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Optimization of ultrasound-assisted extraction of phenolic compounds: Oleuropein, phenolic acids, phenolic alcohols and flavonoids from olive leaves and evaluation of its antioxidant activities
Industrial Crops & Products 124 (2018) 382–388
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Optimization of ultrasound-assisted extraction of phenolic compounds: Oleuropein, phenolic acids, phenolic alcohols and flavonoids from olive leaves and evaluation of its antioxidant activities
T
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Maria Iraklia, , Paschalina Chatzopouloua, Loukia Ekateriniadoub a b
Hellenic Agricultural Organization–Demeter, Institute of Plant Breeding and Genetic Resources, 5700, Thermi, Thessaloniki, Greece Hellenic Agricultural Organization–Demeter, Veterinary Research Institute, 57001, Thermi, Thessaloniki, Greece
A R T I C LE I N FO
A B S T R A C T
Keywords: Olive leaves Oleuropein Hydroxytyrosol Flavonoids Phenolic acids Antioxidant activity
Olive leaves are well known for many useful pharmacological effects. Some of the health benefits are related to phenolic composition, especially to oleuropein (OLE) and flavonoids (FLs) content. This study aimed to investigate the influence of ultrasound-assisted extraction conditions (solvent type, solvent concentration, extraction time and temperature) on the extract yield of OLE, hydroxytyrosol (HTR), FLs (rutin, luteolin, luteolin-7O-glucoside and apigenin) and phenolic acids (protocatechuic, 4-hydroxybenzoic, vanillic, p-coumaric and ferulic acids) from olive leaves using single factor experiments approach. The total phenolic compounds (TPC) and their antioxidant activity based on 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (RSA) and ferric reducing antioxidant power (FRAP) were also evaluated. The highest yields of OLE and FLs, the major phenolics in olive leaves, were obtained with 50% acetone, at 60 °C for 10 min extraction. However, the yields of HTR and PAs increased when water was used as extraction solvent. Good, positive, correlation coefficients were found between OLE, TPC, RSA and FRAP in olive leaves, especially under the influence of solvent type and solvent concentration.
1. Introduction The olive tree (Olea europaea L.) is one of the most important fruit trees in Mediterranean countries such as Italy, Spain, Greece and Tunisia. Although olive leaves are always used as animal feed, there is a rising interest in their application as a valuable material in various fields. They are regarded as a cheap raw material which can be used as a good source of bioactives and they are also one of the by-products in olive-oil production, representing 10% of the weight of olives collected. Furthermore, they also accumulate in large volumes on olive groves during the pruning of the trees (Herrero et al., 2011). Olive leaves are rich in a wide variety of phenolic compounds, such as secoiridoids (oleuropein, ligstroside, dimethyloleuropein) and flavonoids (apigenin, luteolin, luteolin-7-O-glucoside etc), along with other phenolic compounds (hydroxytyrosol, tyrosol, caffeic acid, ferulic acid etc) (Quirantes-Piné et al., 2013), that are responsible for several biological properties, including antioxidant and anti-inflammatory, antimicrobial, antiviral, anti-carcinogenic, as well as beneficial cardiovascular effects (El and Karakaya, 2009). OLE is the most representative polyphenolic constituent of olive
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leaves, responsible for the bitterness of both table olives and extravirgin olive oil. The majority of studies attribute the biological activities of olive leaves to the total or individual phenolic compounds such as OLE (Al-Azzawie and Alhamdani, 2006), HTR (Bouallagui et al., 2011), and FLs (Goulas et al., 2010). However, the phenolic profile in olive leaves varies depending on the origin and variety of the plant material, the geographical location and the agro-ecological conditions, and especially the seasons (Ranalli et al., 2006). Various methods have been used to isolate the bioactive molecules present in olive leaves, from the most common techniques to the more sophisticated including microwave-assisted extraction (Rafiee et al., 2011; Habibi et al., 2018), pressurized liquid extraction (Xynos et al., 2014) and supercritical fluid extraction (Sahin and Bilgin, 2012). It should be pointed out that most of these techniques suffer from high energy costs as they operate under high pressure. Therefore, the development of an effective, suitable and low-cost extraction method is of major importance. In this sense, ultrasound-assisted extraction (UAE) has the potential to reduce extraction times and extraction solvent volumes, as well as to increase the recoveries of active compounds. It has become a well-established technique both, in laboratories and in
Corresponding author. E-mail address: [email protected] (M. Irakli).
https://doi.org/10.1016/j.indcrop.2018.07.070 Received 3 January 2017; Received in revised form 29 June 2018; Accepted 26 July 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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Initially, samples were extracted with different concentrations of three organic solvents tested ranged from 0 to 90% v/v, fixing extraction time and extraction temperature constant at 30 °C for 10 min. Subsequently, the effect of extraction time was investigated by varying the extraction time from 10 to 120 min using the best organic solvent concentration chosen in the initial step and kept the extraction temperature constant at 30 °C. Lastly, the effect of extraction temperature was investigated using the best organic solvent concentration and extraction time determined in the previous stage with extraction temperature ranged from 30 to 65 °C.
industrial scale as well. Several authors found that UAE method is faster, simpler and more efficient than maceration/stirring for the extraction of OLE from olive leaves (Ahmad-Qasem et al., 2013; Xie et al., 2015; Cifá et al., 2018). A recent review highlighted the extraction methods and potential application of the bioactive components of olive leaves (Rahmanian et al., 2015). Although the effect of extraction conditions on the extraction yield of OLE, has been extensively investigated, few studies reporting the effect of extracting parameters on the recovery of other class of phenolic compounds from olive leaves (Delgado-Povedano et al., 2017). The aim of the present study was to maximize the extraction yields of OLE, HTR, FLs and PAs from olive leaves in respect to decrease operating costs with the possibility of lower volumes of solvent and lower extraction times and temperatures. We investigated the application of UAE and the optimization of extraction parameters such as solvent type, solvent concentration, extraction time and temperature in order to obtain extract rich in bioactive compounds with high antioxidant activities as evaluated by RSA and FRAP tests using single factor experiments approach.
2.5. Determination of total phenolic content (TPC) The amount of TPC in olive leaves extracts was determined according to the Folin-Ciocalteu method (Singleton et al., 1999) with minor modifications. Briefly, 200 μL of each extract was reacted with 800 μL of Folin-Ciocalteu reagent (diluted 10-fold) for 2 min. Then, 2 mL of sodium carbonate (7.5% w/v) was added and the volume was adjusted to 10 mL with distilled water. The mixture was allowed to stand for 1 h at room temperature in the dark and the absorbance was measured at 765 nm against blank. The results were expressed as mg of GA equivalents per g of the dried sample (mg GAE/ g dw).
2. Materials and methods 2.1. Chemicals
2.6. Antioxidant activity
OLE, TYR, HTR, luteolin-7-O-glucoside (LUTG), protocatechuic acid (PRCA), p-coumaric acid (pCA), ferulic acid (FA), vanillic acid (VA) and p-hydroxy-benzoic acid (pHBA) were supplied by Sigma-Aldrich (Steinheim, Germany). Apigenin (API), rutin (RUT), luteolin (LUT) and gallic acid (GA) were obtained from Extrasynthese (Genay Cedex, France). Analytical grade of Folin-Ciocalteu, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 6-hydroxyl-2,5,7,8- tetramethychromane-2-carboxylic acid (Trolox) and 2, 4, 6-tripyridyl-s-triazine (TPTZ) were from SigmaAldrich (Steinheim, Germany). All other solvents/chemicals obtained from Chem-Lab (Zedelgem, Belgium) were of analytical grade or highperformance liquid chromatography (HPLC) grade.
2.6.1. DPPH free radical scavenging activity (RSA) The RSA was based on the protocol described by Yen and Chen (1995) with some modifications. Aliquots (150 μL) of extracts were reacted with 2.85 mL DPPH solution in methanol (0.1 mM). After agitation, the reaction mixture was incubated in the dark at room temperature for 5 min and the absorbance was measured at 516 nm. The free radical scavenging capacity (in percentage) was calculated by using the following equation: RSA (%) = (Ao − As) /Ao × 100 where Ao is the absorbance of the blank (methanol) and As is the absorbance of the sample. Results were expressed as mg Trolox equivalents per g of dried sample (mg TE/g dw).
2.2. Plant material Fresh green olive leaves (Olea europaea L., variety Chalkidiki), were collected from the trees grown in north of Greece. Collected leaves were air dried in oven at 40 °C and then were milled using a laboratory mill equipped with a 0.5 mm sieve and finally stored at 4 °C prior to extraction.
2.6.2. Ferric reducing antioxidant power assay (FRAP) The FRAP assay was performed according to Benzie and Strain (1999) with some modifications. Briefly, the fresh FRAP reagent included 10 mL of 10 mM TPTZ solution in 40 mM HCl plus 10 mL of 20 mM FeCl3.6H2O and 100 mL of 300 mM acetate buffer pH 3.6. Aliquot of extract (100 μL) was reacted with the FRAP solution (3 mL) at 37 °C for exactly 4 min under dark conditions. Readings of the colored product were then taken at 593 nm against blank and the results were expressed as mg Trolox equivalents per g of dried sample (mg TE/g dw).
2.3. Ultrasound-assisted extraction of phenolic compounds Powdered and dried olive leaves (250 mg) were extracted with solvent (20 mL) in an ultrasound bath (frequency 37 kHz, model FB 15051, Thermo Fisher Scientific Inc. Loughborough, England) for the different times and temperatures. Then, the crude extracts were centrifuged at 1500 × g for 10 min (Universal 320R, Hettich, Germany), the supernatants were filtered using 0.45-μm syringe filters and used directly for estimation of TPC, assessment of antioxidant capacities and HPLC analysis of phenolic compounds. Each extraction was triplicated and all analysis were performed in three replications.
2.7. HPLC profile of phenolic compounds The analyses were performed on an HPLC Agilent 1200 system (Agilent Technology, Urdorf, Switzerland) equipped with a 250 × 4.6 mm i.d., 5 μm Nucleosil 100 C18 column (MZ, Mainz, Germany) maintained at 30 °C, a 20 μL loop and diode-array detector (DAD). Mobile phase consists of three solvents: (A) 1% acetic acid in water, (B) acetonitrile and (C) methanol and the following gradient program was performed: 0 min, 90% A-0% B; 10 min, 80% A-4% B; 25 min, 75% A-5% B; 30 min, 65% A-5% B; 31 min, 40% A-0% B; 37 min, 35% A-20% B; 50 min, 20% A-80% B. The flow rate of mobile phase was 1.3 mL/min. The DAD recorded the spectra at 260, 280, 320, and 360 nm and the chromatograms were analysed using the Agilent Chemstation software (version B.04.01, Agilent Technologies). Identification of phenolics in olive leaves extract was obtained by comparison of retention times and UV/VIS spectra with those of
2.4. Experimental design In the present study, single factor experiments was used to determine the optimum conditions for extracting phenolic compounds from olive leaves. Four extraction solvents were used: ethanol, methanol, acetone and water. Three independent variables were studied, namely organic solvent concentration, extraction time and extraction temperature in order to optimize the extraction conditions. The level for each independent variable was chosen based on the process responses, OLE, HTR, FLs and PAs as well as TPC, RSA and FRAP. 383
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Results illustrated in Fig. 2, showed similar influence of solvent concentration on the extraction yield of OLE, HTR, PAs and FLs of the obtained extracts. Extraction yield of OLE, the major phenolic compound in olive leaves extract, increased significantly (p < 0.05) as the concentration of all solvents used increased up to 50% and then followed by a considerable drop with further increases in solvent. This is expected due to polar hydroxyl groups of OLE and needed thus polar solvent for extraction. Additionally, the solvent mixtures are better as they deactivate the enzymes which are responsible for conversion of OLE into other compounds which have high protein-denaturing, and protein cross linking activities (Yateem et al., 2014). However, this trend declined when the ethanol and methanol concentration varied from 70 to 90%. This observation could be explained by the fact that a strong polarity might increase the impurities, which may prevent the dissolution of OLE or that the medium polarity solvent was appropriate for extracting OLE according to Xie et al. (2015). The maximum achieved OLE yields were recorded for 50% concentration, 3fold greater than those obtained by water, which represents the lowest value. A similar trend was observed in the yield of total identified FLs. The FLs yield of all extracts increased as the solvent concentration increased up to 50%, whereas further increase up to 90% leads to a rapidly decrease. The highest yield of total FLs was recorded for 50% concentration for all solvents, more than 2-fold of aqueous extracts. On the other hand, the yield of HTR was maximized at 0% organic solvent and then decreased significantly with the increase of the solvent concentration up to 90% (Fig. 2). This may be linked to the fact that HTR is more soluble in water and polar organic solvents than OLE, due to different chemical structures (Habibi et al., 2018). Solvent concentrations between 20–90% showed non-significant differences or slight increase at 50% concentration for the most of the solvent systems. Similarly, the yield of PAs was maximized at 0% organic solvent concentration with significant or non-significant effects with the level of 20%, depending on the kind of solvent. Other concentrations showed non-significant differences or slight decrease. Solvent concentration had also significant effect (p < 0.05) on RSA and FRAP of olive leaves extract (Fig. 3) at most of the levels assessed. The highest values of RSA for all mixtures solvents were obtained at 50% and 70% concentration, whereas the water extract possessed the lowest one. Extract at 50% organic solvent concentration showed significantly higher FRAP values than other concentration levels, whereas no significant differences were observed at concentration levels > 50% for ethanol extracts. This observation was in good agreement with the results of TPC indicating that polyphenolics were the principal compounds responsible for the antioxidant potential of tested extracts. Our results are in accordance with those of González-Montelongo et al.
authentic standards. The extraction yield for each analyte was calculated by the following equation: Extraction yield (mg/g dw) = [(C × V)/W]/1000 where C is the concentration of analyte in each solvent calculated by calibration curves (μg/mL), V is the volume of the extract (mL), and W is the dried weight (dw) of sample (g). 2.8. Statistical analysis The results were expressed as means ± standard deviation (SD) of replicate solvent extractions and triplicate of assays. One way analysis of variance (ANOVA) was used to compare the means. The means were separated by Least Significant Difference (LSD). The statistical significant differences were defined at p < 0.05. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) 19.0. 3. Results and discussion 3.1. Selection of solvent concentration Many parameters can influence the UAE efficiency of phenolics from olive leaves, such as temperature, solvent composition, solid–solvent ratio, extraction time and solid phase particle size (Xie et al., 2015). In the present study, assays were carried out in olive leaves with particle size of 0.5 mm and solid–solvent ratio was adjusted to 1:80. In the present study, extractions with different mixtures of aqueous solvents at concentrations ranging from 0 to 90% (v/v) were performed in order to find the optimal conditions in terms of extraction yield of OLE, HTR, FLs and PAs as well as TPC, RSA and FRAP of the obtained extracts. Similar studies dealing with the extraction of polyphenols from olive leaves have recommended similar proportions of ethanolwater (Mylonaki et al., 2008; Cifá et al., 2018). The individual constituents of the olive leaves extract obtained by 0, 20, 50, 70 and 90% acetone, or ethanol, or methanol were assessed by using HPLC analysis. Eleven phenolic compounds were separated and quantified, as presented in Fig. 1, showing similar chromatographs in terms of the type of compounds found in each extract, although the quantities extracted varied. OLE was the most abundant phenolic compound in olive leaves extract, representing approximately the 95% of the identified phenolics. Among the FLs identified, LUT, LUTG, RUT and API accounted for about 3% of the overall identified phenolics. The major PAs identified was FA, followed by pCA, whereas PRCA, pHBA and VA were minor constituents.
Fig. 1. HPLC chromatograms of water, acetone, ethanol and methanol olive leaves extracts. HTR, hydroxytyrosol; PRCA, protocatechuic acid; TYR, tyrosol; pHBA, phydroxybenzoic acid; VA, vanillic acid; pCA, p-coumaric acid; FA, ferulic acid; LUTG, luteolin-7-O-glucoside; RUT, rutin; OLE, oleuropein; LUT, luteolin; API, apigenin. 384
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Fig. 2. Effect of A) acetone B) ethanol and C) methanol concentration on the extraction of OLE, HTR, FLs and PAs from olive leaves. Results are mean ± SD, n = 3; bars marked with different letters are significantly different (p < 0.05). OLE, oleuropein; HTR, hydroxytyrosol; PAs, phenolic acids; FLs, flavonoids.
(2010) who showed that 50% acetone was more efficient for extracting banana peel phenolics and therefore, produced extracts with higher antioxidant activity. On the contrary, Altıok et al. (2008) reported that 90% acetone was the best extraction solvent for the isolation of bioactive compounds from olive leaves extract. This discrepancy may be due to different extraction methods. Thus, in our experimentation, a concentration of 50% was selected as the best for the following steps.
3.2. Selection of solvent extraction The present study showed that water and mixtures of organic solvents at 50% concentration were able to extract efficiently phenolic compounds from olive leaves (Fig. 4). Acetone extract showed the highest OLE and FLs yields with values of 106.50 mg/g dw and 2.94 mg/g dw, respectively, followed by ethanol and methanol, whereas water was found an insufficient extraction solvent, appearing the lowest extraction yield. However, water was significantly the best solvent for extracting HTR and PAs from olive leaves, followed by other solvents presenting non-significant yield values (Fig. 4B). The extraction solvent systems used, affected similarly the TPC of olive leaves extract; 50% acetone extract yielded significantly higher TPC than methanol and ethanol extracts (Fig. 4A), whereas water extract resulted the lowest one. The same trend was observed for the antioxidant activity of extracts; acetone extract exhibited significantly the highest RSA and FRAP values, followed by methanol and ethanol extracts, exhibiting non-significant effects. Acetone has been also demonstrated to be more effective than other organic solvents for extracting phenolics from many raw materials such as buckwheat (Sun and Ho, 2015), soybean (Lien et al., 2015) etc. Generally, it is strongly believed that the higher molecular weight of the solvent, the lower polarity, which enable other substances, of about the same molecular weight, to be easily extracted (Mokrani and Madani, 2016). Therefore, 50% acetone extract was selected for extracting phenolics from olive leaves, since OLE, the major phenolic compounds class present in olive
Fig. 4. Effect of solvent type on the extraction of A) TPC, RSA and FRAP and B) OLE, HTR, FLs and PAs from olive leaves. Results are mean ± SD, n = 3; bars marked with different letters are significantly different (p < 0.05). Abbreviations as Figs. 2 and 3.
Fig. 3. Effect of A) acetone B) ethanol and C) methanol concentration on the extraction of TPC, RSA and FRAP from olive leaves. Results are mean ± SD, n = 3; bars marked with different letters are significantly different (p < 0.05). TPC, total phenolic content; RSA, DPPH radical scavenging activity; FRAP, ferric reducing antioxidant power. 385
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Fig. 5. Effect of time on the extraction of A) TPC, RSA and FRAP and B) OLE, HTR, FLs and PAs from olive leaves. Results are mean ± SD, n = 3; bars marked with different are significantly different (p < 0.05). Abbreviations as Figs. 2 and 3.
Fig. 6. Effect of temperature on the extraction of A) TPC, RSA and FRAP and B) OLE, HTR, FLs and PAs from olive leaves. Results are mean ± SD, n = 3; bars marked with different letters are significantly different (p < 0.05). Abbreviations as Figs. 2 and 3.
Table 1 Correlation coefficients between different assays under the influence of extraction conditions. Solvent type RSA TPC RSA FRAP
0.955
Solvent concentration
FRAP ***
OLE ***
0.974 0.973***
FLs ***
0.961 0.959*** 0.995***
RSA ***
0.985 0.962*** 0.991***
0.739
FRAP ***
Extraction time OLE
***
0.786 0.888***
FLs ***
0.811 0.773*** 0.853***
RSA ***
0.678 0.417*** 0.614***
Extraction temperature
FRAP ***
0.699
OLE ***
0.807 0.646***
0.134 0.365* 0.125
FLs ***
0.727 0.680*** 0.614***
RSA
FRAP
0.300
−0.014 0.028
OLE 0.933 0.404 0.035
FLs ***
0.382 0.442 0.162
TPC, total phenolic content; OLE, oleuropein; FLs, flavonoids; RSA, DPPH radical scavenging activity; FRAP, ferric-reducing antioxidant power. * Significant at p < 0.05. *** Significant at p < 0.001.
leaves, exhibited the highest yield and antioxidant activity.
consequence, 50% acetone gave the best extraction yield of TPC after 30 min; however, owing to the fact that reducing the extraction time is a crucial parameter in industry and due to detection of no significant difference in phenolic concentration between 10, 30 and 60 min, 10 min seems to be the optimal time for extracting TPC from olive leaves. According to the results presented in Fig. 5A, RSA was found
3.3. Selection of time extraction As shown in Fig. 5A, the rate of TPC extraction was relatively important during the first 30 min and then decreased progressively. As a 386
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observed between TPC and FRAP (r = −0.014). This evidence may be due to the fact that phenolic compounds, especially OLE, present in olive leaves had low stability at high temperatures.
constant between 10 and 30 min extraction time in acetone extracts, though it was further decreased. Similarly, FRAP values increased until 30 min extraction time and then decreased progressively. In general, prolonged time improves the extraction efficiency by completely rupturing the plant cell and also enables the diffusion of solvent to dissolve the phenolic compounds. Furthermore, prolonged extraction time always involves the risk of degradation of phenolics by heating due to light or oxygen exposure (Naczk and Shahidi, 2006). On the other hand, the highest recovery of OLE, the main phenolic components in olive leaves, was achieved within the first 10 min extraction (Fig. 5B). Applying extraction times longer than 10 min, a constant decrease in OLE yield is noticed. However, the yields of other phenolic compounds (HTR, PAs and FLs) were not changed after 10 min extraction. Our results are in accordance with those of Xie et al. (2015) who demonstrated that 3 min was the best time for extracting OLE from olive leaves. For this reason, the optimum extraction time of 10 min was selected as the optimal extraction condition.
4. Conclusions In conclusion, single factor experiments were employed to optimize the extraction parameters of phenolics from olive leaves in respect to OLE, HTR, FLs and PAs yields as well as TPC, RSA and FRAP values. This study suggests that the best extraction conditions were using 50% acetone for 10 min at 60 °C. Under these conditions, 10.65% OLE and 0.29% FLs containing in olive leaves extract as well as high TPC (37.44 mg GAE/g dw) and antioxidant capacity, as confirmed by RSA and FRAP values, were obtained. However, water extract contained more HTR and PAs contents than the other organic solvents. Positive correlation was found between OLE, FLs, TPC, RSA and FRAP in olive leaves, especially under the influence of solvent type and solvent concentration. Therefore, UAE with the above extraction conditions could be a simple, rapid and inexpensive extraction method compared to other novel extraction technologies to obtain olive leaves extract rich in polyphenols with a high antioxidant capacity.
3.4. Selection of temperature extraction Applying the best solvent (50% acetone) and the best extraction time (10 min), the application of different extraction temperatures affected significantly (p < 0.05) the extraction of TPC (Fig. 6A). The results showed that TPC increased as the temperature increased from 25 to 60 °C. However, no significant difference was found for RSA and FRAP values with increasing temperature extraction. Nevertheless, Chew et al. (2011) reported a positive relationship between TPC and extraction temperature, but a negative correlation between antioxidant activity and extraction temperature. It should be noted that increasing temperature beyond a certain value may lead to decomposition of some phenolic compounds. In Fig. 6B it is also demonstrated that the maximum OLE yield was achieved at an extraction temperature of 60 °C. The same trend was observed for the HTR, PAs and FLs yields: 60 °C was significantly (p < 0.05) the most efficient temperature for extracting HTR, PAs and FLs from olive leaves extract, approximately more than 1.5-times the one of 25 °C. Similar positive effect of temperature on total polyphenols recovery from olive leaves has been also observed in previous studies (Xie et al., 2015). However, it should be noted that extremely high temperature extraction may promote possible degradation of phenolic compounds, or may enhance solvent loss through vaporization (Khemakhem et al., 2017). Thus, an extraction temperature of 60 °C was selected, as the optimal extraction condition.
Funding sources This research did not receive any specificgrant from funding agencies in the public, commercial, or not-for-profit sectors. References Ahmad-Qasem, M.H., Cánovas, J., Barrajón-Catalán, E., Micol, V., Cárcel, J.A., GarcíaPérez, J.V., 2013. Kinetic and compositional study of phenolic extraction from olive leaves (var. Serrana) by using power ultrasound. Innov. Food Sci. Emerg. Technol. 17, 120–129. Al-Azzawie, H.F., Alhamdani, M.S.S., 2006. Hypoglycemic and antioxidant effect of oleuropein in alloxan-diabetic rabbits. Life Sci. 78, 1371–1377. Altıok, E., Baycin, D., Bayraktar, O., Ülkü, S., 2008. Isolation of polyphenols from the extracts of olive leaves (Olea europaea L.) by adsorption on silk fibroin. Sep. Purif. Technol. 62, 342–348. Benzie, F., Strain, J., 1999. Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol. 299, 15–23. Bouallagui, Z., Han, J., Isoda, H., Sayadi, S., 2011. Hydroxytyrosol rich extract from olive leaves modulates cell cycle progression in MCF-7 human breast cancer cells. Food Chem. Toxicol. 49, 179–184. Chew, K.K., Khoo, M.Z., Ng, S.Y., Thoo, Y.Y., Aida, W.M., Ho, W., Ho, C.W., 2011. Effect of ethanol concentration, extraction time and extraction temperature on the recovery of phenolic compounds and antioxidant capacity of Orthosiphon stamineus extracts. Int. Food Res. J. 8, 1427–1435. Cifá, D., Skrt, M., Pittia, P., Mattia, C.D., Ulrih, N.P., 2018. Enhanced yield of oleuropein from olive leaves using ultrasound-assisted extraction. Food Sci. Nutr. 6, 1128–1137. Delgado-Povedano, M.M., Priego-Capote, F., Castro, M.D.L., 2017. Selective ultrasoundenhanced enzymatic hydrolysis of oleuropein to its aglycon in olive (Olea europaea L.) leaf extracts. Food Chem. 220, 282–288. El, S.N., Karakaya, S., 2009. Olive tree (Olea europaea) leaves: potential beneficial effects on human health. Nutr. Rev. 67, 632–638. González-Montelongo, R., Lobo, M.G., González, M., 2010. Antioxidant activity in banana peel extracts: testing extraction conditions and related bioactive compounds. Food Chem. 119, 1030–1039. Goulas, V., Papoti, V.T., Exarchou, V., Tsimidou, M.Z., Gerothanassis, I.P., 2010. Contribution of flavonoids to the overall radical scavenging activity of olive (Olea europaea L.) leaf polar extracts. J. Agric. Food Chem. 58, 3303–3308. Habibi, H., Mohammadi, A., Farhoodi, M., Jazaer, S., 2018. Application and optimization of microwave-assisted extraction and dispersive liquid-liquid microextraction followed by high-performance liquid chromatography for the determination of oleuropein and hydroxytyrosol in olive pomace. Food Anal. Methods 1–11. Herrero, M., Temirzoda, T.N., Segura-Carretero, A., Quirantes, R., Plaza, M., Ibañez, E., 2011. New possibilities for the valorization of olive oil by-products. J. Chromatogr. A 1218, 7511–7520. Khemakhem, I., Ahmad-Qasem, H.M., Catalán, E.B., Vicente, M., García-Pérez, J.V., Ayadi, M.A., Bouaziz, M., 2017. Kinetic improvement of olive leaves’ bioactive compounds extraction by using power ultrasound in a wide temperature range. Ultrason. Sonochem. 34, 466–473. Lien, D.T.P., Tram, P.T.B., Toan, H.T., 2015. Effects of extraction process on phenolic content and antioxidant activity of soybean. J. Food Nutr. Sci. 3, 33–38. Mokrani, A., Madani, K., 2016. Effect of solvent, time and temperature on the extraction of phenolic compounds and antioxidant capacity of peach (Prunus persica L.) fruit.
3.5. Correlation analysis The correlations between antioxidant activity (RSA and FRAP) and the contents of TPC, OLE and FLs of olive leaves under different extraction conditions were analyzed (Table 1). Under the factor of solvent type, high significantly correlations (r = 0.955–0.995) were observed between TPC, OLE, FLs, RSA and FRAP values at p < 0.001. The same trend was noticed under the influence of solvent concentration, indicating correlations range from 0.739 (TPC-RSA) to 0.888 (RSA-FRAP) at p < 0.001. However, low correlations were observed between the pairs of FLs-TPC, FLs-RSA and FLs-FRAP (0.417-0.678, p < 0.001). As it concerns the factor of extraction time, the TPC of olive leaves were significantly correlated with their antioxidant activities (r = 0.699 and 0.807 for RSA and FRAP methods, respectively), however, no significant correlations were found between TPC and OLE contents, as well as between FRAP and OLE contents. The OLE content in olive leaves were correlated significantly, but poorly with RSA values (r = 0.365, p < 0.05), whereas FLs content were good correlated with TPC, RSA and FRAP values at p < 0.001. Concerning the influence of extraction temperature condition, only TPC was correlated positively with OLE content (r = 0.933, p < 0.001). No significant correlations were found between the other assays and furthermore, a negative correlation was 387
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Sahin, S., Bilgin, M., 2012. Study on oleuropein extraction from olive tree (Olea europaea) leaves by means of SFE: comparison of water and ethanol as co-solvent. Sep. Sci. Technol. 347, 2391–2398. Singleton, V.L., Orthofer, R., Lamuela-Raventos, R.M., 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagents. Methods Enzymol. 299, 152–178. Sun, T., Ho, C.T., 2015. Antioxidant activities of buckwheat extracts. Food Chem. 90, 743–749. Xie, P.J., Huang, L.X., Zhang, C.H., Feng, Y., Zhang, Y.L., 2015. Reduced pressure extraction of oleuropein from olive leaves (Olea europaea L.) with ultrasound assistance. Food Bioprod. Process. 93, 29–38. Xynos, N., Papaefstathiou, G., Gikas, E., Argyropoulou, A., Aligiannis, N., Skaltsounis, A.L., 2014. Design optimization study of the extraction of olive leaves performed with pressurized liquid extraction using response surface methodology. Sep. Purif. Technol. 122, 323–330. Yateem, H., Afaneh, I., Al-Rimawi, F., 2014. Optimum conditions for oleuropein extraction from olive leaves. Int. J. Appl. Sci. Technol. 4, 153–157. Yen, G.C., Chen, H.Y., 1995. Antioxidant activity of various tea extracts in relation to their antimutagenicity. J. Agric. Food Chem. 43, 27–32.
Sep. Purif. Technol. 162, 68–76. Mylonaki, S., Kiassos, E., Makris, D.P., Kefalas, P., 2008. Optimization of the extraction of olive (Olea europaea) leaf phenolics using water/ethanol- based solvent systems and response surface methodology. Anal. Bioanal. Chem. 392, 977–985. Naczk, M., Shahidi, F., 2006. Phenolics in cereals, fruits and vegetables: occurrence, extraction and analysis. J. Pharm. Biomed. Anal. 41, 1523–1542. Quirantes-Piné, R., Lozano-Sánchez, J., Herrero, M., Ibáñez, E., Segura-Carreteroa, A., Fernández-Gutiérrez, A., 2013. HPLC–ESI–QTOF–MS as a powerful analytical tool for characterizing phenolic compounds in olive-leaf extracts. Phytochem. Anal. 24, 213–223. Rafiee, Z., Jafari, S.M., Alami, M., Khomeiri, M., 2011. Microwave-assisted extraction of phenolic compounds from olive leaves; a comparison with maceration. J. Anim. Plant Sci. 21, 738–745. Rahmanian, N., Jafaric, S.M., Wani, T.A., 2015. Bioactive profile, dehydration, extraction and application of the bioactive components of olive leaves. Trends Food Sci. Technol. 42, 150–172. Ranalli, A., Contento, S., Lucera, L., Di Febo, M., Marchegiani, D., Di Fonzo, V., 2006. Factors affecting the contents of iridoid oleuropein in olive leaves (Olea europaea L.). J. Agric. Food Chem. 54, 434–440.
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Optimization of ultrasound-assisted extraction of phenolic compounds: Oleuropein, phenolic acids, phenolic alcohols and flavonoids from olive leaves and evaluation of its antioxidant activities
T
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Maria Iraklia, , Paschalina Chatzopouloua, Loukia Ekateriniadoub a b
Hellenic Agricultural Organization–Demeter, Institute of Plant Breeding and Genetic Resources, 5700, Thermi, Thessaloniki, Greece Hellenic Agricultural Organization–Demeter, Veterinary Research Institute, 57001, Thermi, Thessaloniki, Greece
A R T I C LE I N FO
A B S T R A C T
Keywords: Olive leaves Oleuropein Hydroxytyrosol Flavonoids Phenolic acids Antioxidant activity
Olive leaves are well known for many useful pharmacological effects. Some of the health benefits are related to phenolic composition, especially to oleuropein (OLE) and flavonoids (FLs) content. This study aimed to investigate the influence of ultrasound-assisted extraction conditions (solvent type, solvent concentration, extraction time and temperature) on the extract yield of OLE, hydroxytyrosol (HTR), FLs (rutin, luteolin, luteolin-7O-glucoside and apigenin) and phenolic acids (protocatechuic, 4-hydroxybenzoic, vanillic, p-coumaric and ferulic acids) from olive leaves using single factor experiments approach. The total phenolic compounds (TPC) and their antioxidant activity based on 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (RSA) and ferric reducing antioxidant power (FRAP) were also evaluated. The highest yields of OLE and FLs, the major phenolics in olive leaves, were obtained with 50% acetone, at 60 °C for 10 min extraction. However, the yields of HTR and PAs increased when water was used as extraction solvent. Good, positive, correlation coefficients were found between OLE, TPC, RSA and FRAP in olive leaves, especially under the influence of solvent type and solvent concentration.
1. Introduction The olive tree (Olea europaea L.) is one of the most important fruit trees in Mediterranean countries such as Italy, Spain, Greece and Tunisia. Although olive leaves are always used as animal feed, there is a rising interest in their application as a valuable material in various fields. They are regarded as a cheap raw material which can be used as a good source of bioactives and they are also one of the by-products in olive-oil production, representing 10% of the weight of olives collected. Furthermore, they also accumulate in large volumes on olive groves during the pruning of the trees (Herrero et al., 2011). Olive leaves are rich in a wide variety of phenolic compounds, such as secoiridoids (oleuropein, ligstroside, dimethyloleuropein) and flavonoids (apigenin, luteolin, luteolin-7-O-glucoside etc), along with other phenolic compounds (hydroxytyrosol, tyrosol, caffeic acid, ferulic acid etc) (Quirantes-Piné et al., 2013), that are responsible for several biological properties, including antioxidant and anti-inflammatory, antimicrobial, antiviral, anti-carcinogenic, as well as beneficial cardiovascular effects (El and Karakaya, 2009). OLE is the most representative polyphenolic constituent of olive
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leaves, responsible for the bitterness of both table olives and extravirgin olive oil. The majority of studies attribute the biological activities of olive leaves to the total or individual phenolic compounds such as OLE (Al-Azzawie and Alhamdani, 2006), HTR (Bouallagui et al., 2011), and FLs (Goulas et al., 2010). However, the phenolic profile in olive leaves varies depending on the origin and variety of the plant material, the geographical location and the agro-ecological conditions, and especially the seasons (Ranalli et al., 2006). Various methods have been used to isolate the bioactive molecules present in olive leaves, from the most common techniques to the more sophisticated including microwave-assisted extraction (Rafiee et al., 2011; Habibi et al., 2018), pressurized liquid extraction (Xynos et al., 2014) and supercritical fluid extraction (Sahin and Bilgin, 2012). It should be pointed out that most of these techniques suffer from high energy costs as they operate under high pressure. Therefore, the development of an effective, suitable and low-cost extraction method is of major importance. In this sense, ultrasound-assisted extraction (UAE) has the potential to reduce extraction times and extraction solvent volumes, as well as to increase the recoveries of active compounds. It has become a well-established technique both, in laboratories and in
Corresponding author. E-mail address: [email protected] (M. Irakli).
https://doi.org/10.1016/j.indcrop.2018.07.070 Received 3 January 2017; Received in revised form 29 June 2018; Accepted 26 July 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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Initially, samples were extracted with different concentrations of three organic solvents tested ranged from 0 to 90% v/v, fixing extraction time and extraction temperature constant at 30 °C for 10 min. Subsequently, the effect of extraction time was investigated by varying the extraction time from 10 to 120 min using the best organic solvent concentration chosen in the initial step and kept the extraction temperature constant at 30 °C. Lastly, the effect of extraction temperature was investigated using the best organic solvent concentration and extraction time determined in the previous stage with extraction temperature ranged from 30 to 65 °C.
industrial scale as well. Several authors found that UAE method is faster, simpler and more efficient than maceration/stirring for the extraction of OLE from olive leaves (Ahmad-Qasem et al., 2013; Xie et al., 2015; Cifá et al., 2018). A recent review highlighted the extraction methods and potential application of the bioactive components of olive leaves (Rahmanian et al., 2015). Although the effect of extraction conditions on the extraction yield of OLE, has been extensively investigated, few studies reporting the effect of extracting parameters on the recovery of other class of phenolic compounds from olive leaves (Delgado-Povedano et al., 2017). The aim of the present study was to maximize the extraction yields of OLE, HTR, FLs and PAs from olive leaves in respect to decrease operating costs with the possibility of lower volumes of solvent and lower extraction times and temperatures. We investigated the application of UAE and the optimization of extraction parameters such as solvent type, solvent concentration, extraction time and temperature in order to obtain extract rich in bioactive compounds with high antioxidant activities as evaluated by RSA and FRAP tests using single factor experiments approach.
2.5. Determination of total phenolic content (TPC) The amount of TPC in olive leaves extracts was determined according to the Folin-Ciocalteu method (Singleton et al., 1999) with minor modifications. Briefly, 200 μL of each extract was reacted with 800 μL of Folin-Ciocalteu reagent (diluted 10-fold) for 2 min. Then, 2 mL of sodium carbonate (7.5% w/v) was added and the volume was adjusted to 10 mL with distilled water. The mixture was allowed to stand for 1 h at room temperature in the dark and the absorbance was measured at 765 nm against blank. The results were expressed as mg of GA equivalents per g of the dried sample (mg GAE/ g dw).
2. Materials and methods 2.1. Chemicals
2.6. Antioxidant activity
OLE, TYR, HTR, luteolin-7-O-glucoside (LUTG), protocatechuic acid (PRCA), p-coumaric acid (pCA), ferulic acid (FA), vanillic acid (VA) and p-hydroxy-benzoic acid (pHBA) were supplied by Sigma-Aldrich (Steinheim, Germany). Apigenin (API), rutin (RUT), luteolin (LUT) and gallic acid (GA) were obtained from Extrasynthese (Genay Cedex, France). Analytical grade of Folin-Ciocalteu, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 6-hydroxyl-2,5,7,8- tetramethychromane-2-carboxylic acid (Trolox) and 2, 4, 6-tripyridyl-s-triazine (TPTZ) were from SigmaAldrich (Steinheim, Germany). All other solvents/chemicals obtained from Chem-Lab (Zedelgem, Belgium) were of analytical grade or highperformance liquid chromatography (HPLC) grade.
2.6.1. DPPH free radical scavenging activity (RSA) The RSA was based on the protocol described by Yen and Chen (1995) with some modifications. Aliquots (150 μL) of extracts were reacted with 2.85 mL DPPH solution in methanol (0.1 mM). After agitation, the reaction mixture was incubated in the dark at room temperature for 5 min and the absorbance was measured at 516 nm. The free radical scavenging capacity (in percentage) was calculated by using the following equation: RSA (%) = (Ao − As) /Ao × 100 where Ao is the absorbance of the blank (methanol) and As is the absorbance of the sample. Results were expressed as mg Trolox equivalents per g of dried sample (mg TE/g dw).
2.2. Plant material Fresh green olive leaves (Olea europaea L., variety Chalkidiki), were collected from the trees grown in north of Greece. Collected leaves were air dried in oven at 40 °C and then were milled using a laboratory mill equipped with a 0.5 mm sieve and finally stored at 4 °C prior to extraction.
2.6.2. Ferric reducing antioxidant power assay (FRAP) The FRAP assay was performed according to Benzie and Strain (1999) with some modifications. Briefly, the fresh FRAP reagent included 10 mL of 10 mM TPTZ solution in 40 mM HCl plus 10 mL of 20 mM FeCl3.6H2O and 100 mL of 300 mM acetate buffer pH 3.6. Aliquot of extract (100 μL) was reacted with the FRAP solution (3 mL) at 37 °C for exactly 4 min under dark conditions. Readings of the colored product were then taken at 593 nm against blank and the results were expressed as mg Trolox equivalents per g of dried sample (mg TE/g dw).
2.3. Ultrasound-assisted extraction of phenolic compounds Powdered and dried olive leaves (250 mg) were extracted with solvent (20 mL) in an ultrasound bath (frequency 37 kHz, model FB 15051, Thermo Fisher Scientific Inc. Loughborough, England) for the different times and temperatures. Then, the crude extracts were centrifuged at 1500 × g for 10 min (Universal 320R, Hettich, Germany), the supernatants were filtered using 0.45-μm syringe filters and used directly for estimation of TPC, assessment of antioxidant capacities and HPLC analysis of phenolic compounds. Each extraction was triplicated and all analysis were performed in three replications.
2.7. HPLC profile of phenolic compounds The analyses were performed on an HPLC Agilent 1200 system (Agilent Technology, Urdorf, Switzerland) equipped with a 250 × 4.6 mm i.d., 5 μm Nucleosil 100 C18 column (MZ, Mainz, Germany) maintained at 30 °C, a 20 μL loop and diode-array detector (DAD). Mobile phase consists of three solvents: (A) 1% acetic acid in water, (B) acetonitrile and (C) methanol and the following gradient program was performed: 0 min, 90% A-0% B; 10 min, 80% A-4% B; 25 min, 75% A-5% B; 30 min, 65% A-5% B; 31 min, 40% A-0% B; 37 min, 35% A-20% B; 50 min, 20% A-80% B. The flow rate of mobile phase was 1.3 mL/min. The DAD recorded the spectra at 260, 280, 320, and 360 nm and the chromatograms were analysed using the Agilent Chemstation software (version B.04.01, Agilent Technologies). Identification of phenolics in olive leaves extract was obtained by comparison of retention times and UV/VIS spectra with those of
2.4. Experimental design In the present study, single factor experiments was used to determine the optimum conditions for extracting phenolic compounds from olive leaves. Four extraction solvents were used: ethanol, methanol, acetone and water. Three independent variables were studied, namely organic solvent concentration, extraction time and extraction temperature in order to optimize the extraction conditions. The level for each independent variable was chosen based on the process responses, OLE, HTR, FLs and PAs as well as TPC, RSA and FRAP. 383
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Results illustrated in Fig. 2, showed similar influence of solvent concentration on the extraction yield of OLE, HTR, PAs and FLs of the obtained extracts. Extraction yield of OLE, the major phenolic compound in olive leaves extract, increased significantly (p < 0.05) as the concentration of all solvents used increased up to 50% and then followed by a considerable drop with further increases in solvent. This is expected due to polar hydroxyl groups of OLE and needed thus polar solvent for extraction. Additionally, the solvent mixtures are better as they deactivate the enzymes which are responsible for conversion of OLE into other compounds which have high protein-denaturing, and protein cross linking activities (Yateem et al., 2014). However, this trend declined when the ethanol and methanol concentration varied from 70 to 90%. This observation could be explained by the fact that a strong polarity might increase the impurities, which may prevent the dissolution of OLE or that the medium polarity solvent was appropriate for extracting OLE according to Xie et al. (2015). The maximum achieved OLE yields were recorded for 50% concentration, 3fold greater than those obtained by water, which represents the lowest value. A similar trend was observed in the yield of total identified FLs. The FLs yield of all extracts increased as the solvent concentration increased up to 50%, whereas further increase up to 90% leads to a rapidly decrease. The highest yield of total FLs was recorded for 50% concentration for all solvents, more than 2-fold of aqueous extracts. On the other hand, the yield of HTR was maximized at 0% organic solvent and then decreased significantly with the increase of the solvent concentration up to 90% (Fig. 2). This may be linked to the fact that HTR is more soluble in water and polar organic solvents than OLE, due to different chemical structures (Habibi et al., 2018). Solvent concentrations between 20–90% showed non-significant differences or slight increase at 50% concentration for the most of the solvent systems. Similarly, the yield of PAs was maximized at 0% organic solvent concentration with significant or non-significant effects with the level of 20%, depending on the kind of solvent. Other concentrations showed non-significant differences or slight decrease. Solvent concentration had also significant effect (p < 0.05) on RSA and FRAP of olive leaves extract (Fig. 3) at most of the levels assessed. The highest values of RSA for all mixtures solvents were obtained at 50% and 70% concentration, whereas the water extract possessed the lowest one. Extract at 50% organic solvent concentration showed significantly higher FRAP values than other concentration levels, whereas no significant differences were observed at concentration levels > 50% for ethanol extracts. This observation was in good agreement with the results of TPC indicating that polyphenolics were the principal compounds responsible for the antioxidant potential of tested extracts. Our results are in accordance with those of González-Montelongo et al.
authentic standards. The extraction yield for each analyte was calculated by the following equation: Extraction yield (mg/g dw) = [(C × V)/W]/1000 where C is the concentration of analyte in each solvent calculated by calibration curves (μg/mL), V is the volume of the extract (mL), and W is the dried weight (dw) of sample (g). 2.8. Statistical analysis The results were expressed as means ± standard deviation (SD) of replicate solvent extractions and triplicate of assays. One way analysis of variance (ANOVA) was used to compare the means. The means were separated by Least Significant Difference (LSD). The statistical significant differences were defined at p < 0.05. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) 19.0. 3. Results and discussion 3.1. Selection of solvent concentration Many parameters can influence the UAE efficiency of phenolics from olive leaves, such as temperature, solvent composition, solid–solvent ratio, extraction time and solid phase particle size (Xie et al., 2015). In the present study, assays were carried out in olive leaves with particle size of 0.5 mm and solid–solvent ratio was adjusted to 1:80. In the present study, extractions with different mixtures of aqueous solvents at concentrations ranging from 0 to 90% (v/v) were performed in order to find the optimal conditions in terms of extraction yield of OLE, HTR, FLs and PAs as well as TPC, RSA and FRAP of the obtained extracts. Similar studies dealing with the extraction of polyphenols from olive leaves have recommended similar proportions of ethanolwater (Mylonaki et al., 2008; Cifá et al., 2018). The individual constituents of the olive leaves extract obtained by 0, 20, 50, 70 and 90% acetone, or ethanol, or methanol were assessed by using HPLC analysis. Eleven phenolic compounds were separated and quantified, as presented in Fig. 1, showing similar chromatographs in terms of the type of compounds found in each extract, although the quantities extracted varied. OLE was the most abundant phenolic compound in olive leaves extract, representing approximately the 95% of the identified phenolics. Among the FLs identified, LUT, LUTG, RUT and API accounted for about 3% of the overall identified phenolics. The major PAs identified was FA, followed by pCA, whereas PRCA, pHBA and VA were minor constituents.
Fig. 1. HPLC chromatograms of water, acetone, ethanol and methanol olive leaves extracts. HTR, hydroxytyrosol; PRCA, protocatechuic acid; TYR, tyrosol; pHBA, phydroxybenzoic acid; VA, vanillic acid; pCA, p-coumaric acid; FA, ferulic acid; LUTG, luteolin-7-O-glucoside; RUT, rutin; OLE, oleuropein; LUT, luteolin; API, apigenin. 384
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Fig. 2. Effect of A) acetone B) ethanol and C) methanol concentration on the extraction of OLE, HTR, FLs and PAs from olive leaves. Results are mean ± SD, n = 3; bars marked with different letters are significantly different (p < 0.05). OLE, oleuropein; HTR, hydroxytyrosol; PAs, phenolic acids; FLs, flavonoids.
(2010) who showed that 50% acetone was more efficient for extracting banana peel phenolics and therefore, produced extracts with higher antioxidant activity. On the contrary, Altıok et al. (2008) reported that 90% acetone was the best extraction solvent for the isolation of bioactive compounds from olive leaves extract. This discrepancy may be due to different extraction methods. Thus, in our experimentation, a concentration of 50% was selected as the best for the following steps.
3.2. Selection of solvent extraction The present study showed that water and mixtures of organic solvents at 50% concentration were able to extract efficiently phenolic compounds from olive leaves (Fig. 4). Acetone extract showed the highest OLE and FLs yields with values of 106.50 mg/g dw and 2.94 mg/g dw, respectively, followed by ethanol and methanol, whereas water was found an insufficient extraction solvent, appearing the lowest extraction yield. However, water was significantly the best solvent for extracting HTR and PAs from olive leaves, followed by other solvents presenting non-significant yield values (Fig. 4B). The extraction solvent systems used, affected similarly the TPC of olive leaves extract; 50% acetone extract yielded significantly higher TPC than methanol and ethanol extracts (Fig. 4A), whereas water extract resulted the lowest one. The same trend was observed for the antioxidant activity of extracts; acetone extract exhibited significantly the highest RSA and FRAP values, followed by methanol and ethanol extracts, exhibiting non-significant effects. Acetone has been also demonstrated to be more effective than other organic solvents for extracting phenolics from many raw materials such as buckwheat (Sun and Ho, 2015), soybean (Lien et al., 2015) etc. Generally, it is strongly believed that the higher molecular weight of the solvent, the lower polarity, which enable other substances, of about the same molecular weight, to be easily extracted (Mokrani and Madani, 2016). Therefore, 50% acetone extract was selected for extracting phenolics from olive leaves, since OLE, the major phenolic compounds class present in olive
Fig. 4. Effect of solvent type on the extraction of A) TPC, RSA and FRAP and B) OLE, HTR, FLs and PAs from olive leaves. Results are mean ± SD, n = 3; bars marked with different letters are significantly different (p < 0.05). Abbreviations as Figs. 2 and 3.
Fig. 3. Effect of A) acetone B) ethanol and C) methanol concentration on the extraction of TPC, RSA and FRAP from olive leaves. Results are mean ± SD, n = 3; bars marked with different letters are significantly different (p < 0.05). TPC, total phenolic content; RSA, DPPH radical scavenging activity; FRAP, ferric reducing antioxidant power. 385
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Fig. 5. Effect of time on the extraction of A) TPC, RSA and FRAP and B) OLE, HTR, FLs and PAs from olive leaves. Results are mean ± SD, n = 3; bars marked with different are significantly different (p < 0.05). Abbreviations as Figs. 2 and 3.
Fig. 6. Effect of temperature on the extraction of A) TPC, RSA and FRAP and B) OLE, HTR, FLs and PAs from olive leaves. Results are mean ± SD, n = 3; bars marked with different letters are significantly different (p < 0.05). Abbreviations as Figs. 2 and 3.
Table 1 Correlation coefficients between different assays under the influence of extraction conditions. Solvent type RSA TPC RSA FRAP
0.955
Solvent concentration
FRAP ***
OLE ***
0.974 0.973***
FLs ***
0.961 0.959*** 0.995***
RSA ***
0.985 0.962*** 0.991***
0.739
FRAP ***
Extraction time OLE
***
0.786 0.888***
FLs ***
0.811 0.773*** 0.853***
RSA ***
0.678 0.417*** 0.614***
Extraction temperature
FRAP ***
0.699
OLE ***
0.807 0.646***
0.134 0.365* 0.125
FLs ***
0.727 0.680*** 0.614***
RSA
FRAP
0.300
−0.014 0.028
OLE 0.933 0.404 0.035
FLs ***
0.382 0.442 0.162
TPC, total phenolic content; OLE, oleuropein; FLs, flavonoids; RSA, DPPH radical scavenging activity; FRAP, ferric-reducing antioxidant power. * Significant at p < 0.05. *** Significant at p < 0.001.
leaves, exhibited the highest yield and antioxidant activity.
consequence, 50% acetone gave the best extraction yield of TPC after 30 min; however, owing to the fact that reducing the extraction time is a crucial parameter in industry and due to detection of no significant difference in phenolic concentration between 10, 30 and 60 min, 10 min seems to be the optimal time for extracting TPC from olive leaves. According to the results presented in Fig. 5A, RSA was found
3.3. Selection of time extraction As shown in Fig. 5A, the rate of TPC extraction was relatively important during the first 30 min and then decreased progressively. As a 386
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observed between TPC and FRAP (r = −0.014). This evidence may be due to the fact that phenolic compounds, especially OLE, present in olive leaves had low stability at high temperatures.
constant between 10 and 30 min extraction time in acetone extracts, though it was further decreased. Similarly, FRAP values increased until 30 min extraction time and then decreased progressively. In general, prolonged time improves the extraction efficiency by completely rupturing the plant cell and also enables the diffusion of solvent to dissolve the phenolic compounds. Furthermore, prolonged extraction time always involves the risk of degradation of phenolics by heating due to light or oxygen exposure (Naczk and Shahidi, 2006). On the other hand, the highest recovery of OLE, the main phenolic components in olive leaves, was achieved within the first 10 min extraction (Fig. 5B). Applying extraction times longer than 10 min, a constant decrease in OLE yield is noticed. However, the yields of other phenolic compounds (HTR, PAs and FLs) were not changed after 10 min extraction. Our results are in accordance with those of Xie et al. (2015) who demonstrated that 3 min was the best time for extracting OLE from olive leaves. For this reason, the optimum extraction time of 10 min was selected as the optimal extraction condition.
4. Conclusions In conclusion, single factor experiments were employed to optimize the extraction parameters of phenolics from olive leaves in respect to OLE, HTR, FLs and PAs yields as well as TPC, RSA and FRAP values. This study suggests that the best extraction conditions were using 50% acetone for 10 min at 60 °C. Under these conditions, 10.65% OLE and 0.29% FLs containing in olive leaves extract as well as high TPC (37.44 mg GAE/g dw) and antioxidant capacity, as confirmed by RSA and FRAP values, were obtained. However, water extract contained more HTR and PAs contents than the other organic solvents. Positive correlation was found between OLE, FLs, TPC, RSA and FRAP in olive leaves, especially under the influence of solvent type and solvent concentration. Therefore, UAE with the above extraction conditions could be a simple, rapid and inexpensive extraction method compared to other novel extraction technologies to obtain olive leaves extract rich in polyphenols with a high antioxidant capacity.
3.4. Selection of temperature extraction Applying the best solvent (50% acetone) and the best extraction time (10 min), the application of different extraction temperatures affected significantly (p < 0.05) the extraction of TPC (Fig. 6A). The results showed that TPC increased as the temperature increased from 25 to 60 °C. However, no significant difference was found for RSA and FRAP values with increasing temperature extraction. Nevertheless, Chew et al. (2011) reported a positive relationship between TPC and extraction temperature, but a negative correlation between antioxidant activity and extraction temperature. It should be noted that increasing temperature beyond a certain value may lead to decomposition of some phenolic compounds. In Fig. 6B it is also demonstrated that the maximum OLE yield was achieved at an extraction temperature of 60 °C. The same trend was observed for the HTR, PAs and FLs yields: 60 °C was significantly (p < 0.05) the most efficient temperature for extracting HTR, PAs and FLs from olive leaves extract, approximately more than 1.5-times the one of 25 °C. Similar positive effect of temperature on total polyphenols recovery from olive leaves has been also observed in previous studies (Xie et al., 2015). However, it should be noted that extremely high temperature extraction may promote possible degradation of phenolic compounds, or may enhance solvent loss through vaporization (Khemakhem et al., 2017). Thus, an extraction temperature of 60 °C was selected, as the optimal extraction condition.
Funding sources This research did not receive any specificgrant from funding agencies in the public, commercial, or not-for-profit sectors. References Ahmad-Qasem, M.H., Cánovas, J., Barrajón-Catalán, E., Micol, V., Cárcel, J.A., GarcíaPérez, J.V., 2013. Kinetic and compositional study of phenolic extraction from olive leaves (var. Serrana) by using power ultrasound. Innov. Food Sci. Emerg. Technol. 17, 120–129. Al-Azzawie, H.F., Alhamdani, M.S.S., 2006. Hypoglycemic and antioxidant effect of oleuropein in alloxan-diabetic rabbits. Life Sci. 78, 1371–1377. Altıok, E., Baycin, D., Bayraktar, O., Ülkü, S., 2008. Isolation of polyphenols from the extracts of olive leaves (Olea europaea L.) by adsorption on silk fibroin. Sep. Purif. Technol. 62, 342–348. Benzie, F., Strain, J., 1999. Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol. 299, 15–23. Bouallagui, Z., Han, J., Isoda, H., Sayadi, S., 2011. Hydroxytyrosol rich extract from olive leaves modulates cell cycle progression in MCF-7 human breast cancer cells. Food Chem. Toxicol. 49, 179–184. Chew, K.K., Khoo, M.Z., Ng, S.Y., Thoo, Y.Y., Aida, W.M., Ho, W., Ho, C.W., 2011. Effect of ethanol concentration, extraction time and extraction temperature on the recovery of phenolic compounds and antioxidant capacity of Orthosiphon stamineus extracts. Int. Food Res. J. 8, 1427–1435. Cifá, D., Skrt, M., Pittia, P., Mattia, C.D., Ulrih, N.P., 2018. Enhanced yield of oleuropein from olive leaves using ultrasound-assisted extraction. Food Sci. Nutr. 6, 1128–1137. Delgado-Povedano, M.M., Priego-Capote, F., Castro, M.D.L., 2017. Selective ultrasoundenhanced enzymatic hydrolysis of oleuropein to its aglycon in olive (Olea europaea L.) leaf extracts. Food Chem. 220, 282–288. El, S.N., Karakaya, S., 2009. Olive tree (Olea europaea) leaves: potential beneficial effects on human health. Nutr. Rev. 67, 632–638. González-Montelongo, R., Lobo, M.G., González, M., 2010. Antioxidant activity in banana peel extracts: testing extraction conditions and related bioactive compounds. Food Chem. 119, 1030–1039. Goulas, V., Papoti, V.T., Exarchou, V., Tsimidou, M.Z., Gerothanassis, I.P., 2010. Contribution of flavonoids to the overall radical scavenging activity of olive (Olea europaea L.) leaf polar extracts. J. Agric. Food Chem. 58, 3303–3308. Habibi, H., Mohammadi, A., Farhoodi, M., Jazaer, S., 2018. Application and optimization of microwave-assisted extraction and dispersive liquid-liquid microextraction followed by high-performance liquid chromatography for the determination of oleuropein and hydroxytyrosol in olive pomace. Food Anal. Methods 1–11. Herrero, M., Temirzoda, T.N., Segura-Carretero, A., Quirantes, R., Plaza, M., Ibañez, E., 2011. New possibilities for the valorization of olive oil by-products. J. Chromatogr. A 1218, 7511–7520. Khemakhem, I., Ahmad-Qasem, H.M., Catalán, E.B., Vicente, M., García-Pérez, J.V., Ayadi, M.A., Bouaziz, M., 2017. Kinetic improvement of olive leaves’ bioactive compounds extraction by using power ultrasound in a wide temperature range. Ultrason. Sonochem. 34, 466–473. Lien, D.T.P., Tram, P.T.B., Toan, H.T., 2015. Effects of extraction process on phenolic content and antioxidant activity of soybean. J. Food Nutr. Sci. 3, 33–38. Mokrani, A., Madani, K., 2016. Effect of solvent, time and temperature on the extraction of phenolic compounds and antioxidant capacity of peach (Prunus persica L.) fruit.
3.5. Correlation analysis The correlations between antioxidant activity (RSA and FRAP) and the contents of TPC, OLE and FLs of olive leaves under different extraction conditions were analyzed (Table 1). Under the factor of solvent type, high significantly correlations (r = 0.955–0.995) were observed between TPC, OLE, FLs, RSA and FRAP values at p < 0.001. The same trend was noticed under the influence of solvent concentration, indicating correlations range from 0.739 (TPC-RSA) to 0.888 (RSA-FRAP) at p < 0.001. However, low correlations were observed between the pairs of FLs-TPC, FLs-RSA and FLs-FRAP (0.417-0.678, p < 0.001). As it concerns the factor of extraction time, the TPC of olive leaves were significantly correlated with their antioxidant activities (r = 0.699 and 0.807 for RSA and FRAP methods, respectively), however, no significant correlations were found between TPC and OLE contents, as well as between FRAP and OLE contents. The OLE content in olive leaves were correlated significantly, but poorly with RSA values (r = 0.365, p < 0.05), whereas FLs content were good correlated with TPC, RSA and FRAP values at p < 0.001. Concerning the influence of extraction temperature condition, only TPC was correlated positively with OLE content (r = 0.933, p < 0.001). No significant correlations were found between the other assays and furthermore, a negative correlation was 387
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Sahin, S., Bilgin, M., 2012. Study on oleuropein extraction from olive tree (Olea europaea) leaves by means of SFE: comparison of water and ethanol as co-solvent. Sep. Sci. Technol. 347, 2391–2398. Singleton, V.L., Orthofer, R., Lamuela-Raventos, R.M., 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagents. Methods Enzymol. 299, 152–178. Sun, T., Ho, C.T., 2015. Antioxidant activities of buckwheat extracts. Food Chem. 90, 743–749. Xie, P.J., Huang, L.X., Zhang, C.H., Feng, Y., Zhang, Y.L., 2015. Reduced pressure extraction of oleuropein from olive leaves (Olea europaea L.) with ultrasound assistance. Food Bioprod. Process. 93, 29–38. Xynos, N., Papaefstathiou, G., Gikas, E., Argyropoulou, A., Aligiannis, N., Skaltsounis, A.L., 2014. Design optimization study of the extraction of olive leaves performed with pressurized liquid extraction using response surface methodology. Sep. Purif. Technol. 122, 323–330. Yateem, H., Afaneh, I., Al-Rimawi, F., 2014. Optimum conditions for oleuropein extraction from olive leaves. Int. J. Appl. Sci. Technol. 4, 153–157. Yen, G.C., Chen, H.Y., 1995. Antioxidant activity of various tea extracts in relation to their antimutagenicity. J. Agric. Food Chem. 43, 27–32.
Sep. Purif. Technol. 162, 68–76. Mylonaki, S., Kiassos, E., Makris, D.P., Kefalas, P., 2008. Optimization of the extraction of olive (Olea europaea) leaf phenolics using water/ethanol- based solvent systems and response surface methodology. Anal. Bioanal. Chem. 392, 977–985. Naczk, M., Shahidi, F., 2006. Phenolics in cereals, fruits and vegetables: occurrence, extraction and analysis. J. Pharm. Biomed. Anal. 41, 1523–1542. Quirantes-Piné, R., Lozano-Sánchez, J., Herrero, M., Ibáñez, E., Segura-Carreteroa, A., Fernández-Gutiérrez, A., 2013. HPLC–ESI–QTOF–MS as a powerful analytical tool for characterizing phenolic compounds in olive-leaf extracts. Phytochem. Anal. 24, 213–223. Rafiee, Z., Jafari, S.M., Alami, M., Khomeiri, M., 2011. Microwave-assisted extraction of phenolic compounds from olive leaves; a comparison with maceration. J. Anim. Plant Sci. 21, 738–745. Rahmanian, N., Jafaric, S.M., Wani, T.A., 2015. Bioactive profile, dehydration, extraction and application of the bioactive components of olive leaves. Trends Food Sci. Technol. 42, 150–172. Ranalli, A., Contento, S., Lucera, L., Di Febo, M., Marchegiani, D., Di Fonzo, V., 2006. Factors affecting the contents of iridoid oleuropein in olive leaves (Olea europaea L.). J. Agric. Food Chem. 54, 434–440.
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