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The Separation and Purification of Microcystin-RR from Cyanobacterial Bloom and Its Effects of Acute Toxicity

Author: ZhangYongLu
Tutor: GuKangDing
School: Huazhong University of Science and Technology
Course: Health Inspection and Quarantine
Keywords: microcystin-RR purification solid phase extraction gel chromatography acute toxicity median lethal dose(LD50)
CLC: R114
Type: Master's thesis
Year: 2013
Downloads: 10
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Abstract

Objective Recently the occurrences of cyanobacterial blooms have become more and morefrequent. Some genera of cyanobacteria may produce microcystins (MCs) which can lead tohealth problems. So the toxicology, immunology and technologies for degradation andremoval of microcystins have been the focus in current studies. The demand for purifiedmicrocystins is on the rise, but they are very expensive and difficult to obtain, which isbecoming a bottleneck for the research.The objectives of this study were to develop a simple and effective method for extractionand purification of microcystin-RR (MC-RR) from cyanobacterial scums by combination of70%methanol extraction, solid phase extraction and Sephadex LH-20gel chromatographyand then to perform the acute toxicity test in mice by using the purified MC-RR forpreliminary assessment of the characteristic toxicity effect of the toxin, target organs,dose-response relation and for providing a basic reference of dose design and observationindex of the other toxicity tests.Methods1. Three pre-treatments, the repeated freezing and thawing way, the water bathway and the70%methanol extraction method, were compared in the process of extractingMC-RR from cyanobacterial bloom scum. The use of70%methanol was recommended forthe extraction of MC-RR from field cyanobacteria samples because of its high efficiency andbetter reproducibility. For70%methanol extraction method, the main factors, such asrepeated times of extraction, temperatures, reaction time and so on, were studied. After themethanol in the crude extract was removed by rotary evaporator, the solid phase extractionand Sephadex LH-20gel chromatography were used for preliminary and further purification,respectively. The absorbance of the elution which was collected by tube fractometer from the Sephadex LH-20column was detected at238nm. The elution curve was plotted by the tubenumbers as X-axis and the absorbance at238nm as Y-axis. The purity and the spectralcharacteristic of the final extracts were identified with HPLC and spectrophotometer,respectively.2. Determination of LD50value of MC-RR by intraperitoneal injection adopts Karber’sMethod. According to preliminary results, the animal groups were divided into five ones interms of different dosages:400μg/kg、282.84μg/kg、200μg/kg、141.42μg/kg、100μg/kg. Thecontrol group was given normal saline. Each group had10Kunming mice, in which thenumbers of the female and male animals were equal. Acute poisoning symptoms, number ofdeaths and the survival time of animals were recorded during the course of experiments.Meanwhile, the hearts, livers and kidneies of the dead animals were surgically separatedfrom the bodies to calculate organ/body weight index and to examine histopathologicalchanges by HE staining. LD50value of MC-RR by oral route administration using a canulawas measured by an up and down method. The serum biochemical parameters, liver/bodyweight index, histopathological changes were determined at the beginning time of toxiceffects and the mean time of death after one LD50dose was given to mice.Results1. Thirty five gram of cyanobacterial bloom scum was extracted twice with70%aqueous methanol for45min at room temperature. After solid phase extraction，4.92mg ofcrude MC-RR, and0.53mg of crude MC-LR were obtained in7.5mL eluent. There werethree elution peaks in elution curve and the MC-RR was determined at the first elution peak.Three point six five milligram of MC-RR, with a final yield of74.1%, was obtained and itspurity was more than90%, with characteristic UV absorption spectra at238nm.2. The LD50via intraperitoneal injection was192.75μg/kg, and the95%confidence intervalwas176.59~206.89μg/kg. Cumulative mortality rates were10/10,9/10,6/10,1/10, and0/10from400to100μg/kg. Liver/body weight indices grew with and histopathological changesbecame more and more serious with the increasing dosages. The LD50by oral canulaadministration was12.10mg/kg, and the95%confidence interval was10.75~13.45mg/kg.After one LD50dose was given to mice, the toxic effects begun at45min and the mean timeof death was4h. Serum levels of AST、ALT、ALP and LDH showed significant increasecompared with the control group at45min and were further enhanced at4h. Liver/bodyweight index was consistent with the serum biochemical variables in alteration trend. Histopathological changes displayed hepatocellular spotty necrosis and mild congestion inthe hepatic sinus at45min, and hepatic necrosis and congestion became more pronounced at4h. No pathological changes were observed in the kidney and the heart of the animals.Conclusions This study established an effective method to purify MC-RR fromcyanobacterial bloom scum by a suitable combination of75%aqueous methanol extraction,solid phase extraction and Sephadex LH-20gel chromatography. The MC-RR could induceliver toxicity, but the nephrotoxicity and the heart lesions were not observed at the acutetoxicity.

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