**1.2 Basic sources of free radicals**

*Strawberry - Pre- and Post-Harvest Management Techniques for Higher Fruit Quality*

fibrosis and consequently cirrhosis [6]. For this reason, using molecules with antioxidant properties has been proposed as a treatment for not only fibrosis but also oxidative stress-related cirrhosis. Liver diseases are considered a major medical problem worldwide. There are known to be a large number of liver diseases caused by different insults. Furthermore, the disease type depends on lifestyle factors. For example, the main causes of liver diseases are reported to be viral and parasitic infections in regions like Africa and Asia. For Europe and America, alcohol consumption is thought to be the most important cause of this disease. However, viral hepatitis has showed an increase in recent times in most of the countries [7]. Lifestyle and unhealthy diet is the leading cause of liver diseases in almost all western countries. Until today, no medication is approved for the treatment of this disease; however, improving diet habits and physical exercise works if the disease is not accompanied by inflammation. On the other hand, biologically active food compounds that regulate gene expressions in lipogenesis, fibrosis, and inflammation serve as good therapeutic means to ameliorate these pathological states observed in liver [1] (**Figure 1**).

Oxidative stress is recognized as a disproportion between the production of free radicals (FR) and the antioxidant defenses [8]. Increased levels of prooxidants result in damage to the cell in terms of lipid peroxidation as well as oxidative DNA damage

*Molecular mechanisms explaining the hepatoprotective effect of food bioactives. Development of NAFLD/ NASH is induced by different risk factors, such as Western-type diet, physical inactivity, and genetic predisposition. In the presence of obesity and IR, there is an increased flux of FFAs to the liver. These FFAs are stored as TG in lipid droplets leading to hepatic fat accumulation or undergo β-oxidation increasing oxidative stress and the inflammatory pathway. The damaged hepatocyte leads to a further increase of inflammatory signaling (IL-1, TNFa, IL-6) and the recruitment of circulating and residual macrophages (KCs). All of these mechanisms can directly induce the activation of HSCs, the major cell type involved in extracellular matrix deposition and liver fibrosis. The bioactive compounds may exert beneficial effects on NAFLD development and progression by inhibiting lipogenesis, β-oxidation of FFAs, inflammation, and HSCs activation. In the cartoon, we have listed the food bioactives indicating the putative mechanisms by which they may improve liver* 

**124**

*damage in NAFLD [1].*

**Figure 1.**

**1.1 Oxidative stress**

Cells produce FR as a result of metabolic events; however, this is not the only source that can cause oxidative stress in body. The pollutants in the environment such as toxic chemicals as well as radiation cause a significant increase in amount of FR, ROS, and reactive nitrogen species (RNS) [10]. In the body, variety of different cell types and chemical reactions produce ROS, the most important metabolism is the cytochrome P450 metabolism and mitochondria-catalyzed electron transport reactions. Most of the inflammatory conditions are also responsible from ROS production, and important cell types in these processes are neutrophils, eosinophils,

#### **Figure 2.**

*Mechanisms of enhanced ROS production during hepatocyte damage. Ethanol metabolism promotes strong ROS production in the ER by the inducible CYP. It impairs GSH import in the mitochondria, preventing ROS removal. It also impairs B-oxidation promoting lipid accumulation. ETOH induces lipid-raft clustering and increases iron uptake, promoting Fe2+ leakage from lysosomes and increased Fe2+ loads in mitochondria and ER, resulting in ROS production. Ethanol also reduced the autophagic removal of damaged cellular components. Viral infection challenges the ER protein folding process leading to ROS production and Ca2+ leakage in the cytosol and mitochondria. Increased MAMs formation promotes Ca2+ efflux from ER into mitochondria, increasing mitochondrial ROS production [15].*

and macrophages [16, 17]. The chief molecule responsible for the reduction of oxygen in mitochondria is ubisemiquinone. Mitochondria is such an important organelle to produce ROS and hydrogen peroxide (H2O2) as it produces 2–3 nmol of superoxide/ min per mg of protein, [17]. Different tissues of mammals and different species of mammals have an enzyme called xanthine oxidase, an enzyme belonging to molybdenum, iron-sulfur, flavin hydroxylases that play an important role in the hydroxylation of purines by the oxidation of hypoxanthine to xanthine. Resultant xanthine then oxidized to uric acid. Oxygen reduction takes place in both of these reactions and the first one produces O2 <sup>−</sup>, while the second one produces H2O2 [16]. Inflammation serves as another source of ROS generation. During inflammation, activated macrophages increase their oxygen uptake, and this process results in production of O2 <sup>−</sup>, nitric oxide (NO), and H2O2 [18]. Another mechanism of O2 <sup>−</sup> production during inflammation is by neutrophils; the enzyme nicotine adenine dinucleotide phosphate [NAD(P) H] oxidase generates O2 <sup>−</sup> that is used to destroy bacteria and this nonphagocytic NAD(P)H oxidases produce O2 <sup>−</sup> in a range of 1–10% [19]. Cytochrome P450 (CYP) enzymes are also important in the production of ROS by the breakdown and/or uncoupling of the P450 catalytic cycle. Hyperoxia would trigger 80% of the H2O2 synthesis by microsomes, and under normoxic conditions, peroxisomes produce H2O2 but not O2 <sup>−</sup>, and most of the peroxisomal H2O2 production takes place in liver [16]. Arginine is reduced to citrulline in a five-electron oxidative reaction by nitric oxide synthases (NOSs) and this reaction gives rise to NO. Immune cells can also produce NO in the oxidative burst during inflammation. NO can react with oxygen and water in an extracellular environment in order to form nitrate and nitrite anions. Also, the NO and O2 <sup>−</sup> can react together and cause a more reactive FR called peroxynitrite anion (ONOO<sup>−</sup>) which can cause lipid peroxidation and fragmentation of DNA [20].

## **1.3 Antioxidants**

Antioxidants are molecules that can help prevent or delay oxidation of an oxidizable substrate when in low concentrations and they have a high affinity to FR [21]. Antioxidants play an important role to maintain health of the organism by scavenging FR by donating electrons to it. This reduces the reactivity of FR and helps maintain prooxidant/antioxidant balance in cell. A lot of different molecules that have antioxidant activity have been identified. Different natural compounds have so far been studied extensively especially in liver diseases (**Table 1**).

## **1.4 Curcumin**

Curcumin, diferuloylmethane or 1,7-bis (4-hydroxy-3-methoxyphenyl)1,6 hepadieno-3,5-dione is obtained from the rhizomes of *Curcuma longa* (turmeric). Curcumin has many pharmacological properties as it is a strong antioxidant, antifibrogenic, anti-inflammatory, antimicrobial, and anticarcinogenic agent and it also aids in a wound healing [22]. The Food and Drug Administration (FDA) has classified turmeric as a safe substance and toxicity assays done on animals have shown curcumin to be safe even when used in high doses. On the other hand, prolonged high-dose intake of turmeric has been associated with incidences of hepatotoxicity in mice and rats [3]. Curcumin is known to have low bioavailability when administered orally. Arcaro et al. [23] used piperine (inhibitor of hepatic and intestinal absorption) together with curcumin. Even in the presence of piperine, antidiabetic and antioxidant activity of curcumin was not altered. But when higher dose of piperine (40 mg/kg) was used, the beneficial effects of curcumin vanished. On the other hand, Sehgal et al. [24] showed the effect of piperine on curcumin in benzo(a)pyrene toxicity in the liver. They found that pretreatment with 100 mg/kg of curcumin protects against

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*Dietary Antioxidants in Experimental Models of Liver Diseases*

**Antioxidant Main clinical effects Clinical relevance**

Curcumin Antioxidant No studies available in human hepatic disorders

Resveratrol Antioxidant Current data are conflicting, so more clinical studies are needed Anti-inflammatory

Coffee Antinecrotic Inverse relationship between coffee-cirrhosis

Quercetin Chelation of transition metal ions No studies available in human hepatic disorders

Silymarin Antioxidant Silymarin has been shown to be effective, but it

Naringenin Antioxidant No studies available in human hepatic disorders

survival rates of patients with cirrhosis Antifibrotic

and prospective clinical trials are necessary Antifibrotic

has been demonstrated, but more basic research

is necessary to do more clinical trials focused on

*DOI: http://dx.doi.org/10.5772/intechopen.83485*

Antifibrotic Anti-inflammatory Antimicrobial Wound healing Anticarcinogenic

Anticarcinogenic Lipid modulation Antifibrotic

Antioxidant Anticholestatic Chemoprotective

Anticarcinogenic Cardioprotective Bacteriostatic Antioxidant Antifibrotic Anti-inflammatory Antiapoptotic Antiaggregatory Vasodilating

Anti-inflammatory Anticarcinogenic Immunomodulation

Hypocholesterolemic Anti-estrogenic Hypolipidemic Antihypertensive Anti-inflammatory Antifibrotic Anticarcinogenic Antiatherogenic


*Strawberry - Pre- and Post-Harvest Management Techniques for Higher Fruit Quality*

first one produces O2

H] oxidase generates O2

but not O2

NO and O2

**1.3 Antioxidants**

**1.4 Curcumin**

NAD(P)H oxidases produce O2

and macrophages [16, 17]. The chief molecule responsible for the reduction of oxygen in mitochondria is ubisemiquinone. Mitochondria is such an important organelle to produce ROS and hydrogen peroxide (H2O2) as it produces 2–3 nmol of superoxide/ min per mg of protein, [17]. Different tissues of mammals and different species of mammals have an enzyme called xanthine oxidase, an enzyme belonging to molybdenum, iron-sulfur, flavin hydroxylases that play an important role in the hydroxylation of purines by the oxidation of hypoxanthine to xanthine. Resultant xanthine then oxidized to uric acid. Oxygen reduction takes place in both of these reactions and the

as another source of ROS generation. During inflammation, activated macrophages

tion is by neutrophils; the enzyme nicotine adenine dinucleotide phosphate [NAD(P)

<sup>−</sup>, and most of the peroxisomal H2O2 production takes place in liver [16].

<sup>−</sup> can react together and cause a more reactive FR called peroxynitrite anion (ONOO<sup>−</sup>) which can cause lipid peroxidation and fragmentation of DNA [20].

Antioxidants are molecules that can help prevent or delay oxidation of an oxidizable substrate when in low concentrations and they have a high affinity to FR [21]. Antioxidants play an important role to maintain health of the organism by scavenging FR by donating electrons to it. This reduces the reactivity of FR and helps maintain prooxidant/antioxidant balance in cell. A lot of different molecules that have antioxidant activity have been identified. Different natural compounds have so

Curcumin, diferuloylmethane or 1,7-bis (4-hydroxy-3-methoxyphenyl)1,6 hepadieno-3,5-dione is obtained from the rhizomes of *Curcuma longa* (turmeric). Curcumin has many pharmacological properties as it is a strong antioxidant, antifibrogenic, anti-inflammatory, antimicrobial, and anticarcinogenic agent and it also aids in a wound healing [22]. The Food and Drug Administration (FDA) has classified turmeric as a safe substance and toxicity assays done on animals have shown curcumin to be safe even when used in high doses. On the other hand, prolonged high-dose intake of turmeric has been associated with incidences of hepatotoxicity in mice and rats [3]. Curcumin is known to have low bioavailability when administered orally. Arcaro et al. [23] used piperine (inhibitor of hepatic and intestinal absorption) together with curcumin. Even in the presence of piperine, antidiabetic and antioxidant activity of curcumin was not altered. But when higher dose of piperine (40 mg/kg) was used, the beneficial effects of curcumin vanished. On the other hand, Sehgal et al. [24] showed the effect of piperine on curcumin in benzo(a)pyrene toxicity in the liver. They found that pretreatment with 100 mg/kg of curcumin protects against

far been studied extensively especially in liver diseases (**Table 1**).

enzymes are also important in the production of ROS by the breakdown and/or uncoupling of the P450 catalytic cycle. Hyperoxia would trigger 80% of the H2O2 synthesis by microsomes, and under normoxic conditions, peroxisomes produce H2O2

Arginine is reduced to citrulline in a five-electron oxidative reaction by nitric oxide synthases (NOSs) and this reaction gives rise to NO. Immune cells can also produce NO in the oxidative burst during inflammation. NO can react with oxygen and water in an extracellular environment in order to form nitrate and nitrite anions. Also, the

increase their oxygen uptake, and this process results in production of O2

oxide (NO), and H2O2 [18]. Another mechanism of O2

<sup>−</sup>, while the second one produces H2O2 [16]. Inflammation serves

<sup>−</sup> that is used to destroy bacteria and this nonphagocytic

<sup>−</sup> in a range of 1–10% [19]. Cytochrome P450 (CYP)

<sup>−</sup>, nitric

<sup>−</sup> production during inflamma-

**126**


#### **Table 1.**

*Nutritional antioxidants.*

a single dose of benzo(a)pyrene; and at this dose, coadministration of piperine had a much better effect than did curcumin alone showing enhancer activity of piperine. In acute and chronic liver injury, curcumin has been shown to have hepatoprotective effects [25]. In 2007, Reyes-Gordillo et al. [26] showed that curcumin is able to inhibit the release of TNF-α, IL-1B, and IL-6. Additionally, curcumin reduces carbon tetrachloride (CC14)-mediated oxidative stress inactivating the nuclear factor-kB (NF-kB) pathway. Moreover, curcumin's hepatoprotective effect takes place by its interactions with Fe3+ and Cu2+. A study by Jiao et al. [27] suggested that curcumin could serve as an iron chelator since transferrin receptor 1 and iron regulatory proteins, indicators of iron depletion, showed an increase with curcumin administration. Charoensuk et al. [28] have indicated that curcumin increases antioxidant capacity of cells by increasing mRNA and protein levels of factors and enzymes such as nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), glutamate cysteine ligase (GCL), transcription factor-3, peroxiredoxin 3 (Prdx3), and Prdx6. Curcumin also increases the activity of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) activity [29, 30]. Curcumin also interacts with enzymes or genes that are important in liver cirrhosis. Hassan et al. [31] showed that curcumin modulates miRNA 199 and 200 which are associated with liver fibrosis in CC14-induced experimental fibrosis model and that curcumin reduced these miRNAs levels close to their basal levels. Finally, in alcohol-induced liver damage, curcumin inhibits the activity of cytochrome P450 2E1 (Cyp2e1) and also its protein levels [32].

#### **1.5 Resveratrol**

The phytoalexin resveratrol (3,5,4′-trans-trihydroxystilbene) is a polyphenol mostly found in red grapes, red wine, peanuts, and berries [33]. Resveratrol has effects on lipid metabolism, and it also has antioxidant, anti-inflammatory,

**129**

**1.6 Coffee**

*Dietary Antioxidants in Experimental Models of Liver Diseases*

anticarcinogenic, and antifibrogenic properties [34]. The rate of absorption of resveratrol is about 75% following an oral administration [35]. Resveratrol is metabolized to resveratrol sulfate, and in its low concentrations, it is converted into resveratrol glucuronide [36] by enzymes glucuronosyltransferase (UGT) or sulfotransferase (ST) [37]. In 2007, Chávez et al. [34] showed that under CCl4, resveratrol decreased cytokine TGF-β levels and prevented hepatic fibrosis. It also inhibited NF-kB translocation to the nucleus. Resveratrol, as an antioxidant, has protective effects against ethanol-induced lipid peroxidation, toxicity by acetaminophen (APAP), and oxidative stress in animal models of cholestasis [38]. Important player in resveratrol's antioxidant activity is suggested to be run by the OH groups [7]. Blocking OH group methylation showed that resveratrol and trimethylated resveratrol provide some degree of protection, but the latter one has a better protective effect [39]. Another hepatoprotection mechanism of resveratrol comes from its ability to activate genes related to antioxidant system or from its ability to inhibit enzymes. A study by Cheng et al. [40] suggested that resveratrol could activate extracellular signal-regulated kinase (ERK) signaling pathway, which may, in turn, enhance the activation and translocation of Nrf2 to the nucleus, thus increasing the expression of HO-1 and glyoxalase. Price et al. [41] found that resveratrol activates AMP-activated protein kinase (AMPK) and increased nicotinamide adenine dinucleotide (NAD) levels in mice. Zhu et al. [42] have also shown that, in mice, administration of resveratrol increased the antioxidant system (SOD, GPx, and GSH) and also the levels of SIRTI and p-AMPK were upregulated in liver. Resveratrol has also been shown to inhibit the activity of Cyp2e1 in microsomes of rat liver [43]. Resveratrol also inhibited the activity of P450 isoform APAP-induced liver injury model [44] and activity of Cyp2e1 was also inhibited in diethylnitrosamine (DEN)-induced hepatocarcinogenesis model [45]. The only clinical study that was performed to determine the resveratrol hepatoprotective effect demonstrated that a 500 mg resveratrol dose administrated for 12 weeks caused a significant reduction in inflammatory cytokines, serum cytokeratin-18, NF-kB activation, liver alanine aminotransferase (ALT), and hepatic steatosis when compared to the placebo group in patients with nonalcoholic fatty liver disease (NAFLD) [46].

Coffee is a mixture of several different molecules such as carbohydrates, vitamins, lipids, nitrogenous molecules, alkaloids, and phenolic compounds [47]. Caffeine, diterpene alcohols (cafestol and kahweol), and chlorogenic acid are the three major compounds found in coffee [48]. Coffee consumption has been linked to the reduction of several chronic diseases [49], probably due to the pharmacological properties that have antinecrotic, antifibrotic, anticholestatic, chemoprotective, and antioxidant functions [50]. Caffeine is the best-known active component of coffee, which is absorbed very rapidly once it has been taken orally (5 min), reaching its peak blood levels after 30 min. When consumed in high amounts, it may produce some side effects. Recommendations from Health Canada in 2013 demanded that the daily caffeine intake for children should not exceed 2.5 mg/kg of the body weight. What is more, tachycardia and arrhythmia typically arise when more than 200 mg of caffeine are ingested [51]. Smith et al. [52] reported in 2002 that the intake of 300 mg of caffeine resulted in a rise in anxiety and tension. Caffeine gets metabolized in the liver. The principal metabolite of caffeine is paraxanthine [53]. An important property of caffeine is that it can easily pass through the blood-brain barrier [54]. Coffee-cirrhosis relationship was shown by Klatsky et al. for the first time [55]. The study showed that the odds ratio for liver cirrhosis tend to decrease from 1.0 for people abstaining from coffee to 0.47, 0.23, 0.21, and 0.16 for 1, 2, 3, or 4 cups of coffee daily, respectively. Although coffee is generally beneficial to the liver, this study failed to show a causative

*DOI: http://dx.doi.org/10.5772/intechopen.83485*

#### *Dietary Antioxidants in Experimental Models of Liver Diseases DOI: http://dx.doi.org/10.5772/intechopen.83485*

anticarcinogenic, and antifibrogenic properties [34]. The rate of absorption of resveratrol is about 75% following an oral administration [35]. Resveratrol is metabolized to resveratrol sulfate, and in its low concentrations, it is converted into resveratrol glucuronide [36] by enzymes glucuronosyltransferase (UGT) or sulfotransferase (ST) [37]. In 2007, Chávez et al. [34] showed that under CCl4, resveratrol decreased cytokine TGF-β levels and prevented hepatic fibrosis. It also inhibited NF-kB translocation to the nucleus. Resveratrol, as an antioxidant, has protective effects against ethanol-induced lipid peroxidation, toxicity by acetaminophen (APAP), and oxidative stress in animal models of cholestasis [38]. Important player in resveratrol's antioxidant activity is suggested to be run by the OH groups [7]. Blocking OH group methylation showed that resveratrol and trimethylated resveratrol provide some degree of protection, but the latter one has a better protective effect [39]. Another hepatoprotection mechanism of resveratrol comes from its ability to activate genes related to antioxidant system or from its ability to inhibit enzymes. A study by Cheng et al. [40] suggested that resveratrol could activate extracellular signal-regulated kinase (ERK) signaling pathway, which may, in turn, enhance the activation and translocation of Nrf2 to the nucleus, thus increasing the expression of HO-1 and glyoxalase. Price et al. [41] found that resveratrol activates AMP-activated protein kinase (AMPK) and increased nicotinamide adenine dinucleotide (NAD) levels in mice. Zhu et al. [42] have also shown that, in mice, administration of resveratrol increased the antioxidant system (SOD, GPx, and GSH) and also the levels of SIRTI and p-AMPK were upregulated in liver. Resveratrol has also been shown to inhibit the activity of Cyp2e1 in microsomes of rat liver [43]. Resveratrol also inhibited the activity of P450 isoform APAP-induced liver injury model [44] and activity of Cyp2e1 was also inhibited in diethylnitrosamine (DEN)-induced hepatocarcinogenesis model [45]. The only clinical study that was performed to determine the resveratrol hepatoprotective effect demonstrated that a 500 mg resveratrol dose administrated for 12 weeks caused a significant reduction in inflammatory cytokines, serum cytokeratin-18, NF-kB activation, liver alanine aminotransferase (ALT), and hepatic steatosis when compared to the placebo group in patients with nonalcoholic fatty liver disease (NAFLD) [46].
