Metabolic fate in adult and pediatric population of steviol glycosides produced from stevia leaf extract by different production technologies.

More than 60 naturally occurring steviol glycosides in the Stevia rebaudiana Bertoni plant share a similar molecular structure with an aglycone steviol backbone conjugated with ß- and α-glycosidic bonds to different sugar moieties. These glycosides are naturally produced in different quantities within the stevia leaf. Certain minor glycosides with superior sensory attributes, such as Reb D and Reb M, are found less than 0.1% in traditional stevia leaves. New technologies can now produce better tasting steviol glycosides by using enzymatic conversion of stevioside and Reb A, which are abundant in stevia leaf. Several regulatory authorities recently evaluated steviol glycosides produced by enzymatic conversion of stevia leaf extract and approved them safe for human consumption. Steviol glycosides undergo microbial hydrolysis in the colon to generate steviol, which is absorbed and metabolized into steviol glucuronide, and excreted primarily via human's urine. Previous studies have shown the hydrolysis of highly purified individual steviol glycosides extracted from stevia leaf are converted to steviol in the presence of colonic microbiota of adults. Since colonic microbiota of children may be different from adults, this study investigates the metabolic fate in the colonic microbiota of adults and children of the minor steviol glycosides produced by extraction and enzymatic conversion of major steviol glycosides from stevia leaf. Several in vitro incubation tests were conducted in human fecal homogenates collected from adult and pediatric populations with steviol glycoside test samples comprised of a complex stevia leaf extract, a blend of minor glycosides isolated from stevia extract and two mixtures of steviol glycosides produced by enzymatic conversion of Reb A to larger molecules by attaching glucose units via ß- or α-glycosidic bonds. Results from these studies clearly demonstrate steviol glycosides produced by extraction from stevia leaf, or enzymatic conversion of stevia leaf extract, share the same metabolic fate in the human gut microbiota from adults and children. Considering a common metabolite structure and a shared metabolic fate in all ages, safety data for individual steviol glycosides can be used to support safety of all steviol glycosides produced by extraction and enzymatic conversion of stevia leaf extract.

Chemical-specific adjustment factors (inter-species toxicokinetics) to establish the ADI for steviol glycosides.

The acceptable daily intake (ADI) of commercially available steviol glycosides is currently 0-4 mg/kg body weight (bw)/day, based on application of a 100-fold uncertainty factor to a no-observed-adverse-effect-level value from a chronic rat study. Within the 100-fold uncertainty factor is a 10-fold uncertainty factor to account for inter-species differences in toxicokinetics (4-fold) and toxicodynamics (2.5-fold). Single dose pharmacokinetics of stevioside were studied in rats (40 and 1000 mg/kg bw) and in male human subjects (40 mg/kg bw) to generate a chemical-specific, inter-species toxicokinetic adjustment factor. Tmax values for steviol were at ∼8 and ∼20 h after administration in rats and humans, respectively. Peak concentrations of steviol were similar in rats and humans, while steviol glucuronide concentrations were significantly higher in humans. Glucuronidation in rats was not saturated over the dose range 40-1000 mg/kg bw. The AUC0-last for steviol was approximately 2.8-fold greater in humans compared to rats. Chemical-specific adjustment factors for extrapolating toxicokinetics from rat to human of 1 and 2.8 were established based on Cmax and AUC0-last data respectively. Because these factors are lower than the default value of 4.0, a higher ADI for steviol glycosides of between 6 and 16 mg/kg bw/d is justified.

Steviol glycosides in purified stevia leaf extract sharing the same metabolic fate.

The safety of steviol glycosides is based on data available on several individual steviol glycosides and on the terminal absorbed metabolite, steviol. Many more steviol glycosides have been identified, but are not yet included in regulatory assessments. Demonstration that these glycosides share the same metabolic fate would indicate applicability of the same regulatory paradigm. In vitro incubation assays with pooled human fecal homogenates, using rebaudiosides A, B, C, D, E, F and M, as well as steviolbioside and dulcoside A, at two concentrations over 24-48 h, were conducted to assess the metabolic fate of various steviol glycoside classes and to demonstrate that likely all steviol glycosides are metabolized to steviol. The data show that glycosidic side chains containing glucose, rhamnose, xylose, fructose and deoxy-glucose, including combinations of α(1-2), ß-1, ß(1-2), ß(1-3), and ß(1-6) linkages, were degraded to steviol mostly within 24 h. Given a common metabolite structure and a shared metabolic fate, safety data available for individual steviol glycosides can be used to support safety of purified steviol glycosides in general. Therefore, steviol glycosides specifications adopted by the regulatory authorities should include all steviol glycosides belonging to the five groups of steviol glycosides and a group acceptable daily intake established.

In vitro metabolism of rebaudioside E under anaerobic conditions: Comparison with rebaudioside A.

The hydrolysis of the steviol glycosides rebaudioside (Reb) A and E, as well as steviolbioside (a metabolic intermediate) to steviol was evaluated in vitro using human fecal homogenates from healthy Caucasian and Asian donors. Incubation of each of the Rebs in both groups resulted in a rapid hydrolysis to steviol. Metabolism of 0.2mg/mL sample was complete within 24h, with the majority occurring within the first 16 h. There were no clear differences in the rate or extent of metabolism of Reb E relative to the comparative control Reb A. The hydrolysis of samples containing 2.0mg/mL of steviol glycosides Reb A and Reb E tended to take slightly longer than 0.2mg/mL samples. Herein, we report for the first time that there were no apparent gender or ethnicity differences in the rate of metabolism of any of the Rebs, regardless of the concentrations tested. Steviolbioside, an intermediate in the hydrolysis of Reb E to steviol was also found to be rapidly degraded to steviol. These results demonstrate Reb E is metabolized to steviol in the same manner as Reb A. These data support the use of toxicology data available on steviol, and on steviol glycosides metabolized to steviol (i.e., Reb A) to underpin the safety of Reb E.

In vitro metabolism of rebaudioside B, D, and M under anaerobic conditions: comparison with rebaudioside A.

The hydrolysis of the steviol glycosides rebaudioside A, B, D, and M, as well as of steviolbioside (a metabolic intermediate) to steviol was evaluated in vitro using human fecal homogenates from healthy donors under anaerobic conditions. Incubation of each of the rebaudiosides resulted in rapid hydrolysis to steviol. Metabolism was complete within 24h, with the majority occurring within the first 8h. There were no clear differences in the rate or extent of metabolism of rebaudioside B, D, or M, relative to the comparative control rebaudioside A. The hydrolysis of samples containing 2.0mg/mL of each rebaudioside tended to take slightly longer than solutions containing 0.2mg/mL. There was no apparent gender differences in the amount of metabolism of any of the rebaudiosides, regardless of the concentrations tested. An intermediate in the hydrolysis of rebaudioside M to steviol, steviolbioside, was also found to be rapidly degraded to steviol. The results demonstrate that rebaudiosides B, D, and M are metabolized to steviol in the same manner as rebaudioside A. These data support the use of toxicology data available on steviol, and on steviol glycosides metabolized to steviol (i.e., rebaudioside A) to substantiate the safety of rebaudiosides B, D, and M.