Characterization of fig achenes’ oil of Ficus carica grown in Tunisia

Characterization of fig achenes’ oil of Ficus carica grown in Tunisia

Accepted Manuscript Characterization of fig achenes’ oil of Ficus carica grown in Tunisia Hala Soltana, Meriem Tekaya, Zahra Amri, Sinda El-Gharbi, Am...

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Accepted Manuscript Characterization of fig achenes’ oil of Ficus carica grown in Tunisia Hala Soltana, Meriem Tekaya, Zahra Amri, Sinda El-Gharbi, Amel Nakbi, Arij Harzallah, Beligh Mechri, Mohamed Hammami PII: DOI: Reference:

S0308-8146(15)30054-6 http://dx.doi.org/10.1016/j.foodchem.2015.10.053 FOCH 18248

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

10 July 2015 6 October 2015 11 October 2015

Please cite this article as: Soltana, H., Tekaya, M., Amri, Z., El-Gharbi, S., Nakbi, A., Harzallah, A., Mechri, B., Hammami, M., Characterization of fig achenes’ oil of Ficus carica grown in Tunisia, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.10.053

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Characterization of fig achenes' oil of Ficus carica grown in Tunisia Hala Soltana1,*, Meriem Tekaya1 , Zahra Amri1, Sinda El-Gharbi1, Amel Nakbi1, Arij Harzallah1, Beligh Mechri1, Mohamed Hammami1

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Biochemistry Laboratory, LR12ES05 "Nutrition- Functional Foods and vascular Health",

Faculty of Medicine, University of Monastir (Tunisia)

* Corresponding Authors: Hala Soltana Tel: +21673462200 Fax: +21673460737 Email Address: [email protected]

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Abstract This work investigated the composition of the oil extract from achenes of "Kholi" variety of Ficus carica, grown in Tunisia. Fatty acid and sterol compositions were analysed by gas chromatography (GC) coupled to flame ionisation detector (FID). Furthermore, the antioxidant capacity in fig achenes' oil was assessed by employing two different in vitro assays such as DPPH., ABTS.+ radical scavenging capacities. Our results indicated that the fig achenes' oil is a rich source of bioactive molecules. The soxhlet n-Hexane extraction of these achenes produced a total oil yield of 16.24%. The predominant fatty acid was linolenic acid. Concerning phytosterols, the total amount reached 1061.45 mg/100g with a predominance of ∆5,23-Stigmastadienol (73.78%). Regarding antioxidant activities, The half maximal inhibitory concentration (IC50) was 215.86 µg/ml and Trolox equivalent antioxidant capacity (TEAC) was 95.25 mM. These data indicate that fig achenes oil of Ficus carica could be potentially useful in food and pharmaceutical applications.

Keywords: Ficus carica, Achenes oil, Antioxidants, Fatty acids, Sterols, Antioxidant activity

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1. Introduction Currently, worldwide interest is oriented for the recovery and exploitation of oils from natural plant resources. Vegetable oils with a high relative amount of minor lipid components are of great importance for human health (Carvalho, Miranda, & Pereira, 2010). Plant phenolics and sterols (Phytosterols) are secondary metabolites playing several physiological roles in plants. Phenols are recognized for their high antioxidant activity and phytosterols are known for their capacity to decrease the levels of plasma cholesterol (Conforti, Modesto, Menichini, Statti, Uzuvo, & Solimene, 2011; Vallverdú-Queralt, Medina-Remón, Martínez-Huélamo, Jauregui, Andres-Lacueva, & Lamuela-Raventos, 2011). Besides, phenols contribute to the color and sensory characteristics of fruits and vegetables, and are used as chemical markers (Conforti et al., 2011; Vallverdú-Queralt et al., 2011). Nowadays, plant achenes constitute new oil sources, especially the achenes of Ficus carica. Little is known about the biochemical composition of achenes of Ficus carica. Its fruits have high phenolic antioxidant concentrations, and antioxidants from these fruits can protect lipoproteins in human plasma from oxidation and produce a significant increase in plasma antioxidant capacity after consumption (Vinson, Zubik, Bose, Samman, & Proch, 2005). When fig fruits are processed to give juice and puree, substantial waste material that contains high levels of achenes is generated. This processing waste could be a potential source of nutraceuticals instead of being fed to livestock or sent to sanitary landfill. To be able to evaluate fig achenes as a source of natural antioxidants, it is important to know their biochemical composition and antioxidant properties. While the polyphenolic composition and antioxidant properties of ficus carica have been the subject of several investigations,

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quantitative and qualitative comparisons of their distribution in the flesh and achenes are missing. The present paper aims to investigate for the first time the contents of principal antioxidant compounds such as total phenols, tocopherols, flavonoids, orthodiphenols, chlorophylls and carotenoids, and also the acidic and sterolic compositions of achenes oil of "Kholi" variety of Ficus carica. The antioxidant activity of the phenolic extract from the oil achenes was also evaluated using its capability to scavenge the free-radical DPP· and ABTS test. The potential dietary importance of their achenes was then discussed. 2. Materiel and methods 2.1. Standards and reagents Reagents and solvents were supplied by Sigma-Aldrich (Steinheim, Germany). The Folin–Ciocalteu reagent was purchased from Merck Schuchardt OHG, Hohenbrunn, Germany. The standards of sterols and fatty acids were obtained from Sigma-Aldrich (USA). DPPH (2, 2- Diphenyl-1-picrylhydrazyl), 2,6-ditert-butyl-4-hydroxy-boxylic acid (BHT), 2,2'azinobis

(3-ethylbenzothiazoline-6-sulfonate)

(ABTS),

and

6-hydroxy-2,5,7,8-

tetramethylchroman-2-carboxylic acid (Trolox), were also purchased from Sigma-Aldrich (Chemie Gmbh, Steinhein, Germany). 2. 2. Plant Materiels Fig fruits (Ficus carica) of variety "Kholi" were collected at full maturity from the region: Mahdia located at the Central East of Tunisia (Altiude: 7m; Latitude: 35°30'16" N; Longitude: 11°03'43" E). The samples were peeled and immediately frozen at -20 °C until extraction. After that, the pulpes were partly thawed (2 h at room temperature) before blending in a food processor. The achenes were then separated from the pulpes by pressing through a strainer. The collected achenes were rinsed several times with running, cold water

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and left to dry at room temperature. Dried fig achenes were powdred and stored at -20°C before extraction. 2. 3. Achenes oil extraction The oil was extracted from the achenes powder with n-hexane in a Soxhlet extractor (Quichfit, England) for 5 h. The organic phase was then removed using a rotary evaporator under reduced pressure. The oils were flushed with nitrogen stream and stored at -20°C in sealed tubes. Results were expressed as the percentage of lipids in the dry matter of achenes powder. 2.4. Extraction of the phenolic fraction The extraction was carried out with the same conditions reported by Pirisi, Cabras, Falqui, Migliorini, & Mugelli (2000). Briefly, 2 ml of n-hexane and 4 ml of a methanol/water (60:40, v/v) solution were added to 4 g of oil sample. After vigorous mixing, they were centrifuged for 3 min at 1490g. The hydroalcoholic phase was collected, and the hexanic phase was re-extracted twice with 4 ml of methanol/ water (60:40, v/v) solution each time. Finally, the hydroalcoholic fractions were combined, washed with 4 ml of n-hexane to remove the residual oil, then concentrated and dried by evaporative centrifuge (Mivac Duo of Genevac Inc., Valley Cottage, NY, USA) in vaccum at 35 °C. 2.5. Determination of total phenol and o-diphenol contents Total phenolic and o-diphenol contents were determined according to the method of Montedoro, Servili, Baldioli, & Miniati (1992) with minor modifications. For total phenols, 0.4 ml of oil extract and 10 ml of diluted Folin–Ciocalteu reagent were mixed. After 1 min incubation, 8 ml of sodium carbonate (75 g/l) was added and the mixture was incubated for 1 h. The absorbance was measured at 765 nm. The same extract was used to determine total odiphenols, as follows: 1 ml of a solution of HCl (0.5 N), 1 ml of a solution of a mixture of NaNO2(10 g) and NaMoO4·2H2O (10 g) in 100 ml H2O and finally 1 ml of a solution of 5

NaOH (1 N) were added to 100 µl of the extract. After 30 min, the absorbance was measured at 500 nm. The total phenols and o-diphenols were expressed on a dry weight basis as mg hydroxytyrosol equivalents/kg of sample. 2.6. Determination of total flavonoids Total flavonoid contents (TF) of the oil extracts were determined according to the colorimetric assay developed by Zhishen, Mengcheng, & Jianming (1999). One ml of properly diluted extract was mixed with 4 ml of distilled water. At zero time, 0.3 ml of (5% w/v) NaNO2 was added. After 5 min, 0.3 ml of (10% w/v) AlCl3 was added. At 6 min, 2 ml of 1 M solution of NaOH were added. Finally, the volume was made up to 10 ml, immediately by the addition of 2.4 ml of distilled water.The mixture was shaken vigorously and the absorbance was read at 510 nm. The results were also expressed on a dry weight basis as mg catechin equivalents (CEQ)/Kg of oil sample. 2. 7 Chlorophylls, Pheophytin-a and Beta-Carotene Contents The total chlorophyll content was calculated at 630, 670 and 710 nm, using carbon tetrachloride as blank and a spectrophotometer (Perkin Elmer Lambda 25). The calculation of the total chlorophyll content is as follows (Kiritssakis et al., 1998):

Chlorophyll (mg/ kg oil) =

ಲలయబశಲళభబ ቁ మ

஺଺଻଴ିቀ

଴.ଽ଴ଵ×௅

where A is the absorbance of the oil at the respective wavelength and L is the cell thickness (cm). Pheophytin-a, an important compound from chlorophyll pigments, was calculated according to the equation:

Pheophytin-a (mg/Kg oil) =

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ଷସହ.ଷ×[஺଺଻଴ି ௅

ಲలయబశಲళభబ ] మ

where A is the absorbance of the oil at the respective wavelength and L is the cell thickness (mm) as described by Psomiadou and Tsimido (2001). Beta-carotene was measured with a UV–visible spectrophotometer (Perkin Elmer Lambda 25) at wave lengths between 440 and 480 nm, according to the method previously described by Dhibi et al. (2014). The β-carotene content was expressed using the following equation: β carotene = A ‫ ג‬max × (105 / 2.650) A ‫ ג‬max : maximum of absorption between 440 and 480 nm 2.8. Gama-Tocopherol Determination Gama-Tocopherol was extracted according to the method of Gimeno, Castellote, Lamuela-Raventós, de la Torre, & López-Sabater (2000). HPLC analysis was carried out on a Hewlett-Packard system (Waldbronn, Germany) comprising an HP-1100 pump, a Rheodyne model 7725 injector (Cotati, CA, USA, loop volume 20 µl), an UV detector (292 nm) and a SUPELCOSIL LC-18-DB column (15 cm × 4.6 mm × 5 µm) thermostated at 45 °C. The mobile phase consisted of methanol/water (96:4, v/v) at a flow rate of 2 ml min-1 during 18 min. The data were stored and processed by an HPLC Chemstation (Dos Series) (Hewlett-Packard). γ-Tocopherol was quantified by the internal standard method. Results are given as µg of γ-tocopherol per gram of oil. 2.9. Fatty acid composition Fatty acid composition was determined by gas chromatography (GC) after conversion to fatty acid methyl esters (FAMEs) with 2 M KOH in methanol at room temperature according to the IUPAC standard method (1992). Analytical gas chromatography was carried out on a Hewlett–Packard 6890 gas chromatograph series II (Agilent Technologies, Palo Alto, California, USA) equipped with VARIAN CP-SIL88 (50 m × 0.25 mm, 0.25 mm film thickness) capillary column. One µl of each sample was injected with a split ratio of 1:200

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and a continuous flow rate of 0.7 ml/min of chromatographic grade helium was used. The oven temperature was initially held for 13,5 min at 175 °C, ramped at 2°C/min up to 185 °C and held isothermal for 3 min. Injector and FID detector temperature were held at 220 and 280°C. FAMEs were identified by comparison of their retention time with respect to pure standards analyzed under the same conditions. FAMEs were quantified according to their percentage area in the lipid fraction. 2.10. Sterols analysis The analysis of sterols was conducted according to the method descriped by Lukic, Lukic, Krapac, Sladoja & Pilizota (2013). α-cholestanol (0.2%) (Supelco, Bellefonte, USA), used as internal standard, was added to 5g of fig achenes oil and were saponified with a potassium hydroxide ethanolic solution. After boiling, water was added and the extraction of the unsaponifiable fraction was carried out with diethyl ether. Following purification with water and drying over sodium sulphate, diethyl ether was evaporated under vacuum and nitrogen. The unsaponifiable fraction was dissolved in chloroform, and approximately 20 mg were loaded on a basic silica TLC plate (Fluka, Buchs, Switzerland). The sterol fraction was separated by elution with a mixture of hexane and diethyl ether 65:35 (v/v). The corresponding band was visualised under UV light after being sprayed with a 2 ',7'dichlorofluorescein 0.2% ethanolic solution, than scraped off with a spatula, and extracted with chloroform and diethyl ether. After the extract was evaporated to dryness, sterols were converted to trimethylsilyl ethers by the addition of pyridine-hexamethyldisilizane-trimethylchlorosilane (9:3:1, v/v/v) (Supelco, Bellefonte, USA), left for 15 min, and then centrifuged. Identification and quantification of sterols as trimethylsilyl ethers was carried out by capillary gas chromatography on a Varian 3350 GC (Varian Inc., Harbour city, USA) equipped with a 30 m × 0.25 mm i.d. × 0.25 µm film thickness Varian VF-5 ms capillary column (5% phenyl:95% dimethylpolysiloxane) and a flameionisation detector. Injector, oven 8

and detector temperatures were 280, 260 and 290°C, respectively, for 40 min. One µl was injected in split mode (1:50). Helium was used as a carrier gas with a flow rate of 1.27 ml/min. Thirteen sterols (cholesterol, Brassicasterol, 24-methylene-cholesterol, campesterol, campestanol, stigmasterol, ∆7-campesterol, ∆5,23-stigmastadienol, clerosterol, β-sitosterol, sitostanol, ∆5-avenasterol, ∆5,24-stigmastadienol,) in oil were identified based on their relative retention times with respect to the internal standard, cholestanol, according to the standardized reference method (EEC, 1991, Annexes V and VI). Sterol concentrations were expressed as mg/100 g of oil with respect to internal standard, assuming a response factor equal to one. Relative amounts were expressed as proportions (%) of total sterols. 2.11. Antioxidant activities 2.11.1. DPPH Radical Scavenging Assay The capacity to scavenge the free radical 2, 2- diphenyl-1-picrylhydrazyl (DPPH) was monitored according to the method described by Bouaziz, Grayer, Simmonds, Damak & Sayadi (2005).The extract solution (0.25 mL) was mixed with 0.5 mL of methanolic solution containing DPPH radicals (6 × 10 -6 M). The mixture was shaken vigorously and left to stand for 30 min at room temperature in the dark (until stable absorbance values were obtained). The reduction of the DPPH-radical was measured by continuous monitoring of the absorption decrease at 517 nm. DPPH scavenging effect was calculated as the percentage of DPPH discoloration using the following equation: % scavenging effect = [(ADPPH − AE)/ADPPH] × 100 where AE is the absorbance of the solution when the sample extract is added at a particular level, and ADPPH is the absorbance of the DPPH solution. The extract concentration providing 50% inhibition (IC50) was calculated from the graph of scavenging effect percentage against extract concentration in the solution. 2.11.2 ABTS Assay 9

The free-radical scavenging capacity was measured using the ABTS decoloration method described by Re, Pellegrini, Proteggente, Pannalla, Yang, & Rice-Evans (1999) with some modifications. Briefly, ABTS radical (ABTS.+) was produced by reacting a solution of the ABTS (7 mM) with a 2.45 mM K2S2O8 solution and allowing the mixture to stand in the dark at room temperature for 12 to 16 h before use. The ABTS.+ solution was diluted with methanol to an absorbance of 0.70 ± 0.02 at 730 nm. After the addition of 100 µL of sample, methanol as a blank, or Trolox standard to 2.9 mL of diluted ABTS.+ solution, absorbance readings were taken after 6 min. Methanolic solutions of known concentrations of Trolox were used for calibration. This standard curve was linear between 0.2 and 0.8 mM of Trolox (y = 0.6792x). The results were expressed in Trolox equivalent antioxidant capacity (TEAC; mM). The results of the present work were represented as mean values ± standard deviation of tree replicate analyses. 3. RESULTS AND DISCUSSION 3.1 Pigment contents in fig achenes' oil Chlorophylls, pheophytins and β-carotene are the pigments present in vegetable oils. The presence of various pigments depends on different factors, such as the fruit ripeness, the plant cultivar, the climatic conditions and the type of soil, and the extraction procedures (Nehdi, Sbihi, Tan, & Al-Resays, 2014). Ficus carica achenes oil contains a notable amount of β-carotene (81.08 mg/kg), but much lower contents of chlorophylls (1.05 mg/kg) and pheophytins (32.97 mg/kg) (Table 1), which justifies the yellow color of the achenes oil. Carotenoids are not only precursors of vitamin A but are also known as primary antioxidants by preventing free radical chain reaction (Akoh, & Min, 2008). In foods such as vegetable oils, carotenoids are typically

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secondary antioxidants by quenching singlet oxygen. Furthermore, at low oxygen pressures carotenoids can neutralize free radicals and act as primary antioxidants in vitro. 3.2. Phenolic contents in fig achenes oil The total phenolic, flavonoid and o-diphenol contents of oil achenes of Ficus carica were determined by spectrophotometric means. These compounds constitute the major class of secondary plant metabolites with bioactive potential attributed to their antioxidant activity (Ahmad et al., 2011). Table 1 showed the concentrations of phenols, o-diphenols and flavonoids in the oil achenes extract. The oil extract has 144.89 mg/kg of phenolic content, and 47.27 mg/kg of odiphenols. Total flavonoids content was 124.5 mg /kg. To our knowledge this is the first work that studied the contents of these secondary metabolites in fig achenes' oil. Similar work has been done on other different plant achenes. Aaby, Skrede, & Wrolstad (2005) showed an average value of 3.6 g/100g FW of flavonoids in F. x ananassa achenes manually separated from freeze-dried berries. Tekaya et al. (2013) reported that the content of olive oil from Picholine variety was 140.89 mg/kg. Sevim and Tuncay (2013), in their study on olive oil from Ayvalık and Memecik varieties in the year of 2009, reported that total phenol contents were 122.34 and 154.98 mg CAE/kg, respectively. The above results suggest that, in comparaison with olive oil, the oil of variety "Kholi" achenes shows also a wealth of phenolic compounds. Thereby, this oil may have positive effects on health and can be used as a food supplement. 3.3. Determination of Gama-tocopherol content Tocopherols play an important role in the quality of vegetable oils because of their antioxidant activity, being more efficient at relatively lower concentrations. HPLC analysis showed that γ-tocopherol was the predominant isomer in fig achenes oil. As shown in Table 1, the γ-tocopherol content in fig achenes oil was 2.23 µg/g. In the same vein, Pande et al.

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(2010) found that γ-tocopherol and α-tocopherol are the two tocopherol isomers present in fig fruit with 0.3 and 0.2 mg/100 g, respectively. γ-Tocopherol can have equal or stronger antioxidant properties than α-tocopherol, and foods containing high concentration of γtocopherol such as nuts are considered to be able to lower the risks of cardiovascular diseases (Li, Tsao, Yang, Kramer, & Hernandez, 2007; Saldeen, Li, & Mehta, 1999). 3.3. Fatty acids composition Total lipid content, expressed as percentage on dry weight basis (dw%) was 16.24 dw % on fig achenes of "Kholi" variety. The fig achenes oil showed a high unsaturated/ saturated fatty acid ratio of 7.62 (Table 2). The high unsaturated/saturated value of Ficus carica achenes oil is due to its low content (11.6 %) of saturated fatty acids (SFA), whose deleterious effects on low-density lipoprotein (LDL) cholesterol have been previously demonstrated (Rivellese et al., 2003), and a higher content of unsaturated fatty acids (88.39%) which is higher than that (~70-85%) in vegetable oils from many important oily seeds, such as sunflower, soybean, peanut, and sesame (Namiki et al., 1995). The two major fatty acids were linolenic acid (47.77%) and cis-linoleic acid (25.48%), followed by cis-oleic (14.7%), palmitic (8.3%), and stearic (2.94%) acids. Additionally, the F.carica achenes oil contains also gadoleic, palmitoleic and arachidic acids, but in much smaller quantities (0.15–0.29%). Trace amounts (<0.1%) of margaric, trans-oleic, tanslinoleic and margaroleic acid were also detected. In other studies, Chua et al. (2008) showed also that the predominant fatty acids in neutral lipids of oil bodies isolated from jelly fig achenes were linolenic acid (62.65%), linoleic acid (18.24%) and oleic acid (10.62%), with a total of 91.51% unsaturated fatty acids. α-Linolenic acid, a member of omega-3 polyunsaturated fatty acids and the dietary essential fatty acid (Burdge and Calder, 2005), may protect against coronary artery disease (Wijendran and Hayes, 2004). In addition, linoleic acid has become increasingly popular in the industry of

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beauty products for its beneficial properties on the skin, such as antiinflammatory, acne reduction, and moisture retention (Darmstadt et al., 2002; Letawe, Boone, & Pierard, 1998). Harris et al. (2009) in a recent review from the American Heart Association Nutrition Commission, showed evidence that PUFA lowers cardiovascular risk and reinforced that their consumption should be encouraged; therefore, we suggest that fig achenes oil, with its high PUFA/SFA ratio of 6.32 may be recommended as dietetic oil. 3.4. Phytosterol composition Sterols are important nonglyceridic constituents of oils and are widely used to check authenticity (Bouaziz, Fki, Jemai, Ayadi, & Sayadi, 2008). Every vegetable oil has a specific sterol composition. Thus, they have a great importance in adulteration detection (Feinberg, Favier, & Ripert, 1987; Gordan and Griffith, 1989). These chemical components are also used for varietal characterizations (Castang, Olle, Derbesy, & Estienne, 1976) and are reported to be indicators of the best period of harvest (Fiorino and Nizzigrifi, 1991). The fig achenes oil is very rich of sterols (table 3). The total sterol content is 1061.45 mg/100g. To our knowledge, this is the first work which studied the sterol composition of achenes oil and the results of our study confirm the high nutritional value of the achenes of Ficus carica, since sterols are known to decrease the risk of certain types of cancer and enhance immune function (Bouic et al., 2001). Phytosterols are also known to reduce serum low density lipoprotein (LDL)-cholesterol level, and food products containing these compounds are widely used as a therapeutic dietary option to reduce plasma cholesterol and atherosclerotic risk (Calpe-Berdiel, Escol-Gil, & Blanco-Vaca, 2009). For Ficus carica achenes oil, the most abundant sterol components present in the sterol fraction are ∆

5,23

-

Stigmastadienol (73.78%) and β-sitosterol (10.12%). These are followed by 24-Methylenecholesterol (4.89%) and campestanol (3.95%) in abundance. All other sterols are present with amounts lower than 3.5% (Table 3). This composition is strongly different with that of latex

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and leaves of other characterised F.carica cultivars. In fact, they are predominantly composed of β-sitosterol as suggested Jeong and Lachance (2001); Oliveira, Silva, Andrade, Valentão, Silva, & Gonçalves (2010). Also, Oliveira et al. (2012) found that the leaves of Ficus carica cultivars contained high amounts of Lanosterol. 3.5. Antioxidant activity Antioxidants have recently become a topic of increasing interest to health and food science researchers and medical experts (Ben Mansour, Poter, Kite, Simmonds, Abdelhedi, & Bouaziz, 2015). The antioxidant activities of the fig achenes oil extract were measured (DPPH and ABTS) and compared to that of commercial synthetic antioxidants such as BHT (IC50 = 9.12 µg/mL, TEAC = 1.59mM). The results are shown in Table 4. The lowest IC50 values indicated the highest free radical scavenging activity of the sample. Consequently, the fig achenes oil extract showed a strong antioxidant activity and was the most effective with an IC50 value of 215.86 µg/ml and TEAC value of 95.25 mM. This high antioxidant activity of the fig achenes oil extract may be attributed mainly to its richness in phenolic content (144.89 mg/kg). In a previous wok of Obeid, Bedgood, Prenzler, & Robards (2007), the effect of phenolic compounds in preventing radical scavenging was studied and it is generally assumed the ability of these compounds to act as hydrogen donors. 4. Conclusion To our knowledge, the present work is the first study which investigated biochemical composition of extracted oil from "Kholi" fig achenes. The studied fig achenes seem to be quite rich in lipids. Biochemical analyses of oil showed that it contains very high levels of carotenoids and phytosterols. Fatty acid composition demonstrated the predominance of the linolenic acid known for its several beneficial properties. Fig achenes oil is also a valuable source of antioxidant compounds such as phenols and tocopherols. As these minor compounds are known to have a wide range of beneficial biological activities and physical

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properties, the oil from fig achenes confirms its nutritional value and dietary importance. The results of our study open new prospects for the valorization of substantial waste material generated from fig fruit processing, as a dietetic and nutritional product, and also may be exploited in the industry of beauty products. Acknowledgments The authors wish to thank M. Imed cheraief for his help in GC/MS analysis. This study was supported by the Ministère de l’Enseignement Supérieur, Ministère de la Recherche Scientifique de la technologie et de développement des compétences (UR03/ES08 Nutrition et Désordres Métaboliques) and CRDA de Mahdia. References Aaby, K., Skrede, G., & Wrolstad, R. E. (2005). Phenolic composition and antioxidant activities in flesh and achenes of strawberries (F. x ananassa). Journal of Agricultural and Food Chemistry, 53, 4032–4040. Ahmad, R., Hashim, H. M., Noor, Z. M., Ismail, N. H., Salim, F., Lajis, N. H., & Shaari, K. (2011). Antioxidant and Antidiabetic Potential of Malaysian Uncaria. Research Journal of Medicinal Plant, 5, 587-595. Akoh, C. C., & Min, D. B. (2008). Food Lipids, Chemistry, Nutrition and Biotechnology, CRC Press, Boca Raton. Ben Mansour, A., Poter, E. A., Kite, G. C., Simmonds, M. S. J., Abdelhedi, R., & Bouaziz, M. (2015). Phenolic Profile Characterization of Chemlali Olive Stones by Liquid Chromatography-Ion Trap Mass Spectrometry. Journal of Agricultural and Food Chemistry, 25,63(7), 1990-5. Bouaziz, M., Fki, I., Jemai, H., Ayadi, M., & Sayadi, S. (2008). Effect of storage on refined and husk olive oils composition stabilization by addition of natural antioxidants from chemlali olive leaves. Food chemistry, 108, 253-262.

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Bouaziz, M., Grayer, R. J., Simmonds, M. S. J., Damak, M., & Sayadi, S. (2005). Identification and Antioxidant Potential of Flavonoids and Low Molecular Weight Phenols in Olive Cultivar Chemlali Growing in Tunisia. Journal of Agricultural and Food Chemistry, 53, 236−241. Bouic, P. J. (2001). The role of phytosterols and phytosterolins in immune modulation: a review of the past 10 years. Current Opinion Clinical Nutrition and Metabolic Care, 4, 471–475. Burdge, G. C., & Calder, P. C. (2005). Conversion of alpha-linolenic acid to longerchain polyunsaturated fatty acids in human adults. Reproduction Nutrition Development, 45, 581-597. Calpe-Berdiel, L., Escol-Gil, J. C., & Blanco-Vaca, F. (2009). New insights into the molecular

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Atherosclerosis, 203, 18–31. Carvalho, I. S., Miranda, I., & Pereira, H. (2010). Evaluation of oil composition of some crops suitable for human nutrition. Industrial Crop Production, 24, 75–78. Castang, J., Olle, M., Derbesy, M., & Estienne, J. (1976). Composition de la fraction stérolique de quelques huiles alimentaires. Extrait des annales des falsifications, 737, 1-29. Chua, A. C. N., Jiang, P. L., Shi, L. S., Chou, W. M., & Tzen, J. T. C. (2008). Characterization of oil bodies in jelly fig achenes. Plant Physiology and Biochemistry, 46, 525-532. Conforti, F., Modesto, S., Menichini, F., Statti, G. A., Uzunov, D., & Solimene, U. (2011). Correlation between environmental factors, chemical composition, and antioxidative properties of caper species growing wild in Calabria (South Italy). Chemistry & Biodiversity, 8, 518–531.

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Table 1 Mean values of the contents of antioxidant compounds (phenols, o-diphenols, flavonoids and pigments) in fig achenes oil of Kholi variety. Concentrations Phenols (mg /kg) 144.89 ± 4.16 Flavonoids (mg /kg) 124.5 ± 2.83 O-diphenols ( mg /kg) 47.27 ± 1.20 β-carotene(mg/ Kg) 81.08 ± 0.39 Chlorophylls (mg/Kg) 1.05 ± 0.31 Pheophytin-a (mg/Kg) 32.97 ± 9.84 γ-tocopherol (µg/g) 2.23 ± 0.26 Results are represented as mean values ± standard deviation of tree replicate analyses.

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Table 2 Fatty acid composition (%) of the studied fig achenes oil. Fatty acids Concentrations C16:0 8.3 ± 0.03 C16:1 0.17 ± 0.00 C17:0 0.07 ± 0.04 C17:1 0.03 ± 0.01 C18:0 2.94 ± 0.03 C18:1 trans 0.03 ± 0.02 C18:1 cis 14.7 ± 0.18 C18:2 trans 0.06 ± 0.01 C18:2 cis 25.48 ± 0.03 C18:3 47.77 ± 0.1 C20:0 0.29 ± 0.04 C20:1 0.15 ± 0.02 ∑ SFA 11.6 ± 0.03 ∑ UFA 88.39 ± 0.15 ∑ PUFA 73.31 ± 0.05 UFA/SFA 7.62 ± 0.05 PUFA/SFA 6.32 ± 0.04 Values are the means of the three replicates analyses (n=3) ± standard deviations. SFA: Saturated fatty acids; UFA: Unsaturated fatty acids; PUFA: Polyunsaturated fatty acids.

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Table 3 Concentrations (mg/100 g) and relative amounts (%) of sterols determined in fig oil of ‘Kholi’ variety. Sterols

Cholesterol Brassicasterol 24-Methylene-cholesterol Campesterol Campestanol Stigmasterol

Contents Concentrations (mg/100 g) 3.01 ± 0.01 0.49 ±0.02 51.9 ± 0.01 0.18 ± 0.00 41.92 ± 0.00 2.5 ± 0.12

∆7-Campesterol

5.48 ± 0.02

∆ 5.23-Stigmastadienol Clerosterol β-Sitosterol Sitostanol

783.10 ± 0.01 2.33 ± 0.00 107.37 ± 0.01 13.55 ± 0.01

∆5-Avenasterol

12.04 ± 0.13

∆5.24-Stigmastadienol Totals sterols

Cholesterol Brassicasterol 24-Methylene-cholesterol Campesterol Campestanol Stigmasterol

37.58 ± 0.01 1061.45 ± 0.04 Relatives amounts (%) 0.28 ± 0.01 0.05 ± 0.01 4.89 ± 0.03 0.02 ± 0.02 3.95 ± 0.00 0.24 ± 0.03

∆7-Campesterol

0.52 ± 0.02

5.23

∆ -Stigmastadienol Clerosterol β-Sitosterol Sitostanol

73.78 ± 0.01 0.22 ± 0.01 10.12 ± 0.03 1.28 ± 0.00

∆5-Avenasterol

1.13 ± 0.04

5.24



-Stigmastadienol

3.54 ± 0.02

Values are the means of the three replicates analyses (n=3) ± standard deviations.

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Table 4.

Radical-Scavenging and ABTS Activities of fig achenes oil extract compared to those of BHT IC50 (µg/mL) (DPPH assay) TEAC (mM) (ABTS assay) fig achenes oil 215.86 95.25 BHT 9.12 1.59

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Highlights * The fig achenes' oil is a rich source of bioactive molecules. * The predominant fatty acid in fig achenes'oil of variety ''Kholi" is linolenic acid. * The sterol analysis of achenes' oil shows the predominance of ∆5,23-Stigmastadienol. * The oil from fig achenes confirms its nutritional value and dietary importance.

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