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Identification of Steviol Glucuronide in Human Urine
JAN M. C. GEUNS,*,† JOHAN BUYSE,‡ANNELIES VANKEIRSBILCK,§
ELISABETH H. M. TEMME,§FRANS COMPERNOLLE,|AND SUZANNE TOPPET|
Laboratory of Functional Biology, KULeuven, Kasteelpark Arenberg 31, B-3001 Leuven, Faculty of
Applied Bioscience and Engineering, Laboratory of Physiology and Immunology of Domestic
Animals, Kasteelpark Arenberg 30, B-3001 Leuven, Faculty of Medicine, Department of Public
Health, Division of Nutritional Epidemiology, Kapucijnenvoer 33-35, B-3000 Leuven, and Laboratory
of Organic Synthesis, KULeuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium
Stevioside (250 mg capsules) was given three times daily to 10 healthy subjects. Steviol glucuronide
(steviol 19-O-β-D-glucopyranosiduronic acid; MM, 494.58; melting point, 198-199 °C) was character-
ized 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.
KEYWORDS:
Stevia rebaudiana
(Bertoni) Bertoni (Asteraceae); stevioside degradation; steviol; steviol
glucuronide
INTRODUCTION
The natural sweetener stevioside is a diterpene glycoside
extracted from the plant SteVia rebaudiana (Bertoni) Bertoni,
which belongs to the Asteraceae family and is native to Brazil
and Paraguay. Stevioside tastes about 300 times sweeter than
0.4 M sucrose and is noncaloric. In some parts of the world,
including Japan, South Korea, Israel, Mexico, Paraguay, Brazil,
Argentina, and Switzerland, stevioside is used to sweeten food
products and beverages. In the United States, powdered SteVia
leaves and refined extracts from the leaves have been used as
a dietary supplement since 1995 (1). Recently, the Joint FAO/
WHO Expert Committee on Food Additives (JECFA) accepted
a temporary allowable daily intake (ADI) of 0-2 mg steviol
equivalents/kg body weight (BW) (2). However, the U.S. Food
and Drug Administration (FDA), European Food Safety Agency
(EFSA) (European Union), and Food Standards Australia and
New Zealand do not accept this temporary ADI.
Bacteria isolated from the human colon are able to effect in
vitro transformation of stevioside into steviol (3-5). In vivo
degradation of stevioside into steviol occurs by bacterial action
in the colon of pigs (6) and humans (7). Among the selected
intestinal groups, bacteroidaceae were the most effective in
hydrolyzing SteVia sweeteners to steviol (5). In the colon of
pigs and humans, oral stevioside was completely degraded into
steviol, which was the only metabolite found in the faeces (6-
8). We report the identification of steviol glucuronide (steviol
19-O-β-D-glucopyranosiduronic acid) as an urinary excretion
product after oral intake of stevioside by human volunteers.
MATERIALS AND METHODS
Chemicals. A commercial mixture of steviol glycosides was
crystallized repeatedly from MeOH affording stevioside [19-O-β-D-
glucopyranosyl-13-O(β-D-glucopyranosyl(1f2)-β-D-glucopyranosyl-
steviol] in over 97% purity; impurities were 2.8% steviolbioside and a
trace of rebaudioside A (0.2%). Steviol was made according to ref 9
and repeatedly crystallized from MeOH to more than 99% purity. All
high-performance liquid chromatography (HPLC) grade solvents (H2O,
acetonitrile, CHCl3, MeOH, EtOH, and N,N-dimethylformamide) were
obtained from Acros Organics (Geel, Belgium), and acetone was
obtained from Biosolve (Valkenswaard, The Netherlands). Triethyl-
amine was from Acros, and 4-(bromomethyl)-7-methocoumarin [IUPAC
name: 4-(bromomethyl)-7-methoxy-2H-chromen-2-one] was from
Fluka (VWR, Haasrode, Belgium). β-Glucuronidase/sulfatase type H-2
from Helix pomatia digestive juice was from Sigma (Bornem, Belgium).
Subjects. To be included in this study, subjects had to be between
21 and 29 years old, be healthy as assessed by a medical questionnaire,
and have visible veins facilitating blood take, and the women could
not be pregnant, as determined from a pregnancy test at the beginning
of the investigation. Persons using medications known to affect the
blood pressure and those with diabetes were excluded (this was assessed
by determining glucose in a urine sample and by a medical question-
naire). Before the start of the study, the weights and heights of the
subjects were measured. The Medical Ethical Committee of the
University Hospital Gasthuisberg Leuven approved the study protocol.
After the purpose of this study was explained to the volunteers, all
gave written informed consent to the protocol.
Ten healthy volunteers, female (five) and male (five), participated
in the study. The volunteers were all between 21 and 29 years old. On
average, the women were 23 (1 years old, 172 (5 cm in height, and
weighed 65 (7 kg. On average, the men were 26 (2 years old, 175
(5 cm in height, and weighed 74 (11 kg. The body mass index
* To whom correspondence should be addressed. Tel: 032-16-321510.
Fax: +32-16-321509. E-mail: Jan.Geuns@bio.kuleuven.be.
†Laboratory of Functional Biology, KULeuven.
‡Laboratory of Physiology and Immunology of Domestic Animals.
§Division of Nutritional Epidemiology.
|Laboratory of Organic Synthesis, KULeuven.
2794
J. Agric. Food Chem.
2006,
54,
2794
−
2798
10.1021/jf052693e CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/04/2006
(BMI) averaged 23.1 (0.9 kg/m2. No power calculation was performed
before the start of the study.
Study Design. After the selection procedure, the volunteers collected
a 24 h control urine. Then, capsules with 250 mg of stevioside were
given to the subjects three times a day with 8 h intervals for a period
of 3 days. The volunteers were instructed to take the capsules with a
glass of water. On the third day, the volunteers were asked to collect
a 24 h urine sample (stevioside urine). The calorie intake between
subjects was not considered, nor was their physical activity, as this
was beyond the aim of this metabolism study.
From the 24 h control and stevioside urine, 20 mL was taken for
the detection of different markers. Creatinine was determined using
the Jaffe´ method (10). Sodium and potassium were detected by in-
direct potentiometric determination by means of ion selective elec-
trodes (Roche/Hitachi Modular analyzers; Roche Diagnostics Belgium,
Vilvoorde, Belgium). For the calcium determination, a complexometric
method was used, based on the reaction of calcium with o-cresol-
phthalein complex in alkaline solution (11). For the analysis of urea, a
kinetic UV assay was applied based on the coupled urease/glutamate
dehydrogenase enzyme system (12).
Urine Fractionation. The total 24 h urine fraction (between 1124
and 2494 mL) was run over an Amberlite XAD-2 column at about 30
mL/min. The column bed volume was 200 mL, which is sufficient for
the adsorption of all amphipathic molecules in a 24 h urine (Geuns et
al., unpublished). The columns were then rinsed with1Lofdistilled
water and then eluted with 400 mL of MeOH:acetone (50:50, v/v).
The eluate was divided into four equal fractions, and the solvent was
evaporated at reduced pressure at 50 °C.
Derivatization of Steviol-Containing Fractions. Fractions contain-
ing steviol and/or dihydroisosteviol used as an internal standard (IS)
were completely dried; the residues were taken up in dry acetone and
derivatized to form the 7-methoxy-coumarinyl esters as described (13).
After derivatization, the samples were purified by thin-layer chroma-
tography (TLC) using CHCl3:MeOH (98:2) as the eluent. The blue
fluorescent TLC band (UV 366 nm) corresponding with the ester
derivative of steviol was scraped off and eluted with CHCl3:MeOH
(80:20). After evaporation of the solvent, the residue was dissolved in
a known amount of MeOH and HPLC was done using a fluorescence
detector (λexc, 321 nm; λem, 391 nm) (13). The identity of the steviol
7-methoxy coumarinyl ester was checked by MS.
Analysis of Bound Steviol. In preliminary experiments, steviol
possibly bound as glucuronide and sulfate conjugates was obtained after
hydrolysis by β-glucuronidase/sulfatase from H. pomatia digestive juice.
Urine fractions were dissolved in 10 mL of MeOH:acetone (50:50)
and dihydroisosteviol (200 µg) (IS) was added to 250 µL of the solution.
The samples were then evaporated, and the residues were dissolved
into 500 µL of acetate buffer, pH 5. Then, 50 µLofβ-glucuronidase/
sulfatase (5000 U/375 U) was added. The mixture was incubated for 6
hat37°C. Under these conditions, a complete conversion to steviol
occurred. After hydrolysis, the samples were purified on C18 Extract
Clean Columns (500 mg, Alltech, Belgium) that were conditioned
before use with 3 mL of MeOH followed by 3 mL of H2O. After
application of the enzyme mixtures, the columns were rinsed with 3
mL of water and 3 mL of 50% MeOH. The steviol and IS were eluted
with 5 mL of MeOH. A 250 µL amount of the latter fraction was
evaporated under a stream of nitrogen at 50 °C. The completely dried
residue was then derivatized as described above.
Isolation of Larger Amounts of Steviol Glucuronide. A column
(35 mL bed volume) was prepared with 50 g of silica gel for column
chromatography (Machery-Nagel Silica gel 60, 0.063-0.2 mm) sus-
pended in ethyl acetate. About 600 mg of urine residue isolated from
the Amberlite XAD-2 purification step (see above) was dissolved in 1
mL of MeOH, and this solution was adsorbed to1gofsilica gel by
evaporating the solvent under a gentle flow of N2and under continuous
mixing. The silica gel-bound sample was applied onto the top of the
column, which was eluted with a solvent mixture of ethyl acetate:
ethanol:water (80:30:20). Fractions of 5 mL were collected. Samples
of each fraction were analyzed by TLC using ethyl acetate:ethanol:
water (80:30:20) as the solvent and compared with steviol glucuronide
as a reference compound. In addition, each fraction was tested for the
occurrence of steviol glucuronide and/or sulfate conjugates. To this
end, 20 µL samples of each fraction were evaporated in an Eppendorf
tube. Then, 150 µL of acetate buffer (pH 5) was added, followed by
20 µLofβ-glucuronidase/sulfatase (2000 U/150 U). After enzymatic
reaction for 15 h at 37 °C, the reaction mixture was freeze-dried and
the residue was derivatized by reaction with 4-(bromomethyl)-7-
methocoumarin (see above). All fractions containing enzyme-sensitive
steviol conjugates eluted as a single large peak from the silica gel
column, indicating the presence of only one steviol conjugate in the
urine samples. Therefore, all of the fractions containing this steviol
conjugate were pooled and the solvent was evaporated. The residue
was subjected to preparative TLC, and about 10 mg of white crystalline
material was isolated following elution of the TLC band with MeOH.
Melting Point. The melting point (uncorrected) was measured on a
Thermovar 9200 (type 300429) apparatus of Reichert-Jung (VWR).
Spectroscopic Techniques. IR. The IR spectrum was recorded in a
KBr pellet (FTIR 1600 series of Perkin-Elmer, Zaventem, Belgium).
MS. Mass spectral analysis of steviol glucuronide was carried out
using the LCQ Advantage ion trap mass spectrometer of Thermo
Finnigan (San Jose, CA) in both positive ESI (+) and negative ESI
(-) electrospray ionization modes. MS/MS spectra were generated by
CID (collision-induced decomposition) of the [M +2Na -H]+adduct
ion formed by ESI (+) and of the [M -H]-molecular ion obtained
by ESI (-).
NMR. 1H and 13C NMR spectra of steviol glucuronide were recorded
on a Bruker AMX 400 spectrometer equipped with an inverse 1H
multinuclei probe and operating at 400.13 MHz for 1H and 100.62 MHz
for 13C measurements (Bruker, Rheinstetten, Germany). The spectra
were run in CD3OD as a solvent. The chemical shifts are reported in
ppm vs TMS (tetramethylsilane) as an internal reference.
In the 13C spectra, the septet of the CD3OD signal was used as an
internal reference and placed at δ49 vs TMS. Besides the fully
decoupled 13C spectrum, the DEPT 135 pulse sequence was used to
differentiate between C, CH, CH2, and CH3signals.
Statistical Analysis. Data were analyzed using the general linear
models procedure of SAS (SAS software; version 8.1; SAS Institute,
Inc., Cary, NC) to test differences between stevioside and control groups
with repeated measurements for the different time points. Results are
expressed as means (standard errors of the mean (SEM). Differences
were considered statistically significant when the pvalue was less than
or equal to 0.05.
RESULTS
The volume of 24 h urine samples averaged 36% higher after
the stevioside intake (1561 (489 mL) as compared to the
control condition (1150 (488 mL). However, this difference
did not reach statistical significance (p)0.06) because of the
large interindividual variations. No significant differences were
detected in electrolytes excreted in the 24 h urine (Table 1).
Markers of tissue damage did not significantly differ between
the control and the stevioside treatment (Table 1).
Following metabolic conversion of ingested stevioside, steviol
glucuronide was isolated as the only metabolite detected in the
Table 1.
Determinants of Tissue Damage before and after Stevioside
Administration
a
stevioside
before after 250 mg of
stevioside with water
plasma (U/L) 0 h 1 h 3 h 7 h
alkaline phosphatase 79
±
980
±
7.3 81
±
787
±
7
ALT/GPT
b
14
±
2.6 11
±
310
±
3.66 12
±
3.33
creatine kinase 101
±
31 74
±
14.6 74
±
13.6 76
±
15.3
lactate dehydrogenase 184
±
41.6 131
±
27.3 132
±
32.6 81
±
23.6
a
Blood was taken on the third day of the experiment. Values are means
±
SEM (
n
)
9).
b
ALT/GPT, alanine aminotransferase or glutamic pyruvate trans-
aminase.
Identification of Steviol Glucuronide in Human Urine
J. Agric. Food Chem.,
Vol. 54, No. 7, 2006 2795
collected urine. The purified, crystalline material had a melting
point of 198-199 °C (uncorrected). The UV spectrum showed
a maximum absorbance at 208 nm (methylene). The IR spectrum
displays bands corresponding to the hydroxyl functions at 3416
(broad) and 1057 (broad) cm-1, to the ester carbonyl group at
1725 cm-1, and to the carboxylic acid at 1616 cm-1.
ESI spectra were run in both positive ESI (+) and negative
ESI (-) modes. In the ESI (+) spectrum, the disodium adduct
ion [M +2Na -H]+was the main peak observed at m/z539.
Further MS/MS analysis of this adduct ion revealed cleavage
of the glycosidic ester linkage to form both the [C(19)O2Na2]+
disodium adduct ion of steviol (m/z363) and the complementary
disodium adduct of the glucuronic acid moiety (m/z221).
The ESI (-) spectrum displayed the molecular ion [M -H]-
at m/z493 as the main ion species, corresponding to depro-
tonation of steviol glucuronide. Other interesting ions were due
to ion-molecule associations providing the dimer and trimer
ion species [2M -H]-, [2M +Na -2H]-, and [3M +2Na -
3H]-, observed at m/z987, 1009, and 1526, respectively.
Separate MS/MS analysis of the [M -H]-ion at m/z493 led
to cleavage of the glycosidic bond of the ester conjugate to
produce two complementary ions at m/z317 and m/z175,
representing the [C(19)O2]-carboxylate anion of steviol and
that of the glucuronic acid part, respectively.
The 19-O-acyl ester glycoside structure (Figure 1) proposed
for steviol glucuronide is fully confirmed by the 1H and 13C
NMR spectra, in which relevant δand Jvalues can be discerned
for the terpene and glucuronide moieties. The 1H NMR spectrum
reveals the two exocyclic vinylidene protons on C17 at δ4.93
and 4.77 (obscured by the OH of methanol at 25 °C). The
methylene and methine protons absorb as overlapping multiplets
between δ2.3 and 0.7. The C18 and C20 methyl groups display
two sharp singlets at δ1.24 and 0.97.
The anomeric proton of the glucuronide appears as a
downfield doublet at δ5.46 (3JH1′-H2′)7.8 Hz); this chemical
shift value is consistent with an 19-O-acyl glycosidic ester
linkage while the vicinal coupling indicates diaxial coupling of
H1′and H2′. Proton H5′is observed as a doublet at δ3.68
(3JH5′-H4′)9 Hz); the signals for H2′,H3′, and H4′appear as
a multiplet (3 H) between δ3.5 and 3.35.
The 13C NMR spectrum of steviol glucuronide displays the
following absorptions for the terpene moiety: two methyls, nine
methylenes, two methines, four quaternary carbon atoms, two
vinylic carbons for the exocyclic C16-C17 double bond, and
an ester carbonyl carbon for C19. Two-dimensional (2D) 1H,
13C correlation spectroscopy via one bond 1JCH coupling
(heteronuclear multiple quantum coherence, HMQC) or via two
and three bonds coupling (heteronuclear multiple-bond correla-
tion, HMBC) allowed unequivocal assignment of all C, CH,
CH3, and two CH2(C1′and C3) carbon atoms. The other CH2
signals were assigned by comparison with the values for free
steviol reported (14). All 13C absorptions found for the terpene
moiety are fully comparable with those reported for steviol
(Table 2).
For the glucuronide part, five absorptions in the 13C spectrum
are identified as CHO carbon atoms by their correlation with
the corresponding protons via the 2D HMQC method. Finally,
2D HMBC correlated spectroscopy was applied to reveal
diagnostic 2Jand 3JCH couplings. Thus, the ester glycoside
linkage CO(19)fO(1′) clearly appears from two 3JCH correla-
tions observed between C19 (δ178) and (i) the C18 methyl
group (δ1.24) and (ii) the anomeric proton (δ5.46). The free
carboxyl group (COOD) of the glucuronide moiety at δ176.6
is identified by its 2JCH correlation with the H5′doublet at δ
3.68.
DISCUSSION
Because of its molecular size, the uptake of stevioside by
the intestinal tract is expected to be extremely low, as suggested
by experiments with everted gastrointestinal sacs of rats (15)
and Caco-2 cell layers in which the uptake of stevioside was
less than 0.16% (6). Moreover, stevioside is not degraded by
the enzymes of the intestinal tract (3,4). However, stevioside
is degraded by bacteria commonly found in the colon resulting
in free steviol that is easily absorbed (5-7,15).
No free steviol was detected in urine. After enzymatic
hydrolysis of urine extracts by β-glucuronidase/sulfatase, steviol
was found as the only aglycone present. There was no indication
for the occurrence of, e.g., steviol sulfates. From in vitro
Figure 1.
Structure of steviol glucuronide and important HMBC interactions.
Table 2.
Assignment of Signals in the
13
C NMR Spectra of Steviol
(
18
) and Steviol Glucuronide (This Work);
δ
Values All Relative to
Internal SiMe
4
steviol in
CDCl
3
(
18
)steviol glucuronide in
CD
3
OD (this work)
13
C
13
C
1
H correlation
CH
3
C18 28.8 29.0 1.24 (s)
C20 15.4 16.4 0.97 (s)
CH
2
C1 40.5 41.9 1.89 and 0.85
C2 19.0 20.2 1.42
C3 37.8 39.1 2.21 and 1.04
C6 21.8 22.9 1.87
C7 41.2 42.8 1.54 and 1.43
C11 20.5 21.4 1.76
C12 39.5 40.6 1.46
C14 47.4 47.3 2.13 and 1.28
C15 47.0 48.9 2.18 and 2.07
CH C5 56.9 58.7 1.12
C9 53.8 55.4 0.99
C C4 43.6 45.1
C8 41.8 42.8
C10 39.5 40.7
C13 80.4 80.8
sp
2
C C16 155.7 157.1
C17 103.0 103.3 4.97 and 4.77 (br. s)
C19 183.5 178.0
glucuronide CH1
′
95.4 5.46 (d)
part of steviol
glucuronide CH5
′
77.4 3.68 (d)
CH2
′
, CH3
′
, CH4
′
78.6, 73.9, 73.5
δ
3.5 to 3.35 (m)
CO
2
D 176.6 (br)
2796
J. Agric. Food Chem.,
Vol. 54, No. 7, 2006 Geuns et al.
incubations of steviol with human liver microsomes, it was
concluded that the transformation of steviol by human mi-
crosomes was very low and about four times lower than that
by rat microsomes (4). As no other metabolites were found,
the following excretion route is suggested (Figure 2). After
degradation of stevioside to steviol by bacteria of the colon,
part of the steviol is absorbed by the colon and transported to
the liver by portal blood. In the liver, steviol glucuronide is
formed, which is released into the blood and filtered out by the
kidneys into the urine. Of the daily dose of 750 mg of stevioside,
300 mg of free steviol is formed in the colon (complete
degradation). About 23 (2.7 mg of free steviol is directly
excreted in the feces. About 277 mg of free steviol is taken up
by the colon, of which about 101.8 (16.4 mg are recovered in
the blood as steviol glucuronide and about 101.8 (21.3 mg
are are excreted into the urine, also as steviol glucuronide. The
total recovery of steviol is 226.6 (23.5 mg (75.5%). This is a
recovery similar to the methodological recovery. The steviol
glucuronide present in the blood is expected to be excreted in
the urine during the next 24 h. The results suggest that there is
no accumulation of steviol derivatives in the human body. In a
metabolism study with volunteers using liquid chromatography-
mass spectrometry, similar results were found, i.e., degradation
of stevioside to steviol in the colon and excretion of a compound
with the same molecular weight as steviol glucuronide (7). No
other metabolites of steviol were detected in urine. However,
no positive identification of the steviol glucuronide was done.
It is known that glucuronide formation easily happens in the
liver as is also the case with soy isoflavones that, after uptake,
were metabolized to compounds, which were hydrolyzable with
a combined β-glucuronidase and sulfatase enzyme preparation
(16).
ABBREVIATIONS USED
ADI, allowable daily intake; BMI, body mass index; BW,
body weight; EFSA, European Food Safety Agency (Europe);
FDA, Food and Drug Administration (United States); HMQC,
heteronuclear multiple quantum coherence; HMBC, hetero-
nuclear multiple-bond correlation.
ACKNOWLEDGMENT
We thank the volunteers for their participation in the study. We
thank the personnel of the division of Youth Health Care
(University of Leuven) and the central laboratory of the
University Hospital Gasthuisberg of Leuven for the analyses
of the different markers in the urine: creatinine, sodium,
potassium, calcium, and ureum. We acknowledge Hilde Ver-
linden, Tom Struyf, Rene´ De Boer, and Christine Vergauwen
for their excellent technical assistance.
Supporting Information Available: Figures of the IR spec-
trum, the ESI-MS, and the 1H NMR spectrum. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Hypothetical route from dietary stevioside to steviol glucuronide in human urine.
Identification of Steviol Glucuronide in Human Urine
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Vol. 54, No. 7, 2006 2797
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Received for review October 31, 2005. Revised manuscript received
February 9, 2006. Accepted February 10, 2006. We acknowledge
Onderzoeksraad KULeuven for Grant OT/00/15, the FWO for Grant
G.0111.01, and Brulo Beheer for its financial support.
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J. Agric. Food Chem.,
Vol. 54, No. 7, 2006 Geuns et al.