Honeybee Pollen From Southern Chile: Phenolic Profile, Antioxidant Capacity, Bioaccessibility, and Inhibition of DNA Damage.

Honeybee pollen (HBP) chemical composition is highly variable conforming to the floral and geographical origin of the pollen grains. The beneficial effects and functional properties of the HBP are well-known and have been mainly attributed to their high content of antioxidant polyphenols. In this work, twelve HBPs samples from the Southern region of Chile (X Región de Los Lagos) were characterized for the first time according to their botanical origin, phenolic composition, and antioxidant activity. The in vitro gastrointestinal digestion assay was done to simulate the human upper digestive tract. Selected honeybee pollen extracts (HBPEs) were assessed as bioaccessible fractions during an in vitro gastrointestinal digestion. Contents of phenolic compounds, antioxidant capacity, and recovery index of quercetin, myricetin, and cinnamic acid were monitored in different steps of gastrointestinal digestion. Furthermore, the protective effect of in vitro digested HBP towards DNA damage induced by peroxyl radicals was evaluated. The introduced species Brassica rapa L. (Brassicaceae), Lotus pedunculatus Cav. (Fabaceae), and Ulex europaeus L. (Fabaceae) predominated in all the HBPs analyzed, while the native species Buddleja globosa Hope (Scrophulariaceae), Luma apiculata (DC.) Burret (Myrtaceae), Embothrium coccineum J.R. Forst. & G. Forst. (Proteaceae) and Eucryphia cordifolia Cav. (Cunoniaceae) appeared less frequently. The content of polyphenols and antioxidant capacity in HBPEs achieved full bioaccessibility at the end of the intestinal digestion step. However, results obtained by a state-of-the-art technique (i.e. HPLC-DAD) demonstrated relatively low values of bioaccessible quercetin and cinnamic acid after the digestion process. In contrast, myricetin showed a high bioaccessibility in the intestinal digestion steps. The protective effect of in vitro digested HBP towards DNA damage induced by peroxyl radicals showed promising results (up to 91.2% protection). In conclusion, HBPs from the X Region de Los Lagos are rich sources of phenolic antioxidants that protect DNA from strand breakage. Therefore, the potential of HBPEs in preventing gastric and/or intestinal cancer should be further considered.

Protection against indomethacin-induced loss of intestinal epithelial barrier function by a quercetin oxidation metabolite present in onion peel: In vitro and in vivo studies.

Oxidative stress is directly implicated in the loss of intestinal epithelial barrier function (IEBF) induced by non-steroidal anti-inflammatory drugs (NSAIDs). Previous studies by our research team demonstrated that 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone (BZF), a quercetin oxidation metabolite that naturally occurs in onion peels, exhibits an antioxidant potency notably higher than quercetin. Thus, we assessed the potential of BZF and a BZF-rich onion peel aqueous extract (OAE) to protect against the loss of IEBF in Caco-2 cell monolayers and in rats exposed to indomethacin. In vitro, pure BZF and OAE standardized in BZF (100 nM), protected against the drop in transepithelial electrical resistance by 70 - 73%. Likewise, it prevented the increase in fluorescein-isothiocyanate labelled dextran (FITC-dextran) paracellular transport by 74% and oxidative stress by 84 - 86%. In vivo, BZF, given orally at a dose 80 µg/Kg bw as OAE, totally abolished a 30-fold increase in FITC-dextran serum concentration induced by indomethacin. This effect was dose-dependent and largely conserved (85%) when OAE was given 180-min prior to indomethacin. The IEBF-protective effect of OAE was accompanied by a full prevention of the NF-ĸB activation, and the increases in interleukine-8 secretion and myeloperoxidase activity induced by indomethacin. The protection was also associated with a 21-fold increase in Nrf2, and a 7-fold and 9-fold increase in heme oxygenase-1 and NAD(P)H-quinone oxidoreductase 1, respectively. The IEBF-protecting effect of OAE involves, most likely, its dual capacity to activate Nrf2 while inhibiting NF-ĸB activation. The extremely low doses of BZF needed to promote such actions warrants extending its IEBF-protective effects to other NSAIDs.

Quercetin Oxidation Metabolite Present in Onion Peel Protects Caco-2 Cells against the Oxidative Stress, NF-kB Activation, and Loss of Epithelial Barrier Function Induced by NSAIDs.

The potential of 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone (BZF), a quercetin oxidation metabolite, and that of a BZF-rich onion peel aqueous extract (OAE) to protect Caco-2 monolayers against the oxidative stress (OS) and an increased permeability (IP) induced by five nonsteroidal anti-inflammatory drugs (NSAIDs) (indomethacin, diclofenac, piroxicam, ibuprofen, and metamizole) were investigated. Under identical OS conditions, the NSAIDs substantially differed in their ability to induce an IP and/or NF-kB activation. The OAE (100 nM BZF) protected in identical magnitude (84-86%) against OS but in a highly dissimilar manner against the IP (18-73%). While all NSAIDs activated NF-kB, the OAE prevented only that induced by indomethacin. Results reveal that the IP has no direct relationship with the OS and that with the exception of indomethacin, the prevention of NSAIDs-induced OS and/or NF-kB activation plays no fundamental role in the IP-protecting effect of OAE. These results warrant the in vivo evaluation of OAE against indomethacin-induced loss of intestinal barrier function.

Low nanomolar concentrations of a quercetin oxidation product, which naturally occurs in onion peel, protect cells against oxidative damage.

The occurrence of the quercetin oxidation metabolite 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone (BZF), whose antioxidant potency is notably higher than the antioxidant potency of quercetin, was investigated in twenty quercetin-rich plant foods. BZF was identified (HPLC-DAD-ESI-MS/MS) only in the dry outer scales of onions and shallots. Aqueous extracts of onions (OAE) and shallots (SAE) were evaluated for their antioxidant and cytoprotective properties. OAE, whose potency did not differ from SAE, protected ROS-exposed Caco2 cells against oxidative (78%) and cellular (90%) damage at a 3 µg/L concentration (corresponding to 0.03 nM of BZF). After chromatographic resolution of OAE, the BZF peak accounted fully and exclusively for its antioxidant effect. The antioxidant effects of OAE and of a pure BZF were described by two perfectly overlapping curves whose concentration-dependence was within the 3 × 10-4 to 102 nM BZF range. Such unprecedented low concentrations place BZF-containing plants on the frontier of the search for novel sources of antioxidants.

Honeybee Pollen Load: Phenolic Composition and Antimicrobial Activity and Antioxidant Capacity.

Honeybee pollen loads result from the agglutination of pollen grains and salivary secretions of bees. The potential use of honeybee pollen as a food supplement greatly depends on its chemical composition, which varies depending on the botanical and geographical origin of the pollen grains. This study aimed to characterize the botanical origin, chemical composition, and antioxidant and antibacterial activities of honeybee pollen from the V Region of Chile. The introduced species Brassica rapa and Eschscholzia californica predominated in the bee pollen analyzed. The honeybee pollen extracts showed antioxidant and antibacterial properties, specifically against the pathogenic microorganism Streptococcus pyogenes. Quercetin and myricetin were found in all samples in large concentrations. The separation of pollen loads from a multifloral sample demonstrated that E. californica pollen loads are responsible for antibacterial activity. This sample also showed a high concentration of quercetin (304.8 mg/100 g of bee pollen). Based on the present results, honeybee pollen from the V Region of Chile has been found to exhibit antioxidant and antimicrobial activities. Furthermore, it is proposed to use quercetin as a quality indicator for honeybee pollen from this region of Chile. These results should help establish better quality control criteria for Chilean honeybee pollen and its potential use as a functional ingredient.

Quercetin Oxidation Paradoxically Enhances its Antioxidant and Cytoprotective Properties.

Quercetin oxidation is generally believed to ultimately result in the loss of its antioxidant properties. To test this assertion, quercetin oxidation was induced, and after each of its major metabolites was identified and isolated by HPLC-DAD-ESI-MS/MS, the antioxidant (dichlorodihydrofluorescein oxidation-inhibiting) and cytoprotective (LDH leakage-preventing) properties were evaluated in Hs68 and Caco2 cells exposed to indomethacin. Compared to quercetin, the whole mixture of metabolites (QOX) displayed a 20-fold greater potency. After resolution of QOX into 12 major peaks, only one (peak 8), identified as 2,5,7,3',4'-pentahydroxy-3,4-flavandione or its 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone tautomer, could account for the antioxidant and cytoprotective effects afforded QOX. Peak 8 exerted such effects at a 50 nM concentration, revealing a potency 200-fold higher than that of quercetin. The effects of peak 8 were seen regardless of whether it was added to the cells 40 min before or simultaneously with the oxygen-reactive species-generating agent, suggesting an intracellular ability to trigger early antioxidant responses. Thus, the present study is the first to reveal that in regard to the intracellular actions of quercetin, attention should be extended toward some of its oxidation products.

Quercetin and related flavonoids conserve their antioxidant properties despite undergoing chemical or enzymatic oxidation.

Oxidation of a phenolic group in quercetin is assumed to compromise its antioxidant properties. To address this assumption, the ROS-scavenging, Folin-Ciocalteau- and Fe-reducing capacities of quercetin and thirteen structurally related flavonoids were assessed and compared with those of mixtures of metabolites resulting from their chemical and enzymatic oxidation. Regardless of the oxidation mode, the metabolites mixtures largely conserved the antioxidant properties of the parent molecules. For quercetin, 95% of its ROS-scavenging and over 77% of its Folin-Ciocalteau- and Fe-reducing capacities were retained. The susceptibility of flavonoids to oxidative disappearance (monitored by HPLC-DAD) and that of the mixtures to retain their antioxidant capacity was favourably influenced by the presence of a catechol (ring-B) and enol (ring C) function. This is the first study to report that mixtures resulting from the oxidation of quercetin and its analogues largely conserve their antioxidant properties.

International Regulations of Propolis Quality: Required Assays do not Necessarily Reflect their Polyphenolic-Related In Vitro Activities.

Propolis has been proposed as a polyphenolic-rich natural product potentially able to be used for human consumption or even for medicinal proposes. To guarantee a minimum phenolic and flavonoid content and as consequence of their related-biological activities, international requirements of propolis quality are commonly applied. In this work we assessed phenolic and flavonoid contents of propolis; the antioxidant capacity (toward peroxyl radicals and hypochlorous acid); the ability to generate nitric oxide (NO); and, finally the antimicrobial activity of 6 propolis samples from the VI region of Chile. Our results show that the total phenolic and flavonoid content of propolis samples are not always in agreement with their polyphenolic-associated in vitro activities. For example, P03 and P06 samples showed the lowest (25 ± 4 GAE/g propolis) and the highest (105 ± 3 GAE/g propolis) total phenolic content, respectively. This was in agreement with flavonoid content and their Oxygen Radical Absorbance Capacity (ORAC) activity. However, this dependence was not observed toward HOCl, NO release and antimicrobial activity. Based on our results, we consider that, in order to guarantee the antioxidant or antimicrobial in vitro effects, the international regulations of propolis quality should contemplate the convenience of incorporating other simple analytical test such as ORAC or antimicrobial tests.

Use of pyrogallol red and pyranine as probes to evaluate antioxidant capacities towards hypochlorite.

Hypochlorite is a strong oxidant able to induce deleterious effects in biological systems. The goal of this work was to investigate the use of PGR and PYR as probes in assays aimed at evaluating antioxidant activities towards hypochorite and apply it to plant extracts employed in Chilean folk medicine. The consumption of PGR and PYR was evaluated from the decrease in the visible absorbance and fluorescence intensity, respectively. Total phenolic content was determined by the Folin Ciocalteau assay. PGR and PYR react with hypochlorite with different kinetics, being considerably faster the consumption of PGR. Different stoichiometric values were also determined: 0.7 molecules of PGR and 0.33 molecules of PYR were bleached per each molecule of added hypochlorite. Both probes were protected by antioxidants, but the rate of PGR bleaching was too fast to perform a kinetic analysis. For PYR, the protection took place without changes in its initial consumption rate, suggesting a competition between the dye and the antioxidant for hypochlorite. Plant extracts protected PYR giving a PYR-HOCl index that follows the order: Fuchsia magellanica ≈ Marrubium vulgare ≈ Tagetes minuta > Chenopodium ambrosoides ≈ Satureja montana > Thymus praecox. Based on both the kinetic data and the protection afforded by pure antioxidants, we selected PYR as the best probe. The proposed methodology allows evaluating an antioxidant capacity index of plant extracts related to the reactivity of the samples towards hypochlorite.