Metabolically activated steviol, the aglycone of stevioside, is mutagenic.

Abstract

Stevioside, a constituent of Stevia rebaudiana, is commonly used as a noncaloric sugar substitute in Japan. Consistent with reports in the literature, we have found that stevioside is not mutagenic as judged by utilization of Salmonella typhimurium strain TM677, either in the presence or in the absence of a metabolic activating system. Similar negative results were obtained with several structurally related sweet-tasting glycosides. However, steviol, the aglycone of stevioside, was found to be highly mutagenic when evaluated in the presence of a 9000 X g supernatant fraction derived from the livers of Aroclor 1254-pretreated rats. Expression of mutagenic activity was dependent on both pretreatment of the rats with Aroclor 1254 and addition of NADPH; unmetabolized steviol was not active. The structurally related species, isosteviol, was not active regardless of metabolic activation. Similarly, chemical reduction of the unsaturated bond linking the carbon-16 and -17 positions of steviol resulted in the generation of two isomeric products, dihydrosteviol A and B, that were not mutagenic. In addition, ent-kaurenoic acid was found to be inactive. It is therefore clear that a metabolite of an integral component of stevioside is mutagenic; structural features of requisite importance for the expression of mutagenic activity include a hydroxy group at position 13 and an unsaturated bond joining the carbon atoms at positions 16 and 17. A potential metabolite of steviol, steviol-16a,17-epoxide, was synthesized chemically and found to be ineffective, as. a direct-acting mutagen. Thus, although stevioside itself 'appears innocuous, it would seem prudent to expeditiously and unequivocally establish the human metabolic disposition of this substance.Stevia rebaudiana Bertoni (Compositae) is a sweet herb indigenous to elevated terrain in the northeastern corner of Paraguay, adjacent to its frontier with Brazil (1). Extracts of this plant and stevioside, its major sweet constituent, are commercially available in Japan, where they have been used for nearly a decade to sweeten a variety of foods including sea foods, pickled vegetables, dessert items, soft drinks, and confectionery (2-4). Recently, S. rebaudiana products were cleared for sale in Brazil (5, 6), and they are also used in Paraguay for the treatment of hyperglycemia (1).Stevioside is one of eight known sweet ent-kaurene glycoside constituents of S. rebaudiana, with the others being steviolbioside, rebaudiosides A-E, and dulcoside A (7). The results of several studies to assess the safety for human consumption of certain of these compounds and S. rebaudiana extracts have appeared in the literature. For example, neither stevioside nor two different S. rebaudiana extracts were found to be significantly active during acute toxicity tests in mice and subacute toxicity tests in rats (8). Also, no dose-related abnormalities in an extensive range of blood chemistry values were observed after subacute oral administration to rats of a S. rebaudiana extract containing 50% ...

Mutagenicity of Steviol and Its Oxidative Derivatives in Salmonella typhimurium TM677.

“…The results were shown in Table 1 as their specific mutation ratio (mutation frequency/mg sample/ plate). Steviol indicated almost similar levels of mutagenicity under the presence of S9 mixture, as reported by Pezzuto et al 6) or Matsui et al 8) Of the eight synthesized derivatives, four compounds [steviol-16,17-epoxide (3), steviol methylester (6), 13,16-seco-13-oxo-steviol methylester (8), and steviol methylester 8,13-lactone (10)] were found to be mutagenic at nearly the same level as steviol (2) itself only under the presence of S9 mixture, and the other three compounds has no activity since over the detection limit. Regardless of the presence or absence of the rat liver ribosomal fraction, 15-hydroxy steviol (4), 16-oxo-steviol methylester (7) and 3,16-seco-13-hydroxy-steviol methylester (9) were found to be non-mutagenic or below detection limit in the test system.…”

“…4) However, it is known that stevioside is converted to its aglycone, steviol, by intestinal bacteria when orally administered to rats. 5) Pezzuto et al 6) demonstrated that steviol causes a dose-dependent positive response in the forward mutation assay using S. typthimurium TM677 in the presence of a metabolic activation system (Aroclor-induced rat liver S9 fraction).…”

A simple synthesis of kaurenoic esters and other derivatives and evaluation of their antifungal activity

“…Infrared spectra were acquired with a Perkin-Elmer FT-IR 1600 spectrometer using KBr for solids and film for liquid samples (range 4000-400 cm -1 ). 1 H NMR (200 MHz) and 13 C NMR (50 MHz, fully decoupled) spectra were recorded with a Bruker AC-200F spectrometer. Samples were prepared in CDCl 3 solution containing 1-2% tetramethylsilane (TMS) as internal standard.…”

“…6 Kaurenoic acid (1), along with several related kauranes, 7 exhibits a wide variety of biological activities such as antimicrobial, anti-inflammatory, anti-fertility, cytotoxic, and trypanocidal. [8][9][10][11][12][13][14][15][16] The relatively high natural abundance of kaurenoic acid (1) and the inexistence of a general method for the synthesis of alkyl kaurenoates by esterification of 1 provides further motivation to investigate its chemical modification 17 in order to obtain derivatives and test their pharmacological activity. 18 This paper describes a general synthesis of kaurenoic esters and derivatives as well as their antifungal activity, evaluated against a panel of human opportunistic pathogenic fungi including yeasts, hialohyphomycetes and dermatophytes.…”

Steviol effect, a glycoside of Stevia rebaudiana, on glucose clearances in rats

“…Stevioside was isolated from a 1-butanol-soluble extract of S. rebaudiana by absorption column chromatography (Kinghorn and Soejarto, 1991). Steviol (molecular weight = 804.80) was obtained by enzymatic hydrolysis of stevioside with pectinase (Pectinol 50 L, Corning Biosystems) (Pezzuto et al, 1985).…”