High-performance liquid chromatographic determination of quercetin and isorhamnetin in rat tissues using β-glucuronidase and acid hydrolysis

doi:10.1016/S0378-4347(99)00520-4

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 738, Issue 2, 11 February 2000, Pages 413-417

https://doi.org/10.1016/S0378-4347(99)00520-4 Get rights and content

Abstract

Quercetin is a plant polyphenol which is present in the diet as an aglycone and as sugar conjugates. Despite potent vasodilatory and antioxidant effects in vitro, destruction by intestinal organisms has been assumed to limit its nutritional relevance in the rat. However, we have refined extraction techniques using β-glucuronidase followed by acid hydrolysis. Following this with HPLC methodology with post-column derivatisation, we have detected significant concentrations of quercetin and its metabolite, isorhamnetin, in tissues of rats maintained on quercetin-rich diets. Percentage recoveries are greater than 95% and intra-batch variation does not exceed 7% suggesting that the method may be useful in further studies of the biological role of this flavonoid.

Introduction

Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one; 3,3′,4′,5,7-pentahydroxy flavone) is a polyphenolic product of the phenylpropanoid biosynthetic pathway in plants and is therefore an integral part of the mammalian diet both as an aglycone and as β-glycosidic conjugates [1]. In vitro models indicate that this flavonoid has numerous biological roles, including antioxidant function, vasodilatory and platelet anti-aggregation effects and the maintenance of capillary integrity [2]. Early studies with rats indicated that quercetin had little nutritional relevance as the aglycone and the β-glycosides appeared to be destroyed by intestinal microorganisms in the small intestine and lower bowel [3], [4]. Recently, however, quercetin and its methylated derivative, isorhamnetin, have been detected in plasma and urine of rats maintained on quercetin-rich diets [5]. This suggests that a significant proportion of the aglycone may be absorbed and further metabolised by tissues. Consequently, to further elaborate on the bioavailability of quercetin, we have developed methodology for extraction using β-glucuronidase incubation and acid hydrolysis with subsequent HPLC detection to quantify tissue concentrations of quercetin and isorhamnetin in rats supplemented with the aglycone for 2 weeks.

Section snippets

Animals and diets

Ten individually-housed weanling male rats of the Rowett Hooded Lister strain were offered, ad libitum, for 10 weeks a standard torula-yeast based semisynthetic diet [6]. Five rats were then offered the diet containing quercetin at a concentrations of 5 g/kg for a further 2 weeks and the remainder were maintained on the original ration. Diets were stored at −40°C before use.

Rats were anaesthetised with ether and blood removed by cardiac puncture into heparinised evacuated tubes (Becton

Results and discussion

The chromatographic peaks of quercetin and its methylated derivative, isorhamnetin, were well separated (retention times 13.07±0.02 and 28.54±0.79 min, respectively) (Fig. 1). There was some indication of the coelution of endogenous components with quercetin, although the peaks were comparatively small. Moreover, a peak with a retention time of 24.82±0.20 min may be kaempferol which differs from quercetin only by the absence of an hydroxyl group on position 5 of the B-ring. Although the spiking

Acknowledgements

We are grateful for financial support from the Scottish Executive for Rural Affairs Department (SERAD).

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Quercetin is a dietary polyphenolic compound with potentially beneficial effects on health. Claims that quercetin has biological effects are based mainly on in vitro studies with quercetin aglycone. However, quercetin is rapidly metabolized, and we have little knowledge of its availability to tissues. To assess the long-term tissue distribution of quercetin, 2 groups of rats were given a 0.1 or 1% quercetin diet [~50 or 500 mg/kg body weight (wt)] for 11 wk. In addition, a 3-d study was done with pigs fed a diet containing 500 mg quercetin/kg body wt. Tissue concentrations of quercetin and quercetin metabolites were analyzed with an optimized extraction method. Quercetin and quercetin metabolites were widely distributed in rat tissues, with the highest concentrations in lungs (3.98 and 15.3 nmol/g tissue for the 0.1 and 1% quercetin diet, respectively) and the lowest in brain, white fat, and spleen. In the short-term pig study, liver (5.87 nmol/g tissue) and kidney (2.51 nmol/g tissue) contained high concentrations of quercetin and quercetin metabolites, whereas brain, heart, and spleen had low concentrations. These studies have for the first time identified target tissues of quercetin, which may help to understand its mechanisms of action in vivo.
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Citation Excerpt :
Another important aspect is whether they are absorbed in sufficient quantity to play their antioxidant role [30–32]. So analytical methods are being developed to determine phenolic compounds in human fluids [33,34]. Pharmacological doses of phenolic compounds used in some studies are as follows (i.e. 20 mg/kg of resveratrol) [35], 25 mg of trans-resveratrol, 25 mg of (+)-catechin and 10 mg of quercetin [36].
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The compounds studied were six phenolic acids, five flavonoids, trans-resveratrol and tyrosol. The effect of the following factors was explored: temperature, pH, the addition of antioxidants and the addition of anticoagulants.
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Short communicationHigh-performance liquid chromatographic determination of quercetin and isorhamnetin in rat tissues using β-glucuronidase and acid hydrolysis

Abstract

Introduction

Section snippets

Animals and diets

Results and discussion

Acknowledgements

Food Chem. Toxicol.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Nutr. Biochem.

FEBS Lett.

Short communication
High-performance liquid chromatographic determination of quercetin and isorhamnetin in rat tissues using β-glucuronidase and acid hydrolysis