Abstract : Pomegranate seed oil was investigated for possible skin cancer chemopreventive efficacy in mice. In the main experiment, two groups consisting each of 30, 4-5-week-old, female CD1 mice were used. Both groups had skin cancer initiated with an initial topical exposure of 7,12-dimethyl benzanthracene and with biweekly promotion using 12-O-tetra deca noylphorbol 13-acetate (TPA). The experimental group was pretreated with 5% pomegranate seed oil prior to each TPA application. Tumor incidence, the number of mice containing at least one tumor, was 100% and 93%, and multiplicity, the average number of tumors per mouse, was 20.8 and 16.3 per mouse after 20 weeks of promotion in the control and pomegranate seed oil-treated groups, respectively (P < .05). In a second experiment, two groups each consisting of three CD1 mice were used to assess the effect of pomegranate seed oil on TPAstimulated ornithine decarboxylase (ODC) activity, an important event in skin cancer promotion. Each group received a single topical application of TPA, with the experimental group receiving a topical treatment 1 h prior with 5% pomegranate seed oil. The mice were killed 5 h later, and ODC activity was assessed by radiometric method. The experimental group showed a 17% reduction in ODC activity. Pomegrante seed oil (5%) significantly decreased (P < .05) tumor incidence, multiplicity, and TPA-induced ODC activity. Overall, the results highlight the potential of pomegranate seed oil as a safe and effective chemopreventive agent against skin cancer.
Chemopreventive Effects of Pomegranate Seed Oil on Skin Tumor Development in CD1 Mice - Justin J. Hora, Emily R. Maydew, Ephraim P. Lansky, and Chandradhar Dwivedi. Journal of Medicinal Food. July 2004, 6(3): 157-161. doi 10.1089 / 10966200360716553

Multiple strands of research provide growing evidence that diet, nutrition, and life style play a role in the development and the course of urological diseases. Numerous micronutrients and polyphenols found in soy, green tea, and many fruits and vegetables have been described to impact diseases including erectile dysfunction, benign prostatic hyperplasia, and prostate cancer. However, oftentimes these reports lack both a scientific rationale and supportive evidence base. The efficacy of pomegranate, on the other hand, in the modulation of central biological processes like inflammation, hypoxia, and oxidative stress that are important in the pathogenesis of urological maladies has been robustly demonstrated in preclinical in vitro and in vivo studies. Moreover, clinical trials have further supported its use in the treatment of several diseases, in particular in themanagement of prostate cancer. Herein, we critically review the scientific knowledge about the current role and future prospects for the use of pomegranate extracts in the therapy of erectile dysfunction, benign prostatic hyperplasia, and prostate cancer.   The biological processes of inflammation, hypoxia, and oxidative stress have a crucial function in the natural biology of men’s urological diseases including ED, BPH, and PCA. In vitro and in vivo preclinical experiments provide evidence supporting that pomegranate extracts are able to :
(i) inhibit proliferation, invasion, metastatic spread, development of castration-resistant PCA growth, and angiogenesis,
(ii) modulate inflammatory pathways, and
(iii) reduce oxidative stress. Clinical biologic activities of pomegranate have been tested in subjects with PCA and ED. Further randomized, double-blind, controlled trials are under way and will be completed soon.
Pomegranate Extracts in the Management of Men’s Urologic Health: Scientific Rationale and Preclinical and Clinical Data Kroeger N., Belldegrun A. S., and Pantuck A. J. - Evidence-Based Complementary and Alternative Medicine - Volume 2013

Punica granatum, commonly known as pomegranate, is a member of the monogeneric family, Punicaceae, and is mainly found in Iran which is considered to be its primary centre of origin. Pg and its chemical components possess various pharmacological and toxicological properties including antioxidant, anti-inflammatory (by inhibiting pro-inflammatory cytokines), anti-cancer and anti-angiogenesis activities. They also show inhibitory effects on invasion/motility, cell cycle, apoptosis, and vital enzymes such as cyclooxygenase (COX), lipooxygenase (LOX), cytochrome P450 (CYP450), phospholipase A2 (PLA2), ornithine decarboxylase (ODC), carbonic anhydrase (CA), 17 beta-hydroxysteroid dehydrogenase (17β-HSDs) and serine protease (SP). Furthermore, they can stimulate cell differentiation and possess anti-mutagenic effects. Pg can also interfere with several signaling pathways including PI3K/AKT, mTOR, PI3K, Bcl-X, Bax, Bad, MAPK, ERK1/2, P38, JNK, and caspase. However, the exact mechanisms for its pharmacological and toxicological properties remain to be unclear and need further evaluation. These properties strongly suggest a wide range use of pomegranate for clinical applications. This review will discuss the areas for which pomegranate has shown therapeutic properties in different mechanisms.
This suggests that Pg can be extensively used as a possible therapy for prevention and treatment of several types of diseases including prostate cancer, colon cancer, breast cancer, lung cancer, skin cancer, leukemia, anti-atherosclerosis, hyperlipidemia, hypertension, myocardial ischemia, myocardial perfusion, diabetes, oral inflammation, infection, anti-erectile dysfunction, male infertility, neonatal hypoxia-ischemic brain injury, alzheimer and obesity.
Keywords: Punica granatum; Chemical components; Toxicological properties; Signaling pathway; Clinical applications. [PTPOMEG40P]
(...to be continued)
A Comprehensive Review of Punica granatum (Pomegranate) Properties inToxicological, Pharmacological, Cellular and Molecular Biology Researches - Iranian Journal of Pharmaceutical Research (2012), 11 (2): 385-400 - Hamid Reza Rahimia, Mohammad Arastoob and Seyed Nasser Ostada

Abstract : Normalization of dyslipidemia has been shown to prevent or reduce the risk of cardiovascular disease. In order to avoid the adverse effects associated with pharmacologic therapies, research has been focusing on non-pharmacologic hypolipidemic alternatives. In vitro and in vivo studies have shown that punicic acid (PA), the conjugated fatty acid which is the main constituent of pomegranate (Punica granatum) seed oil (PSO), has anti-atherogenic effects. PSO consists of about 80% conjugated octadecatrienoic fatty acids, with a high content of 9-cis-, 11-trans-, 13-cisacid or PA, one of the isomers of conjugated linolenic acid (CLN).1 This double-blind, placebo-controlled, randomized clinical trial was aimed at determining the effect of PSO treatment on serum lipid profiles of hyperlipidemic subjects.
Forty-five hyperlipidemic subjects aged over 20 years, with body mass index (BMI) max 35 kg/m2, serum total cholesterol min 5.2 mmol/l, and serum triacylglycerol (TAG) min 1.65 mmol/l were randomly assigned to the treatment (n = 25) or the control (n = 26) groups, and received 400 mg of PSO (Pometane; Vitane Pharma Gmbh Inc; Wolfratshausen, Germany) or placebo twice daily for 4 weeks. Mean values for baseline body weight and age were 74.2 [standard deviation (SD) 10.0] and 75.7 (SD 12.2) kg, and 51 (SD 9) and 55 (SD 9) years for the PSO and placebo groups, respectively. No significant differences between the groups were seen for age, sex, weight, height, dietary intake, physical activity, and consumption of lipid lowering drugs. Serum concentrations of lipids and lipoproteins, as well as waist:hip ratio and body composition, were measured before and 4 weeks after intervention. Differences between the 2 groups at baseline were tested with Student's t test and the Mann-Whitney test. Paired Student's t test and Wilcoxon ranked test were used to compare the baseline and 4-week values in each group. Analysis of covariance was used to distinguish treatment effect between the groups following adjustment of their baseline values.
Oral Administration of Pomegranate Seed Oil Shows Favorable Effects on Lipid Profiles of Hyperlipidemic Subjects Mirmiran P, Fazeli MR, Asghari G, Shafiee A, Azizi F, Br J Nutr. 2010;104: 402-406.

Prostate cancer is the second leading cause of cancer deaths in men in the United States. There is a major need for less toxic but yet effective therapies to treat prostate cancer. Pomegranate fruit from the tree Punica granatum has been used for centuries for medicinal purposes and is described as “nature’s power fruit”. Recent research has shown that pomegranate juice (PJ) and/or pomegranate extracts (PE) significantly inhibit the growth of prostate cancer cells in culture. In preclinical murine models, PJ and/or PE inhibit growth and angiogenesis of prostate tumors. More recently, we have shown that three components of PJ, luteolin, ellagic acid and punicic acid together, have similar inhibitory effects on prostate cancer growth, angiogenesis and metastasis. Results from clinical trials are also promising. PJ and/or PE significantly prolonged the prostate specific antigen (PSA) doubling time in patients with prostate cancer. In this review we discuss data on the effects of PJ and PE on prostate cancer. We also discuss the effects of specific components of the pomegranate fruit and how they have been used to study the mechanisms involved in prostate cancer progression and their potential to be used in deterring prostate cancer metastasis.
Keywords: natural products; metastasis; luteolin; ellagic acid; punicic acid
In summary, the biological activity of pomegranate-derived products, especially the chemotherapeutic and chemopreventive properties, has been investigated in cell, animal and clinical studies. The findings discussed in this review show that pomegranate and its components interfere with multiple biological processes involved in tumor growth, angiogenesis and metastasis of PCa. Because many of the molecular mechanisms are shared by different types of cancers, and the fact that PE has been shown to be effective against breast, lung, colon and skin cancer [105] further enhances the therapeutic potential of PE. Therefore, further studies are warranted, including clinical trials with appropriate control groups using well-characterized and standardized amounts of PJ, PE and specific components as primary or adjuvant therapy in men with PCa. Many of the molecular mechanisms involved in the PJ/PE or L + E + P are amenable to drug treatment and to the development of small inhibitory molecules and therefore allow for combination therapy. Therefore, pomegranate and its components can potentially be used to prevent development and progression of PCa as well as other cancers. What is not known and is of great importance is whether the power of PJ/PE or L + E + P can be used as preventive therapies.
Pomegranate and Its Components as Alternative Treatment for Prostate Cancer - Lei Wang and Manuela Martins-Green - Department of Cell Biology and Neuroscience, University of California Riverside, Riverside, CA 92521, USA

Abstract : Prostate cancer is a commonly diagnosed cancer in men, and dietary chemoprevention by pomegranate (Punica granatum) extracts has shown noticeable benefits. In this study, we investigated the growth inhibitory, antiandrogenic, and pro-apoptotic effects of 13 pure compounds found in the pomegranate in androgen-dependent LNCaP human prostate cancer cells. Cells deprived of steroid hormones were exposed to increasing concentrations (1−100 μM) of pomegranate compounds in the presence of 0.1 nM dihydrotestosterone (DHT), and the inhibition of cell growth was measured by WST-1 colorimetric assay after a 4 day exposure.
Four compounds, epigallocatechin gallate (EGCG), delphinidin chloride, kaempferol, and punicic acid, were found to inhibit DHT-stimulated cell growth at concentrations of 10 μM and above. These four pomegranate compounds inhibited DHT-stimulated androgen receptor nuclear accumulation and the expression of the androgen receptor-dependent genes prostate specific antigen and steroid 5α-reductase type 1 at concentrations ≥10 μM. We determined the possible contribution of apoptosis to the observed decrease in cell growth and found that three compounds, EGCG, kaempferol, and, in particular, punicic acid, induced DNA fragmentation after a 24 h treatment, at concentrations in the 10−100 μM range. Punicic acid, an important fatty acid in pomegranate seeds, was further found to induce intrinsic apoptosis via a caspase-dependent pathway.
In conclusion, punicic acid, the main constituent of pomegranate seed (70−80%), exhibited potent growth inhibitory activities in androgen-dependent LNCaP cells, which appear to be mediated by both antiandrogenic and pro-apoptotic mechanisms.
Keywords : apoptosis; LNCaP; Pomegranate; PSA; punicic acid; SRD5A1
Growth Inhibitory, Antiandrogenic, and Pro-apoptotic Effects of Punicic Acid in LNCaP Human Prostate Cancer Cells - Jihane Gasmi and J. Thomas Sanderson - J. Agric. Food Chem., 2010, 58 (23), pp 12149– 12156, DOI: 10.1021/ jf103306k, Publication Date (Web): November 10, 2010

Abstract : The pomegranate seed oil (PSO) presents a typical fatty acid profile which includes high percentages of the punicic acid (PA) – a conjugated isomer of alpha-Linolenic acid. Conjugated alpha-Linolenic acids (CLnAs) are a collective term for the positional and geometric isomers of octadecatrienoic acid (C18:3) with conjugated double bonds. Recently, conjugated fatty acids have attracted significant attention due to reports of its health benefits in a variety of models of metabolic diseases and chronic inflammatory diseases. However, some work is controversial and there is still no consensus in the literature regarding their effects on animal and human organisms. This article presents a review under the pomegranate seed oil and the potential effects of conjugated a-linolenic acid to health.
Pomegranate Seed Oil (Punica Granatum L.): A Source of Punicic Acid (Conjugated alpha-Linolenic Acid) - Journal of Human Nutrition and Food Science, Illana Louise Pereira de Melo, Eliane Bonifácio Teixeira de Carvalho, Jorge Mancini-Filho

Abstract : Prostate cancer (PCa) is the second cause of cancer deaths in men in the USA. When the cancer recurs, early stages can be controlled with hormone ablation therapy to delay the rate of cancer progression but, over time, the cancer overcomes its hormone dependence, becomes highly aggressive and metastasizes. Clinical trials have shown that pomegranate juice (PJ) inhibits PCa progression. We have previously shown that the PJ components luteolin (L), ellagic acid (E) and punicic acid (P) together inhibit growth of hormonedependent and -independent PCa cells and inhibit their migration and chemotaxis towards CXCL12, a chemokine that is important in PCa metastasis. On the basis of these findings, we hypothesized that L+E+P inhibit PCa metastasis in vivo. To test this possibility, we used a severe combined immunodeficiency mouse model in which luciferase-expressing human PCa cells were injected subcutaneously near the prostate. Tumor progression was monitored with bioluminescence imaging weekly. We found that L+E+P inhibits PC-3M-luc primary tumor growth, inhibits the CXCL12/ CXCR4 axis for metastasis and none of the tumors metastasized.
In addition, L+E+P significantly inhibits growth and metastasis of highly invasive Pten−/−;K-rasG12D prostate tumors. Furthermore, L+E+P inhibits angiogenesis in vivo, prevents human endothelial cell (EC) tube formation in culture and disrupts preformed EC tubes, indicating inhibition of EC adhesion to each other. L+E+P also inhibits the angiogenic factors interleukin-8 and vascular endothelial growth factor as well as their induced signaling pathways in ECs.
In conclusion, these results show that L+E+P inhibits PCa progression and metastasis.
Luteolin, ellagic acid and punicic acid are natural products that inhibit prostate cancer metastasis - Lei Wang, Wenfang Li, Muqing Lin, Monika Garcia, David Mulholland, Michael Lilly and Manuela Martins-Green, Carcinogenesis vol.35 no.10 pp.2321–2330, 2014

Cancer is the second leading cause of death and is becoming the leading one in old age. Vegetable and fruit consumption is inversely associated with cancer incidence and mortality. Currently, interest in a number of fruits high in polyphenols has been raised due to their reported chemopreventive and/or chemotherapeutic potential. Pomegranate has been shown to exert anticancer activity, which is generally attributed to its high content of polyphenols. This review provides a comprehensive analysis of known targets and mechanisms along with a critical evaluation of pomegranate polyphenols as future anticancer agents. Pomegranate evokes antiproliferative, anti-invasive, and antimetastatic effects, induces apoptosis through the modulation of Bcl-2 proteins, upregulates p21 and p27, and downregulates cyclin-cdk network. Furthermore, pomegranate blocks the activation of inflammatory pathways including, but not limited to, the NF-𝜅B pathway.The strongest evidence for its anticancer activity comes from studies on prostate cancer. Accordingly, some exploratory clinical studies investigating pomegranate found a trend of efficacy in increasing prostatespecific antigen doubling time in patients with prostate cancer. However, the genotoxicity reported for pomegranate raised certain concerns over its safety and an accurate assessment of the risk/benefit should be performed before suggesting the use of pomegranate or its polyphenols for cancer-related therapeutic purposes.
Potential Effects of Pomegranate Polyphenols in Cancer, Prevention and Therapy, Eleonora Turrini, Lorenzo Ferruzzi, and Carmela Fimognari, Oxidative Medicine and Cellular Longevity, Volume 2015, Article ID 938475, 19 pages

Abstract : The consumption of pomegranate juice (PJ), a rich source of antioxidant polyphenols, has grown tremendously due to its reported health benefits. Pomegranate extracts, which incorporate the major antioxidants found in pomegranates, namely, ellagitannins, have been developed as botanical dietary supplements to provide an alternative convenient form for consuming the bioactive polyphenols found in PJ. Despite the commercial availability of pomegranate extract dietary supplements, there have been no studies evaluating their safety in human subjects. A pomegranate ellagitannin-enriched polyphenol extract (POMx) was prepared for dietary supplement use and evaluated in two pilot clinical studies. Study 1 was designed for safety assessment in 64 overweight individuals with increased waist size. The subjects consumed either one or two POMx capsules per day providing 710 mg (435 mg of gallic acid equivalents, GAEs) or 1420 mg (870 mg of GAEs) of extracts, respectively, and placebo (0 mg of GAEs). Safety laboratory determinations, including complete blood count (CBC), chemistry, and urinalysis, were made at each of three visits. Study 2 was designed for antioxidant activity assessment in 22 overweight subjects by administration of two POMx capsules per day providing 1000 mg (610 mg of GAEs) of extract versus baseline measurements. Measurement of antioxidant activity as evidenced by thiobarbituric acid reactive substances (TBARS) in plasma were measured before and after POMx supplementation. There was evidence of antioxidant activity through a significant reduction in TBARS linked with cardiovascular disease risk. There were no serious adverse events in any subject studied at either site. These studies demonstrate the safety of a pomegranate ellagitannin-enriched polyphenol dietary supplement in humans and provide evidence of antioxidant activity in humans.
Keyword : Pomegranate extract; polyphenol; POMx; ellagitannins; safety; TBARS; human; Pomegranate; Punicosides
Safety and Antioxidant Activity of a Pomegranate Ellagitannin-Enriched Polyphenol Dietary Supplement in Overweight Individuals with Increased Waist Size, David Heber, Navindra P. Seeram, Holly Wyatt, 10050 J. Agric. Food Chem. 2007, 55 10050– 10054

ABSTRACT : Punicic acid (PA) is a polyunsaturated fatty acid (18:3 n-5), which is classified as a conjugated linolenic acid. PA is also referred as a "super CLnA" whose effect is even more potent than that of an ordinary CLnA. It is found mainly in the seeds of pomegranate fruit (Punica granatum) and Trichoxanthes kirilowii and some other minor sources. It possesses a wide array of biological properties including antidiabetic, antiobesity, antiproliferative, and anticarcinogenic activity against various forms of cancer. In spite of this, PA has not been explored as a nutraceutical or as an ingredient of food products which can be aimed at specific consumer target groups. This review details the various health-beneficial properties of PA and explores the possibilities of its utilization as an active ingredient in various food products.
CONLUSION :Pomegranate per se has attracted human attention due to its array of health benefits, including antioxidant, anticancer, anti-inflammatory, and antidiabetic and also many other properties.The peel, membranes, and seeds are inadvertently produced bythe processing industries (known as “Marc”; Qu and others 2010),which becomes a burden in terms of environmental pollution, ow-ing to its high biological oxygen demand, and it is used as eithercattle feed or is discarded. The above description focuses on thehealth-beneficial aspects of seeds as seed oil with a high contentof PA. This, in turn, could be utilized for the development ofhealth-beneficial (including food) products, targeted for preciseconsumers in particular and also general consumers. A cursorysurvey of the literature reveals that it has not yet moved food in-dustries to see it as a functional ingredient. The exploitation ofPSO by industries would not only pave a mode for waste utiliza-tion but also toward the development of value-added products.PSO with a very high concentration of NOAEL, does not haveany safety issues. Only 1 or 2 reports describing the pro- andantioxidant role of PA at different concentrations may be neces-sary to start its judicious incorporation in food products; and suchproducts developed with PA may then be evaluated for their healthbenefits. Studies pertaining to the stability of PA in food products could be another line of future research.
Keywords: conjugated linolenic acid, eleostearic acid, pomegranate seed oil, punicic acid
Health Benefits of Punicic Acid: A Review P. Aruna, D. Venkataramanamma, Alok Kumar Singh, R.P. Singh, First published: 21 October 2015, DOI: 10.1111/ 1541-4337 . 12171

Abstract : We completed a multicenter study of the effects of pomegranate cold-pressed (Oil) or supercritical CO2-extracted (S) seed oil, fermented juice polyphenols (W), and pericarp polyphenols (P) on human prostate cancer cell xenograft growth in vivo, and/or proliferation, cell cycle distribution, apoptosis, gene expression, and invasion across Matrigel, in vitro. Oil, W, and P each acutely inhibited in vitro proliferation of LNCaP, PC-3, and DU 145 human cancer cell lines.
Growth of the prostate gland is governed by a complex milieu of hormonal factors signaling, in part, via a range of nuclear receptors. The receptors act as a homeostatic mechanism by sensing a diverse range of dietary, xenobiotic, and environmental factors. For example, phytoestrogens and flavonoids are able to regulate estrogen and peroxisome proliferator-activated receptors. The pomegranate fractions may target nuclear receptor- mediated gene regulation and thereby regulate cell proliferation.
The dose of P required to inhibit cell proliferation of the prostate cancer cell line LNCaP by 50% (ED50) was 70 mg/mL, whereas normal prostate epithelial cells (hPrEC) were significantly less affected (ED50 5 250 mg/mL). These effects were mediated by changes in both cell cycle distribution and induction of apoptosis. For example, the androgenindependent cell line DU 145 showed a significant increase from 11% to 22% in G2/M cells (P , .05) by treatment with Oil (35 μg/mL) with a modest induction of apoptosis. In other cell lines/treatments, the apoptotic response predominated, for example, in PC-3 cells treated with P, at least partially through a caspase 3-mediated pathway.
These cellular effects coincided with rapid changes in mRNA levels of gene targets. Thus, 4-hour treatment of DU 145 cells with Oil (35 μg/mL) resulted in significant 2.3 ± 0.001-fold (mean ± SEM) up-regulation of the cyclin-dependent kinase inhibitor p21(waf1/cip1) (P max .01) and 0.6 ± 0.14-fold down-regulation of c-myc (P max .05). In parallel, all agents potently suppressed PC-3 invasion through Matrigel, and furthermore P and S demonstrated potent inhibition of PC-3 xenograft growth in athymic mice. Overall, this study demonstrates significant antitumor activity of pomegranatederived materials against human prostate cancer.
Pomegranate Extracts Potently Suppress Proliferation, Xenograft Growth, and Invasion of Human Prostate Cancer Cells - Martin Albrecht, Wen guo Jiang, James Kumi-Diaka, Ephraim P. Lansky, Lyndon M. Gommersall, Amit Patel, Robert E. Mansel, Ishak Neeman, Albert A. Geldof, and Moray J. Campbell. Journal of Medicinal Food. September 2004

Abstract : Breast cancer is the most common cancer and the second leading cause of cancer death and morbidity among women in the western world. Pomegranate juice (PJ) and three of its specific components have been shown to inhibit processes involved in prostate cancer metastasis. If this also proves to be true for breast cancer, these natural treatments will be promising agents against breast cancer that can serve as potentially effective and nontoxic alternatives or adjuncts to the use of conventional selective estrogen receptor modulators for breast cancer prevention and treatment. To test this possibility, we have used two breast cancer cell lines, MDA-MB-231 cells (ER ) and MCF7 (ER ), and the non-neoplastic cell line MCF10A. We show that, in addition to inhibiting growth of the breast cancer cells, PJ or a combination of its components luteolin (L) + ellagic acid (E) + punicic acid (P) increase cancer cell adhesion and decrease cancer cell migration but do not affect normal cells. These treatments also inhibit chemotaxis of the cancer cells to SDF1α, a chemokine that attracts breast cancer cells to the bone. We hypothesized that PJ and L + E + P stimulate expression of genes that increase adhesion and inhibit genes that stimulate cell migration and inhibit chemotaxis to SDF1α. Using qPCR, we confirmed these proposed effects on gene expression and in addition we found that a gene important in epithelial- to- meshenchymal transitions is decreased. We also found that proinflammatory cytokines/chemokines are significantly reduced by these treatments, thereby having the potential to decrease inflammation and its impact on cancer progression. Discovery that PJ and L + E + P are inhibitory of metastatic processes in breast cancer cells in addition to prostate cancer cells indicate that they are potentially a very effective treatment to prevent cancer progression in general.
Pomegranate juice and specific components inhibit cell and molecular processes critical for metastasis of breast cancer Breast Cancer, Research and Treatment December 2012, Volume 136, Issue 3, pp 647–658 Ana Rocha, Lei Wang, Manuel Penichet, Manuela Martins-Green

Abstract This article aims to determine the phenolic, tocopherol contents, and antioxidant capacities from fruits (juices, peels, and seed oils) of 6 Tunisian pomegranate ecotypes. Total anthocyanins were determined by a differential pH method. Hydrolyzable tannins were determined with potassium iodate. The tocopherol (α-tocopherol, γ -tocopherol, and δ-tocopherol) contents were, respectively, 165.77, 107.38, and 27.29 mg/100 g from dry seed. Four phenolic compounds were identified and quantified in pomegranate peel and pulp using the high-performance liquid chromatography - ultraviolet method: 2 hydroxybenzoic acids (gallic and ellagic acids) and 2 hydroxycinnamic acids (caffeic and p-coumaric acids). Juice, peel, and seed oil antioxidants were confirmed by ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) methods. The highest values were recorded in peels with 25.63 mmol trolox equivalent/100 g and 22.08 mmol TE/100 g for FRAP and ORAC assay, respectively.
Results showed that the antioxidant potency of pomegranate extracts was correlated with their phenolic compound content. In particular, the highest correlation was reported in peels. High correlations were also found between peel hydroxybenzoic acids and FRAP ORAC antioxidant capacities. Identified tocopherols seem to contribute in major part to the antioxidant activity of seed oil. The results implied that bioactive compounds from the peel might be potential resources for the development of antioxidant function dietary food.
Keyword antioxidant capacities, hydrolyzable tannins, Punica granatum L., tocopherols, total phenolics.
Antioxidant Capacities of Phenolic Compounds and Tocopherols from Pomegranate (Punica granatum) Fruits, 2011 Institute of Food Technologists, doi: 10.1111/ j.1750 - 3841. 2011. 02179.x, Walid Elfalleh, Nizar Tlili, Nizar Nasri, Yassine Yahia, H´edia Hannachi, Nizar Chaira, Ma Ying, and Ali Ferchichi

The objective of this study was to investigate the incorporation and metabolism of punicic acid (PA, cis9,trans11,cis13-18:3) in healthy young humans. The study was a randomized controlled trial. After 7 days adaptation with sunflower seed kernels supplementation, 30 subjects were then divided into the control and test group (n = 15). The test group was supplemented with Trichosanthes kirilowii (TK) seed kernels containing 3 g of PA per day in the form of triacylglycerols for 28 days. The control group was provided with sunflower seed kernels. After consumption of TK seeds containing 3 g PA per day for 28 days, the proportion of PA was increased from 0.00 to 0.47% in plasma and 0.00 to 0.37% in red blood cell membranes (RBCM), respectively. The proportion of cis9,trans11-18:2 was increased from 0.05 to 0.23% in plasma and 0.03 to 0.17% in RBCM after 28 days of intervention, respectively. Our results suggest that PA can be effectively incorporated into human plasma and RBCM, and is also associated with the increasing proportion of cis9,trans11-18:2 in humans, presumably as a result of metabolism by a saturation reaction. Edible TK seeds could be a potential dietary source of conjugated linoleic acids.
Incorporation and metabolism of punicic acid in healthy young humans - Mol Nutr Food Res. 2009 Oct;53(10):1336-42. doi: 10.1002/ mnfr. 200800520.

Abstract : Inhibition of lipid peroxidation contributes to the attenuation of macrophage cholesterol accumulation, foam-cell formation and atherosclerosis. Evidence suggests that nutritional antioxidants such as pomegranate juice (PJ) can contribute to the reduction of oxidative stress and atherogenesis. The goals of the present study were to determine whether such beneficial effects of PJ exist when supplemented to apolipoprotein E-deficient (E0) mice with advanced atherosclerosis and to analyze the antiatherosclerotic activity of a tannin-fraction isolated from PJ. Mice (4-mo-old) were supplemented with PJ in their drinking water for 2 mo and compared with age-matched placebo-treated mice, as well as to young (4-mo-old) control mice, for their mouse peritoneal macrophage (MPM) oxidative state, cholesterol flux and mice atherosclerotic lesion size. PJ supplementation reduced each of the proatherogenic variables determined in the present study compared with age-matched placebo-treated mice. It significantly induced serum paraoxonase activity and reduced MPM lipid peroxide content compared with placebo-treated mice and control mice. PJ administration to E0 mice significantly reduced the oxidized (Ox)-LDL MPM uptake by 31% and MPM cholesterol esterification and increased macrophage cholesterol efflux by 39% compared with age-matched, placebo-treated mice. PJ consumption reduced macrophage Ox-LDL uptake and cholesterol esterification to levels lower than those in 4-mo-old, unsupplemented controls. PJ supplementation to E0 mice with advanced atherosclerosis reduced the lesion size by 17% compared with placebo-treated mice. In a separate study, supplementation of young (2-mo-old) E0 mice for 2 mo with a tannin fraction isolated from PJ reduced their atherosclerotic lesion size, paralleled by reduced plasma lipid peroxidation and decreased Ox-LDL MPM uptake. PJ supplementation to mice with advanced atherosclerosis reduced their macrophage oxidative stress, their macrophage cholesterol flux and even attenuated the development of atherosclerosis. Moreover, a tannin-fraction isolated from PJ had a significant antiatherosclerotic activity.
In conclusion, PJ supplementation to E° mice possesses very impressive antiatherogenic properties, which could be related to its potent antioxidative activity and beneficial effect on macrophage cholesterol flux, which results in decreased macrophage cholesterol accumulation. The effect of PJ consumption on atherosclerosis was shown not only when supplementation of PJ to E° mice started before they developed atherosclerotic lesions, but also in mice with extensive atherosclerosis. The above antiatherosclerotic properties of PJ could be related to the presence of a tannin fraction in PJ with potent antioxidative characteristics.
Pomegranate Juice Supplementation to Atherosclerotic Mice Reduces Macrophage Lipid Peroxidation, Cellular Cholesterol Accumulation and Development of Atherosclerosis, 2001 American Society for Nutritional Sciences, J. Nutr. 131: 2082–2089, 2001

Atherosclerosis is enhanced in arterial segments exposed to disturbed flow. Perturbed shear stress increases the expression of oxidation-sensitive responsive genes (such as ELK-1 and p-JUN) in the endothelium. Evidence suggests that polyphenolic antioxidants contained in the juice derived from the pomegranate can contribute to the reduction of oxidative stress and atherogenesis.
The aim of the present study was to evaluate the effects of intervention with pomegranate juice (PJ) on oxidation-sensitive genes and endothelial NO synthase (eNOS) expression induced by high shear stress in vitro and in vivo. Cultured human coronary artery endothelial cells (EC) exposed to high shear stress in vitro and hypercholesterolemic mice were used in this study. PJ concentrate reduced the activation of redox-sensitive genes (ELK-1 and p-JUN) and increased eNOS expression (which was decreased by perturbed shear stress) in cultured EC and in atherosclerosis-prone areas of hypercholesterolemic mice. Moreover, oral administration of PJ to hypercholesterolemic mice at various stages of disease reduced significantly the progression of atherosclerosis. This experimental study indicates that the proatherogenic effects induced by perturbed shear stress can be reversed by chronic administration of PJ. This approach may have implications for the preventionor treatment of atherosclerosis and its clinical manifestations.
Beneficial effects of pomegranate juice on oxidation-sensitive genes and endothelial nitric oxide synthase activity at sites of perturbed shear stress, 4896–490, PNAS March 29, 2005 vol. 102 no. 13 Filomena de Nigris, Sharon Williams-Ignarro, Lilach O. Lerman, Ettore Crimi, Chiara Botti

Several mechanistic studies in cell culture and mouse models suggest possible estrogen receptor-mediated and non-estrogen receptor-mediated benefits of pomegranate juice with respect to breast cancer risk. These studies demonstrate that various constituents of pomegranates can inhibit aromatase and 17β-hydroxysteroid dehydrogenase enzymes or have antiestrogenic activity. Additional large, well-controlled human studies are warranted to elucidate the effects of pomegranate juice intake on serum hormone levels. Clarifying the effects of pomegranate constituents on key hormones known to be involved in breast cancer could result in important information for consumers and shed further light on the impact of diet on breast cancer risk
Pomegranate and breast cancer: possible mechanisms of prevention - Susan R Sturgeon, Alayne G Ronnenberg

Abstract : Pomegranate (Punica granatum L.) fruit is widely consumed as fresh fruit and juice. Because of its potential for health benefits, pomegranate fruit extracts have been commonly marketed as dietary supplements in recent years. The objective of the present study was to investigate potential adverse effects, if any, of a standardized pomegranate fruit extract in rats following subchronic administration. The extract was standardized to 30% punicalagins, the active anomeric ellagitannins responsible for over 50% of the antioxidant potential of the juice. The oral LD50 of the extract in rats and mice was found to be greater than 5 g/kg body weight. The intraperitoneal LD50 in rats and mice was determined as 217 and 187 mg/kg body weight, respectively. In the subchronic study, Wistar strain rats (10/sex/group) were administered via gavage 0 (control), 60, 240 and 600 mg/kg body weight/day of the extract for 90 days. Two additional groups received 0 and 600 mg/kg/day of the extract for 90 days, followed by a 28 day recovery phase. Compared to the control group, administration of the extract did not result in any toxicologically significant treatment-related changes in clinical observations, ophthalmic examinations, body weights, body weight gains, feed consumption, clinical pathology evaluations and organ weights. The hematology and serum chemistry parameters that showed statistical significant changes compared to control group were within the normal laboratory limits and were considered as biological variations and not the toxic effect of the extract. Terminal necropsy did not reveal any treatment-related gross or histopathology findings. Based on the results of this study, the no observed-adverse-effect level (NOAEL) for this standardized pomegranate fruit extract was determined as 600 mg/kg body weight/day, the highest dose tested.
(...to be continued)
Safety assessment of pomegranate fruit extract: Acute and subchronic toxicity studies - by Chintan Patel, Paresh Dadhaniya, Lal Hingorani, M.G. Soni, Food and Chemical Toxicology 46 (2008) 2728– 2735

Abstract : Current investigation focuses on the toxicity evaluation of whole fruit hydroalcoholic extract of Punica granatum L. Punicaceae), used in Cuban traditional medicine a.o. for the treatment of respiratory diseases. Previous findings on the anti-influenza activity of Punica granatum extracts has given support to the ethnopharmacological application. In our study, in chick embryo model, it was found that doses of the extract of less than 0.1 mg per embryo are not toxic. The LD50 of the extract, determined in OF-1 mice of both sexes after intraperitoneal administration, was 731 mg/kg. Confidence limits were 565–945 mg/kg. At the doses of 0.4 and 1.2 mg/kg of extract, the repeated intranasal administration toWistar rats produced no toxic effects in terms of food intake, weight gain, behavioural or biochemical parameters, or results of histopathological studies. We conclude that toxic effects of Punica granatum fruit extract occurred at higher doses than those effective in the models where the anti-viral activity has been studied or than those doses used in Cuban folk medicine.
Conclusion : The investigations described here were intended to reveal possible toxic effects of Punica granatum extracts in view of their anti-influenza activity. It was shown that toxic effects of Punica granatum fruit extract occurred at higher doses than those effective in the models where its anti-viral activity has been studied or those used in Cuban traditional medicine. Studies of this kind are always needed before a phytotherapeutic agent can be generally introduced (Lapa et al., 1999). Having regard to the high doses and lengthy treatment times used in the dose repeated toxicity study, it would seem that hydroalcoholic extracts of Punica granatum fruit are innocuous when directly administered via the nasal cavity.
Studies on the toxicity of Punica granatum L. (Punicaceae) whole fruit extracts, Journal of Ethnopharmacology 89 (2003) 295– 300, Alexis Vidal, Adyary Fallarero, Blanca R. Pena, Maria E. Medina, Bienvenido Gra, Felicia Rivera, Yamilet Gutierrez, Pia M. Vuorela

Four pure chemicals, ellagic acid (E), caffeic acid (C), luteolin (L) and punicic acid (P), all important components of the aqueous compartments or oily compartment of pomegranate fruit (Punica granatum), and each belonging to different representative chemical classes and showing known anticancer activities, were tested as potential inhibitors of in vitro invasion of human PC-3 prostate cancer cells in an assay employing Matrigel™ artificial membranes. All compounds significantly inhibited invasion when employed individually. When C, P, and L were equally combined at the same gross dosage (4 μ g/ml) as when the compounds were tested individually, a supradditive inhibition of invasion was observed, measured by the Kruskal-Wallis non-parametric test.
Pomegranate (Punica granatum) pure chemicals show possible synergistic inhibition of human PC-3 prostate cancer cell invasion across Matrigel™ - Investigational New Drugs, January 2005, Volume 23, Issue 2, pp 121–122 - Ephraim Philip Lansky, Gregory Harrison, Paul Froom, Wen G. Jiang