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Precipitation of antioxidant fine particles from Olea europaea leaves using supercritical antisolvent process
Accepted Manuscript Title: Precipitation of antioxidant fine particles from Olea europaea leaves using Supercritical Antisolvent process Author: C. Chinnarasu A. Montes M.T. Fern´andez- Ponce L. Casas C. Mantell C. Pereyra E.J. Mart´ınez de la Ossa PII: DOI: Reference:
S0896-8446(14)00372-6 http://dx.doi.org/doi:10.1016/j.supflu.2014.11.008 SUPFLU 3162
To appear in:
J. of Supercritical Fluids
Received date: Revised date: Accepted date:
14-7-2014 10-11-2014 10-11-2014
Please cite this article as: C. Chinnarasu, A. Montes, M.T.F.- Ponce, L. Casas, C. Mantell, C. Pereyra, E.J.M. de la Ossa, Precipitation of antioxidant fine particles from Olea europaea leaves using Supercritical Antisolvent process, The Journal of Supercritical Fluids (2014), http://dx.doi.org/10.1016/j.supflu.2014.11.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Precipitation of antioxidant fine particles from Olea europaea leaves using Supercritical Antisolvent process C. Chinnarasu,* † § A. Montesa, M.T. Fernández- Ponce†, L. Casas†, C. Mantell†, C. Pereyra†,
Department of Chemical Engineering and Food Technology,
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†
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E. J. Martínez de la Ossa†
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Faculty of Sciences, International Excellence Agrifood Campus (CeiA3),
Department of Environmental Science, PSG College of Arts and Science,
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§
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University of Cadiz, 11510 Puerto Real (Cadiz), Spain.
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Coimbatore, Tamil Nadu, India. 641 014.
Corresponding author:
Chandrasekar Chinnarasu PhD student
Department of Chemical Engineering and Food Technology University of Cadiz, Spain. Email: [email protected] Fax No: +34 956 016 411
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Highlights SAS process led to successful precipitation of spherical olive leaves fine particles
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SAS processed particles had stronger antioxidant activity than the extract
ABSTRACT
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The extract concentration is the most important key to get a successful precipitation
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A Supercritical Antisolvent (SAS) process was carried out to obtain precipitates with potent antioxidant activity. An extract obtained by Supercritical Fluid Extraction (SFE) of olive leaves was used in this study. The effects of different parameters on the outcome of the SAS process were analyzed: concentration (16 and 32 mg/mL), temperature (35 to 60 ºC), pressure (100 to 200 bar), CO2 flow rate (11 to 30 g/min) and solution flow rate (2 to 8 mL/min).The antioxidant activities of both the extract and the fine particles were evaluated by the DPPH assay. The antioxidant activities were much higher for the fine particles obtained by SAS precipitation than for the extracts obtained from olive leaves by SFE.
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Keywords: Olive leaves; Supercritical Fluid Extraction; Supercritical Antisolvent
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1. Introduction
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precipitation; Antioxidant Activity
Sustainable nanotechnology concerns the design of sustainable chemical products and
processes that eradicate the use and invention of harmful substances. This is a subject of increasing importance in the food, personal care product, textile, pharmaceutical, and chemical industries due to concerns over environmental, health and economic issues [1, 2]. The versatility of supercritical fluids (SCFs) in green technology has led to innovative approaches for the design of micro- and nanoparticles [3]. More specifically, Supercritical Fluid Extraction (SFE) and Supercritical Antisolvent (SAS) processing of natural matter are very effective techniques. SFE is an important alternative as it has several advantages, including environmental compatibility, increased selectivity, the possibility of automation,
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the markedly reduced need for organic solvents, and low levels of degradation of chemical compounds [4-6]. The SAS process is a semi-continuous precipitation technique that produces fine or
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ultrafine particles using a supercritical fluid as an antisolvent under conditions above the critical pressure (Pc) and critical temperature (Tc). CO2 is the most commonly used
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antisolvent due to its low cost, natural abundance, non-flammable and non-toxic nature,
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environmental compatibility, and its non-corrosive nature to the apparatus. Furthermore, materials that are sensitive to mechanical or thermal stress can be micronized due to the
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moderate critical pressure (Pc = 73.8 bar) and temperature (Tc = 304.1 K) [7, 8]. More recently, olive leaves have attracted increasing interest from the scientific
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community and industries worldwide due to their health-promoting benefits. Olive leaves contain plentiful bioactive compounds, including oleuropein, hydroxytyrosol, verbascoside,
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apigenin-7-glucoside, luteolin-7-glucoside, flavonoids, tocopherol, and triterpenes [9]. Olive
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leaf extracts (OLE) have exhibited anti-HIV activity [10], anti-tumor activity in breast cancer
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cells [11], antioxidative, anti-ischemic, hypoglycaemic activity [12], neuroprotective effects [13], and antinociceptive activity [14]. Most conventional extraction techniques suffer from several drawbacks such as lack
of selectivity, the need for large amounts of organic solvent, high temperatures, and long processing times. SFE is therefore a good alternative to obtain extracts that have high concentrations of olive leaf polyphenols. Extracts enriched in oleuropein [15, 16] and tocopherol [9] have been already produced from olive leaves on using SFE. The use of other extraction methods such as microwave-assisted (MAE) extraction [17], homogenizer-assisted extraction (HAE), and ultrasound-assisted extraction (UAE) [18], among others, has
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overcome or reduced most of the drawbacks associated with conventional extraction methods. The precipitation of olive leaf antioxidant fine particles (OLA FPs) from supercritical
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extracts has been carried out by SAS processes, with the particle size and particle size distribution controlled by other micro- or nanoparticle formation methods [19] using
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extremely high temperatures and organic solvents that are not recommended for the
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production of sensitive nanoparticles. To the best of our knowledge, studies in which SAS precipitation has been applied to olive leaf antioxidants (OLA) have not been reported to
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date. Pharmaceutical compounds such as ampicillin, amoxicillin, and naproxen obtained by SAS processes were extensively investigated in our previous studies [8, 20-23]. The
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preparation of these pharmaceutical compounds by conventional techniques may lead to environmental pollution.
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The proposed OLA FPs/SAS system should avoid the use and generation of toxic and
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environmentally damaging substances. The antioxidant activities of the resulting materials
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were evaluated by the DPPH assay in conjunction with UV-Visible spectrophotometry. 2. Materials and methods 2.1. Solvents
CO2 with a minimum purity of 99.8% was provided by Linde (Spain). Ethanol (HPLC
gradient grade) was obtained from Panreac (Barcelona, Spain). 2.2. Plant material Olea europea leaves were collected in 2013 in the region of Jaén (Spain). The leaves were dried in a well-ventilated shady place and the samples were stored. Prior to extraction
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the leaves were ground using Bosch 6000 W grinder fitted with a sieve of approximately 5 mm. 2.3. Supercritical fluid extraction (SFE)
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The supercritical fluid extraction was performed in a high pressure apparatus supplied
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by Thar Technology (Pittsburgh, PA, USA, model SF1000). A schematic diagram of the equipment used in this work is shown in Figure 1 and the system was described in detail in a
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previous publication [24]. For the test, the extraction vessel was loaded with approximately 150 g of olive leaves. The extraction process was performed under the following operating
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conditions: pressure of 100 bar, temperature of 55 ºC, supercritical CO2 flow rate of 16 g/min and ethanol flow rate of 4 mL/min as a co-solvent. These extraction conditions were chosen
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on the basis of previous studies on SFE [24, 25]. The ethanolic extract was recovered in a cyclonic separator in a concentration of 32 mg/mL and then collected in brown glass bottles
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prior to the SAS processes. A portion of the extract was diluted to 16 mg/mL with ethanol
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and another small portion was evaporated to dryness using a rotary evaporator (IKA® RV 10)
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at 40 ºC and the residue was studied by scanning electron microscopy to analyze the olive extract prior to the SAS process. The remaining sample was used in the SAS process. 2.4. Supercritical antisolvent precipitation (SAS) A schematic diagram of the pilot plant, purchased from Thar Technologies® (model
SAS 200), is shown in Figure 2 and the system was described in detail in a previous publication [8]. This equipment was used to carry out the experiments listed in Table 1. The SAS 200 system comprises the following main components: two high-pressure pumps, one for the CO2 (P1) and the other for the solution (P2), which incorporate low-deadvolume head and check valves to provide efficient pumping of CO2 and a range of solvents; a
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stainless steel precipitator vessel (V1) with a volume of 2 L, which consists of two parts, the main body and the frit, all surrounded by an electrical heating jacket (V1-HJ1); an automated high precision back-pressure regulator (ABPR1) attached to a motor controller with a
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position indicator; and a jacketed (CS1-HJ1) stainless steel cyclone separator (CS1) with a volume of 0.5 L to separate the solvent and CO2 once the pressure is released by the manual
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back-pressure regulator (MBPR1). The following auxiliary elements were also required: a low pressure heat exchanger (HE1), cooling lines, and a cooling bath (CWB1) to keep the
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CO2 inlet pump cold and to chill the pump heads; an electrical high-pressure heat exchanger
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(HE2) to preheat rapidly the CO2 in the precipitator vessel to the required temperature; thermocouples placed inside (V1-TS2) and outside (V1-TS1) the precipitator vessel, inside
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the cyclone separator (CS1-TS1), and on the electric high pressure heat exchanger to obtain continuous temperature measurements; and a FlexCOR coriolis mass flowmeter (FM1) to
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measure the CO2 mass flow rate and other parameters such as total mass, density,
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temperature, volumetric flow rate, and total volume. The influence of different parameters on the particle size (PS) and particle size
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distribution (PSD) of the olive leaf extract was studied: concentration (Cc) levels of 16 and 32 mg/mL, pressures (P) between 100 and 200 bar, temperatures (T) between 35 and 60 ºC, CO2 mass flow rate (QCO2) of 11 to 30 g/min and liquid solution flow rate (QL) between 2 and 8 mL/min. The nozzle diameter and washing time were kept constant at 100 µm and 60 min respectively. The operating conditions for the SAS experiments are shown in Table 1. All experiments were performed using the same procedure. CO2 was pumped into the vessel and, when supercritical conditions were achieved, the ethanolic extract was pumped and sprayed into the vessel through the nozzle. The small drops of ethanol were dissolved by the supercritical CO2 and this caused supersaturation of the extract and the consequent
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precipitation of the OLA in the form of a powder; the precipitate was recovered from both the inner wall and the frit of the precipitator vessel. 2.5.
Sample characterization
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Scanning electron microscopy (SEM) images of the dry extract and OLA FPs were
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obtained using a SIRION FEG scanning electron microscope. Prior to analysis, the dry extract and OLA FPs were placed on carbon tape and then covered with a coating of gold
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using a sputter coater. The SEM images were processed using Scion image analysis software (Scion Corporation) to obtain the particle sizes. The mean particle size and particle size
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distribution, as a measurement of the distribution width, were calculated using Statgraphics plus 5.1 software. Around 300 particles were counted to perform the analysis in each
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experiment.
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High pressure liquid chromatography (HPLC) was performed on an Agilent HPLC
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series 1100 system (Agilent, Germany) equipped with a degasser, a quaternary pump, an autosampler, and a UV/vis detector. ChemStation® HP software was used for data analysis. A
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Lichrospher 100 RP-18 column (5 μm) (Agilent Technologies, Germany) was used. Gradient elution was carried out with water/2% v/v acetic acid (solvent A) and MeOH/2% v/v acetic acid (solvent B) at a constant flow rate of 0.45 mL/min. A linear gradient profile with the following proportions (v/v) of solvent B was applied [t (min), %B]: (10, 10), (18, 20), (20, 20), (30, 40), (40, 50), (50, 100). Finally, washing and reconditioning steps for the column were included as follows [t (min), %B]: (95, 100), (97, 10). The injection volume for all samples was 20 µL. Compounds were detected at 280 nm according to the retention time. 2.6. Antioxidant activity assay with DPPH
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The antioxidant activities of the extract and fine particles were determined by the 2,2diphenyl-1-picrylhydrazyl radical (DPPH) assay. The method employed was designed by taking in account the methods described by Brand-Williams and Scherer and Godoy [26, 27].
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Ethanolic solutions of the samples at different concentrations were each added to 3.9 mL of a 6 × 10–5 mol/L DPPH solution in ethanol. The absorbance of DPPH was monitored
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spectrophotometrically at 515 nm at 0 min and every 2 min until the reaction reached the steady state. The DPPH concentration (CDPPH) in the reaction medium was calculated from a
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calibration curve determined by linear regression with Equation 1.
(1)
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Abs = 12.709 x CDPPH + 0.002
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The percentage of DPPH remaining was calculated using Equation 2. % DPPH remaining = CDPPHt/ CDPPHo x100
(2)
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The EC50 (efficient concentration providing 50% inhibition) was calculated
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graphically using a non-linear curve fitting by plotting the sample concentration against the
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% DPPH remaining on steady state. The antioxidant activity was expressed as the Antioxidant Activity Index (AAI), which was calculated considering the final concentration of DPPH and the EC50 of the tested compound in the reaction as follows: AAI = Final concentration of DPPH (µg/mL) / EC50 (µg/mL)
(3)
The final concentration of DPPH was calculated with respect to the concentration of
DPPH in the reaction medium. The antioxidant activity was considered poor when AAI ≤ 0.5, moderate when AAI was between 0.5 and 1.0, strong when AAI was between 1.0 and 2.0, and very strong when AAI ≥ 2.0 [22]. The assays were carried out in triplicate. 3. Results and Discussion
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3.1. Supercritical antisolvent precipitation Eight of the fifteen experiments (1, 4, 5, 6, 7, 8, 9 and 10) led to the successful precipitation of olive leaf antioxidants. The morphology of the unprocessed olive leaf extract
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(Figure 3) was improved by the SAS process: SEM images of OLA particles formed in the experiments mentioned above showed the formation of spherical microparticles with mean
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the PSD values were in the range 0.02–0.51 µm (see Figure 5).
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particles sizes in the range 300–1060 nm (Figure 4). The sizes were uniformly distributed and
The distribution of the OLA fine particles can be fitted to log normal distributions and
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the standard deviations (SD) are also shown. It can be seen from Figures 4 and 5 that the smallest PS with the narrowest distribution was obtained in experiment 9, followed by those
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in experiments 8, 5, 1, 7 and 10, respectively. All of the precipitates were recovered from the
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6.
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single phase on the inner wall of the precipitator vessel and on the nozzle, as shown in Figure
The initial concentration of the extract seems to play a major role in the precipitation
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of particles. The solution with the lowest initial concentration (16 mg/mL) was used in experiments 2, 3, 11, 12, 13, 14 and 15 but particles were not formed in any of these cases. Particle formation was successfully achieved on increasing the concentration of the extract to 32 mg/mL, i.e., experiments 1, 4, 5, 6, 7, 8, 9 and 10. The concentration of the extract was therefore considered to be the parameter that had the most marked influence on the precipitation of olive leaf antioxidants. As can be seen in Figure 4 (runs 1 and 5), an increase in the pressure led to a decrease in both the PS and PSD. This result can be explained by considering that an increase in pressure at constant temperature enhances the solvent power of supercritical CO2 towards the solvent. As a consequence, the liquid solvent molecules are more strongly captured by the
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CO2 and this reduces the possible interaction between solvent and antisolvent compounds [28]. Furthermore, it was observed that run 4 gave a smaller particle size without agglomerates in comparison to run 6, indicating that a lower temperature leads to a smaller
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particle size. The solubility increases with temperature in this range of solvent concentrations and thus higher temperatures reduce the level of supersaturation and larger particles are
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formed [29].
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The liquid flow rate and CO2 flow rate had the most marked influence on the PS and PSD. It can be observed in Figure 4 (run 9) that the higher CO2 flow rate gave a lower
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particle size in comparison with run 8. This fact can be explained as being due to a hydrodynamic effect, meaning that turbulence increases at higher flow rates and higher
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supersaturations are reached, which in turn leads to the formation of smaller particles. Moreover, if the flow rate of CO2 is increased, the bulk fluid has a smaller amount of ethanol
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and the solubility of the antioxidant compounds in these fluids is lower, thus leading to the
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formation of smaller particles [29].
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It was noted that the higher liquid solution flow rate used in run 10 gave a larger particle size when compared with runs 4 and 7. The degree of mixing can be improved at higher liquid flow rates under miscible conditions and that results in a higher supersaturation and the formation of smaller particles [28]. However, in our case the trend was opposite. This finding can be explained because an increase in the flow rate means that the fluid phase formed in the precipitator vessel contained a high quantity of ethanol and this, in turn, reduced the rate of the antisolvent effect of the CO2. The micronization process was therefore shifted towards the growth process and a larger PS was produced. 3.2. Antioxidant activity of OLE and OLA FPs
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Supercritical OLE and SAS precipitation of OLA FPs were evaluated for their radical scavenging activity with the DPPH assay. The radical scavenging activity was evaluated in two SAS samples and significant variations were not observed. The results from OLE and
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run 9 are shown in Table 2. It can be observed in Figure 7 that the AAI activity of the OLA FPs is superior to that of the olive extract.
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Chromatographic analysis of the products obtained indicates that the majority of the
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compounds extracted are present in the particles precipitated by the SAS process. The compounds observed at the initial retention time of the chromatogram (8 min) (Figure 8)
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belong to the tyrosol family and these derivatives have a high antioxidant activity. At a retention time of around 30 min oleuropein was observed and this is a major component in
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both products. Finally, after around 38 min a family of compounds that are not in the SAS precipitated particles was observed. Flavone glycosides also appear in this region of the
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chromatogram and these generally have a lower antioxidant activity than tyrosol and
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oleuropein. Taking into account the conditions under which the chromatograms were obtained, the less polar compounds would appear at longer retention times and, therefore,
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these compounds would not be expected to precipitate in the SAS process but would be carried by CO2 and solvent. The results of the chromatographic analysis indicate that the compounds with higher antioxidant activity are more concentrated in the SAS precipitate particles than in the initial extract, a finding that explains the higher antioxidant activity of this product [30, 31]. 4. Conclusions The supercritical antisolvent (SAS) process is a feasible approach for the formation of spherical olive antioxidant fine particles in the range of 300–1060 nm from supercritical OLE. In comparison to supercritical OLE, the SAS precipitation of OLA FPs leads to higher
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antioxidant activity, indicating that the compounds with higher antioxidant activity are more concentrated in the SAS precipitate particles than in the initial extract. The initial concentration of the solution is a key factor in achieving a successful precipitation. The
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temperature and pressure both have significant impact on the particle size. The liquid solution and SCCO2 flow rates had a marked influence on PS and PSD in that lower and
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higher rates, respectively, are recommended to obtain smaller particles. In the present study a green precipitation procedure has been developed that affords antioxidant-rich natural fine
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particles. The method also minimizes precipitation times, avoids the use of harmful organic
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solvents and eliminates environmental pollution. It is also important to note that the resulting fine particles had desirable properties and are suitable for human consumption.
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ACKNOWLEDGMENTS
We gratefully acknowledge a grant (EIDA3-CEIA3) for the professional development
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of a Co-Supervised Doctoral Thesis for Foreign PhD Students and the Spanish Ministry of
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support.
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Science and Technology (Project CTQ2011-22974v and CTQ2013-47058-R) for financial
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Table legend P (bar)
T (˚C)
QCO2 (g/min)
QL (mL/min)
Cc (mg/mL)
¶ PS (nm)
Success
1
100
35
20
2
32
590
+
2
100
40
11
5
16
-
3
150
35
11
5
16
4
150
50
20
5
5
150
35
20
2
6
150
60
20
7
150
50
20
8
150
50
9
150
50
cr
-
us
-
700
+
32
410
+
5
32
Agglo
+
5
32
840
+
11
2
32
340
+
30
2
32
300
+
an
32
M
d
te
Ac ce p
ip t
runs
10
150
50
20
8
32
1060
+
11
150
50
20
5
16
-
-
12
150
60
20
5
16
-
-
13
200
50
30
5
16
-
-
14
200
35
30
5
16
-
-
15
200
60
20
5
16
-
-
Table 1. SAS precipitation of OLA FPs
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Sample
AAI
EC50 (µg/mL)
1
OLE
0.32
3.16
2
OLA FPs
1.35
0.74
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Test No.
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Table 2. AAI results
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Graphical abstract for formation of OLA FPs from Olea europaea leaves
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SFE
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SAS
SFE: P 100 bar, T 55ºC, CO2 flow rate 16 g/min, Co-solvent (C2H5OH) flow rate 4 g/min, t 180 min
SAS: P 150 bar, T 50ºC, CO2 flow rate 20 g/min, Co-solvent (C2H5OH) flow rate 2 g/min, t 30 min
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Figure captions
Figure 1. Supercritical fluid extraction (SFE)
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Figure 2. Supercritical Antisolvent Process (SAS)
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Figure 3. SEM image of unprocessed olive leaf extract (OLE)
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Figure 4. SEM images of olive leaf antioxidant fine particles (OLA FPs) Figure 5. Particle size distribution of OLA FPs
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Figure 6. Photographs of (a) wall precipitator and (b) nozzle with OLA FPs precipitated by
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SAS process.
Figure 7. Antioxidant activity of OLE and OLA FPs
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Figure 8. HPLC chromatograms at 280 nm of OLE and OLA FPs
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Figure 1. Supercritical fluid extraction (SFE)
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Figure 2. Supercritical Antisolvent Process (SAS)
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Figure 3. SEM image of unprocessed olive leaf extract (OLE)
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run 4
run 5
run 6
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run 7
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run 1
run 9
run 8
run 10
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run 1
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run 4
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Figure 4. SEM images of olive leaf antioxidant fine particles (OLA FPs)
run 7
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run 5
run 9
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run 8
run 10
Figure 5. Particle size distribution of OLA FPs
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a)
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b)
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Figure 6. Photographs of (a) wall precipitator and (b) nozzle with OLA FPs precipitated by SAS process.
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Figure 7. Antioxidant activity of OLE and OLA FPs
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OLE OLA FPs
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Figure 8. HPLC chromatograms at 280 nm of OLE and OLA FPs
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S0896-8446(14)00372-6 http://dx.doi.org/doi:10.1016/j.supflu.2014.11.008 SUPFLU 3162
To appear in:
J. of Supercritical Fluids
Received date: Revised date: Accepted date:
14-7-2014 10-11-2014 10-11-2014
Please cite this article as: C. Chinnarasu, A. Montes, M.T.F.- Ponce, L. Casas, C. Mantell, C. Pereyra, E.J.M. de la Ossa, Precipitation of antioxidant fine particles from Olea europaea leaves using Supercritical Antisolvent process, The Journal of Supercritical Fluids (2014), http://dx.doi.org/10.1016/j.supflu.2014.11.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Precipitation of antioxidant fine particles from Olea europaea leaves using Supercritical Antisolvent process C. Chinnarasu,* † § A. Montesa, M.T. Fernández- Ponce†, L. Casas†, C. Mantell†, C. Pereyra†,
Department of Chemical Engineering and Food Technology,
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†
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E. J. Martínez de la Ossa†
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Faculty of Sciences, International Excellence Agrifood Campus (CeiA3),
Department of Environmental Science, PSG College of Arts and Science,
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§
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University of Cadiz, 11510 Puerto Real (Cadiz), Spain.
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Coimbatore, Tamil Nadu, India. 641 014.
Corresponding author:
Chandrasekar Chinnarasu PhD student
Department of Chemical Engineering and Food Technology University of Cadiz, Spain. Email: [email protected] Fax No: +34 956 016 411
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Highlights SAS process led to successful precipitation of spherical olive leaves fine particles
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SAS processed particles had stronger antioxidant activity than the extract
ABSTRACT
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The extract concentration is the most important key to get a successful precipitation
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A Supercritical Antisolvent (SAS) process was carried out to obtain precipitates with potent antioxidant activity. An extract obtained by Supercritical Fluid Extraction (SFE) of olive leaves was used in this study. The effects of different parameters on the outcome of the SAS process were analyzed: concentration (16 and 32 mg/mL), temperature (35 to 60 ºC), pressure (100 to 200 bar), CO2 flow rate (11 to 30 g/min) and solution flow rate (2 to 8 mL/min).The antioxidant activities of both the extract and the fine particles were evaluated by the DPPH assay. The antioxidant activities were much higher for the fine particles obtained by SAS precipitation than for the extracts obtained from olive leaves by SFE.
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Keywords: Olive leaves; Supercritical Fluid Extraction; Supercritical Antisolvent
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1. Introduction
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precipitation; Antioxidant Activity
Sustainable nanotechnology concerns the design of sustainable chemical products and
processes that eradicate the use and invention of harmful substances. This is a subject of increasing importance in the food, personal care product, textile, pharmaceutical, and chemical industries due to concerns over environmental, health and economic issues [1, 2]. The versatility of supercritical fluids (SCFs) in green technology has led to innovative approaches for the design of micro- and nanoparticles [3]. More specifically, Supercritical Fluid Extraction (SFE) and Supercritical Antisolvent (SAS) processing of natural matter are very effective techniques. SFE is an important alternative as it has several advantages, including environmental compatibility, increased selectivity, the possibility of automation,
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the markedly reduced need for organic solvents, and low levels of degradation of chemical compounds [4-6]. The SAS process is a semi-continuous precipitation technique that produces fine or
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ultrafine particles using a supercritical fluid as an antisolvent under conditions above the critical pressure (Pc) and critical temperature (Tc). CO2 is the most commonly used
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antisolvent due to its low cost, natural abundance, non-flammable and non-toxic nature,
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environmental compatibility, and its non-corrosive nature to the apparatus. Furthermore, materials that are sensitive to mechanical or thermal stress can be micronized due to the
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moderate critical pressure (Pc = 73.8 bar) and temperature (Tc = 304.1 K) [7, 8]. More recently, olive leaves have attracted increasing interest from the scientific
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community and industries worldwide due to their health-promoting benefits. Olive leaves contain plentiful bioactive compounds, including oleuropein, hydroxytyrosol, verbascoside,
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apigenin-7-glucoside, luteolin-7-glucoside, flavonoids, tocopherol, and triterpenes [9]. Olive
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leaf extracts (OLE) have exhibited anti-HIV activity [10], anti-tumor activity in breast cancer
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cells [11], antioxidative, anti-ischemic, hypoglycaemic activity [12], neuroprotective effects [13], and antinociceptive activity [14]. Most conventional extraction techniques suffer from several drawbacks such as lack
of selectivity, the need for large amounts of organic solvent, high temperatures, and long processing times. SFE is therefore a good alternative to obtain extracts that have high concentrations of olive leaf polyphenols. Extracts enriched in oleuropein [15, 16] and tocopherol [9] have been already produced from olive leaves on using SFE. The use of other extraction methods such as microwave-assisted (MAE) extraction [17], homogenizer-assisted extraction (HAE), and ultrasound-assisted extraction (UAE) [18], among others, has
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overcome or reduced most of the drawbacks associated with conventional extraction methods. The precipitation of olive leaf antioxidant fine particles (OLA FPs) from supercritical
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extracts has been carried out by SAS processes, with the particle size and particle size distribution controlled by other micro- or nanoparticle formation methods [19] using
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extremely high temperatures and organic solvents that are not recommended for the
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production of sensitive nanoparticles. To the best of our knowledge, studies in which SAS precipitation has been applied to olive leaf antioxidants (OLA) have not been reported to
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date. Pharmaceutical compounds such as ampicillin, amoxicillin, and naproxen obtained by SAS processes were extensively investigated in our previous studies [8, 20-23]. The
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preparation of these pharmaceutical compounds by conventional techniques may lead to environmental pollution.
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The proposed OLA FPs/SAS system should avoid the use and generation of toxic and
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environmentally damaging substances. The antioxidant activities of the resulting materials
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were evaluated by the DPPH assay in conjunction with UV-Visible spectrophotometry. 2. Materials and methods 2.1. Solvents
CO2 with a minimum purity of 99.8% was provided by Linde (Spain). Ethanol (HPLC
gradient grade) was obtained from Panreac (Barcelona, Spain). 2.2. Plant material Olea europea leaves were collected in 2013 in the region of Jaén (Spain). The leaves were dried in a well-ventilated shady place and the samples were stored. Prior to extraction
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the leaves were ground using Bosch 6000 W grinder fitted with a sieve of approximately 5 mm. 2.3. Supercritical fluid extraction (SFE)
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The supercritical fluid extraction was performed in a high pressure apparatus supplied
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by Thar Technology (Pittsburgh, PA, USA, model SF1000). A schematic diagram of the equipment used in this work is shown in Figure 1 and the system was described in detail in a
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previous publication [24]. For the test, the extraction vessel was loaded with approximately 150 g of olive leaves. The extraction process was performed under the following operating
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conditions: pressure of 100 bar, temperature of 55 ºC, supercritical CO2 flow rate of 16 g/min and ethanol flow rate of 4 mL/min as a co-solvent. These extraction conditions were chosen
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on the basis of previous studies on SFE [24, 25]. The ethanolic extract was recovered in a cyclonic separator in a concentration of 32 mg/mL and then collected in brown glass bottles
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prior to the SAS processes. A portion of the extract was diluted to 16 mg/mL with ethanol
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and another small portion was evaporated to dryness using a rotary evaporator (IKA® RV 10)
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at 40 ºC and the residue was studied by scanning electron microscopy to analyze the olive extract prior to the SAS process. The remaining sample was used in the SAS process. 2.4. Supercritical antisolvent precipitation (SAS) A schematic diagram of the pilot plant, purchased from Thar Technologies® (model
SAS 200), is shown in Figure 2 and the system was described in detail in a previous publication [8]. This equipment was used to carry out the experiments listed in Table 1. The SAS 200 system comprises the following main components: two high-pressure pumps, one for the CO2 (P1) and the other for the solution (P2), which incorporate low-deadvolume head and check valves to provide efficient pumping of CO2 and a range of solvents; a
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stainless steel precipitator vessel (V1) with a volume of 2 L, which consists of two parts, the main body and the frit, all surrounded by an electrical heating jacket (V1-HJ1); an automated high precision back-pressure regulator (ABPR1) attached to a motor controller with a
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position indicator; and a jacketed (CS1-HJ1) stainless steel cyclone separator (CS1) with a volume of 0.5 L to separate the solvent and CO2 once the pressure is released by the manual
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back-pressure regulator (MBPR1). The following auxiliary elements were also required: a low pressure heat exchanger (HE1), cooling lines, and a cooling bath (CWB1) to keep the
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CO2 inlet pump cold and to chill the pump heads; an electrical high-pressure heat exchanger
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(HE2) to preheat rapidly the CO2 in the precipitator vessel to the required temperature; thermocouples placed inside (V1-TS2) and outside (V1-TS1) the precipitator vessel, inside
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the cyclone separator (CS1-TS1), and on the electric high pressure heat exchanger to obtain continuous temperature measurements; and a FlexCOR coriolis mass flowmeter (FM1) to
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measure the CO2 mass flow rate and other parameters such as total mass, density,
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temperature, volumetric flow rate, and total volume. The influence of different parameters on the particle size (PS) and particle size
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distribution (PSD) of the olive leaf extract was studied: concentration (Cc) levels of 16 and 32 mg/mL, pressures (P) between 100 and 200 bar, temperatures (T) between 35 and 60 ºC, CO2 mass flow rate (QCO2) of 11 to 30 g/min and liquid solution flow rate (QL) between 2 and 8 mL/min. The nozzle diameter and washing time were kept constant at 100 µm and 60 min respectively. The operating conditions for the SAS experiments are shown in Table 1. All experiments were performed using the same procedure. CO2 was pumped into the vessel and, when supercritical conditions were achieved, the ethanolic extract was pumped and sprayed into the vessel through the nozzle. The small drops of ethanol were dissolved by the supercritical CO2 and this caused supersaturation of the extract and the consequent
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precipitation of the OLA in the form of a powder; the precipitate was recovered from both the inner wall and the frit of the precipitator vessel. 2.5.
Sample characterization
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Scanning electron microscopy (SEM) images of the dry extract and OLA FPs were
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obtained using a SIRION FEG scanning electron microscope. Prior to analysis, the dry extract and OLA FPs were placed on carbon tape and then covered with a coating of gold
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using a sputter coater. The SEM images were processed using Scion image analysis software (Scion Corporation) to obtain the particle sizes. The mean particle size and particle size
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distribution, as a measurement of the distribution width, were calculated using Statgraphics plus 5.1 software. Around 300 particles were counted to perform the analysis in each
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experiment.
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High pressure liquid chromatography (HPLC) was performed on an Agilent HPLC
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series 1100 system (Agilent, Germany) equipped with a degasser, a quaternary pump, an autosampler, and a UV/vis detector. ChemStation® HP software was used for data analysis. A
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Lichrospher 100 RP-18 column (5 μm) (Agilent Technologies, Germany) was used. Gradient elution was carried out with water/2% v/v acetic acid (solvent A) and MeOH/2% v/v acetic acid (solvent B) at a constant flow rate of 0.45 mL/min. A linear gradient profile with the following proportions (v/v) of solvent B was applied [t (min), %B]: (10, 10), (18, 20), (20, 20), (30, 40), (40, 50), (50, 100). Finally, washing and reconditioning steps for the column were included as follows [t (min), %B]: (95, 100), (97, 10). The injection volume for all samples was 20 µL. Compounds were detected at 280 nm according to the retention time. 2.6. Antioxidant activity assay with DPPH
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The antioxidant activities of the extract and fine particles were determined by the 2,2diphenyl-1-picrylhydrazyl radical (DPPH) assay. The method employed was designed by taking in account the methods described by Brand-Williams and Scherer and Godoy [26, 27].
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Ethanolic solutions of the samples at different concentrations were each added to 3.9 mL of a 6 × 10–5 mol/L DPPH solution in ethanol. The absorbance of DPPH was monitored
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spectrophotometrically at 515 nm at 0 min and every 2 min until the reaction reached the steady state. The DPPH concentration (CDPPH) in the reaction medium was calculated from a
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calibration curve determined by linear regression with Equation 1.
(1)
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Abs = 12.709 x CDPPH + 0.002
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The percentage of DPPH remaining was calculated using Equation 2. % DPPH remaining = CDPPHt/ CDPPHo x100
(2)
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The EC50 (efficient concentration providing 50% inhibition) was calculated
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graphically using a non-linear curve fitting by plotting the sample concentration against the
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% DPPH remaining on steady state. The antioxidant activity was expressed as the Antioxidant Activity Index (AAI), which was calculated considering the final concentration of DPPH and the EC50 of the tested compound in the reaction as follows: AAI = Final concentration of DPPH (µg/mL) / EC50 (µg/mL)
(3)
The final concentration of DPPH was calculated with respect to the concentration of
DPPH in the reaction medium. The antioxidant activity was considered poor when AAI ≤ 0.5, moderate when AAI was between 0.5 and 1.0, strong when AAI was between 1.0 and 2.0, and very strong when AAI ≥ 2.0 [22]. The assays were carried out in triplicate. 3. Results and Discussion
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3.1. Supercritical antisolvent precipitation Eight of the fifteen experiments (1, 4, 5, 6, 7, 8, 9 and 10) led to the successful precipitation of olive leaf antioxidants. The morphology of the unprocessed olive leaf extract
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(Figure 3) was improved by the SAS process: SEM images of OLA particles formed in the experiments mentioned above showed the formation of spherical microparticles with mean
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the PSD values were in the range 0.02–0.51 µm (see Figure 5).
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particles sizes in the range 300–1060 nm (Figure 4). The sizes were uniformly distributed and
The distribution of the OLA fine particles can be fitted to log normal distributions and
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the standard deviations (SD) are also shown. It can be seen from Figures 4 and 5 that the smallest PS with the narrowest distribution was obtained in experiment 9, followed by those
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in experiments 8, 5, 1, 7 and 10, respectively. All of the precipitates were recovered from the
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single phase on the inner wall of the precipitator vessel and on the nozzle, as shown in Figure
The initial concentration of the extract seems to play a major role in the precipitation
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of particles. The solution with the lowest initial concentration (16 mg/mL) was used in experiments 2, 3, 11, 12, 13, 14 and 15 but particles were not formed in any of these cases. Particle formation was successfully achieved on increasing the concentration of the extract to 32 mg/mL, i.e., experiments 1, 4, 5, 6, 7, 8, 9 and 10. The concentration of the extract was therefore considered to be the parameter that had the most marked influence on the precipitation of olive leaf antioxidants. As can be seen in Figure 4 (runs 1 and 5), an increase in the pressure led to a decrease in both the PS and PSD. This result can be explained by considering that an increase in pressure at constant temperature enhances the solvent power of supercritical CO2 towards the solvent. As a consequence, the liquid solvent molecules are more strongly captured by the
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CO2 and this reduces the possible interaction between solvent and antisolvent compounds [28]. Furthermore, it was observed that run 4 gave a smaller particle size without agglomerates in comparison to run 6, indicating that a lower temperature leads to a smaller
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particle size. The solubility increases with temperature in this range of solvent concentrations and thus higher temperatures reduce the level of supersaturation and larger particles are
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formed [29].
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The liquid flow rate and CO2 flow rate had the most marked influence on the PS and PSD. It can be observed in Figure 4 (run 9) that the higher CO2 flow rate gave a lower
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particle size in comparison with run 8. This fact can be explained as being due to a hydrodynamic effect, meaning that turbulence increases at higher flow rates and higher
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supersaturations are reached, which in turn leads to the formation of smaller particles. Moreover, if the flow rate of CO2 is increased, the bulk fluid has a smaller amount of ethanol
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and the solubility of the antioxidant compounds in these fluids is lower, thus leading to the
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formation of smaller particles [29].
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It was noted that the higher liquid solution flow rate used in run 10 gave a larger particle size when compared with runs 4 and 7. The degree of mixing can be improved at higher liquid flow rates under miscible conditions and that results in a higher supersaturation and the formation of smaller particles [28]. However, in our case the trend was opposite. This finding can be explained because an increase in the flow rate means that the fluid phase formed in the precipitator vessel contained a high quantity of ethanol and this, in turn, reduced the rate of the antisolvent effect of the CO2. The micronization process was therefore shifted towards the growth process and a larger PS was produced. 3.2. Antioxidant activity of OLE and OLA FPs
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Supercritical OLE and SAS precipitation of OLA FPs were evaluated for their radical scavenging activity with the DPPH assay. The radical scavenging activity was evaluated in two SAS samples and significant variations were not observed. The results from OLE and
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run 9 are shown in Table 2. It can be observed in Figure 7 that the AAI activity of the OLA FPs is superior to that of the olive extract.
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Chromatographic analysis of the products obtained indicates that the majority of the
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compounds extracted are present in the particles precipitated by the SAS process. The compounds observed at the initial retention time of the chromatogram (8 min) (Figure 8)
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belong to the tyrosol family and these derivatives have a high antioxidant activity. At a retention time of around 30 min oleuropein was observed and this is a major component in
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both products. Finally, after around 38 min a family of compounds that are not in the SAS precipitated particles was observed. Flavone glycosides also appear in this region of the
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chromatogram and these generally have a lower antioxidant activity than tyrosol and
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oleuropein. Taking into account the conditions under which the chromatograms were obtained, the less polar compounds would appear at longer retention times and, therefore,
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these compounds would not be expected to precipitate in the SAS process but would be carried by CO2 and solvent. The results of the chromatographic analysis indicate that the compounds with higher antioxidant activity are more concentrated in the SAS precipitate particles than in the initial extract, a finding that explains the higher antioxidant activity of this product [30, 31]. 4. Conclusions The supercritical antisolvent (SAS) process is a feasible approach for the formation of spherical olive antioxidant fine particles in the range of 300–1060 nm from supercritical OLE. In comparison to supercritical OLE, the SAS precipitation of OLA FPs leads to higher
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antioxidant activity, indicating that the compounds with higher antioxidant activity are more concentrated in the SAS precipitate particles than in the initial extract. The initial concentration of the solution is a key factor in achieving a successful precipitation. The
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temperature and pressure both have significant impact on the particle size. The liquid solution and SCCO2 flow rates had a marked influence on PS and PSD in that lower and
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higher rates, respectively, are recommended to obtain smaller particles. In the present study a green precipitation procedure has been developed that affords antioxidant-rich natural fine
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particles. The method also minimizes precipitation times, avoids the use of harmful organic
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solvents and eliminates environmental pollution. It is also important to note that the resulting fine particles had desirable properties and are suitable for human consumption.
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ACKNOWLEDGMENTS
We gratefully acknowledge a grant (EIDA3-CEIA3) for the professional development
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of a Co-Supervised Doctoral Thesis for Foreign PhD Students and the Spanish Ministry of
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support.
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Science and Technology (Project CTQ2011-22974v and CTQ2013-47058-R) for financial
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Fluids 81 (2013) 236-244.
[24] L. Casas, C. Mantell, M. Rodriguez, A. Torres, F.A. Macias, E.J. Martinez de la Ossa, Supercritical fluid extraction of bioactive compounds from sunflower leaves with carbon dioxide and water on a pilot plant scale, Journal of Supercritical Fluids 45 (2008) 37-42. [25] M.T. Fernandez-Ponce, L.Casas, C. Mantell, M. Rodriguez, E.J. Martinez de la Ossa, Extraction of antioxidant compounds from different varieties of Mangifera indica leaves using green technologies, Journal of Supercritical Fluids 72 (2012) 168-175. [26] W. Brand-Williams, M.E. Cuvelier, C. Berset, Use of a free radical method to evaluate antioxidant activity, Lebensmittel Wissenschaft and Technonologies 28 (1995) 25-30.
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[27] R. Scherer, H. Teixeira Godoy, Antioxidant activity index (AAI) by the 2, 2-diphenyl-1picrylhydrazyl method, Food Chemistry 112 (2009) 654-658. [28] W.K. Sanvely, B. Subramaiam, R.A. Rajeswski, M.R. Defelippis, Micronization of
antisolvent, J. Pharmaceutical Sciences 91 (2002) 2026-2039.
ip t
insulin from halogenated alcohol solution using supercritical carbon dioxide as an
cr
[29] A. Martin, M. J. Cocero, Numerical modelling of jet hydrodynamics, mass transfer, and crystallization kinetics in the supercritical antisolvent (SAS) process, Journal of Supercritical
us
Fluids 32 (2004) 203-219.
an
[30] N. Xynos, G. Papaefstathiou, M.Psychis, A. Argyropoulou, N. Aligiannis, A.-L. Skaltsounis, Development of a green extraction procedure with super/subcritical fluids to
M
produce extracts enriched in oleuropein from olive leaves, Journal of Supercritical Fluids 67 (2012) 89-93.
d
[31] D. Ryan, M. Antolovich, T. Herlt, P.D. Prenzler, S. Lavee, K. Robards, Identification of
te
Phenolic Compounds in Tissues of the Novel Olive Cultivar Hardy’s Mammoth, Journal of
Ac ce p
Agricultural and Food Chemistry 50 (2002) 6716-6724.
Page 17 of 29
Table legend P (bar)
T (˚C)
QCO2 (g/min)
QL (mL/min)
Cc (mg/mL)
¶ PS (nm)
Success
1
100
35
20
2
32
590
+
2
100
40
11
5
16
-
3
150
35
11
5
16
4
150
50
20
5
5
150
35
20
2
6
150
60
20
7
150
50
20
8
150
50
9
150
50
cr
-
us
-
700
+
32
410
+
5
32
Agglo
+
5
32
840
+
11
2
32
340
+
30
2
32
300
+
an
32
M
d
te
Ac ce p
ip t
runs
10
150
50
20
8
32
1060
+
11
150
50
20
5
16
-
-
12
150
60
20
5
16
-
-
13
200
50
30
5
16
-
-
14
200
35
30
5
16
-
-
15
200
60
20
5
16
-
-
Table 1. SAS precipitation of OLA FPs
Page 18 of 29
Sample
AAI
EC50 (µg/mL)
1
OLE
0.32
3.16
2
OLA FPs
1.35
0.74
cr
ip t
Test No.
Ac ce p
te
d
M
an
us
Table 2. AAI results
Page 19 of 29
us
cr
ip t
Graphical abstract for formation of OLA FPs from Olea europaea leaves
an
SFE
Ac ce p
te
d
M
SAS
SFE: P 100 bar, T 55ºC, CO2 flow rate 16 g/min, Co-solvent (C2H5OH) flow rate 4 g/min, t 180 min
SAS: P 150 bar, T 50ºC, CO2 flow rate 20 g/min, Co-solvent (C2H5OH) flow rate 2 g/min, t 30 min
Page 20 of 29
Figure captions
Figure 1. Supercritical fluid extraction (SFE)
ip t
Figure 2. Supercritical Antisolvent Process (SAS)
cr
Figure 3. SEM image of unprocessed olive leaf extract (OLE)
us
Figure 4. SEM images of olive leaf antioxidant fine particles (OLA FPs) Figure 5. Particle size distribution of OLA FPs
an
Figure 6. Photographs of (a) wall precipitator and (b) nozzle with OLA FPs precipitated by
M
SAS process.
Figure 7. Antioxidant activity of OLE and OLA FPs
Ac ce p
te
d
Figure 8. HPLC chromatograms at 280 nm of OLE and OLA FPs
Page 21 of 29
ip t cr us an M Ac ce p
te
d
Figure 1. Supercritical fluid extraction (SFE)
Page 22 of 29
ip t cr us an M d
Ac ce p
te
Figure 2. Supercritical Antisolvent Process (SAS)
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ip t cr us an M
Ac ce p
te
d
Figure 3. SEM image of unprocessed olive leaf extract (OLE)
Page 24 of 29
run 4
run 5
run 6
te
Ac ce p
run 7
d
M
an
us
cr
ip t
run 1
run 9
run 8
run 10
Page 25 of 29
run 1
an
us
cr
run 4
ip t
Figure 4. SEM images of olive leaf antioxidant fine particles (OLA FPs)
run 7
te
d
M
run 5
run 9
Ac ce p
run 8
run 10
Figure 5. Particle size distribution of OLA FPs
Page 26 of 29
ip t
a)
an
us
cr
b)
Ac ce p
te
d
M
Figure 6. Photographs of (a) wall precipitator and (b) nozzle with OLA FPs precipitated by SAS process.
Page 27 of 29
ip t cr us an M
Ac ce p
te
d
Figure 7. Antioxidant activity of OLE and OLA FPs
Page 28 of 29
ip t cr M
an
us
OLE OLA FPs
Ac ce p
te
d
Figure 8. HPLC chromatograms at 280 nm of OLE and OLA FPs
Page 29 of 29