Sweetness and Sweeteners - American Chemical Society

Chapter 37

Downloaded by UNIV OF MONTANA on December 14, 2014 | http://pubs.acs.org Publication Date: March 4, 2008 | doi: 10.1021/bk-2008-0979.ch037

Steviol Glucuronide as Excretion Product of Stevioside in Human Volunteers: Lack of Carcinogenic Properties of Steviol Glycosides and Steviol Jan M. C. Geuns Laboratory Functional Biology, K U Leuven, 3001 Leuven, Belgium

Absorption studies with Caco-2 cell monolayers revealed that steviol glycosides are barely absorbed by the intestines. Metabolism studies in healthy volunteers have shown that stevioside is completely degraded by bacteria of the colon into steviol and that part of this steviol is absorbed and glucuronated in the liver. The glucuronide is released in the blood and filtered by the kidneys into the urine. No accumulation of steviol glycosides or derivatives has been observed. As mutagenic effects of steviol were described, a thorough literature study has been made to evaluate possible risks when using steviol glycosides as a sweetener. The conclusion is that there are no indications that steviol glycosides used as a sweetener are not safe.

© 2008 American Chemical Society

In Sweetness and Sweeteners; Weerasinghe, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

573

574


Introduction Steviol glycosides (mainly stevioside, rebaudioside A ) are sweeteners obtained from Stevia rebaudiana (Bertoni) Bertoni. Oral stevioside is not absorbed by the intestines or degraded by stomach juice (/, 2, 3). Only bacteria of the cecum (rats, hamsters, mice) or the colon of pigs and man degrade stevioside into steviol (2-5). As no metabolism studies were published with volunteers, and these studies are required by JECFA, F D A and EFSA, we organised research with volunteers. One of the aims of the study was to know i f steviol, the aglycone of steviol glycosides, is further metabolised and/or accumulating in the body or i f it is excreted in feces and/or urine.

Experimental Setup Transport studies Transport studies were done with Caco-2 monolayers as described (4). Stevioside and rebaudioside A were tested at 1 m M , steviol at 30, 100, 300 and 1000 μΜ concentrations in 2% D M S O .

Metabolism studies Gellules containing 250 mg pure stevioside were administered thrice daily for 3 days with time intervals of 8 h. On the third day, blood samples (2x4 ml) were taken before and at 0.5, 1, 3, 5 and 7 h after the first dose of day 3. A 24 h urine was collected during the third day and the feces of the third day was collected on the fourth day. To correct for losses of steviol during sample clean-up, dihydroisosteviol (Figure 1) was synthesized as internal standard (IS; 6). Very sensitive analysis of steviol and its possible metabolites was possible after derivatisation with 4(bromomethyl)-7-methoxycoumarin (Figure 2, detection limit between 50 and 100 pg). Sample clean-up was as described (2). Steviol and its derivatives were searched for as the free form or after splitting possible glucuronides respectively sulfates by enzymatic hydrolysis with β-glucuronidase/sulfatase of Helix pomatia digestive juice (2).



Figure I. Breakdown of stevioside and synthesis of dihydroisosteviol


(il -α ut


Figure 2. Derivatization of steviol with 4-(bromomethyl)-7-methoxycoumarin.

(7-methoxy-4-coumaiinyl)methyl ester of steviol


577

Results Transport studies Transport studies of stevioside, rebaudioside A and free steviol were done with Caco-2 monolayers (Figure 3).


50 45 40 35 30 25 20 15 10 5 0 lmMST

lmMrebA

0,03 itiM S V

0,1 m M S V

0,3mM S V

ImM SV

IM Absorptive Β Secretory |

Figure 3. Permeability coefficients for 1 mM stevioside, 1 mM rebaudioside A and steviol (0.03, 0.1, 0.3 and 1 mM) (Geuns unpublished).

Absorptive transport of stevioside and rebaudioside A was below 0.16% and 0.1% respectively of the amounts administered in the apical vial. The absorptive transport of steviol was much higher (around 30%). In the secretory direction, steviol transport was significantly lower (around 5%) than in the absorptive direction (only measured for 0.03 and 0.1 mM). These results are in agreement with those obtained with everted sacs of rat intestines ( 7 ) and with human organic anion transporters (hOATi and hOAT ) expressed in Xenopus laevis oocytes (8). 3

Metabolism studies Feces Analysis N o stevioside was detected in the feces. Free steviol was detected and was the only degradation product of stevioside found. These results are in agreement


578 with those of in vitro incubations with bacteria of human feces and of a metabolism study (9, 10).


Blood No stevioside was detected in the blood after H P L C analysis and monitoring by U V at 210 nm (detection limit ~ 50 ng). No free steviol was detected after derivatisation and H P L C analysis (detection limit ~ 100 pg). After enzymatic hydrolysis of plasma samples, between 0.3 and 21.3 μg steviol/ml plasma was detected. After an oral dose, the steviol glucuronide present in the plasma increased to a maximum, and decreased again after it was filtered out by the kidneys (Figure 4). The rate of metabolism in each volunteer was slightly different, giving an average steviol glucuronide concentration of about 5.65 μg steviol/ml present in the glucuronide.

Urine No free steviol or stevioside were detected by the methods used. However, after enzymatic hydrolysis of urine extracts, between 25 and 205 mg steviol/24 h urine was released from the conjugates. There was no evidence of the occurrence of other steviol metabolites or derivatives (Figure 5). After sample clean-up of large urine fractions, steviol glucuronide could be crystallised and fully characterised by MS, IR and H an C N M R (Figure 6). Its M W was 494.58, its melting point 198-199°C. It showed U V absorption at 208 nm (2). It was suggested that stevioside is fully degraded by bacteria of the colon. Part of the steviol is then absorbed and transported by portal blood to the liver, where the steviol is glucuronated to steviol glucuronide. This is released into the blood and filtered by the kidneys and excreted in the urine (Figure 6). !

13

Quantitative aspects of stevioside metabolism The daily oral dose was 750 mg stevioside. After complete degradation in the colon, the theoretical amount of steviol formed is 300 mg (40% of the stevioside). After hydrolysis, in the urine about 102 mg steviol equivalents were found, in the blood about 102 mg (assuming the blood volume is about 7 % of the B W and the plasma volume about 56 % of the blood volume). After the first dose of the day, 34 mg steviol was found as the maximum amount in the plasma, and this value was multiplied by 3 as 3 doses were given a day (hence 102 mg/total plasma). The feces contained 23 mg. The total amount of steviol detected was 227, i.e. 76 % of the total steviol formed. This recovery is similar



579

Figure 4. Steviol glucuronide concentration in the plasma after the first dose of day 3. Adapted with permission from reference 3. Copyright 2006 The Society for Experimental Biology and Medicine.

Figure 5. Example of a HPLC analysis of the derivatives of a urine fraction, with the ester of steviol around 13 min, that ofIS around 23 min. Adapted with permission from reference 3. Copyright 2006 The Society for Experimental Biology and Medicine.



Figure 6. Route for the formation and excretion of steviol glucuronide.


581 to the methodological recovery found after spiking feces and blood samples with stevioside or steviol.


Comparison of the metabolism study at KULeuven and at the Milan ITB-CNR Research Institute In the study of Milan (10) one single dose of 375 free steviol, stevioside, steviol glucuronide, steviol steviol and 15-oxo-steviol were searched for Chromatography (TIC). In the experiment done at the doses of250 mg each were administered for 3 days.

mg was administered and 16,17

Sweetness and Sweeteners - American Chemical Society

Recommend Documents