Metabolism of stevioside in pigs and intestinal absorption characteristics of stevioside, rebaudioside A and steviol
Introduction
Stevioside, the main sweet component in the leaves of Stevia rebaudiana (Bertoni) Bertoni (Compositae), tastes about 300 times sweeter than sucrose (0.4% solution). Structures of the sweet components of Stevia occurring mainly in the leaves are given in Fig. 1. Their content varies between 4 and 20% of the dry weight of the leaves depending on the cultivar and growing conditions. Stevioside 3 is the main sweet component. Other compounds present but in lower concentration are: steviolbioside 2, rebaudioside A 4, B 5, C 6, D 7, E 8, F 9 and dulcoside A 10 (Kennelly, 2002, Starrat et al., 2002). The presence of steviolbioside and rebaudioside B in extracts might be due to artifacts of the extraction procedure (refs. in Kennelly, 2002).
Both the Stevia plant, its extracts, and stevioside have been used for several years as a sweetener in South America, Asia, Japan, China, and in different countries of the EU. In Brazil, Korea and Japan Stevia leaves, stevioside and highly refined extracts are officially used as a low-calorie sweetener. In the USA, powdered Stevia leaves and refined extracts from the leaves have been used as a dietary supplement since 1995. In 2000, the European Commission refused to accept Stevia or stevioside as a novel food because of a lack of critical scientific reports on Stevia and the discrepancies between cited studies with respect to possible toxicological effects of stevioside and especially its aglycone steviol 1 (Fig. 1) (Kinghorn, 2002; Geuns, unpublished). The advantages of stevioside as a dietary supplement for human subjects are manifold: it is stable, it is non-calorific, it helps maintain good dental health by reducing the intake of sugar and opens the possibility for use by diabetic and phenylketonuria patients and obese persons.
Many papers describe the safety of stevioside used as a sweetener (see Geuns, 2002, for a review). In humans, an acceptable daily intake (ADI) of 7.9 mg stevioside/kg BW was calculated (Xili et al., 1992). However, this ADI should be considered as a minimum value as the authors did not test concentrations of stevioside higher than 793 mg/kg BW (safety factor 100). Considering many reports from the literature, an ADI of more than 20 mg stevioside/kg BW is likely (Geuns, 2002). However, mutagenic effects of steviol, the aglycone of stevioside, and/or its metabolites were reported in the forward mutation test using Salmonella typhimurium TM677 (Pezzuto et al., 1985, Compadre et al., 1988, Matsui et al., 1996a, Temcharoen et al., 1998, Terai et al., 2002). After metabolic activation it was shown that so far unknown steviol metabolites caused mutations in S. typhimurium TM677, i.e. transitions, transversions, duplications and deletions at the guanine phosphoribosyltransferase (gpt) gene (Matsui et al., 1996b). However, stevioside and steviol were inactive in various TA strains of S. typhimurium, Escherichia coli WP2 uvrA/pKM101 and the rec-assay using Bacillus subtilis even when activation S9 mix was present (Matsui et al., 1996a, Klongpanichpak et al., 1997). The direct mutagenic activity of 15-oxo-steviol was refuted by Procinska et al. (1991), but confirmed by Terai et al. (2002). The activity of steviol in S. typhimurium TM677 was only about 1/3000 that of 3,4-benzopyrene and that of steviol methyl ester 8,13 lactone was 1/24,500 that of furylfuramide (Terai et al., 2002). Although a weak activity of steviol and some of its derivatives was found in the very sensitive S. typhymurium TM677 strain, the authors concluded that the daily use of stevioside as a sweetener is safe. Moreover, the presence in the blood of the chemically synthesised steviol derivatives after feeding stevioside has not been demonstrated so far. Very high doses of steviol (90% purity) intubated to hamsters (4 g/kg bw), rats and mice (8 g/kg bw) did not induce micronucleus in bone marrow erythrocytes of both male and female animals. However, these doses showed some cytotoxic effect to the female, but not to the male of all treated animal species (Temcharoen et al., 2000). It is not excluded that the toxicity is due to the 10% impurities present. After metabolic activation of steviol, some gene mutation and chromosomal aberration was found in Chinese hamster lung fibroblasts (Matsui et al., 1996a). It has to be said that of all animals tested hamsters had the most sensitive response. Moreover, in hamster, several metabolites of stevioside were found that are not formed in rats or humans. Therefore, the relevance of experiments with hamsters should be questioned.
It has been shown that oral stevioside is not taken up by the human body (Yamamoto et al., 1985, Bracht et al., 1985) and none of the digestive enzymes from the gastro-intestinal tract of different animals and man are able to degrade stevioside into steviol, the aglycone of stevioside (Wingard et al., 1980, Hutapea et al., 1997, Koyama et al., 2001, Koyama et al., 2003a, Koyama et al., 2003b). Nevertheless, in feeding experiments with rats and hamsters stevioside was metabolised to steviol by the bacterial flora of the caecum. Steviol was found in the blood of the animals with the maximum concentration occurring after 8 h (Nakayama et al., 1986, Koyama et al., 2003a, Koyama et al., 2003b). In the cited studies, it was not indicated that coprophagy, occurring in rodents, was prevented, so it is not clear whether the steviol occurring in the blood was taken up directly from the colon or indirectly from the ingested faeces (after passing through the intestines again). Although bacteria isolated from the human colon are able to transform stevioside into steviol in vitro (Hutapea et al., 1997, Koyama et al., 2001, Koyama et al., 2003a, Koyama et al., 2003b), it has never been proven that this is also the case in vivo nor that the steviol possibly formed is taken up directly from the colon. Moreover, studies with roosters (Pomaret and Lavieille, 1931) and chickens (laying hens and broylers, Geuns et al., 2003) indicated that stevioside was rapidly eliminated from the body, largely untransformed. Only the bacteria from the caecum or colon were able to degrade stevioside into steviol (caecum of mice, rats, hamsters and chickens; colon of pigs and man). The bacteria from the human colon also formed steviol 16,17α-epoxid in vitro, that was again metabolised to steviol. However, in vivo this epoxid formation most probably will not occur due to the anaerobic conditions of the human colon. It was correctly concluded that steviol is the only metabolite in faeces and is not further metabolised (Hutapea et al., 1997, Koyama et al., 2001, Koyama et al., 2003a, Gardana et al., submitted for publication, Geuns et al., 2003). Anyway, steviol epoxid has been tested in mutagenicity studies and showed to be inactive (Pezzuto et al., 1985). In hamsters the LD50 of steviol (90% purity) was 5.2 and 6.1 g/kg bw for respectively, male and female animals. In rats and mice the LD50 was above 15 g/kg bw demonstrating that of the tested animals hamsters are more sensitive to steviol (Toskulkao et al., 1997).
As mutagenic effects of steviol and/or its metabolites were published, one of the most urgent problems to solve is the possible degradation of stevioside into steviol and other metabolites in vivo. In this study, pigs were used because their metabolism resembles that of humans. A second important issue is whether steviol, if produced, is taken up by the intestine and to what extent. Therefore, in addition we studied the transport of steviol, stevioside and rebaudioside A through Caco-2 monolayers as more information is needed with respect to the possible uptake of steviol by the colon. The Caco-2 cell layer is a valuable transport model for the small intestinal epithelium (Hidalgo et al., 1989).
Section snippets
Chemicals
The experiments were performed using stevioside that was purified by repeated crystallisation from MeOH to a purity level of more than 96%. Steviolbioside (around 3%) and rebaudioside A (around 0.5%) were the main impurities. Steviol was made and repeatedly crystallised from MeOH as described (Ogawa et al., 1980). Solvents of HPLC grade were from Acros (H2O, acetonitrile, CHCl3), BDH (MeOH, EtOH, N,N-dimethylformamide) and Biosolve (acetone). N,N-diisopropylethylamine was from Acros and
Detection limits and recovery experiments
The detection limit of stevioside was 50 ng per injection. The recovery of stevioside from spiked faeces samples (1, 6.5 or 20 mg g−1 dry wt.) was 72.1±1.3% (n=20). Recovery could not be improved by using absolute EtOH or 97:3 EtOH:diethylether as extraction solvent. The recovery of stevioside from spiked blood samples (6.67 mg g−1 dry wt.) after TLC clean-up was 68.4±2.6% (n=9). In order to obtain such a recovery it is very important to rinse the sample vials containing the stevioside extracts
Discussion
Steviol was the only compound that we could detect in pig faeces. This is in agreement with published results where steviol was found as the only possible metabolite produced by the intestinal microflora from various animal species and humans under strictly anaerobic conditions (Hutapea et al., 1997, Koyama et al., 2003a, Gardana et al., submitted for publication, Geuns et al., 2003). The steviol formed in the faeces was not found in the blood. These results suggest that steviol, which is only
Acknowledgements
The authors acknowledge Professor D. Kinghorn for the gift or pure stevioside and Specchiasol for financial support. Professor R. Geers is acknowledged for use of the Zootechnical Centre and Jan M.C. Geuns and Patrick Augustijns acknowledge the “Onderzoeksraad KULeuven” for grants OT/00/15 and OT/01/14, respectively and the FWO for grant number G.0111.01. We thank Hilde Verlinden and Tom Struyf for their skilful technical assistance.
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