Evaluation of different extraction methods from pomegranate whole fruit or peels and of the antioxidant and antiproliferative activity of the polyphenolic fraction
Alessandra Mascia*, Andrea Cocciab, Eugenio Lendarob,
Luciana Moscac, Patrizia Paolicellid, Stefania Cesad
aDipartimento di Medicina Sperimentale, Unità di Ricerca di Scienza dell’Alimentazione e Nutrizione Umana, Università degli Studi di Roma “La Sapienza”, viale del Policlinico, 155 – 00161 Roma, Italy
bDipartimento di Scienze e Biotecnologie Medico-Chirurgiche, Università degli Studi di Roma “La Sapienza”, corso della Repubblica, 79 – 04100 Latina, Italy
cDipartimento di Scienze Biochimiche, Università degli Studi di Roma “La Sapienza”, piazzale Aldo Moro, 5 – 00185 Roma, Italy
dDipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, piazzale Aldo Moro, 5 – 00185 Roma, Italy
Pomegranate is a functional food of great interest, due to its multiple beneficial effects on human health. This fruit is rich in anthocyanins and ellagitannins, which exert a protective role towards degenerative diseases. The aim of the present work was to optimize the extraction procedure, from different parts of the fruit, to obtain extracts enriched in selected polyphenols while retaining biological activity. Whole fruits or peels of pomegranate cultivars, with different geographic origin, were subjected to several extraction methods. The obtained extracts were analyzed for polyphenolic content, evaluated for antioxidant capacity and tested for antiproliferative activity on human bladder cancer T24 cells. Two different extraction procedures, employing ethyl acetate as a solvent, were useful in obtaining extracts enriched in ellagic acid and/or punicalagins. Antioxidative and antiproliferative assays demonstrated that the antioxidant capability is directly related to the phenolic content, whereas the antiproliferative activity is to be mainly attributed to ellagic acid.
Plant-based diets have long been associated with increased life expectancy and a reduced risk of chronic and degenerative diseases. Many fruit and vegetable juices are considered of interest as nutraceuticals for their high content in bioactive phytochemicals. Due to their properties, the intake of polyphenols is associated with a lower incidence of several human diseases and lower mortality rates1.
Pomegranate fruits comprise of four parts (exocarp, mesocarp, arils and seeds), all of them containing interesting bioactive molecules, such as anthocyanins in the arils, hydrolysable tannins in the peel and punicic acid in the seeds. All these substances make whole-fruit extracts very interesting as dietary supplements and nutraceuticals, as they are responsible for the antioxidant, anti-mutagenic, anticancer, anti-inflammatory, anti-diabetic and health promoting role of pomegranate. They act as radical scavengers, thus preventing the degradation of food, while protecting human body towards reactive oxygen species, whose role in the pathogenesis of human diseases is widely recognized2.
These phytochemicals display an antiatherogenic effect correlated with the high antioxidant potential of pomegranate extracts. Pomegranate juice consumption in humans decreases lipid peroxidation and LDL susceptibility to aggregation.
Ellagic acid, in its free and bound forms, is one of the most abundant polyphenols in pomegranate and displays a wide spectrum of antitumor properties through multiple pathways.
Given the variety of health promoting activities of pomegranate and the enormous interest that bioactive components isolated from this fruit have raised in the scientific community, we focused our efforts in optimizing the extraction procedure of these phytochemicals in order to obtain extracts enriched in polyphenols, while retaining antioxidant and antiproliferative activities.
Therefore, the aim of this work was to find the best extraction method for either total polyphenols or specific polyphenolic compounds from pomegranate whole fruits or peels, comparing these fractions in terms of antioxidant and antiproliferative capacity. Particular attention was paid to the peels, as they generally represent a waste of pomegranate juice production, although they are an abundant source of polyphenols.
The extraction procedures were carried out on pomegranate whole fruit or on manually separated peels (exocarp and mesocarp), using either a 2-step process, composed of a first extraction with hydroalcoholic solvents and a subsequent repartition with ethyl acetate, or a direct Soxhlet extraction with ethyl acetate (Scheme 1). The obtained extracts were characterized for composition and biological activity. In particular, total phenols, total flavonoids and total anthocyanins were quantified by in batch spectrophotometric measurements, whereas punicalagins and ellagic acid content were determined by HPLC-DAD analysis. The biological activity was evaluated in terms of in vitro antioxidant capacity (NBT assay, ABTS assay, DPPH assay and ORAC assay) and antiproliferative activity, tested on T24 cells of human urinary bladder carcinoma.
The extractions carried out on the peels with ethanol gave yields slightly lower compared to those obtained from whole fruits, but a higher amount of polyphenols was obtained in the first case due to the higher concentration of these molecules in this part of the fruit. In fact, our results show that extracts obtained from the peel have a phenolics content two to three times higher than the corresponding extracts obtained from the whole fruit; the ethanol extract and the aqueous fraction show a 3-fold increase, and the ethyl acetate repartition a 2-fold increase in polyphenolic content. Ethyl acetate fraction showed the greatest concentration of phenolic compounds and the direct extraction with ethyl acetate using Soxhlet apparatus showed similar results in terms of yield, but the extract composition revealed some differences.
The flavonoids content was always higher in pomegranate peel extracts with respect to whole fruit; the ethanol extract and the aqueous fraction show a 3-fold increase, whereas the ethyl acetate fraction a 1.5-fold increase. The higher content of flavonoids is also obtained in the repartition with ethyl acetate. The extraction of pomegranate peel with ethyl acetate using Soxhlet apparatus showed a slight decrease of flavonoid concentration compared to the ethyl acetate repartition. All the different types of extract obtained from the whole fruit showed a 30% increase in content of flavonoids with respect to total polyphenols. For the peels, the content in flavonoids was more variable, ranging from 25% to 40%.
The anthocyanins, as expected, were almost completely transferred in the aqueous fraction, whereas they were not found in the ethyl acetate fraction, but the content of anthocyanins in the Soxhlet extract with ethyl acetate, was sometimes comparable to that of the ethanolic extract.
The HPLC qualitative–quantitative analysis demonstrated the predominant presence of punicalagin α and β anomers and ellagic acid in the phenolic compounds fraction, thus proving a selective extraction of these two compounds in the different extracts. Taking the sum of punicalagins and ellagic acid as 100%, punicalagins represent 95% of the ethanolic extract from whole fruit, whereas ellagic acid represents the remaining 5%. The punicalagins and the ellagic acid contents in the ethanolic extract from whole fruit, were increased 3-fold and 20-fold, respectively, when the extract was obtained from peels, with ellagic acid representing a 30% of the total amount. By subsequent water:ethyl acetate repartition, both in whole fruit and peels the punicalagins are distributed between the two phases, whereas ellagic acid is mainly concentrated in the organic one. In fact, the repartition in ethyl acetate from whole fruit, gave a 2.4-fold increase for punicalagins concentration and even a 40-fold increase in the ellagic acid concentration. The repartition in ethyl acetate from peel gave a comparable punicalagins enrichment with respect to the ethanolic extract, whereas the ellagic acid had a 4-9 fold increase according to the analyzed sample. The extracts in ethyl acetate from peels showed the highest amount of punicalagins and ellagic acid in total, although with different ratios.
In the Soxhlet procedure, in which peels are extracted in the absence of water, the ellagic acid is selectively extracted with respect to punicalagins and represents the main component, being 84–90%, always considering the sum of ellagic acid and punicalagins. The absolute amount of ellagic acid ranged between 40–60 mg/g of dry Soxhlet extract.
The antioxidant activity of pomegranate extracts was evaluated by four different methods, each directed towards a specific free radical. All analytical methods indicated a higher antioxidant activity of extracts prepared from peel rather than whole fruit, as a consequence of their different content in total polyphenols, confirming previous results in literature3. The ethyl acetate fraction showed the greatest antioxidant activity, compared to that from the ethanol extract and the aqueous fraction, which were almost comparable to each other. The direct extraction of peel by Soxhlet, gave a free radical scavenging activity quite comparable to that of the repartition by ethyl acetate, with one exception for the NBT assay; in any case, the antioxidant activity appears to be higher than that of the ethanol extract as well as the aqueous fraction.
The antioxidant potential measured by these assays essentially follow the differences in content of total polyphenols where the increase of the concentration of polyphenolic compounds corresponds to an increase of the antioxidant capacity of the extracts. A good correlation with the antioxidant activity of pomegranate extracts was also appreciable with respect to total flavonoids and, within certain limits, to ellagic acid concentration.
The antiproliferative activity was evaluated by measuring the percent growth inhibition of T24 cells after 48 h of incubation in the presence of 50 μg/ml of the various extracts. The most efficacious were the Soxhlet extracts with a 50% of growth inhibition (Figure 1), whereas the less efficacious were the aqueous extracts from whole fruit, which show a negligible inhibition on cell proliferation. The statistical analysis reveals a strikingly high correlation between percent growth inhibition and ellagic acid amount, less relevant for total polyphenols whereas no correlation was evidenced for punicalagins. These results demonstrated that the temperature adopted in the Soxhlet extraction (ethyl acetate Teb. 77 °C) is compatible with a selective extraction of ellagic acid, as confirmed by tests on the biological activity.
To verify whether the biological activity of the extract could be mainly attributed to ellagic acid, viability experiments were also performed with the purified polyphenol. The comparison between the dose–response curve of ellagic acid present alone or in the extract indicate that purified ellagic acid activity was significantly lower than that exerted when the same amount of polyphenol was present in the extract. The percent inhibition of purified ellagic acid was about 25% after 48 h of treatment, whereas it was more than 50% for the same amount as extract (Figure 2).
A large body of evidence in the literature demonstrates that pomegranate bioactive molecules can exert anti-cancer activities by inhibiting tumor growth, progression and angiogenesis by modulating intracellular pathways mediated by NF-κB, MAP kinases and mTOR, and these properties have been mainly attributed to its high content in antioxidant tannins and flavonoids. The most efficacious extracts were those in which the polyphenolic content was enriched, and most notably the antiproliferative effect was strongly correlated with the ellagic acid content. This confirms that ellagic acid is the main molecule responsible for the anticancer activity of pomegranate and indicate that the different extraction procedures do not affect the biological activity of the extract. The evidence that complex pomegranate extracts possess greater bioactivity than purified ellagic acid indicate a multifactorial effect and chemical synergy of the action for multiple compounds compared to single purified active components.
Concluding, in the present work, for the first time, extractions were performed on blended pomegranate whole fruit with respect to conventional squeezing and juice’s analysis. The extraction procedures, applied on whole fruit and peels, provided the selection of the polyphenolic fraction with a different distribution of its components. A higher content of polyphenols, flavonoids, punicalagins and ellagic acid were found in the peel, when compared to whole fruit, and the highest polyphenol concentration was gained in the repartition in ethyl acetate. A highly significant correlation was shown between total polyphenols and antioxidant capacity and between ellagic acid content and antiproliferative activity on human bladder cancer T24 cells. Moreover, the enhanced antiproliferative activity observed when extracts were compared to the purified ellagic acid used as reference indicate a synergistic effect among the polyphenolic compounds in the complex mixture.
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2) Fischer, U. A., Jaksch, A. V., Carle, R., & Kammerer, D. R. (2013) European Food Research and Technology, 237(2), 209–221.
3) Gil, M. I., Tomas-Barberan, F. A., Hess-Pierce, B., Holcroft, D. M., & Kader, A. A. (2000) Journal of Agriculture and Food Chemistry, 48(10), 4581–4589.