UCLA Center for Human Nutrition

Pomegranate (Punica granatum L.) fruits are widely consumed fresh and in the forms of juice, concentrate, wine and jam. Pomegranate fruit husk is a rich source of hydrolyzable tannins called ellagitannins (ETs). During processing methods in the commercial pomegranate juice (PJ) industry, ETs are extracted from the fruit husk in significant quantities into the juice. Pomegranate husk, a by-product of the PJindustry, is therefore an inexpensive and abundant source of ETs which are present in PJ. Previous methods to isolate pomegranate ETs included labor intensive and time-consuming solid phase extractions by column chromatography (C-18, polyamides, cellulose, Sephadex Lipophilic LH-20, Diaion HP20) and/or use of specialized instruments such as preparative-high performance liquid chromatography (HPLC). We have used an Amberlite XAD-16 resin vacuum-aspirated column to rapidly purify an aqueous extract of pomegranate husk to afford total pomegranate tannins (TPT) in substantial yields (58-60 g TPT/Kg husk; time <1h). Using analytical HPLC, NMR and tandem LC-ES/MS, evaluation of TPT showed that it contains the major fruit husk ET, punicalagin (85% w/w), and ellagic acid (EA; 1.3% w/w) as well as unquantified amounts of punicalin and EA glycosides (hexoside, rhamnoside and pentoside). Since ETs are reported to show potent antioxidant, antiatherosclerotic and anticancer activities, this method can be used for the large-scale production of TPT for future in vitro and in vivo biological studies. This method is practical for industrial applications and could provide a low-cost means to use a currently underutilized food by-product to develop phytoceuticals with potential health benefits or to develop products for use in the cosmetic and food biopreservative industries.

Background: Ellagic acid (EA) and hydrolyzable ellagitannins (ETs) are dietary polyphenols found in fruits and nuts and implicated with potent antioxidant, anticancer and antiatherosclerotic biological properties. Unfortunately, there are no reports on bioavailability studies of EA or ETs in the human body. Therefore we conducted in vivo studies whereby a human subject consumed pomegranate juice (180 ml) containing EA (25 mg) and ETs (318 mg, as punicalagins, the major fruit ellagitannin). Methods: A rapid plasma extraction procedure utilizing acidic precipitation of proteins, followed by HPLC-UV analyses was employed. Results: EA was detected in human plasma at a maximum concentration (31.9 ng/ml) after 1h post ingestion but was rapidly eliminated by 4h. The calibration curve for quantification of EA was linear (r2 = 0.9975) over the concentration range from 1000 to 15.6 ng/ml. Conclusions: Since EA has reportedly strong affinity for proteins and poor absorption in small animals, further studies to investigate whether the presence of free EA in human plasma may be due to its release from the hydrolysis of ETs, facilitated by physiological pH and/or gut microflora action, is warranted. EA can be considered as a biomarker for future human bioavailability studies involving consumption of ETs from food sources.

Pomegranate (Punica granatum L.) fruits are widely consumed as juice (PJ). The potent antioxidant and anti-atherosclerotic activities of PJ are attributed to its polyphenols including punicalagin, the major fruit ellagitannin, and ellagic acid (EA). Punicalagin, EA, a standardized total pomegranate tannin extract (TPT) and PJ were evaluated for in vitro antioxidant, antiproliferative and apoptotic activities. The antioxidative bioassays used included inhibition of lipid peroxidation and Trolox Equivalent Antioxidant Capacity (TEAC) assays. The antiproliferative assays targeted human oral (KB, CAL27), colon (HT-29, HCT116, SW480, SW620) and prostate (RWPE-1, 22Rv1) tumor cells. Apoptotic effects were evaluated against the HT-29 and HCT116 colon cancer cell lines. Punicalagin, EA and TPT were evaluated at 12.5-100 g/mL for antiproliferative assays. However, to evaluate the synergistic and/or additive contributions from other PJ phytochemicals, PJ was tested at concentrations normalized to deliver equivalent amounts of punicalagin (w/w). Punicalagin, EA, TPT and PJ were all evaluated at 10 g/mL concentrations for antioxidant properties and at 100 g/mL concentrations for apoptotic effects. PJ showed greatest antiproliferative activity against all cell lines by inhibiting proliferation from 30-100%. At 100 g/mL, PJ, EA, punicalagin and TPT induced apoptosis in HT-29 colon cells. However, in the HCT116 colon cells, EA, punicalagin and TPT but not PJ induced apoptosis. The trend in antioxidant activity was PJ>TPT>punicalagin>EA. The superior bioactivity of PJ compared to its purified polyphenols illustrated the multifactorial effects and chemical synergy of the action of multiple compounds compared to single purified active ingredients.

Strawberry (Fragaria x ananassa Duch.) fruits contain phenolic compounds that have antioxidant, anticancer, antiatherosclerotic and anti-neurodegenerative properties. Identification of food phenolics is necessary since their nature, size, solubility, degree and position of glycosylation and conjugation influence their absorption, distribution, metabolism and excretion in humans. Freeze-dried whole strawberry fruit powder and strawberry fruit extracts were analyzed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) methods. Phenolics were identified as ellagic acid (EA), EA-glycosides, ellagitannins, gallotannins, anthocyanins, flavonols, flavanols and coumaroyl glycosides. The anthocyanidins were pelargonidin and cyanidin, found predominantly as their glucosides and rutinosides. The major flavonol aglycons were quercetin and kaempferol found as their glucuronides and glucosides. LC-ESI-MS/MS methods differentiated EA from quercetin conjugates since both aglycons have identical molecular weights (302 g/mol). The identification of strawberry phenolics is necessary to generate standardized materials for in vitro and in vivo studies and for the authentication of strawberry-based food products.

Cranberries (Vaccinium macrocarpon Ait.) are an excellent dietary source of phytochemicals that include flavonol glycosides, anthocyanins, proanthocyanidins (condensed tannins), and organic and phenolic acids. Using C-18 and Sephadex Lipophilic LH-20 column chromatography, HPLC and tandem LC-ES/MS, we have analyzed, quantified and separated total cranberry extract (TCE) into fractions enriched in sugars, organic acids, total polyphenols, proanthocyanidins and anthocyanins (39.4, 30.0, 10.6, 5.5, 1.2% composition, respectively). Using a luminescent ATP cell viability assay, the antiproliferative effects of TCE (200 g/mL) vs. all fractions were evaluated against human oral (KB, CAL27), colon (HT-29, HCT116, SW480, SW620) and prostate (RWPE-1, RWPE-2, 22Rv1) cancer cell lines. The total polyphenol fraction was the most active fraction against all cell lines with 96.1 and 95% inhibition of KB and CAL27 oral cancer cells, respectively. For the colon cancer cells, the antiproliferative activity of this fraction was greatest against HCT116 (92.1%) than HT-29 (61.1%), SW480 (60%) and SW620 (63%). TCE and all fractions, showed 50% antiproliferative activity against prostate cancer cells with total polyphenols being the most active fraction (RWPE-1, 95%; RWPE-2, 95%; 22Rv1, 99.6%). Cranberry sugars (78.8 g/mL) did not inhibit the proliferation of any cancer cell lines. The enhanced antiproliferative activity of total polyphenols compared to TCE and its individual phytochemicals suggests synergistic or additive antiproliferative interactions of the anthocyanins, proanthocyanidins and flavonol glycosides within the cranberry extract.