Evaluation of the genotoxic and antigenotoxic potential of Melissa officinalis in mice

Melissa officinalis (L.) (Lamiaceae), a plant known as the lemon balm, is native to the east Mediterranean region and west Asia. Also found in tropical countries, such as Brazil, where it is popularly known as "erva-cidreira" or "melissa", it is widely used in aqueous- or alcoholic-extract form in the treatment of various disorders. The aim was to investigate in vivo its antigenotoxicity and antimutagenicity, as well as its genotoxic/mutagenic potential through comet and micronucleus assaying. CF-1 male mice were treated with ethanolic (Mo-EE) (250 or 500 mg/kg) or aqueous (Mo-AE) (100 mg/kg) solutions of an M. officinalis extract for 2 weeks, prior to treatment with saline or Methyl methanesulfonate (MMS) doses by intraperitoneal injection. Irrespective of the doses, no genotoxic or mutagenic effects were observed in blood and bone-marrow samples. Although Mo-EE exerted an antigenotoxic effect on the blood cells of mice treated with the alkylating agent (MMS) in all the doses, this was not so with Mo-AE. Micronucleus testing revealed the protector effect of Mo-EE, but only when administered at the highest dose. The implication that an ethanolic extract of M. officinalis has antigenotoxic/antimutagenic properties is an indication of its medicinal relevance.     

Dietary flavonoids as potential neuroprotectants

There is an increasing awareness of the role of certain nutritional components, including dietary flavonoids found in fruit, vegetables and beverages, in the maintenance of health and prevention of chronic diseases. In this regard, recent studies highlight an exciting role with respect to their potential neuroprotective actions, in particular towards deficits commonly observed with aging, such as reduced performance of cognitive, memory and learning tasks. These neurological functions, and possible mechanisms involved in controlling them, can be influenced by supplementation of single dietary flavonoids, or as part of a flavonoid-rich preparation. With this, a renewed emphasis is aimed at further understanding their modes and sites of action. Moreover a common theme among many in vitro studies examining mechanisms of neuroprotection is the failure to include biologically relevant metabolites of the flavonoids known to enter the circulation, and thus most likely to be bioavailable to cells and tissues. This oversight will ultimately influence the mechanisms of action proposed to explain the neuroprotection observed in animals and human studies. As such, emerging findings suggest a variety of potential mechanisms of action of flavonoids and their bioavailable metabolites in cytoprotection against oxidative stress, which may be independent of conventional antioxidant reducing activities. Such mechanisms might involve their interaction with cell signalling cascades, their influence on gene expression and the down regulation of pathways leading to cell death.     

Pharmacokinetics and metabolism of dietary flavonoids in humans

Flavonoids are components of fruit and vegetables that may be beneficial in the prevention of disease such as cancer and cardiovascular diseases. Their beneficial effects will be dependent upon their uptake and disposition in tissues and cells. The metabolism and pharmacokinetics of flavonoids has been an area of active research in the last decade. To date, approximately 100 studies have reported the pharmacokinetics of individual flavonoids in healthy volunteers. The data indicate considerable differences among the different types of dietary flavonoids so that the most abundant flavonoids in the diet do not necessarily produce the highest concentration of flavonoids or their metabolites in vivo. Small intestinal absorption ranges from 0 to 60% of the dose and elimination half-lives (T_1/2) range from 2 to 28 h. Absorbed flavonoids undergo extensive first-pass Phase II metabolism in the small intestine epithelial cells and in the liver. Metabolites conjugated with methyl, glucuronate and sulfate groups are the predominant forms present in plasma. This review summarizes the key differences in absorption, metabolism and pharmacokinetics between the major flavonoids present in the diet. For each flavonoid, the specific metabolites that have been identified so far in vivo are indicated. These data should be considered in the design and interpretation of studies investigating the mechanisms and potential health effects of flavonoids.     

The neuroprotective potential of flavonoids: a multiplicity of effects

Flavonoids exert a multiplicity of neuroprotective actions within the brain, including a potential to protect neurons against injury induced by neurotoxins, an ability to suppress neuroinflammation, and the potential to promote memory, learning and cognitive function. These effects appear to be underpinned by two common processes. Firstly, they interact with critical protein and lipid kinase signalling cascades in the brain leading to an inhibition of apoptosis triggered by neurotoxic species and to a promotion of neuronal survival and synaptic plasticity. Secondly, they induce beneficial effects on the vascular system leading to changes in cerebrovascular blood flow capable of causing angiogenesis, neurogenesis and changes in neuronal morphology. Through these mechanisms, the consumption of flavonoid-rich foods throughout life holds the potential to limit neurodegeneration and to prevent or reverse age-dependent loses in cognitive performance. The intense interest in the development of drugs capable of enhancing brain function means that flavonoids may represent important precursor molecules in the quest to develop of a new generation of brain enhancing drugs.     

Human metabolic pathways of dietary flavonoids and cinnamates

Flavonoids and cinnamates are widespread phenolic secondary metabolites synthesized by plants for defensive purposes. Many foods and beverages contain high levels of phenolic compounds. Certain phenolics in the diet are particularly bioactive and have pronounced effects on mammalian cells. These effects, together with epidemiological studies and animal models, have led to the hypothesis that dietary phenolics contribute to the health benefits of a diet rich in fruit and vegetables. This paper examines the biochemistry of the uptake and metabolic route of two groups of plant phenolics, the flavonols and hydroxycinnamates.     

Inhibition of beta-carotene-15,15'-dioxygenase activity by dietary flavonoids

Beta-carotene-15,15'-dioxygenase is an enzyme responsible for providing vertebrates with vitamin A by catalyzing oxidative cleavage of beta-carotene at its central double bond to two molecules of retinal in intestinal cells. However, little data have been reported regarding regulation of the enzyme activity. We have evaluated the effects of antioxidants and dietary flavonoids on the beta-carotene dioxygenase activity in vitro using a pig intestinal homogenate as the enzyme source. 2,6-Di-tert-butyl-4-methylphenol (BHT), a synthetic antioxidant, strongly inhibited the activity at the level of 10(-6) M (a mixed-type inhibition), whereas butylated hydroxyanisole, nor-dihydroguaiaretic acid, n-propyl gallate, and curcumin were moderately inhibitory. Flavonoids such as luteolin, quercetin, rhamnetin, and phloretin remarkably inhibited the dioxygenase activity noncompetitively, whereas flavanones, isoflavones, catechins, and anthocyanidins were less inhibitory. The structure-activity relationship indicated that catechol structure of ring B and a planar flavone structure were essential for inhibition. The enzyme inhibition was also indicated in the cultured Caco-2 cells by the significantly reduced conversion of beta-carotene to retinol when incubated with BHT and rhamnetin at 2 microM and 5 microM, respectively. The results suggest that some dietary antioxidants derived from food sources modulate conversion of beta-carotene to vitamin A in intestinal cells.     

Influence of dietary flavonoids on the glycation of plasma proteins

It has been suggested that the increasing glycation in diabetes can influence the ability of plasma proteins to bind to small molecules. Herein, the influence of flavonoids on the glycation of plasma proteins was investigated. After being incubated with glucose at 37 °C, the levels of glycated albumin (HGA) were significantly improved in healthy human plasma proteins (HPP). The inhibitory effects of flavonoids against the formation of advanced glycation products (AGEs) in HPP were determined as: galangin > apigenin > kaempferol ≈ luteolin > myricetin > quercetin. After being combined with 20 μmol L?1 of quercetin for 11 days, the fresh plasma with δ-glucose caused 323.05-32.07% inhibition of HGA formation in type II diabetes plasma proteins (TPP). Luteolin showed weak inhibition of HGA formation in TPP. However, kaempferol, galangin and apigenin hardly inhibited the formation of HGA in TPP. These results showed that more hydroxyl groups on ring B of flavonoids will enhance the inhibitory effects on the HGA formation in TPP.     

Recent advances in understanding the anti-diabetic actions of dietary flavonoids

Flavonoids are polyphenolic compounds that are abundant in fruits and vegetables, and increasing evidence demonstrates a positive relationship between consumption of flavonoid-rich foods and disease prevention. Epidemiological, in vitro and animal studies support the beneficial effects of dietary flavonoids on glucose and lipid homeostasis. It is encouraging that the beneficial effects of some flavonoids are at physiological concentrations and comparable to clinically-used anti-diabetic drugs; however, clinical research in this field and studies on the anti-diabetic effects of flavonoid metabolites are limited. Flavonoids act on various molecular targets and regulate different signaling pathways in pancreatic β-cells, hepatocytes, adipocytes and skeletal myofibers. Flavonoids may exert beneficial effects in diabetes by (i) enhancing insulin secretion and reducing apoptosis and promoting proliferation of pancreatic β-cells; (ii) improving hyperglycemia through regulation of glucose metabolism in hepatocytes; (iii) reducing insulin resistance, inflammation and oxidative stress in muscle and fat and (iv) increasing glucose uptake in skeletal muscle and white adipose tissue. This review highlights recent findings on the anti-diabetic effects of dietary flavonoids, including flavan-3-ols, flavanones, flavonols, anthocyanidins, flavones and isoflavones, with particular emphasis on the studies that investigated the cellular and molecular mechanisms involved in the beneficial effects of the compounds.     

The interaction of flavonoids with membranes: potential determinant of flavonoid antioxidant effects

Twenty six phenolic substances including representatives of the families, flavanones, flavanols and procyanidins, flavonols, isoflavones, phenolic acids and phenylpropanones were investigated for their effects on lipid oxidation, membrane fluidity and membrane integrity. The incubation of synthetic phosphatidylcholine (PC) liposomes in the presence of these phenolics caused the following effects: (a) flavanols, their related procyanidins and flavonols were the most active preventing 2,2'-azo-bis (2,4-dimethylvaleronitrile) (AMVN)-induced 2-thiobarituric acid-reactive substances (TBARS) formation, inducing lipid ordering at the water-lipid interface, and preventing Triton X-100-induced membrane disruption; (b) all the studied compounds inhibited lipid oxidation induced by the water-soluble oxidant 2,2'-azo-bis (2-amidinopropane) (AAPH), and no family-related effects were observed. The protective effects of the studied phenolics on membranes were mainly associated to the hydrophilicity of the compounds, the degree of flavanol oligomerization, and the number of hydroxyl groups in the molecule. The present results support the hypothesis that the chemical structure of phenolics conditions their interactions with membranes. The interactions of flavonoids with the polar head groups of phospholipids, at the lipid-water interface of membranes, should be considered among the factors that contribute to their antioxidant effects.     

Phytoestrogens as natural prodrugs in cancer prevention: dietary flavonoids

There are many reasons why vegetables and fruits may protect against cancer. As well as containing vitamins and minerals, which help keep the body healthy and strengthen the immune system, they are also good sources of biologically active compounds, which can help to protect cells in the body from damage that can lead to cancer. Notably, dietary flavonoids and other polyphenols are thought to have an important role as chemopreventive agents. Most studies on the possible mechanism of the chemopreventive action of dietary compounds have assumed that free hydroxyl groups of flavonoids and other polyphenols are necessary for their biological effects. However, in the human body dietary polyphenols are rapidly conjugated by glucuronosyltransferases and sulfotransferases, two enzymes that are abundantly present in the small intestine and liver, through which all of the oral dose must pass. Thus, most polyphenols that have been studied, e.g. quercetin, kaempferol, diosmetin, and resveratrol, would not be expected to reach internal organs beyond sites directly along the gastrointestinal tract. When the hydroxyl groups in polyphenols are methylated, the resulting compounds are much less prone to glucuronidation and sulfation. Thus methoxylated compounds are more metabolically stable, increasing their bioavailablity. The peel of various Citrus species can contain high concentrations of polymethoxyflavones, whereas the juice mainly contains hydroxylated flavones. At present, very little is known about the mechanisms by which methoxylated flavones may affect growth and development of tumour cells. Recently, it was shown that tumour specific enzymes can catalyze the O-demethylation of methoxylated flavones, resulting in the formation of flavones with free hydroxyl groups. We propose that demethylation of methoxylated flavones is another example of bioactivation of naturally occurring prodrugs.     

Metabolic conversion of dietary flavonoids alters their anti-inflammatory and antioxidant properties

The notion that dietary flavonoids exert beneficial health effects in humans is often based on in vitro studies using the glycoside or aglycone forms of these flavonoids. However, flavonoids are extensively metabolized in humans, resulting in the formation of glucuronide, methyl, and sulfate derivatives, which may have different properties than their parent compounds. The goal of this study was to investigate whether different chemical modifications of the same flavonoid molecule affect its biological and antioxidant activities. Hence, we studied the anti-inflammatory effects of several major human metabolites of quercetin and (-)-epigallocatechin-3-O-gallate (EGCG) by assessing their inhibitory effects on tumor necrosis factor α (TNFα)-induced protein expression of cellular adhesion molecules in human aortic endothelial cells (HAEC). HAEC were incubated with 1-30 μM quercetin, 3'- or 4'-O-methyl-quercetin, quercetin-3-O-glucuronide, and quercetin-3'-O-sulfate or 20-100 μM EGCG, 4'-O-methyl-EGCG, and 4',4'-di-O-methyl-EGCG, prior to coincubation with 100 U/ml of TNFα. 3'-O-Methyl-quercetin, 4'-O-methyl-quercetin, and their parent aglycone compound, quercetin, all effectively inhibited expression of intercellular adhesion molecule-1 (ICAM-1) with IC(50) values (concentration required for 50% inhibition) of 8.0, 5.0, and 4.4 μM, respectively; E-selectin expression was suppressed to a somewhat lesser but still significant degree by all three compounds, whereas vascular cell adhesion molecule-1 (VCAM-1) was not affected. In contrast, quercetin-3-O-glucuronide (20-100 μM), quercetin-3'-O-sulfate (10-30 μM), and phenolic acid metabolites of quercetin (20-100 μM) did not inhibit adhesion molecule expression. 4',4'-Di-O-methyl-EGCG selectively inhibited ICAM-1 expression with an IC(50) value of 94 μM, whereas EGCG (20-60 μM) and 4'-O-methyl-EGCG (20-100 μM) had no effect. The inhibitory effects of 3'-O-methyl-quercetin and 4',4'-di-O-methyl-EGCG on adhesion molecule expression were not related either to inhibition of NF-κB activation or to their antioxidant reducing capacity. Our data indicate that flavonoid metabolites have different biological and antioxidant properties than their parent compounds, and suggest that data from in vitro studies using nonmetabolites of flavonoids are of limited relevance in vivo.     

Effects of dietary flavonoids on apoptotic pathways related to cancer chemoprevention

Epidemiological studies have described the beneficial effects of dietary polyphenols (flavonoids) on the reduction of the risk of chronic diseases, including cancer. Moreover, it has been shown that flavonoids, such as quercetin in apples, epigallocatechin-3-gallate in green tea and genistein in soya, induce apoptosis. This programmed cell death plays a critical role in physiological functions, but there is underlying dysregulation of apoptosis in numerous pathological situations such as Parkinson's disease, Alzheimer's disease and cancer. At the molecular level, flavonoids have been reported to modulate a number of key elements in cellular signal transduction pathways linked to the apoptotic process (caspases and bcl-2 genes), but that regulation and induction of apoptosis are unclear. The aim of this review is to provide insights into the molecular basis of the potential chemopreventive activities of representative flavonoids, with emphasis on their ability to control intracellular signaling cascades responsible for regulating apoptosis, a relevant target in cancer-preventive approach.     

Abyssinones and related flavonoids as potential steroidogenesis modulators

Abyssinones and related flavonoids were screened against 3 enzymes (3βHSD, 17βHSD and Aromatase) of steroidogenesis pathway. The virtual screening experiment shows high affinity for flavonones than their respective chalcones. A 4'' ­OH blocked prenylated flavonone 2b (2­(2'', 2''­dimethyl chroman­6''­yl)­7­hydroxy chroman­4­one) had consistent binding affinity to all the three enzymes used in this study showing higher binding affinity to aromatase. A good correlation was observed between cytotoxic data (MCF­7, breast cancer cell line) and docking results indicating flavonone as a better steroidogenesis modulator in hormone dependent cancer.     

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.     

The effects of flavonoids on human phenolsulphotransferases: potential in drug metabolism and chemoprevention

Flavonoids are frequently found in fruits and vegetables, and are currently under investigation as potential chemopreventive agents. The present study reports the inhibitory effects of six flavonoids, quercetin, genistein, daidzein, equol, (+)-catechin and flavone, on sulphation of p-nitrophenol, a model substrate for the P-form of PST (thermostable, TS) and dopamine, a model substrate for the M-form of PST (thermolabile, TL). Kinetic studies of the PST activity and the inhibitory effects of flavonoids on the P-form of PST activity (using Hanes-Wolf and Dixon plots) show low Km and Ki values. Quercetin was found to be the most potent inhibitor and flavone was the least active inhibitor among the flavonoids. Kinetic analysis showed that the inhibition was non-competitive and Ki values were determined for each flavonoid. These observations suggest the potential for clinically important pharmacological and toxicological interactions by flavonoids.     

In vivo antigenotoxic potential and possible mechanism of action of selected 4-hydroxy-2H-chromen-2-one derivatives

The in vivo sex‐linked recessive lethal test was carried out in Drosophila melanogaster to investigate whether or not five substituted 4‐hydroxy‐2H‐chromen‐2‐ones can modulate the genotoxicity of the well‐established mutagenic agent ethyl methanesulfonate (EMS). For this purpose, 3 days old Canton S males were treated with the potent mutagen EMS alone in concentration of 0.75 ppm, as well as in combination with one of the five 4‐hydroxycoumarins, namely diethyl 2‐(1‐(4‐hydroxy‐2‐oxo‐2H‐chromen‐3‐yl)ethylidene)malonate ( 2b ), 3‐(1‐(4‐hydroxy‐2‐oxo‐2H‐chromen‐3‐yl)ethylidene)pentane‐2,4‐dione ( 6b ), 4‐(4‐(4‐hydroxy‐2‐oxo‐2H‐chromen‐3‐yl)thiazol‐2‐ylamino) benzenesulfonic acid ( 4c ), 4‐hydroxy‐3‐(2‐(2‐nitropheny lamino)thiazol‐4‐yl)‐2H‐chromen‐2‐one ( 9c ), and (E)‐4‐hydroxy‐3‐(1‐(m‐tolylimino)ethyl)‐2H‐chromen‐2‐one ( 5d ), in concentration of 70 ppm. The frequency of germinative mutations increased significantly after the treatment with EMS and decreased after treatments with coumarins. The maximum reduction was observed after treatments with 2b , 6b , 4c , and 5d . By the formation of hydrogen bonds or electrostatic interactions with O⁶ of DNA guanine, tested coumarins prevent EMS‐induced alkylation. The results indicate a protective role of five 4‐hydroxycoumarins under the action of a strong mutagen. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:322–330, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21426     

Prenylation enhances the biological activity of dietary flavonoids by altering their bioavailability

Flavonoids are distributed across the plant kingdom and have attracted substantial attention owing to their potential benefits for human health. Several studies have demonstrated that flavonoids prenylation enhances various biological activities, suggesting an attractive tool for developing functional foods. This review provides an overview of the current knowledge on how prenylation influences the biological activity and bioavailability of flavonoids. The enhancement effect of prenylation on the biological activities of dietary flavonoids in mammals was demonstrated by comparing the effect of 8-prenyl naringenin (8PN) with that of parent naringenin in the prevention of disuse muscle atrophy in mice. This enhancement results from higher muscular accumulation of 8PN than naringenin. As to bioavailability, despite the lower absorption of 8-prenyl quercetin (8PQ) compared with quercetin, higher 8PQ accumulation was found in the liver and kidney. These data imply that prenylation interferes with the elimination of flavonoids from tissues.