**4.3.2 Cardiovascular health**

348 Cancer Prevention – From Mechanisms to Translational Benefits

cell differentiation and proliferation. *In vitro* assays confirmed that both the pomegranate fermented juice and pericarp extracts strongly promoted cellular differentiation and inhibited proliferation in HL-60 cell cultures; the effect of pomegranate juice on cellular differentiation was less significant. In view of the observations the authors stated the hypothesis of another mechanism by which pomegranate constituents impart an

Mertens-Talcott & Percival (2005) investigated the interactions of ellagic acid and quercetin with resveratrol, with the hypothesis that the selected polyphenols would interact synergistically in the induction of apoptosis and reduction of cell growth in human leukemia cells (MOLT-4). They found significant interaction for the combination of ellagic acid with resveratrol, and alterations in cell cycle kinetics induced by single compounds and combinations were also observed. The authors concluded that the anticarcinogenic potential of foods containing polyphenols may not be based on the effects of individual compounds,

Epidemiological evidence points that many cancers arise from sites of infection, chronic irritation and inflammation (Rakoff-Nahoum, 2006; Heber, 2008). The inflammatory response result in persistent oxidative stress orchestrates tumour microenvironment to microbial infection and mediates tissue repair and regeneration, which may occur due to

Pomegranate and the selected chemical constituents isolated from juice, peel, and seed have been found to have a large range of effects: (i) inhibition of Cyclooxygenase-2 (COX-2) expression and ultimately eicosanoid biosynthesis (Schubert et al., 1999; Shukla et al., 2008); (ii) synergistic suppression of inflammatory cytokine expression (Adams et al., 2006); (iii) inhibition of matrix MMPs (Okamoto et al., 2004; Ahmad et al., 2005; Aslam et

In view of the antioxidant, anticarcinogenic and antiinflammatory properties of pomegranate phenolics and/or its derived metabolites, one could hypothesize that pomegranate and/or its derived metabolites have a beneficial effect on inflammation. Larrosa et al. (2010) evaluated the effects of pomegranate intake and its main microbiotaderived metabolite urolithin-A (UROA) on colon inflammation in a dextran sodium sulfate (DSS)-induced colon inflammation rat model and to assess whether UROA is the main antiinflammatory compound. In addition, they examined the effect of the inflammation on the phenolic metabolism. DSS (5%) was administered for the five last days to Male Fisher rats, fed with 250 mg/kg day pomegranate extract or 15 mg/kg day UROA for 25 days. In both groups inflammation markers (iNOS, COX-2, PTGES and PGE2 in colonic mucosa) were decreased, the gut microbiota was modulated and the G1 to S cell cycle pathway was upregulated. UROA group showed various down-regulated pathways, including that of the inflammatory response. Pomegranate extract, but not UROA, decreased oxidative stress in plasma and colon mucosa. Only UROA preserved colonic architecture. The normal formation of urolithins in pomegranate extract-fed rats was prevented during inflammation suggesting UROA could be the most active anti-inflammatory compound derived from

but may involve a synergistic enhancement of the anticancer effects.

anticarcinogenic effect.

**4.3 Other health effects** 

al., 2006).

**4.3.1 Antiinflammatory activity** 

infectious or non-infectious tissue damage.

Cardiovascular diseases (CVDs) are a leading cause of death and disability worldwide. Hypertension and atherosclerosis, a chronic inflammatory disease characterized by plaque formation in the large arteries, are major risk factors for CVDs, such as stroke, myocardial infarction and heart failure. In addition to genetic factors, age, body weight, blood pressure, dyslipidemia, physical inactivity and behavioural risk factors such as tobacco or alcohol use, diets that include high fat, salty food are thought to play an important role in the development of cardiovascular disease. Epidemiological data have clearly shown that independent risk factors for CVD are serum total cholesterol and low-density lipoprotein cholesterol (LDL-C) (Kannel et. al., 1986; Mirmiran et al., 2009). A large number of clinical trials have demonstrated in order to prevent these cardiovascular diseases from occurring, control of a patient's blood pressure is necessary, either by lifestyle modifications, medication(s) such as use of cholesterol-lowering statins, antihypertensive drugs and antiplatelet agents or both.

While modification of dietary patterns and increased physical activity constitute the primary preventive intervention in lowering coronary heart disease (CHD) and stroke, the role of plant-based bioactive compounds or phytochemicals has attracted much attention since

The Therapeutic Potential of Pomegranate and Its Products for Prevention of Cancer 351

hypercholesterolemic mice significantly reduced the progression of atherosclerosis and

In a randomized, double-blind, parallel trial Davidson et al. (2009) assessed the influence of pomegranate juice consumption on anterior and posterior carotid intima-media thickness (CIMT) progression rates in subjects at moderate risk for coronary heart disease. Participants consumed 240 ml/day of pomegranate juice or a control beverage for up to 18 months. No significant difference in overall CIMT progression rate was observed between pomegranate juice and control treatments. In exploratory analyses, in subjects in the most adverse tertiles for baseline serum lipid peroxides, triglycerides (TGs), highdensity lipoprotein (HDL) cholesterol, TGs/HDL cholesterol, total cholesterol/HDL cholesterol, and apolipoprotein-B100, those in the pomegranate juice group had significantly less anterior wall and/or composite CIMT progression versus control subjects. They concluded that in subjects at moderate coronary heart disease risk, pomegranate juice consumption had no significant effect on overall CIMT progression rate but may have slowed CIMT progression in subjects with increased oxidative stress and disturbances in the TG-rich lipoprotein/HDL

Endothelial apoptosis is a driving force in atherosclerosis development. Oxidized lowdensity lipoprotein (oxLDL) promotes inflammatory and thrombotic processes and is highly atherogenic, as it stimulates macrophage cholesterol accumulation and foam cell formation. In recent years ellagic acid has been the subject of intense research within the fields of cancer and inflammation, however, its protective effect against oxidized LDL-induced injury in vascular endothelial cells have not been clarified. Ou et al. (2010) investigated the antiapoptotic effect of ellagic acid in human umbilical vein endothelial cells (HUVECs) exposed to OxLDL and explored the possible mechanisms. Pretreatment with ellagic acid (5–20 μM) significantly attenuated OxLDL-induced cytotoxicity, apoptotic features, and generation of ROS. In addition, the antiapoptotic effect of ellagic acid was partially inhibited by wortmannin (a PI3K inhibitor) and cavtratin (a specific endothelial NO synthase inhibitor). The alterations induced by OxLDL, however, were attenuated by pretreatment with ellagic acid. The inhibition of OxLDL-induced endothelial apoptosis by ellagic acid is due to its anti-oxidant activity and its ability to modulate the PI3K/Akt/eNOS signaling pathway

According to several review articles (Jurenka, 2008; Li et al., 2008; Yun, 2010) pomegranate flowers, containing abundant ellagitannins, was already prescribed in Unani and Ayurvedic systems of medicine as a remedy for diabetes. The protective effect of pomegranate flowers' extracts (PFLE) was investigated by some authors on blood glucose level, serum lipid profile, total cholesterol, LDL, pancreatic lipid peroxidation and activities of both enzymatic and non-enzymatic antioxidant status in diabetic rats (Huang et al., 2005a,b; Lei et al., 2007; Lan et al., 2009; Bagri et al., 2009). The authors reported that the increase in blood glucose level, total cholesterol, triglycerides, LDL-cholesterol, very low density lipoproteins, lipid peroxidation level with decrease in high density lipoprotein (HDL)-cholesterol, glutathione content and antioxidant enzymes namely, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, superoxide dismutase and catalase can be reversed by administration of aqueous PFLE. PFLE was shown to activate PPAR-a, a cardiac

**4.3.3 Antidiabetic properties (glucose and lipid metabolism activity)** 

isoprostane levels and increased nitrates.

axis.

upto a point.

there is a negative relationship between their consumption and CVDs (Hu, 2003; Dauchet et al., 2005; Nothlings et al., 2008). Pomegranate, being rich in flavonoids and ellagitannins, are potent antioxidants and antiinflammatory agents, thereby counteracting oxidative damage and inflammation which underlie the pathogenesis of CVD (Kaplan et al., 2001). Oxidative stress, the major contributor to CVD, is the build-up of highly reactive free radical species or the decrease of defence mechanisms to protect against biological damage by free radicals due to the imbalance between free radical formation and antioxidant status. Oxidative stress induces inflammation by acting on the pathways that generate inflammatory mediators like adhesion molecules and pro-inflammatory cytokines. The effect of reactive oxygen species (ROS) and reactive nitrogen species (RNS) on human health has been studied for decades, with results indicating increasing the risk of cancer, arthritis, degenerative eye and neurological disorders, as well as general aging (Aruoma, 1998). However, the attention has turned to the effect of these free radicals on CVD and related disorders; such as atherosclerosis, hypertension, hypercholesterolemia, type 2 diabetes, and heart failure (Hamilton et al., 2004).

Sumner et al. (2005) investigated the effect of daily consumption of pomegranate juice for 3 months on myocardial perfusion in patients who had coronary heart disease and myocardial ischemia in a randomized, placebo-controlled, double-blind study. The patients were given either 240 mL pomegranate juice (polyphenol content not specified) or a sports beverage of similar color, flavor, and caloric content daily for three months. Although both control and treatment patients demonstrated similar levels of stress-induced ischemia at baseline, at three months stress-induced ischemia decreased in the treatment group (from 4.5±3.1 to 3.7±3.7). In addition, angina episodes decreased 50% percent in the treatment group but increased 38% in the placebo group. The researchers concluded that pomegranate juice consumption resulted in a reduction in myocardial ischemia and improvement in myocardial perfusion.

Rosenblat et al. (2006) studied the antiatherosclerotic effects of a pomegranate by-product (PBP, which includes the whole pomegranate fruit left after juice preparation). Fourmonth-old E° mice with significant atherosclerosis were given PBP (17 or 51.5 μg of gallic acid equiv/kg/day) with an eight-fold higher polyphenol concentration than pomegranate juice for three months. Consumption of PBP by the mice resulted in a significant reduction in atherosclerotic lesion size by up to 57% and in MPM oxidative status as evidenced by a 27% decrease in total macrophage peroxide levels, a 42% decrease in cellular lipid peroxide levels, and a 19% decrease in peritoneal macrophage uptake of oxidized LDL (Ox-LDL).

Through *in vitro* and *in vivo* studies de Nigris et al. (2006) stated that the proatherogenic effects induced by perturbed shear stress can be also reversed by chronic administration of pomegranate fruit extract (PFE). The researcher investigated the effects of intervention with the PFE rich in polyphones (punicalagin, which is a potent antioxidant) on ELK-1, p-CREB, and endothelial nitric oxide synthase (eNOS) expression induced by high shear stress*.* Both the PFE and the regular pomegranate juice concentrate reduced the activation of ELK-1 and p-CREB and increased eNOS expression in cultured human endothelial cells and in atherosclerosis-prone areas of hypercholesterolemic mice. PFE and pomegranate juice increased cyclic GMP levels while there was no significant effect of both compounds on the conversion of L-arginine to L-citrulline. Administration of these compounds to

there is a negative relationship between their consumption and CVDs (Hu, 2003; Dauchet et al., 2005; Nothlings et al., 2008). Pomegranate, being rich in flavonoids and ellagitannins, are potent antioxidants and antiinflammatory agents, thereby counteracting oxidative damage and inflammation which underlie the pathogenesis of CVD (Kaplan et al., 2001). Oxidative stress, the major contributor to CVD, is the build-up of highly reactive free radical species or the decrease of defence mechanisms to protect against biological damage by free radicals due to the imbalance between free radical formation and antioxidant status. Oxidative stress induces inflammation by acting on the pathways that generate inflammatory mediators like adhesion molecules and pro-inflammatory cytokines. The effect of reactive oxygen species (ROS) and reactive nitrogen species (RNS) on human health has been studied for decades, with results indicating increasing the risk of cancer, arthritis, degenerative eye and neurological disorders, as well as general aging (Aruoma, 1998). However, the attention has turned to the effect of these free radicals on CVD and related disorders; such as atherosclerosis, hypertension, hypercholesterolemia, type 2 diabetes, and heart failure

Sumner et al. (2005) investigated the effect of daily consumption of pomegranate juice for 3 months on myocardial perfusion in patients who had coronary heart disease and myocardial ischemia in a randomized, placebo-controlled, double-blind study. The patients were given either 240 mL pomegranate juice (polyphenol content not specified) or a sports beverage of similar color, flavor, and caloric content daily for three months. Although both control and treatment patients demonstrated similar levels of stress-induced ischemia at baseline, at three months stress-induced ischemia decreased in the treatment group (from 4.5±3.1 to 3.7±3.7). In addition, angina episodes decreased 50% percent in the treatment group but increased 38% in the placebo group. The researchers concluded that pomegranate juice consumption resulted in a reduction in myocardial ischemia and improvement in

Rosenblat et al. (2006) studied the antiatherosclerotic effects of a pomegranate by-product (PBP, which includes the whole pomegranate fruit left after juice preparation). Fourmonth-old E° mice with significant atherosclerosis were given PBP (17 or 51.5 μg of gallic acid equiv/kg/day) with an eight-fold higher polyphenol concentration than pomegranate juice for three months. Consumption of PBP by the mice resulted in a significant reduction in atherosclerotic lesion size by up to 57% and in MPM oxidative status as evidenced by a 27% decrease in total macrophage peroxide levels, a 42% decrease in cellular lipid peroxide levels, and a 19% decrease in peritoneal macrophage

Through *in vitro* and *in vivo* studies de Nigris et al. (2006) stated that the proatherogenic effects induced by perturbed shear stress can be also reversed by chronic administration of pomegranate fruit extract (PFE). The researcher investigated the effects of intervention with the PFE rich in polyphones (punicalagin, which is a potent antioxidant) on ELK-1, p-CREB, and endothelial nitric oxide synthase (eNOS) expression induced by high shear stress*.* Both the PFE and the regular pomegranate juice concentrate reduced the activation of ELK-1 and p-CREB and increased eNOS expression in cultured human endothelial cells and in atherosclerosis-prone areas of hypercholesterolemic mice. PFE and pomegranate juice increased cyclic GMP levels while there was no significant effect of both compounds on the conversion of L-arginine to L-citrulline. Administration of these compounds to

(Hamilton et al., 2004).

myocardial perfusion.

uptake of oxidized LDL (Ox-LDL).

hypercholesterolemic mice significantly reduced the progression of atherosclerosis and isoprostane levels and increased nitrates.

In a randomized, double-blind, parallel trial Davidson et al. (2009) assessed the influence of pomegranate juice consumption on anterior and posterior carotid intima-media thickness (CIMT) progression rates in subjects at moderate risk for coronary heart disease. Participants consumed 240 ml/day of pomegranate juice or a control beverage for up to 18 months. No significant difference in overall CIMT progression rate was observed between pomegranate juice and control treatments. In exploratory analyses, in subjects in the most adverse tertiles for baseline serum lipid peroxides, triglycerides (TGs), highdensity lipoprotein (HDL) cholesterol, TGs/HDL cholesterol, total cholesterol/HDL cholesterol, and apolipoprotein-B100, those in the pomegranate juice group had significantly less anterior wall and/or composite CIMT progression versus control subjects. They concluded that in subjects at moderate coronary heart disease risk, pomegranate juice consumption had no significant effect on overall CIMT progression rate but may have slowed CIMT progression in subjects with increased oxidative stress and disturbances in the TG-rich lipoprotein/HDL axis.

Endothelial apoptosis is a driving force in atherosclerosis development. Oxidized lowdensity lipoprotein (oxLDL) promotes inflammatory and thrombotic processes and is highly atherogenic, as it stimulates macrophage cholesterol accumulation and foam cell formation. In recent years ellagic acid has been the subject of intense research within the fields of cancer and inflammation, however, its protective effect against oxidized LDL-induced injury in vascular endothelial cells have not been clarified. Ou et al. (2010) investigated the antiapoptotic effect of ellagic acid in human umbilical vein endothelial cells (HUVECs) exposed to OxLDL and explored the possible mechanisms. Pretreatment with ellagic acid (5–20 μM) significantly attenuated OxLDL-induced cytotoxicity, apoptotic features, and generation of ROS. In addition, the antiapoptotic effect of ellagic acid was partially inhibited by wortmannin (a PI3K inhibitor) and cavtratin (a specific endothelial NO synthase inhibitor). The alterations induced by OxLDL, however, were attenuated by pretreatment with ellagic acid. The inhibition of OxLDL-induced endothelial apoptosis by ellagic acid is due to its anti-oxidant activity and its ability to modulate the PI3K/Akt/eNOS signaling pathway upto a point.

#### **4.3.3 Antidiabetic properties (glucose and lipid metabolism activity)**

According to several review articles (Jurenka, 2008; Li et al., 2008; Yun, 2010) pomegranate flowers, containing abundant ellagitannins, was already prescribed in Unani and Ayurvedic systems of medicine as a remedy for diabetes. The protective effect of pomegranate flowers' extracts (PFLE) was investigated by some authors on blood glucose level, serum lipid profile, total cholesterol, LDL, pancreatic lipid peroxidation and activities of both enzymatic and non-enzymatic antioxidant status in diabetic rats (Huang et al., 2005a,b; Lei et al., 2007; Lan et al., 2009; Bagri et al., 2009). The authors reported that the increase in blood glucose level, total cholesterol, triglycerides, LDL-cholesterol, very low density lipoproteins, lipid peroxidation level with decrease in high density lipoprotein (HDL)-cholesterol, glutathione content and antioxidant enzymes namely, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, superoxide dismutase and catalase can be reversed by administration of aqueous PFLE. PFLE was shown to activate PPAR-a, a cardiac

The Therapeutic Potential of Pomegranate and Its Products for Prevention of Cancer 353

progression of atherosclerosis by upregulating the hepatic expression with concomitant increased serum PON1 activity (Gouédard et al., 2004; Leckey et al., 2010). Similarly, pomegranate polyphenols seem to have a specific transcriptional role in hepatocyte PON1 expression upregulation (Khateeb et al., 2010). Although it is known that diabetes is associated with increased oxidative stress and the development of atherosclerosis (Mooradian, 2009), no expression studies have been examined in a diabetic model that is fed with high fat. Therefore, Betanzos-Cabrera et al. (2011) investigated whether pomengranate juice induces PON1 gene expression and activity, especially in conditions known for affecting PON1 enzymatic function. The feeding of streptozotocin-induced diabetic mice with a high-fat diet supplemented daily with pomegranate juice significantly induced PON1 gene expression and activity. Interestingly, animals supplemented with pomegranate juice showed the lowest bodyweight. In addition, the pomegranate juice significantly reduced blood glucose but not triacylglycerols and cholesterol levels, demonstrating that

Due to the increasing interest in natural antimicrobials and antioxidants derived from plant sources the investigation of pomegranate has also been an interesting scientific field for researchers, since the capacity of preventing infections of pomegranate extracts was well documented. Food-borne illnesses are still an important concern for both consumers, the food industry and food safety authorities, thus, the ongoing search for natural antimicrobials for prevention of food-borne illnesses is a vast exploring area for scientists. Antimicrobial activities of pomegranate have been studied by some researchers and the extent of inhibitory effect is always attributed to the pomegranate antioxidant activity that depends mainly on the phenolic and anthocyanin content of the fruit (Holetz et al., 2002; Braga et al., 2005; Mathabe et al., 2006; McCarrell et al., 2008; Al-Zoreky, 2009; Duman et al., 2009; Gould et al., 2009; Parashar et al., 2009; Panichayupakaranant et al., 2010, Orak et al., 2011). In a previous study, Opara et al. (2009) reported that the best activity against *Staphyloccocus aureus* and *Pseudomonas aeruginosa* were found in fruit peel compound punicalagin, particularly from Oman, which was coincident with the highest levels of vitamin C detected in these samples. Similar findings were reported by Salgado et al. (2009) and Dahham et al. (2010) in which antibacterial and antifungal activities of pomegranate peel extract (rind), seed extract, juice and whole fruit on the selected bacteria and fungi were investigated. The antimicrobial effectiveness of the extracts depends on the species of bacteria evaluated, the more sensitive being the Gram-positive species *S. aureus* and *Bacillus*  sp. *along* with *Aspergillus niger.* Voravuthikunchai et al. (2006) tested pomegranate and seven other Thai medicinal plant extracts for *in vitro* activity against enterohemorrhagic *Escherichia coli (E. coli* O157:H7). An ethanolic pomegranate peel extract was shown to be both bacteriostatic and bacteriocidal, indicating it may be an effective adjunct treatment for *E. coli* 

The only human trials examining the antibacterial properties of pomegranate extracts have focused on oral bacteria (Sastravaha et al., 2003; Menezes et al., 2006). However, several *in vitro* assays demonstrate its bacteriocidal activity against several highly pathogenic and sometimes antibiotic-resistant organisms. Machado et al. (2002) evaluated the synergistic effect of a pomegranate methanolic extract with five antibiotics on 30 clinical isolates of methicilin-resistant *S. aureus* (MRSA) and methicillin-sensitive *S. aureus*. Antibiotics tested were. Although synergistic activity between the pomegranate extract and tested antibiotics

pomegranate juice has a hypoglycemic effect.

**4.3.4 Antimicrobial properties** 

O157:H7 infection.

transcription factor involved in myocardial energy production via fatty acid uptake and oxidation. PPAR-a activation decreased cardiac uptake and circulation of Iipids. Decreases were observed in cardiac tissue triglycéride content at the end of the study and in plasma total cholesterol and NEFA after four weeks of treatment.These findinds suggest that pomegranate could be used as dietary supplement in the treatment and prevention of chronic diseases characterised by atherogenic lipoprotein profile, aggravated antioxidant status and impaired glucose metabolism.

Rosenblat et al. (2006) investigated the effect of 50 mL/day pomegranate juice for three months on oxidative stress, blood sugar, and lipid profiles in 10 type 2 diabetic patients, with a history of diabetes for 4–10 years, and 10 healthy controls. In diabetic patients, triglyceride levels were 2.8 times greater, (HDL) cholesterol was 28% lower, and hemoglobin AlC (HbAlC) values were 59% higher than in control patients. They stated that consuming pomegranate juice for three months did not significantly affect triglyceride, HDL cholesterol, HbAlC, glucose, or insulin values, but did lower serum C-peptide values by 23%, suggesting improved insulin sensitivity. Researchers concluded that despite the sugars naturally present in pomegranate juice, consumption did not adversely affect diabetic parameters but had a significant effect on atherogenesis via reduced oxidative stress.

Esmailzadeh et al. (2006) investigated the cholesterol-lowering effects of 40 g concentrated pomegranate juice on 22 type 2 diabetic patients (8 men and 14 women) for eight weeks. Statistically significant decreases were observed in total cholesterol, LDL cholesterol, total/HDL cholesterol ratio, and LDL/HDL ratio, which due in part to decreased absorption and increased fecal excretion of cholesterol, as well as possible affects on HMG-CoA reductase and sterol O-acyltransferase, two enzymes key to cholesterol metabolism.

Oleanolic acid, ursolic acid and gallic acid, active components contained in pomegranate flower (Li et al., 2008), have long been recognized to have antihyperlipidemic properties (Liu, 1995; Jang et al., 2008). Xu et al. (2009) speculated that PFLE might improve diabetes and obesity-induced fatty liver, and investigated the effects and underlying mechanisms of action of PFLE on hepatic lipid accumulation in ZDF rats with severe fatty liver and in human liver-derived HepG2 cell line. PFLE-treated ZDF rats showed reduced ratio of liver weight to tibia length, hepatic triglyceride contents and lipid droplets. These effects were accompanied by enhanced hepatic gene expression of peroxisome proliferator-activated receptor (PPAR)-alpha, carnitine palmitoyltransferase-1 and acyl-CoA oxidase (ACO), and reduced stearoyl-CoA desaturase-1. Incontrast, PFLE showed minimal effects on expression of genes responsible for synthesis, hydrolysis or uptake of fatty acid and triglycerides. PGF treatment also increased PPAR-alpha and ACO mRNA levels in HepG2 cells. The authors concluded that PFLE, an Unani medicine, ameliorates diabetes and obesity-associated fatty liver, at least in part, by activating hepatic expression of genes responsible for fatty acid oxidation.

There is growing evidence that paraoxnase (PON1) plays an important role in lipid metabolism, particularly in protecting LDL and HDL from oxidation in vitro, and thus lowering the risk of developing atherosclerosis, and the onset of cardiovascular disease (Mackness et al., 2000,2002). PON1 knockout mice exhibit about a two-fold increase in atherosclerosis (Rozenberg et al., 2003), whereas PON1 expression and activity can be modulated by dietary polyphenols i.e. LDL receptor deficient mice supplemented with quercitine (a polyphenol contained in pomegranate) and moderate ethanol inhibited the progression of atherosclerosis by upregulating the hepatic expression with concomitant increased serum PON1 activity (Gouédard et al., 2004; Leckey et al., 2010). Similarly, pomegranate polyphenols seem to have a specific transcriptional role in hepatocyte PON1 expression upregulation (Khateeb et al., 2010). Although it is known that diabetes is associated with increased oxidative stress and the development of atherosclerosis (Mooradian, 2009), no expression studies have been examined in a diabetic model that is fed with high fat. Therefore, Betanzos-Cabrera et al. (2011) investigated whether pomengranate juice induces PON1 gene expression and activity, especially in conditions known for affecting PON1 enzymatic function. The feeding of streptozotocin-induced diabetic mice with a high-fat diet supplemented daily with pomegranate juice significantly induced PON1 gene expression and activity. Interestingly, animals supplemented with pomegranate juice showed the lowest bodyweight. In addition, the pomegranate juice significantly reduced blood glucose but not triacylglycerols and cholesterol levels, demonstrating that pomegranate juice has a hypoglycemic effect.

#### **4.3.4 Antimicrobial properties**

352 Cancer Prevention – From Mechanisms to Translational Benefits

transcription factor involved in myocardial energy production via fatty acid uptake and oxidation. PPAR-a activation decreased cardiac uptake and circulation of Iipids. Decreases were observed in cardiac tissue triglycéride content at the end of the study and in plasma total cholesterol and NEFA after four weeks of treatment.These findinds suggest that pomegranate could be used as dietary supplement in the treatment and prevention of chronic diseases characterised by atherogenic lipoprotein profile, aggravated antioxidant

Rosenblat et al. (2006) investigated the effect of 50 mL/day pomegranate juice for three months on oxidative stress, blood sugar, and lipid profiles in 10 type 2 diabetic patients, with a history of diabetes for 4–10 years, and 10 healthy controls. In diabetic patients, triglyceride levels were 2.8 times greater, (HDL) cholesterol was 28% lower, and hemoglobin AlC (HbAlC) values were 59% higher than in control patients. They stated that consuming pomegranate juice for three months did not significantly affect triglyceride, HDL cholesterol, HbAlC, glucose, or insulin values, but did lower serum C-peptide values by 23%, suggesting improved insulin sensitivity. Researchers concluded that despite the sugars naturally present in pomegranate juice, consumption did not adversely affect diabetic

parameters but had a significant effect on atherogenesis via reduced oxidative stress.

reductase and sterol O-acyltransferase, two enzymes key to cholesterol metabolism.

Esmailzadeh et al. (2006) investigated the cholesterol-lowering effects of 40 g concentrated pomegranate juice on 22 type 2 diabetic patients (8 men and 14 women) for eight weeks. Statistically significant decreases were observed in total cholesterol, LDL cholesterol, total/HDL cholesterol ratio, and LDL/HDL ratio, which due in part to decreased absorption and increased fecal excretion of cholesterol, as well as possible affects on HMG-CoA

Oleanolic acid, ursolic acid and gallic acid, active components contained in pomegranate flower (Li et al., 2008), have long been recognized to have antihyperlipidemic properties (Liu, 1995; Jang et al., 2008). Xu et al. (2009) speculated that PFLE might improve diabetes and obesity-induced fatty liver, and investigated the effects and underlying mechanisms of action of PFLE on hepatic lipid accumulation in ZDF rats with severe fatty liver and in human liver-derived HepG2 cell line. PFLE-treated ZDF rats showed reduced ratio of liver weight to tibia length, hepatic triglyceride contents and lipid droplets. These effects were accompanied by enhanced hepatic gene expression of peroxisome proliferator-activated receptor (PPAR)-alpha, carnitine palmitoyltransferase-1 and acyl-CoA oxidase (ACO), and reduced stearoyl-CoA desaturase-1. Incontrast, PFLE showed minimal effects on expression of genes responsible for synthesis, hydrolysis or uptake of fatty acid and triglycerides. PGF treatment also increased PPAR-alpha and ACO mRNA levels in HepG2 cells. The authors concluded that PFLE, an Unani medicine, ameliorates diabetes and obesity-associated fatty liver, at least in part, by activating hepatic expression of genes responsible for fatty acid

There is growing evidence that paraoxnase (PON1) plays an important role in lipid metabolism, particularly in protecting LDL and HDL from oxidation in vitro, and thus lowering the risk of developing atherosclerosis, and the onset of cardiovascular disease (Mackness et al., 2000,2002). PON1 knockout mice exhibit about a two-fold increase in atherosclerosis (Rozenberg et al., 2003), whereas PON1 expression and activity can be modulated by dietary polyphenols i.e. LDL receptor deficient mice supplemented with quercitine (a polyphenol contained in pomegranate) and moderate ethanol inhibited the

status and impaired glucose metabolism.

oxidation.

Due to the increasing interest in natural antimicrobials and antioxidants derived from plant sources the investigation of pomegranate has also been an interesting scientific field for researchers, since the capacity of preventing infections of pomegranate extracts was well documented. Food-borne illnesses are still an important concern for both consumers, the food industry and food safety authorities, thus, the ongoing search for natural antimicrobials for prevention of food-borne illnesses is a vast exploring area for scientists. Antimicrobial activities of pomegranate have been studied by some researchers and the extent of inhibitory effect is always attributed to the pomegranate antioxidant activity that depends mainly on the phenolic and anthocyanin content of the fruit (Holetz et al., 2002; Braga et al., 2005; Mathabe et al., 2006; McCarrell et al., 2008; Al-Zoreky, 2009; Duman et al., 2009; Gould et al., 2009; Parashar et al., 2009; Panichayupakaranant et al., 2010, Orak et al., 2011). In a previous study, Opara et al. (2009) reported that the best activity against *Staphyloccocus aureus* and *Pseudomonas aeruginosa* were found in fruit peel compound punicalagin, particularly from Oman, which was coincident with the highest levels of vitamin C detected in these samples. Similar findings were reported by Salgado et al. (2009) and Dahham et al. (2010) in which antibacterial and antifungal activities of pomegranate peel extract (rind), seed extract, juice and whole fruit on the selected bacteria and fungi were investigated. The antimicrobial effectiveness of the extracts depends on the species of bacteria evaluated, the more sensitive being the Gram-positive species *S. aureus* and *Bacillus*  sp. *along* with *Aspergillus niger.* Voravuthikunchai et al. (2006) tested pomegranate and seven other Thai medicinal plant extracts for *in vitro* activity against enterohemorrhagic *Escherichia coli (E. coli* O157:H7). An ethanolic pomegranate peel extract was shown to be both bacteriostatic and bacteriocidal, indicating it may be an effective adjunct treatment for *E. coli*  O157:H7 infection.

The only human trials examining the antibacterial properties of pomegranate extracts have focused on oral bacteria (Sastravaha et al., 2003; Menezes et al., 2006). However, several *in vitro* assays demonstrate its bacteriocidal activity against several highly pathogenic and sometimes antibiotic-resistant organisms. Machado et al. (2002) evaluated the synergistic effect of a pomegranate methanolic extract with five antibiotics on 30 clinical isolates of methicilin-resistant *S. aureus* (MRSA) and methicillin-sensitive *S. aureus*. Antibiotics tested were. Although synergistic activity between the pomegranate extract and tested antibiotics

The Therapeutic Potential of Pomegranate and Its Products for Prevention of Cancer 355

USA. The published safety data is limited and no clinical or laboratory adverse events were reported. However, there are some publications on occurrence of allergic reactions when handling or ingestion of pomegranate fruit/seeds due to eliciting a type I hypersensitivity reaction, and thus it is crucial to advise consumers the side effects

Nowadays, it is widely accepted that the beneficial health effects of fruits and vegetables in

Based on the explosion of interest in the numerous therapeutic properties over the last decade and *in vitro*, animal, and clinical trials pomegranate seems to be a promising food with well-defined therapeutic benefits. The epidomiological data suggests that the pomegranate fruit and its associated bioactive compounds such as phenolic acids, flavonoids, and tannins may possess a strong potential as a chemopreventive and possibly as new tools for preventive and possibly therapeutic interventions against various human cancers. This could have a direct practical implication to cancer patients if consumption of fruits like pomegranate can inhibit the process of carcinogenesis. Further studies should focus on the potential clinical usefulness of the agent through issues such as determining the optimal period and route of administration, systemic bioavailability, potent anticancer activity, optimal dosing and toxicity (if any) of the agent and single or combinatorial approach. In addition, the possible use of pomegranate extracts as a therapy or adjunct for prevention and treatment of several disease processes, such as diabetes, cardiovascular disease, atherosclerosis, inflammation, microbial infection, obesity, male infertility, Alzheimer underscores the need for more clinical research. Therefore, ongoing studies should focus on developing novel pomegranate derived products such as ready-to-eat pomegranate seeds, single-strength juices, juice concentrates, seeds in syrup, frozen seeds, and traditional products such as pomegranate pekmez, leather and molasses, to benefit from

Adams, L.S., Seeram, N.P., Aggarwal, B.B., Takada, Y., Sand, D. & Heber, D. (2006).

Adams, L.S., Zhang, Y., Seeram, N.P., Heber, D. & Chen, S. (2010). Pomegranate

Adhami, V.M., Khan, N. & Mukhtar, H. (2009). Cancer Chemoprevention by Pomegranate:

Adhami, V.M., Khan, N. & Mukhtar, H. (2010). Prevention of Cancer with Pomegranate and

Seerelam, N.P. (Eds), pp.209-226, ISBN 1441975535, Springer

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Pomegranate Juice, Total Pomegranate Ellagitannins, and Punicalagin Suppress Inflammatory Cellsignaling in Colon Cancer Cells. *Journal of Agricultural and Food* 

Ellagitannin-derived Compounds Exhibit Antiproiferation and Antiaromatase Activity in Breast Cancer Cells In vitro. *Cancer Prevention Research,* Vol.3, No.1,

Laboratory and Clinical Evidence. *Nutrition and Cancer,* Vol.61, No.6, pp.811-815,

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the prevention of diseases are due to the bioactive compounds they contain.

these constituents throughout a healthy life cycle.

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ISSN 0163-5581

(McCutcheon et al., 2008).

**6. Conclusion** 

**7. References** 

(chloramphenicol, gentamicin, ampicillin, tetracycline, and oxacillin) was noted in the *S. aureus* isolates, synergy with ampicillin was the most pronounced. A combination of the two increased the lag time to bacterial growth by three hours and was also bacteriocidal as evidenced by a 72.5% reduction in methicillin-sensitive organisms and a 99.9% reduction in MRSA. Bialonska et al. (2009b) stated that commercial extract of pomegranate by-product provided by POM Wonderful (Los Angeles, CA) and punicalagins inhibited the growth of pathogenic clostridia and *S. aureus*. Nevertheless the probiotic lactobacilli and bifidobacteria were not affected by ellagitannins. These findings lead to the conclusion that the growth inhibition toward pathogenic bacteria could be attributed to the accumulation of ellagitannins in the large intestines, where they interact with complex gut microflora, and lower media pH due to the presence of punicalagins.

Su et al. (2010) stated that the combination of pomegranate juice and pomegranate polyphenols was also effective against food-borne viral infectivity and appear to be promising natural remedies for preventing or reducing human norovirus infections. In addition, pomegranate purified polyphenol extract inhibited influenza virus having also a synergistic effect with oseltamivir, since influenza continues to be a major cause of mortality and morbidity eventhough the applications of the vaccines and antiviral therapies (Haidari et al., 2009).

Johann et al. (2010) studying the activity of extracts of some plants used in Brazilian traditional medicine against the pathogenic fungus that causes this Paracoccidioidomycosis, *Paracoccidioides brasiliensis*, reported that the hexane extract of pomegranate stem exhibited better antifungal activity against the three clinical isolated than other parts of the plant or other fractions of the same plant.

*Candida* species, a normal component of the human biota in the gastrointestinal tract and oral and vaginal mucosa, can cause superficial infections such as thrush and vaginitis. Endo et al. (2010) reported that punicalagin isolated from pomegranate peels possessed strong activity against *Candida albicans* and *C. parapsilosis* as well as the combination of punicalagin and fluconazole showed a synergistic interaction. Tayel & El-Tras (2009) demonstrated that methanol, ethanol and water extracts of pomegranate peels were effective against *C. albicans*  growth. In addition, they also proved that pomegranate peel extract aerosol was an efficient method for complete sanitizing of semi-closed places against *C. albicans* growth, and thereby could contribute for preventing *C. albicans* contamination and growth in suspected places. Ethnobotanical studies performed in Brazil had demonstrated the utilization of pomegranate in oral health since denture stomatitis is commonly associated with *C. albicans*  and some other *Candida* species (Santos et al., 2009).

Dell'Agli et al. (2009) studied the *in vitro* antiplasmodial and antimalarial activity of methanolic extracts of a tannin-enriched fraction and of metabolites to estimate their curative efficacy and mechanisms of action. They conclude that methanolic extracts of pomegranate inhibited *Plasmodium falciparum* and *P. vivax* growth *in vitro*, and suggested that these might be attributed to the low bioavailability as well as the kinetic of conversion of ellagic acid to inactive metabolites urolithins.
