Availability and Metabolic Fate of Olive Phenolic Alcohols Hydroxytyrosol and Tyrosol in the Human GI Tract Simulated by the In Vitro GIDM-Colon Model.

Hydroxytyrosol (HTyr) and tyrosol (Tyr) are the most well studied phenolic alcohols of olive oil and olive products demonstrating numerous and significant beneficial health effects. However, their activity in the human organism as food bioactives is strongly associated with their bioavailability and metabolism, while manifested through their metabolites. Nevertheless, there are limited studies investigating their biotransformation and mainly catabolism by gut microflora under a holistic interpretation close to the human organism. Thus, in the present study, the GastroIntestinal Dialysis (GIDM)-colon model, a continuous flow in vitro dialysis system mimicking physiological conditions during human gastrointestinal digestion, was used to explore the metabolism of HTyr and Tyr as pure compounds. The GIDM-colon model simulates absorption from the lumen to the mucosa, followed by the colon phase using pooled human fecal suspensions. Samples were collected at different time points and analyzed via LC-Orbitrap MS. An integrated approach combining Multivariate Data Analysis (MVA) and thorough dereplication procedures led to the identification of HTyr and Tyr metabolites in different phases (gastric, small intestine, and colon), yielding also valuable information about metabolites kinetics. To our knowledge, this is the first study reporting full spectrometric data of HTyr and Tyr metabolites along with possible transformation mechanisms in the GI tract.

Mitigation of benznidazole toxicity and oxidative stress following ascorbic acid supplementation in an adult traveller with chronic indeterminate Chagas' disease.

BACKGROUND: Benznidazole is an effective drug in the trypanocidal treatment of acute and chronic indeterminate Chagas' disease (CD). However, adverse drug reactions (ADR) are common and frequently cause patients to discontinue treatment. OBJECTIVES: We hypothesized that antioxidant supplementation could mitigate benznidazole-induced toxicity. METHODS: We co-supplemented an adult traveller with chronic indeterminate CD who experienced benznidazole ADR with ascorbic acid (AA), 1000 mg/day. We measured selected serum biomarkers of oxidative stress [total antioxidant status (TAS), total oxidative status (TOS), nuclear factor erythroid 2-related factor 2 (Nrf2), malondialdehyde (MDA), extracellular glutathione peroxidase (GPX3), catalase (CAT) and total superoxide dismutase (T-SOD)] at timepoints before and throughout benznidazole treatment and after AA co-supplementation. RESULTS: AA co-supplementation effectively mitigated benznidazole-induced ADR during the aetiological treatment of chronic indeterminate CD. The kinetics of serum biomarkers of oxidative stress suggested significantly decreased oxidative insult in our patient. CONCLUSIONS: We hypothesize that the key pathophysiological mechanism of benznidazole-associated toxicity is oxidative stress, rather than hypersensitivity. AA co-supplementation may improve adherence to benznidazole treatment of chronic indeterminate (or acute) CD. Oxidative stress biomarkers have the potential to guide the clinical management of CD. Prospective studies are needed to establish the benefit of antioxidant co-supplementation to benznidazole treatment of CD in reducing benznidazole toxicity, parasite clearance and the prevention of end-organ damage.

Effects of medicagenic acid metabolites, originating from biotransformation of an Herniaria hirsuta extract, on calcium oxalate crystallization in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Herniaria hirsuta is traditionally used in Moroccan folk medicine for treatment of urinary stones and as a diuretic. It is rich in saponins, which are known to be deglycosylated in the colon, whereafter aglycones such as medicagenic acid are absorbed and further metabolized in the liver. AIM OF THE STUDY: A sample of hepatic metabolites of medicagenic acid, with medicagenic acid glucuronide as the most abundant one, was evaluated for in vitro activity against urinary stones. A crystallization assay and a crystal-cell interaction assay were used to evaluate in vitro activity of hepatic metabolites of medicagenic acid on CaC2O4 (calciumoxalate) crystals, present in the majority of urinary stones. MATERIALS AND METHODS: In the crystallization assay the effects on nucleation of Ca2+ and C2O42- and aggregation of the CaC2O4 crystals are studied. In the crystal-cell interaction assay crystal retention is investigated by determining the amount of Ca2+ bound to injured monolayers of MDCK I cells. RESULTS: Results of the crystallization assay showed a tentative effect on crystal aggregation. The crystal-cell interaction assay showed a significant inhibition of crystal binding, which may reduce crystal retention in the urinary tract. CONCLUSIONS: As both formation of crystals by inhibiting aggregation and retention of crystals is affected, the beneficial effect of H. hirsuta against urinary stones may at least in part be attributed to medicagenic acid metabolites, indicating that saponins containing medicagenic acid may act as prodrugs.

Simulated Gastrointestinal Biotransformation of Chlorogenic Acid, Flavonoids, Flavonolignans and Triterpenoid Saponins in Cecropia obtusifolia Leaf Extract.

It is well known that biotransformation processes in the human body are crucial to form potentially bioactive metabolites from particular classes of natural products. However, little research has been conducted concerning the bioavailability of polyphenols, especially in the colon. The gastrointestinal stability and colonic biotransformation of the crude extract of the leaves of Cecropia obtusifolia, rich in flavone C-glycosides, was investigated under in vitro conditions, and the processing and interpretation of results were facilitated by using an automated machine learning model. This investigation revealed that flavone C-glycosides and flavonolignans from C. obtusifolia were stable throughout their passage in the simulated gastrointestinal tract including the colon phase. On the other hand, the colon bacteria extensively metabolized chlorogenic acid, flavonol, and triterpenoid O-glycosides. This investigation revealed that the colonic microbiota has an important role in the biotransformation of some chemical constituents of this extract.

Magnesium, Iron, Zinc, Copper and Selenium Status in Attention-Deficit/Hyperactivity Disorder (ADHD).

In this study, we critically review the literature concerning the relation of Mg, Fe, Zn, Cu and Se and attention-deficit/hyperactivity disorder (ADHD). Elemental status is estimated using peripheral blood parameters, hair, urine, daily intake and response to supplementation. The observed associations between concentration levels of the elements Mg, Fe, Zn, Cu and Se and ADHD symptoms are contradictory. This is partly due to the heterogeneity and complexity of the disorder. As a trend, lower ferritin and zinc levels can be observed. However, this correlation is not causative, as illustrated by placebo-controlled trials reporting conflicting evidence on the efficacy of supplementation. Well-defined studies on changes in concentration levels of the elements in relation to ADHD symptoms before and after treatment with therapeutics it will be possible to shed more light on the significance of these elements in this behavioral disorder. The discussion on whether a change in concentration of an element is cause or consequence of ADHD is not within the scope of this article.

Toll-Like Receptor-Dependent Immunomodulatory Activity of Pycnogenol®.

BACKGROUND: Pycnogenol® (PYC), an extract of French maritime pine bark, is widely used as a dietary supplement. PYC has been shown to exert anti-inflammatory actions via inhibiting the Toll-like receptor 4 (TLR4) pathway. However, the role of the other receptors from the TLR family in the immunomodulatory activity of PYC has not been described so far. AIM: The aim of this study was to investigate whether PYC might exert its immunomodulatory properties through cell membrane TLRs (TLR1/2, TLR5, and TLR2/6) other than TLR4. Moreover, the effect of gastrointestinal metabolism on the immunomodulatory effects of PYC was investigated. FINDINGS: We showed that intact non-metabolized PYC dose-dependently acts as an agonist of TLR1/2 and TLR2/6 and as a partial agonist of TLR5. PYC on its own does not agonize or antagonize TLR4. However, after the formation of complexes with lipopolysaccharides (LPS), it is a potent activator of TLR4 signaling. Gastrointestinal metabolism of PYC revealed the immunosuppressive potential of the retentate fraction against TLR1/2 and TLR2/6 when compared to the control fraction containing microbiota and enzymes only. The dialyzed fraction containing PYC metabolites revealed the capacity to induce anti-inflammatory IL-10 secretion. Finally, microbially metabolized PYC affected the colonic microbiota composition during in vitro gastrointestinal digestion. CONCLUSIONS: This study showed that gastrointestinal metabolism of PYC reveals its biological activity as a potential inhibitor of TLRs signaling. The results suggest that metabolized PYC acts as a partial agonist of TLR1/2 and TLR2/6 in the presence of the microbiota-derived TLR agonists (retentate fraction) and that it possesses anti-inflammatory potential reflected by the induction of IL-10 from THP-1 macrophages (dialysate fraction).

A red yeast rice-olive extract supplement reduces biomarkers of oxidative stress, OxLDL and Lp-PLA2, in subjects with metabolic syndrome: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Metabolic syndrome (MetS) refers to clustered cardiovascular risk factors (abdominal obesity, pre-diabetes, high blood pressure, dyslipidaemia). Therapies targeting oxidative stress may delay progression to atherosclerosis and diabetes. We investigated the anti-oxidative effect of a supplement combining red yeast rice and olive extract in patients with MetS. METHODS: A double-blind, placebo-controlled, randomised trial was conducted with 50 patients with MetS as defined by National Cholesterol Education Program Adult Treatment Panel III criteria. Forty-nine subjects randomly assigned to red yeast rice-olive extract (RYR-olive extract; 10.82 mg of monacolins and 9.32 mg of hydroxytyrosol per Cholesfytolplus capsule) or placebo completed the 8-week trial. Whereas effects on cardiovascular risk parameters of MetS have been reported recently, the observed significant 20% increase in oxidised low-density lipoprotein (OxLDL) prompted us to investigate other oxidative stress-related parameters: malondialdehyde (MDA), lipoprotein-associated phospholipase A2 (Lp-PLA2) and 8-hydroxy-2'-deoxyguanosine (8-OHdG). Statistical calculations included univariate quantitative analysis, multivariate linear regression and correlation analysis. RESULTS: The updated results indicate that an RYR-olive extract supplement significantly reduced Lp-PLA2 by 7% (p < 0.001), but it failed to show a significant decrease in plasma MDA and 8-OHdG (p > 0.05). Reductions in OxLDL (20%) and Lp-PLA2 (7%) were associated with each other (r = 0.740, p < 0.001). CONCLUSIONS: RYR-olive extract significantly reduced Lp-PLA2 in correlation with the marked reduction in plasma OxLDL, which may lead to a reduced risk for cardiovascular disease in patients with MetS. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02065180 . Registered on 13 February 2014.

In Vitro and In Vivo Study of the Gastrointestinal Absorption and Metabolisation of Hymenocardine, a Cyclopeptide Alkaloid.

Hymenocardine is a cyclopeptide alkaloid present in the root bark of Hymenocardia acida. In traditional African medicine, the leaves and roots of this plant are used to treat malaria, and moderate in vitro antiplasmodial activity has been reported for hymenocardine. However, in view of its peptide-like nature, potential metabolisation after oral ingestion has to be taken into account when considering in vivo experiments. In this study, the stability and small intestinal absorption of hymenocardine was assessed using an in vitro gastrointestinal dialysis model. In addition, potential liver metabolisation was investigated in vitro by incubation with a human S9 fraction. Moreover, hymenocardine was administered to rats per os, and blood and urine samples were collected until 48 and 24 h after oral administration, respectively. All samples resulting from these three experiments were analyzed by LC-MS. Analysis of the dialysate and retentate, obtained from the gastrointestinal dialysis model, indicated that hymenocardine is absorbed unchanged from the gastrointestinal tract, at least in part. After S9 metabolisation, several metabolites of hymenocardine could be identified, the major ones being formed by the reduction and/or the loss of an N-methyl group. The in vivo study confirmed that hymenocardine is absorbed from the gastrointestinal tract unchanged, since it could be identified in both rat plasma and urine, together with hymenocardinol, its reduction product.

Development and Validation of an in vitro Experimental GastroIntestinal Dialysis Model with Colon Phase to Study the Availability and Colonic Metabolisation of Polyphenolic Compounds.

The biological effects of polyphenols depend on their mechanism of action in the body. This is affected by bioconversion by colon microbiota and absorption of colonic metabolites. We developed and validated an in vitro continuous flow dialysis model with colon phase (GastroIntestinal dialysis model with colon phase) to study the gastrointestinal metabolism and absorption of phenolic food constituents. Chlorogenic acid was used as model compound. The physiological conditions during gastrointestinal digestion were mimicked. A continuous flow dialysis system simulated the one-way absorption by passive diffusion from lumen to mucosa. The colon phase was developed using pooled faecal suspensions. Several methodological aspects including implementation of an anaerobic environment, adapted Wilkins Chalgren broth medium, 1.10(8) CFU/mL bacteria suspension as inoculum, pH adaptation to 5.8 and implementation of the dialysis system were conducted. Validation of the GastroIntestinal dialysis model with colon phase system showed a good recovery and precision (CV < 16 %). Availability of chlorogenic acid in the small intestinal phase (37 ± 3 %) of the GastroIntestinal dialysis model with colon phase is comparable with in vivo studies on ileostomy patients. In the colon phase, the human faecal microbiota deconjugated chlorogenic acid to caffeic acid, 3,4-dihydroxyphenyl propionic acid, 4-hydroxybenzoic acid, 3- or 4-hydroxyphenyl acetic acid, 2-methoxy-4-methylphenol and 3-phenylpropionic acid. The GastroIntestinal dialysis model with colon phase is a new, reliable gastrointestinal simulation system. It permits a fast and easy way to predict the availability of complex secondary metabolites, and to detect metabolites in an early stage after digestion. Isolation and identification of these metabolites may be used as references for in vivo bioavailability experiments and for investigating their bioactivity in in vitro experiments.

Investigation of the in vivo antioxidative activity of Cynara scolymus (artichoke) leaf extract in the streptozotocin-induced diabetic rat.

The in vivo antioxidant activity of a quantified leaf extract of Cynara scolymus (artichoke) was studied. The aqueous artichoke leaf extract (ALE), containing 1.5% caffeoylquinic acid with chlorogenic acid being most abundant (0.30%), and luteolin-7-O-glucoside as major flavonoid (0.15%), was investigated by evaluating the effect on different oxidative stress biomarkers, after 3 wk oral supplementation in the streptozotocin-induced diabetic rat model. Apart from two test groups (0.2 g ALE/kg BW/day and 1 g ALE/kg BW/day, where BW is body weight), a healthy control group, untreated oxidative stress group, and vitamin E treated group (positive control) were included. A 0.2 g/kg BW/day of ALE decreased oxidative stress: malondialdehyde and 8-hydroxydeoxyguanosine levels significantly diminished, whereas erythrocyte glutathione levels significantly increased. A 1.0 g/kg BW/day ALE did not show higher antioxidant activity.

Antihepatotoxic activity of a quantified Desmodium adscendens decoction and D-pinitol against chemically-induced liver damage in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The isolation of D-pinitol (or 3-O-methyl-D-chiro-inositol) from an aqueous decoction of Desmodium adscendens (Fabaceae) leaves and twigs is reported. The protective and curative effect of this decoction, in which d-pinitol was quantified, and of pure D-pinitol, against chemically-induced liver damage in rats has been evaluated. MATERIALS AND METHODS: Enzyme levels of aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP), which are among the usual biomarkers for liver damage, were determined in serum samples of experimental animals. The protective effects against D-galactosamine-induced and ethanol-induced liver damage of a decoction of D. adscendens, quantified on its main constituent D-pinitol, was investigated in rats. In addition, the curative effects of pure D-pinitol and D. adscendens against chronic D-galactosamine-induced and acute acetaminophen-induced hepatotoxicity in rats was studied. Silymarin was used as positive control. RESULTS: In a first experiment evaluating the protective effect against acute D-galactosamine-induced liver damage in rats, a significant decrease of AST and ALT was observed for the D. adscendens decoction at a dose equivalent to 5 mg/kg/day and 20 mg/kg/day D-pinitol, as well as 20 mg/kg/day pure D-pinitol. With respect to chronic ethanol-induced liver damage in rats, the protective effects of D. adscendens at doses equivalent to 2 mg/kg/day and 10 mg/kg/day D-pinitol, were not observed for serum AST and ALT levels. However, with respect to reduced mortality of animals, statistical analysis showed a trend towards significance for the Desmodium group receiving a dose equivalent to 10 mg/kg/day D-pinitol, versus the untreated hepatotoxic animals. Additional experiments in rat models of acute acetaminophen-induced and chronic D-galactosamine-induced liver damage indicated that D. adscendens decoction and pure D-pinitol had no curative effect when given in a dose equivalent to 10 mg/kg/day D-pinitol, or up to 20 mg/kg/day as a pure compound daily, respectively. CONCLUSIONS: The aqueous decoction of D. adscendens showed a protective effect in rats against liver damage induced by D-galactosamine and ethanol, and this effect is at least in part due to the presence of D-pinitol. However, no curative effect of D. adscendens decoction or D-pinitol on liver damage induced by the tested chemicals could be demonstrated.

Evaluation of the anti-angiogenic activity of saponins from Maesa lanceolata by different assays.

Angiogenesis, in which a vascular network is established from pre-existing vessels, is a complex multistep process. Mechanisms underlying angiogenesis can be investigated using a variety of in vitro, ex vivo and in vivo approaches. Evaluation of several promising plants and plant metabolites, including terpenoids, revealed promising anti-angiogenic activity. Since the maesasaponins displayed anti-angiogenic activity in the chick chorioallantoic membrane (CAM) assay, their activity was further investigated in several test systems. The rat aorta ring assay was compared with the placental vein assay and then selected for the ex vivo investigation of the saponins. Besides their effect on the viability of HUVEC, the anti-angiogenic capacity of the compounds was also investigated in an in vivo zebrafish assay. The activity of the saponins in the viability assay was more pronounced than in the rat aorta ring assay and similar to the effect observed in the CAM assay. The use of different test systems, however, implies different results in the case of saponins.