The presence of human steroid hormones, including estrone, estriol, estradiol, and testosterone, in pomegranate seeds was reported previously based on TLC separations and colorimetric assays. However, HPLC-DAD- and gas chromatography -MS-based analysis showed that these steroid hormones could not be identified in pomegranate seeds using the more sensitive analytical methods. Multi-target drugs derived from mixtures of plant natural products have increasingly been pursued nowadays to contradict drug tolerance and resistance in cancer therapy. Although several studies have suggested synergistic interactions among pomegranate phytochemicals, this is a promising, but currently under-explored topic. Fresh arils and juice products of pomegranate fruit, as well as seeds of the soft-seeded cultivars, are mostly consumed. Phytochemicals in pomegranate fruit peel extracts, fermented pomegranate juice, blueberry box and pomegranate seed oil exhibited cooperative interactions toward limiting the proliferation, metastasis, and invasiveness of human prostate cancer cells in vitro.
A subsequent analysis with pure phytochemicals, including EA, caffeic acid, luteolin, and punicic acid, also showed synergistic interactions on suppressing the invasion of prostate cancer cells. Interestingly, commercial pomegranate juice demonstrated more antioxidant and anti-proliferative activities than the purified pomegranate polyphenols in colon cancer cells, suggesting that inherent synergies exist among polyphenols and other phytochemicals in pomegranate juice. When the commercial pomegranate-nectarine juice was separated into predominately sugar, organic acid, neutral phenol, and AT fractions, complex antagonistic or synergistic effects were observed among different fractions on the total phenol or total antioxidant content. The antagonistic or synergistic interactions depended on the concentrations of the chemical constituents in the juice product. The polyphenol extracts of pomegranate fruit also synergistically interacted with the antibacterial drug ciprofloxacin, though various bacterial strains responded differently to the phytochemical-drug synergy, and the underlying mechanism of such synergy remains unknown. ATs are colorful, water-soluble polyphenol pigments that are found in many plant foods, such as berries and pomegranate fruits. Plant ATs are often investigated collectively as a group of phytochemicals for their bioactivities, and have been linked to many aspects of human disease prevention and treatment.
The anti-inflammatory and cardioprotective activities of ATs are attributed by their antioxidant properties via various underlying mechanisms. ATs can quench free radicals, inhibit the activity of xanthine oxidase that generates free radicals, and chelate metal ions that are involved in oxidation of low-density lipoproteins. In addition, ATs induce the expression of nuclear factor-erythroid 2-related factor-2 that regulates the expression of endogenous antioxidant enzymes, such as hemeoxygenase-1. Besides reactive oxygen species , ATs also inhibit the production of reactive nitrogen species , particularly nitric oxide , as well as their associated oxidative processes. Furthermore, release of pro-inflammatory mediators and adhesion molecules is suppressed by ATs via targeting of the respective signaling pathways, e.g., the arachidonic acid and the tumor necrosis factor -α, nuclear factor -κB pathways. Aside from cardioprotection, the anti-inflammatory property of ATs also contributes to their anti-obesity effect and action in adipose tissue. Studies using AT-rich fruit extracts showed that ATs prevented the upregulation of inflammatory response in adipose tissue, when triggered with consumption of a high-fat diet, by regulating the NF-κB stress signaling pathway . ATs also reduce the level of pro-inflammatory cytokines and modulate the expression of adipocytokines. Although studies using cell lines and animal models support the anti-obesity effect of ATs, a causal relationship between AT consumption and reduction of body mass index has yet to be conclusively established by clinical studies. This could be due to one or more parameters that are associated with different clinical studies, such as the amount and types of ATs ingested, the food matrix of ATs, and the inherent difference of the study subjects.
In addition to their antioxidant and anti-inflammatory activities, ATs act in cancer chemoprevention by inducing terminal differentiation of tumor cells and thus impeding tumorigenesis. Prevention of malignant cell transformation and inhibition of cancer cell proliferation by ATs have also been reported. ATs can further interfere with cancer development by activating caspases and inducing apoptosis of cancer cells. However, the cancer chemotherapeutic effect of ATs still requires strong supporting evidence from clinical studies. In addition, the bioconversion of ATs by human intestinal microbiome should be rigorously investigated. Similar to other AT-rich fruits, the pomegranate also has a cardioprotective effect by targeting two major causal factors of atherosclerotic lesion and cardiovascular disease, namely the accumulation of cholesterol and oxidized lipids, and arterial macrophage foam cell formation. Pomegranate juice and fruit extracts are also associated with antidiabetic activities, largely due to the antioxidant properties of HTs and ATs in reducing oxidative stress and lipid peroxidation. Supplements of pomegranate juice or fruit extracts at 1 g/kg/day for five weeks, effectively enhanced endothelial NO synthase expression, as well as plasma nitrate and nitrite levels in plasma in obese Zucker rats fed with an atherogenic diet. In addition, supplements of juice at 100 mg/kg/day or 300 mg/kg/day for four weeks prevented the development of high blood pressure in diabetic rats. Consumption of pomegranate juice rich in ATs and HTs also lowered systolic and diastolic blood pressure in hypertensive patients. The bioactivities of pomegranate ATs have mostly been studied in the context of pomegranate juice and fruit extracts. However, these AT-rich sources are abundant in vitamin C, carotenoids, and HTs, which can also contribute to the ascribed bioactivities. To this end, “white” pomegranate cultivars are available, which lack the accumulation of ATs in leaves, fruits and flowers. Comparative analysis of the white and AT-rich pomegranate cultivars will be informative as to the specific role of ATs in the health-promoting activities of pomegranate juice and fruit extracts.ETs are abundant in pomegranate fruit peel, as well as juice and extracts that are produced commercially from the whole fruit. Besides pomegranate, ETs are also present in a wide range of medicinal and food plants, such as many berries, nuts, and herbs. As such, the knowledge of biotransformation and bioconversion of pomegranate ETs by human and microbial enzymes will inform nutritional and pharmacological studies on ETs that are isolated from other plants. EA, a hydrolysis product of ETs , can be detected in the blood circulation of healthy volunteers within half an hour of ingesting pomegranate juice or extracts. The human gut microbiota can further convert EA into urolithins prior to their absorption by the intestinal cells. Aromatase that converts androgens to estrogens has been considered to be a therapeutic target for treating the hormone-sensitive type of breast cancer. Although several urolithins exhibited anti-aromatase activities in a placental microsome-based enzyme assay, blueberry package only UB competitively inhibited and most effectively suppressed the aromatase activity in an aromatase-overexpressing breast cancer cell line. Consistent with “in cell” aromatase-inhibitory activity, UB also significantly arrested testosterone-induced proliferation of MCF-7aro cells, suggesting an underlying mechanism of aromatase inhibition. Interestingly, estrogen-induced proliferation of MCF-7aro cells was also inhibited by urolithins. However, inhibition was was likely effected through mechanisms independent of the aromatase activity. Besides breast cancer, urolithins can also reduce the risks of another sex steroid hormone-related cancer, endometrial cancer. UA and UB caused over 50% reduction in the proliferation of human endometrial cancer cells. UA arrested cell cycle at the G2/M phase and regulated the expression of cell cycle-related proteins at this phase. On the other hand, UA was shown to act as an estrogen agonist and modulate the estrogen-receptor α -dependent gene expression in the ER-positive endometrial cancer cells.
It remains to be determined whether and how estrogen signaling could be involved in the suppression of endometrial cancer cell growth by urolithins. The canonical Wnt/β-catenin signaling pathway has been shown to activate T-cell factor transcription and function in colon carcinogenesis . To determine the potential involvement of urolithins in colon carcinogenesis through Wnt signaling, phenolic extracts of several fruits rich in ETs, including pomegranate, strawberry , and Jamun berry , as well as pure EA and UA compounds, were tested in the human embryonic kidney 293T cells that express a reconstructed canonical Wnt signaling pathway. Although the fruit extracts and chemicals all inhibited Wnt signaling, only UA exhibited an IC50 value that was physiologically relevant in the lumen of colons when taking enterohepatic circulation of urolithins into consideration. During the initiation stage of the HT-29 colon cancer cells, urolithins A–D inhibited TCDD-induced, CYP1-mediated EROD activities. At the cancer progression stage, UA and UB impaired the proliferation of HT-29 cells and led to cell cycle arrest at the G2/M phase, as well as activation of CDKN1A expression. In addition, treatment with urolithins resulted in activation of caspases 3, 8, and 9, suggesting that urolithins induced both the extrinsic and intrinsic apoptotic pathways in HT-29 cells. UA, UB, and mUA inhibited the proliferation of the T24 human bladder cancer cells in vitro, with IC50 values of 43.9, 35.2, and 46.3 µM, respectively, comparable to the UA inhibition of Wnt signaling at an IC50 of 39 µM. The transcript and protein levels of Phospho-p38 mitogen-activated protein kinase were increased by the urolithin treatment, while those of MAP kinase kinase kinase1 and Phospho-c-Jun were decreased in the T24 cells. Furthermore, these urolithins reduced the level of H2O2-induced oxidative stress and induced apoptosis through activation of caspase 3 and PPAR-γ protein expression. Inflammatory responses involving the activation of neutrophils and monocytes play a central role in the development of cardiovascular diseases. Intriguingly, the number of free hydroxyl functional groups on urolithins appeared to impact how they modulate the inflammatory functions of neutrophils. Among the urolithins tested , UA exhibited the most potent antioxidant activities against the release of ROS from the pro-inflammatory triggered neutrophils. UB significantly affected several inflammatory biomarkers that are associated with cardiovascular events, by inhibiting the production of interleukin 8 and metalloproteinase-9 , and preventing the shedding of selectin CD62L triggered by the pro-inflammatory factor cytochalasin A/formyl-met-leu-phenylalanine . UC , on the other hand, inhibited the release of elastase, a pro-inflammatory mediator responsible for extracellular matrix degradation, from the f-MLP-stimulated neutrophils. In addition to recruitment of neutrophils, monocyte adhesion to endothelial cells represents another key event in inflammatory responses. A mixture of UA and UB restricted the adhesion of Tamm-Horsfall protein-1 monocytes to the human umbilical vein endothelial cells. UA glucuronide , but not UA, UB or UB glucuronide, inhibited monocyte adhesion to TNFα-stimulated human aortic endothelial cells ; a dosage-dependent inhibition of TNFα-induced migration of endothelial cells was also shown for the above-mentioned urolithins. NO plays multifaceted roles in combatting cardiovascular diseases. Although UA, UB, and UB glucuronide did not show any effect individually at 15 µM on NO bioavailability, a mixture of the three urolithins at equal concentrations activated the expression of eNOS after a 5-min incubation and increased NO production in primary HAECs after a 24-h incubation. Overall, these in vitro studies with neutrophils, monocytes, and NO suggested the potential anti-inflammatory and cardiovascular-protective functions of urolithins.Based on the urinary excretion of urolithins by healthy volunteers after ingesting ET-rich foods or fruit extracts, three urolithin metabolic types have been defined, including A , B , and 0. Interestingly, the population of the human gut bacteria Gordonibacter urolithinfaciens correlated positively with the in vivo production of UA, but inversely with that of UB and isoUA . A recent study observed an interlinked relationship among gut dysbiosis , ET metabolism, and obesity. A relatively high percentage of metabotype B was found in the overweight-obese group, while metabotype A had a higher presentation in the normoweight than the overweight-obese group. In addition, G. urolithinfaciens levels were higher in the metabotype A than the metabotype B individuals. Further investigations should provide a mechanistic understanding of how consuming the polyphenol precursors of urolithins, in the presence of UA-producing bacteria, may reduce the risks of diseases associated with obesity. Large inter-individual variations in the cardiovascular risk biomarkers were observed in healthy overweight-obese individuals after consuming pomegranate supplement. However, after clustering the different urolithin metabotypes in these individuals, improved blood lipid profiles were evident in the metabotype B group, in a dose-dependent fashion, while there was no significant effect in the metabotype A group. Interestingly, several metabotype 0 individuals shifted to metabotype A or B after consuming pomegranate extracts. Together with the study by Selma et al. , these results suggest that consumption of pomegranate extracts may have personalized effects that are associated with the gut microbiota and the urolithin metabotypes of the individuals.Of the various pomegranate phenolic metabolites exhibiting anti-Alzheimer activities in in vitro assays, only urolithins, including UA, UB, mUA, and methyl-urolithin B , were predicted to be capable of crossing the blood-brain barrier.