This work presents a chemical (the minerals, chlorophyll contents and fatty acids) and thermo-physical investigation (DSC profile) of four varieties of olive leaves grown in Tunisia. The total chlorophy1l contents of olive leaves ranged from 1132.33 to 1795.93 ppm. The results showed that linolenic acid (C18:3) is the major fatty acid in olive leaves (from 30.02 to 42.16%), followed by oleic acid (C18:1) and palmitic acid (C16:0). The thermal profiles of olive leaf extracts determined by their DSC melting curves revealed simple thermograms with a single peak after melting. The hexane extract of the Chemchali variety, which contained relatively high unsaturated fatty acids and low saturated fatty acid levels, exhibited the lowest peak temperature value (54.59 °C) and required the smallest amount of energy for melting (31.57 J·g−1). This study showed that olive leaves possessed physicochemical properties and a fatty acid composition that may become interesting for industrial applications.
The olive is one of the most extensively cultivated fruit crops in the world. The worldwide planted area dedicated to olive trees is about 9.9 million hectares with 95% of them in the Mediterranean basin (FAOSTAT,
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The thermal stability of esters depends on their chemical structure and fatty acid composition. In this respect, differential scanning calorimetry (DSC) gives valuable information on the thermal properties of fats and their suitability for a particular application. Indeed, DSC measures the temperatures and heat flows associated with transitions in materials as a function of time and temperature in a controlled atmosphere. Any endothermic or exothermic event is registered as a peak in the chart, and its area is proportional to the enthalpy gained or lost, respectively (Gloria and Aguilera,
In this work, we aimed to investigate the mineral composition of olive leaves, to establish the fatty acid composition and to study the thermal behaviour of olive leaf extracts by differential scanning calorimetry in an effort to achieve efficient uses of such by-products for industrial applications.
All chemicals and reagents were of analytical reagent grade. Potassium hydroxide, Hydrochloric acid and Nitric acid were purchased from Panreac (Spain). Acetone, methanol, ether and hexane were supplied from Carlo Erba (France).
Olive leaves were harvested from the Olive Tree Institute farm of Sfax, Tunisia (34°43N, 10°41E) in April 2011. Nineteen-year-old olive trees (
Samples of each olive leaf powder (1 g) were ashed at 550 °C in a muffle furnace for 4 hours. The ashes were then cooled in a desiccator and used for the determination of calcium, potassium, magnesium, sodium, iron, zinc and copper contents (AOAC,
Total chlorophyll, chlorophyll a and b contents were determined by the spectrophotometric method according to AOAC (
The olive leaf powder (50 g) was placed in a dark flask and homogenized with 250 mL of hexane (1:5, w/v). After mixing for 4 h in a shaker (Selecta, Spain), the mixture was centrifuged for 15 min at 1000 g. The supernatant was then filtered through a filter paper (Whatman no. 2). The extraction procedure was repeated twice. The solvent was removed by a vacuum rotary evaporation at 40 °C. The extract was pooled, drained under a stream of nitrogen and then stored in a freezer until analysis.
The hexane extracts of olive leaves were filtered with a millipore membrane filter (0.45 μm) prior to gas liquid chromatography analysis. Samples (1 mg) were converted into their corresponding fatty acid methyl esters using a methanolic solution of potassium hydroxide (2 M). The mixture was maintained at 100 °C for 1 h. The reaction was stopped with 0.5 mL of distilled water. Then, the extracted fatty acid methyl esters were dissolved in heptane. GC analyses were performed on a GC-17-A SHIMADZU (Japan), equipped with a hydrogen flame ionization detector and a capillary column: Carbowax (15 m, 0.25 mm).The column temperature was fixed at 180 °C and the injector and detector temperatures were set at 230 °C and 250 °C, respectively. Nitrogen was the carrier gas. Fatty acid methyl esters were identified by comparison of their retention times with respect to pure standards purchased from Sigma and analyzed under the same conditions. Fatty acid methyl esters were quantified according to their percentage area, obtained by the integration of the peaks. The results were expressed as a percentage of individual fatty acids in the extract.
The thermal properties of olive leaf extracts in hexane were determined using a differential scanning calorimeter (NETZSCH-Gerätebau GmbH Thermal analysis DSC 204, Germany). The hexane extract (8±0.1 mg) was weighed in a DSC-aluminium pan. An empty DSC-pan was used as an inert reference. The sample and the reference pans were then placed inside the calorimeter and were quickly cooled to −60 °C at a speed of 5 °C·min−1, held at this temperature for 15 min, and heated to 100 °C with a heating speed of 5 °C·min−1 and the DSC thermographs were recorded during the melting transition. The peak temperature is the temperature maximum of a thermal transition. The onset temperature is the temperature where the extrapolated leading edge of the endotherm intersects with the baseline.
Analyses were carried out in triplicate. The values of different parameters were expressed as the mean ± standard deviation. The results were statistically analyzed by one-way analysis of variance (ANOVA) and Tukey tests using SPSS (Version 11, SPSS Inc., Chicago, USA). Statistical significance was accepted at a level of p<0.05.
The mineral compositions of the olive leaf varieties namely Chemlali (CL), Chemchali (CH), Chetoui (CT) and Zarrazi (ZR) are shown in
Mineral composition of olive leaves (mg·g−1 dry matter)
Minerals | Variety | |||
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Chemlali | Chemchali | Chetoui | Zarrazi | |
8.47a±0.14 | 9.82b±0.09 | 8.05a±0.36 | 6.60c±0.23 | |
10.39a±0.32 | 9.66a,b±0.33 | 9.25b±0.36 | 9.37b±0.29 | |
7.87a±0.23 | 4.47b±0.45 | 9.14a±0.35 | 5.33b±1.61 | |
1.50a±0.25 | 3.00b±0.13 | 1.55a±0.10 | 2.75b±0.00 | |
0.82a±0.03 | 1.51b±0.10 | 0.35c±0.03 | 0.34c±0.02 | |
traces | traces | traces | traces |
(% dry matter).
Different letters in the same row indicate that means are significantly different (p<0.05).
The mineral composition of olive leaves showed that calcium [9.25 mg·g−1 d.m. (CT)−10.39 mg·g−1 d.m. (CL)] was the predominant mineral. Similar values (9.296 mg·g−1 d.m.) were reported by Lee
Potassium amounts in the olive leaves ranged from 4.47 to 9.14 mg·g−1 d.m. Potassium is a micronutrient that plays an important role in the regulation of the heartbeat. Besides, it maintains fluid balances and helps muscles contract. An adequate intake of calcium and potassium contributes to preventing cardiovascular diseases (McCarron and Reusser,
The potassium concentrations of CL and CT olive leaf varieties were significantly (p<0.05) different from those of CH and ZR. The mineral contents of olive leaves could be influenced by the differences in the nutrient status of the soils in which the olive tree grows and by the environmental effects around the tree. Higueras
The effects of soil chemistry on plants were also studied by Lynch
The magnesium contents varied from 1.5 to 3 mg·g−1 in olive leaves. The magnesium average value measured for Spanish olive leaves was 1.5 mg·g−1 (Higueras
Chlorophylls are very common pigments, which give color to plants and several algae. The color of olive leaves is mainly related to their chlorophyll content, as this compound is the main pigment of green vegetables and masks the bright color of carotenoids.
The total chlorophyll, chlorophyll a and chlorophyll b contents of olive leaves are presented in
Total chlorophyll, chlorophyll a and chlorophyll b contents of olive leaves (CL): Chemlali, (CH): Chemchali, (CT): Chetoui and (ZR): Zarrazi. Bars with the same letter are not significantly different (p>0.05).
Chlorophylls are used as additives to food products due to their color and physico-chemical properties. In fact, chlorophyll molecules are extracted and used as natural pigments in processed foods (Gutiérrez-Rosales
The hexane extract yields of olive leaves are shown in
Fatty acid composition of four olive leaf varieties (%)
Fatty acid | Variety | |||
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Chemlali | Chemchali | Chetoui | Zarrazi | |
3.11a±0.26 | 3.58a±0.00 | 3.88a±0.53 | 3.21a±0.41 | |
Myristic acid (C14:0) | 3.15a±0.35 | 2.49a.b±0.65 | 1.80b±0.32 | 3.27a±0.27 |
Palmitic acid (C16:0) | 22.42a±0.53 | 18.22b±1.27 | 20.39a,b±0.95 | 21.19a±0.71 |
Palmitoleic acid (C16:1) | 0.15a±0.00 | 0.13a,b±0.01 | 0.10b±0.02 | 0.17a±0.01 |
Stearic acid (C18:0) | 3.42a±0.41 | 2.59a±0.22 | 3.09a±0.41w | 3.88a±0.39 |
Oleic acid (C18:1) | 26.36a±1.39 | 18.28b±3.46 | 25.07a±0.34 | 25.15a±0.44 |
Linoleic acid (C18:2) | 14.48a±1.11 | 16.13b±0.61 | 16.52b±0.56 | 15.84b±0.53 |
Linolenic acid (C18:3) | 30.02a±1.95 | 42.16b±4.66 | 33.03a±1.19 | 30.50a±1.44 |
28.99±0.42 | 23.30 ±1.31 | 25.29±0.91 | 28.34±0.79 | |
26.51±1.40 | 18.41±3.45 | 25.17±0.35 | 25.32±0.44 | |
44.50±1.28 | 58.29±4.06 | 49.54±0.84 | 46.35±1.22 | |
71.01±0.42 | 76.70±1.31 | 74.71±0.91 | 71.66±0.79 |
Different letters in the same row indicate that means are significantly different (p<0.05).
The fatty acid composition of olive leaf extracts in hexane is shown in
Linolenic acid is a polyunsaturated omega-3 fatty acid which is metabolized to eicosapentaenoic acid, a precursor of eicosanoids with anti-inflammatory and antithrombotic activity (Ruiz
The percentage of polyunsaturated fatty acids [44.50–58.29%] in olive leaves was higher than that of saturated fatty acids [23.30–28.99%]. The olive leaves exhibited higher levels of saturated fatty acids than olive oil (21.22%) (chemlali variety) (Dabbou
The polyunsaturated fatty acids including the omega-3 and omega-6 families detected in the plants constitute an important class of phytochemicals due to their generalized beneficial health effects (Guimarães
Linoleic acid is observed at appreciable percentages in olive leaf extracts [14.48–16.52%]. This essential fatty acid has become increasingly popular because of its beneficial properties for the skin, including anti-inflammatory, acne reduction and moisture retention properties (Darmstadt
In addition, the use of olive leaves in animal feeding appears to increase the content of unsaturated fatty acids and lower the content of saturated fatty acids in animal milk (Fegeros
DSC is a fast and direct way to assess the quality of fats and to study their physical properties. DSC heating profiles help to explore the nature of the phase transition taking place during the melting of fats and oils (Gloria and Aguilera,
Olive leaf extracts in hexane showed the same melting profile (
DSC melting profile of olive leaf extracts in hexane. Olive leaf varieties: CL: Chemlali, CH: Chemchali, CT: Chetoui and ZR: Zarrazi.
Thermal parameters from the DSC melting curves of olive leaf extracts
Parameter | Variety | |||
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Chemlali | Chemchali | Chetoui | Zarrazi | |
29.10a±0.07 | 26.50b±0.06 | 30.10c±0.03 | 29.55d±0.02 | |
54.79a±0.04 | 54.59b±0.10 | 54.70c±0.07 | 54.80a±0.07 | |
54.97a±0.19 | 31.57b±0.11 | 43.24c±0.01 | 42.12d±0.03 |
Different letters in the same row indicate that means are significantly different (p<0.05).
The enthalpy of melting is the heat energy required for melting. This is calculated by integrating the area of the DSC peak on a time basis. Significant differences (p<0.05) were also encountered for the melting enthalpy of the samples and values ranged from 31.57 to 54.97 J·g−1. The thermal parameters and the phase transitions in fats may be affected by the compositional changes between the samples such as fatty acid chain length, degree of unsaturation and nature of distribution of fatty acids in triacylglycerol species. The DSC application upon heating allowed for discriminating among oil samples from olives of different cultivars and/or harvesting periods. The thermal properties of monovarietal extra virgin olive oil samples were found to correlate well with the chemical composition i.e. triacylglycerols, diacylglycerols, total and free fatty acids and oxidation status (Chiavaro
A mathematical model based on a simple regression procedure was also developed to correlate melting parameters to mass fractions of mono and polyunsaturated fatty acids (Fasina
Regarding the peak temperatures of olive leaf extracts, the values ranged from 54.59 to 54.80 °C. The CH variety exhibited the lowest peak temperature value (54.59 °C) and it required a smaller amount of energy for melting (31.57 J·g−1). This can be due to the higher level of unsaturated fatty acids and lower level of saturated fatty acids in the CH olive leaf extract. Indeed, the fatty acid composition influences the melting point values of the samples. Fats containing higher amounts of saturated triacylglycerol species commonly demonstrate a higher melting point than those which are highly unsaturated. Further, the peak temperatures of CL (54.79 °C) and ZR (54.80 °C) were statistically similar (p>0.05). This could be attributed to the similarities observed in the fatty acid composition between the two varieties.
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Data resulting from this work improve the fundamental knowledge about the chemical composition of olive leaves and such a determination is very important to explore varietal changes. The olive leaf is a promising source of calcium and potassium which contribute together with other phytochemicals to the health benefits of olive leaves. Total chlorophy1l contents ranged from 1132.33 to 1795.93 ppm. Chlorophylls can be used as natural additives to food products.
The present study proved that the olive leaf extract in hexane constitute a source of beneficial fatty acids, namely linolenic acid, oleic acid and linoleic acid. Olive leaf extracts exhibited a simple thermogram with a single peak after melting in the DSC. The hexane extract of the CH variety exhibited the lowest peak temperature value (54.59 °C) and it required the smallest amount of energy for melting (31.57 J·g−1) in comparison with the other samples. This was due to the fatty acid composition of the CH variety which exihibited the highest unsaturated fatty acid and lowest saturated fatty acid levels.
Olive leaves are renewable resource which provide adding value to agricultural products and potentially create new rural jobs when used for industrial products.
The authors would like to thank Mrs Naziha Kammoun, researcher in the Olive Tree Institute of Sfax (Tunisia), who provided us with the supply of olive leaves.