Microcystins, which are cyclic heptapeptides produced by some cyanobacterial species from algal blooms, strongly inhibit serine/threonine protein phosphatase and are known as hepatotoxins. Microcystins have many structural variations, yet insufficient information is available on the differences in the cytotoxic potentials among the structural variants. In this study, the cytotoxicities of 16 microcystin variants at concentrations of 0.03–10 μg/mL to primary cultured rat hepatocytes were determined by measuring cellular ATP content, and subsequently determined by their 50% inhibitory concentration (IC50). Differences in the amino acid constituents were associated with differences in cytotoxic potential. [d-Asp3, Z-Dhb7] microcystin-LR exhibited the strongest cytotoxicity at IC50 of 0.053 μg/mL among the microcystin variants tested. Furthermore, [d-Asp3, Z-Dhb7] microcystin-HtyR was also highly cytotoxic. These results suggest that both d-Asp and Z-Dhb residues are important in determining the cytotoxic potential of microcystin variants.
The extract produced from the leaves of Stevia rebaudiana Bertoni (Asteraceae) contains sweet steviol glycosides, mainly stevioside and rebaudioside A (Fig. 1), and has long been used as a sweetener in Japan. In 2008, the specifications for "steviol glycosides" were established by the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) Joint Expert Committee on Food Additives (JECFA), and "stevia extracts" were approved as generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (FDA). Thereafter, the purified stevia extracts specified in JECFA and FDA became popular worldwide.According to the assay for steviol glycocides in the JECFA specifications, 1) the total content of nine types of steviol glycosides (Fig. 2) should not be less than 95%. In the assay method, rebaudioside A is quantified from the peak area on LC using the rebaudioside A standard, and each steviol glycoside except rebaudioside A is quantified from their respective peak areas and absorption coefficients using the stevioside standard as the quantification standard.Various commercial standard reagents of stevioside and rebaudioside A are available, although their purities are different and their exact purities are not indicated in the product literatures. Therefore, if the claimed values are employed in the quantitative analysis of a sample, the quantitated contents of the stevioside and rebaudioside A in the identical sample will be assigned different measured values according to the standard used for the quantification.Quantitative NMR (qNMR) using an internal standard with traceability to the International System of Units (SI units) has recently been used to determine the absolute purities of various chemical substances.2-7) The qNMR method permits the absolute quantification of a target compound without the need for a standard of that compound. In addition, it is rapid and easy. The compound is quantified from the ratios of the integral values of the proton signals of the internal standard and the target compound. In addition, various compounds can be quantified using the same internal standard.In this study, we utilized the qNMR method to determine the purity of stevioside and rebaudioside A in commercial standards. The knowledge of their absolute purities will increase the accuracy of other analytical methods that use standards, such as LC.
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