Safety evaluation of certain food additives - ipcs inchem

188 STEVIOL GLYCOSIDES (addendum)
Five male and five female healthy volunteers (aged 21–29 years) were provided
with capsules containing 250 mg stevioside (99% purity) to be taken 3 times
per day for 3 days. Doses, expressed as steviol, were calculated to be 294 mg/day
or 4.52 mg/kg bw per day for females and 3.97 mg/kg bw per day for males. Twentyfour-hour
urine samples were taken at enrolment and after dosing. A 24-h faeces
sample was collected on the 4th day of the study. Fasting blood samples were
taken before and after dosing, and fasting blood pressure measurements were
taken before the first capsule and at six different time intervals after the first dose.
Urine was analysed for creatinine, sodium, potassium, calcium and urea. Blood was
analysed for plasma glucose, plasma insulin, alkaline phosphatase, ALT, GPT,
creatine kinase and lactate dehydrogenase. Urine, blood and faeces samples were
analysed for free and bound steviol and stevioside. The clinical analyses of blood,
blood pressure and urine showed no differences between samples taken before or
after dosing. No stevioside or free steviol was detected in either blood or urine
samples at any time. Glucuronide-bound steviol was found in blood plasma, with a
mean peak occurring around 30 min after ingestion of the first dose and in 24-h urine
samples. No stevioside or steviol conjugates were found in the faeces, but low levels
of steviol (13–40 mg per 24-h sample) were detected. This study was approved by
the local ethics committee (Geuns et al., 2007).
In a number of studies looking at the ingestion of a single dose of stevioside
in humans (varying doses and numbers of participants), blood, urine and faeces
samples were collected. In all studies, small amounts of unchanged stevioside or
steviol were excreted in the faeces, but the majority of the stevioside dose was
metabolized to steviol, then conjugated with glucuronide and excreted in the urine
and bile (Kraemer & Maurer, 1994; Simonetti et al., 2004).
2.1.3 Effects on enzymes and other biochemical parameters
The effects of stevioside, steviolbioside and one of their metabolites,
isosteviol (purity of each substance 99%), on L-glutamate dehydrogenase extracted
from rat liver mitochondria and purified beef L-glutamate dehydrogenase were
studied. Both enzymes were incubated with the three substances (concentrations
up to 1.5 mmol/l, dissolved in 50% glycerol suspension or diluted with albumin
containing Tris buffer), and reaction rates were measured spectrophotometrically.
No enzyme inhibition was observed with stevioside at any of the concentrations
tested. Steviolbioside at a concentration of 1.5 mmol/l caused 70% inhibition of the
enzymes, and isosteviol caused 45% inhibition at a concentration of 1.5 mmol/l, but
only when the enzymes were diluted in bovine serum albumin and not in glycerol
(Levy et al., 1994).
In several studies using isolated rat cells, rebaudioside A and stevioside
(purity not specified) were found to stimulate secretion of insulin in pancreatic cells
and to interfere with glucose metabolism in adipocyte cells, leading to insulin resistance
in adipocytes, which has not been observed in pancreatic cells (Costa et al.,
2003a, 2003b; Abudula et al., 2004; Chen et al., 2006a). In two studies using isolated
aortic muscle from rats, isosteviol (99.8% purity in one study) at 10 8 –10 5 mol/l
was found to reduce vasopressin-induced contraction of the muscle cells and to
inhibit angiotensin II–induced cell proliferation and endothelin-1 secretion (Wong et