Flavonoids

Flavonoids have been the object of intensive investigation for a number of reasons: (i) Based on their abundance in fruits and vegetables, these substances may explain some of the health-promoting effects of these foodstuffs; (ii) some foods are particularly rich in flavonoids, such as red wine, tea, onions, apples or dark chocolate, whose favorable cardiovascular effects are well known; (iii) medicinal plants rich in flavonoids or their extracts (e.g. an extract of Ginkgo biloba leaves) are used within alternative as well as academic medicine; (iv) specific flavonoids (e.g. quercetin) are also available in certain countries as nutraceuticals⁽Reference Ferrires³⁵⁾.

As far as the effects of flavonoids on the immune system are concerned, primary macrophages have been studied as potential cellular targets of these substances⁽Reference Perez-Vizcaino, Duarte and Jimenez³⁶⁾. A number of flavonoids inhibit macrophage proliferation (but not cell viability) and some additionally reduce TNFα and inducible nitric oxide synthase (iNOS) expression, probably by interfering with the NF-κB pathway. The structural determinants of activity include the C2–C3 double bond, the catechol group in the B ring and the 2-position of the B ring. Most of these flavonoids are glycosides, which are known to be hydrolyzed by bacterial enzymes in the gut. Since luteolin and quercetin are not active in vivo and aglycone flavonoids are absorbed in the small intestine, it is likely that glycosides act as prodrugs, releasing the biologically active aglycone in the lumen and preventing their premature absorption, which has been proven in the case of quercetin⁽Reference Comalada, Ballester and Bailon³⁷⁾.

Over recent years, our group has contributed to the knowledge of flavonoid mechanisms of action in an in vitro model using healthy human peripheral blood mononuclear cells (PBMC). In preliminary studies, three different types of Italian red wine (Primitivo, Lambrusco and Negroamaro) were screened for modulating nitric oxide (NO) production from monocytes. The effects of flavonoids were evaluated in an indirect manner by using wine samples that have previously been deprived of these substances. NO production was maximally obtained in the presence of Negroamaro and this datum was also confirmed by iNOS expression, as assessed by Western blot. In flavonoid-deprived samples, both activities (NO release and iNOS expression) were present at negligible levels. The same was true in samples containing only ethanol⁽Reference Comalada, Camuesco and Sierra³⁸⁾. The evidence that Negroamaro is the strongest inducer of NO seems to suggest a different composition of the polyphenols present in this wine, as also reported in other studies⁽Reference Magrone, Tafaro and Jirillo^39, Reference Vogel⁴⁰⁾, which have related the different vasodilating ability of red wine to the type of grape and country of origin. The above results represent the first evidence of the ability of red wine to generate NO from human monocytes, despite previous reports about the endothelial origin of NO as a regulator of vascular homeostasis⁽Reference Wallerath, Poleo and Li⁴¹⁾. According to our working hypothesis⁽Reference Balzer, Heiss and Schroeter⁴²⁾, in response to acute consumption of flavonoids in red wine, gut-associated macrophages can generate NO that, in turn, reaches the blood via the lymphatic route. As an alternative, flavonoids that are absorbed at the intestinal level and released into the circulation are able to stimulate monocytes to produce NO. In fact, there is evidence that in healthy volunteers, anthocyanins from red grape juice are absorbed at the intestinal level⁽Reference Magrone, Candore and Caruso⁴³⁾. In synthesis, it appears that moderate intake of red wine via NO release can prevent atherogenesis by virtue of its vasodilatory capacities⁽Reference Comalada, Camuesco and Sierra³⁸⁾. As far as release of cytokines from PBMC stimulated with Negroamaro is concerned, this wine was able to skew the immune response toward the T helper-1 cell type via the release of IL-12 and consequentially of interferon (IFN)-γ⁽Reference Bitsch, Netzel and Frank⁴⁴⁾. Furthermore, production of IL-1β and IL-6 occurred, thus suggesting a switch toward the inflammatory pathway. On the other hand, release of the anti-inflammatory cytokine IL-10 was also triggered by Negroamaro, thus indicating that the production of IL-6 and IL-1β was still under the control of IL-10⁽Reference Magrone, Tafaro and Jirillo⁴⁵⁾. In turn, IL-6 stimulates IL-10 production, generating a local feedback loop in order to limit the pro-inflammatory pathway⁽Reference Couper, Blount and Riley⁴⁶⁾. This event accounts for the maintenance of the inflammatory:anti-inflammatory ratio.

As far as B cell responses are concerned, in our test system flavonoids favoured the production of both IgA (mucosal response) and IgG (systemic response). In particular, at the gut-associated lymphoid tissue level, one can speculate that flavonoid-activated B cells may migrate to distant organs, such as the spleen and the secretory glands in the body⁽Reference Balzer, Heiss and Schroeter⁴²⁾, thus representing an amplification loop of the immune response. These immunological mechanisms are able to protect the host against pathogens as well as against neoplastic events via cytotoxic T cell and natural killer cell activation⁽Reference Steensberg, Fisher and Keller^47, Reference Brien, Uhrlaub and Nikolich-Zugich⁴⁸⁾. Based on these concepts, moderate red wine assumption might be useful to consider in age-related disorders because it is known that immunosenescence is characterized by a status of dysregulated immunity that leads to the increased susceptibility of the elderly to infections and autoimmune disease⁽Reference Newman and Riley^49, Reference Hayflick⁵⁰⁾. In this framework, it is worth mentioning that macrophages from old individuals produce much less hydrogen peroxide and NO than young individuals⁽Reference Candore, Colonna-Romano and Balistrieri⁵¹⁾. Interestingly, it has been reported that aged mice express less total p38 and C-Jun N-terminal kinase⁽Reference Pawelec, Solana and Remarque⁵²⁾ and, at the same time, an elevation of p38 and extracellular regulated kinase in red-wine-treated PBMC was observed⁽Reference Bohemer, Goral and Faunce⁵³⁾. Therefore, the increased expression of p38 by flavonoids⁽Reference Magrone, Panaro and Jirillo⁵⁴⁾ may trigger the induction of the IFN-γ pathway, increase the release of pro-inflammatory cytokines and promote Ig class switch in B cells, since p38 is a common denominator for all these pathways⁽Reference Merritt, Enslen and Diehl⁵⁵⁾. In conclusion, moderate red wine consumption or intake of flavonoids can be advantageous in the elderly via increased production of NO by macrophages and p38-induced IFN-γ release.

In our experiments, we found that the expression of phosphorylated p38 was enhanced by lipopolysaccharide (LPS) alone, while in samples simultaneously stimulated with LPS and Negroamaro, a decreased expression of activated p38 was observed. This may indicate that flavonoids tend to down-regulate the LPS-induced p38 phosphorylation pathway⁽Reference Balzer, Heiss and Schroeter⁴²⁾. Furthermore, this fact may be beneficial in the course of Gram-negative infections, where inhibition of p38α activity by the inhibitor SB203580 reduced mortality related to LPS-dependent shock and occurrence of collagen-induced arthritis⁽Reference Cook, Wu and Kang^56, Reference Pietersma, Tilly and Gaestel⁵⁷⁾. These findings have recently been confirmed by the demonstration that macrophage deletion of p38α mitigates noxious effects triggered by LPS⁽Reference Badger, Bradbeer and Votta⁵⁸⁾.

Interestingly, p38 per se may be responsible for NO release and iNOS expression, probably through another nuclear factor, such as signal transducer and activator of transcription-1 (Stat-1) or activator protein-1. In other experiments, we reported an elevated production of IFN-γ by monocytes treated with flavonoids⁽Reference Bitsch, Netzel and Frank⁴⁴⁾. In this context, IFN-γ could act together with p38 as an activation signal of Stat-1⁽Reference Kang, Chen and Otsuka^59, Reference Ganster, Taylor and Shao⁶⁰⁾ that, in turn, by binding to motifs at −5·2 and −5·8 kb in the iNOS promoter, might determine iNOS expression and, therefore, NO synthesis.

The NF-κB family of transcription factors consists of five members of the mammalian NF-κB family: p50/p105, p52/p100, p65, c-Rel and RelB, which are kept inactive in the cytoplasm by association with inhibitors (I)κB proteins⁽Reference Guo, Shao and Du⁶¹⁾ identified as IκBα, IκBβ and IκBε. They maintain NF-κB dimers in the cytoplasm and are crucial to signal responsiveness. IκBα is rapidly degraded during activation, of canonical NF-κB signalling pathways, leading to the release of multiple NF-κB dimers. Binding to IκBα prevents the translocation of NF-κB into the nucleus, thereby maintaining NF-κB in an inactive state. In response to an inflammatory stimulus, cytokines or viral infections, IκB proteins are rapidly degraded by the multicatalytic proteasome⁽Reference Ghosh, May and Kopp⁶²⁾. IκBα has the capacity to both prevent NF-κB binding and dissociate NF-κB from specific DNA consensus sequences. Nuclear localization of IκBα is induced by stimuli activating NF-κB and can be considered as part of a physiological mechanism regulating NF-κB-dependent transcription⁽Reference Renard, Percherancier and Kroll⁶³⁾.

The main observation related to IκBα is that this factor enters the nucleus steadily in cells continuously exposed to stimulation, but is constantly degraded in the nuclear compartment as long as stimulation persists⁽Reference Renard, Percherancier and Kroll⁶³⁾. In our previous study⁽Reference Bohemer, Goral and Faunce⁵³⁾, the phosphorylated form of IκBα was expressed at lower levels in PBMC treated with Negroamaro compared with the unphosphorylated form. However, there is evidence that more molecules of IκBα are generated with time and, therefore, this molecule participates in a feedback loop, where newly synthesized IκBα inhibits the activity of NF-κB⁽Reference Turpin, Hay and Dargemont⁶⁴⁾. These data suggest that flavonoids switch off the NF-κB pathway, thus reducing the release of pro-inflammatory cytokines (e.g. IL-1β).

In another series of results obtained by immunofluorescence analysis, we demonstrated that cells underwent a marked expression of p65 after treatment with whole wine and flavonoids alone, although this was lower than that observed with LPS-treated PBMC⁽Reference Bohemer, Goral and Faunce⁵³⁾. This finding reinforces the concept of the beneficial effects of flavonoids on human health, since an excessive production of p65/NF-κB would lead to induction of several genes that ultimately generate noxious effects into the host⁽Reference Bohemer, Goral and Faunce⁵³⁾. In conclusion, molecular mechanisms elicited by flavonoids on human PBMC seem to play a beneficial role in the host. One can postulate that NO release following activation of extracellular regulated kinase and p38 kinase is able to inhibit platelet aggregation, thus reducing the influx of atherogenic monocytes and LDL through endothelial cells into the wall of arteries and ultimately limiting atherogenesis. Furthermore, NO production may reduce infectious events, such as Chlamydia pneumoniae infection, which represents an important etiological factor in atherosclerosis⁽Reference Hayden and Ghosh^65, Reference Sawayama, Tatsukawa and Maeda⁶⁶⁾.

As far as the role of LPS in atherogenesis is concerned, inhibition of phosphorylated p38 expression in the presence of LPS, as well as the regulatory role of Iκβα mediated by flavonoids, should be taken into consideration in the arrest of the proinflammatory cytokine pathway in a variety of diseases.

In Table 1, the major effects of flavonoids from Negroamaro wine are listed.

Table 1. The salient effects of Negroamaro flavonoids on human healthy mononuclear cells

NO, nitric oxide, NOS, inducible NO synthase; ERK, extracellular regulated kinase.

Resveratrol

RSV (3,4′,5-trihydroxystilbene) is contained in the skins of red fruits, such as mulberries and red grapes⁽Reference Watson and Alp^67, Reference Holme and Pervaiz⁶⁸⁾, and therefore its intake occurs along with flavonoids.

Over recent years, increasing interest in RSV from red wine in relation to human health has been reported. In fact, RSV prevents or delays the onset of age-related diseases, such as diabetes, inflammation, Alzheimer's disease and CVD, also inducing neuroprotection and inhibiting neoplastic growth⁽Reference Pervaiz^69–Reference Vidavalur, Otani and Singal⁷⁷⁾. The therapeutic potential of RSV has been demonstrated by the extended lifespan and ameliorated motor function in mice fed a high-energy diet⁽Reference Aggarwal, Bhardwaj and Aggarwal⁷⁸⁾. The potential benefits to human health include the full range of CVD-associated conditions, e.g. myocardial infarction, arrhythmias, hypertension, hypertrophy, inflammation leading to fibrosis, atherosclerosis and thrombosis.

Although RSV acts as an antioxidant, primarily by increasing NO bioavailability, it can also exhibit pro-oxidant properties in the presence of transition metal ions such as Cu, thus leading to oxidative breakage of cellular DNA⁽Reference Baur, Pearson and Price⁷⁹⁾, which seems to be the common denominator for anticancer and chemopreventive properties of the polyphenol family⁽Reference Alarco´n de la Lastra and Villegas⁸⁰⁾. In fact, plant polyphenols are able to modulate the effects of deregulated cell cycle checkpoints, as a basis for cancer chemoprevention⁽Reference Khan, Afaq and Mukhtar⁸¹⁾.

Low-grade inflammation is implicated in various diseases such as the metabolic syndrome⁽Reference Meeran and Katiyar⁸²⁾, rheumatoid arthritis and neurodegenerative disorders⁽Reference Dandona, Aljada and Chaudhuri⁸³⁾. The cardioprotection and cancer prevention of RSV may depend on its anti-inflammatory effects, such as the inhibition of synthesis and release of pro-inflammatory cytokines and of eicosanoids and the inhibition of immune cell activation. Enzyme inhibition of cyclooxygenase-1 or cyclooxygenase-2 by the inhibitory effects of RSV on transcription factors such as NF-κB or activator protein-1 has also been reported⁽Reference Stolp and Dziegielewska^84, Reference Das and Das⁸⁵⁾. Furthermore, RSV associated with moderate wine intake activates platelet endothelial NOS and, in this way, blunts the pro-inflammatory pathway linked to p38 mitogen-activated protein kinase, thus inhibiting reactive oxygen species production and ultimately platelet function. In another study, RSV⁽Reference Udenigwe, Ramprasath and Aluko⁸⁶⁾ was able to reduce oxidative burst, chemotaxis and degranulation in human neutrophils as well as pro-inflammatory cytokines, such as IL-6, IL-1β, TNF-α and chemokine macrophage inflammatory protein-1α in vitro, as well as in a murine model of acute peritonitis. In this regard, two important targets of RSV were identified, namely the lipid mediator phospholipase D and the proinflammatory kinase SphK.

Other effects of RSV are listed in Table 2.

Table 2. Effects of RSV on different systems