The differential tissue distribution of the citrus flavanone naringenin following gastric instillation

Citrus flavonoids have been investigated for their biological activity, with both anti-inflammatory and -carcinogenic effects being reported. However, little information is known on the bioavailability of these compounds in vivo. The objectives of this study were to determine the tissue distribution of naringenin after gastric gavage of [³H]-naringenin to rats. Unlabelled naringenin was also used to quantify the levels of naringenin and its major metabolites in tissues and eliminated in the urine and faeces. Significant radioactivity was detected in the plasma as well as all tissues examined 2 h post-gavage. After 18 h, higher levels of radioactivity were retained in plasma and tissues (55% of the administered radioactivity). Investigation of the nature of metabolites, using unlabelled naringenin, revealed that the glucuronides were the major components in plasma, tissues and urine, in addition to the colonic metabolite 3-(4-hydroxyphenyl) propionic acid, detected in the urine. The aglycone was the form extensively retained in tissues after 18 h post-gavage. Total identified metabolites detected after 18 h in most tissues were only 1-5% of the levels detected after 2 h. However, the brain, lungs and heart retained 27, 20 and 11%, respectively, relative to the total metabolites detected at 2 h. While radioactive detection suggests increased levels of breakdown products of naringenin after 18 h versus 2 h, the products identified using unlabelled naringenin are not consistent with this, suggesting that a predominant proportion of the naringenin breakdown products at 18 h are retained as smaller decomposition molecules which cannot yet be identified.     

The reaction of flavanols with nitrous acid protects against N-nitrosamine formation and leads to the formation of nitroso derivatives which inhibit cancer cell growth

Studies have suggested that diets rich in polyphenols such as flavonoids may lead to a reduced risk of gastrointestinal cancers. We demonstrate the ability of monomeric and dimeric flavanols to scavenge reactive nitrogen species derived from nitrous acid. Both epicatechin and dimer B2 (epicatechin dimer) inhibited nitrous acid-induced formation of 3-nitrotyrosine and the formation of the carcinogenic N-nitrosamine, N-nitrosodimethylamine. The reaction of monomeric and dimeric epicatechin with nitrous acid led to the formation of mono- and di-nitroso flavanols, whereas the reaction with hesperetin resulted primarily in the formation of nitrated products. Although, epicatechin was transferred across the jejunum of the small intestine yielding metabolites, its nitroso form was not absorbed. Dimer B2 but not epicatechin monomer inhibited the proliferation of, and triggered apoptosis in, Caco-2 cells. The latter was accompanied by caspase-3 activation and reductions in Akt phosphorylation, suggesting activation of apoptosis via inhibition of prosurvival signaling. Furthermore, the dinitroso derivative of dimer B2, and to a lesser extent the dinitroso-epicatechin, also induced significant toxic effects in Caco-2 cells. The inhibitory effects on cellular proliferation were paralleled by early inhibition of ERK 1/2 phosphorylation and later reductions in cyclin D1 levels, indicating modulation of cell cycle regulation in Caco-2 cells. These effects highlight multiple routes in which dietary derived flavanols may exert beneficial effects in the gastrointestinal tract.     

Urinary excretion of hydroxycinnamates and flavonoids after oral and intravenous administration.

The urinary recoveries of the hydroxycinnamates, ferulic acid (3-methoxy, 4-hydroxy cinnamic acid), and chlorogenic acid (the quinic acid ester of 3,4-dihydroxycinnamic acid), and three structurally related flavonoids were studied in the rat. For the latter, the aglycone quercetin was compared with its 3-glucoside (isoquercitrin) and 3-rhamnoglucoside (rutin). Doses of 50 mg/kg were administered via the oral and intravenous routes and urine collected over the subsequent 24-h period. Reverse phase HPLC with photo-diode array detection was used to analyze the unchanged compound and their metabolites excreted in the urine. Ferulic acid and isoquercitrin were orally absorbed (5.4 and 0.48% of administered dose, respectively) and are therefore bioavailable. In contrast, neither unchanged chlorogenic acid, rutin, quercetin, nor the conjugated metabolites in the form of glucuronide or sulphate were detected in the urine after oral dosing. All the flavonoids studied produced low total urinary recoveries after intravenous administration, 9.2% for quercetin-3-rhamnoglucoside, 6.7% for the 3-glucoside, and 2.4% for the aglycone, indicating that extensive metabolism to low molecular weight compounds or excretion via other routes may be occurring. Overall it can be stated that renal excretion is not a major pathway of elimination for intact flavonoids and hydroxycinnamates in the rat.     

Streptozotocin diabetes and sugar transport by rat ileal enterocytes: evidence for adaptation caused by an increased luminal nutrient load.

Preparations of villus enterocytes and brush border membrane vesicles have been used to study the effects of streptozotocin-induced diabetes mellitus in rats on sugar transport across the brush border and basolateral membranes of ileal epithelial cells. In isolated cells, diabetes increased Na(+)-dependent galactose transport across the brush border of mid-villus but not upper villus cells. Galactose transport across the basolateral membrane was, however, enhanced by diabetes in both cell populations. Kinetic analysis of vesicle data suggested the presence of two transporters for Na(+)-dependent glucose transport. Diabetes induced a 5-fold increase in both KT and Vmax of the high-affinity/low-capacity system together with a 2-fold increase in the Vmax of the low-affinity/high-capacity transporter. Glucose was almost undetectable in the lumen of the upper and lower ileum in control animals but was present at high levels (26.1 +/- 4.3 mM and 6.5 +/- 1.3 mM) in diabetic rats. The possible significance of these changes in luminal sugar concentration in relation to the adaptation of transport across ileal enterocytes is discussed.     

Interactions of the flavonoid naringenin in the gastrointestinal tract and the influence of glycosylation

Previous studies have documented the direct antioxidant effects of estradiol, and it is tempting to ascribe the antiapoptosis effects of estradiol to its scavenging of reactive oxygen species. However, recent reports have also demonstrated that long-term exposure of MCF-7 human breast cancer cells to estradiol results in estrogen receptor- and estradiol dose-dependent overexpression of the antiapoptosis gene, bcl-2. We have used the pattern of protection of membrane phospholipids from oxidation as a probe to separate these direct and indirect effects of estradiol from one another. Immediate exposure to estradiol non-specifically protects all membrane phospholipids from oxidation by the diazo radical initiator, AMVN. This implies the direct antioxidant activity of estradiol in this system. In contrast, long-term exposure, with associated increased expression of bcl-2, protects only phosphatidylserine, the oxidation of which is a critical component of the final common pathway for apoptosis. This bcl-2-mediated indirect effect of estradiol is accompanied by prevention of apoptosis in MCF-7 cells.     

Resveratrol is absorbed in the small intestine as resveratrol glucuronide

We have studied the absorption and metabolism of resveratrol in the jejunum in an isolated rat small intestine model. Only small amounts of resveratrol were absorbed across the enterocytes of the jejunum and ileum unmetabolised. The major compound detected on the serosal side was the glucuronide conjugate of resveratrol (96.5% +/- 4.6 of the amount absorbed) indicating the susceptibility of resveratrol to glucuronidation during transfer across the rat jejunum. The presence of the glucuronide was confirmed using HPLC-PDA and nanoES-MS/MS techniques. These findings suggest that resveratrol is most likely to be in the form of a glucuronide conjugate after crossing the small intestine and entering the blood circulation. This will have important implications for the biological functions of resveratrol in vivo.     

Decomposition of cocoa procyanidins in the gastric milieu

There is considerable interest in the bioavailability of flavonoids and phenolic components of the diet and their bioactivity in vivo. However, little is known of pre-absorption events in the gastric lumen. The effects of the acidic environment, as found in the gastric milieu, on procyanidin oligomers of catechin polyphenols has been investigated. The results show that under these conditions the procyanidin oligomers (trimer to hexamer) are hydrolysed to mixtures of epicatechin monomer and dimer, thus enhancing the potential for their absorption in the small intestine.     

Upregulation of ACE2-ANG-(1-7)-Mas axis in jejunal enterocytes of type 1 diabetic rats: implications for glucose transport

The inhibitory effects of the angiotensin-converting enzyme (ACE)-ANG II-angiotensin type 1 (AT(1)) receptor axis on jejunal glucose uptake and the reduced expression of this system in type 1 diabetes mellitus (T1DM) have been documented previously. The ACE2-ANG-(1-7)-Mas receptor axis is thought to oppose the actions of the ACE-ANG II-AT(1) receptor axis in heart, liver, and kidney. However, the possible involvement of the ACE2-ANG-(1-7)-Mas receptor system on enhanced jejunal glucose transport in T1DM has yet to be determined. Rat everted jejunum and Caco-2 cells were used to determine the effects of ANG-(1-7) on glucose uptake and to study the ACE2-ANG-(1-7)-Mas receptor signaling pathway. Expression of target gene and protein in jejunal enterocytes and human Caco-2 cells were quantified using real-time PCR and Western blotting. T1DM increased jejunal protein and mRNA expression of ACE2 (by 59 and 173%, respectively) and Mas receptor (by 55 and 100%, respectively) in jejunum. One millimolar ANG-(1-7) reduced glucose uptake in jejunum and Caco-2 cells by 30.6 and 30.3%, respectively, effects that were abolished following addition of 1 μM A-779 (a Mas receptor blocker) or 1 μM GF-109203X (protein kinase C inhibitor) to incubation buffer for jejunum or Caco-2 cells, respectively. Finally, intravenous treatment of animals with ANG-(1-7) significantly improved oral glucose tolerance in T1DM but not control animals. In conclusion, enhanced activity of the ACE2-ANG-(1-7)-Mas receptor axis in jejunal enterocytes is likely to moderate the T1DM-induced increase in jejunal glucose uptake resulting from downregulation of the ACE-ANG II-AT(1) receptor axis. Therefore, altered activity of both ACE and ACE2 systems during diabetes will determine the overall rate of glucose transport across the jejunal epithelium.     

The fate of olive oil polyphenols in the gastrointestinal tract: Implications of gastric and colonic microflora-dependent biotransformation

We have conducted a detailed investigation into the absorption, metabolism and microflora-dependent transformation of hydroxytyrosol (HT), tyrosol (TYR) and their conjugated forms, such as oleuropein (OL). Conjugated forms underwent rapid hydrolysis under gastric conditions, resulting in significant increases in the amount of free HT and TYR entering the small intestine. Both HT and TYR transferred across human Caco-2 cell monolayers and rat segments of jejunum and ileum and were subject to classic phase I/II biotransformation. The major metabolites identified were an O-methylated derivative of HT, glucuronides of HT and TYR and a novel glutathionylated conjugate of HT. In contrast, there was no absorption of OL in either model. However, OL was rapidly degraded by the colonic microflora resulting in the formation of HT. Our study provides additional information regarding the breakdown of complex olive oil polyphenols in the GI tract, in particular the stomach and the large intestine.