Study On Active Constituents From The Roots Of Dryopteris Crassirhizoma Against Dactylogyrus

As the largest producer with its output,China accounts for about 72.28%in terms of aquatic product from the whole word.The extension of intensive aquaculture model made a series of infectious diseases caused by bacteria,virus and parasite.Of these pathogenic organism,the Dactylogyrus intermedius is one of common parasites of fish,and this kind of monogenean parasitise on the gills of fish,causing fish mortality at high density of infection.Recently,toltrazuril,praziquantel,and albendazole is usually used to control Dactylogyrus,however,the long time use,overuse and abuse of these drugs led to serious resistance,residue relate to the large amount of drugs and food safety issues impel the search to find new safe drugs.Because of the properties of low drug residue and resistance,degradable and friendly to environment,plant derived products have being received concerns.So,scanning active components from natural plants is an efficient way to find an effective anthelmintic.Base on these points,the present study investigated the anthelmintic activity of Dryopteris crassirhizoma(named Mianma Guanzhong in Chinese),the isolation and identification of active compounds,toxicity to fish,and electron microscopic observation of parasite.Below are main findings:1.Study of active extracts from D.crassirhizoma against Dactylogyrus.Liquid-solid extraction of crude extract with petroleum ether,ethyl acetate,ethyl acetate:methanol(1:1),methanol led to four different extracts.Anthelmintic efficacy of each extracts was valued and the result showed that ethyl acetate extract of D.crassirhizoma exhibited a 100%efficacy at the concentration of 125 mg/L after 48 h,being non-toxic to fish,and it was selected to be isolated at the next stage.2.Isolation and structural identification of active constituents of D.crassirhizoma,electron microscopic observation on surface topography of parasite.Ethyl acetate extract was fractionated into three fractions on silica gel CC eluting with chloroform:methanol(1:0?0:1,v/v),and Fraction B was testified to be responsible for the anthelmintic activity.Four compounds were obtained after successive purifications,compound ???exhibited 100%efficacy at 9.0 mg/L and 11.0 mg/L.Based on spectroscopy analysis and comparing with reported spectral data,these two compounds were identified as albaspidin AP and kaempferitrin.The other two compounds were protocatechuic acid and sutchuenoside A.The medium effective concentration(EC50)values of albaspidin AP and kaempferitrin were 2.30?2.71 mg/L.3.The acute toxicity of albaspidin AP and kaempferitrin on fish were examined.This experiment was conducted on healthy goldfish whose body weight lighter than 5.0 g,and the results showed that the median lethal concentration(LC50)values of albaspidin AP and kaempferitrin were 30.4 and 33.1 mg/L respectively.4.Ultrastructural micrographs revealed shrinkage of body surface,dense tegumental folds and dishevelled protuberances.The structural deformities in the treated parasites were indicative of an efficient anthelmintic activity of the isolated compound.