Steviol Glucuronide Research Articles

Relative validity of habitual sugar and low/no-calorie sweetener consumption assessed by food frequency questionnaire, multiple 24-h dietary recalls and urinary biomarkers: an observational study within the SWEET project

BackgroundStudies investigating associations between sweeteners and health yield inconsistent results, possibly due to subjective self-report dietary assessment methods. ObjectivesWe compared the performance of a food frequency questionnaire (FFQ), multiple 24-h dietary recalls (24hRs), and urinary biomarkers to estimate intake of sugars and low/no-calorie sweeteners (LNCSs). MethodsParticipants (n = 848, age 54 ± 12 y) from a 2-y observational study completed 1 semiquantitative FFQ and ≥ 3 nonconsecutive 24hRs. Both methods assessed intake of sugars (mono- and disaccharides, sucrose, fructose, free and added sugars) and sweetened foods and beverages (sugary foods, fruit juice, and sugar or LNCS-containing beverages [sugar-sweetened beverages and low/no-calorie sweetened beverages (LNCSBs)]); 24hRs also included LNCS-containing foods and tabletop sweeteners (low/no-calorie sweetened foods [LNCSFs]). Urinary excretion of sugars (fructose+sucrose) and LNCSs (acesulfame K+sucralose+steviol glucuronide+cyclamate+saccharin) were simultaneously assessed using ultrapressure liquid chromatography coupled to tandem mass spectrometry in 288 participants with 3 annual 24-h urine samples. Methods were compared using, amongst others, validity coefficients (correlations corrected for measurement error). ResultsMedian (interquartile range) FFQ intakes ranged from 0 (0–7) g/d for LNCSBs to 94 (73–117) g/d for mono- and disaccharides. LNCSB use was reported by 32% of participants. Median LNCSB+LNCSF intake using 24hRs was 1 (0–50) g/d and reported by 58%. Total sugar excretions were detected in 100% of samples [56 (37–85) mg/d] and LNCSs in 99% of urine samples [3 (1–10) mg/d]. Comparing FFQ against 24hRs showed VCs ranging from 0.38 (fruit juice) to 0.74 (LNCSB). VCs for comparing FFQ with urinary excretions were 0.25 to 0.29 for sugars and 0.39 for LNCSBs; for 24hR they amounted to 0.31–0.38 for sugars, 0.37 for LNCSBs, and 0.45 for LNCSFs. ConclusionsThe validity of the FFQ against 24hRs for the assessment of sugars and LNCSBs ranged from moderate to good. Comparing self-reports and urine excretions showed moderate agreement but highlighted an important underestimation of LNCS exposure using self-reports.

Development and validation of a UPLC-MS/MS method for the quantification of sugars and non-nutritive sweeteners in human urine

High-intensity sweeteners (‘sweeteners’), such as sucralose, saccharine, acesulfame, cyclamate and steviol, are replacing sugars in many food products, but biomarker-based data on their population-wide exposure, as well as analytical methods that can quantify urinary concentrations of sugars and sweeteners simultaneously, are lacking. Here, we developed and validated an ultra-pressure liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method to quantify glucose, sucrose, fructose, sucralose, saccharine, acesulfame, cyclamate and steviol glucuronide in human urine. Urine samples were prepared by a simple dilution step containing the internal standards in water and methanol. Separation was achieved on a Shodex Asahipak NH2P-40 hydrophilic interaction liquid chromatography (HILIC) column using gradient elution. The analytes were detected using electrospray ionization in negative ion mode, and selective reaction monitoring was optimized using the [M−H]- ions. Calibration curves ranged between 34 and 19,230 ng/mL for glucose and fructose, and 1.8 to 1,026 ng/mL for sucrose and the sweeteners. The method has acceptable accuracy and precision, which depends on the application of appropriate internal standards. Storage of urine samples in lithium monophosphate gives the best overall analytical performance, and storage at room temperature without any preservatives should be avoided since this leads to reduced glucose and fructose concentrations. With the exception of fructose, all analytes were stable throughout 3 freeze–thaw cycles. The validated method was applied to human urine samples, demonstrating quantifiable concentrations of the analytes which were in the expected range. It is concluded that the method has acceptable performance to quantitatively determine dietary sugars and sweeteners in human urine.

Steviol, the aglycon of steviol glycosides, does not reduce hyperglycemia in mice with type 2 diabetes

BackgroundSteviol glycosides, constituents of Stevia rebaudiana Bertoni, are high-intensity sweeteners that enhance glucose-induced insulin secretion and decrease hyperglycemia in mice and rats. Given the extreme sweetness and bitter aftertaste of these compounds, the non-tasting aglycon steviol might be a promising alternative food additive that also harbors anti-hyperglycemic effects. Furthermore, it has been suggested that steviol glucuronide is the main bioactive metabolite of steviol and steviol glycosides that appears in blood. AimTo investigate whether steviol and/or steviol glucuronide reduce hyperglycemia in a high-fat diet (HFD)-induced diabetic mouse model. MethodsSteviol was administered acute per os (p.o.), chronic via drinking water and intravenous (i.v.) to HFD-induced diabetic mice. The effect on glucose homeostasis was assessed using glucose tolerance tests (GTTs). Following chronic and i.v. steviol administration, steviol and steviol glucuronide plasma levels were determined. Steviol glucuronide was administered i.v. and the effect on glycemia was assessed with GTTs. ResultsAcute and chronic oral steviol administration did not lower glycemia. This is due to the very limited and short-lived bioavailability of steviol in plasma. Only after high dosage i.v. steviol, glycemia was transiently reduced shortly after administration (Δ 15 min: 76.4 ± 30.9 mg dL−1, p = 0.035). In contrast, i.v. steviol glucuronide resulted in a 16.4 ± 5.9% lower AUCGlucose(0–120 min) compared to mice that received vehicle (p = 0.040). Moreover, steviol glucuronide enhanced the glucose-induced calcium oscillation frequency in insulin-secreting pancreatic islets. ConclusionOur findings demonstrate that steviol is not a useful derivative of steviol glycosides to improve hyperglycemia in type 2 diabetes. On the other hand, steviol glucuronide has significant anti-hyperglycemic effects in living mice with type 2 diabetes.

Non-Nutritive Sweeteners in Human Amniotic Fluid and Cord Blood: Evidence of Transplacental Fetal Exposure.

This study aimed to investigate human fetal exposure to non-nutritive sweeteners (NNS) by analyzing amniotic fluid and umbilical cord blood. Concentrations of four NNS (acesulfame-potassium [ace-K], saccharin, steviol glucuronide, and sucralose) were measured in amniotic fluid (n = 13) and cord blood samples (n = 15) using liquid chromatography-mass spectrometry. Amniotic fluid samples were obtained for research purposes at the time of term elective cesarean birth or clinically indicated third trimester amnioreduction at Mercy Hospital for Women (Melbourne, Australia). All except four women were in the fasting state. Cord blood samples were obtained from an independent cohort of newborns whose mothers were enrolled in a separate clinical trial at the National Institutes of Health. Ten of 13 amniotic fluid samples contained at least one NNS (ace-K, saccharin, steviol glucuronide, and/or sucralose). Maximum amniotic fluid NNS concentrations of ace-K, saccharin, steviol glucuronide, and sucralose were 78.9, 55.9, 93.5, and 30.6 ng/mL, respectively. Ace-K and saccharin were present in 100% and 80% of the cord blood samples, with maximal concentrations of 6.5 and 2.7 ng/mL, respectively. Sucralose was not detected and steviol glucuronide was not measurable in any of the cord blood samples. Our results provide evidence of human transplacental transmission of NNS. Based on results predominantly obtained from rodent models, we speculate that NNS exposure may adversely influence the offsprings' metabolic health. Well-designed, prospective clinical trials are necessary to understand the impact of NNS intake during pregnancy on human development and long-term health. · NNS consumption during pregnancy has increased in recent years.. · Maternal NNS intake during pregnancy is associated with preterm birth and higher infant weight gain in epidemiologic studies.. · In rodents, in utero NNS exposure induces metabolic abnormalities in mothers and their offspring, alters offspring gut microbiota composition, and promotes sweet taste preference in adulthood.. · It is presently unknown whether and to what degree maternal NNS ingestion in humans leads to direct in utero exposure.. · This study provides the first evidence of in utero NNS exposure in humans and highlights the urgent need to investigate clinical consequences of early life NNS exposure on metabolism, weight, taste preference, and general health..

A Systematic Review of Metabolomic Biomarkers for the Intake of Sugar-Sweetened and Low-Calorie Sweetened Beverages

Intake of added sugars (AS) is challenging to assess compared with total dietary sugar because of the lack of reliable assessment methods. The reliance on self-reported dietary data in observational studies is often cited as biased, with evidence of AS intake in relation to health outcomes rated as low to moderate quality. Sugar-sweetened beverages (SSBs) are a major source of AS. A regular and high intake of SSBs is associated with an overall poor diet, weight gain, and cardiometabolic risks. An elevated intake of low-calorie sweetened beverages (LCSBs), often regarded as healthier alternatives to SSBs, is also increasingly associated with increased risk for metabolic dysfunction. In this review, we systematically collate evidence and provide perspectives on the use of metabolomics for the discovery of candidate biomarkers associated with the intake of SSBs and LCSBs. We searched the Medline, Embase, Scopus, and Web of Science databases until the end of December 2020. Seventeen articles fulfilled our inclusion criteria. We evaluated specificity and validity of the identified biomarkers following Guidelines for Biomarker of Food Intake Reviews (BFIRev). We report that the 13C:12C carbon isotope ratio (δ13C), particularly, the δ13C of alanine is the most robust, sensitive, and specific biomarker of SSBs intake. Acesulfame-K, saccharin, sucralose, cyclamate, and steviol glucuronide showed moderate validity for predicting the short-term intake of LCSBs. More evidence is required to evaluate the validity of other panels of metabolites associated with the intake of SSBs.

Consumption of the Sugar Substitute Stevia Leads to Cross-Reactivity of CEDIA® Buprenorphine II Immunoassay.

Buprenorphine is a semisynthetic opioid that is often used in opiate maintenance therapy. For this purpose, regular toxicological analyses of urine samples are mandatory. For fast analytical results, analyses are commonly performed by immunoassay, for example, Thermo Scientific™ CEDIA® Buprenorphine or Buprenorphine II assay. One drawback of immunoassay-based methods is the possible cross-reaction with other substances. Several drugs have already been checked for cross-reactivity to CEDIA® Buprenorphine II immunoassay. In contrast, cross-reactivities have not been checked for any food additives. In the present study, a cross-reaction of CEDIA® Buprenorphine II assay to steviol glucuronide was investigated. Steviol glucuronide is a phase II metabolite of the sugar substitute stevia. For our study, 32 urine samples of patients in rehabilitation centers were collected. These samples were tested positive with the CEDIA® Buprenorphine II immunoassay. These findings were suspicious, because it was highly unlikely that the patients in those institutions had access to buprenorphine. The absence or presence of buprenorphine in urine samples was evaluated by a validated gas chromatography-mass spectrometry method. In order to determine the concentration of steviol glucuronide in urine samples, a liquid chromatography-tandem mass spectrometry method has been developed and fully validated according to the respective guidelines of the German Society of Toxicological and Forensic Chemistry. The cross-reactivity of steviol glucuronide in the CEDIA® Buprenorphine II immunoassay was observed at concentrations above 15,000 µg/L. These findings demonstrate that food additives should also be considered as compounds that may reduce the selectivity of immunoassays and emphasize the importance of confirming implausible results by selective analytical methods.

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 β- 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 β- 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.

Stevia Metabolites in Biosamples Ranging from Fetal Life to Adulthood

Abstract Objectives Stevia rebaudiana (Bertoni) is a perennial herb native to South America. Its sweetness (∼200–400 times sweeter than sucrose) results from steviol glycosides, particularly stevioside and rebaudioside A. Steviol glycosides are hydrolyzed in the gastrointestinal tract resulting in steviol, which is incompletely absorbed in the colon. In the liver, steviol is converted into its glucuronide derivative and renally excreted. While the use of stevia leaves and crude extracts is still prohibited in the US, steviol glycosides have been ‘generally recognized as safe’ (GRAS) by the Food and Drug Administration (FDA) in 2008. We aimed to determine whether steviol glycosides and glucuronidation products can be found in biosamples collected as early as 2004. Methods In 38 adults, steviol glycosides and glucuronide were analyzed in plasma and in corresponding cerebrospinal fluid samples (CSF); additional 2 persons had only CSF tested. Prenatal exposure was determined in biosamples from 28 individuals (13 amniotic fluid, 15 cord blood). We used ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) utilizing a Thermo Scientific Vanquish UPLC and a Thermo Scientific Altis triple quadruple mass spectrometer with heated electrospray ionization (HESI-II, Thermo Scientific) in negative ion mode (2500 V). Results Seven of 38 adults (18%) had detectable steviol glucuronide concentrations (5 in plasma only, 2 in both plasma and CSF). Maximal concentrations in plasma were 805.4 ng/mL and in CSF 3.3 ng/mL. Two of 13 amniotic fluid samples were positive for steviol glucuronide (max. conc. 93.5 ng/mL) and 1 of 15 cord blood samples contained a trace. In contrast to steviol glucuronide, steviol glycoside could not be measured. Conclusions Steviol glucuronide was found in all types of biosamples (plasma, CSF, amniotic fluid and cord blood), most commonly in plasma (18%). This indicates that exposure to steviol metabolites starts in prenatal life and that these metabolites cross various barriers (e.g., blood-CSF, blood-amniotic fluid). Only samples obtained in and after 2008 were positive for steviol glucuronide, which coincides with the FDA approval. Potential health consequences of exposure to stevia metabolites require further study. Funding Sources N/A.

Steviol glucuronide, a metabolite of steviol glycosides, potently stimulates insulin secretion from isolated mouse islets: Studies in vitro.

AimsSteviol glycosides are the sweet components extracted from medicinal plant Stevia rebaudiana Bertoni, which have antihyperglycaemic effects. Steviol glucuronide (SVG) is the metabolite excreted in human urine after oral administration of steviol glycosides. We aimed to clarify whether SVG exerts direct insulin stimulation from pancreatic islets and to explore its mode of action.Materials and MethodsInsulin secretion was measured after 60 minutes static incubation of isolated mouse islets with (a) 10−9‐10−5 mol/L SVG at 16.7 mmol/L glucose and (b) 10−7 mol/L SVG at 3.3‐16.7 mmol/L glucose. Islets were perifused with 3.3 or 16.7 mmol/L glucose in the presence or absence of 10−7 mol/L SVG. Gene transcription was measured after 72 hours incubation in the presence or absence of 10−7 mol/L SVG.ResultsSVG dose‐dependently increased insulin secretion from mouse islets with 10−7 mol/L exerting the maximum effect in the presence of 16.7 mmol/L glucose (P < .001). The insulinotropic effect of SVG was critically dependent on the prevailing glucose concentration, and SVG (10−7 mol/L) enhanced insulin secretion at or above 11.1 mmol/L glucose (P < .001) and showed no effect at lower glucose concentrations. During perifusion of islets, SVG (10−7 mol/L) had a long‐acting and apparently reversible insulinotropic effect in the presence of 16.7 mmol/L glucose (P < .05). Gene‐transcript levels of B2m and Gcgr were markedly altered.ConclusionThis is the first report to demonstrate that SVG stimulates insulin secretion in a dose‐ and glucose‐dependent manner from isolated mouse islets of Langerhans. SVG may be the main active metabolite after oral intake of steviol glycosides.

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.

Transmembrane transport of steviol glucuronide and its potential interaction with selected drugs and natural compounds

Steviol glucuronide (SVG) is the major metabolite derived from steviol, the aglycone of stevioside and rebaudioside A. After the ingestion of stevioside and rebaudioside A, SVG is formed and excreted into the urine in humans. In the present study, transporter mediated efflux and uptake of SVG was investigated in order to understand molecular mechanisms underlying its renal clearance. Results showed that SVG was not a substrate of efflux transporters BCRP, MRP2, MATE1 or P-gp. In contrast, OAT3 played a predominant role in the uptake of SVG in comparison to OATP1B1, OATP1B3, or OATP2B1. Quercetin, telmisartan, diclofenac, and mulberrin displayed a relatively strong inhibition against OAT3 mediated uptake of SVG with IC50 values of 1.8, 2.9, 8.0, and 10.0 μM, respectively. Because OAT3 is a major uptake transporter in the kidney, inhibition of OAT3 activity may alter SVG's renal clearance by drugs and natural compounds that are used concomitantly with stevia leaf extracts.

Radical scavenging activity of steviol glycosides, steviol glucuronide, hydroxytyrosol, metformin, aspirin and leaf extract of Stevia rebaudiana

Introduction: Reactive oxygen species (ROS), generated in many bioorganic redox processes, are the most dangerous by-products in the aerobic environment. The aim of this study was to compare the in vitro antioxidant activity of some chemical compounds and leaf extract of Stevia rebaudiana as natural antioxidant. Methods: The radical scavenging activity of ascorbic acid, quercetine, stevioside (ST), rebaudioside A (Reb A), steviol glucuronide (SVglu), glucose, sucrose, hydroxytyrosol, metformin and aspirin were measured and expressed as the inhibitory concentration in mM giving 50% reduction of radicals (IC50). Results: Ascorbic acid, quercetine, ST, Reb A, SVglu, glucose, sucrose, hydroxytyrosol, metformin and aspirin were active OH scavengers. Superoxide radicals were efficiently scavenged by ascorbic acid, SVglu, quercetine, hydroxytyrosol, ST, Reb A and aspirin. Quercetine, hydroxytyrosol, ascorbic acid and aspirin were active in limiting the amount of thiobarbituric acid (TBA) reactive material. Only ascorbic acid, quercetine and hydroxytyrosol showed DPPH and NO scavenging activity. Leaf extract of S. rebaudiana had an excellent ROS scavenging activity. Treatment of leaf extracts with PVPP and active charcoal removed a part of their scavenging activity. Results showed that sugars were not efficient ROS scavengers. Conclusion: Radical scavenging activity of steviol derivatives, aspirin, metformin and hydroxytyrosol might explain most of beneficial pharmacological effects on ROS related diseases, such as hypertension, type 2 diabetes, atherosclerosis, inflammation and certain forms of cancers. The results obtained in this study indicate that leaf extract of S. rebaudiana has a great potential to use as a natural antioxidant agent.

Synthesis of ent-Kaurane Diterpene Monoglycosides

Synthesis of two ent-kaurane diterpene glycosides, steviol 19-O-β-D-glucopyranosiduronic acid (steviol glucuronide, 5 ), and 13-hydroxy ent-kaur-16-en-19-oic acid-β-D-glucopyranosyl ester (7) has been achieved from a common starting material, steviol, using phase transfer catalyst. Also, synthesis of an additional 17-nor-ent-kaurane glycoside, namely 13-methyl-16-oxo-17-nor-ent-kauran-19-oic acid-β-D-glucopyranosyl ester (10) was performed using the starting material isosteviol and similar synthetic methodology. Synthesis of all three steviol glycosides was performed using straightforward chemistry and their structures were characterized on the basis of 1D and 2D NMR as well as mass spectral (MS) data.

Pharmacokinetics of rebaudioside A and stevioside after single oral doses in healthy men

This randomized, double-blind, cross-over study assessed the comparative pharmacokinetics of steviol and steviol glucuronide following single oral doses of rebaudioside A and stevioside in healthy adult male subjects. Steviol glucuronide appeared in the plasma of all subjects after administration of rebaudioside A or stevioside, with median t max values of 12.0 and 8.00 h post-dose, respectively. Steviol glucuronide was eliminated from the plasma, with similar t 1/2 values of approximately 14 h for both compounds. Administration of rebaudioside A resulted in a significantly (approximately 22%) lower steviol glucuronide geometric mean C max value (1472 ng/mL) than administration of stevioside (1886 ng/mL). The geometric mean AUC 0– t value for steviol glucuronide after administration of rebaudioside A (30,788 ng h/mL) was approximately 10% lower than after administration of stevioside (34,090 ng h/mL). Steviol glucuronide was excreted primarily in the urine of the subjects during the 72 h collection period, accounting for 59% and 62% of the rebaudioside A and stevioside doses, respectively. No steviol glucuronide was detected in feces. Pharmacokinetic analysis indicated that rebaudioside A and stevioside underwent similar metabolic and elimination pathways in humans with steviol glucuronide excreted primarily in the urine and steviol in the feces. No safety concerns were noted as determined by reporting of adverse events, laboratory assessments of safety or vital signs.

Comparative toxicokinetics and metabolism of rebaudioside A, stevioside, and steviol in rats

The toxicokinetics and metabolism of rebaudioside A, stevioside, and steviol were examined in rats for comparative purposes to determine whether toxicological studies conducted previously with stevioside would be applicable to the structurally-related glycoside, rebaudioside A. Single, oral doses of the radiolabelled compounds were extensively and rapidly absorbed with plasma concentration–time profiles following similar patterns for stevioside and rebaudioside A. Elimination of radioactivity from plasma was essentially complete within 72 h. All plasma samples had similar metabolite profiles; the predominant radioactive component in all samples was steviol, with lower amounts of steviol glucuronide(s) and low levels of one or two other metabolites. Rebaudioside A, stevioside, and steviol were metabolized and excreted rapidly, with the majority of the radioactivity eliminated in the feces within 48 h. Urinary excretion accounted for less than 2% of the administered dose for all compounds in both intact and bile duct-cannulated rats, and the majority of the absorbed dose was excreted via the bile. After administration of the compounds to intact and bile duct-cannulated rats, radioactivity in the feces was present primarily as steviol. The predominant radioactive compound detected in the bile of all cannulated rats was steviol glucuronide(s), indicating de-conjugation in the lower intestine. Overall, the data on toxicokinetics and metabolism indicate that rebaudioside A and stevioside are handled in an almost identical manner. These studies support the use of toxicological safety studies conducted with stevioside for the safety assessment of rebaudioside A.

An improved HPLC method for the analysis of diterpenoid glycosides in Stevia rebaudiana

Stevia rebaudiana, also known as sweetleaf, has been used as a sweetner for centuries in South America. Extracts are up to 300 times sweeter than table sugar, which is attributed to the diterpenoid glyscosides, stevioside and rebaudioside A. Although widely used in Japan and Brazil, most other countries do not allow the use of Stevia as a food additive. In the US, it can only be labelled as a dietary supplement. Many companies are now focusing on isolation of each of the individual diterpenoid glycosides, not just stevioside and rebaudioside A, for use in products as a low-calorie sugar alternative. Current HPLC methods for the determination of diterpenoid glycosides have been on NH2-columns under isocratic conditions [1]. An improved HPLC method utilizing a Phenomenex Synergi Hydro-RP column has been developed on a gradient reverse-phase system of 0.1% trifluoroacetic acid in water and acetonitrile at 202 nm which overcomes the limitations of the reported NH2 method with regards to separation of the minor components of Stevia. The method successfully separates rebaudiosides A, B, C, D and F, dulcoside A, isoteviol, steviol, steviol glucuronide, stevioside, steviolbioside, and several unidentified compounds.

Identification of Steviol Glucuronide in Human Urine

Stevioside (250 mg capsules) was given three times daily to 10 healthy subjects. Steviol glucuronide (steviol 19-O-beta-D-glucopyranosiduronic acid; MM, 494.58; melting point, 198-199 degrees C) was characterized in the 24 h urine as the only excretion product of oral stevioside by MS, NMR, IR, and UV spectroscopy. This is the first report on the unambiguous identification of steviol glucuronide in human urine.

47 Risk characterization

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.