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Steviol effect, a glycoside of Stevia rebaudiana, on glucose clearances in rats/O efeito do esteviol, um glicosideo da Stevia rebaudiana, no clearance da glicose em ratos.

1. Introduction

Stevia rebaudiana Bertoni is one of the 154 members of the genus Stevia and a herb native to South America. The leaves of the shrub contain specific glycosides, which produce a sweet flavour but have no caloric value and are about 30-45 times as sweet as sucrose. For centuries, this herbal sweetener has been used by native Guarani Indians to counteract the bitter flavor of various plant-based medicines and beverages. Many countries have shown interest in its cultivation, and research activities have been initiated. The leaves contain a complex mixture of natural sweet diterpene glycosides: stevioside (4-13% dry weight), steviolbioside (trace), rebaudiosides A (2-4%), B (trace), C (1-2%), D (trace), E (trace) and dulcoside A (0.4-0.7%) (Kinghorn and Soejarto, 1991).

Stevioside is the predominant sweetening component and contains three glucose molecules and steviol, a diterpenic carboxylic alcohol (Kinghorn and Soejarto, 1991). Stevioside has been reported to have acute antihyperglycemic and blood pressure-lowering effects. Curi et al., (1986) observed that extracts of S. rebaudiana leaves significantly decreased plasma glucose levels in normal adult humans and Raskovic et al., (2004) that blood glucose levels in mice treated with S. rebaudiana and stevioside were lower compared with control treated physiological solution. Pure stevioside causes both bradycardia and hypotension, in humans and rats (Melis, 1992; Schmandke, 2004). In addition, it is able to induce a discrete shortening of the duration of electrical systole, suggesting a positive inotropic effect (Boeckh and Humboldt, 1981). A slight hypotensive effect was also noted in human subjects treated with a S. rebaudiana tea administered daily for 30 days (Boeckh, 1992).

We have shown that stevioside induces hypotension, diuresis, natriuresis and kaliuresis (Melis et al., 1986) and a fall in renal tubular reabsorption of glucose in rats (Melis, 1992). These effects are probably dependent on prostaglandin activity (Melis and Sainati, 1991a). Stevioside might be a calcium antagonist as is the case for verapamil (Melis and Sainati, 1991b) and thus could change mean arterial pressure and renal function. S. rebaudiana extract, in doses higher than that used for sweetening purposes, is a vasodilator agent, causing hypotension, diuresis and natriuresis in rats (Melis, 1995). On the basis of the data, stevioside and S. rebaudiana extracts could produce changes in renal function parameters. In addition, it is possible that other compounds in S. rebaudiana may be involved in the vasodilator response seen in rats treated with a concentrated aqueous extract of this plant (Melis, 1996). On the other hand, steviol, the aglycone of several natural products extracted from the leaves of S. rebaudiana, is a diuretic agent without hypotensive activity (Melis, 1997), but has been the subject of relatively few investigations.

The present study employed clearance methodology to evaluate the renal excretion of steviol and clarify the actual participation of this compound on the renal excretion of the same substances in rats. These data may contribute to a better understanding of the effect of steviol on renal function.

2. Material and Methods

Plant material was collected from its natural habitat near Amambai (Parana, Brazil). The plant was authenticated and a voucher specimen has been deposited in the herbarium of the Department of Botany (FFCLRP), at the University of Sao Paulo, Brazil.

The total aqueous extract of S. rebaudiana leaves was prepared by extraction with hot water. Dried leaves (100 g) were suspended in 1000 mL of hot water (100 [degrees]C). After 30 minutes, the suspension was filtered through several layers of cheesecloth, and the filtrate used for the preparation of the compounds. Stevioside was isolated from a 1-butanol-soluble extract of S. rebaudiana by absorption column chromatography (Kinghorn and Soejarto, 1991). Steviol (molecular weight = 804.80) was obtained by enzymatic hydrolysis of stevioside with pectinase (Pectinol 50 L, Corning Biosystems) (Pezzuto et al., 1985).

Thirty normal male Wistar rats with an average weight of 345 g and with free access to food and water were used. Animals were anesthetised with 30 mgx[kg.sup.-1] (ip) of sodium pentobarbital, and placed on a heated table, and a tracheotomy was performed. One jugular vein was catheterised for administration of priming doses and sustained infusion of inulin and p-aminohippuric acid (PAH) according to classical clearance measurement techniques (Smith et al., 1945; Fuhr et al, 1955). Isotonic Ringer's solution containing 2% PAH and 10% inulin was infused at the rate of 0.03 mL/min throughout the course of the experiment. Steviol was infused iv through another catheterised jugular vein, which was not used during the control period. One carotid artery was cannulated for collection of blood samples. A catheter was introduced into the urinary bladder for timed urine collection. The animals were distributed in three groups of ten animals each; according to the priming and infusion doses of 0.5, 1.0 and 3.0 mgx[kg.sup.-1]/h verified by curve dose-response. The experiments were divided into two periods of 30 minutes each, one a control and the other a period of infusion of different steviol concentrations. In the control and steviol infusion periods, the animals received a similar perfused volume per g of body weight (0.5, 1.0 and 3.0 mgx[kg.sup.-1]/h).

Inulin concentration in plasma and urine was determined by the anthrone method (Fuhr et al., 1995). Plasma and urinary PAH concentrations were measured by colorimetric (Smith et al., 1945). The sodium and potassium concentrations in urine and plasma were determined through a Klina flame photometer (Beckman Instruments). Glucose determinations in plasma and urine were obtained by colorimetric estimations by anthrone reagent (Metiver and Viana, 1979). Steviol concentration in plasma and urine was determined by chromatography (Kinghorn et al., 1982). The Tukey test was used for statistical analysis of the data, and the results are presented as the Means [+ or -]SEM, with the critical level of significance set at P < 0.05.

3. Results

Table 1 summarises the results in rats of the measurements of clearances of a number of substances in control and steviol-treated rats at three doses (0.5, 1.0 and 3.0 mgx[kg.sup.-1]/h).

No significant differences were detected in inulin clearance ([C.sub.in]) or p-aminohippuric acid clearance ([C.sub.PAH]), between the periods (C and S), at any concentration of steviol tested. The mean value of the steviol clearance ([C.sub.S]), at all doses employed, is higher than the [C.sub.in] and lower than the [C.sub.PAH]. It can be seen that steviol at all doses employed, except the lowest, induced a statistically significant increase in glucose clearance ([C.sub.G]) when compared to controls and exhibited a dose-dependent effect. Thus, steviol produces a fall in renal tubular reabsorption of glucose.

There was no effect of steviol on Fe[Na.sup.+] and Fe[K.sup.+] at a dose of 0.5 mg.kg-1/h. On the other hand, Fe[Na.sup.+] and Fe[K.sup.+] increased significantly during steviol infusions (1.0 and 3.0 mgx[kg.sup.-1]/h), showing that steviol increases solute excretion and that this effect is dissociated from changes in the inulin clearance ([C.sub.in]).

4. Discussion

The p-aminohippuric acid clearance ([C.sub.PAH]) values, on estimation of renal plasma flow and glomerular filtration rate measured by inulin clearance ([C.sub.in]), did not change in our experiments (Table 1) at any concentration of steviol tested. These data taken together suggest that steviol did not affect the renal hemodynamic. On the other hand, we have shown that stevioside induces systemic and renal vasodilatation, causing hypotension, diuresis and natriuresis per mL of glomerular filtration rate, which are probably dependent on prostaglandin activity (Melis and Sainati, 1991a). In addition stevioside acts on mean arterial pressure and renal function as a calcium antagonist, as is the case for verapamil (Melis and Sainati, 1991a; Melis, 1992).

It can be seen (Table 1) that steviol at all doses employed, except the lowest, induced a statistically significant increase in glucose clearance ([C.sub.G]) when compared to controls and exhibited a dose-dependent effect. These data are in agreement with previous evidence that stevioside and other S. rebaudiana natural products exert an inhibitory effect on monosaccharide transport in the intact rat liver, as is the case for phlorizin and phloretin in the renal tubules (Ishii et al., 1987). This effect presumably occurs at the level of the cell membrane inhibiting the glucose transport system.

The precise mechanism by which steviol affects sodium and potassium transport is not known (Melis, 1997). The proximal convoluted tubule normally reabsorbs 90% of filtered glucose coupled with sodium and the remaining 10% is reabsorbed by more distal nephron segments (Lang, 1986). The fact that sodium excretion increased following steviol infusion, in spite of an unchanged glomerular filtration rate, may be explained by inhibition of the glucose reabsorption in the proximal tubules. In more distal nephron segments, changes in salt transport may be elicited by this inhibitory mechanism of the glucose carrier and by the salt load. Nevertheless, it is speculative that the observed effects in the present experiments are due only to the inhibition of glucose reabsorption on the renal tubules. Moreover, steviol may act directly on another step of the mechanism of tubular sodium reabsorption in the present experiments. Another possibility was the osmotic diuresis provoked by the increase of steviol concentrations in renal tubules, increasing Fe[Na.sup.+]. Therefore, the model used in the present study is not suitable for discriminating between the direct or indirect effects on renal sodium reabsorption in the distal tubule.

Percent fraction potassium excretion (Fe[K.sup.+]) increased after treatment with steviol in all doses employed, except the lowest. These results may be interpreted as follows: the increase in Fe[Na.sup.+] after steviol administration indicates elevated sodium concentrations and water travelling in the direction of the distal tubule. This promotes a sodium-potassium exchange, with potassium being extruded and sodium being reabsorbed by the peritubular membrane of the distal tubule. These results agree with data obtained by others in experimental situations involving elevated sodium concentrations (Malnic et al., 1964; Engbretson and Stoner, 1987).

Another point that deserves to be mentioned is the fact that the mean value of steviol clearance (Cs), at all doses employed, is higher than the [C.sub.in] and lower than the [C.sub.PAH] (Table 1). Renal excretion of PAH is the result of glomerular filtration and tubular secretion, although there is a small amount of tubular reabsorption. Also, some PAH may be protein-bound and/or metabolised. It seems possible that at least part of the excretion of steviol in the urine is due to a secretory mechanism at the level of the renal tubular epithelium.

Acknowledgements--We are grateful to CNPq for financial support.

Received June 25, 2007 - Accepted November 29, 2007 - Distributed May 31, 2009

References

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BOECKH, EMA., 1992. Pharmacological drial of a concentrated crude extract of Stevia rebaudiana Bertoni in healthy volunteers. Arquivos de Biologia e Tecnologia, vol. 35, p. 299-314.

CURI, R., ALVAREZ, M., BAZOTTE, RB., BOTION, LM., GODOY, JL. and BRACHT, A., 1986. Effect of Stevia rabaudiana on glucose tolerance in normal adult humans. Brazilian Journal of Medical and Biological Research, vol. 19, no. 6, p. 771-774.

ENGBRETSON, BGJ. and STONER, LC., 1987. Flowdependent potassium secretion by rabbit cortical collecting tubules in vitro. American Journal of Physiology, vol. 235, p. 869-903.

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ISHII, EL., SCHWAB, AJ. and BRACHT, A., 1987. Inhibition of monosaccharide transport in the intact rat liver by stevioside. Biochemical Pharmacology, vol. 36, no. 9, p. 1417-1433. KINGHORN, AD. and SOEJARTO, DD., 1991. Stevioside. In O'BRIEN, NL. and GELARDI, RC. (Eds.). Alternative Sweetners. New York; Basel; Hong Kong: Marcell Dekker Inc. p. 157-171.

KINGHORN, AD., NANAYAKKARA, NPD., SOEJARTO, DD., MEDON, PJ. and KAMATH, S., 1982. Potential sweetening agents of plant origin. I. Purification of Stevia rebaudiana sweet constituents by dropled counter-current chromatography. Journal of Chromatography, vol. 237, p. 478-483.

KINGHORN, AD., 1992. Food ingredient safety review: Stevia rebaudiana leaves. USA: Herb Research Foundation.

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MELIS, MS., SAINATI, AR. and MACIEL, RE., 1986. Effects of two concentrations of stevioside on renal function and mean arterial pressure in rats. IRCS Medical Science, vol. 14, no. 10, p. 973-974.

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MELIS, MS. & SAINATI, AR., 1991a. Participation of prostaglandins in the effect of stevioside on renal function and arterial pressure in rats. Brazilian Journal of Medical and Biological Research, vol. 24, no. 12, p. 1269-1276.

--, 1991b. Effect of calcium and verapamil on renal function of rats during treatment with stevioside. Journal of Ethnopharmacology, vol. 33, p. 257-262.

MELIS, MS., 1992. Renal excretion of stevioside in rats. Journal of Natural Products, vol. 55, n. 5, p. 688-690.

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METIVER, J. and VIANA, AM., 1979. Determination of microgram quantities of stevioside from leaves of Stevia rebaudiana Bert. by two-dimensional thin layer chromatography. Journal Experimental Biology, vol. 30, p. 805-809.

PEZZUTO, JM., COMPADRE, CM., SWANSON, SM., NANAYAKKARA, NPD. and KINGHORN, AD., 1985. Metabolically activated steviol, the aglycone of the stevioside is mutagenic. Proceedings of the Academy of Natural Science, vol. 82, no. 8, p. 2478-2482.

RASKOVIC, A., GAVRILOVIC, M. and SABO, J., 2004. Glucose concentration in the blood of intact and alloxan-treated mice after pretreatment with commercial preparations of Stevia rebaudiana (Bertoni). European Journal of Drug Metabolism and Pharmacokinetics, vol. 29, n. 2, p. 87-90.

SCHMANDKE, H., 2004. Sweet-tasting steviol glycoside derivatives with antihyperglycaemic and antihypertensive effects. Ernahrungs-Umschau, vol. 51, no. 11, p. 455-462.

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Melis, MS., Rocha, ST. and Augusto, A.*

Departamento de Biologia, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto--FFCLRP, Universidade de Sao Paulo--USP Av. Bandeirantes, 3900, CEP 14030-000, Ribeirao Preto, SP, Brazil *e-mail: aaugusto@usp.br
Table 1. Effect of infusion of steviol (0.5, 1.0 and 3.0
mg.[kg.sup.-1]/h) on clearance of different substances
in rats

Dose and
time period           Inulin clearance        PAH clearance
(mg.[kg.sup.1]/h)   (mL/min.[kg.sup.-1])   (mL/min.[kg.sup.-1])

0.5                          --                     --
(C)                  5.96 [+ or -] 0.85    16.44 [+ or -] 2.45
(S)                  6.02 [+ or -] 0.44    18.00 [+ or -] 0.04
n                            10                     10
1.0                          --                     --
(C)                  6.33 [+ or -] 0.35    18.91 [+ or -] 3.80
(S)                  6.29 [+ or -] 0.71    15.33 [+ or -] 3.10
n                            10                     10
3.0                          --                     --
(C)                  6.55 [+ or -] 0.83    17.39 [+ or -] 4.33
(S)                  6.23 [+ or -] 0.44    17.68 [+ or -] 3.29
n                            10                     10

Dose and
time period           Steviol clearance       Glucose clearance
(mg.[kg.sup.1]/h)   (mL/min.[kg.sup.-1])    (mL/min.[kg.sup.-1])

0.5                           --                      --
(C)                          0.0                     0.0
(S)                 6.96 [+ or -] 1.02 **            0.0
n                             10                      10
1.0                           --                      --
(C)                          0.0                     0.0
(S)                 8.42 [+ or -] 1.04 **   1.25 [+ or -] 1.31 **
n                             10                      10
3.0                           --                      --
(C)                          0.0                     0.0
(S)                 9.36 [+ or -] 1.41 **   1.81 [+ or -] 1.69 **
n                             10                      10

Dose and                 Fractional              Fractional
time period            urinary sodium          urinary sodium
(mg.[kg.sup.1]/h)         excretion             excretion (%)

0.5                          --                      --
(C)                  0.75 [+ or -] 0.06         23 [+ or -] 3.20
(S)                  0.82 [+ or -] 0.04         24 [+ or -] 3.04
n                            10                      10
1.0                          --                      --
(C)                  0.48 [+ or -] 0.11       23.7 [+ or -] 5.2
(S)                  1.62 [+ or -] 0.55 **    69.8 [+ or -] 4.3 **
n                            10                      10
3.0                          --                      --
(C)                  0.64 [+ or -] 0.09       24.7 [+ or -] 4.36
(S)                  0.83 [+ or -] 0.15 **    72.9 [+ or -] 5.25 **
n                            10                      10

Abbreviations: C, control period; S, steviol period; n, number of
animals. Significant relative to control values (C) (Tukey test):
** p < 0.01.
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Author:Melis, M.S.; Rocha, S.T.; Augusto, A.
Publication:Brazilian Journal of Biology
Date:May 1, 2009
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