Study On Active Constituents From Pseudolarix Kaempferi Against Dactylogyrus Infecting Fish

The global aquaculture industry which provided15%of animal protein per year for over2.9billion people developed more fast than before, but the losses caused by aquatic diseaseswere enormous. The disease caused by Dactylogyrus which was commonly seen inaquaculture caused great economic losses every year. The most effective drugs that currentlyused for the treatment of Dactylogyrus were trichlorfon, praziquantel, and mebendazole, etc.however, the overuse and long time use had led to serious problem, such as drug resistance,the destruction of the ecological environment, the problem of food safety. Traditional Chinesemedicine is a potential fishery drugs which has a lot of advantages, such as little side effect,did not exist drug resistance, environmental friendly and safety to aquatic animals. But itcannot be compared with the traditional chemical drugs which have high efficience due tocomplicated compounds and the high dosage in the treatment of Dactylogyrus. If we canisolate the active compounds from traditional Chinese medicine, and then improve theanthelmintic activity and cut down the toxicity through the study of anthelmintic activity andstructure-activity relationship, this will greatly reduce the dosage of medicine and provide thenew ideas for the creation of pollution-free fishery drugs. This study has important applicationprospect.The goal of this study is to screen traditional Chinese medicine which has anthelminticactivity, to find out which dissolvent could dissolve anthelmintic compounds, and the study ofbio-guided isolation and identification of the active components. Combining with chemicalseparation, spectrum analysis and structure analysis technology, we successfully isolated theactive compound, and the make the assessment of acute toxicity. The results of theseexperiments in this paper were as follow:1. The screening of42kinds of methanol extract of traditional Chinese medicine for invivo anthelmintic activity against Dactylogyrus. The organic solvent of methanol was appliedin this experiment for the extraction of the powders of medicine, In vivo anthelmintic activityof these extractions were tested. The results showed that cinnamomum cassia, linderaaggregata, and pseudolarix kaempferi exhibited100%activity with no fish died at theconcentration of200mg/L,40mg/L, and100mg/L.2. The study of active extract of cinnamomum cassia, lindera aggregata, and pseudolarix kaempferi. Five solvents with different polarity including petroleum ether, chloroform, ethylacetate, methanol, and water were applied in this experiment. In vivo athelmintic activity ofthese extractions were tested. The results showed that the methanol extract of Cinnamomumcassia showed100%activity with no fish died at the concentration of30mg/L, EC₅₀valuewas12.25mg/L. The methanol extract of lindera aggregata exhibited100%activity with notoxicity to fish at the concentration of60mg/L, EC₅₀value was23.53mg/L. The ethyl acetateextract of pseudolarix kaempferi showed100%activity with no toxicity to fish at theconcentration of50mg/L, EC₅₀value was17.10mg/L.3. Bio-guided isolation and structural identification of active compound of pseudolarixkaempferi. The methanol extract was partitioned with petroleum ether, chloroform, and ethylacetate successively, then we got the extract of ethyl acetate which had been proved that hadhigh athelmintic activity. Through the method of column chromatograph, LH-20, successivecrystallization, and spectral analysis, we successfully isolate the active compound called（+）-Epicatechin which exhibited93.1%activity with no toxicity to fish at the concentration of15mg/L.4. The result of the acute toxicity test showed that （+）-epicatechin showed no toxicity togoldfish at the concentration of10mg/L,20%mortality of goldfish at the concentration of20mg/L,60%mortality of goldfish at the concentration of30mg/L,80%mortality of goldfish atthe concentration of40mg/L,100%mortality of goldfish at the concentration of50mg/L, the48h LC₅₀and LC90value were27.447mg/L and42.532mg/L, respectively.In this study, we successfully isolated the active compound from traditional Chinesemedicine pseudolarix kaempferi through screening test, active extract test, and bio-guidedisolation test. The compound was identified as （+）-epicatechin through spectrum analysis andcan be developed as a novel drug for the treatment of Dactylogyrus.