α-tocopherol inhibits atherogenesis and improves cardiac function in mice independently of its antioxidant properties.

The impact of α-tocopherol on atherosclerosis is unclear and controversial. While some studies suggest potential benefits, such as antioxidant properties that may reduce oxidative stress, other research indicates no significant preventive effects. The intricate interplay of various factors, including dosage, individual differences, and study methodologies, contributes to the ongoing uncertainty surrounding α-tocopherol's role in atherosclerosis. Further research is needed to clarify its impact and establish clearer guidelines. Therefore, we aimed to evaluate the impact of α-tocopherol on atherogenesis in ApoE-/- fibrillin (Fbn)1C1039G/+ mice, which is a unique mouse model of advanced atherosclerosis with typical features such as large necrotic cores, high levels of inflammation and intraplaque neovascularization that resemble the unstable phenotype of human plaques. ApoE-/- Fbn1C1039G+/- mice were fed a western-type diet (WD) supplemented with a high dose of α-tocopherol (500 mg/kg diet), while control mice were fed a WD containing a low dose of α-tocopherol (50 mg/kg diet). The high dose of α-tocopherol reduced plaque thickness and necrotic core area in the right common carotid artery (RCCA) after 24 weeks WD. Moreover, α-tocopherol decreased plaque formation and intraplaque neovascularization in the RCCA. In addition to its antiatherogenic effect, chronic supplementation of α-tocopherol improved cardiac function in ApoE-/- Fbn1C1039G/+ mice. However, chronic supplementation of α-tocopherol did not decrease lipid peroxidation. On the contrary, α-tocopherol acted as a prooxidant by increasing plasma levels of oxidized LDL and plaque malondialdehyde, an end product of lipid peroxidation. Our data indicate that α-tocopherol inhibits atherogenesis and improves cardiac function independent of its antioxidant properties.

GPX4 overexpression does not alter atherosclerotic plaque development in ApoE knock-out mice.

Ferroptosis is a type of regulated necrosis that is associated with iron-dependent accumulation of lipid hydroperoxides. Given that iron deposition and lipid peroxidation initiate ferroptosis in atherosclerosis and contribute to further plaque development, we hypothesized that inhibition of ferroptosis could be of value in the treatment of atherosclerosis. Glutathione peroxidase 4 (GPX4) is the only enzyme known capable of reducing lipid hydroperoxides. Previous studies have demonstrated that inactivation of GPX4 results in ferroptosis while overexpression of GPX4 confers resistance to ferroptosis. In the present study, we examined the impact of GPX4 overexpression on the development of atherosclerotic plaques. GPX4-overexpressing mice (GPX4Tg/+) were crossbred with ApoE-/- mice and fed a western-type diet for 16 weeks. Atherosclerotic plaques of GPX4Tg/+ ApoE-/- mice showed increased GPX4 expression and a reduced amount of lipid hydroperoxides. However, plaque size and composition were not different as compared to control animals. Similarly, GPX4-overexpressing vascular smooth muscle cells and bone-marrow derived macrophages were not protected against lipid peroxidation and cell death triggered by the ferroptosis inducers erastin and 1S,3RRSL3. We concluded that GPX4 overexpression reduces lipid peroxidation in plaques of ApoE-/- mice, yet GPX4 overexpression is not sufficiently powerful to change plaque size or composition.

Can Biomarkers of Oxidative Stress in Serum Predict Disease Severity in West Nile Virus Infection? A Pilot Study.

West Nile virus (WNV) can cause asymptomatic infection in humans, result in self-limiting febrile illness, or lead to severe West Nile Neuroinvasive disease (WNND). We conducted a pilot study to compare selected biomarkers of oxidative stress in sera of viremic West Nile virus patients and asymptomatic infected blood donors to investigate their potential as predictors of disease severity. We found that total oxidant status was elevated in WNND and in uncomplicated WNV infections (median 9.05 (IQR 8.37 to 9.74) and 7.14 (7.03 to 7.25) µmol H2O2 equiv./L, respectively) compared to asymptomatic infections (0.11 (0.07 to 0.19) µmol H2O2 equiv./L) (p = 0.048). MDA levels showed a similar trend to TOS, but differences were not significant at α = 0.05. Total antioxidant status did not differ significantly between different disease severity groups. Oxidative stress appears to be associated with more severe disease in WNV-infected patients. Our preliminary findings warrant prospective studies to investigate the correlation of oxidative stress with clinical outcomes and severity of WNV infection.

Spontaneous Riboflavin-Overproducing Limosilactobacillus reuteri for Biofortification of Fermented Foods.

Riboflavin-producing lactic acid bacteria represent a promising and cost-effective strategy for food biofortification, but production levels are typically insufficient to support daily human requirements. In this study, we describe the novel human isolate Limosilactobacillus reuteri AMBV339 as a strong food biofortification candidate. This strain shows a high natural riboflavin (vitamin B2) overproduction of 18.36 µg/ml, biomass production up to 6 × 1010 colony-forming units/ml (in the typical range of model lactobacilli), and pH-lowering capacities to a pH as low as 4.03 in common plant-based (coconut, soy, and oat) and cow milk beverages when cultured up to 72 h at 37°C. These properties were especially pronounced in coconut beverage and butter milk fermentations, and were sustained in co-culture with the model starter Streptococcus thermophilus. Furthermore, L. reuteri AMBV339 grown in laboratory media or in a coconut beverage survived in gastric juice and in a simulated gastrointestinal dialysis model with colon phase (GIDM-colon system) inoculated with fecal material from a healthy volunteer. Passive transport of L. reuteri AMBV339-produced riboflavin occurred in the small intestinal and colon stage of the GIDM system, and active transport via intestinal epithelial Caco-2 monolayers was also demonstrated. L. reuteri AMBV339 did not cause fecal microbiome perturbations in the GIDM-colon system and inhibited enteric bacterial pathogens in vitro. Taken together, our data suggests that L. reuteri AMBV339 represents a promising candidate to provide riboflavin fortification of plant-based and dairy foods, and has a high application potential in the human gastrointestinal tract.

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.

Optimization of an in vitro gut microbiome biotransformation platform with chlorogenic acid as model compound: From fecal sample to biotransformation product identification.

Recent data clearly show that the gut microbiota plays a significant role in the biotransformation of many endogenous molecules and xenobiotics, leading to a potential influence of this microbiotic metabolism on activation, inactivation and possible toxicity of these compounds. To study the colonic biotransformation of xenobiotics by the gut microbiome, in vitro models are often used as they allow dynamic and multiple sampling overtime. However, the pre-analytical phase should be carefully optimized to enable biotransformation product identification representative for the in vivo situation. During this study, chlorogenic acid was used as a model compound to optimize a ready-to-use gut microbiome biotransformation platform using an in vitro gastrointestinal dialysis-model with colon phase together with an instrumental platform using liquid chromatography coupled to high resolution mass spectrometry (LC-QTOF-MS). Identification of the biotransformation products of chlorogenic acid was performed using complementary suspect and non-targeted data analysis approaches (MZmine + R and MPP workflow). Concerning the pre-analytical phase, (i) the influence of different incubation media (Wilkins-Chalgren Anaerobic Broth (WCB) and (versus) phosphate buffer) and different incubation times (prior to implementation in the colonic stage of the dialysis model) on fecal bacterial composition and concentration were investigated and (ii) four different sample preparation methods (centrifugation, extraction, sonication and freeze-drying) were evaluated targeting colonic biotransformation of chlorogenic acid. WCB as incubation medium showed to introduce substantial variation in the bacterial composition of the fecal samples, while the sterile phosphate buffer guaranteed a closer resemblance to the in vivo composition. Furthermore, incubation during 24 h in sterile phosphate buffer as medium showed no significant increase or decrease in anaerobic bacterial concentration, concluding that incubation prior to the colonic stage is not needed. Concerning sample preparation, centrifugation, sonication and extraction gave similar results, while freeze-drying appeared to be inferior. The extraction method was selected as an optimal sample preparation method given the quick execution together with a good instrumental sensitivity. This study optimized a ready-to-use platform to investigate colonic biotransformation of xenobiotics by using chlorogenic acid as a model compound. This platform can be used in the future to study differences in colonic biotransformation of xenobiotics using fecal samples of different patient groups.

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).

Oxidative stress and immune aberrancies in attention-deficit/hyperactivity disorder (ADHD): a case-control comparison.

The objective of this study is to compare oxidative stress and immune biomarkers between attention-deficit/hyperactivity disorder (ADHD) patients and controls without ADHD. A case-control comparison between 57 paediatric (6-12 years) untreated ADHD patients from the Antwerp University Hospital and 69 controls without ADHD from random schools in Flanders, Belgium, was conducted. Erythrocyte glutathione (GSH) and plasma lipid-soluble antioxidants (retinol, α-tocopherol, γ-tocopherol, retinyl palmitate, ß-carotene, and co-enzyme Q10) were determined by HPLC with electrochemical detection, plasma malondialdehyde (MDA) by HPLC with fluorescence detection, plasma cytokines (interleukin (IL)-1ß, IL-5, IL-6, IL-8, IL-10, tumour necrosis factor (TNF) and interferon (INF)-γ) and immunoglobulins (IgE, IgG and IgM) by flow cytometry and urinary 8-hydroxy-2'deoxyguanosine (8-OHdG) levels by ELISA assay. Dietary habits were determined by a food frequency questionnaire. Plasma MDA levels were on average 0.031 µM higher in patients than in controls (p < 0.05), and a trend for higher urinary 8-OHdG was observed. Erythrocyte GSH and plasma retinyl palmitate levels, as well as IgG and IgE levels, were higher in patients than in controls as well (on average 93.707 µg/ml, 0.006 µg/ml, 301.555 µg/ml and 125.004 µg/ml, resp., p < 0.05). Finally, a trend for lower plasma IL-5 levels was observed. After Bonferroni correction for multiple testing, the difference in GSH levels remained statistically significant (nominally significant for retinyl palmitate), while significance was lost for MDA, IgG and IgE levels. Dietary habits do not appear to cause the observed differences. These results point at the potential involvement of slight oxidative stress and immune disturbances in ADHD.

In vitro gastrointestinal biotransformation and characterization of a Desmodium adscendens decoction: the first step in unravelling its behaviour in the human body.

OBJECTIVES: The isolation and identification of the flavonoids present in a decoction of Desmodium adscendens was performed. In view of the oral use of the decoction, this work focused on the stability in gastrointestinal conditions and biotransformation by intestinal microflora in the colon of D-pinitol, vitexin and the flavonoid fraction of the decoction, as a first step in unravelling its behaviour in the human body. METHODS: The freeze-dried decoction was first subjected to column chromatography. Subsequently an enriched flavonoid fraction, was separated by repeated semi-preparative high-performance liquid chromatography (HPLC) or by HPLC-SPE. The isolated compounds were elucidated by NMR. Biotransformation experiments were carried in an in vitro gastrointestinal dialysis model. KEY FINDINGS: The major flavonoids of a decoction of D. adscendens were characterized as vicenin-2, isoschaftoside, schaftoside, 2″-O-xylosylvitexin, 2″-O-pentosyl-C-hexosyl apigenin and a O-hexosyl-C-hexosyl apigenin, tentatively identified as 2″-O-glucosyl-vitexin. During their passage in the gastrointestinal dialysis model, vitexin and C-glycosides thereof were found to be stable. Only the O-glycosidic bonds of O-glycosides of vitexin or isovitexin were hydrolysed during the colonic phase. CONCLUSIONS: A D. adscendens decoction was found to be rich in vitexin and isovitexin glycosides from which vitexin and the C-glycosides thereof were found to be stable in the simulated gastrointestinal tract.

Vegetable relishes, high in ß-carotene, increase the iron, zinc and ß-carotene nutritive values from cereal porridges.

Iron, zinc and vitamin A deficiencies are serious public health problems in sub-Saharan Africa, which can be alleviated by dietary diversification. The effects of adding cowpea leaf (CL) and orange-fleshed sweet potato (OFSP) relishes to sorghum and maize porridges on iron, zinc and ß-carotene contents and bioaccessibilities were determined. Despite the high iron content of the CL relish (14.59 mg/100 g), the vegetable relishes had little effect on the iron bioaccessibility from the cereal porridges. Importantly, the addition of the CL relish increased the percentage and amount of bioaccessible zinc 2- and 3-fold, respectively. Addition of CL and OFSP relishes resulted in ß-carotene contents of 10-13 mg/100 g. The ß-carotene from the OFSP relish meals was double as bioaccessible than that from the CL relish meals. Addition of the vegetable relishes has real potential to improve especially the vitamin A and zinc nutritive value of cereal diets.

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.

NecroX-7 reduces necrotic core formation in atherosclerotic plaques of Apoe knockout mice.

BACKGROUND AND AIMS: A large necrotic core is a key feature of atherosclerotic plaque instability. Necrotic cellular debris accumulates in the lipid-rich core and promotes inflammation, destabilization and ultimately rupture of the plaque. Although the role of necrosis in atherosclerosis is rather clear-cut, not many strategies have been performed up till now to specifically target plaque necrosis. In the present study, we tested the plaque stabilizing potential of NecroX-7, a novel compound with antioxidative and anti-necrotic properties. METHODS: Male apolipoprotein E (Apoe) knockout mice were treated with NecroX-7 (30 mg/kg) or vehicle, 3 times per week, via intraperitoneal injections for 16 weeks. Meanwhile, mice were fed a western-type diet to induce plaque formation. RESULTS: NecroX-7 reduced total plaque burden in the thoracic aorta as compared to vehicle-treated mice, without affecting total plasma cholesterol. Plaques in the aortic root of NecroX-7-treated mice showed a significant decrease in necrotic core area, 8-oxodG, iNOS and MMP13 expression, while collagen content and minimum fibrous cap thickness were increased. Moreover, NecroX-7 treatment reduced the expression of multiple inflammation markers such as TNFα, IL1ß, iNOS, HMGB1 and RAGE in a NF-κB-dependent manner. In vitro, NecroX-7 prevented tert-butyl hydroperoxide (tBHP)-induced mitochondrial ROS formation, necrosis, iNOS expression and HMGB1 release in primary macrophages. CONCLUSIONS: NecroX-7 improves features of plaque stability in Apoe knockout mice by reducing necrotic core formation, oxidative stress and inflammation, and by increasing collagen deposition and fibrous cap thickness. Therefore, NecroX-7 could be a promising pleiotropic drug for the treatment of atherosclerosis.

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.