Quercetin and methylquercetin are present in a variety of sulfate and glucuronide conjugates in the plasma of quercetin-fed rats and humans. Quercetin conjugates exhibit various physiological activities, depending on the type and position of conjugation. However, little is known regarding the type and position of isomers of quercetin conjugates in the plasma, their accumulation in the liver and kidneys, and their excretion via urine. Using authentic standards of quercetin conjugates and liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis, we identified and quantified various quercetin conjugates in blood plasma, urine, liver, and kidney tissues 1, 4, and 10 h after orally administering 33.1 μmol kg⁻¹ quercetin glucosides to rats. The profiles of quercetin conjugates were largely different among plasma, urine, liver, and kidneys. Very limited heteroconjugates (7-O-glucuronide-4′-O-sulfate) of quercetin and methylquercetin dominated in the plasma, but these heteroconjugates were much less excreted via urine and did not largely accumulate in the liver and kidneys. Heteroconjugates constituting sulfates other than 4′ position sulfate, 7-O-glucuronide-3′-O-sulfate, 4′-O-glucuronide-7-O-sulfate, and 3′-O-glucuronide-7-O-sulfate were major metabolites in urine, but were minimally detected in the plasma. We also found that mono-sulfate conjugates were abundant in the liver and renal tissues. These results suggest that excretion of quercetin conjugates, especially heteroconjugates, into urine is highly selective. The heteroconjugates with 4′-O-sulfate may be scarcely excreted via urine, and thus accumulate in the blood plasma. Further research is necessary to evaluate the physiological effects of heteroconjugates accumulated in the plasma.

Quercetin is a flavonoid with many physiological effects. Absorbed quercetin is rapidly conjugated in the intestinal epithelium and liver. Different positional isomers of quercetin conjugates have different physiological properties. However, the mechanisms of quercetin conjugation in the intestine are not fully clarified. We examined the regioselective quercetin conjugate formation in the intestine after oral administration of quercetin glycosides, by simultaneous sampling of blood from the portal vein and superior vena cava, and quantifying various positional isomers of quercetin glucuronides and sulfates in conscious rats. Concentrations of quercetin glucuronides were higher in blood from the portal vein than the superior vena cava, showing that glucuronidation mainly occurred in the intestine. Such differences were not observed for quercetin sulfates. Regioselectivity of the intestinal glucuronidation in quercetin hydroxyl groups were 7- >3′- >3- >4′-OH. Quercetin was mainly sulfated on 3′-OH at 30 min, but on 4′-OH at 240 min.

Oxidative stress (OS) contributes to nonalcoholic steatohepatitis (NASH) and hepatocarcinogenesis. We investigated whether antioxidative self-assembling nanoparticles (SMAPo^TN) could reduce the development of NASH and hepatocellular carcinoma (HCC) in p62/Sqstm1 and Nrf2 double knockout (DKO) mice and studied protective mechanisms. We measured disease development in male DKO mice fed a normal chow (NASH model) or a 60% high-fat diet (HFD; HCC model) with or without SMAPo^TN administration for 26 weeks. SMAPo^TN inhibited liver fibrosis in both groups and prevented HCC development (0% vs. 33%, p < 0.05) in the HFD group. SMAPo^TN reduced OS, inflammatory cytokine signaling, and liver fibrosis. RNA-sequencing revealed that SMAPo^TN decreased endoplasmic reticulum stress signaling genes in both groups, HCC driver genes, and cancer pathway genes, especially PI3K-AKT in the HFD groups. In the SMAPo^TN treatment HFD group, serum lipopolysaccharide levels and liver lipopolysaccharide-binding protein expression were significantly lower compared with those in the nontreatment group. SMAPo^TN improved the α-diversity of gut microbiota, and changed the microbiota composition. Oral SMAPo^TN administration attenuated NASH development and suppressed hepatocarcinogenesis in DKO mice by improving endoplasmic reticulum stress in the liver and intestinal microbiota. SMAPo^TN may be a new therapeutic option for NASH subjects and those with a high HCC risk.

A separation process was established to sequentially fractionate and recover three anti-inflammatory components derived from sugars, phycobiliprotein, and chlorophyll from the hot-air-dried thalli of the red alga dulse (Palmaria palmata). The developed process consisted of three steps, without the use of organic solvents. In Step I, the sugars were separated by disrupting the cell wall of the dried thalli with a polysaccharide-degrading enzyme, and a sugar-rich extract (E1) was obtained by precipitating the other components, which were simultaneously eluted by acid precipitation. In Step II, the residue suspension from Step I was digested with thermolysin to obtain phycobiliprotein-derived peptides (PPs), and a PP-rich extract (E2) was obtained by separating the other extracts using acid precipitation. In Step III, solubilized chlorophyll was obtained by heating the residue, which was acid-precipitated, neutralized, and re-dissolved to concentrate the chlorophyll-related components (Chls)-rich extract (E3). These three extracts suppressed inflammatory-cytokine secretion by lipopolysaccharide (LPS)-stimulated macrophages, confirming that the sequential procedure had no negative effects on the activities of any of the extracts. The E1, E2, and E3 were rich in sugars, PPs, and Chls, respectively, indicating that the anti-inflammatory components were effectively fractionated and recovered through the separation protocol.