Olive mill and olive processing residues are attractive sources of natural antioxidants. An important part of these residues is olive tree leaves (usually 5%, but possibly reaching up to 10% of the total olives’ weight depending on practices applied). In addition, during olive tree cultivation, the pruning step generates a considerable volume of olive leaves, which are usually used as animal feed, and which could also be used for antioxidant or olive-leaf extract production [1
]. Olive leaves themselves have been used as a remedy against various diseases, while olive-leaf extract has been reported to have antioxidant capacity, antimicrobial activity, anti-HIV properties, vasodilator effect, and hypoglycaemic effect [1
]. In the past few years, the demand for olive leaf extract has increased for use in foodstuffs, food additives and functional food materials. Although the antioxidant activities of some single phenolic compounds in olive leaf are well known, antioxidant activities of its extract from various solvents, and especially from wild olive varieties for which higher phenolics content is expected [7
], have not been clearly investigated.
As each plant material has its unique properties in terms of phenolic extraction, it is very important to develop the optimal extraction conditions and afterwards the extract evaluation in terms of antioxidant activity and composition, as well as further utilization. Solvent extraction is a process designed to separate soluble phenolic compounds by diffusion from olive leaves (solid matrix) using a solvent (liquid matrix). Many factors contribute to the efficiency of the solvent extraction process, such as solvent type, pH, temperature, the number of extraction steps, solvent/solid ratio, and particle size of the solid matrix [8
]. Furthermore, supercritical fluid extraction (SFE) can be used to obtain various bioactive compounds without any solvent residue and safety hazard. CO2
-SFE is the most favored method for phenolic compounds isolation, which leads to higher phenol recoveries than sonication in liquid solvents. SFE extraction yield was low (only 45%), using liquid methanol [11
]. More recently, during oleuropein extraction from olive leaves, the use of CO2
-SFE alone had been characterized as not satisfactory, needing a polar modifier to improve yield and selectivity of the process [12
Considering that the extractability depends mainly on solvent type and on extraction method, and that it is difficult to recommend a suitable extracting solvent for individual plant materials, due to variation of bioactives in different plant materials, the present investigation was undertaken to study and determine phenols extraction conditions from the wild olive tree leaves using various solvents, as well as to evaluate and compare the phenolic content and the antioxidant activity of solvent and SFE phenolic extracts and best extract application to oil protection.
2. Experimental Section
2.1. Samples and Reagents
Olive leaves were collected from wild olive trees, variety Agrielia (Olea europaea
(Hoffm. & Link.), Synonyme: Olea europaea
(Mill.)), from Attica area, Greece. The olive leaves were analyzed for moisture, total solids, fat, and ash according to AOAC [13
Analytical grade methanol, ethanol, n-propanol, isopropanol and ethyl acetate, as well as HPLC grade acetonitrile, acetic acid and water were purchased from Merck (Darmstadt, Germany). Folin-Ciocalteau phenol reagent and 2,2-diphenyl-1-picryl-hydrazyl (DPPH) were purchased from Sigma Chemical Co. (Sigma-Aldrich Company Ltd., Dorset, Great Britain), while SFC-grade carbon dioxide was purchased from Air Liquide (Paris, France).
2.3. Phenolic Content Determination
The total phenol content of olive leaves extracts was determined colorimetrically, using the Folin-Ciocalteau reagent according to Lafka et al.
] which is a modification of Gutfinger’s [14
] method. Measurements were performed at 725 nm by using a double-beam UV-VIS spectrophotometer Hitachi U-3210 (Hitachi, Ltd., Tokyo, Japan). Caffeic acid served as standard for preparing the calibration curve ranging from 60 to 140 μg/25 ml assay solution.
2.4. Antioxidant Activity
2.4.1. DPPH Radical Scavenging Method
The antioxidant activity of phenolic extracts was evaluated by using the stable 2,2-diphenyl-1-picryl-hydrazyl radical (DPPH) according to the method of Bandoniene et al.
] following a modification by Lafka et al.
] and using a double-beam UV-VIS spectrophotometer Hitachi U-3210 (Hitachi, Ltd., Tokyo, Japan). The radical scavenging activities of the tested samples, was expressed as percentage inhibition of DPPH.
2.4.2. Rancimat Method
Ethanol extracts of olive leaves were freeze-dried (Virtis 5L, Gardiner, NY, USA) and the freeze-dried extracts were added into commercial sunflower oil without any added antioxidant at concentrations ranging from 40 to 240 ppm. The antioxidant potential of these extracts was investigated and compared to the antioxidant potential of samples of commercial sunflower oil containing synthetic (BHT, ascorbyl palmitate) and natural (vitamin E) antioxidants. The measurements were performed in a Rancimat 679 Instrument (Metrohm, Herisau, Switzerland) with air flow-rate and temperature set at 20 l/h and 100 °C, respectively.
2.4.3. Peroxide Value Determination
All solvent and SFE extracts were added at different concentrations (100 and 150 ppm) to commercial virgin olive oil and sunflower oil. Then, all the samples were put in an oven at 85 °C where thermal oxidation took place. Every 24 h the samples were analyzed for peroxide value in order to monitor the oxidation process. The peroxide value was determined according to the EEC [16
] method. The peroxide value was expressed as mM of active oxygen per kg of sample.
2.5. Statistical Analysis
Data were analyzed with one-way ANOVA Post Hoc Tests and pairwise multiple comparisons were conducted with the Tukey’s honestly significant difference test. Model fitting and analysis were performed using Statistica software (Statistica Release 7, Statsoft Inc., Tulsa, OK, USA).
The second order kinetic model was proven as the best in correlating the experimental data, followed by the Elovich model. The nature of the solvent affects the mechanism of extraction. Ethanol was proven and selected as the most appropriate solvent for the extraction of phenolic compounds from olive leaves for production of extracts with high phenol content and high antioxidant activity. For constant temperature, at pH 2.0 and solvent to sample ratio 5:1 v/w the optimum time was 180 min. CO2-SFE of wild olive leaves led to recovery of phenolic compounds with relatively high antioxidant activity, compared to some of the solvents used. Olive leaves could be a low-cost, renewable and abundant source of phenolic antioxidants, with potent use in fatty foods.