Andrea J. Day

Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver β-glucosidase activity

Flavonoid and isoflavonoid glycosides are common dietary phenolics which may be absorbed from the small intestine of humans. The ability of cell‐free extracts from human small intestine and liver to deglycosylate various (iso)flavonoid glycosides was investigated. Quercetin 4′‐glucoside, naringenin 7‐glucoside, apigenin 7‐glucoside, genistein 7‐glucoside and daidzein 7‐glucoside were rapidly deglycosylated by both tissue extracts, whereas quercetin 3,4′‐diglucoside, quercetin 3‐glucoside, kaempferol 3‐glucoside, quercetin 3‐rhamnoglucoside and naringenin 7‐rhamnoglucoside remained unchanged. The K m for hydrolysis of quercetin 4′‐glucoside and genistein 7‐glucoside was ∼32±12 and ∼14±3 μM in both tissues respectively. The enzymatic activity of the cell‐free extracts exhibits similar properties to the cytosolic broad‐specificity β‐glucosidase previously described in mammals.

Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase

Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane‐bound, family 1 β‐glycosidase found on the brush border of the mammalian small intestine. LPH, purified from sheep small intestine, was capable of hydrolysing a range of flavonol and isoflavone glycosides. The catalytic efficiency (k cat/K m) for the hydrolysis of quercetin‐4′‐glucoside, quercetin‐3‐glucoside, genistein‐7‐glucoside and daidzein‐7‐glucoside was 170, 137, 77 and 14 (mM−1 s−1) respectively. The majority of the activity occurred at the lactase and not phlorizin hydrolase site. The ability of LPH to deglycosylate dietary (iso)flavonoid glycosides suggests a possible role for this enzyme in the metabolism of these biologically active compounds.

Human metabolism of dietary flavonoids: Identification of plasma metabolites of quercetin

The position of conjugation of the flavonoid quercetin dramatically affects biological activity in vitro, therefore it is important to determine the exact nature of the plasma metabolites. In the present study, we have used various methods (HPLC with diode array detection, LCMS, chemical and enzymic synthesis of authentic conjugates and specific enzymic hydrolysis) to show that quercetin glucosides are not present in plasma of human subjects 1.5 h after consumption of onions (a rich source of flavonoid glucosides). All four individuals had similar qualitative profiles of metabolites. The major circulating compounds in the plasma after 1.5 h are identified as quercetin-3-glucuronide, 3′-methyl-quercetin-3-glucuronide and quercetin-3′-sulfate. The existence of substitutions in the B and/or C ring of plasma quercetin metabolites suggests that these conjugates will each have very different biological activities.

Flavonoid glucuronides are substrates for human liver β‐glucuronidase

Quercetin glucuronides are the main circulating metabolites of quercetin in humans. We hypothesise that the potential availability of the aglycone within tissues depends on the substrate specificity of the deconjugating enzyme β‐glucuronidase towards circulating flavonoid glucuronides. Human tissues (small intestine, liver and neutrophils) exhibited β‐glucuronidase against quercetin glucuronides. The various quercetin glucuronides were deconjugated at similar rates, but liver cell‐free extracts were the most efficient and the activity was completely inhibited by saccharo‐1,4‐lactone (a β‐glucuronidase inhibitor). Furthermore, pure recombinant human β‐glucuronidase hydrolysed various flavonoid glucuronides, with a 20‐fold variation in catalytic efficiency (k cat/K m=1.3×103 M−1 s−1 for equol‐7‐O‐glucuronide and 26×103 M−1 s−1 for kaempferol‐3‐O‐glucuronide). Similar catalytic efficiencies were obtained for quercetin O‐glucuronides substituted at different positions. These results show that flavonoid glucuronides can be deconjugated by microsomal β‐glucuronidase from various human cells.

Absorption of kaempferol from endive, a source of kaempferol-3-glucuronide, in humans

Objective: To determine the absorption, excretion and metabolism of kaempferol in humans.Design: A pharmacokinetic study of kaempferol from endive over 24 h.Subjects: Four healthy males and four healthy females.Results: Kaempferol, from a relatively low dose (9 mg), was absorbed from endive with a mean maximum plasma concentration of 0.1 μM, at a time of 5.8 h, indicating absorption from the distal section of the small intestine and/or the colon. Although a 7.5-fold interindividual variation between the highest and lowest maximum plasma concentration was observed, most individuals showed remarkably consistent pharmacokinetic profiles. This contrasts with profiles for other flavonoids that are absorbed predominantly from the large intestine (eg rutin). An average of 1.9% of the kaempferol dose was excreted in 24 h. Most subjects also showed an early absorption peak, probably corresponding to kaempferol-3-glucoside, present at a level of 14% in the endive. Kaempferol-3-glucuronide was the major compound detected in plasma and urine. Quercetin was not detected in plasma or urine indicating a lack of phase I hydroxylation of kaempferol.Conclusions: Kaempferol is absorbed more efficiently than quercetin in humans even at low oral doses. The predominant form in plasma is a 3-glucuronide conjugate, and interindividual variation in absorption and excretion is low, suggesting that urinary kaempferol could be used as a biomarker for exposure.Sponsorship: Biotechnology and Biological Sciences Research council for core strategic funding and the University of Leeds for a departmental fellowship (AJD).

Biomarkers for exposure to dietary flavonoids: a review of the current evidence for identification of quercetin glycosides in plasma

Quercetin, a polyphenol with potential health effects, is absorbed by humans and measurement in plasma can be used as a biomarker for intake. However, the chemical nature of the quercetin in blood is still not known, although one possibility is that glucosides are found in an unchanged form from the original food. We propose that the existence of quercetin glucosides in plasma is unlikely, since the metabolic beta-glucosidase capacity of the small intestine and of the liver is too great for quercetin glucosides to escape deglycosylation. We critically examine the limited number of studies which purport to detect quercetin glycosides in blood and the current evidence for the absorption of these compounds from the gastrointestinal tract. We emphasise the need for comprehensive identification of circulating compounds, since polyphenol glucuronides, the expected metabolites in plasma, have almost identical chromatographic properties to the glucosides at acid pH. Studies on the nature of quercetin metabolites in plasma are urgently needed so that the proposed biological activities of quercetin can be re-assessed and that a suitable biomarker of exposure can be established.

Particle-Stabilizing Effects of Flavonoids at the Oil―Water Interface

It has been shown that some common food flavonoids can act as excellent stabilizers of oil-in-water emulsions through their adsorption as water-insoluble particles to the surface of the oil droplets, i.e., Pickering emulsions are formed. Flavonoids covering a wide range of octanol-water partition coefficients (P) were screened for emulsification behavior by low shear mixing of flavonoid+n-tetradecane in a vortex mixer. Most flavonoids with very high or very low P values were not good emulsifiers, although there were exceptions, such as tiliroside, which is very insoluble in water. When a high shear jet homogenizer was used with 20 vol% oil in the presence of 1 mM tiliroside, rutin, or naringin, much finer emulsions were produced: the average droplet sizes (d32) were 16, 6, and 5 μm, respectively. These results may be highly significant with respect to the delivery of such insoluble compounds to the gut, as well as their digestion and absorption.

Effects of pH on the ability of flavonoids to act as Pickering emulsion stabilizers

The flavonoids tiliroside, rutin and naringin have been investigated as stabilizers of Pickering oil-in-water (O/W) emulsions. The mean droplet size of tetradecane emulsions was considerably smaller at higher pH, especially for rutin. The solubility of flavonoids in the aqueous phase was 4-6 times higher at pH 8 compared to pH 2 for tiliroside and rutin, although all absolute solubilities remained low (<1 mM). This agreed with a slight increase in surface activity of tiliroside and rutin at the O-W interface at pH 8 compared to pH 2. However, improved emulsion stabilization at higher pH is better explained by the significant increase in ζ-potential of the flavonoid particles to more negative values at pH 8, which will improve particle dispersion and increase the charge on the droplets stabilized by them. A buckwheat tea extract, rich in rutin, was also shown to be an effective stabilizer of sunflower O/W emulsions.

Phytonutrient intakes in relation to European fruit and vegetable consumption patterns observed in different food surveys

Fruit and vegetables make an important contribution to health, partly due to the composition of phytonutrients, such as carotenoids and polyphenols. The aim of the present study was to quantify the intake of fruit and vegetables across different European countries using food consumption data of increasing complexity: food balance sheets (FBS); the European Food Safety Authority (EFSA) Comprehensive Database; individual food consumption data from the UK National Diet and Nutrition Survey (NDNS). Across Europe, the average consumption of fruit and vegetables ranged from 192 to 824 g/d (FBS data). Based on EFSA data, nine out of fourteen countries consumed < 400 g/d (recommended by the WHO), although even in the highest-consuming countries such as Spain, 36 % did not reach the target intake. In the UK, the average consumption of fruit and vegetables was 310 g/d (NDNS data). Generally, phytonutrient intake increased in accordance with fruit and vegetable intake across all European countries with the exception of lycopene (from tomatoes), which appeared to be higher in some countries that consumed less fruit and vegetables. There were little differences in the average intake of flavanols, flavonols and lycopene in those who did or did not meet the 400 g/d recommendation in the UK. However, average intakes of carotenoid, flavanone, anthocyanidin and ellagic acid were higher in those who consumed >400 g/d of fruit and vegetables compared with those who did not. Overall, intakes of phytonutrients are highly variable, suggesting that while some individuals obtain healthful amounts, there may be others who do not gain all the potential benefits associated with phytonutrients in the diet.

Human metabolism of dietary quercetin glycosides.

Flavonoids, along with other phytochemicals, are thought to have a role in reducing the risk of chronic diseases such as coronary heart disease and cancers.1,2 Fruits and vegetables undoubtedly afford some protection against these diseases,3 but the effects of individual compounds that may play a role is unclear. Evidence has been accumulating from both in vivo and in vitro studies that flavonoids have biological activity that may be beneficial. They are good antioxidants and can act by scavenging free radicals, chelation of metal ions or inhibiting lipid peroxidation,4 and they can inhibit platelet aggregation.5 Flavonols also have the ability to induce phase II detoxification enzymes6 and have been shown to inhibit the growth of certain human cancer cells such as those of the colon,7 ovary,8 and gastrointestinal tract.9