GSI Bibliography - Global Stevia Institute

GSI Bibliography - Global Stevia Institute

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Stevia studies
master list
(The studies are listed in alphabetical order by last name of the first author. This list primarily includes research and reviews related
to health and safety of stevia. Studies relating to chemical analysis and extraction methods or agricultural processes related to
stevia or the stevia plant are not included.)
Abudula R, Jeppesen PB, Rolfsen SE, Xiao J, Hermansen K. Rebaudioside A potently stimulates
insulin secretion from isolated mouse islets: studies on the dose-, glucose-, and calciumdependency. Metabolism. 53(10):1378-81, 2004. Extracts of leaves of the plant Stevia rebaudiana
Bertoni (SrB), have been used for many years in traditional treatment of diabetes in South America.
Stevia leaves contain diterpene glycosides, stevioside and rebaudioside A being the most abundant.
Recently, it was demonstrated that stevioside stimulates the insulin secretion both in vitro and in vivo.
Subsequently, we wanted to elucidate the influence of rebaudioside A on the insulin release from mouse
islets using static incubations, as well as perifusion experiments. Rebaudioside A (10(-16) to 10(-6) mol/L)
dose-dependently stimulated the insulin secretion in the presence of 16.7 mmol/L glucose (P < .05). The
stimulation of insulin release occurs at a concentration of 10(-14) mol/L rebaudioside A, and maximal
insulin response was obtained at 10(-10) mol/L (P < .01). Rebaudioside A stimulates insulin secretion in a
glucose-dependent manner (3.3 to 16.7 mmol/L) and only potentiated insulin secretion at glucose > 6.6
mmol/L. The effect of rebaudioside A is critically dependent on the presence of extracellular Ca2+, ie,
rebaudioside A-induced insulin stimulation at high glucose disappears in the absence of extracellular
Ca2+. In conclusion, rebaudioside A possesses insulinotropic effects and may serve a potential role as
treatment in type 2 diabetes mellitus.
Abudula R, Matchkov VV, Jeppesen PB, Nilsson H, Aalkjaer C, Hermansen K. Rebaudioside A
directly stimulates insulin secretion from pancreatic beta cells: a glucose-dependent action via
inhibition of ATP-sensitive K-channels. Diabetes Obes Metab. 10(11):1074-85, 2008. Recently, we
showed that rebaudioside A potently stimulates the insulin secretion from isolated mouse islets in a dosedependent manner. Little is known about the mechanisms underlying the insulinotropic action of
rebaudioside A. The aim of this study was to define the signalling system by which, rebaudioside A acts.
Isolated mouse islets were used in the cAMP[(125)I] scintillation proximity assay to measure total cAMP
level, and in a luminometric method to measure intracellular ATP and ADP concentrations. Conventional
and permeabilized whole-cell configuration of the patch-clamp technique was used to verify the effect of
rebaudioside A on ATP-sensitive K(+)-channels from dispersed single beta cells from isolated mouse
islets. Insulin was measured by radioimmunoassay from insulinoma MIN6 cells. In the presence of 16.7
mM glucose, the addition of the maximally effective concentration of rebaudioside A (10(-9) M) increased
the ATP/ADP ratio significantly, while it did not change the intracellular cAMP level. Rebaudioside A (10(9) M) and stevioside (10(-6) M) reduced the ATP-sensitive potassium channel (K(ATP)) conductance in a
glucose-dependent manner. Moreover, rebaudioside A stimulated the insulin secretion from MIN6 cells in
a dose- and glucose-dependent manner. In conclusion, the insulinotropic effect of rebaudioside A is
mediated via inhibition of ATP-sensitive K(+)-channels and requires the presence of high glucose. The
inhibition of ATP-sensitive K(+)-channels is probably induced by changes in the ATP/ADP ratio. The
results indicate that rebaudioside A may offer a distinct therapeutic advantage over sulphonylureas
because of less risk of causing hypoglycaemia.
AFSSA (Agence française de sécurité sanitaire des aliments), 2007. Avis de l’Agence française de
sécurité sanitaire des aliments relatif à une authorisation provisoire, pour une durée de deux ans,
d’emploi de steviol, extraits de Stevia rebaudiana, en tant qu’édulcorant en alimentation humaine
dans le cadre de l’article 5 de la directive 89/107/EEC. Maison-Alfort, le 12 octobre 2007.
AFSSA (Agence française de sécurité sanitaire des aliments), 2008. Opinion on a provisional twoyear authorisation for the use of steviol, an extract of Stevia rebaudiana, as a food sweetener
under article 5 of Directive 89/107/EEC, further to Afssa’s opinion of 12 October 2007.
AFSSA (Agence française de sécurité sanitaire des aliments), 2009. Avis de l’Agence française de
sécurité sanitaire des aliments sur un projet d’arrêté modifiant l‘arrêté du 26 aout 2009 relatif à
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l’emploi du rébaudioside A extrait de Stevia rebaudiana comme additif alimentaire. Maison-Alfort,
le 11 Décembre 2009.
Agency Response Letter GRAS Notice No. GRN 000287 Available from:
http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListi
ngs/ucm181937.htm. Accessed June 26, 2010
Agency Response Letter GRAS Notice for Rebaudioside A (Reb-A). Available from:
http://www.accessdata.fda.gov/scripts/fcn/gras_notices/804837A.PDF. Accessed June 26, 2010.
Agency Response Letter GRAS Notice No. Grn 000252 Accessed at
http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListi
ngs/ucm154988.htm.
Akashi H, Yokoyama Y. Security of dried-leaf extracts of Stevia. Toxicological tests. Food
Industry 18, 34-43. 1975.
Anton SD, Martin CK, Han H, Coulon S, Cefalu WT, Geiselman P, Williamson DA.Effects of stevia,
aspartame, and sucrose on food intake, satiety, and postprandial glucose and insulin levels.
Appetite. 55(1): 37-43 , 2010. Consumption of sugar-sweetened beverages may be one of the dietary
causes of metabolic disorders, such as obesity. Therefore, substituting sugar with low calorie sweeteners
may be an efficacious weight management strategy. We tested the effect of preloads containing stevia,
aspartame, or sucrose on food intake, satiety, and postprandial glucose and insulin levels. Design: 19
healthy lean (BMI=20.0-24.9) and 12 obese (BMI=30.0-39.9) individuals 18-50 years old completed three
separate food test days during which they received preloads containing stevia (290kcal), aspartame
(290kcal), or sucrose (493kcal) before the lunch and dinner meal. The preload order was balanced, and
food intake (kcal) was directly calculated. Hunger and satiety levels were reported before and after meals,
and every hour throughout the afternoon. Participants provided blood samples immediately before and
20min after the lunch preload. Despite the caloric difference in preloads (290kcal vs. 493kcal),
participants did not compensate by eating more at their lunch and dinner meals when they consumed
stevia and aspartame versus sucrose in preloads (mean differences in food intake over entire day
between sucrose and stevia=301kcal, p<.01; aspartame=330kcal, p<.01). Self-reported hunger and
satiety levels did not differ by condition. Stevia preloads significantly reduced postprandial glucose levels
compared to sucrose preloads (p<.01), and postprandial insulin levels compared to both aspartame and
sucrose preloads (p<.05). When consuming stevia and aspartame preloads, participants did not
compensate by eating more at either their lunch or dinner meal and reported similar levels of satiety
compared to when they consumed the higher calorie sucrose preload.
Atteh JO, Onagbesan OM, Tona K, Decuypere E, Geuns JM, Buyse J. Evaluation of supplementary
stevia (Stevia rebaudiana, bertoni) leaves and stevioside in broiler diets: effects on feed intake,
nutrient metabolism, blood parameters and growth performance. J Anim Physiol Anim Nutr (Berl).
92(6):640-9, 2008. A perennial schrub, 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 schrub 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 2 week starter and
2 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.
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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 SCFA 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 (T(3)) but had no effect on non-esterified fatty acids. In contrast,
stevioside only decreased T(3). 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 has no beneficial effect on
the performane of broilers.
Aze Y, Toyoda K, Imaida K, Hayashi S, Imazawa T, Hayashi Y, Takahashi M. Subchronic oral
toxicity study of stevioside in F344 rats. Bull. Natl. Inst. Hyg., 48-54 (in Japanese). 1991. A 13-week
subchronic oral toxicity study of stevioside was carried out in F344 rats at dose levels of 0, 0.31, 0.62,
1.25, 2.5 and 5% in diet, to determine appropriate dose levels for a 2-year carcinogenicity study. The rats
were randomly allocated to 6 groups, each consisting of 10 males and 10 females. No animals died
during the administration period. Between the control and treated groups, there were no differences in
body weight gain during the administration period and in food consumption in the later period of the study.
LDH on biochemical investigation and single cell necrosis in the liver revealed by histopathological
examination were increased in all male treated groups. These were not considered specific changes,
because of the lack of any clear dose response, the relatively low severity and the limitation to males.
Other parameters that were found to demonstrate significant differences on hematological and
biochemical investigations were of minor toxicological significance. From these results, a concentration of
5% in diet was concluded to be a suitable maximum tolerable dose of stevioside for a 2-year
carcinogenicity study in rats.
Barriocanal LA, Palacios M, Benitez G, Benitez S, Jimenez JT, Jimenez N, Rojas V. Apparent lack
of pharmacological effect of steviol glycosides used as sweeteners in humans. A pilot study of
repeated exposures in some normotensive and hypotensive individuals and in Type 1 and Type 2
diabetics. Regul Toxicol Pharmacol. 51(1):37-41,2008. Steviol glycosides, isolated from the plant
Stevia rebaudiana (Bertoni) Bertoni, have been used as safe sweetening agents for more than 30 years.
Beneficial effects of high doses of steviol glycosides on hyperglycemia and hypertension have been
previously described when these abnormalities are present. This study was designed to evaluate the
effects of steviol glycosides on blood glucose and on blood pressure (BP) in 3 groups of individuals. This
was a randomized, double-blind, placebo-controlled, long-term study in three groups of patients: Group 1:
subjects with Type 1 diabetes; Group 2: subjects with Type 2 diabetes; and Group 3: subjects without
diabetes and with normal/low-normal BP levels. The subjects in each group were randomly allocated to
active treatment (the steviol glycoside stevioside: 250mg t.d.s.) or to placebo treatment and followed-up
for 3 months. Post-treatment systolic BP, diastolic BP, glucose and glycated hemoglobin (HbA1c) were
not significantly different from baseline measurements, except for the placebo Type 1 diabetics group
where a significant difference was observed for systolic BP and glucose. No side effects were observed in
the two treatment groups. This study shows that oral steviol glycosides, taken as sweetener are well
tolerated and have no pharmacological effect.
Boonkaewwan C, Ao M, Toskulkao C, Specific immunomodulatory and secretory activities of
stevioside and steviol in intestinal cells. Rao MC.J Agric Food Chem.; 56(10):3777-84, 2008.
Stevioside, isolated from Stevia rebaudiana, is a commercial sweetener. It was previously demonstrated
that stevioside attenuates NF-kappaB-dependent TNF-alpha and IL-1beta synthesis in LPS-stimulated
monocytes. The present study examined the effects of stevioside and its metabolite, steviol, on human
colon carcinoma cell lines. High concentrations of stevioside (2-5 mM) and steviol (0.2-0.8 mM)
decreased cell viability in T84, Caco-2, and HT29 cells. Stevioside (2 mM) potentiated TNF-alphamediated IL-8 release in T84 cells. However, steviol (0.01-0.2 mM) significantly suppressed TNF-alphainduced IL-8 release in all three cell lines. In T84 cells, steviol attenuated TNF-alpha-stimulated IkappaB -> NF-kappaB signaling. Chloride transport was stimulated by steviol (0.1 mM) > stevioside (1 mM) at 30
min. Two biological effects of steviol in the colon are demonstrated for the first time: stimulation of Cl(-)
secretion and attenuation of TNF-alpha-stimulated IL-8 production. The immunomodulatory effects of
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steviol appear to involve NF-kappaB signaling. In contrast, at nontoxic concentrations stevioside affects
only Cl(-) secretion.
Boonkaewwan C, Toskulkao C, Vongsakul M. Anti-Inflammatory and Immunomodulatory Activities
of Stevioside and Its Metabolite Steviol on THP-1 Cells. J Agric Food Chem. 54(3):785-9, 2006.
Stevioside, a natural noncaloric sweetener isolated from Stevia rebaudiana Bertoni, possesses antiinflammatory and antitumor promoting properties; however, no information is available to explain its
activity. The aim of this study was to elucidate the anti-inflammatory and immunomodulatory activities of
stevioside and its metabolite, steviol. Stevioside at 1 mM significantly suppressed lipopolysaccharide
(LPS)-induced release of TNF-alpha and IL-1beta and slightly suppressed nitric oxide release in THP-1
cells without exerting any direct toxic effect, whereas steviol at 100 microM did not. Activation of IKKbeta
and transcription factor NF-kappaB were suppressed by stevioside, as demonstrated by Western
blotting. Furthermore, only stevioside induced TNF-alpha, IL-1beta, and nitric oxide release in
unstimulated THP-1 cells. Release of TNF-alpha could be partially neutralized by anti-TLR4 antibody.
This study suggested that stevioside attenuates synthesis of inflammatory mediators in LPS-stimulated
THP-1 cells by interfering with the IKKbeta and NF-kappaB signaling pathway, and stevioside-induced
TNF-alpha secretion is partially mediated through TLR4.
Bornia EC, do Amaral V, Bazotte RB, Alves-Do-Prado W. The reduction of arterial tension
produced by stevioside is dependent on nitric oxide synthase activity when the endothelium is
intact. 23. J Smooth Muscle Res. 44(1):1-8, 2008. In endothelium-intact rat aortic ring preparations precontracted with norepinephrine or KCl, NG-nitro L-arginine (L-NOARG, 0.1 mM) and 1H-[1,2,4] oxidiazolo
[4,3-a] quinoxalin-1-one (ODQ, 10 microM) antagonized the reduction of the vascular tone induced by
stevioside, but this antagonism did not occur when the experiment was performed with endotheliumdenuded aortic rings. The data indicates that the vasodilatation produced by stevioside is dependent on
nitric oxide synthase and guanylate cyclase activities when the endothelium is not damaged.
Bracht AK, Alvarez M, Bracht A. Effects of Stevia rebaudiana natural products on rat liver
mitochondria. Biochem Pharmacol ;34 (6): 873-82. 1986.
Brambilla E, Cagetti MG, Ionescu A, Campus G, Lingström P. An in vitro and in vivo Comparison
of the Effect of Stevia rebaudiana Extracts on Different Caries-Related Variables: A Randomized
Controlled Trial Pilot Study.Caries Research; 48(1):19-23, 2013. The effect of Stevia extracts on in
vitro Streptococcus mutans biofilm formation and in vivo plaque pH was evaluated in this paper. Three
10% solutions containing stevioside, rebaudioside A or sucrose were prepared. MTT assay was used to
evaluate microbiological counts in vitro. Twenty volunteers rinsed for 1 min with each solutions, and
plaque pH was measured at 7 time points after each rinse. Higher in vitro S. mutans biofilm formation was
observed in sucrose solution (p < 0.01). After 5, 10, 15 and 30 min, the sucrose in vivo rinse produced a
statistically significantly lower pH value compared to the Stevia extracts (F = 99.45, p < 0.01).Stevia
extracts can be considered nonacidogenic.
Brandle JE, Starratt AN, Gijzen M. Stevia rebaudiana: its agricultural, biological and chemical
properties. Can. J. of Plant Sci. 78, 527-536, 1998.
Braguini WL, Gomes MA, de Oliveira BH, Carnieri EG, Rocha ME, de Oliveira MB. Activity of
isosteviol lactone on mitochondrial metabolism.Toxicol Lett. 143(1):83-92, 2003. Isosteviol lactone
(LAC), a lactone derivative of the diterpenic acid isosteviol (ISO) was evaluated for its effect on the
oxidative metabolism of mitochondria isolated from rat liver. In this model, LAC (1 mM) depressed the
phosphorylation efficiency, as shown by the decreased respiratory control coefficient (RCC) and ADP/O
ratio. LAC (1 mM) inhibited NADH oxidase (45%), succinate oxidase (34%) and promoted low-level
inhibitions on succinate dehydrogenase (13%), succinate-cytochrome c oxide-reductase (23%),
cytochrome c oxidase (10%), and NADH dehydrogenase (13%). Glutamate dehydrogenase was also a
target for LAC, as it was 85% inhibited by 1 mM LAC. Cyclic voltammetry data showed that LAC, as well
as ISO, does not undergo redox reactions under current experimental conditions. LAC (0.05-0.75 mM)
inhibited the swelling dependent on the glutamate oxidation, 50% of the effect occurring at 0.5 mM LAC.
Swelling supported by KNO(3) and valinomycin was also inhibited over all concentrations used of LAC
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and ISO, the effect being of a lower intensity for LAC, suggesting that the modification of the structure of
ISO by lactonization diminished its interaction with the membrane. This could contribute to attenuation of
the toxic effects described for ISO on mitochondrial function, such as those on respiratory chain
enzymatic complexes and phosphorylating activity.
Bridel M, Lavielle R. Le principe a’ saveur sucre’e du Kaa’-he’-e’ (Stevia rebaudiana) Bertoni.
Bull. Soc. Chim. Biol., 13, 636-655. 1931.
Bridel M, Lavieille R. The sweet principle of kaa-he-e (Stevia rebaudiana). II. Products of diastatic
hydrolysis of stevioside glucose and steviol. C R Acad Sci ;193:72. 1931.
Bridel M, Lavieille R. The sweet principle of kaa-he-e (Stevia rebaudiana). J Pharm Chim ;14:99.
1931.
Brusick DJ A critical review of the genetic toxicity of steviol and steviol glycosides. Food Chem
Toxicol. 46 Suppl 7:S83-91, 2008. Extracts of the leaves of the stevia plant (Stevia rebaudiana Bertoni)
are used to sweeten food and beverages in South America, Japan and China. The
components responsible for the sweet properties of the plant are glycosides of steviol, primary stevioside
(ent-13-hydroxykaur-16-en-18-oic acid), which is 250-300 times sweeter than sucrose and rebaudiosides
A and C. Stevioside and steviol have been subjected to extensive genetic testing. The majority of the
findings show no evidence of genotoxic activity. Neither stevioside nor its aglycone steviol have been
shown to react directly with DNA or demonstrate genotoxic damage in assays relevant to human risk. The
mutagenic activity of steviol and some of its derivatives, exhibited in strain TM677, was not reproduced in
the same bacteria having normal DNA repair processes. The single positive in vivo study
measuring single-strand DNA breaks in Wistar rat tissues by stevioside, was not confirmed in
experiments in mice and appears to be measuring processes other than direct DNA damage. Neither
stevioside nor steviol-induced clastogenic effects at extremely high dose levels in vivo. Application of a
Weight-of-Evidence approach to assess the genetic toxicology database concludes that these
substances do not pose a risk of genetic damage following human consumption.
Carakostas MC, Curry LL, Boileau AC, Brusick DJ. Overview: the history, technical function and
safety of rebaudioside A, a naturally occurring steviol glycoside, for use in food and beverages.
Food Chem Toxicol. Suppl 7:S1-S10, 2008. Rebaudioside A is a sweet tasting steviol glycoside
extracted and purified from Stevia rebaudiana (Bertoni). Steviol glycosides can currently be used as a
food ingredient in only a handful of countries. Questions on specifications, safety and special population
effects have prevented steviol glycosides from obtaining a legal status permitting their use as a
sweetener in most countries. A set of papers reporting results of research studies and reviews has been
compiled in this Supplement to definitively answer unresolved questions. Specifically, recently completed
studies on the general and reproductive toxicity of rebaudioside A corroborate studies carried out with
purified steviol glycosides demonstrating safety at high dietary intake levels. Comparative metabolism
studies provide further affirmation of the common metabolic pathway for all steviol glycosides and the
common metabolism between rats and humans. Finally, clinical studies provide further evidence that
purified rebaudioside A has no effect on either blood pressure or glucose homeostasis. This paper
summarizes the information used to conclude that high purity rebaudioside A (rebiana) produced to foodgrade specifications and according to Good Manufacturing Practices is safe for human consumption
under its intended conditions of use as a general purpose sweetener.
Cargill GRAS Notification for Rebaudioside A, Submitted to the US Food and Drug
Administration, Washington, DC and identified as GRAS Notification 253; see FDA website at
http://www.cfsan.fda.gov/~rdb/opa-grsn.html. 2008
Cariño-Cortés R, Hernández-Ceruelos A, Torres-Valencia JM, González-Avila M, Arriaga-Alba
M, Madrigal-Bujaidar E. Antimutagenicity of Stevia pilosa and Stevia eupatoria evaluated with the
Ames test.Toxicol In Vitro. ;21(4):691-7, 2007.Stevia pilosa and Stevia eupatoria are plants used for
various purposes in traditional medicine. In this report we studied the antimutagenic effect of methanolic
extracts obtained from leaves, root, and flowers of the two species using the Ames test with and without
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metabolic activation. We tested the effect of the extracts on the damage induced by three mutagens with
the following results: 1 - we found an inhibitory effect of both species on the mutagenicity induced by 2aminoanthracene in the strain TA98. The best antimutagenic effect was obtained with leaves of both
species and the flowers of S. eupatoria (99%), 2 - the mutations induced with N-ethyl-N'-nitro-Nnitrosoguanidine in the strain TA100 was also reduced. The flowers of S. pilosa and the root of S.
eupatoria showed about 93% of inhibition, 3 - finally, the mutations induced by mitomycin-C on the strain
TA102 had a reduction of 87% with the leaves of S. eupatoria. Besides, we determined the radical
scavenging potential of the extracts with the DPPH method, and found a potent effect produced by all
extracts, with an efficacy of more than 90%. The present study showed both antimutagenic and
antioxidant potential of the tested extracts, and suggest the pertinence to confirm these effects in other
models, and to accurately determine their mechanism of action.
Cargill GRAS Notification for Rebaudioside A, 2008, Submitted to the US Food and Drug
Administration, Washington, DC and identified as GRAS Notification 253; see FDA website at
http://www.cfsan.fda.gov/~rdb/opa-grsn.html.
Cavalcante da Silva GE, Assef AH, Cordeiro Albino C, de Araujo Funari Ferri L, Tasin G, Takahashi
MH, Filho WE and Barbosa Bazotte R. Investigation of the tolerability of oral stevioside in Brazilian
hyperlipidemic patients. Brazilian Archives of Biology and Technology ;49: 583-7. 2006
Chagas AM, Tabarelli Z, Simoes SRM, Azzolin ELC. Effects of total aqueous extract of Steviarebaudiana and its stevioside upon renal parameters in vagrant dogs and dogs with water
overload. Revista de Ciencias Biomedicas; 11:1-11. 1990.
Chan P, Xu D-Y, Liu J-C, Chen Y-J, Tomlinson B, Huang W-P, Cheng J-T. The effect of stevioside
on blood pressure and plasma catecholamines in spontaneously hypertensive rats. Life Sciences
;63 1679-84. 1998. Stevioside is a sweet-tasting glycoside, composed of stevia, a diterpenic carboxylic
alcohol with three glucose molecules, mainly used as a substitute for non-alcoholic sweetener. It has
previously been shown to reduce blood pressure in studies in animals and human. The effect of
intravenous stevioside on the blood pressure was studied in spontaneously hypertensive rats (SHR). The
hypotensive effect on both systolic and diastolic blood pressure was dose-dependent for intravenous
doses of 50, 100 and 200 mg/kg in conscious SHR. The maximum reductions in systolic and diastolic
blood pressure were 31.4 +/- 4.2% and 40.8 +/- 5.6% (mean +/- SEM) respectively and the hypotensive
effect lasted for more than 60 min with a dose of 200 mg/kg. Serum dopamine, norepinephrine and
epinephrine levels were not changed significantly 60 min after intravenous injection of stevioside 100
mg/kg in anesthetized SHR. The present data show that stevioside given intravenously to conscious SHR
was effective in blood pressure reduction and there was no change in serum catecholamines in
anaesthetized animals with this natural compound.
Chan P, Tomlinson B, Chen YJ, Liu JC, Hsieh MH, Cheng JT. A double-blind placebo-controlled
study of the effectiveness and tolerability of oral stevioside in human hypertension. Br J Clin
Pharmacol. 50(3):215-20, 2000. AIMS: Stevioside is a natural plant glycoside isolated from the plant
Stevia rebaudiana which has been commercialized as a sweetener in Japan for more than 20 years.
Previous animal studies have shown that stevioside has an antihypertensive effect. This study was to
designed to evaluate the effect of stevioside in human hypertension. METHODS: A multicentre,
randomized, double-blind, placebo-controlled study was undertaken. This study group consisted of 106
Chinese hypertensive subjects with diastolic blood pressure between 95 and 110 mmHg and ages
ranging from 28 to 75 years with 60 subjects (men 34, women 26; mean +/- s.d., 54.1+/-3.8 years)
allocated to active treatment and 46 (men 19, women 27; mean +/- s.d., 53.7+/-4.1 years) to placebo
treatment. Each subject was given capsules containing stevioside (250 mg) or placebo thrice daily and
followed-up at monthly intervals for 1 year. RESULTS: After 3 months, the systolic and diastolic blood
pressure of the stevioside group decreased significantly (systolic: 166.0+/-9.4-152.6+/-6.8 mmHg;
diastolic: 104.7 +/- 5.2-90.3+/-3.6 mmHg, P<0.05), and the effect persisted during the whole year. Blood
biochemistry parameters including lipid and glucose showed no significant changes. No significant
adverse effect was observed and quality of life assessment showed no deterioration. CONCLUSIONS:
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This study shows that oral stevioside is a well tolerated and effective modality that may be considered as
an alternative or supplementary therapy for patients with hypertension.
Chang SS, Cook JM, Stability studies of stevioside and rebaudioside A in carbonated beverages.
J. Agric. Food Chem. 31, 409-414. 1983.
Chang JC, Wu MC, Liu IM, Cheng JT. Increase of insulin sensitivity by stevioside in fructose-rich
chow-fed rats. Horm Metab Res. 37(10):610-6, 2005. The intake of dietary fructose has undergone a
marked increase around the world, especially the developed countries, in recent times. Stevioside, a
glycoside contained in the leaves of Stevia rebaudiana Bertoni (Compositae), was used to screen the
effect induced by a diet containing 60% fructose on insulin resistance in rats. Single oral administration of
stevioside for 90 min decreased plasma glucose concentrations in a dose-dependent manner in rats
receiving fructose-rich chow for four weeks. In addition, insulin action on glucose disposal rate was
measured using the glucose-insulin index, the product of the areas under the curve of glucose, and
insulin during the intraperitoneal glucose tolerance test. Oral administration of stevioside (5.0 mg/kg) in
rats given four weeks of fructose-rich chow for 90 min reversed the value of glucose-insulin index,
indicating that stevioside has the ability to improve insulin sensitivity in this insulin-resistant animal model.
Time for the loss of plasma glucose lowering response to tolbutamide (10.0 mg/kg, i. p.) in fructose-rich
chow fed rats was also markedly delayed by repeated stevioside treatment three times daily compared to
the vehicle-treated group. The plasma glucose-lowering activity of tolbutamide was introduced to account
for varying levels of endogenous insulin secretion, and is widely used as the indicator of insulin resistance
development. Thus, it provided the supportive data that repeated oral administration of stevioside delayed
the development of insulin resistance in rats on a high-fructose diet. Increased insulin sensitivity by
stevioside administration was further identified using the plasma glucose-lowering action of exogenous
insulin in streptozotocin-induced diabetic rats (STZ-diabetic rats). Oral administration of stevioside at 0.2
mg/kg three times daily into STZ-diabetic rats for ten days increased the response to exogenous insulin.
Taken together, this demonstrated that oral administration of stevioside improves insulin sensitivity, and
seems suitable as an adjuvant for diabetic patients and/or those that consume large amounts of fructose.
Chang SF, Yang LM, Hsu FL, Hsu JY, Liaw JH, Lin SJ. Transformation of steviol-16a,17-epoxide by
Streptomyces griseus and Cunninghamella bainieri. J Nat Prod ;69:1450–55. 2006. Eight new entbeyerane metabolites, 5-8, 12, and 14-16, and four new ent-kaurane metabolites, 3, 10, 11, and 13,
together with two known metabolites, 4 and 9, were isolated from the microbial transformations of steviol16alpha,17-epoxide using Streptomyces griseus ATCC 10137 and Cunninghamella bainieri ATCC 9244.
The structures of the metabolites were characterized by IR, HRFABMS, and 1D and 2D NMR data. In
addition, a GRE (glucocorticoid response element)-mediated luciferase reporter assay was used to initially
screen for the biological activity of the 11 metabolites and stevioside. Steviol (1), steviol-16alpha,17-epoxide
(2), ent-11alpha,13,16alpha,17-tetrahydroxykauran-19-oic acid (3), ent-17-hydroxy-16-ketobeyeran-19-oic
acid (4), ent-9alpha,13-dihydroxy-16beta,17-epoxykauran-19-oic acid (10), ent-9alpha,17-dihydroxy-16ketobeyeran-19-oic acid (12), ent-1beta,17-dihydroxy-16-ketobeyeran-19-oic acid (14), and stevioside
showed significant effects; in particular, stevioside showed almost equal potency as dexamethasone.
Chatsudthipong V, Thongouppakarn P. Effect and mechanism of stevioside on rat renal function.
FASEB J. 9, A917 [Abstract No. 5322]. 1995.
Chatsudthipong V, Jutabha P. Effect of steviol on para-aminohippurate transport by isolated
perfused rabbit renal proximal tubule. J Pharmacol Exp Ther. 298(3):1120-7, 2001. An inhibitory
effect of steviol, metabolite of the natural sweetener stevioside, on transepithelial transport of paminohippurate (J(PAH)) was observed in isolated S(2) segments of rabbit renal proximal tubules using in
vitro microperfusion. Addition of steviol (0.01--0.25 mM) to the bathing medium significantly depressed
J(PAH) (approximately 50--90%). This inhibitory effect was dose-dependent and was maximum at a
concentration of 0.05 mM. To further examine this effect, a steviol concentration (0.01 mM) that produced
approximately 50% inhibition of J(PAH), was chosen. Addition of 0.01 mM steviol to the bathing medium
significantly depressed J(PAH) by about 50 to 60%. Steviol at the same concentration (0.01 mM), when
present in the tubule lumen, had no significant effect on J(PAH). Addition of 0.01 mM steviol to lumen and
bath simultaneously, produced a slightly greater inhibitory effect compared with addition to bath alone (60
page 8 of 64
versus 70%). A higher concentration of steviol, 0.05 mM (which maximally inhibited J(PAH) when on the
basolateral side), was required on the luminal side than on the basolateral side before an inhibitory effect
was observed. To further examine the mechanism by which steviol inhibited J(PAH), its effect on Na(+)K(+) ATPase activity and ATP content was determined. Steviol at concentrations of 0.01 and 0.05 mM
had no effect on Na(+)-K(+) ATPase activity or cell ATP content. Kinetic analyses indicated that steviol
can competitively inhibit PAH transport at the basolateral membrane. The present study clearly showed
that steviol can have a direct inhibitory effect on renal tubular transport by competitive binding with
organic anion transporter.
Chatsudthipong V, Lungkaphin A, Kaewmokul S. The interaction of steviol with rabbit OCT1 and
OCT2. FASEB J; 17: A476 (Abstract No. 331.4). 2003.
Chatsudthipong V, Muanprasat C. Stevioside and related compounds: therapeutic benefits
beyond sweetness. Pharmacol Ther. 121(1):41-54, 2009. Stevioside, an abundant component of
Stevia rebaudiana leaf, has become well-known for its intense sweetness (250-300 times sweeter than
sucrose) and is used as a non-caloric sweetener in several countries. A number of studies
have suggested that, beside sweetness, stevioside along with related compounds, which include
rebaudioside A (second most abundant component of S. rebaudiana leaf), steviol and isosteviol
(metabolic components of stevioside) may also offer therapeutic benefits, as they have antihyperglycemic, anti-hypertensive, anti-inflammatory, anti-tumor, anti-diarrheal, diuretic, and
immunomodulatory actions. It is of interest to note that their effects on plasma glucose level and blood
pressure are only observed when these parameters are higher than normal. As steviol can interact with
drug transporters, its role as a drug modulator is proposed. This review summarizes the current
knowledge of the pharmacological actions, therapeutic applications, pharmacokinetics and safety of
stevioside and related compounds. Although much progress has been made concerning their biological
and pharmacological effects, questions regarding chemical purity and safety remain unsolved. These
issues are discussed to help guide future research directions.
Chen J, Jeppesen PB, Abudula R, Dyrskog SE, Colombo M, Hermansen K. Stevioside does not
cause increased basal insulin secretion or beta-cell desensitization as does the sulphonylurea,
glibenclamide: studies in vitro. Life Sci. 78(15):1748-53, 2006. We have shown that stevioside (SVS)
enhances insulin secretion and thus may have a potential role as antihyperglycemic agent in the
treatment of type 2 diabetes mellitus. However, whether SVS stimulates basal insulin secretion (BIS)
and/or cause desensitization of beta cells like sulphonylureas (SU), e.g. glibenclamide (GB), is not
known. To explore and compare the effects of SVS pretreatment with those of GB and glucagon-like
peptide-1 (GLP-1), we exposed isolated mouse islets to low or high glucose for 1 h after short-term (2 h)
or long-term (24 h) pretreatment with SVS, GB or GLP-1, respectively. BIS at 3.3 or 5.5 mM glucose were
not changed after short-term pretreatment with SVS (10(-7) M), while it increased about three folds after
pretreatment with GB (10(-7) M). Glucose stimulated insulin secretion (GSIS) (16.7 mM) increased dosedependently after long-term pretreatment with SVS at concentrations from 10(-7) to 10(-5) M.
Pretreatment for 24 h with GB (10(-7) M) increased the subsequent BIS (3.3 mM glucose) (p < 0.001), but
decreased GSIS (16.7 mM glucose) (p < 0.001). In contrast SVS (10(-7) M) and GLP-1 (10(-7) M) did not
stimulate BIS but both enhanced the subsequent GSIS (16.7 mM glucose) (p < 0.05 and p < 0.05,
respectively). While SVS pretreatment increased the intracellular insulin content, GB pretreatment
decreased the insulin content. Our study suggests that SVS pretreatment does not cause a stimulation of
BIS and does not desensitize beta-cells, i.e. SVS seems to have advantageous characteristics to GB as a
potential treatment of type 2 diabetes.
Chen J, Jeppesen PB, Nordentoft I, Hermansen K. Stevioside counteracts the glyburide-induced
desensitization of the pancreatic beta-cell function in mice: studies in vitro. Metabolism.
55(12):1674-80, 2006. The sulfonylurea glyburide (GB) is one of the most frequently used drugs in
diabetes treatment. Long-term pretreatment with GB causes elevated basal insulin secretion (BIS) and
decreased glucose-stimulated insulin secretion (GSIS). These characteristics may play an important role
for the development of hypoglycemia and secondary failure. Stevioside (SVS), a substance extracted
from leaves of Stevia rebaudiana Bertoni, enhances GSIS but not BIS. The aim of the present study was
to clarify whether 24-hour exposure of isolated mouse islets to GB causes dose-dependent decrease in
page 9 of 64
the GSIS and whether it is possible to counteract this desensitization by SVS. We also tested the impact
of the incretin glucagon-like peptide-1 (GLP-1) on the GB-induced desensitization. After 24-hour
preincubation with GB in combination with SVS or GLP-1, we measured the basal and glucose-stimulated
insulin responses and the total islet insulin content. We also determined the fold change in gene
expression of pancreatic and duodenal homeobox 1 and glucose transporter isoform 2. After 24-hour
preincubation in 11.1 mmol/L glucose, GB (10(-11)-10(-3) mol/L) caused a dose-dependent decrease in
GSIS (16.7 mmol/L glucose) (P < .001). GB (10(-7) mol/L) pretreatment elevated BIS, but neither SVS
(10(-7) mol/L) nor GLP-1 (10(-7) mol/L) could reverse this. Interestingly, the GB-induced desensitization
of GSIS was counteracted by both SVS (P < .05) and GLP-1 (P < .05). SVS reversed the decrease in
insulin content caused by GB pretreatment (P < .05). GB pretreatment did not change gene expression of
pancreatic and duodenal homeobox 1 nor glucose transporter isoform 2, whereas SVS significantly upregulated the expression of both genes by more than 2-fold (P < .05). Our results showed that SVS in
combination with GB did not reverse GB-induced increase in BIS, whereas both SVS and GLP-1
counteracted GB-induced desensitization of GSIS. SVS is able to counteract the desensitizing effects of
GB and may be a putative new drug candidate for the treatment of type 2 diabetes mellitus.
Chen J, Jeppesen PB, Nordentoft I, Hermansen K. Stevioside counteracts beta-cell lipotoxicity
without affecting acetyl CoA carboxylase. The Review of Diabetic Studies ;3:178-88. 2006b.
Chronic exposure to high levels of free fatty acids impairs beta-cell function (lipotoxicity). Then basal
insulin secretion (BIS) is increased and lucose-stimulated insulin secretion (GSIS) is inhibited. Acetyl CoA
carboxylase (ACC) acts as the sensor for insulin secretion in pancreatic beta-cells in response to glucose
and other nutrients. Stevioside (SVS), a diterpene glycoside, has recently been shown to prevent
glucotoxic effect by regulating ACC activity. The aim of this study was to investigate whether SVS can
alleviate impaired beta-cell function by regulating ACC activity. We exposed isolated rat islets and the
clonal beta-cell line, INS-1E, to palmitate concentrations of 1.0 or 0.6 mM, respectively, for a period of 24
h to 120 h. The results showed that lipotoxicity occurred in rat islets after 72 h exposure to 1.0 mM
palmitate. The lipotoxicity was counteracted by 10(-6) M SVS (n = 8, p < 0.001). Similar results were
obtained in INS-1E cells. Neither SVS nor palmitate had any effect on the gene expression of ACC,
insulin 2, and glucose transporter 2 in INS-1E cells. In contrast, palmitate significantly increased the gene
expression of carnitine palmitoyl transporter 1 (n = 6, p = 0.003). However, the addition of SVS to
palmitate did not counteract this effect (n = 6, p = 1.0). During lipotoxicity, SVS did not alter levels of ACC
protein, phosphorylated-ACC, ACC activity or glucose uptake. Our results showed that SVS counteracts
the impaired insulin secretion during lipotoxicity in rat islets as well as in INS-1E cells without affecting
ACC activity.
Chen J, Jeppesen PB, Nordentoft I, Hermansen K. Stevioside improves pancreatic beta-cell
function during glucotoxicity via regulation of acetyl-CoA carboxylase. Am J Physiol Endocrinol
Metab. 292(6):E1906-16, 2007. Chronic hyperglycemia is detrimental to pancreatic beta-cells, causing
impaired insulin secretion and beta-cell turnover. The characteristic secretory defects are increased basal
insulin secretion (BIS) and a selective loss of glucose-stimulated insulin secretion (GSIS). Several recent
studies support the view that the acetyl-CoA carboxylase (ACC) plays a pivotal role for GSIS. We have
shown that stevioside (SVS) enhances insulin secretion and ACC gene expression. Whether glucotoxicity
influences ACC and whether this action can be counteracted by SVS are not known. To investigate this,
we exposed isolated mouse islets as well as clonal INS-1E beta-cells for 48 h to 27 or 16.7 mM glucose,
respectively. We found that 48-h exposure to high glucose impairs GSIS from mouse islets and INS-1E
cells, an effect that is partly counteracted by SVS. The ACC dephosphorylation inhibitor okadaic acid
(OKA, 10(-8) M), and 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR, 10(-4) M), an
activator of 5'-AMP protein kinase that phosphorylates ACC, eliminated the beneficial effect of SVS. 5Tetrade-cyloxy-2-furancarboxylic acid (TOFA), the specific ACC inhibitor, blocked the effect of SVS as
well. During glucotoxity, ACC gene expression, ACC protein, and phosphorylated ACC protein were
increased in INS-1E beta-cells. SVS pretreatment further increased ACC gene expression with strikingly
elevated ACC activity and increased glucose uptake accompanied by enhanced GSIS. Our studies show
that glucose is a potent stimulator of ACC and that SVS to some extent counteracts glucotoxicity via
increased ACC activity. SVS possesses the potential to alleviate negative effects of glucotoxicity in betacells via a unique mechanism of action.
page 10 of 64
Chen SY, Li QR. Influence of growth regulatory substances on stevioside content of Stevia
rebaudiana callus. Zhiwu Shenglixue Tongxun ;29(4):265-267. 1993.
Cheng TF, Chang WH, Chang TR. A study on the post-harvest changes in steviosides contents of
Stevia leaves and stems. Nat Sci Counc Monthly Roc ;9(9):775-82. 1981.
Cheng TF, Chang WH. Studies on the nonstevioside components of Stevia extracts. K'o Hsueh Fa
Chan Yueh K'an; 11(2):96-108. 1983.
Chen TH, Chen SC, Chan P, Chu YL, Yang HY, Cheng JT. Mechanism of the hypoglycemic effect of
stevioside, a glycoside of Stevia rebaudiana. Planta Med. 71(2):108-13, 2005. We have studied the
effects of stevioside on the glucose and insulin metabolism in 2 models of diabetes in rats, STZ-induced
diabetic rats and NIDDM diabetic rats induced by feeding with fructose. Stevioside (0.5 mg/kg), lowered
the blood glucose levels in STZ-induced diabetic rats, peaking at 90 min. Stevioside administered twice
daily also demonstrated dose-dependent effects in lowering the glucose levels in both diabetic rat
models. Stevioside reduced the rise in glucose during glucose tolerance testing in normal rats. Stevioside
dose-dependently decreased protein levels of phosphoenol pyruvate carboxykinase (PEPCK) and
PEPCK mRNA after 15 days of treatment. Stevioside also reduced insulin resistance in the diabetic
animals as shown by the glucose lowering effects of tolbutamide. In conclusion, stevioside was able to
regulate blood glucose levels by enhancing not only insulin secretion, but also insulin utilization in insulindeficient rats; the latter was due to decreased PEPCK gene expression in rat liver by stevioside's action
of slowing down gluconeogenesis. Further studies of this agent for the treatment of diabetes appear
warranted.
Chen WS, Yeh CS. Preliminary report on the examination of stevioside by high-pressure liquid
chromatography. Taiwan Tang Yeh Yen Chiu So Yen Chiu Hui Pao ;79:43-48. 1978.
Chueh WJ. A new natural sweetening agent-stevioside. Shih P'in Kung Yeh(Taiwan) ;9:34-35.
1977.
Chung MH, Lee MY. Studies on the development of hydrangea and Stevia as natural sweetening
products. Korean J Biochem ;9(3):149-156. 1978.
Chung MS, Suh HJ, Yoo W, Choi SH, Cho YJ, Cho YH, Kim CJ. Daily intake assessment of
saccharin, stevioside, D-sorbitol and aspartame from various processed foods in Korea. Food
Addit Contam. (11):1087-97, 2005. This study was carried out to estimate the daily intakes (EDIs) of
artificial sweeteners such as saccharin, stevioside, D-sorbitol and aspartame in order to evaluate the
safety of the artificial sweeteners in Korea. A total of 274 food samples were selected from the foods
considered to be representative sources of artificial sweeteners in the Korean diet and analysed by using
HPLC withevaporative light scattering and ultraviolet detectors. In case of aspartame, the reference
values were used without instrumental analysis. The EDIs of saccharin, stevioside, D-sorbitol and
aspartame for average consumers were 0.028, 0.008, 4.9 and 0.14 mg kg-1 body weight day-1,
respectively, and as a proportion of the acceptable daily intake (ADI) were not higher than 1% of ADI of
the Joint FAO/WHO Expert Committee on Food Additives (JECFA). For 90th percentile consumers, the
EDIs of saccharin, stevioside, D-sorbitol and aspartame were 2.0, 0.20, 141 and 4.6 mg kg-1 body weight
day-1, respectively, and as a proportion of the ADI, the EDIs of saccharin and aspartame were 40.7% and
11.4% of the ADI set by the JECFA, respectively. Because JECFA did not assign ADIs for stevioside and
D-sorbitol, the values for these sweeteners were not compared. According to these results, the EDIs of
artificial sweeteners such as saccharin and aspartame in Korea are significantly lower than ADI set by the
JECFA.
Clos JF, DuBois GE, Prakash I. Photostability of rebaudioside A and stevioside in beverages. J
Agric Food Chem.;56(18):8507-13, 2008..The Coca-Cola Company and Cargill, Inc. have initiated the
development and commercialization of the Stevia rebaudiana (Bertoni) derived sweetener rebaudioside
A. Efforts were focused on high purity rebaudioside A (>97% by HPLC), commonly known as rebiana. In
the course of the development program, extensive stability studies were carried out on rebiana, all
page 11 of 64
supporting good stability for use in all food and beverage applications, including conditions where
rebiana-sweetened beverages were exposed to light. Our findings on rebiana light stability refute those of
an earlier study that suggested rebaudioside A to be unstable to sunlight exposure, while the structurally
homologous stevioside is stable. We replicated the earlier study and found no significant
photodegradation for either rebaudioside A or stevioside.
Codex Alimentarius Commission (2007) Codex General Standard for Food Additives (CODEX STAN
192-1995 Accessed at http://www.codexalimentarius.net/gsfaonline/CXS_192e.pdf.
Compadre, C.M., Hussain, R.A., Nanayakkara, N.P., Pezzuto, J.M., Kinghorn, A.D., Mass spectral
analysis of some derivatives and in vitro metabolites of steviol, the aglycone of the natural
sweeteners, stevioside, rebaudioside A, and rubusoside. Biomed. Environ. Mass Spectrom., 15,
211-222. 1988. Steviol (ent-13-hydroxykaur-16-en-19-oic acid), the aglycone of various plant-derived
glycoside sweeteners consumed by human populations, is known to be mutagenic toward Salmonella
tymphimurium strain TM677 when metabolically activated using a 9000 x g supernatant fraction derived
from the liver of Aroclor 1254-pretreated rats. Mass spectral analysis of this diterpenoid and some
analogs revealed characteristic patterns reflecting differential stereochemistry at the C/D rings and
variations in the nature of the substituents present. Such information has been used to help identify
several in vitro metabolites of steviol in conditions known to produce a mutagenic response, when
analyzed by gas chromatography/mass spectrometry. The major pathways of such steviol mammalian
metabolism proved to be allylic oxidation and epoxidation. 15-Oxosteviol, a product of oxidation of the
major steviol metabolite, 15alpha-hydroxysteviol, was found to be a direct-acting mutagen [corrected].
Costa C, Costa S, Rocha M, Peres S, Dacomi A, Piccinato C, Carpinelli A, Lima F. Rebaudioside A,
a glycoside of the Stevia rebaudiana, stimulates insulin secretion in rat isolated pancreatic islets.
Diabetes ;52(1):A370 [Abstract No. 1606-P]. 2003a
Costa C, Costa S, Peres S, De Morales SF, Takada J, Brito L, Alonso MI, Andreotti S, Machado M,
Borges C, Lima F. Rebaudioside A, a diterpene glycoside from Stevia rebaudiana, causes insulin
resistance in rat periepididymal isolated adipocytes. Diabetes ;52(1): A532 [Abstract No. 2307-PO].
2003b.
Constantin J, Ishii-Iwamoto EL, Ferraresi-Filho O, Kelmer-Bracht AM, Bracht A. Sensitivity of
ketogenesis and citric acid cycle to stevioside inhibition of palmitate transport across the cell
membrane. Brazilian Journal of Medical Biological Research ;24:767-771. 1991.
Crammer B, Ikan R. Properties and synthesis of sweetening agents. Chem Soc Rev ;6:431-565.
1977.
Crosby GA. New sweeteners. Crit Rev Food Sci Nutr ;297-323. 1976.
Curi, R., Alvarez, M., Bazotte, R.B., Botion, L.M., Godoy, J.L., Bracht, A. Effect of Stevia rebaudiana
on glucose tolerance in normal adult humans. Braz.J. Med. Biol. Res.19, 771-774 (In Portugese,
English abstract only). 1986 The effect of aqueous extracts of Stevia rebaudiana leaves on a glucose
tolerance test was investigated in 16 normal volunteers. Aqueous extracts of 5 grams of leaves were
administered to volunteers at regular 6-h intervals for 3 days. Glucose tolerance tests were performed
before and after extract administration. A second group of 6 normal volunteers who ingested an aqueous
arabinose solution was also studied to eliminate possible stress effects. The extract of Stevia rebaudiana
increased glucose tolerance. The extract significantly decreased plasma glucose levels during the test
and after overnight fasting in all volunteers.
Curry, L.L., Roberts, A. Subchronic toxicity of rebaudioside A. Food Chem. Toxicol., 46(7)(Suppl.
1), S11-S20. 2008. The safety of the stevia-derived sweetener, rebaudioside A (CAS No. 58543-16-1),
was evaluated in two oral toxicity studies. In a 4-week study, Wistar rats were administered rebaudioside
A at dietary concentrations of 0, 25,000, 50,000, 75,000 and 100,000ppm. The NOAEL, including an
evaluation of testes histopathology, was determined to be 100,000 ppm. In the 13-week study, Wistar rats
page 12 of 64
were administered rebaudioside A at dietary concentrations of 0, 12,500, 25,000 and 50,000ppm.
Reductions in body weight gain attributable to initial taste aversion and lower caloric density of the diet
were observed in high-dose male and females groups. Inconsistent reductions in serum bile acids and
cholesterol were attributed to physiological changes in bile acid metabolism due to excretion of high
levels of rebaudioside A via the liver. All other hepatic function test results and liver histopathology were
within normal limits. Significant changes in other clinical pathology results, organ weights and functional
observational battery test results were not observed. Macroscopic and microscopic examinations of all
organs, including testes and kidneys, were unremarkable with respect to treatment-related findings. The
NOAEL in the 13-week toxicity study was considered to be 50,000ppm or approximately 4161 and
4645mg/kg body weight/day in male and female rats, respectively.
Curry LL, Roberts A, Brown N. Rebaudioside A: two-generation reproductive toxicity study in rats.
Food Chem Toxicol. 46 Suppl 7:S21-30, 2008. Rebaudioside A was administered via the diet to male
and female Han Wistar rats at 0, 7500, 12,500, and 25,000ppm for two generations. Rebaudioside A
treatment was not associated with any signs of clinical toxicity or adverse effects on body weight, body
weight gain, or food consumption. No treatment-related effects of rebaudioside A were observed in either
the F0 or F1 generations on reproductive performance parameters including mating performance, fertility,
gestation lengths, oestrous cycles, or sperm motility, concentration, or morphology. The survival and
general condition of the F1 and F2 offspring, their pre-weaning reflex development, overall body weight
gains, and the timing of sexual maturation, were not adversely affected by rebaudioside A treatment. The
NOAEL for reproductive effects was 25,000ppm and the NOAEL for the survival, development, and
general condition of the offspring also was considered to be 25,000ppm or 2048-2273mg/kg body
weight/day.
Curry LL, Roberts A. Subchronic toxicity of rebaudioside A. Food Chem Toxicol. 46 Suppl 7:S1120, 2008. The safety of the stevia-derived sweetener, rebaudioside A (CAS No. 58543-16-1), was
evaluated in two oral toxicity studies. In a 4-week study, Wistar rats were administered rebaudioside A at
dietary concentrations of 0, 25,000, 50,000, 75,000 and 100,000ppm. The NOAEL, including an
evaluation of testes histopathology, was determined to be 100,000 ppm. In the 13-week study, Wistar rats
were administered rebaudioside A at dietary concentrations of 0, 12,500, 25,000 and 50,000ppm.
Reductions in body weight gain attributable to initial taste aversion and lower caloric density of the diet
were observed in high-dose male and females groups. Inconsistent reductions in serum bile acids and
cholesterol were attributed to physiological changes in bile acid metabolism due to excretion of high
levels of rebaudioside A via the liver. All other hepatic function test results and liver histopathology were
within normal limits. Significant changes in other clinical pathology results, organ weights and functional
observational battery test results were not observed. Macroscopic and microscopic examinations of all
organs, including testes and kidneys, were unremarkable with respect to treatment-related findings. The
NOAEL in the 13-week toxicity study was considered to be 50,000ppm or approximately 4161 and
4645mg/kg body weight/day in male and female rats, respectively.
D’Agostino M, De Simone F, Pizza C, Aquino R. Sterols from Stevia rebaudiana Bertoni. Boll Soc
Ital Biol Sper; 60 (12):2237-40. 1984. The sterol fraction of Stevia rebaudiana Bertoni contains,
essentially, the following sterols: stigmasterol (45,8%), beta-sitosterol (39,4%) and campesterol (13,1%).
The individual components were separated, after acetylation, by HPLC with absolute methanol as eluant.
The identification of the compounds has been carried out through NMR and MS, while the corresponding
percentages have been desumed from the GLC data.
Dao KN, Le VH. Biological properties of flavonoids from Stevia rebaudiana Bert. Tap Chi Duoc Hoc
2: 17/18-21. 1995.
Das S, Das AK, Murphy RA, Punwani IC, Nasution MP, Kinghorn AD. Evaluation of the cariogenic
potential of the intense natural sweeteners stevioside and rebaudioside A. Caries Research
;26:363- 66. 1992. Stevioside and rebaudioside A, two intense natural sweeteners, that are constituents
of the South American plant Stevia rebaudiana, were tested for cariogenicity in albino Sprague-Dawley
rats. Sixty rat pups colonized with Streptococcus sobrinus were divided into four groups and fed
stevioside, rebaudioside A or sucrose added to basal diet 2000 as follows: group 1, 30% sucrose; group
page 13 of 64
2, 0.5% stevioside; group 3, 0.5% rebaudioside A, and group 4, no addition. All four groups were
sacrificed after 5 weeks. S. sobrinus counts were made and caries was evaluated according to Keyes'
technique. There were no differences in food and water intake and weight gains between the four groups.
There were significant differences in sulcal caries scores (p < 0.02) and S. sobrinus counts (p < 0.05)
between group 1 and the other three groups. There were no significant differences between the
stevioside, rebaudioside A and no-addition groups. It was concluded that neither stevioside nor
rebaudioside A is cariogenic under the conditions of this study.
Darise M, Kohda H, Mizutani K, Kasai R, Tanaka O. Chemical constituents of flowers of Stevia
rebaudiana Bertoni. Agr Biol Chem ;47(1):133-5. 1983.
De Cernadas RR, Pryluka M. A method for the isolation of stevioside from leaves of Stevia
rebaudiana Bert. Rev Agroquim Tecnol Aliment ;25(2):268-72. 1985.
De Levy RH. Stevia rebaudiana Bertoni: An excellent natural sweetening agent. Acta Farm
Bonaerense ;3(1):47-50. 1984.
Derkach AI, Kovalyov IP, Bublik NP. Diterpene Glycosides and Phenylpropanoids of Stevia
Rebaudiana Bertoni (Asteraceae). CITATION?
De-Yi X, Hong C, Yuan-Yuan L, 1990. The antihypertensive effects by stevioside in the conscious
normal and hypertensive rats. European Journal of Pharmacology 183, 1822 [Abstract No.
P.th.182].
Dieterich K. The constituents of eupatorium rebaudianum, kaa-he-e, and their pharmaceutical
value. Pharm Zentralhalle Dtschl ;50:435. 1913.
Dyrskog SE, Jeppesen PB, Chen J, Christensen LP, Hermansen K. The diterpene glycoside,
rebaudioside A, does not improve glycemic control or affect blood pressure after eight weeks
treatment in the Goto-Kakizaki rat. Rev Diabet Stud. 2(2):84-91, 2005. The plant, Stevia rebaudiana
Bertoni (SrB), has been used for the treatment of diabetes in traditional medicine. Previously, we have
demonstrated that long-term administration of the glycoside stevioside has insulinotropic,
glucagonostatic, anti-hyperglycemic and blood pressure-lowering effects in type 2 diabetic animal models.
The aim of this study was to elucidate if long-term administration of rebaudioside A, another glycoside
isolated from the plant SrB, could improve glycemic control and lower blood pressure in an animal model
of type 2 diabetes. We divided male Goto-Kakizaki (GK) rats into two groups which were fed a standard
laboratory chow diet for eight weeks. The diet was supplemented with oral rebaudioside A (0.025 g/kg
BW/day) in the experimental group. Blood glucose, weight, blood pressure and food intake were
measured weekly. Animals were equipped with an intra-arterial catheter, and at week eight the conscious
rats underwent an intra-arterial glucose tolerance test (IAGTT) (2.0 g/kg BW). During the IAGTT, the
level of glucose, glucagon, and insulin responses did not differ significantly between the two groups.
Fasting levels of glucose, glucagon, insulin or levels of blood lipids did not differ between the groups
throughout the study period. We observed no effect on blood pressure or weight development. In
conclusion, oral supplementation with rebaudioside A (0.025 g/kg BW/day) for eight weeks did not
influence blood pressure or glycemic control in GK rats. Rebaudioside A failed to show the beneficial
effects in diabetic animals previously demonstrated for stevioside.
Dyrskog SE, Jeppesen PB, Colombo M, Abudula R, Hermansen K. Preventive effects of a soybased diet supplemented with stevioside on the development of the metabolic syndrome and type
2 diabetes in Zucker diabetic fatty rats. Metabolism. 54(9):1181-8, 2005. The world witnesses an
explosive increase in diabetes, demanding intensified prevention and treatment not least for the lowincome population. The plant, Stevia rebaudiana Bertoni, has been used for the treatment of diabetes in
traditional medicine. We have previously demonstrated that stevioside, a diterpene glycoside isolated
from the plant Stevia rebaudiana Bertoni, possesses insulinotropic, glucagonostatic, antihyperglycemic,
and blood pressure-lowering effects in animal studies. We have also found that a dietary supplement,
Abalon, of soy protein, isoflavones, and cotyledon fiber has beneficial effects on cardiovascular risk
page 14 of 64
markers in type 2 diabetes. The aim of this study was to investigate if the combination of stevioside and a
dietary supplement of soy protein possesses beneficial qualities in the treatment of type 2 diabetes and
the metabolic syndrome. We randomized male Zucker diabetic fatty rats into 4 groups and fed them the
different test diets for 10 weeks: (A) standard carbohydrate-rich laboratory diet (chow), (B)
chow+stevioside (0.03 g/kg body weight [BW] per day), (C) 50% soy (Abalon)+50% chow (adjusted for
vitamins and minerals), and (D) 50% soy (Abalon)+50% chow+stevioside 0.03 g/kg BW per day. We
measured plasma glucose, blood pressure, weight, and food intake once weekly. The animals were
equipped with an intra-arterial catheter, and at week 10, the conscious rats underwent an intra-arterial
glucose tolerance test (2.0 g/kg BW). Stevioside exerts beneficial effects in type 2 diabetic Zucker
diabetic fatty rats, that is, lowers blood glucose (area under the glucose curve [AUC(30min)]: group A vs
B, a 19% reduction; and group C vs D, a 12% reduction; P<.001). We did not detect any effect on insulin
or glucagon responses. After 2 weeks of treatment, a decrease in the systolic blood pressure was
observed in the stevioside-treated groups (P<.01). Abalon had beneficial effects on cardiovascular risk
markers, that is, (1) lowers total cholesterol (P<.01), (2) reduces triglycerides (P=.01), and (3) reduces
free fatty acids (P<.001). The combination of stevioside and soy supplementation appears to possess the
potential as effective treatment of a number of the characteristic features of the metabolic syndrome, that
is, hyperglycemia, hypertension, and dyslipidemia. A long-term human study of the concept in type 2
diabetic subjects is needed to verify these promising results in animal diabetes.
EC (European Commission). Report on Methodologies for the Monitoring of Food Additive Intake
Across the European Union. Final Report Submitted by the Task Coordinator, 16 January 1998.
European Commission, 1999a. Opinion on Stevia Rebaudiana Bertoni plants and leaves.
Scientific Committee on Food (CS/NF/STEV/3 Final, 17 June 1999).
European Commission, 1999b. Opinion on stevioside as a sweetener. Scientific Committee on
Food(CS/ADD/EDUL/167Final, 17 June 1999).
European Food Safety Authority, 2008. Steviol glycosides (New submission), EFSA Question
Number EFSA-Q-2008-041). EFSA in Focus – Food, Issue 02, December, p. 12.
European Food Safety Authority (EFSA), Parma, Italy. Scientific Opinion on the safety of steviol
glycosides for the proposed uses as a food additive. EFSA Journal 2010;8(4):1537
EFSA (European Food Safety Authority), 2007. Opinion of the Panel on Food Additives,
Flavourings, Processing Aids and Food Contact Materials (AFC) following a request form the
Commission on Neotame as a sweetener and flavour enhancer. The EFSA Journal 581, 1-43.
EFSA (European Food Safety Authority), 2008. Concise European Food Consumption Database.
Accessible at: http://www.efsa.europa.eu/EFSA/ScientificPanels/datex/efsa_locale1178620753812_ConciseEuropeanConsumptionDatabase.htm Safety of steviol glycosides as a
food additive.
European Food Safety Authority (EFSA), Parma, Italy. Scientific Opinion on the safety of steviol
glycosides for the proposed uses as a food additive. EFSA Journal 2010;8(4):1537.
Felippe GM, Randi AM. Germination and endogenous growth substances of Stevia rebaudiana.
First Brazilian Seminar on Stevia Rebaudiana Inst Tecnol Aliment (Campinas) Brazil June 25-26 :
V.1-V.2.1981.
Felippe GM. Stevia rebaudiana. A review. Cienc Cult (Sao Paulo) ;29:1240. 1977.
Ferreira EB, de Assis Rocha Neves F, da Costa MA, do Prado WA, de Araújo Funari
Ferri L, Bazotte RB. Comparative effects of Stevia rebaudiana leaves and stevioside on glycaemia
and hepatic gluconeogenesis. Planta Med. 72(8):691-6, 2006. The purpose of the present study was to
compare the effect of the oral treatment (gavage) with Stevia rebaudiana (Bert.) Bertoni (SRB) and
page 15 of 64
stevioside (STV) on glycaemia and gluconeogenesis of 15-h fasted rats. For this purpose, the rats
received SRB (20 mg/kg x day), STV (5.5 mg/kg x day) or an equal volume of water (controls) during 15
days. To measure hepatic gluconeogenesis, liver perfusion and isolated epatocytes were used.
Glycaemia and gluconeogenesis from L-alanine (5 mM), L-glutamine (5 mM) and L-lactate (2 mM) were
decreased (P < 0.05) after pre-treatment with SRB. However, the treatment with STV did not influence
glycaemia and gluconeogenesis. Moreover, to get further information about the mechanism by which
SRB leaves inhibit gluconeogenesis their potential role as a PPARgamma agonist was investigated. The
data showed absence of activation of PPARgamma receptors. In summary, our results showed that the
reduction of glycaemia promoted by the treatment with SRB leaves was mediated, at least in part, by an
inhibition of hepatic gluconeogenesis. However, this effect did not involve stevioside and the activation of
PPARgamma receptors.
Ferri LA, Alves-Do-Prado W, Yamada SS, Gazola S, Batista MR, Bazotte RB. Investigation of the
antihypertensive effect of oral crude stevioside in patients with mild essential hypertension.
Phytother Res. 20(9):732-6, 2006. The antihypertensive effect of crude stevioside obtained from the
leaves of Stevia rebaudiana (Bertoni) Bertoni (Compositae) on previously untreated mild hypertensive
patients was examined. Patients with essential hypertension were submitted to a placebo phase for 4
weeks. The volunteers selected in this phase were randomly assigned to receive either capsules
containing placebo during 24 weeks or crude stevioside 3.75 mg/kg/day (7 weeks), 7.5 mg/kg/day (11
weeks) and 15.0 mg/kg/day (6 weeks). All capsules were prescribed twice a daily (b.i.d.), i.e. before lunch
and before dinner. After the placebo phase and after each dose of crude stevioside, body mass index,
electrocardiogram and laboratory tests were performed. During the investigation blood pressure (BP) was
measured biweekly and the remaining data were collected at the end of each stevioside dose step. All
adverse events were prospectively recorded but no major adverse clinical effects were observed during
the trial. Systolic and diastolic BP decreased (p < 0.05) during the treatment with crude stevioside, but a
similar effect was observed in the placebo group. Therefore, crude stevioside up to 15.0 mg/kg/day did
not show an antihypertensive effect. Moreover, the results suggest that oral crude stevioside is safe and
supports the well-established tolerability during long term use as a sweetener in Brazil.
Figlewicz DP, Ioannou G, Bennett Jay J, Kittleson S, Savard C, Roth CL. Effect of moderate intake
of sweeteners on metabolic health in the rat. Physiol Behav. 98(5):618-24, 2009. The rise in
prevalence of obesity, diabetes, metabolic syndrome, and fatty liver disease has been linked to increased
consumption of fructose-containing foods or beverages. Our aim was to compare the effects of moderate
consumption of fructose-containing and non-caloric sweetened beverages on feeding behavior, metabolic
and serum lipid profiles, and hepatic histology and serum liver enzymes, in rats. Behavioral tests
determined preferred (12.5-15%) concentrations of solutions of agave, fructose, high fructose corn syrup
(HFCS), a combination of HFCS and Hoodia (a putative appetite suppressant), or the non-caloric
sweetener Stevia (n=5/gp). HFCS intake was highest, in preference and self-administration tests. Groups
(n=10/gp) were then assigned to one of the sweetened beverages or water as the sole source of liquid at
night (3 nights/wk, 10wks). Although within the normal range, serum cholesterol was higher in the
fructose and HFCS groups, and serum triglycerides were higher in the Agave, HFCS, and HFCS/Hoodia
groups (vs. water-controls, p<0.05). Liver histology was normal in all groups with no evidence of
steatosis, inflammation, or fibrosis; however serum alanine aminotransferase was higher in the fructose
and HFCS groups (vs. water-controls, p<0.05). Serum inflammatory marker levels were comparable
among Stevia, agave, fructose, HFCS, and water-consuming groups, however levels of IL-6 were
significantly lower in association with the ingestion of Hoodia. There were no differences in terminal body
weights, or glucose tolerance assessed by 120-min IVGTTs performed at the end of the 10-week
regimen. We conclude that even moderate consumption of fructose-containing liquids may lead to the
onset of unfavorable changes in the plasma lipid profile and one marker of liver health, independent of
significant effects of sweetener consumption on body weight.
Flecher JR, HG. The sweet herb of Paraguay (Review). Chemurgic Digest;14(4):7-18. 1955.
Flores RZ, Cechin STZ, Rodrigues da Silva AC. Absence of mutagenesis induced by the stevioside
from Stevia rebaudiana (Bert.) Bertoni. Ciencia e Cultura;39:417-418. 1987.
page 16 of 64
FAO (Food and Agriculture Organization), 2007a. Steviol Glycosides. FAO JECFA Monographs 4.
FAO (Food and Agriculture Organization), 2007b. Chemical and Technical Assessment: Steviol
Glycosides. Revised by Paul M. Kuznesof, PhD for the 68th JECFA Meeting.
FAO (Food and Agriculture Organization), 2008. Steviol Glycosides. FAO JECFA Monographs 5.
FAO/WHO, 2009. List of Substances Scheduled for Evaluation and Request for Data. Food and
Agriculture Organization of the United Nations/ World Health Organization, Joint FAO/WHO Expert
Committee on Food Additives. Seventy-third meeting Food additives and Contaminants.Geneva,
8 to 17 June 2010. Published 14 Sept 2009. Accessed at www.who.int/ipcs/food/jecfa/jecfa73.pdf.
FDA (Food and Drug Administration), 2008. Center for Food Safety and Applied Nutrition
CFSAN)/Office of Food Additive Safety, December 17, 2008. Agency Response Letter GRAS Notice
No. GRN 000253.
FDA (Food and Drug Administration). GRAS Notice Inventory. Accessed at
http://www.accessdata.fda.gov/scripts/fcn/fcnNavigation.cfm?filter=Stevia&sortColumn=&rpt=gra
sListing
Food Standards Agency Stevioside import ban. Accessed at
http://www.food.gov.uk/foodindustry/imports/banned_restricted/stevioside.
Food Standards Agency Stevioside import ban. Accessed at
http://www.food.gov.uk/foodindustry/imports/banned_restricted/stevioside.
Food Standards Australia New Zealand. Application A540 - Stevol Glycosides as Intense
Sweeteners Accessed at
http://www.foodstandards.gov.au/foodstandards/applications/applicationa540stevi3096.cfm
FSANZ, (Food Standards Australia New Zealand), 2008. Final Assessment Report, Application
A540, Steviol Glycosides as Intense Sweeteners.
French Ministry of Economy, Finance, and Employment. Arrêté du 26 août 2009 relatif à l’emploi
du rébaudioside A (extrait de Stevia rebaudiana) comme additif alimentaire. Le Journal officiel de
la République française, Edition n° 0206, 6 September 2009. Accessed at http://www.afssa.fr/
(Afssa's opinions, reports and summaries in English: AAAT2009sa0012EN.pdf)
Fuh WS, Chiang BH. Purification of steviosides by membrane and ion exchange processes. J
Food Sci 1990;55(5): 1454-57. 1990.
Fujita Y, Wideman RD, Speck M, Asadi A, King DS, Webber TD, Haneda M, Kieffer TJ. Incretin
release from gut is acutely enhanced by sugar but not by sweeteners in vivo. Am J Physiol
Endocrinol Metab. 296(3):E473-9, 2009. Glucose-dependent insulinotropic polypeptide (GIP) and
glucagon-like peptide-1 (GLP-1) are released during meals from endocrine cells located in the gut
mucosa and stimulate insulin secretion from pancreatic beta-cells in a glucose-dependentmanner.
Although the gut epithelium senses luminal sugars, the mechanism of sugar sensing and its downstream
events coupled to the release of the incretin hormones are not clearly elucidated. Recently, it was
reported that sucralose, a sweetener that activates the sweet receptors of taste buds, triggers incretin
release from a murine enteroendocrine cell line in vitro. We confirmed that immunoreactivity of alphagustducin, a key G-coupled protein involved in taste sensing, is sometimes colocalized with GIP in rat
duodenum. We investigated whether secretion of incretins in response to carbohydrates is mediated via
taste receptors by feeding rats the sweet-tasting compounds saccharin, acesulfame potassium, dtryptophan, sucralose, or stevia. Oral gavage of these sweeteners did not reduce the blood glucose
excursion to a subsequent intraperitoneal glucose tolerance test. Neither oral sucralose nor oral stevia
reduced blood glucose levels in Zucker diabetic fatty rats. Finally, whereas oral glucose increased plasma
GIP levels approximately 4-fold and GLP-1 levels approximately 2.5-fold postadministration, none of the
page 17 of 64
sweeteners tested significantly increased levels of these incretins. Collectively, our findings do not
support the concept that release of incretins from enteroendocrine cells is triggered by carbohydrates via
a pathway identical to the sensation of "sweet taste" in the tongue.
Fullas F, Kim J, Compadre CM, Kinghorn AD. Separation of natural product sweetening agents
using overpressured layer chromatography. J Chromatogr; 464(1):213-19. 1989.
Gardana C, Simonetti P, Canzi E, Zanchi R, Pietta P. Metabolism of stevioside and rebaudioside A
from Stevia rebaudiana extracts by human microflora. J Agric Food Chem. 51(22):6618-22, 2003.
Stevia rebaudiana standardized extracts (SSEs) are used as natural sweeteners or dietary supplements
in different countries for their content of stevioside or rebaudioside A. These compounds possess up to
250 times the sweetness intensity of sucrose, and they are noncaloric and noncariogenic sweeteners.
The aim of this study was to investigate the in vitro transformation of stevioside and rebaudioside A after
incubation with human microflora, the influence of these sweeteners on human microbial fecal community
and which specific groups metabolize preferentially stevioside and rebaudioside A. The experiments were
carried out under strict anaerobic conditions in batch cultures inoculated with mixed fecal bacteria from
volunteers. The hydrolysis was monitored by HPLC coupled to photodiode array and mass spectrometric
detectors. Isolated bacterial strains from fecal materials incubated in selective broths were added to
stevioside and rebaudioside A. These sweeteners were completely hydrolyzed to their aglycon steviol in
10 and 24 h, respectively. Interestingly, the human intestinal microflora was not able to degrade steviol.
Furthermore, stevioside and rebaudioside A did not significantly influence the composition of fecal
cultures; among the selected intestinal groups, bacteroides were the most efficient in hydrolyzing Stevia
sweeteners to steviol.
Gardana C, Scaglianti M and Simonetti P. Evaluation of steviol and its glycosides in Stevia
rebaudiana leaves and commercial sweetener by ultra-high-performance liquid
chromatographymass spectrometry. Journal of Chromatography A; 1217:1463-1470. 2010. Stevia
rebaudiana leaves contain non-cariogenic and non-caloric sweeteners (steviol-glycosides) whose
consumption could exert beneficial effects on human health. Steviol-glycosides are considered safe;
nonetheless, studies on animals highlighted adverse effects attributed to the aglycone steviol. The aim of
the present study was to develop and validate two different ultra-high-performance liquid chromatography
methods with electrospray ionization mass spectrometry (UHPLC-MS) to evaluate steviol-glycosides or
steviol in Stevia leaves and commercial sweetener (Truvia). Steviol-glycosides identity was preliminarily
established by UV spectra comparison, molecular ion and product ions evaluation, while routine analyses
were carried out in single ion reaction (SIR) monitoring their negative chloride adducts. Samples were
sequentially extracted by methanol, cleaned-up by SPE cartridge and the analytes separated by UHPLC
HSS C18 column (150 mm x 2.1 mm I.D., 1.8 microm). The use of CH2Cl2 added to the mobile phase as
source of Cl- enhance sensitivity. The LLOD for stevioside, rebaudioside A, steviolbioside and steviol was
15, 50, 10 and 1 ng ml(-1), respectively. Assay validation demonstrated good performances in terms of
accuracy (89-103%), precision (<4.3%), repeatability (<5.7%) and linearity (40-180 mg/g). Stevioside
(5.8+/-1.3%), rebaudioside A (1.8+/-1.2%) and rebaudioside C (1.3+/-1.4%) were the most abundant
steviol-glycosides found in samples of Stevia (n=10) from southern Italy. Rebaudioside A was the main
steviol-glycosides found in Truvia (0.84+/-0.03%). The amounts of steviol-glycosides obtained by the
UHPLC-MS method matched those given by the traditional LC-NH2-UV method. Steviol was found in all
the leaves extract (2.7-13.2 mg kg(-1)) but was not detected in Truvia (<1 microg kg(-1)). The proposed
UHPLC-MS methods can be applied for the routine quality control of Stevia leaves and their commercial
preparations.
Geeraert B, Crombe F, Hulsmans M, Benhabiles N, Geuns JM, Holvoet P. Stevioside inhibits
atherosclerosis by improving insulin signaling and antioxidant defense in obese insulin-resistant
mice. Int J Obes (Lond). 34(3):569-77, 2010. OBJECTIVE: Stevioside is a non-caloric natural
sweetener that does not induce a glycemic response, making it attractive as sweetener to diabetics and
others on carbohydrate-controlled diets. Obesity is frequently associated with insulin resistance and
increased inflammation and oxidative stress. Therefore, we investigated its effects on insulin resistance,
inflammation and oxidative stress related to atherosclerosis in obese insulin-resistant mice. RESEARCH
DESIGN: Twelve-week-old mice were treated with stevioside (10 mg kg(-1), n=14) or placebo (n=20) for
page 18 of 64
12 weeks. RESULTS: Stevioside had no effect on weight and triglycerides, but lowered glucose and
insulin. Stevioside treatment improved adipose tissue maturation, and increased glucose transport, insulin
signaling and antioxidant defense in white visceral adipose tissues. Together, these increases were
associated with a twofold increase of adiponectin. In addition, stevioside reduced plaque volume in the
aortic arch by decreasing the macrophage, lipid and oxidized low-density lipoprotein (ox-LDL) content of
the plaque. The higher smooth muscle cell-to-macrophage ratio was indicative for a more stable plaque
phenotype. The decrease in ox-LDL in the plaque was likely due to an increase in the antioxidant defense
in the vascular wall, as evidenced by increased Sod1, Sod2 and Sod3. Circulating adiponectin was
associated with improved insulin signaling and antioxidant defense in both the adipose tissue and the
aorta of stevioside-treated mice. CONCLUSION: Stevioside treatment was associated with improved
insulin signaling and antioxidant defense in both the adipose tissue and the vascular wall, leading to
inhibition of atherosclerotic plaque development and inducing plaque stabilization.
Geuns JM, Buyse J, Vankeirsbilck A, Temme EH, Compernolle F, Toppet S. Identification of steviol
glucuronide in human urine. J Agric Food Chem.;54(7):2794-8, 2006. Stevioside (250 mg capsules)
was given three times daily to 10 healthy subjects. Steviol glucuronide (steviol 19-O-beta-Dglucopyranosiduronic acid; MM, 494.58; melting point, 198-199 degrees C) was characterized in the 24 h
urine as the only excretion product of oral stevioside by MS, NMR, IR, and UV spectroscopy. This is the
first report on the unambiguous identification of steviol glucuronide in human urine.
Geuns JM. Safety of Stevia and stevioside. Recent Res Develop Phytochem; 4:75-88. 2000.
Geuns JM. Stevioside. Phytochemistry Nov;64(5):913-21. 2003. 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
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.
Geuns, JM Comments to the paper by Nunes et al., Analysis of genotoxic potential of stevioside by
comet assay, Food and Chemical Toxicology;45:662–666. 2007.
Geuns JM. Analysis of Steviol glycosides: validation of the methods. pp. 59-78 in Proceedings of
the EUSTAS Stevia Symposium, June 27th, KULeuven, Belgium Ed: Jan M.C. Geuns, ISBN:
D/2008/6045/50. 2008.
Geuns JM, Augustijns P, Mols R, Buyse JG, Driessen B. Metabolism of stevioside in pigs and
intestinal absorption characteristics of stevioside, rebaudioside A and steviol. Food Chem
Toxicol. 41(11):1599-607, 2003. 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 0.16x10(-6) and 0.11x10(-6) cm/s, respectively. The Papp value for the absorptive
transport of steviol was about 38.6x10(-6) cm/s while the Papp value for the secretory transport of steviol
was only about 5.32x10(-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
page 19 of 64
steviol probably is adsorbed to the compounds present in the colon of which the contents is being
concentrated by withdrawal of water.
Geuns JM, Bruggeman V, Buyse JG. Effect of stevioside and steviol on the developing broiler
embryos. J Agric Food Chem. 51(17):5162-7, 2003. At day 7 of incubation, fertile broiler eggs were
injected with different amounts of stevioside and steviol of 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 1 week later, not any influence of the different
treatments could be found on embryonic mortality, body weight of the hatchlings, deformations (e.g.,
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.
Geuns JM, Buyse J, Vankeirsbilck A, Temme EH. Metabolism of stevioside by healthy subjects.
Exp Biol Med (Maywood). 232(1):164-73, 2007. 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 24-hr 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 conditions. 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 3
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 micro g/ml (21.3 micro g 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-hr urine (205 mg steviol equivalents/24 hrs). 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.
Geuns JM, Malheiros RD, Moraes VM, Decuypere EM, Compernolle F, Buyse JG.Metabolism of
stevioside by chickens. J Agric Food Chem. 51(4):1095-101, 2003. In intubation experiments (6431168 mg per animal), most of the stevioside administered to chickens 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 of stevioside/kg of feed) with laying hens and meat-type chickens, no
significant differences were found in feed uptake, weight gain, and feed conversion as the result of
stevioside administration. The egg production and egg composition of laying hens were not influenced.
Most ofthe 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.
Ghanta S, Banerjee A, Poddar A, Chattopadhyay S. Oxidative DNA damage preventive activity and
antioxidant potential of Stevia rebaudiana (Bertoni) Bertoni, a natural sweetener. J Agric Food
Chem. 55(26):10962-7, 2007. At 0.1 mg/mL, the ethyl acetate extract (EAE) of the crude 85%
methanolic extract (CAE) of Stevia rebaudiana leaves exhibited preventive activity against DNA strand
scission by *OH generated in Fenton's reaction on pBluescript II SK (-) DNA. Its efficacy is better than
that of quercetin. The radical scavenging capacity of CAE was evaluated by the DPPH test
(IC50=47.66+/-1.04 microg/mL). EAE was derived from CAE scavenged DPPH (IC50=9.26+/-0.04
microg/mL), ABTS+(IC50=3.04+/-0.22 microg/mL) and *OH (IC50=3.08+/-0.19 microg/mL). Additionally,
inhibition of lipid peroxidation induced with 25 mM FeSO 4 on rat liver homogenate as a lipid source was
noted with CAE (IC50=2.1+/-1.07 mg/mL). The total polyphenols and total flavonoids of EAE were 0.86
page 20 of 64
mg gallic acid equivalents/mg and 0.83 mg of quercetin equivalents/mg, respectively. Flavonoids, isolated
from EAE, were characterized as quercetin-3-O-arabinoside, quercitrin, apigenin, apigenin-4-O-glucoside,
luteolin, and kaempferol-3-O-rhamnoside by LC-MS and NMR analysis. These results indicate that Stevia
rebaudiana may be useful as a potential source of natural antioxidants.
Gorbenko N, Poltorak V, Gladkih A, Ivanova O, Khudyakova E, Gorbenko K, Gorshunska M. Natural
sweetener stevioside improves lipid profile and ameliorates oxidative stress in diabetic rabbits.
Diabetologia;48 (Suppl. 1), A273 (Abstract No. 754). 2005.
Goyal SK, Samsher, Goyal RK. Stevia (Stevia rebaudiana) a bio-sweetener: a review. Int J Food Sci
Nutr. Feb;61(1):1-10. 2010. Studies revealed that Stevia has been used throughout the world since
ancient times for various purposes; for example, as a sweetener and a medicine. We conducted a
systematic literature review to summarize and quantify the past and current evidence for Stevia. We
searched relevant papers up to 2007 in various databases. As we know that the leaves of Stevia plants
have functional and sensory properties superior to those of many other high-potency sweeteners, Stevia
is likely to become a major source of high-potency sweetener for the growing natural food market in the
future. Although Stevia can be helpful to anyone, there are certain groups who are more likely to benefit
from its remarkable sweetening potential. These include diabetic patients, those interested in decreasing
caloric intake, and children. Stevia is a small perennial shrub that has been used for centuries as a biosweetener and for other medicinal uses such as to lower blood sugar. Its white crystalline compound
(stevioside) is the natural herbal sweetener with no calories and is over 100-300 times sweeter than table
sugar.
Gregersen S, Jeppesen PB, Holst JJ, Hermansen K. Antihyperglycemic effects of stevioside in
type 2 diabetic subjects.Metabolism. 53(1):73-6, 2004. Stevioside is present in the plant Stevia
rebaudiana Bertoni (SrB). Extracts of SrB have been used for the treatment of diabetes in, for example,
Brazil, although a positive effect on glucose metabolism has not been unequivocally demonstrated. We
studied the acute effects of stevioside in type 2 diabetic patients. We hypothesize that supplementation
with stevioside to a test meal causes a reduction in postprandial blood glucose. Twelve type 2 diabetic
patients were included in an acute, paired cross-over study. A standard test meal was supplemented with
either 1 g of stevioside or 1 g of maize starch (control). Blood samples were drawn at 30 minutes before
and for 240 minutes after ingestion of the test meal. Compared to control, stevioside reduced the
incremental area under the glucose response curve by 18% (P =.013). The insulinogenic index
(AUC(i,insulin)/AUC(i,glucose)) was increased by approximately 40% by stevioside compared to control
(P <.001). Stevioside tended to decrease glucagon levels, while it did not significantly alter the area under
the insulin, glucagon-like peptide 1, and glucose-dependent insulinotropic polypeptide curves. In
conclusion, stevioside reduces postprandial blood glucose levels in type 2 diabetic patients, indicating
beneficial effects on the glucose metabolism. Stevioside may be advantageous in the treatment of type 2
diabetes.
Gregersen S, Hermansen K, Jeppesen PB and Holst JJ. Acute effects of the diterpene glycoside
stevioside in type II diabetic patients. Diabetologia; 44 (1), 236 [Abstract No. 905]. 2001.
Hagiwara A, Fukushima S, Kitaori M, Shibata M, Ito N. Effects of three sweeteners on rat urinary
bladder carcinogenesis initiated by N-butyl-N-(4-hydroxybutyl) nitrosamine. Gann;75:763-768
(English abstract only). 1984. The effects of three sweeteners, sodium saccharin, aspartame and
stevioside, on urinary bladder carcinogenesis in rats initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine
(BBN) were evaluated. Male F344 rats were given 0.01% BBN in their drinking water for 4 weeks and
then the test sweeteners in their diet for 32 weeks. All surviving rats were sacrificed after 36 weeks, and
examined histologically. Treatment with sodium saccharin significantly increased the incidence and extent
of preneoplastic lesions, papillary or nodular (PN) hyperplasia, in rats treated with BBN for 4 weeks.
Administration of 5% aspartame or 5% stevioside in the diet did not, however, affect the incidence or
extent of PN hyperplasia in BBN-treated rats. No preneoplastic or neoplastic lesions of the urinary
bladder were observed in rats treated with the test sweeteners only. The results with sodium saccharin
were consistent with those in our previous experiments. The data also suggest that aspartame and
stevioside do not promote bladder carcinogenesis.
page 21 of 64
Hashimoto Y, Moriyasu M, Nakamura S, Ishiguro S, Komuro M. High-performance liquid
chromatographic determination of Stevia components on a hydrophilic packed column. J
Chromatogr;161:403. 1978.
Hashimoto Y, Moriyasu M. Determination of sweet components in Stevia rebaudiana by highperformance liquid chromatography. Ultraviolet detection. Shoyakugaku Zasshi;32:209-11. 1978.
Health Canada. Revised Guidelines for the Use of Stevia in Natural Health Products. Accessed at
http://www.hc-sc.gc.ca/dhp-mps/prodnatur/legislation/docs/notice-avis-stevia-eng.php.
Hellfritsch C, Brockhoff A, Stähler F, Meyerhof W, Hofmann T. Human Psychometric and Taste
Receptor Responses to Steviol Glycosides. J Agric Food Chem. 60 (27):6782–679, 2012. Steviol
glycosides, the sweet principle of Stevia Rebaudiana (Bertoni) Bertoni, have recently been approved as a
food additive in the EU. The herbal non-nutritive high-potency sweeteners perfectly meet the rising
consumer demand for natural food ingredients in Europe. We have characterized the
organoleptic properties of the most common steviol glycosides by an experimental approach combining
human sensory studies and cell-based functional taste receptor expression assays. On the basis of their
potency to elicit sweet and bitter taste sensations, we identified glycone chain length, pyranose
substitution, and the C16 double bond as the structural features giving distinction to the gustatory profile
of steviol glycosides. A comprehensive screening of 25 human bitter taste receptors revealed that two
receptors, hTAS2R4 and hTAS2R14, mediate the bitter off-taste of steviol glycosides. For some test
substances, e.g., stevioside, we observed a decline in sweet intensity at supra-maximum concentrations.
This effect did not arise from allosteric modulation of the hTAS1R2/R3 sweet taste receptor but might be
explained by intramolecular cross-modal suppression between the sweet and bitter taste component of
steviol glycosides. These results might contribute to the production of preferentially sweet and least bitter
tasting Stevia extracts by an optimization of breeding and postharvest downstream processing.
Höhn S, Zankl H, Mutagenic effects of stevioside in vitro and in vivo, Mutagenesis 5, 622 Abstract
No. 16. 1990.
Hong J, Chen L, Jeppesen PB, Nordentoft I, Hermansen K. Stevioside counteracts the alpha-cell
hypersecretion caused by long-term palmitate exposure. Am J Physiol Endocrinol Metab. 290(3):
E416-22, 2006. Long-term exposure to fatty acids impairs beta-cell function in type 2 diabetes, but little is
known about the chronic effects of fatty acids on alpha-cells. We therefore studied the prolonged impact
of palmitate on alpha-cell function and on the expression of genes related to fuel metabolism. We also
investigated whether the antihyperglycemic agent stevioside was able to counteract these effects of
palmitate. Clonal alpha-TC1-6 cells were cultured with palmitate in the presence or absence of stevioside.
After 72 h, we evaluated glucagon secretion, glucagon content, triglyceride (TG) content, and changes in
gene expression. Glucagon secretion was dose-dependently increased after 72-h culture, with palmitate
at concentrations >or=0.25 mM (P< 0.05). Palmitate (0.5 mM) enhanced TG content of alpha-cells by
73% (P< 0.01). Interestingly, stevioside (10(-8) and 10(-6) M) reduced palmitate-stimulated glucagon
release by 22 and 45%, respectively (P< 0.01). There was no significant change in glucagon content after
72-h culture with palmitate and/or stevioside. Palmitate increased carnitine palmitoyltransferase I (CPT I)
mRNA level, whereas stevioside enhanced CPT I, peroxisome proliferator-activated receptor-gamma, and
stearoyl-CoA desaturase gene expressions in the presence of palmitate (P<0.05). In conclusion, longterm exposure to elevated fatty acids leads to a hypersecretion of glucagon and an accumulation of TG
content in clonal alpha-TC1-6 cells. Stevioside was able to counteract the alpha-cell hypersecretion
caused by palmitate and enhanced the expression of genes involved in fatty acid metabolism. This
indicates that stevioside may be a promising antidiabetic agent in treatment of type 2 diabetes.
Hong Kong Government, 2002. Updated July 2002. [Online] Risk in Brief, issue no. 10,
"Stevioside in Foods", Hong Kong Government, Risk Assessment Section at:
http://www.info.gov.hk/fehd/textmode/safefood/report/ stevioside/stevioside.html.
page 22 of 64
Horio T. Effect of physical exercise on human preference for solutions of various sweet
substances. Percept Mot Skills. 99(3 Pt 1):1061-70, 2004. The effect of physical exercise on taste
preference for various sweet solutions was examined with 44 healthy university students (19 men, 25
women; M age=20, SD=0.9). None had participated in regular exercise programs during the previous
year. After 30 min. of exercise using a bicycle ergometer at the heart-rate intensity corresponding to 50%
VO2 max (maximal oxygen uptake), a rating scale for taste hedonic tone was performed. The test
solutions included various concentrations of sucrose, glucose, stevioside, D-sorbitol, erythritol, and
saccharin. The preference ratings for sucrose, glucose, stevioside, D-sorbitol, and erythritol increased
after exercise, whereas the preference ratings for saccharin were unchanged after exercise at all the
concentrations tested. Taste preference for many sweet substances increased after physical exercise.
Hsieh MH, Chan P, Sue YM, Liu JC, Liang TH, Huang TY, Tomlinson B, Chow MS, Kao PF, Chen
YJ. Efficacy and tolerability of oral stevioside in patients with mild essential hypertension: a twoyear, randomized, placebo-controlled study. Clin Ther. 25(11):2797-808, 2003. BACKGROUND:
Stevioside, a natural glycoside isolated from the plant Steviarebaudiana Bertoni, has been used as a
commercial sweetening agent in Japan and Brazil for >20 years. Previous animal and human studies
have indicated that stevioside has an antihypertensive effect. OBJECTIVES: This study was undertaken
to investigate the long-term (2-year) efficacy and tolerability of stevioside in patients with mild essential
hypertension. Secondary objectives were to determine the effects of stevioside on left ventricular mass
index (LVMI) and quality of life (QOL). METHODS: This was a multicenter, randomized, double-blind,
placebo-controlled trial in Chinese men and women aged between 20 and 75 years with mild essential
hypertension (systolic blood pressure [SBP] 140-159 mm Hg and diastolic blood pressure [DBP] 90-99
mm Hg). Patients took capsules containing 500 mg stevioside powder or placebo 3 times daily for 2
years. Blood pressure was measured at monthly clinic visits; patients were also encouraged to monitor
blood pressure at home using an automated device. LVMI was determined by 2-dimensional
echocardiography at baseline and after 1 and 2 years of treatment. QOL was assessed using the Medical
Outcomes Study 36-Item Short-Form Health Survey. Electrocardiographic, laboratory, and QOL
parameters were assessed at the beginning of treatment, and at 6 months, 1 year, and 2 years.
RESULTS: One hundred seventy-four patients (87 men, 87 women) were enrolled in the study, and 168
completed it: 82 (42 men, 40 women; mean [SD] age, 52 [7] years) in the stevioside group and 86 (44
women, 42 men; mean age, 53 [7] years) in the placebo group. After 2 years, the stevioside group had
significant decreases in mean (SD) SBP and DBP compared with baseline (SBP, from 150 [7.3] to 140
[6.8] mm Hg; DBP, from 95 [4.2] to 89 [3.2] mm Hg; P < 0.05) and compared with placebo (P < 0.05).
Based on patients' records of self-monitored blood pressure, these effects were noted beginning
approximately 1 week after the start of treatment and persisted throughout the study. There were no
significant changes in body mass index or blood biochemistry, and the results of laboratory tests were
similar in the 2 groups throughout the study. No significant difference in the incidence of adverse effects
was noted between groups, and QOL scores were significantly improved overall with stevioside
compared with placebo (P < 0.001). Neither group had a significant change in mean LVMI. However, after
2 years, 6 of 52 patients (11.5%) in the stevioside group had left ventricular hypertrophy (LVH), compared
with 17 of 50 patients (34.0%) in the placebo group (P < 0.001). Of those who did not have LVH at
baseline, 3 of 46 patients (6.5%) in the stevioside group had developed LVH after 2 years, compared with
9 of 37 patients (24.3%) in the placebo group (P < 0.001). CONCLUSIONS: In this 2-year study in
Chinese patients with mild hypertension, oral stevioside significantly decreased SBP and DBP compared
with placebo. QOL was improved, and no significant adverse effects were noted.
Hsin YY, Yang YW, Chang WC. Recent studies on Stevia rebaudiana. K'o Hsueh Fa Chan Yueh
K'an;7(10):1049-55. 1979.
Hsing YL, Su WF, Chang WC. Accumulation of stevioside and rebaudioside a in callus culture of
Stevia rebaudiana Bertoni. Bot Bull Acad Sin;24(2):115-19. 1983.
Hsu YH, Liu JC, Kao PF, Lee CN, Chen YJ, Hsieh MH, Chan P. Antihypertensive effect of
stevioside in different strains of hypertensive rats. Zhonghua Yi Xue Za Zhi (Taipei). 65(1):1-6,
2002. BACKGROUND: Stevioside is a natural sweet-tasting glycoside isolated from the herb Stevia
rebaudiana, composed of stevia, a diterpenic carboxylic alcohol with three glucose molecules, mainly
page 23 of 64
used commercially as sugar substitute. Previous study has shown that it can lower blood pressure in
anesthetized spontaneously hypertensive rats (SHR). This study was undertaken to evaluate the
antihypertensive effect of stevioside in different strains of hypertensive rats and to observe whether there
is difference in blood pressure lowering effect. METHODS: Noninvasive tail-cuff method was employed to
measure blood pressure. Stevioside at the concentrations of 50, 100 and 200 mg/kg were administered
intraperitoneally (ip) to normotensive Wistar-Kyoto rats (NTR), SHR, deoxycorticosterone acetate-salt
(DOCA-NaCl) sensitive hypertensive rats (DHR) and renal hypertensive rats (RHR). RESULTS:
Significant hypotensive effect of stevioside administered ip was noted in different strains of rats at the
dose of 50 mg/kg. When stevioside was increased to the concentrations of 100 and 200 mg/kg, ip, it also
caused slow and persistent lowering of blood pressure in SHR and NTR. Data also showed that
stevioside given at the concentrations of 100, 200 and 400 mg/kg ip resulted in lowering of blood
pressure in SHR dose-dependently. Blood pressure returned to previous levels after the drug was
discontinued for 2-3 days. Drinking of 0.1% stevioside solution in mature SHR could have
antihypertensive effect and also prevented hypertension in immature SHR. CONCLUSIONS: This study
reconfirmed stevioside has hypotensive effect and the effect is more prominent in hypertensive rats.
Huang BL. Recent progress in studies on stevioside. Shipin Kexue (Beijing) ;24:1-4. 1984.
Huang BL. Preliminary studies on laboratory isolation of stevioside crystals from Stevia
rebaudiana. Shih P'in K'o Hsueh (Beijing);28:1-2. 1982.
Huang B. Present status on the application of sweetening constituent of Stevia rebaudiana
Bertoni in non-staple foods. Tiaowei Fushipin Keji (12):8-10. 1982.
Huang YS, Gus A, Qian Y, Chen LY, Cu HF. Variation of steviosides content and selection of typeR-A in Stevia rebaudiana. Zhiwu Ziyuan Yu Huanjing;4( 3):28-32. 1995.
Hübler MO, Bracht A, Kelmer-Bracht AM. Influence of stevioside on hepatic glycogen levels in
fasted rats. Research Communications in Chemical Pathology and Pharmacology;84:111-18. 1994.
The influence of stevioside, the sweet glycoside of Stevia rebaudiana leaves, on the glycogen levels of
fasted rats was investigated. In one set of experiments, single doses of stevioside (200 mumol) or steviol
(200 mumol) were given orally to 24-hours fasted rats, either alone or simultaneously with fructose. Under
these conditions both stevioside and steviol increased the initial glycogen deposition in the liver. In
another set of experiments, stevioside was given to the rats in the drinking water at the beginning of the
fasting periods (5:00 p.m.) of 24 and 48 hours. Two different concentrations were given, 1.0 and 2.0 mM.
Increased hepatic glycogen levels were found at 48 hours with stevioside (1.0 mM) and at 24 hours with
stevioside (2.0 mM). Steviol had no effect on hepatic glycogen levels when given in the drinking water. It
can be concluded that stevioside exerts a stimulatory action on hepatic glycogen synthesis under
gluconeogenic conditions.
Hutapea AM, Toskulkao C, Buddhasukh D, Wilairat P, Glinsukon T. Digestion of stevioside, a
natural sweetener, by various digestive enzymes. Journal of Clinical Biochemistry and Nutrition;
23: 177-86. 1997.
Inglett GE. A potential saccharin replacement stevioside. Botanicals. C R Acad Sci;192:1123-1931.
1972.
Inglett GE. A history of sweeteners. Natural and synthetic. J Toxicol Environ Health; 2:207-13.
1976. Sweetness for the prehistoric man was the taste sensation obtained from sweet berries and honey.
Man's quest for other sweet things led to sucose, starch-derived sugars, and synthetic sweeteners. An
unusual source of sweet taste is a West African berry known as miracle fruit (Synsepalum dulcificum).
This fruit possesses a taste-modifying substance that causes sour foods--e.g., lemons, limes, or
grapefruit--to taste sweet. The active principle was found to be a glycoprotein. Until this time, only small
molecules were considered sweet-evoking substances, but now macromolecules are considered capable
of participating in taste perception. The intense sweetener of the fruit of Dioscoreophyllum cumminsii,
called the serendipity berry, was revealed to be a protein. The intensely sweet principle of
page 24 of 64
Thaumatococcus daniellii, called katemfe, was reported in 1972 to contain two proteins having intense
sweetness. Since intensely sweet protein sweeteners act directly on taste buds as a probe, a peptide
linkage analogous to the aspartic acid sweeteners may be partly responsible for their sweetness.
Ishii EL, Bracht A. Stevioside, the sweet glycoside of Stevia rebaudiana, inhibits the action of
atractyloside in the isolated perfused rat liver. Research Communications in Chemical Pathology
and Pharmacology;53:79-91. 1986. Stevioside inhibits the action of atractyloside on energy metabolism
in the isolated perfused rat liver. The effects of atractyloside on glycolysis, glycogenolysis,
gluconeogenesis and oxygen uptake are decreased by stevioside. The concentration for half-maximal
action is 0.5 mM. The site of the action is located on the outside of the cell. Possibly, stevioside affects
the transport of atractyloside across the cell membrane.
Ishii EL, Schwab AJ, Bracht A. Inhibition of monosaccharide transport in the in tact rat liver by
stevioside. Biochemical Pharmacology;36:1417-33. 1987. The transport and metabolism of D-glucose
and D-fructose in the isolated perfused rat liver and the influence of stevioside and its derivatives were
investigated. The transport parameters were measured by the multiple indicator dilution technique. The
maximal exchange rate of D-glucose was 700 mumol X min-1 X ml-1 and the Km was 38 mM. Stevioside
and its derivatives (isosteviol and steviolbioside) inhibited D-glucose and D-fructose transport across the
cell membrane. The half-maximal effect at 1 mM D-glucose occurred at 0.8 mM stevioside. The inhibitory
action of stevioside was of mixed type. Isosteviol was more potent than stevioside (half-maximal effect at
0.4 mM), whereas steviolbioside was less active (50% inhibition at 2.5 mM). Stevioside was without effect
on D-glucose metabolism, except for transient changes in D-glucose release, reflecting changes in the
intracellular concentration. D-Fructose consumption, however, was specifically affected (half-maximal
effect at 2.8 mM), as well as all parameters depending on D-fructose transformation (D-glucose
production, L-lactate and pyruvate production, and extra oxygen uptake). In livers releasing D-glucose
from endogenous glycogen, strong inhibition of transport increased the intracellular to extracellular Dglucose concentration ratio (Ci/Ce). The control values of Ci/Ce, representing an average over the total
intracellular water space, were always smaller than unity. The latter observation may indicate that Dglucose does not have access to the whole intracellular water space.
Ishidate M, Sofuni T, Yoshikawa K, Hayashi M, Nohmi T, Sawada M, Matsuoka A. Primary
mutagenicity screening of food additives currently used in Japan. Food and Chemical
Toxicology;22:623-36. 1984. Salmonella/microsome tests (Ames tests) and chromosomal aberration
tests in vitro using a Chinese hamster fibroblast cell line were carried out on 190 synthetic food additives
and 52 food additives derived from natural sources, all of which are currently used in Japan. Fourteen out
of 200 tested in the Ames assay showed positive effects and 54 out of 242 were positive in the
chromosome test. Three additives (erythorbic acid, chlorine dioxide and beet red) were positive only in
the Ames test, although their mutagenic potentials were relatively weak, while 43 additives were positive
only in the chromosome test. Eleven additives (calcium hypochlorite, cinnamic aldehyde, L-cysteine
monohydrochloride, Food Green No. 3 (Fast Green FCF), hydrogen peroxide, potassium bromate,
sodium chlorite, sodium hypochlorite, sodium nitrite, cacao pigment and caramel) were positive in both
the Ames test and the chromosome test. Th e usefulness of such primary screening tests combining two
different genetic end-points, gene mutation and chromosomal aberration, and some correlation between
mutagenicity and carcinogenicity of food additives are discussed.
Jaitak V, Gupta AP, Kaul VK, Ahuja PS. Validated high-performance thin-layer chromatography
method for steviol glycosides in Stevia rebaudiana. J Pharm Biomed Anal; 47(4-5):790-4. 2008. A
high-performance thin-layer chromatographic (HPTLC) method was developed and validated as per ICH
(International Conferences on Harmonization) guidelines for simultaneous quantification of three steviol
glycosides, i.e. steviolbioside, stevioside and rebaudioside-A in Stevia rebaudiana leaves. For achieving
good separation, mobile phase of ethyl acetate-ethanol-water (80:20:12, v/v/v) on pre-coated silica gel 60
F254 HPTLC plates were used. The densitometric quantification of steviol glycosides was carried out at
lambda=510 nm in reflection-absorption mode after spraying with acetic anhydride:sulphuric acid:ethanol
reagent. The calibration curves were linear in the range of 160-960 ng/spot for steviolbioside, 1-6
microg/spot for stevioside and 0.5-3 microg/spot for rebaudioside-A with good correlation coefficients
(0.998-0.999). The method was found to be reproducible for quantitative analysis of steviol glycosides in
page 25 of 64
S. rebaudiana leaves collected from ten different locations and will serve as a quality control indicator to
monitor the commercial production of stevioside and its allied molecules during different stages of its
processing.
Jakinovich JR, Moon C, Choi YH, Kinghorn AD. Evaluation of plant extracts for sweetness using
the mongolian gerbil. J Nat Prod;53(1):190-5. 1990. Extracts of Thladiantha grosvenorii fruits, Stevia
rebaudiana leaves, and Abrus precatorius leaves were investigated using Mongolian gerbil
electrophysiological and conditioned taste aversion procedures, which were designed to respond to
sucrose. A close correlation was observed between extracts of these sweet plants known to contain
sweet principles and those extracts indicated as being sweet by a combination of these gerbil bioassays.
The methods employed seem to be suitable for use in aiding the purification of highly sweet compounds
of plant origin.
Jeppesen PB, Gregersen S, Alstrup KK, Hermansen K. Stevioside induces antihyperglycaemic,
insulinotropic and glucagonostatic effects in vivo: studies in the diabetic Goto-Kakizaki (GK) rats.
Phytomedicine. 9(1):9-14, 2002. Extracts of leaves from the plant Stevia rebaudiana Bertoni have been
used in the traditional treatment of diabetes in Paraguay and Brazil. Recently, we demonstrated a direct
insulinotropic effect in isolated mouse islets and the clonal beta cell line INS-1 of the glycoside stevioside
that is present in large quantity in these leaves. Type 2 diabetes is a chronic metabolic disorder that
results from defects in both insulin and glucagon secretion as well as insulin action. In the present study
we wanted to unravel if stevioside in vivo exerts an antihyperglycaemic effect in a nonobese animal
model of type 2 diabetes. An i.v. glucose tolerance test (IVGT) was carried out with and without
stevioside in the type 2 diabetic Goto-Kakizaki (GK) rat, as well as in the normal Wistar rat. Stevioside
(0.2 g/kg BW) and D-glucose (2.0 g/kg BW) were administered as i.v. bolus injections in anaesthetized
rats. Stevioside significantly suppressed the glucose response to the IVGT in GK rats (incremental area
under the curve (IAUC): 648 +/- 50 (stevioside) vs 958 +/- 85 mM x 120 min (control); P < 0.05) and
concomitantly increased the insulin response (IAUC: 51116 +/- 10967 (stevioside) vs 21548 +/- 3101
microU x 120 min (control); P < 0.05). Interestingly, the glucagon level was suppressed by stevioside
during the IVGT, (total area under the curve (TAUC): 5720 +/- 922 (stevioside) vs 8713 +/- 901 pg/ml x
120 min (control); P < 0.05). In the normal Wistar rat stevioside enhanced insulin levels above basal
during the IVGT (IAUC: 79913 +/- 3107 (stevioside) vs 17347 +/- 2882 microU x 120 min (control); P <
0.001), however, without altering the blood glucose response (IAUC: 416 +/- 43 (stevioside) vs 417 +/- 47
mM x 120 min (control)) or the glucagon levels (TAUC: 5493 +/- 527 (stevioside) vs 5033 +/- 264 pg/ml x
120 min (control)). In conclusion, stevioside exerts antihyperglycaemic, insulinotropic, and
glucagonostatic actions in the type 2 diabetic GK rat, and may have the potential of becoming a new
antidiabetic drug for use in type 2 diabetes.
Jeppesen PB, Gregersen S, Rolfsen SE, Jepsen M, Colombo M, Agger A, Xiao J, Kruhøffer M,
Orntoft T, Hermansen K. Antihyperglycemic and blood pressure-reducing effects of stevioside in
the diabetic Goto-Kakizaki rat. Metabolism. 52(3):372-8, 2003. Stevioside, a glycoside present in the
leaves of the plant, Stevia rebaudiana Bertoni (SrB), has acute insulinotropic effects in vitro. Its potential
antihyperglycemic and blood pressure-lowering effects were examined in a long-term study in the type 2
diabetic Goto-Kakizaki (GK) rat. Rats were fed 0.025 g x kg(-1) x d(-1) of stevioside (purity > 99.6%) for 6
weeks. An intra-arterial catheter was inserted into the rats after 5 weeks, and conscious rats were
subjected to arterial glucose tolerance test (2.0 g x kg(-1)) during week 6. Stevioside had an
antihyperglycemic effect (incremental area under the glucose response curve [IAUC]): 985 +/- 20
(stevioside) versus 1,575 +/- 21 (control) mmol/L x 180 minutes, (P <.05), it enhanced the first-phase
insulin response (IAUC: 343 +/- 33 [stevioside] v 136 +/- 24 [control] microU/mL insulin x 30 minutes, P
<.05) and concomitantly suppressed the glucagon levels (total AUC: 2,026 +/- 234 [stevioside] v 3,535 +/282 [control] pg/mL x 180 minutes, P <.05). In addition, stevioside caused a pronounced suppression of
both the systolic (135 +/- 2 v 153 +/- 5 mm Hg; P <.001) and the diastolic blood pressure (74 +/- 1 v 83 +/1 mm Hg; P <.001). Bolus injections of stevioside (0.025 g x kg(-1)) did not induce hypoglycemia.
Stevioside augmented the insulin content in the beta-cell line, INS-1. Stevioside may increase the insulin
secretion, in part, by induction of genes involved in glycolysis. It may also improve the nutrient-sensing
mechanisms, increase cytosolic long-chain fatty acyl-coenzyme A (CoA), and downregulate
phosphodiesterase 1 (PDE1) estimated by the microarray gene chip technology. In conclusion, stevioside
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enjoys a dual positive effect by acting as an antihyperglycemic and a blood pressure-lowering substance;
effects that may have therapeutic potential in the treatment of type 2 diabetes and the metabolic
syndrome.
Jeppesen P, Gregersen S, Poulsen CR, Hermansen K. Stevioside acts directly on pancreatic b
cells to secrete insulin: actions independent of cyclic adenosine monophosphate and adenosine
triphosphate-sensitive K+-channel activity. Metabolism;49:208-14. 2000. The natural sweetener
stevioside, which is found in the plant Stevia rebaudiana Bertoni, has been used for many years in the
treatment of diabetes among Indians in Paraguay and Brazil. However, the mechanism for the blood
glucose-lowering effect remains unknown. To elucidate the impact of stevioside and its aglucon steviol on
insulin release from normal mouse islets and the beta-cell line INS-1 were used. Both stevioside and
steviol (1 nmol/L to 1 mmol/L) dose-dependently enhanced insulin secretion from incubated mouse islets
in the presence of 16.7 mmol/L glucose (P < .05). The insulinotropic effects of stevioside and steviol were
critically dependent on the prevailing glucose concentration, ie, stevioside (1 mmol/L) and steviol (1
micromol/L) only potentiated insulin secretion at or above 8.3 mmol/L glucose (P < .05). Interestingly, the
insulinotropic effects of both stevioside and steviol were preserved in the absence of extracellular Ca2+.
During perifusion of islets, stevioside (1 mmol/L) and steviol (1 micromol/L) had a long-lasting and
apparently reversible insulinotropic effect in the presence of 16.7 mmol/L glucose (P < .05). To determine
if stevioside and steviol act directly on beta cells, the effects on INS-1 cells were also investigated.
Stevioside and steviol both potentiated insulin secretion from INS-1 cells (P < .05). Neither stevioside (1
to 100 micromol/L) nor steviol (10 nmol/L to 10 micromol/L) influenced the plasma membrane K+
adenosine triphosphate ((K+)ATP)-sensitive channel activity, nor did they alter cyclic adenosine
monophosphate (cAMP) levels in islets. In conclusion, stevioside and steviol stimulate insulin secretion
via a direct action on beta cells. The results indicate that the compounds may have a potential role as
antihyperglycemic agents in the treatment of type 2 diabetes mellitus.
Jeppesen PB, Dyrskog SE, Agger A, Gregersen S, Colombo M, Xiao J, Hermansen K. Can
stevioside in combination with a soy-based dietary supplement be a new useful treatment of type
2 diabetes? An in vivo study in the diabetic go to-kakizaki rat. The Review of Diabetic Studies; 3:
189-99. 2006a. The diterpene glycoside stevioside (SVS) and soy bean protein isolate have both been
shown to have beneficial effects in diabetes treatment. As they each show different benefits we
investigated whether the combination of both substances shows an improvement in the treatment of
diabetes in Goto-Kakizaki (GK) rats. Over the course of 4 wk, the rats were fed with the following four test
diets (n = 12 per group): 1. Standard carbohydrate-rich laboratory diet (chow), 2. chow + SVS (0.03 g/kg
BW/day), 3. 80% SPI + 20% chow and 4. 80% SPI + 20 % chow + SVS (0.03 g/kg BW/day). At the end of
the course conscious rats underwent an intra-arterial glucose tolerance test (IAGTT) (2.0 g glucose/kg
BW). Compared to normal chow diet, stevioside in combination with SPI shows the following beneficial
effects in GK rats with mild type 2 diabetes: 1. a 56% reduction in plasma glucose (p < 0.001), 2. a 118%
increase in first-phase insulin (p < 0.005), 3. a 20% reduction in glucagons (p < 0.05), 4. a 28% reduction
in total cholesterol (p < 0.001), 5. a 13% reduction in FFA (p < 0.01), 6. a 49% reduction in TG (p < 0.001)
and 7. a 11% reduction in the systolic blood pressure (p < 0.001). In conclusion, the combination of
stevioside and SPI has synergistic positive effects on the characteristic features of the metabolic
syndrome, i.e. hyperglycemia, hypertension and dyslipidemia.
Jeppesen PB, Barriocanal L, Meyer MT, Palacios M, Canete F, Benitez S, Logwin S, Schupmann Y,
Benitez G, Jimenez JT. Efficacy and tolerability of oral stevioside in patients with type 2 diabetes: a
long-term, randomized, doubleblinded, placebo-controlled study. Diabetologia;49(Suppl. 1): 511–
512 (Abstract No. 0843). 2006.
JECFA (Joint FAO/WHO Expert Committee on Food Additives), 2000. Evaluation of Certain Food
Additives. Fifty-first Report of the Joint FAO/WHO Expert Committee on Food Additives. Geneva,
Switzerland. WHO Technical Report Series, No. 891, 35-37.
JECFA (Joint WHO/FAO Report). Diet Nutrition and the Prevention of Chronic Diseases. Disease
Specific Recommendations. World Health Organization 2003. Accessed at
http://www.who.int/hpr/NPH/docs/who_fao_expert_report.pdf. May 27, 2010.
page 27 of 64
JECFA (Joint FAO/WHO Expert Committee on Food Additives), 2005. Evaluation of Certain Food
Additives. Sixty-third Report of the Joint FAO/WHO Expert Committee on Food Additives, Geneva,
Switz. WHO Technical Report Series, No. 928, 34-39 and 138.
JECFA, 2005. Steviol glycosides. In: 63rd Meeting of the Joint FAO/WHO Expert Committee on
Food Additives. World Health Organization (WHO), Geneva, Switzerland, WHO Technical Report
Series 928, pp. 34-39
JECFA (Joint FAO/WHO Expert Committee on Food Additives), 2006. Safety evaluation of certain
food additives. Prepared by the 63rd meeting of the Joint FAO/WHO Expert Committee on Food
Additives. WHO Food Additives Series, No. 54, 117-144.
JECFA (Joint FAO/WHO Expert Committee on Food Additives), 2007. Evaluation of Certain Food
Additives and Contaminants. Sixty-eighth Report of the Joint FAO/WHO Expert Committee on
Food Additives. World Health Organization (WHO), Geneva, Switzerland. WHO techical report
series No. 947, 50-54.
JECFA (Joint FAO/WHO Expert Committee on Food Additives), 2008. Compendium of Food
Additive Specifications. Monograph 5. Steviol glycosides. Available at:
http://www.fao.org/ag/agn/jecfa-additives/details.html?id=898
JECFA (Joint FAO/WHO Expert Committee on Food Additives), 2009. Safety evaluation of certain
food additives. Prepared by the 69th meeting of the Joint FAO/WHO Expert Committee on Food
Additives. WHO Food Additives Series, No. 60, 183-220.
Joint WHO/FAO Report. Diet Nutrition and the Prevention of Chronic Diseases. Disease Specific
Recommendations. World Health Organization 2003. Accessed at
http://www.who.int/hpr/NPH/docs/who_fao_expert_report.pdf. May 27, 2010.
Joint FAO/WHO Expert Committee on Food Additives. Evaluation of certain food additives.World
Health Organ Tech Rep Ser. (952):1-208, 2009. This report represents the conclusions of a Joint
FAO/WHO Expert Committee convened to evaluate the safety of various food additives, including
flavouring agents, with a view to recommending acceptable daily intakes (ADIs) and to preparing
specifications for identity and purity. The first part of the report contains a general discussion of the
principles governing the toxicological evaluation and assessment of intake of food additives (in particular,
flavouring agents). A summary follows of the Committee's evaluations of technical, toxicological and
intake data for certain food additives (asparaginase from Aspergillus niger expressed in A. niger, calcium
lignosulfonate (40-65), ethyl lauroyl arginate, paprika extract, phospholipase C expressed in Pichia
pastoris, phytosterols, phytostanols and their esters, polydimethylsiloxane, steviol glycosides and sulfites
[assessment of dietary exposure]) and 10 groups of related flavouring agents (aliphatic branched-chain
saturated and unsaturated alcohols, aldehydes, acids and related esters; aliphatic linear alpha,betaunsaturated aldehydes, acids and related alcohols, acetals and esters; aliphatic secondary alcohols,
ketones and related esters; alkoxy-substituted allylbenzenes present in foods and essential oils and used
as flavouring agents; esters of aliphatic acyclic primary alcohols with aliphatic linear saturated carboxylic
acids; furan-substituted aliphatic hydrocarbons, alcohols, aldehydes, ketones, carboxylic acids and
related esters, sulfides, disulfides and ethers; miscellaneous nitrogen-containing substances; monocyclic
and bicyclic secondary alcohols, ketones and related esters; hydroxy- and alkoxy-substituted
benzyl derivatives; and substances structurally related to menthol). Specifications for the following food
additives were revised: canthaxanthin; carob bean gum and carob bean gum (clarified); chlorophyllin
copper complexes, sodium and potassium salts; Fast Green FCF; guar gum and guar gum (clarified);
iron oxides; isomalt; monomagnesium phosphate; Patent Blue V; Sunset Yellow FCF; and
trisodium diphosphate. Re-evaluation of flavouring agents for which estimated intake was based on
anticipated poundage data was carried out for 2-isopropyl- N,2,3-trimethylbutyramide (No. 1595) and Lmonomenthyl glutarate (No. 1414). Annexed to the report are tables summarizing the Committee's
recommendations for intakes and toxicological evaluations of the food additives considered.
page 28 of 64
Jooken E, Amery R, Struyf T, Duquenne B, Geuns J, Meesschaert B. Stability of steviol glycosides
in several food matrices. J Agric Food Chem. 60(42):10606-12, 2012. As steviol glycosides are now
allowed as a food additive in the European market, it is important to assess the stability of these steviol
glycosides after they have been added to different food matrices. We analyzed and tested the stability of
steviol glycosides in semiskimmed milk, soy drink, fermented milk drink, ice cream, full-fat and skimmed
set yogurt, dry biscuits, and jam. The fat was removed by centrifugation from the dairy and soy drink
samples. Proteins were precipitated by the addition of acetonitrile and also removed by centrifugation.
Samples of jam were extracted with water. Dry biscuits were extracted with ethanol. The resulting
samples were concentrated with solid-phase extraction and analyzed by high-performance liquid
chromatography on a C18 stationary phase and a gradient of acetonitrile/aqueous 25 mM phosphoric
acid. The accuracy was checked using a standard addition on some samples. For assessing the stability
of the steviol glycosides, samples were stored in conditions relevant to each food matrix and analyzed
periodically. The results indicate that steviol glycosides can be analyzed with good precision and
accuracy in these food categories. The recovery was between 96 and 103%. The method was also
validated by standard addition, which showed excellent agreement with the external calibration curve. No
sign of decomposition of steviol glycosides was found in any of the samples.
JORF (Journal Officiel de la République Française), Ministère de l’Èconomie, de l’Industrie et de
l’Emploi. Arrêté du 26 août 2009 relatif à l’emploi du rébaudioside A (extrait de Stevia rebaudiana)
comme additif alimentaire. 6 septembre 2009, texte 6 sur 35. 2009
Jutabha P, Toskulkao C, Chatsudthipong V. Effect of stevioside on PAH transport by isolated
perfused rabbit renal proximal tubule. Can J Physiol Pharmacol. 78(9):737-44, 2000. Stevioside, a
non-caloric sweetening agent, is used as a sugar substitute. An influence of stevioside on renal function
has been suggested, but little is known about its effect on tubular function. Therefore, the present study
was designed to explore the direct effect of stevioside on transepithelial transport of p-aminohippurate
(PAH) in isolated S2 segments of rabbit proximal renal tubules using in vitro microperfusion. Addition of
stevioside at a concentration of 0.45 mM to either the tubular lumen, bathing medium, or both at the
same time had no effect on transepithelial transport of PAH. Similarly, a concentration of 0.70 mM
(maximum solubility in the buffer) when present in the lumen, had no effect on PAH transport. However,
this concentration in the bathing medium inhibited PAH transport significantly by about 25-35%. The
inhibitory effect of stevioside was gradually abolished after it was removed from the bath. Addition of 0.70
mM stevioside to both lumen and bathing medium at the same time produced no added inhibitory effect.
Stevioside at this concentration has no effect on Na+/K+-ATPase activity as well as cell ATP content.
These findings suggest that stevioside, at a pharmacological concentration of 0.70 mM, inhibits
transepithelial transport of PAH by interfering with the basolateral entry step, the rate-limiting step for
transepithelial transport. The lack of effect of stevioside on transepithelial transport of PAH on the luminal
side and its reversible inhibitory effect on the basolateral side indicate that stevioside does not
permanently change PAH transport and should not harm renal tubular function at normal human intake
levels.
Kaneda N, Kasai R, Yamasaki K, Tanaka O. Chemical studies on sweet diterpene-glycosides of
Stevia rebaudiana: Conversion of stevioside into rebaudioside-A. Chem Pharm Bull;25:2466. 1977.
Kasai R, Yamaguchi H, Tanaka O. High-performance liquid chromatography of glycosides on a
new type of hydroxyapatite column. J Chromatogr;407(1):205-10. 1987. High-performance liquid
chromatography on a column of newly developed hard spherical hydroxyapatite was applied to the
separation of a variety of glycosides of tri- and diterpenes including Ginseng saponins and Stevia sweet
glycosides, affording satisfactory results by using a simple solvent system, aqueous acetonitrile. This
normal phase chromatography is superior to chromatography on a silica gel column for the separation of
water-soluble glycosides.
Kato I. Utilization and safety of stevioside. Shokuhin Kogyo;18(20):44-9. 1975.
page 29 of 64
Kawamori T, Tanaka T, Hara A, Yamahara J, Mori H. Modifying effects of naturally occurring
products on the development of colonic aberrant crypt foci induced by azoxymethane in F344
rats. Cancer Research;55:1277-82. 1995. Modifying effects of dietary exposure of seven naturally
occurring products on the development of colonic aberrant crypt foci (ACF) induced by azoxymethane
(AOM) were investigated in male F344 rats. The effects of these compounds on proliferation biomarkers
such as the number of silver-stained nucleolar organizer region protein, ornithine decarboxylase activity,
and polyamine concentration in the colon were also estimated. The naturally occurring products tested
included four terpenoids (rebaudioside A, oleanolic acid, costunolide, and soyasaponin A2), one flavonoid
(liquiritin), and two isocoumarins (phyllodulcin and hydrangenol). Animals were given 3 weekly s.c.
injections of AOM (15 mg/kg body weight) to induce ACF. These rats were fed the diet containing 200
ppm of each test chemical for 5 weeks, starting 1 week before the first dosing of AOM. All rats were
sacrificed 2 weeks after the last AOM injection to estimate their modulatory effects on the occurrence of
ACF and the cell proliferation biomarkers in the colon. In groups of rats given AOM and hydrangenol,
oleanolic acid, or costunolide, the frequencies of ACF/colon were significantly lower than that of AOM
alone (P < 0.05, P < 0.005, and P < 0.05, respectively). In groups of rats given AOM and costunolide and
those treated with AOM and soyasaponin A2, both ornithine decarboxylase activity and polyamine
concentration of the colonic mucosal tissue were significantly decreased compared with those in rats
given AOM alone (P < 0.05 and P < 0.001 for costunolide and P < 0.001 and P < 0.05 for soyasaponin
A2, respectively). In groups of rats given AOM and liquiritin, oleanolic acid, or costunolide, the numbers of
silver-stained nucleolar organizer regions/nucleus were significantly lower than that of AOM alone (P <
0.05, P < 0.01, and P < 0.05, respectively). Costunolide decreased four AOM-induced biomarkers, such
as the frequencies of ACF/colon, ornithine decarboxylase activity, polyamine concentration level, and
silver-stained nucleolar organizer region number in the colon. These results indicate that, among the test
chemicals, costunolide has blocking effects against rat colon carcinogenesis and is a possible
chemopreventive agent against colon tumorigenesis. Also, the short-term model described here could be
a very useful prescreening tool for chemopreventive agents against colon cancer.
Kawatani T, Kaneki Y, Tanabe T, Takahashi T. On the cultivation of kaa-he-e (Stevia rebaudiana
Bertoni). VI. Response of kaa-he-e to potassium fertilization rates and to the three major elements
of fertilizer. Nettai Nogyo;24(3):105-12. 1980.
Kelmer-Bracht AM, Alvarez M, Bracht A. Effects of Stevia rebaudiana natural products on rat liver
mitochondria. Biochemical Pharmacology; 34: 873-882. 1985. The effects of several natural products
extracted from the leaves of Stevia rebaudiana on rat liver mitochondria were investigated. The
compounds used were stevioside (a non-caloric sweetener), steviolbioside, isosteviol and steviol. Total
aqueous extracts of the leaves were also investigated. S. rebaudiana natural products inhibited oxidative
phosphorylation, ATPase activity NADH-oxidase activity, succinate-oxidase activity, succinate
dehydrogenase, and L-glutamate dehydrogenase. The ADP/O ratio was decreased. Substrate respiration
(state II respiration) was increased at low concentrations (up to 0.5 mM) and inhibited at higher
concentrations (1 mM or more). In uncoupled mitochondria, inhibition of substrate respiration was the only
effect observed. Net proton ejection induced by succinate and swelling induced by several substrates
were inhibited. Of the compounds investigated, the sweet principle stevioside was less active. It was
concluded that, in addition to the inhibitory effects, S. rebaudiana natural products may also act as
uncouplers of oxidative phosphorylation. The possible physiologic consequences of the ingestion of
stevioside and S. rebaudiana aqueous extracts are discussed.
Kerr WE, Mello ML, Bonadio E. Mutagenicity tests on the stevioside from Stevia rebaudiana (Bert.)
Bertoni. Brazilian Journal of Genetics; 1:173-76. 1983.
Khramov VA, Dmitrienko NV. Chlorogenic acid in leaves and lyophilized extracts of Stevia. Pharm
Chem J: 34(11):605-06. 2000.
Kim HS, Lee HJ. Acceptability of stevioside as a natural sweetener. Han'guk Sikp'um Kwahakhoe
Chi;11(1):56-62. 1979.
Kimata H. Anaphylaxis by stevioside in infants with atopic eczema. Allergy;62:565-66. 2007.
page 30 of 64
Kinghorn D. Ed. Stevia: The Genus Stevia. London, England/New York (NY): Taylor and Francis,
2002.
Kinghorn AD, Soejarto DD, Nanayakkara NP, Compadre CM, Makapugay HC, Hovanec-Brown JM,
Medon PJ, Kamath SK. Potential sweetening agents of plant origin. Part IV. A phytochemical
screening procedure for sweet ent-kaurene glycosides in the genus Stevia. J Nat Prod; 47(3):43944 1984.
Altogether, 110 species of the genus Stevia, comprising both herbarium and fresh leaf samples, were
screened for the presence of sweet ent-kaurene glycosides, using a combination of tlc and hplc, followed
by gc/ms. Stevioside and rebaudiosides A and C were detected in a Stevia rebaudiana herbarium
specimen collected in Paraguay in 1919, and stevioside was observed as a constituent of a Stevia
phlebophylla herbarium specimen collected in Mexico in 1889. Steviol glycosides were not detected in
any of the other 108 Stevia species studied. The phytochemical results obtained in this study are
correlated with those of preliminary organoleptic tests on the sweetness of these Stevia samples, and the
chemotaxonomic implications of the present findings are discussed.
Kinghorn AD, Compadre CM. Naturally occurring intense sweeteners. Pharm Int;6(8): 201-204.
1985.
Kinghorn AD, Soejarto DD. Stevioside. Food Sci Technol; 48(2)ED:157-71. 1991.
Kinghorn AD, Kaneda N, Baek NI, Kennelly EJ, Soejarto DD. Noncariogenic intense natural
sweeteners. Medicinal Research Reviews;18:347-60. 1998. There is a definite relationship between
the dietary consumption of sucrose and the incidence of dental caries. Noncaloric sucrose substitutes for
use in the sweetening of foods, beverages, and medicines may be either synthetic compounds or natural
products. In the United States, four potently sweet artificial sweeteners are approved, namely, saccharin,
aspartame, acesulfame potassium, and sucralose. Highly sweet plant constituents are used in Japan and
some other countries, including the diterpene glycoside stevioside and the protein thaumatin. Recent
progress in a research project oriented towards the discovery and evaluation of novel potentially
noncariogenic sweeteners from plants has focused on substances in the sesquiterpenoid, diterpenoid,
triterpenoid, steroidal saponin, and proanthocyanidin structural classes. The feasibility of using Mongolian
gerbil electrophysiological and behavioral assays to monitor the sweetness of plant extracts,
chromatographic fractions, and pure isolates has been investigated. An in vivo cariogenicity study on the
commercially available natural sweeteners stevioside and rebaudioside A has been carried out.
Klages A. Stevia rebaudiana, a Paraguayian sweet-tasting plant. Pharm Zentralhalle Dtschl;90:257.
1951.
Klongpanichpak S, Temcharoen P, Toskulkao C, Apibal S, Glinsukon T. Lack of mutagenicity of
stevioside and steviol in Salmonella typhimurium TA 98 and TA 100. Journal of the Medical
Association of Thailand; 80(1),S121-S128. 1997. Stevioside, a sweet-tasting diterpene glycoside
derived from Stevia rebaudiana, and steviol, a product from enzymatic hydrolysis of stevioside, were
tested for mutagenic activity by the in vitro Ames test, a preincubation method, using Salmonella
typhimurium TA 98 and TA 100 as the tester strains, either in the presence or absence of metabolic
activating system derived from the sodium phenobarbital and 5,6-benzoflavone pretreated liver S9
fractions from various animal species including rat, mouse, hamster and guinea pig. Stevioside and
steviol at the concentrations up to 50 mg and 2 mg per plate, respectively showed no mutagenic effect on
both tester strains either in the presence or absence of metabolic activating system. However, at the high
concentration both stevioside and steviol showed some toxic effects on both tester strains. The toxic
effect was decreased in the presence of the metabolic activating system.
Kobayashi M, Horikawa S, Degrandi IH, Ueno J, Mitsuhashi H. Dulcosides A and B, new diterpene
glycosides from Stevia rebaudiana. Phytochemistry 1977;16:1405-08. 1977.
KoBert R. Two sweet tasting drugs. Ber Pharm Dtsch Ges;25:162. 1915.
page 31 of 64
Kohda H, Kasai R, Yamasaki K, Murakami K, Tanako O. New sweet diterpene glucosides from
Stevia rebaudiana. Phytochemistry;15:981-83. 1976.
Korir MW, Wachira FN, Wanyoko JK, Ngure RM, Khalid R.The fortification of tea with sweeteners
and milk and its effect on in vitro antioxidant potential of tea product and glutathione levels in an
animal model.Food Chemistry. 145:145-53, 2014. Several studies have demonstrated that tea
flavonoids protect cells and tissues against free radicals which have been implicated in the etiology of
oxidative stress-related disease disorders. However, black tea is commonly consumed with additives that
could otherwise affect the bioavailability of the active tea molecules. In this study, the biochemical
parameters of Kenyan teas were determined and the effect of added milk and sweeteners on the
antioxidant activity of Kenyan teas was investigated. The effect of tea antioxidants on glutathione (GSH)
was also evaluated in vivo in a time series study using Swiss mice. Green teas had the highest levels of
total polyphenols, total and individual catechins, while black teas had high levels of total thearubigins,
total theaflavins and theaflavin fractions. The antioxidant activity was high in green teas though some of
the black teas were as efficacious as the green teas. The addition of milk, sugar and honey significantly
(p<0.05) decreased the antioxidant activity of tea in a concentration-dependent manner. Addition of the
sweetener, stevia (Stevia rebaudiana Bertoni), showed no significant (p>0.05) influence on the
antioxidant activity of tea and therefore can be recommended as a preferred sweetener for tea.
Significantly (p<0.001) higher levels of GSH were observed in plasma than in other tissues. GSH levels
were generally highest 2 h after tea consumption, which indicates the need to repeatedly take tea every 2
h to maximise its potential health benefits.
Kolb N, Herrera JL, Ferreyra DJ, Uliana RF. Analysis of sweet diterpene glycosides from Stevia
rebaudiana: Improved HPLC method. J Agr Food Chem; 49(10):4538-41. 2001. An improved
analytical method was developed which may be applied to quality control of stevioside and rebaudioside
A contents in dried leaves of Stevia rebaudiana before processing; in a selective sampling program
searching for plants of higher yield in diterpene glycosides content; or when a large number of samples
are sent to the laboratory for analysis. The procedure developed involves two steps: solvent extraction
followed by an isocratic HPLC analysis. The sample, 1 g of dried leaves of S. rebaudiana, is ground and
solvent-extracted with EtOH 70% (w/w) in Erlenmeyer flasks by shaking for 30 min in a 70 degrees C
water bath. After the extract was cooled, it was filtered and analyzed by HPLC using an NH(2) column
(250 x 4.6 mm) and a mixture of acetonitrile/water (80:20, v/v) as mobile phase, pH 5 adjusted with acetic
acid. The detection was in the UV range at 210 nm (0.04 AUFS). Quantitation was performed by means
of an external standard calibration curve for each analyte which had been obtained from standard
solutions of pure stevioside and rebaudioside A. Working under these conditions there were no observed
interference effects. The method saves time in sample preparation, and reduces sample handling and
chromatographic analysis time, while having little loss of precision [coefficient of variation (CV%) between
1.8% and 3.0%] and recovery [between 98.5% and 100.5%]. The method was applied to 30 samples of
S. rebaudiana from Misiones (Northeastern Argentina), and the stevioside content found ranged between
3.78 and 9.75% (weight) whereas Rebaudioside A content ranged between 1.62 and 7.27% (weight).
Komai K, Iwamura J. Effects of stevioside and its related compounds on growth of rice and lettuce
seedlings. Nippon Noyaku Gakkaishi;8(4):445-50. 1983.
Komissarenko NF, Putieva ZM, Saatov Z. Flavonoids of the leaves of Stevia rebaudiana. Chem Nat
Comp 33 4:494-495 (1997) Rast Resur;30 1/2:53-64. 1994.
Konoshima T and Takasaki M. Cancer-chemopreventive effects of natural sweeteners and related
compounds. Pure and Applied Chemistry;74:1309-16. 2002.
Koyama E, Kitazawa K, Ohori Y, Izawa O, Kakegawa K, Fujino A, Ui M. In vitro metabolism of the
glycosidic sweeteners, stevia mixture and enzymatically modified stevia in human intestinal
microflora. Food Chem Toxicol. 41(3):359-74, 2003. Stevia mixture, sweeteners extracted from the
leaves of Stevia rebaudiana Bertoni, consists mainly of stevioside and rebaudioside A (glycosides of the
diterpene derivative steviol). The aim of this study was to investigate human intestinal metabolism of
page 32 of 64
stevia mixture and its alpha-glucose derivative (known in Japan as enzymatically modified stevia) by
LC/MS/ESI analysis. Degradation was examined by incubating stevia mixture, enzymatically modified
stevia, stevioside, rebaudioside A, alpha-monoglucosylstevioside, alpha-monoglucosylrebaudioside A
and the aglycone, steviol with pooled human faecal homogenates (obtained from five healthy volunteers)
for 0, 8 and 24 h under anaerobic conditions. Stevia mixture, enzymatically modified stevia, stevioside
and rebaudioside A (0.2 mg/ml) were completely eliminated within 24 h, whereas no degradation of
steviol (0.08 and 0.2 mg/ml) appeared to be found during the incubation period. Stevia mixture, stevioside
and rebaudioside A appeared to be hydrolyzed to steviol by human intestinal microflora: this observation
is consistent with previous rat metabolism studies. Similarly, enzymatically modified stevia appeared to be
metabolized via stevia components and, finally, to steviol. This study suggests that there are apparently
no species differences in intestinal metabolism of stevia mixture between rats and humans.
Koyama E, Sakai N, Ohori Y, Kitazawa K, Izawa O, Kakegawa K, Fujino A, Ui M. Absorption and
metabolism of glycosidic sweeteners of stevia mixture and their aglycone, steviol, in rats and
humans. Food Chem Toxicol. 41(6):875-83, 2003. Stevia mixture, sweeteners extracted from the
leaves of Stevia rebaudiana Bertoni, consists mainly of the glycosides of the diterpene derivative steviol.
The aims of this study were to investigate the absorption (in rats) and the hepatic metabolism (in rats and
humans) of both stevia mixture and steviol. Absorption was investigated both in vivo and ex vivo. In ex
vivo experiments using the rat everted sac method, no absorption of stevia mixture was observed, but
significant absorption of steviol was noted (equivalent to approximately 70% of the absorption referencesalicylic acid- value). In the in vivo experiment, rats received a single oral administration of either steviol
or stevia mixture; a peak steviol concentration in plasma was observed 15 min after its oral
administration, demonstrating rapid absorption. However, after oral administration of stevia mixture, the
steviol concentration in plasma increased steadily over 8 h, suggesting that stevia mixture components
are first degraded and then absorbed as steviol in the rat intestine. Steviol metabolism in humans and
rats was examined by incubating steviol with liver microsomes from the two species. Oxidative
(monohydroxy and dihydroxy) metabolites of steviol were observed by LC-ESI/MS after incubation with
both human and rat liver microsomes. The intrinsic clearance of steviol in human liver microsomes was
4-times lower than that found in rat liver microsomes. In conclusion, this study suggests that there are no
major species differences in steviol hepatic metabolism between rats and humans. Absorption from the
human intestine can be predicted to occur in an analogous manner to that from the rat intestine.
Kraemer T, Maurer HH. On the metabolism of the sweetener stevioside in humans. European
Journal of Pharmaceutical Sciences;2:103 [Abstract No. FC12]. 1994.
Kroyer, GTh, 1999. The low calories sweetener stevioside: stability and interaction with food
ingredients. Lebensmittel, Wissenschaft und Technologie;32:509-512. 1999.
Lailerd N, Saengsirisuwan V, Sloniger JA, Toskulkao C, Henriksen EJ. Effects of stevioside on
glucose transport activity in insulin-sensitive and insulin-resistant rat skeletal muscle.Metabolism.
53(1):101-7, 2004. Stevioside (SVS), a natural sweetener extracted from Stevia rebaudiana, has been
used as an antihyperglycemic agent. However, little is known regarding its potential action on skeletal
muscle, the major site of glucose disposal. Therefore, the purpose of the present study was to determine
the effect of SVS treatment on skeletal muscle glucose transport activity in both insulin-sensitive lean
(Fa/-) and insulin-resistant obese (fa/fa) Zucker rats. SVS was administered (500 mg/kg body weight by
gavage) 2 hours before an oral glucose tolerance test (OGTT). Whereas the glucose incremental area
under the curve (IAUC(glucose)) was not affected by SVS in lean Zucker rats, the insulin incremental
area under the curve (IAUC(insulin)) and the glucose-insulin index (product of glucose and insulin IAUCs
and inversely related to whole-body insulin sensitivity) were decreased (P<.05) by 42% and 45%,
respectively. Interestingly, in the obese Zucker rat, SVS also reduced the IAUC(insulin) by 44%, and
significantly decreased the IAUC(glucose) (30%) and the glucose-insulin index (57%). Muscleglucose
transport was assessed following in vitro SVS treatment. In lean Zucker rats, basal glucose transport in
type I soleus and type IIb epitrochlearis muscles was not altered by 0.01 to 0.1 mmol/L SVS. In contrast,
0.1 mmol/L SVS enhanced insulin-stimulated (2 mU/mL) glucose transport in both epitrochlearis (15%)
and soleus (48%). At 0.5 mmol/L or higher, the SVS effect was reversed. Similarly, basal glucose
transport in soleus and epitrochlearis muscles in obese Zucker rats was not changed by lower doses of
page 33 of 64
SVS (0.01 to 0.1 mmol/L). However, these lower doses of SVS significantly increased insulin-stimulated
glucose transport in both obese epitrochlearis and soleus (15% to 20%). In conclusion, acute oral SVS
increased whole-body insulin sensitivity, and low concentrations of SVS (0.01 to 0.1 mmol/L) modestly
improved in vitro insulin action on skeletal muscle glucose transport in both lean and obese Zucker rats.
These results indicate that one potential site of action of SVS is the skeletal muscle glucose transport
system.
Lee CN, Wong KL, Liu JC, Chen YJ, Cheng JT, Chan P. Inhibitory effect of stevioside on calcium
influx to produce antihypertension. Planta Med. 67(9):796-9, 2001. Stevioside is a sweet-tasting
glycoside occurring abundantly in the leaves ofStevia rebaudiana (Compositae). It has been used
popularly in Japan and Brazil as a sugar substitute for decades. Previous study has shown that it lowered
blood pressure in spontaneously hypertensive rats (SHRs) when administered intravenously. This study
shows that intraperitoneal injection of stevioside 25 mg/kg also has antihypertensive effect in SHRs. In
isolated aortic rings from normal rats, stevioside could dose-dependently relax the vasopressin-induced
vasoconstriction in both the presence and absence of endothelium. However, stevioside had no effect on
phenylephrine- and KCl-induced phasic vasoconstriction. In addition, stevioside lost its influence on
vasopressin-induced vasoconstriction in Ca(2+)-free medium. The results indicate that stevioside caused
vasorelaxation via an inhibition of Ca(2+) influx into the blood vessel. This phenomenon was further
confirmed in cultured aortic smooth muscle cells (A7r5). Using 10(-5) M methylene blue for 15 min,
stevioside could still relax 10(-8) M vasopressin-induced vasoconstriction in isolated rat aortic rings,
showing that this vasorelaxation effect was not related to nitric oxide. The present data show that the
vasorelexation effect of stevioside was mediated mainly through Ca(2+) influx inhibition.
Lee KR, Park JR, Choi BS, Han JS, Oh SL, Yamada Y, Kim KS, Tchai BS. A study on the safety of
stevioside as a new sweetening source. Hanguk Sikpum Kwahakhoe Chi;11:224-31. 1979.
Levy NM, Bracht A and Kelmer-Bracht AM. Effects of Stevia rebaudiana natural products on the
mitochondrial L-glutamate dehydrogenase. Brazilian Archives of Biology and Technology;37:67380. 1994.
Liu JC, Kao PK, Chan P, Hsu YH, Hou CC, Lien GS, Hsieh MH, Chen YJ, Cheng JT. Mechanism of
the antihypertensive effect of stevioside in anesthetized dogs. Pharmacology. 67(1):14-20, 2003.
Stevioside is a sweet-tasting glycoside isolated from the leaves of Stevia rebaudiana. It has been used as
a noncaloric sugar substitute in Japan and Brazil for decades. Previous studies have shown that it
lowered blood pressure in spontaneously hypertensive rats by intravenous injection. This study was
designed to evaluate the hypotensive effect of stevioside in dogs and to define the underlying
mechanism. After nasogastric administration of stevioside powder (200 mg/kg), the blood pressure of
healthy mongrel dogs began to significantly decrease at 60 min and returned to baseline level at 180 min.
The reduction of blood pressure was more rapid (at 5-10 min) and effective after intravenous injection.
However, no significant change of blood pressure was noted after injection through left vertebral artery,
implicating that the hypotensive effect is not related to the central nervous system. Stevioside also
showed significant hypotensive effects in renal hypertensive dogs, in a dose-dependent manner. In
cultured rat aortic smooth muscle cells (A7r5 cell line), stevioside can dose-dependently inhibit the
stimulatory effects of vasopressin and phenylephrine on intracellular Ca(2+) in a calcium-containing
medium. However, no intracellular Ca(2+) inhibitory effect was observed in calcium-free medium,
implicating that stevioside may inhibit the Ca(2+) influx from extracellular fluid. Our present data show that
stevioside did not influence the calcium ionophore (A23187) induced Ca(2+) influx, indicating that the
antagonistic effect was through Ca(2+) channels. This study confirmed that stevioside is an effective
antihypertensive natural product, and its hypotensive mechanism may be probably due to inhibition of the
Ca(2+) influx.
Li j, Jiang H, Shi R., A new acylated quercetin glycoside from the leaves of Stevia rebaudiana
Bertoni. Nat Prod Res;23(15):1378-83. 2009. A new acylated quercetin glycoside quercetin-3-O-(4'''-Otrans-caffeoyl)-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-gala copyranoside (1), along with luteolin (2),
quercetin (3), luteolin-7-O-beta-D-glucoside (4), apigenin-7-O-beta-D-glucoside (5), quercitrin (6),
quercetin-3-O-beta-D-arabinoside (7) and 4,5-di-O-caffeoyl quinic acid (8) have been isolated from the
page 34 of 64
leaves of Stevia rebaudiana Bertoni. The structures of these compounds were determined by
spectroscopic methods (1H- and 13C-NMR, IR and MS) and by 2D-NMR experiments.
Ma J, Ma Z, Wang J, Milne RW, Xu D, Davey AK, Evans AM. Isosteviol reduces plasma glucose
levels in the intravenous glucose tolerance test in Zucker diabetic fatty rats. Diabetes, Obesity and
Metabolism;9:597-99. 2007. AIM: The aim of this study was to test the effect of isosteviol on blood
glucose and insulin levels during the intravenous glucose tolerance test (IVGTT) in Wistar and Zucker
diabetic fatty (ZDF) rats. METHODS: ZDF rats were divided into a control and three isosteviol treatment
(1, 5 and 10 mg/kg) groups. Wistar rats were divided into a control group and an isosteviol treatment
group (10 mg/kg). The rats were fasted for 12 h prior to infusion of isosteviol and glucose (1.0 g/kg).
Blood samples were taken at 0, 5, 15, 30, 60, 90 and 120 min after the injection of glucose. Glucose
concentrations were determined by the glucose oxidase method, and plasma insulin was analysed by
radioimmunoassay. The area under the curve (AUC) of the net change in plasma glucose concentration
was used to compare the isosteviol treatment and control groups. RESULTS: In ZDF rats, isosteviol at 5
and 10 mg/kg caused a significant (p <0.05) reduction in the AUC of glucose during the IVGTT. However,
isosteviol did not increase plasma insulin concentrations in ZDF rats. In Wistar rats,isosteviol did not
significantly affect plasma glucose or insulin levels during the IVGTT.CONCLUSION: Isosteviol exerts an
antihyperglycaemic effect during IVGTT in ZDF rats but not in Wistar rats. Isosteviol has no significant
effect on plasma insulin concentrations. The glucose-lowering effect of isosteviol may be due to changes
in the sensitivity of peripheral tissues to insulin.
Maier, V, Mermann K, Kienle U. Isolated perfused gut absorption of glucose is inhibited by steviol,
a degradation product of the sweetener stevioside. Diabetes;52(1):A554 (Abstract No. 2401-PO).
2003.
Maki KC, Curry LL, Carakostas MC, Tarka SM, Reeves MS, Farmer MV, McKenney JM, Toth PD,
Schwartz SL, Lubin BC, Boileau AC, Dicklin MR, Carakostas MC, Tarka SM. Chronic consumption
of rebaudioside A, a steviol glycoside, in men and women with type 2 diabetes mellitus. Food
Chem Toxicol. 46 Suppl 7:S47-53, 2008. This trial evaluated the effects of 16 weeks of consumption of
1000mg rebaudioside A (n=60) a steviol glycoside with potential use as a sweetener, compared to
placebo (n=62) in men and women (33-75 years of age) with type 2 diabetes mellitus. Mean+/-standard
error changes in glycosylated hemoglobin levels did not differ significantly between the rebaudioside A
(0.11+/-0.06%) and placebo (0.09+/-0.05%; p=0.355) groups. Changes from baseline for rebaudioside A
and placebo, respectively, in fasting glucose (7.5+/-3.7mg/dL and 11.2+/-4.5mg/dL), insulin (1.0+/0.64microU/mL and 3.3+/-1.5microU/mL), and C-peptide (0.13+/-0.09ng/mL and 0.42+/-0.14ng/mL) did
not differ significantly (p>0.05 for all). Assessments of changes in blood pressure, body weight, and
fasting lipids indicated no differences by treatment. Rebaudioside A was well-tolerated, and records of
hypoglycemic episodes showed no excess vs. placebo. These results suggest that chronic use of
1000mg rebaudioside A does not alter glucose homeostasis or blood pressure in individuals with type 2
diabetes mellitus.
Maki KC, Curry LL, Carakostas MC, Tarka SM, Reeves MS, Farmer MV, McKenney JM, Toth PD,
Schwartz SL, Lubin BC, Dicklin MR, Boileau AC, Bisognano JD. The hemodynamic effects of
rebaudioside A in healthy adults with normal and low-normal blood pressure. Food Chem Toxicol.
46 Suppl 7:S40-6, 2008. Rebaudioside A and stevioside are steviol glycosides extracted from the plant
Stevia rebaudiana Bertoni and are used in several countries as food and beverage sweeteners. This
randomized, double-blind trial evaluated the hemodynamic effects of 4weeks consumption of 1000mg/day
rebaudioside A vs. placebo in 100 individuals with normal and low-normal systolic blood pressure (SBP)
and diastolic blood pressure (DBP). Subjects were predominantly female (76%, rebaudioside A and 82%,
placebo) with a mean age of approximately 41 (range 18-73) years. At baseline, mean resting, seated
SBP/DBP was 110.0/70.3mmHg and 110.7/71.2mmHg for the rebaudioside A and placebo groups,
respectively. Compared with placebo, rebaudioside A did not significantly alter resting, seated SBP, DBP,
mean arterial pressure (MAP), heart rate (HR) or 24-h ambulatory blood pressures responses. These
results indicate that consumption of as much as 1000mg/day of rebaudioside A produced no clinically
important changes in blood pressure in healthy adults with normal and low-normal blood pressure.
page 35 of 64
Maki KC, Curry LL, Reeves MS, Toth PD, McKenney JM, Farmer MV, Schwartz SL, Lubin BC, Melis
MS, Rocha ST, Augusto A. Steviol effect, a glycoside of Stevia rebaudiana, on glucose clearances
in rats. Braz J Biol. 69(2):371-4, 2009. Stevia rebaudiana, a South American plant normally used as a
natural herbal sweetener, has been suggested as exerting beneficial effects on human health, including
as an antihypertensive and antihyperglycemic. The present experiment was undertaken to evaluate the
renal excretion of steviol, the aglycone of several natural products extracted from the leaves of S.
rebaudiana, and to clarify the actual participation of this compound on the renal excretion of glucose in
rats, which has been previously suggested as the preferential action of steviol on the Na+-glucose renal
tubular transport system. Steviol was obtained by enzymatic hydrolysis of stevioside with pectinase. Thirty
normal male Wistar rats weighing 345 g were used. After a control period, steviol was infused iv at three
doses (0.5, 1.0 and 3.0 mg.kg-1/h), according to classical clearance techniques. During all the
experiments no significant changes in inulin clearance (Cin) and p-aminohipuric acid clearance (C PAH)
were observed. Administration of steviol resulted in a statistically significant increase in the fractional
sodium excretion (FeNa+), fractional potassium excretion (FeK+), urinary flow as percent of glomerular
filtration rate (V/GFR) and glucose clearance (C G) when compared to controls, but these effects were
absent with the dose of 0.5 mg.kg-1/h. The steviol clearance (C S) was higher than the Cin and lower
than the C PAH at all the doses employed in this study. The data suggest that steviol is secreted by renal
tubular epithelium, causing diuresis, natriuresis, kaliuresis and a fall in renal tubular reabsorption of
glucose.
Matsui M, Matsui K, Kawasaki Y, Oda Y, Noguchi T, Kitagawa Y, Sawada M, Hayashi M, Nohmi T,
Yoshihira K, Ishidate M, Sofuni T. Evaluation of the genotoxicity of stevioside and steviol using
six in vitro and one in vivo mutagenicity assays. Mutagenesis; 11:573-579. 1996. Stevioside, a
constituent of Stevia rebaudiana, is commonly used as a non-caloric sugar substitute in Japan. The
genetic toxicities of stevioside and its aglycone, steviol, were examined with seven mutagenicity tests
using bacteria (reverse mutation assay, forward mutation assay, umu test and rec assay), cultured
mammalian cells (chromosomal aberration test and gene mutation assay) and mice (micronucleus test).
Stevioside was not mutagenic in any of the assays examined. The aglycone, steviol, however, produced
dose-related positive responses in some mutagenicity tests, i.e. the forward mutation assay using
Salmonella typhimurium TM677, the chromosomal aberration test using Chinese hamster lung fibroblast
cell line (CHL) and the gene mutation assay using CHL. Metabolic activation systems containing 9000 g
supernatant fraction (S9) of liver homogenates prepared from polychlorinated biphenyl or phenobarbital
plus 5,6-benzoflavone-pretreated rats were required for mutagenesis and clastogenesis. Steviol was
weakly positive in the umu test using S.typhimurium TA1535/pSK1002 either with or without the metabolic
activation system. Steviol, even in the presence of the S9 activation system, was negative in other
assays, i.e. the reverse mutation assays using S.typhimurium TA97, TA98, TA100, TA102, TA104,
TA1535, TA1537 and Escherichia coli WP2 uvrA/pKM101 and the rec-assay using Bacillus subtilis.
Steviol was negative in the mouse micronucleus test. The genotoxic risk of steviol to humans is
discussed.
Matsukubo T, Takazoe I. Sucrose substitutes and their role in caries prevention. Int Dent J.
56(3):119-30, 2006.Many non- or low-cariogenic sucrose substitutes are currently available and are found
as ingredients of a variety of candy, chewing gum, and drinks. Recently the role of sugar alcohols in
promoting remineralisation of enamel has attracted much attention. Thus, the dental profession needs to
understand the general characteristics and features of sugar substitutes to provide advice on oral health
to patients as well as the general public. There are two critical requirements for sucrose substitutes,
namely, being nutritionally appropriate and not being detrimental to the overall general health of the
individual. The use of a greater variety of confectionary containing sucrose substitutes and the
development of new substitutes with high nutritional value are essential in the battle against caries. In this
paper we review in detail the characteristics of sucrose substitutes currently in use, their role in caries
prevention and promotion of oral health.
Mazzei-Planas G, Kuc J. Contraceptive properties of Stevia rebaudiana. Science; 162:1007. 1968.
Medon PJ, Pezzuto JM, Hovanec-Brown JM, Nanayakkara NP, Soejarto DD, Kamath SK, Kinghorn
AD. Safety assessment of some Stevia rebaudiana sweet principles. Federal Procedure; 41:1568.
1982.
page 36 of 64
Melis MS, Rocha ST, Augusto A. Steviol effect, a glycoside of Stevia rebaudiana, on glucose
clearances in rats. Braz J Biol. 69(2):371-4, 2009. Stevia rebaudiana, a South American plant normally
used as a natural herbal sweetener, has been suggested as exerting beneficial effects on human
health, including as an antihypertensive and antihyperglycemic. The present experiment was undertaken
to evaluate the renal excretion of steviol, the aglycone of several natural products extracted from the
leaves of S. rebaudiana, and to clarify the actual participation of this compound on the renal excretion
of glucose in rats, which has been previously suggested as the preferential action of steviol on the Na+glucose renal tubular transport system. Steviol was obtained by enzymatic hydrolysis of stevioside with
pectinase. Thirty normal male Wistar rats weighing 345 g were used. After a control period, steviol was
infused iv at three doses (0.5, 1.0 and 3.0 mg.kg-1/h), according to classical clearance techniques.
During all the experiments no significant changes in inulin clearance (Cin) and p-aminohipuric acid
clearance (C PAH) were observed. Administration of steviol resulted in a statistically significant increase
in the fractional sodium excretion (FeNa+), fractional potassium excretion (FeK+), urinary flow as
percent of glomerular filtration rate (V/GFR) and glucose clearance (C G) when compared to controls, but
these effects were absent with the dose of 0.5 mg.kg-1/h. The steviol clearance (C S) was higher than the
Cin and lower than the C PAH at all the doses employed in this study. The data suggest that steviol is
secreted by renal tubular epithelium, causing diuresis, natriuresis, kaliuresis and a fall in renal tubular
reabsorption of glucose.
Melis MS, Sainati AR. Participation of prostaglandins in the effect of stevioside on rat renal
function and arterial pressure. Brazilian Journal of Medical and Biological Research;24:1269-76.
1991. 1. The effect of stevioside on renal function was evaluated by clearance techniques in Wistar rats
simultaneously with the effect of indomethacin on the renal action of stevioside. The indomethacin
experiments consisted of four consecutive periods: (C) control; (S), in which stevioside (16 mg/kg)
was continuously infused; (S+I1) and (S+I2) in which indomethacin was infused systemically without
interrupting stevioside infusion. Mean arterial pressure (MAP) and renal function parameters were
measured. 2. Administration of stevioside resulted in a statistically significant dose-related decrease in
MAP (121 +/- 2.30, N = 7 for 4 mg/kg stevioside to 72 +/- 4.79 mmHg, N = 7 for 16 mg/kg stevioside) and
an increase in renal plasma flow (RPF) (10.27 +/- 1.21, N = 7 for 4 mg/kg stevioside to 26.28 +/- 2.87 ml
min-1 kg-1, N = 7 for 16 mg/kg stevioside), with no change in glomerular filtration rate (GFR). Stevioside
also increased fractional sodium (FeNa+) and potassium (FeK+) excretion as well as urine flow (V/GFR).
3. The decrease in MAP (control, 121 +/- 0.93, N = 7; stevioside, 91 +/- 2.48 mmHg) and increase in RPF
(control, 14.21 +/- 1.41, N = 7; stevioside, 32.53 +/- 2.84 mmHg) induced by stevioside (16 mg/kg)
were inhibited by simultaneous administration of indomethacin (2 mg/kg), but GFR was not affected. The
diuretic, natriuretic and kaliuretic effects of stevioside were also abolished by indomethacin. 4. We
conclude that stevioside behaves like a typical vasodilator substance, causing changes in MAP, diuresis,
natriuresis and kaliuresis per ml of GFR, and these effects probably depend on prostaglandins.
Melis MS, Sainati AR. Effect of calcium and verapamil on renal function of rats during treatment
with stevioside. Journal of Ethnopharmacology;33:257-62. 1991b. A study conducted on rats using
classical clearance techniques and arterial pressure measurements showed that stevioside from Stevia
rebaudiana leaves produced a fall in systemic blood pressure, as well as diuresis and natriuresis per
milliliter of glomerular filtration rate. Verapamil tended to increase the renal and systemic effects of
stevioside. In contrast, an infusion of CaCl2 in rats prepared with stevioside induced a marked attenuation
of the vasodilating responses of stevioside. These data are consistent with the possibility that stevioside
may act as a calcium antagonist, as is the case for verapamil.
Melis MS. Renal excretion of stevioside in rats. Journal of Natural Products; 55:688-90. 1992a. The
renal excretion of stevioside, a glycoside extracted from the leaves of Stevia rebaudiana, and its effect on
renal excretion of several substances, was studied through clearance techniques in Wistar rats. After a
control period, stevioside was infused iv at four concentrations (4, 8, 12, and 16 mg/kg). During all the
experiments no significant changes in inulin clearance (CIn) were observed. The stevioside infusion
induced a significant increase in the p-aminohippuric acid clearance (CPAH), fractional sodium excretion
(FeNa+), urinary flow as percent of glomerular filtration rate (V/GFR), and glucose clearance (CG) when
compared to controls, but these effects were absent with the dose of 4 mg/kg. The stevioside clearance
page 37 of 64
(CS) was higher than the CIn and lower than the CPAH at all the doses employed in this study. These
results indicate that the stevioside is secreted by renal tubular epithelium and induces diuresis and
natriuresis and a fall in renal tubular reabsorption of glucose.
Melis MS. Influence of calcium on the blood pressure and renal effects of stevioside. Brazilian
Journal of Medical and Biological Research;25:943-49. 1992b. The effects of verapamil (V, 0.015
mg/min, i.v.) or CaCl2 (800 mEq/l, 0.025 ml kg-1 min-1, i.v.) on renal function and mean arterial pressure
(MAP) were evaluated in male Wistar rats weighing 280-320 g during treatment with stevioside (S, 16 mg
kg-1 h-1, i.v.). 2. Verapamil administered to 10 rats significantly increased the hypotensive effect of
stevioside on MAP (control, 124 +/- 0.77; S, 96 +/- 1.50; S+V, 67 +/- 0.70 mmHg) and on fractional
sodium excretion (control, 0.76 +/- 0.05; S, 1.56 +/- 0.10; S+V, 2.72 +/- 0.25%). Urinary flow, reported
as percent glomerular filtration rate (V/GFR), and renal plasma flow (RPF) increased slightly but not
significantly during stevioside plus verapamil administration. 3. In contrast, infusion of CaCl2 in 10 rats
pretreated with stevioside induced a marked attenuation of MAP (control, 119 +/- 1.83; S, 70 +/- 1.12;
S+CaCl2, 109 +/- 1.60 mmHg) and RPF (control, 16.73 +/- 3.76; S, 34.33 +/- 2.55; S+CaCl2, 17.20 +/2.87 ml min-1 kg-1). The diuresis and natriuresis induced by stevioside were also inhibited by
simultaneous administration of CaCl2. 4. These data are consistent with the view that stevioside acts on
arterial pressure and renal function as a calcium antagonist, as is the case for verapamil.
Melis MS. Stevioside effect on renal function of normal and hypertensive rats. Journal of
Ethnopharmacology; 36:213-17. 1992c. Physiological and pharmacological experiments have
suggested that stevioside from the leaves of Stevia rebaudiana acts as a typical systemic vasodilator.
The effect of stevioside on renal function in both normal and with experimental renal hypertension rats
(GII) was evaluated using clearance techniques. Stevioside provoked hypotension, diuresis and
natriuresis in both the normal and hypertensive rats. Normal rats presented an increase in renal plasma
flow (RPF) and glomerular filtration rate (GFR) constant following stevioside administration. The last effect
is in part due to vasodilation of both the afferent and efferent arterioles. Moreover, stevioside infusion in
hypertensive rats caused an increase in RPF and GFR. These data are consistent with impairment of a
renal autoregulation mechanism in this experimental hypertensive model.
Melis MS. Chronic administration of aqueous extract of Stevia rebaudiana in rats: Renal effects.
Journal of Ethnopharmacology;47:129-34. 1995. The effects of administration of Stevia rebaudiana
extracts for 20, 40 and 60 days on renal function and mean arterial pressure in normal Wistar rats
were evaluated. Results showed that the Stevia rebaudiana treated rats group for 20 days did not
significantly differ from the control group. Chronic administration of a crude extract for 40 and 60 days
induced hypotension, diuresis and natriuresis with glomerular filtration rate (GFR) constant. An increase
of the renal plasma flow (RPF) was exclusively observed for the group treated for 60 days. The results
suggests that oral administration to rats of an aqueous extract of Stevia dried leaves induce systemic and
renal vasodilation, causing hypotension, diuresis and natriuresis.
Melis MS. A crude extract of Stevia rebaudiana increases the renal plasma flow of normal and
hypertensive rats. Brazilian Journal of Medical and Biological Research;29:669-75. 1996. The effect
of S. rebaudiana extract on renal function was evaluated in normotensive and in experimental renal
hypertensive rats (GII) using clearance techniques. Experiments were performed on male Wistar rats
weighing 300-330 g (10 animals per group). Goldblatt GII experimental hypertension was induced
by placing a silver clip with an internal gap of 0.25 mm around the left renal artery under ether anesthesia.
The contralateral kidney was left untouched. Stevia was administered 10-12 weeks after clipping. Oraladministration of Stevia extract, corresponding to 2.67 g dry leaves/day for 30 days, resulted in
a significant decrease in mean arterial pressure in both the normo-(N) and hypertensive rats (H) (N rats:
113 +/- 3.0 mmHg in the control (C) group vs 69.5 +/- 4.0 mmHg in the Stevia (S) group; H rats: 155 +/3.0 mmHg in C vs 108 +/- 4.0 mmHg in S; P < 0.05). Glomerular filtration rate was constant in the N
rats and increased significantly in the H rats after Stevia treatment 16.47 +/- 1.29 vs 14.2 +/- 1.33 ml min1 kg-1 in the C and S groups, respectively, P < 0.05). Normo- and hypertensive rats presented an
increase in renal plasma flow following oral Stevia administration (N rats: 16.4 +/- 3.10 ml min-1 kg-1 in
the C group vs 33.3 +/- 3.20 ml min-1 kg-1 in the S group. P < 0.05; H rats: 19.30 +/- 2.45 ml min-1 kg-1
in the C group vs 37.0 +/- 3.93 ml min-1 kg-1 in the S group, P < 0.05). Stevia administration provoked an
page 38 of 64
increase in urinary flow in both N and H animals (1.37 +/- 0.08% vs 2.32 +/- 0.11%, P < 0.05 and 1.47 +/0.07% vs 2.96 +/- 0.13%, P < 0.05 in N and H rats, respectively). Sodium excretion increased in N and H
animals after Stevia treatment (N rats: 0.61 +/- 0.07% in the C group vs 1.55 +/- 0.20% in the S group, P
< 0.05; H rats: 0.70 +/- 0.10% in the C group vs 2.22 +/- 0.45% in the S group, P < 0.05). These results
are consistent with impairment of a renal autoregulation mechanism in this hypertensive model
after Stevia administration. In conclusion, it was shown that Stevia extract, at doses higher than used for
sweetening purposes, is a vasodilator agent in normo- and hypertensive animals.
Melis MS. Effect of crude extract of Stevia rebaudiana on renal water and electrolytes excretion.
Phytomedicine; 6(4):247-50. 1999. To evaluate the effect of crude extract of Stevia rebaudiana on renal
water, Na+ and K+ excretion, male Wistar rats (250-350 g each) under antidiuresis or water diuresis
conditions, were evaluated. During intravenous infusion of the extract (0.05 mg/min/100 g) no significant
differences were detected in mean arterial pressure or renal hemodynamics parameters. In contrast,
fractional water and sodium excretion and solute clearance increased significantly, in both groups
of animals. In antidiuresis rats the extract significantly increased reabsorption of water by the collecting
duct and in water diuresis animals the extract significantly increased free water clearance. The data
suggest preferential action of the extract in the proximal tubular cells involved with salt
transport mechanism.
Melis MS. Effects of chronic administration of Stevia rebaudiana on fertility in rats. Journal of
Ethnopharmacology;167:157-61. 1999. A study conducted on prepubertal male rats showed that
chronic administration (60 days) of a Stevia rebaudiana aqueous extract produced a decrease in final
weight of testis, seminal vesicle and cauda epididymidis. In addition, the fructose content of the
accessory sex glands and the epididymal sperm concentration are decreased. Stevia treatment tended to
decrease the plasma testosterone level, probably by a putative affinity of glycosides of extract for a
certain androgen receptor, and no alteration occurred in luteinizing hormone level. These data
are consistent with the possibility that Stevia extracts may decrease the fertility of male rats.
Metivier J, Viana AM. The effect of long and short day length upon the growth of whole plants and
the level of soluble proteins, sugars, and stevioside in leaves of Stevia rebaudiana Bert. J Exp
Bot;30:1211-22. 1979.
Minamisono H, Azuma K. Determination of stevioside. Kagoshima-Ken Kogyo Shikenjo
Nempo;24:66-8.1978.
Misawa M. Production of natural substances by plant cell cultures described in Japanese patents.
Plant Tissue Culture Its Bio-Technol Appl Int Congr First;1976:17-26. 1977.
Mitsuhashi H, Ueno J, Sumita T, Yakugaku. Studies on the cultivation of Stevia rebaudiana.
ZASSHI;95:127-30.1975.
Mitsuhashi H, Ueno J, Sumita T. Studies on the cultivation of Stevia rebaudiana: determination of
stevioside. II. Yakugaku Zasshi;95:1501-3.1975.
Miyazaki Y, Watanabe H, Watanabe T. The cultivation of Stevia rebaudiana. III. Yield and stevioside
content of 2-year-old plants. Eisei Shikensho Hokoku;96:86-89. 1978.
Mizukami H, Shiba K, Ohashi H. Enzymatic determination of stevioside in Stevia rebaudiana.
Phytochemistry;21:1927-1930. 1982.
Mizukami H, Shiba K, Inoue S, Ohashi H. Effect of temperature on growth and stevioside formation
of Stevia rebaudiana Bertoni. Shoyakugaku Zasshi;37(2):175-9.1983.
Mizushina Y, Akihisa T, Ukiya M, Hamasaki Y, Murakami-Nakai C, Kuriyama I, Takeuchi T,
Sugawara F, Yoshida H. Structural analysis of isosteviol and related compounds as DNA
polymerase and DNA topoisomerase inhibitors. Life Sci; 77(17):2127-40. 2005 Isosteviol (ent-16-
page 39 of 64
ketobeyeran-19-oic acid) is a hydrolysis product of stevioside, which is a natural sweetener produced in
the leaves of Stevia rebaudiana (Bertoni) Bertoni. In this report, we prepared isosteviol and
related compounds from stevioside by microbial transformation and chemical conversion and assayed
the inhibitory activities toward DNA metabolic enzymes and human cancer cell growth. Among twelve
compounds obtained, only isosteviol (compound 3) potently inhibited both mammalian DNA polymerases
(pols) and human DNA topoisomerase II (topo II), and IC50 value for pol alpha was 64.0 microM.
This compound had no inhibitory effect on higher plant (cauliflower) pols, prokaryotic pols, human topo I,
and DNA metabolic enzymes such as human telomerase, T7 RNA polymerase, and bovine
deoxyribonuclease I. With pol alpha, isosteviol acted non-competitively with the DNA template-primer and
nucleotide substrate. Isosteviol prevented the growth of human cancer cells, with LD50 values of 84167 microM, and 500 microg of the compound caused a marked reduction in TPA (12-Otetradecanoylphorbol-13-acetate)-induced inflammation (inhibitory effect, 53.0%). The relationship
between the structure of stevioside-based compounds and these activities were discussed.
Mizutani K, Lee KH. Cancer preventive agents. Part 8: Chemopreventive effects of stevioside and
related compounds. Bioorg Med Chem. 17(2):600-5, 2009. In a search for potential cancer
chemopreventive agents from natural resources, stevioside (1), a sweetener, and six related compounds,
including two aglycones steviol (6) and isosteviol (7), were screened in an in vitro assay for inhibitory
effects on Epstein-Barr virus early antigen activation. Compounds 1, 6 and 7 showed significant activity in
this assay and also exhibited strong inhibitory effects in a two-stage carcinogenesis test using mouse skin
induced by 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA).
The inhibitory effects of these three compounds were greater than that of glycyrrhizin. Furthermore, these
three compounds significantly inhibited mouse skin carcinogenesis initiated by peroxynitrite and promoted
by TPA. Their activities were comparable to that of curcumin. These results suggested that 1, as well as 6
and 7, could be valuable as chemopreventive agents for chemical carcinogenesis.
Mohd-Radzman NH, Ismail WI, Adam Z, Jaapar SS, Adam A. Potential Roles of Stevia rebaudiana
Bertoni in Abrogating Insulin Resistance and Diabetes: A Review. Evid Based Complement
Alternat Med. 2013:718049, 2013. Insulin resistance is a key factor in metabolic disorders like
hyperglycemia and hyperinsulinemia, which are promoted by obesity and may later lead to Type II
diabetes mellitus. In recent years, researchers have identified links between insulin resistance and many
noncommunicable illnesses other than diabetes. Hence, studying insulin resistance is of particular
importance in unravelling the pathways employed by such diseases. In this review, mechanisms involving
free fatty acids, adipocytokines such as TNF α and PPAR γ and serine kinases like JNK and IKK β ,
asserted to be responsible in the development of insulin resistance, will be discussed. Suggested
mechanisms for actions in normal and disrupted states were also visualised in several manually
constructed diagrams to capture an overall view of the insulin-signalling pathway and its related
components. The underlying constituents of medicinal significance found in the Stevia rebaudiana Bertoni
plant (among other plants that potentiate antihyperglycemic activities) were explored in further depth.
Understanding these factors and their mechanisms may be essential for comprehending the progression
of insulin resistance towards the development of diabetes mellitus.
Mori N, Sakanoue M, Takeuchi M., Shimpo K, Tanabe T. Effect of stevioside on fertility in rats.
Journal of the Food Hygienic Society of Japan;22:409-14 (in Japanese).1981.
Morita E. Outlook for Stevia natural sweetening agents. Shokuhin To Kagaku;19(4):83-87.1977.
Morris JA, Lloyd IA. Sweetening agents from natural sources;39(4):25-38. 1976. Sweetness is an
important taste sensation to humans. The absence of suitable sweeteners as alternatives to cyclamates
and saccharin has led to a renewed interest in sweeteners form natural sources. A brief review of the
history of sweetener usage provides a basis for understanding our present heavy consumption of sweet
substances. The structure of naturally-occurring compounds possessing a sweet taste range from simple
sugars to complex, intensely sweet proteins. The structural types include monoterpenes, diterpenes,
triterpenes, flavonoids, steroid saponins, dipeptides, and proteins. Some of these substances are
not, strictly-speaking, natural but are derived from natural sources by relatively minor chemical
modification. The properties of two non-sweet substances, miraculin and gymnemic acid, are included
because of their close relationship to the subject of sweeteners. Miraculin causes sour substances to
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taste sweet and gymnemic acid selectively blocks sweet taste perception. The second part of the paper
presents some of the work on monellin, the intensely sweet protein from "serendipity berries"
(Dioscoreophyllum cumminsii). The physico-chemical studies of monellin provide convincing evidence
that it is, indeed, a protein. Structural studies using denaturants and specific chemical modifications have
provided a beginning of our understanding of the molecular basis of the sweet taste of monellin.
Nadamitsu S, Segawa M, Sato Y, Kondo K. Effects of stevioside on the frequencies of
chromosomal aberrations and sister chromatid exchanges in the D-6 cell of Chinese hamster.
Hiroshima Daigaku Sogo Kagakubu Kiyo;10, 57-62 [Abstract only]. 1985
Nakayama K, Kasahara D, Yamamoto F. Absorption, Distribution, Metabolism and Excretion of
Stevioside in Rats. Journal of the Food Hygienic Society of Japan;27:1-8. 1986.
Nakamura S, Tamura Y. Variation in the main glycosides of Stevia (Stevia rebaudiana Bertoni).
Nettai Nogyo;29(2):109-15. 1985.
Nakamura M, Kodama N, Sataoh N, Nakamura T, Shingo. Effect of stevioside on the expression of
insulin receptor substrate-1 and -2. Journal of Pharmacological Sciences 91, 115P [Abstract No.
1P014]. 2003.
Nepovim A, Drahosova H, Valicek P, Vanek T. The effect of cultivation conditions on the content of
stevioside in Stevia rebaudiana Bertoni plants cultivated in the Czech Republic. Pharm Pharmacol
Lett;8(1):19-21. 1998.
Nguyen DT. Isolation of stevioside from Stevia rebaudiana. Hoa Hoc Cong Nghiep Hoa Chat;2:6-9.
1996.
Nikiforov AI, Eapen AK. A 90-day oral (dietary) toxicity study of rebaudioside A in Sprague-Dawley
rats. Int J Toxicol. 27(1):65-80, 2008. Rebaudioside A is one of several glycosides found in the leaves of
Stevia rebaudiana (Bertoni) Bertoni (Compositae) stevia that has been identified as a potential
sweetener. The present study (initiated in April 2006 and completed in October 2006) evaluated the
safety of this sweetener when administered as a dietary admix at target exposure levels of 500, 1000,
and 2000 mg/kg/day to Sprague-Dawley rats for 90 days. There were no treatment-related effects on the
general condition and behavior of the animals as determined by clinical observations, functional
observational battery, and locomotor activity assessments. Evaluation of clinical pathology parameters
revealed no toxicologically relevant, treatment-related effects on hematology, serum chemistry, or
urinalysis. Macroscopic and microscopic findings revealed no treatment-related effects on any organ
evaluated. Lower mean body weight gains were noted in males in the 2000 mg/kg/day group throughout
the study, which was considered to be test article related; however, given the small magnitude of the
difference as compared to controls, this effect was not considered to be adverse. Results of this study
clearly demonstrate that dietary administration of high concentrations of rebaudioside A for 90
consecutive days to Sprague-Dawley rats was not associated with any signs of toxicity.
Nikiforov AI, Rihner MO, Eapen AK, Thomas JA. Metabolism and Toxicity Studies Supporting the
Safety of Rebaudioside D. Int J Toxicol. Jun 13, 2013. Rebaudioside D (Reb D) is one of the several
glycosides found in the leaves of Stevia rebaudiana (Bertoni) Bertoni (Compositae) which has been
identified as a potential sweetener. The metabolism of Reb A and Reb D was evaluated in various in vitro
matrices (simulated gastrointestinal fluids, rat liver microsomes, and rat cecal contents) and through
analysis of plasma collected from rats in a dietary toxicity study. Reb A and Reb D showed similar stability
when exposed to simulated stomach and small intestine fluids, with susceptibility to hydrolytic
degradation by enteric bacteria collected from the cecum. Incubations with rat liver microsomes indicated
that neither compound is expected to be metabolized by the liver enzymes. Plasma concentrations of Reb
D, Reb A, and/or the final hydrolysis product of each compound, free/conjugated steviol, were
consistent between animals administered either Reb D or Reb A in the diet. A repeated exposure dietary
toxicity study was conducted to compare the safety of Reb D, when administered at target exposure
levels of 500, 1000, and 2000 mg/kg body weight (bw)/d to Sprague-Dawley rats for 28 days, to that of
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Reb A administered at a target exposure level of 2000 mg/kg bw/d. There were no treatmentrelated effects on the general condition and behavior of the animals and no toxicologically relevant,
treatment-related effects on hematology, serum chemistry, or urinalysis. Macroscopic and microscopic
findings revealed no treatment-related effects on any organ evaluated. Results were comparable
between the group administered 2000 mg/kg/d Reb D and the group administered 2000 mg/kg/d Reb A.
Nikolova-Damyanova B, Bankova V, Popov S. Separation and quantitation of stevioside and
rebaudioside a in plant extracts by normal-phase high performance liquid chromatography and
thin-layer chromatography: A comparison. Phytochem Anal;5(2):81-5. 1994.
Nishiyama P, Kusaumoto IT, Costa SC, Alvarez M, Vieira LG. Correlation between the content of
total carbohydrates and stevioside in leaves of Stevia rebaudiana. Arq Biol Tecnol;34 (3/4):42534.1991.
Nunes P, Pereira NA. Efeito do Caá-heê (Stevia rebaudiana)(Bert) Bertoni sobre a fertilidade de
animais experimentais = [The effect of Stevia rebaudiana on the fertility of experimental animals].
Revista Brasileira de Farmacia;69:46-50. 1988.
Nunes AP, Ferreira-Machado SC, Nunes RM, Dantas FJ, De Mattos JC, Caldeira-de-Araújo A.
Analysis of genotoxic potentiality of stevioside by comet assay. Food Chem Toxicol. 45(4):662-6,
2007. Stevioside is a natural non-caloric sweetener extracted from Stevia rebaudiana (Bertoni) leaves. It
has been widely used in many countries, including Japan, Korea, China, Brazil and Paraguay, either as a
substitute for sucrose in beverages and foods or as a household sweetener. The aim of this work was to
study its genotoxic potentiality in eukaryotic cells. Wistar rats were treated with stevioside solution
(4mg/mL) through oral administration (ad libitum) and the DNA-induced damage was evaluated using the
single cell gel electrophoresis (comet assay). The results showed that treatment with stevioside
generates lesions in peripheral blood, liver, brain and spleen cells in different levels, the largest effect
being in liver. Therefore, these undesired effects must be better understood, once the data present here
point to possible stevioside mutagenic properties.
Oh H, Han E, Choi D, Kim J, Eom M, Kang I, Kang H, Ha K. In vitro and in vivo evaluation of
genotoxicity of stevioside and steviol, natural sweetener. Journal of the Pharmaceutical Society of
Korea;43: 614-622. 1999a.
Oh HY, Han ES, Sohn SJ, Kim JW, Park CH, Eom MO, Ha KW. Evaluation of the genotoxicity of
stevioside and steviol using in vitro mouse lymphoma L5178Y gene mutation assay and in vivo
hepatocyte micronucleus assay. Environmental and Molecular Mutagenesis; 33 (33): 48 [Abstract
No. 153]. 1999b.
Okamoto H, Yoshida D, Mizusaki S. Inhibition of 12-O-tetradecanoylphorbol-13- acetateinduced
induction in Epstein-Barr virus early antigen in Raji cells. Cancer Letters;19:47-53. 1983. Retinol, 5
flavonoids, 3 steroids and 7 sweetening agents were studied for their effects on 12-Otetradecanoylphorbol-13-acetate (TPA)-induced early antigen (EA) of Epstein-Barr virus (EBV) in Raji
cells. Concomitant treatment of Raji cells with TPA and retinol showed inhibition of EA induction. Among
flavonoids, quercetin resulted in effective inhibition of EA induction by TPA and alpha-naphthoflavone
showed the weakly inhibitory effect. None of the other flavonoids such as rutin, catechin and betanaphthoflavone affected the induction of EBV-EA by TPA. beta-Estradiol obviously inhibited EBV-EA
induction by TPA, but hydrocortisone did not show any inhibitory effect on it. Glycyrrhetinic acid, steviol,
phyllodulcin and perrillartine also showed the remarkable inhibition of EBV-EA induction. On the other
hand, glycyrrhizin and stevioside, glycosides of glycyrrhetinic acid and steviol, did not inhibit the induction
of EBV-EA by TPA. Some of the inhibitors reported here may be effective on the inhibition of the in vivo
tumor promotion by TPA.
Okumura M, Fujita Y, Imamura M, Aikawa K. Studies on the safety of stevioside with recassay and
reversion test. Shokuhin Eiseigaku Zasshi;19:486-90. 1978.
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Oliveira-Filho RM, Uehara OA, Minett CASA, Valle LBS. Chronic Administration of Aqueous
Extract of Stevia rebaudiana (Bert.) Bertoni in Rats: Endocrine Effects. General
Pharmacology;20(2):187-91. 1989.
Ong KL, Cheung BM, Man YB, Lau CP, Lam KS. Prevalence, awareness, treatment, and control of
hypertension among United States adults 1999–2004. Hypertension;49:69–75. 2007. Detection of
hypertension and blood pressure control are critically important for reducing the risk of heart attacks and
strokes. We analyzed the trends in the prevalence, awareness, treatment, and control of hypertension in
the United States in the period 1999-2004. We used the National Health and Nutrition Examination
Survey 1999-2004 database. Blood pressure information on 14 653 individuals (4749 in 1999-2000, 5032
in 2001-2002, and 4872 in 2003-2004) aged >or=18 years was used. Hypertension was defined as blood
pressure >or=140/90 mm Hg or taking antihypertensive medications. The prevalence of hypertension
in 2003-2004 was 7.3+/-0.9%, 32.6+/-2.0%, and 66.3+/-1.8% in the 18 to 39, 40 to 59, and >or=60 age
groups, respectively. The overall prevalence was 29.3%. When compared with 1999-2000, there were
nonsignificant increases in the overall prevalence, awareness, and treatment rates of hypertension. The
blood pressure control rate was 29.2+/-2.3% in 1999-2000 and 36.8+/-2.3% in 2003-2004. The ageadjusted increase in control rate was 8.1% (95% CI: 2.4 to 13.8%; P=0.006). The control rates increased
significantly in both sexes, non-Hispanic blacks, and Mexican Americans. Among the >or=60 age group,
the awareness, treatment, and control rates of hypertension had all increased significantly (P<or=0.01).
The improvement in blood pressure control is encouraging, although the prevalence of hypertension has
not declined.
Oshima Y, Saito J, Hikino H. Sterebins E, F, G and H, diterpenoids of Stevia rebaudiana leaves.
Phytochemistry;27(2):624-26. 1988.
Oshima Y, Saito JI, Hiniko H. Sterebins A, B, C and D, bisnorditerpenoids of Stevia rebaudiana
leaves. Tetrahedron;42(23):6443-46.1986.
Oviedo CA, Fronciani G, Moreno R, Maas LC. Hypoglycemic action of Stevia rebaudiana. Excerpta
Medica;209:92.1970.
Pariwat P, Homvisasevongsa S, Muanprasat C, Chatsudthipong V. A natural plant-derived
dihydroisosteviol prevents cholera toxin-induced intestinal fluid secretion. J Pharmacol Exp Ther.
324(2):798-805, 2008. Stevioside and its major metabolite, steviol, have been reported to affect ion
transport in many types of tissues, such as the kidney, pancreas, and intestine. The effect of stevioside,
steviol, and its analogs on intestinal Cl(-) secretion was investigated in a human T84 epithelial cell line.
Short-circuit current measurements showed that steviol and analogs isosteviol, ihydroisosteviol, and
isosteviol 16-oxime inhibited in a dose-dependent manner forskolin-induced Cl(-) secretion with IC(50)
values of 101, 100, 9.6, and 50 microM, respectively, whereas the parent compound stevioside had no
effect. Apical Cl(-) current measurement indicated that dihydroisosteviol targeted the cystic fibrosis
ransmembrane regulator (CFTR). The inhibitory action of dihydroisosteviol was reversible and was not
associated with changes in the intracellular cAMP level. In addition, dihydroisosteviol did not affect
calcium-activated chloride secretion and T84 cell viability. In vivo studies using a mouse closed-loop
model of cholera toxin-induced intestinal fluid secretion showed that intraluminal injection of 50 microM
dihydroisosteviol reduced intestinal fluid secretion by 88.2% without altering fluid absorption. These
results indicate that dihydroisosteviol and similar compounds could be a new class of CFTR inhibitors that
may be useful for further development as antidiarrheal agents.
Park JE, Cha YS. Stevia rebaudiana Bertoni extract supplementation improves lipid and carnitine
profiles in C57BL/6J mice fed a high-fat diet. J Sci Food Agric. 90(7):1099-105, 2010.
BACKGROUND: Stevia (Stevia rebaudiana Bertoni) is a non-caloric natural-source alternative to
artificially produced sugar substitutes. This study investigated the effect of stevia extract on lipid profiles
in C57BL/6J mice. Forty mice were divided into four groups: N-C (normal diet and distilled water), H-C
(high-fat diet and distilled water), H-SC (high fat diet and sucrose, 1 mL kg(-1) per day), and H-SV (highfat diet and stevia extract, 1 mL kg(-1) per day). RESULTS: Body weight gain was significantly higher in
the H-SC group than in the H-SV group. Triglyceride concentrations in serum and liver were lower in the
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H-SV group than in the H-SC group. Serum total cholesterol concentrations were lower in the H-SV and
H-C groups compared to the H-SC group. The concentrations of acid-insoluble acylcarnitine (AIAC) in
serum were higher in the H-SV group than in the H-C and H-SC groups and the acyl/free carnitine level in
liver was significantly higher in the H-SV group than in the N-C group. These results were supported by
mRNA expression of enzymes related to lipid metabolism (ACO, PPARalpha, ACS, CPT-I, ACC)
assessed by real-time polymerase chain reaction. CONCLUSION: These results suggest that the
supplementation of stevia extract might have an anti-obesity effect on high-fat diet induced obese mice.
Parker KJ. Natural high-intensity sweeteners. Bnf Bull;16(3,4):240-45.1976.
Pezzuto JM, Compadre CM, Swanson SM, Nanayakkara D, Kinghorn AD. Metabolically activated
steviol, the aglycone of stevioside, is mutagenic. Proceedings of the National Academy of
Sciences USA;82:2478-82.1985.
Pezzuto JM, Nanayakkara NPD, Compadre CM, Swanson SM, Kinghorn AD, Guenther TM,
Sparnins VL, Lam LK. Characterization of bacterial mutagenicity mediated by 13-hydroxy-entkaurenoic acid (steviol) and several structurally related derivatives and evaluation of potential to
induce glutathione-s-transferase in mice. Mutat Res;169:93-103.1986. Stevioside is a sweet-tasting
diterpene glycoside that is derived from Stevia rebaudiana (Bertoni) Bertoni (Compositae). It is used
commercially in Japan and other parts of the world as a sucrose substitute. Whereas stevioside
demonstrates no mutagenic activity in a variety of test systems, the aglycone, steviol (13-hydroxy-entkaurenoic acid), is mutagenic toward Salmonella typhimurium strain TM677 in the presence of a
metabolic activating system derived from the liver of Aroclor 1254-pretreated rats. The required activating
component is localized in the microsomal fraction of rat liver, suggestive of a cytochrome P-450-mediated
reaction. Partially purified epoxide hydrolase does not inhibit steviol-induced mutagenicity, indicating that
an active metabolite is not an epoxide that serves as a substrate for this enzyme preparation. The 13hydroxy group of steviol is required for the expression of mutagenicity since ent-kaurenoic acid is
nonmutagenic, and acetylation of steviol at this position negates mutagenicity. Similarly, diterpenes
bearing a strong structural resemblance to steviol, cafestol and kahweol, were found to demonstrate
no mutagenic activity toward Salmonella typhimurium TM677, as were their respective acetates and
palmitic acid esters. Conversely, 19-O-beta-D-glucopyranosyl steviol, a potential hydrolysis product of
stevioside, is mutagenic and bactericidal in the presence of a metabolic activating system. Additionally,
in contrast to the nonmutagenic diterpenes cafestol and kahweol that are effective as inducers of
glutathione S-transferase activity, evaluation by administration to mice proved steviol, isosteviol and
various steviol glycosides to be inactive in this process. Thus, structural differences among these
naturally occurring and semi-synthetic diterpenes appear to impart major differences in biological activity
that may relate to human health upon dietary ingestion.
Pinheiro CE, Gasparini OT. Effect of stevioside on the glycemic level of diabetic rabbit and on the
captation of glucose in vitro by adipose and muscular tissue of the rat. First Brazilian Seminar On
Stevia Rebaudiana Inst Tecnol Aliment (Campinas) Brazil: 15.1-15.4, 1981.
Pinheiro CE, Oliveira SS, Da Silva SM, Poletto MI, Pinheiro GJ. Effect of guarana and Stevia
rebaudiana Bertoni (leaves) extracts, and stevioside, on the fermentation and synthesis of
extracellular insoluble polysaccharides of dental plaque. Rev Odont Usp;1(4):9-13.1987.
Planas GM, Kuc J. Contraceptive propertiesof Stevia rebaudiana. Science;162:1007. 1968. A water
decoction of the plant Stevia rebaudiana Bertoni reduces fertility in adult female rats of proven fertility.
The decoction continues to descrease fertility for at least 50 to 60 days after intake is stopped. The
decoction did not affect appetite and apparently did not affect the health of adults rats.
Pomaret M and Lavieille R. Le Principe à saveur sucrée du Kaà-he-é (Stevia rebaudiana Bertoni).
IV. Quelques propriétés physiologiques du Stévioside. Bulletin de Société de Chimie Biologique
(Paris);13:1248-52. 1931.
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Prakash I, Dubois GE, Clos JF, Wilkens KL, Fosdick LE. Development of rebiana, a natural, noncaloric sweetener. Food Chem Toxicol.;46 Suppl 7:S75-82, 2008. Rebiana is the common name for
high-purity rebaudioside A, a natural non-calorie sweetener 200-300 times more potent than sucrose. It
provides zero calories and has a clean, sweet taste with no significant undesirable taste characteristics. It
is functional in a wide array of beverages and foods and can be blended with other non-calorie or
carbohydrate sweeteners. It is stable under dry conditions, and has much better stability than aspartame
or neotame in aqueous food systems. Studies undertaken for the development of a purification process
and for the full characterization of the properties of rebiana are reported here.
Procinska E, Bridges BA, Hanson JR. Interpretation of results with the 8-azaguanine resistance
system in Salmonella typhimurium: No evidence for direct acting mutagenesis by 15-oxosteviol, a
possible metabolite of steviol. Mutagenesis;6:165-7.1991. 15-Oxosteviol, postulated to be the
mutagenic metabolite of steviol, was observed to be non-mutagenic in preliminary experiments using a
number of different systems. Repetition of the original experiment in Salmonella TM677 failed to
show any significant induction of 8-azaguanine resistant mutants by 15-oxosteviol even when the number
of bacteria tested was greatly increased. Examination of the earlier positive result showed that it could not
be justified from the data and revealed a commonly applied way of mishandling data obtained with the
TM677 system.
Purkayastha S, Pugh Jr G,Lynch B, Roberts A, Kwok D, Tarka Jr SM. In vitro metabolism of
rebaudioside B, D, and M under anaerobic conditions: Comparison with rebaudioside A.
Regulatory Toxicology and Pharmacology. Available online 18 December 2013. The hydrolysis of
the steviol glycosides rebaudioside A, B, D, and M, as well as of steviolbioside (a metabolic intermediate)
to steviol was evaluated in vitro using human fecal homogenates from healthy donors under anaerobic
conditions. Incubation of each of the rebaudiosides resulted in rapid hydrolysis to steviol. Metabolism was
complete within 24 h, with the majority occurring within the first 8 h. There were no clear differences in the
rate or extent of metabolism of rebaudioside B, D, or M, relative to the comparative control rebaudioside
A. The hydrolysis of samples containing 2.0 mg/mL of each rebaudioside tended to take slightly longer
than solutions containing 0.2 mg/mL. There was no apparent gender differences in the amount of
metabolism of any of the rebaudiosides, regardless of the concentrations tested. An intermediate in the
hydrolysis of rebaudioside M to steviol, steviolbioside, was also found to be rapidly degraded to steviol.
The results demonstrate that rebaudiosides B, D, and M are metabolized to steviol in the same manner
as rebaudioside A. These data support the use of toxicology data available on steviol, and on steviol
glycosides metabolized to steviol (i.e., rebaudioside A) to substantiate the safety of rebaudiosides B, D,
and M.
Putieva ZM, Saatov Z. Flavonoids of the leaves of Stevia rebaudiana. Chem Nat Comp;33(4):1945.1997.
Rajbhandari A, RoBerts MF. The flavonoids of Stevia rebaudiana. J Nat Prod;46(2):194-5.1983.
Randi AM, Felippe GM. Substances promoting root growth from the achenes of Stevia rebaudiana
(Bert.) Bertoni. Rev Brasil Bot;4:49-51.1981.
Rasenack P. Sweet substances of eupatorium rebaudianum and of licorice. Arb Kais Biol Anst
Land Fortwirtsch;28:420. 1908.
Raskovic A, Gavrilovic M, Jakovljevic V, Sabo J. Glucose concentration in the blood of intact and
alloxan-treated mice after pretreatment with commercial preparations of Stevia rebaudiana
(Bertoni). Eur J Drug Metab Pharmacokinet. 29(2):87-90, 2004. The study was concerned with the
effect of mice pretreatment with two commercial products of Stevia rebaudiana Bertoni on the blood
glucose concentration. One group of mice was pretreated four days with 200 mg/kg of Stevita (Stevita Co,
INC, Arlington Texas) (stevia) and the other with 20 mg/kg of Clear Steviosides liquid (Stevita Co, INC,
Herbal supplement, Brazil) (stevioside), whereas the animals of control group received at the same time
physiological solution. Blood glucose concentration was measured before pretreatment and four days
after that. The changes in glucose level were provoked by glucose-tolerance test (500 mg/kg, p.o.) and
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subcutaneous injection of adrenaline (0.2 mg/kg). The same procedure of measuring blood glucose was
applied on the mice with alloxan-induced diabetes mellitus (two doses of 100 mg/kg with a 24-hour
interval). Blood glucose levels in mice pretreated with stevia and stevioside were lower compared with
control (7.82:6.82:8.01). Also, a smaller increase in this parameter compared to control was registered
with pretreated mice in the glucose-tolerance test, pretreatment with stevioside being again more
effective (8.68:6.36:5.82). Pretreatment with stevioside caused no significant increase in blood glucose
concentration after administering adrenaline, which was not the case with the animals pretreated with
stevia and control. Pretreatment with stevia, and to a greater extent with stevioside, protected test
animals from the toxic action of alloxan compared with controls.
Raskovic A, Jakovljevic V, Mikov M, Gavrilovic M. Joint effect of commercial preparations of
Stevia rebaudiana Bertoni and sodium monoketocholate on glycemia in mice. Eur J Drug Metab
Pharmacokinet. 29(2):83-6, 2004. A study was made of the combined effect of two commercial products
of Stevia rebaudiana Bertoni and sodium monoketocholate (mkc) on blood glucose Concentration in
mice. One group of animals was treated four days with mkc, 4 mg/kg, s.c., second with 200 mg/kg, i.p., of
Stevita (Stevita Co, INC, Arlington, Texas) (stevia), third with 20 mg/kg, i.p., of Clear Steviosides Liquid
(Stevita Co, INC, Herbal supplement, Brazil) (stevioside), fourth with the combination of stevia and mkc,
and the fifth with stevisode and mkc. Blood glucose concentration was measured before treatment, after
the first and fourth dose, as well as after subjecting animals to glucose-tolerance test (500 mg/kg, p.o.) or
provoking glycemia by injecting adrenaline (0.2 mg/kg, s.c.). It was found that one dose of stevioside
combined with mkc caused a significant increase of glycemia with respect of mkc alone and control
(10.80:7.90:8.01). However, when repeated four days, the same pretreatment resulted in a significant
decrease of glycemia compared with single-dose pretreatment (10.80:7.20). The increase in glycemia
with the mice that received four doses of stevioside and mkc and then were subjected to glucosetolerance test was significantly lower compared to that inmice that were pretreated four days only with
mkc before receiving glucose (6.33:7.80). Analogous difference was observed between the animals given
mkc alone and mkc plus stevioside after injecting adrenaline (13.33:10.54). As for the interaction of mkc
and stevia it was found that the combined pretreatment yielded lower values of glycemia compared with
that measured after treatment with stevia alone (6.40:7.82).
Raskovic A, Mikov M, Jakovljevic V, Stilinovic N, Posa M, Kuhajda K, Kevresan S. Effects of
stevioside and sodium salt of monoketocholic acid on glycemia in rats. Journal of Hypertension;
23, (Suppl. 2), S311 [Abstract No. P3.121]. 2005.
Raskovic A, Jakovljevic V, Mikov M. The influence of commercial preparations of Stevia
rebaudiana (Bertoni) on glucose metabolism in mice. Acta Pharmacologica Sinica;27(1):339-40.
2006.
Raskovic A, Mikov M, Skrbic R, Jakovljevic V, Vasovic V, Posa M, Kuhajda K, Kevresan S, Tomic
Z, Siladji D. Effect of stevioside and sodium salt of monoketocholic acid on glycemia in
normoglycemic and diabetic rats. Eur J Drug Metab Pharmacokinet. 33(1):17-22, 2008. This study
investigated the effect of a commercial preparation of stevioside and a synthetic compound, sodium salt
of monketocholic acid (MKC), administered per os (p.o.) and also adminstered via an osmotic pump, on
glycemia in normoglycemic and diabetic Wistar rats. Diabetes was induced with alloxan, 100 mg/kg, i.p.
Normoglycemic and diabetic rats were treated p.o. for five days either with physiological solution (1 ml/kg,
controls), stevioside (20 mg/kg), MKC (4 mg/kg) and a combination of stevioside (20 mg/kg) and MKC (4
mg/kg). Apart from p.o. adminstration, stevioside and MKC were also administered via a subcutaneously
(s.c.) implanted osmotic pump. During treatment and upon termination of the latter, glycemia was
measured and the rats that were treated p.o. were subjected to the oral glucose tolerance test (OGTTT)
at a dose of 1 g/kg. Following this, animals were anesthetized with urethane (0.75 g/kg, i.p.) and killed by
cardiopunction to determine C-peptide levels in the serum. In all three groups of normoglycemic rats
highest decrease in glucose levels was observed on the fourth day of the experiment. The stevioside +
MKC combination showed a stronger hypoglycemic effect compared to individual treatments with
stevioside and MKC (3.73:4.80:4.73 mmol/L). In the group of diabetic rats that received both substances
via the osmotic pump, the hypoglycemic action was also stronger compared to the individual treatments
with stevioside and MKC (16.15:18.89:18.75 mmol/L). The treatment of healthy rats with both substances
page 46 of 64
p.o. caused no statistically significant difference in glycemia, whereas in diabetic rats the combination of
stevioside + MKC showed a statistically significant decrease in glycemia compared to control values. In
both groups of rats, treatment with stevioside and MKC and their combination prevented an increase in
glucose concentrations in the OGTT. Only the administration of stevioside by osmotic pump yielded a
statistically significant increase in the concentrations of C-peptide in the serum of healthy rats. Compared
to controls, the concentrations of C-peptide in diabetic rats were significantly higher after treatment with
either stevioside or its combination with MKC, irrespective of the mode of administration.
Renwick AG, Tarka SM. Microbial hydrolysis of steviol glycosides.Food Chem Toxicol;46 Suppl
7:S70-4, 2008. A review of the role of gut microbiota in the metabolism of the steviol glycosides,
stevioside and rebaudioside A, indicates that they are not absorbed intact but undergo hydrolysis by the
intestinal microflora to steviol. Steviol is not metabolized by the intestinal flora and is absorbed from the
intestine. The rate of hydrolysis for stevioside is greater than for rebaudioside A. Recent studies using
mass spectrometry have shown that steviol-16,17-epoxide is not a microbial metabolite of steviol
glycosides. Bacteroides species are primarily responsible for hydrolysis via their beta-glucosidase activity.
Fecal incubation studies with both human and animal mixed flora provide similar results, and this
indicates that the rat is an appropriate model for studies on steviol glycosides. Given the similarity in the
microbial metabolism of stevioside and rebaudioside A with the formation of steviol as the single
hydrolysis product that is absorbed from the intestinal tract, the toxicological data on stevioside are
relevant to the risk assessment of rebaudioside A.
Renwick AG. The use of a sweetener substitution method to predict dietary exposures for the
intense sweetener rebaudioside A. Food Chem Toxicol. 46 Suppl 7:S61-9, 2008. There are more
published dietary exposure data for intense sweeteners than for any other group of food additives. Data
are available for countries with different patterns of sweetener approvals and also for population groups
with high potential intakes, such as children and diabetic subjects. These data provide a secure basis for
predicting the potential intakes of a novel intense sweetener by adjustment of the reported intakes of
different sweeteners in mg/kg body weight by their relative sweetness intensities. This approach allows
the possibility that a novel sweetener attains the same pattern and extent of use as the existing
sweeteners. The intakes by high consumers of other sweeteners allows for possible brand loyalty to the
novel sweetener. Using this method, the estimated dietary exposures for rebaudioside A in average and
high consumers are predicted to be 1.3 and 3.4mg/kg body weight per day for the general population, 2.1
and 5.0mg/kg body weight per day for children and 3.4 and 4.5mg/kg body weight per day for children
with diabetes. The temporary ADI defined by the JECFA for steviol glycosides [JECFA, 2005. Steviol
glycosides. In: 63rd Meeting of the Joint FAO/WHO Expert Committee on Food Additives. World Health
Organization (WHO), Geneva, Switzerland, WHO Technical Report Series 928, pp. 34-39] was set at 02mg/kg body weight (expressed as steviol equivalents); after correction for the difference in molecular
weights, these estimated intakes of rebaudioside A are equivalent to daily steviol intakes of less than
2mg/kg. In consequence, this analysis shows that the intakes of rebaudioside A would not exceed the
JECFA temporary ADI set for steviol glycosides.
Reports of a Working Group on Scientific Cooperation on Questions Relating to Food, Task 4.2.
SCOOP/INT/REPORT/2 (Brussels: European Commission Directorate General I11 Industry).
Rizzo B, Zambonin L, Angeloni C, Leoncini E, Vieceli Dalla Sega F, Prata C, Fiorentini D, Hrelia S.
Steviol glycosides modulate glucose transport in different cell types. Oxid Med Cell Longev. Nov.
2013. Extracts from Stevia rebaudiana Bertoni, a plant native to Central and South America, have been
used as a sweetener since ancient times. Currently, Stevia extracts are largely used as a noncaloric highpotency biosweetener alternative to sugar, due to the growing incidence of type 2 diabetes mellitus,
obesity, and metabolic disorders worldwide. Despite the large number of studies on Stevia and steviol
glycosides in vivo, little is reported concerning the cellular and molecular mechanisms underpinning the
beneficial effects on human health. The effect of four commercial Stevia extracts on glucose transport
activity was evaluated in HL-60 human leukaemia and in SH-SY5Y human neuroblastoma cells. The
extracts were able to enhance glucose uptake in both cellular lines, as efficiently as insulin. Our data
suggest that steviol glycosides could act by modulating GLUT translocation through the PI3K/Akt pathway
since treatments with both insulin and Stevia extracts increased the phosphorylation of PI3K and Akt.
page 47 of 64
Furthermore, Stevia extracts were able to revert the effect of the reduction of glucose uptake caused by
methylglyoxal, an inhibitor of the insulin receptor/PI3K/Akt pathway. These results corroborate the
hypothesis that Stevia extracts could mimic insulin effects modulating PI3K/Akt pathway.
Roberts A, Renwick AG. Comparative toxicokinetics and metabolism of rebaudioside A,
stevioside, and steviol in rats. Food Chem Toxicol. 46 Suppl 7:S31-9, 2008. The toxicokinetics and
metabolism of rebaudioside A, stevioside, and steviol were examined in rats for comparative purposes to
determine whether toxicological studies conducted previously with stevioside would be applicable to the
structurally-related glycoside, rebaudioside A. Single, oral doses of the radiolabelled compounds were
extensively and rapidly absorbed with plasma concentration-time profiles following similar patterns for
stevioside and rebaudioside A. Elimination of radioactivity from plasma was essentially complete within
72h. All plasma samples had similar metabolite profiles; the predominant radioactive component in all
samples was steviol, with lower amounts of steviol glucuronide(s) and low levels of one or two other
metabolites. Rebaudioside A, stevioside, and steviol were metabolized and excreted rapidly, with the
majority of the radioactivity eliminated in the feces within 48h. Urinary excretion accounted for less than
2% of the administered dose for all compounds in both intact and bile duct-cannulated rats, and the
majority of the absorbed dose was excreted via the bile. After administration of the compounds to intact
and bile duct-cannulated rats, radioactivity in the feces was present primarily as steviol. The predominant
radioactive compound detected in the bile of all cannulated rats was steviol glucuronide(s), indicating deconjugation in the lower intestine. Overall, the data on toxicokinetics and metabolism indicate that
rebaudioside A and stevioside are handled in an almost identical manner. These studies support the use
of toxicological safety studies conducted with steviosidefor the safety assessment of rebaudioside A.
Saenphet K, Aritajat S, Saenphet S, Manosroi J, Manosroi A. Safety evaluation of aqueous extracts
from Aegle marmelos and Stevia rebaudiana on reproduction of female rats. Southeast Asian J
Trop Med Public Health. 37 Suppl 3:203-5, 2006. The purpose of this study was to evaluate the safety
of a Thai medicinal plant, Aegle marmelos, and a non-caloric sweetener, Stevia rebaudiana, on the
reproduction of female rats. Female rats were treated orally with aqueous extract of A. marmelos (6%)
and S. rebaudiana at various concentrations (0, 0.2, 1, or 10%) for 60 days (1 ml/day) before mating. The
control rats received only distilled water. At the end of the treatment period, treated females were mated
with untreated males and the effects on reproduction were examined at day 14 of pregnancy. No notable
abnormalities were observed in any of the pregnant rats. The number of corpus lutea, implanted and
dead fetuses, as well as the sizes of the fetuses in the treated rats were not significantly different from
those of the controls. Based on these results, it may be concluded that aqueous extracts of A. marmelos
and S. rebaudiana at the concentrations used in this study do not alter the reproduction of female rats.
Sainati AR, Melis MS, Maciel RE. Effects of stevioside and verapamil on renal-function in rats.
Brazilian Journal of Medical Biological Research;19:A532. 1986.
Saitsuga H. Use of steviosides in processed foods. Jap Fudo Saiensu;21(7):24-30.1982.
Sakaguchi M, Kan T. Japanese researches on Stevia rebaudiana (Bert.) Bertoni and stevoside.
Cienc Cult; 34:235-48.1948.
Sakamoto I, Kohda H, Murakami K, Tanaka O, Yakugaku. Quantitative analysis of stevioside.
Zasshi;95:1507.1975.
Sakamoto I, Yamasaki K, Tanaka O. Application of 13-C NMR spectroscopy to chemistry of natural
glycosides: rebaudioside-C, a new sweet diterpene glycoside of Stevia rebaudiana. Chem Pharm
Bull;25:844-6.1977.
Sakamoto I. Quantitative determination of Stevia glycosides in a soft drink. Hiroshima-Ken Eisei
Kenkyusho Kenkyu Hokoku;30:11-14. 1983.
Sasaki K. Application of Stevia sweetener to soft drinks. New Food Ind; 25(4):38-43. 1983.
page 48 of 64
Sasaki YF, Kawaguchi S, Kamaya A, Ohshita M, Kabasawa K, Iwama K, Taniguchi K, Tsuda S. The
comet assay with 8 mouse organs: results with 39 currently used food additives. Mutation
Research;519:103-19. 2002. We determined the genotoxicity of 39 chemicals currently in use as
food additives. They fell into six categories-dyes, color fixatives and preservatives, preservatives,
antioxidants, fungicides, and sweeteners. We tested groups of four male ddY mice once orally with each
additive at up to 0.5xLD(50) or the limit dose (2000mg/kg) and performed the comet assay on the
glandular stomach, colon, liver, kidney, urinary bladder, lung, brain, and bone marrow 3 and 24h
after treatment. Of all the additives, dyes were the most genotoxic. Amaranth, Allura Red, New Coccine,
Tartrazine, Erythrosine, Phloxine, and Rose Bengal induced dose-related DNA damage in the glandular
stomach, colon, and/or urinary bladder. All seven dyes induced DNA damage in the gastrointestinal
organs at a low dose (10 or 100mg/kg). Among them, Amaranth, Allura Red, New Coccine, and
Tartrazine induced DNA damage in the colon at close to the acceptable daily intakes (ADIs). Two
antioxidants (butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)), three fungicides
(biphenyl, sodium o-phenylphenol, and thiabendazole), and four sweeteners (sodium cyclamate,
saccharin, sodium saccharin, and sucralose) also induced DNA damage in gastrointestinal organs. Based
on these results, we believe that more extensive assessment of food additives in current use is
warranted.
Schvartzman JB, Krimer DB, Moreno AR. Stevia rebaudiana (ka'aa-he'e) and the cell cycle of
allium cepa. Rev Soc Cient;15:51. 1975.
Schvartzman JB, Krimer DB, Moreno AR. Cytological effects of some medicinal plants used in the
control of fertility. Experientia;33:663.1977.
Sclafani A, Bahrani M, Zukerman S, Ackroff K. Stevia and Saccharin Preferences in Rats and Mice.
Chem Sneses. Apr 22 2010. Use of natural noncaloric sweeteners in commercial foods and beverages
has expanded recently to include compounds from the plant Stevia rebaudiana. Little is known about the
responses of rodents, the animal models for many studies of taste systems and food intake, to stevia
sweeteners. In the present experiments, preferences of female Sprague-Dawley rats and C57BL/6J mice
for different stevia products were compared with those for the artificial sweetener saccharin. The stevia
component rebaudioside A has the most sweetness and least off-tastes to human raters. In ascending
concentration tests (48-h sweetener vs. water), rats and mice preferred a high-rebaudioside, lowstevioside extract as strongly as saccharin, but the extract stimulated less overdrinking and was much
less preferred to saccharin in direct choice tests. Relative to the extract, mice drank more pure
rebaudioside A and showed stronger preferences but still less than those for saccharin. Mice also
preferred a commercial mixture of rebaudioside A and erythritol (Truvia). Similar tests of sweet receptor
T1R3 knockout mice and brief-access licking tests with normal mice suggested that the preferences were
based on sweet taste rather than post-oral effects. The preference response of rodents to stevia
sweeteners is notable in view of their minimal response to some other noncaloric sweeteners (aspartame
and cyclamate).
SCF (Scientific Committee on Food), 1984. Reports of the Scientific Committee for Food
Concerning Sweeteners (Opinion Expressed by the SCF on 14 September 1984). In: Food Science
and Techniques. Brussels, Belgium: Commission of the European Communities (EEC), Health &
Consumer Protection Directorate-General, Scientific Committee on Food, 1985. (Reports of the
Scientific Committee for Food (16th series).
SCF (Scientific Committee on Food), 1989. Reports of the Scientific Committee for Food on
Sweeteners. 21th series. Opinion expressed 11 December 1987 and 10 November 1988, and
adopted 10 November 1988.
SCF (Scientific Committee on Food), 1999. Opinion on Stevioside as a Sweetener (Adopted on
17/6/99). Belgium: European Commission, Health & Consumer Protection Directorate-General,
Scientific Committee on Food. [CS/ADD/EDUL/167 Final].
Sekihashi K, Saitoh H, Sasaki Y. Genotoxicity studies of Stevia extract and steviol by the comet
assay. J Toxicol Sci;27 Suppl 1: 1-8.2002 The genotoxicity of steviol, a metabolite of stevia extract, was
page 49 of 64
evaluated for its genotoxic potential using the comet assay. In an in vitro study, steviol at 62.5, 125, 250,
and 500 micrograms/ml did not damage the nuclear DNA of TK6 and WTK1 cells in the presence and
absence of S9 mix. In vivo studies of steviol were conducted by two independent organizations. Mice
were sacrificed 3 and 24 hr after one oral administration of steviol at 250, 500, 1000, and 2000 mg/kg.
DNA damage in multiple mouse organs was measured by the comet assay as modified by us. After oral
treatment, stomach, colon, liver, kidney and testis DNA were not damaged. The in vivo genotoxicity of
stevia extract was also evaluated for its genotoxic potential using the comet assay. Mice were sacrificed 3
and 24 hr after oral administration of stevia extract at 250, 500, 1000, and 2000 mg/kg. Stomach, colon
and liver DNA were not damaged. As all studies showed negative responses, stevia extract and steviol
are concluded to not have DNA-damaging activity in cultured cells and mouse organs.
Seidemann J. Stevioside, an interesting natural sweetening agent. Nahrung;20:675.1976.
Seidemann J. Naturally occurring sweetening agents. Lebensm Ind;23:553.1976.
Sekihashi K, Saitoh H, Sasaki YF. Genotoxicity studies of Stevia extract and steviol by the comet
assay. Journal of Toxicological Sciences;27:1-8. 2002. The genotoxicity of steviol, a metabolite of
stevia extract, was evaluated for its genotoxic potential using the comet assay. In an in vitro study, steviol
at 62.5, 125, 250, and 500 micrograms/ml did not damage the nuclear DNA of TK6 and WTK1 cells in the
presence and absence of S9 mix. In vivo studies of steviol were conducted by two independent
organizations. Mice were sacrificed 3 and 24 hr after one oral administration of steviol at 250, 500, 1000,
and 2000 mg/kg. DNA damage in multiple mouse organs was measured by the comet assay as modified
by us. After oral treatment, stomach, colon, liver, kidney and testis DNA were not damaged. The in vivo
genotoxicity of stevia extract was also evaluated for its genotoxic potential using the comet assay. Mice
were sacrificed 3 and 24 hr after oral administration of stevia extract at 250, 500, 1000, and 2000 mg/kg.
Stomach, colon and liver DNA were not damaged. As all studies showed negative responses, stevia
extract and steviol are concluded to not have DNA-damaging activity in cultured cells and mouse organs.
Sehar I, Kaul A, Bani S, Pal HC, Saxena AK. Immune up regulatory response of a non-caloric
natural sweetener, stevioside. Chem Biol Interact. 173(2):115-21, 2008. Immunomodulation is a
process, which alters the immune system of an organism by interfering with its functions. This
interference results in either immunostimulation or immunosuppression. An immunomodulator is any
substance that helps to regulate the immune system. This "regulation" is a normalization process, so that
an immunomodulator helps to optimise immune response. Immunomodulators are becoming very popular
in the worldwide natural health industry as these do not tend to boost immunity, but to normalize it.
Keeping this in view, major efforts have to be directed to modulate the immune responses, to permit
effective treatment of various ailments associated with immune system and thus the development of a
safe and effective immunomodulator for clinical us. Leaves of Stevia rebaudiana are a source of several
sweet glycosides of steviol. The major glycoside, stevioside, diterpenoid glycoside--is used in oriental
countries as a food sweetener. Its medical use is also reported as a heart tonic. Besides, it is used
against obesity, hypertension, and stomach burn and to lower uric acid levels. Here in this study,
stevioside was tested for its immunomodulatory activity on different parameters of the immune system at
three different doses (6.25, 12.5 and 25 mg/kg p.o.) on normal as well as cyclophosphamide treated
mice. Stevioside was found effective in increasing phagocytic activity, haemagglutination antibody titre
and delayed type hypersensitivity. In parallel, stevioside substantially increase proliferation in the LPS
and Con A stimulated B and T cells, respectively. Present study, therefore, reveals that the drug holds
promise as immunomodulating agent, which acts by stimulating both humoral as well as cellular immunity
and phagocytic function.
Shiotsu S. Fertility study of Stevia decoction in rats. Technical Journal of Food Chemistry and
Chemicals;4: 108-113. 1996.
Shiozaki K, Fujii A, Nakano T, Yamaguchi T, Sato M. Inhibitory effects of hot water extract of the
Stevia stem on the contractile response of the smooth muscle of the guinea pig ileum. Biosci
Biotechnol Biochem. 70(2):489-94, 2006. The effects of a hot water extract of the stem of Stevia
rebaudiana on the smooth muscle of isolated guinea pig ileum were investigated. The butyl alcohol layer
page 50 of 64
of the extract antagonized the contractions of the isolated guinea pig ileum induced by histamine (1 x 10(5) M) and acetylcholine (1 x 10(-5) M) in a concentration-dependent manner. The butyl alcohol layer of
the extract also showed inhibition of CaCl(2) (1 x 10(-3)-3.8 x 10(-1) M)-induced contractions. The
antagonism of the extract was considered to be non-specific, but this action might be related to an influx
of extracellular Ca(2+). With column chromatography preparation, the active component was assumed to
be as stevioside. The antagonistic effects exerted by the stem extract of Stevia rebaudiana contributed to
the gastroprotective activity of the extract in animals fed dietary histamine.
Shirakawa T, Onishi T. Quantitative analysis of stevioside in soy sauce and of vegetable products.
Kagawa-Ken Hakko Shokuhin Shikenjo Hokoku;71:35-39.1979.
Sholichin M, Yamasaki K, Miyama R, Yahara S, Tanaka O. Labdane-type diterpenes from Stevia
rebaudiana. Phytochemistry;19:326-7.1980.
Shukla S, Mehta A, Bajpai VK, Shukla S. In vitro antioxidant activity and total phenolic content of
ethanolic leaf extract of Stevia rebaudiana Bert. Food Chem Toxicol. 47(9):2338-43, 2009. The aim
of this study was to assess the in vitro potential of ethanolic leaf extract of Stevia rebaudiana as a natural
antioxidant. The DPPH activity of the extract (20, 40, 50, 100 and 200 microg/ml) was increased in a dose
dependent manner, which was found in the range of 36.93-68.76% as compared to ascorbic acid 64.2682.58%. The IC(50) values of ethanolic extract and ascorbic acid in DPPH radical scavenging assay were
obtained to be 93.46 and 26.75 microg/ml, respectively. The ethanolic extract was also found to scavenge
the superoxide generated by EDTA/NBT system. Measurement of total phenolic content of the ethanolic
extract of S. rebaudiana was achieved using Folin-Ciocalteau reagent containing 61.50 mg/g of phenolic
content, which was found significantly higher when compared to reference standard gallic acid. The
ethanolic extract also inhibited the hydroxyl radical, nitric oxide, superoxide anions with IC(50) values of
93.46, 132.05 and 81.08 microg/ml, respectively. However, the IC(50) values for the standard ascorbic
acid were noted to be 26.75, 66.01 and 71.41 microg/ml respectively. The results obtained in this study
clearly indicate that S. rebaudiana has a significant potential to use as a natural antioxidant agent.
Sincholle D, Macroelles P. Etude de l'activité anti-androgénique d'un extrait de Stevia rebaudiana
Bertoni = [The anti-androgenic activity of Stevia rebaudiana Bertoni extract]. Plant Medical
Phytotherapy;23:282-287.1989.
Sinchomi D, Marcorities P. Etude de l'activité anti-androgénique d'un extrait de Stevia rebaudiana
Bertoni. Plantes médicinales et phytothérapie;23(4):282-87. 1989.
Soejarto DD, Kinghorn AD, Farnsworth NR. Potential sweetening agents of plant origin. III.
Organoleptic evaluation of Stevia leaf herbarium specimens for sweetness. J Nat Prod;45(5):59099.1982.
Soejarto DD, Compadre CM, Medon PJ, Kamath SK, Kinghorn AD. Potential sweetening agents of
plant origin. 2. Field search for sweet-tasting Stevia species. Econ Bot;37(1):71-9.1983. A total of
184 Stevia leaf samples taken from herbarium specimens, representing 110 species and 121 taxa, were
screened organoleptically for their taste sensation. Fragments of a 62-year-old leaf of S. rebaudiana
exhibited a potent and prolonged sensation of sweetness, thereby indicating the stability of its sweet entkaurene glycoside constituents to drying, preservation, mounting and storage. No other leaf samples
exhibited an intensity of sweetness equivalent to that of S. rebaudiana, though 18 species and varieties
were considered to exhibit a sweet taste. These taxa appear to be promising candidates for
future phytochemical investigation for new and known ent-kaurene glycosides.
Soejarto DD, Compadre CM, Kinghorn AD. Ethnobotanical notes on Stevia. Bot Mus Leafl Harv
Univ;29(1):1-25.1983.
Srimaroeng C, Chatsudthipong V, Aslamkhan AG, Pritchard JB. Transport of the natural
sweetener stevioside and its aglycone steviol by human organic anion transporter (hOAT1;
SLC22A6) and hOAT3 (SLC22A8). J Pharmacol Exp Ther. 313(2):621-8, 2005. The natural sweetening
page 51 of 64
agent stevioside and its aglycone metabolite, steviol, have been shown to inhibit transepithelial transport
of para-aminohippurate (PAH) in isolated rabbit renal proximal tubules by interfering with basolateral
entry. The aim of the present study was to determine which of the cloned basolateral organic anion
transporters were involved in the renal transport of stevioside and steviol. This question was addressed in
Xenopus laevis oocytes expressing human organic anion transporter 1 (hOAT1), 3 (hOAT3), and winter
flounder OAT (fOat1). The parent compound, stevioside, had no inhibitory effect on either PAH (hOAT1)
or ES (estrone sulfate; hOAT3) uptake. In contrast, steviol showed significant, dose-dependent inhibition
of PAH and ES uptake in hOAT1- or hOAT3-expressing oocytes, respectively. The IC(50) of steviol for
hOAT1-mediated PAH transport was 11.1 microM compared with 62.6 microM for hOAT3-mediated ES
uptake. The Michaelis-Menten inhibition constants (K(i)) for steviol transport mediated by hOAT1 and
hOAT3 were 2.0 +/- 0.3 and 5.4 +/- 2.0 microM, respectively. Trans-stimulation of PAH efflux by steviol
was assessed to determine whether steviol itself was transported by hOAT1 or hOAT3. A low
concentration of 1 microM steviol increased the efflux of [(3)H]PAH (trans-stimulated) via both hOAT1 and
hOAT3. In addition, it was shown by electrophysiology that steviol entry induced inward current in fOat1expressing oocytes. In conclusion, stevioside had no interaction with either hOAT1 or hOAT3, whereas
hOAT1, hOAT3, and fOat1 were all shown to be capable of steviol transport and thus, can play a role in
its renal transport and excretion.
Srimaroeng C, Jutabha P, Pritchard JB, Endou H, Chatsudthipong V. Interactions of stevioside
and steviol with renal organic anion transporters in S2 cells and mouse renal cortical slices.
Pharm Res. 22(6):858-66, 2005. PURPOSE: Our previous studies have shown that both stevioside and
steviol inhibited transepithelial transport of para-aminohippurate (PAH) in isolated rabbit renal proximal
tubules by interfering with organic anion transport system. The current study examined the direct
interactions of stevioside and steviol with specific organic anion transporters. METHODS: S2 cells
expressing human organic anion transporters (hOAT1, hOAT2, hOAT3, and hOAT4) and an intact renal
epithelium were used to determine the inhibitory effect of stevioside and steviol on organic anion
transport. RESULTS: Stevioside at 0.5-1 mM showed no interaction with any OAT. In contrast, steviol
markedly inhibited substrate uptake in all S2hOAT cells. Steviol had low IC50 for hOAT1 (11.4 microM)
and hOAT3 (36.5 microM) similar to that of probenecid, whereas IC50 for hOAT2 (1000 microM) and
hOAT4 (285 microM) was much higher. Results obtained in mouse renal cortical slices were very similar;
that is, stevioside was without inhibitory effect and steviol was a potent inhibitor of PAH and estrone
sulfate (ES) transport.CONCLUSIONS: Stevioside has no interaction with human or mouse OATs. In
contrast, steviol interacts directly with human OATs, in particular, hOAT1 and hOAT3, with a potency
approximating probenecid, suggesting that the inhibition of OAT-mediated transport by steviol could alter
renal drug clearance.
Starratt AN, Kirby CW, Pocs R, Brandle JE. Rebaudioside F, a diterpene glycoside from Stevia
rebaudiana. Phytochemistry;59(4):367-370.2002. The sweet diterpenoid glycoside, rebaudioside F,
was isolated from leaves of a high rebaudioside C producing line of Stevia rebaudiana, and its structure
was established by chemical and spectral studies.
Striedner J, Gutjahr E, Czygan FC, Braunegg G. Contributions to the biotechnological production
of sweeteners from Stevia rebaudiana Bertoni. II. Induction of stevioside accumulation in cell
cultures by variation of the nutrient medium and the analysis of small amounts of stevioside. Acta
Biotechnol; 11(5):501-4.1991.
Suanarunsawat T, Chaiyabutr N. The effect of stevioside on glucose metabolism in the rat.
Canadian Journal of Physiology and Pharmacology;75:976-82.1997. This study was conducted to
determine the effect of stevioside (SVS) on glucose metabolism. The experiments were performed in
male Wistar rats treated with SVS either by intravenous infusion or feeding. SVS infusion (150 mg/mL)
was carried out in doses of 0.67, 1.00, and 1.33 mL.kg-1 body weight.h-1. The plasma glucose level
significantly increased both during and after SVS infusion, whereas it was not affected by SVS feeding
(13.3 mL.kg-1 body weight). The glucose turnover rate (GTR) of [14C(U)]glucose and [3(-3)H]glucose
was not significantly different between control and SVS infusion animals. Percent glucose carbon
recycling and glucose clearance were reduced from 28.7 +/- 1.3 to 23.0 +/- 1.6% (p < 0.05) and from
6.46 +/- 0.34 to 4.99 +/- 0.20 mL.min-1.kg-1 body weight (p < 0.01), respectively. The plasma insulin level
page 52 of 64
did not change, whereas the plasma glucose level significantly increased from 120.3 +/- 5.9 to 176.8 +/10.8 mg% (p < 0.01) during SVS infusion. Animals pretreated with angiotensin II and arginine vasopressin
showed no significant effect, while animals pretreated with prazosin had an attenuated hyperglycemic
effect of SVS infusion. Pretreatment with indomethacin or N omega-nitro-L-arginine methyl ester (LNAME) alleviated the plasma glucose level during the second period of SVS infusion. Pretreatment
with the combination infusion of indomethacin and L-NAME reduced the plasma glucose level from 117.0
+/- 1.8 to 109.0 +/- 1.7 mg% (p < 0.001), and normalized the plasma glucose level in the second period of
SVS infusion. Insulin infusion inhibited the hyperglycemic effect of SVS infusion. The present results
show that the elevation of the plasma glucose level during SVS infusion is not due to the reduction of the
insulin level. It is probably the effect of SVS on glucose transport across the cell. Insulin response to a
high plasma glucose level is suppressed during SVS infusion. Several interactions among
norepinephrine, prostaglandin, and nitric oxide are involved in modulating the hyperglycemia during SVS
infusion.
Sugisawa H, Kasai T, Suzuki H. The modified quantitative analysis of stevioside. Nippon Nogei
Kagaku Kaishi;51:175-77.1977.
Sumida T. Reports on Stevia rebaudiana introduced from Brazil as a new sweetness resource in
Japan. Hokkaido Agr Exp Sta Bull;2:69-83.1973.
Suttajit M, Vinitketkaumnuen U, Meevatee U, Buddhasukh D. Mutagenicity and human
chromosomal effect of stevioside, a sweetener from Stevia rebaudiana Bertoni. Environmental
Health Perspectives;101:53-6. 1993. Leaves of Stevia rebaudiana Bertoni have been popularly used as
a sweetener in foods and beverages for diabetics and obese people due to their potent
sweetener stevioside. In this report, stevioside and steviol were tested for mutagenicity in Salmonella
typhimurium strains TA98 and TA100 and for chromosomal effects on cultured human lymphocytes.
Stevioside was not mutagenic at concentrations up to 25 mg/plate, but showed direct mutagenicity to
only TA98 at 50 mg/plate. However, steviol did not exhibit mutagenicity in either TA98 or TA100, with or
without metabolic activation. No significant chromosomal effect of stevioside and steviol was observed in
cultured blood lymphocytes from healthy donors (n = 5). This study indicates that stevioside and steviol
are neither mutagenic nor clastogenic in vitro at the limited doses; however, in vivo genotoxic tests and
long-term effects of stevioside and steviol are yet to be investigated.
Suzuki H, Kasai T, Sumiiara M, Sugisawa H. Influence of oral administration of stevioside on levels
of blood glucose and liver glycogen of intact rats. Nippon Nogeikagaku Kaishi;51:171-3.1977.
Suzuki H, Kasai T, Sumihara M, Suginawa H. Influence of the oral administration of stevioside on
the levels of blood glucose and liver glycogen in intact rats. Nogyo Kagaku Zasshi;51(3):45.1977.
Switzerland Office of Public Health, 2008,
http.www.bag.admin.ch/themen/lebensmittel/04861/04972/index.html?lang=fr.
Takahashi K, Matsuda M, Ohashi K, Taniguchi K, Nakagomi O, Abe Y, Mori S, Sato N, Okutani K,
Shigeta S. Analysis of anti-rotavirus activity of extract from Stevia rebaudiana. Antiviral Res.
49(1):15-24, 2001. Anti-human rotavirus (HRV) activity of hot water extracts from Stevia rebaudiana (SE)
was examined. SE inhibited the replication of all four serotypes of HRV in vitro. This inhibitory effect of SE
was not reduced on the prior exposure of SE to HCl for 30 min at pH 2. Binding assay with radiolabeled
purified viruses indicated that the inhibitory mechanism of SE is the blockade of virus binding. The SE
inhibited the binding of anti-VP7 monoclonal antibody to HRV-infected MA104 cells. The inhibitory
components of SE were found to be heterogeneous anionic polysaccharides with different ion charges.
The component analyses suggested that the purified fraction named as Stevian with the highest inhibitory
activity consists of the anionic polysaccharide with molecular weight of 9800, and contains Ser and Ala as
amino acids. Analyses of sugar residues suggest uronic acid(s) as sugar components. It did not contain
amino and neutral sugars and sulfate residues. These findings suggest that SE may bind to 37 kD VP7
and interfere with the binding of VP7 to the cellular receptors by steric hindrance, which results in the
blockade of the virus attachment to cells.
page 53 of 64
Takasaki M, Konoshima T, Kozuka M, Tokuda H, Takayasu J, Nishino H, Miyakoshi M,
Takasaki M, Konoshima T, Kozuka M, Tokuda H, Takayasu J, Nishino H, Miyakoshi M, Mizutani K,
Lee KH. Cancer preventive agents. Part 8: Chemopreventive effects of stevioside and related
compounds. Bioorg Med Chem. 17(2):600-5, 2009. In a search for potential cancer chemopreventive
agents from natural resources, stevioside (1), a sweetener, and six related compounds, including two
aglycones steviol (6) and isosteviol (7), were screened in an in vitro assay for inhibitory effects on
Epstein-Barr virus early antigen activation. Compounds 1, 6 and 7 showed significant activity in this assay
and also exhibited strong inhibitory effects in a two-stage carcinogenesis test using mouse skin induced
by 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The
inhibitory effects of these three compounds were greater than that of glycyrrhizin. Furthermore, these
three compounds significantly inhibited mouse skin carcinogenesis initiated by peroxynitrite and promoted
by TPA. Their activities were comparable to that of curcumin. These results suggested that 1, as well as 6
and 7, could be valuable as chemopreventive agents for chemical carcinogenesis.
Takaki M, De Campos Takaki GM, De Santana Diu MB, De Andrade MSS, Da Silva EC.
Antimicrobial activity in leaves extracts of Stevia rebaudiana Bert. Rev Inst Antibiot Univ Fed
Pernambuco Recife;22 1/2:33-9.1985.
Tanaka O. Chemistry of Stevia rebaudiana Bertoni, new source of natural sweetners (abstract).
Ann Rep Nat Prod Res Inst Seoul Natl Univ;18:146-B.1979.
Tanaka O. Chemistry of Stevia rebaudiana Bertoni. New source of natural sweeteners. Korean J
Pharmacog;11:219-27.1980.
Tanaka O. Steviol Glycosides: A New Natural Sweetner. Trends Anal Chem;1(11):246-8.1982.
Temcharoen P, Klopanichpah S, Glinsukon T, Suwannatrai M, Apibal S, Toskulkao C. Evaluation
of the effect of steviol on chromosomal damage using micronucleus test in three laboratory
animal species. Journal of the Medical Association of Thailand;83: s101-s108. 2000. The
chromosomal damage activity of steviol, a product of enzymatic alteration of stevioside, a natural noncaloric sweetener was reevaluated by using a bone marrow micronucleus test in both male and female
hamsters, rats and mice. The micronucleus test is used widely as a rapid and efficient alternative
in chromosome analysis for detecting in vivo cytogenetic damage. Steviol at the dose of 4 g/kg body
weight for hamsters and 8 g/kg body weight for rats and mice showed no effect on the frequencies of
micronucleus formation in bone marrow erythrocytes of both male and female hamsters, rats and mice.
Moreover, there was also no apparent change in the PCEs:NCEs (polychromatic erythrocytes:
normochromatic erythrocytes) ratio of the male animals of all three treated species at 24, 30, 48 and 72
hour intervals. However, steviol at the given dose can cause significant reduction of PCEs to NCEs ratio
of the female hamsters at 72 hours and female rats and mice at 48 and 72 hours after receiving steviol
orally. From these results, it could be proposed that steviol at the given dose to the treated animals
produced adverse metabolites and these metabolites could reach the bone marrow, the target organ for
micronucleus test. These metabolites also exhibited a slightly cytotoxic effect but not clastogenic effect
to the bone marrow erythrocytes.
Terai T, Ren H, Mori G, Yamaguchi Y, Hayashi T. Mutagenicity of steviol and its oxidative
derivatives in Salmonella typhimurium TM677. Chem Pharm Bull (Tokyo). 50(7):1007-10, 2002.
Stevioside is natural non-caloric sweetner isolated from Stevia rebaudiana BERTONI, which has been
used as a non-caloric sugar substitute in Japan. Pezzuto et al. demonstrated that steviol shows a dosedependent positive response in forward mutation assay using Salmonella typhimurium TM677 in the
presence of metabolic activation system (Aroclor induced rat liver S9 fraction). Our studies were carried
out to identify the genuine mutagenic active substance from among the eight steviol derivatives. Steviol
indicate almost similar levels of mutagenicity under the presence of S9 mixture, as reported by Pezzuto et
al.15-Oxo-steviol was found to be mutagenic at the one tenth the level of steviol itself under the presence
page 54 of 64
of S9 mixture. Interestingly, specific mutagenicity of the lactone derivative under the presence of S9
mixture was ten times lower than that of the lactone derivative without the addition of S9 mixture.
Tomita T, Sato N, Arai T, Shiraishi H, Sato M, Takeuchi M, Kamio Y. Bactericidal activity of a
fermented hot-water extract from Stevia rebaudiana Bertoni towards enterohemorrhagic
escherichia coli 0157:H7 and other food-borne pathogenic bacteria. Microbiol
Immunol;41(12):1005-9.1997. A fermented aqueous extract from Stevia rebaudiana Bertoni showed
strong bactericidal activity towards a wide range of food-borne pathogenic bacteria including
enterohemorrhagic Escherichia coli O157:H7. The colony-forming ability of the food-borne pathogenic
bacteria tested so far was reduced to < 10(-7) when exposed to > or = 40% (v/v) solutions of the
fermented extract at 37 C for 2 hr. Secretion of verocytotoxin 1 and 2 by enterohemorrhagic E. coli was
also diminished by fermented extract at a concentration of > or = 10% (v/v). In contrast, the fermented
extract did not significantly kill Bifidobacteria or Lactobacilli. The active principle(s) of the fermented
Stevia extract were bactericidal under acidic conditions.
Tomoyoshi E, Yamamoto S, Ikeda T. Degradation of stevioside in raw soy sauce and chemical
structure of the degraded product. Nippon Jozo Kyokaishi; 86(1):68-74. (Japanese) Abstract only.
1991.
Toskulkao C, Deechakawan WV, Temcharoen P, Buddhasukh D, Glinsukon T. Nephrotoxic effects
of stevioside and steviol in rat renal cortical slices. Journal of Clinical Biochemistry and
Nutrition;16:123-131. 1994a.
Toskulkao C, Sutheerawattananon M. Effects of stevioside, a natural sweetener, of intestinal
glucose absorption in hamsters. Nutrition Research; 14:1711-20. 1994b.
Toskulkao C, Deechakawan W, Leardkamolkarn V, Glinsukon T, Buddhasukh D. The low calorie
natural sweetener stevioside: nephrotoxicity and its relationship to urinary enzyme excretion in the
rat. Phytother Res 8:281-6.1994a.
Toskulkao C, Sutheerawatananon M, Wanichanon C, Saitongdee P, Suttajit M. Effects of
stevioside and steviol on intestinal glucose absorption in hamsters. J Nutritional Science and
Vitaminology; 41:105-13.1995a. The effects of stevioside and steviol (a product of enzymatic hydrolysis
of stevioside) on intestinal glucose absorption were examined in the hamster jejunum in vitro. By using
the jejunal rings technique, we found that stevioside at a high dose of 5 mM had no inhibitory effect on
glucose absorption. In contrast, glucose absorption was inhibited 43% by 1 mM steviol. The inhibition of
glucose absorption by steviol was related to steviol concentration and incubation time. The inhibitory
effect of steviol compared to phlorizin and ouabain was also investigated. Steviol, which caused a
decrease in glucose accumulation in the intestinal ring tissues, possibly acts on the brush border
membrane as does phlorizin. Furthermore, it was also found that steviol altered the morphology of the
intestinal absorptive cells. These results suggest that the possible site of inhibitory action of steviol might
be on the mucosal side and/or at the intracellular organelles of intestinal absorptive cells.
Toskulkao C, Sutheerawattananon M, Piyachaturawat P. Inhibitory effect of steviol, a metabolite of
stevioside, on glucose absorption in everted hamster intestine in vitro. Toxicology Letters;80:1539. 1995b. The effects of stevioside and steviol (a product of enzymatic hydrolysis of stevioside) on
intestinal glucose absorption were examined in hamster jejunum. By using the everted sac technique, we
found that stevioside (1 and 5 mM) had no inhibitory effect on glucose absorption. In contrast, glucose
absorption was inhibited 29% by 1 mM steviol. The inhibition of glucose absorption by steviol was related
to steviol concentration and incubation time. The possible mechanism of steviol inhibitory action of
glucose absorption was also investigated. Reductions in the intestinal mucosal ATP content and
absorptive surface area were responsible for the inhibition of glucose absorption by steviol. The decrease
in the intestinal mucosal ATP content was accompanied by a decrease in the activities of mitochondrial
NADH cytochrome c reductase and cytochrome oxidase. Moreover, no inhibitory effects of steviol on the
activity of intestinal Na+,K(+)-ATPase and glucose uptake in the intestinal brush-border
membrane vesicles were seen. These results suggest that inhibition of intestinal glucose absorption by
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steviol in hamsters is due to the reduction in mucosal ATP content and an alteration of the morphology of
the intestinal absorptive cells.
Toskulkao C, Chaturat L, Temcharoen P, Glinsukon T. Acute toxicity of stevioside, a natural
sweetener, and its metabolite, steviol, in several animal species. Drug and Chemical
Toxicology;20:31-44.1997. The acute toxicity of stevioside and steviol (a product of enzymatic
hydrolysis of stevioside) was investigated in three animal species including rat, mouse and hamster. The
susceptibility to stevioside and steviol acute toxicity in both sexes of these animal species was compared.
The animals were treated intragastrically with stevioside or steviol and general signs and symptoms
were observed. The numbers of dead animals were recorded within a period of 14 days after
administration for estimation of LD50. Stevioside at a dose as high as 15 g/kg BW was not lethal to either
mice, rats or hamsters. Hamsters were found to be more susceptible to steviol than rats or mice. LD50
values of steviol in hamsters were 5.20 and 6.10 g/kg BW for males and females, respectively. In
rats and mice, LD50 values of steviol were higher than 15 g/kg BW in both sexes. Histopathological
examination in the kidney of hamsters induced by steviol revealed severe degeneration of the proximal
tubular cells. These structural alterations were correlated with the increases in serum blood urea nitrogen
(BUN) and creatinine. Therefore, the possible cause of death induced by steviol might be due to acute
renal failure.
Toyoda K, Matsui H, Shoda T, Uneyama C, Takada K, Takahashi M. Assessment of the
carcinogenicity of stevioside in F344 rats. Food and Chemical Toxicology;35:597-603. 1997. The
carcinogenic potential of stevioside, a compound that is used as a sweetener for food and drink, was
examined in F344 rats of both sexes. Stevioside was added to powdered diet at concentrations of 0
(control), 2.5 and 5%. The doses were selected on the basis of results from a 13-wk subchronic toxicity
study and administered to groups of 50 male and 50 female rats ad lib. for 104 wk. All surviving rats were
killed at wk 108. Body weight gains were slightly depressed in line with the dose of stevioside, in both
sexes, and a significant decrease in the final survival rate was observed for the 5% treated
males. Histopathologically, however, there was no significantly altered development of neoplastic or nonneoplastic lesions attributable to the stevioside treatment in any organ or tissue, except for a decreased
incidence of mammary adenomas in females and a reduced severity of chronic nephropathy in males. It
is concluded that stevioside is not carcinogenic in F344 rats under the experimental conditions described.
Tsanava VP, Sardzhyeladze GP, Kharebava LG. Effect of technological procedures on the
composition of volatile substances in Stevia rebaudiana. Subtrop Kult;3:64-70. 1991.
Ukiya M, Sawada S, Kikuchi T, Kushi Y, Fukatsu M, Akihisa T. Cytotoxic and apoptosis-inducing
activities of steviol and isosteviol derivatives against human cancer cell lines. Chem Biodivers.
10(2):177-88, 2013. Seventeen steviol derivatives, i.e., 2-18, and 19 isosteviol derivatives, i.e., 19-37,
were prepared from a diterpenoid glycoside, stevioside (1). Upon evaluation of the cytotoxic activities of
these compounds against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer
cell lines, nine steviol derivatives, i.e., 5-9 and 11-14, and five isosteviol derivatives, i.e., 28-32, exhibited
activities with single-digit micromolar IC(50) values against one or more cell lines. All of these active
compounds possess C(19)-O-acyl group, and among which, ent-kaur-16-ene-13,19-diol 19-O-4',4',4'trifluorocrotonate (14) exhibited potent cytotoxicities against four cell lines with IC(50) values in the range
of 1.2-4.1 μM. Compound 14 induced typical apoptotic cell death in HL60 cells upon evaluation of
the apoptosis-inducing activity by flow-cytometric analysis. These results suggested that acylation of the
19-OH group of kaurane- and beyerane-type diterpenoids might be useful for enhancement of their
cytotoxicities with apoptosis-inducing activity.
Ulbricht C, Isaac R, Milkin T, Poole EA, Rusie E, Grimes Serrano JM, Weissner W, Windsor RC,
Woods J. An evidence-based systematic review of stevia by the natural standard research
collaboration. Cardiovasc Hematol Agents Med Chem ;8(2):113-27, 2010. The objective of this study
was to evaluate the scientific evidence on stevia, including expert opinion, folkloric precedent, history,
pharmacology, kinetics/dynamics, interactions, adverse effects, toxicology, and dosing. This review
serves as a clinical support tool. Electronic searches were conducted in 10 databases, 20 additional
journals (not indexed in common databases), and bibliographies from 50 selected secondary references.
page 56 of 64
No restrictions were placed on the language or quality of the publications. All literature collected pertained
to efficacy in humans, dosing, precautions, adverse effects, use in pregnancy and lactation, interactions,
alteration of laboratory assays, and mechanisms of action. Standardized inclusion and exclusion criteria
were used for selection. Grades were assigned using an evidence-based grading rationale. Based on the
availability of scientific data, two indications are discussed in this review: hypertension and
hyperglycemia. Evaluation of two long-term studies (1 and 2 years in length, respectively) indicates that
stevia may be effective in lowering blood pressure in hypertensive patients, although data from shorter
studies (1-3 months) did not support these findings. A pair of small studies also report positive results with
respect to glucose tolerance and response, although the relatively low methodological rigor of these
experiments limits the strength of these findings. Further investigation is warranted in both indications.
USDA Center for Nutrition Policy and Promotion. Americans Consume Too Many Calories From
Solid Fat, Alcohol,and Added Sugar. Nutrition Insight 33. June 2006. Accessed at
http://www.cnpp.usda.gov/Publications/NutritionInsights/Insight33.pdf#xml=http://65.216.150.153/t
exis/search/pdfhi.txt?query=discretionary+calorie&pr=MyPyramid&rdepth=0&sufs=2&order=r&cq
=&id=4bc8cd0b112. May 27, 2010.
USDA Food and Inspection Service. Report of the U.S. Delegate, 41st Session, Codex Committee
on Food Additives (CCFA). Accessed at
http://www.fsis.usda.gov/codex_alimentarius/Delegate_Report_41CCFA/index.asp.
USDA Food & Nutrition Information Center. Accessed at
http://fnic.nal.usda.gov/nal_display/index.php?info_center=4&tax_level=1
US Food and Drug Administration. Has stevia been approved by the USA to be used as a
sweetener? Accessed at http://www.fda.gov/AboutFDA/Basics/ucm194320.htm.
Urban JD, Carakostas MC, Brusick DJ. Steviol glycoside safety: is the genotoxicity database
sufficient? Food Chem Toxicol. 51:386-90, 2013. The safety of steviol glycoside sweeteners has been
extensively reviewed in the literature. National and international food safety agencies and approximately
20 expert panels have concluded that steviol glycosides, including the widely used sweeteners stevioside
and rebaudioside A, are not genotoxic. However, concern has been expressed in recent publications that
steviol glycosides may be mutagenic based on select studies representing a small fraction of the overall
database, and it has been suggested that further in vivo genotoxicity studies are required to complete
their safety profiles. To address the utility of conducting additional in vivo genotoxicity studies, this review
evaluates the specific genotoxicity studies that are the sources of concern, and evaluates the
adequacy of the database including more recent genotoxicity data not mentioned in those publications.
The current database of in vitro and in vivo studies for steviol glycosides is robust and does not indicate
that either stevioside or rebaudioside A are genotoxic. This, combined with a lack of evidence for
neoplasm development in rat bioassays, establish the safety of all steviol glycosides with respect to their
genotoxic/carcinogenic potential.
Usami M, Sakemi K, Kawashima K, Tsuda M, Ohno Y. Teratogenicity study of stevioside in rats.
Bulletin of the National Institute Hygienic Sciences;113:31-5 [in Japanese]. 1995. Teratogenicity of
stevioside was examined in rats. Stevioside dissolved in distilled water was given to pregnant Wistar rats
by gavage once a day from day 6 through 15 of pregnancy at doses of 0, 250, 500 and 1000 mg/kg/day.
The pregnant rats were sacrificed on day 20 of pregnancy and their fetuses were examined
for malformation. Stevioside caused no increased incidences of fetal malformation, and no toxic signs in
the pregnant rats and the fetuses. It was concluded that stevioside has no teratogenicity in rats when
given by gavage. The no observable adverse effect level was estimated to be over 1000 mg/kg/day for
both pregnant rats and rat fetuses.
Valio IF, Rocha RF. Physiological effects of steviol. Z Pflazenphysiol;73:90-4.1976.
Vanek T, Nepovim A, Valicek P. Determination of stevioside in plant material and fruit teas. J Food
Compos Anal:14(4):383-8. 2001.
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Vasovic V, Vukmirovic S, Posa M, Mikov M, Raskovic A, Jakovljevic V. Effect of rat pre-treatment
with aqueous solutions of stevioside and bile acids on the action of certain cardiovascular drugs.
Eur J Drug Metab Pharmacokinet;31(4):311-14. 2006. The interaction of aqueous solutions of
stevioside and bile acids with cardioactive drugs was studied in rats by registering changes in
their electrocardiograms (ECG). Wistar rats of both sexes received daily doses of 20 mg/kg (i.p.) of an
aqueous solution of stevioside or physiological solution (controls), then were narcotized with urethane
and connected to the ECG apparatus for the first recording. The jugular vein was prepared and
connected to an infusion pump to administer one of the drugs: adrenaline (0.1 mg/ml), verapamil (2.5
mg/ml) or metoprolol (1 mg/ml) to rats in both groups, while recording their ECGs. In the second part of
the study, the animals were treated in the same way but instead of the stevioside solution received a
single dose of 4 mg/kg of monoketocholic acid methyl ester (ME) or sodium salt of the same bile
acid (MKHNa), 30 minutes before cardioactive drug infusion. The infusion rate of cardioactive drugs was
0.2 ml/min, except for verapamil (0.1 ml/min). The events observed on ECG recordings were the first
myocardial reaction to drug infusion, the second longer-lasting reaction (observed as more extended
extrasystoles, decrease in intensity of the QRS complex, or changes in heart rate frequency), and toxicity
effect. In the control animals, adrenaline induced a decrease in heart rate frequency at a dose of 0.094
mg/kg, while with stevioside-pretreated rats this effect appeared significantly earlier (at a dose of 0.018
mg/kg). No toxic effect of adrenaline was observed, either in control or stevioside-pretreated group. Bile
acids caused no changes in myocardial reaction to adrenaline. Only in the group of animals that received
MKHNa, a significant decrease in the QRS complex was observed. Finally, the infusion of stevioside
to intact animals at doses of 45 and 55 mg/kg caused no significant changes in the ECG patterns. The
myocardial reaction to metoprolol remained unchanged in rats of all groups when compared with controls
except for a mild decrease in heart rate frequency. Stevioside induced/produced a significant increase in
myocardial sensitivity to verapamil, but no toxic effect was observed in any of the cases. A similar
conclusion also holds for the interaction with MKHNa, whereas ME caused an increase in the toxicity of
verapamil.
von Schmeling GA, Carvalho FN, Espinoza AD. Stevia rebaudiana Bertoni. Evaluation of the
hypoglycemic effect on alloxanized rabbits. Ciencia e Cultura;29: 599-601. 1977.
Wasuntarawat C, Temcharoen P, Toskulkao C, Mungkornkarn P, Suttajit M, Glinsukon T.
Developmental toxicity of steviol, a metabilite of stevioside, in the hamster. Drug and Chemical
Toxicology;21:207-22. 1998. The developmental toxicity of steviol, a metabolite of stevioside, was
studied in hamsters. Pregnant hamsters were intubated with steviol at dose levels of 0, 0.25, 0.5, 0.75
and 1.0 g/kg BW/day on days 6-10 of gestation. Steviol at doses of 0.75 and 1.0 g/kg BW/day were highly
toxic to both dams and fetuses. Significant decrease of maternal body-weight gain during the
experimental period (days 6-14) and high percentage of maternal mortality indicated the general toxicity
of these two high doses. The number of live fetuses per litter and mean fetal weight also significantly
decreased in the steviol-treated animals at doses of 0.75 and 1.0 g/kg BW day. The animals treated with
an intermediate dose (0.50 g/kg BW/day) exhibited less signs of maternal and developmental toxicity
than the two high doses (0.75 and 1.0 g/kg BW/day). One craniomeningocele was found in a fetus under
the maternal toxic condition in steviol-treated at a dose of 0.75 g/kg BW/day. Neither the skeleton nor
visceral development of the offspring was affected by steviol treatment except delayed ossification of the
xiphoid (bifid) and long bones of the limbs and supernumerary thoracic ribs (14th ribs) tended to be
increased at doses of 0.5 to 1.0 g/kg BW/day steviol. No dose-related teratogenesis was detected. From
the result of the present study concerning maternal toxic condition and embryotoxicity, an oral dose of
0.25 g steviol/kg BW/day is regarded as having no observable effect. This steviol-treated dose is derived
from stevioside 625 mg/kg BW/day which is approximately 80 times higher than the suggested
acceptable daily intake of stevioside for humans (7.938 mg/kg BW/day).
Wei Y. A new method for the determination of total glycosides in Stevia rebaudiana. Shih P'in K'o
Hsueh(Beijing);43:25-7.1983.
Wheeler A, Boileau AC, Winkler PC, Compton JC, Prakash I, Jiang X, Mandarino DA.
Pharmacokinetics of rebaudioside A and stevioside after single oral doses in healthy men. Food
page 58 of 64
Chem Toxicol. 46 Suppl 7:S54-60, 2008. This randomized, double-blind, cross-over study assessed the
comparative pharmacokinetics of steviol and steviol glucuronide following single oral doses of
rebaudioside A and stevioside in healthy adult male subjects. Steviol glucuronide appeared in the plasma
of all subjects after administration of rebaudioside A or stevioside, with median tmax values of 12.0 and
8.00h post-dose, respectively. Steviol glucuronide was eliminated from the plasma, with similar t1/2
values of approximately 14h for both compounds. Administration of rebaudioside A resulted in a
significantly (approximately 22%) lower steviol glucuronide geometric mean Cmax value (1472ng/mL)
than administration of stevioside (1886ng/mL). The geometric mean AUC0-t value for steviol glucuronide
after administration of rebaudioside A (30,788ngh/mL) was approximately 10% lower than after
administration of stevioside (34,090ngh/mL). Steviol glucuronide was excreted primarily in the urine of the
subjects during the 72h collection period, accounting for 59% and 62% of the rebaudioside A and
stevioside doses, respectively. No steviol glucuronide was detected in feces. Pharmacokinetic analysis
indicated that rebaudioside A and stevioside underwent similar metabolic and elimination pathways in
humans with steviol glucuronide excreted primarily in the urine and steviol in the feces. No safety
concerns were noted as determined by reporting of adverse events, laboratory assessments of safety or
vital signs.
White JR, Kramer J, Campbell RK, Bernstein R. Oral use of a topical preparation containing an
extract of Stevia rebaudiana and the chrysanthemum flower in the management of hyperglycemia.
Diabetes Care;17(8):940. 1994.
Williams LD, Burdock GA Genotoxicity studies on a high-purity rebaudioside A preparation. Food
Chem Toxicol. 47(8):1831-6, 2009. Rebaudioside A (Reb A) is a steviol glycoside isolated from the
leaves of the Stevia rebaudiana plant. This non-nutritive, natural sweetener is reported to be 250-450
times sweeter than sucrose and has potential for wide use in the US diet, and is used in Japan and South
America today. The safety of Reb A has been investigated in several recently published studies and
information on genotoxicity is described herein. Reb A was investigated for its potential to induce
genotoxicity in three in vitro and two in vivo assays (conducted according to OECD guidelines). Reb A
was non-mutagenic in an Ames test using Salmonella typhimurium and Escherichia coli, in a
chromosomal aberration test using Chinese Hamster V79 cells and in a mouse lymphoma assay using
L5178Y+/- cells, all studies were conducted at concentrations up to 5000 microg/ml, with and
without metabolic activation. Also, Reb A was non-genotoxic in a bone marrow micronucleus test in mice
at doses up 750 mg/kg bw and in an unscheduled DNA synthesis test in rats at 2000 mg/kg bw. These
studies provide additional evidence that Reb A is not genotoxic at the doses tested and further support
the generally recognized as safe determination of Reb A.
Wingard RE, Brown JP, Enderlin FE, Dale JA, Hale RL, Seitz CT. Intestinal degradation and
absorption of the glycosidic sweeteners stevioside and rebaudioside Experientia;36:519-20. 1980.
Contrary to prior indications, the glycosidic sweeteners stevioside and rebaudioside A are degraded to
the diterpenoid aglycone steviol by rat intestinal microflora in vitro. Additional studies with steviol-17-[14C]
show almost total absorption from the rat lower bowel following intracecal administration.
Wölwer-Rieck U, Tomberg W, Wawrzun A. Investigations on the Stability of Stevioside and
Rebaudioside A in Soft Drinks. J Agric Food Chem. 58 (23):12216–12220, 2010. The stability of the
two steviol glycosides stevioside and rebaudioside A and the possible formation of the aglycon steviol in
different soft drinks were analyzed in samples spiked with stevioside or rebaudioside A after 24, 48, and
72 h storage times at 80 °C. Degradation of up to 70% was observed, and stevioside was less stable than
rebaudioside A. Stevioside and rebaudioside A and their degradation products were analyzed by highperformance liquid chromatography with ultraviolet detection (UV-HPLC) on a HILIC analytical column,
and the identity of the degradation products was confirmed by liquid chromatographyelectrospray ionization mass spectrometry (LC-ESI-MS(n)) in negative mode. A UV-HPLC method was
developed using a C18 analytical column to exclude the presence of the aglycon steviol, which gave a
positive response in the forward mutation assay using the sensitive Salmonella typhimurium TM677
strain. The recoveries of steviol with this method ranged from 95.9 to 109.2%, and the calibration
curves were linear from 1 to 100 μg/mL with R(2) = 0.9999. The limit of detection was 1 μg/mL.
page 59 of 64
Confirmation by LC-ESI-MS(n) resulted in a LOD of 6 ng/mL. The absence of steviol in the degraded
samples could be unambiguously confirmed by UV-HPLC and by LC-ESI-MS(n).
Wölwer-Rieck U. The leaves of Stevia rebaudiana (Bertoni), their constituents and the
analyses thereof: a review. J Agric Food Chem. 60(4):886-95, 2012. The plant Stevia rebaudiana is
well-known due to the sweet-tasting ent-kaurene diterpenoid glycosides. Stevioside and rebaudioside A
are the most abundant and best analyzed, but more than 30 additional steviol glycosides have been
described in the scientific literature to date. Most of them were detected in the last two years. This paper
reviews these new compounds and provides an overview about novel trends in their determination,
separation, analysis, detection, and quantification. The detection and analysis of further constituents such
as nonglycosidic diterpenes, flavonoids, chlorogenic acids, vitamins, nutrients, and miscellaneous minor
compounds in the leaves of Stevia rebaudiana are reviewed as well. A critical review of the antioxidant
capacity of Stevia leaves and its analysis is also included. These different aspects are discussed in
consideration of the scientific literature of the last 10 years.
Wong KL, Lin JW, Liu JC, Yang HY, Kao PF, Chen CH, Loh SH, Chiu WT, Cheng TH, Lin JG, Hong
HJ. Antiproliferative effect of isosteviol on angiotensin-II-treated rat aortic smooth muscle cells.
Pharmacology. 76(4):163-9, 2006. Isosteviol is a derivative of stevioside, a constituent of Stevia
rebaudiana, which is commonly used as a noncaloric sugar substitute in Japan and Brazil. The aims of
this study were to examine whether isosteviol alters angiotensin-II-induced cell proliferation in rat aortic
smooth muscle cells. Cultured rat aortic smooth muscle cells were preincubated with isosteviol, then
stimulated with angiotensin II, after which [(3)H]thymidine incorporation and endothelin-1 secretion were
examined. Isosteviol (1-100 micromol/l) inhibits angiotensin-II-induced DNA synthesis and endothelin-1
secretion. Measurements of 2'7'-dichlorofluorescin diacetate, a redox-sensitive fluorescent dye, showed
an isosteviol-mediated inhibition of intracellular reactive oxygen species generated by the effects of
angiotensin II. The inductive properties of angiotensin II on extracellular signal-regulated kinase (ERK)
phosphorylation were found reversed with isosteviol and antioxidants such as N-acetylcysteine. In
summary, we speculate that isosteviol inhibits angiotensin-II-induced cell proliferation and endothelin-1
secretion via attenuation of reactive oxygen species generation. Thus, this study provides important
insights that may contribute to the effects of isosteviol on the cardiovascular system.
Wong KL, Chan P, Yang HY, Hsu FL, Liu IM, Cheng YW, Cheng JT. Isosteviol acts on potassium
channels to relax isolated aortic strips of Wistar rat. Life Sci. Mar 26;74(19):2379-87.2004. Isosteviol
is a derivative of stevioside, a constituent of Stevia rebaudiana, which is commonly used as a noncaloric
sugar substitute in Japan and Brazil. In the present study, the role of potassium channels in the
vasodilator effect of isosteviol was investigated using potassium channel blockers on isosteviol-induced
relaxation of isolated aortic rings prepared from Wistar rats. Isosteviol dose-dependently relaxed the
vasopressin (10(-8) M)-induced vasoconstriction in isolated aortic rings with or without endothelium.
However, in the presence of potassium chloride (3x10(-2) M), the vasodilator effect of isosteviol on
arterial strips disappeared. Only the inhibitors specific for the ATP-sensitive potassium (K(ATP)) channel
or small conductance calcium-activated potassium (SK(Ca)) channel inhibited the vasodilator effect of
isosteviol in isolated aortic rings contracted with 10(-8) M vasopressin. Also; since the isosteviol-induced
relaxation was unchanged by methylene blue, a role of nitric oxide and/or endothelium in the
vasodilatation produced by isosteviol could be ruled out. The obtained results indicated that vasodilatation
induced by isosteviol is related to the opening of SK(Ca) and K(ATP) channels.
Wong KL, Yang HY, Chan P, Cheng TH, Liu JC, Hsu FL, Liu IM, Cheng YW, Cheng JT. Isosteviol as
a potassium channel opener to lower intracellular calciumconcentrations in cultured aortic
smooth muscle cells. Planta Med. Feb;70(2):108-12.2004. Isosteviol is a derivative of stevioside, a
constituent of Stevia rebaudiana, and is commonly used as a non-caloric sugar substitute in Japan and
Brazil. The present study attempted to elucidate the role of potassium (K (+)) channels in the action of
isosteviol on intracellular calcium concentrations ([Ca (2+)]i) in cultured vascular smooth muscle (A7r5)
cells using the Ca (2+)-sensitive dye Fura-2 as an indicator. The increase of [Ca (2+)]i in A7r5 cells
produced by vasopressin (1 micromol/L) or phenylephrine (1 micromol/L) was attenuated by isosteviol
from 0.01 micromol/L to 10 micromol/L. The attenuation by isosteviol of the vasopressin- and
phenylephrine-induced increase in [Ca (2+)]i was inhibited by glibenclamide, apamin and 4-aminopyridine
page 60 of 64
but not by charybdotoxin. Furthermore, the inhibitory action of isosteviol on [Ca (2+)]i was blocked
when A7r5 cells co-treated with glibenclamide and apamin in conjunction with 4-aminopyridine were
present. Therefore, not only did the ATP-sensitive potassium (K (ATP)) channel affect the action of
isosteviol on [Ca (2+)]i modulation in A7r5 cells, but also those on the small conductance calciumactivated potassium (SK (Ca)) channels and voltage-gated (Kv) channels. However, the blockers of largeconductance Ca (2+)-activated potassium channels failed to modify the inhibitory action of isosteviol on
[Ca (2+)]i. The obtained results indicated that a decrease of [Ca (2+)]i in A7r5 cells by isosteviol is mainly
mediated by the selective opening of K (ATP) channel or/and SK (Ca) channel. Alteration in the Kv
channel also plays a critical role in the inhibitory action of isosteviol.
Wood DJ, Lirette A, Crober DC, Ju HY. The effect of Stevia as a feed sweetener on weight gain and
feed consumption of broiler chicken. Canadian Journal of Animal Sciences;76:267-9. 1996.
World Health Organization. Evaluation of certain food additive and contaminants World Health
Organ Tech Rep Ser. (960):1-226, 2011. This report represents the conclusions of a Joint FAO/WHO
Expert Committee convened to evaluate the safety of various flavouring agents, with a view to concluding
as to safety concerns and to preparing specifications for identity and purity. The Committee also
evaluated the risk posed by two food contaminants, with the aim of deriving tolerable intakes where
appropriate and advising on risk management options for the purpose of public health protection. The first
part of the report contains a general discussion of the principles governing the toxicological evaluation of
and assessment of dietary exposure to food additives (particularly flavouring agents) and contaminants.
A summary follows of the Committee's evaluations of technical, toxicological and dietary exposure data
for 12 groups of flavouring agents (alicyclic ketones, secondary alcohols and related esters; alicyclic
primary alcohols, aldehydes, acids and related esters; aliphatic acyclic and alicyclic alpha-diketones and
related alpha-hydroxyketones; aliphatic acyclic and alicyclic terpenoid tertiary alcohols and
structurally related substances; aliphatic and aromatic amines and amides; aliphatic lactones; aliphatic
primary alcohols, aldehydes, carboxylic acids, acetals and esters containing additional oxygenated
functional groups; aliphatic secondary alcohols, ketones and related esters and acetals; aromatic
substituted secondary alcohols, ketones and related esters; benzyl derivatives; phenol and phenol
derivatives; and simple aliphatic and aromatic sulfides and thiols) and two food contaminants (cadmium
and lead). Specifications for the following food additives were revised: activated carbon, cassia gum,
indigotine, steviol glycosides, sucrose esters of fatty acids, sucrose monoesters of lauric, palmitic or
stearic acid and titanium dioxide. Specifications for the following flavouring agents were revised: 4carvomenthol and 5,6,7,8-tetrahydroquinoxaline. Annexed to the report are tables summarizing the
Committee's recommendations for dietary exposures to and toxicological evaluations of the flavouring
agents and contaminants considered.
WHO, 2000. Joint FAO/WHO Expert Committee on Food Additives. WHO Food Additive Series;
42. Safety evaluation of certain food additives. Stevioside.
WHO, 2003. GEMS/Food regional diets (regional per capita consumption of raw and semi
processed agricultural commodities). Geneva: Global Environment Monitoring System 144 steviol
glycosides K2 Food Contamination Monitoring and Assesssment Programme and Food Safety
Department, World Health Organization.
WHO, 2006. Joint FAO/WHO Expert Committee on Food Additives. WHO Food Additive Series;
54. Safety evaluation of certain food additives, Steviol Glycosides, pp.117-144.
WHO, 2007. Joint FAO/WHO Expert Committee on Food Additves. Sixty-eighth meeting,
Summary and Conclusions, Steviol Glycosides. Issued July 12, 2007.
WHO, 2008. Joint FAO/WHO Expert Committee on Food Additves. Sixty-ninth meeting, Summary
and Conclusions, Steviol Glycosides. Issued July 4, 2008.
WHO, 2009. Joint FAO/WHO Expert Committee on Food Additives. WHO Food Additive Series:
60. Safety evaluation of certain food additives. Steviol Glycosides (addendum).
page 61 of 64
World Health Organization Food Additives. Safety evaluation of certain food additives. Accessed
athttp://whqlibdoc.who.int/publications/2009/9789241660600_eng.pdf.
World Health Organization Technical Report Series 952. Evaluation of certain food additives.
Accessed at http://whqlibdoc.who.int/trs/WHO_TRS_952_eng.pdf.
World Health Organization. WHO/FAO release independent Expert Report on diet and chronic
disease. March 3, 2003. Accessed at http://www.who.int/mediacentre/news/releases/2003/pr20/en/.
May 27, 2010.
Xiao J, Hermansen K. The mechanism underlying the insulintropic effect of steviosideactivation of
acetyl-CoA carboxylase. Diabetes;54(1):A131 [Abstract No. 532P]. 2005.
Xiao J, Kjeld H, Jeppesen PB. The insulintropic effect of stevioside is mediated via activation of
acetyl-CoA carboxylase. Diabetes;54(1):A132 [Abstract No. 536P]. 2005.
Xie SP, Ouyang XZ, Hong WL, Chen MC, Wang DY. The growth and differentiation of callus
cultures of Stevia rebaudiana in relation to the stevioside accumulation. Redai Yaredai Zhiwu
Xuebao;6(1):8-14.1998.
Xili L, Chengjiany B, Eryi X, Reiming S, Yuengming W, Haodong S, Zhiyian H. Chronic oral toxicity
and carcinogenicity study of stevioside in rats. Food and Chemical Toxicology;30:957-65.
1992. Groups of 45 male and 45 female inbred Wistar rats were given diets containing stevioside (85%
pure) at 0, 0.2, 0.6 or 1.2% for 2 yr. After 6, 12 and 24 months, five rats from each group were killed for
haematological and clinical biochemical tests. Growth, food utilization and consumption, general
appearance and mortality were similar in treated and control groups. The mean lifespan of rats
given stevioside was not significantly different from that of the controls. No treatment-related changes
were observed in haematological, urinary or clinical biochemical values at any stage of the study. The
incidence and severity of non-neoplastic and neoplastic changes were unrelated to the level of
stevioside in the diet. The maximum no-observed-effect level of stevioside was 1.2%, and an acceptable
daily intake of stevioside for humans of 7.938 mg/kg body weight/day is suggested.
Xu D, Li Y, Wang J, Davey AK, Zhang S, Evans AM. The cardioprotective effect of isosteviol on
rats with heart ischemia–reperfusion injury. Life Sci;80:269-74.2007. This study was designed to
assess the cardioprotective effect of isosteviol on rats with heart ischemia-reperfusion (IR) injury and to
explore the mechanism of action of the compound. Sprague Dawley rats were divided into 8 groups
(n=10-12): a sham-operated control and 7 ischemia-reperfusion groups (IR control, 3 isosteviol pretreated (0.5, 1.0 and 2.0 mg kg(-1)), ligustrazine pre-treated, 5-hydroxydecanoate (5-HD) pre-treated and
5-HD+ isosteviol pre-treated groups). IR was produced by occluding the left coronary artery for 30 min
followed by re-opening the artery for 90 min. The compounds under investigation were administered
intravenously 10 min prior to occluding the artery. Hemodynamic parameters (+/-dp/dt(max), LVSP,
LVDevP, MAP), heart rate, ventricular tachycardia (VT) and ventricular fibrillation (VF) were determined
during the IR period. The myocardial infarct size, activities of serum lactate dehydrogenase and creatine
kinase were determined at the end of the experiment. In the isosteviol pre-treated groups, the
hemodynamic parameters were improved and the myocardial infarct size, the activities of serum
enzymes, and the incidences of VT and VF were all decreased when compared to the control group.
These effects of isosteviol were similar to that of a traditional cardioprotective agent, ligustrazine. The 5HD+ isosteviol group displayed parameters that were between those in the equivalent isosteviol pretreated group and the IR control group. In conclusion, damage due to a standard rat heart IR injury was
reduced by pretreatment with intravenous isosteviol, and this effect was partly attenuated by a
mitochondrial ATP-sensitive potassium channel blocker, 5-HD.
Xu D, Du W, Zhao L, Davey AK, Wang J. The neuroprotective effects of isosteviol against focal
cerebral ischemia injury induced by middle cerebral artery occlusion in rats. Planta Med.
74(8):816-21, 2008. Occlusion of a cerebral artery impairs blood flow leading to neuronal death.
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Reperfusion of the tissue is associated with inflammation, increased reactive oxygen species, necrosis
and apoptosis. Hence, damage to the brain will continue even after the blood flow is restored. Isosteviol
has been demonstrated to have protective effects against ischemia-reperfusion (IR) injury in the rat heart
and the current study was undertaken to determine whether it is also effective in preventing IR injury in
the brain. Rats were divided into six groups: a sham-operation control group and 5 IR groups that were
pre-treated with either isosteviol 5 mg.kg (-1), 10 mg.kg (-1), 20 mg.kg (-1), nimodipine 5 mg.kg (-1), or
saline. Cerebral ischemia was induced for 2 hours. Twenty-two hours after re-perfusion the rats were
assessed for neurobehavioral deficit, infarct volume, histological changes, and malondialdehyde,
superoxide dismutase (SOD), Bcl-2 and NF-kappaB levels in brain tissue. Pre-treatment with isosteviol
reduced infarct volume, ameliorated cell death and infiltration of neutrocytes, improved neuro-locomotor
activity, increased SOD activity, induced Bcl-2, suppressed lipid superoxidation and the expression of NFkappaB, and therefore retarded necrosis and apoptosis of neurons and inflammation. These positive
effects were dose-dependent with an isosteviol dose of 20 mg.kg (-1), thus being as effective as
nimodipine.
Yadav SK, Guleria P. Steviol glycosides from Stevia: biosynthesis pathway review and their
application in foods and medicine. Crit Rev Food Sci Nutr. 52(11):988-98, 2012. Stevia rebaudiana, a
perennial herb from the Asteraceae family, is known to the scientific world for its sweetness and steviol
glycosides (SGs). SGs are the secondary metabolites responsible for the sweetness of Stevia. They
are synthesized by SG biosynthesis pathway operating in the leaves. Most of the genes encoding the
enzymes of this pathway have been cloned and characterized from Stevia. Out of various SGs, stevioside
and rebaudioside A are the major metabolites. SGs including stevioside have also been synthesized by
enzymes and microbial agents. These are non-mutagenic, non-toxic, antimicrobial, and do not show any
remarkable side-effects upon consumption. Stevioside has many medical applications and its role against
diabetes is most important. SGs have made Stevia an important part of the medicinal world as well as the
food and beverage industry. This article presents an overview on Stevia and the importance of SGs.
Yamada A, Ohgaki S, Noda T, Shimizu M. Chronic toxicity of dietary Stevia Extracts. Journal of the
Food Hygienic Society of Japan;26(2):169-83. 1985.
Yamamoto NS, Kelmer Bracht AM, Ishii EL, Kemmelmeier FS, Alvarez M, Bracht A. Effect of steviol
and its structural analogues on glucose production and oxygen uptake in rat renal tubules.
Experientia;41:55-7. 1985. The effect of several natural products of Stevia rebaudiana on glucose
production and oxygen uptake in rat renal cortical tubules was investigated. Steviol, isosteviol and
glucosilsteviol decreased glucose production and inhibited oxygen uptake. The sweet principle
stevioside, and steviolbioside, however, were without effect on gluconeogenesis and oxygen uptake.
Yamazaki T, Flores HE, Shimomura K, Yoshihira K. Examination of steviol glucosides production
by hairy root and shoot cultures of Stevia rebaudiana. J Nat Prod;54(4):986-92. 1991.
Yang PS, Lee JJ, Tsao CW, Wu HT, Cheng JT. Stimulatory effect of stevioside on peripheral mu
opioid receptors in animals. Neurosci Lett. 454(1):72-5, 2009. Stevioside is a dietary supplement
widely used as a sweetener to prevent hyperglycemic disorders. However, the action mechanisms of this
substance for glucose homeostasis remain obscure. In the present study, a dose-related plasma glucose
reduction was observed in Wistar rats receiving intraperitoneally injections of stevioside. Similar to the
regulation of glucose metabolism by the activation of mu opioid receptors, this action of stevioside was
reversed by naloxonazine under the blockade of mu opioid receptors. We also found that stevioside
increased glycogen synthesis in isolated hepatocytes, which was concentration-dependently blocked by
naloxonazine. Stevioside did not modify the plasma beta-endorphin levels in Wistar rats but it directly
increased the phosphorylation of mu opioid receptors in Chinese hamster ovary cells transfected with mu
opioid receptors. Unlike morphine, chronic administration of stevioside did not induce the withdrawal
signs in mice. Furthermore, stevioside by intraperitoneal injections did not influence the feeding behaviors
of rats. By contrast, intracerebroventricular injections of stevioside increased the rats' food intake, which
was also inhibited by pretreatment with naloxonazine. These results showed that it is difficult for
stevioside to enter the brain. Stevioside has the ability to activate peripheral mu opioid receptors for
page 63 of 64
lowering plasma glucose and to increase glycogen synthesis in liver. Thus, the stimulation of peripheral
mu opioid receptors is responsible for the action of stevioside in the regulation of glucose homeostasis.
Yasukawa AK, Yamaguchii A, Arita J, Sakurai S, Ikeda A, Takido M. Inhibitory effect of edible plant
extracts on 12-O-Tetrade Canoylphorbol-13-Acetate-Induced ear oedema in mice. Phytother
Res;7(2):185-9.1993.
Yasukawa K, Kitanaka S, Seo S. Inhibitory effect of stevioside on tumor promotion by 12-Otetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin. Biol Pharm Bull.
25(11):1488-90, 2002. Four steviol (ent-kaurene-type diterpenoid) glycosides, stevioside, rebaudiosides
A and C, and dulcoside A, have been isolated from Stevia rebaudiana BERTONI. These compounds
showed strong inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced
inflammation in mice. The 50% inhibitory dose of these compounds for TPA-induced inflammation was
54.1-291.6 micro g/ear. Furthermore, at 1.0 and 0.1 mg/mouse of stevioside mixture, the mixture of these
compounds markedly inhibited the promoting effect of TPA (1 micro g/mouse) on skin tumor formation
initiated with 7,12-dimethylbenz[a]anthracene (50 micro g/mouse).
Yodyingyuad V, Bunyawong S. Effect of stevioside on growth and reproduction. Human
Reproduction;6(1): 158-65. 1991. The effect on growth and reproduction in hamsters of stevioside,
which is extracted from stevia leaves (Stevia rebaudiana Bertoni) and is currently used as a non-caloric
sweetener, was investigated. Four groups of 20 one-month-old hamsters (10 males and 10 females) were
daily force-fed with stevioside (0.0, 0.5, 1.0 and 2.5 g/kg body wt/day, respectively). No abnormalities
were found in growth and fertility in both sexes. All males mated females efficiently and successfully.
Females showed normal 4-day oestrus cycles and became pregnant after mating. Each female was
mated and allowed to bear three litters during the period of experiment. The duration of pregnancy,
number of fetuses, as well as number of young delivered each time from females in the experimental
groups were not significantly different from those in the control group. The young F1 and F2 hamsters
continuously receiving stevioside via drinking water until one month old and daily force-fed afterwards at
the same doses as their parents showed normal growth and fertility. Histological examinations of
reproductive tissues from all three generations revealed no evidence of abnormality which could be
linked to the effects of consuming stevioside. We conclude that stevioside at a dose as high as 2.5 g/kg
body wt/day affects neither growth nor reproduction in hamsters.
Yoshida S. Production of sweet substances in Stevia rebaudiana. I. Simple determination of sweet
glucosides in Stevia plant with a thin layer chromato-scanner and their accumulation patterns
with plant growth. Nippon Sakumotsu Gakkai Kiji;55(2):189-95.1986.
Yuajit C, Homvisasevongsa S, Chatsudthipong L, Soodvilai S, Muanprasat C, Chatsudthipong V.
Steviol reduces MDCK Cyst formation and growth by inhibiting CFTR channel activity and
promoting proteasome-mediated CFTR degradation. PLoS One. 8(3):e58871, 2013. Cyst
enlargement in polycystic kidney disease (PKD) involves cAMP-activated proliferation of cyst-lining
epithelial cells and transepithelial fluid secretion into the cyst lumen via cystic fibrosis transmembrane
conductance regulator (CFTR) chloride channel. This study aimed to investigate an inhibitory effect and
detailed mechanisms of steviol and its derivatives on cyst growth using a cyst model in Madin-Darby
canine kidney (MDCK) cells. Among 4 steviol-related compounds tested, steviol was found to be the most
potent at inhibiting MDCK cyst growth. Steviol inhibition of cyst growth was dose-dependent; steviol (100
microM) reversibly inhibited cyst formation and cyst growth by 72.53.6% and 38.2±8.5%, respectively.
Steviol at doses up to 200 microM had no effect on MDCK cell viability, proliferation and apoptosis.
However, steviol acutely inhibited forskolin-stimulated apical chloride current in MDCK epithelia,
measured with the Ussing chamber technique, in a dose-dependent manner. Prolonged treatment (24
h) with steviol (100 microM) also strongly inhibited forskolin-stimulated apical chloride current, in part by
reducing CFTR protein expression in MDCK cells. Interestingly, proteasome inhibitor, MG-132, abolished
the effect of steviol on CFTR protein expression. Immunofluorescence studies demonstrated that
prolonged treatment (24 h) with steviol (100 microM) markedly reduced CFTR expression at the plasma
membrane. Taken together, the data suggest that steviol retards MDCK cyst progression in two ways:
first by directly inhibiting CFTR chloride channel activity and second by reducing CFTR expression, in
page 64 of 64
part, by promoting proteasomal degradation of CFTR. Steviol and related compounds therefore represent
drug candidates for treatment of polycystic kidney disease.
Zaidan LB, Dietrich, SMC, Felippe GM. Effect of photoperiod on flowering and stevioside content
in plants of Stevia rebaudiana Bertoni. Jap J Crop Sci;49:560-74.1980.
Zan FS. Method of extraction and purification of stevioside from Stevia rebaudiana. Hua Hsueh
Chih Ji;27(1): 31-3.1986.
Zanela NL, Bijella MF, Rosa OP.The influence of mouthrinses with antimicrobial solutions on the
inhibition of dental plaque and on the levels of mutans streptococci in children. Pesqui Odontol
Bras. 16(2):101-6, 2002. The effect of daily mouthrinses on dental plaque accumulation and on salivary
mutans streptococci was investigated in 200 children. The utilized solutions were: a placebo solution
composed of mentholated deionized water (group I); 0.12% chlorhexidine gluconate associated to 0.05%
sodium fluoride (group II); 0.2% chlorhexidine digluconate (group III), and 0.5% stevioside mixed with
0.05% sodium fluoride, with pH 3.4 (group IV). In order to verify the effect on plaque formation, the
accumulation of plaque was assessed by means of the Löe12 index, at the beginning and at the end of
the experiment, whereas the quantification of cariogenic streptococci was accomplished on three saliva
samples collected at 3 different moments: before the first mouthrinse, 24 hours after the first mouthrinse
and 1 week after the last mouthrinse. The mouthrinsing routine was carried out on a daily basis during 4
weeks. Five milliliters of solution were rinsed during 1 minute. The results revealed 4.10, 26.75, 41.20,
and 5.91% of reduction in plaque accumulation for groups I, II, III, and IV, respectively. Comparisons
between the groups as to plaque reduction revealed that groups II and III were significantly different from
groups I (control) and IV (p < 0.05), but did not differ from each other. The solution utilized by group III
was the least accepted by children and, as the solution utilized by group II, caused mild dental
pigmentation. There were no statistically significant differences as to the levels of mutans streptococci,
probably due to the low initial levels observed in each one of the four groups.
Zhou R, Ran Z, Li Q, Zi X, Rong YX, Li R. Ion exchange methods in extraction and purification of
steviosides from Stevia rebaudiana. Zhongguo Tiaweipin;12:12-13. 1984.
Zhuang JX, Chen J. Isolation of natural sweeteners-steviosides. Tiaowei Fushipin Keji;11:1920.1984.