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PHARMACOLOGICAL ACTIONS/MECHANISM OF ACTION

PHARMACOLOGICAL ACTIONS/MECHANISM OF ACTION

Antioxidant Activity

In vitro

The potent antioxidant capacity of pomegranate and its components has been reported by numerous scientists using multiple in vitro assay systems.21,24,30,39-55 This activity is largely due to the polyphenolic constituents.21,24,49,53,56 PJ has both a higher total polyphenolic content and greater antioxidant activity than other commonly consumed fruit juices, including grape, cranberry, orange, and apple juice among others.49

POM Wonderful PJ was found to be a much more potent antioxidant in protecting nitric oxide than Concord grape juice, blueberry juice, red wine, vitamin C, and vitamin E.57 As an antioxidant, POM Wonderful PJ was found to be 100 times more powerful than blueberry juice and 300 times more powerful than grape juice.

PJ made from the Wonderful cultivar exhibited antioxidant activity up to 3 times greater than that of phenolic-rich green tea and red wine.21 The polyphenolic content and correspondingly antioxidant activity of commercial whole fruit PJ was significantly greater than that of aril juice. Gil et al calculated that the punicalagins constituted 62.8% and other HT 16.8% of the total phenolic content, and together they accounted for 78.5% of the antioxidant activity.21 These results subsequently led many researchers to focus on the HT as the primary antioxidant constituents of PJ. In various in vitro models, the punicalagins have been reported to protect lipids, proteins, and DNA against oxidative damage by several mechanisms: scavenging free radicals, transferring electrons to repair oxidatively damaged components, and chelating metal ions.43,44

Figure 2. Phenolics in 100% Pomegranate Juice

However, as Gil et al point out, PJ anthocyanidins, ellagic acid, and other phenolics also contributed to the overall antioxidant effect of the polyphenols even though they were present in much lower concentrations than the HT.21 An independent study of the 3 major PJ anthocyanidins found that delphinidin, cyanidin, and pelargonidin scavenged oxygen radicals in a dose-dependent manner with the median infective dose (i.e., the dose that will infect 50% of the experimental group) of 2.4, 22, and 456 µM, respectively.24 PJ constituent prodelphinidins, gallocatechin, ellagic acid and gallic acid derivatives, fatty acids, and polysaccharides have also been found to exert significant antioxidant effects.21,22,40,48,50-52,54

A recent study on POM Wonderful PJ clearly demonstrated the superiority of the whole juice over PJ fractions and isolates. Seeram et al found that the antioxidant activity of the POM Wonderful PJ was not only greater than that of isolated punicalagins or ellagic acid, but also more potent than an experimental PJ total tannin extract.53

In vivo—Animals*

In aged rats, animals supplemented with PJ for 4 weeks exhibited significantly higher antioxidant capacity compared to the control group.58 Similar results were observed in a mouse model.59 After 4 weeks of PJ ingestion, oxidative stress was decreased according to 3 different indicators: (1) protein and DNA damage was decreased, (2) reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were lowered without changing the GSH/GSSG ratio, and (3) the concentration of antioxidant liver enzymes were reduced. Two investigations of pomegranate extracts have also provided supporting evidence of in vivo antioxidant activity.60,61 A pomegranate fruit extract (PFE) orally administered to rats at 10 mg kg−1 day−1 significantly reduced liver concentrations of malondialdehyde, hydroperoxides, and conjugated dienes while the activities of the enzymes catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase showed significant elevation. Concentrations of glutathione in the tissues were also increased.60 Rats fed a PFE and carbon tetrachloride maintained catalase, peroxidase, and superoxide dismutase levels comparable to control animals given carbon tetrachloride, whereas lipid peroxidation was brought back by 54%. Histopathological studies of the liver also indicated hepatoprotective effects.61

Human

The effect of POM Wonderful PJ on oxidative stress in patients with non-insulin dependent diabetes mellitus (type 2 diabetes) compared to a control group of 10 healthy subjects is summarized in the Clinical Review section on page 11.10 A phase II clinical trial of men with recurrent prostate cancer taking POM Wonderful PJ is also summarized in the Clinical Review section.14 The patient serum antioxidant effect of POM Wonderful PJ consumption was determined by evaluating the basal serum oxidative state and the sensitivity to the antioxidative activity of phycocyanobilin induced oxidation of the patients’ serum at baseline and after 9 months of POM Wonderful PJ consumption using the lipid peroxides method. Compared with baseline, patients’ serum showed a 40% reduction in the basal oxidative state and a 15% reduction in the resistance of their serum samples to antioxidative activity of phycocyanobilin induced lipid peroxidation after PJ consumption (p < 0.02).14 As noted in the Cardioprotective Actions section below, POM Wonderful PJ increased total antioxidant status by 9% in 13 healthy male volunteers.62

Shorter duration investigations of generic PJ have produced conflicting results. In a randomized, double-blind, placebo-controlled trial with 30 chronic obstructive pulmonary disease patients, no antioxidant benefits were observed after 5 weeks of PJ consumption.63 In a 5-day pharmacokinetic study involving 6 healthy humans, neither punicalagins nor ellagic acid were detectable in their body fluids, and the metabolites of these compounds did not exert antioxidant activity.64 (Note: These 2 studies used a Spanish variety of pomegranate [Punica granatum, cv Mollar de Albatera]). However, a study with 11 healthy volunteers found a 32% increase in antioxidant capacity ½ hour after the consumption of a single acute dose of a PFE.65

Cardioprotective Actions

In vitro

There is evidence that oxidative stress is an important factor in atherogenesis (the development of lipid deposits in the arteries).66–69 Oxidatively damaged macrophages have an increased capacity to oxidize low density lipoprotein (LDL), increase peroxide contents, and decrease glutathione levels.70,71 These factors promote macrophage cholesterol accumulation and foam cell formation,72,73 early indicators of atherogenesis.69 Therefore the inhibition of LDL oxidation may play a key role in the prevention of atherosclerosis.

Figure 3. Constituent Anthocyanins and Anthocyanidins in Pomegranate Juice

 

R1

R2

R3

R4

1. Cyanidin

OH

H

H

H

2. Cyanidin-3-glucoside

OH

H

Glucose

H

3. Cyanidin-3,5-diglucoside

OH

H

Glucose

Glucose

4. Cyanidin-3-rutinoside

OH

H

Rutinose

H

5. Delphinidin

OH

OH

H

H

6. Delphinidin-3-glucoside

OH

Glucose

H

 

7. Delphinidin-3,5-glucoside

OH

OH

Glucose

Glucose

8. Pelargonidin-3-glucoside

H

H

Glucose

H

9. Pelargonidin-3,5-glucoside

H

H

Glucose

Glucose

 

POM Wonderful PJ showed the highest capacity to decrease LDL oxidation and inhibit oxidative stress in macrophages as compared to red wine, green tea, blueberry, or orange juice.56 In macrophages treated with PJ, degradation of oxidized LDL was reduced by 40% and cholesterol synthesis was inhibited by 50%.73 In J774A1 macrophages, PJ treatment decreased peroxide content by 23% more than the PJ phenolic fraction.51 The polysaccharide fraction dose-dependently decreased peroxide concentrations up to 72%, while in marked contrast a white grape juice polysaccharide fraction dose-dependently increased peroxide by up to 72%. In a mouse diabetes model, PJ polysaccharides reduced cellular glutathione by 18% in peritoneal macrophages.51

Nitric oxide is arguably the body’s most important native defense against cardiovascular disease. Oxidized LDL inhibits the production of nitric-oxide synthase, reducing nitric oxide activity in a dose-dependent manner.74 The subsequent application of POM Wonderful PJ significantly decreased oxidized LDL induced down-regulation of nitric-oxide synthase in human endothelial coronary cells.74 POM Wonderful PJ significantly protected nitric oxide against oxygen radical mediated damage and enhanced the anti-proliferative action of nitric oxide on rat aorta smooth muscle cells.57 However, in bovine pulmonary artery endothelial cells it did not affect endothelial nitric oxide synthase (eNOS) expression or eNOS activity and did not stimulate eNOS promoter activity.57

Atherosclerosis is promoted in arterial regions subjected to disturbed blood flow. This shear stress increases the expression of oxidation sensitive genes such as ELK-1, p-JUN, and p-CREB, leading to increased production of free radicals and suppression of eNOS activity. POM Wonderful PJ increased eNOS activity in a dose-dependent manner and significantly reduced ELK-1, p-CREB, and p-JUN in human endothelial coronary cells.75,76

In addition to direct antioxidant activity, there are several other mechanisms by which PJ may convey protective effects against cardiovascular disease. POM Wonderful PJ was found to exert a dose-dependent inhibitory effect (31%) on angiotensin converting enzyme (ACE) activity in vitro.12

Serum paraoxonase 1 (PON1) and PON2 decrease macrophage oxidative stress.25,55,77 PJ and the polysaccharide fraction were reported to dose-dependently increase expression and activity of PON2 and reduce macrophage oxidative stress.25,55

An increase in prostacyclin synthesis may also provide a protective effect against cardiovascular disease. An acute dose of PJ produced an increase in media prostacyclin while chronic exposure resulted in a 61% rise in prostacyclin synthesis in human aortic endothelial cells.78

In vivo—Animals*

The prophylactic effect of POM Wonderful PJ was assessed in a rabbit model of arteriogenic erectile dysfunction (ED).56 Eight weeks of daily supplementation with 3.87 ml of PJ concentrate (equivalent to 112 uM polyphenols daily) increased intracavernous blood flow, improved smooth muscle relaxation and erectile response, but it did not significantly affect nitric-oxide synthase expression. The PJ intake also prevented erectile tissue fibrosis in the ED group. The authors concluded that supplementation may help prevent smooth muscle dysfunction and fibrosis in ED.

Two studies have found that POM Wonderful PJ supplementation exerted cardio-protective effects in hypercholesterolemic mice.75,76 In both an atherosclerosis prevention and a treatment protocol, 24 weeks of POM Wonderful PJ consumption resulted in a suppression of ELK-1, increased eNOS expression, and the progression of atherosclerosis in animals at various stages of disease development was delayed.75 Both the atherosclerotic lesion area and macrophage foam cell formation were reduced approximately 20% in both POM Wonderful PJ protocols. As measured by plasma isoprostanes, plasma lipid peroxidation was significantly reduced while cholesterol levels were not affected.

These results were reproduced and expanded upon in a subsequent evaluation by de Nigris et al.76 POM Wonderful PJ was again found to reduce the activation of ELK-1 (and p-CREB) and increase eNOS expression in atherosclerosis-prone areas. Plasma lipid peroxidation was reduced by 25% and plasma nitrates increased over 44%. Atherosclerotic lesion area and foam cell formation were both decreased by approximately 25% and atherosclerotic disease progression was significantly inhibited. Maximum arterial relaxation was also significantly increased, and other tests indicated an improvement in endothelium-dependent and endothelium-independent vasomotor reactivity. The authors concluded that chronic POM Wonderful PJ consumption reversed the proatherogenic effects induced by perturbed shear stress.

A study employing obese Zucker rats as a model of metabolic syndrome provided evidence that the protective effects of POM Wonderful PJ are attributable to its polar components.79 Expression of vascular inflammation markers, thrombospondin, and cytokine TGFbeta1 was significantly decreased by POM Wonderful PJ intake. Plasma nitrate and nitrite levels were significantly increased (p < 0.05) and eNOS expression rose in the POM Wonderful PJ group. In contrast to the above mouse experiments, only endothelium-dependent (acetylcholine induced) arterial relaxation was increased in this rat model.

In atherosclerotic apolipoprotein E–deficient (E0) mice, 8 weeks of PJ supplementation reduced macrophage lipid peroxides by 37%.80 In contrast, Aviram et al reported that in E0 mice, 11 weeks of supplementation with PJ reduced macrophage LDL oxidation by 90% with an associated reduction in cellular lipid peroxidation and superoxide release.62 The size of atherosclerotic lesions was reduced by 44% and the number of foam cells declined compared to the controls. Uptake of oxidized LDL and native LDL was decreased by 20%.

The latter finding is in general agreement with the findings of Kaplan et al.81 In E0 mice, 8 weeks of POM Wonderful PJ supplementation reduced macrophage oxidized LDL uptake by 31%, decreased cholesterol esterification, and increased cholesterol efflux by 39%. Lipid peroxide content was 42% lower in the 6-month old PJ-treated mice compared to 6-month old placebo-treated control mice and 20% lower than that of 4-month old control mice. The rate of cholesterol esterification in macrophages from the PJ mice was 80% lower than that of age-matched, placebo-treated mice and 57% lower than control mice. At the end of the experiment, the oxidized LDL and cholesterol esterification levels in the 6-month old PJ group were lower than those of untreated 4-month old control group. Serum paraoxonase activity was 43% higher than that of the placebo-control mice and 26% higher than 4-month old control mice. In mice with advanced disease, atherosclerotic lesions were reduced by 17%.

Macrophage oxidative stress levels were reduced in both streptozotocin-induced diabetic Balb/C mice and healthy control mice that were fed PJ polysaccharides for 10 days.25 A decrease in total peroxide content and PON2 activity, and an increase in glutathione levels, were observed in peritoneal macrophages from animals supplemented with PJ sugars. The opposite effect was seen in animals fed white grape juice: a 22% rise in peroxide and 45% decline in glutathione.51 In an 8-week study with E0 mice, PJ consumption increased PON3 activity by 23%.80

PJ may also provide protection against stroke. In a mouse model, maternal consumption of POM Wonderful PJ protected the pups against hypoxic-ischemic brain injury.82 Brain tissue loss was reduced by 60%. Caspase-3 activation, a marker of apotopic death, was reduced by 84% in the hippocampus and 64% in the neonatal brain cortex. A recent study suggested that it may also convey some protective effects against Alzheimer’s disease. Activity guided fractionation of PFE identified ellagic acid and punicalagin as non-competitive beta-secretase inhibitors.83

Human

The cardioprotective effects of POM Wonderful PJ have been investigated in 4 clinical trials.9–12 The Clinical Review section contains a critical overview of these studies.

In 13 healthy male volunteers, 2 weeks of daily POM Wonderful PJ consumption (50 ml PJ concentrate; the concentrated PJ was diluted 1:5 [v:v] with water to obtain a single strength juice equivalent to 8 ounces per day) resulted in an 11% decrease in collagen-induced platelet aggregation compared to baseline values (p < 0.02).62 LDL oxidative susceptibility was decreased by 43%, as measured by the prolongation of the lag time to oxidation initiation. Plasma susceptibility to oxidation was reduced by 6% and total antioxidant status was increased by 9%. A trend towards a reduction in LDL susceptibility to aggregation and retention was observed in 7 of the 13 subjects. The activity of PON1 was increased by 18%.

In another study, PJ (6-9 mL/kg) was provided to 28 fasted, healthy adult subjects (8 men and 20 women).78 After juice consumption, epinephrine/collagen-induced clotting time (p < 0.05) was significantly prolonged, indicating an inhibition of platelet aggregation. However, it did not significantly affect plasma prostacyclin concentrations.

ANTI-CANCER ACTIONS

In vitro

The anti-cancer effects of pomegranate and its components have been observed in a wide variety of in vitro models, including breast, prostate, colon, leukemia, and skin cancer cell lines, among others.23,28,53,84-94

The in vitro antiproliferative effects of POM Wonderful PJ concentrate (1.74 mg/ml punicalagin and 0.14 mg/ml ellagic acid) was evaluated in human colon and oral cancer cell lines using a luminescence assay.53 POM Wonderful PJ exhibited 100% inhibition of the 2 oral cancer cell lines: (1) at a concentration 12.5 ug/ml in the CAL 27 and (2) at 25 ug/ml in KB cells. At a concentration of 25 ug/ml POM Wonderful PJ, a 100% reduction in growth was also observed in 4 colon cancer lines (the non-metastatic SW 460, metastatic SW 620, HT29, and HCT 116). The ability of POM Wonderful PJ to induce apoptosis was assessed in the latter 2 colon cancer lines using a photometric ELISA assay. In HCT 116 cells, apoptosis was only induced 0.7-fold. However, in HT29 cells apoptosis was induced 2.66-fold over the controls.

The anticancer activity of pomegranate tannin fractions and the isolated pure constituents ellagic acid and punicalagins have been reported by a number of investigators.44,89,95,96 Seeram et al also tested the antiproliferative effects of 3 POM Wonderful PJ components: ellagic acid, punicalagins, and a total pomegranate tannins (TPT) extract.53 While all 3 components exhibited some inhibitory effects in the cancer cell lines, in every case their activity was significantly less than that of the POM Wonderful PJ—even though the stock solutions of juice, TPT, and punicalagins had been normalized to provide the equivalent amount of punicalagins w/w. At a concentration of 12.5 ug/ml, ellagic acid inhibition was less than 55% in all lines while POM Wonderful PJ exhibited ≥ 80% inhibition. In comparison, punicalagins and TPT activity did not even reach 20% inhibition. These results suggest that other POM Wonderful PJ components significantly contribute to the juice’s antiproliferative effects, in addition to the tannin constituents. In this regard, it is noteworthy that pomegranate anthocyanidins, flavonoids, and oils have also been reported to exert anticancer effects against leukemia, colon, breast, prostate, skin, and lung tumors.28,88,89,97-102 These 3 components induced apoptosis only at concentrations equivalent to 100 ug/ml in the 2 colon cancer lines. In HT29 cells, ellagic acid, punicalagins, and TPT induced apoptosis 2.44-, 2.65-, and 2.59-fold, respectively, as compared to the controls (cv 2.66 for POM Wonderful PJ). For HCT 116, the values were 2.85-, 1.52-, and 2.87-fold, respectively.53 The fact that POM Wonderful PJ did not significantly induce apoptosis in the HCT 116 line even though it decreased proliferation 100%, suggests that POM Wonderful PJ anti-cancer effects may occur via at least 2 different mechanisms. In this study, the antioxidant activity of POM Wonderful PJ was also significantly greater than that of the 3 components (POM Wonderful PJ > TPT > punicalagins > ellagic acid), implying that direct inhibition of oxidation may be one of these mechanisms.

Ellagic acid and punicalagins were also evaluated in Caco-2 colon cancer and normal colon cells.95 They dose-dependently inhibited Caco-2 proliferation but their effect was additive, not synergistic. Both compounds significantly increased Caco-2 apoptosis but neither induced apoptosis in the normal colon cells. It appeared that ellagic acid was the actual apoptosis inducer though, as punicalagins treatment did not induce apoptosis until its hydrolysis product, ellagic acid, accumulated in the media.

The protein kinase/nuclear factor kappa-B (NF-kB) signaling pathway promotes the production of cyclooxygenase 2 (COX-2) and there is evidence that over expression COX-2 plays an important role in carcinogenesis.102,103 NF-kB inhibition in particular has been identified as a major therapeutic target, as this transcription factor regulates the expression of over 200 genes involved in cancer progression.102,104

The effect of POM Wonderful PJ and its components on COX-2 and protein kinase/NF-kB activity was evaluated in HT29 colon cancer cells.102 At concentrations normalized to provide the equivalent of 50 ug/ml punicalagins, POM Wonderful PJ, TPT, and punicalagins all significantly inhibited COX-2 expression in a dose-dependent manner. POM Wonderful PJ exhibited the greatest effect, inhibiting COX-2 by 79% compared to 55% TPT and 48% punicalagins. POM Wonderful PJ abolished protein kinase B activity completely and also significantly affected NF-kB activity at a concentration of 50 ug/ml. Pretreatment with POM Wonderful PJ resulted in a 6.4-fold reduction in NF-kB (p65) DNA binding and a 92% reduction in phosphorylation of the p65 subunit. Considering POM Wonderful PJ’s superior potency to TPT and punicalagins, the authors concluded that POM Wonderful PJ’s activity is most likely due to significant interactions with other bioactive constituents of the juice.

Pretreatment with the anthocyanidin constituent delphinidin was found to provide protective effects against UV-B induced decreases in cell viability and apotosis induction in immortalized HaCaT keratinocytes. It inhibited numerous markers of UV-B oxidative stress including increased lipid peroxidation, increased the pro-apoptosis Bax, decreased Bcl-2, and down-regulated the anti-apoptosis Bcl- XL.97

In other studies, PJ was also shown to inhibit the proliferation of leukemia and breast cancer cells.28,90 PJ and PJ polyphenol fractions were found to inhibit proliferation and induce differentiation in HL-60 human promyelocytic leukemia cells, as assessed in 4 different assays.90 PJ and 3 fractions all inhibited growth of estrogen sensitive (MCF-7) and estrogen resistant (MDA-MB-231) breast cancer cells.28 The maximum inhibition (80%) of MCF-7 was induced by fermented PJ at a concentration of 50 ug/ml; the same concentration was 3 to 4 times less effective in the MDA-MB-231 cells and produced minimal toxicity in immortalized normal human breast epithelial cells (MCF-10A).

The anti-angiogenic effects of pomegranate polyphenol and oil fractions were evaluated in breast cancer and normal breast cells by measuring vascular endothelial growth factor and migration inhibition factor.94 The angiogenic promoter vascular endothelial growth factor was significantly down-regulated in normal human breast epithelial (MCF-10A) and estrogen sensitive (MCF-7) breast cancer cells but not estrogen resistant (MDA-MB-231) breast cancer cells. Conversely, expression of the angiogenesis suppressor migration inhibition factor was not affected in the normal and estrogen sensitive cells while it increased in the estrogen resistant cells. All extracts inhibited the proliferation of myometrial and amnionotic fluid fibroblasts, while only fermented PJ polyphenols inhibited tubule growth in human umbilical vein endothelial cells. These results suggest that the antiangiogenic effects of pomegranate polyphenols arise via multiple mechanisms.

In vivo—Animals*

A PFE (70% acetone-water extract) was found to significantly improve survival time in athymic male nude mice xenografted with human A549 non-small cell lung cancer.91 Human clinical relevance of the 2 doses assessed (0.1% and 0.2% PFE) was based on the assumption that a typical healthy person (70 kg) would reasonably consume 250 to 500 ml of PJ per day. The latency period before the appearance of solid tumors was extended to 19 days compared to 15 days in the controls, an increase of 27%. An average tumor volume of 1200 mm3 was reached after 55 days in the controls. At this time point, the average tumor volume in the PFE groups was 621 and 540 mm3, respectively. In the verum groups, 1200 mm3 average tumor volume was reached at 67 days (0.1%) and 79 days (0.2%), prolonging survival time 22% and 44%, respectively.

In female A/J mice, the PFE was also found to inhibit the growth and progression of lung tumors induced by 2 chemical carcinogens: NTCU and B(a)P.92 The mice treated with PFE had significantly lower lung tumor multiplicities in both models, with a tumor reduction of 61.6% in the B(a)P group and a decrease of 65.9% in the NTCU mice. The extract inhibited a number of cell survival pathways, including NF-kB and IkBα kinase activation, and phosphorylation of IkBα, MAPK, protein kinase B, and c-met. Markers of cell proliferation and angiogenesis were also significantly inhibited.

Oral feeding of PFE also inhibited markers of UV-B induced carcinogenesis in SKH-1 hairless mice.105 In SKH-1 hairless mice, the PJ constituent delphinidin also inhibited UV-B induced apoptosis and markers of DNA damage.97

PJ and PFEs have also been reported to be radioprotective and chemo-protective against liver and gastric damage.40,60,106–108

Prostate Cancer

In vitro

The antiproliferative effects of POM Wonderful PJ (1.74 mg/ml punicalagin and 0.14 mg/ml ellagic acid) was evaluated in the metastatic 22RV-1 and the immortal epithelial RWPE-1 prostate cancer cell lines.53 At the lowest concentration evaluated in this study (12.5 ug/ml), POM Wonderful PJ significantly inhibited the proliferation of both prostate cancer lines, reducing the growth of RWPE-1 by 90% and that of 22RV-1 by > 95%.

The PJ constituent ellagic acid and its urolithin A metabolites were assessed in 4 prostate cancer cell lines: (1) androgen-dependent LNCaP, (2) androgen-independent LNCaP-AR, (3) DU 145, and (4) 22RV1.109 The compounds exhibited dose-dependent inhibition in all of the cell lines, with urolithin A inducing a lower IC50 than ellagic acid in all cases. The lowest IC50 values were observed with the derivative urolithin A in LNCaP and 22RV1 cells, at 15.9 and 6.2 umol/L, respectively.

A PFE exerted a significant dose-dependent inhibition of proliferation and induced apotosis in androgen-independent PC-3.93 Mechanistic studies showed that it induced the pro-apoptopic Bax and Bak genes; down-regulated the anti-apoptopic BcL-XL and Bcl-2; decreased cyclins D1, D2, and E; and decreased cyclin-dependent kinase (cdk) 2, 4, and 6 expression. In CWR22Rv1 cells, the PFE significantly decreased androgen receptor expression (90%) at a concentration of 100 ug/ml and significantly decreased prostate specific antigen (PSA) protein levels (67% at 100 ug/ml).

In vivo—Animals*

The inhibitory effect of a PFE was evaluated in severe combined immunodeficient mice xenografted with human prostate cancer cells (LAPC-4).109 Oral administration of the PFE significantly inhibited LAPC-4 proliferation, producing a 1.8 cm3 reduction in tumor volume 6 weeks after inoculation.

Clinically relevant doses of a PFE (0.1% and 0.2% wt/vol) significantly inhibited the growth of androgen-dependent CWR22Rv1 tumors in athymic nude mice. Serum prostate specific antigen (PSA) levels also declined significantly, with a 70% and 85% reduction respectively for the 2 doses after 30 days.93

Pomegranate pericarp polyphenol and oil extracts were tested in a PC-3 xenograft model employing athymic nude mice. At a dose of 2ug/g body weight, both fractions reduced tumor volume by 72% compared to controls at 35 days post-inoculation.85

Human

The results of a phase II prostate cancer clinical trial are reviewed in the Clinical Review section on page 11.14

Estrogenic Activity

In vitro

The purported estrogenicity of pomegranate is a potential concern for prostate and breast cancer patients in particular. There are conflicting results regarding the effects of PJ on estrogen-sensitive MCF-7 breast cancer cells. Maru et al reported that PJ stimulated the proliferation of MCF-7 cells,110 while Kim et al asserted that it inhibited proliferation of this line.28 Jeune et al also reported that undescribed PFEs inhibited MCF-7 proliferation via apotopic induction.87 Mixed results have also been reported regarding androgen-sensitive and androgen-resistant prostate cancer cell lines (see Prostate Cancer section above).

In vitro binding assays have also produced conflicting results. Maru et al reported that PJ exhibited estrogen-like activity, competing with 17β-estradiol for estrogen receptor binding.110 Kim et al asserted that fermented PJ, fresh PJ, pericarp polyphenols, and oil significantly inhibited estrogen synthase (aromatase) and 17-β-hydroxysteroid dehydrogenase (17-β-HSD) type 1.28 At a concentration of 1 mg/100ul, the fresh juice by itself displayed only minimal estrogenic action, while the lyophilized juice effected a 55% inhibition of the estrogenic activity of 17-β-estradiol. The fermented PJ induced 51% and the pericarp polyphenol 24% inhibition of aromatase at a concentration of 0.02 ug/ml. All extracts inhibited 17- β-HSD at 1000 ug/ml, but only the oil was active at 100 ug/ml.

However, POM Wonderful PJ did not exhibit estrogenic activity and there was no significant additive effect of POM Wonderful PJ with 17-β-estradiol in an estrogen agonist assay utilizing transfected MCF-7 cells.111 A small antagonistic effect was noted with the highest concentrations of POM Wonderful PJ concentrate tested at estrogen receptor β.

As previously discussed in the Chemistry section, early reports of steroid hormone constituents in pomegranate seed have not been substantiated by mass spectrometry/nuclear magnetic resonance or by more accurate high power liquid chromatography and gas chromatograph mass spectrometer analysis of seed oil or PJ.29,38 PJ does contain non-steroidal “phytoestrogenic” compounds though, which may account for the weak estrogenic effects some researchers have reported.26,29 Using liquid chromatograph-mass spectrometry analysis teamed with an on-line β-estrogen receptor assay, the flavonoids luteolin, kaempferol, and quercetin were identified as the major PJ estrogenic constituents.29

In vivo—Animals*

In 1964, Sharaf and Nigm reported that pomegranate seed oil exhibited estrogenic activity in mouse and rabbit models.112 Heftmann et al also reported estrogenic activity in ovariectomized mice by a seed constituent, which they identified as estrone based upon TLC and chemical tests only.33 However, the identity of this compound as estrone was never further substantiated.

PJ was also reported to have increased uterine weight in ovariectomized rats110 and in an ovariectomized mouse model of menopausal syndrome; a PFE improved bone properties and depression.113 The clinical relevance of the concentrations used in these studies and the preceding in vitro investigations has not been established.

Human

In a small open label study with 11 post-menopausal women, 7 days of POM Wonderful PJ consumption (8 ounces per day) significantly increased serum estrone levels.114 No significant changes in estradiol, follicle stimulating hormone, luteinizing hormone, or pituitary gonadotropins were observed, and estriol remained undetectable. No significant biological estrogenic effects (as measured by vaginal cornification) were noted and the authors pointed out that while the increase in estrone levels was statistically significant, they would be expected to exert physiological effects only at much higher concentrations.

Pharmacokinetics

In vivo—Animals*

The pharmacokinetics of the PJ constituents ellagic acid and punicalagins has been evaluated in rats. Following oral administration of 0.8 g/kg extract, the maximum ellagic acid plasma concentration (213 ng/ml) was observed at 0.55 h.115 In another oral administration study with rats, approximately 10% was excreted in the feces and urine as 3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one (urolithin A) and a second metabolite was detected but not identified.116 Both compounds were apparently of microfloral origin, as their presence was not detected in germ-free rats but was observed when ellagic acid was incubated in vitro with normal rat gastro-intestinal micro-organisms. Ellagic acid itself was not detected in the feces or urine of any normal rat, but small amounts were detected in the feces of germ-free animals.

In another study, oral administration of PFE led to increased ellagic acid plasma levels, but this compound was not detectable in the prostate.109 Intraperitoneal administration produced roughly 10-fold higher ellagic acid plasma levels and detectable amounts in the prostate. Both oral and intraperitoneal administration of urolithin A resulted in significant levels in the prostate, colon, and intestinal tissues.

In animals fed standard rat diet plus 0.6 to 1.2 g punicalagins per day, approximately 3-6% of the punicalagins was excreted as identified metabolites in feces and urine.117 Presumably, the majority of this compound was converted to undetectable metabolites or accumulated in non-analyzed tissues. Only trace amounts of 5 punicalagins metabolites were detected in the liver or kidney.118

Human

A human subject consumed 180 ml POM Wonderful PJ containing 25 mg ellagic acid and 318 mg punicalagins.119 Ellagic acid was detected in the plasma at a maximum concentration (31.9 ng/ml) after 1 hour but was rapidly eliminated by 4 hours.

Eighteen healthy volunteers were given 180 mL of POM Wonderful PJ concentrate, and blood and urine samples were collected.120 Ellagic acid was detected in the plasma of all subjects with a maximum concentration of 0.06 mmol/L, area under the concentration-time curve (AUC) of 0.17 (mmol/h)/L-1, tmax of 0.98 h, and elimination half-life of 0.71 h. Ellagic acid metabolites, including dimethylellagic acid glucuronide and hydroxy-6H-benzopyran-6-one derivatives (urolithins), were also detected in plasma and urine. On day 0, dimethylellagic acid glucuronide was found in the urine obtained from 15 of 18 subjects, but it was not detected on the day prior or the day after consumption. Urolithin A was found in the urine from 11 subjects on day 0 and in the urine from 16 subjects on the following day. Urolithin B was found in the urine of 3 subjects on day 0 and in the urine of 5 subjects on the next day.