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STEVIOSIDE: TOXICOLOGICAL ASPECTS

Mauro Alvarez, PhD
Faculty of Pharmacy and Biochemistry,
State University of Maringa, Maringa, Brazil

The stevioside is the main sweetening compound found in the leaf of the plant Stevia rebaudiana (Bert.) Bertoni (from 5% to 15% dry weight), followed by ribaudioside (3% to 6%). The other glycosides of the plant appear in minimal quantities. The stevioside and the other Stevia glycosides have high chemical stability because of their tridimensional chemical form which produces resistance to acid and enzymatic hydrolysis ensuring their inalterability even under the biochemical and physiological aspects.

Several experimental studies have been performed to demonstrate the safety of Stevia sweeteners. Pioneering studies performed by Ribaudi (1900), Korbert (1915), and Pomaret and Lavieille (1931) classified the stevioside as a natural sweetener acceptable andsafe for human consumption.

The interest on the safety of stevioside was revived in 1970 when the Japanese food industry started large scale production and sale of the stevioside and Stevia extracts to the population of Japan. As a result, many research studies have been performed in that country on the safety of this product since 1970.

Concentrating on acute toxicity, Akashi and Yokoyama (1975) showed an oral LD 50 of 17 g/kg body weight for the Stevia extract (20 % of stevioside) and 15 g/kg for purified stevioside (93.5%). Mitsuhashi et al. (1976) showed an acute toxicity, with rats and mice, for purified stevioside, by oral intake, an LD 50 of 8.2 g/kg, and, intraperitoneally, an LD 2.99 g/kg. These results showed that there is not an acute toxic effect detectable for the natural product of Stevia rebaudiana. The high dosages needed for the experiments did not permit to reach the real LD 50.

Subacute toxicity studies were performed by Akashi and Yokoyama (1975) with purified stevioside supplied to rats' rations in concentrations up to 7% daily during 3 months, and by Mitsuhashi et al. (1976) who fed rats with 2.5g/kg/day of purified stevioside, also by adding it to their daily ration during 3 months. Both studies did not produce any effect related with the stevioside doses over the animals tested.

Several other experiments have been performed to verify the metabolic action of the Stevia diterpenic glycosides and its derivatives. Thus, Virginais et. al. (1965) and Bracht et. al. (1985) showed effects related to the hydrolysis products of Stevia glycosides, the aglycones steviol and isosteviol, over isolated rat liver mitochondria. Bracht et. al. (1985) showed also that pure stevioside causes no effect over that organelle, even in high concentrations. Some other studies carried out by Bracht et. al. (1985,1986,1987) showed that the Stevia diterpenic aglycones effects, at metabolic levels, are reversible when experimented in isolated organs and cells, observing a possible pharmacological rather than a toxic effect.

A singular effect was shown by Bracht et. al. (1986) for pure stevioside. It was shown that this substance inhibited the transport of glucose across cell membranes when studied in perfused rat liver, and that the stevioside does not cross the membrane. Wingard et. al. (1980) observed intestinal degradation of Stevia sweeteners (stevioside and ribaudioside-A) and the absorption of the aglycones. However, these researchers didn't observe the absorption of intact glycosides. They also showed that the steviol is completely excreted by the biliary way, in the feces. These observations were confirmed by Nakayama et. al. (1986) who fed rats with 3H-stevioside and studied the radioactivity distribution in the body. They also showed that the stevioside is not absorbed by the intestinal tract and after bacterial decomposition it produces the metabolites, steviol and glucose, which are absorbed in the cecal region. The steviol is excreted in the feces by the biliary way, completely bounded, probably by biliary mucopolysaccharide. They also demonstrated that there is no increase of steviol in the blood level, and that this metabolite is recirculated by entero-hepatic way and is excreted in the feces.

Bacterial mutation tests (Ames test) on the stevioside and Stevia extracts have been carried out by several Japanese laboratories according to reports of the Drug Safety Center and Ministry of Health and Welfare of Japan, by investigations of Kerr et. al.(1983),by DNA recombination tests made by Okomura et. al. (1977) and Kohoshi et al. (1977), and by chromosome aberration test made by T. Sugiyama at Kobe University, M. Sazaki at Hokaido University and M. Ishidate at the National Institute of Health Sciences of Japan (see Mitsuhashi 1976). All of these studies showed negative results. Pezzuto et. al. (1985) demonstrated that steviol, the aglycone of stevioside, obtained by enzymatic hydrolysis, is mutagenic to Simonela typhimurium when evaluated in the presence of a 9000 x g supernatant fraction derived from livers of Aeroclor 1254 pretreat rats and addition of NADPH. They showed also that unmetabolized steviol was not active. Stevioside and isosteviol are inactive. Pezzuto et. al. (1986) also showed that steviol did not induce the activating 9000 x g supernatant metabolic systems of glutathione~S~transferase in mice. This evidence eliminates the possibility of mutagenic effects in animal tissues.

Several other experiments on mutagenicity were carried out with pure stevioside and stevia extracts. Okomura et. al. (1977) made tests of mutagenicity by host in female rats with oral intake of maximum doses of llg/kg body weight of pure stevioside. When compared with control groups, these researches did not report any significant difference. Similar results were obtained by Y. Tagima at the National Institute of Heredity in Japan, in Bombix cells. Likewise, Kerr et. al. (1983) in studies on the effects of stevioside on the x chromosome of Drosophilas reported no significant results. Dominant lethality tests were considered negative by studies done by Y. Tagima (1976) (see Mitsuhashi 1976).

Extensive studies on chronic toxicity of stevioside and other Stevia products were performed recently by Yamada et. al. (1985). Male and female rats were fed daily with a ration containing 0.3 to 1% of stevioside and rebaudioside for a period of 24 months. Biochemical anatomo-pathological and cariogenic tests were performed in 41 organs. Hematologic and urinary tests also were performed in the test animals. The symptoms and alterations observed in the group tested did not differentiate from the control group. No dose/effect relationship was observed in any animal, either, even under high dose (1%).

About the contraceptive and abortive affects attributed to aqueous Stevia extract, Planas and Kuc (1968) observed that the fertility in female rats fed with the Stevia extract decreased 50 to 57 %. This data has been refuted by several other studies. Akashi and Yokoyamam (1975) and Mori et. al. (1981) administered high doses of 525 mg/kg/day of stevioside equivalent aqueous Stevia extract to male and female mice during copulation and pregnancy periods. Both researchers could not observe any difference in the copulation and conception averages and any change in the fetus and breeds when compared with the control groups. Silva et. al. (1986) administered Stevia leaves aqueous extract in doses from 4% to 10 % and 0.04% of pure stevioside to female rats during copulation and pregnancy periods. Again, no alterations in the conception and breeding results were reported. These researchers concluded that the contraceptive effect of the Stevia extract is only folklore.

Some studies about hormonal activity were performed by Dorfman and Ness (1960) with steviol and dihidrosteviol showing some residual effects. However, Oliveira Filho et. al. (1986) in a detailed work about the effect of Stevia products on the ~ndocrinous fielddemonstrated that the Stevia products are innocuous at this level of animal physiology.

Some effects observed for the stevioside and its derivatives (steviol,isosteviol and steviolbioside) when showed in isolated cells, organelles or in intact organs (Vignais et. al. 1965; Bracht et. al. 1985; Ishii and Bracht 1987) are, in large part, reversible (Ishii and Bracht 1985) expressing more of a pharmacological rather than a toxicological effect. These effects do not reveal action to physiological levels in intact animals mainly by oral way (Pomaret et Lavieille 1931; Akashi and Yokoyama 1975; Yamada et. al. 1985; Bazotte et. al. 1986).


BIBLIOGRAPHY

Akashi, H.and Yokoyama, Y. (1975). Dried-Leaf Extracts of Stevia. Toxicological Tests. Shokuin Kogyo. 18(20):34-43

Bazotte, B.R., Lonardoni, M.T.C., Alvarez, M., Gaeti, P.W. and Amado, A.B. (1986). Determination of the Lethal Dose (LD 50) for the Isosteviol in Laboratory Animals. Arq. Biol. Tecnol. 29(4) :711-722

Dorfman, R.I. and Ness, W.R. (1960). Anti-Androgenic Activity of Diidrosteviol. Endocrinology. 67:282-285.

Ishii, E.L. and Bracht, A. (1987). Glucose Release by the Liver under Conditions of reduced Activity of Glucose G-Phosphatase. Brazilian J. Med. Biol. 20:837-843.

Ishii, E.L. and Bracht, A. (1986). Stevioside, The Sweet Glycoside of Stevia rebaudiana Inhibits the action of Atractyloside in the Isolated Perfused Rat Liver. Research Comm. Chem. Pathol. Pharmacol. 53(l):79-91.

Ishii, E.L., Schwab, A.J. and Bracht, A. (1987).Inhibition of monosaccharide Transport in the Intact Rat Liver by Stevioside. Biochemical Pharmacology 36(9)1417-1433.

Kelmer Bracht, A.M., Kemmelmeir, F.S., Ishii, E.M., Alvarez, M., and Bracht, A., (1985). Effect of Stevia rebaudiana Natural Products on Cellular and Sub-Cellular Metabolism. Arq. Biol. Tecnol. 28(3):431-455.

Kerr, W.E., Mello, M.L. and Bonadio, E. (1983). Testes de ACZo Mutagenica do Steviosideo da Stevia rebaudiana (Bert.) Bertoni. Rev. Bras. de Genetica VI.l:173-176.

Kohoch, S., Kada, T., Nishioka, H., Yahagi, T., Ishidato, M., Tagima, Y., Endo, H., Yoshikawa, K., Sugisawa, T., and Sasaki, M. (1977). Annual Report of Cancer Research, p.773-774. Ministry of Health and Welfare. Japan.

Korbert, R. (1915). Ober Zwei Subschmeekend Dorgen. Ber. d. Deutsch. Pharm. Ges. 28:420-443.

Mitsuhashi, H. (1976). Safety of Stevioside. In Tama Biochemical Co. Ltd. Report on Safety of Stevia pp. 9-10 (1981)

Mori, n>, Sokanone, M., Takenchji, M., Shimpo, K., and Tanabe, T., (1981). Effect of Stevioside on Fertility in Rats.

Nakayama, K., Kasahara, D., and Yamamoto, F. (1986). Absorption, Distribution, Metabolism and Excretion of Stevioside in Rats. Shokuhim Eiseigaku Zasshi. 27(l):l-8.

Okomura, M., Fugita, Y., Imamura, M., and Ayakawa, K. (1978) Studies on the Safety of Stevioside with Rec-Assay and Reversion Test. Shokuhin Eiseigaku Zasshi. 19:486-490.

Oliveira Filho, R.M., Valle, L.B.S., Minetti, C.A.S.A., Vehara, O.A. (1986). Avaliacao dos Efeitos do Extrato Bruto de Stevia rebaudiana na Esfera Endocrina: Estudos em Ratos. III Seminario Brasileiro sobre Stevia rebaudiana (Bert.) Bertoni. 35-1. Campinas - SP.

Pezzuto, J.M., Comprade, C.M., Swanson, S.M., Nanayakkara, N.P.D. and Kinghorn, A.D. (1985). Metabolically activated steviol, the aglycone of stevioside, is mutagenic. Proc. Natl. Acad. Sci. USA. 2:2478-2482.

Pezzuto, J.M., Nanayakkara, N.P.D., Comprade, C.M., Swanson, S.M., M., Kinghorn, A.D., and Guenthner, T.M. (1986). Characterization of Bacterial Mutagenicity Mediated by 13- hydroxyent-Kaurenoic acid (steviol) and several structurally related derivatives and evaluation of potential to induce glutathione 5-transferase in mice. Mutat. Res. 169(3):93-103.

Planas, G.M., and Kuc, J. (1968). Contraceptive Properties of Stevia rebaudiana. Science. 162:1007.

Pomaret, M.M.M. and Lavieille, R. (1931). Le Priciple a Saveur Sucree du Kaa-hee-e (Stevia rebaudiana Bertoni). IV Qualques Proprietes Physiologiques du Stevioside. Bull. Soc. Chem. Biol. (Paris). 13:1248-1253.

Rebaudi, 0. (1900). Note about el Caa-eee. Revista Quimica Farmaceutica. Buenos Aires. 1,2-3.

Samadiego, C.C., (1946). Stevia rebaudiana - "Caa-yee-he" (yerba dulce). Revista Farmaceutica. Buenos Aires. 88:199-202.

Silva, A.R., Sladanha, C.M., Boelter, R., Chagas, A.M. (1986). Fertilidade de Ratas: Extrato Aquoso de Stevia rebaudiana (Bert.) Bertoni e Steviosideo. III Seminario Brasileiro sobre Stevia rebaudiana (Bert.) Bertoni. 34-1-2.

Vignais, P.V., Duee, E.D., Vignais, P.M. and Huet, J (1966). Effects of Atractyligenin and its Structural Analogues on Oxidative Phosphorylation and on the trans location of Adenine Nucleoides in Mitochondria. Biochim. Biophys. Acta. 118:465-483

Von Schmeling, G.A. (1967). Caa-Hee Edulcorante nao calorico (Stevia rebaudiana Bertoni). Boletim do Sanatorio Sao Lucas. 5:1-14.

Wingard, R.E., Brown Jr., J.P., Enderlin J.A., Dale, R.L., Hale, R.L. and Seitz, C.T. (1979). Intestinal Degradation and Absorption of the Glycosidic Sweeteners Stevioside and Rebaudioside-A. Experimentia. 36:519-520.

Yamada, A., Ohgaki, S., Noda, T., Shimizu, M. (1985). Chronic toxicity of Dietary Stevia Extracts. Shokuin Eiseigaku Zasshi. 26(2). 169-183.

Yamamoto, A.M., Kelmer Bracht, A.M., Ishii, E.L., Kemmelmeier, F.S., Alvarez, M. and Bracht, A. (1985). Effect of Steviol and its structural analogues on glucose production and oxygen uptake in rat tubules. Experimentia. 41:55-57.