Green extraction approach for the recovery of polyphenols from Croatian olive leaves (Olea europea)

Accepted Manuscript Title: Green Extraction Approach for the Recovery of Polyphenols from Croatian Olive leaves (Olea europea) ˇ Authors: Predrag Putnik, Francisco J. Barba, Ivana Spani´ c, Zoran Zori´c, Verica Dragovi´c-Uzelac, Danijela Bursa´c Kovaˇcevi´c PII: DOI: Reference:

S0960-3085(17)30100-1 http://dx.doi.org/10.1016/j.fbp.2017.08.004 FBP 894

To appear in:

Food and Bioproducts Processing

Received date: Revised date: Accepted date:

16-5-2017 9-7-2017 15-8-2017

ˇ Please cite this article as: Putnik, Predrag, Barba, Francisco J., Spani´ c, Ivana, Zori´c, Zoran, Dragovi´c-Uzelac, Verica, Bursa´c Kovaˇcevi´c, Danijela, Green Extraction Approach for the Recovery of Polyphenols from Croatian Olive leaves (Olea europea).Food and Bioproducts Processing http://dx.doi.org/10.1016/j.fbp.2017.08.004 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.

Green Extraction Approach for the Recovery of Polyphenols from Croatian Olive leaves (Olea europea)

Predrag Putnik1, Francisco J. Barba2, Ivana Španić1, Zoran Zorić1, Verica Dragović-Uzelac1, Danijela Bursać Kovačević1*

1

Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000

Zagreb, Croatia 2

Universitat de València, Faculty of Pharmacy, Avda. Vicent Andrés Estellés, s/n, Burjassot 46100,

València, Spain

Corresponding author: Danijela Bursać Kovačević, PhD Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia E-mail: [email protected] Phone: +385 (1) 4605128 Fax:

+385 (1) 4605072

Graphical abstract

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Highlights 

Olive leaves extracts (OLE) are underutilized sustainable source of polyphenols



Pressurized liquid extraction is a good choice for green extraction of polyphenols



Total phenols, flavonoids/flavanols, hydroxycinnamic acids were assessed



Flavonoids/hydroxycinnamic acids were best extracted at higher times and temp



Total phenols/flavanols were best extracted at lower times and temp

Abstract Pressurized liquid extraction (PLE) shown as an innovative green technology for the effective extraction of the various phytochemicals from food by-products, therefore the aims of this study were to evaluate the application of PLE to engineer green extracts of Croatian olive leaves (Olea

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europaea, cv. Oblica) for potential industrial production (functional foods/pharmaceuticals). PLE was conducted under various cycle numbers (1, 2), temperature (60, 80, 100°C) and static times (5, 10, 15 min). Obtained extracts were characterized in terms of: (i) total polyphenols (TP); (ii) total flavonoids (TF); hydroxycinnamic acids (HCA); and (iv) flavonols (FLA). Response surface methodology revealed optimal PLE parameters for polyphenols recovery, observing differences in the extraction conditions (number of cycles, temperature and time) according to the specific polyphenol groups. TP optimal extraction conditions (53.15 mg GAE/g) were achieved after PLE (2 cycles, 80°C/5 min,) while 1 cycle with 100°C/15 min were selected as the optimal PLE conditions for TF recovery (16.51 mg QE/g). For HCA, 2 cycle, 91ºC/15-min were the conditions with highest yields (1.66 mg CA/g); and for FLA extraction under 1 cycle, 87ºC/5-min revealed the highest recovery (8.66 mg QE/g). Results indicated PLE as a good choice for green recovering polyphenols from the olive leaves. Keywords: Oblica olive leaves; green extraction; pressurized liquid extraction; total polyphenols; flavonoids; hydroxycinnamic acids 1.

Introduction Olives are one of the most produced crops, with 65%, 16%, and 15% of worldwide production

in Europe, Asia, and Africa, respectively (FAOSTAT-FAO, 2016). In 2014, Spain was the European country with the largest production, with almost 7 million tons of olives. In the same year, Earth’s landmass planted with olive trees would have covered entire area of Cuba (FAOSTAT-FAO, 2016). If standard plantation density for olives is about 50-100 trees/Ha (Gucci et al., 2012), subsequently on an existing 10 million ha grows around 770 million trees worldwide. Further, if there are approximately 11,777 leaves per olive tree (Proietti et al., 2015), that are

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usually disposed as waste, then the leaves will be available as economical raw material for a long time and for continuous large industrial production (Galanakis, 2013; Galanakis et al., 2010a; Moubarik et al., 2015; Rosello-Soto et al., 2015a). Plus, industry that processes them stands very good chances of improving their profit margin as their income will likely include wastemanagement services for olive oil producers. In conclusion, this type of processing is very sustainable industrial production, as it has secured market for the final product (food and pharmaceutical industry) and cheap raw material that even can be a probable source of an income (Galanakis, 2011, 2012; Galanakis, 2017). Today, the public and food producers are paying more attention to natural food additives and if possible, both will choose a food containing natural additives over synthetic ones. Hence, much effort is being made to identify the antioxidants from natural sources such as phenolic compounds. Polyphenols found in the olive tree, olive fruit, olive oil and olive processing by-products (olive tree leaves and olive mill wastewater) possess strong radical scavenging capacities and can play an important role in protecting against oxidative damages and cellular aging thus could find wide applications in food products and cosmetics (Galanakis, 2017). For instance, polyphenols found in olive leaves extracts showed strong antioxidant capacity and display various effects towards human

health,

namely

antifungal,

antimicrobial,

anti-hypertensive,

anti-inflammatory,

hypoglycemic, and hypocholesterolemic activity (Ghanbari et al., 2012). Similarly to sage extracts (Putnik et al., 2015; Putnik et al., 2016b), olive leaf extracts can be used as food preservatives with a simultaneous ability to prevent lipid oxidation and to add nutritive value of foods (Boskou, 2008). Olive leaves’ extracts can be incorporated into functional products such as various spreads, tomato pastes, and dressings (Boskou, 2008). Further, they can be successfully added to breads (Baiano et al., 2016) or bakery/snacks that have good market potential and ability to deliver important

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nutrients to consumer’s daily diet (Čukelj et al., 2016). Moreover, olive leaves’ extracts may be added to refined oils to recover a portion of the bioactive compounds that were lost during the processing (Boskou, 2008) or they can be sold as sole pharmaceutical supplements. Though potential health benefits of olive leaves are mainly ascribed to the presence of low molecular weight polyphenols, the combined phenolic compounds have significantly higher activities than those of the individual phenolics (Lee and Lee, 2010). Therefore, the entire leaf extracts, and not just their particular compound(s) (e.g. oleuropein), are recommended for the production of nutraceuticals, functional foods, and food additives (Ahmed et al., 2014; Hayes et al., 2011; Pereira et al., 2007). As each plant material possess its characteristic properties in terms of phenolic extraction, it is very important to develop the optimal extraction conditions (Putnik et al., 2016a). Success of the extraction is influenced by numerous factors, but mainly those are thermal stability of BAC, extraction technology, pH, type of solvent, temperature, and length of extraction (Rosello-Soto et al., 2015b). Thermal processing and preservation can significantly decrease the polyphenolic content in plant material and spend up to 25-50% of processing expenses (Bursać Kovačevć et al., 2016; Bursać Kovačević et al., 2015; Putnik et al., 2016b). Hence, it is important to evaluate various (novel) technologies that will favor polyphenolic stability during extractions (Barba et al., 2015; Bursać Kovačevć et al., 2016; Bursać Kovačević et al., 2016b) while at the same time being more eco-friendly, efficient, and cost-effective (Ameer et al., 2017). In that point of view, a pressurized liquid extraction (PLE) is broadly recognized as a green extraction approach. It is mainly due to its low organic solvent consumption thus considering as a promising innovative extraction technology for recovering polyphenols from olive leaves (Rosello-Soto et al., 2015b). It was reported that PLE was successful in extracting polyphenols from various plant matrices that

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can be implemented at industrial-scale (Barba et al., 2016). PLE employs highly pressurized and heated common solvents to obtain extracts rich with BAC (Cha et al., 2010). With the exception to pressure, all principles linked to conventional liquid extraction, apply to PLE as well. The most important PLE parameters are: selection of the appropriate solvents, extraction temperatures, number of extraction cycles, and the length of static extraction times. PLE showed promising results as it outperformed other extraction technologies that were used for polyphenolic recovery from Tunisian olive leaves. Moreover, it showed the highest yield, while changes in phenolic profiles were mainly influenced by the type of solvent (Taamalli et al., 2012a) Currently, there are 30 autochthonous olive cultivars in Croatia while Oblica is their most important representative. Oblica, commonly used for production of virgin olive oils, possess high content of biologically active compounds (BAC) (Sarolic et al., 2015). Aside from the impact of cultivar, great variations in the chemical diversity of polyphenolic compounds are observed according to the climate conditions, cultivation practices, storage time, and the extraction process (Allouche et al., 2004; Obied et al., 2005). Therefore, the recovery of polyphenols from Oblica leaves is of particular interest since the leaves of this variety have not yet been researched as potential source of bioactives. Additionally, conducted literature review for this manuscript revealed a lack of reports with systematic evaluation of PLE parameters applicable to green extraction of polyphenolic from olive leaves. In addition, it is also important to evaluate the different types of phenolic groups since, and dependent of their structure, they will present different functions or may present greater or lesser antioxidant and/or biological activities (Rosello-Soto et al., 2015b). As a result, this work intended to fill-up this gap in the research, and provide useful data for industrial recovery of polyphenols from olive leaves.

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In summary, the objectives in this research were to evaluate the influence of PLE parameters in order to obtain green olive leaves extracts rich in polyphenols. Oblica’s extracts were evaluated for their bioactive value by the content of: (i) total polyphenols (TP); (ii) total flavonoids (TF); (iii) total hydroxycinnamic acids (HCA), and (iv) total flavonols (FLA). The last step (objective) in the research was to optimize PLE parameters, namely cycle number, temperature, and length of static extraction time. Here optimization was confined to maximize content of polyphenols in green olive extracts and save the extraction (production) resources for potential industrial applications.

2. 2.1

Materials and methods Reagents, Solvents and Standards

Methanol was purchased from Macron Fine Chemicals (Norway), ethanol from Gram-Mol (Zagreb, Croatia), Folin-Ciocalteu reagent, aluminum chloride from Kemika (Zagreb, Croatia), and anhydrous sodium carbonate from T.T.T. (Sveta Nedjelja, Croatia). Hydrochloric acid was obtained from Carlo Erba reagent (Val-de-Reuil, France), potassium acetate from VWR Chemicals (Radnor, USA), Quercetin-3-O-galactoside from Extrasynthese (Lyon, France), and caffeic acid from Sigma Aldrich (Bucks, USA). 2.2

Plant Materials

Olive leaves (Olea europaea, cv. Oblica) were collected from the orchard at island of Brač (Croatia). Leaves were randomly collected from different parts of tree crowns during mid-March, and immediately transferred in the laboratory. Afterwards, leaves were dried under ambient conditions

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in the dark, then pulverized with blender, extracted and analyzed (Imetec Dolcevita CG1, 150 W). Powder particle distribution size was: d(0.9) ≤ 391.26 μm; d(0.5) ≤ 215.60 μm; d(0.1) ≤ 12.44 μm, and evaluated by Malvern, Mastersizer 2000 particle size analyzer, Germany. 2.3

Pressurized liquid extraction

The extracts were obtained with aqueous ethanol (50:50, v/v) by an accelerated solvent extractor (ASE 350, Dionex, Sunnyvale, CA, USA). Prior use, ethanol was sonicated for 20 min to remove dissolved oxygen from solvent and to avoid any possible oxidations. Extractions were performed at three different temperatures, two different static times, and two cycles (for details see “Experimental methodology and statistical analysis”). At the bottom of extraction’s cell(s) cellulose filter was placed (Thermo Scientific, Dionex™ 350/150 Extraction Cell Filters) to prevent collection of suspended particles from a collection vial. Then, 1 g of powdered leaves was mixed (1:2 ratio) with diatomaceous earth (DE), and placed in 34 mL stainless steel extraction cell. The DE (as dispersion agent, P/N 062819) was used to reduce the volume of aqueous ethanol during extraction. Clean Ottawa sand was added to fill remaining volume within the cell(s). All extraction cells were placed upon a tray, while required extraction conditions were entered at the command console of the ASE extractor (static time, temperature, cycle). Prior extractions, cells were warmed up where heat-up time varied with the set extraction temperatures (e.g. 5 min for 60-80 ºC, or 7 min for 100 ºC). In all experimental runs, extraction pressure was constant at 10.34 MPa, with flushing (solvent) volume set at 60%. To avoid polyphenolic oxidation during extraction, the cells were purged with liquid nitrogen for 90s. Produced extracts were collected in 60 mL vials, then transferred to 50 mL volumetric flasks, and made-up to volume with extraction solvent. Obtained extracts were stored in dark under -18 ºC until analysis. 8

2.4

Determination of Total Polyphenols

The total phenolic content was estimated by Folin–Ciocalteu (FC) colorimetric method previously described (Shortle et al., 2014) and slightly modified. Briefly, 0.1 mL of properly diluted olive leaves’ extracts was mixed with 0.2 mL of FC reagent and 2 mL of distilled water. After 3 min at room temperature, 1 mL of sodium carbonate (20%, w/v) was added to neutralize the solution. The solution was vigorously mixed and transferred to water bath at 50°C for 25 min. Later, the absorbance was measured at 765 nm by spectrophotometer (UV-1600PC Spectrophotometer, Geldenaaksebaan, Leuven). Extraction solvent was used for blank-controls instead of an extract, while the calibration curve was prepared with standard solution of gallic acid (50-250 mg/L). The total phenolic content was expressed as mg of gallic acid equivalents (GAE)/g of dried olive leaves. 2.5

Determination of Total Flavonoids Content

Method for assessment of TF content was previously reported by colorimetric assay with slight modifications (Chang et al., 2002). In short, 0.5 mL of properly diluted extracts with 1.5 mL of 96% ethanol, 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled water, were mixed for 5 min, and then 0.1 mL of 10% aluminum chloride (w/v) was added. The mixture was kept in a dark at room temperature for 30 min, and absorption was measured at 415 nm. The blanks contained 0.5 mL of extraction solvent, and 0.1 mL of distilled water instead of 10% aluminum chloride (w/v). A calibration curve was constructed using quercetin as the calibration standard (10−100 mg/L), and results were expressed as mg of quercetin equivalents (QE)/g of dried olive leaves.

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2.6

Determination of Total Hydroxycinnamic Acids and Total flavonols Content

Evaluation of HCA and FLA in the extracts was done by a method from the literature with some modifications (Howard et al., 2003). In summary, appropriately diluted 0.25 mL of the extracts was mixed with both, 0.25 mL of 1 g/L HCl and 4.55 mL of 2 g/L HCl. The mixture was vortexed for 2 min before the spectrophotometry that was compared against blanks at, 320 nm and 360 nm for HCA and for FL, respectively. The blanks contained 0.25 mL of extraction solvent. Standard curves of caffeic acid (2-66.7 mg/L) and quercetin (10-100 mg/L) were used to individually quantify HCA and FLA. Results were expressed as mg/g of dried olive leaves (for caffeic acid and quercetin equivalents). 2.7

Experimental methodology and statistical analysis

Experiments were designed as a full factorial randomized designs (n=36). Dependent variables were the contents of: (i) TP; (ii) FL; (iii) HCA, and (iv) FLA. Independent variables were: (i) cycle number (1, 2); extraction temperature (60, 80, and 100 °C); and length of static extraction time (5, 10, and 15 minutes) (Table 1). General hypothesis for the research was that with proper selection of cycle numbers, extraction temperatures, and lengths of static extraction times should be possible to optimize polyphenolic recovery form olive leaves. Therefore, tested methodological nullhypothesis was that the average values of dependent variables would be statistically same for the levels of independent variables, as well as their corresponding three-way interactions. Oppositely, alternative hypothesis was that the average values of dependent variables would differ for the levels of independent variables, and their corresponding three-way interactions. Descriptive statistic was used to assess the basic information about the experimental dataset (e.g. to obtain a sample basic metrics, check for normality of distribution). Normality and homoscedasticity were

10

tested with Kolmogorov–Smirnov and Levene’s test, respectively. Hypotheses (continuous variables) were tested by multivariate analysis of variance (three-way ANOVA with interactions). The significance levels for rejection of a null-hypothesis in all tests were α≤0.05. Analyses were performed with IBM SPSS Statistics (v.20), and experimental design was calculated by Statgraphics Centurion (StatPoint Technologies, Inc, VA, USA). 3.

Results and Discussion

Increased interest for obtaining particular groups of BAC from herbs and other natural sources increased the interest for their efficient extraction, which is a complex manufacturing process. Modern green extraction techniques, such as PLE, represent promising approaches that could overcome current limitations and provide their wide-reaching applications on an industrial scale and in emerging global markets (Ameer et al., 2017). Therefore, it is important to identify PLE conditions methodically, so they can be extrapolated to industrial level and provide the foundation for development of such production. Accordingly, this section of the manuscript was organized into four parts. The first three parts will discuss the general significant trends for extracting various polyphenolic groups from olive leaves (listed in Tables 1-3). Building on that data, final part will give the overall best conditions for the PLE as a logical last step of systematic analysis. 3.1

Total Phenolic Content in Olive Leaves Extracts Obtained with Pressurized liquid extraction

The effects of PLE parameters were assessed on polyphenolic recovery from olive leaves by using aqueous ethanol (50%, v/v) as an extraction solvent. Previous reports already confirmed that ethanol, as the non-toxic and affordable GRAS extraction agent for various bioactive compounds from olive leaves with PLE (Herrero et al., 2011; Taamalli et al., 2012a). Likewise, 50% aqueous ethanol solution was found as the most effective in terms of extraction of phenolic compounds

11

with PLE (Xynos et al., 2014). The results for TP content of olive leaves extracts were presented in Table 1, while influences of PLE parameters on TP were given in Table 2. The averages for TP in the extracts ranged from 41.13±0.58 to 62.99±0.043 mg GAE/g of dry material. Obtained results are comparable to those found earlier for similar extracts, but obtained with ultrasound assisted extraction (UAE) (22.16±0.10 to 46.21±0.15 mg GAE/g d.m.) (Ilbay, Sahin, & Buyukkabasakal, 2014). Concerning extraction methods, previously was shown that the extraction type did not significantly influence TP in olive leaves extracts (ultrasound vs. conventional) with average value of 54±17 mg/g d.m. (Ahmad-Qasem et al., 2014), this agreed well with our results (Table 2). Cycle number showed no significant influence on TP, while with increased static extraction time their content significantly decreased. That is somewhat expected as prolonged exposure to higher temperatures may enhance the reactions such as hydrolysis and oxidation, thus degrading portion of thermally instable polyphenols accounted by the evaluation of TP. This was previously reported for various plant materials (Bursać Kovačevć et al., 2016; Bursać Kovačević et al., 2016b; Bursać Kovačević et al., 2015; Galanakis et al., 2010b). Results from previous studies were consistent with further observations for influence of temperature in our samples, where TP increased and peaked at 80 °C then started to decline and bottomed at 100 °C. Recent review reported that temperatures above 100 °C will have tendency to decrease TP content during PLE (Barba et al., 2016). Similarly, Galanakis et al. (2010b) found that recovery of phenols with ethanol from fresh olive mill wastewater previously treated at temperatures <80°C preserved their phenol characteristics and increased their radical scavenging activity during storage. However, Herrero et al. (2011) found that with higher temperatures TP content increased in the PLE extracts, judging by 1.6-2 fold higher yield with temperature increase from 50 to 200 ºC. Possible explanation might

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be that the increase of temperature adversely affects a solvent dielectric constant and polarity, and promotes solubility of fewer soluble non-polar compounds. Such compounds are accounted by the evaluation of total polyphenols by FC-reagent, hence the increase in their quantity (Teo et al., 2010). Simultaneously, high extraction pressures (e.g. 10.34 MPa) force solvent to extraction material and enhances extraction. At temperatures above 160 °C even pressurized hot water is able to degrade cellular walls, built of hemicellulose or lignans, and cleave their covalently bind insoluble phenolic compounds. Meaning, that during the extraction at higher temperatures hemicellulose can be partially hydrolyzed. Such reactions may contribute to the release of phenolics and other compounds (e.g. reducing sugars) and inflate measurement of TP, thus elevating TP content in extracts (Pérez-Jiménez and Torres, 2011). In order to produce the most complete and efficient extraction, effects of static time should be explored within the static cycles (Mottaleb and Sarker, 2012). Therefore, to observe the significance of the interactions among investigated extraction variables (Table 2) it can be seen that 5-10 min extraction duration is the most likely option that will yield extracts with the highest TP content. Xynos et al. (2014) confirmed the existence of significant cumulative influences (i.e. interaction) of extraction temperature, solvent polarity, and number of cycles on polyphenolic recovery from olive leaves extracts by ASE. That is why such interactions were reported in this manuscript. With 56% aqueous ethanol (v/v), the highest polyphenols recovery was achieved at higher temperatures and with one cycle, and also at lower temperatures with three cycles. Static cycles tend to increase the extraction efficiency, especially for the samples that are difficult to penetrate with solvent. Usually, the addition of a cycle provides the fresh solvent to extraction matrices, which maintains favorable extraction equilibrium (Mottaleb and Sarker, 2012). Similarly,

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interaction of cycle number and temperature also showed a significant impact on total polyphenols in our olive leaves’ extracts (Table 2). Here, both cycles showed increase in total polyphenols, but only up until 80°C when their content declined. Further implying that 80 °C might be the best temperature for extracting general polyphenols from olive leaves. The lowest quantity of total polyphenols found in extracts were obtained by 2 cycles at 100 °C. This observation implies that the excessive exposure of thermally instable polyphenolic compounds to high temperatures may result with their degradation. According to the results, the length of static time should be between 5-10 minutes, as there highest TP yield was obtained at 80 °C, and then declined. Consistent with our findings were previously documented results for microwave-assisted extraction and 80% aqueous methanol solvent, which also detected temperature of 80 °C and time of 8 minutes to be optimal for polyphenols from olive leaves (Taamalli et al., 2012b). Temperature did not show the influence on the total polyphenols at 15 minutes of static extraction time. This was very implicative that 15 min length of static extraction time is too long and may adversely influence the polyphenolic content with unnecessary loss of processing resources. The cumulative influences of all extraction parameters on TP were presented in Table 3. As it can be seen, 2 cycles at 80°C for all three static extraction times (5, 10, 15 min) gave the highest TP content. Accordingly, temperature and static extraction time generally showed greater effects on TP recovery than the cycle numbers. 3.2

Total Flavonoids in Olive Leaves Extracts obtained with Pressurized Liquid Extraction

Olive leaves are important sources of flavonoids as they account for 13-27% of entire radical scavenging activity in their extracts (Goulas et al., 2010). In this study, the extracts contained considerable amounts of flavonoids, in the range from 11.80 to 26.52 mg QE/g d.m (Table 1). The average flavonoid content per extracts in this study was 16.51±0.11 mg QE/g that was 31.1% of 14

total polyphenols (Table 2). Similar amounts of flavonoids were previously reported from other sources for conventional extracts with aqueous solutions of 80% methanol (Nashwa et al., 2014), and 70% ethanol (Abaza et al., 2011). Both studies were conducted at room temperature and with extraction times in a range from 15 minutes until 24 hours. All general increases in cycle numbers, static extraction time, and temperature fostered the rise in flavonoid content in studied samples. Recent work highlighted the impact of temperature on the extraction of total flavonoids from Scutellaria baicalensis Georgi where their yield gradually increased with increasing temperature, and reached the maximum at 60 °C, when it finally dropped in a range of 60-90 °C (Liu et al., 2015). In our samples, interaction between cycle numbers and extraction times had a clear effect on flavonoids yield (Table 2). Regardless the cycle number, the largest concentrations of flavonoids were found in extracts with the highest static time (15 min). Observed interactions between cycle number and temperature indicated that for 1 cycle, and temperatures ranging from 60-80 °C flavonoid content will remain constant, while for temperature of 100 °C this yield will be the highest. Similarly, the extraction with two cycles had the same trend. However, here differences were observed even at lower temperature, or between 60 °C and the range of 80-100 °C, where flavonoid content peaked and then remained constant. This indicates that extraction with the higher number of cycles and temperatures yielded greater flavonoid content. Interestingly, interactions between static extraction time and temperature showed that shorter times and lower temperatures were not beneficial for flavonoid extraction, as they were for total polyphenols. It appeared that the highest flavonoids content was obtained with 15 min of extraction at 100 °C (Table 2). One study with subcritical water extraction of flavonoids from flaxseed showed that flavonoids were better recovered even at higher temperatures (180 °C), for the same length of extraction (Kanmaz, 2014). A possible explanation for these high-temperature

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effects can be sought in increased diffusivity with decreased viscosity. This increase favors the kinetics of desorption of the phenolic compounds from the matrix, and results with their larger yields at higher temperatures (Plaza and Turner, 2015). Finally, considering simultaneous threeway interactions (Table 3), obtained results revealed that olive leaves extracts with the greater flavonoid concentrations were indeed obtained for the majority of highest temperatures and with longest extraction times for both cycles. As previously reported, these results implicate temperature as one of the most dominant factors in flavonoids recovery (Kanmaz, 2014). 3.3

Hydroxycinnamic Acids and Total Flavonols Content in Olive Leaves Extracts obtained with Pressurized Liquid Extraction

The polyphenolic content tends to be greater in the leaves than in the fruits for particular plant species. For instance, phenolic acids and flavonols were significantly higher in a number of recently studied plants (Teleszko and Wojdylo, 2015). The olive leaves extracts from this study, had average concentrations of HCA and FLA of 1.66±0.01 mg CAE/g and 8.66±0.05 mg QE/g, respectively (Table 2). This comprises 3% (HCA) and 16.3% (FLA) of total polyphenolic content in the extracts. Our results were consistent with previously reported data for HCA (Xie et al., 2015). However, content of FLA was slightly lower than already published, possibly due to different research methodology (Skerget et al., 2005). Such results coupled with natural abundance of olive leaves (as raw material) confirmed that obtained extracts could be considered as valued source of these polyphenols. Considering the influence of extraction parameters on HCA the results showed that all intensifications in cycle numbers, static extraction times, and temperatures distinctly elevated the HCA content. This was also retained in mutual interactions among those variables. Consequently, it can be concluded that increase of all studied parameters will yield increase in HCA. Meaning, 16

that the highest extraction of the HCA can be done at temperatures close or higher than 100 °C with larger number of cycles, and with times longer or equal to 15 minutes. This implies increased thermal stability of HCA. Likely associated with isomerization of the main representatives, such as neochlorogenic and chlorogenic acids, to a species with higher thermodynamic stability (Dawidowicz and Typek, 2010). Something similar was previously observed during pasteurization that favored increase in quantity of these groups of organic acids (Bursać Kovačević et al., 2016a). Three-way interaction presented in Table 3 supported the fact that the highest HCA yields could be achieved at 100 °C and with 15 min static extraction time. Contrary to the HCA, cycle number and static extraction time showed no influence on the content of flavanols. However, increased temperature positively affected flavanol content, but only up until 80 °C, when it peaked (similar to total polyphenols), and then declined until 100 °C. When these results were broken down by interaction of the cycle and extraction time, it was observed that more of flavanols were obtained at shorter times but only for the two cycles, whereas for one cycle no influence was observed. This observation seemed to follow the trend already identified with total polyphenols, where longer extraction times decreased the content of the polyphenols likely due to thermal degradation of these compounds. This hypothesis was further implied as higher temperatures had larger flavanol content, but only for shorter static extraction times (i.e. less exposure to higher temperatures adds more polyphenolic stability). With elongation of static extraction time, degradation of flavanols seemed intensified with increased temperature. Hence, again it can be seen that the temperature was one of the most important factors for flavonols extraction from olive leaves (Table 3). This strongly implies that in order to successfully extract flavonols form olive leaves, PLE should be rapid with maximum one cycle and temperatures around 80 °C.

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3.4

Optimization of Extraction Parameters for Recovery of Polyphenols from Olive Leaves Extracts Obtained by Pressurized Liquid Extraction

Optimization is a good mathematical tool that can be successfully applied in food engineering to lower manufacturing/experimental expenses and to predict outcomes of naturally occurring processes from a large number of parameters. Depending on industrial requirements, various production aspects can be optimized, but here focus was on manufacturing extracts with highest content of biologically active compounds while saving production resources. All created mathematical models fitted well with the data, as R2adj ranged from 60-78%. Autocorrelation in all models was calculated by variance inflation factors and was lower than the acceptable level (V.I.F.≤3). Each model and all corresponding predictors were significant at p≤0.05, on contrary lack-of-fit and Durbin-Watson statistic tests were insignificant at p≤0.05. Hence, all statistical metrics confirmed validity of mathematical models. Analyzing entire dataset from Tables 1-3, optimization revealed that if the main goal of industrial production is manufacturing extracts rich in polyphenols regardless of their type, the greatest calculated yield of 64.7 mg/g of TP can be obtained with 2 cycles that should last for 5 minutes each at T=80.4 °C (Figure 1a). Further, the PLE extracts with the highest possible yield of flavonoids (25.66 mg/g) should be obtained by one 15 min cycle of extraction at 100 °C (Figure 1b). Similarly, the largest quantities of HCA will be best obtained with 2 PLE cycles that last 15 minutes each, at temperature of 91.3 °C (Figure 1c). Finally, 9.2 mg/g of flavanols would be achieved in the extracts with one PLE cycle for 5 minutes at T=87.4 °C (Figure 1d).

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4.

Conclusions

Due to high natural abundance and content of biologically active compounds that have medicinal benefits, olive leaves green extracts can be effectively used for engineering functional foods and pharmaceutical products. Such extracts can be successfully added to various food products (spreads, oils, breads, crackers etc.) or offered as different pharmaceutical supplements. Oblica olive leaves extracts in this study showed to be good sources of flavonoids/flavonols and hydroxycinnamic acids. If the goal of extraction is to obtain the highest amounts of total polyphenols (64.71 mg/g) from the leaves, then extracts should be conducted under 2 cycles and static extraction time of 5 minutes at 80 °C. Pressurized liquid extraction settings that would yield the greatest possible amounts of flavonoids (25.66 mg/g) would be set to one extraction cycle that lasts for 15 minutes at 100 °C. If hydroxycinnamic acids are the main target group of molecules in extracts (2.40 mg/g) they will be best recovered by 2 extraction cycles that last for 15 minutes at temperature of 91 °C. And lastly, if extraction was undertaken to obtain the most of flavonols (9.22 mg/g), that can be achieved with one extraction cycle, that lasts for 5 minutes at 87 °C. From obtained results, it can be concluded that pressurized liquid extraction is a good choice for the green extraction of phenolic compounds from olive leaves for potential industrial processing and can be efficiently optimized by response surface methodology.

5.

Acknowledgements

This work was supported by grant from the Croatian Science Foundation: ‘‘Application of innovative technologies for production of plant extracts as ingredients for functional food’’ (HRZZ 3035)’’ and “High voltage discharges for green solvent extraction of bioactive compounds from

19

Mediterranean herbs (IP-2016-06-1913)”. All authors declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects.

20

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Figure captions: Figure 1. The response surface plots for pressurized liquid extraction (PLE) extraction parameters and phenolic compounds from olive leaves

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Tables: Table 1. Average concentrations of total phenols, flavonoids, hydroxycinnamic acids and flavonols in olive leaves extracts obtained by pressurized liquid extraction (PLE) Static Temperature

Cycle extraction time

(°C)

TP

TF

HCK

FLA

number (min) 1

49.72±2.40 13.66±0.37

1.35±0.05 7.98±0.07

2

53.81±1.39 13.70±0.13

1.28±0.06 8.35±0.55

1

53.47±1.71 11.94±0.84

0.44±0.06 7.18±0.72

2

53.96±1.41 16.12±0.82

1.71±0.12 8.46±0.13

1

54.91±1.11 14.59±0.48

1.83±0.07 8.42±0.15

2

47.40±1.94 16.27±0.09

1.75±0.04 8.67±0.27

1

55.09±1.18 13.30±0.31

0.58±0.11 8.79±0.20

2

62.99±0.43 16.70±0.84

2.00±0.06 9.52±0.19

1

58.64±1.25 16.07±0.97

1.90±0.05 9.58±0.10

2

60.08±0.95 15.13±0.34

1.70±0.00 9.10±0.03

1

50.10±0.66 11.80±0.61

1.19±0.05 8.72±0.36

2

54.94±1.76 22.58±1.03

2.21±0.06 9.02±0.05

1

51.14±0.85 13.39±0.27

1.36±0.06 9.07±0.03

2

54.62±1.64 13.87±1.02

1.85±0.06 9.19±0.02

1

51.33±0.50 21.14±0.98

1.93±0.10 8.97±0.10

2

41.13±0.58 16.99±0.24

2.01±0.07 7.94±0.00

5

60

10

15

5

80

10

15

5 100 10

29

1

52.80±2.45 26.52±0.46

2.60±0.06 9.01±0.27

2

50.59±1.60 23.42±0.63

2.20±0.09 7.84±0.38

15

*

expressed as mean±standard deviation of mg/g

TP - total phenols TF – total flavonoids HCK - hydroxycinnamic acids FLA – total flavanols

30

Table 2. Influence of pressurized liquid extraction (PLE) extraction parameters on the content of different polyphenols in olive leaves Level

n

Cycle number

TP

TF

HCA

FLA

p=0.60‡

p≤0.01†

p≤0.01†

p=0.63‡

1

18

53.02±0.34a

15.82±0.16a

1.47±0.02a

8.63±0.07a

2

18

53.28±0.34a

17.20±0.16b

1.86±0.02b

8.68±0.07a

p≤0.01†

p=0.06‡

p≤0.01†

Static extraction time (min)

p≤0.01†

5

12

54.56±0.42a

14.10±0.19a

1.40±0.02a

8.82±0.08a

10

12

53.10±0.42a,b

16.23±0.19b

1.62±0.02b

8.54±0.08a

15

12

51.79±0.42b

19.20±0.19c

1.96±0.02b

8.61±0.08a

p≤0.01†

p≤0.01†

p≤0.01†

Temperature (°C)

p≤0.01†

60

12

52.21±0.42a

14.38±0.19a

1.39±0.02a

8.17±0.08a

80

12

56.97±0.42b

15.93±0.19b

1.60±0.02b

9.12±0.08b

100

12

50.27±0.42c

19.22±0.19c

1.99±0.02c

8.67±0.08c

p≤0.01†

p≤0.03†

p≤0.01†

Cycle number by Static extraction time (min)

p≤0.01†

1,5

6

51.98±0.61a

13.45±0.26a

1.10±0.03a

8.61±0.21a

1,10

6

54.48±0.61b

16.38±0.26b

1.42±0.03b

8.58±0.21a

1,15

6

52.60±0.61a,b

17.64±0.26c

1.88±0.03c

8.72±0.21a

2,5

6

57.14±0.57a

14.76±0.28a

1.71±0.03a

9.02±0.18a

2,10

6

51.72±0.57b

16.08±0.28b

1.81±0.03a

8.50±0.18b

2,15

6

50.98±0.57b

20.76±0.28c

2.05±0.03b

8.51±0.18b

p≤0.01†

p≤0.01†

Cycle number by Temperature (°C)

p≤0.01†

p≤0.01†

1,60

6

52.70±0.61a,b

13.40±0.26a

1.21±0.03a

7.86±0.12a

1,80

6

54.61±0.61a

13.73±0.26a

1.22±0.03a

9.03±0.12b

1,100

6

51.76±0.61b

20.35±0.26b

1.96±0.03b

9.02±0.12b

2,60

6

51.72±0.57a

15.36±0.28a

1.58±0.03a

8.49±0.10a

31

2,80

6

59.34±0.57b

18.14±0.28b

1.97±0.03b

9.22±0.10b

2,100

6

48.78±0.57c

18.09±0.28b

2.02±0.03b

8.33±0.10a

p≤0.01†

p≤0.01†

p≤0.01†

Static extraction time (min) by Temperature (°C)

p≤0.01†

5,60

4

51.76±0.73a

13.68±0.33a

1.32±0.04a

8.16±0.13a

5,80

4

59.04±0.73b

15.00±0.33b

1.29±0.04a

9.16±0.13b

5,100

4

52.88±0.73a

13.63±0.33a

1.60±0.04b

9.13±0.13b

10,60

4

53.72±0.58a

14.03±0.33a

1.08±0.04a

7.82±0.15a

10,80

4

59.36±0.58b

15.60±0.33a

1.80±0.04b

9.34±0.15b

10,100

4

46.23±0.58c

19.06±0.33b

1.97±0.04c

8.46±0.15a

15,60

4

51.15±0.84a

15.43±0.33a

1.79±0.03a

8.54±0.19a

15,80

4

52.52±0.84a

17.19±0.33b

1.70±0.03a

8.87±0.19a

15,100

4

51.69±0.84a

24.97±0.33c

2.40±0.03b

8.43±0.19a

GRAND MEAN

36

53.15±0.24

16.51±0.11

1.66±0.01

8.66±0.05

*Expressed as mean±standard error of mean (mg/g)

TP - total phenols TF – total flavonoids HCK - hydroxycinnamic acids FLA – total flavanols

32

Table 3. Three-way influence of pressurized liquid extraction (PLE) extraction parameters on the content of different polyphenols in olive leaves Cycle numbe r 1

Static

Temperatur

extraction 5 time (min) 10

15

5

2

10

15

e60 (°C) 80 100 60 80 100 60 80 100 60 80 100 60 80 100 60 80 100

TP 49.72±1.15a 55.09±1.15a 51.14±1.15a 53.47±0.89a,b 58.64±0.89a 51.33±0.89b 54.91±1.13a 50.10±1.13a 52.80±1.13a 53.81±0.89a 62.99±0.89b 54.62±0.89a 53.96±0.73a 60.08±0.73b 41.13±0.73c 47.40±1.25a 54.94±1.25b 50.59±1.25c

TF 13.67±0.23a 13.30±0.23a 13.39±0.23a 11.94±0.66a 16.07±0.66b 21.14±0.66c 14.59±0.37a 11.81±0.37b 26.52±0.37c 13.70±0.54a 16.70±0.54a 13.87±0.54a 16.12±0.37a 15.13±0.37a 16.99±0.37a 16.27±0.49a 22.59±0.49b 23.42±0.49b

HCA

FLA

1.35±0.06a 7.98±0.09a 0.58±0.06b 8.79±0.09b 1.36±0.06a 9.07±0.09b 0.44±0.05a 7.18±0.30a 1.90±0.05b 9.58±0.30b 1.93±0.05b 8.97±0.30b 1.83±0.04a 8.42±0.19a 1.19±0.04b 8.72±0.19a 2.60±0.04c 9.02±0.19a 1.28±0.04a 8.35±0.24a 2.00±0.04b 9.53±0.24a 1.85±0.04b 9.19±0.24a 1.71±0.06a 8.46±0.05a 1.70±0.06a 9.10±0.05b 2.01±0.06a 7.95±0.05c 1.75±0.05a 8.67±0.19a,b 2.21±0.05b 9.02±0.19a 2.20±0.05b 7.84±0.19b

*

Expressed as mean±standard error of mean (mg/g)

TP - total phenols TF – total flavonoids HCA - hydroxycinnamic acids FLA – total flavanols

33