The scope of this section is to review the current knowledge on the bioactivities of phytochemicals found in the pomegranate fruit. The emphasis is placed on those studies relating the observed bioactivities (in commercial or freshly prepared juice, peels, and seeds, with or without prefermentation) to specific phytochemical constituents of the pomegranate, even where such linkages have not yet been proven. The major in vitro and in vivo bioactivities evaluated to date have been the antioxidant and antiatherogenic properties of pomegranate juice (either fresh, commercial, or fermented), its extracts, and their purified compounds.
1.4.1 Pomegranate Fruit Phytochemicals As Antioxidants and Antiatherogenic Agents
In vitro antioxidant activities in different parts of the pomegranate fruit, described in a number of reports and groundbreaking research initiated and conducted in the Lipid Research Laboratory of Professor Michael Aviram (Haifa, Israel), is discussed in a separate chapter in this book. Some other studies are described below.
Employing the fermentation process, PJ with seeds was first fermented for 10 days and then concentrated to one-tenth its original volume to yield pomegranate fermented juice (PFJ) extract.50 The remaining seeds were separated, dried, and cold-pressed to form an additional product (PCPSO). The antioxidant activities of the PFJ and PCPSO were tested by the P-carotene linoleic acid method, as was their tendency to inhibit cyclooxygenase and lipoxygenase, key enzymes in the eicosanoid pathway catalyzing the oxidation of polyunsaturated fatty acids. The P-carotene linoleic acid method is based on determining the coupled oxidation of P-carotene and linoleic acid, then measuring P-carotene consumption as a result of its oxidation in the reaction mixture.5152 Both fermented products demonstrated moderate activity, less than the commercial synthetic antioxidant butylated hydroxyl anisole, and more than red wine. PFJ was not active as a cyclooxygenase inhibitor and only weakly inhibited lipoxygenase, while PCPSO revealed weak activity against cyclooxygenase and a strong inhibitory effect on lipoxygenase. As previously discussed, the major fatty-acid constituent in PCPSO is punicic acid, which is found in pomegranate seed oil (>60% of the total oil).
The antioxidant activity of polyphenols isolated from pomegranate seeds was determined by monitoring LDL susceptibility to oxidation and malondialdehyde (MDA) levels in the rat brain in vitro.11 Two new compounds were isolated and identified as coniferyl and sinapyl glycoside derivatives. These two compounds moderately decreased conjugated diene (CD) formation and significantly inhibited MDA production in the rat brain in a dose-dependent manner. The other known polyphenols isolated from the seeds were ellagic acid and its derivatives, which also demonstrated appreciable antioxidant activity.
The antioxidant activity in PJ that was freshly prepared from arils was compared to that in commercial PJ extracted from whole pomegranate, red wine, and green tea infusion.32 Antioxidant activity was determined using four different assays, three of them based on evaluations of the free-radical scavenging capacity of the juice (ABTS, DPPH, N,N-dimethyl-p-phenylenediamine-DMPD), and the fourth based on measuring its iron-reducing capacity (FRAP). The commercial juice revealed the highest activity, three times higher than that of red wine or green tea, and twice the antioxidant capacity of juice from fresh arils. An HPLC chromatogram of the two PJs showed that the commercial PJ includes phenolics additional to those present in the juice obtained from the arils, with a high content of punicalagins and ellagic acid derivatives. These results support the notion that the method of juice extraction plays an important role in its constitution and thus in its activity.32 The commercial juice had a significantly higher total phenolics content (2560 mg/L) relative to juice from either frozen or fresh arils (1800 and 2120 mg/L, respectively), consisting mostly of punicalagins and ellagic acid derivatives.
The antioxidant activity as determined by the P-carotene, linoleic acid, and DPPH systems of extract from pomegranate peel was compared with that from seeds. The methanolic extract has been found to be superior to that obtained with ethyl acetate or water.53 The methanolic peel extract was further tested as an inhibitor of lipid peroxidation, as a hydroxyl radical scavenger, and as an inhibitor of LDL oxidation. The antioxidant potency of the different peel extracts was correlated with their polyphenol content. However, no information was provided as to the type of constituents responsible for those activities, although the extracts were reported to contain ellagic and gallic acids that may probably be produced from the hydrolysis of ellagitannins and gallotannins, respectively.
Among the in vivo experiments aimed at evaluating the biological effects of pomegranate juice (PJ), Aviram et al.54 tested the effect of PJ consumption in male healthy and atherosclerotic, apolipoprotein E-deficient (E0) mice. The authors demonstrated that PJ consumption has antiatherogenic properties with respect to all three related components of atherosclerosis: it significantly affected plasma lipoproteins, arterial macrophages, and blood platelets, all of which was attributed to the effects of the PJ's antioxidant constituents, specifically to a fraction containing ellagitannins.54 In another study, Aviram and Dornfeld55 tested the effect of PJ consumption by hypertensive patients on their blood pressure and on serum angiotensin-converting enzyme (ACE) activity. Hypertension is a known risk factor for the development of atherosclerosis, whereas inhibitors of ACE, an enzyme facilitating the conversion of angiotensin I to angiotensin II, have been shown to attenuate the development of atherosclerosis in several animal studies.56 PJ consumption for 2 weeks caused a minimal reduction in blood pressure, but significantly decreased ACE activity by 36%; moreover, a small (5%) but significant reduction in systolic blood pressure was anticipated. It was assumed that the inhibition of ACE activity was either due to the direct action of specific inhibitors of ACE present in the juice and/or a secondary effect of PJ's antioxidants. In a separate randomized, double-blind, placebo-controlled study, PJ consumption for 3 months was shown to decrease stress-induced myocardial ischemia and improve myocardial perfusion in 45 patients with ischemic cardiovascular disease.69
Perturbed shear stress may trigger signal-transduction events that, in turn, can lead to endothelial dysfunction and enhanced atherogenesis.57 De Nigris and colleagues tested, in vitro and in vivo, the effect of PJ on oxidation-sensitive genes and on endothelial nitrous oxide synthase (eNOS) expression, induced by high shear stress, using cultured human coronary artery endothelial cells (EC) and hyper-cholesterolemic mice.58 Administration of PJ, diluted in the cellular medium of cultured human coronary artery EC exposed to laminar shear stress for 24 h, resulted in the reduced activation of redox-sensitive transcription factors (ELK-1, and p-JUN) and increased eNOS expression, both effects associated with antiatherogenic activity. Supplementation of PJ to hypercholesterolemic mice under oxidative stress (a high-fat diet) resulted in significantly lower plasma lipid peroxidation, a reduction in macrophage foam cell formation, and a 20% decrease in lesion area in atherosclerotic-prone regions, consistent with previous findings.54 A second group of hypercholes-terolemic mice, which was first allowed to develop the disease for 6 months and only then given PJ in their drinking water for 24 weeks, showed significantly reduced atherosclerotic progression, indicating that the proatherogenic effects induced by perturbed shear stress can be reversed by prolonged administration of PJ. These findings may have implications for the prevention or treatment of atherosclerosis. All of the aforementioned effects were attributed to polyphenol-rich PJ constituents, mainly its ellagitannins and anthocyanins.
Shear stress also mediated nuclear factor-kappa B (NF-kB) activation in vascular EC: such activation is associated with several pathologies, including atherosclerosis. Schubert et al. studied the ability of PJ fermented with wine yeast and then de-alcoholized and concentrated (PW) to inhibit tumor necrosis factor a (TNF-a) and NF-kB activation in vascular EC.59 PW proved to be a potent inhibitor of NF-kB activation; furthermore, it was shown that different antioxidants might have similar effects on TNF-a and NF-kB activation, but not necessarily through similar mechanisms of action.
Cerda et al. investigated the effects of supplementation with 1 L of juice/day (more than 5 g/day polyphenols, including 4.4 g/day punicalagin isomers) on rats by assaying a large number of hematological and serobiochemical parameters, such as LDL, high-density lipids (HDL), triglycerides and cholesterol, plasma and urine antioxidant activities (ability to donate electrons), and the presence of certain pomegranate constituents.60 None of the major polyphenols in the original juice (ellagi-tannins, ellagic acid derivatives, and anthocyanins) were detected in the human plasma or urine. The most potent in vitro antioxidant in the original PJ, punicalagin, was not detected (intact) in the human plasma or urine, nor was punicalin or ellagic acid conjugates. It is noteworthy that in a separate study, intact ellagic acid, resulting from the in vivo hydrolysis of ellagitannins, was detected in human plasma after the consumption of PJ.61 Cerda et al.60 also reported that despite the high antioxidant activity of the original juice in vitro, supplementation of PJ to healthy human subjects did not show any parallel significant increase in antioxidant activity of the plasma or urine, as assayed by various parameters. These results are in contrast to previous data showing increased plasma antioxidant capacity after PJ supplementation.54 The differences in results may be explained by the following observations: (a) the major known potent antioxidants, present in the original juice, were absent in the plasma or urine of the PJ-supplemented subjects, possibly due to their degradation and metabolism, (b) the antioxidant activity of the microfloral metabolites identified in the plasma or urine of those that consumed PJ are very weak, and (c) differences in antioxidant assay methodologies. For example, 3,8-dihydroxy-6H-dibenzo-pyran-6-one (urolithin A), an ellagitannin metabolite, showed 42- and 3570-fold lower activity than the parent compound, as assayed by the DPPH and ABTS antioxidant methods.60 Other major metabolites were identified as glucuronide derivatives, with also undetectable antioxidant activity.60
Both Aviram et al. and Cerda et al. agreed that pomegranate supplementation decreases LDL cholesterol and P-lipoprotein levels, whereas other atherogenic factors, such as VLDL cholesterol and LDL triglycerides, increase. The latter report concluded that supplementation of 1 L of PJ for 5 days to healthy volunteers does not result in any conclusive evidence of incurred health benefits with regards to antioxidative capacity. These authors also called for caution concerning the output of data regarding the relevance of in vitro antioxidant capacity of foodstuffs to their in vivo extrapolation.54,60
The effect of feeding a methanolic extract of pomegranate peel to albino rats on the toxic effects of carbon tetrachloride (CCl4) was examined using biochemical and histopathological assays.62 Analysis of the methanolic extract showed 42% (w/w) of the total phenol as catechin equivalents, with gallic and ellagic acids as the two major constituents. The levels of various reactive-oxygen-species (ROS)-combatting enzymes, such as catalase, superoxide dismutase (SOD), and peroxidase, as well as the amount of lipid peroxide in the liver homogenates, were examined. Results were compared with the effect of CCl4 on rats prefed the pomegranate peel extract. CCl4 and its metabolites have been extensively studied as liver toxicants. CCl4 reduced the levels of the aforementioned enzymes by 50 to 90%, and lipid peroxide increased about threefold. Pretreating the rats with the extract preserved the enzymes at control levels and reduced the lipid peroxide value to half of its control level. These protective effects could be attributed to the extract's high content of gallic and ellagic acids and possibly their polymers, all being potent free-radical scavengers.
In a recent study on arteriogenic erectile dysfunction done in a rabbit model, animals consuming PJ showed increased blood flow to the penis compared to control animals.70 In addition, the time to maximum pressure (maximum erection) decreased compared to controls, and nitric-oxide (NO)-mediated smooth-muscle relaxation was improved in the treatment animals. The authors attributed the effects to the antioxidant phytochemicals present in pomegranates.
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