Study On Active Constituents Of Antipathogen Of Fishes From Macleaya Cordata

Four alkaloids (Sanguinarine, 6-Methoxyl-dihydro-chelerythrine, Cryptopine, andβ-Allocryptopine) were isolated from aerial parts of Macleaya microcarpa (Maxim) Fedde using an bioassay-guided isolation method, and inhibitory activity of ethanol extract and these four alkaloids against four fish pathogenic bacteria (Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum and Vibrio harveyi) was assessed in vitro using the agar dilution method. A. hydrophila was the most sensitive strain to all the tested compounds. Minimum inhibitory concentration (MIC) values were lower for sanguinarine against all tested Gram-negative strains than other three alkaloids, with MIC values of 12.5 mg/ l for A. hydrophila and 50 mg/ l to other pathogenic bacteria. Followed by 6-methoxyl-dihydro-chelerythrine, which showed considerable antibacterial activity with MIC values of 80 mg/ l for A. hydrophila, 100 mg/ l for V. harveyi, and 125 mg/ l for both V. anguillarum and A. salmonicida. Cryptopine andβ-allocryptopine revealed similar inhibitory activity with MIC values of 0.1 g/ l for A. hydrophila and 0.2 g/ l for other three bacterial species. These finding revealed that extract and active compounds from M. microcarpa have the potential to be exploited as novel antibacterial agents for the treatment of fish infectious diseases.Establish an RP-HPLC method for the determination of sanguinarine in plasma,and study on the pharmacokinetics of sanguinarine in Carassius auratus. Sanguinarine was were extractted from Macleaya Microcarpa by hot ethanol in ultrasonic wave. Acute toxicity test revealed that the 48h LD50 is 1939.22 mg·kg-1, credible limit of ninty-five percent is 1513.56～2511.88 mg·kg-1.fishes was treated with ethanol extracts of 30 mg·kg-1 body weight by orally administration. Blood samples were collected from caudal vein at selected time points. Protein precipitation with acetonitrile was applied to purify plasma samples before analysis. The sanguinarine concentration in plasma was detected by RP-HPLC on Waters XBridge RP C18column(4.6×150mm 5μm) with acetonitrile and 1% aqueous solution(25:75,) as the mobile phase, flow rate of 0.8 mL·min-1 and detected at 270 nm at room temperature. The calibration curve was 1inear within 0.05μg·ml-1～20μg·ml-1 ( r = 0. 9993) . The 1imit of quantitation was 0.03μg·ml-1 and the extraction recovery was over 80%. The relative standard deviations for the intra and inter day were less than 10%. After oral administration of sanguinarine at the dose of 30 mg·kg-1, sanguinarine plasma C-T curve conform to one-compartment model with 1st order absorption.The pharmacokinetic equation wasC = 1.793 e^-0.213t -1.793e^0.426t.The main parameters of pharmacokinetic were as follows:Ka of 0.426 h-1,a half life of elimination T1/2(e) of 3.255 h, the area under the curve(AUC) of 25.180μg·h·mL-1, Vd/F of 5.594 L·kg-1, CL(s) of 1.191 mL·h-1·kg-1, T(peak)of 3.255 and Cmax of 2.681μg·mL-1. The sanguinarine was rapidly absorbed in Carassius auratus,with a shorter T1/2(e) and extensive distribution, which can be used as a new aquatic plant drug that is worth to be extended.