Pomegranate has been used for centuries to confer health benefits in a number of inflammatory diseases. Based on its usage in Ayurvedic and Unani medicine, dietary supplements containing pomegranate extract are becoming popular in the Western world for the treatment and prevention of arthritis and other inflammatory diseases. More recently standardized extracts of pomegranate fruit (PFE) have been shown to possess anti-inflammatory and cartilage sparing effects in vitro . Published studies have shown that constituents of PFE inhibit the proliferation of human cancer cells and also modulate inflammatory subcellular signaling pathways and apoptosis when directly added to the culture medium [2–6]. PFE has also been shown to significantly reduce the growth of prostate tumors and the levels of prostate-specific antigen (PSA) in nude mice implanted with prostate cancer cells . Several groups have reported that consumption of pomegranate may have cholesterol lowering and cardiovascular and other chronic diseases preventing effects in vivo [8–11]. In these studies the major effect of the pomegranate extract consumption was the reduction of oxidative stress, inhibition of p38-mitogen-activated protein kinase (p38-MAPK) pathway and inhibition of the activation of transcription factor NF-κB. Activation of p38-MAPK and NF-κB is intimately associated with the increased gene expression of TNF-α, IL-1β, MCP1, iNOS and COX-2-agents that are critical mediators of inflammation and the pathogenesis of inflammatory and degenerative joint diseases [12, 13]. These and other published studies [, reviewed in [15, 16]] thus demonstrate that PFE possesses strong antioxidant and anti-inflammatory properties and its consumption has the potential to prevent diseases in which redox imbalance and inflammatory stimuli plays a decisive role.
The major class of phytochemical present in pomegranate is the polyphenols and includes flavonoids, condensed tannins and hydrolysable tannins. Hydrolysable tannins are predominant polyphenols found in pomegranate juice and account for 92% of its antioxidant activity . Pomegranate seeds are rich in sugars, polyunsaturated (n-3) fatty acids, vitamins, polysaccharides, polyphenols, and minerals and have high antioxidant activity. When crushed and dried, the seeds produce an oil with 80% punicic acid, the 18-carbon fatty acid, along with the isoflavone genistein, the phytoestrogen coumestrol, and the sex steroid estrone. The seed coat of the fruit contains delphinidin-3-glucoside, delphinidin-3,5-diglucoside, cyanidin-3-glucoside, cyanidin-3,5-diglucoside, pelargonidin-3-glucoside, and pelargonidin-3,5-diglucoside with delphinidin-3,5-diglucoside being the major anthocyanin in pomegranate juice . Studies have also shown that the antioxidant capacity of pomegranate juice is three times that of the popular antioxidant-containing beverages such as red wine and green tea, presumably due to the presence of hydrolyzable tannins in the rind, along with anthocyanins and ellagic acid derivatives . In a comparative analysis, anthocyanins from pomegranate fruit were also shown to possess higher antioxidant activity than vitamin-E (α-tocopherol), ascorbic acid and β-carotene . Pomegranate extract has also been shown to protect from NSAID and ethanol-induced gastric ulceration . Repeated administration of high doses of a hydroalcoholic extract of pomegranate whole fruit or its constituent ellagitannin punicalagin were non toxic in the dosages commonly employed in traditional medicine systems [19, 20].
Flavonoid rich fractions of pomegranate fruit extract have also been shown to exert antiperoxidative effect as their administration significantly decreased the concentrations of malondialdehyde, hydroperoxides and enhanced the activities of catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase in the liver [21, 22]. Anthocyanins were shown to be effective inhibitors of lipid peroxidation, the production of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) activity in different model systems [22–24]. After consumption, anthocyanins are efficiently absorbed as glycosides from the stomach and are rapidly excreted into bile as intact and metabolized forms [25, 26]. Plasma concentration of 30 μg/ml of punicalagin and 213 ng/ml of ellagic acid after oral administration in rats has been reported . In humans it has been shown that ellagic acid is rapidly absorbed and plasma concentrations of 31.9 ng/ml were detected within one hour of oral consumption of pomegranate juice . Cyclooxygenase (COX), an enzyme involved in the mediation of inflammatory process, catalyzes the rate-limiting step in the synthesis of prostaglandins from arachidonic acid [29, 30]. Of its two isoforms, COX-1 is constitutively expressed in most tissues and appears to be responsible for maintaining normal physiological functions whereas COX-2 has been shown to be involved in cutaneous inflammation, cell proliferation, and skin tumor promotion . These data suggest that inhibition of COX-2 activity is important for alleviating inflammation. Other studies have shown that Prodelphinidins isolated from Ribes nigrum inhibit cyclooxygenase-2 (COX-2) and lipoxygenase activity and production of prostaglandins E2 (PGE2) in vitro, suggesting that the primary effect of delphinidins (also present in pomegranate fruit) may be against inflammatory responses . More recently it has been shown that pomegranate extract exerted a powerful influence in inhibiting the expression of inflammatory cytokines IL-1β and IL-6 in adjunctive periodontal therapy . Other in vitro studies have shown that the bioactivity of total pomegranate extract was superior to its purified individual polyphenols illustrating the multifactorial effects and chemical synergy of the action of multiple compounds present therein .
While evidence from in vitro studies does not prove in vivo biological activity, these do provide a rationale and support for the use of pomegranate fruit or its extract to suppress inflammation in vivo. However, it is also important to point out that there are issues that deserve an explanation and require caution in interpreting the data obtained from in vitro studies. One question often raised is whether the concentration of a plant or fruit extract constituent compound that has been used in in vitro experiments would be realistic or achievable in vivo. In majority of the cases this has to be denied because constituents of plant or fruit extracts are typically not completely bioavailable and only certain constituents can be expected to be absorbed and become bioavailable via the hepatic portal system . Another issue to be considered is that the bioeffective compounds do not necessarily need to be present in the original extract, but might be formed in vivo due to intestinal bacterial and/or hepatic metabolism . This is supported by recent studies demonstrating that after ingestion of pomegranate juice by human volunteers ellagic acid metabolites which were not present in the juice consumed such as dimethylellagic acid glucuronide were detected in plasma and urine while Urolithins-formed by intestinal bacteria-were detected in the urine samples .
Pomegranate fruits are popularly consumed throughout the world and fruit and flower extracts are widely used for the treatment of inflammatory diseases in the traditional medicine systems of Asia and Europe. In this study using rabbits we determined whether after oral ingestion of a standardized preparation of pomegranate fruit extract (PFE), blood plasma samples contained PFE-derived metabolites/constituents by HPLC-DAD analysis. To test whether these same plasma samples exert anti-inflammatory effects, we determined whether the presence of these plasma samples in the assay mixture or culture medium can (a) inhibit the enzymatic activity of purified cyclooxygenases ex vivo; and (b) inhibit IL-1β-induced production of nitric oxide (NO) and PGE2 by rabbit articular cartilage chondrocytes in vitro.