Advances in (poly)phenol research: novel sources, bioavailability, bioaccumulation and bioactivity

In the last decades, increasing scientific evidence has correlated the regular consumption of (poly)phenol-rich foods to a potential reduction of chronic disease incidence and mortality. However, epidemiological evidence on the role of (poly)phenol intake against the risk of some chronic diseases is promising, but not conclusive. In this framework a proper approach to (poly)phenol research is requested, using a step by step strategy. The plant kingdom produces an overwhelming array of structurally diverse secondary metabolites, among which flavonoids and related phenolic and (poly)phenolic compounds constitute one of the most numerous and widely distributed group of natural products. To date, more than 8000 structures have been classified as members of the phytochemical class of (poly)phenol, and among them over 4000 flavonoids have been identified. For this reason, a detailed food (poly)phenolic characterization is essential to identify the compounds that will likely enter the human body upon consumption, to predict the metabolites that will be generated and to unravel the potential effects of phenolic rich food sources on human health. In the first part of this work the attention was focused on the phenolic characterization of fruit and vegetable supplements, considering the increasing attention recently addressed to the so called "nutraceuticals", and on the main coffee industry by-product, namely coffee silverskin. The interest oriented toward (poly)phenols is then extended to their metabolism within the human body, paramount in the framework of their putative health promoting effects. Like all nutrients and non-nutrients, once introduced through the diet, (poly)phenols are subjected to an intense metabolism, able to convert the native compounds into similar conjugated, as well as smaller and deeply modified molecules, which in turn could be further conjugated. Although great strides have been made in the last decades, some steps of the (poly)phenol metabolism remain unclear and are interesting points of research. In the second part of this work the research was focused on a specific bran fraction, namely aleurone, added in feed pellets and in bread to investigate the absorption, metabolism and bioavailability of its phenolic compounds in animal and humans, with a preliminary in vitro step to determine their potential bioaccesibility. This part outlines the best approaches to assess the bioavailability of specific phenolics in several experimental models. The physiological mechanisms explaining the epidemiological and observational data on phenolics and health, are still far from being unraveled or understood in full. Many published results on phenolic actions at cell levels are biased by the fact that aglycones or native compounds have been used, not considering the previously mentioned chemical and biological transformations. In the last part of this thesis work, a new approach in (poly)phenol bioactivity investigation is proposed, consisting of a medium-long term treatment of animals with a (poly)phenol source, in this specific case resveratrol, the detection of its metabolites to determine their possible specific tissue accumulation, and the evaluation of specific parameters and/or mechanism of action at target tissue level. To conclude, this PhD work has contributed to advancing the field, as novel sources of (poly)phenols have been described, the bioavailability of (poly)phenols contained in a novel specific bran fraction used as ingredient has been evaluated in animal and in humans, and, finally, the tissue accumulation of specific (poly)phenol metabolites and the evaluation of specific parameters and/or mechanism of action has been carried out. For these reasons, this PhD work should be considered an example of adequate approach to the investigation of (poly)phenols and of their bioactivity, unavoidable in the process of unequivocally defining their effects on human health.

Seasonal variation in coumarin content of Mikania glomerata

Mikania glomerata Spengal extract contains approximately 0.5 percent of coumarin, a substance that displays anti-inflammatory and expectoratory activities. Extracts from different young leaves of plants collected during the early evening of December and July contained the highest levels coumarin. Plants dried in oven with circulating air contained 7.3 mg of coumarin/g of dry weight, demonstrating that this was the most appropriate drying condition for Mikania glomerata.

Effect of isomalt consumption on faecal microflora and colonic metabolism in healthy volunteers

Due to its low digestibility in the small intestine, a major fraction of the polyol isomalt reaches the colon. However, little is known about effects on the intestinal microflora. During two 4-week periods in a double-blind, placebo-controlled, cross-over design, nineteen healthy volunteers consumed a controlled basal diet enriched with either 30 g isomalt or 30 g sucrose daily. Stools were collected at the end of each test phase and various microbiological and luminal markers were analysed. Fermentation characteristics of isomalt were also investigated in vitro. Microbiological analyses of faecal samples indicated a shift of the gut flora towards an increase of bifidobacteria following consumption of the isomalt diet compared with the sucrose diet (P

Characterization of the Phenol Monooxygenase Gene from Chromobacterium violaceum: Potential Use for Phenol Biodegradation

In this work, the biodegradation mechanism of phenol and sub products (such as catechol and hydroquinone) in Chromobacterium violaceum was investigated by cloning and molecular characterization of a phenol monooxygenase gene in Escherichia coli. This gene (Cvmp) is very similar (74 and 59% of similarity and identity, respectively) to the ortholog from Ralstonia eutropha, bacteria capable of utilizing phenol as the sole carbon source. The phenol biodegradation ability of E. coli recombinant strains was tested by cell-growth in a minimal medium containing phenol as the sole source of carbon and release of intermediary metabolites (catechol and hydroquinone). Interestingly, during the growth of these strains on phenol, catechol, and hydroquinone accumulated transiently in the medium. These metabolites were further analyzed by HPLC. These results indicated that phenol can be initially orto or para hydroxylated to produce cathecol or hydroquinone, respectively, followed by meta-cleavage of aromatic rings. To verify this information, the metabolites obtained from HPLC were submitted to LC/MS to confirm their chemical structure, thereby indicating that the recombinant strains utilize two different routes simultaneously, leading to different ring-fission substrates for the metabolism of phenol. (C) KSBB

A Multivariate Calibration Procedure for UV/VIS Spectrometric Monitoring of BHK-21 Cell Metabolism and Growth

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

BENZENE MYELOCLASTOGENICITY AND METABOLISM IN CD-1 MICE: A CORRELATION

Benzene was studied in its target organ of effect, the bone marrow, with the micronucleus test and metaphase chromosomal analysis. Groups of 5 or 10, male and female CD-1 mice were treated with one or two p.o. or i.p. doses of benzene (440 mg/kg) or toluene (430, 860 or 1720 mg/kg) or both, and sacrificed 30 or 54h after the first dose. Benzene-treated animals were pretreated with phenobarbital (PB), 3-methylcholanthrene (3MC), (beta)-naphthoflavone ((beta)NF), SKF-525A, or Aroclor 1254. Toluene showed no clastogenic activity and reduced the clastogenic effect of co-administered benzene. None of the pretreatments protected against benzene clastogenicity. 3MC and (beta)NF greatly promoted benzene myeloclastogenicity. Dose response curves for benzene myeloclastogenicity were much steeper with 3MC induction than without. Micronuclei (MN) were 4-6 times higher by p.o. than i.p. benzene administration. This was not due to bacterial flora since no difference was found between germ-free and conventional males gavaged with benzene. A sensitive high-pressure liquid chromatographic method was developed and used to explore the relation between metabolic profiles of benzene in urine and MN after various pretreatments. Phenol (PH), trans-trans-muconic acid (MA) and hydroquinone (HQ) in the 48h male mouse urine accounted, respectively, for 12.8-22.8, 1.8-4.7 and 1.5-3.7% of the single oral dose of benzene (880, 440 and 220 mg/kg). Catechol (CT) was seen in trace amounts. MA was identified by ultraviolet and infrared spectroscopy and elemental analysis. Urinary metabolites--especially MA, HQ, and phenol glucuronide--correlated well with MN and were dependent on both the dose and the metabolism of benzene. Benzene metabolism was most inducible by cytochrome P-448 enzyme inducers, by p.o. > i.p., in males > females, and inhibited by toluene. Ph, CT or HQ administered p.o., 250, 150 and 250 mg/kg, respectively, or at 150 mg/kg x 2 after 3MC pretreatment, failed to reproduce the potent myeloclastogenicity of benzene. In fact, only HQ was mildly clastogenic. ^

Human sulfotransferases and their role in chemical metabolism

Sulfonation is an important reaction in the metabolism of numerous xenobiotics, drugs, and endogenous compounds. A supergene family of enzymes called sulfotransferases (SULTs) catalyze this reaction. In most cases, the addition of a sulfonate moiety to a compound increases its water solubility and decreases its biological activity. However, many of these enzymes are also capable of bioactivating procarcinogens to reactive electrophiles. In humans three SULT families, SULT1, SULT2, and SULT4, have been identified that contain at least thirteen distinct members. SULTs have a wide tissue distribution and act as a major detoxification enzyme system in adult and the developing human fetus. Nine crystal structures of human cytosolic SULTs have now been determined, and together with site-directed mutagenesis experiments and molecular modeling, we are now beginning to understand the factors that govern distinct but overlapping substrate specificities. These studies have also provided insight into the enzyme kinetics and inhibition characteristics of these enzymes. The regulation of human SULTs remains as one of the least explored areas of research in the field, though there have been some recent advances on the molecular transcription mechanism controlling the individual SULT promoters. Interindividual variation in sulfonation capacity may be important in determining an individual's response to xenobiotics, and recent studies have begun to suggest roles for SULT polymorphism in disease susceptibility. This review aims to provide a summary of our present understanding of the function of human cytosolic sulfotransferases.

The relationship between substrate metabolism, exercise and appetite control : does glycogen availability influence the motivation to eat, energy intake or food choice?

The way in which metabolic fuels are utilised can alter the expression of behaviour in the interests of regulating energy balance and fuel availability. This is consistent with the notion that the regulation of appetite is a psychobiological process, in which physiological mediators act as drivers of behaviour. The glycogenostatic theory suggests that glycogen availability is central in eliciting negative feedback signals to restore energy homeostasis. Due to its limited storage capacity, carbohydrate availability is tightly regulated and its restoration is a high metabolic priority following depletion. It has been proposed that such depletion may act as a biological cue to stimulate compensatory energy intake in an effort to restore availability. Due to the increased energy demand, aerobic exercise may act as a biological cue to trigger compensatory eating as a result of perturbations to muscle and liver glycogen stores. However, studies manipulating glycogen availability over short-term periods (1-3 days) using exercise, diet or both have often produced equivocal findings. There is limited but growing evidence to suggest that carbohydrate balance is involved in the short-term regulation of food intake, with a negative carbohydrate balance having been shown to predict greater ad libitum feeding. Furthermore, a negative carbohydrate balance has been shown to be predictive of weight gain. However, further research is needed to support these findings as the current research in this area is limited. In addition, the specific neural or hormonal signal through which carbohydrate availability could regulate energy intake is at present unknown. Identification of this signal or pathway is imperative if a casual relationship is to be established. Without this, the possibility remains that the associations found between carbohydrate balance and food intake are incidental.

Modulation of depth-dependent properties in tissue-engineered cartilage with a semi-permeable membrane and perfusion : a continuum model of matrix metabolism and transport

The functional properties of cartilaginous tissues are determined predominantly by the content, distribution, and organization of proteoglycan and collagen in the extracellular matrix. Extracellular matrix accumulates in tissue-engineered cartilage constructs by metabolism and transport of matrix molecules, processes that are modulated by physical and chemical factors. Constructs incubated under free-swelling conditions with freely permeable or highly permeable membranes exhibit symmetric surface regions of soft tissue. The variation in tissue properties with depth from the surfaces suggests the hypothesis that the transport processes mediated by the boundary conditions govern the distribution of proteoglycan in such constructs. A continuum model (DiMicco and Sah in Transport Porus Med 50:57-73, 2003) was extended to test the effects of membrane permeability and perfusion on proteoglycan accumulation in tissue-engineered cartilage. The concentrations of soluble, bound, and degraded proteoglycan were analyzed as functions of time, space, and non-dimensional parameters for several experimental configurations. The results of the model suggest that the boundary condition at the membrane surface and the rate of perfusion, described by non-dimensional parameters, are important determinants of the pattern of proteoglycan accumulation. With perfusion, the proteoglycan profile is skewed, and decreases or increases in magnitude depending on the level of flow-based stimulation. Utilization of a semi-permeable membrane with or without unidirectional flow may lead to tissues with depth-increasing proteoglycan content, resembling native articular cartilage.