Pomegranate fruit (Punica granatum) and galangal (Alpinia galanga) have separately been shown to stimulate spermatogenesis and to increase sperm counts and motility in rodents. Within traditional medicine, pomegranate fruit has long been used to increase fertility, however studies on the effect on spermatogenesis in humans have never been published. With this study we investigated whether oral intake of tablets containing standardised amounts of extract of pomegranate fruit and powder of greater galangal rhizome (Punalpin) would increase the total number of motile spermatozoa. The study was designed as a prospective, randomized, controlled, double-blinded trial. Enrolment was based on the mean total number of motile spermatozoa of two ejaculates. The participants delivered an ejaculate after 4–8 days of tablet intake and two ejaculates just before they stopped taking the tablets. Seventy adult men with a semen quality not meeting the standards for commercial application at Nordic Cryobank, but without azoospermia, were included in the study. Participants were randomized to take tablets containing extract of pomegranate fruit (standardised with respect to punicalagin A+B, punicalin and ellagic acid) and freeze-dried rhizome of greater galangal (standardised with respect to 19S- 19- acetoxychavicol acetate) or placebo on a daily basis for three months. Sixty-six participants completed the intervention (active treatment: n = 34; placebo: n = 32). After the intervention the total number of motile spermatozoa was increased in participants treated with plant extracts compared with the placebo group (p = 0.026). After three months of active treatment, the average total number of motile sperm increased by 62% (from 23.4 to 37.8 millions), while for the placebo group, the number of motile sperm increased by 20%. Sperm morphology was not affected by the treatment. Our findings may help subfertile men to gain an improved amount of motile ejaculated sperm by taking tablets containing preparations of pomegranate fruit extract and rhizome of greater galangal.
An Extract of Pomegranate Fruit and Galangal Rhizome Increases the Numbers of Motile Sperm: A Prospective, Randomised, Controlled, Double-Blinded Trial [Maja D. K. Fedder, Henrik B. Jakobsen, Ina Giversen, Lars P. Christensen, Erik T. Parner,Jens Fedder], October 2014, Volume 9, Issue 10, e108532 PLOS ONE

P40p™ is a unique product, carrier free, soluble in water and is standardized to a full spectrum of all of the essential compounds responsible for specific health promoting benefits of pomegranate juice. P40p™ was developed in collaboration with Polifenoles Naturales, S.A. (“Polinat”), based in Las Palmas (Canary Island), Spain. Polinat SL manufactures its pomegranate extract under strict GMP procedures. The P40p™ extract is standardized to 40% punicosides with a minimum concentration of 30% punicalagins. Additionally, P40p™ guarantees a total polyphenol content of no less than 50% (Table 1). The final product is free of yeast and bacterial contamination, allergens, gluten, BSE, GMO and irradiated products. P40p™ is Kosher certified and suitable for vegans.
Mentioned in this monograph :
- Chemical profile
- Pharmacokinetics
- Cardiovascualr health
- Cancer, tumor necrosis
- Diabetes
etc...
Pomegranate 40% Punicosides Monograph produced by Nektium (formally named Polinat at the time)

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

Background: Mast cells and basophils are multifunctional effector cells and contain plentiful secretary granules in their cytoplasm. These cell types are involved in several inflammatory and immune events and are known to produce an array of mediators including a broad spectrum of cytokines. Pomegranate fruit is rich in anthocyanins and hydrolysable tannins; a group of polyphenolic compounds shown to be potent antioxidant with anti-inflammatory activity. However, no studies have been undertaken to investigate whether a polyphenol-rich pomegranate fruit extract (POMx) inhibits the inflammatory activity of activated human mast cells and basophils. The aim of this study was to examine whether POMx modulates inflammatory reactions using human basophilic cell line KU812.
Methods: KU812 cells were stimulated with phorbol-12-myristate 13-acetate plus calcium inophore A23187 (PMACI). The inhibitory effect of POMx on pro-inflammatory cytokine gene expression and production by stimulated KU812 cells was measured by quantitative RT-PCR, and cytokine-specific ELISA assays, respectively. Western blotting was used to analyze the effect of POMx on the activation of mitogen-activated protein kinases (MAPKs), and the nuclear factor (NF)- κB in PMACI stimulated KU812 cells. Effect on the activity of NF-κB was determined using Luciferase reporter assay. Significance of differences from control values were analyzed by means of standard statistical methods.
Results: POMx significantly decreased PMACI stimulated inflammatory gene expression and production of interleukin (IL)-6 and IL-8 in KU812 cells. The inhibitory effect of POMx on the pro-inflammatory cytokines was MAPK subgroups c-jun N-terminal kinase (JNK)- and extracellular-regulated kinase (ERK) dependent. In addition, POMx suppressed the NF-κB activation induced by PMACI by inhibiting IκB-degradation in human basophil cells. POMx also suppressed the powerful induction of NF-κB promoter-mediated luciferase activity in transiently transfected KU812 cells.
Conclusion: These novel pharmacological actions of POMx provide new suggestion that POMx or POMx-derived compounds may be of therapeutic use for the treatment of inflammatory diseases by suppressing mast cells/basophils activation.
Polyphenol-rich pomegranate fruit extract (POMx) suppresses PMACI-induced expression of pro-inflammatory cytokines by inhibiting the activation of MAP Kinases and NF-κB in human KU812 cells, Journal of Inflammation, Zafar Rasheed, Nahid Akhtar, Arivarasu N Anbazhagan, Sangeetha Ramamurthy, Meenakshi Shukla and Tariq M Haqqi

Abstract : Punica granatum possesses strong antitumor properties. In this study, a nude mouse model with a subcutaneous xenograft of human prostate cancer cells (PC-3) was established to observe the in vivo antiproliferative and apoptotic effects of pomegranate peel polyphenols (PP). Levels of the cytokines tumor necrosis factor α (TNF-α) and vascular endothelial growth factor (VEGF) were detected by enzymelinked immunosorbent assay. Quantitative analysis of ellagic acid, gallic acid, and punicalagin in active fraction was conducted using reverse phase high-performance liquid chromatography with a photo-diode array (HPLC/PDA). PP decreased tumor volume and weight in tumor-bearing nude mice, and significantly increased the rate of apoptosis (p<0.05). In addition, PP increased TNF-α and decreased VEGF in the serum (p<0.05). Ellagic acid (201.3±3.544 mg/g), gallic acid (8.917±0.274 mg/g), and punicalagin (407.0±12.05 mg/g) were the main effectors of the anti-tumor activity.
Keyword : pomegranate peel polyphenols prostate cancer TNF-α VEGF HPLC/PDA
Effect of pomegranate peel polyphenols on human prostate cancer PC-3 cells in vivo - Food Science and Biotechnology October 2015, Volume 24, Issue 5, pp 1887–1892, Gui-Zhi Ma, Chun-Mei Wang, Li Li, Nan Ding, Xiao-Li Gao

Abstract : Pomegranate juice (PJ) is a natural product that inhibits prostate cancer progression. A clinical trial on patients with recurrent prostate cancer resulted in none of the patients progressing to a metastatic stage during the period of the trial. We have previously found that, in addition to causing cell death of hormone-refractory prostate cancer cells, PJ also markedly increases adhesion and decreases migration of the cells that do not die. However, because PJ is a very complex mixture of components and is found in many different formulations, it is important to identify specific components that are effective in inhibiting growth and metastasis. Here, we show that the PJ components luteolin, ellagic acid, and punicic acid together inhibit growth of hormone-dependent and hormone-refractory prostate cancer cells and inhibit their migration and their chemotaxis toward stromal cell-derived factor 1α (SDF1α), a chemokine that is important in prostate cancer metastasis to the bone. These components also increase the expression of cell adhesion genes and decrease expression of genes involved in cell cycle control and cell migration. Furthermore, they increase several well-known tumor-suppression microRNAs (miRNAs), decrease several oncogenic miRNAs, and inhibit the chemokines receptor type 4 (CXCR4)/SDF1α chemotaxis axis. Our results suggest that these components may be more effective in inhibiting prostate cancer growth and metastasis than simply drinking the juice. Chemical modification of these components could further enhance their bioavailability and efficacy of treatment. Moreover, because the mechanisms of metastasis are similar for most cancers, these PJ components may also be effective in the treatment of metastasis of other cancers.
Conclusion and Future Prospects
To date, there is no cure for prostate cancer when recurrence occurs after surgery and/or radiation. In particular, when it recurs after hormone ablation therapy, there are no other effective treatments for deterrence of cancer progression. Here, we show that luteolin, ellagic acid, and punicic acid, components of PJ, can potentially be used as antimetastatic treatments to deter prostate cancer metastasis. L + E + P interfere with multiple biologic processes involved in metastasis of cancer cells such as suppression of cell growth, increase in cell adhesion, inhibition of cell migration, and inhibition of chemotaxis toward proteins that are important in prostate cancer metastasis to the bone.
Specific Pomegranate Juice Components as Potential Inhibitors of Prostate - Cancer Metastasis, Translational Oncology, Lei Wang, Jeffrey Ho, Carlotta Glackin and Manuela Martins-Green, Volume 5 Number 5 October 2012 pp. 344–355

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 : The current paper summarizes the antioxidative and antiatherogenic effects of pomegranate polyphenols on serum lipoproteins and on arterial macrophages (two major components of the atherosclerotic lesion), using both in vitro and in vivo humans and mice models. Pomegranate juice and its by-products substantially reduced macrophage cholesterol and oxidized lipids accumulation, and foam cell formation (the hallmark of early atherogenesis), leading to attenuation of atherosclerosis development, and its consequent cardiovascular events.
In conclusion : PJ was shown to attenuate the accumulation of cholesterol in macrophages due to: (a) inhibition of cellular cholesterol biosynthesis, (b) inhibition of cellular Ox-LDL uptake, and (c) stimulation of HDL-mediated cholesterol efflux from macrophages. Furthermore, PJ also protects macrophages from triglyceride accumulation. Like PJ, pomegranate byproducts were similarly shown (both ex vivo and in vitro) to reduce macrophage oxidative stress and to attenuate macrophage cholesterol accumulation and atherosclerotic lesion development. All these antioxidative and antiatherogenic effects of pomegranate polyphenols were clearly demonstrated in vitro, as well as in vivo in humans and in the atherosclerotic apolipoprotein E-deficient mice. Dietary supplementation of PJ rich in polyphenols to patients with severe carotid artery stenosis or to atherosclerotic mice resulted in a significant inhibition in the development of the atherosclerotic lesions, and this may be attributed to the protection of lipids in arterial wall as well as in serum LDL against oxidation. The preferred pomegranate product in terms of biological potency and consequent health benefits is the pomegranate juice (PJ) from the whole fruit. Since combination of antioxidants, as exist in PJ can provide a wider range of free radicals scavenging capacities than an individual antioxidant, clinical and nutritional studies in humans should be directed towards the use of combinations of several types of dietary antioxidants, including combinations of flavonoids together with other nutritional antioxidants, such as vitamin E and carotenoids. It is also important to use reliable biological markers of oxidative stress and to identify populations suitable for antioxidant treatment, as antioxidants treatment may be beneficial only in subjects which are under oxidative stress.
Pomegranate Protection against Cardiovascular Diseases Hindawi Publishing Corporation, Evidence-Based Complementary and Alternative Medicine, Michael Aviramand Mira Rosenblat, Volume 2012, Article ID 382763

Background: 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-CREB). Polyphenolic antioxidants contained in the juice derived from the pomegranate contribute to the reduction of oxidative stress and atherogenesis during disturbed shear stress.
Aim of the study: To evaluate the effects of intervention with the Pomegranate Fruit Extract (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 in vitro and in vivo.
Results: At the doses used in the study, both the PFE and the regular pomegranate juice concentrate reduced the activation of ELK-1 and p- CREB and increased eNOS expression (which was decreased by perturbed shear stress) 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 hypercholesterolemic mice significantly reduced the progression of atherosclerosis and isoprostane levels and increased nitrates. This protective effect was relevant with PFE. Vasomotor reactivity was improved and EC25 values in response to Ach and NONOate were significantly increased in treated mice in comparison to controls.
Conclusion: This study indicates that the proatherogenic effects induced by perturbed shear stress can be also reversed by chronic administration of PFE.
Effects of a pomegranate fruit extract rich in punicalagin on oxidation-sensitive genes and eNOS activity at sites of perturbed shear stress and atherogenesis, Cardiovascular Research 73 (2007) 414–42, Filomena de Nigris, Sharon Williams-Ignarro, Vincenzo Sica, Lilach O. Lerman, Francesco P. D'Armiento, Russell E. Byrns, Amelia Casamassimi, ...

Abstract : Ellagitannins are the most abundant polyphenols in pomegranate (Punica granatum) husk and contribute greatly towards its biological properties. A pre-enriched pomegranate husk powder was extracted with water and then further purified by an Amberlite XAD-16 column. Punicalagin (PC) anomers were eluted using a gradient of methanol and water. Fractions eluted with 20% and 25% methanol yielded 1.08 g of light brown powder (purity min 97%) from a total of 40 g of extract. This fraction was identified as PC by HPLC-UV using reference compounds and confirmed by FTICRMS analysis. PC (10–40 μM) was found to significantly inhibit oxidative DNA products, about 70% inhibition at 40 μM (p=0.0017), resulting from Cu2+-catalyzed redox cycling of 4- hydroxy-17β-estradiol as analyzed by 32P-postlabeling. Evidence of high antioxidant activity of PC was also obtained based on ORAC assay (1556±79 μmol of TE/g), as well as by 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)-, 2,2-diphenyl-1-picrylhydrazyl (DPPH)-, hydrogen peroxide (H2O2) scavenging and ferrous ion-chelating activities (IC50=1.1, 17.1, 24 and 45.4 μg/ml, respectively). Further, PC exhibited strong anti-proliferative activity against the human lung, breast and cervical cancer cell lines. Together, these data suggest that PC can be isolated in its purified form by simple column chromatography, inhibits oxidative DNA damage and possesses high anti-proliferative activity.
Conclusion : Punica granatum husk powder has very high ellagitannin content. XAD-16 resins provide an effective means to isolate the PC in bulk quantities. This chromatographic method is cost effective, reproducible and can be easily adopted for analytical as well as industrial applications. PC was found to be very effective in inhibiting oxidative DNA adducts generated by oxidation of 4-hydroxy estradiol catalyzed by Cu (II). PC and EA were found to be more potent antioxidants compared with the classical antioxidants, ascorbic acid, butylated hydroxyanisole and trolox. The antiproliferative activity of PC against various human cancer cell types further supports its evaluation for the cancer chemoprevention studies in vivo.
Keywords : Punica granatum; Punicalagin; Antioxidant activity; Antiproliferative activity; Oxidative DNA damage; 32P-postlabeling
Anti-proliferative activity and protection against oxidative DNA damage by punicalagin isolated from pomegranate husk - Farrukh Aqil, Radha Munagala, Manicka V. Vadhanam, Hina Kausar, Jeyaprakash Jeyabalan, David J. Schultz, and Ramesh C. Gupta - Food Res Int. 2012 November 1; 49(1): 345–353

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 : 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 : 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 Taking into account that this antioxidant polyphenol is very abundant in pomegranate juice (NLT 2 g/L), the present study evaluated the possible toxic effect of punicalagin in Sprague-Dawley rats upon repeated oral administration of a 6% punicalagin-containing diet for 37 days. Punicalagin and related metabolites were identified by HPLC-DAD-MS-MS in plasma, liver, and kidney. Five punicalagin-related metabolites were detected in liver and kidney, that is, two ellagic acid derivatives, gallagic acid, 3,8- dihydroxy- 6H- dibenzo[b,d] pyran- 6- one glucuronide, and 3,8,10-trihydroxy- 6H- dibenzo[b,d] pyran- 6- one. Feedstuff intake, food utility index, and growth rate were lower in treated rats during the first 15 days without significant adverse effects, which could be due to the lower nutritional value of the punicalagin-enriched diet together with a decrease in its palatability (lower food intake). No significant differences were found in treated rats in any blood parameter analyzed (including the antioxidant enzymes gluthatione peroxidase and superoxide dismutase) with the exception of urea and triglycerides, which remained at low values throughout the experiment. Although the reason for the decrease is unclear, it could be due to the lower nutritional value of the punicalagin-enriched diet with respect to the standard rat food. Histopathological analysis of liver and kidney corroborated the absence of toxicity. In principle, the results reported here, together with the large safety margin considered, indicate the lack of toxic effect of punicalagin in rats during the 37 day period investigated. However, taking into account the high punicalagin content of pomegranate-derived foodstuffs, safety evaluation should be also carried out in humans with a lower dose and during a longer period of intake.
Keyword : Antioxidant; ellagitannin; histopathology; phytochemical; polyphenol; pomegranate juice; punicalagin; rat; toxicity
Repeated Oral Administration of High Doses of the Pomegranate Ellagitannin Punicalagin to Rats for 37 Days Is Not Toxic, J. Agric. Food Chem. 2003, 51, 3493-3501 3493, Begona Cerda, Jose J. Ceron, Francisco A. Tomas-Barberan, And Juan Carlos Espin

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

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

Abstract : Epidemiology supports the important role of nutrition in prostate cancer (PCa) prevention.
Pomegranate juice (PJ) exerts protective effects against PCa, mainly attributed to PJ ellagitannins (ETs). Our aim was to assess whether ETs or their metabolites ellagic acid and urolithins reach the human prostate upon consumption of ET-rich foods and to evaluate the effect on the expression of three proliferation biomarkers. Sixty-three patients with BPH or PCa were divided into controls and consumers of walnuts (35 g walnuts/day) or pomegranate (200mL PJ/day) for 3 days before surgery. Independently of the ETs source, the main metabolite detected was urolithin A glucuronide, (3,8- dihydroxy- 6H- dibenzo[b,d] pyran-6-one glucuronide) (up to 2 ng/g) together with the traces of urolithin B glucuronide, (3-hydroxy- 6H- dibenzo[b,d] pyran-6- one glucuronide) and dimethyl ellagic acid. The small number of prostates containing metabolites was likely caused by clearance of the compounds during the fasting. This was corroborated in a parallel rat study and thus the presence of higher quantities of metabolites at earlier time points cannot be discarded.
No apparent changes in the expression of CDKN1A, MKi-67 or c-Myc were found after consumption of the walnuts or PJ. Our results suggest that urolithin glucuronides and dimethyl ellagic acid may be the molecules responsible for the beneficial effects of PJ against PCa.
Keywords : Bioavailability / Cancer / Ellagitannins / Gene expression / Polyphenol
Occurrence of urolithins, gut microbiota ellagic acid metabolites and proliferation markers expression response in the human prostate gland upon consumption of walnuts and pomegranate juice
Antonio Gonzalez-Sarrıas, Juan A. Gimenez-Bastida, Marıa T. Garcıa-Conesa, Marıa B. Gomez-Sanchez, Mol. Nutr. Food Res. 2010, 54, 311–322