Antiparasitic Efficacy And Mechanism Research Of Magnolol Against Ichthyophthirius Multifiliis In Gold Fish

Ichthyophthiriasis is one of the most serious parasitic diseases caused by Ichthyophthirius multifiliis which feeding on body surface and gill of freshwater fish.However,since malachite green and mercury salt are forbidden to use,lots part of chemicals can kill I.multifiliis at free-living stage.But they have lower anti-parasite efficacy in vivo and they are limited for further using because of a series of problems,such as the toxicity to fish,food safety,environmental pollution and so on.Natural products have the advantages of low toxicity,easy degradation,environmental friendly and rich resources,which is an aquatic antiparasitic drug of ideal safe and harmless.Magnolol,one of the effective components from Magnolia officinalis,can be chosen as a potential lead compound for the development of commercial drug against I.multifiliis due to its simple chemical structure and easily synthesized and further modified.The anti-I.multifiliis efficacy of the nature compound is evaluated by antiparasite experiments in vitro and in vivo.Moreover,the safety assessment of magnolol against goldfish is evaluated and the mechanism of magnolol against I.multifiliis is preliminarily studied.The results obtained in this work are as follows:1.Antiparasitic efficacy of magnolol against I.multifiliisThe magnolol displayed great effective parasiticide against I.multifiliis both in free-living（theront,protomont and tomont）and parasitic（trophont）stage.The results showed that magnolol at 0.6 mg/L could kill all theronts within 4 h exposure,and median lethal concentration(LC₅₀)of magnolol against I.multifiliis theront wais 0.37 mg/L（95%confidence interval was 0.24-0.52 mg/L）within 4 h exposure.Magnolol could prevent nearly48%protomonts to transform into encysted tomonts at 0.8 mg/L.It could terminate reproduction of I.multifiliis post 6 h exposure of protomonts and encysted tomonts to 0.8 and1.0 mg/L,respectively.In vivo trials showed that 5 h exposure of infected fish to 1.5 mg/L of magnolol almost reduced 40%theronts released from tomonts.Moreover,magnolol possibly had a directly effect on the cell membrane according to morphological observation,and magnolol could inhibit the binary fission of tomont.2.The safety assessment of magnolol against goldfish.The acute toxicity of magnolol against goldfish showed that the 96 h LC₅₀ was 6.0 mg/L（95%confidence interval was 5.2-6.7 mg/L）.The goldfish never exhibited any abnormal phenomenon during 96 h bath test,and it was 100%survival when exposure to magnolol less than 4.5 mg/L.All goldfish would die after exposure to magnolol over 7.5 mg/L in 24 h.Furthermore,the changes of stress related genes and physiological indexes of goldfish were detected after exposure to the effective concentration of magnolol.In this study,the expression of genes（cyp1a,hsp70,gst and sod）and alteration of physiological indexes were evaluated.These results showed that the kidney of goldfish was less sensitive than liver and spleen to magnolol,and the penetration of xenobiotics caused the body’s stress response of goldfish in a short period exposure,and with the extension of time it would recover to homeostasis gradually.3.The effect of magnolol on the reproduction of I.multifiliis.According to screening ten type of reproduction related proteins of I.multifiliis by qPCR,the mRNA level of arginase was down regulated more than 90%whether after 3 h exposure to1.5 mg/L or 3.5 mg/L magnolol in vivo,even its expression level was significantly inhibited after 0.5 h at lower concentration（p<0.05）.Using arginase inhibitor NOHA and adding ornithine,the results indicated that magnolol had similar efficacy with inhibitor.Magnolol could reduce the number of released theronts no matter treatment in vitro or in vivo,while the number of released theronts could be significantly improved（p<0.05）according to adding10 mg/L ornithine.Furthermore,the possible combination way between magnolol and arginase was preliminarily explored through the construction of 3D protein model and the method of molecular docking,the results showed that the stable hydrogen bonds could be formed between phenolic hydroxyl group of magnolol and amino acid residues of enzyme（Gln87,Gly192,Asp194）.The results all above indicated that magnolol could inhibit the reproduction of I.multifiliis according to competing with substrate for binding site of arginase.In summary,magnolol can effectively kill I.multifiliis in vitro and can significantly reduce the proliferation of I.multifiliis in vivo.It can inhibit the reproduction of I.multifiliis according to interaction with arginase.Moreover,magnolol is safe to goldfish at the low concentration,so it as a lead compound has wide applications in the development of the anti-I.multifiliis drug.