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Absorption and Modification of Rutin in the Human Stomach
2.9 Absorption and Modification of Rutin in the Human Stomach H. Pforte,' T. Naser,' G. Jacobasch' and H.J. Buhr2 GERMAN INSTITUTE O F HUMAN NUTRITION, POTSDAMREHBRUCKE, GERMANY CLINICAL AND MOLECULAR GASTROENTEROLOGY AND SURGERY, FREIE UNIVERSITAT BERLIN, GERMANY
1 Introduction Flavonoids have a variety of protective effects on human health.' Biological activities result from both systemically generated actions and influences connected with the intestinal microflora.2 Antiatherosclerotic and anticarcinogenic effects are realized mainly sy~temically.~ It is necessary to know the therapeutic doses in order to optimize a continous gastrointestinal absorption of the flavonoids. Since quercetin is the most abundant flavonoid in the human diet and characterized by a high protective metabolic effectiveness, we focussed our investigations on the absorption of this flavonol compound. Recent findings suggest that quercetin is relatively unstable under milieu conditions of about pH7.0 appearing in the human small intestine. It is therefore difficult to achieve a defined continuous dose of flavonoid absorption. The natural dietary compounds of quercetin in plants are stable glycosides. The carbohydrate chain can be cleaved microbially in the gastrointestinal tract, for .~ a instance by Enterococcus cassellflavus, liberating the aglycon q ~ e r c e t i nAs result, the question arises whether and how effectively quercetin rutinoside can be absorbed in the different parts of the stomach?
2 Methods The absorption process of rutin by the stomach mucosa was studied in Ussing chambers. Stomach wall samples of healthy areas of the antrum and corpus were taken from patients during surgical removal of malignomes and immediatly used for the experimental studies. In cold isotonic solution the samples were stripped and clamped within 10 minutes post-operatively as separating
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H . Pforte et al.
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wall between two compartments of the Ussing chamber. The chambers were filled with Ringer solution and additionally containing albumin ( 5 g%) in the serosal side: The chambers had a temperature of 37 "C and were gassed continously with a mixture of 95% 0 2 and 5% C02. The vitality of the tissue samples was evaluated on the basis of voltage, short circuit and epithelial resistance. Absorption and transport experiments were started by adding quercetin rutinoside (0.8 and 1.6 pM) into the mucosal part of the chamber. Samples from both solutions of the serosal and the mucosal sides were freezedried after 0, 15,30,60 and 90 minutes respectively. After that, they were stored together with the tissue samples at - 80 "C until use. Ffavonoid analysis: The frozen mucosa was lyophilized and pulverized in a mortar and defatted with heptane by sonicating for 10 minutes and Soxhletextraction with methanol for 4 h under reduced pressure at about 45 "C. From the mucosa and from the two solutions flavonoids were extracted and dissolved for analysis by HPLC in DMF/H20 (v/v 2:l). The stationary phase was a Nucleosil RP- 18 endcapped column (Macheray-Nagel, Duren Germany) (250 mm x 4.6mm; 5 p M ) at 30 "C with a 4 x 4 mm pre-column. Flavonoid glycosides and aglycones were eluted with a gradient consisting of water, 80% methanol, buffered with phosphate pH 3.4. Detection was carried out by an ESA coularray detector containing 12 pairs of electrodes and a detection range between 0 and 825 mV. To identify the flavonoids retention times and voltamogrammes of standards were used. The flavonoids were quantified by using an external calibration curve.
3 Results and Discussion Only a small number of studies have been carried out to determine where and how effectively flavonoids are absorbed in the gastrointestinal tract. Results of our Ussing chamber experiments prove that human stomach mucosa absorbs quercetin rutinoside. Both absorption rate of rutin and enzymatic release of the aglycon quercetin differ in dependence of the sample localization in the stomach. Data given in Figure 1 show that generally the antrum takes in more rutin than the corpus, but we identified higher and lower absorption rates in both areas. These results do not agree with the conclusion of Kuhnau that only aglycons could pass through the mucosa.' Hydrolysis of rutin does not occur as a result of the intestinal microorganisms but is caused by a stomach specific pglucosidase. Our own unpublished data of germfree rats also confirm that the low activity of the microbial a-glucosidase in stomach is nearly without any physiological relevance. The conclusion that absorption of flavonoid does not require their aglycone formation furthermore agrees well with results of the Hollman group, who demonstrated in a human study with ileostomy subjects an absorption of quercetin glycosides from onions of 52% and of pure rutin of 17%.6 The pattern of flavonoid compounds in both the serosal fluid and the stomach walls suggest a higher enzymatic activity of B-glucosidase in the corpus than in the antrum (Table 1). The corpus is also characterized by a higher activity of enzymatic O-methylation of quercetin. Formation of isorhamnetin is
Absorption and Modification of Rutin in the Human Stomach
86 antrum
-
11 10
9
corpus 11
high absorption rate
8 7 6 5 4 3 2 1
(3 low
absorption rate
-
rate zc
= 0
1 I
absorption 1 rate ~. 1
3 2
.
'
1
~
-
15 30 60 90
Time [min]
15 30 60 90
Tlmo [min]
Figure 1 Time dependence of the rutin transport in the human corpus and antrum mucosa of the stomach
Table 1 Flavonoid concentrations in the human corpus mucosa of the stomach 90 minutes after addition of rutin on the luminal side Rutin [ n M ]
Quercetin [ n M ]
Isorhamnetin [ n M ]
Transport rate
X
+S
X
fs
X
fs
High Low
2.79 1.75
1.20 0.50
0.31 0.17
0.02 0.13
0.02 0.02
0.01 0.01
believed to be catalyzed by distinct classes of O-methyltransferases (OMTs). Until now no OMTs of the human stomach mucosa have been isolated and characterized. Therefore, we can not decide whether the different rates of the isorhamnetin formation reflect a distinct local expression of OMT-isoenzymes or only a higher O-methylation capacity in the corpus. From these results, the following conclusion is drawn, that the absorption rates of rutin in the stomach correspond with those of the small intestine. The absence of flavonoid degrading bacteria in the stomach favours this part of the gastrointestinum for a monitoring of systemic therapeutic effects of quercetin.
4 References 1 J.V. Formica and W. Regelson, Review of the biology of quercetin and related bioflavonoids, Food Client Toxieol., 1995, 33, 1061-1080. 2 H. Schneider, A. Schwiertz, M.D. Collins and M. Blaut, Anaerobic transformation of quercetin-3-glucoside by bacteria from the human intestinal tract, Arch. Microbiol., 1999,171,81-91. 3 D.D. Schramm and J.B. German, Potential effects of flavonoids on the etiology of vascular disease, J . Nutr. Biochem., 1998, 9, 560-566.
H . Pforte et al.
87
4 R. Simmering, B. Kleessen and M. Blaut, Quantification of the flavonoid-degrading bacterium Eubacteriurn rarnulus in human fecal samples with a species-specific oligonucleotide hybridization probe, Appl. Environ. Microbiol., 1999,54, 1079-1084. 5 J. Kuhnau, The flavonoids: a class of semi-essential food components: their role in human nutrition, World Rev.Nutr. Diet., 1976,24, 117-120. 6 P.C.M. Hollman, Bioavailability of flavonoids, Eur. J . Clin. Nufr., 1997,51, 66-69.
1 Introduction Flavonoids have a variety of protective effects on human health.' Biological activities result from both systemically generated actions and influences connected with the intestinal microflora.2 Antiatherosclerotic and anticarcinogenic effects are realized mainly sy~temically.~ It is necessary to know the therapeutic doses in order to optimize a continous gastrointestinal absorption of the flavonoids. Since quercetin is the most abundant flavonoid in the human diet and characterized by a high protective metabolic effectiveness, we focussed our investigations on the absorption of this flavonol compound. Recent findings suggest that quercetin is relatively unstable under milieu conditions of about pH7.0 appearing in the human small intestine. It is therefore difficult to achieve a defined continuous dose of flavonoid absorption. The natural dietary compounds of quercetin in plants are stable glycosides. The carbohydrate chain can be cleaved microbially in the gastrointestinal tract, for .~ a instance by Enterococcus cassellflavus, liberating the aglycon q ~ e r c e t i nAs result, the question arises whether and how effectively quercetin rutinoside can be absorbed in the different parts of the stomach?
2 Methods The absorption process of rutin by the stomach mucosa was studied in Ussing chambers. Stomach wall samples of healthy areas of the antrum and corpus were taken from patients during surgical removal of malignomes and immediatly used for the experimental studies. In cold isotonic solution the samples were stripped and clamped within 10 minutes post-operatively as separating
84
H . Pforte et al.
85
wall between two compartments of the Ussing chamber. The chambers were filled with Ringer solution and additionally containing albumin ( 5 g%) in the serosal side: The chambers had a temperature of 37 "C and were gassed continously with a mixture of 95% 0 2 and 5% C02. The vitality of the tissue samples was evaluated on the basis of voltage, short circuit and epithelial resistance. Absorption and transport experiments were started by adding quercetin rutinoside (0.8 and 1.6 pM) into the mucosal part of the chamber. Samples from both solutions of the serosal and the mucosal sides were freezedried after 0, 15,30,60 and 90 minutes respectively. After that, they were stored together with the tissue samples at - 80 "C until use. Ffavonoid analysis: The frozen mucosa was lyophilized and pulverized in a mortar and defatted with heptane by sonicating for 10 minutes and Soxhletextraction with methanol for 4 h under reduced pressure at about 45 "C. From the mucosa and from the two solutions flavonoids were extracted and dissolved for analysis by HPLC in DMF/H20 (v/v 2:l). The stationary phase was a Nucleosil RP- 18 endcapped column (Macheray-Nagel, Duren Germany) (250 mm x 4.6mm; 5 p M ) at 30 "C with a 4 x 4 mm pre-column. Flavonoid glycosides and aglycones were eluted with a gradient consisting of water, 80% methanol, buffered with phosphate pH 3.4. Detection was carried out by an ESA coularray detector containing 12 pairs of electrodes and a detection range between 0 and 825 mV. To identify the flavonoids retention times and voltamogrammes of standards were used. The flavonoids were quantified by using an external calibration curve.
3 Results and Discussion Only a small number of studies have been carried out to determine where and how effectively flavonoids are absorbed in the gastrointestinal tract. Results of our Ussing chamber experiments prove that human stomach mucosa absorbs quercetin rutinoside. Both absorption rate of rutin and enzymatic release of the aglycon quercetin differ in dependence of the sample localization in the stomach. Data given in Figure 1 show that generally the antrum takes in more rutin than the corpus, but we identified higher and lower absorption rates in both areas. These results do not agree with the conclusion of Kuhnau that only aglycons could pass through the mucosa.' Hydrolysis of rutin does not occur as a result of the intestinal microorganisms but is caused by a stomach specific pglucosidase. Our own unpublished data of germfree rats also confirm that the low activity of the microbial a-glucosidase in stomach is nearly without any physiological relevance. The conclusion that absorption of flavonoid does not require their aglycone formation furthermore agrees well with results of the Hollman group, who demonstrated in a human study with ileostomy subjects an absorption of quercetin glycosides from onions of 52% and of pure rutin of 17%.6 The pattern of flavonoid compounds in both the serosal fluid and the stomach walls suggest a higher enzymatic activity of B-glucosidase in the corpus than in the antrum (Table 1). The corpus is also characterized by a higher activity of enzymatic O-methylation of quercetin. Formation of isorhamnetin is
Absorption and Modification of Rutin in the Human Stomach
86 antrum
-
11 10
9
corpus 11
high absorption rate
8 7 6 5 4 3 2 1
(3 low
absorption rate
-
rate zc
= 0
1 I
absorption 1 rate ~. 1
3 2
.
'
1
~
-
15 30 60 90
Time [min]
15 30 60 90
Tlmo [min]
Figure 1 Time dependence of the rutin transport in the human corpus and antrum mucosa of the stomach
Table 1 Flavonoid concentrations in the human corpus mucosa of the stomach 90 minutes after addition of rutin on the luminal side Rutin [ n M ]
Quercetin [ n M ]
Isorhamnetin [ n M ]
Transport rate
X
+S
X
fs
X
fs
High Low
2.79 1.75
1.20 0.50
0.31 0.17
0.02 0.13
0.02 0.02
0.01 0.01
believed to be catalyzed by distinct classes of O-methyltransferases (OMTs). Until now no OMTs of the human stomach mucosa have been isolated and characterized. Therefore, we can not decide whether the different rates of the isorhamnetin formation reflect a distinct local expression of OMT-isoenzymes or only a higher O-methylation capacity in the corpus. From these results, the following conclusion is drawn, that the absorption rates of rutin in the stomach correspond with those of the small intestine. The absence of flavonoid degrading bacteria in the stomach favours this part of the gastrointestinum for a monitoring of systemic therapeutic effects of quercetin.
4 References 1 J.V. Formica and W. Regelson, Review of the biology of quercetin and related bioflavonoids, Food Client Toxieol., 1995, 33, 1061-1080. 2 H. Schneider, A. Schwiertz, M.D. Collins and M. Blaut, Anaerobic transformation of quercetin-3-glucoside by bacteria from the human intestinal tract, Arch. Microbiol., 1999,171,81-91. 3 D.D. Schramm and J.B. German, Potential effects of flavonoids on the etiology of vascular disease, J . Nutr. Biochem., 1998, 9, 560-566.
H . Pforte et al.
87
4 R. Simmering, B. Kleessen and M. Blaut, Quantification of the flavonoid-degrading bacterium Eubacteriurn rarnulus in human fecal samples with a species-specific oligonucleotide hybridization probe, Appl. Environ. Microbiol., 1999,54, 1079-1084. 5 J. Kuhnau, The flavonoids: a class of semi-essential food components: their role in human nutrition, World Rev.Nutr. Diet., 1976,24, 117-120. 6 P.C.M. Hollman, Bioavailability of flavonoids, Eur. J . Clin. Nufr., 1997,51, 66-69.