Phytochemical content and antioxidant activity of different fruit parts juices of three figs (Ficus carica L.) varieties grown in Tunisia

Phytochemical content and antioxidant activity of different fruit parts juices of three figs (Ficus carica L.) varieties grown in Tunisia

Industrial Crops and Products 83 (2016) 255–267 Contents lists available at ScienceDirect Industrial Crops and Products journal homepage: www.elsevi...

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Industrial Crops and Products 83 (2016) 255–267

Contents lists available at ScienceDirect

Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop

Phytochemical content and antioxidant activity of different fruit parts juices of three figs (Ficus carica L.) varieties grown in Tunisia Arij Harzallah ∗ , Amira Mnari Bhouri, Zahra Amri, Hala Soltana, Mohamed Hammami Biochemistry Laboratory, Research Laboratory LR12ES05: Lab-NAFS ‘Nutrition—Functional Food & Vascular Health’, Faculty of Medicine—University of Monastir, Tunisia

a r t i c l e

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Article history: Received 2 October 2015 Received in revised form 15 December 2015 Accepted 16 December 2015 Keywords: Ficus carica L. Fig juice Phytochemical content Antioxidant activity Fruit color

a b s t r a c t Fig is an important source of bioactive compounds and has been a typical component in the healthpromoting Mediterranean diet for many centuries. This study was conducted to evaluate differences between phytochemical composition and antioxidant properties of juices of peel, pulp and total fruit of figs from three different varieties grown in Tunisia Mediterranean coast and corresponding to three different colors (green, purple and black) as well as the effect of maturation stage on the amounts of phytochemical composition including total phenols content (TPC), total flavonoids content (TFC), total ortho-diphenols content (TOPC), total tannins content total (TTC) and total anthocyanins content (TAC). Antioxidant potential was assessed by two assays: 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging capacity and reducing power (RP) and showed that different fig juices extracts exhibited the same antioxidant capacity in both systems tested and at different concentrations. Black peel juice acted as the greatest antioxidant by having the highest DPPH and RP activities followed by the juice of black fig total fruit. The antioxidant capacities observed were attributed to higher total phenolic, flavonoid and anthocyanin contents according to the chemometric results. Comparison of phytochemical composition of fig fruits during the development stage revealed a significant increase of TPC, TFC, TOPC, TTC and TAC in the ripe fruits of the three tested varieties. This is the first study comparing the phytochemical composition and antioxidant potential of juices of Ficus carica L. peels, pulps and total fruits. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The fig tree (Ficus carica L.) was evidently originated in the Middle East (Mars, 2001). Most of the world’s fig production occurs in the Mediterranean countries (Sadder and Ateyyeh, 2006), where diets are characterized by abundant intake of fig fruit (Solomon et al., 2006). Tunisia is one of the most important consumers of figs. F. carica L. is present all over Tunisia in various environmental conditions. The tree grows well and produces tasteful and flavouring fruits enhanced by warm and shiny conditions during ripening (Trad et al., 2012).

Abbreviations: TPC, total polyphenols content; TFC, total flavonoids content; TTC, total tannins content; TOPC, total O-diphenols content; TAC, total anthocyanins content; (DPPH) scavenging capacity, 1,1-diphenyl-2-picrylhydrazyl scavenging capacity assay; RP, reducing power assay; PCA, principal component analysis. ∗ Corresponding author. E-mail address: [email protected] (A. Harzallah). http://dx.doi.org/10.1016/j.indcrop.2015.12.043 0926-6690/© 2015 Elsevier B.V. All rights reserved.

Figs were analysed for total polyphenols, total flavonoids and exhibited high anti-oxidant capacity (Solomon et al., 2006). It is a very nourishing food and used in industrial products. It is rich in vitamins, mineral elements, water, and fats. It is one of the highest plant sources of calcium and fiber (Joseph and Raj, 2011). They are rich in easily digestible natural sugars, and contain rich amounts of anthocyanins and flavonoids that contribute to figs coloration (Solomon et al., 2006) and to signaling pathways regulation that guide cellular metabolism. Humans have eaten the fruits of this tree from the earliest times, and utilized it and its by-products (leaves, latex, bark, and roots) in various disorders such as gastrointestinal respiratory, inflammatory, cardiovascular disorders, ulcerative diseases, cancers (Canal et al., 2000; Joseph and Raj, 2011), as laxative, antispasmodic remedies (Guarrera, 2005), antiviral, an tibacterial, hypoglycaemic, and anthelmintic effects (Jeong et al., 2005; Joseph and Raj, 2011). In another hand, figs can be eaten dried, fresh, as a jam or also as a juice. Figs are often peeled; the pulp is eaten and the peel discarded

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Fig. 1. Color appearance of the three fig varieties examined in this study.

(Veberic et al., 2008), however, in different countries like Tunisia, consumers prefer to eat the whole fresh fruit. Most of the previous scientific studies have been focused mainly on dried figs (Vinson et al., 2005; Vallejo et al., 2012), on fresh figs to determine their total phenolic profile and antioxidant capacity (Veberic et al., 2008; Vallejo et al., 2012), and also on fig leafs to describe their antimicrobial activity (Jeong et al., 2009; Lee and Cha, 2010; Ahmad, 2013) or anti-HSV effect (Joseph and Raj, 2011), neglecting the other edible parts. However, there is few data comparing the phytochemical content changes occurring in the fig fruit during ripening (Veberic et al., 2008; Crisosto et al., 2010), as well as, in spite of the few researches describing the distribution of the phenols content between figs pulp and peel (Solomon et al., 2006; Del Caro and Piga, 2008), no data described neither the whole phytochemical composition in the fig fruit including total phenols content (TPC), total flavonoids content (TFC), total ortho-diphenols content (TOPC), total tannins content total (TTC) and total anthocyanins content (TAC), nor the antioxidant potential of juices of different fig fruit parts. Thus, to improve the knowledge on fig compositions and to valorise it as a functional food, the aim of this study is firstly to evaluate the entire phytochemical composition as well as the antioxidant capacities of juices of different ripe fig fruit parts including peel, pulp and total fruit of the most abundant varieties in Tunisia corresponding to three different colors: black, purple and green; and secondly, to evaluate the influence of the variety and the crop timing on the phytochemical composition level of fresh ripe fig fruit.

For every variety, three repetitions were carried out (n = 3); each repetition included 10 fruits sampled from three trees. An agricultural technician performed identification of fruit varieties. Fruits were placed in a 4 ◦ C refrigerated box and transported within 3 h to the Herbarium of the Laboratory of Biochemistry, Faculty of Medicine of Monastir, Tunisia. There, fruits were washed with water, wiped completely dry and stored at −20 ◦ C until preparing samples the next day.

2.2. Sample preparation To prepare whole fig juice, 10 ripe figs (2nd crop) from each variety were mixed. An equal portion of ripe figs from each variety was peeled manually with a knife, paying attention not to include the fruit pulp. Peel and pulp were mixed separately too. A blender (Moulinex, France) was used to obtain peel juice, pulp juice and whole fruit juice. In the blender, the three compartments were juiced. The juice was centrifuged at 3.000 rpm for 10 min; the supernatant was kept and filtered through a filter. The filtered juices were stored at −20 ◦ C until preparing methanolic extracts the following day. Different fig juices samples are shown in Fig. 2. To evaluate the effect of ripening stage on the phytochemical content of figs, 10 ripe and 10 partially ripe figs from each variety were homogenized and used.

2. Materials and methods 2.1. Plant material Fresh fig fruits (F. carica L.) were manually picked from a same farm in Bekalta, (coastal town, governorate of Monastir–Central East part of Tunisia–) in 2 different ripening stages: partially ripe at the beginning of July 2013 (1st crop) and at ripening time at the last of August 2013 (2nd crop). Specimens were chosen from three varieties corresponding to three different colors as following (Figs. 1 and 2): • Kohli variety is a dark type, black in color with dark red pulp. Fruit are sweet and juicy. • Hamri variety is a dark type, purple in color with light red pulp. Fruit are sweet and juicy. • Bidhi variety is a light type, green in color with dark red pulp. Fruit are sweet and juicy.

Fig. 2. Appearance of different fruit parts juices of the three fig varieties examined in this study. A1: Bidhi pulp, A2: Bidhi total fruit, A3: Bidhi peel, B1: Hamri pulp, B2: Hamri total fruit, B3: Hamri peel, C1: Kohli pulp, C2: Kohli total fruit, C3: Kohli peel.

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Fig. 3. Bar graphics comparing total polyphenols content (TPC) in juices of different fig compartments. Results are expressed as means ± standarddeviation (n = 3). Bars with different small letters within one variety are significantly different (p < 0.05) according to Duncan test. Bars with different capital letters within the three varieties are significantly different (p < 0.05) according to Duncan test.

Fig. 4. Bar graphics comparing total flavonoids content (TFC) in juices of different fig compartments. Results are expressed as means ± standarddeviation (n = 3). Bars with different small letters within one variety are significantly different (p < 0.05) according to Duncan test. Bars with different capital letters within the three varieties are significantly different (p < 0.05) according to Duncan test.

2.3. Methanolic extracts preparation Each sample (5 g) was homogenized in 5 ml of methanol until uniform consistency, using an Ultra-Turrax homogenizer (IKA, T25, digital). The homogenates were centrifuged at 3000 rpm for 13 min. The supernatants were recovered, filtered using 0.45 ␮m filter and stored at −20 ◦ C until further analysis within a maximum period of one week. 2.4. Determination of total phenols content (TPC) The method of Montedoro et al. (1992) with slight modifications was used to determine total phenolic content. Each extract (0.1 ml) was combined with water 1:4 (v/v) and 2.5 ml Folin–Ciocalteu’s phenol reagent (1/10) and incubated for one minute at room temperature, followed by the addition of 2 ml of sodium carbonate (7.5%). The mixture was well shaking and standing for 6 h. The absorbance of the solutions was measured at 765 nm using a spectrophotometer (Lambda 25, UV/vis Spectrometer). Values of TPC were estimated by comparing the absorbance of each sample with a standard response curve employing six different Gallic acid standard solutions, in the same conditions reported for the methanolic extract. Results are expressed as milli gram gallic acid equivalents per gram of fresh weight (mg GAE/g FW). Samples of each extraction were analysed in triplicate.

2005; Lenucci et al., 2006; Bakar et al., 2009). Extract (1 ml) was mixed with 4 ml of distilled water in a test tube followed by addition of 0.3 ml of 5% sodium nitrate solution and allowed to react for 5 min. Then 0.3 ml of 10% aluminum chloride solution was added. After 6 min, 2 ml of 1 M sodium hydroxide was added. The mixture was diluted with distilled water up to 10 ml and then mixed well using a vortex. The absorbance was immediately recorded at 510 nm. The absorbance was measured immediately at 510 nm using a spectrophotometer and the flavonoid content was expressed as mg of Catechin equivalents per gram of fresh weight (mg CE/g FW). All the tests were carried out in triplicate. 2.6. Determination of total O-diphenols content (TOPC) Total ortho-diphenol content was determined using the method described by Bahorun et al. (1996). Test sample (100 ␮l) was mixed with 1 ml of HCl (0.5N), 1 ml of (NaNo2 + Na2 MoO4 2H2 O) solution and 1 ml of NaOH (1 M). The mixture was stirred and kept for half an hour at room temperature in the dark. The absorbance was measured at 500 nm against the blank. Different concentrations of hydroxytyrosol solution were used for calibration. Results were expressed as milli gram of hydroxytyrosol equivalents per gram of fresh weight (mg hydroxytyrosol/g FW). 2.7. Determination of total tannins content (TTC)

2.5. Determination of total flavonoids content (TFC) Total flavonoid content was quantified using the colorimetric method with aluminum chloride according to (Marinova et al.,

Total tannins content were determined spectrophotometrically according to Broadhurst and Jones (1978). A 0.5 ml of aqueous extract, contained in a test tube covered with aluminum foil, was

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Fig. 5. Bar graphics comparing total ortho-diphenols content (TOC) in juices of different fig compartments. Results are expressed as means ± standarddeviation (n = 3). Bars with different small letters within one variety are significantly different (p < 0.05) according to Duncan test. Bars with different capital letters within the three varieties are significantly different (p < 0.05) according to Duncan test.

Fig. 6. Bar graphics comparing total tannins content (TTC) in juices of different fig compartments. Results are expressed as means ± standarddeviation (n = 3). Bars with different small letters within one variety are significantly different (p < 0.05) according to Duncan test. Bars with different capital letters within the three varieties are significantly different (p < 0.05) according to Duncan test.

mixed with 3 ml of 4% vanillin–methanol solution and then with 1.5 ml of hydrochloric acid. The mixture was allowed to stand for 15 min at 20 ◦ C in the dark. The absorbance of the mixture was measured at 500 nm. A different concentration of tannic acid aqueous solution (30 mg/l) was used for calibration. The final results were expressed as milli gram tannic acid equivalents per gram of fresh weight (mg TAE/g FW).

2.8. Determination of total anthocyanins content (TAC) Total anthocyanin content was quantified according to Padmavati et al. (1997) and Chung et al. (2005). Anthocyanins were extracted from 0.5 g of fresh weight with 20 ml methanol/water: concentrated HCl (80/20/1). Samples were put on a shaker in the dark at room temperature for 24 h; absorbance (A) was measured spectrophotometrically with the following equation: A = A530 − (0.24 × A657) , (Gould et al., 2000) where A530 is the absorbance at 530 nm and A657 is the absorbance at 657 nm. Total anthocyanin content was calculated as mg Cyanidin-3glucoside equivalents per 100 g of fresh weight by the following equation: , (Giusti and Wrolstad, 2001) where TAC = A×MW×V×DF×100 ␧×100 A = absorbance, MW = molecular weight (449.2 g/mol), DF = dilution factor, and ␧ = molar absorbance coefficient (26,900 L/mol/cm). All measurements were done in triplicate.

2.9. DPPH free radical scavenging activity The hydrogen atom or electron-donation ability of the F. carica extracts was determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical as a reagent according to the method of Braca et al. (2001) with minor modification. Briefly, 3 ml of solution of DPPH radical in methanol was mixed with 1 ml of fig juice extract at different concentrations (2–20 mg/ml). The mixture was shaken vigorously and incubated for 30 min in the dark at room temperature before measuring the absorbance. The scavenging capacity was determined spectrophotometrically by monitoring the decrease in absorbance at 517 nm against a blank using a spectrophotometer (Bio-Rad SmartSpec Plus). The percent DPPH scavenging effect was calculated using the following equation: DPPH scavenging effect(%) =

Acontrol − Asample Acontrol

× 100

where Acontrol is the absorbance of the control reaction containing all reagents except the tested compound. Asample is the absorbance of the test compound. The results were reported as EC50 value, the effective concentration of antioxidant agent (extract) providing inhibition 50% of the initial DPPH radical concentration. EC50 was calculated from the graph-plotting inhibition percentage against extract concentration. The lowest EC50 value indicates the strongest ability of sample to act as an antioxidant. Ascorbic acid was used as an antioxidant standard. Tests were carried out in triplicate.

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2.10. Reducing power assay The reducing power of methanolic extracts of fig juices was determined by the method of Oyaizu (1986). Substances, which have reduction potential, react with potassium ferricyanide (Fe3+) to form potassium ferrocyanide (Fe2+), which then reacts with ferric chloride to form ferric ferrous complex that has an absorption maximum at 700 nm according to the following equation: Antioxidant

Potassium ferricyanidine + Ferric chloride−−−−−−→Potassium ferrocyanide + Ferrous chloride Different concentrations (2–20 mg/ml) of fig juice extract (2.5 ml) were mixed with sodium phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K3 Fe (CN)6] (2.5 ml, 1%). The mixture was incubated at 50 ◦ C for 20 min. After cooling, 2.5 ml of trichloroacetic acid (10%) were added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and a freshly prepared ferric chloride solution (0.5 ml, 0.1%). The absorbance was measured at 700 nm in a spectrophotometer. Increased absorbance of the reaction mixture indicates increase in reducing capability. The percentage increase in reducing power was calculated using the following equation: Increasing in reducing power(%) =

A test − A blank × 100 A blank

where A test is absorbance of test solution; A blank is absorbance of blank. Control was prepared in similar manner excluding samples without adding standard or test compound. Ascorbic acid was used as standard at the same concentrations of the samples. Reducing power was measured by varying the concentration of the extract and the contact time. 2.11. Statistical analyses For all the experiments three samples of each fig juice were analysed and all assays was carried out in triplicate. Data analyses were performed using SPSS software version 22, Duncan multiple range test at p < 0.05 probability level. Principal component analysis (PCA) was carried out using XLSTAT (2014) for Windows (Addinsoft, New York, USA). 3. Results and discussion 3.1. Phytochemical composition in (peel, pulp, total fruit) fig juices of different varieties The phytochemical composition defined by the total polyphenol (TPC), total flavonoid (TFC), total ortho-diphenol (TOPC), total tannin (TTC) and total anthocyanin (TAC) contents of peel, pulp and total fruit juices from three ripe figs (2nd crop) varieties was estimated. The total phenolic content (TPC) of juices extracts is shown in Fig. 3. For the black fig, peel juice showed a high TPC, followed by total fruit and pulp. Values varied between 50.57 GAE/g FW and 74.16 mg GAE/g FW. Concerning purple fig, the amount of TPC was higher and near in all juices, especially for peel and total fruit juices (61.47 mg GAE/g FW and 63.11 mg GAE/g FW.). This may suggest that peel is responsible of the higher content of fruit TPC. However, for Bidhi variety (green fig), amount of TPC was the lowest and close in the three juices (p > 0.05) comparing to other varieties. Similar data were reported by Faleh et al. (2012) who found that green varieties of dry fig have higher phenolic content than red ones, but discorded with those founded by Solomon et al. (2006)

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who reported that total phenolic content of Kadota pulp (green variety) was significantly higher compared with peel. Among varieties, dark fruits (black and purple figs) showed higher polyphenols content than the light ones (green fig). Differences observed were statistically significant (p < 0.05). Flavonoids are important for human health because of their high pharmacological activities as radical scavengers (Bakar et al., 2009). The total flavonoid content (TFC) is mentioned in Fig. 4 are expressed in terms of (mg CE/g FW). Peel of Kohli variety showed the highest concentration of flavonoids (12.75 mg CE/g FW). Meanwhile, in Hamri variety flavonoids were concentrated in total fruits (11.30 mg CE/g FW). However, in Bidhi variety, flavonoids exhibited the lowest concentration compared to the other varieties with similar amounts in different juices parts. Statistically significant differences in ortho-diphenol content (TOPC) were found between dark and light varieties (p < 0.05) (Fig. 5). TOPC in the peels ranged from 8.81 mg hydroxytyrosol/g FW for Kohli variety to 1.45 mg hydroxytyrosol/g FW for Bidhi variety; in the total fruits from 6.97 mg hydroxytyrosol/g FW for Kohli variety to 1.74 mg hydroxytyrosol/g FW for Bidhi variety and in the pulps from 3.92 mg hydroxytyrosol/g FW for Kohli to 2.44 mg hydroxytyrosol/g FW for Bidhi variety. Total ortho-diphenol content were concentrated differently according to variety: for Kohli in peel, for Hamri in total fruit and in pulp for Bidhi variety. Total tannin content (TTC) was found being usually more abundant in Bidhi variety (75.98 mg TAE/g FW in peel, 65.87 mg TAE/g FW in pulp and 60.35 mg TAE/g FW in total fruit) than in Kohli and Hamri ones (Fig. 6). All differences observed between varieties for all samples studied were statistically significant. For Kohli and Bidhi varieties, TTC were concentrated in peel. However in Hamri variety, they were abundant in the total fruit. Total anthocyanin content (TAC) was ranged from 429.80 mg cyanidin-3-glucoside/100 g FW (total fruit) to 162 mg cyanidin3-glucoside/100 g FW (pulp) for Hamri and from 344.89 mg cyanidin-3-glucoside/100 g FW (pulp) to 144.62 mg cyanidin-3glucoside/100 g FW (peel) for Bidhi (Fig. 7). Among varieties, our results showed that Hamri and Bidhi varieties had the lowest anthocyanin content (p < 0.05). Peel and total fruit of Kohli variety hold both the higher total anthocyanin content with respectively 3330.84 mg cyanidin-3-glucoside/100 g FW and 1972.47 mg cyanidin-3-glucoside/100 g FW. Our results are similar to those of Piga et al. (2005) who indicated that anthocyanins are found mainly in the peel, except for certain types of red fruit, in which they also occur in the pulp (cherries and strawberries). A significant difference (p < 0.05) between pulp juice and the other parts juices was observed only for Kohli variety. In summary, dark figs in particular, Kohli fig (black variety) had a significantly higher content of four phytochemical classes: TPC, TFC, TOPC and TAC, compared with the green one; with peel being the major contributing part. TPC, TFC, TOPC and TAC contents were significantly different among the three vegetal materials, following the order peel > total fruit > pulp for black fig and the order total fruit > peel> pulp for purple fig. Our results are also consistent with those of C¸alis¸kan and Polat (2011) who reported that some purple and black fig fruits, from Turkey, contained 2.5-fold higher TPC and 15-fold higher TAC than the green and yellow ones. Same observation was shown with Italian fig fruits (Del Caro and Piga, 2008) and Turkish fig fruits (Solomon et al., 2006) where black figs were mentioned to have higher amounts of polyphenols and anthocyanins than the green ones and those amounts were concentrated in the fig peel. The significant difference between peel and pulp content has also been previously found in other consumed fruits, such as nectarines, peaches and plums (Tomás-Barberán et al., 2001) and was mainly related in the anthocyanin content. Anthocyanins are pigments they impart a pink, red, blue, or purple color in the epidermal tissues of flowers and fruit. In human diet, they are found

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Fig. 7. Bar graphics comparing total anthocyanins content (TAC) in juices of different fig compartments. Results are expressed as means ± standarddeviation (n = 3). Bars with different small letters within one variety are significantly different (p < 0.05) according to Duncan test. Bars with different capital letters within the three varieties are significantly different (p < 0.05) according to Duncan test.

in some cereals, leafy, root vegetables and mostly in fruits (Mazza and Miniati, 1993). It was proposed that cyanidin was the sole anthocyanidin skeleton in fig fruits (Solomon et al., 2006). Puech et al. (1975) suggested the presence of three anthocyanins in dark fig fruits, namely, cyanidin-3-rhamnoglucoside, cyanidin 3,5diglucoside, and pelargonidin 3-rhamnoglucoside, with cyanidin3-rhamnoglucoside being the predominant pigment in ripe peels. Recently, (Del Caro and Piga, 2008) reported that cyanidin 3-Orutinoside was present in both peel and pulp of black fig, whereas, cyanidin 3-O-glucoside was dominated in the peel of black fig only. Different expression of genes controlling the anthocyanin pathway may result in differences in fruit color, with the highest expression associated with the dark varieties. Furthermore, (Oliveira et al., 2009) reported the high amount in fig peel of the flavonoid quercetin 3-O-rutinoside compared to fig pulps and leaves. Data indicate that chemical composition is dependent on the fruit part and the variety, thus extracts of darker varieties namely peel, showed higher contents of phytochemicals compared to lighter colored extracts.

3.2. Antioxidant activity in (peel, pulp and total fruit) juices of different fig varieties The antioxidant capacity was evaluated using two commonly used colorimetric methods namely, 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity and reducing power (RP) assay.

3.2.1. DPPH radical scavenging activity The 1,1-diphenyl-2-picrylhydrazyl DPPH is usually used as a reagent to evaluate free radical scavenging activity of antioxidants (Oyaizu, 1986). In the DPPH assay, the antioxidants were able to reduce the stable radical DPPH to the yellow colored diphenylpicrylhydrazine. Radical scavengers decolorize the colored DPPH• , so the loss of absorbance at 517 nm reflects radical scavenging activity. This study attempted to compare the DPPH free radical (DPPH• ) scavenging activity of different juices of fig fruit parts tested (Fig. 8). The scavenging effect was compared to ascorbic acid such as a standard. The free radical scavenging activity is expressed as percentage of DPPH• inhibition and by the antioxidant concentration required for a 50% of radical reduction (EC50), so that a lower value of EC50 indicated a higher antioxidant activity and vice versa (Table 1).

Table 1 EC50 values obtained in DPPH antioxidant assay of different fruit parts juices of three fig varieties with three different phenotypes. Phenotypes

Peel (mg/ml)

Pulp (mg/ml)

Total fruit (mg/ml)

Black Purple Green

4.52 ± 0.71a 9.71 ± 0.83c 26.7 ± 1.92e

15.45 ± 0.98d 12.99 ± 0.74d 10.59 ± 0.74c

8.63 ± 0.15c 7.04 ± 0.15b 14.6 ± 0.62d

Values are expressed as mean ± SD (n = 3) of triplicate measurement. Means with different letters are significantly different (p < 0.05).

There was a dose-dependent relationship in DPPH• scavenging activity of all three parts (peel, pulp, total fruit) juices of three fig varieties within the range of concentrations from 2 to 20 mg/ml. All samples were proven having antioxidant activities with significant differences (p < 0.05) between all juices extracts. The peel juice extract of black fig with a mean value of 4.55 mg/ml for EC50 was almost near to EC50 of ascorbic acid (2.21 mg/ml) (Fig. 8a, Table 1). The total fruit juice extract EC50 of purple variety (mean value 6.96 mg/ml) was lower than those of peel (mean value 9.73 mg/ml) and pulp (mean value 12.98 mg/ml). The juice extract of the purple total fruit was found to act as a stronger secondary antioxidant (Fig. 8b, Table 1). The peel juice extract of green fig present the lowest DPPH• scavenging activity (Fig. 8c) and the highest EC50 value (Table 1) so that the weakest antioxidant capacity (12–14 times less active than ascorbic acid). Antioxidant activity against DPPH was correlated with the concentration, chemical structure, polymerization, and degree of antioxidants (Kumaran and Karunakaran, 2007; Oszmianski et al., 2007).

3.2.2. Reducing power assay The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. In this assay, the yellow color of the test solution changes to various shades of green and blue depending on the reducing power of each compound (Chung et al., 2002). As shown from Fig. 9, fig juices of different parts had effective and powerful reducing power when compared to the standard ascorbic acid. At 20 mg/ml, peel and total fruit juices extracts of Kohli fig, as well as total fruit and peel juices extracts of Hamri fig demonstrated the highest and the nearest absorbance to those of ascorbic acid at the same concentration. Absorbance values were ranged from 3.36 (Kohli peel) to 2.33 (Kohli total fruit) versus 4.17 for the ascorbic acid. Differences observed between peel, pulp and total fruit of each variety were statistically significant (p < 0.05) started from 15 mg/ml. The reducing properties in foods are associated with reductones (Duh, 1998; Jayaprakasha et al., 2001). The

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phenolic compounds present in fig juices may act in a similar fashion as reductones by donating electrons and quenching free radicals. To conclude, it was found that at the same higher concentration, the extracts cited previously had always the powerful antioxidant activities, measured either by reducing power test or by DPPH test, probably because the phenolic compounds in figs react similarly within two methods. Tomás-Barberán et al. (2001) suggested the fact that the varied radical scavenging activity of extracts depended on the amount of total phenolic in each fraction. The antioxidant capacity is highly correlated with the presence of anthocyanins too (Shiow and Lin, 2000). The relative contributions of anthocyanins to the overall antioxidant capacities were estimated in previous study to 28 and 36% in dark fig fruit (Solomon et al., 2006). Peel of green fig, and the pulp of black fig, maintain both the last positions in the antioxidant capacity scale. Black peel holds the higher antioxidant potential. This study is in agreement with other

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studies suggesting that common fruits and vegetables with dark blue or red colors have the highest antioxidant capacity (Liu et al., 2002; Wu et al., 2006; C¸elik et al., 2008). Our results are consistent with those of Solomon et al. (2006) who reported that dark-colored figs exhibited the highest antioxidant capacity. Fig varieties with dark peel contain higher levels of polyphenols, flavonoids anthocyanins, and accompanied by higher anti-oxidant activity compared with fig variety with light peel. Peel of black fig might be rich sources of natural antioxidants. While eating the fruit, consumers tend most often to remove the peel; however fruit peels are clearly the major source of phenolic compounds, acting as antioxidants that should not be discarded; in fact, the consumption of total ripe fruits, fresh or as a juice is recommended. A part the presence of antioxidants, fig juice is a rich source of carbohydrates and organic acids (Oliveira et al., 2009), known for their health promoting effects. Its consumption may contribute to its protective role against diseases related to oxidative stress.

Fig. 8. DPPH free radical scavenging activity at different concentrations (2–20 mg/ml) of a reference antioxidant: ascorbic acid and different fig parts juices of three varieties in Tunisia. (a) Kohli, (b) Hamri, (c) Bidhi. Values performed in triplicate.

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Fig. 9. Reducing power (RP) of different concentrations (2–20 mg/ml) of a reference antioxidant: ascorbic acid and different fig parts juices of three varieties in Tunisia. (a) Kohli, (b) Hamri, (c) Bidhi. The reducing power was estimated based on the absorbance reading at 700 nm. Values performed in triplicate.

3.3. Changes in total phytochemical composition during fruit development The phytochemical composition consisting of the total polyphenol (TPC), total flavonoid (TFC), total ortho-diphenol (TOPC), total tannin (TTC) and total anthocyanin (TAC) levels was compared between 1st crop (partially mature fruit) and 2nd crop (mature fruit) figs. Phytochemical composition level increased in all ripe fruits compared with partially ripe ones. Results are shown in Fig. 10. Amounts of polyphenol and flavonoid contents show a significant increase (p < 0.05) between two crops. Total polyphenol content ranged from 48.0 mg GAE/g FW (Bidhi) to 70.9 mg GAE/g FW (Hamri) in partially ripe fruit and from 88.5 mg GAE/g FW (Bidhi) to 153 mg GAE/g FW (Hamri) in ripe fruit. Total flavonoid content ranged from 6.7 mg CE/g FW (Bidhi) to 9 mg CE/g FW (Hamri) in partially ripe fruit and from 12.2 mg CE/g FW (Bidhi) to 24.7 mg CE/g FW (Hamri) in ripe fruit. A significant (p < 0.01) increase in TFC of Kohli variety was shown. Ripe figs are 2 times richer in polyphenols than partially ripe ones and 2–3 times richer in flavonoids. Total orthodiphenol content values ranged from to 3.8 mg hydroxytyrosol/g FW (Bidhi) to 16.0 mg hydroxytyrosol/g FW (Kohli) for ripe fruit and from 0.3 mg hydroxytyrosol/g FW (Bidhi)

to 4.1 mg hydroxytyrosol/g FW (Hamri) for partially ripe fruit. Total tannin content values ranged from 45.5 mg TAE/g FW (Kohli) to 134.4 mg TAE/g FW (Bidhi) for ripe fruit and from 14.0 mg TAE/g FW (Kohli) to 21.0 mg TAE/g FW (Bidhi) for partially ripe fruit. Results showed that TTC content in total fig fruit was 3 and 6 times higher in ripe fig than in partially ripe ones, respectively for Kohli and Bidhi varieties, however, the increase is very marked (p < 0.01) in ripe fruit of Hamri variety where TTC achieved an amount of 109.73 mg TAE/g FW. Total anthocyanin content in partially ripe fruits was 1150.9 mg cyanidin-3-glucoside/100 g FW for Kohli and was approximately similar and low in Hamri and Bidhi varieties (respectively 637.5 mg cyanidin-3-glucoside/100 g FW and 529.8 mg cyanidin3-glucoside/100 g FW). Total anthocyanin content increased, with different degrees, in all ripe fruits compared with unripe ones. Hamri and Bidhi fruits of 2nd crop were 1.5 times richer in anthocyanins than 1st crop fruits. Kohli ripe fruits were 4 times richer in anthocyanins than Kohli partially ripe fruits. In fact, Kohli variety showed the highest anthocyanin content in ripe fruits (4447.1 mg cyanidin-3-glucoside/100 g FW) with a significant increase (p < 0.01) compared to partially unripe figs. However, there was not a significant difference in anthocyanin amounts between the partially ripe and rip figs of the purple (Hamri) and the light (Bidhi) varieties were TAC values remain very low (respectively

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Fig. 10. Bar graphics comparing total polyphenols content (TPC), total flavonoids content (TFC), total tannins content (TTC), total O-diphenols content (TOPC) and total anthocyanins content (TAC) between ripe fruit (2nd crop: August crop) and partially ripe fruit (1st crop: July crop) in three fig varieties. Results are expressed as means ± standard deviation (n = 3).

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Fig. 11. (a) Principal components analysis (scores and loading plots, biplot) based on different phytochemical compounds analyzed in peel juices of three Tunisian fig varieties and their antioxidant activity (%DPPH and reducing power (RP)). TPC: total polyphenols content; TOPC: total O-diphenols content; TFC: total flavonoids content; TTC: total tannins content; TAC: total anthocyanins content. (b) Principal components analysis (scores and loading plots, biplot) based on different phytochemical compounds analyzed in pulp juices of three Tunisian fig varieties and their antioxidant activity (%DPPH and reducing power (RP)). TPC: total polyphenols content; TOPC: total O-diphenols content; TFC: total flavonoids content; TTC: total tannins content; TAC: total anthocyanins content. (c) Principal components analysis (scores and loading plots, biplot) based on different phytochemical compounds analysed in total fruit juices of three Tunisian fig varieties and their antioxidant activity (%DPPH and reducing power (RP)). TPC: total polyphenols content; TOPC: total O-diphenols content; TFC: total flavonoids content; TTC: total tannins content; TAC: total anthocyanins content.

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949 mg cyanidin-3-glucoside/100 g FW and 697.1 mg cyanidin-3glucoside/100 g FW). The variety (Oliveira et al., 2009) and the maturity stage interact between each other to affect fruit properties (Crisosto et al., 2010). Many changes develop during fruit ripening such as, peel colour, antioxidant capacity (Toor and Savage, 2005) and physicochemical properties (Alkarkhi et al., 2011) in particular acidity decrease (Wu et al., 2007; Crisosto et al., 2010; Zhang et al., 2010), sugar increase (Wu et al., 2007) and aromatic composition (Trad et al., 2012; Braga et al., 2013), therefore the production of phytochemicals is also influenced. Total phenolic (Josefa et al., 2006) and total anthocyanin (Shiow and Lin, 2000) contents were shown increased in ripe fruits. However, the present results are not consist with those of Kubola and Siriamornpun (2011) who mentioned that fruit total phenolic and total flavonoid contents decreased during the fruit development stage. Fruit ripening process is always accompanied by a change in fruit softening and texture. Enzymes are shown to have a role in fruit ripening. Expression of enzymes involved in the degradation of many different cell wall polymers present in different fig tissue is coordinated both in time and amount with the fruit development (Owino et al., 2004). Peroxidase, polyphenol oxidase (PPO), protease and carbohydrases are involved in mango peel ripening (Ajila et al., 2007). Differences in phytochemical content between the two crops can be explained by weather conditions too. At last of August, when 2nd crop was picked, environment was more warm, dry and sunny than at the beginning of July (1st crop), and so, was favorable for fruits growth and development. The sun-exposed peel of apples terminal fruit had higher anthocyanin levels than shaded peel (Awad et al., 2000). Results were in accordance with those of Veberic et al. (2008) for figs varieties from northern Mediterranean region who stated that the sun-shining weather in August is more favorable for figs growth than in September which is marked by cooler and moister weather. However, phenolic concentration levels reported were several times higher than those noticed in the present study. This could be explained by the difference of variety, climate and environment, as Tunisia is located in the south Mediterranean region. The influence of the region of cultivation (north or south) on the concentration of phenolics in bananas was confirmed (Méndez et al., 2003). The level of anthocyanin in the fruit depends on various factors, namely: species, varieties, growing conditions, seasonal variations, maturity index, processing methods, and storage conditions (Melgarejo et al., 2000; Özkan, 2002). In contrast, Vallejo et al. (2012) mentioned that fig fruit from a crop in (May–June) showed even further highest values of total phenolics comparing to a second crop in (August–September). However, this difference is significant in peels and not in the pulps of fig fruit. In general way, during the ripening period, weather conditions could be either favorable or stressful for the fruit growth. Production of flavonoids, classified as “environmental compounds” because of their production in direct response to environmental conditions, is dependent on ultraviolet light and CO2 levels (Daniel et al., 1999; Caldwell et al., 2005).

3.4. Chemometric analysis The chemometric analysis results generated three biplots showing groupings and subgroupings: the principal component analysis (PCA) (Fig. 11) was carried on the data to compare the phytochemical composition of peels (Fig. 11a), pulps (Fig. 11b) and total fruits (Fig. 11c) juices of three studied varieties of figs and to identify the factors influencing each one. The position of each variable in the loading plot describes its relationship with the other variables. Variables that are close to each other have high correlations.

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The first PCA (Fig. 11a) accounted 87.65% of the total variance (99.25%), the second PCA (Fig. 11b) accounted 67.49% of the total variance (93.23%) and the third PCA (Fig. 11c) accounted 81.08% of the total variance (97.74%) on the first component while the second component accounted 16.68%. Comparisons between PCAs plots indicated that for three PCAs, PC1 was dominated by the following variables: TAC, TOPC, TFC and TPC that were correlated positively with Kohli and Hamri varieties and so they were the mainly responsible for the discrimination of dark peels. However, Bidhi group (green variety) was negatively associated with PC1 and positively with PC2 and was characterized by the abundance of tannins. Reducing power and DPPH groups were correlated positively with Kohli and Hamri groups for peel and total fruit PCAs’ with a slightly higher correlation for Kohli variety (Fig. 11a and b). Anthocyanin, flavonoid and polyphenol, concentrated in Kohli variety more than in Hamri one, seem to be the principal contributors to strong the antioxidant activity revealed by the reducing power and %DPPH scavenging activity. However, for PCA’s pulp, RP and DPPH were correlated negatively with PCA1 (Fig. 11c). Tannins provided by green figs were apparently responsible of the antioxidant capacity of fig pulps. Tannins are well known as potent antioxidants. The soluble tannins are gradually converted into non-soluble condensed form as the fruit begins to ripen and advances progressively (Myhara et al., 1999). Chemometric observations were consistent with those obtained during the evaluation of antioxidant activity of juices. The principal component analysis gave further information about the differences among the fig parts as well as their implication on the antioxidant activity revealed by the reducing power and %DPPH scavenging activity. Results clearly indicated that each variety could be distinguishable from the other. Phytochemical compounds accumulate differently depending on the fruit part and the variety.

4. Conclusion This is the first study comparing juices of F. carica peels, pulps and total fruits, contributing to more knowledge about the nutritional aspects of an important fruit of the Mediterranean diet. The content level of phytochemicals (TPC, TFC, TOPC, TTC, and TAC) is usually influenced not only by the variety, but also varies significantly from one fruit part to the other. Antioxidant capacity of fig seemed to be mostly related to the peel part and not the pulp part. Facts suggest that in a general way, the peel color allow distinguishing the fruit phenol and anthocyanin contents. The large amount of tannins contained in different compartments of green fig may cause its non-negligible antioxidant capacity. The phytochemical composition of fig including phenols, flavonoids, ortho-diphenols tannins and anthocyanins contents is influenced by the ripening stage and varieties of fig. Total polyphenol, total flavonoid, total ortho-diphenol, total tannin and total anthocyanin contents can be used as indicators of the antioxidant activity of foods. Results of this study would stimulate further researches on dark figs, especially; fig peel, which is a by-product, to be utilized a useful source of natural food preservative from a nutritional related point of view and other fig parts as a potential new source of natural antioxidants for food and pharmaceutical industries.

Acknowledgments This study was supported by a grant (to Arij Harzallah) from MOBIDOC device launched under the Supported Project to the research and Innovation System (PASRI), funded by the European

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Union and managed by ANPR, Tunisia. It is a part of collaboration with Sanofi Winthrop-Tunisia Pharmaceutical. This study is a part of research program of the Research Unit LR12ES05 “Nutrition-Functional food and Vascular Health LR-NAFS” and “DGRST-USCR-Mass Spectrometry” financed by the Ministry of Higher Education and Scientific Research (Tunisia).

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