Characterisation of metabolites of the putative cancer chemopreventive agent quercetin and their effect on cyclo-oxygenase activity

Abstract

Quercetin (3,5,7,3',4'-pentahydroxyflavone) is a flavone with putative ability to prevent cancer and cardiovascular diseases. Its metabolism was evaluated in rats and human. Rats received quercetin via the intravenous (i.v.) route and metabolites were isolated from the plasma, urine and bile. Analysis was by high-performance liquid chromatography and confirmation of species identity was achieved by mass spectrometry. Quercetin and isorhamnetin, the 3'-O-methyl analogue, were found in both the plasma and urine. In addition, several polar peaks were characterised as sulphated and glucuronidated conjugates of quercetin and isorhamnetin. Extension of the metabolism studies to a cancer patient who had received quercetin as an i.v. bolus showed that (Quercetin removed) isorhamnetin and quercetin 3'-O-sulphate were major plasma metabolites. As a catechol, quercetin can potentially be converted to a quinone and subsequently conjugated with glutathione (GSH). Oxidation of quercetin with mushroom tyrosinase in the presence of GSH furnished GSH conjugates of quercetin, two mono- and one bis-substituted conjugates. However, these species were not found in biomatrices in rats treated with quercetin. As cyclo-oxygenase-2 (COX-2) expression is mechanistically linked to carcinogenesis, we examined whether quercetin and its metabolites can inhibit COX-2 in a human colorectal cancer cell line (HCA-7). Isorhamnetin and its 4'-isomer tamarixetin were potent inhibitors, reflected in a 90% decrease in prostaglandin E-2 (PGE-2) levels, a marker of COX-2 activity. Quercetin was less effective, with a 50% decline. Quercetin 3- and 7-O-sulphate had no effect on PGE-2. The results indicate that quercetin may exert its pharmacological effects, at least in part, via its metabolites.

Figure 1

Structures of quercetin and its metabolites.

Figure 2

High-performance liquid chromatography chromatograms of extracts of bile (A and B), plasma (C and D) or urine (E and F) from control rats (B, D and F) or rats that had received quercetin either 12.5 (A) or 6.25 mg kg⁻¹ (C) and 2.5 g kg⁻¹ (E) via the i.v. (A and C) or oral (E) routes. Bile was collected prior to (control) and for 20 min after the administration of quercetin; plasma samples were collected 5 min after administration of quercetin; urine samples were pooled over 8 h. Control (untreated) animals received the vehicle only via the appropriate administration route. Symbols i and ii denote retention times of quercetin and isorhamnetin, respectively. AU=absorbance units. For details of sample preparation and chromatographic analysis see Materials and Methods. The chromatograms shown are representative of extracts obtained from three separate animals.

Figure 3

Extracted ion chromatogram (m/z 477) of (A) extract of bile from control rat and (B) quercetin glucuronides from bile of rats, which received quercetin (12.5 mg kg⁻¹ i.v.). Retention times are those noted in Table 1.

Figure 4

High-performance liquid chromatography chromatograms of extracts of plasma from a patient obtained before (bottom trace) and 5 min after administration (top trace) of quercetin (280 mg m⁻² i.v.). Peaks were identified on the basis of cochromatography and selected ion monitoring MS, which afforded m/z 301 for quercetin, m/z 315 for isorhamnetin and m/z 381 for quercetin 3-O-sulphate. AU=absorbance units. For details of sample preparation and chromatographic and mass spectrometric analysis see Materials and Methods.

Figure 5

High-performance liquid chromatography chromatogram of an extract of a mixture of quercetin (1 mM), glutathione (1 mM) and mushroom tyrosinase (150 U ml⁻¹). Mass spectral analysis performed in the selected ion monitoring mode suggests the following peak allocation: ‘1’ bis-glutathionyl-S-quercetin (m/z 911), ‘2’ and ‘3’ glutathionyl-S-quercetin (m/z 606) and ‘4’ quercetin. AU=absorbance units. For details of incubation conditions, sample preparation and chromatographic conditions see Materials and Methods. The chromatogram shown is representative of three separate experiments.

Figure 6

Effect of quercetin, rutin quercetin-7-O-sulphate (Q-7-O-SO₄), quercetin-3-O-sulphate (Q-3-O-SO₄), isorhamnetin and tamarixetin (each 10 μM) on COX-2 enzyme activity, as reflected by PGE-2 levels, in cultures of HCA-7 colon cancer cells. The COX complement of HCA-7 cells is made up almost exclusively of COX-2, while COX-1 is present only to a minor extent (Sharma et al, 2001). Cells were incubated with agents for 6 h, and PGE-2 levels were measured by ELISA. Asterisks indicate that values were significantly different from controls (^*P<0.01, ^**P<0.001). For details of incubation conditions and measurement see Materials and Methods. The values are the mean±s.d. of three to five separate experiments, each conducted in triplicate.