Absorption, Conjugation and Efflux of the Flavonoids, Kaempferol and Galangin, Using the Intestinal CACO-2/TC7 Cell Model

Abstract

Flavonoids are biologically active compounds in food with potential health effects. We have used the Caco-2 cell monolayer model to study the absorption and metabolism of two flavonols, a class of flavonoids, specifically kaempferol and galangin. Metabolism experiments allowed identification of 5 kaempferol conjugates: 3-, 7- and 4'-glucuronide, a sulphate and a glucurono-sulphate; and 4 galangin conjugates: 3-, 5- and 7-glucuronides, and a sulphate, using specific enzyme hydrolysis, HPLC-MS, and HPLC with post column metal complexation/tandem MS. Transport studies showed that the flavonols were conjugated inside the cells then transported across the monolayer or effluxed back to the apical side. Sulphated conjugates were preferentially effluxed back to the apical side, whereas glucuronides were mostly transported to the basolateral side. For kaempferol, a small amount of the unconjugated aglycone permeated in both directions, indicating some passive diffusion. When kaempferol-3-glucuronide and quercetin7-sulphate were applied to either side of the cells, no permeation in either direction was observed, indicating that conjugates cannot re-cross the cell monolayer. Formation of apical kaempferol-7- and 4'-glucuronides was readily saturated, whereas formation of other conjugates at the apical side and all at the basolateral side increased with increasing concentration of kaempferol, implying different transporters are responsible at the apical and basolateral sides. The results highlight the important but complex metabolic changes occurring in flavonoids during absorption.

Figure 1. Identification of galangin metabolites from CaCo-2/TC7 cells and associated media

Typical chromatogram showing metabolites of galangin. The identities of metabolites was confirmed using a combination of retention time, selected ion monitoring LC/MS, tandem mass spectrometry with metal complexation, enzyme hydrolysis with sulphatase and glucuronidase, and UV spectra. No pure authentic standards were available for galangin metabolites. Suspected metabolites are labelled G1-G4, with galangin aglycone (identified by use of an authentic standard) labelled G5.

Figure 2. Identification of kaempferol metabolites from CaCo-2/TC7 cells and associated media

Typical chromatogram showing metabolites of kaempferol. The identities of metabolites were confirmed using a combination of retention time, selected ion monitoring LC/MS, tandem mass spec with metal complexation, kaempferol-3-glucuronide pure authentic standard, enzyme hydrolysis with sulphatase and glucuronidase and UV spectra. Suspected metabolites labelled K1-K5, with kaempferol aglycone labelled K6. UV spectra for the various metabolite peaks also shown with UV peak absorbance at around 250-280 nm and 350-400 nm.

Figure 3. Transport of individual galangin conjugates in CaCo-2/TC7 cell monolayers

Graphs are presented for galangin conjugate transport in apical, cellular and basolateral compartments with apical loading of the galangin (40 μM), as well as for the transport of galangin aglycone. N=6 for all data. G-5 = galangin-5-glucuronide, G-3 = galangin-3-glucuronide, G-7 = galangin-7-glucuronide, G-S = galangin sulphate.

Figure 4. Transport of individual kaempferol conjugates in CaCo-2/TC7 cell monolayers

Graphs are presented for kaempferol conjugate transport in apical, cellular and basolateral compartments with apical loading of the kaempferol (40 μM), as well as for the transport of kaempferol aglycone. N=4 for all data. K-S-G = kaempferol-sulpho-glucuronide, K-3 = kaempferol-3-glucuronide, K-7 = kaempferol-7-glucuronide, K-4′ = kaempferol-4′-glucuronide, K-S = kaempferol sulphate.

Figure 5. Aglycone transport in CaCo-2/TC7 monolayers at varying time points

Graphs are presented for the aglycone transport in apical, cellular and basolateral compartments with apical and basolateral loading of the kaempferol (40 μM, various time points). Dark bars = apical loading, light bars = basolateral loading. N = 3 for all data. All values as nmols of analyte.

Figure 6. Aglycone transport in CaCo-2/TC7 monolayers with varying concentrations of kaempferol

Graphs are presented for kaempferol aglycone transport in apical, cellular and basolateral compartments with apical and basolateral loading of the kaempferol (60 min, various concentrations). Dark bars = apical loading, light bars = basolateral loading. N = 3 for all data. All values as nmols of analyte.