Study of Physicochemicals Characteristics and Antioxidant Capacity of Cork Oak Acorns ( Quercus suber L . ) grown in Three Regions in Tunisia

Souad Belghith Igueld, Hatem Abidi, Malika Trabelsi-Ayadi, Jamila K. Chérif 1,3 Laboratory Applications of Chemical Resources, Natural Substances and Environment (LACReSNE), Faculty of Sciences of Bizerte, University of Carthage, 7021 Zarzouna-Bizerte, Tunisia. Preparatory Institute for Engineering Studies of El-Manar B.P.244 El Manar II 2092 Tunis, Tunisia. Preparatory Institute for Engineering Studies of Tunis, 2 rue Jawaharlal Nehru, Monfleury, 1008 Tunis, Tunisia.


INTRODUCTION
The Cork oak is an evergreen tree of the genus Quercus of the Fagaceae family formerly Cupulifère, it is best known for its thick bark used for making caps.The wood of the cork oak is used as charcoal or firewood due to its considerable calorific value of 7000 cal at 25% of moisture (Natividade, 1956).In Europe, Asia, North Africa, Middle East, and North America, acorns were once a staple (Brandis, 1972).They are also a valuable food for pets, birds and wildlife.Currently, cork oak acorns (Quercus Suber.L.) are traditionally marketed and consumed in North Africa.When dried and ground into flour, it can be transformed into a paste to use instead of bread, or it is used as a stomachic and antidiarrheal when mixed with honey (Sijelmassi, 1993).Epidemiological studies in Serbia have suggested that consumption of cork oak acorns may promote general human health (Jevtovic, 1980).Oak kernels (Quercus semen) were traditionally used in medicine, particularly roasted ones (Quercus semen tostum) as astringents, antidiarrhoeals and antidotes (Tucakov, 1971), since it has been established that a diet rich in fruits reduces the risk of cardiovascular diseases, cancer and other diseases caused by oxidative stress.A revaluation of acorns and their use would be desirable.The acorns of all species must be tested and adjusted for their antioxidant activity.Antioxidants are usually used to prevent chronic and degenerative diseases with intermediate scan of free radicals (Rodriguez et al., 2007;Prior and Gu, 2005).The role of free radical scavenging antioxidants has attracted much attention of not only scientists but also general public, above all, the natural antioxidants contained in fruits, vegetables, spices and dietary supplements (Sies, 2010).Domingues, Ammar et al. and Conde et al. cited several bioactive polyphenols, compounds such as flavonoids, tannins and ellagic acid and its diglucoside derivatives that are present in different parts of the cork oak (fruits, leaves and cork) (Domingues, 2009;Ammar et al., 2005;Conde et al., 1998).Flavonoids have not only demonstrated beneficial effects as antioxidants (Garcia-Saura et al., 2005) but also the ability to regulate the expression of adipocytokine gene (Tsuda et al., 2006).Ellagic acid is strongly believed to have antioxidant and antiviral activity.The composition of the cork oak acorns in tocopherol (α-tocopherol and β-tocopherol) are respectively 38 and 74 mg.kg -1 of dry matter (Cantos et al., 2003).Bainbridges (1986) confirmed that cork oak acorns are rich in carbohydrates, lipids, sterols and amino acids and are low in protein.Rodríguez-Estévez et al. (2008) have enabled to determine the composition of kernels and hulls in minerals, ash, fat, fiber, crude protein.Given the richness of the fruit in antioxidant and given the traditional use of the flour for bread making in parts of North Africa, this fruit can replace in case of famine, some seeds such 69as corn wheat or barley.Therefore, we have undertaken this work to explore antioxidant potential minerals and sugar composition of fruits of cork oak.The influence of harvest location and solvent extract was also studied.

Plant material
Samples that are subject of this study (1kg) were collected during the month of December 2011 in three regions of Tunisia Sejnen (202 m altitude), Nefza (500 m altitude) and Ain Drahem (800 m altitude) located in northern Tunisia.Acorns were cleaned to remove all foreign matter (dust, dirt, immature and damaged fruits).Three batches containing thirty fruits for each region were formed; the hull and kernel of fruit of each batch were then separated and cut into small pieces.All samples were preserved in the dark at a dry place until further use.

Physicochemical characterization
The moisture content was determined according to the method of Twidwell et al. (2002).The dry matter content was determined in accordance with French standard NF B 51-004 (AFNOR, 1986).A mass of 5 g of plant material was introduced into a porcelain crucible and dried in an oven at 105 ºC until a constant mass.The mineral matter content in the fruits of the cork oak was determined according to French standard T 211 OM-93.The formol index was evaluated in the fruit extract after soaking overnight.It was determined using the AFNOR method (V.76-102.1985).The titratable acidity was measured by the titrimetric method according to the Tunisian standard NT 52.15 (1982).Titration of free acidity of the aqueous extracts of all cork oak samples was determined by an assay using a sodium hydroxide solution NaOH (0.01 M) in the presence of phenolphthalein as a color indicator.

Ash content and mineral composition
The method NF.EN.1482(March 2003) was used: 5g sample was incinerated with high pressure in a muffle furnace (MLS 1200) for 24 h at 550 °C.The residue of incineration was extracted with 5 ml of HNO 3 65% and 50 ml of water.The mixture was heated a few minutes in a boiling water bath to dissolve the ash.After cooling, the solution was filtered and then placed in a 50mL volumetric flask, the volume is adjusted by adding distilled water, where Fe, Cu, Mn and Zn and other mineral composition were directly measured at the suitable wavelength for each element, using standard solutions for calibration purposes.The concentration of each analyte in the different samples was expressed as mg.100 g -1 of fresh material from their calibration curves taking into account the dilution factor.

Antioxidant Content Preparation of extracts
Different solvent systems were used to evaluate the best extraction solvent on the extraction of phenolic compounds: S 1 methanol/water (80:20, V/V), S 2 ethanol/water (50:50, V/V); S 3 methanol/acetic acid (99:1).100 mg of fresh samples were extracted by 10 mL of different solvents.The supernatant was centrifuged (2500 rev.min -1 ) filtered through Whatman N o 40 (Whatman International England).

Total Phenolic Content
The amount of total phenols was determined using the Folin-Ciocalteu reagent according to the method of Singleton and Rossi Jr (1965), and 1 mL of the extract was added to 5 mL of distilled water and 1 mL of Na 2 CO 3 (20% ; W/V).After 3 min, 1 ml of the Folin-Ciocalteu reagent was added and mixed.After 30 min of incubation in the dark at 40 °C, the absorbance of all samples was measured at 765 nm using the Shimadzu UV-Vis spectrophotometer T60U.The results were expressed in terms of mg of equivalent of Gallic acid per gram of sample, using a calibration curve of the gallic acid.

Total Flavonoid Content.
The total flavonoid content in Quercus Suber was determined according to Zhishen et al ( 2009) using a method based on the formation of complex flavonoid-aluminium.A volume of 125 µL of methanol extract was added to 75 μL of a solution of NaNO 2 (5%; W/V).The mixture was left to stand for 6 minutes, then 150 μL of a solution AlCl 3 6H 2 O (10 %) freshly prepared were added, after 5 min of rest, 500 μL of NaOH (solution 1M) was added to mixture.The final volume of the solution was adjusted to 2500 μL with distilled water.The absorbance was measured at 510 nm (Shimadzu UV-Vis spectrophotometer T60U).The total flavonoid content of acorn was expressed in terms of catechin equivalent (mg.100 g -1 ).

Total Anthocyanin Content
The total anthocyanin contents were determined according to the procedure of Swain and Hillis (1959).5 g of acorn were submitted to extraction with 10 mL of ethanol (80%; V/V) for 2 hours with agitation (magnetic stirrer).Then the samples were filtered.The filtrates were completed to a total volume of 10 mL with 80% aqueous ethanol.Then 0.02 mL of HCl (37%) was immediately added.The absorbance was read at 525 and 595 nm to quantify anthocyanins using the Shimadzu UV-Vis spectrophotometer T60U.The total anthocyanin contents were quantified according the relation: where A 1 is the absorbance measured at 525 nm, A 2 measured at 595 nm, 0.879 is the extinction coefficient of anthocyanins.Ve: is the total volume recovered after extraction 100 g sample, and F is the dilution factor

Determination of total Carotenoid Contents
The total carotenoid contents were determined according to the procedure described by Rousseff et al. (1987).200 mg of fresh samples were extracted by 10 ml of acetone-water (80:20; V/V), then allowed to stand for 30 min at 4 °C.The extract was filtered through Whatman No. 40.The organic phase obtained was extracted repeatedly with hexane (3×10mL).The combined extracts were subjected to a rotary evaporator (Laborota 4000 Germany) under reduced pressure at 30 °C.The absorbances of all samples were measured at 450 nm using the Shimadzu UV-Vis spectrophotometer T60U.The results were expressed in equivalent β-carotene, compared to a range standard prepared starting from a mother ethanolic solution of β-carotene.

Evaluation of Antioxidant Activity
The free radical-scavenging activity was determined using stable 2,2-diphenyl-1-picrylhydrazyl (DPPH • ) radical according to Brand Williams et al (1995). 2 mg of each sample were dissolved in 1mL of methanol.An aliquot of this solution was mixed with 1 mL of 0.5 mM DPPH in methanol and the final volume adjusted up to 5 mL, so that final concentrations of the samples were in a range from 1,25 to 20 μg.mL -1 .Then, the mixtures were shaken vigorously and left 30 min to stand in the dark.The reduction of DPPH radical is accompanied by a color change from violet (DPPH • ) to yellow (DPPH-H).The absorbance of mixture was measured using a spectrophotometer at 517 nm in a Shimadzu UV-2401 PC spectrophotometer using methanol as blank, after establishing the stationary.One milliliter of 0.5 mM DPPH diluted in 4 mL of methanol was used as control.The yellow color of solution indicates the end of the reaction.This reduction capacity is determined by a decrease in absorbance induced by free radical inhibitors (Majhenic et al, 2007).Antioxidant activity was expressed as percentage of inhibition in relation to control according to the equation proposed by Yen and Duh (1994): (%) = 100.( 0 − )/ 0 , where A 0 is the absorbance of the control (containing all reagents except the tested sample), and As is the absorbance of the tested sample.The effectiveness of acorn extracts in scavenging free radicals was evaluated as the concentration of acorn extracts in the reaction mixture that caused a decrease in the initial DPPH concentration by 50%, defined as IC50.These values were calculated from the graph plotting inhibition percentage against extract concentration.2.6.Sugar content.Soluble sugars were determined by High Performance Liquid Chromatography HPLC method AOAC 980-13 982-1 (1990), using a refractive index detector (RID) (Perkin Elmer).The separation was carried out on a SGE SS Exsil amino column NH 2 (150 x 4.6mm I.D.).The elution solvents used were acetonitrile and water (82:18 v/v).The column was operated at 25 ºC with 1 mL/min flow rate.Sample injection volume was 5 μL.The resultant peak areas in the chromatograms were plotted against calibration curves obtained from multiple standard solutions (external standard method), in a concentration range of 1 mg.mL -1 for each compound.All the experiments were run in triplicate.The mean values and standard deviations were calculated using the Microsoft Excel software (Microsoft Corporation, Redmond, WA).

Physicochemical characteristics
Some chemical properties of cork used in this study are presented in table 1.As can be seen from these data, the moisture content in the samples ranges from 38% to 51%.The highest values are those of Ain Drahem where rainfall exceeds 1559 mm per year.There is the weak acidic character in acorns, the measurable acidity varies between 5.2 x 10 -3 and 7.1 x 10 -3 mmol.L -1 .The kernels have higher acidity than the hulls.Formaldehyde indexes were similar, however, the lowest value of the acidity and formol index was observed in Sejnan variety.The mineral content of cork fruits is presented in table 2. Based on these results, the fruits are rich in calcium, magnesium, sodium and especially in potassium (6.942 g.kg -1 ).Besides potassium, the other minerals are present in the hulls.These results were similar to those reported by Özcan and Bayçu (2005) for potassium content (7.849 g.kg -1 ) of fruit of the Turkish variety Quercus petraea subsp.Several epidemiological studies have established a link between a diet rich in potassium and a reduced risk of hypertension (Houston and Lanham-New, 2008); stroke and renal calculus (Bazzano et al, 2001;Curhan et al,1993).In studies that have established these relationships, this contribution was closely associated with the consumption of fruits and vegetables which contain other beneficial compounds for health (He and Mac Gregor, 2008).WHO (2005) recommends increasing dietary potassium intake to lower blood pressure and reduce the risk of cardiovascular disease, stroke and coronary heart disease in adults.

Carotenoids
The content of carotenoids has been quantified (Tab.3).The results concerning the hulls of acorn of the three regions studied show high levels of carotenoids (13.19 to 30.16 mg EβC.100 g -1 DM), kernels are less rich in carotenoids.

Anthocyanins
The anthocyanin contents of the studied acorns are low (0.05 to 0.13 mg.100g -1 ), with hulls having maximum values.The low values observed could be explained by the immature acorns studied which are collected in December.In spring, acorns stain red or orange which is accompanied by an influx of anthocyanins.Indeed, light, temperature, wind and humidity are factors involved in the rate of anthocyanins.

Flavonoids
Were present in very small quantities in all investigated samples, with higher content in kernel of acorn of Sejnan (0.49 mg ECat.g -1 ).This is in agreement with the results of some studies conducted in Spain in 2009 by Domingues et al. on cork oak fruits that showed a content of 0.6 mg ECat.g -1 .

Total Phenolic Content
The total polyphenol contents of the extracts obtained by the three solvents S 1 , S 2 and S 3 are listed in Table 4. Significant  differences were observed, the amounts of total phenolics in cork oak fruits extracts for different parts of cultivars show that the methanol 1% acetic acid provide the highest levels varied widely, ranging from 77 to 49 mg of GAE.g -1 DM.The lowest levels of total polyphenols were recorded for the extracts obtained from ethanol/water (42.9 to 19 mg of GAE.g -1 DM).This study reported the potential effects of extracting solvent on antioxidant activity estimations for acorns.The methanol 1% acetic acid is the recommended solvent to prepare antioxidant extracts from acorns samples.We observe that for all the extraction solvents used, the kernel has higher polyphenol contents that the hulls.The highest content of polyphenols was obtained from Sejnan plant extract.Additional research is required to investigate the influence of extraction solvent on the chemical composition of acorns antioxidant extracts.

Evaluation of Antioxidant Activity
The evaluation of the antioxidant capacity of the samples was carried out by determining the concentrations required to inhibit stable radical by 50% (IC50), which are shown in Table 3.It can be noted that all the extracts showed appreciable free radical scavenging activities in the DPPH assay.However, kernel extracts (IC50 = 6.94-8.11)inhibit DPPH • radical more rapidly than hulls extracts (IC50 = 9.9 -10.3).This is deduced by the results obtained by comparing the IC50 (Figures 1(a  Our results are in agreement with some previous investigations concerning types of Quercus acorns polyphenols and their antioxidant activity.Rakić et al. in 2007 have also suggested Quercus robur and Quercus cerris to be convenient nutritional components, with significant antioxidant effects (resp., IC50 = 8.04 and IC50 = 8.88).
Gallic acid, digallic acid and gallotannin were identified in the ethyl-acetate fraction of Q. acutissima acorns and caused its high antioxidant efficacy (Lee et al, 1992).The obtained results have provided further grounds for establishing Q. suber acorn kernels as a source for functional food preparation

Correlation between Total Polyphenol Content and Anti-radical Activity
The phenolic contents and flavonoid contents of kernel was higher than hulls, and the kernel exhibit good antioxidant activity.A positive correlation (r = 0,925) was found between the free radical scavenging DPPH (IC50) and the total phenolic compounds content.The polyphenols in extracts of cork oak acorns are probably responsible for the anti-radical activity of these extracts.The capacity of free radical scavenging of antioxidant does not always correlate with the capacity of antioxidation such as lipid peroxidation inhibition.The antioxidation capacity can be accessed from the extent of suppression of lipid peroxidation by antioxidant compared with that in its absence.It is determined not only by the capacity for scavenging free radicals but also by the localization of antioxidant, interaction with other antioxidants, and mobility of antioxidant at the microenvironment.These factors should be considered to understand the action and capacity of antioxidants (Niki, 2010).

Sugars
The cork oak acorns are the sweetest and most popular oak acorns, we therefore measured and identified sugars in the Tunisian variety by high performance liquid chromatography HPLC.
Soluble sugars identified and quantified in the acorns are listed in Table 5. Fructose and glucose are the major kinds of sugar while sucrose and maltose are present in smaller quantities.The lower amounts of sucrose found in these fruits may be due to the enzymatic hydrolysis of this sugar into glucose and fructose during ripening process.The kernel of acorns contains more sugars (4.55 % to 7.08% by dry weight) than the hull (0.735 to 1.28%).Acorns grown in Sejnan have the highest sugar levels.The sugars are transformed under the action of invertase, sucrose is converted by enzymatic hydrolysis in fructose (levulose) and glucose (dextrose) at the stage of maturation.Fruits with a high invertase activity, such as prickly pear fruit, kaki or pomegranates, reach a glucose/fructose ratio near 1 (Ayaz et al, 2000).A ratio higher than 1 has been found in some wild fruits, such as wild redbilberries (Vaccinium vitis-idaea L.) or blackthorn fruits (Crataegus monogyna Jacq.), which usually have more than 3-fold more glucose than fructose (Barros et al, 2010;Souci et al, 2008).On the contrary, all oak acorns samples analyzed in this study showed an inverse behavior, with a ratio glucose/fructose between 0.30 and 0.55, which means that fructose content represented twice glucose content.Moreover, this profile can be related to the intense and pleasant sweet taste of oak acorns when they are completely ripe, since fructose is the sweetest of all the naturally occurring carbohydrates (Hanover and White, 1993).

CONCLUSION
This study conducted on the fruit of the Tunisian cork oak has allowed to determine its physical and chemical characteristics and mineral composition.We observe the richness of this plant in minerals (Ca, K); it can therefore compete with wheat, rice and corn.
This plant showed the presence of phenolic compounds and showed a certain level of antioxidant activity in the kernels as well as in the hulls of acorns.It revealed the presence of four sugars, fructose and glucose being the main sugars.
The results showed that the samples with the highest levels of polyphenols have the lowest IC50.Thus, the antioxidant power of the extracts studied is mainly due to the polyphenols.Great variability in antioxidant and sugar contents of these acorns was observed depending on geographic location, stage of maturation, climate and nature of the extraction solvent.
This natural product can be used as an inexpensive source of natural antioxidants and mineral supplements, especially for food industry which is expanding fast nowadays.Further investigations such as isolation and characterization of the active compounds polyphenols and carotenoids are currently in progress.

Table 1 :
Some chemical properties of cork oak varieties.HuA -fruit hull of Ain drahem, HuS-fruit hull of Sejnan KeN -fruit kernel of Nefza, KeA -kernel of Ain drahem, KeS -fruit kernel of Sejnan

Table 2 :
Mineral content of acorn cork oak expressed as mg/kg of fresh mater.

Table 3 :
Bioactives extract content in hulls and kernels of cork oak.

Table 4 :
Antioxidant activity and total phenolic content of acorn cork oak

Table 5 :
Contents of sugars in hulls and kernels of the cork oak (% DM; mean±SD, n=3)