Evaluation of the genotoxicity of stevioside and steviol using six in vitro and one in vivo mutagenicity assays

Abstract

Stevioside, a constituent of Stevia rebaudiana, is commonly used as a non-caloric sugar substitute in Japan. The genetic toxicities of stevioside and its aglycone, steviol, were examined with seven mutagenicity tests using bacteria (reverse mutation assay, forward mutation assay, umu test and rec assay), cultured mammalian cells (chromosomal aberration test and gene mutation assay) and mice (micronucleus test). Stevioside was not mutagenic in any of the assays examined. The aglycone, steviol, however, produced dose-related positive responses in some mutagenicity tests, i.e. the forward mutation assay using Salmonella typhimurium TM677, the chromosomal aberration test using Chinese hamster lung fibroblast cell line (CHL) and the gene mutation assay using CHL. Metabolic activation systems containing 9000 g supernatant fraction (S9) of liver homogenates prepared from polychlorinated biphenyl or phenobarbital plus 5,6-benzoflavone-pretreated rats were required for mutagenesis and clastogenesis. Steviol was weakly positive in the umu test using S.typhimurium TA1535/pSK1002 either with or without the metabolic activation system. Steviol, even in the presence of the S9 activation system, was negative in other assays, i.e. the reverse mutation assays using S.typhimurium TA97, TA98, TA100, TA102, TA104, TA1535, TA1537 and Escherichia coli WP2 uvrA/pKM101 and the rec-assay using Bacillus subtilis. Steviol was negative in the mouse micronucleus test. The genotoxic risk of steviol to humans is discussed.

Characterization of Novel Steviol Glycosides from Leaves of Stevia rebaudiana Morita

“…Because steviol is known to be unstable in acid and gives rise to isosteviol, 21) the steviol seemed the most likely candidate for the genuine aglycon. This was substantiated by the evidence that signals due to the aglycon moiety in 13 C NMR spectra of the predominant fractions were found at almost the same position as those of authentic steviol glycosides. 22 24) The chemical shifts of several compounds are summarized in Table 2.…”

“…The hot water extracts were resolved into 20 fractions by HPLC on an Amide 80 column. Each fraction was subjected to structural analysis by sugar composition analysis, ESI MS, ESI MS MS, 1 H NMR, 13 C NMR and or chemical analyses such as methylation analysis and mild alkaline or acid hydrolysis, and the structures of 21 steviol glycosides were established. The structures and relative amounts of the 21 steviol glycosides, on the basis of their UV absorbance at 210 nm and relative abundance of the fragment ions in ESI MS MS spectra, are compiled in Table 4 together with the composition of the stevia sweeteners of S. rebaudiana Bertoni.…”

“…NMR analyses of the steviol glycosides were performed with a JEOL AL 300 spectrometer ( 1 H, 300 MHz; 13 C, 75 MHz) at 25 C in pyridine d5. The chemical shifts (δ ppm) were measured by reference to tetramethylsilane.…”

“…The chemical shifts (δ ppm) were measured by reference to tetramethylsilane. The 13 C NMR analysis for the coupling constant values of C 1 for rhamnose (JC1-H1) was performed with a JEOL JNM Lambda 500 spectrometer (JEOL Ltd., Tokyo, Japan) at 25 C in D2O.…”

Occurrence, Biological Activities and Synthesis of Kaurane Diterpenes and their Glycosides

“…Although some work has failed to demonstrate carcinogenic, teratogenic, mutagenic or toxic actions of stevioside (78), after oral administration to rats [108][109][110][111], at doses up to 1.2 % of the diet, and considering, therefore, a maximum ingestion recommendable of 7.94 mg·kg -1 ·d -1 for the human organism [112], others have demonstrated that steviol (24), an aglycone obtained from complete hydrolysis of stevioside, showed a high toxicity in pregnant females and embryos of hamsters, at doses of 0.75 and 1.00 mg·kg -1 ·d -1 , administered from 6 th to 10 th day of gestation [112][113][114]. The animals treated with these doses showed acute renal failure, embryotoxic effects such as weight loss and retardation of the process of fetal ossification, and death.…”