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Research Article
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Effects of Irrigation Regimes on Fatty Acid Composition, Antioxidant and Antifungal Properties of Volatiles from Fruits of Koroneiki Cultivar Grown Under Tunisian Conditions |
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Faten Brahmi,
Hechmi Chehab,
Guido Flamini,
Madiha Dhibi,
Manel Issaoui,
Maha Mastouri
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Mohamed Hammami
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ABSTRACT
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The olive tree is generally grown under rain-fed conditions. However, since the yield response to irrigation is great, even with low amounts of water, there is increasing interest in irrigated agriculture. The main goal of this study was, therefore, to investigate the effect of irrigation regimes on olive (Olea europaea L., cv. Koroneiki) obtained from an intensively-managed orchard in a semi-arid area with a Mediterranean climate in Tunisia. Different irrigation treatments 50% ETc, 75% ETc and 100% ETc were applied to the olive orchard. Accordingly, the effects of three irrigation regimes on volatile compounds, fatty acid composition and biological activities of Koroneiki cultivar were studied. The total profile of the volatile constituents of all samples revealed the predominance of 3-ethenylpyridine (from 14.9-19.6%), phenylethyl alcool (from 7.8-19.2%) and benzaldehyde (from 9.0 to 13.8%). During watering level treatments studied, the major fatty acids were oleic, palmitic and linoleic. Antioxidant activity of the fresh fruit volatiles cultivated at a watering level of 100% ETc was higher than that obtained under 50 and 75% Etc. The results of antifungal activity showed that the fruits volatiles of the three irrigation treatments had varying degrees of growth inhibition against the microorganisms tested.
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How
to cite this article:
Faten Brahmi, Hechmi Chehab, Guido Flamini, Madiha Dhibi, Manel Issaoui, Maha Mastouri and Mohamed Hammami, 2013. Effects of Irrigation Regimes on Fatty Acid Composition, Antioxidant and Antifungal Properties of Volatiles from Fruits of Koroneiki Cultivar Grown Under Tunisian Conditions. Pakistan Journal of Biological Sciences, 16: 1469-1478. DOI: 10.3923/pjbs.2013.1469.1478 URL: https://scialert.net/abstract/?doi=pjbs.2013.1469.1478
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Received: January 11, 2013;
Accepted: March 03, 2013;
Published: May 08, 2013
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INTRODUCTION
Aromatic plants and their essential oils, mixtures of natural volatile compounds
are gaining increasing interest because of their biological activities. These
activities have been known for many thousands of years for their antibacterial,
antifungal and antioxidant properties (Deans and Waterman,
1993). The aromatic plants are the source of natural antioxidants (Smelcerovic
et al., 2006). Their main secondary metabolites, (poly)phenolic compounds
can act as antioxidants by donating hydrogen to highly reactive radicals, thereby
preventing further radical formation. They also have metal chelation properties
(Rice-Evans et al., 1997). Thus, the volatile
compounds have great application and demand in food, perfumery, cosmetics and
pharmaceutical industries. In our search for commercially useful volatile compounds,
Olea europaea L. was selected as one of the plants for study.
This plant is one of the main crops of the Mediterranean basin, which are now
increasingly grown elsewhere. In the last decade, the worldwide area devoted
to their cultivation has increased by 10% (FAOSTAT, 2007).
But in areas with a Mediterranean climate, characterized by little or no rainfall
during the most critical phenological stages of production, intensive olive
growing is barely feasible without irrigation. Therefore, irrigation is a vital
factor in improving both production and productivity (Moriana
et al., 2003). In olive, irrigation can increase production (Samish
and Spiegel, 1961; Lavee et al., 1990; Girona,
1996; Moriana et al., 2003) thereby affecting
better the total oil production per tree. However, little and contradictory
data revealed the amount of seasonal water, necessary to obtain quality-quantitatively
good productions from different olive cultivars. Generally, the differences
are influenced by varying degrees of cultivar adaptability to the pedoclimatic
conditions and agronomic practices adopted in the field trials (Dettori
et al., 1989; Patumi et al., 1999).
In recent years, drip irrigation has become increasingly popular to reduce
the amount of water and fertilizer that are applied to the crop, and also to
reduce the amount of labor (Tan, 1995; Hanson
et al., 1997; Yohannes and Tadesse, 1998).
Then, drip irrigation is a vital factor, in areas where water is limited, in
improving production and productivity, since these features make it potentially
much more efficient than other irrigation methods. Therefore, a proper drip
irrigation rate can affect both the minimum amount of water leached from the
root zone and maintains a high soil matrix potential in the rhizosphere to reduce
plant water stress. As a consequence, accurate information on yield responses
in light of the amount of water applied by drip irrigation would be required
to achieve the best drip irrigation management. However, it is very relevant
to remark that any type of irrigation management employed in the olive orchard
must take into account the ultimate effect on the volatile compounds and fatty
acid composition, especially with regard to its content and profiles. Additionally,
the chemical composition of olives depend on many agronomical factors, e.g.,
olive cultivar (Esti et al., 1998; Romani
et al., 1999), pedoclimatic conditions (Vinha
et al., 2005) and irrigation management (Patumi
et al., 2002; Tovar et al., 2002).
In fact, the studies on the effect of irrigation on the fatty acid composition
of the olive fruit are contradictory (Gatto, 1989;
Milella and Dettori, 1987; Lavee
and Wodner, 1991).
Nevertheless, currently there is a lack of sound detailed information about
the effect of different irrigation, on the major and minor composition of olive
(Servili et al., 2007). Therefore, we evaluated
how drip irrigation affects the volatiles produced by fruits of European olive
variety Koroneiki cultivated in the north of Tunisia. For these reasons,
the main goal of the present study is first to determine irrigation regime effects
on the chemical composition of volatile fraction of European olive variety (cv.
Koroneiki); secondly, to determine the fatty acid composition finally,
to evidence the influence of the various drip irrigation rates on antioxidant
and antifungal properties.
MATERIALS AND METHODS
Plant material and growth conditions: Unripe fruits were collected from
Koroneiki olive (Olea europaea L.) cultivar planted in the experimental
farm of Elkef in north- western of Tunisia (Latitude: 36 18 N;
Longitude: 09 07 E; Altitude: 500 m above sea level) during the
crop season 2008-2009. Elkef region is characterized by a mean annual rainfall
of 450 mm, concentrated mainly from autumn to spring and an average evapotranspiration
(ETc) of 1500 mm. The warmer months are July/August and the coldest are December/January
with a mean annual temperature varied from 7.8-28.5°C. In this olive orchard
(20 ha), water was delivered three times per week at a rate of 4 h j-1
using a localized irrigation system with four drip nozzles of 4 l h-1
each per tree (two per side), set in a line along the rows at a distance of
0.5 m around the trunk with a unit flow of 8 l h-1 (total flow per
tree was 16 l h-1). Koroneiki olive oil cultivar, was tested
in a factorial combination with three irrigation levels [three plots of 36 m2
(6 m x 6 m) (+ three trees per treatement, three plot of 3x 36 m2)
each were designed for each irrigation]: stressed (T1), moderated (T2) and well
irrigation (T3) receiving a seasonal water irrigation amount equivalent to 50,
75 and 100% ETc (Dabbou et al., 2010). The calculation
procedures used by this model are based on the Penman-Monteith- FAO method (Allen
et al., 1998) with a single estimated crop coefficient (Kc = 0.6),
a reference of evapotranspiration applied for each irrigation treatment (ETo)
and a coverage coefficient (Kr = 0.5) (DAndria et
al., 2004) where ETc = Kr . Kc . ET0.
The soil was silty (18%) with alkaline pH (8.10) and consisted of 33% calcium
carbonate, 1.20% organic matter, 0.65 N2, 255 mg kg-1
K2O, and 6 mg kg-1 P2O6. The
experimental plot was grown intensively at a planting density of 286 plants
ha-1 and a tree spacing of 6 m x 6 m with olive oil trees of 5-year-old
after planting.
Extraction method: A 500 g of each sample of the fresh fruits was subjected
to hydrodistillation for 3 h using a Clevenger-type apparatus (Clevenger,
1928). 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 use for further analysis. The percentage
of volatile yield was calculated as the weight of volatile divided by the weight
of fresh fruits.
Identification of the volatiles constituents: Volatile compounds analysis
by GC was performed on a gas chromatograph HP-5890 series II equipped with dual
Flame-ionization Detector (FID). HP-Wax and HP-5 capillary columns (30 m x0.25
mm, 0.25 μm film thickness) were used. The oven temperature was kept at
60°C for 10 min followed by a 5°C min-1 ramp to 220°C.
The carrier gas was nitrogen with a flow rate of 2 mL min-1; split
ratio was 1:30. Injector and detector temperatures were maintained at 250°C.
The injected volume was 0.5 μL. The identification of the components was
performed, for both columns, 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 mx 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 at 3°C min-1; 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; ADAMS Adams, 1995) and homemade
library mass spectra built from pure substances and components of known oils
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 the chemical ionization
gas.
Fatty acid methylation and analysis: Triplicate sub-samples of 0.5 g
were extracted using the modified method of Bligh and Dyer
(1959). Thus, fruit samples were kept in boiling water for 10 min to inactivate
lipase (Douce, 1964) and then ground manually using
a mortar and pestle. A chloroform/methanol (Analytical Reagent, LabScan, Ltd.,
Dublin, Ireland) mixture (1:1, v/v) was used for total lipid extraction. After
washing with water and centrifugation at 3000xg for 10 min, the organic layer
containing total lipids was recovered and dried under a nitrogen stream. Total
Fatty Acids (TFA) were converted into their methyl esters using sodium methoxide
solution (Sigma, Aldrich) according to the method described by Cecchi
et al. (1985). Methyl heptadecanoate (C17:0) was used as an internal
standard. Those fatty acids methyl esters (FAMEs) obtained were subsequently
analyzed.
The fatty acid methyl esters were analyzed on a HP 5890 gas chromatograph (Agilent
Palo Alto, CA, USA) equipped with a flame ionization detector (FID). The esters
were separated on a RT-2560 capillary column (100 m length, 0.25 mm i.d., 0.20
mm film thickness). The oven temperature was kept at 170°C for 2 min, followed
by a 3°C min-1 ramp to 240°C and finally held there for an
additional 15 min period. Nitrogen was used as carrier gas at a flow rate of
1.2 mL min-1. The injector and detector temperatures were maintained
at 225°C. A comparison of the retention times of the FAMEs with those of
co-injected authentic standards (Analytical Reagent, LabScan, Ltd., Dublin,
Ireland) was made to facilitate identification.
Evaluation of antiradical activity by dpph assay: The scavenging activities
of the methanolic extracts were measured according to the method described by
Blois, (1958). Volatiles Methanolic extracts (1 mL) at
different concentrations were added to 1 mL of DPPH methanolic solution (0.004%).
The mixture was shaken vigorously and left to stand at room temperature for
30 min in the dark. Then the absorbance was measured at 517 nm against a blank
by a spectrophotometer (Secommam, U-1789, France). Inhibition of free radical,
DPPH, in percent (I%) was calculated according to formula:
where, Ablank is the absorbance of the control reaction (containing
all reagents except the test compound) and Asample is the absorbance
of the test compound. Extract concentration providing 50% inhibition (IC50)
was calculated from the graph plotting inhibition percentage against extract
concentration. Tests were carried out in triplicate. Butylated hydroxytoluene
(BHT) was used as positive control.
Evaluation of antiradical activity by abts assay: The ABTS+
radical cation scavenging activity of each volatile fraction and ascorbic acid
(control) was determined according to Yvonne et al.,
(2005). In brief, 5.0 mL of a 7.0 mM ABTS was reacted with 88.0 μL
of a 140 mM potassium persulfate overnight in the dark to yield the ABTS+
radical cation. Prior to use in the assay, the ABTS+ radical cation
was diluted with ethanol for 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 diluted ABTS+ radical cation with 10 μL of
test sample and monitoring the change in absorbance at 0, 0.5 and 1 min and
again 5 min intervals until a steady state was achieved. The antioxidant capacity
of volatile fraction was expressed as IC50, the concentration necessary
for 50% reduction of ABTS+.
Antifungal activity: Four fungal species were used for the antifungal
testing, namely: Candida glabrata ATCC 90030, Candida kreusei
ATCC 6258, Candida parapsilosis ATCC 22019 and Candida 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 fractions were first dissolved in 10% DMSO and then
diluted to the highest concentration (10 mg mL-1) to be tested, and
then serial two fold 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 of 100 μL
from the stock solutions of the volatiles initially prepared at the concentration
of 10 m mL-1 was added into the first wells. Then, 100 μL from
their serial dilution were transferred into six consecutive wells. 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 for 18 h at 37°C. The MIC was defined as the lowest concentration
of the compounds to inhibit the growth of microorganisms, after incubation.
Results were expressed in microgram per milliliter.
Statistical analysis: All parameters analyzed were carried out in triplicate.
The results are reported as mean values of three repetitions and standard deviation.
Data were subjected to statistical analysis using the SPSS programme, release
11.0 for Windows (SPSS, Chicago, IL, USA). The one-way analysis of variance
(ANOVA) followed by Duncan multiple range test were employed and the differences
between individual means were deemed to be significant at p<0.05.
RESULTS AND DISCUSSION
Volatiles yield and chemical composition: GC/MS analysis was conducted
on the volatile fruits of the three irrigation treatments. The list of detected
compounds with their relative percentages and retention indices are given in
Table 1 in order of their elution on the column.
The volatile fractions correspond to the 5th year of cultivation practices,
in which the volatile yield, vary between 0.25 and 0.33% for 100 and 50% ETc
equivalent, respectively (Table 2).
Table 1: |
Watering level effect on Olea europaea L. cultivar
(Koroneiki) volatile composition a |
 |
aPercentages obtained by flame ionization detector
(FID) peak area normalization (HP-5 column), bLinear retention
indices (DB-5 column), cTraces (Tr) <0.1% |
As reported by Sotomayor et al. (2004) in other
fruit species Thymus zygis ssp., gracilis, increases in volatile
yield are associated with the decrease in amount of water added. Moreover, a
decrease in volatile yield is detected for the olive cultivar Koroneiki
under watering levels higher than 50% ETc. It seems that variation in volatile
yield can be attributed to some factors like conditions of cultivation especially
irrigation (Arganosa et al., 1998). From this,
it can be concluded that the cultivar Koroneiki grown under watering
levels higher than 50% ETc decreased their volatile production quickly with
time. Thus, statistically significant differences were observed among yields
of the three irrigation treatments studied (Table 2). Nineteen
components were obtained in the volatile fraction of Koroneiki cultivar
grown under a 50% ETc watering level.
Table 2: |
Watering level effect on European olive cultivar Koroneiki
volatile fractions yields and radical-scavenging activities |
 |
Data are expressed by mean values ±SD of three independent
experiments, Values in each column with different superscript letters present
significant differences (p<0.05) between the different irrigation strategies
for each parameter |
The major constituents were 3-ethenylpyridine (19.2%), phenylethyl alcool (17.0%)
and benzaldehyde (11.0%). Twenty- seven components were identified in the volatile
fraction for 75% ETc, were the principle components 3-ethenylpyridine (19.6%),
phenylethyl alcool (19.2%) and benzaldehyde (9%). Twenty-nine components were
characterized in the volatile fraction from plants treated 100% ETc with as
major components 3-ethenylpyridine (14.9%), benzaldehyde (13.8%) and phenylethyl
alcool (7.8%). Among the major common components, 3-ethenylpyridine was the
component present in all the three samples (14.9-19.6%). Benzaldehyde and phenylethyl
alcool were the other prominent components found in considerable quantity in
all samples.
Table 1 contains the relative amounts of different compound
families, found in the volatile fruits of the three irrigation treatments. Oxygenated
non terpene derivatives (64.2-65.5%) comprised the most abundant class of the
compounds detected in all studied samples and contained phenylethyl alcool (7.8-19.2%)
and benzaldehyde (9.0-13.8%) as the main compounds. The nitrogen derivatives
(19.2-19.6%) were the second main class of all volatile samples. Therefore,
it is interesting to note, on one hand, that the sesquiterpenes hydrocarbons
and oxygenated monoterpenes were present in minor percentages (1.3-5.5%) in
all volatile of the three watering level treatments studied. On the other hand,
all volatile samples were also poor in monoterpenes hydrocarbons and non-terpene
hydrocarbons. However, it has been suggested that the accumulation of monoterpenes
under watering levels higher than 50% ETc, has physiological and ecological
role such as a photorespiration-like protection (Penuelas
and Llusia, 2002). The secondary metabolite production is believed to be
stimulated by the irrigation treatments. Like all secondary metabolites, the
volatiles compounds are known to have several important functions such as protection
against predators (microorganisms; fungi; insects; herbivores), against UV radiations
but may also serve as secondary functions attracting the natural enemies of
these herbivores, attraction of pollinators and dispersal of diasopres, inhibitors
of germination and growth, etc. (Kessler and Baldwin, 2001).
Fig. 1 show the distribution of C6 alcohols compounds derived
from the LOX pathway, expressed as (%), in olive cultivar Koroneiki samples
according to the different irrigation levels studied.
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Fig. 1: |
Contents of C6 alcohols compounds derived from linolenic acid
(LnA) and linoleic acid (LA) in olive fruit from the introduced cultivar
as affected by irrigation levels. Alcohols C6/LnA represents (E)-hex-3-en-1-ol.
Alcohols C6/LA represents the contents of hexan-1-ol |
Those C6 alcohols compounds are derived from the cascade of enzymatic reactions
starting with the formation, by lipoxygenase (LOX) action, of 13-hydroperoxides
from linolenic and linoleic acid (Olias et al.,
1993). The total volatile C6 alcohols compounds derived from linolenic acid
ranged from 3.4-6.3% and from 2-4.9% in C6 alcohols compounds derived from linoleic
acid. In fact, regarding the levels of C6 alcohols compounds such as (E)-hex-3-en-1-ol
and hexan-1-ol derived from linolenic and linoleic acid, they decreased with
75% Etc and then increased down to 6.3 and 4.9%, respectively with 100% ETc.
Consequently, specific trend with regard to the change in C6 alcohols compounds
as a function of irrigation regime was observed. Nevertheless, the enzymatic
activity is affected by raising the irrigation levels of olive trees. In addition,
irrigation regime may also influence the production of volatiles (Baccouri
et al., 2008). According to the data reported in Fig.
2, the %C6 compounds and total alcohols increased in the volatiles obtained
from Koroneiki fruits with increased the irrigation levels. Both classes
of compounds were affected by the irrigation in the sense that the increase
in the water applied to the Koroneiki olive trees led to an increase
in these volatiles. However, inconsistent changes in the content of the %C6
aldehydes/C6 were observed for Koroneiki fruits.
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Fig. 2: |
Contents of %C6, total alcohols, total aldehydes,
%C6 alcohols/C6, % C6 aldehydes /C6
from the three introduced cultivars as affected by irrigation levels |
Table 3: |
Variations in fatty acids composition (as a percent of TFA)
of total lipids from fresh fruits of Olea europaea L. cultivar (Koroneiki)
grown under three irrigation regimes |
 |
Data are expressed by mean values±SD of three independent
experiments. Values in each row with different superscript letters present
significant differences (p<0.05) between the different irrigation strategies
for each parameter. C16:0: Palmitic acid, C16:1: Palmitoleic acid, C17:0:
Margaric acid, C17:1: Margaroleic acid, C18:0: Stearic acid, C18:1: Oleic
acid, C18:2: Linoleic acid, C18:3: Linolenic acid, C20:0: Arachidic acid,
C20:1: Gadoleic acid, C22:0: Behenic acid, C22:1: Erucic acid and C24:0:
Lignoceric acid, SFA: Saturated fatty acids, MUFA: Monounsaturated fatty
acids, PUFA: Polyunsaturated fatty acids, O/L: Oleic/Linoleic ratio |
Nevertheless, the trend of total aldehydes compounds and %C6 alcohols/C6
decreased with 75% Etc and then increased down to 40% and 72.25%, respectively
with 100% ETc. Hence, these different amounts of total volatiles should be related
to the agronomic conditions (the irrigation regime).
Fatty acid composition: Based on our experimental data, it was shown
that major fatty acids for the three irrigation regimes were oleic and palmitic
(Table 3). Furthermore, the differences were statistically
significant among three watering level treatments studied of the Koroneiki
cv., especially in palmitoleic acid (p<0.05). The percentage of Saturated
Fatty Acids (SFA), mainly that of arachidic acid (C20:0) decreased from 1.24%
under 50% ETc watering level to 0.42 for 100 % ETc (Table 3).
In addition, we noted that drip irrigation decreased the SFA proportions. It
was also mentioned that oleic acid levels increased to reach 73.14 at 50% ETc
watering level and 73.98 under 100% ETc, respectively. Drip irrigation rates
did not elicit significant changes in the oleic acid proportion. Current biochemical
evidence indicates that, in olive and other plant species, the polyunsaturated
fatty acids (C18:2 and C18:3) are produced by the consecutive desaturation of
oleic acid. The major variation consisted in a decrease of the saturated fatty
acids in favor of an increase in monounsaturated ones. Another important fatty
acid is linoleic acid; its content was in the range of 6.37-7.12% for 50 and
100% ETc. Interest in the PUFA, as health-promoting nutrients, has expanded
dramatically in recent years. A rapidly growing literature illustrates the benefits
of PUFA in alleviating cardiovascular, inflammatory, heart diseases, atherosclerosis,
autoimmune disorder, diabetes and other diseases (Finley
and Shahidi, 2001; Riemersma, 2001). The ratio of
the oleic/linoleic acid (O/L) and monounsaturated/polyunsaturated fatty acids
(MUFA/PUFA), decreased (from 11.48-10.39%) and (from 9.67-8.66%), respectively
at the irrigation treatments, except a higher amount detected at 75% ETc (11.96%)
and (10.5%), respectively. On the other hand, Unger (1982)
found a positive correlation between oleic acid content and water use at the
vegetative stage. Differences among three irrigation levels in both the ratio
O/L and MUFA/PUFA are not statistically significant (p<0.05). In addition,
we noted that the oleic/linoleic acid (O/L) and monounsaturated/polyunsaturated
fatty acids (MUFA/PUFA), which are largely responsible for the sapidity and
healthful effects of the Mediterranean diet.
Antioxidant activity: Antioxidant activity of the fresh fruits volatiles
of European olive cultivar Koroneiki subjected to three irrigation treatments
were determined by two different test systems namely DPPH and ABTS+
radical-scavenging assays. All of the data are presented in Table
2.
Antioxidant activity by DPPH: 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 color change from purple to
yellow and a decrease in absorbance. Free radical scavenging properties of the
fresh fruits volatiles are presented in Table 2. The antioxidant
capacity of volatiles was expressed as IC50 (concentration of antioxidant
required to quench 50% of the stable free radical), which was used to acquire
the optimized extraction condition. The IC50 values for Koroneiki cv.
grown under 50, 75 and 100% ETc were 1599.70, 547.50 and 361.91 μg mL-1,
respectively. The volatile fraction for 100% ETc showed higher scavenging ability
on DPPH radicals than those for 50 and 75% ETc. These results suggest that an
increase in the watering level applied favored the radical-scavenging activity
of the volatile fractions. Thus, we reported increases in antioxidant activity
are associated with the increase in the amount of water added. Our results are
in agreement with those of Jordan et al. (2009)
conducted on Thymus zygis ssp. gracilis. Moreover, DPPH scavenging
abilities of the studied samples were lower than that of synthetic antioxidant
BHT (IC50 = 181.20 μg mL-1). These results were found
to be statistically significant (p< 0.05).The DPPH-radical-scavenging activity
of the volatiles compared to BHT decreased in the order of BHT>volatiles
for 100% ETc>volatiles for 75% ETc>volatiles for 50% ETc.
Literature review shows that oxygenated monoterpenes are mainly responsible
for the antioxidant potential of the plant oils which contain them (Baratta
et al., 1998). Monoterpene hydrocarbons could also be taken into
account for the antioxidative activity observed, but obviously, none has stronger
than that of oxygenated monoterpenes. The presence of strongly activated methylene
groups in these molecules is probably the reason for this behaviour (Ruberto
and Baratta, 2000). According to Table 1, the amount of
oxygenated monoterpenes and monoterpene hydrocarbons is higher in volatile fraction
for 75 and 100% ETc than the other. The slight quantitative differences in the
amounts of these compound families might also explain the differences of the
antioxidant activities between the irrigation treatments studied.
Antioxidant activity by ABTS: The free radical scavenging capacity of
volatile fractions from Koroneiki cultivar was evaluated by the ABTS
assay. The radical-scavenging activities of the different volatile fractions,
according to the watering levels applied, are shown in Table 2.
In the present study, the weakest radical scavenging activity was exhibited
by the volatile fraction obtained under 50% ETc (17479 μg mL-1).
Antioxidant activity of the fresh fruit volatiles cultivated at a watering level
of 100% ETc was higher than that obtained under 50 and 75% ETc with an IC50
value of 14431.68 μg mL-1. On the other hand, none of the samples
showed activity as strong as the positive control ascorbic acid (7.45 μg
mL-1). Furthermore, regarding the watering-level effect on radical-scavenging
activity, results were found statistically significant (p<0.05). As a major
conclusion, from a watering level obtained with 100% ETc must be obtained volatile
fractions from Koroneiki cultivar with high radical-scavenging activity.
Antifungal activity: The antifungal activities of the volatiles coming
from fresh fruits of Koroneiki cultivar grown under three watering levels
were assayed in vitro by a broth micro-dilution method against four phytopathogen
strains and the results are reported in Table 4.
Minimal Inhibitory Concentration (MIC) values of the fresh fruits volatiles
towards the four yeast strains were determined in μg mL-1. The
results of antifungal activity showed that the fruits volatiles of the three
Irrigation treatments had varying degrees of growth inhibition against the microorganisms
tested.
Table 4: |
Antifungal activity of volatile fractions from fresh fruits
of Olea europaea L. cultivar (Koroneiki) grown under three
irrigation regimes |
 |
MIC: Minimum Inhibitory Concentration, Not active, cControl
antifungal |
The strongest fungicidal activity was exhibited by the fresh fruits volatile
cultivated at a watering level of 50% ETc against C. albicans (MIC values
78 μg mL-1). The same sample demonstrated moderate activities
against C. kreusei, C. glabrata and C. parapsilosis (Table
4). C. glabrata showed, on one hand, best susceptibility towards
the volatile fraction for 75% ETc with a MIC value of 156 μg mL-1
followed by C. kreusei, C. albicans MIC 1250 μg mL-1
and C. parapsilosis MIC 2500 μg mL-1. On the other hand,
volatiles coming from fresh fruits cultivated under 100% ETc watering level
were presented as the most active against C. glabrata with a MIC value
of 78 μg mL-1. Whereas, no antifungal activity against C.
kreusei was detected by volatile fraction for 100% ETc.
Fruit volatiles of the three irrigation treatments are complex mixtures of
volatile compounds (Table 1). In fact, Matasyoh
et al. (2007) showed that the chemical compounds like linalool and
α-terpineol had antifungal activity. In addition, it is also possible that
the trace/minor components might be involved in some type of antifungal synergism
with other active components of volatiles. These findings are in agreement with
previous reports (Marino et al., 2001; Hutchings
et al., 1996).
CONCLUSION
Regarding first the volatile fraction yield and quality, secondly, the fatty
acid and volatile composition finally, the antioxidant and antifungal activities,
a single watering level cannot be pre-selected in order to obtain the most satisfactory
values for these parameters. On the one hand, high levels of water are required
for achieving lower IC50 values; on the other hand, volatile fraction yield
is favored by lower watering levels. Hence, the olive volatiles compounds described
herein may be suggested as a potential source of natural antioxidant and antifungal
for food industry.
ACKNOWLEDGMENTS
This research was supported by the Tunisian Ministry of higher Education, Scientific
Research (UR03/ES-08). Part of this work was carried out at the Dipartimento
di Chimica Bioorganica e Biofarmacia, Universitadi Pisa, Italy. We wish
to thank the personnel of the laboratory of Human Nutrition and Metabolic
Disorder Faculty of Medicine of Monastir.
|
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