Recent Publications by researchers of the "European Stevia Center"

 

42) Mohamed Amal A. A., Stijn Ceunen, Jan M.C. Geuns, Wim Van den Ende and Marc De Ley (2011)
UDP-dependent glycosyltransferases involved in the biosynthesis of steviol glycosides
.
J. Plant Physiology (in press).

 

Abstract:
A short-term experiment was designed to measure the transcript levels of downstream genes contributing to the biosynthesis of steviol glycosides. Stevia rebaudiana plants were subjected to long- and short-day conditions for different time intervals. Samples from both lower and upper leaves were collected. Using quantitative real-time polymerase chain reaction, the transcript level of three UDP-dependent glycosyltransferases, UGT85C2, UGT74G1 and UGT76G1, was studied. The results were compared with the steviol glycoside contents measured in the leaves, which were quantified by reversed phase HPLC. In the same daylength condition, steviol glycoside concentration and the transcript level of the three UGT genes was higher in upper leaves than in lower leaves. Steviol glycosides accumulated more in plants under short-day conditions. Under these conditions, a highly significant correlation was found between UGT85C2 transcription and total steviol glycoside accumulation in the upper leaves. This suggests that the glycosylation of steviol to form steviolmonoside is the rate-limiting step in the glycosylation pathway of steviol glycosides. In these upper leaves, a relatively higher accumulation of rebaudioside A compared to stevioside was also observed, however, without correlation with the transcription of UGT76G1.
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41) Geuns J.M.C. (2011)
La Stévia et les glycosides de stéviol : Rien que la vérité sur la Stévia ou la politique mise à nu.
Euprint Ed., Parkbosstraat 3, 3001 Heverlee, Belgium, ISBN: 978-90-74253-130, pp.108.

Résumé:
Le livre présente une mise à jour de l’usage des glycosides de stéviol (les édulcorants de la Stévia) dans l’alimentation, avec des taux maximum proposés pour les différents groupes de recettes. Des études toxicologiques ont démontré que les glycosides de stéviol sont très sûrs et qu’il n’y a pas d’ effets secondaires nuisibles. Les histoires indiennes qui se sont répandues dans le monde entier sur des effets possiblement toxiques du stévioside, sont dues à une opinion ancienne du comité scientifique pour la nourriture de l’UE, une opinion qui nous apprend que ces scientifiques ne semblaient pas connaître, par exemple, la différence entre des animaux mâles ou femelles, entres des hommes et des femmes etc… Ou peut-on donc facilement s’imaginer que d’autres intérêts ont pu prendre le pas sur la science?

Les effets pharmacologiques très intéressants des glycosides de stéviol sont aussi discutés. Néanmoins, pour obtenir ces effets, il faut des doses très élevés qu’on ne va pas atteindre en utilisant les glycosides de stéviol comme édulcorant. Les effets les plus intéressants sont: diminution de la pression artérielle uniquement chez les personnes souffrant d’hypertension, diminution du taux de glucose sanguin chez les diabétiques type 2, amélioration de la sensibilité pour l’insuline, diminution de la formation de cancers de la peau et prévention de l’athérosclérose (formation de plaques) par l’inhibition de l’oxydation de lipoprotéines à basse densité (LDL) (« mauvais cholestérol »). Une explication possible de tous ces effets pharmacologiques positifs est présentée à travers la destruction des espèces réactives de l’oxygène ou radicaux.
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40) Mohsen Daneshyar1, Jan M.C. Geuns2, Hilke Willemsen3, Zarbakht Ansari4, Veerle M. Darras5, Johan G. Buyse3, Nadia Everaert3 (2010)
Evaluation of dietary stevioside supplementation on anti-HSA immunoglobulin G, Alpha-1-glycoprotein, body weight and thyroid hormones in broiler chickens.
J. Poult. Sci 47, 71-76. ).

 

Abstract:
At day 7 of incubation, eggs were injected with high levels of steviol (1.5 and 3 mg per egg). At hatch, no effect of steviol injection was observed on plasma thyroid hormone levels, hatchability, body weight and chick quality. Proportional liver weight of chicks receiving the highest steviol dose was significantly higher, while the proportional bursa weight of the steviol injected groups was significantly lower compared to that of the controls. Moreover, plasma lactate dehydrogenase activity of chicks from the highest steviol injected group was significantly higher compared to control values. At 7 days of age, proportional liver weight of chicks of the 3 mg steviol injected group was significantly lower compared to that of the control treatment. It is concluded that injection of a high dose of steviol has no effect on plasma thyroid hormones and has only slight and temporary metabolic effects on the chicken embryo.
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39) Geeraert B., F. Crombé, M. Hulsmans, N. Benhabilès, J.M. Geuns, and P. Holvoet (2010)
Stevioside inhibits atherosclerosis by improving insulin signalling and antioxidant defense in obese insulin-resistant mice.

International Journal of Obesity, 34, 569-577.
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38) Silviya Stoyanova, Jan M.C. Geuns, Wim Van den Ende, Éva Hideg (2010)
The food additives inulin and stevioside counteract oxidative stress.

International Journal of Food Sciences and Nutrition DOI 10.3109/09637486.2010.523416.

Abstract:
Prebiotics such as inulin (Inu)-type fructans and alternative natural sweeteners such as stevioside (Ste) become more popular as food ingredients. Evidence is accumulating that carbohydrates and carbohydrate-containing biomolecules can be considered true antioxidants, capable of scavenging reactive oxygen species (ROS). Here, we report on the ROS scavenging abilities of Inu and Ste in comparison with other sugars, sugar derivatives and arbutin. It is found that Inu and Ste are superior scavengers of both hydroxyl and superoxide radicals, more effective than mannitol and sucrose. Other compounds, such as 1-kestotriose, trehalose, raffinose and L-malic acid, also showed good reactivity to at least one of the two oxygen free radicals. The strong antioxidant properties of Inu and Ste are discussed. Within the plant vacuole, these compounds could play a crucial role in antioxidant defense mechanisms to help survive stresses. Addition to food assists in natural sweetening, food stabilization and maximizes health impact.
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37) Amal A.A. Mohamed, J.M.C. Geuns, W. Van Den Ende, M. De Ley (2010)
Expression patterns of some cytochrome P450 genes in steviol glycosides biosynthesis pathway in Stevia rebaudiana.
Egypt. J. Exp. Biol (Bot.). 2010, 6: 71-78.

Abstract:
Expression patterns of Kaurene oxidase (KO) and Kaurenoic acid 13-hydroxylase (KAH) genes, triggering the intermediate steps in steviol glycosides biosynthetic pathway in Stevia rebaudiana, was studied using quantitative real time polymerase chain reaction technique (Q-RTPCR). Stevia rebaudiana plants were subjected to different day lengths and irradiance conditions: long and short-day conditions in both full (4800 lux) and shady (400 lux) illuminations. Samples from both old and young leaves were collected, weekly, immersed in liquid nitrogen and kept at -80 °C for further extractions of RNA and steviol glycosides. 18S rRNA and actin were used as endogenous controls for normalizing the data. The expression patterns of the target genes were depending on both the age of the leaves and the light conditions under which the plants were grown.
- The changes in the expression of KO showed an increase in both young and old leaves during the experiment in all conditions. The accumulation of the gene at the end of the experiment was noticeable, specifically, for full-irradiance long-day conditions.
- The expression of the second cytP450 gene (KAH) increased during experimental period in both young and old leaves under most conditions. On the other hand, those subjected to severe irradiance stress during long-day conditions, a very small expression was detected
The expression results for the two genes proved their roles as key regulatory factors in the flow of the metabolites to the pathway for steviol glycosides biosynthesis pathway.
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36) Geuns J.M.C. (2010)
Second EUSTAS round-robin testing of steviol glycosides.

pp. 59-68 in Proceedings of the 4th EUSTAS Stevia Symposium Ed. Jan M.C. Geuns: “Stevia, Science no Fiction” June 29 – 30 2010, KULeuven, Belgium Ed.: Jan M.C. Geuns, ISBN: 978-90-742-53079.

Abstract:
A round-robin testing of a steviol glycoside sample was organised. Ten laboratories participated in the testing. So far, only 7 have sent their results. The sample had a purity of 91.1%. The reported purities of the sample varied between 82.74 and 95.86%. About 3 % purified RebB was added to the sample to check the quality of the analysis of this compound possessing a carboxylic group. The sample contained the following steviol glycosides: Reb D, Reb E, Reb A, ST, Reb F, Reb C, Dul A, Reb G, Rub, Reb B, SB and SM (1 lab). No SV was detected. The number of SVgly analysed in the different laboratories varied between 4 and 11. One lab only analysed ST and Reb A and gave a percentage composition of these compounds.
To improve the accuracy of analysis, different suggestions can be made, such as controlling the drying process of samples and standards, purity of standards, injection of sufficient material and use of solvent gradients to shorten run time and reduce integration errors. The results of this second round-robin testing are better than those of the first testing (Geuns and Struyf 2009).
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35) R. Amery, E. Jooken, B. Duquenne, J. Geuns and B. Meesschaert (2010)
Determination of Steviol Glycosides in Various Dairy Matrices and Soy Drink.

pp. 69 – 82 in: Proceedings of the 4th EUSTAS Stevia Symposium Ed. Jan M.C. Geuns: “Stevia, Science no Fiction” June 29 – 30 2010, KULeuven, Belgium Ed.: Jan M.C. Geuns, ISBN: 978-90-742-53079.

Abstract:
EFSA recently expressed the opinion that stevia extracts containing at least 95% rebaudioside A and/or stevioside are safe as a food additive. Approval for these stevia extracts as a food additive is expected very soon. It is therefore important that stevia extracts can be accurately and precisely determined in various food matrices.
The objective of this study was to determine steviol glycosides in dairy products and soy drink. Milk, ice-cream, fermented milk drink and soy drink sweetened with steviol glycosides were analyzed. Fat from the food matrices was removed by centrifugation and proteins were precipitated with acetonitrile. The supernatant was concentrated on a SPE C18 column and analyzed with HPLC.
The results show an excellent recovery and very good agreement between the use of an external calibration curve, the standard addition method and the use of an internal standard which was added before the extraction.
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34) Struyf T. and Geuns J. (2010)
Development of an internal standard and validation of the methods.

pp. 101 – 110 in: Proceedings of the 4th EUSTAS Stevia Symposium Ed. Jan M.C. Geuns: “Stevia, Science no Fiction” June 29 – 30 2010, KULeuven, Belgium Ed.: Jan M.C. Geuns, ISBN: 978-90-742-53079.

Abstract:
The internal standard method is the best method for the analysis of samples, as it is independent of errors in injection volume, changes in sample volumes, changes in sensitivity of the detector, etc. Use of an internal standard allows for the correction of losses due to sample clean-up of complex samples. An ideal IS is a compound with properties very similar to, and that behaves as, the compounds to be analysed. Ideally, only in the last step of analysis (HPLC), the IS should be well separated from the compounds of the mixture to be analysed. After testing several existing compounds with negative results, we decided to synthesise the 19-O-β-D-galactopyranosyl-13-O-β-D-glucopyranosyl-steviol as IS. This is the 19-galactosyl ester of steviolmonoside (13-O-β-D-glucopyranosyl-steviol).
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33) Geuns J. and Struyf T. (2010)
Radical scavenging activity of steviol glycosides and steviol glucuronide.

pp. 191 – 207 in: Proceedings of the 4th EUSTAS Stevia Symposium Ed. Jan M.C. Geuns: “Stevia, Science no Fiction” June 29 – 30 2010, KULeuven, Belgium Ed.: Jan M.C. Geuns, ISBN: 978-90-742-53079.

Abstract:
A radical scavenging activity for hydroxyl radicals of steviol glycosides and steviol glucuronide has been demonstrated in vitro. The activity was measured by the decrease of the fluorescence of hydroxyterephthalate that is formed from terephthalate in the presence of hydroxyl radicals (excitation at 315 nm, emission at 420 nm). Stevioside, rebaudioside A and rubusoside have about the same scavenging activity and were much better (20 x) than glucose or sucrose. It was demonstrated that steviol glucuronide, the excretion product in urine, also has strong ROS (OH) scavenging activity (14 x better than sugar). This activity might explain most of the beneficial pharmacological effects of oral stevioside on ROS related diseases, such as high blood pressure, type 2 diabetes, insulin resistance, atherosleroris, inflammation and certain forms of cancer, as well as certain brain diseases like Parkinson or Alzheimer. More research is still required on this interesting topic.
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32) Geuns J.M.C.:
Stevia and steviol glycosides. (2010)

Euprint Ed., Parkbosstraat 3, 3001 Heverlee, Belgium, ISBN: 978-90-742-53116, pp. 307.
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31) Geuns J.M.C.:
Estevia y glicósidos de esteviol. (2010)

Euprint Ed., Parkbosstraat 3, 3001 Heverlee, Belgium, ISBN: 978-90-742-53079, pp 327.
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30) Geuns J.M.C.:
Stevia en steviolglycosiden (2010): de naakte waarheid over stevia of de politiek in zijn blootje.

Euprint Ed., Parkbosstraat 3, 3001 Heverlee, Belgium, ISBN: 978-90-74253-147, pp.108.

Abstract:
Het boek geeft een algemene inleiding over de problemen geassocieerd met ongezonde voeding en gebrek aan beweging. Dan worden de verschillende zoetstoffen beschreven die kunnen gebruikt worden om de suikeropname te beperken. Een hoofdstuk is gewijd aan de Steviaplant zelf. Het merendeel van het boek handelt over steviolglycosiden, de zoetstoffen uit Stevia. Er wordt een duidelijk onderscheid gemaakt tussen lage doses zoetstoffen om gerechten te zoeten en hoge doses die verschillende gunstige farmacologische effecten hebben zoals verlaging van bloeddruk in geval van hoge bloeddruk, regulering van de bloedglucose bij type 2 diabetes, vermijden van aderverkalking, voorkomen van ontstekingsreacties en mogelijk ook sommige kankers. Het ontgiftigen van actieve zuurstofradicalen door deze zoetstoffen wordt gezien als het onderliggende mechanisme om de farmacologische effecten te verklaren. Naast een overzicht van bestaande toelatingen voor steviolglycosiden wereldwijd, wordt ook ingegaan op de Novel Food (“Nieuw Voedsel”) reglementering, die ogenschijnlijk niet bedoeld is om de veiligheid van het voedsel te garanderen, doch eerder ten dienste staat van de grote voedsellobbies om concurerende producten van de markt te weren. Het boek eindigt met antwoorden op veel gestelde vragen.

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29) Geuns Jan M.C. & Tom Struyf:
EUSTAS Round-Robin Testing of Steviol Glycosides.
pp. 35-48 in Proceedings of the 3rd EUSTAS Stevia Symposium “Stevia in Europe” July 1st – 2nd 2009, KULeuven, Belgium Ed.: Jan M.C. Geuns, ISBN: 978-90-742-53079.

Abstract:
A round-robin testing of 2 steviol glycoside samples was organised. Ten laboratories participated in the testing. The first sample had a purity of 96.2%. The second sample was a 4/5 dilution of sample 1 with NaHCO3. This way, the drying process itself could be checked. The purity of sample 2 was 82.35%. The reported purities of sample 1 varied between 79.8 and 96.2%, those of sample 2 varied between 58.1 and 81.8%. To improve the accuracy of analysis, different suggestions can be made, such as controlling the drying process of samples and standards, purity of standards, injection of sufficient material and use of solvent gradients to shorten run time and reduce integration errors.
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28) Geeraert Benjamine, Florence Crombé, Maarten Hulsmans, Nora Benhabilès, Jan Geuns, Paul Holvoet:
Natural sweetener stevioside inhibits atherosclerosis by increasing the antioxidant defence in obese, insulin-resistant mice
pp. 49-62 in Proceedings of the 3rd EUSTAS Stevia Symposium “Stevia in Europe” July 1st – 2nd 2009, KULeuven, Belgium Ed.: Jan M.C. Geuns, ISBN: 978-90-742-53079.

Abstract:
Objective - Stevioside is a non-caloric natural sweetener that reduces glucose by increasing insulin in non-obese animals. Obesity is frequently associated with insulin resistance and increased oxidative stress. Therefore, we investigated its effects on insulin resistance and oxidative stress related to atherosclerosis in obese, insulin-resistant and hyperlipidemic mice.
Research Design and Results - Twelve-week old mice were treated with stevioside (10 mg/kg, orally; n=14) or placebo (n=17) for 12 weeks. Stevioside had no effect on weight, but lowered fasting glucose (-18%), insulin (-34%), and cholesterol (-21%). Stevioside treatment increased Lxrα, Fabp4, and Glut4, Irs1, Irs2, and Insr in white visceral adipose tissue, supporting increased adipocyte differentiation and improved insulin signaling. Increased adipose tissue differentiation was associated with an increase in adiponectin (+98%). Stevioside reduced plaque volume in the aortic arch (-22%) by decreasing the macrophage (-23%), lipid (-21%) and oxidized LDL (-44%) content of the plaque. Stevioside treatment was associated with an increase in the antioxidative defense in the vascular wall, as evidenced by increased Sod1, Sod2, and Sod3, which was associated with a decrease in oxidized LDL in the aorta. Furthermore, we found a relation between adiponectin, insulin signaling and oxidative stress in the aorta of stevioside-treated mice.
Conclusion - Stevioside treatment inhibited the atherosclerotic plaque development and was associated with improved insulin signaling and antioxidative defense in the vascular wall.
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27) Mohamed Amal A.A., Jan M.C. Geuns, Wim Van den Ende & Marc De Ley:
Molecular aspects of early steps in steviol biosynthesis
pp. 63-83 in Proceedings of the 3rd EUSTAS Stevia Symposium “Stevia in Europe” July 1st – 2nd 2009, KULeuven, Belgium Ed.: Jan M.C. Geuns, ISBN: 978-90-742-53079.

Abstract:
The first committed steps in the shared pathway for synthesis of the gibberellins and steviol glyosides are controlled by ent-copalyl diphosphate synthase and ent-kaurene synthase. The expression of these genes was followed by RT-Q-PCR in Stevia rebaudiana. Plants were subjected to different light conditions:
12 h light/dark and 18 h light/6 h dark in both full and shady illuminations. Samples from both old and young leaves were collected weekly, immersed in liquid nitrogen and kept at -80°C for further extractions of RNA, steviol and steviol glycosides.

To maximise accuracy, housekeeping genes 18S rRNA and actin were both used as a reference to normalize the data in RT-Q-PCR. The results of normalized data were similar for both genes.
The expression patterns of the two genes were considerably variable depending on both the age of the leaves and the light conditions under which the plants were grown.
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26) Smedts Annelies, Stijn Ceunen, Ruis Amery, Jan M.C. Geuns and Boudewijn Meesschaert:
Bacterial consortia from Paraguayan soil samples with b-glucosidase activity degrading stevioside to steviol
pp. 85-101 in Proceedings of the 3rd EUSTAS Stevia Symposium “Stevia in Europe” July 1st – 2nd 2009, KULeuven, Belgium Ed.: Jan M.C. Geuns, ISBN: 978-90-742-53079.

Abstract:
Steviol glycosides are natural sweeteners known to have pharmacological effects in animals when administered in high doses (3×250 mg/day). Its possible active component is steviol glucuronide. Steviol glucuronide can be chemically synthesized from steviol. In this study, an attempt is made to produce steviol with a high yield by incubating stevioside with soil samples from a Paraguayan Stevia plantation. Different incubation conditions resulted in a different yield of steviol, a different rate of stevioside degradation and steviol formation, and different ways of hydrolysis of stevioside to steviol. The influence of parameters like the concentration of yeast extract, pH, stirring and temperature was analyzed.
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25) Geuns J.M.C. 2008.
Analysis of Steviol glycosides: validation of the methods.

pp. 59-78 in Proceedings of the EUSTAS Stevia Symposium, June 27th 2008, KULeuven, Belgium Ed.: Jan M.C. Geuns, ISBN: D/2008/6045/50.

Abstract:
Pure steviol glycoside standards were used for validating the methods of analysis (Struyf et al., 2008).
UV spectra of steviol glycosides and of steviol were measured in water, EtOH, 80% AcCN and 35% AcCN (5 mg/100 ml). The maximum absorption was around 196, 205, 199 and 197 nm in water, EtOH, 80% AcCN and 35% AcCN, respectively.
The calibration curves of steviol glycosides and of steviol were rectilinear between 15 and 1243 µM. The UV signal was monitored at 190 and 210 nm. It was shown that it is possible to make calibration curves with one single pure standard (eg. stevioside or rebaudioside A). The amounts of the other steviol glycosides present were calculated by use of conversion factors that compensated for the different molecular weights.
The detection limit (S/N = 5) of steviol glycosides was about 25 ng; that of steviol after derivatisation to its (7-methoxy-4-coumarinyl) methyl ester about 100 pg. Limits of quantification (S/N=10) are 50 ng and 200 pg for steviol glycosides and steviol respectively. The range of measurements is between 0.025 and 1000 µg/mL for steviol glycosides (or between about 15 and 1250 µM) and between 0.05 and 50 µg/mL for steviol as its fluorescent derivative (or between 15 nM and 160 µM).
Although the separation of steviol glycosides is easier on propylamino silica gel columns, the analysis of crude samples can be done on C18 columns that can easily be rinsed when becoming dirty.
Methods are summarised for the analysis of steviol glycosides and steviol in different foods.
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24) Struyf, T., Nancy P. Chandia, Wim De Borggraeve, Wim Dehaen, Jan M.C. Geuns, 2008.
Preparation of pure standards of steviol glycosides. Identification of steviol glycosides by LC-MS and NMR.
pp. 29-44 in Proceedings of the EUSTAS Stevia Symposium, June 27th 2008, KULeuven, Belgium Ed.: Jan M.C. Geuns, ISBN: D/2008/6045/50.

Abstract:
Pure standards of the steviol glycosides were prepared from a commercial mixture of 70% pure steviol glycosides by silicagel, silicagel-NH2 Flash column chromatography on columns of 300 mm length x 16 mm ID. Solvents used were ethyl acetate: ethanol: water (120:30:20) for silicagel columns and acetonitrile: water (70:30) for NH2-columns. The eluates were checked by TLC. The combined fractions of standards were further purified by preparative HPLC on silicagel and on C18 columns. The obtained fractions were then crystallised from methanol. Identity of the different steviol glycosides was obtained by spectroscopic data: UV, NMR and MS (both in positive and negative ESI mode). Identity of the compounds in the Stevia leaves was confirmed by co-injections with pure standards, as well as by HPLC analysis on columns with totally different separation characteristics (NH2 and C18 columns).
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23) Crawford A. D., Wouter Crets, Peter A. M. de Witte, Jan Geuns. 2008.
Early-life-stage analysis of stevia metabolites in zebrafish: Stevioside, rebaudioside A and steviol glucuronide are non-toxic, and steviol is anti-angiogenic.
pp. 139-150 in Proceedings of the EUSTAS Stevia Symposium, June 27th 2008, KULeuven, Belgium Ed.: Jan M.C. Geuns, ISBN: D/2008/6045/50. ).

 

Abstract:
Glycosidic sweeteners from Stevia rebaudiana are widely consumed by humans as food additives and/or dietary supplements. Despite encouraging progress towards the establishment of an acceptable safety profile, questions remain as to the biological and pharmacological effects of stevia constituents in vivo – particularly stevioside and rebaudioside A, as well as their metabolic products, steviol and steviol glucuronide. To better understand the bioactivity of these molecules, we analyzed their developmental toxicity in zebrafish, which recently emerged as a powerful model organism for the functionation of small molecules. Stevioside, rebaudioside A, rubusoside, steviol monoside, and steviol glucuronide all exhibited no visible effects up to the maximum concentration analyzed (200mM). Only steviol negatively affected the developing zebrafish embryos at higher concentrations, inducing developmental delays, pericardial edema, circulatory defects and lethality at 200mM. Further analysis in transgenic embryos with vasculature-specific expression of green fluorescent protein (GFP) revealed an appreciable anti-angiogenic effect for steviol. Steviol was also found to potentiate the anti-angiogenic activity of the vascular endothelial growth factor (VEGF) inhibitor SU5416. Given the high concentrations at which these effects of steviol were observed, in addition to the lack of activity seen for steviol glucuronide – the final metabolic product of stevioside and rebaudioside A – it appears likely that the consumption of stevia-derived sweeteners poses no risk for early embryonic development.
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22) Geuns J.M.C. 2008. Stevia: Novel Food in Europe? A critical approach.
Proceedings 2nd International Fresenius Conference “Novel food”, 7_1-5; 4-5 November, Cologne, Germany.
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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).

 

Abstract:
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.
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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).

 

Abstract:
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.
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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).

 

Abstract:
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.
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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).

 

Abstract:
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)

 

Abstract:
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).
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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.

Abstract:
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 [1]. 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 [1].
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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.

 

Abstract:
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.
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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. 54 (7), pp 2794–2798


Abstract:
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 (info@euprint.be).

 

Abstract:
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.
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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 (info@euprint.be).

Abstract:
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. (info@euprint.be).

Abstract:
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.
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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. (info@euprint.be).
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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. (info@euprint.be).


Abstract:
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. (info@euprint.be).


Abstract:
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


Abstract:
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.

Abstract:
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.


Abstract:
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.
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4) Geuns J.M.C. (2003)
Molecules of interest: Stevioside
.
Phytochemistry 64, 913-921.


Abstract:
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.


Abstract:
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.
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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.


Abstract:
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).

 

Abstract:
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.
--------------------------------