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Keywords:

  • Antiproliferative activity;
  • HPLC ;
  • phenolic compounds;
  • pomegranate

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. References

The objective of this study was to assess antioxidant and antiproliferative activities of four different Turkish pomegranate varieties (Hatay, Hicaz, Adana and Antalya) using an in vitro HepG2 cancer cell model. All the pomegranate extracts employed in this study significantly diminish the proliferation of HepG2 cells in a dose-dependent manner. The total phenolic acid, anthocyanin and flavonoid contents for each of the four varieties were determined. The Hatay pomegranate variety had the highest total phenolic acid (337.4 ± 2.34 mg/100 g) and flavonoid (58.42 ± 2.25 mg/100 g) contents of the pomegranates examined. Antioxidant activities of the pomegranates were determined using DPPH and ABTS radical scavenging assays. The lack of correlation between colour index value and antioxidant–antiproliferative activities suggested that phenolic acids and flavonoids are predominant compounds influencing pomegranate's bioactivity rather than anthocyanins. Individual phenolic acids found in Hatay pomegranates were determined, using an HPLC system, as gallic acid being the most predominant phenolic compound.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. References

There is compelling epidemiological evidence that fruit and vegetable consumption plays an important role on inhibiting development of chronic disorders such as cancer and cardiovascular diseases (Doll, 1990; Hertog et al., 1996). The chemopreventive role of fruits is associated with their phytonutrient contents bearing antioxidative properties (Davidson & Touger-Decker, 2009). Of the antioxidant compounds, phenolics drew significant attention in recent years for their ability to scavenge free radicals generated within cell (Middleton et al., 2000; Yang et al., 2001). Phenolic compounds protect human body by neutralising destructive effect of free radicals on plasma membrane, organelle membranes and DNA (Issa et al., 2006; Villano et al., 2007). Presence of hydroxylated aromatic ring(s) is a common feature of phenolic compounds that are widely distributed in plants (Boudet, 2007). Hydroxyl groups linked to aromatic rings are associated with the formation of electron-rich environment that scavenges the reactive oxygen species (ROS) excluding them from reacting nucleophilic centres in plasma membrane, cellular proteins and DNA (Issa et al., 2006). Phenolic compounds produced by plants are divided into two major groups: phenolic acids and flavonoids. Cinnamic acids and benzoic acids are the two main groups of phenolic acids. Anthocyanidins, flavons, flavonols, flavonons, catechins and proanthocyanidins are the subgroups of flavonoids (Cemeroğlu, 2009). The general phenylpropanoid pathway is responsible for biosynthesis of a substrate common to many of the phenylpropanoid compounds such as flavonoids, monolignols, hydroxycinnamic acids, sinapoyl esters, coumarins and stilbenes (Vermerris & Nicholson, 2006). Therefore, phenylpropanoid pathway serves as a rich source of natural compounds including flavonoids, coumarins and lignans (Fraser & Chapple, 2011).

Many reports in the literature concentrated on the determination of the natural sources of phenolics and their possible health benefits on the living organisms (Knekt et al., 1996; Gil et al., 2002; Liu et al., 2002). Fruits, especially red ones, were determined as to be the dietary sources containing high amount of bioactive phenolic compounds. Thus, pomegranates have received considerable interest in recent years because of their abundant bioactive natural compound contents such as vitamin C, flavonoids, gallotannins, cyanidin, pelargonidin, delphinidin glycosides (Gil et al., 2000; Seeram et al., 2006; Tzulker et al., 2007; Mousavijenad et al., 2009), and regular consumption of pomegranate is associated with cancer chemotherapeutic effect (Malik et al., 2005) and prevention of chronic inflammation (Newman & Ephraim, 2007). Pomegranate fruits are rich source of bioactive compounds and thus have higher antioxidant activity compared with red wine and green tea (Gil et al., 2000). The most influential factors that could affect the content and composition of bioactive molecules in pomegranate are genetic heterogeneities, varietal factors, climate conditions, soil structure, agricultural practices, water–heat stress, harvesting time and storage conditions. Pomegranate fruit (Punica granatum L.) is native to the area surrounded by Persia, Babylon, Anatolia and has been grown in Iran, Southeastern Europe, Mesopotamia and India since ancient times. Turkey is one of the prominent pomegranate producers, and the total pomegranate production of Turkey reached 217 572 tons in 2011 (Anon, 2012) that corresponds to one of the largest pomegranate economies in the world. Turkish pomegranate fruit is being used for the industrial manufacturing of wide range of food products such as fruit juice, concentrate, jam, candies, toppings and canned arils besides its fresh consumption.

As Turkey is one of the world's biggest pomegranate producers and exporters, it is imperative to characterise the antioxidant properties of Turkish pomegranate varieties and determine their inhibitory activity on the development of cancer cell lines. Although there are some reports in the literature describing antioxidant capacities of Turkish pomegranates (Ozgen et al., 2008; Gözlekçi et al., 2011; Caliskan & Bayazit, 2012.), there is no study assessing antiproliferative activities of pomegranates growing in the region. The objective of this study was to identify relationship between colour, phytochemical content and antioxidant activity of four Turkish pomegranate varieties and evaluate antiproliferative–antioxidant activities of their extracts.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. References

Plant material

Four different pomegranate (Punica granatum L., cvs., Antalya, Hicaz, Hatay and Adana) samples at commercially ripe stage were obtained from Koruklu, Agricultural Research Center, Turkey in September 2011. The average diameters were 76 mm, 79 mm, 77 mm and 92 mm and the average weights were 231.2 g, 245.5 g, 248.8 g and 376.2 g for Antalya, Hicaz, Hatay and Adana pomegranate varieties, respectively. These pomegranate varieties were selected to study the influence of pomegranate extract variability, as affected by varietal factors, on antioxidant activity and cancer cell proliferation rate. Fruits were immediately transported to the laboratory after harvest and stored at 4 °C until the extraction. All the extracts were stored at −20o C.

Chemicals

HepG2 liver cancer cells were obtained from LGC Standards (ATCC). Glucagon, insulin, hydrocortisone, Williams Medium E (WME), foetal bovine serum (FBS), potassium peroxodisulfate, 2,6-dichloroindophenol, ethanol, acetone and the standards (neochlorogenic acid, gallic acid, catechin, caffeic acid, p-coumaric acid, ferulic acid, epicatechin, rutin, quercetin, kaempferol, resveratrol) used in HPLC analysis were obtained from Sigma Chemical Co. (USA). The 2,2-diphenyl-1-picrylhydrazyl-hydrate (DPPH), 2,2′-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), Folin–Ciocalteu reagent, gallic acid, sodium carbonate, sodium acetate, sodium hydroxide, potassium chloride and aluminium chloride were obtained from Merck Co. (Germany). All other chemicals were of analytical grade.

Extraction

Pomegranate extracts were prepared following the method described by Serra et al. (2010) with some modifications. Briefly, 100 g of arils obtained from three pomegranates in each cultivar was extracted with 100 mL 80% acetone over 5 min. in a chilled metal blender. The homogenates were then filtered through Whatman No.1 filter paper with 100 mL 80% acetone to wash filter cake for remaining phenolics, and the solvent was evaporated in a rotary evaporator at 40 °C until total volume decreases to around 30 mL. The remaining extracts were diluted with distilled water to make concentration of 2 g of fresh pomegranate/mL. The extracts were collected in falcon tubes and stored at −20 °C until analyses.

Determination of aril colour, total phenolic (TP), total flavonoid (TF) and total anthocyanin (TA) contents

The aril colours of four pomegranate varieties were determined using a Hunter Lab. Colorimeter (ColorQuest XE, USA). Colour index values were calculated as described by Shwartz et al. (2009).

TP contents of pomegranates were determined by the Folin–Ciocalteu method with gallic acid standard. Briefly, 30 μL of pomegranate extract was pipetted into a test tube containing 2370 μL deionised water, and 150 μL Folin–Ciocalteu reagent was added onto the mixture. The mixture was then vortexed and waited at dark for 8 min. Then, 450 μL saturated Na2CO3 was added and allowed to stand for 30 min in the dark medium (Roby et al., 2013). Same procedure was run to prepare blank using 30 μL deionised water instead of extract. The absorbances of each sample were read at 750 nm in a spectrophotometer (Libra S70, Biochrom, UK) against the blank. Various concentrations of gallic acid solution (50–500 μg/mL) were used to plot a calibration curve. Results were expressed as mg gallic acid equivalent 100 g pomegranate (Karaaslan et al., 2011).

TF contents of pomegranate varieties were determined by a colorimetric method (Bao et al., 2005) with minor modifications. Pomegranate extracts (0.5 mL) were transferred into a test tube containing 2.5 mL distilled water, and then, 150 μL of 5% NaNO2 solution was poured into the solution. After 6-minute incubation, 300 μL of 10% AlCl3.6H2O solution was added and waited for another 5 min. Then, 1 mL 1 m NaOH was added and the mixture volume was made to 5 mL with distilled water. The mixture absorbance was spectrophotometrically measured at 510 nm. Flavonoid content was quantified using catechin calibration curve, and the results were expressed as milligrams of catechin equivalents per 100 g pomegranate.

TA contents of the pomegranate samples were determined using pH differential method as described by Wrolstad (1976). TA contents of each cultivar were calculated using Eq (1, 2).

  • display math(1)
  • display math(2)

Results were expressed as mg cyanidin-3-glucoside, having molar absorptivity (∈) of 26900 and molecular weight (MW) of 449.2 g/mol. Path length (l) was 1 cm, and A and DF correspond to absorbance value and dilution factor, respectively.

Radical DPPH scavenging capacity

The free radical scavenging capacities of four different extracts were measured according to DPPH method reported by Blois (1958) with minor modifications. Pomegranate extracts were diluted to various concentrations (250, 150, 100, 75, 50, 25, 10, 5 mg/mL) with ethanol. 0.1 mL of each dilution was pipetted onto 2.9 mL of DPPH solution (0.1 mm in ethanol). After 30 min of incubation at room temperature, the decrease in absorbance at 517 nm was measured. Radical scavenging capacity was expressed as per cent scavenging effect (Eq(3)). Median effective dose (EC50) was calculated from per cent inhibition vs. extract concentration curve.

  • display math(3)

where Ac is the absorbance of control and As is the absorbance of sample.

Trolox equivalent antioxidant capacity determination

The method described by Çam et al. (2009) was used to measure Trolox equivalent antioxidant capacity (TEAC) by ABTS radical. Thirty milligrams ABTS was dissolved in 7.8 mL of 2.46 mm peroxodisulfate to prepare the ABTS stock solution. Stock solution was diluted to get the absorbance of 0.700 ± 0.020 at 734 nm. Phosphate buffer was used to dilute the samples by 1:20 (v:v). Diluted samples (50 μL) were added into the ABTS solution (1950 μL), and the mixture was incubated for 6 min, and then, the absorbance was measured. The results were expressed as mg TEAC per 100 g of pomegranate.

Measurement of ascorbic acid

Ascorbic acid measurement was carried out using 2,6-dichloroindophenol titrimetric method (AOAC, 1975).

Measurement of HepG2 cell proliferation

Antiproliferative activities of pomegranate extracts belong four cultivars were assessed according to the colorimetric MTS assay (MTS-based cell titre 96 nonradioactivity cell proliferation assay (Promega, Madison, WI) as reported previously (Liu et al., 2002). In a dose–response experiment, limited to 4 days, HepG2 cancer cells were exposed to various pomegranate extracts at different concentrations. The extracts equivalent to 0, 10, 20, 30, 40 and 50 mg/mL concentrations were transferred to the culture medium, and their effect on the cell proliferation was tested. The cell cultures were exposed to various concentrations of the extracts (0–50 mg/mL). Control cultures received water instead of pomegranate extract and blank contained growth medium with no cells. After 96-hour incubation period, per cent cell proliferation was determined using MTS assay with tetrazolium reagent by reading MTS absorbance at 490 nm using a spectrophotometer (Libra S70, Biochrom, UK). Each experiment was conducted in three replicates.

Isolation and HPLC analysis of phenolic compounds

The highest phenolic content and cancer cell inhibitory activity were found in Hatay pomegranates, and thus, simple phenolic acids present in the Hatay pomegranate juice were investigated. The most common phenolic compounds present in pomegranates were investigated. Peak identification were completed by comparing the retention times (RT) of pure standards and the peaks visualised on the HPLC chromatograms. Phenolic compounds were isolated on a C-18 cartridge (Finisterre, Teknokroma™, ES) using a vacuum manifold. Cartridge was activated with 5 mL ethylacetate, 5 mL methanol (0.01% HCl) and 2 mL acidified water (0.01% HCl), respectively. Sample extract (0.5 mL) was loaded into the cartridge, and then, the cartridge was washed with 2 mL acidified water (0.01% HCl) after all the sample has passed through. The residual solvent was evaporated under a nitrogen flux, and the cartridge was washed with 5 mL ethylacetate to elute nonanthocyanin phenolics. The ethylacetate extract was kept in a water bath (40 °C), and ethylacetate was removed under a stream of nitrogen. Phenolics were dissolved in acidified water (0.01% HCl), filtered through 0.45-μm PVDF filter and transferred to vials for chromatography. Identification and quantification of phenolic compounds were performed on Waters 2795 HPLC (Waters Corporation, Beverly, MA, USA) equipped with a Waters 2996 photodiode array (PDA) detector using a Supelcosil (Supelco) LC-18-T (15 cm × 4.6 mm × 3 μm) column. The mobile phase was a mixture of 0.01% (v/v) trifluoroacetic acid in water (A) and 0.01% (v/v) trifluoroacetic acid in acetonitrile (B). Column temperature was 35 °C, and flow rate was 1 mL.min−1. Quantity of each compound was calculated by peak areas and standard curves of corresponding standards. The gradient conditions were 10%, 10%, 20%, 20%, 10% and 10% of solvent (B) at 0, 30, 50, 55, 56 and 60 min, respectively. The retention times of the standards were 3.38, 3.78, 6.72, 8.04, 10.38 and 18.64 min for neochlorogenic acid, gallic acid, catechin, caffeic acid, p-coumaric acid and ferulic acid, respectively.

Statistical analysis

Correlation and regression analyses were performed using SPSS 10.0 software (SPSS Inc., Chicago, IL, USA). Differences at < 0.01 were considered significant according to least significant difference (LSD) test. All data were reported as mean ± standard deviation of three replications.

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. References

Phytochemical content of pomegranate varieties

In this study, four different pomegranate varieties were selected to investigate their phytochemical content, antioxidant and antiproliferative activities. Hicaz, Adana, Hatay and Antalya pomegranate varieties differ in aril and juice colour (Fig. 1). Hicaz had dark red colour and the highest colour index value (CI) as 7.26 ± 0.03, Adana has red colour and 6.77 ± 0.04 CI, Hatay has pink colour and 6.29 ± 0.02 CI, and Adana has light pink colour and 6.22 ± 0.1 CI. Some chemical properties and total phenolic, flavonoid, anthocyanin concentrations of pomegranates are shown in Tables 1 and 2. The ascorbic acid (AA) contents of pomegranates were quite similar in each of the four varieties (Table 1). Even though the highest amount of AA was detected in Hatay pomegranates (11.18 ± 0.45 mg/100 g), there was no statistically significant difference among the tested pomegranate varieties when considering their AA content. The results showed that AA content of the pomegranates is not a useful indicator for distinguishing examined pomegranate samples. Phytochemical contents of the varieties were highly variable with each sample ranging from 178 to 337.4 mg/100 g for phenolics, from 36.46 to 58.42 mg/100 g for flavonoids and from 1.37 to 6.30 mg/100 g for anthocyanins. Hatay variety exhibited the highest phenolic (337.4 mg/100 g) and flavonoid (58.42 mg/100 g) contents among the assayed samples and also possesses 4.37 mg/100 g of total anthocyanins indicating its quite rich bioactive content. On the contrary, Antalya variety comprised the least amount of phytochemicals among the tested samples. The highest anthocyanin content was determined in Hicaz variety as 6.30 mg in 100 g of fruit that has the dark red fruit juice colour (Fig. 1). Adana variety also displayed a bright red colour and had the second highest anthocyanin content as 5.00 mg/100 g. Hatay and Antalya juices displayed pinkish colour rather than red and contained relatively lower amounts of anthocyanins. In addition to this, Antalya variety had almost five times less total anthocyanin in comparison with Hicaz pomegranates. Total anthocyanin concentrations of Hatay and Antalya pomegranates were detected as 4.37 mg/100 g and 1.37 mg/100 g, respectively. There was no correlation between aril colour (CI values) and phytochemical contents. Hatay variety with pink aril colour (lighter than Hicaz and Adana varieties) displayed the highest flavonoid and phenolic contents. The colour index ratings were only well correlated with anthocyanin content, that is, the Hicaz variety with the highest colour index value exhibited also the highest anthocyanin concentration.

Table 1. Some chemical properties of pomegranate fruits (Adana, Hicaz, Antalya and Hatay cvs.) grown in Turkey (n = 3, ± SD, significant at < 0.01), different letters in the same column indicate statistically significant difference
VarietyBrix (%)Total Acidity (Citric acid %)pHAscorbic Acid (mg/100 g)Colour IndexAril Colour
Hatay17.5 ± 0.21a0.45 ± 0.04a3.63 ± 0.02a11.18 ± 0.45a6.29 ± 0.02aPink
Hicaz19.0 ± 0.24b1.78 ± 0.12b3.20 ± 0.01b11.07 ± 0.65a7.26 ± 0.03bDark Red
Antalya16.5 ± 0.32c0.37 ± 0.03c3.39 ± 0.02c10.51 ± 0.47a6.22 ± 0.10aLight Pink
Adana17.0 ± 0.18c2.46 ± 0.14d3.10 ± 0.01d10.43 ± 0.38a6.77 ± 0.04cRed
Table 2. Total phenolic, flavonoid, anthocyanin contents and TEAC (mg/100 g fruit) of pomegranate varieties (n = 3, ± SD, significant at < 0.01), different letters in the same column indicate statistically significant difference
VarietyTotal Phenolic contentTotal Flavonoid contentTotal Anthocyanin contentTEAC*
  1. *Trolox equivalent antioxidant capacity.

Hatay337.4 ± 2.34a58.42 ± 2.25a4.37 ± 0.06a381.2 ± 4.2a
Adana277.2 ± 3.69b54.33 ± 2.66b5.00 ± 0.07b336.5 ± 8.2b
Hicaz214.7 ± 2.63c49.14 ± 2.05c6.30 ± 0.11c344.3 ± 6.3b
Antalya178.0 ± 1.81d36.46 ± 0.91d1.37 ± 0.06d267.3 ± 5.8c
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Figure 1. The colours of the four different pomegranate varieties grown in Turkey.

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Antioxidant and antiproliferative activities of pomegranates

Total antioxidant activities of the pomegranates shown in Fig. 2 are expressed as the median effective dose, EC50 values. The lowest EC50 value was found in Hatay (74.09 ± 2.32 mg/L), followed by Adana (95.40 ± 2.46 mg/L), Hicaz (127.85 ± 2.73 mg/L) and Antalya (150.74 ± 3.08 mg/L) varieties. There is an inverse proportion between antioxidant activity and EC50 value. According to the obtained data, the highest total antioxidant activity was detected in Hatay variety, which was reflected by lower EC50 value, and the lowest antioxidant activity was found in Antalya. The antioxidant activity differences among the varieties were statistically significant (< 0.01). Hatay variety exerts almost twofold higher antioxidant activity in comparison with that of Antalya pomegranate. ABTS radical scavenging assay was also used to evaluate the antioxidant activities of the pomegranates. The radical scavenging ability of the samples was expressed as TEAC. The highest result was observed for Hatay pomegranates as 381.2 ± 4.2 mg/100 g, and the lowest result was found in Antalya pomegranates as 267.3 ± 5.8 mg/100 g (Fig. 2). Higher TEAC value translates into higher antioxidant capacity. And thus, ABTS radical scavenging assay also indicated high antioxidant capacity of Hatay pomegranates and low antioxidant capacity of Antalya pomegranates. These findings show that each variety depending on its phenolic acid and flavonoid contents possesses own characteristic antioxidant activity and displays variety of specific radical scavenging capacity, and the phenolics and flavonoids are the main compounds responsible for the antioxidant activity.

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Figure 2. Total antioxidant activities of four different pomegranate varieties grown in Turkey as displayed by EC50 value (n = 3, ± SD, significant at < 0.01).

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Many studies reported the relation between red fruit colour and high antioxidant activity. However, in this study, we showed that colour is not the sole determinant of the biovalue of the pomegranates. Hatay variety with lower CI (red colour indicator) compared with Hicaz and Adana varieties exhibited the highest phenolic and flavonoid contents. Çam et al. (2009) found significant correlation between the antioxidant activity and anthocyanin content of the pomegranate fruits. By contrary, in the study of Tzulker et al. (2007), 29 different pomegranate fruits were extracted mechanically in a blender, and it was found that total antioxidant level and phenolic content were significantly correlated (r = 0.95, P < 0.01), and the correlation between antioxidant activity and anthocyanin content was insignificant (> 0.05). Taking the data obtained in this study into consideration, one can conclude that factors promoting the antioxidant activity of pomegranates are phenolics and flavonoids. It was reported previously that vitamin C exerted only 0.4% of total antioxidant activity of the apple skin, and majority of the antioxidant activity may come from flavonoids and phenolics (Eberhardt et al., 2000). The data obtained in this study showed that the higher the phenolic–flavonoid content, the higher the DPPH-ABTS scavenging ability and the inhibitory activity on HepG2 cell proliferation.

The pomegranate extracts inhibited the proliferation of HepG2 cells in a dose-dependent manner according to the assessment of cancer cell proliferation rate as detected by MTS assay in vitro conditions. All the assayed pomegranate extracts displayed antiproliferative activity and inhibited the basal increase in the HepG2 proliferation. The antiproliferative activities of the pomegranate varieties at the concentration of 50 mg/mL were Antalya 49.67 ± 0.85%, Adana 71.59 ± 1.03%, Hicaz 80.01 ± 0.3% and Hatay 88.07 ± 0.12%, respectively (Fig. 3). The variety showing the highest antiproliferative activity is the Hatay, and the lowest antiproliferative activity was observed in Antalya variety. There was a statistically significant difference in terms of inhibitory effect of pomegranate extracts on cancer cell proliferation among the assayed samples (< 0.01). The EC50 value of the antiproliferative activity of pomegranate varieties is shown in Fig. 4. The lowest EC50 value corresponds to the highest antiproliferative activity. Significant differences were found among the EC50 values of the varieties (< 0.01). The EC50 values of pomegranate varieties were Antalya 41.71 ± 0.81 mg/mL, Adana 18.37 ± 0.40, Hicaz 11.24 ± 0.78 and Hatay 7.8 ± 0.35, respectively. The highest antiproliferative activity was detected in Hatay pomegranates.

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Figure 3. Time- and dose-dependent per cent inhibition of HepG2 cell proliferation by pomegranate extracts of the assayed varieties (n = 3, ± SD).

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Figure 4. Antiproliferative activities of pomegranate varieties grown in Turkey as displayed by EC50 value (n = 3, ± SD, significant at < 0.01).

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The pomegranate varieties contained the varied amounts of the total anthocyanins, flavonoids and phenolics (< 0.01). The effect of pomegranate varieties on the inhibition of proliferation in HepG2 cells was significant (< 0.01), and the degree of inhibition was connected with the origin and concentration of the pomegranate extract in the medium. The results obtained in this study were comparable to the data presented previously. Antiproliferative activities of the pomegranate extracts were stronger than the most of the common vegetable's activities (Chu et al., 2002) and similar to the antiproliferative activities found in the raspberry fruits and the other common fruits (Liu et al., 2002; Sun et al., 2002). The cell proliferation inhibiting activities of the pomegranates were differed significantly among the tested cultivars. The variety (Hatay) with the highest antioxidant activity and the phenolic–flavonoid content exerted the highest antiproliferative activity. This finding was one of the most remarkable outcomes of this study and in line with the previous reports, indicating that higher phenolic–flavonoid content attributed to higher antioxidative and antiproliferative properties (Eberhardt et al., 2000). There was no detectable relation between anthocyanin contents and the inhibiting activity of the extracts. Therefore, there is no visible relation between colour parameters and the antioxidant characteristics of the pomegranate varieties tested in this study. These findings supported the idea that aril colour is not a useful indicator for empirical assessment of phytochemical content and the antioxidant activity of the pomegranates.

The relationship between phytochemical content, antioxidant activity and antiproliferative activity of pomegranate varieties

Antalya variety has light pink aril colour and contains the lowest amount of total phenolics, flavonoids and anthocyanins among the examined samples. Antalya pomegranates had the lowest TEAC value and the highest EC50 value and thus the weakest antioxidant activity as well. Antalya variety with the lowest CI had the lowest amount of phytochemicals and antioxidant activities at the same time. However, there was no correlation between colour index ratings and antioxidant activities of the remaining samples. The arils of Hicaz variety had dark red colour containing the highest concentrations of anthocyanins but this was not the case for its phenolic–flavonoid contents, or antioxidant activity. There was a negative correlation between EC50 value of antioxidant activity and total phenolic content (Fig. 5). Another negative correlation was determined between total flavonoid content and EC50 value of antioxidant activity (Fig. 6), indicating that the higher total phenolic and flavonoid contents, the higher antioxidant activity. Similarly, many studies in the literature reported high regression between antioxidant activity and total phenolic substance contents (Liu et al., 2002; Tzulker et al., 2007).

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Figure 5. The relation between total phenolic content and EC50 value of antioxidant activity of four pomegranate varieties.

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Figure 6. The relation between flavonoid content and EC50 value of antioxidant activity of four pomegranate varieties.

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Correlations among the data obtained from the analysis of the four pomegranate varieties were calculated using Pearson's correlation coefficient. The correlations between the total phenolics and antioxidant activity (r = −0.98) and total flavonoids and antioxidant activity (r = −0.92) were statistically significant (< 0.01) (Table 3). According to these results, the total phenolic and flavonoid contents of the pomegranate varieties have an important effect on the antioxidant activity of pomegranate varieties. There was no significant correlation between anthocyanin content and antioxidant activity (r = −0.45, > 0.05). Correlation between the cell proliferation, total phenolics (r = −0.82), flavonoids (r = −0.90) and anthocyanins (r = −0.75) was found statistically significant (< 0.01). In addition to this, the correlation between the cell proliferation and antioxidant activity (r = 0.83) was statistically significant (< 0.01). These results show that total phenolic, flavonoid and anthocyanin contents have a significant effect on the inhibition of proliferation of tumour cells in vitro. The study carried by Seeram et al. (2005) on the pomegranate juice and its phenolic compounds showed that the pomegranate juice with highest antioxidant activity exerted the highest antiproliferative activity. In this study, the results show that the variety with the highest antioxidant activity had the maximal inhibition of tumour cell proliferation, while the variety with the lowest antioxidant activity had the minimal inhibition of tumour cell proliferation.

Table 3. The correlation value (r) among the analyses parameters
 Antioxidant Activity (EC50)Total Phenolic contentTotal Anthocyanin contentTotal Flavonoid content
  1. *< 0.01.

Total Phenolic content−0.98*   
Total Anthocyanin content−0.450.37  
Total Flavonoid content−0.92*0.91 *0.68 
Cell Proliferation (EC50)0.83 *−0.82 *−0.75 *−0.90*

Identification and quantification of individual anthocyanin compounds found in pomegranate juice

Figure 7 depicts a typical HPLC chromatogram showing the resolved peaks at 320 AU. The phenolic compounds present in Hatay pomegranate juice, which had the highest antioxidant and antiproliferative activities, were identified. The amount of the detected free phenolic acids is shown in Fig. 7; each data is the mean of three experimental results ± SD. We identified the simple phenolic compounds present in the pomegranate juice as they are one of the main secondary metabolite groups contributing the fruit's antiproliferative activity. The predominant phenolic compounds detected in the aril juice were gallic acid followed by catechin, neochlorogenic acid, caffeic acid, p-coumaric acid and ferulic acid. Presence of gallic acid, catechin, ferulic acid, caffeic acid and p-coumaric acid in Turkish pomegranate juices was reported previously (Poyrazoğlu et al., 2002), which was consistent with the results presented in this study. Gallic acid was shown to have antiproliferative, antitumorigenic and pro-apoptotic activity on the development human prostate cancer cells (Kaur et al., 2009). And the other studies in the literature previously reported that grape seed extracts rich in gallic acid have cytotoxic effect on DU145 and 22RV1 cells (Inoue et al., 2000; Faried et al., 2007). Based on our findings, the high concentration of gallic acid present in Turkish pomegranates (cv. Hatay) would be responsible for its observed anticancerous characteristics. Similarly tumour suppressive activity of catechin was well documented (Menon et al., 1999), which also was found at high concentrations in assayed pomegranate cultivar. It was reported that catechin interferes lung metastasis and melanoma cell migration by inhibiting metalloproteinase activity (Menon et al., 1999). The other major phenolic compound detected in assayed pomegranates was neochlorogenic acid that was shown to be connected to chemopreventive effect on tumour forming cell proliferation and antioxidant activity on MDA-MB-435 and MCF-10A cells (Dongo, 2008). It was found that neochlorogenic acid triggers intrinsic apoptotic pathways bringing about its tumour suppressive activity and might be considered as a therapeutic agent for controlling multiple signalling pathways relevant to spread of metastatic cancer tumours (Dongo, 2008).

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Figure 7. HPLC chromatogram showing the phenolic compounds present in pomegranate juice (Hatay variety). Identified and quantified phenolics were as follows: neochlorogenic acid (18.6 ± 1.2 mg/100 mL), gallic acid (44.1 ± 1.8 mg/100 mL), catechin (39.6 ± 0.9 mg/100 mL), caffeic acid (3.3 ± 0.6 mg/100 mL), p-coumaric acid (2.7 ± 0.3 mg/100 mL) and ferulic acid (21.6 ± 1.5 mg/100 mL).

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Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. References

According to the results, Hatay pomegranate variety displayed the highest antioxidant and antiproliferative activity that is connected to its high phenolic and flavonoid contents. All the pomegranate extracts examined in this study decreased the proliferation of HepG2 cancer cells. This means that pomegranate could slow down the process of carcinogenesis. Therefore, the consumption of pomegranates, especially Hatay variety, may be recommended. The evidence provided in this study is not sufficient, as in vitro studies do not always represents in vivo studies. However, in vitro studies might play an important role in guiding animal and human research. Therefore, this study could be a reference for future investigations in this area. Even though pomegranates were shown to have the ability on reducing the proliferation of HepG2 cells, one should keep in mind that carcinogenesis is a multistep complex process. Therefore, it is important to examine the role of individual phenolic substances on the gene expression and protein translation. In addition to this, as a research project, one can investigate the role of phenolic compounds present in fruit and their individual ability on the inhibition of cell proliferation.

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  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. References
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