Absorption and distribution of steviol glycosides in animal and human models

November 18, 2015

 

Considerable progress in the understanding of steviol glycoside metabolism has occurred recently. Stevioside and rebaudioside A are hydrolyzed to a common metabolite, the aglycone steviol by colonic and/or cecal bacteria, although, some will pass through the intestinal tract completely or partially intact. There is virtually no absorption of intact steviol glycosides in the gastrointestinal tract. Glucose moieties, released by the microbial metabolism of steviol glycosides, are presumably used for energy by colonic bacteria, as there is no evidence glucose is absorbed. Steviol is absorbed from the colon and elimin

ated via the feces through enterohepatic re-circulation, or via urine as a glucuronide. There is no evidence that steviol accumulates in the body from successive ingestions of steviol glycosides (Roberts and Renwick 2008).

In studies in rat models done by Wingard et al(1), Nakayama et al(2), Koyama et al(7), and in other animal models, including chickens, studied by Geuns et al6, hamsters, studied by Hutapea et al(3), and pigs, studied by Geuns et al(5) indicate that stevioside is not readily absorbed from the GI tract.

 

Sung(4) reported detectable levels of stevioside, but not steviol, in plasma after administration of a Stevia product. Groups of male Sprague-Dawley rats were given T100 sunstevia 95% (containing 70% stevioside) at a dose of 0.5 or 2 g/kg bw by gavage. Stevioside was detected in plasma 5 min after dosing. There was considerable variation between animals, with the time to maximum plasma concentration varying from 10 to 300 min. Clearance did not differ significantly between the doses. Reported plasma half-lives were 10.6 ± 8.7 and 6.7 ± 3.7 h at 0.5 and 2.0 g/kg bw, respectively. At 48 h, 5.7–16.9% and 1–6.7% of the total administered dose of stevioside was receovered in the faeces and urine, respectively. Steviol was detected in faeces collected up to 48 h, but not in plasma sampled up to 24 h after dosing (limit of detection, 1 mg/ml).

 

Intestinal transport of stevioside (1 mmol/l), rebaudioside A (1 mmol) and steviol (30 mmol/l - 1 mol/l) has been investigated in a Caco-2 cell monolayer model by Genus et al(3). The integrity of the monolayer was verified with fluorescein. Transport of stevioside and rebaudioside A was very low (apparent permeability coefficients, 0.16 x 10-6 and 0.11 x 10-6 cm/s, respectively). Steviol was transported more effectively, with a higher apparent perm

 

eability coefficient for absorptive transport (44.5 x 10-6 cm/s) than for secretory transport (7.93 x 10-6 cm/s) at a concentration of 100 mmol/l. At concentrations of 300 mmol/l and 1 mol/l, steviol slightly compromised the integrity of the monolayers during transport.

 

The intestinal absorption of a Stevia mixture and the aglycone steviol was investigated by Koyama et al(7) using everted gastrointestinal sacs from four male Sprague-Dawley rats. The Stevia mixture contained rebaudioside A (28.8%), rebaudioside C (25.2%), stevioside (17.0%) and dulcoside A (10.2%). The everted sacs were incubated in Stevia mixture (0.5 mg/ml) or steviol (0.1 mg/ml) for 30 min. Transport of salicyclic acid (10 mg/ml) was used to confirm that the sacs were functional. Steviol was transported in both the duodenum–jejunum and the ileum (76% and 95% of salicyclic acid transport, respectively). The steviol glycosides were poorly absorbed from the Stevia mixture, with more than 93% remaining in the mucosal fluid (Koyama et al., 2003a).

 

Absorption of the Stevia mixture described above was also investigated in vivo by the same authorsibid in four male Sprague-Dawley rats. Stevia mixture (in 2% gum arabic) was administered at a dose of 125 mg/kg bw. Steviol was not detected in plasma at 1 h, but was detected at increasing concentrations between 2 h and 8 h, when the concentration reached a peak of about 5 mg/ml. In contrast, the peak plasma concentration of steviol (18.31 mg/ml) was observed 15 min after a single oral administration of steviol (45 mg/kg in corn oil). These doses were approximately equimolar for steviol (Koyama et al., 2003a).

 

Similarly Wang et al(10) showed that, in male Sprague-Dawley rats given a single oral dose of Stevioside (purity, 95%) at 0.5 g/kg bw, low concentrations of steviol were detected in plasma for the first 8 h, followed by a rapid increase to a concentration of about 1000 ng/ml at 24 h. This study used a highly sensitive method for detection of steviol, but did not examine levels of stevioside or other metabolites.

 

Metabolism of steviol glycosides in humans and rats is the same, but the pattern of metabolite excretion is different (Wheeler et al. 2008). In humans, as in rats, both stevioside and rebaudioside A are metabolized by bacteria in the lower gut to steviol, which is absorbed into the portal blood system and transported to the liver where it is glucuronidated. In humans, however, most steviol glucuronide appears in the plasma instead of the bile. Peak plasma concentrations of steviol glucuronide in humans occur approximately 8 and 12 hours post-dosing for stevioside and rebaudioside A, respectively. Like the rat, peak plasma metabolite concentrations were lower after rebaudioside A ingestion than after stevioside ingestion. The half-life of steviol glucuronide in human plasma is approximately 14 hours. Steviol glucuronide is the major excretion form of absorbed steviol in humans, and excretion occurs primarily via the urine rather than the feces. Only a small amount of steviol excretion occurs via the feces in humans.

 

 

Biotransformation

 

Gardana et al(8) reported that faecal bacterial suspensions from eleven healthy volunteers (six men and five women) were incubated under anaerobic conditions with 40 mg of Stevioside (purity, 85%) and 40 mg of rebaudioside A (purity, 90%) for 72 h. Stevioside and rebaudioside A were completely hydrolysed to the aglycone steviol within 10 and 24 h, respectively. Among cultures of coliforms, bifidobacteria, enterococci and bacteroides, only the bacteroides were able to hydrolyse these compounds. The data indicated that both glycosides were initially hydrolysed to steviolbioside (this occurred more slowly with rebaudioside A), and the steviolbioside was then rapidly metabolized to steviol. Steviol remained unchanged during the 72 h incubation, indicating that bacterial enzymes are not able to cleave the steviol structure.

 

Human faecal metabolism of Stevia compounds was investigated by Koyama et al(9) in pooled faecal homogenates obtained from five healthy Japanese male volunteers. The materials tested were Stevia mixture (main components: rebaudioside A, stevioside, rebaudioside C, dulcoside A), its a-glucose derivative, referred to as enzymatically modified Stevia (main components: a-glucosylrebaudioside A, aglucosylstevioside, a-glucosylrebaudioside C, a-glucosyl dulcoside A), rebaudioside A, stevioside, steviol, rebaudioside C, dulcoside A, rebaudioside B, rubusoside, a-monoglucosylrebaudioside A and a-monoglucosylstevioside. After incubation of the faecal homogenates under anaerobic conditions for 24 h, the Stevia mixture, glycosides and a-glucose derivatives were all rapidly degraded. Stevioside was hydrolysed, with successive removal of glucose units via rubusoside, to the aglycone steviol. The metabolism of a-monoglucosylstevioside was similar to that of stevioside after a-deglucosylation. For rebaudioside there were two pathways, a major pathway in which rebaudioside A was hydrolysed via stevioside to steviol, and a minor pathway that suggested that rebaudioside A is metabolized via rebaudioside B to steviol. The metabolism of amonoglucosylrebaudioside A was similar to that of rebaudioside A after a-deglucosylation. No degradation of steviol was observed over the 24 h incubation

period. The authors concluded that steviol was the only final product of the metabolism of Stevia-related compounds, including enzymatically modified Stevia in human intestinal microflora, and that there were no apparent species differences in the intestinal metabolism of Stevia mixture between rats and humans.

 

Metabolism of steviol in rats and humans has been investigated by Hutapea et al(3) using pooled human liver microsomal preparations from five male and five female donors, and from rat liver microsomal preparations with the same protein content. Metabolite formation required a nicotinamide adenine dinucleotide phosphate, reduced (NADPH)-generating system, indicating cytochrome P450 (CYP)- dependent metabolism. The metabolic profile obtained with human liver microsomal fractions was similar to that obtained with rat liver microsomal preparations; mass spectrometric analysis indicated the presence of two dihydroxy metabolites and four monohydroxy metabolites. One additional monohydroxy metabolite was detected with the rat preparation. The liver microsomal clearance of steviol was approximately four times lower in humans than in rats (Koyama et al., 2003a). Hamsters were given stevioside (purity not specified) at a dose of 1 g/kg bw by gavage and metabolites were measured in the plasma, urine and faeces at 3, 24 and 24 h, respectively. The samples were treated with glucuronidase/sulfatase to hydrolyse conjugated metabolites. Steviol-16,17a-epoxide, stevioside, 15 ahydroxysteviol and steviolbioside were detected in the plasma, urine and faeces. In addition, isosteviol was detected in the urine and faeces, and steviol was detected in the faeces.

 

Metabolism of stevioside by human volunteers has been investigated in a collaborative study conducted in Belgium and Italy by Genus et al (11,12). In Italy, nine healthy men (aged 20–50 years) were given capsules containing 375 mg of stevioside (purity not specified) after an overnight fast. Low concentrations of stevioside were detected in the plasma of seven of the subjects, with a maximum of 0.1 mg/ml. Peak plasma concentrations occurred at 60 to 180 min after dosing. Steviol glucuronide was detected in five of the men. No free steviol, steviol-16,17a-epoxide, 15ahydroxysteviol or 15-oxo-steviol was detected. Steviol glucuronide was detected in the urine of all men, and low concentrations of stevioside were also present in the urine of two men. Free steviol or its unconjugated metabolites were not detected. Only free steviol was detected in the faeces. In Belgium, five male and five female volunteers (aged 24 ± 2 years) were each given nine doses of 250 mg of stevioside (purity, >97%; impurities being other Stevia glycosides) at 8 h intervals on three successive days. No stevioside or free steviol was detected in the blood. After hydrolysis with b-glucuronidase/sulfatase, steviol was detected at concentrations ranging from 0.7 to 21.3 mg/ml, with peak concentrations occurring at varying times up to 5 h. Similarly, stevioside and conjugated steviol were detected in the urine at 24 h. The only compound detected in the faeces was free steviol. The differences between the two studies were considered to be due to the different doses of stevioside administered and the different detection limits of the analytical method for stevioside. The total recovery of steviol metabolites varied between 22% and 86% of the administered daily dose of stevioside (mean total recovery, 52.1 ± 27%) (Geuns & Pietta, 2004).

 

References

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  2. Wingard, R.E., Brown, J.P., Enderlin, F.E., Dale, J.A., Hale, R.L., Seitz, C.T., Intestinal degradation and absorption of the glycosidic sweeteners stevioside and rebaudioside A. Experientia, 1980 36, 51 9-520.

  3. Wheeler, A., Boileau, A.C., Winkler, P.C., Compton, J.C., Prakash, I., Jiang, I., and Mandarino, D.A., Pharmacokinetics of rebaudioside A and stevioside after single oral doses in healthy men. Food Chem Toxicol 46(Suppl. 7S): 2008, S54–S60.

  4. Nakayama, K., Kasahara, D.,Yamamoto, F., Absorption, distribution, metabolism and excretion of Stevioside in rats. J. Food Hyg.,SOC. Jpn.1986, 27, 1-8.

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  10. Gardana, C., Simonetti, P., Canzi, E., Zanchi, R. & Pieta, P. Metabolism of Stevioside and rebaudioside A from Stevia rebaudiana extracts by human microflora. J. Agri. Food Chem.,2003, 51, 6618–6622.

  11. Koyama, E., Ohori, Y., Kitazawa, K., Izawa, O., Kakegawa, K., Fujino, A. & Ui, M., In vitro metabolism of the glycosidic sweeteners, Stevia mixture and enzymically modified Stevia in human intestinal microflora. Food Chem. Toxicol.,2003,  41, 359–374.

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  13. Geuns, J.M.C. & Pietta, P. Stevioside metabolism by human volunteers. Unpublished report from Laboratory Functional Biology, Kuleuven, Leuven Belgium and ITB-CNR, Segrate (MI), Italy. Submitted to WHO by the Federal Ministry of Social Affairs, Public Health and the Environment, Belgium.2004

  14. Geuns, J.M., Buyse, J., Vankeirsbilck, A., Temme, E.H., Compernolle, F., Toppet, S.,. Identification of steviol glucuronide in human urine. J. Agric. Food Chem.,2006, 5: 2794-2798.

 

 

 

 

 

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