Oxidative Stability of Edible Vegetable Oils Enriched

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Oxidative Stability of Edible Vegetable Oils Enriched in Polyphenols with Olive Leaf Extract F.N. Salta, A. Mylona, A. Chiou, G. Boskou and N.K. Andrikopoulos Food Science and Technology International 2007; 13; 413 DOI: 10.1177/1082013208089563 The online version of this article can be found at: http://fst.sagepub.com/cgi/content/abstract/13/6/413

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Oxidative Stability of Edible Vegetable Oils Enriched in Polyphenols with Olive Leaf Extract F.N. Salta, A. Mylona, A. Chiou, G. Boskou and N.K. Andrikopoulos* Laboratory of Chemistry – Biochemistry – Physical Chemistry of Foods Department of the Science of Dietetics – Nutrition, Harokopio University 70 El. Venizelou Ave., 176 71 Kallithea, Athens, Greece Commercially available oils (olive oil, sunflower oil, palm oil, and a vegetable shortening) were enriched in polyphenols, by adding olive leaf extract. Addition of the extract was performed in such way that the oils were enriched with 200 mg/kg polyphenols. Total polyphenols of both enriched and commercial oils were estimated by the Folin–Ciocalteau assay, while identification and quantification of individual simple phenolic compounds was performed by GC/MS and of oleuropein by HPLC analysis. The enrichment resulted in the supplementation of the commercial oils mainly with oleuropein, hydroxytyrosol, and quercetin. Antioxidant capacity and oxidative stability of the enriched oils and the commercial ones were assessed by the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) scavenging assay and the Rancimat method, respectively. Both antioxidant capacity and oxidative stability were substantially improved for all the oils studied after supplementation. By the procedure adopted, oils rich in polyphenols, mainly in oleuropein, can be produced with olive leaf extract supplementation. Key Words: olive leaves, vegetable oils, polyphenols, antioxidants, oleuropein

INTRODUCTION Edible vegetable oils such as sunflower oil, palm oil, rapeseed oil, and olive oil hold an important place in human nutrition. These oils are consumed as salad oils, cooking oils, or frying oils. Vegetable seed oils commonly used for frying are rich in tocopherols and almost free of phenolic compounds. Olive oil, produced from the olive fruit, is used as cooking and salad oil and contains both tocopherols and phenolic compounds as antioxidants. During frying, a gradual deterioration of oil occurs due to oxidative decomposition reactions (Gertz et al., 2000). Lipid oxidation leads to the production of compounds that reduce the quality of frying oils and foods (Warner, 2002). There is an increased interest for sources of natural antioxidants in order to enrich oils towards reducing lipid oxidation (Giese, 1996). For the enrichment of oils used for frying, oregano extracts in palm oil (Lolos et al., 1999), oregano powder in cottonseed oil

*To whom correspondence should be sent (e-mail: [email protected]). Received 5 June 2006; revised 10 January 2007. Food Sci Tech Int 2007;13(6):413–421 ß SAGE Publications 2007 Los Angeles, London, New Delhi and Singapore ISSN: 1082-0132 DOI: 10.1177/1082013208089563

(Houhoula et al., 2003), rosemary and sage extracts in palm oil (Che Man and Jaswir, 1999) and in rapeseed oil (Gordon and Kourimska, 1995), ethanolic extract of summer savory (Saturejae hortensis L.) in sunflower oil (Yanislieva et al., 1997), methanolic extract of tea leaves (Zandi and Gordon, 1999) and oat extracts in cottonseed oil (Tian and White, 1994), spinach powder in soybean oil (Lee et al., 2002), extracts coming from hyssop, catnip, lemon palm, oregano, sage, and thyme in sunflower oil (Abdalla and Roozen, 1999), and leafy vegetable extracts (cabbage, coriander leaves, hongone, and spinach) in sunflower and groundnut oils (Shyamala et al., 2005) have been used as sources of natural antioxidants. The ability of some herb and spice extracts to preserve -tocopherol in sunflower oil when heated has also been examined by Beddows et al. (2000). Polyphenols are potent antioxidants that demonstrate high ability in free radical scavenging (Visioli et al., 1998b). They are important preventative agents against several degenerative diseases, protecting body tissues against oxidative stress. Polyphenols protect LDL from oxidation, a procedure linked to the initiation of atherosclerosis (Andrikopoulos et al., 2002). Numerous studies have shown that polyphenols, known to be protective against several types of cancer, such as breast, prostate, skin, and colon cancer (Trichopoulou and Lagiou, 1997; Simopoulos, 2001; Kris-Etherton et al., 2002; Tapiero et al., 2002), are associated with low incidence of cardiovascular diseases (Keys, 1995; Trichopoulou and Lagiou, 1997; Simopoulos, 2001;


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Tapiero et al., 2002) and provide protection against inflammation (Tapiero et al., 2002; Trichopoulou and Lagiou, 1997). The leaves of the olive tree Olea europaea, member of the family Oleaceae, have been widely used in folk medicine in the Mediterranean countries (Somova et al., 2003). The main constituents of olive leaves are secoiridoids like oleuropein, ligstroside, dimethyloleuropein, and oleoside (Gariboldi et al., 1986). Olive leaves also contain flavonoids (apigenin, kaempferol, luteolin) as well as phenolic compounds (caffeic acid, tyrosol, hydroxytyrosol). Oleuropein is present in high amounts in olive tree leaves, in lower quantities in table olives and in traces in olive oil (Soler-Rivas et al., 2000). In this study, olive leaves have been used as a source of natural polyphenolic antioxidants for the supplementation of vegetable oils widely consumed for catering purposes. Total and individual polyphenols were assessed in olive leaf extract as well as in the commercial oils and in the supplemented ones. Oils enriched with olive leaf polyphenols were evaluated for their oxidative stability and antioxidant capacity.

MATERIALS AND METHODS Materials Methanol, hexane, acetone, acetonitrile, chloroform, ethyl acetate of analytical grade, propanol-2, methanol HPLC grade, Folin–Ciocalteau reagent, and sodium carbonate were obtained from Merck (Darmstadt, Germany). Oleuropein and hydroxytyrosol were obtained from Extrasynthe´se (Genay, France). 1,1-Diphenyl2-picrylhydrazyl radical (DPPH), bis(trimethylsilyl) trifluoroacetamide (BSTFA), quercetin, p-hydroxyphenyl-propanoic acid, chlorogenic acid, 3-(4-hydroxyphenyl)-1-propanol, and cinnamic acid were obtained from Aldrich (Steinheim, Germany). Tyrosol, protocatechuic acid, 3,4-dihydroxy-phenylacetic acid, catechin, epicatechin, sinapic acid, myricetin, trolox, and caffeic acid, were obtained from Fluka (Steinheim, Germany). 4-Hydroxy-benzoic acid, syringic acid, gallic acid, resveratrol, kaempferol, p-hydroxy-phenyl-acetic acid, ursolic acid, oleanolic acid, vanillin, homovanillyl alcohol, o-coumaric acid, p-coumaric acid, propylene glycol, and ferulic acid were obtained from Sigma (Steinheim, Germany). Vanillic acid was obtained from Serva (Heidelberg, Germany). Olive oil, sunflower oil, palm oil, and a vegetable shortening (consisted of sunflower oil, palm oil, and cottonseed oil) were purchased from the local market. Olive oil was selected to contain low polyphenol content. Olive leaves were collected in springtime from the Fthiotida region (Greece), and belonged to the Kalamon cultivar. The olive leaves were kept at 48C until analysis.

ET AL.

Methods Preparation of Olive Leaf Extract Isolation of polyphenols was performed according to the procedure described by Gariboldi (1986) with minor modifications. Briefly, leaves (50 g) were macerated in methanol (250 mL) for 3 days, in the dark, at room temperature. The extract was separated by filtration and the solvent was evaporated under reduced pressure. The residue was redisolved in 50 mL acetone–water (1 : 1), washed with hexane (50 mL, four times) and chloroform (50 mL, four times), followed by ethyl acetate extraction (50 mL, four times). The ethyl acetate extracts were combined, the solvent was evaporated under reduced pressure and the residue, containing mainly the polyphenols, was dissolved in methanol (5 mL). The methanol extracts were kept at 48C until analysis. Preparation of Enriched Vegetable Oils The edible vegetable oils used in this study were enriched with an appropriate quantity of olive leaf extract corresponding to the addition of approximately 200 mg total polyphenols per kg of oil. According to the Folin–Ciocalteau assay the required quantity of methanol extracts, corresponding to this amount of polyphenols, was 11 mL per g of oil. Appropriate volumes of the olive leaf extract were obtained (110 mL) and the solvent was evaporated under a stream of nitrogen. The dry residue was dissolved in propylene glycol (100 mL) followed by the addition of oil (10 g). Propylene glycol was added to homogenize the mixture. The mixture was vigorously shaken for 2 min. For the quantification of the exact polyphenolic content of the enriched oils the extraction procedure described in the following paragraph was performed. Furthermore, standard oleuropein (18.5 mg), dissolved in propylene glycol, was also used for the supplementation of each oil of the study (100 g). Extraction of Polyphenols from Vegetable Oils Commercial and the respective enriched oils (1 g each) were extracted three times with methanol (2 mL). The extracts were combined and methanol was evaporated under reduced pressure. The residue was dissolved in acetonitrile (2 mL) and washed two times with hexane (3 mL). Acetonitrile was evaporated under vacuum and the residue was dissolved in methanol (1 mL). Folin–Ciocalteau Assay Total polyphenol content was estimated by the Folin–Ciocalteau assay (Gutfinger, 1980). All spectrophotometric data were acquired using a Uvikon 931 (Contron, Milano, Italy) UV-V spectrophotometer. All experiments were performed in duplicate. For the


Edible Vegetable Oils Enriched in Polyphenols

olive leaf extract the methanol final extract (5 mL) was diluted (1 : 10) and a 0.05 mL aliquot was used. For the commercial and the enriched oils an aliquot of 0.1 mL of the final methanol extract (1 mL) was used. Results were expressed as mg caffeic acid equivalents (CAE) per mL extract or per kg of oil. High Performance Liquid Chromatography An HPLC system (Agilent Technologies, model 1050, Waldbronn, Germany) combined with quaternary pump, auto-sampler, diode array detector (HP–1050), fluorescence detector (HP-1046A), and data analysis software, was used. For the analysis of oleuropein a binary solvent system consisting of water acidified with phosphoric acid (pH 3) and methanol was used with a gradient elution on a Nucleosil C18 100-5 (125 mm 4.6 mm) column (MZ, Mainz, Germany) at flow rate 1 mL/min as follows: initially 10% methanol to 18% methanol in 10 min; 28% methanol in 10 min; isocratic for 5 min; 31% methanol in 17.5 min; 34% methanol in 2.5 min; isocratic for 10 min; 40% methanol in 5 min; isocratic for 2.5 min; 70% methanol in 7.5 min; 80% methanol in 10 min; and finally to the initial conditions in 10 min. A 10 min post run for the system equilibration was used. UV detection (280 and 254 nm) plus fluorescence detection (ex 275 nm, em 360 nm) was applied. Injections of 10 mL from the methanol extracts of the oil samples (1 mL) were performed while olive leaf extract (5 mL) was diluted with ratio 1 : 50 before the 10 mL injections. External standard quantification was performed based on a series of five different standard oleuropein concentrations. Gas Chromatography/Mass Spectrometry An Agilent (Wallborn, Germany) HP series GC 6890N coupled with a HP 5973 MS detector (EI, 70 eV), split-splitless injector and an HP 7683 autosampler were used for analysis. Prior to GC analysis 0.1 mL of methanol extract of oils was mixed with internal standard (20 mL, 19.2 mg/mL), was evaporated to dryness under nitrogen and derivatized by the addition of 250 mL BSTFA at 708C for 20 min (Soleas et al., 1997). For olive leaf extract (5 mL), a quantity (0.01 mL) was diluted with methanol to final volume 1 mL and an aliquot of 0.05 mL was mixed with internal standard and subjected to BSTFA derivatization as above. An aliquot (1 mL) of each derivatized sample was injected into the gas chromatograph at a split ratio 1 : 20. Separation of sample was achieved using an HP-5 MS capillary column (5% phenyl – 95% methyl siloxane, 30 m 0.25 mm 250 mm). The chromatographic conditions were described elsewhere (Kalogeropoulos et al., 2007). A Selective Ion Monitoring (SIM) GC/MS method was applied for detection of 25 target polyphenolic compounds and

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3 triterpenoic acids. Detection of polyphenols was based on the þ0.05 RT presence of target and qualifier ions of the standard polyphenols at the predetermined ratios. Target and qualifier ions (T, Q1, Q2) for the 25 polyphenolic compounds, the 3 triterpenoic acids and the internal standard were described elsewhere (Kalogeropoulos et al., 2007). Linearity was obtained for all target compounds detected in samples in the range of quantitation limit and up to twenty times higher concentrations for each compound. DPPH Radical Scavenging Assay The antioxidant capacity of tested oils before and after supplementation was measured by the DPPH radical scavenging assay. Antioxidants present in the oil scavenge DPPH and the decrease in DPPH concentration is monitored by the decrease of absorbance at 515 nm. The color from purple, in the initial solution, turns into yellow when all the amount of free radical is blocked by the antioxidants. A solution of oil in chloroform (10% w/v) was prepared, an aliquot (1 mL) was added to the DPPH solution in chloroform (4 mL, 6 105 M), mixed thoroughly, and the absorbance was measured after 30 min. The absorbance of the solution of DPPH in chloroform (6 105 M) used as control was also measured in parallel. Eight solutions (1 mL each) of Trolox standard in chloroform (0.001–25 mg) were also measured after the addition of DPPH solution in chloroform (4 mL, 6 105 M). The obtained data were used to calculate Trolox equivalents that corresponded to oils. DPPH decrease was caculated as follows: % DPPH decrease ¼ [(AcAt¼30)/Ac] 100 where Ac is the absorbance of control and At¼30 the absorbance of a sample after the time necessary to reach the plateau. The plateau of 30 min has been previously determined (Boskou et al., 2006). Antiradical capacity AAC was determined as follows AAC ¼ (% DPPH decrease – 3.6605)/2.7817 determined from linear regression after plotting the % DPPH decrease of known Trolox solution against the quantity of Trolox (mg). Results were expressed as mg Trolox equivalent per g of oil. All spectrophotometric data were acquired with a Uvikon 931 (Contron) UV-V Spectrophotometer. Experiments were performed in duplicate. Rancimat Method Oil stability before and after supplementation was assessed by the Rancimant method at 1108C in a Rancimat 679 apparatus (Metrohm AG, Herisau, Switzerland). The oxidation process was monitored upon 3 g oil sample at air velocity 20 L/h. The effectiveness of commercial and enriched oils was expressed as the protection factor (PF), where


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ET AL.

Table 1. Changes of edible oil stability parameters before and after their enrichment with the olive leaf extract (Olive leaf extract addition corresponded to the addition of 195 mg caffeic acid equivalents/kg oil, containing 185 mg oleuropein/kg oil). Total polyphenols mg CAE/kg oil (F.C. method) Oil Sunflower oil Palm oil Olive oil Vegetable shortening

Antioxidant capacity mg Trolox equivalent/g oil (DPPH method)

Oxidative stability (IT, h) (Rancimat method)

Before

After

Before

After

AC increase (%)

Before

After

Protection Factor (PF)

nd nd 94 nd

155 157 299 175

282 429 140 297

504 715 260 545

79 67 86 84

1.3 17.5 9 4.2

2 21 13.5 5

1.54 1.20 1.50 1.19

PF ¼ ITinh/IT0 where ITinh is the induction time in the presence of an inhibitor and IT0 is the induction time of the non-inhibited system.

RESULTS AND DISCUSSION Four commercial edible vegetable oils (sunflower oil, olive oil, palm oil, and a vegetable shortening) were supplemented with an extract obtained from olive leaves, rich in polyphenols. The oil types were chosen for their different unsaturation degree. The addition of the extract was calculated to correspond to a polyphenol content of approximately 200 mg per kg oil. This complies with the proposed limits by the Joint FAO/ WHO Expert Committee on Food Additives for a given synthetic antioxidant in fats and oils essentially free from water (JECFA, 2005). Both commercial and supplemented oils were analyzed for total and individual polyphenols. Furthermore, they were tested for their antioxidant capacity and oxidative stability. The olive leaf extract, used for the supplementation of the oils, was also analyzed as above. Several parameters, such as olive tree variety, harvest season, and extraction method, may have an effect on the olive leaf polyphenol composition. However, regardless the parameters that might affect the polyphenol content of the extract, the potential use of an olive oil industrial waste for the amelioration of a widely consumed product such as vegetable oils is a matter of concern, especially in the Mediterranean countries. Estimation of Total Polyphenol Content Total polyphenol content of the olive leaf extract was 17.7 mg/mL of extract corresponding to 1770 mg/kg of olive leaf fresh weight. Appropriate quantities of the olive leaf extract, corresponding to the addition of 195 mg polyphenols (CAE) per kg of oil, were used for the preparation of the supplemented oils. Before supplementation polyphenols were detected only in olive oil (Table 1). The olive oil used was selected among others for its low polyphenol concentration (94 mg/kg of oil), compared to the previously reported

average values of olive oil polyphenols (Owen et al., 2000). Extraction of polyphenols from the oils studied after their supplementation, with 195 mg CAE/ kg, revealed a notable polyphenolic concentration following the recoveries: 80% in sunflower oil, 81% in palm oil, 103% in olive oil, and 90% in the vegetable shortening (Table 1). Identification and Quantification of Phenolic Compounds Phenolic compounds identified and quantified in the methanol extracts of oils before and after the enrichment with olive leaf extract are presented in Table 2, together with the phenolic composition of the olive leaf extract. It should also be noted that the analysis of another olive leaf extract obtained from the same region but in a different harvest season revealed the same polyphenol profile even though total polyphenol content was 1350 mg CAE/kg olive leaves, as compared to the 1770 mg CAE/kg oil of the present study. Thus, even if the concentration of the individual polyphenols might vary to some extend the polyphenolic profile is not significantly affected. In our work a specific amount of polyphenols (195 mg CAE/kg oil) was added and its effect on oxidative stability of oils was studied, which is irrelevant to the yield of the polyphenols extraction from olive leaves. Oleuropein was not detected in any of the oil extracts before supplementation, not even in olive oil. Representative HPLC chromatograms for olive oil before and after its supplementation are displayed in Figure 1, while analysis of the other oils resulted in similar chromatograms. In olive leaf extract the concentration of oleuropein was 16.8 mg/mL extract, corresponding to 1680 mg/kg of olive leave fresh weight. After supplementation, oleuropein was detected in all oils at concentrations that ranged from 74.9–116 mg/kg of oil. According to the results obtained, oleuropein recovery in the enriched oils was 41% for sunflower oil, 50% for palm oil, 63% for olive oil, and 60% for vegetable shortening. Oleuropein HPLC recovery was also assessed in the oil samples fortified with standard oleuropein, revealing an average recovery 64%.


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Table 2. Polyphenol species and triterpenoic acids in olive leaf extract and in edible oils before and after supplementation. Leaves mg/kg (f.w.)

Compound Polyphenols (mg/kg of oil) Oleuropein Vanillin Cinnamic acid Tyrosol p-Hydroxy-benzoic acid p-Hydroxy – phenylacetic acid Vannilic acid Hydroxy-tyrosol Protocatechuic acid p-Coumaric acid Ferulic acid Caffeic acid Quercetin Vanilethanediol Triterpenoic acids (mg/kg of oil) Oleanolic acid Ursolic acid Maslinic acid

Vegetable shortening

Before

After

Before

After

Before

After

1680 nd nd 13.8 7.1 9.0 nd 34.1 11.9 15.5 11.2 19.5 52.2 8.5

nd 0.7 nd 0.5 nd nd nd nd nd nd nd nd nd nd

110.8 0.7 nd 0.6 0.3 nd nd 1.3 0.5 0.7 0.9 1.0 2.4 0.4

nd nd nd 0.6 nd nd nd nd nd nd 0.8 nd nd nd

74.9 nd nd 0.6 0.3 nd nd 1.1 0.5 0.8 0.9 1.1 2.4 0.4

nd 0.7 nd 0.6 0.3 nd nd nd nd nd 0.7 nd nd nd

92.2 0.7 nd 0.6 0.3 nd nd 1.1 0.5 0.7 0.9 1.0 2.3 0.4

nd 0.7 0.5 12.3 0.3 0.4 0.6 0.8 nd 0.8 0.8 nd nd nd

116 0.7 0.5 12.8 0.3 0.4 0.6 1.4 0.5 0.8 1.0 1.0 2.3 0.4

18.9 nd 14.9

0.8 nd nd

0.9 nd nd

nd nd nd

0.3 0 0

nd nd nd

0.6 nd nd

125 3.4 72.5

125 3.4 121.3

U

U Olive oil (B)

Olive oil (A)

Standard oleuropein (100 µg/mL) 20

30

Olive oil

After

Olive leaves

10

Palm oil

Before

UV 254 nm 10 mAU

Sunflower oil

40

50

60 min

Figure 1. HPLC chromatograms of oleuropein standard (100 mg/mL), and olive leaf methanol extract together with olive oil methanol extracts: (A) before and (B) after the enrichment with olive leaf extract. For chromatographic conditions see material and methods. U ¼ unknown. In vitro studies have demonstrated that oleuropein acts as an antitumoral compound (Saenz et al., 1998), is able to lower blood pressure and to inhibit platelet-activating factor activity (Andrikopoulos et al., 2002). Oleuropein is also able to enhance nitric oxide production by mouse macrophages (Visioli et al., 1998a) and to decrease inflammatory mediator production (Miles et al., 2005). It has also been found to inhibit in vitro the mycoplasmas (human pathogenic bacteria) (Furneri et al., 2002).

Studies in rats indicate that oleuropein prevents oxidative myocardial injury (Manna et al., 2004). Recent studies demonstrated that oleuropein might be a modulator of metabolism (Polzonetti et al., 2004). Thus, the enrichment of oils with oleuropein increases the nutritional value of oils and could be considered as functional product development. Other polyphenols were also detected and quantified by GC/MS analysis of the oil extracts. Identification of chromatographic peaks was achieved by comparing the retention times and ratios of three fragment ions of each polyphenolic compound with those of reference compounds (SIM method). The corresponding chromatograms for olive oil and palm oil before and after supplementation are presented in Figures 2 and 3, respectively, while the other oils analyzed resulted in similar total ion chromatograms. In olive leaf extract almost the same polyphenols were detected as in olive oil with the exception of quercetin and vanillethanediol, detected only in olive leaves. The main polyphenolic compound in olive leaf extract was oleuropein, in relatively very high quantities (Table 2) followed by quercetin and hydroxytyrosol. In sunflower oil, palm oil, and vegetable shortening before the enrichment, four different polyphenols and one triterpenoic acid were detected (tyrosol, vanillin, p-hydroxy-benzoic acid, ferullic acid, and oleanolic acid). After the enrichment, oleuropein predominated (Table 2) followed by the other target polyphenols in small amounts, while p-hydroxy-phenyl-acetic acid was absent. As far as triterpenoic acids are concerned, only oleanolic acid was present before the enrichment revealing a relatively low increase after the supplementation. In olive oil samples before the


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ET AL.

Olive oil (A)

4000

4000

15

Abundance

16

3

Palm oil (A)

17

3000 6

3000

6

2000

2000

3

7 1000 1 2

4

1000 1

11 12

4

12

8

5

20.00

20.00

40.00

min

40.00

4000

min

Palm oil (B)

4000 15

3 6

3000

12 8

2000 7 1000

12

17

16

8

3000

6 12

2000 3

13 11

1000 1

4

10 9

13 11

14 15

10

4 9

20.00

5 20.00

Abundance

Olive oil (B)

14 40.00

min

Figure 2. GC–MS TIC chromatograms of olive oil methanol extracts: (A) before and (B) after the enrichment with olive leaf extract. For chromatographic conditions see Materials and Methods. Peaks; 1, vanillin; 2, cinnamic acid; 3, tyrosol; 4, p-hydroxy-benzoic acid; 5, p-hydroxy-phenyl-acetic acid; 6, Internal Standard; 7, vanillic acid; 8, hydroxytyrosol; 9; protocatechuic acid; 10, vanilethanediol; 11, p-coumaric acid; 12, ferulic acid; 13, caffeic acid; 14, quercetin; 15, oleanolic acid; 16, ursolic acid; 17, maslinic acid. enrichment, polyphenols were detected in small amounts while triterpenoic acids in relatively high quantities. Supplementation of olive oil with the extract resulted in a concentration increase of some of the already present polyphenols (tyrosol, hydroxytyrosol, and maslinic acid). Moreover, enrichment with polyphenols initially absent from the olive oil (caffeic acid, quercetin, protocatechuic acid, and vanillethanediol) was observed. In the enriched olive oil the concentration of vanillic acid, cinnamic acid, p-hydroxy-benzoic acid, vanillin, p-hydroxy-phenyl-acetic acid, p-coumaric acid, oleanolic acid, and ursolic acid did not alter. Estimation of Antioxidant Capacity The antioxidant capacity of oils was evaluated by the DPPH radical scavenging method. In Table 1 the antioxidant capacity of the oils tested, before and after supplementation with olive leaf extract is presented.

40.00

min

Figure 3. GC–MS TIC chromatograms of palm oil methanol extracts: (A) before and (B) after the enrichment with olive leaf extract. For chromatographic conditions see material and methods. Peaks as numbered in Figure 2. Results are expressed as Trolox equivalents (mg/g oil). Considering the evaluation of total antioxidant capacity by the DPPH scavenging method, the higher the quantity of Trolox equivalents required for scavenging the same quantity of DPPH, the higher the antioxidant capacity of the sample. The DPPH radical is scavenged by the antioxidants present in oils. Thus, the antioxidant capacity of oils is attributed to the tocopherol content (Gordon et al., 1995), polyphenol content (Boskou et al., 2006), and chlorophyll content (Lanfer-Marquez et al., 2005). Total antioxidant capacity before supplementation followed the decreasing order: palm oil, vegetable shortening, sunflower oil, olive oil. After the enrichment the order of antioxidant capacity remained unaltered but an increase of the Trolox equivalent value was detected in all cases, attributed to the polyphenols in olive leaf extract (Table 1). This increase could be attributed mainly to polyphenols, since the olive leaf extract was free of tocopherols, as demonstrated by HPLC analysis of the extract (data not shown). This finding was unavoidable, considering that the olive leaf extract had been repeatedly washed with hexane during its preparation. The highest antioxidant capacity increase (%) was observed for olive oil (86%) followed by vegetable shortening, sunflower oil, and palm oil (67%). Antioxidant capacity was also assessed using oleuropein standard. Supplementation of oils with



standard oleuropein was performed as 185 mg/kg. The amount of 185 mg standard oleuropein was used since this same amount was also found, by HPLC, to be contained in the 195 mg CAE of olive leaf extract added to the oils. Antioxidant capacity increase (%) of oils enriched with standard oleuropein was calculated to be 18% for sunflower oil, 14% for palm oil, 12% for oilve oil, and 11% for the vegetable shortening. According to these values, antioxidant capacity increments of the oils supplemented with standard oleuropein (11–18%) were in all cases lower than those found when supplementation of the oils with the olive leaf extract was performed (67–86%, Table 1). Thus, olive leaf extract contributed to a higher extend on the antioxidant capacity increase than oleuropein standard. As far as the olive leaf extract is concerned, oleuropein contribution (185 mg) to its total polyphenol content (195 mg CAE), assayed by Folin–Ciocalteau, was 95% when expressed as CAE. This remarkably high contribution of oleuropein to the total polyphenol content (95%) would be controversial for the justification of this lower antioxidant capacity increase (11–18%) observed by the addition of oleuropein standard to the oils. However, since the Folin–Ciocalteau assay is a spectrophotometric method, total polyphenol content values calculated are dependent on the polyphenol used for the construction of standard curve. Taking into account that oleuropein was found to be, by HPLC, the main constituent of the olive leaf extract an oleuropein standard curve was additionally constructed. By using this curve the value of 195 mg CAE (olive leaf extract) added per Kg oil was recalculated as 437 mg oleuropein equivalents (OE)/kg oil, and in that case the contribution of oleuropein, calculated by HPLC, to the total polyphenol content expressed as OE, was 42%. Consequently, the differences observed in the antioxidant capacity increase potency among oleuropein standard and olive leaf extract can be attributed to the presence of other polyphenols and the potential synergistic effect occurring among the olive leaf extract components. Among these other polyphenols the unknown peak of Figure 1 might contribute, together with the other simple phenolics of Table 2, to the increase. Assimopoulou et al. (2004) reported that the antioxidant activity of natural extracts increases by the addition of caffeic acid or citric acid due to synergistic effects. Among the oils studied enriched olive oil had better performance. Thus, it appears that olive oil is a better substrate for the natural antioxidants. These findings might be attributed to the stability of olive oil and to its polyphenolic content.

Estimation of Oxidative Stability The oxidative stability of oils was evaluated by the Rancimat method. This method is often used to assess

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the stability of edible oils at high temperatures, i.e., 100–1208C, that are the more preferable for secondary oxidation reactions. In all samples an increase of the induction time (IT) after supplementation was detected in percentages that ranged from 19 to 54% (Table 1), probably due to the polyphenols supplemented into oils. The highest IT increase was observed for sunflower oil (protection factor 1.54) and the lowest for the vegetable shortening (protection factor 1.19). Even though it is very difficult to compare the data obtained with other studies, we tried to make a rough estimation for the evaluation of olive leaf extract contribution on oxidative stability of oils. The results of the present study were in agreement with the results obtained by Bonilla et al. (1999) where the IT of refined olive oil increased after the fortification with antioxidants obtained from red grape marc. According to the results of Bandoniene et al. (2002), the addition of sage, savory, and borage extracts in rapeseed oil in amounts of 0.05% revealed a protection factor of 1.12, 1.00, and 1.12 respectively, while in our study, the addition of 0.02% olive leaf extract resulted in higher protection. Thus, it can be supposed that the olive leaf extract provides higher oxidative stability than the extracts from sage, savory, and borage. The differences in protection factor between the tested oils could be attributed to the type and the quality of oil. Different protection factors could be achieved using the same proportions of antioxidant in different fat qualities (De Leonardis et al., 2007). The efficacy of the extract when added to oils with different stability was studied in olive oil and palm oil. For this purpose new samples of olive oil and palm oil with IT 13.3 and 24 h, respectively, were used for comparison with the respective oils in Table 1. After supplementation with the olive leaf extract the IT values were found 20.8 and 43.1 h, respectively, for the new samples. The protection factors for the new olive and palm oils were 1.56 and 1.27, respectively, values comparable with 1.50 and 1.20, respectively (Table 1). The aforementioned findings indicated that the effect of the extract was not affected by the stability differences of the tested oils. Consequently, the olive leaf extract might increase considerably the oxidative stability of the tested oil, and compared with other extracts provides high protection.

CONCLUSIONS Based on the results of this study olive leaf extract is a good source of polyphenols, mainly oleuropein, hydroxytyrosol and quercetin. Addition of such an extract to edible vegetable oils may contribute to the increase of radical scavenging activity and oxidative stability of oils.


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