| Ichthyophthiriasis is one of the most harmful parasitic diseases of fish,which is caused by Ichthyophthirius multifiliis parasitizing on the body surface and gills of freshwater fish.With the characteristics of wide distribution and host-free specificity,I.multifiliis can not only cause huge economic losses in breeding and ornamental fishery,but also cause large areas of fish death in natural waters.At present,chemical drugs are the main methods and effective strategies for the prevention and treatment of Ichthyophthiriasis.However,no specific drugs have been available since the specific drugs malachite green and mercury salt were banned.Natural products with abundant resources are easy to degrade in the natural environment and are friendly to the environment.They are the ideal source of anti-parasite drugs for safe and pollution-free aquatic products.Magnolol,one of the main active ingredients found in Magnolia officinalis,is an ideal lead compound for drug development because of its simple chemical structure and its ease of synthesis and modification.Previous studies found that arginase is the drug target of magnolol for the prevention and treatment of I.multifiliis.In this study,the optimal enzyme activity reaction system was constructed using prokaryotic expressed arginase,and based on this system,the in vitro inhibitory effect of magnolol and honokiol derivatives on arginase was determined.Study its quantitative structure-activity relationship,reveal the quantitative change between the structure and activity of the compound,provide a basis and theoretical basis for the subsequent design and synthesis of anti-I.multifiliis drugs,and also provide reference for the research and development of other aquatic drugs.The results obtained in this work were as follows:1. Prokaryotic expression of I.multifiliis arginaseIn this study,after optimizing the arginase sequence of I.multifiliis,a recombinant expression plasmid p ET32a-ARG was constructed,which was successfully expressed in E.coli BL21（DE3）and purified to a soluble recombinant protein ARG,with a molecular weight of about 60 k Da.2. Establishment of the reaction system of arginase from I.multifiliisAn in vitro enzyme activity analysis system was established using the purified ARG recombinant protein.The results of the enzyme activity analysis showed that the enzyme activity of the recombinant protein ARG was about 25.23 U/mg.The enzyme activity was significantly increased when 1 m M Mn2+was added to the reaction system,which was about116.06 U/mg.The optimum p H is 9.5,the optimum temperature is 42℃,and the Km value is0.158 mol/L.Low temperature 4℃and high temperature 70℃can make ARG lose enzyme activity.Both magnolol and arginase-specific inhibitor NOHA inhibited ARG.3. Structure-activity relationship of magnolol and honokiol derivatives on arginase inhibitionThe inhibitory effects of 73 magnolol and honokiol derivatives were measured,among which 35 compounds had inhibitory effects on arginase.Twenty compounds were selected as the training set and four compounds as the test set.The most active compound 21（3,5’-dipropyl-[1,1’-biphenyl]-2’,4-diol）was used as the template molecule to build a Comparative molecular field analysis（Co MFA）model.The cross-validation coefficient q2was 0.595,and the correlation coefficient r2 was 0.967.The Co MFA model was used for prediction,and the fitting graph of the experimental value and the predicted value showed that the model had statistical significance and good prediction ability.Partial least squares（PLS）was used to construct a three dimensional isopositive diagram of the model,which indicated that increasing the volume of substituents at the positions of magnolol and honokiol C-9 and C-9’would increase the activity,while increasing the volume of substituents at the positions of C-2,C-3,C-4 and C-5 would decrease the activity.Increasing the volume of the substituents at C-9 and C-3’increases the activity,and increasing the positivity of the substituents at C-4 increases the activity.In summary,the results showed that the introduction of large groups at the C-9 and C-9’positions of magnolol and honokiol was beneficial to the improvement of inhibitory activity,while and the introduction of large groups at the C-3 and C-4 positions was detrimental to the improvement of inhibitory activity.The increase of electronegativity in the c-4 position is not conducive to the improvement of the enzyme activity.This study provides a basis and theoretical basis for the subsequent design and synthesis of anti-I.multifiliis drugs. |
