Recent Publications by researchers of the "European Stevia Center"
21) Geuns J.M.C. 2008. Steviol Glucuronide as Excretion Product of Stevioside in Human Volunteers. Lack of Carcinogenic Properties of Steviol glycosides and Steviol. Proceedings of the ACS Symposium on “Sweetness and Sweeteners”, Atlanta 2006 (in press).
Absorption studies with Caco-2 cell monolayers revealed that steviol glycosides are barely absorbed by the intestines. Metabolism studies in healthy volunteers have shown that stevioside is completely degraded by bacteria of the colon into steviol and that part of this steviol is absorbed and glucuronated in the liver. The glucuronide is released in the blood and filtered by the kidneys into the urine. No accumulation of steviol glycosides or derivatives has been observed. As mutagenic effects of steviol were described, a thorough literature study has been made to evaluate possible risks when using steviol glycosides as a sweetener. The conclusion is, that there are no indications that steviol glycosides used as a sweetener are not safe.
20) Geuns J.M.C. 2008. Stevioside: a safe sweetener and possible new drug for the treatment of the metabolic syndrome. Proceedings of the ACS Symposium on “Sweetness and Sweeteners”, Atlanta 2006 (in press).
Steviol glycosides used in low amounts for sweetening purposes are safe. Their absorption by the intestines is very low. They are degraded to steviol by bacteria of the colon. Part of this steviol is absorbed and transformed into steviol glucuronide that is excreted in the urine. No accumulation in the body seems to exist. No harmful effects of steviol glycosides have been published in the scientific literature. ADI values have been suggested by calculations made from published results. High doses of steviol glycosides (750 – 1500 mg/day) may have beneficial pharmacological effects as lowering the blood pressure of hypertensive patients, lowering the blood glucose in diabetes type 2. In animal models, they have anti-carcinogenic effects. It is not proven that they have similar effects in man, as the intake as a sweetener will be very low.
19) Atteh, J, O. Onagbesan, K. Tona, J. Buyse, E. Decuypere, J. Geuns (2007) Nutritional Profile of Stevia rebaudiana (Bertoni) leaf and stem: Potential use as supplement in animal feed. Archivos de Zootecnia (in press).
A perennial shrub, stevia (Stevia rebaudiana, Bertoni), and its extracts are used as natural sweeteners and possess antimicrobial properties. However, little is known about the nutritional value of the shrub or its potential use as animal feed either as a fodder, a sweetener or as a feed prebiotic supplement. The reported antimicrobial properties of stevia and the extracts suggest that it has a potential for use as a prebiotic feed additive. This study determined the nutritional profile of the leaves and stem and evaluated their potential utilization in an animal model. Analyzed stevia leaves and stem had 16% and 6.7% crude protein respectively. They were low in fat content (2.6 and 1.1% respectively). The fatty acid profile of the extracted fat showed a preponderance of unsaturated fatty acids (65.8% and 71.4% for leaves and stem respectively). Linolenic acid (C18:3) was the most abundant fatty acid in stevia leaf oil (36%) whereas linoleic acid was the highest in stevia stem (38%). The crude fiber contents on dry fat free basis were 6.8% and 45.4% for leaves and stem respectively. The potassium content of stevia leaves and stem were comparable. Calcium, magnesium, iron, copper, zinc and manganese were higher in stevia leaves than in the stem. The opposite was true for sodium. The results of tests with broiler chickens showed that Apparent, Nitrogen corrected and True metabolisable energy values for stevia leaves were 2113, 2098 and 2223 Kcal/kg respectively. The values for stevia stem were 1573, 1554 and 1675 Kcal/kg. Retention of the protein from the leaves and stem by the chickens was 63% and 65.7% respectively. It was concluded that stevia leaves contain other nutritional attributes besides the high concentration of the sweetening components.
18) Atteh JO, Onagbesan OM, Tona K, Decuypere E, Geuns JMC and Buyse J (2008): Evaluation of supplementary Stevia (Stevia rebaudiana Bertoni) leaves and stevioside in broiler diets: effects on feed intake, nutrient metabolism, blood parameters and growth performance. Journal of Animal Physiology and Animal Nutrition (DOI 10.1111/ j.1439-0396.2007.00760.x).
A perennial shrub, stevia, and its extracts are used as a natural sweetener and have been shown to possess antimicrobial properties. Stevia contains high levels of sweetening glycosides including stevioside, which is thought to possess antimicrobial and antifungal properties. Little is known about the nutritional value of the shrub in livestock. This study determined the potential use of the shrub as a prebiotic animal feed supplement in light of the recent ban on the use of antibiotics in animal feed and the role of its constituent stevioside in the effects of the shrub.
Male Cobb broiler chicks were fed a basal broiler diet without antibiotic but with performance enhancing enzyme mix (positive control), a basal diet without antibiotic and enzymes (negative control), or diets in which 2% of the negative control diet was replaced with either dried ground stevia leaves or 130 ppm pure stevioside during two week starter and two week grower periods. Body weight gains, feed conversion, abdominal fat deposition, plasma hormone and metabolites and caecal short chain fatty acids (SCFA) were measured in the broilers at 2 and 4 weeks of age.
There was no significant effect of the treatments on feed intake during the starter period but birds fed diet supplemented with stevia leaves and stevioside consumed more feed (p<0.05) than those fed the positive control diet during the grower period. Weight gain by birds fed the positive control and stevioside diets was higher (p<0.05) than those fed other diets only during the starter period. Feed/gain ratio of birds fed the positive control and stevioside diets was superior (p<0.05) to others. There was no effect of the treatments on nutrient retention and water content of the excreta. Dietary stevia leave and stevioside decreased total concentration of short chain fatty acids and changed their profile in the ceca. There was no effect of the treatments on pancreas weight. Dietary stevia reduced blood levels of glucose, triglycerides and triiodothyronine (T3) but had no effect on non-esterified fatty acids. In contrast, stevioside only decreased T3. Both the stevia leaves and stevioside diets significantly increased abdominal fat content.
It is concluded that dietary enzyme growth promoters are beneficial to the broilers only during the starter stage and that inclusion of stevia leaves or stevioside had no beneficial effect on the performance of broilers.
17) Geuns Jan M.C. Comments to the paper by Nunes et al. (2007), Analysis of genotoxic potentiality of stevioside by comet assay. Food and Chemical Toxicology 45 (12) 2601-2602 (2007).
Nunes et al. (2007) orally administered 1 concentration (4 mg/ml) of stevioside (88.6% purity) to Wistar rats. DNA-damage was evaluated by the comet assay. They reported lesions in peripheral blood, liver, brain and spleen cells, the most pronounced effects being in the liver. Some comments have to be made to this paper. First of all, the structure shown in their Figure 1b is not correct and it is not that of steviol, but that of ent-kaurenate. The authors used stevioside with a purity of only 88.6 % and they administered only 1 concentration, i.e. the dose-dependence was not tested at all. In their Tables 1 and 2 the SD’s are very large, sometimes much larger than the mean itself! There was no positive control included in the experiment. As the authors performed tests over a period of 6 weeks, they should have included an internal standard to check the electrophoresis parameters over that long period. They did not refer to the excellent work by Sekihashi et al. (2002) and Sasaki (2000) who also tested stevioside and a large number of other compounds under strictly standardised conditions including dose dependency, a positive and negative control, and who did not find DNA damage by steviol glycosides nor by steviol. Moreover, Sekihashi et al. (2002) also tested stomach and colon cells, and this is very relevant as steviol glycosides are not absorbed (Koyama et al., 2003, Geuns et al., 2003). The authors refer to a metabolism study of IV injected 131I-stevioside. This metabolism might totally differ from that after oral uptake, and the 131I might give a totally different metabolism. The metabolism of oral stevioside has been thoroughly studied by Simonetti et al. (2004) and Geuns et al. (2004, 2006, 2007) and it was shown that there is no accumulation or metabolism of steviol in the human body, except steviol glucuronide synthesis that is excreted in the urine. The scores of the control blood cells (their Fig. 2) vary from 0.6 ± 1.34 to up to 27 ± 13.3 at 6 weeks and increase and decrease at different time points (observe the values of the SD too). The authors suggest stress as the possible cause. However, this seems unbelievable and a lack of standardisation by use of an internal standard, and a lack of control of the quality of the feed, that might contain mutagenic compounds, seem more likely. Finally, the p-value discussed on p. 664, left column line 11 (p<0.001) is different from that in Table 1 (p<0.01).
16) Geuns J.M.C. (2007) Herevaluatie van de veiligheid van Stevia rebaudiana en zijn inhoudsstoffen stevioside en rebaudioside A. Nederlands Tijdschrift voor Fytotherapie, 20 (4), 7-10.
De plant Stevia rebaudiana (Bertoni) Bertoni, en haar potentiële waarde als suikervervanger in de voeding of –in een heel andere, hogere dosering– als medicijn voor diabetes-patiënten, werd besproken op de NVF/NVGO-congressen in 2003 en 2005. De European Food Safety Authority (EFSA) is nu aan een nieuwe veiligheidsanalyse begonnen en er wordt verwacht dat de Food and Drug Administration (FDA) in de Verenigde Staten binnenkort hetzelfde gaat doen. In de zakenwereld wordt er op gerekend dat er nu wel een toelating gaat komen want grote Amerikaanse firma's zoals Coca Cola hebben allerlei patenten op gebruik van componenten uit de stevia ingediend . In afwachting van toelating, zijn enkele grondstoffen-leveranciers hun productie-proces en -capaciteit al aan het optimaliseren en uitbreiden zodat aan de verwachte vraag kan worden voldaan .
15) Jan M.C. Geuns, Johan Buyse, Annelies Vankeirsbilck, Elisabeth H.M. Temme (2007) Stevioside Metabolism by Human Volunteers. Experimental Biology and Medicine 232(1), 164-173.
Stevioside (250 mg capsules) was given thrice daily for 3 days to 10 healthy subjects. Blood samples were collected and blood pressure measured after nocturnal fasting, before, and at different time points during the third day of the administration of stevioside. No significant differences were found between the control and the stevioside condition for blood pressure and blood biochemical parameters. The twenty four hours urinary volume and urinary excretion of electrolytes were not significantly different. Likewise, no significant difference was found for mean blood glucose and insulin between control and stevioside condition. Thus oral stevioside is not directly effective as a hypotensive or hypoglycemic agent in healthy subjects at the dose administered in this study.
Stevioside, free steviol and steviol metabolites were analyzed in blood, feces and urine after three days of stevioside administration. No uptake was found of stevioside by the gastrointestinal tract or the amounts taken up were very low and below the detection limit of the UV detector. Stomach juice did not degrade stevioside. All the stevioside reaching the colon was degraded by micro-organisms into steviol, the only metabolite found in feces. In blood plasma, no stevioside, no free steviol or other free steviol metabolites were found. However, steviol glucuronide (SV glu) was found in maximum concentrations of 33 mg/ml (21.3 µg steviol equivalents/ml). In urine, no stevioside or free steviol were present, but SV glu was found in amounts of up to 318 mg/24 h urine (205 mg steviol equivalents/24 h). No other steviol derivatives were detected. In feces, besides free steviol, no other steviol metabolites or conjugates were detected. Steviol was excreted as SV glu in urine.
14) Jan M.C. Geuns, Johan Buyse, Annelies Vankeirsbilck, Elisabeth H.M. Temme, Frans Compernolle, Suzanne Toppet (2006) Identification of Steviol Glucuronide in Human Urine. J. Agric. Food Chem. (in press)
Stevioside (250 mg capsules) was given three times daily to 10 healthy subjects. Steviol glucuronide (steviol 19-O-b-D-glucopyranosiduronic acid, MM: 494.58, melting point 198-199 °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.
13) Geuns Jan M.C (2004). Introduction and presentation of the European Stevia Research Center. p. 5-8. In: Geuns J.M.C. and Buyse J. (Eds.). Proceedings of the first symposium "Safety of stevioside" Leuven, April 16, 2004. Euprint Ed., Parkbosstraat 3, 3001 Heverlee, Belgium. ISBN: 9074253024, pp.127 (email@example.com ).
After founding the "European Stevia Research Center" (ESC) in 2003 (www.kuleuven.ac.be/bio/biofys see under "Stevia center"), an international symposium was organised on April 16th 2004 at the University of Leuven (KULeuven - Belgium) entitled "Safety of stevioside". The European Stevia Research Center was presented. The speakers were introduced. Drs. V. Minne, Laboratory of Functional Biology, KULeuven, developed a method including an internal standard for measuring steviol very sensitively. Prof. Dr. P.B. Jeppesen, Aarhus University Hospital, Denmark, discussed the effects of stevioside on Type 2 diabetes. Dr. E. Koyama, Mitsubishi Chemical Safety Institute, Ibaraki, Japan, studied the metabolism and uptake of stevioside in rats and humans. Prof. J. Buyse, Laboratory of Physiology and Immunology of Domestic Animals, KULeuven, studied stevioside metabolism in animals: chickens and pigs. Prof. P. Pietta, ITB-CNR, Rep. Biochimica-Analitica, Milan, Italy, studied the ex vivo and in vivo metabolism of Stevia sweeteners in humans. Prof. L. Temme, Division: Food and Epidemiology, U.Z. St.-Rafaël, KULeuven, studied the short-term effects of stevioside on blood glucose concentration and blood pressure in healthy volunteers. At the end of the symposium Prof. J. Geuns discussed some hot items including safety aspects and risk assessment, and discussed the metabolism of oral stevioside in humans. The general conclusion of the symposium was that stevioside used as a sweetener is safe.
12) Geuns J.M.C. (2004) Review: The safety of stevioside used as a sweetener. p. 85-127. In: Geuns J.M.C. and Buyse J. (Eds.). Proceedings of the first symposium "Safety of stevioside" Leuven, April 16, 2004. Euprint Ed., Parkbosstraat 3, 3001 Heverlee, Belgium. ISBN: 9074253024, pp. 127 (firstname.lastname@example.org ).
Stevioside is a natural sweetener extracted from leaves of Stevia rebaudiana (Bertoni) Bertoni. The literature about Stevia, the occurrence of its sweeteners, their biosynthetic pathway and toxicological aspects are discussed. Injection or perfusion experiments of organs are considered as not relevant for the use of Stevia or stevioside as food additive, and therefore these studies are not included in this review.
The metabolism of stevioside is discussed in relation to the possible formation of steviol in both animals and man. Different mutagenicity studies as well as studies on carcinogenicity are discussed. Acute and sub-acute toxicity studies revealed a very low toxicity of Stevia and stevioside. Fertility and teratogenicity studies are discussed as well as the effects on the bio-availability of other nutrients in the diet.
The conclusion is that stevioside is safe when used as sweetener. It is suitable for both diabetics, and PKU patients, as well as for obese persons intending to lose weight by avoiding sugar supplements in the diet. No allergic reactions seem to exist.
11) Temme E.H.M., Vankeirsbilck A., Buyse J. and Geuns J.M.C. (2004b) A short-term study of stevioside in healthy volunteers p. 63-74. In: Geuns J.M.C. and Buyse J. (Eds.). Proceedings of the first symposium "Safety of stevioside" Leuven, April 16, 2004. Euprint Ed., Parkbosstraat 3, 3001 Heverlee, Belgium. ISBN: 9074253024, pp.127. (email@example.com ).
Stevioside is a natural plant glycoside isolated from the plant Stevia rebaudiana. It is 300 times sweeter than sugar but contains no calories. Stevioside would therefore be suitable for diets of e.g. diabetic and obese persons. In addition, studies suggested hypotensive and hypoglycaemic effects of stevioside when administered in a high dosage. This study was undertaken to evaluate the short-term effects on blood pressure, urinary excretion of electrolytes and blood glucose/insulin concentrations, in healthy subjects.
The study group consisted of 9 healthy subjects aged between 21 and 29 years. Over a period of 3 days, each subject was given capsules containing stevioside (250 mg) thrice daily. A blood sample was collected and blood pressure was measured before (after nocturnal fasting) and at different time-points after 3 days of stevioside. In addition, two 24-hour urine samples (before and after) were collected by the volunteer to evaluate the volume and concentrations of electrolytes.
The average systolic and diastolic blood pressure was 115 mmHg and 72 mmHg for the stevioside and 114 mmHg and 74 mmHg for the control condition, respectively. No significant differences were found between the stevioside and the control condition. Twenty-four hour urinary volume and urinary excretion of electrolytes was not significantly greater in the stevioside compared with the control condition. Mean blood glucose and insulin were 4.63 mmol/L and 5.9 mU/L for the stevioside and 4.60 mmol/L and 5.6 mU/L for the control condition, respectively, there being no difference between them.
Stevioside, when administered orally for three days in three 250 mg capsules, is not directly effective as a hypotensive or hypoglycaemic agent in healthy subjects, although it might stimulate water and sodium excretion via the urine. More information is needed on longer-term and post-prandial effects.
10) Buyse Johan and Geuns Jan M.C. (2004b) The metabolism of Stevioside by animals: chickens and pigs. p. 35-50. In: Geuns J.M.C. and Buyse J. (Eds.). Proceedings of the first symposium "Safety of stevioside" Leuven, April 16, 2004. Euprint Ed., Parkbosstraat 3, 3001 Heverlee, Belgium. ISBN: 9074253024, pp. 127. (firstname.lastname@example.org ).
9) Minne Veerle J.Y., Compernolle Frans, Toppet Suzanne and Geuns Jan M.C. (2004b). Sensitive HPLC determination of steviol in biological fluids and plant material with fluorescence detection. p. 9-24. In: Geuns J.M.C. and Buyse J. (Eds.). Proceedings of the first symposium "Safety of stevioside" Leuven, April 16, 2004. Euprint Ed., Parkbosstraat 3, 3001 Heverlee, Belgium. ISBN: 9074253024, pp. 127. (email@example.com ).
A simple method is described for the determination of steviol (SV) by reversed-phase high-performance liquid chromatography (RP-HPLC) using dihydro-isosteviol (DHISV) as internal standard (IS). SV and DHISV were derivatised with 4-(bromomethyl)-7-methoxycoumarin in an aprotic solvent (N,N-dimethylformamide (DMF) or acetone). Separation of the resulting ester derivatives was achieved on an ODS column (250 ´ 4.6 mm i.d., 5 µm particle size) at a flow-rate of 1 mL.min-1 using acetonitrile-water (80:20 v/v) as the mobile phase. Using fluorescence detection with excitation at 321 nm and emission at 391 nm, a linear relationship was observed for concentrations between 0.5 and 50 µg mL-1 of SV and the detection limit was 100 pg. The intra- and interday variations (n = 9) were 0.64 and 0.88%, respectively. The application of the method to beer, urine and faeces samples involved a simple procedure of extraction by diethyl ether and derivatisation in DMF. Plant samples required preparation of an acid fraction containing the SV analyte, derivatization and sample clean-up using small silica columns made of pipette tips and thin layer chromatography. A sensitive determination of 5.9 µg of SV present in 1 g of dry plant material was done with high precision and accuracy.
8) Geuns J.M.C., Buyse J., Vankeirsbilck A. and Temme L. (2004) About the safety of stevioside used as a sweetener. p. 75-84 In: Geuns J.M.C. and Buyse J. (Eds.). Proceedings of the first symposium "Safety of stevioside" Leuven, April 16, 2004. Euprint Ed., Parkbosstraat 3, 3001 Heverlee, Belgium. ISBN: 9074253024, pp. 127. (firstname.lastname@example.org ).
Stevioside is a natural sweetener extracted from leaves of Stevia rebaudiana (Bertoni) Bertoni. Some of the more persistent rumours about possible harmful effects of stevioside eg. on male fertility and carcinogenicity, are discussed and subsequently refuted.
The metabolism of stevioside by volunteers has been studied. In the faeces only free steviol was found. Concentrations of free steviol or stevioside in blood or urine were below the detection limits. Steviol conjugates were found in blood and urine as typical excretion products.
A risk assessment was made taking the daily sugar consumption in Belgium as an example.
7) Minne V., Compernolle F., Toppet S., Geuns J.M.C. (2004) Steviol Quantification at the picomol level by HPLC. Journal of Agricultural and Food Chemistry 52, 2445-2449
A simple and highly sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of steviol (SV) using dihydro-isosteviol (DHISV) as an internal standard (IS). SV and DHISV were derivatized by reaction of the acids with 4-(bromomethyl)-7-methoxycoumarin in an aprotic solvent (DMF or acetone). The resulting ester derivatives were separated on an ODS column (250 ´ 4.6 mm i.d., 5 µm particle size) using fluorescence detection with excitation at 321 nm and emission at 391 nm. The mobile phase consisted of acetonitrile-water (80:20 v/v) with a flow rate of 1 mL.min-1 . A linear relationship was observed for concentrations between 0.5 and 50 µg/mL of SV and the detection limit was 100 pg. For application of this method to samples of beer fortified with stevioside, a simple procedure for extraction of the beer with diethyl ether and derivatization in DMF was applied. Whereas beer samples spiked with SV gave a linear response over the range 0.1 - 15 µg/mL beer, no SV could be detected in beer samples enriched in stevioside that had been stored for over 3 years. The application of the method to plant samples involved preparation of an acid fraction containing the SV analyte, derivatization and sample clean-up using small silica columns and thin layer chromatography. A sensitive determination of 594 ng of steviol present in 100 mg of dry plant material was performed with high precision and accuracy.
6) Geuns J. M.C., Bruggeman V., Buyse J.G. (2003) Effect of stevioside and steviol on the developing broiler embryo's. J. Science of Food and Agriculture 51, 5162-5167.
At day 7 of incubation, fertile broiler eggs were injected with different amounts of stevioside and steviol ranging from 0.08, 0.8 or 4 mg stevioside/egg, and 0.025, 0.25 or 1.25 mg steviol/egg. At hatch (day 21) and one week later not any influence of the different treatments could be found on embryonic mortality, body weight of the hatchlings, deformations (eg. bone, beak and head malformations, abnormal feathering, open vent) or abnormal development of the gonads. No stevioside or steviol could be detected in the blood of the hatchlings. The hatchlings developed normally. It is concluded that prenatal exposure to stevioside and steviol is not toxic for the chicken embryo.
5) Geuns J.M.C., Malheiros R.D., Moraes V.M.B., Decuypere E. M.-P., Compernolle F., Buyse J.G. (2003) Stevioside metabolism by chickens. J. Agr. Food Chem. 51, 1095-1101.
In intubation experiments (643-1168 mg per animal), most of the administered stevioside was recovered unchanged in the excreta and only about 2 % was converted into steviol. Neither stevioside nor steviol could be found in the blood. In chronic studies (667 mg stevioside/kg feed) with laying hens and meat-type chickens no significant differences were found in feed uptake, weight gain and feed conversion. The egg production and egg composition of laying hens were not influenced. Most of the stevioside taken up was found untransformed in the excreta and about 21.5 % or 7.3 % was converted to steviol by meat-type chickens or laying hens respectively. No stevioside or steviol could be detected in the blood or in the eggs of the different groups of animals. In anaerobic incubation experiments with chicken excreta only a 20 % conversion of stevioside into steviol was found. No harmful effects were observed in the chronic stevioside supplementation experiments nor in the intubation experiments in which very high stevioside doses were given.
4) Geuns J.M.C. (2003) Molecules of interest: Stevioside. Phytochemistry 64, 913-921.
Stevioside is a natural sweetener extracted from leaves of Stevia rebaudiana (Bertoni) Bertoni. The literature about Stevia, the occurrence of its sweeteappners, their biosynthetic pathway and toxicological aspects are discussed. Injection experiments or perfusion experiments of organs are considered as not relevant for the use of Stevia or stevioside as food, and therefore these studies are not included in this review.
The metabolism of stevioside is discussed in relation with the possible formation of steviol. Different mutagenicity studies as well as studies on carcinogenicity are discussed. Acute and subacute toxicity studies revealed a very low toxicity of Stevia and stevioside. Fertility and teratogenicity studies are discussed as well as the effects on the bio-availability of other nutrients in the diet.
The conclusion is that Stevia and stevioside are safe when used as a sweetener. It is suited for both diabetics, and PKU patients, as well as for obese persons intending to lose weight by avoiding sugar supplements in the diet. No allergic reactions to it seem to exist.
3) Geuns, J. M.C., Augustijns P., Mols R., Buyse J.G. and B. Driessen (2003) Metabolism of stevioside in pigs and intestinal absorption characteristics of stevioside, rebaudioside A and steviol. Food Chem. Toxicol. 41, 1599-1607.
Stevioside orally administered to pigs was completely converted into steviol by the bacteria of the colon. However, no stevioside or steviol could be detected in the blood of the animals, even not after converting steviol into the (7-methoxycoumarin-4-yl)methyl ester of steviol, a very sensitive fluorescent derivative with a detection limit of about 50 pg.
The intestinal transport characteristics of stevioside, rebaudioside A and steviol were also studied in the Caco-2 system. Only a minor fraction of stevioside and rebaudioside A was transported through the Caco-2 cell layer giving a Papp value of 0.16-6 and 0.11´ 10 -6 cm/s respectively. The Papp value for the absorptive transport of steviol was about 38.6´ 10 -6 cm/s while the Papp value for the secretory transport of steviol was only about 5.32´ 10 -6 cm/s suggesting carrier-mediated transport. The discrepancy between the relatively high absorptive transport of steviol and the lack of steviol in the blood may be explained by the fact that in the Caco-2 study, steviol is applied as a solution facilitating the uptake, whereas in the colon steviol probably is adsorbed to the compounds present in the colon of which the contents is being concentrated by withdrawal of water.
2) Nicole Totté, Wim Van den Ende, Els J.M. Van Damme, Frans Compernolle, Ilse Baboeuf and Jan M.C. Geuns. (2003) Cloning and heterologous expression of early genes in gibberellin and steviol biosynthesis via the methylerythritol phosphate pathway in Stevia rebaudiana Bertoni. Can. J. Bot. 81, 517-522.
The ent-kaurene skeleton of chloroplast diterpene glycosides, which are produced in large quantities in the leaves of Stevia rebaudiana Bertoni, is formed via the recently discovered 2-C-Methyl-D-erythritol-4-phosphate pathway. The enzymes catalysing the first two steps of this pathway, 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) were characterized. Using reverse transcriptase-PCR, the dxs and dxr cDNAs were cloned, which comprise ORFs of 2148 and 1422 nucleotides, respectively. The cDNA-derived amino acid sequences for DXS and DXR contain 716 and 474 residues, encoding polypeptides of about 76.6 and 51 kDa, respectively. DXS and DXR from Stevia both contain an N-terminal plastid targeting sequence and show high homology to other known plant DXS and DXR enzymes. Furthermore, we demonstrated through heterologous expression in Escherichia coli that the cloned cDNAs encode functional proteins.
1) Totté N., L. Charon, M. Rohmer, F. Compernolle, I. Baboeuf and J.M.C. Geuns: Biosynthesis of the diterpenoid steviol, an ent-kaurene derivative from Stevia rebaudiana Bertoni, via the methylerythritol phosphate pathway. Tetrahedron Letters, 41, 6407-6410. (2000).
As shown from [1-13C]glucose incorporation, steviol, the diterpene aglycone moiety of stevioside, is synthesized in Stevia rebaudiana Bertoni via the mevalonate-independent methylerythritol phosphate pathway.