Polyphenol Content and Bioactivity of Pomegranate Peel and Their Cultivar and Environment Dependencies

Phenolics contents and in vitro bioactivities of 10 pomegranate peels and their cultivar and environment dependencies were investigated. The results showed total free polyphenol contents of the peels ranged in 104.17200.91 mg GAE/gDW, which were 10-12 folds more than total bound ones and punicalagins B was predominant component of them. Total free phenolic content of pomegranate peel were significantly different among some cultivars and all produce regions which have significant different environment, significantly positively correlated with total sunlight time and negatively with total precipitation and average temperature in fruit mature period. They were significantly positively correlate with DPPH· scavenging capacity, ABTS· scavenging capacity, total reducing power and total antioxidant capacity of the peels. The purified phenolic extracts could induce Caco-2 cells apoptosis in vitro in dose dependent manner and through multiple cell signaling pathways. In conclusion, phenolic content of pomegranate peel depend on both cultivar and meteorological condition. Antioxidant and antiproliferative activities of pomegranate peel extract positively correlate with its total phenolic content.


INTRODUCTION
If reactive oxygen species conn't be balanced by antioxidant defenses in human body, they can damage to lipids, proteins and nucleic acids (McCord, 2000;Devasagayam et al., 2004) and cause cardiovascular disease, cancer, diabetes mellitus, neurodegenerative disorders, rheumatoid arthritis and ageing (Valko et al., 2007;Mahantesh et al., 2012).Therefore, antioxidant nutraceutical has been developping to strengthen antioxidant defenses.
In this study, polyphenols were extracted from peels of 10 pomegranate cultivars collected from 4 regions of China.The phenolic content and antioxidant and antiproliferative activities of the extracts were investigated and compared.The results showed that not only cultivar but also local meteorological condition during fruit mature period significantly effected on the polyphenol contents and bioactivities of the extracts.

Highlights:
 Pomegranate peel's polyphenol content existed cultivar and regional differences  Meteorological condition of mature strongly effected on peel's polyphenol content  Free tannins are main phenolic component and antioxidants of pomegranate peel extract  The phenolics could induce cells apoptosis through multiple cell signaling pathways.

Phenolics extraction:
Free phenolics extraction: Two grams of peel powder was extracted with 40 mL petroleum ether and diethyl ether mixture (3/2(v/v)) to remove lipid.Then the powder was extracted with 40 mL of 60% aqueous ethanol and 40 kHZ of ultrasonic aid for 30 min at room temperature.After centrifugal at 4000 r/min for 10 min, the supernatant was collected and the residue was extracted again in same way.The two supernatants were combined together and diluted to 100 mL with 60% aqueous ethanol and then stored at -20°C for late use.The final residue was used in the next step.
Bound phenolics extraction: Based on the method described by Oboh and Ademosun (2012), the residue mentioned above was mixed with 40 mL of 4 N NaOH.
After being shaken for 1.5 h, the mixture was adjusted pH 2 by HCl and diluted to 100 mL with distilled water.After centrifugal at 4000 r/min for 10 min, the supernatant was stored at -20°C for late use.

Phenolics content determination:
Total polyphenols content was determined with Folin-Ciocalteu method and expressed as milligram of gallic acid equivalent per gram of dry peel powder (mg GAE/gDW).Total flavonoids content was determined with the aluminum chloride colorimetric method described by Lin and Tang (2007) and expressed as milligram of quercetin equivalent per gram of dry peel powder (mg QUE/gDW).Total tannin content was determined with the Folin-Denis method and expressed as milligram of tannin acid equivalents per gram of dry peel powder (mg TAE/gDW).

Phenolic composition analysis:
Fifty milliliters of the phenolic extract sample was infused into the D-101 macroporous resin column in the speed of 5 bed volumes per hour (5 BV/h).After equilibrium for 1 h, distilled water was passed through the column at speed of 5 BV/h to remove the impurity.Then the phenolics were eluted out by aqueous ethanol (70%, v/v) at speed of 2 BV/h.The elution was concentrated to almost dry at 40°C in a rotary evaporator and the residue was redispersed in 5 mL of 30% aqueous ethanol and kept -20°C for late use.
The purified phenolic sample was diluted with aqueous methanol (50%, v/v) to the final total polyphenol concentration of 1 mg/mL and filtrated through a 0.22 μm filter.After 15 μL of the filtrate being injected in HPLC system (C18 reversed phase column of 250×21.1 mm i.d., 5 μm particle size; 12.5 nm pore size), the HPLC was carried out in condition of column temperature: 30°C, detector wavelength: 280 nm, mobile phase A: 1% acetic acid, mobile phase B: methanol, total mobile phase flow rate: 0.8 mL/min and gradient elution procedure: 15% B to 25% B in 15 min-25% B for 10 min-25% B to 75% B in 40 min-75% B to 15% B in 15 min-15% B for 5 min.The phenolic compound was identified and quantified by comparing retention time and peak area with that of the standard.The result was expressed as micrograms per milliliter of the purified phenolic extract solution (µg/mL).
In vitro antioxidant activity assay: DPPH Radical Scavenging Capacity (DRSC) was assayed with the method described by Locatelli et al. (2009).ABTS Radical Scavenging Capacity (ARSC) was assayed with the method described by Re et al. (1999).Total Reducing Power (TRC) was assayed with the method described by Amarowicz et al. (2010).Total Antioxidant Capacity (TAC) was assayed with the method described by Prieto et al. (1999).All results were expressed as gram of gallic acid equivalent per gram of dry peel powder (mg GAE/gDW).

Anti proliferative activity assay:
Cell culture and special media preparation: Caco-2 cells were normally cultured in Mem-High Glucose medium with 12.5% Fetal Bovine Serum (FBS) and 2% penicillin/streptomycin at 37°C and 5% (v/v) CO 2 .The purified phenolic sample was dissolved in PBS and diluted with serum-free culture medium to the final total polyphenol concentration of 100 mg/mL and further diluted with serum-free culture medium to be a serial special media with total polyphenol concentrations of 10, 20, 50, 100, 200 and 500 μg/mL, respectively.
Cytotoxic effect assay: Caco-2 cells were seeded at 1×10 5 cells/mL in a 96 wells polystyrene culture plate.After adding a special medium into specified well, the cells were cultured at 37°C and 5% CO 2 for 48 h.Then, the medium was removed and the cells were incubated with 5 mg/mL of MTT at 37°C for 4 h.Finally, 100 μL of DMSO was added into the well to dissolve formazan crystals and the absorbance of the solution was measured at 490 nm with a microplate reader (Bio Rad Laboratories Ltd., China).Meanwhile, control test (use serum-free culture medium instead of the special medium) was done.The result was expressed as cell viability remaining rate, which was defined as the absorbance quotient between test and control test (% of control).
AO/EB double staining and DAPI staining: Caco-2 cells were cultured in the special medium having total polyphenols concentration of 100 μL/mL for 24 h.The collected cells were fixed with 4% formaldehyde for 10 min and stained with 60 μL AO/EB solution (100 μg/mL AO and 100 μg/mL EB) or DAPI solution (10 μg/mL) at room temperature for 10 min.The morphological change of the cells, including reduction in volume and nuclear chromatin condensation, were observed under a fluorescence microscope (Olympus, IX71, Japan).

Western blot analysis:
After being cultived in a special medium for 48 h, the cells were harvested and lysed in cell lysis buffer (P0013, Beyotime Institute of Biotechnology, Jiangsu, China) for 10 min on ice.Then the sample was centrifuged at 15,000 g for 10 min at 4°C to get supernatant.The total protein concentration of the supernatant was determined by BCA Protein Kit (Thermo Fisher, Shanghai, China).And the supernatant was treated with SDS buffer at 95°C for 5 min.The proteins in it were separated by SDS-PAGE and electro-transferred onto a polyvinylidene fluoride membrane (0.45 μm, Millipore) by using a semidry transfer apparatus (Bio Rad, Shanghai, China).Blocking was performed for 2 h in 5% of nonfat dry .6SX-LT, Lintong, Shaanxi; YN-MZ, Mengzi, Yunnan; XJ-KS, Kashi, Xinjiang; SD-ZZ, Zaozhuang, Shandong; TP, total precipitation in the period of pomegranate mature time.(i.e., 10/08/2011~10/10/2011); AP, average atmospheric pressure in the period; AT, average temperature in the period; AH, average relative humidity in the period; TS, total sunlight time in the period milk dispersed in TBST (20 mM Tris, 166 mM NaCl and 0.05% Tween 20, pH 7.5).Then, the membrane was washed by TBST for 3 times and then immersed in the TBST buffer diluted primary antibody solution.
After standing overnight at 4°C and being washed by TBST for another 3 times, the membrane was immersed in the secondary antibodies solution and incubated at 25°C for 2 h.After the membrane was washed with TBST again, the blots were developed by adding chemiluminescent substrate (Thermo Fisher, China) and exposed in a Molecular Imager Chemidoc XRS System (BioRad, Shanghai, China).

Statistical analysis:
Every test was performed in triplicates and the result was expressed as mean ± SD.One-way Analysis of Variance (ANOVA) and correlation analysis were carried out by DPS 6.55 statistical program software.

RESULTS AND DISCUSSION
Phenolic content of the pomegranate peel: As Table 2A shown, Total Free Polyphenol Content (TFPC) of the peel (104.17-200.91 mg GAE/gDW) was about 12 times more than Total Bound Polyphenol Content (TBPC) (7.35-15.94mg GAE/gDW).Different regional peels had significantly different (p<0.05)TFPC and TBPC, but same regional peels had similar TFPC in most cases.YN peels had the lowest TFPC, while XJ and SD peels had much higher TFPC and TBPC.Similarly, Total Free Flavonoid Content (TFFC) (23.12-54.58mg QUE/gDW) of the peel was about 10 times more than total bound flavonoid content (TBFC) (2.89-5.17mg QUE/gDW) and Total Free Tannin SD-ZZ 3801.17 100.00 641.89 100.00 FPC, free phenolic compound content of the purified free phenolics sample; FPRC, relative content of the free phenolic compound compared to total detected free phenolic compounds; BPC, bound phenolic compound content of the purified bound phenolics sample; BPRC, relative content of the bound phenolic compound compared to total detected bound phenolic compounds; SX-LT, Lintong, Shaanxi; YN-MZ, Mengzi, Yunnan; XJ-KS, Kashi, Xinjiang; SD-ZZ, Zaozhuang, Shandong.
Content (TFTC) (155.02-299.95mg TAE/gDW) was about 12 times more than Total Bound Tannin Content (TBTC) (9.01-16.37mg TAE/gDW).Different regional peels had significantly different (p<0.05)TFFC and TFTC, but same regional peels had similar TFFC and TFTC in most cases.YN peels had the lowest TFFC and TFTC, while XJ and SD peels had much higher TFFC and TFTC.These results suggest that produce environment may strongly effect on TFPC, TFFC and TFTC of pomegranate peel.
There are very limited researches having compared phenolic contents of different regional pomegranate peels in China.Chen et al. (2011) reported peel TPC of 6 Xinjiang and 6 other province pomegranates ranged in 172.2-279.3 and 122.3-147.5 GAE/gDW (converted data) respectively, which also showed XJ pomegranate peels had much higher TPC than others.
Relationship between peel phenolic content and meteorology in mature period: As Table 2B shown, total precipitation was significantly negatively correlated with both TFPC (p<0.01) and TFTC (p<0.05).Average temperature was significantly negatively correlated with TFPC (p<0.01),TFFC (p<0.01) and TFTC (p<0.01).And total sunlight time was significantly positively correlated with TFPC (p<0.01),TFFC (p<0.05) and TFTC (p<0.01).While, both average atmospheric pressure and average relative humidity were not significantly correlated with any kinds of phenolic contents.These results suggest that lower total precipitation, lower average temperature and higher total sunlight time during the fruit mature time are conducive to increase TFPC generally and the significant difference of TFPC among different regional pomegranate is at least partly due to environmental difference.
There is very little information available in literature about the effect of produce environment on phenolic content and bioactivity of pomegranate peel as we known.One possible reason is the reported phenolic content data strongly related to the sampling, determination and result expression methods (Ismail et al., 2012;Calín-Sánchez et al., 2013), which usually inconsistent among different researches.Another reason may be most researchers had neglected to collect the environment information.
In the present research, extraction and determination methods kept consistent, the environmental data of the 4 regions, collected from reliable source, were obviously different from each other; and all tested pomegranates belong to different cultivars, Therefore, phenolic content difference between peels is must due to cultivar or environment differences, or both of them.According to the results mentioned above, it is reasonable to believe that mature environment has played more important effect on phenolic content of pomegranate peel than cultivar.2C shown, 11 phenolic compounds were identified in the purified peel phenolic samples.They were punicalagins A&B, gallic acid, catechine, epicatechine, chlorogenic acid, caffeic acid, ferulic acid, ellagic acid, quercetin and keampferol.Amoung them, punicalagins B accounted for 81.7~91.0% of the whole and punicalagins A for 3.9~7.0%.Although the absolute contents of these compounds in these pomegranate peel extracts existed certain difference, their relative contents were similar from each other.These results suggest that all pomegranate peels have similar phenolic elementary composition.Seeram et al. (2005a) reported that purified aqueous extract of pomegranate peel mainly contained punicalagin (80-85% w/w), ellagic acid (EA; 1.3% w/w) and unquantified amounts of punicalin and EAglycosides.Qu et al. (2012) reported the gallic acid, punicalagin A, punicalagin B and ellagic acid concentrations of the pomegranate (c.v.Wonderful) peel water extract were 1.63, 2.02, 2.18 and 4.91 mg/g respectively.These data also suggest punicalagin is the predominant phenolic compound in pomegranate peel.
Some literatures also reported pomegranate peel extracts of some cultivars had significantly different antioxidant capacity.Hajimahmoodi et al. (2008) reported the peel extract of Sweet white cultivar had more antioxidant potential than other cultivars.Shams Ardekani et al. (2011) reported peel extracts of Sour summer, Sweet saveh malas and Black cultivars had higher antioxidant activity than other cultivars.Fawole et al. (2012) reported radical scavenging activities of pomegranate peel extracts of Arakta, Ganesh and Ruby cultivars were significantly higher than other cultivars.However, these literatures had not analyzed environmental effect on polyphenol content and antioxidant capacity of peel.3B shown, DRSC, ARSC, TRC and TAC were all significantly positively correlated with TFPC, TFFC and TFTC (p<0.01).As Table 3C shown, DRSC, ARSC, TRC and TAC were also significantly positively correlate with TBPC, TBPC and TBTC (p<0.01 or p<0.05).These results suggest that all of pomegranate peel phenolics are antioxidants and the free tannins are the main antioxidants followed by free flavonoids.They are consistent with previous researches (Reddy et al.,2007;Viuda-Martos et al., 2010;Tehranifar et al., 2011).

Relationship between antioxidant capacity and phenolic content: As Table
Antiproliferative activity of the purified peel phenolics: As Fig. 1a shown, treatment with the free phenolic sample purified from SX-1 peel decreased the viability of Caco-2 cells in a dose-dependent manner and the viability had dropped to 60.7% through 48 h treatment at 100 μg/mL.As Fig. 1b shown, treatment with phenolic samples purified from other peels had similar efficacy.These results suggest that all phenolic samples purifed from any kind of pomegranate peel have cytotoxicity on Caco-2 cells and the efficacy is dose-dependent.This is agreed with the previous research (Orgil et al., 2014) which found there were positive relationships between high levels of TPC, punicalagin and gallagic acid treatment and MCF-7 proliferation inhibitory activities.As Fig. 2 shown, the cells' morphology changed to spheroid shape, accompanied by chromatin condensation, indicating the cells had apoptosis in certain degree due to phenolics induced toxicity.In Fig. 2A, we can see 90% of Caco-2 cells' chromatin condensed after the cells exposed to the phenolics at 100 μg/mL for 24 h.In Fig. 2B, we can see purified free phenolics had induced more cells into late apoptosis state than purified bound phenolics.These results suggest that PFP are more effective to induce apoptosis than PBP at same dosage and the phenolics' cytotoxicity is mainly performed by apoptosis effect.
As Fig. 3A shown, when Caco-2 cells were exposed to pomegranate peel phenolics, their caspase-8, caspase-3, p53 and the ratio of tBid/Bid all increased and their PARP content also had a little change.These results suggest that the phenolics induced cell apoptosis through a cascade caspases-dependent pathway and p53 apoptotic pathway also involved.These results are consistent with the previous studies which showed that punicalagin exhibited strong anti-proliferative activity against human lung, breast and cervical cancer cell lines (Aqil et al., 2012); punicalagin, elligic acid and total pomegranate tannins all had apoptotic effect on cells at 100 μg/mL (Seeram et al., 2005b); and caspase-independent apoptotic pathway (Fadeel and Orrenius, 2005), caspasesdependent apoptotic pathway (Chen et al., 2004;Zou et al., 2011) and p53 apoptotic pathway (Hofmann and Sonenshein, 2003) all worked.
Antiproliferative activity difference of different phenolic samples: From Fig. 3B to 3F we can see that, different purified phenolics samples at same dosage induced Caco-2 cells to express apoptosis-related proteins with a little different degree, which indicate the modes of apoptosis in these treatments were slightly different from each other.This result suggests that the purified phenolic samples from different pomegranates only had slightly different antiproliferative potential due to their phenolic compositions only having slight difference.

CONCLUSION
This research revealed that free tannins were main components of peel extract and punicalagins B was predominate compound of purified peel phenolic extract for all 10 tested pomegranates.TFPC of pomegranate peel depend on both cultivar and meteorological condition in pomegranate mature period.Antioxidant capacity of the peel extract was significantly positively correlated with TFPC.All of purified peel phenolic samples had similar elementary composition and could induce Caco-2 cells apoptosis with similar efficiency through multiple cell signaling pathways.

Table 1 :
Meteorological data of the 4 regions during pomegranate mature time

Table 2 :
Phenolic content, composition of pomegranate peel and relationship between the content and local meteorology Table2A: Total free and bound phenolic contents of pomegranate peels Culivar code free polyphenol content; TFFC, total free flavonoid content; TFTC, total free tannin content; TBPC, total bound polyphenol content; TBFC, total bound flavonoid content ; TBTC, total bound tannin content.Data were expressed as means ± SD.In same column, data followed by different letters are significantly different (p<0.05) from each other., significant correlation (p<0.05);**, highly significant correlation (p<0.01);TFPC and TBPC, total free and bound polyphenol contents; TFFC and TBFC,, total free and bound flavonoid contents; TFTC and TBTC, total free and bound tannin content; TP and TS, total precipitation and total sunlight time in the period (10/08/2011~10/10/2011); AP, AT and AH, average atmospheric pressure, average temperature and average relative humidity in the period *

Table 2C :
Phenolic constituent of purified polyphenol sample from pomegranate peel Phenolic compound

Table 3 :
In vitro antioxidant capacity of pomegranate peel extract and its relationship with phenolic content

Table 3A :
In vitro antioxidant capacity of pomegranate peel phenolic extract

Table 3C :
DPPC • scavenging capacity; ARSC, ABTS +• scavenging capacity; TRP, total reducing power; TAC, total antioxidant capacity; P, phenolics.Data were expressed as means ± standard deviations.In same column, data followed by different letters indicate their mean values are significantly different (p<0.05) from each other.Highly significant correlation (p<0.01);TFPC, Total Free Polyphenol Content; TFFC: Total Free Flavonoid Content; TFTC: Total Free Tannin Content; DRSC: DPPC Radical Scavenging Capacity; ARSC, ABTS + radical scavenging capacity; TRP: Total Reducing Power; TAC: Total Antioxidant Capacity Correlations between bound phenolic content and antioxidant activity