Anti-Inflammatory Role of Natural Polyphenols and Their Degradation Products

A tight regulation of the immune/inflammatory system is crucial for maintaining the balance between protective and tissue-damaging responses. Systemic inflammatory response syndrome (SIRS) and sepsis are characterized by a loss of control over inflammatory responses, which can be provoked by a variety of causative agents and severe clinical insults. Sepsis is the most common cause of death in intensive care units worldwide and despite the extensive research we do not fully understand the cellular and molecular mechanisms that are involved in triggering and propagation of septic injury. A number of different approaches have been investigated to try to treat and/or prevent septic shock associated with infections caused by Gram-negative bacteria. Antibiotics constitute a necessary part of the treatment of sepsis, but antibiotics alone, even used optimally, are not sufficient to dramatically reduce the mortality of septic patient, because antibiotics cannot control the complex systemic inflammation and dysregulated host responses. For this reason, considerable efforts have been expended in developing non-antibiotic forms of treatment. A wide variety of dietary plants including grains, berries, legumes, tea, beer, grape/wine, olive oil, chocolate/cocoa, coffee, walnuts, peanuts, spices, fruits, vegetables etc. contain polyphenols (Bravo, 1998). Polyphenols, with 8000 structural variants, are characterized by the presence of aromatic rings bearing one or more hydroxyl moieties, which have a pivotal role in mediating of its antioxidant properties. As antioxidants, polyphenols are normally produced by plants for their antibiotic and antifungal features (Leiro et al., 2004). Polyphenols are generally divided into six major groups: hydroxybenzoic acids, phenolic alcohols, hydroxycinnamic acids, lignans, flavonoids and stilbenes (e.g. resveratrol) (D’Archivio et al., 2007). Recently, a number of natural products or ingredients of traditional medicines and healthy foods such as resveratrol, curcumin, and catechins were extensively investigated and subjected to clinical trials as anti-inflammatory agents (Hatcher et al., 2008). Although the knowledge of absorption, bioavailability and metabolism of polyphenols is not entirely known, it appears that some polyphenols are bioactive and are absorbed in their native or modified form. After the metabolization of polyphenols by the microflora of the intestines, their absorbed forms may be detected in plasma in nanomolar concentration (Rahman et al., 2006). The active components of dietary phytochemicals (e.g. curcumin, resveratrol, capsaicin, catechins, vitamins, beta carotene and dietary fiber) are believed to suppress the


Introduction
A tight regulation of the immune/inflammatory system is crucial for maintaining the balance between protective and tissue-damaging responses.Systemic inflammatory response syndrome (SIRS) and sepsis are characterized by a loss of control over inflammatory responses, which can be provoked by a variety of causative agents and severe clinical insults.Sepsis is the most common cause of death in intensive care units worldwide and despite the extensive research we do not fully understand the cellular and molecular mechanisms that are involved in triggering and propagation of septic injury.A number of different approaches have been investigated to try to treat and/or prevent septic shock associated with infections caused by Gram-negative bacteria.Antibiotics constitute a necessary part of the treatment of sepsis, but antibiotics alone, even used optimally, are not sufficient to dramatically reduce the mortality of septic patient, because antibiotics cannot control the complex systemic inflammation and dysregulated host responses.For this reason, considerable efforts have been expended in developing non-antibiotic forms of treatment.A wide variety of dietary plants including grains, berries, legumes, tea, beer, grape/wine, olive oil, chocolate/cocoa, coffee, walnuts, peanuts, spices, fruits, vegetables etc. contain polyphenols (Bravo, 1998).Polyphenols, with 8000 structural variants, are characterized by the presence of aromatic rings bearing one or more hydroxyl moieties, which have a pivotal role in mediating of its antioxidant properties.As antioxidants, polyphenols are normally produced by plants for their antibiotic and antifungal features (Leiro et al., 2004).Polyphenols are generally divided into six major groups: hydroxybenzoic acids, phenolic alcohols, hydroxycinnamic acids, lignans, flavonoids and stilbenes (e.g.resveratrol) (D'Archivio et al., 2007).Recently, a number of natural products or ingredients of traditional medicines and healthy foods such as resveratrol, curcumin, and catechins were extensively investigated and subjected to clinical trials as anti-inflammatory agents (Hatcher et al., 2008).Although the knowledge of absorption, bioavailability and metabolism of polyphenols is not entirely known, it appears that some polyphenols are bioactive and are absorbed in their native or modified form.After the metabolization of polyphenols by the microflora of the intestines, their absorbed forms may be detected in plasma in nanomolar concentration (Rahman et al., 2006).The active components of dietary phytochemicals (e.g.curcumin, resveratrol, capsaicin, catechins, vitamins, beta carotene and dietary fiber) are believed to suppress the Bioavailability and metabolism of resveratrol have been widely studied and its efficacy depends on its absorption and metabolism.In mice, rats and humans resveratrol has been detected in body fluids (urine, bile, and plasma) as well as in kidneys, stomach, intestine, and liver, following oral administration and this wide tissue targeting suggests an efficient absorption (Boocock et al., 2007;Jang et al., 1997;Vitrac et al., 2003;Wenzel & Somoza, 2005;Wenzel et al., 2005;).

Fig. 2. Structure of quercetin
Foods rich in quercetin include black and green tea, capers, lovage, apples, red grapes and a number of berries (Häkkinen et al., 1999).Dietary quercetin is mostly present as its glycoside form and it is absorbed from the intestinal lumen is mostly converted to conjugated metabolites before entering circulation.A recent study regarding the tissue distribution observed that quercetin concentrated in lungs, testes, kidneys, thymus, heart and liver in rats and in pigs (de Boer et al., 2005).After absorption quercetin can undergo microbial degradation in the colon to phenolic acids and CO 2 , which is exhaled in the breath.

Curcumin
Curcumin (diferuloylmethane) (Figure 3), an orange-yellow component of turmeric or curry powder, is a polyphenol natural product isolated from the rhizome of the plant Curcuma longa.

Fig. 3. Structure of curcumin
Since its first use as a drug in 1937 curcumin's therapeutic potential has been explored in inflammatory diseases, neoplastic disease, cardiovascular and neurodegenerative disease, diabetes, cystic fibrosis and other disorders (Egan et al., 2004;Hsu & Cheng, 2007;Miriyala et al., 2007;Weisberg et al., 2008).However, curcumin, a highly pleiotropic molecule with an excellent safety profile targeting multiple diseases with strong evidence on the molecular level, could not achieve its optimum therapeutic outcome in past clinical trials, largely due to its low solubility and poor bioavailability.Animal and human studies have shown that curcumin is rapidly metabolized and conjugated in the liver, and then excreted, therefore having limited systemic bioavailability (Cheng et al., 2001;Ireson et al., 2001;Sharma et al., 2004).

Epigallocatechin gallate
Epigallocatechin gallate (EGCG), a principal antioxidant derived from green tea, is one of the most extensively investigated chemopreventive phytochemicals (Figure 4).

Fig. 4. Structure of epigallocatechin gallate
Recently, green tea has attracted attention for its health benefits, particularly with respect to its potential for preventing and treating cancer, cardiovascular diseases, inflammatory diseases, and neurodegenerative diseases in humans (Tedeschi et al., 2004;Weisburger & Chung, 2002;Yang et al., 1998).The metabolism of green tea catechins has been studied in various animals and in human subjects (Lee et al., 1995;Pietta et al., 2008).Orally administrated catechin to humans is absorbed, metabolized, and excreted within 24 hours (Harada et al., 1999) and green tea consumption increased the plasma levels of EGCG in a dose-dependent manner (Nakagawa et al., 1997).Furthermore, Suganuma et al. demonstrated that after direct administration of labelled EGCG into the stomachs radioactivity appeared in a wide range of target organs in mice, including the digestive tract, liver, lung, pancreas, mammary gland and skin, brain, kidney, uterus and ovary and testes (Suganuma et al., 1998).

Phenolic acids and aldehydes
Natural phenolic compounds are secondary plant metabolites and the most abundant antioxidant resources.They are widely distributed in plants and present in considerable amounts in the human diet.Phenolic acids are hydroxylated derivatives of benzoic and cinnamic acids.The most common hydroxycinnamic acid derivatives are p-coumaric, caffeic, and ferulic acids which frequently occur in foods as simple esters with quinic acid or glucose.Phenolic acids have received considerable attention due to their various biological activities, including antioxidant, anti-apoptotic and anti-inflammatory capacities (Manach et al., 2004).Caffeic acid (Figure 5) is a widespread phenolic acid that occurs naturally in many agricultural products such as fruits, vegetables, wine, olive oil, and coffee (Mattila & Kumpulainen, 2002).

Fig. 5. Structure of caffeic acid
It is a potent antioxidant, metal chelating (Psotova et al., 2003), anti-inflammatory (Chao et al., 2010), free radical scavenger (Gulcin, 2006) and antidiabetic agent.Caffeic acid has been shown to be an inhibitor of the lipoxygenase enzyme and its conjugates such as chlorogenic and caftaric acids were demonstrated to be more powerful antioxidants in a number of different systems (Fukumoto & Mazza, 2000).Caffeic acid and its derivatives are good substrates of polyphenol oxidases, and under certain conditions may undergo oxidation in plant tissues or products of plant origin (Kerry & Rice-Evans, 1998).Ferulic acid (4-hydroxy-3-methoxy cinnamic acid) is a product of the phenylalanine and tyrosine metabolism, and it is produced by the shikimate pathway in plants (Figure 6).It is commonly found in fruits like orange and in vegetables, such as tomato, carrot, sweet corn and rice bran.The wide spectrum of beneficial effects of ferulic acid for human health due to its antibacterial, anti-inflammatory, hepatoprotective, anticancer, antidiabetic, neuroprotective, anti-atherogenic and antioxidant activity (Srinivasan et al., 2007).Partially, because of its antioxidant and anti-inflammatory activity, ferulic acid is considered as a potential therapeutic agent (together with other natural phenolic compounds) against various diseases like cancer, diabetes, cardiovascular dysfunction, inflammatory diseases and neurodegenerative diseases (Soobrattee et al., 2005).Ferulic acid and ferulaldehyde are potential end-products of dietary polyphenol degradation since they were found at a high concentration in human urine after red wine and chocolate consumption (Gonthier, 2003;Rios et al., 2003).Furthermore, ferulic acid was reported to stay in the blood longer than other antioxidants such as vitamin C, and have higher bioavailability than that of other dietary flavonoids and monophenolics studied so far (Beecher et al., 1998).The structural characteristic of ferulic acid and its reduced form, ferulaldehyde mainly resembles, the difference is one functional group (Figure 6).Due to this structural similarity and the presence of the reactive aldehyde group (which can be easily oxidized to carboxylic group), ferulaldehyde is thought to have very similar or maybe better biological activity as ferulic acid (Radnai et al, 2009;Tucsek et al., 2011).

Reactive oxygen and nitrogen species
Oxidative stress is involved in the pathomechanism of sepsis and reactive oxygen and nitrogen species are important mediators of cellular injury during endotoxemia (Cadenas & Cadenas, 2002).Oxidative damage caused by ROS and RNS will lead, among others, to DNA lesions, function loss of enzymes, increased cell permeability, disturbed signalling over the cell and eventually even cell death via necrosis or apoptosis.Consequently, ROS play a key role in enhancing the inflammation through sustained production of various cytokines, activation and phosphorylation of MAP kinases and redox-sensitive transcription factors, such as NF-κB and AP-1 in various inflammatory diseases.ROS also alters nuclear histone acetylation and deacetylation (chromatin remodelling) leading to increased gene expression of proinflammatory mediators (Rahman et al., 2004).ROS are generated during normal cellular metabolism.The respiratory chain in mitochondria is the major source of oxygen radicals.In inflammatory processes, beside the mitochondrial ROS production there are other possible sources of ROS such as metabolic cascade of arachidonic acid (via COX-2), protease-mediated enzyme xanthine-oxidase and membrane-bound enzyme complex NADPH oxidase (Victor et al., 2005).ROS and RNS cause peroxidation of membrane phospholipids, oxidation of proteins and DNA damage (Pattanaik & Prasad 1996).iNOS is expressed and continuously active during inflammation, where it is involved in host- Severe Sepsis and Septic Shock -Understanding a Serious Killer 386 defence against pathogens.iNOS generates NO which can be converted to its stable products, nitrite and nitrate (Gomez-Jimenez et al., 1995).Many studies reconfirmed the fact that resveratrol is a potent free radical scavenger and also a potent antioxidant (Soleas et al., 1997).Peripheral blood mononuclear cells play a critical role as the first defence line during endotoxemia.Activation of these cells by different physiological and non physiological agents causes a massive respiratory burst followed by an increase in oxygen consumption accompanied by the generation of ROS including superoxide, hydrogen peroxide, and hydroxy radicals.The release of ROS is of major importance for host defence but can also induce tissue damage.Resveratrol has been reported to have a strong inhibitory effect on multiple reactive oxygen species produced by polymorphonuclear leukocytes stimulated with formyl methionyl leucyl phenylalanine a chemotactic peptide (Rotondo et al., 1998).Zymosan is a carbohydrate-rich cell wall preparation derived from the yeast Saccharomyces cerevisiae could activate leukocytes to release ROS and resveratrol was shown as a potent inhibitor of ROS production in both unopsonized zymosan-stimulated RAW 264.7 cells and human monocytes and neutrophils (Jang et al., 1999).It was reported that resveratrol exerted a strong inhibitory effect on superoxide radical and hydrogen peroxide produced by macrophages stimulated by lipopolysaccharides (LPS) or phorbol esters (Martinez & Moreno, 2000).Quercetin has been shown to be an excellent in vitro antioxidant.Within the flavonoid family, quercetin is the most potent scavenger of ROS, including superoxide (Hanasaki et al., 1994;Cushnie & Lamb, 2005), and RNS like NO (van Acker et al., 1995) and peroxynitrite (Haenen et al., 1997;Heijnen et al., 2001) and it also contributes to the total plasma antioxidant capacity (Arts et al., 2004).On the other hand, it is known that during its antioxidative activities, quercetin becomes oxidized into various oxidation products and these products like semiquinone radicals and quinones display various toxic effects, such as increased membrane permeability, due to their ability of arylating protein thiols (Kalyanaraman et al., 1987;Monks & Lau, 1992;Metodiewa et al., 1999).However, quercetin quinone-induced toxicity has been shown in various in vitro studies and has recently been defined as the quercetin paradox (Boots et al., 2007), its in vivo formation and possible toxicity has not been demonstrated yet.Free radicals such as superoxide anion, hydrogen peroxide, and nitric oxide have been reported to be scavenged by curcumin (in the micro to millimolar range) both in vitro and in vivo (Aggarwal et al., 2003).The antioxidant properties of curcumin are evident from its ability to lower lipid peroxidation and maintain the activity status of various antioxidant enzymes.Findings of Biswas et al. indicate that curcumin could scavenge ROS, as determined by electron paramagnetic resonance spectroscopy and it was found to be much faster in terms of quenching ROS when compared to resveratrol and quercetin.Curcumin has also been demonstrated to induce antioxidant defences through increases in glutathione production via Nrf2 activation and induction of glutamate cysteine ligase transcription and interacting directly with superoxide anion and hydroxyl radical (Biswas et al., 2005).In addition, curcumin had an effective scavenging activity in various in vitro antioxidant assays, including DPPH radical, ABTS radical, DMPD radical, superoxide anion radical and hydrogen peroxide; ferric ions (Fe 3+ ) reducing power and ferrous ions (Fe 2+ ) chelating activities (Ak & Gülçin, 2008).This suggests that curcumin has multiple anti-inflammatory properties: as an oxygen radical scavenger and as an antioxidant through modulation of glutathione levels.
Tea polyphenols have been reported to be potent scavengers (more efficient than vitamin E and C) (Nanjo et al., 1996;Pannala et al., 1997) of free radicals such as singlet oxygen, superoxide anions, hydroxyl radicals, and peroxy radicals in a number of in vitro systems (Salah et al., 1995;Morel et al., 1999) and EGCG was found to be the strongest antioxidant among tea catechins (Khokhar & Magnusdottir, 2002).EGCG attenuated 3hydroxykynurenine, a potential neurotoxin in several neurodegenerative disorders, induced cell viability reduction and the increase in the concentration of ROS and caspase-3 activity in neuronal culture were also attenuated, presumably via its antioxidant activity (Jeong et al., 2004).In rat brain tissue, green tea and black tea extracts were shown to inhibit lipid peroxidation promoted by iron ascorbate in homogenates of brain mitochondrial membranes (Levites et al., 2002).A similar effect was also reported using brain synaptosomes, in which the four major polyphenol catechins of green tea were shown to inhibit iron-induced lipid peroxidation (Guo et al., 1996).The ability of green tea polyphenols and catechins, in particular, to chelate metal ions such as iron and copper may contribute to their antioxidant activity by inhibiting transition metal-catalyzed free radical formation.Furthermore, EGCG treatment inhibited the enzyme iNOS (Lin & Lin, 1997;Lin et al., 1999) in activated macrophages.On the other hand, EGCG was found to elevate the activity of two major antioxidant enzymes, superoxide dismutase (SOD) and catalase in mice striatum (Levites et al., 2001).Taken together, the inhibition of enzymes, whose activity may promote oxidative stress and an increase in antioxidant enzyme activities by tea polyphenols might have a beneficial significance to anti-inflammatory processes.Phenolic acids and aldehydes have been reported to exert antioxidant and antiinflammatory effects.Phenolic compounds can trap the free radicals directly or scavenge them through a series of coupled reactions with antioxidant enzymes.Caffeic acid has been shown to protect tissues from ROS-mediated oxidative stress and inhibit lipoxygenase activity resulting in suppressed lipid peroxidation (Yilmaz et al., 2004).It was demonstrated that administration of caffeic acid reduced oxidative stress by increasing antioxidant activities and decreasing oxidant status and lipid peroxidation in the intestine of rats in an experimental model of necrotizing enterocolitis (Tayman et al., 2011).Administration of caffeic acid has been also shown to completely block both the production of ROS from activated neutrophils and the XO system (Ma et al., 2006;Yildiz et al., 2009) and to protect intestinal tissues from ROS-mediated oxidative stress and reduce lipid peroxidation (Ek et al., 2008;Koltuksuz et al., 1999).Treatment of rats orally with caffeic acid resulted in a significant decrease in iron nitrilotriacetate-induced xanthine oxidase, lipid peroxidation, cglutamyl transpeptidase, and H 2 O 2 and there was a dose dependent and significant recovery of renal glutathione content and antioxidant enzymes (Rehman & Sultana, 2011).Caffeic acid has also been shown to be an inhibitor of the lipoxygenase enzyme in several experimental systems (Okutan et al., 2005).In addition, caffeic acid is an effective ABTS radical scavenging, DPPH radical scavenging, superoxide anion radical scavenging and it has total reducing power and metal chelating on ferrous ions activities (Gulcin, 2006).In wheat grain ferulic acid is the most abundant phenolic compounds and a correlation between changes in the plasma ferulic acid concentration and changes in the plasma antioxidant capacity was reported in a human ex vivo study (Mateo Anson et al., 2011).The antioxidant potential of ferulic acid can usually be attributed to its structural characteristic.Three distinctive motifs (3-methoxy and 4-hydroxy groups on the benzene ring, and the carboxylic acid group) of ferulic acid are responsible for its free radical scavenging capability.It has been also reported that ferulic acid or related ester derivatives inhibited the release of ROS and RNS via suppression of iNOS (Jiang et al., 2009) and COX-2 (Hirata et al., 2005, Ronchetti et al., 2009) in LPS-stimulated macrophages.Its reduced form, ferulaldehyde possesses the same structural characteristic and main molecular motifs as ferulic acid up to the aldehyde group.Ferulaldehyde was reported to inhibit LPS-induced iNOS expression and NO synthesis in murine macrophage-like RAW 264.7 cells (Kim et al., 1999) and to have a good antioxidant activity in about the same degree as ferulic acid (Nenadis et al., 2003).Furthermore, ferulaldehyde decreased free radical and nitrite production in a concentrationdependent manner in LPS plus interferon-gamma-treated primary mouse hepatocytes and in LPS-induced RAW 264.7 macrophage cells (Radnai et al., 2009;Tucsek et al., 2011).Taken together these studies have shown that polyphenols are more effective in inhibiting the oxidative damage than the conventional antioxidants and has also been shown to scavenge free radicals such as nitric oxide, lipid hydroperoxyl, hydroxyl, and superoxide anion radicals.Since reactive oxygen and nitrogen species have been implicated in the pathogenesis of various chronic and inflammatory conditions, polyphenols therefore have the potential to control these diseases through their potent antioxidant activity.

Inflammatory cytokines
The immune system produces cytokines and other humoral factors to protect the host when threatened by inflammatory agents, microbial invasion, or injury.The pathogenesis of sepsis is characterized by an overwhelming production of proinflammatory cytokines, such as tumor necrosis factor (TNF)-, interleukin (IL)-1 , IL-6, IL-8 and high mobility group box (HMGB)-1.In some cases these cytokines trigger a beneficial inflammatory response that restores normal homeostasis promoting local coagulation to control tissue damage.However, the overproduction of immunoregulatory mediators can be even more dangerous than the original stimulus, overcoming the normal regulation of the immune response and producing pathological inflammatory disorders (Dinarello, 1994;Hotchkiss & Karl, 2003;Tracey & Cerami, 1993;Tracey & Cerami, 1994;Van der Poll & Lowry, 1995).In severe sepsis the excessive production of proinflammatory cytokines causes capillary leakage, tissue injury, multiple organ failure, coma and death.TNF-, a polypeptide cytokine produced during infection, injury, or invasion, has proved pivotal in triggering the lethal effects of septic shock syndrome and other systemic manifestations of disease.If the infection spreads, however, excessive TNF-production and release into the circulation may be catastrophic and trigger a state of lethal shock via cardiovascular collapse.These toxic effects occur by direct action of TNF-on host cells and by the interaction with a cascade of other endogenous mediators including IL-1, IL-6 and interferon-gamma (Tracey & Cerami, 1993;Tracey & Cerami, 1994).Similar to TNF-and HMGB1, several proinflammatory cytokines and factors, such as IL-1 (Dinarello, 1994), IL-6, IL-8 (Calandra et al., 1990;Hotchkiss et al., 2000) macrophage migration inhibitory factor (Parrish et al., 2008) and lysophosphatidylcholine (Kabarowski et al., 2001), contribute to the pathogenesis and progression of sepsis.Sepsis is characterized by a surge of the pro-inflammatory cytokines TNF-and IL-1 at the early stage.However, as the disease progresses, this early stage coverts to the anti-inflammatory state, marked by decreased levels of TNF-and increased levels of IL-10 (Scumpia & Moldawer, 2005).The increased production of IL-10 in the late phase of sepsis is believed to contribute to 'immunosuppression'.In contrast with early cytokines, such as TNF-and IL-1, which are produced within minutes after infection, HMGB-1 is a late mediator of sepsis that might be a potential therapeutic target to treat 'established' sepsis.The effects on the balance between pro-and anti-inflammatory cytokine expressions have been shown to be specific for specific cytokines and influenced by the structures of polyphenols indicating the complex action exerted by these compounds.Since cytokine-based strategies against septic shock and severe sepsis have produced modest effects in clinical trials (Abraham et al., 2001;Fisher et al., 1994) novel therapeutic approaches are needed and polyphenols are perfect candidates for this role.The release of various cytokines after various stimuli from macrophages and lymphocytes, such as IL-6 (Feng et al., 2002;Zhong et al., 1999), IFN , IL-2, TNF-, and IL-12 (Gao et al., 2001), has been shown to be inhibited by resveratrol.Resveratrol was also reported to suppress the activity of T-and B-cells, and macrophages the major cell types responsible for producing cytokines by decreasing the production of proinflammatory proteins, (Sharma et al., 2007) and it was able to reduce TNF-, IL-1 , IL-6, and COX-2 gene expression and to reduce the secretion of IL-6 and PGE2 in TNF--stimulated adipocytes (Gonzales & Orlando, 2008).In an age-related diseases study, mice treated with resveratrol had decreased expression of the inflammatory markers TNF-, IL-1 , IL-6, intercellular adhesion molecule (ICAM)-1 and iNOS (Pearson et al., 2008).In a mouse model of chronic colitis, resveratrol treatment resulted in significant decreases in the inflammatory cytokines TNFand IL-1 , as well as COX-2 and iNOS activity and an increase in the anti-inflammatory, immune-regulatory cytokine, IL-10 (Sánchez-Fidalgo et al., 2010).Resveratrol administration following trauma-hemorrhage decreased IL-6 cytokine production and protected against lung injury and inflammation in rats (Wu et al., 2008).In a rodent model of LPS-induced airway inflammation resveratrol caused a dose-related inhibition of TNF-, IL-1 , MPO, and CINC-1 levels in the lung tissue (Birrell et al., 2005) and in a human study resveratrol inhibited the release of inflammatory cytokines IL-8 and granulocyte-macrophage colonystimulating factor from alveolar macrophages in chronic obstructive pulmonary disease (Culpitt et al., 2003).However, it is interesting to mention that many of the proinflammatory genes inhibited by resveratrol in several different vitro studies, are not impacted on in vivo models indicating the host and tissue specificity of this polyphenol (Birrell et al., 2005).Several in vitro studies using different cell lines have shown that quercetin is also capable of inhibiting LPS-induced cytokine production.For instance, quercetin inhibits LPSinduced TNF-production in macrophages (Manjeet & Ghosh, 1999) and LPS-induced IL-8 production in lung cells (Geraets et al., 2007).Moreover, in glial cells it was shown that quercetin can inhibit LPS-induced mRNA levels of two cytokines, i.e.TNF-and IL-1 (Bureau et al., 2008).It has already been shown that quercetin can inhibit the production as well as the gene expression of TNF-via modulation of NF-κB in human peripheral blood mononuclear cells (Nair et al., 2006).Results indicate that TNF-and IL-6 accumulations were significantly reduced by quercetin treatment, in murine RAW 264.7 macrophages treated with LPS (Jung & Sung, 2004).Cho and colleagues reported that in the same cell line pretreatment of quercetin inhibited iNOS mRNA, iNOS protein, NO production, TNF-, IL-1 and IL-6 (Cho et al., 2003).Quercetin has also been reported to inhibit IgE-mediated release of histamine, tryptase and production and gene expression of inflammatory cytokines, such as TNF-, IL-1 , IL-6 and IL-8 in phorbol 12-myristate 13acetate and calcium ionophore-stimulated cultured human mast cells (Kempuraj et al., 2005;Min et al., 2007).These results are consistent with studies reporting that quercetin inhibits NO and TNF-release from LPS-stimulated rat Kupffer cells (Kawada et al., 1998) and inhibits iNOS mRNA and NO production in LPS/IFN--activated macrophage cells (Kobuchi et al., 1997).
In 1995 it was demonstrated by Chan that curcumin inhibits LPS-induced production of TNF-and interleukin IL-1 in human monocytic macrophage cell line, Mono Mac 6 and it reduced the biological activity of TNF in L929 fibroblast lytic assay (Chan, 1995).Since this original work, several line of evidence appeared about curcumin's inhibitory effect on inflammatory cytokines.In LPS-stimulated BV2 microglia cells curcumin significantly inhibited the release of pro-inflammatory cytokines in a dose-dependent manner (Jin et al., 2007).Moreover, decreased expression of inflammatory cytokines such as IL-1 , IL-6, and TNF-was reported in different cancer cell lines (Cho et al., 2007).In other studies, curcumin inhibited the production of IL-8, MIP-1 , MCP-1, IL-1 and TNF-by PMA-or LPS-stimulated human monocytes and alveolar macrophages in a concentration-and a timedependent manner (Abe et al., 1999;Chan, 1995).The in vivo effects of curcumin on cytokine production are also demonstrated in different model systems.In two rat models of experimentally-induced pancreatitis, curcumin decreased inflammation by markedly inhibiting mRNA induction of IL-6, TNF-and iNOS in the pancreas (Gukovsky et al., 2003).Curcumin decreased the levels of TNF-and interleukin-6 in mouse plasma after endotoxin-induced hepatic dysfunction and oxidative stress (Kaur et al., 2006) and also inhibited the increase of both IL-1 and TNF-in a chronic model of inflammation in rats (Banerjee et al., 2003).Systemic pretreatment with curcumin abrogates the rise in circulating proinflammatory cytokines and body temperature (Lee et al., 2003) and prevents the onset of disseminated intravascular coagulation (Chen et al., 2007).The effects of EGCG on the inflammatory cytokine production are thought to be cell typedependent.EGCG was reported to inhibit TNF-and MIP-2 production from the RAW 264.7 cells treated with LPS (Yang et al., 1998).Okabe et al reported, that EGCG effectively prevented TNF-release in BALB/3T3 cells stimulated with okadaic acid (Okabe et al., 2001).EGCG, recapitulated HMGB1-inhibiting activities of green tea, and dose-dependently inhibited LPS-induced HMGB1 release in macrophage/monocytes cultures, partially attenuated LPS-induced TNF secretion.EGCG prevents HMGB1-mediated cytokine production-potentially by interfering with HMGB1-induced ligand/receptor clustering (Li et al., 2007).Experimental data suggest that EGCG selectively inhibits LPS-induced release of HMGB1, TNF, IL-6, IL-12, and chemokines, including MIP-1a, MIP-1c, MIP-2, RANTES, KC, MCP1 and CXCL16.EGCG did not affect circulating levels of TNF at late stage of sepsis, but specifically attenuated systemic accumulation of HMGB1, as well as IL-6 and KC-two most reliable surrogate markers of lethal sepsis (Osuchowski et al., 2006;Heuer et al., 2004).Effects of EGCG on the production of IL-8, human homolog of murine CXC-chemokines, have also been reported in selected cell lines.It was shown that EGCG inhibited IL-8 production in HMC-1 cells (Shin et al., 2007), A549 bronchial epithelial cells (Kim et al., 2006), HT29, T84 (Porath et al., 2005) and gastrointestinal epithelial cell line Caco-2 cells (Netsch et al., 2006).In vivo, EGCG attenuated the production of TNF-and MIP-2 in the lungs of mice administered with LPS intratracheally (Bae et al., 2010).Li et al provided both evidence of a lifesaving potential and a mechanism of action for an EGCG therapeutic regimen in mice subjected to cecal ligation and puncture (CLP).EGCG reduced the mortality rate and concentrations of IL-6, keratinocyte-derived chemokine (a mouse IL-8 homologue), and HMGB-1, but not those of TNF-, were significantly reduced.Exposure of monocytes and macrophages to EGCG in vitro reduced HMGB-1 release following LPS stimulation and IL-6 production and EGCG inhibited HMGB-1 aggregation on the macrophage cell surface.HMGB-1 antagonism was evident even when EGCG treatment of stimulated cells was delayed by 6 h.Potential therapeutic uses of EGCG is further strengthened by the fact that EGCG treatment is operative even when it is initiated after the onset of full-blown sepsis (Li et al., 2007).Phenolic acids are clinically important inhibitors of inflammatory cytokine production in vitro and in vivo.Caffeic acid phenethyl ester (CAPE) is a potent inhibitor of early and late events in T-cell receptor-mediated T-cell activation.Moreover, it was found that CAPE specifically inhibited both IL-2 gene transcription and IL-2 synthesis in stimulated T-cells (Márquez et al., 2004).In activated human whole blood cultures, caffeic acid decreased the production of IL-1 without affecting IL-6 concentration (Miles et al., 2005) suggesting the specificity of this phenolic compound on interleukin production.In CLP-induced sepsis and lung injury model in rats, similarly to EGCG, CAPE was found to decrease IL-1, IL-6, IL-10, and TNF-levels of blood samples even when it was administered after the onset of sepsis.(Fidan et al., 2007).In another in vivo study, CAPE rescued C57BL/6 mice from lethal LPSinduced septic shock, while decreasing serum levels of TNF-and IL-1 (Jung et al., 2008).The other large family of phenolic acids: ferulic acid and its derivatives were also attributed as potent inhibitors of inflammatory mediators.Indeed, ferulic acid decreased the levels of inflammatory cytokines, e.g., TNF- (Han et al., 2007) in LPS-stimulated macrophages and lowered pro-:anti-inflammatory cytokine ratios (IL-6:IL-10 and IL-1 :IL-10) in the ex vivo LPS-stimulated blood (Mateo Anson et al., 2011).Ferulaldehyde decreased the levels of early pro-inflammatory cytokines such as TNF-, IL-1β and increased the anti-inflammatory IL-10 in the sera of the LPS-treated mice (Radnai et al., 2009;Tucsek et al, 2011) supporting further the importance of these compounds as potential anti-inflammatory agents.

Signaling pathways
Polyphenols can work as modifiers of signal transduction pathways to elicit their beneficial effects.The anti-inflammatory effect of these natural compounds based on the modulation of pro-inflammatory gene expression such as cyclooxygenase, lipoxygenase, nitric oxide synthases and several inflammatory cytokines, mainly by acting through NF-κB and MAPK signalling (Yoon & Baek, 2005).MAPKs and NF-κB have important activities as mediators of cellular responses to extracellular signals.Some of the MAPKs are important to mammalian cells include extracellular signal regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 and are thought to play an important role in the regulation of pro-inflammatory molecules on cellular responses (Azzolina et al., 2003;Baldassare et al., 1999).Because of their essential role in intracellular signalling network, MAPK pathways and the connected transcription factors are appropriate targets for pharmacological treatment of inflammatory disorders (Lewis et al., 1998) and polyphenols are extensively investigated as possible regulatory molecules in this process.Inflammation involves a cross-talk between several transcription factors, kinases and intracellular and intercellular cytokines involving NF-κB, MAPKs, PKC, PI-3-kinases etc.There has been considerable amount of evidence confirming that resveratrol takes active part in the modulation of these cell signalling molecules.Although resveratrol has been shown to target various intracellular signaling molecules in cultured cell lines, the molecular mechanisms underlying anti-inflammatory activity of resveratrol in vivo remain largely unresolved.Since NF-κB activation is critically linked to inflammatory responses and other chronic diseases associated with ROS and RNS production (Karin et al., 2004) the effect of resveratrol on NF-κB has been studied intensively in the last decade (Holmes-McNary & Baldwin, 2000;Manna et al., 2000) Indeed, resveratrol is a potent inhibitor of wide variety of inflammatory agents-induced activation of NF-κB, and this inhibition is not cell type specific.Some molecular targets of resveratrol are identified in toll-like receptor (TLR)mediated signalling pathways.It has been reported that resveratrol acts on NF-κB by the inhibition of IκB kinase, leading to the inhibition of LPS-induced IκB degradation, which results in the prevention of translocation of NF-κB into the nucleus (Holmes- McNary & Baldwin, 2000).It was suggested that IκB degradation induced by TLR4-TRIF pathway is mediated through the interaction between TRIF and tumor necrosis factor receptorassociated factor 6 (TRAF6) because TRAF6 was shown to associate with the N-terminal part of TRIF (Sato et al., 2003;Jiang et al., 2004).Resveratrol was also shown to inhibit MyD88independent signaling pathways and target expression (Youn et al., 2005).One of the major target molecules subjected to NF-κB-driven transactivation is cyclooxygenase-2, which is the enzyme of the rate-limiting step of the pathway that produces mediators of inflammation.Anti-inflammatory activity of resveratrol and some of its proposed mechanisms of action were attributed mostly in inhibition of COX activity (Das & Das, 2007;Kundu et al., 2006).Resveratrol also interferes with the pro-inflammatory signalling of thrombin resulting in the inhibition of adenosine nucleotide secretion from activated platelets and decreased neutrophil functions via inhibition of PAP and P2-receptor signalling through MAPK and cJun and JNK (Kaneider et al., 2004).However there is amply evidence about the modulatory effect of resveratrol on MAPK pathways, the data are so cell type and experimental system specific that it is hard to conclude and draw a coherent picture.It has already been shown that quercetin can inhibit the production as well as the gene expression of TNF-via modulation of NF-κB in human peripheral blood mononuclear cells (Nair et al., 2006).A possible mechanism behind this modulation was reported to be the inhibition of the degradation of the inhibitory part (IκB ) of this transcription factor (Peet & Li, 1999).In addition, quercetin treatment inhibited NF-κB activation through stabilization of the NF-κB/IκB complex leading to inhibition of IκB degradation and proinflammatory cytokines and NO/iNOS expression in RAW 264.7 macrophages (Cho et al., 2003).NF-κB/DNA binding activity induced by PMA and calcium ionophore was also markedly suppressed by quercetin without altering the binding activity of AP-1 in human mast cells (Min et al., 2007).In the same model quercetin attenuated the PMA and A23187-induced phosphorylation of p38 MAPK but not JNK or ERK.However, in an LPS-induced macrophage model quercetin strongly reduced activation of phosphorylated ERK kinase and p38 MAP kinase but not JNK MAP kinase (Cho et al., 2003).Moreover, TNF-secretion in LPS-stimulated RAW macrophages was also shown to be inhibited by quercetin through interfering with the phosphorylation and activation of JNK and its downstream substrates c-Jun and ATF-2, and ERK1/2 and p38 MAPK (Wadsworth et al., 2001).Although NF-κB inhibition by quercetin in general is supported by several line of evidence the signalling events leading to the blockade of this transcription factor differ from cell to cell and model to model.Curcumin has been shown to suppress the activation of NF-κB induced by various proinflammatory stimuli, presumably through inhibition of IKK kinase activity or DNA binding of p65.It is likely that curcumin also interferes with NF-κB activation at other points along this pathway, such as downstream to the various receptors that signal to this transcription factor.However, inhibition of IKK by curcumin plays a central role in this mechanism since the lack of phosphorylation of NF-κB suppresses binding of NF-κB to DNA sequences and as a consequence expression of genes described in vitro in inflammatory and vascular cells stimulated with LPS, staphylococcal enterotoxin A, TNF-, or IL-1 , and in vivo in models of inflammatory diseases (Chan, 1995;Hatcher et al., 2008;Jobin et al., 1999;Joe et al., 2004;Pan et al., 2000;Singh & Aggarwal, 1995).Regarding the LPS/NF-κB pathway, curcumin competes in vitro with LPS for binding to the TLR-4/myeloid differentiation factor-2 complex (Gradisar et al., 2007) and prevents TLR-4 homodimerization (Youn et al., 2006), events necessary for MyD88-dependent signalling.Further studies indicated that curcumin which contains unsaturated carbonyl group, but not resveratrol (with no unsaturated carbonyl group), inhibits TLR4 activation by interfering with receptor dimerization.This conclusion was further supported by the finding that curcumin inhibits ligand-independent dimerization of constitutively active TLR4.Inhibition of receptor dimerization of TLR4 suggests that curcumin could modify the free sulfhydryl groups of cysteine residues in TLR4, leading to interference of disulfide formation.Curcumin also induces intracellular heat-shock protein 70 (Dunsmore et al., 2001;Shen et al., 2007), a protein chaperone that protects IKK from proteasomal degradation, thus inhibiting NF-κB translocation to the nucleus.Downstream to NF-κB curcumin was found to significantly down-regulate the TNF--induced increase in MMP-13 mRNA and protein expression in primary human chondrocytes and in human bone chondrosarcoma cells by a mechanism involving the inhibition of c-Jun and JNK (Liacini et al., 2003).Interestingly, a study on experimental colitis in which NF-κB-dependent gene expression, inflammation, and tissue injury were significantly attenuated by curcumin an increase in the intestinal level of PPAR-was observed (Zhang et al., 2006) indicating a strong connection between the two transcription factor.In conclusion, inactivation of the NF-κB pathway by curcumin appears to occur at multiple levels and to ameliorate the various stages of sepsis-associated inflammation.EGCG is known to inhibit NF-κB activation induced by many pro-inflammatory stimuli such as UV, LPS, TNF-and IL-1β (Barthelman et al., 1998;Yang et al., 1998;Yang et al., 2001;Wheeler et al., 2004;) resulting in the decrease in the expression of inflammatory gene products including lipoxygenase (Yang & Koo, 2000), COX (Soriani et al., 1998), NOS (Chan et al., 1997;Lin & Lin, 1997), and TNF- (Yang et al., 1998).It has been shown that the activation of NF-κB was suppressed by EGCG possibly mediated through the suppression of the kinase activity of IκB kinase in macrophages and the intestinal epithelial cell line (IEC-6) (Pan et al., 2000;Yang et al., 2001).EGCG was shown to inhibit the activity of IKKβ which is the key kinase in the canonical pathway for NF-κB activation in MyD88-dependent pathway of TLRs.Moreover, EGCG inhibited the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C], or the overexpression of TRIF.The inhibition of IRF3 activation by EGCG was mediated through the suppression of the kinase activity of TANK-binding kinase 1 in TRIF-dependent signalling pathways of TLR3 and TLR4 (Youn et al., 2006).These data indicate that green tea flavonoids, without the structural motif conferring Michael addition, did not inhibit TLR4 dimerization however; they can modulate both MyD88-and TRIF-dependent signalling pathways of TLRs and subsequent inflammatory target gene expression.In addition, Abboud e t a l d e m o n s t r a t e d t h a t E G C G m a y b e beneficial in colitis, which was induced by rectal administration of trinitrobenzene sulfonic acid in C57/BL6 mice, through selective immunomodulatory effects, which may be mediated, at least in part, by inhibition of NF-κB and AP-1 (Abboud et al., 2008).The effects of EGCG on the cellular kinase cascade are rather diversified.In RAW 264.7 cells stimulated with LPS EGCG inhibited the production of TNF-and MIP-2, and attenuated phosphorylation levels of ERK1/2 and JNK, but not p38.Also, EGCG attenuated the production of TNF-and MIP-2, and the phosphorylation of ERK1/2 and JNK in the lungs of mice administered with LPS intratracheally (Bae et al., 2010).Recently, Yun, et al. showed that EGCG inhibited TNF-induced phosphorylation of MAPKs family, such as ERK1/2, p38 and JNK in synovial fibroblast (Yun et al., 2008), while others showed that EGCG inhibited LPS-induced activation of p38, but augmented phosphorylation of ERK1/2 in J774.1 macrophage cells (Ichikawa et al., 2004).Inhibition of protein tyrosine kinase activity, reducing c-jun mRNA expression and inhibition of JNK1 activation by EGCG were reported by Yokozawa et al. (Yokozawa & Dong, 1997).Moreover, green tea polyphenols inhibit p44/42 MAP kinase expression (Lu et al., 1998) and induce the death of smooth muscle cells in a p53-and NF-κB-dependent manner (Ouyang et al., 2004).Taken together, the effects of EGCG on MAPKs phosphorylation are thought to be highly cell type-dependent.It has been demonstrated that CAPE is a potent and specific inhibitor of NF-κB activation (Natarajan et al., 1996).CAPE inhibits LPS-induced nitric oxide and prostaglandin E2 production in a concentration-dependent manner and inhibits iNOS and COX-2 in RAW 264.7 cells, without significant cytotoxicity.CAPE treatment significantly reduced NF-κB translocation and DNA-binding in LPS-stimulated RAW264.7 cells.This effect was mediated through the inhibition of the degradation of IκB and by inhibition of both p38 mitogenactivated protein kinase and ERK phosphorylation, at least in part by inhibiting the generation of reactive oxygen species (Jung et al., 2008).This biological activity of CAPE could also explain by the finding that this phenolic compound prevented the binding to DNA of the p50/p65 NF-κB in vitro and in vivo (Natarajan et al., 1996).In addition to NF-κB, CAPE also targets the nuclear factor of activated T-cells (NFAT) signaling pathway that is known to play a critical role in the immune response.CAPE is a potent inhibitor of the NFAT pathway, and the results of Marquez et al. suggest that the calcineurin phosphatase can represent one of the major targets for CAPE, since this compound inhibits NFAT dephosphorylation and nuclear binding to DNA (Márquez et al., 2004).Other phenolic acids (gallic, caffeic, protocatechic, paracoumaric, sinapic, and ferulic acids) were reported to inhibit the activity of AP-1 transcription factor on MCF-7 cells after stimulation by phorbol 12-myristate 13-acetate and NF-κB activity on LPS/IFN-gamma-stimulated RAW 264.7 cells (Chao et al., 2010;Maggi-Capeyron et al., 2001).Similarly to phenolic acids ferulaldehyde also inhibited the activation and nuclear translocation of NF-κB in the liver of LPS-treated mice.In the same model ferulaldehyde significantly prevented the activation of JNK and Akt, but failed to attenuate LPS-induced activation of ERK1/2 (Radnai et al., 2009).Suppressing both LPS-induced JNK and Akt activation, ferulaldehyde inhibited the most important pathways leading to NF-κB activation, namely the LPS/TLR4/JNK and LPS/ PI-3K/Akt pathways.The observation that FA did not show any effect on the LPS-induced activation of ERK1/2 and p38 MAP kinase pathways indicates that FA's inhibitory target(s) is necessarily downstream of the TLR4 receptors.However, unlike the in vivo model, ferulaldehyde attenuated LPS-induced activation of all three MAP kinases suggesting a uniform regulation of MAPK activation in LPS-stimulated macrophages.These results may arise from the differences of the inflammatory models used, and cell-and tissue-specificity of the LPS-induced processes (Veres et al., 2004).MAP kinase phosphatases (MKP), responsible for dephosphorylation and deactivation of MAP kinases, have a central role in the restraint of innate immune response and the prevention of septic shock syndrome during pathogenic microbial infection (Zhao et al., 2006).It is known that MAPK activation is followed by increased MKP-1 expression probably as a compensatory regulatory mechanism.In line with this evidence Tucsek et al found in an in vitro experimental model that ferulaldehyde shifted increased expression of MKP-1 forward in time which in turn attenuated activation of MAP kinases (Tucsek et al., 2011).

Conclusion
A number of different approaches have been investigated to try to treat and/or prevent septic shock and sepsis.Despite advances in elucidating its pathophysiology, severe sepsis remains a leading cause of death in the critically ill.It has been widely shown that many plant-derived compounds present anti-inflammatory effects.Polyphenols and their degradation products can exert their anti-inflammatory properties at multiple levels, through the modulation of MAPK, Akt and NF-κB signalling pathways, inhibition the production of inflammatory cytokines and chemokines, suppressing the activity of COX and iNOS and decreasing the production of ROS/RNS.Importantly, efficacy is maintained in some cases even when treatment is initiated hours after the onset of sepsis.These agents would be useful not only for the treatment of inflammatory disorders, but also for the control of some other diseases which present an inflammatory origin.However, the majority of the anti-inflammatory studies on plant-derived compounds have been carried out in vitro the polyphenols reviewed in this chapter appear to be safe and to exert anti-inflammatory effects in in vivo studies and even in humans.Thus, polyphenols and their degradation products represent a new, effective family of anti-inflammatory drugs that may help to prevent and treat sepsis.