Evaluation of Bioactive Compounds in Pomegranate Fruit Parts as an Attempt for Their Application as an Active Edible Film

Pomegranate fruit contains high content of phytochemical constituents which have many health benefits. Peel and rind are wastes of pomegranate fruit processing, represent up to 50% of its weight. This study aimed to evaluate the ethanolic extract of pomegranate fruit parts: arils, rind and peel as sources of bioactive compounds as well as their antioxidant and antimicrobial activities for their application as an active edible film. Results clearly demonstrated that peel extract (PE) had the highest content of total phenolics and flavonoids (342 mg GAE /g and 82.33mg catechol /g, respectively ) followed by rind extract (RE) containing 213.00 mg GAE/g and 70.50 mg catechol /g, respectively, and finally arils extract (AE) ( 108.22 mg GAE /g and 55.58 mg catechol/g), respectively. Results indicated that total anthocyanins content was concentrated in PE (15.24mg Cynidian-3-glycoside/g) and AE (11.04 mg Cynidian-3-glycoside/g), while RE (6.51 mg Cynidian-3-glycoside/g) had the lowest value. Peel extract exhibited the highest antioxidant activity followed by RE and were significantly higher than that of AE. These results were confirmed with the DPPH and ABTS assays. Consequently, PE followed by RE had higher antimicrobial activity against several pathogenic strains than AE and can be used as natural preservative for food. Peel extract and RE were incorporated into pectin film at concentration of 15 mg/ml to develop an active edible film. Pectin film without the tested fruit parts extract was used as the control film. The obtained results revealed that the film prepared from pectin with PE and RE was successfully developed and considers as an active edible film with antioxidant and antimicrobial properties.


Introduction
Pomegranate tree is considered as a medicinal plant and the fruit is better known as nature's power fruit recognized worldwide for pleasant taste and excellent health benefits [1]. Fruits of pomegranate are considered one of the most important deciduous shrubs grown successfully in Egypt, not only for local consumption but also for exportation [2]. An increasing demand of pomegranate for industrial processing to make juice, jams, syrup, sauce, flavouring, colouring agents and nutraceuticals is needed, in addition, to the growing demand for fresh consumption, where the rind or peel and seeds of the fruits are discarded [3]. Pomegranate possesses enormous antioxidants activity [4] and is rich in bioactive compounds, mainly polyphenols and anthocyanins, with known health benefits, and is effective in prevention of atherosclerosis, low-density lipoprotein oxidation, prevention of cancer, treatment of cardiovascular diseases and dental conditions, and protection from ultraviolet radiation [5].
The nutraceutical properties of pomegranate fruit are not limited to the edible part: in fact the non-edible fractions of fruit and tree (i.e. peel, seeds, flowers, bark, buds and leaves), although considered as waste, contain even higher amounts of specific nutritionally valuable and biologically active components as compared to the edible fruit [6][7][8].
Pomegranate peel represents about 40-50% of the total fruit weight [9] and this may lead to various environmental problems [10]. It is produced as by product in huge amounts by the food industry and it is an important source of bioactive compounds [11]. Pomegranate peel has been known for many years for its health benefit, including antibacterial activity. The high level of bioactive compounds in the peel as well as the reported health benefits to date make these desirable byproducts as functional ingredients in food, nutraceuticals and pharmaceutics [12]. Pomegranate rind is rich in tannins and polyphenols that demonstrate remarkable antimicrobial activity [13].
Edible coatings and edible films belong to the modern food protection system; over the past few years, interest in the use of edible coatings for perishable foods has considerably increased due to their advantages and potential applications [14]. Edible active films can function either by releasing the active agents into the surrounding atmosphere or through the absorption of components that can deteriorate the quality of food such as oxygen, moisture and free radicals [15]. Active agents with antimicrobial properties may help to extend the shelf life and maintain quality and food safety by increasing the lag phase and slowing the growth phase of microorganisms [16].
The objectives of the present research were to determine and identify the major phytochemical compounds found in the ethanolic extracts of pomegranate fruit parts (arils, rind and peel). The antioxidant and antimicrobial activities of each extracts as well as pectin-based edible films containing ethanolic extracts were also studied.

Materials
Pomegranate (Punica granatum L.) fruits variety "Wonderful" were obtained from local markets in Alexandria, Egypt. The fruits were selected on the basis of size uniformity, maturity stage "based on skin color and firmness "and absence of physical damage.

Chemicals
All chemicals used were of analytical grade, and were purchased from El-Gomhouria Co. for Chemical and Medical Requisites, Alexandria, Egypt. Chemicals used in HPLC were purchased from Bio-Rad chemical Co., CA, and U.S.A. Figure 1 shows pomegranate fruit parts, while Figure 2 shows the outline of preparing pomegranate fruit parts powder arils (A), rind (R) and peel (P) of pomegranate fruits. The fruits were washed with tap water, drained, cut manually to quarters and separated to arils, peel (leathery skin) and rind (spongy mesocarp), all cut into small pieces using a sharp knife and dried in an air oven at 50ºC for 24 hr. Then were ground using an electrical mill (SEB 21260), sieved to obtained particle size of 60 mesh and kept in low density polyethylene bags and stored at 5°C.

Ethanolic Extraction of Dried Powder of
Pomegranate Fruit Parts Dried powder of A, R and P were extracted by ethanol 80% (1:5 w/v) at room temperature (25°C ± 2) for 24 hr. The mixture of ethanol with each of them was filtered, and the rough particles were removed by strainer. Then, the residue was reextracted using the same conditions. The collected extracts were concentrated in a rotary evaporator (40°C at 700 mmHg). The entire procedure was performed in the dark. The concentrated ethanolic extract for each powder was subjected to chemical analysis.
pectin-based edible film was prepared by casting in 2 stages: i. Dissolving 2 g pectin in 100 ml of distilled water, and glycerol (0.5 g/g of pectin) and calcium chloride (0.01g/g of pectin) were added to the suspension. The suspension was then heated to 70°C under stirring, until all the solids were dissolved and a homogeneous suspension was achieved. The suspension was cooled to 40°C, then the fruit part extract were added at the tested concentration (15 mg/ml) according preliminary study.
A control treatment without the tested fruit parts extract was prepared for comparison. ii. The film-forming solution was casted on an acryliccoated plate and then dried for 10-12 h in an oven at about 35°C to obtain a constant weight. Then, the film could be easily removed from the plate. Before analysis, the films were conditioned in desiccators under 58% RH, at 25°C, for 48 h.

Total Phenolics Content
Total phenolic contents of extracts were determined using the method developed by Abirami et al. [18]. One and half milliliters of Folin-Ciocalteu's reagent (diluted 10 times) and 1.2 ml of Na2CO3 (7.5% w/v) were added to 300 µl of sample extract. Mixtures were shaken and kept at room temperature for 30 min before measuring absorbance at 765 nm using a spectrophotometer (Pg T80+, England), testes were carried out in triplicate. Total phenol content (TPC) was expressed as Gallic acid equivalent (GAE) in mg/g plant material or extract.

Total Flavonoids Content
The total flavonoids content of extracts were determined according to Barros et al. [19]. Shortly, 0.5 ml of sample extract was mixed with 2 ml of distilled water followed by addition of 150 µl of NaNO2 (5%) solution. After 6 min, 150 µl of AlCl3 (10% w/v) was added and allowed to stand for another 6 min before 2 ml of NaOH (4% w/v) was added. The mixture was brought to 5 ml with distilled water. Then the mixture was allowed to stand for 15 min at room temperature. The absorbance was measured at 510 nm using a spectrophotometer (Pg T80+, England). A calibration curve of Rutin was prepared and total flavonoids content were determined.

Determination of Total Anthocyanins Content (TA)
TA content was determined by pH differential method using two buffer systems: potassium chloride buffer (pH 1.0, 0.025 M) and sodium acetate buffer (pH 4.5, 0.4 M) according to Elfalleh et al. [20]. Methanolic extract were mixed with 3.6 ml of corresponding buffers and read against water as a blank at 510 and 700 nm. Absorbance (A) was calculated using this formula A = [(A510 -A700) pH1.0 -(A510 -A700) pH4.5] with a molar extinction coefficient of 29600. Results were expressed as mg of cyanidin-3-glucoside equivalents per g dw (mg CGE/g dw).

HPLC Determination of Phenolic and Flavonoid
Compounds HPLC analysis was carried out using an Agilent 1260 series. The separation was carried out using C18 column (4.6 mm x 250 mm i.d., 5 µm). The mobile phase consisted of water (A) and 0.02% tri-floro-acetic acid in acetonitrile (B) at a flow rate 1 ml/min. The mobile phase was programmed consecutively in a linear gradient as follows: 0 min (80% A); 0-5 min (80% A); 5-8 min (40% A); 8-12 min (50% A); 12-14 min (80% A) and 14-16 min (80% A). The multiwavelength detector was monitored at 280 nm. The injection volume was 10 µl for each of the sample solutions. The column temperature was maintained at 35°C.

Scavenging Activity by DPPH Assay
Scavenging activity of the stable 1, 1-diphenyl-2picrylhydrazyl (DPPH) radical was determined according to the procedure based on Brand-Williams et al. [21]. Two milliliters of 0.15 mM DPPH were added to 1 ml of extracts in different dilutions. A control was prepared by adding 2 ml of DPPH to 1 ml of methanol. The contents of the tubes were mixed and allowed to stand for 30 min, and absorbance was measured at 517 nm using a spectrophotometer (Pg T80+, England). The results were expressed as% radical scavenging activity according to equation (1).

Radical scavenging activity%= (A control -A sample )/A control x100 (1)
Where: A control is the absorbance of the control reaction (containing all reagents except the test compound).
A sample is the absorbance with the test compound.

Determination of Radical ABTS + Scavenging Activity
The stock solutions of ABTS + reagent was prepared according to Hwang and Do Thi [22] by reacting equal quantities of a 7 mM aqueous solution of ABTS* with 2.45 mM potassium persulfate for 16 h at room temperature (25°C) in the dark. The working solution was then prepared by diluting 1 mL ABTS* solution with 60 mL of ethanol: water (50:50, v/v) to obtain an absorbance of 1.0± 0.02 units at 734 nm using the spectrophotometer. Extracts (50 µL) were allowed to react with 4.95 mL of the ABTS + solution for 1 h in a dark condition. Then the absorbance was read at 734 nm using the spectrophotometer.% inhibition of the ABTS + free radical was calculated by equation (2) Where: A control is the absorbance of the control reaction (containing all reagents except the test compound).
A sample is the absorbance with the test compound. The standard curve was prepared using Trolox. The results were expressed as mg Trolox equivalents (TE)/g sample).
Additional dilution was needed if the ABTS + value measured was over the linear range of the standard. Application as an Active Edible Film

ii. Determination of minimum inhibitory concentration (MIC)
To examine antimicrobial activity of sample extracts, agar well diffusion assay was used against pathogenic bacteria and yeast as described by Hamad et al. [23]. The bacterial strains were grown in nutrient broth at 37°C, while, yeast strain was grown on Potato Dextrose Agar (PDA) at 29°C for 24 h.
Briefly, 100 µL of overnight activated culture of each pathogen strain (10 6 CFU/mL) were aseptically spread over nutrient agar plates. About 100 µL of 100% extract was transferred into each agar well individually. The plates were then incubated at 37°C for 18 h and the formed clear zones (if found) were measured and recorded. A set of 5 concentrations of sample extracts (50, 25, 12.5, 6.25 and 3.1 mL / 100 mL), were examined to determine the minimum inhibitory concentration (MIC) of each against a specific pathogenic strain [24]. The zones of inhibition were calculated by measuring the diameter of the inhibition zone around the well (mm), including the well diameter. The readings were taken in three different fixed directions in all duplicates and the average values were tabulated.

Statistical Analysis
Data were statistically analyzed by a general linear model procedure of the Fisher's protected least-significant difference (PLSD) test using SAS, 2004 (SAS Institute, Inc ,. Cary, NC) [25]. This test combines ANOVA with a comparison of differences between the means of the treatments at the significance level of P ≤ 0.05.

Bioactive Compounds
Total phenolics, total flavonoids and total anthocyanins content of the ethnolic extracts of pomegranate fruit parts (PE, RE and AE) are shown in Table 1.
Values of total phenolics and flavonoids in the ethanolic extract of pomegranate fruit parts can be arranged as follow: PE had the highest values (342 mg GAE /g dw) of phenolics followed by RE (213.00 mg GAE /g dw), while, AE had the lowest value (108.22 mg GAE /g dw). Also, flavonoids content of PE, RE and AE had the same trend and their values were 82. 33, 70.50 and 55.58 mg catechol /g dw, respectively. These results revealed that PE had the highest content of total phenolics and flavonoids followed by RE and finally AE. Orak et al. [26] reported that pomegranate peel, seed and juice contained considerable amounts of phenolic compounds. Mphahlele et al. [12] stated that these compounds are mainly concentrated in fruit peels and mesocarp. Statistically, the obtained data of total phenolics and flavonoids showed high significant variation between the three tested extracts. The data of total phenolics for PE in the present study are compatible with that mentioned by Derakhshan et al. [27] and higher than that mentioned by Rosas-Burgas et al. [28] and Sumaiya et al. [29]. The results of flavonoid obtained in the present study are higher than that found by Farag et al [30], Souleman et al. [31] and Sumaiya et al. [29]. These variations may be affected by many factors, such as cultivar source, growing and climatic conditions as well as extraction methods.
Total anthocyanins content in the ethanolic extract of the pomegranate fruit parts were 15.24, 11.04 and 6.51 mg Cynidian-3-glycoside /g dw for PE, AE and RE, respectively. These results showed that PE had the highest values followed by AE, while, RE had the lowest value. These results indicated that total anthocyanins content was concentrated in fruit peels and arils. From the statistical point of view, it can be noted high significant differences among the three values. Zhu et al. [32] confirmed that total anthocyanins content was higher in peel than that found in fruit juice. Hou et al. [33] stated that pomegranate fruit is rich in anthocyanin and it has many health benefits' such as antioxidant ability, antiinflammatory, anti-cancer and other physiological functions.

Identification of Phenolics and Flavonoids in the Ethanolic Extracts of Pomegranate Fruit Parts
Several phenolic and flavonid compounds were identified in ethanolic extracts of the three pomegranate fruit parts (AE, RE and PE) using HPLC separation as shown in Table 2. The number of unknown compounds detected in AE, RE and PE were 14, 19 and 20, respectively, according to the identified standard. Meanwhile, the identified phenolic and flavonoids compounds in PE, RE and AE extracts were 11, 9 and 6 respectively. These results have not been consistent with previous studies [31,[34][35][36][37]. These variation may be due to many reasons, such as, cultivar, agriculture process, solvent used, conditions of extraction and identification methods [37]. The results in Table 2 shows that, PE had six identified phenolic compounds. They were gallic acid (6041.1 µg/g dw) that was the predominant followed by caffeic acid (1220.37 µg/g dw), while, the remaining four compounds were relatively low in their concentrations. These results are in accordance with that found by Souleman and Ibrahim [31] who found that gallic acid and caffeic acid were the most predominant compounds in pomegranate cultivars. RE had four identified compounds, where gallic acid (278.44 µg/g dw) was also the predominant, while, AE had 3 identified compounds where cinnamic acid (43.22 µg/g dw) was the most predominate.
Generally, PE had the highest concentration of phenolic and flavonoid compounds followed by RE, while AE had the lowest one except for cinnamic acid. Also, it was observed that PE was characterized by a high proportion of galic acid, caffeic acid, naringenin and catachin, while, RE was characterized by a high proportion of galic acid and catechin in comparison to the remaining compounds. Table 3 shows the antioxidant capabilities of ethanolic extracts of pomegranate fruit parts (PE, RE and AE) as assessed by DPPH and ABTS radical scavenging.

Antioxidant Activity
The radical-scavenging activity on DPPH was expressed as IC 50 . This value was the concentration of the extract required inhibiting 50% of the initial DPPH free radical. Table 3 revealed that PE exhibited the highest activity followed by RE and AE. The mean values were 1.9, 2.64 and 3.81 µg/ ml, respectively. The lower IC 50 value means stronger scavenging DPPH free radicals. In general the antioxidant activity of PE was significantly higher than that of the other parts. This is consistent with [26]. The ABTS + method was used to confirm the results from the DPPH test since it is based on a similar antioxidant mechanism and the results are shown in Table 3. The ethanolic extracts of pomegranate fruit parts showed that ABTS + activities of PE and RE were 312.29 and 261.34 mg TE/g dw, respectively. These values were significantly higher than that of arils being 20.33 mg TE/g. These results were correlated well with the findings of Jalal et al. [38] who reported significantly higher ABTS + activities of pomegranate peel.
The results clearly confirmed that PE contained more antioxidants followed by RE while AE had the lowest activity which are confirmed with the DPPH and ABTS + assays. The results implied that bioactive compounds from the peel might be potential resources for the development of antioxidant function dietary food. In general, antioxidant activities of PE were higher than that of RE and AE. This fact is consistent with previous studies which indicated high antioxidant activity of pomegranate peel comparing with the other parts [26,39].

Antimicrobial Activity of Ethanolic Extract of Pomegranate Fruit Parts (AE, RE and PE)
The antimicrobial activity of ethanolic extracts of pomegranate fruit parts (AE, RE and PE) were investigated against some pathogenic strains by disc diffusion method. Figures 3, 4 and 5 show antimicrobial activity using various concentrations (3.5, 7.5, 15, 30, 62.5, 125 and 250 mg / ml) of ethanolic extracts. Results revealed that ethanolic extracts of all pomegranate fruit parts can successfully control or inhibit the visible growth of the tested types of pathogenic strains and possessed an inhibitory effect. It was observed that antimicrobial activity differed according to the tested pomegranate part, its concentration and the types of pathogenic strains. PE had the highest inhibitory activity followed by RE, while, AE had the lowest. Increasing the concentration of any fruit part extract caused increase in the inhibition zone. These results are in agreement with several studies [40,41].  This could be due to either quantitative or qualitative difference in the phytochemical arrangement of these parts, as well as total antioxidant activity (Table 1). Generally, comparing the sensitivity of pathogenic strains tested showed that Gram-negative bacteria were more sensitive to pomegranate extract parts used than Gram-positive bacteria. Diameter inhibition zone (DIZ) for Gram-negative ranged between 30 to 7 mm for AE, 37 to 6 mm for RE and 42 to 8 mm for PE, while DIZ for Gram-positive ranged between 21 to 7 mm for AE, 30 to 7 mm for RE and 35 to 9 for PE. Minimum inhibitory concentration (MIC) of ethanolic extracts and it's diameter of inhibition zone (DIZ) are presented in Table 4. In general, PE and RE were more effective even at a concentration of MIC 7.5 mg/ml for all tested strains than AE that had 15, 30 and 62.5 mg/ml MIC.
Khan and Hanee [42] reported that the antibacterial activity of peels of pomegranate may be indicative of presence of metabolic toxins or broad spectrum antimicrobial compounds that act against both Gram-positive and Gramnegative bacteria.  Figure 5 shows the highest activity against Candida albicans (ATCCMYA 2876) was recorded for PE that had inhibition zone ranged between 9 to 35 mm at concentration 7.5 to 250 mg/ml followed by RE that had inhibition zone ranged between 6 to 25 mm at concentration 7.5 to 250 mg/ml. The lowest inhibition zone ranged between 7 to 22 mm at concentration 62.5 to 250 mg/ml was observed for the AE. These results are in agreement with those previously studies [43,44].
The obtained results revealed that all the ethanolic extracts of pomegranate parts especially PE is effective for all tested pathogenic strains. Ali et al. [45] reported that the possible mechanism of action of pomegranate parts, especially the peel against microorganisms can be related with the phenolic toxicity that interacts to sulfhydryl groups of proteins in microorganisms.
The differences in microbial activity of the ethanolic extract of pomegranate fruit parts may be due either to the quantitative or qualitative differences in phytochemical arrangement of these parts, as well as total antioxidant activity (Table 1). Previous studies by Duman [46] revealed that antimicrobial activity of pomegranate fruit extracts correlated to phytonutrient properties, such as total phenolic and anthocyanin compounds as well as the antioxidant power.
It can be concluded that pomegranate (Punica granatum L.) fruit parts, especially PE are as a potential source of antimicrobial activity against several pathogenic strains and it can be used as natural preservative for food. Table 5 shows the theoretical and measured phenolic, flavonoid content and antioxidant activity in the prepared films. The results revealed that films containing PE higher total phenolic, total flavonoid content and antioxidant activity than that containing RE. There is a decrease in the values between theoretical and measured phenolic and flavonoid content. This may be due to occurrence losses during film preparation. The experimental values within each row that have no common superscript are significantly different (p≤0.05).

Antioxidant Activity of Pectin -Based Edible Films
Control: pectin only EP: ethanolic extract of peel ER: ethanolic extract of rind.
IC 50 : the concentration required for inhibiting 50% of the initial DPPH free radical.
These losses may have stemmed during the shaking, drying method, compounds may be leached and oxidation [47]. Table 5 shows that films containing PE showed higher radical scavenging activities than that containing RE. The IC50 values were 37.7 and 48.3 µg/ml, respectively, while, the control film did not have any antioxidant activity. Figure  6 shows the radical-scavenging activity of different concentration of pectin films incorporated with the RE and PE. The high antioxidant activities of films with PE may be attributed to its high contents of total phenolic and total flavonoids. The results clearly indicated that pomegranate PE appeared as a richer in natural antioxidants than the other parts. Pectin-based edible films gained antioxidant activity by the addition of pomegranate RE and PE and their antioxidant properties enhancement was dependent on the concentration used. Table 6 shows the antimicrobial activity of pectin -based edible films incorporated with ethanolic extracts of pomegranate PE and RE at concentration two fold of MIC (15 mg/ml). The results showed remarkable antimicrobial activities. The control film did not show any antimicrobial activity against tested pathogenic strains, while pectin-based edible films incorporated with PE and RE exhibited pronounced inhibition zones against most of the tested pathogenic strains. The films containing PE caused the wider inhibition zone compared to RE. The three tested Gram-positive bacteria were more sensitive to pectin-films based PE than RE. The films with RE had no effect on the growth of Bacillus subtilis DB100, in contrast, films with PE caused 27 mm inhibition zone. As for the tested Gram-negative bacteria, films with RE and PE had no effect on the growth of Klebseilla pneumonia ATCC12296 and Pseudomonas marginalis EMCC1271, while, Proteus hauseri EMCC1227 and Escherichia coli ATCC25922 strains were sensitive to the two extracts. The antimicrobial activity of PE was stronger than RE.

Antimicrobial Activity of Pectin -Based Edible Films
The addition of RE and PE to pectin -based edible films had no effect on the growth of the tested yeast strain (Candida albicans ATCCMYA2876). Two fungi strains were tested, one of them Aspergillus parasiticus EMCC 886T was high sensitive to pectin film based PE than RE, while, the other strain Aspergillus flavus EMCC 274 was resistant to the prepared films.
As it can be seen in Table 6, incorporated pectin film with PE and RE has shown antimicrobial activity against some of the tested pathogenic strains, while, ineffectiveness against the other strains. These variation may be due to the interaction of bioactive components in the pectin film matrix that make it difficult to release from the film or possibility of degradation of active compounds during film preparation [47].

Conclusion
As a conclusion of the previous mentioned data and discussion, ethanolic extracts of pomegranate fruit peel and rind had higher amounts of bioactive compounds as well as higher antioxidant activity. The results clearly confirmed that PE contained more antioxidants followed by RE while AE had the lowest activity which are confirmed with the DPPH and ABTS + assays. The result revealed that ethanolic extracts of all pomegranate fruit parts can successfully control or inhibit the visible growth of the tested types of pathogenic strains and possessed an inhibitory effect. It was observed that antimicrobial activity differed according to the tested pomegranate part, its concentration and the types of pathogenic strains. The minimum inhibitory concentration (MIC) was 7.5 mg/ ml for all tested strains than AE that varied from 15-62.5 mg/ml. The differences in microbial activity may be due either to the quantitative or qualitative differences in the phytochemical arrangement of these parts, as well as total antioxidant activity. The incorporation of PE and RE into pectin film resulted in film with good antioxidant and antimicrobial properties and considers as an active edible film and thus it can extend the shelf life of food products.