**3. The biochemistry and pharmokinetics of pomegranate**

There is little knowledge about the absorption, bioavailability, biodistribution, and metabolism of the bioactive compounds present in pomegranate and in other fruits, although they probably have a similar pathway (Petti & Scully, 2009). The bioavailability of polyphenols varies according to the structure, glycosylation and solubility of the molecules which defined their extractability (Lecerf, 2006; Ozcan et al., 2011). In view of limited human studies, it appears that the bioavailability determinations of pomegranate polyphenols is

seeds are rich source of lipids, and the fatty acid component of pomegranate seed oil comprises over 95% of the oil, of which 99% is triacylglycerols. Minor components of the oil include vitamin E, sterols, steroids, and a key component of mammalian myelin sheaths,

'*Pekmez*', a concentrated and shelf-life extended Turkish product, is generally produced from fruits containing high amounts of sugar such as grape, mulberry, carnob, apple, pomegranate, plum and apricot (Alparslan & Hayta, 2002; Demirozu et al., 2002). The rst steps in pomegranate pekmez production is washing, granulating and crushing of the pomegranates. The pomegranate juice, obtained by pressing the crushed pomegranates by a pneumatical or mechanical press, is boiled with a calcareous substance called '*pekmez earth*'*,* white soil containing 70.40% CaCO3 or technical CaCO3, for deacidification and neutralization. The juice is clarified and concentrated usually in open vessels, and rarely under vacuum at 565 mm Hg and 66°C, up to 65–68° Brix; this product is called '*liquid pekmez*'. The liquid pekmez can be consumed either as liquid or solidified at 6oC for 2-3 days via addition of hydrocolloids, to produce '*solid pekmez*'*,* which has a pasty form that is easily

The '*pomegranate leather (pestil)*' is another Turkish pomegranate derived product that can be stored for a long time without deterioration. Pomegranates are washed, granulated, crushed, pressed and ltered to separate the seeds and skin. Pekmez earth is added to neutralize and clarify the fresh pomegranate juice. Clarified juice is ltered and is mixed with the wheat starch. Nuts such as walnut or hazelnut can be added in small pieces if desired. The juice and starch mixture is concentrated upto 40° Brix by boiling and continuous stirring. The puree is spread on cloths of 0.5–2.00 mm thickness and sun-dried until a mild, tasty, light and chewable leathery product is obtained. The dried pestil is

The '*pomegranate molasses (sour pomegranate pekmez, nar eksisi, pomegranate sauce)*', a traditional seasoning commonly used in salads and many dishes to improve the taste and aroma characteristics in Turkey, is a concentrated product produced simply by boiling, without the addition of further sugar or other additives (Poyrazoglu et al., 2002; Incedayi et al., 2010). Pomegranate molasses is a highly nutritive product since it is more concentrate and the have a high mineral content. Traditional methods are still being used to produce pomegranate molasses, of which requires cleaning, crushing, extraction, filtration, and evaporation (upto 35-65° Brix) in an open vessel or under vacuum. Clarification is not recommended in pomegranate molasses since customers prefer bitterness and sourness that comes from phenolic substances and acidity (Vardin & Abbasoglu, 2004; Kaya & Sozer,

There is little knowledge about the absorption, bioavailability, biodistribution, and metabolism of the bioactive compounds present in pomegranate and in other fruits, although they probably have a similar pathway (Petti & Scully, 2009). The bioavailability of polyphenols varies according to the structure, glycosylation and solubility of the molecules which defined their extractability (Lecerf, 2006; Ozcan et al., 2011). In view of limited human studies, it appears that the bioavailability determinations of pomegranate polyphenols is

folded, cut and stored in dry conditions (Maskan et al., 2002).

**3. The biochemistry and pharmokinetics of pomegranate** 

cerebroside (Tsuyuki et al., 1981).

spread on a slice of bread.

2005).

affected by individual variability, differential processing of pomegranate juice, and the analytical techniques used, which need to be sensitive enough to detect low postprandial concentrations of these metabolites (Basu & Penugonda, 2009). An *in vitro* study of pomegranate juice showed that phenolic compounds are available during the digestion in a quite high amount (29%), however, due to pH, anthocyanins are in large transformed into non-red forms and/or degraded and similar results are obtained for vitamin C (Pérez-Vicente et al., 2002).

The recent interest in pomegranate products is due to the fruit's beneficial role in the prevention of prostate cancer, the prevention of the oxidation of both low density lipoprotein (LDL), high density lipoprotein (HDL), and cholesterol, reductions in blood pressure, arthritis, anemia, diarrhea, inflammation, gynecological diseases, atherosclerosis development, the stimulation of T-cell functions and production of cytokines, Alzheimer's disease, and improvoment of sperm quality (Figure 1). These beneficial effects were attributed to the wide range of phytochemicals found in pomegranate. These phytochemicals are predominantly phenolic compounds as well as to those of sugarcontaining polyphenolic tannins and anthocyanins, including primarily hydrolysable ellagitannins, anthocyanins and other polyphenols. Gil et al. (2000) have demonstrated that one of the ellagitannins, punicalagins, is responsible for over 50% of the antioxidant activity of the pomegranate juice. The same reserachers indicated that as being water-soluble, commercial pomegranate juice obtained by pressing the fruit contain signicant amounts of punicalagins, depending on the cultivar. Seeram et al. (2005) proposed punicalagin as a proper chemical marker for the authentication, quality control and standardization of pomegranate products.

Fig. 1. Bioactive effects of pomegranate constituents.

### **4. The health benefits of pomegranate derived products**

In recent years, the focus is on understanding the mechanisms of nutraceutic and health promoting potentials of the foods nutrients. Cancer, in terms of morbidity and mortality, is a

The Therapeutic Potential of Pomegranate and Its Products for Prevention of Cancer 339

bioactive compounds to enhance reactive oxygen species (ROS) elimination and inhibit ROS generation. Oxidative stress, refers to a cell state characterized by excessive production of ROS, has been given growing attention, as the generation of ROS, thus improved oxidative stress, can induce DNA damage and trigger redox-dependent transcription factors which lead to cancer, inflammatory, cardiovascular and neurodegenerative diseases, and aging (Evans et al., 2004; Franco et al., 2008; Ziech et al., 2010; Sedelnikova et al., 2010; Kryston et al., 2011; Martin et al., 2011). Nishikawa (2008) mentioned that sublethal levels of ROS can induce additional changes in DNA of tumor cells to make those cells malignant, stimulate the proliferation of cancer cells, and activate the expression of various molecules, some of

The effect of pomegranate cultivars on antioxidant activity was target of study by some authors (Borochov-Neori et al., 2009; Mousavinejad et al., 2009; Pande & Akoh, 2009; Sadeghi et al. 2009). All authors reported considerable variation in some of the chemical composition profile (lipids, phenols, organic acids, vitamins, sugars) and antioxidant properties of pomegranate samples, independent on the antioxidant method performed. The antioxidant activity of pomegranate and its products was almost determined via *in vitro* trials and several methods could be used for its determination, however, pomegranate showed an antioxidant activity, independent on the antioxidant test assayed and generally with significant linear correlation between phenolic content and antioxidant capacity (Elfalleh et al., 2009). Seeram et al. (2005) stated that the antioxidant level in pomegranate juice was higher than found in other fruit juices, such as blueberry, cranberry, and orange. Schubert et al. (1999) and Gil et al. (2000) demonstrated that pomegranate juice and seed extracts have 2-3 times the *in vitro* antioxidant capacity of

Rosenblat et al. (2006) have shown that pomegranate extracts scavenge free radicals, and decrease macrophage oxidative stress and lipid peroxidation in animals. Studies in rats and mice confirmed the antioxidant properties of a pomegranate by-product extract made from whole fruit minus the juice, showing a 19% reduction in oxidative stress in mouse peritoneal macrophages (MPMs), a 42% decrease in cellular lipid peroxide content, and a 53% increase in reduced glutathione levels. A study in rats with chemically induced liver damage demonstrated that pretreatment with a methanolic extract of pomegranate peel enhanced or maintained the free-radical scavenging activity of the hepatic enzymes such as catalase, peroxidase, and superoxide dismutase to values comparable with control values, whereas resulted in 54% reduction of lipid peroxidation values compared to controls (Chidambara

Using the FRAP (ferric reducing/antioxidant power) assay, Guo et al. (2008) found that consumption of 250 mL pomegranate pulp juice daily for four weeks by healthy elderly subjects resulted in increased plasma antioxidant capacity, while subjects consuming apple juice experienced no significant increase. In addition, subjects consuming the pomegranate pulp juice exhibited significantly decreased plasma carbonyl content, a biomarker for

Several works have demonstrated that peel, seeds, arils have antioxidant activity, nevertheless, after ingestion those antioxidant compounds, mainly tannin components, are metabolized by gut bacteria into urolithins, which readily enter systemic circulation. Bialonska et al. (2009a) studied the antioxidant activities of seven urolithins derivatives in a

oxidant/antioxidant barrier impairment in various inflammatory diseases.

which assist cancer cells to form metastatic colonies.

either red wine or green tea.

Murthy et al., 2002).

major health issue, eventhough there are advances in early detection and in treatment options. Cancer is an aggressive disease, which if not detected at an early stage can metastasize to other organs of the body. *Carcinogenesis* (cancer development) is a multistage process, influenced by mainly age, dietary habits and hormonal balance. There are three stages of cancer: *initiation*, *promotion* and *progression* (Surh, 2003). The hypothesis of alternative methods to prevent cancer seems to be practical and promising strategy to reduce cancer incidinces since treatment options for metastasized cancers remain inadequate. Chemoprevention focuses on cancer prevention by the administration of one or more synthetic or naturally occurring agents to suppress reverse or prolong the process of carcinogenesis (Mukhtar & Ahmad, 1999).

It is clear that bioactive compounds present in daily diet, mainly in fruits and vegetables, have prevention potential in cancer by inhibiting carcinogenesis through cell-defensive and cell-death mechanism regulation. These chemopreventive effects may be attributed to a complex effect of various phenolic substances of antioxidant capacity (Khan et al., 2008).

Pomegranate is rich in anthocyanins, 3-glucosides, 3,5-diglucosides of delphinidin, cyanidin and pelargonidin, ellagitannins and other phenolic compounds, which are known bioactive compounds with antioxidant and antitumoral activity (Ozgen et al., 2008; Chaturvedula et al., 2011; Zhang et al., 2011). Major hydrolysable tannins in pomegranates are gallotannins, ellagic acid tannins and gallagyl tannins, generally termed as *punicalagins*, and they have been shown to inhibit the proliferation of human cancer cells and modulate inammatory subcellular signaling pathways due to a high antioxidant activity (Seeram et al., 2005).

There are several studies conducted to evaluate the efficacy of pomegranate and its products as an anti-proliferative, anti-invasive, and pro-apoptotic agent in various cancer cell lines such as skin, prostate, breast, column, and blood cancer. Adams et al. (2006) revealed that pomegranate juice suppresses cancer activity through the combined antioxidant and antiinflammatory effects by modulating inflammatory cell signaling in colon cancer cells. Malik et al. (2005) suggested that pomegranate juice may have cancer chemopreventive as well as cancer-chemotherapeutic effects against prostate cancer in humans. Pomegranate fruit extract possesses remarkable antitumor-promoting effects in mouse skin.

Researchers found that daily consumption of pomegranate juice may improve stressinduced myocardial ischemia in patients who have coronary heart disease (CHD) and the pomegranate juice not only prevented hardening of the arteries by reducing blood vessel damage, but also reversed the progression of CHD (Sumner et al., 2005). Hartman et al. (2006) reported that pomegranate juice had beneficial effect on animal model of Alzheimer's disease since polyphenols are responsible for neural protection.

#### **4.1 Antioxidant activity**

Over the past few years, consumer demand-based research on functional foods gave a basis for traditional using of pomegranate, which lead to an increase in number of scientific papers concerning pomegranate and its products with health-improving effects (Mehta & Lansky, 2004; Rettig et al., 2008; Turk et al., 2008; Alam et al., 2010; Dai et al., 2010; Jadeja et al., 2010; Park et al., 2010). The reports have focused on *in vitro, ex vivo,* and *in vivo* antioxidant actions, of pomegranate and its products, which are attributed to the chemical composition. However, many other cellular processes are likely to be involved along with

major health issue, eventhough there are advances in early detection and in treatment options. Cancer is an aggressive disease, which if not detected at an early stage can metastasize to other organs of the body. *Carcinogenesis* (cancer development) is a multistage process, influenced by mainly age, dietary habits and hormonal balance. There are three stages of cancer: *initiation*, *promotion* and *progression* (Surh, 2003). The hypothesis of alternative methods to prevent cancer seems to be practical and promising strategy to reduce cancer incidinces since treatment options for metastasized cancers remain inadequate. Chemoprevention focuses on cancer prevention by the administration of one or more synthetic or naturally occurring agents to suppress reverse or prolong the process of

It is clear that bioactive compounds present in daily diet, mainly in fruits and vegetables, have prevention potential in cancer by inhibiting carcinogenesis through cell-defensive and cell-death mechanism regulation. These chemopreventive effects may be attributed to a complex effect of various phenolic substances of antioxidant capacity (Khan et al., 2008).

Pomegranate is rich in anthocyanins, 3-glucosides, 3,5-diglucosides of delphinidin, cyanidin and pelargonidin, ellagitannins and other phenolic compounds, which are known bioactive compounds with antioxidant and antitumoral activity (Ozgen et al., 2008; Chaturvedula et al., 2011; Zhang et al., 2011). Major hydrolysable tannins in pomegranates are gallotannins, ellagic acid tannins and gallagyl tannins, generally termed as *punicalagins*, and they have been shown to inhibit the proliferation of human cancer cells and modulate inammatory subcellular signaling pathways due to a high antioxidant activity (Seeram et al., 2005).

There are several studies conducted to evaluate the efficacy of pomegranate and its products as an anti-proliferative, anti-invasive, and pro-apoptotic agent in various cancer cell lines such as skin, prostate, breast, column, and blood cancer. Adams et al. (2006) revealed that pomegranate juice suppresses cancer activity through the combined antioxidant and antiinflammatory effects by modulating inflammatory cell signaling in colon cancer cells. Malik et al. (2005) suggested that pomegranate juice may have cancer chemopreventive as well as cancer-chemotherapeutic effects against prostate cancer in humans. Pomegranate fruit

Researchers found that daily consumption of pomegranate juice may improve stressinduced myocardial ischemia in patients who have coronary heart disease (CHD) and the pomegranate juice not only prevented hardening of the arteries by reducing blood vessel damage, but also reversed the progression of CHD (Sumner et al., 2005). Hartman et al. (2006) reported that pomegranate juice had beneficial effect on animal model of Alzheimer's

Over the past few years, consumer demand-based research on functional foods gave a basis for traditional using of pomegranate, which lead to an increase in number of scientific papers concerning pomegranate and its products with health-improving effects (Mehta & Lansky, 2004; Rettig et al., 2008; Turk et al., 2008; Alam et al., 2010; Dai et al., 2010; Jadeja et al., 2010; Park et al., 2010). The reports have focused on *in vitro, ex vivo,* and *in vivo* antioxidant actions, of pomegranate and its products, which are attributed to the chemical composition. However, many other cellular processes are likely to be involved along with

extract possesses remarkable antitumor-promoting effects in mouse skin.

disease since polyphenols are responsible for neural protection.

**4.1 Antioxidant activity** 

carcinogenesis (Mukhtar & Ahmad, 1999).

bioactive compounds to enhance reactive oxygen species (ROS) elimination and inhibit ROS generation. Oxidative stress, refers to a cell state characterized by excessive production of ROS, has been given growing attention, as the generation of ROS, thus improved oxidative stress, can induce DNA damage and trigger redox-dependent transcription factors which lead to cancer, inflammatory, cardiovascular and neurodegenerative diseases, and aging (Evans et al., 2004; Franco et al., 2008; Ziech et al., 2010; Sedelnikova et al., 2010; Kryston et al., 2011; Martin et al., 2011). Nishikawa (2008) mentioned that sublethal levels of ROS can induce additional changes in DNA of tumor cells to make those cells malignant, stimulate the proliferation of cancer cells, and activate the expression of various molecules, some of which assist cancer cells to form metastatic colonies.

The effect of pomegranate cultivars on antioxidant activity was target of study by some authors (Borochov-Neori et al., 2009; Mousavinejad et al., 2009; Pande & Akoh, 2009; Sadeghi et al. 2009). All authors reported considerable variation in some of the chemical composition profile (lipids, phenols, organic acids, vitamins, sugars) and antioxidant properties of pomegranate samples, independent on the antioxidant method performed. The antioxidant activity of pomegranate and its products was almost determined via *in vitro* trials and several methods could be used for its determination, however, pomegranate showed an antioxidant activity, independent on the antioxidant test assayed and generally with significant linear correlation between phenolic content and antioxidant capacity (Elfalleh et al., 2009). Seeram et al. (2005) stated that the antioxidant level in pomegranate juice was higher than found in other fruit juices, such as blueberry, cranberry, and orange. Schubert et al. (1999) and Gil et al. (2000) demonstrated that pomegranate juice and seed extracts have 2-3 times the *in vitro* antioxidant capacity of either red wine or green tea.

Rosenblat et al. (2006) have shown that pomegranate extracts scavenge free radicals, and decrease macrophage oxidative stress and lipid peroxidation in animals. Studies in rats and mice confirmed the antioxidant properties of a pomegranate by-product extract made from whole fruit minus the juice, showing a 19% reduction in oxidative stress in mouse peritoneal macrophages (MPMs), a 42% decrease in cellular lipid peroxide content, and a 53% increase in reduced glutathione levels. A study in rats with chemically induced liver damage demonstrated that pretreatment with a methanolic extract of pomegranate peel enhanced or maintained the free-radical scavenging activity of the hepatic enzymes such as catalase, peroxidase, and superoxide dismutase to values comparable with control values, whereas resulted in 54% reduction of lipid peroxidation values compared to controls (Chidambara Murthy et al., 2002).

Using the FRAP (ferric reducing/antioxidant power) assay, Guo et al. (2008) found that consumption of 250 mL pomegranate pulp juice daily for four weeks by healthy elderly subjects resulted in increased plasma antioxidant capacity, while subjects consuming apple juice experienced no significant increase. In addition, subjects consuming the pomegranate pulp juice exhibited significantly decreased plasma carbonyl content, a biomarker for oxidant/antioxidant barrier impairment in various inflammatory diseases.

Several works have demonstrated that peel, seeds, arils have antioxidant activity, nevertheless, after ingestion those antioxidant compounds, mainly tannin components, are metabolized by gut bacteria into urolithins, which readily enter systemic circulation. Bialonska et al. (2009a) studied the antioxidant activities of seven urolithins derivatives in a

The Therapeutic Potential of Pomegranate and Its Products for Prevention of Cancer 341

after 10 days, whereas in rats treated with 2.5% gel, healing was observed on day 12, in contrast to the positive control animals receiving the blank gel, which took 16–18 days for complete healing. The animals treated with 2.5% gel showed moderate healing (55.8% and 40.8% healing compared with negative and positive controls, respectively), whereas the group treated with 5.0% gel showed good healing (59.5% and 44.5% healing compared with negative and positive controls, respectively). Histopathological studies supported the

Afaq et al. (2005) showed that pretreatment of mouse skin with pomegranate fruit extract modulated the activation of mitogen-activated protein kinase (MAPKs) and nuclear factor kappa B (NF-κB), in the 12-*O*-tetradecanoylphorbol 13-acetate (TPA)-induced or ultra violet-B induced skin carcinogenesis model. Aslam et al. (2006) assessed the cosmeceutical value of pomegranate where aqueous fraction of the peel was shown to stimulate type I procollagen synthesis and inhibit MMP-1 production by human dermal fibroblasts. Syed et al. (2006) reported the remarkable photochemopreventive effects of pomegranate fruit extract (PFE) against UVA using normal human epidermal keratinocytes (NHEK) as a test system. PFE, extracted edible part of fruit with acetone, treatment was shown to inhibit UVA-induced phosphorylation of STAT3, ERK1/2 and AKT1 in human epidermal cells. In addition, the inhibitory effect of PFE on UVA-mediated phosphorylation of mTOR and p70S6K may have a regulatory effect on the rate of protein synthesis and activation of

In a study pretreatment of EpiDerm with pomegranate juice, oil or by-product resulted in marked inhibition in the number of cyclobutane primidine dimers (CPDs) and 8-hydroxy-2 deoxyguanosine (8-OHdG) positive cells, ultimately, showing a protective effect of against UVB-mediated DNA damage. UVB irradiation results in the induction in metalloproteinases (MMPs) which degrade extracellular matrix proteins, and eventually, cause skin wrinkling. It was shown that all three components of pomegranate were able to inhibit UVB-induced expressions of MMPs as well as MMP-2 and MMP-9 activity in the EpiDerm (Zaid et al., 2007). Cell culture and animal studies have also supported that intake of pomegranate is associated with decreased skin cancer risk (Pacheco-Palencia et al., 2008). Afaq et al. (2009) found that pretreatment of human reconstituted skin (EpiDermTM FT-200) with pomegranate-derived products inhibited UVB-induced CPDs and 8-OHdG as well as protein oxidation and proliferating cell nuclear antigen (PCNA) protein expression. In addition they reported an inhibition of UVB-induced metalloproteinases (collagenase,

George et al. (2011) examined the chemopreventive efficacy of pomegranate fruit extract (PFE) and diallyl sulfide (DAS), alone and in combination, using 2-stage mouse skin tumorigenesis model. PFE alone delayed onset and tumor incidence by 55%, while in PFE+DAS combination at low doses synergistically decreased tumor incidence more potentially (84%). In addition, regression in tumor volume was seen with continuous combinatorial treatment (*p* < 0.01). Mechanistic studies revealed that this inhibition was associated with decreased expression of phosphorylated ERK1/2, JNK1 and activated NFκB/p65, IKKα, IκBα phosphorylation and degradation in skin tissue/tumor. Histological and cell death analysis also confirmed that combined PFE and DAS inhibit cellular

wound healing increased on application of the gels.

gelatinase, stromelysin, marilysin, elastase and tropoelastin).

proliferation and markedly induce apoptosis than the single agents.

tumor cell proliferation.

cell-based assay in order to reflect bioavailability of the test compound to the cells, and the antioxidant activity is evaluated in the cellular environment and in terms of inhibition of intracellular generation of reactive oxygen species. They found that urolithins exhibited a significant antioxidant activity correlated with the number of hydroxyl groups as well as lipophilicity of the molecules.
