MATERIALS AND METHODS

Plant materials: Fruits and stems of the cultivar Chemchali of Olea europaea L. were collected from the south (Gafsa) of Tunisia. Olive fruits were isolated manually from the aerial parts in our laboratory to obtain a weight of 500-700 g of each part. The stems were separated from the other parts. Voucher specimens have been deposited in the Herbarium of the Laboratory of Biochemistry, Faculty of Medicine of Monastir, Tunisia.

Extraction method: Each sample (250 g) of the fresh fruits and stems was subjected to hydrodistillation for 3 h using a Clevenger-type apparatus. The volatiles obtained after trapping in diethyl ether were dried over anhydrous sodium sulphate, evaporated and concentrated under a gentle stream of N₂ and stored at 4°C until tested and chemically analyzed.

Identification of the volatile constituents: GC analyses were accomplished with an HP-5890 series II instrument equipped with a HP-5 capillary column (30 mx0.25 mm, 0.25 μm film thickness), working with the following temperature program: 60°C for 10 min, ramp of 5°C/min to 220°C; injector and detector temperatures, 250°C; carrier gas, nitrogen (2 mL min^-1); detector, dual FID; split ratio, 1:30; injection, 0.5 μL. The identification of the components was performed, by comparison of their retention times with those of pure authentic samples and by means of their Linear Retention Indices (LRI) relative to the series of n-hydrocarbons.

Gas chromatography-electron impact mass spectrometry (GC-EIMS) analyses were performed with a Varian CP 3800 gas chromatograph (Varain, Inc. Palo Alto, CA) equipped with a DB-5 capillary column (Agilent Technologies Hewlett-Packard, Waldbronn, Germany; 30 m = 0.25 mm; coating thicknessx0.25 mm) and a Varian Saturn 2000 ion trap mass detector. Analytical conditions were as follows: injector and transfer line temperature at 250 and 240°C, respectively; oven temperature was programmed from 60-240°C at 3°C/min; carrier gas, helium at 1 mL min^-1; splitless injector. Identification of the constituents was based on comparison of the retention times with those of the authentic samples, comparing their linear retention indices relative to the series of n-hydrocarbons and on computer matching against commercial (NIST 98 [U.S. National Institute of Standards and Technology] and ADAMS (Adams, 1995) and homemade library mass spectra built from pure substances and components of known samples and MS literature data (Stenhagen et al., 1974; Massada, 1976; Jennings and Shibamoto, 1980; Swigar and Silvestein, 1981; Davies, 1990; Adams, 1995). Moreover, the molecular weights of all the identified substances were confirmed by gas chromatography-chemical ionization mass spectrometry (GC-CIMS), using methanol as chemical ionization gas.

Antioxidant activity
DPPH radical scavenging assay: Radical scavenging activity was determined by a spectrophotometric method based on the reduction of a methanol solution of DPPH as reported by Blois (1958). One millititre of various concentrations of the volatiles in methanol was added to 1 mL of a 0.004% methanol solution of DPPH. The mixture was shaken vigorously and left to stand at room temperature for 30 min in the dark. Then the absorbance was measured with a UV-VIS a spectrophotometer (Secommam, U-1789, France), at 517 nm against a blank. Percent inhibition of the free radical DPPH, was calculated according to Equation:

I% = (A_blank-A_sample)/A_blankx100

where, A_blank is the absorbance of the control reaction (containing all reagents except the tested compound) and A_sample is the absorbance of the tested compound. Concentration providing 50% inhibition (IC₅₀) was calculated from the graph plotting inhibition percentage against sample concentration. Tests were carried out in triplicate. Butylated hydroxytoluene (BHT) was used as a positive control.

ABTS⁺radical cation scavenging: The ABTS⁺ radical cation scavenging activity of each volatile oil and ascorbic acid (control) was determined according to the literature (Yvonne et al., 2005). In brief, 5.0 mL of a 7.0 mM ABTS solution was treated overnight in the dark with 88.0 μL of a 140 mM potassium persulfate solution to yield the ABTS⁺ radical cation. Prior to use in the assay, the ABTS⁺ radical cation was diluted with ethanol to an initial absorbance of about 0.700 (ratio of 1:88) at 734 nm, with 30°C. Free radical scavenging activity was assessed by mixing 1.0 mL of diluted ABTS⁺ radical cation with 10 μL of sample and monitoring the change in absorbance after 0, 0.5 and 1 min and than after 5 min intervals, until a steady state was achieved. The antioxidant capacity of the volatile fractions was expressed as IC₅₀, the concentration necessary for 50% reduction of ABTS⁺.

Antibacterial and antifungal activities
Microbial strains: The employed bacterial strains were Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 27950, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27950. In the case of yeasts, Candida glabrata ATCC 90030, C. kreusei ATCC 6258, C. parapsilosis ATCC 22019 and C. albicans ATCC 90028.

Micro-well dilution assay: Minimum Inhibitory Concentration (MIC) values were determined by micro-titre plate dilution method (Sahin et al., 2004). The inocula of the bacteria and yeasts were prepared from 12 h broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. The volatile oils were first dissolved in 10% DMSO and then diluted to the highest concentration (10 mg mL^-1) to be tested and then serial twofold dilutions were made in 10 mL sterile test tubes containing nutrient broth.

In brief, the 96-well plates were prepared by dispensing into each well 95 μL of nutrient broth and 5 μL of the inocula. An aliquot (100 μL) from the stock solutions of volatiles initially prepared at the concentration of 10 mg mL^-1, was added into the first wells. Then 100 μL from their serial dilutions were transferred into each consecutive well. The last well containing 195 μL of nutrient broth without compound and 5 μL of the inocula on each strip was used as negative control. The final volume in each well was 200 μL. The plate was covered with a sterile plate sealer and then incubated for18 h at 37°C. The MIC was defined as the lowest concentration of the compounds able to inhibit the growth of microorganisms, after incubation. The results were expressed in microgram per milliliter (Smania et al., 2006; Zacchino, 2001).

Statistical analysis: All experiments were performed in triplicate. Data were recorded as means±standard deviations and were analyzed with SPSS (2003). Differences were considered significant at p<0.05.

RESULTS

Chemical composition of volatile oils: The volatile oil yields of Olea europaea L. var. chemchali varied between different parts with 0.90% MS in fruits and 0.92% MS in stems. GC/MS analysis of the volatiles of olive fruits and stems led to the identification of their components which are listed in Table 1 among their percentages and retention indices. The fruits volatile oil composition was characterized by high levels of 3-ethenylpyridine, (E)-2-decenal and 2-ethylbenzaldehyde with, respectively 12.5, 11.4 and 7.7%. Nonanal, (E)-2-decenal and benzyl alcohol were the main components in the stem volatile oil with, respectively 9.9, 9.6 and 9.0%. Other, representative compounds were found as nonanal (6.6%), (E,E)-2,4-decadienal (5.2%) and (Z)-2-heptenal (4.9%) in the fruit and phenol (8.0%), linalool (5.7%) and methyl salicylate (4.9%) in the stem. The minor fraction in the fruits contains other compounds whose rates do not exceed 1%; globulol, heptanal, (Z)-4-decenal, tetradecane, hexadecane and (E)-β-damascenone.

A total of 38 volatile c ompounds were detected, namely 3 esters, 8 alcohols, 2 terpenoids, 17 aldehydes, 3 ketones, 3 saturated hydrocarbons, 1 pyridine and 1 carboxylic acid (Table 1). The identified compounds in fruits and stems represented 88.5 and 73.6% of the total volatile oil, respectively. Quantitatively, alcohols and aldehydes were found to be the most important groups of volatiles in the fruit and stem of Olea europaea L. var. chemchali. The percentage of alcohols represented 15.7 for fruits and 19.9% for stems (Table 1). This class was characterized by the presence of appreciable relative percentage of phenylethyl alcohol (3.1%) in fruit and benzyl alcohol in stems (9.0%). The fruits of var. chemchali were characterized by the highest percentage of aldehydes (52.7%) because their important amounts of (E)-2-decenal (11.4%) and 2-ethylbenzaldehyde (7.7%) (Table 1). In comparison to fruits, the stems of the variety Chemchali are characterized by their superiority in total C6 aldehyde and total C6 alcohol (Fig. 1). The main ester was represented by methyl salicylate, produced in the stems in percentage of 4.9% (Table 1). In fact, the ester (Z)-3-hexenyl acetate was found only in the fruits. The highest relative percentage of terpenoids was found in stems (8.5%), with linalool (5.7%) as the most abundant component. However, the carboxylic acids and pyridine were present only in the fruits, with an important percentage of pyridine (12.5%).

Table 1:	Volatile compoundsa (%) evaluated by GC-MS in the fruit and stem samples from the olive cultivar grown in the area studied
aPercentages obtained by flame ionization detector (FID) peak area normalization (HP-5 column). bLinear retention indices (DB-5 column). cTraces (tr)<0.1%

Table 2:	Antioxidant activities of the volatile oils from fruit and stem of the Olea europaea L. cultivar chemchali
Values are the means of the three different samples (n = 3). *p<0.05; **p<0.01

Fig. 1:

Total alcohols, total C6 alcohols, total aldehydes and total C6 aldehydes in the volatile oils from fruit and stem of the Olea europaea L. cultivar chemchali

Antioxidant activity: The effects of antioxidants in the DPPH-radical-scavenging test reflect the hydrogen-donating capacity of a compound. When the radical form of DPPH is scavenged by an antioxidant through the donation of hydrogen to form a stable DPPH molecule, this leads to a colour change from purple to yellow and a decrease in absorbance. The DPPH-radical-scavenging activity of the volatile oils is shown in Table 2. Lower IC₅₀ value indicates higher antioxidant activity. The fruit (IC₅₀ = 2.63 mg mL^-1) showed higher scavenging ability on DPPH radicals than the stem (IC₅₀ = 3.54 mg mL^-1). DPPH scavenging abilities of the volatile oils from fruit and stem were lower than that of synthetic antioxidant BHT (IC₅₀ = 0.034 mg mL^-1). The same results were observed with the other assay, with the volatile oils from fruit and stem showed moderate activity (Table 2). The lowest EC50 value was detected in volatile oil from stem. These results were found to be statistically significant (p<0.05).

Table 3:	Antibacterial and antivities of volatile oils from fruit and stem of Olea europaea L. cultivar chemchali
aMIC: Minimum inhibitory concentration. b:Not active. c:Not determined. d:Control antifungal. e:Control antibiotic

Antibacterial and antifungal activity: The antibacterial and antifungal activities of the volatile oils from Olea europaea L. fruit and stem were assayed in vitro by a broth micro-dilution method against four pathogenic bacteria and four phytopathogen strains. Table 3 summarizes the microbial growth inhibition by each volatile oil and their combination effects. Minimal Inhibitory Concentration (MIC) values of the volatiles oils toward the selected bacteria and fungi were determined in mg mL^-1. The comparison with the control antibiotic, led to the conclusion that the volatiles were able to inhibit the bacterial and fungal growths but with different effectiveness. The antibacterial activity of the fruits was seen only against Bacillus cereus with a MIC value of 0.31 mg mL^-1. On the other hand, Staphylococcus aureus and P. aeruginosa showed best susceptibility towards the volatile oil of stem with a MIC value of 0.07 mg mL^-1 followed by Escherichia coli with a MIC value of MIC 0.62 mg mL^-1. As shown in Table 3, the antifungal activity against the phytopathogen strains of fruit and stem volatile oils were relatively good. For comparison, the stem of Olea europaea L. demonstrated higher activities against tested fungi.

DISCUSSION

In the present study, the fruit volatile oil of Olea europaea L. var. chemchali was rich in 3-ethenylpyridine, (E)-2-decenal and 2-ethylbenzaldehyde; therefore the stem volatile oil was rich in nonanal, (E)-2-decenal and benzyl alcohol. This result is not in accordance with Wannes et al. (2010), who showed that the volatile oils of the stems and fruits of two varieties of myrtle (Baetica and italica) are similar. The stems volatile oil had a difference qualitative and quantitative composition that of fruits. Data showed that the fruit of var. chemchali was characterized by the highest percentage of aldehydes because their important amounts of (E)-2-decenal; aldehyde typical from olive fruit flavorings; this bioactive compound is known for its antibacterial activity (Kubo et al., 1995). On the other hand, the stems of the variety chemchali are characterized by their superiority in total C6 aldehyde and total C6 alcohol. This result is confirmed by Angerosa et al. (2000) who showed that the proportions of the C6 aldehydes, C6 alcohols and their acetate (LOX products) are mainly dependent on different enzymatic reactions present in different tissues. These compounds are formed by the lipoxygenase pathway that is present in various tissues in plants and involves different reactions enzyme (Gargouri et al., 2008). Moreover, the ester (Z)-3-hexenyl acetate was detected only in the fruits. This ester is synthesized also by means of lipoxygenase (LOX) where the enzymatic reduction of aldehydes catalyzed by ADH, gives rise to the corresponding alcohols. These alcohols serve as substrates for Alcohol Acyltransferase (AAT) for producing esters (Bartley et al., 1985). Thus, it is important to note that the enzymatic activity is strictly dependent of organ of olive. Result showed the heterogeneous distribution of volatile compounds between the different olive parts.

Antioxidant activities of the volatiles oils from olive fruit and stem of the variety chemchali were tested by the DPPH and ABTS-radical-scavenging test. The volatile oils from fruit and stem showed moderate activity. The results showed that the volatile oil from fruit showed higher scavenging ability on DPPH and ABTS radicals when compared to those reported for stems. Volatiles oils are, from the chemical point of view, quite complex mixtures constituted by several tens of components and this complexity makes it often difficult to explain the activity pattern. Even many reports on the antioxidant potentials of the essential oils often refer to concepts such as synergism, antagonism and additivity. Both minor and major compounds should make a significant contribution to the oil’s activity (Wang et al., 2008). Moreover, trying to correlate the observed activity with the chemical composition of the oils, it is noteworthy to cite that considering the molecular characteristics of aldehydes present in a good amount in the volatile oil from fruit, these compounds may contribute to the observed antioxidant activity of the tested volatile oils as far as they were described by Ricci et al. (2005) to be implied in such activities.

The antibacterial and antifungal activities of the volatile oils from Olea europaea L. fruit and stem toward the selected bacteria and fungi were determined. For comparison, the volatile oils from fruit were less active against gram-negative bacteria than g-positive bacteria. This might be due to the protection by a hydrophilic outer membrane of the g-negative bacteria which suppressed the passage of the lipophilic volatile oil (Mann et al., 2000). The highest antibacterial activity in the volatile oil stem could, in part, be associated with the superiority of percentages of terpenoids in stem with comparison from fruit. As described previously by other authors, the essential oils containing terpenoids are active against bacteria (Djoukeng et al., 2005). Thus, the antifungal activity against the phytopathogen strains of fruit and stem volatile oils were relatively good. Compared with fruits, the stem of Olea europaea L. demonstrated higher activities against tested fungi and this is attributed to the high content of monoterpene alcohol, linalool (Pattnaik et al., 1997).

CONCLUSION

The present study provides data about the stems olive volatiles and reveals the interesting character of the Tunisian olive in its volatiles content. As far as it concerns the antioxidant and antimicrobial activities, it should be noted that the volatile oils from fruit and stem showed moderate activity; therefore the stem sample showed higher activities against tested fungi and bacteria than those of the fruit. This also underlines the importance of the ethnobotanical approach for the selection of organ olive in the discovery of new bioactive substances and places these species among the most promising of indigenous drugs.

ACKNOWLEDGMENTS

This research was supported by the Tunisian Ministry of Enseignement Superieur, Scientific Research and Technology (UR03/ES-08). Part of this work was carried out at the Dipartimento di Chimica Bioorganica e Biofarmacia, Universita‘di Pisa, Italy. We wish to thank the personnel of the laboratory of “Human Nutrition and Metabolic Disorder” Faculty of Medicine of Monastir”.