Method for separating and purifying toxin of microcapsule alga
A technology for the separation and purification of microcystins, applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., can solve the problems of low sample loading, high cost, and inability to separate, and achieve the effect of high purity of toxins
Inactive Publication Date: 2006-03-01
INST OF AQUATIC LIFE ACAD SINICA
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[0004] The main types of microcystins in my country's bloom cyanobacteria are MC-RR, [Dha 7 ] MC-RR and MC-LR, wherein the first two toxins have very little structural difference (see molecular structure diagram), only the 7th amino acid Mdha has one less methyl group, which is the same chromatographic peak under the commonly used HPLC chromatographic conditions, and cannot Separation has become a difficult problem in chromatographic separation
Among the existing separation methods, molecular exclusion chromatography (gel chromatography), ion chromatography and flash chromatography cannot solve this problem. From the existing research, only Japanese scientists use preparative thin layer chromatography (HPTLC) The two toxins can be separated, but there are also disadvantages that the thin-layer chromatography plate can only be used once, the cost is high and the sample volume is low
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[0025] 1. Algal toxin extraction: Weigh 10g of cyanobacteria dry powder harvested from Dianchi Lake, add 250ml of 75% methanol, shake on a shaker at room temperature for 60min or continuously stir on a stirrer for 60min, take it out and centrifuge it at 4000r / min for 25min, take it out supernatant. Repeat this step for one, two or three extractions, and combine the supernatants from several extractions.
[0026] 2. Preliminary purification of algal toxins: Concentrate the supernatant by rotary evaporation at 34°C to an oily state, perform preliminary purification of microcystins according to the flash chromatography method proposed by Edwards (1996), and collect microcystins containing MC-RR and [Dha 7 ] The components of MC-RR were concentrated to dryness by rotary evaporation at 34°C, and made up to volume with 20ml of 70% methanol.
[0027] Edwards C., Lawton L., et al..Laboratory-scale purification of microcystins using flashchromatography and reversed-phase high-performa...
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The invention discloses a method for separating and purifying microcystin. Said method uses bloom cyanophyte of eutrophic lake as main raw material, and adopts the processes of organic solvent extraction, rapid chromatographic primary separation and C18 reversed phase preparation liquid chromatography so as to implement separation and purification of microcystin MC-RR and [Dha7]MC-RR. Said method adopts isogradient elution, its stability is good, under the condition of preparation chromatography two microcystins can be completely separated. The single sample quantity can be up to 200 micrograms, and the purity of the obtained MC-RR and [Dha7]MC-RR can be detected by using HPLC-UV up to above 95%.
Description
technical field [0001] The invention relates to a method for separating and purifying microcystins, which is suitable for harvesting two kinds of microcystins MC-RR and [Dha 7 ] Purification of MC-RR. Background technique [0002] The harm of cyanobacterial toxins to the environment and human health has become one of the major environmental issues of global concern. The World Health Organization (WHO) and the United Nations Educational, Cultural and Health Organization (UNESCO) have shown great concern about this issue. In the past ten years, international conferences on toxic cyanobacteria and cyanotoxins have been held continuously, and the number of papers has increased year by year, among which microcystin is the most widely studied. So far, more than 70 kinds of microcystins have been identified, but commercial standard products are limited to MC-LR, MC-RR and MC-YR, and only a few companies in the United States, Germany, Japan (such as Sigma...
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IPC IPC(8): C07K14/405C07K1/14C07K1/16
Inventor 肖邦定陈晓国刘剑彤刘永定宋立荣方涛
Owner INST OF AQUATIC LIFE ACAD SINICA