirius multifiliis, stimulate Cryptocaryon irritans and other fish parasites through soaking administration or oral administration, and the killing effect is better than that of magnolol. The present application realizes the enhancement of the killing activity of fish parasites on the basis of retaining the original green and non-polluting advantages of magnolol, and provides a green, safe and active medicament for the prevention and control of aquatic diseases.

[0006] To achieve the technical purpose of the present application, in one aspect, the present application provides a magnolol compound having a structure as shown in C1-C4,

[0007] In another aspect, the present application claims the application of the above-mentioned magnolol compound in preparing a medicament for killing fish parasites.

[0008] Further, the magnolol compound can kill fish parasites through soaking administration or oral administration, and the fish parasites include Ichthyophthirius multifiliis Ichthyophthirius multifiliis and Cryptocaryon irritans Cryptocaryon irritans The concentration of the soaking-administered magnolol compound C1 is less than or equal to 2.0 mg / L, the concentration of the soaking-administered magnolol compound C2 is less than or equal to 3.0 mg / L, the concentration of the soaking-administered magnolol compound C3 is less than or equal to 2.0 mg / L, and the concentration of the soaking-administered magnolol compound C4 is less than or equal to 2.5 mg / L.

[0009] Specifically, through the toxicity test of the magnolol compound on goldfish, it is found that when the concentration of compound C1 is 1.5-2.0 mg / L, no goldfish appears to be poisoned, when the concentration of compound C1 is increased to 2.5 mg / L, about 13.3% of the goldfish dies, and when the concentration of compound C1 is 3.0, 3.5 and 4.0 mg / L, the mortality rates of the tested goldfish are 53.3%, 83.3% and 100.0% respectively after 96 h of treatment. When the concentration of compound C2 is 2.0-3.0 mg / L, no death and poisoning of the tested fish occurs after 96 h of treatment. When the concentration of compound C2 is increased to 4.0, 5.0 and 6.0 mg / L, the mortality rates of the tested goldfish are 56.7%, 83.3% and 100% respectively.

[0010] Specifically, the present application finds that, through the toxicity test of magnolol compounds on goldfish, when the concentration of compound C3 is 1.5-2.0 mg / L, no fish in the test group dies of poisoning; when the concentration is increased to 2.5 mg / L, about 8 fish die within 96 hours, and the mortality rate is 26.67%; when the concentration of compound C3 is 3.0, 3.5 and 4.0 mg / L, 66.67%, 76.67% and 100% of the fish die after 96 hours, respectively.

[0011] Further, the feeding amount of the orally administered magnolol compounds C1-C4 is not less than 60 mg / kg·d -1 .

[0012] Specifically, the present application finds that, through the test of the influence of orally administered magnolol compounds on the number of fish infected with Ichthyophthirius multifiliis, after 6 days of introducing the fish infected with Ichthyophthirius multifiliis, the fish in each group begin to be infected, and with the extension of the test time, the peak value of the fish in the control group is 24 Ichthyophthirius multifiliis per visual field, while the peak value of the fish in the magnolol group is 13 Ichthyophthirius multifiliis per visual field; compared with the magnolol group and the control group, the number of Ichthyophthirius multifiliis in the fish in the test groups orally administered with compounds C1-C4 is further reduced to 5-9 Ichthyophthirius multifiliis per visual field.

[0013] Specifically, the present application finds that, through the test of the protective effect of orally administered magnolol compounds on fish infected with Ichthyophthirius multifiliis, after 10 days of introducing the diseased fish, the fish in the control group and the magnolol group begin to die, while the fish in the test groups orally administered with compounds C1 and C2 do not die; after 26 days of continuous oral administration, the survival rate of the fish in the control group is only about 10%, the survival rate of the fish in the magnolol group is increased to 60%, and the survival rate of the fish in the test groups orally administered with compounds C1 and C2 is higher than 79%; compared with the magnolol group and the control group, the survival rate is significantly improved (P<0.001). p <0.001), similarly, the survival rate of the fish in the test groups orally administered with compounds C3 and C4 is also significantly improved, which is 80% and 73.33%, respectively.

[0014] Further, the killing of fish parasites includes killing the predators and / or cysts of fish parasites. The magnolol compounds reduce the number of fish infected with parasites, and improve the survival rate of fish. The fish is infected with a parasite disease, and the parasite disease is Ichthyophthirius multifiliis disease and / or Ichthyophthirius multifiliis disease.

[0015] Specifically, the present application finds, through testing the killing effect of magnolol compounds on fish parasites, that the EC 50 values of the magnolol compounds on Ichthyophthirius multifiliis trophonts are 0.035-0.331 mg / L, the EC 50 values on Ichthyophthirius multifiliis cysts are 0.024-0.423 mg / L, the EC 50 values on Cryptocaryon irritans trophonts are 0.044-0.203 mg / L, the EC 50 values on Cryptocaryon irritans cysts are 0.053-0.215 mg / L, and the killing activities are all superior to magnolol, and the killing effects on Ichthyophthirius multifiliis and / or Cryptocaryon irritans trophonts are better, and the reproduction of Ichthyophthirius multifiliis and / or Cryptocaryon irritans cysts is effectively inhibited.

[0016] Compared with the prior art, the technical scheme provided by the present application at least has the following beneficial effects or advantages: (1) The magnolol compounds have better effects of killing fish parasites, preventing and treating Ichthyophthirius multifiliis disease and Cryptocaryon irritans disease and other fish parasite diseases, and the present application provides a green, safe and active medicament for the prevention and treatment of aquatic diseases. The present application finds, through testing the killing effect of magnolol compounds on fish parasites, that the EC 50 values of the magnolol compounds on Ichthyophthirius multifiliis trophonts are 0.035-0.331 mg / L, the EC 50 values on Ichthyophthirius multifiliis cysts are 0.024-0.423 mg / L, the EC 50 values on Cryptocaryon irritans trophonts are 0.044-0.203 mg / L, the EC 50 values on Cryptocaryon irritans cysts are 0.053-0.215 mg / L, and the killing activities are all superior to magnolol, and the killing effects on Ichthyophthirius multifiliis and / or Cryptocaryon irritans trophonts are better, and the reproduction of Ichthyophthirius multifiliis and / or Cryptocaryon irritans cysts is effectively inhibited.

[0017] (2) The magnolol compounds have low toxicity to fish. The present application finds, through testing the toxicity of the magnolol compounds to goldfish, that the safe use concentration of compound C1 should be less than or equal to 2.0 mg / L, compound C2 is safe to goldfish at a concentration less than or equal to 3.0 mg / L, the safe use concentration of compound C3 should be less than or equal to 2.0 mg / L, and the safe use concentration of compound C4 should be less than or equal to 2.5 mg / L.

[0018] (3) The oral magnolol compounds can effectively protect the goldfish from the infection of Ichthyophthirius multifiliis and significantly improve the survival rate of the goldfish. After the goldfish infected with Ichthyophthirius multifiliis for 6 days, the test fish in each group began to be infected, and with the extension of the test time, the peak value of the number of Ichthyophthirius multifiliis in the control group was 24, while the peak value of the number of Ichthyophthirius multifiliis in the magnolol group was 13. Compared with the magnolol group and the control group, the number of Ichthyophthirius multifiliis in the test group of the oral compounds C1-C4 was further reduced to 5-9.

[0019] (4) The oral magnolol compounds can effectively improve the survival rate of the goldfish with parasitic diseases. The test of the protective effect of the oral magnolol compounds on the goldfish with Ichthyophthirius multifiliis found that after the diseased goldfish were introduced for 10 days, the goldfish in the control group and the magnolol group began to die, while the goldfish in the test groups of the oral compounds C1 and C2 did not die. After continuous oral administration for 26 days, the survival rate of the goldfish in the control group was only about 10%, while the survival rate of the goldfish in the oral magnolol group was increased to 60%, and the survival rate of the goldfish in the test groups of the oral compounds C1 and C2 was higher than 79%, which was significantly improved compared with the magnolol group and the control group. p <0.001); similarly, the survival rate of the goldfish in the test groups of the oral compounds C3 and C4 was also significantly improved, which was 80% and 73.33%, respectively. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or the prior art description. Obviously, the drawings in the following description are only some embodiments of the present application.

[0021] Figure 1 Influence of oral magnolol compounds on the survival rate of goldfish.

[0022] Figure 2 Influence of oral magnolol compounds on the parasite load of fish. DETAILED DESCRIPTION

[0023] In the following, the technical solutions of the present application will be described in conjunction with the embodiments, but the present application is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0024] Magnolol and honokiol drugs were purchased from Shanghai Maikelin Biochemical Technology Co., Ltd., and the purity was HPLC grade (≥98%).

[0025] Example 1 This example provides the preparation of magnolol compounds.

[0026] Compounds C1-C4 were synthesized by our laboratory, separated by silica gel column chromatography, identified by nuclear magnetic resonance spectroscopy, and the purity was greater than 97.0%.

[0027] 1.1 Synthesis method of compounds C1 and C3 1 mmol of magnolol / honokiol was dissolved in 6 mL of dichloromethane, 2 mmol of N-bromosuccinimide (NBS) was added to the dichloromethane solution of magnolol / honokiol, and the mixture was stirred at room temperature for 6-12 h. The reaction progress was monitored by TLC method. After the reaction was completed, about 20 mL of dichloromethane was added to the system and extracted three times, washed with water, and dried with anhydrous sodium sulfate overnight to obtain the crude product. The derivative C1 / C3 was separated by silica gel column chromatography with petroleum ether and ethyl acetate as the mobile phase (5:1, v / v).

[0028] The nuclear magnetic resonance hydrogen spectrum of compound C1 synthesized by the above method is: 1 H NMR (400 MHz, Chloroform-d) δ 7.30 (d, J = 2.1 Hz, 2H), 7.19 (s, 2H), 6.96 (d, J = 2.1 Hz, 2H), 5.87 (ddt, J = 17.8, 9.3, 6.7 Hz, 2H), 5.08-4.99 (m, 4H), 3.28 (d, J = 6.8 Hz, 4H). The nuclear magnetic resonance carbon spectrum of compound C1 is: 13 C NMR (100 MHz, Chloroform-d) δ 147.68, 136.72, 133.93, 132.22, 131.08, 125.43, 116.52, 111.21, 39.00. 1.2 Synthesis method of compounds C2 and C4 1.07 g of magnolol / honokiol was dissolved in 10 mL of 10% NaOH solution. Five equivalents (five times the molar amount) of 1-chloropropane (molar ratio of magnolol / honokiol to 1-chloropropane 1:5) were added to the resulting solution, followed by 0.10 mg of tetrabutylammonium bromide as a catalyst. The mixture was stirred at room temperature and refluxed at 80 °C for 24 h. After the reaction was complete, dichloromethane was added for extraction three times, and the lower organic phases were combined. 10–20 g of anhydrous sodium sulfate was added to the resulting organic phase, and the mixture was allowed to stand and dry overnight. After filtering to remove the sodium sulfate solid, the reaction solvent was removed by vacuum distillation (70 °C, 0.07 MPa negative pressure) to obtain the crude product. The crude product was purified by dry loading using silica gel column chromatography (petroleum ether:dichloromethane = 1:1, v / v). TLC was used to detect and collect the eluent containing the target compound. After evaporating the solvent, a yellowish-brown solid product C2 / C4 was obtained.

[0029] The proton NMR spectrum of compound C2 synthesized by the above method is as follows: 1 ¹H NMR (500 MHz, CDCl₃) δ 7.15 (d, J = 1.8 Hz, 2H), 7.11 (dd, J = 8.4, 1.8 Hz, 2H), 6.89 (d, J = 8.3 Hz, 2H), 6.02 – 5.92 (m, 2H), 5.08 (dd, J = 22.0, 5.2 Hz, 4H), 4.14 (t, J = 6.1 Hz, 4H), 3.63 (t, J = 6.1 Hz, 4H), 3.37 (d, J = 6.6 Hz, 4H). The carbon NMR spectrum of compound C₂ is as follows: 13 C NMR (125 MHz, CDCl3) δ 154.11, 137.89, 132.90, 132.38, 128.62, 128.18, 115.72, 113.38, 69.18, 42.05, 39.55. Example 2 This embodiment provides a toxicity test of magnolol compounds on goldfish.

[0030] 2.1 Experimental Animals goldfish( Carassius auratus The goldfish (purchased from the Zhuque Road Flower and Bird Market in Shaanxi Province) were healthy, uniform in size, and weighed 6-8 g. After purchase, they were temporarily kept in a laboratory aquarium for 2 weeks, fed twice a day with goldfish-specific food (purchased from the Zhuque Road Flower and Bird Market in Shaanxi Province) to allow them to adapt to the laboratory environment before conducting experiments.

[0031] 2.2 Test Drugs An appropriate amount of compounds C1, C2, C3 and C4 were weighed into centrifuge tubes containing 1.8 mL of DMSO to make a compound mother liquor with a final concentration of 10,000 mg / L, and filtered using a 0.22 μm sterile filter and stored in a 4°C refrigerator for later use.

[0032] 2.3 Test method The static water test method was used, with fully aerated tap water, pH 7.2, and the water temperature controlled at 22±1°C. The compound C1 mother liquor was first added and mixed thoroughly, and then the test fish were added, with 30 fish per tank. The dissolved oxygen in the water was required to be maintained above 5 mg / L throughout the test. The approximate range of compound concentrations for the formal test was determined by a preliminary test. The preliminary test showed that the fish died within 24 h when the compound concentration was 4.0 mg / L, and no fish died within 96 h when the compound concentration was 1.5 mg / L. On the basis of the preliminary test, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mg / L were set as the test concentrations in the range of 1.5-4.0 mg / L, with an equal difference (tolerance d=0.5). The fish were observed for poisoning and death at 12 h, 24 h, 48 h and 96 h at each compound concentration, and the results are shown in Table 1. The fish were observed at any time during the test, and any dead fish were removed immediately to avoid affecting the water quality and the test results. The fish were judged to be dead when they stopped breathing (gill cover movement stopped) and did not respond to a glass rod or forceps tapping the tail.

[0033] The acute toxicity test of compounds C2-C4 on goldfish was carried out according to the above method, and the test results are shown in Tables 2-4.

[0034] Table 1 Acute toxicity of different concentrations of compound C1 on goldfish

[0035] As shown in Table 1, when the concentration of compound C1 was 1.5-2.0 mg / L, no poisoning of the fish occurred. When the concentration of compound C1 was increased to 2.5 mg / L, about 13.3% of the fish died. After 96 h of treatment with compound C1 at concentrations of 3.0, 3.5 and 4.0 mg / L, the mortality rates of the test fish were 53.3%, 83.3% and 100.0%, respectively, indicating that the safe use concentration of compound C1 should be less than or equal to 2.0 mg / L.

[0036] Table 2 Acute toxicity of different concentrations of compound C2 on goldfish

[0037] From Table 2, it can be seen that when the concentration of compound C2 is 2.0-3.0 mg / L, the goldfish is not poisoned and dies after being treated for 96 h. When the concentration of compound C2 is increased to 4.0, 5.0 and 6.0 mg / L, the mortality of the goldfish is 56.7%, 83.3% and 100% respectively, which indicates that the safe concentration of compound C2 is less than or equal to 3.0 mg / L.

[0038] Table 3 Acute toxicity of compound C3 with different concentrations to goldfish

[0039] From Table 3, it can be seen that when the concentration of compound C3 is 1.5-2.0 mg / L, the goldfish is not poisoned and dies after being treated for 96 h. When the concentration of compound C3 is increased to 2.5 mg / L, about 8 goldfishes die within 96 h, and the mortality is 26.67%. When the concentration of compound C3 is 3.0, 3.5 and 4.0 mg / L, the mortality of the goldfish is 66.67%, 76.67% and 100% respectively, which indicates that the safe concentration of compound C3 is less than or equal to 2.0 mg / L.

[0040] Table 4 Acute toxicity of compound C4 with different concentrations to goldfish

[0041] From Table 4, it can be seen that when the concentration of compound C4 is 2.0-2.5 mg / L, the goldfish is not poisoned and dies after being treated for 96 h. When the concentration of compound C4 is increased to 3.0 mg / L, 1 goldfish dies within 96 h, and the mortality is 3.33%. When the concentration of compound C4 is 4.0, 5.0 and 6.0 mg / L, the mortality of the goldfish is 66.67%, 83.33% and 100% respectively, which indicates that the safe concentration of compound C4 is less than or equal to 2.5 mg / L.

[0042] Example 3 In this example, the killing effect of magnolol compounds on P. peniculata is determined.

[0043] 3.1 Test animals The goldfish (purchased from the flower and bird market in Zhuque Road, Shaanxi Province) weighs 5.7-8.2 g.

[0044] 3.2 Parasites The Ichthyophthirius multifiliis was isolated from sick goldfish in Zhuque Road Flower and Bird Market in Shaanxi Province. The propagation method was as follows: the goldfish was placed in several 40 L water tanks with a temperature of 22.0±2.0℃, and the oxygen was increased by an oxygen pump and the sewage was pumped by siphon method, and 1 / 3 of the water was changed every other day. The method for collecting Ichthyophthirius multifiliis was as follows: the goldfish severely parasitized by Ichthyophthirius multifiliis was placed in a beaker containing 300 mL filtered water for 30 min. Because the goldfish kept swimming, the mature Ichthyophthirius multifiliis fell off the body surface of the goldfish, and the Ichthyophthirius multifiliis cysts were collected by a pipette. Then the collected cysts were cultured at a temperature of 23.5±0.5℃ for 18-20 h to obtain Ichthyophthirius multifiliis predator suspension. The method for counting Ichthyophthirius multifiliis was as follows: 1 μL of Ichthyophthirius multifiliis predator suspension was taken by a pipette and placed on a glass slide, and the number of Ichthyophthirius multifiliis was counted under a dissecting microscope. The average value was taken as the concentration of Ichthyophthirius multifiliis predator suspension, and the concentration of Ichthyophthirius multifiliis was counted according to the concentration.

[0045] 3.3 Test drug The purities of compounds C1-C4 were all greater than 97.0%. An appropriate amount of compound C1, C2, C3 and C4 was weighed and dissolved in a centrifuge tube containing 1.8 mL of DMSO to make the final concentration of the compound mother liquor 10000 mg / L, and a 0.22 μm sterile filter was used for filtration, and the sample was stored in a 4℃ refrigerator for standby.

[0046] 3.4 Killing effect of magnolol phenolic compounds on Ichthyophthirius multifiliis predator (larvae) The killing effect of magnolol phenolic compounds on Ichthyophthirius multifiliis predator was determined by the fixed method. About 300 larvae were placed in each well of a 96-well plate, and then compound C1-C4 mother liquor was added to make the final concentration of magnolol phenolic compounds reach the concentration to be tested, and the test was repeated 3 times. The test was carried out in an environment with a temperature of 23.5±0.5℃, and at 4 h after the action of magnolol phenolic compounds, the dead Ichthyophthirius multifiliis predators were observed and recorded by a dissecting microscope. The Ichthyophthirius multifiliis predators with abnormal morphology or unable to move were determined as dead. The test was repeated at least three times, and the killing effect was verified by using Ichthyophthirius multifiliis predators from different hosts and cultured at different times. The EC 50 values of magnolol phenolic compounds for killing Ichthyophthirius multifiliis predator were calculated and shown in Table 5.

[0047] Table 5 Killing effect of magnolol phenolic compounds on Ichthyophthirius multifiliis predator

[0048] As shown in Table 5, the EC 50 values of magnolol phenolic compounds for killing Ichthyophthirius multifiliis predator were 0.035-0.331 mg / L, and the killing effect was better than that of magnolol, which had a better effect on killing Ichthyophthirius multifiliis predator.

[0049] 3.5 Killing effect of magnolol compounds on P. multigermic cysts In each well of a 24-well plate, 30 P. multigermic cyst precursors were placed, and then compound C1-C4 mother liquor was added to make the final concentration of magnolol compounds reach the concentration to be tested (the final volume of the system was 1 mL). After the 24-well plate was placed in a constant temperature incubator (23.5±0.5℃) for 18-20 h, it was taken out, and the number of dead P. multigermic cysts in each well was recorded under a dissecting microscope (determined as dead P. multigermic cysts that could not complete reproduction), and the number of hatched P. multigermic predators was counted, the reproduction rate of P. multigermic cysts was calculated, the whole test was repeated for 3 times, and it was found by analysis and calculation that the EC 50 values of magnolol compounds for killing P. multigermic cysts were shown in Table 6.

[0050] The reproduction rate of P. multigermic (%) = (the number of P. multigermic predators in each well / the number of P. multigermic predators in the control group in each well) x 100.

[0051] Table 6 Killing effect of magnolol compounds on P. multigermic cysts

[0052] It could be seen from Table 6 that the EC 50 values of magnolol compounds for killing P. multigermic cysts were 0.024-0.423 mg / L, and the killing effect was better than that of magnolol. After treatment with magnolol compounds, the number of predators released by P. multigermic cysts was significantly reduced, indicating that magnolol compounds could effectively inhibit the reproduction of P. multigermic cysts.

[0053] 3.6 Influence of oral administration of magnolol compounds on the number of P. multigermic infected goldfish and the protection effect on diseased goldfish Each day, the goldfish (30 in each group) were fed with medicated feed at 3% of their body weight, and the test drug groups (compound C1-C4) and the magnolol group were administered with drugs at a dose of 60 mg / kg·d -1 . The control group was fed with the same amount of feed without drugs. After 5 days of feeding, the same number of diseased goldfish (each infected with about 200 P. multigermic) were introduced, and then 26 days of continuous feeding was performed. The number of dead goldfish and the parasite load on the fish body were observed and counted every day, and the test was repeated for 3 times. The dead goldfish were promptly removed to avoid affecting other goldfish. After the test, the survival curve of goldfish in each group and the parasite load curve were drawn, and the significance was analyzed using statistical software, and the results were shown in Figure 1 and Figure 2 .

[0054] From Figure 1It was found that 10 days after the introduction of diseased goldfish, mortality began to occur in the control group and the magnolol group, while no mortality occurred in the experimental groups treated with oral compounds C1 and C2. After 26 days of continuous oral administration, the survival rate of goldfish in the control group was only about 10%, while the survival rate of goldfish in the magnolol group increased to 60%, and the survival rate of goldfish in the experimental groups treated with oral compounds C1 and C2 was higher than 79%, showing a significant improvement in survival rate compared with the magnolol group and the control group. p <0.001). Furthermore, the survival rates of goldfish in the oral compound C3 and C4 groups also increased to 80% and 73.33%, respectively. These results indicate that oral compounds C1–C4 can significantly improve the survival rate of goldfish and effectively protect them from infection by *Ichthyophthirius multifiliis*.

[0055] Depend on Figure 2 It was found that infection began to appear in the test fish in each group 6 days after the introduction of the diseased goldfish. As the experimental time was extended, the peak infection count in the control group was 24 *Ichthyophthirius multifiliis* per field of view, while the peak infection count in the magnolol group was 13 *Ichthyophthirius multifiliis* per field of view. Compared with the magnolol group and the control group, the *Ichthyophthirius multifiliis* infection count in the goldfish in the groups that were orally administered compounds C1 and C2 was further reduced to 5-8 *Ichthyophthirius multifiliis* per field of view, indicating that oral administration of magnolol compounds can significantly reduce the number of *Ichthyophthirius multifiliis* infected goldfish. The number of *Ichthyophthirius multifiliis* infected goldfish in the groups that were orally administered compounds C3 and C4 was also reduced to 6-9 trophozoites per field of view. These results confirm that oral administration of magnolol compounds can effectively protect goldfish from *Ichthyophthirius multifiliis* infection, thereby improving the survival rate of goldfish.

[0056] Example 4 This embodiment determined the killing effect of magnolol compounds on Cryptocaryon irritans.

[0057] 4.1 Parasites Irritating Cryptocaryon ( Cryptocaryon irritans Brown The cysts of Cryptocaryon irritans were isolated from the body surface of diseased large yellow croaker and collected in a beaker. Sterilized seawater was added and the cysts were incubated in a 27°C biochemical incubator. After 3-4 days, the larvae were released. The larval suspensions that hatched within 2 hours were collected, mixed, and 10 portions were aspirated onto a glass slide and observed and counted under an optical microscope (40×).

[0058] 4.2 Test Drugs Same as Example 3.

[0059] 4.3 The killing effect of magnolol compounds on Cryptocaryon irritans predatory larvae In each well of the 24-well plate, 1 mL of sterilized filtered seawater containing about 250 Cryptocaryon irritans predators was added, and the test drug solution was added to each well and supplemented with different volumes of sterilized seawater to reach the test drug concentration, the total volume of the test drug solution and the sterilized seawater was 20 μL, and 20 μL of sterile seawater was added as a negative control, then the 24-well plate was placed in a light incubator at 27±0.5°C for 30 min, and then counted under a microscope, and the immobile predators were identified as dead. The whole test was repeated three times, and the EC 50 values of the thick oleanol compounds for killing Cryptocaryon irritans predators are shown in Table 7.

[0060] Table 7 Killing effect of thick oleanol compounds on Cryptocaryon irritans predators

[0061] As can be seen from Table 7, the EC 50 values of the thick oleanol compounds for killing Cryptocaryon irritans predators are 0.044~0.203 mg / L, and the killing activity is better than that of thick oleanol, and the compounds have better effects on killing Cryptocaryon irritans predators.

[0062] 4. Killing effect of thick oleanol compounds on Cryptocaryon irritans cysts Cryptocaryon irritans non-division cysts were collected, washed several times until clean and free of impurities, and placed in a 24-well plate, about 20 in each well. The sterile seawater was used as a control group, and different volumes of sterilized seawater and test drug solution were added to make the total volume of each well 2 mL. Then the 24-well plate was placed in a 27°C constant temperature incubator for 3~4 days, and the seawater was replaced once a day. After the test was completed, the number of un-hatched cysts in each well was counted under an inverted microscope (40x), the whole test was repeated three times, the cyst mortality rate was calculated, and the results are shown in Table 8. The cyst mortality rate was calculated according to the following formula: cyst mortality rate = (number of un-hatched cysts / total number of cysts) x 100.

[0063] Table 8 Killing effect of thick oleanol compounds on Cryptocaryon irritans cysts

[0064] As can be seen from Table 8, the EC 50 values of the thick oleanol compounds for killing Cryptocaryon irritans cysts are 0.053~0.215 mg / L, and compounds C1~C4 can effectively inhibit the reproduction of Cryptocaryon irritans cysts and effectively reduce the release amount of predators.

[0065] The above-described embodiments are merely some of the embodiments of the present application, but not all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but merely represents selected embodiments of the application. All other embodiments obtained by persons of ordinary skill in the art based on the concept of the present application, without making creative labor, are within the scope of the present application.

Claims

1. A magnolol compound, characterized in that, having a structure as shown in C1-C4, 。 2. The magnolol compound of claim 1 for use in the preparation of a medicament for killing fish parasites.

3. Use according to claim 2, characterized in that, The magnolol compound is administered by immersion or orally to kill fish parasites.

4. Use according to claim 3, characterized in that, The fish parasites include Ichthyophthirius multifiliis Ichthyophthirius multifiliis and Cryptocaryon irritans Cryptocaryon irritans .

5. Use according to claim 3, characterized in that, The concentration of the immersion-administered magnolol compound C1 is less than or equal to 2.0 mg / L; The concentration of the immersion-administered magnolol compound C2 is less than or equal to 3.0 mg / L; The concentration of the immersion-administered magnolol compound C3 is less than or equal to 2.0 mg / L; The concentration of the immersion-administered magnolol compound C4 is less than or equal to 2.5 mg / L.

6. Use according to claim 3, characterized in that, The feeding amount of the magnolol compound C1-C4 for oral administration is not less than 60 mg / kg·d -1 .

7. Use according to claim 2, characterized in that, The killing of fish parasites includes killing of fish parasites' predators and / or cysts.

8. The use according to claim 2, characterized in that, The magnolol compound reduces the number of fish infected with parasites.

9. The use according to claim 2, characterized in that, The magnolol compound increases the survival rate of fish infected with a parasitic disease.

10. Use according to claim 9, characterized in that, The parasitic disease is Ichthyophthirius multifiliis and / or Cryptocaryon irritans.

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