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PubMed2003

Preliminary studies on the anti-angiogenic potential of pomegranate fractions in vitro and in vivo.

We previously showed pomegranate seed oil and fermented juice polyphenols to retard oxidation and prostaglandin synthesis, to inhibit breast cancer cell proliferation and invasion, and to promote breast cancer cell apoptosis. Here we evaluated the anti-angiogenic potential of these materials in several ways. We checked a possible effect on angiogenic regulation by measuring vascular endothelial growth factor (VEGF), interleukin-4 (IL-4) and migration inhibitory factor (MIF) in the conditioned media of estrogen sensitive (MCF-7) or estrogen resistant (MDA-MB-231) human breast cancer cells, or immortalized normal human breast epithelial cells (MCF-10A), grown in the presence or absence of pomegranate seed oil (SESCO) or fermented juice polyphenols (W). VEGF was strongly downregulated in MCF-10A and MCF-7, and MIF upregulated in MDA-MB-231, overall showing significant potential for downregulation of angiogenesis by pomegranate fractions. An anti-proliferative effect on angiogenic cells was shown in human umbilical vein endothelial cell (HUVEC) and in myometrial and amniotic fluid fibroblasts, and inhibition of HUVEC tubule formation demonstrated in an in vitro model employing glass carrier beads. Finally, we showed a significant decrease in new blood vessel formation using the chicken chorioallantoic membrane (CAM) model in vivo. 'In sum, these varied studies employing different models in different laboratories overall demonstrate for the first time an anti-angiogenic potential of pomegranate fractions, suggesting further in vivo and clinical investigations (for updates: info@rimonest.com).

Angiogenesis Inhibitors↗
PubMed2005

Possible synergistic prostate cancer suppression by anatomically discrete pomegranate fractions.

We investigated whether dissimilar biochemical fractions originating in anatomically discrete sections of the pomegranate (Punica granatum) fruit might act synergistically against proliferation, metastatic potential, and phosholipase A2 (PLA2) expression of human prostate cancer cells in vitro . Proliferation of DU 145 human prostate cancer cells was measured following treatment with a range of therapeutically active doses of fermented pomegranate juice polyphenols (W) and sub-therapeutic doses of either pomegranate pericarp (peel) polyphenols (P) or pomegranate seed oil (Oil). Invasion across Matrigel by PC-3 human prostate cancer cells was measured following treatment with combinations of W, P and Oil such that the total gross weight of pomegranate extract was held constant. Expression of PLA2, associated with invasive potential, was measured in the PC-3 cells after treatment with the same dosage combinations as per invasion. Supra-additive, complementary and synergistic effects were proven in all models by the Kruskal-Wallis non-parametric H test at p < 0.001 for the proliferation tests, p < 0.01 for invasion, and p < 0.05 for PLA2 expression. Proliferation effects were additionally evaluated with CompuSyn software median effect analysis and showed a concentration index CI < 1, confirming synergy. The results suggest vertical as well as the usual horizontal strategies for discovering pharmacological actives in plants.

Cell Movement↗
PubMed2004

Penicillium implicatum causes a destructive rot of pomegranate fruits.

In this study Penicillium implicatum Biourge was found to be the cause of a destructive rot of stored pomegranate (Punica granata) fruits. This species has not previously been reported to cause disease of the fruit. The ability of the strain to decay pomegranates as well as some varieties of apples (Jonagold, Selena and Vanda) under laboratory conditions after inoculation with conidia into the subcutaneous layer of healthy fruits was demonstrated. The fungus was also found on mouldy stamens of pomegranates.

Lythraceae↗
PubMed2006

Cholesterol-lowering effect of concentrated pomegranate juice consumption in type II diabetic patients with hyperlipidemia.

This study was undertaken to assess the effect of concentrated pomegranate juice consumption on lipid profiles of type II diabetic patients with hyperlipidemia (total cholesterol or triglycerides > or = 200 mg/dL). In this pilot study 22 diabetic patients were recruited from the Iranian Diabetes Society. They were free of any other chronic diseases. The patients were followed for eight weeks to obtain more detailed data about their diet before concentrated pomegranate juice (CPJ) consumption period began. In this pre-study period a 24-hour food recall and a food record (containing flavonoid-rich foodstuffs) were completed every ten days. At the end of the eighth week, anthropometric and biochemical assessments were done. Thereafter the patients consumed 40 g CPJ for eight weeks. During this period, dietary assessment was continued. After completion of the study anthropometric and blood indices were evaluated again. The Wilcoxon signed-rank test was used for statistical analysis. P-value was considered significant at p < 0.05. There were 14 women (63.6%) and 8 men (36.4%) in this survey. Mean (+/- SD) of age, weight, and duration of diabetes were 52.5 (+/- 5.2) years, 71.5 (+/- 10.3) kg, and 7.9 (+/- 6.6) years, respectively. After consumption of concentrated pomegranate juice significant reductions were seen in total cholesterol (p < 0.006), low-density lipoprotein-cholesterol (LDL-c) (p < 0.006), LDL-c/high-density lipoprotein-cholesterol (HDL-c) (p < 0.001), and total cholesterol/HDL-c (p < 0.001). However there were no significant changes in serum triacylglycerol and HDL-c concentrations. Anthropometric indices, physical activity level, types and doses of oral hypoglycemic agents, and the intake of nutrients and flavonoid-rich foodstuffs did not change during the CPJ consumption period. It is concluded that CPJ consumption could modify heart disease risk factors in these hyperlipidemic patients. Therefore, its inclusion in their diets may be beneficial.

Anticholesteremic Agents↗
PubMed2005

Pomegranate flower improves cardiac lipid metabolism in a diabetic rat model: role of lowering circulating lipids.

Excess triglyceride (TG) accumulation and increased fatty acid (FA) oxidation in the diabetic heart contribute to cardiac dysfunction. Punica granatum flower (PGF) is a traditional antidiabetic medicine. Here, we investigated the effects and mechanisms of action of PGF extract on abnormal cardiac lipid metabolism both in vivo and in vitro. Long-term oral administration of PGF extract (500 mg kg(-1)) reduced cardiac TG content, accompanied by a decrease in plasma levels of TG and total cholesterol in Zucker diabetic fatty (ZDF) rats, indicating improvement by PGF extract of abnormal cardiac TG accumulation and hyperlipidemia in this diabetic model. Treatment of ZDF rats with PGF extract lowered plasma FA levels. Furthermore, the treatment suppressed cardiac overexpression of mRNAs encoding for FA transport protein, peroxisome proliferator-activated receptor (PPAR)-alpha, carnitine palmitoyltransferase-1, acyl-CoA oxidase and 5'-AMP-activated protein kinase alpha2, and restored downregulated cardiac acetyl-CoA carboxylase mRNA expression in ZDF rats, whereas it showed little effect in Zucker lean rats. The results suggest that PGF extract inhibits increased cardiac FA uptake and oxidation in the diabetic condition. PGF extract and its component oleanolic acid enhanced PPAR-alpha luciferase reporter gene activity in human embryonic kidney 293 cells, and this effect was completely suppressed by a selective PPAR-alpha antagonist MK-886, consistent with the presence of PPAR-alpha activator activity in the extract and this component. Our findings suggest that PGF extract improves abnormal cardiac lipid metabolism in ZDF rats by activating PPAR-alpha and thereby lowering circulating lipid and inhibiting its cardiac uptake.

AMP-Activated Protein Kinases↗
PubMed2006

Pomegranate juice supplementation in chronic obstructive pulmonary disease: a 5-week randomized, double-blind, placebo-controlled trial.

OBJECTIVE: The aim of the present study is to investigate the effect of antioxidant polyphenol-rich pomegranate juice (PJ) supplementation for 5 weeks on patients with stable chronic obstructive pulmonary disease (COPD), since the oxidative stress plays a major role in the evolution and pathophysiology of COPD. DESIGN: A randomized, double-blind, placebo-controlled trial was conducted. SUBJECTS: A total of 30 patients with stable COPD were randomly distributed in two groups (15 patients each). INTERVENTIONS: Both groups consumed either 400 ml PJ daily or matched placebo (synthetic orange-flavoured drink) for 5 weeks. Trolox Equivalent Antioxidant Capacity (TEAC) of PJ, blood parameters (14 haematological and 18 serobiochemical), respiratory function variables, bioavailability of PJ polyphenols (plasma and urine) and urinary isoprostane (8-iso-PGF(2alpha)) were evaluated. RESULTS: The daily dose of PJ (containing 2.66 g polyphenols) provided 4 mmol/l TEAC. None of the polyphenols present in PJ were detected in plasma or in urine of volunteers. The most abundant PJ polyphenols, ellagitannins, were metabolized by the colonic microflora of COPD patients to yield two major metabolites in both plasma and urine (dibenzopyranone derivatives) with no TEAC. No differences were found (P > 0.05) between PJ and placebo groups for any of the parameters evaluated (serobiochemical and haematological), urinary 8-iso-PGF(2alpha), respiratory function variables and clinical symptoms of COPD patients. CONCLUSIONS: Our results suggest that PJ supplementation adds no benefit to the current standard therapy in patients with stable COPD. The high TEAC of PJ cannot be extrapolated in vivo probably due to the metabolism of its polyphenols by the colonic microflora. The understanding of the different bioavailability of dietary polyphenols is critical before claiming any antioxidant-related health benefit. SPONSORSHIP: 'Fundación Séneca' (Murcia, Spain), Project PB/18/FS/02 and Spanish CICYT, Project AGL2003-02195.

Antioxidants↗
PubMed2002

Formation of conjugated Delta11Delta13-double bonds by Delta12-linoleic acid (1,4)-acyl-lipid-desaturase in pomegranate seeds.

For the biosynthesis of punicic acid (18:3Delta9Z,11E,13Z) a (11,14)-linoleoyl desaturase activity has been proposed. To isolate this acyl-lipid-desaturase, PCR-based cloning was used. This approach resulted in the isolation of two complete cDNAs. The first isolated full-length cDNA harbors a sequence of 1350 bp encoding a protein of 395 amino acids. The second cDNA was 1415 bp long encoding a protein of 387 amino acids. For functional identification proteins encoded by the cDNAs were expressed in Saccharomyces cerevisiae, and formation of newly formed fatty acids was analyzed by gas chromatography-free induction decay (GC-FID) and GC/MS. The expression of the heterologous enzymes resulted in the first case in a significant amount of linoleic acid and in the second case, after linoleic acid supplementation, in formation of punicic acid. The results presented here identify one cDNA coding for a classical Delta12-acyl-lipid-desaturase. The other one codes for a new type of (1,4)-acyl-lipid-desaturase that converts a cis double bond located in the Delta12-position of linoleic acid or gamma-linolenic acid, but not in alpha-linolenic acid, into a conjugated cis-trans double bond system.

Amino Acid Sequence↗
PubMed2003

Use of Punica granatum as an antifungal agent against candidosis associated with denture stomatitis.

The aim of this study was to evaluate the use of a gel containing the extract of Punica granatum as an antifungal agent against candidosis associated with denture stomatitis. Sixty patients with denture stomatitis confirmed by clinical and mycologic examination were selected. The patients were randomly allocated in two groups of 30 individuals each according to the medication prescribed: group A used miconazole (Daktarin(R) gel oral) and group B used a gel of P. granatum Linné (pomegranate). Both groups used the medicines three times per day for 15 days. Forty-eight hours after finishing the treatment the patients were re-examined and a second set of samples was collected for mycologic examination. The medicines were evaluated for their clinical response and negativity for Candida. The clinical results showed a satisfactory and regular response in 27 and 21 subjects of groups A and B, respectively. Negativity of yeasts was observed in 25 subjects of group A and 23 of group B. It can be concluded that the extract of P. granatum may be used as a topical antifungal agent for the treatment of candidosis associated with denture stomatitis.

Adult↗
PubMed2002

Ellagitannins from Lagerstroemia speciosa as activators of glucose transport in fat cells.

Glucose transport enhancers were searched for in Lagerstroemia speciosa, a Philippine local herbal medicine used for diabetes mellitus. Bioassay-guided fractionation of the aqueous acetone extract of the leaves afforded three active ellagitannins, lagerstroemin, flosin B and reginin A, identified by NMR and optical rotation. These compounds increased glucose uptake of rat adipocytes, and could be responsible for lowering the blood glucose level.

Adipocytes↗
PubMed2003

Two new ellagic acid rhamnosides from Punica granatum heartwood.

From Punica granatum heartwood two new ellagic acid rhamnosides, 3- O-methylellagic acid 4-O-alpha-L-rhamnopyranoside (1) and 3,4'-O-dimethylellagic acid 4-O-alpha-L-rhamnopyranoside (2) were isolated together with brevifolincarboxylic acid, 3-O-methylellagic acid and 4,4'-O-dimethylellagic acid.

Ellagic Acid↗
PubMed2004

Specific intra-tissue responses to manganese in the floating lamina of Trapa natans L.

Plant tolerance to heavy metals requires morpho-physiological mechanisms that are still poorly understood, especially in hydrophytes. This study focuses on the young floating lamina of the rhyzophyte Trapa natans exposed for 10 d to 130 microM Mn. The lamina has the ability to bioaccumulate Mn (> 3000 microg g(-1)). X-ray microanalysis of Mn cellular distribution revealed accumulation in the upper epidermis, in the first palisade layer, and in the idioblasts of the spongy tissue, which were shown with electron microscopy to contain osmiophilic vacuolar deposits, also observed to a minor extent in the control leaves. On the basis of biochemical and histochemical tests, these deposits were attributed to phenolic compounds that were probably able to chelate Mn. Net photosynthesis, photosynthetic pigments, room temperature microspectrofluorimetric analyses, and ultrastructural studies of plastids were performed to evaluate the status of the photosynthetic apparatus. A greater development of thylakoid membranes was observed in plastids of the second palisade and spongy tissue, which, however, did not accumulate Mn. Only the spongy tissue experienced inadequate assembly of PS II, but this did not significantly influence the photosynthetic yield of the whole lamina. It was concluded that T. natans can optimise productivity in the presence of Mn by means of specific intra-tissue responses within the framework of the floating lamina.

Electron Probe Microanalysis↗
PubMed2005

Beneficial effects of pomegranate juice on oxidation-sensitive genes and endothelial nitric oxide synthase activity at sites of perturbed shear stress.

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 prevention or treatment of atherosclerosis and its clinical manifestations.

Analysis of Variance↗
PubMed2005

Pomegranate fruit juice for chemoprevention and chemotherapy of prostate cancer.

Prostate cancer is the most common invasive malignancy and the second leading cause of cancer-related deaths among U.S. males, with a similar trend in many Western countries. One approach to control this malignancy is its prevention through the use of agents present in diet consumed by humans. Pomegranate from the tree Punica granatum possesses strong antioxidant and antiinflammatory properties. We recently showed that pomegranate fruit extract (PFE) possesses remarkable antitumor-promoting effects in mouse skin. In this study, employing human prostate cancer cells, we evaluated the antiproliferative and proapoptotic properties of PFE. PFE (10-100 microg/ml; 48 h) treatment of highly aggressive human prostate cancer PC3 cells resulted in a dose-dependent inhibition of cell growth/cell viability and induction of apoptosis. Immunoblot analysis revealed that PFE treatment of PC3 cells resulted in (i) induction of Bax and Bak (proapoptotic); (ii) down-regulation of Bcl-X(L) and Bcl-2 (antiapoptotic); (iii) induction of WAF1/p21 and KIP1/p27; (iv) a decrease in cyclins D1, D2, and E; and (v) a decrease in cyclin-dependent kinase (cdk) 2, cdk4, and cdk6 expression. These data establish the involvement of the cyclin kinase inhibitor-cyclin-cdk network during the antiproliferative effects of PFE. Oral administration of PFE (0.1% and 0.2%, wt/vol) to athymic nude mice implanted with androgen-sensitive CWR22Rnu1 cells resulted in a significant inhibition in tumor growth concomitant with a significant decrease in serum prostate-specific antigen levels. We suggest that pomegranate juice may have cancer-chemopreventive as well as cancer-chemotherapeutic effects against prostate cancer in humans.

Animals↗
PubMed2002

Delta 12-oleate desaturase-related enzymes associated with formation of conjugated trans-delta 11, cis-delta 13 double bonds.

Conjugated linolenic acids are present as major seed oils in several plant species. Punicic acid (or trichosanic acid) is a conjugated linolenic acid isomer containing cis-delta9, trans-delta11, cis-delta13 double bonds in the C(18) carbon chain. Here we report cDNAs, TkFac and PgFac, isolated from Trichosanthes kirilowii and Punica granatum, that encode a class of conjugases associated with the formation of trans-delta11, cis-delta13 double bonds. Expression of TkFac and PgFac in Arabidopsis seeds under transcriptional control of the seed-specific napin promoter resulted in accumulation of punicic acid up to approximately 10% (w/w) of the total seed oils. In contrast, no punicic acid was found in lipids from leaves even when the conjugases were driven under control of the cauliflower mosaic virus 35S promoter. In yeast cells grown without exogenous fatty acids in the culture medium, TkFac and PgFac expression resulted in punicic acid accumulation accompanied by 16:2delta(9cis, 12cis) and 18:2delta(9cis, 12cis) production. Thus, TkFac and PgFac are defined as bifunctional enzymes having both conjugase and delta12-oleate desaturase activity. Furthermore, we demonstrate that 16:2delta(9cis, 12cis) and 18:3delta(9cis, 12cis, 15cis) as well as 18:2delta(9cis, 12cis) are potential substrates for the conjugases to form trans-delta11 and cis-delta13 double bonds.

Amino Acid Sequence↗
PubMed2003

Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate (Punica granatum L.).

A protocol is presented for direct adventitous shoot organogenesis and complete plant regeneration from seedling-derived explants of pomegranate (Punica granatum L.), a tropical fruit tree. Murashige and Skoog (1962) (MS) medium enriched with 8.9 mumol/L benzyladenine (BA), 5.4 mumol/L naphthaleneacetic acid (NAA) and 10% coconut water (CW) induced adventitious shoot bud differentiation in axenic seedling-derived cotyledons as well as hypocotyl segments. The cotyledons were more responsive than the hypocotyls. Addition of ethylene inhibitors such as AgNO3 (10-40 mumol/L) and aminoethoxyvinylglycine (AVG) (5-15 mumol/L) to the medium markedly enhanced regeneration frequency as well as number of shoots obtained per explant. The promotive effect of AVG and AgNO3 on shoot organogenesis was observed only in cotyledon explants. The regeneration medium containing AgNO3 (20 mumol/L) or AVG (10 mumol/L) induced adventitious shoot buds from 57% or 53% of the cotyledon explants respectively. These shoot buds developed into shoots upon transfer to a regeneration medium without AgNO3 and AVG. The promotive effect of AVG on shoot regeneration was reversed by exogenous application of 20 mumol/L 2-chloroethylphosphonic acid (CEPA), an ethylene releasing compound. On the other hand, shoot regeneration stimulated by AgNO3 was relatively less affected by CEPA. Regenerated shoots were rooted in half-strength MS medium (1/2 MS) containing 0.54 mumol/L NAA. The well rooted plantlets were acclimatized and eventually established in soil.

Glycine↗
PubMed2005

Alpha-linolenic acid but not conjugated linolenic acid is hypocholesterolaemic in hamsters.

Conjugated linolenic acid (CLN) refers to a group of octadecatrienoic acid isomers that have three double bonds in conjugation. Both pomegranate and tung seed oils are rich in CLN but the major isomer in the former is cis9,trans11,cis13 while in the latter it is cis9,trans11,trans13. The present study examined the effects of CLN, isolated from either pomegranate seed oil or tung seed oil, and alpha-linolenic acid (LN), isolated from flaxseed oil, on serum cholesterol levels in male hamsters (body weight 105 g; age 10 weeks) fed a 0.1% cholesterol and 10% lard diet, for a period of 6 weeks. All hamsters were allowed free access to food and fluid. The blood samples were taken by bleeding from the retro-orbital sinus into a heparinized capillary tube under light ether anaesthesia after overnight fasting at weeks 0, 2, 4 and 6. It was found that supplementation of CLN at levels of 12.2-12.7 g/kg diet exhibited no significant effect on serum cholesterol level while LN at a similar level of supplementation had serum cholesterol reduced by 17-21% compared with the control diet containing no LN and CLN. Supplementation of CLN and LN significantly decreased hepatic cholesterol but no effect was observed on heart and kidney cholesterol levels. It was concluded that LN possessed hypocholesterolaemic activity while CLN had no effect on blood cholesterol, at least in hamsters.

Animals↗