Isolation And Identification Of The Active Ingredient From Sargentodoxa Cuneata And Its Preliminary Study On The Antiparasitic Efficacy Against Ichthyophthirius Multifiliis

Ichthyophthirius multifiliis is a highly pathogenic protozoan,causes ichthyophthiriasis.The disease has the characteristic of rapid onset and high mortality,posing a serious threat to the healthy development of the freshwater aquaculture industry globally.Conventional anti-I.multifiliis agents have many problems such as high toxicity,environmental pollution and drug residues.It is imperative to search for safe and effective alternative medicine for the treatment of ichthyophthiriasis.Medicinal plants have characteristics of low toxicity to non-target organisms and environmental friendliness,which are an essential source for the discovery and development of new drugs.Herein,the antiparasitic activity of 56 kinds of medicinal plants ethanol extracts against I.multifiliis theronts were evaluated.The active ingredients of medicinal plant with the strongest antiparasitic activity were further isolated by bioassay-guided fractionation.The structure of the active ingredient was identified by NMR,FT-IR,HR-MS.The in vitro and in vivo antiparasitic efficacy of the active ingredient against I.multifiliis and the safety assessment to goldfish were comprehensively evaluated.The main results and conclusions are as follows:1.In vitro screening of medicinal plants against I.multifiliis theront.56 kinds of medicinal plants with antibacterial,antiparasitic or antiviral effects were selected,and the plant extracts were obtained by grinding,ethanol extraction,and vacuum concentration.The extract solution was incubated with theronts suspension in equal volume,and the survival of theronts in each treatment group was observed after 4hours.The results of in vitro experiments were as follows.The extracts of Rosmarinus officinalis（ROE）and Sargentodoxa cuneata（SCE）showed potent antiparasitic activity against I.multifiliis.The lethal concentration of ROE and SCE against theronts were 3.13 and 1.57 mg/L,respectively.The median effective concentration(EC₅₀)of ROE against I.multifiliis theronts,protomonts and tomonts were 1.7,5.2and 10.8 mg/L,respectively.The median lethal concentration(LC₅₀)of ROE to goldfish was 48.7 mg/L,which was 4.5-28.6 times the effective concentration against I.multifiliis in vivo.The EC₅₀ of SCE against I.multifiliis theronts,protomonts and tomonts were 0.1,5.6 and 16.1 mg/L,respectively.The LC₅₀ of SCE to goldfish was 70.3 mg/L,which was 4.4-703.0 times the effective concentration against I.multifiliis in vitro.These results indicated that SCE was a safer medicinal plant with significant antiparasitic activity against I.multifiliis.2.The isolation and identification of the active ingredient from Sargentodoxa cuneata stems against I.multifiliis.The ethanol extract of S.cuneata was extracted with petroleum ether,ethyl acetate,and n-butyl alcohol saturated with water after suspension with water.Bioactivity tests showed that the n-butanol extraction possessed the potent antiparasitic efficacy.The mortality of protomonts exposed to 4.0mg/L of the n-butyl alcohol fraction within 6 h was 64.7%.The active ingredients in n-butyl alcohol fraction were further separated and purified by silica gel column chromatography,thin-layer chromatography,and high-performance liquid chromatography,and the optimum chromatographic conditions were determined.An active ingredient Fraction Cc₃ was obtained by semi-preparative high performance liquid chromatography.2.0 mg/L Fraction Cc₃ could cause 82.2%mortality of protomonts with 6 hours.Through spectral analysis and comparison of literature data,the active ingredient was identified as epicatechin gallate（ECG）.3.Antiparasitic efficacy of ECG against I.multifiliis in vitro and in vivo and its safety assessment to goldfish.In vitro assays revealed that ECG was effective against all the free-living stages of I.multifiliis.The 100%lethal concentrations of ECG against theronts and protomonts were 0.3 and 2.0 mg/L,respectively.10.0 mg/L ECG could terminate tomonts reproduction.Morphologically,2.0 mg/L ECG treatment for2 h induced the loss of cell membrane integrity and leakage of cytoplasmic contents of the protomont.Moreover,an in vivo parasite-goldfish infection model was applied to evaluate the efficacy of ECG on prevention and treatment of ichthyophthiriasis.The results showed that 2.0 mg/L ECG continuous treatment for 12 days could cure goldfish ichthyophthriasis and significantly increase the survival rate of the infected goldfish by 86.7%.The healthy goldfish was less susceptible to I.multifiliis infection.Acute toxicity test revealed that the LC₅₀ of ECG to goldfish was 63.7 mg/L,which was 31.9 times the effective concentration against I.multifiliis in vivo,and it was safe to fish with the effective range of antiparasitic activity against I.multifiliis.Histopathological observations suggested that ECG treatment did not result in significant pathological changes in the gills,liver,kidney,and fins of goldfish.Overall,the present study presents the first isolation of ECG from S.cuneata.In vitro and in vivo assays showed that ECG exhibited potent anti-I.multifiliis activity,and showed low toxicity to goldfish,indicating ECG had the potential to develop into agent for the prevention and treatment of ichthyophthiriasis.